[{"id":"10.64898/2026.05.29.721935","pub_date":"2026-5-29","title":"A 667-nucleotide sequence in the SARS-CoV-2 nsp15 coding region promotes genome encapsidation","abstract":"Coronavirus genome encapsidation depends on cis-acting RNA elements that interact with viral structural proteins. While such packaging signals have been characterized in several coronaviruses, their definition in SARS-CoV-2 remains incomplete. Using synthetic defective SARS-CoV-2 genomes, we identify a 667-nucleotide region within the nsp15 coding sequence that preferentially binds SARS-CoV-2 nucleoprotein and enhances the accumulation of defective viral genomes both in vitro and in vivo. Sequential and targeted deletion analyses further delineate candidate RNA secondary structures within this region that contribute to this enrichment. These structures show similarity to elements within the putative packaging signal of SARS-CoV but are not conserved across other coronaviruses. Together, these findings support the presence of a structured RNA element within nsp15 that contributes to SARS-CoV-2 genome encapsidation and provide a framework for further structural and functional dissection of coronavirus packaging signals. This study identifies a 667-nt region within the SARS-CoV-2 nsp15 coding sequence that binds nucleoprotein and promotes accumulation of defective viral genomes, revealing a previously unrecognized contributor to genome encapsidation. Mapping of candidate RNA structures within this region links SARS-CoV-2 packaging activity to conserved structural features observed in SARS-CoV, while highlighting key differences from other coronaviruses. These findings refine understanding of cis-acting packaging signals in SARS-CoV-2 and provide a foundation for further structural and functional analysis of coronavirus genome encapsidation. A part of the nsp15 coding sequence of SARS-CoV-2 promotes efficient transmission of defective viral genomes in vitro and in vivo. Using a sequential deletion library and targeted deletions within this region we identify RNA structures that may function as packaging signals.","version":"1.1","doi":"10.64898/2026.05.29.721935","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.27.728081","pub_date":"2026-5-29","title":"The biodistribution and effect of post-exposure neutralising monoclonal antibody treatment in a mouse model of SARS-CoV-2 infection with viral spread to the brain","abstract":"Ronapreve, a combination of two neutralising monoclonal antibodies, casirivimab and imdevimab, was amongst the authorised treatments against SARS-CoV-2 early in the COVID-19 pandemic. Ronapreve has lost some of its efficiency with the rise of new virus variants, however, it remains a valuable tool for experimental studies to gain insights into the mechanisms and effects of anti-viral drugs. In this study we combined morphological, pharmacokinetic and molecular approaches (including multiomics) to investigate the biodistribution of Ronapreve in the K18-hACE2 murine model of SARS-CoV-2 neuroinvasion, as well as possible consequences for the brain. We also investigated the effect of the treatment on the infection status. Our results showed that after intraperitoneal injection, Ronapreve accumulates in the serum and is unable to cross the blood-brain barrier, thus not reaching the brain parenchyma; treatment has only a minimal effect on the brain transcriptome, with no significant changes in the brain lipidome or metabolome. Nonetheless, post-exposure Ronapreve treatment resulted in reduced viral loads in the lung and, in particular, the brain, with markedly reduced tissue response in the brain, as shown by the transcriptomic analysis. The results suggest a peripheral mode of action of Ronapreve to block brain infection, possibly by lowering viral replication in the nasal epithelium, reducing a subsequent spread to the brain.","version":"1.1","doi":"10.64898/2026.05.27.728081","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.29.728639","pub_date":"2026-5-29","title":"Late induction of IgG4 following SARS-CoV-2 mRNA vaccination in pregnant and non-pregnant individuals includes clonotypes raised early in the response","abstract":"To better understand how pregnancy impacts humoral immunity, we conducted an in-depth longitudinal analysis of the kinetics and characteristics of vaccine responses in a prospective cohort of pregnant and non-pregnant women. Humoral immune responses observed among pregnant participants who received the mRNA-delivered SARS-CoV-2 vaccination, including their effector functions, were in some cases marginally lower than those among non-pregnant controls, while prior infection was associated with some potentiation in humoral responses. Importantly, vaccine-induced antibodies were efficiently transferred across the placenta, providing the fetus with passive immunity and underscoring the dual benefit of maternal vaccination for both mother and neonate against COVID-19. Delayed induction of spike-specific IgG4 following the primary two-dose vaccination series was observed in vaccine recipients, independent of pregnancy status. In a subset (n=6) of pregnant women whose spike-specific serum IgG repertoires were extensively profiled at the clonotypic level over time as part of another study, we proteomically identified secreted IgG clonotypes that had class-switched to IgG4. Matching of these clonotypes detected as IgG4 to those defined as SARS-CoV-2 spike-specific revealed that, while a minority of total clonotypes, they were elicited early in the immunization series and tended to be more highly mutated, more prevalent, and more persistent than clonotypes in the serological repertoire that were not detected as IgG4. Consistent with the increase in secreted vaccine-specific IgG4 over time, but its poorer placental transfer, these clonotypes were detected at greater levels in maternal but not cord blood at the time of delivery as compared to 28 days post the second vaccine dose. These findings indicate some impact in the kinetics, characteristics, and functions of the humoral response that may be associated with pregnancy-related immune modulation. Conservation of the late class-switch recombination to IgG4 that has previously been associated with mRNA-based SARS-CoV-2 vaccines raises questions about how different immunological states and vaccine components influence short- and long-term characteristics of the humoral immune response.","version":"1.1","doi":"10.64898/2026.05.29.728639","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.29.728794","pub_date":"2026-5-29","title":"Preorganized RdRp-Thumb Dynamics Drives SARS-CoV-2 Polymerase Function","abstract":"The SARS-CoV-2 RNA-dependent RNA polymerase drives viral genome replication and is a major antiviral target. How intrinsic conformational dynamics organize functional states of the polymerase, however, remains incompletely understood. Here, molecular dynamics (MD) simulations combined with free-energy landscape analysis reveal that the apo polymerase samples preexisting conformational states defined by coordinated thumb-subdomain motions. Projection of experimental structures, representative of the polymerase nucleic acid cycle, onto the conformational landscape identified discrete basins spanning apo-like and elongation-like states and revealed a coherent structural axis coupling global polymerase compaction (radius of gyration, Rg) with thumb\u2013interface separation (center-of-mass distance, COM). These motions connect catalytic motifs, RNA-binding regions, and distal regulatory elements across the polymerase ensemble. The observed conformational organization is not apparent from static structures alone and supports a model in which functional transitions arise from intrinsic collective dynamics of the apo enzyme. Intermediate conformational ensembles combine structural stability with retained inter-domain flexibility, identifying mechanically responsive states favorable for allosteric modulation. Together, these findings define structurally coupled regulatory regions within the coronavirus polymerase and support conformational trapping of thumb-subdomain dynamics as a potential strategy for antiviral design targeting RNA virus replication machinery.","version":"1.1","doi":"10.64898/2026.05.29.728794","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.135046","pub_date":"2026-5-29","title":"Real-time sharing of drug screening data on the NCATS OpenData Portal accelerates translational research","abstract":"The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform \u2013 named the NCATS OpenData Portal (ODP) \u2013 for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.","version":"1.2","doi":"10.1101/2020.06.04.135046","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.28.728498","pub_date":"2026-5-29","title":"Evaluating Spike Antigenicity across Endemic Human Coronavirus Models using Flow Virometry","abstract":"While SARS-CoV-2 research has advanced rapidly since COVID-19, endemic human coronaviruses (HCoVs) remain comparatively understudied. Tools to phenotype spike (S), the primary antigenic target on coronaviruses, at the single-virion level could improve vaccine design by capturing variation in epitope availability and spike abundance. Here, we establish a calibrated flow virometry (FV) platform to quantify S antigenicity on native endemic (HCoV-229E, HCoV-OC43) and epidemic (SARS-CoV-2) coronaviruses directly in cell culture supernatants. FV revealed cell line\u2013dependent differences in S antigenicity, including receptor-induced changes in epitope accessibility. Comparison of virion-associated S with recombinant stabilized S by ELISA and biolayer interferometry showed consistent binding for HCoV-OC43, MERS-CoV, and SARS-CoV-2, but differences for HCoV-229E, with FV resolving heterogeneity not captured by bulk assays. Finally, FV showed that HCoV-229E from patient-derived air\u2013liquid interface cultures exhibited reduced antibody binding and distinct S antigenicity compared to cell line\u2013derived virions. Together, these findings establish FV as a platform for single-virion analysis of HCoV antigenicity.","version":"1.1","doi":"10.64898/2026.05.28.728498","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.27.728148","pub_date":"2026-5-29","title":"Dysregulated dsRNA sensor signaling and viral infection during onset of pediatric autoimmune interferonopathy","abstract":"Juvenile dermatomyositis (JDM) is characterized by a type I interferon (IFN-I) signature associated with disease activity. We previously identified a link between SARS-CoV-2 infection and the onset or relapse of JDM. Here, we show that newly diagnosed JDM patients display an overexpression of IFIH1 (encoding MDA5 protein) at baseline, coupled with an altered response to dsRNA stimulation at proteomic and transcriptomic levels, indicating abnormal activation of this antiviral sensing pathway. Single-cell transcriptomic and chromatin accessibility profiling of peripheral blood mononuclear cells (PBMCs) further revealed myeloid-specific enrichment of interferon-stimulated genes (ISGs) and preferential disruption of this pathway at disease onset, supporting a dysregulated IFN-I state in this cell type. We identified SARS-CoV-2 RNA in muscle biopsies of two Covid-19 pandemic-onset JDM patients, strongly implicating viral infection as a potential trigger of the dysregulated MDA5 immune response. To extend these observations beyond SARS-CoV-2, we screened two independent retrospective cohorts for antibodies against 27 common childhood infections. In our discovery cohort JDM patients showed significantly increased exposure to 4 RNA viruses in line with our immunological findings. Increased exposure to RSV B was confirmed in an independent replication cohort supporting a robust association with JDM pathophysiology. Together, these findings integrate systemic, single-cell, and tissue-level analyses implicating RNA viral infection and biased antiviral sensing in shaping IFN-I responses at JDM onset, providing mechanistic insight into environmentally triggered pathogenesis. Type I interferon dysregulation at juvenile dermatomyositis onset implicates altered dsRNA sensing and RNA viral exposure as potential disease triggers.","version":"1.1","doi":"10.64898/2026.05.27.728148","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.14.643374","pub_date":"2026-5-27","title":"Systemic Multi-Omics Analysis Reveals Interferon Response Heterogeneity and Links Lipid Metabolism to Immune Alterations in Severe COVID-19","abstract":"The immune response to SARS-CoV-2 infection is highly heterogeneous, and interferon (IFN)-stimulated genes (ISGs) play a central but context-dependent role in antiviral defense and immune dysregulation. To investigate how ISG heterogeneity relates to immune and metabolic states, we performed an integrated analysis of whole-blood transcriptomics, plasma proteomics, metabolomics, and immune activation markers in hospitalized COVID-19 patients and COVID-negative healthy controls and covalescent individuals. Patients segregated into low (LIS), moderate (MIS), and high (HIS) ISG expression endotypes, largely independent of clinical severity. While high ISG expression was associated with systemic inflammation and innate immune activation, severe disease within the HIS endotype was characterized by marked metabolic perturbations, including depletion of tricarboxylic acid cycle intermediates and multiple lipid classes involved in membrane integrity and immunometabolic signaling. Plasma-transfer assays demonstrated that plasma from severe HIS patients impaired neutrophil and monocyte activation ex vivo, indicating functional attenuation of innate immune responses despite elevated ISG expression. These metabolic alterations correlated with reduced immune activation, supporting the existence of an interferon-associated immune-metabolic axis that constrains immune functionality in severe disease. Although type I IFN neutralization was detected in a subset of patients with IFN antigen reactivity, these samples did not account for ISG heterogeneity or disease severity. Together, these findings show that high ISG expression defines a transcriptional endotype permissive for inflammation but insufficient for effective immune function, highlighting the importance of immune\u2013metabolic context in shaping COVID-19 disease outcomes.","version":"1.5","doi":"10.1101/2025.03.14.643374","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.25.727697","pub_date":"2026-5-27","title":"Antibody Transcytosis and Neutralizing Activity in Respiratory Epithelial Cells","abstract":"The neutralizing activity present in human serum is considered a correlate of protection against SARS-CoV-2 infection and disease but the mechanisms by which serum antibodies are transported into the lumen of the respiratory tract, where they are required to interact with virus particles and infected cells remain incompletely understood. The transcytosis and neutralizing activity of serum-derived IgG and IgA antibodies was investigated using an in vitro SARS-CoV-2 infection model with primary differentiated human nasal and basal epithelial cells (hNECs and hBECs) cultures. Expression of the antibody transport receptors neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) in hNECs cultures was confirmed by qPCR, immunofluorescence microscopy, and flow cytometry. Both receptors were expressed throughout the epithelial cultures, with enriched expression observed in ciliated cells compared with goblet and basal cells. Purified IgG and IgA isolated from convalescent plasma demonstrated specificity for SARS-CoV-2 spike protein and inhibited ACE2-Spike interactions, although activity was reduced against later variants. Purified IgG contained higher anti-spike antibody titers than purified IgA. Functional neutralization assays showed that transcytosed IgG and IgA significantly reduced SARS-CoV-2 infection compared with untreated controls. However, serial dilution studies demonstrated that IgG-mediated neutralization was more potent than IgA-mediated neutralization. Similar results were determined for influenza A virus H3N2 subtype. The transcytosis of IgG was more efficient in hBEC cultures while IgA transcytosis was higher in hNEC cultures, reflecting the levels of the corresponding transport proteins. Together, these findings demonstrate that serum-derived IgG and IgA can undergo transepithelial transport across human nasal epithelium while retaining SARS-CoV-2 or influenza A virus neutralizing activity in vitro. These results suggest that FcRn- and pIgR-mediated antibody transport may contribute to mucosal protection following vaccination or infection and may help identify antibody responses associated with protection against SARS-CoV-2. Serum antibody levels are considered correlates of protection for SARS-CoV-2 and Influenza A virus but its unclear how those antibodies are transported to the apical surface of respiratory epithelial cells, where they must be present for optimal activity. We show that IgG and IgA specific for SARS-CoV-2 or influenza A virus is transcytosed across respiratory epithelial cell cultures, the efficiency of which reflects the level of FcRn or pIgR expression levels, suggesting that cells of the upper and lower respiratory tract transport different antibodies from the blood.","version":"1.1","doi":"10.64898/2026.05.25.727697","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.23.727377","pub_date":"2026-5-27","title":"Ordered Gromov-Hausdorff Metric: A New Tool for Comparative Analysis of Protein Structures","abstract":"Classical protein structure comparison metrics such as RMSD and TM-score effectively assess geometric similarity but ignore the linear order of amino acid residues (Zhang and Skolnick, 2004). The Gromov-Hausdorff (GH) metric compares metric spaces by shape but also does not account for order (Gromov, 1981). This can lead to incorrectly identifying proteins with swapped domains as similar. We introduce the Ordered Gromov-Hausdorff (OGH) metric, defined on ordered metric spaces, to incorporate residue order into the comparison. OGH combines coordinate normalization, an exponential penalty for order violations, and a monotonic alignment algorithm with computational complexity O(n\u00b7w), where w is the search window width. It is proven that OGH satisfies all metric axioms for \u03b1 > 0. Analytical properties include invariance under isometries, upper boundedness, Lipschitz continuity under small coordinate perturbations, and concavity in the weight parameter \u03b1. On the VAD dataset (28 viral proteins from HIV-1, SARS-CoV-2, MERS-CoV), OGH increases monotonically with residue shuffling (up to 0.363 at 100% shuffling) and correlates strongly with TM-score (r = 0.706). In the task of separating homologs at fixed global similarity (TM-score \u2248 0.5), OGH achieves AUC = 0.800, whereas TM-score gives AUC = 0.467, demonstrating that OGH detects conserved order even when global geometry is not conserved. The Python source code for OGH is freely available at https://github.com/andytimoffilim/OGH. The VAD dataset (PDB IDs listed in the paper) is publicly accessible from the RCSB Protein Data Bank (Berman et al., 2000; wwPDB, 2019).","version":"1.1","doi":"10.64898/2026.05.23.727377","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.24.727525","pub_date":"2026-5-26","title":"Antigenic characterization of SARS-CoV-2 variants BA.3.2.1 and BA.3.2.2 in three animal models","abstract":"BA.3.2, a variant of SARS-CoV-2 containing \u223c40 mutations in its spike protein compared to its nearest ancestor, has spread globally since its first detection in South Africa in November 2024. Here, we report antigenic characterization of BA.3.2 viruses in three naive animal models, and visualize its antigenic phenotype in the context of SARS-CoV-2 evolution using antigenic cartography. We find that: (1) BA.3.2 is substantially antigenically divergent from existing SARS-CoV-2 variants; (2) infection with BA.3.2 in hamster and mouse animal models produces sera with lower homologous titer than infection with other variants. Both of these results may have implications for the selection of vaccine antigens.","version":"1.1","doi":"10.64898/2026.05.24.727525","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.26.727115","pub_date":"2026-5-26","title":"First evaluation of a human DPP4 transgenic hamster model for MERS-CoV pathogenesis and transmission","abstract":"MERS-CoV poses a constant pandemic risk, as its viral lineages continue evolving, and zoonotic spillover events could lead to random viral polymorphisms that might lead to human adapted variants. Currently, no small animal model reliably recapitulates both disease progression and transmission dynamics, which are critical aspects for counter-viral measures like vaccine development. Although the Syrian hamster is an optimal animal model for SARS-CoV-2 infection and transmission, it is naturally resistant to MERS-CoV infection. Dipeptidyl peptidase-4 (DPP4) is the functional receptor for MERS-CoV infection, and is highly expressed in human kidney, intestine, liver, and lung tissues. Here, we evaluated the suitability of a human DPP4 (hDPP4) transgenic Syrian hamster model for MERS-CoV research. We used two different MERS-CoV strains (EMC/2012 and D10540/2023) for intranasal inoculation of hamsters. Both strains replicated efficiently, led to comparable severe clinical outcomes, and had similar viral transmission efficiencies. MERS-CoV RNA and nucleoprotein antigen were mainly detected in the brain and the respiratory tract. In summary, we validated a novel hDPP4-transgenic hamster as a suitable model for MERS-CoV infection enabling vaccine and transmission research.","version":"1.1","doi":"10.64898/2026.05.26.727115","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.23.726423","pub_date":"2026-5-26","title":"Low seroprevalence of neutralizing antibodies to gorilla adenovirus 32 (GRAd32) in southern African populations supports evaluation of this vector platform for HIV vaccine development","abstract":"Adenoviruses (Ads) are widely used as vaccine vectors. However, pre-existing immunity to commonly used serotypes, like Ad5, can reduce vaccine immunogenicity, with neutralizing antibody titers >200 previously shown to impact vaccine efficacy. Gorilla adenovirus (GRAd) vectors have been developed to evade pre-existing anti-vector responses, but their seroprevalence in southern Africa is poorly defined. Here, we assessed seroprevalence to GRAd32, Ad26 and Ad5 before (baseline) and after COVID-19 vaccination, in cohorts from South Africa and Zimbabwe. Sera from South African participants enrolled in the Sisonke sub-study (n=100, prior to Ad26.COV2.S vaccination) and the follow-up \u201cBooster after Sisonke\u201d (BaSiS) study (n=226) were tested for neutralizing antibodies to Ad5, Ad26, and GRAd32. These samples included paired pre/post boost samples for 27 donors. We also tested sera from the Zimbabwean Mutala cohort (n=131, of which 44 were unvaccinated, and 87 vaccinated with inactivated vaccines). Participants living with HIV (PLWH) comprised 30-50% of each cohort. In the pre-vaccination samples from the Sisonke cohort, geometric mean titers (GMT) for anti-GRAd32, Ad26, and Ad5 antibodies were 78, 142, and 459, with neutralization titers >200 observed in 14%, 32%, and 68% of participants, respectively. Similarly, in the unvaccinated participants in the Mutala cohort, GMTs for GRAd32, Ad26, and Ad5 were 117, 245, and 536, with neutralizing titers >200 in 22%, 42%, and 69% of participants. We observed no significant difference in Ad antibody titers between PLWH and those living without HIV. We next assessed the impact of COVID-19 vaccination on titers. Vaccination with inactivated COVID-19 vaccines (Sinopharm/Sinovac) did not significantly affect Ad5, Ad26 or GRAd32 titers in an unpaired analysis. In contrast, \u223c9 months after Ad26.COV2.S vaccination, anti-Ad26 titers for longitudinally sampled participants (n=27) increased 10-fold from a GMT of 141 to 1,426. By comparison, GRAd32 responses were not significantly altered by Ad26.COV2.S vaccination, while anti-Ad5 responses showed a modest <2-fold increase. Our data support previous findings that, whereas anti-Ad5 neutralizing antibody responses are commonly detected globally, GRAd32 responses are less frequent. Importantly, GRAd32 neutralizing responses remained unchanged after Ad26.COV2.S vaccination. HIV status had no significant effect on antibody titers. These results support the use of the GRAd32 vector in upcoming HIV vaccine trials, including in regions where Ad26-based COVID-19 vaccines were widely deployed.","version":"1.1","doi":"10.64898/2026.05.23.726423","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.26.725122","pub_date":"2026-5-26","title":"Transgenic human dipeptidyl peptidase-4 Syrian hamsters support MERS coronavirus infection and contact transmission","abstract":"Middle East respiratory syndrome coronavirus (MERS-CoV) is a global health concern due to a high fatality rate associated with human infections and no approved vaccines or therapeutics. While Syrian hamsters are a value animal model for coronavirus research, including SARS-CoV-2, MERS-CoV does not infect wild-type hamsters. Here, we generated transgenic Syrian hamsters expressing human dipeptidyl peptidase-4 (hDPP4), the cellular receptor for MERS-CoV., MERS-CoV replicated efficiently in the respiratory tract tissues of hDPP4 hamsters, causing lethal disease. Treatment with the 3CLpro inhibitor nirmatrelvir significantly reduced viral titers in the lower respiratory tract of infected hDPP4 hamsters. While airborne transmission was not observed, direct contact transmission was observed in all contact hDPP4 hamsters cohoused with infected cage mates. Immunization with purified MERS receptor-binding domain protein reduced virus replication and disease severity but did not prevent direct contact transmission. Collectively, our findings demonstrate that hDPP4 transgenic Syrian hamsters are useful for studying MERS-CoV pathogenesis, transmission, and countermeasure efficacy.","version":"1.1","doi":"10.64898/2026.05.26.725122","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.22.727251","pub_date":"2026-5-26","title":"Substrate and target selectivity of 4\u2032-fluoroadenosine against viral and host polymerases","abstract":"Developing safe and effective treatments against emerging RNA viruses is an important goal in pandemic preparedness efforts. 4\u2032-fluorouridine (4\u2032-FlU) is a broad-spectrum antiviral that was shown to inhibit viral RNA-dependent RNA polymerases (RdRps). Given its notable range of antiviral activity, this class of nucleoside analogs warrants further investigation. Here, we studied the antiviral activity and underlying mechanism of inhibition of 4\u2032-fluoroadenosine (4\u2032-FlA). Like 4\u2032-FlU, 4\u2032-FlA demonstrates a broad-spectrum of antiviral activity against eight prototypic viruses representing diverse families. Enzyme kinetics show that the triphosphate (4\u2032-FlA-TP) is efficiently incorporated by viral RdRps. A cryo-EM structure of RdRp of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in complex with double-stranded RNA and the incorporated monophosphate (4\u2032-FlA-MP) characterizes interactions at the active site. The incorporated analog elicits heterogeneous inhibition patterns in primer extension reactions. In contrast, templates with embedded 4\u2032-FlA-MP inhibit incorporation of complementary UTP across the viral RdRps. However, incorporation of 4\u2032-FIA-TP is not limited to viral polymerases and likewise includes human mitochondrial RNA polymerase. These results demonstrate the general potential for 4\u2032-fluorinated nucleotides as antiviral drugs and guide the development of more selective derivatives for medical use in appropriate settings.","version":"1.1","doi":"10.64898/2026.05.22.727251","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.22.727291","pub_date":"2026-5-26","title":"Epistatic evolution drives HLA-dependent CD8+ T Cell escape risk in diverse populations","abstract":"Understanding how viral evolution shapes HLA-dependent T cell escape is crucial to identify individuals at risk of reduced cellular immunity to emerging variants. Nevertheless, we lack frameworks to model HLA diversity and the evolutionary feasibility of T cell\u2013evading mutations. Here, we construct an HLA map capturing variation in epitope specificity across HLA-typed cohorts. Enhancing this framework with SARS-CoV-2 CD8\u207a T cell escape reveals heterogeneous escape across HLA-defined groups, with clusters enriched for HLA-B*07:02, HLA-A*03:01 and HLA-A*02:01 showing higher epitope loss. To assess the evolutionary plausibility of escape, we model viral sequence fitness using an epistasis-aware protein language approach trained on coronaviruses to systematically score mutations across viral lineages. We find that the fitness effect of mutations dynamically changes with evolving sequence context, and that T cell\u2013evading mutations become fitter with additional escape mutations. This study links host HLA diversity to viral fitness landscapes for surveillance and vaccine design.","version":"1.1","doi":"10.64898/2026.05.22.727291","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.21.725708","pub_date":"2026-5-22","title":"Mitigation of imprinted antibody responses in elderly COVID-19 highly vaccinated individuals","abstract":"SARS-CoV-2 continues to evolve from the Omicron serotype, with BA.2.86 sublineage JN.1 and descendants such as KP.2 predominating in 2025-26. By early 2026, the JN.1-derived NB.1.8.1 and XFG variants had largely replaced other variants globally, with a more recent emergence of the highly divergent BA.3.2 saltation variant. Elderly individuals continue to be at greatest risk of clinical complications from COVID-19, yet contemporary data on kinetics of immune potency and breadth following multiple vaccinations remain very limited in this group. We studied a cohort of forty-three healthy older adults (median age = 85 years, IQR 75-88, 40% female). Using both pseudotyped virus (PV) and surrogate virus neutralisation (SVNT) based assays, we demonstrate that JN.1 and KP.2 vaccinations six months apart elicit high potency neutralisation across all studied variants except BA.3.2.2, which escaped neutralisation almost completely in all individuals. Waning of neutralising activity in serum was observed to be modest in the \u223c6 months between vaccine doses, suggesting sustained immunity following multiple vaccines. While absolute neutralisation titres remained highest against ancestral Wu-1 at all timepoints due to multiple historical exposures and accumulation, the recall responses revealed a shift in immunodominance. After the recent KP.2 vaccine dose, neutralisation against full-length Wu-1 spike was not boosted, whereas all tested JN.1 descendants and BA.3.2.2 showed significant boosts, indicating that immune imprinting against ancestral Wu-1 was partially overcome. Interestingly, RBD-specific neutralising responses experienced a boost following KP.2 vaccination, suggesting that RBD responses remain imprinted but that they constitute a small proportion in the overall Wu-1 neutralising response as immune imprinting is alleviated.","version":"1.1","doi":"10.64898/2026.05.21.725708","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.21.727024","pub_date":"2026-5-22","title":"Force-regulated catch bonds and fusion peptide exposure drive coronavirus entry","abstract":"Coronaviruses invade human cells within dynamic mechanical environments through endocytosis and membrane fusion, both mediated by the class I fusion protein spike. In SARS-CoV and SARS-CoV-2, the spike engages the human ACE2 receptor through a catch bond\u2014an interaction whose lifetime increases under tensile force. Concurrently, mechanical pulling facilitates disruption of the S1/S2 subunits of spike, a critical step for membrane fusion. To elucidate how mechanical cues coordinate these processes, we developed a unified elastic-stochastic model that integrates theoretical analysis and computational simulations to trace viral entry. Our results identify the force-regulated catch bond between spike and ACE2 as a key determinant of successful invasion. This catch bond not only enhances receptor-mediated endocytosis but also increases the probability of S1/S2 disengagement, thereby promoting membrane fusion. Importantly, under conditions of strong catch bonding, the force-accelerated separation of S1 and S2 fine-tunes the balance between entry pathways. These findings uncover a potential mechanobiological mechanism that mediates viral cell entry by coupling receptor binding strength with spike disassembly under force. By characterizing these mechanical regulations, this work facilitates the assessment of emerging viral threats and inspires the design of drug delivery systems that leverage catch-bond kinetics for enhanced targeting.","version":"1.1","doi":"10.64898/2026.05.21.727024","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.20.726568","pub_date":"2026-5-21","title":"Spike antibodies targeting GRP78 predispose to cardiovascular complications compared to Dengue","abstract":"One major aftermath of COVID-19 pandemic is cardiovascular consequences. SARS-CoV-2 binds to ACE2 and downregulates vasodilation. Dengue favors hypotension by weakening endothelial glycocalyx leading to plasma leakage. C1q levels, immune complexes (ICs), and proteomic profiles in serum samples from 52 COVID-19 and 19 pre-pandemic Dengue cases were studied. Unlike Dengue, COVID-19 serums showed elevated coagulation proteins promoting vaso-occlusion and peripheral artery diseases. The stress-induced chaperone and atherosclerosis marker, GRP78 (gene/ protein) was found upregulated upon SARS-CoV-2 spike expression in cardiac/ lung cell lines. Elevated GRP78 levels were also observed in serum samples from COVID-19-diagnosed individuals and subjects with myocardial infarction (MI) in post COVID-era. Surprisingly, spike antibodies (Abs) showed cross-binding to GRP78 and possibly contributed to the observed higher-level ICs in COVID-19 serums (cardiovascular embolism?). Co-localization studies showed that spike Abs (analogous to pro-atherosclerotic GRP78 auto-Abs) could directly bind to upregulated cellular GRP78 (type II hypersensitivity?). Both pathways could worsen vascular injury and atherosclerosis, leading to cardiac complications in COVID-19 cases with narrowed vessels.","version":"1.1","doi":"10.64898/2026.05.20.726568","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.20.726704","pub_date":"2026-5-21","title":"Ribophorin-1 Governs Spike Abundance of Highly Pathogenic Coronaviruses by ER-associated degradation","abstract":"The spike protein of highly pathogenic coronaviruses is indispensable for viral entry, pathogenesis, and immune evasion. However, specific host factors governing spike protein degradation and abundance on progeny virions remain largely uncharacterized. Here, we identify Ribophorin-1 (RPN1), a non-catalytic subunit of the oligosaccharyltransferase (OST) complex, as an ER-localized host restriction factor that selectively depletes spike to inhibit coronavirus infection. Conditional knockout of Rpn1 in mouse lung markedly exacerbates SARS-CoV-2 pathogenesis, increasing viral load, immune cell infiltration, and syncytium formation. In cells, RPN1 knockdown enhances susceptibility to diverse variants, whereas its overexpression attenuates infection. Nano-flow cytometry, cryo-EM and LSCM show that RPN1 reduces spike abundance on progeny virions, resulting in reduction in subsequent syncytium formation across SARS-CoV, SARS-CoV-2, and MERS-CoV. Mechanistically, RPN1 promotes VCP/p97-dependent retrotranslocation and AMFR-mediated ubiquitination of spike protein. Heterologous expression of RPN1\u2019s functional domain (residues 180-307) confers significant protection against SARS-CoV-2 both in vitro and in vivo. Collectively, our findings define an RPN1-initiated ERAD pathway that selectively targets coronavirus spike for proteasomal destruction, and provide proof-of-concept evidence that targeting this RPN1-dependent pathway represents a promising broad-spectrum antiviral strategy.","version":"1.1","doi":"10.64898/2026.05.20.726704","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.18.726016","pub_date":"2026-5-20","title":"Mechanochemical Decoupling of ATP Hydrolysis and RNA Translocation in SARS-CoV-2 nsp13 by the L405D Mutation","abstract":"SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved helicase that couples ATP hydrolysis to RNA translocation through long-range allosteric communication between its ATPase and RNA-binding domains. In prior work, we identified L405 as a key regulator of interdomain motions and proposed that the L405D mutation would disrupt this coupling by perturbing conformational translocations required for translocation [J. Phys. Chem. B 2024 v128 492\u2013503]. Subsequent experiments confirmed that L405D attenuates helicase activity while largely preserving ATPase activity, implicating a breakdown in ATP-to-RNA coupling [J. Biol. Chem. 2026 v302 111198]. Here, we provide a data-driven explanation for this decoupling by combining Gaussian accelerated molecular dynamics (GaMD) simulations with Shape-GMM clustering and linear discriminant analysis. Whereas wild-type nsp13 exhibits both conformational selection and induction, L405D collapses the conformational landscape to operate predominantly through selection, eliminating ATP-induced structural transitions required for efficient catalytic cycling. This loss of induction traps the ATP-binding pocket in closed or mid-open conformations, impairing product release and reducing ATP turnover, while simultaneously disrupting coordinated motif\u2013RNA interactions required for inchworm translocation. These findings establish that mutation-induced reshaping of conformational ensembles can modulate access to reaction-competent states, providing a general framework for understanding how targeted mutations disrupt catalytic function through allosteric ensemble remodeling in motor proteins.","version":"1.1","doi":"10.64898/2026.05.18.726016","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.20.725438","pub_date":"2026-5-20","title":"Direct serological antibody discovery by integrative proteomics yields potent neutralizers overlooked by single-cell BCR sequencing","abstract":"Human antibody discovery relies on accessing in vivo-matured repertoires, yet conventional single-cell B cell receptor sequencing (scBCR-seq) often overlooks the most relevant, functional antibodies secreted by plasma cells. Here, we introduce AbDirect, a protein-centric discovery platform that obtains antibody sequences directly from small-volume biofluids. In this proof-of-concept, we apply AbDirect to potent COVID-19 plasma from which an early-pandemic scBCR-seq study did not identify neutralizers. Upfront reactivity screening of anti-SARS-CoV-2 spike protein repertoires revealed diverse clonal profiles with distinct cross-reactivity and subunit specificity. Targeted de novo sequencing via standalone integrative proteomics yielded 14 IgG1 and 4 IgA1 clones that diverged markedly from peripheral B cell counterparts in germline usage and phylogeny, indicating distinct immunological compartments. Validation via recombinant mAbs demonstrated superior binding and highly potent neutralization for multiple sequenced clones (three with IC50 \u22641.4 nM). AbDirect thus yielded potent antibodies overlooked by scBCR-seq, demonstrating serological discovery as a powerful complementary approach for uncovering functional repertoires that may be inaccessible to cell-based methods.","version":"1.1","doi":"10.64898/2026.05.20.725438","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.18.725477","pub_date":"2026-5-20","title":"Phylogenetically estimated neutral rates and fitness effects of mutations to influenza proteins","abstract":"Influenza virus\u2019s rapid evolution is shaped by both neutral mutation and selection. Phylogenetics can be used to study these processes, but this approach has typically only been applied to a few thousand influenza genome sequences at once. Here, we built phylogenetic trees with >100,000 influenza sequences, and then used these trees to estimate neutral rates of mutations to the virus\u2019s genome. Neutral rates varied by up to ~100-fold among the 12 nucleotide mutation types (A\u2192C,A\u2192G, etc.). These rates were highly correlated among influenza, SARS-CoV-2, and HIV, though more nuanced context-dependent patterns showed marked differences between influenza and SARS-CoV-2. We also estimated fitness effects of mutations by comparing the number of times a mutation was observed to occur along the branches of a tree to the number of times we expect it to have occurred under neutrality. We estimated effects for ~33,000 nonsynonymous and ~8,000 synonymous mutations spanning all influenza proteins. This compendium of estimated effects helps map the relationship between sequence and fitness in a natural setting, including regions where synonymous mutations are under functional constraint, and for proteins with limited experimentally measured effects. We built interactive heatmaps of the estimated fitness effects to help readers explore these data (see https://matsen.group/flu-mut-rates). Altogether, this work places influenza\u2019s mutation rates in a broader cross-viral context and deepens our understanding of how mutation and selection shape influenza evolution in nature at a site-specific level.","version":"1.1","doi":"10.64898/2026.05.18.725477","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.15.725339","pub_date":"2026-5-20","title":"KmerSignificance Score: A discriminative and biologically-informed framework for viral k-mer prioritization","abstract":"Distinguishing closely related viral strains requires identifying genomic regions where subtle sequence differences carry biological significance. While k-mer-based approaches offer computational efficiency for genome analysis, existing methods lack standardized frameworks for evaluating which k-mers are most informative. Current selection strategies focus primarily on statistical discriminative power without integrating biological relevance. We introduce KmerSignificance Score (KSS), a k-mer prioritization framework combining three components: an information-theoretic method measuring strain-distinguishing capacity, an optimized amino acid substitution matrix (MIYATA EVO) for mutation impact assessment, and protein-level functional importance scoring derived from UniProt annotations. KSS produces standardized scores in the [0, 1] interval, enabling direct cross-dataset comparison. The discriminative component achieved classification performance comparable or superior to all tested alternatives (mean F1 = 0.880 vs. 0.718\u20130.877 for six established methods) while additionally providing bounded scores with consistent empirical distributions for cross-dataset comparability. MIYATA EVO, optimized via genetic algorithm, improved biophysical property correlations by 28.4% over the original MIYATA matrix. Protein scoring on 17,470 viral proteins showed robust agreement with UniProt annotation scores (Kendall \u03c4 = 0.777) while revealing finer functional distinctions. Literature validation on SARS-CoV-2 (278,738 sequences, 19 variants), HIV-1 (12,223 sequences, 15 subtypes), and human cytomegalovirus (HCMV; 399\u2013646 sequences, 4\u20138 genotypes) confirmed that high-scoring k-mers consistently map to established variant-defining mutations, subtype-specific polymorphisms, and genotype markers. KSS provides a standardized framework for viral k-mer prioritization with applications in variant surveillance, molecular epidemiology, and functional annotation. The tool is available at https://github.com/bioinfoUQAM/KmerSignificanceScore. Identifying genetic differences between closely related viral strains is essential for pandemic preparedness, vaccine development, and understanding disease outbreaks. With millions of viral genomes now sequenced, researchers need tools that can rapidly pinpoint which genomic differences matter most biologically, not just which are statistically distinctive. Current k-mer-based approaches identify patterns that distinguish viral strains but cannot assess whether those differences affect protein function or disease phenotype. We developed KmerSignificance Score (KSS), a framework that we designed to rank short genomic sequences by combining three types of evidence: how well they distinguish viral strains, how much the encoded amino acid changes affect protein properties, and how functionally important the affected protein is. We standardized the resulting scores on a 0-to-1 scale, allowing direct comparison across different viruses and studies. We validated our framework on three major human pathogens (SARS-CoV-2, HIV-1, and human cytomegalovirus) and found that top-scoring positions consistently correspond to sites with documented roles in immune evasion, drug resistance, viral fitness, and strain classification. Our framework can help prioritize genomic features for surveillance of emerging variants, guide experimental validation, and support molecular epidemiology.","version":"1.1","doi":"10.64898/2026.05.15.725339","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.16.725625","pub_date":"2026-5-19","title":"Variant emergence, not vaccine deployment, drives episodic positive selection on the SARS-CoV-2 spike at provincial scale in Canada","abstract":"Mass immunization against SARS-CoV-2 created a heterogeneous landscape of antibody-mediated immune pressure, yet whether this pressure measurably altered episodic positive selection on spike remains unresolved. Using Canadian genomic surveillance data spanning the five major variants of concern (Alpha, Beta, Gamma, Delta, and Omicron), we inferred time-resolved phylogenies from spike-coding sequences and applied site- and branch-level episodic selection models to identify when and where adaptive change occurred. To evaluate whether vaccination intensity was associated with selection, we integrated these phylogenetic analyses with provincial vaccination time series using cross-correlation and lagged panel regression models that accounted for province and time effects, lineage prevalence, and sampling heterogeneity. Episodic positive selection was concentrated at a limited number of spike codons, especially within the N-terminal domain, receptor-binding domain, and furin cleavage region. However, these signals were dominated by substitutions associated with variant emergence, particularly during the Alpha-to-Delta transition, rather than by vaccination rollout. Whole-gene tests provided no evidence that vaccine intensity was associated with elevated episodic selection, and residualized vaccination trajectories did not predict selection at biologically plausible lags. Across provinces, the timing and distribution of selection events were inconsistent with a vaccine-driven escape model. Together, these results indicate that, at provincial resolution in Canada, episodic positive selection on SARS-CoV-2 spike was driven primarily by variant turnover rather than vaccine deployment. More broadly, this study provides a quantitative, VOC-resolved assessment of spike evolution in a structured epidemic and suggests that population-level vaccination intensity was not a detectable determinant of spike adaptation in the period examined.","version":"1.1","doi":"10.64898/2026.05.16.725625","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.15.725542","pub_date":"2026-5-18","title":"Orally Bioavailable SARS-CoV-2 Protease Inhibitors Bearing a Hydroxymethyl Ketone Warhead","abstract":"The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies.","version":"1.1","doi":"10.64898/2026.05.15.725542","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.05.674565","pub_date":"2026-5-16","title":"Epistasis detection and fitness valleys in viral evolution","abstract":"Probabilistic prognosis of virus evolution, vital for the design of effective vaccines and antiviral drugs, requires the knowledge of adaptive landscape including epistatic interactions. Although epistatic interactions can, in principle, be inferred from abundant sequencing data by various methods, it has been shown previously that genetic linkage between evolving sites obscures their signature and requires averaging over several independent populations. Here we probe the limits of epistasis detection based on pairwise correlations conditioned on the state of a third site on synthetic sequences evolved in a Monte Carlo algorithm with known epistatic pairs in two scenarios: an initially diverse population in the presence of recombination and mutation, with a uniform epistatic network, and an initially\u2013uniform population in the presence of mutation and a fitness valley. In either scenario, the detection error decreases with the number of independent populations and increases with the sequence length. In the first scenario, the accuracy is enhanced by moderate recombination and is maximal, when epistasis magnitude approaches the point of full compensation. In the second scenario, simulation predicts the spontaneous crossing of a fitness valley and forming a new large strain.. The method is then applied to several thousands of sequences of SARS-CoV-2. Results obtained under equal sampling from world regions are consistent with the existence of fitness valleys connecting groups of viral variants. The few epistatic pairs of genomic sites hide in genomic data among numerous random correlations caused by common phylogenetic history. We test a method of epistasis detection designed to compensate for this noise. The accuracy is tested using synthetic sequences generated by a Monte Carlo algorithm with known epistatic pairs. The method is applied to several thousands of sequences of SARS-CoV-2 sampled in two different ways. Results obtained under equal sampling from world regions imply the existence of fitness valleys connecting groups of viral variants.","version":"1.3","doi":"10.1101/2025.09.05.674565","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.15.725307","pub_date":"2026-5-15","title":"A Structural Domain in the genomic RNA of SARS-CoV-2 Folds into a Compact Granular Structure without the N protein: A Single-Molecule Fluorescence Spectroscopic Investigation","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) packages its single-stranded genomic RNA (gRNA) having about 30,000 nucleotides into virions by forming 35\u201340 granular ribonucleoprotein (RNP) units. Each RNP unit has a diameter of \u223c15 nm. While it is generally assumed that the assembly of these RNPs is driven by the binding of the nucleocapsid (N) protein to the gRNA in the cytoplasm, the precise molecular mechanism remains to be fully elucidated. In this study, we develop an experimental strategy based on single-molecule fluorescence and fluorescence correlation spectroscopies to examine the formation of long-range base pairing within a candidate structural domain corresponding to nt 12230-12686 of the gRNA (gRNA12k). Our results demonstrate that the 5\u2019 and 3\u2019 regions of gRNA12k autonomously form long-range base pairing in near-physiological buffers containing mono- and divalent cations, independently of the N protein. This domain possesses an extensive secondary structure, is compact, and can unfold and refold reversibly upon heat treatment and cooling. Notably, the addition of the N protein melts the long-range base pairs, and causes the aggregation of multiple molecules of gRNA12k. Based on these observations, we propose a refined mechanism for the genome assembly in SARS-CoV-2: gRNA initially forms autonomous granular structures, which are subsequently reorganized and condensed by the N protein to chaperone the assembly of the entire gRNA. SARS-CoV-2 organizes its exceptionally long genomic RNA (gRNA) having about 30,000 nt into 35\u201340 granular ribonucleoprotein (RNP) units for viral packaging. It has been assumed that the nucleocapsid (N) protein drives the formation of the RNP granules. In this study, we challenge this prevailing view by demonstrating that a specific region of the gRNA sequence inherently encodes the information to fold into a compact, granular architecture independently of any proteins. Unexpectedly, we found that the N protein partially melts the autonomous structures, suggesting that it acts as an RNA chaperone to facilitate flexible genome assembly. Our findings redefine the interplay between viral proteins and gRNA, offering a new perspective on the mechanism of coronavirus replication.","version":"1.1","doi":"10.64898/2026.05.15.725307","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.14.725177","pub_date":"2026-5-15","title":"Expansion Revealing of Pathology Resolves Nanostructures Associated with Inflammatory Phenotypes in COVID-19 Decedent Human Brain Tissue","abstract":"Expansion revealing (ExR) elucidates cellular organization by separating proteins within dense nanostructures by 20x linear expansion, but requires fixation procedures incompatible with human pathology specimens. Here, we report ExR of pathology (ExRPath), which attains \u223c20 nm resolution and decrowding of such tissues, through iterative 20x expansion, adapted to human brain pathology specimens. We also report a single-shot 15x expansion protocol for such tissues (15ExMPath), achieved through one-shot 15x expansion. Applying ExRPath and 15ExMPath to COVID-19-decedent brain tissue reveals periodic amyloid nanoclusters that co-localize with SARS-CoV-2 in a rare minority of patient specimens, pointing to a potential neuroinflammatory phenotype associated with COVID-19, and highlighting the power of high-throughput nanoimaging, empowered by expansion microscopy, for discovering potential novel disease mechanisms.","version":"1.1","doi":"10.64898/2026.05.14.725177","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.13.725047","pub_date":"2026-5-15","title":"Immunological imprinting shapes the cross-reactive antibody responses to the KP.2 and LP.8.1 vaccine doses","abstract":"The emergence of the SARS-CoV-2 Omicron BA.2.86 subvariant, a lineage derived from the BA.2 strain, led to the 2024-2025 COVID-19 vaccine update to include KP.2 or related JN.1-lineage spike antigens. We evaluated the magnitude, breadth, and durability of humoral immune responses following a single KP.2 vaccine dose in a longitudinal cohort of 21 individuals up to six months. KP.2 vaccination increased spike-specific binding and neutralizing antibodies against the ancestral WA1 strain, alongside the BA.5, XBB.1.5, and KP.2 variants. Power law modeling estimated half-lives for WA1- and KP.2-specific IgG responses at 770 and 248 days, respectively. Additionally, the KP.2 dose increased IgG1 and IgG4 subclasses more than IgG2 and IgG3 responses to both spike proteins. Serum depletion experiments using WA1 or KP.2 proteins demonstrated most vaccine-elicited antibodies were cross-reactive. Consequently, KP.2 vaccine-induced antibodies retained broad neutralizing activity against recently circulating Omicron subvariants (BA.2.86, KP.3.1.1, XEC, LP.8.1, LF.7, XFG.3.12, PQ.1, BA.3.2.1, and RE.2). Using a live virus neutralization assay, XFG.3.12 showed the greatest reduction in neutralizing titers relative to KP.2 (4.2-fold). In a small subset, an LP.8.1 vaccine dose increased neutralizing activity against the matched variant while maintaining WA1 and KP.2 cross-reactivity, but only modestly increased antibodies to divergent variants BA.3.2.1 and RE.2. Ultimately, these data indicate the KP.2 mRNA vaccine generates durable, cross-reactive responses against current Omicron subvariants. However, ongoing spike evolution impacts neutralization of emerging lineages, highlighting the need for continued viral monitoring and timely vaccine updates. SARS-CoV-2 continues to evolve, raising ongoing concerns about how well updated vaccines protect against emerging variants. This study evaluates antibody responses after KP.2 spike mRNA vaccine dose and shows that a single dose induces durable and broadly cross-reactive immunity against both earlier strains and recently circulating Omicron subvariants. Despite this breadth, reduced neutralizing activity against certain emerging variants indicates that ongoing antigenic changes can impact vaccine induced antibody effectiveness. These findings provide insight into how current vaccines perform over time and highlight the need to track viral evolution and update vaccine antigens to maintain broad protection against severe disease, hospitalization, and death.","version":"1.1","doi":"10.64898/2026.05.13.725047","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.08.674976","pub_date":"2026-5-14","title":"Nanoscopy Reveals Heparan Sulfate Clusters as Docking Sites for SARS-CoV-2 Attachment and Entry","abstract":"Virus entry is thought to involve binding a unique receptor for cell attachment and cytosolic entry. For SARS-CoV-2 underlying the COVID-19 pandemic, angiotensin-converting enzyme 2 (ACE2) is widely considered the receptor for cell-surface attachment and cytosolic entry. Using advanced light microscopy to resolve individual virions and receptors, we found instead that heparan sulfate (HS), not ACE2, mediates SARS-CoV-2 cell-surface attachment and subsequent endocytosis. ACE2 functions only downstream of HS to enable viral genome expression. Instead of binding single HS molecules that electrostatically interact with viral surface proteins weakly, SARS-CoV-2 binds clusters of \u223c6\u2013137 HS molecules projecting 60\u2013410 nm above the plasma membrane. These tall, HS-rich clusters, present at about one per 6 \u03bcm\u00b2, act as docking sites for viral attachment. Blocking HS binding with the clinically used HS-binding agent pixantrone strongly inhibited an authentic pathogen, the SARS-CoV-2 Omicron JN.1 subvariant, from attaching to and infecting human airway cells. This work establishes a revised entry paradigm in which HS clusters mediate SARS-CoV-2 attachment and endocytosis, with ACE2 acting downstream, thereby identifying HS interactions as a key anti-COVID-19 strategy. This paradigm and its therapeutic implications may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator. Viral entry, a crucial antiviral target, is typically thought to involve binding its unique receptor for the cell surface attachment and subsequent entry. We examined this concept with advanced microscopies to resolve individual receptors and SARS-CoV-2 virions responsible for the COVID-19 pandemic. We discovered two receptors for viral entry: heparan sulfate, a polysaccharide that may bind many viruses, mediates viral attachment and subsequent endocytosis, whereas angiotensin-converting enzyme 2 (ACE2), the generally assumed SARS-CoV-2 receptor, acts only downstream to facilitate viral infection. This model suggests perturbation of HS binding as a more effective anti-COVID-19 strategy than previously recognized. It may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator.","version":"1.2","doi":"10.1101/2025.09.08.674976","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.10.698783","pub_date":"2026-5-14","title":"Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation","abstract":"The nucleocapsid protein of SARS-CoV-2 scaffolds genomic RNA into ribonucleoprotein complexes (RNP) for assembly in the virion, but also fulfills critical intracellular functions in replication and the suppression of host defense. It is comprised of a folded nucleic acid binding domain (NTD) and a dimerization domain, connected by a disordered linker containing a serine/arginine-rich (SR) region and a leucine-rich sequence (LRS). The switch between intracellular and assembly functions of N-protein is controlled by phosphorylation of the SR region, but the molecular details are unclear. Here we describe a model in which two mutually exclusive intramolecular loops bind the NTD and dynamically control self-association and nucleic acid binding properties dependent on the SR linker phosphorylation state. The model is supported by biophysical properties and interactions of full-length protein, point mutants, and peptide fragments. We find SR linker phosphorylation compacts the protein and inhibits nucleic acid binding and RNP formation, while enhancing self-association through promotion of transient coiled-coils in the LRS of the linker. These changes shift the nucleocapsid protein to a configuration poised for multi-valent interactions that support intracellular functions.","version":"1.2","doi":"10.64898/2026.01.10.698783","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.12.724581","pub_date":"2026-5-14","title":"LOCALE: Local-Alignment Embeddings for Noise-Robust DNA Search at SRA Scale","abstract":"Searching petabase-scale repositories of raw sequencing data such as the NIH Sequence Read Archive (SRA) could transform biological discovery, but existing methods either do not scale well or rely on exact k-mer matching that is brittle to sequencing errors and biological divergence. We recast sequence search as dense retrieval: we learn vector embeddings whose inner-product similarity ranks locally aligned sequences above unaligned ones. Our key observation is that effective retrieval does not require accurate regression of global edit distance\u2014it only requires that sequences with better local alignments score higher than sequences with worse ones. We train a DNABERT-2 encoder with an InfoNCE objective on biologically informed augmentations: overlapping crops of parent sequences corrupted with substitutions, insertions, and deletions. On a 50-accession SRA benchmark, LOCALE maintains 62.4% average Recall@Rq at a 10% mutation rate, while every baseline we evaluated falls below 60% Recall@Rq in the noisy-query setting. The advantage holds at scale: on a 500-accession, 15-Gbp benchmark, LOCALE achieves AUPRC 0.508 at 10% mutation versus 0.129 for MetaGraph.","version":"1.1","doi":"10.64898/2026.05.12.724581","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.12.724615","pub_date":"2026-5-12","title":"Recovery of proofreading-impaired SARS-CoV-2 reveals a mutator phenotype and an ExoN activity threshold for viability","abstract":"Coronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3\u2032-to-5\u2032 exoribonuclease (ExoN) formed by nonstructural proteins 14 (nsp14) and 10 (nsp10). Previous studies suggested that inactivation of the ExoN catalytic site in severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is lethal, leaving unresolved whether the virus can tolerate impaired proofreading activity. Here, we investigated the functional requirement for ExoN in SARS-CoV-2 replication by combining a continuous fluorescence-based biochemical assay with an optimized single-bacmid reverse genetics system. Mutational analysis of residues involved in RNA binding or catalysis revealed graded effects on ExoN activity in vitro. Alanine substitution of Lys9, a residue positioned near the RNA-binding interface, did not reduce ExoN activity, whereas charge reversal at this position (K9E) impaired activity more strongly than alanine substitutions of the catalytic motif I residues D90 and E92 (D90A/E92A). Correspondingly, recombinant SARS-CoV-2 carrying K9A was readily recovered, whereas the D90A/E92A mutant was recovered only after an extended delay and K9E could not be rescued despite repeated attempts. The D90A/E92A mutant exhibited reduced replication while maintaining the engineered ExoN substitutions during serial passage. Deep sequencing of viral populations revealed a marked increase in genome-wide sequence variation in the D90A/E92A mutant, demonstrating a stable mutator phenotype. Together, these findings indicate that SARS-CoV-2 can tolerate substantial impairment of ExoN activity but depends on a minimal activity threshold for viability. This system provides a platform for defining how SARS-CoV-2 proofreading controls genome stability, viral fitness, and sensitivity to antiviral strategies that exploit reduced replication fidelity. Coronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened, because earlier studies suggested that loss of the enzyme\u2019s catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading, but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading.","version":"1.1","doi":"10.64898/2026.05.12.724615","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.04.657802","pub_date":"2026-5-12","title":"In silico design and validation of high-affinity RNA aptamers for SARS-CoV-2 comparable to neutralizing antibodies","abstract":"Nucleic acid aptamers hold promise for clinical applications, yet understanding their molecular binding mechanisms to target proteins and efficiently optimizing their binding affinities remain challenging. Here, we present CAAMO (Computer-Aided Aptamer Modeling and Optimization), which integrates in silico aptamer design with experimental validation to accelerate the development of aptamer-based RNA therapeutics. Starting from the sequence information of a reported RNA aptamer, Ta, for the SARS-CoV-2 spike protein, our CAAMO method first determines its binding mode with the spike protein\u2019s receptor binding domain (RBD) through a multi-strategy computational approach. We then optimize its binding affinity via structure-based rational design. Among the six designed candidates, five were experimentally verified and exhibited enhanced binding affinities compared to the original Ta sequence. Furthermore, we directly compared the binding properties of the RNA aptamers to neutralizing antibodies, and found that the designed aptamer TaG34C demonstrated a comparable binding affinity to the RBD compared to the representative neutralizing antibodies analyzed in this study. This highlights its potential as an alternative to existing COVID-19 antibodies. Our work provides a robust approach for the efficient design of a relatively large number of high-affinity aptamers with complicated topologies. This approach paves the way for the development of aptamer-based RNA diagnostics and therapeutics.","version":"1.5","doi":"10.1101/2025.06.04.657802","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.07.723385","pub_date":"2026-5-12","title":"Crude Fucus vesiculosus fucoidan demonstrates superior SARS-CoV-2 antiviral activity compared to its pure form: binding kinetics and functional studies","abstract":"Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 \u00b5M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 \u00b5M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 \u03bcM) for ACE2 were greater than that of Fvp (66.8 \u03bcM), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 \u03bcg/mL) compared to Fvp (IC50 > 239 \u03bcg/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.","version":"1.1","doi":"10.64898/2026.05.07.723385","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.07.723210","pub_date":"2026-5-12","title":"Temperature-Dependent Replication and Sensitivity to Innate Immunity of Human Coronavirus HKU1","abstract":"The human coronavirus HKU1, causing common colds and occasionally severe illness, remains largely uncharacterized because it has not been successfully grown on immortalized cells. Here, we identified Caco2 cells overexpressing TMPRSS2, the HKU1 receptor, as being highly permissive to infection. HKU1 replicated efficiently, formed syncytia and released infectious progeny in these cells at 33\u00b0C, the temperature of the nasal cavity, but was attenuated at 37\u00b0C. Viral entry occurred similarly at both temperatures, but subsequent viral RNA synthesis was enhanced at 33\u00b0C. Released virions displayed higher stability at 33\u00b0C. In Caco2 and primary epithelial nasal cells, HKU1 was sensitive to interferons (IFN), but induction of IFN stimulated genes, such as IFN-Induced Transmembrane Proteins (IFITMs), was delayed at 33\u00b0C. Once expressed, IFITMs comparably inhibited HKU1 fusion at both temperatures. In contrast, SARS-CoV-2 robustly replicated at 37\u00b0C. Thus, cellular permissiveness, innate immunity and viral properties collectively explain why HKU1 replicates more efficiently at nasal temperature. Our results highlight temperature-sensitivity disparities between coronaviruses, likely associated to different pathogenic outcomes.","version":"1.1","doi":"10.64898/2026.05.07.723210","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.08.709729","pub_date":"2026-5-11","title":"Double-edged swords: Anthracyclines inhibit -1 programmed ribosomal frameshifting and restrict HCoV-OC43 infection but show cytotoxicity","abstract":"Human coronavirus OC43 (HCoV-OC43) constitutes one of the most common causes of the seasonal cold but can also cause severe disease among elderly and immuno-compromised. Currently, there are no approved antiviral drugs to combat HCoV-OC43 infection. Coronaviruses are positive-sense single-stranded RNA (+ssRNA) viruses and utilize -1 programmed ribosomal frameshifting (-1 PRF) to produce the correct stoichiometry of viral protein components. Due to its high conservation, the ribosomal frameshifting stimulation element (FSE) is a promising target for antiviral drug discovery. Aminoglycosides and anthracyclines interact with structured nucleic acids and some of these compounds affect -1 PRF in SARS-CoV-2. To get a comprehensive view of their putative efficacy, we examined all commercially available aminoglycosides and anthracyclines for their ability to affect -1 PRF in HCoV-OC43 and their potential to reduce HCoV-OC43 infection. Of 25 candidates tested, we identified 3 anthracyclines (Idarubicin, Nogalamycin and Daunorubicin) to reduce HCoV-OC43 -1 PRF using in vitro assays. We further demonstrate that the active anthracyclines, but not the inactive anthracycline, bind to the HCoV-OC43 FSE. The active anthracyclines did not significantly affect -1 PRF in SARS-CoV-2, suggesting differences in the interactions of the anthracyclines and FSEs, despite being relatively conserved in HCoV-OC43 and SARS-CoV-2 RNAs. Interestingly, these potent anthracyclines also significantly reduced HCoV-OC43 infection in human host cells. Although, the anthracyclines show some toxicity and introduce lesions in cellular DNA they could constitute an important scaffold for further antiviral development, with the aim to increase efficacy and reduce toxicity. The recent Covid-19 pandemic caused by SARS-CoV-2 showed the need for functional antivirals to immediately combat infections at the onset of future pandemics. In addition to SARS-CoV-2, four other human coronaviruses can cause mild to severe infection. Among those is HCoV-OC43, a coronavirus particularly troublesome for immunocompromised patients. Despite this clinical relevance, no approved antiviral therapies are currently available for the prevention or treatment of HCoV-OC43 infections. In this work, we have identified a subset of anthracyclines (Idarubicin, Nogalamycin and Daunorubicin) that selectively and significantly reduce HCoV-OC43 infection. In contrast to a non-active anthracycline, the same anthracyclines that reduce infection also reduce ribosomal frameshifting and interact with a structured RNA in the viral RNA-genome. Interestingly, Idarubicin, a clinically used anticancer drug, shows selectivity for HCoV-OC43, but not for SARS-CoV-2 RNA. Our work demonstrates that anthracyclines could be important for pandemic preparedness and serve as attractive scaffolds when developing new antivirals targeting coronaviruses.","version":"1.2","doi":"10.64898/2026.03.08.709729","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.06.723356","pub_date":"2026-5-08","title":"The Effect of Vaccination on the Evolution of the SARS-CoV-2 B.1.351 Variant","abstract":"Since the initial distribution of the SARS-CoV-19 vaccine, its widespread use has been hypothesized to act as a selective pressure that drives the COVID-19 virus to mutate. This study aims to investigate the correlation between global vaccination rates and the mutation rate of the SARS-CoV-2 Beta variant (B.1.351). From January to July 2021, nucleotide diversity increased in tandem with vaccination rates, demonstrating that the virus evolved more rapidly in response to selective pressure from mass vaccination. Statistical analysis revealed statistically significant positive correlations between both vaccination rates and vaccine doses administered with nucleotide diversity. Thus, our findings indicate a positive correlation between rising vaccination rates and nucleotide diversity, suggesting that increased vaccination coverage acted as a selective pressure that accelerated viral evolution of SARS CoV2.","version":"1.1","doi":"10.64898/2026.05.06.723356","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.01.715984","pub_date":"2026-5-08","title":"SARS-CoV-2 5\u2019-UTR stem-loops activate the antiviral protein oligoadenylate synthetase 1 (OAS1)","abstract":"The innate immune system relies on pathogen recognition receptors, such as the 2\u2019,5\u2019-oligoadenylate synthetase (OAS) proteins, to detect pathogen-associated molecular patterns like viral double-stranded (ds)RNA. A specific splicing variant of OAS1 (OAS1-p46) has been implicated in initiating an immune response that leads to decreased disease severity during SARS-CoV-2 infection. OAS1-p46 has a C-terminal lipid modification motif that allows for anchoring of the protein to intracellular membranes and thus potential colocalization with immunogenic viral RNA regions such as the SARS-CoV-2 5\u2019-untranslated region (5\u2019-UTR). Here, we show that OAS proteins can detect the 5\u2019-structured elements (5\u2019-SE)\u2013comprising the 5\u2019-UTR and three additional stem-loop structures\u2013and activate the cellular RNase L pathway. Through systematic 3\u2019-end truncations of the 5\u2019-SE, we show that the smallest 5\u2019-SE fragment capable of potently activating OAS1 is the conserved and highly structured SL1-4b region, containing the first four stem-loop structures and their intervening linking sequences (SL1-4), as well as an unstructured region (\u201cSL4b\u201d) to the 3\u2019-side of SL4. Analyses of OAS1 activation and RNA secondary structure probing using selective 2\u2019-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) of additional SL1-4b RNA variants suggests a model in which SL4 acts as the primary OAS1 interaction site, while the unstructured SL4b region and two other stem-loops (SL1 and SL3) are necessary for optimal presentation of this region for OAS1 activation. Our findings reveal a structurally complex viral RNA region that potently activates OAS1, underscoring the potential complexity of RNAs that can strongly activate this innate immune sensor.","version":"1.2","doi":"10.64898/2026.04.01.715984","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.07.723425","pub_date":"2026-5-08","title":"Site-Specific Entry Factors Define Cellular Susceptibility to SARS-CoV-2 in Human Tissues","abstract":"SARS-CoV-2 primarily infects the respiratory epithelium through entry factors ACE2 and TMPRSS2. However, alternative viral entry receptors and/or modulators of viral entry factors expression may contribute to infection in respiratory as well as non-respiratory tissues, including the kidneys and colon. Using single-cell analyses, we identified novel candidates for spike-mediated viral entry in epithelial cells from diverse fresh human tissues exposed to a labeled spike-pseudotyped virus. Systematic viral tracking revealed tissue-specific membrane receptors that contribute to viral entry, functionally validated in human tissues and partially predicted by molecular modeling. We found ADAM17 and IL1R1, together with canonical molecules, to significantly facilitate viral entry in the lung parenchyma, while entry into the renal cortex was less dependable on canonical factors, but strongly modulated by JAK1/2 engagement. These findings uncover distinct viral entry mechanisms across tissues and suggest novel organ-specific therapeutic targets for patients at risk.","version":"1.1","doi":"10.64898/2026.05.07.723425","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.06.723370","pub_date":"2026-5-08","title":"vartracker: an end-to-end tool for pathogen longitudinal variant analysis and visualisation","abstract":"Longitudinal sequencing can reveal fine-grained pathogen evolution during acute and chronic infections and inform public health responses. However, integrating ordered pathogen genomic data into a coherent evolutionary and clinical framework can be tedious and error-prone. We present vartracker, an open-source tool for longitudinal pathogen variant analysis and visualisation. Given an ordered sample manifest, vartracker supports three entry points: raw sequence reads, reference-aligned BAM files, or user-supplied VCF and coverage inputs. Raw-read and BAM inputs are processed through an integrated Snakemake workflow, whereas VCF mode starts from precomputed files. Variants are normalised and annotated relative to a reference genome, tracked across timepoints, and classified as original or newly emerging and as transient or persistent. Inferred amino acid changes are reported, and for SARS-CoV-2 analyses, relevant published literature for key mutations can be automatically linked through a functional database. vartracker outputs a schema-documented results table, provenance metadata for reproducibility, publication-quality static figures, and an interactive heatmap for data exploration. Although packaged with SARS-CoV-2 reference assets and initially developed for SARS-CoV-2 datasets, vartracker is pathogen-agnostic when appropriate reference data are supplied. We demonstrate its utility using SARS-CoV-2 and respiratory syncytial virus A (RSV-A) datasets. vartracker is freely available through GitHub, PyPI and Bioconda.","version":"1.1","doi":"10.64898/2026.05.06.723370","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.06.723221","pub_date":"2026-5-08","title":"Helminth-remodeled microbial indole-3-lactic acid drives AhR-dependent disease tolerance","abstract":"Helminths systemically suppress host immunity, yet whether they impose immune tolerance by rewiring host-associated microbial metabolism remains unclear. Here we show that Trichinella spiralis infection remodels intestinal tryptophan metabolism to generate an AhR-dependent regulatory immune state. T. spiralis infection enriched the commensal bacterium Ligilactobacillus murinus, which converted tryptophan into indole-3-lactic acid (ILA), a microbial metabolite that directly engaged the aryl hydrocarbon receptor. Antibiotic-mediated microbiota depletion abolished infection-induced ILA accumulation, AhR activation and Treg/Th17 rebalancing, whereas fecal microbiota transplantation from infected donors or supplementation with L. murinus or ILA restored these effects. Pharmacological blockade or genetic deletion of AhR eliminated the ability of T. spiralis, L. murinus and ILA to restrain LPS-induced cytokine-storm-like lung inflammation, establishing AhR as an essential host node in this circuit. Extending these findings to viral inflammatory disease, oral ILA improved survival and reduced pulmonary immunopathology in SARS-CoV-2-infected K18-hACE2 mice. Re-analysis of human COVID-19 metabolomic data further revealed reduced circulating ILA in severe disease. These findings define a helminth-remodeled microbial tryptophan metabolic pathway that promotes disease tolerance and identify the ILA\u2013AhR axis as a candidate postbiotic strategy for limiting hyperinflammatory tissue injury.","version":"1.1","doi":"10.64898/2026.05.06.723221","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.06.723222","pub_date":"2026-5-07","title":"SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation","abstract":"Despite the end of the COVID-19 pandemic, SARS-CoV-2 continues to circulate endemically, highlighting the need to better understand the viral determinants of pathogenesis. Non-structural protein 2 (Nsp2) has been implicated in host\u2013virus interactions, yet its function remains poorly defined in the context of infection. Here, we generated a recombinant SARS-CoV-2 lacking Nsp2 (\u0394Nsp2) to investigate its role in viral replication and disease. While \u0394Nsp2 replicated comparably to wild-type virus in vitro and in vivo, its deletion resulted in markedly attenuated disease in K18-hACE2 mice. Wild-type infection induced a strong pro-inflammatory response associated with increased recruitment of monocytes and macrophages, whereas \u0394Nsp2 infection promoted a more balanced antiviral response characterized by enhanced lymphocyte and NK cell recruitment. This was accompanied by reduced pulmonary and systemic inflammation and distinct transcriptional programs, including downregulation of pathways related to RNA processing and translation. Mechanistically, CLIP-seq and proximity labeling suggest that Nsp2 interacts with host RNA and components of the translational machinery. Together, our findings identify Nsp2 as a key virulence factor that drives immunopathology by skewing host immune responses, highlighting its role as a regulator of host\u2013pathogen interactions.","version":"1.1","doi":"10.64898/2026.05.06.723222","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.06.723231","pub_date":"2026-5-07","title":"Functional Landscape of Motifs within the Sarbecovirus Spike Cytoplasmic Tail","abstract":"Sarbecoviruses exhibit extensive diversity in host range and zoonotic potential. Although the ectodomain of the viral spike protein has been well characterized, the functional landscape of the cytoplasmic tail (CT) remains poorly defined. To address this gap, we systematically generated CT truncation variants of representative spike proteins from all major ACE2-utilizing clades to define the roles of conserved host factor\u2013interacting motifs associated with COPI, COPII, FERM, and SNX27. Using vesicular stomatitis virus (VSV)- and lentivirus-based pseudotyping systems, we evaluated viral entry in cells expressing varying levels of ACE2 and TMPRSS2. Our results demonstrate that CT-associated motifs differentially regulate viral infectivity. Specifically, truncation of the COPI- or SNX27-binding motifs markedly reduces entry efficiency, whereas disruption of the COPII-binding motif produces the opposite outcome. By contrast, removal of the FERM-binding motif consistently enhances infectivity across lineages. Mechanistically, truncation of this motif increases spike expression, cell surface localization, incorporation into virions, and particle stability. Importantly, despite these pronounced effects on viral infectivity, deletion of the FERM-binding motif does not affect antigenicity, receptor dependence, or sensitivity to protease inhibitors, as demonstrated by neutralization and inhibition assays. In addition, this approach substantially increases spike protein density on virus-like particles (VLPs). Collectively, by extending the analysis beyond SARS-CoV-1 and SARS-CoV-2, our study reveals a generalizable mechanism in which cytoskeletal anchoring mediated by the FERM-binding motif acts as a limiting determinant of viral assembly. These findings provide a practical framework for optimizing pseudovirus platforms and guiding vaccine development against emerging viral threats.","version":"1.1","doi":"10.64898/2026.05.06.723231","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.05.723025","pub_date":"2026-5-06","title":"Tocilizumab induces significant changes in longitudinal proteomes of blood serum from patients with severe COVID-19 pneumonia","abstract":"Coronavirus disease 2019 (COVID-19) shows highly variable clinical outcomes that are not fully explained by age or comorbidities, underscoring the importance of host molecular responses in determining disease severity. Proteomic and multi-omics studies have linked severe COVID-19 to profound dysregulation of immune, inflammatory, and coagulation pathways, and have shown that circulating protein signatures can predict clinical trajectories. Tocilizumab (TCZ), a monoclonal antibody targeting the interleukin-6 receptor (IL-6R), is an established therapy for IL-6\u2013driven inflammatory diseases and can normalize aberrant molecular profiles. Here, we applied longitudinal serum proteomics to patients with severe SARS-CoV-2 pneumonia treated with TCZ to further characterize how IL-6R blockade reshapes the systemic inflammatory milieu. After TCZ administration, several clinical and inflammatory markers, including C-reactive protein (CRP), CCL5 and CXCL10, decreased. Proteomic profiling revealed that TCZ exerts a sustained effect on the serum proteome, with the most pronounced changes emerging 7 days after treatment. These changes were associated with a broad reconfiguration of the proteomic profile toward a pattern resembling a healthy physiological state, characterized by the restoration of key protein abundances to levels comparable to those observed under homeostatic conditions. Collectively, our findings support that TCZ treatment contributes to the normalization of the inflammatory state in severe COVID-19 and represents a viable therapeutic option for managing the acute inflammatory phase of the disease, while also highlighting additional pathways and biomarkers involved in this recovery process.","version":"1.1","doi":"10.64898/2026.05.05.723025","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.04.722452","pub_date":"2026-5-06","title":"Longitudinal analysis reveals myeloid cell contributions to neuroPASC pathogenesis","abstract":"Neurological and neuropsychiatric symptoms, collectively termed neuroPASC, are among the most prevalent Post-Acute Sequelae of COVID-19 (PASC). Neuroinflammation \u2013 particularly microglia reactivity \u2013 has been implicated in neuroPASC. Current insights are largely derived from post-mortem tissues of acutely infected patients and may not reflect PASC-related neuropathology. We previously established a PASC model in which SARS-CoV-2-infected mice developed persistent behavioral alterations and prolonged neuroinflammation for up to 120 days post-infection (dpi) in the absence of viral neuroinvasion. Here, we extended these results to a longitudinal single-cell RNA sequencing analysis of brain immune cells collected at 0, 6, 30, and 100 dpi. We identified a coordinated contribution of infiltrating and resident myeloid cells to the initiation and persistence of neuroinflammation. In specific, microglia displayed sustained expansion of subclusters characterized by inflammatory, stress response, and metabolic signatures. Border-associated macrophages upregulated monocyte attractants during acute infection. Concurrently, monocytes and neutrophils showed marked brain recruitment and mounted transient inflammatory responses at 6 dpi, potentially triggering long-term microglial reactivity. Together, these findings provide a high-resolution atlas of brain myeloid immune dynamics during neuroPASC and highlight a central role for microglia in sustaining chronic neuroinflammation.","version":"1.1","doi":"10.64898/2026.05.04.722452","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.17.512569","pub_date":"2026-5-05","title":"N6-Adenosine Methylation of a Single Site in SARS-CoV-2 5\u2032-UTR Promotes Translation","abstract":"The coronavirus disease 2019 (COVID19) led to devastating health outcomes and has continued to spread despite global vaccination efforts. This, alongside the rapid emergence of vaccine resistant variants, creates a need for orthogonal therapeutic strategies targeting more conserved facets of severe acute respiratory syndrome coronavirus (SARS-CoV-2). The viral genome is a single positive RNA strand divided into a genomic and a subgenomic segment. All 16 non-structural viral proteins are translated from the genomic polycistronic ORFs 1a, and 1b using a single 5\u2032-UTR leader. To our surprise, the full length 5\u2032-UTR efficiently initiates protein translation despite its predicted structural complexity. Through a combination of biochemical assays and bioinformatic analyses, we demonstrate that a single METTL3-dependent m6A methylation event in SARS-CoV-2 5\u2032-UTR regulates the rate of translation initiation. We demonstrate that m6A likely exerts this effect by destabilizing the third stem loop (SL3) and increasing overall accessibility to protein complexes. Our discovery provides a foundational insight into the biology of SARS-CoV-2 by linking m6A modification to its translational regulation and, thus, opens a new avenue for potential novel therapeutic strategies.","version":"1.2","doi":"10.1101/2022.10.17.512569","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.04.722688","pub_date":"2026-5-05","title":"The conserved QTQTX motif in the SARS-CoV-2 spike protein is dispensable for cleavage and lung cell entry of the emerging variant BA.3.2","abstract":"The furin motif in the SARS-CoV-2 spike (S) protein is important for lung cell entry. It is embedded in an extended loop structure and preceded by a highly conserved QTQTX motif that is required for efficient furin cleavage of the SARS-CoV-2 WA-1 S protein. BA.3.2 is an emerging SARS-CoV-2 saltation variant that is spreading globally in April 2026 and encodes for a highly mutated S protein. Here, we analyzed whether the QTQTX motif is also required for spike protein cleavage and lung cell entry of BA.3.2. We report that two patient-derived spike sequences of the BA.3.2 subvariant BA.3.2.2 lack the first QT repeat of the QTQTX motif and show that this motif is largely dispensable for both cleavage and lung cell entry of BA.3.2.2, which we found to depend on TMPRSS2. Our results suggest that the reconfiguration of the BA.3.2 S protein during persistent infection may have significantly altered the determinants of furin cleavage. The furin motif in the SARS-CoV-2 spike (S) protein is unique among sarbecoviruses and constitutes a virulence determinant. A QTQTX motif located immediately upstream of the furin motif is required for furin cleavage of the S protein of the virus that circulated early in the pandemic. Here, we show that the QTQTX motif is largely dispensable for S protein processing and S protein-driven lung cell entry of the emerging saltation variant BA.3.2, which is currently spreading globally. Thus, BA.3.2 evolution within immunocompromised individuals may have relaxed the requirements for furin processing of the spike protein.","version":"1.1","doi":"10.64898/2026.05.04.722688","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.05.722935","pub_date":"2026-5-05","title":"Targeted metagenomic recovery of coronaviruses from wildlife samples","abstract":"Coronaviruses (CoVs) are diverse RNA viruses infecting a wide range of hosts, with significant implications for public health, animal production and welfare. Bats are key reservoirs of mammalian CoVs and have contributed to the emergence and circulation of several zoonotic viruses in a wildlife context, while mustelids represent important hosts at the human-animal interface, as highlighted by SARS-CoV-2 outbreaks in farmed mink. Using metagenomic next-generation sequencing, we screened bats and wild mustelids and recovered and characterized full-length CoV genomes. Building on previous findings in Danish wildlife, including alpha-CoVs and a MERS-related beta-CoV in bats, this study expands current knowledge of coronavirus diversity and evolution in wildlife, emphasizing their relevance for zoonotic emergence.","version":"1.1","doi":"10.64898/2026.05.05.722935","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.03.722459","pub_date":"2026-5-04","title":"SARS-CoV-2 (E)-protein induces rapid TLR2-mediated T cell activation in mouse lungs revealed by intravital lung microscopy","abstract":"Mounting evidence indicates that T cells can operate in an innate-like mode challenging the classical description of T cells as strictly adaptive immune effectors. T cells can engage innate pattern recognition receptors to mount rapid but antigen-nonspecific responses to infection or cellular stress. This study observed that CD8+ T cells, and to a lesser extent also CD4+ T cells, responded to viral proteins in the mouse lung quickly in an innate-like fashion. We employed intravital lung microscopy to visualize infiltration of CD8+ T cells into the lung following intratracheal instillation of the SARS-CoV-2 envelope (E)-protein. Here, we demonstrate acute recruitment of CD8+ from the pulmonary microcirculation into the lung as early as 4 and 24 hours after (E)-protein instillation. The acute infiltration of CD8+ T cells was not observed in Tlr2\u2212/\u2212 mice. Immunohistochemistry analysis of mouse lungs revealed T cell accumulation in nodular inflammatory foci (NIF) of the lung at perivascular regions and around large airways. Stimulating spleen-derived CD8+ T cells from wild-type mice with (E)-protein ex vivo in combination with cytokines or TCR agonists significantly upregulated CD69 and activated secretion of interferon (IFN)\u03b3 which was not observed with CD8+ T cells isolated from Tlr2\u2212/\u2212 mice. These findings indicate rapid bystander activation of CD8+ T cells by the SARS-CoV-2 envelope (E)-protein that depends on (E)-protein sensing by TLR2. This innate-like CD8+ T cell response to SARS-CoV-2 (E)-protein may offer novel opportunities for diagnostic and therapeutic development, warranting further investigation.","version":"1.1","doi":"10.64898/2026.05.03.722459","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.30.721889","pub_date":"2026-5-04","title":"Single-cell atlas of transcript usage remodelling in antiviral immune responses across human populations","abstract":"Humans exhibit substantial interindividual variation in their immune responses to infection, yet the contribution of transcript usage \u2014the relative abundance of gene isoforms\u2014 to this variation remains poorly understood. Here, we generate the first single-cell atlas of transcript usage variation during early responses to influenza A virus and SARS-CoV-2 across 160 individuals of African and European ancestry. We show that viral stimulation induces widespread remodelling of transcript usage across all major immune lineages, with changes that are largely lineage-restricted and frequently undetected at the gene expression level. We further find that ancestry-associated effects on transcript usage are predominantly cell type-specific and contribute to population differences in antiviral responses. In addition, the genetic regulation of transcript usage during stimulation differs between influenza A and SARS-CoV-2, pointing to virus-dependent regulatory architectures. Together, our findings establish transcript usage as a dynamic regulatory layer shaping responses to viral infection across immune cell types and human populations, providing new insights into the molecular basis of variation in human antiviral immunity.","version":"1.1","doi":"10.64898/2026.04.30.721889","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.30.721971","pub_date":"2026-5-04","title":"Condensate-Like Organization in Respiratory Aerosols Modulates the Dynamics of an Airborne Virus","abstract":"The molecular behavior of viruses within respiratory aerosols plays a critical role in airborne disease transmission yet remains largely inaccessible to experimental characterization. Here, we use a billion-atom all-atom molecular dynamics simulation of a virus-laden respiratory aerosol to uncover how respiratory proteins, lipids, ions, and water collectively assemble around SARS-CoV-2, giving rise to structured microenvironments that influence viral stability and spike dynamics. We find that respiratory components rapidly evolve into heterogeneous networks characterized by protein-rich aggregates, patchy lipid assemblies, and spatially structured ion and water dynamics. These features create distinct microenvironments that constrain molecular transport and stabilize regions surrounding the virion. Within this crowded aerosol context, we observe sustained and selective interactions between aerosol components and the viral spike protein, including preferential recruitment of surfactant lipids and persistent coordination by divalent cations. These interactions modulate spike conformational dynamics, enhancing domain breathing motions and flexibility at key hinge regions while preserving a stable membrane anchor. Together, these observations reveal a condensate-like physical regime in which multivalent aerosol components coalesce into a soft, heterogeneous matrix that selectively modulates viral protein dynamics under extreme crowding. By framing virus-laden respiratory aerosols within this physical context, this work establishes an in situ molecular framework for understanding how aerosols influence viral persistence and offers a platform for exploring mechanisms relevant to airborne disease transmission and mitigation strategies. Respiratory aerosols exhibit condensate-like physical properties that govern the evolution of the particle and modulate the behavior of airborne SARS-CoV-2.","version":"1.1","doi":"10.64898/2026.04.30.721971","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.30.721865","pub_date":"2026-5-04","title":"Modeling Site-Specific Mutation Patterns in Pandemic-Scale Phylogenetics","abstract":"Models of genome evolution often account for different evolutionary rates at different genome positions due to, e.g., varying selective pressures or mutation rates. Recent evidence from millions of publicly shared SARS-CoV-2 genomes has revealed a more complex mutational landscape than can be modeled with existing approaches. Here, mutation rates are in fact not only highly position-specific, as currently modeled, but also nucleotide-specific; for example, specific mutations can occur very often at certain determined genome positions, while at the same positions other mutations might not be highly recurrent. Here, we propose and investigate a general model of genome evolution where each genome position is allowed to evolve under an independent, non-normalized substitution rate matrix describing site-specific rates of all mutation types (\u201cSite-Specific Matrix\u201d model, or SSM). We implement SSM in the efficient pandemic-scale phylogenetic inference software CMAPLE. Large-scale genomic epidemiological simulations suggest that, given enough data, SSM can accurately infer position- and nucleotide-specific substitution rates for more frequently observed nucleotides (typically the reference nucleotide), while other rates require higher levels of divergence. Simulations also show that SSM has a modest impact on the accuracy of phylogenetic tree estimation. We use SSM to analyze the evolution of millions of SARS-CoV-2 genomes and observe substantial mismatches between the substitution rates of classical rate variation models and our SSM estimates. These results suggest that classical models of rate variation are inadequate for modeling site-specific mutation patterns and that SSM is a useful alternative for large-scale genome analyses.","version":"1.1","doi":"10.64898/2026.04.30.721865","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.08.698513","pub_date":"2026-5-04","title":"A genetically encoded reporter reveals coordinated interferon responses in neurons and non-neuronal cells in the brain","abstract":"Interferons (IFNs) are canonical antiviral cytokines with emerging homeostatic functions across tissues, including in the brain. However, detecting IFN-responsive cells remains challenging, particularly in the immune-restricted brain environment, limiting our understanding of the full breadth of IFN signaling across diverse cell types. Here we developed a novel mouse reporter that detects IFN responses in the brain and peripheral tissues, including in neurons. This tool, IFN-brite, Bright Reporter of Interferon sTimulated gene Expression, has two copies of the fluorophore mGreenLantern downstream of the native Isg15 IFN response gene. We observed IFN responses across multiple cell lineages. Deletion of the Type I interferon receptor Ifnar1 largely abrogated these responses, including to IFN\u03b3, suggesting that Type II signaling can trigger secondary Type I responses. We observed IFN-responsive cells including neurons in a model of ischemic stroke and during experience-dependent neural plasticity. Furthermore, we found that peripheral infection with Sars-CoV2 but not influenza A leads to IFN-responsive neurons in the brain, suggesting differential impacts of these two pathogens in the nervous system. These data define a broadly useful new tool for studying IFN responsive cells and provide evidence that diverse cell types including neurons can respond to IFNs.","version":"1.3","doi":"10.64898/2026.01.08.698513","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.30.721843","pub_date":"2026-5-04","title":"The redesign of the molecular scaffold of viral ion channel blockers","abstract":"The rise of new, rapidly mutating viruses presents increasing challenges for drug developers. Traditional methods, such as high-throughput screening and drug repurposing against mutagenic viral targets, have recently shown their limitations. Our current rational molecular engineering approach offers a sustainable solution by targeting viral ion channels, which generally have low mutation rates. First, extending the amantadine molecule led to the development of new compounds that better match the alternating hydrophobic and hydrophilic patterns of the inner walls of ion channels\u2014a common feature across many viruses. Then, simplifying the structure yielded a cyclohexylamine-based minimalist scaffold that effectively blocks the ion channel and demonstrates improved antiviral activity compared to well-known agents such as amantadine and arterolane. SARS-CoV-2 variants served as test systems in laboratory experiments. The new molecular scaffolds presented here provide a strong foundation for designing potent, broad-spectrum viral ion channel blockers.","version":"1.1","doi":"10.64898/2026.04.30.721843","journal":"bioRxiv","score":null},{"id":"10.64898/2026.05.02.722415","pub_date":"2026-5-04","title":"Connecting Cryo-EM and Crystallographic Views of RNA Folding through Ionic Conditions and Structural Flexibility","abstract":"Discrepancies between biomolecular structures resolved by cryo-electron microscopy (cryo-EM) and X-ray crystallography (XRD) often arise from differences in ionic conditions and construct design, yet their mechanistic impact on RNA folding remains unclear. In the SARS-CoV-2 frameshifting stimulatory element, cryo-EM and XRD structures reveal distinct pseudoknot conformations\u2014a bent and a coaxially stacked state\u2014complicating its structure\u2013function relationship. Here, combining all-atom explicit-solvent simulation results with a structure-based electrostatic model, we show that Mg\u00b2\u207a ions drive transitions between these states by stabilizing long-range tertiary interactions and modulating local dynamical coupling involving the slippery site and stem 3. Energy landscape analysis reveals distinct folding pathways, while deletion of the slippery segment in crystallographic constructs alters intermediates and produces pathways inconsistent with single-molecule optical tweezer experiments. This study demonstrates how condition-dependent experiments encode complementary interaction-level information and how physics-based computational approaches integrate these to yield a coherent, mechanistic picture of RNA folding.","version":"1.1","doi":"10.64898/2026.05.02.722415","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.16.628708","pub_date":"2026-5-03","title":"An unsupervised framework for comparing SARS-CoV-2 protein sequences using LLMs","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic led to 700 million infections and 7 million deaths worldwide. While studying these viruses, scientists developed a large amount of sequencing data that was made available to researchers. Large language models (LLMs) are pre-trained on large databases of proteins and prior work has shown its use in studying the structure and function of proteins. This paper proposes an unsupervised framework for characterizing SARS-CoV-2 sequences using large language models. First, we perform a comparison of several language models previously proposed by other authors. This step is used to determine how clustering and classification approaches perform on SARS-CoV-2 sequence embeddings. In this paper, we focus on surface glycoprotein sequences, also known as spike proteins in SARS-CoV-2 because scientists have previously studied their involvement in being recognized by the human immune system. Our contrastive learning framework is trained in an unsupervised manner, leveraging the Levenshtein distance from pairwise alignment of sequences when the contrastive loss is computed by the Siamese Neural Network. The final part of this paper focuses on a comparison with a previous approach on a test dataset containing data from the latter part of the pandemic. In the prediction of emerging variants, the proposed LLM-based approach shows an improvement of 0.2 in terms of the adjusted rand index clustering compared to a previously proposed approach. This shows the potential of applying large language models to this field.","version":"1.3","doi":"10.1101/2024.12.16.628708","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.13.711660","pub_date":"2026-5-03","title":"SARS-CoV-2 virus infection of Peromyscus leucopus demonstrates that infection tolerance is not limited to agents for which deermice are reservoirs","abstract":"The North American deermouse Peromyscus leucopus is reservoir for several zoonotic agents, including bacterial, protozoan, and viral. It is remarkable for indiscernible or limited fitness consequences of these infections, a trait known as infection tolerance. But experimental infections have largely been of pathogens that P. leucopus naturally harbors. We asked whether infection tolerance extended to an agent, like SARS-CoV-2 virus, it had presumably not encountered before. Following protocols for experiments with mice and hamsters, we infected 8 female and 8 male P. leucopus of heterogeneous stock and compared responses of these animals on days 3 or 6 to those of 14 controls inoculated with virus-free medium. Serologic and virologic confirmation of infection was obtained for all exposed deermice. Moderate inflammation in lungs was histologically evident in infected animals, but no histological changes were noted in brains, even when viral RNA was present. Fourteen (88%) animals displayed no or only mild sickness; two had more severe illness. Genome-wide RNA-seq revealed an interferon-stimulated response on day 3 superceded mainly by a cell-mediated response by day 6. In brains transcription of the interferon-stimulated genes Isg15 and Mx2 positively correlated with viral RNA levels. The findings confirmed susceptibility of this species of Peromyscus to SARS-CoV-2 virus. For most infected outbred animals the immune response was swift and effective in controlling the pathogen and without evidence of excessive inflammation. Whatever is the basis for P. leucopus\u2019 trait of infection tolerance, it extended to at least one pathogen that for it would be novel. Peromyscus leucopus is North American rodent that is reservoir for several agents of human disease, while exhibiting minimal illness, a phenotype termed infection tolerance. Whether this trait is pathogen-specific or represents a broader strategy has remained uncertain. By experimentally infecting P. leucopus with SARS-CoV-2 virus, which it is unlikely to have encountered, we investigated whether infection tolerance extends to a novel virus. Despite disseminated infection and lung pathology, most animals showed only mild or no disease. Expression analyses revealed early interferon-stimulated responses followed by cell-mediated responses with only limited production of inflammatory mediators interferon-gamma and nitric oxide synthase 2. Compared with results with a mouse model of infection, deermice displayed higher baseline expression of antiviral genes and quicker resolution of interferon responses. These findings suggest that infection tolerance is a strategy that limits immunopathology generally while resisting microbes, which has implications for understanding reservoir competence and host resilience.","version":"1.2","doi":"10.64898/2026.03.13.711660","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.22.720070","pub_date":"2026-4-30","title":"Bridging spatial scales in respiratory viral infection","abstract":"Respiratory viruses spread within the host through both local expansion and occasional long-range dissemination that seeds new infection foci. We present LEAP, an analytically tractable within-host model that captures this two-scale process by coupling local plaque growth to long-range seeding. The model reduces to an age-dependent branching process and yields a closed-form expression for the exponential growth rate during early infection. Using empirical data to parametrize the model, we find that productive dissemination requires only a small number of successful long-range seeding events per infected cell, with distinct values for SARS-CoV-2 and influenza A virus. LEAP further predicts that, in these well-adapted viruses, interferon-mediated restriction only weakly affects exponential growth, while remaining decisive for poorly adapted ones. More broadly, the model provides a flexible framework for experimentally testable predictions of early infection dynamics. Respiratory viral infection is an inherently spatial process, in which the virus must colonize large areas of the airways to optimize reproduction. Recent studies in animal models infected with influenza A or SARS-CoV-2 have documented long-range stochastic jumps of viral populations between distant regions of the respiratory tract. The emerging picture is one of two co-occurring spatial processes: slow, local plaque expansion and long-range seeding events that are rare but crucial to rapid colonization of the airways. We introduce LEAP (Lotka-Euler Airway Pathogen model), a simple mathematical within-host model that captures these two-scale dynamics by coupling local plaque growth to stochastic long-range seeding. Using LEAP, measurements from the petri dish can directly produce predictions of infection dynamics in the body.","version":"1.2","doi":"10.64898/2026.04.22.720070","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.27.721205","pub_date":"2026-4-29","title":"T-cell repertoire response in individuals with post-acute sequelae of COVID-19","abstract":"T-cells are central to SARS-CoV-2 clearance and immunological memory, yet their contribution to the persistence of post-acute sequelae of COVID-19 (PASC) remains poorly understood. The immunological features that distinguish individuals who develop PASC from those who recover fully are unresolved, in part due to the phenotypic heterogeneity of the condition and the likely multiplicity of its underlying mechanisms. Here, we profiled longitudinal bulk TCR\u03b2 repertoires from 120 individuals in the INCOV cohort\u201371 with PASC and 49 without\u2013sampled at two to three time points spanning the acute and post-acute phases of infection. Using robust statistical modeling of repertoire composition and clonal dynamics, we found that global statistics such as V, J gene usage and CDR3 length do not differ between groups, but that locally enriched sequence motifs and differentially dynamic clones reveal distinct T-cell signatures associated with PASC status. Clones contracting following the peak of the acute response were significantly enriched for SARS-CoV-2 specificity in both groups. Interestingly, Influenza A-specific TCRs were disproportionately enriched among contracting clones in PASC+ repertoires, implicating viral co-infection as a potential contributor to early disease severity and, possibly, PASC pathogenesis. Rare public TCR clones were markedly enriched for SARS-CoV-2 specificity, with PASC+ individuals harboring a modestly but significantly higher proportion than PASC\u2212 individuals. Together, we identified over 1,000 candidate TCR\u03b2 receptors potentially discriminating PASC+ from PASC\u2212 immune responses, opening a path toward the identification of disease-relevant T-cell specificities and the development of T-cell-based immunological biomarkers for long COVID.","version":"1.1","doi":"10.64898/2026.04.27.721205","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.27.721131","pub_date":"2026-4-29","title":"Naturally occurring ACE2 stalk variants are differentially released from the cell","abstract":"Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin-aldosterone system (RAAS). It also acts as a receptor for SARS-CoV-2 and stabilises the B0AT1 amino acid transporter at the cell surface. Therefore, surface expression of ACE2 is crucial for these physiological processes. ACE2 is released as a soluble, catalytically active form, partly through ectodomain shedding. This process mainly involves the sheddases ADAM10 and ADAM17, but the exact regulatory mechanisms remain unclear. We assessed 11 naturally occurring single-point mutations in the ACE2 stalk region. Most variants showed significantly reduced release compared to wild-type (WT) ACE2; however, the single point mutations P734L and G726R significantly increased their release. ACE2_P734L also exhibits higher surface expression, directly increasing the surface levels of B0AT1. Despite B0AT1 and ACE2 forming a tight tetrameric complex, this did not affect ACE2 shedding. This suggests that complex formation does not restrict sheddase access. Overall, these data identify the ACE2 stalk region as a major determinant of shedding efficiency. Naturally occurring variants in this region can substantially affect the release of soluble ACE2, potentially contributing to interindividual differences that are relevant for pathophysiological processes.","version":"1.1","doi":"10.64898/2026.04.27.721131","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.28.721328","pub_date":"2026-4-28","title":"Distinct virus-derived circular RNA molecule influences host response during SARS-CoV-2 infection","abstract":"Virus-derived circular RNA molecules (VcircRNAs) are expressed by many RNA viruses during infection. Putative functions include modulating viral replication and interacting with the host immune response. Some function as non-coding RNA fragments that regulate gene expression through binding to complementary RNA sequences, whereas others contain internal ribosomal entry site (IRES) sequences or non-canonical modifications that allow them to be translated. Here, we confirm the expression of a distinct SARS-CoV-2 VcircRNA molecule, circ7b8N, that has not been previously identified. We found that circ7b8N is expressed and detectable in cell culture infections and in acute infections across SARS-CoV-2 variants and shows promise for detection in post-acute clinical samples. Conservation of circ7b8N junctions is limited to the nearest phylogenetic relatives within the betacoronavirus genus but are present in other human and bat-infecting coronaviruses. Host cell gene expression is modulated by the treatment with circ7b8N agnostic of viral infection. The discovery and subsequent confirmation of circ7b8N expressed by SARS-CoV-2 provides a new biomarker for infection, and its conservation across variants suggests functional importance. Circular RNAs are a well-documented class of molecules expressed by mammalian cells. However, circular RNA molecules expressed by RNA viruses remain largely uncharacterized regarding their generation, specific functions, and roles in host-pathogen interactions. Our computational predictions discovered thousands of distinct circular RNA molecules expressed by SARS-CoV-2. Among these, we confirmed the presence of circ7b8N, the most abundant SARS-CoV-2-derived circular RNA identified in our sequencing data. We found that circ7b8N localizes outside the nucleus and is detectable in clinical samples collected both during and after acute SARS-CoV-2 infection. Although overexpression of circ7b8N was not found to alter viral titers, it modulated the expression of host genes related to immune response activation and membrane remodeling. This suggests that circ7b8N may simultaneously provide pro- and anti-viral functions independent of influencing viral replication. Phylogenetic analyses of coronaviruses suggest that the expression of circ7b8N is a relatively recent evolutionary event, and it is conserved across SARS-CoV-2 variants from the first five years of the pandemic. The abundant presence of circ7b8N across variants in both sequencing data and clinical samples implies it plays a multifaceted role in SARS-CoV-2 pathogenesis.","version":"1.1","doi":"10.64898/2026.04.28.721328","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.27.721040","pub_date":"2026-4-27","title":"Inhibiting the interaction between the mitochondrial receptor Tom70 and SARS CoV 2 Orf9b with small molecules","abstract":"The SARS CoV 2 accessory protein Orf9b is in a complex monomer-dimer equilibrium that influences its interactions with the host mitochondrial receptor Tom70. This interaction is critical for viral suppression of a Type-1 interferon response during infection. Modulating this equilibrium with a small molecule, either by stabilizing the Orf9b dimer or blocking its interaction with Tom70, represents a promising strategy for restoring interferon signaling and the antiviral response. To build tool molecules that could test this concept, we performed two screens: a crystallographic fragment screen against the Orf9b homodimer and a high-throughput fluorescence polarization screen for competitors of an Orf9b-derived peptide binding to Tom70. Fragment screening revealed two binding sites with potential to be developed into an inhibitor: one located at the peripheral dimer interface and the other just outside the lipid-binding channel that defines the central dimer interface. Functionalization of the fragments outside of the lipid-binding channel with hydrophobic moieties stabilized the Orf9b dimer thereby indirectly inhibiting association with Tom70. In parallel, the high throughput screen for competitive inhibitors of the Tom70:Orf9b interaction discovered a separate series of molecules. These molecules display dynamic structure activity relationship (SAR) and could be improved in the future to modulate the interaction between Tom70 and potentially a wide range of substrates. Collectively, these results demonstrate the feasibility of two distinct strategies to manipulate the Orf9b-Tom70 equilibrium, which is critical to the host response to SARS CoV 2 infection.","version":"1.1","doi":"10.64898/2026.04.27.721040","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.21.719937","pub_date":"2026-4-27","title":"A High-Throughput Platform for Rapid Adaptation of DNA Aptamers to SARS-CoV-2 Evolution","abstract":"Rapid pathogen evolution threatens public health by eroding the effectiveness of vaccines, therapeutics, and diagnostic tools. Although spike protein targeting monoclonal antibodies (mAbs) were developed within 10-12 months of the initial outbreak to serve as key theranostic agents, their redesign has struggled to keep pace with viral evolution, rendering many neutralizing antibodies ineffective. Here we demonstrate a novel platform that combines a random-rational hybrid library diversification with high-throughput MiSeq screening to rapidly reprogram aptamers against emerging SARS-CoV-2 spike variants. Interactions between 3 different spike proteins and 11,806 unique aptamer variant designs were profiled within a few days. Starting from a 40-nt aptamer originally selected against wild-type (WT) spike protein, our screen identified a Delta-binding mutant with a 4-fold affinity improvement and an Omicron-binding mutant that converted undetectable binding into nanomolar affinity. We also identified a WT-selective mutant with substantially reduced affinity for Delta, as well as previously unrecognized bases that critically contribute to spike recognition. Integrating high-throughput binding data with molecular dynamics simulations further revealed sequence-dependent structural features underlying variant-specific aptamer-spike interactions. Finally, we developed a sensor based on the identified WT-selective aptamer mutant, enabling highly specific detection of the WT spike protein with robust performance. Together, this work establishes a rapid and adaptable aptamer engineering platform for rapid adaptation of aptamers to evolving pathogens in future pandemics.","version":"1.2","doi":"10.64898/2026.04.21.719937","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.23.684253","pub_date":"2026-4-27","title":"Suppressing Primer-Driven Nonspecific Amplification in LAMP Using TrueLAMP","abstract":"Loop-mediated isothermal amplification (LAMP) is widely used for rapid nucleic acid detection but is limited by primer-driven nonspecific amplification that produces false positives. We developed TrueLAMP, a colorimetric LAMP formulation containing an inhibitor that suppresses nonspecific amplification while preserving target-dependent reactions. Using SARS-CoV-2 as a model, TrueLAMP was evaluated across multiple primer sets. TrueLAMP eliminated amplification in no-template controls while maintaining robust target amplification, achieving 100% specificity across all no-template controls and, using the Lamb primer set as a representative benchmark, a limit of detection of 250 copies per reaction with 95% detection probability. Stable endpoint colorimetric signals were maintained over extended incubation periods, enabling flexible readout without time-critical monitoring. TrueLAMP is compatible with readily available equipment, including convection ovens and temperature-controlled smart mugs, supporting simplified and decentralized molecular testing workflows. Colorimetric LAMP reactions targeting SARS-CoV-2 were performed using TrueLAMP polymerase and buffer containing a nonspecific amplification inhibitor. Reactions were carried out using a two-step incubation (60 \u00b0C for 10 min followed by 65 \u00b0C) in either a convection oven or a smart mug. Amplification was monitored by colorimetric change and quantified using smartphone-based magenta or green channel intensity measurements. Orthogonal validation was performed by agarose gel electrophoresis using reactions with and without the inhibitor. TrueLAMP completely suppresses primer-driven nonspecific amplification in no-template controls across multiple primer sets. TrueLAMP achieved 100% specificity across all no-template controls and a limit of detection of 250 RNA copies per reaction with 95% detection probability (95% CI: 77\u201399%) using the Lamb primer set. Agarose gel analysis confirmed elimination of nonspecific amplification products while preserving target-dependent amplification. A two-step (60 \u00b0C \u2192 65 \u00b0C) incubation ensured consistent amplification across different primer sets and heating modalities. Stable endpoint colorimetric signals enable flexible assay readout without time-critical monitoring. The method maintains LAMP simplicity while improving reliability for routine and decentralized testing applications.","version":"1.4","doi":"10.1101/2025.10.23.684253","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.20.719671","pub_date":"2026-4-24","title":"Unique nasal cell states induced by common pediatric respiratory viruses","abstract":"Respiratory viral infections in early childhood are major drivers of acute morbidity and long-term airway disease, yet how distinct viruses remodel the pediatric nasal mucosa at cellular resolution remains unresolved. Here, we generated a single-cell RNA sequencing atlas of 335,174 nasal epithelial and immune cells from 132 children under five years of age with SARS-CoV-2, rhinovirus, or respiratory syncytial virus (RSV) infection, alongside uninfected controls. Mapping viral transcripts to individual cells revealed virus-specific infected epithelial states: an NF-kB-responsive ciliated subset in SARS-CoV-2 and a previously undescribed KRT17+ squamous-like subset in RSV. We delineated divergent mucosal response programs, including a robust interferon (IFN) response in SARS-CoV-2, an IL-13-responsive secretory program in rhinovirus, and heightened inflammatory and cytotoxic immune activation in RSV. In RSV, specific immune subsets and elevated IFN-response signatures were associated with disease severity, whereas rhinovirus-induced wheeze was marked by expansion of a CST1+ goblet cell subset. Integration of asthma genome-wide association data with our atlas revealed a KRT13+ hillock-like squamous epithelial subset enriched for expression of childhood-onset asthma risk loci. Finally, we demonstrate that this resource enables high-resolution annotation of independent pediatric cohorts in Kolkata, India and rural Bangladesh. Together, this atlas establishes a comprehensive view of antiviral immunity in the pediatric nasal mucosa and defines virus-specific mucosal immune programs relevant to disease severity and asthma risk in early life.","version":"1.2","doi":"10.64898/2026.04.20.719671","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.02.703244","pub_date":"2026-4-23","title":"ACE-2-like Enzymatic Activity in Anti-SARS-CoV-2 Spike Protein Monoclonal Antibodies","abstract":"Many people with acute COVID-19 have clinical disease not clearly attributable to viral replication and many COVID-19 convalescents are affected by post-acute sequelae of COVID-19 (PASC, or long COVID, LC). LC has severely affected public health and economies worldwide. Features of LC including blood pressure dysregulation, coagulopathies, high levels of inflammation, and neuropsychiatric complaints. The mechanisms responsible for the pathogenesis of some of COVID-19\u2019s clinical features and LC have not been well established. The host cell receptor for SARS-CoV-2 is human angiotensin converting enzyme 2 (ACE2), which binds the SARS-CoV-2 spike protein receptor-binding domain (RBD) to initiate infection. We hypothesized that some people may produce anti-RBD antibodies that sufficiently resemble ACE2 structure to have ACE2-like catalytic activity after infection. Those antibodies, ACE2-like abzymes, may contribute to the pathogenesis of LC. Our previous studies showed that ACE2-like activity was associated with immunoglobulin in some acute and convalescent COVID-19 patients. ACE2-like catalytic activity correlated with blood pressure changes following a moderate exercise challenge in people convalescing from COVID-19. To further establish that ACE2-like activity could be attributed to antibodies, we screened human monoclonal antibodies (mAbs) against SARS-CoV-2 spike protein from 3 different research centers and others purchased from a commercial source for ACE2-like catalytic activity. We identified 4 human monoclonal antibodies with ACE2-like catalytic activity. The ACE2-like catalytic activity of these mAbs was not inhibited by MLN-4760, a compound that inhibits native human ACE2 catalytic activity, nor by EDTA, unlike native ACE2, a Zinc metalloprotease, but was inhibited by an overlapping pool of spike peptides. Enzyme kinetic studies showed that the mAbs had substantially lower Vmax and Km values than native ACE2. The data therefore suggested that the antibodies cleave ACE2 substrate via a different mechanism than native ACE2. The identification of specific mAbs with ACE2-like catalytic activity supports the hypothesis that antibodies induced by SARS-CoV-2 infection could help mediate the pathogenesis of COVID-19 and LC, and more generally, the hypothesis that catalytic antibodies induced by infectious agents can contribute to disease pathogenesis.","version":"1.3","doi":"10.64898/2026.02.02.703244","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.20.719720","pub_date":"2026-4-23","title":"Assessing the affinity spectrum of an antigen-specific memory B cell repertoire by inverted ImmunoSpot","abstract":"The biological efficacy of an antibody is largely defined by its affinity. Moreover, because the binding affinity of an antibody can span orders of magnitude, each antigen-specific B cell would not be expected to contribute equally to humoral defense: high-affinity antibodies are likely to possess increased potency in comparison to those with lower affinities. Hence, assessing the affinity spectrum of a person\u2019s antigen-specific B cell repertoire would provide valuable information on their immune competence. Currently, cloning and expression of large numbers of monoclonal antibodies (mAbs) per test subject would be required to gain such insights, but this is impractical in the context of large-scale immune monitoring efforts. Here, we introduce a variant of the B cell ImmunoSpot assay that can simultaneously assess the relative affinity distribution of hundreds of individual B cells in a test sample. Additionally, we also demonstrated its suitability for high-throughput assay workflows that require minimal labor and exploit machine-assisted image analysis software tools. Specifically, as proof of principle, we verified that B cell hybridomas secreting mAbs of different affinities for the SARS-CoV-2 Spike protein could readily be distinguished through simple titration of the soluble antigen detection probe. Furthermore, using this assay methodology we provide evidence for affinity maturation within the Spike-specific memory B cell repertoire following a second COVID-19 mRNA vaccination. Collectively, we introduce a high-throughput suitable and scalable methodology with the potential of filling a major gap in the immune monitoring field: characterizing the affinity distribution of antigen-specific B cells in large study cohorts.","version":"1.1","doi":"10.64898/2026.04.20.719720","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.21.719895","pub_date":"2026-4-23","title":"Identification and SAR optimization of FBXO22-mediated TEAD Targeted Glue\u2122 degraders","abstract":"TEAD transcription factors are emerging oncology targets due to their function as key effectors of the Hippo signalling pathway, which is frequently dysregulated in cancer. Here, we report the discovery and development of potent, deep, and rapid-acting TEAD Targeted Glue\u2122 degraders that leverage an aldehyde-mediated degron mechanism. Our compounds demonstrate exceptional on-pathway selectivity profiles and exhibit the expected Hippo signalling modulation. Mechanistically, we demonstrate that amine-based TEAD degrader scaffolds undergo extracellular conversion to the active aldehyde species, which mediate covalent engagement of FBXO22 C326, triggering TEAD proteasomal degradation. The degraders identified were able to be rationally and systematically optimised for both degradation potency and kinetics, achieving enhanced degradation profiles compared to previously reported FBXO22-targeting approaches, establishing design principles for this degrader class. Our findings highlight strategies for the structure activity relationship (SAR) and rational optimization of aldehyde-mediated degrons to generate novel precision molecular glue degraders against TEAD, a high value oncology target.","version":"1.1","doi":"10.64898/2026.04.21.719895","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.22.720112","pub_date":"2026-4-23","title":"Proximity proteomics reveals a role for IFI16 during human coronavirus infection","abstract":"Viruses rely on the infected host cell to ensure successful replication and propagation of infection. This is achieved through interactions between virus-encoded proteins and proteins expressed in infected cells. All human coronaviruses (HCoVs) encode 16 non-structural proteins (NSPs) which exhibit some level of similarity in identity and function among the HCoVs. To identify host proteins that are potential interacting partners of HCoV NSPs, we utilized split-TurboID along with mass spectrometry and identified IFN-\u03b3-inducible protein-16 (IFI16) as a proximal partner of SARS-CoV-2 NSP8 and NSP10. To investigate the significance of the association between the NSP8/NSP10 complex and IFI16, we utilized CRISPR-Cas9 to knockout and CRISPRi to knockdown IFI16 in A549 cells and demonstrated that loss or reduced expression of IFI16 leads to a decrease in human coronavirus infection. We further demonstrated that there is reduced viral RNA replication and viral protein synthesis upon loss of IFI16. Interestingly, the loss of IFI16 results in reduced expression of type I IFNs. Taken together, these data suggests that IFI16 promotes human coronavirus infection, and the role IFI16 plays in coronavirus replication is independent of its role as a regulator of type I IFN gene expression.","version":"1.1","doi":"10.64898/2026.04.22.720112","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.20.719750","pub_date":"2026-4-23","title":"Continual Learning for Emerging Epitope Landscapes in TCR peptide Binding Prediction","abstract":"The TCR\u2013peptide binding landscape evolves continuously: novel pathogens (SARS-CoV-2, emerging influenza variants) and newly characterized tumor neoantigens introduce epitope families with no training precedent. Deploying a static model trained on historical data leads to degraded performance on emerging epitopes, while naive fine-tuning on new data causes catastrophic forgetting\u2014erasing performance on previously learned epitopes. We introduce ContinualTCR, a continual learning framework that combines reservoir replay with Elastic Weight Consolidation (EWC) regularization to balance stability (retaining old-epitope performance) and plasticity (adapting to new epitopes). Evaluated on a temporally partitioned VDJdb\u2013 IEDB benchmark across four sequential epitope arrival tasks, ContinualTCR achieves new-epitope AUROC 0.812 and old-epitope AUROC 0.781 simultaneously\u2014reducing catastrophic forgetting by 62.9% relative to naive fine-tuning. A streaming evaluation protocol with per-task backward transfer (BWT) reporting reveals that replay alone resolves 30.6% of forgetting, EWC alone resolves 57.3%, and their combination achieves synergistic complementarity. These results establish continual learning as a necessary component of production TCR specificity systems that must adapt to evolving pathogen and neoantigen landscapes without requiring full retraining.","version":"1.1","doi":"10.64898/2026.04.20.719750","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.19.719510","pub_date":"2026-4-22","title":"SARS-CoV-2 spike protein-associated sialoglycoconjugates induce nanoscale filipodia to facilitate micro-size platelet clotting","abstract":"COVID-19 disease is associated with thrombosis, but the pathogenic mechanism remains unclear. Here, we investigate how SARS-CoV-2 spike protein causes platelet activation and aggregation. Our three-dimensional ultrastructural analyses showed that invaginated platelet structures, open canalicular system (OCS), expanded upon activation, trapping viral particles in the process. Binding with platelet OCS concealed SAR-CoV-2 spike-coated particles from virion detection in platelet-depleted blood plasma. Both SARS-CoV-2 spike coated-particles and recombinant spikes specifically induced platelet aggregation with nanoscale filipodia extensions, with the terminal sialic acids of the SARS-CoV-2 spike protein-associated sialoglycoconjugates being the key determinant in platelet activation. Our work illustrates that virus-associated sialic acids, not proteins, are functionally responsible for SARS-CoV-2 induced thrombotic events, providing a mechanistic insight on how glycosylation contributes to disease severity in COVID-19. This study lays the foundation for the development of glycan-modified vaccines with reduced risks of thrombosis.","version":"1.2","doi":"10.64898/2026.04.19.719510","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.20.719543","pub_date":"2026-4-22","title":"Long-range allosteric communication, double mutant cycles, and energetic coupling in SARS-CoV-2 spike protein","abstract":"SARS-CoV-2 spike protein is continuously evolving, leading to new variants. While mutations in the receptor-binding domain (RBD) enhance binding to the ACE2 receptor and evade neutralizing antibodies, the function of mutations in the N-terminal domain (NTD) remains poorly understood. Using two independent methods, surface plasmon resonance (SPR) and cryo-EM, we show that NTD mutations increase the population of spike protein with the RBD in the \u201cup\u201d conformational state. SPR association and dissociation kinetics of spike binding to ACE2 and antibodies, analyzed using a coupled equilibrium model, determined the relative populations and indicated that the RBD-up-to-down transition is rate-limiting relative to the RBD-down-to-up transition. Advanced model fitting of cryo-EM Coulomb potential maps confirmed the populations. The combined effect of NTD and RBD mutations exceeds the sum of their individual effects, indicating long-range allosteric communication and energetic coupling within the spike protein.","version":"1.1","doi":"10.64898/2026.04.20.719543","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.03.626570","pub_date":"2026-4-21","title":"Spike conformational tuning rather than epitope erosion drives SARS-CoV-2 resistance to broadly neutralizing antibodies","abstract":"Escape from protective immunity has been a defining feature of the COVID-19 pandemic and is widely attributed to mutations in neutralizing antibody epitopes within the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We investigated whether this paradigm accounts for the escape of Omicron lineage viruses from broadly neutralizing antibodies (bNAbs) targeting conserved RBD epitopes. We found that mutational erosion of bNAb binding epitopes was not a major driver of Omicron immune escape. Viruses carrying RBDs from highly resistant contemporary Omicron variants were potently neutralized by bNAbs when those RBDs were placed into an ancestral Wuhan-like spike backbone. Similarly, sera collected during the pre-Omicron era from individuals vaccinated with Wuhan-based vaccines retained robust RBD-specific neutralizing activity against these chimeric viruses. Mechanistically, bNAb treatment triggered exposure of the S2\u2019 cleavage site in neutralization-sensitive but not resistant spike proteins, indicating that bNAb susceptibility reflects differences in spike propensity for antibody-induced premature conformational transition. These findings show that, rather than escaping through mutation of bNAb epitopes, SARS-CoV-2 has evolved spike variants with altered conformational dynamics that resist bNAb-induced destabilization of the prefusion spike trimer.","version":"1.2","doi":"10.1101/2024.12.03.626570","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.16.719115","pub_date":"2026-4-20","title":"Systematic evaluation of 24 extraction and library preparation combinations for metagenomic sequencing of SARS-CoV-2 in saliva","abstract":"Sequencing the respiratory tract transcriptome has the potential to provide insights into infectious pathogens and the host\u2019s immune response. While DNA-based sequencing is more standard in clinical laboratories due to its stability, RNA assays offer unique advantages. RNA reflects dynamic physiological changes, and for RNA viruses, viral RNA particles directly represent copies of the viral genome, enabling greater diagnostic sensitivity. However, RNA\u2019s susceptibility to degradation remains a significant challenge, particularly in RNase-rich specimens like saliva. To address this, we conducted a systematic, combinatorial evaluation of 24 distinct mNGS workflows, crossing eight nucleic acid extraction methods with three RNA-Seq library preparation protocols. Remnant saliva samples (n = 6) were pooled and spiked with MS2 phage as a control. The SARS-CoV-2 virus was spiked into half of the samples, which were extracted using the eight different extraction methods (n = 3) and compared using RNA Integrity Number equivalent (RINe) scores and RNA concentration. The extracted RNA was then processed across the three library construction methods and subjected to short-read sequencing to assess all 24 combinations head-to-head. We compared methods based on viral read recovery and found that RINe and concentration did not correlate with viral detection. The Zymo Quick-RNA Magbead kit and the Tecan Revelo RNA-Seq High-Sensitivity RNA library kit were the extraction and library-preparation kits that yielded the most SARS-CoV-2 reads, respectively. Importantly, our combinatorial analysis revealed that any small variability attributable to different nucleic acid extraction methods was heavily overshadowed by differences in quality attributable to the RNA-Seq library preparation methods. These findings challenge the reliance on conventional RNA quality metrics for clinical metagenomics and underscore the need to redefine extraction quality standards for mNGS applications. mNGS is a powerful and unbiased approach towards pathogen detection that has mostly been applied to blood and cerebrospinal fluid samples. However mNGS has recently been applied to more areas including the respiratory pathogen detection space, with potential applications in both in-patient diagnostics and public health surveillance. Saliva samples are an ideal sample type for these use cases since they can be collected non-invasively. However, saliva is also a challenging sample type due to its high RNase activity and often yields low-quality nucleic acid. This study explores the feasibility of using saliva specimens in mNGS with contrived SARS-CoV-2 samples to optimize the combination of two factors: nucleic acid extraction and RNA-seq library preparation. Exploration in this area could enhance the sensitivity of saliva-based mNGS assays, with the goal of future expansion of this specimen type in clinical diagnostics and public health surveillance. The choice of RNA-Seq library preparation kit has a greater impact on pathogen detection than the nucleic acid extraction method. The combination of Zymo Quick-RNA Magbead extraction kit and TECAN Revelo RNA-Seq High Sensitivity RNA library kit recovered the highest percentage of total SARS-CoV-2 reads. RNA quantity and RINe score do not correlate with viral read capture, indicating a need for an alternative metric to assess RNA quality for downstream mNGS clinical diagnostics.","version":"1.1","doi":"10.64898/2026.04.16.719115","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.17.716662","pub_date":"2026-4-20","title":"Conserved metabolic vulnerabilities across pathogenic coronaviruses nominate host-directed therapeutic targets","abstract":"Pathogenic coronaviruses profoundly rewire host cell metabolism to support viral replication, yet whether these metabolic alterations expose shared and actionable vulnerabilities remains unclear. By integrating transcriptomic profiles from cells infected with SARS-CoV, SARS-CoV-2, and MERS-CoV with genome-scale metabolic models, we identify conserved and virus-specific metabolic perturbations affecting mitochondrial transport, nucleotide biosynthesis, fatty acid metabolism, and redox balance. Despite distinct transcriptional responses, all three viruses converge on a limited set of metabolic reactions whose flux ranges deviate strongly from healthy states. Using a network-based predictive framework, we systematically identify gene-pair perturbations that restore perturbed reaction fluxes toward non-infected metabolic states. Predicted rescue mechanisms reveal shared metabolic dependencies across coronaviruses, as well as time-dependent virus-specific vulnerabilities, and nominate druggable host targets. Notably, several top predictions align with independent experimental and clinical evidence, including metabolic interventions shown to reduce viral replication or disease severity in COVID-19 patients. Together, our results define conserved metabolic rescue pathways in coronavirus infection and provide a general strategy for identifying host-directed therapeutic opportunities from transcriptomic data. Coronaviruses converge on shared metabolic vulnerabilities in host cells NiTRO predicts gene pairs that rescue viral-induced metabolic states Mitochondrial transport emerges as a key pan-coronavirus target Top predictions validated by clinical trials and in vitro evidence Dohai et al. develop NiTRO, a network-based algorithm that integrates coronavirus-induced transcriptomic changes with genome-scale metabolic models to identify gene-pair perturbations capable of rescuing infected metabolic states. The approach reveals shared and virus-specific druggable metabolic vulnerabilities, with top predictions corroborated by clinical evidence.","version":"1.1","doi":"10.64898/2026.04.17.716662","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.10.717575","pub_date":"2026-4-17","title":"Integrated Computational and Experimental Evaluation of Hesperidin as a Multi-Target Modulator of Viral Entry and Protease Activity","abstract":"Flavonoids have been widely investigated for their antiviral and anti-inflammatory properties, but their mechanisms of action often remain insufficiently defined. In the present study, high-purity flavonoids were evaluated using an integrated workflow combining molecular docking, LigPlot+ interaction mapping, surface plasmon resonance (SPR), fluorescence-based TMPRSS2 inhibition assays, and cell-based viability studies. Docking with AutoDock Vina identified Hesperidin as the strongest overall candidate among the compounds evaluated. Hesperidin showed strong active-site engagement with TMPRSS2, including interactions with catalytic residues His296, Asp345, and Ser441, and stable binding within the SARS-CoV-2 main protease (Mpro) pocket. Comparative docking showed weaker or more peripheral interaction patterns for Rutin and moderate Spike binding for Hesperidin and Rutin. Experimental validation demonstrated dose-dependent inhibition of TMPRSS2 activity with an IC50 of 79.1 \u00b5M for Hesperidin and 43.5 \u00b5M for Hesperetin, while Rutin showed partial inhibition without a defined IC50 in the tested range. In Calu-3 cells, pre-treatment with Hesperidin or Rutin reduced SARS-CoV-2 Spike-induced cytotoxicity by approximately 30% without detectable intrinsic toxicity at the concentrations tested Docking analysis of Hesperidin and Rutin with the SARS-CoV-2 Spike protein revealed moderate interaction patterns involving residues such as Asn343, Ser371, and Val367. Hydrogen bond distances were generally in the range of approximately 2.9\u20133.3 \u00c5, indicating moderate stabilization compared with the stronger active-site interactions observed for Hesperidin in TMPRSS2. The resulting binding poses suggest that these flavonoids can associate with structurally relevant regions of the Spike receptor-binding domain; however, they do not strongly overlap with the key residues required for ACE2 interaction. Rutin, in particular, exhibited a more peripheral and distributed binding mode within the Spike\u2013ACE2 complex, indicating limited potential for direct disruption of the binding interface. In addition to SARS-CoV-2 targets, docking analysis extended to influenza viral proteins revealed moderate interaction of Hesperidin with hemagglutinin (HA) and strong catalytic-pocket binding of Rutin to neuraminidase (NA), involving key residues associated with enzymatic activity. These findings broaden the scope of the study to include influenza viral entry and release mechanisms, supporting a multi-virus, multi-target framework.","version":"1.2","doi":"10.64898/2026.04.10.717575","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.15.718757","pub_date":"2026-4-16","title":"Mechanistic insights into the association and activation of the SARS-CoV-2 2\u2019-O-Methyltransferase (NSP16)","abstract":"The nsp16 2\u2019-O-Methyltransferase is an essential non-structural protein of SARS-CoV-2, which methylates the viral mRNA cap structure, enabling it to evade the host immune response for higher translation efficiency. However, nsp16 is only active when it is bound to its cofactor, namely the non-structural protein-10 (nsp10). Understanding how nsp10 binds to and activates nsp16 function can help to develop targeted inhibitors; however, given the varying degree of disorder in both nsp10 and nsp16, characterizing this interaction has been challenging. Using long-timescale molecular dynamics simulations and AI/ML methods, we posit that the nsp16/nsp10 binding process is mediated by a hydrophobic latch formed with Leu4298 from nsp10 and a hydrophobic concave on the nsp16 protein surface. Our study highlights how the nsp16 S-adenosyl-L-methionine (SAM) pocket closes in its monomer state, which in turn deactivates the MTase function. We also observe that the nsp16/nsp10 complex allows for the RNA binding site to open with the empty SAM pocket. The results reveal how the SAM pocket loops facilitate SAM binding while allowing for the by-product S-adenosyl-L-homocysteine (SAH) to exit. Our study thus provides valuable atomistic-level mechanistic insights into understanding the activation of nsp16 MTase function while highlighting the challenges of studying protein-protein interactions mediated by largely flexible/disordered regions. Nsp16 carries out the methylation of the viral mRNA to gain immune evasion and translation efficiency. Understanding its complex molecular machinery can help us develop better therapeutic treatments. Here, we explore the key activation conditions for the SARS-CoV-2 nsp16 function via molecular dynamics simulation and AI/ML methods. The results demonstrate the role of nsp16 loops in different stages preparing for the methylation reaction from nsp16/nsp10 binding, (de)activation of nsp16 function and how the nsp16 SAM binding pocket can affect the RNA binding loops. This research explains the role of the nsp16 loops, which orchestrate its molecular function, and provides valuable insight to develop more targeted therapeutic approaches to disrupt viral immune evasion activity.","version":"1.1","doi":"10.64898/2026.04.15.718757","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.07.716934","pub_date":"2026-4-16","title":"Intranasal Anti-CD3 Antibody Treatment Attenuates Post\u2013COVID Neuroinflammation and Enhances Hippocampal Neurogenesis and Cognitive Function in Mice","abstract":"Cognitive impairment is a disabling feature of Long COVID, with data supporting neuroinflammation and maladaptive glial responses as primary drivers. Nasal administration of an anti-CD3 monoclonal antibody (aCD3 mAb) has shown therapeutic benefits in autoimmune and CNS disease models. Using a respiratory-restricted mild SARS-CoV-2 mouse model of Long COVID, we show that nasal anti-CD3 mAb, administered shortly after infection or during chronic neuroinflammation, increased brain FoxP3+ IL\u201310+ Tregs, reduced microglial and astrocytic gliosis in the white matter and hippocampus, restored neurogenesis, and improved short-term memory. Nasal aCD3 mAb reprogrammed microglia from an antigen-presenting, NF-\u03baB-driven inflammatory state toward chemokine signaling, phagosome, and TGF \u03b2\u2013related regulatory phenotype. Patients with Long COVID with neurological symptoms had lower circulating Treg populations. These findings identify nasal administration of aCD3 mAb as a noninvasive strategy to control neuroinflammation, restore the neurogenic niche, and offer a novel approach to treating cognitive impairment in Long COVID.","version":"1.2","doi":"10.64898/2026.04.07.716934","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.15.718804","pub_date":"2026-4-16","title":"Repeated SARS-CoV-2 Antigenic Exposures from Prior Vaccinations and Infections Demonstrate Limits of Antibody Durability and Breadth Against Newer Variants","abstract":"Widespread immunity from vaccination and infection has reduced COVID-19 morbidity and mortality, but this immunity varies across the population. Understanding how repeated antigenic exposures influence antibody responses helps to inform future vaccination strategies. Serum samples collected one and six months after XBB.1.5 vaccination from 25 generally healthy healthcare workers with varying exposure histories were assessed for neutralizing activity against a range of variants, from pre-Omicron variants to latest Omicron JN.1 sublineage variants and divergent BA.3.2 variants, using lentiviral pseudoviruses. Participants were stratified by vaccination and infection history. XBB.1.5 vaccination elicited broad neutralizing responses, with strong boosting against previously encountered antigens relative to vaccine-matched XBB.1.5 and newer variants. Geometric mean neutralization titers were generally comparable across exposure groups, indicating limited influence of prior Omicron infection or bivalent vaccination, though intra-group heterogeneity was observed. At six months, overall titers declined by 36-62%. Titers remained highest against the pre-Omicron and lowest against JN.1 sublineage variants. N-terminal glycosylation (DelS31, T22N) modestly affected neutralization. XBB.1.5 vaccination elicited broad neutralizing antibody responses against previously encountered and vaccine-matched antigens regardless of exposure history, but titers waned after six months. This waning, compounded by continued emergence of immune-evasive variants and heterogenous population immunity, underscores the need for continually monitoring neutralizing antibody durability and breadth to guide evidence-based COVID-19 vaccine formulation updates.","version":"1.1","doi":"10.64898/2026.04.15.718804","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.13.718069","pub_date":"2026-4-15","title":"Lifetime-based multiplexed detection of viral RNA using fluorogenic aptamers","abstract":"Fluorogenic aptamers (FAPs) are emerging molecular probes for viral RNA and DNA sensing. However, their use in multiplexed nucleic acid sensing has been hindered by cross-reactivity and overlapping emission spectra. Here we address these limitations by introducing a fluorescence-lifetime-based multiplexed detection strategy using variants of the DNA fluorogenic aptamer Lettuce that exhibits distinct fluorescence lifetimes when complexed with the fluorogen TO1-biotin. To effectively evolve Lettuce for diverse lifetimes, we developed a large-scale screening platform, termed FAP-FLIM-NGS (fluorogenic aptamer-based fluorescence lifetime imaging microscopy on next-generation sequencing chips), which measures the fluorescence lifetimes of \u223c104 Lettuce/TO1-biotin complexes directly on an Illumina MiSeq flow cell. Using this approach, three variants with markedly different lifetimes were identified: a single mutant (smC14T, 6.0 ns) and two double mutants (dmA5T/C14T, 5.2 ns, and dmA5T/T22A, 4.4 ns). To demonstrate the utility of these Lettuce variants in multiplexed detection, a set of split Lettuce probes targeting viral RNA fragments derived from SARS-CoV-2, MERS-CoV, and influenza A were designed and tested. Phasor plot analysis confirmed that these probes can robustly distinguish individual targets as well as mixtures containing any two or all three targets purely based on distinct fluorescence lifetimes of probes, thereby overcoming the challenges of cross-reactivity and spectral overlap. Beyond this proof of concept, our findings establish a generalizable strategy for engineering FAPs with customized photophysical properties, opening new avenues for next-generation diagnostics and molecular sensing technologies.","version":"1.1","doi":"10.64898/2026.04.13.718069","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.10.717462","pub_date":"2026-4-13","title":"Loss of host factor-mediated m6Am methylation of the viral RNA cap impairs SARS CoV-2 replication","abstract":"Eukaryotic mRNAs are co-transcriptionally capped at the 5\u2032 end with a methylated m7G moiety (cap0), which in higher eukaryotes is further methylated on the ribose (Nm) of the transcription start site (TSS) nucleotide to create the cap1 structure (m7GpppNm). Coronaviruses that replicate in the cytoplasm encode their own capping enzymes to acquire this cap1 structure which facilitates translation and shields them from the host innate immune system. Here we report the identification of an additional N6-methyladenosine (m6A) methylation on the 5\u2032 cap (m7Gpppm6Am) of the human coronavirus SARS-CoV-2 RNA. It is catalysed by the host m6A methylase PCIF1 following capping by virus-encoded non-structural protein NSP 14 and NSP16. Human cell cultures lacking PCIF1 accumulate reduced levels of the viral RNA and support reduced viral replication. Furthermore, Pcif1 mutant mice infected with SARS CoV-2 display milder symptoms. We identify the host RNA methyltransferase PCIF1 as a critical ally of SARS CoV-2 for viral replication.","version":"1.1","doi":"10.64898/2026.04.10.717462","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.10.717770","pub_date":"2026-4-13","title":"MIMIQ: Fast mutual information calculation and significance testing for single-cell RNA sequencing analysis","abstract":"Mutual information is a fundamental quantity in information theory that describes the non-linear dependency between two variables, and has numerous applications within bioinformatics and beyond. However, its exploitation is hampered by a trade-off between computational intensity and accuracy. Here we present an adaptive binning approach to computing the pairwise mutual information, optimized for small integer counts such as those observed in single-cell RNA sequencing. By assuming a sampling distribution such as the negative binomial, a \u03c72 test statistic for hypothesis testing can be computed simultaneously via a copula transformation. Using these quantities, we show how gene rewiring of CD4+ naive T-cells during SARS-CoV-2 infection can be studied using a single-cell sequencing dataset of healthy and COVID-19 donors.","version":"1.1","doi":"10.64898/2026.04.10.717770","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.11.716570","pub_date":"2026-4-13","title":"Serial vaccination expands and refines human CD4+ T cell memory","abstract":"CD4+ T cells coordinate protective immunity against pathogens. However, a major unresolved question is how human CD4+ T cell memory is established and evolves following primary and repeated vaccination. Using COVID-19 mRNA vaccination as a model, we tracked 50 distinct antigen-specific populations directly ex vivo with peptide\u2013MHC class II tetramers in eight SARS-CoV-2\u2013na\u00efve individuals from pre-vaccine baseline through memory time points after three mRNA doses. Our findings identify the primary vaccine series as the main driver of memory pool size. It leverages pre-existing memory while preferentially recruiting high-avidity T cells, establishing an immunodominance hierarchy dominated by a small subset of precursors. Booster vaccination refines both the magnitude and quality of T cell memory. It increases select populations and enhances differentiation of subdominant CD4+ T cells. Populations that did not become more abundant after boosting retained their polyfunctional potential. Beyond establishing memory to the ancestral spike, vaccinations broadened responses by recruiting cross-reactive T cells recognizing viral variants. Collectively, these findings reveal how human CD4+ T cell memory evolves through sequential immunizations to generate a functionally diverse and broadly responsive memory repertoire against future viral challenges.","version":"1.1","doi":"10.64898/2026.04.11.716570","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.10.717613","pub_date":"2026-4-11","title":"Protenix-v2: Broadening the Reach of Structure Prediction and Biomolecular Design","abstract":"Advances in biomolecular modeling have broadened the range of problems addressable by structure prediction and design models. Here, we present results from Protenix-v2, a system spanning high-accuracy structure prediction and biomolecular design. On the structure prediction side, Protenix-v2 achieves antibody-antigen success rates with up to 13-point gains over Protenix-v1, while 5-seed performance surpasses previous 1000-seed results. On the design side, Protenix-v2 demonstrates a 100% target-level success rate in novelty-controlled VHH-Fc campaigns, reaching hit rates up to 48%. Crucially, the model enables hit discovery on difficult GPCR targets with hit rates of 16%\u201388% (VHH-Fc) and up to 50% (mAb) under 16\u201330 testing budgets per target. Resulting hits show high developability and diversity. Beyond antibody tasks, we report improved ligand-related plausibility and successful cross-variant SARS-CoV-2 spike RBD mini-binder design. These results establish Protenix-v2 as a robust and powerful model for accelerated drug discovery.","version":"1.1","doi":"10.64898/2026.04.10.717613","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.09.717495","pub_date":"2026-4-10","title":"Phagocytic Clearance of SARS-CoV-2 Nucleocapsid- and RNA-Containing Immune Complexes Drives Inflammatory Cytokine Production and Endothelial Dysfunction","abstract":"The aberrant inflammation that characterizes severe COVID-19 is incompletely understood. Given the persistence of SARS-Cov-2 RNA and nucleocapsid protein (N) and the presence of anti-N antibody during the course of severe infection, we investigated the role of RNA-containing immune complexes (ICs) in driving inflammation. We found that ICs consisting of SARS-CoV-2 RNA, N, and anti-N IgG1 stimulate primary human monocytes in vitro to produce inflammatory cytokines and chemokines in a manner dependent on Fc\u03b3 receptors and partially dependent on toll-like receptor-8. In addition, the inflammatory response induced in monocytes by RNA-containing ICs caused endothelial dysfunction in vascularized micro-organs. Using nasopharyngeal samples from SARS-CoV-2-infected individuals, SARS-CoV-2 RNA and N were captured by anti-N monoclonal antibody in the absence of lysing reagents, indicating that SARS-CoV-2 RNA and N complexes are present outside of virions and cells. Finally, we found that during an early wave of COVID-19, the anti-N IgG:IgM ratio predicted severe clinical outcomes, consistent with a role for inflammatory, IgG-mediated phagocytic clearance of nucleic acid-containing ICs in SARS-CoV-2 pathogenesis, perhaps mitigated by non-inflammatory, IgM-mediated clearance. We conclude that RNA-containing ICs may play a role in the pathogenesis of severe COVID-19. Since all pathogenic viruses encode nucleic acid-binding proteins, such as N, and these proteins often elicit an antibody response, inflammatory clearance of nucleic acid-containing ICs may also contribute to disease severity in other viral infections.","version":"1.1","doi":"10.64898/2026.04.09.717495","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.08.717316","pub_date":"2026-4-09","title":"Role of Nonneutralizing Antibodies and Fc Effector Functions in Inhibiting SARS-CoV-2 Infection","abstract":"Neutralizing monoclonal antibodies (mAbs) are a key component of antiviral therapeutics against SARS-CoV-2; however, the contribution of Fc-mediated effector functions remains underexplored. Here, we compare the antiviral activities of the neutralizing and non-neutralizing mAbs CB6 and CR3022, respectively. The Fc regions of both plant-produced mAbs carried nonfucosylated, non-galactosylated complex glycans (pCB6 and pCR3022), and CR3022 was also produced with mammalian-typical galactosylated, fucosylated glycans (mCR3022). pCR3022 exhibited markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated virus inhibition (ADCVI) compared to mCR3022, indicating a significant impact of Fc glycosylation on antiviral activity despite the lack of neutralization. pCB6 exhibited potent neutralization while further enhancing virus clearance through synergistic Fc effector activity. Our findings suggest that Fc-mediated mechanisms, especially ADCC and ADCVI, can contribute substantially to viral control and may be particularly valuable against immune-evasive variants. These results advance our understanding of the functional roles that non-neutralizing antibodies can play in SARS-CoV-2 infection and highlight the potential of Fc glycoengineering to modulate the antiviral efficacy of both neutralizing and non-neutralizing mAbs.","version":"1.1","doi":"10.64898/2026.04.08.717316","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.07.717019","pub_date":"2026-4-08","title":"SKIN AS A POTENTIAL ENTRY POINT FOR SARS-COV-2","abstract":"The primary route of SARS-CoV-2 entry is via respiratory epithelium. However, many COVID-19 patients developed dermatological lesions, and SARS-CoV-2 RNA has been detected in the patients\u2019 skin. Inflammatory skin diseases, psoriasis and atopic dermatitis (AD), significantly increased the risk of COVID-19. To evaluate the potential role of skin in SARS-CoV-2 host interactions, we utilized 3D human skin organoids (HSO) generated from human epidermal keratinocytes, as well as neonatal skin explants. HSO were treated with cytokines involved in acute and chronic skin inflammation and cytokine storm in severe COVID-19 disease, TNF-\u03b1, IL-6, IL-1\u03b2, and IFN-\u03b3, individually and in combination. HSO were also treated with Th1 (TNF-\u03b1 + IL-17) and Th2 (IL-4 + IL-13) cocktails inducing pro-psoriasis and pro-AD HSO changes, respectively. All individual cytokines, and especially their combinations, elevated the expression of ACE2 and TMPRSS2 at mRNA/protein levels. The Th2 induced only TMPRSS2, the Th1 predominantly induced ACE2. Topically applied Spike-pseudotyped lentiviral Tomato reporter, which binds ACE2 similarly to SARS-CoV-2, successfully infected control and cytokine-treated HSO as well as neonatal skin explants. Cytokine treatment, especially TNF-\u03b1 + IL-6 + IL-1\u03b2 + IFN-\u03b3 and the Th1, significantly increased viral entry. Transcriptomic analysis further revealed partial overlap between gene expression signatures induced by Spike-mediated entry in inflamed HSO and those observed in lung tissue from COVID-19 patients, supporting the biological relevance of skin models. Together, these findings demonstrate that inflammation enhances the permissiveness of human skin to SARS-CoV-2 entry, suggesting that the skin may represent a previously underappreciated interface in viral host interactions.","version":"1.1","doi":"10.64898/2026.04.07.717019","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.25.690586","pub_date":"2026-4-08","title":"How endosomal PIKfyve inhibition prevents viral membrane fusion and entry","abstract":"Enveloped viruses enter cells by membrane fusion. The viral membrane fuses with a host membrane, either at the cell surface or within endocytic compartments. For endocytic entry, fusion is typically triggered by low pH and often requires proteolytic priming by compartment-specific host proteases, which together define the site and mechanism of fusion and shape viral tropism. Inhibition of the lipid kinase PIKfyve, which generates PI(5)P and PI(3,5)P\u2082 in late endosomes and lysosomes, swells those compartments and blocks infection by a subset of enveloped viruses, including Ebola virus, Marburg virus, coronaviruses (SARS-CoV-2), and VSV chimeras bearing Ebola, SARS-CoV-2, or Lassa glycoproteins, while showing minor effects on H1N1 influenza and no effect on VSV or VSV\u2013rabies chimeras. In the work reported here, we have determined the basis for selectivity. We show that swelling of late endosomes/lysosomes, independent of changes in lipid composition or altered virion trafficking, is sufficient to block virus\u2013endosome fusion and genome release, even when endosomal acidity is preserved. Acute PIKfyve inhibition with apilimod or brief hypotonic treatment produced endosomal swelling and impaired infection by interrupting a late endosomal entry step. Live-cell 3D lattice light-sheet fluorescence microscopy imaging tracked fluorescent virions accumulating and arresting in late endosomes prior to fusion, and single-cell, single-round assays confirmed loss of infectivity. These data support a simple biophysical mechanism: endo-lysosomal swelling, likely increasing endosomal membrane tension, creates an energy barrier to fusion and genome release. Inducing such swelling may offer a general strategy to inhibit viruses that depend on late endosomal entry. Why does inhibiting the endosomal lipid kinase PIKfyve, which generates PI(5)P and PI(3,5)P2, block entry of some enveloped viruses but spare VSV-G? Using single-round infectivity and live-cell 3D imaging, we show that acute PIKfyve inhibition traps incoming VSV chimeras bearing SARS-CoV-2 spike or Ebola GP in swollen late endosomes/lysosomes before fusion and genome release. Hypotonic swelling phenocopies the block, and removing glutamine prevents apilimod-induced swelling and largely restores infection, arguing that swelling\u2014not altered phosphoinositides\u2014restricts entry. We propose a physical mechanism: increased endosomal membrane tension raises the energetic barrier for fusion-pore formation and genome release. Modulating endolysosomal volume may therefore inhibit viruses that rely on late endosomal entry.","version":"1.2","doi":"10.1101/2025.11.25.690586","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.25.714173","pub_date":"2026-4-08","title":"Horse, not zebra: accounting for lineage abundance in maximum likelihood phylogenetics","abstract":"Maximum likelihood phylogenetic methods are popular approaches for estimating evolutionary histories from genome data. These methods do not make prior assumptions regarding strategies used for deciding which genomes were sequenced. However, in genomic epidemiology the sequencing rate is often agnostic to the specific pathogen strain considered. In this scenario, a pathogen strain prevalence should be reflected in its relative abundance in the genome data. Here, I show that this simple assumption, when appropriate and incorporated within maximum likelihood phylogenetics, greatly improves the accuracy of phylogenetic inference. I introduce and assess two separate approaches to achieve this. The first approach rescales the likelihood of a phylogenetic tree by the number of distinct binary topologies obtainable by arbitrarily resolving multifurcations in the tree. This approach interprets multifurcations as the result of lack of signal for resolving a bifurcating topology rather than as an instantaneous multifurcating event. The second approach instead includes a tree prior that assumes that genomes are sequenced at a rate proportional to their abundance. Both approaches favor phylogenetic placement at abundant lineages, and dramatically improve the accuracy of phylogenetic inference in scenarios like SARS-CoV-2 phylogenetics, where large multifurcations are common. This considerable impact is also observed in real pandemic-scale SARS-CoV-2 genome data, where accounting for lineage prevalence reduces phylogenetic uncertainty by around one order of magnitude. Both approaches were implemented in the open source phylogenetic software MAPLE v0.7.5.4 (https://github.com/NicolaDM/MAPLE).","version":"1.2","doi":"10.64898/2026.03.25.714173","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.07.637145","pub_date":"2026-4-07","title":"The K18-hACE2 mouse model of SARS-CoV-2 infection to illustrate the role and response of the vasculature in neurotropic viral infection","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) primarily affects the respiratory tract and lungs; however, the associated disease, coronavirus disease 2019 (COVID-19), can involve the central nervous system (CNS) in both its acute and long-term (Long COVID) clinical manifestation. The pathomechanisms underlying neurological impairments in COVID-19 are not yet fully understood, hence experimental studies to clarify the direct effect of SARS-CoV-2 in the brain can provide further insight. In the present study we used the K18-hACE2 model, intranasally challenged with SARS-CoV-2 ancestral and Delta isolates at low or medium doses, to address the hypothesis that the inflammatory response raised in the brain of infected mice is secondary to neuronal infection. Our data confirmed that the virus reaches the brain even after low dose (102 PFU/mouse, Delta isolate) infection where it targets the neurons without overt neuropathic effect, sparing the blood vessels. In situ investigation of the resulting inflammatory response showed the recruitment of leukocytes via postcapillary venules, with their accumulation in the perivascular space and occasional migration into the neuroparenchyma, without targeting and/or damage to the vessel wall. These changes were reflected in the brain transcriptome and proteome which showed positive enrichment of pathways and up-regulation of genes involved in the inflammatory response including the recruitment (including adhesion and migration) and activity of leukocytes. Additionally, morphological and transcriptome/proteome changes suggest minimal associated blood-brain barrier dysfunction. The brain metabolome and lipidome showed minimal changes; these were consistent with oxidative stress and inflammatory and immune/antiviral responses. The results obtained from our model indicate that SARS-CoV-2 infection of the neurons can result in limited neuroinflammation. These data can help to understand more fully the reaction of the CNS in COVID-19 patients, and neurotropic virus infections in general.","version":"1.2","doi":"10.1101/2025.02.07.637145","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.03.716319","pub_date":"2026-4-06","title":"High-speed 3D single-virus tracking reveals actin-aided viral trafficking of SARS-CoV-2 on the plasma membrane","abstract":"Early interactions between viruses and live cells are difficult to resolve due to rapid extracellular motion, 3D nature of the cell membrane, and the fast, nanoscale interactions involved. While actin is a central regulator of viral entry, direct observations of actin-aided trafficking have been restricted to membrane protrusions on glass surfaces given the limitations of conventional methods. Here, high-speed 3D Tracking and Imaging microscopy (3D-TrIm) is integrated with highly photostable StayGold-labeled SARS-CoV-2 virus-like particles to capture long-term, high-resolution single-virus trajectories in live cells. This approach revealed distinct regimes of viral dynamics, including extracellular diffusion, protrusion-based surfing, and an unreported linear trafficking mode along the plasma membrane that precedes viral internalization. This work demonstrates that this membrane trafficking is actin-driven and positively correlated with ACE2 expression. These findings reveal new actin exploitation by viruses and demonstrate the utility of 3D-TrIm for dissecting dynamic virus\u2013cell interactions at high spatiotemporal resolution.","version":"1.1","doi":"10.64898/2026.04.03.716319","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.07.611841","pub_date":"2026-4-06","title":"Discovery of novel quinoline papain-like protease inhibitors for COVID-19 through topology constrained molecular generative model","abstract":"The rapid emergence of drug-resistant SARS-CoV-2 variants poses a persistent challenge to current antiviral strategies. Mutations in key viral targets, including RNA-dependent RNA polymerase (RdRp), main protease (3CLpro), and papain-like protease (PLpro), have been shown to markedly reduce the efficacy of approved therapeutics, highlighting the urgent need for next-generation antivirals capable of overcoming resistance. Here, we report the discovery of a novel class of PLpro inhibitors through an AIDD strategy based on a topology-constrained molecular generative model (Tree-Invent) integrated with structure-guided optimization. Scaffold hopping from a previously reported lead enabled the identification of a quinoline-based chemical series with substantially improved metabolic stability and antiviral potency. Structure-guided optimization yielded compound GZNL-2016, which exhibited potent enzymatic inhibition of PLpro (IC50 = 10.5 nM), robust antiviral activity against multiple SARS-CoV-2 variants, including Omicron BA.5 and XBB.1, and favorable pharmacokinetic properties following oral administration. Notably, GZNL-2016 retained substantial inhibitory activity against the clinically relevant drug-resistant mutant PLpro E167K (IC50 = 480.2 nM; Ki = 439.3 nM), in contrast to previously reported inhibitors that exhibit markedly reduced potency. In a SARS-CoV-2 infection mouse model, oral administration of GZNL-2016 significantly reduced pulmonary viral titers, demonstrating in vivo antiviral efficacy. Collectively, this study establishes an AI-enabled strategy for rapid antiviral discovery and identifies GZNL-2016 as a promising lead compound to address the threat of coronavirus infections caused by drug-resistant mutant SARS-CoV-2 variants. We leveraged an AI generative model to discover a novel PLpro inhibitor with excellent liver stability, low CYP, hERG inhibition and reasonable oral PK properties. At the same time, the compound 16 exhibits high efficacy for the resistance mutation E167K, which resulted in severe resistance to the previously reported inhibitor Jun12682 and PF-07957472.","version":"1.3","doi":"10.1101/2024.09.07.611841","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.03.716256","pub_date":"2026-4-03","title":"The transmembrane domain regulates the kinetics of the SARS-CoV-2 spike conformational transition","abstract":"The homotrimeric SARS-CoV-2 spike glycoprotein comprises two subunits: S1, which recognizes host-receptors through its receptor-binding domains (RBDs), and S2, anchored to the viral membrane through its transmembrane domain (TMD), which facilitates the fusion of the viral envelope with the host cell membrane. Upon host-receptor engagement and proteolytic activation, S1 dissociates and triggers a large conformational transition in S2, involving structural rearrangements in the S2 ectomembrane-domains and the TMD. While studies have focused on the ectomembrane-domain dynamics, the TMD has typically been modeled as being in a trimeric state. Here, we use molecular dynamics simulations of a coarse-grained structure-based model (SBM) with an implicit membrane to investigate the role of TMD dynamics in modulating S2 conformational conversion. We first recapitulate previous results from an all-atom SBM with a trimeric TMD and re-emphasize that the extended pre-hairpin intermediate state of S2, which brings the two membranes into apposition, is a byproduct of the prefusion-to-postfusion transition. Next, by introducing dynamics into the TMD, we find a late fusion intermediate structurally similar to a recent cryo-EM structure. A dynamic TMD also makes the conformational transition faster. Simulations including the S1/S2 complex reveal coupled RBD-TMD dynamics: when all three RBDs are in the closed state, they can stabilize the TMD in a trimeric configuration, whereas the opening of a single RBD can trigger a transition to a dynamic TMD. So, the dynamics and the conformational preferences of the TMD can be tuned by the presence and conformation of S1. There is some evidence that the TMDs of class I viral fusion proteins, such as spike, contribute to viral fusion by modulating membrane properties. Our simulations indicate an expanded role for the function of the TMD, where it can directly regulate the kinetics of S2 conformational transitions. The SARS-CoV-2 fusion protein, spike, undergoes a large conformational transition, which facilitates the fusion of the viral and host-cell membranes and the delivery of the viral genome into the host cell. Despite extensive studies of the spike conformational conversion, its transmembrane domain (TMD) has largely been viewed as a viral membrane anchor. Using coarse-grained structure-based model simulations, we show that TMD dynamics can modulate the timing of the spike-S2 (fusion subunit) prefusion-to-postfusion conformational conversion. The presence of spike-S1 (receptor-recognition subunit) can suppress TMD dynamics, potentially reducing the rate of spike conformational conversion and viral fusion. Thus, the spike TMD regulates the kinetics of spike-mediated membrane fusion, and TMD-targeting strategies can be an additional avenue for antiviral intervention.","version":"1.1","doi":"10.64898/2026.04.03.716256","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.02.716254","pub_date":"2026-4-03","title":"Frustration Landscapes of Broadly Neutralizing SARS-CoV-2 Spike Antibodies Targeting Conserved Epitopes Reveal Energetic Logic of Escape-Proof and Escape-Prone Mechanisms","abstract":"The continued evolution of SARS-CoV-2 has enabled escape from most monoclonal antibodies, yet a subset of broadly neutralizing antibodies targeting three newly identified super-conserved RBD epitopes\u2014SCORE-A, SCORE-B, and SCORE-C\u2014retains remarkable activity against even the most recent JN.1-derived sublineages. Here we employed an integrated computational framework combining conformational dynamics, mutational scanning, MM-GBSA binding energetics, and frustration profiling to dissect the molecular mechanisms by which XGI antibodies achieve broad neutralization and resistance to immune escape. Structural analysis revealed that all three SCORE epitopes share a common architecture: a highly conserved, minimally frustrated core that provides stable anchoring, flanked by peripheral regions that accommodate antibody-specific variations. Conformational dynamics showed that SCORE-A antibodies (XGI-183) rigidify the lateral epitope while leaving the RBM partially mobile; SCORE-B antibodies (XGI-198, XGI-203) clamp the RBM apex, directly blocking ACE2; and SCORE-C antibodies (XGI-171) allosterically loosen the RBM loop, impairing receptor engagement indirectly. Mutational scanning identified a hierarchical hotspot organization where primary hotspots (e.g., K356, T500, Y380, T385) are evolutionarily constrained and minimally frustrated, while secondary hotspots (e.g., V503, Y508, S383) are neutrally frustrated and represent the principal sites of immune-driven mutations. MM-GBSA decomposition revealed that van der Waals-driven hydrophobic packing dominates binding, with electrostatic interactions providing auxiliary stabilization. Critically, frustration analysis demonstrated that immune escape hotspots reside precisely in zones of neutral frustration\u2014\u201cenergetic playgrounds\u201d that permit mutational exploration without destabilizing the RBD\u2014while minimally frustrated cores are evolutionarily locked. The comparative analysis of conformational versus mutational frustration distributions revealed a unifying principle: aligned neutral frustration yields permissive, escape-prone interfaces; decoupling enables targeting of constrained cores; and convergence of minimal frustration in both distributions creates invulnerable interfaces. These findings establish that broad neutralization arises not from ultra-high-affinity anchors but from strategic energy distribution across rigid, evolutionarily informed interfaces, providing a roadmap for designing next-generation therapeutics that target the invulnerable cores of viral surface proteins.","version":"1.1","doi":"10.64898/2026.04.02.716254","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.02.716099","pub_date":"2026-4-03","title":"Novel African Rhinolophus bat ACE2 sequences reveal the determinants of Afro-Eurasian sarbecovirus entry","abstract":"Sarbecoviruses, including SARS-CoV and SARS-CoV-2, are frequently linked to Rhinolophus bats as their putative natural reservoirs. Angiotensin-converting enzyme 2 (ACE2), a host carboxypeptidase widely expressed in mammalian tissues, plays a critical role in sarbecovirus infection by serving as the cellular receptor for the viral spike (S) protein. Given recent human outbreaks and pandemics caused by members of sarbecoviruses, and the wide distribution of Rhinolophus bats, it is essential to maintain surveillance of these viruses while improving our understanding of their interactions with bat hosts, particularly the ACE2 receptor. However, while Rhinolophus bats from Asia have been relatively well studied, African Rhinolophus bats remain underrepresented and require further investigation. In this study, five Rhinolophus bat lung samples were obtained from Zambia, and ACE2 genes from these individuals were cloned and sequenced. We further evaluated the susceptibility of ACE2 variants to a panel of sarbecoviruses, revealing key residues that influence viral infectivity. ACE2 polymorphism was observed among Rhinolophus simulator individuals, revealing multiple ACE2 genotypes within the sampled population. However, R. simulator ACE2s did not permit infection by the clade 3 Afro-Eurasian sarbecoviruses tested in this study. Notably, RhGB01 and BM48-31 virus utilized only Rhinolophus blasii ACE2. Mutational analyses further suggested that ACE2 residues 31 and 41 play important roles in modulating spike-ACE2 interactions. This study reports 4 unique ACE2 sequences of R. simulator and R. blasii, and provides new insights into the molecular interactions between African Rhinolophus species ACE2s and the S protein of sarbecoviruses circulating in Africa and Europe. As putative natural reservoirs of sarbecoviruses, including SARS-CoV and SARS-CoV-2, Rhinolophus bats play a critical role in the emergence of zoonotic coronaviruses, making it essential to understand their interactions with these viruses for future pandemic preparedness. While Asian Rhinolophus bats have been relatively well studied, African species remain underrepresented, highlighting the need for further investigation. In this study, we cloned and sequenced ACE2 genes of five Rhinolophus bats collected in Zambia, Africa. We identified ACE2 polymorphism among Rhinolophus simulator individuals, although this variation was not associated with susceptibility to the clade 3 Afro-Eurasian sarbecoviruses examined. In addition, we identified key ACE2 residues that govern SARS-CoV-2 spike-ACE2 interactions and contribute to distinct infectivity patterns across species. These findings expand our understanding of the molecular determinants of sarbecovirus host range and support improved surveillance and risk assessment of emerging coronaviruses.","version":"1.1","doi":"10.64898/2026.04.02.716099","journal":"bioRxiv","score":null},{"id":"10.64898/2026.04.02.716024","pub_date":"2026-4-02","title":"TF-IDF k-mer\u2013based Classical and Hybrid Machine Learning Models for SARS-CoV-2 Variant Classification under Imbalanced Genomic Data","abstract":"Accurate classification of SARS-CoV-2 genomic variants is essential for effective genomic surveillance, yet it is challenged by extreme class imbalance, limited representation of rare variants, and distribution shifts in real-world sequencing data. In this study, we employed hybrid RF-SVM framework designed for robust detection of rare SARS-CoV-2 variants. It integrates a random forest and a polynomial-kernel based support vector machine to enhance sensitivity to minority classes while maintaining overall predictive stability. We systematically compared classical machine learning models, deep learning approaches, and hybrid strategies under both standard and distribution-shifted evaluation settings. Our results show that classical models using TF-IDF\u2013based k-mer features outperform deep learning methods on macro-averaged performance metrics. The Random Forest classifier using TF-IDF Feature achieved the best overall performance, with a macro-averaged F1-score of 0.8894 and an accuracy of 96.3%. The model also demonstrated strong generalization ability, as evidenced by stable cross-validation performance (CV accuracy = 0.9637). Hybrid RF-SVM model further improves rare variant detection under severe class imbalance. Calibration analysis indicates reliable probability estimates for common variants, although challenges persist for minority classes. Overall, this study highlights the limitations of deep learning in highly imbalanced genomic settings and demonstrates that carefully designed hybrid machine learning approaches provide an effective and interpretable solution for rare SARS-CoV-2 variant detection.","version":"1.1","doi":"10.64898/2026.04.02.716024","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.09.707564","pub_date":"2026-4-01","title":"Persistent SARS-CoV-2 Spike is Associated with Localized Immune Dysregulation in Long COVID Gut Biopsies","abstract":"SARS-CoV-2 persistence is a proposed driver of Long COVID (LC), but the in-situ relationship between residual viral antigen and immune dysregulation remains poorly defined. To address this critical gap, we employed a high-resolution, multi-modal approach\u2014combining RNAscope, GeoMx Digital Spatial Profiling (DSP), spatial transcriptomics, and multiplex immunofluorescence\u2014on 25 terminal ileum and left colon biopsies from a clinical cohort of 8 LC participants and 5 healthy controls. We confirmed the persistence of SARS-CoV-2 Spike transcript and protein in the gut tissue of all LC cases and controls tested. Yet, comparison of Spike-positive (Spike+) regions in LC versus healthy control colon tissues revealed a differential, symptomatic state-associated signature, with 57 differentially expressed genes (DEGs) (26 upregulated, 31 downregulated), revealing genes that disrupt the immune response in LC subjects. LC colon Spike+ regions demonstrated increased expression of AQP8 and other absorptive-related genes (SLC26A3, SLC26A2, and CLCA4) which are involved with Chron\u2019s disease along with transcripts involved in tumorigenesis (GUCA2A, S100P, TSPAN1). Simultaneous downregulation of key homeostatic chemokines (CXCL13, CCL19, CCL21), and other transcripts reported to exhibit low expression in colorectal cancers (TMEM88B, NIBAN3, DMBT1), suggesting a paradox of epithelial tissue stress yet dysfunctional immune trafficking. Further analysis comparing Spike+ versus Spike-regions within LC colon tissue demonstrated an active, localized, antigen-driven immune microenvironment, identifying 122 DEGs (82 upregulated, 40 downregulated), including tumorigenesis genes. Cellular deconvolution of Spike+ regions revealed a statistically significant focal enrichment of myeloid-derived cells (macrophages, non-classical/intermediate monocytes), plasma cells, and regulatory T cells, coupled with significant enrichment in T-cell-related pathways, including \u201cAntigen processing and presentation,\u201d and \u201cTh1/Th2/Th17 cell differentiation.\u201d The ileum displayed a similar, though less pronounced, signature, demonstrating these statistically significant findings are specific to the colon of LC subjects. In contrast, corresponding Spike+ vs. Spike-analysis in healthy control colon tissues showed a more modest transcriptional response with 38 DEGs. Our data provide robust evidence that persistent SARS-CoV-2 Spike protein detection in the gut is not immunologically inert. Instead, it is actively associated with distinct, immune cell composition shifts and a dysfunctional pro-inflammatory transcriptional profile, supporting the hypothesis that retained viral antigen drives chronic immune dysregulation in tissue of Long COVID subjects.","version":"1.2","doi":"10.64898/2026.03.09.707564","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.31.715496","pub_date":"2026-4-01","title":"Omicron-Enhanced Immunosuppressive Effects of SARS-CoV-2 ORF3a and ORF9b Accessory Proteins on Monocytic Inflammatory Response","abstract":"This study investigates the poorly understood roles of SARS-CoV-2 accessory proteins using monocytic THP-1 cells expressing individual viral ORFs. ORF3a, ORF7b, and ORF9b were identified as major immunomodulators that suppress host inflammatory signaling. Specifically, cells expressing ORF3a or ORF9b exhibited reduced Toll-like receptor 4 (TLR4)-mediated production of key proinflammatory molecules\u2014CCL2, CCL4, and IL-1\u03b2\u2014resulting in diminished immune cell recruitment. Importantly, Omicron-associated mutations in ORF3a (T223I) and ORF9b (P10S\u2370+\u2370\u0394E27N28A29) amplified this immunosuppressive effect, leading to stronger transcriptomic suppression consistent with Omicron\u2019s reduced pathogenicity and clinical outcomes. These findings suggest that SARS-CoV-2 accessory proteins, particularly ORF3a and ORF9b, play pivotal roles in modulating monocytic immune responses. Enhanced suppression in Omicron variants highlights an evolutionary adaptation contributing to immune evasion and milder disease manifestations.","version":"1.1","doi":"10.64898/2026.03.31.715496","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.31.715419","pub_date":"2026-4-01","title":"VYD2311 is a promising candidate for passive immunization against COVID-19 in immunocompromised individuals","abstract":"For millions of immunocompromised individuals, vaccines may not elicit adequate protection from infections, so alternative strategies for pre-exposure prophylaxis are essential. There is only one non-vaccine product authorized in the U.S. as pre-exposure prophylaxis against COVID-19: the monoclonal antibody pemivibart. We previously showed that pemivibart had lower neutralizing activity in vitro against many recent dominant SARS-CoV-2 variants, such as KP.3.1.1, NB.1.8.1, and LP.8.1.1, than it had against JN.1, which was dominant when the antibody was first authorized. The manufacturer of pemivibart (Invivyd) recently initated clinical testing of a new monoclonal antibody derived from pemivibart, VYD2311, but there are no available studies of the activity of VYD2311 against dominant and emerging SARS-CoV-2 variants. Here, using pseudovirus neutralization assays, we measured the neutralizing activity of laboratory-synthesized VYD2311 and pemivibart against dominant and emerging SARS-CoV-2 variants, including XFG, NB.1.8.1, and the genetically distant BA.3.2.2. We found that VYD2311 potently neutralized all tested variants in vitro, dramatically more so than pemivibart. Combined with interpretation of earlier clinical trials of a parental antibody product, we conclude that VYD2311 is a promising candidate for passive immunoprophylaxis against COVID-19, particularly for those who do not respond well to vaccination.","version":"1.1","doi":"10.64898/2026.03.31.715419","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.30.715311","pub_date":"2026-3-31","title":"Intranasal immunization with live-attenuated RSV-vectored SARS-CoV-2 vaccines elicits antigen-specific systemic and mucosal immunity and protects against viral challenge and natural infection","abstract":"The emergence of new SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission to others. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from either the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8\u207a lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Therefore, intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.","version":"1.1","doi":"10.64898/2026.03.30.715311","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.20.614085","pub_date":"2026-3-30","title":"Nucleobase Methylation Enhances SARS-CoV-2 Chain Terminator Evasion of Exonuclease Proofreading","abstract":"Nucleoside analogues (NuAs) targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) represent a key antiviral strategy. However, their efficacy is fundamentally limited by the viral proofreading exoribonuclease nsp14, which excises misincorporated NuAs from nascent viral RNA. To identify NuAs capable of achieving chain termination while resisting nsp14-nsp10-mediated excision, we sequentially assessed a panel of nucleotide analogues encompassing ribose 2\u2032, 3\u2032, and nucleobase modifications for RdRp chain termination followed by nsp14-nsp10 ExoN resistance. Among the candidates, 5-methyl-3\u2032-dUTP emerges as a standout inhibitor. It functions as an efficient immediate chain terminator, and the 5-methyl modification confers substantial resistance to nsp14-nsp10-mediated proofreading excision, notably outperforming its unmodified counterpart 3\u2032-dUTP. RdRp also exhibited improved incorporation efficiency for this analogue. RNA chains terminated with 5-methyl-3\u2032-dUTP exhibit barely detectable primer extension even following nsp14-nsp10-mediated exonucleolytic cleavage. The superior chain termination and ExoN resistance of 5-methyl-3\u2032-dUTP were independently corroborated using an smFRET assay. Mechanistically, molecular dynamics simulations reveal that this enhanced ExoN resistance arises from destabilization of F146 loop stacking in the nsp14-nsp10 active site. Collectively, these findings establish 5-methyl-3\u2032-dUTP as a promising antiviral lead compound and provide a structural framework for designing next-generation nucleoside analogues capable of evading coronavirus proofreading surveillance.","version":"1.3","doi":"10.1101/2024.09.20.614085","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.29.711974","pub_date":"2026-3-30","title":"Panmap: Scalable phylogeny-guided alignment, genotyping, and placement on pangenomes","abstract":"Pangenomes capture population-level variation but remain computationally challenging at scale. We present Panmap, a tool that leverages evolutionary structure to place, align, and genotype sequencing reads against mutation-annotated pangenomes containing up to millions of genomes. Panmap introduces a phylogenetically compressed k-mer index that stores only sequence differences along branches, enabling efficient comparison of reads to both sampled genomes and inferred ancestors. This approach reduces index size by up to 600-fold and construction time by over three orders of magnitude relative to existing tools. Panmap places a 100\u00d7 coverage SARS-CoV-2 sample onto 20,000 genomes in 0.4 seconds and onto 8 million genomes in under two minutes. Furthermore, it enables accurate haplotype identification and abundance estimation in metagenomic samples and sensitive placement of ancient environmental DNA without prior alignment. Our approach makes large-scale pangenomes directly amenable to read mapping, genome assembly, alignment-free phylogenetic placement, and metagenomic analysis.","version":"1.1","doi":"10.64898/2026.03.29.711974","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.28.714969","pub_date":"2026-3-29","title":"Structure of SARS-CoV-2 spike in complex with its co-receptor the neuronal cell adhesion protein contactin 1","abstract":"The emergence of SARS-CoV-2 has caused millions of deaths and excess morbidity in the worldwide population. In addition to its respiratory symptoms, SARS-CoV-2 has become known for its neurotropism and long-term neurological sequelae, with a post-acute infection syndrome commonly referred to as long-COVID. Next to the host receptor angiotensin-converting enzyme 2 (ACE2) additional interactions of the SARS-CoV-2 spike (S) protein have been described for neuronal co-receptors specific to the nervous system including cell adhesion protein contactin 1 (CNTN1). Details of the spike-CNTN1 interaction have remained elusive. Here, we quantified the spike-CNTN1 interaction by surface plasmon resonance and resolved the structure of the complex by single particle cryo-electron microscopy (cryo-EM). Spike and CNTN1 interact with nanomolar affinity, driven by an avidity effect and mediated by the horseshoe moiety of CNTN1. The cryo-EM structure reveals that the CNTN1 Ig1-4 horseshoe is wedged in between two receptor binding domains (RBDs) and interacts, through Ig3, with a unique receptor interface at the base of the RBD in the up-conformation. This receptor interface is not previously described for other spike receptors but overlaps with the epitopes of several neutralizing monoclonal antibodies. Comparison of our data with available spike structures suggests one spike trimer can bind three CNTN1 molecules, or alternatively, different co-receptors such as ACE2 and CNTN1, simultaneously. These findings shed new light on the molecular determinants of SARS-CoV-2 neurotropism.","version":"1.1","doi":"10.64898/2026.03.28.714969","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.27.714475","pub_date":"2026-3-27","title":"Impact of viral membrane oxidation on SARS-CoV-2 spike protein transmembrane anchoring stability","abstract":"Reactive oxygen species generated during inflammation can oxidize viral envelope lipids, with outcomes ranging from modulated infectivity to viral inactivation. For SARS-CoV-2, the molecular mechanisms by which membrane lipid oxidation influences spike protein anchoring remain poorly understood. We use all-atom molecular dynamics (MD) simulations to quantify how graded oxidation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) affects the anchoring of the SARS-CoV-2 spike transmembrane (TM) region in an endoplasmic-reticulum\u2013Golgi intermediate compartment (ERGIC)-like multicomponent membrane. Viral envelopes containing 0, 25, 50, 75, and 100% oxidized POPC (PoxnoPC) corresponding to 0 \u2212 55% oxidation of all PO-type phospholipids were simulated with the spike TM helix and cytoplasmic tail embedded in a POPC/POPE/POPI/POPS/cholesterol mixture. Steered MD and umbrella sampling were used to calculate the potential of mean force (PMF) for extracting the TM+CT region along the membrane normal. Partial oxidation (25 \u2212 75% POPC) produced reductions in the detachment barrier that were not statistically distinguishable from the native system within the sampling uncertainty, whereas full POPC oxidation lowered the anchoring free energy by about 23% (from 606 \u00b1 39 to 464 \u00b1 38 kJ mol\u22121), indicating that oxidation of roughly half of the glycerophospholipids can measurably weaken spike-membrane coupling. Despite this reduction, the remaining barrier (about 180kBT ) is still large, suggesting that oxidation alone may be insufficient for spontaneous spike detachment and likely acts synergistically with mechanical forces during fusion or immune engagement. Analysis of acyl-chain order parameters, area per lipid, membrane thickness, number-density profiles, and lateral lipid clustering reveals that POPC peroxidation decreases lipid order, thins and softens the bilayer, and disrupts cholesterol-stabilized clusters that refer to large cooperative lipid assemblies (>10 lipids) identified via RDF-based clustering. These oxidation-induced changes reduce hydrophobic matching around the TM helix and facilitate its extraction from the viral envelope. Our results provide a mechanistic link between lipid peroxidation, membrane nanostructure, and spike anchoring, supporting lipid oxidation for example during cold atmospheric plasma or ozone treatment as a physically grounded contributing antiviral mechanism against SARS-CoV-2.","version":"1.1","doi":"10.64898/2026.03.27.714475","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.12.653592","pub_date":"2026-3-27","title":"Evaluating SARS-CoV-2 Antibody Resilience via Prediction and Design of Escape Viral Variants","abstract":"The evolutionary trajectory of SARS-CoV-2 is shaped by competing pressures for ACE2 binding, viability, and escape from neutralizing antibodies targeting its receptor-binding domain (RBD). Here, we present EscapeMap, a modular framework that enables prediction and design of variants escaping antibodies. EscapeMap integrates deep mutational scanning data for ACE2 and 31 monoclonal antibodies with a generative sequence model trained on pre-pandemic Coronaviridae. To experimentally probe escape potential, we designed RBD variants under pressure from four clinically relevant antibodies (SA55, S2E12, S309, VIR-7229). Among these designs, bearing up to 21 mutations from wildtype, 50% expressed as stable proteins. Binding assays confirm that S309 and VIR-7229 retain recognition across diverse mutation combinations. EscapeMap accurately forecasts which antibodies are vulnerable to escape by our designed sequences. Finally, by identifying correlated escape routes, we predict and experimentally verify, antibody combinations less prone to simultaneous escape, offering a quantitative basis for guiding therapeutic strategies.","version":"1.3","doi":"10.1101/2025.05.12.653592","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.24.713966","pub_date":"2026-3-25","title":"SARS-CoV-2 PLpro Drives Epithelial Barrier Disruption Across Drosophila and Mammalian Epithelia","abstract":"Disruption of epithelial homeostasis and barrier integrity, compounded by maladaptive inflammation, is central to SARS-CoV-2 pathogenesis. Prior reductionist studies relying on mammalian cell lines, which lack the capacity to form architecturally faithful epithelium in vitro, were ill-suited to identify the viral proteins that proximally underpin these epithelial perturbations. We therefore screened all SARS-CoV-2 NSPs and ORFs in the Drosophila larval imaginal disc\u2014an intact epithelium whose conserved junctional architecture and stress signaling circuits closely recapitulate those of human epithelium. This systemic filter identified the papain-like protease (PLpro) of NSP3 as a primary driver of junctional disruption. In imaginal disc epithelium, PLpro expression induced elevated oxidative stress (ROS) and hyperactivation of Akt, JNK, and JAK-STAT pathways\u2014a self-amplifying pathological network that acts as a \u2018cog-in-the-wheel\u2019 to derail tissue homeostasis. PLpro further induced organ-specific junctional perturbations and dysregulation of stress signaling in the larval tracheal system and the adult midgut, extending its epithelial-disrupting role across three morphologically distinct organs. Targeted genetic suppression of this network restored integrity, establishing these pathways as causal drivers. Critically, PLpro expression in mammalian MDCK epithelium recapitulated these defects, revealing cross-species conservation. These findings redefine PLpro as a \u2018dual-threat\u2019 pathogen\u2014not merely an immune-evasion factor, but a direct \u2018architect of injury\u2019 and a previously unrecognized determinant of COVID-19 pathogenesis.","version":"1.1","doi":"10.64898/2026.03.24.713966","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.21.713333","pub_date":"2026-3-25","title":"IL-1\u03b2 and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19","abstract":"Vascular dysfunction and coagulopathy are hallmarks of severe COVID-19. How SARS-CoV-2 infection drives endothelial dysfunction, despite the virus not infecting or replicating in endothelial cells, remains controversial. Here, we used an in vitro co-culture model of the human pulmonary epithelial-endothelial cell barrier to investigate which inflammatory mediators drive endothelial dysfunction during SARS-CoV-2 infection. SARS-CoV-2 infection of primary human bronchial epithelial cells increased adjacent endothelial cell expression of the leukocyte adhesion marker ICAM-1, disrupted endothelial VE-cadherin junctions, promoted endothelial cell death, and promoted platelet adherence to gaps in the endothelial monolayers. Dexamethasone treatment rescued these dysregulated endothelial phenotypes in infected co-cultures, confirming that inflammatory signalling was the primary driver of SARS-CoV-2-induced endothelial dysfunction. Specifically, epithelial-derived TNF and IL-1\u03b2 promoted endothelial dysfunction, as inhibition of TNF or IL-1R signalling blocked SARS-CoV-2-induced endothelial dysfunction in co-cultures. SARS-CoV-2-infected wild-type mice, but not TNF, IL-1\u03b2, or TNF/IL-1\u03b2\u2212 deficient mice, displayed increased endothelial ICAM-1 expression, while an anti-IL-1\u03b2 monoclonal antibody prevented SARS-CoV-2-induced ICAM-1 expression and fibrin deposition in aged K18-ACE2 mice. Our data indicate that TNF and IL-1\u03b2 are the specific cytokines that drive multiple aspects of endothelial dysfunction during acute SARS-CoV-2 infection, and that inhibiting their signalling pathways may provide therapeutic benefit in preventing vascular complications of COVID-19.","version":"1.1","doi":"10.64898/2026.03.21.713333","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.20.713312","pub_date":"2026-3-25","title":"Somatic evolution of a cross-reactive germline antibody that expands its breadth to neutralize new SARS-CoV-2 variants","abstract":"Rapid antigenic drift of the SARS-CoV-2 receptor-binding domain (RBD) underlies immune escape and continues to challenge the durability of antibody-mediated protection. Among the major classes of RBD-directed antibodies, germline-encoded IGHV3-53 responses are highly potent against early SARS-CoV-2 variants but are generally compromised by Omicron-associated mutations. Here, we identify an intrinsically cross-reactive IGHV3-53 germline antibody that recognizes multiple pre-Omicron variants, including SARS-CoV-2 wild-type, Alpha, and Delta. Notably, we demonstrate that targeted somatic evolution can further expand this breadth to overcome the immune escape of different Omicron variants. Guided by integrated structural and sequence analyses, we introduce four somatic mutations (G26E, T28I, S53P, and Y58F) into the germline antibody, resulting in markedly enhanced binding and neutralization of Omicron BA.1, BA.2, and BA.4/5. High-resolution crystal structures reveal that these mutations re-establish critical interactions disrupted by substitutions on Omicron RBD and optimize affinity at a remodeled epitope interface. Collectively, our findings delineate a structural and mechanistic pathway through which an inherently cross-reactive germline antibody lineage can be adaptively refined to counter highly divergent SARS-CoV-2 variants. This work highlights the underappreciated breadth encoded within the na\u00efve B-cell repertoire and provides a conceptual framework for engineering and eliciting antibody responses resilient to future antigenic drift.","version":"1.1","doi":"10.64898/2026.03.20.713312","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.19.712934","pub_date":"2026-3-25","title":"Experimental SARS-CoV-2 infection using horseshoe bats","abstract":"Horseshoe bats are known as the natural reservoir of sarbecoviruses. To understand how horseshoe bats coexist with sarbecoviruses in nature, experimental infection can provide direct evidence. However, in vivo infection studies using horseshoe bats have been lacking because of the difficulty of maintaining insectivorous bats in a laboratory setting. Here, we established a stable husbandry system for greater horseshoe bats (Rhinolophus ferrumequinum) and performed experimental infection with SARS-CoV-2. In contrast to Syrian hamsters which showed substantial viral replication, infected horseshoe bats exhibited low-level but persistent viral replication in the lung without overt disease. Histological analyses revealed that inflammatory lesions in the bat lungs were spatially restricted and temporally delayed compared with those in hamsters. Transcriptomic analyses further showed preferential activation of tissue repair pathways but limited inflammatory responses following infection. Notably, bats expressed several interferon-stimulated genes prior to infection. Our results suggest that a host strategy combining constitutive antiviral state, limited inflammation and enhanced tissue repair may result in controlled viral replication without overt disease, likely enabling horseshoe bats to coexist with sarbecoviruses.","version":"1.1","doi":"10.64898/2026.03.19.712934","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.23.713300","pub_date":"2026-3-25","title":"Cross-coronavirus host susceptibility loci influence disease severity through immune mediators","abstract":"Severe disease following infection with SARS-CoV or SARS-CoV-2 is driven in part by genetically regulated immune responses that promote lung injury. Previously, we showed that genetic risk for severe disease is conserved across viruses and between mouse and human, identifying the HrS43 locus as a shared determinant of severity. Here, to resolve immune pathways linking host loci to disease outcomes, we apply an integrative statistical framework combining Bayesian identification of predictive immune traits with QTL mapping and mediation analysis across infection conditions. This approach identifies immune predictors of disease severity across both viruses, reveals extensive genetic control of the immune system at homeostasis and during infection, and supports locus-specific causal mechanisms of immunopathology. Notably, HrS43 appears to influence disease severity through distinct immune mediators in SARS-CoV versus SARS-CoV-2, demonstrating that conserved genetic susceptibility can drive virus-specific immunopathology with translational relevance across species.","version":"1.1","doi":"10.64898/2026.03.23.713300","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.24.713916","pub_date":"2026-3-25","title":"Coronavirus envelope protein drives iron sensing disorder by hijacking the TAp73-FDXR axis","abstract":"Iron overload is increasingly recognized as a critical contributor to coronavirus pathogenesis, yet the underlying induction mechanisms remain unclear. Here, we uncover a fundamental pathway by which coronavirus drives IRP1 RNA-binding activity to induce iron accumulation via targeting the TAp73-FDXR axis. Specifically, coronavirus infection represses transcription of FDXR (encoding the key rate-limiting enzyme in host iron-sulfur cluster synthesis), thereby impairing host iron-sulfur cluster generation to trigger the functional conversion of the cytosolic aconitase 1 (ACO1) into iron-regulatory protein 1 (IRP1), ultimately leading to the host\u2019s persistently false perception of iron deficiency. We identify TAp73 as the primary transcription factor governing FDXR expression, and demonstrate that the coronavirus envelope protein (CoV-E) orchestrates TAp73 nuclear export. Subsequently, CoV-E binds TAp73 through a critical valine residue within its C-terminal PBM domain, inducing the K48-linked ubiquitination and proteasomal degradation of TAp73. Furthermore, we developed a CoV-E\u2013targeting molecule, DPTP-FC, which blocks CoV-E\u2013TAp73 interaction via forming steric hindrance and effectively alleviates iron accumulation and tissue damage caused by PEDV, PDCoV, and SARS-CoV-2 infection. Our study reveals the central role of the TAp73-FDXR axis in CoV-induced iron accumulation, highlighting CoV-E as an attractive antiviral target and DPTP-FC as a promising therapeutic candidate.","version":"1.1","doi":"10.64898/2026.03.24.713916","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.17.628831","pub_date":"2026-3-25","title":"Fitness translocation: improving variant effect prediction with biologically-grounded data augmentation","abstract":"Data scarcity limits the characterization of protein fitness landscapes and the development of accurate variant effect prediction models. To address this challenge, we introduce fitness translocation, a data augmentation strategy that generates synthetic variants for a target protein by leveraging variant fitness data previously measured in homologous proteins. Using embeddings from protein language models, the method computes the difference between each homolog variant and its wild type and applies these offsets to the target wild-type embedding to create synthetic variants in embedding space. We illustrate the utility of fitness translocation in the context of variant effect prediction on three protein families: IGPS, GFP, and SARS-CoV-2 spike proteins, across different models and training data sizes. Fitness translocation consistently improves predictive performance, particularly under limited training data, and is effective even when augmenting with remote homologs sharing as little as 35% sequence identity. These results illustrate how biologically grounded data augmentation can expand and diversify protein fitness landscapes, supporting more data-efficient protein engineering. The code is available at https://github.com/adrienmialland/ProtFitTrans.","version":"1.4","doi":"10.1101/2024.12.17.628831","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.19.695307","pub_date":"2026-3-23","title":"Structural determinants of IGHV1-69 public antibodies conferring resilience to SARS-CoV-2 antigenic escape","abstract":"R1-32-like public antibodies, characterized by shared IGHV1-69/IGLV1-40 usage, are elicited in more than 50% of individuals with COVID-19 and have been implicated in driving recurrent mutations at L452SARS2 and F490SARS2 within their convergent epitope in the SARS-CoV-2 spike receptor-binding domain. These mutations effectively mediate escape from non-affinity-matured R1-32-like antibodies with germline-like sequences. Here, we characterize four affinity-matured R1-32-like antibodies, C092, C807, BD56-104, and BD56-597, that tolerate L452SARS2 and F490SARS2 mutations. We show that this tolerance arises from residues introduced by somatic hypermutation at convergent positions across multiple CDR loops and surrounding regions, thereby creating additional contacts that reinforce epitope binding. An unusual N354SARS2 glycosylation site, which emerged in BA.2.86 and became fixed in its descendants, is linked to escape from affinity-matured R1-32-like antibodies, implying ongoing selection by this public antibody class. Using an AI model trained on integrated structural, neutralization, and binding data, we further identified ZL525, an ultrapotent R1-32-like antibody with pan-SARS-CoV-2 variant activity, including against the highly evasive KP.3 variant carrying the N354SARS2 glycosylation, and broad sarbecovirus cross-reactivity extending to SARS-CoV-1. Together, these findings show how affinity maturation enables public antibodies to adapt to viral antigenic drift, reveal their role in shaping SARS-CoV-2 antigenic evolution, and demonstrate the potential of AI-empowered strategies for discovering broadly neutralizing antibodies.","version":"1.2","doi":"10.64898/2025.12.19.695307","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.24.696405","pub_date":"2026-3-23","title":"VINE: Variational inference for scalable Bayesian reconstruction of species and cell-lineage phylogenies","abstract":"Bayesian methods are now widely used in reconstructing both species and cell-lineage phylogenies, but they remain heavily reliant on computationally intensive Markov chain Monte Carlo sampling. Phylogenetic variational inference (VI) circumvents this dependency but so far has been limited in speed and scalability. Here we introduce Variational Inference with Node Embeddings (Vine), a computational method that combines an embedding of taxa in a high-dimensional space and a distance-based \u201cdecoder\u201d with several algorithmic innovations to dramatically improve phylogenetic VI. Vine supports both standard DNA substitution models and CRISPR barcode-mutation models for inference of cell-lineage trees and tissue-migration histories. In extensive simulation experiments, we show that Vine is comparable in accuracy to the best available Bayesian methods with speeds orders of magnitude faster. We then apply Vine to \u223c1,000 complete SARS-CoV-2 genomes and \u223c900 lung-cancer cell barcodes, showing reductions in compute time from days to hours or minutes.","version":"1.2","doi":"10.64898/2025.12.24.696405","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.19.712870","pub_date":"2026-3-20","title":"SARS-CoV-2 Defective Viral Genomes from Distinct Genomic Regions Drive Divergent Interferon Responses","abstract":"Defective viral genomes (DVGs) are naturally generated during genomic replication of many RNA viruses. When produced early in infection or supplemented at the onset of infection, DVGs can attenuate viral pathogenesis by stimulating IFN responses and antagonizing wild type (WT) virus replication, highlighting their potential as antiviral therapeutics. However, during natural infection DVGs can exert both antiviral and proviral effects depending on their generation kinetics, species, and abundance, underscoring the need to better understand their roles in viral pathogenesis. Coronaviruses (CoVs) remain a major global health threat and ubiquitously generate DVGs, yet DVGs\u2019 roles during CoV infection are largely unknown. Using SARS-CoV-2 as a model, we previously identified DVGs in vitro and in patient samples and discovered two major genomic hotspots (A and B) for their generation. Here, we first showed that overall DVG abundance tended to positively correlate with COVID-19 severity, with approximately 40% of DVGs originating from a specific genomic region designated hotspot B. Analysis of a publicly available single-cell RNA-seq datasets revealed that DVGs from hotspot B, but not hotspot A, were associated with elevated IFN responses, suggesting that DVGs derived from different genomic regions vary in their ability to stimulate innate immunity. To test this directly, we constructed two representative DVGs corresponding to hotspots A and B. Both DVGs suppressed the replication of co-infecting WT virus; however, only DVG-B induced robust IFN responses, exceeding those triggered by WT virus alone. This was further confirmed in human precision-cut lung slices. Mechanistically, DVG-B\u2013derived dsRNA exhibited a distinct subcellular distribution compared with WT virus. Complementation with the nucleocapsid (N) partially restored dsRNA organization but did not alter the IFN response. Together, our findings demonstrate that DVGs arising from distinct genomic hotspots differentially regulate IFN responses, potentially contributing to varied pathogenic outcomes during SARS-CoV-2 infection. Defective viral genomes (DVGs) are naturally produced during RNA virus infection and can suppress viral pathogenesis by stimulating innate immune responses. However, their roles in coronavirus infection, particularly SARS-CoV-2, remain poorly understood. This study investigated the species-specific function of DVGs generated during SARS-CoV-2 infection and their impact on disease outcomes. Cohort analysis revealed that overall DVG abundance tended to positively correlate with COVID-19 severity, with approximately 40% of DVGs originating from a specific genomic region designated hotspot B. Single-cell RNA sequencing showed that DVGs from hotspot B, but not hotspot A, were associated with elevated IFN responses, suggesting that DVGs from different genomic regions vary in their immunostimulatory capacity. To directly test this, we constructed representative DVGs from both hotspots. While both suppressed wild-type virus replication, only DVG-B induced robust IFN responses both in vitro and ex vivo. Mechanistically, DVG-B produced dsRNA with distinct subcellular distribution compared to wild-type virus. Interestingly, complementation with the viral nucleocapsid protein partially restored dsRNA organization but did not alter IFN responses. These findings demonstrate that SARS-CoV-2 DVGs arising from different genomic hotspots differentially regulate innate immunity, potentially contributing to varied pathogenic outcomes during infection.","version":"1.1","doi":"10.64898/2026.03.19.712870","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.19.712895","pub_date":"2026-3-20","title":"Natural selection driven by escape from shifting antibody classes shapes SARS-CoV-2 evolution","abstract":"The phenotypic fitness landscape defines the action of natural selection on pathogens, linking changes in their phenotypes to transmission and evolution. The rapidly changing nature of epidemic spread and antigenic landscapes pushes viruses to evolve on fitness seascapes. As a result, evolution of viruses such as SARS-CoV-2 proceeds in a neverending series of waves, driven by epistatic interactions and by the arms race between viral adaptation and human immunity. Phenotypic characterisation of these rapidly changing fitness seascapes is an open challenge. Using a Phenotypic Selection Inference framework that links phylogenetic estimates of mutation fitness effects with deep mutational scanning data, we traced how selective pressures on viral phenotypes have shifted throughout the COVID-19 pandemic. Natural selection has favoured enhanced ACE2 binding since the emergence of SARS-COV-2, with relatively constant selective pressure even for the most recent variants. The strength of selection for antibody escape was comparable to ACE2 binding during early evolution, but as population immunity rose, escape from class 3 and then class 2 antibodies became dominant. For variants circulating in 2024, natural selection shifted toward class 3 antibody escape, while those circulating in 2025 have experienced dynamic, rapidly changing pressures for escape from all antibody classes. These transitions reflect an ongoing arms race between viral adaptation and human immunity. Our findings reveal that SARS-CoV-2 antigenic evolution is governed by dynamic, class-specific immune pressures, and that selection for replication capacity has been continuously present during the pandemic, presumably to compensate for the effects of antigenic escape on viral replication. Our approach for the inference of phenotypic selection provides a framework to understand and anticipate the evolution of future variants.","version":"1.1","doi":"10.64898/2026.03.19.712895","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.18.712394","pub_date":"2026-3-20","title":"Duplex Reverse Transcription Loop-Mediated Isothermal Amplification on a Nanofluidic Digital Chip (Nano-dChip)","abstract":"Over the past decades, the frequency of viral outbreaks has increased substantially worldwide, driven in part by global travel and resulting in millions of deaths each year. This trend underscores the urgent need for rapid, simple, and accessible diagnostic tools for infectious disease detection. Here, we present a nanofluidic digital chip (Nano-dChip) for point-of-care viral RNA detection that delivers results within 30 minutes at a cost of less than $0.50 per chip. The Nano-dChip employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for highly sensitive and specific target amplification. Reaction reagents are compartmentalized into numerous nanofluidic reservoirs, enabling highly quantitative detection while minimizing contamination risks. Using a single chip, we successfully detect both SARS-CoV-2 and Influenza H3 RNA with a detection limit of 10 fM, demonstrating the Nano-dChip\u2019s potential as a rapid, low-cost, and scalable diagnostic platform for timely outbreak control.","version":"1.1","doi":"10.64898/2026.03.18.712394","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.25.696505","pub_date":"2026-3-18","title":"Large scale prospective evaluation of co-folding across 557 Mac1-ligand complexes and three virtual screens","abstract":"Accurate prediction of ligand-bound protein complexes and ranking them by affinity are central problems in drug discovery. While deep learning co-folding methods can help address these challenges, their evaluation has been hampered by the difficulties in assessing independence from training data and insufficiently large test sets. Here we test the ability of co-folding methods to predict the structures of 557 ligands bound to the SARS-CoV-2 NSP3 macrodomain (Mac1) that were determined after the training cut-off dates. AlphaFold3 (AF3), Boltz-2, and Chai-1 each reproduced >50% of the Mac1 ligand poses to better than 2 \u00c5 RMSD of experiment. Despite the potential for co-folding to describe protein conformational changes that stabilize ligand binding, we did not find that common conformational rearrangements, including peptide flip and a large loop opening, were recapitulated by the co-folding prediction. For AF3 and Chai-1, ligand pose prediction confidence weakly, but significantly, tracked experimental potency, while DOCK3.7 energies were only weakly correlated. Boltz-2 affinity predictions showed the strongest correlation with measured potency and, after calibration, achieved lower mean absolute error than a baseline predictor. We next assessed whether co-folding scores could rescore docking hit-lists to distinguish true ligands from non-binders among hundreds of molecules prospectively experimentally tested against AmpC \u03b2-lactamase, the dopamine D4 and the \u03c3\u2082 receptors. AF3 ligand pose confidence values did not separate true ligands from high-scoring false-positives as effectively as docking scores or Boltz-2 affinity predictions did. Taken together, the modest, but independent correlations of docking score and co-folding confidence or affinity suggests that integrating physics-based and deep-learning and approaches may help with hit prioritization and subsequent optimization in structure-based ligand discovery.","version":"1.3","doi":"10.64898/2025.12.25.696505","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.10.710948","pub_date":"2026-3-16","title":"Constrained Diffusion as a Paradigm for Evolution","abstract":"A foundational question in computational biology is how to utilize data to describe the forces driving evolution. Here, we view evolution as a novel diffusion process constrained by many biological, physical, and environmental factors affecting organism viability at any given time. We introduce DiffEvol, a framework that models evolution as constrained diffusion over a discrete genotype space. Using real-world genomic sequence data alone, DiffEvol estimates complex evolutionary constraints by inverting the diffusion dynamics to recover a constrained subspace representing the viable genotype manifold, as well as its evolution over time. Applied to SARS-CoV-2 sequence data from 2020\u20132024, DiffEvol reconstructs constraint functions that recapitulate known viral fitness trends, including a pronounced \u201cphase transition\u201d that occurred following the widespread adoption of the SARS-CoV-2 vaccine. Our constraint subspace representation of the data characterizes such features and trends more clearly. This framework could be used not only to improve forecasting of emergent pathogenic strains, but also to produce more accurate reverse time analyses of their evolutionary dynamics to help identify ancestral variants and the forces having shaped a pathogen\u2019s evolutionary trajectory. More generally, this formulation provides a method for linking observed sequence mutations to an evolving fitness landscape. Thus, constrained subspace diffusion offers a mathematical language for evolutionary dynamics in any system where random variation interacts with slowly-changing structural or global constraints, and can be applied to more complex evolutionary phenomena such as vaccine resistance, viral escape, and protein evolution.","version":"1.2","doi":"10.64898/2026.03.10.710948","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.14.711762","pub_date":"2026-3-15","title":"TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant respiratory disease, particularly in vulnerable populations. Although ACE2 is the primary receptor for viral entry, previous studies have identified a naturally occurring, ACE2-independent entry pathway in certain airway-derived cell lines. Utilization of this pathway depends on surface heparan sulfates and requires the E484D substitution within the receptor-binding domain of the viral Spike (S) protein. In this study, we expand the panel of airway-derived cell lines that support ACE2-independent, S-E484D-dependent replication and identify the host lysosomal transmembrane protein TMEM106B as a critical host factor for infection. Knockout of TMEM106B completely abolishes infection by SARS-CoV-2 SE484D in NCI-H522 and NCI-H661 cells. Moreover, ectopic expression of the luminal C-terminal domain (CTD) of TMEM106B-either alone or redirected to the plasma membrane - is sufficient to enable viral entry and infection in otherwise non-permissive cells. We further show that Fc-TMEM106B-CTD decoy protein blocks infection by SARS-CoV-2 SE484D, supporting a direct interaction between the S-E484D protein and TMEM106B-CTD. Finally, passaging experiments with a chimeric VSV-SARS-CoV-2 SE484D identify additional mutations within the heptad repeat 1 that enhance TMEM106B utilization and viral spread in the ACE2-independent cell models. Together, these findings demonstrate that TMEM106B is a key mediator of a naturally occurring ACE2-independent pathway in multiple airway-derived cells lines and suggest that variation in the Spike protein can expand receptor usage by SARS-CoV-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to acquire mutations in the viral spike (S) protein as it circulates in humans. How these mutations influence the cell and tissue tropism of SARS-CoV-2 remains poorly understood. While ACE2 is the canonical host cell receptor for SARS-CoV-2, previous studies revealed the presence of a naturally occurring ACE2-independent entry pathway in multiple airway-derived cell lines that can be utilized only by a SARS-CoV-2 variant bearing the E484D substitution within the viral S protein. Here we expand the airway-derived cell line models that support ACE2-independent and S-E484D-dependent entry and show that the host lysosomal transmembrane protein, TMEM106B, is an essential viral entry factor. Although the E484D substitution is currently rare in human isolates, our findings raise the possibility that small mutations in SARS-CoV-2 S can allow the utilization of alternative entry pathways that broadens its cell and tissue tropism.","version":"1.1","doi":"10.64898/2026.03.14.711762","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.13.711570","pub_date":"2026-3-14","title":"ISG15-USP18 signaling restrains viperin-dependent metabolic antiviral restriction","abstract":"Type I interferon (IFN-I) responses are tightly regulated to balance antiviral defense with cellular homeostasis. In humans, interferon-stimulated gene 15 (ISG15) functions as a critical negative regulator of IFN-I signaling by stabilizing the IFN negative regulator USP18, yet the functional consequences of ISG15 deficiency remain elusive. Here, we show that the loss of ISG15 exaggerates the JAK-STAT activation and, downstream, amplifies multiple ISGs including the nucleotide-modifying enzyme RSAD2 (viperin). Our quantitative proteomics, genetic reconstitution, and signaling analyses establish that defective USP18 stabilization skews the IFN response towards viperin expression. This amplified ISG network promotes viperin-catalyzed accumulation of the antiviral nucleotide analog ddhCTP, resulting in enhanced inhibition of viral RNA synthesis and the replication of Crimean-Congo hemorrhagic fever virus and SARS-CoV-2. Together, these findings demonstrate an ISG15-USP18-viperin axis that can be targeted to boost the metabolic antiviral restriction.","version":"1.1","doi":"10.64898/2026.03.13.711570","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.28.651145","pub_date":"2026-3-14","title":"MAVS Safeguards Mitochondrial Integrity to Drive a Potent Intrinsic Antiviral Program","abstract":"Intrinsic antiviral defenses can restrict infection independent of paracrine interferon (IFN) signaling. While mitochondrial homeostasis is essential for immunity, its direct role in viral restriction remains incompletely understood. Here, we identify mitochondrial antiviral signaling protein (MAVS) as a central effector of a potent IFN-independent antiviral immunity orchestrated though mitochondrial safeguarding. We demonstrate that MAVS is critical for mitochondrial integrity as its loss leads to mitochondrial fragmentation, depolarization, and elevated mitophagy, alongside impaired bioenergetics and protein import. Mechanistically, MAVS maintains mitochondrial integrity by stabilizing the translocase of the outer membrane (TOM) complex and sustaining the abundance of its core components. Remarkably, this mitochondria-driven immunity restricts SARS-CoV-2 replication even under IFN-deficient conditions, and operates alongside the IFN pathways during JEV infection. Our findings redefine MAVS as a mitochondrial guardian that preserves organellar stability to enact host defense. These results highlight mitochondrial integrity as a fundamental determinant of broad antiviral immunity, integrating intrinsic and IFN-dependent mechanisms to counteract viral pathogenesis.","version":"1.2","doi":"10.1101/2025.04.28.651145","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.12.711354","pub_date":"2026-3-13","title":"Epistasis and the changing fitness landscapes of SARS-CoV-2","abstract":"Since its emergence in late 2019, millions of SARS-CoV-2 genomes have been generated as part of global efforts to monitor the evolution and spread of the virus. This unprecedented volume of data provides a unique opportunity to study viral evolution at unparalleled resolution. In particular, individual genomic sites can be observed to have mutated independently thousands of times. These mutation counts have been used to estimate site-specific mutation rates and fitness effects for most mutations across the viral genome. Here, we use these data to investigate how the landscape of mutational fitness costs has changed over the course of the pandemic. SARS-CoV-2 evolution over the past six years has been characterized by the emergence of distinct variants separated by long branches corresponding to evolutionary saltations involving up to 50 mutations. We compare inferred fitness landscapes across these variants and find that shifts in the estimated effects of non-synonymous mutations are linked to genetic differences between them. Sites with altered fitness costs are enriched near positions where the genetic backgrounds differ. To explain the observed changes, we introduce a model with pairwise epistatic interactions between mutations and residues that differ between variants. This model is able to explain about half of the variance in the shifts of fitness effects and suggests that each mismatch between variants substantially alters mutation effects at typically 1 to 3 additional positions.","version":"1.1","doi":"10.64898/2026.03.12.711354","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.13.711550","pub_date":"2026-3-13","title":"Identification and Masking of Artefactual and Misleading Within-Host Variants in Deep-Sequencing SARS-CoV-2 Data","abstract":"Deep sequencing data are increasingly used to study within-host viral diversity and to inform evolutionary inference. For SARS-CoV-2, analyses based on intra-host single-nucleotide variants (iSNVs) have been widely applied to quantify within-host diversity and infer transmission dynamics. However, these applications critically depend on the reliable identification of low-frequency variants, which remain vulnerable to systematic and technical artefacts. In this study, we show that recurrent artefactual iSNVs are common in large-scale SARS-CoV-2 sequencing data and can persist even under conservative minor allele frequency (MAF) thresholds. Using data from the UK\u2019s Office for National Statistics COVID-19 Infection Survey, we demonstrate that such artefacts are predominantly sequencing centre-rather than protocol-specific. Each centre exhibits a modest, distinct set of recurrent artefactual variants showing little overlap with sites routinely masked at the consensus level. To address this, we developed a systematic, dataset-aware framework that uses recurrence within sequencing datasets to identify small, noise-adapted sets of artefactual iSNVs to mask. Applying this framework reduces spurious sharing of low-frequency variants between samples and qualitatively alters downstream inferences, including estimates of within-host diversity and transmission bottleneck sizes. Together, these findings highlight the importance of explicit, dataset-aware artefact control for robust inference from within-host variation, particularly as genomic studies increasingly seek to exploit sub-consensus diversity in rapidly evolving pathogens such as SARS-CoV-2.","version":"1.1","doi":"10.64898/2026.03.13.711550","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.13.711527","pub_date":"2026-3-13","title":"Spike Antibody Fc Drives Protection from SARS-CoV-2 Challenge in Macaques","abstract":"A definitive correlate of protection (CoP) for SARS-CoV-2 has yet to be formally established. Previously, using data from a series of non-human primate vaccine challenge studies, we reported that neutralising antibodies (NAbs) are the strongest candidate for clinical protection against COVID-19 and that spike binding antibody is the strongest candidate CoP for viral burden post-challenge. In this study, we further characterised the protective binding antibody profile by analysing spike antibody-dependent complement deposition, Fc\u03b3R binding, isotype and antibody glycosylation. Using the machine learning platform SIMON, we demonstrate that antibody-dependent complement deposition (ADCD) and Fc\u03b3R binding are strong candidate co-correlates for each of the post-challenge outcomes; viral load and lung pathology. We found that spike antibody sialylation closely followed by Fc\u03b3R2A, was the spike antibody feature with the strongest negative correlation with histopathology score. Spike antibody ADCD, Fc\u03b3R binding, isotype and glycosylation significantly differed by immunisation regimen and sex, which demonstrates the heterogeneity of immune mechanisms induced by different immunisation platforms. We conclude that spike binding antibody, with the protective functional characteristics described herein, is a candidate CoP that captures both protection from severe clinical disease and protection against a high viral burden. These findings should be taken into consideration for future SARS-CoV-2 vaccine development.","version":"1.1","doi":"10.64898/2026.03.13.711527","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.04.709710","pub_date":"2026-3-13","title":"Investigating the use of human COVID-19 rapid assays to detect antibody and antigen in domesticated dogs (Canis lupus familiaris) and cats (Felis catus)","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes coronavirus disease-2019 (COVID-19) in humans, is also known to infect animals including dogs (Canis lupus familiaris) and cats (Felis catus). This study evaluated the efficacy of human COVID-19 rapid antigen and antibody tests in dogs and cats. Nasal/oral swabs from 60 animals (32 dogs, 28 cats) and serum from 40 animals (20 dogs, 20 cats) were tested. Rapid antigen tests used on respiratory swabs showed low-to-moderate sensitivity (75% dogs, 57% cats) and moderate-to-high specificity (79% dogs, 95% cats) compared to RT-PCR. Rapid antibody tests used on serum samples demonstrated low-to-moderate sensitivity (70% dogs, 50% cats) and moderate-high specificity (60% dogs, 100% cats) compared to PRNT. While imperfect, these test kits may have some utility for field surveillance studies, particularly when species-specific rapid SARS-CoV-2 assays for dogs and cats are unavailable. These test characteristics in dogs and cats are similar to the findings from studies of the same types of tests in humans which have found an average sensitivity and specificity of common commercially available kits in the US range from 50.0-84.3% and 64.5-74.3%, respectively, when used with human samples (1,2).","version":"1.2","doi":"10.64898/2026.03.04.709710","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.11.710959","pub_date":"2026-3-13","title":"Mechanistic insights into the color transformation of a non-FRET substrate for RNase activity detection","abstract":"DNA-templated silver nanoclusters (DNA/AgNCs) have created a new class of non-FRET DNase substrates, termed Subak, that exhibits a color change upon DNase digestion. Although Subak substrates offer advantages such as ratiometric readouts and low manufacturing costs over traditional FRET substrates, the mechanism governing AgNC color switching remains unclear. Here, using a site-specific cleavage strategy, we identify color-switching hotspots and demonstrate that AgNC transformation can be controlled by the cleavage positions within the nucleic acid host. Our data support a cleavage-driven reorganization of the AgNC coordination environment, converting a non-emissive precursor into a red-emitting cluster, rather than direct enzyme-cluster interactions. Leveraging this insight, we engineer rSubak, an RNA-incorporated Subak that displays 95 nm red shift (530 to 625 nm) upon RNase cleavage. In amplification-free CRISPR/Cas13 assays for SARS-CoV-2, influenza A (A/H5N1), and measles viruses (MV) detection, rSubak achieved a limit of detection of 0.3 pM, superior to that of the commercial RNaseAlert (\u223c250 pM). Collectively, our results establish Subak as a generalizable, non-FRET platform for sensitive ratiometric reporting the activities of diverse nucleases.","version":"1.1","doi":"10.64898/2026.03.11.710959","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.11.711159","pub_date":"2026-3-12","title":"Discovery of Semicarbazone and Thiosemicarbazone Analogs as Competitive SARS-CoV-2 Virus Main Protease (Mpro) Inhibitors","abstract":"Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 Mpro. From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 Mpro. Among them, compound 25, a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC50) of 99 \u03bcM. Additionally, two thiosemicarbazone analogs (compounds 50 and 51) inhibited SARS-CoV-2 Mpro with IC50 values of 61 \u03bcM and 70 \u03bcM. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 Mpro. Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 Mpro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 Mpro inhibitors.","version":"1.1","doi":"10.64898/2026.03.11.711159","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.11.711036","pub_date":"2026-3-12","title":"Preliminary stability studies of a \u00df-SARS-CoV-2 virus-like particle vaccine","abstract":"We aimed to study the stability of a \u00df-SARS-CoV-2 virus-like particle (VLP) vaccine in a series of preliminary experiments using select stabilising excipients. \u00df-SARS-CoV-2 VLPs were produced and purified using established methodologies. The thermostability of VLPs was tested at 4\u00b0C and -30\u00b0C in the presence or absence of stabilizers polysorbate 80, sorbitol or L-histidine in the presence of a physiological NaCl concentration of 137mM. The integrity of VLPs was assessed using ELISA, Western immunoblot and dynamic light scatter (DLS). \u00df-SARS-CoV-2 VLPs were stable at 4\u00b0C for 14 days and the addition of stabilizing excipients improved stability compared to VLPs in PBS alone. Storage of VLPs at -80\u00b0C maintained particle integrity by DLS analysis for up to 2 years. Excipients helped to maintain the immunogenicity of the VLPs by ELISA and Western immunoblot and DLS analysis revealed that VLPs retained their particulate structure. SARS-CoV-2 continues to circulate globally and cause significant illness. The problem of waning immunity to mRNA/LNPs has necessitated frequent boosters to keep pace of emerging variants. The development of alternative vaccines therefore remans a priority. Protein based vaccines, like VLPs, offer a safe alternative able to produce longer lasting immune responses. In this preliminary stability analysis, the \u00df-SARS-CoV-2 VLPs were found to be stable at 4\u00b0C and the addition of excipients improved VLP stability. Storage of VLPs at -30\u00b0C and -80\u00b0C also showed that the VLPs are stable for very long periods. Our findings will be of importance for the ongoing development of a SARS-CoV-2 VLP based vaccine.","version":"1.1","doi":"10.64898/2026.03.11.711036","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.18.670983","pub_date":"2026-3-12","title":"Bivalent mRNA booster encoding virus-like particles elicits potent polyclass receptor-binding domain antibodies in pre-vaccinated mice","abstract":"mRNA vaccines emerged as a leading vaccine technology during the COVID-19 pandemic. However, their sustained protective efficacies were limited by relatively short-lived antibody responses and the emergence of SARS-CoV-2 variants, necessitating frequent and variant-updated boosters. We recently developed the ESCRT- and ALIX-binding region (EABR) mRNA vaccine platform, which encodes engineered immunogens that induce budding of enveloped virus-like particles (eVLPs) from the plasma membrane, thereby resulting in presentation of immunogens on cell surfaces and eVLPs. Prior studies showed that spike (S)-EABR mRNA-LNP immunizations elicited enhanced neutralizing antibody responses against ancestral and variant SARS-CoV-2 compared with conventional S mRNA-LNP in na\u00efve mice, but the effectiveness of S-EABR mRNA-LNP boosters in the context of pre-existing immunity has not been investigated. Here, we evaluated monovalent Wuhan-Hu-1 (Wu1) and bivalent (Wu1/BA.5) S-EABR mRNA-LNP boosters in mice pre-vaccinated with conventional Wu1 S mRNA-LNP. Compared to conventional S mRNA-LNP boosters, the EABR approach enhanced monovalent and bivalent mRNA-LNP booster-induced neutralizing responses against Omicron subvariants BA.1, BA.5, BQ.1.1, and XBB.1, with bivalent S-EABR mRNA-LNP consistently eliciting the highest titers. Epitope mapping of polyclonal antisera by deep mutational scanning revealed that bivalent S-EABR mRNA-LNP boosted diverse \u201cpolyclass\u201d anti-receptor-binding domain (RBD) responses, suggesting balanced targeting of multiple RBD epitope classes. In contrast, monovalent S, bivalent S, and monovalent S-EABR mRNA-LNP boosters elicited less diverse polyclonal serum responses primarily targeting immunodominant RBD epitopes. Cryo-EM structures demonstrated that bivalent mRNA immunizations promote S heterotrimer formation, potentially enhancing bivalent S-EABR mRNA-LNP booster-induced antibody breadth and polyclass epitope targeting by activating cross-reactive B cells through intra-S crosslinking. These findings support the future design of bivalent or multivalent S-EABR mRNA-LNP boosters as a promising strategy to confer broader, and therefore potentially more durable, protection against emerging SARS-CoV-2 variants and other rapidly evolving viruses.","version":"1.3","doi":"10.1101/2025.08.18.670983","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.11.711006","pub_date":"2026-3-11","title":"Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues","abstract":"Deep mutational scans across receptor-binding domains (RBDs) of diverging SARS-CoV-2 variants reveal ongoing changes to the effects of mutations, a phenomenon known as epistasis. Careful accounting for these altered mutational effects is important in viral surveillance and forecasting, and more broadly, for understanding the impacts of epistasis on real-world viral evolutionary trajectories. Using a yeast-display RBD deep mutational scanning (DMS) platform, we measure the impacts of virtually all single amino acid mutations and single-residue deletions in the Omicron KP.3.1.1 and LP.8.1 RBDs on folded RBD expression and binding affinity for the human ACE2 receptor. Our comprehensive maps reveal patterns of evolutionary accessibility and constraint at single-residue resolution and when compared to prior datasets, highlight sites whose amino acid preferences continue to change across viral variants. Notably, sites 455, 456, and 493 \u2013 which have exhibited repeated substitutions and epistatic dependencies across Omicron subvariants going back to BA.1 \u2013 again demonstrate altered patterns of mutational accessibility and constraint. Therefore, it appears that these hotspots of repeated RBD evolution have not yet converged on fixed amino acid solutions, but instead remain sites of ongoing epistatic reconfiguration. We compare our measurements of direct RBD:ACE2 affinity with recently published measurements of mutation impacts on ACE2 binding in the full quaternary spike context, which also integrates the effects of spike conformational dynamics; our analysis uncovers mutations like H505W that could favor adoption of the down/closed RBD conformation as a viral strategy for future antigenic evolution.","version":"1.1","doi":"10.64898/2026.03.11.711006","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.09.709174","pub_date":"2026-3-11","title":"Unsupervised identification of low-frequency antigen-specific TCRs using distance-based anomaly scoring","abstract":"Identifying antigen-specific T cell receptors (TCRs) within the diverse human repertoire remains challenging due to their extremely low frequencies, often as rare as one per million cells. Here, we propose a novel unsupervised approach that detects low-frequency antigen-specific TCRs through distance-based anomaly detection in TCR sequence space. Our method is based on the observation that antigen-specific TCRs preferentially localize at the periphery of V gene clusters rather than cluster centers. Using TCRdist3 to quantify sequence distances, we identify query TCRs that are anomalous compared to reference repertoires within their V-J gene combinations. We validated this approach across three immunological contexts: COVID-19 infection, influenza vaccination, and yellow fever vaccination. For SARS-CoV-2-specific TCR detection in a COVID-19 patient, our method demonstrated 34.3% accuracy, significantly outperforming similarity-based (ALICE: 8.0%) and frequency-based methods (edgeR: 5.8%, the Pogorelyy method: 6.3%), and uniquely detected low-frequency antigen-specific TCRs at clone count one. The minimal overlap with conventional approaches (\u22646.7%) indicates our method captures distinct TCR clones overlooked by existing analyses. This spatial distribution-based paradigm provides a complementary strategy for TCR specificity detection, particularly valuable for identifying rare antigen-specific clones essential for understanding immune responses.","version":"1.1","doi":"10.64898/2026.03.09.709174","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.25.684418","pub_date":"2026-3-11","title":"Mechanistic Evaluation of Amplification Lag in Paper-Based Colorimetric Loop Mediated Isothermal Amplification (LAMP) and Its Reduction by BSA Pre-Coating","abstract":"Colorimetric loop-mediated isothermal amplification (LAMP) on microfluidic paper-based analytical devices (\u03bcPADs) offers a low-cost, disposable, and equipment-free alternative to liquid LAMP assays. However, amplification on \u03bcPADs is consistently slower, by 5\u201346%, than reactions in tubes. To identify the origin of this delay, we evaluated heat transfer, diffusion in porous cellulose, and nonspecific adsorption of LAMP components across both high- and low-copy input regimes. Our results show that once thermal equilibrium is reached, reduced effective diffusion is the dominant contributor to the kinetic lag at low copy numbers, whereas nonspecific adsorption becomes the primary barrier at higher template concentrations. Pre-coating the paper with bovine serum albumin (BSA) mitigates adsorption. It narrows the tube-to-paper gap, thereby accelerating amplification of the SARS\u2013CoV-2 ORF7ab synthetic gene by an average of 6 minutes, from 1E3 to 1E5 copies per reaction. These findings provide a mechanistic basis for the copy-number-dependent behavior of \u03bcPAD LAMP and offer simple, low-cost strategies to improve the speed and reliability of \u03bcPAD nucleic acid assays.","version":"1.2","doi":"10.1101/2025.10.25.684418","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.09.709481","pub_date":"2026-3-10","title":"One and Done: A safe, adaptable single-cycle SARS-CoV-2 vaccine platform blocks XBB.1.5 infection and transmission","abstract":"A next-generation SARS-CoV-2 vaccine must address the currently inadequate prevention of virus transmission, particularly against emerging variants of concern, a challenge that none of the licensed commercial vaccines fully meet. Effective control of respiratory pandemics necessitates vaccines that 1) can be rapidly adapted, 2) have high patient compliance with simple and non-invasive administration, and 3) block transmission in a virus challenge. We describe here the characterization of an updated single-cycle SARS-CoV-2 vaccine candidate (scVac), engineered as a replication-defective virus with targeted deletions of the E gene and ORF6 and ORF7a, along with a truncation of ORF3a. The candidate carries an Omicron XBB.1.5 Spike (scVacXBB), maintaining all essential antigenic properties. The vaccine demonstrated an excellent safety profile in K18-hACE2 transgenic mice, the most sensitive virulence model, with no clinical signs or adverse events observed. In the Syrian hamster model, potent systemic and mucosal immune responses were induced, along with a strong neutralizing antibody response. Notably, there was no virus transmission to co-housed na\u00efve animals, which outperforms a bivalent Omicron mRNA vaccine reference. Our results demonstrate that scVacXBB-induced immunity not only prevents disease but also effectively blocks transmission. Furthermore, the successful introduction of the XBB.1.5 Spike protein into the scVac platform demonstrates the pipeline\u2019s ability to adapt quickly to any emerging variant. These findings highlight the potential of this single-cycle concept as a next-generation COVID-19 vaccine, offering robust protection with a strong safety profile.","version":"1.1","doi":"10.64898/2026.03.09.709481","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.09.710460","pub_date":"2026-3-10","title":"The serum from critical COVID-19 patients induces proteomic changes in olfactory neuroepithelial cells that resemble post-covid neurological complications","abstract":"Post-acute sequelae of SARS-CoV-2 infection (PASC), commonly referred to as Long COVID, comprise a constellation of persistent, recurrent, or newly emerging symptoms that may endure for months or years following acute infection. Beyond respiratory impairment, PASC is characterized by a wide spectrum of extrapulmonary manifestations, among which neurological and neuropsychiatric symptoms are highly prevalent. Reported features include olfactory dysfunction with loss of smell and taste, fatigue, neuroinflammation, cognitive and memory impairment, depression, and anxiety, with some symptoms persisting up to one year post-infection. Despite increasing recognition of these complications, the molecular mechanisms underlying post-COVID neurological sequelae remain poorly defined. In this study, we employed a label-free quantitative (LFQ) proteomics approach to investigate protein alterations in olfactory neuroepithelium\u2013derived stem cells (ONEs), a unique population of neural progenitors located in the olfactory mucosa at the interface between the respiratory system and both the peripheral and central nervous systems. Due to their anatomical exposure and susceptibility to SARS-CoV-2, ONEs represent a highly relevant translational model for exploring virus-associated neurobiological processes. ONEs derived from healthy donors were incubated with serum from either asymptomatic PCR-positive individuals (AS; n=4) or critically ill hospitalized patients (CR; n=6). Proteomic profiling revealed a distinct differential protein expression pattern in ONEs exposed to CR serum compared with AS serum. Altered pathways were associated with viral infection responses, respiratory and cardiovascular dysfunction, and notably, cerebrovascular and nervous system disorders. These findings highlight the vulnerability of ONEs to systemic factors associated with severe COVID-19 and provide molecular insight into mechanisms potentially contributing to persistent neurological sequelae in PASC.","version":"1.1","doi":"10.64898/2026.03.09.710460","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.03.697434","pub_date":"2026-3-10","title":"LP.8.1-directed COVID-19 mRNA vaccines durably boost neutralizing antibodies and mitigate ancestral immune imprinting","abstract":"As SARS-CoV-2 evolves, it evades existing immunity elicited by exposure to earlier strains of the virus. In response, vaccine manufacturers have updated COVID-19 vaccines annually since 2022, though immune imprinting to the ancestral strain has blunted antibody responses to modern viral variants. In early 2025, the JN.1 subvariant LP.8.1 was dominant and manufacturers updated mRNA vaccine formulations to target LP.8.1 (LP.8.1 MV). However, by late 2025, other subvariants were dominant (XFG and NB.1.8.1) or emerging (e.g., PE.1.4, BA.3.2, PY.1.1.1) around the world. It is critical to understand the extent to which updated vaccine boosters elicit titers against both their target strain and recent variants. Further, it is important to quantify the extent to which immune imprinting continues to shape antiviral immune responses. Using pseudoviruses, we measured neutralizing antibody titers against a panel of 11 SARS-CoV-2 variants in serum samples from 36 adult participants in the United States before and approximately 1 month after LP.8.1 MV booster. We found that neutralizing antibody titers were substantially increased by the boost, with the greatest increases elicited against LP.8.1 and XFG. For the first time since 2022, post-boost titers were higher against the homologous vaccine target (LP.8.1) than against D614G (representing the ancestral strain). Combined, these results indicate that ancestral immune imprinting is mitigated to the greatest extent observed to date by LP.8.1 MV. Lastly, for a subset of participants, we measured neutralizing titers at approximately 4 months post-booster and found that LP.8.1-directed antibody titers were durable, with an estimated average half-life of approximately 66 days.","version":"1.2","doi":"10.64898/2026.01.03.697434","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.03.709420","pub_date":"2026-3-09","title":"Computational Study of Antibody Binding to SARS-CoV-2 Variants","abstract":"The unprecedented structural and binding data for antibodies to the SARS-COV2 virus taken together with the mutations for the spike protein allows for a broad simulation study of antibody-spike protein binding. This provides an understanding of the co-evolution of human immunity and viral immunity escape. We utilized the YASARA molecular dynamics program to generate initial antibody-spike structures and simulate to equilibration for six SARS-COV2 variants and 10 different antibodies sampling two different binding regions to the receptor binding domain of the spike (especially for the Class I antibodies in the same part of the spike which attaches to the ACE2 receptor protein) and one to the N-terminal of the spike. Starting structures for antibody binding to variant spike proteins are perturbatively achieved through point mutations and insertions/deletions in the YASARA program. We employed YASARA to measure interfacial hydrogen bound counts between antibodies and variant spike proteins, and the HawkDock MMGBSA program to characterize trends in binding energies with mutation for four of the antibodies. We utilized the VMD program to analyze the time course of hydrogen bond populations. As seen in previous studies, interfacial hydrogen bond counts serve as an excellent proxy for binding energies without the large systematic error inherent in the latter. We find that there is generally a decline in antibody binding strength, as measured by interfacial hydrogen bond counts, with viral evolution, but that a modest re-entrance of binding strength is present for most antibodies studied. Generically, the antibody heavy chain binds more strongly to the spike protein, through for approximately half the antibodies the light chain binding strength converges to the heavy chain strength with viral evolution. The key conclusion is that the identified re-entrant immunity, speculatively arising from a balancing of maintenance of ACE2-spike binding while escaping antibodies through mutation, allows for some maintenance and even strengthening of immunity for later viral strains from early infection or vaccination.","version":"1.1","doi":"10.64898/2026.03.03.709420","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.07.583884","pub_date":"2026-3-09","title":"Visualizing the Rapid Degradation and Differential Binding of Membrane-Bound Human ACE2 Protein Upon Binding and Internalization of the SARS-CoV-2 Spike RBD Protein","abstract":"The SARS-CoV-2 betacoronavirus infects humans through binding the Angiotensin Converting Enzyme 2 (ACE2) protein that lines the nasal cavity and lungs, followed by import into a cell utilizing the Transmembrane Protease, Serine 2 (TMPRSS2) cofactor. ACE2 binding is mediated by an approximately 200-residue portion of the SARS-CoV-2 extracellular spike protein, the receptor binding domain (RBD). Robust interactions are shown using a novel cell-based assay between an RBD membrane tethered-GFP fusion protein and a membrane-bound ACE2-Cherry fusion protein. Several observations were not predicted, including rapid and sustained interactions leading to internalization of the RBD fusion protein into ACE2-expressing cells and rapid downregulation of ACE2-Cherry fluorescence, suggesting that a membrane-associated form of RBD found on the viral coat may have long-term system-wide consequences on ACE2-expressing cells. Targeted mutation in the RBD disulfide Loop 4 led to a loss of internalization for several variants tested. However, a secreted RBD did not cause ACE2 downregulation of ACE2-Cherry fluorescence. Omicron BA.1 and BA.2 variants have altered their dependency on the amino terminus (Nt) of the ACE2 protein. In contrast, the H-CoV-NL63 RBD is only dependent on the ACE2 internal region for binding, leading to the conclusion that the RBD binding surface of ACE2 appears relatively fluid and amenable to internalizing a range of novel variants.","version":"1.2","doi":"10.1101/2024.03.07.583884","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.18.558353","pub_date":"2026-3-07","title":"IgG-inspired, multivalent protein-DNA nanostructures for high-affinity, tunable, and reversible binding to biomolecular targets","abstract":"Multivalency enables nanostructures to bind molecular targets with high affinity. Although IgG antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we use computational simulation to guide the design and synthesis of a DNA nanostructure-based synthetic antibody (\u201cnano-synbody\u201d). The nano-synbody is comprised of a three-helix bundle DNA nanostructure with three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The structure was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance binding through avidity effects. Moreover, the design allowed for the display of one, two, or three protein-displaying arms, thereby systematically probing the effect of multivalency on binding affinity. The binding strength of the nano-synbody increased with the increasing number of arms, yielding 11.2 pM affinity (\u223c100-fold enhancement over monovalent binding) for the wild-type spike protein for the three-arm structure. Moreover, the multivalency was able to yield a 95 pM affinity for the Omicron variant, a mutant against which the monovalent protein was ineffective. The nano-synbody could also block infection of a spike protein-bearing pseudovirus, and similarly demonstrated effective inhibition of the Omicron variant when trimerized. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms bound to the RBD domains, confirming the intended trivalent attachment. Finally, nano-synbody binding could be reversed by removing one, two, or three arms in a programmable fashion, via toehold-mediated strand displacement. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach (and then remove) two or more different binding ligands, will enable the high-affinity binding of a range of proteins, and pave the way towards their manipulation using DNA-based nano-robotic devices.","version":"1.2","doi":"10.1101/2023.09.18.558353","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.07.709392","pub_date":"2026-3-07","title":"Dirty mice better recapitulate key features of mRNA vaccine immunogenicity observed in humans","abstract":"Although specific pathogen free (SPF) mice have traditionally been used to test candidate vaccines, recent work has demonstrated that \u201cdirty\u201d mice with broad microbial exposure more appropriately recapitulate human immune responses. Using a model where lab mice are co-housed with pet store mice, we modeled SARS-CoV-2 mRNA vaccine responses in dirty and traditional SPF models. We found that dirty mice show reduced serum spike-binding antibody titers after prime and require a second booster dose to reach SPF-level spike antibody titers. Additionally, spike antibodies showed faster waning in dirty mice through 5 months post-vaccination, and neutralizing activity of these antibodies were reduced against Omicron variants, directly comparable with observations in humans. We further investigated the seasonality and consistency of pathogens in cohoused mice, and the impact of serial microbial exposure on our animal model system. We found that pathogen exposure and T cell activation remained consistent over time, and that a single co-housing event was sufficient to provide broad microbial exposure. This work demonstrates that the dirty mouse co-housing system is a promising, translationally representative approach to screen candidate mRNA vaccines for efficacy and durability prior to human clinical trials. The development of mRNA vaccines during the COVID-19 pandemic dramatically reduced hospitalization and death rates for infected individuals. However, booster vaccinations were required to achieve full efficacy, and protection waned over time. Our research leveraged a \u201cdirty\u201d mouse model to test whether SARS-CoV-2 mRNA vaccinations in animals with previous microbial exposure better modelled human immune responses. We found that dirty mice require a booster vaccination for full efficacy and experienced waning serum antibody titer over time. We propose this approach as a future model for robust preclinical mRNA vaccine testing.","version":"1.1","doi":"10.64898/2026.03.07.709392","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.04.703990","pub_date":"2026-3-06","title":"A macrocyclic peptide-based fusion inhibitor targeting SARS-CoV-2 Spike S2 subunit","abstract":"Continuous emergence of SARS-CoV-2 variants carrying mutations in Spike presents a significant challenge for durable antiviral agents. Here we screen for random 13-amino acid non-mimetic macrocyclic peptides that bind to Spike and identify PA-001 that inhibits SARS-CoV-2 infection with high potency at 0.23\u20132.9 nM as 50% inhibitory concentration (IC50). PA-001 bound to Spike S2 subunit and inhibited the membrane fusion during virus entry. Through drug-resistant selection, we revealed that PA-001 targeted the fusion peptide proximal region (FPPR) in S2, which has not been recognized as a drug target to date. Consistent with its highly conserved amino acid sequences beyond strains, PA-001 exhibited broad antiviral activity against all tested SARS-CoV-2 variants, in contrast to clinically-approved S1-targeting antibodies that lost activity to Omicron variants. PA-001 suppressed SARS-CoV-2 propagation and disease progression in mouse- and hamster-infection models, both by administration prophylactically and therapeutically. Combination therapy with remdesivir further enhanced antiviral profiles. In clinical phase-I trial, PA-001 was well-tolerated and showed high systemic exposure, with 4,300\u201310,300-fold concentration of IC50 as maximum plasma concentration by single administration to healthy volunteers. These evidence propose FPPR as an unexpected antiviral drug target accessible by macrocyclic peptides and identify PA-001 as a potent anti-SARS-CoV-2 fusion inhibitor.","version":"1.2","doi":"10.64898/2026.03.04.703990","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.06.710084","pub_date":"2026-3-06","title":"Molecular Characterization of SARS-CoV-2 N Protein Interfaces: Implications for Oligomerization, RNA Binding, and Phase Separation","abstract":"The SARS-CoV-2 nucleocapsid (N) protein is central to genomic RNA recognition, condensation, and packaging, yet the molecular organization of its multivalent N\u2013N and N\u2013RNA interaction network involved in this process remains unclear. Here, we define the oligomerization and RNA-binding interfaces of the C-terminal domain (CTD) and its flanking intrinsically disordered regions (IDRs), the leucine-rich helix (LH) and the C-terminal IDR (C-IDR), using size-exclusion chromatography (SEC), cross-linking, mutational studies and NMR spectroscopy. We identify discrete oligomerization interfaces within the CTD and C-IDR that drive higher-order assembly, and show, through liquid\u2013liquid phase separation (LLPS) and electron microscopy (EM), that C-IDR residues are essential for RNA-induced condensate formation. Moreover, the mapping of RNA-binding residues highlights Arg277 as a conserved determinant of CTD\u2013RNA recognition. Notably, the two IDRs exert opposing regulatory effects on RNA binding, with the C-IDR enhancing and the LH attenuating CTD-RNA interactions. Together, these findings reveal how cooperative interfaces between the CTD and its flanking IDRs orchestrate N-protein oligomerization and RNA condensate formation and highlight potential intervention sites for disrupting SARS-CoV-2 ribonucleoprotein assembly.","version":"1.1","doi":"10.64898/2026.03.06.710084","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.02.708985","pub_date":"2026-3-04","title":"Microfilaremic loiasis is associated with T cell hyporesponsiveness against SARS-CoV-2","abstract":"Loiasis is a chronic filarial infection endemic to Central and West Africa. Although long considered benign, increasing evidence links loiasis to substantial morbidity and mortality. The infection is associated with immune modulation, including Th2-skewed responses and elevated regulatory cytokines. Clinically, loiasis is classified as microfilaremic (presence of circulating microfilariae) or amicrofilaremic (\u201coccult\u201d) disease, the latter defined by a history of eyeworm migration without detectable microfilaremia. This study investigated how chronic L. loa infection influences antibody and T cell responses to SARS-CoV-2 following natural infection. Between 2022 and 2024 this cross-sectional study was done in Lambar\u00e9n\u00e9 and surrounding rural areas of Gabon. Study procedures included diagnostics for loiasis and immunological assays. Microfilaremia was confirmed by stained blood smear microscopy, and occult disease was identified using the Rapid Assessment Procedure for Loiasis. SARS-CoV-2-specific IgG responses to spike and nucleocapsid proteins were measured by ELISA, and IFN-\u03b3 responses to spike antigen were assessed using an interferon-gamma release assay. Overall, 192 participants were categorized as microfilaremic (n=43), occult loiasis (n=59), or without evidence of active loiasis (n=90). IFN-\u03b3 responses were reduced in microfilaremic individuals compared with other participants (p= 0.031), whereas IgG responses did not differ. Subsequent analysis across the three groups confirmed that IFN-\u03b3 responses were lower in microfilaremic compared with occult participants (p= 0.012). These findings suggest that microfilaremic loiasis may impair proinflammatory T cell responses to viral antigens, highlighting the need for further research into the broader immunological effects of Loa loa infection in endemic populations. Loiasis is a parasitic infection caused by the worm Loa loa and is common in parts of Central and West Africa. Although long considered relatively benign, growing evidence indicates that loiasis is associated with substantial morbidity. The immunological consequences of chronic Loa loa infection remain poorly understood. A small number of studies suggest that Loa loa may influence immune regulation, but its broader impact on antiviral immunity is largely unknown. The COVID-19 pandemic provided a unique opportunity to examine immune responses to a newly emerging virus in a population where loiasis is endemic. We therefore investigated how different forms of Loa loa infection influence immune responses after natural SARS-CoV-2 infection. We compared individuals with circulating microfilariae in their blood (microfilaremic), individuals with occult loiasis (history of eye worm), and individuals without signs of active infection. We found that microfilaremic individuals had weaker virus-specific IFN-\u03b3 T cell responses, while antibody levels were similar across groups. These findings suggest that active loiasis may dampen certain antiviral immune functions. Understanding the underlying mechanisms is important, as such immune modulation could affect responses to vaccines and other infectious diseases in endemic regions.","version":"1.1","doi":"10.64898/2026.03.02.708985","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.03.709398","pub_date":"2026-3-04","title":"Defining the Antigenic Topology and Prospective Binding Breadth of Vaccination-induced SARS-CoV-2 Neutralizing Antibodies","abstract":"Antibodies that neutralize SARS-CoV-2 primarily target the viral spike glycoprotein, yet the breadth of these responses is continually challenged by viral evolution. While extensive structural studies have defined epitopes across the spike protein, how antibodies elicited by the initial mRNA vaccination campaigns perform against subsequently emerging variants remains an important question. Here, we structurally and functionally characterize a panel of early plasmablast-derived human monoclonal antibodies isolated following primary mRNA vaccination, targeting both the receptor-binding domain (RBD) and the N-terminal domain (NTD) of spike. Using cryo\u2013electron microscopy, variant-binding analyses, and viral-fusion inhibition assays, we observe that antibodies directed against immunodominant regions of the RBD and NTD are highly potent but more frequently impacted by variant-associated mutations. In contrast, antibodies engaging a conserved hydrophobic pocket within the NTD exhibit broader reactivity and neutralize through distinct molecular mechanisms. Together, these findings extend prior structural studies of spike-directed antibodies by prospectively assessing the breadth of vaccine-elicited antibodies against later variants and identifying structural features associated with differential escape sensitivity. These results contribute to a growing understanding of how early vaccine-induced antibody repertoires relate to subsequent viral evolution. Antibody epitopes on SARS-CoV-2 spike determine prospective breadth and vulnerability to viral evolution.","version":"1.1","doi":"10.64898/2026.03.03.709398","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.03.709384","pub_date":"2026-3-04","title":"Exposure route drives SARS-CoV-2 infection patterns in non-human primates","abstract":"Public health policy and clinical interventions against infectious diseases rely on understanding the factors that govern the initiation and progression of infections inside hosts. Animal infection experiments are essential tools to study these complex, multi-scale processes under controlled conditions, and they have shown that the dose and route of exposure can influence within-host disease dynamics. However, observed differences are difficult to quantify and to disentangle from confounding factors because small sample sizes limit the statistical power and scientific scope of individual studies. Here, by compiling and analyzing the largest published database of non-human primate challenge experiments (107 studies; 721 animals; 22,183 observations), we quantify how exposure conditions and demographic factors shape within-host SARS-CoV-2 infection kinetics in the respiratory and gastrointestinal tracts. We show that exposure route has stronger effects on kinetics than dose, age, sex, or species, with directly exposed tissues exhibiting distinct spatiotemporal kinetics from non-exposed tissues. We estimate 50% infectious doses for different tissues and show that they vary greatly (from <101.2 up to >107.4 pfu) depending on the exposure route. We find that dose effects on kinetics are also route-and tissue-specific, primarily influencing nasally-inoculated animals. Our results suggest that exposure route drives infection kinetics more strongly than dose in a critical model system for translational medicine, and they demonstrate the untapped potential for meta-analysis to extract additional insights from costly animal experiments.","version":"1.1","doi":"10.64898/2026.03.03.709384","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.04.624804","pub_date":"2026-3-03","title":"Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus","abstract":"SARS-CoV-2 infection poses a major threat to public health, and understanding the mechanism of viral replication and virion release would help identify therapeutic targets and effective drugs for combating the virus. Herein, we identified E3 ubiquitin-protein ligase Itchy homolog (ITCH) as a central regulator of SARS-CoV-2 at multiple steps and processes. ITCH enhances the ubiquitination of viral envelope and membrane proteins and mutual interactions of structural proteins, thereby aiding in virion assembly. ITCH-mediated ubiquitination also enhances the interaction of viral proteins to the autophagosome receptor p62, promoting their autophagosome-dependent secretion. Additionally, ITCH disrupts the trafficking of the protease furin and the maturation of cathepsin L, thereby suppressing their activities in cleaving and destabilizing the viral spike protein. Furthermore, ITCH exhibits robust activation during the SARS-CoV-2 replication stage, and SARS-CoV-2 replication is significantly decreased by genetic or pharmacological inhibition of ITCH. These findings provide new insights into the mechanisms of the SARS-CoV-2 life cycle and identify a potential target for developing treatments for the virus-related diseases.","version":"1.2","doi":"10.1101/2024.12.04.624804","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.02.709177","pub_date":"2026-3-03","title":"A new mRNA antigen vaccine induces potent B and T cell responses and in vivo protection against SARS-CoV-2","abstract":"The SARS-CoV-2 mRNA vaccine provides effective protection against viral infection and severe disease by inducing efficient adaptive immunity. However, vaccine efficacy is decreased against emerging variants, and immune memory is relatively short-lived. Here, we added new T cell epitopes to the RBD (receptor-binding domain) mRNA vaccine and identified a SARS-CoV-2 membrane epitope that significantly improved vaccine-induced immunity and protection in vivo. That new vaccine, designated G1-C, induced 8.2-fold higher levels of RBD-specific antibodies than did RBD and enhanced spike-specific T cell and B cell responses. Remarkably, the G1-C modulated hematopoietic stem cell (HSC) differentiation and increased levels of B and NK cells by regulating multiple signaling pathways in bone marrow potentially via Fos, Klf4, and Klf6 transcription factors. Altogether, these findings identify a new vaccine candidate to control viral infection by affecting the lymphoid-myeloid lineage bias and suggest the potential role of T cell epitopes in vaccine design and development.","version":"1.1","doi":"10.64898/2026.03.02.709177","journal":"bioRxiv","score":null},{"id":"10.64898/2026.03.02.709067","pub_date":"2026-3-03","title":"An Integrated Computational Antigen Discovery Pipeline with Hierarchical Filtering for Emerging Viral Variants","abstract":"Emerging and evolving viral diseases, such as SARS-CoV-2, continue to pose significant global health challenges, underscoring the urgent need for rapid and scalable antigen discovery pipelines. This work presents a computational pipeline that integrates diverse computational tools and machine learning models to accelerate the identification and optimization of antigen candidates. The pipeline employs efficient filtering and consensus-based strategies to highlight epitopes with high therapeutic potential. We demonstrate its utility by significantly narrowing the antigen search space for Rift Valley fever virus (RVFV) and Mayaro virus (MAYV), and by effectively identifying conserved neutralizing epitopes in SARS-CoV-2. Our proposed computational antigen pipeline offers a powerful framework for expediting the development of future vaccines and therapeutics in response to emerging pathogens.","version":"1.1","doi":"10.64898/2026.03.02.709067","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.27.708159","pub_date":"2026-2-27","title":"Antigenic landscape of a highly mutated SARS-CoV-2 Spike in ongoing viral evolution","abstract":"The ongoing emergence of SARS-CoV-2 variants in an increasingly immune-experienced population is largely enabled by the plasticity of the Spike protein, which lies at the frontline of host immune pressure. Here, we investigated how extensive remodeling of the N-terminal domain (NTD) in Spike influences its antigenic properties. Using BA.2.87.1, a variant heavily mutated in this domain, we found that even large deletions do not substantially disrupt overall Spike structure or pseudovirus infectivity. However, our structural and binding analyses revealed that the NTD exhibits increased flexibility and that interaction with a heme-metabolite, contributing to assembly of an immunodominant epitope, is lost. These conformational effectscoupled with mutations, compromised the ability of human convalescent antibodies to engage this domain, contributing to their reduced neutralizing capacity. Consequently, BA.2.87.1-like variants may escape recognition by the pre-existing NTD-targeting antibodies, potentially reducing protection. Together, our results highlight the intrinsic adaptability of the Spike beyond the receptor-binding domain, with important implications for immune escape during viral evolution. Structural flexibility of the Spike and immune escape of a SARS-CoV-2 variant with public health implications.","version":"1.1","doi":"10.64898/2026.02.27.708159","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.26.706090","pub_date":"2026-2-27","title":"Transition from infectivity and immune escape to pure escape as an evolutionary strategy during the COVID-19 pandemic","abstract":"New SARS-CoV-2 variants have undergone repeated selective sweeps since the beginning of the COVID-19 pandemic, but the fitness advantages and mechanisms driving these sweeps are not fully understood. We developed a probabilistic modeling framework to analyze pandemic growth, infectivity, and immune escape, explicitly accounting for seven immune exposure histories in 5,732 experiments and estimating the effects of 835 mutations. We found infectivity was important for early variants, but as gains became zero-sum, growth became driven by consistent increase in immune escape conferred by a primarily additive effect of mutational accumulation. While phenotypic tradeoffs exist for individual mutations, successful viral strains boast assemblages of mutations that do not sacrifice infectivity for escape. Thus, during an apparent transition to endemicity, SARS-CoV-2 evolution ascended along an evolutionary ridge in the mutational space defined by infectivity and escape, with infectivity reaching an early peak and antigenicity continuing to evolve.","version":"1.1","doi":"10.64898/2026.02.26.706090","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.03.692155","pub_date":"2026-2-26","title":"Serum IL-6 and IL-18 Responses in Pediatric COVID-19: Role of Vitamin D Status in a Cohort from Azerbaijan","abstract":"This study investigated the effects of COVID-19 infection and vitamin D status on inflammatory cytokine levels in children during the pandemic. A cohort of 170 children aged 1\u201317 years with PCR-confirmed SARS-CoV-2 infection was enrolled. Serum levels of IL-6 and IL-18 were analyzed in relation to vitamin D status and COVID-19 diagnosis. Our analysis revealed that children with vitamin D deficiency exhibited a trend toward increased levels of IL-6 and IL-18 compared to those with normal vitamin D levels; however, this association did not reach statistical significance. In contrast, COVID-19 infection was associated with significantly higher cytokine levels relative to healthy controls. These findings suggest a potential modulatory role of vitamin D in pediatric inflammatory responses to SARS-CoV-2, meriting further investigation through prospective studies.","version":"1.2","doi":"10.64898/2025.12.03.692155","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.26.708234","pub_date":"2026-2-26","title":"Identifying severe COVID-19 risk variants modulating enhancer reporter activity in lung cells","abstract":"Common genetic variants contribute to risk for complex human diseases. However, despite thousands of associations, variants modulating disease risk and their functional impact remain largely unknown. This includes SARS-CoV-2 infection, where outcomes range from asymptomatic to fatal. Most host risk variants associated with COVID-19 disease, identified through genome wide association studies, are located in the non-coding genome and may function by altering gene expression in disease-relevant cells and tissues. To address this at scale, we tested >4800 severe COVID-19-associated variants to determine the impact of individual variants and variant combinations on regulatory activity using Self-Transcribing Active Regulatory Region sequencing, a massively-parallel reporter assay, in a lung epithelial cell line (A549). We identify 166 variants within active sequences, of which 29 modulate activity allele-specifically. Evaluating variant combinations, we observe both additive and non-additive effects on regulatory activity. We employ state-of-the-art deep learning models to interpret allele-specific variant effects on regulatory activity and endogenous genomic features. Our work provides a set of prioritised severe COVID-19-associated variants that modulate regulatory activity in lung epithelial cells, candidate transcription factors, and candidate target genes with potential to be disease modifying.","version":"1.1","doi":"10.64898/2026.02.26.708234","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.25.707691","pub_date":"2026-2-25","title":"Identification of MED13 and DDX60 as critical host factors for SARS-CoV-2 infections","abstract":"The virus responsible for COVID-19, SARS-CoV-2, continues to spread through the world. The ongoing emergence of new variants with increased viral transmission and immune evasion continue to pose a challenge. Although large-scale genetic screens have identified numerous host factors required for SARS-CoV-2 infection, however the function of these hits remain incompletely understood. In this study, we performed a haploid forward genetic screen in chemically mutagenized mouse embryonic stem cells overexpressing human ACE2 and identified MED13 and DDX60 as important host factors involved in the modulation of SARS-CoV-2 infection. In this study, we have identified and characterized the function of these key element factors for SARS-CoV2 infection. Knockdown of CKM subunits\u2014with the exception of CDK8\u2014or the helicase DDX60 was sufficient to reduce SARS-CoV-2 infection across Vero E6, A549, and Calu-3 cells. During SARS-CoV-2 infection, MED13 was found to function downstream of the JAK/STAT interferon pathway, but also showed another function independently of the interferon response pathway. Surprisingly, while DDX60 is traditionally involved in the interferon response pathway, its knockdown reduces SARS-CoV-2 infection, suggesting DDX60 can promote SARS-CoV-2 infection Interestingly, while inactivation of MED13 or DDX60 markedly reduced SARS-CoV-2 and SARS-CoV infection, it did not affect MERS-CoV. Collectively, these results identify MED13 and DDX60 as critical host determinants for SARS-CoV-2 related-coronaviruses with pandemic potential. Using an unbiased haploid genetic screening approach, this study identifies for the first time MED13 and DDX60 as host factors that influence coronavirus replication through transcriptional and interferon-associated pathways. While DDX60 has previously been linked to antiviral signaling, our findings suggest it can promote coronavirus infection. Most interestingly, the effects of these factors extend beyond SARS-CoV-2 to other coronaviruses. These findings broaden the current understanding of coronavirus-host interaction by highlighting transcriptional control and interferon-associated pathways as modulators of infection rather than focusing solely on viral entry. The work provides mechanistic insight into how host regulatory networks influence coronavirus replication and suggests potential targets for host-directed therapies with activity against multiple present and future coronavirus threats. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. The data generated in this study are provided in the Supplementary Information/Source Data file. Sequencing data are available on NCBI Sequence Read Archive under the accession number BioProject PRJNA1271794. The next generation sequencing data generated in this study has been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRX29485992, SRX29485993, SRX29485994, SRX29485995, SRX29485996, SRX29485997, SRX29485998, SRX29485999, SRX29486000, SRX29486001, SRX29486002, SRX29486003, SRX29486004, SRX29486005, SRX29486006, SRX29486007, SRX29486010, SRX29486011, SRX29486012, SRX29486013, SRX29486014, SRX29486015, SRX29486016, SRX29486017, SRX29486018, SRX29486019, SRX29486021, SRX29486022, SRX29486023, SRX29486024","version":"1.1","doi":"10.64898/2026.02.25.707691","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.24.707668","pub_date":"2026-2-25","title":"Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages","abstract":"Circadian rhythms, driven by 24-hour molecular oscillators, or \u201cclocks\u201d, widely tune physiology to the daily rhythms of light and dark to enhance organismal fitness. In mammals, the cellular immune response is tightly regulated by these rhythms such that immunometabolic output is coordinated across the day, consolidating macrophage physiology into temporally distinct phases that determine the macrophage response to stimuli. Importantly, key proteins in the macrophage response to viral infection have been found to be under circadian control, and time of day of adjuvant application is known to affect the efficacy of vaccinations, including in the case of the SARS-CoV-2 virus. However, little is known about the molecular changes that underly the temporal response to vaccine application. Therefore, to investigate the circadian response of macrophage physiology to adjuvant exposure, we exposed primary mouse and human macrophages to the SARS-CoV-1 and CoV-2 spike proteins at different times over the circadian day. To further explore the time-of-day effect, we performed a multi-omics analysis and in vitro tissue culture assays examining macrophage responses over circadian time. We found that, conserved across the species, the timing of spike protein exposure dictated two distinct temporal responses which were characterized by hallmarks of immunometabolic suppression and modest immunometabolic activation. Intriguingly, these temporal responses were driven by central metabolic and mitochondrial changes rather than classical immune activation, suggesting immunometabolic control is a primary regulator of the temporal response of immune cells to stimuli.","version":"1.1","doi":"10.64898/2026.02.24.707668","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.17.706397","pub_date":"2026-2-25","title":"TMPRSS2 reduces antibody recognition of SARS-CoV-2 spike","abstract":"The serine protease TMPRSS2 acts as a cofactor for SARS-CoV-2 entry by cleaving the viral spike (S) to initiate fusion. Whether TMPRSS2 has an impact on humoral immune response against S remains poorly characterized. Here, we show that TMPRSS2 impairs antibody binding to S. In S-expressing and infected cells, TMPRSS2 decreases monoclonal antibody (mAb) and immune serum binding, as well as antibody-dependent cellular cytotoxicity (ADCC) induction. Using a panel of 39 mAbs targeting various S regions, we observe that those binding to the S2 subunit are the most affected by TMPRSS2. TMPRSS2 promotes a partial shedding of S1 and changes S2 conformation. This processing reduces Angiotensin-Converting Enzyme 2 (ACE2) binding while increasing cell-cell fusion. We further observe that the capacity of TMPRSS2 to decrease antibody recognition is conserved across coronaviruses and shared with other TMPRSS proteins. However, TMPRSS2 expression in infected cells does not impact significantly virions\u2019 infectivity or the antibody recognition, as measured by flow virometry. Collectively, our findings suggest that TMPRSS2 processing of S favors a fusion intermediate conformation which is less sensitive to antibody recognition.","version":"1.2","doi":"10.64898/2026.02.17.706397","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.24.707865","pub_date":"2026-2-25","title":"Cytosolic interaction with RNA-helicase DDX39A titrates viral RNA G-quadruplex mediated \u03b1-Synuclein amyloidogenesis","abstract":"Amyloid aggregates of \u03b1-Synuclein are hallmark of Parkinson\u2019s Disease (PD) and related neurodegenerative diseases. \u03b1-Synuclein, being an non-canonical RNA-binding protein (RBP), associates with other RBPs within cytosolic RNA-protein granules to modulate mRNA-stability. Conversely, mRNA G-quadruplexes (rG4s) expedite \u03b1-Synuclein amyloidogenesis. However, spatiotemporal control on \u03b1-Synuclein amyloidogenesis by other RBPs remains unexplored. Here, we report that RNA-dependent cytosolic interaction with DEAD-box RNA-helicase DDX39A decelerates \u03b1-Synuclein amyloidogenesis. Viral infections transiently elevate rG4s in cytoplasm. Perturbing interactions between \u03b1 Synuclein and DDX39A using viral rG4s from H1N1-influenza and SARS-CoV-2 genomes expedites intracellular amyloidogenesis. Conversely, DDX39A overexpression alleviates \u03b1-Synuclein amyloidogenesis in mouse primary neurons triggered by SARS-CoV-2 infection. We demonstrate that while DDX39A unwinds viral rG4s to mitigate \u03b1-Synuclein sol-gel transition, its reciprocal cooperative phase separation with \u03b1-Synuclein enhances the helicase\u2019s rG4-unwinding activity. We propose that accelerated \u03b1-Synuclein amyloidogenesis represents a trade-off within this RNA\u2013protein interaction equilibrium, contributing to the viral etiology of PD.","version":"1.1","doi":"10.64898/2026.02.24.707865","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.24.707628","pub_date":"2026-2-25","title":"Highly sensitive enzyme- and amplification-free, quantitative DNA detection using YVO4:Eu luminescent nanoparticle probes","abstract":"The sensitive detection of nucleic acids is crucial for the accurate diagnosis of infections. In this context, amplification-based methods, such as the quantitative Polymerase Chain Reaction (qPCR) are the gold standard for ultrasensitive DNA or RNA detection and quantification. However, despite its widespread use in developed countries during the COVID-19 pandemic, qPCR remains a costly tool, difficult to implement into low-infrastructure locations. Efforts for the development of alternative tools have yielded high sensitivity approaches but sensitivity is typically reached at the expense of complexity. We here report the development of a simple, sensitive, amplification-, and enzyme-free nucleic acid detection technique using YVO4:Eu luminescent nanoparticles. We established an optimized interaction scheme to efficiently reveal target DNA fragments with nanoparticles. By exploiting the extremely strong absorption of the vanadate matrix in the UV to excite the nanoparticles inducing the characteristic Eu3+ emission at 617 nm via energy transfer, we achieved a highly sensitive (down to 500 particles/mm2; 17,000 particles/well) read-out in standard microplates using a home-made optical reader with light-emitting diode (LED), 275-nm excitation. We reached a 50-aM (30,000 copies/mL) sensitivity for the detection of the 72-base DNA fragment of the SARS-CoV-2 n1 gene. Our new quantitative analytical method detects nucleic acids without amplification with performances close to standard PCR (10,000 copies/mL), and could be the basis for a transportable alternative for the diagnosis of infectious diseases.","version":"1.1","doi":"10.64898/2026.02.24.707628","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.23.707522","pub_date":"2026-2-24","title":"Genetic Evidence Indicates the Evolutionary Importance of the SARS-CoV-2 ORF9b protein","abstract":"All known betacoronaviruses possess an overlapping alternate-frame gene within the nucleocapsid gene. In SARS-CoV-2, the gene for this \u201cinternal protein\u201d is ORF9b. The WHO Variants of Concern (VOC) Alpha, Delta, and Omicron all possess noncoding mutations that increase ORF9b expression. We show that with two exceptions, every major variant of the VOC era has had similar noncoding mutations to increase ORF9b expression and that these mutations are also frequently seen in long-branch, anachronistic, posited chronic-infection (PCI) sequences. Furthermore, we show that the amino acid substitution rate in ORF9b is higher than for any other SARS-CoV-2 gene, both in high-quality circulating sequences and in PCI sequences. This suggests that, as immunity to SARS-CoV-2 has grown in the population, increased ORF9b expression has conferred an evolutionary advantage, likely due to its ability to antagonize the antiviral type-I interferon response. We also show that PCI sequences are marked by distinct mutational patterns in ORF9b, some of which later became prominent in major variants. This evidence points to the importance of ORF9b for both viral transmission and within-host persistence.","version":"1.1","doi":"10.64898/2026.02.23.707522","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.23.707600","pub_date":"2026-2-24","title":"AVE0991, a Mas receptor agonist, increases influenza and COVID-19 severity in vivo","abstract":"Dysregulation of the renin-angiotensin system (RAS) contributes to severe influenza and COVID-19, potentially via impaired ACE2/Ang-(1-7)/Mas receptor (MasR) signalling. AVE0991, an orally bioavailable MasR agonist, protects against non-infectious lung inflammation, but its effects in viral respiratory disease are unknown. We evaluated AVE0991 in human lung epithelial cells and murine models of influenza A virus (IAV) and SARS-CoV-2 infection. In vitro, AVE0991 suppressed cytokines IL-6 and TNF-\u03b1 to both IAV and SARS-CoV-2 and reduced IAV titres. Unexpectedly, in vivo treatment worsened disease. These findings highlight discordant in vitro and in vivo effects and underscore the need for careful evaluation of RAS-targeted therapies in acute viral infection.","version":"1.1","doi":"10.64898/2026.02.23.707600","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.09.675101","pub_date":"2026-2-24","title":"Dissecting epigenome dynamics in human immune cells upon viral and chemical exposure by multimodal single-cell profiling","abstract":"Environmental and pathogen exposure can lead to profound remodeling of the gene-regulatory landscape across human immune cell populations. Here, we present a single-nucleus chromatin accessibility atlas of human immune cells comprising 271,299 cells and 319,420 candidate regulatory elements from individuals exposed to HIV-1, COVID-19, Influenza virus, organophosphates, as well as healthy controls. Our longitudinal HIV cohort reveals epigenetic signatures of T cell exhaustion manifested in changes in the accessibility at binding sites for the FOXP family transcription factors. Upon severe exposure to SARS-Cov2 we identified changes in chromatin accessibility manifesting in a regulatory switch in cytokine networks characterized by the downregulation of the NF-\u03baB motif family, alongside concordant changes in AP-1 and IRF factor networks in CD14+ classical monocytes. By integrating single-cell profiles of DNA methylation from matched samples, we created a multimodal epigenome atlas of human immune cell states using the accessibility-derived candidate regulatory elements. Both modalities exhibit complementary epigenetic signatures at transcription factor binding sites associated with cell state, as exemplified in the process of memory formation in T-cells, where BATF, AP-1, and ETS motifs exhibit significant epigenetic covariance across both epigenomic layers. Finally, by linking potentially regulatory DNA methylation signatures to changes in chromatin accessibility in monocytes, we observe that severe COVID-19 involves selective, multiomics remodeling of epigenetic profiles at TF binding sites manifested in concordant DNA methylation and accessibility dynamics at inflammation-associated regulatory TFs.","version":"1.2","doi":"10.1101/2025.09.09.675101","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.23.677998","pub_date":"2026-2-23","title":"Redirection of SARS-CoV-2 to Phagocytes by Intranasal sACE2-Fc as a Universal Decoy Confers Complete Prophylactic Protection","abstract":"The rapid evolution of SARS-CoV-2 and other respiratory RNA viruses limits the success of current vaccines and antibody-based therapies. Engineered decoy receptors based on soluble angiotensin-converting enzyme 2 (sACE2) offer promising alternatives. Clinical-grade recombinant sACE2 inhibits SARS-CoV-2 replication in vitro but shows limited clinical success. This study reports an optimized sACE2 mutant fused to human IgG1 Fc (B5-D3), which redirects virus\u2013decoy complexes to lysosomal degradation in macrophages. Intranasal prophylactic delivery of B5-D3 confers complete protection in SARS-CoV-2-infected K18-hACE2 mice. Abrogation of Fc effector functions compromises antiviral protection, indicating that Fc-mediated uptake of virus\u2013decoy complexes is critical. Transcriptomic analysis suggests that B5-D3 induces early immune activation in lungs of infected mice. Bio-distribution and flow cytometry reveal selective targeting of airway phagocytes. In vitro assays confirm lysosomal degradation of virus\u2013decoy complexes by macrophages without productive infection. These findings reveal a distinct antiviral mechanism via phagocytic clearance, supporting refined regimens for decoy treatments against SARS-CoV-2 and potentially other respiratory viruses.","version":"1.3","doi":"10.1101/2025.09.23.677998","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.20.706927","pub_date":"2026-2-23","title":"Genomic Evolution of SARS-CoV-2 Delta Variants Pre- and Post-Omicron Emergence using Alignment-free Machine Learning models","abstract":"The SARS-CoV-2 Delta variant (B.1.617.2), initially classified as a variant of concern due to its enhanced transmissibility and vaccine-escape mutations, underwent further genomic changes following the emergence of the Omicron variant (B.1.1.529). This study investigates the genomic differences in Delta variant spike gene sequences collected before and after the emergence of Omicron. A total of 190 sequences were analyzed using an alignment-free approach incorporating k-mer-based feature extraction and machine learning models, including convolutional neural networks (CNN), K-means clustering, and random forest classification. The random forest model achieved 93% accuracy, with significant F1 scores, effectively distinguishing the two Delta variant groups. Comparative analysis revealed 157 persistent mutations and four vanished mutations in the post-Omicron group. Cluster analysis showed notable shifts, indicating stable yet evolving genomic patterns over time. The study demonstrates the advantage of alignment-free methods in detecting subtle sequence variations that alignment-based approaches may overlook. These findings enhance our understanding of SARS-CoV-2 evolution and provide a framework for identifying key genomic signatures relevant to public health. The methodology and insights gained offer potential applications in variant surveillance, vaccine design, and viral evolutionary studies, supporting preparedness for future SARS-CoV-2 variant emergence.","version":"1.1","doi":"10.64898/2026.02.20.706927","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.21.707085","pub_date":"2026-2-23","title":"PFOA induced metabolic and immune perturbations in a SARS-2 infection model","abstract":"This study evaluates the impact of PFOA exposure on the metabolome and immune response to SARS-2 using a ferret model. Ferrets were separated into control or PFOA-exposed groups (10/mg/kg/day) and challenged with SARS-2. Longitudinal analyses encompassing clinical assessments, serological profiling, histopathology, and untargeted nuclear magnetic resonance (NMR) metabolomics revealed significant metabolic and immunological perturbations. We found prominent effects of PFOA exposure on metabolism, which resulted in altered metabolic responses to SARS-2 infection. PFOA exposure was also associated with impaired immune function, as evidenced by decreased serum IgG levels, increased viral loads, and prolonged peak infectivity. These findings underscore the consequences of PFOA exposure on host metabolism and immunity during infectious diseases.","version":"1.1","doi":"10.64898/2026.02.21.707085","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.20.707093","pub_date":"2026-2-22","title":"The magnitude of the secondary B cell response is primarily defined by antibody feedback inhibition rather than the number of memory B cells present","abstract":"Clonal expansion of memory lymphocytes after each antigen encounter is the primary mechanism for amplifying immunity. For most vaccines, boosters are common practice and are expected to stimulate proliferation of pre-existing memory B cells (Bmem), thereby expanding the antigen-specific Bmem pool, along with driving their differentiation into antibody-producing plasma cells that replenish antibody titers. It is widely assumed that the number of Bmem present in the body prior to administration of a booster vaccination will define the magnitude of the ensuing response. However, due to technical limitations hampering reliable detection of rare antigen-specific Bmem in human subjects, the extent to which Bmem numbers are actually modulated following a booster vaccination remains unclear. By comparing Bmem frequencies and antibody titers in the same individuals after primary and secondary vaccination with SARS-CoV-2 Spike (S-antigen)-encoding mRNA we found that expansion of Bmem and the magnitude of the secondary antibody response were not determined by the number of Bmem measured before the second vaccine inoculation. Instead, both were inversely correlated with levels of S-antigen-specific serum IgG prior to the secondary antigen exposure. Collectively, the data suggest that secondary B cell responses are constrained by antibody feedback inhibition of Bmem, rather than their paucity.","version":"1.1","doi":"10.64898/2026.02.20.707093","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.18.706653","pub_date":"2026-2-19","title":"The buried S2 apex of SARS-CoV-2 spike elicits an immunodominant germline-restricted public antibody response","abstract":"The continued mutational pressure on the SARS-CoV-2 S1 subunit underscores the need to target the conserved S2 region for pan-coronavirus vaccine development. A detailed molecular understanding of S2-directed immune responses is therefore essential. In this study, we identified the S2 apex as the most immunodominant epitope within the S2 subunit, eliciting robust antibody responses despite occlusion by S1, using electron-microscopy-based polyclonal epitope mapping (EMPEM) of plasma from infected and vaccinated individuals. Structure-guided sequence analysis with antibody databases revealed that antibodies targeting a poorly characterized S2 Apex-B site form a convergent public clonotype, which is predominantly derived from the IGHV3-30 germline with a 14-residue CDRH3 containing a G/S-G-S/N-Y motif. This clonotype is extensively expanded, accounting for up to 40% of total spike-reactive antibody sequence counts in individual vaccinated donors. This study elucidates the molecular basis the high-frequency elicitation of this non-neutralizing clonotype emphasizing that its immunodominance acts as a primary hurdle for universal coronavirus vaccines and underscore the need for precision antigen design to redirect immunity toward more potent neutralizing targets.","version":"1.1","doi":"10.64898/2026.02.18.706653","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.18.706716","pub_date":"2026-2-19","title":"Minipoa: A minimizer-based method for fast and memory-efficient partial order alignment","abstract":"Partial order alignment (POA) has emerged as a fundamental component in long-read error correction, assembly and pangenomics. However, conventional POA algorithms are limited by high time and memory requirements, making them inefficient for large-scale datasets. Here, we present minipoa, a fast and memory-efficient POA tool that incorporates seed-chain-align heuristics, adaptive or static banding strategies, and single-instruction multiple-data optimizations. Minipoa achieves up to a 5-fold speedup over abPOA, reduces memory usage by up to 16-fold, and improves correction accuracy, while maintaining strong performance on both PacBio and ONT simulated datasets, and can be readily integrated into existing long-read error correction and assembly workflows. In multiple sequence alignment datasets, minipoa demonstrates superior computational efficiency and alignment accuracy compared with all other tested tools, achieving Total Column scores up to 2.5-fold higher than MAFFT in low-similarity scenarios. Moreover, minipoa enables multiple sequence alignment of megabase-long genomes and million-sequence datasets, demonstrated by 342 Mycobacterium tuberculosis sequences and one million SARS-CoV-2 sequences respectively. Collectively, minipoa is well positioned to become a cornerstone in the era of large-scale pangenomics.","version":"1.1","doi":"10.64898/2026.02.18.706716","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.16.706083","pub_date":"2026-2-17","title":"Cross-reactivity of SARS-CoV-2-specific T cells against tumor-associated antigens via molecular mimicry","abstract":"We have recently described SARS-COV-2 antigens showing sequence and conformational homology to tumor associated antigens (TAAs). Moreover, cross-reactive T cells have been identified in individuals either infected by the SARS-CoV-2 virus or vaccinated with the BNT162b2 preventive vaccine. In the present study, we analyzed the specific cross-binding TCRs by single cell RNA TCR sequencing. The paired SARS-CoV-2 epitope LLLDDFVEI (VIR) and the PRDX5 tumor associated antigen LLLDDLLVS (TAA) were selected to elicit cross-reacting T cells ex vivo. PBMCs from 5 healthy individuals were cultured for 10 days with 10 ug every 3 days of one of the two peptides and cells were selected for single cell RNA TCR sequencing. Results in CD8+ T Effector cells (TTE) showed the amplification or the de novo identification of a handful number of TRAV/TRBV genes and of CDR3\u03b1\u03b2 motifs upon treatment ex vivo with both epitopes, which are specific for each subject in the analysis. The very same clonotypes were identified also in the CD8+ T proliferating subset, confirming that both epitopes induced a highly activated and plastic state. Conformational prediction analyses of pMHC-TCR complexes showed perfect structural overlap, supporting the functional cross-reaction of CD8+ T cells with both the viral and the tumor antigens. Our results describe for the first time the TCR CDR3\u03b1\u03b2 motifs amplified or de novo expanded by induction with a viral antigen showing a molecular mimicry with a tumor antigen. They are strictly individual and do not match with any motif in the publicly available TCR repository. However, considering the significant degeneracy in the TCR binding to the same epitope, the finding of identical TCR CDR3\u03b1\u03b2 motifs elicited by two homologous epitopes is of the highest functional relevance. Such results provide a clear experimental validation proof that microbial epitopes mimicking TAAs can be used to develop off-the-shelf preventive/therapeutic vaccine formulations. Indeed, such non-self antigens are much stronger immunogens and may elicit a potent cross-reacting anti-cancer T cell response.","version":"1.1","doi":"10.64898/2026.02.16.706083","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.15.705976","pub_date":"2026-2-17","title":"Antibody Responses Against Coronaviruses in the Wuhan Population","abstract":"Prolonged circulation of SARS-CoV-2 may broaden population-level neutralizing antibody responses to diverse coronaviruses; however, the extent of this breadth remains unclear. To address this gap, we assessed serum neutralizing activity in 869 individuals from Wuhan, China, including 78 pre-pandemic samples collected in 2017 and 791 post-pandemic samples obtained in 2025. Specifically, using pseudovirus neutralization assays, sera were tested against a panel of coronaviruses, including circulating SARS-CoV-2 Omicron subvariants XFG and NB.1.8.1. Neutralization was measured at a fixed dilution for all samples and by determination of 50% inhibitory dilution (ID50) for selected sera. In parallel, antigenic cartography and genetic distance analyses were applied to relate functional antigenic relationships to spike protein divergence. Our results showed that, compared with pre-pandemic sera, post-pandemic sera exhibited enhanced neutralizing activity against multiple sarbecoviruses and the merbecovirus MjHKU4r-CoV-1, with mean inhibition increases ranging from 1.7% to 76.5% at a 1:20 dilution. Notably, the largest increases from 51.8% to 76.5% were observed against clade 1b sarbecoviruses. In contrast, neutralizing activity against human alphacoronaviruses and MERS-CoV showed no meaningful enhancement. Moreover, neutralizing titers in post-pandemic sera were strongly correlated across sarbecoviruses, particularly among clade 1b viruses. Antigenic mapping further revealed that several zoonotic sarbecoviruses were antigenically closer to ancestral SARS-CoV-2 than contemporary Omicron subvariants, despite substantially greater genetic divergence. Taken together, these findings demonstrate that cumulative SARS-CoV-2 exposure promotes broadly cross-neutralizing antibody responses within the sarbecovirus subgenus and provide a quantitative framework to inform the rational design of pan-sarbecovirus vaccines and antibody-based countermeasures for future coronavirus preparedness.","version":"1.1","doi":"10.64898/2026.02.15.705976","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.04.703885","pub_date":"2026-2-16","title":"Development and characterization of mouse-adapted recombinant SARS-CoV-2 expressing reporter genes","abstract":"Transgenic K18-hACE2 mice are a standard model for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), albeit with limitations. A mouse-adapted 30 (MA30) SARS-CoV-2 has been developed to allow infection of wild-type (WT) mice strains. However, SARS-CoV-2 MA30 cannot be easily tracked in vitro, ex vivo, or in vivo. To address the problem, we developed a recombinant (r)SARS-CoV-2 based on the MA30 strain expressing fluorescent (mCherry) and luciferase (nanoluciferase, Nluc) reporter genes, alone or in combination, that enable tracking of viral infection in WT C57BL/6 and BALB/c mice. Insertion of the reporter genes resulted in minor viral attenuation in vitro, with \u223c0.5-1.0-log lower titers than rSARS-CoV-2 MA30 WT in A549 hACE2 cells, while maintain similar plaque morphology and replication kinetics in Vero AT cells. In vivo, reporter-expressing rSARS-CoV-2 MA30 caused transient weight loss, contrasting with lethal rSARS-CoV-2 MA30 WT infection. Bioluminescence imaging of rSARS-CoV-2 MA30 Nluc in C57BL/6 and BALB/c mice revealed peak pulmonary replication at 2 days post-infection, with resolution by day 4, and correlated with tissue viral loads. Our results demonstrate the feasibility of using rSARS-CoV-2 MA30 expressing reporter genes to track viral infection in vitro, ex vivo, and in vivo without a need for secondary approaches to monitor viral infection as are required for rSARS-CoV-2 MA30 WT. Our system is highly suitable to evaluate prophylactic vaccines and therapeutic antibodies or antiviral approaches in WT or transgenic C57BL/6 and BALB/c mice without the shortcomings of K18-hACE2 mice and with the added advantage of non-invasive monitoring of treatment efficacy. The K18-hACE2 transgenic mouse model limits the capability to study SARS-CoV-2. While a mouse adapted 30 (MA30) has been developed to study SARS-CoV-2 in wild-type (WT) mice, it does not allow non-invasive tracking of viral infections. Recombinant viruses expressing reporter genes enable real-time monitoring of infection dynamics, opening an avenue to study viral tropism and easily evaluate prophylactic and therapeutic approaches. They furthermore support longitudinal studies, which reduces the number of research animals required. Here, we show that a recombinant (r)SARS-CoV-2 expressing fluorescent (mCherry) and nanoluciferase (Nluc) reporter genes, alone or in combination, can be used to track viral infections in vitro, ex vivo, and in vivo without the need for secondary approaches that are required to detect SARS-CoV-2 MA30 in WT mice. These reporter-expressing rSARS-CoV-2 MA30 may accelerate vaccine development and antiviral drug discovery in WT or transgenic mice bypassing the need for hACE2 overexpression in K18-hACE2 transgenic mice.","version":"1.1","doi":"10.64898/2026.02.04.703885","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.13.705720","pub_date":"2026-2-16","title":"Macrophage Migration Inhibitory Factor (MIF) Overexpression Induced by SARS-CoV-2 Infection Promotes Neural Regeneration in Human Brain Organoids","abstract":"While SARS-CoV-2 primarily targets the respiratory system, its neurological effects have become a significant clinical concern. Postmortem analyses reveal astrogliosis, neuronal death, and blood-brain barrier dysfunction, yet the interplay between neural injury and endogenous repair remains unclear. Here, we employed human embryonic stem cell\u2013derived brain organoids to examine viral tropism, bystander effects, and regenerative responses following infection. Single-cell transcriptomics and histological assays showed that SARS-CoV-2 productively infects neurons, neural progenitors, astroglia, and choroid plexus cells, triggering widespread apoptosis and senescence in both infected and neighboring cells. Despite low infection rates, organoids activated robust regenerative programs, including axon guidance, Wnt pathway signaling in mature neurons, and radial glia proliferation. Importantly, macrophage migration inhibitory factor (MIF) emerged as a key mediator, being strongly upregulated in both infected and uninfected cells, particularly in the choroid plexus. Recombinant MIF promoted dendritic outgrowth and cortical progenitor activation in uninfected organoids. Computational analyses indicated that MIF stimulates neural regenerative via EGFR signaling and upregulates its own expression in non-infected cells. These findings identify MIF as a molecular link between SARS-CoV-2-induced neural damage and regenerative activation in cortical cells.","version":"1.1","doi":"10.64898/2026.02.13.705720","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.13.705786","pub_date":"2026-2-16","title":"\u201cIt\u2019s not just about the science\u201d. The impact of undergraduate research projects and COVID-19 on graduate attributes and employability","abstract":"Over the past two decades, Higher Education Institutions have increasingly prioritised transferrable skills to enhance graduate employability. Graduate Attributes (GAs) now act as key indicators of student competencies for both learners and employers. Final-year research projects, typically high in credit value, represent capstone experiences that promote subject expertise and GA development through research, written work, and oral presentations. This study analyses pre- and post-project survey data from RQF Level 6 biomedical and biomolecular science students at a Russell Group University over four years (2019\u20132023). Most projects were laboratory-based, though the 2020\u20132021 cohort completed theirs remotely due to COVID-19. Students reflected on expectations and experiences of GA development, subject knowledge, and employability. Initial responses revealed anxiety and uncertainty, particularly among the 2020\u20132021 cohort, but most anticipated gains in skills and employability. Post-project feedback confirmed this, identifying critical thinking, confidence, resilience, collaboration, and future focus as key outcomes. Digital capability was notably strengthened, especially during remote delivery. The findings emphasise the importance of a shared understanding of GAs in bioscience education and the value of embedding structured reflection and preparatory support to help students recognise and articulate their evolving skills.","version":"1.1","doi":"10.64898/2026.02.13.705786","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.12.705501","pub_date":"2026-2-13","title":"Disentangled multimodal evolutionary representations for cross-virus predictive modeling of antigenic change","abstract":"Antigenic evolution drives immune escape and complicates early assessment of emerging pathogens, especially when genomic surveillance is sparse and functional measurements lag behind real-world spread. Early prediction and generalization to unknown viruses require faithful and information-rich representations of viral evolution, a need that is not fully addressed by existing protein language model\u2013based approaches. Here we introduce DERIVE, a flow-based generative framework that learns a disentangled latent representation of multimodal evolutionary history by integrating sequence homology with physicochemical and structural features. Using only pre-pandemic SARS-CoV-2 sequences, DERIVE prioritizes high-risk mutations, reconstructs strain-level evolutionary trajectories, forecasts immune escape and prevalence trends, and produces mutation-level effect maps. The learned representation transfers robustly across viral families, with strong concordance to functional data for influenza virus, HIV, rabies lyssavirus and chikungunya virus. Together, these results position DERIVE as a generalizable and interpretable framework for anticipating antigenic evolution and supporting early, actionable assessment of future \u201cDisease X\u201d threats.","version":"1.1","doi":"10.64898/2026.02.12.705501","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.11.705256","pub_date":"2026-2-12","title":"White-tailed deer milk exhibits SARS-CoV-2 neutralizing antibodies and synergistic mechanisms that contribute to rapid viral RNA degradation","abstract":"White-tailed deer (WTD) represent the most significant SARS-CoV-2 wildlife reservoir in North America, yet the role of antiviral mechanisms in vertical transmission remains unexplored. We investigated SARS-CoV-2 antibody responses and viral stability in milk from lactating WTD and humans to characterize species-specific antiviral mechanisms. SARS-CoV-2 neutralizing antibodies were detected in milk and serum in WTD specimens using complementary immunoassays, providing the first evidence of humoral immune responses in wildlife milk. Despite antibody presence indicating prior SARS-CoV-2 exposure, viral RNA was undetectable in all WTD milk samples. This pattern aligns with observations in human milk, where viral RNA was also undetectable both during active infection (when nasal swabs were positive) and during antibody-positive periods following recovery. In vitro stability studies revealed striking species differences: all SARS-CoV-2 variants (A, B.1.1.7, BA.1.1.529) rapidly degraded in WTD milk within 30 min at physiological temperatures, while remaining mostly stable in human milk for up to 60 min. Biochemical characterization identified multifactorial degradation mechanisms in WTD milk, including 5-20 fold elevated mineral concentrations (sodium, magnesium, phosphorus, and potassium), enhanced protease activity, and increased lactoperoxidase levels. Individual mineral supplementation revealed variant-specific susceptibilities, with B.1.1.7 showing pronounced sensitivity to ionic stress. Mechanistic studies demonstrated synergistic effects between elevated ionic concentrations and proteolytic activity, with heat-labile and heat-stable degradation pathways contributing to viral inactivation. These findings reveal that WTD milk possesses intrinsic antiviral properties fundamentally different from human milk, representing an evolutionary adaptation that may impact viral persistence and transmission dynamics in wildlife populations. These findings reveal antiviral mechanisms in WTD milk that represent a previously unrecognized component of pathogen control in wildlife reservoirs, with important implications for understanding wildlife-pathogen interactions and zoonotic risk assessment. White-tailed deer (WTD) have become the primary wildlife reservoir for SARS-CoV-2, with millions of infected animals across North America. Despite this significance, the presence of protective antibodies and viral behavior in WTD milk remained unexplored. We collected milk samples from lactating WTD during hunting seasons to investigate whether WTD produce neutralizing antibodies similar to those found in human milk and to examine how the virus behaves in this biological fluid. Our analysis revealed that WTD milk contains antibodies capable of neutralizing SARS-CoV-2. When we compared viral stability between WTD and human milk, we observed that WTD milk rapidly degrades viral genetic material within 30-40 min, while the same virus remains stable in human milk for over an hour. We identified that WTD milk contains mineral concentrations 5-20 times higher than human milk, including elevated levels of sodium, magnesium, and potassium, along with enhanced enzyme activity that breaks down viral components. These findings indicate that WTD milk functions as a protective barrier rather than a transmission route. This has implications for understanding viral persistence in wildlife populations and assessing potential risks to human health. Our work demonstrates that deer milk possesses multiple biological defense mechanisms that may protect offspring from viral infections, contributing to our understanding of wildlife immunity and pandemic preparedness.","version":"1.1","doi":"10.64898/2026.02.11.705256","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.11.705426","pub_date":"2026-2-12","title":"Antibody-Dependent Heterotypic Syncytia Drive COVID-19 Inflammation and Disease Progression","abstract":"Severe COVID-19 is characterized by profound immune dysregulation and excessive inflammation. Aberrant myeloid responses drive the hyperinflammation, yet the precise mechanisms remain elusive. Here, we demonstrate that certain spike antibodies promote the formation of heterotypic syncytia between monocytes/macrophages and virus-infected pneumocytes, leading to excessive inflammatory cytokine release. This antibody-dependent syncytium formation requires Fc\u03b3RI (CD64) and ADAM10, as well as the assembly of the six-helix bundle by spike S2 subunit. Notably, these syncytia exhibit a more potent pro-inflammatory signature compared to virus-infected epithelial cells. In vivo, sub-neutralizing antibody concentrations amplified inflammation and exacerbated disease in SARS-CoV-2-infected mice without increasing viral burden. Furthermore, scRNA-seq of postmortem lung tissues from COVID-19 patients implicated the monocytes/macrophages-derived syncytia as a key cellular source of inflammatory cytokines. Together, these findings define a novel mechanism of antibody-dependent enhancement of inflammation that helps explain the hyperinflammatory responses in severe COVID-19, and suggest new therapeutic opportunities targeting this pathway.","version":"1.1","doi":"10.64898/2026.02.11.705426","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.10.705130","pub_date":"2026-2-12","title":"Splicer: Phylogenetic Placement in Sub-Linear Time","abstract":"Phylogenetic placement is an established approach for rapidly classifying new genetic sequences and updating a phylogeny without fully recomputing it. Popular maximum-likelihood placement methods, such as pplacer and EPA-ng, tend to struggle computationally when the size of the reference tree increases to tens or hundreds of thousands of sequences. As a more scalable alternative, distance-based and parsimony-based placement methods were introduced such as UShER. These methods, in principle, scale linearly as the size of the reference tree grows. However, as the scale of genetic and genomic sequences continues to grow nearly exponentially, developing algorithms that can perform placement in sub-linear time while maintaining accuracy becomes more crucial. Here, we develop Splicer, the first such algorithm that can perform placement in guaranteed  time. To achieve this performance, Splicer first decomposes the original reference tree into \u2018blobs\u2019 and constructs a phylogenetic scaffold tree linking representatives from different blobs. Every blob in such decomposition has at most  taxa, and the scaffold tree has at most  leaves, where c is any constant. Then, given the query sequences for placement, they are first placed onto a scaffold tree using pplacer or EPA-ng, and then placed more precisely within the respective blobs. We demonstrate the high accuracy of Splicer on an empirical influenza A virus dataset that has sparse coverage due to limited genomic surveillance. We also show that Splicer can, for the first time, apply maximum-likelihood placement to COVID-19 pandemic-scale data using a dataset with over 12 million SARS-CoV-2 reference genomes. Splicer scales the highly accurate maximum-likelihood approaches implemented in pplacer and EPA-ng to trees with millions of taxa and eliminates the necessity to curate and subsample genomic datasets for real-time classifications. Splicer tool and source code are freely available at https://github.com/flu-crew/splicer.","version":"1.1","doi":"10.64898/2026.02.10.705130","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.09.704753","pub_date":"2026-2-10","title":"Viral haplotype reconstruction from long reads with virCHap","abstract":"Resolving genomes at the haplotype level for viral populations is crucial for understanding the prevalence of viral diseases and for the development of effective therapeutic treatments. However, viral haplotype reconstruction still presents challenges, such as an unknown number of strains, high inter-strain similarity, repetitive regions, and difficulties in abundance estimation. Here, we developed virCHap, a new reference-based haplotype phasing algorithm for viruses, which applies graph partitioning followed by iteratively quantifiable cluster merging on long-read sequencing data. Benchmarking on simulated and real datasets demonstrates that virCHap outperforms current tools in terms of recall, accurate abundance estimates and read clustering accuracy. On the simulated large-genome VZV experiment, virCHap had a 96.5% recall, 14% higher than the second-best method, and had the most accurate abundance estimates. On a real 5-strain PVY dataset, virCHap had a precision exceeding 92.9%, a recall of over 97%, and a read clustering accuracy of 82%, outperforming the second-best method by 32%. On a real 6-strain SARS-CoV-2 dataset, virCHap achieved >96.9% accuracy, and the most accurate abundance estimates within the spike gene.","version":"1.1","doi":"10.64898/2026.02.09.704753","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.06.704035","pub_date":"2026-2-09","title":"A Novel Secretome Rewrites the Immune Response in Viral Acute Respiratory Distress Syndrome","abstract":"Hyperinflammatory syndromes such as viral acute respiratory distress syndrome (ARDS) demand immunotherapies that are safe and effective at suppressing cytokine storm. As a novel treatment for ARDS, PRS CK STORM is proposed as a next-generation cell-free secretome derived from co-cultures of M2 macrophages and mesenchymal stromal cells. We performed a double-blind, randomized controlled trial to test the effects of PRS CK STORM in K18-hACE2 transgenic mice infected intranasally with 105 PFU of SARS-CoV-2 BE.1.1. This was complemented by a mechanistic analysis of this secretome using transcriptomic and COX2 enzymatic activity studies, as well as a compositional analysis of its proteomic and miRNA profile. In a lethal SARS-CoV-2 ARDS mouse model, PRS CK STORM significantly improved lung histopathology to a degree on par with corticosteroids, while stimulating angiogenesis in damaged lung tissue. Early and late cytokine profiling showed marked reductions in IFN-\u03b3. In response to PRS CK STORM treatment, transcriptomic analyses in inflamed macrophages revealed robust downregulation of key proinflammatory drivers (MyD88, TRAF6, IKK2, NF-\u03baB, COX2). In vitro enzymatic assays confirmed potent, dose-dependent inhibition of COX2, with high inter-batch reproducibility. A compositional analysis revealed this secretome to be rich in anti-inflammatory miRNAs, immune-modulating proteins, and regenerative factors. PRS CK STORM operates as a multi-target immune recalibrator, enabling broad downregulation of pathological inflammation while promoting tissue repair. Its off-the-shelf, GMP-manufactured format ensures reproducibility and scalability, offering a novel resolution pharmacology approach for cytokine storm syndromes originated from ARDS and beyond.","version":"1.1","doi":"10.64898/2026.02.06.704035","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.26.672080","pub_date":"2026-2-05","title":"Epitope-Driven Polyfunctional Divergence of SARS-CoV-2 RBD Antibodies","abstract":"SARS-CoV-2 antibodies targeting the receptor binding domain (RBD) of the spike protein can potently neutralize infection and exert additional antiviral functions. Here, we characterize the functional profiles of human RBD-specific memory B-cell antibodies elicited by ancestral SARS-CoV-2 infection. While SARS-CoV2 neutralizing antibodies, mainly class 1 and 3 anti-RBD antibodies, often lose binding and neutralizing capacities against Omicron variants, most non-neutralizers remain broadly reactive. Despite their restricted cross-reactivity, neutralizing antibodies mediate Fc-effector functions including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. Most neutralizers also enhance binding of antibodies that target the SARS-CoV-2 spike fusion peptide via receptor-mimetic allostery. In contrast, broadly reactive non-neutralizers fail to trigger phagocytic or allosteric effects. Thus, viral escape compromises antibody neutralizing as well as other antiviral activities, leaving broadly reactive, non-polyfunctional antibodies that exert minimal immune pressure less effective at controlling infection.","version":"1.2","doi":"10.1101/2025.08.26.672080","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.04.703921","pub_date":"2026-2-05","title":"Intranasal Replicating Adenovirus type 4-SARS-CoV-2 Recombinants Induce Superior Immune Response Durability and Efficacy in Preclinical Testing Compared to Standard Intramuscular Vaccines","abstract":"The portfolio of next generation COVID-19 vaccines would benefit from candidates that induce durable systemic and mucosal immune responses that would lessen person-to-person transmission. We constructed an intranasal (IN) replication-competent adenovirus type 4 recombinant platform to express SARS-CoV-2 Spike variants (Ad4-S) and assessed immunogenicity and efficacy in the Syrian hamster model. Although both IN Ad4-S and intramuscular (IM) vaccines (Ad26.CoV2.S and mRNA-1273) induced serum binding antibodies, only Ad4-S induced a robust mucosal response in the nasal cavity. IN Ad4-S vaccination induced serum neutralizing titers equivalent to or greater than IM vaccination but more durable up to 6 months. Upon challenge, IN immunization also resulted in less weight loss, greater breadth and durability of restriction of viral replication, and less lung pathology than IM immunization up to 268 days after immunization. These data support the potential of the IN Ad4 vaccine platform to reduce transmission of SARS-CoV-2 and other respiratory viruses with pandemic potential.","version":"1.1","doi":"10.64898/2026.02.04.703921","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.26.684604","pub_date":"2026-2-05","title":"Constrained Evolutionary Funnels Shape Viral Immune Escape","abstract":"Understanding how viral proteins adapt under immune pressure while preserving viability is crucial for anticipating antibody-resistant variants. We present a probabilistic framework that predicts viral escape trajectories and shows that immune evasion is channeled into a small set of viable \u201cescape funnels\u201d within the vast mutational space. These escape funnels arise from the combined constraints of protein viability and antibody escape, modeled using a generative model trained on homologs and deep mutational scanning data. We derive a mean-field approximation of evolutionary path ensembles, enabling us to quantify both the fitness and entropy of escape routes. Applied to SARS-CoV-2 receptor binding domain, our framework reveals convergent evolution patterns, predicts mutation sites in variants of concern, and explains differences in antibody-cocktail effectiveness. In particular, cocktails with de-correlated escape profiles slow viral adaptation by forcing longer, higher-cost escape paths. Viruses evolve to evade our immune defenses, but with constraints. Like navigating a minefield, each step toward immune escape comes at the potential cost of structural stability and functionality. We show that despite the vast mutational space, immune escape is funneled into a small set of predictable pathways. Using a statistical-physics model grounded in antibody experiments and SARS-CoV-2 epidemiology data, we identify these escape funnels\u2014enabling therapies designed to block them before they are ever used.","version":"1.2","doi":"10.1101/2025.10.26.684604","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.03.703458","pub_date":"2026-2-04","title":"Bayesian neural networks enable inference of complex phylodynamic processes","abstract":"Phylogenetic branching patterns carry essential information about population and diversification dynamic processes, including speciation, extinction, and epidemiological transmission. Phylodynamic models offer a rigorous mathematical framework for quantifying these dynamics from phylogenetic trees. Extensions of these models enable the incorporation of external covariates as predictors of phylodynamic parameters, for instance allowing us to link traits or environmental variables with changes in speciation, extinction, or transmission rates. However, the dependencies between predictors and phylodynamic parameters are typically restricted to linear, additive effects and may thus fail to capture complex, potentially non-linear relationships underlying evolutionary dynamics. To address this limitation, we propose a new framework, BELLA, which leverages unsupervised Bayesian neural networks (BNNs) to flexibly model functional relationships between key phylodynamic parameters and a broad set of predictors, including categorical traits, quantitative variables, and time series data. Based on these covariates, the BNN weights are estimated through Markov chain Monte Carlo and can be inferred jointly with the phylogenetic tree topology and branching times, obtained directly from sequence alignment data. Using extensive simulations, we demonstrate that this approach accurately recovers predictor-parameter relationships, mitigates overfitting, and remains robust across both macroevolutionary and epidemiological contexts. By incorporating tools from explainable artificial intelligence, we further show that our framework reliably identifies the most influential predictors and yields interpretable descriptions of their impact on phylodynamic rates. Finally, we apply our method to two empirical analyses: linking SARS-CoV-2 migration dynamics with travel data during its early spread in Europe, and inferring trait and time-dependent speciation and extinction rates in the Cenozoic diversification of platyrrhines. Our unsupervised BNN framework substantially expands the capabilities of phylodynamic inference providing a powerful and flexible approach to model complex macroevolutionary and epidemiological processes. Understanding how species diversify or pathogens spread requires linking phylogenetic trees to biological traits and environmental characteristics. We present BELLA, an unsupervised Bayesian neural network that learns complex, nonlinear links between predictors\u2014such as traits or environmental variables\u2014and key rates such as speciation, extinction, transmission, and migration directly from sequence data\u2014which in turn inform on the phylogenetic relationships. BELLA does not require any training data, enabling inferences that were previously not tractable. Across simulated epidemics and macroevolutionary histories, BELLA is more accurate than state-of-the-art linear models relating predictors and rates, while remaining interpretable through explainable artificial intelligence. Empirical applications in epidemiology reveal nonlinear effects of travel among countries on the early spread of SARS-CoV-2 in Europe, while a macroevolutionary application shows selective extinction events in the diversifcation history of New World monkeys. The BELLA framework expands the reach of phylodynamics in biology and public health.","version":"1.1","doi":"10.64898/2026.02.03.703458","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.05.583578","pub_date":"2026-2-03","title":"Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants","abstract":"The open reading frame 8 (ORF8), an accessory protein of SARS-CoV-2, is prone to deletions and mutations across different viral variants, which was first described in several Singapore variants. The reason why viral evolution favors loss or inactivation of ORF8 is not fully understood, although the effects of ORF8 on inflammation, immune evasion, and disease severity have been described. Here we show \u2013using clinical ORF8-deficient viral isolates, virus-like particles (VLPs) and viral replicons\u2013 that ORF8 expression dampens viral particle production. ORF8 physically interacts with the viral Spike protein and induces Golgi fragmentation, overall contributing to less virus particle production. Using systematic ORF8 deletions, we mapped the particle-reducing function to its N-terminal signal peptide. Interestingly, this part of ORF8 is severely truncated in the recent XBB.1.5 variant, and when restored, suppresses viral particle production in the context of the entire viral genome. Collectively, our data supports the model that evolutionary pressure exists to delete ORF8 sequence and expression across SARS-CoV-2 variants to fully enable viral particle production. SARS-CoV-2 variants continue to emerge worldwide with advantages in replication and immune evasion. Many variants have acquired distinct mutations in independent lineages to abolish ORF8 expression. To understand the molecular mechanisms behind this evolutionary trend, we utilized reverse genetics, molecular virology, and confocal microscopy to show that ORF8 has antiviral functions by dampening viral particle production and inducing Golgi stress during infection. Our data demonstrate that SARS-CoV-2 is continuing its adaptation to optimize viral particle production and other unknown aspects of viral infection.","version":"1.2","doi":"10.1101/2024.03.05.583578","journal":"bioRxiv","score":null},{"id":"10.64898/2026.02.02.702852","pub_date":"2026-2-03","title":"Functional and Structural Basis of Omicron BA.3.2.1 Spike","abstract":"SARS-CoV-2 Omicron descendant BA.3.2, derived from BA.3 and carrying 39 additional spike substitutions, prompts concerns about altered fitness and antigenicity. Using BA.3.2.1 as a representative sublineage, we have engineered live-attenuated mNeonGreen SARS-CoV-2 encoding spikes from BA.3.2.1 and the JN.1-derived LP.8.1 and XEC, and benchmarked them against BA.3 and KP.3 in Calu-3 cells and primary human airway epithelium. BA.3.2.1 outcompetes BA.3 yet replicates more slowly than JN.1-descendants. Despite increased RBD-hACE2 affinity, BA.3.2.1 spike binds hACE2 less efficiently than LP.8.1, and shows the greatest resistance to neutralization by sera from KP.2/KP.3 infections. Cryo-EM reveals a predominantly closed, compact, asymmetric trimeric spike with a remodeled N-terminal domain, a distinct fusion-peptide-proximal region and altered N-glycosylation. BA.3.2.1 spike adopts a fine-tuned receptor-binding interface and shifted monoclonal-antibody footprints. These findings identify a trade-off between immune evasion and replication fitness driven by closed spike conformation, rationalizing BA.3.2\u2019s limited prevalence and underscoring the necessity for continued variant surveillance.","version":"1.1","doi":"10.64898/2026.02.02.702852","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.29.702655","pub_date":"2026-1-30","title":"Displayed and Encoded Antigens on Adenovirus Vectors Optimize Humoral and Cellular Immune Responses in Rhesus Macaques","abstract":"Adenovirus vector-based vaccines were deployed widely during the COVID-19 pandemic. In this study, we explored the potential of displaying an antigen on the surface of the adenovirus capsid as well as encoding an antigen as a transgene in the adenovirus vector to optimize both humoral and cellular immune responses. We show that displaying SARS-CoV-2 Spike receptor biding domain (RBD) on the Ad5 capsid while simultaneously encoding Spike as a transgene induced robust antibody and T cell responses in rhesus macaques. These data demonstrate that adenoviruses can be utilized simultaneously as both nanoparticle scaffolds and viral vectors.","version":"1.1","doi":"10.64898/2026.01.29.702655","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.28.702465","pub_date":"2026-1-29","title":"Functional genomic analysis reveals mechanisms of epigenetic interference in SARS-CoV-1 and SARS-CoV-2","abstract":"Small viral RNAs (svRNA) are key factors in host adaptation and virulence and are previously identified in SARS-CoV-2 and SARS-CoV-1. Little is known about svRNA conservation among high-risk coronaviruses (subgenus Sarbecovirus) or how svRNAs might contribute to COVID-19. Here, using functional genomics we identify six novel svRNA-triplex-forming oligonucleotides (svRNA-TFOs) in SARS-CoV-2, and through comparative analyses with SARS-CoV-1, delineate evolutionary pathways for svRNA-TFOs in Sarbecovirus. In addition to significant enrichment of the svRNA-TFO enhancer-associated gene targets among differentially expressed gene sets collected during SARS-CoV-2 infection, we find statistical support for non-random associations with >25 % of recombination breakpoints and hotspots in the Wuhan-Hu-1 genome. Small RNA-sequencing reveals highly abundant svRNA-TFO-N derived from the nucleocapsid of SARS-CoV-2. Using synonymous site conservation analysis, we find svRNA-TFO-N, and other svRNA-TFOs are conserved among both SARS-CoV-2- and SARS-CoV-1-related lineages. Furthermore, BLASTn reveals svRNA-TFO-N, svRNA-TFO-S.1 and svRNA-TFO-E share sequence homology against various mammalian genomes supporting a potential mechanism of host adaptation. Finally, we identify potential links between the putative svRNA-TFO-S.1 and svRNA-TFO-N RNA progenitor structures with variant of concern-associated S:D614G and N:D377Y mutations, respectively. Collectively these findings support a role for epigenetic interference with potential to reveal critical insights into the evolution of SARS-CoV-2 and SARS-CoV-1.","version":"1.1","doi":"10.64898/2026.01.28.702465","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.27.701989","pub_date":"2026-1-28","title":"Diminished EBV-Specific Humoral Immunity is Associated with Neuropsychiatric Long COVID Development up to 12 Months Post-COVID-19 Symptom Onset","abstract":"Infection with SARS-CoV-2 can lead to long COVID, a chronic multisystemic condition estimated to affect approximately 400 million people worldwide. Although underlying mechanisms remain elusive, aberrant ongoing inflammation driven by Epstein-Barr virus (EBV) reactivation and persistent SARS-CoV-2 viral reservoirs have been hypothesized. We compared cellular and humoral immune responses to SARS-CoV-2 and EBV between participants with neuropsychiatric long COVID and recovered individuals. Peripheral blood mononuclear cells (PBMCs) and sera were collected from 27 long COVID individuals with \u22652 neuropsychiatric symptoms and 27 matched recovered participants at 3-6 months post-COVID-19 symptom onset (PSO). PBMCs were assessed for IFN-\u03b3, IL-2, TNF\u237a, and granzyme B T-cell responses against SARS-CoV-2, EBV, and human cytomegalovirus (HCMV). Sera were evaluated for neutralizing activity against live ancestral SARS-CoV-2 and EBV, and EBV reactivation was assessed by early antigen-diffuse IgG. We observed no significant differences in SARS-CoV-2-, EBV-, or HCMV-specific T-cell responses or live virus neutralization between long COVID and recovered groups at 3-6 months PSO. EBV reactivation was additionally only detected in one neuropsychiatric long COVID participant. However, reduced EBV neutralizing capacity at 3-6 months significantly associated with fatigue at 12 months PSO. Anti-EBV viral capsid antigen IgG levels were also significantly diminished in long COVID participants and similarly trended lower in those reporting fatigue at 12 months PSO. We therefore detected no differences in SARS-CoV-2- or EBV-specific T-cell responses or serological neutralizing capacity between neuropsychiatric long COVID and recovered participants; however, diminished EBV-specific humoral immunity may serve as a prognostic marker for neuropsychiatric long COVID development. We performed a comprehensive analysis of cellular and humoral immune responses to SARS-CoV-2, Epstein-Barr virus, and human cytomegalovirus in individuals with neuropsychiatric long COVID, a subgroup that remains poorly characterized. Although no differences in virus-specific T-cell immunity were observed between long COVID and recovered individuals, diminished Epstein-Barr virus neutralization at 3-6 months was associated with persistent or relapsing fatigue at 12 months post-COVID-19 symptom onset. Anti-viral capsid antigen IgG antibody levels were also significantly lower in neuropsychiatric long COVID participants at 3-6 months and similarly trended lower in those reporting fatigue at 12 months post-symptom onset. Together, these findings suggest that impaired humoral immunity to Epstein-Barr virus may contribute to the development or persistence of neuropsychiatric long COVID and highlight a promising future direction for mechanistic studies.","version":"1.1","doi":"10.64898/2026.01.27.701989","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.20.633897","pub_date":"2026-1-28","title":"In silico evidence suggests that the SARS-CoV-2 Spike protein may target coiled-coil regions of numerous cytoskeletal and cytoskeleton-associated proteins","abstract":"Understanding the interactions between host and viral envelope proteins is essential to get insights into the dynamics of viral infection. To investigate more closely the mechanisms governing SARS-CoV-2 entry and intracellular trafficking, I reanalyzed the most extensive SARS-CoV-2\u2013human protein\u2013protein interactome dataset currently available. My investigation centered on the Spike S protein, a key player in initiating viral infection by binding to the host cell membrane receptor Angiotensin-Converting Enzyme 2 (ACE2). I first present evidence demonstrating the statistical overrepresentation of actin-binding proteins among the Spike S partners/interactors. Next, I show that a majority of these partners contains a structural domain sharing high similarity with the C-terminal region of the Myosin II heavy chain, Myosin II being known for its roles in muscle contraction and various cellular motility processes. I subsequently demonstrate that this domain is particularly prevalent in actin-binding proteins, intermediate filaments proteins and kinesins, which all are related to the cytoskeleton known to be involved in diverse cellular functions, including endocytosis and intracellular transport \u2014 processes crucial for viral infections. Finally, I highlight that the structural domain mentioned above is a bonafide coiled-coil region. I therefore conclude that Spike S might target proteins possessing such regions. Collectively, my findings suggest that the interactions between SARS-CoV-2 Spike S and human proteins, potentially mediated by coiled-coil regions, may have been underestimated. As this work relies on in silico evidence, direct biological extrapolations require caution.","version":"1.4","doi":"10.1101/2025.01.20.633897","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.27.702130","pub_date":"2026-1-28","title":"Coupling high-throughput protease enzymology with viral replication reveals biochemical constraints of viral fitness","abstract":"Proteases govern essential biological processes and are key drug targets, yet how protease sequence variation quantitatively reshapes biochemical parameters and constrains biological fitness remains poorly understood. Here, we integrate high-throughput in vitro enzymology with cellular assays to link protease sequence, biochemistry, and fitness. We extend a microfluidic platform for high-throughput protease enzymology (HT-MEKpro), which is broadly applicable across protease families and catalytic classes, enabling measurement of catalytic turnover (kcat), Michaelis constant (KM), inhibitor potency (IC50), and relative substrate specificity for 102-103 variants. Applied to the SARS-CoV-2 main protease (Mpro), HT-MEKpro generated parallel catalytic and inhibitory landscapes for >400 variants. Integration with viral replication and in-cell cleavage assays reveals that variants with altered substrate specificity fail to support replication, suggesting imbalanced polyprotein processing as a constraint on viral fitness. More broadly, these data can enable mechanistically grounded modeling of protease sequence\u2013property relationships and inform strategies for pharmacological modulation beyond active-site inhibition.","version":"1.1","doi":"10.64898/2026.01.27.702130","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.26.701894","pub_date":"2026-1-27","title":"Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies","abstract":"The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the protein\u2019s structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential. SARS-CoV-2 and emerging coronaviruses remain a major global health threat, yet critical gaps persist in our understanding of their molecular pathogenesis. The nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 antigen, plays essential roles beyond genome packaging, including immune evasion and intracellular organization. Here, we generate and characterize a panel of domain-specific nanobodies (VHHs) that enable precise dissection of N\u2019s functional architecture. Using integrated biochemical, structural, and virological approaches, we uncover distinct mechanisms of viral inhibition, including disruption of phase condensation through a conserved linker region. These findings address long-standing knowledge gaps about a multifunctional viral protein and establish VHHs as powerful, modular tools for probing coronavirus biology, with broad potential for therapeutic, diagnostic, and cell biology applications.","version":"1.1","doi":"10.64898/2026.01.26.701894","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.24.700018","pub_date":"2026-1-26","title":"A bireporter recombinant SARS-CoV-2 Omicron BA.5 for in vitro and in vivo studies","abstract":"The continuous emergence of variants of concern (VoC) represent a significant challenge to effectively control SARS-CoV-2. Although FDA-approved vaccines and antivirals have been successfully developed and implemented for the prophylactic and therapeutic intervention of SARS-CoV-2 infection, recent VoC could escape protection garnered by previous vaccine and antiviral approaches. Determining the efficacy of prophylactics and/or therapeutics against recent VoC will assist in efficiently controlling currently circulating SARS-CoV-2 strains. We used our previously described bacterial artificial chromosome (BAC)-based reverse genetics approach for Omicron BA.5 to generate a recombinant SARS-CoV-2 BA.5 encoding a fusion of ZsGreen to Nanoluciferase (rBA.5 ZsG-Nluc) from the locus of the viral nucleocapsid (N) protein separated by the porcine teschovirus-1 (PTV-1) 2A proteolytic cleavage site. The rBA5 ZsG-Nluc replicates to levels comparable to recombinant BA.5 wild-type (rBA.5 WT) and expresses high levels of ZsG and Nluc in cultured cells. This facilitates tracking viral infection and the identification of antivirals and neutralizing antibodies (NAbs) with EC50 and NT50 values, respectively, similar to those obtained with rBA.5 WT. Importantly, in K18 hACE2 mice, rBA.5 ZsG-Nluc retains the same pathogenicity and ability to replicate in the lungs of infected mice as rBA.5 WT. Using rBA.5 ZsG-Nluc, we detected Nluc activity systemically and Nluc and ZsG expression in the lungs of infected mice using an in vivo imaging system (IVIS). Our results demonstrate the feasibility of using rBA.5 ZsG-Nluc to track viral infections and identify prophylactics and therapeutics against recent SARS-CoV-2 VoC in vitro, ex vivo, and in vivo. SARS-CoV-2, the causative virus of the COVID-19 pandemic, is continually evolving to escape immunity acquired by previous natural infections or vaccinations. Moreover, recent SARS-CoV-2 variants of concern (VoC) have acquired antiviral resistant mutations to FDA-approved drugs. The emergence of these VoC highlights the importance of identifying new prophylactics and therapeutics against currently circulating SARS-CoV-2 strains. We generated a recombinant bireporter Omicron BA.5 SARS-CoV-2 (rBA.5 ZsG-Nluc) that expresses reporter proteins, which are useful for cellular and whole animal studies and has similar viral replication and pathogenicity to a wild-type (WT) recombinant Omicron BA.5 SARS-CoV-2 (rBA.5 WT). In K18 hACE2 mice, rBA.5 ZsG-Nluc infection can be tracked systemically or in the lungs of infected mice using an in vivo imaging system (IVIS). We establish a proof-of-concept platform of rBA.5 ZsG-Nluc in combination with an ancestral SARS-CoV-2 strain expressing mCherry to simultaneously identify antivirals and neutralizing antibodies against original and recent SARS-CoV-2 strains.","version":"1.1","doi":"10.64898/2026.01.24.700018","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.26.701742","pub_date":"2026-1-26","title":"Trophoblast ferroptosis restricts SARS-CoV-2 spread in the placenta","abstract":"Prenatal SARS-CoV-2 infection is associated with adverse pregnancy outcomes, but placental mechanisms that restrict viral spread remain unclear. Here we show that SARS-CoV-2 exposure induces ferroptosis-linked iron dysregulation in the placenta as a host defense. Human placentas from early gestation SARS-CoV-2\u2013exposed pregnancies exhibited persistent viral protein expression at term, iron accumulation, disrupted localization of iron transport proteins, and reduced expression of the ferroptosis inhibitor, GPX4. In trophoblast cells and newly generated stem cell-derived trophoblast organoids (SC-TOs) with physiological apical-out polarity, infection with live SARS-CoV-2 Delta variant suppressed expression of iron efflux transporter, ferroportin and ferroptosis inhibitors, GPX4 and PLA2G6, promoting lipid peroxidation and ferroptotic signaling. Sub-lethal pharmacological activation of ferroptosis reduced viral titers in trophoblasts, indicating an antiviral function. Together, these results uncover a new mechanism through which the placenta attempts to restrict SARS-CoV-2 replication. However, this protective response is accompanied by placental iron sequestration, which may compromise maternal-fetal iron transfer and help explain iron deficiency and anemia reported in infants born after prenatal SARS-CoV-2 exposure, highlighting a delicate balance between iron and ferroptosis-mediated protection and damage with implications for pregnancy outcomes.","version":"1.1","doi":"10.64898/2026.01.26.701742","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.24.701458","pub_date":"2026-1-26","title":"The Unexpected Visibility of the SARS-CoV-2 Nucleocapsid Protein Reveals a Hidden Route of Surface Trafficking","abstract":"The SARS-CoV-2 Nucleocapsid (N) protein, long regarded as an internal structural component of the virion, unexpectedly localizes to the plasma membrane of infected cells. Here, we show that N is actively trafficked to the cell surface via a ceramide-dependent unconventional secretory pathway. Live-stained imaging and kinetic analyses revealed that N surface association begins early in infection, before Spike (S) expression and viral release, and persists after enzymatic removal of heparan sulfate. Pharmacological disruption of phosphoinositide or phosphatidylserine interactions had minimal effect, whereas inhibition of neutral sphingomyelinase with GW4869 markedly reduced surface N, identifying a ceramide-regulated route as essential for its export. This mechanism distinguishes N from canonical transmembrane viral proteins and explains how N-specific antibodies mediate potent Fc-effector responses across SARS-CoV-2 variants. Our findings redefine the spatiotemporal dynamics of coronavirus structural proteins and reveal an unanticipated axis of immune visibility within the infected cell. Internal viral proteins are generally thought to remain confined to intracellular compartments, yet several viruses display such proteins at the surface of infected cells through mechanisms that remain incompletely defined. In this study, we characterize a host-regulated trafficking route that contributes to the delivery of the SARS-CoV-2 nucleocapsid (N) protein, a non-membrane viral protein, to the plasma membrane. Our findings indicate that N protein surface expression occurs independently of virion assembly, membrane integration, or extracellular rebinding, and instead involves host vesicular processes outside the classical secretory pathway. By elucidating how a leaderless coronavirus protein can access the cell surface, this work addresses a key gap in coronavirus cell biology and provides a framework for understanding how internal viral proteins may exploit host trafficking pathways. More broadly, these results highlight unconventional host trafficking pathways as determinants of viral protein localization and provide a framework for understanding how viruses exploit cellular export mechanisms beyond the classical secretory system.","version":"1.1","doi":"10.64898/2026.01.24.701458","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.26.701677","pub_date":"2026-1-26","title":"Multifaceted Functional Complexity of SARS-CoV-2 Helicase Nsp13 Underlies Its Integrated Motor and Remodeling Activities","abstract":"SARS-CoV-2 nonstructural protein 13 (Nsp13) is a superfamily 1 helicase essential for viral replication. Although its canonical ATP-dependent unwinding activity is well established, the broader functional repertoire of Nsp13 remains unclear. Here, we show that Nsp13 encodes a high degree of complexity by acting as a tunable nucleic acid remodeler that integrates motor and non-motor activities within a single protein. Nsp13 operates in multiple mechanistically distinct regimes, including a canonical ATP-dependent helicase mode and a Mg\u00b2\u207a-primed, ATP-independent remodeling state capable of destabilizing short duplexes, hairpins, and G-quadruplexes. Mg\u00b2\u207a binding allosterically stabilizes a compact RecA1\u2013RecA2 configuration, priming the enzyme for ATP-independent remodeling. Unwinding polarity is substrate-dependent, with duplexes supporting bidirectional remodeling, whereas G-quadruplexes are preferentially resolved in the 5\u2032\u21923\u2032 direction. Beyond strand separation, Nsp13 also exhibits robust strand annealing and nucleic acid chaperone activities. Cofactors, substrate topology, and enzyme concentration dynamically regulate these activities. Together, our findings establish Nsp13 as a highly integrated nucleic acid remodeling system and reveal how a single viral helicase switches between motor-driven and remodeling-dominated states to meet the structural demands of replication and transcription.","version":"1.1","doi":"10.64898/2026.01.26.701677","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.23.701152","pub_date":"2026-1-26","title":"DISCO-LAMP: A Novel discontinuous LAMP assay for isothermal antigen detection","abstract":"Proximity ligation assay (PLA), in which the ligation of two DNA probes is greatly accelerated by the associating target molecules, has emerged as a highly sensitive technique for protein detection. The detection of the ligated DNA typically relies on PCR, which requires temperature cycling. In this study, we report on a novel discontinuous (DISCO)-LAMP assay that enables the wash-free detection of PLA products via loop-mediated isothermal amplification (LAMP). Due to the exponential amplification nature of LAMP, a careful balance between efficient amplification of the ligated full-length DNA and minimal background amplification from the individual constituent probes is essential but often challenging to achieve. After extensive template/primer design and assay optimization, DISCO-LAMP assay achieved a detection limit of 1 fM for the ligated DNA probe while maintaining undetectable background amplification at 1 nM of each individual probe. DISCO-LAMP detected Shiga toxin 2 (Stx2) with a limit of detection (LoD) of 100 fM when functionalized with Stx2-binders, as well as both Wuhan-1 and Omicron spike protein when functionalized with DS16, a newly engineered DARPin targeting a conserved epitope on the SARS-CoV-2 Spike protein. We believe DISCO-LAMP represents a versatile and efficient LAMP-based PLA technology that is readily adaptable for sensing diverse targets.","version":"1.1","doi":"10.64898/2026.01.23.701152","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.23.701408","pub_date":"2026-1-25","title":"Biodistribution of mRNA vaccines in rats: Enrichment in injection site and lymph tissues and rapid clearance without tissue persistence","abstract":"Messenger RNA (mRNA) vaccines using lipid nanoparticles (LNPs) are well-established and globally approved with acceptable safety profiles for preventing respiratory disease. Other mRNA-LNP product concepts are also emerging as novel treatments for broader clinical use. Here, we describe mRNA-LNP vaccine tissue distribution and kinetics after intramuscular dosing using three products formulated with same LNP matrix: mRNA-1273 (Spikevax\u2122), mRNA-1647 (a candidate cytomegalovirus [CMV] vaccine), and a reporter mRNA (nascent peptide-luciferase) drug product. Consistent biodistribution patterns were observed across studies: tissues with highest exposures were the injection site, draining lymph nodes, and spleen, with minimal distribution to non-lymphoid tissues. Vaccine components cleared rapidly from circulation and tissues, with complete elimination simulated to occur by \u223c2 weeks. Following mRNA-1273 vaccination, Spike protein levels were transiently observed (elimination <5 days) and did not accumulate with repeated dosing. The ionizable lipid in the LNP matrix, Lipid H, underwent biotransformation and was excreted renally and hepatically, with no human-specific metabolites. Collectively, these results indicate that the LNP composition, not mRNA cargo, governs biodistribution. Furthermore, in a SARS-CoV-2 infection-free model, there was no evidence of Spike protein persistence. Overall, the data establish a framework that justifies leveraging biodistribution data across products and supports eliminating redundant animal studies.","version":"1.1","doi":"10.64898/2026.01.23.701408","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.23.701355","pub_date":"2026-1-24","title":"Hepatocyte growth factor activator inhibitor-2 rapidly inactivates airway-expressed human Type II Transmembrane Serine Proteases","abstract":"Human Type II Transmembrane Serine Proteases (TTSPs) are essential entry factors for various influenza A and coronaviruses, and drive cancer metastasis when they are overexpressed by tumor cells. However, the natural inhibition mechanisms that regulate these proteases are not well understood. One natural transmembrane protease inhibitor, hepatocyte growth factor activator inhibitor-2 (HAI-2), has been shown to block TMPRSS2 activity and can prevent SARS-CoV-2 infection and reduce TMPRSS2-driven prostate cancer metastasis when overexpressed. In this study, we present biochemical and biophysical evidence showing that HAI-2 effectively inactivates TMPRSS2 and other TTSPs only after they have undergone zymogen activation. Through mutagenesis and ligand binding assays, we demonstrate that Kunitz Domain 1 (KD1) and KD2 can form stable ternary complexes with TMPRSS2 and other TTSPs, but do not employ the typical Laskowski inhibitor mechanism found for other macromolecular serine protease inhibitors. We also show that HAI-2 proteins do not inhibit the coagulation protease thrombin and that multivalent human IgG-tagged HAI-2 proteins are highly potent TMPRSS2 inhibitors. Our findings provide a mechanistic understanding of how TTSP activity is regulated in human airway cells and offer a foundation for developing engineered soluble HAI-2 proteins as anti-TTSP antivirals and anti-cancer therapeutics.","version":"1.1","doi":"10.64898/2026.01.23.701355","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.22.701138","pub_date":"2026-1-24","title":"Polymer-lipid hybrid nanoparticle enhances mRNA delivery and T cell-mediated immunity","abstract":"mRNA vaccines have transformed prophylactic immunization against infectious diseases as well as therapeutic interventions for cancer. However, their effectiveness against emerging viral variants and a range of malignancies continues to be hindered by suboptimal induction of T cell-mediated immunity. To overcome this limitation, here we developed a polymer-lipid hybrid nanoparticle (PLNP) platform engineered to improve mRNA delivery to antigen-presenting cells (APCs) and to potentiate T cell responses. Relative to conventional mRNA lipid nanoparticle (LNP) vaccines, mRNA PLNP vaccines demonstrated markedly improved lymph node targeting, APC activation, Th1-biased pro-inflammatory cytokine response, and antigen-specific T cell expansion while retaining robust humoral immunity. Remarkably, mRNA-PLNP vaccines generated approximately 50% more antigen-specific CD8+ T cells than mRNA-LNP vaccines across multiple antigens, including SARS-CoV-2 spike, influenza hemagglutinin, and ovalbumin. In prophylactic applications, mRNA PLNP vaccine provided complete protection against SARS-CoV-2 variants. As a therapeutic approach in a melanoma model, mRNA PLNP vaccination resulted in enhanced tumor control and significantly prolonged survival compared to LNP-based formulations. Collectively, these results establish PLNP as a versatile and broadly applicable platform for augmenting mRNA vaccine efficacy through improved mRNA delivery and T cell priming, offering promising implications for infection prevention and cancer immunotherapy.","version":"1.1","doi":"10.64898/2026.01.22.701138","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.22.701182","pub_date":"2026-1-23","title":"Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology","abstract":"Long COVID frequently presents with persistent neurological symptoms, including cognitive impairment, fatigue, and sleep disturbances; however, its underlying mechanisms remain unclear. Here, we show that SARS-CoV-2 infection induces lasting cortical neuronal injury and hypothalamic orexin (hypocretin) dysfunction in vivo. In K18-hACE2 and wild-type BALB/c mice, viral RNA persisted in the brain and coincided with focal loss of Neuronal Nuclei (NeuN)-positive cortical neurones beyond acute infection. SARS-CoV-2, but not the influenza A virus, triggered rapid and sustained suppression of hypothalamic orexin expression, defining a virus-specific neuropathological signature. Considering the downregulation of orexin and focal cortical NeuN expression, we showed that augmenting orexin signalling using recombinant orexin-A/B restored NeuN expression in vitro and in vivo. Overall, these findings identify the orexin system as a selective neural vulnerability to SARS-CoV-2 and define orexinergic circuit disruption as a mechanistic axis underlying the neurological manifestations of Long COVID.","version":"1.1","doi":"10.64898/2026.01.22.701182","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.22.701202","pub_date":"2026-1-23","title":"Comparative profiling of SARS-CoV-2 variant infections reveals diverse impacts on host cell RNA and RNA binding protein distribution and regulation","abstract":"RNA viruses perturb cellular RNA metabolism to promote viral transcript expression and genome replication. However, the extent of host cell RNA reprogramming and the consequences on RNA binding protein (RBP) expression and localization remain poorly understood. Here, we utilized an integrated set of transcriptomics, proteomics, and high-content imaging approaches to compare the impact of different SARS-CoV-2 variants, including an early viral isolate and later emerging variants of concern (Alpha, Delta and Omicron) on the host cellular RNA and RBP landscapes. While all variants deeply remodelled the host transcriptome in a subcellular compartment-specific manner, we found that Omicron variants express lower levels of viral RNA products compared to earlier variants, while still driving efficient production of viral proteins. The surge in viral RNA expression correlated with altered subcellular RNA biotype distribution, cytoplasmic mRNA exclusion, the selective disruption of the localization of polyA-tail interacting proteins, and the nuclear accumulation of non-coding transcripts such as RN7SL family members and MALAT-1. Unexpectedly, we found many RBPs that exhibiting altered subcellular localization following abiotic innate immune stimulation with poly(I:C) but remained unaltered in cells infected with SARS-CoV-2 variants, revealing a capacity for these viruses to limit the overall impact on the RBP subcellular distribution. Our results further indicate that Omicron infection sustains higher expression levels of proteins components of the signal recognition particle and translation factors, which may enable a more effective production of immune response and viral components. Altogether, this work highlights the deep impact of SARS-CoV-2 infection on host cell RNA regulatory landscape and provides new insights to explain the differential impact of Omicron compared to early SARS-CoV-2 variants. SARS-CoV-2 variants drive varying degrees of viral RNA expression in infected Calu-3 cells Infection reshapes the host cell RNA landscape in a cell-compartment specific manner RN7SL RNA exhibits a strong increase in relative abundance and accumulates in the nucleus of infected cells SARS-CoV-2 infections impact the localization of polyA-tail binding proteins while blocking the nuclear accumulation of immune response-sensitive RBPs Omicron infections are less disruptive to cellular function and sustain the production of translation factors and components of the SRP complex","version":"1.1","doi":"10.64898/2026.01.22.701202","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.22.700386","pub_date":"2026-1-23","title":"Heterologous betacoronavirus spike immunization in nonhuman primates elicits cross-reactive antibodies that neutralize both sarbeco- and merbecoviruses","abstract":"In anticipation of future coronavirus (CoV) pandemics, developing vaccines that elicit broadly neutralizing antibodies (bnAbs) against diverse CoVs is critical. Here, we vaccinated rhesus macaques with SARS-CoV-2 spike (S)-protein, then boosted with heterologous \u03b2-CoV S-proteins to focus responses to common conserved S2 bnAb epitopes. Initial SARS-CoV-2 priming elicited receptor-binding domain (RBD)-focused responses, while MERS-CoV boosting redirected responses toward the S2 region, including the stem-helix bnAb site. Although S2-directed serum cross-neutralization was undetectable and most isolated cross-reactive monoclonal antibodies (mAbs) targeted non-neutralizing epitopes, two S2 stem-helix mAbs were identified from memory B cells. These bnAbs neutralized diverse sarbeco- and merbecoviruses, including MERS-CoV, and conferred robust in vivo protection against SARS-CoV-2 challenge. Structural studies revealed that these macaque bnAbs closely mimic human S2-stem bnAbs induced by infection. These findings provide proof-of-principle for vaccination strategies that elicit broadly protective \u03b2-coronavirus responses and highlight non-human primates as a translational model for evaluating S2-targeted immunogens.","version":"1.1","doi":"10.64898/2026.01.22.700386","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.19.700281","pub_date":"2026-1-20","title":"SARS-CoV-2 membrane protein biogenesis","abstract":"Viral protein biogenesis underpins every viral life cycle stage, and elucidating these processes could reveal fundamental principles of virus\u2013host interaction, and vulnerabilities amenable to therapeutic targeting. Here we apply biophysical, molecular, and cell biology techniques to investigate the insertion, folding, and oligomerization of the SARS-CoV-2 M protein. We describe the sequential co-translational insertion of the hydrophobic core, and demonstrate that the cytosolic C-terminal domain undergoes slower adoption of its tertiary structure. Additionally, we characterize how the transmembrane domain bundle facilitates M-protein oligomerization. Our results reveal a hydrophobic residue cluster that is essential for protein folding and co-translational dimerization. Additionally, we identify the cellular machinery responsible for targeting and inserting the M protein into the ER membrane, and chaperones and cofactors that may contribute to proper folding.","version":"1.1","doi":"10.64898/2026.01.19.700281","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.01.691496","pub_date":"2026-1-20","title":"Alphaviral capsid proteins inhibit stress granule assembly via competitive RNA binding with G3BP1","abstract":"Viral infection is one of the conditions that induces stress granule (SG) formation, a cellular defense mechanism that exerts antiviral effects. To counteract this host response, viruses have evolved a broad spectrum of strategies to inhibit SG formation. However, the molecular mechanisms underlying SG inhibition remain poorly understood. The nucleocapsid proteins play a critical role in virus replication and host interaction. Here, using Semliki Forest Virus (SFV) as a model, we uncover the function of the alphavirus nucleocapsid in SG inhibition. This inhibitory function depends on oligomerization mediated by an N-terminal \u03b1-helix and with a positively charged intrinsically disordered region (IDR). We show that SFV capsid directly competes with G3BP1 for RNA binding, thereby disrupting G3BP1-RNA liquid-liquid phase separation (LLPS) in vitro and SG assembly in cells. This mechanism is conserved across the alphavirus family but is not shared by the nucleocapsid of SARS-CoV-2 or other endemic viruses examined. Notably, expression of a peptide from SFV capsid is sufficient to inhibit SG formation induced by Amyotrophic Lateral Sclerosis (ALS)-associated mutations, suggesting potential therapeutic applications. Our findings reveal mechanistic insight into SG modulation by the viral capsid protein and provide a possible bioengineering tool for probing SG dynamics in health and disease.","version":"1.2","doi":"10.64898/2025.12.01.691496","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.15.699801","pub_date":"2026-1-19","title":"Ultra-deep sequencing reveals intra-host diversity and co-infection-driven evolution of SARS-CoV-2","abstract":"As COVID-19 enters an endemic phase, SARS-CoV-2 continues to diversify under ongoing immune pressure, with Omicron sublineages and episodic emergent variants sustaining reinfections worldwide. Intra-host evolution represents the earliest stage of this diversification, yet remains undercharacterized, particularly in regions with limited genomic surveillance. Here, we conducted high-throughput sequencing on 96 nasopharyngeal swab samples from Chilean individuals (2020\u20132022), achieving an average per-base genome coverage of \u223c60,000x across the viral genome. This ultra-deep sequencing coverage enabled the identification of intra-host single-nucleotide variants (iSNVs) and co-infection events with high sensitivity and accuracy. Co-infections, especially with Omicron, significantly increased iSNV frequency and recombination, driving viral diversity. Evolutionary analysis based on the non-synonymous to synonymous ratio (dN/dS) shows that Omicron is under extensive purifying selection (global dN/dS \u223c 0.55). However, Omicron co-infection cases exhibited higher dN/dS ratios (\u223c0.58), suggesting a lower level of purifying selection and increased genetic diversity. Notably, the Spike gene showed dN/dS ratios indicative of positive selection (dN/dS > 1), which are more pronounced in co-infection cases than in Omicron alone. This suggests that co-infections are providing the substrate for the emergence of new variants with enhanced transmissibility and immune evasion capabilities. Together, these findings demonstrate that ultra-deep sequencing is crucial for mapping the evolutionary forces driving SARS-CoV-2 intra-host adaptation and the emergence of new variants.","version":"1.1","doi":"10.64898/2026.01.15.699801","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.16.699918","pub_date":"2026-1-19","title":"Synthetic RNA-protein decoy granules to prevent SARS-CoV-2 infection","abstract":"Synthetic receptor decoys offer a promising strategy to block viral entry but are often limited by instability and rapid clearance. Here we introduce AntiCoV decoy particles: phase-separated synthetic RNA-protein nanoparticles that display multivalent human ACE2 and act as robust decoy receptors for SARS-CoV-2. The granules remain structurally stable for at least two weeks at room temperature. Using confocal imaging and FRET, we show that the SARS-CoV-2 receptor-binding domain is absorbed into the granule matrix with nanometer-scale proximity to ACE2. In viral entry assays with Delta and Omicron (BA.1) variants, AntiCoV decoy particles achieve complete inhibition of infection at low micromolar concentrations and outperform soluble ACE2, which also exhibits infection enhancement at low doses. A minimal kinetic model suggests a multistep spike-priming mechanism that reconciles this biphasic response. AntiCoV decoy particles therefore provide a stable, modular, and pan-variant antiviral platform with potential for low-cost, room-temperature-stable prophylaxis.","version":"1.1","doi":"10.64898/2026.01.16.699918","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.01.651662","pub_date":"2026-1-16","title":"Revisiting Endothelial Tropism of SARS-CoV-2 Using a Cell-Specific hACE2 Mouse Model","abstract":"Severe COVID-19 is frequently associated with vascular complications, raising ongoing debate about whether SARS-CoV-2 can directly infect endothelial cells and thereby contribute to disease pathogenesis. Although endothelial cells express angiotensin-converting enzyme 2 (ACE2), the in vivo relevance of endothelial-restricted viral tropism remains unclear. To directly assess the consequences of endothelial-restricted SARS-CoV-2 tropism in vivo, we generated a transgenic mouse model expressing human ACE2 under control of the endothelial-specific Cdh5 promoter (Cdh5-hACE2). Despite confirmed pulmonary endothelial expression and protein presence of hACE2, SARS-CoV-2 infection of Cdh5-hACE2 mice did not induce clinical illness, detectable viral replication, immune cell influx in the lung, or histopathological abnormalities in the lung or brain. These findings indicate that endothelial-restricted SARS-CoV-2 tropism alone is insufficient to drive productive infection and clinical disease in vivo, suggesting that endothelial involvement in COVID-19 likely arises in the context of broader cellular infection or systemic host responses rather than from primary endothelial infection.","version":"1.2","doi":"10.1101/2025.05.01.651662","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.15.699647","pub_date":"2026-1-16","title":"Joint mechanistic modeling of viral and antibody responses to vaccines in non-human primates to quantify SARS-CoV-2 mechanistic correlates of protection","abstract":"In a global SARS-CoV-2 landscape of hybrid immunity resulting from a vaccinated worldwide population and the emergence of new variants able to escape immunity, the identification and quantification of correlates of protection (CoPs) is crucial for the adaptation of next-generation SARS-CoV-2 vaccines. Antibodies and their neutralizing capacity have been identified as reliable mechanistic CoPs. Here, we proposed an original mechanistic model jointly describing viral and antibody dynamics, and their mutual interactions, observed after SARS-CoV-2 infection in non-human primates (NHPs) with distinct immunological backgrounds. From the model, the concentration of neutralizing antibodies being protective against viral infection spreading was derived using the reproduction number. Counterfactual simulations were also performed to better understand and validate immune mechanisms driving immune control. The model was estimated on viral, binding (bAb), and neutralizing (nAb) antibody dynamics, with data collected in 34 naive and convalescent NHPs. Animals were involved in a preclinical study evaluating two next-generation protein-based vaccines targeting the RBD of the Spike protein to CD40-expressing cells, and the original BNT162b2 mRNA vaccine against Delta SARS-CoV-2 infection. Our model validated the functionality of nAb to neutralize viruses as the primary mechanism of protection. An inhibitory antibody concentration against Delta variant of 20 AU/mL was deemed protective against Delta infection in naive animals. Moreover, we showed the strong benefit of hybrid immunity to induce faster and more protective antibody responses pointing out the crucial role of the memory B-cell immune response in viral control. Finally, an additional effect of natural immunity beyond antibodies and enhancing the elimination of infected cells was identified, suggesting the potential role of the T-cell response in viral control. Our model showed the benefit of combining information from both viral and antibody responses to better qualitatively and quantitatively inform on their protective capacity against Delta SARS-CoV-2 infection.","version":"1.1","doi":"10.64898/2026.01.15.699647","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.19.639030","pub_date":"2026-1-13","title":"Possible exposure to unidentified coronaviruses in roe deer (Capreolus capreolus) populations suggested by SARS-CoV-2 serological investigation in France","abstract":"The risk of viral transmissions from domestic/wild animals to humans is a major public health concern. Humans can also transmit viruses back to domestic and wild animals, acting as reservoir for virus maintenance and sources of epidemic diseases re-emergence. The SARS-CoV-2, causing COVID-19, likely originated from wildlife and has been evidenced to transmit from humans to captive, domestic and wild animals. White-tailed deer (Odocoileus virginianus) show high SARS-CoV-2 prevalence following human contamination, suggesting they could act as an emerging virus reservoir. We completed recent research on European cervid species by investigating whether SARS-CoV-2 had emerged in longitudinally-monitored European roe deer (Capreolus capreolus) populations in direct contact with humans in France. We performed indirect tests (serological ELISAs and seroneutralization) on sera collected before and after the virus emergence in humans, and direct RT-PCR tests on nasal swabs collected in 2022. We also investigated the virus exposure and prevalence in three other cervid species. ELISA tests were positive for 2.20% of sera, pre- and post-pandemic, but seroneutralization and PCR tests were negative. Although one population showed increased seroprevalence post-2020, results suggest that SARS-CoV-2 has not emerged in those populations, and that ELISA cross-reaction with one or several unidentified circulating coronaviruses is possible.","version":"1.6","doi":"10.1101/2025.02.19.639030","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.12.698941","pub_date":"2026-1-13","title":"Loss of nsp14-exonuclease activity impairs the replication, proofreading, fitness and pathogenesis of SARS-CoV-2","abstract":"Coronaviruses (CoVs) replicate their RNA genomes with increased fidelity than other RNA viruses, a mechanism mediated by the proofreading and recombination activities of the exoribonuclease domain of replicase nonstructural protein 14 (nsp14-ExoN). Both murine hepatitis virus (MHV) and SARS-CoV tolerate nsp14-ExoN loss-of-function mutations (ExoN-) (D90A and E92A) but have impaired replication fidelity and pathogenesis yet identical substitutions in MERS-CoV and SARS-CoV-2 have been reported to be lethal. Here, we report a saturation mutagenesis approach facilitating the recovery and analysis of several constellations of SARS-CoV-2 nsp14 ExoN-inactivating, loss-of-function substitutions, including the canonical D90A and E92A. Biochemical assays with purified WT or ExoN- nsp10-14 fusion proteins confirmed that active site substitutions abolished ExoN activity (ExoN-). SARS-CoV-2 ExoN- viruses had impaired replication, RNA synthesis, and recombination, as well as decreased replication fidelity and loss of fitness in vitro. ExoN- viruses were significantly attenuated for replication in human primary airway epithelial cells and were attenuated for replication and pathogenesis in WT mice as well as the highly susceptible K18 transgenic mice. In the absence of interferon signaling in vivo, SARS-CoV and SARS-CoV-2 ExoN- viral replication could be nearly fully restored. These results demonstrate that SARS-CoV-2 ExoN- are viable but highly impaired for replication, fitness, and fidelity in vitro as well as innate immune antagonism and pathogenesis in vivo. Collectively, our results solidify the multiple critical roles for nsp14-ExoN across CoV genera and establish new approaches for rescue and analysis of loss-of-function substitutions for studies of CoV replication, pathogenesis, and evolution.","version":"1.1","doi":"10.64898/2026.01.12.698941","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.12.699133","pub_date":"2026-1-13","title":"Engineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogen","abstract":"Vaccines capable of eliciting broadly neutralising antibodies (bnAbs) are a major goal for pandemic preparedness. A persistent challenge across vaccine Wields is how to deliberately recruit the rare B cell clones that recognise conserved epitopes shared across diverse viral variants. BnAbs have been known to frequently emerge through extensive somatic hypermutation during afWinity maturation, here we describe an alternative, structure-driven mechanism for bnAb selection. We designed an mRNA vaccine in which two antigenically distinct SARS-CoV-2 variant\u2019s (Omicron and Delta; O-\u0394) receptor binding domains (RBDs) are physically fused on a single polypeptide. This design is predicted to favour B cell antigen receptors capable of engaging conserved epitopes on both RBDs with enhanced avidity. A matched non-divergent tandem RBD (Delta-Delta; \u0394-\u0394) served as a control. The divergent (O-\u0394) immunogen was robustly expressed and retained high-afWinity ACE2 binding. In mice, immunisation elicited potent antibody responses and increased the frequency of antigen-speciWic cross-reactive B cells, recognising Delta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Using multicolour RBD tetramers and single-cell B cell receptor sequencing, we show that breadth arises via two distinct pathways. The divergent vaccine preferentially enriches clonally distinct cross-reactive B cells (not present within non-cross-reactive B cell pools) with low levels of somatic hypermutation (SHM), consistent with selection of germline-biased precursors. In contrast, the matched control vaccine yields cross-reactivity primarily within existing clonal lineages (clonal overlap between cross-reactive and non-cross-reactive cells) and at higher mutational burdens, consistent with afWinity-maturation-driven acquisition of breadth. Together, these Windings demonstrate that antigen structure can bias B cell selection towards cross-reactive speciWicities without requiring extensive SHM. This work establishes a simple, modular antigen-design principle in which juxtaposing appropriately divergent antigens on a single scaffold promotes the enrichment of bnAb-prone B cells, providing a scalable strategy for vaccine development against rapidly evolving pathogens.","version":"1.1","doi":"10.64898/2026.01.12.699133","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.12.699045","pub_date":"2026-1-13","title":"Molecular, cellular and network mapping of brain structural deviations in patients with Post-COVID19 syndrome","abstract":"Post-COVID-19 syndrome encompasses persistent cognitive, neurological, and psychiatric symptoms following SARS-CoV-2 infection, profoundly affecting global quality of life. Clarifying the neurobiological basis of these symptoms is vital for effective therapeutic interventions. This study utilized normative modelling of brain structure (\u201cCentileBrain\u201d) to quantify individualized deviations in cortical thickness, surface area, and subcortical volumes among 20 patients experiencing persistent fatigue following mild COVID-19, compared to 20 matched healthy controls. Group-level analyses on deviation scores revealed subtle yet distinct regional alterations in cortical thickness, specifically decreased thickness within orbitofrontal cortices and increased thickness in occipital/sensory cortices. Although at the individual regional level, the proportion of patients exhibiting infranormal or supranormal thickness values was relatively low (<35%) and comparable to controls, deviations frequently clustered within structurally connected circuits, affecting up to 50% more of patients. Spatial analysis of regional cortical thickness alterations correlated significantly with the constitutive expression patterns of TMPRSS2, an essential protein facilitating SARS-CoV-2 cellular entry. Canonical correlation analyses further identified specific cell-type distributions and neuroreceptor densities predictive of regional thickness changes, highlighting neurons and molecular targets associated with serotoninergic, cannabinoid, cholinergic, and glutamatergic signalling pathways. Network-diffusion modelling constrained by a canonical structural connectome significantly outperformed null models based on permuted connectomes and Euclidean distance metrics, identifying posterior-parietal regions as probable initiation points (\u201cseeds\u201d) for network-wide structural changes. Seed likelihood correlated positively with TMPRSS2 expression levels, suggesting that these posterior-parietal regions may be particularly susceptible to SARS-CoV-2 infection. This highlights a plausible mechanism where structural alterations could propagate through connected neural networks, although direct evidence of such propagation requires further investigation. These findings provide novel insights into potential mechanisms underlying neural circuit disruptions in post-COVID-19 fatigue and suggest avenues for therapeutic neuromodulation. Normative modelling revealed heterogenous individualised cortical structural deviations in patients with PCS. Regional deviations in cortical thickness and surface area clustered within anatomically connected circuits, affecting up to 50% more of PCS participants. Structural changes were significantly associated with regional TMPRSS2 expression, implicating SARS-CoV-2 entry pathways in PCS-related brain alterations. Network diffusion modelling identified posterior-temporoparietal regions as likely epicentres of pathology propagation through the structural connectome. Cortical deviations were linked to neuronal and microglial cell-type densities and receptor systems including serotonergic, glutamatergic, cannabinoid and cholinergic pathways, suggesting targets for neuroimmune-informed interventions.","version":"1.1","doi":"10.64898/2026.01.12.699045","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.12.699018","pub_date":"2026-1-13","title":"Multimodal imaging suggests potential immune-vascular contributions to altered regional brain perfusion and oxygen metabolism in Post-COVID-19 Syndrome","abstract":"Post-COVID-19 Syndrome (PCS) frequently presents with persistent fatigue, cognitive impairment, and emotional symptoms. Although structural brain changes remain subtle, growing evidence implicates functional and metabolic disruptions in ongoing symptomatology. We used multimodal MRI to investigate cerebral perfusion and oxygen metabolism in PCS and examined their associations with cognitive function and peripheral biomarkers. We enrolled 40 individuals with prior mild SARS-CoV-2 infection, including 20 with persistent fatigue and 20 recovered controls matched for age, sex, BMI, and acute COVID-19 severity. Participants underwent structural MRI, arterial spin labelling (ASL) to quantify regional cerebral blood flow (CBF), and asymmetric spin echo imaging to estimate oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen consumption (CMRO\u25a1). We assessed cognition using an online battery and measured serum levels of TNF-\u03b1, IL-6, IL-8, IL-13, IFN-\u03b3, GFAP, and S100\u03b2, alongside blood routine tests. We performed ANCOVAs on predefined regions of interest (hippocampus, anterior cingulate cortex [ACC], insula, amygdala, striatum), followed by Bayesian inference and exploratory whole-brain analyses. PCS participants showed increased CMRO\u25a1 in the hippocampus and decreased CMRO\u25a1 in the ACC. Subfield analysis revealed elevated OEF and CMRO\u25a1 across most hippocampal regions, excluding the entorhinal cortex. Whole-brain analyses identified increased perfusion in salience-related regions (insula, ACC, thalamus) and decreased perfusion in posterior cortical and cerebellar areas, in the absence of grey matter volume differences. Higher hippocampal metabolism positively correlated with cognitive performance, suggesting compensatory adaptation to sustain function. In contrast, lower ACC CMRO\u25a1 correlated with depressive symptoms, reduced motivation, and elevated TNF-\u03b1 and GFAP, consistent with neurovascular uncoupling possibly driven by immune-glial activation. These findings reveal distinct physiological disruptions in PCS, with potential implications for stratified, metabolism-focused interventions.","version":"1.1","doi":"10.64898/2026.01.12.699018","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443207","pub_date":"2026-1-12","title":"SARS-CoV-2 Infection Causes Strong mTORC1 Inhibition and Massive Polysome Collapse","abstract":"Viruses employ distinct strategies to ensure efficient translation of their mRNAs over the host transcripts. SARS-CoV-2 targets host mRNAs and ribosomes to favor its own protein synthesis. However, the modulation of the key signal pathways that control the host protein translation machinery during SARS-CoV-2 infection has not been sufficiently addressed. Here, by employing an early variant and a Delta variant isolate that evolved later in the pandemic demonstrates that SARS-CoV-2 infection results in massive polysome collapse starting from 24 hpi, a hallmark of global translation inhibition. Unexpectedly, eIF2\u03b1 phosphorylation, commonly targeted by viruses to induce translation arrest, was not involved in the translation arrest, suggesting that SARS-CoV-2 countermeasures by the virus to suppress ISR. eIF4E phosphorylation remained unaltered during the infection, ruling out its involvement in the preferential translation of SARS-CoV-2 transcripts. We find that SARS-CoV-2 infection consistently causes mTORC1 inhibition in a comparable manner across both variants indicating that the virus likely targets mTORC1 pathway to suppress host translation. Interestingly, mTORC1 inhibition by SARS-CoV-2 did not impact the polysomal loading of ribosomal protein transcripts rpS3 and rpL26, suggesting that 5\u2019TOP mRNAs are spared from the translation suppression and that ribosomal protein synthesis remains active during the infection. Pharmacological activation of mTORC1 did not significantly impact viral replication, suggesting that mTORC1 inhibition might be selectively restricting the host mRNAs from accessing the translation machinery, facilitating a more robust translation of viral transcripts. This study provides new insights into the molecular interactions by which SARS-CoV-2 variants, despite their different clinical outcomes, converge on a conserved mechanism to manipulate host translation regulatory pathways.","version":"1.2","doi":"10.1101/2021.05.08.443207","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.09.698728","pub_date":"2026-1-12","title":"A Mammalian Surface Display Platform to Optimize the Antigenicity of Viral Proteins for Vaccine Design","abstract":"Vaccine development often involves modifying native viral proteins to enhance their stability and antigenicity, as seen in approved Covid-19 vaccines and multiple HIV vaccine candidates currently under investigation. High throughput screening on the surface of mammalian cells enables the rapid evaluation of oligomeric, glycosylated viral proteins and the identification of mutations that improve their properties for vaccine design. Here, we developed an experimental platform that uses the PiggyBac transposon system to display libraries of viral protein variants on the surface of mammalian cells for efficient screening. This approach addresses common challenges in existing mammalian display systems, including low transfection efficiency and the development of stable cell lines for iterative selection rounds. The new platform was validated by expressing and characterizing antigenically diverse viral proteins from influenza, SARS-CoV-2, and HIV. In a further application, library screening of influenza haemagglutinin libraries identified mutations that increased binding of broadly cross-reactive antibodies to a conserved, but partially occluded, epitope of interest for the development of a universal influenza vaccine. These results demonstrate the potential of this mammalian display platform to rapidly engineer immunogens with desired antigenic properties for vaccine design.","version":"1.2","doi":"10.64898/2026.01.09.698728","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.09.698592","pub_date":"2026-1-10","title":"MOV10 inhibits SADS-CoV replication by enhancing TRIM24-mediated K63-linked TRAF3 ubiquitination and this inhibition is antagonized by viral N protein","abstract":"The global outbreak of SARS-CoV-2 has resulted in a renewed focus on coronaviruses with the potential for cross-species transmission and mutation risks. In particular, swine acute diarrhea syndrome coronavirus (SADS-CoV), potentially originating from intermediate horseshoe bat (Rhinolophus affinis), is a novel coronavirus that causes acute diarrhea, vomiting, and high mortality in suckling piglets. Innate immunity plays a crucial role in defending the host against invading pathogens. Previous studies have identified several host factors that inhibit SADS-CoV replication through innate immunity; however, research on SADS-CoV remains insufficient compared to that on other coronaviruses. In this study, the host factor Moloney leukemia virus 10 protein (MOV10) inhibited SADS-CoV replication by promoting interferon (IFN) production. Mechanistically, MOV10 exerted its antiviral effect primarily through its N-terminal domain, which regulated the TNF receptor-associated factor 3 (TRAF3)-mediated innate immune pathway. Specifically, we reveal that MOV10 enhanced tripartite motif-containing 24 (TRIM24)-mediated K63-linked ubiquitination of TRAF3, thereby promoting IFN production and inhibiting SADS-CoV replication. Furthermore, we identified the strategies by which SADS-CoV evaded innate immunity. The viral N protein inhibited the antiviral effect of MOV10 by disrupting interaction between MOV10 and TRAF3, and by promoting K48-linked polyubiquitination of TRAF3, leading to TRAF3 degradation via the ubiquitin-proteasome pathway. Collectively, our findings reveal the role of MOV10 in antiviral immunity and how SADS-CoV evades host immune defense.","version":"1.1","doi":"10.64898/2026.01.09.698592","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.18.665531","pub_date":"2026-1-09","title":"Covariance-Based MD Simulation Analysis Pinpoints Nanobody Attraction and Repulsion Sites on SARS-CoV-2 Omicron Spike Protein","abstract":"Omicron\u2019s heavily mutated SARS-CoV-2 spike receptor-binding domain (RBD) enables broad escape from neutralizing antibodies and nanobodies (Nbs), yet the atomistic basis of epitope-dependent Nb loss remains unclear. We performed >8 \u03bcs of all-atom molecular dynamics (MD) simulations of 13 Nbs (H11-D4, H11-H4, Huo-H3, MR17, NB21, NM1230, RE5D06, SB14, SB23, SB45, Ty1, VHH-E, WNB2) bound to the Omicron RBD to characterize binding-pose stability and interfacial dynamics. Principal-component analysis of MD trajectories yields binding-pose free-energy landscapes: most Nbs occupy single dominant basins yet often deviate from WTbound poses (orientation shifts), whereas NB21 and SB14 show pose plasticity. Using our covariance-matrix\u2013based interaction-mapping method, we map stabilizing versus destabilizing residue\u2013residue couplings and classify the underlying attractive interactions (salt bridges, hydrogen bonds, hydrophobic contacts) and repulsive interactions (like-charge repulsion or polarity mismatches). Attractive contacts converge on recurrent hydrophobic \u201canchor\u201d patches on the receptor-binding motif (RBM) (\u223cV445-G456 and \u223cF490-Y501) alongside binder-specific, complementarity-determining-region (CDR)-shaped interactions. Omicron substitutions rewire contact networks, shift binding orientations, and can introduce unfavorable repulsion, directly at mutation sites (e.g., E484A) or indirectly via mutation-driven reorientation, weakening binding. Mutation-induced repulsion reshapes correlated interfacial motions and reduces interfacial stability, providing a mechanistic basis for diminished binding robustness of multiple Nbs that were effective against WT and/or earlier variants. Low-speed steered MD pulling (AFM comparable) of ACE2 and selected Nbs shows lower unbinding work for Nbs than ACE2 (\u223c16\u201342 vs \u223c45 kcal/mol), with NM1230 and SB23 most mechanically robust. These Nb\u2013RBD interaction fingerprints pinpoint epitope-specific determinants and mutation-induced clash sites, providing a blueprint for structure-guided CDR engineering of Nbs with sustained potency against ongoing SARS-CoV-2 evolution.","version":"1.2","doi":"10.1101/2025.07.18.665531","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.08.698330","pub_date":"2026-1-08","title":"Nanobodies against the S2 region of the spike protein potently neutralize SARS-CoV-2 viruses and show resistance to virus escape","abstract":"Entry of coronaviruses into cells is mediated by the viral spike (S) glycoproteins each consisting of S1 receptor binding and S2 membrane fusion subunits. The sequence of the S2 region is very highly conserved amongst variants of SARS-CoV-2 and compared to the S1 unit shares significant sequence identity amongst different beta-coronavirus lineages. By targeting the S2 of SARS-CoV-2 we have identified two selective and potent neutralizing nanobodies (BA.1-C2 and BA.1-D3) that bind to two different quaternary epitopes in the S2 formed by the Heptad Repeat 2 (HR2) trimer at the base of the spike protein. The HR2 sequence is identical in SARS-CoV and SARS-CoV-2 but differs in other beta-coronaviruses explaining the lack of binding to the spike proteins of MERS-CoV or HuCoV-OC43. No viral escape was observed following serial passaging of SARS-CoV-2 (JN.1) with a combination of BA.1-C2 and BA.1-D3 and the most potent of these nanobodies reduced viral load in the hamster model of COVID-19, following intranasal administration. Overall, the results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.","version":"1.1","doi":"10.64898/2026.01.08.698330","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.08.687354","pub_date":"2026-1-08","title":"Hidden Markov models detect recombination and ancestry of SARS-CoV-2","abstract":"When individuals are co-infected with distinct SARS-CoV-2 lineages, homologous recombination can generate mosaic genomes carrying mutations from both parental lineages. A variety of methods exist to detect recombinant sequences and their parental lineages in surveillance-scale datasets comprised of millions of SARS-CoV-2 genomes. However, these methods often rely on user-specified parameters, such as the probability a recombination breakpoint occurs between adjacent positions on the query sequence. In this study, we devise a hidden Markov model that detects recombinant SARS-CoV-2 sequences and identifies their parental lineages within a test set of sequences. Our method does not depend on user-specified parameters and can accommodate de novo mutations on the query sequence that are not present in the predicted parental lineages. To achieve this, we use maximum likelihood to estimate parameters that characterize the transition and emission probabilities in our hidden Markov model. Applying our method to 440,307 SARS-CoV-2 sequences sampled in England between September 2020 and March 2024, we detect 7,619 recombinant sequences corresponding to 1.73% (95% CI: [1.69%, 1.77%]) of all sampled sequences. We observe a positive association between the proportion of query sequences detected as recombinant in each week and community SARS-CoV-2 prevalence. This is consistent with higher prevalence increasing the risk of co-infection by distinct lineages and promoting the emergence of recombinant sequences. Finally, we observe localized clusters of recombination breakpoints within spike and in intergenic regions.","version":"1.2","doi":"10.1101/2025.11.08.687354","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.08.698347","pub_date":"2026-1-08","title":"Linkage-aware inference of fitness from short-read time-series genomic data","abstract":"Inferring the fitness effect of mutations is a basic problem in understanding the evolution of populations over time. When multiple mutations are present in a population simultaneously, genetic linkage comes into play, and the fate of an individual mutation depends on both its fitness as well as the background on which it occurs. Accurate inference of fitness effects for evolutionary systems with multiple competing mutations is therefore contingent on resolving the confounding effects of genetic linkage, captured by the covariance between allele-pairs. Increasingly, evolutionary studies are using short-read sequencing technologies to produce detailed snapshots of evolving populations. This presents a problem as the frequencies of allele-pairs are not known beyond the read-length, hampering any attempt to resolve the effects of genetic linkage between pairs of loci residing on different reads. Here we present a computationally efficient pipeline for inferring selection from short-read time-series data with partial allele-pair frequency information, while accounting for linkage. Simulation results show that the method has good performance and is scalable to systems with several thousand variants. Additionally, we demonstrate the pipeline\u2019s utility on real datasets of within-host HIV and SARS-CoV-2 evolution, showcasing its applicability in resolving linkage effects from complex evolutionary histories.","version":"1.1","doi":"10.64898/2026.01.08.698347","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.07.697678","pub_date":"2026-1-07","title":"Single-antibody mutational scanning reveals poly-specificity and immune constraints in vaccination responses","abstract":"Current repertoire technologies with single-antibody resolution usually assess binding to only one or a few recombinant antigens, neglecting variant diversity of antigens and poly-specificity of antibodies. Here, we introduce a method for analyzing antibody repertoires at single-antibody resolution against a large library of SARS-CoV-2 receptor-binding domain (RBD) variants, enabling the identification and analysis of recognized RBD variants by rare, poly-specific antibodies. This technique provided unique insights into single-antibody binding and escape profiles within a murine immunization model with different RBDs, offering valuable data useful to optimize vaccine design against emerging variants and study antibody poly-specificity.","version":"1.1","doi":"10.64898/2026.01.07.697678","journal":"bioRxiv","score":null},{"id":"10.64898/2026.01.03.697510","pub_date":"2026-1-05","title":"Coronavirus Nucleocapsid Proteins Hijack Host Protein Kinase A Catalytic Subunit \u03b1 into Nucleus to Evade from STAT1 Signaling","abstract":"The role of the cAMP/PKA pathway in antiviral innate immunity, including during coronavirus infections, remains unclear. We discovered coronavirus N proteins initiate cAMP-ADCY10-PKA cascade. Cytoplasmic PKA activates STAT1 independently of canonical JAK/TYR2 signaling. Coronavirus N proteins, via a conserved arginine (e.g., PEDV R58, SARS-CoV-2 R92), directly bind and sequester PKA C\u03b1 into the nucleus to evade STAT1 activation. Using PEDV as a model, mutant viruses with NmutNLS and NR58A were generated. Wildtype PEDV infection suppressed STAT1 activation in cells expressing PKA C\u03b1/PKA C\u03b1S339A deficient in STAT1 phosphorylation. However, in rXS0101mutNLS- or rXS0101R58A-infected cells, only PKA C\u03b1S339A expression inhibited STAT1 activation. Downregulation of STAT1 activation is accompanied with increased viral replication. This study first elaborates that PKA C\u03b1 activates STAT1 in the cytoplasm of infected cells distinctly from canonical JAK/STAT1 signaling, while coronaviruses evade this antiviral response by sequestering PKA C\u03b1 into the nucleus via direct N protein interaction.","version":"1.1","doi":"10.64898/2026.01.03.697510","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.03.686211","pub_date":"2026-1-04","title":"The Coronavirus Envelope Is Modulated by Host Inflammation and Enriched in Bioactive Lipids Required for Replication","abstract":"How inflammation or disease regulates coronavirus lipid membranes is currently unknown, while patient-derived viral envelopes have never been structurally characterized. Here, we show that four cultured SARS-CoV-2 strains (England2, Alpha, Beta, and Delta) possess conserved, phospholipid- and cholesterol-rich envelopes, with pro-thrombotic and infection-promoting aminophospholipids (aPL) displayed predominantly on the outer leaflet (approximately 70\u201380%). Exposure to interleukin-4 (IL-4) markedly altered envelope fatty acyl composition, whereas interleukin-6 (with or without its soluble receptor IL-6R\u03b1) and dexamethasone had no detectable effect. Viral envelopes were susceptible to hydrolysis by secretory phospholipase A2 (sPLA2), an enzyme associated with adverse clinical outcomes. SARS-CoV-2 isolated directly from patient saliva exhibited cholesterol-enriched envelopes that were highly conserved across clinical isolates. In addition, clinical samples contained pro-coagulant oxidized phospholipids and bioactive lipoxygenase (LOX)-derived oxylipins. The dominance of external facing pro-coagulant aPL and eoxPL may support known thrombotic complications of severe COVID19 viremia. Last, gene-silencing experiments demonstrated that 15-LOX2 is required for replication of related coronaviruses. Together, these findings reposition the coronavirus envelope as an active, dynamic structure rather than a passive scaffold, and challenge the protein-centric view of viral function. The lipid envelope is proposed as a potential therapeutic target through modulation of host innate immunity, and dampening thrombotic potential. Viruses such as SARS-CoV-2 are surrounded by a host-derived lipid envelope. Little is known about how this changes during infection/inflammation. We determined the lipid composition of the SARS-CoV-2 envelope using both laboratory-grown viruses and patient isolates. Across several pandemic strains, the envelope was rich in cholesterol and phospholipids and showed a consistent structure. Lipids linked to thrombosis and infection were mainly exposed on the outer virus surface. The inflammatory cytokine interleukin-4 altered the envelope\u2019s fatty acid composition, while other treatments did not. Patient-derived viruses contained additional bioactive lipids, and blocking an enzyme that generates these lipids reduced coronavirus replication. In summary, the envelope is an active component of infection and potential target for new treatments to dampen infectivity and thrombosis.","version":"1.2","doi":"10.1101/2025.11.03.686211","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.31.697156","pub_date":"2026-1-02","title":"Post-COVID impairment of memory T cell responses to community-acquired pathogens can be rectified by activating cellular metabolism","abstract":"Infection rates involving bacterial and viral pathogens have increased precipitously after the COVID-19 pandemic. While it has been speculated that higher infection rates resulted from increased hospitalizations throughout the pandemic or greater use of antibiotics, precisely why rates remain high today has remained unexplained. Mitochondrial dysfunction is known to occur post-COVID and may disrupt immune responses. Within T cells, SARS-CoV-2 infection is linked to low mitochondrial membrane potential, increased mitochondrial apoptosis, and decreased mitochondrial respiration, which together impact cellular activation and function beyond the acute phase of illness. Here, we demonstrate that decreased mitochondrial function in antigen-specific T cells post-COVID may contribute to higher infection susceptibility by metabolically immobilizing T cell memory responses. Using donor-matched peripheral blood samples from 31 COVID-na\u00efve individuals who subsequently contracted COVID-19, we tracked how influenza A (IAV), Staphylococcus aureus (SA), and Varicella-zoster virus (VZV) T cell responses were impacted by COVID-19 infection. We found that gene expression linked to T cell activation decreased but mitochondrial redox pathways increased in CD4 memory T cells post-COVID. However, mitochondrial flux and reactive oxygen species production were limited in a plurality of post-COVID memory T cells after stimulation with IAV, SA, and VZV. Furthermore, we found a disordered relationship between memory T cell mobilization of glycolysis, fatty acid metabolism, and oxidative phosphorylation pathways post-COVID which resulted in diminished use of catabolic pathways including glycolysis and fatty acid oxidation in antigen-specific T cells. Modulation of mitochondrial function with metformin and ubiquinol partially rescued the post-COVID decline in T cell catabolism. Collectively, these findings indicate that COVID-19 infection may have lasting effects on inhibiting T cell memory responses to commonly encountered community-acquired pathogens which can be corrected with commonly available medications. This has significant implications for the clinical care of immunologically vulnerable populations in the post-pandemic era.","version":"1.1","doi":"10.64898/2025.12.31.697156","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.30.696971","pub_date":"2026-1-02","title":"Oral Administration of the carboxylic acid ester prodrug NHC SN_9 protects mice from lethal Orthopoxvirus challenge: Implications for the Treatment of Monkeypox","abstract":"The global resurgence of monkeypox (Mpox) since 2022 has highlighted an urgent need for effective antiviral therapeutics against orthopoxvirus infections. While vaccines provide prophylactic protection, therapeutic options remain limited, particularly for immunocompromised individuals. Previously, we demonstrated potent antiviral activity of N4-hydroxycytidine (NHC) analogs against coronaviruses, including SARS-CoV-2. In the present study, we evaluated the antiviral efficacy of one such analog, NHC conjugate with phenylpropionic acid \u2013 SN_9, against orthopoxviruses using Vaccinia virus (VACV) as a model. SN_9 demonstrated superior in vitro antiviral activity compared with EIDD-2801 (molnupiravir), significantly inhibiting VACV replication. In vivo, oral administration of SN_9 provided substantial protection in murine models of orthopoxvirus infection, achieving 90% survival in a sublethal infection model and 70% survival in a lethal challenge model. Animals receiving treatment showed a reduction in disease severity and faster weight recovery. Given the high genetic and antigenic similarity among orthopoxviruses, including VACV, variola virus, and monkeypox virus (MPXV), these findings suggest that SN_9 represents a promising candidate for the treatment and prophylaxis of Mpox.","version":"1.1","doi":"10.64898/2025.12.30.696971","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.27.696524","pub_date":"2025-12-30","title":"Molecular signature of COVID-19 prior to its exacerbation by multi-omics survey","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic due to its high transmissibility and ability to evade innate immune responses. Comprehensive characterization of the disease is essential for elucidating its pathophysiology and clinical progression. In this study, we performed multi-omics analyses of plasma samples collected from SARS-CoV-2-positive patients prior to clinical deterioration of coronavirus disease 2019 (COVID-19). These samples revealed the potential of previously reported clinical parameters, including CRP and neutrophil level, to predict COVID-19 exacerbation in the early stage. Our analysis identified a novel panel of molecules that precede the clinical manifestations associated with COVID-19 progression. These candidate biomarkers exhibited strong correlations with previously reported clinical and immunological parameters. Notably, several inflammation-related markers showed inverse associations with specific interferon subtypes, including IFN-\u03b16 and IFN-\u03b18, potentially reflecting mechanisms of SARS-CoV-2-mediated immune evasion. Our findings contribute to the understanding of virus-induced acute exacerbation and offer a valuable foundation for future pandemic research.","version":"1.1","doi":"10.64898/2025.12.27.696524","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.29.696828","pub_date":"2025-12-30","title":"Post-translational modifications within fibrinaloid microclot complexes distinguish Pre-COVID-19 Postural Orthostatic Tachycardia Syndrome (POTS), Long COVID, and Long COVID-POTS and reveal disease-specific molecular pathways","abstract":"Pre-COVID-19 Postural orthostatic tachycardia syndrome (PC-POTS), Long COVID, and their overlap (LC-POTS) are chronic post-viral conditions marked by debilitating symptoms despite frequently normal routine laboratory results. We previously identified insoluble fibrinaloid microclot complexes (FMCs) in Long COVID. It is not known whether FMCs are also present in PC-POTS, or whether post-translational modifications (PTMs) within FMC-entrapped proteins contribute to disease mechanisms. Platelet-poor plasma from healthy controls, PC-POTS patients (collected prior to the COVID-19 pandemic), Long COVID (without POTS) and LC-POTS patients underwent fluorescence imaging flow cytometry to quantify FMCs. Proteomic analyses were performed on insoluble protein fractions using a double trypsin digestion strategy and data-independent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential protein abundance, PTMs, and amyloidogenicity were compared across groups. Measured with imaging flowcytometry in objects/mL, higher levels of FMCs were present in disease groups compared to controls, although not statistically significant. Statistically significant differences potentially lay within FMC sizes and composition. Furthermore, despite only a few dysregulated proteins, FMC proteomics revealed extensive and disease-specific peptides with PTM dysregulation across coagulation, immune, and metabolic pathways. Long COVID displayed FMCs with PTMs of coagulation proteins including prominent advanced glycation end-products (AGE)- and oxidation-based modifications of fibrinogen subunits, particularly fibrinogen subunit A (FIBA), resembling diabetic glycation profiles. FMCs in PC-POTS showed fewer fibrinogen PTMs but markedly increased modifications in metabolic proteins, including oxidised apoA1 and apoB, and immune patterns with complement-related proteins (C3, C4A/B, IC1), immunoglobulin G1 (IGG1) and alpha 2 macroglobulin (A2MG). LC-POTS shared coagulation pathology with Long COVID and immune pathology with PC-POTS. Many dysregulated peptides were determined by in silco methods to be highly amyloidogenic, consistent with FMCs as \u03b2-sheet-rich aggregates. Protein-level differences were minimal compared with PTM changes. This study provides the first evidence that post-translational modifications (PTMs) within fibrinaloid microclots complexes (FMCs) uniquely distinguish pre-COVID-19 POTS, Long COVID, and Long COVID-POTS. Because PC-POTS samples were collected before SARS-CoV-2, their PTM patterns reflect intrinsic disease biology, allowing a clear separation from Long COVID-related changes. PTM profiling revealed pro-coagulant fibrinogen modifications in Long COVID and LC-POTS, metabolic-oxidative disruptions in Long COVID and PC-POTS, and immune dysregulation in PC-POTS and LC-POTS. None of these is detectable with routine assays, and all are independent of protein abundance. The consistent presence of amyloidogenic peptides suggests a contribution to microvascular dysfunction. These findings define disease-specific PTM landscapes and support new diagnostic and therapeutic avenues across autonomic and post-viral disorders.","version":"1.1","doi":"10.64898/2025.12.29.696828","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.28.696697","pub_date":"2025-12-29","title":"SARS-CoV-2 and MERS-CoV disrupt host protein synthesis via nsp1 with differential effects on the integrated stress response","abstract":"Coronaviruses pose a serious threat to public health, driving the need for antiviral therapeutics and vaccines. Therefore, it is paramount to understand how this family of viruses evades cellular antiviral responses and establishes productive infection. The conserved coronavirus non-structural protein (nsp)1 has been shown to inhibit host protein synthesis and promote host mRNA degradation while viral mRNAs are protected. We showed previously that SARS-CoV-2 induces activation of host integrated stress response (ISR) kinases PKR and PERK, which promote phosphorylation of eIF2\u03b1 and consequent inhibition of host protein synthesis. In contrast, eIF2\u03b1 remains unphosphorylated during MERS-CoV infection. To investigate the interactions of nsp1 and the ISR kinases, we utilized recombinant SARS-CoV-2 and MERS-CoV expressing nsp1 with mutations in each of two conserved domains. Upon infection with SARS-CoV-2 nsp1 mutants, translation was shut down in wildtype (WT) and PKR knockout (KO) cells but rescued in PERK KO cells, likely due to reduced p-eIF2\u03b1. In contrast, translation was rescued during infection with the analogous MERS-CoV nsp1 mutants even in WT cells. Moreover, SARS-CoV-2 WT suppressed expression of GADD34, a negative regulator of eIF2\u03b1 phosphorylation, while SARS-CoV-2 nsp1 mutants induced GADD34. In contrast MERS-CoV WT induced GADD34. Utilizing single-molecule fluorescence in situ hybridization, we found that SARS-CoV-2 and MERS-CoV nsp1 promote host mRNA degradation during WT, but not nsp1 mutant, infection. Finally, while SARS-CoV-2 WT suppressed stress granule formation, nsp1 mutants induced stress granules containing host RNA. Thus, SARS-CoV-2 and MERS-CoV differ in interactions with the ISR and nsp1 control of host protein synthesis. Coronaviruses cause disease across a wide range of animal species, and the human coronaviruses SARS-CoV-2 and MERS-CoV have caused epidemics of severe respiratory illness. Thus, it is imperative to understand how these viruses antagonize host responses and cause lethal disease. We show here that the betacoronavirus non-structural protein (nsp)1 promotes shutdown of host protein synthesis while preserving viral protein synthesis and, in addition, promotes degradation of host mRNAs. However, SARS-CoV-2 and MERS-CoV differ in their ability to manipulate the host integrated stress response, indicating that it is important to understand detailed coronavirus-host interactions and how they differ even between lethal coronaviruses. Such insights will inform the development of antiviral therapeutics to treat and prevent current and future coronavirus outbreaks.","version":"1.1","doi":"10.64898/2025.12.28.696697","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.25.696538","pub_date":"2025-12-29","title":"The SARS-CoV-2 nonstructural protein 15 collapses the host cytoskeleton by interacting with the host keratin type II cytoskeletal 1","abstract":"COVID-19, the deadliest recorded pandemic of the 21st century, was caused by a novel coronavirus, SARS-CoV-2. To explore why this coronavirus was significantly more pathogenic than its predecessors, more information is needed about the functions of the viral proteins that contributed to this uptick in virulence. This study explored how one of these viral proteins, the nonstructural protein 15 (NSP15), interacts with the host. Our findings revealed a novel protein-protein interaction between NSP15 and the host keratin type II cytoskeletal 1 in Vero E6 cells, as determined by mass spectrometry, and subsequently validated by immunoprecipitation. We observed that when NSP15 was delivered into Vero E6 cells, the fibrous network of keratin intermediate filaments was disrupted, and the nuclear structure was lost. This disruption of the keratin cytoskeleton was then shown to cause a statistically significant reduction in cell viability of Vero E6 cells. These results indicate that the presence of NSP15 in host cells leads to a collapse of the keratin cytoskeleton. This cytoskeletal collapse could be a mechanism the virus employs to escape the infected host cell via cell lysis, once significant amounts of viral protein and progeny have accumulated.","version":"1.1","doi":"10.64898/2025.12.25.696538","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.23.696280","pub_date":"2025-12-26","title":"Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates","abstract":"SARS-CoV-2 3CL-Mpro is essential for viral replication. Several circulating variants carry mutations distant from the catalytic residues yet maintain proteolytic function. The Omicron substitution P132H exemplifies this tolerance. However, preservation of overall activity does not exclude subtler effects on substrate recognition or selectivity, which can be influenced by distal structural perturbations. To define whether this mutation alters enzymatic activity at a cleavage junction distinct from the Nsp4-Nsp5 site, we measured and compared the biochemical properties of Wuhan with Omicron (e.g. steady-state kinetics) and determined a high-resolution structure of the Omicron (P132H) 3CL-Mpro in complex with an Nsp8-Nsp9 peptide. Catalytic efficiency and Nirmatrelvir sensitivity were comparable to wild type across both cleavage-site substrates, and all variants retained the characteristic pH optimum. In contrast, P132H displayed reduced thermal stability at elevated temperature. Crystallographic analysis showed that His132 adopts a distinct conformation upon substrate binding that reorganizes interactions with Glu240 and neighboring residues while preserving active-site geometry. Together, these results define how 3CL-Mpro tolerates variant-associated mutations without compromising activity at a noncanonical cleavage junction.","version":"1.1","doi":"10.64898/2025.12.23.696280","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.18.694353","pub_date":"2025-12-24","title":"IGHV3-53 antibody abundance drives divergent SARS-CoV-2 immune imprinting","abstract":"The mechanisms driving divergent SARS-CoV-2 immune imprinting in populations primed with different COVID-19 vaccines remain unclear. Recipients of inactivated vaccines readily develop Omicron-specific antibodies through repeated breakthrough infections, whereas mRNA-vaccinated individuals exhibit severe ancestral-strain imprinting that suppresses de novo Omicron-specific responses. These differences could result in distinct antibody landscapes, leading to regional epidemiological divergence and necessitating region-specific vaccine update strategies. Importantly, conventional wild-type mouse models fail to recapitulate strong human SARS-CoV-2 imprinting, which significantly hinders imprinting-related mechanistic investigation and vaccine update evaluation. Here, we surprisingly found that V(D)J-humanized mice could faithfully recapitulate human severe SARS-CoV-2 immune imprinting phenotypes. Comprehensive antibody repertoire and epitope mapping of 583 monoclonal antibodies from these models revealed that the abundance of pre-existing human IGHV3-53/66-encoded SARS-CoV-2 antibody responses determine imprinting severity following Omicron exposure through antibody-mediated masking of Omicron-specific epitopes. Both passive transfer of IGHV3-53/66 antibodies and knock-in of the human IGHV3-53 gene were sufficient to induce severe SARS-CoV-2 imprinting in wild-type mice. Concordantly, head-to-head comparison also showed that mRNA vaccine recipients retained higher IGHV3-53/66 antibody abundance and thus stronger imprinting than inactivated vaccine recipients. Consequently, compared to NB.1.8.1, XFG exhibits greater immune evasion in mRNA-vaccinated individuals but not in inactivated vaccine recipients. This explains the regional predominance of XFG in mRNA-vaccinated populations, while NB.1.8.1 prevails in inactivated vaccine-dominated countries. Together, these findings demonstrate that the V(D)J germline repertoire\u2014even a single germline-encoded antibody response\u2014can profoundly shape humoral imprinting severity. Accordingly, we constructed a human IGHV3-53 knock-in mouse model that can accurately recapitulate human SARS-CoV-2 antibody landscape, providing a valuable tool for guiding future COVID-19 vaccine updates.","version":"1.2","doi":"10.64898/2025.12.18.694353","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.18.695048","pub_date":"2025-12-23","title":"Single-Particle Tracking and Positional Phenotyping Reveals Variant-Specific Early Checkpoints in SARS-CoV-2 Cell Entry","abstract":"SARS-CoV-2 entry is governed by Spike (S) protein-mediated engagement of ACE2 and subsequent activation of either plasma membrane fusion mediated by TMPRSS2 or endocytic uptake. Currently, most insights into these pathways come from bulk assays that obscure the fate of individual virions, thereby concealing intricate mechanistic details that can inform on therapeutic intervention strategies. Here, we applied single-particle fluorescence imaging to directly observe the early checkpoints of SARS-CoV-2 cell entry pathways and separate binding from internalization. Fluorescent virus-like particles (VLPs) pseudotyped with either G614 or Omicron BA.5 S protein variants were imaged on HEK293T-ACE2 (TMPRSS2-negative) and classified at the single-particle level as surface-interacting, crossing, or internal. At baseline, G614 VLPs show higher binding and a larger internalized share than BA.5 VLPs, revealing general divergence in early entry behavior between variants. A trivalent anti-S receptor-binding domain aptamer reduces G614 binding and lowers its internalization. Conversely, the aptamer does not block BA.5 VLP cell binding but increases its internalization efficiency. Pitstop 2, an inhibitor of clathrin-mediated endocytosis, causes no significant change in this observation window, consistent with early clathrin-sensitive events having already progressed. Quantification of trajectories reveals variant-specific mobility: BA.5 displays higher step length than G614, consistent with greater lateral scanning and surface retention. Together, these compact single-particle readouts expose variant-resolved early checkpoints in entry and provide a simple platform to test how ligands and pathway probes shift binding and internalization.","version":"1.2","doi":"10.64898/2025.12.18.695048","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.25.690589","pub_date":"2025-12-23","title":"Crystallographic Ensembles Reveal the Structural Basis of Binding Entropy in SARS-CoV2 Macrodomain","abstract":"Structure-based drug design has traditionally focused on optimizing static, enthalpic interactions between ligands and proteins or on displacing binding site solvent molecules to entropically favor binding. A potentially large contributor to binding thermodynamics is the difference in conformational entropy of the protein upon binding a ligand; however, this has been difficult to quantify especially in high throughput. Here, through multiconformer ensemble modeling of hundreds of ligand-bound SARS-CoV-2 Macrodomain (Mac1) X-ray structures, we show how ligand binding reorganizes both protein conformational entropy and water molecules. By applying an optimal transport\u2013based clustering algorithm, we show how specific protein\u2013ligand interactions patterns drive the magnitude and spatial redistribution of conformational entropy and solvent networks. Using isothermal titration calorimetry (ITC), we demonstrate a correlation between experimental binding thermodynamics and conformational entropy estimated from structural ensemble models, showing that increased conformational heterogeneity and a less connected hydrogen-bonded water network lead to more entropic binding. These results establish a framework for extracting thermodynamically meaningful information from crystallographic ensembles, enabling the integration of entropic effects into prospective, ensemble-aware drug discovery.","version":"1.2","doi":"10.1101/2025.11.25.690589","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.19.695600","pub_date":"2025-12-22","title":"Infection of 5xFAD mice with a mouse-adapted SARS-CoV-2 does not alter Alzheimer\u2019s disease neuropathology yet induces wide-spread changes in gene expression across diverse cell types","abstract":"Alzheimer\u2019s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia. It is characterized by cognitive decline and accumulation of amyloid beta (A\u03b2) plaques and neurofibrillary tangles. Accumulating evidence indicates that viral infection may worsen and/or increase development of established AD pathology. The COVID-19 pandemic has brought attention to the link between SARS-CoV-2 infection and neurologic conditions that vary in severity and duration, as well as the worsening of clinical symptoms in elderly people with dementia. To better understand potential mechanisms by which SARS-CoV-2 infection impacts AD neuropathology, aged 5xFAD and wildtype (WT) mice were intranasally infected with mouse-adapted SARS-CoV-2 (MA10). Intranasal infection of aged-matched (10-14 month) 5xFAD or wild type (WT) C57BL/6 mice with MA10 resulted in viral infection of the lungs that correlated with acute viral pneumonia characterized by lymphocyte inflammation and antiviral immune responses. Viral RNA was not detected within the central nervous system (CNS) of either WT or 5xFAD mice at days 7 or 21 post-infection (p.i.), nor were there signs of overt glial activation or neuroinflammation. There were no differences in either A\u03b2 plaque volume or number within the brains of MA10-infected 5xFAD mice compared to uninfected 5xFAD mice. However, bulk RNA sequencing and spatial transcriptomics revealed evidence of altered expression of genes associated with neuronal and glial dysfunction, as well as reduced expression of genes encoding adhesion molecules in vascular endothelial cells. Collectively, these findings demonstrate that MA10 infection did not affect A\u03b2 plaque size or numbers in 5xFAD mice, yet in both WT and 5xFAD mice, there were numerous down-stream effects on gene expression associated with resident CNS cell function.","version":"1.1","doi":"10.64898/2025.12.19.695600","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.19.695420","pub_date":"2025-12-22","title":"Identification of allo- or orthosteric VHH/single-domain antibodies that enhance or block pathogen binding to Siglec-1 on dendritic cells","abstract":"Siglec-1 (Sialoadhesin/CD169) is expressed on myeloid cells and plays a key role in host defences by capturing incoming sialylated-pathogens such as Campylobacter jejuni. However, binding to Siglec-1 has also been exploited by pathogens such as SARS-CoV-2 for further dissemination. Here we identified high-affinity VHHs also known as single-domain antibodies or Nanobodies that bind to Siglec-1 and allo- or orthosterically modulate ligand binding. VHH 2C2 was shown to bind directly to the ligand binding site of Siglec-1 and blocked binding of ganglioside liposomes and Campylobacter jejuni to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1+ DCs. VHH 2C2 also blocked SARS-CoV-2 binding of moDCs. In contrast, the VHHs 1B5 and 1C1 interacted with Siglec-1 outside the ligand binding site and acted as positive allosteric modulators of Siglec-1 ligand interactions, as was illustrated by increased ganglioside liposome and Campylobacter jejuni binding by moDCs. Our data suggests that mechanistically, the VHH 1B5 and 1C1 interfere with the cis-binding sialic acids present on the Siglec-1-expressing cell and thereby enhance trans-interactions with ligands. In conclusion, we have isolated VHH that enhance or block Siglec-1 ligand binding to a variety of sialylated-pathogens enabling further interrogation of Siglec-1 function. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH could potentially serve as broad-spectrum pathogen blocking agents.","version":"1.1","doi":"10.64898/2025.12.19.695420","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.17.694727","pub_date":"2025-12-19","title":"The pharmacokinetics, bio-distribution and therapeutic efficacy of a trimeric nanobody against SARS-CoV-2 in the Syrian golden hamster COVID-19 model","abstract":"The rapid emergence of SARS-CoV-2 prompted the development of anti-viral therapies and vaccines to combat the COVID-19 pandemic. The continuous emergence of variants of concern has necessitated the development of platforms that can be rapidly adapted. Nanobodies, offer advantages over conventional monoclonal antibodies, including low molecular weight, high antigen-binding affinity, enhanced tissue penetration, blood\u2013brain barrier permeability, and simplified production. Previous studies have demonstrated the efficacy of nanobodies against respiratory viruses such as SARS-CoV-2, respiratory syncytial virus, and influenza A virus in animal models. We previously reported the protective effects of nanobody trimers against SARS-CoV-2. However, pharmacokinetic and biodistribution data for nanobodies remain limited. To address this, we evaluated the efficacy, biodistribution and longevity of action of a nanobody trimer (A8) administered intranasally (IN) or intraperitoneally (IP) in Syrian golden hamsters. Our findings revealed that the A8 trimer reached peak concentrations shortly after administration and was subsequently cleared from the system via both routes. Importantly, early administration of A8 trimer reduced virus mediated weight loss and viral load compared with untreated controls supporting its potential as a therapeutic candidate for SARS-CoV-2 infection.","version":"1.1","doi":"10.64898/2025.12.17.694727","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.19.695342","pub_date":"2025-12-19","title":"An Oral Combination therapy against SARS-CoV-2 based on Synergistic Action of Auranofin and Remdesivir","abstract":"The combination of direct-acting and host-directed antivirals targeting SARS-CoV-2 represents an attractive treatment strategy to combat COVID-19. In our previous work, we showed that the FDA-approved anti-arthritis drug Auranofin restricts SARS-CoV-2 replication and pathology in an animal model. Here, we report that Auranofin inhibits SARS-CoV-2 by targeting viral entry and main protease (Mpro) activity without affecting viral transcription. Time-of-addition studies combined with functional assays of viral entry, protease activity, and cell\u2013cell fusion delineated its inhibitory effects at both early and late stages of the viral life cycle. Molecular docking and isothermal titration calorimetry analyses of Auranofin and the known Mpro inhibitor Nirmatrelvir indicated competitive binding within the Mpro active-site pocket. In addition, Auranofin attenuated NF-\u03baB-dependent signalling and suppressed proinflammatory cytokine production. Combination studies with SARS-CoV-2 targeting nucleoside analogues, revealed the strongest synergistic antiviral activity with remdesivir in vitro. Comparable synergy was observed between auranofin and GS-621763, the orally bioavailable derivative of remdesivir, and was further validated in a preclinical animal model. Collectively, these findings provide a rationale for the further development of auranofin-nucleoside analog combinations targeting SARS-CoV-2. This research has been supported by ICMR (IIRPIG-2023-0000978) and BIRAC grant (BT/CS0070/06/22) to ST. We acknowledge the infrastructure and research support provided to IISc by the Crypto Relief Fund, L&T Trust, DST-FIST program, Institute of Eminence Fund, Ministry of Education, and the DBT-IISc partnership program (Phase II). RN acknowledges DBT-RA fellowship, SK acknowledges PMRF fellowship, and RS acknowledges FICCI-PMRF fellowship. Multiple previous studies have provided evidence showing that auranofin is an antiviral agent targeting SARS-CoV-2 through a host-targeting mechanism, involving the inhibition of thioredoxin reductase and redox homeostasis. Mixed results have been reported regarding the effect of this drug on inhibiting virus-induced syncytia. A prior study from our lab demonstrated the drug\u2019s effectiveness in reducing SARS-CoV-2 viral loads in both cell and animal models. Our study firmly establishes the synergistic use of Auranofin and Remdesivir GS 621763 in the treatment of SARS-CoV-2 infection.","version":"1.1","doi":"10.64898/2025.12.19.695342","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.17.694980","pub_date":"2025-12-19","title":"Moremi Bio Agent: Using Neisseria meningitidis Reference Data For The Double Blinded Validation of A General Purpose Biology-Trained Reasoning Model for Pathogen and Antigen Discovery","abstract":"Antibodies serve as vital diagnostic and therapeutic agents due to their exceptional specificity toward antigenic targets. Mapping antibody\u2013antigen interactions is essential for understanding immune responses and developing vaccines or biologics. Traditional antigen identification relies on labor-intensive wet-lab techniques such as phage display, peptide microarrays, and ELISA, while computational methods employ sequence alignment, epitope mapping, and structure prediction. Despite progress, to our knowledge, no existing AI framework has demonstrated the ability to blindly inference\u2014predicting an antibody\u2019s antigen target solely from its amino acid sequence without prior biological context. This research employed Moremi Bio Nano, a general agentic reasoning large language model (LLM), to infer the antigen and pathogen targets of an anonymized monoclonal antibody sequence from Imperial College London. The model received only the VH and VL chain sequences and autonomously hypothesized, ranked, and validated probable targets. Of ten independent inference tests, four were completed successfully, with three correctly identifying the experimentally validated antigen and pathogen; with SARS-CoV-2 Spike RBD, Neisseria meningitidis fHbp v1.1, and SARS-CoV Spike emerging as the top 3-ranked candidates across both reranking strategies. Validation of the model\u2019s predictions with experimental wet-lab data confirmed its capacity for correct antigen inference, marking Moremi Bio Nano as a first-of-its-kind AI system demonstrating reasoning-driven antigen discover; complementing experimental immunology and advancing automated biological inference.","version":"1.1","doi":"10.64898/2025.12.17.694980","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.17.694833","pub_date":"2025-12-18","title":"Oncolytic Reovirus mediates innate-driven SARS-CoV-2 elimination in the absence of cell toxicity","abstract":"Interplay between type I interferon (IFN) driven innate responses and viral antagonism strongly influences SARS-CoV-2 transmission and the COVID-19 disease course. Hence, variant adaptation includes diminished induction of IFN stimulated genes (ISG) and/or evasion of their effector functions. Exogenous IFN treatment \u201crewires\u201d innate responses to drive virus elimination, yet therapeutic trials to date have been unremarkable. Resolving this paradox could translate to variant-agnostic innate immunotherapy. By contrast, oncolytic viruses (OV) exhibit profoundly attenuated innate antagonism, resulting in potent IFN responses despite the inherently immunosuppressive nature of tumour microenvironments. Moreover, OV only undergo lytic replication within innate-deficient malignant cells, and not in cells where sufficient innate responses exist. This, combined with previous studies showing that OV suppressed replication of underlying oncogenic viruses in tumours, we explored whether clinical grade oncolytic Orthoreovirus (Reo) superinfection could eliminate SARS-CoV-2 from immune-competent lung epithelial cell lines in the absence of toxicity. Reo exerted profound activation of innate responses, including when SARS-CoV-2 infection was already established, rewiring cells towards an antiviral state emulating that of Reo infection alone. Both intracellular and paracrine mechanisms induced ISG repertoires including multiple known anti-SARS-CoV-2 effectors, as well as others that remain unvalidated. Amongst these, we demonstrate the first direct evidence that MX2 and XAF1 restrict SARS-CoV-2 replication. Thus, with an excellent safety record, self-amplification, and respiratory tract tropism, we propose that Reo superinfection may provide a tractable alternative to recombinant cytokines for innate antiviral immunotherapy.","version":"1.2","doi":"10.64898/2025.12.17.694833","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.16.694738","pub_date":"2025-12-18","title":"\u03b2-Coronavirus Nsp6 hijacks host ER translocation machineries into viral replication centers","abstract":"\u03b2-coronaviruses evade host immune detection by replicating their genomes within double membrane vesicles (DMVs) derived from endoplasmic reticulum (ER) membranes. For example, SARS-CoV and CoV-2 encode three non-structural membrane proteins (Nsp 3, 4, and 6) which can remodel the ER to form DMVs. Here we test whether Nsps also function to recruit key host machineries required for viral replication and assembly within ER-derived DMVs. We use mouse hepatitis virus to study whether \u03b2-coronavirus Nsps coordinate ER remodeling with host machinery recruitment. We demonstrate that Nsp6 generates Nsp6-remodeled ER domains that sequester host ER insertases including the Sec61 translocon, EMC, and GEL complexes. FRAP and FLIP experiments confirm that Nsp6 domains remain continuous with the ER and do not restrict membrane protein diffusion, except for those insertases that are sequestered there by Nsp6. Together, these data demonstrate a dual role for Nsp6 in remodeling ER membranes and sequestering host translocation machinery away from the general ER and into DMVs.","version":"1.1","doi":"10.64898/2025.12.16.694738","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.15.694351","pub_date":"2025-12-16","title":"Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2\u2013induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome\u2013plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.","version":"1.1","doi":"10.64898/2025.12.15.694351","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.15.694478","pub_date":"2025-12-16","title":"Preclinical immunogenicity of the LP.8.1-adapted BNT162b2 COVID-19 vaccine","abstract":"SARS-CoV-2 evolution toward antigenically distinct lineages drives escape from host immunity. JN.1 lineage derivatives have recently dominated the global epidemiologic landscape. In preclinical models, an LP.8.1-adapted BNT162b2 vaccine elicited higher serum neutralizing antibody responses against contemporary, circulating JN.1 sublineages, including the currently dominant XFG, as compared to JN.1-, KP.2- and XEC-adapted vaccines. These findings supported the selection of an LP.8.1-adapted vaccine for the composition of the 2025-26 COVID-19 vaccine formula.","version":"1.1","doi":"10.64898/2025.12.15.694478","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.16.688752","pub_date":"2025-12-16","title":"Distinct Mechanisms for Inhibition of SARS-CoV-2 Main Protease: Dimerization Promoted by Peptidomimetic Inhibitors and Disrupted by Ebselen","abstract":"The SARS-CoV-2 main protease (Mpro) is a key target for antiviral drugs. Given its conserved sequence across coronaviruses and its essential role in viral replication, numerous inhibitors have been developed to target its active site. Mpro exists in equilibrium between the active dimer and inactive monomer, rendering the targeting of dimerization as a promising alternative strategy for drug development. This study addresses knowledge gaps regarding the monomer-dimer equilibrium and conformational changes of Mpro induced by inhibitor binding. We utilized 13C labeling combined with native mass spectrometry to assess how different types of inhibitors (including peptidomimetic inhibitors PF-07321332, PF-00835231, GC376, boceprevir; non-peptidomimetic inhibitors carmofur, ebselen and its analog MR6-31-2; and allosteric inhibitors AT7519 and pelitinib) influence the monomer-dimer equilibrium and subunit exchange of Mpro. Additionally, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to investigate the conformational dynamics of Mpro and its interactions with these inhibitors. Key findings revealed divergent mechanisms: peptidomimetic inhibitors significantly shifted the equilibrium towards the dimeric state, suppressing subunit exchange dynamics and rigidifying the dimer interface. In contrast, ebselen impaired the dimer form and increased the flexibility of the dimer interface. Notably, we identified a novel covalent binding site for ebselen at C300 by tandem mass spectrometry, with molecular dynamics simulations further indicating that this modification allosterically altered the hydrogen bond network of the Mpro dimer interface. Overall, this study reveals distinct inhibitory modes between peptidomimetic inhibitors and ebselen, highlighting the potential of targeting allosteric sites at the dimer interface for the design of next-generation Mpro inhibitors.","version":"1.2","doi":"10.1101/2025.11.16.688752","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.02.679942","pub_date":"2025-12-16","title":"Rapid discovery of antiviral targets through dimensionality reduction of genome-scale metabolic models","abstract":"The COVID-19 pandemic underscored the urgent need for rapid and broadly applicable strategies to identify antiviral targets against emerging pathogens. Conventional approaches, which rely on detailed viral characterization and large-scale drug screening, are too slow to address this challenge. Here, we introduce a transcriptome-based computational framework that integrates genome-scale metabolic models with dimensionality reduction to uncover host metabolic vulnerabilities that support viral replication. Applying this approach to bulk and single-cell RNA-seq data from HCoV-OC43\u2013infected cells and organoids identified oxidative phosphorylation as a key vulnerability, and pharmacological inhibition of complex I effectively curtailed viral replication. Extending the framework to SARS-CoV-2 and MERS-CoV revealed pyrimidine catabolism as a conserved antiviral pathway, with inhibition of its rate-limiting enzyme DPYD suppressing replication in organoid models. Re-analysis of SARS-CoV-2 patient metabolome data further confirmed elevated DPYD activity, underscoring its clinical relevance. Together, these findings establish a generalizable and rapid strategy for host-directed antiviral discovery, providing a foundation for precision therapeutics and pandemic preparedness. Host-directed antiviral therapies offer several advantages in antiviral research, but identifying key host factors poses a significant challenge. By integrating genome-scale metabolic models with single-gene knockout simulation and dimensionality reduction, we developed a computational framework based on single and bulk RNA-seq data that can systematically pinpoint host pathways whose downregulation is predicted to rewire virus-induced metabolic alterations. Applying this approach to multiple human coronaviruses reveals unique metabolic vulnerabilities, and we experimentally demonstrate that inhibiting these host metabolic pathways reduces viral replication. This framework provides a generalizable antiviral strategy to discern effective targets and can be further extended to investigate virus\u2013host metabolic interactions.","version":"1.2","doi":"10.1101/2025.10.02.679942","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.14.694192","pub_date":"2025-12-15","title":"Thymidine Phosphorylase Drives SARS-CoV-2 Spike Protein-Induced Lung Tumorigenesis","abstract":"COVID-19 survivors exhibit increased interstitial lung fibrosis, a known risk factor for lung cancer. We investigated whether SARS-CoV-2 Spike protein (SP)\u2013induced lung injury and elevated thymidine phosphorylase (TYMP) promote lung tumorigenesis. A TriNetX retrospective cohort analysis was combined with mechanistic studies in K18-hACE2TG and K18-hACE2TG/Tymp\u2212/\u2212 mice. Mice received intratracheal SP or control lysate followed by a urethane-induced lung cancer protocol. Lung injury, inflammation, thrombosis, fibrosis, STAT3 activation, cytokine profiles, and tumor burden were assessed. In vitro assays evaluated SP- and RBD-induced ACE2 processing. Propensity score\u2013matched TriNetX cohorts demonstrated an increased lung cancer risk after COVID-19, particularly among current smokers (n = 166,807; RR 1.22; HR 1.50; P < .001). In mice, SP induced acute lung injury, neutrophil infiltration, and microthrombi, which were reduced in TYMP-deficient mice. SP markedly increased lung tumor incidence and aggressiveness, whereas TYMP deficiency reduced tumor formation from 50% to 18% of lung lobes. SP-induced STAT3 upregulation and collagen deposition were significantly attenuated in K18-hACE2TG/Tymp\u2212/\u2212 mice. Cytokine profiling revealed a tumor-promoting, myeloid-dominant inflammatory milieu in K18-hACE2TG mice, in contrast to a T cell\u2013inflamed, anti-tumor profile in K18-hACE2TG/Tymp\u2212/\u2212 mice. SP and RBD altered ACE2 processing, generating lower\u2013molecular-weight fragments consistent with enhanced turnover. SARS-CoV-2 SP drives lung injury, fibrosis, and tumorigenesis through a TYMP-dependent mechanism involving STAT3 signaling and inflammatory microenvironment remodeling. COVID-19 significantly increases lung cancer risk, especially in current smokers. TYMP represents a potential therapeutic target to mitigate long-term pulmonary consequences of COVID-19.","version":"1.1","doi":"10.64898/2025.12.14.694192","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.12.694004","pub_date":"2025-12-15","title":"SARS-CoV-2 Intra-host Variation Shows Evidence of Transmission and Convergent Evolution in a University Surveillance Cohort","abstract":"Monitoring and understanding the transmission and evolution of SARS-CoV-2 remains a significant pub-lic health priority. Within-host genetic variation provides insight into viral evolution during infection and may help infer transmission events. In this study, we analyzed intrahost variation in SARS-CoV-2 genome sequences from Boston University\u2019s testing mandate. Focusing on intrahost single nucleotide variants (iSNVs), we inferred transmission events and assessed the selective forces shaping within-host viral evolution. To minimize false-positive iSNVs resulting from systematic biases, we implemented stringent data filtering and developed a heuristic to exclude contamination-derived artifacts arising from batched sequencing. We find that intrahost variation is limited and infrequently transmitted during acute infections, suggesting that shared iSNVs serve as highly specific but insensitive markers of transmission. We also observed incomplete purifying selection shaping within-host diversity, with the loci most affected changing among variants of concern. Finally, we identified a highly recurrent iSNV (G11083T) which may represent a site of positive selection. Our results highlight that within host variation provides insight towards within host pathogen evolution, in spite of a limited use towards genomic epidemiology. SARS-CoV-2 is the most extensively sequenced pathogen to date, yet much of its genomic data remains underutilized. Intrahost variation, in particular, is less studied than consensus-level variation, partly because most datasets lack technical sequencing replicates to control false-positive signals. Using genomic data from a university testing mandate and applying rigorous filtering to systematically minimize false-positive iSNVs in a data-driven manner, we obtain insights into SARS-CoV-2 evolution and transmission from intrahost variation. Our work underscores the potential to use existing, large-scale datasets to better understand pathogen evolution in situ. All sequence data have been deposited in the Sequence Read Archive (SRA) of the national center for biotechnology information (NCBI), under the project accession number PRJNA892225. All code is open-access and available in GitHub at: https://github.com/Leacavalli/Sars-cov-2-Intrahost-Variation. Any additional supporting data has been provided within the article.","version":"1.1","doi":"10.64898/2025.12.12.694004","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.12.694002","pub_date":"2025-12-15","title":"Development of a Bacterial Colorimetric Reporter System for Functional Screening of SARS-Cov-2 Main Protease Inhibitors Using Plant Preparations (juices): A Proof-of-Concept Study","abstract":"SARS-CoV-2 main protease (Mpro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on biochemical and computational approaches that lack the biological context of living cells, or costly mammalian-cell based models. Therefore, there remains a shortage of simple and biosafe cellular models enabling rapid, functional screening of potential Mpro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter system was developed that directly links SARS-CoV-2 Mpro activity to \u03b2-galactosidase function in Escherichia coli. To the best of our knowledge, the developed system represents the first bacterial colorimetric model for direct monitoring of SARS-CoV-2 Mpro inhibition in living cells. The system enables real-time visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective, biologically relevant, biosafe alternative to existing screening assays. Functional validation was performed using pomegranate juice as a representative natural inhibitor source. The system provides a simple, scalable, and biosafe platform for the primary screening of antiviral candidates, including phytochemicals, under standard laboratory conditions.","version":"1.1","doi":"10.64898/2025.12.12.694002","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.12.693972","pub_date":"2025-12-15","title":"Structural Dynamics and Allosteric Communication of a SARS-Like Bat Coronavirus Spike Glycoprotein","abstract":"SARS-like bat coronaviruses (CoVs) pose ongoing public health risks due to their zoonotic potential, making it important to understand the molecular pathways driving their evolution. We recently showed that SHC014-CoV can infect human cell lines in an ACE2-dependent manner after acquiring two spike ectodomain mutations (F294L and A835D). However, how the wild-type (WT) SHC014 spike differs dynamically from these mutants remains unclear. Here, we built fully glycosylated ectodomain models of WT and three mutants (F294L, A835D, and the double mutant, DM) and performed triplicate 1-\u03bcs all-atom molecular dynamics (MD) simulations for each variant. The two mutations exhibit epistasis, altering structural rearrangements relative to WT. Notably, the DM receptor binding domain (RBD) begins sampling the open conformation in our conventional MD. At the atomic level, the DM spike mitigates the dense negative packing introduced by A835D through a salt-bridge network, while F294L disrupts \u03c0-mediated interactions, together enhancing RBD opening propensity\u2014critical for viral entry. Increased flexibility of the subdomain-2 \u201c620-loop\u201d further modulates DM RBD openness. Dynamical network analysis identified three allosteric communication pathways. In WT and F294L, \u201cPathway 1\u201d forms the baseline route linking the 620-loop to the RBD, whereas in A835D and DM it extends to the FPPR, reshaping long-range communication. \u201cPathway 2\u201d is conserved across variants but is most prominent in WT and F294L. \u201cPathway 3\u201d appears only in A835D and DM, compensating for reduced communication along Pathway 2. Overall, this work provides an atomistic perspective on SHC014 molecular adaptation during host-to-host transmission and highlights mechanistic features that may inform future therapeutic and pandemic-preparedness efforts. Bat coronaviruses are an important source of future pandemic threats, but we still know little about how small genetic changes help them infect humans. In this study, we used detailed computer simulations to watch how tiny mutations in a bat coronavirus spike protein change its motion and shape. We found that two specific mutations work together to make the spike more likely to open\u2014a step required for the virus to enter human cells. By revealing how these molecular changes increase infection potential, our work helps improve understanding of coronavirus evolution and may guide strategies to prepare for future outbreaks.","version":"1.1","doi":"10.64898/2025.12.12.693972","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.10.693352","pub_date":"2025-12-13","title":"DISCO-seq: 3D single-cell transcriptomics of intact biological systems","abstract":"Single-cell transcriptomics has transformed tissue analysis, yet current methods struggle to integrate whole-tissue 3D architecture. Conventional techniques restrict molecular profiling to pre-selected 2D sections, losing systemic context and introducing anatomical bias by sampling less than 0.001% of a whole organism. To overcome these challenges, we developed DISCO-seq, a tissue-clearing chemistry that enables superior RNA accessibility compared to fresh or fixed tissues. DISCO-seq integrates whole-organ or organism 3D imaging with both untargeted and targeted transcriptomics, yielding high-quality RNA from cleared tissues comparable to standard samples. We demonstrate its versatility by investigating tumor heterogeneity in a syngeneic glioblastoma mouse model, using 3D imaging to identify spatially distinct microenvironments and characterize their unique transcriptomic signatures. Moreover, DISCO-seq enabled unbiased, whole-body mapping of SARS-CoV-2 S1 protein deposition in mice, followed by transcriptomic profiling of spatially defined niches. By bridgingmesoscale 3D imaging with single-cell transcriptomics, DISCO-seq establishes a paradigm for anatomically contextualized, hypothesis-free tissue interrogation. DISCO-seq integrates RNA-preserving tissue-clearing chemistry with whole-organ or organism 3D imaging, enabling anatomically unbiased single-cell transcriptomics. DISCO-seq yields RNA quality and transcriptome profiles equivalent to those obtained from matched fresh or fixed tissues. DISCO-seq identifies discrete glioblastoma microenvironments and defines their transcriptomic states within the intact brain. DISCO-seq enables whole-body mapping of SARS-CoV-2 S1 and uncovers region-specific immune and metabolic responses across anatomical niches. Supplementary movies can be seen at: http://discotechnologies.org/DISCO-seq/","version":"1.1","doi":"10.64898/2025.12.10.693352","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.11.693810","pub_date":"2025-12-12","title":"Humoral immunity induced by XEC monovalent vaccines against SARS-CoV-2 variants including XEC, LP.8.1, NB.1.8.1, XFG, and BA.3.2","abstract":"After the emergence of SARS-CoV-2 Omicron variant at the end of 2021, Omicron has highly diverged into various sublineages: e.g., BA.5 in 2022, XBB.1.5 in 2023, JN.1 in 2024. Currently, several JN.1 subvariants including XEC, LP.8.1, NB.1.8.1 and XFG are circulating worldwide. Additionally, recent studies show that BA.3.2, a descendant of Omicron BA.3, exhibits profound immune evasion potential. On December 5, 2025, BA.3.2, was designated a variant under monitoring by the WHO. To prevent COVID-19, several countries including Japan have continuously developed and approved variant-adapted vaccines: e.g., ancestral/BA.5 bivalent vaccine in 20223, XBB.1.5-based monovalent vaccine in 20234, JN.1-based monovalent vaccine in 2024, and LP.8.1-based monovalent vaccine in 2025. Since XEC was more prevalent than LP.8.1 at the beginning of 2025 in Japan, two Japanese pharmaceutical companies, Daiichi Sankyo and Meiji Seika Pharma, have produced XEC-based vaccines. Here, we investigated the antiviral immunity induced by XEC-based monovalent vaccines against recently circulating SARS-CoV-2 variants, as well as BA.3.2, in the Japanese population. To assess the neutralizing antibody response induced by XEC-based vaccines, we obtained sera from individuals who had been vaccinated with the XEC-based mRNA monovalent vaccine produced by Daiichi Sankyo (N=22) or the XEC-based self-amplifying replicon vaccine produced by Meiji Seika Pharma (N=20). We collected sera before and 3\u20134 weeks after vaccination and then performed a neutralization assay using these sera with lentivirus-based pseudoviruses harboring spike proteins of B.1.1, BA.5, XBB.1.5, JN.1, XEC, LP.8.1, NB.1.8.1, XFG and BA.3.2. The 50% neutralization titers of the sera against all variants tested were significantly increased in after vaccination in both Daiichi Sankyo cohort (2.1-fold to 11.9-fold, P<0.0001) and Meiji Seika Pharma cohort (1.4-fold to 3.9-fold, P<0.0005). Consistent with our recent study using the sera from LP.8.1-based vaccinees, the humoral immunity induced by XEC-based vaccines against JN.1 and its subvariants (XEC, LP.8.1, NB.1.8.1 and XFG) was greater than that against other variants (B.1.1, BA.5, XBB.1.5 and BA.3.2). Notably, our result showed that the XEC-based mRNA vaccine induces a stronger humoral immunity compared to the XEC-based replicon vaccine, and even the LP.8.1-adapted vaccines. However, it should be noted that our cohorts are relatively small and may include confounding factors that affect the results, such as age, sex, history of natural infection and vaccination status. Future investigations with larger cohorts are required to better understand this possibility. To compare the immune status induced by XEC- and LP.8.1-based vaccines, we analyzed the cross-neutralization induced by these vaccines using antigenic cartography. The antigenic map was depicted based on the 50% neutralization titers values obtained from this study and our recent study using LP.8.1-based vaccine sera. The cartography showed that the immune status induced by XEC-based vaccines was similar to that induced by LP.8.1-based vaccines against the nine SARS-CoV-2 variant antigens tested. In sum, our investigations, including the recent one8, suggest that all 4 of the JN.1 subvariants-based vaccines that we tested induce profound humoral immunity against a broad range of SARS-CoV-2 variants.","version":"1.1","doi":"10.64898/2025.12.11.693810","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.10.693372","pub_date":"2025-12-12","title":"AI recognizes convergent somatic hypermutation signatures to allow the discovery of variant-resilient broadly neutralizing antibodies","abstract":"Elucidating the mechanisms by which broadly neutralizing antibodies (bnAbs) develop to confer durable immunity in humans is pivotal for the rational design of next-generation vaccines and therapeutics. VH3-53/3-66-encoded public antibodies are widely elicited in the population after the COVID-19 pandemic. Here, we isolated 15 VH3-53/3-66-encoded bnAbs from elite neutralizers who experienced sequential SARS-CoV-2 Omicron breakthrough infections. These bnAbs exhibit exceptional potency and breadth against circulating variants and, as members of a long-lived public antibody lineage, likely contribute to durable humoral protection. Genetic analysis of VH3-53/3-66-encoded bnAbs reveals a convergent somatic hypermutation pattern at seven positions in and around the CDR loops that emerges with repeated viral exposures and distinguishes them from non-bnAbs. Grafting these combined mutations onto clinically escaped antibodies broadens their breadth and restores neutralization activity. Structural analysis shows that these convergent mutations cooperatively remodel CDRs to bind and tolerate virus receptor-binding domain mutations. Using an AI-based antibody language model trained on data linking somatic hypermutations to binding affinity and neutralization, the model is able to identify mutational patterns predictive of breadth, enabling the discovery of a rare bnAb with protective activity against the latest variants from early-pandemic antibody repertoires. This demonstrates the feasibility of establishing an AI-empowered pipeline to identify mutation-tolerant bnAbs from early-pandemic antibody repertoires to fight fast-evolving newly introduced viruses.","version":"1.2","doi":"10.64898/2025.12.10.693372","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.08.692901","pub_date":"2025-12-08","title":"A Secreted Subunit SARS-CoV-2 RBD-CpE Yeast Oral Vaccine Induces an Adaptive Immune Response in BALB/c Mice","abstract":"Despite their differences, all the current COVID-19 vaccines need to be refrigerated and administered intramuscularly by a health care worker. They are also relatively expensive. These characteristics often make COVID-19 vaccine storage, distribution, and administration relatively complex, especially in low- and middle-income countries where refrigeration and healthcare workers are limited. To address these challenges, we are developing a COVID-19 oral vaccine utilizing the yeast Saccharomyces boulardii as a delivery platform. We have successfully engineered recombinant strains of Saccharomyces boulardii to express and secrete the Receptor Binding Domain (RBD) of the Spike protein from the original Wuhan-Hu-1 strain of the SARS- CoV-2 virus, fused to the C-terminal fragment of Clostridium perfringens enterotoxin (CpE). Animal trials suggest that our candidate secreted oral yeast vaccine can elicit detectable RBD- specific IgG and IgA, as well as an IFN-\u03b3 but not an IL-4 response, in BALB/c mice. As such, our data suggest that Saccharomyces boulardii remains a promising and novel vaccine delivery platform not only for COVID-19 but also for other infectious diseases.","version":"1.1","doi":"10.64898/2025.12.08.692901","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.08.692995","pub_date":"2025-12-08","title":"Conserved phosphorylatable residues in motif G of positive-stranded virus RdRps regulate polymerase activity and suggest targets for drug design","abstract":"RNA viruses rely on an RNA-dependent RNA polymerase (RdRp) to replicate their genome. RdRps share a conserved core replicase structure described as a right hand within which RNA replication occurs. RNA polymerases contain a series of conserved motifs (A-G) that are essential for catalysis. Motif G, located at the RNA entry channel, guides the incoming RNA into the catalytic centre and and holds it in place during catalysis. Although RdRp phosphorylation has been reported, it has been scarcely studied. In most studied cases, phosphomimetic mutations reduced viral replication. In this study, we identified Theiler\u2019s murine encephalomyelitis virus (TMEV) polymerase (3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 located in motif G are highly conserved in the sequences of picornavirus polymerases and in the structure of many positive-stranded virus polymerases, including nsp12 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using mutagenesis and reporter viruses, we show that phosphomimetic mutation of either residue abrogates viral replication, for both TMEV and SARS-CoV-2. Mutations of 3Dpol residues 109 and 110 into all other possible residues shows that, besides negatively charged phosphomimetic residues, bulky residues strongly inhibit replication, suggesting that phosphorylation inhibits polymerase activity by steric hindrance and/or through charge repulsion with RNA entering the catalytic core. Because these phosphorylatable residues are surface-exposed and conserved among viral polymerases, they represent promising targets for the rational design of broad-spectrum antiviral agents. RNA viruses require an RNA-dependent RNA polymerase to replicate their genome. We identified in Theiler\u2019s murine encephalomyelitis virus polymerase (TMEV 3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 are located in the entry channel of the polymerase, a region important for directing the RNA into the polymerase and locking it in place during catalysis. Incidentally, Thr109 and Ser110 are highly conserved in the sequences of picornavirus polymerases and in the structures of many positive-stranded virus polymerases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp12. Our study revealed that, in both TMEV 3Dpol and SARS-CoV-2 nsp12, mutation of either residue into a negatively charged amino acid that mimics phosphorylation abrogates viral replication, suggesting phosphorylation would block polymerase activity. As these phosphorylated residues are accessible and conserved, they provide important candidate targets for the design of antiviral molecules.","version":"1.1","doi":"10.64898/2025.12.08.692995","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.04.692282","pub_date":"2025-12-05","title":"The analysis of a large dataset of PCR-test results in the community reveals the impact of age, variants and vaccination status on SARS-CoV-2 viral dynamics","abstract":"The COVID-19 pandemic has shown the value of large-scale community PCR tests for epidemic surveillance, but the viral load measurements they provide have seldom been exploited to reconstruct within-host trajectories. Because these data are collected for diagnostic rather than research purposes, they are characterized by sparse longitudinal follow-up, heterogeneous sampling, and missing metadata, raising uncertainty about their usefulness to reconstruct with-host viral dynamics. We first conducted a simulation study to assess the feasibility of estimating the viral dynamics patterns from such datasets. Across multiple scenarios replicating realistic sampling patterns, we found that peak viral load and clearance time could be estimated with good accuracy, although uncertainty tended to be underestimated. Parameters driving early viral kinetics, e.g. incubation and proliferation time, were mostly estimated with poor precision, as most community tests are conducted after symptom onset, later in infection. We then applied this framework to a large dataset of 322,218 PCR tests associated with symptomatic SARS-CoV-2 infections in France between July 2021 and March 2022, encompassing both Delta-variant circulation and the emergence of first Omicron variants. We quantified the effects of age, vaccination, and variants on viral load trajectories. Age \u226565 years was consistently associated with longer clearance time, extending the duration of detectable viral load by 2 to 6 days. Vaccination shortened the clearance time by 2 to 4 days but had minimal impact on peak viral load. Infections by Omicron-variants were associated with a lower peak viral load and shorter clearance times compared with pre-Omicron (Delta) infections. Thus, community PCR tests can be leveraged to identify key parameters of viral dynamics. As multiplex PCR testing becomes increasingly widespread, establishing robust frameworks for data collection, sharing, and privacy protection will be essential to support the use of these data. In this study, we explored whether viral load data collected routinely in community laboratories can help us understand how factors like age, vaccination, and viral variant influence the course of infection. These data, collected for diagnostic purposes rather than research, are large but often incomplete, with most individuals tested only after the time of symptoms onset. We first used simulations to assess whether, despite these limitations, key aspects of viral dynamics can be reliably estimated. We then applied our approach to millions of test results collected in France during periods dominated by Delta and Omicron variants. We found that older age was consistently associated with longer infection duration, while vaccination reduced the time the virus remained detectable without affecting peak viral levels. Omicron infections were generally associated with lower peak viral load and somewhat faster clearance than pre-Omicron infections. Overall, our work demonstrates that community testing data, can provide valuable insights into viral dynamics and help monitor the effects of new variants or interventions. As multiplex tests become more common, facilitating and improving data collection, sharing, and privacy protection will be essential to make the most of these resources in future outbreaks.","version":"1.1","doi":"10.64898/2025.12.04.692282","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.21.649858","pub_date":"2025-12-05","title":"A flaw in using pre-trained pLLMs in protein-protein interaction inference models","abstract":"With the growing pervasiveness of pre-trained protein large language models (pLLMs), pLLM-based methods are increasingly being put forward for the protein-protein interaction (PPI) inference task. Here, we identify and confirm that existing pre-trained pLLMs are a source of data leakage for the downstream PPI task. We characterize the extent of the data leakage problem by training and comparing small and efficient pLLMs on a dataset that controls for data leakage (\u201cstrict\u201d) with one that does not (\u201cnon-strict\u201d). While data leakage from pre-trained pLLMs cause measurable inflation of testing scores, we find that this does not necessarily extend to other, non-paired biological tasks such as protein keyword annotation. Further, we find no connection between the context-lengths of pLLMs and the performance of pLLM-based PPI inference methods on proteins with sequence lengths that surpass it. Furthermore, we show that pLLM-based and non-pLLM-based models fail to generalize in tasks such as prediction of the human-SARS-CoV-2 PPIs or the effect of point mutations on binding-affinities. This study demonstrates the importance of extending existing protocols for the evaluation of pLLM-based models applied to paired biological datasets and identifies areas of weakness of current pLLM models.","version":"1.2","doi":"10.1101/2025.04.21.649858","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.03.692161","pub_date":"2025-12-04","title":"Persistent CD4+ T cell functional deficits during recovery from prolonged symptomatic SARS-CoV-2 infection","abstract":"Symptoms of acute SARS-CoV-2 infection often resolve quickly but are sometimes associated with persistent immune dysfunction. The factors that predispose individuals to compromised immune function have not been well defined. We investigated CD4+ T cell phenotype and function in a small cohort of individuals who recovered from mild to moderate SARS-CoV-2 infection without hospitalization and were divided into short or prolonged symptom duration groups. Five individuals with prolonged symptom duration showed marked downregulation of CD4 on CD3+CD8\u2212 T cells (CD4low group) and a poor response to TCR stimulation with the superantigen Staphylococcal enterotoxin B (SEB), as shown by weak upregulation of the activation markers CD134 and CD69. CD4 surface intensities recovered to normal levels in four of these individuals within 3-12 months. Selected cytokines (IL-1RA, IL-7, and VEGF) were elevated in individuals with low CD4, but plasma levels of anti-S1 IgG did not correlate with CD4 defects. Bulk RNA sequencing of unstimulated and SEB-treated CD3+CD8\u2212 T cells revealed a >50% reduction in the number of differentially expressed genes in the CD4low group compared to the same individuals after CD4 levels were recovered and a healthy control group. Upstream regulator analysis of differentially expressed genes in unstimulated CD4low cells suggested a response to IFN, while SEB-stimulated CD4low cells showed reduced functionality of IL-2, CD28, and SATB1 regulated pathways. In summary, prolonged symptomatic recovery from SARS-CoV-2 infection was associated with a global CD4+ T cell response defect, defined by low surface CD4 expression, evidence of IFN signaling, and defective T cell activation.","version":"1.1","doi":"10.64898/2025.12.03.692161","journal":"bioRxiv","score":null},{"id":"10.64898/2025.12.01.690145","pub_date":"2025-12-02","title":"Development and structure-guided characterization of a novel ACE2-binding macrocyclic peptide","abstract":"Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation, and the main protein receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63, developed using mRNA display, with high ACE2-binding affinity. The peptide was characterized in vitro in terms of purity, stability, hydrophilicity and ACE2 binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 \u00c5 resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the wide catalytic cleft of the ACE2 peptidase domain, with no direct contact to the conserved zinc ion site. WJL-63 residues Q4, R7, R11 and R14 anchor the peptide deep inside the binding pocket. The opposite edges of the peptide were found to be in contact with subdomain 1 and subdomain 2 of the peptidase domain. This upright binding mode requires an open ACE2 conformation, in contrast to small molecule carboxypeptidase inhibitors, which typically bind to the closed conformation of the enzyme. As a consequence of the open conformation binding mode, the front edge of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells on which it revealed relatively strong binding with a KD value of 90 \u00b1 28 nM. WJL-63 provides a strong basis for the development of new classes of compounds for the modulation of ACE2 conformation, and for the development of imaging agents for the visualization of ACE2, for example in fluorescence or electron microscopy, or positron emission tomography (PET) imaging.","version":"1.1","doi":"10.64898/2025.12.01.690145","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.28.691153","pub_date":"2025-12-02","title":"Biodiversity databases as underutilized resources for pathogen discovery: a quantitative synthesis of bat and rodent tissue collections in natural history museums","abstract":"Zoonotic spillovers are becoming increasingly frequent, and the devastating effects of the SARS-CoV-2 pandemic demonstrate our continued inability to combat their consequences effectively. Natural history museums can enhance the study of zoonoses by serving as valuable resources for understanding the ecology and evolutionary history of pathogens and their wildlife hosts. Despite the growing interest in the role museums can play in pathobiology research and zoonotic risk assessment, there remains a lack of centralized resources for locating tissue samples that may be leveraged for pathogen discovery. Using the world\u2019s most significant global aggregator of museum specimens, The Global Biodiversity Information Facility (GBIF), we examined how such tools could be adopted to identify specimens that might be sources of viral genetic material. Focusing on tissue samples from the mammalian orders Rodentia and Chiroptera, speciose taxa that host a high diversity of known zoonotic viruses, we examined temporal, spatial, and taxonomic gaps and patterns in the available tissue samples. Our analyses reveal a heavy bias toward tissue samples collected from the Americas (and consequently, taxonomic groups found in the Americas), with most collected samples housed in North American institutions. This limits the scope of future pathogen discovery efforts and presents a barrier to pandemic preparedness in the Global South. We also examine gaps in metadata quality (e.g., descriptions of preservation method and storage medium) and outline recommendations for GBIF to facilitate future biosurveillance projects and effectively incorporate natural history museums into One Health disease research. The SARS-CoV-2 pandemic has driven a greater research focus on understanding wildlife reservoirs of zoonotic pathogens. As a complement to field-based sampling of wildlife, natural history museum collections house millions of specimens that could be used by researchers to study pathogens quickly, safely, and cost-effectively. Digital databases of museum specimens and their associated tissue samples were originally created for biodiversity research, but these could be adapted to guide pathogen discovery research. Using the largest of these databases, The Global Biodiversity Information Facility, we explored tissue samples from rodents and bats, which have been shown to carry a disproportionate number of zoonotic pathogens. We searched for keywords that would indicate the presence of relevant tissue samples and then mapped the results visually. This revealed disproportionate distributions of tissue samples across time, space, and taxonomy. Our study is the first attempt to assess how a biodiversity database can be used for this novel purpose. We suggest changes that would improve these databases for zoonotic disease research.","version":"1.2","doi":"10.1101/2025.11.28.691153","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.28.691177","pub_date":"2025-12-01","title":"Development of a Recombinant Single-Cycle Influenza Viral Vector as an Intranasal Vaccine against SARS-CoV-2 and SARS-like Betacoronaviruses","abstract":"The COVID-19 pandemic has demonstrated the detrimental potential of zoonotic coronavirus transmission to human populations. Effective vaccines capable of eliciting immunity to SARS-CoV-2 have been pivotal in mitigating the spread of the virus. In this study, we describe the generation of a non-replicating pseudotyped influenza A virus (S-FLU), where the native haemagglutinin (HA) sequence is replaced with the coding sequence of either a membrane-anchored form (TM) or secretory form (Sec) of the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 Wuhan (S-RBD Wuhan). We showed that both S-RBD-TM and S-RBD-Sec viruses can be generated via reverse genetics and grown to high titre. Intranasal immunisation in mice with S-RBD-TM elicits robust serum binding and neutralisation activity against SARS-CoV-2, superior to S-RBD-Sec. Furthermore, we demonstrate that a heterologous prime-boost immunisation regime in mice with S-RBD-TM Wuhan and S-RBD-TM BM48-31 (a distant Clade 3 SARS-like betacoronavirus (sarbecovirus)) increases antibody breadth against mismatched sarbecoviruses compared to homologous prime-boost with S-RBD-TM Wuhan. These results suggest that S-RBD-TM is a promising intranasal vaccine candidate against SARS-CoV-2 and may offer potential as a broadly protective sarbecovirus vaccine.","version":"1.1","doi":"10.1101/2025.11.28.691177","journal":"bioRxiv","score":null},{"id":"10.1101/2025.12.01.691535","pub_date":"2025-12-01","title":"SARS-CoV-2 Spike Peptides Trigger Nociceptive Responses Through Spinal TLR4 Pathways","abstract":"COVID-19 has affected over 700 million people worldwide, with a significant portion of the population experiencing severe pulmonary and circulatory complications, often accompanied by symptoms such as prostration and pain. Moreover, the manifestation of these symptoms and other COVID-19-related complications varies depending on viral mutations, particularly those occurring in the spike (S) protein of SARS-CoV-2. Therefore, the present study aimed to investigate the effects of three different S proteins on nociceptive threshold, as well as the spinal involvement of TLR4 and microglia in this process. Male C57BL/6 mice received intrathecal administration of three synthetic peptides (PSPD2001, PSPD2002 and PSPD2003) derived from the SARS-CoV-2 S protein or saline. Nociceptive threshold was assessed using the von Frey filament test before and after peptide administration. The spinal involvement of Toll-like receptor 4 (TLR4), microglia, p38 MAPK, and NF-\u03baB was evaluated using specific antagonists and inhibitors. mRNA expression of TLR4 was assessed by RT-PCR, pro-inflammatory cytokine levels by ELISA, and microglial activation in the dorsal horn of the spinal cord was analyzed by immunofluorescence in wild-type, CX3CR1GFP\u25a1/\u25a1, and TLR4\u25a1/\u25a1 mice. In addition, molecular dynamics analysis was performed to assess the temporal stability of the PSPD2003\u2013TLR4 complex. Pharmacological data demonstrated that the peptides induced nociception involving TLR4, microglia, p38 MAPK, and NF-\u03baB. Notably, PSPD2003 increased TLR4 mRNA expression and elevated TNF-\u03b1 and IL-6 levels in the spinal cord. PSPD2003 also enhanced microglial activation in the spinal cord, which was abolished in TLR4\u25a1/\u25a1 mice. Molecular dynamics analysis results robustly demonstrate that PSPD2003 forms a stable and functionally relevant complex with TLR4. These findings suggest that SARS-CoV-2 S protein-derived peptides contribute to pain during COVID-19 infection, with spinal TLR4 and microglia playing key roles in this process.","version":"1.1","doi":"10.1101/2025.12.01.691535","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.30.691454","pub_date":"2025-12-01","title":"IgG-Bridging\u2013Seeded Synergistic Aggregation of SARS-CoV-2 Spikes Underlies Potent Neutralization by A Low-Affinity Antibody","abstract":"Mechanistic studies of viral neutralization typically prioritize high-affinity antibodies, relegating low-affinity binders to the sidelines. We report P5-1C8, a Class 1 SARS-CoV-2 antibody that exemplifies this underexplored \u201clow-affinity yet high-potency\u201d phenotype, retaining strong neutralization of Omicron JN.1 despite markedly weakened trimer binding (KD = 225 nM; IC50 = 0.06 nM). Structural and biophysical analyses reveal that P5-1C8 engages WT and BA.1 spikes through canonical intra-spike bivalency, but with JN.1 it induces aggregation. Using virion-like nanoparticles displaying multiple spikes, we show that IgG remains bound with no detectable dissociation and triggers pronounced aggregation. Coarse-grained molecular dynamics delineate the stepwise pathway in which weak IgG-spike contacts seed aggregation via transient inter-spike bridging. Together, these findings establish the first mechanistic framework demonstrating how weak-binding antibodies can nonetheless achieve potent neutralization through higher-order aggregation, thereby expanding the conceptual landscape of antibody function and opening new directions for antibody evaluation and design. Low-affinity antibodies are frequently disregarded in discovery pipelines. This work reports P5-1C8, a Class 1 SARS-CoV-2 antibody with weak trimer binding (KD-to-IC50 > 3,700-fold) yet potent neutralization of Omicron JN.1. Structural, biophysical, functional and coarse-grained simulations collectively demonstrate that transient inter-spike IgG bridging seeds higher-order aggregation, which in turn drives neutralization and provides a mechanistic framework.","version":"1.1","doi":"10.1101/2025.11.30.691454","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.15.688605","pub_date":"2025-11-28","title":"Evolutionary rewiring of host metabolism and interferon signalling by SARS-CoV-2 variants","abstract":"SARS-CoV-2 variants differ in transmissibility and immune evasion, but their effects on host-cell metabolism and signalling remain less defined. Using integrated transcriptomic, phosphoproteomic, and amino acid profiling in primary nasal epithelial cells, we compared early and late host responses to pre-Omicron variants (Alpha, Beta), Delta, and Omicron subvariants (BA.1, BA.5). Pre-Omicron strains broadly suppressed antiviral interferon-stimulated gene expression and reprogrammed metabolism by reducing mitochondrial oxidative phosphorylation and \u03b2-oxidation. Delta infection was associated with extensive transcriptional and metabolic remodelling, characterised by activation of stress- and growth-related kinases and selective retention of biosynthetic amino acids, consistent with a host response to stress and viral modulation of interferon-associated signalling. In contrast, Omicron infection elicited a more restrained response dominated by cytokine and survival pathways, with limited metabolic activation and interferon suppression. Together, these findings suggest SARS-CoV-2 has progressively evolved toward a strategy that maintains efficient upper-airway replication while minimising epithelial stress and inflammation.","version":"1.3","doi":"10.1101/2025.11.15.688605","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.28.691101","pub_date":"2025-11-28","title":"Interaction and co-phase separation of SARS-CoV-2 nucleocapsid protein and human hnRNPA1 and its implications for viral life cycle","abstract":"The nucleocapsid (N) protein of SARS-CoV-2 is central to viral assembly and replication. It binds viral RNA to form a helical nucleocapsid and enabling genome packaging and release into host cells. Human hnRNPA1, one of the most abundant RNA-binding proteins in eukaryotes, regulates key aspects of RNA metabolism, including splicing, transcription, localisation, and transport. Here, we report a direct physical interaction between SARS-CoV-2 N protein and hnRNPA1 with moderate affinity (Kd \u223c 0.18 \u03bcM), primarily mediated through their intrinsically disordered regions. Furthermore, we found that these proteins co-phase separate in vitro and co-localise within stress granules in cells. In vivo studies reveal that hnRNPA1 suppresses viral replication, suggesting that the N protein \u2013 hnRNPA1 interaction plays an important role in modulating the viral life cycle.","version":"1.1","doi":"10.1101/2025.11.28.691101","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.19.608527","pub_date":"2025-11-28","title":"Enhanced metagenomics-enabled transmission inference with TRACS","abstract":"Coexisting strains of the same species within the human microbiota pose a substantial challenge to inferring the host-to-host transmission of both pathogenic and commensal microbes. Here, we present TRACS, a highly accurate algorithm for estimating genetic distances between strains at the level of individual SNPs, which is robust to intra-species diversity within the host. Analysis of well-characterised Faecal Microbiota Transplantation datasets, along with extensive simulations, demonstrates that TRACS substantially outperforms existing strain aware transmission inference methods. We use TRACS to infer transmission networks in patients colonised with multiple strains, including SARS-CoV-2 amplicon sequencing data from UK hospitals, deep population sequencing data of Streptococcus pneumoniae and single-cell genome sequencing data from malaria patients infected with Plasmodium falciparum. Applying TRACS to gut metagenomic samples from a large cohort of 176 mothers and 1,288 infants born in UK hospitals revealed species-specific transmission rates between mothers and their infants. Notably, TRACS identified increased persistence of Bifidobacterium breve in infants, a finding missed by previous analyses due to the presence of multiple strains.","version":"1.2","doi":"10.1101/2024.08.19.608527","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.25.690547","pub_date":"2025-11-26","title":"Sustained Humoral Activation through self-amplifying mRNA Vaccination Enhances Longitudinal Antibody Function in a Phase III Trial","abstract":"Sustained and functional antibody responses to respiratory pathogens through vaccination is critical for global public health. The development and deployment of mRNA vaccines during the coronavirus disease 2019 (COVID19) pandemic was a landmark achievement in modern medicine and ushered in a new age of vaccine innovation. The mRNA-based vaccines elicited strong antibody responses, both neutralizing and extra-neutralizing, against the viral Spike protein. The antibody levels waned with time since vaccination, and that coupled with the antigenic drift of the virus prompted updates to the mRNA vaccine composition and evaluation of other mRNA modalities. Self-amplifying mRNA (sa-mRNA) vaccines such as ARCT-154 can prolong antigen production and durability of humoral immune response post-immunization, and can thus be administered at a lower dose. How this translates into the overall humoral architecture compared to that shaped by conventional mRNA vaccinations, however, is unclear. Here, we analyze serum-based antibody responses from a recent Phase III trial comparing humoral responses elicited by ARCT-154 and mRNA BNT162B2. All participants had received three doses of mRNA COVID-19 vaccines and were randomized to receive a booster dose of ARCT-154 or BNT162B2. Primary outcomes were to quantify waning responses against ancestral/wild type SARS-CoV-2 Spike (WT Spike) and a panel of antigenically SARS-CoV-2 variants Spikes. Through a systems serology approach, we identified that the sa-mRNA vaccine ARCT-154 elicited a unique antibody response compared to BNT162B2 defined by a sustained, activating profile to the vaccine-encoded Spike protein and a broad spectrum of drifted Spikes. Notably, potently activating Fc\u03b3RIIIA-binding antibodies showed a sustained stimulation in the ARCT-154-treatment arm, and this translated to an enhanced natural killer (NK) cell activation. The NK-activation through ARCT-154 was present for both target WT Spike and the antigenically drifted BA.5 Spike, which was the predominant form of SARS-CoV-2 during the observation period. Our results support a model whereby prolonged antigen expression and presentation moves immune profiles towards activating phenotypes with broad antigenic coverage.","version":"1.1","doi":"10.1101/2025.11.25.690547","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.08.544212","pub_date":"2025-11-25","title":"A Pandemic-Scale Ancestral Recombination Graph for SARS-CoV-2","abstract":"Millions of SARS-CoV-2 genome sequences were collected during the COVID-19 pandemic, forming a dataset of unprecedented richness. Estimated genealogies are fundamental to understanding this ocean of data and form the primary input to many downstream analyses. A basic assumption of methods to infer genealogies from viral genetic data is that recombination is negligible and the genealogy is a tree. However, recombinant lineages have risen to global prevalence, and simple tree representations are therefore incomplete and potentially misleading. We present sc2ts, a method to infer reticulate genealogies as an Ancestral Recombination Graph (ARG) in real time at pandemic scale. We infer an ARG for 2.48 million SARS-CoV-2 genomes, which leverages the widely used tskit software ecosystem to support further analyses and visualisation. This rich and validated resource clarifies the relationships among recombinant lineages, quantifies the rate of recombination over time, and provides a lower bound on detectable recombination.","version":"1.3","doi":"10.1101/2023.06.08.544212","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.22.689758","pub_date":"2025-11-24","title":"Key residues in SARS-CoV-2 NSP3 hyper variable region are necessary to modulate early stress granule activity","abstract":"Antagonism of the host responses that limits viral replication is critical to the success of infection. Recently, we identified that the hypervariable region (HVR) of SARS-CoV-2 NSP3 binds to FXR1 and disrupts stress granule formation during the early stages of infection. Despite variation across the rest of the HVR, a 20-amino acid region, highly conserved in the Sarbecovirus family, is required for NSP3-FXR1 binding, but the critical residues remained unresolved. In this study, we explore the individual residues in NSP3 driving FXR1 binding and determine their impact on viral replication, pathogenesis, and stress granule formation. Our results indicate that the tyrosine at position 138 (Y138) and a phenylalanine at position 145 (F145) are required for FXR1 binding and affinity. Using reverse genetics, we showed mutating NSP3 Y138A/F145A (YF mutant) reduced viral replication in vitro and in vivo. Importantly, we demonstrate that attenuation is not due to differential type I interferon responses, but rather loss of stress granule control by the NSP3 mutant as compared to WT. Together, our findings demonstrate the importance of Y138 and F145 within the NSP3-HVR in regulating stress granule formation at the early times post infection. Stress granules play a key role in host-antiviral defenses and viruses have developed strategies to antagonize their activity. For SARS-CoV-2, the virus has two proteins that antagonize stress granules with NSP3 acting early and nucleocapsid acting at late times. Here, we show that key NSP3 residues Y138 and F145, conserved across the Sarbecovirus family, are necessary to bind FXR1 and disrupt its activity in stress granule formation. Mutating these residues results in attenuation of SARS-CoV-2 replication and induces stress granule formation at early times post infection. These results show the importance of these NSP3 residues in disrupting stress granule formation early and highlight multiple approaches SARS-CoV-2 uses to antagonize stress granule activation.","version":"1.1","doi":"10.1101/2025.11.22.689758","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.21.689747","pub_date":"2025-11-24","title":"Fragment-Based Development of NSP14 Exonuclease Inhibitors Confounded by Batch-to-Batch Variability","abstract":"Point mutations in the exonuclease (ExoN) site of non-structural protein 14 (NSP14) compromise the fitness of betacoronaviruses like SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy. However, there are no advanced compounds that inhibit NSP14\u2019s ExoN activity. Building upon the reported crystal structures of two fragments bound to NSP14\u2019s ExoN site, we identified a series of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN. However, upon resynthesis, we discovered that these putative leads were false positives, perhaps due to contaminating divalent cations which potently inhibit NSP14 ExoN. Our results provide a cautionary tale to the field about the sensitivity of NSP14 to divalent cations and illustrate the challenges associated with directly targeting the NSP14 ExoN site via fragment merging.","version":"1.1","doi":"10.1101/2025.11.21.689747","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.24.689034","pub_date":"2025-11-24","title":"Non-clinical safety of GRAd vector-based COVID-19 and HIV vaccines supports a platform regulatory approach","abstract":"The rapid development of safe and efficacious vaccines is often hindered by extensive, mandated non-clinical safety evaluations in animals. Here we present the complete non-clinical studies for two investigational vaccines based on the GRAd platform, a gorilla-derived group C adenoviral vector. When administered intramuscularly, GRAd-COV2 and GRAdHIVNE1 were well tolerated. Studies in rats and rabbits showed localized distribution and transient, non-adverse inflammatory responses, while successfully inducing expected immune responses to their respective antigens. Notably, both vaccines demonstrated a consistent safety profile despite transgene and backbone differences, comparable to other replication-defective adenoviral vectors. The established non-clinical safety profile of the GRAd platform provides a robust foundation for a more efficient and streamlined regulatory pathway. By leveraging this prior knowledge, future GRAd-based vaccines can achieve accelerated clinical development while fully adhering to the ethical principles of replacement, reduction, and refinement of animal use in research.","version":"1.1","doi":"10.1101/2025.11.24.689034","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.19.688845","pub_date":"2025-11-21","title":"Antigen-reactive CD4+ T cells after SARS-CoV-2 vaccination show divergent phenotypic states with or without restimulation bias","abstract":"Understanding antigen reactivity is crucial for characterizing CD4+ T helper (Th) cell fate, yet conventional peptide restimulation assays introduce phenotypic bias by activating cells ex vivo. However, by performing single-cell RNA and T cell receptor (TCR) sequencing on both antigen-stimulated and unstimulated samples, clonotypes can be tracked across conditions to identify antigen-reactive CD4+ T cells and simultaneously be assessed for their phenotypes in the unperturbed state. Using this \u2018reverse phenotyping\u2019 strategy, complemented by DNA-barcoded peptide-HLA (pHLA) class II multimers, we here tracked SARS-CoV-2 spike-reactive CD4+ T cells longitudinally after repeated mRNA vaccination. Without stimulation, reactive clones showed more Th-neutral features and less of an activated Th1-like state than would be assessed after antigen restimulation. Furthermore, transgenic TCR re-expression guided separation of antigen-specific from bystander-activated clones. These results uncover unbiased phenotypes of antigen-reactive CD4+ T cells and highlight that cell state classification can differ fundamentally when judged by phenotype versus function.","version":"1.1","doi":"10.1101/2025.11.19.688845","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.21.689519","pub_date":"2025-11-21","title":"Increased mannosylation of extracellular vesicles in Long COVID plasma provides a potential therapeutic target for Galanthus nivalis agglutinin (GNA) affinity resin","abstract":"There is no proven therapy for Long COVID, a post-acute illness characterized by a myriad of diverse symptoms including fatigue, dyspnea, and brain fog following SARS-CoV-2 infection. Extracellular vesicles (EVs) have been implicated in Long COVID pathogenesis by promoting viral and inflammatory signaling with their molecular cargo. In this study, we investigated whether EV abundance and glycome characteristics are altered in plasma from people with Long COVID and whether they can be targeted for removal using a glycan-binding affinity resin. Large (100\u2212500 nm) and small (40\u2212200 nm) EVs were isolated from plasma of participants in the post-acute phase of COVID-19 and analyzed by nanoparticle flow cytometry to measure concentration and glycan characteristics. Plasma of those with Long COVID contained elevated levels of both large and small EVs, and mannose-positive large EVs were significantly increased in comparison to recovered controls (p < 0.05). EV capture assays using Galanthus nivalis agglutinin (GNA) affinity resin demonstrated small EV removal positively correlated with mannose-positive EV abundance (r = 0.341, p < 0.05). NanoString analyses identified seven EV-associated miRNAs significantly depleted by GNA affinity resin treatment of plasma. PROGENy pathway inference of validated miRNA-mRNA interactions suggests these reductions may lead to a downregulation of JAK-STAT signaling and upregulation of Estrogen, VEGF, and PI3K pathways, resulting in a favorable rebalancing of immune and tissue-repair networks. These findings reveal specific glycome EV-miRNA cargo signatures in Long COVID and the potential clinical benefits of a lectin capture therapeutic strategy to remove these pathogenic vesicles and their inflammatory cargo.","version":"1.1","doi":"10.1101/2025.11.21.689519","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.18.689152","pub_date":"2025-11-20","title":"Humoral immunity induced by LP.8.1 monovalent vaccines against a broad range of SARS-CoV-2 variants including XEC, NB.1.8.1, XFG, and BA.3.2","abstract":"In the spring of 2025, multiple SARS-CoV-2 Omicron JN.1 subvariants were circulating, with LP.8.1 among the major variants. Pharmaceutical companies such as Pfizer/BioNTech, Moderna, and Novavax/Takeda adopted monovalent LP.8.1 for their 2025\u20132026 season vaccines, following recommendations issued by the WHO in May 2025. As of November 2025, SARS-CoV-2 variants including LP.8.1, XEC, NB.1.8.1, and XFG\u2014all designated as variants under monitoring\u2014were circulating. In terms of the spike gene, these recent variants as well as LP.8.1 are derived from JN.1. Moreover, BA.3.2, a BA.3 descendant with multiple mutations in the spike gene, has recently emerged and exhibits robust immune evasion. In Japan, the rollout of the LP.8.1-based vaccination has progressed since the end of September 2025. We previously reported the humoral immunity induced by the XBB.1.5-based monovalent vaccine in 2023 and the JN.1-based monovalent vaccine in 2024 in the Japanese population. Here, we investigated the efficiency of humoral immunity induced by two LP.8.1-based vaccines, the mRNA vaccine from Pfizer/BioNTech and the recombinant protein-based vaccine from Novavax/Takeda, in Japan. We performed neutralization assays using sera obtained from individuals who received the LP.8.1 mRNA vaccine from Pfizer/BioNTech (N=29) or the LP.8.1 recombinant protein vaccine from Novavax/Takeda (N=20) with pseudoviruses harboring spike proteins of B.1.1, BA.5, XBB.1.5, JN.1, LP.8.1, XEC, NB.1.8.1, XFG and BA.3.2. In both mRNA and protein-based vaccinee groups, the change in 50% neutralizing titer (NT50) against variants that were predominant before JN.1 (i.e., B.1.1, BA.5 and XBB.1.5) were smaller than those against JN.1 and its subvariants, including LP.8.1, XEC, NB.1.8.1 and XFG. Consistent with recent studies, neutralizing antibodies against BA.3.2 were induced by both vaccines. However, the induction fold change of BA.3.2 was smaller than those of JN.1 and its subvariants. Next, we tested humoral immune response of participants who received both JN.1-based vaccine in 2024 and LP.8.1-based vaccine in 2025 (N=15). Approximately a year after the JN.1-based vaccination, neutralization titer has waned against all variants tested. However, when we compare the NT50s of pre-vaccination sera between 2025 and 2024, those in 2025 against all variants except for B.1.1 were significantly higher than those last year. This suggests that the cross-neutralizing antibodies induced by JN.1-based vaccination were still maintained for a year. Furthermore, the neutralization abilities against the JN.1 sublineages tested and BA.3.2 were significantly reboosted after the LP.8.1-based vaccination. Our study shows immune boosting by the LP.8.1-based vaccine is effective in achieving cross-neutralization against a broad range of JN.1 sublineages in a JN.1-na\u00efve population and in recalling waning humoral immunity against these subvariants.","version":"1.1","doi":"10.1101/2025.11.18.689152","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.18.689118","pub_date":"2025-11-19","title":"Epistasis and background dependence in the evolution of Omicron variants of the SARS-CoV-2 Spike protein","abstract":"The rapid and repeated emergence of SARS-CoV-2 variants, particularly within the Omicron lineage, highlights the virus\u2019s remarkable ability to adapt under shifting immune pressures. A central molecular battleground in this evolutionary arms race is the spike receptor-binding domain (RBD), which must simultaneously maintain high affinity for the human ACE2 receptor while evading recognition by neutralizing antibodies. In this study, we construct and analyze multiple combinatorial libraries of SARS-CoV-2 RBD variants spanning major branches of Omicron evolution, including BA.1, BA.2, BA.5, XBB, and JN.1. Using high-throughput yeast display and binding assays, we map the effects of thousands of mutations and their combinations on ACE2 binding and antibody evasion. Our results reveal that while many RBD mutations exhibit additive effects, several mutations interact epistatically in a background-dependent manner. In particular, we identify synergistic interactions between BA.1 and BA.5 mutations that enhance antibody evasion, likely facilitating the rise of recombinant variants and convergent evolution. Conversely, some mutations show lineage-restricted compatibility, suggesting potential constraints on future evolutionary trajectories. Our comprehensive genotype-to-phenotype maps uncover both rugged and smooth regions of the viral fitness landscape and underscore the importance of epistasis in shaping SARS-CoV-2 evolution. These findings improve our ability to anticipate future viral variants and provide a framework for understanding how host-pathogen co-evolution unfolds at the molecular level.","version":"1.1","doi":"10.1101/2025.11.18.689118","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.19.689107","pub_date":"2025-11-19","title":"A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation","abstract":"The SARS-CoV-2 spike protein binds its receptor ACE2 to initiate target-cell infection. To engage ACE2, at least one of the three receptor-binding domains (RBDs) of the spike must adopt the up orientation. Here we describe S22, a potent, bioavailable, and non-toxic inhibitor of BA.2 and all subsequent Omicron variants. Cryo-EM analyses showed that S22 assembled as a trimer in a previously uncharacterized pocket of the spike apex, stabilizing all three RBDs in the down orientation, thereby preventing ACE2 association. Binding studies, especially those using mixed S22-sensitive and -resistant spikes, imply a cooperative assembly of three S22 molecules with three RBDs, resulting in an unusually slow S22 off-rate. Consistent with its slow dissociation and favorable pharmacokinetics, S22 suppressed viral replication 100-fold in the lungs of XBB.1.5-infected mice. Thus, S22 potently inhibits Omicron entry through a distinct mechanism whereby a small compound assembles cooperatively as a trimer to stabilize spike in an inactive conformation.","version":"1.1","doi":"10.1101/2025.11.19.689107","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.17.661973","pub_date":"2025-11-19","title":"Taste dysfunction in Long COVID","abstract":"Persistent taste dysfunction is frequently reported in individuals with post-acute sequelae of infection by SARS-CoV-2 (Long COVID). The mechanisms and pathological correlates underlying this taste dysfunction are unknown. This study investigates the underlying pathology in 28 non-hospitalized subjects diagnosed with COVID-19 and who experienced taste disturbances more than 12 months after testing positive for SARS-CoV-2. To objectively establish the nature of taste deficit, we used the WETT taste test, which quantifies the subject\u2019s ability to taste each of the five taste qualities: sweet, umami, bitter, sour, and salty. We then biopsied five to eight fungiform taste papillae (FP) in 20 of the 28 subjects. The FPs were analyzed histologically for overall taste bud structure and innervation, and by quantitative PCR (qPCR) for mRNA expression of markers for different taste receptor cells. Although all subjects had reported taste dysfunction, only three showed overall taste scores below the 10th percentile for a normal population adjusted for age and sex. However, 11 of the 28 subjects exhibited total loss of one or more taste qualities. Loss of PLC\u03b22-dependent taste qualities (sweet, umami, bitter) was significantly more common and was correlated with reduced expression of PLC\u03b22 and Tas1R3 mRNAs. Histological analysis revealed generally preserved taste bud structure and innervation, but with occasional disorganized taste buds and abnormal, isolated PLC\u03b22-positive cells in the epithelium. Our findings suggest long-term taste dysfunction after COVID-19 occurs rarely -- more frequently involving PLC\u03b22-dependent taste qualities -- but is not due to wholesale disruption of the taste periphery.","version":"1.2","doi":"10.1101/2025.07.17.661973","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.19.689216","pub_date":"2025-11-19","title":"Unconventional linkers facilitate potent stabilized coronavirus stem antibody responses following nanoparticle vaccination","abstract":"Vaccine technologies that protect against a range of related pathogens within viral families, such as human immunodeficiency virus (HIV), influenza, and coronaviruses (CoVs) represent the future of viral vaccine development. Towards developing broad-spectrum CoV and influenza vaccines, we and others previously designed and evaluated CoV and influenza stem antigens; but these elicited relatively weak and sub-neutralizing antibody (Ab) responses. Multivalent antigen display on nanoparticles (NPs) is an established strategy to enhance and shape immunogenicity. However, one facet of NP vaccines has been largely overlooked: the indispensable linker segment between the antigen and NP core. Here, we introduce de novo\u2013designed rigid (L2) and rarely used long flexible (L6) linkers to optimally display antigens on NPs, target occluded epitopes, and enhance cross-reactive Ab responses, using prefusion-stabilized Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S-2P) and stem (SS) antigens as prototype antigens. Antigenic characterization of L2-NPs confirmed enhanced Ab binding and exposure of cross-reactive epitopes compared with L6-NPs and soluble antigens. Immunization with SS-L2-NPs elicited broader, more potent cross-reactive Ab responses across the seven human-infecting CoVs and pandemic threat WIV1-CoV, whereas SS-L6-NPs induced stronger neutralizing Ab responses against MERS-CoV, SARS-CoV-2, and WIV1-CoV. Ab competition and systems serology analyses revealed that SS-L2-NPs elicit robust Fc-mediated effector functions. By improving CoV-targeting Ab functionality, these linker approaches have the potential to confer broad-spectrum CoV protection and represent a promising strategy against hypervariable influenza and HIV viruses \u2013 as well as other broad viral families with pandemic potential.","version":"1.1","doi":"10.1101/2025.11.19.689216","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.18.689095","pub_date":"2025-11-18","title":"Molecular mechanism by which SARS-CoV-2 Orf9b suppresses the Tom70-Hsp90 interaction to evade innate immunity","abstract":"The Tom70-Hsp90 interaction is critical for activating MAVS-mediated interferon (IFN) production. Upon RNA virus infection, cytosolic Hsp90 recruits key innate immune signaling proteins to MAVS on mitochondria through its interaction with Tom70. To evade this innate immune response, the SARS-CoV-2 protein Orf9b binds to Tom70, thereby disrupting the Tom70-Hsp90 interaction and suppressing IFN production. Despite its importance, the molecular mechanism underlying Orf9b-mediated inhibition of IFN signaling remains unclear. Here, using an integrative approach combining cryo-electron microscopy, 19F NMR spectroscopy, and isothermal titration calorimetry (ITC), we show that Orf9b inhibits Hsp90 binding to Tom70 through a bipartite mechanism. The helix and intrinsically disordered tail of Orf9b sterically block the access of two distinct structural units of Hsp90 to Tom70. We also find that Orf9b-mediated allosteric conformational changes in Tom70 do not contribute to the inhibition of the Hsp90 binding. Comprehensive structural, thermodynamic, and kinetic analyses further reveal that Orf9b primarily slows the association kinetics between Hsp90 and Tom70. Collectively, our results provide a high-resolution mechanistic framework for understanding Orf9b-mediated suppression of the host innate immune response.","version":"1.1","doi":"10.1101/2025.11.18.689095","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.18.688681","pub_date":"2025-11-18","title":"MAAD: Multidimensional Antiviral Antibody Database","abstract":"Antibodies have emerged as central components of therapeutic strategies against viral infectious diseases, functioning as key effectors in both prevention and treatment. While traditional antibody discovery has relied heavily on high-throughput screening, the field is now shifting toward rational antibody design, which requires integrative insights into sequence-structure-function relationships. However, the absence of a standardized and well-annotated antibody database integrating these multidimensional features hampers systematic exploration, cross-pathogen comparison, and rational antibody design. Here, we introduce a \u201cMultidimensional Antiviral Antibody Database\u201d (MAAD; http://www.xxx), a curated platform dedicated to antibody nanobody and single-chain variable fragment targeting three high-impact RNA virus families, Coronaviridae (SARS-CoV-1, SARS-CoV-2, MERS-CoV), Orthomyxoviridae (influenza virus), and Pneumoviridae (respiratory syncytial virus, human metapneumovirus), due to the large, high-quality datasets accumulated in recent years. MAAD further incorporates a suite of interactive analysis modules, including CDR annotation, similarity-based CDR3 sequence analysis, V/J gene usage profiling, sequence-based clustering and structure-based antigen-antibody interfaces residues with per-site entropy and mutation rate profiling. These features enable in-depth exploration of antibody sequence characteristics, thereby facilitating functional and structural insights for rational antibody design. Together, by bridging antibody sequence, structure and function, MAAD offers an open and standardized platform that advances comparative antiviral research and supports therapeutic antibody discovery.","version":"1.1","doi":"10.1101/2025.11.18.688681","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.22.613454","pub_date":"2025-11-18","title":"Accurate Multiple Sequence Alignment of Ultramassive Genome Sets","abstract":"With ever increasing sequencing efficiency, there is a pressing need to tackle a presently intractable task of accurate multiple sequence alignment (MSA) for ultramassive genome sets of millions and beyond. Additionally, efficient graph and probabilistic representations for downstream analysis are in dire lack. In light of these challenges, we develop a set of essentially linearly scalable algorithms, including that for constructing directed acyclic graphs, for training tiled profile hidden Markov models and for conducting alignment on such graphs. The power of these algorithms is demonstrated by both significantly improved accuracy and tremendous acceleration of SAR-CoV-2 MSA by three observed to five projected orders of magnitude for genome set sizes ranging from 40,000 to 4 million when compared with widely utilized MAFFT. Future application to other viral species and extension to more complex genomes will prove this algorithm set as a cornerstone for the coming era of ultramassive genome sets.","version":"1.2","doi":"10.1101/2024.09.22.613454","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.17.688702","pub_date":"2025-11-17","title":"pH-induced structural changes in SARS-CoV-2 spike variants","abstract":"pH critically influences SARS-CoV-2 infectivity and stability by, for example, triggering pH-dependent conformational changes in the spike glycoprotein that can facilitate membrane fusion and viral entry into host cells. Using the emerging dynamical nonequilibrium molecular dynamics (D-NEMD) simulations approach, we investigated how biologically relevant pH shifts affect the functional dynamics of the fully glycosylated spike from the ancestral, Delta, and Omicron BA.1 variants of concern. For this, over 1100 nonequilibrium simulations were conducted to capture the pH-induced structural and dynamic changes that occur following transitions from physiological to acidic and alkaline conditions, with the former mimicking the low pH environment within endosomes, and the latter the high pH conditions accessible to nascent exhaled aerosols. D-NEMD reveals that pH changes trigger distinct, variant-specific conformational responses in key regions of the spike, including the receptor binding domain (RBD), fusion peptide proximal region (FPPR), and C-terminal domain (CTD). The ancestral spike shows broad pH sensitivity, characterised by directional motions and region-specific structural rearrangements that depend on the pH conditions. The spike of the Delta variant displays increased reactivity to alkaline pH, potentially explaining its reduced stability in alkaline aerosols. The spike of Omicron BA.1, in contrast, responds strongly to acidic conditions with spontaneous RBD opening and pronounced structural rearrangements in the FPPR and CTD. This behaviour aligns with this variant\u2019s preference for endosomal entry and reduced reliance on TMPRSS2-mediated fusion. The Omicron BA.1 spike also shows increased resilience to alkaline pH, suggesting greater environmental stability. Our findings further emphasise the key role of glycans in spike activation, with glycan N234 stabilising the RBD \u201cup\u201d conformation during pH-induced transitions in Omicron under acidic conditions. These insights, together, highlight pH as a potential evolutionary pressure for SARS-CoV-2 and underscore the importance of glycosylation and environmental pH variability in shaping the behaviour of viral fusion proteins.","version":"1.1","doi":"10.1101/2025.11.17.688702","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.17.688795","pub_date":"2025-11-17","title":"Dynamics of interactions between three major respiratory pathogens in reconstituted human epithelium","abstract":"Respiratory viral coinfections pose a substantial global health burden, yet the underlying virus\u2013virus interactions remain incompletely understood. Here, we systematically examined the interplay among influenza A virus (IAV), SARS-CoV-2, and respiratory syncytial virus (RSV) using a reconstituted human airway epithelium model. We monitored viral replication dynamics and host transcriptional responses under both simultaneous and sequential infection conditions. Our results reveal complex, asymmetric interactions strongly influenced by infection timing and sequence. IAV exerted a pronounced inhibitory effect on SARS-CoV-2, mediated mainly by a robust type III interferon response. However, transient early enhancement of SARS-CoV-2 by IAV and subsequent bidirectional inhibition were also observed. Transcriptomic profiling identified coinfection-specific gene expression signatures enriched for metabolic and cell death pathways. In IAV\u2013RSV coinfections, IAV generally suppressed RSV replication; strikingly, prior RSV infection significantly enhanced subsequent IAV replication, potentially through RSV-induced cellular remodeling or syncytia formation. Overall, host responses to coinfection were integrated and non-additive, with distinct transcriptional programs suggesting activation of unique cellular pathways beyond canonical antiviral responses. Together, these findings highlight the pivotal role of innate immunity and the order of infection in determining the outcome of respiratory viral coinfections, providing mechanistic insights with implications for clinical management and epidemic modeling. Respiratory viruses often circulate simultaneously; however, their interactions within the same host remain poorly understood. We used a reconstituted human airway epithelium model to examine coinfections involving influenza A virus (IAV), SARS-CoV-2, and respiratory syncytial virus (RSV). Our findings reveal that these viruses do not simply compete or coexist: their interactions are dynamic, asymmetric, and highly dependent on the order in which infections occur. IAV generally suppresses SARS-CoV-2 replication through a strong type III interferon response, although brief early enhancement and later mutual inhibition can occur. Transcriptomic profiling reveals that coinfection triggers a complex array of host responses, including alterations in metabolic and cell-death pathways. In IAV/RSV coinfections, IAV consistently inhibits RSV; however, prior RSV infection markedly boosts subsequent IAV replication\u2014likely due to RSV-induced cellular remodeling, such as syncytia formation. These transcriptional signatures point to altered cell adhesion and motility as key features of such interactions. Overall, our results demonstrate that innate immunity and the sequence of infection shape coinfection outcomes in non-additive ways, driving distinct cellular responses beyond classical antiviral pathways. Understanding these mechanisms is essential for improving clinical management and guiding the development of targeted antiviral strategies.","version":"1.1","doi":"10.1101/2025.11.17.688795","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.10.561643","pub_date":"2025-11-16","title":"Native spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions","abstract":"Understanding the molecular properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for tackling future outbreaks. Current structural knowledge of the trimeric spike protein relies on truncated recombinant proteins and/or inactivated full-length forms, which may suffer from overstabilization. Here, we apply cryo-electron tomography (cryo-ET) at a Biosafety Level 3 facility to study the virus structure in its native, active state. The virus particles exhibit variable shapes and sizes with diffusible spikes, with the majority in typical prefusion conformations. Notably, we identified unprecedented, a transient open-trimer prefusion states, revealing a hidden flexibility with opened S1 conformation. Subtomogram averaging of the prefusion spikes indicates a loosely packed trimeric architecture that may facilitate the formation of open-trimer state. A cryo-EM map of recombinant Omicron BA.2.75 spike protein further confirms the presence of this loosely packed trimer as a minor conformational state. The observed dynamics uncover conserved cryptic regions that can be targeted for broadly effective vaccines. Structural analysis of active viruses profoundly impacts our understanding of the overlooked fusion mechanism and vaccine, antibody/drug design.","version":"1.3","doi":"10.1101/2023.10.10.561643","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.15.688659","pub_date":"2025-11-16","title":"Sequence-aware Prediction of Point Mutation-induced Effects on Protein-Protein Binding Affinity using Deep Learning","abstract":"Amino acid mutations may lead to significant changes in the binding affinity of protein complexes, thereby causing a series of cellular dysfunctions. Therefore, accurate prediction of protein-protein binding affinity changes (\u0394\u0394G) induced by amino acid mutations is of great importance for understanding protein-protein interactions (PPIs). In this study, we propose SAMAffinity, a protein sequence-aware deep learning architecture for predicting changes in protein-protein binding affinity caused by amino acid mutations. SAMAffinity predicts mutation-induced \u0394\u0394G by integrating multi-source sequence features, leveraging a Mutation-Site Identification (MSI) module to highlight local semantic shifts and a Binding-Interface Awareness (BIA) module to capture interaction changes. Benchmark evaluations on public datasets show that under the mutation-level data splitting strategy, SAMAffinity outperforms the state-of-the-art sequence-based method AttABseq by 33.3%, 72.3%, 31.8%, and 30.5% on S1131, S4169, S645, and M1101 datasets, respectively. Moreover, under the complex-level data splitting strategy, SAMAffinity surpasses the structure-based method MpbPPI by 22.9%, 22.7%, 5.0%, and 11.4% on the corresponding datasets. Beyond predictive accuracy, the strong consistency between the model\u2019s predicted distribution and natural amino-acid mutation tendencies indicates that SAMAffinity effectively captures the underlying mutational landscape shaped by intrinsic biochemical and evolutionary factors. Based on this capability, SAMAffinity demonstrated strong generalization in a study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases, suggesting its potential for optimizing therapeutic antibody design.","version":"1.1","doi":"10.1101/2025.11.15.688659","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.15.688481","pub_date":"2025-11-15","title":"Clade C MERS-CoV camel strains vary in protease utilization during viral entry","abstract":"MERS-CoV is a lethal pathogen with pandemic potential. Clade A and B MERS-CoV viruses have caused outbreaks in the Middle East since 2012 when they initially spilled over from camels to humans. Clade C viruses, however, are only found in camels across Africa and the spillover potential of these viruses seems to be lower than for clade A/B strains but remains to be fully understood. Here, we report that clade C spikes are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes and that most clade C spikes induce reduced syncytium formation. Additionally, we demonstrate that several East African clade C strains are less able to utilize the TMPRSS2-mediated pathway for viral entry in both cell lines and primary nasal epithelial cultures. We map the molecular basis of this reduced TMPRSS2 usage to the N-terminal domain (NTD) and subdomain 2 (SD2) of East African clade C MERS-CoV. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may underlie the reduced replication of East African clade C strains in humans, while the reduced replication of West African strains remains to be further investigated. Overall, we suggest that altered protease usage may contribute to differential tropism of East African clade C strains and indicate geographically distinct selection pressures on spike between MERS-CoV strains circulating in camels. Clade A/B MERS-CoV outbreaks have caused 957 deaths since the first spillover in 2012; meanwhile, Clade C MERS-CoV strains have been found in camels across Africa but have not been reported to cause outbreaks. Investigating why these viruses do not successfully transmit to humans will be key to understanding the pandemic potential of the African MERS-CoV camel reservoir. Our study indicates that clade C viruses exhibit less spike cleavage and that East African clade C isolates are less able to utilize the TMPRSS2-mediated pathway during viral entry of both human cell lines and primary nasal cells. Differences in viral entry pathways could alter cellular and organ tropism and inform our understanding of the pandemic potential of these viruses.","version":"1.1","doi":"10.1101/2025.11.15.688481","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.14.688437","pub_date":"2025-11-15","title":"An optimized contact map for G\u014dMartini 3 enabling conformational changes in protein assemblies","abstract":"Advances in structural biology, particularly cryo-electron microscopy, have enabled high-resolution characterization of complex biomolecular assemblies. These developments emphasize the need for computational approaches capable of describing biologically relevant conformational changes over extended timescales. G\u014dMartini 3 is a coarse-grained approach that demonstrates computational efficiency and versatility across several systems, from protein-binding membranes and soluble proteins to intrinsically disordered proteins, while preserving key physicochemical features. In this work, we introduce an optimized approach that integrates dynamic contact information from AA-MD simulations to refine the contact map in G\u014dMartini simulations. Specifically, we define high-frequency contacts (HFC), which reduce the number of original G\u014d contacts set by \u224820\u201330%, thereby improving the representation of conformational states beyond the original approach. Benchmarking different contact-selection criteria revealed that including intra- and interchain HFC captures structural flexibility and domain dynamics. The method was tested on three small soluble proteins and on the SARS-CoV-2 spike protein. Overall, the optimized contact map improves sampling efficiency and expands the accessible conformational landscape relative to the original G\u014dMartini 3 implementation. The full framework is available as an open-source resource for large-scale simulations of biomolecular assemblies.","version":"1.1","doi":"10.1101/2025.11.14.688437","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.12.688084","pub_date":"2025-11-12","title":"Mutation and ACE2-induced Allosteric Network Rewiring in Delta and Omicron SARS-CoV-2 Spike Proteins","abstract":"The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates viral entry by binding its receptor-binding domain (RBD) to the host receptor ACE2. Spike mutations in different variants have been experimentally shown to influence the rate of conformational transitions and alter viral infectivity. In parallel, both experimental and computational studies have reported the presence of long-range allosteric communication within the spike protein, suggesting that such mutations may also affect allosteric signaling pathways involved in viral function. A detailed understanding of the allosteric residue network is essential for rational antiviral drug design. In this study, we performed extensive atomistic molecular dynamics (MD) simulations of the spike proteins from the Delta and Omicron variants in both ACE2-bound and unbound states. By integrating linear mutual information (LMI) calculations and graph theory-based analysis, we delineated the long-range allosteric communication networks embedded within the spike protein. Betweenness centrality metrics enabled the identification of residues that act as key mediators of information flow. Notably, ACE2 binding markedly enhances allosteric coupling throughout the spike. We identified three key linkers, Link1 (NTD-RBD), Link2 (RBD-SD1), and Link3 (SD2-FP), as primary mediators of allosteric communication. Delta exhibits stronger signaling through Link1 and Link2, whereas Omicron redirects communication via Link3. While Delta maintains localized connectivity within the S1 domain but loses long-range contact with the S2 core, Omicron forms a broader yet weaker S1 network and establishes long-range coupling. We propose that the N856K and T547K mutations reshape the conformational landscape, reconfiguring allosteric communication pathways in Omicron. Furthermore, our analysis reveals distinct domain-level allosteric couplings in Delta and Omicron, pointing to variant-specific differences in fusogenicity and immune evasion. By mapping key allosteric sites and mutation-induced conformational shifts, our study may provide a framework for developing robust antiviral strategies resilient to future emerging SARS-CoV-2 variants. Receptor engagement at the RBD rewires long range allostery in the SARS CoV 2 spike. Using LMI and graph theory-based analyses, we map communication paths and pinpoint residues that govern spike opening and infection. ACE2 binding globally strengthens coupling, but energy and signals propagate along variant specific routes: Delta biases toward openness, channeling binding energy into RBD opening; Omicron remains less open, routing energy to the S2 core to prime fusion. Information flows through three linkers - Link1 (NTD to RBD), Link2 (RBD to SD1), and Link3 (SD2 to FP), with Delta emphasizing Link1/Link2 and Omicron shifting to Link3 and CD connections. We suggest that the N856K and T547K mutations reshape this landscape in Omicron.","version":"1.1","doi":"10.1101/2025.11.12.688084","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.06.687090","pub_date":"2025-11-10","title":"Integrated Antibody and DIA\u2013Based Plasma Proteomics Uncover Temporal Host Responses to SARS\u2013CoV\u20132 Infection","abstract":"This is a single\u2013case longitudinal study in which we monitored antibody responses and plasma proteome dynamics over a 310\u2013day period in a single individual who underwent COVID\u201319 vaccination and SARS\u2013CoV\u20132 infection. To evaluate COVID\u201319 antibody responses, human plasma samples collected over 310 days (August 18, 2021, to June 22, 2022) were analyzed by DIA\u2013LC\u2013MS/MS proteomics. This led to the acquisition of a quantitative protein profile and allowed validation of the temporal behavior of expressed proteins within the samples. A total of 1,502 proteins were identified from the plasma samples. Before vaccination, after the first dose, after the second dose, and during the period after contracting the novel coronavirus, protein quantification values during each event interval were compared. Despite minimal changes before and after COVID\u201319 vaccination, notable proteins exhibiting distinct high\u2013expression and low\u2013expression patterns were identified after SARS\u2013CoV\u20132 infection.","version":"1.1","doi":"10.1101/2025.11.06.687090","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.10.687698","pub_date":"2025-11-10","title":"Susceptibility of Human Neural Stem Cells to SARS-CoV-2: Entry Mechanisms and Glycocalyx Influence","abstract":"Previous work demonstrated that ectodermal cells exhibit greater susceptibility to SARS-CoV-2 infection than endodermal and mesodermal cells, raising concerns about potential vulnerability of the developing nervous system. We hypothesized that neural stem cells (NSCs) derived from human ectoderm are susceptible to SARS-CoV-2 infection. Using pseudotyped viral particles representing both wild-type and Omicron spike variants, we confirmed efficient infection of NSCs, with Omicron variants preferentially utilizing endocytosis-mediated entry. Inhibition of endocytosis with filipin and OcTMAB significantly reduced infection across all spike variants. Low glycosylation levels on NSCs facilitated viral entry, and enzymatic removal of glycosylation increased their susceptibility. Ectodermal infection by SARS-CoV-2 raises serious concern for potential teratogenic effects on the nervous system, possibly causing latent or subclinical anomalies not immediately evident at birth. Therefore, future clinical studies and long-term surveillance of infants and children exposed in utero are necessary to investigate and identify potential neurodevelopmental deficits.","version":"1.1","doi":"10.1101/2025.11.10.687698","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.03.673802","pub_date":"2025-11-10","title":"Coronavirus NSP14 Drives Internal m7G Modification to Rewire Host Splicing and Promote Viral Replication","abstract":"SARS-CoV-2, the causative agent of COVID-19, manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral nonstructural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of guanosine triphosphate (GTP) to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14\u2019s N7-methyltransferase (N7-MTase) domain and is enhanced by interaction with NSP10. Internal m7G modification by NSP14 predominantly occurs in the nucleus and is conserved across alpha-, beta- and gamma-coronaviruses. Mechanistically, we show that this RNA modification disrupts normal splicing by promoting intron retention and generating novel splice junctions, particularly in genes regulating genome stability, RNA metabolism and nuclear processes. Importantly, inhibition of m7G modification, through pharmacological targeting of NSP14 or RNA polymerase II, impairs SARS-CoV-2 replication, indicating that the virus hijacks host transcriptomic machinery to support infection. Our findings reveal a previously unrecognized epitranscriptomic mechanism by which coronaviruses reprogram host gene expression and suggest that NSP14-induced m7G modification is a potential therapeutic target.","version":"1.2","doi":"10.1101/2025.09.03.673802","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.07.687132","pub_date":"2025-11-07","title":"Immune transcriptomic differences in paediatric patients with SARS-CoV-2 compared to other lower respiratory tract infections","abstract":"The clinical severity of SARS-CoV-2 infection in children varies, with asymptomatic or mild illness predominating and a minority developing severe disease. Understanding the immunological responses that underlie severity of disease may guide future development of preventive or therapeutic interventions. This study compared whole blood transcriptomes of healthy children (N=127), children with mild/asymptomatic SARS-CoV-2 infection (N=71) and children hospitalised with severe SARS-COV-2 (N=41), lower respiratory tract illness (LRTI) or LRTI due to Respiratory Syncytial Virus (RSV-LRTI) (N=47) or Pulmonary Tuberculosis (PTB) (N=47). We identified >5000 differentially expressed genes including: OLFM4, IFI27, CBX7, IGF2BP3, OTOF for severe SARS-CoV-2; IFI27, OTOF, SIGLEC1, IFI44L and USP18 for RSV-LRTI, and MMP8, LTF, IGF2BP3, GPR84, CD177, C1QC and DEFA4 for PTB, at false discovery rate (FDR) <0.05. Pathway analysis identified enrichment for neutrophil degranulation, interferon gamma signalling, overexpression of ribosomal proteins and depletion of immune response in severe SARS-CoV-2 compared to healthy (SAR-COV-2 uninfected) children. Weighted Gene Co-expression Network Analysis (WGCNA) identified 10 correlated gene modules shared between LRTI showing similar underlying response mechanisms. Cellular decomposition analysis identified the depletion of 22 cell types in severe SARS-CoV-2, 16 for RSV-LRTI and 21 for PTB compared to healthy SARS-CoV-2 uninfected control children. We identified 82 genes important for discriminating asymptomatic/mild from severe SARS-CoV-2 including CBX7, TRAF1, ZNF324 and CASS4; 93 healthy from severe SARS-CoV-2 including RORC, CBX7, NR3C2, MID2 and ADAMTS2; 110 genes for RSV-LRTI and 95 for PTB children which can be used for future therapeutic targets.","version":"1.1","doi":"10.1101/2025.11.07.687132","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.07.687137","pub_date":"2025-11-07","title":"The TEAIV model: Extending the standard TEIV model to account for viral budding ramp up","abstract":"Understanding how viruses replicate and spread within a host is fundamental to predicting disease progression, timing, and dosage of effective therapeutic and prophylactic interventions. A target-cell limited approach is often used to model within-host viral kinetics to characterize disease infection dynamics. The standard target-cell limited model, the TEIV model, has been instrumental for understanding SARS-CoV-2 within-host viral kinetics, however, its core assumptions of instantaneous viral budding and exponentially distributed cell lifetimes oversimplify fundamental biological processes, potentially limiting predictive power. In this work, we consider a novel model approach, the TEAIV model: an extension to the TEIV model that considers an infected partially-productive cell state that ramps up through n stages to a fully productive state. We fit the TEAIV model to various SARS-CoV-2 viral load datasets, separated by hospitalized (severe infection) and non-hospitalized (mild infection) individual data, consider multiple different ramping functions, and compare to standard TEIV model fits. We further compare TEAIV and TEIV model fits with and without infected cell death incorporated in the eclipse (E) and partially infected (A) compartments and investigate best-fit frameworks up to 10 A states. We find that linear budding ramp-up dynamics minimizes the BIC (with \u0394BIC > 2) across all model formulations when the total number of eclipse and budding compartments exceeds three. Furthermore, we find that the inclusion or exclusion of cell death applied to eclipse or ramping compartments does not substantially affect this result. Finally, we analytically consider the most general TEAIV model and discuss future modelling considerations. Our results demonstrate that accounting for non-instantaneous viral budding provides a better fit to SARS-CoV-2 viral load data and establishes a foundation for more mechanistically informed within-host models.","version":"1.1","doi":"10.1101/2025.11.07.687137","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.04.686558","pub_date":"2025-11-05","title":"Temporal Dynamics of Antigen-Specific T Cell Expansion in Primary SARS-CoV-2 Infection","abstract":"Quantifying T cell response during primary infection in humans is crucial for understanding adaptive immunity. Leveraging a controlled human challenge to SARS-CoV-2, we characterized antigen-specific T cell response within and across individuals. Notably, individual clones reached similar maximum frequencies despite differences in the timing of their peak expansion. Mathematical modeling showed that this observation is consistent with precursor frequency, but not TCR signal strength, as the source of inter-clonal variability. Single-cell profiling revealed distinct temporal programs for CD4+ and CD8+ T cells, with CD4+ cells expanding earlier but contracting to a lower frequency. Clones with similar receptors, likely recognizing the same antigen, expanded at similar times. Together, these findings highlight how clone-intrinsic properties such as precursor frequency and lineage shape T cell clonal kinetics. These insights provide a quantitative framework for understanding T cell response in humans, with implications for vaccine design.","version":"1.1","doi":"10.1101/2025.11.04.686558","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.20.614179","pub_date":"2025-11-04","title":"Machine Learning Driven Simulations of the SARS-CoV-2 Fitness Landscape from Deep Mutational Scanning Experiments","abstract":"Predicting protein variant effects is a key challenge in preparing for pathogenic viral strains, understanding mutation-linked diseases, and designing new proteins. Protein sequence-structure-function relationships are difficult to model due to complex allosteric and epistatic effects. To investigate efficient modeling strategies, we trained supervised machine learning (ML) models with deep mutational scanning (DMS) libraries of SARS-CoV-2 receptor binding domain (RBD) sequences labeled with angiotensin converting enzyme 2 (ACE2) binding affinity. These models demonstrate superior performance predicting combinatorial mutation effects compared to adding or averaging the effects of point mutations and exhibit strong extrapolative performance ranking omicron variants when training only on wild type (WT) variants. We characterize the RBD fitness landscape combining ML with Markov Chain Monte Carlo simulations to predict evolutionary patterns from the WT sequence, and generate comparable sequence profiles to high fitness sequences in DMS data predicting mutations in unseen omicron variants. These models provide insight into the relationship between RBD sequence elements, and offer a new perspective on the use of DMS to predict emerging viral strains, which we anticipate will be applicable to other evolutionary prediction tasks. To facilitate application and future development of this strategy, we introduce Mavenets: https://github.com/SztainLab/mavenets.","version":"1.2","doi":"10.1101/2024.09.20.614179","journal":"bioRxiv","score":null},{"id":"10.1101/2025.11.01.684729","pub_date":"2025-11-03","title":"Caspase-11 Mediated Hyperinflammation Impairs CD8\u207a T Cell Immunity and Viral Clearance in Severe SARS-CoV-2 Infection","abstract":"Severe SARS-CoV-2 infection is characterized by lung hyperinflammation, impaired interferon responses, and defective T-cell activation, yet the molecular drivers of these immune dysregulations remain incompletely understood. Caspase-11 (CASP11), a key mediator of the non-canonical inflammasome, has been shown to mediate an innate hyperinflammatory response and cytokine release in a non-severe, non-lethal SARS-CoV-2 infection model. However, the role played by CASP11 in severe SARS-CoV-2 disease and how it impacts adaptive immunity is not identified. Here, we newly discover that CASP11 exacerbates severe SARS-CoV-2 pathogenesis by amplifying early innate immune responses while concurrently impairing antiviral CD8 T-cell immunity. Using global knockouts, reciprocal bone marrow chimeras, and phagocyte-monocyte system (PMS) cell-specific CASP11 deletion models, we show that CASP11 deletion in monocyte-derived cells reduces lung inflammation, enhances type I and II interferon signaling, and promotes robust virus-specific effector CD8\u207a T-cell response. This was associated with enhanced viral clearance and improved survival, even under lethal infection conditions. Importantly, CASP11 KO mice also exhibited faster resolution of post-viral inflammation, suggesting a role in long-term immune remodeling. These findings position CASP11 as a promising immunomodulatory target for acute and delayed manifestations of severe SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.11.01.684729","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.31.685956","pub_date":"2025-11-03","title":"Neutral Frustration Landscape Architecture of SARS-CoV-2 Spike-Antibody Interfaces Shapes Immune Evasion Mechanisms for Ultrapotent Neutralizing Antibodies and Determines Pathways of Viral Adaptation: Insights from Integrative Computational Approach","abstract":"The relentless evolution of SARS-CoV-2 underscores the urgent need to decipher the molecular principles that enable certain antibodies to maintain exceptional breadth and resilience against immune escape. In this study, we employ a multi-pronged computational framework integrating structural analysis, conformational dynamics, mutational scanning, MM-GBSA binding energetics, and conformational and mutational frustration profiling to dissect the mechanisms of ultrapotent neutralization by a cohort of broadly reactive Class 1 antibodies (BD55-1205, 19-77, ZCP4C9, ZCP3B4) and the Class 4/1 antibody ADG20. We reveal a unifying biophysical architecture: these antibodies bind via rigid, pre-configured interfaces that distribute binding energy across extensive epitopes through numerous suboptimal yet synergistic interactions, predominantly with backbone atoms and conserved side chains. This distributed redundancy enables tolerance to mutations at key sites like F456L or A475V without catastrophic loss of affinity. Mutational scanning identifies a hierarchical hotspot organization where primary hotspots (e.g., H505, Y501, Y489, Y421) which overlap with ACE2-contact residues and incur high fitness costs upon mutation are buffered by secondary hotspots (e.g., F456, L455) that are more permissive to variation. MM-GBSA energy decomposition confirms that van der Waals-driven hydrophobic packing dominates binding, with primary hotspots contributing disproportionately to affinity, while electrostatic networks provide auxiliary stabilization that mitigates mutational effects. Critically, both conformational and mutational frustration analyses demonstrate that immune escape hotspots reside in neutral-frustration landscape that permit mutational exploration without destabilizing the RBD, explaining the repeated emergence of convergent mutations across lineages. Our results establish that broad neutralization arises not from ultra-high-affinity anchors, but rather from strategic energy distribution across rigid, evolutionarily informed interfaces. By linking distributed binding, neutral frustration landscapes, and viral fitness constraints, this framework provides a predictive blueprint for designing next-generation therapeutics and vaccines capable of withstanding viral evolution.","version":"1.1","doi":"10.1101/2025.10.31.685956","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.31.685854","pub_date":"2025-11-03","title":"NSeqVerify: An Easy-to-Use Desktop Suite for Integrated NGS Data Analysis, from Raw Reads to Taxonomic Assignment","abstract":"The proliferation of next-generation sequencing (NGS) data has created a computational bottleneck, especially for researchers lacking specialized bioinformatics training. Standard analysis workflows require mastering multiple command-line tools, hindering exploratory data analysis and delaying scientific discovery. This work presents NSeqVerify, a new cross-platform, open-source desktop software developed in Java, designed to overcome these barriers. NSeqVerify implements a fully integrated genomic workflow within a single, intuitive graphical user interface (GUI). The suite includes: (1) a preprocessing module for quality control and filtering of FASTQ files; (2) a de novo assembler employing a sophisticated De Bruijn graph algorithm with an iterative multi-k-mer strategy to maximize contiguity; and (3) a taxonomic assignment module that automates BLAST searches against NCBI databases and displays the results in an easily interpretable tabular format. The tool was validated through controlled use cases, demonstrating its ability to accurately reconstruct reference viral genomes (HIV-1) and to deconvolute metagenomic mixtures (HIV-1 and SARS-CoV-2). The final test consisted of analyzing a real elephant fecal virome (SRA: SRR35776009), where NSeqVerify successfully assembled contigs \u2014 two of which overlapped and appeared to form a partial 1555 bp genome of a putative Smacovirus, enabling the identification of its capsid protein and the prediction of its 3D structure using AlphaFold. NSeqVerify democratizes NGS data analysis, providing a robust \u201call-in-one\u201d solution that empowers molecular biologists, students, and clinicians to perform end-to-end genomic analyses. The software is freely available under the GNU GPLv3 license at (https://github.com/roberto117343/NSeqVerify). roberto117343@gmail.com","version":"1.1","doi":"10.1101/2025.10.31.685854","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.12.664533","pub_date":"2025-11-01","title":"Predicting Future SARS-CoV-2 Mutations using Deep Learning","abstract":"SARS-CoV-2 continues to spread over the world steadily as opposed to many earlier estimations that it would disappear in less than two years. Even though SARS-CoV-2 vaccines have reduced the speed of the infection significantly, they could not fully stop it. On the contrary, the World Health Organization has recently published cautionary statements that infection counts are on the rise, and a huge wave is expected in winter. Vaccines mostly target specific regions of the virus. The high mutation rate of SARS-CoV-2 is one essential tool that the virus exploits to escape from the available vaccines. Therefore, researchers have been working on designing next-generation vaccines against the new variants of the virus. Nevertheless, SARS-CoV-2 acquires new mutations faster than we can adapt our vaccines due to long clinical trial periods. Hence, there is a need for computational tools that can predict future SARS-CoV-2 mutations before they even emerge. In this paper, we propose several deep-learning-based methods to estimate the possible future mutations in SARS-CoV-2 genome. We design and evaluate various ensemble and bagging architectures enriched with a large set of genomic, biochemical, and phylogenetic features. We evaluate our models on the GISAID data and demonstrate that the best-performing methods achieve an F1-Macro score of 0.78.","version":"1.2","doi":"10.1101/2025.07.12.664533","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.30.685644","pub_date":"2025-10-31","title":"Multimodal single-cell analysis following SARS-CoV-2 breakthrough infection reveals distinct B cell populations","abstract":"B cells are crucial to mediating durable immunity by generating high-affinity, antigen-specific antibodies and memory B cells upon pathogen exposure and re-exposure. While advances in single-cell technologies have expanded the understanding of B cell heterogeneity, the precise developmental trajectories and functional roles of memory B cell subsets remain poorly characterized. This study employs single-cell RNA sequencing to analyze B cell responses after SARS-CoV-2 re-exposure. The integration of B cell gene expression, surface protein profiles, antigen specificity, and BCR repertoire data from 12 individuals vaccinated against COVID-19 and subsequently infected with SARS-CoV-2 variants reveals dynamic transitions between B cell populations, from activated states to more quiescent phases. Furthermore, the profiling of key surface proteins and mRNA markers enhances the knowledge of B cell-mediated immunity following antigen exposure.","version":"1.1","doi":"10.1101/2025.10.30.685644","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.30.685646","pub_date":"2025-10-31","title":"In vitro Characterization of Peptidomimetic Proteolysis Targeting Chimera (PROTAC) as a Degrader of 3-Chymotrypsin-Like Protease (Mpro/3CLpro) against SARS-CoV-2","abstract":"The SARS-CoV-2 main protease (3CLpro) is a key target for antiviral development. We investigated FT235, a peptidomimetic PROTAC linking a GC-376 warhead to pomalidomide for targeted degradation. FT235 bound 3CLpro, inhibiting activity (IC50 = 21.2 \u00b5M), and reducing protease levels in cells. In vitro data showed no cytotoxicity up to 100 \u00b5M and variant-dependent antiviral activity, with increased potency in the presence of a P-gp inhibitor. These results support PROTAC-based antivirals as promising therapeutic candidates.","version":"1.1","doi":"10.1101/2025.10.30.685646","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.31.685727","pub_date":"2025-10-31","title":"Mechanism of substrate binding by the SARS-CoV-2 NiRAN domain and modulation of its activities during replication","abstract":"The SARS-CoV-2 Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain initiates viral genome capping by RNAylating nsp9 with the 5\u2032-pppA-end of the genome followed by GDP-dependent deRNAylation to form the core capped GpppA-genome. Additionally, it cycles nsp9 through NMPylation-deNMPylation to generate GpppN. It is unclear how the distinct substrates, 5\u2032-pppA-RNA and NTP, are bound, and how NiRAN balances RNAylation versus NMPylation. Earlier models proposed a common base-up pose for both the substrates. Here, structure-guided mutagenesis and reconstitution assays show that 5\u2032-pppA indeed binds base-up during RNAylation, revealing that nsp12-Asp711 confers adenine selectivity, whereas, NTP adopts a perpendicular base-out pose during NMPylation. NiRAN intrinsically favors NMPylation over RNAylation, but nsp13 NTPase activity flips this preference. RNAylation weakens when RdRp is RNA-bound or replicating it, suggesting that a trans-acting NiRAN associated with an RNA-free RdRp performs capping. These findings provide insights into the orchestration of NiRAN activities and potential druggable sites for anti-viral therapeutics.","version":"1.1","doi":"10.1101/2025.10.31.685727","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.30.685465","pub_date":"2025-10-31","title":"Fine-tuned protein language model identifies antigen-specific B cell receptors from immune repertoires","abstract":"Scalable identification of antigen-specific antibodies from whole immune repertoire V(D)J sequences is a central challenge in biomedical engineering. We show that protein language models (PLMs) fine-tuned on antibody heavy-chain sequences can directly predict antigen specificity from unselected immune repertoires. We assessed our model, Antigen Specificity Predictor (ASPred), against SARS-CoV-2, influenza, and HIV-AIDS antigens, observing comparable predictive performance. In the whole immune repertoire V(D)J sequences of mice immunized with the SARS-CoV-2 spike protein\u2019s receptor-binding domain (RBD), ASPred identified antibody sequences specific to RBD. Several candidate sequences were validated, including one as a heavy chain-only nanobody with 20.7 nM dissociation constant. Molecular dynamics simulations supported the predicted interactions at coarse-grained and atomic levels. Benchmarking against Barcode-Enabled Antigen Mapping (BEAM) of B cell receptor sequence data had highly significant overlaps with ASPred predictions, suggesting scalability. The predicted SARS-CoV-2 binders differed substantially from training sequences, demonstrating generalization beyond sequence memorization. Together, we establish that heavy chain antibody sequences encode sufficient information for PLMs to infer specificity, offering a scalable framework for antibody discovery with broad applications.","version":"1.1","doi":"10.1101/2025.10.30.685465","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.30.685528","pub_date":"2025-10-30","title":"SARS-CoV-2 Variant-Dependent Alterations in Nasopharyngeal Microbiota and Host Inflammatory Response","abstract":"The SARS-CoV-2 pandemic saw multiple outbreaks occur over short periods. This was linked to the virus\u2019s high infectivity and rapid mutation rate, which hindered the development of effective treatments and a comprehensive understanding of COVID-19 pathophysiology. The nasopharyngeal tract, the main entry site of SARS-CoV-2, interacts with angiotensin-converting enzyme 2 (ACE2) receptors via the viral spike glycoprotein, triggering pro-inflammatory responses that affect both tissue integrity and the resident microbiota. Viral infection induces dysbiosis by modulating microbiome diversity. Although distinct variants produce independent symptoms and viral loads, their impact on the nasopharyngeal microbiota and host inflammatory profile remains poorly understood. To address this, we analyzed nasopharyngeal samples from Chilean individuals collected during different phases of the pandemic. The microbiota was characterized by 16S rRNA gene sequencing (V3\u2013V4 region), and cytokine expression was quantified by RT-qPCR. Finally, applied rigorous data processing and machine-learning models Random Forest and K-Nearest Neighbors to identify associations between SARS-CoV-2 variants, inflammatory markers, and opportunistic bacterial genera. Our results reveal that SARS-CoV-2 infection promotes distinct immunomicrobial signatures marked by TNF-\u03b1-driven inflammation and the expansion of opportunistic taxa. These variant-dependent alterations indicate that host inflammatory responses and microbial dysbiosis are closely intertwined in the nasopharyngeal environment. This study provides a comprehensive framework integrating microbial and host factors to better understand the mechanisms underlying COVID-19 pathogenesis and highlights the potential of combining microbiome and cytokine profiling with machine-learning approaches to differentiate infection outcomes across viral variants. Understanding how SARS-CoV-2 infection alters the upper respiratory microbiota and host immune responses is essential to uncover the mechanisms underlying COVID-19 severity. In this study, we integrate bacterial 16S rRNA sequencing, inflammatory markers, and machine learning to identify key descriptors of infection such as TNF-\u03b1, Acinetobacter, Prevotella, and Staphylococcus. These markers define a conserved immunomicrobial signature across viral variants, linking inflammatory activation with microbial dysbiosis in the nasopharyngeal environment. Our findings highlight the value of the correct and robust application of machine learning models and nested cross-validation strategies in high-dimensional (wide) datasets to improve variant discrimination, reveal host\u2013microbe interactions, and support the development of therapeutic strategies for respiratory viral infections.","version":"1.1","doi":"10.1101/2025.10.30.685528","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.27.25338874","pub_date":"2025-10-28","title":"Untargeted longitudinal ultra deep metagenomic sequencing of wastewater provides a comprehensive readout of expected and unexpected viral pathogens","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Wastewater surveillance has become a powerful tool to monitor circulating viruses at a community level. Currently, most wastewater surveillance efforts use target-based approaches such as quantitative PCR techniques or hybrid capture. This study explores the feasibility of using untargeted ultra-deep metagenomic sequencing as a comprehensive approach to wastewater surveillance. To test this, composite influent wastewater samples were collected weekly at a single site from January 2024 through June 2025. Sequencing was performed using random hexamers on all samples, with an average depth of 1.1 billion reads per sample. Human enteric viruses such as rotaviruses, astroviruses, and noroviruses were detected at high levels in virtually every sample. SARS-CoV-2 was also detected in most samples and the counts per sample positively correlated with digital PCR (dPCR) measurements. Less abundant respiratory pathogens such as influenza A and B, rhinoviruses, parainfluenzaviruses, and human coronaviruses 229E, OC43, NL63, and HKU1 were also regularly detected. However, those pathogens displayed distinct and reproducible winter and spring seasonality. Several unexpected viruses were also detected, such as several detections of highly pathogenic avian influenza H5N1 (HPAI H5N1) genotype B3.13, a month-long surge of hepatitis A virus, and a large season-specific surge in influenza C virus. The most abundant known virus detected was the\n                  <jats:italic>Tobamovirus</jats:italic>\n                  tomato brown rugose fruit virus, which was present stably year-round at high abundance. However, other tobamoviruses such as tomato mosaic virus were detected primarily in the late growing season. This eighteen-month study highlights that ultra deep sequencing enables detection of expected and unexpected viral pathogens without targeted enrichment.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Importance</jats:title>\n                  <jats:p>This study demonstrates that untargeted ultra deep metagenomic sequencing can provide a comprehensive tool for wastewater surveillance of viral pathogens. By generating approximately 1 billion reads per sample across 78 consecutive weeks, we captured expected pathogens such as SARS-CoV-2, noroviruses, and influenza viruses. Additionally, we captured unexpected viral signals such as influenza C and highly pathogenic avian influenza H5N1. The wide range of viral taxa captured in this study also displays epidemiologically relevant seasonality. We also observed a correlation between metagenomic SARS-CoV-2 read counts and dPCR values to validate this method against other wastewater surveillance methods currently in use. Our findings highlight how ultra deep metagenomics can enhance pandemic preparedness, enable early detection of non-standard and clinically overlooked species, and broaden the scope of One Health monitoring by capturing human, animal, and plant viral signatures from a composite wastewater sample.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2025.10.27.25338874","journal":"medRxiv","score":null},{"id":"10.1101/2025.10.27.683653","pub_date":"2025-10-27","title":"Design and development of a SARS and MERS Combination Vaccine","abstract":"This work was conducted during the COVID-19 pandemic prior to the licensing of any vaccines against COVID-19. Although several COVID-19 vaccines are now commercially available, this research on the development of a combination Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS) is still relevant and shows how a combination vaccine can be designed, produced and rapidly tested in the laboratory. We present the development of a combination vaccine designed to provide immunity against Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS). The primary objective of this vaccine design is twofold: to mitigate the burden of coronavirus and to address the specific vulnerability of regions prone to recurrent MERS outbreaks. Our combination vaccine incorporates antigenic components from zoonotic sources, specifically SARS-CoV, SARS-CoV-2, and MERS. We assess the impact of combining different Spike protein\u2019s S1 subunit antigens, due to its recognised immunogenic potential, and adjuvants on serum antibody titres, virus neutralizing capabilities, and inter-antigen immune responses. We report a robust and broad antibody response against SARS-CoV-2 and related coronaviruses, which was amplified by different adjuvant formulations, including alum, MPLA, CpG, and Squalene-in-Water Emulsion.","version":"1.1","doi":"10.1101/2025.10.27.683653","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.27.684767","pub_date":"2025-10-27","title":"Structural constraints acting on the SARS-CoV-2 spike protein reveal limited space for viral adaptation","abstract":"The SARS-CoV-2 pandemic resulted in an unprecedented scientific response. The enormous scale of global genome sequencing, protein structural determination and targeted studies of protein and variant dynamics has resulted in a unique dataset which provides a valuable resource for understanding of viral evolutionary dynamics. Previous analysis of SARS-CoV-2 evolution has revealed apparently saltatory dynamics, with viral variants arising following large evolutionary jumps without genetic intermediates represented in the sequence database. We utilise rich SARS-CoV-2 datasets to interrogate the role of protein structural constraint in SARS-CoV-2 evolution and whether these evolutionary jumps may result from the viral spike protein accessing new regions of viable sequence space. We apply multiple computational predictors of structural constraint across different structural backgrounds and assess how constraint has changed during SARS-CoV-2 variant evolution. These predictions are validated using substitution data from the SARS-CoV-2 global sequence database. We find that all predictive methods suggest that the structural constraint experienced by specific sites has undergone very limited change, despite significant phenotypic evolution of the SARS-CoV-2 S protein. Signature mutations for variants of concern are not found to be under structural constraint by any computational predictor regardless of which viral variant structure is used to calculate predictions. We also develop a machine learning model to assess substitution viability, combining predictors of evolutionary constraint with information about local structural context. This confirms our conclusions, with model performance largely unaffected by the use of different viral variant structures. We also find no reduction in the shared proportion of accessible substitutions over evolutionary time, as would be expected if the S protein had entered and explored novel sequence space during variant evolution. These results suggest that despite its rapid rate of mutation, the SARS-CoV-2 S protein is subject to strict structural constraints and shows that viral genomes exhibit limited plasticity following infection of a new host.","version":"1.1","doi":"10.1101/2025.10.27.684767","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.24.684489","pub_date":"2025-10-27","title":"A divergent betacoronavirus with a functional furin cleavage site in South American bats","abstract":"Bats are natural reservoirs for a wide range of RNA viruses. Members of the genus Betacoronavirus, including Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome virus (MERS-CoV), have attracted particular attention due to their recent zoonotic emergence. However, much of the known diversity of betacoronaviruses is based on data from Asia, Africa, and the Middle East, with limited genomic information available from the Americas. Herein, we report the complete genome of a novel bat betacoronavirus identified from a Pteronotus parnellii bat sampled in Brazil. Phylogenetic analysis revealed that this virus is sufficiently distinct from the five recognized Betacoronavirus subgenera to represent a new subgenus. Of note, the spike protein of this novel bat coronavirus possesses a functional furin cleavage site at the S1/S2 junction with a unique amino acid sequence motif (RDAR) that differs from that found in SARS-CoV-2 (RRAR) by only one amino acid. Comparative structural analysis identified other betacoronaviruses in bats with furin cleavage sites at the S1/S2 junction, suggesting that this region is a structurally permissive \u201chotspot\u201d for cleavage site incorporation. Our study provides a broader understanding of the phylogenetic and functional diversity of bat coronaviruses as well as their zoonotic potential.","version":"1.1","doi":"10.1101/2025.10.24.684489","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.23.684143","pub_date":"2025-10-26","title":"SAMHD1 promotes SARS-CoV-2 infection by enhancing HNF1-dependent ACE2 expression in lung epithelial cells","abstract":"Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts a board spectrum of viruses through multifaceted mechanisms. It also limits spontaneous- and virus-induced innate immune responses by suppressing proinflammatory cytokine and type-I interferon (IFN-I) production. Some viruses escape SAMHD1 restriction by utilizing SAMHD1-mediated innate immune suppression to establish effective infection through viral antagonism. Our previous studies showed that SAMHD1 is a proviral factor facilitating replication of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in human macrophages, monocytic THP-1 and epithelial-like HEK293T cell lines by suppressing IFN responses. However, it is unclear about the function of SAMHD1 in lung epithelial cells during SARS-CoV-2 infection. Here, we report that SAMHD1 facilitates SARS-CoV-2 replication in lung epithelial Calu-3 cells by enhancing endogenous expression of the viral receptor angiotensin-converting enzyme 2 (ACE2) via hepatocyte nuclear factor 1-alpha (HNF1\u03b1) and HNF1\u03b2. Using pseudotyped SARS-CoV-2 and lentiviral vectors, we found that SARS-CoV-2 spike protein-mediated viral entry was suppressed in Calu-3 cells with SAMHD1 knockout (KO). SAMHD1 KO repressed ACE2 expression in Calu-3 cells at mRNA and protein levels. Functional analyses revealed that HNF1\u03b1 and HNF1\u03b2 were crucial for the endogenous ACE2 expression in Calu-3 cells. Additionally, SAMHD1 KO led to a reduction in the expression levels and ACE2-promoting function of HNF1\u03b1 and HNF1\u03b2. Inhibition of IFN antiviral response by baricitinib, a Janus kinase 1 and 2 (JAK 1/2) inhibitor, did not revert the suppression of SARS-CoV-2 in SAMHD1 KO Calu-3 cells. Our findings demonstrate that SAMHD1 facilitates HNF1-mediated ACE2 expression and SARS-CoV-2 replication in Calu-3 cells via a novel mechanism beyond its IFN-suppressive function. During viral infection, SAMHD1 acts as a viral restriction factor and a suppressor of the innate immune system, controlling viral replication while also ensuring immune homeostasis. The innate immune suppressive function of SAMHD1 can be proviral for some viruses. SAMHD1 has been shown to facilitate SARS-CoV-2 infection in human macrophages, THP-1 and HEK293T cell lines by promoting IFN antagonism, but its role in lung epithelial cells is unclear. Here, we demonstrated that SAMHD1 promotes SARS-CoV-2 replication in human lung epithelial Calu-3 cells by enhancing expression of the major viral receptor ACE2. We found that SAMHD1 facilitated HNF1-medaited ACE2 expression that was required for spike protein-mediated SARS-CoV-2 entry. However, inhibiting IFN signaling in SAMHD1 KO Calu-3 cells was not sufficient to revert SARS-CoV-2 replication. Our findings shed light on the differential proviral function of SAMHD1 in ACE2 expressing cells and suggest that SAMHD1 can facilitate SARS-CoV-2 infection beyond enhancing IFN antagonism.","version":"1.2","doi":"10.1101/2025.10.23.684143","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.13.659551","pub_date":"2025-10-26","title":"Susceptible host dynamics explain pathogen resilience to perturbations","abstract":"Interventions to slow the spread of SARS-CoV-2 significantly disrupted the transmission of other pathogens. As interventions lifted, whether and when human pathogens would eventually return to their pre-pandemic dynamics remains to be answered. Here, we present a framework for estimating pathogen resilience based on how fast epidemic patterns return to their pre-pandemic dynamics. By analyzing time series data from Hong Kong, Canada, Korea, and the US, we quantify the resilience of common respiratory pathogens and further predict when each pathogen will eventually return to its pre-pandemic dynamics. Our predictions are able to distinguish which pathogens should have returned already, and deviations from these predictions reveal long-term impacts of pandemic perturbations. We find a faster rate of susceptible replenishment underlies pathogen resilience and sensitivity to both large and small perturbations. Overall, our analysis highlights the persistent nature of common respiratory pathogens compared to vaccine-preventable infections, such as measles. COVID-19 interventions slowed the transmission of many respiratory pathogens in different ways, raising questions about the mechanisms driving the variation in responses to interventions. To address this gap, we characterized the sensitivity of pathogen transmission to perturbations by quantifying how fast each pathogen returned to its pre-pandemic circulation patterns. We analyzed data from Hong Kong, Canada, Korea, and the US, and showed that common respiratory pathogens are far less sensitive to perturbations than measles, a vaccine-preventable infection. Finally, we showed that the speed of replenishment of the susceptible population\u2014for example, through waning immunity\u2014largely determines this sensitivity, suggesting that the persistence of common respiratory pathogens is likely driven by rapid susceptible replenishment.","version":"1.3","doi":"10.1101/2025.06.13.659551","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.22.684004","pub_date":"2025-10-24","title":"SARS-CoV-2 infection induced alterations in ADAR editing patterns differ between patients who developed critical compared to non-critical COVID-19","abstract":"COVID-19, caused by the SARS-CoV-2 virus, has a wide spectrum of clinical presentations even among individuals with similar demographics. Disease severity has been linked with viral recognition-triggered expression of Interferons (IFNs) and Interferon stimulated genes (ISGs). Among these ISGs, ADARp150 is a member of adenosine deaminases acting on RNA (ADARs) enzyme family. ADARs are RNA editing enzymes that contribute to transcriptome diversity and modulate immune response during viral infections. While previous studies have identified altered ADAR expression and editing patterns during SARS-CoV-2 infection, it remains unknown whether ADAR expression and activity differ between patients with varying severities of COVID-19, specifically, in individuals who developed critical compared to non-critical COVID-19. We address this question by analyzing a publicly available, deeply sequenced whole blood RNA-seq dataset from individuals with either critical or non-critical COVID-19, matched for age, sex, and presence of comorbidities. Our results show differential expression of thousands of genes, including those involved in neutrophil degranulation, and upregulation of ADAR1 and its isoform ADARp110 in patients with critical COVID-19. We further identify global differences in the total number of edits, driven by ADAR1 and ADAR2 expression levels in critical but not in non-critical patients. ADAR activity also differed within Alu elements and in the proportion of edits with varying functional consequences. We further identified severity specific editing events, including nonsynonymous edits, within distinct biological pathways. Moreover, we identified 140 high confidence editing sites within 126 genes, that are differentially edited between the two patient groups. These genes were enriched in infectious disease, cell cycle, signal transduction, RNA and protein metabolism in addition to inflammatory pathways such as neutrophil degranulation and signaling by interleukins. Enrichment/modulation of neutrophil degranulation pathway at transcriptional and post transcriptional levels suggest the importance, complex regulation, and contribution of this pathway in COVID-19 disease severity. Finally, using a random forest classifier, we identified a set of differentially edited sites that could serve as molecular markers for COVID-19 disease severity. Together, our study demonstrates varying expression and editing patterns of ADARs between critical and non-critical patients, suggesting a potential role of ADAR editing in varying severity of COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2025.10.22.684004","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.22.683849","pub_date":"2025-10-22","title":"Co-administration of intranasal parainfluenza virus vaccines expressing antigenically distinct SARS-CoV-2 S antigens elicits broad and durable immunity in hamsters","abstract":"Intranasal COVID-19 vaccines with the ability to induce broad and durable mucosal and systemic immunity would be useful as stand-alone vaccines or in combination with injectable vaccines. Here, we evaluated in the hamster model the immunogenicity, breadth of immunity, and durability of protection elicited by co-administration of two live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3) vectors expressing antigenically distinct prefusion stabilized S proteins of the ancestral SARS-CoV-2 isolate (B/HPIV3/S-6P) or the Omicron/BA.5 variant (B/HPIV3/S-BA.5-2P). These vectors are being developed as bivalent pediatric vaccines against HPIV3 and SARS-CoV-2 and are based on bovine PIV3 with the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins replaced by those of human PIV3. To broaden the S-specific antibody response, we evaluated co-administration of these B/HPIV3 S-expressing vectors. Each B/HPIV3 S-expressing vector induced robust serum anti-S IgG and IgA antibody levels to the antigen-matched S protein that were sustained for at least five months. Co-administration increased the breadth of the S-specific antibody response, spanning the antigenic breadths of the response elicited by each B/HPIV3 S-expressing vector individually. Animals that had received the mixture of vectors developed neutralizing antibodies to ancestral as well as recently circulating SARS-CoV-2 strains. Hamsters immunized intranasally were protected against Omicron/BA.5 challenge 5 months after immunization, with no weight loss, SARS-CoV-2 challenge virus replication, or increase in host inflammatory cytokines in the upper and lower airways detectable after the challenge, indicating durable protection. Thus, intranasal co-administration of live-attenuated B/HPIV3 expressing antigenically distinct S proteins induced broad and durable antibody responses and long-term protection against Omicron/BA.5 challenge. This approach warrants further development and may better protect against emerging SARS-CoV-2 variants. Current SARS-CoV-2 vaccines protect against severe disease but are less efficient at blocking infection. Intranasal SARS-CoV-2 vaccines, however, have been shown to induce local immunity that better blocks infection at the nasal portal of entry and have been proposed as booster vaccines to induce better protection against SARS-CoV-2 variants. In the present study, we evaluated in hamsters an intranasal live-attenuated chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) as a bivalent pediatric vaccine against PIV3 and SARS-CoV-2. Co-administration in a single intranasal dose of two B/HPIV3 vectors, one expressing the spike protein S from the antigenically distinct ancestral Wuhan-Hu-1 strain and one expressing S of Omicron/BA.5, induced broad and durable serum anti-S IgG and IgA antibody responses that remained strong for at least five months. Hamsters challenged with the Omicron/BA.5 strain five months after immunization were protected from weight loss, inflammatory responses, and challenge virus replication in the upper and lower airways after challenge. Thus, in the hamster model, intranasal immunization with live-attenuated B/HPIV3 expressing SARS-CoV-2 S can provide durable protection for several months, and combining two antigenically distinct B/HPIV3 S-expressing vectors substantially broadens the antibody response. This mucosal immunization approach may better protect against infection from emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2025.10.22.683849","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.21.683815","pub_date":"2025-10-22","title":"ADAPT-M: A workflow for rapid, quantitative in vitro measurements of enriched protein libraries","abstract":"Protein-protein interactions underpin most cellular interactions, and engineered binders present powerful tools for probing biology and developing novel therapeutics. One bottleneck in binder generation is the scalable, quantitative characterization of these interactions. We present ADAPT-M (Affinity Determination by Adaptation of ProTein binders for Microfluidics), a streamlined workflow that connects yeast surface display (YSD) with in vitro affinity and kinetic measurements using the high-throughput STAMMPPING microfluidic platform. ADAPT-M quantifies Kds and dissociation kinetic parameters for hundreds of enriched protein variants in under one week without requiring hands-on protein purification. We applied ADAPT-M to a computationally designed library targeting the SARS-CoV-2 Omicron BA.1 receptor binding domain, successfully recovering and measuring Kds for most highly enriched YSD variants. Measurements correlate strongly with biolayer interferometry and yeast titration assays. ADAPT-M further enabled selection of lead candidates for structural and mutational analysis, which revealed designed paratopes were preserved despite binding to off-target epitopes. By bridging YSD screening and in vitro validation, ADAPT-M accelerates protein binder discovery and supports data-driven protein engineering.","version":"1.1","doi":"10.1101/2025.10.21.683815","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.21.683784","pub_date":"2025-10-21","title":"Interactomics of SARS-CoV-2 Macrodomain 1 Reveals Putative Clients of ADP-ribosyl Hydrolase Activity","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well-conserved across variants and is critical for suppression of host immune response to infection, making Mac1 a promising target for therapeutic development. Mac1 binds and cleaves the post-translational modification ADP-ribose and is hypothesized to have a downstream effect on host interferon response, but the exact cellular targets of Mac1 are still unknown. Characterizing the substrates of Mac1 ADP-ribosyl hydrolase activity using a catalytically inactive mutant N40D can reveal critical virus-host interactions to identify protein targets of Mac1 and reveal mechanisms of host interferon suppression. Here, we co-immunoprecipitated WT Mac1 and Mac1 N40D from HEK293T and A549 cell lines and quantified changes in protein interactions by TMT-multiplexed tandem mass spectrometry. We identified interactions between Mac1 and ADP-ribosylated substrates involved in DNA damage response, cytoskeletal components, and cell cycle regulation. Additionally, several members of the TRiC complex involved in protein folding were selectively enriched with mutant Mac1 from A549 cells. These findings suggest a novel role of Mac1 in regulating host protein folding.","version":"1.1","doi":"10.1101/2025.10.21.683784","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.17.683096","pub_date":"2025-10-20","title":"A Human Alveolus-on-Chip Recapitulates SARS-CoV-2-mediated Lung Injury in an Organ-relevant Context for Pre-clinical Applications","abstract":"Respiratory viruses pose a constant threat to public health as highlighted by the pandemic outbreak of COVID-19. The most severe manifestation of COVID-19 is observed in the distal lung, where SARS-CoV-2 infection can result in massive inflammation and barrier breakdown. To date, few therapies are approved for clinical use in COVID-19 patients partially due to the lack of highly translational pre-clinical models of the alveoli. Human-derived microphysiological systems pose a promising new class of in vitro models to study viral infection in a relevant context. Therefore, we aimed to develop a Lung-on-Chip (LOC) model for studying SARS-CoV-2 infection at the alveolar barrier. Using an immortalized alveolar epithelial cell line, AXiAEC, and human lung microvascular endothelial cells (hLMVEC) we established a SARS-CoV-2 infection model on a LOC system. The LOC models the alveolar epithelial/endothelial barrier under physiological breathing motion. Our results demonstrated that AXiAEC maturation at the air-liquid interface (ALI) is essential for SARS-CoV-2 infection. SARS-CoV-2 infected the breathing LOC model and induced breakdown of the air-blood barrier. Finally, we evaluated the application of the LOC SARS-CoV-2 infection model for efficacy testing and demonstrated the antiviral effect of remdesivir. Drug treatment not only inhibited viral replication but also protected the alveolar barrier from damage and partially reverted SARS-CoV-2-mediated transcriptional dysregulation in AXiAEC. This novel LOC infection model recapitulates aspects of COVID-19. Our results highlight the importance of physiological cues such as ALI and stretch to accurately model host-pathogen interactions in the distal lung. Application of LOC models in pre-clinical drug testing may facilitate candidate compound selection at an early stage, allowing the allocation of resources to a few promising candidate compounds raising the potential to accelerate drug development and reduce costs and animal testing.","version":"1.1","doi":"10.1101/2025.10.17.683096","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.16.682699","pub_date":"2025-10-20","title":"A cocktail of SARS-CoV-2 spike stem helix domain and receptor binding domain human monoclonal antibodies prevent the emerge of viral escape mutants","abstract":"Neutralizing antibodies (NAbs) targeting the spike (S) glycoprotein remain a crucial therapeutic strategy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, emerging viral variants have escaped all Food and Drug Administration (FDA)-approved NAb treatments, underscoring the urgent need for effective therapeutic alternatives. Using a nanoluciferase (Nluc)-expressing attenuated recombinant SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b (\u03943a7b-Nluc), we characterized resistance profiles of two broadly protective NAbs targeting the S receptor binding domain (RBD) in S1 (1301B7) and the stem helix domain (SH) in S2 (1249A8). Serial passaging of \u03943a7b-Nluc under selective pressure identified a 1301B7 antibody-resistant mutants (ARM-B7) harboring an RBD mutation (S371F) that conferred resistance to 1301B7 and other RBD-directed NAbs (Casirivimab, SC27 and Sotrovimab). In contrast, no ARM emerged under treatment with 1249A8, or an antibody cocktail of 1301B7 (RBD) + 1249A8 (SH). These findings demonstrate that S2 SH-targeting NAbs shows higher genetic barrier to resistance than S1 RBD-targeting NAbs, and that a NAbs cocktail therapy targeting the SARS-CoV-2 S1 RBD and S2 SH offers the most effective strategy to prevent the emergence of escape mutations. Together, our findings provide critical insights into developing next-generation resistance-evading NAb therapies against SARS-CoV-2, and potentially other coronaviruses, and demonstrate the value of using our attenuated viral platforms for the safe identification of ARM without the potential biosafety concerns of doing these experiments using wild-type (WT) forms of SARS-CoV-2. The clinical efficacy of early SARS-CoV-2 NAbs has been challenged by the emergence of escape viral variants, highlighting an urgent need to anticipate resistance. Using a luminescent attenuated SARS-CoV-2 platform, we profiled resistant mutations against two broadly protective SARS-CoV-2 NAbs. Passage of \u03943a7b-Nluc in the presence of a NAb targeting the RBD S1 domain (1301B7) readily selected for an ARM, whereas passage in the presence of an SH S2 domain NAb (1249A8) did not. Notably, a cocktail of 1301B7 and 1249A8 created a high barrier of selecting SARS-CoV-2 ARM, preventing the emergence of resistant variants. We identified an S371F mutation in the S1 RBD of ARM-B7 that confers resistance to 1301B7 and other S1 RBD-targeting NAbs. These results highlight the importance of combination therapies targeting both variable RBD S1 and conserved SH S2 domain for the efficient treatment of SARS-CoV-2 and to prevent the emerge of NAb-induced escape mutations.","version":"1.1","doi":"10.1101/2025.10.16.682699","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.16.682893","pub_date":"2025-10-20","title":"Repeated COVID-19 vaccine boosters elicit variant-specific memory B cells in humans","abstract":"The first exposure to a pathogen or an antigen profoundly impacts immune responses upon subsequent encounter with related pathogens. This immune imprinting explains that infection or vaccination with currently circulating SARS-CoV-2 variants primarily recalls cross-reactive memory B cells and antibodies induced by prior Wu spike (S) glycoprotein exposure rather than priming de novo responses. The magnitude and persistence of immune imprinting in mRNA vaccinated populations and the prospect to overcome it are not understood. To understand the impact of immune imprinting, we investigated memory B cell and plasma antibody responses after administration of multiple doses of XBB.1.5 and JN.1/KP.2 updated COVID-19 vaccine boosters. We found that administration of the JN.1/KP.2 booster elicited broadly neutralizing antibody responses against recently circulating SARS-CoV-2 variants that were accounted for by recall of Wu S-induced immunity. We detected an increased fraction of serum antibodies and particularly memory B cells recognizing XBB.1.5 S and KP.2 S, but not Wu S, relative to individuals who received a single XBB.1.5 booster a year prior. These findings suggest that repeated exposures to antigenically divergent S trimers contribute to progressively overcoming immune imprinting and support vaccine updates and innovation to provide continued protection against COVID-19. Immune imprinting due to repeated SARS-CoV-2 Wuhan-Hu-1 spike exposures is widely observed in humans. Tortorici et al. show that the humoral immune response is dominated by recall of pre-existing Wu S-induced serum antibodies and memory B cells after administration of multiple XBB.1.5 and JN.1/KP.2 COVID-19 vaccine boosters. However, the detection of an appreciable fraction of serum antibodies and particularly memory B cells binding the updated vaccine antigens, but not Wu, suggests a path towards overcoming immune imprinting XBB.1.5, JN.1 and KP.2 S COVID-19 vaccine boosters elicit neutralizing antibodies against current variants. Serum neutralizing activity against circulating variants elicited after multiple doses of updated COVID-19 vaccine boosters derive from recall of Wu S-elicited antibodies. Non-neutralizing antibodies specific for the updated spike antigens were detected after multiple, updated COVID-19 boosters.","version":"1.1","doi":"10.1101/2025.10.16.682893","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.17.683094","pub_date":"2025-10-20","title":"Predicting SARS-CoV-2 evolution dynamics with spatiotemporal resolution by DMS-empowered protein language model","abstract":"Early identification of emerging dominant SARS-CoV-2 variants is essential for effective pandemic preparedness, yet existing methodologies face significant limitations. Experimental characterizations are costly and not feasible for real-time surveillance, whereas existing computational approaches cannot achieve satisfactory precision in predicting future dominant lineages and fail to capture the spatiotemporal dynamics of fitness under evolving host immune pressures. Here, we introduce DeepCoV (DMS-Empowered Evolution Prediction of CoronaVirus), a deep-learning framework for the dynamic identification of novel variants with high potential to become prevelent. It integrates deep mutational scanning (DMS)-derived mutation phenotypes with epidemiological surveillence data reflecting historical viral evolution and the dynamic fitness landscape. DeepCoV accurately forecasted the dominance of recently circulating lineages a month in advance, achieving a 90% reduction in false discovery rate while capturing temporal and geographic dynamics of variant spread and reconstructing their regional prevalence trajectories. Moreover, DeepCoV identified mutational hotspots of Omicron-derived backbones in silico, revealing convergent evolution trends. This scalable solution enables timely identification of immune-evasive variants and prospective alert of critical mutations, providing actionable insights for vaccine updates and pandemic surveillance.","version":"1.1","doi":"10.1101/2025.10.17.683094","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.18.683252","pub_date":"2025-10-18","title":"Thermodynamic Modeling of mRNA with the Addition of Precipitants","abstract":"Nucleic acid therapeutics (NATs) have recently emerged as an exciting therapeutic modality for a range of indications, most notably as vaccines for SARS-CoV-2. In many cases, the thermodynamics of a system containing nucleic acids (such as in the downstream purification of mRNA from solution or within the lipid nanoparticle) can significantly influence the properties and efficacy of that system. Consequently, an accurate thermodynamic description of the system is essential for understanding and optimizing that system. In this work, the SAFT-\u03b3 Mie equation of state was used to predictively model mRNA solubility. Experimental measurements of the solubility of two different mRNA sequences in various conditions (namely choice of precipitant(s), precipitant concentration, and temperature) were obtained and used to validate the model. Not only was the thermodynamic model able to quantitatively predict the solubility of mRNA in solution under different conditions, it was also able to yield mechanistic insight into the factor driving precipitation, namely the disruption of water-mRNA hydrogen bonding. The developed model can be extended to other mRNA sequences in a range of conditions beyond the experimental data presented in this work.","version":"1.1","doi":"10.1101/2025.10.18.683252","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.17.682775","pub_date":"2025-10-17","title":"Serological evidence of SARS-CoV-2 infection in red and fallow deer in Great Britain","abstract":"SARS-CoV-2 is the viral agent of COVID-19 disease in humans and has been shown to infect a wide range of mammals, including white-tailed deer in North America and fallow deer in the Republic of Ireland. There are six species of deer in the UK which inhabit both urban and rural areas, providing a broad interface for human-deer interaction. Little is known if British deer species act as a reservoir for zoonotic spread of consequence to human health. Here, we report a high seroprevalence of SARS-CoV-2 neutralising antibodies in British deer, adding native red deer to the list of susceptible species. 62% of fallow and 25% of red deer sampled between 2024 and 2025 from one British deer park were positive for SARS-CoV-2 infection by serology. Data suggest that deer may function as permissive, reservoir-capable hosts and highlight the risks of anthroponosis and zoonosis from unregulated human-deer contact. While there is currently no evidence to suggest that these viruses are persisting within deer populations in Great Britain, the animal origins of SARS-CoV-2 highlight the importance of appropriate and limited interactions with wildlife, to minimise transmission risks and safeguard both animal and human health.","version":"1.1","doi":"10.1101/2025.10.17.682775","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.15.682635","pub_date":"2025-10-17","title":"Genome-wide Screening Identifies Unique Host-Directed Drugs and Pro-viral Signalling Pathways for SARS-CoV-2","abstract":"SARS-CoV-2 is a positive-sense RNA virus and was responsible for the devastating COVID-19 pandemic. Although the current disease burden is less severe, there are limited treatment options, significant gaps in knowledge, and a looming threat of the emergence of variants and future pandemics. To address these challenges, we performed genome-wide CRISPR knockout screens in a novel human lung cell line NCI-H23ACE2, as well as in HEK293TACE2 cells, with SARS-CoV-2 Wuhan virus, with the aim of identifying host-dependency factors that could predict effective antivirals. We identified four host-directed drugs, donepezil, dH-ergocristine, trametinib and sorafenib, that could potentially be repurposed to treat coronavirus infections. Three of the drugs inhibited SARS-CoV-2, HCoV-229E, and HCoV-OC43, suggesting they could be used as pan-coronavirus antivirals. We also confirmed that SARS-CoV-2 relies on the NRAS/Raf/MEK/ERK signaling pathway for its replication. Our study highlights the robustness and efficiency of a bilateral approach of gene silencing and antiviral screening to identify host-dependency factors and effective antivirals.","version":"1.1","doi":"10.1101/2025.10.15.682635","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.03.680348","pub_date":"2025-10-17","title":"Cathepsin K as a Key Protease in Processing of SARS-CoV-2 Spike Activation Sites and a Target of Dual-Inhibition","abstract":"SARS-CoV-2 relies on host proteases to prime its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized yet functionally important mediator of spike activation. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease for spike processing, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral infection in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of spike processing during the endosomal viral entry. Comprehensive enzymatic profiling demonstrated that hCatK exhibits 24- to 63-fold higher catalytic efficiency toward the Furin-cleavage site (FCS) sequence than hCatL and displays a distinct substrate-recognition pattern at the Omicron FCS relative to the Wuhan variant. We further demonstrate that hCatK is an off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, with a sub-micromolar potency (IC50 = 0.6 \u00b1 0.1 \u00b5M). A 1.9 \u00c5 crystal structure of the hCatK\u2013Nirmatrelvir complex delineates the molecular basis of inhibitor binding and supports the rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases involved in SARS-CoV-2 spike activation and establish hCatK as a previously overlooked but strategic target for antiviral intervention.","version":"1.2","doi":"10.1101/2025.10.03.680348","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.16.682991","pub_date":"2025-10-17","title":"A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2","abstract":"Protection from pathogenic organisms relies heavily on the adaptive immune response, for which key regulators are CD4 T cells. CD4 T cells, notable in the complexity of their repertoire and functional potential, can most easily be dissected with the ability to identify, quantify, characterize and isolate epitope-specific cells. In the study reported here, we present a systematic and unbiassed strategy that has enabled identification of highly immunogenic peptide epitopes derived from influenza virus and SARS-CoV-2, presented by human HLA-DR proteins. Coupling the use of HLA-DR transgenic mice with infection and vaccination with highly sensitive epitope specific cytokine ELISpot assays, we have narrowed the potential epitopes from 450-600 peptides to 5-15 peptides, by an iterative process of elimination and selection which we have termed a funnel approach. These epitopes have been validated in HLA-DR typed human CD4 T cells directly ex vivo and enabled derivation and implementation of HLA-DR peptide tetramers. Tetramer staining of human PBMCs enriched CD4 T memory populations from healthy adult subjects highlighting this approach as a sensitive and specific method of identifying novel epitopes and subsequent CD4 T cell responses to human viral infections. Tracking single epitope-specific CD4 T cells enables sophisticated analyses of the human response to infectious pathogens, vaccines and probing the human CD4 T cell immune memory compartment. The studies presented here provide a unbiased strategy for accomplishing this goal and provide a verified compilation of candidate HLA-DR restricted CD4 T cell peptide epitopes for future studies by researchers in the field of human immunology.","version":"1.1","doi":"10.1101/2025.10.16.682991","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.16.682984","pub_date":"2025-10-17","title":"The effects of denifanstat on hepatitis E virus replication and triggered inflammatory response","abstract":"Metabolic dysfunction\u2013associated steatotic liver disease (MASLD) is initiated by intra-cellular fatty acid accumulation, with macrophage-mediated inflammatory responses playing a pivotal role in driving disease progression. MASLD frequently coexists with viral infections, for example in the context of hepatitis E virus (HEV) infection which explores host lipid metabolism for its replication and secretion. But no targeted therapies currently exist for this complex comorbidity. Denifanstat, a fatty acid synthase (FASN) inhibitor developed for treating metabolic dysfunction-associated steatohepatitis (MASH), has shown to have antiviral activity against SARS-CoV-2 infection. In this study, we aim to assess the effects of denifanstat on HEV infection in the context of steatotic liver disease. We first employed human liver-derived primary organoids exposed to fatty acids and inoculated with HEV. Treatment with denifanstat had no major effect on HEV replication in the steatotic organoids. However, we found that denifanstat moderately inhibited viral replication in macrophages, and regulated HEV-triggered inflammatory responses. These results highlight the importance of understanding how the emerging MASH treatment may affect viral infections that coexist in patients.","version":"1.1","doi":"10.1101/2025.10.16.682984","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.15.682549","pub_date":"2025-10-15","title":"Durability of DNA-LNP and mRNA-LNP Vaccine-Induced Immunity Against SARS-CoV-2 XBB.1.5","abstract":"mRNA-lipid nanoparticle (LNP) vaccines induce robust adaptive immune responses and have proven highly effective against SARS-CoV-2. However, their long-term effectiveness is limited by waning humoral responses, which decline substantially within the first six months post-boost vaccination. DNA-LNPs are being investigated as an alternative vaccine platform, offering prolonged antigen expression and robust immunity. Here, we present the first comparison of SARS-CoV-2 DNA- and mRNA-LNP vaccines in a long-term in vivo challenge model. Both nucleic acid platforms induced strong neutralizing antibody responses and conferred equivalent protection in Syrian hamsters challenged three weeks post-boost. Notably, DNA-LNP vaccination maintained high binding and neutralizing antibody titers six months post-boost, whereas mRNA-LNPs exhibited a marked decline. Correspondingly, while DNA-LNPs completely protected from weight loss, viral replication, and lung pathology at this late timepoint, mRNA-LNP vaccination conferred minimal protection. These findings demonstrate that DNA-LNPs can sustain durable immunity, highlighting their potential as a next-generation vaccine platform that could reduce the need for frequent boosters.","version":"1.1","doi":"10.1101/2025.10.15.682549","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.14.682277","pub_date":"2025-10-15","title":"Murine Toll-like receptor 8 is a nucleic acid multi-sensor detecting 2\u2019,3\u2019-cyclic monophosphate guanosine as well as combinations of ribo-, deoxy-, cyclic nucleotides, and nucleosides","abstract":"Toll-like receptor 8 (TLR8) in humans senses RNA degradation products and elicits an inflammatory immune response. In contrast, the ligand specificity and function of its murine counterpart mTLR8, long considered non-functional, remain poorly defined. Here, we established an agonist combination model of poly-deoxythymidine (poly-dT) DNA and TLR7/8 binding site 1 agonists such as uridine or the benzazepine compound TL8-506, which activates mTLR8, while suppressing mTLR7 signaling. Extensive agonist analysis based on this model revealed that 2\u2019,3\u2019-cyclic guanosine monophosphate (2\u2019,3\u2019-cGMP) serves as a natural ligand for mTLR8, suggesting functionality of its binding site 1 without engagement of site 2. In addition, 2\u2019,3\u2019-cyclic uridine monophosphate, bacterial single-stranded (ss) DNA, double-stranded (ds) DNA fragments, microRNAs, ssRNA derived from HIV1, SARS-CoV-2, or bacterial sources all potentiate mTLR8 sensing of site 1 agonists. All these stimuli induce distinct inflammatory responses from murine macrophages and microglia via TLR8. In vivo, intrathecal administration of TL8-506 and poly-dT led to microglial accumulation and neuronal injury in the murine cerebral cortex through TLR8, highlighting the potential neuropathological consequences of mTLR8 activation. Taken together, our study defines mTLR8 as a nucleic acid sensor detecting 2\u2019,3\u2019-cGMP as well as combinations of ssDNA, dsDNA, ssRNA fragments, 2\u2019,3\u2019-cyclic nucleotide monophosphates, and nucleosides, with implications for host defense and neuroinflammation.","version":"1.1","doi":"10.1101/2025.10.14.682277","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.09.557914","pub_date":"2025-10-14","title":"SARS-CoV-2 spike antibodies cross-react with dengue virus and enhance infection in vitro and in vivo","abstract":"The presence of non-neutralizing antibodies of any dengue serotype, increase the severity of subsequent infection by other dengue serotypes. During SARS-CoV-2 pandemic, the number of symptomatic dengue cases increased in India. We describe that antibodies isolated from convalescent plasma from COVID-19 patients enhances DENV2 infection in vitro. CR3022, one antibody against SARS-CoV-2 spike protein, also showed elevated DENV2 infection in vitro. In silico protein-protein interactions between the spike antibodies and the DENV2 E-protein revealed significant interactions. Likewise, few monoclonal/polyclonal antibodies against SARS-CoV-2 showed increased dengue infection in vitro. Importantly, the AG129 mice infected with SARS-CoV-2 three-weeks prior to DENV2 infection, showed elevated dengue pathogenesis. Thus, highlighting the possibilities of elevated infection and symptomatic dengue disease in COVID-19 survivors.","version":"1.5","doi":"10.1101/2023.10.09.557914","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.11.681833","pub_date":"2025-10-14","title":"Discovery of AVI-6451, a Potent and Selective Inhibitor of the SARS-CoV-2 ADP-Ribosylhydrolase Mac1 with Oral Efficacy in vivo","abstract":"The COVID-19 pandemic made plain the need for effective antivirals acting on novel antiviral targets, among which viral macrodomains have attracted considerable attention. We recently described AVI-4206 (1), a potent and selective inhibitor of the SARS-CoV-2 ADP-ribosylhydrolase Mac1 based on a 9H-pyrimido[4,5-b]indole core, the first Mac1 inhibitor to demonstrate antiviral efficacy in mouse models of SARS-CoV-2 infection, but requiring IP administration and frequent dosing. Herein we describe an extensive, structurally enabled medicinal chemistry effort to identify orally bioavailable Mac1 inhibitors by addressing permeability and efflux liabilities of 1 and many of its analogs. Multiple strategies were pursued to overcome these issues, including replacing a urea function to reduce hydrogen bond donor count. While heterocyclic urea mimetics could deliver analogs like AVI-6318 (3) with potencies and ADME profiles similar to 1, abrogation of the P-gp liability was finally achieved with entirely non-polar substituents in place of urea. Thus, AVI-6451 (4) is a potent Mac1 inhibitor lead with low intrinsic clearance, high oral bioavailability, and antiviral efficacy with once-daily oral administration in a mouse model of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2025.10.11.681833","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.13.682000","pub_date":"2025-10-14","title":"The hyaluronan receptor CD44 drives COVID-19 severity through its regulation of neutrophil migration","abstract":"The novel respiratory disease COVID-19 caused by the coronavirus SARS-CoV-2 continues to be a public health emergency worldwide, and there is a need for more effective therapy for patients. The relationship between the extracellular matrix and the host immune response to infection is severely understudied. Deposition of the polysaccharide hyaluronan (HA) into the lungs is associated with more severe COVID-19 disease outcomes. HA is a major component of the extracellular matrix in connective tissues and is abundant in many parts of the body, including cartilage, skin, brain, and vitreous body. CD44 is the primary receptor for HA and is found on almost all immune cells in the lung. Known functions of CD44 include mediation of immune cell migration, activation, and differentiation. We hypothesized that increased HA deposition during COVID-19 increases CD44-mediated immune cell infiltration into lungs and results in more severe pathology. Here, we report that in mice infected with a mouse-adapted strain of SARS-CoV-2, treatment with a combination of two anti-CD44 monoclonal antibodies confers a significant survival benefit and reduces weight loss and clinical score of the mice on Day 4 post infection. We show that anti-CD44 treatment decreases many key cytokines and chemokines in the bronchoalveolar lavage fluid on Day 4. With flow cytometry, we show that anti-CD44 reduces the numbers of neutrophils in infected lungs. We also show through immunofluorescence that treatment with anti-CD44 antibodies reduces colocalization of HA and CD45 in lung sections, indicating that HA\u2019s interaction with immune cells contributes to pathology. Our findings demonstrate that disruption of HA-receptor interactions is a way to prevent inflammatory pathology in pulmonary infection.","version":"1.1","doi":"10.1101/2025.10.13.682000","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.13.682101","pub_date":"2025-10-14","title":"Mechanisms of sex differences in acute and long COVID sequelae in mice","abstract":"While males are more likely to suffer severe outcomes during acute COVID-19, a greater proportion of females develop post-acute sequalae of COVID-19 (PASC) despite similar rates of infection. To identify mechanisms of PASC, mice were infected with SARS-CoV-2 and viral, inflammatory, and behavioral outcomes were evaluated through 84 days post infection. Sex differences were not observed in virus replication or persistence of viral RNA in pulmonary or extrapulmonary tissues in acute or PASC phases. Following recovery from infection, female mice exhibited persistent neurocognitive and behavioral impairments, along with greater frequencies of inflammatory myeloid subsets, neuroinflammation, and dysregulated T cell subsets, including Tregs. Sex differences in inflammation and cognitive phenotypes during PASC were mediated by the presence of two X chromosomes. XX animals independent of chromosome Y presented with neuroinflammation and PASC along with infection-induced upregulation of the X-linked genes Xist and Tlr7 that regulate inflammation and chronic disease outcomes.","version":"1.1","doi":"10.1101/2025.10.13.682101","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.10.681726","pub_date":"2025-10-12","title":"In silico analysis of the Chikungunya virus and SARS-CoV-2 Macrodomain","abstract":"The macrodomain of the Chikungunya virus NSP3 protein (ChikV Mac1) hydrolyzes mono-ADP ribose post-translational modifications on the human host proteins. Mutations in Mac1 reduce ChikV virulence. Thus, ChikV Mac1 is a viral drug target. While no potent ChikV Mac1 inhibitors are available, high-affinity inhibitors were developed for Mac1 in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we rationalize this difference in the ligand binding affinity in terms of three differences in the binding site structure and dynamics. First, the apo ChikV Mac1 binding site exhibits substantially more conformational heterogeneity than that of SARS-CoV-2, based on microsecond-scale molecular dynamics simulations; it also has a less druggable binding site, based on program SiteMap. Second, water binding sites overlapping with the ligand binding site in ChikV Mac1 are predicted by program WaterMap to have a stronger affinity for water molecules than the corresponding sites in SARS-CoV-2 Mac1, thus decreasing ligand binding affinity. Finally, a smaller number of Mac1 residues interacts persistently with ADP-ribose in holo ChikV Mac1 than in SARS-CoV-2. With these rationalizations in hand, we designed ligands of ChikV Mac1 using a fragment growth strategy; subsequent molecular dynamics simulations of a representative ligand in complex with ChikV Mac1 substantiated our design.","version":"1.1","doi":"10.1101/2025.10.10.681726","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.30.578034","pub_date":"2025-10-10","title":"Stratification of viral shedding patterns in saliva of COVID-19 patients","abstract":"Living with COVID-19 requires continued vigilance against the spread and emergence of variants of concern (VOCs). Rapid and accurate saliva diagnostic testing, alongside basic public health responses, is a viable option contributing to effective transmission control. Nevertheless, our knowledge regarding the dynamics of SARS-CoV-2 infection in saliva is not as advanced as our understanding of the respiratory tract. Here we analyzed longitudinal viral load data of SARS-CoV-2 in saliva samples from 144 patients with mild COVID-19 (a combination of our collected data and published data). Using a mathematical model, we quantified individual-level viral dynamics and stratified them into three groups using a clustering approach. Notably, the three groups exhibited distinct differences viral RNA detection durations: 11.5 days (95% CI: 10.6 to 12.4), 17.4 days (16.6 to 18.2), and 30.0 days (28.1 to 31.8), respectively. Surprisingly, this stratified grouping remained unexplained despite our analysis of 47 types of clinical data, including basic demographic information, clinical symptoms, results of blood tests, and vital signs. Additionally, we quantified the expression levels of 92 micro-RNAs in a subset of saliva samples, but these also failed to explain the observed stratification, although the mir-1846 level may have been weakly correlated with peak viral load. Our study provides insights into SARS-CoV-2 infection dynamics in saliva, highlighting the challenges in predicting the duration of viral RNA detection without indicators that directly reflect an individual\u2019s immune response, such as antibody induction. Given the significant individual heterogeneity in the kinetics of saliva viral shedding, identifying biomarker(s) for viral shedding patterns will be crucial for improving public health interventions in the era of living with COVID-19.","version":"1.2","doi":"10.1101/2024.01.30.578034","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.09.681538","pub_date":"2025-10-10","title":"Clustering of SARS-CoV-2 membrane proteins in lipid bilayer membranes","abstract":"The accumulation of viral structural proteins along the ER-Golgi intermediate compartment (ERGIC) membrane leads to SARS-CoV-2 self-assembly and budding, driven by the interactions between these proteins, RNA and the ERGIC membrane. The membrane protein (M) is believed to interact with other structural proteins and form clusters needed for the induction of membrane curvature that facilitates virion formation. However, the role played by direct and membrane-mediated interactions between M proteins and their interactions with other proteins in the clustering process remains unclear. Here, we utilize a combination of all-atom molecular dynamics (MD) simulations, continuum modeling and experiments to show that M-M interactions are sufficient to drive clustering in ERGIC-like lipid bilayers in the absence of other proteins or RNA. Using all-atom MD simulations we were able to estimate the membrane thinning induced by M proteins and the resulting membrane-mediated M-M interaction. Combining this with a continuum model that describes the evolution of M protein density in a planar lipid membrane, we identified the existence of a critical, direct M-M interaction energy needed for cluster assembly at a given density. By comparing the model predictions with analysis of atomic force microscopy images of M protein clusters in supported lipid bilayers, we were able to estimate the direct M-M interaction energy and found it to be significantly larger than the membrane mediated interaction energy. Our work therefore establishes that M protein interactions are sufficient to drive clustering and provides a quantitative understanding of the role played by direct and membrane-mediated interactions of M proteins in viral assembly and budding.","version":"1.1","doi":"10.1101/2025.10.09.681538","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.09.681417","pub_date":"2025-10-09","title":"A novel vaccination strategy induces vaccine-specific mucosal responses at port of viral entry and exit: using systemic SARS-CoV-2 vaccination as a test case","abstract":"Respiratory infectious diseases are associated with substantial morbidity and mortality rates worldwide, especially at the extremes of age and in immunocompromised individuals. Over the past two decades, respiratory viruses have driven nine epidemics and pandemics (including the COVID-19 pandemic); and their ongoing emergence, persistence and evolution continue to threat global health security. The upper respiratory tract (URT) represents the primary access point for respiratory viruses, where initial host infection occurs. Vaccine-mediated URT mucosal memory responses can control infection, prevent transmission and limit viral evolution. However, vaccines against respiratory viruses predominantly have systemic administration routes that elicit strong responses in the circulation to prevent severe respiratory disease, but do not effectively block infection and onward community transmission. To overcome the limitations of systemic vaccination alone, we present a novel intervention combining systemic vaccination with targeted non-antigenic inflammatory stimulation of the URT, to induce vaccine-specific immune responses in the URT mucosa. Using SARS-CoV-2 vaccination as a test case, we demonstrate for the first time, that intranasal coadministration of exogenous IFN-\u03b1 as a targeted inflammatory URT signal, alongside systemic vaccination, induces vaccine-specific T-cell responses in the URT.","version":"1.1","doi":"10.1101/2025.10.09.681417","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.16.628620","pub_date":"2025-10-09","title":"ConvMut: Exploration of viral convergent mutations along phylogenies","abstract":"Convergent evolution in protein antigens is common across pathogens and has also been documented in SARS-CoV-2 (hCoV-19); the most likely reason is the need to evade the selective pressure exerted by previous infection- or vaccine-elicited immunity. There is a pressing need for tools that allow automated analysis of convergent mutations. In response to this need, we developed ConvMut, a tool to analyze genetic sequence data to identify patterns of recurrent mutations in SARS-CoV-2 evolution. To this end, we exploited the granular phylogenetic tree representation developed by PANGO, allowing us to observe what we call deltas, i.e., groups of mutations that are acquired on top of the immediately upstream tree nodes. Deltas comprise amino acid substitutions, insertions, and deletions. ConvMut can perform individual protein analysis to identify the most common single mutations acquired independently in a given subtree (starting from a user-selected root). Such mutations are represented in a barplot that can be sorted by frequency or position, and filtered by region of interest. Lineages are then gathered into clusters according to their sets of shared mutations. Finally, an interactive graph orders the evolutionary steps of clusters, details the acquired amino acid changes for each sublineage, and allows us to trace the evolutionary path until a selected lineage. Other unique tools are paired with the main functionality of ConvMut to support a complete analysis, such as a frequency analysis for a given nucleotide or amino acid changes at a given residue across a selected phylogenetic subtree. ConvMut will facilitate the design of antiviral anti-Spike monoclonal antibodies and Spike-based vaccines with longer-lasting efficacy, minimizing development and marketing failures.","version":"1.2","doi":"10.1101/2024.12.16.628620","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.08.680531","pub_date":"2025-10-09","title":"A searchable database of publications using fluorescent probes and flow cytometry to study antigen-specific B cells","abstract":"The study of antigen-specific B cells has resulted in important advances in all fields of immunology, the development of experimentally and/or clinically useful antibodies, and as a starting point for rationally designed vaccine antigens. A key innovation allowing for widespread study of antigen-specific B cells was the development of fluorescent antigen probes for use with flow cytometry. Initially these studies were mostly focused upon B cells specific for a variety of model antigens, but over the past decade focus has shifted towards the study of B cells specific for antigens from pathogens such as SARS-CoV-2, HIV, and Influenza virus. Importantly however, these types of approaches have been used for hundreds of different antigens and could be used for thousands more. Unfortunately, studies of B cells specific for an antigen of interest are not easily searchable on current publication databases since these assays are often a small portion of a larger publication. To overcome this, we built a searchable database of studies analyzing antigen-specific B cells by flow cytometry using fluorescent antigen probes that is located at www.immunology.virginia.edu/Taylor/Bcell/Database.php. Using this database, we assessed the number of publications per year revealing rapid growth in the use of this approach in recent years. While much of this rapid growth was focused upon the assessment of B cells specific for SARS-CoV-2, HIV-1, or Influenza virus, studies assessing B cells specific for hundreds of different antigens derived from numerous microbes, animals, plants, or other sources can be found in the database. Combined, the antigen-specific B cell database was built to facilitate identification of studies assessing these cells and for analysis of the field as a whole.","version":"1.1","doi":"10.1101/2025.10.08.680531","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.08.681090","pub_date":"2025-10-08","title":"PERK inhibition rewires translational and CMGC protein kinase networks into an antiviral state","abstract":"Protein kinases (PKs) are central regulators of cellular signaling, yet only a small fraction of the human kinome is targeted therapeutically, and kinase\u2013substrate relationships remain incompletely defined. Here, we systematically characterize kinome regulation during human coronavirus 229E (HCoV-229E) infection across transcriptomic, translational, proteomic, and phospho-proteomic layers. We reveal that pharmacological inhibition of the ER stress sensor kinase PERK reprograms host protein biosynthesis and phospho-proteomic landscapes, simultaneously blocking viral nucleocapsid phosphorylation and modulating multiple host kinases. This rewiring antagonizes virus-induced translational shutdown, along with pronounced regulation of the CMGC kinase family, a pattern conserved in SARS-CoV and SARS-CoV-2 infected cells. Comparative analyses with PERK depletion distinguish on-target from off-target effects of PERK inhibition. Our findings uncover the kinome-scale consequences of PERK perturbation in coronavirus infection and demonstrate how the polypharmacology of PERK inhibitors can be harnessed to establish a potent antiviral state, revealing new avenues for host-directed antiviral strategies.","version":"1.1","doi":"10.1101/2025.10.08.681090","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.19.677134","pub_date":"2025-10-08","title":"Methionine matters: a common mechanism of viral inhibition of host defense identified via AI-assisted molecular dynamics","abstract":"Diverse groups of viruses infecting higher eukaryotes inhibit mRNA export via physical blockage of the Rae1-Nup98 complex within the host cell\u2019s nucleopore. This is thought to most often involve the critical placement of hydrophilic flanked single methionine residues along polypeptide extensions that reach into the nucleopore. However, it is unknown how this presumably conserved mechanism might function across diverse viral taxa. Here we employ a comparative molecular dynamics (MD) approach comparing motions of wild-type and mutant viral proteins in Rae-Nup98 bound vs. unbound states. Our comparisons of MD simulations are enhanced by kernel-based denoising allowing the isolation of non-random functional dynamics from random thermal noise. We demonstrate that despite large structural differences, three evolutionarily distinct viral systems (i.e. VSV M protein, SARS-CoV2 ORF 6, and KSHV ORF 10) share nearly identical single methionine-dependent functional dynamics related to the host cell inhibition of nuclear transport. This finding strongly supports a convergently-evolved common functional mechanism across viruses involving specific structural placement of non-polar residues like methionine and potentially providing a common therapeutic target for broad spectrum anti-viral treatment.","version":"1.2","doi":"10.1101/2025.09.19.677134","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.30.679586","pub_date":"2025-10-07","title":"Identification and characterization of a SARS-CoV-2 Mpro G23 deletion ensitrelvir-resistant mutant","abstract":"Ensitrelvir is an antiviral drug that specifically targets the main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been approved for the treatment of coronavirus disease 2019 (COVID-19) due to the conservation of its target protein which is essential in the viral lifecycle. However, SARS-CoV-2 could introduce mutations in the viral proteins to confer resistance to antivirals. Thus, screening for drug-resistant SARS-CoV-2 mutants and elucidating their resistant mechanisms are critical for guiding the selection of effective antiviral therapies. Here, we utilized a luminescent attenuated SARS-CoV-2 (\u03943a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a G23 deletion (G23del) in Mpro with high resistance (>1,000 fold) to ensitrelvir, but not to the other Mpro inhibitor (nirmatrelvir) or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del was confirmed by generating a recombinant luminescent attenuated SARS-CoV-2 containing G23del in the non-structural protein 5 (NSP5) gene (\u03943a7b-Nluc G23del). \u03943a7b-Nluc G23del exhibited significant resistance to ensitrelvir in both cultured cells an in K18 hACE2 transgenic mice. Binding affinity revealed that G23del mutation substantially altered Mpro binding affinity for ensitrelvir but not nirmatrelvir. In conclusion, our results demonstrate that G23del in Mpro can confer high resistance to ensitrelvir. Positively, G23del in Mpro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating infections with SARS-CoV-2 containing G23del with these other approved antivirals. The clinical use of SARS-CoV-2 antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for FDA-approved antivirals. Our study addresses this by employing a luminescent attenuated virus platform (\u03943a7b-Nluc WT) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we have identified a G23 deletion (G23del) in SARS-CoV-2 Mpro, which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer more than 1,000-fold increased resistance to ensitrelvir, SARS-CoV-2 containing G23del remained sensitive to other Mpro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using \u03943a7b-Nluc to safely identify and characterize drug resistant viruses without the biosafety concern of using virulent SARS-CoV-2 and advance the design of next-generation antiviral drugs.","version":"1.1","doi":"10.1101/2025.09.30.679586","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.02.679237","pub_date":"2025-10-06","title":"PM2.5 Exposure Facilitates SARS-CoV-2 Infection through ACE2/TMPRSS2 Regulation and Suppression of Anti-Viral Response","abstract":"Epidemiological studies suggest an interaction between air pollution including particulate matter <2.5 \u00b5m (PM2.5) and coronavirus disease 2019 (COVID-19) mortality and morbidity; however, the underlying mechanisms are not clear. The aim of our study was to investigate effects of PM2.5 on viability, epithelial integrity, and cellular entry of SARS-CoV-2 into airway epithelial cells, and the mechanisms involved. We exposed Calu-3 airway epithelial cell cultures to PM2.5 (10, 50, and100 \u00b5g/ml) and SARS-CoV-2 (MOI 0.01) for 24 h. The viability of Calu-3 cells and epithelial barrier integrity were determined using MTT assay and immunofluorescence staining for Zonula Occludens-1, respectively. mRNA expression for viral entry-related genes such as angiotensin converting enzyme (ACE)2 and transmembrane protease, serine (TMPRSS)2, and inflammatory and inflammasomal genes, including interleukin (IL)-8,IL-6, nuclear factor (NF)-\u03baB p65 (RELA), JNK, c-JUN, Caspase-1, IL-1\u03b2, NLRP3, was analyzed by qRT-PCR. Intracellular viral spike protein intensity and RNA-dependent RNA polymerase (RdRP) expression were determined using immunofluorescence staining and qRT-PCR, respectively. ELISA was used to analyze the release of inflammatory cytokines (IL-8, IL-6, and GM-CSF). Higher concentrations of 100\u00b5g/ml PM2.5 decreased Calu-3 cell viability (p=0.02) and deteriorated epithelial barrier integrity, while 50 \u00b5g/ml of PM2.5 (p<0.01) induced mRNA expression for ACE2 and TMPRSS2. Although PM2.5 alone decreased c-JUN, it did not alter the expression of mRNA for JNK and RELA. In contrast, a combination of SARS-CoV-2 and PM2.5 led to a significant increase in mRNA for both JNK and RELA (p < 0.05 and p < 0.01, respectively) and attenuated c-JUN expression. Moreover, our results indicated an increase in the expression of IL-1\u03b2, IL-6, and GM-CSF following exposure to PM2.5 and PM2.5 + SARS-CoV-2, whereas IL-8 was induced only by SARS-CoV-2 exposure. Co-incubation of Calu-3 cells with PM2.5 and SARS-CoV-2 leads to a decrease in IL-8, IL-1\u03b2, Caspase-1 (CASP-1), and Interferon gamma (IFNG) expression. Finally, the viral load (RdRP) also increased in the presence of both PM2.5 and the SARS-CoV-2 group. Our findings have demonstrated that PM2.5 impaired epithelial integrity and cell viability, whereas it increased the mRNA expression for ACE2 and TMPRSS2, and induced inflammatory changes in Calu-3 cells incubated with SARS-CoV-2. These findings suggest that PM2.5 can facilitate the entry of SARS-CoV-2 into airway epithelial cells, and that both PM2.5 and SARS-CoV-2 can decrease the inflammatory and antiviral responses of the host cell.","version":"1.2","doi":"10.1101/2025.10.02.679237","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.08.606661","pub_date":"2025-10-06","title":"The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2","abstract":"SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Standard cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner. However, a stronger antiviral effect for AVI-4206 is observed in human airway organoids and peripheral blood monocyte-derived macrophages. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.","version":"1.4","doi":"10.1101/2024.08.08.606661","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.03.679858","pub_date":"2025-10-06","title":"Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains","abstract":"The ease with which emerging SARS-CoV-2 variants escape neutralizing antibodies limits protection afforded by a prior exposure, be it infection or vaccination. While rare, broadly neutralizing antibodies with activity towards diverse sarbecoviruses have been detected in convalescent serum. Motivated by findings that plasma responses show increased neutralization breadth and potency with continued antigen exposure, we isolated monoclonal antibodies (mAbs) after a SARS-CoV-2 re-infection and compared them to those isolated one year prior, after the first breakthrough infection. Among clonal lineage members identified at both time points, mAbs from the later time point showed improved neutralization potency and breadth. One mAb isolated after re-infection, C68.490, targets a conserved region in the receptor binding domain core and shows remarkable activity not only against SARS-CoV-2 variants, but also diverse sarbecoviruses from more distant clades present in animal reservoirs. These findings suggest that a focus on individuals with diverse and repeated antigen exposure could lead to identification of antibodies with therapeutic utility not just towards current and future SARS-CoV-2 variants, but also distant sarbecoviruses in the event of a future spillover.","version":"1.1","doi":"10.1101/2025.10.03.679858","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.31.644992","pub_date":"2025-10-06","title":"Modeling viral and bacterial infections in human lung organotypic systems reveals strain specific host responses","abstract":"In this study, we developed novel lung organoid-on-chip models that elucidate differential human tissue response to various strains of respiratory pathogens: Streptococcus pneumoniae and SARS-CoV-2. We show that human fetal-derived distal lung epithelial cells are readily expandable in 3D as organoids, thereby providing a highly sustainable source of lung progenitor cells. These 3D organoid progenitors can then be induced to produce airway and alveolar organoids on microfluidic devices. Upon challenge with Streptococcus pneumoniae, a bacterium known to cause pneumonia, a rapid and strain-dependent colonization was observed at the epithelial surface of alveolar chips. We also assessed SARS-CoV-2 infection in the alveoli-on-chip system and observed that the Delta variant exhibited greater infectivity as compared to the Omicron BA.5. Both SARS-CoV-2 variants induced potent interferon responses and triggered the expression of different interferon-stimulated genes. Our results demonstrate that strain-specific host defense mechanisms can be recapitulated in human-organoid-based microfluidic systems, paving the way for the use of such platforms for more targeted assessments of human response to novel emergent pathogen strains. Human fetal epithelial lung stem cells can be expanded as multipotent organoids and differentiated into both airway or alveolar organoids Multipotent lung organoids efficiently produce functional epithelia of small airway or alveoli when grown on-chip. Streptococcus pneumoniae inoculation in alveoli-on-chip mimics the early stages of bacterial colonization in lung epithelia Alveoli on-chip system recapitulates variant-specific interactions. SARS-CoV-2 Delta replicates but not Omicron BA.5. Robust interferon response upon SARS-CoV-2 infection shows Alveoli on-chip can model innate immune responses.","version":"1.3","doi":"10.1101/2025.03.31.644992","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.04.680210","pub_date":"2025-10-06","title":"Bioengineered human tonsil organoids as an immuno-engineering platform for evaluating immune functions","abstract":"Although animal models remain the preclinical gold standard, they are constrained by interspecies differences in adaptive immunity, including antibody class-switching, affinity maturation, and multicellular interactions. To address this gap, we established a more physiologically relevant platform, a bioengineered tonsil organoid (BTO) system that combines autologous tonsil-derived stromal cells with immune cells to generate highly uniform organoids, preserving multicellular complexity and sustained stromal-immune interactions. BTO cultures mounted robust, antigen-specific recall responses to influenza, tetanus, and COVID-19 antigens. Notably, stimulation with ovalbumin and SARS-CoV-1 spike protein expanded antigen-recognizing B cells, indicating capacity for na\u00efve responses. Digital spatial transcriptomics and proteomics analysis confirmed immune-stromal crosstalk underpinning both humoral and cellular responses of BTO after challenge. These data highlight stromal cells as active scaffolds that not only maintain 3D architecture but also facilitate immune activation, memory recall, and recognition of na\u00efve antigens. Together, BTO offers a human-relevant model for mechanistic immunology and for pre-clinical evaluation on immunotherapies and vaccines not achievable with 2D cultures or animal models.","version":"1.1","doi":"10.1101/2025.10.04.680210","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.03.680366","pub_date":"2025-10-05","title":"Targeting a macrophage stemness factor to mitigate diseases post respiratory viral infection","abstract":"Tissue-resident alveolar macrophages (AMs) rely on intrinsic stem-like programs for self-renewal and maintenance, yet the transcriptional networks that support these functions and their relevance to post-viral lung disease remain largely unknown. Here, we identify TCF4 (Tcf7l2) as a critical transcription factor that governs AM maturation and stemness. Loss of TCF4 impaired AM proliferation, shifted their identity toward a pro-inflammatory phenotype, and exacerbated host morbidity following influenza or SARS-CoV-2 infection. Conversely, enforced TCF4 expression promoted the expansion of mature AMs, and supported lung recovery, thereby protecting against severe acute viral disease. Mechanistically, TCF4 antagonized \u03b2-catenin-driven inflammatory transcription while preserving oxidative phosphorylation, defining a reciprocal regulatory axis essential for AM function. Notably, respiratory viral infections and exuberant interferon signaling suppressed TCF4 expression, which remains chronically reduced in murine and human lungs with post-COVID fibrosis. This downregulation is associated with persistent KRT8hi dysplastic epithelium and collagen deposition. Moreover, aging diminished TCF4 levels and enforced TCF4 expression dampened age-associated decline of AM self-renewal. Furthermore, in vivo TCF4 overexpression after viral clearance enhanced mature AM accumulation, promoted lung epithelium regeneration, attenuated chronic tissue fibrosis and restored pulmonary physiologial function in aged lungs in a model of persistent pulmonary fibrosis post-acute viral infection. These findings have established TCF4 as a key regulator of AM stemness and identified a promising therapeutic target for long COVID and related chronic lung diseases through the modulation of embryonic-derived macrophage regenerative capacity by targeting TCF4.","version":"1.1","doi":"10.1101/2025.10.03.680366","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.03.680220","pub_date":"2025-10-03","title":"Investigation of anti-SARS CoV-2 multimeric bicyclic peptide inhibitors in a range of pre-clinical therapeutic settings","abstract":"The spread of respiratory viruses, such as Influenza and SARS-CoV-2 has presented significant challenges over the last 30 years with few effective therapeutic options available to this day. Bi-cyclic peptides represent a unique, modular, modality in the antiviral armamentarium against future pandemics. This study provides a deeper evaluation of multivalent bi-cyclic (Bicycle\u00ae) molecule efficacy in several preclinical SARS-CoV-2 challenge settings. We explore both pre-exposure prophylaxis and post-exposure therapeutic settings via subcutaneous and intranasal routes of administration. We contextualize this further in bespoke scenarios of immune compromisation, and viral transmission. Promisingly, in all studies we observe efficacy, significantly reducing infectious viral burden at each study endpoint. These data further support candidacy of Bicycle molecules as a differentiated antiviral therapeutic class in the context of pandemic preparedness. The COVID-19 pandemic, triggered a rapid wave of innovation, accelerating the delivery of new vaccine technology and anti-viral treatments. In our first paper, we described the discovery and molecular optimization of Bicycle molecules as a novel drug class for the potential treatment of SARS CoV-2. Here, we have performed deeper characterization of these molecules in established animal models that simulate SARS-CoV-2 transmission, testing more convenient delivery routes, such as intra-nasal. The Bicycle molecules demonstrated positive outcomes in each of these studies and suggest that Bicycle molecules, as convenient and effective anti-viral treatments, could be an important addition to help future preparedness against new viral pandemics.","version":"1.1","doi":"10.1101/2025.10.03.680220","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.03.680253","pub_date":"2025-10-03","title":"Immune Cell Dysfunction of SARS-CoV-2: Mathematical Modeling of the Within-Host Immune Dynamics","abstract":"The COVID-19 pandemic has spurred extensive research into viral transmission and control, yet the mechanisms of the human immune response to SARS-CoV-2 remain incompletely understood, particularly the role of natural killer (NK) cells and cytokine regulation in disease severity. Mathematical modeling provides a powerful approach to bridge this gap by linking viral dynamics with immune interactions. In this work, we develop a mechanistic within-host model, formulated in a system of coupled ordinary and delayed differential equations, to investigate the contributions of NK cell activity, interferon signaling, and pro-inflammatory cytokines to viral clearance and disease outcome. Model parameters are estimated from experimental data, and computational simulations are used to explore how dysregulated NK responses and cytokine feedback loops may drive divergent clinical outcomes. Local sensitivity analysis identifies the most influential parameters shaping host\u2013pathogen dynamics, highlighting potential control points for intervention. In addition, knockdown simulations are performed to mimic potential therapeutic interventions, allowing us to evaluate their advantages and limitations in silico. These findings provide mechanistic insights into COVID-19 immune dynamics and offer a foundation for guiding the design of future treatment strategies. We develop a within-host mathematical model of the SARS-CoV-2 immune response. The model incorporates key cytokines and immune cells including Natural Killer cells. Numerical simulations reproduce cytokine storms and NK cell dysfunction in severe disease. Sensitivity analysis identifies parameter impact and potential therapeutic interventions.","version":"1.1","doi":"10.1101/2025.10.03.680253","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.22.677528","pub_date":"2025-10-03","title":"Computational Analysis of Silent Mutation Effects on SARS-CoV-2 RNA\u2013Host RNA-Binding Protein Interactome","abstract":"RNA-Binding proteins (RBPs) play critical roles in host-virus interaction. They facilitate the regulation of viral RNA (vRNA) turnover by recognizing and forming complexes with the vRNA structure via specific RNA motifs-RNA binding domain interaction. However, due to consistent evolving nature of viruses, silent mutations in the viral genome can impact RBP-vRNA binding thereby altering the RNA processing. While efforts have been made in characterizing other forms of mutations leading to changes in amino acids sequence in SARS-CoV-2 variants, details on how silent mutations impact RBP-vRNA interaction remain limited. Here, we use extensive in silico mutagenesis to introduce silent mutations in the SARS-CoV-2 genome to generate four different synthetic variants and map the interaction of the variants and the wild-type with a catalogue of human RBPs. Our result shows variation in accumulation and reduction of the RBPs binding motifs in the variants compared to the virus reference sequence on a global scale and at the UTRs. The majority of the RBPs with AU-rich binding motifs are reduced in the variants, while RBPs with mostly GC-rich motifs accumulate more binding positions, suggesting that a single change from U/A to G/C and vice versa can impact RBP- viral interactions. Furthermore, we use structural analysis to show the interaction of the vRNA with PUF60 and KHDRBS3 proteins, two RBPs that have not been previously implicated in SARS-CoV- 2 interactome. Our findings show that loss to the conserved poly(U) in PUF60 binding motifs in some of the variants affects its interaction with the protein at the 5\u2032 end, which may disrupt the function of the protein as an anti-viral RNA regulator. We also predicted the key residues in KHDRBS3 interacting with its binding motif in the wild-type at the 3\u2032 end, while noting that the vRNA structural changes in the variants may contribute to the loss of this interaction. Overall, our predictions contribute to the insights into virus evolution and pathogenicity of potential new variants due to the impact of synonymous changes in the nucleotide sequences on protein-RNA interaction.","version":"1.1","doi":"10.1101/2025.09.22.677528","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.28.569052","pub_date":"2025-10-03","title":"Identification of Antigen-Specific T Cell Receptors with Combinatorial Peptide Pooling","abstract":"T cell receptor (TCR) repertoire diversity enables the antigen-specific immune responses against the vast space of possible pathogens. Identifying TCR-antigen binding pairs from the large TCR repertoire and antigen space is crucial for biomedical research. Here, we introduce copepodTCR, an open-access tool to design and interpret high-throughput experimental TCR specificity assays. copepodTCR implements a combinatorial peptide pooling scheme for efficient experimental testing of T cell responses against large overlapping peptide libraries, that can be used to identify the specificity of (or \u201cdeorphanize\u201d) TCRs. The scheme detects experimental errors and, coupled with a hierarchical Bayesian model for unbiased interpretation, identifies the response-eliciting peptide sequence for a TCR of interest out of hundreds of peptides tested using a simple experimental set-up. Using in silico simulations, we demonstrate the varied experimental settings in which copepodTCR yields efficient and interpretable TCR specificity results. We validated our approach on a library of 253 overlapping peptides covering the SARS-CoV-2 spike protein, split across 12 pools. A single stimulation with combinatorial pools identified the correct epitope of two TCRs with known specificity and then deorphanized two SARS-CoV-2 associated TCRs shared among a large cohort of COVID-19 patients. We provide experimental guides to efficiently design larger screens covering thousands of peptides which will be crucial to identify antigen-specific T cells and their targets from limited clinical material.","version":"1.3","doi":"10.1101/2023.11.28.569052","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.30.675572","pub_date":"2025-10-03","title":"Discovery of rare antigen-specific TCRs via replicate profiling","abstract":"Development of effective vaccines and targeted immunotherapies for cancer, autoimmunity, allergy, and infectious diseases requires comprehensive understanding of functionality and antigenic specificity of involved T cell clones. A major technical challenge remains the high-throughput identification of antigen-specific T cells. Here, we present a rapid cost-efficient TCR discovery assay starting from PBMC that enables ultra-sensitive discovery of clonal alpha-beta paired TCRs responding to individual or pooled peptides. In a small-scale experiment with a single donor, assay identified over 90 SARS-CoV-2-specific CD4+ and CD8+ TCR\u03b2 clonotypes, validated by clonal tracking and comparison against known SARS-CoV-2-specific TCRs. Positioning within the scRNA-Seq map revealed distinct helper T cell subsets involved in primary and secondary response. Further validation in a cohort of five donors identified nearly 1,000 CD4+ and CD8+ TCR clonotypes specific to viral and fungal peptide antigens. The assay demonstrated exceptional sensitivity in capturing low-frequency clones and allowed accurate TCR\u03b1/TCR\u03b2 pairing, validated using single-cell transcriptomics. The ability to capture low-frequency antigen-specific TCRs, combined with detailed scRNA-Seq annotation, establishes an integrated pipeline that links antigen-responsive clones to their precise functional phenotypes. This platform provides a robust foundation for dissecting T cell roles in health and disease and accelerates the development of vaccines and immunotherapies.","version":"1.1","doi":"10.1101/2025.09.30.675572","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.30.679491","pub_date":"2025-10-02","title":"Oral Microbiota Composition in Children and Adults During Spanish COVID-19 Lockdown: Impact of Home Self-Confinement and SARS-CoV-2 infection","abstract":"The COVID-19 pandemic changed society\u2019s habits and customs due to the social restrictions and health measures imposed during the first half of 2020. This study analyzes the composition of the oral microbiota in relation to age, household cohabitation, SARS-CoV-2 infection, and COVID-19 severity among children and adults under home confinement in Barcelona, Spain. A prospective study conducted involving children and adults confined during the COVID-19 pandemic in the Barcelona Metropolitan Area between April and June 2020 included multiple cases of several participants living within the same family household. Saliva samples were collected from all participants, and microbiota composition was characterized through 16S rRNA gene sequencing. A total of 142 adults and 265 children living in 121 family households were included in the study. All 142 adults had a prior confirmed SARS-CoV-2 infection, and 20 (14.08%) of them had a history of severe COVID-19. SARS-CoV2 infection was detected in 58/265 (21.89%) of children; all of them were asymptomatic. Oral microbiota composition and diversity did not differ by SARS-CoV-2 infection status in children. In contrast, adults with severe COVID-19 exhibited lower microbiota diversity and distinct microbiota composition compared to those with mild disease symptoms. Age-related differences in oral microbiota composition were marked in the younger children groups. Additionally, cohabiting individuals shared more Amplicon Sequence Variants (ASVs) than non-cohabitants. Age and cohabitation strongly influenced oral microbial composition. Our study demonstrates that oral microbiota composition in adults varies according to COVID-19 severity, whereas such microbial shifts are not observed in asymptomatic pediatric populations, regardless of infection status.","version":"1.1","doi":"10.1101/2025.09.30.679491","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.01.679787","pub_date":"2025-10-02","title":"Memory B cell Development in Response to mRNA SARS-CoV-2 and Nanoparticle Immunization in Mice","abstract":"Nanoparticle immunogens excel at rapidly inducing high levels of circulating antibodies and are being deployed as part of several novel vaccines. However, their ability to elicit memory B cell responses is less well understood. Here we compared serologic and memory B cell responses to prime boost vaccination with either SARS-CoV-2 Wuhan-Hu-1 mRNA vaccine, or protein nanoparticles: SARS-CoV-2 B.1.351 homotypic containing a single receptor binding domain (RBD); (homotypic beta) or a combination of different Sarbecovirus RBDs (mosaic 8b), respectively. The memory B cells elicited by the 3 vaccine regimens showed closely related antibody sequences, similar levels of somatic mutation and clonal diversity. The breadth of serologic responses elicited by the mosaic nanoparticles were comparable to the homotypic nanoparticle and superior to the mRNA vaccine for some mismatched strains. However, serum neutralizing titers to SARS-CoV-2 were highest after mRNA vaccination. The three vaccines elicited memory B cells that produced antibodies specific to a broad range of epitopes on the RBD that differed in a way that may reflect epitope masking. Monoclonal antibodies derived from memory B cells elicited by the mosaic 8b nanoparticle showed greater breadth against a panel of SARS-CoV-2 variants and SARS-CoV. Nanoparticle vaccines are promising next-generation vaccine candidates, yet their capacity to generate durable memory B cell responses remains incompletely understood. We compared immune responses following SARS-CoV-2 mRNA, homotypic beta nanoparticle, or mosaic 8b nanoparticle vaccination in mice. Serum antibody neutralizing responses against a panel of SARS-CoV-2 variants elicited by an mRNA vaccine were equivalent or superior to those elicited by mosaic 8b nanoparticle vaccines. However, the monoclonal antibodies derived from memory B cells elicited by the mosaic 8b nanoparticle showed better neutralizing breadth against heterologous pseudoviruses. These findings highlight individual strengths of mRNA and nanoparticle vaccines and show that mosaic 8b nanoparticle immunogens can enhance the breadth of memory B cell-derived antibodies.","version":"1.1","doi":"10.1101/2025.10.01.679787","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.01.679928","pub_date":"2025-10-02","title":"Leveraging Multimodal Large Language Models to Extract Mechanistic Insights from Biomedical Visuals: A Case Study on COVID-19 and Neurodegenerative Diseases","abstract":"The COVID-19 pandemic has intensified concerns about its long-term neurological impact, with growing evidence linking SARS-CoV-2 infection to neurodegenerative diseases (NDDs) such as Alzheimer\u2019s (AD) and Parkinson\u2019s (PD). Patients with these conditions not only face higher risk of severe COVID-19 outcomes but may also undergo accelerated cognitive and motor decline following infection. Proposed mechanisms\u2014ranging from neuroinflammation and blood\u2013brain barrier disruption to abnormal protein aggregation\u2014closely mirror core features of neurodegenerative pathology. Yet, current knowledge is fragmented across text, figures, and pathway diagrams, hindering integration into computational models capable of uncovering systemic patterns. To address this gap, we applied GPT-4 Omni (GPT-4o), a multimodal large language model, to extract mechanistic insights from biomedical figures. Over 10,000 images were retrieved through targeted searches on COVID-19 and neurodegeneration; after automated and manual filtering, a curated subset was analyzed. GPT-4o extracted biological relationships as semantic triples, which were grouped into six mechanistic categories\u2014including microglial activation and barrier disruption\u2014using ontology-guided similarity and assembled into a Neo4j knowledge graph. Accuracy was evaluated against a gold-standard dataset of expert-annotated images using BioBERT-based semantic matching. This evaluation also enabled prompt tuning, threshold optimization, and hyperparameter assessment. Results demonstrate that GPT-4o successfully recovers both established and novel mechanisms, yielding interpretable outputs that illuminate complex biological links between SARS-CoV-2 and neurodegeneration. This study showcases the potential of multimodal LLMs to mine biomedical visual data at scale. By complementing text mining and integrating figure-derived knowledge, our framework advances understanding of COVID-19\u2013related neurodegeneration and supports future translational research.","version":"1.1","doi":"10.1101/2025.10.01.679928","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.02.680065","pub_date":"2025-10-02","title":"Directed evolution of a stem-helix\u2013targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity","abstract":"Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report on the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that optimize binding and stabilize epitope engagement. Our findings demonstrate the potential of rational antibody engineering to expand bnAb breadth across divergent betacoronaviruses. This work supports the development of engineered bnAbs and S2-targeted vaccines for broad betacoronavirus countermeasures and highlights strategies to achieve cross-lineage immunity for future pandemic threats. The persistent emergence of new SARS-CoV-2 variants of concern that evade neutralizing antibody responses and other zoonotic betacoronaviruses with pandemic potential have provided strong motivation to develop broadly neutralizing antibodies (bnAbs) that target more conserved regions of the spike protein in sarbecoviruses and other betacoronaviruses. Here, we employed a directed evolution strategy to engineer the sarbecovirus-neutralizing antibody CC65.1, which targets the conserved S2 stem helix, to enhance its binding affinity with the MERS-CoV stem helix region, thereby conferring MERS-CoV neutralization. High-resolution structural studies of engineered CC65.1 revealed that key mutations reshape the paratope to better accommodate and stabilize the MERS-CoV S2 stem helix, resulting in increased binding affinity and neutralization potency. This study emphasizes the critical role of affinity maturation in expanding neutralization breadth and provides valuable insights for design of bnAbs to prevent and treat pandemic threats by betacoronaviruses.","version":"1.1","doi":"10.1101/2025.10.02.680065","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.30.679521","pub_date":"2025-10-01","title":"Crystallographic characterisation and development of bi-substrate inhibitors of coronavirus nsp14 methyltransferase","abstract":"SARS-CoV-2 non-structural protein 14 (nsp14) is essential for viral mRNA cap guanine-N7 methylation and represents a promising but underexplored antiviral target. Herein we describe a structure-guided campaign based on a hit from a focussed SAM mimetic library. Systematic SAR exploration guided by six X-ray co-crystal structures in complex with SARS-CoV-2 led to compound 26, a bi-substrate inhibitor that bridges the SAM and RNA cap binding sites. Compound 26 achieved nanomolar potency against nsp14 from SARS-CoV-2 (IC50 = 53 nM), SARS-CoV-1, and two alphacoronaviruses, with excellent selectivity over human RNMT and flaviviral MTase. In general, the compounds demonstrated favourable metabolic stability, passive permeability, and no HepG2 cytotoxicity. However, cellular antiviral activity was limited, revealing disconnects between enzyme inhibition and phenotypic response. These findings provide a structural framework for optimizing bi-substrate methyltransferase inhibitors against coronaviruses with a view for pan-coronaviral activity.","version":"1.1","doi":"10.1101/2025.09.30.679521","journal":"bioRxiv","score":null},{"id":"10.1101/2025.10.01.679649","pub_date":"2025-10-01","title":"Rational Design of Multiclade Coronavirus Spike Immunodominant Domain Nanoparticles to Elicit Broad Antibody Responses","abstract":"Four seasonal endemic human coronaviruses (EhCoVs), HKU1-CoV, OC43-CoV, 229E-CoV, and NL63-CoV, are culprits of mild upper respiratory and periodic severe diseases in vulnerable populations. Despite their prevalence, understanding EhCoVs\u2019 antigenic and immune signatures remains elusive. SARS-CoV-2 has evolved as the fifth EhCoV, requiring seasonal vaccination in most parts of the world, and currently, no other EhCoV vaccines are available. SARS-CoV-2 co-infection with EhCoVs increases disease severity; thus, combined vaccination may provide increased protection against seasonal EhCoVs overall. Here, we explored Spike (S) receptor binding domain (RBD) vs. N-terminal domain (NTD) B-cell immunodominance in EhCoV-positive convalescent donors and immunogenicity in mice. We found that while antibody and B-cell isotypes were relatively dominant to S NTD, mice immunized with S RBD elicited significantly higher binding and neutralizing antibody (nAb) responses. With that knowledge, we used computational methods to infer that EhCoV S sequences evolve into two main clades and designed chimeric immunodominant domains (IDDs) from both clades for each EhCoV. IDDs were scaffolded onto two-component nanoparticles (NPs) displaying each IDD separately (monovalent IDD NP); three \u00df-EhCoV IDDs (Mosaic-3 IDD NP); or five EhCoVs IDDs (Mosaic-5 IDD NP). Mice immunized with mosaic IDD NPs, but not soluble IDD antigens nor monovalent IDD NPs, elicited potent, broadly cross-reactive binding and neutralizing antibody (Ab) responses against SARS-CoV-2 variants, other EhCoVs, and Sarbecoviruses. System serology revealed that all four IDD immunogens elicited distinct Ab subclasses and Fc-effector functions, with mosaic-5 IDD NPs eliciting the most de novo Ab subclasses, distributions, and broader Fc-mediated immune mechanisms. Dissection of vaccine-immune sera revealed polyclonal Ab responses against multiple non-overlapping cross-reactive S epitopes. Due to elicitation of broad Ab responses with combinatory functionality, IDD NPs open new horizons for developing first-in-class supraseasonal EhCoV vaccine candidates, with potential to decrease frequent SARS-CoV-2 sequence updates and protect against other EhCoVs. Moreover, elicitation of Ab breadth that spans pandemic-threat Sarbecoviruses gives mosaic IDD NPs promise towards pandemic preparedness.","version":"1.1","doi":"10.1101/2025.10.01.679649","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.30.679433","pub_date":"2025-10-01","title":"Glycans modulate the adsorption of RBD Glycoproteins on polarizable surfaces","abstract":"The complex interplay between glycans and protein conformational dynamics during adsorption onto polarizable surfaces opens several routes to exploring the glycans potential as molecular interactions modulators. Molecular simulations are able to dissect the interactions of Receptor Binding Domain (RBD) glycoproteins for different SARS-CoV-2 variants of concern (VoC), in both open and closed conformations, with polarizable planar interfaces. Advanced analysis projected on 2D revealed distinct adsorption mechanisms depending on the initial loci of the glycan within the protein wall. Hydrophobic surfaces facilitated stable adsorption for both RBD conformations. Conversely, hydrophilic surfaces exhibited reduced adsorption, particularly for the closed-RBD, where glycans predominantly formed hydrogen bonds. Glycans significantly modulated closed-RBD adsorption, either enhancing it by permanent tethering or impeding it depending on the two initial conformations and protein mutations (omicron). Results for the individual RBDs are shown to be consistent with simulations for the complete S1 spike glycoprotein. Our findings unveil novel glycan-mediated adsorption phenomena and provide fundamental insights into glycoprotein-surface interactions, paving the way for understanding glycan roles in protein aggregation and recognition at polarizable biological interfaces.","version":"1.1","doi":"10.1101/2025.09.30.679433","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.18.671001","pub_date":"2025-9-30","title":"Spike mutations that affect the function and antigenicity of recent KP.3.1.1-like SARS-CoV-2 variants","abstract":"SARS-CoV-2 is under strong evolutionary selection to acquire mutations in its spike protein that reduce neutralization by human polyclonal antibodies. Here we use pseudovirus-based deep mutational scanning to measure how mutations to the spike from the recent KP.3.1.1 SARS-CoV-2 strain affect cell entry, binding to ACE2 receptor, RBD up/down motion, and neutralization by human sera and clinically relevant antibodies. The spike mutations that most affect serum antibody neutralization sometimes differ between sera collected before versus after recent vaccination or infection, indicating these exposures shift the neutralization immunodominance hierarchy. The sites where mutations cause the greatest reduction in neutralization by post-vaccination or infection sera include receptor-binding domain (RBD) sites 475, 478 and 487, all of which have mutated in recent SARS-CoV-2 variants. Multiple mutations outside the RBD affect sera neutralization as strongly as any RBD mutations by modulating RBD up/down movement. Some sites that affect RBD up/down movement have mutated in recent SARS-CoV-2 variants. Finally, we measure how spike mutations affect neutralization by three clinically relevant SARS-CoV-2 antibodies: VYD222, BD55-1205, and SA55. Overall, these results illuminate the current constraints and pressures shaping SARS-CoV-2 evolution, and can help with efforts to forecast possible future antigenic changes that may impact vaccines or clinical antibodies. This study measures how mutations to the spike of a SARS-CoV-2 variant that circulated in early 2025 affect its function and recognition by both the polyclonal antibodies produced by the human immune system and monoclonal antibodies used as prophylactics. These measurements are made with a pseudovirus system that enables safe study of viral protein mutations using virions that can only infect cells once. The study identifies mutations that decrease recognition by current human antibody immunity; many of these mutations are increasingly being observed in new viral variants. It also shows the importance of mutations that move the spike\u2019s receptor binding domain up or down. Overall, these results are useful for forecasting viral evolution and assessing which newly emerging variants have reduced recognition by immunity and antibody prophylactics.","version":"1.2","doi":"10.1101/2025.08.18.671001","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.29.679146","pub_date":"2025-9-29","title":"Entry, replication and innate immunity evasion of BANAL-236, a SARS-CoV-2-related bat virus, in Rhinolophus and human cells","abstract":"Asian Rhinolophus bats are considered the natural reservoirs of an ancestral SARS-CoV-2. However, the biology of SARS-CoV-2-related viruses in bat cells is not well understood. Here, we investigated the replication of BANAL-236, the only bat-derived SARS-CoV-2 relative isolated to date, in Rhinolophus cells. BANAL-236 did not replicate in wild-type Rhinolophus cell lines. Entry assays using pseudoviruses expressing the spike proteins (S) of SARS-CoV-2, BANAL-236, and BANAL-52 revealed that efficient S-mediated entry depends on the expression of human ACE2 (hACE2) and human TMPRSS2 (hTMPRSS2) in human and Rhinolophus cells. Expression of Rhinolophus entry factors, either alone or in combination, did not facilitate SARS-CoV-2 or BANAL-236 entry in human cells, suggesting that the S protein of BANAL-236 interacts more efficiently with hACE2 than with its Rhinolophus counterpart (rACE2). Through biochemical, virological, and electron microscopy analyses, we showed that BANAL-236 and SARS-CoV-2 completed their replication cycles in a Rhinolophus cell line engineered to express high levels of hACE2 and hTMPRSS2. Despite efficient viral replication in modified Rhinolophus and human cells, no induction of interferon (IFN)-stimulated genes was detected. Using a screening approach, we identified several BANAL-236 proteins that antagonize IFN production and signaling in human cells. Our findings thus show that BANAL-236 possesses critical features that enabled zoonotic spillover: hACE2 usage and potent evasion of human IFN responses. The Rhinolophus cellular model we established offers a platform for further investigating the interactions between bat coronaviruses and their reservoir hosts. Bats are known reservoirs for viruses that cause severe diseases in humans, such as coronaviruses and filoviruses. Bat species naturally or experimentally infected with these viruses rarely exhibit clinical symptoms, suggesting an evolved tolerance to viral infections. To elucidate the mechanisms underlying viral tolerance and to identify factors that could facilitate zoonotic spillover, it is essential to study the replication of bat-borne viruses in relevant bat cellular models. Here, we investigated the replication of BANAL-236, a SARS-CoV-2 related virus isolated from fecal samples of Rhinolophus bats in Northen Laos, in a novel cell line derived from Rhinolophus ferrumequinum lung fibroblasts. Our findings reveal that BANAL-236 can efficiently use human entry factors and potently evade the human innate immune response, two traits that may have contributed to its zoonotic transmission. Furthermore, the R. ferrumequinum cell lines we developed is a valuable model for investigating the molecular interactions between sarbecoviruses and their natural hosts.","version":"1.1","doi":"10.1101/2025.09.29.679146","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.25.678525","pub_date":"2025-9-29","title":"Plasma concentrations of nirmatrelvir and molnupiravir required for inhibition of SARS-CoV-2 replication differ between rhesus macaques and humans","abstract":"Early during the COVID-19 pandemic, non-human primate (NHP) infection models emerged as highly useful tools for preclinical screening of antiviral drugs. However, it is uncertain whether NHP models can be used to precisely inform optimal dosing in humans. We previously established and validated mathematical models which were fit to SARS-CoV-2 viral loads from human clinical trials. These models identified that plasma drug concentrations required to inhibit viral replication by 50% in humans (in vivo EC50) differ substantially from in vitro EC50 estimates in cell culture systems. Here we apply models to sequential viral load data from SARS-CoV-2 infected rhesus macaques (RM) that were untreated or treated with nirmatrelvir/ritonavir, molnupiravir, or both drugs. We identify that equivalent plasma drug concentrations correspond to greater antiviral potency in lungs compared to nasal passages for nirmatrelvir and molnupiravir. Average nirmatrelvir antiviral efficacy in RM (30% in nasal passages and 46% in lungs) was estimated to be less than in humans (82%) due to shorter plasma drug half-life. Molnupiravir efficacy in RM (95% in nasal and 99% in lungs) is estimated to be similar to efficacy in humans against omicron variants. Our model estimates that 10-fold higher plasma nirmatelvir concentrations are needed in humans versus RM to achieve 50% reduction in viral replication, whereas 20-fold lower plasma molnupiravir concentrations are needed. Our results suggest that dose optimization in humans based on modeling of NHP viral loads is limited by drug-specific differences in pharmacokinetic, pharmacodynamic and virologic profiles, and that data from human phase 1 and 2 trials is better suited for this task.","version":"1.1","doi":"10.1101/2025.09.25.678525","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.22.607124","pub_date":"2025-9-29","title":"A modular encapsulation system for precision delivery of proteins, nucleic acids and small molecules","abstract":"Targeted nanoparticles have the potential to revolutionize therapeutics for medical applications. Here, we demonstrate the utility of a flexible precision nanovesicle delivery system for functional delivery of DNA, RNA, proteins and drugs into target cells. Nanovesicles generated by the membrane sculpting protein caveolin, termed caveospheres, can be loaded with RNA, DNA, proteins or drugs post-synthesis or incorporate genetically-encoded cargo proteins during production without the need for protein purification. Functionalized fluorescently-labeled caveospheres form a modular system that shows high stability in biological fluids, specific uptake by target-positive cells, and can deliver proteins, drugs, DNA, and mRNA directly to the cytoplasm and nuclei of only the target cells. The negligible level of off-target transduction and uniform levels of targeted expression demonstrates advantages of the system over lipid-mediated gene delivery. Caveospheres can also be engineered to mimic viral particles by displaying the SARS-CoV-2-RBD protein, enabling targeted delivery to human bronchial epithelial cells. We demonstrate their application as a targeted transfection system for cells in culture, and critically, their efficacy in precision tumor killing in vivo.","version":"1.3","doi":"10.1101/2024.08.22.607124","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.26.672567","pub_date":"2025-9-26","title":"A Comparative Nanomechanical Study of Antibody and Nanobody Binding to SARS-CoV-2 Variants","abstract":"The receptor-binding domain of the SARS-CoV-2 spike protein is the principal target of neutralizing antibodies (Abs) and nanobodies (Nbs). Although their thermodynamic binding properties have been extensively characterized, their stability under mechanical force remains less understood. Here, we perform a comparative nanomechanical analysis of three Abs (PDI-231, S2X259, and R1-32) and three Nbs (R14, C1, and n3113.1) bound to the RBD from the WT strain and the Omicron BA.4 and JN.1 variants. Using coarse-grained steered molecular dynamics within the G\u014dMartini 3 framework, we identified distinct force\u2013response behaviors shaped by epitope topology, binding architecture, and variant-specific mutations. Ab/RBD dissociation was characterized by asymmetric rupture events, variant-dependent unfolding of RBD segments, and occasional deformation of antibody constant domains. Analysis of single-chain systems revealed that the heavy chain acts as the main load-bearing element, while the light chain sustains a consistent but weaker mechanical response. For the two-chain Ab system, the cooperative action of both chains enhances stability, enabling complexes to withstand rupture forces in the range of 500 pN. By contrast, Nb/RBD complexes dissociated primarily through rigid-body mechanisms, transmitting force more directly to the RBD interface with minimal structural disruption. Together, these results demonstrate that mechanical resilience emerges from immune complex topology and inter-chain cooperation, providing complementary insights beyond affinity into the design of therapeutics resilient to viral evolution.","version":"1.1","doi":"10.1101/2025.09.26.672567","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.24.678304","pub_date":"2025-9-25","title":"DYNAMICS OF RHINOVIRUS SARS-COV-2 COINFECTIONS AND SUPERINFECTIONS IN HUMAN AIRWAY CULTURES REVEAL TYPE-DEPENDENT VIRAL INTERFERENCE","abstract":"Coinfections between respiratory viruses are frequent but their outcomes are poorly understood. Rhinoviruses (RVs) and SARS-CoV-2 are two clinically relevant respiratory pathogens circulating year-round. We used differentiated human bronchial air\u2013liquid interface (ALI) tissue cultures to study coinfections and staggered superinfections between SARS-CoV-2 BA.2 and two RVs (RV-A1, RV-A16). RV-A16 exerted strong and sustained interference on SARS-CoV-2 replication, whereas RV-A1 showed transient effects. SARS-CoV-2 had limited impact on RVs but persisted long-term despite interference. Superinfection demonstrated that pre-established infection with either virus reduced subsequent replication of the other. RNA-FISH revealed spatially distinct infection foci with few dual-infected cells. Although coinfections prolonged interferon and cytokine secretion, functional assays showed that SARS-CoV-2 BA.2 replication was resistant to IFN, in contrast to Wuhan strains. Pleconaril inhibition of RV-A16 spread reduced its interference, highlighting the role of viral spreading. These findings highlight the complexity of respiratory viral interactions and their potential influence on transmission dynamics during viral co-circulation.","version":"1.1","doi":"10.1101/2025.09.24.678304","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.25.678349","pub_date":"2025-9-25","title":"Whole-genome DNA methylation profiling in COVID-19 positive patients reveals alterations in pathways linked to neurological dysfunction","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA betacoronavirus, causing coronavirus disease-19 (COVID-19). Infection with SARS-CoV-2 can result in a broad spectrum of clinical outcomes, ranging from asymptomatic or mild to a severe, deadly illness. Emerging evidence suggests SARS-CoV-2 affects host gene regulation through epigenetic mechanisms, such as DNA methylation, potentially contributing to immune dysregulation and post-acute sequelae, including neurological and psychiatric disorders. However, the extent and functional relevance of these epigenetic changes remain uncertain. We employed whole-genome bisulfite sequencing to profile DNA methylation in peripheral blood from SARS-CoV-2-positive patients across a spectrum of symptom severity, ranging from asymptomatic to severe (n=101), in comparison to SARS-CoV-2-negative individuals (n=105). We observed a widespread hypomethylation in the genomes of infected individuals, which was more pronounced in severe cases. Notably, we identified differentially methylated genes in patients with mild (19 genes), moderate (19 genes), and severe (35 genes) symptoms. These genes included those involved in canonical immune responses as well as known to be linked to neurodegenerative diseases. Subsequent pathway enrichment analysis further supported the significant association between the differentially methylated genes and those implicated in Alzheimer\u2019s and Parkinson\u2019s disease, as well as neuropsychiatric conditions, suggesting potential epigenetic links between acute SARS-CoV-2 infection and long-term neurological outcomes. This is one of the first studies to comprehensively map severity-stratified genome-wide DNA methylation changes in COVID-19 patients. Our findings underscore the potential importance of epigenetic regulation in the acute responses to SARS-CoV-2 infection and highlight an overlap with epigenetic mechanisms relevant for neuropsychiatric disease processes.","version":"1.1","doi":"10.1101/2025.09.25.678349","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.25.678549","pub_date":"2025-9-25","title":"SARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I Inhibition","abstract":"SARS-CoV-2 reprograms host metabolism to promote viral replication and evade immune responses. While infection is known to impair mitochondrial function and enhance glycolysis, the role of viral accessory proteins in these alterations remains unclear. Here, we investigate the metabolic impact of the accessory protein ORF7a. Lentiviral expression of ORF7a in human lung epithelial (A549) and monocytic (THP1) cells, coupled with integrated transcriptomic, proteomic, and metabolomic analyses, revealed profound dysregulation of glucose and lipid metabolism. ORF7a impaired oxidative phosphorylation, reducing basal and maximal respiration, inducing mitochondrial depolarization, and increasing reactive oxygen species. Mechanistically, ORF7a upregulated pyruvate dehydrogenase kinase 4 (PDK4), promoting pyruvate dehydrogenase (PDH) complex phosphorylation and suppressing pyruvate oxidation. However, pharmacological PDK4 inhibition did not restore respiration. High-resolution respirometry in frozen samples revealed impaired complex I function, while Blue Native-PAGE demonstrated defective respiratory supercomplex assembly. By linking enzymatic inhibition with structural destabilization, our study uncovers a functional vulnerability of the mitochondrial respiratory chain to viral manipulation. These findings establish ORF7a as a key modulator of host metabolic reprogramming and highlight mitochondrial pathways as potential therapeutic targets in COVID-19.","version":"1.1","doi":"10.1101/2025.09.25.678549","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.24.678199","pub_date":"2025-9-25","title":"Late treatment initiation leads to reduced antiviral potency","abstract":"The timing of initiation is critical in antiviral treatment and viral dynamic (VD) modeling is a powerful tool to study the within-host viral load changes and evaluate antiviral treatment effects using mathematical equations. Previous simulation studies have shown that early treatment initiation is critical to maximize the therapeutic response in antiviral treatment in an acute viral infection such as influenza and SARS-CoV-2. A recent experimental study demonstrated that late therapy initiation can lead to diminished antiviral potency. However, most VD model simulations with varying treatment initiation time accounted only for the effect of initiation condition (i.e., state of different cell populations when the therapy started), the loss of drug potency has been under-investigated. This may overestimate the antiviral effect, potentially resulting in suboptimal dose selection. To this end, we aimed to characterize relationship between the drug potency (EC50) and the timing of drug addition, using nirmatrelvir and GS-441524 against SARS-CoV-2 as an example. Viral load data were obtained from in vitro experiments with various drug concentrations and treatment initiated between 0 to 3 days post infection. EC50 values were fitted for each treatment initiation group and were found to vary with the timing of treatment initiation in both drugs. Also, a VD model with time-varying EC50 provided better fits than a constant EC50 model (BIC = 1667.90 vs. 1677.84). Further simulations also indicated that a constant EC50 model overestimated the antiviral efficacy when treatment started late. These findings highlighted the importance of considering EC50 shift when optimizing dosage regimens for patients presenting late.","version":"1.1","doi":"10.1101/2025.09.24.678199","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.22.677856","pub_date":"2025-9-24","title":"VSA-2, a novel plant-derived adjuvant for SARS-CoV-2 subunit vaccine","abstract":"QS-21, a key component of several licensed vaccines is facing limited supply, dose-limiting toxicity and other drawbacks which together limit its broader usage. Development of saponin alternatives to QS-21 that retain its desirable adjuvant activity without its drawbacks is in high need. Incorporating an amide side chain into the more sustainable Momordica saponins (MS) I and II led to the recent discovery of two semisynthetic immunostimulatory adjuvants VSA-1 and VSA-2. Here, we showed that the receptor-binding protein (RBD) of ancestral SARS-CoV-2 adjuvanted with VSA-2 (VSA-2-RBD) induced high titers of SARS-CoV-2-specific humoral and T helper-1 prone immune responses in mice comparable to that triggered by QS-21-RBD. Vaccination with VSA-2-RBD provided strong protection against SARS-COV-2, Delta and Omicron variants infection and the virus-induced lung inflammation and pathology similarly as QS-21-RBD vaccination. Overall, our results suggest that VSA-2 adjuvant can potentially complement the clinically proven saponin adjuvant QS-21 in vaccines against infectious diseases.","version":"1.1","doi":"10.1101/2025.09.22.677856","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.19.677428","pub_date":"2025-9-24","title":"How many crystal structures do you need to trust your docking results?","abstract":"Structure-based drug discovery technologies generally require the prediction of putative bound poses of protein:small molecule complexes to prioritize them for synthesis. The predicted structures are used for a variety of downstream tasks such as pose-scoring functions or as a starting point for binding free energy estimation. The accuracy of downstream models depends on how well predicted poses match experimentally-validated poses. Although the ideal input to these downstream tasks would be experimental structures, the time and cost required to collect new experimental structures for synthesized compounds makes obtaining this structure for every input intractable. Thus, leveraging available structural data is required to efficiently extrapolate new designs. Using data from the open science COVID Moonshot project\u2014where nearly every compound synthesized was crystallographically screened\u2014we assess several popular strategies for generating docked poses in a structure-enabled discovery program using both retrospective and prospective analyses. We explore the tradeoff between the cost of obtaining crystal structures and the utility for accurately predicting poses of newly designed molecules. We find that a simple strategy using molecular similarity to identify relevant structures for template-guided docking is successful in predicting poses for the SARS-CoV-2 main viral protease. Further efficiency analysis suggests template-based docking of a scaffold series is a robust strategy even when the quantity of available structural data is limited. The resulting open source pipeline and curated datasets should prove useful for automated modeling of bound poses for downstream scoring, machine learning, and free energy calculation tasks for structure-based drug discovery programs.","version":"1.1","doi":"10.1101/2025.09.19.677428","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.19.677384","pub_date":"2025-9-23","title":"A Novel dual Influenza virus and SARS-CoV-2 neutralisation assay","abstract":"Influenza and SARS-CoV-2 are co-circulating during the traditional influenza season giving rise to potential co-infections; currently estimated at 2.4%. Additionally, this means that the uptake of both vaccines (including SARS-CoV-2 boosters) will be encouraged within the same timeframe. Currently, patients receive separate vaccines in different arms on the same or different days. Induction of neutralising antibodies (NAbs) is used as the measure of efficacy for SARS-CoV-2 vaccinations while, the hemagglutination inhibition (HI) assay is the gold standard for seasonal influenza vaccine assessment. However, with the advent of novel universal influenza vaccine and dual SARS-CoV-2/Influenza vaccine approaches it is possible that functional NAb assays will become an essential component of a vaccine assessment toolbox. This assay is capable of distinguishing NAb responses during both dual vaccination development, and as part of co-infection and imprinting studies. Taking advantage of existing pseudotyped viruses developed by us, we present herein a dual neutralisation assay, which utilises 2 different luciferase (Renilla and Firefly) reporter lentiviral vectors. We configured assays with various combinations of SARS-CoV-2 and influenza subtype/variant combinations and compared sensitivity of this dual approach to neutralisation levels seen in single virus pseudotype Micro-neutralisation (pMN) in a double-blinded test of monoclonal antibody cocktails, and subsequently with a pre-screened serum panel. Mono and dual neutralisation pMN identified correctly positive and negative samples and IC50 values were all within 2-fold of each other, suggesting that specificity and sensitivity are retained post-multiplex. As we have taken a pseudovirus based approach there is the potential to tailor to specific vaccination candidates or currently circulating strains of each virus. The use of this versatile assay could form part of the toolbox of analysis of dual vaccination candidates and investigating responses during co-infection including studies assessing the impact of immune imprinting in the future.","version":"1.1","doi":"10.1101/2025.09.19.677384","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.23.676511","pub_date":"2025-9-23","title":"Long-read single-cell RNA sequencing uncovers cell-type specific transcript regulation in COVID-19","abstract":"SARS-CoV-2 infection leads to extensive host transcriptomic changes, but the role of alternative splicing in shaping the immune response remains underexplored. Here, we present the first application of long-read single-cell RNA sequencing on nasopharyngeal swabs from COVID-19 patients and healthy controls to resolve transcript-level changes across cell types. Our analysis identified major epithelial cell types and pronounced immune infiltration, with cell-type annotations concordant with those from short-read data. By enabling isoform-level resolution, our nanopore sequencing approach revealed cell-type specific alternative splicing, undetectable with short-read sequencing. For example, although gene-level expression of the key immune and apoptosis regulators, IFNAR2 and FAIM, did not differ between COVID-19 patients and healthy controls we identified marked shifts in isoform usage. Between moderate and critical cases, we observed cell-type specific differential transcript usage in the T cell signaling kinase FYN and the immune-regulatory transcription factor IRF2. As some of these splicing alterations yield functionally distinct isoforms, we hypothesize that alternative splicing modulates immune signaling and apoptosis, fine-tuning the host response to SARS-CoV-2 infection. Our study demonstrates the unique power of long-read single-cell transcriptomics to uncover isoform-resolved regulatory changes, offering novel insights into the role of alternative splicing in shaping immune responses to viral infections.","version":"1.1","doi":"10.1101/2025.09.23.676511","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.23.678000","pub_date":"2025-9-23","title":"High-throughput cell-free profiling of SARS-CoV-2 RBD variants enables rapid and quantitative in vitro affinity landscape mapping","abstract":"SARS-CoV-2 variants continue to threaten public health, necessitating the study of cumulative and epistatic effects of receptor-binding domain (RBD) mutations on antibody evasion. We present a high-throughput platform combining cell-free protein synthesis and microfluidics to quantify the affinity of a large number of RBD triplet mutants covering the evolutionary space between wild-type and Omicron against two therapeutic antibodies and one engineered binder. Using rapid in vitro gene assembly and cell-free synthesis, we expressed 518 RBD variants and obtained 31,740 quantitative affinity measurements to generate three comprehensive binding energy landscapes. This approach enables rapid and large-scale in vitro affinity profiling and machine learning-based predictions, providing a valuable tool for studying emerging variants.","version":"1.1","doi":"10.1101/2025.09.23.678000","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.22.677712","pub_date":"2025-9-22","title":"Mechanisms of superior respiratory IgA responses against SARS-CoV-2 after mucosal vaccination","abstract":"Mucosal immunization and respiratory IgA offer significant promise in protecting against airborne pathogens, including SARS-CoV-2. However, the conditions and mechanisms that lead to the robust induction of respiratory IgA responses following mucosal vaccination remain poorly understood. It is also currently debatable whether mucosal vaccination is still warranted given that most individuals in developed countries have established a hybrid immunity from vaccination and infection. Here we characterized respiratory mucosal immune responses after SARS-CoV-2 infection, vaccination or both in humans. We found that hybrid immunity resulted in moderately increased respiratory IgA and neutralizing antibody responses compared to infection or vaccination alone. However, a direct comparison of hybrid immunity and a mucosal adenovirus-based booster vaccination in animal models revealed that respiratory booster immunization elicited markedly stronger and more durable respiratory IgA, T cell response, and protective immunity against SARS-CoV-2, supporting the promise of respiratory mucosal vaccination. Mechanistically, we found that mucosal booster immunization induced local IgA- secreting cells in the respiratory mucosa, aided by pulmonary CD4+ T cells in situ. Strikingly, local IL-21-producing Blimp-1+ Th1 effector cells were critical in mediating the CD4+ T cell help for respiratory IgA production. Furthermore, lung macrophages were important for this respiratory IgA response via the production of TGF-\u03b2. Consequently, we demonstrated delivery of adenoviral booster to the lower airway was necessary to generate robust upper and lower airway IgA responses. Collectively, our results uncover a local cellular network supporting enhanced respiratory IgA responses, with implications for the development of optimal mucosal immunization strategies against SARS-CoV-2 and other respiratory pathogens.","version":"1.1","doi":"10.1101/2025.09.22.677712","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.21.677675","pub_date":"2025-9-22","title":"Knocking out two polyphenol oxidase genes significantly improves recombinant protein purification in Nicotiana benthamiana","abstract":"Efficient purification remains one of the major bottlenecks in the development of plant-based systems for recombinant protein production. The complex metabolites, particularly polyphenols, which usually cause recombinant protein aggregation during purification. In this study, we identified two key polyphenol oxidase genes, PPOa and PPOb from N.benthamiana as responsible for these effects. Using CRISPR/Cas9, we generated two ppoa;ppob double knockout lines that significantly improved the purification of functional proteins like SARS-CoV-2 Spike trimer and influenza HA trimer. These lines showed reduced polyphenol-protein interactions, minimized aggregation, and higher purification yields. Our work establishes a clean, high-efficiency N. benthamiana chassis for scalable recombinant protein production.","version":"1.1","doi":"10.1101/2025.09.21.677675","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.16.676697","pub_date":"2025-9-21","title":"mRNA-Based Designer APCs Elicit Robust CD8\u207a and CD4\u207a T Cell Responses","abstract":"mRNA-based therapeutics have demonstrated notable success in SARS-CoV-2 vaccines and are emerging in cancer immunotherapy. However, conventional mRNA cancer vaccines are limited by the low immunogenicity of tumor-associated and neoantigens. We addressed this limitation by formulating a modular, liposome-based mRNA cocktail comprising three distinct mRNAs encoding tumor antigen, the co-stimulatory molecule CD80, and membrane-tethered IL-2. Administration of this mRNA mixture transforms somatic cells into \u2018designer antigen-presenting cells (APCs)\u2019 in vivo, which simultaneously express the antigen, a co-stimulatory molecule, and a cytokine. These designer APCs more effectively activated tumor antigen-specific CD8\u207a T cells than mRNA encoding the antigen alone and elicited robust anti-tumor immune responses. In addition, substituting IL-2 in the mRNA mixtures with membrane-tethered IL-12 led to the expansion and differentiation of endogenous antigen-specific Th1 helper T cells in vivo. Importantly, this platform activated NY-ESO-1-specific CD8\u207a T cells both in human PBMCs in vitro and in HLA-A*02:01-transgenic mice, highlighting its translational potential. This modular mRNA strategy reprograms somatic cells in situ into designer APCs, providing a flexible and translatable platform for precision immunotherapy.","version":"1.2","doi":"10.1101/2025.09.16.676697","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.13.623299","pub_date":"2025-9-20","title":"SARS-CoV-2 ORF3a blocks lysosomal cholesterol egress by disrupting VPS39-regulated NPC2 trafficking and BMP metabolism","abstract":"Cholesterol homeostasis relies on lysosomes, which release free cholesterol from degraded lipids. We show that SARS-CoV-2 blocks lysosomal cholesterol export through its protein ORF3a. ORF3a binds the HOPS subunit VPS39, and disrupting this interaction restores cholesterol trafficking. Two mechanisms underlie this defect. First, ORF3a\u2013VPS39 interaction traps the sorting receptor CI-MPR and the retrieval complex retromer in endosomes/lysosomes, impairing trafficking of the cholesterol transporter NPC2. Retromer deletion reproduced these defects. Second, ORF3a reduces bis(monoacylglycerol)phosphates (BMPs), lysosomal lipids required for cholesterol export. Lipidomics and proteomics revealed altered metabolism of BMP precursors, mitochondrial phosphatidylglycerols (PGs), and reduced mitochondrial proteins at lysosomes. ORF3a\u2013VPS39 interaction decreased lysosome\u2013mitochondrion membrane contact sites (MCS), excluding autophagy or mitochondrion-derived vesicles as routes for PG transfer. VPS39 deletion decreased the MCS and BMPs. These findings identify VPS39 as a regulator of NPC2 trafficking and BMP biosynthesis and reveal that ORF3a disrupts both pathways to block cholesterol egress.","version":"1.2","doi":"10.1101/2024.11.13.623299","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.18.677166","pub_date":"2025-9-19","title":"Single dose alum adjuvanted RBD protein vaccine provides protection against homologous challenge with SARS-CoV-2 Washington strain and heterologous rechallenge with Delta and Omicron BA.5 variants in K18 hACE2 mouse model","abstract":"Most COVID-19 vaccination strategies require at least two doses-a primary vaccine followed by a booster dose with the updated variant-specific vaccine. However, vaccine and booster dose hesitancy make it challenging to administer multiple doses of vaccines in the unvaccinated and vaccinated populations, respectively. Thus, it is important to determine if vaccinated individuals exposed to SARS-CoV-2 infection develop immune responses that may protect them against emerging variants of concern (VoCs). We have developed mouse models to understand the protective efficacy of an RBD-based vaccine against challenge and rechallenge with SARS-CoV-2 VoCs. Mice were vaccinated with RBD protein vaccine formulated in 2% Alhydrogel (alum) adjuvant by subcutaneous route. To determine the efficacy of RBD vaccine, mice were challenged approximately 4 weeks post-vaccination with SARS-CoV-2 variants by intranasal route. To determine the efficacy of RBD vaccine against SARS-CoV-2 rechallenge, mice were rechallenged with SARS-CoV-2 Delta and Omicron BA.5 variants at day 14 post SARS-CoV-2 Washington (WA) strain challenge. Our data suggest that single dose alum adjuvanted RBD protein from Wuhan strain provides protection against homologous challenge with SARS-CoV-2 WA strain but failed to provide protection against heterologous challenge with Delta and Omicron BA.5 variants. Interestingly, vaccinated mice that survived homologous challenge with the WA strain showed protection against heterologous rechallenge with Delta and omicron BA.5 variants. Furthermore, infectious viral loads of Delta and Omicron BA.5 were not detected in the lung tissues collected from the rechallenged mice at 3 days post-rechallenge. The data suggest that a single dose RBD vaccine from the ancestral Wuhan strain together with survival from WA strain challenge induces protective immune responses against Delta and Omicron BA.5 variants rechallenge. These mouse models will be useful to determine the immune responses that correlate with protection against challenge and rechallenge with SARS-CoV-2 VoCs.","version":"1.1","doi":"10.1101/2025.09.18.677166","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.19.677199","pub_date":"2025-9-19","title":"Regulation of NFKBIZ by precise Regnase-1 endoribonuclease cleavage and subsequent uridylation","abstract":"A conserved sequence in the 3\u2032UTR of NFKBIZ mRNA has long been recognized as a regulator of cytokine production and interferon responses. We show that the endoribonuclease Regnase-1 controls NFKBIZ expression through a precise and modular RNA degradation mechanism. The structured core element undergoes specific endonucleolytic cleavage, while flanking upstream and downstream stem\u2013loop modules, previously implicated in Regnase-1 recognition, act cooperatively to enhance cleavage efficiency by \u223c25-fold. Following cleavage, the upstream fragment is rapidly uridylated, accelerating decay of the NFKBIZ open reading frame. This pathway explains how driver mutations \u2013 found in this RNA region \u2013 responsible for diffuse large B-cell lymphoma elevate NFKBIZ expression and how a segment of the SARS-CoV-2 genome \u2013 previously linked to NFKBIZ activation \u2013 suppresses Regnase-1 cleavage via hybridization to this regulatory RNA segment. Together, these findings define a mechanistic framework for Regnase-1\u2013mediated control of NFKBIZ, linking its cleavage activity to both lymphomagenesis and viral pathogenesis.","version":"1.1","doi":"10.1101/2025.09.19.677199","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.11.675561","pub_date":"2025-9-19","title":"Geno2pheno[bNAbs]: Interpretable and accurate HIV antibody resistance prediction","abstract":"Antiretroviral therapy (ART) is a life saving option for people living with HIV-1 (PLWH) and is effective against many viral strains. The most common ARTs involve combinations of drugs targeting viral or cellular proteins. Most of these drugs have to be taken daily. An alternative to ARTs with established inhibitors comprises broadly neutralizing antibodies (bNAbs). However, bNAbs share the problem of viral resistance with protein inhibitors. We developed a web service geno2pheno[bNAbs] that allows users to upload viral genotypes and estimates the respective resistance to many common bNAbs. The service uses trained statistical models to classify the virus into sensitive and resistant, respectively or to regress the IC50. We used two linear models as well as two neural nets for each task and multi-task (MT) learning to train both models for IC50 prediction and classification simultaneously. During multi-task learning we penalize divergence of class and IC50 score in addition to the loss individual to each of the models. We compared the linear models of geno2pheno[bNAbs] to other state-of-the-art methods like recurrent neural nets and self-attention, and found them to be competitive in regard to accuracy and have the benefit of fast computation and being easily interpretable in regard to features, i.e., positions on the envelope. We developed a web service for the prediction of antibody resistance (geno2pheno[bNAbs]) to HIV-1, which is free to use and can be extended to other viruses, like Sars-Cov2, in the future.","version":"1.2","doi":"10.1101/2025.09.11.675561","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.17.676934","pub_date":"2025-9-18","title":"Potent broad-spectrum anti-coronaviral frameshift inhibitors from virtual screen of RNA binding","abstract":"Coronavirus genomes contain an RNA pseudoknot that directs \u22121 programmed ribosomal frame-shifting (\u22121 PRF) to control expression of viral proteins crucial for replication. Ligands that inhibit \u22121 PRF can thus attenuate viral propagation and have potential as drugs for limiting corona-virus infections. To search for novel small-molecule frameshift inhibitors with anticoronaviral activity, we computationally screened over 14 million compounds for binding to the SARS-CoV-2 pseudoknot, followed by experimental validation of the top hits for inhibition of \u22121 PRF and viral replication. We identified multiple potent \u22121 PRF inhibitors, effective at nM concentrations, some of which significantly suppressed SARS-CoV-2 replication in cell culture. Several compounds also inhibited \u22121 PRF in multiple representative bat coronaviruses, indicating broad-spectrum activity. These results showcase the promise of viral RNA structures like frameshift-stimulatory pseudoknots as targets for broad-spectrum antiviral drugs.","version":"1.1","doi":"10.1101/2025.09.17.676934","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.06.622014","pub_date":"2025-9-17","title":"Impact of different synonymous codon substitution strategies on SARS-CoV-2 nucleocapsid protein expression in Escherichia coli","abstract":"Synonymous codon substitution, a gene engineering approach in synthetic biology, has been effective in improving the codon composition of recombinant genes of interest based on various criteria without altering the amino acid sequence. The SARS-CoV-2 virus nucleocapsid (N) protein is a stable, conserved and highly immunogenic that is less prone to mutation during infection, making it a key antigen in in vitro diagnosis, vaccine development, immunological and structural studies. While reports have focused on applying optimized N protein for different applications, the basic parameters used by different optimization tools for choosing the best approach for the N gene synonymous codon substitution are often neglected. Here, we analyzed the influence of different synonymous codon substitution strategies on SARS-CoV-2 N-protein expression in E. coli. Using different codon optimization (CO) and harmonization (CH) tools, we predicted and compared how parameters such as GC content, Codon Adaptation Index, codon quality and number of rare codons present in these sequences affect the N-protein expression. Our results also show that Minimum Free Energy (MFE) and RNA structure of N-term and C-tail of the N-protein coding sequence influence protein folding. We then predicted that the SR-rich region of the N-protein may contribute to slowing down the elongation rate during translation. This work presents a fundamental analysis of how different optimization tools affect SARS-CoV-2 N-protein expression and folding and suggests a basic approach to choosing the best strategy for optimal expression and folding of the protein for further studies. Codon substitution affects SARS-CoV-2 nucleocapsid (N) protein expression. SARS-CoV-2 N-protein expression varies with different codon optimization tools. RNA structures of N- and C-term impact RNA stability, protein expression and folding. SR-rich region of the N-protein may slow down elongation rate during translation.","version":"1.3","doi":"10.1101/2024.11.06.622014","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.16.676634","pub_date":"2025-9-17","title":"mRNA-1273.251 and mRNA-1283.251 vaccines expressing SARS-CoV-2 variant LP.8.1 antigens broadly neutralize contemporary JN.1-lineage viruses","abstract":"The continued evolution of the SARS-CoV-2 Omicron JN.1 lineage has led to the emergence of antigenically distinct subvariants including KP.2, KP.3, XEC, and LP.8.1, which became the dominant strains in the Americas and Europe by mid-2025. LP.8.1 was designated a Variant Under Monitoring by the WHO in January 2025 due to its potential to displace prior circulating variants. Informed by early growth modeling and antigenic analysis, we selected LP.8.1 as a candidate strain for the 2025-2026 vaccine season. Here, we describe the development of updated LP.8.1-matched mRNA vaccine compositions encoding either the full-length spike protein for mRNA-1273 (monovalent) or the membrane-anchored receptor-binding and N-terminal domains for the mRNA-1283 vaccine. Initial in vitro characterization, including structural analysis, demonstrated robust antigen expression and intact antigenic features. Immunogenicity of both vaccines were evaluated in murine models following immunization as either a primary series in na\u00efve animals or as a booster dose. LP.8.1-matched vaccines elicited strong neutralizing antibody responses against the homologous LP.8.1 strain and more recently emerging JN.1-lineage subvariants, including XFG and NB.1.8.1. Notably, the mRNA-1283 vaccine expressing LP.8.1 induced higher mean neutralization titers than the mRNA-1273 version across multiple variants. These data demonstrate the immunogenicity and breadth of both LP.8.1-based mRNA-1273 and mRNA-1283 vaccines in the context of ongoing JN.1 lineage evolution and support the selection of LP.8.1 as the updated vaccine antigen for the 2025-2026 season.","version":"1.1","doi":"10.1101/2025.09.16.676634","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.17.674891","pub_date":"2025-9-17","title":"Mechanisms for cross-neutralisation of diverse bat sarbecoviruses","abstract":"The continuing evolution of SARS-CoV-2 variants of concern, and the increasing spillover potential of sarbecoviruses into the human population presents an important and urgent need to discover cross-reactive monoclonal antibodies (mAbs) for future therapeutic use and identify conserved neutralising epitopes that can be used for rationale design of broadly protective sarbecovirus vaccines. Here we study the neutralising epitopes on WIV-1 Spike of three mAbs that confer broad sarbecovirues and SARS-CoV-2 variant neutralisation, including XEC and JN.1. mAb V1WT_06 binds a highly conserved RBD site V epitope that is mediated by the heavy chain alone. V1WT_06 contact residues are highly conserved in circulating viruses suggesting that the epitope is evolutionarily and functionally constrained. mAbs V1WT_41 and VA14_26 bind overlapping RBD class 4 epitopes with differing angles of approach that impact on the degree of ACE2 competition. We show that neutralisation by these mAbs is maintained when virus entry is via Japanese horseshoe bat and Halcyon horseshoe bat ACE2. These mAbs are ideal candidates for therapeutic antibody development and inform the rational design of pan-coronavirus vaccines.","version":"1.1","doi":"10.1101/2025.09.17.674891","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.12.675949","pub_date":"2025-9-17","title":"PDLIM2 Repression: A Common Mechanism in Viral Lung Infection","abstract":"PDLIM2, a PDZ-LIM domain-containing protein expressed highest in the lung and immune cells, serves as a unique tumor suppressor and immune modulator, mainly by turning off the activation of the master transcription factors NF-\u03baB and STAT3. While its role in cancer is established, the involvement of PDLIM2 in viral infection remains unclear. Here, we analyzed public gene expression data of blood leukocytes, bronchoalveolar lavage cells, and lung tissues from uninfected healthy humans and those infected with the respiratory virus SARS-CoV-2 or influenza. We found that PDLIM2 expression was repressed by viral infection, and notably, this repression correlated with the severity of infectious diseases. Consistently, the expression level of PDLIM2 was negatively associated with NF-\u03baB and STAT3 activity across a diverse range of cell types, such as macrophages, monocytes, neutrophils, T cells, alveolar type 1 and 2 epithelial cells, airway epithelial cells, and fibroblasts. Accordingly, cells with low PDLIM2 expression exhibited aberrant activation of signaling pathways essential for cellular functions and immune responses. These findings highlight PDLIM2 repression as a common mechanism underlying human viral infectious diseases and suggest PDLIM2 as a potential biomarker and therapeutic target for disease prognosis, prevention, and treatment.","version":"1.2","doi":"10.1101/2025.09.12.675949","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.13.675985","pub_date":"2025-9-16","title":"Identification of protective human monoclonal antibodies using a K18 hACE2 transgenic mouse SARS-CoV-2 challenge model","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses human angiotensin converting enzyme 2 (hACE2) as its obligate receptor for cell entry. The K18 hACE2 transgenic mouse line, which expresses hACE2 under control of the human keratin 18 (K18) promoter, is used as an animal model for the study of COVID-19 pathogenesis. Here, we evaluate this model in the screening of human monoclonal antibody (hmAb) therapies against SARS-CoV-2. We included 206 hmAbs from the Coronavirus Immunotherapeutic Consortium Database (CoVIC-DB) and identified many that protected against a lethal challenge with the virus. Our data showed that mouse weight change from day 5 onward highly correlated with survival. Many of the protective hmAb candidates we identified also showed strong viral neutralization and spike protein (SP) binding when measured in vitro; however, in many cases, in vitro assays failed to identify protective hmAbs, suggesting that the mouse model may capture characteristics of the hmAbs that other methods cannot. Our findings demonstrate the relevance of including in vivo models for the characterization of therapeutics against SARS-CoV-2, as these improve both accuracy and expediency in the screening process.","version":"1.1","doi":"10.1101/2025.09.13.675985","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.12.675971","pub_date":"2025-9-16","title":"Metabolic Signatures of Pulmonary Embolism in COVID-19: Insights from Longitudinal Intensive Care Unit Profiles","abstract":"Pulmonary embolism is a severe complication of COVID-19 infection, associated with a hypercoagulable state and heightened risk of blood clots. As SARS-CoV-2 has become endemic, understanding pulmonary embolism\u2019s metabolic effects in COVID-19 patients is warranted. This study investigated the longitudinal metabolic profiles of 66 Intensive Care Unit-admitted COVID-19 patients at Erasmus Medical Center to identify metabolites and mechanisms associated with pulmonary embolism. A total of 1209 metabolic species were measured, including amines and lipids. Metabolic changes were analysed across four timeframes: i) general analysis of pulmonary embolism, ii) 72 hours prior to pulmonary embolism, iii) 48 hours prior and the day of pulmonary embolism, and iv) the day of and 48 hours post-pulmonary embolism. The general analysis revealed significant upregulation of amines, triglycerides, phosphatidylethanolamines, ether-linked phosphatidylethanolamines, and eicosanoids in patients who developed a pulmonary embolism. Phosphatidylethanolamines containing the 20:3 fatty acid side chain were notably elevated. Minimal metabolic dysregulation was observed 72 hours before pulmonary embolism, with subtle increases in lysophosphatidylcholines and lysophosphatidylethanolamines. In contrast, there was a strong metabolic response during and post-pulmonary embolism, phosphatidylethanolamines (47%), ether-linked phosphatidylethanolamines(96%) and sphingosines(40%). These findings underscore the critical role of lipid metabolism in pulmonary embolism, particularly triglycerides and specific lipid species. The limited metabolic perturbations before pulmonary embolism suggest early prediction challenges, emphasising the need for further research into temporal metabolic changes and their clinical applications.","version":"1.1","doi":"10.1101/2025.09.12.675971","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.15.676388","pub_date":"2025-9-16","title":"Evaluation of Antigen Expression and Early Immune Response following Cutaneous Suction-mediated DNA Delivery","abstract":"Suction-based in vivo cutaneous DNA transfection is a newly developed, cost-effective method that produces high transfection efficiency. This method has shown robust immunogenic responses following SARS-CoV-2 DNA vaccination in both pre-clinical studies and clinical trials. The current work investigates suction-based transfection and immune activation on a detailed, cellular level. The spatiotemporal patterns of antigen expression in rat skin following suction-induced delivery of a pEGFP-N1 plasmid and a SARS-CoV-2 DNA vaccine are evaluated via immunofluorescence staining, which demonstrates early and prolonged expression. The epidermis is identified as the primary location of transfection, and the transfected cells are primarily epidermal keratinocytes. Early immune response is assessed by detection of antigen presenting cells (APCs) following suction-induced DNA vaccination.","version":"1.1","doi":"10.1101/2025.09.15.676388","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.12.675771","pub_date":"2025-9-16","title":"A Generative Foundation Model for Antibody Design","abstract":"Antibodies are indispensable components of the immune system, yet the design of high-affinity antibodies remains a time-consuming and experimentally intensive process. To address this challenge, we present IgGM, a novel generative foundation model designed to accelerate high-affinity antibody engineering. IgGM learns the complex relationships underlying the binding interactions between antigens and antibodies, as well as the mapping between antibody sequences and structures. By conditioning on different inputs, IgGM supports a wide range of antibody design tasks, including complex structure prediction, inverse design, affinity maturation, framework optimization, humanization, and de novo antibody design. It is compatible with both conventional antibodies and nanobodies, and allows user-defined CDR loop lengths for flexible design. To prioritize candidates, we introduce a frequency-based computational screening strategy that enhances design efficiency. Extensive evaluation through both in silico benchmarks and in vitro experiments across diverse antigens such as PD-L1, Protein A, TNF-\u03b1, IL-33, SARS-CoV-2 RBD and its variants demonstrates that IgGM consistently generates antibodies or nanobodies with high measured affinity. These results underscore IgGM\u2019s versatility and effectiveness as a powerful tool for next-generation antibody discovery and optimization.","version":"1.1","doi":"10.1101/2025.09.12.675771","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.14.676173","pub_date":"2025-9-15","title":"High-stability, clamp-free soluble Sarbecovirus spike trimers and their potential for pan-Sarbecovirus vaccine development","abstract":"Broadly effective vaccines are needed to protect against future pandemics caused by severe acute respiratory syndrome coronavirus (SARS CoV)-like coronaviruses (sarbecoviruses). The development of simple trimeric subunit vaccines based on the Sarbecovirus spike (S) has proven problematic due to the unstable nature of the S trimer. Here we developed clamp-free, highly stable soluble S trimers by truncating the stem helix to maximize yield and covalently linking the 3 monomers via engineered disulfides to increase thermostability. In K18hACE2 mice, covalently linked SARS CoV-2 S trimers elicited >10-fold higher neutralizing antibody (NAb) titres than parental unlinked trimers and protected the mice against viral challenge. A trivalent vaccine formulation comprised of covalently stabilized spikes derived from 3 divergent ACE2-using Sarbecovirus clades elicited broad and potent neutralizing activity in mice. The covalently linked S trimers were stable at 37\u00b0C for 112 days and remained intact following lyophilization and storage at ambient temperature for 6 months. This study establishes a framework for producing simple and stable highly immunogenic pan-Sarbecovirus S subunit vaccines that can be stored and distributed in the absence of a cold chain.","version":"1.1","doi":"10.1101/2025.09.14.676173","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.09.675039","pub_date":"2025-9-14","title":"Computational screening and automatic filtering for the discovery of novel inhibitors of TMPRSS2, a type II transmembrane serine protease","abstract":"Transmembrane Serine Protease 2 (TMPRSS2) is a membrane protein of the type II serine protease family of enzymes implied in epithelial homeostasis. It is involved in several diseases, notably prostate cancer and SARS-CoV-2 infections. Over the years, only a few tested TMPRSS2 inhibitors showed consistent results. This prompted us to select it as target of structure-based virtual screening, to search for novel inhibitors among a library of 475,770 small molecules. Two sets of TMPRSS2 structures were selected, one taken from molecular dynamics simulations, the other from recently solved X-ray crystallographic structures. We designed a workflow to filter docking results in a reproducible way, allowing for a faster and more reliable selection. The program uses four metrics: the pose consistency of the ligand, docking score, number of interactions with key protein residues, and cluster analysis. This led to the selection and visual inspection of two sets of 500 compounds, which yielded 10 reasonable hit candidates.","version":"1.1","doi":"10.1101/2025.09.09.675039","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.12.675877","pub_date":"2025-9-12","title":"Immunoregulatory effect of metformin in monocytes exposed to SARS-CoV-2 spike protein subunit 1","abstract":"Severe COVID-19 is characterized by a hyperinflammatory state associated with an exacerbated inflammatory activation of monocytes and macrophages in the respiratory tract. Metformin has been identified as a potent monocyte inflammatory suppressor, and it has been demonstrated to attenuate inflammation in COVID-19. The mechanisms underlying metformin anti-inflammatory effects are, however, unclear. We thus sought to investigate metformin\u2019s main interactions and their respective isolated effects in modulating monocyte inflammatory response to SARS-CoV-2 stimulation. Classical human monocytes were isolated from healthy 18-40-year-old individuals and stimulated in vitro with recombinant spike protein subunit 1 (rS1) to assess glycolytic and oxidative metabolic responses by Seahorse extracellular flux analysis, and inflammatory gene expression by qPCR. Stimulated monocytes were either pre-treated with metformin, rotenone, S1QEL, or A769662. Monocytes stimulated in vitro with rS1 showed an increased glycolytic response associated with production of pro-inflammatory cytokines. Metformin pre-treatment reduced glycolytic activation while partially suppressing inflammation. Rotenone-dependent mitochondrial complex I inhibition was not able to replicate the same effect, and neither complex I specific ROS scavenging. Conversely, A769662 induced AMPK activation led to suppressed glycolytic inflammatory response and cytokine expression pattern similar to metformin, thus suggesting AMPK modulation as a possible central component for metformin\u2019s mode of action upon S1 stimulation. In summary, further investigation into the interactions underlying AMPK activity on monocytes in the context of SARS-CoV-2 may provide a better elucidation of metformin\u2019s anti-inflammatory effect.","version":"1.1","doi":"10.1101/2025.09.12.675877","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.12.675721","pub_date":"2025-9-12","title":"Mechanism of SARS-CoV-2 Nucleocapsid Protein Phosphorylation-induced Functional Switch","abstract":"The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed WT Np formed discrete complexes on viral 5\u02b9 UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1-4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs. WT Np also compacted and irreversibly bound single-stranded DNA; this activity was significantly reduced by phosphorylation. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants.","version":"1.1","doi":"10.1101/2025.09.12.675721","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.07.674729","pub_date":"2025-9-12","title":"Releasing RNA from formalin: novel 20th century rotavirus alphagastroenteritidis strains identified in Australian microbat voucher specimens","abstract":"Archival specimens held in biorepositories (e.g. natural history collections) offer rare temporal snapshots of global biodiversity. These collections not only preserve species morphology and aspects of ecology, but increasingly provide access to historical molecular data, including insights into wildlife disease. As several pandemics have originated from animal viruses spilling over into the human population (i.e., SARS-CoV-2/COVID-19, 2009 H1N1 influenza, and HIV/AIDS), characterising the diversity of viruses circulating in wildlife populations is essential for proactive pandemic preparedness. Yet, current surveillance remains biased toward contemporary viruses of economic importance. One solution to bridging spatiotemporal gaps in wildlife virus knowledge is retrospective screening of vouchered wildlife specimens. However, such efforts have been hindered by formalin fixation of specimens, which degrades and cross-links nucleic acids. Here we demonstrate that formalin-fixed vouchered wildlife specimens retain both host and viral RNA fragments after being stored for up to sixty years. We recovered fragments of divergent strains of Rotavirus alphagastroenteritidis from two Australian microbat species; Nyctophilus geoffroyi (lesser long-eared bat) and Rhinolophus megaphyllus (smaller horseshoe bat), representing the first characterisation of Rotavirus alphagastroenteritidis in Australian bats, and the oldest identification of the virus to date. Concurrently, we sequenced endogenous host RNA, providing a proof-of- concept for dual host-virus transcript recovery from vouchered specimens. This study highlights the role biorepositories can play in reconstructing historical viral landscapes and enabling spatiotemporal host-virus insight to advance both biodiversity science and global pandemic preparedness.","version":"1.1","doi":"10.1101/2025.09.07.674729","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.11.675496","pub_date":"2025-9-11","title":"Climatic Influence on COVID-19: Investigating the Role of Temperature and Humidity in the Spread of the Omicron Variant","abstract":"Understanding how climate modulates infectious disease dynamics is critical for anticipating epidemic patterns. This study examines the association between climatological variables\u2014specifically temperature and relative humidity\u2014and the incidence of the SARS-CoV-2 Omicron variant (B.1.1.529) during its global wave (2021\u20132022). Using global epidemiological and climate data, we applied Scale-Dependent Correlation (SDC) analysis to detect transient, scale-specific associations across regions and periods. We identified consistent negative correlations between incidence and both temperature and humidity, especially in mid-latitudes during colder months. These findings were compared with predictions from stochastic population-based compartmental models incorporating climate-dependent transmission parameters. Among the tested formulations, the temperature-based model achieved the best fit to observed case trajectories. Our results highlight a robust climatological influence on Omicron transmission dynamics and underscore the importance of integrating climate indicators into epidemic modeling and preparedness strategies.","version":"1.1","doi":"10.1101/2025.09.11.675496","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.11.675615","pub_date":"2025-9-11","title":"Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection","abstract":"Broad spectrum antivirals are critical to respond rapidly to the threat posed by newly emerging RNA viruses. One potential candidate is the natural compound thapsigargin (Tg). Tg potently induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Recent studies have demonstrated that Tg has robust antiviral activity against several human coronaviruses (CoVs), including SARS-CoV-2, although the specific antiviral mechanism(s) have remained unclear. Here, we aimed to characterize the role of the UPR in the antiviral activity of Tg against HCoV-229E, a model common cold CoV. Consistent with previous findings, we show that a short 30-minute priming of A549 cells with Tg potently inhibits HCoV-229E infection. Time-of-addition assays showed that Tg is most effective when added up to 8 hours post-infection. Furthermore, Tg inhibits the accumulation of double-stranded RNA in infected cells, suggesting that Tg inhibits early stages of viral RNA replication. Using selective UPR pathway inhibitors to narrow down the role of these pathways in mediating the antiviral effect of Tg, we show that the inhibition of IRE1 or ATF6 does not impair the ability of Tg to inhibit HCoV-229E infection. The use of stable knockdown A549 cells in which IRE1, PERK, or ATF6 expression was silenced further revealed that the antiviral activity of Tg is not dependent on the expression of any of the three UPR sensors individually. However, HCoV-229E replication is inhibited in A549-shIRE1 cells, or in cells treated with the IRE1 inhibitor (KIRA6), suggesting that IRE1 activation may play a pro-viral role during HCoV-229E infection. Selective UPR pathway activators were used to further probe down the role of each pathway during HCoV-229E infection. Selective activation of the PERK pathway, but not IRE1 or ATF6 pathways, inhibits HCoV-229E infection. Lastly, to more broadly test the antiviral role of PERK against CoV RNA replication, we used BHK-21 cells that stably express a SARS-CoV-2 replicon. We show that selective PERK activation robustly inhibits SARS-CoV-2 replication, comparable to Tg. Overall, these findings provide insight into the antiviral mechanism(s) of Tg against CoV infection and demonstrate that modulation of the UPR may be exploited as an antiviral strategy.","version":"1.1","doi":"10.1101/2025.09.11.675615","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.10.675275","pub_date":"2025-9-11","title":"Cutaneous suction-mediated transfection in mice for delivery of DNA-encoded vaccines and proteins","abstract":"An important step to fulfill the functionalities of DNA vaccines and therapeutics is transfection in vivo to produce the encoded antigens or therapeutic proteins. A cutaneous suction-based method has demonstrated effectiveness in many animal models and has been successfully applied in human clinical trials, but has not been extended to mouse models, where numerous disease models, transgenic strains, and murine-specific reagents exist. The current work establishes and optimizes methods for cutaneous suction-mediated DNA transfection in mice. By adapting a smaller cup diameter and smaller injection volume, the challenges of skin hyperelasticity and decreased skin thickness can be effectively addressed, and vaccinating mice with the GLS-5310 SARS-CoV-2 DNA vaccine yielded high levels of binding antibody and T cell responses. Additionally, suction following injection of a novel pVAX1-based expression vector yielded systemic levels of a SEAP transgene. Thus, suction-mediated delivery of nucleic acid-based therapies and vaccines can be a valuable tool for the study in pre-clinical mouse models.","version":"1.1","doi":"10.1101/2025.09.10.675275","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.09.658611","pub_date":"2025-9-10","title":"The Susceptibility of Airborne SARS-CoV-2 to Far-UVC Irradiation","abstract":"Far-ultraviolet-C (far-UVC) irradiation has emerged as a breakthrough disinfection technology for the treatment of indoor air. Far-UVC wavelengths (222 nm) from filtered krypton-chloride excimer lamps are effective at inactivating airborne viruses and safe for human exposure, thus enabling the continuous treatment of bulk air in occupied settings. This study quantifies the susceptibility of airborne SARS-CoV-2, aerosolized in human saliva, to far-UVC radiation. We measured fluence rate-based Z value susceptibility constants (\u00b1 std. err.) of 4.4 \u00b1 0.6 and 6.8 \u00b1 0.7 cm2 mJ-1 for airborne SARS-CoV-2 under 40% and 65% relative humidity (RH) levels, respectively. At modeled far-UVC irradiation levels corresponding to 25% of the maximum safe human exposure limit, the resulting far-UVC equivalent air changes per hour (eACH) exceeded 62 hr \u00b9 at 65% RH and were significantly greater than the corresponding airborne SARS-CoV-2 natural decay rate (( std. err.) of 5.4 \u00b1 1.1 hr \u00b9, measured in the absence of far-UVC. These results define first-order loss rates for airborne SARS-CoV-2 under far-UVC exposure and support quantitative risk assessments and rational disinfection system implementation. Quantified the ability of far-UVC to facilitate airborne SARS-CoV-2 inactivation, guiding the implementation of far-UVC to promote transmission risk reduction in occupied spaces.","version":"1.2","doi":"10.1101/2025.06.09.658611","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.05.674529","pub_date":"2025-9-09","title":"Structure-based discovery of inhibitors of Mac1 domain of nonstructural protein-3 of SARS-CoV-2 by machine learning-augmented screening of chemical space","abstract":"Significant efforts have been recently dedicated to the discovery of small molecule inhibitors against the Macrodomain 1 (Mac1) of nonstructural protein 3 (NSP3) as potential antivirals for SARS-CoV-2. Thus, Mac1 has also been selected as the target for the Critical Assessment of Hit-finding Experiments (CACHE) challenge #3. As contestants in that challenge, we developed a computational strategy that ranked on the top among all 23 participants in the competition and resulted in the discovery of a novel chemical series of non-charged Mac1 inhibitors. Those have been identified through the combination of machine learning-accelerated virtual screening of Enamine REAL Diversity Subset of approximately 25 million compounds and consequent hit expansion into the entire Enamine REAL Space library. In particular, the initially identified hit compound CACHE3-HI_1706_56 (KD = 20 \u00b5M) was explored by probing 17 close analogues from a library of 44 billion molecules from the Enamine REAL. All those analogues effectively displaced the Mac1-binding ADP-ribose peptide, and 12 were confirmed to engage with Mac1 by the Surface Plasmon Resonance experiments, revealing a new chemical series of compounds for hit-to-lead optimization. The structure of the CACHE3-HI_1706_56-Mac1 complex was further determined at high resolution with crystallography, confirming initial computational predictions. Our results illustrate the effectiveness of ML-accelerated docking to rapidly identify novel chemical series and provide a strong foundation for the development of SARS-CoV-2 NSP3 Mac1 inhibitors.","version":"1.2","doi":"10.1101/2025.09.05.674529","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.08.674928","pub_date":"2025-9-09","title":"Dysregulation of innate immunity and cellular metabolism through virus-induced deISGylation","abstract":"Interferon-stimulated gene 15 (ISG15) regulates diverse cellular responses including antiviral immunity through its conjugation to proteins, a process known as ISGylation. Several pathogens, including SARS-CoV-2, subvert ISGylation by encoding deISGylating enzymes. However, the direct targets and physiological consequences of coronaviral deISGylation remain poorly defined. Here, we ablated the deISGylating activity of the SARS-CoV-2 papain-like protease (PLpro) and found that loss of deISGylation boosted innate immune activation, attenuated virus replication, and promoted viral clearance in human cells and in mice. Through untargeted metabolomics and ISGylome proteomics analyses, combined with functional studies, we discovered in molecular detail how the activities of key metabolic enzymes in glycolysis, the pentose phosphate pathway, and oxidative stress are controlled by PLpro deISGylation. These findings provide fundamental new insight into how reversible ISGylation regulates immunity and metabolic processes at the molecular level and highlight viral deISGylation as a major viral tactic for rewiring immunometabolism.","version":"1.1","doi":"10.1101/2025.09.08.674928","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.03.673949","pub_date":"2025-9-08","title":"Understanding the future risk of bat coronavirus spillover into humans \u2013 correlating sarbecovirus receptor usage, host range, and antigenicity","abstract":"Sarbecoviruses interact with their receptor, angiotensin converting enzyme 2 (ACE2), via the receptor binding domain (RBD) of Spike, the immunodominant target for neutralising antibodies. Understanding the interplay and correlation between ACE2-determined host range and antigenicity is vitally important for understanding the zoonotic potential of related bat sarbecoviruses. Using binding assays, pseudotype-entry assays and a diverse panel of mammalian ACE2 proteins, we examined the host range and related antigenicity of multiple bat coronaviruses. Broad bat ACE2 usage (a generalist phenotype) was most common in clade 1 sarbecoviruses, including SARS-CoV-2 and the BANAL isolates from Laos. In contrast, clade 3 (e.g., RhGB07) and 5 (e.g., Rc-o319) sarbecoviruses exhibited more restricted ACE2 usage (a specialist phenotype). A novel structure for RhGB07 Spike further helped to identify RBD residues associated with this receptor specialism. Interestingly, the generalist phenotypes were largely maintained with more diverse mammalian receptor libraries, including human, non-human primate, livestock, rodent ACE2 and potential intermediate reservoir hosts (e.g., civet, racoon dog, pangolin), while specialists, like RhGB07, exhibited wider phenotypic diversity. The impact of SARS-CoV-2\u2019s continued evolution in humans was also examined, identifying an expanding and/or shifting pattern of generalism for variants, especially Omicron and its sub-lineages. Furthermore, we compared and correlated these entry phenotypes with antigenicity using sera from SARS-CoV-2 convalescent individuals. Clade 1 viruses, phylogenetically related to SARS-CoV-2, were antigenically the most similar, with robust evidence for cross-neutralisation; however, there was still evidence for limited cross-neutralisation across the entire sub-genus. Finally, using monoclonal antibodies, derived from COVID-19 vaccinees with breakthrough infections, we pin-pointed the antibody epitope classes responsible for wider neutralisation. Our research indicates that generalist ACE2-using sarbecoviruses are phylogenetically and antigenically related to SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.09.03.673949","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.05.674545","pub_date":"2025-9-08","title":"Coronavirus M proteins disperse the trans-Golgi network and inhibit anterograde protein trafficking in the secretory pathway","abstract":"Coronaviruses (CoVs) encode Spike, Membrane (M), and Envelope (E) transmembrane proteins that are translated and processed at the endoplasmic reticulum (ER) and traverse the secretory pathway to converge at sites of virus assembly. Three transmembrane ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK), sense the accumulation of unfolded proteins in the ER and initiate the unfolded protein response (UPR) to increase ER protein folding capacity. We observed UPR modulation by numerous CoV proteins, including Spike, which broadly activated all three arms of the UPR. By contrast, M selectively inhibited the ATF6 arm of the UPR, either when it was activated by CoV proteins like Spike, or when it was activated by chemical stimuli of ER stress; however, M was unable to inhibit Spike-mediated activation of IRE1 or PERK. ATF6 inhibition was conserved amongst all human CoV M proteins. Amongst the UPR sensors, ATF6 has a unique activation mechanism whereby ER stress triggers translocation to the Golgi where ATF6 is processed by resident proteases to release the ATF6-N bZIP transcription factor. Because M had no effect on the function of the ATF6-N transcription factor, we reasoned that it may act upstream by inhibiting ATF6 trafficking in the secretory pathway. Indeed, we observed that ectopically expressed M inhibited several processes that require ER-to-Golgi transport, including sterol regulatory element binding protein-2 (SREBP2)-mediated activation of sterol responses and stimulator of interferon response cGAMP interactor 1 (STING1)-mediated activation of interferon responses. M also inhibited the secretion of a soluble Gaussia luciferase reporter protein. Using a Retention Using Selective Hooks (RUSH) cargo sorting assay, we observed that M accumulated in the cis-Golgi and inhibited further anterograde transport of a transmembrane reporter protein beyond this compartment, while dispersing the trans-Golgi network (TGN). We also observed a conserved TGN dispersal phenotype in cells infected with SARS-CoV-2, hCoV-OC43, or hCoV-229E. We determined that M is present in both detergent resistant and detergent soluble membranes and that M increased cholesterol abundance at the cis-Golgi. Together, these observations suggest that CoV M proteins disrupt the TGN and impede normal anterograde traffic in the canonical secretory pathway, potentially by increasing cholesterol levels at the cis-Golgi . Because CoV egress does not require the TGN, this mechanism could allow the virus to selectively interfere with host responses to infection without impeding egress of nascent virions. Many viruses encode proteins that limit host antiviral responses. Coronaviruses encode a remarkably diverse array of proteins that antagonize host responses at discrete steps including the detection of viral products, signal transduction, host mRNA processing and nuclear export, and protein synthesis. Here, we describe a new form of viral antagonism of host antiviral responses by remodelling the secretory pathway, dispersing a distal portion of the Golgi network and causing accumulation of proteins early in the Golgi. This is achieved by an abundant viral structural transmembrane glycoprotein M, which is best known for its role as the central player in the assembly of new viruses. This newly discovered function of M allows it to limit host antiviral responses that depend on trafficking in the late secretory pathway, while maintaining its role in virus assembly in the early secretory pathway.","version":"1.1","doi":"10.1101/2025.09.05.674545","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.26.672356","pub_date":"2025-9-05","title":"The 2\u2019-endo conformation of arabinose-CTP and arabinose-UTP inhibit viral polymerases by inducing long pauses","abstract":"Key to supporting human health in the face of evolving viruses is the development of novel antiviral drug scaffolds with the potential for broad inhibition of viral families. Nucleoside analogs are a key class of drugs that have demonstrated potential for the inhibition of several viral species. Here, we evaluate arabinose nucleotides (ara-NTP) as inhibitors of the SARS-CoV-2 and poliovirus polymerases using biochemistry, biophysics and structural biology. Ara-NTPs compete poorly with their natural counterparts for incorporation into RNA by viral polymerases. However, upon incorporation, ara-NMPs induce long polymerase pausing in both SARS-CoV-2 and poliovirus polymerase RNA elongation. Our studies suggest that subsequent nucleotide incorporation is inhibited at the catalytic step due to the 2\u2019-endo sugar pucker of the incorporated ara-NMP.","version":"1.2","doi":"10.1101/2025.08.26.672356","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.04.674295","pub_date":"2025-9-04","title":"Structural basis for selective remdesivir incorporation by SARS-CoV-2 RNA polymerase, and S759A resistance","abstract":"Nucleoside analogs (NAs) have been successfully used to treat viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analogs, such as remdesivir, can inhibit as delayed chain terminators or when in the template strand. Determining the frequency of remdesivir triphosphate (RTP) incorporation in the presence of the competing ATP can help in understanding different modes of viral RNA-dependent RNA polymerase (RdRp) inhibition by NTP analogs. We employed enzymatic assays, mass spectrometry, and cryo-EM to show that SARS-CoV-2 RdRp preferentially incorporates RTP, outcompeting 10-fold higher ATP concentration; however, successive RTP incorporations are less favoured when ATP is present. Structures of SARS-CoV-2 RdRp in this and previous studies demonstrate resilience of remdesivir:UMP base pair to translocation, explaining the reduced preference for conjugate incorporations. Together, the RTP versus ATP incorporation is driven by their relative concentration and structural rigidity of remdesivir:UMP, ultimately limiting the number of incorporated remdesivir in a fully synthesized RNA strand. The S759A mutant confers RTP resistance, and the structures of S759A RdRp catalytic complexes reveal that altered ribose-ring conformation and repositioning of the primer 3\u2032-end nucleotide contribute to RTP resistance. These findings enhance our understanding of non-obligate NTP analogs and provide insight into S759A resistance mechanism.","version":"1.1","doi":"10.1101/2025.09.04.674295","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.01.673077","pub_date":"2025-9-04","title":"Viral evolution during primary infection in immunocompromised hosts","abstract":"The immune response to viral infection is a delicate balance. By perturbing this balance, immunodeficiencies are expected to influence within-host viral evolution. Indeed, the presence of immunocompromised hosts has been argued to be a source of novel viral variants in some infectious diseases, including SARS-CoV-2. However, these arguments rest upon between-host models and so the role of immunodeficiencies on within-host evolution in primary infections is poorly understood. Using a mechanistic immunological model, here we consider how different immunodeficiencies shape the orchestration of the immune response during primary infection. We study how this alters the viral fitness landscape, thus speeding and slowing viral evolution. We show that during acute infections, while immunodeficiencies in neutrophils and interferon initially speed viral evolution, by the time the infection is cleared, mutations are at lower frequencies than in immunocompetent hosts. In persistent infections, we show that while T cell deficiencies slow viral evolution, interleukin-6 and macrophage deficiencies speed viral evolution. Finally, we show that positive epistatic interactions arising due to the immunological response will accelerate the evolution of viral mutations affecting the ability of virions to evade different aspects of the immune response and to enter host cells.","version":"1.1","doi":"10.1101/2025.09.01.673077","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.02.673804","pub_date":"2025-9-03","title":"Capturing the Mutational Dynamics of SARS-CoV-2 with Graphs","abstract":"The rapid evolution of SARS-CoV-2 presents significant challenges for modeling viral dynamics, driven by lineage diversification and region-specific mutation patterns. While phylogenetic trees are traditionally used for evolutionary inference, the massive volume of SARS-CoV-2 genomic data, with many similar sequences and few distinguishing mutations, poses computational and methodological limitations. The quasispecies theory instead models viral evolution as a cloud of mutants, motivating a graph-based representation that better captures the complexity of mutational events. Geographic variation adds another critical layer to this complexity. Mutation trends often differ across regions due to local transmission dynamics, host population structures, and selective pressures. In this study, we present the Mutation Learning Graph (MLG), a directed graph framework that organizes SARS-CoV-2 variants based on their cumulative mutation profiles relative to the reference genome (NC_045512.2), thereby capturing the dynamics of mutation propagation. This structure captures fine-grained mutational transitions and encodes plausible evolutionary relationships among variants. To construct these graphs, we introduce an alignment-aware mutation profiling method and a novel Ancestor-Joining algorithm, which incorporates ancestral variants as inferred intermediate nodes to connect observed genomes through biologically coherent mutational paths. We generate MLG datasets for ten geographically and epidemiologically diverse regions and benchmark them on two graph-based tasks: node-level lineage classification and edge-level mutational transition prediction. Using baseline graph neural network architectures (GCN, GraphSAGE, GAT, GGNN, VGAE), we demonstrate how mutation-centric graph structures expose key biological challenges, such as lineage imbalance and location-specific mutation spectra. For node classification, GraphSAGE and GGNN consistently achieved high accuracy (up to 0.96) and AUROC (up to 0.98). In contrast, VGAE and GraphSAGE led the way in link prediction, with AUPRCs of up to 0.96. These results highlight the effectiveness of MLG for capturing biologically meaningful mutation patterns and underscore the importance of localized, mutation-aware modeling for predicting viral mutations and future variant emergence.","version":"1.1","doi":"10.1101/2025.09.02.673804","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.02.672566","pub_date":"2025-9-03","title":"Respiratory viral infections prime accelerated lung cancer growth","abstract":"The COVID-19 pandemic has highlighted long-term health concerns of viral pneumonia, yet its potential impact on cancer development and growth remains poorly understood. Here, we demonstrate that prior infection with SARS-CoV-2 or influenza virus promoted lung tumor progression by reprogramming the local immune landscape. Retrospective clinical analysis revealed that patients hospitalized with COVID-19 exhibited increased lung cancer incidence. Using multiple murine lung cancer models, we show that prior severe respiratory viral infections accelerated tumor growth and reduced survival. Mechanistically, prior viral pneumonia epigenetically remodeled the lung to establish a pro-tumor microenvironment, including the local accumulation of SiglecFhi tumor-associated neutrophils, a transcriptionally reprogrammed, immunosuppressive population whose signature predicted poor prognosis in human lung adenocarcinoma. In parallel, epithelial compartments exhibited altered differentiation trajectories, with persistence of injury-associated alveolar intermediates positioned along tumorigenic lineages. We observe sustained chromatin remodeling at key cytokine loci in immune and structure cells, linking inflammatory memory to persistent immune suppression. Therapeutically, combined inhibition of neutrophil recruitment via CXCR2 and PD-L1 signaling restored CD8\u207a T cell infiltration and suppressed tumor growth. Together, our findings establish a direct causal relationship between viral pneumonia, including COVID-19, and lung tumorigenesis, highlighting the urgent need to monitor survivors for elevated cancer risk and to develop targeted interventions and therapies aimed at preventing potential cancer bursts in COVID-19 convalescents.","version":"1.1","doi":"10.1101/2025.09.02.672566","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.17.663733","pub_date":"2025-9-02","title":"Airway epithelial SARS-CoV-2 infectious and repair responses: relationships to age, sex, and post-COVID pulmonary syndromes","abstract":"The long-term pulmonary sequelae of SARS-CoV-2 respiratory infections reflect infection severity, innate and adaptive immunity, and respiratory epithelial repair. This study investigated the acute and reparative responses as a function of age and sex in primary human bronchial epithelial (HBE) cultures utilizing a 14-day SARS-CoV-2 infection protocol. SARS-CoV-2 infection peaked at 3 days post-infection (dpi) with an \u223c 2 log titer suppression at 14 dpi. SARS-CoV-2 infection induced interferon, interferon-induced gene, and cell damage responses. No age- or sex-dependent effects on SARS-CoV-2 infection were detected. Airway epithelia repaired to an abnormal mucus metaplastic/inflammatory state that reflected potentially beneficial and adverse consequences at 14 dpi. Repair processes were infection severity-dependent, not sex-dependent, and were more robust in young donor cultures. Analyses of long-COVID subjects with persistent pulmonary fibrosis or persistent bronchitic airway diseases exhibited expression of HBE 14 dpi failed repair gene signatures, including ISG gene signatures. Human airway epithelial repair post-SARS-CoV-2 is prolonged and incomplete in vitro over 14 days, and persistently abnormal repair may contribute to phenotypes of people with long-COVID pulmonary syndrome.","version":"1.2","doi":"10.1101/2025.07.17.663733","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.01.673483","pub_date":"2025-9-02","title":"Computational and Experimental Identification of Potential Neutralizing Peptides Derived from Human ACE2 Against SARS-CoV-2 Infection","abstract":"The human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for the entry of SARS-CoV-2. Some human alleles of ACE2 exhibit an improved affinity for the SARS-CoV-2 Spike protein. However, the impact of ACE2 polymorphisms on SARS-CoV-2 infection remains unclear. Our previous study predicted that G431 and S514 in the receptor-binding domain (RBD) od SARS-CoV-2 S1 domain are important for S protein stability, and that S protein residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD\u2013ACE2 interaction (1). In this study, we explored the potential of hACE2-derived neutralizing peptides as a therapeutic strategy against SARS-CoV-2 and investigated how ACE2 polymorphisms affect RBD\u2013ACE2 binding affinity. We applied computational saturation mutagenesis to systematically screen the binding affinity changes among all possible ACE2 missense mutations within the ACE2\u2013Wuhan-S1 complex. Mutations at ACE2 residues D355 and Y41 were predicted to weaken binding affinity, whereas those at N330 and D30 enhanced it. We identified six ACE2 regions (19\u201349, 65\u2013102, 320\u2013333, 348\u2013359, 378\u2013395, 552\u2013563) to be vital for ACE2\u2013RBD interaction. We synthesized peptides corresponding to these six regions and tested them using a pseudotyped viral particle system and dot blot assay. Three peptides were confirmed to bind with S protein, and four exhibited inhibitory effects. We aligned ACE2\u2013Wuhan-S1 and ACE2\u2013Omicron-S1 complexes, conducted correlation analysis, and observed similar binding patterns, suggesting that these peptides also have potential to neutralize Omicron strains. SARS-CoV-2 continues its global spread. In this research, we identified six regions within ACE2 that are vital for interaction with the viral S RBD and have potential to neutralize SARS-CoV-2 infection. Among the six peptides derived from ACE2, three were confirmed to bind with S protein of Wuahan strain, and four exhibited inhibitory effects on Wuahn strain SARS-CoV-2. We also found ACE2 residues D355 and Y41 as weakening affinity, and N330 and D30 as enhancing it. We also aligned this complex with the ACE2\u2013Omicron-S1 complex, performed correlation analyses, and compared their patterns of stability changes upon mutations, and obtained similar results, indicating that these peptides may also be effective against Omicron variants. These results provide insight into the role of ACE2 polymorphism in viral entry and suggest that hACE2-derived peptides may offer a promising therapeutic strategy against SARS-CoV-2, demonstrating strong consistency between our computational predictions and experimental outcomes.","version":"1.1","doi":"10.1101/2025.09.01.673483","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.01.673409","pub_date":"2025-9-02","title":"Targeted degradation of SARS-CoV-2 via the autophagy-lysosome system using chemical mimetics of the N-degron pathway","abstract":"In the N-degron pathway, ATE1 transfers the amino acid L-arginine (L-Arg) from Arg-tRNAArg to N-terminal (Nt) residues of cellular proteins. The resulting Arg/N-degrons bind the autophagic receptor p62/SQSTSM-1/Sequestosome-1 to induce lysosomal degradation of various biomaterials. Here, we demonstrate that the chemical mimetics of Arg/N-degrons, termed autophagy-targeting ligands (ATLs), can induce lysosomal degradation of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) via p62-mediated macroautophagy. In Vero E6 cells infected with SARS-CoV-2, ATLs promoted p62 self-polymerization and enhanced LC3 synthesis and lipidation, leading to viral sequestration within autophagosomes for lysosomal degradation. In transgenic mice overexpressing human angiotensin-converting enzyme 2 (ACE2), oral administration of ATL1014 inhibited viral replication and increased viability. In a Syrian hamster model, ATL1014 attenuated viral replication in the lungs and demonstrated efficacy in inflammatory lesions and pulmonary congestions. These results identify the N-degron pathway as a potential target for a host-targeting strategy (HTS) against a broad spectrum of viruses.","version":"1.1","doi":"10.1101/2025.09.01.673409","journal":"bioRxiv","score":null},{"id":"10.1101/2025.09.02.673669","pub_date":"2025-9-02","title":"Design of a novel multi-epitope mRNA vaccine against BtHKU5-CoV-2 using immunoinformatics","abstract":"Bat HKU5-CoV-2 (BtHKU5-CoV-2), a recently discovered bat-infecting merbecovirus, was found to infect human cell lines by utilizing the human angiotensin-converting enzyme 2 (ACE2) receptor, similar to SARS-CoV-2, which caused millions of deaths. Moreover, its broad host tropism has raised significant concerns about potential human spillover risk. Therefore, there is an urgent need to develop vaccines to combat the potential outbreak of BtHKU5-CoV-2. However, research focusing on BtHKU5-CoV-2 remains limited. In this study, we designed a novel multi-epitope vaccine against BtHKU5-CoV-2 using an immunoinformatic approach. Eight cytotoxic T lymphocyte (CTL) epitopes, seven helper T lymphocyte (HTL) epitopes, and five linear B lymphocyte (LBL) epitopes were screened from the spike glycoprotein of BtHKU5-CoV-2. The selected epitopes were joined together with an appropriate linker, and \u03b2-defensin II and MHC I-targeting domain (MITD) were incorporated into the construct to enhance vaccine immunogenicity. Biological characteristic analysis revealed that the designed vaccine exhibited strong antigenicity and immunogenicity while being non-toxic and non-allergenic. The tertiary structure of the multi-epitope vaccine was modeled, refined, and validated, demonstrating its structural stability and near-native conformation. Molecular docking studies showed that the vaccine successfully docked with Toll-like receptor 2 (TLR2) and TLR4. Moreover, its mRNA exhibits strong interactions with TLR3, TLR7, and TLR8 receptors. Additionally, in silico immune simulations have suggested that vaccination could trigger robust humoral and cellular immunity. These findings suggest that the proposed mRNA vaccine is a potential candidate for targeting BtHKU5-CoV-2. Further experiments are necessary to validate its protective efficacy. BtHKU5-CoV-2, a newly discovered merbecovirus isolated from bats, exhibits potential for spillover into humans. It was found to utilize human ACE2 as functional receptors for infection. A functional receptor acts like a \u201ckey\u201d that fits into the \u201clock\u201d on the host cell, enabling viral entry. BtHKU5-CoV-2 warrants significant attention, because it shares the same functional receptor with SARS-CoV-1 and SARS-CoV-2, which caused the 2003 SARS epidemic and the 2019 pandemic, respectively. Thus, developing vaccines to prevent potential global outbreaks of BtHKU5-CoV-2 is urgently needed. Theoretically, within the body\u2019s immune surveillance system, proteins from BtHKU5-CoV-2 are processed via proteasomal degradation into short peptides. The peptides with immunogenicity bind to MHC molecules and are presented on the cell surface. These peptides, known as epitopes, can initiate immune reaction. In this study, we designed a multi-epitope mRNA vaccine against BtHKU5-CoV-2 using immunoinformatics methods. Epitopes were screened from the spike glycoprotein, a promising target of BtHKU5-CoV-2. Our results suggest that the vaccine is safe and capable of inducing strong humoral and cellular immunity. Therefore, this mRNA vaccine represents a promising candidate for preventing furture BtHKU5-CoV-2 outbreak.","version":"1.1","doi":"10.1101/2025.09.02.673669","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.31.673412","pub_date":"2025-9-02","title":"Excitability as a Design Principle in the Immune System","abstract":"Growing datasets and mechanistic detail in immunology have outpaced the development of unifying concepts. Such concepts are required to explain the primary goals of immune circuits - strong response to pathogens, tolerance to self, and prevention of collateral damage. A principle that achieves these goals across diverse immune circuits could unify our understanding of the immune system. Here, we propose that excitability, a concept from dynamical systems, serves this role. We screen thousands of circuits to identify those that generate excitable dynamics, and find a single robust design. We scan the human immune network to find this circuit architecture in dozens of innate and adaptive subsystems. We provide evidence for excitability in data on longitudinal responses to SARS-CoV-2. Similar motifs underlie T cell activation, autoimmune flares, and tumor immune responses. This conserved motif provides therapeutic targets and suggests that excitability is a core design principle of immunity, bridging molecular and cellular levels.","version":"1.1","doi":"10.1101/2025.08.31.673412","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.28.672949","pub_date":"2025-8-29","title":"Determinants of Natural Killer Cell-Mediated Antibody Dependent Cellular Cytotoxicity in SARS-CoV-2 Antibodies","abstract":"A growing body of evidence underscores the role of antibody-dependent cellular cytotoxicity (ADCC) in antiviral immunity. Yet, the mechanisms underlying the ability of certain antibodies (Abs) to mediate potent ADCC activity remain poorly characterized \u2013 in particular the contribution of features within the antigen binding Fab region remain largely unexplored. In this study, we leveraged a collection of 142 SARS-CoV2 monoclonal Abs to systematically dissect the determinants of ADCC activity. We analyzed their epitope domain target, binding characteristics, neutralization potency, somatic hypermutation (SHM) and CDR3 length to determine the contribution of these features to ADCC activity. We found that ADCC activity is primarily shaped by epitope target-particularly targeting of the S2 domain of the Spike glycoprotein. ADCC potency was not associated with the degree of SHM or neutralization. Notably, ADCC activity was not correlated with binding affinity and moderate binding to antigen was sufficient for ADCC activity. By integrating these analyses, we provide a comprehensive framework for understanding the molecular and functional determinants of ADCC. Together, these findings offer novel insights into the mechanisms that underpin ADCC functions, with implications for vaccine design and therapeutic Abs development. While antibodies are best known for stopping viruses from entering cells, they also activate other arms of the immune system. One important function is their ability to signal immune cells to destroy virus-infected targets, a process called antibody-dependent cellular cytotoxicity (ADCC). In this study, we examined 142 antibodies directed against the spike protein of the virus that causes COVID-19. We compared their binding patterns, neutralizing ability, level of maturation, and other features to determine what drives their capacity to trigger ADCC. We found that the region of the spike protein targeted by the antibody was the main factor shaping this activity, with antibodies that recognized the S2 region being particularly effective. In contrast, the amount of antibody maturation, the strength of binding, or the ability to neutralize the virus did not predict immune cell activation. Our findings show that antibodies can contribute to antiviral defense through distinct mechanisms and provide new insights for guiding vaccine and antibody therapy design.","version":"1.1","doi":"10.1101/2025.08.28.672949","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.28.672883","pub_date":"2025-8-28","title":"Structural and functional analysis of VYD222: a broadly neutralizing antibody against SARS-CoV-2 variants","abstract":"Extensive mutations in SARS-CoV-2 spike protein have rendered most therapeutic monoclonal antibodies (mAbs) ineffective. However, here we describe VYD222 (pemivibart), a human mAb re-engineered from ADG20 (adintrevimab), which maintains potency despite substantial virus evolution. VYD222 received FDA Emergency Use Authorization for pre-exposure prophylaxis of COVID-19 in certain immunocompromised adults and adolescents. Here we show potent neutralization of this antibody against a broad range of emerging variants, including Omicron KP.3 and KP.3.1.1. X-ray crystal structures of VYD222 complexed with the receptor-binding domains of prototype SARS-CoV-2 and Omicron BA.5 demonstrate the binding epitope spans from the receptor binding site to the conserved CR3022 site. Notably, many of the matured residues between ADG20 and VYD222 occur outside the paratopic region. Deep mutational scanning indicates that SARS-CoV-2\u2019s ability to escape VYD222 is constrained by structural compatibility and the need to maintain receptor binding. These findings provide crucial insights into the escape-resistant neutralization of VYD222 against a broad panel of clinically relevant SARS-CoV-2 variants and offer valuable guidance for risk assessment of emergent variants.","version":"1.1","doi":"10.1101/2025.08.28.672883","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.27.672781","pub_date":"2025-8-28","title":"12-Lipoxygenase (12-LOX) Plays a Key Role in Hyperinflammatory Response Caused by SARS-CoV-2","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. The severe disease outcomes are often linked to a hyperinflammatory response known as a \u201ccytokine storm.\u201d The underlying mechanisms responsible for this exaggerated immune response remain incompletely understood. This study aimed to investigate the molecular pathways contributing to the severe inflammatory damage and mortality associated with COVID-19. SARS-CoV-2 hijacks host lipid metabolism, particularly the phospholipase A2 (PLA2) pathway, which leads to the production of the bioactive molecules, including the 12-Lipoxygenase (12-LOX)-derived lipid mediators in platelets, as well as in lung and vascular cells. We hypothesized that 12-LOX drives the hyperinflammatory response and disease severity, and that its inhibition could reduce inflammation and improve outcomes. Analysis of autopsy lung samples from COVID-19 decedents and SARS-CoV-2-infected K18-hACE2 transgenic mice revealed increased 12-LOX expression. We evaluated VLX-1005, a selective small-molecule 12-LOX inhibitor, in infected mice. Treatment initiated 48 hours post-infection significantly improved survival, reduced body weight loss, and decreased lung inflammation compared to controls. Notably, male mice showed higher survival rates than females. VLX-1005 treatment also suppressed key chemokines and cytokines associated with the cytokine storm, and reduced lung damage. These findings identify 12-LOX as a critical mediator of the hyperinflammatory response in severe COVID-19 and support its inhibition as a promising therapeutic strategy to mitigate inflammatory damage and reduce mortality. This study provides critical insights into the mechanisms underlying severe COVID-19, identifying 12-Lipoxygenase (12-LOX) as a key driver of the hyperinflammatory response that contributes to disease severity and mortality. By demonstrating that SARS-CoV-2 hijacks host lipid metabolism to elevate pro-inflammatory lipid mediators, the research uncovers a novel pathogenic pathway that exacerbates lung inflammation. The use of VLX-1005, a selective 12-LOX inhibitor, significantly improved survival and reduced inflammatory damage in a mouse model, highlighting its therapeutic potential. These findings not only deepen our understanding of COVID-19 pathogenesis but also position 12-LOX as a promising target for intervention, offering a new avenue for mitigating the effects of cytokine storms in severe cases.","version":"1.1","doi":"10.1101/2025.08.27.672781","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.28.672752","pub_date":"2025-8-28","title":"SARS-CoV-2 nucleocapsid induces hyperinflammation and vascular leakage through the Toll-like receptor signaling axis in macrophages","abstract":"Tens of thousands of severe COVID-19 cases are hospitalized weekly in the U.S., often driven by an imbalance between antiviral responses and inflammatory signaling, leading to uncontrolled cytokine secretion. The SARS-CoV-2 nucleocapsid (N) protein is a known immune antagonist, but its role in macrophage-driven cytokine storms is unclear. We demonstrate that N functions in a pathway-specific manner, specifically amplifying nuclear factor \u03baB-related transcripts upon Toll-like receptor 7/8 stimulation. Moreover, we show that this is a conserved feature of pathogenic coronaviruses, with the delta variant N being the most pro-inflammatory. Our interaction networks suggest the delta variant N drives inflammation through interactions with several stress granule-related proteins. Profiling of secreted cytokines revealed that supernatants from the delta variant N-expressing macrophages disrupt brain and heart endothelial barriers, implicating N in COVID-19-associated cognitive and cardiac complications. Our findings highlight N-mediated immune imbalance as a driver of severe COVID-19 and identify N as a promising therapeutic target to mitigate hyperinflammation.","version":"1.1","doi":"10.1101/2025.08.28.672752","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.28.672794","pub_date":"2025-8-28","title":"Morphological cell profiling for drug repurposing against SARS-CoV-2 infection","abstract":"Antiviral drug discovery has traditionally focused on targeting viral proteins, while host-directed strategies remain largely underexplored. Here, present a systematic drug repurposing strategy leveraging morphological profiling to identify host-targeting antivirals. Our image-based approach combines viral protein immunostaining with high-content Cell Painting analysis to simultaneously assess viral replication and provide in-depth analysis of host cell responses. By screening 5,275 repurposable drugs against SARS-CoV-2 infected cells, we identified compounds that reversed the infected cell phenotype, including ones not detected by conventional cytopathicity and antibody-based assays. A counter-screen excluded compounds whose antiviral activity was likely driven by drug-induced phospholipidosis (DIPL). Pathway enrichment analysis of compounds validated by both Cell Painting dose-response and DIPL assays, revealed host processes frequently hijacked by viruses, including innate immune responses and kinases. Among the top hits, both novel candidates, such as serdemetan, and previously reported broad-spectrum antivirals, such as sunitinib, were identified. Our approach constitutes an adaptable and scalable platform suited for diverse viral pathogens and cell systems. We provide a resource of open access screening data, images, and automated analysis pipelines to advance both antiviral discovery and pandemic preparedness.","version":"1.1","doi":"10.1101/2025.08.28.672794","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.27.672735","pub_date":"2025-8-28","title":"Affinity Maturation and Light-Chain-Mediated Paratope Diversification Anticipate Viral Evolution","abstract":"A key goal of vaccinology is to train the immune system to combat current pathogens while simultaneously preparing it for future evolved variants. Understanding factors contributing to anticipatory breadth, wherein affinity maturation against an ancestral strain yields neutralization capacity against evolved variants, is therefore of great importance. Here, we investigated the mechanism of anticipatory breadth development in a public antibody family targeting the functionally restricted ACE2 binding site on SARS-CoV-2. IGHV3-53/66 antibodies isolated from memory B cells of infection-na\u00efve individuals vaccinated with the ancestral Wuhan-strain mRNA vaccine frequently neutralized evolved Omicron variants and contained several hallmark mutations previously shown to enhance neutralization breadth. Comparative analyses with antibodies from Omicron breakthrough infections revealed that breadth-associated patterns of somatic hypermutation emerged independently of variant exposure. However, Omicron infection had a marked impact on light chain pairing frequencies, suggestive of variant-imposed selection of favorable light chains. Analysis of available IGHV3-53/66 antibody structures complexed with SARS-CoV-2 receptor binding domain (RBD) clarified these findings; convergent somatic mutations on the heavy chain largely refined contacts with invariant RBD residues, while light chain pairings shifted epitopes to avoid steric challenges posed by Omicron mutations. These findings support a model of anticipatory breadth with three key elements: (1) targeting of a functionally restricted epitope, (2) affinity maturation to establish an affinity buffer, and (3) variable chain pairing to generate paratope diversity. These elements each serve to compensate for a distinct consequence of viral mutagenesis, offering a mechanistic framework for anticipating viral evolution.","version":"1.1","doi":"10.1101/2025.08.27.672735","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.27.672717","pub_date":"2025-8-28","title":"Integrated phenotypic screening and chemical proteomics identifies ETF1 ligands that modulate viral translation and replication","abstract":"Emerging and re-emerging viruses pose a significant threat to global health. Although direct-acting antivirals have shown success, their efficacy is limited by the rapid emergence of drug-resistant viral variants. Hence, there is an urgent need for additional broad spectrum antiviral therapeutic strategies. Here, we identify by phenotypic screening a set of stereochemically defined photoreactive small molecules (photo-stereoprobes) that stereoselectively suppress SARS-CoV-2 replication in human lung epithelial cells. Structure-activity relationship-guided chemical proteomics identified the eukaryotic translation termination factor 1 (ETF1) as a target of the photo-stereoprobes, and this interaction was recapitulated with recombinant purified ETF1. We found that the photo-stereoprobes modulate programmed ribosomal frameshifting mechanisms essential for SARS-CoV-2 infection without causing ETF1 degradation, thus distinguishing the photo-stereoprobes from other known ETF1-directed small molecules. We finally show that the photo-stereoprobes also inhibit the replication of additional viruses with non-canonical ribosomal frameshifting mechanisms. Our findings thus identify a mechanistically distinct class of ETF1 ligands that implicate host translation termination processes as a potential target for antiviral development. The identification of broad-spectrum antivirals that target host proteins is a desirable strategy to combat emerging viral infections given the rapid escape potential of viruses and the need to develop new countermeasures for clinically significant pathogens. Here, we integrate phenotypic screening and chemical proteomics to identify photo-stereoprobe small molecules that inhibit the replication of multiple viruses. We show that these compounds bind the protein eukaryotic peptide chain release factor subunit 1 (ETF1) and modulate programmed ribosomal frameshifting. Unlike previously described ligands for ETF1, which lead to proteasomal destruction of the protein, we did not find that the photo-stereoprobes altered ETF1 content in cells. Our findings thus point to an opportunity to pharmacologically modulate a host protein implicated in programmed ribosomal frameshifting as a strategy to combat infection of viruses from different families.","version":"1.1","doi":"10.1101/2025.08.27.672717","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.26.672456","pub_date":"2025-8-26","title":"The Effect of SARS-COV-2 Protein Fragments on the Dimerization of \u03b1-Synuclein","abstract":"There is evidence that amyloidogenic segments in SARS-COV-2 proteins can induce aggregation of \u03b1-synuclein (\u03b1S), the main component of brain-located amyloids whose presence is connected with Parkinson\u2019s Disease (PD). Using molecular dynamic simulations, we could show in earlier work that SARS-COV-2 protein fragments shift the ensemble of \u03b1S chains toward more aggregation-prone conformations. However, the mechanism by which these chains assemble into fibrils, the presumed neurotoxic agent in PD, is not clear. The first step on that route are dimers. For this reason, we have now, using again molecular dynamics simulations, studied how the fragment 194FKNIDGYFKI203 (FI10) of the SARS-COV-2 spike protein, and the fragment 54SFYVYSRVK62 (SK9) of the envelope protein, alter the ensemble of \u03b1-synuclein dimers. Our simulations suggest a differential stabilization of such dimers that would preferentially seed rod-like fibrils over the competing twister-like structures.","version":"1.1","doi":"10.1101/2025.08.26.672456","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.20.671191","pub_date":"2025-8-26","title":"Enhancing the outcome of crystallographic screening for efficient drug discovery by choosing the right crystal form","abstract":"In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign.","version":"1.2","doi":"10.1101/2025.08.20.671191","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.21.671674","pub_date":"2025-8-25","title":"SARS-CoV-2 NSP14 inhibitor exhibits potent antiviral activity and reverses NSP14-driven host modulation","abstract":"The emergence of SARS-CoV-2 variants and drug-resistant mutants highlights the urgent need for novel antiviral therapeutics. SARS-CoV-2 NSP14, an N7-guanosine methyltransferase, plays a critical role in viral RNA capping, enabling viral replication and immune evasion. While NSP14 has emerged as a promising drug target, its role in host-virus crosstalk and the cellular consequences of NSP14 inhibition remain poorly understood. Here, we present the identification and characterization of C10, a highly potent and selective first-in-class non-nucleoside inhibitor of the NSP14 S-adenosylmethionine (SAM)-binding pocket. C10 demonstrates robust antiviral activity against SARS-CoV-2, including its variants, with EC50 values ranging from 64.03 to 301.9 nM, comparable to the FDA-approved drug remdesivir in our cell-based assays. C10 also exhibits broad-spectrum activity against other betacoronaviruses and inhibits SARS-CoV-2 at the replication stage. C10 suppresses viral translation and exhibits immunostimulatory effect. Additionally, C10 specifically reversed NSP14-mediated alterations in host transcriptome. The antiviral efficacy of C10 was further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings highlight C10 as a promising candidate for the development of effective treatments against SARS-CoV-2 and its emerging variants. This study also uncovers a novel mechanism of NSP14 in SARS-CoV-2 pathogenesis and its therapeutic potential, providing insights that may extend to other viral capping methyltransferases.","version":"1.1","doi":"10.1101/2025.08.21.671674","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.22.671713","pub_date":"2025-8-22","title":"Pediatric Long COVID Is Characterized by Myeloid CCR6 Suppression and Immune Dysregulation","abstract":"The biological mechanisms underlying long COVID in the pediatric population are poorly understood. Our study aimed to characterize the immune pathophysiology of long COVID in children and young people (CYP). We analyzed major immune cell compartments in PBMCs, as well as specific SARS-CoV-2 antibody response in CYP with (n=99) and without (n=18) long COVID at three months following acute infection. Our findings indicate that pediatric long COVID is associated with a dysregulated immune response characterized by altered innate immunity and overactivated T-, B- and NK-cell responses. Furthermore, CYP with long COVID had an impaired humoral response to SARS-CoV-2 marked by a dysregulated B-cell compartment and lower levels of anti-RBD IgG and IgA. This correlated with reduced neutralizing capacity against SARS-CoV-2. Random forest analysis identified CCR6 expression on myeloid cells as the most relevant biomarker that distinguishes long COVID from control individuals with 79% accuracy.","version":"1.1","doi":"10.1101/2025.08.22.671713","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.17.670707","pub_date":"2025-8-19","title":"Monoclonal Antibodies from COVID-19 Convalescent Patients Target Cryptic Epitopes for Universal SARS-CoV-2 Neutralization","abstract":"The COVID-19 pandemic, which has resulted in over seven million global fatalities, poses a substantial threat to public health and precipitated a global economic crisis. Emerging variants of concern (VOCs) with enhanced transmissibility and improved immune evasion may compromise the efficacy of current antiviral and immunotherapies, necessitating comprehensive investigations into the immune response to SARS-CoV-2. The conformational dynamics of the receptor binding domain (RBD) in SARS-CoV-2 spike and the presentation of neutralizing antibody epitopes influence viral transmission and infection rates. In this study, we have identified highly conserved non-RBM epitopes for two potent monoclonal antibodies (mAbs), TAU-1109 and TAU-2310, isolated from convalescent human patients, which contribute to the broad neutralizing activity of these mAbs against all the circulating VOCs, including the recently emerged Omicron subvariants. We employed high- resolution structural data in conjunction with systematic biochemical investigation to elucidate the neutralization mechanism of TAU-1109 and TAU-2310. The mechanism involves antibody-mediated destabilization of the spike trimer, resulting in the premature shedding of the S1 subunit and rendering the spike incapable of mediating host cell entry. The identification of conserved cryptic epitopes in our study advances the mechanistic understanding of immune response against SARS-CoV-2, providing novel avenues for the development of universal therapeutic antibodies and vaccines to combat COVID-19.","version":"1.1","doi":"10.1101/2025.08.17.670707","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.18.670887","pub_date":"2025-8-19","title":"SARS-CoV-2 S protein activates the HIV latent reservoir through the mTOR pathway","abstract":"During the global COVID-19 pandemic, mRNA vaccines using the S protein as antigen were widely used.Vaccine-induced S proteins can persist in vivo for weeks, triggering low-level immune activation. HIV latent reservoir maintenance is a major challenge for ART therapy, especially when immune pressure is waning. This then raises critical questions for HIV-infected patients: does prolonged exposure to S proteins affect HIV latent reservoir stability? Recent studies have pointed out that S proteins may activate the mTOR signaling pathway, which in turn affects the immune response and metabolic processes of cells. And the mTOR pathway is closely related to the maintenance and activation of HIV latent reservoir. However, how S proteins affect the HIV latent reservoir and the mechanism of activation are unclear. To explore the mechanism of how SARS-CoV-2 S proteins regulate the HIV latent reservoir and to explore whether S proteins regulate the HIV latent reservoir through the mTOR pathway, we constructed an in vitro HIV latent reservoir model for our experiments.To evaluate the potential role of S protein in HIV latent reservoir activation, relevant markers of HIV latent reservoir activation were detected using ELISA, flow cytometry, and RT-qPCR; and the relationship between S protein and mTOR was also detected by WB, CO-IP, and IFC.It was found that S proteins activated the HIV latent reservoir while increasing mTOR expression. It was further observed that mTOR inhibitors significantly inhibited S protein-induced activation of the HIV latent reservoir, and mTOR activators reversed the inhibitory effect of mTOR inhibitors on HIV latent reservoir activation. In summary, we found that S proteins activated the HIV latent reservoir through the mTOR pathway. S protein interacts with mTOR and activates the mTOR-p-p70S6K-pS6 pathway, which promotes HIV transcription","version":"1.1","doi":"10.1101/2025.08.18.670887","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.18.670908","pub_date":"2025-8-19","title":"Persistent Immune Dysregulation during Post-Acute Sequelae of COVID-19 is Manifested in Antibodies Targeting Envelope and Nucleocapsid Proteins","abstract":"Post-Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome or \u201cLong COVID\u201d represents a widespread health challenge that necessitates the development of novel diagnostic approaches and targeted therapies that can be readily deployed. Immune dysregulation has been reported as one of the hallmarks of PASC, but the extent of PASC immune dysregulation in patients over time remains unclear. We therefore assessed SARS-CoV-2-specific antibody responses, peripheral immune cell profiles, autoantibody profiles and circulating cytokines for up to 6 months in participants with a SARS-CoV-2 infection who either convalesced or developed PASC. Compared to convalescent, PASC participants with a broad range of PASC phenotypes exhibited persistently elevated IgG titers for SARS-CoV-2 Envelope and Nucleocapsid proteins over the 6 months of study duration. In contrast, the IgG responses to Spike protein were significantly lower in the PASC cohort with predominantly IgG1 and IgG3 class-switched bias. Using CyTOF analysis, we show elevated numbers of circulating T follicular helper cells (cTFH) and mucosa-associated invariant T cells (MAIT), which also correlated with high anti-Envelope IgG titers. Persistent immune activation was accompanied by augmented serum cytokine profiles with LIF, IL-11, Eotaxin-3, and HMGB-1 in PASC participants, who also demonstrated significantly higher rates of autoantibodies. These findings highlight the persistence of immune dysregulation in PASC, underscoring the need to explore targeted therapies addressing viral persistence, dysregulated antibody production, and autoimmunity.","version":"1.1","doi":"10.1101/2025.08.18.670908","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.15.668720","pub_date":"2025-8-18","title":"Dose-dependent interferon programs in myeloid cells after mRNA and adenovirus COVID-19 vaccination","abstract":"The SARS-CoV-2 pandemic provided a rare opportunity to study how human immune responses develop to a novel viral antigen delivered through different vaccine platforms. However, to date, no study has directly compared immune responses to all three FDA-approved COVID-19 vaccines at single-cell multi-omic resolution. We longitudinally profiled SARS-CoV-2\u2013na\u00efve adults (n=31) vaccinated with BNT162b2, mRNA-1273, or Ad26.COV2.S, integrating plasma cytokines, antibody titers, and single-cell multi-omic data (DOGMA-seq). We discovered a distinct, transient interferon program (ISG-dim) that emerged specifically 1-2 days after the first mRNA dose in \u223c10% of myeloid cells. This state was characterized by ISGF3 complex activation and its target genes (e.g., MX1, MX2, DDX58), with transcriptional and epigenetic profiles distinct from the robust interferon program observed after mRNA boosting or a single Ad26.COV2.S dose (ISG-high). In vitro stimulation of human monocytes showed that IFN-\u03b1 alone recapitulates ISG-dim, whereas both IFN-\u03b1 and IFN-\u03b3 are required for ISG-high. These findings define dose-dependent interferon programming in human myeloid cells, highlight mechanistic differences between priming and boosting, with implications for optimizing vaccine platform choice, dose scheduling, and formulation.","version":"1.1","doi":"10.1101/2025.08.15.668720","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.15.670620","pub_date":"2025-8-18","title":"A Super-Resolution Spatial Atlas of SARS-CoV-2 Infection in Human Cells","abstract":"The spatial organization of viral and host components dictates the course of infection, yet the nanoscale architecture of the SARS-CoV-2 life cycle remains largely uncharted. Here, we present a comprehensive super-resolution Atlas of SARS-CoV-2 infection, systematically mapping the localization of nearly all viral proteins and RNAs in human cells. This resource reveals that the viral main protease, nsp5, localizes to the interior of double-membrane vesicles (DMVs), challenging existing models and suggesting that polyprotein processing is a terminal step in replication organelle maturation. We identify previously undescribed features of the infection landscape, including thin dsRNA \u201cconnectors\u201d that physically link DMVs, and large, membrane-less dsRNA granules decorated with replicase components, reminiscent of viroplasms. Finally, we show that the antiviral drug nirmatrelvir induces the formation of persistent, multi-layered bodies of uncleaved polyproteins. This spatial Atlas provides a foundational resource for understanding coronavirus biology and offers crucial insights into viral replication, assembly, and antiviral mechanisms.","version":"1.1","doi":"10.1101/2025.08.15.670620","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.18.670804","pub_date":"2025-8-18","title":"Metatranscriptomic Insights into Host-Microbiome Interactions Underlying Asymptomatic COVID-19 Cases","abstract":"In recent years, the research on Coronavirus disease 2019 (COVID-19) has surged rapidly due to its infectious nature and epidemiological importance as a pandemic. Association of other pathogenic microbes or different human microbiomes with COVID-19 severity is well established. While much is known about COVID-19, studies exploring how host-pathogen interactions at the transcriptomic level influence disease severity are still limited. This research focuses on metatranscriptomic perspective of COVID-19 patients from different Bangladeshi cohorts. Metatranscriptomic sequencing was performed using the extracted RNA of forty different nasopharyngeal samples. After preprocessing and assembly of the genome sequence data, taxonomic identification and diversity along with antibiotic resistance pattern was analyzed using different bioinformatic pipelines. COVID-19 positive and asymptomatic positive patients had a higher abundance of pathogenic and multidrug resistant bacteria whereas the Healthy or recovered patients had higher fungal population. Differential gene expression analysis was also performed to identify the upregulated and downregulated genes responsible for different biological and immunological pathways of humans. Here, immunological response related genes were mostly upregulated in positive cases which was also evident in the proinflammatory cytokines upregulation. Moreover, asymptomatic positive cases showed low TLR-4 expression, which is key to recognizing pathogen-associated molecular patterns (PAMPs) and triggering pro-inflammatory immune responses. This study can clarify the gene expression and signaling pattern of COVID-19 patients with different severity. This may also suggest that some populations exhibit reduced basal expression of TLR-4, which may suppress innate immune activation following infection and contribute to asymptomatic clinical outcomes; however, this hypothesis requires further investigation. Future studies should aim to validate these associations using larger, ethnically diverse cohorts with comprehensive clinical and demographic data.","version":"1.1","doi":"10.1101/2025.08.18.670804","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.14.670296","pub_date":"2025-8-14","title":"Cross-Neutralizing Monoclonal Antibodies with Broad Activity Against Human and Bat-Derived SARS-Related Coronaviruses","abstract":"Effective therapeutics for severe cases of SARS-CoV-2 are still needed. As new variants of concern emerged an increase in hospitalizations was observed, especially in non-vaccinated individuals, immunocompromised individuals and the elderly, whereby treatment options became challenging. Several monoclonal antibodies (mAb) are being evaluated for approval due to their ability to neutralize the virus. Here, we report monoclonal antibodies targeting the Spike (S) with a strong neutralization profile against lentiviral and VSV pseudotypes displaying the Spikes of SARS-CoV-1, SARS-CoV-2 and variants of concern, in addition to two bat coronaviruses. We found several mAbs that were able to bind and neutralize a broad set of variants, with one mAb able to neutralize SARS-CoV-1, SARS-CoV-2, RaTG13 and WIV16. Their binding affinities were also characterized, and several mAbs were in the picomolar range against different variants. These results indicate that their use as a cocktail of monoclonal antibodies to treat patients infected with variants associated with severe disease and immune escape can be explored as an option. Furthermore, the cross reactivity observed by one mAb may reveal further insight into vulnerable portions of the Spike protein, which may become valuable future targets.","version":"1.1","doi":"10.1101/2025.08.14.670296","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.05.641771","pub_date":"2025-8-14","title":"WITHDRAWN: Investigation of the Mechanism of Action of JinHuangJieDu against SARS-CoV-2 Infection and Inflammation Based on Network Pharmacology and Experimental Validation","abstract":"The authors have withdrawn this manuscript because additional experiments are required to enhance the completeness and clarity of the study. Therefore, the authors do not wish this work to be cited as a reference for any project. If you have any questions, please contact the corresponding author at lcchenlong@163.com.","version":"1.2","doi":"10.1101/2025.03.05.641771","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.13.670105","pub_date":"2025-8-13","title":"A Mouse Model of SARS-CoV-2-Driven Acute Maladaptive Responses and Chronic Systemic Diseases","abstract":"Our understanding of SARS-CoV-2 acute and post-acute pathogenesis is hindered by the lack of adequate small animal models. We present RAB/6N, a mouse model prone to severe disease after exposure to SARS-CoV-2 clinical isolates, with lethal cases showing no widespread brain infection typical of the widely used K18-hACE2 mouse model. Lung viral replication in RAB/6N mice remains steady for several days before a decline in viral titers. Delayed initiation of infection clearance is marked by increased lung T-cell extravasation and type-2 immune responses, leading to maladaptive lung consolidation. While systemic antiviral cytokine responses only correlate with SARS-CoV-2 brain infection in K18-hACE2 mice, they are concomitant with pulmonary immune dynamics in infected RAB/6N mice. Convalescent RAB/6N mice display systemic inflammation and decreased antibody titers against SARS-CoV-2 spike RBD, persistent viral RNA and prolonged lymphoid infiltration in the lungs. These animals also exhibit signatures of multi-organ dysfunction, cognitive impairment, cardiac inflammation, hyper- immunoglobulin production, and various autoimmune disorders, illuminating the molecular correlates of various pathologies associated with post-acute sequelae of COVID-19 (PASC). RAB/6N mice pave the way for dissecting the molecular drivers underlying SARS-CoV-2-induced acute maladaptive responses and subsequent post-acute systemic diseases. This preclinical platform also opens opportunities for the exploration of therapeutic interventions against systemic PASC and for anticipating the emergence of PASC-associated comorbidities. We generated a hACE2-transgenic mouse model that develops maladaptive lung immune responses upon acute SARS-CoV-2 infection, leading to fatal outcomes or post-acute systemic disease syndromes in convalescent animals.","version":"1.1","doi":"10.1101/2025.08.13.670105","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.13.669859","pub_date":"2025-8-13","title":"Human outbreak detection and best practice MPXV analysis and interpretation with squirrel","abstract":"High numbers of reported mpox cases and recent identification of multiple sustained human outbreaks of MPXV have highlighted the need for robust, best-practice genomic surveillance tools. In light of the SARS-CoV-2 pandemic, many labs across the globe developed the capacity to do virus genome sequencing, however MPXV presents additional analytical challenges due to its large genome size, tracts of low-complexity or repeat regions, genetically distinct clades, and the need to perform bespoke APOBEC3-mutation reconstruction. We present squirrel (Some Quick Reconstruction to Resolve Evolutionary Links), an open source bioinformatic tool that can perform clade-aware alignment, mutation quality assessment, phylogenetic inference and automated APOBEC3-mutation classification on branches of the phylogeny. Squirrel can be run on the command line or launched through the EPI2ME GUI through the squirrel-nf workflow, enabling robust analysis without need for the command line. With the interactive output report produced and publication-ready APOBEC3-reconstruction visualisation, squirrel enables researchers to distinguish between zoonotic and sustained human outbreaks and help accurately inform public health responses.","version":"1.1","doi":"10.1101/2025.08.13.669859","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.12.669990","pub_date":"2025-8-13","title":"Toehold-VISTA: A machine learning approach to decipher programmable RNA sensor-target interactions","abstract":"RNA-based biosensors have emerged as essential tools in synthetic biology and diagnostics, enabling precise and programmable responses to diverse RNA inputs. However, the time to design, produce, and screen high-performance RNA sensors remains a critical challenge. The fundamental rules governing RNA-RNA interactions\u2014specifically the structure-function relationships that determine sensor performance\u2014remain poorly understood. Here, we present a method enabling versatile in-silico RNA-targeting analysis (VISTA), a machine learning-guided framework for the rapid design of RNA sensors. VISTA integrates biophysical modeling of both sensor and target RNAs with a partial least squares discriminant analysis (PLS-DA) machine learning framework. Using high-throughput experimental measurements with sequence-structure feature extraction to train predictive models, we capture the key determinants of RNA sensor performance. We find that by using toehold switches as a model RNA sensor, Toehold-VISTA successfully designs RNA sensors with improved function against SARS-CoV-2 RNA. These findings establish a broadly applicable, target-aware design strategy for accelerating RNA sensor engineering across biotechnology and diagnostic applications.\n\n\n","version":"1.1","doi":"10.1101/2025.08.12.669990","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.08.669385","pub_date":"2025-8-11","title":"Modulation of A\u03b21-42 Aggregation by a SARS-COV-2 Protein Fragment","abstract":"A number of studies have pointed out to the possibility that SARS-COV-2 infections could trigger amyloid diseases such as Parkinson\u2019s disease or type-II diabetes. In the present study we probe this question for Alzheimer\u2019s disease which is connected with presence of amyloids rich in A\u03b2-peptides. For this purpose, we study by way of molecular dynamics simulations the interaction between the fragment FKNIDGYFKI of the Spike protein with A\u03b21-42 monomer and two fibril models, one patient-derived and one synthetic. Our results are compared with previous studies of other amyloid-forming proteins to identify commonalities and differences in the modulation of amyloid-formation by the viral protein fragment.","version":"1.1","doi":"10.1101/2025.08.08.669385","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.02.597051","pub_date":"2025-8-11","title":"The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis","abstract":"Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus \u2013 a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered but retains association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.","version":"1.3","doi":"10.1101/2024.06.02.597051","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.07.669152","pub_date":"2025-8-08","title":"One Thousand SARS-CoV-2 Antibody Structures Reveal Convergent Binding and Near-Universal Immune Escape","abstract":"Since the emergence of SARS-CoV-2, understanding how antibodies recognize and adapt to viral evolution has been central to vaccine and therapeutic developments. To date, over 1,100 SARS-CoV-2 antibody structures\u201416% of all known antibody\u2013antigen complexes\u2014have been resolved, marking the largest structural biology effort towards a single pathogen. Here, we present a comprehensive analysis of this landmark dataset to investigate the principles of antibody recognition and immune escape. Human immunoglobulins (IgGs) and camelid single-chain antibodies dominate the dataset, collectively mapping 99% of the receptor-binding domain surface. Despite remarkable sequence and conformational diversity, antibodies exhibit striking convergence in their paratope structures, revealing evolutionary constraints in epitope selection. Structural and functional analyses reveal near-universal immune escape of antibodies, including all clinical monoclonals, by advanced variants such as KP3.1.1. On average, over one-third of antibody epitope residues are mutated. These findings support pervasive immune escape, underscoring the need to effectively leverage multi-epitope targeting strategies to achieve durable immunity.","version":"1.1","doi":"10.1101/2025.08.07.669152","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.05.666673","pub_date":"2025-8-06","title":"In Vivo Evolution of Monoclonal Antibody CR3022 to Achieve Cross-Neutralization of SARS-CoV-2 and Implications for Vaccine Strategies Against SARS-related Viruses","abstract":"The epitope that monoclonal CR3022 binds to represents a promising target for broad protection against a wide range of human and zoonotic coronaviruses. We developed a powerful model to evaluate antibody affinity maturation in vivo using immunoglobulin (Ig)-humanized mice that express the predicted germline heavy chain of antibody CR3022. SARS-CoV/SARS-CoV-2 sequential immunization led to the convergent evolution of the germline CR3022 through somatic hypermutation (SHM) that resembled the affinity-matured CR3022 from a human, but now also adapted to key variants and divergent sarbecoviruses. While simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol elicited dominant responses to other epitopes. X-ray crystal structures revealed that SARS-CoV-2-neutralizing CR3022-like antibodies exhibit enhanced affinity by increasing polar and electrostatic interactions. Overall, these findings show CR3022-like clones can be readily adapted through SHM to increase breadth and potency to sarbecoviruses by relatively minor shifts in affinity with appropriate vaccination strategies.","version":"1.1","doi":"10.1101/2025.08.05.666673","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.04.668557","pub_date":"2025-8-05","title":"Escape from SARS-CoV-2 Nsp1-mediated host shutoff by TIAR transcript reveals general features of Nsp1 resistance","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape strategies include general inhibition of host gene expression referred to as host shutoff. Viral non-structural protein 1 (Nsp1) is the main host shutoff factor that blocks protein translation and induces messenger RNA (mRNA) cleavage and degradation. Viral mRNAs are resistant to the translation shutoff and cleavage induced by Nsp1, and the 5\u2019 leader sequence present in all viral mRNAs has been shown to confer resistance. However, the exact molecular mechanism for escape from Nsp1 host shutoff has not been demonstrated. In our previous work, we analyzed the effects of Nsp1 on the expression and function of cellular proteins important for stress granule formation. We discovered that the host transcript for the TIA1 cytotoxic granule-associated RNA binding protein like 1 (TIAL1, commonly referred to as TIAR) is resistant to SARS-CoV-2 Nsp1 host shutoff. In this work, using reporter shutoff assays, we examined sequence and structural features of the TIAR 5\u2019 untranslated region (UTR) and discovered that the first 23 nucleotides of the TIAR transcript are both necessary and sufficient to confer resistance to the Nsp1. Furthermore, our work revealed that the lack of guanosines within a window of 10 to 18 nucleotides downstream from the 5\u2019 end is a defining feature of Nsp1-resistant transcripts shared between the SARS-CoV-2 leader sequence and the TIAR 5\u2019 UTR. Our findings are consistent with the model in which sequence features of 5\u2019 UTRs, rather than their secondary structure, confer resistance to Nsp1 host shutoff to both viral and cellular mRNAs.","version":"1.1","doi":"10.1101/2025.08.04.668557","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.03.668320","pub_date":"2025-8-05","title":"Integrative genomic study of mutation dynamics and Evolutionary trends in SARS-CoV-2 omicron BA.3","abstract":"This study investigates the evolutionary dynamics of the SARS-CoV-2 BA.3 lineage with an emphasis on saltation-driven adaptation. Using 81 high-coverage BA.3 genome sequences and 1,011 complete patient status records obtained from GISAID, we conducted a comprehensive analysis that integrated temporal, mutational, phylogenetic, and selection pressure assessments. Temporal analysis of patient records revealed that BA.3 sequences were predominantly collected between November 2021 and March 2022, with highly mutated variants emerging in the last quarter of 2024. Pairwise alignment of the spike gene demonstrated near-identical sequences among recent isolates from Gauteng Province and subtle yet significant differences in a KwaZulu Natal isolate when compared to the Wuhan Hu-1 reference strain. Domain-specific mutation mapping showed that mutations were concentrated in key functional regions of the spike protein, particularly within the receptor-binding domain and its binding motif. Phylogenetic reconstruction and pervasive selection analysis further revealed that recent BA.3 variants form a divergent clade characterized by extensive adaptive mutations. These findings indicate that saltatory events play a critical role in shaping the genetic landscape of BA.3, with important implications for viral infectivity, immune escape, and the design of next-generation vaccines.","version":"1.1","doi":"10.1101/2025.08.03.668320","journal":"bioRxiv","score":null},{"id":"10.1101/2025.08.03.668365","pub_date":"2025-8-04","title":"Therapeutic Potential of Cyclodextrins Targeting Dengue Virus and SARS-CoV-2 Infection and Pathogenesis","abstract":"Cyclodextrins (CDs) are cyclic oligosaccharides with promising therapeutic applications, including antiviral activity. During viral infections, pathogenesis arises not only from viral replication but also from viral proteins that act as \u201ctoxins\u201d, disrupting cellular barriers and inducing endothelial dysfunction, a hallmark of severe diseases such as dengue and COVID-19. Dengue virus (DENV) NS1 and SARS-CoV-2 Spike proteins induce endothelial hyperpermeability, contributing to severe complications. Here we explored the potential of a panel of 18 CDs in mitigating endothelial dysfunction caused by these viral proteins and evaluated the CDs\u2019 antiviral activity in vitro and in vivo. The effect of CDs on endothelial hyperpermeability was assessed using a trans-endothelial electrical resistance assay with human pulmonary microvascular endothelial cells exposed to DENV NS1 and SARS-CoV-2 Spike proteins. Antiviral efficacy of CDs was evaluated in Vero cells infected with DENV2 and Calu-3 cells infected with SARS-CoV-2, and in vivo protection was assessed in a lethal DENV2 mouse model. CDs effectively inhibited DENV NS1-induced endothelial hyperpermeability in vitro, demonstrating their potential to counteract NS1-mediated barrier disruption. In the murine model, CD1 treatment provided partial protection against DENV-induced morbidity and mortality. Further, CDs significantly reduced SARS-CoV-2 infection in vitro and inhibited Spike-induced endothelial dysfunction. These findings indicate that CDs can prevent endothelial hyperpermeability induced by DENV NS1 and SARS-CoV-2 Spike proteins and exhibit antiviral activity against SARS-CoV-2, positioning them as promising candidates for mitigating endothelial complications associated with viral infections. Further research is needed to explore the clinical relevance of CDs and their mechanisms of action.","version":"1.1","doi":"10.1101/2025.08.03.668365","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.16.638509","pub_date":"2025-8-01","title":"Coupled equilibria of dimerization and lipid binding modulate SARS Cov 2 Orf9b interactions and interferon response","abstract":"Open Reading Frame 9b (Orf9b), an accessory protein of SARS-CoV and -2, is involved in innate immune suppression through its binding to the mitochondrial receptor Translocase of Outer Membrane 70 (Tom70). Previous structural studies of Orf9b in isolation revealed a \u03b2-sheet-rich homodimer, however, structures of Orf9b in complex with Tom70 revealed a monomeric helical fold. Here, we developed a biophysical model that quantifies how Orf9b switches between these conformations and binds to Tom70, a requirement for suppressing the type 1 interferon response. We used this model to characterize the effect of lipid binding and mutations in variants of concern to the Orf9b:Tom70 equilibrium. We found that the binding of a lipid to the Orf9b homodimer biases the Orf9b monomer:dimer equilibrium towards the dimer by reducing the dimer dissociation rate \u223c100-fold. We also found that mutations in variants of concern can alter different microscopic rate constants without significantly affecting binding to Tom70. Together our results highlight how perturbations to different steps in these coupled equilibria can affect the apparent affinity of Orf9b to Tom70, with potential downstream implications for interferon signaling in coronavirus infection.","version":"1.3","doi":"10.1101/2025.02.16.638509","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.31.667456","pub_date":"2025-7-31","title":"Cooperativity and Communication between the Active Sites of the Dimeric SARS-CoV-2 Main Protease","abstract":"The coronaviral main protease (Mpro) is essential for the replication of the virus, and has been the subject of various biochemical, structural and enzymatic studies, as well as a drug target against SARS-CoV-2 infections. SARS-CoV-2 Mpro is known to be active as a dimer, with the N terminus of one protomer completing a key active site pocket of the other protomer. Despite apparent cooperativity in catalytic activity, how the two distal active sites in the dimer communicate and might be modulating binding and/or catalysis at the other remain to be clarified. Here, we have investigated the interplay between cooperativity, dimerization, and substrate cleavage in SARS-CoV-2 Mpro through a combination of enzymatic assays, crystal structures, and protein characterization. To disentangle the contribution of each active site to the observed enzymatic activity, we developed a cleavage assay involving heterodimers of active and inactive (C145A or inhibitor-bound) monomers. Strikingly, we found that heterodimerization increased cleavage efficiency per active monomer. Additionally, we mapped a network of critical residues bridging the two active sites and probed this network through engineered mutations. By dissecting the cooperativity and communication between the active sites, we provide new insights into the Mpro reaction cycle and functional significance of its dimeric architecture.","version":"1.1","doi":"10.1101/2025.07.31.667456","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.29.667219","pub_date":"2025-7-31","title":"A Genome Sequence Variant Monitoring Program for Seasonal Influenza A H3N2 and Respiratory Syncytial Virus A using Wastewater-Based Surveillance in Ontario, Canada","abstract":"Seasonal respiratory viruses, such as the Influenza A virus and the respiratory syncytial virus, are responsible for over a billion infections worldwide each year resulting in a substantial burden on health care systems. Surveillance of these viruses, including their prevalence in communities and their evolution, are essential for informing public health decisions and recommending vaccine formulations and schedules. Typically, these viruses are monitored using clinical samples from patients seeking medical attention. Recently, wastewater-based surveillance (WBS) has been leveraged to understand transmission dynamics and genome evolution of SARS-CoV-2 and seasonal respiratory viruses. To further the utility of WBS we developed and implemented novel tiled-amplicon sequencing assays to identify and track Influenza A virus H3N2 and respiratory syncytial virus A circulating in Southern Ontario, Canada. We also developed virus specific deconvolution tools to estimate the abundance of mixed lineages in wastewater. These assays were able to accurately determine which lineages were circulating in wastewater with high sensitivity and specificity. If implemented in regular surveillance programs, they could be used to inform real-time public health decisions and determine potential disease surge with impact on emergency room visits and hospitalization, as well as track which emerging strains will become predominant in the future and determine which strains should be the focus of seasonal vaccines.","version":"1.1","doi":"10.1101/2025.07.29.667219","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.29.667267","pub_date":"2025-7-30","title":"A Structure-Based Computational Pipeline for Broad-Spectrum Antiviral Discovery","abstract":"The rapid emergence of viruses with pandemic potential continues to pose a threat to public health worldwide. With the typical drug discovery pipeline taking an average of 5\u201310 years to reach clinical readiness, there is an urgent need for strategies to develop broad-spectrum antivirals that can target multiple viral family members and variants of concern. We present a structure-based computational pipeline designed to identify and evaluate broad-spectrum inhibitors across viral family members for a given target in order to support spectrum breadth assessment and prioritization in lead optimization programs. This pipeline comprises three key steps: (1) an automated search to identify viral sequences related to a specified target construct, (2) pose prediction leveraging any available structural data, and (3) scoring of protein-ligand complexes to estimate antiviral activity breadth. The pipeline is implemented using the drugforge package: an open-source toolkit for structure-based antiviral discovery. To validate this framework, we retrospectively evaluated two overlapping datasets of ligands bound to the SARS-CoV-2 and MERS-CoV main protease (Mpro), observing useful predictive power with respect to experimental binding affinities. Additionally, we screened known SARS-CoV-2 Mpro inhibitors against a panel of human and non-human coronaviruses, demonstrating the potential of this approach to assess broad-spectrum antiviral activity. Our computational strategy aims to accelerate the identification of antiviral therapies for current and emerging viruses with pandemic potential, contributing to global preparedness for future outbreaks.","version":"1.1","doi":"10.1101/2025.07.29.667267","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.29.667457","pub_date":"2025-7-29","title":"A DNA scaffold approach facilitates 5\u2032 labelling of the SARS-CoV-2 RNA pseudoknot for smFRET investigation","abstract":"Single-molecule F\u00f6rster resonance energy transfer (smFRET) studies of highly structured RNA molecules are often frustrated by issues with efficient dye conjugation. Here, we develop a DNA scaffold-based labelling strategy, and apply it to the frameshift-stimulating RNA pseudoknot from the SARS-CoV-2 genome. We prepare FRET-active reporters containing both Cy3 (donor) and Cy5 (acceptor) molecules and conduct measurements on freely diffusing single molecules, enabling the evaluation of conformational heterogeneity via smFRET population distributions. We identify that freely diffusing pseudoknots, modified at the base of stem 1, display a broad range of NaCl-dependent FRET states in solution, consistent with conformational freedom that extends beyond the static X-ray and cryo-EM structures. This work is a proof-of-principle demonstration of the feasibility of our DNA scaffold approach in enabling smFRET studies on this important class of biomolecule. Together, this work outlines new biochemical and biophysical approaches towards the study of RNA conformational dynamics in pseudoknots, riboswitches and other structured RNA elements.","version":"1.1","doi":"10.1101/2025.07.29.667457","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.28.667286","pub_date":"2025-7-29","title":"Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation","abstract":"Members of the TRIM E3 ligase family are effectors of the host innate or intrinsic defense against various viruses; however, how specific TRIM proteins antagonize coronavirus infection is still largely elusive. Through an RNAi screen targeting 71 human TRIM genes, we identified multiple TRIM proteins with antiviral or proviral activity against SARS-CoV-2. TRIM32 potently restricted SARS-CoV-2 replication in a RING E3 ligase-dependent but interferon-independent manner. Mechanistically, TRIM32 binds to and SUMOylates the 3\u2032-to-5\u2032 exoribonuclease (ExoN) of NSP14, which is essential for SARS-CoV-2 replication. TRIM32-mediated NSP14 SUMOylation at K9 and K200 inhibits RNA binding and NSP10 cofactor recruitment, respectively, ultimately suppressing ExoN activity. Our study further revealed that NSP14 SUMOylation by TRIM32 and its antiviral activity are broadly conserved for coronaviruses. These results identify the coronaviral NSP14 protein as a direct target of host restriction via SUMOylation, which may uncover novel ways to therapeutically inhibit coronavirus infections in humans.","version":"1.1","doi":"10.1101/2025.07.28.667286","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.03.626536","pub_date":"2025-7-29","title":"The mammalian SKI complex is a broad-spectrum antiviral drug target that upregulates cellular cholesterol to inhibit viral replication","abstract":"There is a need for the development of broad-spectrum antiviral compounds that can act as first line therapeutic countermeasures to emerging viral infections. Host-directed approaches present a promising avenue of development and carry the benefit of mitigating risks of viral escape mutants. We have previously found the SKI (super killer) complex to be a broad-spectrum, host-target with our lead compound ('UMB18') showing activity against influenza A virus, coronaviruses, and filoviruses. The SKI complex is a cytosolic RNA helicase and we previously found that UMB18 inhibited viral RNA production but did not further define the mechanism. Here, we demonstrate that UMB18 directly binds to SKIC8 of the SKI complex and through transcriptomic analysis of UMB18 treated A549 cells revealed an upregulation of genes in the mevalonate pathway which drives cholesterol synthesis. Further investigation validated the genetic upregulation and confirmed an increase in total cellular cholesterol. This upregulation was dependent on the sterol regulatory element binding proteins (SREBPs) and their regulator SCAP, the major regulators for cholesterol and fatty acid synthesis. Depletion of the SREBPs or SCAP with siRNA, or extraction of cholesterol with methyl \u03b2-cyclodextrin attenuated UMB18 antiviral activity, emphasizing the role of increased cholesterol synthesis in this mechanism of action. Our findings further define the antiviral mechanism of a developmental host-directed therapeutic approach with broad applicability against emerging viral pathogens. The COVID-19 pandemic has underscored the urgent need for effective countermeasures to novel and emerging viral pathogens. Our research presented here builds upon our previously published data on an experimental novel antiviral compound termed UMB18. We have found this compound capable of inhibiting replication of influenza A virus, coronaviruses and the filoviruses Marburg and Ebola virus, but did not fully define a mechanism of action. In this work, we demonstrate that UMB18 exerts antiviral activity by modulating cellular cholesterol levels. By targeting the SKI complex, UMB18 triggers an increase in endogenous cellular cholesterol which disrupts the fine balance viruses rely on for efficient infection. We demonstrate that this mechanism inhibits replication of SARS-CoV-2, revealing a previously undescribed host-directed strategy for antiviral intervention. These findings highlight UMB18\u2019s potential as a broad-spectrum antiviral agent and pave the way for further research into its mechanism and therapeutic applications, offering a promising avenue for development of antiviral countermeasures to current, novel and emerging pathogens.","version":"1.2","doi":"10.1101/2024.12.03.626536","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.28.667129","pub_date":"2025-7-28","title":"Predicting interactions between the SARS-CoV-2 spike glycoprotein and the human proteome using AphaFold and molecular dynamics simulations","abstract":"Since the COVID-19 pandemic began, the SARS-CoV-2 virus has caused over 775 million cases and more than 7 million deaths worldwide. Despite progress in treatments and vaccines, we still need better ways to prevent and treat the disease. To do this, a clearer understanding of how SARS-CoV-2 interacts with human proteins is needed. We developed a new computational tool to predict and study these protein-protein interactions. Our method uses two stages of AlphaFold, an AI tool, to predict how SARS-CoV-2 proteins bind to human proteins, followed by molecular dynamics simulation to refine these predictions. We tested this method in a small study focusing on the S1 subunit of the SARS-CoV-2 spike protein interacting with human cell junction and synaptic proteins, and in a larger study screening the spike S1 N- terminal domain against the entire human proteome. We validated the method using experimental virus\u2013human protein interactions. The results provide valuable insights into how SARS-CoV-2 interacts with human proteins, guiding future experiments to better understand COVID-19\u2019s short- and long-term effects. This developed method could be utilized to study protein-protein interactions in other biological systems.","version":"1.1","doi":"10.1101/2025.07.28.667129","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.28.667187","pub_date":"2025-7-28","title":"SIMPLICITY: an agent-based, multi-scale mathematical model to study SARS-CoV-2 intra- and between-host evolution","abstract":"Computational tools are frequently used to describe pathogen evolutionary dynamics either within infected hosts or at the population level. However, there is a lack of models that capture the complex interplay between within-host and between-host evolutionary dynamics, leaving a knowledge gap with regard to realistic evolutionary dynamics as observed within molecular surveillance programs. We present SIMPLICITY, a multi-scale mathematical model that combines within-host disease progression and viral evolution, with a population-level model of virus transmission and immune evasion. We parameterize SIMPLICITY based on SARS-CoV-2 within-host viral dynamics, observed evolutionary rates, as well as dynamics of immune waning. We then apply SIMPLICITY to study the dynamics and mechanisms driving SARS-CoV-2 evolution at the population level. We compare a model of gradually increasing transmission fitness with an adaptive fitness landscape model that accounts for temporal changes in transmission fitness due to infection history and immune waning in the population. Our simulations demonstrate that escape from population immunity generates evolutionary dynamics encompassing selective sweeps, which resembles actual SARS-CoV-2 evolution. To the contrary, the model of gradual fitness fails to resemble realistic SARS-CoV-2 evolutionary dynamics. Overall, this demonstrates that infection history and immune waning should be considered in models of viral fitness, including machine learning approaches. In short, SIMPLICITY can be used to investigate mechanisms driving viral evolution and constitutes a versatile tool for creating synthetic datasets of viral evolution to e.g. benchmark and challenge phylogenetic pipelines used in outbreak investigation.","version":"1.1","doi":"10.1101/2025.07.28.667187","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.26.666918","pub_date":"2025-7-28","title":"Coronavirus protein interaction mapping in bat and human cells identifies molecular and genetic switches for immune evasion and replication","abstract":"Coronaviruses, including SARS-CoV-2, can cause severe disease in humans, whereas reservoir hosts like Rhinolophus bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat-origin relative RaTG13 using affinity purification-mass spectrometry (AP-MS) in human and Rhinolophus ferrumequinum (RFe) bat cells. This approach identified both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 required a non-synonymous mutation in nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Analysis of the viral protein Orf9b revealed differential interactions with mitochondrial proteins Tom70 and MTARC2. A single residue difference in Orf9b between SARS-CoV-2 and RaTG13 functions as a molecular switch, weakening Tom70 binding and immune evasion in human cells while enhancing interaction with the bat-specific restriction factor MTARC2. These findings demonstrate how a single-residue substitution can reshape virus-host interactions and contribute to immune evasion and host adaptation.","version":"1.1","doi":"10.1101/2025.07.26.666918","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.09.612081","pub_date":"2025-7-26","title":"The Impact of Tokenizer Selection in Genomic Language Models","abstract":"Genomic language models have recently emerged as a new method to decode, interpret, and generate genetic sequences. Existing genomic language models have utilized various tokenization methods, including character tokenization, overlapping and non-overlapping k-mer tokenization, and byte-pair encoding, a method widely used in natural language models. Genomic sequences differ from natural language because of their low character variability, complex and overlapping features, and inconsistent directionality. These features make sub-word tokenization in genomic language models significantly different from both traditional language models and protein language models. This study explores the impact of tokenization in genomic language models by evaluating their downstream performance on forty-four classification fine-tuning tasks. We also perform a direct comparison of byte pair encoding and character tokenization in Mamba, a state-space model. Our results indicate that character tokenization outperforms sub-word tokenization methods on tasks that rely on nucleotide level resolution, such as splice site prediction and promoter detection. While byte-pair tokenization had stronger performance on the SARS-CoV-2 variant classification task, we observed limited statistically significant differences between tokenization methods on the remaining downstream tasks.","version":"1.3","doi":"10.1101/2024.09.09.612081","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.25.666822","pub_date":"2025-7-25","title":"Omicron Subvariants Infection Kinetics and Nirmatrelvir Efficacy in Transgenic K18-hACE2 Mice","abstract":"The persistent evolution of SARS-CoV-2 has led to the emergence of antigenically distinct Omicron subvariants exhibiting increased transmissibility, immune evasion, and altered pathogenicity. Among these, recent subvariants like JN.1, KP.3.1.1, and LB.1 possess unique antigenic and virological features, underscoring the need for continued surveillance and therapeutic evaluation. As vaccines and commercial monoclonal antibodies show reduced effectiveness against these variants, the role of direct-acting antivirals, such as Nirmatrelvir, targeting conserved viral elements like the main protease inhibitor, becomes increasingly crucial. In this study, we investigated the replication kinetics, host immune responses, and therapeutic susceptibility of three recently circulating Omicron subvariants in the K18-hACE2 transgenic mouse model, using the SARS-CoV-2 parent WA1/2020 strain as a reference. Omicron subvariants exhibited a marked temporal shift in viral infection kinetics characterized by an early lung viral titer peak (\u223c7-8 Log PFU) at 2 days post-infection (dpi), followed by a decline (1\u20133 Log PFU) by 4 dpi. Pulmonary cytokine and chemokine responses (GM-CSF, TNF-\u03b1, IL-1\u03b2, IL-6) showed an earlier increase in subvariant-infected mice compared to a gradual response in WA1/2020 infection. Notably, Nirmatrelvir treatment led to significant reduction in lung viral titers in subvariant-infected mice than in WA1/2020, surpassing its efficacy against the parent strain. These findings highlight that Omicron subvariants infection yields a broad dynamic range in viral burden with minimum variability, while retaining a prominent therapeutic response to Nirmatrelvir. This study provides insights to the emerging subvariants pathogenesis and therapeutic responsiveness, reinforcing the importance of continued variant monitoring and the development of effective countermeasures. The emergence of SARS-CoV-2 Omicron subvariants still poses a significant public health challenge due to their antigenic drift, altered pathogenesis, and immune evasion capabilities. This study comprehensively highlights the distinct replication kinetics, immune responses, and therapeutic susceptibility of the recently circulating Omicron subvariants JN.1, KP.3.1.1, and LB.1 using the K18-hACE2 mouse model. We demonstrate that while these subvariants exhibit altered virological profiles and immune activation patterns compared to the parent WA1/2020 strain, they remain susceptible to Nirmatrelvir which is a direct-acting main protease inhibitor. Notably, Nirmatrelvir demonstrated greater in vivo antiviral efficacy in Omicron subvariant infections than in the ancestral WA1/2020 strain. These findings underscore the enduring therapeutic value of protease-targeting antivirals and emphasize the critical need for ongoing variant-specific preclinical assessments.","version":"1.1","doi":"10.1101/2025.07.25.666822","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.23.666463","pub_date":"2025-7-24","title":"De novo administration of antiviral monoclonal antibodies against SARS-CoV-2 or influenza using mRNA lipid nanoparticles","abstract":"Monoclonal antibodies (mAbs) are an emerging class of therapeutics for the prevention and treatment of viral infections. Recent advances in mRNA/lipid nanoparticle (LNP) technology provide a potential new modality for the expression of mAbs in vivo, potentially bypassing the need for recombinant manufacturing of mAb proteins. In this study, we compared traditional infusion of neutralising mAbs targeting SARS-CoV-2 or influenza to mRNA-based induction of de novo mAb expression in treated mice. High serum concentrations of mAbs were achieved upon delivery of a single mRNA encoding both heavy and light chains via intravenous or intramuscular routes using prototypic LNP formulations. However, pharmacokinetics were heavily influenced by the induction of anti-drug antibody responses directed against the encoded mAbs, driving reductions in in vivo half-life and compromising protective capacity against SARS-CoV-2 Omicron BA.1 infection. Overall, mRNA/LNP delivery comprises a feasible and attractive pathway to speed the development and deployment of antiviral antibodies, however optimisation of LNP formulation, dosing and administration routes is required to maximise protective potential.","version":"1.1","doi":"10.1101/2025.07.23.666463","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.21.664397","pub_date":"2025-7-24","title":"Characterizing Co-Circulating Respiratory Virus Genomic Diversity in Switzerland with Hybrid-Capture Sequencing and Phylogenetic Reconstructions: Insights into the 2023/24 Season","abstract":"Respiratory viruses circulate yearly with strain-specific patterns. Although SARS-CoV-2 and Influenza A/B genomic surveillance is well-developed, most respiratory viruses are unevenly monitored, lacking geographical diversity to capture wider population dynamics. Consequently, insights into respiratory virus evolution are limited. We investigated the genetic diversity of these viruses within one country. During the 2023/24 season, we conducted whole-genome sequencing of 1129 clinical samples using a hybrid-capture protocol. These samples were pre-tested by real-time PCR panels throughout Switzerland. Leveraging publicly-available full-length genomes, we constructed background datasets representative of geographical diversity and built phylogenies for all viruses with more than 40 new high-quality genomes from this study. We detected 981 viruses and recovered 461 high-quality genomes, including 18 co-infections, from 437 PCR-positive and 6 PCR-negative samples. All viruses detected by PCR were also detected by sequencing in 56% of samples. The four most prevalent viruses were Influenza A/H1N1, SARS-CoV-2, RSV-A, and HPIV-3, and their seasonal spread was consistent with wastewater monitoring and influenza-like illness reports. Swiss viral genomes were representative of the global genomic diversity, with evidence for multiple introductions into Switzerland, and we identified putative Swiss clusters. In this proof-of-concept study, we focus on 3 viruses (Influenza A/H1N1, RSV-A/B, and HPIV-3), and we demonstrate the streamlined implementation of a broad respiratory virus genomic surveillance workflow with an off-the-shelf protocol and publicly-available software. In addition, we highlight additional evolutionary insights that can only be derived from genomic surveillance. Going forward, this dataset will be a useful resource for future investigations into respiratory viral genomic diversity.","version":"1.1","doi":"10.1101/2025.07.21.664397","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.18.665436","pub_date":"2025-7-23","title":"TCR2HLA: calibrated inference of HLA genotypes from TCR repertoires enables identification of immunologically relevant metaclonotypes","abstract":"T cell receptors (TCRs) recognize peptides presented by polymorphic human leukocyte antigen (HLA) molecules, but HLA genotype data are often missing from TCR repertoire sequencing studies. To address this, we developed TCR2HLA, an open-source tool that infers HLA genotypes from TCR\u03b2 repertoires. Expanding on work linking public TRBV-CDR3 sequences to HLA genotypes, we incorporated \u201cquasi-public\u201d metaclonotypes \u2013 composed of rarer TCR\u03b2 sequences with shared amino acid features \u2013 enriched by HLA genotypes. Using four TCR\u03b2seq datasets from 3,150 individuals, we applied TRBV gene partitioning and locality-sensitive hashing to identify \u223c96,000 TCR\u03b2 features strongly associated with specific HLA alleles from 71M input TCRs. Binary HLA classifiers built with these features achieved high balanced accuracy (>0.9) across common HLA-A (9/12), B (9/12), C (6/13), DRB1 (11/11) alleles and prevalent DPA1/DPB1 (6/10), DQA1/DQB1 (8/17) heterodimers. We also introduced a high-sensitivity calibration to support predictions in samples with as few as 5,000 unique clonotypes. Calibrated predictions with confidence filtering improved reliability. Beyond genotype imputation, TCR2HLA enables the discovery of novel HLA- and exposure-associated TCRs, as shown by the identification of SARS-CoV-2 related TCRs in a large COVID-19 dataset lacking HLA data. TCR2HLA provides a scalable framework for bridging the gap between TCRseq data and HLA genotype for biomarker discovery.","version":"1.1","doi":"10.1101/2025.07.18.665436","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.18.662329","pub_date":"2025-7-21","title":"Antibody evasion and receptor binding of SARS-CoV-2 LP.8.1.1, NB.1.8.1, XFG, and related subvariants","abstract":"SARS-CoV-2 continues to evolve, causing repeated waves of infections around the world. It is critical to understand the features of the virus that explain its growth advantages. Recently, the SARS-CoV-2 Omicron JN.1 subvariants KP.3.1.1 and XEC were outcompeted by later JN.1 progenies, most prominently LP.8.1 and LP.8.1.1. Other recent JN.1 subvariants, such as LF.7.2.1, which became prevalent in Asia, and MC.10.1, have also been under monitoring. Subsequently, NB.1.8.1 and XFG subvariants began increasing in prevalence, as well. We found that serum neutralizing antibody titers against LP.8.1, LP.8.1.1, LF.7, LF.7.2.1, MC.10.1 were similar to XEC in a cohort of 20 KP.2-based monovalent mRNA vaccine (KP.2 MV) recipients and in a cohort 20 adults who did not receive KP.2 MV. NB.1.8.1 and XFG were more evasive of serum neutralization than LP.8.1.1. We then characterized subvariant susceptibility to monoclonal antibody (mAb) neutralization using a panel of 12 mAbs spanning several epitopes on the SARS-CoV-2 spike, and found that LP.8.1 and XFG, MC.10.1 and NB.1.8.1, and LF.7.2.1 evade different classes of mAbs relative to earlier JN.1 subvariants, even if the tested polyclonal serum neutralizing antibody titers were not different overall. Next, we found that the receptor-binding affinity of LP.8.1 to ACE2 was the highest among the tested viruses, while that of LF.7.2.1 was lowest. Therefore, unlike most prior SARS-CoV-2 sublineage evolutionary trajectories, receptor-binding affinity, possibly reflecting enhanced transmissibility\u2013and not increased antibody evasion\u2013better explained the rise of LP.8.1, while the expansion of NB.1.8.1 and XFG again appear correlated with their enhanced antibody evasion.","version":"1.1","doi":"10.1101/2025.07.18.662329","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.18.649534","pub_date":"2025-7-21","title":"SARS-CoV E protein couples asymmetric leaflet thickness and curvature deformations","abstract":"The Envelope protein (E protein) of SARS-CoVs 1 and 2 has been implicated in the viral budding process and maintaining the spherical shape of the virus, but direct evidence linking the protein to long-range membrane deformation is still lacking. Computational predictions from molecular simulation have offered conflicting results, some showing long-range E-induced membrane curvature and others showing only local deformations. In the present study, we determine the mechanism driving these deformations by modulating the degree of hydrophobic mismatch between protein and membrane. We observe that certain barostat and restraint settings, common in coarse-grained MD simulations, can prevent equilibration of the membrane area. Our results indicate that the E protein does not induce long-range curvature, but does exhibit severe local deformations that are exacerbated by hydrophobic mismatch. These deformations occur in conjunction with local leaflet thickness asymmetry, suggesting asymmetry and curvature couple to reduce the free energy cost of a deformed membrane. E protein pentamers from SARS-CoV-1 and SARS-CoV-2 do not induce long-range curvature in membranes when simulated in isolation. Previous findings documenting long-range membrane deformation may reflect the use of restraint and barostat settings that trap the membrane in a compressed state. E proteins induce large, asymmetric membrane deformations local to the protein, but these deformations do not propagate into the bulk. Membrane leaflet thickness asymmetry may be non-negligible around proteins that are not cylindrical. Membrane leaflet thickness asymmetry and mean curvature couple to alleviate free energy cost of deformation.","version":"1.2","doi":"10.1101/2025.04.18.649534","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.21.665885","pub_date":"2025-7-21","title":"Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection","abstract":"Human in vitro lung models represent advanced tools for studying respiratory infections, particularly those caused by emerging respiratory pathogens. Despite scientific advances, vaccine and therapeutics pre-clinical development has yet to fully adopt human-relevant testing platforms due in part to a lack of validation. In this study, we characterised how static and dynamic flow culture conditions influence microphysiological systems (MPS) generated using primary bronchial and alveolar epithelial cells. We assessed epithelial structure, functional differentiation, and infection dynamics. This study represents the first direct comparison of how dynamic flow and endothelial co-culture influence viral tropism, replication kinetics, and host responses across anatomically distinct regions of the respiratory tract in vitro. Dynamic flow promoted formation of more physiologically relevant tissue architecture, pseudostratified bronchial epithelium and alveolar sac-like structures, with enhanced epithelial differentiation and retention of region-specific cell phenotypes at the transcriptomic level. Both static and dynamic flow models demonstrated responsiveness to inflammatory stimuli (poly(I:C), LPS), producing distinct, tissue-specific cytokine profiles and supporting infection with multiple SARS-CoV- 2 variants. Differences in infection efficiency, viral replication, and host gene expression were observed between variants, with dynamic flow models offering enhanced sensitivity and resolution. In alveolar tissues, dynamic flow increased infection efficiency and reduced variability, enabling more robust and consistent transcriptional responses. This facilitated the identification of interferon signalling pathways as key targets of the host response. Among the variants tested, Delta induced the most extensive tissue damage and strongest transcriptional response, whereas Omicron BA.5 exhibited greater infectivity in alveolar models compared to earlier variants. Our findings demonstrate that dynamic flow MPS more closely replicate human lung tissue architecture and cellular diversity, while also enhancing the predictive power and clinical relevance of airway models for ex vivo studies of SARS-CoV-2 infection. These improvements strengthen the reliability of data generated for the study of host\u2013pathogen interaction studies and support the use of dynamic systems for evaluating novel anti-infectives, immunomodulators, and functional characterisation of immune sera generated by next-generation vaccines. Collectively, our results highlight the value of integrating dynamic in vitro models into preclinical pipelines for emerging respiratory pathogens.","version":"1.1","doi":"10.1101/2025.07.21.665885","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.20.665262","pub_date":"2025-7-21","title":"Charged Scanning Mutagenesis as a High Throughput Approach for Epitope Mapping","abstract":"Identifying neutralizing epitopes is important for developing vaccines and inhibitors against viral pathogens. We describe a rapid method for epitope mapping, employing barcoded charged scanning mutagenesis libraries displayed on the yeast surface, and screening using flow cytometry coupled with deep sequencing. Prior scanning mutagenesis data suggest that mutations to a charged residue, such as Aspartic acid or Arginine, will be well tolerated at exposed positions of an antigen, and minimally affect protein stability and expression. Yet such substitutions at epitope residues strongly perturb binding to a cognate partner. We constructed an Aspartate scanning library of SARS-CoV-2 RBD and linked every mutation in the library to a defined unique barcode. The approach was used to map epitopes targeted in polyclonal sera of mice immunized with different SARS-CoV-2 immunogens. In contrast to complete mutational scans, charged scanning mutagenesis with the introduced barcoding strategy employs libraries with >50-fold lower diversity, facilitating library construction, screening, and downstream analysis, and also allowing for further multiplexing of samples, thus accelerating interaction site identification, as well as vaccine and inhibitor development.","version":"1.1","doi":"10.1101/2025.07.20.665262","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.14.664835","pub_date":"2025-7-18","title":"Humanized mice enable in vivo evaluation of engineered plasma cell biology and therapeutic function","abstract":"Engineered plasma cells (ePCs) offer a durable strategy for in vivo delivery of therapeutic antibodies, but standard immunodeficient mouse models lack human immune factors critical for plasma cell survival and function. We utilized a humanized mouse model in which NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were engrafted with human CD34+ stem cells as recipients for infusions with autologous ePCs. In this setting, ePCs localized to plasma cell niches and stably secreted antibodies for over three months. To improve the selection of antibodies for secretion, we developed a B cell receptor surface display screen that identified candidate antibody sequences with high secretion potential. An anti-SARS-CoV-2 antibody (clone 297) selected by this method showed robust secretion both in vitro and vivo, and serum from ePC-engrafted mice potently neutralized SARS-CoV-2 pseudovirus. Together, these findings establish a physiologically relevant model for testing human ePCs, and offer a generalizable strategy for optimizing antibody selection to support long-term therapeutic delivery.","version":"1.1","doi":"10.1101/2025.07.14.664835","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.17.665346","pub_date":"2025-7-17","title":"CoV-UniBind: A Unified Antibody Binding Database for SARS-CoV-2","abstract":"Since the emergence of SARS-CoV-2, numerous studies have investigated antibody interactions with viral variants in vitro, and several datasets have been curated to compile available protein structures and experimental measurements. However, existing data remain fragmented, limiting their utility for the development and validation of machine learning models for antibody\u2013antigen interaction prediction. Here, we present CoV-UniBind, a unified database comprising over 75,000 entries of SARS-CoV-2 antibody\u2013antigen sequence, binding, and structural data, integrated and standardised from three public sources and multiple peer-reviewed publications. To demonstrate its utility, we benchmarked multiple protein folding and inverse folding models across tasks relevant to antibody design and vaccine development. We expect CoV-UniBind to facilitate future computational efforts in antibody and vaccine development against SARS-CoV-2. The curated datasets, structures, model scores and antibody synonyms are free to download at https://huggingface.co/datasets/InstaDeepAI/cov-unibind. Folded structures are available upon request.","version":"1.1","doi":"10.1101/2025.07.17.665346","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.16.665240","pub_date":"2025-7-17","title":"A novel chimeric coronavirus spike vaccine combining SARS-CoV-2 RBD and scaffold domains from HKU-1 elicits potent neutralising antibody responses","abstract":"The receptor binding domain (RBD) of the SARS-CoV-2 spike is the major target for neutralising antibodies elicited by current vaccines. Using small domains such as the RBD as vaccine immunogens, however, may constrain the availability of CD4 T follicular helper (TFH) cells and impact immunogenicity. We engineered a novel chimeric trimeric RBD (CTR) glycoprotein, replacing the RBD of human coronavirus HKU-1 spike with SARS-CoV-2 RBD of either ancestral (WT) or Omicron BA.2 strains. This strategy maintains a native trimeric conformation of the RBD, while providing additional sources of CD4 T cell help via the HKU-1 spike scaffold. In C57BL/6 mice, CTR-BA.2 prime-boost vaccination elicited high anti-BA.2-RBD IgG and neutralising titres, matching responses in animals immunised with native SARS-CoV-2 spike proteins. GC B cells elicited by CTR-BA.2 were predominantly WT+/BA.2+ cross- reactive, and TFH cells predominantly recognised HKU-1 epitopes, demonstrating scaffold-directed T cell help. Macaques prime-boost immunised with CTR-WT similarly elicited high anti-RBD IgG, anti-spike IgG and neutralising responses, comparable to native spike-vaccinated animals. In draining lymph nodes of CTR-WT vaccinated macaques, RBD-specific GC B cells were present at elevated levels. In contrast to the murine studies, lymph node-draining TFH responses in macaques were broadly elicited against RBD, NTD/S2 or HKU-1-derived peptides. Although native SARS- CoV-2 spike was also highly immunogenic in animal models, our findings establish the chimeric glycoprotein design as a strategy to overcome the poor immunogenicity of the SARS-CoV-2 RBD by engaging CD4 TFH cells, while maintaining the ability to elicit protective neutralising responses. A chimeric glycoprotein design preserves SARS-CoV-2 RBD antigenic conformation enabling elicitation of neutralising responses, while allowing recruitment of HKU-1 scaffold-directed CD4 helper responses to support the humoral response.","version":"1.1","doi":"10.1101/2025.07.16.665240","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.03.631215","pub_date":"2025-7-17","title":"An ultra-long heavy chain bovine antibody neutralizes SARS-CoV-2 and reacts broadly with sarbecoviruses","abstract":"The ongoing threat of new SARS-CoV-2 variants and other sarbecoviruses has driven efforts to develop broadly neutralizing monoclonal antibodies (mAbs). This study used immunized cattle, known for producing antibodies with ultra-long CDRH3 domains, to generate 33 mAbs, ten of which had ultra-long CDRH3s (>50 amino acids). Of these, mAbs P7 and 99, demonstrated broad and potent neutralization. Notably, mAb P7 neutralized all tested variants, including SARS-CoV-1, with IC50 values between 0.05 and 9.2 \u00b5g/mL, and showed cross-reactivity with RBDs from various sarbecoviruses. Structural analysis revealed that mAb 99 binds the spike protein\u2019s RBD at the ACE2 binding site. Although the exact binding of P7 wasn\u2019t resolved, evidence suggests it targets a hidden epitope, promoting spike trimer dissociation via its extended CDRH3. In Syrian hamsters, both mAbs significantly reduced lung viral loads. These results support the potential of bovine-derived mAbs, particularly those with ultra-long CDRH3s, for future antiviral therapies.","version":"1.2","doi":"10.1101/2025.01.03.631215","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.16.665198","pub_date":"2025-7-17","title":"Molecular Insights into Long COVID: Plasma Proteomics Reveals Oxidative Stress, Coagulation Cascade Activation, and Glycolytic Imbalance","abstract":"Persistent symptoms following SARS-CoV-2 infection are the hallmark of post-COVID condition (PCC), also referred to as long COVID. However, our knowledge is limited on the underlying molecular mechanisms. In this study, we performed data-independent acquisition mass spectrometry plasma proteomics (DIA-MS) to identify molecular alterations associated with PCC. DIA-MS proteomic analysis revealed a few proteins linked to oxidative stress that had altered expression. Notably, PCC samples exhibited downregulation of the antioxidant protein peroxiredoxin 6 (PRDX6) and upregulation of oxidative stress-associated proteins particularly vanin-1 (VNN1) and paraoxonase-3 (PON3). Additionally, the PCC group showed significantly higher levels of six proteins (PCSK9, CST3, C1Q, CPB2, KNG1 GAPDH), which were linked to pathways involving glycolysis, complement and coagulation cascades, and inflammation. Oxidative stress analysis confirmed that PCC samples had significantly higher levels of DNA damage (8-OHdG) than the convalescent group, whereas antioxidant markers, such as reduced and oxidized glutathione (GSH and GSSG), were significantly lower in PCC samples than in uninfected controls. Our observations point towards ongoing oxidative and inflammatory processes in PCC and suggest potential targets for biomarker development and therapeutic intervention.","version":"1.1","doi":"10.1101/2025.07.16.665198","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.14.664650","pub_date":"2025-7-16","title":"Genomic and epidemiologic characteristics of SARS-CoV-2 persistent infections in California, January 2021 - July 2023","abstract":"Novel SARS-CoV-2 variants demonstrating considerable intra-host evolution emerged throughout the pandemic. The persistent infections thought to give rise to these variants, however, have been difficult to identify at scale. This study sought to detect and characterize persistent infection cases in California using routine epidemiologic and genomic surveillance data. We identified 69 persistent infection cases with collection dates between January 2021 and July 2023 ranging from 21 to 400 days in duration, with an average of 44 days. Significant differences were identified in age distribution, sex, hospitalizations, and deaths between persistent infection cases and all sequenced California SARS-CoV-2 cases. Underlying health conditions were identified for the majority of cases with available medical records. The mutations found in these cases were suggestive of positive selection in the Spike receptor binding domain and convergent evolution toward immune evasion and residues observed in previous persistent infections. We describe a 400-day B.1.429 infection that demonstrates substantial intra-host evolution, and a BA.5.11 persistent infection revealing apparent competition between two intra-host viral subpopulations. By establishing a framework for detecting persistent infections, this study lays the groundwork for other public health organizations to monitor and investigate highly divergent SARS-CoV-2 viruses. Genomic surveillance has been used to monitor the evolution and spread of SARS-CoV-2 variants throughout the pandemic. When a new variant emerges, it is often due to the accumulation of mutations during a persistent infection, i.e. in an individual who was unable to clear the virus after an infection. Using genomic and epidemiologic surveillance data, we identify 69 of these persistent infections in California and provide demographic and clinical characteristics of these infections compared to the broader population of SARS-CoV-2 infections. The identification of risk factors for persistent infections provides important insight into the epidemiology of SARS-CoV-2, while the identification of shared mutations between these infections enhances our understanding of SARS-CoV-2 evolution that may result in new variants. Ultimately, our work may help public health labs to better monitor persistent infections in the future, prior to the potential emergence and spread of novel variants into the community.","version":"1.1","doi":"10.1101/2025.07.14.664650","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.16.665136","pub_date":"2025-7-16","title":"HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2","abstract":"The SARS-CoV-2 E protein through its C-terminal PDZ-binding motif (PBM) interacts with several host PDZ-containing proteins, including the ZO-1 protein via its PDZ2 domain, thereby contributing to viral pathogenesis. Targeting this interaction represents a potential therapeutic strategy. In this study, we determined the X-ray structure of the E PBM peptide in complex with the ZO-1 PDZ2 domain at 1.7 \u00c5 resolution. The structure revealed a domain-swapped dimer conformation of ZO-1 PDZ2, with the E PBM peptide conventionally bound within the PDZ domain\u2019s canonical binding groove exhibiting key interactions characteristic of type II PBM\u2013PDZ interactions. To identify potential inhibitors of the E PBM/ZO-1 PDZ2 interaction, we performed a HTRF screening using a protein-protein interaction-focused library of 1,000 compounds. This led to the identification of 36 hits that disrupted this interaction. Subsequent cytotoxicity and dose- response assays narrowed the selection to 14 promising compounds. Docking simulations showed that some compounds bind within or near the PBM-binding pocket, supporting a competitive mechanism of interaction inhibition, while others bind at a central interface between the two PDZ monomers, suggesting an inhibition of dimerization, which in turn prevents PBM binding. Thus, the ZO-1 PDZ2\u2013E PBM interaction can be inhibited through both direct and indirect mechanisms. Finally, antiviral assays using a NanoLuciferase-expressing recombinant SARS-CoV-2 demonstrated that one compound, C19, significantly reduced viral replication, highlighting its potential as a candidate for further therapeutic development. Crystal structure reveals the binding determinant of E protein PBM to ZO-1 PDZ2 dimer HTRF screening identified 36 inhibitors of the E\u2013ZO-1 protein interaction Docking revealed dual mechanisms: PBM groove binding and dimer interface disruption Compound C19 reduced SARS-CoV-2 replication in a NanoLuc reporter assay Targeting E\u2013ZO-1 interaction may prevent barrier loss and systemic COVID-19 effects","version":"1.1","doi":"10.1101/2025.07.16.665136","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.16.665104","pub_date":"2025-7-16","title":"Mouse Adapted Omicron BA.5 Induces A Fibrotic Lung Disease Phenotype in BALB/c Mice","abstract":"Following SARS-CoV-2 Omicron BA.1, subsequent Omicron sub-lineages have continued to emerge, challenging the development of intervention and prevention strategies, including monoclonal antibodies and vaccines. To better understand the pathogenic effects caused by Omicron BA.5 infection, we developed a mouse-adapted virus with overt disease burden in BALB/c mice. Acute disease was characterized by significant weight loss and lung dysfunction following high-dose challenges. In survivor animals that were followed through 107 days post-infection, subpleural fibrosis with associated tertiary lymphoid structures was noted. Serum from these mice demonstrated potent neutralization against BA.5, with substantially reduced neutralization titers against early epidemic, zoonotic, and more recent contemporary XBB.1.5 variants. Intervention with pre-clinical monoclonal antibodies revealed that robust protection from BA.5-induced lung disease was possible after prophylactic administration. Together, this model enables the investigation of therapeutic approaches for both acute and post-acute sequelae of COVID-19. In order to best combat the evolving landscape of SARS-CoV-2 variants of interest and variants of concern the development of effective small animal models is of critical importance. Herein, we describe the development of a model system in BALB/c mice to study the effects of SARS-CoV-2 BA.5 in both acute and chronic disease manifestations. Intriguingly, we determined that fibrotic lung disease with tertiary lymphoid structures was a prominent feature in the lungs of mice that survived through the acute phase of infection. This is a prominent concern in human patients that survive the initial infection insult. As such, and most critically, the model system presented here provides researchers with an effective pathway in which long COVID manifestations and potential interventions can be studied.","version":"1.1","doi":"10.1101/2025.07.16.665104","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.15.664547","pub_date":"2025-7-16","title":"Programmable translational inhibition by a molecular glue-oligonucleotide conjugate","abstract":"Selective inhibition of mRNA translation is a promising strategy for modulating the activity of disease-associated genes, yet achieving both high potency and specificity remains challenging. Rocaglamide A (RocA), a molecular glue, inhibits translation by clamping eIF4A onto polypurine motifs found in many transcripts, thereby limiting RocA\u2019s specificity. Here, we developed RocASO, a chemical conjugate that links RocA to an antisense oligonucleotide (ASO) capable of base-pairing with defined mRNA sequences, thus directing RocA\u2019s clamping mechanism to chosen targets and enhancing overall potency and specificity. We show that RocASOs are compatible with various types of ASO modalities, including gapmers that induce the degradation of target RNAs. RocASOs were designed to effectively knock down endogenous genes (PTGES3, HSPA1B) and SARS-CoV-2 viral RNA, the latter conferring potent antiviral activity in cells. These findings establish RocASO as a versatile platform for programmable translational inhibition with therapeutic potential.","version":"1.1","doi":"10.1101/2025.07.15.664547","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.10.664163","pub_date":"2025-7-15","title":"Heterotypic Protein Interactions Modulate the Condensate Dynamics and Aggregation of \u03b1-Synuclein","abstract":"Multicomponent biomolecular condensates formed by diverse multivalent proteins underlie numerous cellular processes, from ribosome biogenesis to stress response regulation, and have recently been implicated in disease-related protein aggregation. Understanding how heterotypic protein interactions modulate condensate dynamics and transitions to amyloid states remains a major challenge. Here, we combine fluorescence -based ensemble and single-molecule approaches, molecular simulations, and systematic domain deletions to investigate how charge patterning and domain structure influence co-condensation between \u03b1-synuclein, a disordered neuronal protein linked to Parkinson\u2019s disease, and the SARS-CoV-2 nucleocapsid protein (NP), a structured viral RNA-binding protein. We find that co-condensation is driven by multivalent electrostatic interactions and occurs when the heterotypic affinity exceeds a threshold, resulting in restricted protein dynamics and altered condensate material properties. These changes promote the formation of dense amyloid fibrils, as confirmed by atomic force microscopy and fluorescence assays. Our results elucidate how heterotypic interactions within multiphasic condensates can modulate phase behavior and aggregation, providing insight into broader mechanisms linking condensate dysregulation with neurodegenerative diseases.","version":"1.1","doi":"10.1101/2025.07.10.664163","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.11.664150","pub_date":"2025-7-14","title":"Integrated Systems Vaccinology Reveals Distinct Metabolic Responses to SARS-CoV-2 Infection and DNA-Based Vaccines in Ferrets","abstract":"Understanding systemic effects of vaccination and infection is central to defining correlates of protection against SARS-CoV-2. We used untargeted serum metabolomics to profile the immunometabolic landscape of ferrets after SARS-CoV-2 infection and DNA-/protein-based vaccination. Ferrets were vaccinated with either a multigenic DNA vaccine encoding SARS-CoV-2 RBD, M, and N (OC2), an N-only DNA vaccine (OC12), a recombinant spike protein with QS-21 adjuvant (S+QS21), or a hepatitis B/D control construct (Hep-B/D), and subsequently challenged with SARS-CoV-2. Serum was analyzed longitudinally at baseline, post-vaccination, and post-challenge. SARS-CoV-2 infection induced broad metabolic reprogramming, involving TCA cycle, glutathione metabolism, and nucleotide turnover, reflecting inflammation and cellular activation. OC2 vaccination induced strong metabolic shifts in amino acid and mitochondrial pathways despite low pre-challenge anti-S antibodies. Post-challenge, these shifts extended to redox and nucleotide pathways, correlating with robust anti-S and very strong anti-N antibody responses and complete viral clearance in BAL, but with marked airway pathology, consistent with T cell-mediated clearance of infected cells. S+QS21 and OC12 induced distinct, immunogen-specific signatures with partial protection, while Hep-B/D showed minimal systemic engagement. Metabolite-antibody correlations revealed vaccine-specific associations, highlighting lipid and amino acid pathways as potential immunogenicity biomarkers. Overlap and heatmap analyses showed that metabolic trajectories reflect both the magnitude and quality of immune training. These findings underscore the value of systems vaccinology in resolving mechanistic differences in vaccine responses and support metabolic profiling as a tool for evaluating immune efficacy in preclinical vaccine studies.","version":"1.1","doi":"10.1101/2025.07.11.664150","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.10.664240","pub_date":"2025-7-11","title":"Deciphering the Mechanistic Continuum of Broadly Neutralizing Class 4 Antibodies Targeting Conserved Cryptic Epitopes of the SARS-CoV-2 Spike Protein : Operating at the Intersection of Binding, Allostery and Immune Escape","abstract":"Understanding atomistic basis of multi-layer mechanisms employed by broadly reactive neutralizing antibodies of SARS-CoV-2 spike protein without directly blocking receptor engagement remains an important challenge in coronavirus immunology. Class 4 antibodies represent an intriguing case: they target a deeply conserved, cryptic epitope on the receptor-binding domain yet exhibit variable neutralization potency across subgroups F1 (CR3022, EY6A, COVA1-16), F2 (DH1047), and F3 (S2X259). In this study, we employed a multi-modal computational approach combining atomistic and coarse-grained simulations, mutational scanning of binding interfaces, binding free energy calculations, and allosteric modeling using dynamic network analysis to map the allosteric landscapes and binding hotspots of these antibodies. Through this approach, our data revealed that distal binding can influence ACE2 engagement and immune escape traits through the confluence of direct interfacial interactions and allosteric effects. We found that group F1 antibodies can operate via classic allostery by modulating flexibility in the receptor binding domain loop regions and indirectly interfering with ACE2 binding using long-range effects. Group F2 antibody DH1047 represents an intermediate mechanism, engaging residues T376, R408, V503, and Y508 hotspots which are both critical for ACE2 binding and under immune pressure. Mutational scanning and rigorous binding free energy calculations highlight the synergy between hydrophobic and electrostatic interactions, while dynamic network modeling reveals a shift toward localized communication pathways connecting the cryptic site to the ACE2 interface. Our results demonstrate how group F3 antibody S2X259 achieves efficient synergistic mechanism through confluence of direct competition with ACE2 and localized allosteric effects leading to stabilization of the spike protein at the cost of increased escape vulnerability. Dynamic network analysis identifies a shared \u201callosteric ring\u201d embedded in the core of the receptor binding domain and serving a conserved structural scaffold mediating long-range signal propagation with antibody-specific extensions propagating toward the ACE2 interface. The findings of this study support a modular model of antibody-induced allostery where neutralization strategies evolve via refinement of peripheral network connections, rather than complete redesign of the epitope itself. Taken together, this study establishes a robust computational framework for atomistic understanding of mechanisms underlying neutralization activity and immune escape for class 4 antibodies which harness conformational dynamics, binding energetics, and allosteric modulation to influence viral entry. The findings highlight the modular evolution of neutralization strategies, where progressive refinement of peripheral interactions enhances potency but increases susceptibility to immune pressure.","version":"1.1","doi":"10.1101/2025.07.10.664240","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.08.556703","pub_date":"2025-7-09","title":"Contrasting Effects of SARS-CoV-2 Vaccination vs. Infection on Antibody and TCR Repertoires","abstract":"Antibodies and helper T cells play important roles in SARS-CoV-2 infection and vaccination. We sequenced B- and T-cell receptor repertoires (BCR/TCR) from the blood of 251 infectees, vaccinees, and controls to investigate whether features of these repertoires could predict subjects\u2019 SARS-CoV-2 neutralizing antibody titer (NAbs), as measured by enzyme-linked immunosorbent assay (ELISA). We sequenced recombined immunoglobulin heavy-chain (IGH), TCR\u03b2 (TRB), and TCR\u03b4 (TRD) genes in parallel from all subjects, including select B- and T-cell subsets in most cases, with a focus on their hypervariable CDR3 regions, and correlated this AIRRseq data with demographics and clinical findings from subjects\u2019 electronic health records. We found that age affected NAb levels in vaccinees but not infectees. Intriguingly, we found that vaccination and infection have an effect on non-productively recombined IGHs, suggesting an effect that precedes clonal selection. We found that repertoires\u2019 binding capacity to known SARS-CoV-2-specific CD4+ TRBs performs as well as the best hand-tuned approximate or \u201cfuzzy\u201d matching at predicting a protective level of NAbs, while also being more robust to repertoire sample size and not requiring hand-tuning. The overall conclusion from this large, unbiased, clinically well annotated dataset is that B- and T-cell adaptive responses to SARS-CoV-2 infection and vaccination are surprising, subtle, and diffuse. We discuss methodological and statistical challenges faced in attempting to define and quantify such strong-but-diffuse repertoire signatures and present tools and strategies for addressing these challenges.","version":"1.2","doi":"10.1101/2023.09.08.556703","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.08.661948","pub_date":"2025-7-09","title":"Dissecting the effect of single- and co-infection of TB and COVID-19 pathogens on the sputum microbiome","abstract":"Tuberculosis (TB) and COVID-19 are both respiratory diseases and understanding their interaction is important for effective co-infection management. Although some studies have investigated TB and COVID-19 co-infection in terms of immune responses, microbial dysbiosis in such cases remains unexplored. In this study, we understand the interface between TB and COVID-19 by systematically inspecting the microbial composition of sputum samples collected from four groups of individuals: TB only, COVID-19 only, and both TB and COVID-19 (TBCOVID) infected patients, and uninfected group (Controls). Besides metagenomic analysis of the microbiome of these sputum samples, we also performed whole genome sequencing analysis of a subset of TB-positive samples. Different bioinformatic analyses ensured data quality and revealed significant differences in the microbial composition between Control vs. disease groups. To understand the effect of COVID-19 on TB, we compared TBCOVID vs. TB samples and observed (i) higher read counts of TB-causing bacteria in the TBCOVID group, and (ii) differential abundance of several taxa such as Capnocytophaga gingivalis, Escherichia coli, Prevotella melaninogenica and Veillonella parvula. Functional profiling with PICRUSt2 revealed significantly elevated pathways in the TBCOVID group relative to TB group, some of which are related to metabolism of pulmonary surfactant lipids (e.g., Cytidine diphosphate diacylglycerol (CDP-DAG) biosynthesis pathway with fold change of 7.46). Further clustering of these significantly elevated pathways revealed a sub-cluster of individuals with adverse treatment outcomes. Two individuals in this sub-cluster showed prevalence of respiratory pathogens like Stenotrophomonas maltophilia \u2013 knowing such information can help personalize the antibiotic treatment to match the pathogen profile of the individual. Overall, our study reveals the effect of COVID-19 in the airway microbiome of TB patients, and encourages the use of co-microbial/co-pathogen profiling to personalize TB treatment. The community of microbes in an individual\u2019s airway tract can play a complex role in respiratory diseases like TB and COVID-19. Although changes in microbial composition in TB and COVID-19 patients have been studied separately, we present a first-of-its-kind investigation of the airway tract microbiome of individuals simultaneously infected with TB and COVID-19 pathogens. Our results highlight that co-infection with COVID-19 in TB patients alters the abundance of certain bacterial species and their related pathways. For instance, Capnocytophaga gingivalis is abundant in co-infected patients, but not present in the TB-only patient group. This species and other differentially abundant species that we identified in the co-morbid condition, if replicated in independent cohorts, can help explain how COVID-19 could exacerbate the severity of lung infection in TB patients. Our study also stimulates future longitudinal studies using expanded datasets to understand the role of concomitant pathogens, and assess whether adjusting the antibiotic regimen accordingly can improve TB treatment outcomes.","version":"1.1","doi":"10.1101/2025.07.08.661948","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.07.663401","pub_date":"2025-7-08","title":"A conserved long-range RNA interaction in SARS-CoV-2 recruits ADAR1 to enhance virus proliferation","abstract":"Long-range RNA-RNA pairing impacts the genome structure and function of SARS-CoV-2 variants. To understand the structure and function relationships of different SARS-CoV-2 variants that have emerged during the COVID-19 pandemic, we performed high-throughput structure probing and modelling of the genomic structures of the wildtype (WT), Alpha, Beta, Delta and Omicron variants of SARS-CoV-2. We observed that genomes of SARS-CoV-2 variants are generally structurally conserved, and that single-nucleotide variations and interactions with RNA binding proteins can impact RNA structures across the viruses. Importantly, using proximity ligation sequencing, we identified many conserved ultra-long-range RNA-RNA interactions, including one that spans more than 17 kb in both the WT virus and the Omicron variant. We showed that mutations that disrupt this 17 kb long-range interaction reduce viral fitness at later stages of its infection cycle, while compensatory mutations partially restore virus fitness. Additionally, we showed that this ultra-long-range RNA-RNA interaction structure binds directly to ADAR1 to alter the RNA editing levels on the viral genome. These studies deepen our understanding of RNA structures in the SARS-CoV-2 genome and their ability to interact with host factors to facilitate virus infectivity.","version":"1.1","doi":"10.1101/2025.07.07.663401","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.07.663519","pub_date":"2025-7-08","title":"BST-2 inhibits SARS-CoV-2 egress at intracellular membranes and is neutralized by ORF7a","abstract":"Bone marrow stromal antigen 2 (BST-2, or tetherin) is an interferon-inducible host restriction factor that inhibits the release of enveloped viruses by tethering nascent virions to cellular membranes. While its antiviral function is well established in retroviral systems, its role in SARS-CoV-2 egress remains unclear. Here, we used a virus-like particle (VLP) system composed of SARS-CoV-2 structural proteins M, E, and N to investigate the impact of BST-2 on viral particle release. BST-2 significantly inhibited VLP release in HEK293T and Calu-3 lung epithelial cells. Confocal microscopy revealed that BST-2 colocalizes with viral structural proteins at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), the main site of coronavirus assembly. We next evaluated the roles of the SARS-CoV-2 accessory proteins ORF3a and ORF7a in overcoming this restriction. ORF3a localized to endolysosomal compartments and promoted VLP release through a BST-2-independent mechanism, without altering BST-2 expression or localization. In contrast, ORF7a colocalized with both BST-2 and ERGIC markers and restored VLP release by promoting BST-2 degradation. Notably, ORF7a also relieved BST-2-mediated restriction of HIV-1 VLP release, suggesting a conserved antagonistic function. These findings identify BST-2 as an intracellular inhibitor of SARS-CoV-2 particle release and establish ORF7a as viral accessory antagonist that neutralizes this host defense.","version":"1.1","doi":"10.1101/2025.07.07.663519","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.07.663433","pub_date":"2025-7-08","title":"Replication of SARS-CoV-2 Omicron lineages is defined by TMPRSS2 use in environments where ACE2 is complexed with solute carriers SLC6A19 and SLC6A20","abstract":"The Omicron variant of SARS-CoV-2 emerged in late 2021 and since then Omicron subvariants have continued to evolve and dominate globally. The viral S protein evolved towards highly efficient antibody evasion and replicative capacity in the upper respiratory tract resulting in high transmissibility. At the same time, the mutations acquired in the S protein diminish infection of the lung epithelium and pathogenic potential. The changing entry requirements for Omicron sub-lineages that lead to this shift in tropism remain poorly understood. We resolve the changing replication requirements of SARS-CoV-2 to be related to two distinct pools of ACE2. The first pool relates to ACE2\u2019s role in the renin angiotensin system (RAS) and this pool can complex with TMPRSS2 (RAS-ACE2). The second pool relates to ACE2\u2019s role as a protein solute carrier chaperone than cannot complex with TMPRSS2 (Chaperone ACE2). Here, we demonstrate that pre-Omicron lineages replicate in a TMPRSS2 dependent manner across both ACE2 pools, whilst Omicron lineages can only spread and replicate using chaperone ACE2. This provides a mechanistic basis for the evolving infectivity requirements of SARS-CoV-2 and furthermore provides approaches to track and monitor ACE2 utilizing coronaviruses. Mechanistic basis for shift in SARS-CoV-2 tropism with the arrival of Omicron. A. Chaperone ACE2 is defined structurally as a heterodimer of dimers with a solute carrier protein-SLC6A19 or SLC6A20. Here this ACE2 structure can exist uncomplexed from TMPRSS2 and enables TMPRSS2 use by both pre-Omicron and Omicron lineages. B. Renin Angiotensin ACE2 is defined by ACE2 with an exposed collectrin-like domain (CLD), which enables binding of TMPRSS2 or ADAM-17. Here ACE2 can form a complex with TMPRSS2 in a manner that allows pre-Omicron but not Omicron lineages to utilize TMPRSS2 to facilitate infection. Here Omicron lineages are heavily attenuated as they cannot use TMPRSS2 to spread. C. to E. Based on single cell profiles, ACE2 can exist as a chaperone with SLC6A20 in C. the Nasal Cavity or D. primarily as RAS-ACE2 in the lung to respond to acute lung injury. E. The largest pool of ACE2 in our body resides within the small intestine on enterocytes and this further facilitates replication in this tissue by pre-Omicron and Omicron lineages.","version":"1.1","doi":"10.1101/2025.07.07.663433","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.07.663218","pub_date":"2025-7-08","title":"Shedding of mitochondrial Voltage-Dependent Anion Channel-1 (VDAC1) Reflects COVID-19 Severity and Reveals Macrophage Dysfunction","abstract":"COVID-19 severity correlates with lymphopenia and increased pro-inflammatory cytokines. However, the dysfunction of tissue macrophages in COVID-19 patients during the inflammatory cytokine storm has not been fully elucidated. Hospitalized COVID-19 patients were divided into three groups based on their symptomatic severity: exhibiting mild, moderate, or severe symptoms. Patients exhibited successively increased serum levels of mitochondrial voltage-dependent anion channel 1 (VDAC1) at days 0, 3, 7, 10, and 14, returning to those of non-infected subjects at day 28. Serum level of VDAC1 was positively correlated with COVID-19 severity and with increased white blood cell (WBC), neutrophil, lymphocyte, procalcitonin (PCT), and gamma-glutamyltransferase (GT) levels. Peripheral blood mononuclear cells (PBMCs) from hospitalized COVID-19 patients showed increased VDAC1 content concomitant with a reduced ATP content. Culture of monocytes, isolated from healthy individuals, and differentiated into polarized M1 macrophages, together with a cytokine mixture (IL-1\u03b2, IFN-\u03b3, and TNF-\u03b1), to mimic the inflammatory cytokine storm, for 24 h markedly increased VDAC1 and Monocyte chemoattractant protein-1 (MCP-1) release in culture medium. The presence of the cytokine mixture reduced the ATP content, cell viability, and the phagocytic capability of macrophages. Co-staining of VDAC1 and the plasma membrane marker Na+/K+-ATPase showed that cytokine-treatment mistargeted VDAC1 to the cell surface of macrophages. All these effects were prevented by VDAC1 inhibition using VBIT-4, VDAC1-specific antibody (VDAC1-ab), or metformin. Our findings indicate that increased VDAC1 expression and cell surface mistargeting in immune cells might be associated with cell dysfunction, potentially contributing to the severity of COVID-19 infection. The data also indicate serum VDAC1 as a biomarker of COVID-19 severity and the use of VDAC1 inhibitors as potential drug candidates restoring macrophages and PBMCs function in individuals severely affected by COVID-19.","version":"1.1","doi":"10.1101/2025.07.07.663218","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.02.662833","pub_date":"2025-7-07","title":"Respiratory syncytial virus infection confers heterologous protection against SARS-CoV-2 via induction of \u03b3\u03b4 T cell-mediated trained immunity and SARS-CoV-2 reactive mucosal T cells","abstract":"The respiratory viruses can concurrently or sequentially infect a host and influence the trajectory of each other. The underlying immune mechanisms are not well understood. Here, we investigated whether respiratory syncytial virus (RSV) infection affects host vulnerability to subsequent SARS-CoV-2 infection in two murine models of SARS-CoV-2 infection. We found that prior RSV infection-induced heterologous protection against subsequent SARS-CoV-2 infection was dose and time dependent. RNA-seq and immunological analyses revealed that RSV triggered the activation of lung antigen presenting cells (APC)s and SARS-CoV-2 reactive mucosal T cells at day 9, which declined at 1 month. RSV also induced the expansion of lung \u03b3\u03b4 T cells and the upregulation of their cellular metabolic pathways. Furthermore, RSV infection in TCR\u03b4-/- mice, which are deficient of \u03b3\u03b4 T cells, resulted in a reduced SARS-CoV-2 reactive mucosal T cell response and subsequent increased viral loads and higher levels of virus-induced inflammatory responses in the lung upon SARS-CoV-2 challenge compared to the wild-type mice. In summary, our findings suggest that RSV infection provides heterologous protection against the subsequent SARS-CoV-2 infection via induction of \u03b3\u03b4 T cell-mediated trained immunity in the lung and SARS-CoV-2 reactive mucosal T cell responses.","version":"1.1","doi":"10.1101/2025.07.02.662833","journal":"bioRxiv","score":null},{"id":"10.1101/2025.07.03.663092","pub_date":"2025-7-07","title":"A chimeric Ad5-Envp-VLP vaccine platform confers broad-spectrum immunity against emerging and re-emerging pathogens","abstract":"Integrating complementary vaccine modalities is essential for combating emerging infectious threats. Here, we developed Ad5-Envp-VLP, a chimeric adenoviral platform synergizing adenoviral delivery efficiency with virus-like particle (VLP) structural mimicry. This system stably produces self-assembling VLPs in suspension HEK293 cultures, exhibiting enhanced immunogenicity over soluble antigens. Following intramuscular immunization, the platform induces early B cell expansion and sustains germinal center reactions, driven by the upregulation of B cell cycle-related genes (Cdc6, Cdc45, Cdc20, Cdc25C, Aurka, Aurkb, and Ccnb1/2) and robust T follicular helper (Tfh) cell differentiation, generating durable neutralizing antibodies against both influenza virus and rabies virus. These effects are conserved across mouse, canine, and feline models. Crucially, integrated flow cytometry and scRNA-seq demonstrate that intranasal delivery recruits and functionally reprograms lung innate immune cells (notably alveolar macrophages and dendritic cells), driving mucosal sIgA secretion and CTL responses. A single nasal dose confers lasting protection against homologous and heterologous influenza A strains. The platform also elicits cross-neutralizing antibodies against SARS-CoV-2 variants. Together, Ad5-Envp-VLP thus establishes a modular vaccine platform for antigenically plastic pathogens by combining in vivo self-assembly with dual pulmonary-muscular delivery. Novel mucosal and systemic vaccine delivers self-assembling particles, triggering strong lung immunity and broad virus protection across species.","version":"1.1","doi":"10.1101/2025.07.03.663092","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.30.662440","pub_date":"2025-7-02","title":"Human pluripotent stem cell-derived bronchial airway organoids provide insights into differential innate immune and long-term responses to SARS-CoV-2 infection in healthy and COPD","abstract":"Respiratory infections are a major global health concern, as underscored by the COVID-19 pandemic. To better understand bronchial tissue responses to viral infection, we have developed a preclinical in vitro model mimicking the multiciliated airway epithelium, from induced pluripotent stem cell (iPSC) and cultured in an air-liquid interface (iALI). By using iPSCs reprogrammed from patients with chronic obstructive pulmonary disease (COPD), we successfully generated a fully differentiated and functional bronchial epithelium exhibiting key COPD features with goblet and basal cell hyperplasia and tissue inflammation. SARS-CoV-2 could infected and replicated for several weeks in both healthy and COPD models, with a recurrent peak at 3 days after infection. Infected iALI exhibited cilia destruction and increased mucus secretion. Innate immune response of different infected iALI reveals a differential expression of interferon-stimulated genes (ISGs) and pro-inflammatory cytokine secretion. Notably, COPD iALI displayed an earlier innate immune response to SARS-CoV-2 infection as compared to healthy iALI, suggesting a genetic susceptibility of COPD iALI towards inflammation induced by SARS-CoV-2 infection, and a less efficient response to antivirals. In conclusion, our study demonstrates that the iALI bronchial organoid model is a powerful tool for investigating bronchial tissue responses to long term respiratory viral infections, antivirals, and patients with COPD or other airway pathology. hiPSC-derived COPD airway organoids SARS-CoV-2 productively infects induced pluripotent stem-cell derived bronchial organoids iALI and persisted over the long term SARS-CoV-2 iALI infection results in cilia destruction, increased mucus secretion and a strong innate immune response SARS-CoV-2 infection elicited a higher and earlier innate immune response in iCOPD SARS-CoV-2 infection in iCOPD respond less to antivirals SARS-CoV-2 causes severe lower respiratory tract infection in COVID-19 patients, which can persist over time. Here, we used an in-house developed in vitro airway organoid derived from induced human pluripotent stem cells (iALI) to study SARS-CoV-2 infection over long term in healthy or COPD patients whom respiratory failure is at risk during infection. Our results show that SARS-CoV-2 infection results in high and lethal infection of bronchial epithelial cells, that persist over time, inducing mucus secretion, destruction of ciliated cells and specific cytokine release. A late innate immune response is observed in the healthy iALI, while in iCOPD, it appears earlier and stronger, suggesting a different sensing of SARS-CoV-2 in COPD patients, accompanied by a reduce sensitivity to antivirals. In conclusion, our study demonstrates that the iALI organoid model is a powerful tool for investigating bronchial tissue responses to long term respiratory viral infections, from healthy to pathologic patients.","version":"1.1","doi":"10.1101/2025.06.30.662440","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.14.643358","pub_date":"2025-7-02","title":"Dissecting the effects of single amino acid substitutions in SARS-CoV2 Mpro","abstract":"Successfully predicting the effects of amino acid substitutions on protein function and stability remains challenging. Recent efforts to improve computational models have included training and validation on high-throughput experimental datasets, such as those generated by deep mutational scanning (DMS) approaches. However, DMS signals typically conflate a substitution\u2019s effects on protein function with those on in vivo protein abundance; this limits the resolution of mechanistic insights that can be gleaned from DMS data. Distinguishing functional changes from abundance-related effects is particularly important for substitutions that exhibit intermediate outcomes (e.g., partial loss-of-function), which are difficult to predict. Here, we explored changes in in vivo abundance for substitutions at representative positions in the SARS-CoV-2 Main Protease (Mpro). For this study, we used previously published DMS results to identify \u201crheostat\u201d positions, which are defined by having substitutions that sample a broad range of intermediate outcomes. We generated 10 substitutions at each of six positions and separately measured effects on function and abundance. Results revealed an \u223c45-fold range of change for abundance, demonstrating that it can make significant contributions to DMS outcomes. Moreover, the six tested positions showed diverse substitution sensitivities for function and abundance. Some positions influenced only one parameter. Others exhibited rheostatic effects on both parameters, which to our knowledge, provides the first example of such behavior. Since effects on function and abundance may arise through different biophysical bases, these results underscore the need for datasets that independently measure these parameters in order to build predictors with enhanced mechanistic insights. Changing one amino acid in a protein can affect its function, its abundance, or both. Understanding these separate effects will help scientists predict how protein changes alter biology, which is important for understanding pathogen evolution, improving personalized medicine, and bioengineering. This work reports a study to experimentally separate these effects for the main protease of SARS-CoV-2 and suggests strategies for building better prediction models for emerging variants of this key viral protein.","version":"1.2","doi":"10.1101/2025.03.14.643358","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.30.661938","pub_date":"2025-7-01","title":"SARS-CoV-2 spike protein amyloid fibrils impair fibrin formation and fibrinolysis","abstract":"Long COVID, also known as post-acute sequelae of COVID-19 (PASC) from SARS-CoV-2 infection, is a debilitating and persistent disease of multiple systems and organs. WHO reports a total of 778 million registered SARS-CoV-2 infections as of June 2025. Recent studies indicate that as many as 25% of COVID patients will experience at least one symptom of Long COVID. Long COVID pathophysiology is a complex and not fully established process. One prevailing theory is that the formation of fibrin amyloid microclots (fibrinaloids), due to SARS-CoV-2 infection, can induce persistent inflammation and capillary blockage. An association between the amyloidogenic Spike protein of SARS-CoV-2 and impaired fibrinolysis has previously been made when it was observed that fibrin clots formed in the presence of a mixture of amyloid fibrils from the spike protein displayed a resistance to plasmin-mediated lysis. Here we investigated the molecular processes of impaired fibrinolysis using seven amyloidogenic SARS-COV-2 Spike peptides. Five out of seven Spike amyloid fibrils appeared not to substantially interfere with the fibrinogen-fibrin-fibrinolysis process in vitro, while two spike fibrils were active in different ways. Spike601 amyloid fibrils (sequence 601-620) impaired thrombin mediated fibrin formation by binding and sequestering fibrinogen but did not affect fibrinolysis. On contrary fibrin clots formed in the presence of Spike685 amyloid fibrils (sequence 685-701) exhibited a marked resistance to plasmin mediated fibrinolysis. We conclude that Spike685 amyloid fibrils can induce dense fibrin clot networks, as well as incorporate fibrin into aggregated structures that resist fibrinolysis. These results demonstrate how the Spike protein of SARS-CoV-2 could contribute to the formation fibrinolysis-resistant microclots observed in long COVID.","version":"1.1","doi":"10.1101/2025.06.30.661938","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.29.662219","pub_date":"2025-7-01","title":"Back-projection improves inference from sparsely sampled genomic surveillance data","abstract":"Highly transmissible SARS-CoV-2 variants have emerged throughout the COVID-19 pandemic, driving new waves of infections. Genomic surveillance data can provide insights into the virus\u2019s evolution and biology. However, delayed and limited regional data can introduce biases in epidemiological models, potentially obscuring transmission patterns. To address this issue, we used a novel, variant-specific back-projection model to estimate a distribution of likely infection times from sample collection times. We combined this approach with epidemiological modeling to estimate selection for increased transmission in a way that accounts for the uncertainty in infection times. Tests in simulations demonstrated that our method can make the inference of selection more reliable. We also applied our approach to SARS-CoV-2 data, where it excelled in smoothing and extending data from geographic regions or times with poor sampling. Overall, our method can aid in the reliable identification of mutations and variants with higher transmission rates.","version":"1.1","doi":"10.1101/2025.06.29.662219","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.28.662162","pub_date":"2025-7-01","title":"Exploring Dynamic Modulation of Binding, Allostery and Immune Resistance in the SARS-CoV-2 Spike Complexes with Classes of Antibodies Targeting Cryptic Binding Sites: Antibody-Specific Augmentations of Conserved Allosteric Architecture Can Influence Evolution of Viral Escape","abstract":"The ongoing evolution of SARS-CoV-2 variants has underscored the need to understand not only the structural basis of antibody recognition but also the dynamic and allosteric mechanisms that could underlie complexity of broad and escape-resistant neutralization. In this study, we employed a multi-scale approach integrating structural analysis, hierarchical molecular simulations, mutational scanning and network-based allosteric modeling to dissect how Class 4 antibodies (represented by S2X35, 25F9, and SA55) and Class 5 antibodies (represented by S2H97, WRAIR-2063 and WRAIR-2134) can modulate conformational behavior, binding energetics, allosteric interactions and immune escape patterns of the SARS-CoV-2 spike protein. Using hierarchical simulations of the antibody complexes with the spike protein and ensemble-based mutational scanning of binding interactions we showed that these antibodies through targeting conserved cryptic sites can exert allosteric effects that influence global conformational dynamics in the RBD functional regions. The ensemble-based mutational scanning of binding interactions revealed an excellent agreement with experimentally derived deep mutational scanning (DMS) data accurately recapitulating the known binding hotspots and escape mutations across all studied antibodies. The predicted destabilization values in functional sites are consistent with experimentally observed reductions in antibody binding affinity and immune escape profiles demonstrating that computational models can robustly reproduce and forecast mutation-induced immune escape trends. Using dynamic network modeling we characterized the antibody-induced changes in residue interaction networks and long-range interactions. The results revealed that class 4 antibodies can exhibit distinct patterns of allosteric influence despite targeting overlapping regions, while class 5 antibodies elicit consistently dense and broadly distributed allosteric networks and long-range stabilization of the RBD conformations. Dynamic network analysis identifies a conserved allosteric network core that mediates long-range interactions and incudes antibody specific allosteric extensions that connect the binding interface hotspots with allosteric hubs. This study suggests that mechanisms of binding and immune escape for classes of antibodies targeting cryptic binding sites may be determined by confluence of multiple factors including high-affinity binding and long-range allosteric effects that modulate RBD adaptability and propagation of dynamic constraints which can influence mechanisms of viral evolution and escape.","version":"1.1","doi":"10.1101/2025.06.28.662162","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.30.662263","pub_date":"2025-7-01","title":"The impact of clade B lineage 5 MERS coronaviruses spike mutations from 2015 to 2023 on virus entry and replication competence","abstract":"Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus that can cause zoonotic disease in humans with lethal severe viral pneumonia. Dromedary camels are the source of zoonotic infection. As of June 2025, MERS-CoV has resulted in a total of 2626 reported cases, 36% of these being fatal. The number of reported human cases has been on a decreasing trend since 2016 and reached a minimum level during the COVID-19 pandemic. The reason for the reduction of cases is unclear and may be multifactorial. We hypothesized that mutations accumulating in the virus spike protein may have reduced zoonotic potential. Here, we investigate the impact of recently emerged virus spike-protein mutations on virus replication competence using pseudoviruses and replication-competent recombinant viruses. We found that two spike variants detected in 2019 show a reduced cell entry and lower viral replication in human cells. However, spike variants detected in 2023 sequences, did not show significant changes in cell entry and viral replication. All the MERS-CoV spikes tested showed a cell-entry pathway preference via the cell-surface TMPRSS2 route. Our data suggests that spike protein mutations are not a major determinant of the fewer MERS-CoV human cases observed. MERS-CoV is identified by the World Health Organization (WHO) as a potential pandemic candidate. The ability of coronaviruses to mutate and adapt in new hosts raises concerns about the impact of virus genetic changes on human zoonotic potential. There has been a notable decline in human MERS cases reported to the WHO since 2018, but the underlying reasons remain unclear. Here, we focus on investigating whether the recently emerged virus spike mutations may contribute to this observation. We found that while some spike mutations detected in 2019 reduce cell entry and viral replication, more recent viruses do not share this phenotype. This study highlighted a need for comprehensive genomic surveillance and phenotyping of recent MERS-CoV isolates to understand the potential role, if any, of other non-spike virus mutations on viral zoonotic competence and to explore alternate hypothesis, such as cross-reactive immunity from COVID-19 contributing to reduced human MERS-CoV disease.","version":"1.1","doi":"10.1101/2025.06.30.662263","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.27.662024","pub_date":"2025-6-30","title":"SARS-CoV-2 Viral Pseudoparticles Preferentially Infect Ectoderm In Human Embryonic Tissues","abstract":"Early stages in human development are difficult to study in pregnant women. We used a \u201cdisease-in-a-dish\u201d model to investigate SARS-CoV-2 infection of human embryonic stem cells and the three germ layers. Ectoderm had a significantly higher infection than the other cell types. This was due to: (1) the use of two entry pathways by the ectoderm (fusion and endocytosis), (2) high levels of TMPRSS2 in the ectoderm, and (3) a much-reduced ectodermal glycocalyx, which facilitated viral attachment to the ACE2 receptor. Our findings provide strong evidence that cells in young postimplantation human embryos are susceptible to SARS-CoV-2 infection, which could be embryo lethal or teratogenic in surviving embryos. The high level of infection in ectoderm is a concern as its derivatives may also be affected by SARS-CoV-2. Future clinical work should investigate the functioning of the nervous system in infants born to mothers who had COVID-19 during pregnancy. SARS-CoV-2 pseudoparticles infected human embryonic stem cells, endoderm, mesoderm and ectoderm Ectoderm was significantly more susceptible to infection than the other three cell types Factors accounting for increased susceptibility and tissue tropism were identified SARS-CoV-2 virus can adversely affect early stages of human development","version":"1.1","doi":"10.1101/2025.06.27.662024","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.27.661568","pub_date":"2025-6-30","title":"SARS-CoV-2 infection disrupts syncytial and endothelial integrity and alters PLGF levels in the placenta","abstract":"SARS-CoV-2 infection during pregnancy has been associated with an increased risk for several pregnancy-related disorders, particularly preeclampsia (PE). However, there are limited studies determining the impact of SARS-CoV-2 on placental physiology and function. Placental samples were acquired from two large prospective cohorts: STOP-COVID19 and REBRACO studies. Placental villous tissues (VTs) were collected from pregnant women who tested positive for SARS-CoV-2 without PE during pregnancy. Immunohistochemistry and immunofluorescence were used to assess pathological features known to be altered in PE, including 1) syncytial knot formation; 2) alterations in renin-angiotensin system components; 3) and endothelial integrity. Maternal serum was collected to examine AT1 autoantibodies levels using an immunoassay. SARS-CoV-2 viral proteins spike, nucleocapsid, and ORF3a were observed in the syncytiotrophoblast layer and stroma of placental VT. SARS-CoV-2-infected placentas exhibited increased numbers of syncytial knots, which were positive for Flt-1 and SARS-CoV-2 viral proteins. In addition, the presence of placental infarctions and excessive fibrin deposits was also observed in infected placentas. Infection was associated with decreased placental expression of PlGF and an increase in the placental Flt-1/PlGF expression ratio, mostly driven by PlGF. No significant changes in maternal serum AT1AA levels were observed. Finally, SARS-CoV-2-infected placentas exhibited a significant decrease in vimentin expression. SARS-CoV-2 infection negatively impacts placental integrity in the form of increased syncytial knots, dysregulated RAS components, and endothelial damage. Since all these features are similarly disrupted in PE, this could be a mechanism through which SARS-CoV-2 infection during pregnancy increases the risk of a PE-like syndrome.","version":"1.1","doi":"10.1101/2025.06.27.661568","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.26.661659","pub_date":"2025-6-27","title":"A Novel \u2018Three-in-one\u2019 Mucosal Vaccine Elicits Broad Protection against Three Distinct Clusters of ACE2-using Sarbecoviruseses","abstract":"With the persistent risk of coronavirus epidemics, the development of universal vaccines is urgently needed and challenging to achieve. In this study, we aimed to design a multivalent immunogen to provide broad protection against diverse ACE2-using sarbecoviruses, which pose a significant threat to global health. Our analysis revealed that the receptor-binding domains (RBDs) of ACE2-using sarbecoviruses segregate into three antigenically distinct clusters, including one cluster that encompasses viruses related to SARS-CoV-2. Based on these findings, we engineered a \u2018three-in-one\u2019 immunogen, designated as 3Rs-NC, which incorporates representative RBDs from all three clusters. 3Rs-NC preserved the natural epitope configuration of each monomeric RBD component, and efficiently elicited high levels of neutralizing antibodies against representative viruses and closely related sarbecoviruses. When administered intranasally with flagellin-based mucosal adjuvant KFD, 3Rs-NC induced robust and durable RBD-specific serum IgG and mucosal IgA responses in mice. Furthermore, KFD-adjuvanted 3Rs-NC conferred sustained protection in both the upper and lower respiratory tracts against SARS-CoV-2 Omicron BA.1 and SARS-like coronavirus WIV1. Additionally, 3Rs-NC immunization protected mice from lethal challenge of SARS-like coronavirus rRsSHC014S, with more efficient protection observed in female mice than male mice. This needle-free \u2018three-in-one\u2019 vaccine represents a promising candidate for a universal mucosal vaccine against ACE2-using sarbecoviruses and could serve as a foundational component of \u2018three-in-one\u2019 vaccine series to form a comprehensive coronavirus vaccine package. novel \u2018three-in-one\u2019 immunogen 3Rs-NC induced a high level of neutralizing antibodies against three distinct ACE2-using Sabecovirus clusters. administration of 3Rs-NC plus KFD adjuvant (3Rs-NC+KFDi.n) generated potent and long-lasting RBD-specific serum IgG and mucosal IgA. 3Rs-NC+KFDi.n provided long-term protection in both upper- and lower-respiratory tracts in mice. 3Rs-NC+KFDi.n could serve as a foundational component for next-generation coronavirus vaccine strategies.","version":"1.1","doi":"10.1101/2025.06.26.661659","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.25.661044","pub_date":"2025-6-25","title":"Comprehensive investigation of SARS-CoV-2 intestinal pathogenesis in Drosophila","abstract":"Gastrointestinal (GI) manifestations have been increasingly reported in COVID-19 patients. Here, we use the Drosophila melanogaster midgut model to investigate SARS-CoV-2-induced GI pathogenesis. The fly midgut exhibits susceptibility to orally administered virus, resulting in disrupted epithelial architecture, reduced organ size, and altered visceral muscle dynamics. These effects are accompanied by sustained proliferation of intestinal stem cells alongside decreased replenishment and viability of differentiated cells. Transcriptomic profiling reveals biphasic perturbations in midgut gene expression, particularly in pathways related to lipid metabolism. Intriguingly, SARS-CoV-2 elicits a dichotomous effect on lipid homeostasis, with lipid droplet accumulation in the posterior midgut and depletion in anterior segments. Treatment with Plitidepsin, a COVID-19 drug candidate, mitigates most SARS-CoV-2 pathogenic features in both the Drosophila midgut and human pulmonary cells, while modulating basal lipid droplet homeostasis in uninfected conditions. These findings establish the Drosophila midgut as a potent model for studying SARS-CoV-2 GI pathogenesis and evaluating antiviral compounds.","version":"1.1","doi":"10.1101/2025.06.25.661044","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.25.661409","pub_date":"2025-6-25","title":"The Renin\u2013Angiotensin System Modulates SARS-CoV-2 Entry via ACE2 Receptor","abstract":"The RAS plays a central role in cardiovascular regulation and has gained prominence in the pathogenesis of COVID-19 due to the critical function of ACE2 as the entry receptor for SARS-CoV-2. Angiotensin IV, but not angiotensin II, has recently been reported to enhance the binding between the viral spike protein and ACE2. To investigate the virological significance of this effect, we developed a single-round infection assay using SARS-CoV-2 viral-like particles expressing the spike protein. Our results demonstrate that while angiotensin II does not affect viral infectivity across concentrations ranging from 40LnM to 400LnM, angiotensin IV enhances viral entry at a low concentration but exhibits dose-dependent inhibition at higher concentrations. These findings highlight the unique dual role of angiotensin IV in modulating SARS-CoV-2 entry. In silico molecular docking simulations indicate that angiotensin IV was predicted to associate with the S1 domain near the receptor-binding domain in the open spike conformation. Given that reported plasma concentrations of angiotensin IV range widely from 17 pM to 81 nM, these levels may be sufficient to promote, rather than inhibit, SARS-CoV-2 infection. This study identifies a novel link between RAS-derived peptides and SARS-CoV-2 infectivity, offering new insights into COVID-19 pathophysiology and informing potential therapeutic strategies.","version":"1.1","doi":"10.1101/2025.06.25.661409","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.23.661193","pub_date":"2025-6-25","title":"A single extracellular vesicle-based platform supporting both RBD protein and mRNA vaccination against SARS-CoV-2","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syn-drome coronavirus 2 (SARS-CoV-2) has highlighted the need for novel, effective, and safe vaccine platforms. Extracellular vesicles (EVs) facilitate cell-to-cell communication and can modulate immune responses by delivering antigens. Here, we propose a versatile EV-based vaccine plat-form in which immune-stimulating EVs, engineered using acoustic shock waves (SW), are used to deliver either protein or mRNA antigens. Immunostimulatory EVs derived from LPS-activated THP-1 monocytes (aTEVs) were loaded with SARS-CoV-2 receptor-binding domain (RBD) protein or RBD-encoding mRNA via SW post-loading. Both aTEV-RBD-Pro and aTEV-RBD-mRNA elicited robust RBD-specific humoral and cellular immune responses in mice without the need for external adjuvants. Moreover, lyophilized aTEV-RBD-Pro vaccines retained immunogenicity after storage, supporting their chain-independent stability. These results demonstrate that a single EV-based platform can independently support both protein- and mRNA-based vaccination, highlighting its flexibility for next-generation vaccine development.","version":"1.1","doi":"10.1101/2025.06.23.661193","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.12.575398","pub_date":"2025-6-25","title":"Exploring the Human-Coronavirus protein-protein interaction network from the perspective of a novel host-virus association","abstract":"Host-pathogen interaction is the best example of an evolutionary arms race where pathogen and host continuously coevolve to survive and exert negative effects on each other. The adaptability of both host and pathogen is critical for this association. In this study, we explored the association of severe acute respiratory syndrome (SARS) coronaviruses (CoVs) with their human host from the genomic and evolutionary perspectives based on a comparative analysis of SARS and MERS coronaviruses. We observed that human proteins that are part of the SARS-CoV2-human association are enriched in hubs and bottlenecks. Again, these proteins take part in more protein complexes and show slower evolutionary rates compared to the human proteins associated with the two other coronaviruses, SARS-CoV and MERS-CoV. Moreover, the human proteins involved in the interaction with SARS-CoV2 are mostly longer proteins harboring long intrinsically disordered stretches and a higher level of disordered protein binding sites. Codon usage analysis revealed that the novel coronavirus is least adapted to codons used in housekeeping and lung-specific genes, compared to the other two coronaviruses. We conclude that the signatures showed by the SARS-CoV2-human protein interaction network revealed the virus\u2019s association with vital human proteins and pathways, via interactions mediated by protein complexes and intrinsically disordered protein binding sites, which may have assisted the higher infectivity of SARS-CoV2 in its human host than the other two less-virulent human coronaviruses, despite having a lower optimization to its host\u2019s codons.","version":"1.2","doi":"10.1101/2024.01.12.575398","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.23.661172","pub_date":"2025-6-24","title":"PUM2 binds SARS-CoV-2 RNA and PUM1 mildly reduces viral RNA levels, but neither protein affects progeny virus production","abstract":"Pumilio proteins (PUM1 and PUM2) are essential post-transcriptional regulators of gene expression found across plants, animals, and yeast. They bind Pumilio Response Elements (PREs) on messenger RNAs (mRNAs) to modulate mRNA stability and translation. PUMs have been implicated in diverse cellular processes, including stem cell maintenance, neurogenesis, and cell cycle regulation. They have also been reported to negatively regulate innate immunity genes and to participate in viral RNA sensing. Previous high-throughput interactome studies revealed that PUMs bind SARS-CoV-2 RNA. We found that SARS-CoV-2 transcripts contain multiple conserved PREs, some of which are preferentially bound by PUM2. Surprisingly, altering PUM levels does not affect the production of progeny virions. However, depletion of PUM1 slightly increases intracellular viral RNA levels, suggesting that PUM1 either plays a mild antiviral role against SARS-CoV-2 or regulates a host factor that promotes viral replication. Notably, PUM1 also negatively regulates innate immunity gene expression both at steady state and during SARS-CoV-2 infection. Our findings support a complex immunomodulatory role for PUM1, acting both as a negative regulator of innate immunity genes and a mild inhibitor of SARS-CoV-2 RNA accumulation. However, in cell culture, these roles appear negligible based on viral progeny output. Whether the multiple PREs found in the SARS-CoV-2 genome contribute to evasion of PUM1 activity remains an open question.","version":"1.1","doi":"10.1101/2025.06.23.661172","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.23.654375","pub_date":"2025-6-24","title":"Identifying Key Hub Genes that Attribute Varying Host Responses: A Longitudinal RNA-seq Analysis of SARS-CoV-2 Delta and Omicron Infections","abstract":"Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has severely impacted global health, with different Variants of Concern (VOCs) resulting in varied clinical outcomes. Among these, the Delta and Omicron variants have drawn significant attention due to their distinct characteristics\u2014Delta is associated with higher virulence and Omicron has greater transmissibility but reduced severity. This study compares host immune responses to the Delta and Omicron variants using RNA-seq data from infected primary human airway epithelial cells. Both variants triggered a robust antiviral innate immune response by 2 days post-infection (dpi). However, Omicron was found to elicit a more rapid immune response, showing pathway enrichment at 1 dpi. In contrast, Delta displayed no immune-related pathway activation within the first 24 hours, suggesting it may evade early immune detection, promoting increased viral replication. By 3 dpi, Delta induced a more aggressive immune response, particularly in pathways related to cell death and pro-inflammatory signaling, such as \u201cprogrammed cell death\u201d and \u201cregulation of cell death.\u201d Weighted gene co-expression network analysis revealed distinct immune-related genes: Delta infections were characterized by hub genes like MYD88 and IL1R1 involved in pro-inflammatory responses, HLA-A & B, NLRC5 and PSMB9 involved in antigen presentation and TNSF10 and IFR1 involved in pro-apoptotic processes. Conversely, Omicron infections were marked by hub genes such as CXCL1 and CXCL8 involved in immune cells recruitment, MET and LYN involved in reducing hyperinflammatory responses and maintaining immune balance, and IFI44, EIF2AK2 and IFIT5 responsible for the sensing of viral RNA among others.","version":"1.1","doi":"10.1101/2025.06.23.654375","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.23.661149","pub_date":"2025-6-24","title":"Host microRNA Target Prediction in SARS-CoV-2 and Hepatitis E Virus Genomes: Insights from RNAhybrid Analysis","abstract":"MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that can influence viral replication and pathogenesis by binding to viral RNA genomes. This study uses RNAhybrid to predict binding sites of three abundant human miRNAs\u2014hsa-miR-155-5p, hsa-miR-21-5p, and hsa-let-7a-5p\u2014within the genomes of SARS-CoV-2 (Bangladesh isolate OM967280.1) and Hepatitis E Virus genotype 1 (NC_001434.1). Our results show stable hybridization events with minimum free energy (mfe) values ranging from \u221223.8 to \u221228.1 kcal/mol, indicating strong potential interactions. These findings suggest that host miRNAs may modulate viral RNA stability or translation, impacting viral pathogenesis. Further experimental validation is necessary to confirm these regulatory relationships and explore their therapeutic potential.","version":"1.1","doi":"10.1101/2025.06.23.661149","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.04.611305","pub_date":"2025-6-24","title":"Nanoparticle-Supported, Rapid, and Electronic Detection of SARS-CoV-2 Antibodies and Antigens at Sub-Femtomolar Level","abstract":"Major challenges remain to precisely detect low-abundance proteins from diverse biofluids in a rapid and cost-effective manner. Here we present a gold nanoparticle (AuNP)-supported, rapid electronic detection (NasRED) platform with sub-femtomolar sensitivity and high specificity. Surface-functionalized AuNPs act as multivalent detectors to recognize target antigens and antibodies through high-affinity binding, subsequently forming aggregates precipitated in a microcentrifuge tube and producing a solution color change. The optical extinction of residual floating AuNPs is digitized using a customized circuitry incorporating inexpensive optoelectronic elements and feedback mechanisms for stabilized readout. Uniquely, NasRED introduces active fluidic forces through engineered centrifugation and vortex agitation, effectively promoting protein detection at low concentrations and accelerating signal generation. Using SARS-CoV-2 as a demonstration, NasRED enables detection of both antibodies and antigens from a small sample volume (6 \u00b5L), distinguishes the viral antigens from those of human coronaviruses, and delivers test results in a short time (as fast as <15 min). The limits of detection (LoDs) for antibody detection are approximately 49 aM (7 fg/mL) in phosphate-buffered saline, or >3,000 times more sensitive than Enzyme-Linked Immunosorbent Assay (ELISA), \u223c76 aM (11 fg/mL) in human pooled serum and in the femtomolar range in diluted whole blood. For nucleocapsid protein detection, NasRED LoDs are \u223c190 aM (10 fg/mL) in human saliva and \u223c2 fM (100 fg/mL) in nasal fluid. Unlike laboratory-based ELISA platforms, NasRED is a one-pot, in-solution assay that eliminates the needs for washing, labeling, expensive instrumentation or highly trained operators. With low reagent costs and a compact system footprint, this modular digital platform is well-suited for accurate, near-patient diagnosis and screening of a wide range of infectious and chronic diseases.","version":"1.2","doi":"10.1101/2024.09.04.611305","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.22.660949","pub_date":"2025-6-23","title":"Targeting PI3K Signaling for Broad Inhibition of \u03b2-Coronavirus Infection","abstract":"The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a central role in regulating key cellular processes such as survival, metabolism, and immune responses. Aberrant activation of this pathway is associated with tumorigenesis, and several PI3K inhibitors have been developed as anticancer agents. Emerging evidence suggests that viruses, including \u03b2-coronaviruses, have evolved mechanisms to exploit host PI3K signaling for their replication and immune evasion. In this study, we evaluated the antiviral efficacy of a panel of PI3K inhibitors against \u03b2-coronaviruses, including mouse hepatitis virus (MHV), human OC43 (HuCoV-OC43) and four major SARS-CoV-2 variants using both cell line and organoid models. Our findings reveal that these compounds exhibit low micromolar potency in inhibiting viral replication. Notably, the inhibitor C20 (PWT33597) demonstrated broad-spectrum activity against multiple \u03b2-coronaviruses, including SARS-CoV-2, MHV, and HuCoV-OC43, in conventional cell lines as well as in air\u2013liquid interface (ALI)-cultured, differentiated primary human nasal and bronchial epithelial cells. Given that cytokine storm is a major contributor to SARS-CoV-2\u2013related multiorgan failure and mortality, we further explored the impact of PI3K inhibition on host inflammatory responses. We found that MHV infection markedly increased cytokine expression in 17CL-1 fibroblasts and RAW264.7 macrophages. Interestingly, treatment with C20 further amplified cytokine production in this context, suggesting complex immunomodulatory effects that warrant further investigation. Together, our findings support the therapeutic potential of repurposing PI3K inhibitors as broad-spectrum antivirals. These compounds not only suppress viral replication but may also influence host immune responses, providing a promising avenue for intervention against current and emerging coronavirus threats.","version":"1.1","doi":"10.1101/2025.06.22.660949","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.19.25329920","pub_date":"2025-06-20","title":"Quantum-enhanced nanodiamond rapid test advances early SARS-CoV-2 antigen detection in clinical diagnostics","abstract":"<jats:p>\n                  Worldwide, the urgent need for more sensitive rapid lateral flow tests (LFTs) for early disease diagnosis is driving advances in quantum technologies. Spin-enhanced nanodiamond LFTs offer the potential for a sensitivity step change, yet to date only model systems have been studied with few clinical samples. Herein, we report the largest spin-enhanced LFT clinical evaluation, focusing on SARS-CoV-2 antigen as an exemplar. The limit of detection for recombinant antigens was 0.67 pg/ml, and inactivated whole virus wild-type and Omicron variants were 13 TCID\n                  <jats:sub>50</jats:sub>\n                  /mL and 8.8 TCID\n                  <jats:sub>50</jats:sub>\n                  /mL, respectively. Our blinded clinical study with 103 patient upper respiratory tract swab samples showed 95.1% sensitivity (Ct \u2264 30) and 100% specificity benchmarked to RT-qPCR, with no cross-reactivity to influenza A, RSV, and Rhinovirus. Analysis of trial data indicates spin-enhanced LFTs could diagnose SARS-CoV-2 on average 2.0 days earlier than conventional gold nanoparticle LFTs with identical antibodies, and just 0.6 days after RT-qPCR. Quantum-enhanced LFT sensitivity would have detected ~69,400 more patients in a single day at the peak of Omicron, reducing the transmission risk and protecting populations. Our findings mark an important milestone in the emerging field of quantum-enhanced diagnostics, with the potential for significant health-economic benefits to patients, populations, health systems and society.\n                </jats:p>","version":null,"doi":"10.1101/2025.06.19.25329920","journal":"medRxiv","score":null},{"id":"10.1101/2025.06.18.660454","pub_date":"2025-6-20","title":"Uncovering the Pathophysiological Pattern of Expression from Integrated Analysis across Uniformly Processed RNA Sequencing COVID-19 Datasets","abstract":"Post-acute sequelae of SARS-CoV-2 infection (PASC) affects millions globally, yet the molecular mechanisms underlying acute COVID-19 and its chronic sequelae remain poorly understood. We performed an integrative transcriptomic analysis of three independent RNA-seq datasets, capturing the complete COVID-19 pathophysiology from health through acute severe infection to post-acute sequelae and mortality (n=142 total samples). We implemented a containerized analytical pipeline from data download, quantification, differential gene expression to uniformly process these three RNA-seq datasets. Our analysis reveals striking molecular dichotomies contrasting disease phases with profound clinical implications. Acute severe/critical COVID-19 reveals predominant enrichment of TNF-\u03b1 signaling via NF-\u03baB pathways (normalized enrichment score >2.5, FDR <0.001), reflecting a cytokine storm pathophysiology characterized by rapid inflammatory developments involving IL-6, TNF-\u03b1, and anti-apoptotic responses. In contrast, PASC patients exhibit dominant enrichment of Myc Targets V1 and Oxidative Phosphorylation pathways (NES >2.2, FDR <0.005), indicating important shifts toward cellular adaptation. Pathway signature analysis identifies core differentially expressed genes that reliably distinguish disease phases, thereby offering objective biomarkers for precision diagnosis and monitoring. These findings establish a comprehensive molecular framework distinguishing acute inflammatory from chronic metabolic COVID-19 phases, with potential clinical applicability. TNF-\u03b1/NF-\u03baB pathway signatures identify patients at risk for severe disease progression, while Myc/OXPHOS signatures allow objective PASC diagnosis, addressing current reliance on subjective and eliminative diagnosis. This integrative analytical framework has utility beyond COVID-19, offering an applicable approach for precision medicine implementation across other diseases processes. This study transforms COVID-19 from a symptom-based to a molecularly-defined disease spectrum, enabling precision diagnosis, prognostic monitoring, classification, and targeted therapeutic possibilities based on pathway-specific biomarkers rather than subjective clinical assessments.","version":"1.1","doi":"10.1101/2025.06.18.660454","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.30.569413","pub_date":"2025-6-19","title":"SARS-CoV-2 NSP13 interacts with TEAD to suppress Hippo-YAP signaling","abstract":"The Hippo pathway controls organ development, homeostasis, and regeneration primarily by modulating YAP/TEAD-mediated gene expression. Although emerging studies report Hippo-YAP dysfunction after viral infection, it is largely unknown in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we analyzed RNA sequencing data from induced pluripotent stem cell\u2013derived cardiomyocytes (iPSC-CMs) and SARS-CoV-2-infected human lung samples, and observed a decrease in YAP target gene expression. In screening SARS-CoV-2 nonstructural proteins, we found that nonstructural protein 13 (NSP13), a conserved coronavirus helicase, inhibits YAP transcriptional activity independent of the upstream Hippo kinases LATS1/2. Consistently, introducing NSP13 into cardiomyocytes suppresses an active form of YAP (YAP5SA) in vivo. Subsequent investigations on NSP13 mutants revealed that NSP13 helicase activity, including DNA binding and unwinding, is crucial for suppressing YAP transactivation. Mechanistically, TEAD4 serves as a platform to recruit NSP13 and YAP. NSP13 likely inactivates the YAP/TEAD4 transcription complex by remodeling chromatin to recruit proteins, such as transcription termination factor 2 (TTF2), to bind the YAP/TEAD/NSP13 complex. These findings reveal a novel YAP/TEAD regulatory mechanism and uncover molecular insights into Hippo-YAP regulation after SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2023.11.30.569413","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.19.624333","pub_date":"2025-6-19","title":"The sequence and structural integrity of the SARS-CoV-2 Spike protein transmembrane domain is crucial for viral entry","abstract":"The Spike (S) protein of SARS-CoV-2 is a type I membrane protein that mediates target cell recognition and membrane fusion. While its transmembrane domain (TMD) is traditionally viewed as a passive anchor to the viral envelope, emerging evidence suggests that TMDs often play active roles in the biogenesis and function of membrane proteins. Here, we investigated the functional role of the SARS-CoV-2 S protein TMD during viral entry. To this end, we introduced a series of amino acid substitutions and insertions within the hydrophobic core of the TMD and assessed their impact on S protein activity. Our findings reveal that the SARS-CoV-2 S protein is susceptible to alterations in its TMD. Functional determinants, including sequence features and structural parameters critical for viral entry, are distributed throughout the TMD, with a more pronounced contribution from its N-terminal region. We also demonstrate that the relative orientation of the regions flanking the TMD influences viral entry. Finally, our data suggest that the TMD mediates homo-oligomerization through a motif enriched in small residues, underscoring its functional importance beyond membrane anchoring.","version":"1.3","doi":"10.1101/2024.11.19.624333","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.17.657579","pub_date":"2025-6-18","title":"Poldip2 deficiency attenuates disease severity in a mouse model of COVID-19","abstract":"The lungs are the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), with the infection resulting in lung inflammation, pulmonary vascular leakage and diffuse alveolar damage. Polymerase delta-interacting protein-2 (Poldip2) mediates lung inflammation and vascular permeability after lipopolysaccharide-induced acute respiratory distress syndrome; however, its role in regulating lung permeability, vascular inflammation and tissue damage following SARS-CoV-2 infection is completely unknown. Here, we assessed the role of Poldip2 in inflammation, immune cell infiltration and lung tissue damage in response to SARS-CoV-2 infection. Our data shows that while deletion of Poldip2 does not affect the susceptibility to SARS- CoV-2 infection, mice heterozygous for Poldip2 exhibit reduced lung tissue damage, reduced cytokine and chemokine induction and decreased infiltration of myeloperoxidase (MPO)-positive neutrophils into inflamed lung tissue. These data reveal that Poldip2 depletion mitigates inflammation and immune cell infiltration following SARS-CoV-2 infection, highlighting the therapeutic potential of Poldip2 inhibition to attenuate severe lung injury.","version":"1.1","doi":"10.1101/2025.06.17.657579","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.18.660355","pub_date":"2025-6-18","title":"A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications","abstract":"T7 RNA polymerase is ubiquitously used in the fields of synthetic biology and biotechnology. Yet the ability to precisely and modularly regulate T7 RNAP remains surprisingly limited. Here, we developed a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables the scalable design of T7 RNAP based gene regulatory networks and enables the rapid and sensitive detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins. By integrating a protein design pipeline, we generated biosensors using fully synthetic binders, demonstrating the potential for rapid development of novel protein-based sensors. We constructed a diagnostic cell-free system combining SARS-CoV-2 Spike protein sensing, gene regulatory based amplification, enzymatic amplification, and glucose based detection demonstrating the potential for point-of-care detection with high sensitivity. This work establishes a flexible and expandable framework for constructing gene circuits responsive to a wide range of biomolecules and demonstrates the potential for engineering point-of-care cell-free diagnostic assays.","version":"1.1","doi":"10.1101/2025.06.18.660355","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.16.659836","pub_date":"2025-6-17","title":"SARS-CoV-2 ORF8 sequence conservation and mutational analysis \u2014 insight into the influence of dataset size on identifying top mutations","abstract":"Given how quickly the SARS-CoV-2 virus mutates, the COVID-19 pandemic has been a major source of concern. The ORF8 accessory protein is one such protein, which is reported to have undergone many mutations. This makes ORF8 an intriguing protein to investigate how these mutations might play a role in overall ORF8 activity. In this study, we have performed conservation and mutational analysis on SARS-CoV- 2 ORF8 protein sequences to identify the conserved and mutated residues. We have also split the ORF8 sequence data into SARS-CoV-2 variant datasets to further identify top mutations across each of them. The mutated and conserved residues were visualised on the available structure of ORF8 to highlight the conserved and mutated sites, which might hold some biological significance. Finally, our study also investigated the significance of sequence dataset size in capturing top mutations following multiple sequence alignments. The COVID-19 pandemic was caused by the SARS-CoV-2 virus, which is known to change over time, i.e., it gets mutated, resulting in the generation of different variants. The ORF8 accessory protein of the SARS-CoV- 2 genome is known to undergo these changes more frequently. In our study, we used SARS-CoV-2 ORF8 protein sequences from various variants to identify mutations among them. Furthermore, we have discovered sites that remain unchanged over time, a phenomenon known as conservation. We think that these unchanged and changed sites could be important for biology and studying them will help in understanding the underlying mechanism of how ORF8 interacts with partner proteins based on existing experimental data. Lastly, we have looked at how much sequence data is sufficient for identifying the top mutated sites.","version":"1.1","doi":"10.1101/2025.06.16.659836","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.16.660018","pub_date":"2025-6-17","title":"Open-science discovery of DNDI-6510, a compound that addresses genotoxic and metabolic liabilities of the COVID Moonshot SARS-CoV-2 Mpro lead inhibitor","abstract":"The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.","version":"1.1","doi":"10.1101/2025.06.16.660018","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.13.659539","pub_date":"2025-6-16","title":"Dual Role of Plasmacytoid Dendritic Cells in Humoral and CD8\u207a T Cell Memory Post COVID-19 mRNA Vaccination","abstract":"The Pfizer-BioNTech coronavirus vaccine (BNT162b2), one of the first nanoparticle-based vaccines approved by the World Health Organisation (WHO), demonstrated 95% efficacy in preventing against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the precise mechanism of action underlying its effectiveness remains poorly understood. This study investigated the early immune responses in the draining lymph node (dLN) and its role in mediating antiviral protection following vaccination. Here, we focused on the involvement of antigen-presenting cells (APCs) in adaptive immunity. In this study, we demonstrated that the Pfizer-BioNTech coronavirus vaccine is rapidly transported to the dLN and is primarily captured by leukocytes that initiate the expression of the viral antigenic spike protein. Notably, we demonstrated that plasmacytoid dendritic cells (pDCs) are key orchestrators of the inflammatory and humoral response, as their specific depletion led to impaired antibody production and diminished neutralization capacity. Furthermore, single-cell transcriptomic analysis revealed an interaction between pDCs and CD8+ T cells that facilitates T cell activation. In vivo experiments confirmed that pDCs expressing the viral spike protein directly engage with CD8+ T cells, promoting their differentiation and expansion. Moreover, the absence of pDCs affected the formation of antigen-specific memory T cells. Overall, these findings highlight that pDCs are essential players in mediating both adaptive and humoral responses to the Pfizer-BioNTech coronavirus vaccine, providing insights into the mechanistic functioning of mRNA vaccines and establishing a novel role for pDCs as professional APCs.","version":"1.1","doi":"10.1101/2025.06.13.659539","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.28.625962","pub_date":"2025-6-13","title":"Generative prediction of real-world prevalent SARS-CoV-2 mutation with in silico virus evolution","abstract":"Predicting the mutation prevalence trends of emerging viruses in the real world is an efficient means to update vaccines or drugs in advance. It is crucial to develop a computational method for the prediction of real-world prevalent SARS-CoV-2 mutations considering the impact of multiple selective pressures within and between hosts. Here, a deep-learning generative framework for real-world prevalent SARS-CoV-2 mutation prediction, named ViralForesight, is developed on top of protein language models and in silico virus evolution. Through the paradigm of host-to-herd in silico virus evolution, ViralForesight reproduced previous real-world prevalent SARS-CoV-2 mutations for multiple lineages with superior performance. More importantly, ViralForesight correctly predicted the future prevalent mutations that dominate the COVID-19 pandemic in the real world more than half a year in advance with in vitro experimental validation. Overall, ViralForesight demonstrates a proactive approach to the prevention of emerging viral infections, accelerating the process of discovering future prevalent mutations with the power of generative deep learning.","version":"1.2","doi":"10.1101/2024.11.28.625962","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.12.659257","pub_date":"2025-6-13","title":"SARS-CoV-2 envelope protein induces LC3 lipidation via the V-ATPase-ATG16L1 axis","abstract":"Coronaviruses encode envelope (E), a structural component of the virion that is important for assembly and egress. E has proton channel activity that prevents premature rearrangement of the spike glycoprotein as virions encounter acidic compartments as they exit the cell. How infected cells respond to this pH disruption during coronavirus infection is unknown. Here we show that SARS-CoV-2 E ion channel activity triggers the proton pump V-ATPases to recruit the ATG16L1 complex during infection. This results in ATG8 molecules such as LC3B decorating perturbed compartments. This recruitment of autophagy machinery does not inhibit viral replication, rather SARS-CoV-2 exploits this response. Inhibition of the V-ATPase/ATG16L1 interaction using the Salmonella effector SopF inhibits SARS-CoV-2 replication. Careful distinction between use of the autophagic machinery from canonical macroautophagy is required in order to better understand coronavirus replication and for rational targeting of any potential host-directed therapies.","version":"1.1","doi":"10.1101/2025.06.12.659257","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.09.658367","pub_date":"2025-6-13","title":"HAVEN: Hierarchical Attention for Viral protEin-based host iNference","abstract":"It is crucial to accurately predict hosts of viruses to understand and anticipate human infectious diseases that originate from animals. There is a lack of versatile models that handle out-of-distribution factors such as unseen hosts and viruses. We develop a machine learning model for predicting the host infected by a virus, given only the sequence of a protein encoded by the genome of that virus. Our approach, HAVEN, is the first to apply to multiple hosts and to generalize to unseen hosts and viruses. HAVEN is a transformer-based architecture coupled with hierarchical self-attention that can accept sequences of highly diverse lengths. We integrate HAVEN with a prototype-based few-shot learning classifier to predict rare classes. We demonstrate the accuracy, robustness, and generalizability of HAVEN through a comprehensive series of experiments. In particular, we show that HAVEN can achieve a median AUPRC of 0.67 while predicting common hosts. Moreover, HAVEN retains this AUPRC value even for rare hosts (median prevalence as low as 0.09%). Our model performs on par with state-of-the-art foundation models, which are 65 to 5, 000 times larger in size, and outperforms them in identifying hosts of SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2025.06.09.658367","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.10.659002","pub_date":"2025-6-12","title":"Garlic-Derived Allicin Modulates Expression of the SARS-CoV-2 Spike Receptor-Binding Domain in Human Splenic Fibroblasts","abstract":"Allicin, a natural sulfur-rich compound, is formed when garlic is crushed or chopped. It has been used for centuries as a natural defence against bacteria and fungi. However, we do not fully understand how well it works against viruses, such as SARS-CoV-2. In this study, we investigated the effect of allicin on the expression of the SARS-CoV-2 receptor-binding domain of the spike protein in human primary splenic fibroblasts. These cells were transfected with a plasmid encoding RBD-S protein fused to a superfolder green fluorescent protein (sfGFP), a bright, stable marker. When cells were treated with 50\u25a1\u00b5M allicin, either 30 min before or 6 h after transfection, a pronounced drop in fluorescence was observed, indicating reduced spike protein expression. Interestingly, the cell morphology, growth, and behavior remained normal, indicating that allicin could minimize spike protein levels without being toxic to the cells. These results open the door to the use of allicin as a gentle, natural antiviral agent and raise critical questions regarding the regulation of protein expression after transcription. Further research is required to understand these effects better.","version":"1.1","doi":"10.1101/2025.06.10.659002","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.10.658990","pub_date":"2025-6-12","title":"Biophysics of the Coronavirus\u2014Membrane Interaction\u2014Role of Nonequilibrium Binding Energy","abstract":"Scientists seemed not to have explored mechanical kinetic energy, a path function for the determination of nonequilibrium binding energy (NEBE). The study explored mechanical kinetic energy, a path function in particles that requires nonequilibrium binding energy (NEBE) to counteract it. It aimed to show that there was a minimally sufficient NEBE to counteract mechanical kinetic energy, using literature data to evaluate derived equations and computations. As is typical, diffusivities increase with temperature; however, near binding, the binding limitations cause the diffusivities (1.519 \u2192 2.784 exp. (\u2212 15) m2/s for D variant; 1.415\u21922.577 exp. (\u2212 15) m2/s for G variant) to be lower than when they are far from binding (4.36 \u21927.99 exp. (\u2212 15) m2/s for D variant; 4.151\u21927.611 exp. (\u2212 15) m2/s for G variant). With breath emission equal to 1.29 exp. (7)/m3, the NEBEs were 656.020 and 663.212 kcal/mol for Delta and Omicron variants of SARS-CoV-2, respectively; and with 27.9 exp. (7)/m3, the corresponding values were 568.921 and 633 kcal/mol; with a breath emission rate equal to 9.31 exp. (+6)/hr., the NEBEs for the Omicron variant in 1 hr. and in 1 min. were 651.703 and 683.353 kcal/mol, respectively; with 201 exp. (+6)/hr., the corresponding values were 444.157 and 663.212 kcal/mol. The G variant of SARS-CoV-2 showed higher NEBEs (952\u2192671 kcal/mol) than D variants (834\u2192588 kcal/mol), corresponding to 293.15\u2192318.15 K. The decreasing trend in maximum NEBE  with rising temperature implies that the binding affinity of the virus may be attenuated at higher temperatures. It is, therefore, medically plausible to administer airborne, parenteral, oral, etc., drugs at temperatures above body temperatures that are tolerable and in a controlled fashion. Future studies should be directed to a definite determination of the size and molar mass of variants of SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.06.10.658990","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.11.658579","pub_date":"2025-6-12","title":"Reducing Supply Chain Dependencies for Viral Genomic Surveillance: Get by with a Little HELP from Commercial Enzymes already in your Lab Freezer","abstract":"The COVID-19 pandemic exposed vulnerabilities in global laboratory supply chains, disrupting genomic surveillance efforts essential to epidemic response. To address this challenge, we developed ARTIC HELP (Homebrew Enzymes for Library Preparation), a practical, open-source adaptation of the widely adopted ARTIC nanopore sequencing protocol for viral genomic surveillance. We describe generic, cost-effective alternatives to all enzyme mixes used in tiling multiplex RT-PCR amplification of the virus genome, and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL), making it broadly applicable to any laboratory. Through systematic evaluation, we identified a wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases as effective alternatives when standard reagents are unavailable due to high cost or limited supply: B-family Pfu-based polymerases with a fused Sso7d DNA-binding domain, and blends combining A-family (Taq-based) and B-family (Pfu-based) polymerases. Validation on clinical samples of SARS-CoV-2 and Norovirus GII confirmed that the HELP workflow achieves genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct \u226428), the wild-type M-MLV RT combined with selected Pfu or A+B polymerases, along with optimised HELP mixes (EP, BL, AL), achieved genome coverage of 84.0\u201399.6%. For Norovirus GII (Ct \u226432), the HELP workflow using one of the Pfu polymerases achieved genome coverage of >85% for six out of eight genotypes tested. Notably, several of the other polymerases tested showed reduced performance at higher Ct values. However, they still achieved strong coverage at Ct <24, supporting their use as emergency alternatives in rapid outbreak-response sequencing when viral input is high and RNA quality is sufficient. Our approach, ARTIC HELP, provides a framework which can be implemented to address supply chain disruptions, while maintaining robust genomic sequencing capabilities. A cost analysis highlights the well-known significant global disparities in reagent pricing, driven not by protocol differences but by import fees and supply barriers. Thus, our findings highlight the need for fairer global pricing models and support for local sourcing strategies like HELP, to promote equity in genomic research and ensure preparedness for future public health challenges.","version":"1.1","doi":"10.1101/2025.06.11.658579","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.10.658224","pub_date":"2025-6-11","title":"American mink as an animal model to study SARS-CoV-2 and vaccine response","abstract":"Selecting a suitable animal model is crucial in understanding infectious diseases and developing vaccines. Here, we developed a receptor-binding domain -based SARS-CoV-2 vaccine with mouse Fc an immunopotentiator in a mink model. Four different variations of the vaccine were tested in groups of 30-31 American mink and followed for IgG and neutralizing antibodies (nAb) up to 27 weeks. Subcutaneous version induced a strong IgG and nAb response within two weeks and was still detectable at 27 weeks. Intranasal version also caused a detectable, although weaker, immune response. A simultaneously given subcutaneous vaccine against virus enteritis, botulism and hemorrhagic pneumonia potentially caused a lower SARS-CoV-2 antibody response, highlighting the need for further studies on co-effects of vaccines. In virus challenge with Alpha variant (B.1.1.7), vaccinated mink had a stronger antibody response than unvaccinated mink. Despite not preventing the infection, vaccinated mink had milder clinical signs and less virus in saliva. Another challenge of unvaccinated mink with Omicron variant showed similar results to alpha (BA.1) variant. Virus RNA was detected in the brain of unvaccinated mink but not vaccinated mink by in situ hybridization, indicating a suitability of mink to study neurological effects of SARS-CoV-2 and potentially long COVID as well. Finding a good animal model is very important in studying infectious diseases and their treatment. The recent SARS-CoV2 pandemic highlighted this dilemma. We have developed an animal model based on mink due to their close match to humans in the symptomology of this disease. Such model will enable the study of relative susceptibility, transmission, tissue tropism, complex pathogenesis and long COVID, as well as prophylaxis and vaccines. This model will also help reduce the use of primates in this research. We have further developed a new vaccine for SARS-CoV2 based on the receptor binding domain of the S-protein with an inbuilt immune-enhancer. This vaccine underwent extensive testing in mink to determine response, long term protection, and safety. The vaccine was found to provide excellent titers of neutralizing antibodies with wide range in target variants. It also reduced the severity and duration of visible symptoms in the animals significantly. We propose this vaccine candidate for further study and future commercialization.","version":"1.1","doi":"10.1101/2025.06.10.658224","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.09.658648","pub_date":"2025-6-11","title":"SARS-CoV-2-induced dysregulation in ADAR editing patterns persists post viral clearance in individuals with mild COVID-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections remain a public health concern worldwide. Viral antigen triggered innate immune response leads to induction of interferons (IFNs) and interferon stimulated genes (ISGs) including ADAR1 p150 isoform, which edits adenosine (A) residues within double stranded RNAs in both the virus and the host. Such RNA editing mediated by ADARs plays a crucial role in innate immune responses during viral infections through modulation of host-virus interactions. Additionally, ADAR editing acts post-transcriptionally, and serves as a mechanism of dynamic regulation of transcriptome and proteome diversity. While evidence points to changes in ADAR editing during infection, we do not know whether editing targets change over the course of the infection. Here, we explored temporal changes in ADAR expression and editing patterns, across three distinct stages of SARS-CoV-2 infection. Furthermore, we examined whether infection-triggered dysregulation in ADAR editing persists or returns to pre-infection states post-viral clearance. We addressed this question by analyzing publicly available whole blood RNA sequencing samples from forty-five, age-matched individuals. The individuals selected had no documented comorbidities, developed mild COVID-19, and were sampled across three distinct stages of SARS-CoV-2 infection: pre-, mid-, and post-infection. Our results demonstrate dynamic changes in ADAR expression and editing across the three stages. We further identified unique editing sites resulting from SARS-CoV-2 infection, across all three stages of infection, within genes involved in immune response pathways. Noteworthy, genes within neutrophil degranulation pathway appear to be edited, suggesting they may play a role in inflammation and sequelae observed post-SARS-CoV-2 infection. Our results demonstrate a consistent trend of elevated ADAR expression and reduced overall ADAR editing within each individual mid-infection. Subsequently, in some post-infection samples ADAR expression returns to approximately pre-infection levels, while in others it remains dysregulated. These differences may be contributing to heterogeneity in disease outcomes seen in individuals post-SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2025.06.09.658648","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.04.626800","pub_date":"2025-6-10","title":"Optimizing a Human Monoclonal Antibody for Better Neutralization of SARS-CoV-2","abstract":"SARS-CoV-2 has largely evolved to resist antibody pressure, with each successive viral variant becoming more and more resistant to serum antibodies in the population. This evolution renders all previously authorized anti-spike therapeutic monoclonal antibodies inactive, and it threatens the remaining pipelines against COVID-19. We report herein the isolation of a human monoclonal antibody with a broad but incomplete SARS-CoV-2 neutralization profile, but structural analyses and mutational scanning lead to the engineering of variants that result in greater antibody flexibility while binding to the viral spike. Three such optimized monoclonal antibodies neutralize all SARS-CoV-2 strains tested with much improved potency and breadth, including against subvariants XEC and LP.8.1. The findings of this study not only present antibody candidates for clinical development against COVID-19, but also introduce a unique engineering approach to improve antibody activity via increasing conformational flexibility.","version":"1.2","doi":"10.1101/2024.12.04.626800","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.17.618867","pub_date":"2025-6-10","title":"Phosphorylation Toggles the SARS-CoV-2 Nucleocapsid Protein Between Two Membrane-Associated Condensate States","abstract":"The Nucleocapsid protein (N) of SARS-CoV-2 plays a critical role in the viral lifecycle by regulating RNA replication and packaging the viral genome. N and RNA phase separate to form condensates that may be important for these functions. Both functions occur at membrane surfaces, but how N toggles between these two membrane-associated functional states is unclear. Here, we reveal that phosphorylation switches how N condensates interact with membranes, partly by modulating condensate material properties. Phosphorylated N forms liquid condensates that wet membranes, reminiscent of N\u2019s role in RNA replication. In contrast, unmodified N forms viscoelastic condensates that can be engulfed by membranes, evoking viral genome packaging. These results suggest that phosphorylation serves as a regulatory switch within the viral replication cycle by modulating N\u2019s association with membranes. We gained mechanistic insight through structural analysis and molecular simulations, which suggest phosphorylation induces a conformational change that softens condensate material properties. Our studies also show that phosphorylation alters N\u2019s interaction with viral membrane proteins. Together, our findings uncover a novel aspect of SARS-CoV-2 biology by identifying membrane association as a key feature of N condensates and providing mechanistic insights into the regulatory role of phosphorylation. Understanding this mechanism suggests potential therapeutic targets for COVID infection.","version":"1.2","doi":"10.1101/2024.10.17.618867","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.22.655348","pub_date":"2025-6-09","title":"Testing for a wildlife reservoir of divergent SARS-CoV-2 in white-tailed deer","abstract":"The 2021 discovery of a divergent lineage (B.1.641) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer (Odocoileus virginianus) from Ontario raised concerns that deer were a potential reservoir. To assess whether white-tailed deer continued to be infected with B.1.641 and to test for spillover into other species, we established a surveillance program in Ontario by sampling wildlife via existing monitoring programs and through active surveillance of captive and wild animals. Between 2022 to 2024, we tested 2,839 animals, identifying one active SARS-CoV-2 infection (a likely spillover of a recombinant XBB.2.3.11.3 lineage), but no cases of B.1.641. Overall, 93 animals (6.8%) tested positive for SARS-CoV-2 antibodies, including 89 white-tailed deer, two Virginia opossums (Didelphis virginiana), one American mink (Neogale vison), and one river otter (Lontra canadensis). In Southwestern Ontario, where B.1.641 was originally detected, 15.2% of deer samples were seropositive. Generalized Linear Models demonstrated that seropositive deer were more likely to be found in areas with a higher fall deer harvest and human population density, and closer to previous B.1.641 cases. Our data suggest that deer-associated B.1.641 may have caused a relatively localized epizootic without forming a stable reservoir. This study underscores the importance of One Health-focused surveillance.","version":"1.2","doi":"10.1101/2025.05.22.655348","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.08.658496","pub_date":"2025-6-09","title":"SARS-CoV-2 reshapes m\u2076A methylation in long non-coding RNAs of human lung cells","abstract":"N\u2076-Methyladenosine (m\u2076A) is a key base modification that regulates RNA stability and translation during viral infection. While m\u2076A methylation of host mRNAs has been studied in SARS-CoV-2-infected cells, its role in long non-coding RNAs (lncRNAs) is unknown. Here, we analyzed direct RNA sequencing (dRNA-seq) data from infected human lung cells (Calu-3) using a machine learning m\u2076A detection framework. We observed a global increase in m\u2076A levels across ten antiviral response\u2013 associated lncRNAs, with UCA1, GAS5, and NORAD\u2014regulators of interferon (IFN) signaling\u2014 showing the most pronounced changes. This might, in part, explain the attenuated IFN expression observed in infected cells. We identified methylated DRACH motifs in predicted lncRNA duplex-forming regions, which may favor Hoogsteen base-pairing, which destabilize secondary structures and target interaction sites. These results provide new perspectives on how SARS-CoV-2 could impact lncRNAs to modulate host immunity and viral persistence through m\u2076A-dependent mechanisms. Peter et al. show that SARS-CoV-2 infection alters m\u2076A methylation of host lncRNAs by analysis of direct RNA sequencing data with machine learning. Key immune-regulatory lncRNAs show m\u2076A alterations in RNA pairing regions, suggesting a novel mechanism by which m\u2076A may modulate antiviral responses and promote viral persistence. Direct RNA-seq and machine learning reveal m\u2076A changes in lncRNAs SARS-CoV-2 infection changes m\u2076A patterns in antiviral response-associated lncRNAs LncRNAs UCA1, GAS5, and NORAD exhibit pronounced m\u2076A remodeling upon infection m\u2076A sites overlap RNA duplex-forming regions in immune-regulatory lncRNAs m\u2076A may impact lncRNA duplexes via Hoogsteen pairing","version":"1.1","doi":"10.1101/2025.06.08.658496","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.07.658468","pub_date":"2025-6-09","title":"Conformational Landscaping and Dynamic Mutational Profiling of Binding Interactions and Immune Escape for Broadly Neutralizing Class I Antibodies with SARS-CoV-2 Spike Protein: Distributed Binding Hotspot Networks Underlie Mechanism of Viral Resistance Against Existing Variants","abstract":"The rapid evolution of SARS-CoV-2 has underscored the need for a detailed understanding of antibody binding mechanisms to combat immune evasion by emerging variants. In this study, we investigated the interactions between Class I neutralizing antibodies BD55-1205, BD-604, OMI-42, P5S-1H1, and P5S-2B10 and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using multiscale modeling which combined coarse-grained simulations and atomistic reconstruction of conformational landscapes together with mutational scanning of the binding interfaces, dynamic profiling of binding and immune escape using molecular mechanics generalized Born surface area (MM-GBSA) analysis. A central theme emerging from this work is the critical role of epitope breadth and interaction diversity in determining an antibody resilience to mutations. BD55-1205 antibody exemplifies the advantages of broad epitope coverage and distributed hotspot mechanisms. By engaging an extensive network of residues across the RBD, BD55-1205 minimizes its dependence on individual side-chain conformations, allowing it to maintain robust binding even when key residues are mutated. This adaptability is particularly evident in its tolerance to mutations at positions such as L455 and F456, which severely compromise other antibodies. The ability of BD55-1205 to sustain cumulative interactions underscores the importance of targeting diverse epitopes through multiple interaction mechanisms, a strategy that enhances resistance to immune evasion while maintaining functional integrity. In contrast, BD-604 and OMI-42, with localized binding mechanisms, are more vulnerable to escape mutations at critical positions such as L455, F456, and A475. P5S-1H1 and P5S-2B10 exhibit intermediate behavior, balancing specificity and adaptability but lacking the robustness of BD55-1205. Mutational scanning identified key residues Y421, Y489, and F456 as critical hotspots for RBD stability and antibody binding, highlighting their dual role in viral fitness and immune evasion. The computational predictions generated through mutational scanning and MM-GBSA analysis demonstrate excellent agreement with experimental data on average antibody escape scores. This study underscores the diversity of binding mechanisms employed by different antibodies and molecular basis for high affinity and excellent neutralization activity of the latest generation of antibodies.","version":"1.1","doi":"10.1101/2025.06.07.658468","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.05.658000","pub_date":"2025-6-09","title":"A high-throughput immunopeptidome platform for MHC II alleles to characterize antigen-specific CD4+ T cells","abstract":"CD4+ T cells play a pivotal role in adaptive immunity, recognizing peptide antigens presented by MHC II molecules during infections and tumor development. Identifying immunodominant MHC II epitopess is essential for understanding CD4+ T cell responses; however, current methods such as mass spectrometry, suffer from low sensitivity and throughput, while computational algorithms show variable accuracy. To overcome these challenges, we developed EliteMHCII, a high-throughput immunopeptidome profiling platform that identifies antigen-derived MHC II epitopes and measures peptide binding affinity across 24 globally common MHC II alleles. Using EliteMHCII, we assessed the immunodominant epitopes of the SARS-CoV-2 RBD protein. Validation in vaccinated individuals and humanized mouse models revealed a strong correlation between high-affinity peptides and robust CD4+ T cell responses, while low-affinity peptides failed to elicit responses. Therefore, our immunopeptidome profiling platform, EliteMHCII, serves as a rapid, high throughput, feasible platform for CD4+ T cell epitope discovery at a global populational level in the context of infectious diseases and cancer immunotherapy.","version":"1.1","doi":"10.1101/2025.06.05.658000","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.04.657798","pub_date":"2025-6-08","title":"Designing a broad-spectrum four-helix bundle targeting different strains of SARS-CoV-2 Spike Receptor Binding Domains with ACE2-like binding interface","abstract":"One major strategy for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to evade antibody drugs or preventive vaccines is high mutation rate of the spike receptor binding domain (RBD). Because variable RBDs of different SARS-CoV-2 strains must bind to the same human receptor angiotensin-converting enzyme 2 (hACE2) for viral cell entry and infection, we hypothesize that designing a protein with the same or very similar hACE2 binding interface might have a broad-spectrum effect against various SARS-CoV-2 strains. The designed protein binds specifically to the WT-RBD (with micromolar affinity) but not to RBDs from other SARS-CoV-2 strains. However, two rounds of the E. coli display and Magnetic Cell Sorting (MACS) selection are sufficient to yield a protein named CYN1 with nanomolar binding affinities not only to the WT-RBD but also to those of Omicron BA.1, XBB.1.16, and JN.1. Molecular dynamics simulations and free-energy hotspot analysis revealed that CYN1\u2019s broader spectrum capability stems from its engagement of essentially all ACE2 hotspot residues critical for WT-RBD binding, unlike the designed protein. The discovery of CYN1, differing by only four mutations from the designed protein, confirms that targeting the small interface of human viral receptors\u2014rather than the entire receptor\u2014offers a viable strategy for developing broad-spectrum inhibitors. This approach minimizes potential off-target effects arising from receptor multifunctionality.","version":"1.2","doi":"10.1101/2025.06.04.657798","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.20.594961","pub_date":"2025-6-08","title":"Plasmacytoid dendritic cells are dispensable or detrimental in murine systemic or respiratory viral infections","abstract":"Plasmacytoid dendritic cells (pDCs) are major producers of type I/III interferons. As interferons are crucial for antiviral defense, pDCs are assumed to play an essential role in this process. However, robust evidence supporting this dogma is scarce. Genetic or pharmacological manipulations that eliminate pDC or disrupt their interferon production often affect other cells, confounding interpretation. To overcome this issue, we engineered pDC-less mice that are specifically and constitutively devoid of pDCs by expressing diphtheria toxin under coordinated control of the Siglech and Pacsin1 genes, uniquely co-expressed in pDCs. pDC-less mice mounted protective immunity against systemic infection with mouse Cytomegalovirus and showed higher survival and less lung immunopathology to intranasal infection with influenza virus and SARS-CoV2. Thus, contrary to the prevailing dogma, we revealed that pDCs and their interferons are dispensable or deleterious during several viral infections. pDC-less mice will enable rigorously reassessing the roles of pDCs in health and disease.","version":"1.2","doi":"10.1101/2024.05.20.594961","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.05.658001","pub_date":"2025-6-06","title":"Rationally Designed, ACE2 Mimetic Binder to the SARS Cov-2 Associated Spike Protein for COVID-19 Therapeutics and Beyond","abstract":"We have developed a 23 residue, optimized helical peptide biomimetic from the SARS-CoV-2 Spike protein binding partner ACE2 that demonstrated a Therapeutic Index (TI) of >\u223c20 in authentic viral cell challenge assays, including WT and Omicron strains. The therapeutic is an optimized \u201cpeptide decoy\u201d based on the virus\u2019s human cell target ACE2 and, as such, may have more general applicability across coronavirus family members that use ACE2 for cellular entry. We experimentally verify a comprehensive, rational optimization strategy of the peptide through improved binding, helical content, and solubility from its native sequence. These techniques may have general applicability for helical peptide optimization for other therapeutic targets as well. Importantly, techniques also exist for protecting helical peptides in vivo for improved delivery. Peptides are readily modifiable with single residue substitutions for quick response to mutated targets, and they typically have relatively low toxicity and ease of manufacturing, making peptides extremely attractive as biological therapeutics against viral pathogens. The general concept of using peptide decoys across other viral human cell targets is also discussed. Although the COVID-19 pandemic has ended, SARS-CoV-2 virus still causes hundreds of deaths each week world-wide. The virus uses a human cell receptor called ACE2 to latch on and enter cells. Here, the small critical attachment segment of ACE2 is developed as a \u201cdecoy\u201d molecule thereby protecting host cells and significantly reducing viral replication, which is the role of a therapeutic agent. The effective segment in this case is called a helical peptide and it was optimized for better binding, solubility, and stability using rational methods based on our understanding of this class of molecule. Moreover, future viruses from this family of coronaviruses may likely use ACE2 as their host cell receptor, as recently demonstrated in the Middle Eastern Respiratory Syndrome Virus of bats and, therefore, the ACE2 decoy therapeutic may have future applications as well. Development of a broader range of decoy molecules for different viruses using different human cell receptors may represent a forward-thinking approach to prepare for future pandemics and outbreaks.","version":"1.1","doi":"10.1101/2025.06.05.658001","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.02.657400","pub_date":"2025-6-03","title":"The Role of SARS-CoV-2 Nucleocapsid Protein Persistence in Inducing Chronic Type I Interferon and Mitochondrial Dysfunction","abstract":"The potential mechanisms that link SARS-CoV-2 infection to post-acute sequelae of SARS-CoV-2 symptoms and ultimately transition to new onset of autoimmune disease remain poorly understood. Here, we report the consequences of SARS-CoV-2 nucleocapsid (N) protein persistence in the absence of detectable SARS-CoV-2 replication in otherwise healthy individuals, a set of systemic lupus erythematosus (SLE) patients, and a case of a 60-year-old man who developed a new onset of lupus nephritis seven months following mild COVID-19 disease. We have identified that N protein persistence in peripheral blood mononuclear cells (PBMC) did not correlate with detectable SARS-CoV-2 RNA in blood but is associated with significantly increased secretion of type I interferons (IFN) and presence of tubuloreticular structures in peripheral leukocytes. We further demonstrate that the N protein colocalizes in the mitochondrial fraction of PBMCs from individuals positive for N protein alongside mitochondrial antiviral signaling protein (MAVS). In vitro expression of the N protein in a monocytic cell line showed that the N protein itself was directly capable of interacting with MAVS in the absence of viral RNA, and this interaction was enhanced if the cell was exposed to oxidative stress. We show that the type I IFN signature in the presence of N protein expression was MAVS, but not STING signaling pathway-dependent. Our findings suggest a mechanism for the onset and promotion of type I IFN signature after COVID-19; in our model, the SARS-CoV-2 N protein can independently trigger sustained type I IFN production via direct activation of MAVS and its spontaneous oligomerization. This long-lasting type I IFN generation may create a chronic inflammatory milieu, favoring autoimmunity with SLE-like symptoms in susceptible individuals. The persistence of SARS-CoV-2 nucleocapsid in the absence of viral replication is associated with an induction of mitochondria-mediated inflammatory milieu, which can favor autoimmunity in susceptible individuals.","version":"1.1","doi":"10.1101/2025.06.02.657400","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.02.657371","pub_date":"2025-6-03","title":"Multi-Omic Profiling Identifies Conserved Metabolic Pathways Critical for SARS-CoV-2 Variants Infection","abstract":"The rapid evolution of SARS-CoV-2 has led to the emergence of numerous variants with enhanced transmissibility and immune evasion. Despite widespread vaccination, infections persist, and the mechanisms by which SARS-CoV-2 reprograms host metabolism remain incompletely understood. Here, we investigated whether virus-induced lipid remodeling is conserved across variants and whether changes in lipid abundance correlate with alterations in lipid biosynthetic enzymes. Using global untargeted lipidomics and quantitative proteomics, we analyzed A549-ACE2 cells infected with the Delta (B.1.617.2) or Omicron (B.1.1.529) variants and compared them to cells infected with the ancestral WA1 strain. In parallel, we conducted quantitative proteomics to assess virus-induced changes in the host proteome. Our results reveal that SARS-CoV-2 drives a remarkably consistent pattern of metabolic rewiring at both the lipidomic and proteomic levels across all three variants. We mapped changes in the expression of host metabolic enzymes and compared these to corresponding shifts in lipid abundance. This integrative analysis identified key host proteins involved in virus-mediated lipid remodeling, including fatty acid synthase (FASN), lysosomal acid lipase (LIPA), and ORM1-like protein 2 (ORMDL2). Together, these findings highlight conserved metabolic dependencies of SARS-CoV-2 variants and underscore host lipid metabolism as a potential target for broad-spectrum antiviral strategies.","version":"1.1","doi":"10.1101/2025.06.02.657371","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.30.657095","pub_date":"2025-6-02","title":"Diverse tendencies in codon usage evolution of SARS-CoV-2 genes","abstract":"The dynamic evolution of SARS-CoV-2 virus since the COVID-19 outbreak in late 2019 has raised questions about potential evolutionary trends in protein-coding sequences and their adaptation to the human host. To address this, we compiled a dataset of 94,571 complete genomes with known collection dates, spanning from January 2020 to October 2024. Using a novel representation of codon usage, we recoded SARS-CoV-2 protein-coding sequences to strings of labels reflecting the human synonymous codon usage. Our analysis reveals different evolutionary pathways in the codon usage between structural, non-structural and accessory protein-coding sequences from the coronavirus. The genes coding for structural proteins tend to exhibit a less optimal adaptation to the human codon usage, whereas open reading frames ORF1a and ORF1ab encoding non-structural proteins show an opposite trend. The sequences for the accessory proteins demonstrated a variable tendency to change the codon preferences. The evolution of the more optimal codon usage in ORF1a and ORF1ab sequences can be associated with a higher speed and efficiency of translation of the coded polyproteins. Following their cleavage, the products play important roles in viral replication and transcription. Thus, the adaptation of their codons can increase the virus\u2019 proliferation. In contrast, alterations in codon usage within structural protein-coding sequences may be associated with changes in their less accurate translation and folding during the synthesis, which can provide an advantage in evading the host immune response. The results show that codon usage adaptations to the human host differ based on the gene type and function, reflecting a balance between their conflicting evolutionary pressures. Our findings on variations in codon usage among coronavirus genes provide valuable insights that can aid in developing new strategies for the optimization of codons in vaccine mRNA and DNA for emerging strains.","version":"1.1","doi":"10.1101/2025.05.30.657095","journal":"bioRxiv","score":null},{"id":"10.1101/2025.06.02.657353","pub_date":"2025-6-02","title":"Engineering a mouse-adapted SADS-CoV and establishing a neonatal mouse model to study its infection","abstract":"Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging bat-origin alphacoronavirus causing severe disease in neonatal piglets, with significant economic losses to the swine industry as a consequence. The virus exhibits a broad species tropism, infecting cells derived from pigs, humans and mice, highlighting its potential for cross-species transmission. Due to drawbacks associated with the use of young piglets, there is a need for an appropriate small animal model to study SADS-CoV biology. Here we established a mouse infection model based on a murinized mutant of the virus, mSADS-CoV, in which the ectodomain of the viral spike protein was replaced by that of the murine coronavirus mouse hepatitis virus (MHV). This chimeric virus, generated through targeted RNA recombination, replicated efficiently in murine cell cultures and exhibited an age-dependent infection in neonatal mice that was lethal in 2-day-old BALB/c mice affecting various organs, notably the intestine. We validated our infection model by successfully verifying the efficacy of the RNA-dependent RNA polymerase inhibitor remdesivir (RDV). The model will serve as a valuable tool for studying SADS-CoV pathogenesis and for elucidating the roles of host factors in viral replication as well as for preclinical evaluation of vaccine candidates and antiviral compounds targeting the viral replication machinery. SADS-CoV poses a dual threat to the swine industry and public health because of its broad species tropism and potential for cross-species transmission. The emergence of other bat-derived coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV), underscores the need for robust models to study these pathogens. The successful rescue of mSADS-CoV and the development of a mouse infection model represent significant advancements in SADS-CoV research. This model not only enables the evaluation of antiviral therapeutics such as RDV but also provides a powerful platform for investigating viral replication mechanisms and host-pathogen interactions, offering critical insights for pandemic preparedness.","version":"1.1","doi":"10.1101/2025.06.02.657353","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.28.656516","pub_date":"2025-5-30","title":"A non-spike nucleocapsid R204P mutation in SARS-CoV-2 Omicron XEC enhances inflammation and pathogenicity","abstract":"The global circulation of SARS-CoV-2 in human populations has driven the emergence of Omicron subvariants, which have become highly diversified through recombination. In late 2024, SARS-CoV-2 Omicron XEC variant emerged from the recombination of two JN.1 progeny, KS.1.1 and KP.3.3, and became predominant worldwide. Here, we investigated virological features of the XEC variant. Epidemic dynamics modeling suggested that spike substitutions in XEC mainly contribute to its increased viral fitness. Additionally, four licensed antivirals were effective against XEC. Although the fusogenicity of XEC spike is comparable to that of the JN.1 spike, the intrinsic pathogenicity of XEC in hamsters was significantly higher than that of JN.1. Notably, we found that the nucleocapsid R204P mutation of XEC enhanced inflammation through NF-\u03baB activation. Recent studies suggest that the evolutionary potential of spike protein is reaching its limit. Indeed, our findings highlight the critical role of non-spike mutations in the future evolution of SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.05.28.656516","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.13.584850","pub_date":"2025-5-30","title":"Engineered Migrasomes: Harnessing Core Migrasome Machinery and Hypotonic Shock to Develop a Robust and Thermally Stable Vaccine Platform","abstract":"The increasing ability of pathogens and tumor cells to evade immune detection underscores the urgent need for novel vaccine platforms leveraging diverse biological mechanisms. Additionally, logistical challenges associated with cold-chain transportation significantly limit vaccine accessibility, especially in resource-limited regions. Recently, we identified migrasomes, specialized organelles generated during cell migration, which are inherently stable and enriched with immune-modulating molecules. To address the low yield of natural migrasomes, we engineered migrasome-like vesicles (eMigrasomes) using hypotonic shock combined with cytoskeletal disruption to enhance vesicle formation. The biogenesis of eMigrasomes relies on the core migrasome machinery, faithfully recapitulating the biophysical attributes of native migrasomes while significantly improving production efficiency. We demonstrate that eMigrasomes loaded with a model antigen elicit potent antibody responses and maintain structural integrity and immunogenic potential at room temperature. Furthermore, eMigrasomes displaying the SARS-CoV-2 Spike protein induce robust humoral immune responses, providing effective protection against viral infection. Our findings highlight the potential of utilizing migrasome biology and hypotonic shock-driven vesicle generation as an innovative, stable, and broadly accessible vaccine platform.","version":"1.2","doi":"10.1101/2024.03.13.584850","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.28.656328","pub_date":"2025-5-29","title":"Predictive Modeling of Immune Escape and Antigenic Grouping of SARS-CoV-2 Variants","abstract":"The ongoing adaptive evolution of SARS-CoV-2 is characterized by the continued emergence of novel variants that escape from previously acquired infection- and/or vaccination-derived immunity. This continued SARS-CoV-2 variant evolution has necessitated annual vaccine updates to better match circulating viral variants. To optimize protection against emerging variants of interest and concern, a reliable means of predicting the immune escape of novel variants is needed to enable at-risk preparation of new vaccines. Herein, we describe the development and applications of a risk calculator that uses statistical modeling to predict the immune escape of emerging variants. The calculator utilizes previously published spike-antibody epitope and escape profiles and in vitro neutralization assessment of a large panel of pseudotyped SARS-CoV-2 variants evaluated against clinical sera. The calculator enables the grouping of antigenically related SARS-CoV-2 variants to guide strain selection for at-risk vaccine design and preparation, in anticipation of potential future requests by the global public health agencies. Here, we demonstrated the strain selection exercises for the XBB.1.5- and JN.1/KP.2-adapted mRNA-1273 COVID-19 vaccines in the 2023-2024 and 2024-2025 seasons, respectively, which were supported by both the risk calculator and preclinical and clinical immunogenicity data and were later recommended by the global public health agencies.","version":"1.1","doi":"10.1101/2025.05.28.656328","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.28.656620","pub_date":"2025-5-29","title":"Tracing SARS-CoV-2 Evolution in Algeria: Insights from 2020\u20132023","abstract":"SARS-CoV-2 continues to circulate globally, leading to the emergence of new viral sublineages. This study examined 449 full-length genomic sequences from Algeria (March 2020 to May 2023) to explore SARS-CoV-2 evolution in this region. Our analysis revealed multiple introductions of viral strains, which were marked by significant genomic changes over time. Algeria-specific mutations were identified, providing insights into regional adaptations and evolutionary dynamics. Furthermore, a statistically significant recombination event was detected using the RDP 5 software. EPI_ISL_15920753 was recombinant, whereas EPI_ISL_15790700 and EPI_ISL_12156732 constituted the major and minor parents, respectively. Notably, haplotype analysis revealed 179 distinct haplotypes, including a highly connected node (H91) associated with a superspreading event later in the pandemic (July\u2013September 2022), suggesting rapid transmission likely driven by mobile individuals and large gatherings. These findings emphasize the ongoing evolution of SARS-CoV-2 in local settings and highlight the importance of genomic surveillance for tracking emerging mutations and their potential impact. Bridging a critical gap in North African genomic surveillance, this study presents the first comprehensive evolutionary analysis of SARS-CoV-2 in Algeria using a significantly larger dataset of 449 sequences, a substantial increase from the only previous study of 29 genomes conducted at the onset of the pandemic. Our results provide essential insights into how the virus was introduced, disseminated, and adapted within the country, thereby guiding regional public health initiatives and supplying important data for global comprehension of the evolution of SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.05.28.656620","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.18.629144","pub_date":"2025-5-29","title":"Integrated analysis of COVID-19 multi-omics data for eQTLs reveals genetic mechanisms underlying disease severity","abstract":"The global pandemic caused by the SARS-CoV-2 virus provided an unprecedented opportunity to investigate genetic factors influencing the disease severity of the viral infection. Despite a plethora of recent research on both SARS-CoV-2 and COVID-19, few have taken a systems biology approach to address individual-level variation, especially based on non-European populations. Accordingly, we analyzed multi-omics data generated at three timepoints from 193 Korean COVID-19 patients with mild or severe symptoms, composed of whole genome sequencing, blood-based single-cell RNA-sequencing (2.15M cells), 195 cytokine profiles, and human leukocyte antigen (HLA) allele data. We identified expression quantitative trait loci (eQTLs), disease severity interacting eQTLs (n = 388), and disease progression interacting eQTLs (n = 945) for various cell types. We elucidated a complex regulatory mechanism involving HLA genes and their targets, and identified genetic determinants of cytokine levels. Finally, we show how regulation of ieQTLs is established by upstream transcription factors (TFs), illustrating complex regulation of the IGFBP7 ieQTL by a combined action of two TFs, which is potentially important in conferring differential severity. This study illuminates an efficient molecular interrogation framework that can be applied toward understanding infectious disease progression in individuals of different genotypes.","version":"1.2","doi":"10.1101/2024.12.18.629144","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.28.656401","pub_date":"2025-5-29","title":"Immunological evaluation in African Green Monkeys of two nasal vaccine candidates to induce a broad immunity against coronaviruses","abstract":"The COVID-19 pandemic has had a profound impact on the world. While the disease is currently under control, the emergence of new coronavirus variants with pandemic potential underscores the need for more universal coronavirus vaccines. This study evaluates the immunogenicity in non-human primates of two broad-spectrum nasal vaccine candidates called PanCoV. The vaccine preparations are based on highly conserved regions of SARS-CoV-2, specifically the nucleocapsid (N) and the S2 subunit from Spike protein, which include several relevant T-cell epitopes and have the potential to form virus-like particles. PanCoV1 candidate contains the N protein, while the PanCoV2 comprises a chimeric protein containing the C-terminal domain of N protein fused to a fragment of S2 subunit. Both vaccine candidates also include the receptor-binding domain (RBD) from Spike protein and the ODN-39M as CpG adjuvant. The results demonstrate that both nasal vaccine candidates can boost the anti-RBD immune response and induce anti-N immunity, this systemic response shows cross-reactivity with SARS-CoV, MERS-CoV, and H-CoV antigens. In addition, a neutralizing response against SARS-CoV-2 that persisted for almost three months after the last dose was induced. A mucosal-specific and cross-reactive IgA response was also observed, mainly in monkeys vaccinated with PanCoV2. Furthermore, the specific IFN\u03b3-secreting response observed in the vaccinated groups peripheral blood mononuclear cells highlights the ability of these candidates to stimulate cellular immunity with a Th1 bias. PanCoV vaccines emerge as promising candidates for use as a booster alternative, with the potential to amplify and broaden the scope of the immune response against coronaviruses.","version":"1.1","doi":"10.1101/2025.05.28.656401","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.26.656146","pub_date":"2025-5-28","title":"Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites","abstract":"The global impact of the COVID-19 pandemic underscores the critical need for a comprehensive understanding of SARS-CoV-2 replication mechanisms. While the central roles of the RNA dependent RNA polymerase (NSP12), primase protein (NSP8), and nucleocapsid protein (N) in the virus life cycle are extensively studied, the precise nature of their interactions with the full-length viral RNA genome remain incompletely characterized. In this study, we sought to address this knowledge gap by employing enhanced crosslinking and immunoprecipitation (eCLIP) to map the binding sites of NSP8, NSP12, and N proteins across the SARS-CoV-2 genome at early stages of viral RNA and protein synthesis and late stages of virion assembly. Our findings revealed interactions of NSP8 and NSP12 to the 5\u2019 and 3\u2019 untranslated regions (UTRs) of both positive and negative sense RNA, regions known to regulate viral replication, transcription, and translation. We identified a surprising and essential NSP12 binding site within the RNA sequence encoding the conserved Y1 domain of NSP3, which regulates RNA abundance upstream of the site. Additionally, we found that N protein interacts with the 5\u2019 UTR and influences translation efficiency. Finally, we report a novel regulatory function of N protein in modulating ribosomal frameshifting proximal to the frameshift element, a crucial process for maintaining viral protein stoichiometry. Our results provide a detailed molecular map of SARS-CoV-2 protein-RNA interactions, revealing potential therapeutic targets for attenuating viral fitness and informing the development of next-generation antiviral strategies.","version":"1.1","doi":"10.1101/2025.05.26.656146","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.24.655874","pub_date":"2025-5-28","title":"High-throughput screening for class I peptide MHC binding via yeast surface display","abstract":"T cells rely on short peptides presented by highly polymorphic major histocompatibility complexes (MHCs) to selectively initiate adaptive immune responses. Despite its importance, few techniques can systemically evaluate stable peptide presentation across diverse MHC alleles. Here, we describe a yeast display pipeline that can be deployed to rapidly screen proteomic space to identify class I pMHC binders across many alleles. Through this, we capture unique biological phenomena such as interference with peptide presentation via type IV drug-induced hypersensitivity. We apply this approach to multiple pathogen proteomes (Mtb Type 7S substrates, SARS-CoV-2, Dengue, and Zika) to create a high-resolution catalog of potential T cell antigens. Altogether, this platform acts as a flexible tool to generate large unbiased datasets for class I peptide presentation at a speed and scale competitive with the biological systems they represent.","version":"1.1","doi":"10.1101/2025.05.24.655874","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.23.655815","pub_date":"2025-5-27","title":"COVID-19 induces persistent transcriptional changes in adipose tissue that are not associated with Long COVID","abstract":"Long COVID is a heterogeneous condition characterized by a wide range of symptoms that persist for 90 days or more following SARS-CoV-2 infection. Now more than five years out from the onset of the SARS-CoV-2 pandemic, the mechanisms driving Long COVID are just beginning to be elucidated. Adipose tissue has been proposed as a potential reservoir for viral persistence and tissue dysfunction contributing to symptomology seen in Long COVID. To test this hypothesis, we analyzed subcutaneous adipose tissue (SAT) from two cohorts: participants with subacute COVID-19 (28\u201389 days post-infection) compared to pre-pandemic controls, and participants with Long COVID compared to those with those classified as \u201cindeterminate\u201d based on the RECOVER-Adult Long COVID Research Index (12-47 months post-infection). We found no evidence of persistent SARS-CoV-2 RNA in adipose tissue in any participant. SAT from participants with subacute COVID-19 displayed significant transcriptional remodeling, including depleted immune activation pathways and upregulated Hox genes and integrin interactions, suggesting resident immune cell exhaustion and perturbations in tissue function. However, no consistent changes in gene expression were observed between Long COVID samples and samples from indeterminant participants. Thus, SAT may contribute to inflammatory dysregulation following COVID-19, but does not appear to play a clear role in Long COVID pathophysiology. Further research is needed to clarify the role of adipose tissue in COVID-19 recovery.","version":"1.1","doi":"10.1101/2025.05.23.655815","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.24.655938","pub_date":"2025-5-24","title":"Identification and characterization of a non-peptidic cyclophilin ligand with antiviral activity against feline and porcine \u03b1-coronaviruses","abstract":"Coronaviruses (CoVs) are emerging pathogens that have been extensively studied over the last 20 years and can cause acute respiratory diseases in humans, as exemplified by the SARS-CoV-2 pandemic. Coronaviruses are also known for their importance in veterinary medicine, being responsible for severe pathologies in pets and livestock. These include Feline Infectious Peritonitis Virus (FIPV), which causes a fatal disease in cats. In livestock, porcine coronaviruses such as Transmissible Gastroenteritis Virus (TGEV) and Porcine Epidemic Diarrhoea Virus (PEDV) are the causative agents of an acute enteric disease in piglets with a high mortality rate and a significant impact on the pork industry. In addition, animal coronaviruses may represent a zoonotic reservoir. Therefore, efficient antiviral strategies are required to inhibit the multiplication of coronaviruses infecting various animal species. Here, we synthesized 20 small-molecule ligands that target cyclophilins, a family of cellular chaperons hijacked by several viruses including CoVs. We screened their antiviral activity against feline and porcine alpha-CoVs, and identified a compound, F83233, as a potent inhibitor of FIPV, TGEV and PEDV replication at micromolar concentrations that was effective in feline, porcine and simian cells. As cyclophilins are highly conserved among mammals, F83233 could be a promising antiviral to treat different animal and zoonotic coronaviruses.","version":"1.1","doi":"10.1101/2025.05.24.655938","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.20.655126","pub_date":"2025-5-23","title":"Deletion of the Envelope gene attenuates SARS-CoV-2 infection by altered Spike localization and increased cell-to-cell transmission","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a highly transmissible acute respiratory infection that can result in severe pneumonia and death. Many details of SARS-CoV-2 infection are not fully understood, including the cell biology and host-virus interactions involved in coronavirus assembly and release, in which the Envelope (E) structural protein is instrumental. Deletion of E in other coronaviruses has been shown previously to either attenuate or abrogate infection. To determine the role of E on SARS-CoV-2 virus production and infectivity, we produced reporter SARS-CoV-2 with or without the E gene deleted using a bacterial artificial chromosome. Replication of \u0394E SARS-CoV-2 was attenuated in Vero E6 cells expressing human ACE2 and TMPRSS2 and in human epithelial cell lines. Electron and immunofluorescence microscopy and virology assays showed that \u0394E SARS-CoV-2 increased cell surface expression of Spike (S) glycoprotein, leading to reduced S incorporation into \u0394E SARS-CoV-2 particles and promotion of increased cell-to-cell transmission that evades neutralizing antibody inhibition. Trans-complementation of E partially rescued \u0394E SARS-CoV-2 S incorporation and restored cell-free transmission. In addition to validating the role of E in retention of S in the ER-Golgi intermediate complex (ERGIC), our results showed that a lack of E led to reorganization of the ERGIC during SARS-CoV-2 infection. Improved understanding of E in SARS-CoV-2 replication and host pathogenesis may help development of novel therapeutics. Non-S coronavirus structural proteins, including E, are conserved, making them potential pan-coronavirus therapeutic targets. Many details about these proteins and their roles in viral replication and host pathogenesis are unknown. In this study, we showed that SARS-CoV-2 replicates without E but is attenuated and impaired for virus particle formation, with less S incorporated into virions and more S expressed on the cell surface compared to wild-type virus. SARS-CoV-2 lacking E spread primarily via cell fusion and evaded neutralizing antibodies. In addition, the absence of E resulted in the reorganization of the ERGIC cell secretory compartment during SARS-CoV-2 infection. A better understanding of how E influences SARS-CoV-2 replication could guide directed design of novel therapeutics for treatment of COVID-19 patients, as well as the potential for pan-coronavirus protection against future coronavirus outbreaks.","version":"1.2","doi":"10.1101/2025.05.20.655126","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.18.654751","pub_date":"2025-5-23","title":"Unmasking complex kinetics in viral entry by inferring hypoexponential models","abstract":"Single-event completion times, such as are estimated in viral entry, offer both promise and challenge to kinetic interpretation. The promise is that they are able to constrain underlying kinetic models much more efficiently than bulk kinetics, but the challenge is that completion times alone can incompletely determine complex reaction topologies. Gamma distributions or mechanistic models have often been used to estimate kinetic parameters for such data, but the gamma distribution relies on homogenous processes contributing to the rate-limiting behavior of the system. Here, we introduce hypoexponential analysis to estimate heterogeneous kinetic processes. We demonstrate that hypoexponential fitting can indeed estimate rate constants separated by 2-3 orders of magnitude. We then apply this approach to measurements of SARS-CoV-2 entry, showing that ACE2 reduces the number of rate-limiting steps but does not change the rates of these kinetic processes. We propose a kinetic model whereby SARS-CoV-2 entry is driven by a mixture of ACE2-accelerated and ACE2-independent spike protein activation events. Inferring such models requires the capability to detect heterogeneous kinetic processes, provided by robust estimation of hypoexponential distributions.","version":"1.1","doi":"10.1101/2025.05.18.654751","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.20.655086","pub_date":"2025-5-22","title":"SARS-CoV-2 Nonstructural Protein 3 Remodels the Phosphorylation of Target Proteins via Protein-Protein Interactions","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with a significant impact on human health. The molecular basis of its pathogenicity remains incompletely understood. The viral nucleocapsid (N) protein, the most abundant protein expressed during SARS-CoV-2 infection, is thought to contribute to disease progression. Yet, its interaction network in the context of viral infection remains largely unexplored. Here, we generated a recombinant (r)SARS-CoV-2 expressing a Strep-tagged N protein by using a reverse genetics system. Affinity purification and mass spectrometry identified an interaction between SARS-CoV-2 N protein and the nonstructural protein 3 (NSP3). Domain mapping revealed that the N dimerization domain and the N-terminal region of NSP3 mediate this interaction. Notably, an N protein mutant lacking its N-terminal domain exhibited enhanced binding to NSP3 and underwent dephosphorylation, implicating NSP3 as a potential viral phosphatase. We further found that NSP3 interacts with Interferon Regulatory Factor 3 (IRF3), a key transcription factor involved in host type I interferon (IFN-\u03b1/\u03b2) antiviral response. SARS-CoV-2 NSP3 expression suppressed poly(I:C)-induced IRF3 phosphorylation and broadly reduced cellular phosphorylation levels in a dose-dependent manner. These findings suggest that SARS-CoV-2 NSP3 modulates host phosphorylation dynamics to subvert antiviral signaling and facilitate viral replication. Understanding virus-host and virus-virus interactions is essential for elucidating the mechanisms of viral replication and immune evasion. Previous studies using individually expressed SARS-CoV-2 proteins have identified host interacting factors but have largely overlooked interactions between viral proteins. Here, we engineered a recombinant SARS-CoV-2 virus expressing Strep-tagged nucleocapsid (N) protein, allowing the identification of both viral and host proteins interacting with N during live infection. We discovered an interaction between N and nonstructural protein 3 (NSP3), revealing a previously unrecognized role for NSP3 in modulating protein phosphorylation, interacting with Interferon Regulatory Factor 3 (IRF3), and regulating the innate immune response. This work demonstrates a powerful strategy for dissecting protein interaction networks during SARS-CoV-2 infection and identifies potential targets for therapeutic intervention.","version":"1.1","doi":"10.1101/2025.05.20.655086","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.20.654373","pub_date":"2025-5-22","title":"A conserved region T-cell vaccine for Sarbecoviruses","abstract":"The rapid development of vaccines was a critical part of the global response to the COVID-19 pandemic. SARS-CoV-2 (a Sarbecovirus and member of the Betacoronavirus genus responsible for the pandemic) virus was first detected in Wuhan, China in late 2019. Effective mRNA vaccines based on the viral Spike protein were designed from the earliest isolates and available by December of 2020. SARS-CoV-2 has continued to evolve in the human population, accruing neutralizing antibody resistance mutations that have necessitated updating the vaccine periodically to better match contemporary variants. Neutralizing antibody cross-reactivity is generally very limited among the diverse members of the betacoronavirus genus that are of clinical importance in people. Here, we present an alternative vaccine strategy based on eliciting T-cell responses targeting four highly conserved regions shared across the betacoronavirus proteomes. We hypothesized that cross-reactive responses to these regions could temper disease severity. Focusing immune responses on highly conserved epitopes could be beneficial as SARS-CoV-2 continues to evolve, or if a novel betacoronavirus should enter the human population. Vaccination with these highly conserved regions induced robust T-cell responses in mice and rhesus macaques. Vaccinated hamsters were significantly protected against weight loss and lung inflammation after challenge with the SARS-CoV-2 Omicron variant. After a SARS-CoV-2 Delta challenge in rhesus macaques, 3 out of 4 animals in the control group had infectious virus in their bronchoalveolar lavage samples, while the 4 animals in the vaccinated group did not.","version":"1.1","doi":"10.1101/2025.05.20.654373","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.20.655128","pub_date":"2025-5-22","title":"Three-dimensional single-particle reconstruction by atomic force microscopy allows rapid structural-based validation of recombinant SARS-CoV-2 Spike protein from a single topology image","abstract":"Atomic force microscopy (AFM) is a versatile multi-modal imaging method frequently used for structural characterisation of biological surfaces at the nanoscale. However, AFM-based three-dimensional single-particle reconstruction has hitherto not been possible due to the tip-sample convolution artifact that distorts AFM images of individual molecules, and the disconnect between two-dimensional AFM images of surface deposited molecules and their three-dimensional structures. Here, three-dimensional single-particle analysis was developed for rapid structure-based validation of protein structures using as few as a single AFM topology image, based on contact-point reconstruction AFM (CPR-AFM) in an integrative approach with cryo-electron microscopy maps available in the Electron Microscopy Data Bank by template matching. This approach was demonstrated on the structural validation of recombinant trimeric ectodomain of SARS-CoV-2 Spike glycoprotein to show its immediate utility as a rapid structure-based sample quality control method in the recombinant expression and purification of the Spike protein samples that can be used in vaccines and therapeutics research. These results show that three-dimensional single-particle reconstruction by AFM is possible, that high signal-to-noise AFM imaging offers a rapid and cost-effective way of validation or identification of three-dimensional protein structures at single particle level, and that AFM can be linked to structural data derived from methods such as cryo-electron microscopy, resulting in integrative methodologies with new capabilities for structural biology.","version":"1.1","doi":"10.1101/2025.05.20.655128","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.19.654917","pub_date":"2025-5-22","title":"Exploring Spike-Dependent and ACE2-Independent Viral Entry into Salivary Epithelial Cells in the Absence of ACE2","abstract":"Salivary gland infection by SARS-CoV-2 requires viral entry via routes and mechanisms that remain unresolved. This study examined the expression of the angiotensin- converting enzyme 2 (ACE2) receptor in salivary tissues and basal cell-derived human salivary progenitor cells (hS/PCs), an unstudied potential entry point for SARS-CoV-2. Multiple detection modalities, including immunocytochemistry, western blotting, flow cytometry and RT- PCR, demonstrated a consistent lack of ACE2 protein and transcript in both tissue specimens and primary salivary epithelial cells. Antigen retrieval at pH 9 was determined to be optimal for immunodetection protocols, yet ACE2 remained undetectable. Small intestine tissue served as a positive control, confirming the validity of the methods and reagents we used. Considering there can be other receptors for SARS-CoV-2, flow cytometric analyses demonstrated that recombinant SARS-CoV-2 spike protein failed to bind to salivary epithelial cells, in contrast to HEK293 cells engineered to overexpress ACE2, which showed robust spike binding. These findings strongly support our conclusion that salivary cells do not serve as major targets for SARS-CoV-2 infection via ACE2 or spike protein, whether through direct exposure to viral particles in ductal fluids or via access to basal cells across the basement membrane.","version":"1.2","doi":"10.1101/2025.05.19.654917","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.20.655152","pub_date":"2025-5-22","title":"Deconstructing natural products to develop synthetic small molecule attachment inhibitors with broad spectrum antiviral activity","abstract":"The continuous emergence of new viruses and the number of viruses that are each highly consequential for few people raise a need for broad spectrum antivirals. Most human and emerging viruses first attach to cellular glycosaminoglycans (GAG) or sialylated glycans (SG), a potential target for broad spectrum antivirals. Attachment to the former is through polar interactions between the negatively charged glycans and positively charged domains in virion proteins and typically inhibited by negatively charged polymers. Attachment to the latter is through specific interactions at binding pockets and inhibited by molecules that bind to these pockets. Surprisingly, EGCG inhibits viruses that attach to GAG and SG. However, it does so with widely differing potencies, is not a pharmacologically desirable molecule, and is limited by solubility. We tested whether it was possible to develop small synthetic molecules to inhibit viruses that attach to SG or GAG. We first identified the EGCG moieties responsible for the antiviral activity. The two polyhydroxylated phenyl groups were essential while the central benzopyran linker was not. We thus designed a series of gallate compounds to explore the minimal pharmacophore required for broad-spectrum antiviral activity. By exploring the linkers and number of galloyls, we identified small molecule inhibitors of herpes simplex virus 1, influenza A virus, and the coronaviruses hCoV OC43 and SARS-CoV-2. These compounds have low micromolar to submicromolar potency and no limiting cytotoxicity. These molecules are still not pharmacologically optimized and limited by solubility, but they define a minimal pharmacophore that confers broad spectrum antiviral activity. The SARS-CoV-2 pandemic demonstrated the importance of antivirals in managing emerging viruses. Although vaccines were successfully developed in less than two years, there was resistance to vaccination while infected or sick people were far more willing to take antivirals. It is impossible to develop antivirals specific for unknown viruses, but broad spectrum antivirals could control viral spread until more specific and potent drugs are developed. Human pathogenic and emerging viruses commonly attach to glycans, providing a target for broad spectrum antivirals. However, inhibitors of attachment to glycosaminoglycans do not typically inhibit viruses attaching to sialylated glycans, and vice-versa. We had found that EGCG has the unique property of inhibiting viruses that attach both glycans, with quite different potencies. Here, deconstructed a natural compound, EGCG, to identify the moieties responsible for its antiviral activity to then produce broad spectrum antiviral compounds against established and emerging viruses that attach to either glycan.","version":"1.1","doi":"10.1101/2025.05.20.655152","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.19.25327882","pub_date":"2025-05-21","title":"Antibody responses following COVID-19 vaccination and breakthrough infections in na\u00efve and convalescent individuals suggests imprinting to the ancestral strain of SARS-CoV-2","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The binding and neutralising activity of SARS-CoV-2 antibodies are important correlates of protection of current COVID-19 vaccines. SARS-CoV-2 exposure status and COVID-19 vaccine types can influence these responses and the breadth of cross-reactivity to variants. In this longitudinal cohort study, we used SARS-CoV-2-specific multiplex Luminex\n                  <jats:sup>\u00ae</jats:sup>\n                  antibody assays and live virus neutralisation of ancestral (VIC01/2020), Delta and Omicron (BA1, BA2 and BA5) SARS-CoV-2 variants to compare antigen-specific binding and neutralising antibody (nAb) responses to primary vaccination (two doses) of adenovirus vectored (AdVV) or mRNA vaccines followed by a booster dose of mRNA vaccine in convalescent (n=51) and infection-na\u00efve individuals (n=47). In a subset of individuals, we performed additional analysis of antibody responses following breakthrough infection.\n                </jats:p>\n                <jats:p>We found that titres of anti-SARS-CoV-2 nAb following primary vaccination (2 doses) with AdVV vaccine were significantly lower than those following mRNA vaccine, irrespective of prior SARS-CoV-2 infection status. However, an mRNA vaccine booster dose resulted in equivalent binding and nAb titres to the ancestral virus in all individuals, irrespective of primary vaccine type. Notably, vaccinated infection-na\u00efve, but not convalescent individuals required the third dose of vaccine (mRNA) to induce nAbs to Omicron subvariants BA1, BA2 and BA5, though titres against the variants were lower than those against the ancestral strain. Importantly, breakthrough infection with Omicron strains induced higher nAb titre rises against the ancestral strain than against Omicron variants consistent with imprinting of the immunologic response and recall of pre-existing immunity to the ancestral strain.</jats:p>","version":null,"doi":"10.1101/2025.05.19.25327882","journal":"medRxiv","score":null},{"id":"10.1101/2025.05.02.651777","pub_date":"2025-5-19","title":"Do Existing COVID-19 Vaccines Need to Be Updated in 2025?","abstract":"COVID-19 vaccines have been updated each year since 2022 to improve protection against evolving SARS-CoV-2 variants. However, it is unclear whether a reformulation will be necessary for 2025. KP.2-based monovalent COVID-19 mRNA vaccines (KP.2 MV) were authorized for use in 2024, and they conferred substantial protection against hospitalizations caused by viral variants that emerged and dominated later, such as KP.3.1.1 and XEC. Today, LP.8.1 and its subvariant LP.8.1.1 have become dominant worldwide, particularly so in North America. Other variants, such as the LF.7 subvariant LF.7.2.1, have emerged with a growth advantage in Asia. To characterize the antigenicity of LP.8.1, LP.8.1.1, LF.7, LF.7.2.1, and another variant under monitoring, MC.10.1, we tested serum samples from 20 individuals who recently received KP.2 MV in neutralization assays against JN.1, KP.2, KP.3, KP.3.1.1, XEC, LP.8.1, LP.8.1.1, LF.7, LF.7.2.1, or MC.10.1 pseudoviruses. Serum neutralizing antibody titers against LP.8.1, LP.8.1.1, LF.7, LF.7.2.1, and MC.10.1 were comparable to those against KP.3.1.1 and XEC, indicating that LP.8.1.1 and other recently dominant subvariants are antigenically similar to their predecessors. Therefore, the currently authorized KP.2 MV may not need to be updated for 2025, if the vaccine manufacturers could demonstrate comparable immunogenicity for KP.2 MV and LP.8.1-based mRNA vaccines and, of course, in the absence of an antigenically divergent SARS-CoV-2 variant emerging.","version":"1.2","doi":"10.1101/2025.05.02.651777","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.23.650113","pub_date":"2025-5-19","title":"COVID-19 in Space: Possible Health Risks and Preparedness Guidelines","abstract":"Background The COVID-19 pandemic of 2020 resulted in over 705 million infections and more than 7 million deaths worldwide. The virus primarily spreads through aerosol droplets released during breathing, coughing, or sneezing, leading to symptoms ranging from mild fever and cough to severe outcomes, including death. Given the high risk associated with COVID-19, understanding its behaviour in diverse geographical and environmental conditions is critical. Space exploration and tourism represent an emerging industry, projected to reach a market value of $1.8 trillion. With numerous space missions planned by space agencies such as NASA, SpaceX, and ISRO, it is vital to address potential health risks for astronauts and space tourists. With the expansion of human exploration into space, there is an urgent need to assess the risks posed by COVID-19 in extraterrestrial environments. This study reviews existing literature on airborne infections in space, identifies key knowledge gaps, and enhances preparedness for potential COVID-19 outbreaks during space missions. A systematic literature review was conducted to identify studies examining airborne infectious diseases in space and their health effects under microgravity. Databases searched included PubMed and NASA\u2019s Open Data Portal. To compare these findings with Earth-based data, additional systematic reviews were performed to analyze the known effects of these diseases on Earth, using Pathogen Safety Data Sheets. A separate systematic review was conducted using PubMed to explore similarities between COVID-19 and the selected airborne infectious diseases. Using a comparative approach, disease effects observed on Earth and in space were analyzed to predict COVID-19\u2019s potential behavior in microgravity. Existing guidelines for managing airborne diseases in space and on Earth were reviewed and compared to develop a set of preparedness recommendations for COVID-19 in space. The airborne infectious diseases occurring in space found in this study include Aspergillus fumigatus, Beauveria bassiana, Epstein-Barr Virus (EBV), Escherichia coli, Klebsiella pneumoniae infections, Pseudomonas aeruginosa, Roseolovirus (Human Herpesvirus 6 & 7), Salmonella Typhimurium infection, Serratia marcescens infection, Staphylococcus aureus, Staphylococcus epidermidis, and Varicella-Zoster Virus (VZV). The relationship between the aforementioned diseases and COVID-19 was used in regard to theorizing the effects of COVID-19 in space. Six Tentative effects of COVID-19 in a microgravity environment could be theorized in this study. Along with that, recommendations to improve the current space travel health guidelines have also been referred to. The results of this study will change the course of human space exploration by assisting in the protection of space travelers and guiding the development of new designs for spacecraft that include extra safety features.","version":"1.2","doi":"10.1101/2025.04.23.650113","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.15.654199","pub_date":"2025-5-15","title":"Specificity profiling of SARS-CoV-2 PLpro using proteome-derived libraries of linear peptides suggests secondary preference for basic motifs","abstract":"SARS-CoV-2 papain-like protease (PLpro) is essential for viral replication and immune modulation. Here, a proteomic identification of protease cleavage sites (PICS) approach was applied using proteome-derived peptide libraries to determine the enzyme\u2019s substrate specificity. PLpro exhibited a yet unreported preference for basic amino acids at P1, primarily arginine or lysine. This secondary specificity frequently involved cleavage between two basic residues (e.g., K|K, K|R or R|K). Experiments with in-house and commercial PLpro in GluC-generated peptide libraries from Escherichia coli and HEK293 proteomes confirmed this preference, though with lower overall efficiency compared to typical trypsin-like proteases. SARS-CoV-1 PLpro likewise displayed this basic-site specificity, underscoring its conservation across related coronaviruses. Site-directed mutagenesis of acidic residues near the catalytic triad to neutral variants altered cleavage preferences, indicating the involvement of these side chains in substrate binding and potential alternative binding modes. We also evaluated wild-type PLpro specificity on intact protein lysates rather than peptide libraries to assess how structure influences cleavage patterns. Notably, P1 arginine specificity persisted at the protein level, whereas lysine specificity was reduced, suggesting additional structural constraints in complex substrates. A strong presence of glycine on the prime side further suggests a bias toward unstructured regions. These findings reveal an expanded substrate recognition repertoire for SARS-CoV-2 PLpro, which may be relevant for the design of targeted inhibitors and understanding of viral protease function.","version":"1.1","doi":"10.1101/2025.05.15.654199","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.14.654028","pub_date":"2025-5-15","title":"Pharmacokinetic-Pharmacodynamic Trade-offs in SARS-CoV-2 Main Protease Inhibitors Unveiled through Machine Learning and Molecular Dynamics Simulations","abstract":"The SARS-CoV-2 main protease (Mpro) is a validated therapeutic target for inhibiting viral replication. Despite the screening of over 55,000 compounds, few candidates have advanced clinically, underscoring the difficulty in optimizing both target affinity and drug-like properties. Thus, developing effective Mpro inhibitors requires balancing high-affinity binding with favorable pharmacokinetic (PK) properties, such as solubility and permeability. To address this challenge, we integrated machine learning (ML) and molecular dynamics (MD) simulations to investigate the trade-offs between pharmacodynamic (PD) and PK properties in Mpro inhibitor design. We developed ML models to classify Mpro inhibitors based on experimental IC50 data, combining molecular descriptors with structural insights from MD simulations. Our Support Vector Machine (SVM) model achieved strong performance (training accuracy = 0.84, ROC AUC = 0.91; test accuracy = 0.79, ROC AUC = 0.86), while our logistic regression model (training accuracy = 0.78, ROC AUC = 0.85; test accuracy = 0.76, ROC AUC = 0.83) identified key molecular features influencing activity, including quantitative estimation of drug\u2013likeness (QED), Log P and molecular weight (ExactMolWt). Notably, PK descriptors often exhibited opposing trends to binding affinity: hydrophilic features enhanced binding affinity but compromised PK properties, whereas hydrogen bonding, hydrophobic and \u03c0\u2013\u03c0 interactions in subsites S2 and S3/S4 are fundamental for binding affinity. Our findings highlight the need for a balanced approach in Mpro inhibitor design, strategically targeting these subsites may balance PD and PK properties. This study provides a computational framework for rational Mpro inhibitor discovery, combining ML and MD to investigate the complex interplay between enzyme inhibition and drug\u2013likeness. These insights may guide in hit-to-lead optimization of the novel next-generation Mpro inhibitors of the SARS-CoV-2 with preclinical and clinical potential.","version":"1.1","doi":"10.1101/2025.05.14.654028","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.14.654036","pub_date":"2025-5-15","title":"AI-based decoding of long covid cognitive impairments in mice using automated behavioral system and comparative transcriptomic analysis","abstract":"Long COVID (LC) following SARS-CoV-2 infection affects millions of individuals world-wide and manifests with a variety of symptoms including cognitive dysfunction also known as \u201cbrain fog\u201d. This is characterized by difficulties in executive functions, planning, decision-making, working memory, impairments in complex attention, loss of ability to learn new skills and perform sophisticated brain tasks. No effective treatment options currently exist for LC-related cognitive dysfunction. Here, we use the IntelliCage, which is an automated tracking system of cognitive functions, following SARS-CoV-2 infection in mice, measuring the ability of each mouse within a group to perform tasks that mimic complex human behaviors, such as planning, decision-making, cognitive flexibility, and working memory. Artificial intelligence and machine learning analyses of the tracking data classified LC mice into distinct behavioral categories from non-infected control mice, permitting precise identification and quantification of complex cognitive dysfunction in a controlled, replicable manner. Importantly, we find that brains from LC mice with cognitive dysfunction exhibit transcriptomic alterations similar to those observed in humans suffering from LC-related cognitive impairments, including altered expression of genes involved in learning, executive functions, synaptic functions, neurotransmitters and memory. Together, our findings establish a validated murine model and an automated unbiased approach to study LC-related cognitive dysfunction for the first time, and providing a valuable tool for screening potential treatments and therapeutic interventions.","version":"1.1","doi":"10.1101/2025.05.14.654036","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.12.653443","pub_date":"2025-5-15","title":"Discovery and structure\u2013activity relationship analysis of 2-Pyridyl Thieno[3,2-d]pyrimidine derivatives as promising therapeutic candidates for the treatment of Buruli ulcer","abstract":"Mycobacterium ulcerans, the bacterium causing Buruli ulcer (BU), can potentially develop resistance to existing antibiotics (rifampicin - clarithromycin/ moxifloxacin), underscoring the need for new antimycobacterial treatments. This study screened the Pathogen Box from Medicines for Malaria Venture (MMV) to identify M. ulcerans inhibitors. Four hit compounds were found, including the 2-(6-methylpyridin-2-yl)-N-(pyrimidin-4-yl)thieno[3,2-d]pyrimidin-4-amine MMV688122 as a novel anti-M. ulcerans chemotype. Synthesis of structural analogues of MMV688122 allowed the identification of 2-(4-methylpyridin-2-yl)-N-(pyrimidin-4-yl)thieno[3,2-d]pyrimidin-4-amine MMV1578877 as the most potent, with submicromolar activity. Importantly, this analogue was non-cytotoxic up to 100 \u00b5M in human fibroblasts. Structure-activity relationship (SAR) studies indicated the crucial role of the methylpyridin-2-yl group in inhibiting M. ulcerans and the possibility to replace the thienopyrimidine core by a quinazoline. While MMV1578877 showed better metabolic stability than MMV688122, further improvement and testing in real-world M. ulcerans clinical isolates are still required. Further metabolite identification and SAR data should guide the optimization of this novel chemotype to enable in vivo testing. Buruli ulcer is a neglected tropical disease that causes severe skin ulcers and long-term disability, mostly affecting people in remote African communities. Current treatments rely on antibiotics that are not always effective and may lead to resistance. In this study, we searched for new drug candidates by testing a library of compounds provided by the Medicines for Malaria Venture (MMV). We discovered a promising new chemical family that can kill the bacteria responsible for Buruli ulcer in the lab. One compound, in particular, showed strong activity without harming human cells. This compound also showed better stability and effectiveness. These findings bring us closer to developing a new, safer, and more effective treatment for Buruli ulcer.","version":"1.1","doi":"10.1101/2025.05.12.653443","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.13.653138","pub_date":"2025-5-14","title":"IL-4 and TGF-\u03b2 Regulate Inflammatory cytokines and Cellular Infiltration in the Lung in Mouse-adapted SARS-CoV-2 Infection","abstract":"The pathology of severe COVID-19 is due to a hyperinflammatory immune response persisting after viral clearance. To understand how the immune response to SARS-CoV-2 is regulated to avoid severe COVID-19, we tested relevant immunoregulatory cytokines. TGF-\u03b2, IL-10 and IL-4 were neutralized upon infection with mouse-adapted SARS-CoV-2 (CMA3p20), a model of mild disease; and lung inflammation was quantified by histology and flow cytometry at early and late time points. Mild weight loss, and lung inflammation including consolidation and alveolar thickening were evident 3 days post-infection (dpi) and inflammation persisted to 7 dpi. Coinciding with early monocytic infiltrates, CCL2 and granulocyte-colony stimulating factor (G-CSF) were transiently produced 3 dpi, while IL-12 and CCL5 persisted to 7 dpi, modeling viral and inflammatory phases of disease. Neutralization of TGF-\u03b2, but not IL-10 or IL-4, significantly increased lung inflammatory monocytes and elevated serum but not lung IL-6. Neutralization of IL-4 prolonged weight loss and increased early perivascular infiltration without changing viral titer. Anti-IL-4 reduced expression of Arg1, a gene associated with alternative activation of macrophages. Neutralizing TGF-\u03b2 and IL-4 had differential effects on pathology after virus control. Lung perivascular infiltration was reduced 7 dpi by neutralization of IL-4 or TGF-\u03b2, and peri-airway inflammation was affected by anti-TGF-\u03b2, while alveolar infiltrates were not affected by either. Anti-IL-4 prolonged IL-12 to 7 dpi along with reduced IL-10 in lungs. Overall, the immunoregulatory cytokines TGF-\u03b2 and IL-4 dampen initial inflammation in this maSARS-CoV-2 infection, suggesting that promotion of immunoregulation could help patients in early stages of disease.","version":"1.1","doi":"10.1101/2025.05.13.653138","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.13.653844","pub_date":"2025-5-14","title":"Uridine cytidine kinases dictate the therapeutic response of molnupiravir via its bioactivation","abstract":"Molnupiravir is a nucleoside analogue antiviral drug with activities against a broad spectrum of RNA viruses, including its clinical indication SARS-CoV-2. Whilst its antiviral mechanism-of-action is well defined, host pharmacogenetic factors that regulate its therapeutic responses \u2013 efficacy as well as selectivity \u2013 have not been mechanistically deciphered and characterized. Here we identified that uridine cytidine kinase (UCK), the first and rate-limiting kinase of the pyrimidine salvage pathway, could effectively phosphorylate N4-hydroxycytidine (NHC), the active compound of molnupiravir, and thereby dictate its anti-SARS-CoV-2 efficacy, as well as its selectivity index. Using target engagement and enzyme kinetic assays, we demonstrate that recombinant UCK isoform 1 (UCK1) and 2 (UCK2) effectively bind and phosphorylate NHC, with UCK2 displaying 9-fold higher catalytic efficiency. Accordingly, in SARS-CoV-2-infected cells, downregulation of UCK2 via siRNA hampered the intracellular accumulation of the triphosphate antiviral metabolite of NHC, resulting in 10-fold reduction of antiviral efficacy. Furthermore, we could recapitulate this using a pan-UCK small molecule inhibitor. Critically, both UCK downregulation and inhibition significantly reduced the selectivity index of molnupiravir/NHC. Altogether, this work underscores the pivotal roles of UCK enzymes in upholding molnupiravir efficacy and therapeutic window, and furthermore, highlights their potential as pharmacologically tractable targets for tailoring the therapeutic response to this antiviral agent.","version":"1.1","doi":"10.1101/2025.05.13.653844","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.16.589454","pub_date":"2025-5-13","title":"Viral evolution prediction identifies broadly neutralizing antibodies against existing and prospective SARS-CoV-2 variants","abstract":"Monoclonal antibodies (mAbs) targeting the SARS-CoV-2 receptor-binding domain (RBD) are used to treat and prevent COVID-19. However, the rapid evolution of SARS-CoV-2 drives continuous escape from therapeutic mAbs. Therefore, the ability to identify broadly neutralizing antibodies (bnAbs) against future variants is needed. Here, we use deep mutational scanning (DMS) to predict viral RBD evolution and to select for mAbs neutralizing both existing and prospective variants. A retrospective analysis of 1,103 SARS-CoV-2 wildtype-elicited mAbs shows that this method can increase the probability of identifying effective bnAbs against the XBB.1.5 strain from 1% to 40% in an early pandemic setup. Among these bnAbs, BD55-1205 exhibited potent activity against all tested variants. Cryo-EM structural analyses revealed the receptor mimicry of BD55-1205, explaining its broad reactivity. Delivery of mRNA-LNPs encoding BD55-1205-IgG in mice resulted in ~5,000 serum NT50 against XBB.1.5, HK.3.1, and JN.1 variants. Combining bnAb identification using viral evolution prediction with the versatility of mRNA delivery technology can enable rapid development of next-generation antibody-based countermeasures against SARS-CoV-2 and potentially other pathogens with pandemic potential.","version":"1.2","doi":"10.1101/2024.04.16.589454","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.09.653029","pub_date":"2025-5-13","title":"A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants","abstract":"The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 \u03943a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging \u03943a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 \u03943a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 \u03943a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2. Small-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins (\u03943a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using \u03943a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 \u03943a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 \u03943a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.05.09.653029","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.11.652904","pub_date":"2025-5-13","title":"Phylogeny-driven design of broadly protective sarbecovirus receptor-binding domain nanoparticle vaccines","abstract":"Vaccines against emerging SARS-CoV-2 variants and sarbecoviruses with pandemic potential must elicit a robust humoral immune response in a population imprinted with the SARS-CoV-2 spike (S) protein. Here, we designed protein nanoparticle (NP) vaccines co-displaying the SARS-CoV-2 BA.5, SARS-CoV-1, and BtKY72 receptor-binding domains (RBDs) with or without the Wuhan-Hu-1 (Wu) RBD. We show that these vaccines elicit cross-reactive and broadly neutralizing plasma antibody responses against SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune animals. Immunization with multivalent RBD-NPs overcomes immune imprinting and elicits neutralizing antibodies and memory B cells specific for the BA.5, SARS-CoV-1, and BtKY72 RBDs in mRNA-1273-vaccinated non-human primates. Multivalent RBD-NPs outperform a monovalent Wu RBD-NP vaccine by providing superior protection in mice and non-human primates challenged with the vaccine-mismatched SARS-CoV-2 XBB.1.5 or the pre-emergent RsSHC014. These data support the use of multivalent RBD-NP vaccines for SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune populations.","version":"1.1","doi":"10.1101/2025.05.11.652904","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.16.649172","pub_date":"2025-5-12","title":"Ex vivo recapitulation of intramuscular mRNA vaccination with na\u00efve and recall antigens using a human Lymphoid Follicle Chip platform","abstract":"Predicting the efficacy and toxicity of intramuscular mRNA vaccines remains challenging. Here, we describe an ex vivo human cell-based model that replicates immune responses to lipid nanoparticle (LNP)-based mRNA vaccines that require intramuscular injection. Vaccines are administered into a biomimetic muscle module containing human skeletal myoblasts and antigen-presenting cells (APCs) to mimic intramuscular vaccination, followed by transfer of the APCs and soluble factors to a microfluidic human lymphoid follicle chip (LF Chip) to mimic lymphatic drainage. Non-replicating mRNA vaccines directly induce antigen expression in APCs, whereas self-amplifying mRNA vaccines require muscle cell-APC contact within the intramuscular vaccination module. Transfer of APCs and soluble factors to the LF Chip induces LF expansion, de novo antigen-specific IgG production against a na\u00efve antigen (rabies virus glycoprotein), and cytokine release, with responses varying depending on LNP type. Vaccination of LF chips against SARS- COV-2 Spike recall antigen using the Moderna Spikevax vaccine generates neutralizing antibodies and induces somatic hypermutation. This biomimetic platform offers an all-human alternative for evaluating vaccine-induced immunity, potentially obviating the need for non-human primates and accelerating vaccine development.","version":"1.2","doi":"10.1101/2025.04.16.649172","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.09.653001","pub_date":"2025-5-09","title":"A nasal vaccine candidate, based on S2 and N proteins from SARS-CoV-2, generates a broad antibody response systemic and at lower respiratory tract","abstract":"Since the beginning of the COVID-19 pandemic various groups around the world have intensively worked in the development of vaccine candidates against SARS-CoV-2. Several vaccines have been approved in the past years, the majority are based on the Spike or RBD proteins and employs parenteral administration routes. Considering the recent history of Coronavirus zoonotic events, causing serious human health problems, the generation of vaccines with a broad scope of protection and the potential to cut / reduce the transmission remains in the spotlight. The current global pandemic preparedness initiatives have promoted also the preclinical evaluation of a new group of Coronavirus vaccines. In the present work a nasal vaccine candidate based on two highly conserved Sarbecovirus proteins, S2 and nucleocapsid (N), is evaluated in two different mice strains. The vaccine preparation, containing a CpG ODN as adjuvant, was able to generate high antibody titers against both antigens, in sera and bronchoalveolar lavages. This antibody response results cross-reactive to S2 from SARS-CoV-1 and MERS-CoV, and to N from SARS-CoV-1. However, a very low neutralizing capacity was found in the sera of the immunized mice when a pseudoviral system assay was used. On the other hand, the vaccine preparation induces, at systemic compartment, IFN \u03b3 secretion, and a marked IgG2a response, specific against both proteins; a profile consistent with the development of a Th1 pattern. Although further evaluations should be done, including protection assays, the demonstrated cross-reactivity level and mucosal response constitutes promising features of this vaccine candidate.","version":"1.1","doi":"10.1101/2025.05.09.653001","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.09.652710","pub_date":"2025-5-09","title":"Inhibitors of membrane associated serine proteases block replication of coronavirus SARS-CoV-2 and influenza virus H1N1","abstract":"TMPRSS2 is a membrane associated serine protease which is important in the viral pathogenesis of coronaviruses and influenza viruses. We developed mechanism-based covalent \u03b1-ketobenzothiazole (kbt) inhibitors using substrate specificity PS-SCL screening of TMPRSS2 as a rational guide for inhibitor design. Three distinct focused libraries of tetrapeptide kbts were synthesized and evaluated for their inhibition of TMPRSS2, matriptase and other serine proteases. We also investigated different capping groups for the previously reported tripeptide inhibitor Ac-QFR-kbt (MM3144) to increase its selectivity over the blood coagulation protease factor Xa. The most potent compounds were tested for their ability to inhibit viral replication of SARS-CoV-2 coronavirus and the H1N1 influenza virus. The most active compounds were profiled for their pharmacokinetics (PK) in mice. Several promising new compounds were identified with improved potency, selectivity, and drug-like properties including Bz-QFR-kbt (CA1043) and Cbz-QFR-kbt (ZFH9141) with an IC50 of 150 nM and 60 nM for H1N1, respectively.","version":"1.1","doi":"10.1101/2025.05.09.652710","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.08.652856","pub_date":"2025-5-08","title":"No Evidence of Direct Activation of Human Neutrophil Responses by Multivalent Prefusion Trimeric SARS-CoV-2 Spike Protein ex vivo","abstract":"The SARS-CoV-2 Spike (S) protein is essential for viral entry and serves as the primary immunogen in most COVID-19 vaccines. While its role in adaptive immunity is well defined, its potential to contribute directly to innate immune activation and remains incompletely understood. Neutrophils, in particular, are prominent effectors in COVID-19 severity, yet how they respond directly to the S protein presented in a multivalent format is unclear. Here, we investigated whether the S protein can directly activate human neutrophils ex vivo using two biologically relevant models: nanoparticles displaying multivalent stabilized prefusion trimeric S glycoprotein, and purified \u03b2-propiolactone-inactivated SARS-CoV-2 virions. Neutrophils were exposed to nanoparticles or inactivated virus, either alone or pre-coated with monoclonal or polyclonal anti-S antibodies. Nanoparticles displaying Respiratory Syncytial Virus (RSV) Fusion (F) protein and purified \u03b2-propiolactone-inactivated RSV served as comparators. Across all models and conditions tested, the S protein did not induce significant neutrophil responses. No consistent effects were observed on cell viability, surface marker expression, reactive oxygen species production, neutrophil extracellular trap formation, cytokine release, or inflammatory gene expression\u2014even in the presence of anti-S antibodies mimicking immune complexes. Results with F-nanoparticles and inactivated RSV were similarly modest. These findings indicate that the trimeric prefusion S protein, whether displayed multivalently on nanoparticles or in the context of inactivated viral particles, is insufficient to trigger robust neutrophil activation. This work provides insight into the innate immune profile of the S protein and suggests that its use in vaccine platforms is unlikely to directly provoke neutrophil-mediated inflammatory responses.","version":"1.1","doi":"10.1101/2025.05.08.652856","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.07.652638","pub_date":"2025-5-08","title":"Multi-step antibody class switching in a primary human response is restricted after IGHG2 and dependent on B cell maturation stage","abstract":"Class switch recombination (CSR) allows the formation of functionally specialized antibodies. Understanding of CSR dynamics is key for better design and prediction of vaccines to protect mucosal surfaces. To investigate CSR in a primary human immune response under a controlled setting, we sampled healthy volunteers without COVID-19 history every other day during the first three weeks after SARS-CoV-2 vaccination, with additional time points up to six months. Leveraging bulk and single-cell B cell receptor repertoires, single-cell transcriptomics, and immunophenotyping, we uncover paradigm-shifting insights into CSR. Newly activated B cells produce sterile transcripts of all antibody isotype constant regions (IGHC) simultaneously up to IGHG2, challenging the view that sterile transcription occurs for only a single IGHC gene at a time. CSR follows a multistep progression along the IGHC locus; in this challenge vaccine-induced B cells switch from IGHM to IGHG3 and IGHG1, followed by a subsequent switching to IGHA1 and IGHG2 after secondary immunization. IGHA2 clones require pre-switching to IGHA1 clones. Notably, switching tendency, measured by IGHC sterile transcription, is memory B cell subtype dependent, particularly beyond IGHG2. Contrary to other vaccines, antigen-specific B cells are enriched in DN2, Cmem2 and DN4 subtypes after immunization. We also observe a temporal decoupling of CSR and somatic hypermutation (SHM), with the latter detectable only after six months post-immunization. Our data describes the dynamics between CSR, SHM, sterile transcription and B cell memory development during a human primary response, challenges textbook models of CSR and offers new insights to aid control of CSR direction. Class switch recombination occurs independently of somatic hypermutation and in a multistep fashion up to IGHG2 during a primary response in humans.","version":"1.1","doi":"10.1101/2025.05.07.652638","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.06.652338","pub_date":"2025-5-07","title":"Beyond Natural Antibodies: Scaffold-Based Generation of Novel Anti-3CLpro Nanobody Nb01","abstract":"In the post-pandemic era, continuous mutations and persistent infections of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pose a significant threat to global health, making the normalization of preventive measures imperative. Due to its high conservation, the 3CLpro is relatively stable across all these variants without significant changes, suggesting that drugs targeting this enzyme could be effective against all variant viruses. And compared to traditional antibodies, nanobodies show significant advantages against SARS-CoV-2 and its variants. Traditional antibodies often lose their inhibitory activity due to viral mutation. Nanobodies are characterised by their small size, high stability, high affinity for antigen binding, and high water solubility, which enable them to capture viruses quickly and effectively address the continuous mutations and persistent infections of SARS-CoV-2. In this research, we devised a strategy to produce nanobodies by utilizing a fragment-generating large language model, resulting in the identification of a nanobody named Nb01. Nb01 exhibits potent and broad-spectrum neutralizing activity against numerous SARS-CoV-2 variants. The nanobody Nb01, directed against the SARS-CoV-2 3CLpro, demonstrated efficacy against the majority of prevalent SARS-CoV-2 variants. Notably, it has a higher affinity than the current best performing nanobodies (S43, bn03, R14, and 3-2A2-4) for most of the variants, including the Alpha (27.5%), Gamma (29.7%), Omicron BA.2 (32.2%), BA.4/5 (81.2%), BF.7 (64.2%) and XBB (5.5%) variants. For other variants, Nb01 displayed affinities that were on par with these benchmark nanobodies. In summary, the exceptional specificity, low toxicity, robust stability, and extensive spectrum of Nb01 indicate its potential to be developed as a nanobody therapeutic for the management of SARS-CoV-2 infections and its diverse variants.","version":"1.1","doi":"10.1101/2025.05.06.652338","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.06.651076","pub_date":"2025-5-07","title":"LAMPrey: a standardised method for analysing quantitative LAMP (qLAMP) and qPCR reactions using the inflection cycle threshold iCt","abstract":"Quantitative Loop-mediated isothermal amplification (qLAMP) is a relatively new method that has gained popularity in recent years, particularly in disease identification, including during the recent SARS-CoV-2 pandemic. Unlike conventional quantitative PCR (qPCR), qLAMP features a linear amplification phase before the exponential phase. Determining cycle threshold (Ct) values through automatic thresholding may therefore produce inaccurate results, and the nature of these thresholds complicates comparability between studies and softwares. We introduce a new method for transforming sigmoidal amplification curves into inflection threshold curve (iCt) to address issues with auto thresholds and analysis of qLAMP. This method is implemented as a collection of R functions named LAMPrey, suitable for analysis of both qPCR and qLAMP reactions performed in the two most commonly used real-time thermocyclers. We simulate qLAMP amplification differences, demonstrate that iCt and Ct methods perform equivalently for conventional qPCR with an Illumina library quantitation kit, and show that iCt values outperform Ct values for quantifying qLAMP reactions in zebrafish embryos. All scripts developed for this paper are available at https://github.com/dodged13/LAMPrey","version":"1.1","doi":"10.1101/2025.05.06.651076","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.04.652125","pub_date":"2025-5-06","title":"A Cluster of Acidic Residues in the Cytoplasmic Domain of SARS-CoV-2 Spike is Required for Virion-Incorporation and Infectivity","abstract":"Like all coronaviruses, the infectivity of SARS-CoV-2 virus particles (virions) requires incorporation of the Spike glycoprotein. Yet, the mechanisms that support the virion-incorporation of Spike are incompletely defined. We noted an unusual feature of human sarbecovirus Spike proteins: their cytoplasmic domains (CDs) contain a stretch of acidic amino acids (DEDDSE). This sequence resembles a cluster of acidic residues, or acidic cluster (AC) motif, found in the cytoplasmic domain of the cellular endoprotease Furin. In Furin, the acidic cluster acts as a protein sorting signal, supporting its intracellular localization at the trans-Golgi network (TGN). We tested the contribution of the acidic cluster motif in the Spike CD to protein interactions and to the infectivity of SARS-CoV-2. We used virus-like particles (VLPs) as a model for viral \u201cinfection\u201d (transduction). The SARS-CoV2 VLPs were produced by co-expressing Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and deliver an RNA encoding luciferase to target cells expressing the ACE2 receptor. Remarkably, when all five acidic residues of the DEDDSE sequence were replaced with alanines, the VLPs were rendered non-infectious. The N-terminal DE residues provided most of the activity of the acidic cluster. These virologi-cally-impaired Spike mutants were able to reach the cell surface and induce the formation of syncytia, indicating that they are fusogenic and capable of anterograde traffic through the biosynthetic pathway to the plasma membrane. Despite this, they failed to efficiently incorporate into virions. We observed acidic cluster motif-dependent interactions of the Spike CD with several cellular proteins that could potentially support its role in virion-incorporation, including the ERM proteins Ezrin, Radixin, and Moesin; the retromer subunit Vps35, and the medium subunits of the clathrin adaptor complexes AP1 and AP2. While the key cofactor and mechanism of action remains to be defined, this region of acidic residues in the Spike CD appears to be a novel determinant of SARS-CoV-2 infectivity.","version":"1.1","doi":"10.1101/2025.05.04.652125","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.03.652024","pub_date":"2025-5-05","title":"Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature","abstract":"The COVID-19 pandemic was marked by successive waves of SARS-CoV-2 variants with distinct properties. The Omicron variant that emerged in late 2021 showed a major antigenic shift and rapidly spread worldwide. Since then, Omicron-derived variants have maintained their global dominance, for reasons that remain incompletely understood. We report that the original Omicron variant BA.1 evolved several traits that converged in facilitating viral spread. First, Omicron displayed an early replicative advantage over previous variants when grown in a reconstructed nasal epithelium model based on primary human cells. The increase in Omicron replication was more marked at the 33\u00b0C temperature characteristic of human nasal passages, resulting in a physiologically relevant advantage. Omicron also caused a decrease in epithelial integrity, as measured by transepithelial electrical resistance and caspase-3 activation. Furthermore, Omicron caused a more marked loss of motile cilia at 33\u00b0C than other variants, suggesting a capacity to impair mucociliary clearance. RNAseq analysis showed that Omicron induced a broad transcriptional downregulation of ciliary genes but only a limited upregulation of host innate defense genes at 33\u00b0C. The lower production of type I and type III interferons in epithelia infected by Omicron compared to those infected by the Delta variant, at 33\u00b0C as well as 37\u00b0C, confirmed the increased capacity of Omicron to evade the innate antiviral response. Thus, Omicron combined replication speed, motile cilia impairment, and limited induction of innate antiviral responses when propagated in reconstructed nasal epithelia at physiological temperature. Omicron has the capacity to propagate efficiently but stealthily in the upper respiratory tract, which likely contributed to the evolutionary success of this SARS-CoV-2 variant. The COVID-19 pandemic was initially characterized by a rapid succession of viral variants that emerged independently of each other, with each of these variants outcompeting previous ones and rising to regional or global dominance. A major evolutionary shift occurred in late 2021, with the emergence of the highly divergent Omicron BA.1 variant. Since then, all the dominant SARS-CoV-2 variants have been derived from Omicron, for reasons that remain incompletely understood. In this study, we chose to compare the replication of SARS-CoV-2 variants in a human nasal epithelium model grown at 37\u00b0C but also at 33\u00b0C, a more physiological temperature that approximates that found in the nasal cavity. In this model, Omicron showed an early replicative advantage that was more marked at nasal physiological temperature. Omicron also markedly impaired the layer of motile cilia that normally contributes to the clearance of inhaled particles from the nasal mucosa. Even though it caused tissue damage, Omicron triggered only a minimal antiviral interferon response from epithelia grown at 33\u00b0C. Thus, Omicron has the capacity to propagate rapidly but stealthily in the nasal epithelium at physiological temperature, which helps account for the efficient dissemination of this variant worldwide.","version":"1.1","doi":"10.1101/2025.05.03.652024","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.03.652023","pub_date":"2025-5-05","title":"Discovery of an Antiviral PROTAC Targeting the SARS-CoV-2 Main Protease Using an Allosteric Warhead","abstract":"Targeted protein degradation represents a new paradigm in drug development. By hijacking the cellular ubiquitin-proteasome-system pathogenic proteins are degraded via heterobifunctional molecules, referred to as proteolysis targeting chimeras (PROTACs). However, to date, only few PROTACs targeting viral or proviral proteins have been developed. To explore the possibilities and advantages of antiviral PROTACs, we have developed an antiviral PROTAC against the SARS-CoV-2 main protease (MPro) using an allosteric warhead. Here, we present the design of ten MPro degraders that were developed using pelitinib as a warhead, an allosteric binder of MPro. Among several candidates, LLP019 emerged as the most potent molecule, capable of degrading up to 90% of MPro after ectopic expression in HEK293F cells. LLP019 displayed significant antiviral activity against several variants of concern of SARS-CoV-2 in infected Calu3 cells. In conclusion, we show that the development of antiviral PROTACs using an allosteric warhead represents a promising antiviral strategy, expanding the range of possible target proteins and ligands. We present a proof-of-concept study to construct a PROTAC targeting the SARS-CoV-2 main protease (MPro) using a warhead that binds outside the catalytic pocket.","version":"1.1","doi":"10.1101/2025.05.03.652023","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.02.651855","pub_date":"2025-5-05","title":"Single-cell alternative polyadenylation analysis reveals mechanistic insights of COVID-19-associated neurological and psychiatric effects","abstract":"COVID-19 is associated with increased risks of neurological and psychiatric sequelae. Alternative polyadenylation (APA) is ubiquitous in human genes, resulting in mRNA diversity, and has been validated to play a pivotal regulatory role in the onset and progression of a variety of diseases, including viral infections. Here, we analyzed the APA usage across different cell types in frontal cortex cells from non-viral control group and COVID-19 patients, and identified functionally related APA events in COVID-19. According to our study, the poly(A) site (PAS) usage is different among cell types and following SARS-COV-2 infection. Moreover, we found the genes with significant PAS level changes affected pathways related to RNA splicing, and neuronal development and function, suggesting that survivors of COVID-19 will have a high risk of these diseases and that alternative splicing functions cause these changes. Additionally, APA usage and its correlation with gene expression levels varied across genes, some prefer short isoform that is more stable to produce more proteins, while others may be regulated by different mechanisms. A total of 267 risk genes targeted by microRNAs for common neurological and psychiatric disorders were found to undergo significant changes in APA following infection. In conclusion, our comprehensive analysis of APA in neural cells from COVID-19 patients at the single-cell level elucidated changes in APA levels in the brains of SARS-COV-2-infected patients and confirmed that these changes impair the function of the nervous system, providing important insights for COVID-19-associated sequelae. We analyzed the changes in single-cell APA level in the frontal cortex after SARS-COV-2 infection and found that PAS usage is different among cell types and changed after infection. We find that APA changes are related to RNA splicing, and neuronal development and function, such as synaptic plasticity, nervous system development, learning or memory and cognition, and the brain function may be impaired due to global gene expression and APA changes. A total of 267 risk genes for common neurological and psychiatric disorders were found to undergo significant changes in APA following infection, implying that APA alterations in specific genes could be a potential therapeutic target and predictive biomarker for neurological and psychiatric disease. APA in infection samples results in the removal/addition of miRNA target sites in the 3\u2032-UTR of genes. A total of 2404 miRNAs were predicted which suggests that the miRNAs\u2019 target site information can indicate the impact of APA events and has the potential to be used as a predictive biomarker.","version":"1.1","doi":"10.1101/2025.05.02.651855","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.11.648405","pub_date":"2025-5-04","title":"Alpha-BET: Functional labeling of envelope glycoproteins with single domain antibodies for in-virus single molecule imaging","abstract":"We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.","version":"1.2","doi":"10.1101/2025.04.11.648405","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.01.651552","pub_date":"2025-5-02","title":"Dissecting serum polyclonal antibody escape to SARS-CoV-2 variants by deep mutational learning","abstract":"The rapid emergence of SARS-CoV-2 variants harboring multiple receptor-binding domain (RBD) mutations continues to challenge the efficacy of vaccines and antibody therapeutics. While deep mutational scanning (DMS) has been instrumental in mapping single-mutation effects on antibody binding and immune escape, it remains limited in its ability to assess combinatorial mutational landscapes. Here, we extend deep mutational learning (DML), a method integrating combinatorial mutagenesis, yeast surface display, deep sequencing, and machine learning, to analyze serum polyclonal antibody escape. Human sera from COVID-19-vaccinated individuals were screened against diverse RBD variant libraries, validating 300 serum-variant interactions across 10 individuals by comparing predicted and observed binding to 30 RBD variants, confirming accurate mapping of binding and escape profiles. Model performance remained consistent across machine learning architectures, suggesting that serum binding and escape are governed by distinct, localized RBD sequence features. Notably, escape profiles were highly individualized, whereas binding signatures were more conserved, reflecting convergent epitope targeting. This approach highlights the potential of DML to generalize beyond observed RBD variants, assess cohort-specific immune breadth, and inform vaccine and therapeutic design in the face of viral evolution.","version":"1.1","doi":"10.1101/2025.05.01.651552","journal":"bioRxiv","score":null},{"id":"10.1101/2025.05.01.651343","pub_date":"2025-5-02","title":"Expression and Characterization of SARS-CoV-2 Spike Protein in Thermothelomyces heterothallica C1","abstract":"The COVID-19 pandemic demonstrated a pressing need for rapid, adaptive, and scalable manufacturing of vaccines and reagents. With the transition into an endemic disease and rising threats of other emerging pandemics, production of these biologicals requires a stable and sustainable supply chain and accessible distribution methods. In this study, we demonstrate the strength of an engineered filamentous fungal platform, Thermothelomyces heterothallica C1, for high volumetric productivity of the full-length spike glycoprotein. Spike protein produced in this system is highly thermostable and immunization of mice with spike made in C1 or mammalian platforms resulted in a similar humoral response. Additionally, it was shown that the native N-glycan profile can be redecorated with complex sialylated structures, if necessary, resulting in a more human-like glycan profile, without impacting binding characteristics as shown experimentally and in simulations. Through extensive physicochemical analysis, the C1 produced spike performs similarly to spike proteins produced in other commercially available systems. The data presented is evidence that C1 can be a strong platform for production of complex glycosylated recombinant proteins such as subunit antigen vaccines.","version":"1.1","doi":"10.1101/2025.05.01.651343","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.30.651462","pub_date":"2025-5-01","title":"Antigenic and Virological Characteristics of SARS-CoV-2 Variant BA.3.2, XFG, and NB.1.8.1","abstract":"The emergence of the SARS-CoV-2 saltation variant BA.3.2, which harbors over 50 mutations relative to its ancestral BA.3 lineage, has raised concerns about its potential to drive outbreaks similar to BA.2.86/JN.1. Concurrently, variants such as NB.1.8.1, LF.7.9, XEC.25.1, XFH, and XFG exhibit enhanced growth advantages over LP.8.1.1, necessitating a comparative analysis of their antigenic and virological characteristics. Here, we evaluated the infectivity, ACE2-binding efficiency, and immune evasion of these variants. Pseudovirus assays revealed BA.3.2\u2019s robust antibody evasion, including resistance to Class 1/4 monoclonal antibodies; however, its ACE2 engagement efficiency was markedly reduced due to a closed spike conformation, leading to low infectivity. While XFG and LF.7.9 demonstrated strong immune escape associated with A475V and N487D mutations, their reduced receptor-binding efficiency suggested a need for compensatory adaptations. In contrast, NB.1.8.1 retained high ACE2 affinity and humoral immune evasion, supporting its potential for future dominance. Collectively, BA.3.2\u2019s current profile limits its ability to compete with emerging variants like NB.1.8.1. Sustained monitoring of BA.3.2\u2019s evolution\u2014 particularly for mutations stabilizing an open RBD conformation or enhancing escape from Class 1 antibodies\u2014is essential to assess its outbreak potential.","version":"1.1","doi":"10.1101/2025.04.30.651462","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.19.545534","pub_date":"2025-4-30","title":"SARS-CoV-2 variants exhibit differential gut tropism and dysbiosis in Syrian golden hamsters","abstract":"Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) is a respiratory virus that uses angiotensin-converting enzyme 2 (ACE2) protein as an entry receptor. Thus, ACE2 expression levels in different tissues should dictate viral tropism. Notably, human ACE2 mRNA and protein levels are most abundant in the gastrointestinal tract, a pattern mirrored in Syrian golden hamsters. This prompted us to investigate the gastrointestinal tropism of SARS-CoV-2 variants of concern, including the Wuhan-like Hong Kong strain, highly pathogenic Delta, and highly transmissible but mildly pathogenic Omicron variants in hamsters. Delta was the most pathogenic in the respiratory and gastrointestinal tracts, followed by ancestral Wuhan-like and Omicron strains. In the gastrointestinal tract, viral RNA load was significant in the proximal organs such as the oesophagus and stomach, highest in the SI, and minimal to undetectable in the colon. Additionally, all three variants reduced fecal microbial diversity, with the Delta causing the highest decrease in observed features and phylogenetic diversity. Our findings highlight Delta\u2019s stronger preference for the gastrointestinal tract, suggesting a link between high virulence and gastrointestinal tropism of SARS-CoV-2 variants of concern.","version":"1.2","doi":"10.1101/2023.06.19.545534","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.01.621580","pub_date":"2025-4-30","title":"ImmunoStruct: a multimodal neural network framework for immunogenicity prediction from peptide-MHC sequence, structure, and biochemical properties","abstract":"Epitope-based vaccines are promising therapeutic modalities for infectious diseases and cancer, but identifying immunogenic epitopes is challenging. The vast majority of prediction methods only use amino acid sequence information, and do not incorporate wide-scale structure data and biochemical properties across each peptide-MHC. We present ImmunoStruct, a deep-learning model that integrates sequence, structural, and biochemical information to predict multi-allele class-I peptide-MHC immunogenicity. By leveraging a multimodal dataset of \u223c27,000 peptide-MHCs, we demonstrate that ImmunoStruct improves immunogenicity prediction performance and interpretability beyond existing methods, across infectious disease epitopes and cancer neoepitopes. We further show strong alignment with in vitro assay results for a set of SARS-CoV-2 epitopes, as well as strong performance in peptide-MHC-based cancer patient survival prediction. Overall, this work also presents a new architecture that incorporates equivariant graph processing and multimodal data integration for the long standing task in immunotherapy.","version":"1.2","doi":"10.1101/2024.11.01.621580","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.29.651261","pub_date":"2025-4-30","title":"RECUR: Identifying recurrent amino acid substitutions from multiple sequence alignments","abstract":"Identifying recurrent changes in biological sequences is important to multiple aspects of biological research -from understanding the molecular basis of convergent phenotypes, to pinpointing the causative sequence changes that give rise to antibiotic resistance and disease. Here, we present RECUR, a method for identifying recurrent amino acid substitutions from multiple sequence alignments that is fast, easy to use, and scalable to thousands of sequences. We demonstrate the utility and performance characteristics of RECUR on a data set of surface glycoprotein (S) protein sequences from SARS-CoV-2 \u2013 identifying widespread recurrent evolution throughout the protein. Structural analysis of the recurrently evolving sites revealed significant enrichment in the exposed receptor-binding S1 subunit and at the interface with the human angiotensin-converting enzyme 2 (hACE2), whereas recurrent substitutions were depleted at the trimeric interface of the S protein. Finally, in silico modelling showed that recurrent substitutions have primarily acted to stabilise the trimeric interface, but had no consistent effect at the hACE2 interface, suggesting that evolution at these sites has been shaped by opposing selection pressures \u2013 balancing the need to maintain or enhance hACE2 binding with pressures to diversify and evade host immune responses. A standalone implementation of the algorithm is available under the GPLv3 licence at https://github.com/OrthoFinder/RECUR.","version":"1.1","doi":"10.1101/2025.04.29.651261","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.28.650947","pub_date":"2025-4-29","title":"Highly potent antiviral drug candidates targeting SARS-CoV-2 nsp3 and nsp5","abstract":"Faced with the emergence of three epidemics linked to a virus from the coronavirid\u00e6 family over the last 20 years (SARS-CoV in China in 2002, MERS-CoV in Arabia in 2012, and SARS- CoV-2 worldwide in 2019), the identification of new antiviral treatments is of major public health concern. As part of the development of new drugs, several molecular modeling tools such as docking, virtual screening and molecular dynamics were combined with databases to decipher potential inhibitors of coronavirus targets. We performed a structure-based design of antiviral drugs targeting SARS-CoV-2 non-structural proteins 3 and 5 (nsp3 and nsp5) based on a high-throughput virtual screening of the ZINC15 database tranches for ligand docking followed by click chemistry with a particular attention paid to relaxed structures mimicking potential in vivo interactions. Based on the above in silico approaches, we selected two small molecules with high affinity binding for nsp3 and nsp5 named Amb929 and Amb701, respectively. We then assessed the antiviral activity of both the Amb929 and Amb701 against SARS-CoV-2 infection taking into account their potential cytotoxicity. Although both compounds inhibited SARS-CoV-2 replication in vitro, Amb929 displayed the most efficient anti-SARS-CoV-2 activity. Among these two drugs, Amb929 was less cytotoxic compared to Amb701, demonstrating an optimal anti-SARS-CoV-2 response with a high selectivity index in cell culture and a high inhibition of SARS-CoV-2 replication compared to untreated condition on a model of human airway epithelium (HAE), paving the way for the development of drugs with very potent anti-coronavirus activity.","version":"1.1","doi":"10.1101/2025.04.28.650947","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.28.650951","pub_date":"2025-4-29","title":"mRNA-delivered neutralizing antibodies confer protection against SARS-CoV-2 variant in the lower and upper respiratory tract","abstract":"Monoclonal antibodies (mAbs) have been developed as effective biological countermeasures against a range of human diseases. The high cost of antibody production and manufacturing limits its clinical application and widespread use. The mRNA-lipid nanoparticle (mRNA-LNP) is a versatile platform for development of vaccines and protein-replacement therapeutics. Since the COVID-19 pandemic, a number of neutralizing mAbs against SARS-CoV-2 have been identified with several being used clinically under emergency authorization. Herein, we report the design and generation of mRNA-LNPs expressing two SARS-CoV-2 neutralizing mAbs, 76E1 and LY1404, which respectively target the viral spike protein\u2019s fusion peptide (FP) epitope within the S2 subunit and the receptor-binding domain (RBD) within the S1 subunit. We show a single intramuscular administration of mRNA-LNPs results in efficient LY1404 and 76E1 mAb production in mice which is sustained for 7-14 days. Further, we evaluate the protective efficacy of mRNA-LNP formulations encoding the two antibodies in mouse and hamster models challenged with different SARS-CoV-2 viral strains. The data demonstrate that a single administration of mRNA-LNP encoding the more broadly neutralizing antibody 76E1 confers significant protection against the immune-evasive SARS-CoV-2 Omicron variant BQ.1 in both the upper and lower respiratory tract of the hamsters, indicating its potential impact on limiting both viral disease and viral acquisition. Together, our study expands the potential of the mRNA-LNP platform to deliver therapeutic antibodies for rapid prevention or treatment of pathogenic infections.","version":"1.1","doi":"10.1101/2025.04.28.650951","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.27.650832","pub_date":"2025-4-28","title":"Breakthrough infection elicits synergistic broadly neutralizing antibodies targeting non-dominant epitopes on RBD and NTD in an individual of inactivated SARS-CoV-2 vaccine","abstract":"Identification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. Through single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were respectively measured with ELISA and pseudovirus-based neutralization assay. Their epitopes were mapped by competition ELISA and site- directed mutation. Among a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13, and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20, and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. These four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection.","version":"1.1","doi":"10.1101/2025.04.27.650832","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.18.619070","pub_date":"2025-4-28","title":"Intrahost dynamics, together with genetic and phenotypic effects predict the success of viral mutations","abstract":"Predicting the fitness of mutations in the evolution of pathogens is a long-standing and important, yet largely unsolved problem. In this study, we used SARS-CoV-2 as a model system to explore whether the intrahost diversity of viral infections could provide clues on the relative fitness of single amino acid variants (SAVs). To do so, we analysed \u223c15 million complete genomes and nearly \u223c8000 sequencing libraries generated from SARS-CoV-2 infections, which were collected at various timepoints during the COVID-19 pandemic. Across timepoints, we found that many of the SAVs that went on to reach high frequency could be detected in the intrahost diversity of samples collected at a median of 3-22 months prior. Additionally, we found that genetic linkage patterns observed at the interhost level can also be observed in the intrahost diversity of infections. Application of machine learning models allowed us to learn highly generalisable intrahost, physiochemical and phenotypic patterns to forecast the future fitness of intrahost SAVs (r2=0.48-0.63). Most of these models performed significantly better when considering genetic linkage between mutations (r2=0.53-0.67), pointing to epistasis being an important determinant in the evolution of SARS-CoV-2. Overall, our results highlight the predictive power of intrahost diversity data, and document the evolutionary forces shaping the fitness of mutations. Such insights offer potential to forecast the emergence of future variants and ultimately inform the design of vaccine targets.","version":"1.2","doi":"10.1101/2024.10.18.619070","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.26.650794","pub_date":"2025-4-28","title":"OrfViralScan 3.0: An intuitive tool for the identification and tracking of open reading frames in viral genomes","abstract":"The identification and analysis of open reading frames (ORFs) are fundamental steps in genome annotation, which requires accurate bioinformatics tools. OrfViralScan 3.0 is presented, a desktop application developed in Java (requires Java 11 to run), featuring an intuitive graphical user interface (GUI) designed to facilitate the annotation and tracking of ORFs. The program offers functionalities to search for ORFs (initiated by ATG) in individual sequences (ORF Search), track specific ORFs based on length and location across multiple genomes or sequence sets (Track Specific ORF), preprocess FASTA files to standardize formatting (Preprocess File), and split large genomes into manageable fragments (Divide Into Fragments). The utility of OrfViralScan 3.0 is demonstrated through the analysis of the SARS-CoV-2 reference genome (NC_045512.2), the successful tracking of the Spike protein in 983 out of 1000 complete viral genomes, and the preparation of the Escherichia coli genome (NC_000913.3) for fragmented analysis. The software\u2019s capabilities, limitations, and potential future applications are discussed. An example is also included featuring the SARS-CoV-2 Spike protein, showing the folded ORF obtained with OrfViralScan 3.0 using AlphaFold 3. The program\u2019s source code is available on GitHub under the GNU GPLv3 license. roberto117343@gmail.com","version":"1.1","doi":"10.1101/2025.04.26.650794","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.23.650164","pub_date":"2025-4-24","title":"K5 polysaccharides inhibit SARS-CoV-2 infection by preventing spike-proteolytic priming","abstract":"SARS-CoV-2 spike glycoprotein is a promising drug target due to its crucial role in viral infection. Heparin, a long linear polysaccharide that inhibits SARS-CoV-2 infection by acting on spike, has limited antiviral applications due to its anticoagulant effect. E. coli K5 polysaccharides share the same structure as the heparin precursor and can be chemically modified to devoid anticoagulant activity. Here, biochemical assays and computer simulations reveal that K5 with a high degree of sulfation at O-(K5OSH) or N- and O-positions (K5NOSH) bind spike with higher affinity than heparin, preventing its binding to ACE2 and cleavage by furin. This mechanism is supported by a cell syncytia assay showing that K5OSH and K5NOSH inhibit viral infection by blocking membrane fusion. Infection assays for SARS-CoV-2 Wuhan-Hu-1 and Omicron BA.1 variants corroborate their antiviral activity. These results support the therapeutic potential of K5OSH and K5NOSH against SARS-CoV-2, with K5OSH displaying the more promising activity profile.","version":"1.1","doi":"10.1101/2025.04.23.650164","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.23.650148","pub_date":"2025-4-24","title":"In Vitro and Viral Evolution Convergence Reveal the Selective Pressures Driving Omicron Emergence","abstract":"In vitro protein evolution provides powerful insights into the amino acid sequences that underlie key biological functions. Here, we used this approach to explore the evolutionary trajectories of the SARS-CoV-2 spike protein receptor-binding domain (RBD) constrained to engage the human ACE2 receptor\u2014an essential first step in viral infection. Applying mild (LSS) or stringent (HSS) selection pressures starting from the ancestral Wuhan strain, we found that HSS, but not LSS rapidly converged on mutations characteristic of the Omicron variant. HSS resulted in fewer, but dominant, non-synonymous mutations mirroring Omicron mutations and its advanced sub-lineages. Conversely, LSS produced only some Omicron-like mutations at much lower frequencies and with incomplete representation. Notably, initiating evolution from Omicron itself resulted in high-fidelity maintenance of Omicron-defining mutations under both HSS and LSS conditions. This evolutionary pattern parallels global SARS-CoV-2 mutation trends as well as in silico simulations, emphasizing the critical role of receptor-binding constraints in shaping viral adaptation, which may be a frequent driver during zoonosis. Predominantly immune evasion associated mutations not selected in vitro. Our findings demonstrate the predictive capacity of in vitro evolution, suggesting Omicron\u2019s abrupt emergence resulted from rare, high-stringency selection, superimposed on a background of broader, milder pressures, with Omicron being the humanized SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.04.23.650148","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.23.650176","pub_date":"2025-4-24","title":"Characterization of betacoronavirus HKU-1 and OC43 internal proteins using a prototypic coronavirus","abstract":"Coronaviruses express a repertoire of accessory proteins for evading host immune responses. A small internal (I) accessory protein is expressed by the genus Betacoronavirus. Previous studies reported that the I proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 inhibit type I interferon (IFN-I) expression through distinct mechanisms and have different roles in pathogenesis. Human coronaviruses (hCoV) HKU1 and OC43 are betacoronaviruses that also encode the I protein as an accessory protein. Although hCoV-HKU1 and hCoV-OC43 predominantly cause common cold in healthy adults, susceptible individuals infected with these viruses can develop severe disease. However, the virulence factors contributing to pathogenesis after infection with common cold CoVs (CCCoVs) have not been fully characterized. In particular, the functions of the hCoV-HKU1 and hCoV-OC43 I proteins have not been previously reported. The lack of robust reverse genetic systems, tissue culture and animal models limit the study of hCoV-HKU1 and hCoV-OC43 pathogenesis. Here, we examine the role of the hCoV-HKU1 and hCoV-OC43 I proteins in pathogenesis using a prototypic coronavirus. We introduce the I proteins of hCoV-HKU1 and hCoV-OC43 independently to a neurotropic strain of mouse hepatitis virus (J2.2). J2.2 infection is well characterized with clearly defined immune responses which allows the study of I proteins in the context of authentic coronavirus infection. We show that the I protein of hCoV-HKU1, but not that of hCoV-OC43, ameliorates MHV-J2.2 pathogenesis while the I protein of MERS-CoV exacerbates disease. The presence of the hCoV-HKU1 I protein decreases virus titers and cytokine expression while the I protein of MERS-CoV leads to increased immune cell infiltration and virus titers in mice after J2.2 infection. Moreover, the I proteins of hCoV-HKU1 and hCoV-OC43 show different patterns of subcellular localization. Overall, our findings suggest that the I protein of different betacoronaviruses play unique roles in pathogenesis. Factors governing the differences in the pathogenicity between highly pathogenic human coronaviruses (SARS-CoV, MERS-CoV and SARSR-CoV-2) and seasonal coronaviruses (hCoV-HKU1 and hCoV-OC43) are not completely understood. These differences are at least in part contributed to by the accessory proteins encoded between these two groups of human coronaviruses. The use of a heterologous coronavirus infection model provides an isogenic background for the direct comparison of viral proteins encoded by various coronaviruses. In this study, we compare the role of one of the accessory proteins encoded by betacoronaviruses, the I protein, in mediating disease outcome using a prototypic coronavirus. We demonstrate that the I protein of the highly pathogenic MERS-CoV but not that of the seasonal coronaviruses HKU1 and OC43 contributes to enhanced disease in the context of MHV-J2.2 infection, highlighting that virus-specific functions of accessory proteins encoded by different hCoVs.","version":"1.1","doi":"10.1101/2025.04.23.650176","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.30.610469","pub_date":"2025-4-23","title":"SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remains a global public health threat with considerable economic consequences. The non-structural protein 16 (nsp16), in complex with nsp10, facilitates the final viral mRNA capping step through its 2\u2032-O-methylase activity, helping the virus to evade host immunity and prevent mRNA degradation. However, nsp16 regulation by host factors remains poorly understood. While various E3 ubiquitin ligases interact with SARS-CoV-2 proteins, their roles in targeting nsp16 for degradation are unclear. In this study, we demonstrate that nsp16 undergoes ubiquitination and proteasomal degradation mediated by the host E3 ligases UBR5 and MARCHF7. UBR5 induces K48-linked ubiquitination, whereas MARCHF7 promotes K27-linked ubiquitination, independently suppressing SARS-CoV-2 replication in cell cultures and in mice. Notably, UBR5 and MARCHF7 also degrade nsp16 variants from different viral strains, exhibiting broad-spectrum antiviral activity. Our findings reveal novel antiviral mechanisms of the ubiquitin-proteasome system (UPS) and highlight their potential therapeutic targets against COVID-19.","version":"1.5","doi":"10.1101/2024.08.30.610469","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.23.650177","pub_date":"2025-4-23","title":"SARS-CoV-2 causes chronic lung inflammation and impaired respiratory capacity in aged Roborovski dwarf hamsters","abstract":"Roborovski dwarf hamsters are permissive for SARS-CoV-2 infection and progress to acute viral pneumonia with profound lung tissue injury, recapitulating hallmarks of severe COVID-19 in vulnerable patient groups such as older adults. In this study, we established dwarf hamster whole body plethysmography and assessed disease severity and propensity for long-term compromise of lung recovery from severe COVID-19-like disease in young, adult, and aged animals. Aged dwarf hamsters infected intranasally with variant of concern (VOC) omicron BA.4 experienced more severe clinical signs, carried a higher lung virus load, and had a greater risk of succumbing to infection. Resting airway hypersensitivity was transiently increased in aged, but not young, dwarf hamsters 3-4 days post infection (dpi). Pharmacologically induced respiratory distress revealed compromised lung capacity in animals of both age groups at peak disease. Aged animals showed impaired respiratory function for 45 days, mounted a weaker antiviral response, and developed chronic pneumonia with lasting tissue damage. Treatment of acute disease with approved antivirals, paxlovid-like nirmatrelvir+ritonavir or molnupiravir, prevented long-term respiratory sequelae in aged animals. Nirmatrelvir+ritonavir fully suppressed transient respiratory distress and mediated complete survival of aged animals. This study shows a high positive correlation between host age and SARS-CoV-2 disease severity in dwarf hamsters, establishes a model for chronic pneumonia with impaired respiratory capacity in at-risk hosts, and demonstrates benefit of antiviral therapy of acute disease for long-term respiratory health. In the COVID-19 pandemic, the frequency of chronic respiratory insufficiency after acute SARS-CoV-2 infection was positively linked to patient age. Roborovski dwarf hamsters recapitulate hallmarks of life-threatening COVID-19 in at-risk patients that present with acute respiratory failure and prolonged respiratory incapacitation. In this study, we monitored disease progression and lung function in young and aged dwarf hamsters infected with a VOC omicron isolate and assessed the effect of antiviral treatment on long-term lung function. We established a strong correlation between host age and SARS-CoV-2 disease severity in dwarf hamsters, identified a high propensity of aged animals to develop chronic lung inflammation, and demonstrated a long-term loss of respiratory capacity in the subset of aged animals that survived the acute infection. Antiviral treatment suppressed the development of late sequelae and preserved lung function. These results have important implications for effective SARS-CoV-2 management in aged hosts at high risk of developing severe viral pneumonia with long-term impaired lung function.","version":"1.1","doi":"10.1101/2025.04.23.650177","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.22.25326224","pub_date":"2025-04-23","title":"COVID-19 Vaccination Timing, Relative to Acute COVID-19, and Subsequent Risk of Long COVID","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>Long COVID is a debilitating condition that impacts millions of Americans, but patients and clinicians have little information on how to prevent this disorder. Vaccination is a vital tool in preventing acute COVID-19 and may confer additional protection against Long COVID. There is limited evidence regarding the optimal timing of COVID-19 vaccination (i.e., vaccination schedule) to minimize the risk of Long COVID.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We applied Longitudinal Targeted Maximum Likelihood Estimation to electronic health record (EHR) data from a retrospective cohort of patients vaccinated against COVID-19 between December 2021 and September 2022. We evaluated the association between binary COVID-19 vaccination status (two or more doses vs. zero doses) and 12-month Long COVID risk among patients diagnosed with acute COVID-19 between December 2021 and September 2022. In addition, we compared the 12-month cumulative risk of Long COVID (ICD-10 code U09.9) among patients diagnosed with acute COVID-19 one to three months after vaccination, three to five months after vaccination, or five to seven months after vaccination while adjusting for relevant high-dimensional baseline and time-dependent covariates.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    We analyzed EHR data from a retrospective cohort of 1,558,018 patients. In our binary cohort (\n                    <jats:italic>n</jats:italic>\n                    = 519,980), we found that vaccinated patients had a lower risk of Long COVID than unvaccinated patients (adjusted marginal risk ratio 0.84 (0.81, 0.88)). In our longitudinal cohort (\n                    <jats:italic>n</jats:italic>\n                    = 1,085,291), we did not find a significant difference in Long COVID risk comparing patients who were diagnosed with acute COVID-19 one to three months after vaccination versus patients who were diagnosed with COVID-19 three to five months (adjusted marginal risk ratio 0.93 (95% CI 0.62, 1.41) or 5 to 7 months (adjusted marginal risk ratio 1.06 (95% CI 0.72, 1.56)) after vaccination.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>We found that COVID-19 vaccination before SARS-CoV-2 infection was protective against Long COVID, and we did not find that this protection significantly waned within 7 months after vaccination. These findings suggest that COVID-19 vaccination protects against Long COVID.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2025.04.22.25326224","journal":"medRxiv","score":null},{"id":"10.1101/2025.01.02.631145","pub_date":"2025-4-23","title":"Cross-reactive sarbecovirus antibodies induced by mosaic RBD-nanoparticles","abstract":"Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD-nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b\u2013immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike-antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD-nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs. SARS-CoV-2 variants and potential zoonotic sarbecovirus infections continue to threaten human health. Anti-SARS-CoV-2 mAbs that recognize conserved epitopes could be used prophylactically or therapeutically. We present approaches to elicit and identify cross-reactive mAbs using immunizations in animals with mosaic RBD-nanoparticles. We show that human and other mammalian immune systems can utilize different antibody gene segments to arrive at similar modes of antigen recognition, underscoring the flexibility of mammalian antibody repertoires and suggesting that experimental animals can be used to generate therapeutically-useful cross-reactive anti-RBD mAbs. The combination of mosaic-8b RBD-nanoparticles to focus the immune response and a multiplexed assay to select cross-reactive mAbs can be applied at larger scale, or against other pathogens, to identify mAbs of therapeutic and scientific potential.","version":"1.2","doi":"10.1101/2025.01.02.631145","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.22.649918","pub_date":"2025-4-23","title":"Interprotomer Crosstalk in Mosaic Viral Glycoprotein Trimers Provides Insight into Polyvalent Immunogen Co-assembly","abstract":"SARS-CoV-2 variants have demonstrated the ability to evade immune responses, leading to waves of infection throughout the pandemic. In response, bivalent mRNA vaccines, encoding the original Wuhan-Hu-1 and emerging variants, were developed to display both spike antigens. To date, it has not been determined whether co-transfection and co-translation of different SARS-CoV-2 variants results in co-assembly of mosaic heterotrimer antigens and how this may affect trimer stability, dynamics, and antigenicity. Understanding whether such mosaic heterotrimers can form and their implications for antigen structure can provide important information to guide future polyvalent vaccine design where multiple variants of an antigen are co-formulated. To investigate this, we purified mosaic spike assemblies of both genetically close (Omicron BA.2 and XBB) and distant (Omicron BA.2 and Wuhan-Hu-1 G614) strains. We found that the stability and integrity of mosaic spike trimers were maintained without misfolding or aggregation. Glycosylation profiles likewise were preserved relative to the homotrimer counterparts. Hydrogen/deuterium-exchange mass spectrometry and biolayer-interferometry were used to investigate the mosaic spike dynamics and any impact on epitope presentation and receptor binding. The Omicron-XBB heterotrimer, sharing a common fusion subunit sequence, retained protomer-specific dynamics similar to the corresponding homotrimers in antigenically important regions. The Omicron-G614 heterotrimer, co-assembling from protomers of divergent fusion subunit sequences, likewise showed overall similar dynamics and conformations in the receptor-binding subunit compared to the homotrimers. However, the incorporation of the Wuhan-Hu-1 G614 protomer led to a stabilizing effect on the relatively unstable Omicron fusion subunit in the heterotrimer. These findings reveal structural dynamic crosstalk in mosaic trimers, suggesting a potential for enhanced immunogen display and important considerations to be aware of in the use of polyvalent nucleic acid vaccines. Here we investigated the possibility of immunogen co-assembly from bivalent nucleic acid vaccines with a focus on probing potential impacts on antigen stability and epitope display. We purified mosaic heterotrimers composed of Omicron BA.2 and Wuhan-Hu-1 G614 protomers as well as those composed of Omicron BA.2 and XBB protomers. Both mosaic heterotrimers maintained their structural integrity, morphology, and N-glycosylation profiles. Using hydrogen/deuterium-exchange mass spectrometry, we demonstrated that both types of heterotrimers preserved strain-specific dynamics in critical antigenic regions, comparable to their homotrimer counterparts. Crosstalk between fusion subunits of Omicron BA.2 and Wuhan-Hu-1 G614 revealed a stabilizing effect of the G614 protomer on the inherently less stable fusion subunit in Omicron. Our findings provide insight into the structural dynamic profiles of co-expressed immunogen display, highlighting important considerations for polyvalent formulations that are being pursued to provide broad protection against highly variable pathogens.","version":"1.1","doi":"10.1101/2025.04.22.649918","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478476","pub_date":"2025-4-23","title":"WITHDRAWN: SARS-CoV-2 invades cognitive centers of the brain and induces Alzheimer\u2019s-like neuropathology","abstract":"The authors have withdrawn this manuscript because the data is being questioned by the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.4","doi":"10.1101/2022.01.31.478476","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.18.649623","pub_date":"2025-4-22","title":"CXCL13/CXCR5 Chemokine Axis Promotes CXCR5+CD19+ B-Cell and Follicular/Effector CXCR5+CD4+ T-Cell Responses in the Lungs Associated with Protection from Severe and Fatal COVID-19 Following Infection with Pathogenic SARS-CoV-2 Delta Variant","abstract":"Chemokines play an important role in shaping lung innate and adaptive immunity to pulmonary infections and diseases. However, the role of CXC ligand 13 (CXCL13), a chemokine homeostatically produced by various lung cell types, in the protection from SARS-CoV-2 infection and disease remains controversial. Some studies reported that asymptomatic patients who survived severe COVID-19 had CXCL13-dominated mucosal immune responses in the lungs early during infection. In contrast, other studies reported that a high level of CXCL13 was associated with severity and mortality in COVID-19 patients. In this study, to determine the direct role of CXCL13 in SARS-CoV-2 infection and disease, we generated CXCL13-/-K18-hACE2 mice, that are both transgenic for ACE2 and deficient in CXCL13 and compared their infection and COVID-19-like disease symptoms with those in wild-type K18-hACE2 transgenic mouse littermates following intranasal inoculation with the pathogenic SARS-CoV-2 delta variant (B.1.617.2). Compared to age- and gender-matched SARS-CoV-2 infected wild-type K18-hACE2 mice, SARS-CoV-2 infected CXCL13-/-K18-hACE2 deficient mice exhibited (i) higher viral load in the lungs; (ii) severe COVID-19-like lung pathology; (iii) exacerbated weight loss; (iv) increased mortality. The apparent severe COVID-19-like symptoms in CXCL13-/-K18-hACE2 deficient mice were associated with: (i) significantly lower frequencies of functional lung-resident C-X-C chemokine receptor 5+ (CXCR5)+CD19+ B cells, follicular CXCR5+CD4+ helper T cells (Tfh cells), and IFN-\u03c8+TNF-\u03b1+GzmB+Ki67+effector CD4+ Th1 cells; and (ii) a significant reduction in the levels of SARS-CoV-2-Spike specific Th1 associated IgG1 and IgG2b antibody isotypes. These findings corroborate previous human reports suggesting a critical role of the CXCL13/CXCR5 chemokine axis in the protective B- and T-cell mucosal immunity to SARS-CoV-2 infection and disease, offering a potential new immunotherapeutic target for treatment.","version":"1.1","doi":"10.1101/2025.04.18.649623","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.17.648944","pub_date":"2025-4-22","title":"Antibody escape drives emergence of diverse spike haplotypes resembling variants of concern in persistent SARS-CoV-2 infections","abstract":"Evolution of SARS-CoV-2 in long-term persistent infections is hypothesised to be a major source of variants of concern (VOC). However, determination of viral subpopulations by linkage of intra-host variants into haplotypes has been limited by commonly used genomic sequencing techniques. We developed a sequencing and analysis methods for identifying full-length intra-host spike haplotypes and analysed their diversification over the course of persistent infections in individuals with inherited or acquired immunodeficiencies. Mutations frequently emerged in positions found in VOCs that confer escape from neutralising antibodies, while selection analyses showed strong evidence of positive selection and identified specific amino acid positions undergoing selection. In a single chronic infection lasting more than 500 days from the first wave of the pandemic, we detailed the evolution of spike as it gradually acquired mechanisms to evade both autologous and heterologous neutralising antibodies, redolent of Omicron variants. This provides one of the strongest pieces of evidence for persistent infections being the source of immune-evasive variants with accelerated evolution, underscoring their impact on the evolutionary trajectory of SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.04.17.648944","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.16.649090","pub_date":"2025-4-22","title":"Frequency Chaos Game Representation - Singular Value Decomposition for Alignment-Free Phylogenetic Analysis","abstract":"This paper introduces \u201dSVD-FCGR,\u201d a scalable and efficient frame-work for phylogenetic analysis using Singular Value Decomposition (SVD) on Frequency Chaos Game Representation (FCGR). Unlike traditional MSA techniques, SVD-FCGR handles large datasets with lower computational complexity. It supports both genome-wide and gene-specific analyses, as demonstrated with datasets of Japanese Encephalitis Virus (JEV), Hepatitis B Virus (HBV), Human Immunodeficiency Virus(HIV-1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). For COVID-19, gene-level analysis of surface glycoprotein highlighted mutations affecting viral adaptability, while the envelope gene remained conserved. The method produced detailed phylogenetic trees, surpassing tools like MEGA in resolution and scalability. Validation from the AF Project ranked it 11th among alignment-free methods, confirming its reliability and adaptability.","version":"1.1","doi":"10.1101/2025.04.16.649090","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.17.649446","pub_date":"2025-4-21","title":"Recombination alters the receptor binding and furin cleavage site in novel bat-borne HKU5-CoV-2 coronavirus","abstract":"HKU5-CoV-2 is a new bat-infecting coronavirus phylogenetically related to MERS-CoV. It has recently been confirmed that HKU5-CoV-2 can enter human cells and organoids in vitro via the ACE2 receptor, raising concerns about its pandemic potential due to zoonotic spillover. Whether recombination has an influence on HKU5-CoV-2 is completely unclear to date. Here we report the first evidence of HKU5-CoV-2 viral recombination, in association with mutations at the receptor binding domain (RBD) and S1/S2 furin cleavage site (FCS) of the spike protein. Using linkage disequilibrium and haploblock analysis, we identified that 167 recombination hotspots and 27 haploblocks. SNP23016/23043/23064/23156/23193/23285 at the RBD and SNP23833/23847 at the FCS are recombinant hotspots. Our results suggest that recombination may lead to the substitution at RBD residue 498 (Thr498Val/Val498Thr, Thr498Ile/Ile498Thr), which Thr498 directly contacts the ACE2 receptor. Recombination also causes Ser723 deletion/insertion and Ser729Ala substitution at the FCS. These mutations could affect host tropism and change furin cleavage activity. Our results indicate that recombination has played a critical role in HKU5-CoV-2 evolution and infectivity.","version":"1.2","doi":"10.1101/2025.04.17.649446","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.17.638720","pub_date":"2025-4-21","title":"JAK inhibitors remove innate immune barriers facilitating viral propagation","abstract":"Janus kinase (JAK) inhibitors are small-molecule therapeutics that reduce inflammation in autoimmune and inflammatory diseases by modulating the JAK-STAT pathway. While effective in alleviating immune-mediated conditions, JAK inhibitors can impair antiviral defences by suppressing interferon (IFN) responses, potentially increasing susceptibility to viral infections. This study investigates the pro-viral mechanism of JAK inhibitors, focusing on baricitinib, across various cell lines, organoids, and viral strains, including a recombinant Rift Valley fever virus, influenza A virus, SARS-CoV-2, and wild type adenovirus. Our findings demonstrate that baricitinib suppresses transcription of IFN-stimulated genes in non-infected cells (ISGs) which is triggered by type I IFNs produced by infected cells, facilitating viral propagation. The pro-viral effects were influenced by viral load, inhibitor concentration, and structural characteristics of the compound. These results underscore the dual effects of JAK inhibitors: reducing inflammation while potentially exacerbating viral infections. Additionally, the findings highlight opportunities to leverage JAK inhibitors for viral research, vaccine production, and drug screening.","version":"1.2","doi":"10.1101/2025.02.17.638720","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.17.649456","pub_date":"2025-4-18","title":"SM3DD with Segmented PCA: A Comprehensive Method for Interpreting 3D Spatial Transcriptomics","abstract":"We developed Standardised Minimum 3D Distance (SM3DD), an entirely cell segmentation/annotation-free approach to the analysis of spatial RNA datasets, using it to compare lung tissue from 16 clinically normal individuals to those of 18 SARS-CoV-2 patients who died from acute respiratory distress syndrome. RNA spatial coordinates were determined using the CosMx\u2122 Spatial Molecular Imager (Bruker Spatial Biology, US). For each individual transcript location, we calculated the three-dimensional distances to the nearest transcript of each transcript type, standardising the distances to each transcript type. Mean SM3DDs were compared between normal and SARS-CoV-2 patients. Notably, hierarchical clustering of the directional log10(P) values organized genes by functionality, making it easier to interpret biological contexts and for FKBP11, where a decrease in distance to MZT2A was the most significant difference, suggesting a role in interferon signaling. Using a segmented principal components analysis of the entire SM3DD dataset, we identified multiple pathways, including \u2018SARS-CoV-2 infection\u2019, even though the assay did not include any SARS-CoV-2 transcripts.","version":"1.1","doi":"10.1101/2025.04.17.649456","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.16.649178","pub_date":"2025-4-17","title":"Deletion of a KSF Motif Attenuates NSP1 Host Cell Translation Shutoff and Impairs SARS-CoV-2 Virulence","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with profound social and economic consequences. The viral spike (S) protein has been identified as a key determinant of SARS-CoV-2 pathogenicity. In this study, we demonstrate that the Omicron BA.4 and BA.5 variants, which have closely related S proteins, exhibit different virulence in K18-hACE2 transgenic mice. A comparison of genomic sequences revealed key differences between variants BA.4 and BA.5, including a three amino acid deletion (\u0394KSF) in the linker region of the non-structural protein 1 (NSP1) in BA.4. Using reverse genetic systems, we engineered a recombinant (r)SARS-CoV-2 BA.5 expressing BA.4 NSP1, which was significantly attenuated in vivo, similar to the natural BA.4 isolate, compared to rBA.5 wild-type (WT). This finding indicates that NSP1 is responsible, at least in part, for the differences in virulence between BA.4 and BA.5. Mechanistically, BA.4 NSP1 showed a reduced ability to inhibit host gene translation compared to BA.5 NSP1. Notably, a rSARS-CoV-2 WA1 original strain containing the same \u0394KSF in NSP1 was also attenuated in vivo compared to rWA1 WT. Together, these findings highlight the contributions of the NSP1 linker region to inhibiting host gene expression and SARS-CoV-2 pathogenicity, as well as the feasibility of targeting NSP1 for the rational design of live-attenuated vaccines and/or antivirals. Understanding why some SARS-CoV-2 variants cause more severe disease than others is crucial for proper management or for the rational design of prophylactic and therapeutic treatments. While most studies focus on the spike (S) protein, we found that another viral protein, NSP1, also plays a key role in disease severity among SARS-CoV-2 variants. A small deletion in NSP1, present in the Omicron BA.4 variant, weakens the virus ability to shut down the host\u2019s immune response, making BA.4 less severe than BA.5. When we introduced the same deletion into the original SARS- CoV-2 WA1 strain, the virus also became less harmful. This discovery suggests that NSP1 is an important virulence factor and supports the feasibility of targeting NSP1 for the development of new prophylactic and therapeutic treatments against SARS-CoV-2. By uncovering NSP1\u2019s role in pathogenesis, our study provides insights that could help in designing better strategies to combat future variants of SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.04.16.649178","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.15.649027","pub_date":"2025-4-17","title":"Exploring Diverse Binding Mechanisms of Broadly Neutralizing Antibodies S309, S304, CYFN-1006 and VIR-7229 Targeting SARS-CoV-2 Spike Omicron Variants: Integrative Computational Modeling Reveals Balance of Evolutionary and Dynamic Adaptability in Shaping Molecular Determinants of Immune Escape","abstract":"Evolution of SARS-CoV-2 has led to the emergence of variants with increased immune evasion capabilities, posing significant challenges to antibody-based therapeutics and vaccines. The cross-neutralization activity of antibodies against Omicron variants is governed by a complex and delicate interplay of multiple energetic factors and interaction contributions. In this study, we conducted a comprehensive analysis of the interactions between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and four neutralizing antibodies S309, S304, CYFN1006, and VIR-7229. Using integrative computational modeling that combined all-atom molecular dynamics (MD) simulations, mutational scanning, and MM-GBSA binding free energy calculations, we elucidated the structural, energetic, and dynamic determinants of antibody binding. Our findings reveal distinct dynamic binding mechanisms and evolutionary adaptation driving broad neutralization effect of these antibodies. We show that S309 targets conserved residues near the ACE2 interface, leveraging synergistic van der Waals and electrostatic interactions, while S304 focuses on fewer but sensitive residues, making it more susceptible to escape mutations. The analysis of CYFN-1006.1 and CYFN-1006.2 antibody binding highlights broad epitope coverage with critical anchors at T345, K440, and T346, enhancing its efficacy against variants carrying the K356T mutation which caused escape from S309 binding. Our analysis of broadly potent VIR-7229 antibody binding to XBB.1.5 and EG.5 Omicron variants emphasized a large and structurally complex epitope, demonstrating certain adaptability and compensatory effects to F456L and L455S mutations. Mutational profiling identified key residues crucial for antibody binding, including T345, P337, and R346 for S309, and T385 and K386 for S304, underscoring their roles as evolutionary 'weak spots' that balance viral fitness and immune evasion. The results of this energetic analysis demonstrate a good agreement between the predicted binding hotspots and critical mutations with respect to the latest experiments on average antibody escape scores. The results of this study dissect distinct energetic mechanisms of binding and importance of targeting conserved residues and diverse epitopes to counteract viral resistance. Broad-spectrum antibodies CYFN1006 and VIR-7229 maintain efficacy across multiple variants and achieve neutralization by targeting convergent evolution hotspots while enabling tolerance to mutations in these positions through structural adaptability and compensatory interactions at the binding interface. The results of this study underscore the diversity of binding mechanisms employed by different antibodies and molecular basis for high affinity and excellent neutralization activity of the latest generation of antibodies.","version":"1.1","doi":"10.1101/2025.04.15.649027","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.02.565396","pub_date":"2025-4-16","title":"Long-term serial passaging of SARS-CoV-2 reveals signatures of convergent evolution","abstract":"Understanding viral evolutionary dynamics is crucial to pandemic responses, prediction of virus adaptation over time, and virus surveillance for public health strategies. Whole-genome sequencing (WGS) of SARS-CoV-2 has enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a complementary controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. In this study, nine virus lineages, including four \u201cvariants of concern\u201d and three former \u201cvariants under investigation\u201d, were sampled over \u226533 serial passages (range 33-100) in Vero E6 cells. WGS was used to examine virus evolutionary dynamics and identify key mutations with implications for fitness and/or transmissibility. Viruses accumulated mutations regularly during serial passaging. Many low-frequency variants were lost but others became fixed, suggesting either in vitro benefits, or at least a lack of deleterious effect. Mutations arose convergently both across passage lines, and when compared with contemporaneous SARS-CoV-2 clinical sequences. These mutations included some hypothesised to drive lineage success through host immune evasion (e.g. S:A67V, S:H655Y). The appearance of these mutations in vitro suggested key mutations can arise convergently even in the absence of a multicellular host immune response through mechanisms other than immune-driven mutation. Such mutations may provide other benefits to the viruses in vitro, or arise stochastically. Our quantitative investigation into SARS-CoV-2 evolutionary dynamics spans the greatest number of serial passages to date, and will inform measures to reduce the effects of SARS-CoV-2 infection on the human population. The ongoing evolution of SARS-CoV-2 remains a challenge for long term public health efforts to minimise the effects of COVID-19. Whole-genome sequencing of outbreak cases has enabled global contact tracing efforts and the identification of mutations of concern within the virus\u2019 genome. However, complementary approaches are necessary to inform our understanding of virus evolution and clinical outcomes. Here we charted evolution of the virus within a controlled cell culture environment, focusing on nine different virus lineages. Our approach demonstrates how SARS-CoV-2 continues to evolve readily in vitro, with changes mirroring those seen in outbreak cases globally. Findings of the study are important for i) investigating the mechanisms of how mutations arise, ii) predicting the future evolutionary trajectory of SARS-CoV-2, and iii) informing treatment and prevention design.","version":"1.3","doi":"10.1101/2023.11.02.565396","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.15.648942","pub_date":"2025-4-16","title":"Characterization and evolutionary history of novel SARS-CoV-2-related viruses in bats from Cambodia","abstract":"Circulating bat coronaviruses present a significant pandemic threat, yet our understanding of their genetic diversity and evolutionary dynamics remains limited. Over 3 years, we sampled 1,462 bats in Cambodia\u2019s Steung Treng province, identifying extensive and diverse coronaviruses co-circulation. Using metatranscriptomic and amplicon sequencing, we generated 33 complete sarbecovirus genomes, revealing novel lineages that cluster into four distinct groups, each associated with different Rhinolophus bat species. Our analysis highlights rapid migration and recombination of sarbecovirus lineages over short distances and timescales. Of note, the receptor-binding domains of two novel viral groups exhibit high similarity to SARS-CoV-2, and pseudovirus assays confirmed the ability of this spike protein to mediate entry into cells expressing human ACE2, suggesting a potential zoonotic risk. The observed genetic diversity underscores the urgent need for continuous surveillance to identify high-risk animal-to-human interfaces and inform pandemic preparedness.","version":"1.1","doi":"10.1101/2025.04.15.648942","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.19.629473","pub_date":"2025-4-16","title":"A Large Language Model Guides the Affinity Maturation of Antibodies Generated by Combinatorial Optimization Algorithms","abstract":"The ability of an antibody to bind an antigen with high specificity and strength (i.e., its binding affinity) are critical properties in the design of neutralizing antibodies. Recent technical advances in AI and a surge of experimental data on antigen-antibody interaction are driving innovations in the design and optimization of antibodies via affinity maturation. Here we introduce Ab-Affinity, a novel large language model which can accurately predict the binding affinity of specific antibodies against a target peptide within the SARS-CoV-2 spike protein. When used in conjunction with a genetic algorithm and simulated annealing, Ab-Affinity can generate novel antibodies with more than a 160-fold increase in predicted binding affinity compared to those obtained experimentally. Our experimental results show that the synthetic antibodies produced by Ab-Affinity have strong predicted biophysical properties. Molecular docking and molecular dynamics simulation of binding interactions of the best synthetic antibodies show enhanced interactions and stability on the target peptide epitope. In general, antibodies generated by Ab-Affinity are superior to those obtained with other existing computational methods.","version":"1.2","doi":"10.1101/2024.12.19.629473","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.13.648564","pub_date":"2025-4-15","title":"Role of Dengue in SARS-CoV-2 Evolution in Dengue Endemic Regions","abstract":"Evidence suggests that dengue virus (DV) antibodies (Abs) and SARS-CoV-2 Abs were cross-reactive, resulting in reciprocal serological cross-interaction and providing cross-protection in populations in co-endemic regions. It became apparent from the present study that SARS-CoV-2 variants were preferentially selecting mutation(s)/deletions in the spike to evade interaction with DV Abs. We looked at mutations in the SARS-CoV-2 spike protein and how they affected the antigenicity, focusing on successively emergent major variants such as B.1.1.7 (Kent), B.1.617 (Delta), B.1.617.2.1 (Delta-Plus), B.1.1.529 (Omicron-BA.1) and B.1.1.529 (Omicron-BA.2). To further understand the effect of cross-reactive DV Abs on SARS-CoV-2 spike mutation(s), structural studies and docking simulations were performed using DV-2 envelope (E) Abs. Signature mutation (s) in spike variants implied that majority of the above changes in the spike were driven by DV Abs rather than immune pressure of SARS-CoV-2 (preceding variants). This was supported by the fact that the lately emergent SARS-CoV-2 variants like Omicrons (2022-23) were at least 50% less cross-reactive to DV compared to preceding predominant strains. This was further supported by the observed cross-binding of pre-pandemic DV Ab-positive serums to spike synthetic peptides in ELISA, designed from certain regions of the spike RBD which showed maximum cross-interactions with DV E Abs.","version":"1.1","doi":"10.1101/2025.04.13.648564","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.13.648648","pub_date":"2025-4-15","title":"A Pan-Beta-Coronavirus Vaccine Bearing Conserved and Asymptomatic B- and T-Cell Epitopes Protect Against Highly Pathogenic Delta and Highly Transmissible Omicron SARS-CoV-2 Variants of Concern","abstract":"SARS CoV-2 continues to evolve into new viral variants due to mutation, primarily in the Spike protein. Existing Spike-based vaccines are less effective because these variants can be more transmissible and evade vaccine-induced immunity. By targeting more conserved, Spike, and non-Spike, viral antigens using both arms of the adaptive immune system, i.e. B and T cells, we aim to reduce the reliance on neutralizing antibodies and avoid potential mismatches between the COVID-19 vaccines and circulating virus strains. In this way, enhanced immune memory function and broad-spectrum protection against existing and evolving virus variants can be attained. We have developed a mRNA-LNP-based multi-epitope vaccine incorporating conserved CD8+ T-cell, CD4+ T-cell, and B-cell epitopes. These conserved epitopes were selected as being highly recognized by B- and T-cells from unvaccinated asymptomatic COVID-19 patients. To evaluate the effectiveness of this multi-epitope \u201casymptomatic\u201d vaccine, we utilized a novel triple transgenic h-ACE-2-HLA-A2/DR mouse model to enable the assessment of human T cell epitopes. Key observations include induction of: (i) robust protection against infection and disease caused by SARS-CoV-2 Delta (B.1.617.2) and Omicron (XBB.1.5) variants, as measured by reduced weight loss, virus replication, and lung pathology; (ii) strong antibody responses,; and (iii) potent SARS-CoV-2 epitope-specific IFN-\u03b3-producing CD4+/CD8+ T cells and Follicular helper CD4+ T (TFH) cells. These data support the strategy of targeting B-cells and T-cells directed toward highly conserved and \u201casymptomatic\u201d epitopes, from both structural and non-structural viral protein antigens, to generate a broad-spectrum protective immunity to minimize disease impact across multiple SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2025.04.13.648648","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.14.648681","pub_date":"2025-4-15","title":"Bat sarbecovirus WIV1-CoV bears an adaptive mutation that alters spike dynamics and enhances ACE2 binding","abstract":"SARS-like betacoronaviruses (sarbecoviruses) endemic in bats pose a significant zoonotic threat to humans. Genetic pathways associated with spillover of bat sarbecoviruses into humans are incompletely understood. We previously showed that the WT spike of the rhinolophid bat coronavirus SHC014-CoV has poor entry activity and uncovered two distinct genetic pathways outside the receptor-binding domain (RBD) that increased spike opening, ACE2 binding, and cell entry. Herein, we show that the widely studied bat sarbecovirus WIV1-CoV is likely a cell culture-adapted variant of Rs3367-CoV, which was sequenced from the same population of rhinolophid bats as SHC014-CoV. We demonstrate that the acquisition of a single amino-acid substitution in the \u2018630 loop\u2019 of the S1 subunit was the key spike adaptation event during the successful isolation of WIV1-CoV, and that it enhances spike opening, virus-receptor recognition, and cell entry in much the same manner as the substitutions we previously identified in SHC014-CoV using a pseudotype system. The conformational constraints on both the SHC014-CoV and Rs3367-CoV spikes could be alleviated by pre-cleaving them with trypsin, suggesting that the spike-opening substitutions arose to circumvent the lack of S1\u2013S2 cleavage. We propose that the \u2018locked-down\u2019 nature of these spikes and their requirement for S1\u2013S2 cleavage to engage ACE2 represent viral optimizations for a fecal-oral lifestyle and immune evasion in their natural hosts. These adaptations may be a broader property of bat sarbecoviruses than currently recognized. The acquisition of a polybasic furin cleavage site at the S1\u2013S2 boundary is accepted as a key viral adaptation for SARS-CoV-2 emergence that overcame a host protease barrier to viral entry in the mammalian respiratory tract. Our results suggest alternative spillover scenarios in which spike-opening substitutions that promote virus-receptor binding and entry could precede, or even initially replace, substitutions that enhance spike cleavage in the zoonotic host. Recent epidemic-causing coronaviruses, including SARS-CoV-2, originated in bats. Large numbers of such viruses circulate in bats and pose clear and present risks to humans. However, our incomplete understanding of the variables that influence viral \u2018spillover\u2019 into new hosts challenges attempts to stratify viruses by threat level. We showed previously that the entry spike of the bat coronavirus SHC014-CoV, closely related to SARS-CoV-1 and SARS-CoV-2, exists largely in a closed conformation that is incompatible with its binding to the viral receptor, ACE2, and that genetic changes in key control sequences \u2018open\u2019 the spike and unlock its entry activity. Here, we extend these findings to a second bat coronavirus. We demonstrate that WIV1-CoV, a highly studied virus that was previously isolated from the same population of Chinese horseshoe bats, is in fact a variant of the bat coronavirus Rs3367-CoV that acquired a genetic change in the same hotspot region of the spike during its propagation in cell culture. Our findings support the idea that the closed spikes of at least some (and likely, many) bat coronaviruses, while exquisitely adapted to their natural milieu in the bat digestive system, suffer poor functional activity outside this milieu, imposing a barrier to viral spillover that must be overcome through viral adaptation. Further, we identify spike genetic changes that overcome this deficit and may have value as prognostic markers of zoonotic risk.","version":"1.1","doi":"10.1101/2025.04.14.648681","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.14.648815","pub_date":"2025-4-15","title":"Covering All Bases: A Computational Method to Design Broad-spectrum T-cell-inducing Vaccines Applied to Betacoronaviruses","abstract":"Antigenically diverse pathogens, such as coronaviruses, pose substantial threats to global health. This highlights the need for effective broad-spectrum vaccines that elicit robust immune responses in a large proportion of the human population against a wide array of pathogen variants. Here, we introduce Spectravax, an AI-enabled computational method to design broad-spectrum vaccines that account for genetic diversity in both the host and pathogen populations. Using Spectravax, we designed a nucleocapsid (N) antigen to elicit cross-reactive immune responses to viruses from the Sarbecovirus and Merbecovirus subgenera of Betacoronaviruses. In silico analyses demonstrated superior predicted host and pathogen coverage for Spectravax compared to wild-type sequences and existing computational designs. Experimental validation in mice supported these predictions: Spectravax N elicited robust immune responses to SARS-CoV, SARS-CoV-2, and MERS-CoV\u2014the three coronaviruses responsible for major outbreaks in humans since 2002\u2014while wild-type and existing computational designs elicited limited responses. Furthermore, we were able to identify the MERS-CoV N epitopes responsible for Spectravax\u2019s cross-reactivity. Thus, we advance the rational design of broad-spectrum vaccines for pandemic preparedness.","version":"1.1","doi":"10.1101/2025.04.14.648815","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.27.645784","pub_date":"2025-4-15","title":"Energy calculations for atomic oxygen liberation from hypohalous acids biosynthesized by mammalian peroxidases","abstract":"The ultimate process of the mammalian immunological system is destruction of invasive microbes by peroxidase (PO) activities. Three enzymes, lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) require two substrates for this task. The first is H2O2 and the second either I\u2212, Br\u2212, Cl\u2212 or SCN\u2212. All POs are able to synthesize hypoiodous acid and OSCN\u2212 but two can create HOBr (EPO and MPO) and only MPO can biosynthesize HOCl. Using density-functional theory (DFT) and following first principles of molecular modelling we investigated the energetics of hypohalous acid breakdowns by calculating the internal energy differences between the reactants and products. Atomic oxygen (ATOX) and simple halide acids are the energetically favoured products with halide cations and hydroxide ion being minor products. There is nothing subtle about ATOX, it is a very destructive species. ATOX production explains the documented ability of POs to indiscriminately and rapidly destroy invasive microbes and the role of MPO initiating inflammation. Previously unrecognised, the biosynthesis of ATOX also explains why mammalian POs are restricted to surfaces or special cells which are themselves destroyed after PO activation. This chemistry is the probable reason why dietary iodine, uniquely provided by the Japanese diet, helped to significantly reduce deaths from the SARS-CoV-2 viral agent during the COVID-19 pandemic in Japan. The energetics of hypohalous acid, HOI, HOBr and HOCl, breakdown into atomic oxygen (ATOX) or halide cation were calculated using density-functional theory The release of neutral ATOX from all three hypohalous acids is greatly preferred over halide cation and hydroxide production ATOX produced by mammalian peroxidases is incredibly powerful and largely responsible for the ultimate destruction of most invasive, especially airborne microbes \n\n\n\n","version":"1.2","doi":"10.1101/2025.03.27.645784","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.11.648353","pub_date":"2025-4-14","title":"Customizable host and viral transcript enrichment using CRISPR-Cas9 long-read sequencing for isoform discovery and validation","abstract":"Long-read RNA sequencing has been broadly utilized to examine the diversity of transcriptomes, understand differential expression and discover novel transcript isoforms. One of the major limitations of whole transcriptome sequencing is the difficulty in obtaining sufficient depth for low abundant transcripts. Methods which address this are either difficult to scale or customize: long- range PCR is customizable but difficult to scale beyond a few targets; probe hybridization panels are suited for scaling but require substantial investment to customize. In this study, we adopted RNA-guided CRISPR-Cas9 nuclease-based enrichment to target specific human and SARS-CoV-2 transcripts followed by long-read sequencing, utilizing minimal number of guide RNAs per target isoform. Our findings demonstrate that the CRISPR-Cas system is a highly effective method for customizable long-read sequencing of target transcripts while maintaining the accuracy of relative gene expression levels. The results highlight a valuable method for future research on transcript enrichment for isoform identification and low abundance transcript detection in infectious disease diagnosis.","version":"1.1","doi":"10.1101/2025.04.11.648353","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.13.648591","pub_date":"2025-4-13","title":"Zinc ions inactivate influenza virus hemagglutinin and prevent receptor binding","abstract":"Influenza A viruses (IAV) cause seasonal flu and occasional pandemics. In addition, the potential for the emergence of new strains presents unknown challenges for public health. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of these viruses. Metal ions embedded into PPE have been demonstrated to inactivate respiratory viruses, but the underlying mechanism of inactivation and potential for resistance is presently not well understood. In this study, we found that zinc ions directly impact the binding of influenza virus to host cell sialic acid receptors using hemagglutination assays. Quantifying this effect, we observed that zinc ions inhibit IAV receptor binding within 1 minute of exposure in a concentration-dependent manner. Maximum inhibition was achieved within 1 hour and irreversible for at least 24 hours. Serial passaging of IAV in the presence of zinc did not result in resistance. Overall, these findings are in line with previous observations indicating that zinc-embedded materials impact the IAV hemagglutinin and SARS-CoV-2 spike proteins, and support work toward developing robust, passive, self-cleaning antiviral barriers in PPE.","version":"1.1","doi":"10.1101/2025.04.13.648591","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.11.648322","pub_date":"2025-4-11","title":"The Role of Ribose Modifications on the Structural Stability of Nucleotide Analogs with \u03b1-thiotriphosphate at the Active Site of SARS-CoV-2 RdRp","abstract":"As a promising drug target, RNA-dependent RNA polymerase (RdRp) has attracted much attention recently due to its notable conserved active site, especially in the context of COVID-19 spreading. To inhibit the function of RdRp, nucleotide analog is a common choice for acting as a chain terminator or RNA corruptor. Although some nucleotide analogs have shown the ability to terminate the extension of a nascent strand of SARS-CoV-2, most of them are likely to be excised due to the proofreading of SARS-CoV-2 nsp14/nsp10. A previous experimental study found that introducing sulfur modification into analogs\u2019 phosphate moieties (\u03b1-thiotriphosphate; \u201cthio\u201d modification) can break the bottleneck. For instance, Sofosbuvir with \u03b1-thiotriphosphate modification successfully escaped from the excision of nsp14/10. However, it is unknown how the \u03b1-thiotriphosphate affects the structural stability of nucleotide analogs with different ribose modifications at the active site of SARS-CoV-2 RdRp. Thus, in this study, we performed extensive molecular dynamics simulations on four nucleotide analogs with \u03b1-thiotriphosphate to elucidate what kind of ribose modification combined with \u201cthio\u201d modification would benefit the analog\u2019s structural stability at the active site. We found that the \u201cthio\u201d modification led to the torsion of phosphate moieties, profoundly affecting the overall conformation of analogs and surrounding residues, determining the Watson-Crick base pairing and catalytic efficiency. Interestingly, chemical modification on the ribose, especially the 1\u2019 and 3\u2019-ribose positions, increases the structural stability of analogs through hydrogen bond interactions. Our results revealed nucleotide analogs\u2019 structural and dynamical features with \u201cthio\u201d modification at the active site, which may contribute to future drug design or repurposing aimed at the SARS-CoV-2 RdRp.","version":"1.1","doi":"10.1101/2025.04.11.648322","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.10.648177","pub_date":"2025-4-11","title":"Mucin anchoring of SARS-CoV-2 neutralizing nanobinders increases intranasal antiviral efficacy","abstract":"Respiratory viruses represent a major threat for human health, and despite effectiveness to limit the severity of the disease, they fail to block transmission. Recent advances have shown that the nasal cavity is the primal infection site of respiratory viruses, and thus represents a very attractive target for prophylactic treatment with antivirals in order to limit virus dissemination. However, mucociliary clearance limits the efficiency of a local treatment in the nasal cavity. We have previously developed a potent anti-Spike nanobinder blocking SARS-CoV-2 entry. Here, it was used as a proof of concept to show the strength of anchoring this synthetic antiviral to the mucin layer with a mucin-binding domain, increasing its residence time in the nasal cavity up to 6 hours post-instillation. This was very effective as a prophylactic treatment to limit infection of sentinel hamsters. Our strategy could be extended to antivirals against other major respiratory viruses such as RSV or influenza viruses, but also in other diseases by targeting specific epithelia increasing residence time and local concentration of the drug.","version":"1.1","doi":"10.1101/2025.04.10.648177","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.10.647895","pub_date":"2025-4-11","title":"Spatial Clustering of Interface Residues Enhances Few-Shot Prediction of Viral Protein Binding","abstract":"Predicting protein binding affinities across large combinatorial mutation spaces remains a critical challenge in molecular biology, particularly for understanding viral evolution and antibody interactions. While combinatorial mutagenesis experiments provide valuable data for training predictive models, they are typically limited due to experimental constraints. This creates a significant gap in our ability to predict the effects of more extensive mutation combinations, such as those observed in emerging SARS-CoV-2 variants. We present PROXICLUST, which strategically combines smaller combinatorial mutagenesis experiments to enable accurate predictions across larger combinatorial spaces. Our approach leverages the spatial proximity of amino acid residues to identify potential epistatic interactions, using these relationships to optimize the design of manageable-sized combinatorial experiments. By combining just two small combinatorial datasets, we achieve accurate binding affinity predictions across substantially larger mutation spaces (R2 \u2248 0.8), with performance strongly correlated with capture of high-order epistatic effects. We validated our method in five different protein-protein interaction datasets, including binding of SARS-CoV-2 receptor binding domain (RBD) to various antibodies and cellular receptors, as well as influenza RBD- antibody interactions. This work provides a practical framework for extending the predictive power of combinatorial mutagenesis beyond current experimental constraints, offering applications in viral surveillance and antibody engineering.","version":"1.1","doi":"10.1101/2025.04.10.647895","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.09.647941","pub_date":"2025-4-10","title":"The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection","abstract":"Here the infection dynamics, replication, and pathogenicity of a recombinant virus containing a deletion of ORF6 (rWA1\u0394ORF6) on the backbone of the highly virulent SARS-CoV-2 WA1 virus (rWA1) were investigated and compared to the parental rWA1 virus. While both rWA1 and rWA1\u0394ORF6 viruses replicated efficiently in cultured cells, the rWA1\u0394ORF6 virus produced smaller plaques, suggesting reduced cell-to-cell spread. Luciferase reporter assays revealed immune suppressing effects of ORF6 on interferon and nuclear factor kappa B (NF-\u03baB) signaling pathways. Pathogenesis assessment in cats revealed that animals inoculated with rWA1 were lethargic and presented with fever on days 2 and 4 post-infection (pi), whereas rWA1\u0394ORF6-inoculated animals developed subclinical infection. Additionally, animals inoculated with rWA1\u0394ORF6 presented reduced infectious virus shedding in nasal and oral secretions and broncho-alveolar lavage fluid when compared with the rWA1-inoculated cats. Similarly, the rWA1\u0394ORF6-inoculated cats presented reduced virus replication in the respiratory tract as evidenced by lower viral loads and reduced lung inflammation on day 3 and 5 pi when compared to rWA1-inoculated animals. Host gene transcriptomic analysis revealed marked differences in differentially expressed genes (DEG) in the nasal turbinate of animals infected with rWA1 when compared to rWA1\u0394ORF6. Importantly, type I interferon signaling was significantly upregulated in rWA1\u0394ORF6 infected cats when compared to rWA1-inoculated animals, which is correlated to the reduced replication of rWA1\u0394ORF6 in the upper and lower respiratory tracts of infected animals. Collectively, these results demonstrate that the SARS-CoV-2 ORF6 is an important virulence determinant of the virus contributing to the modulation of host antiviral immune responses. SARS-CoV-2 encodes several proteins that inhibit host interferon responses. The accessory protein ORF6 antagonizes interferon signaling by blocking the nucleocytoplasmic trafficking of key transcription factors. In this study, we showed that ORF6 plays an important role in SARS-CoV-2 pathogenesis. While both rWA1 and rWA1\u0394ORF6 viruses replicated efficiently in cell culture, the rWA1\u0394ORF6 presented impaired cell-to-spread and reduced innate immune inhibition as compared to the parental rWA1. Pathogenicity study in the feline model revealed an attenuated phenotype of the rWA1\u0394ORF6 indicating that the ORF6 is a major virulence determinant of SARS-CoV-2. These results also demonstrate that ORF6 contributed to the feline host range of SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.04.09.647941","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.23.619918","pub_date":"2025-4-09","title":"T cell epitope mapping reveals immunodominance of evolutionarily conserved regions within SARS-CoV-2 proteome","abstract":"As SARS-CoV-2 variants continue to emerge capable of evading neutralizing antibodies, it has become increasingly important to fully understand the breadth and functional profile of T cell responses to determine their impact on the immune surveillance of variant strains. Here, sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains of SARS-CoV-2 suggested that epitope immunodominance and cross-reactivity are major predictive determinants of T cell immunity. To identify immunodominant epitopes across the viral proteome, we generated a comprehensive map of CD4+ and CD8+ T cell epitopes within non-spike proteins that induced polyfunctional T cell responses in convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by mutations in emerging variants. Conservation analysis across historical human coronaviruses combined with in silico alanine scanning mutagenesis of non-spike proteins underscored the functional importance of mutationally-constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the mutationally-constrained SARS-CoV-2 proteome, potentially providing immune surveillance against emerging variants, and inform the design of next-generation vaccines targeting antigens throughout SARS-CoV-2 proteome for broader and more durable protection. Polyfunctional CD8+ and CD4+ T cells directed against SARS-CoV-2 target mutationally constrained regions of the viral proteome.","version":"1.2","doi":"10.1101/2024.10.23.619918","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.07.647508","pub_date":"2025-4-08","title":"Differential association of SARS-CoV-2 IgG responses with anti-OC43 IgG in a Senegalese cohort","abstract":"Numerous studies elucidated the kinetics of the humoral immune response post-SARS-CoV-2 infection. However, in sub-Saharan Africa, the evolution of SARS-CoV-2 IgG antibody responses and their interaction with pre-existing seasonal human coronavirus (HCoVs: OC43, 229E, NL63, HKU1) immunity remain underexplored. A prospective cohort study was conducted in Senegal during the first year of the COVID-19 pandemic (March to December 2020). A total of 204 patients with laboratory-confirmed COVID-19 were included. Patients were classified as symptomatic (n=157) or asymptomatic (n=47) based on clinical presentation. Plasma samples (n=705) were collected over 6 months from SARS-CoV-2 positive individuals. IgG levels against SARS-CoV-2 and HCoVs were measured using a multiplex bead-based assay. Among the 204 participants included (95 [46.6%] female, median age, 44 [7\u201395]), SARS-CoV-2 IgG were detectable 6 months post-infection, peaking at 1 month for most antigens, except for Spike (S), which peaked at 3 months. Elderly patients (>60 years) exhibited higher IgG levels against both SARS- CoV-2 and HCoVs. Symptomatic patients had higher IgG levels than asymptomatic individuals, especially for WTS, RBD, S2, and N. Anti-HCoV IgG levels remained stable post-infection, with OC43 peaking at week 3 in symptomatic patients. A positive correlation was found between anti-SARS-CoV- 2 and anti-OC43 IgG in symptomatic patients. The study highlights persistent SARS-CoV-2 IgG antibodies for up to 6 months and suggests a link between pre-existing HCoV-OC43 immunity and COVID-19 outcomes in Senegal. These findings could help shape future vaccine strategies, considering the influence of circulating HCoVs on long- term protection against SARS-CoV-2. Understanding how our immune system responds to SARS-CoV-2, the virus responsible for COVID- 19, is essential for guiding public health countermeasures and informing vaccine development strategies. In our study, we monitored, in COVID-19 patients, the evolution of IgG antibody responses against SARS-CoV-2 structural proteins over a six-month period. Additionally, we examined how previous exposure to common seasonal coronaviruses might influence immune responses to SARS-CoV-2. Conducting this research in an African context is particularly important, as data on immune responses to SARS-CoV-2 in this region are scarce. Our results provide valuable insights into the complex interplay between immune responses elicited by SARS-CoV-2 and pre-existing immunity from seasonal circulating coronaviruses. These findings enhance our understanding of immune memory and cross-reactivity, two critical factors for assessing long-term protection and optimizing vaccine strategies. By shedding light on the dynamics of antibody responses over time within a sub-Saharan population, our research contributes to the global effort aimed at developing effective interventions against COVID-19 and preparing for future coronavirus outbreaks.","version":"1.1","doi":"10.1101/2025.04.07.647508","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.04.647203","pub_date":"2025-4-07","title":"COVID-19 vaccination induces cross-neutralisation of sarbecoviruses related to SARS-CoV-2","abstract":"Close relatives of SARS-CoV-1 and SARS-CoV-2 continue to circulate in wildlife, posing an ongoing threat of zoonotic spillover. While vaccination played a key role in controlling the COVID-19 pandemic, variants of concern (VOC) with immune evasive substitutions emerged on multiple occasions, causing widespread breakthrough infections. The combined threats of zoonosis and newly emerging VOCs, coupled with the potential for recombination, underscore the need to assess the breadth of existing vaccine-mediated protection. Here, we investigate a cohort of older individuals (median age 68.5 years) for the potential of cross-neutralisation against Omicron lineage VOC and animal sarbecoviruses induced by four COVID-19 vaccine doses. Despite the recent use of a bivalent mRNA vaccine dose (encoding spike from Wu-1 and omicron), we observed that neutralisation of Omicron lineage VOCs such as BA.1 and BA.2 were reduced compared to SARS-CoV-2 Wu-1, suggesting an imprinted immune response from pre-Omicron lineage viruses. Similarly, both SARS-CoV-1 and a SARS-CoV-1-related bat CoV were neutralised less efficiently than SARS-CoV-2 Wu-1. Unexpectedly, however, we observed that two animal SARS-CoV-2-related viruses, BANAL-20-52 (from bats) and a pangolin-CoV, were more sensitive to serum neutralising antibodies than SARS-CoV-2 Wu-1 itself. These surprising findings suggest that vaccine-mediated adaptive immunity may provide efficient cross-protection against certain animal sarbecoviruses.","version":"1.1","doi":"10.1101/2025.04.04.647203","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.05.647275","pub_date":"2025-4-07","title":"An updated dataset of early SARS-CoV-2 diversity supports a wildlife market origin","abstract":"The origin of SARS-CoV-2 has been intensely scrutinized, and epidemiological and genomic evidence has consistently pointed to Wuhan\u2019s Huanan Seafood Wholesale Market as the epicenter of the COVID-19 pandemic. Early cases were associated with this market, and environmental sequencing placed the common ancestor of SARS-CoV-2 genomic diversity within the market. Phylogenetic analysis also suggested separate introductions of lineages A and B into the human population, a finding that can be tested with additional data. Here, we curated an expanded sequence dataset of early SARS-CoV-2 viral genomes, including newly available sequences from mid-January 2020. In this dataset, we found no additional support for previously proposed alternative progenitor sequences, or for any evolutionary intermediates between lineages A and B in the human population. Instead, we identified SARS-CoV-2 lineages that may have spread from the market, and additional samples of a sublineage of lineage A with three mutations, including one found in closely related bat coronaviruses. Although our analysis of early pandemic genomes suggests that this mutation is unlikely to characterize the immediate SARS-CoV-2 ancestor, it is more plausible than two previously proposed ancestral genomes. These findings reinforce the proposed emergence of SARS-CoV-2 from the wildlife trade at the Huanan market, demonstrating how new data continues to both solidify and clarify our understanding of how the pandemic began.","version":"1.1","doi":"10.1101/2025.04.05.647275","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.04.647209","pub_date":"2025-4-07","title":"Stoichiometric Insights into SARS-CoV-2 Spike\u2013ACE2 Binding Across Variants","abstract":"The SARS-CoV-2 spike protein binds to the angiotensin-converting enzyme 2 (ACE2) receptor to mediate viral entry, with mutations in different variants influencing binding affinity and conformational dynamics. Using large-scale molecular dynamics simulations, we analyzed the Spike\u2013ACE2 complex in the wild-type (WT), Beta, and Delta variants. Our findings reveal significant conformational rearrangements at the inter-face in Beta and Delta compared to WT, leading to distinct interaction networks and changes in complex stability. Binding free energy analysis further highlights variant-specific differences in ACE2 affinity, with alternative binding modes emerging over the simulation. The results enhance our understanding of spike\u2013ACE2 stoichiometry across variants, providing implications for viral infectivity and therapeutic targeting.","version":"1.1","doi":"10.1101/2025.04.04.647209","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.16.628663","pub_date":"2025-4-07","title":"High-dimensional mediation analysis to elucidate the role of metabolites in the association between PFAS exposure and reduced SARS-CoV-2 IgG in pregnancy","abstract":"We previously found that per- and polyfluoroalkyl substances (PFAS) mixture exposure is inversely associated with SARS-CoV-2 IgG (IgG) antibody levels in pregnant individuals. Here, we aim to identify metabolites mediating this relationship to elucidate the underlying biological pathways. This cross-sectional study included 59 pregnant participants from a US-based pregnancy cohort. Untargeted metabolomic profiling was performed using Liquid Chromatography-High Resolution Sass spectrometry (LC-HRMS), and weighted Quantile Sum (WQS) regression was applied to assess the PFAS and metabolites mixture effects on IgG. Metabolite indices positively or negatively associated with IgG levels were constructed separately and their mediation effects were examined independently and jointly. The PFAS- index was negatively associated with IgG levels (beta=-0.315, p<0.001), with PFHpS and PFHxS as major contributors. Two metabolite-indices were constructed, one positively (beta=1.249, p<0.001) and one negatively (beta=-1.200, p<0.001) associated with IgG. Key contributors for these indices included trigonelline, adipate, p-octopamine, and n-acetylproline. Analysis of a single mediator showed that 74.6% (95% CI: 45.9%, 98.0%) and 68.6% (95% CI: 41.8%, 94.1%) of the PFAS index-IgG total effect were mediated by the negative and positive metabolites-indices, respectively. Joint analysis of the metabolite-indices indicated a cumulative mediation effect of 83.8% (95% CI: 58.1%, 98.7%). Enriched pathways associated with these metabolites indices were phenylalanine, tyrosine, and tryptophan biosynthesis and arginine metabolism. We observed significant mediation effects of plasma metabolites on the PFAS-IgG relationship, suggesting that PFAS is associated with alteration in the balance of plasma metabolites that contributes to reduced plasma IgG production.","version":"1.2","doi":"10.1101/2024.12.16.628663","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.04.647326","pub_date":"2025-4-07","title":"Intranasal sarbecovirus vaccine booster elicits cross-clade, durable and protective systemic and mucosal immunity","abstract":"Short-lived, clade-specific immune responses with limited mucosal priming are limitations faced by current COVID-19 mRNA vaccines against sarbecoviruses. We have developed a nasal booster vaccine candidate that induced robust and sustained, cross-clade, systemic and mucosal protective immunity. Two recombinant Clec9A-specific monoclonal antibodies fused to the Receptor Binding Domain (RBD) from Omicron XBB.1.5 and SARS-CoV-1, respectively were generated. In Comirnaty mRNA-vaccinated mice, boosting with each individual Clec9A-RBD construct induced immune responses that either were limited in breadth or waned over time; while boosting with both constructs combined (Clec9AOMNI) elicited robust cross-clade neutralizing antibodies (nAb) and T cell responses that were significantly more sustained compared to Bivalent Comirnaty (BC) mRNA vaccine booster. The persistence of RBD-specific follicular helper CD4+ T cells, germinal centre B cells, and long-lived plasma cells that facilitated affinity maturation in Clec9AOMNI-boosted mice, correlated with the detection of triple cross-reactive B cells that bind to ancestral SARS-CoV-2 ancestral, SARS-CoV-2 XBB.1.5 and SARS-CoV-1 RBD. Remarkably, intranasal boosting with Clec9AOMNI generated robust and sustained mucosal immune responses in the upper and lower respiratory compartments, including RBD-specific IgA, cross-clade nAb and cellular immunity together with functional tissue-resident memory T cells, without compromising the systemic immune responses. Correspondingly, Clec9AOMNI booster conferred superior protection against Omicron BA.1 compared to BC booster when challenge was performed at six months post-boost. Hence, Clec9AOMNI is a promising nasal booster vaccine candidate that has the potential to mitigate pandemic threats from emerging sarbecoviruses. Nasal booster immunization with dendritic cell-targeting vaccine candidate in mRNA-vaccinated mice induced cross-clade, sustained, systemic and mucosal protective immunity.","version":"1.1","doi":"10.1101/2025.04.04.647326","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.02.646864","pub_date":"2025-4-04","title":"Infant CD4 T-cell response to SARS-CoV-2 mRNA vaccination is restricted in cytokine production and modified by vaccine manufacturer","abstract":"Safe and effective vaccines are a key preventative measure to protect infants from SARS-CoV-2 infection and disease. Although mRNA vaccines induce robust antibody titers in infants, little is known about the quality of CD4 T-cell responses induced by vaccination. CD4 T-cell responses are important in orchestrating coordinated immune responses during infection and may help to limit disease severity. METHODS: To characterize the CD4 T-cell response to SARS-CoV-2 mRNA vaccination in infants, we sampled blood from 13 infants before and after primary SARS-CoV-2 mRNA vaccine series; samples from 12 historical vaccinated adults were used for comparisons. PBMC were stimulated with Spike peptide pools and the ability of CD4 T-cells to secrete Th1, Th2, and Th17 cytokines was quantified. A measure of polyfunctionality was generated using the COMPASS algorithm. RESULTS: We observed a significant increase in CD4 T-cells producing IL-2 (0.01% vs. 0.08%, p=0.04) and TNF-\u03b1 (0.007% vs. 0.07%, p=0.007) following vaccination in infants but a more muted induction of IFN-\u03b3 production (0.01% vs 0.04%, p=0.08). This contrasted with adults, in whom vaccination induced robust production of IFN-\u03b3, IL-2, and TNF-\u03b1. Th2 and Th17 responses were limited in both infants and adults. In infants, CD4 T-cell responses post-vaccination were greater in those who received mRNA-1273 versus BNT162b. In contrast to CD4 T-cell responses, Spike-specific IgG titers were similar in infants and adults. CONCLUSIONS: These data suggest that infants have restricted induction of cytokine producing CD4 T-cells following SARS-CoV-2 mRNA vaccination relative to adults.","version":"1.1","doi":"10.1101/2025.04.02.646864","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.03.646589","pub_date":"2025-4-04","title":"Optimized pipeline for generating highly potent neutralizing Affitins: application to SARS-CoV-2 spike protein","abstract":"Viral infections represent a global health threat, causing three million deaths annually. Antiviral strategies primarily target viral genome replication or prevent virus entry into host cells. While most FDA-approved antiviral drugs are small chemical molecules, protein-based therapies like monoclonal antibodies (mAbs) offer advantages such as broad and high neutralizing activities, and ability to recruit immune responses to enhance viral clearance. Additionally, alternative protein scaffolds with favorable properties have been developed to substitute or complement mAbs. We have developed Affitins, a novel class of small artificial affinity proteins (7 kDa) derived from hyperthermophilic archaea. Affitins, selected from high-diversity libraries (\u223c1012 variants) against any target protein, are highly stable, easy to engineer, and cost-effective to produce. Here, we developed a pipeline to generate Affitins in different multimerization formats targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein through ribosome display selection and assessed their efficacy. The most potent candidates, dimerized or trimerized into 17\u201327 kDa proteins, displayed high thermal stability, strong binding affinities, and potent neutralization against various SARS-CoV-2 variants, with IC50 values as low as 32 pM. We also report the first hexameric Affitins, formed by dimerizing trimers through Fc fragment fusion (\u223c106 kDa), achieving potent neutralization with an IC50 of 0.8 pM, ranking them among the most potent B1.351 SARS-Cov-2 neutralizing proteins. Our findings highlight Affitins as promising antiviral agents, demonstrating their versatility for engineering affinity proteins with higher valencies than that of antibodies while retaining a lower molecular weight, thereby expanding the toolkit for combating viral threats.","version":"1.1","doi":"10.1101/2025.04.03.646589","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.03.646981","pub_date":"2025-4-04","title":"Crosstalk Between Disrupted Blood-Brain Barrier, Neuroinflammation, and Coagulopathy in AC70 hACE2 Tg Mice with Prolonged Neurological Manifestations Induced by the Omicron BA.1 Variant of SARS-CoV-2","abstract":"Long COVID, or post-acute sequela of COVID (PASC), has emerged as a major post-pandemic challenge, with lasting neurological effects in a substantial number of patients. The underlying pathophysiological mechanisms of PASC remains elusive, largely due to the lack of suitable animal models that replicate key clinical and pathological features, especially in survivors of acute infection. Here, we employed the Omicron BA.1-infected AC70 hACE2 Tg mice to explore the pathogenesis of COVID-19. Our findings in the AC70/BA.1 model demonstrated that surviving mice developed lasting neurological sequelae of COVID-19, making it a promising platform for studying the pathogenesis of neuro- or long-COVID. Histopathological examinations revealed a transient pulmonary inflammatory response that correlated with the levels of live virus, whereas neuroinflammation within the brains persisted and progressed beyond the acute phase, without a clear linkage to direct virus effects. We showed that the severity of neurological sequelae directly correlated with the degree of blood-brain-barrier (BBB) disruption, alterations in tight junctions (TJs) and fibrinogen extravasation into the brain parenchyma. Additionally, BA.1-infected mice elicited a sustained systemic prothrombotic state, with dysregulated coagulation and fibrinolytic pathways in an organ-specific manner, as evidenced by distinct pattern of intracellular fibrin(ogen) depositions, elevated d-dimers, and tissue-plasminogen activator (t-PA) expression in the lungs and brains. We also found a positive correlation between progressive neuroinflammation and t-PA expression, which was closely co-localized with Iba-1-positive cells, indicating that activated microglia may serve as an additional source of t-PA in the CNS. Lastly, we showed that BA.1 variant triggered prolonged anti-Annexin A2 (ANXA2) autoantibody production. ANXA2 is essential for the neurovascular system, coordinating multiply dynamic processes within the endothelium, including cell surface fibrinolysis and TJ assembly. Our analysis revealed that impaired TJ structures coexisted with diminished ANXA2 levels around the brain vessels, suggesting its involvement in BBB dysfunction and neuroinflammation. Long COVID symptoms, especially neurological complications, remains a key public health concern worldwide. Alterations in the cerebrovascular system, along with ongoing coagulation disorders, may play a crucial role in the pathogenesis of this condition. Here, we established murine model for study post-acute sequela of COVID (PASC). We demonstrated that AC70 Tg mice infected with a high dose of BA.1 Omicron variant developed prolonged neurological symptoms, accompanied by progressive neuroinflammation in survivors. Our findings clearly showed the involvement of blood-brain-barrier (BBB) disfunction in the severity of neuropathology induced by SARS-CoV-2 infection. We also showed that the bidirectional interaction between dysregulated coagulation/ fibrinolysis and inflammatory systems may exhibit a distinct pattern in the CNC, significantly contributing to persistent neurological symptoms. Finally, we highlighted a potential role of malfunctioning Annexin A2 (ANXA2) in this phenomenon that may serve as a promising drug target for neuro-COVID interventions.","version":"1.1","doi":"10.1101/2025.04.03.646981","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.29.573647","pub_date":"2025-4-04","title":"DNA origami vaccine (DoriVac) nanoparticles improve both humoral and cellular immune responses to infectious diseases","abstract":"Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine nanotechnology, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific heptad repeat 2 (HR2) peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in na\u00efve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular and humoral immune responses in human cells. Moreover, DoriVac bearing full-length SARS-CoV-2 spike protein achieved immune responses comparable to current mRNA vaccine platforms while potentially reducing storage constraints. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities, underscoring its potential utility in addressing future pandemics.","version":"1.2","doi":"10.1101/2023.12.29.573647","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.02.646838","pub_date":"2025-4-03","title":"Effect of the S2\u2019 site cleavage on SARS-CoV-2 spike","abstract":"SARS-CoV-2 initiates infection of host cells by fusing its envelope lipid bilayer with the cell membrane. To overcome kinetic barriers for membrane fusion, the virus-encoded spike (S) protein refolds from a metastable prefusion state to a lower energy, stable postfusion conformation. The protein is first split into S1 and S2 fragments at a proteolytic site after synthesis, and presumably further cleaved at a second site, known as the S2\u2019 site, before membrane fusion can occur. We report here a cryo-EM structure of S2 fragment after the S2\u2019 cleavage, possibly representing a late fusion intermediate conformation, in which the fusion peptide and transmembrane segment have yet to pack together, distinct from the final, postfusion state. Functional assays demonstrate that the S2\u2019 cleavage accelerates membrane fusion, probably by stabilizing fusion intermediates. These results advance our understanding of SARS-CoV-2 entry and may guide intervention strategies against pathogenetic coronaviruses.","version":"1.1","doi":"10.1101/2025.04.02.646838","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.31.646276","pub_date":"2025-4-03","title":"Minimal impact of ivermectin on immune response and transcriptional profiles in na\u00efve adults with mild COVID-19","abstract":"Ivermectin (IVM), an antiparasitic drug, was repurposed to treat COVID-19 based on its in vitro antiviral effects. However, it was abandoned after multiple clinical trials reported a lack of efficacy. Immunomodulatory effects have been proposed but remain unclear, yet they may be relevant given IVM use for other infections. We assessed the IVM immunomodulatory effect in 24 participants from a clinical trial evaluating its potential to reduce COVID-19 transmission in mild cases within 48 hours of symptoms onset. The IVM-treated patients showed non-significant lower viral loads, and a significantly shorter duration of hyposmia/anosmia. We measured IgG, IgA, and IgM against five SARS-CoV-2 antigens, and 30 cytokines by Luminex, alongside whole blood RNA sequencing, pan-leukocyte immunophenotyping, and SARS-CoV-2-specific T cell analysis by flow cytometry from day 1 to day 28 post-treatment. All antibody responses increased from day 4, while 13 cytokines significantly decreased over time (adjusted p<0.05). IVM-treated patients had only significantly higher anti-nucleocapsid IgG levels at day 4 (adjusted p=0.041) and 7 (adjusted p=0.045) compared to placebo. SARS-CoV-2-specific CD4+ and CD8+ T cells increased over time, with significantly higher effector memory CD4+ T cells at day 7 compared to day 1 (p=0.027) and the only difference between groups was lower frequencies of spike-specific na\u00efve CD4+ T cells at day 7 in IVM-treated participants (0.006% vs 0.036% p=0.02). Transcriptomic data showed downregulation of innate and antiviral blood transcriptional modules (BTMs) over time, with an increase in adaptive immune related BTMs. While no differential gene expression was detected, the IVM-treated had upregulated innate and downregulated T cell and cell cycle BTMs compared to placebo. Overall, our comprehensive longitudinal analysis of early immune responses in mild COVID-19 revealed no robust immunological effects of IVM, consistent with clinical trials results and suggesting a lack of efficacy of IVM in COVID-19 treatment. Ivermectin (IVM), an antiparasitic drug, was tested in clinical trials as a potential treatment for COVID-19 due to its in vitro antiviral properties and hypothetical immunomodulatory effects. In this study, we explored the immunomodulatory effects of IVM in a clinical trial involving 24 mild COVID-19 patients who received IVM within 48 hours of symptoms onset. The IVM group showed a trend towards lower viral loads post-treatment, and a significantly shorter duration of hyposmia/anosmia. We comprehensively analyzed immune responses by measuring antibody levels, cytokine profiles, immune cell subsets and whole blood gene expression over 28 days. The IVM group only had increased levels of IgG against the SARS-CoV-2 nucleocapsid compared to the control group. IVM did not affect the kinetics of SARS-CoV-2 T cells, despite a slight decrease in naive CD4+ T cells. Additionally, gene expression analysis showed a decrease in innate and antiviral responses and an increase of adaptive responses over time that were slightly stronger in the placebo compared to the IVM group. In conclusion, despite some differences in IVM treated participants, our detailed analyses do not support significant immunomodulatory effects that could benefit disease progression.","version":"1.1","doi":"10.1101/2025.03.31.646276","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.02.646796","pub_date":"2025-4-03","title":"Development of pyrazolo[1,5-a]pyrimidine based macrocyclic kinase inhibitors targeting AAK1","abstract":"Since the outbreak of SARS-CoV-2 in recent years, our society has become more aware that zoonotic diseases pose a real threat. Therefore, the demand for small molecules that target host proteins, essential for viral entry and replication, has increased as an interesting strategy for the development of antiviral agents, as these agents may be effective against several different pathogens. NAK kinases is one such potential target family because they are involved in a variety of cellular functions, hijacked by viruses to invade host cells, such as clathrin-mediated endocytosis. A large number of different inhibitors have already been reported targeting NAK kinases, but there are still no compounds that selectively target AAK1 over other NAK family members, in particular the closely related family member BIKE. Here, we developed a series of pyrazolo[1,5-a]pyrimidine-based macrocyclic NAK inhibitors, starting from the acyclic AAK1 inhibitor LP-935509. Through a structure-guided activity relationship study within the NAK family, we identified potent AAK1 inhibitors 16, 18 and 27, which show promising selectivity within the NAK family. The inhibitors showed a potent inhibition of the phosphorylation of the AP-2 complex and the antiviral activity of the compounds was evaluated against various RNA viruses.","version":"1.1","doi":"10.1101/2025.04.02.646796","journal":"bioRxiv","score":null},{"id":"10.1101/2025.04.01.646621","pub_date":"2025-4-02","title":"Genetic diversity of pangolin coronaviruses reveals a key immuno-evasive substitution at spike residue 519","abstract":"Malayan pangolins are unprecedented hosts for several SARS-CoV-2-related coronaviruses, which have previously been known to only infect Rhinolophus bats. Much debate has hence surrounded their possible role as intermediate hosts in the emergence of SARS-CoV-2, but the virological phenotypes of most pangolin coronaviruses (pCoVs) remain unclear. Here, we comprehensively analyze all pCoVs to date identified from trafficked pangolins seized in the Guangdong province of China, which are remarkably similar to SARS-CoV-2 in the spike protein. We explore an unknown genetic diversity within these viruses and uncover how this diversity translates to different virological phenotypes. Strikingly, several Guangdong pCoVs harbor a lysine substitution at residue 519 of spike protein, which contributes to marked immune evasion potentially by modulating the conformational state of spike protein. Furthermore, we highlight that a similar immuno-evasive mutation at residue 519 of the spike protein was acquired by SARS-CoV-2. These findings support that pangolin\u2013 and human-infecting coronaviruses represent independent spillover events from natural bat reservoirs, and that immuno-evasive mutations at residue 519 may be a common vector of viral evolution in coronaviruses that infect non-bat hosts.","version":"1.1","doi":"10.1101/2025.04.01.646621","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.29.646083","pub_date":"2025-4-01","title":"Ketamine inhibition of SARS-CoV-2 replication in astrocytes is associated with COVID-19 disease severity in a variant-dependent manner","abstract":"Severe coronavirus infections, including SARS-CoV-2, can cause neurological symptoms, but the underlying neurotropic mechanisms are unclear. Experiments with SARS-CoV-2 variants B.1.258.17, B.1.1.7, and BA.5.3.2 (termed wild-type, alpha and omicron, respectively) revealed that human astrocytes, not neurons, support viral proliferation. During the COVID-19 pandemic, new virus variants exhibited milder disease progression. A retrospective study of patients with COVID-19 infected by wild-type or alpha variants was conducted to test whether ketamine, an anaesthetic that inhibits endocytosis, affects COVID-19. At admission, patients infected with the wild-type showed greater disease severity than alpha variant patients, but the disease course was similar. This may be due to distinct ketamine-mediated SARS-CoV-2 variant-dependent effects, revealing stronger ketamine inhibition of the wild-type variant than the alpha variant mediating astrocyte responses involving the expression of ACE2, a viral cell entry site, viral proteins RNA-dependent RNA polymerase and envelope protein-E in infected cells. Overexpression of SARS-CoV-2 protein 3a attenuated astroglial lysosomal traffic, and 3a and nsp6 differentially modulated lipid droplet accumulation and initiation of autophagy, where ketamine predominantly affected vesicle dynamics. In summary, human astrocytes, but not neurons, contribute to SARS-CoV-2 neurotropism, highlighting the potential benefits of ketamine treatment in coronavirus infections.","version":"1.1","doi":"10.1101/2025.03.29.646083","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.11.623100","pub_date":"2025-4-01","title":"Vaccine-induced and hybrid immunity against SARS-CoV-2 variants of concern in two cohorts in Queensland, Australia (2021-2022)","abstract":"The spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for vaccine development, as antibodies generated against the spike protein are the most immunodominant and neutralizing against the virus. However, variants of concern (VOC), often containing multiple mutations within neutralizing epitopes, confer immune evasion of the response generated by current SARS-CoV-2 vaccines. To assess the immunogenicity and virus-neutralisation ability of antibodies induced by individual or combination of Vaxzevria (AstraZeneca: AZ) and Comirnaty (Pfizer: PZ) vaccines and of a hybrid immunity induced in people who were also infected with SARS-CoV-2, longitudinal sera samples of participants recruited through the David Serisier Research biobank (Mater Research Hospital) or at the University of Queensland were collected. ELISA with a panel of purified Spike proteins from ancestral, alpha, delta and omicron BA.1 and BA.2 VOCs showed significantly (by \u223c2 to 6-fold) reduced IgG antibody titres against Spike proteins from Omicron BA.1 and BA.2 compared to ancestral strain regardless of the number of vaccinations or presence of infection. Neutralisation assays showed reduced activity against delta and omicron BA.1, BA.5 and BA.5 VOCs. However, the differences were in general less pronounced than in the ELISA assay and some were not statistically significant, particularly after four (two AZ and two PZ) vaccinations. We also generated by circular polymerase extension reaction an attenuated SARS-CoV-2 strain with deletion of all accessory genes, ORF 3, 6, 7 and 8, based on the ancestral (QLD02) virus backbone (QLD02\u03943678) and validated it in virus-neutralization assays with our panel of sera samples. We showed the attenuation of the QLD02\u03943678 virus in Vero E6 and human Caco2 cells. We demonstrated that neutralization assays with the wild-type QLD02 virus and QLD02\u03943678 virus were concordant, providing a safe platform for neutralisation assays in BSL2/PC2 settings.","version":"1.2","doi":"10.1101/2024.11.11.623100","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.30.646166","pub_date":"2025-4-01","title":"A NanoBRET-based assay monitoring interactions in the USP18 signaling hub identifies the first cell-penetrant small molecule compromising USP18/ISG15 binding","abstract":"Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico, this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro. To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators.","version":"1.1","doi":"10.1101/2025.03.30.646166","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.28.645941","pub_date":"2025-3-31","title":"The CCR2 inflammatory pathway is a target for improving severe disease and pulmonary inflammation in experimental COVID-19","abstract":"SARS-CoV2 can induce an acute respiratory distress syndrome (ARDS), provoked by a dysregulated hyper-inflammatory pulmonary immune response. Here we used the keratinocyte-18 humanized angiotensin converting enzyme-2 (K18-hACE2) mouse model of SARS-CoV2, where expression of the CoV2 spike protein receptor, hACE-2, is restricted to epithelia, to characterize inflammatory pulmonary immune responses post-intranasal infections with the delta isolate SARS-CoV2(\u0394 B.1.617.2). Immune-profiling by focused transcript analysis, inflammatory protein array, and multi-color flow cytometry, confirmed that clinically relevant markers of COVID-19 (IL-6, GM-CSF, neutrophils, inflammatory monocytes) were significantly elevated in lungs of mice at day 5 post-infection and that remdesivir anti-viral active metabolite (GS441524) treatment significantly modified SARS-CoV2\u0394 viral loads and pulmonary-specific inflammation. Chemokine ligands of CCR2 (CCL2/7/8) were amongst the top 5% upregulated pulmonary transcripts in a focused human infection response array to SARS-CoV2\u0394. CCL2 was confirmed as elevated in protein assays in SARS-CoV2\u0394 infected lungs. To address functional relevance of the CCR2 pathway of inflammatory cell recruitment to the lungs mediating disease, mice were administered with anti-CCR2 antibody daily at point of infection for up to six days. Anti-CCR2 treated mice showed significant improved welfare scores, were protected from weight loss, modified myeloid pneumonitis, and displayed significantly blunted cytokine and chemokine response in the lungs, despite not affecting pulmonary viral loads. Our data supports therapeutic benefit of modifying CCR2-dependent cell recruitment in the treatment of viral-induced ARDS.","version":"1.1","doi":"10.1101/2025.03.28.645941","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.28.645914","pub_date":"2025-3-31","title":"Gut barrier integrity biomarkers are associated with increased inflammation and predict disease status in hospitalized COVID-19 patients","abstract":"The COVID-19 global pandemic persists as an endemic disease with frequent case spikes and a significant continued burden on public health. Although most COVID-19 cases are asymptomatic or mild, severe infections requiring hospitalization have resulted in likely more than 14 million cumulative deaths to date. One hallmark of severe COVID-19 is a dysregulated immune response that leads to systemic inflammation and contributes to disease severity and mortality but is not explained by viral replication alone. Severe COVID-19 has been shown to disrupt the gut microbiome and increase intestinal permeability which may contribute to immune dysregulation and systemic inflammation. In this study, we investigated the differences in plasma biomarkers for microbial translocation and gut barrier damage as well as circulating cytokines between healthy volunteers and patients hospitalized with COVID-19. We then performed a correlation analysis to understand how the relationships between these plasma biomarkers differed and used a random forest model to assess their accuracy in distinguishing between these two groups. Our results demonstrated that hospitalized COVID-19 patients have elevated concentrations of pro-inflammatory cytokines and markers of microbial translocation, and that the relationships between these biomarkers were significantly altered compared to healthy volunteers, especially those related to the mucosal associated homeostatic cytokines IL-17A and IL-23. Furthermore, IL-6 and LBP were the top biomarkers for prediction accuracy in our random forest model, highlighting the importance of managing microbial translocation in COVID-19 and its potential utility as a biomarker for disease severity. COVID-19 continues to be a burden on public health. Understanding how plasma biomarkers differ between healthy and acutely infected individuals can help in understanding pathogenesis and predict disease severity. It has been demonstrated that COVID-19 disrupts the gut microbiome and intestinal permeability, which contributes to systemic inflammation. Our study highlights the link between gut barrier integrity and inflammation in hospitalized SARS-CoV-2 cases, demonstrating the usefulness of gut barrier integrity biomarkers in predicting disease severity, and offers insights for therapeutic interventions in acute COVID-19 infection.","version":"1.1","doi":"10.1101/2025.03.28.645914","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.30.646023","pub_date":"2025-3-31","title":"Functional and structural characterization of a combination of pan-sarbecovirus antibodies with potent antiviral activity","abstract":"Sarbecoviruses, a subgenus of coronaviruses, include strains with zoonotic spillover risk as exemplified by recent outbreaks (SARS-CoV-1, SARS-CoV-2). Monoclonal antibodies targeting conserved spike protein regions (RBD class 4, S2 fusion machinery) exhibit broad sarbecovirus neutralization, but their utility has been impacted by immune selection leading to escape and suboptimal neutralization. Using structure-conditioned machine-learning protein design we optimized two broadly neutralizing sarbecovirus antibodies to rescue the activity of a class 4 anti-RBD antibody against Omicron variants and improve the neutralization profile of an anti-S2 stem antibody, resulting in the identification of the lead candidates PRO-37587 and GB-0669. Both antibodies displayed remarkable resilience to SARS-CoV-2 evolution. Structural analyses elucidated the mechanistic basis of their broad neutralization profiles. In combination, these antibodies exhibited improved SARS-CoV-2 neutralization in vitro and in vivo and a higher barrier to resistance. These findings support further evaluation of such antibody combination as countermeasure against current and emerging sarbecoviruses.","version":"1.1","doi":"10.1101/2025.03.30.646023","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.29.646087","pub_date":"2025-3-31","title":"Divalent cations govern the activity of coronavirus nsp15","abstract":"Coronavirus nsp15 is an endoribonuclease that limits the accumulation of viral dsRNA in cytosol and plays a crucial role in the evasion of host immunity. Here, we show that Co2+ or Ni2+ potently activates nsp15 of human and animal coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, MHV-A59, and PEDV. Whereas Zn2+ strongly inhibits nsp15 of these coronaviruses. Cryo-EM structures of the Co2+-bound SARS-CoV-2 nsp15/dsRNA complexes reveal that Co2+ binds to the nsp15 active site and is coordinated by three catalytic residues H234, H249, K289, and the 2\u2019 hydroxyl of the flipped base from dsRNA, suggesting that Co2+ stabilizes the catalytic core and increases substrate binding. Although our data indicated that Ni2+ and Zn2+ bind to the same site as Co2+, Zn2+ lacks the catalytic capability and inhibits nsp15. These findings reveal that divalent cations govern coronavirus nsp15\u2019s activity and provide basis for new therapeutic strategies.","version":"1.1","doi":"10.1101/2025.03.29.646087","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.07.622419","pub_date":"2025-3-26","title":"SARS-CoV-2 infection enhancement by Amphotericin B: Implications for disease management","abstract":"Severe coronavirus disease 2019 (COVID-19) patients who require hospitalisation are at high risk of invasive pulmonary mucormycosis. Amphotericin B (AmB), which is the first line therapy for invasive pulmonary mucormycosis, has been shown to promote or inhibit replication of a spectrum of viruses. In this study, we first predicted that AmB and Nystatin had strong interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins using in silico screening, indicative of drugs with potential therapeutic activity against this virus. Subsequently, we investigated the impact of AmB, Nystatin, Natamycin, Fluconazole and Caspofungin on SARS-CoV-2 infection and replication in vitro. Results showed that AmB and Nystatin actually increased SARS-CoV-2 replication in Vero E6, Calu-3 and Huh7 cells. At optimal concentrations, AmB and Nystatin increase SARS-CoV-2 replication by up to 100- and 10-fold in Vero E6 and Calu-3 cells, respectively. The other antifungals tested had no impact on SARS-CoV-2 infection in vitro. Drug kinetic studies indicate that AmB enhances SARS-CoV-2 infection by promoting viral entry into cells. Additionally, knockdown of genes encoding for interferon-induced transmembrane (IFITM) proteins 1, 2, and 3 suggests AmB enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of the IFITM3 protein. This study further elucidates the role of IFITM3 in viral entry and highlights the potential dangers of treating COVID-19 patients, with invasive pulmonary mucormycosis, using AmB. AmB and Nystatin are common treatments for fungal infections but were predicted to strongly interact with SARS-CoV-2 proteins, indicating their potential modulation or inhibition against the virus. However, our tests revealed that these antifungals, in fact, enhance SARS-CoV-2 infection by facilitating viral entry into cells. The magnitude of enhancement could be up to 10- or 100-fold depending on cell lines used. These findings indicate that AmB and Nystatin have the potential to enhance disease when given to patients infected with SARS-CoV-2 and therefore should not be used for treatment of fungal infections in active COVID-19 cases.","version":"1.2","doi":"10.1101/2024.11.07.622419","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.25.644515","pub_date":"2025-3-26","title":"Comprehensive longitudinal profiling of SARS-CoV-2-specific CD8+ T-cells reveal strong functional impairment and recognition bias as markers for disease severity","abstract":"CD8+ T-cells are essential for controlling and resolving SARS-CoV-2 infection, yet their antigen-specific resolution in relation to disease severity, functional dynamics during acute infection, and long-term memory formation remain incompletely understood. Using comprehensive longitudinal profiling of 553 SARS-CoV-2 immunogenic antigens across globally prevalent HLAs, we identified antigen-specific CD8+ T-cell responses that were either critical for early viral clearance or associated with severe disease outcomes. During acute infection, patients with severe COVID-19 exhibited a broader and more robust CD8+ T-cell response than those with mild disease. Notably, we identified HLA-A1-restricted immunodominant antigen-specific T-cells strongly associated with severe disease. These T-cells were present at extremely high frequencies but showed significantly reduced expression of cytotoxic molecules at both the transcriptomic (PRF1, GZMB, GZMH, GNLY) and protein levels (IFN-\u03b3, TNF-\u03b1, IL-2), as revealed by multidimensional single-cell and cytokine profiling. In contrast, patients with mild disease had T-cells that recognized a more restricted set of antigens, showed only partial overlap with those in severe cases, and showed enhanced cytotoxicity, along with enrichment in gene sets associated with cytotoxic function, hypoxia, and glycolysis. Furthermore, the long-term memory CD8+ T-cells were maintained for a limited subset of immunodominant antigens, with their persistence correlating with their initial frequency during infection. Importantly, SARS-CoV-2 vaccination following infection expanded the long-term T-cell repertoire by enhancing pre-existing responses and generating de novo responses, regardless of prior disease severity. These findings resolve the antigen-specific kinetics and durability of CD8+ T-cells in SARS-CoV-2 infection and provide key insights into their functional landscape. This knowledge could inform future vaccine strategies and therapeutic interventions to enhance protective immunity against emerging viral threats.","version":"1.1","doi":"10.1101/2025.03.25.644515","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.25.645210","pub_date":"2025-3-26","title":"Long-Term Intra-Host Evolution of SARS-CoV-2 in an Immunocompromised Patient: Recombination and Within-Host Mutations Driving Viral Adaptation","abstract":"An immuno-compromised patient with lymphoma experienced a prolonged SARS-CoV-2 infection lasting 14 months, initially infected with B.1.160, followed by B.1.1.7. This study focused on intra-host single nucleotide variants (iSNVs) and single nucleotide polymorphisms (SNPs), distinguishing their origin as either within-host mutations or parental. The whole genome analysis revealed accelerated evolution, with positive selection detected in key genes such as Spike, N, ORF9b, and nsp13, all involved in viral replication and immune evasion. Of the two evolutionary mechanisms involved, host-driven mutation has played a dominant role in this evolutionary story. C>T transitions emerged as the most widespread mutational signature, consistent with host-driven RNA editing mechanisms. Two host-internal mutations, A28271T in the translation initiation region of the N Kozak gene and C26858T in the M gene, were highly shared among the deposited SARS-CoV-2 sequences. Recombination with parental lineages played a major role particularly in Spike, ORF3a and M genes. In Spike, the B.1.1.7 sequence was selected, wild-type ORF3a and M were restored, suggesting a selective advantage in returning to an ancestral sequence. In addition, the temporary emergence of intra-host convergent mutations in Spike, notably L5F, D796H and T572I, underlines the strong selective pressures exerted on this gene. A 126-nucleotide deletion in ORF8 resulted in a truncated protein, reinforcing its uselessness for viral replication, as observed in circulating variants such as B.1.1.7. The present case highlights the complex interplay between viral recombination and mutations within the host in chronic infections and further underscores the remarkable evolutionary plasticity of SARS-CoV-2 and its potential to generate highly adapted viral strains.","version":"1.1","doi":"10.1101/2025.03.25.645210","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.20.629482","pub_date":"2025-3-26","title":"Generation of antigen-specific paired chain antibody sequences using large language models","abstract":"The traditional process of antibody discovery is limited by inefficiency, high costs, and low success rates. Recent approaches employing artificial intelligence (AI) have been developed to optimize existing antibodies and generate antibody sequences in a target-agnostic manner. In this work, we present MAGE (Monoclonal Antibody GEnerator), a sequence-based Protein Language Model (PLM) fine-tuned for the task of generating paired human variable heavy and light chain antibody sequences against targets of interest. We show that MAGE can generate novel and diverse antibody sequences with experimentally validated binding specificity against SARS-CoV-2, an emerging avian influenza H5N1, and respiratory syncytial virus A (RSV-A). MAGE represents a first-in-class model capable of designing human antibodies against multiple targets with no starting template.","version":"1.3","doi":"10.1101/2024.12.20.629482","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.25.645253","pub_date":"2025-3-26","title":"Delphy: scalable, near-real-time Bayesian phylogenetics for outbreaks","abstract":"Pathogen genomic analysis is central to tracking, understanding, and containing outbreaks, but complexity and high costs of state-of-the-art (SOTA) phylogenetic tools limit global access and impact. We introduce Delphy, an exact reformulation of Bayesian phylogenetics designed to transform its speed, scalability and accessibility while retaining SOTA accuracy. Delphy\u2019s central data structure, an Explicit Mutation Annotated Tree, exploits the high sequence similarity in large-scale epidemic datasets for efficient tree exploration and convergence. By reproducing key analyses from recent major epidemics (Ebola, Zika, SARS-CoV-2, mpox, and H5N1), we demonstrate SOTA accuracy with up to 1,000x speedups. Assessing Delphy\u2019s scalability, we show that a simulated dataset of 100,000 sequences can be analyzed in under a day\u2013the largest such computation to date. We distribute Delphy as a client-side web application, enabling users worldwide to turn raw data into interactive results within minutes, without the data ever leaving the user\u2019s machine. Delphy automatically identifies key viral lineages and mutations, as well as their emergence and prevalence through time, all with quantified uncertainties derived from a solid theoretical foundation. Delphy shows the power of Bayesian phylogenetics as a fast, accessible frontline tool for tackling future outbreaks.","version":"1.1","doi":"10.1101/2025.03.25.645253","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.21.644583","pub_date":"2025-3-25","title":"AMULETY: A Python package to embed adaptive immune receptor sequences","abstract":"Large language models have been developed to capture relevant features of adaptive immune receptors, each with unique potential applications. However, the diversity in available models presents challenges in accessibility and usability for downstream applications. Here we present AMULETY (Adaptive imMUne receptor Language model Embedding Tool), a Python-based software package to generate language model embeddings for adaptive immune receptor sequences, enabling users to leverage the strengths of different models without the need for complex configuration. AMULETY offers functions for embedding adaptive immune receptor amino acid sequences using pre-trained protein or antibody language models for paired heavy and light chain or single chain sequences. We showcase the variability on the embedding space for several embeddings on a dataset of antibody binders to several SARS-CoV-2 epitopes and showed that different models may be effective at capturing different aspects of the distinctions between epitope groups. AMULETY is available under GPL v3 license from https://github.com/immcantation/amulety or via pip from the Python Package Index (PyPI) from https://pypi.org/project/amulety/.","version":"1.1","doi":"10.1101/2025.03.21.644583","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.21.644627","pub_date":"2025-3-24","title":"Deep learning-based 3D spatial transcriptomics with X-Pression","abstract":"Spatial transcriptomics technologies currently lack scalable and cost-effective options to profile tissues in three dimensions. Technological advances in microcomputed tomography enabled non-destructive volumetric imaging of tissue blocks with sub-micron resolution at a centimetre scale. Here, we present X-Pression, a deep convolutional neural network-based frame-work designed to reconstruct 3D expression signatures of cellular niches from volumetric microcomputed tomography data. By training on a singular 2D section of a paired spatial transcriptomics experiment, X-Pression achieves high accuracy and is capable of generalising to out-of-sample examples. We utilised X-Pression to demonstrate the benefit of 3D examination of tissues on a paired SARS-CoV-2 vaccine efficacy spatial transcriptomics and microcomputed tomography cohort of a recently developed live attenuated SARS-CoV-2 vaccine. By applying X-Pression to the entire mouse lung, we visualised the sites of viral replication at the organ level and the simultaneous collapse of small alveoli in their vicinity. In addition, we assessed the immunological response following vaccination and virus challenge infection. X-Pression offers a valuable and cost-effective addition to infer expression signatures without the need for consecutive 2D sectioning and reconstruction, providing new insights into transcriptomic profiles in three dimensions.","version":"1.1","doi":"10.1101/2025.03.21.644627","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.20.644166","pub_date":"2025-3-21","title":"Immune Antibodies Recognizing the Stem Region of SARS-CoV-2 Spike Protein: Molecular Modelling and In Vitro Study of Synthetic Peptides Presentation to the Antibodies","abstract":"Antibodies to peptide 1147 (amino acids 1147-61) of the SARS-CoV-2 protein S are highly diagnostic. Peptide 1147, although located in a region that is partly spatially hidden in the intact protein, is not subject to mutations, suggesting therapeutic potential. The aim of this study was to elucidate the architecture of this region and the way in which it is presented to antibodies. As a model system, this peptide carrying a single lipophilic tail and the same peptide carrying a lipophilic tail at both ends (pseudocyclic) were incorporated into lipid membrane. Isolated anti-1147 antibodies interacted with it regardless of how the peptide was presented, be that freely exposed via the N-terminus, organized as a pseudocycle, or adsorbed on the surface. MDS showed that peptide 1147 is capable of closely approaching the membrane. Analysis of the surface properties of peptide 1147 in membrane-bound states and in available conformations in the full-sized S protein reveals interface for interaction with antibodies. Interestingly, the latter bears similarities to one published peptide-antibody complex. However, these antibodies, in spite of their high diagnostic significance, show no virus-neutralizing activity, indicating that peptide 1147 has no therapeutic value as a synthetic vaccine.","version":"1.1","doi":"10.1101/2025.03.20.644166","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.20.644376","pub_date":"2025-3-21","title":"TGF-\u03b2 inhibitor SB431542 suppresses coronavirus replication through multistep inhibition","abstract":"The COVID-19 pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-\u03b2 receptor I (ALK5) inhibitor, exhibits potent antiviral activity against SARS-CoV-2 through unprecedented multitargeted mechanisms. Through comprehensive in vitro, and in silico analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupt its canonical function in inhibiting autophagosome-lysosome fusion. This interaction restored lysosomal acidification and normalized perinuclear LAMP-1 localization, significantly impairing virion assembly as evidenced by disrupted nucleocapsid-RNA association and reduced intracellular viral titers. Additionally, SB431542 downregulated the CLEAR network genes responsible for lysosomal biogenesis, further restricting viral egress pathways. Our temporal analyses revealed that at later infection stages (36-48 hpi), SARS-CoV-2 exploits TGF-\u03b2-induced lysosomal membrane permeabilization (LMP) and apoptosis for viral release\u2014processes effectively inhibited by SB431542 through suppression of GADD45b and BAX expression. These multiple mechanisms resulted in an exceptional EC50 of 515 nM against SARS-CoV-2. In vivo efficacy was demonstrated in embryonated chicken eggs, where SB431542 conferred dose-dependent protection against lethal infectious bronchitis virus (IBV) challenge, with a favourable therapeutic index of 34.54. Remarkably, sequential passaging of SARS-CoV-2 for 50 generations under SB431542 selection pressure failed to generate resistant variants, contrasting sharply with the rapid resistance emergence typical of direct-acting antivirals. These findings establish SB431542 as a promising broad-spectrum coronavirus inhibitor with a unique triple-mechanism approach that simultaneously targets viral entry via TGF-\u03b2/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-\u03b2-induced apoptosis during late-stage infection\u2014 collectively imposing multiple selective constraints that impede escape mutation development. The COVID-19 pandemic highlighted the urgent need for antiviral drugs with high barriers to resistance. This study reveals that SB431542, a drug previously developed to inhibit TGF-\u03b2 signaling, exhibits remarkable effectiveness against SARS-CoV-2 through an unprecedented triple-mechanism approach. Unlike conventional antivirals that target a single viral component, SB431542 simultaneously disrupts viral entry, assembly, and release by binding to the viral ORF3a protein and modulating host cellular processes. Most importantly, SARS-CoV-2 failed to develop resistance against SB431542 even after 50 generations of exposure\u2014a significant advantage over current therapeutics that quickly lose effectiveness due to viral mutations. Our findings also uncover that coronaviruses exploit both lysosomal dysfunction and programmed cell death to spread efficiently, providing new targets for therapeutic intervention. This research establishes SB431542 as a promising broad-spectrum coronavirus inhibitor and demonstrates the value of targeting host-virus interactions to overcome antiviral resistance.","version":"1.1","doi":"10.1101/2025.03.20.644376","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.30.615772","pub_date":"2025-3-20","title":"Design of nanobody targeting SARS-CoV-2 spike glycoprotein using CDR-grafting assisted by molecular simulation and machine learning","abstract":"The design of proteins capable to effectively bind to specific protein targets is crucial for developing therapies, diagnostics, and vaccine candidates for viral infections. Here, we introduce a complementarity-determining regions (CDRs)-grafting approach for designing nanobodies (Nbs) that target specific epitopes, with the aid of computer simulation and machine learning. As a proof-of-concept, we designed, evaluated, and characterized a high-affinity Nb against the spike protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. The designed Nb, referred to as Nb Ab.2, was synthesized and displayed high-affinity for both the purified receptor-binding domain protein and to the virus-like particle, demonstrating affinities of 9 nM and 60 nM, respectively, as measured with microscale thermophoresis. Circular dichroism showed the designed protein\u2019s structural integrity and its proper folding, whereas molecular dynamics simulations provided insights into the internal dynamics of Nb Ab.2. This study shows that our computational pipeline can be used to efficiently design high affinity Nbs with diagnostic and prophylactic potential, which can be tailored to tackle different viral targets. In this study, we present a pipeline for designing a high-affinity nanobody (Nb) targeting the SARS-CoV-2 spike protein using enhanced sampling molecular dynamics simulations and CDR-grafting. To address the challenges of CDR grafting in Nbs, including the need for structural similarity between the CDR motif of interest and the scaffold region, we utilized the Nb scaffold cAbBCII10, known for its versatility in accommodating various CDRs. We generated a library based on the cAbBCII10 framework with diverse, unrelated CDRs and applied machine learning to identify the most promising candidates. Our approach enabled successful engineering of a Nb that binds to the SARS-CoV-2 spike protein with high affinity, demonstrating the effectiveness of our design pipeline for potential therapeutic applications.","version":"1.2","doi":"10.1101/2024.09.30.615772","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.18.643769","pub_date":"2025-3-19","title":"Machine Learning Reveals Distinct Immunogenic Signatures of Th1 Imprinting in ART-Treated Individuals with HIV Following Repeated SARS-CoV-2 Vaccination","abstract":"The human immune system is intrinsically variable and remarkably diverse across a population. The immune response to antigens is driven by a complex interplay of time-dependent interdependencies across components of the immune system. After repeated vaccination, the humoral and cellular arms of the immune response display highly heterogeneous dynamics, further complicating the attribution of a phenotypic outcome to specific immune system components. We employ a random forest (RF) approach to classify informative differences in immunogenicity between older people living with HIV (PLWH) on ART and an age-matched control group who received up to five SARS-CoV-2 vaccinations over 104 weeks. RFs identify immunological variables of importance, interpreted as evidence for Th1 imprinting, and suggest novel distinguishing immune features, such as saliva-based antibody screening, as promising diagnostic features towards classifying responses (whereas serum IgG is not). Additionally, we implement supervised and unsupervised Machine Learning methods to produce physiologically accurate synthetic datasets that conform to the statistical distribution of the original immunological data, thus enabling further data-driven hypothesis testing and model validation. Our results highlight the effectiveness of RFs in utilizing informative immune feature interdependencies for classification tasks and suggests broad impacts of ML applications for personalized vaccination strategies among high-risk populations.","version":"1.1","doi":"10.1101/2025.03.18.643769","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.17.643667","pub_date":"2025-3-18","title":"Development of a thermostable and broadly neutralizing pan-sarbecovirus vaccine candidate","abstract":"Zoonotic spillover of sarbecoviruses to humans resulted in theSARS-CoV-1 outbreak in 2003 and the current COVID-19 pandemic caused by SARS-CoV-2. In both cases, the viral spike protein (S) is the principal target of neutralising antibodies that prevent infection. Within spike, the immunodominant receptor-binding domain (RBD) is the primary target of neutralising antibodies in COVID-19 convalescent sera and vaccine recipients. We have constructed stabilized RBD derivatives of different sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02 (Clade 2) and BtKY72 (Clade 3). Stabilization enhanced yield by an 3-23-fold. The RBD derivatives were conformationally intact as assayed by binding to multiple broadly neutralizing antibodies. The stabilized RBDs show significant enhancement in apparent Tm, exhibit resistance to a 2-hour incubation at temperatures up to 60\u2103 in PBS in contrast to corresponding WT RBDs, and show prolonged stability of over 15 days at 37\u2103 after lyophilization. In mice immunizations, both stabilization and trimerization significantly enhanced elicited neutralization titers by \u223c100 fold. The stabilized RBD cocktail elicited high neutralizing titers against both homologous and heterologous pseudoviruses. The immunogenicity of the vaccine formulation was assessed in both na\u00efve and SARS-CoV-2 pre-immunized mice, revealing an absence of immune imprinting, thus indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics. The COVID-19 pandemic was caused by the sarbecovirus SARS-CoV-2. Phylogenetically, sarbecoviruses are divided into four clades: Clade 1a, Clade 1b, Clade 2 and Clade 3 and within these clades, are many other sarbecovirus strains with pandemic or epidemic potential. It is therefore important to develop a broadly protective, pan-sarbecovirus vaccine formulation that can be cheaply and rapidly produced. While mRNA vaccine formulations are efficacious, they have stringent low temperature storage requirements and there is limited manufacturing expertise in low and middle income countries for this modality. Neutralizing antibodies are important for protection and in the case of SARS-CoV-2 are primarily directed against the Receptor Binding Domain (RBD) of the surface spike protein. In this study, we have designed and developed an adjuvanted, protein subunit, pan sarbecovirus vaccine formulation using stabilized RBD derivatives from diverse sarbecoviruses as immunogens. We demonstrate that the stabilized RBD derivatives have considerably enhanced yield and thermal stability relative to corresponding WT proteins and that the formulation remains stable for up to two weeks at 37\u00b0C. The formulation was found to be highly immunogenic in both na\u00efve and pre-immunized mice, eliciting neutralizing titers well above the known protective threshold, indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics.","version":"1.1","doi":"10.1101/2025.03.17.643667","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.09.642188","pub_date":"2025-3-18","title":"Learning the language of protein-protein interactions","abstract":"Protein Language Models (PLMs) trained on large databases of protein sequences have proven effective in modeling protein biology across a wide range of applications. However, while PLMs excel at capturing individual protein properties, they face challenges in natively representing protein\u2013protein interactions (PPIs), which are crucial to understanding cellular processes and disease mechanisms. Here, we introduce MINT, a PLM specifically designed to model sets of interacting proteins in a contextual and scalable manner. Using unsupervised training on a large curated PPI dataset derived from the STRING database, MINT outperforms existing PLMs in diverse tasks relating to protein-protein interactions, including binding affinity prediction and estimation of mutational effects. Beyond these core capabilities, it excels at modeling interactions in complex protein assemblies and surpasses specialized models in antibody-antigen modeling and T cell receptor\u2013epitope binding prediction. MINT\u2019s predictions of mutational impacts on oncogenic PPIs align with experimental studies, and it provides reliable estimates for the potential for cross-neutralization of antibodies against SARS-CoV-2 variants of concern. These findings position MINT as a powerful tool for elucidating complex protein interactions, with significant implications for biomedical research and therapeutic discovery.","version":"1.2","doi":"10.1101/2025.03.09.642188","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.14.643408","pub_date":"2025-3-17","title":"Demonstration and structural basis of a therapeutic DNA aptamer for SARS-CoV-2 spike protein detection","abstract":"At the onset of the COVID-19 pandemic, the absence of a rapid and highly specific diagnostic method for the SARS-CoV-2 virus led to significant delays in detection, adversely affecting clinical outcomes. This shortfall highlights the urgent need for adaptable, scalable, and reusable diagnostic technologies to improve future pandemic responses. To address this challenge, we developed a renewable electrochemical impedance biosensor device employing a synthetic nucleotide-based therapeutic aptamer (termed \u2018aptasensor\u2019) targeting the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We demonstrate that our aptasensor can detect the Omicron BA.2 S protein within one hour and possesses concentration-dependent sensitivity at biologically relevant levels. Notably, the aptasensor is reusable after regeneration by a simple pH 2 buffer treatment. Aptamer binding to the S protein was confirmed by immunogold labeling and visualization by negative-stain electron microscopy. We used cryogenic electron microscopy (cryo-EM) to resolve high-resolution maps of the S protein in both the open and closed conformations and characterized aptamer binding to the up RBD in the open conformation. Taken together, these results establish the versatility and scalability of aptamer-based biosensors, presenting them as a potential transformative diagnostic platform for emerging pathogens. This combination of rapid detection, specificity, and renewable capabilities in a single diagnostic solution marks a significant advance in pandemic preparedness.","version":"1.1","doi":"10.1101/2025.03.14.643408","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.02.641033","pub_date":"2025-3-17","title":"Hydroxychloroquine-functionalized Ionizable Lipids Mitigate Inflammatory Responses in mRNA Therapeutics","abstract":"Lipid nanoparticle (LNP)-based mRNA therapeutics, highlighted by the success of SARS-CoV-2 vaccines, face challenges due to inflammation caused by ionizable lipids. These ionizable lipids can activate the immune system, particularly when co-delivered with nucleic acids, leading to undesirable inflammatory responses. We introduce a novel class of anti-inflammatory ionizable lipids functionalized with hydroxychloroquine (HCQ), which suppresses both lipid-induced and nucleic acid-induced immune activation. These HCQ-functionalized LNPs (HL LNPs) exhibit reduced proinflammatory responses while maintaining efficient mRNA delivery. Structural and physicochemical analyses revealed that HCQ-functionalization results in a distinct particle structure with significantly improved stability. The efficacy of HL LNPs was demonstrated across various therapeutic contexts, including a prophylactic vaccination model against varicella-zoster virus (VZV) and CRISPR-Cas9 gene editing targeting PCSK9. Notably, HL LNPs showed robust mRNA expression after repeated administration, addressing concerns of inflammation and ensuring sustained therapeutic effects. These findings highlight the potential of HCQ-functionalized LNPs in expanding the safe use of mRNA therapeutics, particularly for applications requiring repeated dosing and in scenarios where inflammation-induced side effects must be minimized.","version":"1.2","doi":"10.1101/2025.03.02.641033","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.14.643398","pub_date":"2025-3-17","title":"Immune-Coagulation Dynamics in Severe COVID-19: Insights from Autoantibody Profiling and Transcriptomics","abstract":"Severe COVID-19 is characterized by immune dysregulation and coagulation abnormalities, leading to complications such as thromboembolism and multi-organ failure. This study explores the relationship between autoantibodies targeting coagulation-related factors and gene expression in severe COVID-19. Whole-blood transcriptomics revealed upregulation of coagulation-related genes, including VWF and Factor V, in severe patients compared to mild cases and healthy controls. Autoantibody profiling against seven coagulation-related proteins (ADAMTS13, Factor V, Protein S, SERPINC1, Apo-H, PROC1, and Prothrombin) showed reactivities below established positivity thresholds, but mean-fluorescent intensities were elevated numerically in severe (Protein S) and convalescent (SERPINC1) patients. Correlation analysis revealed trends of negative associations between autoantibody reactivities and coagulation gene expression in severe cases, suggesting a potential role for autoantibodies in modulating immune-coagulation interactions warranting further orthogonal validation. Furthermore, age-dependent increases in subthreshold autoantibody reactivities were observed in severe cases, highlighting the potential impact of immunosenescence on disease severity. These findings do not exclude the possibility that subthreshold autoantibodies may contribute indirectly to immune-coagulation dynamics in severe COVID-19 through mechanisms beyond direct transcriptional regulation. This study highlights the complexity of immune-coagulation interactions and provides foundation for future research into their biological and clinical relevance, particularly for identifying biomarkers and therapeutic targets in thromboinflammatory diseases.","version":"1.1","doi":"10.1101/2025.03.14.643398","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.14.640671","pub_date":"2025-3-14","title":"Intranasal AAV Vaccination of SARS-CoV-2 Induce Strong and Sustained Neutralizing Antibodies in Mice","abstract":"The COVID-19 pandemic continues to pose significant health challenges, despite existing vaccines. This study evaluates the immunogenicity of recombinant adeno-associated viruses (AAV) expressing SARS-CoV-2 spike proteins, administered intramuscularly and intranasally in mice. Both delivery methods of AAV5-spike, AAV5-spike stabilized trimer as well as AAV44.9-spike elicited robust serum anti-spike antibodies within 8-12 weeks, with high levels of anti-spike antibodies sustained for over a year. Comparison of mouse serum antibodies 16 weeks post intramuscular or intranasal AAV5 administration demonstrated similar SARS-CoV-2 spike binding neutralizing activity in vitro. Analysis of changes in cellular immunity by ELISpot at 12 weeks post-AAV spike transduction revealed interferon-\u03b3 induction in response to peptide challenge. Despite a decline in AAV vector DNA at the injection site, the persistence of anti-spike antibodies demonstrated that AAV-vectors can elicit lasting immune responses, highlighting nasal AAV administration as a potential strategy to block respiratory virus infections.","version":"1.1","doi":"10.1101/2025.03.14.640671","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.12.642945","pub_date":"2025-3-14","title":"Cleaved vs. Uncleaved: How Furin Cleavage Reshapes the Conformational Landscape of SARS-CoV-2 Spike","abstract":"The SARS-CoV-2 Spike protein is the primary target for vaccine design, with immunogens typically engineered to enhance stability by introducing proline mutations (2P) and mutating or deleting the Furin Cleavage Site (FCS). While these modifications improve structural integrity, studies suggest that furin cleavage can play a functional role in Spike protein dynamics, potentially enhancing ACE2 receptor binding. However, the impact of this cleavage on the unbound form of the Spike protein remains unclear. In this study, we use extensive all-atom molecular dynamics (MD) simulations to compare the structural and dynamic properties of cleaved and uncleaved Spike proteins in their pre-fusion, unbound state. Our results show that Furin cleavage significantly alters allosteric communication within the protein, increasing correlated motions between the Receptor Binding Domain (RBD) and N-terminal Domain (NTD), which may facilitate receptor engagement. Principal Component Analysis (PCA) reveals that the cleaved and uncleaved Spike proteins sample distinct conformational landscapes, with the cleaved form displaying enhanced flexibility and a broader range of RBD tilt angles. Additionally, Furin cleavage primes the S2 subunit by expanding the central helix, potentially influencing the transition to the post-fusion state. Glycan clustering patterns further suggest an adaptive structural response to cleavage, particularly in the NTD and RBD regions. These findings highlight the potential functional consequences of FCS deletion in immunogen design and underscore the importance of considering the native cleavage state in vaccine and therapeutic development.","version":"1.1","doi":"10.1101/2025.03.12.642945","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.12.642872","pub_date":"2025-3-13","title":"The proton-activated chloride channel inhibits SARS-CoV-2 spike protein-mediated viral entry through the endosomal pathway","abstract":"SARS-CoV-2 binds to its obligatory receptor, angiotensin-converting enzyme 2 (ACE2) and capitalizes on decreasing endosomal acidity and cathepsin-mediated spike protein cleavage to enter cells. Endosomal acidification is driven by V-ATPase which pumps protons (H+) into the lumen. The driving force for H+ is maintained by the import of chloride (Cl-) which is mediated by intracellular CLC transporters. We have recently identified the Proton-Activated Chloride (PAC) channel as a negative regulator of endosomal acidification. PAC responds to low pH and releases Cl- from the lumen to prevent endosomal hyperacidification. However, its role in SARS-CoV-2 viral entry remains unexplored. Here, we show that overexpressing the PAC channel in ACE2 expressing HEK 293T cells markedly inhibited the SARS-CoV-2 spike-mediated viral entry. Several lines of evidence suggest that this effect was due to the suppression of the endosomal entry pathway. First, the abilities of PAC to regulate endosomal acidification and inhibit pseudoviral entry were both dependent on its endosomal localization and channel activity. Second, the inhibitory effect on viral entry was similar to the suppression mediated by E64-d, a cathepsin inhibitor, while no major additive effect for both treatments was observed. Third, this inhibition was also attenuated in cells expressing TMPRSS2, which provides the alternative entry pathway through cell surface. Importantly, PAC overexpression also inhibited the number and size of plaques formed by two live SARS-CoV-2 isolates (B.1 and Omicron XBB.1.16) in Vero E6 cells. Altogether, our data indicates that PAC plays a vital role in inhibiting SARS-CoV-2 viral entry and identifies this endosomal channel as a potential novel target against the infection of SARS-CoV-2 and other viruses, which rely on the endosomal pathway.","version":"1.1","doi":"10.1101/2025.03.12.642872","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.12.642881","pub_date":"2025-3-13","title":"Few-Shot Viral Variant Detection via Bayesian Active Learning and Biophysics","abstract":"The early detection of high-fitness viral variants is critical for pandemic response, yet limited experimental resources at the onset of variant emergence hinder effective identification. To address this, we introduce an active learning framework that integrates protein language model ESM3, Gaussian process with uncertainty estimation, and a bio-physical model to predict the fitness of novel variants in a few-shot learning setting. By benchmarking on past SARS-CoV-2 data, we demonstrate that our methods accelerates the identification of high-fitness variants by up to fivefold compared to random sampling while requiring experimental characterization of fewer than 1% of possible variants. We also demonstrate that our framework benchmarked on deep mutational scans effectively identifies sites that are frequently mutated during natural viral evolution with a predictive advantage of up to two years compared to baseline strategies, particularly those enabling antibody escape while preserving ACE2 binding. Through systematic analysis of different acquisition strategies, we show that incorporating uncertainty in variant selection enables broader exploration of the sequence landscape, leading to the discovery of evolutionarily distant but potentially dangerous variants. Our results suggest that this framework could serve as an effective early warning system for identifying concerning SARS-CoV-2 variants and potentially emerging viruses with pandemic potential before they achieve widespread circulation.","version":"1.1","doi":"10.1101/2025.03.12.642881","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.11.642706","pub_date":"2025-3-12","title":"Informing antiviral effectiveness for influenza A and SARS-CoV-2 by quantifying within-host interaction between transmission and immunity","abstract":"Antiviral therapies are among the most effective pharmaceutical interventions in treatment of a variety of viral pathogens. To optimize the antiviral effectiveness it is crucial to characterize the relationship between multiple cellular modes of antiviral action and the complex response of the host\u2019s innate immune system relative to the within host dynamics of a proliferating virus. Since their introduction in 1968 and 2019, Influenza A virus (IAV) H3N2 and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), respectively, have caused unprecedented damage on the public health infrastructure globally. In addition to the substantial burden of morbidity and mortality around the world, both viruses have the potential of undergoing evolution leading to antigenic escape from the prevailing interventions. These biological characteristics advocate for urgent development of effective antivirals for the treatment of IAV and SARS-CoV-2. In this multi-stage study, we develop a suite of within-host models encompassing a number of hypotheses regarding virus-specific innate host functional responses and their impacts on the proliferation of IAV H3N2 and SARS-CoV-2 viruses. We use likelihood-based statistical inference to confront these hypotheses with infection data on IAV H3N2 and SARS-CoV-2 from infection experiments in ferrets. Upon identifying the best-fitting model of within-host dynamics, we can quantify the potential impact of antiviral drug therapy as a function of effectiveness and timing of initiation. We find significant mechanistic differences between the infection dynamics of H3N2 IAV and SARS-CoV-2 and associated model parameters. The treatment consequences of these differences are that SARS-CoV-2 is harder to control with antivirals, requiring earlier initiation and a more effective drug. Antiviral drugs are prophylactic chemical agents that are used to contain several viral infections. Influenza A H3N2 virus (H3N2) and Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) are very important viral pathogens that have caused unprecedented, global public health damage in the recent times. This makes development of antiviral drugs crucial along with other pharmaceutical prophylactics like vaccination. To optimize the pathogen specific effectiveness, however, it is necessary to simultaneously explore the relationship among the intra-host viral kinetics, immune dynamics and modes of antiviral action. In this article we theoretically analyze antiviral action of a drug in union with effect of the host\u2019s innate immune system in containing infections of SARS-CoV-2 and H3N2 in infection experiments with ferrets. We find fundamental differences in the requisite antiviral effectiveness which, we posit, is due to substantially different inter-cellular proliferation potential between the two viruses.","version":"1.1","doi":"10.1101/2025.03.11.642706","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.11.642564","pub_date":"2025-3-12","title":"Development of a General Purpose Targeted LC-MS Method for Accurate Quantification of the SARS-CoV-2 Spike Protein Expression","abstract":"The COVID-19 pandemic has catalyzed interest in immuno-multiple reaction monitoring (immuno-MRM) methods, with the detection of peptides unique to the nucleocapsid protein in nasopharyngeal swabs. While current applications predominantly focus on disease biomarkers, the pandemic has unveiled new opportunities, namely for the quantification of antigen expression following mRNA vaccination. Here, we present an optimized immuno-MRM method for quantifying SARS-CoV-2 spike protein fusion peptide, SFIEDLLFNK, for several practical applications. The method is versatile, applicable to multiple biological matrices, including plasma, and can be extended to nasopharyngeal swabs. It also offers a high-precision tool for assessing protein expression following plasmid and mRNA transfection. Moreover, in parallel to enabling accurate antigen quantification, the flow-through can be used to determine the proteome profile of the infected cells, providing insights into the intracellular immune response. This dual capability supports the rapid optimization of mRNA vaccines, thereby driving advancements in vaccine development strategies.","version":"1.1","doi":"10.1101/2025.03.11.642564","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.11.642723","pub_date":"2025-3-12","title":"Allosteric Control and Glycan Shielding Adaptations in the SARS-CoV-2 Spike from Early to Peak Virulence","abstract":"The SARS-CoV-2 Spike glycoprotein is central to viral infectivity and immune evasion, making it a key target for vaccine and therapeutic design. This trimeric peplomer undergoes dynamic conformational changes, particularly in its Receptor Binding Domain (RBD), which transitions between closed (down) and ACE2-accessible (up) states relative to the rest of the protein, to facilitate host cell entry. Structural understanding of such critical inter-domain motions, as well as epitope exposure quantification, is essential for obtaining an effective molecular handle over this protein and, in turn, exploiting it towards improved immunogen development. Focusing on the early circulating D614G form and the later emerging Delta (B.1.617.2) variant with higher virulence, we performed large-scale molecular dynamics simulations of the soluble form of the Spike in both \u2018down\u2019 and \u2018up\u2019 conformations of the RBD. Guided by differences in overall fluctuations, we described reaction coordinates based on domain rotations and tilting to extract features that distinguish D614G versus Delta structural behavior of the N-terminal Domain (NTD) and RBD. Using reaction coordinate analysis and Principal Component Analysis (PCA), we identify allosteric coupling between the N-terminal Domain (NTD) and RBD, where NTD tilting influences RBD gating. While some of these motions are conserved across variants, Delta exhibits an optimized RBD-gating mechanism that enhances ACE2 accessibility. Additionally, glycan remodeling in Delta enhances shielding at the NTD supersite, contributing to reduced sensitivity to neutralizing antibodies. Finally, we uncover the impact of the D950N mutation in the HR1 region, which modulates downstream Spike dynamics and immune evasion. Together, our findings reveal variant-specific and conserved structural determinants of SARS-CoV-2 Spike function, providing a mechanistic basis for allosteric modulation, glycan-mediated immune evasion, and viral adaptation. These insights offer valuable guidance for rational vaccine and therapeutic design against SARS-CoV-2 and emerging variants.","version":"1.1","doi":"10.1101/2025.03.11.642723","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.10.642264","pub_date":"2025-3-11","title":"The furin cleavage site is required for pathogenesis, but not transmission of SARS-CoV-2","abstract":"The SARS-CoV-2 spike, key to viral entry, has two features that differentiate it from other sarbecoviruses: the presence of a furin cleavage site (FCS; PRRAR sequence) and an extended S1/S2 loop characterized by an upstream QTQTN amino acid motif. Our prior works show that shortening the S1/S2 loop by deleting either the FCS (\u0394PRRA) or deleting an upstream sequence (\u0394QTQTN), ablates spike processing, alters host protease usage, and attenuates infection in vitro and in vivo. With the importance of the loop length established, here we evaluated the impact of disrupting the FCS, but preserving the S1/S2 loop length. Using reverse genetics, we generated a SARS-CoV-2 mutant that disrupts the FCS (PQQAR) but maintains its extended S1/S2 loop. The SARS-CoV-2 PQQAR mutant has reduced replication, decreased spike processing, and attenuated disease in vivo compared to wild-type SARS-CoV-2. These data, similar to the FCS deletion mutant, indicate that loss of the furin cleavage site attenuates SARS-CoV-2 pathogenesis. Importantly, we subsequently found that the PQQAR mutant is transmitted in the direct contact hamster model despite lacking an intact FCS. However, competition transmission showed that the mutant was attenuated compared to WT SARS-CoV-2. Together, the data argue that the FCS is required for SARS-CoV-2 pathogenesis but is not strictly required for viral transmission.","version":"1.1","doi":"10.1101/2025.03.10.642264","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.09.642200","pub_date":"2025-3-10","title":"The protease inhibitor Nirmatrelvir synergizes with inhibitors of GRP78 to suppress SARS-CoV-2 replication","abstract":"Nirmatrelvir, the active compound of the drug Paxlovid, inhibits the Main protease of SARS-CoV-2 (MPro, 3CLPro, NSP5). Its therapeutic application reduces but does not abolish the progression of COVID-19 in humans. Here we report a strong synergy of Nirmatrelvir with inhibitors of the ER chaperone GRP78 (HSPA5, BiP). Combining Nirmatrelvir with the GRP78-antagonizing drug candidate HA15 strongly inhibits the replication of SARS-CoV-2, to a far greater extent than either drug alone, as observed by diminished cytopathic effect, levels of detectable virus RNA, TCID50 titers, accumulation of the non-structural protein 3 (NSP3), as well as Spike and N proteins. The original SARS-CoV-2 strain as well as an Omicron variant were similarly susceptible towards the drug combination. Other GRP78 inhibitors or siRNAs targeting GRP78 also fortified the antiviral effect of Nirmatrelvir. In a hamster model of COVID-19, the combination of Nirmatrelvir with HA15 alleviated pneumonia-induced pulmonary atelectasis more effectively than the single drugs. In conclusion, inhibition of the virus Main protease and cellular GRP78 cooperatively diminishes virus replication and may improve COVID-19 therapy.","version":"1.1","doi":"10.1101/2025.03.09.642200","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.11.598471","pub_date":"2025-3-10","title":"A human-ACE2 knock-in mouse model for SARS-CoV-2 infection recapitulates respiratory disorders but avoids neurological disease associated with the transgenic K18-hACE2 model","abstract":"Animal models have been instrumental in elucidating the pathogenesis of SARS-CoV-2 infection and testing COVID-19 vaccines and therapeutics. Wild-type (WT) mice are not susceptible to many SARS-CoV-2 variants, therefore transgenic K18-hACE2 mice have emerged as a standard model system. However, this model is characterized by severe disease, particularly associated with neuroinfection, which leads to early humane endpoint euthanasia. Here, we established a novel knock-in (KI) mouse model by inserting the original K18-hACE2 transgene into the collagen COL1A1 locus using a recombinase mediated cassette exchange (RMCE) system. Once the Col1a1-K18-hACE2 mouse colony was established, animals were challenged with a B.1 SARS-CoV-2 (D614G) isolate and were monitored for up to 14 days. Col1a1-K18-hACE2 mice exhibited an initial weight loss similar to the K18-hACE2 transgenic model but did not develop evident neurologic clinical signs. The majority of Col1a1-K18-hACE2 mice did not reach the preestablished humane endpoint, showing progressive weight gain after 9 days post-infection (dpi). Importantly, despite this apparent milder pathogenicity of the virus in this mouse model compared to the K18-hACE2 transgenic model, high levels of viral RNA were detected in lungs, oropharyngeal swab, and nasal turbinate. Remaining lesions and inflammation in lungs were still observed after 14 dpi. In contrast, although low level viral RNA could be detected in a minority of Col1a1-K18- hACE2 animals, no brain lesions were observed at any timepoint. Overall, Col1a1-K18- hACE2 mice constitute a new model for investigating SARS-CoV-2 pathogenesis and treatments, with potential implications for studying long-term COVID-19 sequelae. K18-hACE2 mice express high levels of the human protein ACE2, the receptor for SARS- CoV-2, and can therefore be infected by this virus. These animals have been crucial to understand viral pathogenesis and to test COVID-19 vaccines and antiviral drugs. However, K18-hACE2 often die after infection with initial SARS-CoV-2 variants likely due to a massive brain infection that does not occur in humans. Here, we used a technology known as knock-in that allows for the targeted insertion of a gene into a mouse and we have generated a new hACE2-mouse. We have characterized this new animal model demonstrating that the virus replicates in the respiratory tract, damaging lung tissue and causing inflammation. In contrast to K18-hACE2 mice, only limited or no brain infection could be detected, and most animals recovered from infection with remaining lung lesions. This new model could be instrumental for the study of specific disease aspects such as post-COVID condition, sequelae, and susceptibility to reinfection.","version":"1.3","doi":"10.1101/2024.06.11.598471","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.09.642282","pub_date":"2025-3-10","title":"From Plasmid to Pure Protein: Production and Characterization of SARS-CoV-2 PLpro","abstract":"Papain-like protease (PLpro) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. We present protocols for PLpro production in Escherichia coli. PLpro expressed as a fusion with the Saccharomyces cerevisiae Smt3 protein (SUMO), is purified and obtained in its native form upon hydrolysis, with yields as high as 38 mg L-1. The protocol also provides isotope-enriched samples suitable for NMR studies. Protocols are also presented for PLpro characterization by mass spectrometry, 1D 19F-NMR and 2D heteronuclear NMR, and a fluorescence-based enzyme assay. Production, purification, and biochemical analysis of native N- and C-termini PLpro High yields in E. coli, up to 38 mg L-1 using lysogeny broth. Supports labeled samples for inhibitor interaction studies. 19F NMR and fluorescence assays for inhibitor screening and IC50 determination. SARS-CoV-2 PLpro is a key cysteine protease involved in viral replication and immune evasion, making it an important target for antiviral drug development. This study presents a detailed protocol for PLpro production, purification, and biochemical analysis, achieving high yields in E. coli. The workflow includes fusion expression with a His-SUMO tag, isotope labeling for inhibitor studies, and assays for screening and quantifying inhibitors. This comprehensive guide facilitates large-scale production of active PLpro for drug discovery and structural studies.","version":"1.1","doi":"10.1101/2025.03.09.642282","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.09.642192","pub_date":"2025-3-10","title":"Investigating endothelial cell transduction and hexon:PF4 binding of ChAdOx1 in the context of VITT","abstract":"Vaccines against SARS-CoV2 have been essential in controlling COVID-19 related mortality and have saved millions of lives. Adenoviral (Ad) based vaccines have played an integral part in this vaccine campaign, with licensed vaccines based on the simian Y25 isolate (Vaxzevria, Astrazeneca) and human Ad type 26 (Jcovden, Janssen) widely adopted. As part of the largest global vaccination programme ever undertaken, ultrarare thromboembolic events have been described in approximately 1:200,000 vaccinees administered with Ad based SARS-CoV2 vaccines. The mechanism underpinning these adverse events remain to be completely delineated, but is characterised by elevated autoantibodies against PF4 which, in complex with PF4, cluster, bind FC\u03b3RIIa on platelets and induce thrombus formation. Here we investigated the ability of ChAdOx1 to transduce and activate endothelial cells. Using protein sequence alignment tools and in vitro transduction assays, the ability of ChAdOx1 to infect endothelial cells was assessed. Furthermore, the ability of ChAdOx1 infection to activate endothelial cells was determined. Finally, using surface plasmon resonance we assessed the electrostatic interactions between the ChAdOx1 hexon and PF4. Despite lacking the primary cell entry receptor, Coxsackie and Adenovirus Receptor (CAR), ChAdOx1 efficiently transduced endothelial cells in a CAR-independent manner. This transduction did not result in endothelial cell activation. Purified hexon protein from ChAdOx1 preps did, however, bind PF4 with a similar affinity to that previously reported for the whole ChAdOx1 capsid. These data confirm the need to develop non-PF4 binding adenoviral capsids to reduce the potential adverse events associated with VITT.","version":"1.1","doi":"10.1101/2025.03.09.642192","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.07.642108","pub_date":"2025-3-10","title":"Point-Of-Need One-Pot Multiplexed RT-LAMP Test For Detecting Three Common Respiratory Viruses In Saliva","abstract":"Respiratory viral infections pose a significant global public health challenge, partly due to the difficulty in rapidly and accurately distinguishing between viruses with similar symptoms at the point of care, hindering timely and appropriate treatment and limiting effective infection control and prevention efforts. Here, we developed a multiplexed, non- invasive saliva-based, reverse transcription loop-mediated isothermal amplification (RT- LAMP) test that enables the simultaneous detection of three of the most common respiratory infections, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza (Flu), and respiratory syncytial virus (RSV), in a single reaction via specific probes and monitored in real-time by a machine-learning-enabled compact analyzer. Our results demonstrate that the multiplexed assay can effectively detect three target RNAs with high accuracy. Further, testing with spiked saliva samples showed strong agreement with reverse transcription polymerase chain reaction (RT-PCR) assay, with area under the curve (AUC) values of 0.82, 0.93, and 0.96 for RSV, Influenza, and SARS-CoV-2, respectively. By enabling the rapid detection of respiratory infections from easily collected saliva samples at the point of care, the device presented here offers a practical and efficient tool for improving outcomes and helping prevent the spread of contagious diseases. This research presents an innovative approach to respiratory infection diagnostics by combining a one-pot isothermal molecular test with machine learning-based analysis to simultaneously detect SARS-CoV-2, Influenza, and RSV in saliva samples. The battery- powered portable analyzer features novel machine-learning-assisted fluorescence detection for multiplexed reporter quantification, eliminating the need for traditional filter- based optical components and enabling adaptation to new targets without hardware changes. The test demonstrates high accuracy in detecting single and co-infections in spiked saliva samples, providing a rapid, cost-effective point-of-need solution. This tool can expand testing access, improve patient outcomes, and support more effective disease control, particularly in resource-limited or decentralized healthcare settings.","version":"1.1","doi":"10.1101/2025.03.07.642108","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.07.642080","pub_date":"2025-3-09","title":"SARS-CoV-2 Exploits Host Translation and Immune Evasion Pathways via Viral RNA\u2013Host Protein Interactions","abstract":"RNA viruses, including SARS-CoV-2, Influenza A Virus (IAV), Zika Virus, and Dengue Virus (DENV) pose serious global health challenges by manipulating host cellular mechanisms. SARS-COV-2, in particular exploits host translational machinery to enhance replication and evade immune response. Here, we investigated how SARS-CoV-2 circumvents host immune defenses through RNA - host protein interactions. By integrating multiple datasets, ClusterProfiler, KEGG, Reactome, WikiPathways, and Gene Ontology, we performed functional enrichment analyses on host protein interactions with SARS-CoV-2 RNA. Our results identified key pathways involved in viral replication, translation regulation, and immune evasion. Comparing SARS-CoV-2 interactomes from IAV, Zika, and DENV, we uncovered a subset of 275 common host proteins serving as promising targets for broad-spectrum antiviral strategies. Network analysis highlighted critical translation factors (EEF1A1, EIF4A1, EIF3H) and RNA-binding proteins (NCL, ILF3) as key nodes in viral replication. These findings provide insights into RNA virus pathogenesis and support the development of targeted therapeutics.","version":"1.1","doi":"10.1101/2025.03.07.642080","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.07.642081","pub_date":"2025-3-09","title":"D614G reshapes allosteric networks and opening mechanisms of SARS-CoV-2 spikes","abstract":"The SARS-CoV-2 spike glycoprotein binds human epithelial cells and enables infection through a key conformational transition that exposes its receptor binding domain (RBD). Experimental evidence indicates that spike mutations, particularly the early D614G variant, alter the rate of this conformational shift, potentially increasing viral infectivity. To investigate how mutations reshape the conformational landscape, we conducted extensive weighted ensemble simulations of the Ancestral, Delta, and Omicron BA.1 spike strains along the RBD opening pathway. We observe that Ancestral, Delta, and Omicron BA.1 spike RBDs open differently, with Omicron BA.1 following a more direct opening profile until it reaches a \u201csuper-open\u201d state wherein it begins to \u201cpeel\u201d, suggesting increased S1 flexibility. Via dynamical network analysis, we identified two allosteric communication networks uniting all S1 domains: the established N2R linker and a newly discovered anti-parallel R2N linker. In Delta and Omicron BA.1 variant spikes, RBD opening is facilitated by both linkers, while the Ancestral strain relies predominantly on the N2R linker. In the ancestral spike, the D614-K854 salt bridge impedes allosteric communication through the R2N linker, whereas the loss of this salt bridge in all subsequent VOCs alleviates local frustration and, we believe, accelerates RBD opening. Hydrogen-deuterium mass spectrometry experiments validate these altered dynamics in the D614 region across Ancestral, D614G, and Omicron BA.1 spikes. This study unveils a \u2018hidden\u2019 allosteric network, connecting the NTD to the RBD via the 614-proximal region, and the D614G mutation reshapes the fitness landscape of these critical viral glycoproteins. Our work reveals how the D614G mutation in the SARS-CoV-2 spike protein reshapes its internal communication pathways and speeds up receptor binding domain (RBD) opening, providing mechanistic insight into the evolution and enhanced infectivity of SARS-CoV-2 variants of concern. We also describe differences in opening pathways and relative rates of opening for Delta and Omicron BA.1 spike RBDs relative to the original (Ancestral) coronavirus strain from Wuhan, China.","version":"1.1","doi":"10.1101/2025.03.07.642081","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.06.641885","pub_date":"2025-3-07","title":"SARS-CoV-2 Infects Peripheral Sensory Neurons and Promotes Axonal Degeneration via TRPV1 Activation","abstract":"Common neurological symptoms of COVID-19, such as anosmia, headaches, and cognitive dysfunction, depend on interactions between the peripheral and central nervous systems. However, the molecular mechanisms by which SARS-CoV-2 affects the peripheral nervous system remain poorly understood, with ongoing debate about whether sensory neurons can be directly infected by the virus. In this study, human iPSC-derived sensory neurons were exposed to the SARS-CoV-2 BA.5 variant, a mutant virus, or viral S1 proteins. Under control conditions, sensory neurons exhibited low expression of ACE2. However, exposure to BA.5 or S1 proteins significantly upregulated ACE2 expression in peripherin-positive sensory neurons. Virological analysis confirmed that SARS-CoV-2 directly infects TRPV1-expressing sensory neurons, including olfactory neurons. Moreover, exposure to the live virus or S1 proteins induced TRPV1 upregulation and translocation from the nucleus to the cytosol, resulting in axonal destruction. Single-nucleus transcriptomic analysis revealed that viral exposure enhanced cAMP signaling, virus receptor and transmembrane transporter activities, and inflammatory regulation of TRP channels, which collectively contributed to synaptic and axonal damage. Importantly, treatment with a TRPV1 antagonist demonstrated neuroprotective effects. These findings underscore the need for further research into the interaction between SARS-CoV-2 and TRPV1, as well as its downstream signaling pathways, to develop therapeutic strategies for preventing sensory neuron loss during viral infections. iPSC technology was employed to generate peripheral sensory neurons from human induced pluripotent stem cells (iPSCs), providing a valuable platform for studying the impact of SARS-CoV-2 on peripheral sensory neurons. Our findings demonstrated that the SARS-CoV-2 Omicron BA.5 variant exerted both direct and indirect effects on peripheral sensory neurons. Virus exposure upregulated the angiotensin-converting enzyme 2 (ACE2) receptor in peripherin-positive neurons. Additionally, exposure to the virus or its S1 spike protein increased transient receptor potential vanilloid 1 (TRPV1) expression and trafficking, leading to axonal degeneration. Single-nucleus RNA sequencing revealed that BA.5 exposure enhanced cAMP signaling pathway, virus receptor and transmembrane transporter activities, and inflammatory regulation of TRP channels that led to the significantly damage on synapses and axon guidance. The TRPV1 antagonist capsazepine inhibited TRPV1 activation and mitigated axonal damage, offering neuroprotective effects for sensory neurons exposed to SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.03.06.641885","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.06.641801","pub_date":"2025-3-06","title":"DMBT1 promotes SARS-CoV-2 infection and its SRCR-derived peptide inhibits SARS-CoV-2 infection","abstract":"DMBT1 is a large scavenger receptor cysteine rich (SRCR) B protein that has been reported as a tumor suppressor gene and a co-receptor for HIV-1 infection. Here we found DMBT1 is a major mucosal protein bound to SARS-CoV-2. Overexpression of DMBT1 in 293T cells enhanced infection by SARS-CoV-2 in ACE2 dependent manner. Blocking experiments using overlapping peptide library of SRCR domain of DMBT1 showed that CQGRVEVLYRGSWGTV peptide, which contains bacteria-binding VEVLXXXXW motif, could inhibit SARS-CoV-2 infection. High concentration of the peptide can significantly inhibit the replication of SARS-CoV-2 in hamsters. Single cell sequencing analysis showed the highest expression abundance of DMBT1 at airway submucosal glands. These results demonstrate that membrane DMBT1 can promote SARS-CoV-2 infection, and serve as a candidate target for antiviral development. We discovered that a protein named as DMBT1, which is found in our mucous membranes, plays a significant role in SARS-CoV-2 infection. DMBT1 is a large glycoprotein that has previously reported linked to tumor suppression and HIV-1 infection. Our single-cell sequencing analysis revealed that DMBT1 is most abundant in the airway submucosal glands. In our study, we found that when DMBT1 is overexpressed in cells, it enhances SARS-CoV-2 infection. Using a peptide library from DMBT1, we pinpointed a specific peptide, CQGRVEVLYRGSWGTV, which can inhibit SARS-CoV-2 infection. At high concentrations, it significantly reduces the virus\u2019s ability to replicate in hamsters. These findings suggest that DMBT1 on the cell membrane can promote SARS-CoV-2 infection and highlight DMBT1 as a potential target for developing antiviral treatments.","version":"1.1","doi":"10.1101/2025.03.06.641801","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.06.641790","pub_date":"2025-3-06","title":"Synergistic interference with SARS-CoV-2 replication by Molnupiravir-derived N4 hydroxycytidine and inhibitors of CTP synthetase","abstract":"N4-hydroxycytidine (NHC), the active metabolite of Molnupiravir, is incorporated into nascent RNA of SARS-CoV-2 and interferes with subsequent virus replication. We have previously described synergy between NHC and inhibitors of dehydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine synthesis. Upon DHODH inhibition, the lack of endogenous pyrimidines conceivably enhances NHC incorporation. However, the question remains whether preventing the synthesis of just one pyrimidine base, cytidine, might as well augment the antiviral efficacy of NHC. We tested this by inhibiting CTP synthetases (CTPSs), the cellular enzymes that directly catalyze the synthesis of a cytidine nucleotide. We observed that inhibitors of CTP synthetase (CTPSis), namely cyclopentenyl cytosine (CPEC) as well as STP938 and STP720, display a strong synergy with NHC for diminishing SARS-CoV-2 replication in cell culture, as shown earlier for DHODH inhibitors. NHC and CTPSis in combination prevented the cytopathic effect of SARS-CoV-2 and strongly reduced the release of viral RNA and infectious particles, as well as the synthesis of viral proteins. This combination was also active against an Omicron variant of SARS-CoV-2. Addition of cytidine, but not uridine, rescued virus growth under these conditions. Of note, treating SARS-CoV-2-infected hamsters with the CTPS1 inhibitor STP938 strongly diminished COVID pathology. We propose that CTPS inhibition has the potential to increase the efficacy of antiviral cytidine analogues and to treat coronavirus infections. The efficacy of NHC against SARS-CoV-2 replication in cell culture models is intensified by several orders of magnitude through targeting cellular CTP-Synthetase. The drug combination still displays its effect against SARS-CoV-2 replication in the presence of uridine, suggesting that serum uridine cannot counteract its efficacy. CTPS inhibition diminishes COVID-19-like pathology in an established animal model.","version":"1.1","doi":"10.1101/2025.03.06.641790","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.05.641671","pub_date":"2025-3-06","title":"Unveiling the Immune Landscape of COVID-19 and prolonged Long-COVID through Single-Cell RNA Sequencing","abstract":"Long-COVID affects at least 10% of COVID-19 survivors, displaying debilitating symptoms across multiple organ systems. Despite the increasing prevalence, the underlying causes remain unclear. This study presents a unique analysis of the PBMC transcriptomic landscape of COVID-19 and Long-COVID patients at a single-cell resolution. We reconstructed the cell state and communication using differentially expressed gene profiling and ligand-receptor interaction analyses. Our results reveal altered T and NK cell subset proportions, diminished proliferating lymphocyte and B cell signalling capacity, and the expression of exhaustion and cytotoxicity associated genes 1.5 \u2013 2 years post-infection, suggesting incomplete immune recovery. Collectively, these findings provide insights into the immune processes underlying the progression of COVID-19 into a chronic Long-COVID state.","version":"1.1","doi":"10.1101/2025.03.05.641671","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.04.641425","pub_date":"2025-3-05","title":"SARS CoV-2 spike adopts distinct conformational ensembles in situ","abstract":"Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.","version":"1.1","doi":"10.1101/2025.03.04.641425","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.05.641480","pub_date":"2025-3-05","title":"No baby boom of social mammals in European zoos resulting from COVID-19 lockdowns","abstract":"Most zoo animals are exposed daily to human visitors, which could be a major disturbance on reproduction. Although few studies have examined the relationship between number of births and crowd size by comparing high and low visitor days, the effect of zoo visitors on animal reproduction over longer timescales has been overlooked. We assessed whether number of births in 34 social mammal species hosted in zoos from 23 European countries differed after prolonged periods of closure to the public (COVID-19 lockdowns) as compared with preceding years. An analysis restricted to the periods following the lockdowns and corresponding to any conception during closure (from 2020 to 2022), compared with the same periods in the two previous years (2018 and 2019), showed no effect of zoo closure on number of births, even when considering each species\u2019 life history traits and whether they were managed under a breeding programme. We found no evidence that a complete and prolonged absence of visitors affected the ability to reproduce in social mammals housed across European zoos. By analysing birth variation in a large sample of mammalian species, this study contributes to a better knowledge of the natality of mammals in captive settings opened to the public.","version":"1.1","doi":"10.1101/2025.03.05.641480","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.05.606569","pub_date":"2025-3-04","title":"The Impact of Coronavirus Nsp1 on Host mRNA Degradation Is Independent of Its Role in Translation Inhibition","abstract":"When host cells are infected with coronaviruses, the first viral protein produced is Nsp1. This protein inhibits host protein synthesis and induces host mRNA degradation to enhance viral proliferation. Despite its critical role, the mechanism by which Nsp1 mediates cellular mRNA degradation remains unclear. In this study, we use cell-free translation to address how the host mRNA stability is regulated by Nsp1. We reveal that SARS-CoV-2 Nsp1 binding to the ribosome is enough to trigger mRNA degradation independently of ribosome collisions or active translation. MERS-CoV Nsp1 inhibits translation without triggering degradation, highlighting mechanistic differences between the two Nsp1 counterparts. Nsp1 and viral mRNAs appear to co-evolve, rendering viral mRNAs immune to Nsp1-mediated degradation in SARS-CoV-2, MERS-CoV and Bat-Hp viruses. By providing new insights into the mode of action of Nsp1, our study helps to understand the biology of Nsp1 better and find new strategies for therapeutic targeting against coronaviral infections. Cell-free assays allow the decoupling of Nsp1-mediated translation inhibition from RNA degradation. Nsp1 interaction with the ribosome is crucial for mRNA degradation, but active translation is not required. SARS-CoV-2 Nsp1 induces mRNA degradation, while MERS-CoV Nsp1 inhibits translation without triggering degradation. 5\u2019UTR-specific protection of viral mRNAs from Nsp1 indicates a co-evolutionary adaptation between the two features","version":"1.4","doi":"10.1101/2024.08.05.606569","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.01.639360","pub_date":"2025-3-03","title":"Longitudinal analysis of antibody titers after primary and booster mRNA COVID-19 vaccination can identify individuals at risk for breakthrough infection","abstract":"A key issue in the post-COVID-19 era is the ongoing administration of COVID-19 vaccines. Repeated vaccination is essential for preparing against currently circulating and newly emerging SARS-CoV-2 variants while enabling people to continue with daily life. Optimizing vaccination strategies is crucial to efficiently manage medical resources and establish an effective vaccination framework. Therefore, it is important to quantitatively understand vaccine-induced immunity dynamics and to be able to identify poor responders with lower sustained antibody titers as potential priorities for revaccination. We investigated longitudinal antibody titer data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to November 2022 for whom basic demographic and health information was available. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers after 2 primary doses and 1 booster dose of mRNA COVID-19 vaccines. We identified 3 notable populations, which we refer to as the durable, the vulnerable, and the rapid-decliner populations, approximately half of which remained in the same population after the booster dose. Notably, the rapid-decliner population experienced earlier infections than the others. Furthermore, when comparing IgG(S) titers, IgA(S) titers, and T-spot counts between participants who experienced breakthrough infections after booster vaccination and those who did not, we found that IgA(S) titers were significantly lower in breakthrough infected participants during the early stage after booster vaccination. Our computational approach is adaptable to various types of vaccinations. This flexibility can inform policy decisions on vaccine distribution to enhance immunity both in future pandemics and in the post-COVID-19 era.","version":"1.1","doi":"10.1101/2025.03.01.639360","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.28.640788","pub_date":"2025-3-02","title":"Cryo-ET of IgG bivalent binding on SARS-CoV-2 provides structural basis for antibody avidity","abstract":"The bivalent nature of IgG enhances its neutralization potency against enveloped viruses; however, on-virion structural details of IgG bivalent binding with antigens remain elusive. Here we used cryo-ET to investigate how two potent IgGs P17 and S309 interact with S-trimers on the SARS-CoV-2 surface. We found that these antibodies exploit the mobility of S-trimers to form diverse bivalent binding patterns. P17 stabilizes S-trimers in a one-RBD-up conformation and gathers S-trimer into linear multimers within minutes, whereas S309 primarily forms circular S-trimer assemblies that extend into lattice-like structures. Additionally, both IgGs can facilitate inter-virion coupling through bivalent binding of opposing S-trimers. These findings provide a structural basis for understanding IgG avidity and offer insights for antibody engineering and vaccine design.","version":"1.1","doi":"10.1101/2025.02.28.640788","journal":"bioRxiv","score":null},{"id":"10.1101/2025.03.01.640975","pub_date":"2025-3-02","title":"Global Distribution of Coronaviruses among Bat Populations detected using Molecular techniques, A Systematic Review","abstract":"Surveillance of bat coronaviruses (CoVs) is of public health importance, as accumulating evidence suggests that bats are hosts of the three significant pandemic viruses, namely Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2. Studies focused on identifying different species of bat CoVs may have information cardinal for effective prevention and control of emerging zoonotic diseases. We conducted a systematic review using selected keywords (Surveillance, detection, identification, discovery, isolation, characterization, molecular methods, and Bat coronaviruses) to evaluate molecular studies on CoVs in bats. A total of 790 articles were found using the advanced search strategy of the PubMed database. Following the selection criteria, a total of 127 articles were finally chosen for full-text evaluation. Out of the total of 54 countries examined, China emerged as the country with the highest number of studies, accounting for 26% (n = 33). The sample categories consisted of faecal, urine, guano, blood, tissues, oral, and rectal swabs. The molecular detection approaches included polymerase chain reaction (PCR)-based techniques using species-specific, genus-specific, or broad-range primers. Approximately 94.5% (n = 120) of studies used PCR assays that amplified the partial RdRp gene of length ranging from 123 to 440 bp, followed by amplicon sequencing using either Sanger or next-generation sequencing technologies. Full genome sequencing was only performed in approximately 33.9% (n = 43), with metagenomics approaches being used in 15.7% (n = 20) of the studies. The higher positivity rate of bat CoVs were detected in Asia. Globally, the most predominant bat species which tested positive for CoVs were Rhinolophus, Myotis, Miniopterus, Scotophilus, Eidolon, Chaerephon, Hipposideros, and Desmodus. Continuous bat coronavirus surveillance using molecular methods and full genome sequencing is of utmost importance in detecting and characterizing viruses at molecular level and establishing the genetic diversity of new and circulating viruses.","version":"1.1","doi":"10.1101/2025.03.01.640975","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.26.639208","pub_date":"2025-2-28","title":"Discovery of The Clinical Candidate S-892216: A Second-Generation of SARS-CoV-2 3CL Protease Inhibitor for Treating COVID-19","abstract":"The coronavirus disease 2019 (COVID-19) pandemic crisis has been mitigated by worldwide efforts to develop vaccines and therapeutic drugs. However, there remains concern regarding public health and an unmet need for therapeutic options. Herein, we report the discovery of S-892216, a second-generation SARS-CoV-2 3C-like protease (3CLpro) inhibitor, to treat COVID-19. S-892216 is a reversible covalent 3CLpro inhibitor with highly potent antiviral activity and an EC50 value of 2.48 nM against SARS-CoV-2 infected cells. Structure-based design of a covalent modifier for compound 1 revealed that introducing a nitrile warhead increased 3CLpro inhibition activity by 180-fold. Subsequent optimization efforts yielded S-892216, which combined a favorable pharmacokinetic profile and high off-target selectivity. S-892216 exhibited antiviral activity against diverse SARS-CoV-2 variants, with no cross-resistance to major mutations reducing antiviral activities of nirmatrelvir and ensitrelvir. In SARS-CoV-2-infected mice, S-892216 inhibited viral replication in the lungs similar to ensitrelvir, although at a 30-fold lower dose.","version":"1.1","doi":"10.1101/2025.02.26.639208","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.26.640194","pub_date":"2025-2-28","title":"Preclinical evaluation of the efficacy of \u03b1\u2212Difluoromethylornithine and Sulindac against SARS-CoV-2 infection","abstract":"Despite numerous research efforts and several effective vaccines and therapies developed against COronaVIrus Disease 2019 (COVID-19), drug repurposing remains an attractive alternative to identify new treatments for SARS-CoV-2 virus variants and other viral infections that may emerge in the future. Cellular polyamines support viral propagation and tumor growth. Here we tested the antiviral activity of an irreversible inhibitor of polyamine biosynthesis, \u03b1-difluoromethylornithine (DFMO) and a non-steroidal anti-inflammatory drug (NSAID) Sulindac, which have been previously evaluated for colon cancer chemoprevention. The drugs were tested as single agents and in combination in human Calu-3 lung adenocarcinoma and Caco-2 colon adenocarcinoma cell lines and the K18-hACE2 transgenic mouse model of severe COVID-19. DFMO/Sulindac combination significantly suppressed SARS-CoV-2 N1 Nucleocapsid mRNA and ACE2 mRNA levels in the infected human cell lines by interacting synergistically when cells were pretreated with drugs and additively when treatment was applied to the infected cells. The antiviral activity of DFMO and Sulindac was tested in vivo as prophylaxis (drug supplementation at the doses equivalent to the human chemoprevention trial started 7 days before infection) or as treatment (drug supplementation started 24 hours post-infection). Prophylaxis with DFMO and Sulindac as single agents significantly increased survival rates in the young male mice (p=0.01, and p=0.027, respectively), and the combination was effective in the aged male mice (p=0.042). Young female mice benefited the most from the prophylaxis with Sulindac alone (p=0.001) and DFMO/Sulindac combination (p=0.018), while aged female mice did not benefit significantly from any interventions. The treatment regime was ineffective in suppressing SARS-CoV-2 infection in K18-hACE2 mice. Overall, animal studies demonstrated the protective age- and sex-dependent antiviral efficacy of DFMO and Sulindac against SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.02.26.640194","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.26.640439","pub_date":"2025-2-28","title":"Dynamics of natural selection preceding human viral epidemics and pandemics","abstract":"Using a phylogenetic framework to characterize natural selection, we investigate the hypothesis that zoonotic viruses require adaptation prior to zoonosis to sustain human-to-human transmission. Examining the zoonotic emergence of Ebola virus, Marburg virus, influenza A virus, SARS-CoV, and SARS-CoV-2, we find no evidence of a change in the intensity of natural selection immediately prior to a host switch, compared with typical selection within reservoir hosts. We conclude that extensive pre-zoonotic adaptation is not necessary for human-to-human transmission of zoonotic viruses. In contrast, the reemergence of H1N1 influenza A virus in 1977 showed a change in selection, consistent with the hypothesis of passage in a laboratory setting prior to its reintroduction into the human population, purportedly during a vaccine trial. Holistic phylogenetic analysis of selection regimes can be used to detect evolutionary signals of host switching or laboratory passage, providing insight into the circumstances of past and future viral emergence.","version":"1.1","doi":"10.1101/2025.02.26.640439","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.27.640555","pub_date":"2025-2-28","title":"Construction and characterization of coronavirus nonstructural protein 3-host protein interaction networks unravel an important role of cleavage and polyadenylation specificity factor 6 in regulation of viral RNA replication","abstract":"Coronavirus nonstructural protein 3 (nsp3) plays a crucial role in viral replication and immune evasion. However, functional and proteomic characterization of this protein, especially the interaction networks between nsp3 from different coronaviruses and host cell factors, is hindered by its huge size, complex structural feature and the presence of multiple transmembrane domains. In this study, we report the application of a high-performance cytoplasmic expression system to efficiently and accurately express the full-length nsp3 from betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and gammacoronavirus infectious bronchitis virus (IBV), to investigate their interactions with host proteins using proteomics approaches. Our study identified 1,150 host proteins that interact with IBV nsp3 and 920 with SARS-CoV-2 nsp3. Among them, 658 are shared by the two nsp3 proteins. Further validation and preliminary characterization of seven selected candidates, DDX5, DDX39, DHX9, elF4A3, SRRT and CPSF6, demonstrated the reproducibility and reliability of the proteomics data. More interestingly, an important regulatory role of the nsp3-CPSF6 interaction in the replication and transcription of IBV gRNA and sgRNA was unraveled. The construction of nsp3-host protein interaction networks from two distantly related coronaviruses would have provided a foundation for future studies of host cell factors in the regulation of coronavirus replication and pathogenesis.","version":"1.1","doi":"10.1101/2025.02.27.640555","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.25.640151","pub_date":"2025-2-27","title":"Unveiling the Complete Spectrum of SARS-CoV-2 Fusion Stages by In Situ Cryo-ET","abstract":"SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM has revealed stable prefusion and postfusion conformations of the spike, the transient intermediate states during the fusion process have remained poorly understood. Here, we designed a near-native viral fusion system that recapitulates SARS- CoV-2 entry and used cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that is dependent on protease cleavage and inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2\u2019 cleavage concurrently transition to postfusion conformations encircling the hemifusion and pre-fusion pores in a distinct conical arrangement. Subtomogram averaging revealed that the WS6 S2 antibody binds to the spike\u2019s stem-helix, crosslinks and clusters prefusion spikes and inhibits refolding of fusion intermediates. These findings elucidate the complete process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.","version":"1.1","doi":"10.1101/2025.02.25.640151","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.24.639954","pub_date":"2025-2-27","title":"Impact of cell culture model systems on SARS-CoV-2 and MERS-CoV infection dynamics and antiviral responses","abstract":"Cell cultures are widely used to study infectious respiratory diseases and to test therapeutics; however, they do not faithfully recapitulate the architecture and complexity of the human respiratory tract. Lung organoids have emerged as an alternative model that partially overcomes this key disadvantage. Lung organoids can be cultured in various formats that offer potential for studying highly pathogenic viruses. However, the effects of these different formats on virus infection remain unexplored, leaving their relative value unclear. In this study, we generated primary lung organoids from human donor cells and used them to derive monolayers and air-liquid interface (ALI) cultures with the goal of comparing the replication kinetics of two circulating highly pathogenic coronaviruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV). Infection studies revealed that organoid-derived monolayers displayed limited infection and the innate immune response was impaired against bacterial lipopolysaccharide (LPS) but not against virus-like double-stranded dsRNA or poly(I:C). Meanwhile, organoids and organoid-derived ALI cultures retained viral permissivity, with ALI cultures displaying diverse antiviral immune responses against both coronaviruses. SARS-CoV-2 and MERS-CoV demonstrated differential replication kinetics in organoid and organoid-derived ALI cultures. Therefore, primary organoid-derived cells in two-dimensional monolayer or three-dimensional ALI formats influence virus infection and host antiviral responses. Our study informs the selection of culture conditions for organoid-based respiratory disease research and therapeutic testing. The COVID-19 pandemic heralded the upsurge in human-derived lung organoid based studies due to their cellular heterogeneity that emulates the cellular complexity of the respiratory tract. A major disadvantage of organoid models resides in their apical-in conformation that \u201chides\u201d cells and proteins that are typically exposed to the air-liquid interface (ALI) in the airways and are targets of viruses. Here, we generated monolayers and ALI cultures to facilitate cell exposure to highly relevant pathogens and compared them to parental organoids. Organoids at the ALI captured infection and immune responses better than organoids and organoid-derived monolayer cultures. Organoids at the ALI are a viable approach to improve identification and characterization of virus infection, host responses, and therapeutic testing.","version":"1.1","doi":"10.1101/2025.02.24.639954","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.25.639252","pub_date":"2025-2-27","title":"Structural and Conformational Impact of Deleterious Spike Protein Mutations in SARS-CoV-2 Omicron Lineages","abstract":"A large number of mutations in the Spike (S) protein of the SARS-CoV-2 omicron variant have been noted to alter the receptor binding domain (RBD) and increase the binding surface and enhance the opening of the binding pocket. The cumulative effect of S1 and S2 subunit mutations can influence the conformational dynamics of the binding surface, facilitating the release of viral genome into host cells. This study investigates the deleterious mutations across all Omicron lineages identified in our analysis and their effect on the conformational stability of RBD opening. Whole Genome Sequencing of 231 SARS-CoV-2 positive patients in Karachi, Pakistan, were performed using Illumina Miseq instrument and raw reads were analyzed using viralrecon pipeline. The mutational effects of omicron variant on the stability of S protein, including wild-type (7FG7), close (6VXX) and open (6VYB) states, were assessed through MD simulations. Four deleterious missense mutations (Tyr505His, Asn764Lys, Asp950Asn, Asn969Lys) were identified in the S1 and S2 subunit of the S protein of omicron variant. In the wildtype and open state mutant models, Tyr505His, Asp950Asn and Asn969Lys caused destabilizing effects, higher RMSDs vs. wild-type, and fluctuations in the RBD (438-510) region and S2 subunit (946-1010), compared to the native structure. These mutations increased the binding pocket propensity to open in mutant model compared to the native open conformation (6VYB). This structural change promoted trimer opening in the open state through \u03b1-helix movement in the S2 subunit away from the RBD region. In the closed state, only S2 subunit mutations (Asp950Asn and Asn969Lys) lead to predicted destabilization through the movement of protomer C towards protomer B (RBD region). These S2 subunit mutations are predicted to stabilize the RBD \u201cdown\u201d conformation potentially enhancing spike antigenic heterogeneity. This study highlighted the cumulative effect of S1 (Tyr505His) and S2 (Asp950Asn and Asn969Lys) subunits mutations on different S protein states, potentially controlling its conformational dynamics and presentation to host receptors. Future experimental studies are needed to elucidate the biological significance of these alterations, particularly by establishing a link between the identified mutations and their impact on viral biology.","version":"1.1","doi":"10.1101/2025.02.25.639252","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.31.474032","pub_date":"2025-2-27","title":"Combining antigenic data from public sources gives an early indication of the immune escape of emerging virus variants","abstract":"The rapid spread of the Omicron BA.1 (B.1.1.529.1) SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) variant in 2021 resulted in international efforts to quickly assess its escape from immunity generated by vaccines and previous infections. Numerous laboratories published BA.1 neutralization data as preprints and reports. We collated this data in real time and regularly presented updates of the aggregated results in US, European and WHO research and advisory settings. Here, we retrospectively analyzed the accuracy of these aggregations from 85 different sources published during a time period from 2021/12/08 up to 2022/08/14. We found that the mean titer fold change from wild type-like variants to BA.1, a standard measure of a variant\u2019s immune escape, remained stable after the first 15 days of data reporting in people who were twice vaccinated, and incoming data increased the confidence in this quantity. Further, it is possible to build reliable, stable antigenic maps from this collated data already after one month of incoming data. We here demonstrate that combining early reports from variable, independent sources can rapidly indicate a new virus variant\u2019s immune escape and can therefore be of immense benefit for public health.","version":"1.4","doi":"10.1101/2021.12.31.474032","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.15.638421","pub_date":"2025-2-26","title":"A truncated SARS-CoV-2 nucleocapsid protein enhances virus fitness by evading antiviral responses","abstract":"Viruses face a selective pressure to evade cellular antiviral responses to control the outcome of an infection. However, due to their limited genome size, viruses must adopt unique strategies to confront cellular sensors. Since emerging in humans, SARS-CoV-2 has accrued multiple mutations throughout its genome, some of which enhanced virus replication and led to the rise of viral variants. However, the biological consequences of many of these changes remain to be discovered. Here, we show that SARS-CoV-2 produces a truncated form of the nucleocapsid protein, called N*M210. Due to the acquisition of a viral transcription regulatory sequence (TRS) in the N gene, certain variants, such as Omicron, produce a new viral mRNA that markedly increases N*M210 expression. We show that N*M210 is a dsRNA binding protein, which inhibits multiple arms of the cellular antiviral response, including blocking interferon induction and inhibiting stress granule formation. We created a panel of recombinant SARS-CoV-2 viruses (rSARS-2) with mutations in the N gene that increased or decreased N*M210 production. We show that N*M210 production increases virus fitness, as viruses that produce more N*M210 outcompeted wild-type rSARS-2. We demonstrate that the fitness advantage provided by N*M210 is partly due to its ability to potently block stress granules. We propose a model where, to evade the cellular antiviral response, SARS-CoV-2 has evolved a mechanism to increase the production of a truncated form of the N protein, which broadly limits the activation of dsRNA-induced antiviral responses, tipping the balance in favour of the virus in the battle for control of the cell.\n\n\n SARS-CoV-2 variants evolved to upregulate truncated N (N*) synthesis to increase virus fitness N*M210 is a potent dsRNA-binding protein that blocks cellular dsRNA sensing N*M210 inhibits stress granule formation independent of G3BP1 binding","version":"1.1","doi":"10.1101/2025.02.15.638421","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.05.611549","pub_date":"2025-2-26","title":"Signatures of omicron-like adaptation in early SARS-CoV-2 variants and chronic infection","abstract":"Omicron emergence represented a seismic event in the COVID-19 pandemic, demonstrating what was essentially antigenic shift in a virus that cannot reassort its genome as influenza can. Understanding the success of Omicron is essential, and yet we have little understanding of the biological underpinnings of its ability to accommodate diverse mutations and bring together deleterious mutations to generate a highly successful new serotype. Persistent SARS-CoV-2 infections are a likely source of new variants and may provide valuable insight into past and future evolutionary trajectories, in particular those involving allosteric interactions that defy genotype to phenotype prediction. Here we observe upper airway specific evolution of SARS-CoV-2 demonstrating fusion peptide (FP) domain mutation S:P812S adjacent to the S2\u2019 cleavage site that emerged during a chronic infection in an immunocompromised individual. Indeed, this mutation had previously emerged in an ancestral B lineage as well as the delta variant lineage and transmitted successfully in populations globally, though remains uncharacterised. P812S in spike pseudotyped virus particles did not impact entry efficiency across cell lines expressing endogenous ACE2 and TMPRSS2. However, efficiency of spike cleavage at S1/S2 was reduced and molecular dynamics simulation demonstrated altered S1/S2 loop conformations that possibly impacted furin mediated cleavage. Consistent with impaired S1/S2 cleavage, and reminiscent of Omicron BA.1, cell-cell fusogenicity was severely impaired by introduction of P812S. The mutation also introduced significant perturbations to the FP region and also affected protomer-protomer packing. P812S conferred evasion of a neutralising monoclonal antibody targeting the fusion peptide, consistent with significant structural rearrangements in the FP region. Finally, P812S bearing viruses showed evasion of polyclonal neutralising antibodies in sera from vaccinated individuals at 32\u00b0C (simulating upper respiratory tract) and to a lesser extent at 37\u00b0C. Thus we report a novel mutational adaptation to the upper airway allowing enhanced immune evasion to fusion peptide targeting neutralising antibodies that also incurs a defect in ability to induce syncytia. These data shed light on the balance between upper airway adaptation/immune evasion by SARS-CoV-2, ability to induce syncytia formation, and disease severity given the established link between syncytia and severe COVID-19.","version":"1.2","doi":"10.1101/2024.09.05.611549","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.08.622746","pub_date":"2025-2-26","title":"WITHDRAWN: Neutralizing Activity and Viral Escape of Pemivibart by SARS-CoV-2 JN.1 sublineages","abstract":"The authors have withdrawn this manuscript because of a conflict of interest. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.2","doi":"10.1101/2024.11.08.622746","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.12.638008","pub_date":"2025-2-26","title":"WITHDRAWN: SA55 broadly neutralizes SARS-CoV-2 and robustly prevents viral escape by JN.1 sublineages","abstract":"The authors have withdrawn this manuscript because of a conflict of interest. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.2","doi":"10.1101/2025.02.12.638008","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.22.639619","pub_date":"2025-2-25","title":"SARS-CoV-2 nucleocapsid uniquely disrupts chromatin over pathophysiologically relevant gene promoters","abstract":"SARS-CoV-2, the causative agent of COVID-19, is a positive-sense, single-stranded RNA virus that causes a spectrum of disease severity, from asymptomatic infection to severe illness to long-term sequelae. Similar to other human coronaviruses, SARS-CoV-2 proteins modulate host genomic responses through epigenomic modifications, facilitating viral replication and immune evasion. While the nucleocapsid protein is well known for its role in RNA stability and immune modulation, its impact on host chromatin organization remains unclear. To investigate this, we generated stable human alveolar cell lines expressing nucleocapsid proteins from endemic and pandemic human coronaviruses. Our analysis revealed that nucleocapsid proteins from all tested coronaviruses induced changes in nucleosome positioning and occupancy at specific gene promoters involved in coagulation pathways, hormone signaling, and innate immune responses. Additionally, SARS-CoV-2-specific alterations were identified in genes dysregulated in severe infections, suggesting a direct role for epigenomic modifications in disease pathophysiology. We also observed extensive changes in nucleosome susceptibility to nuclease digestion in SARS-CoV and SARS-CoV-2 samples that were not observed in common cold cell lines. Promoters with altered sensitivity and resistance to nuclease were linked to innate immune, metabolic, olfactory, and signaling pathways known to be dysregulated in severe COVID-19 and post-acute sequelae (PASC). These findings demonstrate that nucleocapsid protein expression alters chromatin structure at specific loci, implicating viral proteins in host genome dysregulation. Furthermore, we identified both shared and unique chromatin targets of SARS-CoV-2 and common cold coronaviruses, highlighting pathways for further investigation and potential therapeutic intervention. Host chromatin is known to be modulated by coronaviruses during infections. However, the role of the nucleocapsid protein in these alterations are unknown. Here, we show that nucleocapsids from seven human coronaviruses alter nucleosome distribution and susceptibility to enzymatic digestion over specific gene promoters in a human lung cell line. Nucleocapsids from SARS-CoV and SARS-CoV-2 have the most prominent effects which are seen over genes involved in immune responses, metabolism, hormone signaling, and other pathways that are known to be dysregulated in severe COVID-19 and post-acute sequelae of COVID-19.","version":"1.2","doi":"10.1101/2025.02.22.639619","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.20.639218","pub_date":"2025-2-25","title":"Identification of novel cis-acting elements, E-box and ISRE, regulating IFN\u03b3-IRF1 axis-mediated NLRC5 expression","abstract":"NLRC5 and CIITA are the primary transcriptional regulators of MHC class I and MHC class II, respectively, and play essential roles in adaptive immunity. While the regulatory mechanisms of CIITA have been extensively characterized, the transcriptional control of NLRC5 remains incompletely understood. In this study, we identified two novel conserved cis-regulatory elements within the NLRC5 promoter, an E-box and an ISRE. Furthermore, we revealed IRF1 as a novel transcriptional regulator of NLRC5, binding directly to the ISRE within the NLRC5 promoter. Using the newly identified NLRC5 ISRE, we established a screening platform to identify modulators of the IFN\u03b3-IRF1-NLRC5 axis. This system corroborated the inhibitory effects of known viral antagonists and led to the identification of novel SARS-CoV-2 viral factors that suppress IFN\u03b3- mediated IRF1 nuclear translocation, thereby inhibiting the MHC class I pathway. By elucidating the previously unproven molecular mechanism underlying IFN\u03b3-mediated NLRC5 regulation, our study provides critical insights into viral immune evasion strategies and the modulation of antigen presentation. These findings may facilitate the development of MHC class I-targeted therapeutics by modulating the IRF1-NLRC5 axis.","version":"1.1","doi":"10.1101/2025.02.20.639218","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.24.639813","pub_date":"2025-2-24","title":"NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1","abstract":"Nuclear factor erythroid 2\u2013related factor 2 (NRF2) is a master regulator of anti-oxidative and detoxifying cell responses. In addition, it plays important roles in host cell defenses against pathogenic viruses, and small molecules that activate NRF2 signaling can exert potent antiviral effects. We recently found that the NRF2 activators 4-octyl itaconate (4OI), bardoxolone (BARD), and sulforaphane (SFN) interfere with influenza A virus replication by blocking the nuclear export factor exportin 1 (XPO1), which did not require NRF2 signaling. Here, we have assessed their potential to inhibit highly pathogenic (SARS-CoV-2) and seasonal (hCoV-229E) coronaviruses and begun to elucidate the involved mechanisms of action. Using human cell lines and iPSC-derived vascular endothelial cells, we find that NRF2 knock-out or knock-down enhances infection by both viruses, indicating that physiologic NRF2 signaling restricts human coronavirus infection. 4OI, BARD, SFN, as well as the XPO1 blocker Selinexor (SEL), greatly limit infection by both viruses, but in an NRF2-independent manner. Strikingly, the compounds (particularly 4OI) downregulate ACE2 and TMPRSS2 mRNA and protein in Calu3 cells, leading to a >10-fold reduction in viral cell entry by 4OI and SEL, as assessed using SARS-CoV-1 and -2 spike protein VSV pseudotypes. A cycloheximide chase experiment revealed that 4OI dramatically reduces ACE2 half-life, which requires the E3 ligases NEDD4L and MCM1, suggesting that 4OI targets ACE2 for destruction by the proteasome. Moreover, 4OI and SEL reduce XPO1 protein levels, and all compounds reduce XPO1 mRNA levels. Co-incubation experiments of 4OI and the transcription blocker actinomycin D in A549 cells suggest that 4OI acts primarily by interfering with transcription of the XPO1 gene. XPO1 knock-down markedly reduces 229E replication. All four compounds interfere with 229E infection, but do not alter expression of ANPEP, the cellular receptor for this virus. Their anti-229E efficacy depends on expression of XPO1 in host cells in the order of SEL (most dependent) >4OI >SFN >BARD (least dependent), suggesting that especially BARD interferes with 229E infectivity via yet another, unknown, target. Taken together, these results suggest that \u201cNRF2 activators\u201d act as potent antivirals against human coronaviruses by targeting diverse host factors which are critical for viral infectivity. Host-directed antiviral compounds act by a variety of mechanisms. For instance, they stimulate cellular antiviral immune responses and target host cell factors which are required for the viral life cycle. Pharmacologic activation of the NRF2 signaling pathway is a particularly attractive antiviral strategy, as this pathway restricts replication of a variety of viruses and also protects cells from excessive inflammation and oxidative stress resulting from accumulation of reactive oxygen species. In our previous study of the NRF2 activators bardoxolone, sulforaphane, and 4-octyl itaconate as host-directed treatments for influenza A virus infection, we found that these compounds interfered with replication of the virus. Unexpectedly, this antiviral activity was completely independent of NRF2 signaling, but resulted from blocking the nuclear export factor XPO1. In the present study, we find that these compounds limit infection by SARS-CoV-2 and hCoV-229E and that, again, this antiviral effect is NRF-independent. Instead, it depends to a large extent on downregulating ACE2 and TMPRSS2 (the major host cell receptors for SARS-CoV-1 and 2) and blocking/downregulating XPO1. Our results underscore the potential of \u201cNRF2 activators\u201d as adjunct treatments for viral infections, as they protect the host by anti-oxidative, anti-inflammatory, and cytoprotective mechanisms and also interfere with diverse host factors required for the viral life cycle.","version":"1.1","doi":"10.1101/2025.02.24.639813","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.21.639485","pub_date":"2025-2-24","title":"Flagellin in the human gut microbiome is a diet-adjustable adjuvant for vaccination","abstract":"The intestinal microbiota is thought to modulate immune responsiveness to vaccines. Human studies on this topic, however, have yielded inconsistent results. We hypothesized that the microbiome would influence innate immune responses, and thus vaccine reactogenicity, more directly than vaccine immunogenicity. To test this, we established the \u00b5HEAT (Microbial-Human Ecology And Temperature) study, which longitudinally profiled the fecal microbiota, oral body temperature and serum antibody responses of 171 healthy adults (18-40 years old) before and after vaccination for SARS-CoV-2. Increased temperature after vaccination (\u0394T) was associated with habitual diet and with baseline metabolic and immune markers. The microbiomes of \u0394T-high (\u0394Thi) participants were characterized by high expression of flagellin and an overabundance of the flagellated bacterium Waltera. Fecal samples from \u0394Thi participants induced more inflammation in human cells and stronger post-vaccine temperature responses in mice compared to \u0394Tlo samples, suggesting a causal role for the microbiome. Moreover, Waltera flagellin replicated the inflammatory phenotypes in vitro and was modulable via a dietary additive. Overall, these data suggest that flagellin from the gut microbiome stimulates innate immunity and vaccine reactogenicity, and that this axis can be manipulated via diet. These findings have implications for improving human vaccine tolerance and immunogenicity.","version":"1.1","doi":"10.1101/2025.02.21.639485","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.18.638792","pub_date":"2025-2-22","title":"Effect of obesity on the acute response to SARS-CoV-2 infection and development of post-acute sequelae of COVID-19 (PASC) in nonhuman primates","abstract":"Long-term adverse consequences of SARS-CoV-2 infection, termed \u201clong COVID\u201d or post-acute sequelae of COVID (PASC), are a major component of overall COVID-19 disease burden. Prior obesity and metabolic disease increase the severity of acute disease, but SARS-CoV-2 infection also contributes to the development of new-onset metabolic disease. Since the COVID pandemic occurred in the context of the global obesity epidemic, an important question is the extent to which pre-existing obesity modifies long-term responses to SARS-CoV-2 infection. We utilized a nonhuman primate model to compare the effects of infection with the SARS-CoV-2 delta variant in lean and obese/insulin-resistant adult male rhesus macaques over a 6-month time course. While some longitudinal responses to SARS-CoV-2 infection, including overall viral dynamics, SARS-CoV-2-specific IgG induction, cytokine profiles, and tissue persistence of viral RNA, did not appreciably differ between lean and obese animals, other responses, including neutralizing Ab dynamics, lung pathology, body weight, degree of insulin sensitivity, adipocytokine profiles, body temperature, and nighttime activity levels were significantly different in lean versus obese animals. Furthermore, several parameters in lean animals were altered following SARS-CoV-2 infection to resemble those in obese animals. Notably, persistent changes in multiple parameters were present in most animals, suggesting that PASC may be more prevalent than estimated from self-reported symptoms in human studies.","version":"1.2","doi":"10.1101/2025.02.18.638792","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.21.639446","pub_date":"2025-2-21","title":"Human monoclonal antibodies that target clade 2.3.4.4b H5N1 hemagglutinin","abstract":"The highly pathogenic avian influenza H5N1 virus clade 2.3.4.4b has been spreading globally since 2022, causing mortality and morbidity in domestic and wild birds and mammals, including infection in humans, raising concerns about its pandemic potential. We aimed to generate a panel of anti-hemagglutinin (HA) human monoclonal antibodies (mAbs) against the H5 protein of clade 2.3.4.4b. H2L2 Harbour Mice\u00ae, which express human immunoglobulin germline genes, were immunized with H5 and N1 recombinant proteins from A/mallard/New York/22-008760-007-original/2022 H5N1 virus, enabling the generation of human chimeric antibodies. Through hybridoma technology, sixteen full human mAbs were generated, most of which showed cross-reactivity against H5 proteins from different virus variants. The functionality of the sixteen mAbs was assessed in vitro using hemagglutination inhibition and microneutralization assays with viruses containing a clade 2.3.4.4b HA. Fourteen out of the sixteen mAbs neutralized the virus in vitro. The mAbs with the strongest hemagglutination inhibition activity also demonstrated greater neutralizing capacity and showed increased protective effects in vivo when administered prophylactically or therapeutically in a murine H5N1 challenge model. Using cryo-electron microscopy, we identified a cross-clonotype conserved motif that bound a hydrophobic groove on the head domain of H5 HA. Akin to mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the coronavirus 2019 (COVID-19) pandemic, these mAbs could serve as important treatments in case of a widespread H5N1 epidemic or pandemic.","version":"1.1","doi":"10.1101/2025.02.21.639446","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.18.638747","pub_date":"2025-2-21","title":"Seek-a-Seq: Optical detection of target nucleic acids with attomolar sensitivity","abstract":"We introduce Seek-a-Seq, an enzyme-free, PCR amplification-free, optical method to detect microbial nucleic acids based on fluorescent readout of hybridization between target sequences and complementary probes immobilized on the surface of customized sample chambers. We demonstrate the ability to detect ultralow concentration (~ 1 attomolar) of short target DNA and synthetic SARS-CoV-2 RNA with ease and specificity. Seek-a-Seq can enormously reduce costs, accelerate diagnostic workflows, and outperform existing enzyme-dependent methods.","version":"1.1","doi":"10.1101/2025.02.18.638747","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.21.639439","pub_date":"2025-2-21","title":"EpiMolBio: A Novel User-Friendly Bioinformatic Program for Genetic Variability Analysis","abstract":"Genetic sequence analysis has become essential in many medicine, biology, and epidemiology fields. However, the currently available tools can pose a challenge for users without advanced computational skills. We present EpiMolBio, a free-to-use software designed with an intuitive, user-friendly interface that enables a broad spectrum of users to explore genetic variability. Its diverse toolkit encompasses sequence processing, conservation and variability analysis, consensus sequence generation, and genome mutation or amino acid changes identification, including specialized tools for HIV and SARS-CoV-2 analysis. Freely available on the web at https://www.epimolbio.com and https://github.com/EpiMolBio/EpiMolBio. africa.holguin@salud.madrid.org; roberto117343@gmail.com Supplementary Table 1, 2, and Supplementary Text 1.","version":"1.1","doi":"10.1101/2025.02.21.639439","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.17.638623","pub_date":"2025-2-19","title":"Evolution of BA.2.86 to JN.1 reveals functional changes in non-structural viral proteins are required for fitness of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is still circulating among humans, leading to the continuous evolution. SARS-CoV-2 Omicron JN.1 evolved from a distinct SARS-CoV-2 lineage, BA.2.86, spread rapidly worldwide. It is unclear why BA.2.86 did not become dominant and was quickly replaced by JN.1, which possesses one amino acid substitution in the spike protein (S:L455S) and two in the non-spike proteins NSP6 and ORF7b (NSP6:R252K and ORF7b:F19L) compared to BA.2.86. Here, we utilized recombinant viruses to elucidate the impact of these mutations on the virological characteristics of JN.1. We found that the mutation in the spike attenuated viral replication, but the non-spike mutations enhanced replication, suggesting the mutations in the non-spike proteins compensate for the one in the spike to improve viral fitness, as the mutations in the spike contribute to further immune evasion. Our findings suggest that functional changes in both the spike and non-spike proteins are necessary in the evolution of SARS-CoV-2 to enable evasion of adaptive immunity within the human population while sustaining replication. Because the spike protein is strongly associated with certain virological properties of SARS-CoV-2, such as immune evasion and infectivity, most previous studies on SARS-CoV-2 variants have focused on spike protein mutations. However, the non-spike proteins also contribute to infectivity, as observed throughout the evolution of Omicron subvariants. In this study, we demonstrate a \u201ctrade-off\u201d strategy in SARS-CoV-2 Omicron JN.1 in which the reduced infectivity caused by spike mutation is compensated by non-spike mutations. Our results provide insight into the evolutionary scenario of the emerging virus in the human population.","version":"1.1","doi":"10.1101/2025.02.17.638623","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.18.638851","pub_date":"2025-2-19","title":"Immunological and clinical markers of post-acute sequelae of COVID-19: Insights from mild and severe cases six months post-infection","abstract":"Post-acute sequelae of COVID-19 (PASC) is a complex and multifaceted clinical challenge requiring to emphasize its underlying pathophysiological mechanisms. This study assessed hundreds of virological, serological, immunological, and tissue damage biomarkers in two patient cohorts who experienced mild (n=270) or severe (n=188) COVID-19, 6 to 9 months post-initial infection, and in which 40% and 57.4% of patients, respectively, developed PASC. Blood analysis showed that mains differences observed in humoral, viral, and biological biomarkers were associated with the initial COVID-19 severity, rather than being specifically linked to PASC. However, patients with PASC displayed altered CD4+ and CD8+ memory T-cell subsets, with higher cytokine-secreting cells and increased terminally differentiated CD45RA+ effector memory T cells (TEMRA). Elevated SARS-CoV-2-specific T cells responsive to nucleocapsid/membrane proteins with a TEMRA phenotype were also observed. A random forest model identified these features and initial symptom duration as top variables discriminating PASC, achieving over 80% classification accuracy. Nine months post-initial SARS-CoV-2 infection, over 40% of patients developed PASC Regardless of PASC, the initial disease form influenced the main immune differences PASC led to altered memory T-cell subsets with elevated TEMRA phenotype PASC presence is associated with disease form, initial symptom count and duration","version":"1.1","doi":"10.1101/2025.02.18.638851","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.17.638734","pub_date":"2025-2-19","title":"Oral Cavity Serves as Long-Term COVID-19 Reservoir with Increased Periodontal and Viral Disease Risk","abstract":"SARS-CoV-2 infection can lead to long-term health problems affecting multiple body systems termed long COVID. Currently, limited information exists about long-term oral health manifestations in COVID-19 patients with limited healthcare access. We conducted a sequential, cross-sectional study (December 2020\u2013March 2024) to assess how racial/ethnic differences (Black/Hispanic vs White/Asian) and health disparities affect oral and non-oral long COVID symptoms and their relationship with COVID-19 vaccination. We retrospectively reviewed patients\u2019 oral health record from University of Illinois Chicago dental clinics before vaccination (December 2020; N=1150; Covid+/- N=575/group) and after vaccination (December 2021; N=592; Covid+/- N=292/group). Participants were recruited in two separate prospective groups of COVID-19 positive subjects (February\u2013April 2021; pre-vaccination: N=158; January\u2013March 2024; post-vaccination: N=171), we examined clinical indicators of oral (periodontal and salivary glands) and non-oral (neurologic) sequalae 3\u20136 months after initial exposure. We measured viral S protein by flow cytometry and quantified inflammatory markers, viral entry receptors, and oral viral load to correlate molecular, and cellular changes in COVID-19 positive subjects before and after vaccination. Our results identified racial disparity indicating oral associated post-acute sequelae (PASC) primarily manifested as periodontal (gum) disease (COVID-19 positive: 73.1\u00b118.9% vs COVID-19 negative: 33.1\u00b114.3%) and correlated with higher rates of dry mouth (57.5%), taste disturbance (47%), and smell loss (20%). Vaccination reduced oral PASC in COVID-19 positive subjects; however, periodontal disease indicators persisted compared to the COVID-19 negative group. Notably, 3-6 months post-infection, while SARS-CoV-2 Spike (S) transcript was rarely detected in saliva (\u223c6%), its protein was commonly detected (\u223c70%) in the COVID-19 positive subjects indicating incomplete viral clearance. This correlates with significantly higher salivary expression of viral entry receptors (ACE2, and TRMPSS2), and inflammatory mediators (IL-6, IL-8 and MMP-8), in COVID-19 positive subjects. This finding was further supported by higher prevalence of other oral viruses including Epstein-Barr Virus (70.5%), Herpes Simplex Virus (8.1%), and Human Papillomavirus (17.5%) in COVID-19 positive subjects. COVID-19 history significantly correlates with severe oral health complications in predominantly Black communities, while vaccination reduced but did not eliminate these issues. The oral cavity serves as a long-term viral reservoir, and periodontal inflammation with increased oral viral presence in COVID-positive patients may increase susceptibility to oral and non-oral viral diseases and identify risk for long COVID.","version":"1.1","doi":"10.1101/2025.02.17.638734","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.17.638432","pub_date":"2025-2-19","title":"In a pathophysiologic state due to SARS-CoV-2, viscosity and cholesterol are two-edged swords","abstract":"Much attention has been paid to the genetic composition and molecular biology of viral particles, infection, and micro-anatomical impacts that culminate in fatalities; vaccines have been in continuous development and production; less attention is paid to the fundamental issues of thermodynamics and activation energy characterization of viral RNA replication and cell death. The study aimed at deriving equations that can be fitted to both theoretically and empirically derived data for the quantitation of other thermodynamic parameters and its cognate dimensionless equilibrium constant; some of the derived equations addressed the issue of viscosity and the concentration of cholesterol in particular as they affect translational velocity needed for the delivery of biomolecules to the site of need. The instantaneous velocities before terminal velocity are: \u223c 0.046674 m/s (cytosol); 0.141837 m/s (water). The terminal velocities were approximately equal to 3.548614 nm/s for the cytosol and 99.590626 nm/s for the water; these values were computed using literature values of translational diffusion coefficients (Di) of glucose in cytoplasm and in water. The value in water is higher than in the cytosol because of higher cytosolic viscosity than aqueous viscosity. These support the view that cholesterol and viscosity have a dual-edged effect on the pathophysiologic state orchestrated by SARS-CoV-2; higher viscosities in the membrane and in the cytoplasm enhance binding and infection and can diminish the progress of infection, respectively. Higher feasibility and rates were observed at lower thermodynamic temperatures than at higher ones, according to the outcome of the analysis of the viral binding free energy and activation energy, respectively. The dimensionless constant values were higher at the earlier time of the infection and decreased with time, exhibiting a power law relationship. It is advised, among others, that pharmaceuticals (including airborne surfactants) and drugs in solution be given at temperatures above body temperature. Swab testing should be performed on a regular basis to detect significant infections early. Future in vitro and in vivo studies on viral infection might focus on various time periods at different temperatures, above and below body temperature. With vaccine and/or drug the viral infection can regress; without treatment there could be infection and progression into disease state; discontinuation of treatment can cause a reinfection.","version":"1.1","doi":"10.1101/2025.02.17.638432","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.16.638411","pub_date":"2025-2-17","title":"Age- and Virus-Specific Signatures of In Vitro Reconstituted Human Airway Epithelia in the Presence and Absence of Respiratory Viral Infections","abstract":"While Influenza Virus and Respiratory Syncytial Virus (RSV) are considered as a significant health burden in children, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes milder diseases in this age group compared to adults. To investigate the involvement of the upper respiratory tract human airway epithelium (HAE) in this pattern, we established an in-house model of reconstituted HAE cultured in air-liquid interface from nasal swabs of children and adults and characterised it before and after ex vivo respiratory viral infections using focused and unbiased approaches. Fully differentiated paediatric HAE exhibited an increasing induction level of genes related to mucociliary clearance, while higher expression of innate immune pathways was found in the ones from adults. While similar viral replication kinetics in both age groups were shown for SARS-CoV-2, Influenza A Virus (IAV), RSV and Rhinovirus (RV) infection, transcriptomic analysis showed stronger and earlier induction of IFN-related pathways in SARS-CoV-2-infected HAE from children compared to IAV, RSV and RV. IAV and RSV had the weakest innate immune response increase in HAE from children versus adults. RV infection showed an intermediate pattern, resembling SARS-CoV-2 more than RSV or IAV. Our work demonstrates a distinct sensing of SARS-CoV-2 compared to other respiratory viruses ex vivo, which may contribute to the milder course of disease in SARS-CoV-2 infected children and argues for a role of early virus-HAE interaction in shaping viral pathogenesis. Furthermore, we show that innate immune responses towards respiratory viruses are virus-specific and differ between age groups. Hence, findings on SARS-CoV-2 cannot be extrapolated to other respiratory viruses.","version":"1.1","doi":"10.1101/2025.02.16.638411","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.16.636315","pub_date":"2025-2-17","title":"VIRUS-MVP: A framework for comprehensive surveillance of viral mutations and their functional impacts","abstract":"As viruses evolve, they accumulate genetic mutations that can influence disease severity, transmissibility, and the effectiveness of vaccines and therapeutics. Real-time tracking of viral mutations and their functional impacts is essential to understand these changes and assess their implications for public health responses. VIRUS-MVP is an interactive, portable platform designed for the comprehensive surveillance of viral mutations. Initially developed for SARS-CoV-2, it now fully supports mpox and is expanding to include influenza and RSV. The platform links viral mutations to functional annotations, providing insights into their predicted effects on viral infectivity, immune evasion, and protein functionality. It features an interactive interface for visualizing mutation distributions, a modular and reproducible genomics workflow, and a curated annotation resource that captures known impacts on viral proteins and host interactions. Users can also import custom functional annotations to tailor analyses to specific research needs or emerging pathogens. Developed collaboratively with public health and academic partners, VIRUS-MVP enhances understanding of viral evolution and its public health impact by bridging genomic data with biological insights. The platform is open-source, adaptable, and accessible on GitHub.","version":"1.1","doi":"10.1101/2025.02.16.636315","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.14.638187","pub_date":"2025-2-14","title":"Magnetoencephalography Reveals Neuroprotective Effects of COVID-19 Vaccination in Non-Human Primates","abstract":"COVID-19, caused by the SARS-CoV-2 virus, can lead to widespread neurological complications, including cognitive deficits and neurodegenerative symptoms, even in the absence of significant structural brain abnormalities. The potential neuroprotective effects of SARS-CoV-2 vaccination remain underexplored. Here, we demonstrate the neuroprotective effects of a psoralen-inactivated SARS-CoV-2 vaccine in a non-human primate model using resting-state magnetoencephalography (MEG), a non-invasive neurophysiological recording technique with sub-millisecond temporal and submillimeter spatial resolution. MEG scans demonstrated substantial preservation of neural activity across multiple brain regions in vaccinated subjects compared to unvaccinated controls following viral challenge. This approach not only underscores the role of vaccination in mitigating severe neurological outcomes but also highlights the capability of MEG to detect subtle yet significant changes in brain function that may be overlooked by other imaging modalities. These findings advance our understanding of vaccine-induced neuroprotection and establish MEG as a powerful tool for monitoring brain function in the context of viral infections.","version":"1.1","doi":"10.1101/2025.02.14.638187","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.12.637926","pub_date":"2025-2-14","title":"Identification of a multi-omics factor predictive of long COVID in the IMPACC study","abstract":"Following SARS-CoV-2 infection, \u223c10-35% of COVID-19 patients experience long COVID (LC), in which often debilitating symptoms persist for at least three months. Elucidating the biologic underpinnings of LC could identify therapeutic opportunities. We utilized machine learning methods on biologic analytes and patient reported outcome surveys provided over 12 months after hospital discharge from >500 hospitalized COVID-19 patients in the IMPACC cohort to identify a multi-omics \u201crecovery factor\u201d. IMPACC participants who experienced LC had lower recovery factor scores compared to participants without LC. Biologic characterization revealed increased levels of plasma proteins associated with inflammation, elevated transcriptional signatures of heme metabolism, and decreased androgenic steroids in LC patients. The recovery factor was also associated with altered circulating immune cell frequencies. Notably, recovery factor scores were predictive of LC occurrence in patients as early as hospital admission, irrespective of acute disease severity. Thus, the recovery factor identifies patients at risk of LC early after SARS-CoV-2 infection and reveals LC biomarkers and potential treatment targets.","version":"1.1","doi":"10.1101/2025.02.12.637926","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.13.638030","pub_date":"2025-2-14","title":"Human type-I interferon omega holds potent antiviral properties and promotes cytolytic CD8+ T cell responses","abstract":"The type-I interferon family is well known for its critical role in innate immunity. It comprises several members, among which IFN-\u03b12 and IFN-\u03b2 are the most extensively studied, with important antiviral and immune-modulatory functions. Recent findings linking autoantibodies against type-I interferons to severe COVID-19 suggest a potential role for IFN-\u03c9 in combating SARS-CoV-2 infection. However, little is known about human IFN-\u03c9, as most research on this interferon has been conducted in feline models. Here, we demonstrate that human IFN-\u03c9 is secreted at levels comparable to those of IFN-\u03b12 or IFN-\u03b2 upon stimulation with inflammatory agonists and triggers a robust antiviral response, inhibiting SARS-CoV-2 infection in vitro. Moreover, IFN-\u03c9 enhances the effector functions of antigen-specific CD8+ T cells primed de novo from healthy donor cells, highlighting its capacity to promote strong cellular immunity. Our results position IFN-\u03c9 as a key member of the type-I interferon family, with promising potential for therapeutic and vaccine applications. Type-I interferons are pleiotropic cytokines, including IFN-\u03b1 and IFN-\u03b2, which are well known for their antiviral activities. Here, we report on the functional characteristics of human IFN-\u03c9, a neglected member of the type-I IFN family, which has primarily been studied in feline models. We show here that human IFN-\u03c9 induces intense downstream signaling in cells resulting in the upregulation of antiviral genes, and efficient restriction of SARS-CoV-2 replication. IFN-\u03c9 also promotes the acquisition of strong cytolytic functions by antigen primed CD8+ T cells. Overall, our findings portray human IFN-\u03c9 as a major antiviral molecule, similar to the well-studied and highly effective IFN-\u03b12. The secretion of IFN-\u03c9 upon infection is likely to be crucial for effective control of multiple viruses, advocating for its use in therapeutic approaches in humans.","version":"1.1","doi":"10.1101/2025.02.13.638030","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.13.638027","pub_date":"2025-2-14","title":"Design of Protein Sequences with Precisely Tuned Kinetic Properties","abstract":"Recent advances in computational biology have enabled solutions to the inverse folding problem - finding an amino acid sequence that folds into a target structure. An open question concerns the design of proteins that in addition to having the correct target structure also have precisely tuned kinetic properties, such as folding and unfolding rates. To address this problem, we formulate the inverse folding problem as a quest for a sequence with a target free energy landscape. To propose a procedure to address this problem, here we describe the Inverse Folding Molecular Dynamics (IF-MD) method, which combines inverse folding with enhanced sampling molecular dynamics and Bayesian optimization. IF-MD leverages ensemble averages from molecular dynamics simulations, reweighted according to a Bayesian framework, to guide the design of sequences exhibiting specific kinetic properties. We demonstrate the methodology by optisising the binding kinetics of H11, a nanobody against the SARS-CoV-2 spike receptor-binding domain (RBD), thus identifying nanobody variants with slower unbinding kinetics than H11. Mechanistic analysis reveals that this kinetic property arises from a shift towards configurations closer to the bound state and increased free energy barriers for dissociation. These findings highlight the power of IF-MD for efficiently navigating the vast sequence space to design proteins with a tailored free energy landscape.","version":"1.1","doi":"10.1101/2025.02.13.638027","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.24.630260","pub_date":"2025-2-13","title":"IL-1\u03b2-driven NF-\u03baB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19), caused by infection with the enveloped RNA betacoronavirus, SARS-CoV-2, led to a global pandemic involving over 7 million deaths. Macrophage inflammatory responses impact COVID-19 severity; however, it is unclear whether macrophages are infected by SARS-CoV-2. We sought to identify mechanisms regulating macrophage expression of ACE2, the primary receptor for SARS-CoV-2, and to determine if macrophages are susceptible to productive infection. We developed a humanized ACE2 (hACE2) mouse whereby hACE2 cDNA was cloned into the mouse ACE2 locus under control of the native promoter. We validated the susceptibility of hACE2 mice to SARS-CoV-2 infection relative to wild-type mice and an established K18-hACE2 model of acute fulminating disease. Intranasal exposure to SARS-CoV-2 led to pulmonary consolidations with cellular infiltrate, edema, and hemorrhage, consistent with pneumonia, yet unlike the K18-hACE2 model, hACE2 mice survived and maintained stable weight. Infected hACE2 mice also exhibited a unique plasma chemokine, cytokine, and growth factor inflammatory signature relative to K18-hACE2 mice. Infected hACE2 mice demonstrated evidence of viral replication in infiltrating lung macrophages, and infection of macrophages in vitro revealed a transcriptional profile indicative of altered RNA and ribosomal processing machinery as well as activated cellular antiviral defense. Macrophage IL-1\u03b2-driven NF-\u03baB transcription of ACE2 was an important mechanism of dynamic ACE2 upregulation, promoting macrophage susceptibility to infection. Experimental models of COVID-19 that make use of native hACE2 expression will allow for mechanistic insight into factors that can either promote host resilience or increase susceptibility to worsening severity of infection.","version":"1.2","doi":"10.1101/2024.12.24.630260","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.12.637095","pub_date":"2025-2-13","title":"Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses","abstract":"The rapid development of vaccines against SARS-CoV-2 during the COVID-19 pandemic proved vital in controlling viral spread and reducing mortality and morbidity. Both neutralising activity and effector function activity of Spike-specific antibodies have been shown to be important for their protective and therapeutic activity. However, several recent studies have reported that vaccination with mRNA based COVID-19 vaccines can lead to elevated levels of Spike-specific IgG4, an isotype which is often considered anti-inflammatory due to its reduced binding to Fc\u03b3 receptors on immune cells. Here we show that the level of Spike-specific IgG4 produced following BNT162b2 vaccination is impacted by the interval between and frequency of vaccines boosts, prior SARS-CoV-2 infection (hybrid immunity), breakthrough infection and bivalent vaccine boosters. Despite the increase in Spike-specific IgG4 between the 2nd and 3rd BNT162b2 vaccine dose, neutralisation, ADCD and ADCP activity all increased. Through expression of SARS-CoV-2 monoclonal antibodies cloned as IgG1, IgG2, IgG3 and IgG4, we demonstrated that whilst Spike-specific IgG4 had reduced effector function activity, including ADCC, ADCD and ADCP, IgG4 was only inhibitory when directly competing with functional IgG subclasses binding to an overlapping epitope. In the context of polyclonal plasma, ADCC and ADCD activity could not be depleted by addition of high concentrations of a Spike-specific IgG4 mAb cocktail suggesting the non-stimulatory effect of Spike-specific IgG4 may be hidden in more complex scenarios, such as polyclonal mixes in serum.","version":"1.1","doi":"10.1101/2025.02.12.637095","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.12.637941","pub_date":"2025-2-13","title":"Annexin A1 as a key modulator of lung inflammation during coronavirus infections","abstract":"Exacerbated inflammation is a major contributor to tissue damage and mortality in infectious diseases, including SARS-CoV-2. The resolution phase of inflammation is critical for restoring tissue homeostasis following an injury. Annexin A1 (AnxA1) is a ubiquitous protein that plays a fundamental role in the resolution of inflammation, including in preclinical models of infectious disease. Here, we investigated the role of AnxA1 in coronavirus infection and its potential as a host-targeted therapeutic strategy against SARS-CoV-2. Wild-type (WT) and AnxA1 knockout (AnxA1KO) mice were intranasally infected with the murine betacoronavirus MHV-3 to study the endogenous role of AnxA1. Immunohistochemistry and Western blot analyses in the lungs of MHV-3-infected mice revealed increased AnxA1 expression and its cleavage, which was associated with neutrophilic infiltration (Ly6G+ cells) mainly in peribronchiolar and perivascular regions. AnxA1-deficient mice exhibited higher neutrophilic infiltration and lung damage, alongside increased CXCL1 production in the lungs, when compared to WT-infected mice. In a murine model of SARS-CoV-2 infection in K18-hACE2 mice, we found increased AnxA1 cleavage associated with lung inflammation. Treatment of SARS-CoV-2-infected K18-hACE2 mice with the AnxA1-mimetic peptide, Ac2-26, reduced lung damage and lethality, without altering the host ability to deal with viral replication. Notably, Ac2-26-treated mice exhibited similar levels of protection to that afforded by the nucleotide analogue Remdesivir, following SARS-CoV-2 infection. Our findings highlight the protective role of the endogenous AnxA1 in mitigating coronavirus-induced lung inflammation and underscore the therapeutic potential of AnxA1 mimetic Ac2-26 as a host-targeted therapy against SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.02.12.637941","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.06.565757","pub_date":"2025-2-12","title":"Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike","abstract":"The trimeric spike protein plays an essential role in the SARS-CoV-2 virus lifecycle, facilitating virus entry through binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating viral-host membrane fusion. The SARS-CoV-2 spike contains a fatty acid (FA) binding site at the interface between two neighbouring receptor-binding domains. This site, also found in some other coronaviruses, binds free fatty acids such as linoleic acid. Binding at this site locks the spike in a non-infectious, closed conformation. This site is coupled to functionally important regions, but the effects of glycans on these allosteric effects have not been investigated. Understanding allostery and how this site modulates the behaviour of the spike protein could potentiate the development of promising alternative strategies for new coronavirus therapies. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to investigate allosteric effects of the FA site in the fully glycosylated spike of the original SARS-CoV-2 ancestral variant. The results show allosteric networks that connect the FA site to important functional regions of the protein, including some more than 40 \u00c5 away, including the receptor binding motif, an antigenic supersite in the N-terminal domain, the furin cleavage site, regions surrounding the fusion peptide, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected connection between different allosteric sites. Notably, 65% of amino acid substitutions, deletions and insertions in the Alpha, Beta, Delta, Gamma and Omicron variants map onto or close to the identified allosteric pathways. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spikes revealed that the presence of glycans does not qualitatively change the internal allosteric pathways within the protein, with some glycans facilitating the transmission of the structural changes within and between subunits. The spike protein is crucial for the SARS-CoV-2 virus, enabling the fusion of the viral and host cell membranes. This protein contains several allosteric sites, including a fatty acid binding site at the interface between every two neighbouring receptor-binding domains. This site modulates the behaviour of the protein, with the binding of various free fatty acids and other small molecules influencing the spike\u2019s structure. In particular, the binding of linoleic acid, an essential fatty acid molecule, stabilizes the protein in a non-infectious locked conformation, thus making it inaccessible for binding to human receptors. Here, we investigate how the fatty acid site modulates the structural and dynamical behaviour of the fully glycosylated protein. Our work reveals complex patterns of communication between the fatty acid site and functionally important regions of the spike (including the receptor binding motif, the antigenic supersite in the N-terminal domain, the heme/biliverdin site, furin cleavage site and the fusion-peptide surrounding regions) and shed new light on the roles of glycans in this protein.","version":"1.3","doi":"10.1101/2023.11.06.565757","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.08.637262","pub_date":"2025-2-11","title":"Accelerated memory T cell decline and tolerogenic recall responses to SARS-CoV-2 vaccination in diabetes","abstract":"Type 1 and type 2 diabetes are associated with increased severity and mortality from respiratory virus infections, including SARS-CoV-2. Vaccination in the general population significantly reduces the risk of severe respiratory viral infection and triggers a strong, polyfunctional and lasting T cell response in healthy individuals. However, vaccine effectiveness in people with diabetes is unclear. Here we studied the magnitude and functional characteristics of vaccine-specific CD4+ and CD8+ T cell responses to the full vaccination protocol, and the recall response after a third booster dose of SARS-CoV-2 vaccine in people with type 1 and type 2 diabetes, and compared them to those of people without diabetes. We found defects in both CD4+ and CD8+ T cell memory maintenance and the functionality of the vaccine specific T cells in people with diabetes compared to people without. In those individuals with diabetes that harbored detectable vaccine-specific T cells, they displayed an unfocused, tolerogenic phenotype characterized by increased expression of IL-13 and IL-10 in T1D and T2D compared to people without diabetes. These results have implications for vaccination strategies for people with diabetes.","version":"1.1","doi":"10.1101/2025.02.08.637262","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.08.637239","pub_date":"2025-2-11","title":"Effects of protein boosters on antibody responses","abstract":"SARS-CoV-2 has infected a large fraction of the human population. Currently, most individuals have developed immunity either through vaccination or natural infection. Despite this, SARS-CoV-2 booster immunizations are still recommended to reduce the risk of reinfections, but there is still limited understanding on how different booster vaccine platforms influence antibody responses. We conducted immunological studies in mice to evaluate the boosting effects of different vaccine platforms on antibody responses. C57BL/6 mice were first primed with an adenovirus serotype 5 (Ad5) vaccine expressing the SARS-CoV-2 spike protein. The mice were then boosted with the same Ad5-based vaccine (homologous boosting) or with a protein-based vaccine (heterologous boosting). Interestingly, the heterologous regimen (Ad5 prime + protein boost) elicited superior antibody responses, relative to the homologous regimen (Ad5 prime + Ad5 boost). Similar potentiation of antibody responses was reported when mice were primed with poxvirus or rhabdovirus vectors and then boosted with protein. These findings highlight a potential advantage of protein booster immunizations to potentiate humoral immunity.","version":"1.1","doi":"10.1101/2025.02.08.637239","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.08.637211","pub_date":"2025-2-10","title":"Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E","abstract":"Coronaviruses, including SARS-CoV-2, rely on host factors for their replication and pathogenesis, while hosts deploy defense mechanisms to counteract viral infections. Although numerous host proviral factors have been identified, the landscape of host restriction factors and their underlying mechanisms remain less explored. Here, we conducted genome-wide CRISPR knockout screens using three distinct coronaviruses\u2014SARS-CoV-2, HCoV-OC43 (a common cold human virus from the genus Betacoronavirus) and porcine epidemic diarrhea virus (Alphacoronavirus) to identify conserved host restriction factors. We identified glycosylphosphatidylinositol (GPI) biosynthesis as the pan-coronavirus host factor that restrict viral entry by disrupting spike protein-mediated membrane fusion at both endosomal and plasma membranes. GPI biosynthesis generates GPI moieties that covalently anchor proteins (GPI-anchored proteins [GPI-APs]) to the cell membrane, playing essential roles in various cellular processes. Through focused CRISPR knockout screens targeting 193 GPI-APs, we identified LY6E as the key downstream effector mediating the antiviral activity of the GPI biosynthesis pathway. These findings reveal a novel role for GPI biosynthesis as a conserved host defense mechanism against coronaviruses and highlight LY6E as a critical antiviral effector. This study provides new insights into virus-host interactions and the development of host-directed antiviral therapies.","version":"1.1","doi":"10.1101/2025.02.08.637211","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.04.636569","pub_date":"2025-2-05","title":"DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication","abstract":"The SARS-CoV-2 pandemic and the emergence of novel variants underscore the need to understand host-virus interactions and identify host factors that restrict viral infection. Here, we perform a genome-wide CRISPR knockout screen to identify host restriction factors for SARS-CoV-2, revealing DAZAP2 as a potent antiviral gene. DAZAP2, previously implicated in SARS-CoV-2 restriction, is found to inhibit viral entry by blocking virion fusion with both endolysosomal and plasma membranes. Additionally, DAZAP2 suppresses genomic RNA replication without affecting the primary translation of viral replicases. We demonstrate that DAZAP2 functions as a pan-coronavirus restriction factor across four genera of coronaviruses. Importantly, knockout of DAZAP2 enhances SARS-CoV-2 infection in mouse models and in human primary airway epithelial cells, confirming its physiological relevance. Mechanistically, antiviral activity of DAZAP2 appears to be indirect, potentially through the regulation of host gene expression, as it primarily localizes to the nucleus. Our findings provide new insights into the host defense system against coronaviruses and highlight DAZAP2 as a potential target for host-directed antiviral therapies. During viral infection, the host defense response is mediated by a variety of host factors through distinct mechanisms that have yet to be fully elucidated. Although DAZAP2 was previously implicated in SARS-CoV-2 restriction, its mechanisms of action and in vivo relevance remain unclear. In this study, we identify the DAZAP2 as a potent pan-coronavirus restriction factor that inhibits viral infection through dual mechanisms: blocking virion fusion with both endolysosomal and plasma membranes, and suppressing genomic RNA replication. We confirm its physiological relevance in host defense using mouse models and primary cell cultures. This study advances our understanding of host-pathogen interactions. Targeting DAZAP2 or its regulatory pathways could provide a new approach to enhance host defense against current and future coronavirus threats.","version":"1.1","doi":"10.1101/2025.02.04.636569","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.03.636361","pub_date":"2025-2-04","title":"Active surveillance of cats and dogs from households with human COVID-19 cases reveals over one quarter of pets infected with SARS-CoV-2 in 2020-2021 in Texas, United States","abstract":"Households where people have COVID-19 are high risk environments for companion animals that are susceptible to SARS-CoV-2. We sampled 579 pets from 281 households with one or more laboratory-confirmed person with COVID-19 in central Texas from June 2020 to May 2021. Nineteen out of 396 (4.8%) dogs and 21 out of 157 (13.4%) cats were positive for SARS-CoV-2 by RT-qPCR. Additionally, 95/382 (25%) dogs and 52/146 (36%) cats harbored SARS-CoV-2 neutralizing antibodies. Twenty-six companion animals of ten other species were negative. Overall, 164 (29%) pets were positive for SARS-CoV-2 by molecular and/or serological tests; a total of 110 (39%) out of 281 households had at least one animal with active or past SARS-CoV-2 infection. Cats were more likely to be infected by SARS-CoV-2 and had higher endpoint antibody titers than dogs. Through viral isolation from a subset of respiratory swabs, we documented 6 different lineages in dogs and cats, including the B.1.1 lineage in a cat one month prior to the first known human case in the country. We observed animal and human-pet interaction factors associated with higher risk of infection for dogs and cats, such as days after COVID-19 diagnosis and sharing food. Frequency of clinical signs of disease reported by owners of pets with active infections did not differ from uninfected ones, suggesting that not all reported signs are attributed to SARS-CoV-2 infection. Characterizing animal infections using active SARS-CoV-2 surveillance in pets at risk of infection may aid in One Health pandemic prevention, response, and management.","version":"1.1","doi":"10.1101/2025.02.03.636361","journal":"bioRxiv","score":null},{"id":"10.1101/2025.02.03.636181","pub_date":"2025-2-04","title":"Differential time-restricted sensitivity of enveloped viruses to Sec61 translocon blockade","abstract":"The morphogenesis of enveloped viruses relies on the trafficking of transmembrane proteins through the secretory pathway to sites of virus envelopment. The first step in this pathway, their translocation into the endoplasmic reticulum, is therefore an attractive target for broad-spectrum intervention. Here, we tested if blockade of the Sec61 translocon by the Mycobacterium ulcerans exotoxin mycolactone, a potent inhibitor of Sec61, could block the production of virus glycoproteins and subsequent production of infectious virus from a range of human enveloped viruses: the DNA virus herpes simplex virus 1 (HSV1), and the RNA viruses, respiratory syncytial virus (RSV), influenza A virus (IAV), SARS coronavirus 2 (SARS CoV2) and Zika virus (ZIKV). In line with known translocation mechanisms, mycolactone blocked in vitro translocation and ectopic expression of type I transmembrane proteins but not type III, multipass or cytosolic proteins. Translocation of the type II protein RSV G was also blocked and although ectopically expressed G protein was detected, it was not glycosylated. Pretreatment of cells with mycolactone also blocked the synthesis of type I transmembrane proteins in infected cells and either the synthesis or glycosylation of type II transmembrane proteins, and the production of progeny from all viruses tested, while having no effect on virus entry or downstream synthesis of cytosolic proteins. While mycolactone treatment of HSV1 infected cells at various times after infection resulted in the immediate inhibition of virus production at the point of addition, IAV, RSV and ZIKV became resistant to the action of mycolactone surprisingly early in infection, and before virus glycoprotein synthesis was even detectable or virus production had begun. We therefore conclude that although inhibition of the translocation of virus transmembrane proteins through the Sec61 translocon can in principle block virus production, the morphogenesis of many enveloped RNA viruses requires only limited amounts of envelope proteins for successful propagation, providing novel insight into the biology of these viruses. Many circulating human pathogens are enveloped viruses that all use the cellular secretory pathway to target their envelope proteins to cellular sites of virus particle assembly. The potential to target this pathway could therefore offer a novel broad-spectrum therapy for existing, emerging and as yet unknown human pathogens. Here we have targeted the initial step in this pathway using a highly potent Sec61 inhibitor, mycolactone, to carry out the first comprehensive assessment of translocation disruption on a range of enveloped human viruses from different virus families, including herpes simplex virus, influenza A virus and SARS-CoV2. Our results have shown that Sec61 inhibition blocks the onward trafficking of many virus envelope proteins that are essential to produce infectious virus at assembly sites. However, unexpectedly, we found that several of the viruses were resistant to the effects of this toxin when it was added early in infection, indicating that the synthesis of these essential virus proteins occurs earlier in infection than previously recognised. Hence, while this approach may not be suitable as a broad intervention strategy, it has revealed new information on virus biology and provides us with a novel tool for exploring a wide range of enveloped viruses.","version":"1.1","doi":"10.1101/2025.02.03.636181","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.23.575696","pub_date":"2025-2-03","title":"Genome data artifacts and functional studies of deletion repair in the BA.1 SARS-CoV-2 spike protein","abstract":"Mutations within the N-terminal domain (NTD) of the spike (S) protein are critical for the emergence of successful SARS-CoV-2 viral lineages. The NTD has been repeatedly impacted by deletions, often exhibiting complex and dynamic patterns, such as the recurrent emergence and disappearance of deletions in dominant variants. This study investigates the influence of repair of NTD lineage-defining deletions found in the BA.1 lineage (Omicron variant) on viral success. We performed comparative genomic analyses of more than 10 million SARS-CoV-2 genomes from GISAID to decipher the transmission success of viruses lacking S:\u0394H69/V70, S:\u0394V143/Y145, or both. These findings were contrasted against a screening of publicly available raw sequencing data, revealing substantial discrepancies between data repositories, suggesting that spurious deletion repair observations in GISAID may result from systematic artifacts. Specifically, deletion repair events were approximately an order of magnitude less frequent in the read-run survey. Our results suggest that deletion repair events are rare, isolated events with limited direct influence on SARS-CoV-2 evolution or transmission. Nevertheless, such events could facilitate the emergence of fitness-enhancing mutations. To explore potential drivers of NTD deletion repair patterns, we characterized the viral phenotype of such markers in a surrogate in vitro system. Repair of the S:\u0394H69/V70 deletion reduced viral infectivity, while simultaneous repair with S:\u0394V143/Y145 led to lower fusogenicity. In contrast, individual S:\u0394V143/Y145 repair enhanced both fusogenicity and susceptibility to neutralization by sera from vaccinated individuals. This work underscores the complex genotype-phenotype landscape of the spike NTD in SARS-CoV-2, which impacts viral biology, transmission efficiency, and immune escape potential, offering insights with direct relevance to public health, viral surveillance, and the adaptive mechanisms driving emerging variants.","version":"1.2","doi":"10.1101/2024.01.23.575696","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.29.635498","pub_date":"2025-1-31","title":"Fast, flexible analysis of differences in cellular composition with crumblr","abstract":"Changes in cell type composition play an important role in human health and disease. Recent advances in single-cell technology have enabled the measurement of cell type composition at increasing cell lineage resolution across large cohorts of individuals. Yet this raises new challenges for statistical analysis of these compositional data to identify changes in cell type frequency. We introduce crumblr (DiseaseNeurogenomics.github.io/crumblr), a scalable statistical method for analyzing count ratio data using precision-weighted linear mixed models incorporating random effects for complex study designs. Uniquely, crumblr performs statistical testing at multiple levels of the cell lineage hierarchy using a multivariate approach to increase power over tests of one cell type. In simulations, crumblr increases power compared to existing methods while controlling the false positive rate. We demonstrate the application of crumblr to published single-cell RNA-seq datasets for aging, tuberculosis infection in T cells, bone metastases from prostate cancer, and SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2025.01.29.635498","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.17.633602","pub_date":"2025-1-30","title":"Tryptophan metabolism reprogramming contributes to the prothrombotic milieu in mice and humans infected with SARS-CoV-2","abstract":"SARS-CoV-2 infection disturbs the coagulation balance in the blood, triggering thrombosis and contributing to organ failure. The role of prothrombotic metabolites in COVID-19-associated coagulopathy remains elusive. Leveraging K18-hACE2 mice infected with SARS-CoV-2, we observed higher levels of the tryptophan metabolite, kynurenine, compared to controls. SARS-CoV-2 infected mice showed a significant upregulation of enzymes controlling Kynurenine biogenesis, such as indoleamine 2,3-dioxygenase (IDO-1) and tryptophan 2,3-dioxygenase levels in kidneys and liver, respectively, as well as changes in the enzymes involved in kynurenine catabolism, including kynurenine monooxygenase and kynurinase. Consistent with the agonistic role of these metabolites in Aryl Hydrocarbon Receptor (AHR) signaling, AHR activation and its downstream mediator, tissue factor (TF), a highly potent procoagulant factor, was observed in endothelial cells (ECs) of lungs and kidneys of infected mice. These findings were validated in humans, where compared to controls, sera of COVID-19 patients showed increased levels of Kynurenine, kynurenic acid, anthranilic acid, and quinolinic acid. Activation of the AHR-TF axis was noted in the kidneys and lungs of COVID-19 patients, and COVID-19 sera showed higher IDO-1 activity than controls. Levels of Kyn in COVID-19 patients correlated strongly with the TF-inducing activity of COVID-19 sera on ECs. A specific IDO-1 inhibitor or AHR inhibitor separately or in combination suppressed COVID-19 sera-induced TF activity in ECs. Together, we identified IDO-1 as upregulated by SARS-CoV-2 infection, resulting in augmented Kyn and its prothrombotic catabolites, thereby suggesting the Kyn-AHR-TF axis as possibly a new diagnostic and/or therapeutic target. SARS-CoV-2-infection upregulates kynurenine biogenesis in the liver and diminishes kynurenine catabolism in the lungs and kidneys. An increase in kynurenine stimulates the AHR-TF axis in the microvasculature in COVID-19 patients, which is inhibited by pharmacological manipulation.","version":"1.2","doi":"10.1101/2025.01.17.633602","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.27.635150","pub_date":"2025-1-29","title":"Accelerated Adaptation of SARS-CoV-2 Variants in Mice Lacking IFITM3 Preserves Distinct Tropism and Pathogenesis","abstract":"Here we investigated whether interferon induced transmembrane protein 3 (IFITM3), a key antiviral protein deficient in certain human populations, affects interspecies adaptation of SARS-CoV-2. We found that SARS-CoV-2 Beta and Omicron variants passaged through IFITM3-deficient versus wild type mice exhibit enhanced replication and pathogenesis in this new host species. Enhancements associated with amino acid substitutions in the viral genome, suggesting that IFITM3 limits accumulation of adaptive mutations. Mouse-adapted viruses enabled comparative studies of variants in mice. Beta caused lung dysfunction and altered cilia-associated gene programs, consistent with broad viral antigen distribution in lungs. Omicron, which shows low pathogenicity and upper respiratory tract preference in humans, replicated to high nasal titers while showing restrained spatial distribution in lungs and diminished lung inflammatory responses compared to Beta. Our findings demonstrate that IFITM3 deficiency accelerates coronavirus adaptation and reveal that intrinsic SARS-CoV-2 variant traits shape tropism, immunity, and pathogenesis across hosts. IFITM3 is a critical barrier to SARS-CoV-2 adaptation in new host species Mouse-adapted SARS-CoV-2 strains enable comparative pathology Omicron favors nose and large airways, leading to mild lung pathology Beta exhibits broad lung replication, driving severe inflammation and dysfunction","version":"1.2","doi":"10.1101/2025.01.27.635150","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.28.635280","pub_date":"2025-1-29","title":"Allostery links hACE2 binding, pan-variant neutralisation and helical extension in the SARS-CoV-2 Spike protein","abstract":"The SARS-CoV-2 spike protein is highly antigenic, with epitopes in three distinct regions of the receptor binding domain (RBD) alone that have known mechanisms of neutralization. In previous work, we predicted a fourth RBD epitope based on allosteric conformational perturbations measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS) upon complexation with the canonical spike protein target, human angiotensin-converting enzyme 2 (hACE2). We subsequently identified a pan-neutralizing antibody (ICO-hu104) with the predicted epitope, however, as the epitope was somewhat distant from the hACE2 binding interface, and our previous work limited to the spike RBD, the neutralization mechanism was unclear. Using HDX-MS, we investigated the binding of ICO-hu104 to the full-length SARS-CoV-2 spike protein from Wuhan, Delta and Omicron variants. We demonstrate that binding of ICO-hu104 at its epitope results in an increase in deuterium uptake in the distant HR1 domain for variants of concern, which in a biological context could be indicative of destabilisation of the helices within this region, promoting S1 shedding or failure of helical extension during S2-mediated fusion. This is supported by our computational modelling, highlighting propagation of allosteric effects to the S2 coiled-coil region. Collectively, this work demonstrates an alternative neutralization mechanism for ICO-hu104 which is distinct from its first-generation predecessors and thus opens alternative avenues targeting non-RBD epitopes through assessment of allosteric perturbations. HDX-MS reveals decreased deuterium uptake within the HR1 region of S2 for SARS-CoV-2 spike protein for variants of concern when bound to ICO-hu104. Computational modelling validates high allosteric coupling between ICO-hu104 epitope and HR1 region. Suggests an alternative neutralization mechanism from its predecessor ICO-hu23, whereby destabilisation of helices within the HR1 region promotes S1 shedding and/or failure of helical extension during S2-mediated fusion.","version":"1.1","doi":"10.1101/2025.01.28.635280","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.28.635305","pub_date":"2025-1-29","title":"Statistical crystallography reveals an allosteric network in SARS-CoV-2 Mpro","abstract":"To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics with atomic resolution remains a key challenge. Here, using thousands of crystals of the main protease (Mpro) from SARS-CoV-2, we were able to infer a model of the protein\u2019s correlated motions. Mpro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the correlated motions that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally linked to the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography can determine protein correlated motions and rationalize their biological function. Crystallography at scale goes beyond a single structure, revealing native-state protein dynamics.","version":"1.1","doi":"10.1101/2025.01.28.635305","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.27.635166","pub_date":"2025-1-28","title":"The Interplay of Furin Cleavage and D614G in Modulating SARS-CoV-2 Spike Protein Dynamics","abstract":"We report a detailed analysis of the full-length SARS-CoV-2 spike dynamics within a native-like membrane environment and variants inaccessible to studies on soluble constructs by conducting hydrogen-deuterium exchange mass spectrometry (HDX-MS) on enveloped virus-like particles (eVLPs) displaying various spike constructs. We find that the previously identified open-interface trimer conformation is sampled in all eVLP-displayed spike variants studied including sequences from engineered vaccine constructs and native viral sequences. The D614G mutation, which arose early in the pandemic, favors the canonical \u2018closed-interface\u2019 prefusion conformation, potentially mitigating premature S1 shedding in the presence of a cleaved furin site and providing an evolutionary advantage to the virus. Remarkably, furin cleavage at the S1/S2 boundary allosterically increases the flexibility of the S2\u2019 site, which may facilitate increased TMPRSS2 processing, enhancing viral infectivity. The use of eVLPs in HDX-MS studies provides a powerful platform for studying viral and membrane proteins in near-native environments.","version":"1.1","doi":"10.1101/2025.01.27.635166","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.26.634807","pub_date":"2025-1-27","title":"SARS-CoV-2 cellular coinfection is limited by superinfection exclusion","abstract":"The coinfection of individual cells is a requirement for exchange between two or more virus genomes, which is a major mechanism driving virus evolution. Coinfection is restricted by a mechanism known as superinfection exclusion (SIE), which prohibits the infection of a previously infected cell by a related virus after a period of time. SIE regulates coinfection for many different viruses, but its relevance to the infection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was unknown. In this study, we investigated this using a pair of SARS-CoV-2 variant viruses encoding distinct fluorescent reporter proteins. We show for the first time that SARS-CoV-2 coinfection of individual cells is limited temporally by SIE. We defined the kinetics of the onset of SIE for SARS-CoV-2 in this system, showing that the potential for coinfection starts to diminish within the first hour of primary infection, and then falls exponentially as the time between the two infection events is increased. We then asked how these kinetics would affect the potential for coinfection with viruses during a spreading infection. We used plaque assays to model the localised spread of SARS-CoV-2 observed in infected tissue, and showed that the kinetics of SIE restrict coinfection, and therefore sites of possible genetic exchange, to a small interface of infected cells between spreading viral infections. This indicates that SIE, by reducing the likelihood of coinfection of cells, likely reduces the opportunities for genetic exchange between different strains of SARS-CoV-2 and therefore is an underappreciated factor in shaping SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2025.01.26.634807","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.23.634579","pub_date":"2025-1-27","title":"Addressing SARS-CoV-2 Evolution: Neutralization of Emerging Variants of Concern by the AVX/COVID-12 \u2018Patria\u2019 Vaccine Based on HexaPro-S Ancestral Wuhan Spike or Its Updated BA.2.75.2 Version","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global health challenge, causing severe morbidity and mortality, particularly in vulnerable groups such as the elderly, immunocompromised individuals, and those with comorbidities. In low- and middle-income countries (LMICs), vaccine access is hindered by high costs and inequitable distribution. To tackle these issues, Mexico developed the AVX/COVID-12 (V-Wu) vaccine, a recombinant Newcastle disease virus (NDV)-based platform expressing a stabilized ancestral Wuhan spike protein (HexaPro-S). Locally manufactured after rigorous testing and regulatory approval, V-Wu aims to enhance self-sufficiency and equity in immunization. This study evaluates an updated vaccine version, AVX/COVID-12 (V-BA), designed to combat Omicron subvariants by expressing the HexaPro-S protein of BA.2.75.2. Both vaccines were administered intramuscularly in K18-hACE2 transgenic and BALB/c mouse models using a prime-boost regimen. Immunogenicity was analyzed by measuring antibodies against Omicron S proteins BA.2.75.2 and XBB.1.5, as well as neutralizing antibodies against Wuhan, BA.1, XBB.1.16, and JN.1 variants. Both vaccines were safe, eliciting robust antibody responses against Omicron S proteins and neutralizing antibodies against multiple emerging SARS-CoV-2 variants of concern (VOCs). V-BA demonstrated superior protection against current Omicron variants, while V-Wu offered broader coverage, including the ancestral Wuhan strain and emerging variants like JN.1. These findings underscore the adaptability of NDV-based platforms in addressing the evolving SARS-CoV-2 landscape and reaffirm the ongoing utility of the ancestral Patria vaccine. Together, they demonstrate the potential of these platforms to drive the development of next-generation vaccines tailored to emerging viral threats, contributing to global health equity.","version":"1.1","doi":"10.1101/2025.01.23.634579","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.24.634813","pub_date":"2025-1-27","title":"Molecular Mechanisms of Drug Resistance and Compensation in SARS-CoV-2 Main Protease: The Interplay Between E166 and L50","abstract":"The SARS-CoV-2 main protease (Mpro) is essential for viral replication, and a primary target for COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, target the Mpro active site to block viral polyprotein cleavage and thus replication. However, drug resistance mutations at the active site residue Glu166 (E166) have emerged in in vitro selection studies, raising concerns about the durability of current antiviral strategies. Here, we investigate the molecular basis of drug resistance conferred by E166A and E166V mutations against nirmatrelvir and the related PF-00835231, individually and in combination with the distal mutation L50F. We found that E166 mutations reduce nirmatrelvir potency by up to 3000-fold while preserving substrate cleavage, with catalytic efficiency reduced by only up to 2- fold. This loss of catalytic efficiency was compensated for by the addition of L50F in the double- mutant variants. We have determined three cocrystal structures of the E166 variants (E166A, E166V, and E166V/L50F) bound to PF-00835231. Comparison of these structures with wild- type demonstrated that E166 is crucial for dimerization and for shaping the substrate-binding S1 pocket. Our findings highlight the mutability of E166, a prime site for resistance for inhibitors that leverage direct interactions with this position, and the potential emergence of highly resistant and active variants in combination with the compensatory mutation L50F. These insights support the design of inhibitors that target conserved protease features and avoid E166 side- chain interactions to minimize susceptibility to resistance.","version":"1.1","doi":"10.1101/2025.01.24.634813","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.28.610042","pub_date":"2025-1-27","title":"MINERVA FAIR assessment fosters open science & scientific crowd-sourcing in systems biomedicine","abstract":"The Disease Maps Project (https://disease-maps.org) focuses on the development of diseasespecific comprehensive structured knowledge repositories supporting translational medicine research. These disease maps require continuous interdisciplinary collaboration and should be reusable and interoperable. Adhering to the Findable, Accessible, Interoperable and Reusable (FAIR) principles enhances the utility of such digital assets. We used the RDA FAIR Data Maturity Model and assessed the FAIRness of the Molecular Interaction NEtwoRk VisuAlization (MINERVA) Platform. MINERVA is a standalone webserver that allows users to manage, explore and analyse disease maps and their related data manually or programmatically. We exemplify the FAIR assessment on the Parkinson\u2019s Disease Map (PD map) and the COVID-19 Disease Map, which are large-scale projects under the umbrella of the Disease Maps Project, aiming to investigate molecular mechanisms of the Parkinson\u2019s disease and SARS-CoV-2 infection, respectively. We discuss the FAIR features supported by the MINERVA Platform and we outline steps to further improve the MINERVA FAIRness and to better connect this resource to other ongoing scientific initiatives supporting FAIR in computational systems biomedicine.","version":"1.2","doi":"10.1101/2024.08.28.610042","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.25.634704","pub_date":"2025-1-27","title":"A pan-respiratory virus attachment inhibitor with high potency in human airway models and in vivo","abstract":"Respiratory viruses can cause severe infections, including bronchiolitis and pneumonia, often leading to hospitalization or death. Due to their ease of transmission, they are also scrutinized for their pandemic potential. No broad-spectrum antiviral is currently available. However most respiratory viruses use sialic acid or heparan sulfates as attachment receptors. Here, we report the identification of a pan-respiratory antiviral strategy based on mimicking both glycans. We synthesized and characterized a unique modified cyclodextrin that simultaneously mimics heparan sulfate and sialic acid. This novel compound demonstrated broad-spectrum antiviral activity against important human pathogens: parainfluenza virus 3, respiratory syncytial virus, influenza virus H1N1, SARS-CoV-2. Additionally, the compound is active against different avian strains of influenza virus, revealing its importance for pandemic preparedness. The compound retains broad- spectrum activity in ex vivo models of respiratory tissues and in vivo experiments against RSV and Influenza virus, using both prophylactic and therapeutic strategies. These findings represent a significant step forward in the development of future treatments and preventive measures for respiratory viral infections. Following the SARS-CoV-2 pandemic, Influenza A H5N1 has become widespread in poultry and has already begun to spill over into mammals, posing a potential risk for the next pandemic. Indeed, respiratory viruses represent a major threat for future global health crises. Unfortunately, there is a significant lack of broad-spectrum antivirals available for such scenarios. To address this gap, our study developed a single molecule with the capacity to inhibit a wide range of clinically relevant human respiratory viruses including avian strains of influenza virus. This antiviral demonstrated verified efficacy not only in cellular systems but also in human-derived respiratory tissues and animal models.","version":"1.1","doi":"10.1101/2025.01.25.634704","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.13.632836","pub_date":"2025-1-26","title":"MDL-001: An Oral, Safe, and Well-Tolerated Broad-Spectrum Inhibitor of Viral Polymerases","abstract":"The death toll and financial stress posed by the recent COVID-19 pandemic have highlighted the pressing need to develop safe and effective, broad-spectrum inhibitors to treat viral infections. To accelerate the antiviral drug discovery process, we developed GALILEO\u2122, a computational platform that interfaces with a customizable bioinformatics pipeline with a geometric deep learning algorithm we named ChemPrint\u2122 for in silico drug screening. Combining these algorithms with a large chemical repositioning library, we discovered MDL-001, which interacts with the Thumb pocket 1 subdomain of multiple single-stranded RNA viruses. For MDL-001, we demonstrate potent in vitro activity against a broad spectrum of pathogenic viruses, and we demonstrate potent in vivo efficacy in a mouse model of SARS-CoV-2 infection. In clinical trials, orally administered MDL-001 has been shown to be safe and well tolerated. These data underline both the effectiveness of the GALILEO\u2122 platform for drug discovery and the promise of MDL-001 as a novel broad-spectrum antiviral clinical candidate.","version":"1.3","doi":"10.1101/2025.01.13.632836","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.22.634352","pub_date":"2025-1-24","title":"A trans-amplifying mRNA vaccine with consensus spike elicits broadly cross-neutralizing antibody response against multiple SARS-CoV-2 variants","abstract":"SARS-CoV-2 continues to evolve and evade vaccine immunity necessitating vaccines that offer broad protection across variants. Conventional mRNA vaccines face cost and scalability challenges, prompting the exploration of alternative platforms like trans-amplifying (TA) mRNA that offer advantages in safety, manufacturability, and antigen dose optimization. Using consensus sequences of immunodominant antigens is a promising antigen design strategy for board cross-protection. Combining these two features, we designed and evaluated a TA mRNA vaccine encoding a consensus spike protein from SARS-CoV-2. Mice receiving the TA mRNA vaccine produced neutralizing antibody levels comparable to a conventional mRNA vaccine using 40 times less antigen mRNA. In hACE2 transgenic mice challenged with the Omicron BA.1 variant, the TA mRNA vaccine reduced lung viral titers by over 10-fold and induced broadly cross-neutralizing antibodies against multiple variants. These findings highlight the potential of TA mRNA vaccines with consensus antigen design, to improve efficacy and adaptability against SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2025.01.22.634352","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.22.634135","pub_date":"2025-1-24","title":"Clonal interference and changing selective pressures shape the escape of SARS-CoV-2 from hundreds of antibodies","abstract":"SARS-CoV-2 rapidly evolves to evade human immunity. While the virus\u2019s overall resistance to human polyclonal antibody responses has steadily increased over time, the dynamics by which it escaped individual monoclonal antibodies within these responses have not been thoroughly explored. Recently, a series of studies by Cao et al. [1, 2, 3] used deep mutational scanning (DMS) to identify which mutations allow the Wuhan-Hu-1 receptor-binding domain to escape binding by individual antibodies, doing so for thousands of antibodies. Here, we sought to use these data to retrospectively examine the evolutionary dynamics of escape from a set of 1,603 antibodies. For each antibody, we used the DMS data to predict an antibody-escape score for each of thousands of globally circulating viral sequences from the first 3.5 years of the pandemic, and then computed an escape trajectory that quantifies how the population\u2019s average escape score changed over time. We use pseudovirus neutralization data from Cao et al. and Wang et al. [4] to validate common patterns in escape trajectories. While some trajectories increase monotonically over time, others show large fluctuations as a result of clade-displacement events that reduce the frequency of antibody-escape mutations in the viral population. Fitness effects of mutations estimated from natural sequences suggest that the mutations are displaced due to clonal interference. Further, these estimates suggest that the order in which escape mutations arose is shaped by changing selective pressures. Overall, this work helps describe how SARS-CoV-2 evaded the individual components of a polyclonal immune response in nature, and suggests that evasion occurred via complex evolutionary dynamics.","version":"1.1","doi":"10.1101/2025.01.22.634135","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.23.634602","pub_date":"2025-1-24","title":"A single dimer of the SARS-CoV-2 N protein can associate with multiple fragments of single-stranded and stem-loop RNAs","abstract":"The nucleocapsid (N) protein of SARS-CoV-2 associates with the viral genomic RNA (gRNA) and forms the ribonucleoprotein (RNP) granules. However, the detailed molecular structures of RNP and their formation mechanism are largely unknown. We used circular dichroism (CD) spectroscopy, fluorescence correlation spectroscopy (FCS) and single-molecule F\u00f6rster resonance energy transfer (sm-FRET) spectroscopy to understand the interaction between the N protein and different structural units of RNA. We chose polyadenylate chains with 40, 30 and 20 bases possessing a single-stranded structure and three stem loops with 50, 41 and 29 bases selected from gRNA, and labeled their 5\u2019 and 3\u2019 ends by Alexa488 and Alexa647, respectively, for the FCS and sm-FRET measurements. We found that the N protein started to bind to the single-stranded RNAs at the concentrations between 10 and 100 nM. The binding of the N protein to one of the stem loops occurred at the concentration less than 10 nM without melting the stem loop. For all the samples, the binding of multiple molecules of the RNA fragments to a single dimer of the N protein was observed. These results demonstrate that the N protein acts as a non-specific binder to both single-stranded and stem-loop structures of RNA, and that the N protein might contract a long RNA chain by bridging its multiple segments. We propose that the RNP granules might be folded by the association of the numerous stem loops of gRNA triggered by the assembly of the N protein.","version":"1.1","doi":"10.1101/2025.01.23.634602","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.11.622903","pub_date":"2025-1-23","title":"Increased preference for lysine over arginine in spike proteins of SARS-CoV-2 BA.2.86 variant and its daughter lineages","abstract":"The COVID-19 pandemic offered an unprecedented glimpse into the evolution of its causative virus, SARS-CoV-2. It has been estimated that since its outbreak in late 2019, the virus has explored all possible alternatives in terms of missense mutations for all sites of its polypeptide chain. Spike protein of the virus exhibits the largest sequence variation in particular, with many individual mutations impacting target recognition, cellular entry, and endosomal escape of the virus. Moreover, recent studies unveiled a significant increase in the total charge on the spike protein during the evolution of the virus in the initial period of the pandemic. While this trend has recently come to a halt, we perform a sequence-based analysis of the spike protein of 2665 SARS-CoV-2 variants which shows that mutations in ionizable amino acids continue to occur with the newly emerging variants, with notable differences between lineages from different clades. What is more, we show that within mutations of amino acids which can acquire positive charge, the spike protein of SARS-CoV-2 exhibits a prominent preference for lysine residues over arginine residues. This lysine-to-arginine ratio increased at several points during spike protein evolution, most recently with BA.2.86 and its sublineages, including the recently dominant JN.1, KP.3, and XEC variants. The increased ratio is a consequence of mutations in different structural regions of the spike protein and is now among the highest among viral species in the Coronaviridae family. The impact of high lysine-to-arginine ratio in the spike proteins of BA.2.86 and its daughter lineages on viral fitness remains unclear; we discuss several potential mechanisms that could play a role and that can serve as a starting point for further studies.","version":"1.2","doi":"10.1101/2024.11.11.622903","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.17.633612","pub_date":"2025-1-20","title":"SARS-CoV-2 neutralizing antibody specificities differ dramatically between recently infected infants and immune-imprinted individuals","abstract":"The immune response to viral infection is shaped by past exposures to related virus strains, a phenomenon known as imprinting. For SARS-CoV-2, much of the population has been imprinted by a viral spike from an early strain, either through vaccination or infection during the early stages of the COVID-19 pandemic. As a consequence of this imprinting, infection with more recent SARS-CoV-2 strains primarily boosts cross-reactive antibodies elicited by the imprinting strain. Here we compare the neutralizing antibody specificities of imprinted individuals versus infants infected with a recent strain. Specifically, we use pseudovirus-based deep mutational scanning to measure how spike mutations affect neutralization by the serum antibodies of adults and children imprinted by the original vaccine versus infants with a primary infection by a XBB* variant. While the serum neutralizing activity of the imprinted individuals primarily targets the spike receptor-binding domain (RBD), serum neutralizing activity of infants only infected with XBB* mostly targets the spike N-terminal domain (NTD). In these infants, secondary exposure to the XBB* spike via vaccination shifts more of the neutralizing activity towards the RBD, although the specific RBD sites targeted are different than for imprinted adults. The dramatic differences in neutralization specificities among individuals with different exposure histories likely impact SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2025.01.17.633612","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.17.633662","pub_date":"2025-1-20","title":"A phylogenetic method identifies candidate drivers of the evolution of the SARS-CoV-2 mutation spectrum","abstract":"The molecular processes that generate new mutations evolve, but the causal mechanisms are largely unknown. In particular, the relative rates of mutation types (e.g., C>T), the mutation spectrum, sometimes vary among closely related species and populations. I present an algorithm for subdividing a phylogeny into distinct mutation spectra. By applying this approach to a SARS-CoV-2 phylogeny comprising approximately eight million genome sequences, I identify 10 shifts in the mutation spectrum. I find strong enrichment consistent with candidate causal amino-acid substitutions in the SARS-CoV-2 polymerase, and strikingly three appearances of the same homoplasious substitution are each associated with decreased C>T relative mutation rates. With rapidly growing genomic datasets, this approach and future extensions promises new insights into the mechanisms of evolution of mutational processes.","version":"1.1","doi":"10.1101/2025.01.17.633662","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.18.633743","pub_date":"2025-1-20","title":"Recommendations for Automating Hydrogen/Deuterium Exchange Mass Spectrometry Measurements using Data-Independent Acquisition Methods","abstract":"Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS is limited in throughput because manual data analysis is still the norm. We previously developed HX-MS technology to add a second dimension of deuteration data and promote automated data processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse for illustration, we show that ion optics and collision energy settings typical of a proteomics DIA experiment generate maximal peptide retrieval from the DIA library. As few as three MS sequence ions are sufficient to generate a deuteration measurement with a precision that exceeds what is possible in traditional HX-MS. DIA window sizes are based on the chromatographic resolution of the method. An inter-scan window offset method is the recommended default configuration for most HX-DIA applications, but an intra-scan overlap method can be tuned for highest performance and is recommended when maximum peptide retrieval is desired. A fully automated HX-MS solution consists of Trajan HDX automation technology, an Orbitrap Eclipse mass spectrometer and AutoHX software. We demonstrate its robustness on an extensive time-course analysis of phosphorylase B and an epitope analysis of single domain antibodies (VHHs, nanobodies) specific to the receptor binding domain of SARS-CoV2 spike protein.","version":"1.1","doi":"10.1101/2025.01.18.633743","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.16.633443","pub_date":"2025-1-18","title":"Structure-based discovery of highly bioavailable, covalent, broad-spectrum coronavirus-MPro inhibitors with potent in vivo efficacy","abstract":"The main protease (MPro) of SARS-CoV-2 is crucial for viral replication and is the target of nirmatrelvir (the active ingredient of Paxlovid) and ensitrelvir. The identification of new agents with differentiated pharmacokinetic and drug resistance profiles will increase therapeutic options for COVID-19 patients and bolster pandemic preparedness generally. Starting with a lead-like dihydrouracil chemotype from a large-library docking campaign, we improved MPro inhibition >1,000-fold by engaging additional sub-sites in the MPro active site, most notably by employing a latent propargyl electrophile to engage the catalytic Cys145. Advanced leads from this series, including AVI-4516 and AVI-4773 show pan-coronavirus antiviral activity in cells, very low clearance in mice, and for AVI-4773 a rapid reduction in viral titers more than a million-fold after just three doses, more rapidly and effectively than the approved drugs, nirmatrelvir and ensitrelvir. Both AVI-4516 and AVI-4773 are well distributed in mouse tissues, including brain, where concentrations ten or fifteen-thousand times the EC90, respectively, are observed eight hours after an oral dose. As exemplar of the series, AVI-4516 shows minimal inhibition of major CYP isoforms and human cysteine and serine proteases, likely due to its latent\u2013electrophilic warhead. AVI-4516 also exhibits synergy in cellular infection models in combination with the RdRp inhibitor molnupiravir, while related analogs strongly inhibit nirmatrelvir-resistant MPro mutant virus in cells. The in vivo and antiviral properties of this new chemotype are differentiated from existing clinical and pre-clinical MPro inhibitors, and will advance new therapeutic development against emerging SARS-CoV-2 variants and other coronaviruses. This manuscript describes the discovery of a new class of potent inhibitors of the SARS-CoV-2 major proteases (MPro) with a unique mechanism of inhibition, pan coronaviral activity in cellulo, exquisite selectivity vs. the human proteome, and exceptional in vivo efficacy in SARS-CoV-2 infection models that surpasses that of currently approved agents.","version":"1.1","doi":"10.1101/2025.01.16.633443","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.16.633331","pub_date":"2025-1-16","title":"Optimizing SARS-CoV-2 RBD Boundaries for Enhanced E. coli Expression","abstract":"The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a significant risk to global health, warranting the formulation of efficient preventive and therapeutic measures to tackle its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of coronaviruses plays a pivotal role in viral attachment and entry into host cells. The receptor-binding domain (RBD) of the SARS-CoV-2 S protein has demonstrated a robust binding affinity to ACE2 receptors in humans. Consequently, it has become a prime target for therapeutic interventions using antibodies, vaccines, or other designed inhibitors. This paper presents an optimized RBD sequence that can be efficiently expressed in Escherichia coli and refolded to yield a functional protein. Using optimized refolding procedures, we obtained 10-12 mg of active protein from a one-liter LB culture. The biological activity of the refolded RBD was confirmed by monitoring its interaction with the designed LCB1 miniprotein ligand by surface plasmon resonance, wherein they exhibited significant affinity levels as reflected by their dissociation constants (KDs < 10 nM). The resulting RBD could be an ideal target for designing potent COVID-19 antivirals.\n\n\n","version":"1.1","doi":"10.1101/2025.01.16.633331","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.09.611961","pub_date":"2025-1-16","title":"A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and selects for interferon-susceptible mutant viruses","abstract":"The approval of COVID-19 vaccines and antiviral drugs has been crucial to end the global health crisis caused by SARS-CoV-2. However, to prepare for future outbreaks from drug-resistant variants and novel zoonotic coronaviruses (CoVs), additional therapeutics with a distinct antiviral mechanism are needed. Here, we report a novel guanidine-substituted diphenylurea compound that suppresses CoV replication by interfering with the uridine-specific endoribonuclease (EndoU) activity of the viral non-structural protein-15 (nsp15). This compound, designated EPB-113, exhibits strong and selective cell culture activity against human coronavirus 229E (HCoV-229E) and also suppresses the replication of SARS-CoV-2. Viruses, selected under EPB-113 pressure, carried resistance sites at or near the catalytic His250 residue of the nsp15-EndoU domain. Although the best-known function of EndoU is to avoid induction of type I interferon (IFN-I) by lowering the levels of viral dsRNA, EPB-113 was found to mainly act via an IFN-independent mechanism, situated during viral RNA synthesis. Using a combination of biophysical and enzymatic assays with the recombinant nsp15 proteins from HCoV-229E and SARS-CoV-2, we discovered that EPB-113 enhances the EndoU cleavage activity of hexameric nsp15, while reducing its thermal stability. This mechanism explains why the virus escapes EPB-113 by acquiring catalytic site mutations which impair compound binding to nsp15 and abolish the EndoU activity. Since the EPB-113-resistant mutant viruses induce high levels of IFN-I and its effectors, they proved unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. Our findings suggest that antiviral targeting of nsp15 can be achieved with a molecule that induces a conformational change in this protein, resulting in higher EndoU activity and impairment of viral RNA synthesis. Based on the appealing mechanism and resistance profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target. Despite major achievements in ending the global COVID-19 crisis through vaccines and antiviral drugs, SARS-CoV-2 infection remains a serious health threat for vulnerable individuals, such as elderly and immunocompromised patients. These populations could benefit from innovative treatments that target the virus from multiple angles. In this study, we focus on the nsp15 endoribonuclease (EndoU), a poorly understood coronavirus protein that aids in viral evasion from the host innate immune response and is also assumed to regulate viral RNA synthesis. Our research centers around a newly identified, first-in-class inhibitor of coronavirus nsp15, designated EPB-113. Although nsp15 is best-known for its function in interferon evasion, EPB-113 mainly acts via an interferon-independent mechanism that is situated during viral RNA synthesis. When incubated with nsp15 protein, EPB-113 induces a structural change and enhances its EndoU enzyme activity. EPB-113 selects for escape mutants that carry substitutions in the core of EndoU, leading to a severe replication defect. Hence, our study introduces a novel drug concept to disturb the multifunctional nsp15 protein and suppress coronavirus replication on multiple fronts.","version":"1.2","doi":"10.1101/2024.09.09.611961","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.14.633005","pub_date":"2025-1-15","title":"SARS-CoV-2 nsp1 mediates broad inhibition of translation in mammals","abstract":"SARS-CoV-2 nonstructural protein 1 (nsp1) promotes innate immune evasion by inhibiting host translation in human cells. However, the role of nsp1 in other host species remains elusive, especially in bats which are natural reservoirs of sarbecoviruses and possess a markedly different innate immune system than humans. Here, we reveal that SARS-CoV-2 nsp1 potently inhibits translation in bat cells from Rhinolophus lepidus, belonging to the same genus as known sarbecovirus reservoirs hosts. We determined a cryo-electron microscopy structure of SARS-CoV-2 nsp1 bound to the Rhinolophus lepidus 40S ribosome and show that it blocks the mRNA entry channel via targeting a highly conserved site among mammals. Accordingly, we found that nsp1 blocked protein translation in mammalian cell lines from several species, underscoring its broadly inhibitory activity and conserved role in numerous SARS-CoV-2 hosts. Our findings illuminate the arms race between coronaviruses and mammalian host immunity (including bats), providing a foundation for understanding the determinants of viral maintenance in bat hosts and spillovers.","version":"1.1","doi":"10.1101/2025.01.14.633005","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.15.633120","pub_date":"2025-1-15","title":"Metabolic syndrome enhances SARS-CoV-2 disease severity and reduces mRNA vaccine efficacy in a mouse model","abstract":"Metabolic syndrome (MetS) is a cluster of pathophysiological conditions linked to the disruption of metabolic processes associated with energy storage and consumption. Approximately one-third of adults in the United States are currently diagnosed with MetS. Patients with MetS experience higher mortality rates following SARS-CoV-2 infection and exhibit poor vaccine efficacy following influenza virus vaccination compared to metabolically healthy individuals. However, the specific impact of MetS on immune responses to SARS-CoV-2 infection and vaccination has not been widely studied. To address this gap, we utilized high-fat diet feeding to establish a murine model of MetS, in which mice exhibit the same diagnostic criteria for MetS as human patients. We then used high-fat diet-induced MetS mice and regular chow diet-fed wild-type mice to analyze immune responses to SARS-CoV-2 infection and vaccination. Following SARS-CoV-2 infection, we monitored mice for disease severity, measured levels of virally induced inflammation, and quantified viral titers in various tissues. Our results indicate that MetS mice exhibit accelerated mortality post-infection, accompanied by elevated mRNA levels of inflammatory cytokine transcripts at sites of infection. Additionally, MetS alters the degree of viral replication across various tissues. Furthermore, our vaccination studies revealed that MetS reduces the potency of vaccine-induced neutralizing antibodies against both the ancestral SARS-CoV-2 strain and the Delta variant. Overall, our findings suggest that MetS exacerbates SARS-CoV-2 disease severity and diminishes vaccine efficacy, underscoring the need for tailored strategies to protect individuals with MetS from severe outcomes following infection and vaccination. This study represents the first detailed account of the failure to generate protective neutralizing antibody responses to SARS-CoV-2 in mice with metabolic syndrome (MetS) following vaccination. The insights gained from this research inform future vaccine design and aid in identifying individuals at greater risk of breakthrough infections. By specifically isolating the risk associated with MetS, rather than obesity alone, these findings lay the groundwork for future investigations aimed at enhancing immune responses in individuals with MetS. This work is highly relevant to a broad audience, as it addresses a critical unmet need in vaccine development and provides essential guidance for the rational design of vaccines to protect vulnerable populations affected by MetS.","version":"1.1","doi":"10.1101/2025.01.15.633120","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.14.632961","pub_date":"2025-1-15","title":"Deciphering the Host-Pathogen Interface in COVID-19: The precision molecular insight into epitranscriptomic modifications of high-impact transcripts","abstract":"The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a profound global impact since its emergence in late 2019. Characterized by a wide spectrum of clinical manifestations, ranging from asymptomatic infection to severe respiratory distress and death, COVID-19 has necessitated extensive research into the host-pathogen interactions that drive disease progression. Understanding the molecular mechanisms underlying the host response to SARS-CoV-2 infection is crucial for the development of effective therapeutic interventions and preventative strategies. This study employed a multi-omic approach that combined direct RNA sequencing (DRS) and Illumina cDNA sequencing to investigate whole blood transcriptomic profiles in COVID-19 patients. By leveraging the unique capabilities of Nanopore DRS, which provides long-read sequencing data, we were able to capture not only gene expression levels but also crucial RNA modifications, including poly(A) tail length, non-adenine residue (non-A), pseudouridylation (psU), and 5-methylcytosine (m5C) methylation. This comprehensive analysis allowed us to identify differentially expressed genes (DEGs) and explore the impact of these RNA modifications on gene expression and function within the context of COVID-19. Our findings reveal significant alterations in gene expression patterns, poly(A) tail lengths, non-A and the prevalence of psU and m5C modifications in COVID-19 patients compared to healthy controls. These results provide valuable insights into the complex interplay between viral infection, host immune response, and RNA processing, contributing to a deeper understanding of COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2025.01.14.632961","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.14.632924","pub_date":"2025-1-15","title":"Unlocking the Secrets of NSP3: AlphaFold2-assisted Domain Determination in SARS-CoV-2 Protein","abstract":"Non-structural protein 3 (nsp3) is crucial for the SARS-CoV-2 infection cycle. It is the largest protein of the virus, consisting of roughly 2000 residues, and a major drug target. However, due to its size, disordered regions, and transmembrane domains, the atomic structure of the whole protein has not yet been established. Only 10 out of its 16 domains were individually determined in experiments. Here, we demonstrate how structural bioinformatics, AI-based fold prediction, and traditional experiments complement each other and can shed light on the makeup of this important protein, both in SARS-CoV-2 and related viruses. Our method can be generalized for other multi-domain proteins, so we describe it in detail. Our prediction-based approach reveals a previously undescribed folded domain, which we could confirm experimentally. Our research also suggests a potential function of the nidovirus-wide conserved domain Y1: This domain may be involved in the assembly of nsp3, nsp4, and nsp6 into the hexameric pore, which was discovered by electron tomography and exports RNA into the cytosol. The Y1-hexamer, however, could not be expressed and purified on its own. We also provide a revised domain segmentation and nomenclature of nsp3 domains based on a compilation of previous research and our own findings.","version":"1.1","doi":"10.1101/2025.01.14.632924","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.14.632953","pub_date":"2025-1-15","title":"Trypstatin as a Novel TMPRSS2 Inhibitor with Broad-Spectrum Efficacy Against Corona and Influenza Viruses","abstract":"Respiratory viruses, such as SARS-CoV-2 and influenza, exploit host proteases like TMPRSS2 for entry, making TMPRSS2 a prime antiviral target. Here, we report the identification and characterization of Trypstatin, a 61-amino acid Kunitz-type protease inhibitor derived from human hemofiltrate. Trypstatin inhibits TMPRSS2 and related proteases, with IC50 values in the nanomolar range, comparable to the small molecule inhibitor camostat mesylate. In vitro assays demonstrated that Trypstatin effectively blocks spike-driven entry of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and hCoV-NL63, as well as hemagglutinin-mediated entry of influenza A and B viruses. In primary human airway epithelial cultures, Trypstatin significantly reduced SARS-CoV-2 replication and retained activity in the presence of airway mucus. In vivo, intranasal administration of Trypstatin to SARS-CoV-2-infected Syrian hamsters reduced viral titers and alleviated clinical symptoms. These findings highlight Trypstatin\u2019s potential as a broad-spectrum antiviral agent against TMPRSS2-dependent respiratory viruses.","version":"1.1","doi":"10.1101/2025.01.14.632953","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.12.632164","pub_date":"2025-1-14","title":"Distinct CD8+ T-cell types Associated with COVID-19 Severity in Unvaccinated HLA-A2+ Patients","abstract":"Although emerging data have revealed the critical role of memory CD8+ T cells in preventing and controlling SARS-CoV-2 infection, virus-specific CD8+ T-cell responses against SARS-CoV-2 and its memory and innate-like subsets in unvaccinated COVID-19 patients with various disease manifestations in an HLA-restricted fashion remain to be understood. Here, we show the strong association of protective cellular immunity with mild COVID-19 and unique cell types against SARS-CoV-2 virus in an HLA-A2 restricted manner. ELISpot assays reveal that SARS-CoV-2-specific CD8+ T-cell responses in mild COVID-19 patients are significantly higher than in severe patients, whereas neutralizing antibody responses against SARS-CoV-2 virus significantly correlate with disease severity. Single-cell analyses of HLA-A2-restricted CD8+ T cells, which recognize highly conserved immunodominant SARS-CoV-2-specific epitopes, demonstrate divergent profiles in unvaccinated patients with mild versus severe disease. CD8+ T-cell types including cytotoxic KLRB1+ CD8\u03b1\u03b1 cells with innate-like T-cell signatures, IFNGhiID3hi memory cells and IL7R+ proliferative stem cell-like memory cells are preferentially observed in mild COVID-19, whereas distinct terminally-differentiated T-cell subsets are predominantly detected in severe COVID-19: highly activated FASLhi T-cell subsets and early-terminated or dysfunctional IL4R+ GATA3+ stem cell-like memory T-cell subset. In conclusion, our findings suggest that unique and contrasting SARS-CoV-2-specific CD8+ T-cell profiles may dictate COVID-19 severity. Potent memory CD8+ T-cell subtypes with gene signature in mild COVID-19 patients (upper) and dysfunctional CD8+ T-cell subtypes with gene signature in severe COVID-19 patients (lower).","version":"1.1","doi":"10.1101/2025.01.12.632164","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.13.632691","pub_date":"2025-1-14","title":"A Fine-tuned ProtGPT2 (transformer model) for Predicting more Virulent SARS-CoV-2 variants and understanding its virulence by biophysical methods","abstract":"The emergence of Variants of Concern (VOCs) of SARS-COV2 with increased virulence and transmissibility has been linked to multiple mutations in the RBD region, altering their antigenic properties. In this study, we used a specialized ProtGPT2 model trained on the SARS-COV2 spike protein to forecast probable mutations on the spike protein that have not yet emerged. Upon prediction, we systematically studied the stability of single-site and multisite mutations using unbiased molecular dynamics simulations. Binding free energies were used to study the physicochemical significance of the mutations and their affinity to human ACE2 receptor. Our results show that there are specific hot-spots that mutate in the spike protein that enhance binding affinity through electrostatic and improved non-bonded interactions and highlight the role of specific energetic contributions in viral adaptation and infectivity. Our analysis revealed that the reduction of a disulphide bridge within sites 480-488 lowered the binding free energy and increased the flexibility of the loop region, enhancing its interface interaction with ACE, leading to a more virulent variant than Omicron.","version":"1.1","doi":"10.1101/2025.01.13.632691","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.12.632508","pub_date":"2025-1-13","title":"Extracellular stiffness regulates site-specific lung development","abstract":"Extracellular matrix (ECM) stiffness plays a crucial role in regulating cell fate and maturation, but its influence on lung development is limited known. Here we utilized stiffness-tunable gelatin methacryloyl (GelMA) hydrogels to investigate how ECM stiffness influences site-specific lung development in a stem cell-derived lung organoid model. We found increased stiffness promoted NKX2-1+ lung progenitor cells (LPCs) generation. In airway organoids (hAWOs), stiff hydrogels directed proximal airway differentiation enriched with goblet, ciliated, and basal cells; whereas the decreased stiffness favored emergence of secretory cells in the proximal-distal transition zone and distal airway. In alveolar organoids (hALOs), increased stiffness enhanced AT2 and AT1 cells transition. Moreover, infection assays with Omicron BA.1.1 and Delta variants recapitulated the proximal-to-distal tropism of SARS-CoV-2 in the lung. Transcriptomic sequencing revealed ECM stiffness regulates lung development via Hippo, TGF-\u03b2, HIF and Wnt pathways. These findings advance mechanism understanding of ECM stiffness on lung development and provide a novel mechanical regulation for generating site-specific lung organoids.","version":"1.1","doi":"10.1101/2025.01.12.632508","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.10.632425","pub_date":"2025-1-10","title":"Processing of Genomic RNAs by Dicer in Bat Cells Limits SARS-CoV-2 Replication","abstract":"Bats are reservoirs for numerous viruses that cause serious diseases in other animals and humans. Several mechanisms are proposed to contribute to the tolerance of bats to these pathogens. This study investigates the response of bat cells to double-stranded RNA generated by SARS-CoV-2 replication. Here, we found the involvement of Dicer in the processing of viral genomic RNAs during SARS-CoV-2 infection. Examining RNA sequencing of infected cells, small-interfering RNA (siRNA)-like fragments were found derived from viral RNAs. Depletion of Dicer showed a reduction in these RNAs and an increase in viral loads suggesting unlike other mammals, bats may use Dicer to limit viral replication. This prompted the exploration of key dsRNA sensors in bat cells. Our analysis showed significant upregulation of OAS1 and MX1 in response to dsRNA, while PKR levels remained low, suggesting alternative dsRNA-response mechanisms are present that eschew the common PKR-based system. These results further show how bats employ distinct strategies for antiviral defense that may contribute to tolerating viral infections. They suggest the involvement of Dicer in antiviral mechanisms in bats, a function not observed in other mammals. This highlights a mechanism for bat originating viruses to evolve features that in other animals could cause extreme antiviral responses such as is seen with SARS-CoV-2.","version":"1.1","doi":"10.1101/2025.01.10.632425","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.09.632180","pub_date":"2025-1-10","title":"Design of SARS-CoV-2 RBD Immunogens to Focus Immune Responses Towards Conserved Coronavirus Epitopes","abstract":"SARS-CoV-2 continues to evolve, with new variants emerging that evade pre-existing immunity and limit the efficacy of existing vaccines. One approach towards developing superior, variant-proof vaccines is to engineer immunogens that preferentially elicit antibodies with broad cross-reactivity against SARS-CoV-2 and its variants by targeting conserved epitopes on spike. The inner and outer faces of the Receptor Binding Domain (RBD) are two such conserved regions targeted by antibodies that recognize diverse human and animal coronaviruses. To promote the elicitation of such antibodies by vaccination, we engineered \u201cresurfaced\u201d RBD immunogens that contained mutations at exposed RBD residues outside the target epitopes. In the context of pre-existing immunity, these vaccine candidates aim to disfavor the elicitation of strain-specific antibodies against the immunodominant Receptor Binding Motif (RBM) while boosting the induction of inner and outer face antibodies. The engineered resurfaced RBD immunogens were stable, lacked binding to monoclonal antibodies with limited breadth, and maintained strong interactions with target broadly neutralizing antibodies. When used as vaccines, they limited humoral responses against the RBM as intended. Multimerization on nanoparticles further increased the immunogenicity of the resurfaced RBDs immunogens, thus supporting resurfacing as a promising immunogen design approach to rationally shift natural immune responses to develop more protective vaccines.","version":"1.1","doi":"10.1101/2025.01.09.632180","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.10.632212","pub_date":"2025-1-10","title":"Single-molecule assay reveals the impact of composition, RNA duplex, and inhibitors on the binding dynamics of SARS-CoV-2 polymerase complex","abstract":"The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multi-subunit replication complex consisting of non-structural proteins (nsps) 12, 7 and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein-induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or co-expressed replication complex and RNA. Increasing binding times were observed, in this order, with nsp7 (none) nsp8 and nsp12, in nsp8-nsp12 mixtures and in reconstituted mixtures bearing all three proteins. Unstable, transient, and stable binding modes were recorded in the latter case, indicating that complexation is dynamic, and the correct conformation must be achieved before stable RNA binding can occur. Notably, the co-expressed protein yields mostly stable binding even at low concentrations, while the reconstituted proteins exhibit unstable binding indicating inefficient complexation with reduced protein. The SM-PIFE assay distinguishes inhibitors that impact protein binding from those that prevent replication, as demonstrated with suramin and remdesivir, respectively. The data reveals a correlation between binding lifetime/affinity, and protein activity, and underscores differences between co-expressed vs reconstituted mixtures, suggesting the existence of trapped conformations that may not evolve to productive binding.","version":"1.1","doi":"10.1101/2025.01.10.632212","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.10.632299","pub_date":"2025-1-10","title":"A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent","abstract":"Coronaviruses (CoV) encode sixteen non-structural proteins (nsps), most of which form the replication-transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles into a processive polymerase remains poorly understood. We show that the core RTC preferentially assembles by first having nsp12-polymerase bind to the RNA template, followed by the subsequent association of nsp7 and nsp8. Once assembled on the RNA template, the core RTC requires hundreds of seconds to undergo a conformational change that enables processive elongation. In the absence of RNA, the (apo-)RTC requires several hours to adopt its elongation-competent conformation. We propose that this obligatory activation step facilitates the recruitment of additional nsp\u2019s essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.","version":"1.1","doi":"10.1101/2025.01.10.632299","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.07.631679","pub_date":"2025-1-09","title":"Identification and Functional Characterization of Regulatory Variants in DPP9 Associated with COVID-19 Severity","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to a wide-range of clinical outcomes, which have been extensively studied through genome-wide association studies (GWAS). Starting from lead genetic variants associated with COVID-19 infection and severity, we identified a subset of non-coding candidate variants with potential regulatory functions. Using bioinformatics analysis and functional screening in three cell lines, we prioritized two DPP9 variants within a haplotype that increases the risk of severe COVID-19. This haplotype exhibited increased regulatory activity and altered transcription factor binding, suggesting its role in influencing COVID-19 severity through modulation of DPP9 expression in immune and lung cell types. The interest of our study lies in the functional characterization of regulatory variants responsible for the higher levels of DPP9 and lung damage observed in patients with severe COVID-19. These findings advance our understanding of genetic risk factors for COVID-19 and highlight functional SNPs that may guide future therapeutic research.","version":"1.1","doi":"10.1101/2025.01.07.631679","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.08.631842","pub_date":"2025-1-09","title":"Evolution of SARS-CoV-2 T cell responses as a function of multiple COVID-19 boosters","abstract":"The long-term effects of repeated COVID-19 vaccinations on adaptive immunity remain incompletely understood. Here, we conducted a comprehensive three-year longitudinal study examining T cell and antibody responses in 78 vaccinated individuals without reported symptomatic infections. We observed distinct dynamics in Spike-specific humoral and cellular immune responses across multiple vaccine doses. While antibody titers incrementally increased and stabilized with each booster, T cell responses rapidly plateaued, maintaining remarkable stability across CD4+ and CD8+ subsets. Notably, approximately 30% of participants showed CD4+ T cell reactivity to non-Spike antigens, consistent with asymptomatic infections. Single-cell RNA sequencing revealed a diverse landscape of Spike-specific T cell phenotypes, with no evidence of increased exhaustion or significant functional impairment. However, qualitative changes were observed in individuals with evidence of asymptomatic infection, exhibiting unique immunological characteristics, including increased frequencies of Th17-like CD4+ T cells and GZMKhi/IFNR CD8+ T cell subsets. Remarkably, repeated vaccinations in this group were associated with a progressive increase in regulatory T cells, potentially indicating a balanced immune response that may mitigate immunopathology. By regularly stimulating T cell memory, boosters contribute to a stable and enhanced immune response, which may provide better protection against symptomatic infections.","version":"1.1","doi":"10.1101/2025.01.08.631842","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.08.631993","pub_date":"2025-1-09","title":"APMAT analysis reveals the association between CD8 T cell receptors, cognate antigen, and T cell phenotype and persistence","abstract":"Elucidating the relationships between a class I peptide antigen, a CD8 T cell receptor (TCR) specific to that antigen, and the T cell phenotype that emerges following antigen stimulation, remains a mostly unsolved problem, largely due to the lack of large data sets that can be mined to resolve such relationships. Here, we describe Antigen-TCR Pairing and Multiomic Analysis of T-cells (APMAT), an integrated experimental-computational framework designed for the high-throughput capture and analysis of CD8 T cells, with paired antigen, TCR sequence, and single-cell transcriptome. Starting with 951 putative antigens representing a comprehensive survey of the SARS-CoV-2 viral proteome, we utilize APMAT for the capture and single cell analysis of CD8 T cells from 62 HLA A*02:01 COVID-19 participants. We leverage this unique, comprehensive dataset to integrate with peptide antigen properties, TCR CDR3 sequences, and T cell phenotypes to show that distinct physicochemical features of the antigen-TCR pairs strongly associate with both T cell phenotype and T cell persistence. This analysis suggests that CD8+ T cell phenotype following antigen stimulation is at least partially deterministic, rather than the result of stochastic biological properties.","version":"1.1","doi":"10.1101/2025.01.08.631993","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.07.631013","pub_date":"2025-1-08","title":"The mutation rate of SARS-CoV-2 is highly variable between sites and is influenced by sequence context, genomic region, and RNA structure","abstract":"RNA viruses like SARS-CoV-2 have a high mutation rate, which contributes to their rapid evolution. The rate of mutations depends on the mutation type (e.g., A\u2192C, A\u2192G, etc.) and can vary between sites in the viral genome. Understanding this variation can shed light on the mutational processes at play, and is crucial for quantitative modeling of viral evolution. Using the millions of available SARS-CoV-2 full-genome sequences, we estimate rates of synonymous mutations for all 12 possible nucleotide mutation types and examine how much these rates vary between sites. We find a surprisingly high level of variability and several striking patterns: the rates of four mutation types suddenly increase at one of two gene boundaries; the rates of most mutation types strongly depend on a site\u2019s local sequence context, with up to 56-fold differences between contexts; consistent with a previous study, the rates of some mutation types are lower at sites engaged in RNA secondary structure. A simple log-linear model of these features explains \u223c15-60% of the fold-variation of mutation rates between sites, depending on mutation type; more complex models only modestly improve predictive power out of sample. We estimate the fitness effect of each mutation based on the number of times it actually occurs versus the number of times it is expected to occur based on the model. We identify several small regions of the genome where synonymous or noncoding mutations occur much less often than expected, indicative of strong purifying selection on the RNA sequence that is independent of protein sequence. Overall, this work expands our basic understanding of SARS-CoV-2\u2019s evolution by characterizing the virus\u2019s mutation process at the level of individual sites and uncovering several striking mutational patterns that arise from unknown mechanisms.","version":"1.1","doi":"10.1101/2025.01.07.631013","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.04.567060","pub_date":"2025-1-06","title":"Sexual Dimorphism in Systemic Inflammatory Responses to Femur Fracture in Mice Infected with SARS-CoV-2-Like Virus","abstract":"Patients with femur fractures who are concurrently infected with COVID-19 face a threefold increase in mortality, likely due to a compounded inflammatory response. Furthermore, sex-specific differences in immune responses to COVID-19 have been documented, implicating gender as a potential modulator of disease severity in these comorbid conditions. Understanding the inflammatory interplay underlying this association is critical for the development of effective, targeted therapies to mitigate mortality. In this study, we investigated the systemic, sex-specific inflammatory response in mice that sustain a fracture while infected with a murine coronavirus (MHV), which belongs to the same genus as SARS-CoV-2. Our findings reveal that the combined inflammatory incidents of MHV infection and fracture disrupt the systemic immune response in both female and male mice, leading to immune dysregulation characterized by altered cell recruitment and disruption of the normal inflammatory cascade. Notably, the study identifies sex-specific differences in immune response, with female subjects exhibiting significantly elevated levels of inflammatory cytokines, including IL-18 and TNF\u03b1, while males exhibit a diminished response. These sexually dimorphic differences are also reflected in the systemic immune cell populations, suggesting that the quantity of immune factors released may contribute to the observed discrepancies. Notably, these differences were minimal or moderate in animals that either got an MHV infection or fracture alone. Our findings indicate that the overproduction of proinflammatory cytokines, such as IFN\u03b3, IL-18, and TNF\u03b1\u2014reminiscent of cytokine storm syndrome\u2014drives immune dysregulation, exacerbating outcomes in patients with these comorbidities. The observed sex-specific responses may be influenced by factors such as sex hormones, including estrogen, highlighting the importance of considering gender in therapeutic approaches. These insights provide a foundation for the development of tailored interventions to improve outcomes for COVID-19 patients with musculoskeletal trauma, including fractures.","version":"1.2","doi":"10.1101/2023.12.04.567060","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.04.25319877","pub_date":"2025-01-05","title":"Freeze-Drying as a Novel Concentrating Method for Wastewater Detection of SARS-CoV-2","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Detecting viral RNA from wastewater has emerged as a cost-effective approach for community-level surveillance during the recent SARS-CoV-2 pandemic. Although various concentrating methods have been developed, none are optimal for all key requirements for wastewater viral detection. Freeze-drying, a technique widely used for concentrating and preserving biological materials, remains underexplored for this purpose. This study compared the performance of freeze-drying and centrifugal ultrafiltration in terms of recovery efficiency, detection limit, and other key parameters. Early pandemic samples in this study, with extremely low viral concentrations, offered an ideal benchmark to assess their suitability for early-warning applications. Statistical analyses showed that freeze-drying achieved significantly higher recovery efficiency (0.338% \u00b1 0.065% vs. 0.149% \u00b1 0.046%), superior detection ratio (81.6% vs. 36.8%), and lower detection limit (0.06 vs. 0.36 copies/mL) compared to centrifugal ultrafiltration. To our knowledge, this is the first study to apply freeze-drying for wastewater-based viral detection. Despite its longer processing time, freeze-drying offers multiple advantages, including the elimination of pretreatment steps, a flexible workflow, reduced RNA degradation under cryogenic conditions, minimal pathogen exposure, lower labor demands, and less human interference during processing. These features position freeze-drying as a novel alternative for wastewater-based viral surveillance, particularly for decision-making when establishing such systems.</jats:p>\n                <jats:sec>\n                  <jats:title>Synopsis</jats:title>\n                  <jats:p>Freeze-drying is a new wastewater virus concentrating method that outperforms centrifugal ultrafiltration, providing a simpler, safer, and more sensitive approach for community surveillance.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2025.01.04.25319877","journal":"medRxiv","score":null},{"id":"10.1101/2025.01.02.631052","pub_date":"2025-1-03","title":"Broad-Spectrum Antiviral Efficacy of 7-Deaza-7-Fluoro-2\u2019-C-Methyladenosine Against Multiple Coronaviruses In Vitro and In Vivo","abstract":"The Coronaviridae family has been implicated in several major epidemics over the past two decades, including those caused by SARS-CoV, MERS-CoV, and, most recently, SARS-CoV-2. The COVID-19 pandemic, driven by SARS-CoV-2, has led to over seven million deaths worldwide and has been associated with prolonged symptoms, chronic sequelae, and substantial socioeconomic disruptions. The limited availability of effective antiviral treatments, coupled with the ability of coronaviruses to mutate and evade immune defenses, underscores the urgent need for innovative antiviral agents. This study explores the efficacy of the nucleoside analogue DFMA as a potential antiviral agent against multiple Coronaviridae family members, including SARS-CoV-2 and two strains of murine hepatitis viruses (MHV-3 and MHV-A59). In vitro analyses demonstrated that DFMA effectively reduced the viral load in the supernatant of infected cells and enhanced cell viability for both MHV-3 and MHV-A59. Against SARS-CoV-2, DFMA showed a significant reduction in viral load, with a calculated Selectivity Index (SI) of 6.2. In vivo investigations further confirmed the antiviral potential of DFMA. In three distinct murine models\u2014a severe COVID-19 model using MHV-3, a mild COVID-19 model employing MHV-A59, and a transgenic K18-hACE2 mouse model infected with SARS-CoV-2\u2014DFMA administration significantly reduced viral loads in the lungs of infected mice. Additionally, DFMA mitigated inflammatory responses in all models by lowering levels of key inflammatory mediators, such as CXCL1, CCL2, and IL-6. These findings suggest that DFMA possesses broad-spectrum antiviral activity against coronaviruses and may serve as a promising therapeutic candidate for current and future coronavirus outbreaks. Further research is warranted to elucidate its mechanism of action and evaluate its efficacy in clinical settings. Coronaviruses have caused significant outbreaks over the past two decades. Since 2020, COVID-19 has resulted in millions of deaths and lasting global impacts. The limited availability of effective antivirals and the virus\u2019s ability to mutate and evade vaccines and monoclonal antibody therapy emphasize the urgent need for new treatments. This study investigates DFMA, a promising antiviral candidate, targeting SARS-CoV-2 and two related coronaviruses.Our promising results demonstrated significant antiviral activity of DFMA, not only against SARS-CoV-2 but also against other similar coronaviruses, indicating potential future use against COVID-19 and other possible coronavirus-related diseases.","version":"1.1","doi":"10.1101/2025.01.02.631052","journal":"bioRxiv","score":null},{"id":"10.1101/2025.01.01.631001","pub_date":"2025-1-02","title":"Enzyme kinetics model for the coronavirus main protease including dimerization and ligand binding","abstract":"The coronavirus main protease (MPro) plays a pivotal role in viral replication and is the target of several antivirals against SARS-CoV-2. In some species, CRCs of MPro enzymatic activity can exhibit biphasic behavior in which low ligand concentrations activate the enzyme whereas higher ones inhibit it. While this behavior has been attributed to ligand-induced dimerization, quantitative enzyme kinetics models have not been fit to it. Here, we develop a kinetic model integrating dimerization and ligand binding. We perform a Bayesian regression to globally fit the model to multiple types of biochemical and biophysical data. The reversible covalent inhibitor GC376 strongly induces dimerization and binds to the dimer with no cooperativity. In contrast, the fluorescent peptide substrate has a minor effect on dimerization but binds to the dimer with positive cooperativity. The biphasic concentration response curve occurs because compared to substrate, the inhibitor accelerates turnover in the opposite catalytic site.","version":"1.1","doi":"10.1101/2025.01.01.631001","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.31.630868","pub_date":"2025-1-01","title":"Efficient searches in protein sequence space through AI-driven iterative learning","abstract":"Protein sequence space is vast. This fact, together with the prevalence of epistasis, hampers the engineering of novel enzymes through library screening and is a major obstacle to any attempt to predict natural protein evolution. Recently, specialized methodologies have been used to determine fitness data on \u223c260000 sequences for the gene of the enzyme dihydrofolate reductase and antibody affinity data for all combinations of the mutations present in the receptor binding domain (RBD) of the Omicron strain of SARS-CoV-2 (\u223c30000 variants). We show that, upon iterative training on a total of just a few hundred variants, various state-of-the-art AI tools (multi-layer perceptron, random forest and XGBoost algorithms) find very high fitness variants of the enzyme and predict the antibody-evasion patterns of the RBD. This work provides a basis for efficient, widely applicable, low-throughput experimental approaches to assess viral protein evolution and to engineer enzymes for biotechnological applications.","version":"1.1","doi":"10.1101/2024.12.31.630868","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.11.578752","pub_date":"2024-12-30","title":"Pathways in the brain, heart and lung influenced by SARS-CoV-2 NSP6 and SARS-CoV-2 regulated miRNAs: an in silico study hinting cancer incidence","abstract":"The influence of SARS-CoV-2 non-structural protein in the host\u2019s tissue-specific complexities remains a mystery and needs more in-depth attention because of COVID-19 recurrence and long COVID. Here we investigated the influence of SARS-CoV-2 transmembrane protein NSP6 (Non-structural protein 6) in three major organs \u2013 the brain, heart, and lung in silico. To elucidate the interplay between NSP6 and host proteins, we analyzed the protein-protein interaction network of proteins regulated after SARS-CoV-2 infection and that are interacting with NSP6 interacting proteins. Pathway enrichment analyses provided global insights into biological pathways governed by differentially regulated genes in the three tissues after COVID-19 infection. Many drugs targeting hub genes of tissue-specific protein interactome were found that could be candidates for COVID-19 management. MiRNA-gene network for the tissue-specific regulated proteins was also deduced and comparing gene list targeted by SARS-CoV-2 regulated miRNAs, we found three and two common genes in the brain and lung respectively. Among the five common proteins revealed as potential therapeutic targets across the three tissues, Galectin3 (LGALS3) that was upregulated in the heart and brain after COVID-19 infection is reported to be influencing all the ten hallmarks of cancer positively and is found in multiple cancers. COVID-19 infection also causes myocardial inflammation and heart failure (HF). HF is observed to be increasing cancer incidence. Our bioinformatics and systems study hints probable effect of COVID-19 infection in cancer incidence and warrants in-depth studies in this direction and cancer surveillance especially with the present scenario of long COVID-19 and recurrent COVID-19 infections. \n\n","version":"1.2","doi":"10.1101/2024.02.11.578752","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.29.630646","pub_date":"2024-12-30","title":"Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection","abstract":"Viruses exploit host metabolism for replication and immune-regulation. Understanding how SARS-CoV-2 alters the host metabolism may lead to treatments for COVID-19. We report that a ubiquitous gaseous molecule, hydrogen sulfide (H2S), regulates redox, metabolism, and mitochondrial bioenergetics to control SARS-CoV-2. Virus replication is associated with down-regulation of the H2S-producing enzymes cystathionine-\u03b2-synthase (CBS), cystathionine-\u03b3-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (3-MST), resulting in diminished endogenous H2S levels. Inhibition of CTH resulted in SARS-CoV-2 proliferation. A slow-releasing H2S donor, GYY4137, diminished virus replication by inducing Nrf2/Keap1 pathway, restoring redox balance and mitochondrial bioenergetics. Treatment of SARS-CoV-2-infected animals with GYY4137 suppressed viral replication, ameliorated respiratory pathology, and restored antioxidant gene expression. Notably, whole-body plethysmography showed improved pulmonary function variables, including pulmonary obstruction and end-expiratory pause upon GYY4137 treatment in vivo. Data extend our understanding of H2S-mediated regulation of viral-infections, and open new avenues for investigating the pathogenic mechanisms and therapeutic opportunities for coronavirus-associated disorders.","version":"1.1","doi":"10.1101/2024.12.29.630646","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.30.628795","pub_date":"2024-12-30","title":"Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6","abstract":"The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation enhanced host lipid droplet consumption by NSP6 without impacting its known ER-zippering function or double-membrane vesicle morphology. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6\u2019s key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications. As SARS-CoV-2 continues to spread and adapt in humans, viral variants with enhanced spread and immune evasion have emerged throughout the COVID-19 pandemic. While most of the mutations occur in the Spike protein and have been extensively studied, non-Spike mutations have been accumulating and are not as well understood. Here, we constructed Spike-defective genomes of recent Omicron variants and systematically compared their RNA replication capabilities independently from Spike. We also performed single-round infection assays with various Omicron variant Spike proteins to quantify viral entry capabilities. Interestingly, viral entry and RNA replication were negatively correlated, suggesting that as variants evolved reduced entry functions under growing immune pressure on Spike, RNA replication increased as a compensatory mechanism. We focused on a viral protein NSP6 that acquired mutations that significantly enhanced RNA replication. We validated that a frequently accessed L260F mutation in NSP6 enhanced viral infection in cells and increased pathogenesis in mice. While the mutation did not alter NSP6\u2019s role in maintaining a membranous network of viral replication factories, the mutation enhanced viral hijacking of the host lipid droplet machinery. Collectively, we highlight the important role of non-Spike mutations in the evolutionary trajectories of SARS-CoV-2 variants with possible monitoring and therapeutic implications.","version":"1.1","doi":"10.1101/2024.12.30.628795","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.27.630511","pub_date":"2024-12-30","title":"LC-MS/MS characterization of SOBERANA\u00ae02, a receptor binding domain-tetanus toxoid conjugate vaccine against SARS-CoV-2","abstract":"SOBERANA\u00ae02 is a safe and effective conjugate vaccine against SARS-CoV-2, produced using the maleimide-thiol chemistry. In this vaccine, the Cys538 in the recombinant receptor binding domain (RBD) of SARS-CoV-2 is linked, through a thiosuccinimide linker, to lysine residues of tetanus toxoid (TT) preparation. LC-MS/MS analysis revealed that TT is a complex mixture of proteins, similar to other TTs where the detoxified tetanus neurotoxin (d-TeNT) has been shown to be the most abundant protein (30-56%), regardless the quantification method used. The fifteen most abundant proteins account for approximately 78% of the total proteins. LC-MS/MS analysis of the activation process showed that 102 out of 107 lysine residues in the d-TeNT incorporated a maleimide group. In contrast, when tryptic peptides isolated by Ni2+-NTA affinity chromatography, were analyzed by LC-MS/MS, only 22 Lys residues in the d-TeNT were cross-linked to the RBD C-terminal tryptic peptide (538CVNF541-HHHHHH), probably due to steric hindrance. Twelve and eighteen conjugation sites were assigned based on the identification of linear peptides carrying a conjugated lysine residues (\u0394m = +1454.58 Da or \u0394m = +1472.59 Da) and cross-linked peptides with stabilized linker forms, respectively. Eight conjugation sites were coincidently assigned by both strategies. The assignment of the conjugation sites was manually validated by observed regularities (z\u22653+, XIC, immonium ions, specific linker fragment ions) not considered by the identification software (PEAKS, Kojak and pLink2). The RBD was also conjugated, but to a lesser extent, to ten other low abundance carrier proteins. To our knowledge, this work is the first report of conjugation site assignment in a TT-based conjugate vaccine.","version":"1.1","doi":"10.1101/2024.12.27.630511","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.26.630404","pub_date":"2024-12-27","title":"The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice","abstract":"COVID-19 (Coronavirus disease 19) is caused by infection with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in the respiratory system and other organ systems. Tissue injuries resulting from viral infection and host hyperinflammatory responses may lead to moderate to severe pneumonia, systemic complications, and even death. While anti-inflammatory agents have been used to treat patients with severe COVID-19, their therapeutic effects are limited. GPR4 (G protein-coupled receptor 4) is a pro-inflammatory receptor expressed on vascular endothelial cells, regulating leukocyte infiltration and inflammatory responses. In this study, we evaluated the effects of a GPR4 antagonist, NE-52-QQ57, in the SARS-CoV-2-infected K18-hACE2 transgenic mouse model. Our results demonstrated that GPR4 antagonist treatment increased the survival rate in this severe COVID-19 mouse model. The inflammatory response, characterized by proinflammatory cytokines and chemokines, was reduced in the GPR4 antagonist group compared with the vehicle group. Additionally, both SARS-CoV-2 RNA copy numbers and infectious viral titers in the mouse lung were decreased in the GPR4 antagonist group. The percentage of SARS-CoV-2 antigen-positive mouse brains was also decreased in the GPR4 antagonist group compared to the vehicle group. Furthermore, the GPR4 antagonist inhibited SARS-CoV-2 propagation in Vero E6 cells. Together, these results suggest that GPR4 antagonism may be explored as a novel approach for the treatment of COVID-19 and other similar viral diseases.","version":"1.1","doi":"10.1101/2024.12.26.630404","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.27.630350","pub_date":"2024-12-27","title":"Virological and antigenic characteristics of SARS-CoV-2 variants LF.7.2.1, NP.1, and LP.8.1","abstract":"XEC and KP.3.1.1 have surpassed KP.3 to become the globally dominant lineages due to their unique NTD mutations. However, several emerging JN.1 sublineages, such as LF.7.2.1, MC.10.1, NP.1, and, especially, LP.8.1, have demonstrated superior growth advantages compared to XEC. It is critical to access the virological and antigenic characteristics of these emerging SARS-CoV-2 variants. Here, we found that LF.7.2.1 is significantly more immune invasive than XEC, primarily due to the A475V mutation, which enabled the evasion of Class 1 neutralizing antibodies. However, LF.7.2.1\u2019s weak ACE2 binding affinity substantially impaired its fitness. Likewise, MC.10.1 and NP.1 exhibited strong antibody immune evasion due to the A435S mutation, but their limited ACE2 engagement efficiency restricted their growth advantage, suggesting that A435S may regulate the Spike conformation, similar to the NTD glycosylation mutations found in KP.3.1.1 and XEC. Most importantly, we found that LP.8.1 showed comparable humoral immune evasion to XEC but demonstrated much increased ACE2 engagement efficiency, supporting its rapid growth. These findings highlight the trade-off between immune evasion and ACE2 engagement efficiency in SARS-CoV-2 evolution, and underscore the importance of monitoring LP.8.1 and its descend lineages.","version":"1.1","doi":"10.1101/2024.12.27.630350","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.24.629478","pub_date":"2024-12-26","title":"mRNA-1273 vaccines adapted to JN.1 or KP.2 elicit cross-neutralizing responses against the JN.1 sublineages of SARS-CoV-2 in mice","abstract":"The continued diversification of SARS-CoV-2 omicron lineage has given rise to the JN.1 variant and descendant strains (KP.2, KP.3, and XEC) that have prolonged the JN.1 infection wave. JN.1 and KP.2 show decreased susceptibility to neutralization sera in recipients of XBB.1.5 vaccine boosters, supporting the recent authorization of JN.1- and KP.2-matched mRNA vaccines in the United States, Europe, and other regions. We evaluated the immunogenicity of two updated monovalent variant-containing formulations of mRNA-1273 vaccines encoding the spike protein of the omicron subvariants JN.1 (mRNA-1273.167) and KP.2 (mRNA-1273.712) as compared with the monovalent XBB.1.5 vaccine (mRNA-1273.815). The vaccines were administered either as a two-dose primary series in naive mice or as a booster (third) dose in mice previously immunized with two-dose primary series of mRNA-1273 (ancestral strain). The neutralizing antibody response elicited by these vaccines against JN.1 subvariants (KP.3 and LA.2) and the recombinant strain (XEC), which achieved dominance in the United States during late 2024, was evaluated. Primary series immunization with either JN.1- or KP.2-matched vaccine elicited robust neutralizing antibody titers against the matched strains and effectively cross-neutralized KP.3, LA.2, and XEC, but not the antigenically distant XBB.1.5. Similarly, JN.1- and KP.2-matched vaccines administered as a booster (third) dose increased titers against the corresponding strains and JN.1-related subvariants, but not against XBB.1.5. These data suggest these strains are antigenically similar with relatively few spike differences between JN.1 and KP.2/JN.1-related subvariants. Our results demonstrate the potency of JN.1- and KP.2-containing mRNA-1273 vaccines in neutralizing the matched variants and their utility in cross-neutralizing JN.1-related subvariants KP.3, LA.2, and XEC. Taken together, these data suggest that the licensed JN.1 and KP.2 mRNA vaccines are likely to continue to protect against the emerging strains as the JN.1 lineage further evolves.","version":"1.1","doi":"10.1101/2024.12.24.629478","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.18.604213","pub_date":"2024-12-25","title":"Early evolution of BA.2.86 sheds light on the origins of highly divergent SARS-CoV-2 lineages","abstract":"Since the emergence of SARS-CoV-2 in late 2019, highly divergent variants with novel constellations of mutations have periodically emerged to displace previously co-circulating virus lineages. The evolutionary mechanisms behind this process remain unclear, but the prevailing hypothesis supports emergence through single events followed by a rapid accumulation of mutations in individual long-term infections. In July 2023, the Omicron-descending BA.2.86 Variant of Interest (VOI) emerged to outcompete other virus lineages, with descending sublineages still dominating as of December 2024. We use BA.2.86 as a case study to test the hypothesis that highly divergent variants emerge during a single chronic infection. By applying a comprehensive suite of evolutionary analyses to independent high-resolution datasets, we identify a cluster of approximately 100 BA.2 genomes that fall directly ancestral and shorten the branch leading to BA.2.86, carrying some BA.2.86-specific lineage defining mutations. These genomes, sampled from multiple countries and at different timepoints, share a single ancestor dating back to December 2021. Additionally, we detect over 2600 earlier BA.2, BA.2.75, and XBB.1.5 genomes with multiple mutations later associated with BA.2.86. These genomes represent a standing genetic variation that likely facilitated a gradual evolution in the direct ancestry of this variant. We further uncover a complex evolutionary process involving recombination between the BA.2.75 and XBB.1.5 parental lineages. Together, our results challenge the notion of a single emergence event, and instead support a more complex scenario, with BA.2.86 likely having evolved over multiple transmission chains.","version":"1.2","doi":"10.1101/2024.07.18.604213","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.04.578544","pub_date":"2024-12-25","title":"Enhanced Antibody Response to the Conformational Non-RBD Region via DNA Prime-Protein Boost Elicits Broad Cross-Neutralization Against SARS-CoV-2 Variants","abstract":"Preventing immune escape of SARS-CoV-2 variants is crucial in vaccine development to ensure broad protection against the virus. Conformational epitopes beyond the RBD region are vital components of the spike protein but have received limited attention in the development of broadly protective SARS-CoV-2 vaccines. In this study, we used a DNA prime-protein boost regimen to evaluate the broad cross-neutralization potential of immune response targeting conformational non-RBD region against SARS-CoV-2 viruses in mice. Mice with enhanced antibody responses targeting conformational non-RBD region show better performance in cross-neutralization against the Wuhan-01, Delta, and Omicron subvariants. Via analyzing the distribution of conformational epitopes, and quantifying epitope-specific binding antibodies, we verified a positive correlation between the proportion of binding antibodies against the N-terminal domain (NTD) supersite (a conformational non-RBD epitope) and SARS-CoV-2 neutralization potency. The current work highlights the importance of high ratio of conformational non-RBD-specific binding antibodies in mediating viral cross-neutralization and provides new insight into overcoming the immune escape of SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2024.02.04.578544","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.23.630161","pub_date":"2024-12-25","title":"Biomolecular condensates control and are defined by RNA-RNA interactions that arise in viral replication","abstract":"Cells must limit RNA\u2013RNA interactions to avoid irreversible RNA entanglement. Cells may prevent deleterious RNA-RNA interactions by genome organization to avoid complementarity however, RNA viruses generate long, perfectly complementary antisense RNA during replication. How do viral RNAs avoid irreversible entanglement? One possibility is RNA sequestration into biomolecular condensates. To test this, we reconstituted critical SARS-CoV-2 RNA\u2013RNA interactions in Nucleocapsid condensates. We observed that RNAs with low propensity RNA\u2013RNA interactions resulted in more round, liquid-like condensates while those with high sequence complementarity resulted in more heterogeneous networked morphology independent of RNA structure stability. Residue-resolution molecular simulations and direct sequencing-based detection of RNA\u2013RNA interactions support that these properties arise from degree of trans RNA contacts. We propose that extensive RNA\u2013RNA interactions in cell and viral replication are controlled via a combination of genome organization, timing, RNA sequence content, RNA production ratios, and emergent biomolecular condensate material properties.","version":"1.2","doi":"10.1101/2024.12.23.630161","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.08.583965","pub_date":"2024-12-23","title":"Immune Signatures of SARS-CoV-2 Infection Resolution in Human Lung Tissues","abstract":"While human autopsy samples have provided insights into pulmonary immune mechanisms associated with severe viral respiratory diseases, the mechanisms that contribute to a clinically favorable resolution of viral respiratory infections remain unclear due to the lack of proper experimental systems. Using mice co-engrafted with a genetically matched human immune system and fetal lung xenograft (fLX), we mapped the immunological events defining successful resolution of SARS-CoV-2 infection in human lung tissues. Viral infection is rapidly cleared from fLX following a peak of viral replication, histopathological manifestations of lung disease and loss of AT2 program, as reported in human COVID-19 patients. Infection resolution is associated with the activation of a limited number of hematopoietic subsets, including inflammatory monocytes and non-canonical double-negative T-cells with cytotoxic functions, which are highly enriched in viral RNA and dissipate upon infection resolution. Activation of specific human fibroblast and endothelial subsets also elicit robust antiviral and monocyte chemotaxis signatures, respectively. Notably, systemic depletion of human CD4+ cells, but not CD3+ cells, abrogates infection resolution in fLX and induces persistent infection, supporting evidence that peripheral CD4+ monocytes are important contributors to SARS-CoV-2 infection resolution in lung tissues. Collectively, our findings unravel a comprehensive picture of the immunological events defining effective resolution of SARS-CoV-2 infection in human lung tissues, revealing markedly divergent immunological trajectories between resolving and fatal COVID-19 cases.","version":"1.3","doi":"10.1101/2024.03.08.583965","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.20.629131","pub_date":"2024-12-23","title":"LEF1-AS1 deregulation in the peripheral blood of patients with persistent post-COVID symptoms","abstract":"Long COVID denotes the persistence of symptoms after acute SARS-CoV-2 infection lasting for at least two months without another identifiable cause. Affecting an estimated 15% of COVID-19 patients, long COVID manifests in a wide range of symptoms. Despite extensive research on its one-year effects, limited data exist beyond 12 months. Due to the different manifestations of long COVID, its diagnosis can be challenging. Identifying potential mechanistic contributors and biomarkers would be highly valuable. Recent studies have highlighted the potential of noncoding RNAs (ncRNAs) as biomarkers for disease stratification in COVID-19. Specifically, we have recently identified miR-144-3p and a subset of lncRNAs as candidates for assessing disease severity and outcomes in COVID-19. This study extends such investigations to 98 long COVID patients recruited 18 months after hospitalization, exploring the relationship between circulating ncRNA expression and persistent symptoms. While miR-144-3p, HCG18, and lncCEACAM21 expression did not differ between symptomatic and asymptomatic patients, LEF1-AS1 was downregulated in peripheral blood mononuclear cells (PBMCs) of symptomatic patients. Of note, multiple LEF1-AS1 isoforms and LEF1 sense transcript levels were reduced and negatively correlated with relevant clinical markers. While further studies are needed, our discoveries offer new perspectives for the diagnosis and management of persistent long COVID.","version":"1.1","doi":"10.1101/2024.12.20.629131","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.20.629611","pub_date":"2024-12-22","title":"PrimalScheme: open-source community resources for low-cost viral genome sequencing","abstract":"Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, Covid-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses, and has generated over 67,000 primer schemes for the global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online \u223c250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primer schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.","version":"1.1","doi":"10.1101/2024.12.20.629611","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.22.629961","pub_date":"2024-12-22","title":"Structural Insights into the Activation and Inhibition of the ADAM17-iRhom2 Complex","abstract":"The endopeptidase activity of ADAM (a disintegrin and metalloprotease) -17, the primary processor of several EGFR ligands and tumor necrosis factor-alpha (TNF-\u03b1), is essential for proper embryonic development and immune regulation. Dysregulated ADAM17 activity is prevalent in a wide array of human diseases, including cancer, chronic inflammation, and SARS-CoV-2 viral progression. Initially translated as an inactive enzyme zymogen, ADAM17 maturation and enzymatic function are tightly regulated by its obligate binding partners, the inactive rhomboid proteins (iRhom) -1 and -2. Here, we present the cryo-EM structure of the ADAM17 zymogen bound to iRhom2. Our findings elucidate the interactions within the ADAM17-iRhom2 complex, the inhibitory mechanisms of the therapeutic MEDI3622 antibody and ADAM17 prodomain, and the previously unknown role of a membrane-proximal cytoplasmic re-entry loop of iRhom2 involved in the mechanism of activation. Importantly, we perform cellular assays to validate our structural findings and provide further insights into the functional implications of these interactions, paving the way for developing therapeutic strategies targeting this biomedically critical enzyme complex.","version":"1.1","doi":"10.1101/2024.12.22.629961","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.18.629000","pub_date":"2024-12-20","title":"Study of the immunogenicity, efficacy and safety of recombinant RBD SARS-CoV-2 vaccine with CpG adjuvant in rodent, non-rodent and Maccaca fascicularis using Indonesian Strain Virus","abstract":"SARS-CoV-2 is the leading cause of the COVID-19 pandemic that causes acute respiratory syndrome, emerged in late 2019, and was declared a global pandemic on March 11th, 2020. A safe and effective vaccine that prevents SARS-CoV-2 infection or minimize SARS-CoV-2 disease burden is needed. However, in 2021, Low- and middle-income countries (LMICs) face challenges regarding supply of COVID-19 vaccines. Indonesia, as a public sector vaccine manufacturing in developing countries was developed COVID-19 vaccine using a platform based on recombinant subunit proteins, a Receptor Binding Domain (RBD) of SARS-CoV-2 formulated with combination of Alhydrogel and CpG Oligodeoxynucleotides 1018 (CpG). In this study, we report the preclinical study including immunogenicity, toxicity and efficacy of vaccine in animal models. The vaccine immunogenicity tested in mice and non-human primates, the toxicity was done in rodents and non-rodents and challenged study and efficacy was done in non-human primates (NHPs) model. The animal model was vaccinated intramuscularly (IM). The serology result in mice and non-human primates showed significant antibody titers and neutralizing antibody responses compared to the RBD formulations adjuvanted with Alhydrogel only. Safety study in Wistar rats and New Zealand rabbits for single-dose (acute toxicity) and repeated-dose (sub-chronic toxicity) showed no abnormalities in the animal\u2019s organs and behaviors and no deaths were reported in tested animals. Two doses of vaccination have been shown to protect NHPs against SARS-CoV-2 infection, as detected by drastic viral reduction from sample swab in nasal, anal, trachea and nasal wash in 7 days after virus challenged, also viral load measurement from lung and BAL tissue showed negative result, which gave better result than negative control and control vaccine group. No evidence of disease enhancement was observed. These results support clinical development of SARS-CoV-2 vaccine, and in 2022 this vaccine has been approved for emergency use in Indonesia.","version":"1.1","doi":"10.1101/2024.12.18.629000","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.19.629569","pub_date":"2024-12-20","title":"Infectious potential and circulation of SARS-CoV-2 in wild rats","abstract":"Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a wide range of animal species (pets, mink\u2026) have been naturally infected with this betacoronavirus. The emergence of new variants has increased the ability of SARS-CoV-2 to infect species that were not susceptible to the \u201coriginal\u201d SARS-CoV-2, such as mice and rats. This work attempted to evaluate the role of urban rats in the SARS-CoV-2 transmission by combining surveillance studies of rat populations in urban environments, in vivo experimental inoculation of SARS-CoV-2 and comparative viral-receptor interaction in silico analyses. We studied the circulation of SARS-CoV-2 in wild Rattus norvegicus (n=401) captured in urban areas and sewage systems of several French cities. Except for 3 inconclusive samples (2/75 from Bordeaux and 1/261 from Lyon) none of the 353 sera tested showed anti-SARS-CoV-2 antibodies by microsphere immunoassay. However, the 3 inconclusive sera samples were negative by virus neutralisation assay. No SARS-CoV-2 viral RNA was detected in all lungs collected from the 401 captured urban brown rats. In complement, four rat groups (two wild-type colonies, Rattus norvegicus and Rattus rattus, and two laboratory strains, Sprague-Dawley and Wistar) were inoculated with the SARS-CoV-2 Omicron BA.5. At 4 days post-inoculation, no infectious viral particles were detected in the lungs and upper respiratory tract (URT) while viral RNA was detected at a low level only in the URT of all groups. In addition, seroconversion was observed 14 days after inoculation in the four groups. By molecular modelling, the Omicron BA.5 receptor binding domain (RBD) had lower affinities for Rattus norvegicus and Rattus rattus ACE2 than Homo sapiens ACE2. Based on these results the SARS-CoV-2 Omicron BA.5 was unable to infect laboratory and wild type rats. In addition, Rattus norvegicus collected for this study in different areas of France were not infected with SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.12.19.629569","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.10.536311","pub_date":"2024-12-20","title":"Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein","abstract":"Intranasal vaccination is an attractive strategy for preventing COVID-19 disease as it stimulates the production of multimeric secretory immunoglobulin A (IgA), the predominant antibody isotype in the mucosal immune system, at the target site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry. Currently, intranasal vaccine efficacy is evaluated based on the measurement of polyclonal antibody titers in nasal lavage fluid. However, how individual multimeric secretory IgA protects the mucosa from SARS-CoV-2 infection remains to be elucidated. To understand the precise contribution and molecular nature of multimeric secretory IgA induced by intranasal vaccines, we developed 99 monoclonal IgA clones from nasal mucosa and 114 monoclonal IgA or IgG clones from nonmucosal tissues of mice that were intranasally immunized with the SARS-CoV-2 spike protein. The nonmucosal IgA clones exhibited shared origins and common and unique somatic mutations with the related nasal IgA clones, indicating that the antigen-specific plasma cells in the nonmucosal tissues originated from B cells stimulated at the nasal mucosa. Comparing the spike protein binding reactivity, angiotensin-converting enzyme-2-blocking and in vitro SARS-CoV-2 virus neutralization of monomeric and multimeric secretory IgA pairs recognizing different epitopes showed that even non-neutralizing monomeric IgAs, which represents 70% of the nasal IgA repertoire, can protect against SARS-CoV-2 infection when expressed as multimeric secretory IgAs. We also demonstrated that the intranasal administration of multimeric secretory IgA delivered as prophylaxis in the hamster model reduced infection-induced weight loss. Our investigation is the first to demonstrate the function of nasal IgA at the monoclonal level, showing that nasal immunization can provide effective immunity against SARS-CoV-2 by inducing multimeric secretory IgAs at the target site of the virus infection.","version":"1.3","doi":"10.1101/2023.04.10.536311","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.27.582110","pub_date":"2024-12-20","title":"The effect of molnupiravir and nirmatrelvir on SARS-CoV-2 genome diversity in severe models of COVID-19","abstract":"Immunocompromised individuals are susceptible to severe COVID-19 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on SARS-CoV-2 infection in k18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection. Mice were immunosuppressed using cyclophosphamide and infected with a B daughter lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered and viral load and viral sequence diversity was assessed. Treatment of infected but immune compromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion (Ts/Tv) ratio, representative of G>A and C>U mutations and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir. Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristic of variants of concern (VOCs). Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimised public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model.","version":"1.3","doi":"10.1101/2024.02.27.582110","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.19.628950","pub_date":"2024-12-20","title":"Structural Insights into the Assembly and Regulation of 2\u2019-O RNA Methylation by SARS-CoV-2 nsp16/nsp10","abstract":"2\u2019-O-ribose methylation of the first transcribed base (adenine or A1 in SARS-CoV-2) of viral RNA mimics the host RNAs and subverts the innate immune response. How nsp16, with its obligate partner nsp10, assembles on the 5\u2019-end of SARS-CoV-2 mRNA to methylate the A1 has not been fully understood. We present a \u223c 2.4 \u00c5 crystal structure of the heterotetrameric complex formed by the cooperative assembly of two nsp16/nsp10 heterodimers with one 10-mer Cap-1 RNA (product) bound to each. An aromatic zipper-like motif in nsp16 and the N-terminal regions of nsp10 and nsp16 orchestrate an oligomeric assembly for efficient methylation. The front catalytic pocket of nsp16 stabilizes the upstream portion of the RNA while the downstream RNA remains unresolved, likely due to its flexibility. An inverted nsp16 dimer extends the positively charged surface area for longer RNA to influence the catalysis. Additionally, a non-specific nucleotide-binding pocket on the backside of nsp16 plays a critical role in catalysis, further contributing to its enzymatic activity.","version":"1.1","doi":"10.1101/2024.12.19.628950","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.19.629520","pub_date":"2024-12-20","title":"Quantitative Characterization and Prediction of the Binding Determinants and Immune Escape Hotspots for Groups of Broadly Neutralizing Antibodies Against Omicron Variants: Atomistic Modeling of the SARS-CoV-2 Spike Complexes with Antibodies","abstract":"The growing body of experimental and computational studies suggested that the cross-neutralization antibody activity against Omicron variants may be driven by balance and tradeoff of multiple energetic factors and interaction contributions of the evolving escape hotspots involved in antigenic drift and convergent evolution. However, the dynamic and energetic details quantifying the balance and contribution of these factors, particularly the balancing nature of specific interactions formed by antibodies with the epitope residues remain scarcely characterized. In this study, we performed molecular dynamics simulations, ensemble-based deep mutational scanning of SARS-CoV-2 spike residues and binding free energy computations for two distinct groups of broadly neutralizing antibodies : E1 group (BD55-3152, BD55-3546 and BD5-5840) and F3 group (BD55-3372, BD55-4637 and BD55-5514). Using these approaches, we examine the energetic determinants by which broadly potent antibodies can largely evade immune resistance. Our analysis revealed the emergence of a small number of immune escape positions for E1 group antibodies that correspond to R346 and K444 positions in which the strong van der Waals and interactions act synchronously leading to the large binding contribution. According to our results, E1 and F3 groups of Abs effectively exploit binding hotspot clusters of hydrophobic sites critical for spike functions along with selective complementary targeting of positively charged sites that are important for ACE2 binding. Together with targeting conserved epitopes, these groups of antibodies can lead to the expanded neutralization breadth and resilience to antigenic shift associated with viral evolution. The results of this study and the energetic analysis demonstrate excellent qualitative agreement between the predicted binding hotspots and critical mutations with respect to the latest experiments on average antibody escape scores. We argue that E1 and F3 groups of antibodies targeting binding epitopes may leverage strong hydrophobic interactions with the binding epitope hotspots critical for the spike stability and ACE2 binding, while escape mutations tend to emerge in sites associated with synergistically strong hydrophobic and electrostatic interactions.","version":"1.1","doi":"10.1101/2024.12.19.629520","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.05.602305","pub_date":"2024-12-20","title":"Generation of an inflammatory niche in an injectable hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines","abstract":"Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due in part to scalability, adaptability, and potency. Yet there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel depot technology which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available SARS-CoV-2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.","version":"1.3","doi":"10.1101/2024.07.05.602305","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.19.629550","pub_date":"2024-12-20","title":"Tiled Amplicon Sequencing Enables Culture-free Whole-Genome Sequencing of Pathogenic Bacteria From Clinical Specimens","abstract":"Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens.","version":"1.1","doi":"10.1101/2024.12.19.629550","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.16.628601","pub_date":"2024-12-19","title":"Long-term immune changes after COVID-19 and the effect of BCG vaccination and latent infections on disease severity","abstract":"Several years after the COVID-19 pandemic, the role of trained immunity in COVID-19 remains controversial, and questions regarding the long-term effects of COVID-19 on immune cells remain unresolved. We investigated the roles of Bacillus Calmette\u2013Gu\u00e9rin (BCG) vaccination and latent infections in the progression of COVID-19 and sepsis. We conducted a prospective analysis of 97 individuals recovering from mild-to-critical COVID-19 and 64 sepsis patients. Immune cell frequencies, expression of functional markers, and plasma titres of anti-Toxoplasma gondii/cytomegalovirus/BCG antibodies were assessed and their impact on disease severity and outcomes were determined. To examine monocyte responses to secondary challenge, monocytes isolated from COVID-19 convalescent patients, BCG vaccinated and unvaccinated volunteers were stimulated with SARS-CoV-2 and LPS. Post COVID-19 patients showed immune dysregulation regardless of disease severity characterized mainly by altered expression of activation and functional markers in myeloid (CD39, CD64, CD85d, CD11b) and lymphoid cells (CD39, CD57, TIGIT). Strikingly, post-critical COVID-19 patients showed elevated expression of CD57 in CD8+ T cells compared to other severity groups. Additionally, a higher frequency of CMV and T. gondii seropositive-alongside a lower frequency of BCG seropositive-patients were associated with severe and critical COVID-19. However, the monocyte response to stimulation was unaffected by the severity of COVID-19. These findings highlight the long-term alterations of immune cells in post-COVID-19 patients emphasizing the substantial impact of COVID-19 on immune function. However, our data showed no relationship between previous BCG vaccination and protection against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.12.16.628601","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.18.629172","pub_date":"2024-12-19","title":"Tattoo ink induces inflammation in the draining lymph node and impairs the immune response against a COVID-19 vaccine","abstract":"Despite safety concerns regarding the toxicity of tattoo ink, no studies have reported the consequences of tattooing on the immune response. In this work, we have characterized the transport and accumulation of different tattoo inks in the lymphatic system using a murine model. Upon quick lymphatic drainage, we observed that macrophages mainly capture the ink in the lymph node (LN). An initial inflammatory reaction at local and systemic levels follows ink capture. Notably, the inflammatory process is maintained over time as we observed clear signs of inflammation in the draining LN two months following tattooing. In addition, the capture of ink by macrophages was associated with the induction of apoptosis in both human and murine models. Furthermore, the ink accumulated in the LN altered the immune response against a COVID-19 vaccine. We observed a reduced antibody response following vaccination with a mRNA-based SARS-CoV-2 vaccine, which was associated with a decreased expression of the Spike protein in macrophages in the draining LN. Considering the unstoppable trend of tattooing in the population, our results are crucial in informing the toxicology programs, policymakers, and the general public regarding the potential risk of the tattooing practice associated with an altered immune response.","version":"1.1","doi":"10.1101/2024.12.18.629172","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.16.628561","pub_date":"2024-12-16","title":"A novel SARS-CoV-2-derived infectious vector system","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. The development of antiviral drugs for COVID-19 has been hampered by the requirement of a biosafety level 3 (BSL3) laboratory for experiments related to SARS-CoV-2, and by the lack of easy and precise methods for quantification of infection. Here, we developed a SARS-CoV-2 viral vector composed of all four SARS-CoV-2 structural proteins constitutively expressed in lentivirally transduced cells, combined with an RNA replicon deleted for SARS-CoV-2 structural protein genes S, M and E, and expressing a luciferase-GFP fusion protein. We show that, after concentrating viral stocks by ultracentrifugation, the SARS-CoV-2 viral vector is able to infect two human cell lines expressing receptors ACE2 and TMPRSS2. Both luciferase activity and GFP fluorescence were detected, and transduction was remdesivir-sensitive. We also show that this vector is inhibited by three type I interferons (IFN-I) subtypes. Although improvements are needed to increase infectious titers, this vector system may prove useful for antiviral drug screening and SARS-CoV-2-related investigations.","version":"1.1","doi":"10.1101/2024.12.16.628561","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.14.628346","pub_date":"2024-12-16","title":"Highly recurrent multi-nucleotide mutations in SARS-CoV-2","abstract":"Multi-nucleotide mutations (MNMs) simultaneously replace multiple nu-cleotides. They are a significant contributor to evolution and disease, as well as to misdiagnosis, misannotation and other biases in genome data analysis. MNMs are generally thought to be rare and random events. However, by processing millions of publicly shared genomes, we show that certain MNMs are highly recurrent in SARS-CoV-2: they repeatedly and consistently modify the same multiple nucleotides at the same genome position in the same way. The most frequent of these MNMs have independently occurred hundreds of times across all SARS-CoV-2 lineages. The vast majority of these recurrent MNMs are linked to transcription regulatory sequences. We propose a mechanism that explains them through template switching as part of the natural transcription process of the virus. This previously unknown mutational pattern increases our understanding of the evolution of SARS-CoV-2 and potentially many other nidoviruses. It also has important consequences for computational evolutionary biology: we show that for example recurrent MNMs cause approximately 14% of false positives during inference of recombination in SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.12.14.628346","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.23.624482","pub_date":"2024-12-16","title":"Diverse patterns of intra-host genetic diversity in chronically infected SARS-CoV-2 patients","abstract":"In rare individuals with a severely immunocompromised system, chronic infections of SARS-CoV-2 may develop, where the virus replicates in the body for months. Sequencing of some chronic infections has uncovered dramatic adaptive evolution and fixation of mutations reminiscent of lineage-defining mutations of variants of concern (VOCs). This has led to the prevailing hypothesis that VOCs emerged from chronic infections. To examine the mutation dynamics and intra-host genomic diversity of SARS-CoV-2 during chronic infections, we focused on a cohort of nine immunocompromised individuals with chronic infections and performed longitudinal sequencing of viral genomes. We show that sequencing errors may cause erroneous inference of high genetic diversity, and to overcome this we used duplicate sequencing across patients and time-points, allowing us to distinguish errors from low frequency mutations. We further find recurrent low frequency mutations that we flag as most likely sequencing errors. This stringent approach allowed us to reliably infer low frequency mutations and their dynamics across time. We inferred a synonymous divergence rate of the virus of \u223c2\u00d710\u22126 mutations/base/day, consistent with the SARS-CoV-2 mutation rate estimated in tissue culture. The rate of non-synonymous divergence varied widely among the different patients. We highlight two patients with opposing patterns: in one patient the rate of divergence was zero, yet this patient harbored multiple presumably defective viruses at low frequencies throughout the infection. Another patient exhibited dramatic adaptive evolution, including clonal competition. Overall, our results suggest that the emergence of highly divergent variants from chronic infections is likely a very rare event and this emphasizes the need to better understand the conditions that allow such emergence events.","version":"1.2","doi":"10.1101/2024.11.23.624482","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.16.628627","pub_date":"2024-12-16","title":"Hamsters with long Covid exhibits a neurodegenerative signature in the brainstem","abstract":"After infection with SARS-CoV-2, patients may present with one or more symptoms that appear or persist over time, including fatigue, respiratory, cardiovascular and neurological disorders. Neurological symptoms include anxiety, depression and impaired short-term memory. However, the exact underlying mechanisms of long Covid are not yet decrypted. Using the golden hamster as a model, we provide further evidence that SARS-CoV-2 is neuroinvasive and can persist in the central nervous system, as we found viral RNA and replicative virus in the brainstem after 80 days of infection. Infected hamsters presented a neurodegenerative signature in the brainstem, with overexpression of innate immunity genes, impacted dopaminergic and glutamatergic synapses, altered energy metabolism. Finally, the infected hamsters manifested persistent signs of depression and impaired short-term memory, as well as late-onset signs of anxiety, as a valuable model to study long Covid. Conclusively, we provide evidence that virus-related and neurodegenerative and immunometabolic mechanisms coexist in the brainstem of infected hamsters and contribute to the manifestation of neuropsychiatric and cognitive symptoms. SARS-CoV-2 infects and persists in the brainstem of intranasally-inoculated hamsters Persistent neuropsychiatric and cognitive consequences are observed in SARS-CoV-2-infected hamsters The brainstem present distinct transcriptome profiles in acute and in long Covid The dopaminergic and glutamatergic systems are affected in long Covid The SARS-CoV-2 infection affects the expression of genes related to neurodegenerative processes in acute and in long Covid","version":"1.1","doi":"10.1101/2024.12.16.628627","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.12.628247","pub_date":"2024-12-13","title":"Angiotensin peptides enhance SARS-CoV-2 spike protein binding to its host cell receptors","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that caused the Coronavirus Disease 2019 (COVID-19) pandemic, has a spike glycoprotein that is involved in recognizing and fusing to host cell receptors, such as angiotensin-converting enzyme 2 (ACE2), neuropilin-1 (NRP1), and AXL tyrosine-protein kinase. Since the major spike protein receptor is ACE2, an enzyme that regulates angiotensin II (1-8), this study tested the hypothesis that angiotensin II (1-8) influences the binding of the spike protein to its receptors. While angiotensin II (1-8) did not influence spike-ACE2 binding, we found that it significantly enhances spike-AXL binding. Our experiments showed that longer lengths of angiotensin, such as angiotensin I (1-10), did not significantly affect spike-AXL binding. In contrast, shorter lengths of angiotensin peptides, in particular, angiotensin IV (3-8), strongly increased spike-AXL binding. Angiotensin IV (3-8) also enhanced spike protein binding to ACE2 and NRP1. The discovery of the enhancing effects of angiotensin peptides on spike-host cell receptor binding may suggest that these peptides could be pharmacological targets to treat COVID-19 and post-acute sequelae of SARS-CoV-2 (PASC), which is also known as long COVID.","version":"1.1","doi":"10.1101/2024.12.12.628247","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.12.628253","pub_date":"2024-12-13","title":"Histopathological Evaluation of Pulmonary Arterial Remodeling in COVID-19","abstract":"A positive-sense single-stranded RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused the coronavirus disease 2019 (COVID-19) pandemic that devastated the world. While this is a respiratory virus, one feature of the SARS-CoV-2 infection was recognized to cause pathogenesis of other organs. Because the membrane fusion protein of SARS-CoV-2, the spike protein, binds to its major host cell receptor angiotensin-converting enzyme 2 (ACE2) that regulates a critical mediator of cardiovascular diseases, angiotensin II, COVID-19 is largely associated with vascular pathologies. In fact, we have previous reported that postmortem lung tissues collected from patients who died of COVID-19 exhibited thickened pulmonary vascular walls and reduced vascular lumen. The present study extended these findings by further characterizing the pulmonary vasculature of COVID-19 patients using larger sample sizes and providing mechanistic information through histological observations. The examination of 56 autopsy lung samples showed thickened vascular walls of small pulmonary arteries after 14 days of disease compared to H1N1 influenza patients who died before COVID- 19 pandemic started. Pulmonary vascular remodeling in COVID-19 patients was associated with hypertrophy of the smooth muscle layer, perivascular fibrosis, edema and lymphostasis, inflammatory infiltration, perivascular hemosiderosis and neoangiogenesis. We found a correlation between the duration of hospital stay and the thickness of the muscular layer of pulmonary arterial walls. These results further confirm that COVID-19 affects the pulmonary vasculature and warrants an evaluation of patients that survived COVID-19 for possible future development of pulmonary arterial hypertension.","version":"1.1","doi":"10.1101/2024.12.12.628253","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516107","pub_date":"2024-12-13","title":"Activity profiling of deubiquitinating inhibitors-bound to SARS-CoV-2 papain like protease with antiviral efficacy in murine infection model","abstract":"SARS-CoV-2 papain-like protease (PLpro) is a key antiviral target as it plays a dual role in viral replication and in modulation of innate immune responses by deubiquitinating or deISGylating host proteins. Thus, therapeutic targeting of PLpro serves as a two-pronged approach to abate SARS-CoV-2. Interestingly, PLpro shares structural and functional similarities with the cellular deubiquitinating enzymes (DUBs) and in this study this fact has been exploited to identify DUBs inhibitors that target the Ubiquitin/ISG15 binding site and the known catalytic substrate binding pocket of PLpro. Among these identified compounds, flupenthixol, lithocholic acid, teneligliptin, and linagliptin markedly inhibited the proteolytic activity of purified PLpro and demonstrated potent antiviral efficacies against SARS-CoV-2 infection in a dose dependent manner. Treatment with lithocholic acid and linagliptin suppressed the expression levels of inflammatory mediators, thereby, restoring immune responses. Crystal structures of SARS-CoV-2 PLpro in complex with linagliptin and with lithocholic acid determined in this study, revealed insights into the inhibition mechanism with unique interactions within the Ubiquitin/ISG15 binding site (S2 site; Phe69, His73, Asn128, His175) and the substrate binding cleft. Additionally, oral and intraperitoneal treatments with linagliptin increased survival, reduced lung viral load, and ameliorated histopathological damage in mouse-adapted model of SARS-CoV-2 infection. The study for the first time demonstrates a two-pronged strategy using DUB inhibitors that target the proteolytic activity of PLpro and simultaneously reinstates the host\u2019s immune response against SARS-CoV-2.","version":"1.5","doi":"10.1101/2022.11.11.516107","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.12.628214","pub_date":"2024-12-13","title":"SARS-CoV-2 Nsp14 binds Tollip and activates pro-inflammatory pathways while downregulating interferon-\u03b1 and interferon-\u03b3 receptors","abstract":"SARS coronavirus 2 (SARS-CoV-2) non-structural protein 14 (Nsp14) possesses an N-terminal exonuclease (ExoN) domain that provides a proofreading function for the viral RNA-dependent RNA polymerase and a C-terminal N7-methyltransferase (N7-MTase) domain that methylates viral mRNA caps. Nsp14 also modulates host functions. This includes the activation of NF-\u03baB and downregulation of interferon alpha/beta receptor 1 (IFNAR1). Here we demonstrate that Nsp14 exerts broader effects, activating not only NF-\u03baB responses but also ERK, p38 and JNK MAP kinase (MAPK) signaling, promoting cytokine production. Further, Nsp14 downregulates not only IFNAR1 but also IFN-\u03b3 receptor 1 (IFNGR1), impairing cellular responses to both IFN\u03b1 and IFN\u03b3. IFNAR1 and IFNGR1 downregulation is via a lysosomal pathway and also occurs in SARS-CoV-2 infected cells. Analysis of a panel of Nsp14 mutants reveals a consistent pattern. Mutants that disable ExoN function remain active, whereas N7-MTase mutations impair both pro-inflammatory pathway activation and IFN receptor downregulation. Innate immune modulating functions also require the presence of both the ExoN and N7-MTase domains likely reflecting the need for the ExoN domain for N7-MTase activity. We further identify multi-functional host protein Tollip as an Nsp14 interactor. Interaction requires the phosphoinositide-binding C2 domain of Tollip and sequences C-terminal to the C2 domain. Full length Tollip or regions encompassing the Nsp14 interaction domain are sufficient to counteract both Nsp14-mediated and Nsp14-independent activation of NF-\u03baB. Knockdown of Tollip partially reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infected cells, suggesting relevance of Nsp14-Tollip interaction for Nsp14 innate immune evasion functions. SARS-CoV-2 protein Nsp14 both activates NF-\u03baB, which promotes virus replication and inflammation, and downregulates IFNAR1, which can render infected cells resistant to the antiviral effects of IFN-\u03b1/\u03b2. Our study demonstrates that Nsp14 also activates MAPK signaling and downregulates IFNGR1, causing broader impacts than previously recognized. Data from a panel of Nsp14 mutants suggests a common underlying effect of Nsp14 may be responsible for its multiple innate immune activities. We further describe a novel interaction between Nsp14 and Tollip, a selective autophagy receptor. We show that Tollip expression downregulates Nsp14 activation of NF-\u03baB and that Tollip knockdown reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infection, suggesting that Tollip functions as a regulator of Nsp14 innate immune modulation.","version":"1.1","doi":"10.1101/2024.12.12.628214","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.12.628120","pub_date":"2024-12-13","title":"Subtle changes at the RBD/hACE2 interface during SARS-CoV2 variant evolution: a molecular dynamics study","abstract":"The SARS-CoV-2 Omicron variants present a different behavior compared to the previous variants, all particularly in respect to the Delta variant, as it seems to promote a lower morbidity although being much more contagious. In this perspective, we performed new molecular dynamics (MD) simulations of the various spike RBD/hACE2 complexes corresponding to the WT, Delta and Omicron variants (BA.1 up to BA.4/5) over 1.5 \u00b5s timescale. Then, carrying out a comprehensive analysis of residue interactions within and between the two partners, allowed us to draw the profile of each variant by using complementary methods (PairInt, hydrophobic potential, contact PCA). Main results of PairInt calculations highlighted the most involved residues in electrostatic interactions that represent a strong contribution in the binding with highly stable contacts between spike RBD and hACE2 (importance of mutated residues at positions 417, 493 and 498). In addition to the swappable arginine residues (493/498), the apolar contacts made a substantial and complementary contribution in Omicron with the detection of two hydrophobic patches, one of which was correlated with energetic contribution calculations. This study brings new highlights on the global dynamics of spike RBD/hACE2 complexes resulting from the analysis of contact networks and cross-correlation matrices able to detect subtle changes at point mutations. The results of our study are also consistent with alternative approaches such as binding free energy calculations but are more informative and sensitive to transient or low-energy interactions. Nevertheless, the energetic contributions of residues at positions 501 and 505 were in good agreement with hydrophobic interactions measurements. The contact PCA networks could identify the intramolecular incidence of the S375F mutation occurring in all Omicron variants and likely conferring them an advantage in binding stability. Collectively, these data revealed the major differences observed between WT/Delta and Omicron variants at the RBD/hACE2 interface, which may explain the greater persistence of Omicron. The evolution of SARS-CoV-2 was extremely rapid, leading to the global predominance of Omicron variants, despite the many mutations identified in the spike protein. Some of these were introduced to evade the immune system, but many others were located in the Receptor Binding Domain (RBD) without affecting its efficient binding to hACE2 and preserving the high infectivity of this variant. To unravel the mechanism by which this protein-protein connection remains strong or stable, it is necessary to study the different types of interactions at the atomic level and over time using molecular dynamics (MD) simulations. Indeed, in contrast to crystal or cryo-EM structures providing only a fixed image of the binding process, MD simulations have allowed to unambiguously identify the sustainability of some interactions mediated by key residues of spike RBD. This study could also highlight the interchangeable role of certain residues in compensating for a mutation, which in turn allows the virus to maintain durable binding to the host cell receptor.","version":"1.1","doi":"10.1101/2024.12.12.628120","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.11.627986","pub_date":"2024-12-12","title":"A Label-free Nanowell-based Impedance Sensor for Ten-minute SARS-CoV-2 Detection","abstract":"This work explores label-free biosensing as an effective method for biomolecular analysis, ensuring the preservation of native conformation and biological activity. The focus is on a novel electronic biosensing platform utilizing micro-fabricated nanowell-based impedance sensors, offering rapid, point-of-care diagnosis for SARS-CoV-2 (COVID-19) detection. The nanowell sensor, constructed on a silica substrate through a series of microfabrication processes including deposition, patterning, and etching, features a 5\u00d75 well array functionalized with antibodies. Real-time impedance changes within the nanowell array enable diagnostic results within ten minutes using small sample volumes (<5 \u00b5L). The research includes assays for SARS-CoV-2 spike proteins in Phosphate-buffered saline (PBS) and artificial saliva buffers to mimic real human SARS-CoV-2 samples, covering a wide range of concentrations. The sensor exhibits a detection limit of 0.2 ng/mL (1.5 pM) for spike proteins. Middle East Respiratory Syndrome (MERS-CoV) spike proteins are differentiated from SARS-CoV-2 spike proteins, demonstrating specificity.","version":"1.1","doi":"10.1101/2024.12.11.627986","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.11.627892","pub_date":"2024-12-11","title":"Lipid specificity of action of SARS-CoV-2 fusion peptide fragments on model membranes","abstract":"The study focuses on investigating the interaction of SARS-CoV-2 fusion peptide fragment with model membranes of various lipid composition to elucidate the molecular mechanisms of peptide-derived membrane fusion. The work utilized the short fragment of SARS-CoV-2 fusion peptide which is homologous to 816-827 region of the native SARS-CoV-2 FP (FP816-827) and contains the highly conserved LLF motif responsible for membrane fusion, and its ineffective analogue (mFP816-827), where LLF motif was replaced for AAA. Using fluorescence fusion assay, it was demonstrated that the LLF motif plays a key role in inducing liposome fusion, whereas its replacement completely abolishes this capability. The fusogenic activity of the peptide strictly depended on the vesicle lipid composition. It was potentiated by phosphatidylethanolamine and inhibited by phosphatidylserine. Molecular dynamics revealed that both peptides predominantly adopt an \u03b1-helical conformation; however, the native peptide interacts more strongly with the hydrophobic core of the membrane by increasing peptide-lipid hydrophobic contacts, while the mutant version exhibits a more superficial localization. Differential scanning microcalorimetry data indicated that the ability of FP816- 827 to disturb lipid packing increased with decreasing membrane lipid tail length. The molecular mechanisms underlying the fusogenic activity of the SARS-CoV-2 fusion peptide were identified, specifically its ability to cluster phospholipid head groups in its own vicinity. As a result, local regions with positive spontaneous curvature are formed in the outer monolayer, facilitating membrane fusion. The findings highlight the role of membrane composition and lipid architecture in the mechanism of viral fusion with host cells.","version":"1.1","doi":"10.1101/2024.12.11.627892","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.10.626852","pub_date":"2024-12-11","title":"Gut Microbiome Signatures During Acute Infection Predict Long COVID","abstract":"Long COVID (LC), manifests in 10-30% of non-hospitalized individuals post-SARS-CoV-2 infection leading to significant morbidity. The predictive role of gut microbiome composition during acute infection in the development of LC is not well understood, partly due to the heterogeneous nature of disease. We conducted a longitudinal study of 799 outpatients tested for SARS-CoV-2 (380 positive, 419 negative) and found that individuals who later developed LC harbored distinct gut microbiome compositions during acute infection, compared with both SARS-CoV-2\u2013positive individuals who did not develop LC and negative controls with similar symptomatology. However, the temporal changes in gut microbiome composition between the infectious (0\u20131 month) and post-infectious (1\u20132 months) phases was not different between study groups. Using machine learning, we showed that microbiome composition alone more accurately predicted LC than clinical variables. Including clinical data only marginally enhanced this prediction, suggesting that microbiome profiles during acute infection may reflect underlying health status and immune responses thus, help predicting individuals at risk for LC. Finally, we identified four LC symptom clusters, with gastrointestinal and fatigue-only groups most strongly linked to gut microbiome alterations.","version":"1.1","doi":"10.1101/2024.12.10.626852","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.10.627186","pub_date":"2024-12-11","title":"Alignment-Free Viral Sequence Classification at Scale","abstract":"The rapid increase in nucleotide sequence data generated by next-generation sequencing (NGS) technologies demands efficient computational tools for sequence comparison. Alignment-based methods, such as BLAST, are increasingly overwhelmed by the scale of contemporary datasets due to their high computational demands for classification. This study evaluates alignment-free (AF) methods as scalable and rapid alternatives for viral sequence classification, focusing on identifying techniques that maintain high accuracy and efficiency when applied to extremely large datasets. We employed six established AF techniques to extract feature vectors from viral genomes, which were subsequently used to train Random Forest classifiers. Our primary dataset comprises 297,186 SARS-CoV-2 nucleotide sequences, categorized into 3502 distinct lineages. Furthermore, we validated our models using dengue and HIV sequences to demonstrate robustness across different viral datasets. Our AF classifiers achieved 97.8% accuracy on the SARS-CoV-2 test set, and 99.8% and 89.1% accuracy on dengue and HIV test sets, respectively. Despite the high-class dimensionality, we show that word-based AF methods effectively represent viral sequences. Our study highlights the practical advantages of AF techniques, including significantly faster processing compared to alignment-based methods and the ability to classify sequences using modest computational resources.","version":"1.1","doi":"10.1101/2024.12.10.627186","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.09.527884","pub_date":"2024-12-11","title":"Effect of exportin 1/XPO1 nuclear export pathway inhibition on coronavirus replication","abstract":"Nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting the XPO1-mediated nuclear export pathway with selective inhibitors has a diverse effect on virus replication by regulating antiviral, proviral, and anti-inflammatory pathways. The XPO1 inhibitor, Selinexor, is an FDA-approved anticancer drug predicted to have antiviral or proviral functions against viruses. Here, we observed that pretreatment of cultured cell lines from human or mouse origin with nuclear export inhibitor Selinexor significantly enhanced protein expression and replication of Mouse Hepatitis Virus (MHV), a mouse coronavirus. Knockdown of cellular XPO1 protein expression also significantly enhanced the replication of MHV in human cells. However, for SARS-CoV-2, selinexor treatment had diverse effects on virus replication in different cell lines. These results indicate that XPO1-mediated nuclear export pathway inhibition might affect coronavirus replication depending on cell types and virus origin.","version":"1.2","doi":"10.1101/2023.02.09.527884","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.10.627777","pub_date":"2024-12-10","title":"SARITA: A Large Language Model for Generating the S1 Subunit of the SARS-CoV-2 Spike Protein","abstract":"The COVID-19 pandemic has profoundly impacted global health, economics, and daily life, with over 776 million cases and 7 million deaths from December 2019 to November 2024. Since the original SARS-CoV-2 Wuhan strain emerged, the virus has evolved into variants such as Alpha, Beta, Gamma, Delta, and Omicron, all characterized by mutations in the Spike glycoprotein, critical for viral entry into human cells via its S1 and S2 subunits. The S1 subunit, binding to the ACE2 receptor and mutating frequently, affects infectivity and immune evasion; the more conserved S2, on the other hand, facilitates membrane fusion. Predicting future mutations is crucial for developing vaccines and treatments adaptable to emerging strains, enhancing preparedness and intervention design. Generative Large Language Models (LLMs) are becoming increasingly common in the field of genomics, given their ability to generate realistic synthetic biological sequences, including applications in protein design and engineering. Here we present SARITA, an LLM with up to 1.2 billion parameters, based on GPT-3 architecture, designed to generate high-quality synthetic SARS-CoV-2 Spike S1 sequences. SARITA is trained via continuous learning on the pre-existing protein model RITA. When trained on Alpha, Beta, and Gamma variants (data up to February 2021 included), SARITA correctly predicts the evolution of future S1 mutations, including characterized mutations of Delta, Omicron and Iota variants. Furthermore, we show how SARITA outperforms alternative approaches, including other LLMs, in terms of sequence quality, realism, and similarity with real-world S1 sequences. These results indicate the potential of SARITA to predict future SARS-CoV-2 S1 evolution, potentially aiding in the development of adaptable vaccines and treatments.","version":"1.1","doi":"10.1101/2024.12.10.627777","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.10.627775","pub_date":"2024-12-10","title":"Structural and Functional Insights into the Evolution of SARS-CoV-2 KP.3.1.1 Spike Protein","abstract":"The JN.1-sublineage KP.3.1.1 recently emerged as the globally prevalent SARS-CoV-2 variant, demonstrating increased infectivity and antibody escape. We investigated how mutations and a deletion in the KP.3.1.1 spike protein (S) affect ACE2 binding and antibody escape. Mass spectrometry revealed a new glycan site at residue N30 and altered glycoforms at neighboring N61. Cryo-EM structures showed that the N30 glycan and rearrangement of adjacent residues did not significantly change the overall spike structure, up-down ratio of the receptor-binding domains (RBDs), or ACE2 binding. Furthermore, a KP.3.1.1 S structure with hACE2 further confirmed an epistatic effect between F456L and Q493E on ACE2 binding. Our analysis shows SARS-CoV-2 variants that emerged after late 2023 are now incorporating reversions to residues found in other sarbecoviruses, including the N30 glycan, Q493E, and others. Overall, these results inform on the structural and functional consequences of the KP.3.1.1 mutations, the current SARS-CoV-2 evolutionary trajectory, and immune evasion.","version":"1.1","doi":"10.1101/2024.12.10.627775","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.10.627528","pub_date":"2024-12-10","title":"The SARS-CoV-2 envelope PDZ Binding Motif acts as a virulence factor disrupting host\u2019s epithelial cell-cell junctions","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, has significantly impacted global health, emphasizing the need to understand its pathogenicity and virulence mechanisms. SARS-CoV-2 disrupts the alveolar epithelial barrier and exacerbates airway inflammation, leading to acute respiratory failure, but the molecular details remain unclear. Additionally, SARS-CoV-2 infection causes neurological symptoms, potentially due to its weakly understood ability to cross the blood-brain barrier. The viral multifunctional Envelope (E) protein is crucial for its virulence, playing a key role in virus assembly, budding, and release. The E protein contains a PDZ-binding motif (PBM) that interacts with host PDZ domain-containing proteins, potentially affecting host signaling pathways and contributing to pathogenicity. This study focuses on the E protein PBM and its role in virulence, disrupting respiratory epithelial barriers and exacerbating airway inflammation. We generated recombinant mutant viruses lacking the PBM and conducted both in vitro and in vivo experiments to elucidate its impact on viral fitness, pathogenicity, and effects on the epithelial integrity. In vitro, the viral mutants showed delayed replication and reduced cytopathic effects. In vivo, experiments with hamsters revealed that PBM-deficient viruses caused less weight loss, lower viral loads, and reduced inflammation, indicating decreased pathogenicity. Histological analyses confirmed less airway damage in these hamsters compared to those infected with the wild-type virus. Additionally, PBM-deficient viruses had impaired interactions with tight junction proteins like ZO-1, a PDZ-containing protein, crucial for maintaining epithelial barrier integrity. Our findings also demonstrate that the PBM does not play a significant role in neuroinvasion during the acute phase of infection, as evidenced by comparable viral RNA loads across brain regions in infected hamsters, regardless of PBM presence. Histopathological and transcriptomic analyses further support this observation, suggesting that the PBM primarily affects specific epithelial barriers. Additionally, RNA-seq analysis on lung and brainstem from infected hamsters reveals that the PBM modulates inflammatory and immune responses, with a stronger impact in lung tissue than in the brainstem. PBM-deficient viruses induce lower levels of inflammation and cytokine expression, suggesting PBM\u2019s specific role in enhancing viral pathogenicity through the activation of pathways such as NF-\u03baB and TNF. Thus, the E protein PBM plays a critical role in SARS-CoV-2\u2019s fitness, virulence, and pathogenicity, through the disruption of cell junctions and inflammation, underscoring its potential as a target for therapeutic interventions.","version":"1.1","doi":"10.1101/2024.12.10.627528","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.10.627725","pub_date":"2024-12-10","title":"Longitudinal cellular and humoral immune responses following Covid-19 BNT162b2-mRNA-based booster vaccination of craft and manual workers in Qatar","abstract":"In March 2020, the rapid spread of SARS-CoV-2 prompted global vaccination campaigns to mitigate COVID-19 disease severity and mortality. The 2-dose BNT162b2- mRNA vaccine effectively reduced infection and mortality rates, however, waning vaccine effectiveness necessitated the introduction of a third vaccine dose or booster. To assess the magnitude and longevity of booster-induced immunity, we conducted a longitudinal study of SARS-CoV-2 specific cellular and humoral immune responses among Qatar\u2019s vulnerable craft and manual worker community. We also investigated the impact of prior naturally acquired immunity on booster vaccination efficacy. Seventy healthy participants were enrolled in the study, of whom half had prior SARS-CoV-2 infection. Blood samples were collected before and after booster vaccination to evaluate immune responses through SARS- CoV-2 specific ELISpots, IgG ELISA, neutralization assays, and flow cytometric immunophenotyping T cell analysis revealed increased Th1 cellular responses, marked by enhanced IFN-\u03b3 release, in recently infected participants, which was further enhanced by booster vaccination for up to 6-months. Furthermore, booster vaccination stimulated cytotoxic T cell responses in infection-na\u00efve participants, characterized by granzyme B production. Both natural SARS-CoV-2 infection and booster vaccination induced robust and durable SARS-CoV-2 specific humoral immune responses, with high neutralizing antibody levels. Prior natural infection was also linked to an increased number of class- switched B cells prior to booster vaccination. These findings underscore the importance of booster vaccination in enhancing anti-viral immunity across both infection-na\u00efve and previously infected individuals, enhancing distinct arms of the anti-viral immune response and prolonging naturally acquired immunity.","version":"1.1","doi":"10.1101/2024.12.10.627725","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.09.626962","pub_date":"2024-12-10","title":"Differential control of mycobacteria among COVID-19 patients is associated with CD28+ CD8+ T cells","abstract":"Diseases caused by SARS-CoV-2 and Mycobacterium tuberculosis (M.tb) represent two public health emergencies. In severe disease, both pathogens may share a biological niche in the lower respiratory tract. There is significant potential for SARS-CoV-2 and M.tb infections to be co-present within individuals and enhance or moderate the respective outcomes of either infection. Here, we investigated how whole blood samples, as well as CD4+ and CD8+ T cells, from individuals hospitalised with acute COVID-19 disease respond to mycobacterial challenge. To do this, samples were assessed by ex vivo mycobacterial growth inhibition assays, immune cell phenotyping by mass cytometry, and whole blood cytokine responses to mycobacterial antigens assessed by flow cytometry. These studies identified a subgroup of COVID-19 patients whose blood had an enhanced capacity to inhibit mycobacterial growth. The ability to control mycobacterial growth was associated with the presence of a non M.tb-specific CD28+ CD8+ T cell population, with a particular activation status and migratory phenotype. This work improves our understanding of factors involved in mycobacterial control, and may contribute to the design of novel therapies for TB.","version":"1.1","doi":"10.1101/2024.12.09.626962","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.08.627411","pub_date":"2024-12-10","title":"Orchestration of SARS-CoV-2 Nsp4 and host-cell ESCRT proteins induces morphological changes of the endoplasmic reticulum","abstract":"Upon entry into the host cell, the non-structural proteins 3, 4, and 6 (Nsp3, Nsp 4, and Nsp6) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate the formation of double- membrane vesicles (DMVs) through extensive rearrangement of the host cell endoplasmic reticulum (ER) to replicate the viral genome and translate viral proteins. To dissect the functional roles of each Nsp and the molecular mechanisms underlying the ER changes, we exploited both yeast S. cerevisiae and human cell experimental systems. Our results demonstrate that Nsp4 alone is sufficient to induce ER structural changes. Nsp4 expression led to robust activation of both the unfolded protein response (UPR) and the ER surveillance (ERSU) cell cycle checkpoint, resulting in cortical ER inheritance block and septin ring mislocalization. Interestingly, these ER morphological changes occurred independently of the canonical UPR and ERSU components but were mediated by the endosomal sorting complex for transport (ESCRT) proteins Vps4 and Vps24 in yeast. Similarly, ER structural changes occurred in human cells upon Nsp4 expression, providing a basis for a minimal experimental system for testing the involvement of human ESCRT proteins and ultimately advancing our understanding of DMV formation.","version":"1.1","doi":"10.1101/2024.12.08.627411","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.09.627570","pub_date":"2024-12-10","title":"Accelerated amyloid deposition in SARS-CoV-2 infected mouse models of Alzheimer\u2019s disease","abstract":"Familial Alzheimer\u2019s disease (AD) involving known AD causing genes accounts for a small fraction of cases, the vast majority are sporadic. Neuroinflammation, secondary to viral infection, has been suggested as an initiating or accelerating factor. In this work we tested the hypothesis that SARS-CoV-2 (SCV2) viral infection accelerates the development of AD pathology in mouse models of AD. We profiled transcriptomic changes using transgenic APP/PSEN1 and P301S mouse models that develop AD pathology and k18hACE2 mice that express the humanized ACE2 receptor used by SCV2 to enter cells. This study identified the interferon and chemokine responses constituting key shared pathways between SCV2 infection and the development of AD pathology. Two transgenic mouse models of AD: APP/PSEN1 (develops amyloid pathology) and 3xTg AD (develops both amyloid and tau pathology) were crossed with k18-hACE2 mice to generate hybrid hACE2-3xTg and hACE2-APP/PSEN1 mice. Neuroinflammation and amyloid deposition in the brain of infected mice were imaged in vivo using molecular MRI (mMRI) probes and confirmed postmortem by histopathology. Results show that 11-14-month-old SCV2 infected hACE2-3xTg mice exhibit neuroinflammation 10 days post infection and 4\u20135-month-old hACE2-APP/PS1 hybrid mice develop amyloid deposits, while age-matched uninfected mice exhibit neither phenotype. This suggests that SCV2 infection could induce or accelerate AD when risk factors are present.","version":"1.1","doi":"10.1101/2024.12.09.627570","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.06.627164","pub_date":"2024-12-09","title":"Comprehensive Analysis of SARS-CoV-2 Spike Evolution: Epitope Classification and Immune Escape Prediction","abstract":"The evolution of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has produced unprece-dented numbers of structures of the Spike protein. This study presents a comprehensive analysis of 1,560 published Spike protein structures, capturing most variants that emerged throughout the pandemic and covering diverse heteromerization and interacting complexes. We employ an interaction-energy informed geometric clustering to identify 14 epitopes characterized by their conformational specificity, shared interface with ACE2 binding, and glycosylation patterns. Our per-residue interaction evaluations accurately predict each residue\u2019s role in antibody recognition and as well as experimental measurements of immune escape, showing strong correlations with DMS data, thus making it possible to predict the behaviour of future variants. We integrate the structural analysis with a longitudinal analysis of nearly 3 million viral sequences. This broad-ranging structural and longitudinal analysis provides insight into the effect of specific mutations on the energetics of interactions and dynamics of the SARS-CoV-2 Spike protein during the course of the pandemic. Specifically, with the emergence of widespread immunity, we observe an enthalpic trade-off in which mutations in the receptor binding motif (RBM) that promote immune escape also weaken the interaction with ACE2. Additionally, we also observe a second mechanism, that we call entropic trade-off, in which mutations outside of the RBM contribute to decrease the occupancy of the open state of SARS-CoV-2 Spike, thus also contributing to immune escape at the expense of ACE2 binding but without changes on the ACE2 binding interface. This work not only highlights the role of mutations across SARS-CoV-2 Spike variants but also reveals the complex interplay of evolutionary forces shaping the evolution of the SARS-CoV-2 Spike protein over the course of the pandemic.","version":"1.1","doi":"10.1101/2024.12.06.627164","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.11.617898","pub_date":"2024-12-09","title":"Identification and Targeting of Regulators of SARS-CoV-2-Host Interactions in the Airway Epithelium","abstract":"Although the impact of SARS-CoV-2 in the lung has been extensively studied, the molecular regulators and targets of the host-cell programs hijacked by the virus in distinct human airway epithelial cell populations remain poorly understood. This is in part ascribed to the use of nonprimary cell systems, overreliance on single-cell gene expression profiling that does not ultimately reflect protein activity, and bias toward the downstream effects rather than their mechanistic determinants. Here we address these issues by network-based analysis of single cell transcriptomic profiles of pathophysiologically relevant human adult basal, ciliated and secretory cells to identify master regulator (MR) protein modules controlling their SARS-CoV-2-mediated reprogramming. This uncovered chromatin remodeling, endosomal sorting, ubiquitin pathways, as well as proviral factors identified by CRISPR analyses as components of the host response collectively or selectively activated in these cells. Large-scale perturbation assays, using a clinically relevant drug library, identified 11 drugs able to invert the entire MR signature activated by SARS-CoV-2 in these cell types. Leveraging MR analysis and perturbational profiles of human primary cells represents a novel mechanism-based approach and resource that can be directly generalized to interrogate signatures of other airway conditions for drug prioritization.","version":"1.2","doi":"10.1101/2024.10.11.617898","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.06.625234","pub_date":"2024-12-09","title":"Defining a highly conserved B cell epitope in the receptor binding motif of SARS-CoV-2 spike glycoprotein","abstract":"SARS-CoV-2 mRNA vaccines induce robust and persistent germinal centre (GC) B cell responses in humans. It remains unclear how the continuous evolution of the virus impacts the breadth of the induced GC B cell response. Using ultrasound-guided fine needle aspiration, we examined draining lymph nodes of nine healthy adults following bivalent booster immunization. We show that 77.8% of the B cell clones in the GC expressed as representative monoclonal antibodies recognized the spike protein, with a third (37.8%) of these targeting the receptor binding domain (RBD). Strikingly, only one RBD-targeting mAb, mAb-52, neutralized all tested SARS- CoV-2 strains, including the recent KP.2 variant. mAb-52 utilizes the IGHV3-66 public clonotype, protects hamsters challenged against the EG.5.1 variant and targets the class I/II RBD epitope, closely mimicking the binding footprint of ACE2. Finally, we show that the remarkable breadth of mAb-52 is due to the somatic hypermutations accumulated within vaccine-induced GC reaction. Booster SARS-CoV-2 mRNA vaccine recruits and broadens GC B cell responses targeting a highly conserved site on receptor binding domain of spike glycoprotein.","version":"1.1","doi":"10.1101/2024.12.06.625234","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.24.563704","pub_date":"2024-12-09","title":"Repertoire-based mapping and time-tracking of helper T cell subsets in scRNA-Seq","abstract":"The functional programs selected by CD4+ helper (Th) T cell clones fundamentally determine the architecture of the immune response to distinct challenges. Advances in scRNA-Seq have enhanced our understanding of the diversity of these programs, yet the correspondence between scRNA-Seq clusters and previously characterized Th subsets remains unclear. In this study, we use immune repertoires to position phenotypically sorted Th subsets within scRNA-Seq data from three healthy donors. This approach, termed TCR-Track, and accurately maps Th1, Th1-17, Th17, Th22, Th2a, Th2, Tfh, and Treg subsets, outperforming CITE-Seq-based mapping. Remarkably, the mapping is tightly focused on specific scRNA-Seq clusters despite a four-year interval between the sorting of subsets and the effector CD4+ scRNA-Seq experiment. Thus, while transient T cell plasticity is commonly observed in functionally active T cell populations, TCR-Track reveals high intrinsic program sustainability of Th clones circulating in peripheral blood. Repertoire overlap analysis at the scRNA-Seq level confirms that circulating Th1, Th2, Th2a, Th17, Th22, and Treg subsets are clonally independent. However, a prominent clonal overlap between corresponding clusters indicates that cytotoxic CD4+ T cells differentiate from Th1 clones. More specifically, we demonstrate that sorted CCR10+ Th cells correspond to a specific Th22 scRNA-Seq cluster, while CCR10-CCR6+CXCR3-CCR4+ cells, traditionally sorted as the Th17 subset, represent a mixture of bona fide Th17 and clonally unrelated CCR10low Th22 cells, which may have confounded investigators in previous studies. This clear distinction of Th17 and Th22 subsets should influence vaccine and T cell based therapies development. Additionally, we show that SARS-CoV-2 infection is associated with transient IFN type 1 activation of naive CD4+ T cells, and an increased proportion of effector IFN- induced Th cells is associated with a moderate course of the disease but remains low in critical COVID-19 cases. Using integrated scRNA-Seq, TCR-Track, and CITE-Seq data from 122 donors, we provide a comprehensive Th scRNA-Seq reference that should facilitate further investigation of Th subsets in fundamental and clinical studies.","version":"1.2","doi":"10.1101/2023.10.24.563704","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.06.627182","pub_date":"2024-12-09","title":"Constant-pH MD Simulations of Lipids","abstract":"Constant pH Molecular Dynamics (CpHMD) simulations represent a cutting-edge computational approach for studying biological systems with remarkable realism. Recent advancements have enhanced the accessibility and efficiency of CpHMD, significantly reducing the performance overhead compared to traditional constant-protonation MD simulations. This chapter guides the reader through the application of CpHMD to investigate the pH-dependent behavior of Cationic Ionizable Lipids (CILs) \u2014 a critical component of Lipid Nanoparticles (LNPs), which are among the most promising platforms for drug delivery. LNPs, including those employed in mRNA-based vaccines, played a pivotal role in the global response to the SARS-CoV-2 pandemic, underscoring their potential in modern medicine. The chapter begins with a comprehensive introduction to the fundamental concepts of LNPs and provides a step-by-step protocol for setting up simulations of membranes containing CILs to calculate their apparent pKa. This parameter is crucial for governing the in vivo behavior of LNPs, where precise control is essential to optimize delivery efficiency while minimizing toxicity. By showcasing the ability of CpHMD simulations to unravel the intricate relationship between pH-dependent protonation, membrane structure, and lipid distribution, this chapter highlights their potential to inform the rational design of novel LNP formulations.","version":"1.1","doi":"10.1101/2024.12.06.627182","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.05.626967","pub_date":"2024-12-06","title":"SARS-CoV-2 predation of Golgi-bound PI4P primes the massive activation of the DNA Damage Response kinase ATM in the cytoplasm","abstract":"Like all viruses, SARS-CoV-2, the causative agent of COVID-19, relies on host cell resources to replicate. Our study reveals that, among these resources, SARS-CoV-2 hijacks the oxysterol-binding protein 1 (OSBP1) transporter to exploit the Golgi-bound phosphatidylinositol-4-phosphate (PI4P) pool. This leads to a depletion of Golgi-resident PI4P, triggering the activation of the ATM DNA Damage Response (DDR) kinase in the cytoplasm. As such, ATM, typically anchored to PI4P at the Golgi in an inactive state, undergoes auto-phosphorylation and cytoplasmic release upon SARS-CoV-2-induced PI4P depletion. Conversely, pharmacological inhibition of ATM auto-phosphorylation, which stabilizes its interaction with PI4P, significantly impairs SARS-CoV-2 replication. The requirement for PI4P and impact of ATM inhibition might be conserved across coronaviruses, as similar effects were observed with HCoV-229E. Finally, SARS-CoV-2-induced, cytoplasmic ATM pre-activation primes cells for an accelerated response to DNA damage, which might contribute to the severe outcomes of COVID-19 observed in cancer patients undergoing chemo- or radiotherapy. Therefore, this study uncovers a DNA damage-independent mode of ATM activation and highlights the potential of ATM inhibitors as therapeutic agents against COVID-19.","version":"1.1","doi":"10.1101/2024.12.05.626967","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.05.627124","pub_date":"2024-12-06","title":"Capture of fusion-intermediate conformations of SARS-CoV-2 spike requires receptor binding and cleavage at either the S1/S2 or S2\u2019 site","abstract":"Although the structures of pre- and post-fusion conformations of SARS-CoV-2 spikes have been solved by cryo-electron microscopy, the transient spike conformations that mediate virus fusion with host cell membranes remain poorly understood. In this study, we used a peptide fusion inhibitor corresponding to the heptad repeat 2 (HR2) in the S2 transmembrane subunit of the spike to investigate fusion-intermediate conformations that involve exposure of the highly conserved heptad repeat 1 (HR1). The HR2 peptide disrupts the assembly of the HR1 and HR2 regions of the spike, which form six-helix bundle during the transition to the post-fusion conformation. We show that binding of the spike S1 subunit to ACE2 is sufficient to trigger conformational changes that allow the peptide to capture a fusion-intermediate conformation of S2 and inhibit membrane fusion. When TMPRSS2 is also present, an S2\u2019 fusion intermediate is captured though the proportion of the S2\u2019 intermediate relative to the S2 intermediate is lower in Omicron variants than pre-Omicron variants. In spikes lacking the natural S1/S2 furin cleavage site, ACE2 binding alone is not sufficient for trapping fusion intermediates; however, co-expression of ACE2 and TMPRSS2 allows trapping of an S2\u2019 intermediate. These results indicate that, in addition to ACE2 engagement, at least one spike cleavage is needed for unwinding S2 into an HR2-sensitive fusion-intermediate conformation. Our findings elucidate fusion-intermediate conformations of SARS-CoV-2 spike variants that expose conserved sites on spike that could be targeted by inhibitors or antibodies. The SARS-CoV-2 spike protein undergoes two proteolytic cleavages and major conformational changes that facilitate fusion between viral and host membranes during virus infection. Spike is cleaved to S1 and S2 subunits during biogenesis, and S2 is subsequently cleaved to S2\u2019 as the virus enters host cells. While structures of pre-fusion and post-fusion spike conformations have been extensively studied, transient fusion-intermediate conformations during the fusion process are less well understood. Here, we use a peptide fusion inhibitor corresponding to a heptad repeat domain in the S2 subunit to investigate fusion-inducing conformational changes. During spike-mediated cell-cell fusion, we show that the peptide binds to spike only after spike engages ACE2 and is cleaved at the S1/S2, S2\u2019, or both sites. Thus, S2 needs at least one cleavage to refold to a peptide-sensitive fusion intermediate. SARS-CoV-2 variants differed in the proportion of S2 and S2\u2019 fusion intermediates captured after receptor binding, indicating that the virus has evolved not only to alter its entry pathway but also to modulate S2 unfolding. This work informs the development of antiviral strategies targeting conserved sites in fusion-intermediate conformations of spike and contributes more broadly to the understanding of the entry mechanisms of viral fusion proteins.","version":"1.1","doi":"10.1101/2024.12.05.627124","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.08.602485","pub_date":"2024-12-05","title":"Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants","abstract":"During the COVID-19 pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here we report an analysis of 164 serum samples isolated from breakthrough infection patients in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wildtype, Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping using charged scanning mutagenesis coupled to yeast surface display and Next Generation Sequencing. All of these sera primarily targeted the recently identified class 5 cryptic epitope along with the class 1 and class 4 epitopes, targeted to a lesser extent. The class 5 epitope is completely conserved across all SARS-CoV-2 variants and for the majority of the Sarbecoviruses. In line with these observations, a major fraction of the serum samples, including the selected three, show broad neutralizing activity against recent Omicron subvariants including XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variant of concerns was observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population was sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern, thus paves the path towards the development of future vaccine candidates. Worldwide implementation of COVID19 vaccine and parallel emergence of newer SARS-CoV-2 variants has shaped the humoral immune response in a population specific manner. While characterization of this immune response is important for monitoring the disease progression at population level, it is also imperative for the development of effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the world\u2019s second largest COVID19 vaccination drive and also encountered large number of post vaccination \u201cbreakthrough\u201d infections. From a cohort of breakthrough infection patients, we identified individuals whose sera showed broadly cross-reactive immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope which was not identified in previous population level studies conducted in western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants including the recently emerged ones and also for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.","version":"1.2","doi":"10.1101/2024.07.08.602485","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.03.626676","pub_date":"2024-12-04","title":"Widespread gene-environment interactions shape the immune response to SARS-CoV-2 infection in hospitalized COVID-19 patients","abstract":"Genome-wide association studies performed in patients with coronavirus disease 2019 (COVID-19) have uncovered various loci significantly associated with susceptibility to SARS-CoV-2 infection and COVID-19 disease severity. However, the underlying cis-regulatory genetic factors that contribute to heterogeneity in the response to SARS-CoV-2 infection and their impact on clinical phenotypes remain enigmatic. Here, we used single-cell RNA-sequencing to quantify genetic contributions to cis-regulatory variation in 361,119 peripheral blood mononuclear cells across 63 COVID-19 patients during acute infection, 39 samples collected in the convalescent phase, and 106 healthy controls. Expression quantitative trait loci (eQTL) mapping across cell types within each disease state group revealed thousands of cis-associated variants, of which hundreds were detected exclusively in immune cells derived from acute COVID-19 patients. Patient-specific genetic effects dissipated as infection resolved, suggesting that distinct gene regulatory networks are at play in the active infection state. Further, 17.2% of tested loci demonstrated significant cell state interactions with genotype, with pathways related to interferon responses and oxidative phosphorylation showing pronounced cell state-dependent variation, predominantly in CD14+ monocytes. Overall, we estimate that 25.6% of tested genes exhibit gene-environment interaction effects, highlighting the importance of environmental modifiers in the transcriptional regulation of the immune response to SARS-CoV-2. Our findings underscore the importance of expanding the study of regulatory variation to relevant cell types and disease contexts and argue for the existence of extensive gene-environment effects among patients responding to an infection.","version":"1.1","doi":"10.1101/2024.12.03.626676","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.26.624714","pub_date":"2024-12-04","title":"The identification of a SARs-CoV2 S2 protein-derived peptide with super-antigen-like stimulatory properties on T-cells","abstract":"Severe COVID-19 can trigger a cytokine storm, leading to acute respiratory distress syndrome (ARDS) with similarities to superantigen-induced toxic shock syndrome. An outstanding question is whether SARS-CoV-2 protein sequences can directly induce inflammatory responses. In this study, we identify a region in the SARS-CoV-2 S2 spike protein with sequence homology to bacterial super-antigens (termed P3). Computational modeling predicts P3 binding to sites on MHC class I/II and the TCR that partially overlap with sites for the binding of staphylococcal enterotoxins B and H. Like SEB and SEH peptides, P3 stimulated 25-40% of human CD4+ and CD8+ T cells, increasing IFN-\u03b3 and granzyme B production. viSNE and SPADE profiling identified overlapping and distinct IFN-\u03b3 and GZMB subsets. The super-antigenic properties of P3 were further evident by its selective expansion of T cells expressing specific TCR V\u03b1 and V\u03b2 chain repertoires. In vivo experiments in mice revealed that the administration of P3 led to a significant upregulation of proinflammatory cytokines IL-1\u03b2, IL-6, and TNF-\u03b1. While the clinical significance of P3 in COVID-19 remains unclear, its homology to other mammalian proteins suggests a potential role for this peptide family in human inflammation and autoimmunity.","version":"1.3","doi":"10.1101/2024.11.26.624714","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.03.625388","pub_date":"2024-12-04","title":"An accelerating, decreasing phylogenetic trend in SARS-CoV-2 genome compositional heterogeneity during the pandemic","abstract":"The rapid evolution of SARS-CoV-2 during the pandemic, driven by a plethora of mutations, many of which enable the virus to evade host resistance, has likely altered its genome\u2019s compositional structure (i.e. the arrangement of compositional domains of varying lengths and nucleotide frequencies within the genome). To explore this hypothesis, we summarize the evolutionary effects of these mutations by computing the Sequence Compositional Complexity (SCC) in random datasets of fully sequenced genomes. Phylogenetic ridge regression of SCC against time reveals a striking downward evolutionary trend, as well as an increasing rate of change, suggesting the ongoing adaptation of the virus\u2019s genome structure to the human host. Other genomic features, such as strand asymmetry, the effective number of K-mers, and the depletion of CpG dinucleotides, each linked to the virus\u2019s adaptation to its human host, also exhibit decreasing phylogenetic trends over the course of the pandemic, along with strong phylogenetic correlations to SCC. Overall, our findings suggest an accelerated, genome-wide evolutionary trend toward a more symmetric and homogeneous genome compositional structure in SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.12.03.625388","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.02.626472","pub_date":"2024-12-03","title":"KP.2-based monovalent mRNA vaccines robustly boost antibody responses to SARS-CoV-2","abstract":"In response to the ongoing evolution of SARS-CoV-2, COVID-19 mRNA vaccines were recently updated to encode the spike protein of the KP.2 subvariant of the JN.1 sublineage. However, the immunogenicity of KP.2-based monovalent mRNA vaccines (KP.2 MV) has yet to be fully evaluated and reported, particularly against dominant and growing viral variants KP.3.1.1 and XEC, which bear some distinct mutations from KP.2. Here we report that KP.2 MV boosters elicit robust neutralizing antibody titers in a cohort of 16 healthy adult participants against all tested variants in pseudovirus neutralization assays. The highest post-boost geometric mean titers were against older variants D614G (17,293) and BA.5 (14,358), suggestive of immune imprinting, but the post-boost titers against currently dominant or growing viruses KP.3.1.1 (1,698) and XEC (1,721) were still robust. Fold-changes in titers were highest against recent JN.1 subvariants, including JN.1, KP.2, KP.3, KP.3.1.1, and XEC, (5.8-to-7.8-fold), compared to older variants D614G and BA.5 (1.6- and 2.5-fold), which suggests that KP.2 MV boosters have at least partially mitigated immune imprinting. Overall, these results show that KP.2 MV boosters elicit robust neutralizing antibodies against dominant SARS-CoV-2 viruses.","version":"1.1","doi":"10.1101/2024.12.02.626472","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.13.618110","pub_date":"2024-12-03","title":"B cells imprinted by ancestral SARS-CoV-2 develop pan-sarbecovirus neutralization in immune recalls","abstract":"A key question on ancestral SARS-CoV-2 immune imprinting is to what extent imprinted B cells can develop neutralizing breadth and potency in immune recalls. Here, we longitudinally tracked B cells recognizing wild-type spike in two individuals, who were sequentially infected by Omicron variants after receiving mRNA vaccines. Functional and genetic analysis of 632 monoclonal antibodies (mAbs) from those B cells reveals that mAbs cloned after second infection have dramatically enhanced neutralizing breadth and potency, which is attributed to recall and maturation of pre-existing memory B cells. Among the 11 mAbs that potently neutralize SARS-CoV-2 variants from wild-type to KP.3, 5 mAbs are classified into public clonotypes encoded by IGHV3-53 or IGHV3-66, whereas the rest belong to a rarely reported clonotype encoded by IGHV3-74. Notably, IGHV3-74 mAbs can also broadly neutralize other sarbecoviruses by targeting a novel epitope on receptor-binding domain of spike. These results support that ancestral SARS-CoV-2 immune imprinting can be harnessed in developing pan-SARS-CoV-2 and even pan-sarbecovirus vaccines. Chen et al. demonstrate that B cells imprinted by ancestral SARS-CoV-2 have tremendous potential to develop neutralizing breadth and potency in repeated immune recalls driven by Omicron variants, implicating that ancestral SARS-CoV-2 immune imprinting can be harnessed in developing pan-SARS-CoV-2 and even pan-sarbecovirus vaccines.","version":"1.2","doi":"10.1101/2024.10.13.618110","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.02.626375","pub_date":"2024-12-02","title":"Environmental exposures and familial background alter the induction of neuropathology and inflammation after SARS-CoV-2 infection","abstract":"Basal ganglia disease has been reported as a post-infection sequela of several viruses, with documentation of this phenomenon from the H1N1 Spanish flu to the recent COVID-19 (SARS-CoV-2) pandemic. SARS-CoV-2 infection leads to multisystem deficits, including those affecting the nervous system. Here, we investigated whether a SARS-CoV-2 infection alone increases the susceptibility to develop parkinsonian phenotypes in C57BL/6J mice expressing the human ACE2 receptor, or in addition to two well-known toxin exposures, MPTP and paraquat. Additionally, we examined mice carrying a G2019S mutation in the LRRK2 gene. We also examined if vaccination with either an mRNA- or protein-based vaccine can alter any observed neuropathology. We find that the infection with the WA-1/2020 (alpha) or omicron B1.1.529 strains in ACE2 and G2019S LRRK2 mice both synergize with a subtoxic exposure to the mitochondrial toxin MPTP to induce neurodegeneration and neuroinflammation in the substantia nigra. This synergy appears toxin-dependent since we do not observe this following exposure to the direct redox-inducing compound paraquat. This synergistic neurodegeneration and neuroinflammation is rescued in WT mice that were vaccinated using either mRNA- and protein- based vaccines directed against the Spike protein of the SARS-CoV-2 virus. However, in the G2019S LRRK2 mutant mice, we find that only the protein-based vaccine but not the mRNA- based vaccine resulted in a rescue of the SARS-CoV-2 mediated neuropathology. Taken together, our results highlight the role of both environmental exposures and familial background on the development of parkinsonian pathology secondary to viral infection and the benefit of vaccines in reducing these risks.","version":"1.1","doi":"10.1101/2024.12.02.626375","journal":"bioRxiv","score":null},{"id":"10.1101/2024.12.02.626213","pub_date":"2024-12-02","title":"Dynamic ensembles of SARS-CoV-2 N-protein reveal head-to-head coiled-coil-driven oligomerization and phase separation","abstract":"The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral lifecycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo liquid-liquid phase separation (LLPS) is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein\u2019s dynamic self-assembly. We show that the N-protein\u2019s interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that the IDL exists in a concentration-dependent equilibrium between monomeric, dimeric, and trimeric states. The CC motif facilitates parallel, head-to-head oligomerization of N-protein dimers, transitioning between compact (closed) and extended (open) configurations depending on the interaction network within the IDL. This linker-driven assembly modulates LLPS, impacting the size, stability, and dynamics of biomolecular condensates. Here, we present the most comprehensive conformational landscape analysis of the N-protein to date, providing a detailed model of its self-assembly and LLPS. Our findings highlight how the structural plasticity of the IDL and CC-mediated interactions are pivotal to its roles in the SARS-CoV-2 lifecycle.","version":"1.1","doi":"10.1101/2024.12.02.626213","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.19.585703","pub_date":"2024-11-30","title":"Concepts and methods for predicting viral evolution","abstract":"The seasonal human influenza virus undergoes rapid evolution, leading to significant changes in circulating viral strains from year to year. These changes are typically driven by adaptive mutations, particularly in the antigenic epitopes, the regions of the viral surface protein haemagglutinin targeted by human antibodies. Here we describe a consistent set of methods for data-driven predictive analysis of viral evolution. Our pipeline integrates four types of data: (1) sequence data of viral isolates collected on a worldwide scale, (2) epidemiological data on incidences, (3) antigenic characterization of circulating viruses, and (4) intrinsic viral phenotypes. From the combined analysis of these data, we obtain estimates of relative fitness for circulating strains and predictions of clade frequencies for periods of up to one year. Furthermore, we obtain comparative estimates of protection against future viral populations for candidate vaccine strains, providing a basis for pre-emptive vaccine strain selection. Continuously updated predictions obtained from the prediction pipeline for influenza and SARS-CoV-2 are available on the website previr.app.","version":"1.3","doi":"10.1101/2024.03.19.585703","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.28.625932","pub_date":"2024-11-29","title":"Unraveling Microglial Spatial Organization in the Developing Human Brain with DeepCellMap, a Deep Learning Approach Coupled to Spatial Statistics","abstract":"Mapping cellular organization in the developing brain presents significant challenges due to the multidimensional nature of the data, characterized by complex spatial patterns that are difficult to interpret without high-throughput tools. We developed DeepCellMap, a deep-learning-assisted tool that integrates multi-scale image processing with advanced spatial and clustering statistics. This pipeline was designed to map microglial organization during normal and pathological brain development but can be adapted to any cell type. Using DeepCellMap, we capture the morphological diversity of microglia, identify strong coupling between proliferative and phagocytic phenotypes, and show that distinct spatial clusters rarely overlap as human brain development progresses. Additionally, we uncover a novel association between microglia and blood vessels in fetal brains exposed to maternal SARS-CoV-2. These findings offer insights into whether various microglial phenotypes form networks in the developing brain to occupy space, and in conditions involving haemorrhages, whether microglia respond to, or influence changes in blood vessel integrity. DeepCellMap is available as open-source software and is a powerful tool for extracting spatial statistics and analyzing cellular organization in large tissue sections, accommodating various imaging modalities. This platform could open new avenues for studying brain development and related pathologies.","version":"1.1","doi":"10.1101/2024.11.28.625932","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.27.625579","pub_date":"2024-11-28","title":"The IL-33/ST2 signaling axis drives pathogenesis in acute SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remains a significant threat to global public health. Immunopathological damage plays a role in driving pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan failure in severe COVID-19. Therefore, dissecting the pulmonary immune response to SARS-CoV-2 infection is critical to understand disease pathogenesis and identify immune pathways targetable by therapeutic intervention. Considering that the type 2 cytokine IL-13 enhances COVID-19 disease severity, therapeutic targeting of upstream signals that drive type 2 immunity may confer further protection. In this study, we investigate the role of the IL-33/ST2 signaling axis, a potent inducer of type 2 immunity in the lung, in a mouse model of COVID-19. Upon infection with mouse-adapted SARS-CoV-2 MA10, ST2-/- mice had significantly improved weight loss and survival (69.2% vs 13.3% survival; P = 0.0005), as compared to wild-type mice. In a complementary pharmacologic approach, IL-33/ST2 signaling was inhibited using HpBARI_Hom2, a helminth derived protein that binds to mouse ST2 and blocks IL-33 signaling. In SARS-CoV-2 MA10 infection, HpBARI_Hom2-treated mice had significantly improved weight loss and survival (60% vs 10% survival; P = 0.0035), as compared to inert control-treated mice. These data demonstrate that loss of IL-33/ST2 signaling confers protection during acute SARS-CoV-2 MA10 infection, implicating the IL-33/ST2 signaling axis as an enhancer of COVID-19 disease severity. The protection conferred by pharmacologic blockade of IL-33/ST2 signaling was independent of viral control, as HpBARI_Hom2-treated mice had no reduction in viral titers. This finding suggests an immunopathogenic role for IL-33/ST2 signaling. One potential mechanism through which IL-33/ST2 signaling may drive severe disease is through enhancement of type 2 immune pathways including IL-5 production, as pulmonary IL-5 concentrations were found to depend on IL-33/ST2 signaling in acute SARS-CoV-2 MA10 infection.","version":"1.1","doi":"10.1101/2024.11.27.625579","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.26.625543","pub_date":"2024-11-27","title":"Systemic exposure to COVID-19 virus-like particles modulates firing patterns of cortical neurons in the living mouse brain","abstract":"Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) causes a systemic infection that affects the central nervous system. We used virus-like particles (VLPs) to explore how exposure to the SARS-CoV-2 proteins affects brain activity patterns in wild-type (WT) mice and in mice that express the wild-type human tau protein (htau mice). VLP exposure elicited dose-dependent changes in corticosterone and distinct chemokine levels. Longitudinal two-photon microscopy recordings of primary somatosensory and motor cortex neurons that express the jGCaMP7s calcium sensor tracked modifications of neuronal activity patterns following exposure to VLPs. There was a substantial short-term increase in stimulus-evoked activity metrics in both WT and htau VLP-injected mice, while htau mice showed also increased spontaneous activity metrics and increase activity in the vehicle-injected group. Over the following weeks, activity metrics in WT mice subsided, but remained above baseline levels. For htau mice, activity metrics either remain elevated or decreased to lower levels than baseline. Overall, our data suggest that exposure to the SARS-CoV-2 VLPs leads to strong short-term disruption of cortical activity patterns in mice with long-term residual effects. The htau mice, which have a more vulnerable genetic background, exhibited more severe pathobiology that may lead to more adverse outcomes.","version":"1.1","doi":"10.1101/2024.11.26.625543","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.06.552160","pub_date":"2024-11-26","title":"SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection","abstract":"Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), nearly 20% of hospitalized patients are at risk for thromboembolic events. This prothrombotic state is considered a key factor in the increased risk of stroke, which is observed clinically during both acute infection and long after symptoms clear. Here we develop a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells (ECs), pericytes (PCs), and smooth muscle cells (SMCs) to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly SMCs, are a susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterize the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, PCs, and ECs. We observe that infected SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we show human ECs exposed to the secretome of infected SMCs produce hemostatic factors that contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level.","version":"1.2","doi":"10.1101/2023.08.06.552160","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.26.625333","pub_date":"2024-11-26","title":"Association between COVID-19 Disease Severity and T Cell Receptor Repertoire","abstract":"During the COVID-19 pandemic, while most infected individuals experienced mild to moderate symptoms, a significant subset developed severe illness. A clinical test distinguishing between mild and severe cases could inform effective treatment strategies. Toward the latter stages of the pandemic, it became evident that vaccination or prior infection cannot entirely prevent reinfection. However, they are crucial in reducing the risk of severe disease by inducing T-cell memory. T cell receptors (TCRs), which can be obtained from human blood, serve as valuable biomarkers for monitoring T cell responses to SARS-CoV-2 infection. In this study, we investigated the associations between TCR metrics and COVID-19 severity and found significant associations. Furthermore, such associations could depend on the subset of TCRs used (e.g., TCRs from CD8+ T or CD4+ T cells) and when the TCRs were collected.","version":"1.1","doi":"10.1101/2024.11.26.625333","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.22.624893","pub_date":"2024-11-25","title":"Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine","abstract":"The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5\u2019 RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.","version":"1.1","doi":"10.1101/2024.11.22.624893","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.23.624996","pub_date":"2024-11-25","title":"Naturally occurring N-terminal mutations in SARS-CoV-2 nsp1 impact innate immune modulation but do not affect virus virulence","abstract":"The non-structural protein 1 (nsp1) of SARS-CoV-2 plays a key role in host innate immune evasion. We identified two deletion variants (\u039482-85 and \u039483-86) in the N-terminal region of the nsp1 of a SARS-CoV-2 BA.5.2.1 variant recovered from a human patient. Analysis of the sequence databases revealed a frequency of 0.5% of these mutations amongst available SARS-CoV-2 sequences. Structural analysis of the deletion mutant nsp1\u039482-85 and nsp1\u039483-86 revealed a distortion in the protein pocket when compared to the wild-type nsp1 which may affect protein function. To evaluate the functional relevance of these mutations, we cloned the mutant BA.5.2.1 nsp1\u039482-85 and nsp1\u039483-86 and wild-type nsp1 proteins in expression plasmids and performed luciferase reporter-based assays to assess activation of the interferon and nuclear factor kappa B (NF-\u03baB) signalling pathways. Both nsp1\u039482-85 and nsp1\u039483-86 mutants showed marked decreased ability to inhibit the interferon beta (IFN-\u03b2) and NF-\u03baB pathway activation. To assess the relevance of these deletions in the context of SARS-CoV-2 infection, we generated recombinant viruses carrying the wild type BA.5.2.1 nsp1 or the BA.5.2.1 nsp1\u039482-85 and nsp1\u039483-86 deletions in the backbone of WA1 strain. In vitro characterization of the recombinant SARS-CoV-2 viruses revealed that the recombinant viruses containing the nsp1\u039482-85 and nsp1\u039483-86 deletions presented similar plaque size and morphology to those produced by the wild-type rWA1-BA.5.2.1-nsp1 virus, indicating a similar ability of the mutant viruses to spread from cell to cell. Importantly, pathogenesis studies revealed that these mutations did not affect virus virulence and pathogenesis in a hamster model of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.11.23.624996","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.06.515367","pub_date":"2024-11-25","title":"Mathematical Modeling of Impacts of Patient Differences on Renin-Angiotensin System and Applications to COVID-19 Lung Fibrosis Outcomes","abstract":"Patient-specific premorbidity, age, and sex are significant heterogeneous factors that influence the severe manifestation of lung diseases, including COVID-19 fibrosis. The renin-angiotensin system (RAS) plays a prominent role in regulating the effects of these factors. Recent evidence shows patient-specific alterations of RAS homeostasis concentrations with premorbidity and the expression level of angiotensin-converting enzyme 2 (ACE2) during COVID-19. However, conflicting evidence suggests decreases, increases, or no changes in RAS peptides after SARS-CoV-2 infection. In addition, detailed mechanisms connecting the patient-specific conditions before infection to infection-induced RAS alterations are still unknown. Here, a multiscale computational model was developed to quantify the systemic contribution of heterogeneous factors of RAS during COVID-19. Three submodels were connected\u2014an agent-based model for in-host COVID-19 response in the lung tissue, a RAS dynamics model, and a fibrosis dynamics model to investigate the effects of patient-group-specific factors in the systemic alteration of RAS and collagen deposition in the lung. The model results indicated cell death due to inflammatory response as a major contributor to the reduction of ACE and ACE2. In contrast, there were no significant changes in ACE2 dynamics due to viral-bound internalization of ACE2. The model explained possible mechanisms for conflicting evidence of patient-group-specific changes in RAS peptides in previously published studies. Simulated results were consistent with reported RAS peptide values for SARS-CoV-2-negative and SARS-CoV-2-positive patients. RAS peptides decreased for all virtual patient groups with aging in both sexes. In contrast, large variations in the magnitude of reduction were observed between male and female virtual patients in the older and middle-aged groups. The patient-specific variations in homeostasis RAS peptide concentrations of SARS-CoV-2-negative patients also affected the dynamics of RAS during infection. The model results also showed that feedback between RAS signaling and renin dynamics could restore ANGI homeostasis concentration but failed to restore homeostasis values of RAS peptides downstream of ANGI. In addition, the results showed that ACE2 variations with age and sex significantly altered the concentrations of RAS peptides and led to collagen deposition with slight variations depending on age and sex. This model may find further applications in patient-specific calibrations of tissue models for acute and chronic lung diseases to develop personalized treatments.","version":"1.4","doi":"10.1101/2022.11.06.515367","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.22.624784","pub_date":"2024-11-25","title":"Unraveling SARS-CoV-2 Host-Response Heterogeneity through Longitudinal Molecular Subtyping","abstract":"Hospitalized COVID-19 patients exhibit diverse immune responses during acute infection, which are associated with a wide range of clinical outcomes. However, understanding these immune heterogeneities and their links to various clinical complications, especially long COVID, remains a challenge. In this study, we performed unsupervised subtyping of longitudinal multi-omics immunophenotyping in over 1,000 hospitalized patients, identifying two critical subtypes linked to mortality or mechanical ventilation with prolonged hospital stay and three severe subtypes associated with timely acute recovery. We confirmed that unresolved systemic inflammation and T-cell dysfunctions were hallmarks of increased severity and further distinguished patients with similar acute respiratory severity by their distinct immune profiles, which correlated with differences in demographic and clinical complications. Notably, one critical subtype (SubF) was uniquely characterized by early excessive inflammation, insufficient anticoagulation, and fatty acid dysregulation, alongside higher incidences of hematologic, cardiac, and renal complications, and an elevated risk of long COVID. Among the severe subtypes, significant differences in viral clearance and early antiviral responses were observed, with one subtype (SubC) showing strong early T-cell cytotoxicity but a poor humoral response, slower viral clearance, and greater risks of chronic organ dysfunction and long COVID. These findings provide crucial insights into the complex and context-dependent nature of COVID-19 immune responses, highlighting the importance of personalized therapeutic strategies to improve both acute and long-term outcomes.","version":"1.1","doi":"10.1101/2024.11.22.624784","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.27.581995","pub_date":"2024-11-24","title":"Secondary structure of the SARS-CoV-2 genome is predictive of nucleotide substitution frequency","abstract":"Accurate estimation of the effects of mutations on SARS-CoV-2 viral fitness can inform public-health responses such as vaccine development and predicting the impact of a new variant; it can also illuminate biological mechanisms including those underlying the emergence of variants of concern (Carabelli et al., 2023). Recently, Lan et al. reported a model of SARS-CoV-2 secondary structure and its underlying dimethyl sulfate (DMS) reactivity data (Lan et al., 2022). I investigated whether base reactivities and secondary structure models derived from them can explain some variability in the frequency of observing different nucleotide substitutions across millions of patient sequences in the SARS-CoV-2 phylogenetic tree. Nucleotide basepairing was compared to the estimated \u201cmutational fitness\u201d of substitutions, a measurement of the difference between a substitution\u2019s observed and expected frequency that is correlated with other estimates of viral fitness (Bloom and Neher, 2023). This comparison revealed that secondary structure is often predictive of substitution frequency, with significant decreases in substitution frequencies at basepaired positions. Focusing on the mutational fitness of C\u2192U, the most common type of substitution, I describe C\u2192U substitutions at basepaired positions that characterize major SARS-CoV-2 variants; such mutations may have a greater impact on fitness than appreciated when considering substitution frequency alone.","version":"1.2","doi":"10.1101/2024.02.27.581995","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.21.624622","pub_date":"2024-11-22","title":"SARS-CoV-2 infection in hiPSC-derived neurons is cathepsin-dependent and causes accumulation of HIF1\u0251 and phosphorylated tau","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to infect the human brain and a subset of human neurons in vitro. We have previously demonstrated that the virus enters the human induced pluripotent stem cell (hiPSC)-derived neurons via an endosomal-lysosomal pathway, which is dependent on low levels of angiotensin-converting enzyme 2 (ACE2) and independent of transmembrane serine protease 2 (TMPRSS2). Here, we use hiPSC-derived neurons overexpressing ACE2 in co-culture with human astrocytes to show that the infection with both SARS-CoV-2 Wuhan and Omicron XBB.1.5 variants is dependent on cathepsins and can be efficiently blocked by an inhibitor of cathepsin B (CA-074-ME). The result was reproducible in non-transgenic hiPSC-derived cortical organoids. The cathepsin L inhibitor SB412515 was less effective against the Wuhan strain but equally effective against the Omicron variant. Using PCR and reinfection assays, we show that SARS-CoV-2 can replicate in neurons in 2D co-cultures. Interestingly, the infectivity of the newly produced virions declined at 24 hours post-infection despite a further increase in released viral RNA at later time points, suggesting the possible activation of an antiviral response in neurons and/or astrocytes, which is supported by a correspondent increase in the levels of secreted cytokines. Furthermore, the number of infected neurons decreased within five days, suggesting that SARS-CoV-2 infection eventually leads to the death of the target neuronal cell in vitro. The infection also caused the accumulation of the hypoxia-inducible stress factor HIF1-\u03b1 in infected neurons under normoxia. Finally, we confirm and expand the previous finding that in SARS-CoV-2 infected neurons, the microtubule-associated protein tau is hyperphosphorylated at multiple loci, including S202/T205, and mislocalized to the soma of the infected neurons. Hyperphosphorylation and mislocalization of tau are hallmarks of Alzheimer\u2019s disease (AD) and other \u2018tauopathies\u2019. Our data provides further evidence supporting the neurodegenerative potential of SARS-CoV-2 infection. The recent COVID-19 pandemic has raised concerns about the potential for SARS-CoV-2 to infect the brain and worsen brain diseases like Alzheimer\u2019s disease. Research has shown that SARS-CoV-2 can indeed infect the human brain, including a small number of neurons and other brain cells in laboratory settings. In our previous studies, we identified the endosomal pathway as the route the virus uses to enter neuronal cells. In this study, we build on that work by demonstrating that inhibitors of endo-lysosomal cathepsin proteases can block this neuronal infection. We also found that infectious progeny virions are released from the infected neuronal cells. Importantly, the infection proves harmful to the host cells, as evidenced by a decrease in the number of infected cells in experimental cultures over a five-day period. Additionally, we confirm and expand on earlier findings that SARS-CoV-2 infection leads to the phosphorylation and altered localization of the tau protein, a process associated with brain diseases like Alzheimer\u2019s. Finally, we observed an increase in the production of inflammatory cytokines following neuronal infection with SARS-CoV-2, along with an accumulation of the stress marker protein HIF-1\u03b1 in neurons. This protein has been linked to other viral infections and Alzheimer\u2019s disease. Overall, our data suggest that SARS-CoV-2 exhibits neurodegenerative characteristics.","version":"1.1","doi":"10.1101/2024.11.21.624622","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.20.624003","pub_date":"2024-11-21","title":"The nucleocapsid (N) proteins of different human coronaviruses demonstrate a variable capacity to induce the formation of cytoplasmic condensates","abstract":"To date, seven human coronaviruses (HCoVs) have been identified. Four of these viruses typically manifest as a mild respiratory disease, whereas the remaining three can cause severe conditions that often result in death. The reasons for these differences remain poorly understood, but may be related to the properties of individual viral proteins. The nucleocapsid (N) protein plays a crucial role in the packaging of viral genomic RNA and the modification of host cells during infection, in part due to its capacity to form dynamic biological condensates via liquid-liquid phase separation (LLPS). In this study, we investigated the capacity of N proteins derived from all HCoVs to form condensates when transiently expressed in cultured human cells. A fraction of the transfected cells were observed to contain cytoplasmic granules in which the most of the N proteins were accumulated. Notably, the N proteins of SARS-CoV and SARS-CoV-2 showed a significantly reduced tendency to form cytoplasmic condensates. The condensate formation was not a consequence of overexpression of N proteins, as is typical for LLPS-inducing proteins. These condensates contained components of stress granules (SGs), indicating that the expression of N proteins caused the formation of SGs, which integrate N proteins. Thus, the N proteins of two closely related viruses, SARS-CoV and SARS-CoV-2, have the capacity to antagonize SG induction and/or assembly, in contrast to all other known HCoVs.","version":"1.1","doi":"10.1101/2024.11.20.624003","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.03.587973","pub_date":"2024-11-20","title":"Within-host genetic diversity of SARS-CoV-2 across animal species","abstract":"Infectious disease transmission to different host species makes eradication very challenging and expands the diversity of evolutionary trajectories taken by the pathogen. Since the beginning of the ongoing COVID-19 pandemic, SARS-CoV-2 has been transmitted from humans to many different animal species, within which viral variants of concern could potentially evolve. Previously, using available whole genome consensus sequences of SARS-CoV-2 from four commonly sampled animals (mink, deer, cat, and dog) we inferred similar numbers of transmission events from humans to each animal species. Using a genome-wide association study (GWAS), we identified 26 single nucleotide variants (SNVs) that tend to occur in deer \u2013 more than any other animal \u2013 suggesting a high rate of viral adaptation to deer. The reasons for this rapid adaptive evolution remain unclear, but within-host evolution \u2013 the ultimate source of the viral diversity that transmits globally \u2013 could provide clues. Here we quantify intra-host SARS-CoV-2 genetic diversity across animal species and show that deer harbor more intra-host SNVs (iSNVs) than other animals, providing a larger pool of genetic diversity for natural selection to act upon. Mixed infections involving more than one viral lineage are unlikely to explain the higher diversity within deer. Rather, a combination of higher mutation rates, longer infections, and species-specific selective pressures are likely explanations. Combined with extensive deer-to-deer transmission, the high levels of within-deer viral diversity help explain the apparent rapid adaptation of SARS-CoV-2 to deer.","version":"1.2","doi":"10.1101/2024.04.03.587973","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.20.624471","pub_date":"2024-11-20","title":"Robust antiviral humoral immunity induced by JN.1 monovalent mRNA vaccines against a broad range of SARS-CoV-2 Omicron subvariants including JN.1, KP.3.1.1 and XEC","abstract":"As of November 2024, SARS-CoV-2 Omicron JN.1 subvariants, such as KP.2 (JN.1.11.1.2), KP.3 (JN.1.11.1.3), KP.3.1.1 (JN.1.11.1.3.1.1), and XEC \u2014 a recombinant lineage between KS.1.1 (JN.13.1.1.1) and KP.3.3 (JN.1.11.1.3.3) \u2014 have been circulating in several countries. To control the infection with SARS-CoV-2 Omicron JN.1 subvariants, JN.1 monovalent mRNA vaccines have been developed. Some previous reports showed that the JN.1 monovalent mRNA vaccine of Pfizer/BioNTech (US/Germany) increased antiviral humoral immunity against JN.1 subvariants and XEC. However, the efficacy of other available JN.1 monovalent mRNA vaccines (e.g., Daiichi-Sankyo, Japan) remains unassessed. To validate the antiviral efficacy induced by JN.1 mRNA vaccines, sera were collected from individuals vaccinated with Pfizer/BioNTech JN.1 mRNA vaccine (N=15) or Daiichi-Sankyo JN.1 mRNA vaccine (N=19) before and 3-4 weeks after vaccination. We then performed a neutralization assay using these sera and pseudoviruses. Both Pfizer/BioNTech JN.1 vaccine (2.4-to 8.0-fold, P=0.0001) and Daiichi-Sankyo JN.1 vaccine (2.3-to 13-fold, P=0.0001) boosted antiviral humoral immunity against all variants tested with statistical significance. While the Pfizer/BioNTech mRNA vaccine encodes the full-length JN.1 spike (S), the Daiichi-Sankyo mRNA vaccine encodes the receptor-binding domain of JN.1 S. Our data suggest that the receptor-binding domain of JN.1 S can effectively induce antiviral humoral immunity against JN.1 subvariants and XEC comparable to the full-length JN.1 S. However, it should be considered that the sizes of our cohorts are relatively small (<20 donors per cohort), and donor characteristics, such as age, sex, underlying disease status, and previous SARS-CoV-2 infection, may critically affect the experimental results. Future investigations with larger cohorts will address this concern. When compared to vaccination with JN.1 mRNA vaccines, our previous investigations showed that the natural infection of JN.1 and KP.3.3 elicited poorer antiviral humoral immunity against JN.1 and its subvariants. Our results suggest that the JN.1 mRNA vaccination more robustly induces antiviral humoral immunity against recent JN.1 subvariants than the natural infection of JN.1 subvariants regardless of manufacturer. Moreover, as we reported last year, the humoral immunity induced by XBB.1.5 monovalent mRNA vaccine against XBB.1.5 was weaker than that against ancestral B.1.1. However, in the case of JN.1 monovalent mRNA vaccine, here we showed that the 50% neutralization titer against XBB.1.5 is greater than that against ancestral B.1.1. These observations imply that immune imprinting has shifted from that biased toward pre-Omicron to that biased toward Omicron, depending on the time and/or number of immune stimuli (e.g., infection and/or vaccination).","version":"1.1","doi":"10.1101/2024.11.20.624471","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.18.623742","pub_date":"2024-11-19","title":"Low levels of neutralizing antibodies against SARS-CoV-2 KP.3.1.1 and XEC in serum from seniors in May 2024","abstract":"New immune evasive variants of SARS-CoV-2 may increase infections and hospitalizations in risk groups, such as the elderly. In this study we evaluated neutralizing antibodies against KP.3.1.1 and XEC, virus variants that are either widely distributed or on the rise globally, in sera from a cohort of seniors aged 68 - 82 years from April/May 2024. Neutralizing responses were low against both KP.3.1.1 and XEC, supporting the recommendation of an updated covid-19 vaccine booster in this age group.","version":"1.1","doi":"10.1101/2024.11.18.623742","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.19.623967","pub_date":"2024-11-19","title":"Scalable cell-free production of active T7 RNA polymerase","abstract":"The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a one-liter cell-free gene expression (CFE) reaction that achieves over 80% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags and agitation to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than 4 orders of magnitude reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.","version":"1.1","doi":"10.1101/2024.11.19.623967","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.06.543855","pub_date":"2024-11-18","title":"Re-annotation of SARS-CoV-2 proteins using an HHpred-based approach opens new opportunities for a better understanding of this virus","abstract":"Since the publication of the genome of SARS-CoV-2 \u2013 the causative agent of COVID-19 \u2013 in January 2020, many bioinformatic tools have been applied to annotate its proteins. Although effcient methods have been used, such as the identification of protein domains stored in Pfam, most of the proteins of this virus have no detectable homologous protein domains outside the viral taxa. As it is now well established that some viral proteins share similarities with proteins of their hosts, we decided to explore the hypothesis that this lack of homologies could be, at least in part, the result of the documented loss of sensitivity of Pfam Hidden Markov Models (HMMs) when searching for domains in \u201cdivergent organisms\u201d. In order to improve the annotation of SARS-CoV-2 proteins, we used the HHpred protein annotation tool. To avoid \u201cfalse positive predictions\u201d as much as possible, we designed a robustness procedure to evaluate the HHpred results. In total, 6 robust similarities involving 6 distinct SARS-CoV-2 proteins were detected. Of these 6 similarities, 3 are already known and well documented, and one is in agreement with recent crystallographic results. We then examined carefully the two similarities that have not yet been reported in the literature. We first show that the C-terminal part of Spike S (the protein that binds the virion to the cell membrane by interacting with the host receptor, triggering infection) has similarities with the human prominin-1/CD133; after reviewing what is known about prominin-1/CD133, we suggest that the C-terminal part of Spike S could both improve the docking of Spike S to ACE2 (the main cell entry receptor for SARS-CoV-2) and be involved in the delivery of virions to regions where ACE2 is located in cells. Secondly, we show that the SARS-CoV-2 ORF3a protein shares similarities with human G protein-coupled receptors (GPCRs), such as Lutropin-choriogonadotropic hormone receptor, primarily belonging to the \u201cRhodopsin family\u201d. To further investigate these similarities, we compared Prominin 1 and Lutropin-choriogonadotropic hormone receptor to a set of viral proteins using HHPRED. Interestingly, Prominin 1 showed similarities with 6 viral Spike glycoproteins, primarily from coronaviruses. Equally interestingly, Lutropin-choriogonadotropic hormone receptor showed similarities with 23 viral G-protein coupled receptors, particularly from Herpesvirales. We conclude that the approach described here (or similar approaches) opens up new avenues of research to better understand SARS-CoV-2 and could be used to complement virus annotations, particularly for less-studied viruses.","version":"1.4","doi":"10.1101/2023.06.06.543855","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.17.624037","pub_date":"2024-11-18","title":"Antibody evasiveness of SARS-CoV-2 subvariants KP.3.1.1 and XEC","abstract":"SARS-CoV-2 continues to evolve and spread around the world, and it remains critical to understand the functional consequences of mutations that lead to dominant viral variants. KP.3.1.1 is currently the most prevalent subvariant worldwide, while the recombinant subvariant XEC is exhibiting the fastest growth rate. Here we measured the in vitro neutralization of KP.3.1.1 and XEC by human sera, monoclonal antibodies, and soluble hACE2 receptor relative to their parental subvariants KP.3 and JN.1. KP.3.1.1 and XEC were slightly more resistant (1.3-1.6-fold) than KP.3 to serum neutralization, and the resultant antigenic map showed that the new subvariants are antigenically similar. Both also demonstrated greater resistance to neutralization by select monoclonal antibodies and soluble hACE2, all of which target the top of the viral spike. Our findings suggested that upward motion of the receptor-binding domain in spike is partially hindered by the N-terminal-domain mutations found KP.3.1.1 and XEC, thereby allowing these subvariants to better evade serum antibodies that target the viral spike when it is in the up position and thus having a growth advantage in the population.","version":"1.1","doi":"10.1101/2024.11.17.624037","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.14.622799","pub_date":"2024-11-16","title":"Chronic Viral Reactivation and Associated Host Immune Response and Clinical Outcomes in Acute COVID-19 and Post-Acute Sequelae of COVID-19","abstract":"Chronic viral infections are ubiquitous in humans, with individuals harboring multiple latent viruses that can reactivate during acute illnesses. Recent studies have suggested that SARS- CoV-2 infection can lead to reactivation of latent viruses such as Epstein-Barr Virus (EBV) and cytomegalovirus (CMV), yet, the extent and impact of viral reactivation in COVID-19 and its effect on the host immune system remain incompletely understood. Here we present a comprehensive multi-omic analysis of viral reactivation of all known chronically infecting viruses in 1,154 hospitalized COVID-19 patients, from the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study, who were followed prospectively for twelve months. We reveal significant reactivation of Herpesviridae, Enteroviridae, and Anelloviridae families during acute stage of COVID-19 (0-40 days post- hospitalization), each exhibiting distinct temporal dynamics. We also show that viral reactivation correlated with COVID-19 severity, demographic characteristics, and clinical outcomes, including mortality. Integration of cytokine profiling, cellular immunophenotyping, metabolomics, transcriptomics, and proteomics demonstrated virus-specific host responses, including elevated pro-inflammatory cytokines (e.g. IL-6, CXCL10, and TNF), increased activated CD4+ and CD8+ T-cells, and upregulation of cellular replication genes, independent of COVID-19 severity and SARS-CoV-2 viral load. Notably, persistent Anelloviridae reactivation during convalescence (\u22653 months post-hospitalization) was associated with Post-Acute Sequelae of COVID-19 (PASC) symptoms, particularly physical function and fatigue. Our findings highlight a remarkable prevalence and potential impact of chronic viral reactivation on host responses and clinical outcomes during acute COVID-19 and long term PASC sequelae. Our data provide novel immune, transcriptomic, and metabolomic biomarkers of viral reactivation that may inform novel approaches to prognosticate, prevent, or treat acute COVID- 19 and PASC.","version":"1.1","doi":"10.1101/2024.11.14.622799","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.01.596790","pub_date":"2024-11-14","title":"iHDSel software: The Price equation and the population stability index to detect genomic patterns compatible with selective sweeps. An example with SARS-CoV-2","abstract":"A large number of methods have been developed and continue to be developed for detecting the signatures of selective sweeps in genomes. Significant advances have been made, including the combination of different statistical strategies and the incorporation of artificial intelligence (machine learning) methods. Despite these advances, several common problems persist, such as the unknown null distribution of the statistics used, necessitating simulations and resampling to assign significance to the statistics. Additionally, it is not always clear how deviations from the specific assumptions of each method might affect the results. In this work, allelic classes of haplotypes are used along with the informational interpretation of the Price equation to design a statistic with a known distribution that can detect genomic patterns caused by selective sweeps. The statistic consists of Jeffreys divergence, also known as the population stability index, applied to the distribution of allelic classes of haplotypes in two samples. Results with simulated data show optimal performance of the statistic in detecting divergent selection. Analysis of real SARS-CoV-2 genome data also shows that some of the sites playing key roles in the virus\u2019s fitness and immune escape capability are detected by the method. The new statistic, called JHAC, is incorporated into the iHDSel (informed HacDivSel) software available at https://acraaj.webs.uvigo.es/iHDSel.html.","version":"1.2","doi":"10.1101/2024.06.01.596790","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.13.622718","pub_date":"2024-11-14","title":"Characteristics of early career health researchers and experiences of burnout during the COVID-19 pandemic in Canada","abstract":"The COVID-19 pandemic disrupted research globally. How it impacted Canadian early-career health researchers (ECHRs) remains unclear. We administered a survey to understand the composition of ECHRs in Canada, their job experiences, and experiences of burnout during the COVID-19 pandemic. A cross-sectional survey was conducted in May 2023 of Canadian ECHRs defined as within 7 years of their first independent research position. Quantitative analyses included a description of respondents by research pillar, socio-demographic and workplace characteristics, and the prevalence of burnout, disengagement or exhaustion. Sample characteristics were compared to national data on ECHRs from a Canadian funding agency. Thematic analysis of free-text responses was also conducted. A total of 225 respondents met the eligibility criteria. Most respondents were assistant professors and characteristics of our sample were like the national data. The COVID-19 pandemic posed many challenges to student recruitment, and emotional support of students, with over half of the respondents reporting a moderate to significant decline in mental health compared to pre-pandemic. A significant proportion of respondents were experiencing high burnout (62%, 95%CI:56-67%), exhaustion (64%, 95%CI: 57-70%) or disengagement (91%, 95%CI: 87-95%). Thematic analysis identified three themes: ongoing benefits/problems preceding the pandemic, unintended outcomes of strategies to manage/prevent/contain COVID-19, and reasons to stay in their current position. Our survey revealed that Canadian ECHRs reported many diverse challenges during the COVID-19 pandemic and high burnout, putting the sustainability of this workforce at risk. Improved systems are needed to understand the long-term impacts and support the future of the Canadian health research ecosystem.","version":"1.1","doi":"10.1101/2024.11.13.622718","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.12.623177","pub_date":"2024-11-13","title":"COMPARATIVE CHARACTERISTICS OF THE ACCUMULATION OF DIFFERENT VARIANTS OF THE SARS-COV-2 VIRUS (WUHAN, DELTA, OMICRON) IN THE ORGANS OF MODEL ANIMALS","abstract":"The genotypic variability of the SARS-CoV-2 virus has proven to be extremely high, and the emergence of new strains raises concerns about their possible high virulence, transmissibility, and ability to bypass responses of the body\u2019s immune system induced by previous infection or vaccination. Therefore, one of the main tasks is to study the pathogenesis of various variants of the virus using experimental animal biomodels of SARS-CoV-2 to quickly find methods and approaches to fighting new viruses. 60 humanized mice of the C57BL/6-Tgtn (CAG-human AEC2-IRES-Luciferase-WPRE-polyA) line (hACE2) were used. Mice were infected intranasally at different doses with three variants of the SARS-CoV-2 virus: Wuhan, Delta and Omicron. We showed that humanized hACE2 mice, when infected with all three variants of the SARS-CoV-2 virus, showed typical pathological changes in lung consistency comparable to those found in COVID-19 in humans. All mice developed interstitial pneumonia, characterized by inflammatory cell infiltration and thickening of the alveolar septa, characteristic of vascular damage. At a dose of 4 lg plaque-forming unit (PFU), all variants showed 100% mortality. A dose-dependent effect was established only for the Wuhan and Delta variants. In a comparative assessment of different variants of the SARS-CoV-2 virus in a humanized mouse model of hACE2, it was found that the Delta variant leads to more severe damage compared to Wuhan or Omicron.","version":"1.1","doi":"10.1101/2024.11.12.623177","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.11.623127","pub_date":"2024-11-13","title":"Neutralization of recent SARS-CoV-2 variants by genetically and structurally related mAbs of the pemivibart lineage","abstract":"Pemivibart is a monoclonal antibody therapy currently under Emergency Use Authorization for the for the pre-exposure prophylaxis of coronavirus disease 2019 (COVID-19) in adults and adolescents over 12 years of age with certain immunocompromised conditions. As a part of the overall monitoring strategy for the activity of pemivibart, the antibody is regularly evaluated against emerging variants of SARS-CoV-2 using pseudovirus neutralization assays. Recent clinical data from Invivyd demonstrates that the PhenoSense pseudovirus assays carried out at Monogram Biosciences have been a reliable and consistent predictor of continued pemivibart clinical activity against SARS-COV-2 variants that have predominated across the timespan that includes the CANOPY clinical trial and the post-EUA authorization period. Additionally, new potential antibodies based upon the structural framework of pemivibart are continuously under evaluation. Fifteen of these yeast-produced \u201cpemivibart-like\u201d antibodies were tested for neutralization activity against recent variants KP.3 and KP.3.1.1. Like pemivibart, all 15 maintained activity against KP.3.1.1, with the change in IC50 averaging 2.51-fold +/-0.7 compared to KP.3. Four pemivibart-like antibodies were also tested against the XEC variant, with the change in IC50 averaging 3.01-fold compared to KP.3. These data suggest continued activity for pemivibart and pemivibart-like antibodies against KP.3.1.1 and XEC, recent variants containing N-terminal domain modifications.","version":"1.1","doi":"10.1101/2024.11.11.623127","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.11.622995","pub_date":"2024-11-13","title":"SARS-CoV-2 EndoU-ribonuclease regulates RNA recombination and impacts viral fitness","abstract":"Coronaviruses (CoVs) maintain large RNA genomes that frequently undergoes mutations and recombination, contributing to their evolution and emergence. In this study, we find that SARS-CoV-2 has greater RNA recombination frequency than other human CoVs. In addition, coronavirus RNA recombination primarily occurs at uridine (U)-enriched RNA sequences. Therefore, we next evaluated the role of SARS-CoV-2 NSP15, a viral endonuclease that targets uridines (EndoU), in RNA recombination and virus infection. Using a catalytically inactivated EndoU mutant (NSP15H234A), we observed attenuated viral replication in vitro and in vivo. However, the loss of EndoU activity also dysregulated inflammation resulting in similar disease in vivo despite reduced viral loads. Next-generation sequencing (NGS) demonstrated that loss of EndoU activity disrupts SARS-CoV-2 RNA recombination by reducing viral sub-genomic message but increasing recombination events that contribute to defective viral genomes (DVGs). Overall, the study demonstrates that NSP15 plays a critical role in regulating RNA recombination and SARS-CoV-2 pathogenesis.","version":"1.1","doi":"10.1101/2024.11.11.622995","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.12.623292","pub_date":"2024-11-13","title":"Metaproteomics reveals age-specific alterations of gut microbiome in hamsters with SARS-CoV-2 infection","abstract":"The gut microbiome\u2019s pivotal role in health and disease is well-established. SARS-CoV-2 infection often causes gastrointestinal symptoms and is associated with changes of the microbiome in both human and animal studies. While hamsters serve as important animal models for coronavirus research, there exists a notable void in the functional characterization of their microbiomes with metaproteomics. In this study, we present a workflow for analyzing the hamster gut microbiome, including a metagenomics-derived hamster gut microbial protein database and a data-independent acquisition metaproteomics method. Using this workflow, we identified 32419 protein groups from the fecal microbiomes of young and old hamsters infected with SARS-CoV-2. We showed age-specific changes in the expressions of microbiome functions and host proteins associated with microbiomes, providing further functional insight into the dysbiosis and aberrant cross-talks between the microbiome and host in SARS-CoV-2 infection. Altogether this study established and demonstrated the capability of metaproteomics for the study of hamster microbiomes.","version":"1.1","doi":"10.1101/2024.11.12.623292","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.12.623078","pub_date":"2024-11-13","title":"Immune Evasion, Cell-Cell Fusion, and Spike Stability of the SARS-CoV-2 XEC Variant: Role of Glycosylation Mutations at the N-terminal Domain","abstract":"SARS-CoV-2 continues to evolve, producing new variants that drive global COVID-19 surges. XEC, a recombinant of KS.1.1 and KP.3.3, contains T22N and F59S mutations in the spike protein\u2019s N-terminal domain (NTD). The T22N mutation, similar to the DelS31 mutation in KP.3.1.1, introduces a potential N-linked glycosylation site in XEC. In this study, we examined the neutralizing antibody (nAb) response and mutation effects in sera from bivalent-vaccinated healthcare workers, BA.2.86/JN.1 wave-infected patients, and XBB.1.5 monovalent-vaccinated hamsters, assessing responses to XEC alongside D614G, JN.1, KP.3, and KP.3.1.1. XEC demonstrated significantly reduced neutralization titers across all cohorts, largely due to the F59S mutation. Notably, removal of glycosylation sites in XEC and KP.3.1.1 substantially restored nAb titers. Antigenic cartography analysis revealed XEC to be more antigenically distinct from its common ancestral BA.2.86/JN.1 compared to KP.3.1.1, with the F59S mutation as a determining factor. Similar to KP.3.1.1, XEC showed reduced cell-cell fusion relative to its parental KP.3, a change attributed to the T22N glycosylation. We also observed reduced S1 shedding for XEC and KP.3.1.1, which was reversed by ablation of T22N and DelS31 glycosylation mutations, respectively. Molecular modeling suggests that T22N and F59S mutations of XEC alters hydrophobic interactions with adjacent spike protein residues, impacting both conformational stability and neutralization. Overall, our findings underscore the pivotal role of NTD mutations in shaping SARS-CoV-2 spike biology and immune escape mechanisms.","version":"1.1","doi":"10.1101/2024.11.12.623078","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.12.623198","pub_date":"2024-11-13","title":"Dynamic metabolic modeling of ATP allocation during viral infection","abstract":"Viral pathogens, like SARS-CoV-2, hijack the host\u2019s macromolecular production machinery, imposing an energetic burden that is distributed across cellular metabolism. To explore the dynamic metabolic tension between the host\u2019s survival and viral replication, we developed a computational framework that uses genome-scale models to perform dynamic Flux Balance Analysis of human cell metabolism during virus infections. Relative to previous models, our framework addresses the physiology of viral infections of non-proliferating host cells through two new features. First, by incorporating the lipid content of SARS-CoV-2 biomass, we discovered activation of previously overlooked pathways giving rise to new predictions of possible drug targets. Furthermore, we introduce a dynamic model that simulates the partitioning of resources between the virus and the host cell, capturing the extent to which the competition depletes the human cells from essential ATP. By incorporating viral dynamics into our COMETS framework for spatio-temporal modeling of metabolism, we provide a mechanistic, dynamic and generalizable starting point for bridging systems biology modeling with viral pathogenesis. This framework could be extended to broadly incorporate phage dynamics in microbial systems and ecosystems.","version":"1.1","doi":"10.1101/2024.11.12.623198","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.16.594569","pub_date":"2024-11-12","title":"Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles","abstract":"Innate immune responses triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection play pivotal roles in the pathogenesis of COVID-19, while host factors including pro-inflammatory cytokines are critical for viral containment. By utilizing quantitative and qualitative models, we discovered that soluble factors secreted by human monocytes potently inhibit SARS-CoV-2-induced cell-cell fusion in viral-infected cells. Through cytokine screening, we identified that interleukin-1\u03b2 (IL-1\u03b2), a key mediator of inflammation, inhibits syncytia formation mediated by various SARS-CoV-2 strains. Mechanistically, IL-1\u03b2 activates RhoA/ROCK signaling through a non-canonical IL-1 receptor-dependent pathway, which drives the enrichment of actin bundles at the cell-cell junctions, thus prevents syncytia formation. Notably, in vivo infection experiments in mice confirms that IL-1\u03b2 significantly restricted SARS-CoV-2 spreading in the lung epithelia. Together, by revealing the function and underlying mechanism of IL-1\u03b2 on SARS-CoV-2-induced cell-cell fusion, our study highlights an unprecedented antiviral function for cytokines during viral infection.","version":"1.2","doi":"10.1101/2024.05.16.594569","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.11.623004","pub_date":"2024-11-12","title":"The Virtual Lab: AI Agents Design New SARS-CoV-2 Nanobodies with Experimental Validation","abstract":"Science frequently benefits from teams of interdisciplinary researchers. However, most scientists don\u2019t have access to experts from multiple fields. Fortunately, large language models (LLMs) have recently shown an impressive ability to aid researchers across diverse domains by answering scientific questions. Here, we expand the capabilities of LLMs for science by introducing the Virtual Lab, an AI-human research collaboration to perform sophisticated, interdisciplinary science research. The Virtual Lab consists of an LLM principal investigator agent guiding a team of LLM agents with different scientific backgrounds (e.g., a chemist agent, a computer scientist agent, a critic agent), with a human researcher providing high-level feedback. We design the Virtual Lab to conduct scientific research through a series of team meetings, where all the agents discuss a scientific agenda, and individual meetings, where an agent accomplishes a specific task. We demonstrate the power of the Virtual Lab by applying it to design nanobody binders to recent variants of SARS-CoV-2, which is a challenging, open-ended research problem that requires reasoning across diverse fields from biology to computer science. The Virtual Lab creates a novel computational nanobody design pipeline that incorporates ESM, AlphaFold-Multimer, and Rosetta and designs 92 new nanobodies. Experimental validation of those designs reveals a range of functional nanobodies with promising binding profiles across SARS-CoV-2 variants. In particular, two new nanobodies exhibit improved binding to the recent JN.1 or KP.3 variants of SARS-CoV-2 while maintaining strong binding to the ancestral viral spike protein, suggesting exciting candidates for further investigation. This demonstrates the ability of the Virtual Lab to rapidly make impactful, real-world scientific discovery.","version":"1.1","doi":"10.1101/2024.11.11.623004","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.10.622868","pub_date":"2024-11-11","title":"A Mouse-adapted SARS-CoV-2 Model for Investigating Post-acute Sequelae of COVID infection","abstract":"The coronavirus disease of 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), remains a major health issue after nearly 7 millions of death toll in the last four years. As the world is recovering with improving vaccines and antiviral treatments, the alarming rate of long-COVID, or Post-acute Sequelae of COVID-19 (PASC), calls for further investigations. Among a list of symptoms associated with multi-organ dysfunctions, the neurological complications are particularly intriguing, yet the underlying mechanisms remain elusive. With the recently developed mouse adapted SARS-CoV-2 stain, we are now able to model the mild COVID infection in C57BL/6 mice and study the chronic immune responses and subsequent damages in different organs long after the viruses are clearly naturally in the body. More specifically, we found adult C57BL/6J mice developed neurological impairments, including behavior changes related to sensorimotor coordination, depression- and anxiety-like behaviors, and inflammation in multiple organs including lung, liver and brain, which persisted over at least 4 weeks in mice even with mild infection. Therefore, this model can be used to further explopred the mechanisms of PASC, as well as potential intervention or therapeutic approaches.","version":"1.1","doi":"10.1101/2024.11.10.622868","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.04.621927","pub_date":"2024-11-11","title":"Immunologic and Biophysical Features of the BNT162b2 JN.1- and KP.2-Adapted COVID-19 Vaccines","abstract":"Vaccines remain a vital public health tool to reduce the burden of COVID-19. COVID-19 vaccines that are more closely matched to circulating SARS-CoV-2 lineages elicit more potent and relevant immune responses that translate to improved real-world vaccine effectiveness. The rise in prevalence of the Omicron JN.1 lineage, and subsequent derivative sublineages such as KP.2 and KP.3, coincided with reduced neutralizing activity and effectiveness of Omicron XBB.1.5-adapted vaccines. Here, we characterized the biophysical and immunologic attributes of BNT162b2 JN.1- and KP.2-adapted mRNA vaccine-encoded spike (S) protein immunogens. Biophysical interrogations of S revealed the structural consequences of hallmark amino acid substitutions and a potential molecular mechanism of immune escape employed by JN.1 and KP.2. The vaccine candidates were evaluated for their immunogenicity when administered as fourth or fifth doses in BNT162b2-experienced mice or as a primary series in na\u00efve mice. In both vaccine-experienced and na\u00efve settings, JN.1- and KP.2-adapted vaccines conferred improved neutralizing responses over the BNT162b2 XBB.1.5 vaccine against a broad panel of emerging JN.1 sublineages, including the predominant KP.3.1.1 and emerging XEC lineages. Antigenic mapping of neutralizing responses indicated greater antigenic overlap of JN.1- and KP.2-adapted vaccine responses with currently circulating sublineages compared to an XBB.1.5-adapted vaccine. CD4+ and CD8+ T cell responses were generally conserved across all three vaccines. Together, the data support the selection of JN.1- or KP.2-adapted vaccines for the 2024-25 COVID-19 vaccine formula. The Omicron JN.1- and KP.2-adapted BNT162b2 mRNA vaccines encoding prefusion S proteins elicit similar preclinical neutralizing antibody responses against circulating JN.1 sublineage pseudoviruses that are more potent than those elicited by past iterations of BNT162b2 licensed vaccines, thus demonstrating the importance of annual strain changes to the COVID-19 vaccine.","version":"1.2","doi":"10.1101/2024.11.04.621927","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.10.622838","pub_date":"2024-11-10","title":"Generation of a biosafety mouse model infection for SARS-CoV-2 replicon delivery particles","abstract":"Animal models are essential for understanding the pathogenesis of SARS-CoV-2 and for developing therapeutic strategies. Replicon delivery particles (RDPs) were a component of trans-complementary systems for SARS-CoV-2, which is a safe and convenient tool in researching SARS-CoV-2 in animal biosafety level-II laboratory (ABSL-2). Here, we constructed a mouse model that conditional expressing SARS-CoV-2 N on the background of the K18-hACE2 KI mice. The SARS-CoV-2 N with flanked loxP-stop-loxP sequence was under the CAG promoter, and this cassette was knocked into the Tiger locus of mouse by CRISPR-Cas9 (K18-hACE2-N KI). By mating K18-hACE2-N KI mice with Cre tool mice, the offspring can express SARS-CoV-2 N (Cre-N-hACE2 KI) systemically and in a tissue-specific manner. Cre-N-hACE2 KI exhibited susceptibility to the SARS-CoV-2 \u0394N-GFP/HBiT infection. The viral loads in lung exhibited a mountain-like trend, peaking at 4 days post-infection, and lung injuries can be observed. Overall, we demonstrated a mouse model infection for SARS-CoV-2 \u0394N-GFP/HBiT to understand SARS-CoV-2 pathogenesis.","version":"1.1","doi":"10.1101/2024.11.10.622838","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.08.622602","pub_date":"2024-11-09","title":"Pathogenesis and immune response to respiratory coronaviruses in their natural porcine host","abstract":"Porcine respiratory coronavirus (PRCV) is a naturally occurring pneumotropic coronavirus in the pig, providing a valuable large animal model to study acute respiratory disease. PRCV pathogenesis and the resulting immune response was investigated in pigs, the natural large animal host. We compared two strains, ISU-1 and 135, which induced differing levels of pathology in the respiratory tract to elucidate the mechanisms leading to mild or severe disease. The 135 strain induced greater pathology which was associated with higher viral load and stronger spike-specific antibody and T cell responses. In contrast, the ISU-1 strain triggered mild pathology with a more balanced immune response and greater abundance of T regulatory cells. A higher frequency of putative T follicular helper cells was observed in animals infected with strain 135 at 11 days post-infection. Single-cell RNA-sequencing of bronchoalveolar lavage revealed differential gene expression in B and T cells between animals infected with 135 and ISU-1 at 1 day post infection. These genes were associated with cell adhesion, migration, and immune regulation. Along with increased IL-6 and IL-12 production, these data suggest that heightened inflammatory responses to the 135 strain may contribute to pronounced pneumonia. Among BAL immune cell populations, B cells and plasma cells exhibited the most gene expression divergence between pigs infected with different PRCV strains, highlighting their potential role in maintaining immune homeostasis in the respiratory tract. These findings indicate the potential of the PRCV model for studying coronavirus induced respiratory disease and identifying mechanisms that determine infection outcomes. Understanding how our immune system reacts to respiratory viruses, like SARS-CoV-2, is crucial to developing better treatments. While most COVID-19 infections are mild, some cases lead to severe lung damage, but we do not fully understand why. To study this, we used pigs, which respond more like humans compared to small animals, to explore how the immune system deals with respiratory coronaviruses. We tested two porcine respiratory coronavirus strains that caused different levels of lung damage. The more severe strain triggered a strong immune response and high inflammation, leading to lung pathology similar to that seen in severe COVID-19 cases. By contrast, the milder strain caused a balanced immune response, including more regulatory T cells that help control inflammation. We also found changes in genes related to antibody-producing cells, which may be important for controlling respiratory pathology. Interestingly, changes in immune responses and gene expression lasted long after the virus was cleared, potentially making individuals more vulnerable to future infections - similar to the \u201clong COVID\u201d symptoms seen in people. We propose that this pig model could help us study coronavirus-induced lung damage and test new therapies to prevent severe disease.","version":"1.1","doi":"10.1101/2024.11.08.622602","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.08.622749","pub_date":"2024-11-09","title":"Identification of Covalent Cyclic Peptide Inhibitors Targeting Protein-Protein Interactions Using Phage Display","abstract":"Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein-protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein-protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 Spike-ACE2 protein-protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein-protein interactions with long lasting effects.","version":"1.1","doi":"10.1101/2024.11.08.622749","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.07.622580","pub_date":"2024-11-08","title":"SARS-CoV-2 JN.1 reveals attenuated pathogenicity and airborne transmission","abstract":"JN.1 is a subvariant of SARS-CoV-2 Omicron BA.2.86 lineage that was predominant worldwide in early 2024, of which the in vivo characteristics are largely unknown. Our results demonstrated that the replication of JN.1 was more efficient than that of the parental BA.2 in Vero cells, which demonstrated low dependence on TMPRSS2. Compared to Omicron variants BA.2 and XBB EG.5.1, JN.1 replicated less efficiently in hACE2 mouse lungs of which the intranasal infection was not lethal to hACE2 mice and led to weaker immune dysregulation. On a more sensitive, aged hACE2 hamster model, JN.1 led to a lower mortality rate and no weight loss, corresponding well with the low preference in lower airways. Lower amounts of viruses in nasal washes and exhaled aerosols were detected in JN.1 infected wildtype hamsters than EG.5.1, and consistently, JN.1 also exhibited largely reduced airborne transmission. Moreover, the poor transmission was also clearly demonstrated even by using hamsters expressing hACE2 receptors in the whole airway. Thus both pathogenicity and airborne transmission of JN.1 were demonstrated to be largely attenuated. Currently, SARS-CoV-2 JN.1 and its subvariants have fully replaced the previous dominant XBB lineage around the world. Although the strong immune evasion of JN.1 has been distinctly revealed, its in vivo pathogenicity and airborne transmission remained unclear. By using multiple Omicron-sensitive rodent models, our findings demonstrated that the pathogenicity of JN.1 was largely attenuated. The weak airborne transmission of JN.1 in wildtype and hACE2 hamsters was consistent with the reported relative lower transmissibility in human, and the using of airway-expressing hACE2 hamsters ulteriorly eliminates the potential bias in viral transmission studies induced by receptor divergence between animal models and human. These findings uncover the in vivo virological characteristics of SARS-CoV-2 novel lineage, providing insights for communicable disease control.","version":"1.1","doi":"10.1101/2024.11.07.622580","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.08.622599","pub_date":"2024-11-08","title":"Lineage-specific neutralising antibodies after SARS-CoV-2 mild disease. Immune boosting effect of vaccination","abstract":"We followed a group of 105 non-vaccinated individuals after Alpha or Delta SARS-CoV-2 mild disease, measuring the viral shedding (qRT-PCR, dPCR and subgenomic RNA-E) and humoral response (commercial immunoassay and pseudovirus and live virus neutralisation) up to six months. Sixty nine patients received a vaccination boost during the follow-up period (n=95). Subgenomic RNA-E showed a shorter period until negativity (mean 2.2 weeks) compared to gRNA (mean 5.2 weeks). A high correlation between qRT-PCR and dPCR was found for viral load estimation, even when no nucleic acid extraction was used in dPCR (R2 = 0.87). Post-convalescent sera showed the strongest neutralisation against the variant of natural exposure, while the neutralisation capacity against Omicron variants was significantly lower compared to the other variants. Additionally, the results suggested that commercial immunoassays may not accurately predict protection against a different variant than the variant of exposure. An immune boosting effect of the SARS-CoV-2 vaccination was evident. Variant-specific neutralising antibodies were detected one month after natural infection. Although short lived, maximum igG response was observed after hybrid immunisation (natural infection + vaccination). This study also points to potential improvements in the clinical management of SARS-CoV-2 cases. Firstly, subgenomic RNA-E is a potentially more accurate biomarker of infectivity than current qRT-PCRs using genomic RNA as target. Secondly, accuracy of high-throughput immunoassays must be validated in order to estimate specific protection and organise vaccination campaigns. Our findings could play a role in the current implementation of SARS-CoV-2 vaccine programs. Years after SARS-CoV-2 related infections challenged the healthcare systems of the whole world, the optimal strategy to deal with diagnosis, quarantines or vaccination patterns is still a matter of debate. The interplay between infectivity and immunity in the different circulating variants is complex, and qPCR for diagnosis may extend quarantines, as detection of viral RNA does not necessarily mean that the virus remains infectious. We studied a group of patients infected with pre-Omicron variants to study how their variant-specific antibodies reacted to the past and present variants of SARS-CoV-2. In both Alpha and Delta, antibodies neutralise their own variants better than other variants that came before and after. Perhaps most importantly, neutralisation was lowest against Omicron variants. We followed the viral shedding dynamics of the patients, testing different PCR techniques and targets. Subgenomic RNA was detectable in nasopharyngeal samples for a shorter time than genomic RNA and it has been suggested as a good marker of infectivity. Using sgRNA instead of genomic RNA as a PCR target could reduce hospital bed occupation and quarantine time. Overall, we hope that these results could help guide pandemic and diagnostic control in the future.","version":"1.1","doi":"10.1101/2024.11.08.622599","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.07.622402","pub_date":"2024-11-07","title":"Distinct Binding Modes of Inter-Spike Cross-Linking Suggest a Supplementary Mechanism for SARS-CoV-2 Antibody Neutralization","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its Omicron subvariants drastically amplifies transmissibility, infectivity, and immune escape, mainly due to their resistance to most neutralizing antibodies. Thus, exploring the mechanisms underlying antibody evasion is crucial. Although the full-length native form of antibody, immunoglobulin G (IgG), offers valuable insights into the neutralization, structural investigations primarily focus on the fragment of antigen-binding (Fab). Here, we employ single-particle cryo-electron microscopy (cryo-EM) to characterize a W328-6H2 antibody, in its native IgG form complexed with severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus 2 wild-type (WT) and Omicron variant BA.1 spike protein (S). Three high-resolution structures reveal that the full-length IgG forms a centered head-to-head dimer of trimer when bound fully stoichiometrically with both SARS and WT S, while adopting a distinct offset configuration with Omicron BA.1 S. Combined with functional assays, our results suggest that, beyond the binding affinity between the RBD epitope and Fab, the higher-order architectures of S trimer and full-length IgG play an additional role in neutralization, elucidate a potential molecular basis of Omicron immune escape, and expand our understanding on antibody evasion mechanisms.","version":"1.1","doi":"10.1101/2024.11.07.622402","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.05.622192","pub_date":"2024-11-07","title":"GenomicSign: A computational method to discover unique, specific, and amplifiable signatures of target genomic sequences","abstract":"Molecular diagnostics for the rapid identification of infectious, virulent, and pathogenic organisms are key to health and global security. Such methods rely on the identification and detection of signatures possessed by the organism. In this work, we outline a computational algorithm, GenomicSign, to determine unique and amplifiable genomic signatures of a set of target sequences against a background set of non-target sequences. The set of target sequences might comprise variants of a pathogen of interest, say SARS-CoV2 virus. Unique k-mers of the consensus target sequence for a range of k-values are determined, and the threshold k-value yielding a sharp transition in the number of unique k-mers is identified as kopt. Corresponding unique k-mers for k \u2265 kopt are compared against the set of non-target sequences to identify target-specific unique k-mers. A pair of proximal such k-mers could enclose a potential amplicon. Primers to such pairs are designed and scored using a custom scheme to rank the potential amplicons. The top-ranked resulting amplicons are candidates for unique and amplifiable genomic signatures. The entire workflow is demonstrated using a case study with the SARS-CoV2 omicron genome. A case study distinguishing the SARS-CoV2 omicron target strain against non-target other SARS-CoV2 variants is performed to illustrate the workflow. GenomicSign has been implemented in Python and is available as an open-source software under MIT licence (https://www.github.com/apalania/GenomicSign).","version":"1.1","doi":"10.1101/2024.11.05.622192","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.06.622391","pub_date":"2024-11-07","title":"A Single Chimeric Spike Antigen Induces Pan-Sarbecovirus Immunity","abstract":"Next-generation vaccines are required to address the evolving nature of SARS-CoV-2 and to protect against emerging pandemic threats from other coronaviruses. These vaccines should aim to elicit broad-protection, provide long-lasting immunity and facilitate equitable access for all populations. In this study, a panel of chimeric, full-length spike antigens were developed that incorporate mutations from previous, circulating and predicted SARS-CoV-2 variants. The lead candidate (CoVEXS5) was obtained from a high-yield production process in stable CHO cells with purity of >95%, long-term stability and elicitation of broadly cross-reactive neutralising antibodies when delivered to mice in a squalene emulsion adjuvant (Sepivac SWE\u2122). In both mice and hamsters, CoVEXS5 immunisation reduced clinical disease signs, lung inflammation and organ viral titres after SARS-CoV-2 infection, including challenge with the highly immunoevasive Omicron XBB.1.5 subvariant. In mice previously primed with a licenced protein vaccine (NVX-CoV2373), CoVEXS5 could boost T cell immunity, as well as neutralising antibodies levels against viruses from three sarbecoviruses clades. The breadth of sarbecovirus cross-reactivity elicited by CoVEXS5 exceeded that observed after boosting with the NVX-CoV2373 vaccine. These findings highlight the potential of a chimeric spike antigen, formulated in an open-access adjuvant, as a next-generation vaccine candidate to enhance cross-protection against emerging sarbecoviruses in vaccinated populations globally.","version":"1.1","doi":"10.1101/2024.11.06.622391","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.05.622148","pub_date":"2024-11-06","title":"Nanoparticle-Supported, Rapid, Digital Quantification of Neutralizing Antibodies Against SARS-CoV-2 Variants","abstract":"The measurement of neutralizing immune responses to viral infection is essential, given the heterogeneity of human immunity and the emergence of new virus strains. However, neutralizing antibody (nAb) assays often require high-level biosafety containment, sophisticated instrumentation, and long detection times. Here, as a proof-of-principle, we designed a nanoparticle-supported, rapid, electronic detection (NasRED) assay to assess the neutralizing potency of monoclonal antibodies (mAbs) against SARS-CoV-2. The gold nanoparticles (AuNPs) coated with human angiotensin-converting enzyme 2 (ACE2) protein as nAb potency reporters were mixed with the mAbs to be tested, as well as streptavidin-conjugated multivalent spike (S) protein or their receptor binding domains (RBD). High-affinity and ACE2-competitive nAbs alter the S (or RBD)-to-ACE2 binding level and modulate AuNP cluster formation and precipitation. The amount of free-floating AuNP reporters is quantified by a semiconductor-based readout system that measures the AuNPs\u2019 optical extinction, producing nAb signals that can differentiate SARS-CoV-2 variants (Wuhan-Hu-1, Gamma, and Omicron). The modular design nature, short assay time (less than 30 minutes), and portable and inexpensive readout system make this NasRED-nAb assay applicable to measuring vaccine potency, immune responses to infection, and the efficacy of antibody-based therapies.","version":"1.1","doi":"10.1101/2024.11.05.622148","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.05.622058","pub_date":"2024-11-06","title":"Integrative metagenomics and metabolomics reveal age-associated gut microbiota and metabolite alterations in experimental COVID-19","abstract":"Aging is a key contributor of morbidity and mortality during acute viral pneumonia. The potential role of age-associated dysbiosis on disease outcomes is still elusive. In the current study, we used high-resolution shotgun metagenomics and targeted metabolomics to characterize SARS-CoV-2-associated changes in the gut microbiota from young (2-month-old) and aged (22-month-old) hamsters, a valuable model of COVID-19. We show that age-related dysfunctions in the gut microbiota are linked to disease severity and long-term sequelae in older hamsters. Our data also reveal age-specific changes in the composition and metabolic activity of the gut microbiota during both the acute phase (day 7 post-infection, D7) and the recovery phase (D22) of infection. Aged hamsters exhibited the most notable shifts in gut microbiota composition and plasma metabolic profiles. Through an integrative analysis of metagenomics, metabolomics, and clinical data, we identified significant associations between bacterial taxa, metabolites and disease markers in the aged group. On D7 (high viral load and lung epithelial damage) and D22 (body weight loss and fibrosis), numerous amino acids, amino acid-related molecules, and indole derivatives were found to correlate with disease markers. In particular, a persistent decrease in phenylalanine, tryptophan, glutamic acid, and indoleacetic acid in aged animals positively correlated with poor recovery of body weight and/or lung fibrosis by D22. In younger hamsters, several bacterial taxa (Eubacterium, Oscillospiraceae, Lawsonibacter) and plasma metabolites (carnosine and cis-aconitic acid) were associated with mild disease outcomes. These findings support the need for age-specific microbiome-targeting strategies to more effectively manage acute viral pneumonia and long-term disease outcomes.","version":"1.1","doi":"10.1101/2024.11.05.622058","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.04.621772","pub_date":"2024-11-05","title":"Neutralizing antibody evasion of SARS-CoV-2 JN.1 derivatives KP.3, KP.3.1.1, LB.1, and XEC","abstract":"The emergence of SARS-CoV-2 variants poses ongoing challenges to vaccine efficacy. We evaluated neutralizing antibody responses against JN.1 and its derivatives (KP.3, KP.3.1.1, LB.1, and XEC) in healthcare workers who received seven doses of BNT162b2, including XBB.1.5 monovalent vaccine. In COVID-19-na\u00efve individuals, KP.3.1.1 and LB.1 showed substantial immune escape, while previously infected individuals maintained neutralization activity against all variants. We also demonstrated that JN.1-based immunization induces robust cross-neutralizing activity against emerging variants. A single amino acid deletion at position 31 in the spike protein significantly impacted immune evasion. These findings support the potential effectiveness of JN.1-based vaccines while highlighting the need for continued surveillance and vaccine optimization.","version":"1.1","doi":"10.1101/2024.11.04.621772","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.29.591666","pub_date":"2024-11-05","title":"Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny","abstract":"The SARS-CoV-2 genome occupies a unique place in infection biology \u2013 it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in \u201ceyeballing\u201d trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.","version":"1.3","doi":"10.1101/2024.04.29.591666","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.09.617418","pub_date":"2024-11-05","title":"A Modular Bacteriophage T4 Nanoparticle Platform Enables Rapid Design of Dual COVID-19-Flu Mucosal Vaccines","abstract":"A multivalent, rapidly deployable, mucosal vaccine platform is desperately needed to prevent the acquisition and transmission of respiratory infections during epidemics and pandemics. We present one such bacteriophage T4-based platform, and design of dual COVID-19-Flu mucosal vaccines by exploiting its unique architecture. These include: T4\u2019s natural affinity for nasal mucosa, flexible engineering to incorporate multiple antigens, and repeat and symmetric epitope presentation for enhanced B cell responses. Hundreds of SARS-CoV-2 spike trimers and nucleocapsid proteins, and influenza hemagglutinin trimers and M2e peptides, were incorporated into a single phage, creating the highest density nanoparticle presentation yet reported. Intranasal administration of adjuvant-free vaccine induced robust mucosal immunity in mice including, neutralizing antibody and secretory IgA, lung-resident CD4+/CD8+ T cells, diverse memory B cells, and complete protection against SARS-CoV-2 and influenza challenges. The noninfectious T4 phage offers an extraordinary platform to rapidly design potent mucosal vaccines against emerging bacterial and viral threats.","version":"1.2","doi":"10.1101/2024.10.09.617418","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.04.620124","pub_date":"2024-11-05","title":"Direct lipid interactions control SARS-CoV-2 M protein conformational dynamics and virus assembly","abstract":"M is the most abundant structural membrane protein in coronaviruses and is essential for the formation of infectious virus particles. SARS-CoV-2 M adopts two conformations, Mshort and Mlong, and regulated transition between states is hypothesized to coordinate viral assembly and budding. However, the factors that regulate M conformation and roles for each state are unknown. Here, we discover a direct M-sphingolipid interaction that controls M conformational dynamics and virus assembly. We show M binds Golgi-enriched anionic lipids including ceramide-1-phosphate (C1P). Molecular dynamics simulations show C1P interaction promotes a long to short transition and energetically stabilizes Mshort. Cryo-EM structures show C1P specifically binds Mshort at a conserved site bridging transmembrane and cytoplasmic regions. Disrupting Mshort-C1P interaction alters M subcellular localization, reduces interaction with Spike and E, and impairs subsequent virus-like particle cell entry. Together, these results show endogenous signaling lipids regulate M structure and support a model in which Mshort is stabilized in the early endomembrane system to organize other structural proteins prior to viral budding.","version":"1.1","doi":"10.1101/2024.11.04.620124","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.01.621557","pub_date":"2024-11-04","title":"Lineage Detector: Efficient Tool for Detecting New SARS-Cov-2 Lineages","abstract":"Since the novel virus of SARS-Cov-2\u2019s emergence, it continues to mutate at significant speed. The mutation speed and diversity of the virus has reached a level that is hard to be analysed purely via human tracing even with the help of UShER trees. We create an open-sourced tool of Lineage Detector, an automated tool that helps highlight the most important lineages of interest on Usher trees. Lineage Detector can highlight the most interesting SARS-CoV-2 branches for manual investigation and reduce the work of volunteer variant hunters to 8 \u223c 10%, greatly improve their efficiency. Since its release, it has helped the identification, proposal and designation process of more than 100 SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.11.01.621557","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.02.24316649","pub_date":"2024-11-04","title":"Inconsistent Rebound of Invasive Pneumococcal Disease in Connecticut following the Initial Phase of the SARS-CoV-2 Pandemic","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The incidence of invasive pneumococcal disease (IPD) decreased during the SARS-CoV-2 pandemic and rebounded inconsistently over 2 years, with occasional returns to pre-pandemic levels followed by subsequent declines. We evaluated several explanations including changes in rates of viral infections that could interact with pneumococcus and changes in blood culture practices.</jats:p>","version":null,"doi":"10.1101/2024.11.02.24316649","journal":"medRxiv","score":null},{"id":"10.1101/2023.05.08.539898","pub_date":"2024-11-04","title":"Endo-Lysosome-Targeted Nanoparticle Delivery of Antiviral Therapy for Coronavirus Infections","abstract":"SARS-CoV-2 can infect cells through endocytic uptake, a process which is targeted by inhibition of lysosomal proteases. However, clinically this approach to treat viral infections has afforded mixed results, with some studies detailing an oral regimen of hydroxychloroquine accompanied by significant off-target toxicities. We rationalized that an organelle-targeted approach will avoid toxicity while increasing the concentration of the drug at the target. Here we describe a lysosome-targeted, mefloquine-loaded poly(glycerol monostearate-co-\u03b5-caprolactone) nanoparticle (MFQ-NP) for pulmonary delivery via inhalation. Mefloquine is a more effective inhibitor of viral endocytosis than hydroxychloroquine in cellular models of COVID-19. MFQ-NPs are less toxic than molecular mefloquine, 100-150 nm in diameter, and possess a negative surface charge which facilitates uptake via endocytosis allowing inhibition of lysosomal proteases. MFQ-NPs inhibit coronavirus infection in mouse MHV-A59 and human OC43 coronavirus model systems and inhibit SARS-CoV-2-WA1 and its Omicron variant in a human lung epithelium model. This study demonstrates that organelle-targeted delivery is an effective means to inhibit viral infection.","version":"1.2","doi":"10.1101/2023.05.08.539898","journal":"bioRxiv","score":null},{"id":"10.1101/2024.11.03.621713","pub_date":"2024-11-04","title":"Machine learning engineered PoLixNano nanoparticles overcome delivery barriers for nebulized mRNA therapeutics","abstract":"There continues to be a dearth of competent inhalable mRNA delivery although it holds great potential for addressing a wide variety of refractory diseases. The huge advances seen with parenteral-administered lipid nanoparticle (LNP) have not been translated into nebulized mRNA delivery due to the aggressive nebulization process and insurmountable barriers inherent to respiratory mucosa. Here, we show amphiphilic block copolymers revealed by machine learning (ML) can spontaneously form stabilized nanoparticles (PoLixNano) with the lipids components of LNP and simultaneously impart the PoLixNano with \u201cshield\u201d (shear force-resistant) and \u201cspear\u201d (pulmonary barriers-penetrating abilities) capabilities. We present a ML approach that leverages physicochemical properties and inhaled mRNA transfection profiles of a chemically diverse library of polymeric components to validate the integration of \u201cshield\u201d and \u201cspear\u201d properties as highly predictive indicators of transfection efficiency. This quantitative structure-mRNA transfection prediction (QSMTP) model identifies top-performing amphiphilic-copolymers from more than 10000 candidates and suggests their mucus-penetrating ability outweights the shear force-resistant property in contributing to efficient mRNA transfection. The optimized PoLixNano substantially outperforms the LNP counterpart and mediates up to 1114-times higher levels of mRNA transfection in animal models with negligible toxicities. The PoLixNano promotes overwhelming SARS-CoV-2 antigen-specific sIgA antibody secretion and expansion of TRM cells which collectively confers 100% protection in mice against lethal SARS-CoV-2 challenges and blocks the transmission of Omicron variant between hamsters. PoLixNano also displays versatile therapeutic potential in lung carcinoma and cystic fibrosis models. Our study provides new insights for designing delivery platforms of aerosol-inhaled mRNA therapeutics with clinical translation potential.","version":"1.1","doi":"10.1101/2024.11.03.621713","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.30.621174","pub_date":"2024-10-31","title":"Antigenic imprinting dominates humoral responses to new variants of SARS-CoV-2 in a hamster model of COVID-19","abstract":"The emergence of SARS-CoV-2 variants escaping immunity challenges the efficacy of current vaccines. Here, we investigated humoral recall responses and vaccine-mediated protection in Syrian hamsters immunized with the third-generation Comirnaty\u00ae Omicron XBB.1.5-adapted COVID-19 mRNA vaccine, followed by infection with either antigenically closely (EG.5.1) or distantly related (JN.1) Omicron subvariants. Vaccination with YF17D vector encoding a modified Gamma spike (YF-S0*) served as a control for pre-Omicron SARS-CoV-2 immunity. Our results show that both Comirnaty\u00ae XBB.1.5 and YF-S0* induce robust, however, poorly cross-reactive, neutralizing antibody (nAb) responses. In either case, total antibody and nAb levels increased following infection. Intriguingly, the specificity of these boosted nAbs did not match the respective challenge virus but was skewed towards the primary antigen used for immunization, suggesting a marked impact of antigenic imprinting; confirmed by antigenic cartography. Furthermore, limited cross-reactivity and rapid decline of nAbs induced by Comirnaty\u00ae XBB.1.5 with EG.5.1 and, more concerning, JN.1. raises doubts about sustained vaccine efficacy against recent circulating Omicron subvariants. Future vaccine design may have to address two major issues: (i) to overcome original antigenic sin that limits the breadth of a protective response towards emerging variants, and (ii) to achieve sustained immunity that lasts for at least one season.","version":"1.1","doi":"10.1101/2024.10.30.621174","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.16.613345","pub_date":"2024-10-31","title":"TIRTL-seq: Deep, quantitative, and affordable paired TCR repertoire sequencing","abstract":"\u0251/\u03b2 T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) \u0251 and \u03b2 chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired \u0251\u03b2TCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is \u223c$200. We benchmarked TIRTL-seq against state-of-the-art 5\u2019RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both \u0251 and \u03b2 TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.","version":"1.2","doi":"10.1101/2024.09.16.613345","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.30.620795","pub_date":"2024-10-31","title":"Quality of vaccination-induced T cell responses is conveyed by polyclonality and high, but not maximum, antigen receptor avidity","abstract":"While the quantity of vaccination-induced T cells represents a routine immunogenicity parameter, the quality of such responses is poorly understood. Here, we report on a clinical cohort of 29 human healthy individuals who received three mRNA vaccinations against SARS-CoV-2 before any breakthrough infection. We characterized the magnitude, phenotype and clonal composition of CD8 T cell responses against 16 epitope specificities by ELISpot, flow cytometry as well as single-cell RNA, TCR and surface protein sequencing. To test the functionality of identified clonotypes, 106 T cell receptors (TCR) from five epitope-specific repertoires were re-expressed and tested for peptide sensitivity. While recruited repertoires were overall enriched for high-avidity TCRs, differential clonal expansion was not linked to fine avidity differences. Instead, maintenance of polyclonality ensured robustness in counteracting mutational escape of epitopes. Our findings on the induction and maintenance of high-functionality polyclonal T cell repertoires shed light on T cell quality as a neglected criterion in the assessment of vaccine immunogenicity.","version":"1.1","doi":"10.1101/2024.10.30.620795","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.29.620982","pub_date":"2024-10-30","title":"Deep generative design of neutralizing nanobodies against SARS-CoV-2 variants","abstract":"In recent years, single-domain heavy chain antibodies (nanobodies), with only one-tenth the molecular weight of conventional antibodies, have emerged as important therapeutic proteins in the fight against SARS-CoV-2. However, the rapid mutation of the virus often renders existing nanobodies ineffective, underscoring the need to develop nanobodies that specifically target new variants. Traditional methods for discovering nanobodies are time-consuming and complex, making it difficult to efficiently identify nanobodies that bind to specific epitopes. To address this, we propose a de novo nanobody design method based on a Generative Adversarial Network (GAN). We developed a deep generative model, AiCDR, which consists of three discriminators and one generator. By determining the contribution of these three discriminators to the entire network, we can enhance the discrimination of generated sequences and reduce the similarity of the generated CDR3 sequences with natural peptides and random sequences, thereby ensuring their nature-like properties. These generated CDR3 sequences were then grafted onto humanized nanobody scaffolds, resulting in a structural library of approximately 104 nanobodies with natural-like properties. Using computational methods, we screened this library against the Spike (S) protein of the SARS-CoV-2 Omicron variant and identified 10 candidate nanobodies. Functional assays confirmed that two of these nanobodies exhibited neutralizing activity against the S protein. Our study demonstrates the potential of deep learning in nanobody design and offers a novel approach for developing nanobodies that target rapidly evolving viral variants.","version":"1.1","doi":"10.1101/2024.10.29.620982","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.11.617752","pub_date":"2024-10-29","title":"Functional Activity and Binding Specificity of small Ankyrin Repeat Proteins called Ankyrons against SARS-CoV-2 variants","abstract":"Effective management of COVID-19 requires clinical tools to treat the disease in addition to preventive vaccines. Several recombinant mAbs and their cocktails have been developed to treat COVID-19 but these have limitations. Here, we evaluate small ankyrin repeat proteins called Ankyrons\u2122 that were generated to bind with high affinity to the SARS-CoV-2 virus. Ankyrons are ankyrin repeat proteins comprised of repetitions a structural module. Each module consists of a \u03b2-turn followed by two antiparallel \u03b1-helices. The Ankyrons\u2122 are directly selected in vitro from a highly diverse library of around a trillion clones in ribosome display and like antibodies can bind with high affinity to almost any target. We assessed Ankyrons that were generated against the wild-type SARS-CoV-2 and the Delta and Omicron variants in a binding assay. We determined that all Ankyrons were specific in that they did not bind to MERS, a related coronavirus. While all Ankyrons bound with high affinity to the variant they were generated against, some also showed cross-reactivity to all three SARS-CoV-2 variants. Binding assays are useful for screening analytes but do not provide information about clinical effectiveness. Therefore, we used a pseudovirus-based neutralization assay to show that five of the Ankyrons evaluated neutralized all three strains of SARS-CoV-2. We have provided a workflow for the evaluation of novel Ankyrons against a viral target. This suggests that Ankyrons could be useful for rapidly developing new research tools for studying other emerging infectious diseases rapidly with the optional further potential for developing Ankyrons into diagnostic and even therapeutic applications. Yun-Jong Park: Investigation, Methodology, Format Analysis, Writing \u2013 original draft. Wojciech Jankowski: Methodology, Format Analysis, and Review. Nicholas C Hurst, Jeremy W Fry, Nikolai F Schwabe and Linda C C Tan: Resources, Review & Editing. Zuben E Sauna: Conceptualization, Supervision, Writing \u2013 original draft and Final editing.","version":"1.2","doi":"10.1101/2024.10.11.617752","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.28.620664","pub_date":"2024-10-29","title":"Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication","abstract":"Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. All CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, though the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFN\u03b3, similar to a Mac1 deletion virus. To confirm their specificity, we passaged MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared to WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug-resistance emerged at the expense of viral fitness. Coronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. All CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, though the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug-resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating coronavirus infections.","version":"1.1","doi":"10.1101/2024.10.28.620664","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.27.620493","pub_date":"2024-10-29","title":"Unveiling Humoral and Cellular Immune Responses to SARS-CoV-2 in Head and Neck Cancer: A Comparative Study of Vaccination and Natural Infection in Romania","abstract":"To fill the knowledge gap regarding the antiviral immunity in oncologic patients, we performed a comparative study on natural/vaccine-induced SARS-CoV-2 immunity in head and neck cancer (HNC) in Romania. Blood was collected from HNC (n=49) and controls (n=14), stratified as vaccinated (RNA/adenovirus-based vaccines), convalescent, and hybrid immunity. Plasma IgG/IgA antibodies (Abs) against Spike (S1/S2), receptor binding domain (RBD), and nucleocapsid (NC), and cytokines were quantified using the MILLIPLEX\u00ae technology. The frequency/phenotype/isotype of RBD-specific B-cells were studied by flow cytometry using tetramers (Tet++). Cell proliferation in response to Spike/NC peptides was monitored by carboxyfluorescein succinimidyl ester (CFSE) assay. A longitudinal follow-up was performed on n=25 HNC. Levels of S1/S2/RBD-specific IgG/IgA Abs were similarly high in HNC and controls, but significantly increased in convalescent/hybrid versus vaccinated HNC. NC-specific IgG/IgA Abs were only detected in convalescent/hybrid immunity groups. The frequency of Tet++ B-cells in HNC was similar to controls, irrespective of the immunization status, and correlated positively with RBD IgG/IgA Abs and negatively with the time since immunization (TSI). Compared to total B-cells, Tet++ were enriched in CD27+ memory phenotype and IgG/IgA isotype. A linear regression model identified Spike S2 IgG and NC IgA Abs as strong positive predictors of Tet++ frequencies, while IL-6 was a marginally significant negative predictor. Tet++ frequency remained stable at median TSI of 341 versus 117 days, despite a decline in memory phenotype. HNC participants mount efficient and durable SARS-CoV-2 humoral immunity, with RBD-specific IgG/IgA Abs and Tet++ B-cells representing the major immunization outcomes.","version":"1.1","doi":"10.1101/2024.10.27.620493","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.14.579654","pub_date":"2024-10-29","title":"Immune escape and replicative capacity of Omicron lineages BA.1, BA.2, BA.5.1, BQ.1, XBB.1.5, EG.5.1 and JN.1.1","abstract":"In the 5th year after the emergence of SARS-CoV-2, Omicron lineages continue to evolve and cause infections. Here, we used eight authentic SARS-CoV-2 isolates to assess their capacity to escape immunity of different exposure histories and their replicative capacity in polarized human airway epithelial cells (HAE) derived from the nasal and bronchial epithelium. Using live-virus neutralization assays of 108 human sera or plasma of different immunological backgrounds, progressive immune escape was observed from B.1 (ancestral virus) to EG.5.1, but no significant difference between EG.5.1 and JN.1.1. Vaccinated individuals without natural infection and individuals with a single infection, but no vaccination showed markedly reduced or completely lost neutralization against the latest variants, while in those with hybrid immunity almost all sera showed some neutralization capacity. Furthermore, although absolute titers differed between groups, the pattern of immune escape between the variants remains comparable with strongest loss of neutralization observed for the latest variants. In vitro studies with HAE at 33\u00b0C and 37\u00b0C showed some, but minor differences in virus replication and innate immune responses upon infection. Notably, infection with XBB.1.5, EG.5.1 and JN.1.1 showed slightly increased viral growth in nasal HAE at 33\u00b0C. Altogether, these data underscore increasing immune escape across heterogeneous immunological backgrounds with gradually increasing antibody escape of evolving Omicron lineages until variant EG.5.1, but not any further for the latest dominant lineage JN.1.1. They also suggest that viral dynamics within Omicron lineages are driven by a combination of immune evasion and increase in viral replication.","version":"1.2","doi":"10.1101/2024.02.14.579654","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.03.582873","pub_date":"2024-10-28","title":"Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms","abstract":"mRNA vaccines against SARS-CoV-2 were rapidly developed and effective during the pandemic. However, some limitations remain to be resolved, such as the short-lived induced immune response and certain adverse effects. Therefore, there is an urgent need to develop new vaccines that address these issues. While live-attenuated vaccines are a highly effective modality, they pose a risk of adverse effects, including virulence reversion. In the current study, we constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the NSP14, NSP1, spike and ORF7-8 coding regions. Intranasal inoculation with BK2102 induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs, leading to protection against a SARS-CoV-2 D614G and an Omicron BA.5 strains. The neutralizing antibodies induced by BK2102 persisted for up to 364 days, which indicated that they confer long-term protection against infection. Furthermore, we confirmed the safety of BK2102 using transgenic (Tg) mice expressing human ACE2 (hACE2), that are highly susceptible to SARS-CoV-2. BK2102 did not kill the Tg mice, even when virus was administered at a dose of 106 plaque-forming units (PFU), while 102 PFU of the D614G strain or an attenuated strain lacking the furin cleavage site (FCS) of the spike was sufficient to kill mice. These results suggest that BK2102 is a promising live-vaccine candidate strain that confers long-term protection without significant virulence.","version":"1.2","doi":"10.1101/2024.03.03.582873","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.27.620470","pub_date":"2024-10-28","title":"Disruption of molecular interactions between G3BP1 stress granule host protein and nucleocapsid (NTD-N) protein impedes SARS-CoV-2 virus replication","abstract":"The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of host\u2019s immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1NTF-2) and the intrinsically disordered N-terminal domain (NTD-N1-25) of the N protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N128-138) within the N-terminal domain of the N protein (NTD-N41-174) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide binding pocket in G3BP1NTF-2, not only disrupted the protein-protein interactions (PPIs) between the NTD-N41-174 and G3BP1NTF-2 but also exhibited significant antiviral efficacy against SARS-CoV-2 replication with EC50 values of \u223c1.8 \u00b5M and \u223c1.3 \u00b5M, respectively. The findings of this study, validated by biophysical thermodynamics and biochemical investigations, advance the potential of developing therapeutics targeting the SG host protein against SARS-CoV-2, which may also serve as a broad-spectrum antiviral target.","version":"1.1","doi":"10.1101/2024.10.27.620470","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.24.619902","pub_date":"2024-10-26","title":"A Novel Cyanine-Based Fluorescent Dye for Targeted Mitochondrial Imaging in Neurotoxic Conditions and In Vivo Brain Studies","abstract":"Mitochondrial dysfunction is a key feature of neurodegenerative diseases, often preceding symptoms and influencing disease progression. However, real-time in vivo imaging of mitochondria in the brain is limited by existing dyes like MitoTrackers, which struggle with poor tissue penetration, phototoxicity, and inability to cross the blood-brain barrier (BBB). This study introduces Cy5-PEG4, a novel mitochondrial-targeting dye that overcomes these limitations, enabling high-resolution, non-invasive imaging of mitochondrial dynamics. Cy5-PEG4 effectively labels mitochondria in primary neuronal cells exposed to the SARS-CoV-2 RNYIAQVD peptide, revealing dose-dependent alterations in mitochondrial function that may contribute to COVID-19-related neurodegeneration. Importantly, Cy5-PEG4 crosses the BBB without causing neuroinflammation or toxicity, making it a safe tool for in vivo brain imaging and detailed studies of mitochondrial responses. In 3D cultured cells, Cy5-PEG4 captures dynamic changes in mitochondrial distribution and morphology as cell structures mature, highlighting its potential in neurobiological research, diagnostics, and therapeutic development. These findings support Cy5-PEG4 as a powerful tool for studying disease progression, identifying early biomarkers, and evaluating therapeutic strategies in neurodegenerative disorders and COVID-19.","version":"1.1","doi":"10.1101/2024.10.24.619902","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.24.620087","pub_date":"2024-10-25","title":"Composition of saliva metabolome is significantly associated with SARS-CoV2 infection and with severity of COVID-19 disease","abstract":"The metabolome of COVID-19 patients has been studied sparsely, with most research focusing on a limited number of plasma metabolites or small cohorts. This is the first study to test saliva metabolites in COVID-19 patients in a comprehensive way, revealing significant changes linked to disease severity and highlighting saliva is potential as a non-invasive diagnostic tool. We included 30 asymptomatic subjects with no prior COVID-19 infection or vaccination, 102 patients with mild SARS-CoV-2 infection, and 61 hospitalized patients with confirmed SARS-CoV-2 status. Saliva samples were analyzed using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) in positive and negative ionization modes. Significant changes in metabolites were identified in COVID-19 patients, with distinct patterns based on disease severity. Healthy individuals exhibited a well-regulated bacterial network, while severe cases showed disordered microbial networks. Elevated dipeptides such as Val-Glu and Met-Gln in moderate cases suggest specific protease activity related to SARS-CoV-2. Increased acetylated amino acids like N-Acetylserine and N-Acetylhistidine indicate potential biomarkers for stress and disease severity. Bacterial metabolites, including muramic acid and indole-3-carboxaldehyde, were higher in mild-moderate cases, indicating oral microbiota changes. In severe cases, polyamines and organ damage-related metabolites, such as N-acetylspermine and 3-methylcytidine, were significantly increased. Interestingly, metabolites reduced in moderate cases were elevated in severe cases. Saliva metabolomics offers insight into disease progression and potential biomarkers for COVID-19.","version":"1.1","doi":"10.1101/2024.10.24.620087","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.23.609366","pub_date":"2024-10-25","title":"Comparison of immunogenicity and protection efficacy of self-amplifying and circular mRNA vaccines against SARS-CoV-2","abstract":"Recent advances in vaccine technology have positioned messenger RNA (mRNA) vaccines as safe and reliable options for human use. Conventionally, mRNA vaccines were designed using linear or self-amplifying mRNA (SAM), the latter considered to be superior. However, limited success was achieved with SAM vaccines during the COVID-19 pandemic. Further, studies on Circular mRNA (Circ-RNA) vaccines against the SARS-CoV-2, Ebola and monkey pox proved their efficacy. Circ-RNAs are highly stable, neither they induce inflammatory response nor require any extracellular protein for their function. Here, we compared the efficacy of SAM- and Circ-RNA vaccines using the SARS-CoV-2-RBD (receptor binding domain) as the antigen. Both SAM-RBD and Circ-RBD induced a comparable anti-RBD IgG titer and virus-neutralizing antibody titer. However, the latter induced a significantly higher memory T-cell response. Immunization with SAM- and Circ-RBD showed no mortality and improved lung pathophysiology against acute SARS-CoV-2 infection in mice. The Circ-RBD vaccine is stable for 4 weeks at 40C. A bivalent vaccine containing Circ-RBD of both delta and omicron SARS-CoV-2 variants potently neutralized these viruses. These findings demonstrate Circ-RNA-RBD as an excellent vaccine candidate against COVID-19 and also provide a platform for developing bivalent Circ-RNA vaccine candidates against SARS-CoV-2 or other viruses with rapidly emerging variants.","version":"1.2","doi":"10.1101/2024.08.23.609366","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.25.620241","pub_date":"2024-10-25","title":"Differential Responses of Lung and Intestinal Microbiota to SARS-CoV-2 Infection: A Comparative Study of the Wuhan and Omicron Strains in K18-hACE2 tg Mice","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2, has led to the emergence of viral variants with distinct characteristics. We investigated the differential effects of the original Wuhan strain and the emergent Omicron variant of SARS-CoV-2 using a K18-hACE2 transgenic mouse model. We compared the mortality rates, viral loads, and histopathological changes in lung and tracheal tissues, as well as alterations in the lung and intestinal microbiota following infection. We observed significant differences in disease severity, with the Wuhan strain causing higher mortality and more severe lung damage than the Omicron variant. Furthermore, microbiome analyses revealed distinct shifts in microbiota associated with infection by each variant, suggesting that microbiome-related mechanisms might influence disease outcomes. This comprehensive comparison enhances our understanding of COVID-19 pathogenesis and highlights the importance of microbiome dynamics in viral infections, providing insights for future therapeutic and preventive strategies. Understanding the differential impacts of SARS-CoV-2 variants is crucial for effective public health response and treatment development. This study provides insights into the pathogenesis of the original Wuhan strain and the Omicron variant of SARS-CoV-2, revealing significant differences in host mortality, viral load, and lung pathology. The use of the K18-hACE2 transgenic mouse model enables detailed examination of these differences in a controlled setting. Furthermore, this study highlights the importance of the microbiome in modulating disease severity and host responses to viral infections. By uncovering distinct microbial shifts associated with infection by different SARS-CoV-2 variants, this study suggests potential microbiome-related mechanisms that might be targeted to mitigate disease outcomes.","version":"1.1","doi":"10.1101/2024.10.25.620241","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.25.620187","pub_date":"2024-10-25","title":"Prevalence of Key Spike Protein Mutations and Their Limited Effect on COVID-19 Clinical Manifestations in Sylhet, Bangladesh","abstract":"SARS-CoV-2 is the virus responsible for the COVID-19 pandemic, which has spread rapidly around the world and had a significant impact on public health and the economy worldwide. This study investigated the correlation between SARS-CoV-2 spike protein mutations, clinical outcomes and patient demographics in the Sylhet region of Bangladesh. We looked at the full genome sequences of 37 SARS-CoV-2 samples that were collected between January and June 2020. Specifically, we looked at five major spike protein mutations: D614G, A570D, D1118H, A222V, and P681R. The D614G mutation was the most prevalent (94.6%), followed by A570D and D1118H (both 32.4%), A222V (29.7%), and P681R (13.5%). Despite their high prevalence, we found no statistically significant associations between these mutations and clinical outcomes or demographic variables, except for possible trends for the P681R mutation. We found that age played a decisive role in recovery from COVID-19, with older patients exhibiting slower recovery rates. In terms of predictors of outcome, gender differences were observed: clinical symptoms and viral genetic mutations were more influential for men, while age and disease progression were more important for women. Common nucleotide substitutions (A23403G, C3037T, and C14408T) associated with European strains were identified, suggesting possible routes of transmission. This study contributes to our understanding of the genetics, clinical manifestations and epidemiology of SARS-CoV-2 in the Sylhet region and emphasizes the need for continuous genomic surveillance and adaptive public health strategies.","version":"1.1","doi":"10.1101/2024.10.25.620187","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.25.620289","pub_date":"2024-10-25","title":"Long-term Immunity of a Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice","abstract":"COVID-19 vaccines effectively prevent symptomatic infection and severe disease, including hospitalization and death. However, unequal vaccine distribution during the pandemic, especially in low- and middle-income countries, has led to the emergence of vaccine-resistant strains. This underscores the need for alternative, safe, and thermostable vaccine platforms, such as dissolved microneedle array patches (MAP) delivering a subunit vaccine, which eliminate the need for cold chain and trained healthcare personnel. This study demonstrates that the SARS-CoV-2 S1 monomer with RS09, a TLR-4 agonist peptide, serves as an optimal protein subunit immunogen. This combination stimulates a stronger S1-specific immune response, resulting in binding to the membrane-bound spike on the cell surface and ACE2-binding inhibition, compared to the monomer S1 alone or trimer S1, regardless of RS09. MAP delivery of the rS1RS09 subunit vaccine elicited higher and longer-lasting immunity compared to conventional intramuscular injection. S1-specific IgG levels remained significantly elevated for up to 70 weeks post-administration. Additionally, different doses of 5, 15, and 45 \u03bcg of MAP vaccines induced robust and sustained Th2-prevalent immune responses, suggesting a dose-sparing effect and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants at 15 and 45 \u03bcg dose. Moreover, gamma irradiation as a terminal sterilization method did not significantly affect immunogenicity, with irradiated vaccines maintaining comparable efficacy to non-irradiated ones. The stability of MAP vaccines was evaluated after long-term storage at room temperature and refrigeration for 19 months, showing minimal protein degradation. Further, after an additional one-month of storage at elevated temperature (42\u00b0C), rS1RS09 in both non-irradiated and irradiated MAP degraded less than 3%, while the liquid subunit vaccine degraded over 23%. Overall, these results indicate that gamma irradiation sterilized MAP-rS1RS09 vaccines maintain stability during extended storage without refrigeration, supporting their potential for mass production and widespread use in global vaccination efforts.","version":"1.1","doi":"10.1101/2024.10.25.620289","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.24.620075","pub_date":"2024-10-24","title":"SARS-CoV-2 within-host population expansion, diversification and adaptation in zoo tigers, lions and hyenas","abstract":"SARS-CoV-2 rapidly adapts to new hosts following cross-species transmission; this is highly relevant as novel within-host variants have emerged following infection of susceptible wild and domestic animal species. Furthermore, SARS-CoV-2 transmission from animals (e.g., white-tailed deer, mink, domestic cats, and others) back to humans has also been observed, documenting the potential of novel animal-derived variants to infect humans. We investigated SARS-CoV-2 evolution and host-specific adaptation during an outbreak in Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) at Denver Zoo in late 2021. SARS-CoV-2 genomes from longitudinal samples collected from 16 individuals were evaluated for within-host variation and genomic signatures of selection. The outbreak was likely initiated by a single spillover of a rare Delta sublineage subsequently transmitted from tigers to lions to hyenas. Within-host virus populations rapidly expanded and diversified. We detected signatures of purifying and positive selection, including strong positive selection in hyenas and in the nucleocapsid (N) gene in all animals. Four candidate species-specific adaptive mutations were identified: N A254V in lions and hyenas, and ORF1a E1724D, spike T274I, and N P326L in hyenas. These results reveal accelerated SARS-CoV-2 adaptation following host shifts in three non-domestic species in daily contact with humans.","version":"1.1","doi":"10.1101/2024.10.24.620075","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.24.620034","pub_date":"2024-10-24","title":"Effect of cysteine oxidation in SARS-CoV-2 Spike protein on its conformational changes: insights from atomistic simulations","abstract":"This study investigates the effect of cysteine (Cys) oxidation on the conformational changes of the SARS-CoV-2 Spike (S) protein, a critical factor in viral attachment and entry into host cells. Using targeted molecular dynamics (TMD) simulations, we explore the conformational transitions between the down (inaccessible) and up (accessible) states of the SARS-CoV-2 S protein in both its native and oxidized forms. Our findings reveal that oxidation significantly increases the energy barrier for these transitions, as indicated by the work required to move from the down to the up conformation and vice versa. Specifically, in the oxidized system compared to the native system, the energy required to transition from the down to the up conformation increases by approximately 131 \u00b1 1 kJ.mol\u22121, while the energy required for the reverse transition increases by about 223 \u00b1 6 kJ.mol\u22121. This is due to the stabilizing effect of oxidation on the conformation of the SARS-CoV-2 S protein. Analysis of hydrogen bond and salt bridge formation before and after oxidation provides additional insights into the stabilization mechanisms, showing an increase in salt bridge formation that contributes to conformational stabilization. These results underscore the potential of targeting translational modifications to hamper viral entry or enhance susceptibility to neutralization, offering a novel perspective for antiviral strategy development against SARS-CoV-2. This study adds important knowledge to the field of viral protein dynamics and highlights the critical role of structural and computational biology in uncovering new therapeutic avenues.","version":"1.1","doi":"10.1101/2024.10.24.620034","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.14.598983","pub_date":"2024-10-24","title":"AIVE: accurate predictions of SARS-CoV-2 infectivity from comprehensive analysis","abstract":"An unprecedented amount of SARS-CoV-2 data has been accumulated compared with previous infectious diseases, enabling insights into its evolutionary process and more thorough analyses. This study investigates SARS-CoV-2 features as it evolved to evaluate its infectivity. We examined viral sequences and identified the polarity of amino acids in the Receptor Binding Motif (RBM) region. We detected an increased frequency of amino acid substitutions to lysine (K) and arginine (R) in Variants of Concern (VOCs). As the virus evolved to Omicron, commonly occurring mutations became fixed components of the new viral sequence. Furthermore, at specific positions of VOCs, only one type of amino acid substitution and a notable absence of mutations at D467 was detected. We found that the binding affinity of SARS-CoV-2 lineages to the ACE2 receptor was impacted by amino acid substitutions. Based on our discoveries, we developed APESS, an evaluation model evaluating infectivity from biochemical and mutational properties. In silico evaluation using real-world sequences and in vitro viral entry assays validated the accuracy of APESS and our discoveries. Using Machine Learning, we predicted mutations that had the potential to become more prominent. We created AIVE, a web-based system, accessible at https://ai-ve.org to provide infectivity measurements of mutations entered by users. Ultimately, we established a clear link between specific viral properties and increased infectivity, enhancing our understanding of SARS-CoV-2 and enabling more accurate predictions of the virus.","version":"1.2","doi":"10.1101/2024.06.14.598983","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.09.612101","pub_date":"2024-10-24","title":"The Conformational Space of the SARS-CoV-2 Main Protease Active Site Loops is Determined by Ligand Binding and Interprotomer Allostery","abstract":"The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and is, therefore, an important drug target. Here, we investigate two flexible loops in Mpro that play a role in catalysis. Using all-atom molecular dynamics simulations, we analyze the structural ensemble of Mpro in an apo state and substrate-bound state. We find that the flexible loops can adopt open, intermediate (partly open) and closed conformations in solution, which differs from the partially closed state observed in crystal structures of Mpro. When the loops are in closed or intermediate states, the catalytic residues are more likely to be in close proximity, which is crucial for catalysis. Additionally, we find that substrate binding to one protomer of the homodimer increases the frequency of intermediate states in the bound protomer, while also affecting the structural propensity of the apo protomer\u2019s flexible loops. Using dynamic network analysis, we identify multiple allosteric pathways connecting the two active sites of the homodimer. Common to these pathways is an allosteric hotspot involving the N-terminus, a critical region that comprises part of the binding pocket. Taken together, the results of our simulation study provide detailed insight into the relationships between the flexible loops and substrate binding in a prime drug target for COVID-19.","version":"1.3","doi":"10.1101/2024.09.09.612101","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.23.619886","pub_date":"2024-10-23","title":"Recent Endemic Coronavirus Infection Associates With Higher SARS-CoV-2 Cross-Reactive Fc Receptor Binding Antibodies","abstract":"Recent documented infection with an endemic coronavirus (eCoV) associates with less severe coronavirus disease 2019 (COVID-19), yet the immune mechanism behind this protection has not been fully explored. We measured both antibody and T cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in SARS-CoV-2 na\u00efve individuals classified into two groups, either with or without presumed recent eCoV infections. There was no difference in neutralizing antibodies and T cell responses against SARS-CoV-2 antigens between the two groups. SARS-CoV-2 na\u00efve individuals with recent presumed eCoV infection, however, had higher levels of Fc receptor (FcR) binding antibodies against eCoV spikes (S) and SARS-CoV-2 S2. There was also a significant correlation between eCoV and SARS-CoV-2 FcR binding antibodies. Recent eCoV infection boosts cross-reactive antibodies that can mediate Fc effector functions, and this may play a role in the observed heterotypic immune protection against severe COVID-19.","version":"1.1","doi":"10.1101/2024.10.23.619886","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.23.619479","pub_date":"2024-10-23","title":"Rewired type I IFN signaling is linked to age-dependent differences in COVID-19","abstract":"Advanced age is the most important risk factor for severe disease or death from COVID-19, but a thorough mechanistic understanding of the molecular and cellular underpinnings is lacking. Multi-omics analysis of samples from SARS-CoV-2 infected persons aged 1 to 84 years, revealed a rewiring of type I interferon (IFN) signaling with a gradual shift from signal transducer and activator of transcription 1 (STAT1) to STAT3 activation in monocytes, CD4+ T cells and B cells with increasing age. Diversion of interferon IFN signaling was associated with increased expression of inflammatory markers, enhanced release of inflammatory cytokines, and delayed contraction of infection-induced CD4+ T cells. A shift from IFN-responsive germinal center B (GCB) cells towards CD69high GCB and atypical B cells corresponded to the formation of IgA in children while complement fixing IgG was dominant in adults. Our data provide a mechanistic basis for inflammation-prone responses to infections and associated pathology during aging.","version":"1.1","doi":"10.1101/2024.10.23.619479","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.23.619754","pub_date":"2024-10-23","title":"Enhanced immune evasion of SARS-CoV-2 KP.3.1.1 and XEC through NTD glycosylation","abstract":"KP.3.1.1 has surpassed KP.3 to become the new globally dominant strain, while XEC, a recombinant variant of KS.1.1/KP.3.3, is rapidly expanding across Europe and North America. Notably, both variants carry mutations, S31del of KP.3.1.1 and T22N of XEC, that could introduce new N-linked glycans on the Spike N-terminal domain (NTD), emphasizing the urgent need to assess their potential changes in viral characteristics. Here, we found that both KP.3.1.1 and XEC maintained the high ACE2-Spike binding affinity and pseudovirus infectivity of KP.3. Importantly, compared to KP.3, KP.3.1.1, and especially XEC, could further evade the neutralizing antibodies in convalescent plasma, even those elicited by KP.2-like breakthrough infections. Interestingly, both variants demonstrated increased resistance against monoclonal neutralizing antibodies targeting various epitopes on the receptor-binding domain (RBD). These suggest that the additional NTD glycosylation of KP.3.1.1 and XEC could enhance immune evasion via allosteric effects, and supports the future prevalence of XEC.","version":"1.1","doi":"10.1101/2024.10.23.619754","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.22.604276","pub_date":"2024-10-23","title":"Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing","abstract":"Nanopore direct RNA sequencing (dRNA-seq) enables unique insights into (epi-)transcriptomics. However, applications are currently limited by the lack of accurate and cost-effective sample multiplexing. We introduce WarpDemuX, an ultra-fast and highly accurate adapter-barcoding and demultiplexing approach. WarpDemuX enhances speed and accuracy by fast processing of the raw nanopore signal, use of a lightweight machine-learning algorithm and design of optimized barcode sets. We demonstrate its utility by performing a rapid phenotypic profiling of different SARS-CoV-2 viruses, crucial for pandemic prevention and response, through multiplexed sequencing of longitudinal samples on a single flowcell. This identifies systematic differences in transcript abundance and poly(A) tail lengths during infection. Additionally, integrating WarpDemuX into sequencing control software enables real-time enrichment of target molecules through barcode-specific adaptive sampling, which we demonstrate by enriching low abundance viral RNA. In summary, WarpDemuX is a broadly applicable, high-performance, and economical multiplexing solution for nanopore dRNA-seq, facilitating advanced (epi-)transcriptomic research.","version":"1.2","doi":"10.1101/2024.07.22.604276","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.07.583829","pub_date":"2024-10-22","title":"In silico genomic surveillance by CoVerage predicts and characterizes SARS-CoV-2 Variants of Interest","abstract":"Rapidly evolving viral pathogens such as SARS-CoV-2 continuously accumulate amino acid changes, some of which affect transmissibility, virulence or improve the virus\u2019 ability to escape host immunity. Since the beginning of the pandemic and establishment of SARS-CoV-2 as a human pathogen, multiple lineages with concerning phenotypic alterations, so called Variants of Concern (VOCs), have emerged and risen to predominance. To optimize public health management and to ensure the continued efficacy of vaccines, the early detection of such variants of interest is essential. Therefore, large-scale viral genomic surveillance programs have been initiated worldwide, with data being deposited in public repositories in a timely manner. However, technologies for their continuous interpretation are currently lacking. Here, we describe the CoVerage system (www.sarscoverage.org) for viral genomic surveillance, which continuously predicts and characterizes novel and emerging potential Variants of Interest (pVOIs) from country-wise lineage frequency dynamics together with their antigenic and evolutionary alterations utilizing the GISAID viral genome resource. In a comprehensive assessment of VOIs, VOCs and VUMs identified, we demonstrate how CoVerage can be used to swiftly identify and characterize such variants, with a lead time of almost three months relative to them reaching their maximal abundances. CoVerage can facilitate the timely identification and assessment of future SARS-CoV-2 variants relevant for public health.","version":"1.2","doi":"10.1101/2024.03.07.583829","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.15.532790","pub_date":"2024-10-22","title":"HuR enhances SARS-CoV-2 non-structural protein translation through the genomic 5\u2019-UTR, by promoting polypyrimidine tract-binding protein binding","abstract":"Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) viral RNA associates with different RNA-binding host proteins at each stage of its life cycle. We found sequence dependent binding of one such important protein, human antigen R (HuR) to SARS-CoV-2 5\u2032UTR and studied its potential role in virus life cycle. The knockdown and knockout studies revealed importance of such binding in viral translation. We identified 5\u2032-UTR mutations in SARS-CoV-2 variants of concern that altered the HuR-binding affinity. Interestingly, HuR enhanced non-structural protein translation through the genomic 5\u2032-UTR, by promoting polypyrimidine tract-binding protein binding to the 5\u2032-UTR. However, HuR suppressed the structural protein translation from sub genomic 5\u2019UTR. HuR knockout increased the sensitivity to remdesivir treatment by decreasing its half-maximal inhibitory concentration. An antisense oligonucleotide (whose binding site overlapped the HuR-binding site) reduced viral RNA levels in wild-type cells but not HuR-knockout cells. Our results indicate that HuR regulates the balance between SARS-CoV-2 structural and non-structural proteins and guides the infection of viral variants, implying that HuR can potentially be targeted for therapeutic interventions. Viruses interacts with various host proteins throughout their life cycle. One significant protein is HuR, an RNA-binding protein that regulates RNA stability and translation. HuR binds to viral RNAs at the 5\u2019UTR or 3\u2019UTR, impacting their translation and replication. We identified conserved HuR binding sites in the SARS-CoV-2 5\u2019UTR across different beta coronaviruses. This binding enhanced the initiation of translation from the genomic 5\u2019 UTR, increasing the production of non-structural proteins essential for viral replication. Additionally, we discovered that another host protein, PTB, promotes HuR binding to the viral 5\u2019 UTR, facilitating its loading onto ribosomes. Conversely, HuR plays an antagonistic role concerning subgenomic RNAs (sgRNAs), which code for structural proteins, by regulating and limiting their levels. This dual regulation indicates that the virus exploits HuR for its benefit while the host employs it to control viral spread. Targeting HuR may help manipulate the SARS-CoV-2 life cycle. We found that HuR knockout increased sensitivity to the antiviral drug Remdesivir. Using an antisense oligonucleotide to block HuR binding effectively reduced viral RNA levels. Our findings highlight the critical role of HuR in regulating viral protein production and its potential as a therapeutic target.","version":"1.2","doi":"10.1101/2023.03.15.532790","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.19.619238","pub_date":"2024-10-22","title":"Inter-laboratory harmonization of microsphere immunoassays for SARS-CoV-2 antibody detection in dried blood spots and oral fluids","abstract":"Dried blood spots (DBS) and oral fluids (OF) are easily attainable biospecimen types that have enabled population scale antibody monitoring for SARS-CoV-2 exposure and vaccination. However, the degree to which the two different biospecimen types can be used interchangeably remains unclear. To begin to address this question, we generated contrived DBS (cDBS) and OF (cOF) from serum panels from SARS-CoV-2 infected, vaccinated, and uninfected individuals. The contrived samples were evaluated using SARS-CoV-2 multiplexed microsphere immunoassays (MIAs) at two different institutions. Intra-laboratory tests revealed near perfect agreement between cDBS and cOF for N and S antigens, as evidenced by \u03ba = 0.97-1 and 98%-100% agreement. Inter-laboratory comparisons were equally robust for both N (\u03ba = 0.94-0.96; 97.5%-98 % agreement) and S (\u03ba = 0.98 -1.0; 99.0%-100%). Furthermore, assays were transferred between labs, including methods and reagents, and a subset of cDBS and cOF samples (n = 52) were tested. Qualitative concordance remained high (\u03ba = 0.94-1.0; 97.5%-100% agreement), confirming that integrity of the assays is retained upon transfer. In summary, our results provide evidence that DBS and OF can be used interchangeably across laboratories and institutions for the qualitative assessment of SARS-CoV-2 antibody determinations.","version":"1.1","doi":"10.1101/2024.10.19.619238","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.22.619639","pub_date":"2024-10-22","title":"Interferon-mediated NK cell activation is associated with limited neutralization breadth during SARS-CoV-2 infection","abstract":"Best known for their ability to kill infected or malignant cells, natural killer (NK) cells are also underappreciated regulators of the antibody response to viral infection. In mice, NK cells can kill T follicular helper (Tfh) cells, decreasing somatic hypermutation and vaccine responses. Although human NK cell activation correlates with poor vaccine response, the mechanisms of human NK cell regulation of adaptive immunity are poorly understood. We found that in human ancestral SARS-CoV-2 infection, broad neutralizers, who were capable of neutralizing Alpha, Beta, and Delta, had fewer NK cells that expressed inhibitory and immaturity markers whereas NK cells from narrow neutralizers were highly activated and expressed interferon-stimulated genes (ISGs). ISG-mediated activation in NK cells from healthy donors increased cytotoxicity and functional responses to induced Tfh-like cells. This work reveals that NK cell activation and dysregulated inflammation may play a role in poor antibody response to SARS-CoV-2 and opens exciting avenues for designing improved vaccines and adjuvants to target emerging pathogens.","version":"1.1","doi":"10.1101/2024.10.22.619639","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.22.619692","pub_date":"2024-10-22","title":"Engineered protein destabilization reverses intrinsic immune evasion for candidate vaccine pan-strain KSHV and SARS-CoV-2 antigens","abstract":"Both Kaposi sarcoma herpesvirus LANA and SARS coronavirus 2 RdRp/nsp12 are highly conserved replication proteins that evade immune processing. By deleting the LANA central repeat 1 domain (LANA\u0394CR1) or by dividing RdRp into two separated fragments (RdRpFrag) to maximize nascent protein mis-folding, cis peptide presentation was increased. Native LANA or RdRp SIINFEKL fusion proteins expressed in MC38 cancer cells were not recognized by activated OT-1 CD8+ cells against SIINFEKL but cytotoxic recognition was restored by expression of the corresponding modified proteins. Immunocompetent syngeneic mice injected with LANA- or RdRp-SIINFEKL MC38 cells developed rapidly-growing tumors with short median survival times. Mice injected with LANA\u0394CR1- or RdRpFrag-SIINFEKL had partial tumor regression, slower tumor growth, longer median survival, as well as increased effector-specific tumor-infiltrating lymphocytes. These mice developed robust T cell responses lasting at least 90 days post-injection that recognized native viral protein epitopes. Engineered vaccine candidate antigens can unmask virus-specific CTL responses that are typically suppressed during native viral infection.","version":"1.1","doi":"10.1101/2024.10.22.619692","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.19.585745","pub_date":"2024-10-22","title":"srahunter: a user-friendly tool to speed up and simplify data downloading from NCBI SRA","abstract":"Easy access and use of vast datasets are paramount for advancing scientific discovery in steadily expanding studies based on high-throughput sequencing (HTS). The Sequence Read Archive (SRA) is a publicly accessible repository currently holding a huge amount of HTS reads, as part of the International Nucleotide Sequence Database Collaboration (INSDC). However, accessing, downloading, and managing data and metadata efficiently can be challenging. Here, we introduce srahunter, a tool designed to simplify data and metadata acquisition from SRA. Developed with Python, srahunter leverages the core functionalities of SRA Toolkit and Entrez Direct, to enable automated downloading, smart data management, and user-friendly metadata integration through an interactive HTML table https://github.com/GitEnricoNeko/srahunter. Compared to existing tools, srahunter increases the efficiency of metadata retrieval by reducing the technical barriers to SRA data and streamlining the handling of SRA datasets, and can therefore accelerate the development of genomics and multiple \u2018omics research.","version":"1.2","doi":"10.1101/2024.03.19.585745","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.21.619398","pub_date":"2024-10-21","title":"This is SPRTA: assessing phylogenetic confidence at pandemic scales","abstract":"Phylogenetics plays a central role in evolutionary biology and genomic epidemiology. Assessing phylogenetic confidence and reliability is therefore crucial and methods to do this, such as Felsenstein\u2019s bootstrap, are among the most used in modern science. However, methods based on Felsenstein\u2019s bootstrap suffer from excessive computational demand, and are unsuitable for large datasets. Furthermore, most of these methods emerge from a cladistic framework which makes their results hard to interpret in the context of genomic epidemiology. We propose SPRTA (\u201c SPR-based Tree Assessment\u201d), an efficient and interpretable approach to assess confidence in phylogenetic trees. SPRTA shifts the paradigm of phylogenetic support measurement from evaluating the confidence in clades (groupings of taxa) to genome evolution histories, for example assessing if a lineage evolved from another considered lineage or not. This focus on evolutionary histories is particularly valuable in genomic epidemiology, where typically the evolutionary and transmission history of a pathogen are of interest, not clade content. We illustrate the use of SPRTA by investigating a global SARS-CoV-2 phylogenetic tree relating > 2M genomes, highlighting plausible alternative evolutionary origins of many SARS-CoV-2 variants. We have implemented SPRTA within the free and open source maximum likelihood phylogenetic software MAPLE, available from https://github.com/NicolaDM/MAPLE.","version":"1.1","doi":"10.1101/2024.10.21.619398","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.21.619361","pub_date":"2024-10-21","title":"Integrative Transcriptomic Profiling of NK Cells and Monocytes: Advancing Diagnostic and Therapeutic Strategies for COVID-19","abstract":"In this study, we use integrated transcriptomic datasets from the GEO repository with the purpose of investigating immune dysregulation in COVID-19. Thus, in this context, we decided to be focused on NK cells and CD14+ monocytes gene expression, considering datasets GSE165461 and GSE198256, respectively. Other datasets with PBMCs, lung, olfactory, and sensory epithelium and lymph were used to provide robust validation for our results. This approach gave an integrated view of the immune responses in COVID-19, pointing out a set of potential biomarkers and therapeutic targets with special regard to standards of physiological conditions. IFI27, MKI67, CENPF, MBP, HBA2, TMEM158, THBD, HBA1, LHFPL2, SLA, and AC104564.3 were identified as key genes from our analysis that have critical biological processes related to inflammation, immune regulation, oxidative stress, and metabolic processes. Consequently, such processes are important in understanding the heterogeneous clinical manifestations of COVID-19\u2014from acute to long-term effects now known as \u2018long COVID\u2019. Subsequent validation with additional datasets consolidated these genes as robust biomarkers with an important role in the diagnosis of COVID-19 and the prediction of its severity. Moreover, their enrichment in key pathophysiological pathways presented them as potential targets for therapeutic intervention.The results provide insight into the molecular dynamics of COVID-19 caused by cells such as NK cells and other monocytes. Thus, this study constitutes a solid basis for targeted diagnostic and therapeutic development and makes relevant contributions to ongoing research efforts toward better management and mitigation of the pandemic.","version":"1.1","doi":"10.1101/2024.10.21.619361","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.18.619137","pub_date":"2024-10-19","title":"Nirmatrelvir-Resistant Mutations in SARS-CoV-2 Mpro Enhance Host Immune Evasion via Cleavage of NF-\u03baB Essential Modulator","abstract":"Nirmatrelvir is a SARS-CoV-2 Mpro inhibitor in Paxlovid. Patients treated with it often produce mutant viruses in which the Mpro resists Nirmatrelvir inhibition. A common interpretation is that the mutations allow the virus to escape inhibition, but here we report that these mutations enable the protease to more effectively cleave the host protein NF-kappa-B essential modulator (NEMO), which weakens the immune response, improves viral replication, and may contribute to long COVID.","version":"1.1","doi":"10.1101/2024.10.18.619137","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.16.618773","pub_date":"2024-10-17","title":"Virological characteristics of the SARS-CoV-2 XEC variant","abstract":"The SARS-CoV-2 JN.1 variant (BA.2.86.1.1), arising from BA.2.86.1 with spike protein (S) substitution S:L455S, outcompeted the previously predominant XBB lineages by the beginning of 2024. Subsequently, JN.1 subvariants including KP.2 (JN.1.11.1.2) and KP.3 (JN.1.11.1.3), which acquired additional S substitutions (e.g., S:R346T, S:F456L, and S:Q493E), have emerged concurrently. As of October 2024, KP.3.1.1 (JN.1.11.1.3.1.1), which acquired S:31del, outcompeted other JN.1 subvariants including KP.2 and KP.3 and is the most predominant SARS-CoV-2 variant in the world. Thereafter, XEC, a recombinant lineage of KS.1.1 (JN.13.1.1.1) and KP.3.3 (JN.1.11.1.3.3), was first identified in Germany on August 7, 2024. XEC acquired two S substitutions, S:T22N and S:F59S, compared with KP.3 through recombination, with a breakpoint at genomic position 21,738\u201322,599. We estimated the relative effective reproduction number (Re) of XEC using a Bayesian multinomial logistic model based on genome surveillance data from the USA, the United Kingdom, France, Canada, and Germany, where this variant has spread as of August 2024. In the USA, the Re of XEC is 1.13-fold higher than that of KP.3.1.1. Additionally, the other countries under investigation herein showed higher Re for XEC. These results suggest that XEC has the potential to outcompete the other major lineage including KP.3.1.1. We then assessed the virological properties of XEC using pseudoviruses. Pseudovirus infection assay showed that the infectivity of KP.3.1.1 and XEC was significantly higher than that of KP.3. Although S:T22N did not affect the infectivity of the pseudovirus based on KP.3, S:F59S significantly increased it. Neutralization assay was performed using three types of human sera: convalescent sera after breakthrough infection (BTI) with XBB.1.5 or KP.3.3, and convalescent sera after JN.1 infection. In all serum groups, XEC as well as KP.3.1.1 showed immune resistance when compared to KP.3 with statistically significant differences. In the cases of XBB.1.5 BTI sera and JN.1 infection sera, the 50% neutralization titers (NT50s) of XEC and KP.3.1.1 were comparable. However, we revealed that the NT50 of XEC was significantly (1.3-fold) lower than that of KP.3.1.1. Moreover, both S:T22N and S:F59S significantly (1.5-fold and 1.6-fold) increased the resistance to KP.3.3 BTI sera. Here we showed that XEC exhibited higher pseudovirus infectivity and higher immune evasion than KP.3. Particularly, XEC exhibited more robust immune resistance to KP.3.3 BTI sera than KP.3.1.1. Our data suggest that the higher Re of XEC than KP.3.1.1 is attributed to this property and XEC will be a predominant SARS-CoV-2 variant in the world in the near future.","version":"1.1","doi":"10.1101/2024.10.16.618773","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.14.617443","pub_date":"2024-10-16","title":"SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo","abstract":"In late 2019, SARS-CoV-2 spilled-over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions, and compare species, sex, age, virus and antibody detection assay, and types, frequency and length of clinical signs, demonstrating broad heterogeneity amongst infections. We then describe a SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for four months. Lions had significantly more viral RNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health. Surveillance and experimental testing have shown many animal species, including companion, wildlife, and conservatory, are susceptible to SARS-CoV-2. Early in the COVID-19 pandemic, big cats at zoological institutions were among the first documented cases of naturally infected animals; however, challenges in the ability to collect longitudinal samples in zoo animals have limited our understanding of SARS-CoV-2 kinetics and clearance in these settings. We measured SARS-CoV-2 infections over three months in lions, tigers and hyenas at Denver Zoo, and detected viral RNA, infectious virus, neutralizing antibodies, and recrudescence after initial clearance. We found lions had longer and higher levels of virus compared to the other species. All animals were infected by a rare viral lineage circulating in the human population, suggesting a single spillover followed by interspecies transmission. These data are important in better understanding natural SARS-CoV-2 spillover, spread and infection kinetics within multiple species of zoo animals.","version":"1.1","doi":"10.1101/2024.10.14.617443","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.15.618435","pub_date":"2024-10-16","title":"Mechanistic insights into the activity of SARS-CoV-2 RNA polymerase inhibitors using single-molecule FRET","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in significant global mortality and disruption. Despite extensive research, the precise molecular mechanisms underlying SARS-CoV-2 replication remain unclear. To address this, we developed a single-molecule F\u00f6rster resonance energy transfer (smFRET) assay to directly visualize and analyse in vitro RNA synthesis by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We purified the minimal replication complex, comprising nsp12, nsp7, and nsp8, and combined it with fluorescently labelled RNA substrates, enabling real-time monitoring of RNA primer elongation at the single-molecule level. This platform allowed us to investigate the mechanisms of action of key inhibitors of SARS-CoV-2 replication. In particular, our data provides evidence for remdesivir\u2019s mechanism of action, which involves polymerase stalling and subsequent chain termination dependent on the concentration of competing nucleotide triphosphates. Our study demonstrates the power of smFRET to provide dynamic insights into SARS-CoV-2 replication, offering a valuable tool for antiviral screening and mechanistic studies of viral RdRp activity.","version":"1.1","doi":"10.1101/2024.10.15.618435","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.10.515993","pub_date":"2024-10-16","title":"Intranasal self-amplifying RNA SARS-CoV-2 vaccine produces protective respiratory and systemic immunity and prevents viral transmission","abstract":"While mRNA vaccines have been effective in combating SARS-CoV-2, waning of vaccine-induced antibody responses and lack of vaccine-induced respiratory tract immunity contribute to ongoing infection and transmission. In this work, we compare and contrast intranasal (i.n.) and intramuscular (i.m.) administration of a SARS-CoV-2 self-amplifying RNA (saRNA) vaccine delivered by a nanostructured lipid carrier (NLC). Both i.m. and i.n. vaccines induce potent systemic serum neutralizing antibodies, bone marrow-resident IgG-secreting cells, and splenic T cell responses. The i.n. vaccine additionally induces robust respiratory mucosal immune responses, including SARS-CoV-2-reactive lung-resident memory and lung-homing T cell populations. As a booster following previous i.m. vaccination, the i.n. vaccine also elicits the development of mucosal virus-specific T cells. Both the i.m. and i.n. administered vaccines durably protect hamsters from infection-associated morbidity upon viral challenge, significantly reducing viral loads and preventing challenged hamsters from transmitting virus to na\u00efve cagemates. This saRNA-NLC vaccine\u2019s potent systemic immunogenicity, and additional mucosal immunogenicity when delivered i.n., may be key for combating SARS-CoV-2 and other respiratory pathogens.","version":"1.3","doi":"10.1101/2022.11.10.515993","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.20.529306","pub_date":"2024-10-15","title":"Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease","abstract":"The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis. Viral proteases can strategically target human proteins to manipulate host biochemistry during infection. Here, we show that the SARS-CoV-2 main protease (Mpro) can specifically recognize and cleave the human tRNA methyltransferase enzyme TRMT1, and that cleavage of TRMT1 cripples its ability to install a key modification on human tRNAs that is critical for protein translation. Our structural and functional analysis of the Mpro-TRMT1 interaction shows how the flexible Mpro active site engages a conserved sequence in TRMT1 in an uncommon binding mode to catalyze its cleavage and inactivation. These studies provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.","version":"1.4","doi":"10.1101/2023.02.20.529306","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.12.617998","pub_date":"2024-10-14","title":"Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water","abstract":"Biogenic silver nanoparticles have been reported as good antimicrobial candidates. Thus, this study investigated the antiviral activity of silver nanoparticles synthesized against SARS-CoV-2. The silver nanoparticle was biosynthesized using leave extracts of Moringa oleifera (AgNPmo) and characterized using UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy, and X-ray diffractometry (XRD). The AgNPmo was first tested on clinical bacterial isolates, Pseudomonas aeruginosa (ATTC 154423) and Staphylococcus aureus (ATTC 209233), to ascertain its antimicrobial potential. In-vitro studies were also conducted to determine the cytotoxicity effect of AgNPmo on Vero cells. The efficacy and concentration of AgNPmo against SARS-CoV-2 were evaluated using a qPCR assay in a dose-dependent manner. The results demonstrated the successful biosynthesis and characterization of AgNPmo and its efficacy against the bacterial isolates. The AgNPmo showed low toxicity on the Vero cells. The IC50 from the cytotoxicity assay demonstrated the antiviral activity of the AgNPmo on the SARS-CoV-2 virus, leading to an increase in the Cycle threshold values, notably at 48 hours of incubation and at low concentrations. The results showed that the biogenic AgNPmo synthesized was cost-effective and showed both antimicrobial and antiviral potentials. These findings suggest that the nanoparticles could be a promising alternative for combating SARS-CoV-2, especially for water purification and preventing transmission.","version":"1.1","doi":"10.1101/2024.10.12.617998","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.13.618053","pub_date":"2024-10-14","title":"Impact of an Oxidative RNA Lesion on in vitro Replication Catalyzed by SARS-CoV-2 RNA-dependent RNA Polymerase","abstract":"The production of reactive oxygen species in response to RNA virus infection results in the oxidation of viral genomic RNA within infected cells. These oxidative RNA lesions undergo replication catalyzed by the viral replisome. G to U transversion mutations are frequently observed in the SARS-CoV-2 genome and may be linked to the replication process catalyzed by RNA-dependent RNA polymerase (RdRp) past the oxidative RNA lesion 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG). To better understand the mechanism of viral RNA mutagenesis, it is crucial to elucidate the role of RdRp in replicating across oxidative lesions. In this study, we investigated the RNA synthesis catalyzed by the reconstituted SARS-CoV-2 RdRp past a single 8-oxo-rG. The RdRp-mediated primer extension was significantly inhibited by 8-oxo-rG on the template RNA. During the blockage of the extension reaction, the rate of product release by RdRp was extremely slow, indicating that the RdRp machinery remained bound to the replicating primer/template RNA. Once RdRp was able to bypass 8-oxo-rG, it preferentially incorporated rCMP, with a lesser amount of rAMP opposite 8-oxo-rG. In contrast, RdRp demonstrated greater activity in extending from the mutagenic rA:8-oxo-rG terminus compared to the lower efficiency of extension from the rC:8-oxo-rG pair. Based on steady-state kinetic analyses for the incorporation of rNMPs opposite 8-oxo-rG and chain extension from rC:8-oxo-rG or rA:8-oxo-rG, the relative bypass frequency for rA:8-oxo-rG was found to be seven-fold higher than that for rC:8-oxo-rG. Therefore, the properties of RdRp indicated in this study may contribute to the mechanism of mutagenesis of the SARS-CoV-2 genome.","version":"1.1","doi":"10.1101/2024.10.13.618053","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.11.617884","pub_date":"2024-10-14","title":"Comparative assessment of machine learning algorithms to predict severity of disease in COVID-19 patients based on eight cofactors","abstract":"Machine learning is one of the important tools to diagnose and predict the diseased state accurately and effectively. The COVID-19 pandemic caused due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become one of the most researched healthcare topics worldwide. Machine learning algorithms can find efficient and reliable ways to predict the COVID-19 from vast amounts of existing health care data, allowing faster, effective, and more accurate diagnosis with lower risk based on the symptoms. Based on the countrywide data published by the Israeli Ministry of Health, we propose a system that detects COVID-19 instances using simple variables. The COVID-19 dataset used in the study consisted of 278848 patients samples with five different symptoms, namely cough, fever, sore throat, shortness of breath, and headache, apart from other basic information like age, gender, and test indication excluding confirmed COVID-19 result. The data was analyzed using traditional supervised machine learning algorithms namely, Decision tree, Support vector machine, Random Forest, Logistic regression, k-nearest neighbor, and Naive Bayes based on eight cofactors with high accuracy rate (\u2265 0.9450). Apart from Support vector machine, all other algorithms displayed better performance based on the AUC score calculated using the receiver operator characteristic (ROC) curve. This study also highlights the significant differences between precision, recall and accuracy for each model.","version":"1.1","doi":"10.1101/2024.10.11.617884","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.11.617727","pub_date":"2024-10-14","title":"Synthesis and Evaluation of Carmofur Analogs as Antiproliferative Agents, Inhibitors to the Main Protease (Mpro) of SARS-CoV-2, and Membrane Rupture-Inducing Agents","abstract":"Initially developed as a derivatized analog of 5-fluorouracil for the treatment of colorectal cancer, carmofur has more recently demonstrated potent covalent inhibition of the main protease (Mpro) of SARS-CoV-2. Harnessing our previously described workflow for the optimized preparation of carmofur using benchtop 19F NMR spectroscopy, here, we prepared and evaluated a synthetic library of nine carmofur analogs with a selection of side chain motifs or single-atom substitution to explore the diversifiability of these compounds as Mpro inhibitors, where we discovered that a hexyl carbamate analog outperformed carmofur, and as antiproliferative agents in model human cell lines to identify differences in potency when the carbonyl electrophilicity and/or alkyl side chains are modified. Finally, we describe a novel workflow for the evaluation of membrane-rupturing small molecules through imaging of fluorescently labeled giant unilamellar vesicles (GUVs), and through this, we identified two lipophilic urethane analogs of carmofur bearing dodecyl urethane and octadecyl urethane side chains that have potent membrane-rupturing capability in the nanomolar range, providing insight into a potential mechanism for the in vitro activities of lipidated 5-fluorouracil analogs.","version":"1.1","doi":"10.1101/2024.10.11.617727","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.11.617877","pub_date":"2024-10-11","title":"Intranasal delivery of a broadly neutralizing single domain antibody targeting ACE2 protects against SARS-CoV-2 infection","abstract":"SARS-CoV-2 accumulates mutations over time leading to the emergence of variants, which become largely resistant to existing vaccines and spike protein-targeted antiviral treatment. Therefore, there is a need for other therapies with broad efficiency. Here, we targeted the angiotensin-converting enzyme 2 (ACE2), the major entry receptor for SARS-CoV-2. We purified three single domain heavy chain antibodies (VHHs) after immunization of an alpaca with the ectodomain of ACE2. These VHHs bound ACE2 with nanomolar affinity and specifically detected membrane-anchored ACE2. Two of them (B07 and B09) neutralized by a competitive mechanism multiple SARS-CoV-2 isolates, including Omicron variants (XBB.1.16.1; EG.5.1.3; BA.2.86.1), without impacting the proteolytic activity of the enzyme. Fusion of B07 with conventional Fc domain markedly improved its binding and neutralizing efficacy. This dimeric Fc-conjugated B07 (B07-Fc) recognized specific residues of the N-terminal helix 1 of ACE2. When administrated prophylactically and intranasally, B07-Fc induced a strong dose-dependent protection of mice expressing human ACE2 (K18-hACE2) from SARS-CoV-2 Omicron. Hamsters were weakly protected due to low binding of B07-Fc to hamster ACE2. These single domain antibodies targeting hACE2 represent potential broad-spectrum therapeutic candidates against any emerging viruses using ACE2 as a receptor. These inhalable neutralizing single domain antibodies also represent a non-invasive approach against respiratory viral infection.","version":"1.1","doi":"10.1101/2024.10.11.617877","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.11.617762","pub_date":"2024-10-11","title":"Structural insights into the RNA binding inhibitors of the C-terminal domain of the SARS-CoV-2 nucleocapsid","abstract":"The SARS-CoV-2 nucleocapsid (N) protein is an essential structural element of the virion, playing a crucial role in enclosing the viral genome into a ribonucleoprotein (RNP) assembly, as well as viral replication and transmission. The C-terminal domain of the N-protein (N-CTD) is essential for encapsidation, contributing to the stabilization of the RNP complex. In a previous study, three inhibitors (ceftriaxone, cefuroxime, and ampicillin) were screened for their potential to disrupt the RNA packaging process by targeting the N-protein. However, the binding efficacy, mechanism of RNA binding inhibition, and molecular insights of binding with N-CTD remain unclear. In this study, we evaluated the binding efficacy of these inhibitors using isothermal titration calorimetry (ITC), revealing the affinity of ceftriaxone (18 \u00b1 1.3 \u03bcM), cefuroxime (55 \u00b1 4.2 \u03bcM), and ampicillin (28 \u00b1 1.2 \u03bcM) with the N-CTD. Further inhibition assay and fluorescence polarisation assay demonstrated RNA binding inhibition, with IC50 ranging from 10.4 to 12.4 \u03bcM and KD values between 24 and 32 \u03bcM for the inhibitors. Additionally, we also determined the inhibitor-bound complex crystal structures of N-CTD-Ceftriaxone (2.0 \u00c5) and N-CTD-Ampicillin (2.2 \u00c5), along with the structure of apo N-CTD (1.4 \u00c5). These crystal structures revealed previously unobserved interaction sites involving residues K261, K266, R293, Q294, and W301 at the oligomerization interface and the predicted RNA-binding region of N-CTD. These findings provide valuable molecular insights into the inhibition of N-CTD, highlighting its potential as an underexplored but promising target for the development of novel antiviral agents against coronaviruses. The inhibitors ceftriaxone, cefuroxime, and ampicillin-demonstrated high-affinity binding to the C-terminal domain (N-CTD) of the SARS-CoV-2 nucleocapsid (N) protein, effectively disrupting the formation of the N-CTD-RNA complex. Complex crystal structures of N-CTD with ceftriaxone and ampicillin revealed previously unobserved distinct binding sites. Structures reveal how the selected inhibitors disrupt the oligomerization of N-CTD and hinder the RNA packaging process of the virus.","version":"1.1","doi":"10.1101/2024.10.11.617762","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.09.617371","pub_date":"2024-10-09","title":"Structural basis of TMPRSS11D specificity and autocleavage activation","abstract":"Transmembrane Protease, Serine-2 (TMPRSS2) and TMPRSS11D are human proteases that enable SARS-CoV-2 and Influenza A/B virus infections, but their biochemical mechanisms for facilitating viral cell entry remain unclear. We demonstrate these proteases can spontaneously and efficiently cleave their own zymogen activation motifs, thereby activating their wider protease activity on other cellular substrates. We determined TMPRSS11D co-crystal structures in complexes with a native TMPRSS11D zymogen activation motif and with an engineered activation motif, providing insights into TMPRSS11D autocleavage activation and revealing unique regions of its substrate binding cleft. We further show that a protease inhibitor that underwent clinical trials for TMPRSS2-targeted COVID-19 therapy, nafamostat mesylate, was rapidly cleaved by TMPRSS11D and converted to low activity derivatives. These insights into human protease viral tropism and into liabilities with existing human serine protease inhibition strategies will guide future drug discovery campaigns for these targets.","version":"1.1","doi":"10.1101/2024.10.09.617371","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.08.617329","pub_date":"2024-10-09","title":"Genome-Wide Identification of Stable RNA Secondary Structures Across Multiple Organisms Using Chemical Probing Data: Insights into Short Structural Motifs and RNA-Targeting Therapeutics","abstract":"Small molecules targeting specific RNA binding sites, including stable and transient RNA structures, are emerging as effective pharmacological approaches for modulating gene expression. However, little is understood about how stable RNA secondary structures are shared across organisms, an important factor in controlling drug selectivity. In this study, I provide an analytical pipeline named RNA Secondary Structure Finder (R2S-Finder) to discover short, stable RNA structural motifs for humans, Escherichia coli (E. coli), SARS-CoV-2, and Zika virus by leveraging existing in vivo and in vitro genome-wide chemical RNA-probing datasets. I found several common features across organisms. For example, apart from the well-documented tetraloops, AU-rich tetraloops are widely present in different organisms. I also found that the 5\u2019 untranslated region (UTR) contains a higher proportion of stable structures than the coding sequences in humans, SARS-CoV-2, and Zika virus. In general, stable structures predicted from in vitro (protein-free) and in vivo datasets are consistent in humans, E. coli, and SARS-CoV-2, indicating that most stable structure formation were driven by RNA folding alone, while a larger variation was found between in vitro and in vivo data with certain RNA types, such as human long intergenic non-coding RNAs (lincRNAs). Finally, I predicted stable three- and four-way RNA junctions that exist both in vivo and in vitro conditions, which can potentially serve as drug targets. All results of stable sequences, stem-loops, internal loops, bulges, and three- and four-way junctions have been collated in the R2S-Finder database (https://github.com/JingxinWangLab/R2S-Finder), which is coded in hyperlinked HTML pages and tabulated in CSV files.","version":"1.1","doi":"10.1101/2024.10.08.617329","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.10.590792","pub_date":"2024-10-09","title":"25-hydroxycholesterol promotes brain endothelial dysfunction by remodelling cholesterol metabolism","abstract":"The antiviral enzyme cholesterol 25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25HC), which modulates cholesterol metabolism during infection, have been previously associated with vascular pathology. Viral infections have been linked to risk of intracerebral haemorrhage (ICH) but the molecular mechanisms leading to brain vessel rupture via antiviral responses remain unknown. We hypothesised that the CH25H/25HC pathway may impact neuroendothelial integrity in the context of infection-associated ICH. Here, using a SARS-CoV-2-spike-induced zebrafish ICH model and foetal human SARS-CoV-2-associated cortical tissue containing microbleeds, we identified an upregulation of CH25H in infection-associated cerebral haemorrhage. Using zebrafish ICH models and human brain endothelial cells, we asked whether 25HC may promote neurovascular dysfunction by modulating cholesterol metabolism. We found that 25HC and pharmacological inhibition of HMGCR by atorvastatin interacted to exacerbate brain bleeding in zebrafish larvae and in vitro brain endothelial dysfunction. In vitro 25HC-induced dysfunction was also rescued by cholesterol supplementation. These results demonstrate that the antiviral factor 25HC can dysregulate brain endothelial function by remodelling cholesterol metabolism. We propose that the CH25H/25HC pathway represents an important component in the pathophysiology of brain vessel dysfunction associated with infection and cholesterol dysregulation in the context of ICH. The antiviral metabolite 25-hydroxycholesterol dysregulates brain endothelial function by remodelling cholesterol metabolism, thereby providing a mechanistic link between viral infection and brain endothelial dysfunction in conditions such as intracerebral haemorrhage.","version":"1.2","doi":"10.1101/2024.05.10.590792","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.12.561935","pub_date":"2024-10-09","title":"Hidden evolutionary constraints dictate the retention of coronavirus accessory genes","abstract":"Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2 to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. In contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which arose via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remains largely intact, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes and extending these findings to viruses currently adapting to humans.","version":"1.3","doi":"10.1101/2023.10.12.561935","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.09.617356","pub_date":"2024-10-09","title":"Cell-free immuno-profiling on a genetically programmed biochip","abstract":"Emerging cell-free synthetic biology approaches provide biosafe, cheap, and versatile genetic tools to advance therapeutic research and development. Combined with micro-fabrication technology, we developed a platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies and human receptors in miniaturized compartments on a silicon chip. Photolithographic surface patterning of protein traps and on chip expression from high density gene brushes generated a continuous surface density gradient of fluorescently labeled antigens. Antibodies binding to the antigen gradient generate a full binding curve in each single compartment for affinity determination. We used the SARS-CoV-2 antigens as a model to profile the specificity and affinity of monoclonal antibodies to > 30 viral epitopes synthesized simultaneously on one chip in a genotype-phenotype linked compartments. We further profiled polyclonal antibodies in minute volumes of human sera, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. Cell-free co-synthesis of the human ACE2 receptor with the viral Receptor-Binding-Domain yielded relative binding affinities to different SARS-CoV-2 variants. This rapid, quantitative, and on-chip genetically programmed approach allows to study complex protein-protein interactions independent of protein purification steps for human immuno-profiling with a fast response time for combating emerging pathogens.","version":"1.1","doi":"10.1101/2024.10.09.617356","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.06.616878","pub_date":"2024-10-07","title":"Chronic infections can generate SARS-CoV-2-like bursts of viral evolution without epistasis","abstract":"Multiple SARS-CoV-2 variants have arisen during the first years of the pandemic, often bearing many new mutations. Several explanations have been offered for the surprisingly sudden emergence of multiple mutations that enhance viral fitness, including cryptic transmission, spillover from animal reservoirs, epistasis between mutations, and chronic infections. Here, we simulated pathogen evolution combining within-host replication and between-host transmission. We found that, under certain conditions, chronic infections can lead to SARS-CoV-2-like bursts of mutations even without epistasis. Chronic infections can also increase the global evolutionary rate of a pathogen even in the absence of clear mutational bursts. Overall, our study supports chronic infections as a plausible origin for highly mutated SARS-CoV-2 variants. More generally, we also describe how chronic infections can influence pathogen evolution under different scenarios.","version":"1.1","doi":"10.1101/2024.10.06.616878","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.04.616743","pub_date":"2024-10-07","title":"Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors","abstract":"The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident a need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e. PIK3i, mTORi, multiple RTKi) that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.","version":"1.1","doi":"10.1101/2024.10.04.616743","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.05.616797","pub_date":"2024-10-07","title":"Spike mRNA Vaccine Encapsulated in a Lipid Nanoparticle Composed of Phospholipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine Induced Potent B- and T-cell Responses Associated with Protection against COVID-19 in Hamsters","abstract":"Lipid nanoparticles (LNPs) have recently emerged as one of the most advanced vehicle platforms for efficient in vivo delivery of nucleoside-modified mRNA vaccine, particularly for COVID-19. LNPs comprise four different lipids: ionizable lipids, helper or neutral lipids, cholesterol, and lipids attached to polyethylene glycol (PEG). Studies on using the mRNA-LNP platform for vaccines have largely focused on the nucleic acid cargo with less attention to the LNP vehicle. While the LNPs protect mRNA from degradation and efficiently deliver the mRNA to antigen-presenting cells the effect of lipid composition and biophysical properties on the immunogenic and protective mRNA vaccine remain to be fully elucidated. In the present study, we used SARS-CoV-2 Spike-mRNA as a prototype vaccine, to study the effect of 4 different of LNPs with various lipid compositions. We demonstrate that when the same Spike-mRNA was delivered in the LNP4 formulation based on phospholipid 1,2-dioleoyl-sn-glycero-3- Phosphoethanolamine it outperformed the immunogenicity and protective efficacy of three LNPs (LNP1, LNP2, and LNP3) that are based on different lipids. Compared to other three LNPs, the LNP4: (i) enhanced phenotypic and functional maturation of dendritic cells; (ii) induced strong T-cell responses, (iii) increased secretion of proinflammatory, pro-follicular T helper (Tfh) cell cytokines; (iv) induced higher neutralization IgG titers; and (v) and provided better protection against SARS-CoV-2 infection and COVID-19 in the hamster model. We discussed the potential mechanisms by which LNP which include the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine may activate protective B- and T-cell responses.","version":"1.1","doi":"10.1101/2024.10.05.616797","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.05.616778","pub_date":"2024-10-06","title":"Inhibitors of dihydroorotate dehydrogenase synergize with the broad antiviral activity of 4\u2032-fluorouridine","abstract":"RNA viruses present a constant threat to human health, often with limited options for vaccination or therapy. Notable examples include influenza viruses and coronaviruses, which have pandemic potential. Filo- and henipaviruses cause more limited outbreaks, but with high case fatality rates. All RNA viruses rely on the activity of a virus-encoded RNA-dependent RNA polymerase (RdRp). An antiviral nucleoside analogue, 4\u2032-Fluorouridine (4\u2032-FlU), targets RdRp and diminishes the replication of several RNA viruses, including influenza A virus and SARS-CoV-2, through incorporation into nascent viral RNA and delayed chain termination. However, the effective concentration of 4\u2032-FlU varied among different viruses, raising the need to fortify its efficacy. Here we show that inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme essential for pyrimidine biosynthesis, can synergistically enhance the antiviral effect of 4\u2032-FlU against influenza A viruses, SARS-CoV-2, henipaviruses, and Ebola virus. Even 4\u2032-FlU-resistant mutant influenza A virus was re-sensitized towards 4\u2032-FlU by DHODH inhibition. The addition of uridine rescued influenza A virus replication, strongly suggesting uridine depletion as a mechanism of this synergy. 4\u2032-FlU was also highly effective against SARS-CoV-2 in a hamster model of COVID. We propose that the impairment of endogenous uridine synthesis by DHODH inhibition enhances the incorporation of 4\u2032-FlU into viral RNAs. This strategy may be broadly applicable to enhance the efficacy of pyrimidine nucleoside analogues for antiviral therapy. Strong synergy of DHODH inhibitors with 4\u2032-FlU Activity of the combination against previously resistant influenza virus Broadly active combination against a diverse set of RNA viruses Successful targets include highly pathogenic Ebola and Nipah viruses","version":"1.1","doi":"10.1101/2024.10.05.616778","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.04.616448","pub_date":"2024-10-04","title":"Impact of JN.1 booster vaccination on neutralisation of SARS-CoV-2 variants KP.3.1.1 and XEC","abstract":"The SARS-CoV-2 KP.3.1.1 lineage is currently the dominating lineage on several continents. In parallel, the XEC lineage, a recombinant of KS.1.1 and KP.3.3, is on track of becoming the next dominant lineage in Europe and North America. Here we performed a rapid virological characterisation of the XEC lineage and studied the impact of JN.1 mRNA booster vaccination on KP.3.1.1 and XEC neutralisation.","version":"1.1","doi":"10.1101/2024.10.04.616448","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.02.616394","pub_date":"2024-10-03","title":"Serum proteomics reveals high-affinity and convergent antibodies by tracking SARS-CoV2 hybrid immunity to emerging variants of concern","abstract":"The rapid spread of SARS-CoV2 and continuing impact on human health has prompted the need for effective and rapid development of monoclonal antibody therapeutics. In this study, we interrogate polyclonal antibodies in serum and B cells from whole blood of three donors with SARS-CoV2 immunity to find high-affinity anti-SARS-CoV2 antibodies to escape variants. Serum IgG antibodies were selected by affinity to the receptor-binding domain (RBD) and non-RBD sites on the spike protein of Omicron subvariant B.1.1.529 from each donor. Antibodies were analyzed by bottom-up mass spectrometry, and matched to single- and bulk-cell sequenced repertoires for each donor. Antibodies observed in serum were recombinantly expressed, and characterized to assess domain binding, cross-reactivity between different variants, and capacity to inhibit RBD binding to host protein. Donors infected with early Omicron subvariants had serum antibodies with subnanomolar affinity to RBD that show binding activity to a newer Omicron subvariant BQ.1.1. The donors also showed a convergent immune response. Serum antibodies and other single- and bulk-cell sequences were similar to publicly reported anti-SARS-CoV-2 antibodies, and characterized serum antibodies had the same variant-binding and neutralization profiles as their reported public sequence. The serum antibodies analyzed were a subset of anti-SARS-CoV2 antibodies in the B cell repertoire, which demonstrates significant dynamics between the B cells and circulating antibodies in peripheral blood.","version":"1.1","doi":"10.1101/2024.10.02.616394","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.21.517338","pub_date":"2024-10-03","title":"Ultrasound inhibits SARS-CoV-2 infectivity in vitro","abstract":"The global spreading of SARS-CoV-2 and the emergence of new variants underscore the ongoing need to develop new vaccines and antiviral drugs. While electromagnetic and acoustic waves have well-known virucidal properties, their application in therapeutic settings has been limited due to harming effects in biological matter. Here, we investigates the potential of ultrasound to interfere with SARS-CoV-2. Specifically, we study the effects of acoustic waves in the 1\u201320 MHz frequency range to determine their impact on the viral envelope of SARS-CoV-2. Our in vitro experiments demonstrate ultrasound exhibits a virucidal effect on SARS-CoV-2 without production of heat or cavitation. This study offers a promising physics-based approach to combat SARS-CoV-2 and potentially other spherical viruses, broadening the scope of antiviral treatments.","version":"1.3","doi":"10.1101/2022.11.21.517338","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.29.615653","pub_date":"2024-10-03","title":"RNA binding protein Musashi1 interacts with the viral genomic RNA and restricts SARS-CoV-2 infection by repressing translation","abstract":"Musashi RNA binding proteins are important post-transcriptional regulators of stem cell homeostasis and are known to be involved in viral infections. However, their role in SARS-CoV-2 infection remains largely unknown. Using computational studies, in vivo RNA immunoprecipitation and biochemical assays, here, we establish that Musashi1 (Msi1) interacts with viral genomic RNA through direct binding to the SARS-CoV-2 3\u2019UTR. Importantly, binding of Msi1 to the viral 3\u2019UTR results in translational repression mediated by inhibition of Poly (A) binding protein (PABP). Conversely, Msi1 knockout promotes robust viral replication and increased viral protein expression. Using 2D cell cultures, stem cells and 3D organoids, we show that depletion of Msi1 in intestinal cells augments infection. This finding explains why the human intestine serves as a reservoir for the SARS-CoV-2 virus, wherein differentiated enterocytes, which have negligible levels of Msi1, are highly affected. Contrarily, stem cells which are enriched for Msi1 expression, are known to be less permissive to SARS-CoV-2 infection despite expressing the entry receptors. Our findings show how translation repression of SARS-CoV-2 by stem cell RNA binding proteins such as Msi1 could help evasion of infection.","version":"1.2","doi":"10.1101/2024.09.29.615653","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.03.616431","pub_date":"2024-10-03","title":"Delayed viral clearance and altered inflammatory responses resulted in increased severity of SARS-CoV-2 infection in aged mice","abstract":"Since the onset of the COVID-19 pandemic, advanced age has emerged as a major predictor of disease severity. Epidemiological investigations consistently demonstrate an overrepresentation of the elderly in COVID-19 hospitalizations and fatalities. Despite this, a comprehensive understanding of the molecular mechanisms explaining how old age constitutes a critical risk factor remains elusive. To unravel this, we designed an animal study, juxtaposing the course of COVID-19 in young adults (2 months) and geriatric (15-22 months) mice. Both groups of K18(hACE2) mice were intranasally exposed to 500 TCID50 of the SARS-CoV-2 Delta variant with a variety of outcomes assessed on days 3, 5, and 7 post-infections (DPI). Analyses included pulmonary cytokines, RNA, viral loads, lipidomic profiles, and histological assessments, with a concurrent evaluation of the percentage of mice reaching humane endpoints. The findings unveiled notable distinctions between the two groups, with aged mice exhibiting impaired viral clearance at 7 DPI, correlating with diminished survival rates together with an absence of weight loss recovery at 6-7 DPI. Additionally, elderly-infected mice exhibited a deficient Th1 response characterized by diminished productions of IFNg, CCL2, CCL3, and CXCL9 relative to younger mice. Furthermore, mass-spectrometry analysis of the lung lipidome indicated altered expression of several lipids with immunomodulatory and pro-resolution effects in aged mice such as Resolvin, HOTrEs, and NeuroP, but also DiHOMEs-related ARDS. Collectively, disease severity implies a dysregulation of the antiviral response in elderly-infected mice relative to younger mice, resulting in compromised viral clearance and a more unfavorable prognosis. This underscores the potential efficacy of immunomodulatory treatments for elderly subjects experiencing symptoms of severe COVID-19. In this study, we investigated why older age is linked to more severe COVID-19 outcomes by comparing the progression of the disease in young (2 months) and elderly (15-22 months) K18(hACE2) mice infected with the SARS-CoV-2 Delta variant. After exposing both groups to the virus, we assessed various factors such as viral loads, immune responses, and lipid profiles in the lungs at different time points. Our findings revealed that elderly mice struggled to clear the virus by day 7 post-infection, leading to higher mortality rates and poorer recovery compared to younger mice. Aged mice showed weaker immune responses, with reduced production of key antiviral proteins like IFNg and certain chemokines. Lipid analysis also highlighted differences in molecules involved in immune regulation and lung protection, such as decreased levels of pro-resolving lipids and increased lipids associated with lung injury. These results suggest that older mice have a compromised antiviral defense, which could inform new therapeutic approaches for elderly patients with severe COVID-19.","version":"1.1","doi":"10.1101/2024.10.03.616431","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.03.616483","pub_date":"2024-10-03","title":"Dysregulation of lung epithelial cell homeostasis and immunity contributes to Middle East Respiratory Syndrome coronavirus disease severity","abstract":"Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoV from past 20 years (SARS-CoV, SARS-CoV-2, MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers and acute lung injury among lethal and sub-lethal virus doses. Virus induced apoptosis of type I and II alveolar epithelial cells suggest that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B-cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness. Emerging coronaviruses like SARS-CoV, SARS-CoV-2 and MERS-CoV cause a range of disease outcomes in humans from asymptomatic, moderate and severe respiratory disease which can progress to death but the factors causing these disparate outcomes remain unclear. Understanding host responses to mild and life-threatening infection provides insight into virus-host networks within and across organ systems that contribute to disease outcomes. We used multi-omics approaches to comprehensively define the host response to moderate and severe MERS-CoV infection. Severe respiratory disease was associated with dysregulation of the immune response. Key lung epithelial cell populations that are essential for lung function get infected and die. Mice lacking key immune cell populations experienced greater virus replication but decreased disease severity implicating the immune system in both protective and pathogenic roles in the response to MERS-CoV. These data could be utilized to design new therapeutic strategies targeting specific pathways that contribute to severe disease.","version":"1.1","doi":"10.1101/2024.10.03.616483","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.01.616085","pub_date":"2024-10-03","title":"Real-Time Spatiotemporal Tracking of Infectious Outbreaks in Confined Environments with a Host-Pathogen Agent-Based System","abstract":"Deadly infection outbreaks in confined spaces, whether it is a COVID-19 outbreak on a cruise ship or at a veteran home, or measles and stomach flu outbreaks in schools, can be characterized by their rapid spread due to the abundance of common spaces, shared airways, and high population density. Preventing future infectious outbreaks and developing efficient mitigation protocols can benefit from advanced computational modeling approaches. Here, we developed an agent-based modeling approach to study the spatiotemporal dynamics of an infection outbreak in a confined environment caused by a specific pathogen and to determine effective infection containment protocols. The approach integrates the 3D geographic information system of a confined environment, the behavior of the hosts, key biological parameters about the pathogen obtained from the experimental data, and the general mechanics of host-pathogen and pathogen-fomite interactions. To assess our approach, we applied it to the historical data of infectious outbreak caused by norovirus, H1N1 influenza A, and SARS-CoV-2 viruses. As a result our model was able to accurately predict the number of infections per day, correctly identify the day when the CDC vessel sanitation protocol would be triggered, single out key biological parameters affecting the infection spread, and propose important changes to existing containment protocols, specific for different pathogens. This research not only contributes to our understanding of infection spread and containment in cruise ships but also offers insights applicable to other similar confined settings, such as nursing homes, schools, and hospitals. By providing a robust framework for real-time outbreak modeling, this study proposes new, more effective containment protocols and enhances our preparedness for managing infectious diseases and emerging pathogens in confined environments.","version":"1.1","doi":"10.1101/2024.10.01.616085","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.02.616254","pub_date":"2024-10-02","title":"Structural basis of SARS-Cov-2 spike recognition by engineered synthetic multivalent VHH antibodies","abstract":"High-throughput technologies such as next-generation sequencing (NGS), microarray-based gene synthesis, and phage display have empowered the discovery and engineering of precisely defined, synthetic antibodies with high avidity and drug-like features. Here, we describe a scalable process for engineering homo- and hetero-hexavalent variable domains of camelid heavy-chain (VHH)-Fc antibodies against the severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein. Overall, we demonstrate that VHH trimerization is an effective and modular approach for increasing the affinity of anti-S1 VHH-Fc antibodies for the highly mutated S proteins of SARS-CoV-2 variants. We show that one specific nanobody (named TB201-1) binds spike trimer protein at the interface of two neighboring RBDs, recognizing one distinct epitope on one RBD but making a set of secondary interactions with the neighboring RBD. From this structure, we determine the epitope-paratope residues responsible for spike-nanobody interaction and how mutations found in the SARS-CoV-2 variants contribute to oblate TB201-1 binding. This approach could be leveraged to improve existing antibody-based diagnostics and therapeutics targeting SARS-CoV-2 as the virus evolves.","version":"1.1","doi":"10.1101/2024.10.02.616254","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.30.613319","pub_date":"2024-10-01","title":"Cxcl10 is required for survival during SARS-CoV-2 infection in mice","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, remains endemic worldwide \u223c5 years since the first documented case. Severe COVID-19 is widely considered to be caused by a dysregulated immune response to SARS-CoV-2 within the respiratory tract. Circulating levels of the chemokine CXCL10 are strongly positively associated with poor outcome; however, its precise role in pathogenesis and its suitability as a therapeutic target have remained undefined. Here, we challenged 4-6 month old C57BL/6 mice genetically deficient in Cxcl10 with a mouse-adapted strain of SARS-CoV-2. Infected male, but not female, Cxcl10-/- mice displayed increased mortality compared to wild type controls. Histopathological damage, inflammatory gene induction and virus load in the lungs of male mice 4 days post infection and before death were not broadly influenced by Cxcl10 deficiency. However, accumulation of B cells and both CD4+ and CD8+ T cells in the lung parenchyma of infected mice was reduced in the absence of Cxcl10. Thus, during acute SARS-CoV-2 infection, Cxcl10 regulates lymphocyte infiltration in the lung and confers protection against mortality. Our preclinical model results do not support targeting CXCL10 therapeutically in severe COVID-19.","version":"1.1","doi":"10.1101/2024.09.30.613319","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.19.608570","pub_date":"2024-10-01","title":"A modular model of immune response as a computational platform to investigate a pathogenesis of infection disease","abstract":"The COVID-19 pandemic significantly transformed the field of mathematical modeling in immunology. International collaboration among numerous research groups yielded a substantial amount of experimental data, which greatly facilitated model validation and led to the development of new mathematical models. The aim of the study is an improvement of system understanding of the immune response to SARS-CoV-2 infection based on the development of a modular mathematical model which provides a foundation for further research on host-pathogen interactions. We utilized the open-source BioUML platform to develop a model using ordinary, delay and stochastic differential equations. The model was validated using experimental data from middle-aged individuals with moderate COVID-19 progression, including measurements of viral load, antibodies, CD4+ and CD8+ T cells, and interleukin-6 levels. Parameter optimization and sensitivity analysis were conducted to refine the model\u2019s accuracy. The model effectively reproduces moderate, severe, and critical COVID-19 progressions, consistent with experimental observations. We investigated the efficiency and contributions of innate and adaptive immunity in response to SARS-CoV-2 infection and assessed immune system behavior during co-infection with HIV and organ transplantation. Additionally, we studied therapy methods, such as interferon administration. The developed model can be employed as a framework for simulating other infectious diseases taking into account follow-up immune response. Despite the significant progress reached in understanding of COVID-19, traditional methods still struggle to analyze and interpret the extensive and sometimes controversial experimental data on SARS-CoV-2 infection. Mathematical and systems biology approaches attempt to address this challenge by developing mathematical models of the immune response. We aimed not only to investigate the disease at a systemic level but also to provide a framework for further research on host-pathogen interactions, both existing and forthcoming. To achieve this, we constructed a model incorporating both innate and adaptive immunity, as well as cellular and humoral components. This together allowed us to conduct a series of in silico experiments, exploring the immune response across various levels and compartments. The results of these investigations offer valuable insights into the complex dynamics of the immune system and can guide future research and therapeutic strategies.","version":"1.2","doi":"10.1101/2024.08.19.608570","journal":"bioRxiv","score":null},{"id":"10.1101/2024.10.01.616060","pub_date":"2024-10-01","title":"Sustained exposure to multivalent antigen-decorated nanoparticles generates broad anti-coronavirus responses","abstract":"The threat of future coronavirus pandemics requires developing cost-effective vaccine technologies that provide broad and long-lasting protection against diverse circulating and emerging strains. Here we report a multivalent liposomal hydrogel depot vaccine technology comprising the receptor binding domain (RBD) of up to four relevant SARS and MERS coronavirus strains non-covalently displayed on the surface of the liposomes within the hydrogel structure. The multivalent presentation and sustained exposure of RBD antigens improved the potency, neutralizing activity, durability, and consistency of antibody responses across homologous and heterologous coronavirus strains in a na\u00efve murine model. When administrated in animals previously exposed to the wild-type SARS-CoV-2 antigens, liposomal hydrogels elicited durable antibody responses against the homologous SARS and MERS strains for over 6 months and elicited neutralizing activity against the immune-evasive SARS-CoV-2 variant Omicron BA.4/BA.5. Overall, the tunable antigen-decorated liposomal hydrogel platform we report here generates robust and durable humoral responses across diverse coronaviruses, supporting global efforts to effectively respond to future viral outbreaks. Rapidly mutating infectious diseases such as influenza, HIV, and COVID-19 pose serious threats to human health. Yet, most vaccines still do not mount durable protection against mutagenic viruses and fail to induce broad responses to protect against emergent strains. Materials approaches to vaccine design, such as employing sustained delivery approaches or decorating nanoparticle constructs with multiple antigens, have shown promise in improving the breadth and potency of vaccines. Yet, these approaches typically require cumbersome chemistries and have not been explored in pre-exposed populations over clinically relevant time scales. Here, we report the development of an injectable liposomal hydrogel depot technology capable of prolonged presentation of multiple coronavirus antigens non-covalently coordinated on the surface of the liposomes forming the hydrogel structure. These hydrogels improve the potency, durability and breadth of vaccine response and are easy to fabricate, enabling the rapid design of next generation vaccines that confer protection against rapidly evolving pandemics.","version":"1.1","doi":"10.1101/2024.10.01.616060","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.29.615295","pub_date":"2024-10-01","title":"Teicoplanin attenuates RNA virus infection in vitro","abstract":"Teicoplanin (TP) is a glycopeptide antibiotic used for Gram-positive bacterial infections, and it has been reported to inhibit SARS-CoV-2 and Ebola virus entry through cathepsin inhibition. Given that TP can inhibit viruses belonging to different virus families, we aimed to expand the potential targets of TP to determine whether TP can be developed as a broad-spectrum antiviral agent. Considering the original indication of TP, we first determined the effects of TP against viruses that cause respiratory tract infections and found that TP inhibits enveloped and non-enveloped RNA viruses, namely: human and avian influenza viruses; representative coronaviruses including porcine epidemic diarrhea virus (PEDV), human coronavirus OC43 (HCoV-OC43), and SARS-CoV-2; measles virus; human respiratory syncytial virus A2; and enterovirus 71 (EV-71). Representative flaviviruses, Zika virus (ZIKV) and dengue virus serotype 2 (DENV2), were also susceptible to inhibition by TP. In contrast, TP did not attenuate infection of human adenovirus 5, a non-enveloped DNA virus. Addition of TP at the endocytosis stage but not at the attachment/binding stage of PEDV infection reduced PEDV production in vitro, indicating cathepsin inhibition. Meanwhile, addition of TP during either the attachment/binding or the endocytosis stage of ZIKV infection reduced ZIKV particle production in host cells, and in silico modeling suggested that TP has potential binding pockets in the envelope proteins of ZIKV and DENV2. These results show that TP can be developed as a broad-spectrum antiviral especially against RNA viruses, with potentially different targets in the replication cycle of various viruses.","version":"1.1","doi":"10.1101/2024.09.29.615295","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.14.532012","pub_date":"2024-9-30","title":"Deep physico-chemical characterization of the serum antibody response using a dual-titration microspot assay","abstract":"Antigen specific humoral immunity can be characterized by the analysis of serum antibodies. While serological assays for the measurement of specific antibody levels are available, these are not quantitative in the biochemical sense. Yet, understanding humoral immune responses quantitatively on the systemic level would need a universal, complete, quantitative, comparable measurement method of antigen specific serum antibodies of selected immunoglobulin classes. Here we describe a fluorescent, dual-titration immunoassay, which provides the physico-chemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. We define the theory of the approach that is based on physical chemistry. For validation of theory, we used recombinant receptor binding domain of SARS-CoV-2 as antigen on microspot arrays and varied the concentration of both the antigen and serum antibodies from infected persons to obtain a measurement matrix of binding data. Both titration curves were simultaneously fitted using an algorithm based on the generalized logistic function and adapted for analyzing thermodynamic variables of binding. We obtained equilibrium affinity constants and chemical potentials for distinct antibody classes. These variables reflect the quality and the effective quantity of serum antibodies, respectively. The proposed fluorescent dual-titration microspot immunoassay can generate truly quantitative serological data that is suitable for immunological, medical and systems biological analysis.","version":"1.2","doi":"10.1101/2023.03.14.532012","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.22.558930","pub_date":"2024-9-30","title":"TMPRSS2 activation of Omicron lineage Spike glycoprotein is regulated by Collectrin-like domain of ACE2","abstract":"Continued high-level spread of SARS-CoV-2 has enabled an accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) dependent entry by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on TMPRS22 association with the ACE2 Collectrin-Like Domain (CLD). In engineered cells overexpressing ACE2 and TMPRSS2, ACE2/TMPRSS2 complexes led to either augmentation or attenuation of viral infectivity of pre-Omicron and Omicron lineages, respectively. Mutagenesis of the ACE2-CLD TMPRSS2 cleavage site in ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 activation of viral fusion. Our data supports the evolution of Omicron lineages towards the use of ACE2 unable to form complexes with TMPRSS2 and consistent with ACE2 structure and function as a chaperone for many tissue specific amino acid transport proteins.\n\nGraphical Abstract:\nACE2-TMPRSS2 pool model and evolution of SARS-CoV-2 tropism.\nA. &-B. ACE2-TMPRSS2 pool model to reconcile the evolving entry requirements of SARS-CoV-2 and changes in viral tropism in vivo. A. Both SARS-CoV-1 and early SARS-CoV-2 (pre-Omicron) lineages have molecular dual tropism, with efficient entry when ACE2 (blue protein) can form complexes with TMPRSS2 (green protein) and in settings where TMPRSS2 is excluded from ACE2 (C4-ACE2 CLD). In both settings ACE2 initially engages SARS-CoV-2 spike and fusion is then triggered through TMPRSS2 cleavage of the Spike S2 domain B. Over time, the dual tropism for two distinct pools of ACE2 (with and without TMPRSS2) has been lost, with consolidation towards ACE2 where TMPRSS2 is no longer in a complex. With the arrival of Omicron lineages, ACE2-TMPRSS2 complexes could no longer enable efficient Spike S2 cleavage and fusogenic activation (TMPRSS2 \u201coff\u201d confirmation\u201d). Rather, only TMPRSS2 uncoupled from ACE2 could facilitate the latter cleavage of S2. C. Over time this has further consolidated over generations of omicron lineages from 2022 lineages (BA.1, BA.2 and BA.5) through to 2023 lineages (XBB.1.5) and now in 2024 JN.1 lineages such as KP.3. Overall, this supports the initial molecular tropism of early SARS-CoV-2 clades to be similar to that observed for SARS-CoV-1, with dual tropism across both ACE2 pools and replication proceeding in tissues where ACE2-TMPRSS2 complexes would be prevalent (e.g. Lung). The evolution away from ACE2-TMPRSS2 complexes towards ACE2 where TMPRSS2 is structurally uncoupled (e.g. ACE2 as a chaperone for solute carriers SLCA619 or SLCA620) is consistent selection of this ACE2 pool in a manner that has sustained transmission fitness within the human population.\n\n A. &-B. ACE2-TMPRSS2 pool model to reconcile the evolving entry requirements of SARS-CoV-2 and changes in viral tropism in vivo. A. Both SARS-CoV-1 and early SARS-CoV-2 (pre-Omicron) lineages have molecular dual tropism, with efficient entry when ACE2 (blue protein) can form complexes with TMPRSS2 (green protein) and in settings where TMPRSS2 is excluded from ACE2 (C4-ACE2 CLD). In both settings ACE2 initially engages SARS-CoV-2 spike and fusion is then triggered through TMPRSS2 cleavage of the Spike S2 domain B. Over time, the dual tropism for two distinct pools of ACE2 (with and without TMPRSS2) has been lost, with consolidation towards ACE2 where TMPRSS2 is no longer in a complex. With the arrival of Omicron lineages, ACE2-TMPRSS2 complexes could no longer enable efficient Spike S2 cleavage and fusogenic activation (TMPRSS2 \u201coff\u201d confirmation\u201d). Rather, only TMPRSS2 uncoupled from ACE2 could facilitate the latter cleavage of S2. C. Over time this has further consolidated over generations of omicron lineages from 2022 lineages (BA.1, BA.2 and BA.5) through to 2023 lineages (XBB.1.5) and now in 2024 JN.1 lineages such as KP.3. Overall, this supports the initial molecular tropism of early SARS-CoV-2 clades to be similar to that observed for SARS-CoV-1, with dual tropism across both ACE2 pools and replication proceeding in tissues where ACE2-TMPRSS2 complexes would be prevalent (e.g. Lung). The evolution away from ACE2-TMPRSS2 complexes towards ACE2 where TMPRSS2 is structurally uncoupled (e.g. ACE2 as a chaperone for solute carriers SLCA619 or SLCA620) is consistent selection of this ACE2 pool in a manner that has sustained transmission fitness within the human population.","version":"1.4","doi":"10.1101/2023.09.22.558930","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.26.615262","pub_date":"2024-9-28","title":"Comparative evaluation of cell-based assay technologies for scoring drug-induced condensation of SARS-CoV-2 nucleocapsid protein","abstract":"Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 hours post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.","version":"1.1","doi":"10.1101/2024.09.26.615262","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.31.526494","pub_date":"2024-9-28","title":"Structural prediction of chimeric immunogens to elicit targeted antibodies against betacoronaviruses","abstract":"Betacoronaviruses pose an ongoing pandemic threat. Antigenic evolution of the SARS-CoV-2 virus has shown that much of the spontaneous antibody response is narrowly focused rather than broadly neutralizing against even SARS-CoV-2 variants, let alone future threats. One way to overcome this is by focusing the antibody response against better-conserved regions of the viral spike protein. Here, we present a design approach to predict stable chimeras between SARS-CoV-2 and other coronaviruses, creating synthetic spike proteins that display a desired conserved region and vary other regions. We leverage AlphaFold to predict chimeric structures and create a new metric for scoring chimera stability based on AlphaFold outputs. We evaluated 114 candidate spike chimeras using this approach. Top chimeras were further evaluated using molecular dynamics simulation as an intermediate validation technique, showing good stability compared to low-scoring controls. Experimental testing of five predicted-stable and two predicted-unstable chimeras confirmed 5/7 predictions, with one intermediate result. This demonstrates the feasibility of the underlying approach, which can be used to design custom immunogens to focus the immune response against a desired viral glycoprotein epitope.","version":"1.2","doi":"10.1101/2023.01.31.526494","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.24.563721","pub_date":"2024-9-26","title":"Forecasting dominance of SARS-CoV-2 lineages by anomaly detection using deep AutoEncoders","abstract":"The coronavirus disease of 2019 (COVID-19) pandemic is characterized by sequential emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, lineages, and sublineages, outcompeting previously circulating ones because of, among other factors, increased transmissibility and immune escape. We propose DeepAutoCoV, an unsupervised deep learning anomaly detection system to predict future dominant lineages (FDLs). We define FDLs as viral (sub)lineages that will constitute more than 10% of all the viral sequences added to the GISAID database on a given week. DeepAutoCoV is trained and validated by assembling global and country-specific data sets from over 16 million Spike protein sequences sampled over a period of about 4 years. DeepAutoCoV successfully flags FDLs at very low frequencies (0.01% \u2013 3%), with median lead times of 4-17 weeks, and predicts FDLs \u223c5 and \u223c25 times better than a baseline approach For example, the B.1.617.2 vaccine reference strain was flagged as FDL when its frequency was only 0.01%, more than a year before it was considered for an updated COVID-19 vaccine. Furthermore, DeepAutoCoV outputs interpretable results by pinpointing specific mutations potentially linked to increased fitness, and may provide significant insights for the optimization of public health pre-emptive intervention strategies. Introduction of DeepAutoCoV: The article introduces DeepAutoCoV, an unsupervised deep learning anomaly detection system designed to predict future dominant lineages (FDLs) of SARS-CoV-2. FDLs are defined as viral (sub)lineages that will constitute more than 10% of all viral sequences added to the GISAID database in a given week; Performance and Predictive Capability: DeepAutoCoV successfully flags FDLs at very low frequencies (0.01% to 3%), with median lead times of 4 to 17 weeks before they become dominant. It predicts FDLs approximately 5 to 25 times better than baseline approaches. For instance, the B.1.617.2 vaccine reference strain was identified when its frequency was only 0.01%, over a year before it was considered for vaccine updates; Interpretable Results and Mutation Identification: The system provides interpretable results by pinpointing specific mutations that may be linked to increased fitness, offering insights that can optimize public health interventions. Key FDL mutations, such as those found in Delta and Omicron variants, are identified and analyzed for their potential impact on viral spread and immune escape; Advantages and Applications: DeepAutoCoV is advantageous because it does not require prior assumptions about which protein sites are more likely to mutate. Its application in genomic surveillance systems could significantly reduce the time needed for public health responses to emerging variants, enabling early interventions such as vaccine updates; Evaluation and Comparisons: The performance of DeepAutoCoV was tested over four years of global and national surveillance data, demonstrating superior predictive power compared to other supervised and unsupervised methods. The system is periodically updated to adapt to the evolving viral landscape, making it a robust tool for ongoing surveillance efforts.","version":"1.4","doi":"10.1101/2023.10.24.563721","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.26.615113","pub_date":"2024-9-26","title":"Differential Kinetics of SARS-CoV-2 Proteases Revealed by a Dual-Color, BRET-based Protease Biosensor, DuProSense","abstract":"While SARS-CoV-2 Mpro and PLpro proteases are known to cleave polyproteins pp1a and pp1ab at multiple sites, these have not been comprehensively characterized in living cells. Here we engineered a two-color Bioluminescence Resonance Energy Transfer (BRET)-based, dual protease (DuProSense) biosensor platform relying on a proximity-dependent energy transfer from a luciferase donor to two spectrally separated fluorescent protein acceptors enabling simultaneous monitoring of processing of two cleavage sites in a single assay with high specificity. DuProSense revealed a similar Mpro and PLpro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of various Mpro and PLpro cleavage sites using DuProSense revealed significant differences in cleavage rates and nirmatrelvir potency of Mpro cleavage sites but no correlation between the cleavage rates and nirmatrelvir IC50 values. Overall, our results provide deeper insights into the proteolytic processing of SARS-CoV-2 polyproteins and the dual color BRET platform will find wider applications in the future. Engineered a two-color BRET-based, dual protease biosensor (DuProSense) DuProSense biosensor enabled simultaneous and specific monitoring of Mpro and PLpro activities DuProSense platform revealed differential cleavage kinetics of Mpro cleavage sites in live cells DuProSense platform revealed Mpro cleavage site-dependent nirmatrelvir potency in live cells","version":"1.1","doi":"10.1101/2024.09.26.615113","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.26.613632","pub_date":"2024-9-26","title":"Human NK cell responses to the SARS-CoV-2 Spike269-277 peptide YLQPRTFLL","abstract":"Natural killer (NK) cells act as the first line of defense against virus infections. The effector functions of human NK cells are controlled by inhibitory and activating receptors, including NKG2A and NKG2C, which recognize peptides presented by HLA-E. Recent studies have suggested that the SARS-CoV-2 Spike269-277 peptide YLQPRTFLL may modulate NK cell activity. Here, we show that the YLQPRTFLL peptide is poorly presented by HLA-E. Functional interrogation further revealed that loading of target cells with YLQPRTFLL did not affect the effector functions of NKG2A+ nor NKG2C+ NK cells. Our findings thus indicate that the Spike269-277 peptide YLQPRTFLL has a limited influence on human NK cell responses.","version":"1.1","doi":"10.1101/2024.09.26.613632","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.25.614975","pub_date":"2024-9-26","title":"Betacoronaviruses Differentially Activate the Integrated Stress Response to Optimize Viral Replication in Lung Derived Cell Lines","abstract":"The betacoronavirus genus contains five of the seven human viruses, making it a particularly critical area of research to prepare for future viral emergence. We utilized three human betacoronaviruses, one from each subgenus-HCoV-OC43 (embecovirus), SARS-CoV-2 (sarbecovirus) and MERS-CoV (merbecovirus)- to study betacoronavirus interaction with the PKR-like ER kinase (PERK) pathway of the integrated stress response (ISR)/unfolded protein response (UPR). The PERK pathway becomes activated by an abundance of unfolded proteins within the endoplasmic reticulum (ER), leading to phosphorylation of eIF2\u03b1 and translational attenuation in lung derived cell lines. We demonstrate that MERS-CoV, HCoV-OC43, and SARS-CoV-2 all activate PERK and induce responses downstream of p-eIF2\u03b1, while only SARS-CoV-2 induces detectable p-eIF2\u03b1 during infection. Using a small molecule inhibitor of eIF2\u03b1 dephosphorylation, we provide evidence that MERS-CoV and HCoV-OC43 maximize replication through p-eIF2\u03b1 dephosphorylation. Interestingly, genetic ablation of GADD34 expression, an inducible phosphatase 1 (PP1)-interacting partner targeting eIF2\u03b1 for dephosphorylation, did not significantly alter HCoV-OC43 or SARS-CoV-2 replication, while siRNA knockdown of the constitutive PP1 partner, CReP, dramatically reduced HCoV-OC43 replication. Combining growth arrest and DNA damage-inducible protein (GADD34) knockout with peripheral ER membrane\u2013targeted protein (CReP) knockdown had the maximum impact on HCoV-OC43 replication, while SARS-CoV-2 replication was unaffected. Overall, we conclude that eIF2\u03b1 dephosphorylation is critical for efficient protein production and replication during MERS-CoV and HCoV-OC43 infection. SARS-CoV-2, however, appears to be insensitive to p-eIF2\u03b1 and, during infection, may even downregulate dephosphorylation to limit host translation. Lethal human betacoronaviruses have emerged three times in the last two decades, causing two epidemics and a pandemic. Here, we demonstrate differences in how these viruses interact with cellular translational control mechanisms. Utilizing inhibitory compounds and genetic ablation, we demonstrate that MERS-CoV and HCoV-OC43 benefit from keeping p-eIF2\u03b1 levels low to maintain high rates of virus translation while SARS-CoV-2 tolerates high levels of p-eIF2\u03b1. We utilized a PP1:GADD34/CReP inhibitor, GADD34 KO cells, and CReP-targeting siRNA to investigate the therapeutic potential of these pathways. While ineffective for SARS-CoV-2, we found that HCoV-OC43 seems to primarily utilize CReP to limit p-eIF2a accumulation. This work highlights the need to consider differences amongst these viruses, which may inform the development of host-directed pan-coronavirus therapeutics.","version":"1.1","doi":"10.1101/2024.09.25.614975","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.25.614948","pub_date":"2024-9-25","title":"Placentas from SARS-CoV-2 infection during pregnancy exhibit foci of oxidative stress and DNA damage","abstract":"COVID-19 during pregnancy is linked to increased maternal morbidity and a higher incidence of preterm births, yet the underlying mechanisms remain unclear. Cellular senescence, characterized by the irreversible cessation of cell division, is a critical process in placental function and its dysregulation has been implicated in pregnancy complications like preterm birth. Senescence can be induced by various stressors, including oxidative stress, DNA damage, and viral infections. In this study, we determined whether COVID-19 had an impact on placental senescence. We examined placentas from women infected with SARS-CoV-2 (n=10 term, 4 preterm) compared to uninfected controls (n=10 term, 3 preterm). The placentas were analyzed for SARS-CoV-2 infection/replication (Spike and Nucleocapsid viral proteins), markers of DNA damage (\u03b3H2AX) and oxidative stress (ROS), and senescence (telomere length; cell cycle regulators, SASP). While no overall differences in cellular senescence markers were observed between the COVID-19 positive and negative groups, we found increased secreted SASP markers and confocal microscopy revealed localized areas of oxidative stress and DNA damage in the placentas from COVID-19 positive cases. These findings indicate that SARS-CoV-2 infection induces localized placental damage, warranting further investigation into its impact on maternal and perinatal outcomes.","version":"1.1","doi":"10.1101/2024.09.25.614948","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.22.614369","pub_date":"2024-9-25","title":"Dynamics of Spike-Specific Neutralizing Antibodies Across Five-Year Emerging SARS-CoV-2 Variants of Concern Reveal Conserved Epitopes that Protect Against Severe COVID-19","abstract":"Since early 2020, several SARS-CoV-2 variants of concern (VOCs) continue to emerge, evading waning antibody mediated immunity produced by the current Spike-alone based COVID-19 vaccines. This caused a prolonged and persistent COVID-19 pandemic that is going to enter its fifth year. Thus, the need remains for innovative next generation vaccines that would incorporate protective Spike-derived B-cell epitopes that resist immune evasion. Towards that goal, in this study we (i) Screened the sequences of Spike among many VOCs and identified conserved and non-conserved linear B-cell epitopes; (ii) Compared titers and neutralization antibodies specific to these conserved and non-conserved B-cell epitopes from serum of symptomatic and asymptomatic COVID-19 patients that were exposed to multiple VOCs across the 5-year COVID-19 pandemic, and (iii) Compared protective efficacy of conserved versus non-conserved B-cell epitopes against the most pathogenic Delta variant in a \u201chumanized\u201d ACE-2/HLA transgenic mouse model. We found robust conserved B-cell epitope-specific antibody titers and neutralization in sera from asymptomatic COVID-19 patients. In contrast, sera from symptomatic patients contained weaker antibody responses specific to conserved B-cell epitopes. A multi-epitope COVID-19 vaccine that incorporated the conserved B-cell epitopes, but not the non-conserved B-cell epitopes, significantly protected the ACE2/HLA transgenic mice against infection and COVID-19 like symptoms caused by the Delta variant. These findings underscore the importance of conserved B-cell epitopes in generating robust protective immunity against severe COVID-19 symptoms caused by various VOCs, providing valuable insights for the development of broad-spectrum next generation Coronavirus vaccines capable of conferring cross-variant protective immunity. A persistent COVID-19 pandemic continues to evolve because of a continued emergence of SARS-CoV-2 variants of concern (VOCs) that escape the antibodies induced by the current Spike-alone COVID-19 vaccines. Identifying and characterizing the protective and non-protective Spike-derived B-cell epitopes that resist immune-evasion is a paramount for the development of broad-spectrum next generation Coronavirus vaccines. The present study identified Spike-derived conserved B cell epitopes that (i) are targeted by consistent and strong antibody responses in asymptomatic COVID-19 patients across the 5-year pandemic regardless of VOCs; and (ii) provided strong protection in \u2018humanized\u201d ACE2/HLA transgenic mice against infection and COVID-19 like symptoms caused by the most pathogenic Delta variant. The findings have the potential to inform the design of next generation Coronavirus vaccines capable of conferring cross-variant protective immunity. Protective SARS-CoV-2 Conserved Linear B Cell Epitopes Identified from Spike Protein.","version":"1.1","doi":"10.1101/2024.09.22.614369","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.24.614691","pub_date":"2024-9-25","title":"Exploring the Biological Mechanisms of Severe COVID-19 in the Elderly: Insights from an Aged Mouse Model","abstract":"Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global health crisis, particularly affecting the elderly, who are more susceptible to severe outcomes. However, definitive parameters or mechanisms underlying the severity of COVID-19 in elderly people remain confused. Thus, this study seeks to elucidate the mechanism behind the increased vulnerability of elderly individuals to severe COVID-19. We employed an aged mouse model with a mouse-adapted SARS-CoV-2 strain to mimic the severe symptoms observed in elderly patients with COVID-19. Comprehensive analyses of the whole lung were performed using transcriptome and proteome sequencing, comparing data from aged and young mice. For transcriptome analysis, bulk RNA sequencing was conducted using an Illumina sequencing platform. Proteomic analysis was performed using mass spectrometry following protein extraction, digestion, and peptide labeling. We analyzed the transcriptome and proteome profiles of young and aged mice and discovered that aged mice exhibited elevated baseline levels of inflammation and tissue damage repair. After SARS-CoV-2 infection, aged mice showed increased antiviral and inflammatory responses; however, these responses were weaker than those in young mice, with significant complement and coagulation cascade responses. In summary, our study demonstrates that the increased vulnerability of the elderly to severe COVID-19 can be attributed to an attenuated antiviral response and the overactivation of complement and coagulation cascades.","version":"1.1","doi":"10.1101/2024.09.24.614691","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.25.614910","pub_date":"2024-9-25","title":"Inflammation cellular platform (INCEPLAT) for testing anti-inflammatory compounds for SARS-CoV-2","abstract":"From the early days of the COVID-19 pandemic, an excessive release of proinflammatory cytokines, such as IL6, was detected in serum from patients. As a consequence, several anti-inflammatory drugs, such as Dexamethasone (a strong corticoid), were used to counteract such cytokine storm occurring during severe disease. By contrast, pro-inflammatory interleukin 11 (IL11), a member of the IL6 family, was detected in respiratory tissues from infected patients and in experimental epithelial cellular models. In this work, human A549 lung epithelial cells were individually transduced with SARS-CoV-2 open reading frames (ORFs), resulting in a IL11 increase, which was significantly decreased after Dexamethasone treatment. The use of this cellular platform allowed us to screen for new possible anti-inflammatory compounds from Fasciola hepatica. Our results highlighted the ability of FhNEJ (Fasciola hepatica newly excysted juvenile flukes) somatic extract to decrease IL11 levels in ORF-transduced cells. These results emphasized the role of IL11 in lung epithelial inflammation, making it a potential target for future treatments of lung inflammation which occurs in COVID-19, and validate the use of these ORF-expressing cells as a cellular platform to test anti-inflammatory compounds for COVID-19 disease.","version":"1.1","doi":"10.1101/2024.09.25.614910","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.25.614931","pub_date":"2024-9-25","title":"SARS-CoV-2 ORF3a Protein Impairs Syncytiotrophoblast Maturation, Alters ZO-1 Localization, and Shifts Autophagic Pathways in Trophoblast Cells and 3D Organoids","abstract":"SARS-CoV-2 infection poses a significant risk to placental physiology, but its impact on placental homeostasis is not well understood. We and others have previously shown that SARS-CoV-2 can colonize maternal and fetal placental cells, yet the specific mechanisms remain unclear. In this study, we investigate ORF3a, a key accessory protein of SARS-CoV-2 that exhibits continuous mutations. Our findings reveal that ORF3a is present in placental tissue from pregnant women infected with SARS-CoV-2 and disrupts autophagic flux in placental cell lines and 3D stem-cell-derived trophoblast organoids (SC-TOs), impairing syncytiotrophoblast differentiation and trophoblast invasion. This disruption leads to protein aggregation in cytotrophoblasts (CTB) and activates secretory autophagy, increasing CD63+ extracellular vesicle secretion, along with ORF3a itself. ORF3a also compromises CTB barrier integrity by disrupting tight junctions via interaction with ZO-1, mediated by its PDZ-binding motif, SVPL. Colocalization of ORF3a and ZO-1 in SARS-CoV-2-infected human placental tissue supports our in vitro findings. Deleting the PDZ binding motif in the ORF3a protein (ORF3a-noPBM mutant) restored proper ZO-1 localization at the cell junctions in an autophagy-independent manner. Lastly, we demonstrate that constitutive ORF3a expression induces SC-TOs to transition towards a secretory autophagy pathway likely via the PBM motif, as the ORF3a-NoPBM mutants showed a significant lack of CD63 expression. This study demonstrates the functional impact of ORF3a on placental autophagy and reveals a new mechanism for the activation of secretory autophagy, which may lead to increased extracellular vesicle secretion. These findings provide a foundation for exploring therapeutic approaches targeting ORF3a, specifically focusing on its PBM region to block its interactions with host cellular proteins and limiting placental impact.","version":"1.1","doi":"10.1101/2024.09.25.614931","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.24.613938","pub_date":"2024-9-25","title":"Respiratory shedding of infectious SARS-CoV-2 Omicron XBB.1.41.1 lineage with increased evolutionary rate among captive white-tailed deer","abstract":"White-tailed deer (Odocoileus virginianus) have high value for research, conservation, agriculture and recreation, and may be important SARS-CoV-2 reservoirs with unknown human health implications. In November 2023, we sampled 15 female deer in a captive facility in central Texas, USA. All individuals had neutralizing antibodies against SARS-CoV-2 and 11 had RT-qPCR-positive respiratory swabs; one also had a positive rectal swab. Six of 11 respiratory swabs yielded infectious virus with replication kinetics of most samples displaying lower growth 24-48 h post infection in vitro when compared to Omicron lineages isolated from humans in Texas in the same period. However, virus growth was similar between groups by 72 h, suggesting no strong attenuation of deer-derived virus. All deer viruses clustered in XBB Omicron clade, with more mutations than expected compared to contemporaneous viruses in humans, suggesting that crossing the species barrier to deer was accompanied by a high substitution rate.","version":"1.1","doi":"10.1101/2024.09.24.613938","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.24.614759","pub_date":"2024-9-25","title":"Structural and Functional Glycosylation of the Abdala COVID-19 Vaccine","abstract":"Abdala is a COVID-19 vaccine produced in Pichia pastoris and is based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Abdala is currently approved for use in multiple countries with clinical trials confirming its safety and efficacy in preventing severe illness and death. Although P. pastoris is used as an expression system for protein-based vaccines, yeast glycosylation remains largely uncharacterised across immunogens. Here, we characterise N-glycan structures and their site of attachment on Abdala and show how yeast-specific glycosylation decreases binding to the ACE2 receptor and a receptor-binding motif (RBM) targeting antibody compared to the equivalent mammalian-derived RBD. Reduced receptor and antibody binding is attributed to changes in conformational dynamics resulting from N-glycosylation. These data highlight the critical importance of glycosylation in vaccine design and demonstrate how individual glycans can influence host interactions and immune recognition via protein structural dynamics.","version":"1.1","doi":"10.1101/2024.09.24.614759","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.25.614883","pub_date":"2024-9-25","title":"The dimerization domain of SARS CoV 2 Nucleocapsid protein is partially disordered as a monomer and forms a high affinity dynamic complex","abstract":"The SARS-CoV-2 Nucleocapsid (N) is a 419 amino acids protein that drives the compaction and packaging of the viral genome. This compaction is aided not only by protein-RNA interactions, but also by protein-protein interactions that contribute to increasing the valence of the nucleocapsid protein. Here, we focused on quantifying the mechanisms that control dimer formation. Single-molecule F\u00f6rster Resonance Energy Transfer enabled us to investigate the conformations of the dimerization domain in the context of the full-length protein as well as the energetics associated with dimerization. Under monomeric conditions, we observed significantly expanded configurations of the dimerization domain (compared to the folded dimer structure), which are consistent with a dynamic conformational ensemble. The addition of unlabeled protein stabilizes a folded dimer configuration with a high mean transfer efficiency, in agreement with predictions based on known structures. Dimerization is characterized by a dissociation constant of \u223c 12 nM at 23 OC and is driven by strong enthalpic interactions between the two protein subunits, which originate from the coupled folding and binding. Interestingly, the dimer structure retains some of the conformational heterogeneity of the monomeric units, and the addition of denaturant reveals that the dimer domain can significantly expand before being completely destabilized. Our findings suggest that the inherent flexibility of the monomer form is required to adopt the specific fold of the dimer domain, where the two subunits interlock with one another. We proposed that the retained flexibility of the dimer form may favor the capture and interactions with RNA, and that the temperature dependence of dimerization may explain some of the previous observations regarding the phase separation propensity of the N protein.","version":"1.1","doi":"10.1101/2024.09.25.614883","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.23.614552","pub_date":"2024-9-25","title":"Expression of a constitutively active nitrate reductase increases SARS-CoV-2 Spike protein production in Nicotiana benthamiana leaves that otherwise show traits of senescence","abstract":"The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the Spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana, a process promoted by the co-expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus-like particles (CoVLPs) occurs. We recently used RNAseq and time course sampling to characterize molecular responses of N. benthamiana leaf cells expressing P19 only, or P19 in combination with recombinant S protein. This revealed expression of the viral proteins to deeply affect the physiological status of plant cells, including through the activation of immune responses. Here, transcriptomics shows that the production of CoVLPs also induces leaf senescence, as revealed by the upregulation of senescence-associated genes, activation of senescence-related proteases, and downregulation of genes involved in basic metabolic functions like photosynthesis or nitrogen uptake and assimilation. CoVLP production also upregulated asparagine synthetase genes and led to consequent accumulation of asparagine, a nitrogen-rich amino acid is known to facilitate the reallocation of nitrogen resources from senescent to young growing organs. Hypothesizing these combined host responses to restrain foreign protein accumulation, an attempt was made to support nitrogen reduction in CoVLP-producing leaves by co-expressing a constitutively active, light-insensitive form of the nitrate reductase. We show this strategy to increase S protein accumulation in leaf tissues, thereby suggesting that boosting nitrogen metabolism of agroinfiltrated leaves improves recombinant protein yields in N. benthamiana.","version":"1.1","doi":"10.1101/2024.09.23.614552","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.29.605618","pub_date":"2024-9-25","title":"Resolving the Structure of a Guanine Quadruplex in TMPRSS2 Messenger RNA by Circular Dichroism and Molecular Modeling","abstract":"The presence of a guanine quadruplex in the opening reading frame of the messenger RNA coding for the transmembrane serine protease 2 (TMPRSS2) may pave the way to original anticancer and host-oriented antiviral strategy. Indeed, TMPRSS2 in addition to being overexpressed in different cancer types, is also related to the infection of respiratory viruses, including SARS-CoV-2, by promoting the cellular and viral membrane fusion through its proteolytic activity. The design of selective ligands targeting TMPRSS2 messenger RNA requires a detailed knowledge, at atomic level, of its structure. Therefore, we have used an original experimental-computational protocol to predict the first resolved structure of the parallel guanine quadruplex secondary structure in the RNA of TMPRSS2, which shows a rigid core flanked by a flexible loop. This represents the first atomic scale structure of the guanine quadruplex structure present in TMPRSS2 messenger RNA.","version":"1.2","doi":"10.1101/2024.07.29.605618","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.22.614338","pub_date":"2024-9-24","title":"Direct and ultrasensitive bioluminescent detection of intact respiratory viruses","abstract":"Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of intact virus. Instead, specific detection of intact respiratory virus particles may better assess the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of \u2018free\u2019 viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1 - 0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.","version":"1.1","doi":"10.1101/2024.09.22.614338","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.21.614276","pub_date":"2024-9-23","title":"Neuroinvasive and neurovirulent potential of SARS-CoV-2 in the acute and post-acute phase of intranasally inoculated ferrets","abstract":"Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) can cause systemic disease, including neurological complications, even after mild respiratory disease. Previous studies have shown that SARS-CoV-2 infection can induce neurovirulence through microglial activation in the brains of patients and experimentally inoculated animals, which are models representative for moderate to severe respiratory disease. Here, we aimed to investigate the neuroinvasive and neurovirulent potential of SARS-CoV-2 in intranasally inoculated ferrets, a model for subclinical to mild respiratory disease. The presence of viral RNA, histological lesions, virus-infected cells, and the number and surface area of microglia and astrocytes were investigated. Viral RNA was detected in various respiratory tissue samples by qPCR at 7 days post inoculation (dpi). Virus antigen was detected in the nasal turbinates of ferrets sacrificed at 7 dpi and was associated with inflammation. Viral RNA was detected in the brains of ferrets sacrificed 7 dpi, but in situ hybridization nor immunohistochemistry did not verify evidence of infection. Histopathological analysis of the brains showed no evidence for an influx of inflammatory cells. Despite this, we observed an increased number of Alzheimer type II astrocytes in the hindbrains of SARS-CoV-2 inoculated ferrets. Additionally, we detected an increased microglial activation in the olfactory bulb and hippocampus, and a decrease in the astrocytic activation status in the white matter and hippocampus of SARS-CoV-2 inoculated ferrets. In conclusion, although showed that SARS-CoV-2 has limited neuroinvasive potential in this model for subclinical to mild respiratory disease, there is evidence for neurovirulent potential. This study highlights the value of this ferret model to study the neuropathogenecity of SARS-CoV-2 and reveals that a mild SARS-CoV-2 infection can affect both microglia and astrocytes in different parts of the brain.","version":"1.1","doi":"10.1101/2024.09.21.614276","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.20.24314056","pub_date":"2024-09-23","title":"How floods may affect the spatial spread of respiratory pathogens: the case of Emilia-Romagna, Italy in May 2023","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The negative impact of floods on public health has been increasing, as climate change makes these events more frequent and intense. Floods are known to cause direct injury and favor the spread of many waterborne and vector-borne pathogens. Their effect on the circulation of respiratory pathogens, like influenza and SARS-CoV-2, is, however, still unclear. In this study, we quantify this effect through the analysis of large-scale behavioral data coupled to mathematical models of epidemic spread. We focus on the devastating floods occurred in Italy in 2023 and measure how they impacted human contact patterns within and between communities. We find a substantial increase in contacts occurring 3 weeks after the floods, both among residents of the affected areas and between them and those living in distant, unaffected areas of Italy. Then, through mathematical simulations, we determine that these disrupted contact patterns can carry a circulating pathogen to previously unaffected geographic areas, as well as increasing infection counts across the country. Our findings may help set up protocols to use large-scale human contact data to contain epidemic outbreaks before, during and in the aftermath of floods.</jats:p>","version":null,"doi":"10.1101/2024.09.20.24314056","journal":"medRxiv","score":null},{"id":"10.1101/2022.09.15.508120","pub_date":"2024-9-21","title":"SARS-CoV-2 and HSV-1 Induce Amyloid Aggregation in Human CSF Resulting in Drastic Soluble Protein Depletion","abstract":"The corona virus (SARS-CoV-2) pandemic and the resulting long-term neurological complications in patients, known as long COVID, have renewed the interest in the correlation between viral infections and neurodegenerative brain disorders. While many viruses can reach the central nervous system (CNS) causing acute or chronic infections (such as herpes simplex virus 1, HSV-1), the lack of a clear mechanistic link between viruses and protein aggregation into amyloids, a characteristic of several neurodegenerative diseases, has rendered such a connection elusive. Recently, we showed that viruses can induce aggregation of purified amyloidogenic proteins via the direct physicochemical mechanism of heterogenous nucleation (HEN). In the current study, we show that the incubation of HSV-1 and SARS-CoV-2 with human cerebrospinal fluid (CSF) leads to the amyloid aggregation of several proteins known to be involved in neurodegenerative diseases, such as: APLP1 (amyloid beta precursor like protein 1), ApoE, clusterin, \u03b12-macroglobulin, PGK-1 (phosphoglycerate kinase 1), ceruloplasmin, nucleolin, 14-3-3, transthyretin and vitronectin. Importantly, UV-inactivation of SARS-CoV-2 does not affect its ability to induce amyloid aggregation, as amyloid formation is dependent on viral surface catalysis via HEN and not its ability to replicate. Additionally, viral amyloid induction led to a dramatic drop in the soluble protein concentration in the CSF. Our results show that viruses can physically induce amyloid aggregation of proteins in human CSF and result in soluble protein depletion, and thus providing a potential mechanism that may account for the association between persistent and latent/reactivating brain infections and neurodegenerative diseases. Viruses have generally been excluded from the etiology of amyloid pathologies based on the assumption that amyloid formation requires a proteinaceous conformational template (a prion) to form. Here we show that neuroinvasive viruses induce amyloid aggregation of a plethora of proteins in human CSF even after UV inactivation. Our work illustrates that viruses can induce amyloid aggregation of endogenous human proteins in their native environment by acting as physical catalysts of amyloid nucleation and phase transition. Demonstrating this direct mechanistic link, which is independent of templating, can help better understand the link between viruses and neurodegenerative disorders, especially in the post-COVID-19 era.","version":"1.2","doi":"10.1101/2022.09.15.508120","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.27.586931","pub_date":"2024-9-21","title":"TCR transgenic clone selection guided by immune receptor analysis and single cell RNA expression of polyclonal responders","abstract":"Since the precursor frequency of na\u00efve T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are PCR-cloned into expression vectors. Here, we exploited the rapid advances in single cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice (CORona Spike Epitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.","version":"1.2","doi":"10.1101/2024.03.27.586931","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.19.611578","pub_date":"2024-9-20","title":"Involvement of 5\u2019 and 3\u2019 UTRs on SARS-CoV-2 Genome Packaging","abstract":"The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, packages its large single-stranded RNA genome through a precise yet enigmatic mechanism. A packaging signal (PS) within its genome was proposed to facilitate the assembly of new viral particles. Here in this study, we report the role of the 5\u2019 and 3\u2019 untranslated regions (UTRs) in PS-mediated genome packaging. Utilizing proximity ligation data, we demonstrate direct interactions between the UTRs and the PS9 element, a key packaging signal within the SARS-CoV-2 genome. Multiple evidence confirmed that the presence of UTRs enhance the packaging efficiency of infectious virus-like particles (iVLPs) and recruits more nucleocapsid (N) protein, suggesting a critical role in genome compaction and packaging. These insights into the regulatory mechanisms of SARS-CoV-2 genome packaging provide novel targets for antiviral therapeutics and contribute to the broader understanding of coronavirus assembly.","version":"1.1","doi":"10.1101/2024.09.19.611578","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.19.613860","pub_date":"2024-9-20","title":"Designing and bioengineering of CDRs with higher affinity against receptor-binding domain (RBD) of SARS-CoV-2 Omicron variant","abstract":"The emergence of the SARS-CoV-2 Omicron variant highlights the need for innovative strategies to address evolving viral threats. This study bioengineered three nanobodies H11-H4, C5, and H3 originally targeting the Wuhan RBD, to bind more effectively to the Omicron RBD. A structure-based in silico affinity maturation pipeline was developed to enhance their binding affinities. The pipeline consists of three key steps: high-throughput in silico mutagenesis of complementarity-determining regions (CDRs), protein-protein docking for screening, and molecular dynamics (MD) simulations for assessment of the complex stability. A total of 741, 551, and 684 mutations were introduced in H11-H4, C5, and H3 nanobodies, respectively. Protein-protein docking and MD simulations shortlisted high-affinity mutants for H11-H4(6), C5(5), and H3(6). Further, recombinant production of H11-H4 mutants and Omicron RBD enabled experimental validation through Isothermal Titration Calorimetry (ITC). The H11-H4 mutants R27E, S57D, S107K, D108W, and A110I exhibited improved binding affinities with dissociation constant (KD) values ranging from \u223c8.8 to \u223c27 \u00b5M, compared to the H11-H4 nanobody KD of \u223c32 \u00b5M, representing a three-fold enhancement. This study demonstrates the potential of the developed in silico affinity maturation pipeline as a rapid, cost-effective method for repurposing nanobodies, aiding the development of robust prophylactic strategies against evolving SARS-CoV-2 variants and other pathogens.","version":"1.1","doi":"10.1101/2024.09.19.613860","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.18.613660","pub_date":"2024-9-19","title":"SARS-CoV-2 Survival on Skin and its Transfer from Contaminated Surfaces","abstract":"Understanding the transmission dynamics of SARS-CoV-2, particularly its transfer from contaminated surfaces (fomites) to human skin, is crucial for mitigating the spread of COVID-19. While extensive research has examined the persistence of SARS-CoV-2 on various surfaces, there is limited understanding of how efficiently it transfers to human skin, and how long it survives on the skin. This study investigates two key aspects of SARS-CoV-2 transmission: (1) the transfer efficiency of SARS-CoV-2 from non-porous (plastic and metal) and porous (cardboard) surfaces to a 3D human skin model (LabSkin), and (2) the survival of SARS-CoV-2 on the skin under different temperature conditions. First, we validated LabSkin as a suitable surrogate for human skin by comparing the transfer efficiency of the bacteriophage Phi 6 from surfaces to LabSkin and to human volunteers\u2019 fingers. No significant differences were observed, confirming LabSkin\u2019s suitability for these studies. Subsequently, the transfer of SARS-CoV-2 from surfaces to LabSkin was assessed, showing that plastic and metal surfaces had similar transfer efficiencies (\u223c13%), while no transfer occurred from cardboard once the inoculum had dried on the surface. Finally, the survival of SARS-CoV-2 on skin was assessed, showing a rapid decay at higher temperatures, with a half-life ranging from 2.8 to 17.8 hours depending on the temperature. These findings enhance our understanding of viral transmission via fomites and inform public health strategies to reduce the risk of SARS-CoV-2 transmission through surface contact.","version":"1.1","doi":"10.1101/2024.09.18.613660","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.05.602129","pub_date":"2024-9-18","title":"From a single sequence to evolutionary trajectories: protein language models capture the evolutionary potential of SARS-CoV-2 protein sequences","abstract":"Protein language models (PLMs) capture features of protein three-dimensional structure from amino acid sequences alone, without requiring multiple sequence alignments (MSA). The concepts of grammar and semantics from natural language have the potential to capture functional properties of proteins. Here, we investigate how these representations enable assessment of variation due to mutation. Applied to SARS-CoV-2\u2019s spike protein using in silico deep mutational scanning (DMS) we demonstrate the PLM, ESM-2, has learned the sequence context within which variation occurs, capturing evolutionary constraint. This recapitulates what conventionally requires MSA data to predict. Unlike other state-of-the-art methods which require protein structures or multiple sequences for training, we show what can be accomplished using an unmodified pretrained PLM. We demonstrate that the grammaticality and semantic scores represent novel metrics. Applied to SARS-CoV-2 variants across the pandemic we show that ESM-2 representations encode the evolutionary history between variants, as well as the distinct nature of variants of concern upon their emergence, associated with shifts in receptor binding and antigenicity. PLM likelihoods can also identify epistatic interactions among sites in the protein. Our results here affirm that PLMs are broadly useful for variant-effect prediction, including unobserved changes, and can be applied to understand novel viral pathogens with the potential to be applied to any protein sequence, pathogen or otherwise.","version":"1.2","doi":"10.1101/2024.07.05.602129","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.17.613525","pub_date":"2024-9-18","title":"Acute Viral Infection Accelerates Neurodegeneration in a Mouse Model of ALS","abstract":"While several viral infections have been associated with amyotrophic lateral sclerosis (ALS), the mechanism(s) through which they promote disease has remained almost entirely elusive. This study investigated the impact of common, acute viral infections prior to disease onset on ALS progression in the SOD1G93A mouse model. A single sublethal infection prior to onset of ALS clinical signs was associated with markedly accelerated ALS disease progression characterized by rapid loss of hindlimb function. Prior infection resulted in gliosis in the lumbar spine and upregulation of transcriptional pathways involved in inflammatory responses, metabolic dysregulation, and muscular dysfunction. Therapeutic suppression of gliosis with an anti-inflammatory small molecule, or administration of a direct-acting antiviral, was associated with significantly improved ALS clinical signs, akin to what was observed in uninfected animals. This study provides causal and mechanistic evidence that the immune response elicited by acute viral infections may be an important etiological factor that alters ALS disease trajectory, and provides insight into novel therapeutic and preventative strategies for ALS. Acute viral infection with influenza A virus and SARS-CoV-2 accelerates the progression of ALS in SOD1G93A mice.","version":"1.1","doi":"10.1101/2024.09.17.613525","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.16.613250","pub_date":"2024-9-17","title":"Affinity tag free purification of SARS-Cov-2 N protein and its crystal structure in complex with ssDNA","abstract":"The nucleocapsid (N) protein is one of the four structural proteins in SARS-CoV-2, playing key roles in viral assembly, immune evasion, and stability. One of its primary functions is to protect viral RNA by forming the nucleocapsid. However, the precise mechanisms of how the N protein interacts with viral RNA and assembles into a nucleocapsid remain unclear. Compared to other SARS-CoV-2 components, the N protein has several advantages: higher sequence conservation, lower mutation rates, and stronger immunogenicity, making it an attractive target for antiviral drug development and diagnostics. Therefore, a detailed understanding of the N protein\u2019s structure is essential for deciphering its role in viral assembly and for developing effective therapeutics. In this study, we report the expression and purification of a soluble recombinant N protein, along with a 1.55\u00c5 resolution crystal structure of its nucleic acid-binding domain (N-NTD) in complex with ssDNA. Our structure reveals new insights into the conformation and interaction of the flexible N-arm, which could aid in understanding nucleocapsid assembly. Additionally, we identify residues that are critical for ssDNA interaction.","version":"1.1","doi":"10.1101/2024.09.16.613250","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.14.613008","pub_date":"2024-9-16","title":"In computer explore The neutralization mechanism of Amubarvimab and Romlusevimab against SARS-COV-2 mutants","abstract":"Since the end of 2019, The coronavirus disease 2019 (COVID-19) has been endemic worldwide for three years, causing more than 6.95 million deaths and having a massive impact on the global political economy. With time, the Severe acute respiratory syndrome coronavirus 2(SARS-COV-2) is also constantly mutating. Mutations lead to stronger infectivity or virulence of the virus, and some monoclonal antibodies against wild-type SARS-COV-2 are also challenging to play a role. Amubarvimab and Romlusevimab were originally developed against wild-type SARS-COV-2; however, these monoclonal antibodies\u2019 neutralizing efficacy and mechanism against these mutants are unknown. In this study, the binding ability of Amubarvimab and Romlusevimab to 7 mutant strains were tested by computer method and the interaction mechanism was explored. Our experimental data show that Amubarvimab can effectively bind most mutations and maintain the stability of the complexes mainly through hydrogen bond interaction; However, the binding efficiency of Romlusevimab was lower than that of Amubarvimab, and the stability of the complexes was maintained mainly through electrostatic interaction. Both Amubarvimab and Romlusevimab show low binding potency against E406W and Q498Y mutations, so there is a certain probability of immune escape in the face of variants carrying E406W and Q498Y mutations when Amubarvimab and Romlusevimab are used in combination.","version":"1.1","doi":"10.1101/2024.09.14.613008","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.13.613000","pub_date":"2024-9-16","title":"Modeling suggests SARS-CoV-2 rebound after nirmatrelvir-ritonavir treatment is driven by target cell preservation coupled with incomplete viral clearance","abstract":"In a subset of SARS-CoV-2 infected individuals treated with the oral antiviral nirmatrelvir-ritonavir, the virus rebounds following treatment. The mechanisms driving this rebound are not well understood. We used a mathematical model to describe the longitudinal viral load dynamics of 51 individuals treated with nirmatrelvir-ritonavir, 20 of whom rebounded. Target cell preservation, either by a robust innate immune response or initiation of nirmatrelvir-ritonavir near the time of symptom onset, coupled with incomplete viral clearance, appear to be the main factors leading to viral rebound. Moreover, the occurrence of viral rebound is likely influenced by time of treatment initiation relative to the progression of the infection, with earlier treatments leading to a higher chance of rebound. Finally, our model demonstrates that extending the course of nirmatrelvir-ritonavir treatment, in particular to a 10-day regimen, may greatly diminish the risk for rebound in people with mild-to-moderate COVID-19 and who are at high risk of progression to severe disease. Altogether, our results suggest that in some individuals, a standard 5-day course of nirmatrelvir-ritonavir starting around the time of symptom onset may not completely eliminate the virus. Thus, after treatment ends, the virus can rebound if an effective adaptive immune response has not fully developed. These findings on the role of target cell preservation and incomplete viral clearance also offer a possible explanation for viral rebounds following other antiviral treatments for SARS-CoV-2. Nirmatrelvir-ritonavir is an effective treatment for SARS-CoV-2. In a subset of individuals treated with nirmatrelvir-ritonavir, the initial reduction in viral load is followed by viral rebound once treatment is stopped. We show the timing of treatment initiation with nirmatrelvir-ritonavir may influence the risk of viral rebound. Nirmatrelvir-ritonavir stops viral growth and preserves target cells but may not lead to full clearance of the virus. Thus, once treatment ends, if an effective adaptive immune response has not adequately developed, the remaining virus can lead to rebound. Our results provide insights into the mechanisms of rebound and can help develop better treatment strategies to minimize this possibility.","version":"1.1","doi":"10.1101/2024.09.13.613000","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.13.612805","pub_date":"2024-9-15","title":"Variant Evolution Graph: Can We Infer How SARS-CoV-2 Variants are Evolving?","abstract":"The SARS-CoV-2 virus has undergone mutations over time, leading to genetic diversity among circulating viral strains. This genetic diversity can affect the characteristics of the virus, including its transmissibility and the severity of symptoms in infected individuals. During the pandemic, this frequent mutation creates an enormous cloud of variants known as viral quasispecies. Most variation is lost due to the tight bottlenecks imposed by transmission and survival. Advancements in next-generation sequencing have facilitated the rapid and cost-effective production of complete viral genomes, enabling the ongoing monitoring of the evolution of the SARS-CoV-2 genome. However, inferring a reliable phylogeny from GISAID (the Global Initiative on Sharing All Influenza Data) is daunting due to the vast number of sequences. In the face of this complexity, this research proposes a new method of representing the evolutionary and epidemiological relationships among the SARS-CoV-2 variants inspired by quasispecies theory. We aim to build a Variant Evolution Graph (VEG), a novel way to model viral evolution in a local pandemic region based on the mutational distance of the genotypes of the variants. VEG is a directed acyclic graph and not necessarily a tree because a variant can evolve from more than one variant; here, the vertices represent the genotypes of the variants associated with their human hosts, and the edges represent the evolutionary relationships among these variants. A disease transmission network, DTN, which represents the transmission relationships among the hosts, is also proposed and derived from the VEG. We downloaded the genotypes of the variants recorded in GISAID, which are complete, have high coverage, and have a complete collection date from five countries: Somalia (22), Bhutan (102), Hungary (581), Iran (1334), and Nepal (1719). We ran our algorithm on these datasets to get the evolution history of the variants, build the variant evolution graph represented by the adjacency matrix, and infer the disease transmission network. Our research represents a novel and unprecedented contribution to the field of viral evolution, offering new insights and approaches not explored in prior studies.","version":"1.1","doi":"10.1101/2024.09.13.612805","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.12.612614","pub_date":"2024-9-14","title":"SARS-CoV-2 protein ORF3a induces Atg8ylation of lysosomal membranes","abstract":"Autophagy Conjugation machinery forms a center piece of autophagy and is essential for sequestration of a broad range of cargo destined for degradation. Apart from its role in canonical autophagy, recent evidence suggests an unconventional role of conjugation machinery. Membrane Atg8ylation is one of the manifestations of autophagy, wherein ATG8 conjugation machinery recruit mammalian ATG8s (mATG8s) to the damaged membranes for repair or removal. Herein, we show that SARS-CoV-2 factor ORF3a induces membrane Atg8ylation and selectively inflicts lysophagy, a cellular response to evade apoptotic cell death. mATG8s and SNARE protein syntaxin 17 (STX17) interact with ORF3a and are required for Atg8ylation induced by ORF3a. ORF3a displaces mTOR from the lysosomes and affects nuclear translocation of TFEB, which is dependent on mATG8s and STX17. Despite mTOR inhibition, its conventional target ULK1 is dispensable for ORF3a induced Atg8ylation. In addition, mATG8s and STX17 protected against the cell death induced by ORF3a. Overall, our findings demonstrate ORF3a induced lysosomal membrane Atg8ylation while identifying the unexpected role of STX17 in Atg8ylation.","version":"1.1","doi":"10.1101/2024.09.12.612614","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.12.612455","pub_date":"2024-9-14","title":"Ongoing evolution of Middle East Respiratory Syndrome Coronavirus, Kingdom of Saudi Arabia, 2023-2024","abstract":"Middle East respiratory syndrome coronavirus (MERS-CoV) circulates in dromedary camels in the Arabian Peninsula and occasionally causes spillover infections in humans. Due to lack of sampling during the SARS-CoV-2 pandemic, current MERS-CoV diversity is poorly understood. Of 558 dromedary camel nasal swabs from Saudi Arabia, sampled November 2023 to January 2024, 39% were positive for MERS-CoV RNA by RT-PCR. We generated 42 MERS-CoV and seven human 229E-related CoV by high-throughput sequencing. For both viruses, the sequences fell into monophyletic clades apical to the most recent available genomes. The MERS-CoV sequences were most similar to those from lineage B5. The new MERS-CoVs sequences harbor unique genetic features, including novel amino acid polymorphisms in the Spike protein. The new variants require further phenotypic characterization to understand their impact. Ongoing MERS-CoV spillovers into humans pose significant public health concerns, emphasizing the need for continued surveillance and phenotypic studies.","version":"1.1","doi":"10.1101/2024.09.12.612455","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.11.612489","pub_date":"2024-9-13","title":"PROS1 released by human lung basal cells upon SARS-CoV-2 infection facilitates epithelial cell repair and limits inflammation","abstract":"Factors governing the coagulopathy and pneumonitis associated with severe viral infections remain unresolved. We previously found that the expression of protein S (PROS1) is increased in lung epithelium of patients with mild COVID-19 as compared to severe COVID-19. We hypothesised that PROS1 may exert a local effect that protects the upper airway against severe inflammation by modulating epithelial and myeloid cell responses. To test this, in vitro air-interface cultures, seeded from primary healthy human lung epithelial cells, were infected with different SARS-CoV-2 clades. This model, validated by single-cell RNAseq analysis, recapitulated the dynamic cell-profile and pathogenic changes of COVID-19. We showed that PROS1 was located in the basal cells of healthy pseudostratified epithelium. During SARS-Cov-2 infection, PROS1 was released by basal cells, which was partially mediated by interferon. Transcriptome analysis showed that SARS-CoV-2 infection induced proinflammatory phenotypes (CXCL10/11high, PTGS2posF3high, S100A8/A9high) of basal and transitional cells. PROS1 strongly downregulated these cells and transformed the proinflammatory CXCL10/11high basal cells into the regenerative S100A2posKRThigh basal cell phenotype. In addition, SARS-CoV-2 infection elevated M-CSF secretion from epithelium, which induced MERTK, a receptor for PROS1, on monocytes added into 3D lung epithelial culture. We demonstrated that SARS-CoV-2 drives monocyte phenotypes expressing coagulation (F13A1) and complement (C1\u01ea) genes. PROS1 significantly downregulated these phenotypes and induced higher expression of MHC class II. Overall, this study demonstrated that the epithelium-derived PROS1 during SARS-CoV-2 infection inhibits the proinflammatory epithelial phenotypes, favours basal cell regeneration, and inhibits myeloid inflammation while enhancing antigen presentation. These findings highlight the importance of basal epithelial cells and PROS1 protection from viral infection induced severe lung pathology. 1) SARS-CoV2 infection of the epithelium results in release of IFN. 2) IFN secretion has an autocrine effect on epithelial cells 3) Infection and IFN cause release of PROS1 from the basal cells, as well as M-CSF from the epithelium 4) PROS1 acts on basal cells which express MERTK, a PROS1 receptor 5) PROS1 downregulated the proinflammatory phenotypes expanded by viral infection, while upregulating KRThigh basal cells with repair phenotypes 6) The secreted M-CSF drives MERTK expression on monocytes in cocultures with epithelium. 7) PROS1 induces downregulation of monocyte clusters characteristic of viral infection that express pro-coagulation and complement genes, while upregulating clusters with higher MHC class II. 8) In summary, PROS1 mediates phenotypic switch of SARS-Cov2 induced pathogenic myeloid clusters with complement and coagulation phenotypes into phenotype with efficient antigen presentation, reduces proinflammatory activation of epithelium and induces epithelial barrier repair, resulting in mild COVID-19.","version":"1.1","doi":"10.1101/2024.09.11.612489","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.11.612497","pub_date":"2024-9-13","title":"Differential Immunoregulation by Human Surfactant Protein A Variants Determines Severity of SARS-CoV-2-induced Lung Disease","abstract":"COVID-19 remains a significant threat to public health globally. Infection in some susceptible individuals causes life-threatening acute lung injury (ALI/ARDS) and/or death. Human surfactant protein A (SP-A) is a C-type lectin expressed in the lung and other mucosal tissues, and it plays a critical role in host defense against various pathogens. The human SP-A genes (SFTPA1 and SFTPA2) are highly polymorphic and comprise several common genetic variants, i.e., SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). Here, we elucidated the differential antiviral and immunoregulatory roles of SP-A variants in response to SARS-CoV-2 infection in vivo. Six genetically-modified mouse lines, expressing both hACE2 (SARS-CoV-2 receptor) and individual SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3(1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were challenged intranasally with 103 PFU SARS-CoV-2 or saline (Sham). Infected KO and 1A0 mice had more weight loss and mortality compared to other mouse lines. Relative to other infected mouse lines, a more severe ALI was observed in KO, 1A0, and 6A2 mice. Reduced viral titers were generally observed in the lungs of infected SP-A mice relative to KO mice. Transcriptomic analysis revealed an upregulation in genes that play central roles in immune responses such as MyD88, Stat3, IL-18, and Jak2 in the lungs of KO and 1A0 mice. However, Mapk1 was significantly downregulated in 6A2 versus 1A0 mice. Analysis of biological pathways identified those involved in lung host defense and innate immunity, including pathogen-induced cytokine, NOD1/2, and Trem1 signaling pathways. Consistent with the transcriptomic data, levels of cytokines and chemokines such as G-CSF, IL-6 and IL-1\u03b2 were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. These findings demonstrate that human SP-A variants differentially modulate SARS-CoV-2-induced lung injury and disease severity by differentially inhibiting viral infectivity and regulating immune-related gene expressions.","version":"1.1","doi":"10.1101/2024.09.11.612497","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.12.612598","pub_date":"2024-9-13","title":"Intranasal parainfluenza virus-vectored vaccine expressing SARS-CoV-2 spike protein of Delta or Omicron B.1.1.529 induces mucosal and systemic immunity and protects hamsters against homologous and heterologous challenge","abstract":"The continuous emergence of new SARS-CoV-2 variants requires that COVID vaccines be updated to match circulating strains. We generated B/HPIV3-vectored vaccines expressing 6P-stabilized S protein of the ancestral, B.1.617.2/Delta, or B.1.1.529/Omicron variants as pediatric vaccines for intranasal immunization against HPIV3 and SARS-CoV-2 and characterized these in hamsters. Following intranasal immunization, these B/HPIV3 vectors replicated in the upper and lower respiratory tract and induced mucosal and serum anti-S IgA and IgG. B/HPIV3 expressing ancestral or B.1.617.2/Delta-derived S-6P induced serum antibodies that effectively neutralized SARS-CoV-2 of the ancestral and B.1.617.2/Delta lineages, while the cross-neutralizing potency of B.1.1.529/Omicron S-induced antibodies was lower. Despite the lower cross-neutralizing titers induced by B/HPIV3 expressing S-6P from B.1.1.529/Omicron, a single intranasal dose of all three versions of B/HPIV3 vectors was protective against matched or heterologous WA1/2020, B.1.617.2/Delta or BA.1 (B.1.1.529.1)/Omicron challenge; hamsters were protected from challenge virus replication in the lungs, while low levels of challenge virus were detectable in the upper respiratory tract of a small number of animals. Immunization also protected against lung inflammatory response after challenge, with mild inflammatory cytokine induction associated with the slightly lower level of cross-protection of WA1/2020 and B.1.617.2/Delta variants against the BA.1/Omicron variant. Serum antibodies elicited by all vaccine candidates were broadly reactive against 20 antigenic variants, but the antigenic breadth of antibodies elicited by B/HPIV3-expressed S-6P from the ancestral or B.1.617.2/Delta variant exceeded that of the S-6P B.1.1.529/Omicron expressing vector. These results will guide development of intranasal B/HPIV3 vectors with S antigens matching circulating SARS-CoV-2 variants. Intranasal COVID vaccines have the potential to stimulate respiratory mucosal immunity, effectively restricting replication of SARS-CoV-2 in the respiratory tract, thereby reducing virus shedding and transmission. To develop pediatric vaccines for intranasal immunization against HPIV3 and SARS-CoV-2, we use live-attenuated bovine-human parainfluenza virus vaccine, a pediatric intranasal parainfluenza virus vaccine candidate, designed to express the stabilized SARS-CoV-2 spike protein. We compared the immunogenicity and breadth of protection of the B/HPIV3-expressed ancestral, B.1.617.2/Delta, or B.1.1.529/Omicron variants following intranasal immunization in hamsters. All three B/HPIV3 vectors replicated in the respiratory tract, induced mucosal and serum anti-S IgA and IgG, and were protective in the hamster model against matched or heterologous WA1/2020, B.1.617.2/Delta or BA.1 (B.1.1.529.1)/Omicron SARS-CoV-2 challenge. Serum antibodies elicited by all intranasal vaccine candidates were broadly reactive against 20 antigenic variants of SARS-CoV-2, but the antigenic breadth of antibodies elicited by B/HPIV3-expressed stabilized S from the ancestral or B.1.617.2/Delta variant exceeded that of the S-6P B.1.1.529/Omicron expressing vector. Thus, these intranasal vectored SARS-CoV-2 vaccine candidates induce cross-protective SARS-CoV-2 immunity with antigenic breadth similar to that of injectable SARS-CoV-2 vaccines. These results will guide development of intranasal COVID vaccines based on B/HPIV3 vectors with S antigens matching current SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.09.12.612598","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.12.612662","pub_date":"2024-9-13","title":"Monovalent XBB.1.5 mRNA Vaccine Recalls a More Durable and Coordinated Antibody Response to SARS-CoV-2 Spike than the Bivalent WT/BA.5 mRNA Vaccine","abstract":"In the fall of 2023, the monovalent XBB.1.5 mRNA vaccine for COVID-19 became available. However, the comparative magnitude, durability, and functionality of antibody responses induced by the XBB.1.5 vaccine compared with the 2022-2023 bivalent wildtype (WT) + Omicron BA.5 vaccine remains to be fully determined. In this study, we compared antibody profiles generated by these two vaccines in healthcare workers. We show that the monovalent XBB.1.5 vaccine induced higher magnitude binding, neutralizing, and Fc-gamma receptor (Fc\u03b3R) binding antibodies to the XBB.1.5 spike compared with the bivalent vaccine against the WT and BA.5 spikes, both at both peak immunogenicity and at 6 months post-vaccination. Moreover, antibody interaction architectures and correlations remained more robust at 6 months post-vaccination with the XBB.1.5 vaccine, whereas these correlations were largely lost at 6 months with the bivalent vaccine. Our results suggest that the XBB.1.5 vaccine led to a more durable and functionally coordinated antibody response compared to the bivalent vaccine.","version":"1.1","doi":"10.1101/2024.09.12.612662","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.11.612487","pub_date":"2024-9-12","title":"Electromagnetic waves destabilize the SARS-CoV-2 Spike protein and reduce SARS-CoV-2 Virus-Like Particle (SC2-VLP) infectivity","abstract":"Infection and transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to pose a global public health concern. Using electromagnetic waves represents an alternative strategy to inactivate pathogenic viruses such as SARS-CoV-2 and reduce overall transmission. However, whether electromagnetic waves reduce SARS-CoV-2 infectivity is unclear. Here, we adapted a coplanar waveguide (CPW) to identify electromagnetic waves that could neutralize SARS-CoV-2 virus-like particles (SC2-VLPs). Treatment of SC2-VLPs, particularly at frequencies between 2.5-3.5 GHz at an electric field of 400 V/m for 2 minutes, reduced infectivity. Exposure to a frequency of 3.1 GHz decreased the binding of SC2-VLPs to antibodies directed against the Spike S1 subunit receptor binding domain (RBD). These results suggest that electromagnetic waves alter the conformation of Spike, thereby reducing viral attachment to host cell receptors. Overall, this data provides proof-of-concept in using electromagnetic waves for sanitation and prevention efforts to curb the transmission of SARS-CoV-2 and potentially other pathogenic enveloped viruses.","version":"1.1","doi":"10.1101/2024.09.11.612487","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.11.575201","pub_date":"2024-9-12","title":"The relationship between gut and nasopharyngeal microbiome composition can predict the severity of COVID-19","abstract":"Coronavirus disease 2019 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that displays great variability in clinical phenotype. Many factors have been described to be correlated with its severity, and microbiota could play a key role in the infection, progression, and outcome of the disease. SARS-CoV-2 infection has been associated with nasopharyngeal and gut dysbiosis and higher abundance of opportunistic pathogens. To identify new prognostic markers for the disease, a multicenter prospective observational cohort study was carried out in COVID-19 patients divided into three cohorts based on symptomatology: mild (n=24), moderate (n=51), and severe/critical (n=31). Faecal and nasopharyngeal samples were taken, and the microbiota was analyzed. Linear discriminant analysis identified M. salivarium, P. dentalis, and H. parainfluenzae as biomarkers of severe COVID-19 in nasopharyngeal microbiota, while P. bivia and P. timonensis were defined in faecal microbiota. Additionally, a connection between faecal and nasopharyngeal microbiota was identified, with a significant ratio between P. timonensis (faeces) and P. dentalis and M. salivarium (nasopharyngeal) abundances found in critically ill patients. This ratio could serve as a novel prognostic tool for identifying severe COVID-19 cases.","version":"1.2","doi":"10.1101/2024.01.11.575201","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.07.574522","pub_date":"2024-9-06","title":"A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models","abstract":"Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.","version":"1.2","doi":"10.1101/2024.01.07.574522","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.05.611382","pub_date":"2024-9-05","title":"Genomic Epidemiology of SARS-CoV-2 in Norfolk, UK, March 2020 \u2013 December 2022","abstract":"In the UK, the COVID-19 Genomics UK Consortium (COG-UK) established a real time national genomic surveillance system during the COVID-19 pandemic, producing centralised data for monitoring SARS-CoV-2. As a COG-UK partner, Quadram Institute Bioscience (QIB) in Norfolk sequenced over 87,000 SARS-CoV-2 genomes, contributing to the region becoming densely sequenced. Retrospective analysis of SARS-CoV-2 lineage dynamics in this region may contribute to preparedness for future pandemics. 29,406 SARS-CoV-2 whole genome sequences and corresponding metadata from Norfolk were extracted from the COG-UK dataset, sampled between March 2020 and December 2022, representing 9.9% of regional COVID-19 cases. Sequences were lineage typed using Pangolin, and subsequent lineage analysis carried out in R using RStudio and related packages, including graphical analysis using ggplot2. 401 global lineages were identified, with 69.8% appearing more than once and 31.2% over ten times. Temporal clustering identified six lineage communities based on first lineage emergence. Alpha, Delta, and Omicron variants of concern (VOC) accounted for 8.6%, 34.9% and 48.5% of sequences respectively. These formed four regional epidemic waves alongside the remaining lineages which appeared in the early pandemic prior to VOC designation and were termed \u2018pre-VOC\u2019 lineages. Regional comparison highlighted variability in VOC epidemic wave dates dependent on location. This study is the first to assess SARS-CoV-2 diversity in Norfolk across a large timescale within the COVID-19 pandemic. SARS-CoV-2 was both highly diverse and dynamic throughout the Norfolk region between March 2020 \u2013 December 2022, with a strong VOC presence within the latter two thirds of the study period. The study also displays the utility of incorporating genomic epidemiological methods into pandemic response. The COG-UK collection of SARS-CoV-2 sequences and metadata are available for public download on their archive website under the \u2018Latest sequence data\u2019 heading <https://webarchive.nationalarchives.gov.uk/ukgwa/20230507102210/https://www.cogconsortium.uk/priority-areas/data-linkage-analysis/public-data-analysis/>. Sequence names for all sequences used from this dataset alongside GISAID accession numbers where present are available in Supplementary Table 1. We extracted 29,406 regional Norfolk based SARS-CoV-2 sequences from the COG-UK SARS-CoV-2 dataset and revealed significant regional diversity and dynamic emergence of variant of concern (VOC) epidemic waves \u2013 spanning Alpha, Delta and Omicron lineages. We also applied statistical modelling to complement genomic methodology, with temporal clustering of significant first lineage emergences chronologically matching VOC waves and subwaves. The study highlights the importance of integration of genomic epidemiology into public health strategies for pandemic response, and the utility of using this data for retrospective research.","version":"1.1","doi":"10.1101/2024.09.05.611382","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.05.611469","pub_date":"2024-9-05","title":"SARS-CoV-2 Nsp15 antagonizes the cGAS-STING-mediated antiviral innate immune responses","abstract":"Coronavirus (CoV) Nsp15 is a viral endoribonuclease (EndoU) with a preference for uridine residues. CoV Nsp15 is an innate immune antagonist which prevents dsRNA sensor recognition and stress granule formation by targeting viral and host RNAs. SARS-CoV-2 restricts and delays the host antiviral innate immune responses through multiple viral proteins, but the role of SARS-CoV-2 Nsp15 in innate immune evasion is not completely understood. Here, we generate an EndoU activity knockout rSARS-CoV-2Nsp15-H234A to elucidate the biological functions of Nsp15. Relative to wild-type rSARS-CoV-2, replication of rSARS-CoV-2Nsp15-H234A was significantly decreased in IFN-responsive A549-ACE2 cells but not in its STAT1 knockout counterpart. Transcriptomic analysis revealed upregulation of innate immune response genes in cells infected with rSARS-CoV-2Nsp15-H234A relative to wild-type virus, including cGAS-STING, cytosolic DNA sensors activated by both DNA and RNA viruses. Treatment with STING inhibitors H-151 and SN-011 rescued the attenuated phenotype of rSARS-CoV-2Nsp15-H234A. SARS-CoV-2 Nsp15 inhibited cGAS-STING-mediated IFN-\u03b2 promoter and NF-\u03baB reporter activity, as well as facilitated the replication of EV-D68 and NDV by diminishing cGAS and STING expression and downstream innate immune responses. Notably, the decline in cGAS and STING was also apparent during SARS-CoV-2 infection. The EndoU activity was essential for SARS-CoV-2 Nsp15-mediated cGAS and STING downregulation, but not all HCoV Nsp15 share the consistent substrate selectivity. In the hamster model, rSARS-CoV-2Nsp15-H234A replicated to lower titers in the nasal turbinates and lungs and induced higher innate immune responses. Collectively, our findings exhibit that SARS-CoV-2 Nsp15 serves as a host innate immune antagonist by targeting host cGAS and STING. Host innate immune system serves as the primary defense against pathogens, including SARS-CoV-2. Co-evolving with the hosts, viruses develop multiple approaches to escape the host surveillance. SARS-CoV-2 silences and dysregulates innate immune responses, and the chaos of antiviral IFN responses highly correlates to COVID-19 disease severity. Nsp15 is a conventional innate immune antagonist across coronaviruses, but the biological impact about SARS-CoV-2 Nsp15 is still unclear. Here, we provide a novel insight that SARS-CoV-2 Nsp15 hampers the expression of innate immune regulator \u2013 cGAS and STING via its endoribonuclease activity, then further ameliorates virus replication.","version":"1.1","doi":"10.1101/2024.09.05.611469","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.05.611400","pub_date":"2024-9-05","title":"The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII","abstract":"Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.","version":"1.1","doi":"10.1101/2024.09.05.611400","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.05.611372","pub_date":"2024-9-05","title":"Conserved role of spike S2 domain N-glycosylation across beta-coronavirus family","abstract":"Besides acting as an immunological shield, the N-glycans of SARS-CoV-2 are also critical for viral life cycle. As the S2 subunit of spike is highly conserved across beta-coronaviruses, we determined the functional significance of the five \u2018stem N-glycans\u2019 located in S2 between N1098-N1194. Studies were performed with 31 Asn-to-Gln mutants, beta-coronavirus virus-like particles and single-cycle viral replicons. Deletions of stem N-glycans enhanced S1 shedding from trimeric spike, reduced ACE2 binding and abolished syncytia formation. When three or more N-glycans were deleted, spike expression on cell surface and incorporation into virions was both reduced. Viral entry function was progressively lost upon deleting the N1098 glycan in combination with additional glycosite modifications. In addition to SARS-CoV-2, deleting stem N-glycans in SARS-CoV and MERS-CoV spike also prevented viral entry into target cells. These data suggest multiple functional roles for the stem N-glycans, and evolutionarily conserved properties for these complex carbohydrates across human beta-coronaviruses. Previous work shows that the N-linked glycans of SARS-CoV-2 are essential for viral life cycle. Few natural mutations have been observed in the S2-subunit of the viral spike glycoprotein in GISAID data, and mutations are absent in the five \u2018stem N-glycans\u2019 located between N1098-N1194. In the post-fusion spike structure these glycans lie equidistant, ~4 nm apart, suggesting functional significance. Upon testing the hypothesis that these glycans are critical for SARS-CoV-2 function, we noted multiple roles for the complex carbohydrates including regulation of S1-subunit shedding, spike expression on cells and virions, syncytial formation/cell-cell fusion and viral entry. Besides SARS-CoV-2, these glycans were also critical for other human beta-coronaviruses. Thus, these carbohydrates represent targets for the development of countermeasures against future outbreaks.","version":"1.1","doi":"10.1101/2024.09.05.611372","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.03.608162","pub_date":"2024-9-05","title":"Nsp1 stalls DNA Polymerase \u03b1 at DNA hairpins","abstract":"The human primosome, a four-subunit complex of DNA primase and DNA polymerase alpha (Pol\u03b1), plays a critical role in DNA replication by initiating RNA and DNA synthesis on both chromosome strands. A recent study has shown that a major virulence factor in the SARS-CoV-2 infection, Nsp1 (non-structural protein 1), forms a stable complex with Pol\u03b1 but does not affect the primosome activity. Here we show that Nsp1 inhibits DNA synthesis across inverted repeats prone to hairpin formation. Analysis of current structural data revealed the overlapping binding sites for Nsp1 and the winged helix-turn-helix domain of RPA (wHTH) on Pol\u03b1, indicating a competition between them. Comparison of the inhibitory effect of Nsp1 and wHTH on DNA hairpin bypass by Pol\u03b1 showed an 8-fold lower IC50 value for Nsp1 (1 \u00b5M). This study provides a valuable insight into the mechanism of inhibition of human DNA replication by Nsp1 during a SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.09.03.608162","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.04.611219","pub_date":"2024-9-05","title":"Neutralization and Stability of JN.1-derived LB.1, KP.2.3, KP.3 and KP.3.1.1 Subvariants","abstract":"During the summer of 2024, COVID-19 cases surged globally, driven by variants derived from JN.1 subvariants of SARS-CoV-2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and stability of these subvariants\u2014LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2, and increased resistance to elevated temperatures. Molecular modeling suggests that the DelS31 mutation induces a conformational change that stabilizes the NTD and strengthens the NTD-Receptor-Binding Domain (RBD) interaction, thus favoring the down conformation of RBD and reducing accessibility to both the ACE2 receptor and certain nAbs. Additionally, the DelS31 mutation introduces an N-linked glycan modification at N30, which shields the underlying NTD region from antibody recognition. Our data highlight the critical role of NTD mutations in the spike protein for nAb evasion, stability, and viral infectivity, and suggest consideration of updating COVID-19 vaccines with antigens containing DelS31.","version":"1.1","doi":"10.1101/2024.09.04.611219","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.04.611185","pub_date":"2024-9-05","title":"From tissue to subcellular level : imaging human precision-cut lung slices (PCLS) to gain insight into pandemic bacterial or viral infections","abstract":"We describe a method for the generation and deep imaging of human precision-cut lung slices (PCLS). PCLS bridge the gap between in vivo and in vitro studies, providing a robust system for visualizing events from tissue to subcellular levels in the three-dimensional lung environment, with the preservation of all resident cell types and cell-cell interactions. They also constitute a validated model for studying host cell-pathogen interactions. Here, we detail the generation of human PCLS, followed by their infection and imaging by laser scanning confocal microscopy and transmission electron microscopy (TEM). We establish the conditions for ex vivo infection and replication of two pathogens of relevance to human respiratory health: a virus (SARS-CoV-2) and a bacterium (Mycobacterium tuberculosis, Mtb). PCLS can be obtained in a single day, infected the next day, and were successfully cultivated for up to a week in this study. Imaging was performed on fixed samples. The preparation of PCLS took one day for confocal imaging and five days for TEM imaging. All procedures are readily adaptable to explore other pathogens and other species and are easy to implement by users with experience in tissue culture. Some specialist equipment (an Alabama tissue slicer) is required for PCLS generation.","version":"1.1","doi":"10.1101/2024.09.04.611185","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.01.610733","pub_date":"2024-9-04","title":"Optimisation of a multiplexed, high throughput assay to measure neutralising antibodies against SARS-CoV-2 variants","abstract":"A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of monoclonal antibody EC50 titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay. Evaluation of convalescent human sera confirmed assay validity, with consistent fold drops in EC50 for variant pseudoviruses relative to the ancestral strain observed across single and multiplexed assays. This multiplexed pVNT provides a reliable tool for assessing nAb responses against SARS-CoV-2 variants and could be used to accelerate preclinical vaccine assessment in preparation for the next coronavirus pandemic.","version":"1.1","doi":"10.1101/2024.09.01.610733","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.03.610799","pub_date":"2024-9-04","title":"Structural and molecular basis of the epistasis effect in enhanced affinity between SARS-CoV-2 KP.3 and ACE2","abstract":"The recent emergence of SARS-CoV-2 variants KP.2 and KP.3 has been marked by mutations F456L/R346T and F456L/Q493E, respectively, which significantly impact the virus\u2019s interaction with human ACE2 and its resistance to neutralizing antibodies. KP.3, featuring F456L and Q493E, exhibits a markedly enhanced ACE2 binding affinity compared to KP.2 and the JN.1 variant due to synergistic effects between these mutations. This study elucidated the structures of KP.2 and KP.3 RBD in complex with ACE2 using cryogenic electron microscopy (Cryo-EM) and decipher the structural and thermodynamic implications of these mutations on receptor binding by molecular dynamics (MD) simulations, revealing that F456L mutation facilitates a more favorable binding environment for Q493E, leading to stronger receptor interactions which consequently enhance the potential for incorporating additional evasive mutations. These results underscore the importance of understanding mutational epistatic interactions in predicting SARS-CoV-2 evolution and optimizing vaccine updates. Continued monitoring of such epistatic effects is crucial for anticipating new dominant strains and preparing appropriate public health responses.","version":"1.1","doi":"10.1101/2024.09.03.610799","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.13.580068","pub_date":"2024-9-03","title":"SARS-CoV-2 Serosurvey of healthy, privately owned cats presenting to a New York City animal hospital in the early phase of the COVID-19 pandemic (2020-2021)","abstract":"SARS-CoV-2, the cause of the ongoing COVID-19 pandemic, not only infects humans but is also known to infect various species, including domestic and wild animals. While many species have been identified as susceptible to SARS-CoV-2, there are limited studies on the prevalence of SARS-CoV-2 in animals. Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the U.S. in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health \u201clock-down\u201d. Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and two of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1/1024 and 1/4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV-2 in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our study provide a new context for SARS-CoV-2 transmission events across species. SARS-CoV-2 has a broad animal tropism and can infect a wide range of animal species, leading to an expansion of the viral reservoir. Expansion of this viral reservoir may result in the accumulation of mutations within these species, potentially giving rise to new viral variants and facilitating reverse zoonotic transmission. Domestic cats are particularly noteworthy in this regard due to their close contact with humans. Currently, there are very limited studies on the prevalence of SARS-CoV-2 infection in domestic cats during the early stages of the pandemic, especially in the United States. This retrospective study addresses the gap by investigating seroprevalence of SARS-CoV-2 in cats in New York City, the epicenter of the COVID-19 pandemic in the United States during the early pandemic. Our work underscores the importance of adopting a One Health approach to pandemic prevention and conducting routine surveillance across different animal species","version":"1.2","doi":"10.1101/2024.02.13.580068","journal":"bioRxiv","score":null},{"id":"10.1101/2024.09.02.610835","pub_date":"2024-9-03","title":"SARS-CoV-2 Mediated Inhibition Of Respiratory Syncytial Virus","abstract":"With circulation of SARS-CoV-2, fears about coinfection with other respiratory viruses such as influenza and RSV were significant, but the opposite was observed. Distancing/barriers played a major role in reducing other viral co-infections, however, some infrequent co-infections still occurred. We investigated the relationship between SARS-CoV-2 and RSV during coinfection to understand how they might compete or synergize. We found only RSV\u2019s replication was significantly reduced when coinfected with SARS-CoV-2. Investigation of the mechanism revealed that the SARS-CoV-2 protein Nsp1 disrupts the RSV M2-2 protein but not the upstream M2-1 protein on the same biscistronic mRNA transcript. The impact of Nsp1 on M2-2 was not dependent on M2-2 being the second ORF in a bicistronic mRNA transcript, but likely from prevention of ribosomal termination-reinitiation necessary for M2-2 production. Additional viral ORFs from influenza A, influenza B, or Sendai virus dependent on the same or other ribosomal initiation mechanisms were tested and we found only influenza B M/M2 which likely uses a similar method as M2-2 was disrupted. Various M2-2 constructs, with/without the proposed site of ribosomal termination-reinitiating, co-transfected with Nsp1 and were in agreement that disruption to M2-2 expression occurs if the site of re-initiation was present upstream. These data not only suggest Sars-CoV-2 can outcompete RSV through suppression of M2, but may also point to potential ways to interfere with RSV by targeted therapies.","version":"1.1","doi":"10.1101/2024.09.02.610835","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.08.566182","pub_date":"2024-9-03","title":"Emergence and spread of feline infectious peritonitis due to a highly pathogenic canine/feline recombinant coronavirus","abstract":"Cross-species transmission of coronaviruses (CoVs) poses a serious threat to both animal and human health. Whilst the large RNA genome of CoVs shows relatively low mutation rates, recombination within genera is frequently observed and demonstrated. Companion animals are often overlooked in the transmission cycle of viral diseases; however, the close relationship of feline (FCoV) and canine CoV (CCoV) to human hCoV-229E, as well as their susceptibility to SARS-CoV-2 highlight their importance in potential transmission cycles. Whilst recombination between CCoV and FCoV of a large fragment spanning orf1b to M has been previously described, here we report the emergence of a novel, highly pathogenic FCoV-CCoV recombinant responsible for a rapidly spreading outbreak of feline infectious peritonitis (FIP), originating in Cyprus. The minor recombinant region, spanning spike (S), shows 96.5% sequence identity to the pantropic canine coronavirus NA/09. Infection has rapidly spread, infecting cats of all ages. Development of FIP appears very frequent and sequence identities of samples from cats in different districts of the island is strongly supportive of direct transmission. A near cat-specific deletion in the domain 0 of S is present in >90% of FIP cats. It is unclear as yet whether this deletion is directly associated with disease development and may be linked to a biotype switch. The domain 0 deletion and several amino acid changes in S, particularly the receptor binding domain, indicate potential changes to receptor binding and cell tropism.","version":"1.3","doi":"10.1101/2023.11.08.566182","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.30.610497","pub_date":"2024-8-30","title":"SARS-CoV-2 Delta variant induces severe damages in the nasal cavity from the first day post-infection in the Syrian hamster model","abstract":"SARS-CoV-2 replication initiates in the nasal cavity and can spread to the lower respiratory tract. However, the early physiopathological events that occur in the nasal cavity after infection remain poorly understood. In this study, we investigated the initial steps of viral infection from 1 day post-infection (dpi) in Syrian hamsters infected with SARS-CoV-2 D614G, Delta and Omicron (BA.1) variants and compared them with animals sacrificed at 4 dpi. While the level of viral replication in the nasal turbinates of the three groups of hamsters was equivalent at 4dpi, the amount of viral RNA at 1dpi was higher in D614G- and Delta-infected animals than in the Omicron group. No difference in viral RNA levels or inflammatory markers in the nasal turbinates was observed between D614G- and Delta-infected animals, except for a significantly higher level of IFN-\u03bb in the Delta group at 1dpi. Additionally, histological analysis revealed a more rapid diffusion of the Delta virus reaching the posterior zone of the nasal cavity at 1dpi inducing significant damage to the olfactory epithelium. At the same time, the D614G and Omicron infections were essentially restricted to the anterior part of the nasal cavity with less damage observed. Consistently, viral replication was already effective in the lungs of all Delta- infected hamsters at 1 dpi, but only in two of the six D614G animals. Our results highlight the importance of studying viral infection in the nasal cavity very early after infection with a spatial approach to better understand the physiopathology of the different SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.08.30.610497","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.07.579374","pub_date":"2024-8-29","title":"Dimensionality reduction distills complex evolutionary relationships in seasonal influenza and SARS-CoV-2","abstract":"Public health researchers and practitioners commonly infer phylogenies from viral genome sequences to understand transmission dynamics and identify clusters of genetically-related samples. However, viruses that reassort or recombine violate phylogenetic assumptions and require more sophisticated methods. Even when phylogenies are appropriate, they can be unnecessary or difficult to interpret without specialty knowledge. For example, pairwise distances between sequences can be enough to identify clusters of related samples or assign new samples to existing phylogenetic clusters. In this work, we tested whether dimensionality reduction methods could capture known genetic groups within two human pathogenic viruses that cause substantial human morbidity and mortality and frequently reassort or recombine, respectively: seasonal influenza A/H3N2 and SARS-CoV-2. We applied principal component analysis (PCA), multidimensional scaling (MDS), t-distributed stochastic neighbor embedding (t-SNE), and uniform manifold approximation and projection (UMAP) to sequences with well-defined phylogenetic clades and either reassortment (H3N2) or recombination (SARS-CoV-2). For each low-dimensional embedding of sequences, we calculated the correlation between pairwise genetic and Euclidean distances in the embedding and applied a hierarchical clustering method to identify clusters in the embedding. We measured the accuracy of clusters compared to previously defined phylogenetic clades, reassortment clusters, or recombinant lineages. We found that MDS embeddings accurately represented pairwise genetic distances including the intermediate placement of recombinant SARS-CoV-2 lineages between parental lineages. Clusters from t-SNE embeddings accurately recapitulated known phylogenetic clades, H3N2 reassortment groups, and SARS-CoV-2 recombinant lineages. We show that simple statistical methods without a biological model can accurately represent known genetic relationships for relevant human pathogenic viruses. Our open source implementation of these methods for analysis of viral genome sequences can be easily applied when phylogenetic methods are either unnecessary or inappropriate.","version":"1.2","doi":"10.1101/2024.02.07.579374","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.12.575267","pub_date":"2024-8-29","title":"De novo-designed minibinders expand the synthetic biology sensing repertoire","abstract":"Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled \u201csmart\u201d therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo-designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. We further demonstrate that two other minibinders directed against the clinically relevant epidermal growth factor receptor are able to drive CAR-dependent cytotoxicity with efficacy similar to or better than an existing antibody-based CAR. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.","version":"1.2","doi":"10.1101/2024.01.12.575267","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.28.609965","pub_date":"2024-8-29","title":"Development of ketobenzothiazole-based peptidomimetic TMPRSS13 inhibitors with low nanomolar potency","abstract":"TMPRSS13, a member of the Type II Transmembrane Serine Proteases (TTSP) family, is involved in cancer progression and in cell entry of respiratory viruses. To date, no inhibitors have been specifically developed toward this protease. In this study, a chemical library of 65 ketobenzothiazole-based peptidomimetic molecules was screened against a proteolytically active form of recombinant TMPRSS13 to identify novel inhibitors. Following an initial round of screening, subsequent synthesis of additional derivatives supported by molecular modelling, uncovered important molecular determinants involved in TMPRSS13 inhibition. One inhibitor, N-0430, achieved low nanomolar affinity towards TMPRSS13 activity in a cellular context. Using a SARS-CoV-2 pseudovirus cell entry model, we further show the ability of N-0430 to block TMPRSS13-dependent entry of the pseudovirus. The identified peptidomimetic inhibitors and the molecular insights of their potency gained from this study will aid in the development of specific TMPRSS13 inhibitors.","version":"1.1","doi":"10.1101/2024.08.28.609965","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.28.610093","pub_date":"2024-8-28","title":"Identification of a new ORF3a-E fusion subgenomic RNA of SARS-CoV-2 and its biological features in the infection process","abstract":"Subgenomic RNAs (sgRNAs) are discontinuous transcription products of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are involved in viral gene expression and replication, but their exact functions are still being studied. Here, we report the identification of a new type of sgRNA, the fusion ORF3a-E-sgRNA, involved in the infection process of SARS-CoV-2. This sgRNA codes both ORF3a and E and can be detected throughout the viral life cycle in SARS-CoV-2-infected cells with high copy numbers. ORF3a-E-sgRNA guides more ORF3a translation and promotes the expression of cellular ribosomal protein S3 (RPS3) and the binding of eukaryotic translation initiation factor 4E (eIF4E). Single-cell sequencing of a SARS-CoV-2-infected human bronchial epithelial cell line (16HBE) revealed that maintenance of this stable translational environment by ORF3a-E-sgRNA is important for the SARS-CoV-2 assembly and release capabilities and is also beneficial for viral evasion of host innate immunity. More importantly, the transcription level of ORF3a-E-sgRNA contributes to differences in infection processes between the Wuhan strain and XBB strain of SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.08.28.610093","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.28.610123","pub_date":"2024-8-28","title":"Polyunsaturated fatty acid-derived lipid mediator networks characterize COVID-19 severity and risk for critical illness","abstract":"Severe COVID-19, caused by SARS-CoV-2 infection, is characterized by excessive inflammation leading to the development of pneumonia and acute respiratory distress syndrome. Bioactive lipid mediators (LMs) derived from \u03c96 and \u03c93 polyunsaturated fatty acids are central to the regulation of inflammation, controlling both its initiation and resolution. Still, their role in COVID-19 remains underexplored. By employing a holistic approach involving the analysis of white blood cell transcriptomes, targeted lipidomics, cytokine and immune cell profiling, across the spectrum of disease severity groups, including mild non-hospitalized patients and healthy individuals, we now show that LM networks are profoundly altered in COVID-19, correlate with inflammatory patterns, and stratify patients according to disease severity. Central to this are CYP450-derived LMs such as 20-HETE, lipid peroxidation metabolites such as iPF2a-VI, and lipoxygenase-derived LMs such as 12-HETE, all of which are major vasoactive mediators of inflammation. Among them, 20-HETE appears to be a promising prognostic biomarker for ICU admission and a potential therapeutic target for severe COVID-19 disease. Our study thus underscores the significance of LM networks in COVID-19 pathophysiology and sheds light into the broader mechanisms driving viral pneumonia in humans.","version":"1.1","doi":"10.1101/2024.08.28.610123","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.03.555867","pub_date":"2024-8-26","title":"Meisoindigo: An Effective Inhibitor of SARS-CoV-2 Main Protease Revealed by Yeast System","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has had a significant impact on global health and the global economy. Despite the availability of vaccines, limited accessibility and vaccine hesitancy pose challenges in controlling the spread of the disease. Effective therapeutic strategies, including antiviral drugs, are needed to combat the future spread of new SARS-CoV-2 virus variants. The main protease (Mpro) is a critical therapeutic target for COVID-19 medicines, as its inhibition impairs viral replication. However, the use of substances that inhibit Mpro may induce selection pressure. Thus, it is vital to monitor viral resistance to known drugs and to develop new drugs. Here, we have developed a yeast system for the identification of Mpro inhibitors as an alternative to costly and demanding high-biosecurity procedures. The system is based on stable expression of Mpro and does not require selection media. Yeast can be cultured on a rich carbon source, providing rapid growth and screening results. The designed tool was subsequently used to screen the FDA-Approved Drug Library. Several chemicals with Mpro inhibitory properties were identified. We found that meisoindigo, which was not previously known to have the potential to inhibit Mpro, was highly effective. Our results may promote the development of new derivatives with therapeutic properties against SARS-CoV-2 and other beta-coronaviruses.","version":"1.2","doi":"10.1101/2023.09.03.555867","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.25.609621","pub_date":"2024-8-26","title":"Extensive exploration of structure activity relationships for the SARS-CoV-2 macrodomain from shape-based fragment merging and active learning","abstract":"The macrodomain contained in the SARS-CoV-2 non-structural protein 3 (NSP3) is required for viral pathogenesis and lethality. Inhibitors that block the macrodomain could be a new therapeutic strategy for viral suppression. We previously performed a large-scale X-ray crystallography-based fragment screen and discovered a sub-micromolar inhibitor by fragment linking. However, this carboxylic acid-containing lead had poor membrane permeability and other liabilities that made optimization difficult. Here, we developed a shape- based virtual screening pipeline - FrankenROCS - to identify new macrodomain inhibitors using fragment X-ray crystal structures. We used FrankenROCS to exhaustively screen the Enamine high-throughput screening (HTS) collection of 2.1 million compounds and selected 39 compounds for testing, with the most potent compound having an IC50 value equal to 130 \u03bcM. We then paired FrankenROCS with an active learning algorithm (Thompson sampling) to efficiently search the Enamine REAL database of 22 billion molecules, testing 32 compounds with the most potent having an IC50 equal to 220 \u03bcM. Further optimization led to analogs with IC50 values better than 10 \u03bcM, with X-ray crystal structures revealing diverse binding modes despite conserved chemical features. These analogs represent a new lead series with improved membrane permeability that is poised for optimization. In addition, the collection of 137 X-ray crystal structures with associated binding data will serve as a resource for the development of structure-based drug discovery methods. FrankenROCS may be a scalable method for fragment linking to exploit ever-growing synthesis-on- demand libraries.","version":"1.1","doi":"10.1101/2024.08.25.609621","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.22.609137","pub_date":"2024-8-23","title":"Bronchoalveolar Lavage Single-Cell Transcriptomics Identifies Immune Cells Driving COVID-19 Severity in Patients","abstract":"The continuous threats posed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, including the emergence of potentially more infectious and deadly variants, necessitate ongoing studies to uncover novel and detailed mechanisms driving disease severity. Using single-cell transcriptomics, we conducted a secondary data analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients of varying severities and healthy controls to comprehensively examine immune responses. We observed significant immune cell alterations correlating with disease severity. In severe cases, macrophages showed upregulation of pro-inflammatory genes TNF\u03b1 and IL1\u03b2, contributing to severe inflammation and tissue damage. Neutrophils exhibited increased activation, marked by S100A8, CXCL8, and IL1\u03b2 expression, with extended viability and reduced phagocytosis. Genes such as MCL1 and HIF1\u03b1 supported extended viability, while MSR1 and MRC1 indicated reduced phagocytosis. Enhanced formation of neutrophil extracellular traps (NETs) and reduced clearance, indicated by NET-associated markers, were linked to thrombo-inflammation and organ damage. Both macrophages and neutrophils in severe cases showed impaired efferocytosis, indicated by decreased expression of MSR1 and TREM2 in macrophages and downregulation of FCGR3B in neutrophils, leading to the accumulation of apoptotic cells and exacerbating inflammation. Severe cases were characterized by M1 macrophages with high TNF\u03b1 and IL1\u03b2, while milder cases had M2 macrophages with elevated PPAR\u03b3. Low-density neutrophils (LDNs) increased significantly in severe cases, showing higher CXCR4 and CD274 and lower FCGR3B compared to high-density neutrophils (HDNs). NK and T cells in severe cases demonstrated altered receptor and gene expression, with increased activation markers IFN\u03b3 and ISG15, suggesting a paradoxical state of activation and exhaustion. This imbalance suggests a potential mechanism for immune dysregulation and ineffective antiviral responses in severe COVID-19. This analysis highlights the critical role of dysregulated neutrophil, macrophage, NK, and T cell responses in severe COVID-19, identifying potential therapeutic targets and providing novel insights into the disease. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, poses continuous health threats due to emerging, potentially more infectious and deadly variants. We used single-cell gene analysis from lung fluid samples, known as bronchoalveolar lavage (BAL), from COVID-19 patients to understand why some cases become more severe than others. In severe cases, immune cells called macrophages and neutrophils showed higher levels of genes that trigger inflammation and cause damage to the body. These cells were more active and lived longer but were less capable of clearing away dead cells and debris, leading to prolonged inflammation. Severe cases also had more neutrophils that were less effective in fighting infections. Another type of immune cell, NK and T cells, showed changes indicating an ineffective response to the virus, with signals that were not properly coordinated to fight the infection. This imbalance in the immune response can lead to severe inflammation and organ damage. Our findings highlight potential targets for treatments to help manage severe COVID-19 and improve patient outcomes. Understanding these immune cell behaviors through single-cell gene analysis could guide the development of new therapies and improve strategies for treating severe COVID-19.","version":"1.1","doi":"10.1101/2024.08.22.609137","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.22.24312324","pub_date":"2024-8-23","title":"Unraveling the impact of COVID-19 on urban mobility: A Causal Machine Learning Analysis of Beijing\u2019s Subway System","abstract":"The COVID-19 pandemic has drastically altered urban travel patterns, particularly in public transportation systems like subways. This study examines the effects of the pandemic on subway ridership in Beijing by analyzing the influence of 19 factors, including demographics, land use, network metrics, and weather conditions, before and during the pandemic. Data was collected from June 2019 and June 2020, covering 335 subway stations and over 258 million trips. Using a three-stage analytical framework\u2014comprising Light Gradient Boosting Machine (LightGBM) for fitting, Meta-Learners for causal analysis, and SHapley Additive exPlanations (SHAP) for interpretation\u2014we observed a substantial decline in ridership, with approximately 10,000 fewer passengers per station daily, especially in densely populated areas. Our findings reveal significant shifts in influential factors such as centrality, housing prices, and restaurant density. The spatiotemporal analysis highlights the dynamic nature of these changes. This study underscores the need for adaptive urban planning and provides insights for public health strategies to enhance urban resilience in future pandemics. The COVID-19 pandemic has highlighted the vulnerabilities of urban transportation systems, especially subways, to sudden disruptions. This study explores how various factors influencing subway ridership in Beijing changed during the pandemic, revealing significant shifts in travel patterns. By understanding these changes, we can better prepare for future public health emergencies and improve urban resilience. Our research provides critical insights for urban planners, public health officials, and policymakers, enabling them to make informed decisions that enhance the adaptability and sustainability of urban environments in the face of global challenges.","version":"1.1","doi":"10.1101/2024.08.22.24312324","journal":"medRxiv","score":null},{"id":"10.1101/2024.08.23.609196","pub_date":"2024-8-23","title":"Identifying novel chemical matter against the Chikungunya virus nsP3 macrodomain through crystallographic fragment screening","abstract":"Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.","version":"1.1","doi":"10.1101/2024.08.23.609196","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.15.580500","pub_date":"2024-8-22","title":"A critical reexamination of recovered SARS-CoV-2 sequencing data","abstract":"SARS-CoV-2 genomes collected at the onset of the Covid-19 pandemic are valuable because they could help understand how the virus entered the human population. In 2021, Jesse Bloom reported on the recovery of a dataset of raw sequencing reads that had been removed from the NCBI SRA database at the request of the data generators, a scientific team at Wuhan University (Wang et al., 2020b). Bloom concluded that the data deletion had obfuscated the origin of SARS-CoV-2 and suggested that deletion may have been requested to comply with a government order; further, he questioned reported sample collection dates on and after January 30, 2020. Here, we show that sample collection dates were published in 2020 by Wang et al. together with the sequencing reads, and match the dates given by the authors in 2021. Collection dates of January 30, 2020 were manually removed by Bloom during his analysis of the data. We examine mutations in these sequences and confirm that they are entirely consistent with the previously known genetic diversity of SARS-CoV-2 of late January 2020. Finally, we explain how an apparent phylogenetic rooting paradox described by Bloom was resolved by subsequent analysis. Our reanalysis demonstrates that there was no basis to question the sample collection dates published by Wang et al.. The automatically generated Full Text version of our manuscript is missing footnotes; they are available in the PDF version.","version":"1.3","doi":"10.1101/2024.02.15.580500","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.21.608921","pub_date":"2024-8-21","title":"Fc-independent SARS-CoV-2 infection-enhancing antibodies decouple N-terminal and receptor-binding domains by cross-linking neighboring spikes","abstract":"Antibody dependent enhancement (ADE) is a serious concern in vaccine development. The canonical ADE pathways are dependent on the fragment crystallizable (Fc) region of the antibody. In SARS-CoV-2 several antibodies have been discovered that inflict ADE in vitro. These antibodies target the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. We previously proposed that these NTD-targeting infection-enhancing antibodies (NIEAs) cross-link neighboring spike proteins via their NTDs, and that this results in a decoupling between the NTD and receptor binding domain (RBD), facilitating the \u201cRBD down\u201d to \u201cup\u201d transition. In this study we present a combination of molecular dynamics simulations and cryogenic electron microscopy data that, together, demonstrate that NIEAs are indeed able to cross-link neighboring SARS-CoV-2 spike proteins, and that this cross-linking results in a decoupling of the NTD and RBD domains. These findings provide support for an Fc region independent ADE pathway that is not only relevant for SARS-CoV-2 but also for other viruses of which the spike proteins undergo a conformational change upon host cell entry.","version":"1.1","doi":"10.1101/2024.08.21.608921","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.19.608619","pub_date":"2024-8-21","title":"Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response","abstract":"A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus\u2019 immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.","version":"1.1","doi":"10.1101/2024.08.19.608619","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.20.608835","pub_date":"2024-8-21","title":"Escape of SARS-CoV-2 variants KP1.1, LB.1 and KP3.3 from approved monoclonal antibodies","abstract":"First-generation anti-SARS-CoV-2 monoclonal antibodies (mAbs) used for prophylaxis or therapeutic purposes in immunocompromised patients have been withdrawn because of the emergence of resistant Omicron variants. In 2024, two novel mAbs, Pemivibart and Sipavibart, have been approved by health authorities, but their activity against contemporary JN.1 sublineages is poorly characterized. We isolated authentic JN.1.1, KP1.1, LB.1 and KP3.3 viruses and evaluated their sensitivity to neutralization by these mAbs in two target cell lines. Compared to ancestral strains, Pemivibart remained moderately active against JN.1 sub-variants, with a strong increase of 50% Inhibitory Concentration (IC50), reaching up to 3 to 15 \u03bcg/ml for KP3.3. Sipavibart neutralized JN.1.1 but lost antiviral efficacy against KP1.1, LB.1 and KP3.3. Our results highlight the need for a close clinical monitoring of Pemivibart and raise concerns about the clinical efficacy of Sipavibart.","version":"1.1","doi":"10.1101/2024.08.20.608835","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.21.608923","pub_date":"2024-8-21","title":"Aging and viral evolution impair immunity against dominant pan-coronavirus-reactive T cell epitope","abstract":"Immune evasion by escape mutations subverts immunity against SARS-CoV-2. A role of pan-coronavirus immunity for more durable protection is being discussed but has remained understudied. We here investigated the effects of age, mutations, and homo-/heterologous vaccination regimens on the dominant pan-coronavirus-specific cellular and humoral epitope iCope after SARS-CoV-2 infection and vaccination in detail. In the older, quantitatively, and qualitatively reduced iCope-reactive CD4+ T cell responses with narrow TCR repertoires could not be enhanced by vaccination and were further compromised by emerging spike mutations. In contrast pan-coronavirus-reactive humoral immunity was affected only by mutations and not by age. Our results reveal a distinct deficiency of the dichotomous layer of pan-coronavirus immunity in the older, critical for long-term protection against SARS-CoV-2 variants. Aging and viral evolution impair dominant pan-coronavirus immunity, a hallmark of efficient and broad immune competence against SARS-CoV-2","version":"1.1","doi":"10.1101/2024.08.21.608923","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.21.24312107","pub_date":"2024-08-21","title":"vPro-MS enables identification of human-pathogenic viruses from patient samples by untargeted proteomics","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Viral infections are commonly diagnosed by the detection of viral genome fragments or proteins using targeted methods such as PCR and immunoassays. In contrast, metagenomics enables the untargeted identification of viral genomes, expanding its applicability across a broader spectrum. In this study, we introduce proteomics as a complementary approach for the untargeted identification of human-pathogenic viruses from patient samples. The viral proteomics workflow (vPro-MS) is based on an\n                  <jats:italic>in-silico</jats:italic>\n                  derived peptide library covering the human virome in UniProtKB (331 viruses, 20,386 genomes, 121,977 peptides), which was especially designed for diagnostic purposes. A scoring algorithm (vProID score) was developed to assess the confidence of virus identification from proteomics data (\n                  <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='https://github.com/RKI-ZBS/vPro-MS'>https://github.com/RKI-ZBS/vPro-MS</jats:ext-link>\n                  ). In combination with high-throughput diaPASEF-based data acquisition, this workflow enables the analysis of up to 60 samples per day. The specificity was determined to be &gt; 99,9 % in an analysis of 221 plasma, swab and cell culture samples covering 18 different viruses (e.g. SARS, MERS, EBOV, MPXV). The sensitivity of this approach for the detection of SARS-CoV-2 in nasopharyngeal swabs corresponds to a PCR cycle threshold of 27 with comparable quantitative accuracy to metagenomics. vPro-MS enables the integration of untargeted virus identification in large-scale proteomic studies of biofluids such as human plasma to detect previously undiscovered virus infections in patient specimens.\n                </jats:p>","version":null,"doi":"10.1101/2024.08.21.24312107","journal":"medRxiv","score":null},{"id":"10.1101/2024.08.12.607496","pub_date":"2024-8-19","title":"Pemivibart is less active against recent SARS-CoV-2 JN.1 sublineages","abstract":"Protection from COVID-19 vaccination is suboptimal in many immunocompromised individuals. In March 2024, the Food and Drug Administration issued an Emergency Use Authorization for pemivibart (Permagard/VYD222), an engineered human monoclonal antibody, for pre-exposure prophylaxis in this vulnerable population. However, SARS-CoV-2 has since evolved extensively, resulting in multiple Omicron JN.1 sublineages. We therefore evaluated the in vitro neutralizing activity of pemivibart against the prevalent forms of JN.1, including KP.2, KP.3, KP.2.3, LB.1, and, importantly, KP.3.1.1, which is now expanding most rapidly. A panel of VSV-based pseudoviruses representing major JN.1 sublineages was generated to assess their susceptibility to pemivibart neutralization in vitro. Structural analyses were then conducted to understand the impact of specific spike mutations on the virus-neutralization results. Pemivibart neutralized both JN.1 and KP.2 in vitro with comparable activity, whereas its potency was decreased slightly against LB.1, KP.2.3, and KP.3 but substantially against KP.3.1.1. Critically, the 50% inhibitory concentration of pemivibart against KP.3.1.1 was \u223c6 \u00b5g/mL, or \u223c32.7 fold higher than that of JN.1 in our study. Structural analyses suggest that Q493E and the S31-deletion mutations in viral spike contribute to the antibody evasion, with the latter having a more pronounced effect. Our findings show that pemivibart has lost substantial neutralizing activity in vitro against KP.3.1.1, the most rapidly expanding lineage of SARS-CoV-2 today. Close monitoring of its clinical efficacy is therefore warranted. These results also highlight the imperative to expand our arsenal of preventive agents to protect millions of immunocompromised individuals who could not respond robustly to COVID-19 vaccines.","version":"1.2","doi":"10.1101/2024.08.12.607496","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.25.564014","pub_date":"2024-8-19","title":"Early antiviral CD4 and CD8 T cell responses and antibodies are associated with upper respiratory tract clearance of SARS-CoV-2","abstract":"T cells are involved in protective immunity against numerous viral infections. Data regarding functional roles of human T cells in SARS-CoV-2 (SARS2) viral clearance in primary COVID-19 are limited. To address this knowledge gap, samples were assessed for associations between SARS2 upper respiratory tract viral RNA levels and early virus-specific adaptive immune responses for 95 unvaccinated clinical trial participants with acute primary COVID-19 aged 18-86 years old, approximately half of whom were considered high risk for progression to severe COVID-19. Functionality and magnitude of acute SARS2-specific CD4 and CD8 T cell responses were evaluated, in addition to antibody responses. Most individuals with acute COVID-19 developed SARS2-specific T cell responses within 6 days of COVID-19 symptom onset. Early CD4 T cell and CD8 T cell responses were polyfunctional, and both strongly associated with reduced upper respiratory tract SARS2 viral RNA, independent of neutralizing antibody titers. Overall, these findings provide evidence for protective roles for circulating SARS2\u2013specific CD4 and CD8 T cells during acute COVID-19.","version":"1.3","doi":"10.1101/2023.10.25.564014","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.01.606251","pub_date":"2024-8-18","title":"Sulfoglycodendron Antivirals with Scalable Architectures and Activities","abstract":"Many viruses initiate their cell-entry by binding their multi-protein receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multi-protein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed detail nature of binding between these antivirals and viral protein receptors, and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.","version":"1.2","doi":"10.1101/2024.08.01.606251","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.14.607882","pub_date":"2024-8-15","title":"Profiling serum immunodominance following SARS-CoV-2 primary and breakthrough infection reveals distinct variant-specific epitope usage and immune imprinting","abstract":"Over the course of the COVID-19 pandemic, variants have emerged with increased mutations and immune evasive capabilities. This has led to breakthrough infections (BTI) in vaccinated individuals, with a large proportion of the neutralizing antibody response targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike glycoprotein. Immune imprinting, where prior exposure of an antigen to the immune system can influence the response to subsequent exposures, and its role in a population with heterogenous exposure histories has important implications in future vaccine design. Here, we develop an accessible approach to map epitope immunodominance of the neutralizing antibody response in sera. By using a panel of mutant Spike in a pseudovirus neutralization assay, we observed distinct epitope usage in convalescent donors infected during wave 1, or infected with the Delta, or BA.1 variants, highlighting the antigenic diversity of the variant Spikes. Analysis of longitudinal serum samples taken spanning 3 doses of vaccine and subsequent breakthrough infection (BTI), showed the influence of immune imprinting from the ancestral-based vaccine, where reactivation of existing B cells elicited by the vaccine resulted in the enrichment of the pre-existing epitope immunodominance. However, subtle shifts in epitope usage in sera were observed following BTI by Omicron sub-lineage variants. Antigenic distance of Spike, time after last exposure, and number of vaccine boosters may play a role in the persistence of imprinting from the vaccine. This study provides insight into RBD neutralizing epitope usage in individuals with varying exposure histories and has implications for design of future SARS-CoV-2 vaccines. Throughout the COVID-19 pandemic, the continued emergence of new SARS-CoV-2 variants has resulted in a rise in breakthrough infections (BTIs). Infection with different variants has led to varying exposure histories in the general population. Although the neutralizing response to Spike has been thoroughly characterized, with several key epitopes identified, there is a lack of knowledge of the proportion each epitope contributes to the neutralizing response in sera and how this is affected by exposure history. Here, we use a panel of mutant Spike pseudoviruses to screen epitope usage and immunodominance in polyclonal sera. In a cohort of unvaccinated donors infected with different variants, distinct epitope usage was observed, highlighting the antigenic diversity between the variant Spikes. Furthermore, samples collected spanning multiple vaccine doses and BTI showed the influence of prior immunity from the vaccine on epitope usage. Although a large proportion of the immune response following BTI could be attributed to enrichment of pre-existing immunodominance from the vaccine, subtle shifts in epitope usage were observed with infection by more mutated variants. This work gives more detailed insight into differences in the neutralizing response of individuals with varying exposure histories that may inform next generation SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2024.08.14.607882","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.14.607914","pub_date":"2024-8-15","title":"SARS-CoV-2 and Mycobacterium tuberculosis co-infection in vitro","abstract":"In less than a year, SARS-CoV-2 (SARS2) has managed to displace Mycobacterium tuberculosis (Mtb) as the leading cause of death worldwide due to a single infectious agent. Both pathogens affect the respiratory tract, mainly the lungs. However, the impact that a possible Mtb + SARS2 co-infection can have on the host response is still unknown. Herein we propose (depict) a rigorous system to evaluate the complex interaction between the two infections in vitro in a lung epithelial cell line (A549). Overall, the process includes eight steps: (I) Mtb culture, (II) cell maintenance, (III) preparation of viral stocks, (IV) determination of infectious titers, (V) Mtb and SARS2 co-infection, (VI) determination of intracellular bacterial load, (VII) SARS2 viability test, and (VIII) decontamination of supernatants. This comprehensive protocol will allow experimentalists to study the pathogenesis of co-infection in vitro, and facilitate collaborative work in the literature.","version":"1.1","doi":"10.1101/2024.08.14.607914","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.13.607736","pub_date":"2024-8-15","title":"AAB-seq: An antigen-specific and affinity-readable high-throughput BCR sequencing method","abstract":"B-cell receptor (BCR) sequencing is a powerful antibody discovery tool but current methodology is often inefficient, and lead generation often requires the production and testing of numerous antibody candidates, and it is difficult to provide affinity information for their antibodies at the same time. Here, we introduce AAB-seq (antigen affinity-readable High-throughput BCR sequencing), an efficient antibody screening tool to identify antigen binding affinity of thousands of paired BCRs. It employs fluorophore and DNA barcode-labeled antigen and secondary antibody targeting Ig light chain to label B cells and uses high throughput single cell BCR sequencing and surface protein profiling to obtain the ratio of surface bound antigen to surface BCR in thousands of B-cells. Using AAB-seq, we accurately identified valuable candidate antibodies 1743-3 and 1743-13 from SARS-CoV-2 RBD immunized mouse, providing a basis for further development of SARS-CoV-2 antibody drugs. Thus, AAB-seq allows high throughput identification of antibody sequences paired with antigen affinity, which improves the screening efficiency of functional antibodies and provides an effective solution for the rapid discovery and development of new therapeutic monoclonal antibodies.","version":"1.1","doi":"10.1101/2024.08.13.607736","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.13.607855","pub_date":"2024-8-15","title":"Identification of biomarkers for COVID-19 associated secondary hemophagocytic lymphohistiocytosis","abstract":"We aimed to define and validate novel biomarkers that could identify individuals with COVID-19 associated secondary hemophagocytic lymphohistiocytosis (sHLH) and to test whether fatalities due to COVID-19 in the presence of sHLH were associated with specific defects in the immune system. In two cohorts of adult patients presenting with COVID-19 in 2020 and 2021, clinical lab values and serum proteomics were assessed. Subjects identified as having sHLH were compared to those with COVID-19 without sHLH. Eight deceased patients defined as COVID-sHLH underwent genomic sequencing in order to identify variants in immune-related genes. Two tertiary care hospitals in Seattle, Washington (Virginia Mason Medical Center and Harborview Medical Center). 186 patients with COVID-19 None Nine percent of enrolled COVID-19 subjects met our defined criteria for sHLH. Using broad serum proteomic approaches (O-link and SomaScan), we identified three biomarkers for COVID-19 associated sHLH (soluble PD-L1, TNF-R1, and IL-18BP), supporting a role for proteins previously associated with other forms of sHLH (IL-18BP and sTNF-R1). We also identified novel biomarkers and pathways of COVID-sHLH, including sPD-L1 and the syntaxin pathway. We detected variants in several genes involved in immune responses in individuals with COVID-sHLH, including in DOCK8 and in TMPRSS15, suggesting that genetic alterations in immune-related genes may contribute to hyperinflammation and fatal outcomes in COVID-19. Biomarkers of COVID-19 associated sHLH, such as soluble PD-L1, and pathways, such as the syntaxin pathway, and variants in immune genes in these individuals, suggest critical roles for the immune response in driving sHLH in the context of COVID-19. To define biomarkers that could identify individuals with COVID-19 associated secondary hemophagocytic lymphohistiocytosis (sHLH) and to test whether fatalities due to COVID-19 in the presence of sHLH were associated with specific defects in the immune system. In two independent cohorts using two different platforms, we identified sPD-L1, IL-18BP, and sTNF-R1 as COVID-sHLH biomarkers. We identified the syntaxin pathway as important in COVID-sHLH and variants in immune-related genes in a subset of deceased COVID-sHLH subjects. Immune related proteins and pathways are dysregulated in COVID-sHLH.","version":"1.1","doi":"10.1101/2024.08.13.607855","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.13.607777","pub_date":"2024-8-14","title":"Torsional Twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains","abstract":"Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.","version":"1.1","doi":"10.1101/2024.08.13.607777","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.29.582713","pub_date":"2024-8-13","title":"Direct pharmacological AMPK activation inhibits mucosal SARS-CoV-2 infection by reducing lipid metabolism, restoring autophagy flux and the type I IFN response","abstract":"AMP-activated protein kinase (AMPK) plays a central role in regulating cell energy balance. When activated, AMPK supresses energy-consuming pathways such as lipid and protein synthesis while increasing nutrient availability through the activation of autophagy. These pathways downstream AMPK activation contribute to SARS-CoV-2 infection, which hijacks autophagy and accumulates lipid droplets in viral factories to support viral replication. Here, we assessed the antiviral activity of the direct pan-AMPK allosteric activator MK-8722 in vitro. MK-8722 efficiently inhibited infection of Alpha and Omicron SARS-CoV-2 variants in Vero76 and human bronchial epithelial Calu-3 cells at micromolar concentration. This inhibition relied on restoring the autophagic flux, which redirected newly synthesized viral proteins for degradation, and on a reduction in lipid metabolism, which affected the viral factories. Furthermore, MK-8722 treatment increased the type I interferon (IFN-I) response. Post-infection treatment with MK-8722 was enough to inhibit efficiently viral replication and restore the IFN-I response. Finally, MK-8722 treatment did not alter the SARS-CoV-2-specific CD8+ T cell response mounted upon Spike vaccination. Overall, by activating AMPK, MK-8722 acts as an effective antiviral against SARS-CoV-2 infection, even when applied post-exposure, paving the way for preclinical tests aimed at inhibiting viral replication and improving patients\u2019 symptoms. MK-8722 exerts post-exposure antiviral activity MK-8722 induces a decrease in cellular lipid content MK-8722 promotes an increase in the autophagic flux of viral components MK-8722 promotes the restoration of the IFN-I activity MK-8722 antiviral activity is compatible with virus-specific T cell responses","version":"1.2","doi":"10.1101/2024.02.29.582713","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.08.607190","pub_date":"2024-8-10","title":"Eosinophils protect against SARS-CoV-2 following a vaccine breakthrough infection","abstract":"Waning immunity and the emergence of immune evasive SARS-CoV-2 variants jeopardize vaccine efficacy leading to breakthrough infections. We have previously shown that innate immune cells play a critical role in controlling SARS-CoV-2. To investigate the innate immune response during breakthrough infections, we modeled breakthrough infections by challenging low-dose vaccinated mice with a vaccine-mismatched SARS-CoV-2 Beta variant. We found that low-dose vaccinated infected mice had a 2-log reduction in lung viral burden, but increased immune cell infiltration in the lung parenchyma, characterized by monocytes, monocyte-derived macrophages, and eosinophils. Single cell RNA-seq revealed viral RNA was highly associated with eosinophils that corresponded to a unique IFN-\u03b3 biased signature. Antibody-mediated depletion of eosinophils in vaccinated mice resulted in increased virus replication and dissemination in the lungs, demonstrating that eosinophils in the lungs are protective during SARS-CoV-2 breakthrough infections. These results highlight the critical role for the innate immune response in vaccine mediated protection against SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.08.08.607190","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.08.606885","pub_date":"2024-8-10","title":"A pan-variant miniprotein inhibitor protects against SARS-CoV-2 variants","abstract":"The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.","version":"1.1","doi":"10.1101/2024.08.08.606885","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.09.607288","pub_date":"2024-8-09","title":"SARS-COV-2 nucleocapsid protein hijacks multiple components of the host nuclear transport machinery for distinct functions","abstract":"Many viruses target the host nuclear transport machinery to traffic their own proteins or restrict trafficking of host cargo, including mediators of antiviral immune signalling. Nucleocapsid (N) proteins from several coronaviruses traffic to the nucleus/nucleolus, with roles in cell cycle regulation. While N-protein from severe acute respiratory syndrome virus 2 (SARS-COV-2), the causative agent of the global COVID-19 pandemic, is widely reported to localise to the cytoplasm, we identify multiple, functionally distinct interactions of N-protein with the host\u2019s nuclear transport machinery. Using quantitative cell imaging, including fluorescence recovery after photobleaching, and protein-protein interaction analysis, we describe a sub-population of SARS-COV-2 N-protein that localises more diffusely between the nucleus and cytoplasm, undergoes active nuclear import, and re-localises nuclear import receptors (karyopherins) KPNB1 and KPNA2 to the nucleus and cytoplasm, respectively. Truncation analyses identify at least two distinct KPNA2/KPNB1 binding sites located in the N-terminal and C-terminal regions of N-protein. Interestingly, while mutation of K/R-rich sites within these domains reduces KPNA2/KPNB1 binding and disables re-localisation of KPNA2, re-localisation of KPNB1 and nuclear import of N-protein remain intact, indicating that these are molecularly and functionally distinct mechanisms. siRNA knockdown confirms a role for KPNB1 in N-protein nuclear trafficking, while KPNA2 binding and mislocalisation may be antagonistic. Thus, SARS-COV-2 N-protein binds to karyopherins via multiple distinct sites to facilitate import and other functions.","version":"1.1","doi":"10.1101/2024.08.09.607288","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.09.607279","pub_date":"2024-8-09","title":"Extraction-free colorimetric RT-LAMP for SARS-CoV-2 RNA detection from saliva","abstract":"To develop a rapid and easy diagnostic assay for detection of SARS-CoV-2 RNA presented in saliva samples. The color-based RT-LAMP was used to detect nucleocapsid (N) gene of SARS-CoV-2 without RNA extraction from saliva. RNA spiked saliva can be used directly for cDNA synthesis after heat inactivation of the saliva and diluted 2-fold with either water or PBS. For both PCR and LAMP, 20% of saliva did not have obvious effect on the reaction. Saliva did not interfere with RT-LAMP when the volume of saliva was less than 20% of the total volume. The sensitivity of the RT-LAMP reached to 1.034\u00d710-5 ng/\u00b5l (475 copies/\u03bcl). The RT-LAMP assay was validated by testing 20 RNA spiked saliva samples. The assay specificity was similar to that of data without saliva. The RT-LAMP colorimetric assay can be used as a screening method with the advantages of being rapid, easy to use than the qRT-PCR.","version":"1.1","doi":"10.1101/2024.08.09.607279","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.06.606943","pub_date":"2024-8-07","title":"AMPK/ULK1 activation downregulates TXNIP, Rab5, and Rab7 and inhibits endocytosis-mediated entry of human pathogenic viruses","abstract":"Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran-uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus and SARS-CoV-2 not only in mammalian and insect cells but in precision-cut lung slices and neuronal organoids. However, receptor presentation at the cytoplasmic membrane was unaffected, indicating that receptor-binding remained unaltered and later steps of endocytosis were targeted. Indeed, AMPK pathway activation reduced early endocytic factors TXNIP, Rab5 and the late endosomal marker Rab7 amounts. Furthermore, AMPK activation impaired SARS-CoV-2 late-replication steps by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction of early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acids and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses.","version":"1.1","doi":"10.1101/2024.08.06.606943","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.05.606725","pub_date":"2024-8-06","title":"A COVID-19 Rapid Antigen Test Employing Upconversion Nanoparticles","abstract":"The COVID-19 pandemic has underscored the critical need for rapid and accurate diagnostic tools. Current methods, including PCR and rapid antigen tests (RAT), have limitations in speed, sensitivity, and the requirement for specialized equipment and trained personnel. Nanotechnology, particularly upconversion nanoparticles (UCNPs), offer a promising alternative due to their unique optical properties. UCNPs can convert low-energy near-infrared (NIR) light into higher-energy visible light, making them ideal for use as optical probes in single molecule detection and point of care applications. This study, initiated in early 2020, explores the opportunity of using highly doped UCNPs (40%Yb3+/4%Er3+) in lateral flow assay (LFA) for the early diagnosis of COVID-19. The UCNPs-based LFA testing demonstrated a minimum detection concentration of 100 pg/mL for SARS-CoV-2 antigen and 105 CCID50/mL for inactivated virus. Clinical trials, conducted in Malaysia and Western Australia independently, showed that the technique was at least 100 times more sensitive than commercial RAT kits, with a sensitivity of 100% and specificity of 91.34%. The development process involved multidisciplinary collaborations, resulting in the Virulizer device, an automated strip reader for point-of-care testing. This work sets a reference for future development of highly sensitive and quantitative rapid antigen tests, aiming for the Limits of Detection (LoD) in the range of sub-ng/mL.","version":"1.1","doi":"10.1101/2024.08.05.606725","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.03.606463","pub_date":"2024-8-06","title":"Comparison of Commercially Available Thermostable DNA Polymerases with Reverse-Transcriptase Activity in Coupled Reverse-Transcription Polymerase Chain Reaction Assays","abstract":"Reverse-transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse-transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection, but not for long-fragment RT-PCR amplification.","version":"1.1","doi":"10.1101/2024.08.03.606463","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.06.606761","pub_date":"2024-8-06","title":"Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning","abstract":"Deep mutational scanning (DMS) has emerged as a powerful approach for evaluating the effects of mutations on binding or function. Here, we developed a multiplexed DMS by phage display protocol to define the binding determinants of short linear motifs (SLiMs) binding to peptide binding domains. We first designed a benchmarking DMS library to evaluate the performance of the approach on well-known ligands for eleven different peptide binding domains, including the talin-1 PTB domain. Systematic benchmarking against a gold-standard set of motifs from the eukaryotic linear motif (ELM) database confirmed that the DMS by phage analysis correctly identifies known motif binding determinants. The DMS analysis further defined a non-canonical PTB binding motif, with a putative extended conformation. A second DMS library was designed aiming to provide information on the binding determinants for 19 SLiM-based interactions between human and SARS-CoV-2 proteins. The analysis confirmed the affinity determining residues of viral peptides binding to host proteins, and refined the consensus motifs in human peptides binding to five domains from SARS-CoV-2 proteins, including the non-structural protein (NSP) 9. The DMS analysis further pinpointed mutations that increased the affinity of ligands for NSP3 and NSP9. An affinity improved cell-permeable NSP9-binding peptide was found to exert stronger antiviral effects as compared to the initial wild-type peptide. Our study demonstrates that DMS by phage display can efficiently be multiplexed and applied to refine binding determinants, and shows how DMS by phage display can guide peptide-engineering efforts.","version":"1.1","doi":"10.1101/2024.08.06.606761","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.05.606734","pub_date":"2024-8-06","title":"Human Vascularized Macrophage-Islet Organoids to Model Immune-Mediated Pancreatic \u03b2 cell Pyroptosis upon Viral Infection","abstract":"There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single cell RNA-seq analysis of human islets exposed to SARS-CoV-2 or Coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and \u03b2 cell pyroptosis. To distinguish viral versus proinflammatory macrophage-mediated \u03b2 cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both \u03b2 cells and endothelial cells compared to separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced \u03b2 cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory macrophage-mediated \u03b2 cell pyroptosis. This study established hPSC- derived VMI organoids as a valuable tool for studying immune cell-mediated host damage and uncovered mechanism of \u03b2 cell damage during viral exposure.","version":"1.1","doi":"10.1101/2024.08.05.606734","journal":"bioRxiv","score":null},{"id":"10.1101/2024.08.01.605860","pub_date":"2024-8-02","title":"Using virtual patient cohorts to uncover immune response differences in cancer and immunosuppressed COVID-19 patients","abstract":"The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in millions of deaths globally. Adults with immunosuppression (e.g., solid organ transplant recipients) and those undergoing active cancer treatments experience worse infections and more severe COVID-19. It is difficult to conduct clinical studies in these populations, resulting in a restricted amount of data that can be used to relate mechanisms of immune dysfunction to COVID-19 outcomes in these vulnerable groups. To study immune dynamics after infection with SARS-CoV-2 and to investigate drivers of COVID-19 severity in individuals with cancer and immunosuppression, we adapted our mathematical model of the immune response during COVID-19 and generated virtual patient cohorts of cancer and immunosuppressed patients. The cohorts of plausible patients recapitulated available longitudinal clinical data collected from patients in Montr\u00e9al, Canada area hospitals. Our model predicted that both cancer and immunosuppressed virtual patients with severe COVID-19 had decreased CD8+ T cells, elevated interleukin-6 concentrations, and delayed type I interferon peaks compared to those with mild COVID-19 outcomes. Additionally, our results suggest that cancer patients experience higher viral loads (however, with no direct relation with severity), likely because of decreased initial neutrophil counts (i.e., neutropenia), a frequent toxic side effect of anti-cancer therapy. Furthermore, severe cancer and immunosuppressed virtual patients suffered a high degree of tissue damage associated with elevated neutrophils. Lastly, parameter values associated with monocyte recruitment by infected cells were found to be elevated in severe cancer and immunosuppressed patients with respect to the COVID-19 reference group. Together, our study highlights that dysfunction in type I interferon and CD8+ T cells are key drivers of immune dysregulation in COVID-19, particularly in cancer patients and immunosuppressed individuals.","version":"1.1","doi":"10.1101/2024.08.01.605860","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.28.605443","pub_date":"2024-7-30","title":"A detailed protocol for generating spike trans-complemented SARS-CoV-2 replicons","abstract":"Multiple approaches have been implemented for basic science studies that attempt to investigate SARS-CoV-2 biology or virology in biosafety level-2 setting. These include pseudotyped-virus based on lentivirus and vesicular stomatitis virus, virus-like particles that only contain the SARS-CoV-2 structural proteins, and single-cycle replicons. Among these, the single-cycle replicons most closely resemble the authentic virus as they essentially include the full viral genome, except for essential elements required for active virus multiplication. In this regard, we previously developed a SARS-CoV-2 replicon system where a Gaussia Dura luciferase-P2A-mNeonGreen reporter cassette replaced viral spike. In this short paper, we present an optimized protocol for the use of this reagent that overcomes previous technical limitations. We demonstrate that co-transfection of this bacmid along with spike plasmid, using the improved protocol, yields high-quality spike bearing SARS-CoV-2 virus particles with single-cycle infectivity. Due to the nature of bacmid construction, this approach is particularly useful for studying the impact of spike mutagenesis on virus evolution in BSL-2 setting.","version":"1.1","doi":"10.1101/2024.07.28.605443","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447334","pub_date":"2024-7-30","title":"Sequence of the SARS-CoV-2 spike transmembrane domain makes it inherently dynamic","abstract":"The homotrimeric SARS-CoV-2 spike protein enables viral infection by mediating the fusion of the viral envelope with the host membrane. The spike protein is anchored to the SARS-CoV-2 envelope by its transmembrane domain (TMD), which is composed of three TM helices, each contributed by one of the protomers of the homotrimeric spike. Although the TMD is important for SARS-CoV-2 viral fusion and is well-conserved across the Coronaviridae family, it is unclear whether it is a passive anchor of the spike or actively promotes viral fusion. Specifically, the nature of the TMD dynamics and how these dynamics couple to the large pre- to post-fusion conformational transition of the spike ectomembrane domains remains unknown. Here, we computationally study the SARS-CoV-2 spike TMD in both homogenous POPC and cholesterol containing membranes to characterize its structure, dynamics, and self-assembly. Different tools identify distinct segments of the spike sequence as its TM helix. Atomistic simulations of a spike protomer segment that includes the superset of the TM helix predictions show that the membrane-embedded TM sequence bobs, tilts and gains and loses helicity at the membrane edges. Coarse-grained multimerization simulations using representative TM helix structures from the atomistic simulations exhibit diverse trimer populations whose architecture depends on the structure of the TM helix protomer. Multiple overlapping and conflicting dimerization interfaces stabilized these trimeric populations. An asymmetric conformation is populated in addition to a symmetric conformation and several in-between trimeric conformations. While the symmetric conformation reflects the symmetry of the resting spike, the asymmetric TMD conformation could promote viral membrane fusion through the stabilization of a fusion intermediate. Together, our simulations demonstrate that the SARS-CoV-2 spike TM anchor sequence is inherently dynamic, trimerization does not abrogate these dynamics and the various observed TMD conformations may enable viral fusion.","version":"1.2","doi":"10.1101/2021.06.07.447334","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.30.605768","pub_date":"2024-7-30","title":"Passive infusion of an S2-Stem broadly neutralizing antibody protects against SARS-CoV-2 infection and lower airway inflammation in rhesus macaques","abstract":"The continued evolution of SARS-CoV-2 variants capable of subverting vaccine and infection-induced immunity suggests the advantage of a broadly protective vaccine against betacoronaviruses (\u03b2-CoVs). Recent studies have isolated monoclonal antibodies (mAbs) from SARS-CoV-2 recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other \u03b2-CoVs. Many of these mAbs target the conserved S2 stem region of the SARS-CoV-2 spike protein, rather the receptor binding domain contained within S1 primarily targeted by current SARS-CoV-2 vaccines. One of these S2-directed mAbs, CC40.8, has demonstrated protective efficacy in small animal models against SARS-CoV-2 challenge. As the next step in the pre-clinical testing of S2-directed antibodies as a strategy to protect from SARS-CoV-2 infection, we evaluated the in vivo efficacy of CC40.8 in a clinically relevant non-human primate model by conducting passive antibody transfer to rhesus macaques (RM) followed by SARS-CoV-2 challenge. CC40.8 mAb was intravenously infused at 10mg/kg, 1mg/kg, or 0.1 mg/kg into groups (n=6) of RM, alongside one group that received a control antibody (PGT121). Viral loads in the lower airway were significantly reduced in animals receiving higher doses of CC40.8. We observed a significant reduction in inflammatory cytokines and macrophages within the lower airway of animals infused with 10mg/kg and 1mg/kg doses of CC40.8. Viral genome sequencing demonstrated a lack of escape mutations in the CC40.8 epitope. Collectively, these data demonstrate the protective efficiency of broadly neutralizing S2-targeting antibodies against SARS-CoV-2 infection within the lower airway while providing critical preclinical work necessary for the development of pan\u2013\u03b2-CoV vaccines. In this study, we explore the development of a broadly protective vaccine against betacoronaviruses (\u03b2-CoVs), including SARS-CoV-2. We focused on monoclonal antibodies (mAbs) from individuals who recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other \u03b2-CoVs. Unlike current vaccines that target the S1 region of the virus, these mAbs target a highly conserved S2 region of the spike protein. One antibody, CC40.8, showed promising results in small animal models. To further test its effectiveness, we infused CC40.8 into rhesus macaques at different doses and then challenged them with SARS-CoV-2. We found that higher doses of CC40.8 significantly reduced viral loads and inflammation in the lower airway. Additionally, there were no escape mutations in the targeted region, suggesting that the virus could not easily evade the antibody. Our findings highlight the potential of S2-targeting antibodies to protect against SARS-CoV-2 and support the development of vaccines that can broadly protect against various \u03b2-CoVs. RA, TFR, and DRB are listed as inventors on pending patent applications describing the SARS-CoV-2 and HCoV-HKU1 S cross-reactive antibodies. DRB and RA are listed as inventors on a pending patent application describing the S2 stem epitope immunogens identified in this study. DRB is a consultant for IAVI. All other authors declare that they have no competing interests. Pan-beta-coronavirus neutralizing mAb CC40.8 reduces SARS-CoV-2 viral loads and inflammation within the lower airway of infected rhesus macaques and provides pre-clinical support for S2-directed immunization strategies.","version":"1.1","doi":"10.1101/2024.07.30.605768","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.29.605395","pub_date":"2024-7-30","title":"Mapping protein conformational landscapes from crystallographic drug fragment screens","abstract":"Proteins are dynamic macromolecules. Knowledge of a protein\u2019s thermally accessible conformations is critical to determining important transitions and designing therapeutics. Accessible conformations are highly constrained by a protein\u2019s structure such that concerted structural changes due to external perturbations likely track intrinsic conformational transitions. These transitions can be thought of as paths through a conformational landscape. Crystallographic drug fragment screens are high-throughput perturbation experiments, in which thousands of crystals of a drug target are soaked with small-molecule drug precursors (fragments) and examined for fragment binding, mapping potential drug binding sites on the target protein. Here, we describe an open-source Python package, COLAV (COnformational LAndscape Visualization), to infer conformational landscapes from such large-scale crystallographic perturbation studies. We apply COLAV to drug fragment screens of two medically important systems: protein tyrosine phosphatase 1B (PTP-1B), which regulates insulin signaling, and the SARS CoV-2 Main Protease (MPro). With enough fragment-bound structures, we find that such drug screens also enable detailed mapping of proteins\u2019 conformational landscapes.","version":"1.1","doi":"10.1101/2024.07.29.605395","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.27.605454","pub_date":"2024-7-29","title":"Identification of potential SARS-CoV-2 genetic markers resulting from host domestication","abstract":"We developed a k-mer-based pipeline, namely the Pathogen Origin Recognition Tool using Enriched K-mers (PORT-EK) to identify genomic regions enriched in the respective hosts after the comparison of metagenomes of isolates between two host species. Using it we identified thousands of k-mers enriched in US white-tailed deer and betacoronaviruses in bat reservoirs while comparing them with human isolates. We demonstrated different coverage landscapes of k-mers enriched in deer and bats and unraveled 148 mutations in enriched k-mers yielded from the comparison of viral metagenomes between bat and human isolates. We observed that the third position within a genetic codon is prone to mutations, resulting in a high frequency of synonymous mutations of amino acids harboring the same physicochemical properties as unaltered amino acids. Finally, we classified and predicted the likelihood of host species based on the enriched k-mer counts. Altogether, PORT-EK showcased its feasibility for identifying enriched viral genomic regions, illuminating the different intrinsic tropisms of coronavirus after host domestication. A measure of enriched viral genomic correlates resulting from host domestication as a potential predictor of zoonotic risk.","version":"1.1","doi":"10.1101/2024.07.27.605454","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.28.24311115","pub_date":"2024-07-29","title":"Assessing Healthy Vaccinee Effect in COVID-19 Vaccine Effectiveness Studies: A National Cohort Study in Qatar","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>This study investigated the presence of the healthy vaccinee effect\u2014the imbalance in health status between vaccinated and unvaccinated individuals\u2014in two COVID-19 vaccine effectiveness studies involving primary series and booster vaccinations. It also examined the temporal patterns and variability of this effect across different subpopulations by analyzing the association between COVID-19 vaccination and non-COVID-19 mortality in Qatar.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Two matched, retrospective cohort studies assessed the incidence of non-COVID-19 death in national cohorts of individuals with a primary series vaccination versus no vaccination (two-dose analysis), and individuals with three-dose (booster) vaccination versus primary series vaccination (three-dose analysis), from January 5, 2021, to April 9, 2024.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The adjusted hazard ratio (aHR) for non-COVID-19 death was 0.76 (95% CI: 0.64-0.90) in the two-dose analysis and 0.85 (95% CI: 0.67-1.07) in the three-dose analysis. In the first six months of follow-up in the two-dose analysis, the aHR was 0.35 (95% CI: 0.27-0.46); however, the combined analysis of all subsequent periods showed an aHR of 1.52 (95% CI: 1.19-1.94). In the first six months of follow-up in the three-dose analysis, the aHR was 0.31 (95% CI: 0.20-0.50); however, the combined analysis of all subsequent periods showed an aHR of 1.37 (95% CI: 1.02-1.85). The overall effectiveness of the primary series and third-dose vaccinations against severe, critical, or fatal COVID-19 was 95.9% (95% CI: 94.0-97.1) and 34.1% (95% CI: \u221246.4-76.7), respectively. Subgroup analyses showed that the healthy vaccinee effect is pronounced among those aged 50 years and older and among those more clinically vulnerable to severe COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>A strong healthy vaccinee effect was observed in the first six months following vaccination. This effect may have stemmed from a lower likelihood of vaccination among seriously ill, end-of-life individuals, and less mobile elderly populations.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2024.07.28.24311115","journal":"medRxiv","score":null},{"id":"10.1101/2024.02.05.578888","pub_date":"2024-7-26","title":"The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming","abstract":"Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key novel cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2\u2019 functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection and they provide a mechanistic basis for the development of novel GAG derivatives with anti-SARS-CoV-2 potential. A key to blocking SARS-CoV-2 infection is to understand why it infects some cell types more than others. Heparan sulfate (HS) proteoglycans are differentially expressed on the surface of host cells and, with ACE2 receptors, provide an entry route for SARS-CoV-2. Here, we used computer simulations to investigate how highly sulfated glycosaminoglycans, a model for HS expressed in lungs, impact the interaction between virus spike and host ACE2. The simulations indicate that HS, together with host and spike N-glycans, stabilizes the spike:ACE2 complex and triggers structural changes, including host protease cleavage, contributing to the SARS-CoV-2 infection mechanism. This study lays the basis for a better understanding of the cell-specificity of SARS-CoV-2 infection and for developing strategies for inhibiting SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2024.02.05.578888","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.12.584739","pub_date":"2024-7-25","title":"Tracking inflammation resolution signatures in lungs after SARS-CoV-2 omicron BA.1 infection of K18-hACE2 mice","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which can result in severe disease, often characterised by a \u2018cytokine storm\u2019 and the associated acute respiratory distress syndrome. However, many infections with SARS-CoV-2 are mild or asymptomatic throughout the course of infection. Although blood biomarkers of severe disease are well studied, less well understood are the inflammatory signatures in lung tissues associated with mild disease or silent infections, wherein infection and inflammation are rapidly resolved leading to sequelae-free recovery. Herein we described RNA-Seq and histological analyses of lungs over time in an omicron BA.1/K18-hACE2 mouse infection model, which displays these latter features. Although robust infection was evident at 2 days post infection (dpi), viral RNA was largely cleared by 10 dpi. Acute inflammatory signatures showed a slightly different pattern of cytokine signatures compared with severe infection models, and where much diminished 30 dpi and absent by 66 dpi. Cellular deconvolution identified significantly increased abundance scores for a number of anti-inflammatory pro-resolution cell types at 5/10 dpi. These included type II innate lymphoid cells, T regulatory cells, and interstitial macrophages. Genes whose expression trended downwards over 2 \u2013 66 dpi included biomarkers of severe disease and were associated with \u2018cytokine storm\u2019 pathways. Genes whose expression trended upward during this period were associated with recovery of ciliated cells, AT2 to AT1 transition, reticular fibroblasts and innate lymphoid cells, indicating a return to homeostasis. Very few differentially expressed host genes were identified at 66 dpi, suggesting near complete recovery. The parallels between mild or subclinical infections in humans and those observed in this BA.1/K18-hACE2 mouse model are discussed with reference to the concept of \u201cprotective inflammation\u201d.","version":"1.2","doi":"10.1101/2024.03.12.584739","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.24.604948","pub_date":"2024-7-25","title":"The Impact of SARS-CoV-2 nsp14 Proofreading on Nucleoside Antiviral Activity: Insights from Genetic and Pharmacological Investigations","abstract":"Nucleoside analogues are a class of well-established antiviral agents that act by being directly incorporated into the viral genome during the replication process, resulting in chain termination or the induction of lethal mutations. While many nucleoside analogues have exhibited broad-spectrum activity against a wide range of viruses, their effectiveness against SARS-CoV-2 is limited. The lack of activity is hypothesized to be attributed to the proofreading function of viral nsp14 exonuclease. In this study, the role of the nsp14 proofreading in modulating nucleoside antiviral activity was investigated using genetic and pharmacological approaches. Introduction of exonuclease attenuation or disabling mutations to nsp14 led to either severe replication defect or increased sensitivity of SARS-CoV-2 and SARS-CoV replicons to specific nucleoside analogues. In contrast, repurposing of HCV NS5A inhibitors to suppress nsp14 exonuclease activity is insufficient to enhance the potency of nucleoside analogues. These findings provided further support for nsp14 as a target for SARS-CoV-2 antiviral development and highlighted the complex interplay between nsp14 proofreading and RNA replication.","version":"1.1","doi":"10.1101/2024.07.24.604948","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.13.575537","pub_date":"2024-7-24","title":"Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2","abstract":"Coronavirus disease 2019 (COVID-19) is an immune-related disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The complete pathogenesis of the virus remains to be determined. Unraveling the molecular mechanisms governing SARS-CoV-2 interactions with host cells is crucial for the formulation of effective prophylactic measures and the advancement of COVID-19 therapeutics. We analyzed human lung single-cell RNA sequencing dataset to discern the association of butyrophilin subfamily 3 member A2 (BTN3A2) expression with COVID-19. The BTN3A2 gene edited cell lines and transgenic mice were infected by live SARS-CoV-2 in a biosafety level 3 (BSL-3) laboratory. Immunoprecipitation, flow cytometry, biolayer interferometry and competition ELISA assays were performed in BTN3A2 gene edited cells. We performed quantitative real-time PCR, histological and/or immunohistochemical analyses for tissue samples from mice with or without SARS-CoV-2 infection. The BTN3A2 mRNA level was correlated with COVID-19 severity. BTN3A2 expression was predominantly identified in epithelial cells, elevated in pathological epithelial cells from COVID-19 patients and co-occurred with ACE2 expression in the same lung cell subtypes. BTN3A2 targeted the early stage of the viral life cycle by inhibiting SARS-CoV-2 attachment through interactions with the receptor-binding domain (RBD) of the Spike protein and ACE2. BTN3A2 inhibited ACE2-mediated SARS-CoV-2 infection by reducing ACE2 in vitro and in vivo. These results reveal a key role of BTN3A2 in the fight against COVID-19. Identifying potential monoclonal antibodies which mimic BTN3A2 may facilitate disruption of SARS-CoV-2 infection, providing a therapeutic avenue for COVID-19. This study was supported by the National Natural Science Foundation of China (32070569, U1902215, and 32371017), the CAS \u201cLight of West China\u201d Program, and Yunnan Province (202305AH340006). Our understanding of the pathogenesis of COVID-19, especially key molecular events in the early stage of viral infection, remains incompletely albeit we witnessed many progresses. This knowledge gap hinders the finding for effective and specific antiviral agents against SARS-CoV-2. The entry of SARS-CoV-2 is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2) and is affected by host antiviral defenses. Targeting these universal host factors required for virus replication is the most promising approach for effective prevention and treatment of COVID-19. Our study revealed that BTN3A2, a primate-specific gene, acts as a potent inhibitor of SARS-CoV-2 infection in vitro and in vivo. The up-regulation of BTN3A2 upon SARS-CoV-2 infection competed with the ACE2 receptor for binding to the Spike protein, subsequently reducing ACE2 expression and ACE2-mediated SARS-CoV-2 entry. These data highlighted that BTN3A2 as a novel host factor with protective effects against SARS-CoV-2 infection. The BTN3A2 holds considerable potential as a therapeutic drug for mitigating the impact of SARS-CoV-2 and its variants of concern (VOCs).","version":"1.2","doi":"10.1101/2024.01.13.575537","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.16.580725","pub_date":"2024-7-24","title":"A genome-wide arrayed CRISPR screen identifies PLSCR1 as an intrinsic barrier to SARS-CoV-2 entry that recent virus variants have evolved to resist","abstract":"Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence SARS-CoV-2 infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of COVID-19 patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 over-expression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently.","version":"1.3","doi":"10.1101/2024.02.16.580725","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.23.604853","pub_date":"2024-7-24","title":"Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant","abstract":"Deep mutational scanning experiments aid in the surveillance and forecasting of viral evolution by providing prospective measurements of mutational effects on viral traits, but epistatic shifts in the impacts of mutations can hinder viral forecasting when measurements were made in outdated strain backgrounds. Here, we report measurements of the impact of all single amino acid mutations on ACE2-binding affinity and protein folding and expression in the SARS-CoV-2 Omicron BA.2.86 spike receptor-binding domain (RBD). As with other SARS-CoV-2 variants, we find a plastic and evolvable basis for receptor binding, with many mutations at the ACE2 interface maintaining or even improving ACE2-binding affinity. Despite its large genetic divergence, mutational effects in BA.2.86 have not diverged greatly from those measured in its Omicron BA.2 ancestor. However, we do identify strong positive epistasis among subsequent mutations that have accrued in BA.2.86 descendants. Specifically, the Q493E mutation that decreased ACE2-binding affinity in all previous SARS-CoV-2 backgrounds is reversed in sign to enhance human ACE2-binding affinity when coupled with L455S and F456L in the currently emerging KP.3 variant. Our results point to a modest degree of epistatic drift in mutational effects during recent SARS-CoV-2 evolution but highlight how these small epistatic shifts can have important consequences for the emergence of new SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.07.23.604853","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.24.604889","pub_date":"2024-7-24","title":"The presenting HLA determines fidelity of SARS-CoV-2 spike protein epitope prediction","abstract":"During the course of the COVID-19 pandemic, multiple studies used prediction methods to identify potential epitopes. While additional studies identified epitopes from convalescent and vaccinated subjects, few studies have compared the predicted to identified epitopes. Here we used three methods alone and in combination to predict helper T cell epitopes and compared the results to experimentally determined peptide binding. The correspondence between the results predicted from each method or combination and experimental results depends on the HLA being investigated. We were also able identify the prediction methods which lead to the most consistent results. Lastly, these observations were extended to more HLAs to predict epitopes which may be globally presented. All the predicted epitopes were previously identified as helper T cell epitopes. These results suggest predicting the binding to a larger number of HLAs may lead to higher fidelity identification of epitopes.","version":"1.1","doi":"10.1101/2024.07.24.604889","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.22.604652","pub_date":"2024-7-23","title":"The Molecular Evolution of the SARS-COV-2 Spike Protein: Study of Amino Acid Substitutions and Types","abstract":"The molecular evolution of SARS-COV-2 has been challenging to predict. Emergence of the Omicron Variant of Concern (VOC) and its sublineages indicated that SARS-COV-2 could evolve more rapidly than previously thought. We analyzed the mutation and amino acid substitution patterns in the spike (S) protein of SARS-COV-2 VOCs to assess how they evolved in response to the selective pressure exerted by both natural immunity and vaccination. Our results indicate less evolutionary constraint on the first part of the S protein, allowing more amino acid substitutions, especially in the NTD, RBD, and subdomain 1. Omicron lineages introduced mutations in the FP and HR1 domains for the first time. The NTD, subdomain 1, and FP domains allowed more radical amino acid substitutions, followed by RBD and HR1, possibly due to their function. There were up to nine conservative and one radical substitutions in the amino acids interacting with the Human ACE2 receptor in the RBM of the Omicron sublineages only, a remarkable departure from the previous VOCs. We show that the molecular evolution of SARS-COV-2 S protein was relatively limited up to the Omicron lineage. The selective pressure from previous VOCs and global vaccination potentially accelerated the emergence of the highly transmissible Omicron lineage. This antigenic drift in Omicron is fueled by a high rate of radical amino acid substitutions in the S1 domains, resulting in positive selection with a high potential to change due to adaptive evolution. However, the conservative nature of changes in the RBM may signal a relative stabilization.","version":"1.1","doi":"10.1101/2024.07.22.604652","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.23.604777","pub_date":"2024-7-23","title":"SARS-CoV-2 S, M and E Structural Proteins Down-modulate HIV-1 LTR Activity and Modulate Endoplasmic Reticulum Stress Responses","abstract":"We have previously shown that the Hepatitis C Virus (HCV) E1E2 envelope glycoprotein can down-modulate HIV-1 long-terminal repeat (LTR) activity through disruption to NF-\u03baB activation. This response is associated with up-regulation of the endoplasmic reticulum (ER) stress response pathway. Here we demonstrate that the SARS-CoV-2 S, M and E but not the N structural protein can perform similar down-modulation of HIV-1 LTR activation and in a dose-dependent manner in both HEK293 and lung BEAS-2B cell-lines and interpreted as a result of NF-\u03baB down-modulation. The effect is highest with the SARS-CoV-2 Wuhan S strain and decreases over-time for the subsequent emerging variants of concern (VOC) with omicron providing the weakest effect. We developed pseudo-typed viral particle (PVP) molecular viral tools that allowed for the generation of cell-lines constitutively expressing separately the four SARS-CoV-2 structural proteins and utilising the VSV-g envelope protein to deliver the integrated gene construct. Differential gene expression analysis (DGEA) was performed on cells expressing S, E, M or N to determine cell activation status. It it was determined that gene expression differences were found in a number of interferon-stimulated genes (ISGs), including IF16, IFIT1, IFIT2 and ISG15 as well as for a number of heat shock protein (HSP) genes, including HSPH1, HSPA6 and HSPBP1 with all four SARS-CoV-2 structural proteins. There were also differences observed with expression patterns of transcription factors with both SP1 and MAVS upregulated in the presence of S, M and E but not the N protein. Collectively the results indicate that gene expression patterns associating with ER stress pathways can be identified with SARS-CoV-2 envelope glycoprotein expression. The results suggest the SARS-CoV-2 can modulate activation of an array of cell pathways resulting in disruption to NF-\u03baB signalling hence providing alterations to multiple physiological responses of SARS-CoV-2 infected cells.","version":"1.1","doi":"10.1101/2024.07.23.604777","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.22.604655","pub_date":"2024-7-23","title":"Computationally designed mRNA-launched protein nanoparticle vaccines","abstract":"Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic1\u20136. These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses7\u201310, while mRNA vaccines can be rapidly manufactured11 and elicit antigen-specific CD4 and CD8 T cells12,13. Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies.","version":"1.1","doi":"10.1101/2024.07.22.604655","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.21.604465","pub_date":"2024-7-23","title":"The Impact of Preprints on the Citations of Journal Articles Related to COVID-19","abstract":"To investigate the impact of preprints on the citation counts of COVID-19-related papers, this study compares the number of citations received by drafts initially distributed as preprints and later published in journals with those received by papers directly submitted to journals. The difference in the median number of citations between COVID-19 preprint-distributed papers and COVID-19 directly submitted papers published in 184 journals was tested using the Mann-Whitney U test. The results showed that 129 journals had a statistically significant higher median citation count for COVID-19 preprint-distributed papers compared to directly submitted papers, with a p-value of less than 0.05. In contrast, no journals had a statistically significant higher median citation count for COVID-19 directly submitted papers. This indicates that 70.11% of the journals that published preprint-distributed papers experienced a significant increase in citations. We also identified that among the 184 journals, 13 journals garnered a substantial number of citations. Among the 74,037 COVID-19 papers, preprint-distributed papers (9,028) accounted for only 12.19%. However, among the 2,015,997 citations received by COVID-19 papers, preprint-distributed papers garnered 542,715 citations, representing a substantial 26.92%. These results suggest that distributing preprints prior to formal publication may help COVID-19 research reach a wider audience, potentially leading to increased readership and citations.","version":"1.1","doi":"10.1101/2024.07.21.604465","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.22.604541","pub_date":"2024-7-23","title":"Arabidopsis NIM1-INTERACTING1 (NIMIN1) is a multi-domain protein controlling transition from systemic acquired resistance (SAR) to cell death","abstract":"NIM1-INTERACTING (NIMIN) proteins were identified by interaction with the systemic acquired resistance (SAR) key regulator NPR1. The Arabidopsis family comprises four related, yet distinct members. Ample evidence predicts that NIMIN1 is a multi-domain protein which comes into action after the onset of salicylic acid (SA) signaling prior to induction of the PR-1 defense gene. Bioinformatics, protein-protein interaction assays and expression studies in tobacco were applied to explore functions of NIMIN1. The N-terminal sequence encompassing amino acids 1 to 15 conveyed accumulation of NIMIN1 protein in planta. This newly identified segment and the known conserved NIMIN1 domains, i.e., two distinct NPR1 binding sites and an EAR motif, are neatly separated from each other by disordered regions. Co-expression of NIMIN1 and NPR1 reinforced accumulation of NPR1 protein while ectopic overexpression of NIMIN1 promoted emergence of cell death driven by the EAR motif and disturbed development of tobacco plants. We suggest that NIMIN1 acts as a dynamic signaling agent controlling transition of pathogen-infected leaves from survival to tissue collapse. Initially, NIMIN1 binding renders the NPR1 transcription complex sensitive to the SAR signal SA enabling PR-1 transcription. At high levels of SA, NIMIN1 is outcompeted by SA from the NPR1 C-terminus, and accumulating NIMIN1 engages via its EAR motif with TOPLESS-RELATED3, thereby affecting global hormone signaling and inducing cell death in severely endangered tissue.","version":"1.1","doi":"10.1101/2024.07.22.604541","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.18.604163","pub_date":"2024-7-19","title":"SARS-CoV-2 B Epitope-Guided Neoantigen NanoVaccines Enhance Tumor-Specific CD4/CD8 T Cell Immunity Through B Cell Antigen Presentation","abstract":"Current neoantigen cancer vaccines activate T cell immunity through dendritic cell /macrophage-mediated antigen presentation. It is unclear whether incorporating B cell-mediated antigen presentation into current neoantigen vaccines could enhance CD4/CD8 T cell immunity to improve their anticancer efficacy. We developed a SARS-CoV-2 B cell epitope-guided neoantigen peptide/mRNA cancer nanovaccines (BSARSTNeoAgVax) to improve anticancer efficacy by enhancing tumor-specific CD4/CD8 T cell antitumor immunity through B cell-mediated antigen presentation. BSARSTNeoAgVax crosslinked with B cell receptor, promoted SARS-CoV-2 B cell-mediated antigen presentation to tumor-specific CD4 T cells, increased tumor-specific follicular/non-follicular CD4 T cells, and enhanced B cell-dependent tumor-specific CD8 T cell immunity. BSARSTNeoAgVax achieved superior efficacy in melanoma, pancreatic, and breast cancer models compared to the current neoantigen vaccines. Our study provides a universal platform, SARS-CoV-2 B epitope-guided neoantigen nanovaccines, to improve anticancer efficacy against various cancer types by enhancing CD4/CD8 T cell antitumor immunity through viral-specific B cell-mediated antigen presentation.","version":"1.1","doi":"10.1101/2024.07.18.604163","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.18.604218","pub_date":"2024-7-19","title":"Rapid Detection of Active Coronavirus Infection by Lateral Flow Test Strips: A New Approach to Distinguish Replicating Viruses from Non-Replicating Viruses","abstract":"This manuscript describes the development of an alternative method to detect active coronavirus infection, which targets negative-sense RNA, a product of active viral replication. Few diagnostic methods are capable of discriminating between replicating and non-replicating viruses, complicating decisions related to quarantine and therapeutic interventions. We propose strand-specific nucleic acid diagnostics as a means of distinguishing between active and inactive RNA virus infections and prototype a CRISPR-based lateral flow assay that specifically detects replicating coronaviruses. Such a paradigm in diagnostics could guide more effective public health measures to curb the spread of SARS-CoV-2 and other single-stranded viruses.","version":"1.1","doi":"10.1101/2024.07.18.604218","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.14.603481","pub_date":"2024-7-17","title":"Mutational and evolutionary dynamics of non-structural and spike proteins from variants of concern (VOC) of SARS-CoV-2 in India","abstract":"Monitoring the genetic diversity and emerging mutations of SARS-CoV-2 still remains to be crucial in India. This study extensively analyzes the lineage dynamics, mutation screening, structural analysis, and phylodynamics of SARS-CoV-2 variants of concern (VOC) in India from October 2020 to September 2023. Predominant variants identified include alpha, beta, delta, and omicron, with delta and omicron making up 76.05% of sequenced genomes. The B.1.617.2 lineage of delta was the major contributor to COVID-19 cases before the rapid rise of omicron. Mutation screening of non-structural proteins (NSPs) and spike revealed distinct profiles for each VOC. Co-mutation patterns/networks of the most frequently observed mutations specific for each VOC were also identified and subsequently structural and energetic alteration of the co-mutants were analyzed using rigorous molecular dynamics simulations. Furthermore, comparative analysis of phylogenetic trees based on genomic and mutational data revealed that nsp1, nsp3, nsp4, nsp13, and nsp14 exhibit strongest association with the increased mutation load across the genome of SARS-CoV-2 in Indian population. Our comparative phylogenetic study also revealed that mutability patterns of nsp14 and spike have highest similarity supporting critical role of nsp14 for SARS-CoV-2 infectivity and persistence. This research provides a comprehensive overview of SARS-CoV-2 evolution in India.","version":"1.2","doi":"10.1101/2024.07.14.603481","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.17.603909","pub_date":"2024-7-17","title":"Evolution of SARS-CoV-2 spike trimers towards optimized heparan sulfate cross-linking and inter-chain mobility","abstract":"The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S)-protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. Recent variants of concern (VOCs), notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the S-protein trimer\u2019s interaction with the negatively charged HS polysaccharide chains in the matrix. These increased interactions, however, may reduce Omicron\u2019s ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, and raise the question of how HS-associated virus movement can be mechanistically explained. In this work, we show that Omicron S-proteins have evolved to balance HS interaction stability and dynamics, resulting in enhanced mobility on an HS-functionalized artificial matrix. Both properties are achieved by the ability of Omicrons S-proteins to cross-link at least two HS chains, providing both high avidity to retain the protein inside the HS-rich matrix, and fast dynamics, thus enabling direct S-protein switching between HS chains as a prerequisite for mobility at the cell surface. Optimized HS interactions can be targeted pharmaceutically, because an HS mimetic significantly suppressed surface binding and cellular infection specifically of the Omicron VOC. These findings suggest a robust way to interfere with SARS-CoV-2 Omicron infection and, potentially, future variants.","version":"1.1","doi":"10.1101/2024.07.17.603909","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.16.603835","pub_date":"2024-7-17","title":"Virological characteristics of the SARS-CoV-2 KP.3.1.1 variant","abstract":"The SARS-CoV-2 JN.1 variant (BA.2.86.1.1), arising from BA.2.86.1 with spike protein (S) substitution S:L455S, outcompeted the previously predominant XBB lineages by the beginning of 2024. Subsequently, JN.1 subvariants including KP.2 (JN.1.11.1.2) and KP.3 (JN.1.11.1.3), which acquired additional S substitutions e.g., S:R346T, S:F456L, and S:Q493E, have emerged concurrently. Thereafter, JN.1 subvariants, such as LB.1 (JN.1.9.2.1), KP.2.3 (JN.1.11.1.2.3), and KP.3.1.1 (JN.1.11.1.3.1.1), which convergently acquired a deletion of Serine at the 31st position in S (S:S31del) in addition to the above substitutions, have emerged and spread as of June 2024. We recently reported the virological features of JN.1 subvariants including KP.2, KP.3, LB.1, and KP.2.3.2,3 Here, we investigated the virological properties of KP.3.1.1. First, we estimated the relative effective reproduction number (Re) of KP.3.1.1 using a Bayesian multinomial logistic model4 based on genome surveillance data from Spain, the USA, France, Canada, and the UK, where this variant has spread as of June 2024. In Spain, the Re of KP.3.1.1 is over 1.2-fold higher than that of JN.1 and even higher than those of KP.2, KP.3, LB.1, and KP.2.3. Additionally, the other countries under investigation herein show higher Re for KP.3.1.1. However, it must be noted there is the possibility of overestimation in these countries due to more limited KP.3.1.1 sequence numbers. These results suggest that KP.3.1.1 will spread worldwide along with other JN.1 sublineages. We then assessed the virological properties of KP.3.1.1 using pseudoviruses. The pseudovirus of KP.3.1.1 had significantly higher infectivity than that of KP.3. Neutralization of KP.3.1.1 was tested using i) convalescent sera after breakthrough infection (BTI) with XBB.1.5 or EG.5, ii) convalescent sera after the infection with HK.3 or JN.1, and iii) sera after monovalent XBB.1.5 vaccination. The 50% neutralization titer (NT50) against KP.3.1.1 was significantly lower than KP.3 (1.4\u20131.6-fold) in all four groups of convalescent sera tested. KP.3.1.1 also showed a 1.3-fold lower NT50 against XBB.1.5 vaccine sera than KP.3. Moreover, KP.3.1.1 showed stronger resistance with a 1.3-fold lower NT50 with statistical significances to the convalescent sera infected with EG.5 and HK.3 than KP.2.3. Altogether, KP.3.1.1 exhibited a higher Re, higher pseudovirus infectivity, and higher neutralization evasion than KP.3. These results align with our recent report that the JN.1 subvariants with S:S31del (e.g., KP.2.3 and LB.1) exhibited enhanced Re and immune evasion compared to the other JN.1 subvariants without S:S31del (e.g., JN.1, KP.2, and KP.3), highlighting the evolutionary significance of S:S31del in the JN.1 lineages.","version":"1.1","doi":"10.1101/2024.07.16.603835","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.16.603698","pub_date":"2024-7-16","title":"Functional implications of the interaction of the SARS-CoV-2 Nucleocapsid protein with factors involved in nonsense-mediated mRNA decay","abstract":"The RNA genome of the SARS-CoV-2 virus encodes for four structural proteins, 16 non- structural proteins and nine putative accessory factors. A high throughput analysis of interactions between human and SARS-CoV-2 proteins identified multiple interactions of the structural Nucleocapsid (N) protein with RNA processing factors. The N-protein, which is responsible for packaging of the viral genomic RNA was found to interact with two RNA helicases, UPF1 and MOV10 that are involved in nonsense-mediated mRNA decay (NMD). Using a combination of biochemical and biophysical methods, we investigated the interaction of the SARS-CoV-2 N-protein with NMD factors at a molecular level. Our studies led us to identify the core NMD factor, UPF2, as an interactor of N. The viral N-protein engages UPF2 in multipartite interactions and can negate the stimulatory effect of UPF2 on UPF1 catalytic activity. N also inhibits UPF1 ATPase and unwinding activities by competing in binding to the RNA substrate. We further investigate the functional implications of inhibition of UPF1 catalytic activity by N in mammalian cells. The interplay of SARS-CoV-2 N with human UPF1 and UPF2 does not affect decay of host cell NMD targets but might play a role in stabilizing the viral RNA genome.","version":"1.1","doi":"10.1101/2024.07.16.603698","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.16.603692","pub_date":"2024-7-16","title":"The SARS-CoV-2 nucleocapsid protein inhibits the cellular Nonsense-Mediated mRNA Decay (NMD) pathway preventing the full enzymatic activation of UPF1","abstract":"The Nonsense-mediated mRNA decay (NMD) pathway triggers the degradation of defective mRNAs and governs the expression of mRNAs with specific characteristics. Current understanding indicates that NMD is often significantly suppressed during viral infections to protect the viral genome. In numerous viruses, this inhibition is achieved through direct or indirect interference with the RNA helicase UPF1, thereby promoting viral replication and enhancing pathogenesis. In this study, we employed biochemical, biophysical assays, and cellular investigations to explore the interplay between UPF1 and the Nucleocapsid (Np) protein of SARS-CoV-2. We evaluated their direct interaction and its impact on inhibiting cellular NMD. Furthermore, we characterized how this interaction affects UPF1\u2019s enzymatic function. Our findings demonstrate that Np inhibits the unwinding activity of UPF1 by physically obstructing its access to structured nucleic acid substrates. Additionally, we showed that Np binds directly to UPF2, disrupting the formation of the UPF1/UPF2 complex essential for NMD progression. Intriguingly, our research also uncovered a surprising pro-viral role of UPF1 and an antiviral function of UPF2. These results unveil a novel, multi-faceted mechanism by which SARS-CoV-2 evades the host\u2019s defenses and manipulates cellular components. This underscores the potential therapeutic strategy of targeting Np-UPF1/UPF2 interactions to treat COVID-19.","version":"1.1","doi":"10.1101/2024.07.16.603692","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.15.602781","pub_date":"2024-7-16","title":"Dissecting human monoclonal antibody responses from mRNA- and protein-based XBB.1.5 COVID-19 monovalent vaccines","abstract":"The emergence of highly contagious and immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has required reformulation of coronavirus disease 2019 (COVID-19) vaccines to target those new variants specifically. While previous infections and booster vaccinations can enhance variant neutralization, it is unclear whether the monovalent version, administered using either mRNA or protein-based vaccine platforms, can elicit de novo B-cell responses specific for Omicron XBB.1.5 variants. Here, we dissected the genetic antibody repertoire of 603 individual plasmablasts derived from five individuals who received a monovalent XBB.1.5 vaccination either with mRNA (Moderna or Pfizer/BioNtech) or adjuvanted protein (Novavax). From these sequences, we expressed 100 human monoclonal antibodies and determined binding, affinity and protective potential against several SARS-CoV-2 variants, including JN.1. We then select two vaccine-induced XBB.1.5 mAbs, M2 and M39. M2 mAb was a de novo, antibody, i.e., specific for XBB.1.5 but not ancestral SARS-CoV-2. M39 bound and neutralized both XBB.1.5 and JN.1 strains. Our high-resolution cryo-electron microscopy (EM) structures of M2 and M39 in complex with the XBB.1.5 spike glycoprotein defined the epitopes engaged and revealed the molecular determinants for the mAbs\u2019 specificity. These data show, at the molecular level, that monovalent, variant-specific vaccines can elicit functional antibodies, and shed light on potential functional and genetic differences of mAbs induced by vaccinations with different vaccine platforms.","version":"1.1","doi":"10.1101/2024.07.15.602781","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.13.603361","pub_date":"2024-7-15","title":"SARS-CoV-2 entry and fusion are independent of ACE2 localization to lipid rafts","abstract":"Membrane fusion occurs at the early stages of SARS-CoV-2 replication, during entry of the virus, and later during the formation of multinucleated cells called syncytia. Fusion is mediated by the binding of the viral Spike protein to its receptor ACE2. Lipid rafts are dynamic nanodomains enriched in cholesterol and sphingolipids. Rafts can act as platforms for entry of d\u00ecerent viruses by localizing virus receptors, and attachment factors to the same membrane microdomains. Here, we first demonstrate that cholesterol depletion by methyl-beta-cyclodextrin inhibits Spike mediated fusion and entry. To further study the role of ACE2 lipid raft localization in SARS-CoV-2 fusion and entry, we design a GPI-anchored ACE2 construct. Both ACE2 and ACE2-GPI proteins are similarly expressed at the plasma membrane. Through membrane flotation assays, we show that in d\u00ecerent cell lines, ACE2-GPI localises predominantly to raft domains of the plasma membrane while ACE2 is non-raft associated. We then compare the ability of ACE2 and ACE2-GPI to permit SARS-CoV-2 pseudovirus entry and syncytia formation and replication of d\u00ecerent viral variants. We find little d\u00ecerence in the two proteins. Our results demonstrate that SARS-CoV-2 entry and fusion are a cholesterol dependent and raft-independent process. Rafts are often exploited by viruses and used as platforms to enhance their entry into the cell or spread from cell-to-cell. The membrane localization of ACE2 and the role of lipid rafts in SARS-CoV-2 entry and cell-to-cell spread is poorly understood. The function of lipid rafts in viral fusion is often studied through their disruption by cholesterol-depleting agents. However, this process may have \u00f2-target impacts on viral fusion independently of lipid-raft disruption. Therefore, we created an ACE2 construct that localizes to lipid rafts using a GPI anchor. Conversely, wild-type ACE2 was non-raft associated. We find that the localization of ACE2 to lipid rafts does not modify the fusion dynamics of SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.07.13.603361","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.12.603240","pub_date":"2024-7-15","title":"Rate variation and recurrent sequence errors in pandemic-scale phylogenetics","abstract":"Phylogenetic analyses of genome sequences from infectious pathogens reveal essential information regarding their evolution and transmission, as seen during the COVID-19 pandemic. Recently developed pandemic-scale phylogenetic inference methods reduce the computational demand of phylogenetic reconstruction from genomic epidemiological datasets, allowing the analysis of millions of closely related genomes. However, widespread homoplasies, due to recurrent mutations and sequence errors, cause phylogenetic uncertainty and biases. We present new algorithms and models to substantially improve the computational performance and accuracy of pandemic-scale phylogenetics. In particular, we account for, and identify, mutation rate variation and recurrent sequence errors. We reconstruct reliable and public sequence alignment and phylogenetic tree of > 2 million SARS-CoV-2 genomes encapsulating the evolutionary history and global spread of the virus up to February 2023.","version":"1.1","doi":"10.1101/2024.07.12.603240","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.15.603540","pub_date":"2024-7-15","title":"p16High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy","abstract":"The ability of an organism to overcome infectious diseases has traditionally been linked to killing invading pathogens. Accumulating evidence, however, indicates that, apart from restricting pathogen loads, organismal survival is coupled to an additional yet poorly understood mechanism called disease tolerance. Here we report that p16High immune cells play a key role in establishing disease tolerance. We found that the FDA-approved BNT162b2 mRNA COVID-19 vaccine is a potent and rapid inducer of p16High immune subsets both in mice and humans. In turn, p16High immune cells were indispensable for counteracting different lethal conditions, including LPS-induced sepsis, acute SARS-CoV-2 infection and ionizing irradiation. Mechanistically, we propose that activation of TLR7 or a low physiological activity of STING is sufficient to induce p16High immune subset that, in turn, establishes a low adenosine environment and disease tolerance. Furthermore, containing these signals within a beneficial range by deleting MDA5 that appeared sufficient to maintain a low activity of STING, induces p16High immune cells and delays organ deterioration upon aging with improved healthspan. Our data highlight the beneficial role of p16High immune subsets in establishing a low adenosine environment and disease tolerance.","version":"1.1","doi":"10.1101/2024.07.15.603540","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.24.600393","pub_date":"2024-7-13","title":"Distinct pathway for evolution of enhanced receptor binding and cell entry in SARS-like bat coronaviruses","abstract":"Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N\u2013terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles\u2019 heels that could be targeted with countermeasures.","version":"1.2","doi":"10.1101/2024.06.24.600393","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.02.573939","pub_date":"2024-7-12","title":"Development of robust antiviral assays using relevant apical-out human airway organoids","abstract":"While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput (\u223c10-fold) and fewer cells (\u223c100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.","version":"1.2","doi":"10.1101/2024.01.02.573939","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.03.574082","pub_date":"2024-7-12","title":"Mutation of a highly conserved isoleucine residue in loop 2 of several \u03b2-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental to infection","abstract":"All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. The conserved CoV macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases by removing ADP-ribose from target proteins and is critical for CoV replication and pathogenesis. Mac1 is a potential therapeutic target for CoV disease and several groups are actively developing Mac1 inhibitors. However, we lack a basic knowledge of how many of the key residues in the Mac1 ADP-ribose binding pocket contribute to its biochemical and virological functions. In this study, we engineered alanine mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and recombinant viruses for both MERS-CoV and SARS-CoV-2 to determine their importance in both Mac1 biochemical functions and CoV infection. Interestingly, an isoleucine-to-alanine mutation in loop 2 of both MERS-CoV and SARS-CoV-2 Mac1 proteins enhanced ADP-ribose binding. But surprisingly, that proved to be detrimental to virus infection, indicating that this isoleucine functions to control Mac1 ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding to promote infection and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.","version":"1.2","doi":"10.1101/2024.01.03.574082","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.12.24310301","pub_date":"2024-07-12","title":"RBD amplicon sequencing of wastewater reveals patterns of variant emergence and evolution","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Rapid evolution of SARS-CoV-2 has resulted in the emergence of numerous variants, posing significant challenges to public health surveillance. Clinical genome sequencing, while valuable, has limitations in capturing the full epidemiological dynamics of circulating variants in the general population. This study utilized receptor-binding domain (RBD) amplicon sequencing of wastewater samples to monitor the SARS-CoV-2 community dynamics and evolution in El Paso, TX. Over 17 months, we identified 91 variants and observed waves of dominant variants transitioning from BA.2 to BA.2.12.1, BA.4&amp;5, BQ.1, and XBB.1.5. Our findings demonstrated early detection of variants and identification of unreported outbreaks, while showing strong consistency with clinical genome sequencing data at the local, state, and national levels. Alpha diversity analyses revealed significant periodical variations, with the highest diversity observed in winter and the outbreak lag phases, likely due to lower competition among variants before the outbreak growth phase. The data underscores the importance of low transmission periods for rapid mutation and variant evolution. This study highlights the effectiveness of integrating RBD amplicon sequencing with wastewater surveillance in tracking viral evolution, understanding variant emergence, and enhancing public health preparedness.</jats:p>","version":null,"doi":"10.1101/2024.07.12.24310301","journal":"medRxiv","score":null},{"id":"10.1101/2023.05.16.541023","pub_date":"2024-7-12","title":"CParty: Hierarchically Constrained Partition Function of RNA Pseudoknots","abstract":"Biologically relevant RNA secondary structures are routinely predicted by efficient dynamic programming algorithms that minimize their free energy. Starting from such algorithms, one can devise partition function algorithms, which enable stochastic perspectives on RNA structure ensembles. As most prominent example McCaskill\u2019s partition function algorithm is derived from pseudoknot-free energy minimization. While this algorithm became hugely successful for the stochastic analysis of pseudoknot-free RNA structure, as of yet there exists only one pseudoknotted partition function implementation, which covers only simple pseudoknots and comes with a borderline-prohibitive complexity of O(n5) in the RNA length n. In this article, we develop a partition function algorithm corresponding to the hierarchical pseudoknot prediction of HFold, which performs exact optimization in a realistic pseudoknot energy model. In consequence, our algorithm CParty carries over HFold\u2019s advantages over classical pseudoknot prediction to stochastic analysis. In only cubic time, it computes the hierarchically constrained partition function over pseudoknotted density-2 structures G \u222a G\u2032, composed of pseudoknot-free parts G and G\u2032, where G is given. Thus, it follows the common hypothesis of hierarchical pseudoknot formation, where pseudoknots form as tertiary contacts only after a first pseudoknot-free \u2018core\u2019 G. Like HFold, CParty is very efficient, achieving the low complexity of the pseudoknot-free algorithm. Finally, by computing pseudoknotted ensemble energies, we unveil kinetics features of a therapeutic target in SARS-CoV-2. CParty is available at https://github.com/HosnaJabbari/CParty.","version":"1.3","doi":"10.1101/2023.05.16.541023","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.10.602842","pub_date":"2024-7-10","title":"N-acylethanolamine acid amidase inhibition reduces SARS-CoV-2 infection in Human Precision cut-lung slices and downregulates NF-\u03baB signalling","abstract":"Like other positive-sense RNA viruses, SARS-CoV-2 manipulates host lipid metabolism to facilitate its replication by enhancing lipogenesis and lipid droplet formation. In doing so, SARS-CoV-2 infection perturbs bioactive lipid levels associated with the inflammatory response. One of these, Palmitoylethanolamide (PEA) is suppressed during SARS-CoV-2 infection since it activates the Peroxisome Proliferator-Activated Receptor-\u03b1 (PPAR-\u03b1), a transcription factor that suppresses the nuclear factor-B (NF-\u03baB), which is mandatory to sustain SARS-CoV-2 replication. PEA levels are regulated by N-acylethanolamine acid amidase (NAAA), a lysosomal enzyme responsible for catalysing the breakdown of PEA. We hypothesized that NAAA inhibition might interfere with SARS-CoV-2 replication since it will lead PEA to accumulate, activating PPAR-\u03b1 and, consequently, suppressing NF-\u03baB. Our results reveal that genetic or chemical ablation of NAAA significantly suppresses SARS-CoV-2 replication by three log10 in human-derived precision-cut lung slices. Therefore, we investigated whether inhibiting NAAA could influence NF-\u03baB activation through the activation of PPAR-\u03b1. We observed PPAR-\u03b1 increased expression in NAAA-/-cells, while PPAR-\u03b1 expression remained low in infected parental cells. As expected, the elevated PPAR-\u03b1 expression correlated with a parallel reduction in NF-\u03baB activation when NAAA is ablated. These findings underscore NAAA as an essential host factor for SARS-CoV-2 replication and propose a potential mechanism of action rooted in the attenuation of NF-\u03baB activation during viral replication. Over the past three years, COVID-19 has claimed nearly 7 million lives worldwide, prompting extensive efforts to find effective treatments. While RNA-based vaccines have been developed rapidly, they alone have not completely halted the spread of the virus, making the search for antiviral therapies crucial. One promising approach targets the anti-inflammatory lipid PEA, which has shown some success in COVID-19 clinical trials. PEA is quickly degraded by the enzyme NAAA. Researchers have found that inhibiting NAAA can enhance and prolong PEA anti-inflammatory effects. NAAA inhibitors have already shown effectiveness in reducing chronic pain and lung inflammation in animal models and have also been effective against Zika virus replication. Our research focused on testing the NAAA inhibitor ARN726 against SARS-CoV-2. In human lung cells and lung tissue samples, ARN726 significantly reduced SARS-CoV-2 replication and inflammation. We discovered that this inhibition suppresses the NF-\u03baB pathway, which the virus uses to fuel its replication and sustain Cytokine storm. Overall, our findings suggest that NAAA inhibitors like ARN726 could be repurposed to combat COVID-19 and potentially other coronaviruses, offering a novel and effective antiviral strategy.","version":"1.1","doi":"10.1101/2024.07.10.602842","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.10.602835","pub_date":"2024-7-10","title":"Global siRNA Screen Reveals Critical Human Host Factors of SARS-CoV-2 Multicycle Replication","abstract":"Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.","version":"1.1","doi":"10.1101/2024.07.10.602835","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.10.602843","pub_date":"2024-7-10","title":"Omicron-specific ultra-potent SARS-CoV-2 neutralizing antibodies targeting the N1/N2 loop of Spike N-terminal domain","abstract":"A multitude of functional mutations continue to emerge on the N-terminal domain (NTD) of the spike protein in SARS-CoV-2 Omicron subvariants. Understanding the immunogenicity of Omicron NTD and the properties of antibodies elicited by it is crucial for comprehending the impact of NTD mutations on viral fitness and guiding vaccine design. In this study, we find that most of NTD-targeting antibodies isolated from individuals with BA.5/BF.7 breakthrough infection (BTI) are ancestral (wildtype or WT)-reactive and non-neutralizing. Surprisingly, we identified five ultra-potent neutralizing antibodies (NAbs) that can only bind to Omicron but not WT NTD. Structural analysis revealed that they bind to a unique epitope on the N1/N2 loop of NTD and interact with the receptor-binding domain (RBD) via the light chain. These Omicron-specific NAbs achieve neutralization through ACE2 competition and blockage of ACE2-mediated S1 shedding. However, BA.2.86 and BA.2.87.1, which carry insertions or deletions on the N1/N2 loop, can evade these antibodies. Together, we provided a detailed map of the NTD-targeting antibody repertoire in the post-Omicron era, demonstrating their vulnerability to NTD mutations enabled by its evolutionary flexibility, despite their potent neutralization. These results highlighted the importance of considering the immunogenicity of NTD in vaccine design. COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major global public health concern four years after its emergence. The N-terminal domain (NTD) is a critical component of the spike glycoprotein, which is pivotal for SARS-CoV-2 cellular entry and serves as a primary target for antibody therapeutics and vaccine development. Characterizing the properties of antibodies elicited by NTD of Omicron sublineages is crucial for understanding viral evolution and guiding vaccine design. Here, we show that Omicron infection after vaccination induces majorly non-neutralizing NTD antibodies. Still, we identified a class of ultra-potent neutralizing antibodies (NAbs) which specifically bind to the NTD of Omicron sublineages. These NAbs neutralize the virus by competing with ACE2 and blocking ACE2-mediated S1 shedding. Structural analyses reveal that these antibodies target a unique epitope on the N1/N2 loop of NTD, and intriguingly interact with the receptor-binding domain (RBD) of spike glycoprotein. This class of NAbs with the special binding pattern, are escaped by BA.2.86 and BA.2.87.1 sublineages, shedding light on the role of recently emerged mutations in the N1/N2 loop of NTD. Our findings provide fresh insights into the immunogenicity of Omicron NTD, highlighting its capacity for antibody evasion due to its evolutionary flexibility. This underscores the importance of carefully considering the NTD component in vaccine design.","version":"1.1","doi":"10.1101/2024.07.10.602843","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.09.602810","pub_date":"2024-7-10","title":"Atomistic Prediction of Structures, Conformational Ensembles and Binding Energetics for the SARS-CoV-2 Spike JN.1, KP.2 and KP.3 Variants Using AlphaFold2 and Molecular Dynamics Simulations: Mutational Profiling and Binding Free Energy Analysis Reveal Epistatic Hotspots of the ACE2 Affinity and Immune Escape","abstract":"The most recent wave of SARS-CoV-2 Omicron variants descending from BA.2 and BA.2.86 exhibited improved viral growth and fitness due to convergent evolution of functional hotspots. These hotspots operate in tandem to optimize both receptor binding for effective infection and immune evasion efficiency, thereby maintaining overall viral fitness. The lack of molecular details on structure, dynamics and binding energetics of the latest FLiRT and FLuQE variants with the ACE2 receptor and antibodies provides a considerable challenge that is explored in this study. We combined AlphaFold2-based atomistic predictions of structures and conformational ensembles of the SARS-CoV-2 Spike complexes with the host receptor ACE2 for the most dominant Omicron variants JN.1, KP.1, KP.2 and KP.3 to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and computations of binding affinities, we identified binding energy hotspots and characterized molecular basis underlying epistatic couplings between convergent mutational hotspots. The results suggested that the existence of epistatic interactions between convergent mutational sites at L455, F456, Q493 positions that enable to protect and restore ACE2 binding affinity while conferring beneficial immune escape. To examine immune escape mechanisms, we performed structure-based mutational profiling of the spike protein binding with several classes of antibodies that displayed impaired neutralization against BA.2.86, JN.1, KP.2 and KP.3. The results confirmed the experimental data that JN.1, KP.2 and KP.3 harboring the L455S and F456L mutations can significantly impair the neutralizing activity of class-1 monoclonal antibodies, while the epistatic effects mediated by F456L can facilitate the subsequent convergence of Q493E changes to rescue ACE2 binding. Structural and energetic analysis provided a rationale to the experimental results showing that BD55-5840 and BD55-5514 antibodies that bind to different binding epitopes can retain neutralizing efficacy against all examined variants BA.2.86, JN.1, KP.2 and KP.3. The results support the notion that evolution of Omicron variants may favor emergence of lineages with beneficial combinations of mutations involving mediators of epistatic couplings that control balance of high ACE2 affinity and immune evasion.","version":"1.1","doi":"10.1101/2024.07.09.602810","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.09.602697","pub_date":"2024-7-09","title":"SARS-CoV-2 shedding dynamics in human respiratory tract","abstract":"It is crucial to understand how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) sheds in human respiratory tract, but this question is still elusive to understand due to technical limitations. Here we integrated published human metagenomic data of SARS-CoV-2 and developed a novel algorithm named as RedeCoronaVS to systematically dissect SARS-CoV-2 shedding modes with single-cell data as reference. We identify that SARS-CoV-2 particles are the dominant mode of viral shedding in the very early infection phase (\u226424 hours after hospitalization). Within the first week after hospitalization, SARS-CoV-2 replicons within host cells dominate SARS-CoV-2 shedding together with viral particles. One week later, viral fragments become the dominant mode in patients with mild or moderate symptoms, but viral replicons still dominate in some patients with severe symptoms. In addition to epithelial cells, SARS-CoV-2 replicons in neutrophils, macrophages, and plasma cells also show important roles and are associate with sampling time and disease severity.","version":"1.1","doi":"10.1101/2024.07.09.602697","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.08.602557","pub_date":"2024-7-08","title":"SARS-CoV-2 neutralization and protection of hamsters via nasal administration of a humanized neutralizing antibody","abstract":"Monoclonal antibodies are widely used for the treatment of infectious human diseases, including COVID-19. Since the start of the pandemic, eight monoclonal antibodies against SARS-CoV-2 were granted emergency use authorization. High mutation rate of the SARS-CoV-2 virus has led to the emergence of highly transmissible variants efficiently evading vaccine-induced immunity. This highlights the importance of identifying broadly neutralizing antibodies with therapeutic potential. In this study, we used a panel of murine monoclonal antibodies (mAb) to identify a subset that bound and neutralized a broad spectrum of SARS-CoV-2 variants. Intranasal delivery of XR10, the most promising murine mAb, protected hamsters against infection by Alpha and Delta variants. We next humanized XR10 mAb using a combination of CDR-grafting and Vernier zones preservation approaches (CRVZ) to create a panel of humanized antibody variants. We ranked the variants based on their spike binding ability and virus neutralization. Of these, XR10v48 demonstrated the best ability to neutralize SARS-CoV-2 variants. XR10v48 was protective in hamsters when given as a single 50 \u00b5g/kg intranasal dose at the time of viral challenge. XR10v48 features 34 key amino acid residues retained from the murine progenitor. Our work introduces a potent humanized antibody that demonstrates neutralizing activity in vivo at a low dose.","version":"1.1","doi":"10.1101/2024.07.08.602557","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.05.602217","pub_date":"2024-7-08","title":"NSP4 mutation T492I drives rapid evolution of SARS-CoV-2 toward Omicron","abstract":"T492I, a mutation encountered in SARS-CoV-2 nonstructural protein 4 (NSP4), enhances viral replication and alters nonstructural protein cleavage, causing potential evolutionary impacts. Through comprehensive comparative analyses based on evolve-and-resequence experiments of SARS-CoV-2 wild-type and Delta strains with or without T492I, we demonstrate that NSP4 T492I not only increases the mutation rate, but also accelerates the emergence of many mutations characteristic for Omicron variants. Accordingly, viral populations that evolved from ancestors with T492I, show Omicron-biased selective forces and increases in viral replication, infectivity, immune evasion capacity, potentials for cross-species transmission and receptor-binding affinity. Aside from enhanced replication, we observed stronger epistasis regarding viral replication and infectivity in T492I than in S N501Y and NSP6 \u0394SGF; this facilitates the regulation of mutation types, which can drive fast evolution of Omicron specific mutations. Our results highlight the role of an important replication-enhancing mutation in regulating the evolutionary rates and mutational trends of SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.07.05.602217","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.06.602340","pub_date":"2024-7-08","title":"Structural impact of synonymous mutations in six SARS-CoV-2 Variants of Concern","abstract":"SARS-CoV-2 continues to spread and infect people worldwide. While most effort into characterizing variants of this virus have focused on non-synonymous changes, accumulation of synonymous mutations in different viral variants has also occurred. Here we characterize six Variants of Concern in terms of their mutational content, and make predictions regarding the impact of those mutations on potential genomic RNA secondary structure and stability. We find that synonymous mutations typically have no or modest impact to RNA secondary structure. As synonymous mutations are free from the selective pressure imposed on protein-altering mutations, the impact of synonymous mutations is largely limited to RNA secondary structure considerations. The absence of major, structure-altering synonymous mutations emphasize the importance of RNA structure, including within coding regions, to viral fitness.","version":"1.1","doi":"10.1101/2024.07.06.602340","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.04.602122","pub_date":"2024-7-08","title":"Heli-SMACC: Helicase-targeting SMAll Molecule Compound Collection","abstract":"Helicases have emerged as promising targets for the development of antiviral drugs; however, the family remains largely undrugged. To support the focused development of viral helicase inhibitors we identified, collected, and integrated all chemogenomics data for all available helicases from the ChEMBL database. After thoroughly curating and enriching the data with relevant annotations we have created a derivative database of helicase inhibitors which we dubbed Heli-SMACC (Helicase-targeting SMAll Molecule Compound Collection). The current version of Heli-SMACC contains 20,432 bioactivity entries for viral, human, and bacterial helicases. We have selected 30 compounds with promising viral helicase activity and tested them in a SARS-CoV-2 NSP13 ATPase assay. Twelve compounds demonstrated ATPase inhibition and a consistent dose-response curve. The Heli-SMACC database may serve as a reference for virologists and medicinal chemists working on the development of novel helicase inhibitors. Heli-SMACC is publicly available at https://smacc.mml.unc.edu. We created a curated Helicase-Targeting SMAll Molecule Compound Collection (Heli-SMACC). Heli-SMACC covers 29 human, viral, and bacterial helicases. Twelve of thirty selected compounds demonstrated inhibitory activity in a SARS-CoV-2 NSP13 ATPase Assay. Heli-SMACC is freely available online at https://smacc.mml.unc.edu.","version":"1.1","doi":"10.1101/2024.07.04.602122","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.03.530798","pub_date":"2024-7-05","title":"Inhibition of host N-myristoylation compromises the infectivity of SARS-CoV-2 due to Golgi-bypassing egress from lysosomes and endoplasmic reticulum","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, remains a global health concern despite vaccines, neutralizing antibodies, and antiviral drugs. Emerging mutations can reduce the effectiveness of these treatments, suggesting that targeting host cell factors may be a valuable alternative. N-myristoyltransferases (NMT) are essential enzymes for protein N-myristoylation, affecting stability, interaction, localization, and function of numerous proteins. We demonstrate that selective inhibition of host cell NMT decreases SARS-CoV-2 infection by 90% in human lung and primary nasal epithelial cells, and choroid plexus-cortical neuron organoids. NMT inhibition does not affect viral entry, replication or release, but impairs the maturation and incorporation of viral envelope proteins into newly assembled virions, leading to compromised infectivity of released virions. The inhibition of host NMT triggers a Golgi-bypassing pathway for SARS-CoV-2 progeny virion egress, which occurs through endoplasmic reticulum and lysosomal intermediates.","version":"1.3","doi":"10.1101/2023.03.03.530798","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.03.601941","pub_date":"2024-7-04","title":"IL-13 decreases susceptibility to airway epithelial SARS-CoV-2 infection but increases disease severity in vivo","abstract":"Treatments available to prevent progression of virus-induced lung diseases, including coronavirus disease 2019 (COVID-19) are of limited benefit once respiratory failure occurs. The efficacy of approved and emerging cytokine signaling-modulating antibodies is variable and is affected by disease course and patient-specific inflammation patterns. Therefore, understanding the role of inflammation on the viral infectious cycle is critical for effective use of cytokine-modulating agents. We investigated the role of the type 2 cytokine IL-13 on SARS-CoV-2 binding/entry, replication, and host response in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 infection in vivo. IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2- expressing ciliated cells rather than by neutralization in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened disease severity in mice; the effects were mediated by eicosanoid signaling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. We conclude that IL-13-induced inflammation differentially affects multiple steps of COVID-19 pathogenesis. IL-13-induced inflammation may be protective against initial SARS-CoV-2 airway epithelial infection; however, it enhances disease progression in vivo. Blockade of IL-13 and/or eicosanoid signaling may be protective against progression to severe respiratory virus-induced lung disease. Prior to this study, various pieces of evidence indicated the significant role of cytokines in the pathogenesis and progression of COVID-19. Severe COVID-19 cases were marked by cytokine storm syndrome, leading to immune activation and hyperinflammation. Treatments aimed at modulating cytokine signaling, such as IL-6 receptor antagonists, had shown moderate effects in managing severe COVID-19 cases. Studies also revealed an excessive production of type 2 cytokines, particularly IL-13 and IL-4, in the plasma and lungs of COVID-19 patients, which was associated with adverse outcomes. Treatment with anti-IL-13 monoclonal antibodies improved survival following SARS-CoV-2 infection, suggesting that IL-13 plays a role in disease severity. Type 2 cytokines were observed to potentially suppress type 1 responses, essential for viral clearance, and imbalances between these cytokine types were linked to negative COVID-19 outcomes. These findings highlighted the complex interactions between cytokines and the immune response during viral infections, underscoring the importance of understanding IL-13\u2019s role in COVID-19 and related lung diseases for developing effective therapeutic interventions. In this study, we explored the impact of IL-13-induced inflammation on various stages of the SARS-CoV-2 infection cycle using both murine (in vivo) and primary human airway epithelial (in vitro) culture models. Our findings indicated that IL-13 provided protection to airway epithelial cells against SARS-CoV-2 infection in vitro, partly by reducing the number of ACE2- expressing ciliated cells. Conversely, IL-13 exacerbated the severity of SARS2-N501YMA30-induced disease in mice, primarily through Pla2g2d-mediated eicosanoid biosynthesis. Current evidence indicates that PLA2G2D plays a crucial role in the IL-13-driven exacerbation of COVID-19 in mice, suggesting that targeting the IL-13-PLA2G2D axis could help protect against SARS-CoV-2 infection. These insights are important for clinical research, especially for studies focusing on drugs that modify IL-13 signaling or modulate eicosanoids in the treatment of asthma and respiratory virus-induced lung diseases.","version":"1.1","doi":"10.1101/2024.07.03.601941","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.03.601972","pub_date":"2024-7-04","title":"SARS-CoV-2 vaccination during pregnancy enhances hippocampal neurogenesis and working memory in offspring via IFN-gamma responsive microglia","abstract":"In the face of severe adverse outcomes of pandemic Coronavirus disease 2019 (COVID-19) infection, vaccines proved potently immunogenic and safe in humans and are today strongly recommended in pregnancy. This study investigates, in offspring mice, the effect of maternal COVID-19 vaccination on postnatal physical development, behavior and neurogenesis. After inoculation with inactivated COVID-19 vaccine (Vero Cell) at gestational day 14.5, antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were detected in serum of both dams and pups. At one month of age, pups born to vaccinated dams, but not the offspring of non-vaccinated dams, exhibited greater working memory and more neural cell proliferation, neuroblast formation, neuronal stem cell activity and larger numbers of mature neurons within the dentate gyrus (DG). Luminex multiplex assay revealed elevated levels of hippocampal cytokines/chemokines critical to neurogenesis and memory function, namely interferon-\u03b3 [IFN-\u03b3] and CX3C motif chemokine ligand 1 [CX3CL1]. Conditional knockout technology implicated microglial IFN-\u03b3 receptor 1 (IFN\u03b3R1) and CX3C motif chemokine receptor 1 (CX3CR1) as crucial intercellular participants in the neuronal developmental process, via regulating microglial activation and chemotaxis, respectively. We propose that, rather than posing risk for neurodevelopmental abnormalities, maternal SARS-CoV-2 vaccination transiently enhances hippocampal neurogenesis and working memory in offspring.","version":"1.1","doi":"10.1101/2024.07.03.601972","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.30.596664","pub_date":"2024-7-04","title":"Ancestral SARS-CoV-2 immune imprinting persists on RBD but not NTD after sequential Omicron infections","abstract":"Whether Omicron exposures could overcome ancestral SARS-CoV-2 immune imprinting remains controversial. Here we analyzed B cell responses evoked by sequential Omicron infections in vaccinated and unvaccinated individuals. Plasma neutralizing antibody titers against ancestral SARS-CoV-2 and variants indicate that immune imprinting is not consistently induced by inactivated or recombinant protein vaccines. However, once induced, immune imprinting is not countered by successive Omicron challenges. We compared binding specificities, neutralizing capacities, developing origins and targeting epitopes of monoclonal antibodies from individuals with or without immune imprinting. Although receptor-binding domain (RBD) and N-terminal domain (NTD) of spike are both primary targets for neutralizing antibodies, immune imprinting only shapes antibody responses to RBD by impeding the production of Omicron-specific neutralizing antibodies while facilitating the development of broadly neutralizing antibodies. We propose that immune imprinting can be either neglected by NTD-based vaccines to induce variant-specific antibodies or leveraged by RBD-based vaccines to induce broadly neutralizing antibodies.","version":"1.2","doi":"10.1101/2024.05.30.596664","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.03.601404","pub_date":"2024-7-04","title":"Spatial transcriptomics unveils the in situ cellular and molecular hallmarks of the lung in fatal COVID-19","abstract":"Severe Coronavirus disease 2019 (COVID-19) induces heterogeneous and progressive diffuse alveolar damage (DAD) highly disrupting lung tissue architecture and homeostasis, hampering disease management leading to fatal outcomes. Characterizing DAD pathophysiology across disease progression is of ultimate importance to better understand the molecular and cellular features driving different DAD patterns and to optimize treatment strategies. To contextualize the interplay between cell types and assess their distribution, spatial transcriptomics (ST) techniques have emerged, allowing unprecedented resolution to investigate spatial architecture of tissues. To this end, post-mortem lung tissue provides valuable insights into cellular composition and their spatial relationships at the time of death. Here, we have leveraged VisumST technology in post-mortem COVID-19 induced acute and proliferative DAD lungs including control samples with normal morphological appearance, to unravel the immunopathological mechanisms underlying DAD, providing novel insights into cellular and molecular communication events driving DAD progression in fatal COVID-19. We report a progressive loss of endothelial cell types, pneumocytes type I and natural killer cells coupled with a continuous increase of myeloid and stromal cells, mostly peribronchial fibroblasts, over disease progression. Spatial organization analysis identified variable cellular compartments, ranging from major compartments defined by cell type lineages in control lungs to increased and more specific compartmentalization including immune-specific clusters across DAD spectrum. Importantly, spatially informed ligand-receptor interaction (LRI) analysis revealed an intercellular communication signature defining COVID-19 induced DAD lungs. Transcription factor (TF) activity enrichment analysis identified TGF-B pathway as DAD driver, highlighting SMAD3 and SMAD7 TFs activity role during lung fibrosis. Integration of deregulated LRIs and TFs activity allowed us to propose a downstream intracellular signaling pathway in peribronchial fibroblasts, suggesting potential novel therapeutic targets. Finally, spatio-temporal trajectories analysis provided insights into the alveolar epithelium regeneration program, characterizing markers of pneumocytes type II differentiation towards pneumocytes type I. In conclusion, we provide a spatial characterization of lung tissue architecture upon COVID-19 induced DAD progression, identifying molecular and cellular hallmarks that may help optimize treatment and patient management.","version":"1.1","doi":"10.1101/2024.07.03.601404","journal":"bioRxiv","score":null},{"id":"10.1101/2024.07.02.601716","pub_date":"2024-7-03","title":"Quantification of heterogeneity in human CD8+ T cell responses to vaccine antigens: an HLA-guided perspective","abstract":"Vaccines have historically played a pivotal role in controlling epidemics. Effective vaccines for viruses causing significant human disease, e.g., Ebola, Lassa fever, or Crimean Congo hemorrhagic fever virus, would be invaluable to public health strategies and counter-measure development missions. Here, we propose coverage metrics to quantify vaccine-induced CD8+T cell-mediated immune protection, as well as metrics to characterize immuno-dominant epitopes, in light of human genetic heterogeneity and viral evolution. Proof-of-principle of our approach and methods will be demonstrated for Ebola virus, SARS-CoV-2, and Burkholderia pseudomallei (vaccine) proteins.","version":"1.1","doi":"10.1101/2024.07.02.601716","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.29.601123","pub_date":"2024-7-02","title":"e3SIM: epidemiological-ecological-evolutionary simulation framework for genomic epidemiology","abstract":"Infectious disease dynamics are driven by the complex interplay of epidemiological, ecological, and evolutionary processes. Accurately modeling these interactions is crucial for understanding pathogen spread and informing public health strategies. However, existing simulators often fail to capture the dynamic interplay between these processes, resulting in oversimplified models that do not fully reflect real-world complexities in which the pathogen\u2019s genetic evolution dynamically influences disease transmission. We introduce the epidemiological-ecological-evolutionary simulator (e3SIM), an open-source framework that concurrently models the transmission dynamics and molecular evolution of pathogens within a host population while integrating environmental factors. Using an agent-based, discrete-generation, forward-in-time approach, e3SIM incorporates compartmental models, host-population contact networks, and quantitative-trait models for pathogens. This integration allows for realistic simulations of disease spread and pathogen evolution. Key features include a modular and scalable design, flexibility in modeling various epidemiological and population-genetic complexities, incorporation of time-varying environmental factors, and a user-friendly graphical interface. We demonstrate e3SIM\u2019s capabilities through simulations of realistic outbreak scenarios with SARS-CoV-2 and Mycobacterium tuberculosis, illustrating its flexibility for studying the genomic epidemiology of diverse pathogen types.","version":"1.1","doi":"10.1101/2024.06.29.601123","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.30.601403","pub_date":"2024-7-01","title":"Differential action modes of Neutrophil Extracellular Trap-targeted drugs define T cell responses in SARS-CoV-2 infection","abstract":"Neutrophil extracellular traps (NETs) play a dual role in SARS-CoV-2 infection, aiding early immune defense but also contributing to lung damage. While NET targeting may improve clinical outcomes in SARS-CoV-2 infection, its impact on adaptive immunity, crucial for fighting the virus, remains unclear. Our study demonstrates that both recombinant human DNase (rhDNase), degrading NET structure, and GSK484, inhibiting NET formation, reduce lung NET concentration and improve clinical outcomes in infected mice, yet they differ in their influence on T cell responses. We show that rhDNase does not impact T cell responses, whereas GSK484 diminishes virus-specific T cell responses. In vitro, GSK484 decreases dendritic cell antigen presentation by impairing antigen uptake and reduces IL-2 signaling by affecting its production by T cells. In a model of lung inflammation, GSK484 diminishes antigen-specific T cell activation and proliferation, while rhDNase shows a potential to boost T cell responses via the presence of NET fragments that reduce T cell activation threshold. Our findings suggest that NET targeting with rhDNase or GSK484 holds therapeutic potential for treating SARS-CoV-2 infection, while their distinct modes of action shape T cell responses during the infection.","version":"1.1","doi":"10.1101/2024.06.30.601403","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.29.601315","pub_date":"2024-7-01","title":"PARP14 mediated SQSTM1/p62 cysteine ADP-ribosylation is counteracted by the SARS-CoV-2 macrodomain","abstract":"Several ADP-ribosyltransferases are upregulated during viral infections and are crucial for the cellular immune response. While interferon-induced PARP14 ADP-ribosylates various substrates, viruses such as SARS-CoV-2 counteract this by reversing ADP-ribosylation. The exact mechanism of PARP14\u2019s antiviral activity and the targets of viral macrodomains remain unknown. Here, we observe that PARP14 mono-ADP-ribosylates the selective autophagy adaptor SQSTM1/p62 at cysteine residues 113, 289/90, and 331 following interferon treatment. This correlates with the ADP-ribosylation of cytoplasmic p62 foci that colocalize with ubiquitin and PARP14 but not with LC3, thereby distinguishing them from classical autophagosomes. Moreover, the SARS-CoV-2 macrodomain effectively prevented this p62 modification, suggesting an antiviral function for this ADP-ribosylated target. Furthermore, our results indicate that TRIM21 prevents the autophagic degradation of ADP-ribosylated p62, suggesting that the identified p62 foci may have autophagy-independent roles. This study contributes to our understanding of the molecular dynamics involved in host-virus interactions and highlights the potential role of ADP-ribosylation in the regulation of innate immunity.","version":"1.1","doi":"10.1101/2024.06.29.601315","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.28.601197","pub_date":"2024-6-30","title":"Gut microbial profiling of COVID-19 patients in Uganda","abstract":"While COVID-19 spread globally, the role of the gut microbiota in patient outcomes has remained an area of exploration especially in resource limited settings. This study aimed to comprehensively profile the gut microbiome among Ugandan COVID-19 patients and infer potential implications. Nasopharyngeal swabs, stool, clinical and demographic data were collected from COVID-19 confirmed cases at the COVID-19 isolation and treatment centers in Kampala and Entebbe, Uganda, during the first and second waves of the pandemic in Uganda (i.e., 2020 and 2021, respectively). SARS-CoV-2 presence in the swab samples was confirmed by quantitative real-time RT-PCR assays. 16S rRNA metagenomic next-generation sequencing was performed on the DNA extracted from the stool samples, followed by bioinformatics analysis. Machine learning was used to determine microbes that were associated with disease severity. We observed varied gut microbial composition between COVID-19 patients and healthy controls. Potentially pathogenic bacteria such as Klebsiella oxytoca, Salmonella enterica and Serratia marcescens had an increased presence in COVID-19 disease states, especially severe cases. Enrichment of opportunistic pathogens, such as Enterococcus species, and depletion of beneficial microbes, like Alphaproteobacteria, was observed between mild and severe cases. Machine learning identified age and microbes such as Ruminococcaceae, Bacilli, Enterobacteriales, porphyromonadaceae, and Prevotella copri as predictive of severity. These findings suggest that the microbiome plays a role in the dynamics of SARS-CoV-2 infection in African patients. The shift in abundance of specific microbes can moderately predict severity of COVID-19 in this population. Their direct or indirect roles in determining severity should be investigated further for potential therapeutic interventions.","version":"1.1","doi":"10.1101/2024.06.28.601197","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.24.600468","pub_date":"2024-6-28","title":"Broad-spectrum RNA antiviral inspired by ISG15-/- deficiency","abstract":"Type I interferons (IFN-I) are cytokines with potent antiviral and inflammatory capacities. IFN-I signaling drives the expression of hundreds of IFN-I stimulated genes (ISGs), whose aggregate function results in the control of viral infection. A few of these ISGs are tasked with negatively regulating the IFN-I response to prevent overt inflammation. ISG15 is a negative regulator whose absence leads to persistent, low-grade elevation of ISG expression and concurrent, self-resolving mild autoinflammation. The limited breadth and low-grade persistence of ISGs expressed in ISG15 deficiency are sufficient to confer broad-spectrum antiviral resistance. Inspired by ISG15 deficiency, we have identified a nominal collection of 10 ISGs that recapitulate the broad antiviral potential of the IFN-I system. The expression of the 10 ISG collection in an IFN-I non-responsive cell line increased cellular resistance to Zika, Vesicular Stomatitis, Influenza A (IAV), and SARS-CoV-2 viruses. A deliverable prophylactic formulation of this syndicate of 10 ISGs significantly inhibited IAV PR8 replication in vivo in mice and protected hamsters against a lethal SARS-CoV-2 challenge, suggesting its potential as a broad-spectrum antiviral against many current and future emerging viral pathogens. Human inborn error of immunity-guided discovery and development of a broad-spectrum RNA antiviral therapy","version":"1.1","doi":"10.1101/2024.06.24.600468","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.27.599960","pub_date":"2024-6-27","title":"An intricate balancing act: Upstream and downstream frameshift co-regulatory elements","abstract":"Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against Covid-19. During ribosomal translation, a fraction of elongating ribosomes slips by one base in the 5\u2032 direction and enters a new reading frame for viral protein synthesis. Any interference with this process profoundly affects viral replication and propagation. For Covid-19, two RNA sites associated with ribosomal frameshifting for SARS-CoV-2 are positioned on the 5\u2032 and 3\u2032 of the frameshifting residues. Although much attention has been on the 3\u2032 frameshift element (FSE), the 5\u2032 stem-loop (attenuator hairpin, AH) can play a role. The formation of AH has been suggested to occur as refolding of the 3\u2032 RNA structure is triggered by ribosomal unwinding. However, the attenuation activity and the relationship between the two regions are unknown. To gain more insight into these two related viral RNAs and to further enrich our understanding of ribosomal frameshifting for SARS-CoV-2, we explore the RNA folding of both 5\u2032 and 3\u2032 regions associated with frameshifting. Using our graph-theory-based modeling tools to represent RNA secondary structures, \u201cRAG\u201d (RNA-As-Graphs), and conformational landscapes to analyze length-dependent conformational distributions, we show that AH coexists with the 3-stem pseudoknot of the 3\u2032 FSE (graph 3_6 in our dual graph notation) and alternative pseudoknot (graph 3_3) but less likely with other 3\u2032 FSE alternative folds (such as 3-way junction 3_5). This is because an alternative length-dependent Stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. In addition, we design four mutants for long lengths that stabilize or disrupt AH, AS1 or FSE pseudoknot to illustrate the deduced AH/AS1 roles and favor the 3_5, 3_6 or stem-loop. These mutants further show how a strengthened pseudoknot can result from a weakened AS1, while a dominant stem-loop occurs with a strengthened AS1. These structural and mutational insights into both ends of the FSE in SARS-CoV-2 advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting a sequence of length-dependent folds, which in turn define potential therapeutic intervention techniques involving both elements. Our work also highlights the complexity of viral landscapes with length-dependent folds, and challenges in analyzing these multiple conformations.","version":"1.1","doi":"10.1101/2024.06.27.599960","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.16.549249","pub_date":"2024-6-27","title":"Saponin Nanoparticle Adjuvants Incorporating Toll-Like Receptor Agonists Drive Distinct Immune Signatures and Potent Vaccine Responses","abstract":"Over the past few decades, the development of potent and safe immune-activating adjuvant technologies has become the heart of intensive research in the constant fight against highly mutative and immune evasive viruses such as influenza, SARS-CoV-2, and HIV. Herein, we developed a highly modular saponin-based nanoparticle platform incorporating toll-like receptor agonists (TLRas) including TLR1/2a, TLR4a, TLR7/8a adjuvants and their mixtures. These various TLRa-SNP adjuvant constructs induce unique acute cytokine and immune-signaling profiles, leading to specific Th-responses that could be of interest depending on the target disease for prevention. In a murine vaccine study, the adjuvants greatly improved the potency, durability, breadth, and neutralization of both COVID-19 and HIV vaccine candidates, suggesting the potential broad application of these adjuvant constructs to a range of different antigens. Overall, this work demonstrates a modular TLRa-SNP adjuvant platform which could improve the design of vaccines for and dramatically impact modern vaccine development. Saponin-TLRa nanoadjuvants provide distinct immune signatures and drive potent, broad, durable COVID-19 and HIV vaccine responses.","version":"1.4","doi":"10.1101/2023.07.16.549249","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.24.600376","pub_date":"2024-6-24","title":"Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2","abstract":"Cell entry of severe acute respiratory coronavirus-2 (SARS-CoV-2) and other CoVs can occur via two distinct routes. Following receptor binding by the spike glycoprotein, membrane fusion can be triggered by spike cleavage either at the cell surface in a transmembrane serine protease 2 (TMPRSS2)-dependent manner or within endosomes in a cathepsin-dependent manner. Cellular sialoglycans have been proposed to aid in CoV attachment and entry, although their functional contributions to each entry pathway are unknown. In this study, we used genetic and enzymatic approaches to deplete sialic acid from cell surfaces and compared the requirement for sialoglycans during endosomal and cell-surface CoV entry, primarily using lentiviral particles pseudotyped with the spike proteins of different sarbecoviruses. We show that entry of SARS-CoV-1, WIV1-CoV and WIV16-CoV, like the SARS-CoV-2 omicron variant, depends on endosomal cathepsins and requires cellular sialoglycans for entry. Ancestral SARS-CoV-2 and the delta variant can use either pathway for entry, but only require sialic acid for endosomal entry in cells lacking TMPRSS2. Binding of SARS-CoV-2 spike protein to cells did not require sialic acid, nor was sialic acid required for SARS-CoV-2 entry in TMRPSS2-expressing cells. These findings suggest that cellular sialoglycans are not strictly required for SARS-CoV-2 attachment, receptor binding or fusion, but rather promote endocytic entry of SARS-CoV-2 and related sarbecoviruses. In contrast, the requirement for sialic acid during entry of MERS-CoV pseudoparticles and authentic HCoV-OC43 was not affected by TMPRSS2 expression, consistent with a described role for sialic acid in merbecovirus and embecovirus cell attachment. Overall, these findings clarify the role of sialoglycans in SARS-CoV-2 entry and suggest that cellular sialoglycans mediate endosomal, but not cell-surface, SARS-CoV-2 entry. Thus, it may be important to consider both cell entry pathways when developing sarbecovirus entry inhibitors targeting virus-sialoglycan interactions. The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in over 676 million infections and 6.8 million deaths so far, demonstrating the threat posed by emerging CoVs. In humans, SARS-CoV-2 and related coronaviruses cause respiratory tract infections, such as the common cold, as well as more severe disease in some individuals. To prepare for future outbreaks, conserved steps in the CoV replication could be considered for antiviral prophylactic or therapeutic approaches. One such process is CoV cell entry, which occurs via two main routes: At the cell surface or within endosomes. Cellular receptors, proteases and complex sugars, known as glycans, mediate CoV entry steps. In this study, we compared the role of a specific glycan subset, sialoglycans, in endosomal and cell surface CoV entry. We show that sialoglycans are required for entry of various CoVs that are mainly dependent on the endosomal route, but in the case of SARS-CoV-2, sialoglycans were not required when the cell-surface entry route was available. Our findings contribute to understanding the mechanisms of CoV entry, which could inform development of pan-CoV antivirals that target CoV entry steps.","version":"1.1","doi":"10.1101/2024.06.24.600376","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.23.600238","pub_date":"2024-6-24","title":"ACE-2 Blockade & TMPRSS2 Inhibition Mitigate SARS-CoV-2 Severity Following Cigarette Smoke Exposure in Airway Epithelial Cells In Vitro","abstract":"Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.","version":"1.1","doi":"10.1101/2024.06.23.600238","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.21.600129","pub_date":"2024-6-24","title":"Digital Immunoassay for Rapid Detection of SARS-CoV-2 Infection in a Broad Spectrum of Animals","abstract":"The ability of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to infect a wide-range of species raises significant concerns regarding both human-to-animal and animal-to-human transmission. There is an increasing demand for highly sensitive, rapid, and simple diagnostic assays that can detect viral infection across various species. In this study, we developed a biosensor assay that adapted a monoclonal-antibody (mAb)-based blocking ELISA format into an Activate Capture + Digital Counting (AC + DC)-based immunoassay. The assay employs a photonic crystal (PC) biosensor, gold-nanoparticle (AuNP) tags, SARS-CoV-2 nucleocapsid (N) protein, and specific anti-N mAb to detect antibody responses in animals exposed with SARS-CoV-2. We demonstrated a simple 2-step 15-min test that was capable of detecting as low as 12.5 ng of antibody in controlled standard serum samples. Based on an evaluation of 176 cat serum samples with known antibody status, an optimal percentage of inhibition (PI) cut-off value of 0.588 resulted in a diagnostic sensitivity of 98.3% and a diagnostic specificity of 96.5%. The test is highly repeatable with low variation coefficients of 2.04%, 2.73%, and 4.87% across different runs, within a single run, and on a single chip, respectively. The test was further employed to detect antibody responses in multiple animal species as well as investigate dynamics of antibody response in experimentally infected cats. This test platform provides an important tool for rapid field surveillance of SARS-CoV-2 infection across multiple species.","version":"1.1","doi":"10.1101/2024.06.21.600129","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.21.600102","pub_date":"2024-6-24","title":"EEG Signatures of COVID-19 Survival compared to close contacts and the Cuban EEG normative database","abstract":"The EEG constitutes a powerful neuroimaging technique for assessing functional brain impairment in COVID-19 patients. The current investigation compared the EEG among COVID-19 survivors, close contacts and the Cuban EEG normative database, using semi-quantitative visual EEG inspection, quantitative and the current source density measures EEG analysis. The resting-state EEG activity, quantitative QEEG, and VARETA inverse solution, were evaluated in 173 subjects: 87 patients confirmed cases by the positive reverse transcription polymerase chain reaction (RT-PCR), 86 close contacts (negative PCR) and the Cuban EEG normative database. All patients were physical, neurological, and clinically assessed using neurological retrospective survey and version 2.1 of the Schedules for Clinical Assessment in Neuropsychiatry (SCAN). The GTE score showed significant differences in terms of frequency scores of backgrounds rhythmic activity, diffuse slow activity, and focal abnormality. The QEEG analysis showed a pattern of abnormality with respect to the Cuban EEG normative values, displaying an excess of alpha and beta activities in the fronto-central-parietal areas in both groups. The anomalies, of COVID-19 patients and close contacts, differs in the right fronto-centro parietal area. The COVID 19 group differed-s from the close control group in theta band of the right parieto-central. The symptomatic group of COVID-19 patients differs from asymptomatic patients in delta and theta activities of the parieto-central region. The sources of activation using VARETA showed a difference in cortical activation patterns at alpha and beta frequencies in the groups studied with respect to the normative EEG database. In beta frequency were localized in right middle temporal gyrus in both groups and right angular gyrus in Covid 19 group only. In alpha band, the regions were the left supramarginal gyrus for Covid 19 group and the left superior temporal gyrus for Control group. Greater activation was found in the right middle temporal gyrus at alpha frequency in COVID-19 patients than in their close contacts. Brain functions are impaired in long COVID-19 patients. QEEG and VARETA permit us to comprehend the susceptibility of particular brain regions exposed to viral illness. Background frequency abnormalities diffuse slow activity and focal abnormality associated with a pattern of excess oftheta, alpha and beta energies in in the right fronto-centro-parietal regions in QEEG analysis characterizedCOVID-19 patients. Patients with COVID-19 show more alpha and beta EEG activities related to normative EEG database. Patients with COVID-19 and close contacts show high cortical activation in temporo-parietal areas in alpha and beta bands compared to normative EEG database. Patients with COVID-19 (positive PCR) have high activation in the right middle frontal gyrus for alpha band related to close contacts.","version":"1.1","doi":"10.1101/2024.06.21.600102","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.20.599933","pub_date":"2024-6-21","title":"Large-scale genomic analysis of SARS-CoV-2 Omicron BA.5 emergence in the United States","abstract":"The COVID-19 pandemic is marked by the continuing emergence of novel SARS-CoV-2 variants. Questions remain about the mechanisms with which these lineages establish themselves in new geographical areas. In this study, we performed a discrete phylogeographic analysis on \u223c19,000 SARS-CoV-2 sequences of Omicron BA.5 lineages between February and June 2022 to better understand how it emerged in different regions of the United States (U.S.). We found that the earliest introductions came from Africa, the putative origin of the variant, but the majority were from Europe, correlating with the high volume of air travelers. Additionally, the analysis revealed extensive domestic transmission between different regions of the U.S. driven by population size and cross-country transmission. Results suggest that most of the within-U.S. spread was between three regions that include California, New York, and Florida. Our results form a framework for understanding novel SARS-CoV-2 variant emergence in the U.S.","version":"1.1","doi":"10.1101/2024.06.20.599933","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.20.599956","pub_date":"2024-6-21","title":"ACE2 decoy Fc-fusions and bi-specific killer engager (BiKEs) require Fc engagement for in vivo efficacy against SARS-CoV-2","abstract":"SARS-CoV-2 virus has continued to evolve over time necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a key line of defense for unvaccinated or immunocompromised individuals. However, these mAbs are now ineffective against current SARS-CoV-2 variants. Here, we tested three aspects of \u03b1SARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for in vivo clearance of SARS-CoV-2. Secondly, we tested bi-specific killer engagers (BiKEs) that simultaneously engage SARS-CoV-2 and a specific Fc receptor. Benefits of these engagers include the ease of manufacturing, stability, more cell-specific targeting, and high affinity binding to Fc receptors. Using both mAbs and BiKEs, we found that both neutralization and Fc receptor engagement were necessary for effective SARS-CoV-2 clearance. Thirdly, due to ACE2 being necessary for viral entry, ACE2 will maintain binding to SARS-CoV-2 despite viral evolution. Therefore, we used an ACE2 decoy Fc-fusion or BiKE, instead of an anti-SARS-CoV-2 antibody sequence, as a potential therapeutic that would withstand viral evolution. We found that the ACE2 decoy approach also required Fc receptor engagement and, unlike traditional neutralizing antibodies against specific variants, enabled the clearance of two distinct SARS-CoV-2 variants. These data show the importance of Fc engagement for mAbs, the utility of BiKEs as therapies for infectious disease, and the in vivo effectiveness of the ACE2 decoy approach. With further studies, we predict combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels. ACE2, scFv, mAb, BiKE, COVID-19, Fc, CD16, CD32b, CD64, d.p.i With equal dosing, both neutralization and Fc engagement are necessary for the optimal efficacy of in vivo antibodies and bi-specific killer engagers (BiKEs) against SARS-CoV-2. BiKEs can clear SARS-CoV-2 virus and protect against severe infection in the hACE2-K18 mouse model. ACE2 decoys as part of Fc-fusions or BiKEs provide in vivo clearance of two disparate SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.06.20.599956","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.21.600005","pub_date":"2024-6-21","title":"Molecular basis of Ad5-nCoV Vaccine-Induced Immunogenicity","abstract":"In response to coronavirus disease 2019 (COVID-19), numerous vaccines have been developed to protect against SARS-CoV-2 infection. Ad5-nCoV (Convidecia) is a vaccine listed for emergency use by the WHO and has been administrated to millions of people globally. It comprises a series of human adenovirus 5 (Ad5) replication-incompetent vectored vaccines that transduce the spike protein (S) gene of various SARS-CoV-2 strains. Despite promising clinical data demonstrating its safety and effectiveness, the underlying molecular mechanism of its high immunogenicity and incidence of adverse reactions remains less understood. Here we combined cryo-ET, fluorescence microscopy and mass spectrometry to characterize the in situ structures, density and site-specific glycan compositions of the Ad5-nCoV_Wu and Ad5-nCoV_O vaccine-induced S antigens, which encode the unmodified SARS-CoV-2 Wuhan-Hu-1 S gene and optimized Omicron S gene, respectively. We found that the vaccine-induced S are structurally intact, antigenic and densely distributed on the cell membrane. Compared to Ad5-nCoV_Wu induced S, the Ad5-nCoV_O induced S demonstrate significantly better stability and is less likely to induce syncytia among inoculated cells. Our work demonstrated that Ad5-nCoV is a prominent platform for antigen induction and cryo-ET can be a useful technique for vaccine characterization and development.","version":"1.1","doi":"10.1101/2024.06.21.600005","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.21.600068","pub_date":"2024-6-21","title":"Characterizing neuroinvasion and neuropathology of SARS-CoV-2 by using AC70 human ACE2 transgenic mice","abstract":"COVID-19 presents with a plethora of neurological signs and symptoms despite being characterized as a respiratory disease, including seizures, anxiety, depression, amnesia, attention deficits, and alterations in consciousness. The olfactory nerve is widely accepted as the neuroinvasive route by which the etiological agent SARS-CoV-2 enters the brain, but the trigeminal nerve is an often-overlooked additional route. Based on this consensus, we initially conducted a pilot experiment investigating the olfactory nerve route of SARS-CoV-2 neuroinvasion via intranasal inoculation in AC70 human ACE2 transgenic mice. Notably, we found that the trigeminal ganglion is an early and highly efficient site of viral replication, which then rapidly spread widely throughout the brain where neurons were primarily targeted. Despite the extensive viral infection across the brain, obvious evidence of tissue pathology including inflammatory infiltration, glial activation, and apoptotic cell deaths were not consistently observed, albeit inflammatory cytokines were significantly induced. However, the expression levels of different genes related to neuronal function, including the neurotransmitter dopamine pathway as well as synaptic function, and markers of neuronal damage were altered as compared to mock-infected mice. Our findings suggest that the trigeminal nerve can be a neuroinvasive route complementary to the olfactory nerve and that the ensuing neuroinvasion presented a unique neuropathological profile. This study provides insights into potential neuropathogenic mechanisms utilized by coronaviruses. COVID-19 presents with extrapulmonary signs and symptoms, the most notable of which involve the central nervous system, such as seizures and alterations in consciousness, and can eventually lead to death if severe enough. Some neurological signs and symptoms may continue to persist in some patients even after the resolution of active viral infection in the form of post-acute sequelae. Since the trigeminal nerve is a commonly under-studied route of entry into the brain in studies of coronaviruses and the neuropathogenic mechanisms of COVID-19 are not entirely elucidated, there is a need to thoroughly investigate this route of neuroinvasion. The significance of our research is in providing insights into the possible routes of SARS-CoV-2 neuroinvasion as well as the discovery of potential neuropathogenic mechanisms which may help guide the development of novel medical countermeasures.","version":"1.1","doi":"10.1101/2024.06.21.600068","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.18.599502","pub_date":"2024-6-21","title":"Surviving Medical School During a Pandemic: Experiences of New York Medical Students During the Height of SARS-CoV-2","abstract":"The COVID-19 pandemic dramatically altered the landscape of medical education. While patients overwhelmed hospital systems, lockdowns and social distancing recommendations took priority, and medical education was pushed online. Early in 2020, New York State (NYS) was hit especially hard by COVID-19. This study sought to understand the effect of the COVID-19 pandemic on medical students well-being and education. NYS medical students responded to a six-question survey during April and May 2020. Questions assessed self-reported changes in stress levels, academic performance, and board preparation efforts. Open-ended data was analyzed using a modified grounded theory approach. 488 responses across 11 medical schools were included (response rate of 5.8%). Major themes included: standardized test-related stressors (23%), study-related changes (19%), education and training concerns (17%), financial stressors (12%), and additional family obligations (12%). Second year students reported more stress/anxiety than students in other years (95.9%, p-value< 0.00001). Reported stress/anxiety, effects on exam preparation, and anticipated academic effect varied by geographics. While all NYS medical students reported being greatly affected, those closest to the NY City pandemic epi-center and closest to taking the Step 1 exam were the most distressed. Lack of flexibility of the medical education system during this public health emergency contributed to worsened student well-being. It is time to make plans for supporting the long-term mental health needs of these physicians-in-training and to examine ways the academic medical community can better adapt to the needs of students affected by a large public health emergency in the future.","version":"1.1","doi":"10.1101/2024.06.18.599502","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.18.599635","pub_date":"2024-6-20","title":"C->U transition biases in SARS-CoV-2 \u2013 still rampant four years from the start of the COVID-19 pandemic","abstract":"The evolution of SARS-CoV2 in the pandemic and post-pandemic periods has been characterised by rapid adaptive changes that confer immune escape and enhanced human-to-human transmissibility. Sequence change is additionally marked by an excess number of C->U transitions suggested as being due to host-mediated genome editing. To investigate how therse influence the evolutionary trajectory of SARS-CoV-2, 2000 high quality, coding complete genome sequences of SARS-CoV-2 variants collected pre-September, 2020, and from each subsequently appearing alpha, delta, BA.1, BA.2, BA.5, XBB, EG, HK and JN.1 lineages were downloaded from NCBI Virus in April, 2024. C->U transitions were the most common substitution during diversification of SARS-CoV-2 lineages over the 4-year observation period. A net loss of C bases and accumulation of U\u2019s occurred at a constant rate of approximately 0.2%-0.25% / decade. C->U transitions occurred in over a quarter of all sites with a C (26.5%; range 20.0%-37.2%), around 5 times more than observed for the other transitions (5.3%-6.8%). In contrast to an approximately random distribution of other transitions across the genome, most C->U substitutions occurred at statistically preferred sites in each lineage. However, only the most C->U polymorphic sites showed evidence for a preferred 5\u2019U context previously associated with APOBEC 3A editing. There was a similarly weak preference for unpaired bases suggesting much less stringent targeting of RNA than mediated by A3 deaminases in DNA editing. Future functional studies are required to determine editing preferences, impacts on replication fitness in vivo of SARS-CoV-2 and other RNA viruses and impact on host tropism.","version":"1.1","doi":"10.1101/2024.06.18.599635","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.20.599898","pub_date":"2024-6-20","title":"Time-based quantitative proteomic and phosphoproteomic analysis of A549-ACE2 cells during SARS-CoV-2 infection","abstract":"The outbreak of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2, led to an ongoing pandemic with devastating consequences for the global economy and human health. With the global spread of SARS-CoV-2, multidisciplinary initiatives were launched to explore new diagnostic, therapeutic, and vaccination strategies. From this perspective, proteomics could help to understand the mechanisms associated with SARS-CoV-2 infection and to identify new therapeutic targets for antiviral drug repurposing and/or discovery. A TMT-based quantitative proteomics and phosphoproteomics analysis was performed to study the proteome remodeling of human lung alveolar cells transduced to express human ACE2 (A549-ACE2) after infection with SARS-CoV-2. Targeted PRM analysis was performed to assess the detectability in serum and prognostic value of selected proteins. A total of 6802 proteins and 6428 phospho-sites were identified in A549-ACE2 cells after infection with SARS-CoV-2. Regarding the viral proteome, 8 proteins were differentially expressed after 6 h of infection and reached a steady state after 9 h. In addition, we detected several phosphorylation sites of SARS-CoV-2 proteins, including two novel phosphorylation events at S410 and S416 of the viral nucleoprotein. The differential proteins here identified revealed that A549-ACE2 cells undergo a time-dependent regulation of essential processes, delineating the precise intervention of the cellular machinery by the viral proteins. From this mechanistic background and by applying machine learning modelling, 29 differential proteins were selected and detected in the serum of COVID-19 patients, 14 of which showed promising prognostic capacity. Targeting these proteins and the protein kinases responsible for the reported phosphorylation changes may provide efficient alternative strategies for the clinical management of COVID-19.","version":"1.1","doi":"10.1101/2024.06.20.599898","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.11.598368","pub_date":"2024-6-20","title":"BCR, not TCR, repertoire diversity is associated with favorable COVID-19 prognosis","abstract":"The SARS-CoV-2 pandemic has had a widespread and severe impact on society, yet there have also been instances of remarkable recovery, even in critically ill patients. In this study, we used single-cell RNA sequencing to analyze the immune responses in recovered and deceased COVID-19 patients during moderate and critical stages. The study included three unvaccinated patients from each outcome category. Although expanded T cell receptor (TCR) clones were predominantly SARS-CoV-2-specific, they represented only a small fraction of the total repertoire in all patients. In contrast, while deceased patients exhibited monoclonal B cell receptor (BCR) expansions without COVID-19 specificity, survivors demonstrated diverse and specific BCR clones. These findings suggest that neither TCR diversity nor BCR monoclonal expansions are sufficient for viral clearance and subsequent recovery. Differential gene expression analysis revealed that protein biosynthetic processes were enriched in survivors, but that potentially damaging mitochondrial ATP metabolism was activated in the deceased. This study underscores that BCR repertoire diversity, but not TCR diversity, correlates with favorable outcomes in COVID-19.","version":"1.2","doi":"10.1101/2024.06.11.598368","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.16.585341","pub_date":"2024-6-20","title":"Identification of novel and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries","abstract":"In vitro screening of large compounds libraries with automated high-throughput screening is expensive, time consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECL) can be rapidly performed with routine equipment available in most laboratories. In this study we identified novel inhibitors of SARS-CoV-2 main protease (Mpro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of Mpro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 displaying an IC50 of 30 nM, proving that the rapid exploration of large chemical spaces enabled by DECL technology, allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. Compound MP1, a close analogue of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC50 = 2.3 \u00b5M) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds as well as the substitution of selected residues with D-enantiomers will be explored in the future to improve the antiviral activity of these novel compounds.","version":"1.2","doi":"10.1101/2024.03.16.585341","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.18.599357","pub_date":"2024-6-19","title":"The effect of circulating neutralizing antibodies on the replication of SARS-CoV-2 variants following post-vaccination infections","abstract":"The impact of pre-existing neutralizing antibodies (NAbs) titers on SARS-CoV-2 viral shedding dynamics in post-vaccination infection (PVI) are not well understood. We characterized viral shedding longitudinally in nasal specimens in relation to baseline (pre/peri-infection) serum neutralizing antibody titers in 125 participants infected with distinct SARS-CoV-2 variants. Among 68 participants who had received vaccinations, we quantified the effect of baseline serum NAb titers on maximum viral RNA titers and on the duration of infectivity. Baseline NAb titers were higher and efficiently targeted a broader range of variants in participants who received one or two monovalent ancestral booster vaccinations compared to those with a full primary vaccine series. In participants with Delta variant infections, baseline NAb titers targeting Delta were negatively correlated with maximum viral RNA copies. Per log10 increase in baseline NAb IC50, maximum viral load was reduced -2.43 (95% confidence interval [CI] -3.76, -1.11) log10 N copies and days of infectious viral shedding were reduced -2.79 [95% CI: -4.99, -0.60] days. By contrast, in those with Omicron infections (BA.1, BA.2, BA.4 or BA.5 lineages) baseline NAb responses against Omicron lineages did not predict viral outcomes. Our results provide robust estimates of the effect of baseline NAbs on the magnitude and duration of nasal viral replication after PVI (albeit with an unclear effect on transmission) and show how immune escape variants efficiently evade these modulating effects.","version":"1.1","doi":"10.1101/2024.06.18.599357","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.19.598823","pub_date":"2024-6-19","title":"A Spike Trimer Dimer-Inducing Nanobody with Anti-Sarbecovirus Activity","abstract":"The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7F\u2019s ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses.","version":"1.1","doi":"10.1101/2024.06.19.598823","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.18.599612","pub_date":"2024-6-19","title":"Sex differences and immune correlates of Long COVID development, persistence, and resolution","abstract":"Sex differences have been observed in acute COVID-19 and Long COVID (LC) outcomes, with greater disease severity and mortality during acute infection in males and a greater proportion of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to the pathogenesis of LC. To investigate the immunologic underpinnings of LC development and persistence, we used single-cell transcriptomics, single-cell proteomics, and plasma proteomics on blood samples obtained during acute SARS-CoV-2 infection and at 3 and 12 months post-infection in a cohort of 45 patients who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Specifically, males who would develop LC at 3 months had widespread increases in TGF-\u03b2 signaling during acute infection in proliferating NK cells. Females who would develop LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, and increased IL1 signaling in monocytes at 12 months post infection. Several immune features of LC were also conserved across sexes. Both males and females with LC had reduced co-stimulatory signaling from monocytes and broad upregulation of NF-\u03baB transcription factors. In both sexes, those with persistent LC demonstrated increased LAG3, a marker of T cell exhaustion, reduced ETS1 transcription factor expression across lymphocyte subsets, and elevated intracellular IL-4 levels in T cell subsets, suggesting that ETS1 alterations may drive an aberrantly elevated Th2-like response in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics. This multi-omic analysis of Long COVID reveals sex differences and immune correlates of Long COVID development, persistence, and resolution.","version":"1.1","doi":"10.1101/2024.06.18.599612","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.14.24308523","pub_date":"2024-06-18","title":"Emergence of transmissible SARS-CoV-2 variants with decreased sensitivity to antivirals in immunocompromised patients with persistent infections","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n=15). All patients received remdesivir and some also received nirmatrelvir-ritonavir or monoclonal antibodies. Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient treated with remdesivir and nirmatrelvir-ritonavir.\n                  <jats:italic>In vitro</jats:italic>\n                  characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility\n                  <jats:italic>in vivo</jats:italic>\n                  .\n                </jats:p>","version":null,"doi":"10.1101/2024.06.14.24308523","journal":"medRxiv","score":null},{"id":"10.1101/2024.06.17.599107","pub_date":"2024-6-17","title":"TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication","abstract":"SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7-/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.","version":"1.1","doi":"10.1101/2024.06.17.599107","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.16.599198","pub_date":"2024-6-17","title":"Comparison of three tiled amplicon sequencing approaches for SARS-CoV-2 variant detection from wastewater","abstract":"During the COVID-19 pandemic, the detection and sequencing of SARS-CoV-2 from wastewater proved to be a valuable tool in assessing trends at the community level. Several whole genome enrichment methods have been proposed for sequencing SARS-CoV-2 from the mixed wastewater community, but there is little consensus on the most appropriate sequencing methods for variant detection or abundance estimations. Few studies have elucidated the errors associated with these methods or have established minimum sequencing requirements for correct interpretation of the results. To address these needs, we systematically assessed the efficacy of three tiled amplicon enrichment methods (Freed/Midnight, ARTIC V4, NEB VarSkip) for whole genome sequencing of SARS-CoV-2 variants using mock wastewater communities with variants at known proportions. We found the ARTIC V4 approach yielded the most accurate results for variant identification and variant abundance estimation, followed by the NEB VarSkip approach. Conversely, the NEB VarSkip method obtained the highest genomic coverage, with the ARTIC V4 method achieving the second highest coverage. Finally, we determined that the Freed/Midnight library preparation methods are not well-suited for use with short read sequencing. Based on the present results, the ARTIC V4 workflow appears to be the most robust and cost-effective approach for monitoring circulating SARS-CoV-2 variants with wastewater surveillance. This work is informative for practitioners of wastewater-based epidemiology. Here, we detail a systematic comparison of three tiled amplicon sequencing approaches for enrichment of SARS-CoV-2 variants from wastewater. Using mock communities of known variant composition, we validate the analysis methods previously published by Baaijens et al. in Genome Biology (2022) for estimating variant abundance from wastewater using an RNAseq pipeline, kallisto. We provide recommendations for minimum sequencing requirements for accurate abundance estimates of SARS-CoV-2 variants in wastewater. The sequences generated from the mock communities have been uploaded to NCBI\u2019s Sequence Read Archive and will be useful to other practitioners seeking to validate their sequencing methods or bioinformatic pipelines.","version":"1.1","doi":"10.1101/2024.06.16.599198","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.17.599344","pub_date":"2024-6-17","title":"The differential effect of SARS-COV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage and virus evolution","abstract":"The non-structural protein 1 (NSP1) of SARS-CoV-2 blocks the mRNA entry channel of the 40S ribosomal subunit, causing inhibition of translation initiation and subsequent degradation of host mRNAs. However, target mRNA specificity and the way in which viral mRNAs escape NSP1-mediated degradation have not been clarified to date. Here we found that NSP1 acts as a translational switch capable of blocking or enhancing translation depending on how preinitiation complex 43S-PIC is recruited to the mRNA, whereas NSP1-mediated mRNA degradation mostly depends on codon usage bias. Thus, fast-translating mRNAs with optimal codon usage for human cells that preferentially recruit 43S-PIC by threading, showed a dramatic sensitivity to NSP1. On the contrary, slow-translating mRNAs with suboptimal codon usage and 5\u2019 UTR that enabled slotting on 43S-PIC were resistant to or even enhanced by NSP1. Translation of SARS-CoV-2 mRNAs escapes NSP1-mediated inhibition by a proper combination of suboptimal codon usage and slotting-prone 5\u2019 UTR that also confers efficient translation. Thus, the prevalence of non-optimal codons found in SARS-CoV-2 and other coronavirus genomes is favored by the distinctive effect that NSP1 plays on translation and mRNA stability.","version":"1.1","doi":"10.1101/2024.06.17.599344","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.19.590276","pub_date":"2024-6-17","title":"Evolving antibody response to SARS-CoV-2 antigenic shift from XBB to JN.1","abstract":"The continuous evolution of SARS-CoV-2, particularly the emergence of the BA.2.86/JN.1 lineage replacing XBB lineages, necessitates re-evaluation of current vaccine compositions. Here, we provide a comprehensive analysis of the humoral immune response to XBB and JN.1 human exposures, emphasizing the need for JN.1-lineage-based boosters. We demonstrate the antigenic distinctiveness of XBB and JN.1 lineages in SARS-CoV-2-naive individuals but not in those with prior vaccinations or infections, and JN.1 infection elicits superior plasma neutralization titers against its subvariants. We highlight the strong immune evasion and receptor binding capability of KP.3, supporting its foreseeable prevalence. Extensive analysis of the BCR repertoire, isolating \u223c2000 RBD-specific monoclonal antibodies (mAbs) with their targeting epitopes characterized by deep mutational scanning (DMS), underscores the systematic superiority of JN.1-elicited memory B cells (MBCs). Notably, Class 1 IGHV3-53/3-66-derived neutralizing antibodies (NAbs) contribute majorly within wildtype (WT)-reactive NAbs against JN.1. However, KP.2 and KP.3 evade a substantial subset of them, even those induced by JN.1, advocating for booster updates to KP.3 for optimized enrichment. JN.1-induced Omicron-specific antibodies also demonstrate high potency across all Omicron lineages. Escape hotspots of these NAbs have mainly been mutated in Omicron RBD, resulting in higher immune barrier to escape, considering the probable recovery of previously escaped NAbs. Additionally, the prevalence of broadly reactive IGHV3-53/3-66- encoding antibodies and MBCs, and their capability of competing with all Omicron-specific NAbs suggests their inhibitory role on the de novo activation of Omicron-specific naive B cells, potentially explaining the heavy immune imprinting in mRNA-vaccinated individuals. These findings delineate the evolving antibody response to Omicron antigenic shift from XBB to JN.1, and highlight the importance of developing JN.1 lineage, especially KP.3-based vaccine boosters, to enhance humoral immunity against current and future SARS-CoV-2 variants.","version":"1.3","doi":"10.1101/2024.04.19.590276","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.05.583494","pub_date":"2024-6-15","title":"Biological modifications of the immune response to COVID-19 vaccine in patients treated with rituximab and immune-checkpoint inhibitors","abstract":"Investigating the impact of immune-modulating therapies on mRNA vaccine efficacy transcends the immediate context of the COVID-19 pandemic. This study focuses on the differential immune responses to the third dose of COVID-19 mRNA vaccine among healthy volunteers, cancer patients treated with immune-checkpoint inhibitors (ICIs), and those treated with the anti-CD20 antibody rituximab. Utilizing RNA sequencing, serology, and interferon-\u03b3 release assessment, we charted the temporal dynamics of the immune response in such cohorts. Our findings indicate that ICIs maintain an immune profile similar to that of healthy individuals, whereas treatment with rituximab is associated with impairment of type I interferon response and the upregulation of transcripts pertaining to regulatory T cells, with a global dysfunction of both humoral and cellular immunity. This research deepens our understanding of the sophisticated interplay within the immune system in health and disease states, potentially informing therapeutic strategies across a spectrum of immunological conditions. Our study examines how cancer treatments that modify the immune system affect transcriptional, serological, and cellular responses to a model for repeated antigenic stimulation in humans, represented by the SARS-CoV-2 booster vaccine. Specifically, we investigated patients treated with rituximab (RTX), which impairs antibody production, and immune checkpoint inhibitors (ICI), which can trigger autoimmune disorders. We discovered that RTX-treated patients not only exhibit a reduced antibody response but actually show a diminished interferon-mediated immune response, indicating a broader immune disruption than anticipated. Conversely, ICI-treated patients responded to the vaccine similarly to healthy individuals, suggesting that fears of adverse vaccine reactions in these patients may be unfounded. This research highlights important considerations for the clinical management of cancer patients receiving these treatments.","version":"1.2","doi":"10.1101/2024.03.05.583494","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.14.599031","pub_date":"2024-6-14","title":"Profiling endogenous airway proteases and antiproteases and measuring proteolytic activation of Influenza HA using in vitro and ex vivo human airway surface liquid samples","abstract":"Imbalance of airway proteases and antiproteases has been implicated in diseases such as COPD and environmental exposures including cigarette smoke and ozone. To initiate infection, endogenous proteases are commandeered by respiratory viruses upon encountering the airway epithelium. The airway proteolytic environment likely contains redundant antiproteases and proteases with diverse catalytic mechanisms, however a proteomic profile of these enzymes and inhibitors in airway samples has not been reported. The objective of this study was to first profile extracellular proteases and antiproteases using human airway epithelial cell cultures and ex vivo nasal epithelial lining fluid (NELF) samples. Secondly, we present an optimized method for probing the proteolytic environment of airway surface liquid samples (in vitro and ex vivo) using fluorogenic peptides modeling the cleavage sites of respiratory viruses. We detected 48 proteases in the apical wash of cultured human nasal epithelial cells (HNECs) (n=6) and 57 in NELF (n=13) samples from healthy human subjects using mass-spectrometry based proteomics. Additionally, we detected 29 and 48 antiproteases in the HNEC apical washes and NELF, respectively. We observed large interindividual variability in rate of cleavage of an Influenza H1 peptide in the ex vivo clinical samples. Since protease and antiprotease levels have been found to be altered in the airways of smokers, we compared proteolytic cleavage in ex vivo nasal lavage samples from male/female smokers and non-smokers. There was a statistically significant increase in proteolysis of Influenza H1 in NLF from male smokers compared to female smokers. Furthermore, we measured cleavage of the S1/S2 site of SARS-CoV, SARS-CoV-2, and SARS-CoV-2 Delta peptides in various airway samples, suggesting the method could be used for other viruses of public health relevance. This assay presents a direct and efficient method of evaluating the proteolytic environment of human airway samples in assessment of therapeutic treatment, exposure, or underlying disease.","version":"1.1","doi":"10.1101/2024.06.14.599031","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.13.598952","pub_date":"2024-6-14","title":"Optimization of Soluble Expression of CTA1-(S14P5)4-DD and CTA1-(S21P2)4-DD Fusion Proteins as Candidates for COVID-19 Intranasal Vaccines","abstract":"Developing intranasal vaccines against pandemics and devastating airborne infectious diseases is imperative. The superiority of intranasal vaccines over injectable systemic vaccines is evident, but the challenge in developing effective intranasal vaccines is more substantial. Fusing a protein antigen with the catalytic domain of cholera toxin (CTA1) and the two-domain D of staphylococcal protein A (DD) has significant potential for intranasal vaccines. In the present study, we constructed two fusion proteins containing CTA1, tandem repeat linear epitopes of the SARS-CoV-2 spike protein (S14P5 or S21P2), and DD. The in silico characteristics and solubility of the fusion proteins CTA1-(S14P5)4-DD and CTA1-(S21P2)4-DD were analyzed when overexpressed in Escherichia coli. Structural predictions indicated that each component of the fusion proteins was compatible with its origin. Both fusion proteins were predicted by computational tools to be soluble when overexpressed in E. coli. Contrary to these predictions, the constructs exhibited limited solubility. The solubility did not improve even after lowering the cultivation temperature from 37\u00b0C to 18\u00b0C. Induction with IPTG at the early log phase, instead of the usual mid-log phase growth, significantly increased soluble CTA1-(S21P2)4-DD but not CTA1-(S14P5)4-DD. The solubility of overexpressed fusion proteins significantly increased when a non-denaturing detergent (Nonidet P40, Triton X100, or Tween 20) was added to the extraction buffer. In a scale-up purification experiment, the yields were low, only 1-2 mg/L of culture, due to substantial losses during the purification stages, indicating the need for further optimization of the purification process.","version":"1.1","doi":"10.1101/2024.06.13.598952","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.13.598902","pub_date":"2024-6-14","title":"Evolution of Omicron lineage towards increased fitness in the upper respiratory tract in the absence of severe lung pathology","abstract":"The emergence of the Omicron lineage represented a major genetic drift in SARS-CoV-2 evolution. This was associated with phenotypic changes including evasion of pre-existing immunity and decreased disease severity. Continuous evolution within the Omicron lineage raised concerns of potential increased transmissibility and/or disease severity. To address this, we evaluated the fitness and pathogenesis of contemporary Omicron variants XBB.1.5, XBB.1.16, EG.5.1, and JN.1 in the upper (URT) and lower respiratory tract (LRT). We compared in vivo infection in Syrian hamsters with infection in primary human nasal and lung epithelium cells and assessed differences in transmissibility, antigenicity, and innate immune activation. Omicron variants replicated efficiently in the URT but displayed limited pathology in the lungs compared to previous variants and failed to replicate in human lung organoids. JN.1 was attenuated in both URT and LRT compared to other Omicron variants and failed to transmit in the hamster model. Our data demonstrate that Omicron lineage evolution has favored increased fitness in the URT.","version":"1.1","doi":"10.1101/2024.06.13.598902","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.13.598798","pub_date":"2024-6-13","title":"Historical texts as a potential resource for plant-derived natural products against SARS-CoV-2 \u2013 the example of the Receptarium of Burkhard III von Hallwyl from 16th century Switzerland","abstract":"In the search for more effective prophylactic and possibly curative therapeutics against SARS-CoV-2, an historical-ethnobotanical approach was used to select plants described in the Receptarium of Burkhard III von Hallwyl (RBH), an influential recipe text from 16th century Switzerland. Ten species were identified based on specific historical uses presumably linked with the treatment of viral infections as well as inflammatory conditions. For each plant candidate, aqueous and hydroethanolic extracts have been produced. CellTiter-Glo\u00ae Luminescent Cell Viability Assay was used to assess antiviral activity against SARS-CoV-2 and the effect on cell viability of the extracts. Of the ten plant species tested, four displayed an antiviral activity \u2265 50% at 16.7 \u00b5g/ml with acceptable cell viability (> 75%): Sambucus nigra L. (leaves), Viola odorata L. (leaves), Geranium robertianum L. (arial parts) and Artemisia vulgaris L. (aerial parts). The crude extracts were partitioned in aqueous and organic fractions and further analyzed. The ethyl acetate fractions of S. nigra, V. odorata and G. robertianum expressed significant antiviral activity of nearly 100% at 5.6 \u00b5g/ml (P < 0.05). The most potent inhibitory activity was observed for the ethyl acetate fraction of Viola odorata L. (leaves) with 87% at 1.9 \u00b5g/ml (P < 0.0001). Alongside bioactivity analysis phytochemical fingerprints were made, with the aim to understand important substance classes contained. Further investigations are required to explore the active principles. Our study shows that an ethnopharmacological approach based on historical records of traditional use to select potential herbal candidates coupled with a rational screening process enables an efficient search for plant-derived natural products with antiviral activity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.06.13.598798","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.06.597720","pub_date":"2024-6-12","title":"Potent and broadly neutralizing antibodies against sarbecoviruses elicited by single ancestral SARS-CoV-2 infection","abstract":"Monoclonal antibody (mAb) therapeutics hold promise for both preventing and treating infectious diseases, especially among vulnerable populations. However, the emergence of various variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents challenges for current mAb treatments, emphasizing the need for more potent and broadly neutralizing antibodies. In this study, we employed an unbiased screening approach to discover broadly neutralizing antibodies and successfully isolated two mAbs from individuals with only exposure to ancestral SARS-CoV-2. One of these antibodies, CYFN1006-1, exhibited robust cross-neutralization against a spectrum of SARS-CoV-2 variants, including the latest JN.1 and KP.2 variants, with consistent IC50 values ranging from \u223c1 to 5 ng/mL. Notably, it also displayed broad neutralization activity against SARS-CoV and related sarbecoviruses, such as WIV1, SHC014, RaTG13, and GD-Pangolin. Structural analysis revealed that these mAbs target shared hotspot but mutation-resistant epitopes, with their Fabs locking the RBD in the \u201cdown\u201d conformation through interactions with adjacent Fabs and RBDs, and cross-linking Spike trimers into di-trimers to block viral infection. In vivo studies conducted in a JN.1-infected hamster model validated the protective efficacy of CYFN1006-1, emphasizing its therapeutic potential. These findings suggest that, through meticulous approaches, rare antibodies with cross-neutralization activities against SARS-CoV-2 and related sarbecoviruses can be identified from individuals with exclusively ancestral virus exposure.","version":"1.2","doi":"10.1101/2024.06.06.597720","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.31.596900","pub_date":"2024-6-12","title":"Induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors","abstract":"Viral accessory proteins play critical roles in viral escape form host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, ORF10), strongly activates inflammasome-dependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.","version":"1.2","doi":"10.1101/2024.05.31.596900","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.29.502014","pub_date":"2024-6-12","title":"Diverse Strategies Utilized by Coronaviruses to Evade Antiviral Responses and Suppress Pyroptosis","abstract":"Viral infection triggers inflammasome-mediated caspase-1 activation. Nevertheless, limited understanding exists regarding how viruses use the active caspase-1 to evade host immune response. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of coronaviruses (CoVs) to illustrate the intricate regulation of CoVs to combat IFN-I signaling and pyroptosis. Our findings demonstrate that PEDV infection stabilizes caspase-1 expression via papain-like protease PLP2\u2019s deubiquitinase activity and the enhanced stabilization of caspase-1 disrupts IFN-I signaling by cleaving RIG-I at D189 residue. 6-Thioguanine (6TG), a PLP2 inhibitor, plays a critical role in reversing the inhibitory effect of IFN-I via PLP2 and also inhibiting PEDV replication. Meanwhile, PLP2 can degrade GSDMD-p30 by removing its K27-linked ubiquitin chain at K275 to restrain pyroptosis. Papain-like proteases from other genera of CoVs (PDCoV and SARS-CoV-2) have the similar activity to degrade GSDMD-p30. We further demonstrate that SARS-CoV-2 N protein induced NLRP3 inflammasome activation also uses the active caspase-1 to counter IFN-I signaling by cleaving RIG-I. Therefore, our work unravels a novel antagonistic mechanism employed by CoVs to evade host antiviral response.","version":"1.3","doi":"10.1101/2022.07.29.502014","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.593942","pub_date":"2024-6-11","title":"SARS-CoV-2 Infection of Salivary Glands Compromises Oral Antifungal Innate Immunity and Predisposes to Oral Candidiasis","abstract":"Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most notable is histatin-5 which is exclusively produced in salivary glands with uniquely potent antifungal activity against the opportunistic pathogen Candida albicans. Recently, SARS-CoV-2 was shown to replicate in salivary gland acinar cells eliciting local immune cell activation. In this study, we performed mechanistic and clinical studies to investigate the implications of SARS-CoV-2 infection on salivary histatin-5 production and Candida colonization. Bulk RNA-sequencing of parotid salivary glands from COVID-19 autopsies demonstrated statistically significant decreased expression of histatin genes. In situ hybridization, coupled with immunofluorescence for co-localization of SARS-CoV-2 spike and histatin in salivary gland cells, showed that histatin was absent or minimally present in acinar cells with replicating viruses. To investigate the clinical implications of these findings, salivary histatin-5 levels and oral Candida burden in saliva samples from three independent cohorts of mild and severe COVID-19 patients and matched healthy controls were evaluated. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of C. albicans. Analysis of prospectively recovered samples indicated that the decrease in histatin-5 is likely reversible in mild-moderate disease as concentrations tended to increase during the post-acute phase. Importantly, salivary cytokine profiling demonstrated correlations between activation of the Th17 inflammatory pathway, changes in histatin-5 concentrations, and subsequent clearance of C. albicans in a heavily colonized subject. The importance of salivary histatin-5 in controlling the proliferation of C. albicans was demonstrated using an ex vivo assay where C. albicans was able to proliferate in COVID-19 saliva with low histatin-5, but not with high histatin-5. Taken together, the findings from this study provide direct evidence implicating SARS-CoV-2 infection of salivary glands with compromised oral innate immunity, and potential predisposition to oral candidiasis. Saliva contains antimicrobial peptides part of host innate immunity crucial for protection against colonizing microbial species. Most notable is the antifungal peptide histatin-5 produced in salivary glands cells. SARS-CoV-2 was shown to replicate in salivary gland cells causing tissue inflammation. In this study, we showed decreased expression of histatin genes in salivary glands from COVID-19 autopsies, and co-localization studies of SARS-CoV-2 spike and histatin revealed absence or minimal presence of histatin in acinar cells with replicating virus. To investigate the clinical implications of these findings, we conducted studies using saliva samples from subjects with mild to severe COVID-19, matched with healthy controls. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects with enhanced prevalence of C. albicans. Prospective analysis indicated the decrease in histatin-5 is reversible in mild-moderate disease, and salivary cytokine profiling demonstrated activation of the Th17 inflammatory pathway. The importance of salivary histatin-5 in controlling the proliferation of C. albicans was demonstrated using an ex vivo assay where C. albicans was able to proliferate in saliva with low histatin-5, but not with high histatin-5. Collectively, the findings provide direct evidence implicating SARS-CoV-2 infection of salivary glands with compromised oral innate immunity and predisposition to oral candidiasis.","version":"1.2","doi":"10.1101/2024.05.13.593942","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.10.598174","pub_date":"2024-6-11","title":"Kinetic landscape of single virus-like particles highlights the efficacy of SARS-Cov-2 internalization","abstract":"The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression, pH change, and nucleocapsid release. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into Rab5-negative endosomes via Dynamin-dependent scission. Less than two minutes later, the pH of VLPs drops below 5 followed by an increase in VLP speed, yet these two events are not interrelated. Nucleocapsid release from the VLPs occurs with similar kinetics to the pH drop, suggesting that VLP fusion occurs during endosome acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site altered the rate of VLP internalization events, indicating that they do not affect these processes. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells. Time-lapse videos of the studied internalization events can be accessed in the dedicated COVIDynamics database.","version":"1.1","doi":"10.1101/2024.06.10.598174","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.08.597602","pub_date":"2024-6-11","title":"Barcoded SARS-CoV-2 viruses define the impact of time and route of transmission on the transmission bottleneck in a Syrian hamster model","abstract":"The transmission bottleneck, defined as the number of viruses that transmit from one host to infect another, is an important determinant of the rate of virus evolution and the level of immunity required to protect against virus transmission. Despite its importance, SARS-CoV-2\u2019s transmission bottleneck remains poorly characterized, in part due to a lack of quantitative measurement tools. To address this, we adapted a SARS-CoV-2 reverse genetics system to generate a pool of >200 isogenic SARS-CoV-2 viruses harboring specific 6-nucleotide barcodes inserted in ORF10, a non-translated ORF. We directly inoculated donor Syrian hamsters intranasally with this barcoded virus pool and exposed a paired na\u00efve contact hamster to each donor. Following exposure, the nasal turbinates, trachea, and lungs were collected, viral titers were measured, and the number of barcodes in each tissue were enumerated to quantify the transmission bottleneck. The duration and route (airborne, direct contact, and fomite) of exposure were varied to assess their impact on the transmission bottleneck. In airborne-exposed hamsters, the transmission bottleneck increased with longer exposure durations. We found that direct contact exposure produced the largest transmission bottleneck (average 27 BCs), followed by airborne exposure (average 16 BCs) then fomite exposure (average 8 BCs). Interestingly, we detected unique BCs in both the upper and lower respiratory tract of contact animals from all routes of exposure, suggesting that SARS-CoV-2 can directly infect hamster lungs. Altogether, these findings highlight the utility of barcoded viruses as tools to rigorously study virus transmission. In the future, barcoded SARS-CoV-2 will strengthen studies of immune factors that influence virus transmission.","version":"1.1","doi":"10.1101/2024.06.08.597602","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.10.598324","pub_date":"2024-6-11","title":"Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 sublineage, including HK.3 and EG.5.1","abstract":"With the SARS-CoV-2 Omicron XBB.1.9 sublineage circulating worldwide, two XBB.1.9 variants, EG.5.1 and HK.3 spread rapidly and became dominant from middle 2023. However, the spike features, pathogenicity, and transmissibility of HK.3 are largely unknown. Here, we performed multiscale investigations to reveal the virological features of XBB.1.9 subvariants, especially the newly emerging HK.3. HK.3 revealed high replication efficiency in vitro. The HK.3 spike exhibited enhanced processing, although its infectivity, fusogenicity, and hACE2 binding affinity were comparable to those of the EG.5 and XBB.1 spikes. All XBB.1.9.1, EG.5.1 and HK.3 strains demonstrated efficient transmission in hamsters, although XBB.1.9.1 exhibited stronger fitness in the upper airways. HK.3 and EG.5.1 exhibited greater pathogenicity than XBB.1.9.1 and BA.2 in H11-K18-hACE2 hamsters. Our studies provide insight into the newly emerging pathogens HK.3 and EG.5.1. In animal models, the ongoing attenuated pathogenicity and poor transmission of Omicron subvariants seems to reach a consensus. However, our results revealed that Omicron XBB.1.9 subvariants, including one of the key variants of interest, EG.5 with its another key subvariant HK.3, universally exhibited both increased pathogenicity and highly transmission. This study reemphasized the importance of surveillance in characteristics of epidemic Omicron subvariants.","version":"1.1","doi":"10.1101/2024.06.10.598324","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.02.560570","pub_date":"2024-6-10","title":"SARS-CoV-2 neurotropism-induced anxiety/depression-like behaviors require Microglia activation","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with a wide range of \u201clong COVID\u201d neurological symptoms. However, the mechanisms governing SARS-CoV-2 neurotropism and its effects on long-term behavioral changes remain poorly understood. Using a highly virulent mouse-adapted SARS-CoV-2 strain, denoted as SARS2-N501YMA30, we demonstrated that intranasal inoculation of SARS2-N501YMA30 results in viral dissemination to multiple brain regions, including the amygdala and hippocampus. Behavioral assays indicated a marked elevation in anxiety- and depression-like behaviors post infection. A comparative analysis of RNA expression profiles disclosed alterations in the post-infected brains. Additionally, we observed dendritic spine remodeling on neurons within the amygdala after infection. Infection with SARS2-N501YMA30 was associated with microglial activation and a subsequent increase in microglia-dependent neuronal activity in the amygdala. Pharmacological inhibition of microglial activity subsequent to viral spike inoculation mitigates microglia-dependent neuronal hyperactivity. Transcriptomic analysis of infected brains revealed the upregulation of inflammatory and cytokine-related pathways, implicating microglia-driven neuroinflammation in the pathogenesis of neuronal hyperactivity and behavioral abnormality. Overall, these data provide critical insights into the neurological consequences of SARS-CoV-2 infection and underscore microglia as a potential therapeutic target for ameliorating virus-induced neurobehavioral abnormalities.","version":"1.2","doi":"10.1101/2023.10.02.560570","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.05.597664","pub_date":"2024-6-09","title":"Virological characteristics of the SARS-CoV-2 KP.3, LB.1 and KP.2.3 variants","abstract":"The SARS-CoV-2 JN.1 variant, arising from BA.2.86.1 with a substitution in the spike (S) protein, S:L455S, exhibited increased fitness and outcompeted the previously predominant XBB lineages by the beginning of 2024. Subsequently, JN.1 subvariants including KP.2 and KP.3, which convergently acquired S protein substitutions such as S:R346T, S:F456L, and S:Q493E, have emerged concurrently. Furthermore, JN.1 subvariants such as LB.1 and KP.2.3, which convergently acquired S:S31del in addition to the above substitutions, have emerged and spread as of June 2024. Here we investigated the virological properties of KP.3, LB.1 and KP.2.3. We estimated the relative effective reproduction number (Re) of KP.3, LB.1, and KP.2.3 using a Bayesian multinomial logistic model based on the genome surveillance data from Canada, the UK, and the USA, where these variants have spread from March to April 2024. The Re of KP.3 is more than 1.2-fold higher than that of JN.1 and higher than or comparable to that of KP.2 in these countries. Importantly, the Re values of LB.1 and KP.2.3 are even higher than those of KP.2 and KP.3. These results suggest that the three variants we investigated herein, particularly LB.1, and KP.2.3, will become major circulating variants worldwide in addition to KP.2 and KP.3. The pseudovirus infectivity of KP.2 and KP.3 was significantly lower than that of JN.1. On the other hand, the pseudovirus infectivity of LB.1 and KP.2.3 was comparable to that of JN.1. Neutralization assay was conducted by using four types of breakthrough infection (BTI) sera with XBB.1.5, EG.5, HK.3 and JN.1 infections as well as monovalent XBB.1.5 vaccine sera. In all four groups of BTI sera tested, the 50% neutralization titers (NT50) against LB.1 and KP.2.3 were significantly lower than those against JN.1 (2.2-3.3-fold and 2.0-2.9-fold) and even lower than those against KP.2 (1.6-1.9-fold and 1.4-1.7 fold). Although KP.3 exhibited neutralization resistance against all BTI sera tested than JN.1 (1.6-2.2-fold) with statistical significance, there were no significant differences between KP.3 and KP.2. In the case of infection-naive XBB.1.5 vaccine sera, the NT50 values of JN.1 subvariants were very low. In the case of XBB.1.5 vaccine sera after natural XBB infection, the NT50 values against KP.3, LB.1 and KP.2.3 were significantly lower than those of JN.1 (2.1-2.8-fold) and even lower than KP.2 after infection (1.4-2.0-fold). Overall, our results suggest that the S substitutions convergently acquired in the JN.1 subvariants contribute to immune evasion, and therefore, increase their Re when compared to parental JN.1. More importantly, LB.1 and KP.2.3 exhibited higher pseudovirus infectivity and more robust immune resistance than KP.2. These data suggest that S:S31del is critical to exhibit increased infectivity, increased immune evasion, and therefore, potentially contributes to increased Re.","version":"1.1","doi":"10.1101/2024.06.05.597664","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.04.597396","pub_date":"2024-6-08","title":"Water-Glycan Interactions Drive the SARS-CoV-2 Spike Dynamics: Insights into Glycan-Gate Control and Camouflage Mechanisms","abstract":"To develop therapeutic strategies against COVID-19, we introduce a high-resolution all-atom polarizable model capturing many-body effects of protein, glycans, solvent, and membrane components in SARS-CoV-2 spike protein open and closed states. Employing \u03bcs-long molecular dynamics simulations powered by high-performance cloud-computing and unsupervised density-driven adaptive sampling, we investigated the differences in bulk-solvent-glycan and protein-solvent-glycan interfaces between these states. We unraveled a sophisticated solvent-glycan polarization interaction network involving the N165/N343 residues that provide structural support for the open state and identified key water molecules that could potentially be targeted to destabilize this configuration. In the closed state, the reduced solvent polarization diminishes the overall N165/N343 dipoles, yet internal interactions and a reorganized sugar coat stabilize this state. Despite variations, our glycan-solvent accessibility analysis reveals the glycan shield capability to conserve constant interactions with the solvent, effectively camouflaging the virus from immune detection in both states. The presented insights advance our comprehension of viral pathogenesis at an atomic level, offering potential to combat COVID-19.","version":"1.2","doi":"10.1101/2024.06.04.597396","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.06.597774","pub_date":"2024-6-07","title":"A Nanobody Interaction with SARS-CoV-2 Spike Allows the Versatile Targeting of Lentivirus Vectors","abstract":"While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor binding domain to direct lentivirus infection of Spike-expressing cells. Using three different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. The targeting is dependent on the fusion function of Spike, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion, and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections. We have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein, and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion, and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.","version":"1.1","doi":"10.1101/2024.06.06.597774","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.13.580053","pub_date":"2024-6-06","title":"Emergence and spread of SARS-CoV-2 variants from farmed mink to humans and back during the epidemic in Denmark, June-November 2020","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) not only caused the COVID-19 pandemic but also had a major impact on farmed mink production in several European countries. In Denmark, the entire population of farmed mink (over 15 million animals) was culled in late 2020. During the period of June to November 2020, mink on 290 farms (out of about 1100 in the country) were shown to be infected with SARS-CoV-2. Genome sequencing identified changes in the virus within the mink and it is estimated that about 4000 people in Denmark became infected with these mink virus variants. However, the routes of transmission of the virus to, and from, the mink have been unclear. Phylogenetic analysis revealed the generation of multiple clusters of the virus within the mink. Detailed analysis of changes in the virus during replication in mink and, in parallel, in the human population in Denmark, during the same time period, has been performed here. The majority of cases in mink involved variants with the Y435F substitution and the H69/V70 deletion within the Spike (S) protein; these changes emerged early in the outbreak. However, further introductions of the virus, by variants lacking these changes, from the human population into mink also occurred. Based on phylogenetic analysis of viral genome data, we estimate, using a conservative approach, that about 17 separate examples of mink to human transmission occurred in Denmark but up to 59 such events (90% credible interval: (39-77)) were identified using parsimony to count cross-species jumps on transmission trees inferred using Bayesian methods. Using the latter approach, 136 jumps (90% credible interval: (117-164)) from humans to mink were found, which may underlie the farm-to-farm spread. Thus, transmission of SARS-CoV-2 from humans to mink, mink to mink, from mink to humans and between humans were all observed. (298 words) In addition to causing a pandemic in the human population, SARS-CoV-2 also infected farmed mink. In Denmark, after the first identification of infection in mink during June 2020, a decision was made in November 2020 to cull all the farmed mink. Within this outbreak, mink on 290 farms (out of about 1100 in the country) were found to have been infected. We showed, by analysis of the viruses from the mink, that the viruses on the farms were mainly of three different, but closely related, types (termed Clusters 2, 3 and 4) that shared certain distinctive features. Thus, we found that many outbreaks in mink resulted from transmission of the virus between mink farms. However, we identified that new introductions of other virus variants, presumably from infected humans, also occurred. Furthermore, we showed that spread of the virus from infected mink to humans also happened on multiple occasions. Thus, transmission of these viruses from humans to mink, mink to mink, from mink to humans and between humans were all observed. (172 words)","version":"1.3","doi":"10.1101/2024.02.13.580053","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.05.597541","pub_date":"2024-6-05","title":"Engineering a SARS-CoV-2 vaccine targeting the RBD cryptic-face via immunofocusing","abstract":"The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the main target of neutralizing antibodies. Although they are infrequently elicited during infection or vaccination, antibodies that bind to the conformation-specific cryptic face of the RBD display remarkable breadth of binding and neutralization across Sarbecoviruses. Here, we employed the immunofocusing technique PMD (protect, modify, deprotect) to create RBD immunogens (PMD-RBD) specifically designed to focus the antibody response towards the cryptic-face epitope recognized by the broadly neutralizing antibody S2X259. Immunization with PMD-RBD antigens induced robust binding titers and broad neutralizing activity against homologous and heterologous Sarbecovirus strains. A serum-depletion assay provided direct evidence that PMD successfully skewed the polyclonal antibody response towards the cryptic face of the RBD. Our work demonstrates the ability of PMD to overcome immunodominance and refocus humoral immunity, with implications for the development of broader and more resilient vaccines against current and emerging viruses with pandemic potential. Using the immunofocusing technique protect, modify, deprotect (PMD) we engineer SARS-CoV-2 receptor-binding domain (RBD) immunogens that generate broadly neutralizing antibodies against the conserved cryptic face. The ability to refocus humoral immunity to broadly conserved epitopes has important implications for the design of universal vaccines.","version":"1.1","doi":"10.1101/2024.06.05.597541","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.03.596876","pub_date":"2024-6-04","title":"Tracking the genetic diversity of SARS-CoV-2 variants in Nicaragua throughout the COVID-19 Pandemic","abstract":"The global circulation of SARS-CoV-2 has been extensively documented, yet the dynamics within Central America, particularly Nicaragua, remain underexplored. This study characterizes the genomic diversity of SARS-CoV-2 in Nicaragua from March 2020 through December 2022, utilizing 1064 genomes obtained via next-generation sequencing. These sequences were selected nationwide and analyzed for variant classification, lineage predominance, and phylogenetic diversity. We employed both Illumina and Oxford Nanopore Technologies for all sequencing procedures. Results indicated a temporal and spatial shift in dominant lineages, initially from B.1 and A.2 in early 2020 to various Omicron subvariants towards the study\u2019s end. Significant lineage shifts correlated with changes in COVID-19 positivity rates, underscoring the epidemiological impact of variant dissemination. The comparative analysis with regional data underscored the low diversity of circulating lineages in Nicaragua and their delayed introduction compared to other countries in the Central American region. The study also linked specific viral mutations with hospitalization rates, emphasizing the clinical relevance of genomic surveillance. This research advances the understanding of SARS-CoV-2 evolution in Nicaragua and provide valuable information regarding its genetic diversity for public health officials in Central America. We highlight the critical role of ongoing genomic surveillance in identifying emergent lineages and informing public health strategies.","version":"1.1","doi":"10.1101/2024.06.03.596876","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.31.596896","pub_date":"2024-6-03","title":"Respiratory mucosal administration of DNA aptamer nanomaterials protects against antigenically diverse SARS-CoV-2 variants","abstract":"The ongoing COVID-19 pandemic has highlighted the need for innovative therapeutic strategies to combat rapidly evolving pathogens that challenge the efficacy of traditional vaccines and monoclonal antibody treatments. Here, we explored the potential of TMSA52, a previously described homotrimeric DNA aptamer as a universal prophylactic and therapeutic agent against SARS-CoV-2. TMSA52 demonstrates exceptional binding affinities and broad neutralization against diverse SARS-CoV-2 variant spike proteins that are further enhanced through multimerization onto lamellar iridium nanoplates. Respiratory mucosal delivery of TMSA52 nanomaterials was well-tolerated. Surprisingly, TMSA52 offered potent protection from infection with ancestral SARS-CoV-2 on-par with monoclonal antibodies, and superior protection against antigenically distant SARS-CoV-2 variants. These findings establish DNA aptamers as a promising, cost-effective, and scalable alternative to traditional monoclonal antibody therapies. This study underscores the potential of aptamer-based platforms as a next-generation strategy to enhance global pandemic preparedness and expand our arsenal of infectious disease countermeasures.","version":"1.1","doi":"10.1101/2024.05.31.596896","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.29.596362","pub_date":"2024-6-03","title":"Recurrent SARS-CoV-2 spike mutations confer growth advantages to select JN.1 sublineages","abstract":"The recently dominant SARS-CoV-2 Omicron JN.1 has evolved into multiple sublineages, with recurrent spike mutations R346T, F456L, and T572I, some of which exhibit growth advantages, such as KP.2. We investigated these mutations in JN.1, examining their individual and combined effects on immune evasion, ACE2 receptor affinity, and in vitro infectivity. F456L increased resistance to neutralization by human sera, including those after JN.1 breakthrough infections, and by RBD class-1 monoclonal antibodies, significantly altering JN.1 antigenicity. R346T enhanced ACE2-binding affinity, without a discernible effect on serum neutralization. Individually, R346T and T572I modestly enhanced infectivity of each pseudovirus. Importantly, expanding sublineages such as KP.2 containing R346T, F456L, and V1104L, showed similar neutralization resistance as JN.1 with R346T and F456L, suggesting V1104L does not appreciably affect antibody evasion. Our findings illustrate how certain JN.1 mutations confer growth advantages in the population and could inform the design of the next COVID-19 vaccine booster.","version":"1.2","doi":"10.1101/2024.05.29.596362","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.31.596892","pub_date":"2024-6-03","title":"A predominately pulmonary activation of complement in a mouse model of severe COVID-19","abstract":"Evidence from in vitro studies and observational human disease data suggest the complement system plays a significant role in SARS-CoV-2 pathogenesis, although how complement dysregulation develops in patients with severe COVID-19 is unknown. Here, using a mouse-adapted SARS-CoV-2 virus (SARS2-N501YMA30) and a mouse model of severe COVID-19, we identify significant serologic and pulmonary complement activation following infection. We observed C3 activation in airway and alveolar epithelia, and in pulmonary vascular endothelia. Our evidence suggests that while the alternative pathway is the primary route of complement activation, components of both the alternative and classical pathways are produced locally by respiratory epithelial cells following infection, and increased in primary cultures of human airway epithelia in response to cytokine exposure. This locally generated complement response appears to precede and subsequently drive lung injury and inflammation. Results from this mouse model recapitulate findings in humans, which suggest sex-specific variance in complement activation, with predilection for increased C3 activity in males, a finding that may correlate with more severe disease. Our findings indicate that complement activation is a defining feature of severe COVID-19 in mice and lay the foundation for further investigation into the role of complement in COVID-19.","version":"1.1","doi":"10.1101/2024.05.31.596892","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.02.596989","pub_date":"2024-6-03","title":"Persistent Neurological Deficits in Mouse PASC Reveal Antiviral Drug Limitations","abstract":"Post-Acute Sequelae of COVID-19 (PASC) encompasses persistent neurological symptoms, including olfactory and autonomic dysfunction. Here, we report chronic neurological dysfunction in mice infected with a virulent mouse-adapted SARS-CoV-2 that does not infect the brain. Long after recovery from nasal infection, we observed loss of tyrosine hydroxylase (TH) expression in olfactory bulb glomeruli and neurotransmitter levels in the substantia nigra (SN) persisted. Vulnerability of dopaminergic neurons in these brain areas was accompanied by increased levels of proinflammatory cytokines and neurobehavioral changes. RNAseq analysis unveiled persistent microglia activation, as found in human neurodegenerative diseases. Early treatment with antivirals (nirmatrelvir and molnupiravir) reduced virus titers and lung inflammation but failed to prevent neurological abnormalities, as observed in patients. Together these results show that chronic deficiencies in neuronal function in SARS-CoV-2-infected mice are not directly linked to ongoing olfactory epithelium dysfunction. Rather, they bear similarity with neurodegenerative disease, the vulnerability of which is exacerbated by chronic inflammation.","version":"1.1","doi":"10.1101/2024.06.02.596989","journal":"bioRxiv","score":null},{"id":"10.1101/2024.06.02.597066","pub_date":"2024-6-03","title":"Urban wastewater contains a functional human antibody repertoire of mucosal origin","abstract":"Wastewater-based surveillance of human disease offers timely insights to public health, helping to mitigate infectious disease outbreaks and decrease downstream morbidity and mortality. These systems rely on nucleic acid amplification tests for monitoring disease trends, while antibody-based seroprevalence surveys gauge community immunity. However, serological surveys are resource-intensive and subject to potentially long lead times and sampling bias. We identified and characterized a human antibody repertoire, predominantly secretory IgA, isolated from a central wastewater treatment plant and building-scale wastewater collection points. These antibodies partition to the solids fraction and retain immunoaffinity for SARS-CoV-2 and Influenza A virus antigens. This stable pool could enable real-time tracking of correlates of vaccination, infection, and immunity, aiding in establishing population-level thresholds for immune protection and assessing the efficacy of future vaccine campaigns, particularly those that are designed to induce humoral mucosal immunity.","version":"1.1","doi":"10.1101/2024.06.02.597066","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.30.596396","pub_date":"2024-5-31","title":"Characterizing a Lethal CAG-ACE2 Transgenic Mouse Model for SARS-CoV-2 infection with Using Cas9-Enhanced Nanopore Sequencing","abstract":"The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the primary animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5 kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed for ACE2 expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressed ACE2 in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1-1.0 TCID50). Infected mice from the two lines began to show COVID-19 symptoms three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied Nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against the ACE2 transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.","version":"1.1","doi":"10.1101/2024.05.30.596396","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.31.596818","pub_date":"2024-5-31","title":"Variable rates of SARS-CoV-2 evolution in chronic infections","abstract":"An important feature of the evolution of the SARS-CoV-2 virus has been the emergence of highly mutated novel variants, which are characterised by the gain of multiple mutations relative to viruses circulating in the general global population. Cases of chronic viral infection have been suggested as an explanation for this phenomenon, whereby an extended period of infection, with an increased rate of evolution, creates viruses with substantial genetic novelty. However, measuring a rate of evolution during chronic infection is made more difficult by the potential existence of compartmentalisation in the viral population, whereby the viruses in a host form distinct subpopulations. We here describe and apply a novel statistical method to study within-host virus evolution, identifying the minimum number of subpopulations required to explain sequence data observed from cases of chronic infection, and inferring rates for within-host viral evolution. Across nine cases of chronic SARS-CoV-2 infection in hospitalised patients we find that non-trivial population structure is relatively common, with four cases showing evidence of more than one viral population evolving independently within the host. We find cases of within-host evolution proceeding significantly faster, and significantly slower, than that of the global SARS-CoV-2 population, and of cases in which viral subpopulations in the same host have statistically distinguishable rates of evolution. Non-trivial population structure was associated with high rates of within-host evolution that were systematically underestimated by a more standard inference method.","version":"1.1","doi":"10.1101/2024.05.31.596818","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.31.596857","pub_date":"2024-5-31","title":"Disruption of Immune Responses By Type I Diabetes Exacerbates SARS-CoV-2 Mediated Lung Injury","abstract":"COVID-19 commonly presents as pneumonia, with those most severely affected progressing to respiratory failure. Patient responses to SARS-CoV-2 infection are varied, with comorbidities acting as major contributors to varied outcomes. Focusing on one such major comorbidity, we assessed whether pharmacological induction of Type I Diabetes Mellitus (T1DM) would increase the severity of lung injury in a murine model of COVID-19 pneumonia utilizing wild type mice infected with mouse-adapted SARS-CoV-2. Hyperglycemic mice exhibited increased weight loss and reduced blood oxygen saturation in comparison to their euglycemic counterparts, suggesting that these animals indeed experienced more severe lung injury. Transcriptomic analysis revealed a significant impairment of the adaptive immune response in the lungs of diabetic mice compared to those of control. In order to expand the limited options available for tissue analysis due to biosafety restrictions, we also employed a novel technique to digest highly fixed tissue into a single cell suspension, which allowed for flow cytometric analysis as well as single cell RNA sequencing. Flow immunophenotyping and scRNA-Seq confirmed impaired recruitment of T cells into the lungs of T1DM animals. Additionally, scRNA-Seq revealed a distinct, highly inflammatory macrophage profile in the diabetic cohort that correlates with the more severe infection these mice experienced clinically, allowing insight into a possible mechanism for this phenomenon. Recognizing the near certainty that respiratory viruses will continue to present significant public health concerns for the foreseeable future, our study provides key insights into how T1DM results in a much more severe infection and identifies possible targets to ameliorate comorbidity-associated severe disease. We define the exacerbating effects of Type I Diabetes Mellitus (T1DM) on COVID-19 pneumonia severity in mice. Hyperglycemic mice experienced increased weight loss and reduced oxygen saturation. Transcriptomic analysis revealed impaired immune responses in diabetic mice, while flow cytometry and single-cell RNA sequencing confirmed reduced T cell recruitment and an inflammatory macrophage profile. Additionally, we introduced a novel technique for tissue analysis, enabling flow cytometric analysis and single-cell RNA sequencing on highly fixed tissue samples.","version":"1.1","doi":"10.1101/2024.05.31.596857","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.30.596590","pub_date":"2024-5-30","title":"Transfer of IgG from Long COVID patients induces symptomology in mice","abstract":"SARS-CoV-2 infections worldwide led to a surge in cases of Long COVID, a post-infectious syndrome. It has been hypothesized that autoantibodies play a crucial role in the development of Long COVID and other syndromes, such as fibromyalgia and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In this study, we tested this hypothesis by passively transferring total IgG from Long COVID patients to mice. Using Glial Fibrillary Acidic Protein (GFAP) and type-I interferon expression, we stratified patients into three Long COVID subgroups, each with unique plasma proteome signatures. Remarkably, IgG transfer from the two subgroups, which are characterized by higher plasma levels of neuronal proteins and leukocyte activation markers, induced pronounced and persistent sensory hypersensitivity with distinct kinetics. Conversely, IgG transfer from the third subgroup, which are characterized by enriched skeletal and cardiac muscle proteome profiles, reduced locomotor activity in mice without affecting their motor coordination. These findings demonstrate that transfer of IgG from Long COVID patients to mice replicates disease symptoms, underscoring IgG\u2019s causative role in Long COVID pathogenesis. This work proposes a murine model that mirrors Long COVID\u2019s pathophysiological mechanisms, which may be used as a tool for screening and developing targeted therapeutics.","version":"1.1","doi":"10.1101/2024.05.30.596590","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.29.596469","pub_date":"2024-5-29","title":"Dissecting the ocular impact of SARS-CoV-2 variants: Analysis of eye globe and retina in animal models","abstract":"Neurological and ocular manifestations were reported in COVID-19 patients and in SARS-CoV-2 infected animal models. However, the effects of SARS-CoV-2 variants of concern (VoC) on the eyes and the retina remain unclear. Here, we investigate the cellular and molecular consequences of SARS-CoV-2 VoC infection on the eye and retina in mice and hamsters. Infection with the ancestral SARS-CoV-2 and the Gamma VoC induced a subtle increase in the eye volume of K18-hAce2 mice, but no morphological alteration was observed in hamsters\u2019 eyes. Evaluation of the ocular tropism revealed that distinct SARS-CoV-2 VoC reached the eye globe, but not the retina of K18-hAce2 mice. In contrast, SARS-CoV-2 variants are detected in the hamsters\u2019 retina during both acute infection and after disease recovery. Despite the presence of viral RNA, no inflammation was observed in the hamster retina, as evidenced by unchanged microglial cell density and unaltered gene expression of several immune mediators. Altogether, these findings indicate a limited impact of SARS-CoV-2 variants on the eye and retina.","version":"1.1","doi":"10.1101/2024.05.29.596469","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.29.596308","pub_date":"2024-5-29","title":"Impact of variants and vaccination on nasal immunity across three waves of SARS-CoV-2","abstract":"SARS-CoV-2 infection and COVID-19 disease vary with respect to viral variant and host vaccination status. However, how vaccines, emergent variants, and their intersection shift host responses in the human nasal mucosa remains uncharacterized. We and others have shown during the first SARS-CoV-2 wave that a muted nasal epithelial interferon response at the site of infection underlies severe COVID-19. We sought to further understand how upper airway cell subsets and states associate with COVID-19 phenotypes across viral variants and vaccination. Here, we integrated new single-cell RNA-sequencing (scRNA-seq) data from nasopharyngeal swabs collected from 67 adult participants during the Delta and Omicron waves with data from 45 participants collected during the original (Ancestral) wave in our prior study. By characterizing detailed cellular states during infection, we identified changes in epithelial and immune cells that are both unique and shared across variants and vaccination status. By defining SARS-CoV-2 RNA+ cells for each variant, we found that Delta samples had a marked increase in the abundance of viral RNA+ cells. Despite this dramatic increase in viral RNA+ cells in Delta cases, the nasal cellular compositions of Delta and Omicron exhibit greater similarity, driven partly by myeloid subsets, than the Ancestral landscapes associated with specialized epithelial subsets. We found that vaccination prior to infection was surprisingly associated with nasal macrophage recruitment and activation rather than adaptive immune cell signatures. While patients with severe disease caused by Ancestral or Delta variants had muted interferon responses, Omicron-infected patients had equivalent interferon responses regardless of disease severity. Our study defines the evolution of cellular targets and signatures of disease severity in the upper respiratory tract across SARS-CoV-2 variants, and suggests that intramuscular vaccines shape myeloid responses in the nasal mucosa upon SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.05.29.596308","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.28.24308095","pub_date":"2024-05-29","title":"SARS-CoV-2 correlates of protection from infection against variants of concern","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Serum neutralizing antibodies (nAbs) induced by vaccination have been linked to protection against symptomatic COVID-19 and severe disease. However, much less is known about the efficacy of nAbs in preventing the acquisition of infection, especially in the context of natural immunity and against SARS-CoV-2 immune-escape variants. In this study, we conducted mediation analysis to assess serum nAbs induced by prior SARS-CoV-2 infections as potential correlates of protection (CoPs) against Delta and Omicron BA.1/2 wave infections, in rural and urban household cohorts in South Africa. We find that, in the Delta wave, anti-D614G nAbs mediate 37% (95%CI 34% \u2013 40%) of the total protection against infection conferred by prior exposure to SARS-CoV-2, and that protection decreases with waning immunity. In contrast, anti-Omicron BA.1 nAbs mediate 11% (95%CI 9 \u2013 12%) of the total protection against Omicron BA.1/2 wave infections, due to Omicron\u2019s neutralization escape. These findings underscore that CoPs mediated through nAbs are variant-specific, and that boosting of nAbs against circulating variants might restore or confer immune protection lost due to nAb waning and/or immune escape. However, the majority of immune protection against SARS-CoV-2 conferred by natural infection cannot be fully explained by serum nAbs alone. Measuring these and other immune markers including T-cell responses, both in the serum and in other compartments such as the nasal mucosa, may be required to comprehensively understand and predict immune protection against SARS-CoV-2.</jats:p>","version":null,"doi":"10.1101/2024.05.28.24308095","journal":"medRxiv","score":null},{"id":"10.1101/2024.05.29.596393","pub_date":"2024-5-29","title":"The effects of iron deficient and high iron diets on SARS-CoV-2 lung infection and disease","abstract":"The severity of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often dictated by a range of comorbidities. A considerable literature suggests iron deficiency and iron overload may contribute to increased infection, inflammation and disease severity, although direct causal relationships have been difficult to establish. Here we generate iron deficient and iron loaded C57BL/6J mice by feeding low and high iron diets, with mice on a normal iron diet representing controls. All mice were infected with a primary omicron XXB SARS-CoV-2 isolate and lung inflammatory responses were analyzed by histology, immunohistochemistry and RNA-Seq. Compared with controls, iron deficient mice showed no significant changes in lung viral loads or histopathology, whereas, iron loaded mice showed slightly, but significantly, reduced lung viral loads and histopathology. Transcriptional changes were modest, but illustrated widespread dysregulation of inflammation signatures for both iron deficient vs. controls, and iron loaded vs. controls. Some of these changes could be associated with detrimental outcomes, whereas others would be viewed as beneficial. Diet-associated iron deficiency or overload thus induced modest modulations of inflammatory signatures, but no significant histopathologically detectable disease exacerbations. A diet deficient in iron can lead to anemia, a widespread problem worldwide. A diet with excessive iron is less common, but can be associated with excessive consumption of iron supplements. We investigate herein using a mouse model, whether low or high iron diets predispose to detrimental outcomes in the lungs after infection with SARS-CoV-2. A considerable literature suggests iron dysregulation would promote infection and inflammation. However, we found, although inflammatory responses showed modest modulations, viral loads were unaffected or slightly reduced, and lung histopathology was either unaffected or indicated slightly less severe disease. These findings do not support a view that low or high iron diets represent comorbidities predisposing to overt detrimental outcomes for acute COVID-19 lung disease.","version":"1.1","doi":"10.1101/2024.05.29.596393","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.28.596157","pub_date":"2024-5-29","title":"mRNA Vaccine-Induced SARS-CoV-2 Spike-Specific IFN-\u03b3 and IL-2 T-cell Responses are Predictive of Serological Neutralization and are Transiently Enhanced by Pre-Existing Cross-Reactive Immunity","abstract":"The contributions of SARS-CoV-2-specific T-cells to vaccine efficacy and durability are unclear. We investigated the relationships between mRNA vaccine-induced spike-specific IFN-\u03b3 and IL-2 T-cell responses, anti-spike/RBD IgG/IgA antibodies, and live virus neutralizing capacity in long-term-care-home staff doubly vaccinated with BNT162b2 or mRNA-1273. The impacts of pre-existing cross-reactive T-cell immunity to SARS-CoV-2 on cellular and humoral responses to vaccination were additionally assessed. Mathematical modelling of the kinetics of spike-specific IFN-\u03b3 and IL-2 T-cell responses over 6-months post-second dose was bifurcated into recipients who exhibited gradual increases (54% and 42%, respectively) with doubling times of 173 days, or decreases (46% and 58%, respectively) with half-lives of 115 days. Differences in kinetics did not correlate with any clinical phenotypes, although increases were proposed to be due to subclinical viral exposures. Serological anti-spike/RBD IgG/IgA antibody levels had otherwise decayed in all participants with half-lives of 55, 53, 76, and 59 days, respectively. Spike-specific T-cell responses induced at 2-6 weeks correlated with live viral neutralization at 6-months post-second dose, especially in hybrid immune individuals. Participants with pre-existing cross-reactive T-cell immunity to SARS-CoV-2 exhibited greater spike-specific T-cell responses, reduced anti-RBD IgA antibody levels, and a trending increase in neutralization at 2-6 weeks post-second dose. Non-spike-specific T-cells predominantly targeted SARS-CoV-2 non-structural protein at 6-months post-second dose in cross-reactive participants. mRNA vaccination was lastly shown to induce off-target T-cell responses against unrelated antigens. In summary, vaccine-induced spike-specific T-cell immunity appeared to influence serological neutralizing capacity, with only a modest effect induced by pre-existing cross-reactivity. Our findings provide important insights on the potential contributions of mRNA vaccine-induced spike-specific T-cell responses to the durability of neutralizing antibody levels in both uninfected and hybrid immune recipients. Our study additionally sheds light on the precise impacts of pre-existing cross-reactive T-cell immunity to SARS-CoV-2 on the magnitude and kinetics of cellular and humoral responses to vaccination. This study should ultimately inform the development of novel pan-coronavirus vaccines and vaccine regimens that can maximize the durability and breadth of protection against both current and future human coronaviruses of concern.","version":"1.1","doi":"10.1101/2024.05.28.596157","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.28.596354","pub_date":"2024-5-29","title":"Differential prevalence and risk factors for infection with coronaviruses in bats collected during 2020 in Yunnan Province, China","abstract":"Coronaviruses (CoVs) pose a threat to human health globally, as highlighted by severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and the COVID-19 pandemic. Bats from the Greater Mekong Subregion (GMS) are an important natural reservoir for CoVs. Here we report the differential prevalence of CoVs in bats across biological and ecological factors within Yunnan Province. We also show the coexistence of CoVs in individual bats and identify an additional putative host for SARS-related CoV, with higher dispersal capacity than other known hosts. Notably, 11 SARS-related coronaviruses (SARSr-CoVs) were discovered in horseshoe bats and a Chinese water myotis bat by pan-CoV detection and Illumina sequencing. Our findings facilitate an understanding of the fundamental features of the distribution and circulation of CoVs in nature as well as zoonotic spillover risk in the One health framework.","version":"1.1","doi":"10.1101/2024.05.28.596354","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.28.596359","pub_date":"2024-5-29","title":"The Impact of Preprints on COVID-19 Research Dissemination: A Quantitative Analysis of Journal Publications","abstract":"Preprints have played an unprecedented role in disseminating COVID-19-related science results to the public. The study aims to elucidate the role of preprints during the COVID-19 public health emergency (2020-2023) through a quantitative analysis of journal papers. Among the 247,854 COVID-19-related papers published in PubMed, 12,152 were initially released as preprints and were eventually published in 1,380 journals. This number is more than five times the 246 journals to which submissions can be made directly from bioRxiv through the B2J program. Journals with higher impact factors and Normalized Eigenfactor scores tend to publish a larger number of preprint-derived articles. The proportion of preprints among PubMed papers was 0.049, but this varies significantly by journal. In the top 30 journals, most exceed this proportion, indicating that these journals are preferred by authors for submitting their work. These findings highlight the growing acceptance and impact of preprints in the scientific community, particularly in high-impact journals.","version":"1.1","doi":"10.1101/2024.05.28.596359","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.25.595913","pub_date":"2024-5-27","title":"The hyperphosphorylation of SARS-CoV-2\u2019s nucleocapsid protein by GSK-3 involves a complex and redundant priming mechanism","abstract":"Upon entry into host cells, the nucleocapsid (N) protein of coronavirus becomes heavily phosphorylated within its central Ser-Arg domain by glycogen synthase kinase-3 (GSK-3). Most substrates for GSK-3 need to be phosphorylated, or primed, by another Ser/Thr kinase four residues downstream of the GSK-3 phosphorylation site. It was widely assumed that the hyperphosphorylation of SARS-CoV-2\u2019s N protein requires independent priming at Ser-188 and Ser-206, which initiates sequential phosphorylation by GSK-3. Here we present biochemical and mass spectrometry data that contradicts this simple model, revealing instead a much more complex and highly cooperative mechanism where redundant priming, as well as exosite docking, enables N protein\u2019s efficient phosphorylation by GSK-3. The R203M and R203K/G204R mutations, found in the recent delta and omicron variants of concern, both hinder N hyperphosphorylation by GSK-3. The mechanistic insights generated in this study suggest a novel approach to treat COVID-19 by combining multiple classes of pharmacological agents to inhibit N hyperphosphorylation.","version":"1.1","doi":"10.1101/2024.05.25.595913","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.24.595851","pub_date":"2024-5-27","title":"Cell-free synthesis and purification of recombinant nucleocapsid (N), membrane (M), and envelope (E) proteins","abstract":"Rapid production of soluble recombinant antigens is important for developing in vitro diagnostics, vaccines, and drugs against virus such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this research, hard-to-express nucleocapsid, membrane, and envelope proteins were successfully expressed by an Escherichia coli-based cell-free protein synthesis system. The solubility of the proteins was optimized using various amphipathic molecules. Most of the impurities were easily removed by a one-step Ni-NTA affinity chromatography. This study provides an easy and quick alternative for virus\u2019s trans-membrane and nucleotides associated recombinant protein expression, which has potential downstream application for early screening of newly emerging viruses.","version":"1.1","doi":"10.1101/2024.05.24.595851","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.24.595657","pub_date":"2024-5-24","title":"Immunogenicity and protective efficacy of an influenza virus-like particle-based SARS-CoV-2 hybrid vaccine candidate in rhesus macaques","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus co-infections present a heightened COVID-19 disease and hospitalization cases. Here, we studied the immunogenicity and efficacy of an influenza-A/PR8 virus-like particle (FluVLP)-based hybrid vaccine candidate displaying GPI-anchored SARS-CoV-2 receptor binding domain fused to GM-CSF and GPI-anchored interleukin-12 (FluVLP-RBD) in rhesus macaques. Animals (n=4/group) received two doses of either FluVLP or FluVLP-RBD vaccine four weeks apart and were challenged with SARS-CoV-2 (WA1/2020) infection via intranasal and intratracheal routes. We determined vaccine-induced IgG and neutralizing antibody titers in serum and their association with viral replication in the lower and upper airways (lung, throat, and nose) and lung-associated pathologies. FluVLP-RBD vaccine induced a strong binding IgG in serum against multiple SARS-CoV-2 variants (WA1/2020, Delta and Omicron; BA.1). Both vaccines induced strong influenza A/PR8-specific IgG. Following the SARS-CoV-2 challenge, all four animals in the FluVLP-RBD group showed a profound control of virus replication in all three airway compartments as early as day 2 through day 10 (day of euthanasia). This level of viral control was not observed in the FluVLP group as 2-3 animals exhibited high virus replication in all three airway compartments. The protection in the FluVLP-RBD vaccinated group correlated positively with post challenge neutralizing antibody titer. These results demonstrated that a FluVLP-based hybrid SARS-CoV-2 vaccine induces strong antibody responses against influenza-A/PR8 and multiple SARS-CoV-2 RBD variants and protects from SARS-CoV-2 replication in multiple compartments in macaques. These findings provide important insights for developing multivalent vaccine strategies for respiratory viruses. Co-infection with multiple respiratory viruses poses a greater risk than individual infections, especially for individuals with underlying health conditions. Studies in humans consistently demonstrated that simultaneous infection with SARS-CoV-2 and influenza leads to more severe respiratory illness and an increased rate of hospitalization. Therefore, developing hybrid vaccines targeting multiple respiratory viruses is of high importance. The hybrid vaccines also help to reduce the economic and logistic burden associated with vaccine coverage, distribution and storage. Here, we evaluate the immunogenicity and effectiveness of a novel hybrid flu-SARS-CoV-2 vaccine candidate using a nonhuman primate pre-clinical model. Our findings reveal that this vaccine elicits a strong immune response against influenza and SARS-CoV-2 viruses. Importantly, it provides strong protection against SARS-CoV-2 infection and associated pathological conditions.","version":"1.1","doi":"10.1101/2024.05.24.595657","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.593833","pub_date":"2024-5-24","title":"Enhancing affinity of neutralizing SARS-CoV-2 nanobody through facile structure-guided mutations in CDRs","abstract":"The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for development of the next generation therapeutics. This study delves into the refinement and engineering of CDRs through in silico affinity maturation followed by binding validation using ITC and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of RBD of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Subsequent biophysical studies using ITC provided insights into binding affinity and key thermodynamic parameters. Consistent with in silico findings, seven single mutations resulted in enhanced affinity. The mutants were further tested for neutralization activity against SARS-CoV-2 pseudovirus. L106T, L106Q, S107R, and S107Q generated mutants were more effective in virus-neutralizing with IC50 values of \u223c0.03 \u00b5M, \u223c0.13 \u00b5M, \u223c0.14 \u00b5M, and \u223c0.14 \u00b5M, respectively as compared to the native nanobody (IC50 \u223c0.77 \u00b5M). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.","version":"1.3","doi":"10.1101/2024.05.13.593833","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.451025","pub_date":"2024-5-24","title":"A cGAMP-containing hydrogel for prolonged SARS-CoV-2 RBD subunit vaccine exposure induces a broad and potent humoral response","abstract":"The receptor binding domain (RBD) of the SARS-CoV-2 virus spike protein has emerged as a promising target for generation of neutralizing antibodies. Although the RBD subunit is more stable than its encoding mRNA, RBD-based subunit vaccines have been hampered by RBD\u2019s poor immunogenicity. We hypothesize that this limitation can be overcome by sustained co-administration with a more potent and optimized adjuvant than standard adjuvants. The endogenous innate immune second messenger, cGAMP, holds promise as potent activator of the anti-viral STING pathway. Unfortunately, delivery of cGAMP as a therapeutic exhibits poor performance due to poor pharmacokinetics and pharmacodynamics from rapid excretion and degradation by its hydrolase ENPP1. To overcome these limitations, we sought to create an artificial immunological niche enabling slow release of cGAMP and RBD to mimic natural infections in which immune activating molecules are co-localized with antigen. Specifically, we co-encapsulated cGAMP and RBD in an injectable polymer-nanoparticle (PNP) hydrogel. This cGAMP-adjuvanted hydrogel vaccine elicited more potent, durable, and broad antibody responses with improved neutralization as compared to dose-matched bolus controls and hydrogel-based vaccines lacking cGAMP. The cGAMP-adjuvanted hydrogel platform developed is suitable for delivery of other antigens and may provide enhanced immunity against a broad range of pathogens.","version":"1.2","doi":"10.1101/2021.07.03.451025","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.24.595699","pub_date":"2024-5-24","title":"The HCoV-HKU1 N-terminal domain binds a wide range of 9-O-acetylated sialic acids presented on different glycan cores","abstract":"Coronaviruses recognize a wide array of protein and glycan receptors using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal (S1-NTD) and C-terminal (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possess an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated \u03b12-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GNTI- cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high-mannose-containing NTD proteins revealed a much broader receptor binding profile compared to its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated \u03b12-3 linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding towards 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids, N- or O-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding. (218)","version":"1.1","doi":"10.1101/2024.05.24.595699","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595402","pub_date":"2024-5-24","title":"Interferon-Induced PARP14-Mediated ADP-Ribosylation in p62 Bodies Requires an Active Ubiquitin-Proteasome System","abstract":"Biomolecular condensates are cellular compartments without enveloping membranes, enabling them to dynamically adjust their composition in response to environmental changes through post-translational modifications. A recent study has revealed that interferon-induced ADP-ribosylation (ADPr), which can be reversed by a SARS-CoV-2-encoded hydrolase, is enriched within a condensate. However, the identity of the condensate and responsible host ADP-ribosyltransferase remain elusive. Here, we demonstrate that interferon induces ADPr through transcriptional activation of PARP14, requiring both its physical presence and catalytic activity for condensate formation. Interferon-induced ADPr colocalizes with PARP14, and these PARP14/ADPr condensates contain key components of p62 bodies\u2014including the selective autophagy receptor p62 and its binding partner NBR1, along with K48-linked and K63-linked polyubiquitin chains\u2014but lack the autophagosome marker LC3B. Knockdown of p62 disrupts the formation of these ADPr condensates. Importantly, these structures are unaffected by autophagy inhibition but depend on both ubiquitin activation and proteasome activity. Taken together, these findings demonstrate that interferon triggers PARP14-mediated ADP-ribosylation in p62 bodies, which requires an active ubiquitin-proteasome system.","version":"1.1","doi":"10.1101/2024.05.22.595402","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595414","pub_date":"2024-5-23","title":"SARS-CoV-2 infection unevenly impacts metabolism in the coronal periphery of the lungs","abstract":"COVID-19 significantly decreases amino acids, fatty acids, and most eicosanoids SARS-CoV-2 preferentially localizes to central lung tissue Metabolic disturbance is highest in peripheral tissue, not central like viral load Spatial metabolomics allows detection of metabolites not altered overall SARS-CoV-2, the virus responsible for COVID-19, is a highly contagious virus that can lead to hospitalization and death. COVID-19 is characterized by its involvement in the lungs, particularly the lower lobes. To improve patient outcomes and treatment options, a better understanding of how SARS-CoV-2 impacts the body, particularly the lower respiratory system, is required. In this study, we sought to understand the spatial impact of COVID-19 on the lungs of mice infected with mouse-adapted SARS2-N501YMA30. Overall, infection caused a decrease in fatty acids, amino acids, and most eicosanoids. When analyzed by segment, viral loads were highest in central lung tissue, while metabolic disturbance was highest in peripheral tissue. Infected peripheral lung tissue was characterized by lower levels of fatty acids and amino acids when compared to central lung tissue. This study highlights the spatial impacts of SARS-CoV-2 and helps explain why peripheral lung tissue is most damaged by COVID-19.","version":"1.1","doi":"10.1101/2024.05.22.595414","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.594713","pub_date":"2024-5-23","title":"\u201cRapid SARS-CoV-2 Detection Using High-Sensitivity Thickness Shear Mode Sensors\u201d","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has emphasized the urgent need for accurate and readily available diagnostic tools. Conventional diagnostic methods, such as reverse transcription real-time polymerase chain reaction (RT-qPCR), are often labor-intensive and time-consuming, which highlights the necessity for rapid point-of-care diagnostic solutions. This study introduces an innovative, low-cost, and highly sensitive diagnostic platform for swift COVID-19 detection. Our platform utilizes the mass sensing properties of thickness shear mode (TSM) transducers to detect and quantify the SARS-CoV-2 nucleocapsid protein through polyethylene glycol (PEG)-based chemistry (1). To confirm surface functionalization and evaluate the effects of the virus lysis buffer, we employed surface characterization techniques including Digital Holographic Microscopy (DHM), Scanning Electron Microscopy (SEM) with Energy-Dispersive X-ray spectroscopy (EDX), and Raman spectroscopy. Sensitivity tests with heat-inactivated SARS-CoV-2 samples demonstrated a sensitivity of about 0.256 Hz/TCID50/mL and a limit of detection (LOD) of roughly 150 TCID50/mL. Specificity was verified through cross-reactivity testing. Our detailed characterization and sensitivity analysis underscore the platform\u2019s reliability, making it a promising candidate for efficient and accessible COVID-19 diagnosis at the point of care.","version":"1.1","doi":"10.1101/2024.05.22.594713","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.23.595478","pub_date":"2024-5-23","title":"Efficacy of late-onset antiviral treatment in immune-compromised hosts with persistent SARS-CoV-2 infection","abstract":"The immunocompromised are at high risk of prolonged SARS-CoV-2 infection and progression to severe COVID-19. However, efficacy of late-onset direct-acting antiviral (DAA) therapy with therapeutics in clinical use and experimental drugs to mitigate persistent viral replication is unclear. In this study, we employed an immunocompromised mouse model, which supports prolonged replication of SARS-CoV-2 to explore late-onset treatment options. Tandem immuno-depletion of CD4+ and CD8+ T cells in C57BL/6 mice followed by infection with SARS-CoV-2 variant of concern (VOC) beta B.1.351 resulted in prolonged infection with virus replication for five weeks after inoculation. Early-onset treatment with nirmatrelvir/ritonavir (paxlovid) or molnupiravir was only moderately efficacious, whereas the experimental therapeutic 4\u2019-fluorourdine (4\u2019-FlU, EIDD-2749) significantly reduced virus load in upper and lower respiratory compartments four days post infection (dpi). All antivirals significantly lowered virus burden in a 7-day treatment regimen initiated 14 dpi, but paxlovid-treated animals experienced rebound virus replication in the upper respiratory tract seven days after treatment end. Viral RNA was detectable 28 dpi in paxlovid-treated animals, albeit not in the molnupiravir or 4\u2019-FlU groups, when treatment was initiated 14 dpi and continued for 14 days. Low-level virus replication continued 35 dpi in animals receiving vehicle but had ceased in all treatment groups. These data indicate that late-onset DAA therapy significantly shortens the duration of persistent virus replication in an immunocompromised host, which may have implications for clinical use of antiviral therapeutics to alleviate the risk of progression to severe disease in highly vulnerable patients. Four years after the onset of the global COVID-19 pandemic, the immunocompromised are at greatest risk of developing life-threatening severe disease. However, specific treatment plans for this most vulnerable patient group have not yet been developed. Employing a CD4+ and CD8+ T cell-depleted immunocompromised mouse model of SARS-CoV-2 infection, we explored therapeutic options of persistent infections with standard-of-care paxlovid, molnupiravir, and the experimental therapeutic 4\u2019-FlU. Late-onset treatment initiated 14 days after infection was efficacious, but only 4\u2019-FlU was rapidly sterilizing. No treatment-experienced viral variants with reduced susceptibility to the drugs emerged, albeit virus replication rebounded in animals of the paxlovid group after treatment end. This study supports the use of direct-acting antivirals for late-onset management of persistent SARS-CoV-2 infection in immunocompromised hosts. However, treatment courses likely require to be extended for maximal therapeutic benefit, calling for appropriately powered clinical trials to meet the specific needs of this patient group.","version":"1.1","doi":"10.1101/2024.05.23.595478","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595410","pub_date":"2024-5-23","title":"Starve a cold or feed a fever? Identifying cellular metabolic changes following infection and exposure to SARS-CoV-2","abstract":"Viral infections induce major shifts in cellular metabolism elicited by active viral replication and antiviral responses. For the virus, harnessing cellular metabolism and evading changes that limit replication are essential for productive viral replication. In contrast, the cellular response to infection disrupts metabolic pathways to prevent viral replication and promote an antiviral state in the host cell and neighboring bystander cells. This competition between the virus and cell results in measurable shifts in cellular metabolism that differ depending on the virus, cell type, and extracellular environment. The resulting metabolic shifts can be observed and analyzed using global metabolic profiling techniques to identify pathways that are critical for either viral replication or cellular defense. SARS-CoV-2 is a respiratory virus that can exhibit broad tissue tropism and diverse, yet inconsistent, symptomatology. While the factors that determine the presentation and severity of SARS-CoV-2 infection remain unclear, metabolic syndromes are associated with more severe manifestations of SARS-CoV-2 disease. Despite these observations a critical knowledge gap remains between cellular metabolic responses and SARS-CoV-2 infection. Using a well-established untargeted metabolomics analysis workflow, we compared SARS-CoV-2 infection of human lung carcinoma cells. We identified significant changes in metabolic pathways that correlate with either productive or non-productive viral infection. This information is critical for characterizing the factors that contribute to SARS-CoV-2 replication that could be targeted for therapeutic interventions to limit viral disease.","version":"1.1","doi":"10.1101/2024.05.22.595410","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595397","pub_date":"2024-5-23","title":"Use of Stem Cell-Derived Cardiomyocyte and Nasal Epithelium Models to Establish a Multi-Tissue Model Platform to Validate Repurposed Drugs Against SARS-CoV-2 Infection","abstract":"The novel coronavirus disease (COVID-19) and any future coronavirus outbreaks will require more affordable, effective and safe treatment options to complement current ones such as Paxlovid. Drug repurposing can be a promising approach if we are able to find a rapid, robust and reliable way to down-select and screen candidates using in silico and in vitro approaches. With repurposed drugs, ex vivo models could offer a rigorous route to human clinical trials with less time invested into nonclinical animal (in vivo) studies. We have previously shown the value of commercially available ex vivo/3D airway and alveolar tissue models, and this paper takes this further by developing and validating human nasal epithelial model and embryonic stem cells derived cardiomyocyte model. Five shortlisted candidates (fluvoxamine, everolimus, pyrimethamine, aprepitant and sirolimus) were successfully compared with three control drugs (remdesivir, molnupiravir, nirmatrelvir) when tested against key variants of the SARS-CoV-2 virus including Delta and Omicron, and we were able to reconfirm our earlier finding that fluvoxamine can induce antiviral efficacy in combination with other drugs. Scalability of this high-throughput screening approach has been demonstrated using a liquid handling robotic platform for future \u2018Disease-X\u2019 outbreaks.","version":"1.1","doi":"10.1101/2024.05.22.595397","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.17.580824","pub_date":"2024-5-23","title":"Extracellular vesicles alter trophoblast function in pregnancies complicated by COVID-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) causes placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction and alteration of the placental transcriptome. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro. We found that the gestational timing of COVID-19 is a major determinant of circulating EV function and cargo. In vitro trophoblast exposure to EVs isolated from patients with an active infection at the time of delivery, but not EVs isolated from Controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an active infection, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19 and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.","version":"1.2","doi":"10.1101/2024.02.17.580824","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.15.594334","pub_date":"2024-5-23","title":"Decoding glycosylation potential from protein structure across human glycoproteins with a multi-view recurrent neural network","abstract":"Glycosylation is described as a non-templated biosynthesis. Yet, the template-free premise is antithetical to the observation that different N-glycans are consistently placed at specific sites. It has been proposed that glycosite-proximal protein structures could constrain glycosylation and explain the observed microheterogeneity. Using site-specific glycosylation data, we trained a hybrid neural network to parse glycosites (recurrent neural network) and match them to feasible N-glycosylation events (graph neural network). From glycosite-flanking sequences, the algorithm predicts most human N-glycosylation events documented in the GlyConnect database and proposed structures corresponding to observed monosaccharide composition of the glycans at these sites. The algorithm also recapitulated glycosylation in Enhanced Aromatic Sequons, SARS-CoV-2 spike, and IgG3 variants, thus demonstrating the ability of the algorithm to predict both glycan structure and abundance. Thus, protein structure constrains glycosylation, and the neural network enables predictive in silico glycosylation of uncharacterized or novel protein sequences and genetic variants.","version":"1.2","doi":"10.1101/2024.05.15.594334","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595297","pub_date":"2024-5-22","title":"SARS-CoV-2 sequencing artifacts associated with targeted PCR enrichment and read mapping","abstract":"Protocols and pipelines for SARS-CoV-2 genome sequencing were rapidly established when the COVID-19 outbreak was declared a pandemic. The most widely used approach for sequencing SARS-CoV-2 includes targeted enrichment by PCR, followed by shotgun sequencing and reference-based genome assembly. As the continued surveillance of SARS-CoV-2 worldwide is transitioning towards a lower level of intensity, it is timely to re-visit the sequencing protocols and pipelines established during the acute phase of the pandemic. In the current study, we have investigated the impact of primer scheme and reference genome choice by sequencing samples with multiple primer schemes (Artic V3, V4.1 and V5.3.2) and re-processing reads with multiple reference genomes. We have also analysed the temporal development in ambiguous base calls during the emergence of the BA.2.86.x variant. We found that the primers used for targeted enrichment can result in recurrent ambiguous base calls, which can accumulate rapidly in response to the emergence of a new variant. We also found examples of consistent base calling errors, associated with PCR artifacts and amplicon drop-out. Similarly, misalignments and partially mapped reads on the reference genome resulted in ambiguous base calls, as well as defining mutations being omitted from the assembly. These findings highlight some key limitations of using targeted enrichment by PCR and reference-based genome assembly for sequencing SARS-CoV-2, and the importance of continuously monitoring and updating primer schemes and bioinformatic pipelines.","version":"1.1","doi":"10.1101/2024.05.22.595297","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595176","pub_date":"2024-5-22","title":"MicroRNA-374b regulates SARS-CoV-2 viral protein mediated endothelial to mesenchymal transition by targeting c-FLIP","abstract":"The pathophysiological consequences of COVID-19 disease are still unclear, however, endothelial cell (EC) dysfunction has been observed to play a key role in disease progression and severity. Many reports suggests that SARS-CoV-2 mediated endothelial dysfunction is the result of intracellular signaling initiated by the binding of the spike protein to ACE2, which can modify endothelial cell phenotype. Recent reports suggests endothelial to mesenchymal transition (Endo MT) as a process heavily involved in lung fibrosis of COVID 19 patients. EndoMT is involved in many chronic and fibrotic diseases and appears to be regulated by complex molecular mechanisms and different signaling pathways, in particular microRNAs (miRNAs), which constitute a crucial mediator of EndoMT. MicroRNAs (miRNAs), small endogenous RNA molecules that regulate several physiological processes including endothelial homeostasis, and vascular diseases, can be perturbed by infecting viruses. Based on these facts, this study was designed to decipher the role of miR-374b, which was found to be significantly downregulated upon profiling of SARS-CoV-2 viral protein stimulated endothelial cells. Gene profiling of endothelial cells revealed c-FLIP (CFLAR) to be among the most significantly upregulated gene. In silico target prediction analysis using targetscan revealed c-FLIP as the major target of miR-374b. Further it was identified that miR-374b can reverse c-FLIP mRNA and protein levels in SARS-CoV-2 viral protein stimulated endothelial cells under conditions of miR-374b overexpression. Since vascular dysfunction involve, under many circumstances, loss of vascular tone due to mesenchymal transition of endothelial cells, we next checked if fibrotic events are initiated downstream of c-FLIP pathway. Further mechanistic studies involving identification of the expression pattern of mesenchymal markers in SARS-CoV-2 viral protein stimulated endothelial cells in presence or absence of miR-374b provide evidence for the important role of miR-374b in regulating SARS CoV-2 mediated EndoMT and fibrotic events downstream of c-FLIP pathway and may highlight possible new therapeutic approaches targeted at the damaged endothelium.","version":"1.1","doi":"10.1101/2024.05.22.595176","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.18.594818","pub_date":"2024-5-22","title":"SARS-CoV-2 variants from long-term, persistently infected immunocompromised patients have altered syncytia formation, temperature-dependent replication, and serum neutralizing antibody escape","abstract":"SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates from two COVID-19 patients undergoing immunosuppressive cancer therapy, with all isolates obtained greater than 100 days after the initial COVID-19 diagnoses and compared to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient - who did mount a SARS-CoV-2 IgM response \u2013 showed temperature dependent changes in plaque size as well as increased syncytia formation and escape from serum neutralizing antibody. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.","version":"1.2","doi":"10.1101/2024.05.18.594818","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595089","pub_date":"2024-5-22","title":"An anti-SARS-CoV-2 formula consisting volatile oil from TCM through inhibiting multiple targets in cultured cells","abstract":"At present, coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variant virus are prevalent all over the world, taking a major toll on human lives worldwide. Oral mucosa and saliva are the high-risk routes of transmission. Inactivation of the virus in the mouth is one of the important strategies to reduce the source infectivity of the virus. However, the current preparations for inactivating oral virus are mainly aimed at ACE2 protein, and its effect needs to be improved. In this study, through literature mining, multi-target screening and other methods, we screened and optimized the volatile oil formula (Artemisiae argyi folium, Chrysanthemum morifolium, Trollii chinensis flos, Lonicerae japonicae flos) from the volatile oil of traditional Chinese medicine to inhibit the invasion and replication of SARS-CoV-2. A test using pseudotype virus with S glycoprotein confirmed that this formula could effectively bind to the S glycoprotein to prevent SARS-CoV-2 host cell entry. Molecular docking experiments showed that the m ultiple molecules in the formula might weakly bind to these targets including ACE2, cathepsin L, furin, mpro/3cl. In all, the volatile oil formula designed in this paper offers a general affordable strategy to protect patients from SARS-CoV-2 infections through debulking or minimizing transmission to others.","version":"1.1","doi":"10.1101/2024.05.22.595089","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595309","pub_date":"2024-5-22","title":"SARS-CoV-2 3CLPro Dihedral Angles Reveal Allosteric Signaling","abstract":"In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state-of-the-art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the protein\u2019s C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedrals angles in transmitting allosteric signals.","version":"1.1","doi":"10.1101/2024.05.22.595309","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.25.505316","pub_date":"2024-5-22","title":"Single cell multiomic analyses reveal divergent effects of DNMT3A and TET2 mutant clonal hematopoiesis in inflammatory response","abstract":"DNMT3A and TET2 are epigenetic regulators commonly mutated in age related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematological neoplasms. While individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant CH on COVID-19-related cytokine release severity and mortality. Using single cell proteogenomics we show that DNMT3A mutations involve cells of myeloid and lymphoid lineages. Using single cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of IL32, a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Together, these data provide a mechanistic insight into the poor inflammatory outcomes seen in DNMT3A mutant CH patients infected with Sars-COV2.","version":"1.2","doi":"10.1101/2022.08.25.505316","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.08.576722","pub_date":"2024-5-22","title":"Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals","abstract":"Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.","version":"1.2","doi":"10.1101/2024.02.08.576722","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.22.595230","pub_date":"2024-5-22","title":"Exploring the ability of the MD+FoldX method to predict SARS-CoV-2 antibody escape mutations using large-scale data","abstract":"Antibody escape mutations pose a significant challenge to the effectiveness of vaccines and antibody-based therapies. The ability to predict these escape mutations with computer simulations would allow us to detect threats early and develop effective countermeasures, but a lack of large-scale experimental data has hampered the validation of these calculations. In this study, we evaluate the ability of the MD+FoldX molecular modeling method to predict escape mutations by leveraging a large deep mutational scanning dataset, focusing on the SARS-CoV-2 receptor binding domain. Our results show a positive correlation between predicted and experimental data, indicating that mutations with reduced predicted binding affinity correlate moderately with higher experimental escape fractions. We also demonstrate that better performance can be achieved using affinity cutoffs tailored to distinct antibody-antigen interactions rather than a one-size-fits-all approach. We find that 70% of the systems surpass the 50% precision mark, and demonstrate success in identifying mutations present in significant variants of concern and variants of interest. Despite promising results for some systems, our study highlights the challenges in comparing predicted and experimental values. It also emphasizes the need for new binding affinity methods with improved accuracy that are fast enough to estimate hundreds to thousands of antibody-antigen binding affinities.","version":"1.1","doi":"10.1101/2024.05.22.595230","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.20.595020","pub_date":"2024-5-21","title":"Characteristics of JN.1-derived SARS-CoV-2 subvariants SLip, FLiRT, and KP.2 in neutralization escape, infectivity and membrane fusion","abstract":"SARS-CoV-2 variants derived from the immune evasive JN.1 are on the rise worldwide. Here, we investigated JN.1-derived subvariants SLip, FLiRT, and KP.2 for their ability to be neutralized by antibodies in bivalent-vaccinated human sera, XBB.1.5 monovalent-vaccinated hamster sera, sera from people infected during the BA.2.86/JN.1 wave, and class III monoclonal antibody (Mab) S309. We found that compared to parental JN.1, SLip and KP.2, and especially FLiRT, exhibit increased resistance to COVID-19 bivalent-vaccinated human sera and BA.2.86/JN.1-wave convalescent sera. Interestingly, antibodies in XBB.1.5 monovalent vaccinated hamster sera robustly neutralized FLiRT and KP.2 but had reduced efficiency for SLip. These JN.1 subvariants were resistant to neutralization by Mab S309. In addition, we investigated aspects of spike protein biology including infectivity, cell-cell fusion and processing, and found that these subvariants, especially SLip, had a decreased infectivity and membrane fusion relative to JN.1, correlating with decreased spike processing. Homology modeling revealed that L455S and F456L mutations in SLip reduced local hydrophobicity in the spike and hence its binding to ACE2. In contrast, the additional R346T mutation in FLiRT and KP.2 strengthened conformational support of the receptor-binding motif, thus counteracting the effects of L455S and F456L. These three mutations, alongside D339H, which is present in all JN.1 sublineages, alter the epitopes targeted by therapeutic Mabs, including class I and class III S309, explaining their reduced sensitivity to neutralization by sera and S309. Together, our findings provide insight into neutralization resistance of newly emerged JN.1 subvariants and suggest that future vaccine formulations should consider JN.1 spike as immunogen, although the current XBB.1.5 monovalent vaccine could still offer adequate protection.","version":"1.1","doi":"10.1101/2024.05.20.595020","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.15.594393","pub_date":"2024-5-20","title":"ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis","abstract":"Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host (immune) proteins such as MDA5 and IRF3 in a process called ISGylation, thereby limiting the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.","version":"1.1","doi":"10.1101/2024.05.15.594393","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.16.594608","pub_date":"2024-5-17","title":"Spike protein of the SARS-CoV-2 omicron variant interacts with actin","abstract":"The omicron variant of SARS-CoV-2 is responsible for the COVID-19 pandemic, serving as a significant origin for the variants still being detected today. It affects the spike protein that most vaccines used to target when the Omicron strain was discovered. Here, we demonstrate that the receptor binding domain (RBD) of the Omicron variant of SARS-CoV-2 exhibits an increased affinity for human angiotensin-converting enzyme type 2 (hACE2) as a viral cell receptor compared to the prototype RBD. We also identified that \u03b2- and \u03b3-actin are Omicron-specific binding partners of RBD. Protein complex predictions suggested that many of the Omicron-specific amino acid substitutions might be involved in the affinity of RBD and actin. Accordingly, we highlight the intriguing observation that proteins expected to localize to different cellular compartments exhibit strong binding.","version":"1.1","doi":"10.1101/2024.05.16.594608","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.16.594465","pub_date":"2024-5-16","title":"Cell invasive amyloid assemblies from SARS-CoV-2 peptides can form multiple polymorphs with varying neurotoxicity","abstract":"The neurological symptoms of COVID-19, such as memory loss, cognitive and sensory disruption (neuro-COVID) are well reported. These neurological symptoms frequently persist for months (post-acute sequalae of COVID-19 or PASC). The molecular origins of neuro-COVID and how it contributes to PASC are unknown, however a growing body of research highlights that the self-assembly of protein fragments from SARS-CoV-2 into amyloid nanofibrils may play a causative role. Previously, we identified two fragments from the proteins Open Reading Frame 6 (ORF6) and ORF10 that self-assemble into neurotoxic amyloid assemblies. Here we further our understanding of the self-assembly mechanisms and nano-architectures formed by these fragments as well as performing a more in-depth study of the biological responses of co-cultured neurons. By solubilising the peptides in a fluorinated solvent we eliminate insoluble aggregates in the starting materials (seeds) that change the polymorphic landscape of the assemblies. The resultant assemblies are dominated by structures with higher free energies (e.g. ribbons and amorphous aggregates) that are less toxic to cultured neurons. We also show the first direct evidence of cellular uptake by viral amyloids. This work highlights the importance of understanding the polymorphic behaviour of amyloids particularly in the context of neuro-COVID and PASC. The neurological symptoms of COVID-19 are likely to be, in part, caused by the aggregation of viral proteins into neurotoxic amyloid nanofibrils. Changes in aggregation conditions alters the balance of fibril structures formed (polymorphism), influencing their toxicity to a neuronal cell line. These findings increase our understanding of viral amyloids and highlight the importance of careful choice of experimental protocol when studying these systems.","version":"1.1","doi":"10.1101/2024.05.16.594465","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.15.594386","pub_date":"2024-5-15","title":"Assessing pH-dependent Conformational Changes in the Fusion Peptide Proximal Region of the SARS-CoV-2 spike glycoprotein","abstract":"One of the entry mechanisms of the SARS-CoV-2 coronavirus into host cells involves endosomal acidification. It has been proposed that under acidic conditions the Fusion Peptide Proximal Region (FPPR) of the SARS-CoV-2 spike glycoprotein acts as a pH-dependent switch, modulating immune response accessibility by influencing the positioning of the Receptor Binding Domain (RBD). This would provide an indirect coupling of RBD opening to environmental pH. Here, we explored this possible pH-dependent conformational equilibrium of the FPPR within the SARS-CoV-2 spike glycoprotein. We analyzed hundreds of experimentally determined spike structures from the Protein Data Bank, and carry out pH-Replica Exchange Molecular Dynamics, exploring the extent to which the FPPR conformation depends on pH and the positioning of the RBD. Meta-analysis of experimental structures identified alternate conformations of the FPPR among structures in which this flexible regions was resolved. However, the results did not support a correlation between the FPPR conformation and either RBD position or the reported pH of the cryo-EM experiment. We calculated pKa values for titratable side chains in the FPPR region using PDB structures, but these pKa values showed large differences between alternate PDB structures that otherwise adopt the same FPPR conformation type. This hampers comparison of pKa values in different FPPR conformations to rationalize a pH-dependent conformational change. We supplemented these PDB-based analyses with all-atom simulations, using constant pH-Replica Exchange Molecular Dynamics to estimate pKa values in the context of flexibility and explicit water. The resulting titration curves show good reproducibility between simulations, but also suggest that the titration curves of the different FPPR conformations are the same within error bars. In summary, we were unable to find evidence supporting the previously published hypothesis of FPPR pH-dependent equilibrium, either from existing experimental data, or from constant pH MD simulations. The study underscores the complexity of the spike system and opens avenues for further exploration into the interplay between pH and SARS-CoV-2 viral entry mechanisms.","version":"1.1","doi":"10.1101/2024.05.15.594386","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.14.24307380","pub_date":"2024-05-15","title":"Post-Acute Cardiovascular Outcomes of COVID-19 in Children and Adolescents: An EHR Cohort Study from the RECOVER Project","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The risk of cardiovascular outcomes in the post-acute phase of SARS-CoV-2 infection has been quantified among adults and children. This paper aimed to assess a multitude of cardiac signs, symptoms, and conditions, as well as focused on patients with and without congenital heart defects (CHDs), to provide a more comprehensive assessment of the post-acute cardiovascular outcomes among children and adolescents after COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This retrospective cohort study used data from the RECOVER consortium comprising 19 US children\u2019s hospitals and health institutions between March 2020 and September 2023. Every participant had at least a six-month follow-up after cohort entry. Absolute risks of incident post-acute COVID-19 sequelae were reported. Relative risks (RRs) were calculated by contrasting COVID-19-positive with COVID-19-negative groups using a Poisson regression model, adjusting for demographic, clinical, and healthcare utilization factors through propensity scoring stratification.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>A total of 1,213,322 individuals under 21 years old (mean[SD] age, 7.75[6.11] years; 623,806 male [51.4%]) were included. The absolute rate of any post-acute cardiovascular outcome in this study was 2.32% in COVID-19 positive and 1.38% in negative groups. Patients with CHD post-SARS-CoV-2 infection showed increased risks of any cardiovascular outcome (RR, 1.63; 95% confidence interval (CI), 1.47-1.80), including increased risks of 11 of 18 post-acute sequelae in hypertension, arrhythmias (atrial fibrillation and ventricular arrhythmias), myocarditis, other cardiac disorders (heart failure, cardiomyopathy, and cardiac arrest), thrombotic disorders (thrombophlebitis and thromboembolism), and cardiovascular-related symptoms (chest pain and palpitations). Those without CHDs also experienced heightened cardiovascular risks after SARS-CoV-2 infection (RR, 1.63; 95% CI, 1.57-1.69), covering 14 of 18 conditions in hypertension, arrhythmias (ventricular arrhythmias and premature atrial or ventricular contractions), inflammatory heart disease (pericarditis and myocarditis), other cardiac disorders (heart failure, cardiomyopathy, cardiac arrest, and cardiogenic shock), thrombotic disorders (pulmonary embolism and thromboembolism), and cardiovascular-related symptoms (chest pain, palpitations, and syncope).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Both children with and without CHDs showed increased risks for a variety of cardiovascular outcomes after SARS-CoV-2 infection, underscoring the need for targeted monitoring and management in the post-acute phase.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Clinical Perspective section</jats:title>\n                  <jats:sec>\n                    <jats:title>What is new?</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>We investigated the risks of 18 post-acute COVID-19 cardiovascular outcomes in the pediatric population without Multisystem Inflammatory Syndrome in Children (MIS-C) in over 1 million patients, stratified by congenital heart defects (CHD) status.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>We extended the follow-up period beyond previous pediatric studies, ensuring every participant had at least a six-month follow-up after cohort entry.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>We included a comprehensive cross-section of the US pediatric population across various healthcare settings including primary, specialty, and emergency care, as well as testing and inpatient facilities.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>What are the clinical implications?</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Within the post-acute phase, children and adolescents previously infected with SARS-CoV-2 are at statistically significant increased risk of incident cardiovascular outcomes, including hypertension, ventricular arrhythmias, myocarditis, heart failure, cardiomyopathy, cardiac arrest, thromboembolism, chest pain, and palpitations. These findings are consistent among patients with and without CHDs.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Awareness of the heightened risk of cardiovascular disorders after COVID-19 can lead to a timely referral, investigations, and management of these conditions in children and adolescents.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2024.05.14.24307380","journal":"medRxiv","score":null},{"id":"10.1101/2024.05.14.594200","pub_date":"2024-5-15","title":"The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model","abstract":"The use of fixed dose-combinations of antivirals with different mechanisms of action has proven a key in the successful treatment of infections with HIV and HCV. For the treatment of infections with SARS-CoV-2 and possible future epi-/pandemic coronaviruses, it will be important to explore the efficacy of combinations of different drugs, in particular to avoid resistance development, such as in patients with immunodeficiencies. As a first effort, we studied the antiviral potency of combinations of antivirals. To that end, we made use of primary human airway epithelial cell (HAEC) cultures grown at the air-liquid interface that were infected with the beta coronavirus OC43. We found that the triple combination of GS-441524 (parent nucleoside of remdesivir), molnupiravir, and ribavirin resulted in a more pronounced antiviral efficacy than what could be expected from a purely additive antiviral effect. The potency of this triple combination was next tested in SARS-CoV-2 infected hamsters. To that end, for each of the drugs, intentionally suboptimal or even ineffective doses were selected. Yet, in the lungs of all hamsters that received triple prophylactic therapy with suboptimal/inactive doses of GS-441524, molnupiravir, and ribavirin, no infectious virus was detectable. Our finding indicate that co-administration of approved drugs for the treatment of coronavirus infections should be further explored but also against other families of viruses with epidemic and pandemic potential for which no effective antiviral treatment is available.","version":"1.1","doi":"10.1101/2024.05.14.594200","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.14.594077","pub_date":"2024-5-15","title":"An integrative characterisation of proline cis and trans conformers in a disordered peptide","abstract":"Intrinsically disordered proteins (IDPs) often contain proline residues, which undergo cis/trans isomerisation. While molecular dynamics (MD) simulations have the potential to fully characterise the proline cis and trans sub-ensembles, they are limited by the slow timescales of isomerisation and force field inaccuracies. Nuclear magnetic resonance (NMR) spectroscopy can report on ensemble-averaged observables for both the cis and trans proline states, but a full atomistic characterisation of these sub-ensembles is challenging. Given the importance of proline cis/trans isomerisation for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle X-ray scattering (SAXS) to characterise the two sub-ensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimised for disordered proteins. Each simulation was run for an accumulated time of 180-220 \u00b5s until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamics simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamics simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multi-scale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterised by either approach in isolation. This study employs MD simulations (with metadynamics), NMR spectroscopy, and SAXS to elucidate the individual cis and trans proline conformations of ORF6CTR from SARS-CoV-2. The good agreement on proline cis/trans populations observed in experiments (NMR) and those calculated from simulations in the AMBER03ws force field (with SAXS reweighting) showcases the efficiency of this interdisciplinary approach, which can be used to characterise highly dynamic disordered protein states, even for very slow processes. Furthermore, our study emphasises the importance of considering both computational and experimental methodologies to gain a more holistic understanding of highly dynamic proteins. The presented integrative approach sets a precedent for future studies aiming to explore complex and dynamic biological systems with slow transitions such as proline isomerisations.","version":"1.1","doi":"10.1101/2024.05.14.594077","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.594050","pub_date":"2024-5-15","title":"Classifying Genetic Interactions Using an HIV Experimental Study","abstract":"Current methods of addressing novel viruses remain predominantly reactive and reliant on empirical strategies. To develop more proactive methodologies for the early identification and treatment of diseases caused by viruses like HIV and Sars-CoV-2, we focus on host targeting, which requires identifying and altering human genetic host factors that are crucial to the life cycle of these viruses. To this end, we present three classification models to pinpoint host genes of interest. For each one, we thoroughly analyze the current predictive accuracy, susceptibility to modifications of the input space, and potential for further optimization. Our methods rely on the exploration of different gene representations, including graph-based embeddings and large foundation transformer models, to establish a set of baseline classification models. Subsequently, we introduce an order-invariant Siamese neural network that exhibits more robust pattern recognition with sparse datasets while ensuring that the representation does not capture unwanted patterns, such as the directional relationship of genetic interactions. Through these models, we generate biological features that predict pairwise gene interactions, with the intention of extrapolating this proactive therapeutic approach to other virus families.","version":"1.1","doi":"10.1101/2024.05.13.594050","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.593815","pub_date":"2024-5-14","title":"Exploring respiratory viral pathogens and bacteriome from symptomatic SARS-CoV-2-negative and positive individuals","abstract":"In the COVID pandemic era, increased mortality was seen despite some unknown etiologies other than SARS-CoV2 viral infection. Vaccination targeted to SARS-CoV2 was successful due to infection caused by pathogens of viral origin based on symptomatology. Hence, it is essential to detect other viral and bacterial infections throughout the initial wave of the COVID-19 disease outbreak, particularly in those suffering from a symptomatic respiratory infection with SARS-CoV-2-negative status. This study was planned to explore the presence of bacterial and other respiratory viruses in symptomatic patients with SARS-CoV2-positive or negative status. The study selected128 patient\u2019s samples out of 200 patients\u2019 samples (100 at each time point) collected for routine SARS-CoV-2 detection schedule in December 2020 and June 2021. Considering the seasonal changes responsible for the occurrence of respiratory pathogens, we finalized 64 SARS-CoV-2 tested patients with 32 SARS-CoV-2-negatives and 32 SARS-CoV-2-positives from each collection time to examine them further using real-time PCR for the presence of other viral species and bacterial infection analyzing 16S rRNA metagenome supporting to cause respiratory infections. Along with various symptoms, we observed the co-infection of adenovirus and influenza B(Victoria) virus to two SARS-CoV-2-positive samples. The SARS-CoV-2-negative but symptomatic patient showed Rhinovirus (7/64 i.e. 10.9%) and Influenza (A/H3N2) infection in 4 patients out of 64 patients (6.25%). Additionally, one SARS-CoV-2-negative patient enrolled in June 2021 showed PIV-3 infection. Influenza A/H3N2 and Adenovirus were the cause of symptoms in SARS-CoV-2-negative samples significantly. Thus, the overall viral infections are considerably higher among SARS-CoV-2-negative patients (37.5% Vs 6.25%) compared to SARS-CoV-2-positive patients representing respiratory illness probably due to the abundance of the viral entity as well as competition benefit of SARS-CoV-2 in altering the imperviousness of the host. Simultaneously, 16S rRNA ribosomal RNA metagenomenext-generation sequencing (NGS) data from the same set of samples indicated a higher frequency of Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, fusobacteriota, Patescibacteria, and Campilobacterotaphyla out of 15 phyla, 240 species from positive and 16 phyla, 274 species from negative samples. Exploring co-infecting respiratory viruses and bacterial populations becomes significant in understanding the mechanisms associated with multiple infecting pathogens from symptomatic COVID-positive and negative individuals for initiating proper antimicrobial therapy. Frequent transfer of SARS-CoV-2 events has resulted in the emergence of other viral infections along with several evolutionarily separate viral lineages in the global SARS-CoV-2 population, presenting significant viral variants in various regions worldwide. This variation also raises the possibility of reassortment and the creation of novel variants of SARS-CoV-2, as demonstrated by the COVID pandemic in all the waves, which may still be able to cause illness and spread among people. Still unclear, though, are the molecular processes that led to the adaption of other viral and bacterial pathogens in humans when a human SARS-CoV-2 virus was introduced. In this study, we identified the presence of various other viral infections and bacterial content in symptomatic COVID-19-positive and negative patients, as evidenced by the data obtained using next-generation sequencing of 16S rRNA metagenome and real-time PCR detection technologies. Symptoms might have been induced by bacterial content and various viral entities other than the SARS-CoV-2 viral infection in the COVID-negative population, indicating its importance in detecting and initiating appropriate therapy to recover from all other infections.","version":"1.1","doi":"10.1101/2024.05.13.593815","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.593816","pub_date":"2024-5-14","title":"Adjuvanted subunit intranasal vaccine prevents SARS-CoV-2 onward transmission in hamsters","abstract":"Most COVID-19 vaccine trials have focused on recipient protection, not protection of their contacts, a critical need. As a subunit intranasal COVID-19 vaccine reduced nasopharyngeal virus more than did an intramuscular (IM) vaccine, we hypothesized that this vaccine might reduce onward transmission to others. We vaccinated hamsters with either the IM-administrated Moderna mRNA vaccine twice or one dose of mRNA IM followed by adjuvanted subunit intranasal vaccine. 24 hours after SARS-CoV-2 challenge, these animals were housed with na\u00efve recipients in a contactless chamber that allows airborne transmission. Onward airborne transmission was profoundly blocked: the donor and recipients of the intranasal vaccine-boosted group had lower oral and lung viral loads (VL), which correlated with mucosal ACE2 inhibition activity. These data strongly support the use of the intranasal vaccine as a boost to protect not only the vaccinated person, but also people exposed to the vaccinated person, a key public health goal. Natural transmission of SARS-CoV-2 is primarily airborne, through the respiratory mucosal route. However, current licensed COVID-19 vaccines are all intramuscular and induce more systemic than mucosal immunity. Here, we did a head-to-head comparison of COVID-19 booster vaccines on SARS-CoV-2 onward transmission. We found that compared to boosting with a Moderna mRNA systemic vaccine, a nanoparticle intranasal COVID-19 vaccine much more effectively prevents onward airborne transmission to na\u00efve recipient hamsters. The protection was correlated with local mucosal antibody. Thus, a mucosal nanoparticle vaccine should be considered for preventing onward airborne transmission, a key public health necessity that has not been adequately studied.","version":"1.1","doi":"10.1101/2024.05.13.593816","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.14.594070","pub_date":"2024-5-14","title":"Unique RNA replication characteristics and nucleocapsid protein expression may explain differences in the replication capacity of SARS-COV-2 lineages","abstract":"COVID-19 pandemic in Brazil was characterized by the sequential circulation of the SARS-CoV-2 lineages B.1.1.33, and variants Zeta (P.2), Gamma (P.1/P.1.*), Delta (B.1.617.2/AY.*), and Omicron (BA.*). Our research aimed to compare the biological traits of these lineages and variants by analyzing aspects of viral replication including binding, entry, RNA replication, and viral protein production. We demonstrated that the replication capacity of these variants varies depending on the cell type, with Omicron BA.1 exhibiting the lowest replication in the human pulmonary cells. Additionally, the nucleocapsid proteoforms generated during infection exhibit distinct patterns across variants. Our findings suggest that factors beyond the initial stages of virus entry influence the efficiency of viral replication among different SARS-CoV-2 variants. Thus, our study underscores the significance of RNA replication and the role of nucleocapsid proteins in shaping the replicative characteristics of SARS-CoV-2 variants. The COVID-19 pandemic was characterized by the emergence of different viral variants that presents specific properties such as response to antibodies, pathogenicity and detection by diagnostic tests. The circulation of these variants presented a particular pattern depending on the global geographic regions. Despite the cessation of the pandemic, as officially declared by the World Health Organization in 2023, new viral variants continue to emerge while aspects of the virus-cell interaction that contribute to the replication of these variants have not yet been completely understood. In our study, we compared the biological characteristics of SARS-CoV-2 variants that circulated in Brazil during the pandemic, verifying aspects of entry, viral replication and production of viral RNA and proteins. Our results indicate that Omicron BA.1 variant has reduced replication and protein production in human lung cells. We also observed that the viral nucleocapsid protein presents proteoforms that vary according to the variant. These differences could help to explain the differences observed in viral replication in human pulmonary cells.","version":"1.1","doi":"10.1101/2024.05.14.594070","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.594034","pub_date":"2024-5-14","title":"Omicron-specific naive B cell maturation alleviates immune imprinting induced by SARS-CoV-2 inactivated vaccine","abstract":"SARS-CoV-2 ancestral strain-induced immune imprinting poses great challenges to vaccine updates. Studies showed that repeated Omicron exposures could override immune imprinting induced by inactivated vaccines but not mRNA vaccines, a disparity yet to be understood. Here, we analyzed the underlying mechanism of immune imprinting alleviation in inactivated vaccine (CoronaVac) cohorts. We observed in CoronaVac-vaccinated individuals who experienced BA.5/BF.7 breakthrough infection (BTI), the proportion of Omicron-specific memory B cells (MBCs) substantially increased after an extended period post-Omicron BTI, with their antibodies displaying enhanced somatic hypermutation and neutralizing potency. Consequently, the neutralizing antibody epitope distribution encoded by MBCs post-BA.5/BF.7 BTI after prolonged maturation closely mirrors that in BA.5/BF.7-infected unvaccinated individuals. Together, these results indicate the activation and expansion of Omicron-specific na\u00efve B cells generated by first-time Omicron exposure helped to alleviate CoronaVac-induced immune imprinting, and the absence of this process should have caused the persistent immune imprinting seen in mRNA vaccine recipients. Longitudinal MBC profiling of CoronaVac-vaccinated individuals following BA.5 BTI Omicron-specific MBC proportion rises greatly after extended period post-BA.5 BTI Omicron-specific naive B cell maturation reduces ancestral strain immune imprinting","version":"1.1","doi":"10.1101/2024.05.13.594034","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.593878","pub_date":"2024-5-13","title":"Persistent lung inflammation and alveolar-bronchiolization due to Notch signaling dysregulation in SARS-CoV-2 infected hamster","abstract":"Long COVID or Post-acute sequalae of COVID-19 (PASC) defines the persistent signs, symptoms, and conditions long after initial SARS-CoV-2 infection which affecting over 10% of COVID-19 patients, with 40% of them affecting respiratory system. The lung histopathological changes and underlying mechanism remain elusive. Here we systemically investigate histopathological and transcriptional changes at 7, 14, 42, 84 and 120 days-post-SARS-CoV-2-infection (dpi) in hamster. We demonstrate persistent viral residues, chronic inflammatory and fibrotic changes from 42dpi to 120dpi. The most prominent lung histopathological lesion is multifocal alveolar-bronchiolization observed in every animal from 14dpi until 120dpi. However, none of the above are observed in hamsters recovered from influenza A infection. We show airway progenitor CK14+ basal cells actively proliferate, differentiate into SCGB1A+ club cell or Tubulin+ ciliated cells, leading to alveolar-bronchiolization. Most importantly, Notch pathway is persistently upregulated. Intensive Notch3 and Hes1 protein expression are detected in alveolar-bronchiolization foci, suggesting the association of sustained Notch signaling with dysregulated lung regeneration. Lung spatial transcriptomics show upregulation of genes positively regulating Notch signaling is spatially overlapping with alveolar-bronchiolization region. To be noted, significant upregulation of tumor-related genes was detected in abnormal bronchiolization region by spatial transcriptomics analysis, indicating possible risk of lung carcinoma. Collectively, our data suggests SARS-CoV-2 infection caused chronic inflammatory and fibrotic tissue damages in hamster lung, sustained upregulation of Notch pathway signaling contributed to the dysregulated lung regeneration and CK14+ basal cells-driven alveolar-bronchiolization. The study provides important information for potential therapeutic approaches and probable long-term surveillance of malignancy in PASC management.","version":"1.1","doi":"10.1101/2024.05.13.593878","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.11.593709","pub_date":"2024-5-13","title":"Persistent Activation of Chronic Inflammatory Pathways in Long Covid","abstract":"Long Covid, or Post-Acute Sequelae of COVID-19 (PASC), involves a spectrum of chronic symptoms following resolution of acute SARS-CoV-2 infection. Current hypotheses for the pathogenesis of Long Covid include persistent SARS-CoV-2, activation of other viruses, tissue damage, autoimmunity, endocrine insufficiency, immune dysfunction, and complement activation. We evaluated 142 participants, including uninfected controls (N=35), acutely infected individuals (N=54), convalescent controls (N=25), and Long Covid patients (N=28), by comprehensive immunologic, virologic, transcriptomic, and proteomic analyses. Long Covid was characterized by persistent inflammatory pathways compared with convalescent controls and uninfected controls, including upregulation of IL-6 and JAK-STAT pathways as well as activation of coagulation, complement, metabolism, and T cell exhaustion pathways. Moreover, robust activation of these pathways during acute COVID-19 infection correlated with the subsequent development of Long Covid. In an independent validation cohort (N=47), Long Covid patients had higher levels of plasma IL-6R compared with convalescent controls and uninfected controls. These data demonstrate that Long Covid is characterized by persistent activation of chronic inflammatory pathways, suggesting novel therapeutic targets and biomarkers of disease.","version":"1.1","doi":"10.1101/2024.05.11.593709","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.13.593807","pub_date":"2024-5-13","title":"Supervised fine-tuning of pre-trained antibody language models improves antigen specificity prediction","abstract":"Antibodies play a crucial role in adaptive immune responses by determining B cell specificity to antigens and focusing immune function on target pathogens. Accurate prediction of antibody-antigen specificity directly from antibody sequencing data would be a great aid in understanding immune responses, guiding vaccine design, and developing antibody-based therapeutics. In this study, we present a method of supervised fine-tuning for antibody language models, which improves on previous results in binding specificity prediction to SARS-CoV-2 spike protein and influenza hemagglutinin. We perform supervised fine-tuning on four pre-trained antibody language models to predict specificity to these antigens and demonstrate that fine-tuned language model classifiers exhibit enhanced predictive accuracy compared to classifiers trained on pretrained model embeddings. The change of model attention activations after supervised fine-tuning suggested that this performance was driven by an increased model focus on the complementarity determining regions (CDRs). Application of the supervised fine-tuned models to BCR repertoire data demonstrated that these models could recognize the specific responses elicited by influenza and SARS-CoV-2 vaccination. Overall, our study highlights the benefits of supervised fine-tuning on pre-trained antibody language models as a mechanism to improve antigen specificity prediction. Antibodies are vigilant sentinels of our adaptive immune system that recognize and bind to targets on foreign pathogens, known as antigens. This interaction between antibody and antigen is highly specific, akin to a fitting lock and key mechanism, to ensure each antibody precisely targets its intended antigen. Recent advancements in language modeling have led to the development of antibody language model to decode specificity information in the sequences of antibodies. We introduce a method based on supervised fine-tuning, which enhances the accuracy of antibody language models in predicting antibody-antigen interactions. By training these models on large datasets of antibody sequences, we can better predict which antibodies will bind to important antigens such as those found on the surface of viruses like SARS-CoV-2 and influenza. Moreover, our study demonstrates the potential of the models to \u201cread\u201d B cell repertoire data and predict ongoing responses, offering new insights into how our bodies respond to vaccination. These findings have significant implications for vaccine design, as accurate prediction of antibody specificity can guide the development of more effective vaccines.","version":"1.1","doi":"10.1101/2024.05.13.593807","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.12.593784","pub_date":"2024-5-13","title":"Analysis of differential expression of matrix metalloproteins and defensins in the nasopharyngeal milieu of mild and severe COVID-19 cases","abstract":"A subset of COVID-19 disease patients suffers a severe form of the illness, however, underlying early pathophysiological mechanisms associated with the severe form of COVID-19 disease remain to be fully understood. Several studies showed the association of COVID-19 disease severity with the changes in the expression profile of various matrix metalloproteinases (MMPs) and defensins. However, the link between the changes in the expression of matrix metalloproteinase (MMPs) and defensins (DA) in the nasopharyngeal milieu, during early phases of infection, and disease severity remains poorly understood. Therefore, we performed differential gene expression analysis of matrix metalloproteinases (MMPs) and defensins in the nasopharyngeal swab samples collected from mild and severe COVID-19 cases within three days of infection and examined the association between MMP and DA expression and disease severity. A total of 118 previously collected nasopharyngeal samples from mild and severe COVID-19 patients (as per the WHO criteria) were used in this study. To determine the viral loads and assess the mRNA expression of matrix metalloproteinase (MMPs) and defensins, a real-time qPCR assay was used. To assess statistically significant differences in the mean expression of viral loads and the cytokines in between the severe and mild groups, an unpaired T-test was applied. The Pearson correlation test was used to assess the correlation between cytokine expressions. In addition, a multivariable logistic regression analysis was carried out with all the variables from the data set using \u2018severity\u2019 as the outcome variable. Our results showed that the expression of DA3 and MMP2 to be considerably lower in the severe group than in the mild group. Furthermore, there was a significant association between MMP1 and DA4 and DA6 (r=0.5, p=0.0001); as well as between MMP7 and DA1 and DA6 (r=0.5, p=0.00). Additionally, the regression analysis shows a significant correlation (p 0.05) between MMP2 and the severity of COVID-19 disease. The early detection of changes in the expression of MMPs and defensins may act as a useful biomarker/predictor for possible severe COVID-19 disease, which may be useful in the clinical management of patients to reduce COVID-19-associated morbidity and mortality.","version":"1.1","doi":"10.1101/2024.05.12.593784","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.10.575113","pub_date":"2024-5-13","title":"Generative artificial intelligence performs rudimentary structural biology modeling","abstract":"Natural language-based generative artificial intelligence (AI) has become increasingly prevalent in scientific research. Intriguingly, capabilities of generative pre-trained transformer (GPT) language models beyond the scope of natural language tasks have recently been identified. Here we explored how GPT-4 might be able to perform rudimentary structural biology modeling. We prompted GPT-4 to model 3D structures for the 20 standard amino acids and an \u03b1-helical polypeptide chain, with the latter incorporating Wolfram mathematical computation. We also used GPT-4 to perform structural interaction analysis between nirmatrelvir and its target, the SARS-CoV-2 main protease. Geometric parameters of the generated structures typically approximated close to experimental references. However, modeling was sporadically error-prone and molecular complexity was not well tolerated. Interaction analysis further revealed the ability of GPT-4 to identify specific amino acid residues involved in ligand binding along with corresponding bond distances. Despite current limitations, we show the capacity of natural language generative AI to perform basic structural biology modeling and interaction analysis with atomic-scale accuracy.","version":"1.6","doi":"10.1101/2024.01.10.575113","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.09.593095","pub_date":"2024-5-12","title":"Lateral Flow Assay Sensitivity and Signal Enhancement via Laser \u00b5-Machined Constrains in Nitrocellulose Membrane","abstract":"Multiplex lateral flow assay (LFA) is a handful diagnostic technology that can identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses in one strip, which can be tested at the point-of-care without the need for equipment or skilled personnel outside the laboratory. Although its simplicity and practicality make it an appealing solution, it remains a grand challenge to substantially enhance the colorimetric LFA sensitivity. The local flow rate constraints imposed in nitrocellulose (NC) membranes via a number of vertical femtosecond laser micromachined microchannels are important for prolonged specific binding interactions. Porous NC membrane surfaces were structured with different widths and densities \u03bc-channels employing a second harmonic of the Yb:KGW femtosecond laser and sample XYZ translation over a microscope objective-focused laser beam. The influence of the microchannel parameters on the vertical wicking speed was evaluated from the video recordings. The obtained results indicated that \u03bc-channel length, width, and density in NC membranes controllably increased the immunological reaction time between the analyte and the labeled antibody by 950%. Image analysis of the colorimetric indicators confirmed that the flow rate delaying strategy enhanced the signal sensitives by 40% compared with pristine NC LFA.","version":"1.1","doi":"10.1101/2024.05.09.593095","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.10.593585","pub_date":"2024-5-10","title":"Chloroquine Up-regulates Expression of SARS-CoV-2 receptor Angiotensin Converting Enzyme-2 in Endothelial Cells","abstract":"The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) posed a serious threat to global public health. Hydroxychloroquine (HCQ), which is a derivative of Chloroquine (CQ), was a WHO-recommended drug to treat COVID-19 with mixed effects. The purpose of the present study is to evaluate the plausible mechanisms of HCQ actions behind its observed mixed effect. We demonstrate that CQ-treatment significantly up-regulates mesenchymal markers and SARS-CoV-2 receptor ACE2 in cultured endothelial cells. The detrimental effect of HCQ in seriously ill COVID-19 patients might be due to CQ-induced increased expression of endothelial ACE2 exacerbating the severity of SARS-CoV-2 infection. Our study warrants further investigation in animal models and humans and caution while prescribing HCQ to patients with an impaired renin-angiotensin-aldosterone system, such as in hypertension, cardiovascular diseases, or chronic kidney disease; particularly with ACE-inhibitors or statin therapy.","version":"1.1","doi":"10.1101/2024.05.10.593585","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.09.593388","pub_date":"2024-5-10","title":"High Throughput Screening with a Primary Human Mucociliary Airway Model Identifies a Small Molecule with Anti-SARS-CoV-2 Activity","abstract":"Respiratory viruses (e.g. influenza, RSV, SARS etc.) attack the proximal airway and cause a wide spectrum of diseases for which we have limited therapies. To date, a few primary human stem cell-based models of the proximal airway have been reported for drug discovery but scaling them up to a higher throughput platform remains a significant challenge. Here we present a microscale, primary human stem cell-based proximal airway model of SARS-CoV-2 infection, which is amenable to moderate-to-high throughput drug screening. The model recapitulates the heterogeneity of infection seen among different patients and with different SARS-CoV-2 variants. We applied this model to screen 2100 compounds from targeted drug libraries using an image-based quantification method. While there were heterogeneous responses across variants for host factor targeting compounds, the direct acting antivirals showed a consistent response and we characterized a new antiviral drug that is effective against both the parental strain and the Omicron variant.","version":"1.1","doi":"10.1101/2024.05.09.593388","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.09.593331","pub_date":"2024-5-09","title":"Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5\u2019 UTR of the viral genome","abstract":"Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the Ubiquitin-like 1 (Ubl1), Nucleic Acid Binding (NAB), \u03b2-coronavirus-Specific Marker (\u03b2SM) domains and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5\u2019 UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5\u2019 UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5\u2019 UTR of viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein.","version":"1.1","doi":"10.1101/2024.05.09.593331","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.02.573936","pub_date":"2024-5-09","title":"Longitudinal transcriptional changes reveal genes from the natural killer cell-mediated cytotoxicity pathway as critical players underlying COVID-19 progression","abstract":"Patients present a wide range of clinical severities in response SARS-CoV-2 infection, but the underlying molecular and cellular reasons why clinical outcomes vary so greatly within the population remains unknown. Here, we report that negative clinical outcomes in severely ill patients were associated with divergent RNA transcriptome profiles in peripheral immune cells compared with mild cases during the first weeks after disease onset. Protein-protein interaction analysis indicated that early-responding cytotoxic NK cells were associated with an effective clearance of the virus and a less severe outcome. This innate immune response was associated with the activation of select cytokine-cytokine receptor pathways and robust Th1/Th2 cell differentiation profiles. In contrast, severely ill patients exhibited a dysregulation between innate and adaptive responses affiliated with divergent Th1/Th2 profiles and negative outcomes. This knowledge forms the basis of clinical triage that may be used to preemptively detect high-risk patients before life-threatening outcomes ensue. - Mild COVID-19 patients displayed an early transcriptional commitment with NK cell function, whereas severe patients do so with neutrophil function. - The identified co-expressed genes give insights into a coordinated transcriptional program of NK cell cytotoxic activity being associated with mild patients. - Key checkpoints of NK cell cytotoxicity that were enriched in mild patients include: KLRD1, CD247, and IFNG. - The early innate immune response related to NK cells connects with the Th1/Th2 adaptive immune responses, supporting their relevance in COVID-19 progression.","version":"1.2","doi":"10.1101/2024.01.02.573936","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.09.593284","pub_date":"2024-5-09","title":"Broadening sarbecovirus neutralization with bispecific antibodies combining distinct conserved targets on the receptor binding domain","abstract":"Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2-02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2-02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.","version":"1.1","doi":"10.1101/2024.05.09.593284","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.08.593102","pub_date":"2024-5-09","title":"varVAMP: automated pan-specific primer design for tiled full genome sequencing and qPCR of highly diverse viral pathogens","abstract":"Time- and cost-saving surveillance of viral pathogens is achieved by tiled sequencing in which a viral genome is amplified in overlapping PCR amplicons and qPCR. However, designing pan-specific primers for viral pathogens that have high genomic variability represents a major challenge. Here, we present a bioinformatics command-line tool, called varVAMP (variable virus amplicons). It relies on multiple sequence alignments of highly variable virus sequences and enables automatic pan-specific primer design for qPCR or tiled amplicon whole genome sequencing. The varVAMP software guarantees pan-specificity by two means: it designs primers in regions with minimal variability and introduces degenerate nucleotides into primer sequences to compensate for common sequence variations. We demonstrate varVAMP\u2019s utility by designing and evaluating novel pan-specific primer schemes suitable for sequencing the genomes of SARS-CoV-2, Hepatitis E virus, rat Hepatitis E virus, Hepatitis A virus, Borna-disease-virus-1, and Poliovirus. Moreover, we established highly sensitive and specific Poliovirus qPCR assays that could potentially simplify current Poliovirus surveillance. Importantly, wet-lab and bioinformatic techniques established for SARS-CoV-2 tiled amplicon sequencing were readily transferable to these new primer schemes and will allow sequencing laboratories to extend their established methodology to other human pathogens.","version":"1.1","doi":"10.1101/2024.05.08.593102","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.08.593105","pub_date":"2024-5-08","title":"Covalent DNA-Encoded Library Workflow Drives Discovery of SARS-CoV-2 Non-structural Proteins Inhibitors","abstract":"The global coronavirus disease 2019 (COVID-19) pandemic persists, with the ongoing mutation of the virus. Consequently, the development of inhibitors with diverse binding modes and mechanisms of action, along with the elucidation of novel binding sites is of paramount importance. The 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) are two validated cysteine proteases that cleave the viral polyprotein and are essential for viral replication. In this study, we utilized covalent DNA-Encoded libraries (CoDELs) workflow to identify two series of triazine-based covalent inhibitors targeting 3CLpro and PLpro. Molecular docking facilitated the identification of optimization pathways, further refined through medicinal chemistry efforts, leading to the development of the non-peptide 3CLpro inhibitor LU9, which exhibited an IC50 value of 0.34 \u03bcM, and crystal structure of LU10 revealed a unique binding mode within the active site. Additionally, the X-ray cocrystal structure of SARS-CoV-2 PLpro with XD5 uncovered a previously unexplored binding site, adjacent to the catalytic pocket, providing an opportunity for further development of PLpro inhibitors. Overall, these novel compounds serve as valuable chemical probes for target validation and represent promising drug candidates for the continued development of SARS-CoV-2 antivirals.","version":"1.1","doi":"10.1101/2024.05.08.593105","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.08.593110","pub_date":"2024-5-08","title":"TMEM106B-mediated SARS-CoV-2 infection allows for robust ACE2-independent infection in vitro but not in vivo","abstract":"Angiotensin converting enzyme 2 (ACE2) serves as the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ACE2-independent entry has been observed in vitro for SARS-CoV-2 strains containing the E484D amino acid substitution in the spike protein. In this study, we conducted a whole genome CRISPR-Cas9 knockout screen using a SARS-CoV-2 strain containing the spike-E484D substitution (SARS-CoV-2MA1) to identify the ACE2-independent entry mechanisms. Our findings revealed that SARS-CoV-2MA1 infection in HEK293T cells relied on heparan sulfate and endocytic pathways, with TMEM106B emerging as the most significant contributor. While SARS-CoV-2MA1 productively infected human brain organoids and K18-hACE2 mouse brains, it did not infect C57BL/6J or Ifnar-/- mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is a protein that is predominantly expressed in the brain, did not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in wild-type mice. Importantly, SARS-CoV-2MA1 did not replicate in Ace2-/- mouse respiratory tracts. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2E484D is likely a phenomenon specific to in vitro conditions, with no apparent clinical implications.","version":"1.1","doi":"10.1101/2024.05.08.593110","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.05.592584","pub_date":"2024-5-06","title":"Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern","abstract":"The ongoing emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from COVID-19 convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596\u2019s favorable binding profile, we designed a series of bispecific antibodies (bsAbs) termed CoV2-biRNs, that featured both NTD and RBD specificities. Notably, two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, EG.5.1, and BA.2.86, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 significantly reduced the viral load within the lungs of K18-hACE2 mice following challenge with SARS-CoV-2 XBB.1.5. In conclusion, our NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs. Bispecific antibodies with a highly cross-reactive NTD antibody demonstrate resilience to SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2024.05.05.592584","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.03.592493","pub_date":"2024-5-05","title":"The effect of Citrus reticulata peel extract containing hesperidin on inhibition of SARS-CoV-2 infection based on pseudovirus entry assays","abstract":"Orange (Citrus reticulata Blanco) peel contains a flavonoid glycoside hesperidin (HSD) as the primary component. HSD, upon enzymatic hydrolysis, forms hesperetin (HST) aglycone derivate. These two flavonoids have been predicted to have in-silico affinities for ACE2 and SARS-CoV-2 spike, crucial proteins in SARS-CoV-2 infection mechanisms. However, in vitro antiviral testing of orange peel extract, HSD, and HST has not been reported. This study presents for the first time a pseudovirus entry assay approach to test the anti-SARS-CoV-2 effect of HSD, HST, and orange peel extract prepared by hydrodynamic cavitation (HCV). We used a non-virulent pseudovirus model as an alternative to the original virus to target the entry point and enable research to be conducted outside the BSL-3 facility. Based on HPLC analysis, the test results showed that HCV contained HSD at about 4% w/w. Moreover, HSD 1 and 10 \u03bcM, HST 10 \u03bcM, and HCV 1 \u03bcg/ml showed inhibition of pseudovirus entry in 293/hACE2 cells with percentages inhibition 25.92, 37.40, 27.32, and 38.97 %, respectively. Despite HCV 1 \u03bcg/ml showing about 6 % lower inhibitory activity than HSD 1 \u03bcM in pseudovirus entry assay, it holds potential as a supplement or source of raw material for HSD as a SARS-CoV-2 antiviral.","version":"1.1","doi":"10.1101/2024.05.03.592493","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.01.591558","pub_date":"2024-5-03","title":"Evidence that the SARS-CoV-2 S protein undergoes a conformational change at the Golgi Complex that leads to the formation of virus neutralising antibody binding epitopes in the S1 protein subunit","abstract":"Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralizing antibodies.","version":"1.1","doi":"10.1101/2024.05.01.591558","journal":"bioRxiv","score":null},{"id":"10.1101/2024.05.01.591800","pub_date":"2024-5-03","title":"Interferon-Inducible Guanylate-Binding Protein 5 Inhibits Replication of Multiple Viruses by Binding to the Oligosaccharyltransferase Complex and Inhibiting Glycoprotein Maturation","abstract":"Viral infection induces production of type I interferons and expression of interferon-stimulated genes (ISGs) that play key roles in inhibiting viral infection. Here, we show that the ISG guanylate-binding protein 5 (GBP5) inhibits N-linked glycosylation of key proteins in multiple viruses, including SARS-CoV-2 spike protein. GBP5 binds to accessory subunits of the host oligosaccharyltransferase (OST) complex and blocks its interaction with the spike protein, which results in misfolding and retention of spike protein in the endoplasmic reticulum likely due to decreased N-glycan transfer, and reduces the assembly and release of infectious virions. Consistent with these observations, pharmacological inhibition of the OST complex with NGI-1 potently inhibits glycosylation of other viral proteins, including MERS-CoV spike protein, HIV-1 gp160, and IAV hemagglutinin, and prevents the production of infectious virions. Our results identify a novel strategy by which ISGs restrict virus infection and provide a rationale for targeting glycosylation as a broad antiviral therapeutic strategy. The interferon-stimulated gene GBP5 is induced by SARS-CoV-2 infection in vitro and in vivo. ER-localized GBP5 restricts N-linked glycosylation of SARS-CoV-2 spike protein, leading to protein misfolding and preventing transport to the Golgi apparatus. GBP5 binds to OST complex accessory proteins and potentially blocks access of the catalytic subunit to the spike protein. GBP5 inhibits N-glycosylation of key proteins in multiple viruses, including SARS-CoV-2 Pharmacological inhibition of OST blocks host cell infection by SARS-CoV-2, variants of concern, HIV-1, and IAV. Viral infection induces production of type I interferons and expression of interferon-stimulated genes (ISGs) that play key roles in inhibiting viral infection. We found that the interferon-stimulated gene GBP5 is induced by SARS-CoV-2 infection in vitro and in vivo. GBP5 inhibits N-glycosylation of key proteins in multiple viruses, including SARS-CoV-2. Importantly, pharmacological inhibition of Oligosaccharyltransferase (OST) Complex blocks host cell infection by SARS-CoV-2, variants of concern, HIV-1, and IAV, indicating future translational application of our findings.","version":"1.1","doi":"10.1101/2024.05.01.591800","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.26.591384","pub_date":"2024-5-01","title":"Refining SARS-CoV-2 Intra-host Variation by Leveraging Large-scale Sequencing Data","abstract":"Understanding the evolution of viral genomes is essential for elucidating how viruses adapt and change over time. Analyzing intra-host single nucleotide variants (iSNVs) provides key insights into the mechanisms driving the emergence of new viral lineages, which are crucial for predicting and mitigating future viral threats. Despite the potential of next-generation sequencing (NGS) to capture these iSNVs, the process is fraught with challenges, particularly the risk of capturing sequencing artifacts that may result in false iSNVs. To tackle this issue, we developed a workflow designed to enhance the reliability of iSNV detection in large heterogeneous collections of NGS libraries. We use over 130,000 publicly available SARS-CoV-2 NGS libraries to show how our comprehensive workflow effectively distinguishes emerging viral mutations from sequencing errors. This approach incorporates rigorous bioinformatics protocols, stringent quality control metrics, and innovative usage of dimensionality reduction methods to generate representations of this high-dimensional dataset. We identified and mitigated batch effects linked to specific sequencing centers around the world and introduced quality control metrics that consider strand coverage imbalance, enhancing iSNV reliability. Additionally, we pioneer the application of the PHATE visualization approach to genomic data and introduce a methodology that quantifies how related groups of data points are within a two-dimensional space, enhancing our ability to explain clustering patterns based on their shared genetic characteristics. Our workflow sheds light on the complexities of viral genomic analysis with state-of-the-art sequencing technologies and advances the detection of accurate intra-host mutations, opening the door for an enhanced understanding of viral adaptation mechanisms.","version":"1.2","doi":"10.1101/2024.04.26.591384","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.10.579717","pub_date":"2024-4-30","title":"FABP4 as a Therapeutic Host Target Controlling SARS-CoV2 Infection","abstract":"Host metabolic fitness is a critical determinant of infectious disease outcomes. Obesity, aging, and other related metabolic disorders are recognized as high-risk disease modifiers for respiratory infections, including coronavirus infections, though the underlying mechanisms remain unknown. Our study highlights fatty acid-binding protein 4 (FABP4), a key regulator of metabolic dysfunction and inflammation, as a modulator of SARS-CoV-2 pathogenesis, correlating strongly with disease severity in COVID-19 patients. We demonstrate that loss of FABP4 function, by genetic or pharmacological means, reduces SARS-CoV2 replication and disrupts the formation of viral replication organelles in adipocytes and airway epithelial cells. Importantly, FABP4 inhibitor treatment of infected hamsters diminished lung viral titers, alleviated lung damage and reduced collagen deposition. These findings highlight the therapeutic potential of targeting host metabolism in limiting coronavirus replication and mitigating the pathogenesis of infection.","version":"1.2","doi":"10.1101/2024.02.10.579717","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.29.591599","pub_date":"2024-4-30","title":"L-Shaped Distributions of the Relative Substitution Rates (c/\u00b5) in SARS-COV-2 with or without Molecular Clocks, Challenging Mainstream Evolutionary Theories","abstract":"A definitive test to quantify fitness changes of mutations is required to end a continuing 50-year \u201cneutralist-selectionist\u201d debate in evolutionary biology. Our previous work introduced a substitution-mutation rate ratio c/\u00b5 test (c: substitution rate in Translated Region/TR or UnTranslated Region/UTR; \u00b5: mutation rate) to quantify the selection pressure and thus the proportions of strictly neutral, nearly neutral, beneficial, and deleterious mutations in a genome. Intriguingly, both a L-shaped probability distribution of c/\u00b5 and molecular clock were observed for SARS-COV-2\u2019s genome. We found that the proportion of the different mutation types from the distribution is not consistent with the hypotheses of the three existing evolution theories (Kimura\u2019s Neutral Theory/KNT, Ohta\u2019s Nearly Neutral Theory/ONNT and the Selectionist Theory/ST), and a balance condition explains the molecular clock, thus we proposed a new theory named as Near-Neutral Balanced Selectionist Theory (NNBST). In this study, the c/\u00b5 analysis was extended beyond the genome to 26 TRs, 12 UTRs, and 10 TRSs (Transcriptional Regulatory Sequences) of SARS-COV-2. While L-shaped probability distributions of c/\u00b5 were observed for all of 49 segments, molecular clocks were observed for only 24 segments, supporting NNBST and Near-Neutral Unbalanced Selectionist Theory (NNUST) to explain the molecular evolution of 24/25 segments with/without molecular clocks. Thus, the Near-Neutral Selectionist Theory (NNST) integrates traditional neutral and selectionist theories to deepen our understanding of how mutation, selection, and genetic drift influence genomic evolution. The \u201cneutralist-selectionist\u201d debate in molecular evolution has been unresolved for 50 years due to the three main theories of molecular evolution (Kimura\u2019s Neutral Theory/KNT, Ohta\u2019s Nearly-Neutral Theory/ONNT, Selectionist Theory/ST) disagreeing on the proportion of neutral mutations (KNT), nearly-neutral deleterious mutations (ONNT) and adaptive mutations (ST) within species. We recently developed a robust method, the c/\u00b5 relative substitution rate test, to quantify the proportion of each mutation type within >11K genomic sequences of SARS-COV-2 RNA virus. Our previous analysis revealed an L-shaped c/\u00b5 probability distribution and a constant substitution rate (e.g., molecular clock) for the SARS-COV-2 genome over 19 months, and the proportions of mutation types were inconsistent with those predicted by the three theories. We thus proposed the Near-Neutral Balanced Selectionist Theory (NNBST) to explain the molecular clock-feature and L-shaped probability distribution for SARS-COV-2. In this study, we extended this analysis to the 25 protein-coding gene segments and 24 non-protein-coding segments of SARS-COV-2. We observed that all 49 segments exhibited an L-shaped probability distribution and 24 out of the 49 segments exhibited a molecular clock, however the remaining 25 segments did not exhibit a molecular clock. We thus propose the Near-Neutral Unbalanced Selectionist Theory (NNUST) and NNBST to explain the segments without/with molecular clock features, respectively. We also coin the Near-Neutral Selectionist Theory (NNST) to combine traditional KNT, ONNT and ST to deepen our understanding of how mutation, selection, and genetic drift influence genomic evolution.","version":"1.1","doi":"10.1101/2024.04.29.591599","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.29.591494","pub_date":"2024-4-30","title":"Altered DNA methylation pattern contributes to differential epigenetic immune signaling in the upper respiratory airway of COVID-19 patients","abstract":"The emergence of SARS-CoV-2 has had a profound adverse impact on global health and continues to remain a threat worldwide. The disease spectrum of COVID-19 ranges from asymptomatic to fatal clinical outcomes especially in the elderly population and in individuals with underlying medical conditions. The impact of COVID-19 on host immune responses and immune cells at the protein and DNA levels remains largely ambiguous. In a case-control study, here we explored the impact of COVID-19 on DNA methylation patterns in the upper respiratory airway to determine how SARS-CoV-2 infection altered the immune status of individuals requiring hospitalization for COVID-19. We performed DNA methylation arrays on nasopharyngeal swabs at inclusion/hospitalization as well as 6 weeks post-inclusion. Our study reveals a distinct DNA methylation pattern in COVID-19 patients compared to healthy controls, characterized by 317 779 differentially methylated CpGs. Notably, within the transcription start sites and gene body, COVID-19 patients exhibited a higher number of genes/CpGs with elevated methylation levels. Enrichment analysis of methylated genes at transcription start sites highlighted the impact on genes associated with inflammatory responses and immune functions. Some SARS-CoV-2 -induced CpG methylations were transient, returning to normal levels by 6 weeks post-inclusion. Enriched genes of interest included IL-17A, a pivotal cytokine implicated with inflammation and healing, and NUP93, associated with antiviral innate immunity. Further, six genes in our data set, OAS1, CXCR5, APP, CCL20, CNR2, and C3AR1, were found in enrichment analysis with previous COVID-19 studies. Additionally, RNAse1 and RNAse2 emerged as key regulators, while IL-18 played a role in various biological processes in COVID-19 patients. Overall, our results demonstrates that COVID-19 has a major impact on the upper airway by modifying the methylation pattern of many genes and this could have implications for the conditioning of the airways and how the individual response to future airway infections.","version":"1.1","doi":"10.1101/2024.04.29.591494","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.25.591047","pub_date":"2024-4-30","title":"Essentiality and dynamic expression of the human tRNA pool during viral infection","abstract":"Human viruses depend on the translation resources of the host cell. A significant translation resource is the tRNA pool of the cell, as human viruses do not encode tRNA genes. Through tRNA sequencing, we inspected the human tRNA pool upon infection of human Cytomegalovirus (HCMV) and SARS-CoV-2. HCMV-induced alterations in tRNA expression were predominantly virus-driven, with minimal influence from the cellular immune response. Notably, specific tRNA post-transcriptional modifications appeared to modulate stability and were susceptible to HCMV manipulation. In contrast, SARS-CoV-2 infection did not significantly impact tRNA expression or modifications. We compared the codon usage of viral genes to the proliferation-differentiation codon-usage signatures of human genes. We found a marked difference between the viruses, with HCMV genes aligning with differentiation codon usage and SARS-CoV-2 genes reflecting proliferation codon usage. We further found that codon usage of structural and gene expression-related viral genes displayed high adaptation to host cell tRNA pools. Through a systematic CRISPR screen targeting human tRNA genes and modification enzymes, we identified specific tRNAs and enzymes that improve or reduce HCMV infectivity and cellular growth. These findings highlight the dynamic interplay between the tRNA pool and viral infection dynamics, shedding light on mechanisms governing host-virus interactions.","version":"1.1","doi":"10.1101/2024.04.25.591047","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.27.591446","pub_date":"2024-4-29","title":"Potent neutralization by a receptor binding domain monoclonal antibody with broad specificity for SARS-CoV-2 JN.1 and other variants","abstract":"SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) \u2013 ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1\u00c5 resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.","version":"1.1","doi":"10.1101/2024.04.27.591446","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.26.591329","pub_date":"2024-4-29","title":"Characterization of Collaborative Cross mouse founder strain CAST/EiJ as a novel model for lethal COVID-19","abstract":"Mutations in SARS-CoV-2 variants of concern (VOCs) have expanded the viral host range beyond primates, and a limited range of other mammals, to mice, affording the opportunity to exploit genetically diverse mouse panels to model the broad range of responses to infection in patient populations. Here we surveyed responses to VOC infection in genetically diverse Collaborative Cross (CC) founder strains. Infection of wild-derived CC founder strains produced a broad range of viral burden, disease susceptibility and survival, whereas most other strains were resistant to disease despite measurable lung viral titers. In particular, CAST/EiJ, a wild-derived strain, developed high lung viral burdens, more severe lung pathology than seen in other CC strains, and a dysregulated cytokine profile resulting in morbidity and mortality. These inbred mouse strains may serve as a valuable platform to evaluate therapeutic countermeasures against severe COVID-19 and other coronavirus pandemics in the future.","version":"1.1","doi":"10.1101/2024.04.26.591329","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.25.591033","pub_date":"2024-4-29","title":"Hidden Challenges in Evaluating Spillover Risk of Zoonotic Viruses using Machine Learning Models","abstract":"Machine learning models have been deployed to assess the zoonotic spillover risk of viruses by identifying their human infectivity potential. However, the scarcity of comprehensive datasets poses a major challenge, limiting the predictable range of viruses. Our study addressed this limitation through two key strategies: constructing expansive datasets across 26 viral families and developing new models leveraging large language models pre-trained on extensive nucleotide sequences. Our approaches substantially boosted our model performance. This enhancement was particularly notable in segmented RNA viruses, which are involved with severe zoonoses but have been overlooked due to limited data availability. Furthermore, models trained on data up to 2018 displayed strong generalization capability for viruses emerging post-2018. Nonetheless, we also found remaining challenges in alerting the zoonotic potential of specific viral lineages, including SARS-CoV-2. Our study elaborates on the models and datasets for predicting viral infectivity and highlights the unresolved issues to fully exploit machine learning in preparing for future zoonotic threats.","version":"1.1","doi":"10.1101/2024.04.25.591033","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.09.531875","pub_date":"2024-4-29","title":"Recent evolutionary origin and localized diversity hotspots of mammalian coronaviruses","abstract":"Several coronaviruses infect humans, with three, including the SARS-CoV2, causing diseases. While coronaviruses are especially prone to induce pandemics, we know little about their evolutionary history, host-to-host transmissions, and biogeography. One of the difficulties lies in dating the origination of the family, a particularly challenging task for RNA viruses in general. Previous cophylogenetic tests of virus-host associations, including in the Coronaviridae family, have suggested a virus-host codiversification history stretching many millions of years. Here, we establish a framework for robustly testing scenarios of ancient origination and codiversification versus recent origination and diversification by host switches. Applied to coronaviruses and their mammalian hosts, our results support a scenario of recent origination of coronaviruses in bats and diversification by host switches, with preferential host switches within mammalian orders. Hotspots of coronavirus diversity, concentrated in East Asia and Europe, are consistent with this scenario of relatively recent origination and localized host switches. Spillovers from bats to other species are rare, but have the highest probability to be towards humans than to any other mammal species, implicating humans as the evolutionary intermediate host. The high host-switching rates within orders, as well as between humans, domesticated mammals, and non-flying wild mammals, indicates the potential for rapid additional spreading of coronaviruses across the world. Our results suggest that the evolutionary history of extant mammalian coronaviruses is recent, and that cases of long-term virus\u2013host codiversification have been largely over-estimated.","version":"1.3","doi":"10.1101/2023.03.09.531875","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.25.591137","pub_date":"2024-4-26","title":"An amyloidogenic fragment of the SARS CoV-2 envelope protein promotes serum amyloid A misfolding and fibrillization","abstract":"SARS CoV-2 infection can affect a surprising number of organs in the body and cause symptoms such as abnormal blood coagulation, fibrinolytic disturbances, and neurodegeneration. Our study delves into the intricate pathogenic potential of a SARS-CoV-2 envelope protein peptide, shedding light on its implications for multi-organ effects and amyloid formation. Specifically, we focus on the peptide SK9 or SFYVYSRVK derived from the C-terminus of human SARS coronavirus 2 envelope protein. We demonstrate that SK9 containing peptides readily form classic amyloid structures consistent with predictions of amyloid aggregation algorithms. In vivo, overexpression of proteases such as neutrophil elastase during inflammation can potentially lead to C-terminal peptides containing SK9. We also demonstrate that SK9 can promote the fibrillization of SAA, a protein marker of acute inflammation. Our investigations reveal that the aromatic residues Phe2 and Tyr3 of SK9 play a pivotal role in its amyloidogenic function. We show that the primary sites of SK9-SAA binding lie in the amyloidogenic hotspots of SAA itself. Our results highlight two possible complications of SARS CoV-2 infection in individuals with hyper-inflammation either due to amyloids arising from SK9 containing peptides or SK9-induced AA amyloidosis.","version":"1.1","doi":"10.1101/2024.04.25.591137","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.24.590786","pub_date":"2024-4-26","title":"Virological characteristics of the SARS-CoV-2 KP.2 variant","abstract":"The JN.1 variant (BA.2.86.1.1), arising from BA.2.86(.1) with the S:L455S substitution, exhibited increased fitness and outcompeted the previous dominant XBB lineage by the biggening of 2024. JN.1 subsequently diversified, leading to the emergence of descendants with spike (S) protein substitutions such as S:R346T and S:F456L. Particularly, the KP.2 (JN.1.11.1.2) variant, a descendant of JN.1 bearing both S:R346T and S:F456L, is rapidly spreading in multiple regions as of April 2024. Here, we investigated the virological properties of KP.2. KP.2 has three substitutions in the S protein including the two above and additional one substitution in non-S protein compared with JN.1. We estimated the relative effective reproduction number (Re) of KP.2 based on the genome surveillance data from the USA, United Kingdom, and Canada where >30 sequences of KP.2 has been reported, using a Bayesian multinomial logistic model. The Re of KP.2 is 1.22-, 1.32-, and 1.26-times higher than that of JN.1 in USA, United Kingdom, and Canada, respectively. These results suggest that KP.2 has higher viral fitness and potentially becomes the predominant lineage worldwide. Indeed, as of the beginning of April 2024, the estimated variant frequency of KP.2 has already reached 20% in United Kingdom. The pseudovirus assay showed that the infectivity of KP.2 is significantly (10.5-fold) lower than that of JN.1. We then performed a neutralization assay using monovalent XBB.1.5 vaccine sera and breakthrough infection (BTI) sera with XBB.1.5, EG.5, HK.3 and JN.1 infections. In all cases, the 50% neutralization titer (NT50) against KP.2 was significantly lower than that against JN.1. Particularly, KP.2 shows the most significant resistance to the sera of monovalent XBB.1.5 vaccinee without infection (3.1-fold) as well as those who with infection (1.8-fold). Altogether, these results suggest that the increased immune resistance ability of KP.2 partially contributes to the higher Re more than previous variants including JN.1.","version":"1.1","doi":"10.1101/2024.04.24.590786","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.24.590836","pub_date":"2024-4-25","title":"Delineating the functional activity of antibodies with cross-reactivity to SARS-CoV-2, SARS-CoV-1 and related sarbecoviruses","abstract":"The recurring spillover of pathogenic coronaviruses and demonstrated capacity of sarbecoviruses, such SARS-CoV-2, to rapidly evolve in humans underscores the need to better understand immune responses to this virus family. For this purpose, we characterized the functional breadth and potency of antibodies targeting the receptor binding domain (RBD) of the spike glycoprotein that exhibited cross-reactivity against SARS-CoV-2 variants, SARS-CoV-1 and sarbecoviruses from diverse clades and animal origins with spillover potential. One neutralizing antibody, C68.61, showed remarkable neutralization breadth against both SARS-CoV-2 variants and viruses from different sarbecovirus clades. C68.61, which targets a conserved RBD class 5 epitope, did not select for escape variants of SARS-CoV-2 or SARS-CoV-1 in culture nor have predicted escape variants among circulating SARS-CoV-2 strains, suggesting this epitope is functionally constrained. We identified 11 additional SARS-CoV-2/SARS-CoV-1 cross-reactive antibodies that target the more sequence conserved class 4 and class 5 epitopes within RBD that show activity against a subset of diverse sarbecoviruses with one antibody binding every single sarbecovirus RBD tested. A subset of these antibodies exhibited Fc-mediated effector functions as potent as antibodies that impact infection outcome in animal models. Thus, our study identified antibodies targeting conserved regions across SARS-CoV-2 variants and sarbecoviruses that may serve as therapeutics for pandemic preparedness as well as blueprints for the design of immunogens capable of eliciting cross-neutralizing responses. There is a large collection of sarbecoviruses related to SARS-CoV-2 circulating in animal reservoirs with the potential to spillover into humans. Neutralizing antibodies have the potential to protect against infection, although viral escape is common. In this study, we isolated several monoclonal antibodies that show broad activity against different sarbecoviruses. The antibodies target epitopes in the core of the receptor binding domain that are highly conserved in sequence across sarbecoviruses and emerging SARS-CoV-2 variants. One antibody showed remarkable breadth against both SARS-CoV-1 variants as well as diverse sarbecoviruses. The results of deep mutational scanning suggest that mutations at these predicted sites of escape may functionally constrain viral fitness. Our functional profiling of cross-reactive antibodies highlights vulnerable sites of sarbecoviruses, with some antibodies poised as broadly neutralizing candidates for therapeutic use against future sarbecovirus emergence.","version":"1.1","doi":"10.1101/2024.04.24.590836","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.23.590789","pub_date":"2024-4-24","title":"SARS-CoV-2 Omicron Envelope T9I adaptation confers resistance to autophagy","abstract":"To date, five variants of concern (VOCs) of SARS-CoV-2 have emerged that show increased fitness and/or immune evasion. While the continuously evolving escape from humoral immune responses has been analyzed in detail, adaptation of SARS-CoV-2 to human innate immune defenses such as autophagy is less understood. Here, we demonstrate that mutation T9I in the structural envelope (E) protein confers autophagy resistance of Omicron VOCs (BA.1, BA.5 and XBB.1.5) compared to 2020 SARS-CoV-2 or the Delta VOC. Mechanistic analyses revealed that Omicron-associated E T9I shows increased inhibition of autophagic flux and colocalization/interaction with autophagosomes, thus shielding incoming SARS-CoV-2 S pseudotyped virions from autophagy. Rare Omicron isolates carrying ancestral E T9 remain sensitive towards autophagy whereas recombinant early 2020 SARS-CoV-2 expressing E T9I shows increases resistance against autophagy. Our data indicate that the E T9I mutation drives autophagy resistance of the Omicron variants and thus may have contributed to their effective spread.","version":"1.1","doi":"10.1101/2024.04.23.590789","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.23.24306243","pub_date":"2024-04-24","title":"Development of a simple and highly sensitive virion concentration method to detect SARS-CoV-2 in saliva","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Controlling novel coronavirus pandemic infection (COVID-19) is a global challenge, and highly sensitive testing is essential for effective control. The saliva is a promising sample for high-sensitivity testing because it is easier to collect than nasopharyngeal swab samples and allows large-volume testing.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We developed a simple SARS-CoV-2 concentration method from saliva samples that can be completed in less than 60 min. We performed a spike test using 12 ml of saliva samples obtained from healthy volunteer people, and the developed method performance was evaluated by comparison using a combination of automatic nucleic acid extraction followed by RT-qPCR detection. In saliva spike tests using a 10-fold dilution series of SARS-CoV-2, the developed method was consistently 100-fold more sensitive than the conventional method.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The developed method can improve the sensitivity of the SARS-CoV-2 test using saliva and speed up and save labor in screening tests by pooling many samples. Furthermore, the developed method has the potential to contribute to the highly sensitive detection of various human and animal viral pathogens from the saliva and various clinical samples.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Highlight</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>A method has been developed to detect SARS-CoV-2 from human saliva with 100 times higher sensitivity than conventional methods.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>The developed method combines simple pretreatment within 60 min with conventional nucleic acid extraction and RT-qPCR.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>This method can be applied for more sensitive virus testing from individual saliva.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>This method can potentially be applied to screening more than 100 saliva samples while maintaining the equivalent detection power of conventional methods.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>The method can be adapted to improve the sensitivity of detecting various pathogens from human and animal saliva.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2024.04.23.24306243","journal":"medRxiv","score":null},{"id":"10.1101/2024.04.24.587375","pub_date":"2024-4-24","title":"Bridging genomic gaps: A versatile SARS-CoV-2 benchmark dataset for adaptive laboratory workflows","abstract":"Genomic sequencing\u2019s adoption in public health laboratories (PHLs) for pathogen surveillance is innovative yet challenging, particularly in the realm of bioinformatics. Low- and middle-income countries (LMICs) face increased difficulties due to supply chain volatility, workforce training, and unreliable infrastructure such as electricity and internet services. These challenges also extend to high-income countries (HICs) where bioinformatics is nascent in PHLs and hampered by a lack of specialized skills and computational infrastructure. This underlines the urgency for flexible and resource-aware strategies in genomic sequencing to improve global pathogen surveillance. In response to these challenges, the present research was conducted to identify and analyse key variables influencing the quality and accuracy of amplicon sequence data. An extensive benchmark dataset was developed that encompassed a diverse collection of isolates, viral loads, primer schemes, library preparation methods, sequencing technologies, and basecalling models, totalling 750 sequences. This dataset was analysed with bioinformatic workflows selected for varying levels of technical capacity. The evaluation focused on quality metrics, consensus accuracy, and common genomic epidemiological indicators. The analysis uncovers complex interactions between multiple parameters in laboratory and bioinformatic processes. emphasising resource-constrained PHLs, practical guidelines are proposed. Insights from the benchmark dataset aim to guide the establishment of specific laboratory and bioinformatics protocols for amplicon sequencing in these settings. The findings can also be used to guide the creation of specialised training curricula, further advancing genomic equity. The benchmark dataset itself allows laboratories to customise and evaluate workflows, catering to their distinct requirements and capacities. Such a holistic approach is imperative to build the capacity to monitor pathogens worldwide. This study marks a step toward equity in the field of pathogen genomics, especially for resource-constrained PHLs. It develops and evaluates a comprehensive amplicon sequencing benchmark dataset, offering vital insights for PHLs engaged in genomic surveillance. In particular, the study finds that the choice of basecaller model has a minimal impact on the quality and accuracy of consensus sequences derived from ONT data, which is crucial for labs with limited computational resources. It also highlights the effectiveness of longer amplicons in ensuring consistent coverage and reducing amplicon dropouts at higher viral loads. While Illumina remains a gold standard for data quality, the combination of the Midnight primer scheme with ONT\u2019s Rapid library preparation is shown to be a viable alternative, reducing costs, procedural complexity, and hands-on time. The study synthesises these findings into practical guidelines to aid in the development of amplicon sequencing workflows for SARS-CoV-2 with implications for other pathogens.","version":"1.1","doi":"10.1101/2024.04.24.587375","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.22.590623","pub_date":"2024-4-24","title":"A unified theory for predicting pathogen mutual invasibility and co-circulation","abstract":"A key aim in the dynamics and control of infectious diseases is predicting competitive outcomes of pathogen interactions. Observed pathogen community structure indicates both considerable coexistence of related variants and spectacular instances of replacement, notably in seasonal influenza and SARS-CoV-2. However, an overall comparative quantitative framework for invasion and coexistence remains elusive. Inspired by modern ecological coexistence theory, we address this gap by developing pathogen invasion theory (PIT) and test the resulting framework against empirical systems. PIT predicts near-universal mutual invasibility across major pathogen systems, including seasonal influenza strains and SARS-CoV-2 variants. Predicting co-circulation from mutual invasibility further depends on the extent of overcompensatory susceptible depletion dynamics. Our analyses highlight the central role of immuno-epidemiological factors in determining pathogen coexistence and community structure.","version":"1.1","doi":"10.1101/2024.04.22.590623","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.22.589104","pub_date":"2024-4-23","title":"Deep, unbiased and quantitative mass spectrometry-based plasma proteome analysis of individual responses to mRNA COVID-19 vaccine","abstract":"Global campaign against COVID-19 have vaccinated a significant portion of the world population in recent years. Combating the COVID-19 pandemic with mRNA vaccines played a pivotal role in the global immunization effort. However, individual responses to a vaccine are diverse and lead to varying vaccination efficacy. Despite significant progress, a complete understanding of the molecular mechanisms driving the individual immune response to the COVID-19 vaccine remains elusive. To address this gap, we combined a novel nanoparticle-based proteomic workflow with tandem mass tag (TMT) labeling, to quantitatively assess the proteomic changes in a cohort of 12 volunteers following two doses of the Pfizer-BioNTech mRNA COVID-19 vaccine. This optimized protocol seamlessly integrates comprehensive proteome analysis with enhanced throughput by leveraging the enrichment of low-abundant plasma proteins by engineered nanoparticles. Our data demonstrate the ability of this nanoparticle-based workflow to quantify over 3,000 proteins from 48 human plasma samples, providing the deepest view into COVID-19 vaccine-related plasma proteome study. We identified 69 proteins exhibiting a boosted response to the vaccine after the second dose. Additionally, 74 proteins were differentially regulated between seven volunteers, who contracted COVID-19 despite receiving two doses of the vaccine, and the ones who did not contract COVID-19. These findings offer valuable insights into individual variability in response to vaccination, demonstrating the potential of personalized medicine approaches in vaccine development.","version":"1.1","doi":"10.1101/2024.04.22.589104","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.22.590420","pub_date":"2024-4-22","title":"Novel Automatic Classification of Human Adult Lung Alveolar Type II Cells Infected with SARS-CoV-2 through Deep Transfer Learning Approach","abstract":"SARS-CoV-2 can infect alveoli, inducing a lung injury and thereby impairing the lung function. Healthy alveolar type II (AT2) cells play a major role in lung injury repair as well as keeping alveoli space free from fluids, which is not the case for infected AT2 cells. Unlike previous studies, this novel study aims to automatically differentiate between healthy and infected AT2 cells with SARS-CoV-2 through using efficient AI-based models, which can aid in disease control and treatment. Therefore, we introduce a highly accurate deep transfer learning (DTL) approach that works as follows. First, we downloaded and processed 286 images pertaining to healthy and infected human AT2 (hAT2) cells, obtained from the electron microscopy public image archive. Second, we provided processed images to two DTL computations to induce ten DTL models. The first DTL computation employs five pre-trained models (including DenseNet201 and ResNet152V2) trained on more than million images from ImageNet database to extract features from hAT2 images. Then, flattening and providing the output feature vectors to a trained densely connected classifier with Adam optimizer. The second DTL computation works in a similar manner with a minor difference in which we freeze the first layers for feature extraction in pre-trained models while unfreezing and training the next layers. Compared to TFtDenseNet201, experimental results using five-fold cross-validation demonstrate that TFeDenseNet201 is 12.37 \u00d7 faster and superior, yielding the highest average ACC of 0.993 (F1 of 0.992 and MCC of 0.986) with statistical significance (p < 2.2 \u00d7 10\u221216 from a t-test).","version":"1.1","doi":"10.1101/2024.04.22.590420","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.22.590578","pub_date":"2024-4-22","title":"Structural basis for the inhibition of coronaviral main proteases by PF-00835231","abstract":"The main protease (Mpro) of coronaviruses plays a key role in viral replication, thus serving as a hot target for drug design. It has been proven that PF-00835231 is promising inhibitor of SARS-CoV-2 Mpro. Here, we report the inhibition potency of PF-00835231 against SARS-CoV-2 Mpro and seven Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) from SARS-CoV-2 variants. The results confirm that PF-00835231 has broad-spectrum inhibition against various coronaviral Mpros. In addition, the crystal structures of SARS-CoV-2 Mpro, SARS-CoV Mpro, MERS-CoV Mpro, and seven SARS-CoV-2 Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) in complex with PF-00835231 are solved. A detailed analysis of these structures reveal key determinants essential for inhibition and elucidates the binding modes of different coronaviral Mpros. Given the importance of the main protease for the treatment of coronaviral infection, structural insights into the Mpro inhibition by PF-00835231 can accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.","version":"1.1","doi":"10.1101/2024.04.22.590578","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.19.590267","pub_date":"2024-4-19","title":"Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L","abstract":"Recent discoveries expanding the spectrum of ubiquitylation substrates to include non-proteinaceous molecules have broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is not shared with another DELTEX family member DTX2. DTX3L shows preference for the 3\u2019-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2 and SARS-CoV-2 PLpro. Overall, our study provides evidence for reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications.","version":"1.1","doi":"10.1101/2024.04.19.590267","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.17.589886","pub_date":"2024-4-18","title":"Accurate evaluation of live-virus microneutralization for SARS-CoV-2 variant JN.1","abstract":"Emerging SARS-CoV-2 variants require rapid assessments of pathogenicity and evasion of existing immunity to inform policy. A crucial component of these assessments is accurate estimation of serum neutralising antibody titres using cultured live virus isolates. Here, we report our updated culture methods for Omicron sub-variant JN.1 using Caco-2 cells and the subsequent evaluation of neutralising antibody titres (nAbTs) in recipients of BNT162b2-XBB.1.5 monovalent and the Ancestral/BA.5 containing bivalent vaccines. We compared culture of JN.1 in either Vero V1 cells or Caco-2 cells, finding culture in Vero V1 either resulted in low-titre stocks or induced crucial mutations at the Spike furin cleavage site. Using the sequence-clean culture stocks generated in Caco-2 cells, we assessed serum samples from 71 healthy adults eligible for a COVID-19 vaccination given as a 5th dose booster: all participants had detectable nAbs against JN.1 prior to vaccination, with baseline/pre-existing nAbTs between both vaccine groups comparable (p = 0.240). However, nAbTs against JN.1 post-vaccination were 2.6-fold higher for recipients of the monovalent XBB1.5 vaccine than the BA.4/5 bivalent vaccine (p<0.001). Regular re-appraisal of methods involved in the evaluation of new variants is required to ensure robust data are used to underpin crucial severity assessments as variants arise and vaccine strain selection decisions.","version":"1.1","doi":"10.1101/2024.04.17.589886","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.16.589585","pub_date":"2024-4-18","title":"Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C)","abstract":"Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease. MIS-C is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Limited studies tested cellular function ex vivo to understand the aberrant immune response in MIS-C. We found dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease","version":"1.1","doi":"10.1101/2024.04.16.589585","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.16.589808","pub_date":"2024-4-18","title":"Bond strength between receptor binding domain of spike protein and human angiotensin converting enzyme-2 using machine learning","abstract":"The spike protein (S-protein) of SARS-CoV-2 plays an important role in binding, fusion, and host entry. In this study, we have predicted interatomic bond strength between receptor binding domain (RBD) and angiotensin converting enzyme-2 (ACE2) using machine learning (ML), that matches with expensive ab initio calculation result. We collected bond order result from ab initio calculations. We selected a total of 18 variables such as bond type, bond length, elements and their coordinates, and others, to train ML models. We then trained five well-known regression models, namely, Decision Tree regression, KNN Regression, XGBoost, Lasso Regression, and Ridge Regression. We tested these models on two different datasets, namely, Wild type (WT) and Omicron variant (OV). In the first setting, we used 90% of each dataset for training and 10% for testing to predict the bond order. XGBoost model outperformed all the other models in the prediction of the WT dataset. It achieved an R2 Score of 0.997. XGBoost also outperformed all the other models with an R2 score of 0.9998 in the prediction of the OV dataset. In the second setting, we trained all the models on the WT (or OV) dataset and predicted the bond order on the OV (or WT) dataset. Interestingly, Decision Tree outperformed all the other models in both cases. It achieved an R2 score of 0.997.","version":"1.1","doi":"10.1101/2024.04.16.589808","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.18.590061","pub_date":"2024-4-18","title":"ACE2-independent sarbecovirus cell entry is supported by TMPRSS2-related enzymes and reduces sensitivity to antibody-mediated neutralization","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2, demonstrated that zoonotic transmission of animal sarbecoviruses threatens human health but the determinants of transmission are incompletely understood. Here, we show that most spike (S) proteins of horseshoe bat and Malayan pangolin sarbecoviruses employ ACE2 for entry, with human and raccoon dog ACE2 exhibiting broad receptor activity. The insertion of a multibasic cleavage site into the S proteins increased entry into human lung cells driven by most S proteins tested, suggesting that acquisition of a multibasic cleavage site might increase infectivity of diverse animal sarbecoviruses for the human respiratory tract. In contrast, two bat sarbecovirus S proteins drove cell entry in an ACE2-independent, trypsin-dependent fashion and several ACE2-dependent S proteins could switch to the ACE2-independent entry pathway when exposed to trypsin. Several TMPRSS2-related cellular proteases but not the insertion of a multibasic cleavage site into the S protein allowed for ACE2-independent entry in the absence of trypsin and may support viral spread in the respiratory tract. Finally, the pan-sarbecovirus antibody S2H97 enhanced cell entry driven by two S proteins and this effect was reversed by trypsin. Similarly, plasma from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, and use of the ACE2-independent, trypsin-dependent pathway reduced neutralization sensitivity. In sum, our study reports a pathway for entry into human cells that is ACE2-independent, supported by TMPRSS2-related proteases and associated with antibody evasion.","version":"1.1","doi":"10.1101/2024.04.18.590061","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.18.590031","pub_date":"2024-4-18","title":"A natural ANI gap that can define intra-species units of bacteriophages and other viruses","abstract":"Despite the importance of intra-species variants of viruses for causing disease and/or disrupting ecosystem functioning, there is no universally applicable standard to define these. A 95% whole-genome average nucleotide identity (ANI) gap is commonly used to define species, especially for bacteriophages, but whether a similar gap exists within species that can be used to define intra-species units has not been evaluated yet. Whole-genome comparisons among members of 1,016 bacteriophage species revealed a region of low frequency of pairs around 99.2-99.8% ANI, showing 3-fold or fewer pairs than expected for an even or normal distribution. This second gap is prevalent in viruses infecting various cultured or uncultured hosts, and from a variety of environments, although a few exceptions to this pattern were also observed (\u223c3.7% of the total species evaluated) and are likely attributed to cultivation biases. Similar results were observed for a limited set of eukaryotic viruses that are adequately sampled including SARS-CoV-2, whose ANI-based clusters matched well the WHO-defined Variants of Concern, indicating that they represent functionally and/or ecologically distinct units. The existence of sequence-discrete units appears to be predominantly driven by (high) ecological cohesiveness coupled to either recombination frequency for bacteriophages or selection and clonal evolution for other viruses such as SARS-CoV-2. These results indicate that fundamentally different underlying mechanisms could lead to similar diversity patterns. Based on these results, we propose the 99.5% ANI as a practical, standardized, and data-supported threshold for defining viral intra-species units of bacteriophages, for which we propose the term genomovars. Viral species are composed of an ensemble of intra-species variants whose dynamic may have major implications for human and animal health and/or ecosystem functioning. However, the lack of universally-accepted standards to define these intra-species variants has led researchers to use different approaches for this task, creating inconsistent intra-species units across different viral families and confusion in communication. By comparing hundreds of viral bacteriophage genomes, we show that there is a nearly universal natural gap in whole-genome average nucleotide identities (ANI) among genomes at around 99.5%, which can be used to define intra-species units. Therefore, these results advance the molecular toolbox for tracking viral intra-species units and should facilitate future epidemiological and environmental studies.","version":"1.1","doi":"10.1101/2024.04.18.590031","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.16.589036","pub_date":"2024-4-17","title":"SARS-CoV2 infection triggers reactive astrocyte states and inflammatory conditions in long-term Human Cortical Organoids","abstract":"SARS-CoV2, severe acute respiratory syndrome coronavirus 2, is frequently associated with neurological manifestations. Despite the presence of mild to severe CNS-related symptoms in a cohort of patients, there is no consensus whether the virus can infect directly brain tissue or if the symptoms in patients are a consequence of peripheral infectivity of the virus. Here, we use long-term human stem cell-derived cortical organoids to assess SARS-CoV2 infectivity of brain cells and unravel the cell-type tropism and its downstream pathological effects. Our results show consistent and reproducible low levels of SARS-CoV2 infection of astrocytes, deep projection neurons, upper callosal neurons and inhibitory neurons in 6 months human cortical organoids. Interestingly, astrocytes showed the highest infection rate among all infected cell populations that led to increased presence of reactive states. Further, transcriptomic analysis revealed overall changes in expression of genes related to cell metabolism, astrocyte activation and, inflammation and further, upregulation of cell survival pathways. Thus, local and minor infectivity of SARS-CoV2 in the brain may induce widespread adverse effects and may lead to resilience of dysregulated neurons and astrocytes within an inflammatory environment.","version":"1.1","doi":"10.1101/2024.04.16.589036","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.15.589676","pub_date":"2024-4-17","title":"PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2","abstract":"In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C\u2019s roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.","version":"1.1","doi":"10.1101/2024.04.15.589676","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.568364","pub_date":"2024-4-17","title":"Predicting Antibody and ACE2 Affinity for SARS-CoV-2 BA.2.86 and JN.1 with In Silico Protein Modeling and Docking","abstract":"The emergence of SARS-CoV-2 lineages derived from Omicron, including BA.2.86 (nicknamed \u201cPirola\u201d) and its relative, JN.1, has raised concerns about their potential impact on public and personal health due to numerous novel mutations. Despite this, predicting their implications based solely on mutation counts proves challenging. Empirical evidence of JN.1\u2019s increased immune evasion capacity in relation to previous variants is mixed. To improve predictions beyond what is possible based solely on mutation counts, we conducted extensive in silico analyses on the binding affinity between the RBD of different SARS-CoV-2 variants (Wuhan-Hu-1, BA.1/B.1.1.529, BA.2, XBB.1.5, BA.2.86, and JN.1) and neutralizing antibodies from vaccinated or infected individuals, as well as the human angiotensin-converting enzyme 2 (ACE2) receptor. We observed no statistically significant difference in binding affinity between BA.2.86 or JN.1 and other variants. Therefore, we conclude that the new SARS-CoV-2 variants have no pronounced immune escape or infection capacity compared to previous variants. However, minor reductions in binding affinity for both the antibodies and ACE2 were noted for JN.1. We discuss the implications of the in silico findings and highlight the need for modeling and docking studies to go above and beyond mutation and basic serological neutralization analysis. Future research in this area will benefit from increased structural analyses of memory B-cell derived antibodies and should emphasize the importance of choosing appropriate samples for in silico studies to assess protection provided by vaccination and infection. More-over, the fitness benefits of genomic variation outside of the RBD of BA.2.86 and JN.1 need to be investigated. This research contributes to understanding the BA.2.86 and JN.1 variants\u2019 potential impact on public health. Taken together, this work introduces a paradigm for functional genomic epidemiology in ongoing efforts to combat the evolving SARS-CoV-2 pandemic and prepare for other hazards.","version":"1.5","doi":"10.1101/2023.11.22.568364","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.15.589583","pub_date":"2024-4-17","title":"Exploring Metabolic Anomalies in COVID-19 and Post-COVID-19: A Machine Learning Approach with Explainable Artificial Intelligence","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2, has led to significant challenges worldwide, including diverse clinical outcomes and prolonged post-recovery symptoms known as Long COVID or Post-COVID-19 syndrome. Emerging evidence suggests a crucial role of metabolic reprogramming in the infection\u2019s long-term consequences. This study employs a novel approach utilizing machine learning (ML) and explainable artificial intelligence (XAI) to analyze metabolic alterations in COVID-19 and Post-COVID-19 patients. By integrating ML with SHAP (SHapley Additive exPlanations) values, we aimed to uncover metabolomic signatures and identify potential biomarkers for these conditions. Our analysis included a cohort of 142 COVID-19, 48 Post-COVID-19 samples and 38 CONTROL patients, with 111 identified metabolites. Traditional analysis methods like PCA and PLS-DA were compared with advanced ML techniques to discern metabolic changes. Notably, XGBoost models, enhanced by SHAP for explainability, outperformed traditional methods, demonstrating superior predictive performance and providing different insights into the metabolic basis of the disease\u2019s progression and its aftermath, the analysis revealed several metabolomic subgroups within the COVID-19 and Post-COVID-19 conditions, suggesting heterogeneous metabolic responses to the infection and its long-term impacts. This study highlights the potential of integrating ML and XAI in metabolomics research.","version":"1.1","doi":"10.1101/2024.04.15.589583","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.09.588755","pub_date":"2024-4-17","title":"Major Metabolites from Hypericum Perforatum L., Hyperforin and Hypericin, are both active against Human Coronaviruses","abstract":"COVID-19 pandemic has highlighted the need of antiviral molecules against coronaviruses. Plants are an endless source of active compounds. In the current study, we investigated the potential antiviral effects of Hypericum perforatum L.. Its extract contained two major metabolites belonging to distinct chemical classes, hypericin (HC) and hyperforin (HF). First, we demonstrated that HC inhibited HCoV-229E at the entry step by directly targeting the viral particle in a light-dependent manner. While antiviral properties have already been described for HC, the study here showed for the first time that HF has pan-coronavirus antiviral capacity. Indeed, HF was highly active against Alphacoronavirus HCoV-229E (IC50 value of 1.10 \u00b5M), and Betacoronaviruses SARS-CoV-2 (IC50 value of of 0.24 to 0.98 \u00b5M), SARS-CoV (IC50 value of 1.01 \u00b5M) and MERS-CoV (IC50 value of 2.55 \u00b5M). Unlike HC, HF was active at a post-entry step, most likely the replication step. Antiviral activity of HF on HCoV-229E and SARS-CoV-2 was confirmed in primary human respiratory epithelial cells. Furthermore, in vitro combination assay of HF with remdesivir showed that their association was additive, which was encouraging for a potential therapeutical association. As HF was active on both Alpha- and Betacoronaviruses, a cellular target was hypothesized. Heme oxygenase 1 (HO-1) pathway, a potential target of HF, has been investigated but the results showed that HF antiviral activity against HCoV-229E was not dependent on HO-1. Collectively, HF is a promising antiviral candidate in view of our results and pharmacokinetics studies already published in animal models or in human.","version":"1.2","doi":"10.1101/2024.04.09.588755","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.14.589423","pub_date":"2024-4-16","title":"Aging shapes infection profiles of influenza A virus and SARS-CoV-2 in human lung slices","abstract":"The recent coronavirus disease 2019 (COVID-19) outbreak revealed the susceptibility of elderly patients to respiratory virus infections, showing cell senescence or subclinical persistent inflammatory profiles and favouring the development of severe pneumonia. In our study, we evaluated the potential influence of lung aging on the efficiency of replication of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as determined the pro-inflammatory and antiviral responses of the distal lung tissue. Using precision-cut lung slices (PCLS) from donors of different ages, we found that pandemic H1N1 and avian H5N1 IAV replicated in the lung parenchyma with high efficacy. In contrast to these IAV strains, SARS-CoV-2 early isolate and Delta variant of concern (VOC) replicated less efficiently in PCLS. Interestingly, both viruses showed reduced replication in PCLS from older compared to younger donors, suggesting that aged lung tissue represents a sub-optimal environment for viral replication. Regardless of the age-dependent viral loads, PCLS responded to infection with both viruses by an induction of IL-6 and IP-10/CXCL10 mRNAs, being highest for H5N1. Finally, while SARS-CoV-2 infection was not causing detectable cell death, IAV infection caused significant cytotoxicity and induced significant early interferon responses. In summary, our findings suggest that aged lung tissue might not favour viral dissemination, pointing to a determinant role of dysregulated immune mechanisms in the development of severe disease. PCLS from donors of varying ages were exposed to SARS-CoV-2 or IAV. Notably, the latter exhibited the highest replication efficacy, triggering early interferon responses, elevated IL-6 and IP-10/CXCL10 mRNAs expression, and significant cell death compared to SARS-CoV-2. Overall, across all age groups, the pulmonary environment showed sustained immunocompetence. For both viruses, older donor-derived PCLS displayed reduced viral permissiveness, suggesting aged lung tissue might not favour viral dissemination, implying other factors contribute to severe disease development.","version":"1.1","doi":"10.1101/2024.04.14.589423","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.05.565350","pub_date":"2024-4-16","title":"Universal protection against SARS-CoV-2 viruses by multivalent mRNA vaccine in mice","abstract":"The continual emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants challenges available SARS-CoV-2 vaccines for adequate control of outbreaks. Currently, universal vaccines capable of obviating the need for exact strain matching between mRNA vaccines and circulating viruses are absent. In this study, we designed, manufactured, and evaluated a nucleoside-modified lipid nanoparticle mRNA vaccine, aimed for offering broad-spectrum protection against recent SARS-CoV-2 variants. Additionally, the protection efficiency of monovalent, bivalent, quadrivalent, and XBB.1.5 mRNA vaccines was compared with the proposed universal vaccine. The neutralizing antibody activity against wuhan-1, BA.4/5, XBB.1.5, B.1.1.529, BQ.1.1, EG.5.1 and JN.1 was assessed using enzyme-linked immunosorbent assay, rapid fiber-optic biolayer interferometry-based biosensor, and pseudovirus neutralization test. Our results reveal that the proposed multivalent vaccine affords comprehensive protection against previously circulating, current and previously unidentified SARS-CoV-2 strains.","version":"1.2","doi":"10.1101/2023.11.05.565350","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.12.589299","pub_date":"2024-4-15","title":"Associations between SARS-CoV-2 Infection or COVID-19 Vaccination and Human Milk Composition: A Multi-Omics Approach","abstract":"The risk of contracting SARS-CoV-2 via human milk-feeding is virtually non-existent. Adverse effects of COVID-19 vaccination for lactating individuals are not different from the general population, and no evidence has been found that their infants exhibit adverse effects. Yet, there remains substantial hesitation among this population globally regarding the safety of these vaccines. Herein we aimed to determine if compositional changes in milk occur following infection or vaccination, including any evidence of vaccine components. Using an extensive multi-omics approach, we found that compared to unvaccinated individuals SARS-CoV-2 infection was associated with significant compositional differences in 67 proteins, 385 lipids, and 13 metabolites. In contrast, COVID-19 vaccination was not associated with any changes in lipids or metabolites, although it was associated with changes in 13 or fewer proteins. Compositional changes in milk differed by vaccine. Changes following vaccination were greatest after 1-6 hours for the mRNA-based Moderna vaccine (8 changed proteins), 3 days for the mRNA-based Pfizer (4 changed proteins), and adenovirus-based Johnson and Johnson (13 changed proteins) vaccines. Proteins that changed after both natural infection and Johnson and Johnson vaccine were associated mainly with systemic inflammatory responses. In addition, no vaccine components were detected in any milk sample. Together, our data provide evidence of only minimal changes in milk composition due to COVID-19 vaccination, with much greater changes after natural SARS-CoV-2 infection. The impact of the observed changes in global milk composition on infant health remain unknown. These findings emphasize the importance of vaccinating the lactating population against COVID-19, as compositional changes in milk were found to be far less evident after vaccination compared to SARS-CoV-2 infection. Importantly, vaccine components were not detected in milk after vaccination.","version":"1.1","doi":"10.1101/2024.04.12.589299","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.12.589252","pub_date":"2024-4-15","title":"SARS-CoV-2 spike S2 subunit inhibits p53 activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2 proteins in cancer cells","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike S2-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike S2 were found to have increased cell viability as compared to control cells. Further observations on \u03b3-H2AX expression in spike S2-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway. The preliminary observations reported here warrant further studies to unravel the impact of SARS-CoV-2 and its various encoded proteins including spike on pathways of tumorigenesis and response to cancer therapeutics.","version":"1.1","doi":"10.1101/2024.04.12.589252","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.06.556503","pub_date":"2024-4-15","title":"Analysis of emergent bivalent antibody binding identifies the molecular reach as a critical determinant of SARS-CoV-2 neutralisation potency","abstract":"Key functions of antibodies, such as viral neutralisation, depend on bivalent binding but the factors that influence it remain poorly characterised. Here, we develop and employ a new bivalent model to mechanistically analyse binding between >45 patient-isolated IgG1 antibodies interacting with SARS-CoV-2 RBD surfaces. Our method reproduces the monovalent on/off-rates and enables measurements of the bivalent on-rate and the molecular reach: the maximum antigen separation that supports bivalent binding. We find large variations in these parameters across antibodies, including variations in reach (22-46 nm) that exceed the physical antibody size (\u223c15 nm) due to the antigen size. The bivalent model integrates all parameters, including reach and antigen density, to predict an emergent binding potency for each antibody that matches their neutralisation potency. Indeed, antibodies with similar monovalent affinities to the same RBD-epitope but with different reaches display differences in emergent bivalent binding that match differences in their neutralisation potency. Together, our work highlights that antibodies within an isotype class binding the same antigen can display differences in molecular reach that can substantially modulate their emergent binding and functional properties. Antibodies are soluble proteins that can neutralise pathogens by sticking to them. They contain two identical \u2018arms\u2019 that allow them to simultaneously bind two identical \u2018antigen\u2019 molecules on pathogen surfaces. Although we know that bivalent binding is important for neutralisation, we don\u2019t know how different antibodies achieve it. We developed a new model to analyse the mechanism of bivalent binding and used it to study over 45 antibodies from COVID-19 patients that bind the RBD antigen of SARS-CoV-2. Unexpectedly, we found that the molecular reach of an antibody, which is the maximum antigen separation that supports bivalent binding, varied widely between antibodies and exceeded their physical size. We show how antibody binding emerges from the interplay of multiple factors, including reach, and that this emergent binding predicts their neutralisation function. The ability to analyse and predict bivalent binding should improve our understanding and exploitation of antibodies.","version":"1.2","doi":"10.1101/2023.09.06.556503","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.12.24301191","pub_date":"2024-04-15","title":"Exploring the Spatial Distribution of Persistent SARS-CoV-2 Mutations - Leveraging mobility data for targeted sampling","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Given the rapid cross-country spread of SARS-CoV-2 and the resulting difficulty in tracking lineage spread, we investigated the potential of combining mobile service data and fine-granular metadata (such as postal codes and genomic data) to advance integrated genomic surveillance of the pandemic in the federal state of Thuringia, Germany. We sequenced over 6,500 SARS-CoV-2 Alpha genomes (B.1.1.7) across seven months within Thuringia while collecting patients\u2019 isolation dates and postal codes. Our dataset is complemented by over 66,000 publicly available German Alpha genomes and mobile service data for Thuringia. We identified the existence and spread of nine persistent mutation variants within the Alpha lineage, seven of which formed separate phylogenetic clusters with different spreading patterns in Thuringia. The remaining two are sub-clusters. Mobile service data can indicate these clusters\u2019 spread and highlight a potential sampling bias, especially of low-prevalence variants. Thereby, mobile service data can be used either retrospectively to assess surveillance coverage and efficiency from already collected data or to actively guide part of a surveillance sampling process to districts where these variants are expected to emerge. The latter concept was successfully implemented as a proof-of-concept for a mobility-guided sampling strategy in response to the surveillance of Omicron sublineage BQ.1.1. The combination of mobile service data and SARS-CoV-2 surveillance by genome sequencing is a valuable tool for more targeted and responsive surveillance.</jats:p>","version":null,"doi":"10.1101/2024.04.12.24301191","journal":"medRxiv","score":null},{"id":"10.1101/2024.04.12.589332","pub_date":"2024-4-15","title":"Characterization of spike processing and entry mechanisms of seasonal human coronaviruses NL63, 229E and HKU1","abstract":"Although much has been learned about the entry mechanism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the details of entry mechanisms of seasonal human coronaviruses (HCoVs) remain less well understood. In the present study, we established that 293T cell lines that stably express angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) support high level transduction of lentiviral pseudoviruses bearing spike proteins of seasonal HCoVs, HCoV-NL63, -229E, or -HKU1, respectively. Our results showed that entry of HCoV-NL63, -229E and -HKU1 pseudoviruses is sensitive to endosomal acidification inhibitors (chloroquine and NH4Cl), indicating virus entry via the endocytosis route. Although HCoV-HKU1 pseudovirus infection requires TMPRSS2 expression on cell surface, endocytosis-mediated HCoV-HKU1 entry requires the serine protease domain but not the serine protease activity of TMPRSS2. We also show that amino acids in the predicted S1/S2 junctions of spike proteins of HCoV-NL63, and - 229E are essential for optimal entry but non-essential for spike-mediated entry of HCoV-HKU1. Our findings provide insights into entry mechanism of seasonal HCoVs that may support the development of novel treatment strategies. Details of the entry mechanisms of seasonal human coronaviruses (HCoVs) remain to be fully explored. To investigate the entry of HCoV-NL63, -229E and -HKU1 CoVs, we employed 293T cells that stably express angiotensin converting enzyme (ACE2) aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) to study entry mechanisms of pseudoviruses bearing spike proteins of HCoV-NL63, -229E and - HKU1 respectively. Our results provide new insights into the predicted S1/S2 subunit junctions, cellular receptor, and protease requirements for seasonal HCoV pseudovirus entry via endocytic route and may support the development of novel treatment strategies.","version":"1.1","doi":"10.1101/2024.04.12.589332","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.04.515237","pub_date":"2024-4-12","title":"Widespread exposure to SARS-CoV-2 in wildlife communities","abstract":"Pervasive SARS-CoV-2 infections in humans have led to multiple transmission events to captive animals. While SARS-CoV-2 has a potential broad wildlife host range, most documented infections to date are found in a single species, the white-tailed deer. The extent of SARS-CoV-2 exposure among wildlife species and the factors that influence wildlife transmission risk remain unknown. We sampled 23 wildlife species for SARS-CoV-2 and examined the effects of urbanization and human use on seropositivity. Here, we document positive detections of SARS-CoV-2 RNA in six species, including the deer mouse, Virginia opossum, raccoon, groundhog, Eastern cottontail, and Eastern red bat. In addition, we found that sites with high human activity had three times higher seroprevalence than low human-use areas. We detected SARS-CoV-2 genomic sequences from nine individuals of six species which were assigned to seven Pango lineages of the Omicron variant. The close match to variants circulating in humans at the time suggests at least seven recent human-to-animal transmission events. Our data support that exposure to SARS-CoV-2 has been widespread in wildlife communities and suggests that areas with high human activity may serve as points of contact for cross-species transmission.","version":"1.3","doi":"10.1101/2022.11.04.515237","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.11.587623","pub_date":"2024-4-12","title":"Cardiovascular symptoms of PASC are associated with trace-level cytokines that affect the function of human pluripotent stem cell derived cardiomyocytes","abstract":"Globally, over 65 million individuals are estimated to suffer from post-acute sequelae of COVID-19 (PASC). A large number of individuals living with PASC experience cardiovascular symptoms (i.e. chest pain and heart palpitations) (PASC-CVS). The role of chronic inflammation in these symptoms, in particular in individuals with symptoms persisting for >1 year after SARS-CoV-2 infection, remains to be clearly defined. In this cross-sectional study, blood samples were obtained from three different sites in Australia from individuals with i) a resolved SARS-CoV-2 infection (and no persistent symptoms i.e. \u2018Recovered\u2019), ii) individuals with prolonged PASC-CVS and iii) SARS-CoV-2 negative individuals. Individuals with PASC-CVS, relative to Recovered individuals, had a blood transcriptomic signature associated with inflammation. This was accompanied by elevated levels of pro-inflammatory cytokines (IL-12, IL-1\u03b2, MCP-1 and IL-6) at approximately 18 months post-infection. These cytokines were present in trace amounts, such that they could only be detected with the use of novel nanotechnology. Importantly, these trace-level cytokines had a direct effect on the functionality of pluripotent stem cell derived cardiomyocytes in vitro. This effect was not observed in the presence of dexamethasone. Plasma proteomics demonstrated further differences between PASC-CVS and Recovered patients at approximately 18 months post-infection including enrichment of complement and coagulation associated proteins in those with prolonged cardiovascular symptoms. Together, these data provide a new insight into the role of chronic inflammation in PASC-CVS and present nanotechnology as a possible novel diagnostic approach for the condition.","version":"1.1","doi":"10.1101/2024.04.11.587623","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.10.588851","pub_date":"2024-4-11","title":"Microgliosis, astrogliosis and loss of aquaporin-4 polarity in frontal cortex of COVID-19 patients","abstract":"The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), causing human coronavirus disease 2019 (COVID-19), not only affects the respiratory tract, but also impacts other organs including the brain. A considerable number of COVID-19 patients develop neuropsychiatric symptoms that may linger for weeks and months and contribute to \u201clong-COVID\u201d. While the neurological symptoms of COVID-19 are well described, the cellular mechanisms of neurologic disorders attributed to the infection are still enigmatic. Here, we studied the effect of an infection with SARS-CoV-2 on the structure and expression of marker proteins of astrocytes and microglial cells in the frontal cortex of patients who died from COVID-19 in comparison to non-COVID-19 controls. Most of COVID-19 patients had microglial cells with retracted processes and rounded and enlarged cell bodies in both gray and white matter, as visualized by anti-Iba1 staining and confocal fluorescence microscopy. In addition, gray matter astrocytes in COVID-19 patients were frequently labeled by intense anti-GFAP staining, whereas in non-COVID-19 controls, most gray matter astrocytes expressed little GFAP. The most striking difference between astrocytes in COVID-19 patients and controls was found by anti-aquaporin-4 (AQP4) staining. In COVID-19 patients, a large number of gray matter astrocytes showed an increase in AQP4. In addition, AQP4 polarity was lost and AQP4 covered the entire cell, including the cell body and all cell processes, while in controls, AQP4 immunostaining was mainly detected in endfeet around blood vessels and did not visualize the cell body. In summary, our data suggest neuroinflammation upon SARS-CoV-2 infection including microgliosis and astrogliosis, including loss of AQP4 polarity.","version":"1.1","doi":"10.1101/2024.04.10.588851","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.10.588984","pub_date":"2024-4-11","title":"Coatomer complex I is required for the transport of SARS-CoV-2 progeny virions from the endoplasmic reticulum-Golgi intermediate compartment","abstract":"SARS-CoV-2 undergoes budding within the lumen of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and delivers progeny virions to the cell surface by employing vesicular transport. However, the molecular mechanisms remain poorly understood. Using three-dimensional electron microscopic analysis, such as array tomography and electron tomography, we found that virion-transporting vesicles possessed a coated protein on their membrane and demonstrated that the coated protein was coatomer complex I (COPI). During the later stages of SARS-CoV-2 infection, we observed a notable alteration in the distribution of COPI and ERGIC throughout the cytoplasm. Depletion of COPB2, a key component of COPI, led to the confinement of SARS-CoV-2 structural proteins in the perinuclear region, where progeny virions were accumulated within the ERGIC. While the expression levels of viral proteins within cells were comparable, this depletion significantly reduced the efficiency of virion release, leading to the significant inhibition of viral replication. Hence, our findings suggest COPI as a critical player in facilitating the transport of SARS-CoV-2 progeny virions from the ERGIC. Thus, COPI could be a promising target for the development of antivirals against SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.04.10.588984","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.10.588883","pub_date":"2024-4-11","title":"A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation","abstract":"SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a \u223c1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 \u00b5M. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.","version":"1.1","doi":"10.1101/2024.04.10.588883","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.08.588357","pub_date":"2024-4-11","title":"Reduced selection during sweeps lead to adaptive momentum on rugged landscapes","abstract":"Evolutionary theory seeks to explain the remarkable diversity and adaptability of life on Earth. Current theory offers substantial explanatory power, but it overlooks important transient dynamics that are prominent only when populations are outside equilibrium, such as during selective sweeps. We identify a dynamic that we call \u201cadaptive momentum\u201d whereby lineages with a selective advantage can temporarily sustain more deleterious mutations. This reduction in the strength of purifying selection allows populations to explore fitness valleys that are usually too costly to enter, potentially leading to the discovery of otherwise inaccessible fitness peaks. Using mathematical and agent-based simulations, we demonstrate adaptive momentum and show how periods of disequilibrium become windows of enhanced adaptation. Genetic exploration can occur during these windows without requiring mechanisms such as changing environments or complex landscapes. Adaptive momentum provides a simple potential explanation for bursts of rapid evolution observed in nature, including in pathogens such as SARS-CoV-2 and cancers. (152 words)","version":"1.1","doi":"10.1101/2024.04.08.588357","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.26.583354","pub_date":"2024-4-10","title":"Identifying immune signatures of common exposures through co-occurrence of T-cell receptors in tens of thousands of donors","abstract":"Memory T cells are records of clonal expansion from prior immune exposures, such as infections, vaccines and chronic diseases like cancer. A subset of the receptors of these expanded T cells in a typical immune repertoire are highly public, i.e., present in many individuals exposed to the same exposure. For the most part, the exposures associated with these public T cells are unknown. To identify public T-cell receptor signatures of immune exposures, we mined the immunosequencing repertoires of tens of thousands of donors to define clusters of co-occurring T cells. We first built co-occurrence clusters of T cells responding to antigens presented by the same Human Leukocyte Antigen (HLA) and then combined those clusters across HLAs. Each cross-HLA cluster putatively represents the public T-cell signature of a single prevalent exposure. Using repertoires from donors with known serological status for 7 prevalent exposures (HSV-1, HSV-2, EBV, Parvovirus, Toxoplasma gondii, Cytomegalovirus and SARS-CoV-2), we identified a single T-cell cluster strongly associated with each exposure and used it to construct a highly sensitive and specific diagnostic model for the exposure. These T-cell clusters constitute the public immune responses to prevalent exposures, 7 known and many others unknown. By learning the exposure associations for more T-cell clusters, this approach could be used to derive a ledger of a person\u2019s past and present immune exposures.","version":"1.2","doi":"10.1101/2024.03.26.583354","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.23.554434","pub_date":"2024-4-09","title":"Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling","abstract":"RHIM is a protein motif in cell death proteins that assembles higher-order signaling complexes and triggers regulated cell death, which in itself limits virus spread and additionally triggers inflammation for mounting immune responses. A few DNA viruses employ viral RHIMs mimicking host RHIMs. However, these viral RHIMs counteract host cell death by interacting with host RHIM proteins and blocking complex formation to alleviate antiviral defenses. Whether RNA viruses operate such viral RHIMs remains unknown. RHIM-protein signaling promotes lung damage and cytokine storm in respiratory RNA virus infections, arguing the presence of viral RHIMs. Here, we report the novel viral RHIMs in Nsp13 and Nsp14 of SARS-CoV-2 and other bat RNA viruses, providing the basis for bats as the hosts for their evolution. Nsp13 promoted cell death in bat and human cells, however, viral RHIM of Nsp13 is more critical for human cell death than bat cells, suggesting species-specific regulation. The conformation of RNA-binding channel in Nsp13 is critical for cell death in bat and human cells. Nsp13 showed RHIM-dependent interactions with ZBP1 and RIPK3 and promoted the formation of large insoluble complexes of ZBP1 and RIPK3. Also, Nsp13 promoted ZBP1-RIPK3 signaling-mediated cell death dependent on intracellular RNA ligands. Intriguingly, the SARS-CoV-2 genome consists of bona fide Z-RNA-forming segments. These SARS-CoV-2 Z-RNA segments promoted Nsp13-dependent cell death, further revealing Nsp13\u2019s association with Z-RNA sensing and ZBP1-RIPK3 signaling. Our findings reveal the functional viral RHIMs of bat-originated RNA viruses regulating host cell death associated with Z-RNA sensing and ZBP1-RIPK3 signaling activation. These observations allow the understanding of mechanisms of cellular damage and cytokine storm in SARS-CoV-2 and other bat-originated RNA virus infections. Bat-associated RNA viruses employ viral RHIMs and regulate host cell death.","version":"1.2","doi":"10.1101/2023.08.23.554434","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.05.570076","pub_date":"2024-4-09","title":"#GotGlycans: Role of N343 Glycosylation on the SARS-CoV-2 S RBD Structure and Co-Receptor Binding Across Variants of Concern","abstract":"Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and to structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work we show through over 45 \u03bcs of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N-glycosylation at N343 and how this glycan\u2019s structural role changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2) and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N-glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield.","version":"1.3","doi":"10.1101/2023.12.05.570076","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.31.573251","pub_date":"2024-4-09","title":"Rapid and specific detection of single nanoparticles and viruses in microfluidic laminar flow via confocal fluorescence microscopy","abstract":"Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we report the development of a flow virometer for the specific and rapid detection of single nanoparticles based on confocal microscopy. The combination of laminar flow and multiple dyes enable the detection of correlated fluorescence signals, providing information on nanoparticle volumes and specific chemical composition properties, such as viral envelope proteins. We evaluated and validated the assay using fluorescent beads and viruses, including SARS-CoV-2. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting clinically-relevant virus loads. Based on our results, we envision the use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup.\n\n\n","version":"1.3","doi":"10.1101/2023.12.31.573251","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.03.587743","pub_date":"2024-4-08","title":"Discovery of orally bioavailable SARS-CoV-2 papain-like protease inhibitor as a potential treatment for COVID-19","abstract":"The RNA-dependent RNA polymerase (RdRp), 3C-like protease (3CLpro), and papain-like protease (PLpro) are pivotal components in the viral life cycle of SARS-CoV-2, presenting as promising therapeutic targets. Currently, all FDA-approved antiviral drugs against SARS-CoV-2 are RdRp or 3CLpro inhibitors. However, the mutations causing drug resistance have been observed in RdRp and 3CLpro from SARS-CoV-2, which makes it necessary to develop antivirals with novel mechanisms. Through the application of a structure-based drug design (SBDD) approach, we discovered a series of novel potent non-covalent PLpro inhibitors with remarkable in vitro potency and in vivo PK properties. The co-crystal structures of PLpro with leads revealed that the residues D164 and Q269 around the S2 site are critical for improving the inhibitor\u2019s potency. The lead compound GZNL-P36 not only inhibited SARS-CoV-2 and its variants at the cellular level with EC50 ranging from 58.2 nM to 306.2 nM, but also inhibited HCoV-NL63 and HCoV-229E with EC50 of 81.6 nM and 2.66 \u03bcM, respectively. Oral administration of the compound resulted in significantly improved survival and notable reductions in lung viral loads and lesions in SARS- CoV-2 infection mouse model, consistent with RNA-seq data analysis. Our results indicate that PLpro inhibitor is a promising SARS-CoV-2 therapy.","version":"1.2","doi":"10.1101/2024.04.03.587743","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.05.588051","pub_date":"2024-4-07","title":"Ipsilateral or contralateral boosting of mice with mRNA vaccines confers equivalent immunity and protection against a SARS-CoV-2 Omicron strain","abstract":"Boosting with mRNA vaccines encoding variant-matched spike proteins has been implemented to mitigate their reduced efficacy against emerging SARS-CoV-2 variants. Nonetheless, in humans, it remains unclear whether boosting in the ipsilateral or contralateral arm with respect to the priming doses impacts immunity and protection. Here, we boosted K18-hACE2 mice with either monovalent mRNA-1273 (Wuhan-1 spike) or bivalent mRNA-1273.214 (Wuhan-1 + BA.1 spike) vaccine in the ipsilateral or contralateral leg relative to a two-dose priming series with mRNA-1273. Boosting in the ipsilateral or contralateral leg elicited equivalent levels of serum IgG and neutralizing antibody responses against Wuhan-1 and BA.1. While contralateral boosting with mRNA vaccines resulted in expansion of spike-specific B and T cells beyond the ipsilateral draining lymph node (DLN) to the contralateral DLN, administration of a third mRNA vaccine dose at either site resulted in similar levels of antigen-specific germinal center B cells, plasmablasts/plasma cells, T follicular helper cells and CD8+ T cells in the DLNs and the spleen. Furthermore, ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines conferred similar homologous or heterologous immune protection against SARS-CoV-2 BA.1 virus challenge with equivalent reductions in viral RNA and infectious virus in the nasal turbinates and lungs. Collectively, our data show limited differences in B and T cell immune responses after ipsilateral and contralateral site boosting by mRNA vaccines that do not substantively impact protection against an Omicron strain. Sequential boosting with mRNA vaccines has been effective strategy to overcome waning immunity and neutralization escape by emerging SARS-CoV-2 variants. However, it remains unclear how the site of boosting relative to the primary vaccination series shapes optimal immune responses or breadth of protection against variants. In K18-hACE2 transgenic mice, we observed that boosting with historical monovalent or variant-matched bivalent vaccines in the ipsilateral or contralateral limb elicited comparable levels of serum spike specific antibody and antigen-specific B and T cells responses. Moreover, boosting on either side conferred equivalent protection against a SARS-CoV-2 Omicron challenge strain. Our data in mice suggest that the site of boosting with an mRNA vaccine does not substantially impact immunity or protection against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.04.05.588051","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.05.588295","pub_date":"2024-4-07","title":"Direct genome sequencing of respiratory viruses from low viral load clinical specimens using target capture sequencing technology","abstract":"The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging\u2014especially in the case of respiratory viruses\u2014due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. Here we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/\u00b5L of SARS-CoV-2 or 544 copies/\u00b5L of influenza A virus, respectively. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/\u00b5L, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses, but also contribute to epidemiological studies.","version":"1.1","doi":"10.1101/2024.04.05.588295","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.04.588067","pub_date":"2024-4-07","title":"Spatial proteomics identifies a novel CRTC-dependent viral sensing pathway that stimulates production of Interleukin-11","abstract":"Appropriate cellular recognition of viruses is essential for the generation of effective innate and adaptive antiviral immunity. Viral sensors and their signalling components thus provide a crucial first line of host defence. Many exhibit subcellular relocalisation upon activation, triggering expression of interferon and antiviral genes. To identify novel signalling factors we analysed protein relocalisation on a global scale during viral infection. CREB Regulated Transcription Coactivators-2 and 3 (CRTC2/3) exhibited early cytoplasmic-to-nuclear translocation upon a diversity of viral stimuli, in diverse cell types. This movement was depended on Mitochondrial Antiviral Signalling Protein (MAVS), cyclo-oxygenase proteins and protein kinase A. We identify a key effect of transcription stimulated by CRTC2/3 translocation as production of the pro-fibrogenic cytokine interleukin-11. This may be important clinically in viral infections associated with fibrosis, including SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.04.04.588067","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.05.588359","pub_date":"2024-4-06","title":"Antigenic cartography using hamster sera identifies SARS-CoV-2 JN.1 evasion seen in human XBB.1.5 booster sera","abstract":"Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with five to six-fold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a five-fold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.","version":"1.1","doi":"10.1101/2024.04.05.588359","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.05.24305357","pub_date":"2024-04-06","title":"Systems vaccinology identifies clinical and immunological correlates of SARS-CoV-2 vaccine response in solid-organ transplant recipients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Solid organ transplant (SOT) recipients are at enhanced risk of adverse outcomes following infectious challenges due to immunosuppressive treatment and additional comorbidities. Unfortunately, SOT recipients are also poor responders to the key medical intervention to preventing infection: vaccines. Here we performed a systems vaccinology study on a cohort of 59 kidney transplant recipients and 31 lung transplant recipients who received the mRNA Pfizer-BioNTech COVID-19 vaccine. Analyzing the immunological status of the patients prior to vaccination, we were able to identify multiple immunological associates of relatively improved vaccine responses following two or three doses of mRNA-based SARS-CoV-2 vaccine. These immunological associates predicted, with 95.0% and 93.3% accuracy, vaccine response after the second and third dose, respectively. Comparison of the immunological associates with vaccine response in SOT recipients revealed two distinct immune configurations: a non-classical configuration, distinct from the immune state of healthy subjects, associated with responses to two doses of mRNA vaccine and that could be mediated partly by the presence of double negative B cell subsets which are more prominently represented in responsive SOT recipients, and a \u201cnormalized\u201d configuration, closer to the immune state of healthy subjects, associated with potent antibody responses to three doses of mRNA vaccine. These results suggest that immunosuppression in SOT recipients can result in distinct immune states associated with different trade-offs in vaccine responsiveness. Immune phenotyping of SOT recipients for immune constellation may be an effective approach for identifying patients most at risk of poor vaccine responses and susceptibility to vaccine-preventable diseases.</jats:p>\n                <jats:sec>\n                  <jats:title>One-sentence summary</jats:title>\n                  <jats:p>SOT recipients showed distinct immune states at baseline associated with different profiles of vaccine-associated immune response.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2024.04.05.24305357","journal":"medRxiv","score":null},{"id":"10.1101/2024.04.03.587933","pub_date":"2024-4-04","title":"Comparative analysis of serological assays and sero-surveillance for SARS-CoV-2 exposure in US cattle","abstract":"Coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to pose a significant threat to public health globally. Notably, SARS-CoV-2 demonstrates a unique capacity to infect various non-human animal species, documented in captive and free-living animals. However, experimental studies revealed low susceptibility of domestic cattle (Bos taurus) to ancestral B.1 lineage SARS-CoV-2 infection, with limited viral replication and seroconversion. Despite the emergence of viral variants with potentially altered host tropism, recent experimental findings indicate greater permissiveness of cattle to SARS-CoV-2 Delta variant infection compared to other variants, though with limited seroconversion and no clear evidence of transmission. While some studies detected SARS-CoV-2 antibodies in cattle in Italy and Germany, there is no evidence of natural SARS-CoV-2 infection in cattle from the United States or elsewhere. Since serological tests have inherent problems of false positives and negatives, we conducted a comprehensive assessment of multiple serological assays on over 600 cattle serum samples, including pre-pandemic and pandemic cattle sera. We found that SARS-CoV-2 pseudovirus neutralization assays with a luciferase reporter system can produce false positive results, and care must be taken to interpret serological diagnosis using these assays. We found no serological evidence of natural SARS-CoV-2 infection or transmission among cattle in the USA. Hence, it is critical to develop more reliable serological assays tailored to accurately detect SARS-CoV-2 antibodies in cattle populations and rigorously evaluate diagnostic tools. This study underscores the importance of robust evaluation when employing serological assays for SARS-CoV-2 detection in cattle populations.","version":"1.1","doi":"10.1101/2024.04.03.587933","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.03.587916","pub_date":"2024-4-04","title":"Exploration of the link between COVID-19 and gastric cancer from the perspective of bioinformatics and systems biology","abstract":"Coronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has caused a global pandemic. Gastric cancer (GC) poses a great threat to people\u2019s health, which is a high- risk factor for COVID-19. Previous studies have found some associations between GC and COVID-19, whereas the underlying molecular mechanisms are not well understood. We used a bioinformatics and systems biology approach to investigate the relationship between GC and COVID-19. The gene expression profiles of COVID-19 (GSE196822) and GC (GSE179252) were downloaded from the Gene Expression Omnibus (GEO) database. After identifying the shared differentially expressed genes (DEGs) for GC and COVID-19, functional annotation, protein-protein interaction (PPI) network, hub genes, transcriptional regulatory networks and candidate drugs were analyzed. A total of 209 shared DEGs were identified to explore the linkages between COVID-19 and GC. Functional analyses showed that Immune-related pathway collectively participated in the development and progression of COVID-19 and GC. In addition, there are selected 10 hub genes including CDK1, KIF20A, TPX2, UBE2C, HJURP, CENPA, PLK1, MKI67, IFI6, and IFIT2. The transcription factor/gene and miRNA/gene interaction networks identified 38 transcription factors (TFs) and 234 miRNAs. More importantly, we identified ten potential therapeutic agents, including ciclopirox, resveratrol, etoposide, methotrexate, trifluridine, enterolactone, troglitazone, calcitriol, dasatinib and deferoxamine, some of which have been reported to improve and treat GC and COVID-19. This study also provides insight into the diseases most associated with mutual DEGs, which may provide new ideas for research on the treatment of COVID-19. This research has the possibility to be contributed to effective therapeutic in COVID-19 and GC.","version":"1.1","doi":"10.1101/2024.04.03.587916","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.02.587850","pub_date":"2024-4-03","title":"Predicting Functional Conformational Ensembles and Binding Mechanisms of Convergent Evolution for SARS-CoV-2 Spike Omicron Variants Using AlphaFold2 Sequence Scanning Adaptations and Molecular Dynamics Simulations","abstract":"In this study, we combined AlphaFold-based approaches for atomistic modeling of multiple protein states and microsecond molecular simulations to accurately characterize conformational ensembles and binding mechanisms of convergent evolution for the SARS-CoV-2 Spike Omicron variants BA.1, BA.2, BA.2.75, BA.3, BA.4/BA.5 and BQ.1.1. We employed and validated several different adaptations of the AlphaFold methodology for modeling of conformational ensembles including the introduced randomized full sequence scanning for manipulation of sequence variations to systematically explore conformational dynamics of Omicron Spike protein complexes with the ACE2 receptor. Microsecond atomistic molecular dynamic simulations provide a detailed characterization of the conformational landscapes and thermodynamic stability of the Omicron variant complexes. By integrating the predictions of conformational ensembles from different AlphaFold adaptations and applying statistical confidence metrics we can expand characterization of the conformational ensembles and identify functional protein conformations that determine the equilibrium dynamics for the Omicron Spike complexes with the ACE2. Conformational ensembles of the Omicron RBD-ACE2 complexes obtained using AlphaFold-based approaches for modeling protein states and molecular dynamics simulations are employed for accurate comparative prediction of the binding energetics revealing an excellent agreement with the experimental data. In particular, the results demonstrated that AlphaFold-generated extended conformational ensembles can produce accurate binding energies for the Omicron RBD-ACE2 complexes. The results of this study suggested complementarities and potential synergies between AlphaFold predictions of protein conformational ensembles and molecular dynamics simulations showing that integrating information from both methods can potentially yield a more adequate characterization of the conformational landscapes for the Omicron RBD-ACE2 complexes. This study provides insights in the interplay between conformational dynamics and binding, showing that evolution of Omicron variants through acquisition of convergent mutational sites may leverage conformational adaptability and dynamic couplings between key binding energy hotspots to optimize ACE2 binding affinity and enable immune evasion.","version":"1.1","doi":"10.1101/2024.04.02.587850","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.26.489314","pub_date":"2024-4-03","title":"Disease diagnostics using machine learning of immune receptors","abstract":"Clinical diagnosis typically incorporates physical examination, patient history, and various laboratory tests and imaging studies, but makes limited use of the human system\u2019s own record of antigen exposures encoded by receptors on B cells and T cells. We analyzed immune receptor datasets from 593 individuals to develop MAchine Learning for Immunological Diagnosis (Mal-ID), an interpretive framework to screen for multiple illnesses simultaneously or precisely test for one condition. This approach detects specific infections, autoimmune disorders, vaccine responses, and disease severity differences. Human-interpretable features of the model recapitulate known immune responses to SARS-CoV-2, Influenza, and HIV, highlight antigen-specific receptors, and reveal distinct characteristics of Systemic Lupus Erythematosus and Type-1 Diabetes autoreactivity. This analysis framework has broad potential for scientific and clinical interpretation of human immune responses.","version":"1.5","doi":"10.1101/2022.04.26.489314","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.18.553908","pub_date":"2024-4-02","title":"Host factor PLAC8 is required for pancreas infection by SARS-CoV-2","abstract":"Although COVID-19 initially caused great concern about respiratory symptoms, mounting evidence shows that also the pancreas is productively infected by SARS-CoV-2. However, the severity of pancreatic SARS-CoV-2 infection and its pathophysiology are still under debate. Here we investigated the consequences of SARS-CoV-2 pancreatic infection and the role of the host factor Placenta-associated protein (PLAC8) We analyzed plasma levels of pancreatic enzymes and inflammatory markers in a retrospective cohort study of 120 COVID-19 patients distributed in 3 severity-stratified groups. We studied the expression of SARS-CoV-2 and PLAC8 in the pancreas of deceased COVID-19 patients as well as in non-infected donors. We performed infection experiments in PLAC8 knock-out PDAC cell lines with full SARS-CoV-2 virus. We found that analysis of circulating pancreatic enzymes aided the stratification of patients according to COVID-19 severity and predict outcomes. Interestingly, we found an association between PLAC8 expression and SARS-CoV-2 infection in postmortem analysis of COVID-19 patients. Using full SARS-CoV-2 infectious virus inoculum from Wuhan-1 and BA.1 strains, we demonstrated that PLAC8 is necessary for productive infection of PDAC cell lines. Finally, we observed an overlap between PLAC8 and SARS-CoV-2 immunoreactivities of the pancreas of deceased patients. Our data indicate the human pancreas as a SARS-CoV-2 target with plausible signs of injury and demonstrate that the host factor PLAC8 is required for SARS-CoV-2 pancreatic infection, thus defining new target opportunities for COVID-19-associated pancreatic pathogenesis. Previous studies have shown that the pancreas is infected by SARS-CoV-2. However, none of these studies have described measurable pancreatic damage associated to COVID-19 severity and the pathogenesis of pancreatic SARS-CoV-2 infection remains largely unknown. Novel host factors have been proposed for SARS-CoV-2 infection of mainly the airway epithelium, none of them studied in the pancreas. Our study shows clinically relevant pancreatic damage associated with SARS-CoV-2 infiltration and assesses the predictive potential of circulating pancreatic enzymes to stratify patients according to COVID-19 severity and predict clinical outcomes in a cohort of 120 patients. Our data show that host factor Placenta-associated protein 8 (PLAC8) expression is linked to SARS-CoV-2 infection in postmortem analysis of COVID-19 patients and functionally demonstrated the full requirement of PLAC8 for SARS-CoV-2 pancreatic infection and viral replication. Our data confirm the human pancreas as a SARS-CoV-2 target with signs of injury unveiling the measurement of pancreatic enzymes for prognosis value and demonstrating that host factor PLAC8 is required for SARS-CoV-2 pancreatic infection defining new stratification and target opportunities for COVID-19-associated pancreatic pathogenesis.","version":"1.2","doi":"10.1101/2023.08.18.553908","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.18.585599","pub_date":"2024-4-02","title":"Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots","abstract":"In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure\u2122). DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.","version":"1.2","doi":"10.1101/2024.03.18.585599","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.02.587663","pub_date":"2024-4-02","title":"Single-cell analysis of lung epithelial cells reveals age and cell population-specific responses to SARS-CoV-2 infection in ciliated cells","abstract":"The ability of SARS-CoV-2 to evade antiviral immune signaling in the airway contributes to the severity of COVID-19 disease. Additionally, COVID-19 is influenced by age and has more severe presentations in older individuals. This raises questions about innate immune signaling as a function of lung development and age. Therefore, we investigated the transcriptome of different cell populations of the airway epithelium using pediatric and adult lung tissue samples from the LungMAP Human Tissue Core Biorepository. Specifically, lung lobes were digested and cultured into a biomimetic model of the airway epithelium on an air-liquid interface. Cells were then infected with SARS-CoV-2 and subjected to single-cell RNA sequencing. Transcriptional profiling and differential expression analysis were carried out using Seurat. The clustering analysis identified several cell populations: club cells, proliferating epithelial cells, multiciliated precursor cells, ionocytes, and two biologically distinct clusters of ciliated cells (FOXJ1high and FOXJ1low). Interestingly, the two ciliated cell clusters showed different infection rates and enrichment of processes involved in ciliary biogenesis and function; we observed a cell-type-specific suppression of innate immunity in infected cells from the FOXJ1low subset. We also identified a significant number of genes that were differentially expressed in lung cells derived from children as compared to adults, suggesting the differential pathogenesis of SARS-CoV-2 infection in children versus adults. We discuss how this work can be used to identify drug targets to modulate molecular signaling cascades that mediate an innate immune response and begin to understand differences in COVID-19 outcomes for pediatric vs. adult populations. Viral innate immune evasion leads to uncontrolled viral spread in infected tissues and increased pathogenicity in COVID-19. Understanding the dynamic of the antiviral signaling in lung tissues may help us to understand which molecular signals lead to more severe disease in different populations, particularly considering the enhanced vulnerability of older populations. This study provides foundational insight into the age-related differences in innate immune responses to SARS-CoV-2, identifying distinct patterns of infection and molecular signaling in different cell populations of airway epithelial cells from pediatric and adult lung tissues. The findings provide a deeper understanding of age-related differences in COVID-19 pathology and pave the way for developing targeted therapies.","version":"1.1","doi":"10.1101/2024.04.02.587663","journal":"bioRxiv","score":null},{"id":"10.1101/2024.04.01.587566","pub_date":"2024-4-02","title":"Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance","abstract":"SARS-CoV-2 main protease, Mpro, is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. Mpro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a Mpro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length Mpro protein as substrates, we demonstrate that the binding of Mpro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the Mpro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of Mpro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying Mpro and substrate interactions, and offers important insights into Mpro function, resistance development, and inhibitor design.","version":"1.1","doi":"10.1101/2024.04.01.587566","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.31.586409","pub_date":"2024-4-02","title":"Mapping immunodominant sites on the MERS-CoV spike glycoprotein targeted by infection-elicited antibodies in humans","abstract":"Middle-East respiratory syndrome coronavirus (MERS-CoV) first emerged in 2012 and causes human infections in endemic regions. Most vaccines and therapeutics in development against MERS-CoV focus on the spike (S) glycoprotein to prevent viral entry into target cells. These efforts, however, are limited by a poor understanding of antibody responses elicited by infection along with their durability, fine specificity and contribution of distinct S antigenic sites to neutralization. To address this knowledge gap, we analyzed S-directed binding and neutralizing antibody titers in plasma collected from individuals infected with MERS-CoV in 2017-2019 (prior to the COVID-19 pandemic). We observed that binding and neutralizing antibodies peak 1 to 6 weeks after symptom onset/hospitalization, persist for at least 6 months, and broadly neutralize human and camel MERS-CoV strains. We show that the MERS-CoV S1 subunit is immunodominant and that antibodies targeting S1, particularly the RBD, account for most plasma neutralizing activity. Antigenic site mapping revealed that polyclonal plasma antibodies frequently target RBD epitopes, particularly a site exposed irrespective of the S trimer conformation, whereas targeting of S2 subunit epitopes is rare, similar to SARS-CoV-2. Our data reveal in unprecedented details the humoral immune responses elicited by MERS-CoV infection, which will guide vaccine and therapeutic design.","version":"1.1","doi":"10.1101/2024.03.31.586409","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.28.587189","pub_date":"2024-4-01","title":"Synthetic coevolution reveals adaptive mutational trajectories of neutralizing antibodies and SARS-CoV-2","abstract":"The Covid-19 pandemic showcases a coevolutionary race between the human immune system and SARS-CoV-2, mirroring the Red Queen hypothesis of evolutionary biology. The immune system generates neutralizing antibodies targeting the SARS-CoV-2 spike protein\u2019s receptor binding domain (RBD), crucial for host cell invasion, while the virus evolves to evade antibody recognition. Here, we establish a synthetic coevolution system combining high-throughput screening of antibody and RBD variant libraries with protein mutagenesis, surface display, and deep sequencing. Additionally, we train a protein language machine learning model that predicts antibody escape to RBD variants. Synthetic coevolution reveals antagonistic and compensatory mutational trajectories of neutralizing antibodies and SARS-CoV-2 variants, enhancing the understanding of this evolutionary conflict.","version":"1.1","doi":"10.1101/2024.03.28.587189","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.07.566110","pub_date":"2024-4-01","title":"Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles","abstract":"The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.","version":"1.3","doi":"10.1101/2023.11.07.566110","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.29.587391","pub_date":"2024-4-01","title":"Robotic-inspired approach to multi-domain membrane receptor conformation space: theory and SARS-CoV-2 spike protein case study","abstract":"The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic field\u2019s methods to determine a robotic arm\u2019s forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.","version":"1.1","doi":"10.1101/2024.03.29.587391","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.28.587229","pub_date":"2024-3-29","title":"Broad-Spectrum Coronavirus Inhibitors Discovered by Modeling Viral Fusion Dynamics","abstract":"Broad-spectrum therapeutics capable of inhibiting SARS-CoV-2, its variants, and related coronaviruses hold promise in curbing the spread of COVID-19 and averting future pandemics. Here, we employed a multidisciplinary approach that included molecular dynamics simulation (MDS) and artificial intelligence (AI)-based docking predictions to identify potent inhibitors that target a conserved region within the SARS-CoV-2 spike protein that mediates membrane fusion by undergoing large-scale mechanical rearrangements. In silico binding screens honed in on this region, leading to the discovery of FDA-approved drugs and novel molecules predicted to disrupt spike protein conformational changes. These compounds significantly inhibited SARS-CoV-2 infection and blocked the entry of spike protein-bearing pseudotyped \u03b1, \u03b2, \u03b3, \u03b4 variants as well as SARS-CoV and MERS-CoV in cultured human ACE2-expressing cells. The optimized lead compound significantly inhibited SARS-CoV2 infection in mice when administered orally.","version":"1.1","doi":"10.1101/2024.03.28.587229","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.28.587260","pub_date":"2024-3-29","title":"Enhanced mucosal B- and T-cell responses against SARS-CoV-2 after heterologous intramuscular mRNA prime/intranasal protein boost vaccination with a combination adjuvant","abstract":"Current COVID-19 mRNA vaccines delivered intramuscularly (IM) induce effective systemic immunity, but with suboptimal immunity at mucosal sites, limiting their ability to impart sterilizing immunity. There is strong interest in rerouting immune responses induced in the periphery by parenteral vaccination to the portal entry site of respiratory viruses, such as SARS-CoV-2, by mucosal vaccination. We previously demonstrated the combination adjuvant, NE/IVT, consisting of a nanoemulsion (NE) and an RNA-based RIG-I agonist (IVT) induces potent systemic and mucosal immune responses in protein-based SARS-CoV-2 vaccines administered intranasally (IN). Herein, we demonstrate priming IM with mRNA followed by heterologous IN boosting with NE/IVT adjuvanted recombinant antigen induces strong mucosal and systemic antibody responses and enhances antigen-specific T cell responses in mucosa-draining lymph nodes compared to IM/IM and IN/IN prime/boost regimens. While all regimens induced cross-neutralizing antibodies against divergent variants and sterilizing immunity in the lungs of challenged mice, mucosal vaccination, either as homologous prime/boost or heterologous IN boost after IM mRNA prime was required to impart sterilizing immunity in the upper respiratory tract. Our data demonstrate the benefit of hybrid regimens whereby strong immune responses primed via IM vaccination are rerouted by IN vaccination to mucosal sites to provide optimal protection to SARS-CoV-2.","version":"1.1","doi":"10.1101/2024.03.28.587260","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.28.586935","pub_date":"2024-3-29","title":"Abolished frameshifting for predicted structure-stabilizing SARS-CoV-2 mutants: Implications to alternative conformations and their statistical structural analyses","abstract":"The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.","version":"1.1","doi":"10.1101/2024.03.28.586935","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.25.586578","pub_date":"2024-3-29","title":"Human long noncoding RNA, VILMIR, is induced by major respiratory viral infections and modulates the host interferon response","abstract":"Long noncoding RNAs (lncRNAs) are a newer class of noncoding transcripts identified as key regulators of biological processes. Here we aimed to identify novel lncRNA targets that play critical roles in major human respiratory viral infections by systematically mining large-scale transcriptomic datasets. Using bulk RNA-sequencing (RNA-seq) analysis, we identified a previously uncharacterized lncRNA, named virus inducible lncRNA modulator of interferon response (VILMIR), that was consistently upregulated after in vitro influenza infection across multiple human epithelial cell lines and influenza A virus subtypes. VILMIR was also upregulated after SARS-CoV-2 and RSV infections in vitro. We experimentally confirmed the response of VILMIR to influenza infection and interferon-beta (IFN-\u03b2) treatment in the A549 human epithelial cell line and found the expression of VILMIR was robustly induced by IFN-\u03b2 treatment in a dose and time-specific manner. Single cell RNA-seq analysis of bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients uncovered that VILMIR was upregulated across various cell types including at least five immune cells. The upregulation of VILMIR in immune cells was further confirmed in the human T cell and monocyte cell lines, SUP-T1 and THP-1, after IFN-\u03b2 treatment. Finally, we found that knockdown of VILMIR expression reduced the magnitude of host transcriptional responses to IFN-\u03b2 treatment in A549 cells. Together, our results show that VILMIR is a novel interferon-stimulated gene (ISG) that regulates the host interferon response and may be a potential therapeutic target for human respiratory viral infections upon further mechanistic investigation. Identifying host factors that regulate the immune response to human respiratory viral infection is critical to developing new therapeutics. Human long noncoding RNAs (lncRNAs) have been found to play key regulatory roles during biological processes, however the majority of lncRNA functions within the host antiviral response remain unknown. In this study, we identified that a previously uncharacterized lncRNA, VILMIR, is upregulated after major respiratory viral infections including influenza, SARS-CoV-2, and RSV. We demonstrated that VILMIR is an interferon-stimulated gene that is upregulated after interferon-beta (IFN-\u03b2) in several human cell types. We also found that knockdown of VILMIR reduced the magnitude of host transcriptional responses to IFN-\u03b2 treatment in human epithelial cells. Our results reveal that VILMIR regulates the host interferon response and may present a new therapeutic target during human respiratory viral infections.","version":"1.1","doi":"10.1101/2024.03.25.586578","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.27.24303943","pub_date":"2024-03-29","title":"Repeated vaccination with homologous influenza hemagglutinin broadens human antibody responses to unmatched flu viruses","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The on-going diversification of influenza virus necessicates annual vaccine updating. The vaccine antigen, the viral spike protein hemagglutinin (HA), tends to elicit strain-specific neutralizing activity, predicting that sequential immunization with the same HA strain will boost antibodies with narrow coverage. However, repeated vaccination with homologous SARS-CoV-2 vaccine eventually elicits neutralizing activity against highly unmatched variants, questioning this immunological premise. We evaluated a longitudinal influenza vaccine cohort, where each year the subjects received the same, novel H1N1 2009 pandemic vaccine strain. Repeated vaccination gradually enhanced receptor-blocking antibodies (HAI) to highly unmatched H1N1 strains within individuals with no initial memory recall against these historical viruses. An\n                  <jats:italic>in silico</jats:italic>\n                  model of affinity maturation in germinal centers integrated with a model of differentiation and expansion of memory cells provides insight into the mechanisms underlying these results and shows how repeated exposure to the same immunogen can broaden the antibody response against diversified targets.\n                </jats:p>","version":null,"doi":"10.1101/2024.03.27.24303943","journal":"medRxiv","score":null},{"id":"10.1101/2024.03.27.586885","pub_date":"2024-3-27","title":"The inflammatory microenvironment of the lung at the time of infection governs innate control of SARS-CoV-2 replication","abstract":"SARS-CoV-2 infection leads to vastly divergent clinical outcomes ranging from asymptomatic infection to fatal disease. Co-morbidities, sex, age, host genetics and vaccine status are known to affect disease severity. Yet, how the inflammatory milieu of the lung at the time of SARS-CoV-2 exposure impacts the control of viral replication remains poorly understood. We demonstrate here that immune events in the mouse lung closely preceding SARS-CoV-2 infection significantly impact viral control and we identify key innate immune pathways required to limit viral replication. A diverse set of pulmonary inflammatory stimuli, including resolved antecedent respiratory infections with S. aureus or influenza, ongoing pulmonary M. tuberculosis infection, ovalbumin/alum-induced asthma or airway administration of defined TLR ligands and recombinant cytokines, all establish an antiviral state in the lung that restricts SARS-CoV-2 replication upon infection. In addition to antiviral type I interferons, the broadly inducible inflammatory cytokines TNF\u03b1 and IL-1 precondition the lung for enhanced viral control. Collectively, our work shows that SARS-CoV-2 may benefit from an immunologically quiescent lung microenvironment and suggests that heterogeneity in pulmonary inflammation that precedes or accompanies SARS-CoV-2 exposure may be a significant factor contributing to the population-wide variability in COVID-19 disease outcomes.","version":"1.1","doi":"10.1101/2024.03.27.586885","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.27.584106","pub_date":"2024-3-27","title":"CCQM-P199b: Interlaboratory comparability study of SARS-CoV-2 RNA copy number quantification","abstract":"Nucleic acid amplification tests including reverse transcription quantitative PCR (RT-qPCR) are used to detect RNA from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Standardized measurements of RNA can facilitate comparable performance of laboratory tests in the absence of existing reference measurement systems early on in a pandemic. Interlaboratory study CCQM P199b \u201cSARS-CoV-2 RNA copy number quantification\u201d was designed to test the fitness-for-purpose of developed candidate reference measurement procedures (RMPs) for SARS-CoV-2 genomic targets in purified RNA materials, and was conducted under the auspices of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) to evaluate the measurement comparability of national metrology institutes (NMIs) and designated institutes (DIs), thereby supporting international standardization. Twenty-one laboratories participated in CCQM P199b and were requested to report the RNA copy number concentration, expressed in number of copies per microliter, of the SARS-CoV-2 nucleocapsid (N) gene partial region (NC_045512.2: 28274-29239) and envelope (E) gene (NC_045512.2: 26245-26472) (optional measurements) in samples consisting of in vitro transcribed RNA or purified RNA from lentiviral constructs. Materials were provided in two categories: lower concentration (\u2248 (101-104) /\u03bcL in aqueous solution containing human RNA background) and high concentration (\u2248 109 /\u03bcL in aqueous solution without any other RNA background). For the measurement of N gene concentration in the lower concentration study materials, the majority of laboratories (n = 17) used one-step reverse transcription-digital PCR (RT-dPCR), with three laboratories applying two-step RT-dPCR and one laboratory RT-qPCR. Sixteen laboratories submitted results for E gene concentration. Reproducibility (% CV or equivalent) for RT-dPCR ranged from 19 % to 31 %. Measurements of the high concentration study material by orthogonal methods (isotope dilution-mass spectrometry and single molecule flow cytometry) and a gravimetrically linked lower concentration material were in a good agreement, suggesting a lack of overall bias in RT-dPCR measurements. However methodological factors such as primer and probe (assay) sequences, RT-dPCR reagents and dPCR partition volume were found to be potential sources of interlaboratory variation which need to be controlled when applying this technique. This study demonstrates that the accuracy of RT-dPCR is fit-for-purpose as a RMP for viral RNA target quantification in purified RNA materials and highlights where metrological approaches such as the use of in vitro transcribed controls, orthogonal methods and measurement uncertainty evaluation can support standardization of molecular methods.","version":"1.1","doi":"10.1101/2024.03.27.584106","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.27.586820","pub_date":"2024-3-27","title":"Global variation in prior exposure shapes antibody neutralization profiles of SARS-CoV-2 variants up to BA.2.86","abstract":"The highly mutated SARS-CoV-2 variant, BA.2.86, and its descendants are now the most frequently sequenced variants of SARS-CoV-2. We analyze antibody neutralization data from eight laboratories from the UK, USA, Denmark, and China, including two datasets assessing the effect of XBB.1.5 vaccines, to determine the effect of infection and vaccination history on neutralization of variants up to and including BA.2.86, and produce antibody landscapes to describe these neutralization profiles. We find evidence for lower levels of immune imprinting on pre-Omicron variants in sera collected from Denmark and China, which may be explained by lower levels of circulation of the ancestral variant in these countries, and the use of an inactivated virus vaccine in China.","version":"1.1","doi":"10.1101/2024.03.27.586820","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.26.586802","pub_date":"2024-3-27","title":"Virological characteristics of SARS-CoV-2 Omicron BA.5.2.48","abstract":"With the prevalence of sequentially-emerged sublineages including BA.1, BA.2 and BA.5, SARS-CoV-2 Omicron infection has transformed into a regional epidemic disease. As a sublineage of BA.5, the BA.5.2.48 outbreak and evolved into multi-subvariants in China without clearly established virological characteristics, especially the pathogenicity. Though reduced airborne transmission and pathogenicity of former Omicron sublineages have been revealed in animal models, the virological characteristics of BA.5.2.48 was unidentified. Here, we evaluated the in vitro and in vivo virological characteristics of two isolates of the prevalent BA.5.2.48 subvariant, DY.2 and DY.1.1 (a subvariant of DY.1). DY.2 replicates more efficiently than DY.1.1 in HelahACE2+ cells and Calu-3 cells. The A570S mutation (of DY.1) in a normal BA.5 spike protein (DY.2) leads to a 20% improvement in the hACE2 binding affinity, which is slightly reduced by a further K147E mutation (of DY.1.1). Compared to the normal BA.5 spike, the double-mutated protein demonstrates efficient cleavage and reduced fusogenicity. BA.5.2.48 demonstrated enhanced airborne transmission capacity in hamsters than BA.2. The pathogenicity of BA.5.2.48 is greater than BA.2, as revealed in K18-hACE2 rodents. Under immune selection pressure, DY.1.1 shows stronger fitness than DY.2 in hamster turbinates. Thus the outbreaking prevalent BA.5.2.48 multisubvariants exhibites divergent virological features. Omicron continues to circulate and evolves novel sublineages with indistinguishable pathogenicity and transmission. Therefore humanized Omicron-sensitive animal models must be applied to evaluate the virological characteritics and antiviral therapeutics. By using multiple models including the Omicron-lethal H11-K18-hACE2 rodents, BA.5.2.48 revealed higher pathogenicity in the novel H11-K18-hACE2 rodent models than the previously epidemic BA.2, and thus the models are more adapted to Omicron studies. Moreover, the regional outbreaking of BA.5.2.48 promotes the multidirectional evolution of its subvariants, gaining either enhanced pathogenicity or a fitness in upper airways which is associated with higher transmission, highlighting the importance of surveillance and virological studies on regionally endemic sublineages which represents the short-run evolutionary direction of Omicron.","version":"1.1","doi":"10.1101/2024.03.26.586802","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.18.517133","pub_date":"2024-3-27","title":"In-silico docking platform with serine protease inhibitor (SERPIN) structures identifies host cysteine protease targets with significance for SARS-CoV-2","abstract":"Serine Protease Inhibitors (SERPINs) regulate protease activity in various physiological processes such as inflammation, cancer metastasis, angiogenesis, and neurodegenerative diseases. However, their potential in combating viral infections, where proteases are also crucial, remains underexplored. This is due to our limited understanding of SERPIN expression during viral-induced inflammation and of the SERPINs\u2019 full spectrum of target proteases. Here, we demonstrate widespread expression of human SERPINs in response to respiratory virus infections, both in vitro and in vivo, alongside classical antiviral effectors. Through comprehensive in-silico docking with full-length SERPIN and protease 3D structures, we confirm known inhibitors of specific proteases; more importantly, the results predict novel SERPIN-protease interactions. Experimentally, we validate the direct inhibition of key proteases essential for viral life cycles, including the SERPIN PAI-1\u2019s capability to inhibit select cysteine proteases such as cathepsin L, and the serine protease TMPRSS2. Consequently, PAI-1 suppresses spike maturation and multi-cycle SARS-CoV-2 replication. Our findings challenge conventional notions of SERPIN selectivity, underscore the power of in-silico docking for SERPIN target discovery, and offer potential therapeutic interventions targeting host proteolytic pathways to combat viruses with urgent unmet therapeutic needs. Serine protease inhibitors (SERPINs) play crucial roles in various physiological processes, including viral infections. However, our comprehension of the full array of proteases targeted by the SERPIN family has traditionally been limited, hindering a comprehensive understanding of their regulatory potential. We developed an in-silico docking platform to identify new SERPIN target proteases expressed in the respiratory tract, a critical viral entry portal. The platform confirmed known and predicted new targets for every SERPIN examined, shedding light on previously unrecognized patterns in SERPIN selectivity. Notably, both key proteases for SARS-CoV-2 maturation were among the newly predicted targets, which we validated experimentally. This underscores the platform\u2019s potential in uncovering targets with significance in viral infections, paving the way to define the full potential of the SERPIN family in infectious disease and beyond.","version":"1.2","doi":"10.1101/2022.11.18.517133","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.465994","pub_date":"2024-3-26","title":"Chronumental: time tree estimation from very large phylogenies","abstract":"Phylogenetic trees are an important tool for interpreting sequenced genomes, and their interrelationships. Estimating the date associated with each node of such a phylogeny creates a \u201ctime tree\u201d, which can be especially useful for visualising and analysing evolution of organisms such as viruses. Several tools have been developed for time-tree estimation, but the sequencing explosion in response to the SARS-CoV-2 pandemic has created phylogenies so large as to prevent the application of these previous approaches to full datasets. Here we introduce Chronumental, a tool that can rapidly infer time trees from phylogenies featuring large numbers of nodes. Chronumental uses stochastic gradient descent to identify lengths of time for tree branches which maximise the evidence lower bound under a probabilistic model, implemented in a framework which can be compiled into XLA for rapid computation. We show that Chronumental scales to phylogenies featuring millions of nodes, with chronological predictions made in minutes, and is able to accurately predict the dates of nodes for which it is not provided with metadata.","version":"1.3","doi":"10.1101/2021.10.27.465994","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.22.586249","pub_date":"2024-3-25","title":"How do we respond to the next SARS CoV epidemic/pandemic? A bioinformatics approach with the promise of preventing or reducing the severity of future SARS CoV related pandemics","abstract":"In this work, we develop a Bayesian weighted scheme to generate evolutionary lineages of a particular viral protein sequence of interest and through a process of clustering and choosing representative lineages from the different clusters according to an evolutionary fitness objective function, we demonstrate it is possible to have anticipated the emergence of the SARS-CoV 2 (2019) strain from the SARS-CoV 1(2004) strain and having shown this retrospectively, we discuss the possibility of applying this approach along with continuous genomic surveillance of SARS-CoVs to prevent or reduce severity of future SARS-CoV related pandemics by being prepared with broad neutralization strategies for anticipated future lineages of SARS-CoVs identified through bioinformatics approaches such as that reported in this work.","version":"1.1","doi":"10.1101/2024.03.22.586249","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.25.586528","pub_date":"2024-3-25","title":"System and transcript dynamics of cells infected with severe acute respiratory syndrome virus 2 (SARS-CoV-2)","abstract":"Statistical laws arise in many complex systems and can be explored to gain insights into their structure and behavior. Here, we investigate the dynamics of cells infected with severe acute respiratory syndrome virus 2 (SARS-CoV-2) at the system and individual gene levels; and demonstrate that the statistical frameworks used here are robust in spite of the technical noise associated with single-cell RNA sequencing (scRNA-seq) data. A biphasic fit to Taylor\u2019s power law was observed, and it is likely associated with the larger sampling noise inherent to the measure of less expressed genes. The type of the distribution of the system, as assessed by Taylor\u2019s parameters, varies along the course of infection in a cell type-dependent manner, but also sampling noise had a significant influence on Taylor\u2019s parameters. At the individual gene level, we found that genes that displayed signals of punctual rank stability and/or long-range dependence behavior, as measured by Hurst exponents, were associated with translation, cellular respiration, apoptosis, protein-folding, virus processes, and immune response. Viruses replicate within susceptible cells by exploiting the cellular machinery. Consequently, cells initiate defenses against the virus and signal other cells, notably immune cells. This ongoing battle prompts significant alterations in the cells\u2019 gene expression patterns throughout the infection process. In this study, we apply statistical principles from complex systems theory to analyze gene expression data from individual cells infected with SARS-CoV-2. Our research aims to elucidate how viral infection impacts cells at both systemic and individual gene levels. Our primary findings are twofold: (i) the virus influences the distribution of gene transcripts over the course of infection, varying depending on cell type. (ii) As the infection progresses, numerous genes associated with critical cellular functions and immunity exhibit signs of punctual instability and/or autocorrelation, indicating their response to viral infection at various stages of the process.","version":"1.1","doi":"10.1101/2024.03.25.586528","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.21.585980","pub_date":"2024-3-25","title":"Influenza sequence validation and annotation using VADR","abstract":"Tens of thousands of influenza sequences are deposited into the GenBank database each year. The software tool FLAN has been used by GenBank since 2007 to validate and annotate incoming influenza sequence submissions, and has been publicly available as a webserver but not as a standalone tool. VADR is a general sequence validation and annotation software package used by GenBank for Norovirus, Dengue virus and SARS-CoV-2 virus sequence processing that is available as a standalone tool. We have created VADR influenza models based on the FLAN reference sequences and adapted VADR to accurately annotate influenza sequences. VADR and FLAN show consistent results on the vast majority of influenza sequences, and when they disagree VADR is usually correct. VADR can also accurately process influenza D sequences as well as influenza A H17, H18, H19, N10 and N11 subtype sequences, which FLAN cannot. VADR 1.6.3 and the associated influenza models are now freely available for users to download and use.","version":"1.1","doi":"10.1101/2024.03.21.585980","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.13.540634","pub_date":"2024-3-25","title":"Unrestricted Versus Regulated Open Data Governance: A Bibliometric Comparison of SARS-CoV-2 Nucleotide Sequence Databases","abstract":"Two distinct modes of data governance have emerged in accessing and reusing viral data pertaining to COVID-19: an unrestricted model, espoused by data repositories part of the International Nucleotide Sequence Database Collaboration and a regulated model promoted by the Global Initiative on Sharing All Influenza data. In this paper, we focus on publications mentioning either infrastructure in the period between January 2020 and January 2023, thus capturing a period of acute response to the COVID-19 pandemic. Through a variety of bibliometric and network science methods, we compare the extent to which either data infrastructure facilitated collaboration from different countries around the globe to understand how data reuse can enhance forms of diversity between institutions, countries, and funding groups. Our findings reveal disparities in representation and usage between the two data infrastructures. We conclude that both approaches offer useful lessons, with the unrestricted model providing insights into complex data linkage and the regulated model demonstrating the importance of global representation.","version":"1.3","doi":"10.1101/2023.05.13.540634","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.23.586412","pub_date":"2024-3-24","title":"A human commons cell atlas reveals cell type specificity for OAS1 isoforms","abstract":"We describe an open source Human Commons Cell Atlas comprising 2.9 million cells across 27 tissues that can be easily updated and that is structured to facilitate custom analyses. To showcase the flexibility of the atlas, we demonstrate that it can be used to study isoforms of genes at cell resolution. In particular, we study cell type specificity of isoforms of OAS1, which has been shown to offer SARS-CoV-2 protection in certain individuals that display higher expression of the p46 isoform. Using our commons cell atlas we localize the OAS1 p44b isoform to the testis, and find that it is specific to round and elongating spermatids. By virtue of enabling customized analyses via a modular and dynamic atlas structure, the commons cell atlas should be useful for exploratory analyses that are intractable within the rigid framework of current gene-centric cell atlases.","version":"1.1","doi":"10.1101/2024.03.23.586412","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.16.540953","pub_date":"2024-3-23","title":"Identification of Novel Allosteric Sites of SARS-CoV-2 Papain-Like Protease (PLpro) for the Development of COVID-19 Antivirals","abstract":"Coronaviruses such as SARS-CoV-2 encode a conserved papain-like protease (PLpro) that is crucial for viral replication and immune evasion, making it a prime target for antiviral drug development. In this study, three surface pockets on SARS-CoV-2 PLpro that may function as sites for allosteric inhibition were computationally identified. To evaluate the effects of these pockets on proteolytic activity, 52 residues were separately mutated to alanine. In Pocket 1, located between the Ubl and thumb domains, the introduction of alanine at T10, D12, T54, Y72, or Y83 reduced PLpro activity to <12% of that of WT. In Pocket 2, situated at the interface of the thumb, fingers, and palm domains, Q237A, S239A, H275A, and S278A inactivated PLpro. Finally, introducing alanine at five residues in Pocket 3, between the fingers and palm domains, inactivated PLpro: S212, Y213, Y251, K254, and Y305. Pocket 1 has a higher druggability score than Pockets 2 and 3. MD simulations showed that interactions within and between domains play critical roles in PLpro activity and thermal stability. The essential residues in Pockets 1 and 2 participate in a combination of intra- and inter-domain interactions. By contrast, the essential residues in Pocket 3 predominantly participate in inter-domain interactions. The most promising targets for therapeutic development are Pockets 1 and 3, which have the highest druggability score and the largest number of essential residues, respectively. Non-competitive inhibitors targeting these pockets may be antiviral agents against COVID-19 and related coronaviruses.","version":"1.3","doi":"10.1101/2023.05.16.540953","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.21.586176","pub_date":"2024-3-22","title":"Neutralisation sensitivity of the SARS-CoV-2 BA.2.87.1 variant","abstract":"Against the backdrop of the rapid global takeover and dominance of BA.1/BA.2 and subsequently BA.2.86 lineages, the emergence of a highly divergent SARS-CoV-2 variant warrants characterization and close monitoring. Recently, another such BA.2 descendent, designated BA.2.87.1, was detected in South Africa. Here, we show using spike-pseudotyped viruses that BA.2.87.1 is less resistant to neutralisation by prevailing antibody responses in Sweden than other currently circulating variants such as JN.1. Further we show that a monovalent XBB.1.5-adapted booster enhanced neutralising antibody titers to BA.2.87.1 by almost 4-fold. While BA.2.87.1 may not outcompete other currently-circulating lineages, the repeated emergence and transmission of highly diverged variants suggests that another large antigenic shift, similar to the replacement by Omicron, may be likely in the future.","version":"1.1","doi":"10.1101/2024.03.21.586176","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.21.568093","pub_date":"2024-3-22","title":"Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2","abstract":"Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.","version":"1.2","doi":"10.1101/2023.11.21.568093","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.22.586241","pub_date":"2024-3-22","title":"Design of Antigen-Specific Antibody CDRH3 Sequences Using AI and Germline-Based Templates","abstract":"Antibody-antigen specificity is engendered and refined through a number of complex B cell processes, including germline gene recombination and somatic hypermutation. Here, we present an AI-based technology for de novo generation of antigen-specific antibody CDRH3 sequences using germline-based templates, and validate this technology through the generation of antibodies against SARS-CoV-2. AI-based processes that mimic the outcome, but bypass the complexity of natural antibody generation, can be efficient and effective alternatives to traditional experimental approaches for antibody discovery.","version":"1.1","doi":"10.1101/2024.03.22.586241","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.20.585837","pub_date":"2024-3-21","title":"SARS-CoV-2 infection activates inflammatory macrophages in vascular immune organoids","abstract":"SARS-CoV-2 provokes devastating tissue damage by cytokine release syndrome and leads to multi-organ failure. Modeling the process of immune cell activation and subsequent tissue damage is a significant task. Organoids from human tissues advanced our understanding of SARS-CoV-2 infection mechanisms though, they are missing crucial components: immune cells and endothelial cells. This study aims to generate organoids with these components. We established vascular immune organoids from human pluripotent stem cells and examined the effect of SARS-CoV-2 infection. We demonstrated that infections activated inflammatory macrophages. Notably, the upregulation of interferon signaling supports macrophages\u2019 role in cytokine release syndrome. We propose vascular immune organoids are a useful platform to model and discover factors that ameliorate SARS-CoV-2-mediated cytokine release syndrome.","version":"1.1","doi":"10.1101/2024.03.20.585837","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.24.563841","pub_date":"2024-3-21","title":"Simulation-Driven Design of Stabilized SARS-CoV-2 Spike S2 Immunogens","abstract":"The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2\u2019s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimer\u2019s opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.","version":"1.3","doi":"10.1101/2023.10.24.563841","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.20.585792","pub_date":"2024-3-21","title":"Development of radiofluorinated MLN-4760 derivatives for PET imaging of the SARS-CoV-2 entry receptor ACE2","abstract":"The angiotensin converting enzyme 2 (ACE2) plays a regulatory role in the cardiovascular system and serves SARS-CoV-2 as an entry receptor. The aim of this study was to synthesize and evaluate radiofluorinated derivatives of the ACE2 inhibitor MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were demonstrated to be suitable for non-invasive imaging of ACE2, potentially enabling a better understanding of its expression dynamics. Based on computational molecular modeling, the ACE2-binding modes of F-MLN-4760 and F-Aza-MLN-4760 were similar to that of MLN-4760. Co-crystallization of the hACE2/F-MLN-4760 protein complex was performed for confirmation. Displacement experiments using [3H]MLN-4760 enabled the determination of the binding affinities of the synthesized F-MLN-4760 and F-Aza-MLN-4760 to ACE2 expressed in HEK-ACE2 cells. Aryl trimethylstannane-based and pyridine-based radiofluorination precursors were synthesized and used for the preparation of the respective radiotracers. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were evaluated with regard to the uptake in HEK-ACE2 and HEK-ACE cells and in vitro binding to tissue sections of HEK-ACE2 xenografts and normal organs of mice. Biodistribution and PET/CT imaging studies of [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were performed using HEK-ACE2 and HEK-ACE xenografted nude mice. Crystallography data revealed an equal ACE2-binding mode for F-MLN-4760 as previously found for MLN-4760 and indicated that the same would hold true for F-Aza-MLN-4760. The IC50 values were all in the high nM range, but three-fold lower for F-MLN-4760 and seven-fold lower for F-Aza-MLN-4760 than for MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were obtained in 1.4 \u00b1 0.3 GBq and 0.5 \u00b1 0.1 GBq activity with >99% radiochemical purity in a 5.3% and 1.2% radiochemical yield, respectively. Uptake in HEK-ACE2 cells was higher for [18F]F-MLN-4760 (67 \u00b1 9%) than for [18F]F-Aza-MLN-4760 (37 \u00b1 8%) after 3 h incubation while negligible uptake was seen in HEK-ACE cells (<0.3%). [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 accumulated specifically in HEK-ACE2 xenografts of mice (13 \u00b1 2% IA/g and 15 \u00b1 2% IA/g at 1 h p.i.) with almost no uptake observed in HEK-ACE xenografts (<0.3% IA/g). This was confirmed by PET/CT imaging, which also visualized unspecific accumulation in the gall bladder and intestinal tract. Both radiotracers showed specific and selective binding to ACE2 in vitro and in vivo. [18F]F-MLN-4760 was, however, obtained in higher yields and the ACE2-binding affinity was superior over that of [18F]F-Aza-MLN-4760. [18F]F-MLN-4760 would, thus, be the candidate of choice for further developlment to enable PET imaging of ACE2 in patients.","version":"1.1","doi":"10.1101/2024.03.20.585792","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.19.585194","pub_date":"2024-3-20","title":"Validation of RT-qPCR primers and probes for new and old variants of SARS-CoV-2 in a world scale","abstract":"The demand for molecular diagnosis of pathogens has surged dramatically since the onset of the COVID-19 pandemic. In this context, different diagnostic tests have been developed to identify SARS-CoV-2 in patient samples. The emergence of new variants of SARS-CoV-2 raises questions about whether the molecular tests available for diagnosis continue to be effective in detecting the virus in biological samples. This study analyzed the viability of molecular targets directed to N, E and RdRp genes available against the new variants of SARS-CoV-2. For this, we used bioinformatics tools to analyze SARS-CoV-2 genomic data of different variants deposited in GSAID and NCBI virus genomic databases to assess the accuracy of molecular tests available for the diagnosis of COVID-19. We also developed software for analyzing mutation frequencies in different molecular targets from the mutation database. Mutation frequency analysis revealed a high rate of mutations in the N, E and RdRp genes and targets, although the target regions were more conserved. Only three SNPs were recurrent in the sequences of the variants identified in different continents and all in different targets. On the other hand, the registered mutations are not consistent and do not appear frequently in isolates of the same variant in all regions of the world. Our data suggest that the molecular targets designed for the first SARS-CoV-2 variants remain valid for the identification of new virus variants despite the large number of identified haplotypes. However, false negative test failures can be identified by using more than one molecular target for the same sample. Genomic regions that are under evolutive selective pressure should be avoided in the use of the diagnostic, once the emergence of new variants may affect the efficiency of molecular testing on a global scale.","version":"1.1","doi":"10.1101/2024.03.19.585194","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.20.585861","pub_date":"2024-3-20","title":"Bivalent COVID-19 vaccines boost the capacity of pre-existing SARS-CoV-2-specific memory B cells to cross-recognize Omicron subvariants","abstract":"Bivalent COVID-19 vaccines comprising ancestral Wuhan-Hu-1 (WH1) and the Omicron BA.1 or BA.5 subvariant elicit enhanced serum antibody responses to emerging Omicron subvariants. We characterized the memory B-cell (Bmem) response following a fourth dose with a BA.1 or BA.5 bivalent vaccine, and compared the immunogenicity with a WH1 monovalent fourth dose. Healthcare workers previously immunized with mRNA or adenoviral vector monovalent vaccines were sampled before and one-month after a monovalent, BA.1 or BA.5 bivalent fourth dose COVID-19 vaccine. RBD-specific Bmem were quantified with an in-depth spectral flow cytometry panel including recombinant RBD proteins of the WH1, BA.1, BA.5, BQ.1.1, and XBB.1.5 variants. All recipients had slightly increased WH1 RBD-specific Bmem numbers. Recognition of Omicron subvariants was not enhanced following monovalent vaccination, while both bivalent vaccines significantly increased WH1 RBD-specific Bmem cross-recognition of all Omicron subvariants tested by flow cytometry. Thus, Omicron-based bivalent vaccines can improve recognition of descendent Omicron subvariants by pre-existing, WH1-specific Bmem, beyond that of a conventional, monovalent vaccine. This provides new insights into the capacity of variant-based mRNA booster vaccines to improve immune memory against emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.03.20.585861","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.18.584627","pub_date":"2024-3-18","title":"CORACLE (COVID-19 liteRAture CompiLEr): A platform for efficient tracking and extraction of SARS-CoV-2 and COVID-19 literature, with examples from post-COVID with respiratory involvement","abstract":"During the COVID-19 pandemic there emerged a need to efficiently monitor and process large volumes of scientific literature on the subject. Currently, as the pandemic is winding down, the clinicians encountered a novel syndrome - Post-acute Sequelae of COVID- 19 (PASC) - that affects over 10% of those who contract SARS-CoV-2 and presents a significant and growing challenge in the medical field. The continuous influx of new research publications underscores a critical need for efficient tools for navigating the literature. We aimed to develop an application which will allow monitoring and categorizing COVID-19-related literature through building publication networks and medical subject headings (MeSH) maps to be able to quickly identify key publications and publication networks. We introduce CORACLE (COVID-19 liteRAture CompiLEr), an innovative web application designed for the analysis of COVID-19-related scientific articles and the identification of research trends. CORACLE features three primary interfaces: The \u201cSearch\u201d interface, which displays research trends and citation links; the \u201cCitation Map\u201d interface, allowing users to create tailored citation networks from PubMed Identifiers (PMIDs) to uncover common references among selected articles; and the \u201cMeSH\u201d interface, highlighting current MeSH trends and associations between MeSH terms. Our web application, CORACLE, leverages regularly updated PubMed data to aggregate and categorize the extensive literature on COVID-19 and PASC, aiding in the identification of relevant research publication hubs. Using lung function in PASC patients as a search example, we demonstrate how to identify and visualize the interactions between the relevant publications. CORACLE proves to be an effective tool for the extraction and analysis of literature. Its functionalities, including the MeSH trends and customizable citation mapping, facilitate the discovery of relevant information and emerging trends in COVID-19 and PASC research.","version":"1.1","doi":"10.1101/2024.03.18.584627","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.17.585388","pub_date":"2024-3-18","title":"Binding of SARS-CoV-2 nucleocapsid protein to uninfected epithelial cells induces antibody-mediated complement deposition","abstract":"SARS-CoV-2 infection triggers strong antibody response toward Nucleocapsid-Protein (NP), suggesting extracellular presence beyond its intra-virion RNA binding. Interestingly, NP was found to decorate infected and proximal uninfected cell-surfaces. Here, we propose a new mechanism through which extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated linear-glycosaminoglycans by spatial rearrangement of its RNA-binding sites facilitated by the flexible, positively charged, linker. Coating of uninfected lung-derived cells with purified NP attracted anti-NP-IgG from lung fluids and sera collected from COVID-19 patients. The magnitude of this immune recognition was significantly elevated in moderate compared to mild COVID-19 cases. Importantly, binding of anti-NP-IgG present in sera generated clusters that triggered C3b deposition by the classical complement pathway. Heparin analog enoxaparin outcompeted NP-binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings unveil how extracellular NP may exacerbate COVID-19 tissue damage, and suggest leads for preventative therapy. IgG from patients\u2019 sera target NP-bound cells resulting in complement activation The flexible linker allows NP to both bind linear sulfated GAGs and wrap around RNA Heparin analogs prevent NP surface binding and alleviate complement activation Cell-ELISA anti-NP IgG levels differ between mild and moderate COVID-19","version":"1.1","doi":"10.1101/2024.03.17.585388","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.15.585207","pub_date":"2024-3-18","title":"Modulation of SARS-CoV-2 spike binding to ACE2 through conformational selection","abstract":"The first step of SARS-CoV-2 infection involves the interaction between the trimeric viral spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2). The receptor binding domain (RBD) of S adopts two conformations: open and closed, respectively, accessible and inaccessible to ACE2. Therefore, RBD motions are suspected to affect ACE2 binding; yet a quantitative description of the underlying mechanism has been elusive. Here, using single-molecule approaches, we visualize RBD opening and closing and probe the S/ACE2 interaction. Our results show that RBD dynamics affect ACE2 binding but not unbinding. The resulting modulation is quantitatively predicted by a conformational selection model in which each protomer behaves independently. Our work reveals a general molecular mechanism affecting binding affinity without altering binding strength, helping to understand coronavirus infection and immune evasion.","version":"1.1","doi":"10.1101/2024.03.15.585207","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.15.584819","pub_date":"2024-3-18","title":"A Protein Language Model for Exploring Viral Fitness Landscapes","abstract":"Successively emerging SARS-CoV-2 variants lead to repeated epidemic surges through escalated spreading potential (i.e., fitness). Modeling genotype\u2013fitness relationship enables us to pinpoint the mutations boosting viral fitness and flag high-risk variants immediately after their detection. Here, we introduce CoVFit, a protein language model able to predict the fitness of variants based solely on their spike protein sequences. CoVFit was trained with genotype\u2013fitness data derived from viral genome surveillance and functional mutation data related to immune evasion. When limited to only data available before the emergence of XBB, CoVFit successfully predicted the higher fitness of the XBB lineage. Fully-trained CoVFit identified 549 fitness elevation events throughout SARS-CoV-2 evolution until late 2023. Furthermore, a CoVFit-based simulation was able to predict the higher fitness of JN.1 subvariants before their detection. Our study provides both insight into the SARS-CoV-2 fitness landscape and a novel tool potentially transforming viral genome surveillance.","version":"1.1","doi":"10.1101/2024.03.15.584819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.04.483074","pub_date":"2024-3-15","title":"SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion","abstract":"The COVID-19 pandemic is caused by SARS-CoV-2, an enveloped RNA virus. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Disrupting Golgi function with small molecules strongly inhibits viral infection. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. TGN46 depletion and GRASP55 expression additively inhibit viral secretion, indicating that they act at different stages. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to control viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2022.03.04.483074","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.15.585163","pub_date":"2024-3-15","title":"ONE HEALTH APPROACH ON SARS-COV-2 \u2013 USING SHEEP AS SENTINEL ANIMALS TO INCREASE FUTURE PANDEMIC PREPAREDNESS \u2013 a pilot study","abstract":"Coronaviruses are a family of viruses that can infect a number of species of birds and mammals with great zoonotic potential to cross species barriers and cause spill-over events. SARS-CoV-2 has been shown to cause clinical and inapparent disease and mortality in several animals cohabitating with humans. Sheep are also susceptible to SARS-CoV-2 and have potential to harbor and spread the virus, as well as develop neutralising antibodies due to similarities of virus-receptor interactions to those in humans. The main aim of this study was to investigate the prevalence of SARS-CoV-2 neutralising antibodies in sentinel animals after natural exposure to the virus. The serum samples were collected from sheep in Central Portugal, Serra da Estrela region, both prior to and during the COVID-19 pandemic. The sheep were kept on dairy farms for production of Serra da Estrela cheese, in small herds and in constant contact with farm workers. The sera were tested using already established SARS-CoV-2 pseudovirus systems for multiple SARS-CoV-2 variants including Wuhan, Delta and Omicron. Partial neutralisation activity towards Wuhan and Delta variants was observed, while neutralisating antibody escape was observed in all Omicron variants tested due to the mutations present . Our results indicate that potential SARS-CoV-2 virus cross-species transmission could have been established through contacts between people and animals on sheep farms. Using farm animals as sentinels is of great importance for implementing One Health Approach in zoonotic virus surveillance and control towards increasing future pandemic preparedness.","version":"1.1","doi":"10.1101/2024.03.15.585163","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.14.584985","pub_date":"2024-3-15","title":"Differential Patterns of Cross-Protection against Antigenically Distinct Variants in Small Animal Models of SARS-CoV-2 Infection","abstract":"Continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will likely force more future updates of vaccine composition. Based on a series of studies carried out in human ACE2 transgenic mice (K18-hACE2) and Syrian hamsters, we show that immunity at the respiratory tract, acquired through either previous infection or vaccination with an in-house live attenuate virus, offers protection against antigenically distinct variants in the absence of variant spike-specific neutralizing antibodies. Interestingly, immunity acquired through infection of a modern variant (XBB.1.5) was insufficient in preventing brain infection by the ancestral virus (WA1/2020) in K18-hACE2 mice. Similarly, previous infection with WA1/2020 did not protect against brain infection by XBB.1.5. Our results highlight the importance of immune components other than neutralizing antibodies in maintaining protection against new variants in the respiratory tract, but also paint scenarios where a monovalent vaccine based on a contemporary variant may be less effective against the ancestral strain. Many studies have assessed the cross neutralization of various SARS-CoV-2 variants induced by breakthrough infections or vaccine boosters. Few studies, however, have modeled a more severe type of breakthrough infection. Here, we show that immunity acquired through a previous infection by either a historical virus (WA1/2020) or a contemporary variant (XBB.1.5) failed to protect against brain infection of K18-hACE2 mice by an antigenically distinct virus, although it largely protected the respiratory tract. Our results provided a potential model to investigate the role of different immune components in curbing SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.03.14.584985","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.14.583523","pub_date":"2024-3-15","title":"de Novo Sequencing of Antibodies for Identification of Neutralizing Antibodies in Human Plasma Post SARS-CoV-2 Vaccination","abstract":"We present a method for sequencing polyclonal IgG enriched from human plasma, employing a combination of de novo sequencing, proteomics, bioinformatics, protein separation, sequencing, and peptide separations. Our study analyzes a single patient\u2019s IgG antibody response triggered by the Moderna Spikevax mRNA COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generated 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo sequencing, exhibited similar or higher binding affinities than the original natural polyclonal antibody. Our neutralization tests revealed that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies while highlighting the effectiveness and potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Our proposed approach is an advancement in characterizing the IgG response by directly investigating the circulating pool of IgG without relying exclusively on the B-cell repertoire or population. This is crucial as the B-cell analysis may not accurately represent the circulating antibodies. Interestingly, a large proportion (80 to 90%) of the human antibody sequences generated against SARS-CoV-2 in the literature have been derived solely from B-cell analysis. Therefore, the ability to offer a different perspective is crucial in gaining a comprehensive understanding of the IgG response. We investigate human IgG targeting the receptor binding domain using de novo proteomics. The peripheral B-cell repertoire may not adequately cover all the circulating IgG for human IgG sequencing. Our approach overcomes this limitation by using a de novo protein sequencing on top of standard proteomics. We obtained distinct de novo sequences, showcasing our method\u2019s potential. The recombinant proteins we generate possess traits comparable to or surpassing the naturally occurring polyclonal antibodies (pAbs). This study highlights similarities and differences between IgG populations in blood and circulating B-cells, which is crucial for future biologics development.","version":"1.1","doi":"10.1101/2024.03.14.583523","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.14.585062","pub_date":"2024-3-15","title":"Development of an engineered extracellular vesicles-based vaccine platform for combined delivery of mRNA and protein to induce functional immunity","abstract":"mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs to contain ovalbumin mRNA and protein (EVOvaM+P) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs, natural nanoparticle carriers shed by all cells, that contain mRNA and protein Spike (S) protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.","version":"1.1","doi":"10.1101/2024.03.14.585062","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.15.500232","pub_date":"2024-3-15","title":"Urban birds\u2019 tolerance towards humans was largely unaffected by increased variation in human levels due to COVID-19 shutdowns","abstract":"The coronavirus disease 2019 (COVID-19) pandemic dramatically altered human activities, and, during shutdowns. Here, we evaluated whether urban birds from five countries changed their tolerance towards humans during the COVID-19 shutdowns. We collected 6369 flight initiation distance estimates for 147 bird species and found that human numbers in parks (at a given hour, day, week or year - before and during the COVID-19 shutdowns) had a little effect on birds\u2019 tolerance of approaching humans. Apart from the actual human numbers in the area (hourly scale), the effect of human activity at other temporal scales centered around zero. The results were similar across countries, for most species or when we restricted our analyses only to species sampled both before and during the COVID-19 shutdowns. As expected, the level of daily human presence in parks (measured by Google Mobility Reports) correlated negatively with the stringency of governmental restrictions (a weekly proxy for human presence) and was overall lower during COVID-19 shutdowns than during the post-shutdown year (2022). Our results highlight the resilience of birds to changes in human numbers on multiple temporal scales, the complexities of linking animal fear responses to human behavior, and the challenge of quantifying both simultaneously in situ.","version":"1.2","doi":"10.1101/2022.07.15.500232","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.13.584735","pub_date":"2024-3-14","title":"Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV","abstract":"Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that causes severe and often lethal respiratory illness in humans. The MERS-CoV spike (S) protein is the viral fusogen and the target of neutralizing antibodies, and has therefore been the focus of vaccine design efforts. Currently there are no licensed vaccines against MERS-CoV and only a few candidates have advanced to Phase I clinical trials. Here we developed MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for SARS-CoV-2. Two-component protein nanoparticles displaying MERS-CoV S-derived antigens induced robust neutralizing antibody responses and protected mice against challenge with mouse-adapted MERS-CoV. Electron microscopy polyclonal epitope mapping and serum competition assays revealed the specificities of the dominant antibody responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle vaccine elicited antibodies targeting multiple non-overlapping epitopes in the RBD, whereas anti-NTD antibodies elicited by the S-2P\u2013 and NTD-based immunogens converged on a single antigenic site. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.","version":"1.1","doi":"10.1101/2024.03.13.584735","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.12.571279","pub_date":"2024-3-14","title":"Self-inhibiting percolation and viral spreading in epithelial tissue","abstract":"SARS-CoV-2 induces delayed type-I/III interferon production, allowing it to escape the early innate immune response. The delay has been attributed to a deficiency in the ability of cells to sense viral replication upon infection, which in turn hampers activation of the antiviral state in bystander cells. Here, we introduce a cellular automaton model to investigate the spatiotemporal spreading of viral infection as a function of virus and host-dependent parameters. The model suggests that the considerable person-to-person heterogeneity in SARS-CoV-2 infections is a consequence of high sensitivity to slight variations in biological parameters near a critical threshold. It further suggests that within-host viral proliferation can be curtailed by the presence of remarkably few cells that are primed for IFN production. Thus the observed heterogeneity in defense readiness of cells reflects a remarkably cost-efficient strategy for protection.","version":"1.2","doi":"10.1101/2023.12.12.571279","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.13.584785","pub_date":"2024-3-14","title":"CircRNA-Pro: A Novel Toolkit for High-Precision Detection of Differentially Expressed Circular RNAs and Translatable Circular RNAs","abstract":"With the increasing discovery of circular RNAs (circRNAs) and their critical roles in gene regulation and disease progression, there is a growing need for more accurate and efficient tools for circRNAs research. In response, we have developed an integrated software suite specifically for circRNAs. This all-in-one tool specializes in detecting differentially expressed circRNAs, including those with the potential to be translated into proteins, and allows for comparing against relevant databases, thereby enabling comprehensive circRNA profiling and annotation. To enhance the accuracy in detecting differentially expressed circRNAs, we incorporated three different software algorithms and cross-validated their results through mutual verification. Additionally, this toolkit improves the effectiveness in identifying translatable circRNAs by optimizing Ribo-seq alignment and verifying against public circRNA databases. The performance of circRNA-pro has been evaluated through its application to public RNA-seq and Ribo-seq datasets on breast cancer and SARS-CoV-2 infected cells, and the results obtained have been validated against previous literature and databases. Overall, our integrated toolkit provides a reliable workflow for circRNA research, facilitating insights into their diverse roles across life sciences.","version":"1.1","doi":"10.1101/2024.03.13.584785","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.21.563433","pub_date":"2024-3-13","title":"Changes in total charge on spike protein of SARS-CoV-2 in emerging lineages","abstract":"Charged amino acid residues on the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been shown to influence its binding to different cell surface receptors, its non-specific electrostatic interactions with the environment, and its structural stability and conformation. It is therefore important to obtain a good understanding of amino acid mutations that affect the total charge on the spike protein which have arisen across different SARS-CoV-2 lineages during the course of the virus\u2019 evolution. We analyse the change in the number of ionizable amino acids and the corresponding total charge on the spike proteins of almost 2200 SARS-CoV-2 lineages that have emerged over the span of the pandemic. Our results show that the previously observed trend toward an increase in the positive charge on the spike protein of SARS-CoV-2 variants of concern has essentially stopped with the emergence of the early omicron variants. Furthermore, recently emerged lineages show a greater diversity in terms of their composition of ionizable amino acids. We also demonstrate that the patterns of change in the number of ionizable amino acids on the spike protein are characteristic of related lineages within the broader clade division of the SARS-CoV-2 phylogenetic tree. Due to the ubiquity of electrostatic interactions in the biological environment, our findings are relevant for a broad range of studies dealing with the structural stability of SARS-CoV-2 and its interactions with the environment. The data underlying the article are available in the online Supplementary Material.","version":"1.2","doi":"10.1101/2023.10.21.563433","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.12.584682","pub_date":"2024-3-13","title":"Changes in the senescence profile and immune checkpoints in HIV-infected individuals after COVID-19","abstract":"Both SARS-CoV-2 and HIV infection exhibit alterations in the senescence profile and immune checkpoint (IC) molecules. However, the midterm impact of SARS-CoV-2 on these profiles in people with HIV (PWH) remains unclear. This study aimed to evaluate differences in plasma biomarker levels related to ICs, the senescence-associated secretory phenotype (SASP), and pro- and anti-inflammatory cytokines in PWH following recovery from SARS-CoV-2 infection. We conducted a cross-sectional study of 95 PWH receiving antiretroviral therapy, stratified by SARS-CoV-2 infection status: a) 48 previously infected (HIV/SARS) and b) 47 controls without previous infection (HIV). Plasma biomarkers (n=44) were assessed using Procartaplex Multiplex Immunoassays. Differences were analyzed using a generalized linear model adjusted for sex and ethnicity and corrected for the false discovery rate. Significant values were defined as an adjusted arithmetic mean ratio \u22651.2 or \u22640.8 and a qvalue<0.1. Spearman correlation evaluated relationships between plasma biomarkers (significant correlations, rho\u22650.3 and q value<0.1). The median age of the PWH was 45 years, and 80% were men. All SARS-CoV-2-infected PWH experienced symptomatic infection; 83.3% had mild symptomatic infection, and sample collection occurred at a median of 12 weeks postdiagnosis. The HIV/SARS group showed higher levels of ICs (CD80, PDCD1LG2, CD276, PDCD1, CD47, HAVCR2, TIMD4, TNFRSF9, TNFRSF18, and TNFRSF14), SASP (LTA, CXCL8, and IL13), and inflammatory plasma biomarkers (IL4, IL12B, IL17A, CCL3, CCL4, and INF1A) than did the HIV group. SARS-CoV-2 infection in PWH causes significant midterm disruptions in plasma ICs and inflammatory cytokine levels, highlighting SASP-related factors, which could be risk factors for the emergence of complications in PWH.","version":"1.1","doi":"10.1101/2024.03.12.584682","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.13.583470","pub_date":"2024-3-13","title":"SARS-CoV-2 methyltransferase nsp10-16 in complex with natural and drug-like purine analogs for guiding structure-based drug discovery","abstract":"Non-structural protein 10 (nsp10) and non-structural protein 16 (nsp16) are part of the RNA synthesis complex, which is crucial for the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nsp16 exhibits 2\u2019-O-methyltransferase activity during viral messenger RNA capping and is active in a heterodimeric complex with enzymatically inactive nsp10. It has been shown that inactivation of the nsp10-16 protein complex interferes severely with viral replication, making it a highly promising drug target. As information on ligands binding to the nsp10-16 complex (nsp10-16) is still scarce, we screened the active site for potential binding of drug-like and fragment-like compounds using X-ray crystallography. The screened set of 234 compounds consists of derivatives of the natural substrate S-adenosyl methionine (SAM) and adenine derivatives, of which some have been described previously as methyltransferase inhibitors and nsp16 binders. A docking study guided the selection of many of these compounds. Here we report structures of binders to the SAM site of nsp10-16 and for two of them, toyocamycin and sangivamycin, we present additional crystal structures in the presence of a second substrate, Cap0-analog/Cap0-RNA. The identified hits were tested for binding to nsp10-16 in solution and antiviral activity in cell culture. Our data provide important structural information on various molecules that bind to the SAM substrate site which can be used as novel starting points for selective methyltransferase inhibitor designs.","version":"1.1","doi":"10.1101/2024.03.13.583470","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.11.584210","pub_date":"2024-3-12","title":"Mutational Profiling of SARS-CoV-2 PLpro in human cells reveals requirements for function, structure, and drug escape","abstract":"SARS-CoV-2, the causative agent of COVID-19, is responsible for the recent global pandemic and remains a major source of mortality. Papain-like protease (PLpro) is a target for SARS-CoV-2 inhibitor development, as it is not only essential for viral replication through cleavage of the viral poly-proteins pp1a and pp1ab, but also has de-ubiquitylation and de-ISGylation activities, which can affect innate immune responses. To understand the features of PLpro that dictate activity and anticipate how emerging PLpro variants will affect function, we employed Deep Mutational Scanning to evaluate the mutational effects on enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify all mutations in neighboring residues that support or ablate activity. We characterize residues responsible for substrate binding and demonstrate that although the blocking loop is remarkably tolerant to nearly all mutations, its flexibility is important for enzymatic function. We additionally find a connected network of mutations affecting function but not structure that extends far from the active site. Using our DMS libraries we were able to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.","version":"1.1","doi":"10.1101/2024.03.11.584210","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.12.580004","pub_date":"2024-3-12","title":"SARS-CoV-2 Omicron XBB lineage spike structures, conformations, antigenicity, and receptor recognition","abstract":"A recombinant lineage of the SARS-CoV-2 Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryo-EM structures of XBB.1.5, XBB.1.16, EG.5 and EG.5.1 spike (S) ectodomains to reveal reinforced 3-RBD-down receptor inaccessible closed states mediated by interprotomer receptor binding domain (RBD) interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters including stability, receptor binding and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.","version":"1.3","doi":"10.1101/2024.02.12.580004","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.11.584367","pub_date":"2024-3-11","title":"Exploring the therapeutic potential of defective interfering particles in reducing the replication of SARS-CoV-2","abstract":"SARS-CoV-2 still presents a global threat to human health due to the continued emergence of new strains and waning immunity amongst vaccinated populations. Therefore, it is still relevant to investigate potential therapeutics, such as therapeutic interfering particles (TIPs). Mathematical and computational modelling are valuable tools to study viral infection dynamics for predictive analysis. Here, we expand on the previous work by Grebennikov et al. (2021) on SARS-CoV-2 intra-cellular replication dynamics to include defective interfering particles (DIPs) as potential therapeutic agents. We formulate a deterministic model that describes the replication of wild-type (WT) SARS-CoV-2 virus in the presence of DIPs. Sensitivity analysis of parameters to several model outputs is employed to inform us on those parameters to be carefully calibrated from experimental data. We then study the effects of co-infection on WT replication and how DIP dose perturbs the release of WT viral particles. Furthermore, we provide a stochastic formulation of the model that is compared to the deterministic one. These models could be further developed into population-level models or used to guide the development and dose of TIPs. SARS-CoV-2 continues to evolve, with new strains or sub-strains being identified thanks to efforts to monitor the virus. Consequently, new strains threaten human health as current vaccinations may not adequately protect against future strains. It is therefore important to understand the roles that additional therapeutics could play in protecting against these future strains. Therapeutic interfering particles (TIPs), otherwise referred to as defective interfering particles (DIPs), could provide an additional treatment option against future strains. Previous models have examined the role of DIPs at the within-host level during co-infection with wild-type virus, but have paid little attention to intra-cellular dynamics. Here we extend the previous intra-cellular replication model of SARS-CoV-2 by Grebennikov et al. (2021) to include co-infection of WT virus with DIPs. We show that DIPs lead to a reduction in the WT virus in a dose-dependent manner, with higher doses leading to up to 10-fold reduction in total WT virus released from a cell depending on the multiplicity of infection (MOI). We find these results to be consistent for both deterministic and stochastic formulations of the model. Our approaches could be developed into a within-host model or population-level model, which could then be used to guide therapeutic DIP doses.","version":"1.1","doi":"10.1101/2024.03.11.584367","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.08.584120","pub_date":"2024-3-11","title":"Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation","abstract":"The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.","version":"1.1","doi":"10.1101/2024.03.08.584120","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.10.584306","pub_date":"2024-3-11","title":"SARS-CoV-2 Omicron BA.2.87.1 Exhibits Higher Susceptibility to Serum Neutralization Than EG.5.1 and JN.1","abstract":"As SARS-CoV-2 continues to spread and mutate, tracking the viral evolutionary trajectory and understanding the functional consequences of its mutations remain crucial. Here, we characterized the antibody evasion, ACE2 receptor engagement, and viral infectivity of the highly mutated SARS-CoV-2 Omicron subvariant BA.2.87.1. Compared with other Omicron subvariants, including EG.5.1 and the current predominant JN.1, BA.2.87.1 exhibits less immune evasion, reduced viral receptor engagement, and comparable infectivity in Calu-3 lung cells. Intriguingly, two large deletions (\u039415-26 and \u0394136-146) in the N-terminal domain (NTD) of the spike protein facilitate subtly increased antibody evasion but significantly diminish viral infectivity. Collectively, our data support the announcement by the USA CDC that the public health risk posed by BA.2.87.1 appears to be low.","version":"1.1","doi":"10.1101/2024.03.10.584306","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.02.535277","pub_date":"2024-3-11","title":"Community structure and temporal dynamics of SARS-CoV-2 epistatic network allow for early detection of emerging variants with altered phenotypes","abstract":"The emergence of viral variants with altered phenotypes is a public health challenge underscoring the need for advanced evolutionary forecasting methods. Given extensive epistatic interactions within viral genomes and known viral evolutionary history, efficient genomic surveillance necessitates early detection of emerging viral haplotypes rather than commonly targeted single mutations. Haplotype inference, however, is a significantly more challenging problem precluding the use of traditional approaches. Here, using SARS-CoV-2 evolutionary dynamics as a case study, we show that emerging haplotypes with altered transmissibility can be linked to dense communities in coordinated substitution networks, which become discernible significantly earlier than the haplotypes become prevalent. From these insights, we develop a computational framework for inference of viral variants and validate it by successful early detection of known SARS-CoV-2 strains. Our methodology offers greater scalability than phylogenetic lineage tracing and can be applied to any rapidly evolving pathogen with adequate genomic surveillance data.","version":"1.2","doi":"10.1101/2023.04.02.535277","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.11.583978","pub_date":"2024-3-11","title":"Distinct Patterns of SARS-CoV-2 BA.2.87.1 and JN.1 Variants in Immune Evasion, Antigenicity and Cell-Cell Fusion","abstract":"The rapid evolution of SARS-CoV-2 variants presents a constant challenge to the global vaccination effort. In this study, we conducted a comprehensive investigation into two newly emerged variants, BA.2.87.1 and JN.1, focusing on their neutralization resistance, infectivity, antigenicity, cell-cell fusion, and spike processing. Neutralizing antibody (nAb) titers were assessed in diverse cohorts, including individuals who received a bivalent mRNA vaccine booster, patients infected during the BA.2.86/JN.1-wave, and hamsters vaccinated with XBB.1.5-monovalent vaccine. We found that BA.2.87.1 shows much less nAb escape from WT-BA.4/5 bivalent mRNA vaccination and JN.1-wave breakthrough infection sera compared to JN.1 and XBB.1.5. Interestingly. BA.2.87.1 is more resistant to neutralization by XBB.15-monovalent-vaccinated hamster sera than BA.2.86/JN.1 and XBB.1.5, but efficiently neutralized by a class III monoclonal antibody S309, which largely fails to neutralize BA.2.86/JN.1. Importantly, BA.2.87.1 exhibits higher levels of infectivity, cell-cell fusion activity, and furin cleavage efficiency than BA.2.86/JN.1. Antigenically, we found that BA.2.87.1 is closer to the ancestral BA.2 compared to other recently emerged Omicron subvariants including BA.2.86/JN.1 and XBB.1.5. Altogether, these results highlight immune escape properties as well as biology of new variants and underscore the importance of continuous surveillance and informed decision-making in the development of effective vaccines.","version":"1.1","doi":"10.1101/2024.03.11.583978","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.07.583823","pub_date":"2024-3-08","title":"Antigenicity assessment of SARS-CoV-2 saltation variant BA.2.87.1","abstract":"The recent emergence of a SARS-CoV-2 saltation variant, BA.2.87.1, which features 65 spike mutations relative to BA.2, has attracted worldwide attention. In this study, we elucidate the antigenic characteristics and immune evasion capability of BA.2.87.1. Our findings reveal that BA.2.87.1 is more susceptible to XBB-induced humoral immunity compared to JN.1. Notably, BA.2.87.1 lacks critical escaping mutations in the receptor binding domain (RBD) thus allowing various classes of neutralizing antibodies (NAbs) that were escaped by XBB or BA.2.86 subvariants to neutralize BA.2.87.1, although the deletions in the N-terminal domain (NTD), specifically 15-23del and 136-146del, compensate for the resistance to humoral immunity. Interestingly, several neutralizing antibody drugs have been found to restore their efficacy against BA.2.87.1, including SA58, REGN-10933 and COV2-2196. Hence, our results suggest that BA.2.87.1 may not become widespread until it acquires multiple RBD mutations to achieve sufficient immune evasion comparable to that of JN.1.","version":"1.1","doi":"10.1101/2024.03.07.583823","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.07.583944","pub_date":"2024-3-08","title":"Coronavirus Spike-RBD Variants Differentially Bind to the Human ACE2 Receptor","abstract":"The SARS-CoV-2 betacoronavirus infects people through binding the human Angiotensin Receptor 2 (ACE2), followed by import into a cell utilizing the Transmembrane Protease, Serine 2 (TMPRSS2) and Furin cofactors. Analysis of the SARS-CoV-2 extracellular spike protein has suggested critical amino acids necessary for binding within a 197-residue portion, the receptor binding domain (RBD). A cell-based assay between a membrane tethered RBD-GFP fusion protein and the membrane bound ACE2-Cherry fusion protein allowed for mutational intersection of both RBD and ACE2 proteins. Data shows Omicron BA.1 and BA.2 variants have altered dependency on the amino terminus of ACE2 protein and suggests multiple epitopes on both proteins stabilize their interactions at the Nt and internal region of ACE2. In contrast, the H-CoV-NL63 RBD is only dependent on the ACE2 internal region for binding. A peptide inhibitor approach to this internal region thus far have failed to block binding of RBDs to ACE2, suggesting that several binding regions on ACE2 are sufficient to allow functional interactions. In sum, the RBD binding surface of ACE2 appears relatively fluid and amenable to bind a range of novel variants.","version":"1.1","doi":"10.1101/2024.03.07.583944","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.28.569056","pub_date":"2024-3-08","title":"Human Cytokine and Coronavirus Nucleocapsid Protein Interactivity Using Large-Scale Virtual Screens","abstract":"Understanding the interactions between SARS-CoV-2 and the human immune system is paramount to the characterization of novel variants as the virus co-evolves with the human host. In this study, we employed state-of-the-art molecular docking tools to conduct large-scale virtual screens, predicting the binding affinities between 64 human cytokines against 17 nucleocapsid proteins from six betacoronaviruses. Our comprehensive in silico analyses reveal specific changes in cytokine-nucleocapsid protein interactions, shedding light on potential modulators of the host immune response during infection. These findings offer valuable insights into the molecular mechanisms underlying viral pathogenesis and may guide the future development of targeted interventions. This manuscript serves as insight into the comparison of deep learning based AlphaFold2-Multimer and the semi-physicochemical based HADDOCK for protein-protein docking. We show the two methods are complementary in their predictive capabilities. We also introduce a novel algorithm for rapidly assessing the binding interface of protein-protein docks using graph edit distance: graph-based interface residue assessment function (GIRAF). The high-performance computational framework presented here will not only aid in accelerating the discovery of effective interventions against emerging viral threats, but extend to other applications of high throughput protein-protein screens.","version":"1.2","doi":"10.1101/2023.11.28.569056","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.08.561389","pub_date":"2024-3-07","title":"Protein design for evaluating vaccines against future viral variation","abstract":"Recurrent waves of SARS-CoV-2 infection, driven by the periodic emergence of new viral variants, highlight the need for vaccines and therapeutics that remain effective against future strains. Yet, our ability to proactively evaluate such therapeutics is limited to assessing their effectiveness against previous or circulating variants, which may differ significantly in their antibody escape from future viral evolution. To address this challenge, we develop a deep learning method to predict the effect of mutations on fitness and escape from neutralizing antibodies. We use this model to engineer 83 unique SARS-CoV-2 Spike proteins incorporating novel combinations of up to 46 amino acid changes relative to the ancestral B.1 variant. The designed constructs were infectious and evaded neutralization by nine well-characterized panels of human polyclonal anti-SARS-CoV-2 immune sera (from vaccinated, boosted, bivalent boosted, and breakthrough infection individuals). Designed constructs on contemporary SARS-CoV-2 strains displayed similar levels of antibody neutralization escape and similar antigenic profiles as variants seen subsequently (up to 12 months later) during the COVID-19 pandemic despite differences in exact mutations. Our approach provides targeted datasets of antigenically diverse escape variants for an early evaluation of the protective ability of vaccines and therapeutics to inhibit not only currently circulating but also future variants. This approach is generalizable to other viral pathogens.","version":"1.2","doi":"10.1101/2023.10.08.561389","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.04.583415","pub_date":"2024-3-06","title":"Targeting G9a translational mechanism of SARS-CoV-2 pathogenesis for multifaceted therapeutics of COVID-19 and its sequalae","abstract":"By largely unknown mechanism(s), SARS-CoV-2 hijacks the host translation apparatus to promote COVID-19 pathogenesis. We report that the histone methyltransferase G9a noncanonically regulates viral hijacking of the translation machinery to bring about COVID-19 symptoms of hyperinflammation, lymphopenia, and blood coagulation. Chemoproteomic analysis of COVID-19 patient peripheral mononuclear blood cells (PBMC) identified enhanced interactions between SARS-CoV-2-upregulated G9a and distinct translation regulators, particularly the N-methyladenosine (m6A) RNA methylase METTL3. These interactions with translation regulators implicated G9a in translational regulation of COVID-19. Inhibition of G9a activity suppressed SARS-CoV-2 replication in human alveolar epithelial cells. Accordingly, multi-omics analysis of the same alveolar cells identified SARS-CoV-2-induced changes at the transcriptional, m6A-epitranscriptional, translational, and post-translational (phosphorylation or secretion) levels that were reversed by inhibitor treatment. As suggested by the aforesaid chemoproteomic analysis, these multi-omics-correlated changes revealed a G9a-regulated translational mechanism of COVID-19 pathogenesis in which G9a directs translation of viral and host proteins associated with SARS-CoV-2 replication and with dysregulation of host response. Comparison of proteomic analyses of G9a inhibitor-treated, SARS-CoV-2 infected cells, or ex vivo culture of patient PBMCs, with COVID-19 patient data revealed that G9a inhibition reversed the patient proteomic landscape that correlated with COVID-19 pathology/symptoms. These data also indicated that the G9a-regulated, inhibitor-reversed, translational mechanism outperformed G9a-transcriptional suppression to ultimately determine COVID-19 pathogenesis and to define the inhibitor action, from which biomarkers of serve symptom vulnerability were mechanistically derived. This cell line-to-patient conservation of G9a-translated, COVID-19 proteome suggests that G9a inhibitors can be used to treat patients with COVID-19, particularly patients with long-lasting COVID-19 sequelae.","version":"1.1","doi":"10.1101/2024.03.04.583415","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.05.583594","pub_date":"2024-3-06","title":"MORTALITY-ASSOCIATED SARS-COV-2 GENOMIC VARIANTS FROM PATIENTS HOSPITALIZED FOR SEVERE PNEUMONIA IN AGUASCALIENTES, MEXICO FROM 2020 TO 2023","abstract":"The SARS-CoV-2 virus has caused a global health crisis, resulting in a significant loss of human lives. It is essential to report disease and mutation associations to provide ideas for public health interventions and preventive measures. In this study, to determine the association between genomic variants and the severity of pneumonia caused by SARS-CoV-2, a sequencing analysis of 150 patient samples with confirmed COVID-19 was conducted. These samples were collected between 2021 and 2023 and isolated in Aguascalientes, Mexico. The patient cohort had males and females ranging from 0 to 91 years old. Males accounted for 66% of the population analyzed. The Delta variant was the most prevalent lineage associated with deaths in 2021-2022, while the B.1.1.529 lineages emerged in mid-2022. Currently, the XBB lineage is the most commonly identified in Mexico. New mutations L95M and L46M in ORF 8 and ORF 9 were discovered in 30% and 20% of the sequences and are uniquely present in the studied population. These mutations are positively associated with patient death. This study provides valuable data to aid in understanding the evolution of SARS-CoV-2 in specific populations and explores the severity of the disease and mutation correlations.","version":"1.1","doi":"10.1101/2024.03.05.583594","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.04.583288","pub_date":"2024-3-06","title":"Predicting Phylogenetic Bootstrap Values via Machine Learning","abstract":"Estimating the statistical robustness of the inferred tree(s) constitutes an integral part of most phylogenetic analyses. Commonly, one computes and assigns a branch support value to each inner branch of the inferred phylogeny. The most widely used method for calculating branch support on trees inferred under Maximum Likelihood (ML) is the Standard, non-parametric Felsenstein Bootstrap Support (SBS). Due to the high computational cost of the SBS, a plethora of methods has been developed to approximate it, for instance, via the Rapid Bootstrap (RB) algorithm. There have also been attempts to devise faster, alternative support measures, such as the SH-aLRT (Shimodaira\u2013Hasegawalike approximate Likelihood Ratio Test) or the UltraFast Bootstrap 2 (UFBoot2) method. Those faster alternatives exhibit some limitations, such as the need to assess model violations (UFBoot2) or meaningless low branch support intervals (SH-aLRT). Here, we present the Educated Bootstrap Guesser (EBG), a machine learning-based tool that predicts SBS branch support values for a given input phylogeny. EBG is on average 9.4 (\u03c3 = 5.5) times faster than UFBoot2. EBG-based SBS estimates exhibit a median absolute error of 5 when predicting SBS values between 0 and 100. Furthermore, EBG also provides uncertainty measures for all per-branch SBS predictions and thereby allows for a more rigorous and careful interpretation. EBG can predict SBS support values on a phylogeny comprising 1654 SARS-CoV2 genome sequences within 3 hours on a mid-class laptop. EBG is available under GNU GPL3. github.com/wiegertj/EBG github.com/wiegertj/EBG-train julius-wiegert@web.de","version":"1.1","doi":"10.1101/2024.03.04.583288","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.02.24303242","pub_date":"2024-03-05","title":"The Majority of SARS-CoV-2 Plasma Cells are Excluded from the Bone Marrow Long-Lived Compartment 33 Months after mRNA Vaccination","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The goal of any vaccine is to induce long-lived plasma cells (LLPC) to provide life-long protection. Natural infection by influenza, measles, or mumps viruses generates bone marrow (BM) LLPC similar to tetanus vaccination which affords safeguards for decades. Although the SARS-CoV-2 mRNA vaccines protect from severe disease, the serologic half-life is short-lived even though SARS-CoV-2-specific plasma cells can be found in the BM. To better understand this paradox, we enrolled 19 healthy adults at 1.5-33 months after SARS-CoV-2 mRNA vaccine and measured influenza-, tetanus-, or SARS-CoV-2-specific antibody secreting cells (ASC) in LLPC (CD19\n                  <jats:sup>\u2212</jats:sup>\n                  ) and non-LLPC (CD19\n                  <jats:sup>+</jats:sup>\n                  ) subsets within the BM. All individuals had IgG ASC specific for influenza, tetanus, and SARS-CoV-2 in at least one BM ASC compartment. However, only influenza- and tetanus-specific ASC were readily detected in the LLPC whereas SARS-CoV-2 specificities were mostly excluded. The ratios of non-LLPC:LLPC for influenza, tetanus, and SARS-CoV-2 were 0.61, 0.44, and 29.07, respectively. Even in five patients with known PCR-proven history of infection and vaccination, SARS-CoV-2-specific ASC were mostly excluded from the LLPC. These specificities were further validated by using multiplex bead binding assays of secreted antibodies in the supernatants of cultured ASC. Similarly, the IgG ratios of non-LLPC:LLPC for influenza, tetanus, and SARS-CoV-2 were 0.66, 0.44, and 23.26, respectively. In all, our studies demonstrate that rapid waning of serum antibodies is accounted for by the inability of mRNA vaccines to induce BM LLPC.\n                </jats:p>","version":null,"doi":"10.1101/2024.03.02.24303242","journal":"medRxiv","score":null},{"id":"10.1101/2024.03.05.583547","pub_date":"2024-3-05","title":"A 10-valent composite mRNA vaccine against both influenza and COVID-19","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 viruses has had a persistent and significant impact on global public health for four years. Recently, there has been a resurgence of seasonal influenza transmission worldwide. The co-circulation of SARS-CoV-2 and seasonal influenza viruses results in a dual burden on communities. Additionally, the pandemic potential of zoonotic influenza viruses, such as avian Influenza A/H5N1 and A/H7N9, remains a concern. Therefore, a combined vaccine against all these respiratory diseases is in urgent need. mRNA vaccines, with their superior efficacy, speed in development, flexibility, and cost-effectiveness, offer a promising solution for such infectious diseases and potential future pandemics. In this study, we present FLUCOV-10, a novel 10-valent mRNA vaccine created from our proven platform. This vaccine encodes hemagglutinin (HA) proteins from four seasonal influenza viruses and two avian influenza viruses with pandemic potential, as well as spike proteins from four SARS-CoV-2 variants. A two-dose immunization with the FLUCOV-10 elicited robust immune responses in mice, producing IgG antibodies, neutralizing antibodies, and antigen-specific cellular immune responses against all the vaccine-matched viruses of influenza and SARS-CoV-2. Remarkably, the FLUCOV-10 immunization provided complete protection in mouse models against both homologous and heterologous strains of influenza and SARS-CoV-2. These results highlight the potential of FLUCOV-10 as an effective vaccine candidate for the prevention of influenza and COVID-19. Amidst the ongoing and emerging respiratory viral threats, particularly the concurrent and sequential spread of SARS-CoV-2 and influenza, our research introduces FLUCOV-10. This novel mRNA-based combination vaccine, designed to counteract both influenza and COVID-19, by incorporating genes for surface glycoproteins from various influenza viruses and SARS-CoV-2 variants. This combination vaccine showed highly effective in preclinical trials, generating strong immune responses, and ensuring protection against both matching and heterologous strains of influenza and SARS-CoV-2. FLUCOV-10 represents a significant step forward in our ability to address respiratory viral threats, showcasing potential as a singular, adaptable vaccine solution for global health challenges.","version":"1.1","doi":"10.1101/2024.03.05.583547","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.09.552633","pub_date":"2024-3-05","title":"Iterative In Silico Screening for Optimizing Stable Conformation of Anti-SARS-CoV-2 Nanobodies","abstract":"Nanobodies (Nbs or VHHs) are single-domain antibodies (sdAbs) derived from camelid heavy-chain antibodies. The variable region of these nanobodies has special and unique characteristics, such as small size, good tissue penetration, and cost-effective production, making nanobodies a good candidate for the diagnosis and treatment of viruses. Identifying effective nanobodies against the COVID-19 would help us defeat this dangerous virus or other unknown variants in future. Herein, we introduce an in silico screening strategy for optimizing stable conformation of anti-SARS-CoV-2 nanobodies. Firstly, various complexes containing nanobodies were downloaded from the RCSB database, which were identified from immunized llamas. The primary docking between nanobodies and the SARS-CoV-2 spike protein receptor-binding domain was performed through ClusPro program, with the manually screening that leaving the reasonable conformation to the next step. Then, the binding distances of atoms between the antigen-antibody interfaces were measured through the NeighborSearch algorithm. Finally, filtered nanobodies were acquired according to HADDOCK scores through HADDOCK docking the COVID spike protein with nanobodies under restrictions of calculated molecular distance between active residues and antigenic epitopes less than 4.5 \u00c5. In this way, those nanobodies which with more reasonable conformation and with stronger neutralizing efficacy were acquired. To validate the efficacy ranking of the nanobodies we obtained, we calculated the binding affinities (\u0394G) and dissociation constants (Kd) of all screened nanobodies using the PRODIGY web tool, and predicted the stability changes induced by all possible point mutations in nanobodies using the MAESTROWeb server. Furthermore, we examined the performance of the relationship between nanobodies\u2019 ranking and their number of mutation-sensitive sites (Spearman correlation > 0.68), the results revealed a robust correlation, indicating that the superior nanobodies identified through our screening process exhibited fewer mutation hotspots and higher stability. This correlation analysis demonstrates the validity of our screening criteria, underscoring the suitability of these nanobodies for future development and practical implementation. In conclusion, this three-step screening strategy iteratively in silico greatly improved the accuracy of screening desired nanobodies compared to using only ClusPro docking or default HADDOCK docking settings. It provides new ideas for the screening of novel antibodies and computer-aided screening methods.","version":"1.2","doi":"10.1101/2023.08.09.552633","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.01.581813","pub_date":"2024-3-05","title":"Filopodial Mechanotransduction is regulated by Angiotensin-Converting Enzyme 2 (ACE2) and by SARS-CoV-2 spike protein","abstract":"Filopodia are dynamic, actin-rich cellular protrusions, increasingly linked to cellular mechanotransduction. However, how dynamic filopodia translate external mechanical cues remains poorly understood. Recent studies show that the SARS-CoV-2 spike (S) protein binds the ACE2 receptor on airway multicilia and that cilia are required for viral infection(1) and sufficient to induce filopodial extension and viral binding. To test if spike protein is sufficient to induce filopodial expansion, we employed live-cell single-particle tracking with quantum dots targeting ACE2, to reveal a robust filopodia extension and virus binding mechanism requiring the enzymatic activity of ACE2. Using time-lapse imaging, we reveal that spike protein binding to filopodia is associated with intracellular actin remodeling, alterations in bulk cell stiffness, and an elevation in intracellular calcium levels linked to actin-rearrangement, filopodia initiation, and persistence. We propose the activation of ACE2 creates an active signaling and mechanosensory environment within adherent cells and airway epithelial cells that allows the remodeling of actin in filopodia to trap virus and potentially organize viral exit from cells. [164 words] SARS-CoV-2 spike protein activates calcium and actin dynamics to enable filopodial extension and virus binding","version":"1.1","doi":"10.1101/2024.03.01.581813","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.04.583260","pub_date":"2024-3-05","title":"SARS-CoV-2 modulates NK cell responses via induction of HLA-E and triggers expansion of adaptive NK cells during acute infection","abstract":"HLA-E expression plays a central role for modulation of NK cell function by interaction with inhibitory NKG2A and stimulatory NKG2C receptors on canonical and adaptive NK cells, respectively. Here, we demonstrate that infection of human primary lung tissue with SARS-CoV-2 leads to increased HLA-E expression and show that processing of the peptide YLQPRTFLL from the spike protein is primarily responsible for the strong, dose-dependent increase of HLA-E. Targeting the peptide site within the spike protein revealed that a single point mutation was sufficient to abrogate the increase in HLA-E expression. Spike-mediated induction of HLA-E differentially affected NK cell function: whereas degranulation, IFN-\u03b3 production, and target cell cytotoxicity were enhanced in NKG2C+ adaptive NK cells, effector functions were inhibited in NKG2A+ canonical NK cells. Analysis of a cohort of COVID-19 patients in the acute phase of infection revealed that adaptive NK cells were induced irrespective of the HCMV status, challenging the paradigm that adaptive NK cells are only generated during HCMV infection. During the first week of hospitalization, patients exhibited a selective increase of early NKG2C+CD57- adaptive NK cells whereas mature NKG2C+CD57+ cells remained unchanged. Further analysis of recovered patients suggested that the adaptive NK cell response is primarily driven by a wave of early adaptive NK cells during acute infection that wanes once the infection is cleared. Together, this study suggests that NK cell responses to SARS-CoV-2 infection are majorly influenced by the balance between canonical and adaptive NK cells via the HLA-E/NKG2A/C axis.","version":"1.1","doi":"10.1101/2024.03.04.583260","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.04.583178","pub_date":"2024-3-05","title":"Why is the Omicron main protease of SARS-CoV-2 less stable than its wild-type counterpart? A crystallographic, biophysical, and theoretical study of the free enzyme and its complex with inhibitor 13b-K","abstract":"During the continuing evolution of SARS-CoV-2, the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November that year. Along with its sublineages, it has maintained a prominent role ever since. The Nsp5 main protease (Mpro) of the Omicron virus is characterized by a single dominant mutation, P132H. Here we determined the X-ray crystal structures of the P132H mutant (or O-Mpro) as free enzyme and in complex with the Mpro inhibitor, the alpha-ketoamide 13b-K, and we conducted enzymology, biophysical as well as theoretical studies to characterize the O-Mpro. We found that O-Mpro has a similar overall structure and binding with 13b-K; however, it displays lower enzymatic activity and lower thermal stability compared to the WT-Mpro (with \u201cWT\u201d referring to the original Wuhan-1 strain). Intriguingly, the imidazole ring of His132 and the carboxylate plane of Glu240 are in a stacked configuration in the X-ray structures determined here. The empirical folding free energy calculations suggest that the O-Mpro dimer is destabilized relative to the WT-Mpro due to the less favorable van der Waals interactions and backbone conformation in the individual protomers. The all-atom continuous constant pH molecular dynamics (MD) simulations reveal that His132 and Glu240 display coupled titration. At pH 7, His132 is predominantly neutral and in a stacked configuration with respect to Glu240 which is charged. In order to examine whether the Omicron mutation eases the emergence of further Mpro mutations, we also determined crystal structures of the relatively frequent P132H+T169S double mutant but found little evidence for a correlation between the two sites.","version":"1.1","doi":"10.1101/2024.03.04.583178","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.04.580093","pub_date":"2024-3-05","title":"Yeast-based production platform for potent and stable heavy chain-only antibodies","abstract":"Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.","version":"1.1","doi":"10.1101/2024.03.04.580093","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.01.582987","pub_date":"2024-3-04","title":"Newcastle Disease Virus Vector-Based SARS-CoV-2 Vaccine Candidate AVX/COVID-12 Activates T Cells and Is Recognized by Antibodies from COVID-19 Patients and Vaccinated","abstract":"Several effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and implemented in the population. However, the current production capacity falls short of meeting global demand. Therefore, it is crucial to further develop novel vaccine platforms that can bridge the distribution gap. AVX/COVID-12 is a vector-based vaccine that utilizes the Newcastle Disease virus (NDV) to present the SARS-CoV-2 spike protein to the immune system. This study analyses the antigenicity of the vaccine candidate by examining antibody binding and T-cell activation in individuals infected with SARS-CoV-2 or variants of concern (VOCs), as well as in healthy volunteers who received coronavirus disease 2019 (COVID-19) vaccinations. Our findings indicate that the vaccine effectively binds antibodies and activates T-cells in individuals who received 2 or 3 doses of BNT162b2 or AZ/ChAdOx-1-S vaccines. Furthermore, the stimulation of T-cells from patients and vaccine recipients with AVX/COVID-12 resulted in their proliferation and secretion of interferon-gamma (IFN-\u03b3) in both CD4+ and CD8+ T-cells. In conclusion, the AVX/COVID-12 vectored vaccine candidate demonstrates the ability to stimulate robust cellular responses and is recognized by antibodies primed by the spike protein present in SARS-CoV-2 viruses that infected patients, as well as in the mRNA BNT162b2 and AZ/ChAdOx-1-S vaccines. These results support the inclusion of the AVX/COVID-12 vaccine as a booster in vaccination programs aimed at addressing COVID-19 caused by SARS-CoV-2 and its VOCs.","version":"1.1","doi":"10.1101/2024.03.01.582987","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.25.568670","pub_date":"2024-3-04","title":"Identification of the host reservoir of SARS-CoV-2 and determining when it spilled over into humans","abstract":"Since the emergence of SARS-CoV-2 in Wuhan in 2019 its host reservoir has not been established. Phylogenetic analysis was performed on whole genome sequences (WGS) of 71 coronaviruses and a Breda virus. A subset comprising two SARS-CoV-2 Wuhan viruses and 8 of the most closely related coronavirus sequences were used for host reservoir analysis using Bayesian Evolutionary Analysis Sampling Trees (BEAST). Within these genomes, 20 core genome fragments were combined into 2 groups each with similar clock rates (5.9\u00d710\u22123 and 1.1\u00d710\u22123 subs/site/year). Pooling the results from these fragment groups yielded a most recent common ancestor (MRCA) shared between SARS-COV-2 and the bat isolate RaTG13 around 2007 (95% HPD: 2003, 2011). Further, the host of the MRCA was most likely a bat (probability 0.64 - 0.87). Hence, the spillover into humans must have occurred at some point between 2007 and 2019 and bats may have been the most likely host reservoir.","version":"1.2","doi":"10.1101/2023.11.25.568670","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.03.583187","pub_date":"2024-3-04","title":"B cell maturation restored ancestral germlines to control Omicron BA.2.86","abstract":"The unceasing interplay between SARS-CoV-2 and the human immune system has led to a continuous maturation of the virus and B cell response providing an opportunity to track their evolution in real time. We longitudinally analyzed the functional activity of almost 1,000 neutralizing human monoclonal antibodies (nAbs) isolated from vaccinated people, and from individuals with hybrid and super hybrid immunity (SH), developed after three mRNA vaccine doses and two breakthrough infections. The most potent neutralization and Fc functions against highly mutated variants, including BA.2.86, were found in the SH cohort. Despite different priming, epitope mapping revealed a convergent maturation of the functional antibody response. Neutralization was mainly driven by Class 1/2 nAbs while Fc functions were induced by Class 3/4 antibodies. Remarkably, broad neutralization was mediated by restored IGHV3-53/3-66 B cell germlines which, after heterogenous exposure to SARS-CoV-2 S proteins, increased their level of somatic hypermutations. Our study shows the resilience of the human immune system which restored previously expanded germlines and activated na\u00efve B cells to broaden the antibody repertoire of antibodies to control future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.03.03.583187","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.01.582992","pub_date":"2024-3-04","title":"Time-dependent enhancement of mRNA vaccines by 4-1BB costimulation","abstract":"mRNA vaccines have demonstrated efficacy against COVID-19. However, concerns regarding waning immunity and breakthrough infections have motivated the development of next-generation vaccines with enhanced efficacy. In this study, we investigated the impact of 4-1BB costimulation on immune responses elicited by mRNA vaccines in mice. We first vaccinated mice with an mRNA vaccine encoding the SARS-CoV-2 spike antigen like the Moderna and Pfizer-BioNTech vaccines, followed by administration of 4-1BB costimulatory antibodies at various times post-vaccination. Administering 4-1BB costimulatory antibodies during the priming phase did not enhance immune responses. However, administering 4-1BB costimulatory antibodies after 96 hours elicited a significant improvement in CD8 T cell responses, leading to enhanced protection against breakthrough infections. A similar improvement in immune responses was observed with multiple mRNA vaccines, including vaccines against common cold coronavirus, human immunodeficiency virus (HIV), and arenavirus. These findings demonstrate a time-dependent effect by 4-1BB costimulation and provide insights for developing improved mRNA vaccines.","version":"1.1","doi":"10.1101/2024.03.01.582992","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.02.583082","pub_date":"2024-3-04","title":"Heterogeneous hybrid immunity against Omicron variant JN.1 at 11 months following breakthrough infection","abstract":"A highly transmissible SARS-CoV-2 variant JN.1 is rapidly spreading throughout the nation, becoming the predominant strain in China and worldwide. However, the current immunity against the circulating JN.1 at population level has yet to be fully evaluated. We recruited representative cohorts with stratified age groups and diverse combinations of vaccination and/or infection in recent months, and promptly assessed humoral immunity for these subjects predominantly exhibiting hybrid immunity. We report that at 11 months following BA.5-wave breakthrough infection (BTI), these vaccinated individuals generally showed above-the-threshold yet low level of neutralizing activity against JN.1, with slightly greater potency observed in children and adolescents compared to adults and seniors. Meanwhile, XBB/EG.5-wave reinfection post-BTI significantly boosted the neutralizing antibodies against Omicron variants, including JN.1 in both adults (13.4-fold increase) and seniors (24.9-fold increase). To better understand respiratory mucosal protection against JN.1 over an extended period of months post-BTI, we profiled the humoral immunity in bronchoalveolar lavage samples obtained from vaccinated subjects with or without BTI, and revealed increased potency of neutralizing activity against the BA.5 and JN.1 variants in the respiratory mucosa through natural infection. Notably, at 11 months post-BTI, memory B cell responses against prototype and JN.1 were detectable in both blood and respiratory mucosa, displaying distinct memory features in the circulation and airway compartments. XBB/EG.5-wave reinfection drove the expansion of JN.1-specific B cells, along with the back-boosting of B cells responding to the ancestral viral strain, suggesting the involvement of immune imprinting. Together, this study indicates heterogeneous hybrid immunity over 11 months post-BTI, and underscores the vulnerability of individuals, particularly high-risk seniors, to JN.1 breakthrough infection. An additional booster with XBB-containing vaccine may greatly alleviate the onward transmission of immune-evasive SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.03.02.583082","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.09.527693","pub_date":"2024-3-03","title":"Genotypic-phenotypic landscape computation based on first principle and deep learning","abstract":"The relationship between genotype and fitness is fundamental to evolution, but quantitatively mapping genotypes to fitness has remained challenging. We propose the Phenotypic-Embedding theorem (P-E theorem) that bridges genotype-phenotype through an encoder-decoder deep learning framework. Inspired by this, we proposed a more general first principle for correlating genotype-phenotype, and the Phenotypic-Embedding theorem provides a computable basis for the application of first principle. As an application example of the P-E theorem, we developed the Co-attention based Transformer model to bridge Genotype and Fitness (CoT2G-F) model, a Transformer-based pre-train foundation model with downstream supervised fine-tuning (SFT) that can accurately simulate the neutral evolution of viruses and predict immune escape mutations. Accordingly, following the calculation path of the P-E theorem, we accurately obtained the basic reproduction number (R0) of SARS-CoV-2 from first principles, quantitatively linked immune escape to viral fitness, and plotted the genotype-fitness landscape. The theoretical system we established provides a general and interpretable method to construct genotype-phenotype landscapes, providing a new paradigm for studying theoretical and computational biology.","version":"1.3","doi":"10.1101/2023.02.09.527693","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.29.24303568","pub_date":"2024-03-02","title":"Sex differences in symptomatology and immune profiles of Long COVID","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>\n                  Strong sex differences in the frequencies and manifestations of Long COVID (LC) have been reported with females significantly more likely than males to present with LC after acute SARS-CoV-2 infection\n                  <jats:sup>1\u20137</jats:sup>\n                  . However, whether immunological traits underlying LC differ between sexes, and whether such differences explain the differential manifestations of LC symptomology is currently unknown. Here, we performed sex-based multi-dimensional immune-endocrine profiling of 165 individuals\n                  <jats:sup>8</jats:sup>\n                  with and without LC in an exploratory, cross-sectional study to identify key immunological traits underlying biological sex differences in LC. We found that female and male participants with LC experienced different sets of symptoms, and distinct patterns of organ system involvement, with female participants suffering from a higher symptom burden. Machine learning approaches identified differential sets of immune features that characterized LC in females and males. Males with LC had decreased frequencies of monocyte and DC populations, elevated NK cells, and plasma cytokines including IL-8 and TGF-\u03b2-family members. Females with LC had increased frequencies of exhausted T cells, cytokine-secreting T cells, higher antibody reactivity to latent herpes viruses including EBV, HSV-2, and CMV, and lower testosterone levels than their control female counterparts. Testosterone levels were significantly associated with lower symptom burden in LC participants over sex designation. These findings suggest distinct immunological processes of LC in females and males and illuminate the crucial role of immune-endocrine dysregulation in sex-specific pathology.\n                </jats:p>","version":null,"doi":"10.1101/2024.02.29.24303568","journal":"medRxiv","score":null},{"id":"10.1101/2024.02.28.582613","pub_date":"2024-3-01","title":"Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination","abstract":"Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective.","version":"1.2","doi":"10.1101/2024.02.28.582613","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.01.582951","pub_date":"2024-3-01","title":"Compartment-Specific Antibody Correlates of Protection to SARS-CoV-2 Omicron in Macaques","abstract":"Antibodies represent a primary mediator of protection against respiratory viruses such as SARS-CoV-2. Serum neutralizing antibodies (NAbs) are often considered a primary correlate of protection. However, detailed antibody profiles including characterization of antibody functions in different anatomic compartments are not well understood. Here we show that antibody correlates of protection against SARS-CoV-2 challenge are different in systemic versus mucosal compartments in rhesus macaques. In serum, neutralizing antibodies were the strongest correlate of protection and were linked to Spike-specific binding antibodies and other extra-neutralizing antibody functions that create a larger protective network. In contrast, in bronchiolar lavage (BAL), antibody-dependent cellular phagocytosis (ADCP) proved the strongest correlate of protection rather than NAbs. Within BAL, ADCP was linked to mucosal Spike-specific IgG, IgA/secretory IgA, and Fc\u03b3-receptor binding antibodies. Our results support a model in which antibodies with different functions mediate protection at different anatomic sites. The correlation of ADCP and other Fc functional antibody responses with protection in BAL suggests that these antibody responses may be critical for protection against SARS-CoV-2 Omicron challenge in mucosa.","version":"1.1","doi":"10.1101/2024.03.01.582951","journal":"bioRxiv","score":null},{"id":"10.1101/2024.03.01.582915","pub_date":"2024-3-01","title":"T7 RNA polymerase-independent expression of reporter genes from a T7 promoter-driven SARS-CoV-2 replicon-encoding DNA in human cells","abstract":"Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Surprisingly, however, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.","version":"1.1","doi":"10.1101/2024.03.01.582915","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.29.582263","pub_date":"2024-3-01","title":"Profiling of linear B-cell epitopes against human coronaviruses in pooled sera sampled early in the COVID-19 pandemic","abstract":"Antibodies play a key role in the immune defence against infectious pathogens. Understanding the underlying process of B cell recognition is not only of fundamental interest; it supports important applications within diagnostics and therapeutics. Whereas the nature of conformational B cell epitope recognition is inherently complicated, linear B cell epitopes offer a straightforward approach that potentially can be reduced to one of peptide recognition. Using an overlapping peptide approach representing the entire proteomes of the seven main coronaviruses known to infect humans, we analysed sera pooled from eight PCR-confirmed COVID-19 convalescents and eight pre-pandemic controls. Using a high-density peptide microarray platform, 13-mer peptides overlapping by 11 amino acids were in situ synthesised and incubated with the pooled primary serum samples, followed by development with secondary fluorochrome-labelled anti-IgG and -IgA antibodies. Interactions were detected by fluorescence detection. Strong Ig interactions encompassing consecutive peptides were considered to represent \u201chigh-fidelity regions\u201d (HFRs). These were mapped to the coronavirus proteomes using a 60% homology threshold for clustering. We identified 333 human coronavirus derived HFRs. Among these, 98 (29%) mapped to SARS-CoV-2, 144 (44%) mapped to one or more of the four circulating common cold coronaviruses (CCC), and 54 (16%) cross-mapped to both SARS-CoV-2 and CCCs. The remaining 37 (11%) mapped to either SARS-CoV or MERS-CoV. Notably, the COVID-19 serum was skewed towards recognising SARS-CoV-2-mapped HFRs, whereas the pre-pandemic was skewed towards recognising CCC-mapped HFRs. In terms of absolute numbers of linear B cell epitopes, the primary targets are the ORF1ab protein (60%), the spike protein (21%), and the nucleoprotein (15%) in that order; however, in terms of epitope density the order would be reversed. We identified linear B cell epitopes across coronaviruses, highlighting pan-, alpha-, beta-, or SARS-CoV-2-corona-specific B cell recognition patterns. These findings could be pivotal in deciphering past and current exposures to epidemic and endemic coronavirus. Moreover, our results suggest that pre-pandemic anti-CCC antibodies may cross-react against SARS-CoV-2, which could explain the highly variable outcome of COVID-19. Finally, the methodology used here offers a rapid and comprehensive approach to high-resolution linear B-cell epitope mapping, which could be vital for future studies of emerging infectious diseases.","version":"1.1","doi":"10.1101/2024.02.29.582263","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.29.582705","pub_date":"2024-3-01","title":"Sputum Production and Salivary Microbiome in COVID-19 Patients Reveals Oral-Lung Axis","abstract":"SARS-CoV-2 is a severe respiratory disease that primarily targets the lungs and was the leading cause of death worldwide during the pandemic. Investigating the intricate interplay between the oral microbiome and inflammatory cytokines during the acute phase of infection is crucial for understanding host immune responses. This study aimed to explore the relationship between the oral microbiome and cytokines in COVID-19 patients, specifically examining those with and without sputum production. Saliva and blood samples from 50 COVID-19 patients were subjected to 16S ribosomal RNA gene sequencing to analyze the oral microbiome. Additionally, 65 saliva and serum cytokines were assessed using Luminex multiplex analysis. The Mann-Whitney test compared cytokine levels between individuals with and without sputum production. Our study revealed significant differences in the membership (Jaccard dissimilarity: p=0.016) and abundance (PhILR dissimilarity: p=0.048; metagenomeSeq) of salivary microbial communities between COVID-19 patients with and without sputum production. Seven bacterial genera, including Prevotella, Streptococcus, Actinomyces, Atopobium, Filifactor, Leptotrichia, and Selenomonas, were present in statistically higher proportions of patients with sputum production (p<0.05, Fisher\u2019s exact test). Eight bacterial genera, including Prevotella, Megasphaera, Stomatobaculum, Leptotrichia, Veillonella, Actinomyces, Atopobium, and Corynebacteria were significantly more abundant in the sputum-producing group, while Lachnoacaerobaculum was notably more prevalent in the non-sputum-producing group (p<0.05, ANCOM-BC).We observed a significant positive correlation between salivary IFN-gamma (Interferon-gamma) and Eotaxin2/CCL24 (chemokine ligand 24) with sputum production. Conversely, negative correlations were noted in serum MCP3/CCL7 (monocyte-chemotactic protein 3/Chemokine ligand 7), MIG/CXCL9 (Monokine induced by gamma/Chemokine ligand 9), IL1 beta (interleukin 1 beta), and SCF (stem cell factor) with sputum production (p<0.05, Mann-Whitney test). Substantial distinctions in salivary microbial communities were evident between COVID-19 patients with and without sputum production, emphasizing the notable impact of sputum production on the oral microbiome and cytokine levels during the acute phase of infection.","version":"1.1","doi":"10.1101/2024.02.29.582705","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.28.582480","pub_date":"2024-2-29","title":"Discovery of anti-SARS-CoV-2 S2 protein antibody CV804 with broad-spectrum reactivity with various beta coronaviruses and analysis of its pharmacological properties in vitro and in vivo","abstract":"SARS-CoV-2 pandemic alerts us that spillovers of various animal coronaviruses to human in the future may bring us enormous damages. Thus, there is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses.CV804 against the S2 domain of the spike protein, which is less prone to mutations. CV804 shows not only broad cross-reactivities with representative 20 animal-origin coronaviruses but also with diseases-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and mutant strains of SARS-CoV-2. Other than that, the main characteristics of CV804 are that it has strong antibody-dependent cellular cytotoxicity (ADCC) activity to SARS-CoV2 spike protein-expressed cells in vitro and completely lacks virus-neutralization activity. Comprehensively in animal models, CV804 suppressed disease progression by SARS-CoV-2 infection. Structural studies using HDX-MS and point mutations of recombinant spike proteins revealed that CV804 binds to a unique epitope within the highly conserved S2 domain of the spike proteins of various coronaviruses. Based on the overall data, we suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein expressed on the surface of the infected cells and weakens the viral virulence by supporting host immune cells\u2019 attack through ADCC activity in vivo. CV804 epitope identified in this study is not only useful for the design of pan-corona antibody therapeutics but also to design next-generation coronavirus vaccines and antiviral drugs.","version":"1.1","doi":"10.1101/2024.02.28.582480","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.560070","pub_date":"2024-2-29","title":"Curcumin and turmeric extract inhibit SARS-CoV-2 pseudovirus cell entry and spike-mediated cell fusion","abstract":"Turmeric extract (TE) with curcumin as its main active ingredient has been studied as a potential COVID-19 therapeutic. Curcumin has been studied in silico and in vitro against a naive SARS-CoV-2 virus, yet little is known about TE\u2019s impact on SARS-CoV-2 infection. Moreover, no study reveals the potential of both curcumin and TE on the inhibition of SARS-CoV-2 cell-to-cell transmission. Here, we investigated the effects of both curcumin and TE on inhibiting SARS-CoV-2 entry and cell-to-cell transmission using pseudovirus (PSV) and syncytia models. We performed a PSV entry assay in 293T or 293 cells expressing hACE2. The cells were pretreated with curcumin or TE and then treated with PSV with or without the test samples. Next, we carried out syncytia assay by co-transfecting 293T cells with plasmids encoding spike, hACE2, and TMPRSS2 to be treated with the test samples. The results showed that in PSV entry assay on 293T/hACE/TMPRSS2 cells, both curcumin and TE inhibited PSV entry at concentrations of 1 \u00b5M and 10 \u00b5M for curcumin and 1 \u00b5g/ml and 10 \u00b5g/ml for TE. Moreover, both curcumin and TE reduced syncytia formation compared to control cells. Our study shows that TE and curcumin are potential inhibitors of SARS-CoV-2 infection at entry points, either by direct or indirect infection models.","version":"1.2","doi":"10.1101/2023.09.28.560070","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.28.582665","pub_date":"2024-2-29","title":"Peptide Mold: A Novel Strategy for Mapping Potential Binding Sites in Protein Targets","abstract":"A novel concept titled \u2018Peptide Mold\u2019 for mapping potential binding sites in protein targets is presented. A large multiconformer tetrapeptide library comprising of 32 million conformations of all possible combinations of naturally-occurring amino acids was constructed and used for molecular docking analysis in the substrate-binding site of SARS-CoV-2 PLpro enzyme. The top-ranking, structurally-diverse tetrapeptide docked conformations (symbolizing peptide mold, analogous to a clay mold) were used then for elucidating a five-point pharmacophore. Ligand-based virtual screening of a large, multiconformer library of phytoconstituents using the derived five-point pharmacophore led to identification of potential binders for SARS-CoV-2 PLpro at its substrate-binding site. The approach is based on generating the imprint of a macromolecular binding site (cavity) using tetrapeptides (clay), thereby generating a reverse mold (with definitive shape and size), which can further be used for identifying small-molecule ligands matching the captured features of the target binding site. The approach is based on the fact that the individual amino acids in the tetrapeptide represent all possible drug-receptor interaction features (electrostatic, H-bonding, van der Waals, dispersion and hydrophobic among others). The \u2018peptide mold\u2019 approach can be extended to any protein target for mapping the binding site(s), and further use of the generated pharmacophore model for virtual screening of potential binders. The peptide mold approach is a robust, hybrid computational screening strategy, overcoming the present limitations of structure-based methods, e.g., molecular docking and the ligand-based methods such as pharmacophore search. Exploration of the peptide mold strategy is expected to yield high-quality, reliable and interesting virtual hits in the computational screening campaigns during the hit and lead identification stages.","version":"1.1","doi":"10.1101/2024.02.28.582665","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.27.582258","pub_date":"2024-2-28","title":"Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome","abstract":"The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.","version":"1.1","doi":"10.1101/2024.02.27.582258","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.26.582219","pub_date":"2024-2-28","title":"The S2 subunit of spike encodes diverse targets for functional antibody responses to SARS-CoV-2","abstract":"The SARS-CoV-2 virus responsible for the COVID-19 global pandemic has exhibited a striking capacity for viral evolution that drives continued evasion from vaccine and infection-induced immune responses. Mutations in the receptor binding domain of the S1 subunit of the spike glycoprotein have led to considerable escape from antibody responses, reducing the efficacy of vaccines and monoclonal antibody (mAb) therapies. Therefore, there is a need to interrogate more constrained regions of Spike, such as the S2 subdomain. Here, we describe a collection of S2 mAbs from two SARS-CoV-2 convalescent individuals that target multiple regions in the S2 subdomain and can be grouped into at least five epitope classes. Most did not neutralize SARS-CoV-2 with the exception of C20.119, which bound to a highly conserved epitope in the fusion peptide and showed broad binding and neutralization activity across SARS-CoV-2, SARS-CoV-1, and closely related zoonotic sarbecoviruses. Several of the S2 mAbs tested mediated antibody-dependent cellular cytotoxicity (ADCC) at levels similar to the S1 mAb S309 that was previously authorized for treatment of SARS-CoV-2 infections. Three of the mAbs with ADCC function also bound to spike trimers from HCoVs, such as MERS-CoV and HCoV-HKU1. Our findings suggest there are diverse epitopes in S2, including functional S2 mAbs with HCoV and sarbecovirus breadth that likely target functionally constrained regions of spike. These mAbs could be developed for potential future pandemics, while also providing insight into ideal epitopes for eliciting a broad HCoV response. The early successes of vaccines and antibody therapies against SARS-CoV-2, the virus responsible for the COVID-19 global pandemic, leveraged the considerable antibody response to the viral entry protein, spike, after vaccination or infection. These initial interventions were highly effective at protecting from infection and reducing severe disease or death. However, SARS-CoV-2 has shown no sign of abating, with the continued rise of new variants that have escaped some of the antibody defense due to distinct alterations most significantly in regions of the spike protein that elicit most of the anti-viral, functional antibody response. These findings suggest a critical need to identify vaccine approaches and therapies that provide the broadest possible antibody responses, focused on regions of spike critical for SARS-CoV-2 infection and, therefore, do not undergo changes that could lead to immune evasion. Our study describes a panel of functional antibodies, from individuals after SARS-CoV-2 infection, that recognize the S2 spike subdomain that is responsible for carrying out viral fusion with host cells. These regions in S2 are generally well conserved across SARS-CoV-2 variants and other closely related viruses and thus, could guide more effective vaccine design in the face of continued viral evolution.","version":"1.1","doi":"10.1101/2024.02.26.582219","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.28.582510","pub_date":"2024-2-28","title":"Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model","abstract":"Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function may play a key role in Long COVID. Despite its importance, the impact of SARS-CoV-2 on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and human Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir and the TMPRSS2 inhibitor Camostat prevented SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.","version":"1.1","doi":"10.1101/2024.02.28.582510","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.27.582254","pub_date":"2024-2-28","title":"Virological traits of the SARS-CoV-2 BA.2.87.1 lineage","abstract":"The highly mutated SARS-CoV-2 BA.2.87.1 lineage was recently detected in South Africa, but its transmissibility is unknown. Here, we report that BA.2.87.1 efficiently enters human cells but is more sensitive to antibody-mediated neutralization than the currently dominating JN.1 variant. Acquisition of adaptive mutations might thus be needed for high transmissibility.","version":"1.1","doi":"10.1101/2024.02.27.582254","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.27.582131","pub_date":"2024-2-28","title":"Carboxylated graphene: A novel approach for enhanced IgA-SARS-CoV-2 electrochemical biosensing","abstract":"Biosensors comprise devices that use a material of biological nature as receptors connected to transducers, these devices are capable of capturing biorecognition signals, called a primary signal, and converting it to a measurable signal. In this study, we report the synthesis of carboxylated graphene (CG) through a carboxylation method in acid medium and further characterization of the materials by different techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Raman spectroscopy, thermal gravimetric analysis (TGA), and X-ray diffraction (DRX). Also, the surface of the screen-printed carbon electrodes (SPCEs) was modified with CG for subsequent immobilization of N-protein of SARS-CoV-2, which allowed the detection of antibodies (IgA-SARS-CoV-2). The electrical properties and response of the biosensor were investigated using electrochemical techniques (cyclic voltammetry and electrochemical impedance spectroscopy). Through the chemical characterization techniques, it was possible to confirm the success of the CG synthesis process. The biosensor fabricated shown to be able to detect IgA-SARS-CoV-2 in the range of 1:1000 to1:200 v/v in phosphate buffer solution (PBS) and the limit of detection calculated was 1:1601 v/v. this perspective they comprise a wide range of applications due to its advantages, such as the possibility of a shorter response time, reproducibility, the miniaturization of detection devices such as the use of screen-printed electrodes, the use of small amounts of sample, the high sensitivity and specificity, low limits of detection and the integration of nano materials that make it possible to improve the detected signal.","version":"1.1","doi":"10.1101/2024.02.27.582131","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.28.582544","pub_date":"2024-2-28","title":"Designed mosaic nanoparticles enhance cross-reactive immune responses in mice","abstract":"Using computational methods, we designed 60-mer nanoparticles displaying SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) by (i) creating RBD sequences with 6 mutations in the SARS-COV-2 WA1 RBD that were predicted to retain proper folding and abrogate antibody responses to variable epitopes (mosaic-2COMs; mosaic-5COM), and (ii) selecting 7 natural sarbecovirus RBDs (mosaic-7COM). These antigens were compared with mosaic-8b, which elicits cross-reactive antibodies and protects from sarbecovirus challenges in animals. Immunizations in na\u00efve and COVID-19 pre-vaccinated mice revealed that mosaic-7COM elicited higher binding and neutralization titers than mosaic-8b and related antigens. Deep mutational scanning showed that mosaic-7COM targeted conserved RBD epitopes. Mosaic-2COMs and mosaic-5COM elicited higher titers than homotypic SARS-CoV-2 Beta RBD-nanoparticles and increased potencies against some SARS-CoV-2 variants than mosaic-7COM. However, mosaic-7COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons. These results support using mosaic-7COM to protect against highly mutated SARS-CoV-2 variants and zoonotic sarbecoviruses with spillover potential.","version":"1.1","doi":"10.1101/2024.02.28.582544","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.25.581989","pub_date":"2024-2-27","title":"Tissue-Specific Metabolomic Reprogramming Determines the Disease Pathophysiology of Sars-Cov-2 Variants in Hamster Model","abstract":"Despite significant effort, a clear understanding of host tissue-specific responses and their implications for immunopathogenicity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant infection has remained poorly defined. To shed light on the interaction between organs and specific SARS-CoV-2 variants, we sought to characterize the complex relationship among acute multisystem manifestations, dysbiosis of the gut microbiota, and the resulting implications for SARS-CoV-2 variant-specific immunopathogenesis in the Golden Syrian Hamster (GSH) model using multi-omics approaches. Our investigation revealed increased viremia in diverse tissues of delta-infected GSH compared to the omicron variant. Multi-omics analyses uncovered distinctive metabolic responses between the delta and omicron variants, with the former demonstrating dysregulation in synaptic transmission proteins associated with neurocognitive disorders. Additionally, delta-infected GSH exhibited an altered fecal microbiota composition, marked by increased inflammation-associated taxa and reduced commensal bacteria compared to the omicron variant. These findings underscore the SARS-CoV-2-mediated tissue insult, characterized by modified host metabolites, neurological protein dysregulation, and gut dysbiosis, highlighting the compromised gut-lung-brain axis during acute infection. In hamsters at acute infection, SARS-CoV-2 variant-specific metabolic responses and gut dysbiosis dysregulate synaptic transmission proteins.","version":"1.1","doi":"10.1101/2024.02.25.581989","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.13.580114","pub_date":"2024-2-27","title":"Cytometry Masked Autoencoder: An Accurate and Interpretable Automated Immunophenotyper","abstract":"High-throughput single-cell cytometry data are crucial for understanding involvement of immune system in diseases and responses to treatment. Traditional methods for annotating cytometry data, specifically manual gating and clustering, face challenges in scalability, robustness, and accuracy. In this study, we propose a cytometry masked autoencoder (cyMAE), which offers an automated solution for immunophenotyping tasks including cell type annotation. The cyMAE model is designed to uphold user-defined cell type definitions, thereby facilitating easier interpretation and cross-study comparisons. The cyMAE model operates on a pre-train and fine-tune approach. In the pre-training phase, cyMAE employs Masked Cytometry Modelling (MCM) to learn relationships between protein markers in immune cells solely based on protein expression, without relying on prior information such as cell identity and cell type-specific marker proteins. Subsequently, the pre-trained cyMAE is fine-tuned on multiple specialized tasks via task-specific supervised learning. The pre-trained cyMAE addresses the shortcomings of manual gating and clustering methods by providing accurate and interpretable predictions. Through validation across multiple cohorts, we demonstrate that cyMAE effectively identifies co-occurrence patterns of bound labeled antibodies, delivers accurate and interpretable cellular immunophenotyping, and improves the prediction of subject metadata status. Specifically, we evaluated cyMAE for cell type annotation and imputation at the cellular-level and SARS-CoV-2 infection prediction, secondary immune response prediction against COVID-19, and prediction of the infection stage in COVID-19 progression at the subject-level. The introduction of cyMAE marks a significant step forward in immunology research, particularly in large-scale and high-throughput human immune profiling. This approach offers new possibilities for predicting and interpreting cellular-level and subject-level phenotypes in both health and disease.","version":"1.2","doi":"10.1101/2024.02.13.580114","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.24.581855","pub_date":"2024-2-26","title":"Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase","abstract":"Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13 \u00b0C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. Additionally, some of the examined substitutions in the RdRp subunits notably influenced the sensitivity of RdRp to Remdesivir\u00ae, highlighting their potential implications for therapeutic strategies. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants. While the impact of SARS-CoV-2 spike protein mutations has been extensively explored, our understanding of mutations within the RNA-dependent RNA polymerase (RdRp), crucial for viral replication and a key target for antivirals like Remdesivir, remains limited with studies conducted solely in silico. We focused on selected RdRp mutations identified from December 2019 to June 2022, assessing their effects on enzyme stability, complex assembly, and activity. Advanced biochemical analyses reveal how these mutations can alter RdRp functionality, providing insights into viral evolution and resistance mechanisms. This study, pioneering in assessing the biochemical implications of RdRp mutations, provides invaluable insights into their roles in viral replication and antiviral resistance, hereby opening new pathways for developing therapies against the continuously evolving SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.02.24.581855","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.24.581861","pub_date":"2024-2-26","title":"Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing","abstract":"Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Mxyococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle. This immunogen elicited conformationally-relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.","version":"1.1","doi":"10.1101/2024.02.24.581861","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.23.581661","pub_date":"2024-2-23","title":"Thymidine Phosphorylase Mediates SARS-CoV-2 Spike Protein Enhanced Thrombosis in K18-hACE2TG Mice","abstract":"COVID-19, caused by SARS-CoV-2, is associated with arterial and venous thrombosis, thereby increasing mortality. SARS-CoV-2 spike protein (SP), a viral envelope structural protein, is implicated in COVID-19-associated thrombosis. However, the underlying mechanisms remain unknown. Thymidine phosphorylase (TYMP), a newly identified prothrombotic protein, is upregulated in the plasma, platelets, and lungs of patients with COVID-19 but its role in COVID-19-associated thrombosis is not defined. In this study, we found that wild-type SARS-CoV-2 SP significantly promoted arterial thrombosis in K18-hACE2TG mice. SP-accelerated thrombosis was attenuated by inhibition or genetic ablation of TYMP. SP increased the expression of TYMP, resulting in the activation of signal transducer and activator of transcription 3 (STAT3) in BEAS-2B cells, a human bronchial epithelial cell line. A siRNA-mediated knockdown of TYMP inhibited SP-enhanced activation of STAT3. Platelets derived from SP-treated K18-hACE2TG mice also showed increased STAT3 activation, which was reduced by TYMP deficiency. Activated STAT3 is known to potentiate glycoprotein VI signaling in platelets. While SP did not influence ADP- or collagen-induced platelet aggregation, it significantly shortened activated partial thromboplastin time and this change was reversed by TYMP knockout. Additionally, platelet factor 4 (PF4) interacts with SP, which also complexes with TYMP. TYMP enhanced the formation of the SP/PF4 complex, which may potentially augment the prothrombotic and procoagulant effects of PF4. We conclude that SP upregulates TYMP expression, and TYMP inhibition or knockout mitigates SP-enhanced thrombosis. These findings indicate that inhibition of TYMP may be a novel therapeutic strategy for COVID-19-associated thrombosis. SARS-CoV-2 spike protein, thymidine phosphorylase, and platelet factor 4 form a complex that may promote clot formation. Inhibiting thymidine phosphorylase attenuates SARS-CoV-2 spike protein-enhanced thrombosis, platelet activation, and coagulation.","version":"1.1","doi":"10.1101/2024.02.23.581661","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.22.579423","pub_date":"2024-2-23","title":"A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding","abstract":"The nucleoprotein (N) of SARS-CoV-2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich domain (SR) that is hyperphosphorylated in infected cells. This modification is thought to regulate function of N, although mechanistic details remain unknown. We use time-resolved NMR to follow local and long-range structural changes occurring during hyperphosphorylation by the kinases SRPK1/GSK-3/CK1, thereby identifying a conformational switch that abolishes interaction with RNA. When 8 approximately uniformly-distributed sites are phosphorylated, the SR domain competitively binds the same interface as single-stranded RNA, resulting in RNA binding inhibition. Phosphorylation by PKA does not prevent RNA binding, indicating that the pattern resulting from the physiologically-relevant kinases is specific for inhibition. Long-range contacts between the RNA-binding, linker and dimerization domains are also abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into recruitment of specific host kinases to regulate viral function.","version":"1.1","doi":"10.1101/2024.02.22.579423","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.21.24303159","pub_date":"2024-02-23","title":"Prospective study of machine learning for identification of high-risk COVID-19 patients","abstract":"<jats:p>The Coronavirus Disease 2019 (COVID-19) pandemic constituted a public health crisis with a devastating effect in terms of its death toll and effects on the world economy. Notably, machine learning methods have played a pivotal role in devising novel technological solutions designed to tackle challenges brought forth by this pandemic. In particular, tools for the rapid identification of high-risk COVID-19 patients have been developed to aid in the effective allocation of hospital resources and for containing the spread of the virus. A comprehensive validation of such intelligent technological approaches is needed to ascertain their clinical utility; importantly, it may help develop future strategies for efficient patient classification to be used in future viral outbreaks. Here we present a prospective study to evaluate the performance of state-of-the-art machine-learning models proposed in PloS one 16, e0257234 (2021), which we developed for the identification of high-risk COVID-19 patients across four identified clinical stages. The model relies on artificial neural networks trained with historical patient data from Mexico. To assess their predictive capabilities across the six, registered, epidemiological waves of COVID-19 infection in Mexico, we measure the accuracy within each wave without retraining the neural networks. We then compare their performance against neural networks trained with cumulative historical data up to the end of each wave. Our findings indicate that models trained using early historical data exhibit strong predictive capabilities, which allows us to accurately identify high-risk patients in subsequent epidemiological waves\u2014under clearly varying vaccination, prevalent viral strain, and medical treatment conditions. These results show that artificial intelligence-based methods for patient classification can be robust throughout an extended period characterized by constantly evolving conditions, and represent a potentially powerful tool for tackling future pandemic events, particularly for clinical outcome prediction of individual patients.</jats:p>","version":null,"doi":"10.1101/2024.02.21.24303159","journal":"medRxiv","score":null},{"id":"10.1101/2023.06.06.543529","pub_date":"2024-2-22","title":"Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies","abstract":"SARS-CoV-2 vaccines have been used worldwide to combat COVID-19 pandemic. To elucidate the factors that determine the longevity of spike (S)-specific antibodies, we traced the characteristics of S-specific T cell clonotypes together with their epitopes and anti-S antibody titers before and after BNT162b2 vaccination over time. T cell receptor (TCR) \u03b1\u03b2 sequences and mRNA expression of the S-responded T cells were investigated using single-cell TCR- and RNA-sequencing. Highly expanded 199 TCR clonotypes upon stimulation with S peptide pools were reconstituted into a reporter T cell line for the determination of epitopes and restricting HLAs. Among them, we could determine 78 S epitopes, most of which were conserved in variants of concern (VOCs). After the 2nd vaccination, T cell clonotypes highly responsive to recall S stimulation were polarized to follicular helper T (Tfh)-like cells in donors exhibiting sustained anti-S antibody titers (designated as \u201csustainers\u201d), but not in \u201cdecliners\u201d. Even before vaccination, S-reactive CD4+ T cell clonotypes did exist, most of which cross-reacted with environmental or symbiotic bacteria. However, these clonotypes contracted after vaccination. Conversely, S-reactive clonotypes dominated after vaccination were undetectable in pre-vaccinated T cell pool, suggesting that highly-responding S-reactive T cells were established by vaccination from rare clonotypes. These results suggest that de novo acquisition of memory Tfh-like cells upon vaccination may contribute to the longevity of anti-S antibody titers.","version":"1.3","doi":"10.1101/2023.06.06.543529","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.19.24303026","pub_date":"2024-02-21","title":"Clot Twist \u2013 D-dimer analysis of healthy adults receiving heterologous or homologous booster COVID-19 vaccine after a single prime dose of Ad26.COV2.S in a phase II randomised open-label trial, BaSiS","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  BaSiS (\n                  <jats:bold>B</jats:bold>\n                  ooster\n                  <jats:bold>A</jats:bold>\n                  fter\n                  <jats:bold>Si</jats:bold>\n                  sonke\n                  <jats:bold>S</jats:bold>\n                  tudy) is a prospectively enrolled open-label trial in which healthy adults, with controlled co-morbidities and no prior thrombosis, who received a single Ad26.COV2.S prime vaccination primarily through the Sisonke phase IIIB open label implementation study in South Africa. An exploratory objective evaluated the clotting profiles of participants who were enrolled across 4 sites in South Africa and randomised 1:1:1:1 to receive one of full-dose Ad26.COV2.S, half-dose Ad26.COV2.S, full-dose Comirnaty or half-dose Comirnaty booster. D-dimer testing (INNOVANCE\u00aeD-Dimer Assay), as a coagulopathy marker, was conducted pre-booster (baseline) and 2 weeks post-booster. The median age among 285 participants was 42.2 years (IQR:35.5-48.7), 235/285 (82.5%) were female, 269/285 (94.4%) were Black African. Of the 40.4% (115/285) people living with HIV (PLHIV), 79.1% (91/115) were well-controlled on antiretroviral therapy. At baseline, 39.3% (112/285) had elevated d-dimers; all asymptomatic. Females and obese participants were significantly more likely to have elevated baseline d-dimers (OR=4.17; 95% CI:1.88 to 9.26 and OR=2.64; 95% CI:1.57 to 4.43, respectively). Of 169 with normal baseline d-dimers, 29 (17.2%) became elevated 2 weeks post-booster: median increase 0.23\u00b5g/ml (IQR:0.15-0.42); those receiving full-dose Comirnaty exhibited lower risk of d-dimer elevation post vaccination, compared to other booster vaccination arms (OR:0.26; 95% CI:0.07 to 0.98). PLHIV experienced significantly higher median increases compared to HIV uninfected participants (0.43 vs 0.17, p=0.004). Elevated d-dimers in asymptomatic, low-risk adults were unexpectedly common but were not associated with thromboembolism, supporting the rationale of using d-dimers only if clinically indicated. Trial Registration: South African Clinical Trails Register number DOH-27-012022-7841.\n                </jats:p>","version":null,"doi":"10.1101/2024.02.19.24303026","journal":"medRxiv","score":null},{"id":"10.1101/2024.02.19.581112","pub_date":"2024-2-20","title":"Characterization of the SARS-CoV-2 BA.5 Variants in H11-K18-hACE2 Hamsters","abstract":"This study aims to comprehensively characterize the SARS-CoV-2 BA.5 variants using K18 hACE2 transgenic mice and golden hamsters as model organisms. Previous research on SARS-CoV-2 has utilized both mouse and hamster models, leading to conflicting results concerning the virus\u2019s lethality. In our study, the finding suggests that H11-K18 hACE2 golden hamsters closely mimic the disease progression observed in human COVID-19 cases caused by BA.5 variants, demonstrating consistent severity and symptoms comparable to severe infections. Additionally, hamsters exhibit heightened respiratory viral replication, accurately reflecting the clinical viral kinetics observed in humans. The study emphasizes the critical importance of selecting an appropriate animal model for SARS-CoV-2 research, while also providing robust support for the hypothesis that BA.5 variants contribute to fatal outcomes in COVID-19 cases. These findings highlight the pivotal role of the golden hamster model in advancing our understanding of the pathogenic mechanisms underlying SARS-CoV-2 variants, as well as in the development of targeted therapeutic strategies. Our research work explores groundbreaking insights that could reshape our understanding of COVID-19 and pave the way for targeted therapies. We use golden hamster models to express the possibility of different animal models could contribute to human diseases. We hope this finding could clarify some conflicts existed, and help further development of medication for COVID.","version":"1.1","doi":"10.1101/2024.02.19.581112","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.19.579434","pub_date":"2024-2-20","title":"Prior exposure to malaria decreases SARS-CoV-2 mediated mortality in K18-hACE2 mice without influencing viral load in lungs","abstract":"Epidemiological evidence for decreased prevalence and/or mortality due to SARS-CoV-2 infections in countries endemic for malaria have been reported. However, such associational studies in human population are limited by known and several unknown confounding factors. The current study, the first of its kind, was designed to seek experimental evidence to test the hypothesis if prior exposure to Plasmodial infections cross-protect against SARS-CoV-2 challenge infection in a murine model, K-18 human ACE2 transgenic mice. Mice that had recovered from Plasmodium chabaudi infection 40 days earlier were challenged with a virulent strain of SARS-CoV-2 and viral load in lungs as well as mortality were scored and compared with K18 hACE2 mice that had not experienced prior malaria. The viral load in lungs 6 days post challenge were comparable in malaria recovered mice and controls suggesting no significant generation of anti-viral immunity. However, mice with prior malaria exposure were significantly protected against SARS-CoV-2 induced mortality. Significant differences were observed in several host immune responses between the two groups when cytokines, chemokines and transcription factors were quantified in lungs. The plasma levels of several cytokines and chemokines were also significantly different between the two groups. The results of the study suggest that prior exposure to malaria protects mice against viral induced mortality in K18 hACE2 transgenic mice challenged with a virulent isolate of SARS- CoV-2 in the absence of demonstrable host immunity inhibiting viral growth in lungs.","version":"1.1","doi":"10.1101/2024.02.19.579434","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.16.580687","pub_date":"2024-2-19","title":"Robust SARS-CoV-2 Neutralizing Antibodies Sustained through Three Months Post XBB.1.5 mRNA Vaccine Booster","abstract":"SARS-CoV-2-neutralizing antibodies were substantially expanded one month after a shot of XBB.1.5 monovalent mRNA vaccine (XBB.1.5 MV) booster, but the durability of this response remained unknown. Here, we addressed this question by performing neutralization assays on four viral variants (D614G, BA.5, XBB.1.5, and JN.1) using sera from 39 adult participants obtained at \u223c1 month and \u223c3 months post an XBB.1.5 MV booster. Our findings indicate that the resultant neutralizing antibody titers were robust and generally maintained at stable levels for the study period, similar to those following XBB infection. Importantly, this durability of neutralizing antibody titers contrasts with the decline observed after a booster of the original monovalent or BA.5 bivalent mRNA vaccine. Our results are in line with the recent national data from the Centers for Disease Control and Prevention, showing the efficacy against symptomatic SARS-CoV-2 infection is sustained for up to 4 months after an XBB.1.5 MV booster.","version":"1.1","doi":"10.1101/2024.02.16.580687","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.16.580615","pub_date":"2024-2-16","title":"Robust Neutralization of SARS-CoV-2 Variants Including JN.1 and BA.2.87.1 by Trivalent XBB Vaccine-Induced Antibodies","abstract":"Newly emerged SARS-CoV-2 variants like JN.1, and more recently, the hypermutated BA.2.87.1, have raised global concern. We recruited two groups of participants who had BA.5/BF.7 breakthrough infection post three doses of inactivated vaccines: one group experienced subsequent XBB reinfection, while the other received the XBB-containing trivalent WSK-V102C vaccine. Our comparative analysis of their serum neutralization activities revealed that the WSK-V102C vaccine induced stronger antibody responses against a wide range of variants, notably including JN.1 and the highly escaped BA.2.87.1. Furthermore, our investigation into specific mutations revealed that fragment deletions in NTD significantly contribute to the immune evasion of the BA.2.87.1 variant. Our findings emphasize the necessity for ongoing vaccine development and adaptation to address the dynamic nature of SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.02.16.580615","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.13.580127","pub_date":"2024-2-16","title":"MobileLAMP: A portable, low-cost, open-source device for isothermal nucleic acid amplification","abstract":"Isothermal amplification-based methods for pathogen DNA or RNA detection offer high sensitivity, rapid detection, and the potential for deployment in remote fields and home testing. Consequently, they are emerging as alternatives to PCR and saw a surge in research activity and deployment for the rapid detection of SARS-CoV-2 during the Covid-19 pandemic. The most common isothermal DNA detection methods rely on minimal reagents for DNA amplification and simple hardware that can maintain isothermal conditions and read-out a fluorescent or colorimetric signal. Many researchers globally are working on improving these components based on diverse end-user needs. In this work, we have recognized the need for an open-source hardware device for isothermal amplification, composed of off-the-shelf components that are easily accessible in any part of the world, is easily manufacturable in a distributed and scalable way using 3D printing, and that can be powered using a wide diversity of batteries and power sources. We demonstrate the easy assembly of our device design and demonstrate its efficacy using colorimetric LAMP for both RNA and DNA targets.","version":"1.1","doi":"10.1101/2024.02.13.580127","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.25.517953","pub_date":"2024-2-15","title":"A single-dose MCMV-based vaccine elicits long-lasting immune protection in mice against distinct SARS-CoV-2 variants","abstract":"Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have led to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMV) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant murine CMV (MCMV) vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, S-specific humoral and cellular immunity was not only maintained but even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two immune-evading variants of concern (VoCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19. While widespread vaccination has substantially reduced risks of severe COVID presentations and morbidity, immune waning and continuous immune escape of novel SARS-CoV-2 variants have resulted in a need for numerous vaccine boosters and a continuous adaptation of vaccines to new SARS-CoV-2 variants. We show in proof of principle experiments with a recombinant murine cytomegalovirus expressing the SARS-CoV-2 spike protein (MCMVS) that one immunization with a CMV vaccine vector drives enduring protection in both young and aged mice, with long-term maturation of immune responses that broaden the antiviral effects over time. Hence, this approach resolves issues of immune waning and mitigates the effects of COVID-19 evolution and immune escape, reducing the need for additional immunizations and potentially improving vaccine compliance.","version":"1.2","doi":"10.1101/2022.11.25.517953","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.14.580225","pub_date":"2024-2-15","title":"A Broad-Spectrum Multi-Antigen mRNA/LNP-Based Pan-Coronavirus Vaccine Induced Potent Cross-Protective Immunity Against Infection and Disease Caused by Highly Pathogenic and Heavily Spike-Mutated SARS-CoV-2 Variants of Concern in the Syrian Hamster Model","abstract":"The first-generation Spike-alone-based COVID-19 vaccines have successfully contributed to reducing the risk of hospitalization, serious illness, and death caused by SARS-CoV-2 infections. However, waning immunity induced by these vaccines failed to prevent immune escape by many variants of concern (VOCs) that emerged from 2020 to 2024, resulting in a prolonged COVID-19 pandemic. We hypothesize that a next-generation Coronavirus (CoV) vaccine incorporating highly conserved non-Spike SARS-CoV-2 antigens would confer stronger and broader cross-protective immunity against multiple VOCs. In the present study, we identified ten non-Spike antigens that are highly conserved in 8.7 million SARS-CoV-2 strains, twenty-one VOCs, SARS-CoV, MERS-CoV, Common Cold CoVs, and animal CoVs. Seven of the 10 antigens were preferentially recognized by CD8+ and CD4+ T-cells from unvaccinated asymptomatic COVID-19 patients, irrespective of VOC infection. Three out of the seven conserved non-Spike T cell antigens belong to the early expressed Replication and Transcription Complex (RTC) region, when administered to the golden Syrian hamsters, in combination with Spike, as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) (i.e., combined mRNA/LNP-based pan-CoV vaccine): (i) Induced high frequencies of lung-resident antigen-specific CXCR5+CD4+ T follicular helper (TFH) cells, GzmB+CD4+ and GzmB+CD8+ cytotoxic T cells (TCYT), and CD69+IFN-\u03b3+TNF\u03b1+CD4+ and CD69+IFN-\u03b3+TNF\u03b1+CD8+ effector T cells (TEFF); and (ii) Reduced viral load and COVID-19-like symptoms caused by various VOCs, including the highly pathogenic B.1.617.2 Delta variant and the highly transmittable heavily Spike-mutated XBB1.5 Omicron sub-variant. The combined mRNA/LNP-based pan-CoV vaccine could be rapidly adapted for clinical use to confer broader cross-protective immunity against emerging highly mutated and pathogenic VOCs. As of January 2024, over 1500 individuals in the United States alone are still dying from COVID-19 each week despite the implementation of first-generation Spike-alone-based COVID-19 vaccines. The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs), such as the currently circulating highly mutated BA.2.86 and JN.1 Omicron sub-variants, constantly overrode immunity induced by the first-generation Spike-alone-based COVID-19 vaccines. Here we report a next generation broad spectrum combined multi-antigen mRNA/LNP-based pan-CoV vaccine that consists of nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) that delivers three highly conserved non-Spike viral T cell protein antigens together with the Spike protein B-cell antigen. Compared side-by-side to the clinically proven first-generation Spike-alone mRNA/LNP-based vaccine, the combined multi-antigen mRNA/LNP-based pan-CoV vaccine-induced higher frequencies of lung-resident non-Spike antigen-specific T follicular helper (TFH) cells, cytotoxic T cells (TCYT), effector T cells (TEFF) and Spike specific-neutralizing antibodies. This was associated to a potent cross-reactive protection against various VOCs, including the highly pathogenic Delta variant and the highly transmittable heavily Spike-mutated Omicron sub-variants. Our findings suggest an alternative broad-spectrum pan-Coronavirus vaccine capable of (i) disrupting the current COVID-19 booster paradigm; (ii) outpacing the bivalent variant-adapted COVID-19 vaccines; and (iii) ending an apparent prolonged COVID-19 pandemic.","version":"1.1","doi":"10.1101/2024.02.14.580225","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.15.24302857","pub_date":"2024-02-15","title":"Prevalent and persistent new-onset autoantibodies in mild to severe COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients in five time points over 16 months using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months. We demonstrated associations between distinct new-onset autoantibodies and neuropsychiatric symptoms post-COVID-19. Using epitope mapping, we determined the main epitopes of selected new-onset autoantibodies, validated them in independent cohorts of neuro-COVID and pre-pandemic healthy controls, and identified molecular mimicry between main epitopes and the conserved fusion peptide of the SARS-CoV-2 Spike glycoprotein. Our work describes the complexity and dynamics of the autoantibody repertoire emerging with COVID-19 and supports the need for continued analysis of the new-onset autoantibody repertoire to elucidate the mechanisms of the post-COVID-19 condition.</jats:p>","version":null,"doi":"10.1101/2024.02.15.24302857","journal":"medRxiv","score":null},{"id":"10.1101/2024.02.13.580123","pub_date":"2024-2-14","title":"Pooled PPIseq: screening the SARS-CoV-2 and human interface with a scalable multiplexed protein-protein interaction assay platform","abstract":"Protein-Protein Interactions (PPIs) are a key interface between virus and host, and these interactions are important to both viral reprogramming of the host and to host restriction of viral infection. In particular, viral-host PPI networks can be used to further our understanding of the molecular mechanisms of tissue specificity, host range, and virulence. At higher scales, viral-host PPI screening could also be used to screen for small-molecule antivirals that interfere with essential viral-host interactions, or to explore how the PPI networks between interacting viral and host genomes co-evolve. Current high-throughput PPI assays have screened entire viral-host PPI networks. However, these studies are time consuming, often require specialized equipment, and are difficult to further scale. Here, we develop methods that make larger-scale viral-host PPI screening more accessible. This approach combines the mDHFR split-tag reporter with the iSeq2 interaction-barcoding system to permit massively-multiplexed PPI quantification by simple pooled engineering of barcoded constructs, integration of these constructs into budding yeast, and fitness measurements by pooled cell competitions and barcode-sequencing. We applied this method to screen for PPIs between SARS-CoV-2 proteins and human proteins, screening in triplicate >180,000 ORF-ORF combinations represented by >1,000,000 barcoded lineages. Our results complement previous screens by identifying 74 putative PPIs, including interactions between ORF7A with the taste receptors TAS2R41 and TAS2R7, and between NSP4 with the transmembrane KDELR2 and KDELR3. We show that this PPI screening method is highly scalable, enabling larger studies aimed at generating a broad understanding of how viral effector proteins converge on cellular targets to effect replication.","version":"1.1","doi":"10.1101/2024.02.13.580123","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.13.580056","pub_date":"2024-2-14","title":"Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage","abstract":"Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike\u2019s interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.","version":"1.2","doi":"10.1101/2024.02.13.580056","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.13.580076","pub_date":"2024-2-14","title":"Data mining antibody sequences for database searching in bottom-up proteomics","abstract":"Mass spectrometry (MS)-based proteomics allows identifying and quantifying thousands of proteins but suffers from challenges when measuring human antibodies due to their vast variety. The mainly used bottom-up proteomics approaches rely on database searches that compare experimental values of peptides and their fragments to theoretical values derived from protein sequences in a database. While the human body can produce millions of distinct antibodies, the current databases for human antibodies such as UniProtKB/Swiss-Prot are limited to only 1095 sequences (as of 2024 Jan). This limitation may hinder the identification of new antibodies using mass spectrometry. Therefore, extending the database for mass spectrometry is an important task for discovering new antibodies. Recent genomic studies have compiled millions of human antibody sequences publicly accessible through the Observed Antibody Space (OAS) database. However, this data has yet to be exploited to confirm the presence of these antibodies. In this study, we adopted this extensive collection of antibody sequences for conducting efficient database searches in publicly available proteomics data with a focus on the SARS-CoV-2 disease. Thirty million heavy antibody sequences from 146 SARS-CoV-2 patients in the OAS database were digested in silico to obtain 18 million unique peptides. These peptides were then used to create new databases for bottom-up proteomics. We used those databases for searching new antibody peptides in publicly available SARS-CoV-2 human plasma samples in the Proteomics Identification Database (PRIDE). This approach avoids false positives in antibody peptide identification as confirmed by searching against negative controls (brain samples) and employing different database sizes. We show that the found sequences provide valuable information to distinguish diseased from healthy and expect that the newly discovered antibody peptides can be further employed to develop therapeutic antibodies. The method will be broadly applicable to find characteristic antibodies for other diseases.","version":"1.1","doi":"10.1101/2024.02.13.580076","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.12.579921","pub_date":"2024-2-14","title":"A spring-loaded and leakage-tolerant synthetic gene switch for in-vitro detection of DNA and RNA","abstract":"Nucleic acid tests (NATs) are essential for biomedical diagnostics. Traditional NATs, often complex and expensive, have prompted the exploration of Toehold-Mediated Strand Displacement (TMSD) circuits as an economical alternative. However, the wide application of TMSD-based reactions is limited by \u2018leakage\u2019\u2014the spurious activation of the reaction leading to high background signals and false positives. Here we introduce a new TMSD cascade that recognizes a custom nucleic acid input and generates an amplified output. The system is based on a pair of thermodynamically spring-loaded DNA modules. The binding of a predefined nucleic acid target triggers an intermolecular reaction that activates a T7 promoter, leading to the perpetual transcription of a fluorescent aptamer that can be detected by a smartphone camera. The system is designed to permit the selective depletion of leakage byproducts to achieve high sensitivity and zero-background signal in the absence of the correct trigger. Using Zika virus (ZIKV)- and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived nucleic acid sequences, we show that the assay generates a reliable target-specific readout. Native RNA can be directly detected under isothermal conditions, without requiring reverse transcription, with a sensitivity as low as 200 attomole. The modularity of the assay allows easy re-programming for the detection of other targets by exchanging a single sequence domain. This work provides a low-complexity and high-fidelity synthetic biology tool for point-of-care diagnostics and for the construction of more complex biomolecular computations.","version":"1.1","doi":"10.1101/2024.02.12.579921","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.26.577395","pub_date":"2024-2-13","title":"Discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors with efficacy in a murine infection model","abstract":"Vaccines and first-generation antiviral therapeutics have provided important protection against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease (Mpro) have demonstrated clinical efficacy, known PLpro inhibitors have to date lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Herein, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.","version":"1.2","doi":"10.1101/2024.01.26.577395","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.12.579977","pub_date":"2024-2-13","title":"Identification of SARS-CoV-2 Mpro inhibitors through deep reinforcement learning for de novo drug design and computational chemistry approaches","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease (COVID-19) since its emergence in December 2019. As of January 2024, there has been over 774 million reported cases and 7 million deaths worldwide.[ While vaccination efforts have been successful in reducing the severity of the disease and decreasing the transmission rate, the development of effective therapeutics against SARS-CoV-2 remains a critical need.[2] The main protease (Mpro) of SARS-CoV-2 is an essential enzyme required for viral replication and has been identified as a promising target for drug development. In this study, we report the identification of novel Mpro inhibitors, using a combination of deep reinforcement learning for de novo drug design with 3D pharmacophore/shape-based alignment and privileged fragment match count scoring components followed by hit expansions and molecular docking approaches. Our experimentally validated results show that 3 novel series exhibit potent inhibitory activity against SARS-CoV-2 Mpro, with IC50 values ranging from 1.3 uM to 2.3 uM and a high degree of selectivity. These findings represent promising starting points for the development of new antiviral therapies against COVID-19.","version":"1.1","doi":"10.1101/2024.02.12.579977","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.25.563967","pub_date":"2024-2-13","title":"Discovery of a novel inhibitor of macropinocytosis with antiviral activity","abstract":"Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a new molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using High-Throughput Microscopy, where we identified new chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as Monkeypox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present a new molecule that inhibits viral entry via the endocytic route, offering a new alternative to prevent viral infection.","version":"1.2","doi":"10.1101/2023.10.25.563967","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.09.579701","pub_date":"2024-2-12","title":"Efficacy of Host Cell Serine Protease Inhibitor MM3122 against SARS-CoV-2 for Treatment and Prevention of COVID-19","abstract":"We have developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases including transmembrane protease serine 2 (TMPRSS2), matriptase and hepsin. TMPRSS2 is a membrane associated protease which is highly expressed in the upper and lower respiratory tract and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell receptor binding, entry, replication, and dissemination of new virion particles. We have previously shown that compound MM3122 exhibited sub nanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell-viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Further, we have evaluated MM3122 in a mouse model of COVID-19 and have demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion, and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in pro-inflammatory cytokines and chemokines production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2 infected mice. Therefore, MM3122 is a promising lead candidate small molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host-cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and therapeutic drug for the treatment of COVID-19 in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.","version":"1.1","doi":"10.1101/2024.02.09.579701","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.05.543733","pub_date":"2024-2-12","title":"Data-driven recombination detection in viral genomes","abstract":"Recombination is a key molecular mechanism for the evolution and adaptation of viruses. The first recombinant SARS-CoV-2 genomes were recognized in 2021; as of today, more than ninety SARS-CoV-2 lineages are designated as recombinant. In the wake of the COVID-19 pandemic, several methods for detecting recombination in SARS-CoV-2 have been proposed; however, none could faithfully confirm manual analyses by experts in the field. We hereby present RecombinHunt, a novel, automated method for the identification of recombinant/mosaic genomes purely based on a data-driven approach. RecombinHunt compares favorably with other state-of-the-art methods and recognizes recombinant SARS-CoV-2 genomes (or lineages) with one or two breakpoints with high accuracy, within reduced turn-around times and small discrepancies with respect to the expert manually-curated standard nomenclature. Strikingly, applied to the complete collection of viral sequences from the recent monkeypox epidemic, RecombinHunt identifies recombinant viral genomes in high concordance with manually curated analyses by experts, suggesting that our approach is robust and can be applied to any epidemic/pandemic virus. In conclusion, RecombinHunt represents a breakthrough in the detection of recombinant viral lineages in pandemic/epidemic scenarios and could substantially improve/advance community-based approaches for the detection of recombinant viral genomes based on phylogenetic analyses.","version":"1.2","doi":"10.1101/2023.06.05.543733","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.09.579589","pub_date":"2024-2-10","title":"SARS-COV-2 induces blood-brain barrier and choroid plexus barrier impairments and vascular inflammation in mice","abstract":"The coronavirus disease of 2019 (COVID-19) pandemic that has led to more than 700 million confirmed cases and near 7 million deaths. Although Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains as an important question, which is also centered on the mystery whether the virus is capable of breaching the barriers into the central nervous system. By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. In addition, the blood-cerebrospinal fluid barrier at the choroid plexus was also impaired after infection. Therefore, cerebrovascular and choroid plexus dysfunctions are important aspects of COVID-19 and may contribute to the neurological complications both acutely and in long COVID.","version":"1.1","doi":"10.1101/2024.02.09.579589","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.09.579628","pub_date":"2024-2-10","title":"Human coronavirus 229E infection inactivates pyroptosis executioner gasdermin D but ultimately leads to lytic cell death partly mediated by gasdermin E","abstract":"Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and causes local respiratory symptoms. It has been reported that HCoV-229E can cause cell death in a variety of cells in vitro. However, the molecular pathways that lead to virus-induced cell death remain poorly characterized. Here, we show that the main protease (Mpro) of HCoV-229E can cleave the pyroptosis executioner gasdermin D (GSDMD) within its active N-terminal domain at two different sites (Q29 and Q193) to generate fragments unable to cause pyroptosis. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection leads to lytic cell death. We further demonstrate that virus-induced lytic cell death is partially dependent on the activation of caspases-3 and -8. Interestingly, inhibition of caspases does not only reduce lytic cell death upon infection, but also sustains the release of virus particles over time, which suggests that caspase-mediated cell death is a mechanism to limit virus replication and spread. Finally, we show that pyroptosis is partially dependent on another gasdermin family member, gasdermin E (GSDME). During HCoV-229E infection, GSDME is cleaved to yield its N-terminal pore-forming domain (p30). Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus, whereas this is not the case for GSDMD knockout cells, which aligns with the observation that GSDMD is also inactivated by caspase-3 during infection. These results suggest that GSDMD is inactivated during HCoV-229E infection, and point to GSDME as an important player in the execution of virus-induced cell death. Recently, it has been shown that the Mpros of coronaviruses possess accessory functions other than their main role in the proteolytic processing of the viral polyproteins. Although the SARS-CoV-2 outbreak has fuelled the discovery of host cellular substrates of SARS-CoV-2 Mpro, less is known about the interplay of less pathogenic human coronavirus Mpros with host proteins. We demonstrate that HCoV-229E Mpro cleaves GSDMD at two sites within its pore-forming domain, which disrupts GSDMD-mediated pyroptosis. These results point to a new strategy for HCoV-229E to escape the host antiviral response. Additionally, we show that GSDME contributes to virus-induced lytic cell death upon activation by caspase-3, shedding light on a previously undescribed cell death mechanism occurring in HCoV-229E infected cells.","version":"1.1","doi":"10.1101/2024.02.09.579628","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.20.567873","pub_date":"2024-2-09","title":"Distinct evolution of SARS-CoV-2 Omicron XBB and BA.2.86/JN.1 lineages combining increased fitness and antibody evasion","abstract":"The unceasing circulation of SARS-CoV-2 leads to the continuous emergence of novel viral sublineages. Here, we isolated and characterized XBB.1, XBB.1.5, XBB.1.9.1, XBB.1.16.1, EG.5.1.1, EG.5.1.3, XBF, BA.2.86.1 and JN.1 variants, representing >80% of circulating variants in January 2024. The XBB subvariants carry few but recurrent mutations in the spike, whereas BA.2.86.1 and JN.1 harbor >30 additional changes. These variants replicated in IGROV-1 but no longer in Vero E6 and were not markedly fusogenic. They potently infected nasal epithelial cells, with EG.5.1.3 exhibiting the highest fitness. Antivirals remained active. Neutralizing antibody (NAb) responses from vaccinees and BA.1/BA.2-infected individuals were markedly lower compared to BA.1, without major differences between variants. An XBB breakthrough infection enhanced NAb responses against both XBB and BA.2.86 variants. JN.1 displayed lower affinity to ACE2 and higher immune evasion properties compared to BA.2.86.1. Thus, while distinct, the evolutionary trajectory of these variants combines increased fitness and antibody evasion.","version":"1.3","doi":"10.1101/2023.11.20.567873","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.07.579282","pub_date":"2024-2-08","title":"Traditional uses and pharmacological activities of Tetracera alnifolia (Wild) Drake","abstract":"Tetracera alnifolia (Wild) Drake, is well used in traditional Guinean medicine for the treatment of infectious skin diseases. The present aim was to contribute to the valorization of Tetracera alnifolia leaves, focused on ethnomedical, biological and phytochemical investigations. we conducted an ethnomedical survey across several markets of the city of Conakry to identify 39 healers. Chloroform, methanol, dichloromethane, and aqueous extracts were tested for activities against protozoa, bacteria, fungi, HIV, and SARS-CoV-2. the traditional healers indicated that T. alnifolia is used in the treatment of more than 15 pathologies including Fassa (marasmus/malnutrition), Soukhou kouy\u00e9 (white discharge in women), and T\u00e8mou bankhi (sexual weakness in men). Leaves were the most used part. The modes of preparation included decoction and powder. Data from biological activities identicatied good activities of the methanolic extract against Leishmania infantum (MIC = 8.11 \u03bcg / ml) and a moderate activity on Trypanosoma brucei (MIC = 28.15 \u03bcg / ml) and Staphylococcus aureus (MIC = 29.91 \u03bcg / ml), while dichloromethane extracts acted on live SARS-CoV-2 replication with up to 53.4% inhibition at 50 \u03bcg/mL. these results explain at least in part the traditional use of T. alnifolia","version":"1.1","doi":"10.1101/2024.02.07.579282","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.05.578925","pub_date":"2024-2-06","title":"Second Boost of Omicron SARS-CoV-2 S1 Subunit Vaccine Induced Broad Humoral Immune Responses in Elderly Mice","abstract":"Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death from the disease. However, repeated homologous boosters, while considered a solution for severe forms of the disease caused by new SARS-CoV-2 variants in elderly individuals and immunocompromised patients, cannot provide complete protection against breakthrough infections. This highlights the need for alternative platforms for booster vaccines. In our previous study, we assessed the boost effect of the SARS-CoV-2 Beta S1 recombinant protein subunit vaccine (rS1Beta) in aged mice primed with an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) via subcutaneous injection or intranasal delivery, which induced robust humoral immune responses (1). In this follow-up study, we demonstrated that a second booster dose of a non-adjuvanted recombinant Omicron (BA.1) S1 subunit vaccine with Toll-like receptor 4 (TLR4) agonist RS09 (rS1RS09OM) was effective in stimulating strong S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants in 100-week-old mice. Importantly, the second booster dose elicits cross-reactive antibody responses, resulting in ACE2 binding inhibition against the spike protein of SARS-CoV-2 variants, including Omicron (BA.1) and its subvariants. Interestingly, the levels of IgG and neutralizing antibodies correlated with the level of ACE2 inhibition in the booster serum samples, although Omicron S1-specific IgG level showed a weaker correlation compared to Wuhan S1-specific IgG level. Furthermore, we compared the immunogenic properties of the rS1 subunit vaccine in young, middle-aged, and elderly mice, resulting in reduced immunogenicity with age, especially an impaired Th1-biased immune response in aged mice. Our findings demonstrate that the new variant of concern (VOC) rS1 subunit vaccine as a second booster has the potential to offer cross-neutralization against a broad range of variants and to improve vaccine effectiveness against newly emerging breakthrough SARS-CoV-2 variants in elderly individuals who were previously primed with the authorized vaccines.","version":"1.1","doi":"10.1101/2024.02.05.578925","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.05.578560","pub_date":"2024-2-06","title":"Building Blocks of Understanding: Constructing a Reverse Genetics Platform for studying determinants of SARS-CoV-2 replication","abstract":"To better understand viral pathogenesis, host-virus interactions, and potential therapeutic interventions, the development of robust reverse genetics systems for SARS-CoV-2 is crucial. Here, we present a reverse genetics platform that enables the efficient manipulation, assembly, and rescue of recombinant SARS-CoV-2. The versatility of our reverse genetics system was demonstrated by generating recombinant SARS-CoV-2 viruses. We used this system to generate N501Y and Y453F spike protein mutants. Characterization studies revealed distinct phenotypic effects, impact on viral fitness, cell binding, and replication kinetics. We also investigated a recently discovered priming site for NSP9, which is postulated to produce a short RNA antisense leader sequence. By introducing the U76G mutation into the 5\u2019UTR, we show that this priming site is necessary for the correct production of genomic and subgenomic RNAs, and also for efficient viral replication. In conclusion, our developed reverse genetics system provides a robust and adaptable platform for the efficient generation of recombinant SARS-CoV-2 viruses for their comprehensive characterization. In this study, we present a versatile reverse genetics platform facilitating the efficient manipulation, assembly, and rescue of recombinant SARS-CoV-2. Demonstrating its adaptability, we successfully engineered N501Y and Y453F spike protein mutants, each exhibiting distinct phenotypic effects on viral fitness, cell binding, and replication kinetics. We also investigated a novel negative sense priming site for NSP9, demonstrating a role in RNA production and viral replication. This straightforward reverse genetic system is therefore a powerful tool to generate recombinant viruses for advancing our understanding of SARS-CoV-2 biology.","version":"1.1","doi":"10.1101/2024.02.05.578560","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.03.578771","pub_date":"2024-2-05","title":"XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants","abstract":"The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination (\u223c1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.","version":"1.1","doi":"10.1101/2024.02.03.578771","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.02.578538","pub_date":"2024-2-05","title":"Viral interference between severe acute respiratory syndrome coronavirus 2 and influenza A viruses","abstract":"Some respiratory viruses can cause a viral interference through the activation of the interferon (IFN) pathway that reduces the replication of another virus. Epidemiological studies of coinfections between SARS-CoV-2 and other respiratory viruses have been hampered by non-pharmaceutical measures applied to mitigate the spread of SARS-CoV-2 during the COVID-19 pandemic. With the ease of these interventions, SARS-CoV-2 and influenza A viruses can now co-circulate. It is thus of prime importance to characterize their interactions. In this work, we investigated viral interference effects between an Omicron variant and a contemporary influenza A/H3N2 strain, in comparison with an ancestral SARS-CoV-2 strain and the 2009 pandemic influenza A/H1N1 virus. We infected nasal human airway epitheliums with SARS-CoV-2 and influenza, either simultaneously or 24 h apart. Viral load was measured by RT-qPCR and IFN-\u03b1/\u03b2/\u03bb1/\u03bb2 proteins were quantified by immunoassay. Expression of four interferon-stimulated genes (ISGs; OAS1/IFITM3/ISG15/MxA) was also measured by RT-droplet digital PCR. Additionally, susceptibility of each virus to IFN-\u03b1/\u03b2/\u03bb2 recombinant proteins was determined. Our results showed that influenza A, and especially A/H3N2, interfered with both SARS-CoV-2 viruses, but that SARS-CoV-2 only interfered with A/H1N1. Consistently with these results, influenza, and particularly the A/H3N2 strain, caused a higher production of IFN proteins and expression of ISGs than SARS-CoV-2. The IFN production induced by SARS-CoV-2 was marginal and its presence during coinfections with influenza was associated with a reduced IFN response. All viruses were susceptible to exogenous IFNs, with the ancestral SARS-CoV-2 and Omicron being less susceptible to type I and type III IFNs, respectively. Thus, influenza A causes a viral interference towards SARS-CoV-2 most likely through an IFN response. The opposite is not necessarily true, and a concurrent infection with both viruses leads to a lower IFN response. Taken together, these results help us to understand how SARS-CoV-2 interacts with another major respiratory pathogen. During the COVID-19 pandemic, non-pharmaceutical measures were able to reduce the spread of SARS-CoV-2 and most respiratory viruses. Since the ease of these measures, SARS-CoV-2 variants and other viruses, such as influenza A, have started to co-circulate and can now infect a same host and interact with each other. These interactions can lead to attenuated or aggravated infections and can affect the timing of epidemics. Therefore, it is very important to elucidate how the new SARS-CoV-2 interacts with other viruses to better predict their implications in human health and their epidemic activity. Our work contributes to better understand these interactions using viruses that have likely co-circulated after lifting mitigation interventions, i.e., SARS-CoV-2 Omicron variant and a contemporary influenza A/H3N2 strain. We studied how each virus may affect the other virus\u2019 growth and how these interactions were associated with the innate immune response of the host. We found that a prior infection with influenza A can decrease the growth of SARS-CoV-2 while the latter reduces the innate immune response. Our results help to understand the interplay between SARS-CoV-2 and influenza A in the host and may improve mathematical models predicting epidemics.","version":"1.2","doi":"10.1101/2024.02.02.578538","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.01.577684","pub_date":"2024-2-02","title":"Development of a cell-based DIFF-rGFP assay system for generalized discovery of viral protease Inhibitors","abstract":"Viral protease is an attractive target for antiviral therapeutics, but current viral protease inhibitor screening methods still need to be improved. Here, we systematically investigated the sites that may accommodate exogenous short peptides within Enhanced Green Fluorescent Protein (EGFP) and constructed a series of recombinant green fluorescent proteins (rGFPs). Meanwhile, a cell-based, simple and reliable assay system named DIFF-rGFP was developed relying on the co-expression of rGFP and the protease for protease inhibitor screening with the example of 3CLpro, in which the fluorescence intensity increases with the action of the inhibitor. The DIFF-rGFP assay avoided the requirement of a higher biosafety lab and can be performed in a high-throughput manner. For proof of concept, we demonstrated this method to discover novel inhibitors against SARS-CoV-2. We believe the proposed method, in combination with available drug libraries, may accelerate the identification of novel antivirals.","version":"1.1","doi":"10.1101/2024.02.01.577684","journal":"bioRxiv","score":null},{"id":"10.1101/2024.02.02.578553","pub_date":"2024-2-02","title":"Recapitulating memory B cell responses in a Lymphoid Organ-Chip to evaluate mRNA vaccine boosting strategies","abstract":"Predicting the immunogenicity of candidate vaccines in humans remains a challenge. To address this issue, we developed a Lymphoid Organ-Chip (LO chip) model based on a microfluidic chip seeded with human PBMC at high density within a 3D collagen matrix. Perfusion of the SARS-CoV-2 Spike protein mimicked a vaccine boost by inducing a massive amplification of Spike-specific memory B cells, plasmablast differentiation, and Spike-specific antibody secretion. Features of lymphoid tissue, including the formation of activated CD4+ T cell/B cell clusters and the emigration of matured plasmablasts, were recapitulated in the LO chip. Importantly, myeloid cells were competent at capturing and expressing mRNA vectored by lipid nanoparticles, enabling the assessment of responses to mRNA vaccines. Comparison of on-chip responses to Wuhan monovalent and Wuhan/Omicron bivalent mRNA vaccine boosts showed equivalent induction of Omicron neutralizing antibodies, pointing at immune imprinting as reported in vivo. The LO chip thus represents a versatile platform suited to the preclinical evaluation of vaccine boosting strategies.","version":"1.1","doi":"10.1101/2024.02.02.578553","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.31.578159","pub_date":"2024-1-31","title":"An isothermal calorimetry assay for determining steady state kinetic and enzyme inhibition parameters for SARS-CoV-2 3CL-protease","abstract":"This manuscript describes the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CLpro from the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and its inhibition by Ensitrelvir, a known non-covalent inhibitor. 3CLpro is the main protease that plays a crucial role of producing the whole array of proteins necessary for the viral infection that caused the spread of COVID-19, responsible for millions of deaths worldwide as well as global economic and healthcare crises in recent years. The proposed calorimetric method proved to have several advantages over the two types of enzymatic assays so far applied to this system, namely F\u00f6rster Resonance Energy Transfer (FRET) and Liquid Chromatography-Mass Spectrometry (LC-MS). The developed ITC-based assay provided a rapid response to 3CLpro activity, which was used to directly derive the kinetic enzymatic constants KM and kcat reliably and reproducibly, as well as their temperature dependence, from which the activation energy of the reaction was obtained for the first time. The assay further revealed the existence of two modes of inhibition of 3CLpro by Ensitrelvir, namely a competitive mode as previously inferred by crystallography as well as an unprecedented uncompetitive mode, further yielding the respective inhibition constants with high precision. The calorimetric method described in this paper is thus proposed to be generally and widely used in the discovery and development of drugs targeting 3CLpro.","version":"1.1","doi":"10.1101/2024.01.31.578159","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.30.578038","pub_date":"2024-1-31","title":"A novel microporous biomaterial vaccine platform for long-lasting antibody mediated immunity against viral infection","abstract":"Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude and duration of anti-receptor binding domain antibodies compared to Alum and mRNA-vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.","version":"1.1","doi":"10.1101/2024.01.30.578038","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.14.557827","pub_date":"2024-1-30","title":"Some mechanistic underpinnings of molecular adaptations of SARS-COV-2 spike protein by integrating candidate adaptive polymorphisms with protein dynamics","abstract":"We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 Spike (S) protein. With this approach, we first identified Candidate Adaptive Polymorphisms (CAPs) of the SARS-CoV-2 Spike protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.","version":"1.2","doi":"10.1101/2023.09.14.557827","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.29.577677","pub_date":"2024-1-30","title":"Spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through multiple mechanisms","abstract":"Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity could be recovered in the presence of heparin sulfate, which targets the \u201cN354 pocket\u201d to ease restrictions of conformational transition resulting in a \u201cRBD-up\u201d state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms. N354 glycosylation acts as a conformational control element to modulate infectivity Reduced infectivity could be recovered by altered binding mode of heparin sulfate N354 glycosylation improved fusogenicity and conferred escape from ADCC antibodies N354 glycosylation reduced immunogenicity and conferred immune evasion","version":"1.1","doi":"10.1101/2024.01.29.577677","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.29.24301882","pub_date":"2024-01-30","title":"Possible roles of phytochemicals with bioactive properties in the prevention of and recovery from COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Purpose</jats:title>\n                  <jats:p>There have been large geographical differences in the infection and death rates of COVID-19. Foods and beverages containing high amounts of phytochemicals with bioactive properties were suggested to prevent contracting, to limit the severity of, and to facilitate recovery from COVID-19. The goal of our study was to determine the correlation of the type of foods/beverages people consumed and the risk reduction of contracting COVID-19 and the recovery from COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed an online survey that asked the participants whether they contracted COVID-19, their symptoms, time to recover, and their frequency of eating various types of foods/beverages. The survey was first developed in English and then translated into 10 different languages.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The participants who did not contract COVID-19 consumed vegetables, herbs/spices, and fermented foods/beverages significantly more than the participants who contracted COVID-19 and those who were not tested but became sick most likely from COVID-19. The geographic location of participants corresponded with the language of the survey, except for the English version, thus, nine out of the 10 language versions represented a country. Among the six countries (India, Iran, Italy, Japan, Russia, Spain) with over one hundred participants, we found that in India and Japan the people who contracted COVID-19 showed significantly shorter recovery time, and greater daily intake of vegetables, herbs/spices, and fermented foods/beverages was associated with faster recovery.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Our results suggest that phytochemical compounds included in the vegetables may have contributed in not only preventing contraction of COVID-19, but also accelerating their recovery. (249 words; EJN limit is 250 words)</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2024.01.29.24301882","journal":"medRxiv","score":null},{"id":"10.1101/2024.01.26.577489","pub_date":"2024-1-30","title":"A general platform for targeting MHC-II antigens via a single loop","abstract":"Class-II major histocompatibility complexes (MHC-IIs) are central to the communications between CD4+ T cells and antigen presenting cells (APCs), but intrinsic structural features associated with MHC-II make it difficult to develop a general targeting system with high affinity and antigen specificity. Here, we introduce a protein platform, Targeted Recognition of Antigen-MHC Complex Reporter for MHC-II (TRACeR-II), to enable the rapid development of peptide-specific MHC-II binders. TRACeR-II has a small helical bundle scaffold and uses an unconventional mechanism to recognize antigens via a single loop. This unique antigen-recognition mechanism renders this platform highly versatile and amenable to direct structural modeling of the interactions with the antigen. We demonstrate that TRACeR-II binders can be rapidly evolved across multiple alleles, while computational protein design can produce specific binding sequences for a SARS-CoV-2 peptide of unknown complex structure. TRACeR-II sheds light on a simple and straightforward approach to address the MHC peptide targeting challenge, without relying on combinatorial selection on complementarity determining region (CDR) loops. It presents a promising basis for further exploration in immune response modulation as well as a broad range of theragnostic applications.","version":"1.1","doi":"10.1101/2024.01.26.577489","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.549041","pub_date":"2024-1-29","title":"Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus","abstract":"To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.","version":"1.2","doi":"10.1101/2023.07.14.549041","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.29.577695","pub_date":"2024-1-29","title":"Multiple layers of innate immune response antagonism of SARS-CoV-2","abstract":"Several SARS-CoV-2 proteins have been shown to counteract the host innate immune response, mostly using in vitro protein expression, which may not fully reflect their role in the context of viral infection. In addition, while each viral protein was characterized in a different experimental system, their relative contribution in immunosuppression remains unclear. Here we used a SARS-CoV-2 bacterial artificial chromosome with en passant mutagenesis to recover a panel of twelve infectious recombinant SARS-CoV-2 viruses, each with mutations in either NSP1, NSP2, NSP3, NSP6, NSP12, NSP13, NSP14, NSP15, NSP16, ORF3a, ORF6 or ORF8. We used the interferon-stimulated response element (ISRE)-driven luciferase assay in 293T-ACE2/TMPRSS2 cells to test the panel, demonstrating that mutations in many proteins, especially in NSP1 and NSP15, increased the type I interferon response relative to the parental wild-type virus. RNA-seq analysis of mutant-virus infected Calu-3 cells showed that the mutations in NSP1 or NSP15 lead to higher expression of multiple genes involved in innate immune response, cytokine-mediated signaling and regulation of lymphocyte proliferation. Furthermore, mutations in either NSP1 or NSP15 resulted in a greater maturation of human monocyte-derived dendritic cells in vitro. Infection of K18 hACE2 transgenic mice with either NSP1 or NSP15 mutated viruses demonstrated attentuated respiratory tract replication. Analysis of lung immune cells from infected mice by single-cell RNA-seq identified 15 populations of major myeloid and lymphoid cells with changes in the pattern of their activation associated with viral infection. The effects of mutations in NSP1 or NSP15 on these responses are consistent with differences in the immunosuppressive mechanisms utilized by the two proteins. Overall, these data demonstrate different and redundant mechanisms of innate immune antagonism by SARS-CoV-2 and suppression of activation of antigen presenting cells and T and B lymphocytes mediated by multiple viral proteins. The mechanisms by which SARS-CoV-2 and its proteins modulate host immunity, specifically the interferon response, are still not clear. We generated twelve infectious SARS-CoV-2 viruses with mutations in individual proteins and demonstrated that many of them have interferon-antagonizing activity and immunosuppressive effects in human cells and in the K18 hACE mouse model of infection. We idemtified distinct and redundant mechanisms of immunosuppression of SARS-CoV-2 mediated by multiple individual viral proteins, with 9 out of the 12 tested proteins showing some immunosuppressive effect in at least one experimental system. The demonstrated immunosuppressive effects extend from the innate response to immune cells to pathologic changes in vivo. Importantly, this work shows, for the first time, a comparison of the effects of multiple viral proteins in the context of authentic viral infection, rather than in a surrogate system, and shows the relative contribution of each viral protein under identical experimental conditions. Overall, our data indicates that SARS-CoV-2 antagonizes multiple immune mechanisms, particularly type I interferon signaling, activation of innate immune cells and T and B lymphocyte functions with the greatest effects due to NSP1 and NSP15.","version":"1.1","doi":"10.1101/2024.01.29.577695","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.28.577610","pub_date":"2024-1-29","title":"Low-coverage whole genome sequencing for a highly selective cohort of severe COVID-19 patients","abstract":"Despite advances in identifying genetic markers associated to severe COVID-19, the full genetic characterisation of the disease remains elusive. This study explores the use of imputation in low-coverage whole genome sequencing for a severe COVID-19 patient cohort. We generated a dataset of 79 imputed variant call format files using the GLIMPSE1 tool, each containing an average of 9.5 million single nucleotide variants. Validation revealed a high imputation accuracy (squared Pearson correlation \u22480.97) across sequencing platforms, showing GLIMPSE1\u2019s ability to confidently impute variants with minor allele frequencies as low as 2% in Spanish ancestry individuals. We conducted a comprehensive analysis of the patient cohort, examining hospitalisation and intensive care utilisation, sex and age-based differences, and clinical phenotypes using a standardised set of medical terms developed to characterise severe COVID-19 symptoms. The methods and findings presented here may be leveraged in future genomic projects, providing vital insights for health challenges like COVID-19.","version":"1.1","doi":"10.1101/2024.01.28.577610","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.18.576147","pub_date":"2024-1-28","title":"nf-core/airrflow: an adaptive immune receptor repertoire analysis workflow employing the Immcantation framework","abstract":"Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) is a valuable experimental tool to study the immune state in health and following immune challenges such as infectious diseases, (auto)immune diseases, and cancer. Several tools have been developed to reconstruct B cell and T cell receptor sequences from AIRR-seq data and infer B and T cell clonal relationships. However, currently available tools offer limited parallelization across samples, scalability or portability to high-performance computing infrastructures. To address this need, we developed nf-core/airrflow, an end-to-end bulk and single-cell AIRR-seq processing workflow which integrates the Immcantation Framework following BCR and TCR sequencing data analysis best practices. The Immcantation Framework is a comprehensive toolset, which allows the processing of bulk and single-cell AIRR-seq data from raw read processing to clonal inference. nf-core/airrflow is written in Nextflow and is part of the nf-core project, which collects community contributed and curated Nextflow workflows for a wide variety of analysis tasks. We assessed the performance of nf-core/airrflow on simulated sequencing data with sequencing errors and show example results with real datasets. To demonstrate the applicability of nf-core/airrflow to the high-throughput processing of large AIRR-seq datasets, we validated and extended previously reported findings of convergent antibody responses to SARS-CoV-2 by analyzing 97 COVID-19 infected individuals and 99 healthy controls, including a mixture of bulk and single-cell sequencing datasets. Using this dataset, we extended the convergence findings to 20 additional subjects, highlighting the applicability of nf-core/airrflow to validate findings in small in-house cohorts with reanalysis of large publicly available AIRR datasets. nf-core/airrflow is available free of charge, under the MIT license on GitHub (https://github.com/nf-core/airrflow). Detailed documentation and example results are available on the nf-core website at (https://nf-co.re/airrflow).","version":"1.2","doi":"10.1101/2024.01.18.576147","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.24.577125","pub_date":"2024-1-28","title":"Impact of the COVID-19 Pandemic on Undergraduate Research in the Department of Biology at Western University: Effect on project types, learning outcomes, and student perceptions","abstract":"Undergraduate research is a high impact practice that offers numerous benefits to students, academic institutions, and the wider scientific community. Unfortunately, undergraduate research has faced restrictions due to the COVID-19 pandemic. This study aimed to assess how the COVID-19 pandemic has impacted: (1) the number and types of undergraduate research projects performed in the Department of Biology at the University of Western Ontario, and (2) the satisfaction-levels and perceived learning outcomes of students performing these projects. This study also aimed to incorporate a \u2018One Health\u2019 framework through an emphasis on stakeholder involvement and the need for future action. A survey of 33 students who completed an undergraduate research project in the Department of Biology in the 2020/2021 academic year, and 68 students who completed an undergraduate research project in the 5 years prior was conducted. In keeping with the One Health approach, key stakeholders were identified, and a stakeholder map was constructed. The number of projects performed did not change dramatically despite COVID-19 restrictions. However, a shift towards dry research was observed with 87.9% of students in the 2020/2021 academic year conducting dry research, compared to 16.4% of students in the 5 years prior. Students who conducted research in the 2020/2021 academic year indicated lower overall levels of satisfaction and enjoyment, though their perceived learning outcomes were consistent with students who completed their projects in the 5 years prior. 53 key stakeholders from academia, government, industry, media, and the public were identified. Students provided invaluable feedback on their undergraduate research experiences that can be used to improve the quality of undergraduate research courses in the Department of Biology in the future. Findings may be of use to other departments and educational institutions that are seeking to improve their own undergraduate research courses amidst the COVID-19 pandemic or looking to incorporate experiential-based learning techniques into existing online courses.","version":"1.1","doi":"10.1101/2024.01.24.577125","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.25.577193","pub_date":"2024-1-26","title":"In silico assessment of immune cross protection between BCoV and SARS-CoV-2","abstract":"Humans have long shared infectious agents with cattle, and the bovine-derived human common cold OC-43 CoV is a not-so-distant example of cross-species viral spill over of coronaviruses. Human exposure to the Bovine Coronavirus (BCoV) is certainly common, as the virus is endemic in most high-density cattle-raising regions. Since BCoVs are phylogenetically close to SARS-CoV-2, it is possible that cross-protection against COVID-19 occurs in people exposed to BCoV. This article shows an in silico investigation of human cross-protection to SARS-CoV-2 due to BCoV exposure. We determined HLA recognition and human B lymphocyte reactivity to BCoV epitopes using bioinformatics resources. A retrospective geoepidemiological analysis of COVID-19 was then performed to verify if BCoV/SARS-CoV-2 cross-protection could have occurred in the field. Brazil was used as a model for the epidemiological analysis of the impact of livestock density \u2013 as a proxy for human exposure to BCoV \u2013 on the prevalence of COVID-19 in people. As could be expected from their classification in the same Betacoronavirus genus, we show that several human B and T epitopes are shared between BCoV and SARS-CoV-2. This raised the possibility of cross-protection of people from exposure to the bovine coronavirus. Analysis of field data added partial support to the hypothesis of viral cross-immunity from human exposure to BCoV. There was a negative correlation between livestock geographical density and COVID-19. Whole-Brazil data showed areas in the country in which COVID-19 prevalence was disproportionally low (controlled by normalization by transport infrastructure). Areas with high cattle density had lower COVID-19 prevalence in these low-risk areas. These data are hypothesis-raising indications that cross-protection is possibly being induced by human exposure to the Bovine Coronavirus.","version":"1.1","doi":"10.1101/2024.01.25.577193","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.24.577112","pub_date":"2024-1-26","title":"WRKY1 confers resistance to powdery mildew by accelerating SAR and preventing over-immunity in apple","abstract":"Powdery mildew is one of the most serious diseases in apple production. SAR has a broad-spectrum immunity in plants against pathogen. Plants activate SAR against pathogen invasion and also prevent over-immunity. The relevant mechanism is still unknown in apple. In this study, we isolated and identified powdery mildew pathogen from the field and preserved them on the apple tissue culture seedlings. We performed DAP-seq of powdery mildew-inducible WRKY40. WRKY40 positively regulates NPR3like by directly binding to the W-box element of its promoter. NPR3like represses the expression of the PR1 gene in the presence of SA by competing with TGA2 for binding to NPR1. WRKY1 positively regulates WRKY40 by directly binding to the dual W-box element of its promoter, while WRKY1 positively regulates NPR3like by directly binding to the W-box element of its promoter. The expression trends of WRKY1, WRKY40, and NPR3like were basically the same as that of PR1 within 24 h after powdery mildew and SA treatments. Besides, WRKY1 increased SA content by positively regulating EPS1. After inoculation with powdery mildew, the up-regulation of PR1 in RNAi-silenced plants of WRKY1 was more slowly compared with the wild type, and the number of spores and mycelium increased significantly. In summary, we established a new model of NPR3like inhibition of NPR1 activity positively regulated by the WRKY1-WRKY40 module and found that the WRKY1-EPS1 module accelerated the up-regulation of PR1 by increasing the SA content. Finally, we elucidated WRKY1 confers resistance to powdery mildew by accelerating SAR and preventing over-immunity in apple.","version":"1.1","doi":"10.1101/2024.01.24.577112","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.24.576385","pub_date":"2024-1-25","title":"Investigating Sensitivity, Specificity and Accuracy of Variant Calling Pipelines for Analyzing SARS-CoV-2 Data","abstract":"The rapidly increasing popularity of Next Generation Sequencing and analysis methods in clinical and research settings necessitates an understanding of ideal combinations in identifying genomic variants. Especially with the importance of detecting accurate variants for the development of targeted SARS-CoV-2 vaccines. This research compares the results of two \u2018Mapping Algorithms \u2018, BWA-MEM and Bowtie2, and two \u2018Variant Calling Algorithms \u2018, LoFreq and FreeBayes, and their combinatory Variant Calling Pipelines on the analyses of Next Generation Sequencing (NGS) data of five SARS-CoV-2 samples collected from patients in the USA, India, Italy, and Malawi and sourced for this research from the publicly available NCBI SRA database. Our analysis of mapping algorithms found that BWA-MEM likely has higher sensitivity and specificity than Bowtie2 for mapping reads, and their specificity and sensitivity vary with read length. Furthermore, the accuracy of variant calling algorithms increases with the number of reads, while higher read length possibly leads to divergence in accuracy and sensitivity. Overall, FreeBayes was found to likely be more sensitive to detecting variants when used with Bowtie2 rather than BWA-MEM for analyzing SARS-CoV-2 data.","version":"1.1","doi":"10.1101/2024.01.24.576385","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.24.577015","pub_date":"2024-1-25","title":"Interferon-\u03b3 as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein","abstract":"ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking the innate immune response of the infected cells by inhibiting interferon pathways. Additionally, it binds and immobilises the RAE1 protein onto the cytoplasmic membranes, thereby blocking the transport of mRNA from the nucleus to the cytoplasm. In all these cases the host cell proteins are tethered by the flexible C-terminus of ORF6. A possible strategy to inhibit the biological activity of ORF6 is to bind its C-terminus with suitable ligands. Our in silico experiments suggest that hIFN\u03b3 binds the ORF6 protein with high affinity, thus impairing its interactions with RAE1 and, consequently, its activity in viral invasion. The here reported in vitro studies reveal a shift of the localization of RAE1 in ORF6 overexpressing cells upon treatment with hIFN\u03b3 from predominantly cytoplasmic to mainly nuclear, resulting in restoration of the export of mRNA from the nucleus. We also explored the expression of GFP in transfected with ORF6 cells by means of fluorescence microscopy and qRT-PCR, finding that treatment with hIFN\u03b3 unblocks the mRNA trafficking and reinstates the GFP expression level. The ability of the cytokine to block ORF6 is also reflected in minimising its negative effects on DNA replication by reducing accumulated RNA-DNA hybrids. Our results, therefore, suggest hIFN\u03b3 as a promising inhibitor of the most toxic SARS-CoV-2 protein.","version":"1.1","doi":"10.1101/2024.01.24.577015","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.23.576505","pub_date":"2024-1-24","title":"CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment","abstract":"SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2024.01.23.576505","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.23.575909","pub_date":"2024-1-24","title":"Novel sensor-integrated proteome on chip (SPOC) platform with thousands of folded proteins on a 1.5 sq-cm biosensor chip to enable high-throughput real-time label-free screening for kinetic analysis","abstract":"An automated proteomic platform for producing and screening an array of functional proteins on biosensor surfaces was developed to address the challenges of measuring proteomic interaction kinetics in high throughput (HTP). This technology is termed Sensor-Integrated Proteome On Chip (SPOC\u00ae) which involves in-situ cell-free protein expression in nano-liter volume wells (nanowells) directly from rapidly customizable arrays of plasmid DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins onto a 1.5 sq-cm surface of a single gold biosensor chip. Arrayed SPOC sensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic affinity, avidity data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of the SPOC protein array was validated by antibody binding assay, post-translational modification, mutation-mediated differential binding kinetics, and catalytic activity screening on model SPOC protein arrays containing p53, Src, Jun, Fos, HIST1H3A, and SARS-CoV-2 receptor binding domain (RBD) protein variants of interest, among others. Monoclonal antibodies were found to selectively bind their target proteins on the SPOC array. A commercial anti-RBD antibody was used to demonstrate discriminatory binding to numerous SARS-CoV-2 RBD variants of concern with comprehensive kinetic information. With advantages of HTP, flexibility, low-cost, quick turnaround time, and real-time kinetic affinity profiling, the SPOC proteomic platform addresses the challenges of interrogating protein interactions at scale and can be deployed in various research and clinical applications.","version":"1.1","doi":"10.1101/2024.01.23.575909","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.23.563669","pub_date":"2024-1-23","title":"Migraine inhibitor olcegepant reduces weight loss and IL-6 release in SARS-CoV-2 infected older mice with neurological signs","abstract":"COVID-19 can result in neurological symptoms such as fever, headache, dizziness, and nausea. However, neurological signs of SARS-CoV-2 infection have been hardly assessed in mouse models. Here, we infected two commonly used wildtype mice lines (C57BL/6 and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including motion- related dizziness. We then evaluated whether the Calcitonin Gene-Related Peptide (CGRP) receptor antagonist, olcegepant, used in migraine treatment could mitigate acute neuroinflammatory and neurological responses to SARS-COV-2 infection. We infected wildtype C57BL/6J and 129/SvEv mice, and a 129 \u03b1CGRP-null mouse line with a mouse-adapted SARS- CoV-2 virus, and evaluated the effect of CGRP receptor antagonism on the outcome of that infection. First, we determined that CGRP receptor antagonism provided protection from permanent weight loss in older (>12 m) C57BL/6J and 129 SvEv mice. We also observed acute fever and motion-induced dizziness in all older mice, regardless of treatment. However, in both wildtype mouse lines, CGRP antagonism reduced acute interleukin 6 (IL-6) levels by half, with virtually no IL-6 release in mice lacking \u03b1CGRP. These findings suggest that migraine inhibitors such as those blocking CGRP signaling protect against acute IL-6 release and subsequent inflammatory events after SARS-CoV-2 infection, which may have repercussions for related pandemic and/or endemic coronaviruses. COVID-19 can cause neurological symptoms such as fever, headache, dizziness, and nausea. However, such neurological symptoms of SARS-CoV-2 infection have been hardly assessed in mouse models. Here, we first infected two commonly used wildtype mice lines (C57BL/6 and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including motion-related dizziness. Further, we showed that migraine treatment drug olcegepant could reduce long-term weight loss and IL-6 release associated with SARS-CoV-2 infection. These findings suggest that a migraine blocker can be protective for at least some acute SARS-CoV-2 infection signs and raise the possibility that it may also impact long-term outcomes of infection.","version":"1.5","doi":"10.1101/2023.10.23.563669","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.22.576742","pub_date":"2024-1-23","title":"Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination","abstract":"We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ('hybrid immunity'). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure. We highlight one instance where hybrid immunity arising from breakthrough infection resulted in a marked increase in the breadth and affinity of a highly abundant vaccination-elicited plasma IgG antibody, SC27. With an intrinsic binding affinity surpassing a theoretical maximum (KD < 5 pM), SC27 demonstrated potent neutralization of various SARS-CoV-2 variants and SARS-like zoonotic viruses (IC50 \u223c0.1\u20131.75 nM) and provided robust protection in vivo. Cryo-EM structural analysis unveiled that SC27 binds to the RBD class 1/4 epitope, with both VH and VL significantly contributing to the binding interface. These findings suggest that exceptionally broad and potent antibodies can be prevalent in plasma and can largely dictate the nature of serological neutralization. \n\u25aaInfection and vaccination elicit unique IgG antibody profiles at the molecular level\n\u25aaImmunological imprinting varies between infection (S2/NTD) and vaccination (RBD)\n\u25aaHybrid immunity maintains the imprint of first infection or first vaccination\n\u25aaHybrid immune IgG plasma mAbs have superior neutralization potency and breadth\n Infection and vaccination elicit unique IgG antibody profiles at the molecular level Immunological imprinting varies between infection (S2/NTD) and vaccination (RBD) Hybrid immunity maintains the imprint of first infection or first vaccination Hybrid immune IgG plasma mAbs have superior neutralization potency and breadth","version":"1.1","doi":"10.1101/2024.01.22.576742","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.21.563398","pub_date":"2024-1-23","title":"Evaluation of the cross reactivity of neutralising antibody response in vaccinated human and convalescent hamster sera against SARS-CoV-2 variants up to and including JN.1 using an authentic virus neutralisation assay","abstract":"New vaccines, therapeutics and immunity elicited by natural infection create evolutionary pressure on SARS-CoV-2 to evolve and adapt to evade vaccine-induced and infection-elicited immunity. Vaccine and therapeutics developers thus find themselves in an \u201carms race\u201d with the virus. The ongoing assessment of emerging SARS-CoV-2 variants remains essential as the global community transitions from an emergency response to a long-term management plan. Here, we describe how an authentic virus neutralisation assay using low passage clinical virus isolates has been employed to monitor resistance of emerging virus variants to neutralising antibodies from humans and experimentally infected hamsters. Sera and plasma from people who received three doses of a vaccine as well as those who received a bivalent booster were assessed against SARS-CoV-2 variants, up to and including JN.1. Contemporary or recent virus variants showed substantial resistance to neutralisation by antibodies from those who had received three doses of an ancestral vaccine but were still effectively neutralised by antibodies from individuals who had received a bivalent booster (ancestral/BA.1). In our recent studies, however, the JN.1 VOI was found to be significantly more resistant to neutralisation by antibodies from those who had received the ancestral/BA.1 bivalent boost. Convalescent sera from hamsters that had been experimentally infected with one of seven virus variants (ancestral, BA.1, BA.4, BA.5.2.1, XBB.1.5, XBB.1.16, XBB.2.3) were also tested here. The recent contemporary variant, BA.2.86, was effectively neutralised by sera from hamsters infected with XBB.1.5 and XBB.1.16 but it was not neutralised by sera from those infected with BA.5.2.1. These data support the recommendations given by the WHO that a new vaccine was required and should consist of an XBB sub-lineage antigen.","version":"1.2","doi":"10.1101/2023.10.21.563398","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.20.576353","pub_date":"2024-1-23","title":"Temperature-dependent Spike-ACE2 interaction of Omicron subvariants is associated with viral transmission","abstract":"The continued evolution of SARS-CoV-2 requires persistent monitoring of its subvariants. Omicron subvariants are responsible for the vast majority of SARS-CoV-2 infections worldwide, with XBB and BA.2.86 sublineages representing more than 90% of circulating strains as of January 2024. In this study, we characterized the functional properties of Spike glycoproteins from BA.2.75, CH.1.1, DV.7.1, BA.4/5, BQ.1.1, XBB, XBB.1, XBB.1.16, XBB.1.5, FD.1.1, EG.5.1, HK.3 BA.2.86 and JN.1. We tested their capacity to evade plasma-mediated recognition and neutralization, ACE2 binding, their susceptibility to cold inactivation, Spike processing, as well as the impact of temperature on Spike-ACE2 interaction. We found that compared to the early wild-type (D614G) strain, most Omicron subvariants Spike glycoproteins evolved to escape recognition and neutralization by plasma from individuals who received a fifth dose of bivalent (BA.1 or BA.4/5) mRNA vaccine and improve ACE2 binding, particularly at low temperatures. Moreover, BA.2.86 had the best affinity for ACE2 at all temperatures tested. We found that Omicron subvariants Spike processing is associated with their susceptibility to cold inactivation. Intriguingly, we found that Spike-ACE2 binding at low temperature was significantly associated with growth rates of Omicron subvariants in humans. Overall, we report that Spikes from newly emerged Omicron subvariants are relatively more stable and resistant to plasma-mediated neutralization, present improved affinity for ACE2 which is associated, particularly at low temperatures, with their growth rates.","version":"1.1","doi":"10.1101/2024.01.20.576353","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.23.549087","pub_date":"2024-1-22","title":"Biophysical principles predict fitness of SARS-CoV-2 variants","abstract":"SARS-CoV-2 employs its spike protein\u2019s receptor binding domain (RBD) to enter host cells. The RBD is constantly subjected to immune responses, while requiring efficient binding to host cell receptors for successful infection. However, our understanding of how RBD\u2019s biophysical properties contribute to SARS-CoV-2\u2019s epidemiological fitness remains largely incomplete. Through a comprehensive approach, comprising large-scale sequence analysis of SARS-CoV-2 variants and the discovery of a fitness function based on binding thermodynamics, we unravel the relationship between the biophysical properties of RBD variants and their contribution to viral fitness. We developed a biophysical model that uses statistical mechanics to map the molecular phenotype space, characterized by binding constants of RBD to ACE2, LY-CoV016, LY-CoV555, REGN10987, and S309, onto a epistatic fitness landscape. We validate our findings through experimentally measured and machine learning (ML) estimated binding affinities, coupled with infectivity data derived from population-level sequencing. Our analysis reveals that this model effectively predicts the fitness of novel RBD variants and can account for the epistatic interactions among mutations, including explaining the later reversal of Q493R. Our study sheds light on the impact of specific mutations on viral fitness and delivers a tool for predicting the future epidemiological trajectory of previously unseen or emerging low frequency variants. These insights offer not only greater understanding of viral evolution but also potentially aid in guiding public health decisions in the battle against COVID-19 and future pandemics. This research presents a biophysical model that maps the molecular properties of SARS-CoV-2\u2019s receptor binding domain into an epistatic fitness landscape. By linking the binding affinities of the virus to its epidemic fitness, we offer a powerful tool for understanding and predicting the emergence and success of new viral variants. Our model, validated with real-world data and informed by theoretical insights, provides a foundation for interpreting the evolutionary trajectory of past pandemics and predicting those of the future. The adaptability of this biophysical model extends to the key proteins of other viruses as well, signifying its potential in guiding public health interventions, and advancing our understanding of viral evolution.","version":"1.3","doi":"10.1101/2023.07.23.549087","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.21.576083","pub_date":"2024-1-22","title":"Cytoarchitecture of SARS-CoV-2 infected hamster lungs by X-ray phase contrast tomography: imaging workflow and classification for drug testing","abstract":"X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. In this work, we present a workflow implemented at a laboratory \u03bcCT setup, using semi-automated data acquisition, reconstruction and statistical quantification of lung tissue in an early screen of Covid-19 drug candidates. Different drugs were tested in a hamster model after SARS-CoV-2 infection. To make full use of the recorded high-throughput XPCT data, we then used morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing invaluable insights into the pathological progression and partial recovery due to drug treatment.","version":"1.1","doi":"10.1101/2024.01.21.576083","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.15.575706","pub_date":"2024-1-22","title":"Adsorption-driven deformation and landing-footprints of the RBD proteins in SARS-CoV-2 variants onto biological and inanimate surfaces","abstract":"Respiratory viruses, carried through airborne microdroplets, frequently adhere to surfaces, including plastics and metals. However, our understanding of the interactions between viruses and materials remains limited, particularly in scenarios involving polarizable surfaces. Here, we investigate the role of receptor-binding domain (RBD) mutations on the adsorption of SARS-CoV-2 to hydrophobic and hydrophilic surfaces employing molecular simulations. To contextualize our findings, we contrast the interactions on inanimate surfaces with those on native-biological interfaces, specifically the ACE2 receptor. Notably, we identify a twofold increase in structural deformations for the protein\u2019s receptor binding motif onto the inanimate surfaces, indicative of enhanced shock-absorbing mechanisms. Furthermore, the distribution of amino acids (landing-footprints) on the inanimate surface reveals a distinct regional asymmetry relative to the biological interface. In spite of the H-bonds formed at the hydrophilic substrate, the simulations consistently show a higher number of contacts and interfacial area with the hydrophobic surface, with the WT RBD adsorbed more strongly than the delta or omicron RBDs. In contrast, the adsorption of delta and omicron to hydrophilic surfaces was characterized by a distinctive hopping-pattern. The novel shock-absorbing mechanisms identified in the virus adsorption on inanimate surfaces could lead current experimental efforts in the design of virucidal surfaces.","version":"1.2","doi":"10.1101/2024.01.15.575706","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.03.574008","pub_date":"2024-1-21","title":"An infection and pathogenesis mouse model of SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR)","abstract":"SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it an invaluable surrogate model for evaluating the efficacy of drugs and vaccines against SARS-CoV-2.","version":"1.2","doi":"10.1101/2024.01.03.574008","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.18.24301434","pub_date":"2024-01-20","title":"Evaluation of the impact of concentration and extraction methods on the targeted sequencing of human viruses from wastewater","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of most near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by dPCR compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased by using methods that reduce non-target nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.</jats:p>\n                <jats:sec>\n                  <jats:title>Synopsis</jats:title>\n                  <jats:p>Four concentration/extraction methods combined with probe-capture sequencing of human viruses in raw wastewater were compared. Innovaprep ultrafiltration with solids removal had the best performance for human virus detection sensitivity, richness, and recovery of near-complete genomes.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2024.01.18.24301434","journal":"medRxiv","score":null},{"id":"10.1101/2024.01.17.575851","pub_date":"2024-1-19","title":"Deciphering the Molecular Mechanism of Post-Acute Sequelae of COVID-19 through Comorbidity Network Analysis","abstract":"The post-acute sequelae of COVID-19 presents a significant health challenge in the post-pandemic world. Our study aims to analyze longitudinal electronic health records to determine the impact of COVID-19 on disease progression, provide molecular insights into these mechanisms, and identify associated biomarkers. We included 58,710 patients with COVID-19 records from 01/01/2020 to 31/08/2022 and at least one hospital admission before and after the acute phase of COVID-19 (28 days) as the treatment group. A healthy control group of 174,071 individuals was established for comparison using propensity score matching based on pre-existing diseases (before COVID-19). We built a comorbidity network using Pearson correlation coefficient differences between pairs of pre-existing disease and post-infection disease in both groups. Disease-protein mapping and protein-protein interaction network analysis revealed the impact of COVID-19 on disease trajectories through protein interactions in the human body. The disparity in the weight of prevalent disease comorbidity patterns between the treatment and control groups highlights the impact of COVID-19. Certain specific comorbidity patterns show a more pronounced influence by COVID-19. For each comorbidity pattern, overlapping proteins directly associated with pre-existing diseases, post-infection diseases, and COVID-19 help to elucidate the biological mechanism of COVID-19\u2019s impact on each comorbidity pattern. Proteins essential for explaining the biological mechanism can be identified based on their weights. Disease comorbidity associations influenced by COVID-19, as identified through longitudinal electronic health records and disease-protein mapping, can help elucidate the biological mechanisms of COVID-19, discover intervention methods, and decode the molecular basis of comorbidity associations. This analysis can also yield potential biomarkers and corresponding treatments for specific disease patterns. Ethical approval for this study was granted by the Institutional Review Board of the University of Hong Kong/HA HK West Cluster (UW20-556, UW21-149 and UW21-138). We searched PubMed for research articles up to Nov 30, 2022, with no language restrictions, using the terms \u201cPost-Acute Sequelae of COVID-19\u201d OR \u201cPASC\u201d OR \u201cLong COVID\u201d AND \u201ccomorbidity\u201d OR \u201cmultimorbidity\u201d OR \u201cco-morbidity\u201d OR \u201cmulti-morbidity\u201d. We found most related papers focus on the comorbidity or multimorbidity patterns among PASC. Some papers focus on the associations between specific diseases and PASC. However, no study investigated the biological mechanism of PASC from the perspective of comorbidity network. This study investigated the biological mechanism of PASC based on the comorbidity network including the impact of pre-existing diseases (diseases diagnosed within 730 days before COVID-19) on the development of PASC. We classified pairs of pre-existing disease and post-infection disease (new diseases diagnosed in 28 days to 180 days after COVID-19) as comorbidity associations. Through a comparison of the frequency of comorbidity associations in health people group and patients with COVID-19 infection group, we identified comorbidity patterns that are significantly influenced by COVID-19 infection and constructed a comorbidity network comprising of 117 nodes (representing diseases) and 271 edges (representing comorbidity patterns). These comorbidity patterns suggest COVID-19 patients with these pre-existing diseases have higher risk for post-infection diseases. Through the analysis of the Protein-Protein interaction (PPI) network and associations between diseases and proteins, we identified key proteins in the topological distance of each comorbidity pattern and important biological pathways by GO enrichment analysis. These proteins and biological pathways provide insights into the underlying biological mechanism of PASC. The identification of elevated-risk comorbidity patterns associated with COVID-19 infection is crucial for the effective allocation of medical resources, ensuring prompt care for those in greatest need. Furthermore, it facilitates the recovery process of patients from COVID-19, offering a roadmap for their path back to health. The key proteins identified in our study have the potential to serve as biomarkers and targets for therapeutic intervention, thereby establishing a foundation for the development of new drugs and the repurposing of existing ones. Further research should focus on drug discovery and the development of drug recommendations for patients with COVID-19 infections.","version":"1.2","doi":"10.1101/2024.01.17.575851","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.08.503267","pub_date":"2024-1-18","title":"Revealing the drivers of antibiotic resistance trends in Streptococcus pneumoniae amidst the 2020 COVID-19 pandemic: Insights from mathematical modeling","abstract":"Non-pharmaceutical interventions implemented to block SARS-CoV-2 transmission in early 2020 led to global reductions in the incidence of invasive pneumococcal disease (IPD). By contrast, most European countries reported an increase in antibiotic resistance among invasive Streptococcus pneumoniae isolates from 2019 to 2020, while an increasing number of studies reported stable pneumococcal carriage prevalence over the same period. To disentangle the impacts of the COVID-19 pandemic on pneumococcal epidemiology in the community setting, we propose a mathematical model formalizing simultaneous transmission of SARS-CoV-2 and antibiotic-sensitive and -resistant strains of S. pneumoniae. To test hypotheses underlying these trends five mechanisms were built in into the model and examined: (1) a population-wide reduction of antibiotic prescriptions in the community, (2) lockdown effect on pneumococcal transmission, (3) a reduced risk of developing an IPD due to the absence of common respiratory viruses, (4) community azithromycin use in COVID-19 infected individuals, (5) and a longer carriage duration of antibiotic-resistant pneumococcal strains. Among 31 possible pandemic scenarios involving mechanisms individually or in combination, model simulations surprisingly identified only two scenarios that reproduced the reported trends in the general population. They included factors (1), (3), and (4). These scenarios replicated a nearly 50% reduction in annual IPD, and an increase in antibiotic resistance from 20% to 22%, all while maintaining a relatively stable pneumococcal carriage. Exploring further, higher SARS-CoV-2 R0 values and synergistic within-host virus- bacteria interaction mechanisms could have additionally contributed to the observed antibiotic resistance increase. Our work demonstrates the utility of the mathematical modeling approach in unraveling the complex effects of the COVID-19 pandemic responses on AMR dynamics.","version":"1.4","doi":"10.1101/2022.08.08.503267","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.10.574801","pub_date":"2024-1-18","title":"scRNA-seq reveals persistent aberrant differentiation of nasal epithelium driven by TNF\u03b1 and TGF\u03b2 in post-COVID syndrome","abstract":"Post-COVID syndrome (PCS) currently affects approximately 3-17% of people following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has the potential to become a significant global health burden. PCS presents with various symptoms, and methods for improved PCS assessment are presently developed to guide therapy. Nevertheless, there are few mechanistic insights and treatment options. Here, we performed single-cell RNA transcriptomics on nasal biopsies from 33 patients suffering from PCS with mild, moderate, or severe symptoms. We identified 17 different cell clusters representing 12 unique cell populations, including all major epithelial cell types of the conducting airways and basal, secretory, and ciliated cells. Severe PCS was associated with decreased numbers of ciliated cells and the presence of immune cells. Ensuing inflammatory signaling upregulated TGF\u03b2 and induced an epithelial-mesenchymal transition, which led to the high abundance of basal cells and a mis-stratified epithelium. We confirmed the results in vitro using an air-liquid interface culture and validated TNF\u03b1 as the causal inflammatory cytokine. In summary, our results show that one mechanism for sustained PCS is not through continued viral load, but through the presence of immune cells in nasal tissue leading to impaired mucosal barrier function and repeated infections. These findings could be further explored as a therapeutic option akin to other chronic inflammatory diseases by inhibiting the TNF\u03b1-TGF\u03b2 axis, restoring the nasal epithelium, and reducing respiratory tract-related infections.","version":"1.2","doi":"10.1101/2024.01.10.574801","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520156","pub_date":"2024-1-18","title":"Phase 1 of the National Institutes of Health Preprint Pilot: Testing the viability of making preprints discoverable in PubMed Central and PubMed","abstract":"The National Library of Medicine (NLM) launched a Pilot in June 2020 to: 1) explore the feasibility and utility of adding preprints to PubMed Central (PMC) and making them discoverable in PubMed, and 2) to support accelerated discoverability of National Institutes of Health (NIH)-supported research without compromising user trust in NLM\u2019s widely used literature services. The first phase of the Pilot focused on archiving preprints reporting NIH-supported SARS-CoV-2 virus and COVID-19 research. To launch Phase 1, NLM identified eligible preprint servers and developed processes for identifying NIH-supported preprints within scope in these servers. Processes were also developed for the ingest and conversion of preprints in PMC and to send corresponding records to PubMed. User interfaces were modified for display of preprint records. NLM collected data on the preprints ingested and discovery of preprint records in PMC and PubMed and engaged users through focus groups and a survey to obtain direct feedback on the Pilot and perceptions of preprints. Between June 2020 and June 2022, NLM added more than 3,300 preprint records to PMC (viewed 4 million times) and PubMed (viewed 3 million times) Nearly one-quarter of preprints in the Pilot were not associated with a peer-reviewed published journal article. User feedback revealed that the inclusion of preprints did not have a notable impact on trust in PMC or PubMed. NIH-supported preprints can be identified and added to PMC and PubMed without disrupting existing operations processes. Additionally, inclusion of preprints in PMC and PubMed accelerates discovery of NIH research without reducing trust in NLM literature services. Phase 1 of the Pilot provided a useful testbed for studying NIH investigator preprint posting practices, as well as knowledge gaps among user groups, during the COVID-19 public health emergency, an unusual time with heightened interest in immediate access to research results.","version":"1.2","doi":"10.1101/2022.12.12.520156","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.14.24301293","pub_date":"2024-01-17","title":"Blood transcriptomics reveal persistent SARS-CoV-2 RNA and candidate biomarkers in Long COVID patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  With an estimated 65 million individuals suffering from Long COVID, validated therapeutic strategies as well as non-invasive biomarkers are direly needed to guide clinical management. We used blood digital transcriptomics in search of viral persistence and Long COVID diagnostic biomarkers in a real-world, general practice-based setting with a long clinical follow-up. We demonstrate systemic SARS-CoV-2 persistence for more than 2 years after acute COVID-19 infection. A 2-gene biomarker, including\n                  <jats:italic>FYN</jats:italic>\n                  and SARS-CoV-2 antisense RNA, correctly classifies Long COVID with 93.8% sensitivity and 91.7% specificity. Specific immune transcripts and immunometabolism score correlate to systemic viral load and patient-reported anxiety/depression, providing mechanistic links as well as therapeutic targets to tackle Long COVID.\n                </jats:p>","version":null,"doi":"10.1101/2024.01.14.24301293","journal":"medRxiv","score":null},{"id":"10.1101/2024.01.16.575813","pub_date":"2024-1-17","title":"Discrete and conserved inflammatory signatures drive thrombosis in different organs after Salmonella infection","abstract":"Inflammation-induced thrombosis is a common consequence of bacterial and viral infections, such as those caused by Salmonella Typhimurium (STm) and SARS-CoV-2. The identification of multi-organ thrombosis and the chronological differences in its induction and resolution raises significant challenges for successfully targeting multi-organ infection-associated thrombosis. Here, we identified specific pathways and effector cells driving thrombosis in the spleen and liver following STm infection. Thrombosis in the spleen is independent of IFN-\u03b3 or the platelet C-type lectin-like receptor CLEC-2, while both molecules were previously identified as key drivers of thrombosis in the liver. Furthermore, we identified platelets, monocytes, and neutrophils as core constituents of thrombi in both organs. Depleting neutrophils or monocytic cells independently abrogated thrombus formation. Nevertheless, blocking TNF\u03b1, which is expressed by both myeloid cell types, diminished both thrombosis and inflammation which correlates with reduced endothelial expression of E-selectin and leukocyte infiltration. Moreover, tissue factor and P-selectin glycoprotein ligand 1 inhibition impair thrombosis in both spleen and liver, identifying multiple common checkpoints to target multi-organ thrombosis. Therefore, organ-specific, and broad mechanisms driving thrombosis potentially allow tailored treatments based on the clinical need and to define the most adequate strategy to target both thrombosis and inflammation associated with systemic infections.","version":"1.1","doi":"10.1101/2024.01.16.575813","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.21.517390","pub_date":"2024-1-16","title":"Lineage frequency time series reveal elevated levels of genetic drift in SARS-CoV-2 transmission in England","abstract":"Genetic drift in infectious disease transmission results from randomness of transmission and host recovery or death. The strength of genetic drift for SARS-CoV-2 transmission is expected to be high due to high levels of superspreading, and this is expected to substantially impact disease epidemiology and evolution. However, we don\u2019t yet have an understanding of how genetic drift changes over time or across locations. Furthermore, noise that results from data collection can potentially confound estimates of genetic drift. To address this challenge, we develop and validate a method to jointly infer genetic drift and measurement noise from time-series lineage frequency data. Our method is highly scalable to increasingly large genomic datasets, which overcomes a limitation in commonly used phylogenetic methods. We apply this method to over 490,000 SARS-CoV-2 genomic sequences from England collected between March 2020 and December 2021 by the COVID-19 Genomics UK (COG-UK) consortium and separately infer the strength of genetic drift for pre-B.1.177, B.1.177, Alpha, and Delta. We find that even after correcting for measurement noise, the strength of genetic drift is consistently, throughout time, higher than that expected from the observed number of COVID-19 positive individuals in England by 1 to 3 orders of magnitude, which cannot be explained by literature values of superspreading. Our estimates of genetic drift will be informative for parameterizing evolutionary models and studying potential mechanisms for increased drift. The transmission of pathogens like SARS-CoV-2 is strongly affected by chance effects in the contact process between infected and susceptible individuals, collectively referred to as random genetic drift. We have an incomplete understanding of how genetic drift changes across time and locations. To address this gap, we developed a computational method that infers the strength of genetic drift from time series genomic data that corrects for non-biological noise and is computationally scalable to the large numbers of sequences available for SARS-CoV-2, overcoming a major challenge of existing methods. Using this method, we quantified the strength of genetic drift for SARS-CoV-2 transmission in England throughout time and across locations. These estimates constrain potential mechanisms and help parameterize models of SARS-CoV-2 evolution. More generally, the computational scalability of our method will become more important as increasingly large genomic datasets become more common.","version":"1.3","doi":"10.1101/2022.11.21.517390","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.28.555008","pub_date":"2024-1-16","title":"Reference materials for SARS-CoV-2 molecular diagnostic quality control: validation of encapsulated synthetic RNAs for room temperature storage and shipping","abstract":"The Coronavirus pandemic unveiled the unprecedented need for diagnostic tests to rapidly detect the presence of pathogens in the population. Real-time RT-PCR and other nucleic acid amplification techniques are accurate and sensitive molecular techniques that necessitate positive controls. To meet this need, Twist Bioscience has developed and released synthetic RNA controls. However, RNA is an inherently unstable molecule needing cold storage, costly shipping, and resource-intensive logistics. Imagene provides a solution to this problem by encapsulating dehydrated RNA inside metallic capsules filled with anhydrous argon, allowing room temperature and eco-friendly storage and shipping. Here, RNA controls produced by Twist were encapsulated (RNAshells) and distributed to several laboratories that used them for COVID-19 detection tests by amplification. One RT-LAMP procedure, four different RT-PCR devices and 6 different PCR kits were used. The amplification targets were genes E, N; RdRp, Sarbeco-E and Orf1a/b. RNA retrieval was satisfactory, and the detection was reproducible. RNA stability was checked by accelerated aging. The results for a 10-year equivalent storage time at 25 \u00b0C were not significantly different from those for unaged samples. This room temperature RNA stability allows the preparation and distribution of large strategic batches which can be stored for a long time and used for standardization processes between detection sites. Moreover, it makes it also possible to use these controls for single use and in the field where large temperature differences can occur. Consequently, this type of encapsulated RNA controls, processed at room temperature, can be used as reference materials for the SARS-Cov-2 virus as well as for other pathogens detection.","version":"1.3","doi":"10.1101/2023.08.28.555008","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.15.575741","pub_date":"2024-1-16","title":"Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody","abstract":"Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.","version":"1.1","doi":"10.1101/2024.01.15.575741","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.14.575588","pub_date":"2024-1-15","title":"Intestinal helminth infection impairs vaccine-induced T cell responses and protection against SARS-CoV-2","abstract":"Although vaccines have reduced COVID-19 disease burden, their efficacy in helminth infection endemic areas is not well characterized. We evaluated the impact of infection by Heligmosomoides polygyrus bakeri (Hpb), a murine intestinal hookworm, on the efficacy of an mRNA vaccine targeting the Wuhan-1 spike protein of SARS-CoV-2. Although immunization generated similar B cell responses in Hpb-infected and uninfected mice, polyfunctional CD4+ and CD8+ T cell responses were markedly reduced in Hpb-infected mice. Hpb-infected and mRNA vaccinated mice were protected against the ancestral SARS-CoV-2 strain WA1/2020, but control of lung infection was diminished against an Omicron variant compared to animals immunized without Hpb infection. Helminth mediated suppression of spike-specific CD8+ T cell responses occurred independently of STAT6 signaling, whereas blockade of IL-10 rescued vaccine-induced CD8+ T cell responses. In mice, intestinal helminth infection impairs vaccine induced T cell responses via an IL-10 pathway and compromises protection against antigenically shifted SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.01.14.575588","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.14.575569","pub_date":"2024-1-15","title":"Different vaccine platforms result in distinct antibody responses to the same antigen in haemodialysis patients","abstract":"Generalised immune dysfunction in chronic kidney disease, especially in patients requiring haemodialysis (HD), significantly enhances the risk of severe infections. Moreover, vaccine-induced immunity is typically reduced in HD populations, but the full mechanisms behind this remain unclear. The SARS-CoV-2 pandemic provided an opportunity to examine the magnitude and functionality of antibody responses in HD patients to a previously unencountered antigen, Spike (S)-glycoprotein, after vaccination with different vaccine platforms (viral vector (VV); mRNA (mRV)). Here, we compared total and functional anti-S antibody responses (cross-variant neutralisation and complement binding) in 187 HD patients and 43 healthy controls 21-28 days after serial immunisation. After 2 doses of the same vaccine, HD patients had anti-S antibody levels and complement binding capacity comparable to controls. However, 2 doses of mRV induced greater polyfunctional antibody responses than VV, yet previous SARS-CoV-2 infection or an mRV boost after 2 doses of VV significantly enhanced antibody functionality in HD patients. Therefore, HD patients can generate near-normal, functional antigen-specific antibody responses following serial vaccination to a novel antigen, suggesting largely intact B cell memory. Encouragingly, exploiting immunological memory by using mRNA vaccines and boosting may improve the success of vaccination strategies in this vulnerable patient population.","version":"1.1","doi":"10.1101/2024.01.14.575569","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.03.560722","pub_date":"2024-1-13","title":"Drug Discovery in Low Data Regimes: Leveraging a Computational Pipeline for the Discovery of Novel SARS-CoV-2 Nsp14-MTase Inhibitors","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 \u00b1 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 \u00b1 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.","version":"1.3","doi":"10.1101/2023.10.03.560722","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.21.558814","pub_date":"2024-1-13","title":"Intestinal microbiota programming of alveolar macrophages influences severity of respiratory viral infection","abstract":"Susceptibility to respiratory virus infections (RVIs) varies widely across individuals. Because the gut microbiome impacts immune function, we investigated the influence of intestinal microbiota composition on RVI and determined that segmented filamentous bacteria (SFB), naturally acquired or exogenously administered, protected mice against influenza virus (IAV) infection. Such protection, which also applied to respiratory syncytial virus and SARS-CoV-2, was independent of interferon and adaptive immunity but required basally resident alveolar macrophages (AM). In SFB-negative mice, AM were quickly depleted as RVI progressed. In contrast, AM from SFB-colonized mice were intrinsically altered to resist IAV-induced depletion and inflammatory signaling. Yet, AM from SFB-colonized mice were not quiescent. Rather, they directly disabled IAV via enhanced complement production and phagocytosis. Accordingly, transfer of SFB-transformed AM into SFB-free hosts recapitulated SFB-mediated protection against IAV. These findings uncover complex interactions that mechanistically link the intestinal microbiota with AM functionality and RVI severity. Intestinal segmented filamentous bacteria reprogram alveolar macrophages promoting nonphlogistic defense against respiratory viruses.","version":"1.2","doi":"10.1101/2023.09.21.558814","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.527147","pub_date":"2024-1-12","title":"Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease","abstract":"Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2023.02.10.527147","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.11.574849","pub_date":"2024-1-12","title":"Neural Network-Assisted Humanization of COVID-19 Hamster scRNAseq Data Reveals Matching Severity States in Human Disease","abstract":"Translating findings from animal models to human disease is essential for dissecting disease mechanisms, developing and testing precise therapeutic strategies. The coronavirus disease 2019 (COVID-19) pandemic has highlighted this need, particularly for models showing disease severity-dependent immune responses. Single-cell transcriptomics (scRNAseq) is well poised to reveal similarities and differences between species at the molecular and cellular level with unprecedented resolution. However, computational methods enabling detailed matching are still scarce. Here, we provide a structured scRNAseq-based approach that we applied to scRNAseq from blood leukocytes originating from humans and hamsters affected with moderate or severe COVID-19. Integration of COVID-19 patient data with two hamster models that develop moderate (Syrian hamster, Mesocricetus auratus) or severe (Roborovski hamster, Phodopus roborovskii) disease revealed that most cellular states are shared across species. A neural network-based analysis using variational autoencoders quantified the overall transcriptomic similarity across species and severity levels, showing highest similarity between neutrophils of Roborovski hamsters and severe COVID-19 patients, while Syrian hamsters better matched patients with moderate disease, particularly in classical monocytes. We further used transcriptome-wide differential expression analysis to identify which disease stages and cell types display strongest transcriptional changes. Consistently, hamster\u2019s response to COVID-19 was most similar to humans in monocytes and neutrophils. Disease-linked pathways found in all species specifically related to interferon response or inhibition of viral replication. Analysis of candidate genes and signatures supported the results. Our structured neural network-supported workflow could be applied to other diseases, allowing better identification of suitable animal models with similar pathomechanisms across species. Neural networks can successfully match disease states between animal models and humans using single-cell data as shown for COVID-19 Moderately diseased patients best matched Syrian hamster cells; severely diseased patients best matched Roborovski hamster neutrophils","version":"1.1","doi":"10.1101/2024.01.11.574849","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.10.575114","pub_date":"2024-1-11","title":"SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain","abstract":"SARS-CoV-2, the virus responsible for COVID-19, triggers symptoms such as sneezing, aches and pain. These symptoms are mediated by a subset of sensory neurons, known as nociceptors, that detect noxious stimuli, densely innervate the airway epithelium, and interact with airway resident epithelial and immune cells. However, the mechanisms by which viral infection activates these neurons to trigger pain and airway reflexes are unknown. Here, we show that the coronavirus papain-like protease (PLpro) directly activates airway-innervating trigeminal and vagal nociceptors in mice and human iPSC-derived nociceptors. PLpro elicits sneezing and acute pain in mice and triggers the release of neuropeptide calcitonin gene-related peptide (CGRP) from airway afferents. We find that PLpro-induced sneeze and pain requires the host TRPA1 ion channel that has been previously demonstrated to mediate pain, cough, and airway inflammation. Our findings are the first demonstration of a viral product that directly activates sensory neurons to trigger pain and airway reflexes and highlight a new role for PLpro and nociceptors in COVID-19.","version":"1.1","doi":"10.1101/2024.01.10.575114","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.11.575161","pub_date":"2024-1-11","title":"APNet, an explainable sparse deep learning model to discover differentially active drivers of severe COVID-19","abstract":"Computational analyses of plasma proteomics provide translational insights into complex diseases such as COVID-19 by revealing molecules, cellular phenotypes, and signaling patterns that contribute to unfavorable clinical outcomes. Current in silico approaches dovetail differential expression, biostatistics, and machine learning, but often overlook nonlinear proteomic dynamics, like post-translational modifications, and provide limited biological interpretability beyond feature ranking. We introduce APNet, a novel computational pipeline that combines differential activity analysis based on SJARACNe co-expression networks with PASNet, a biologically-informed sparse deep learning model to perform explainable predictions for COVID-19 severity. The APNet driver-pathway network ingests co-expression and classification weights to aid result interpretation and hypothesis generation. APNet outperforms alternative models in patient classification across three COVID-19 proteomic datasets, identifying predictive drivers and pathways, including some confirmed in single-cell omics and highlighting under-explored biomarker circuitries in COVID-19. APNet\u2019s R, Python scripts and Cytoscape methodologies are available at https://github.com/BiodataAnalysisGroup/APNet ggeorav@certh.gr Supplementary information can be accessed in Zenodo (10.5281/zenodo.10438830).","version":"1.1","doi":"10.1101/2024.01.11.575161","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.568013","pub_date":"2024-1-10","title":"Generation and evaluation of protease inhibitor-resistant SARS-CoV-2 strains","abstract":"Since the start of the SARS-CoV-2 pandemic, the search for antiviral therapies has been at the forefront of medical research. To date, the 3CLpro inhibitor nirmatrelvir (Paxlovid\u00ae) has shown the best results in clinical trials and the greatest robustness against variants. A second SARS-CoV-2 protease inhibitor, ensitrelvir (Xocova\u00ae), has been developed. Ensitrelvir, currently in Phase 3, was approved in Japan under the emergency regulatory approval procedure in November 2022, and is available since March 31, 2023. One of the limitations for the use of antiviral monotherapies is the emergence of resistance mutations. Here, we experimentally generated mutants resistant to nirmatrelvir and ensitrelvir in vitro following repeating passages of SARS-CoV-2 in the presence of both antivirals. For both molecules, we demonstrated a loss of sensitivity for resistance mutants in vitro. Using a Syrian golden hamster infection model, we showed that the ensitrelvir M49L mutation, in the multi-passage strain, confers a high level of in vivo resistance. Finally, we identified a recent increase in the prevalence of M49L-carrying sequences, which appears to be associated with multiple repeated emergence events in Japan and may be related to the use of Xocova\u00ae in the country since November 2022. These results highlight the strategic importance of genetic monitoring of circulating SARS-CoV-2 strains to ensure that treatments administered retain their full effectiveness.","version":"1.2","doi":"10.1101/2023.11.22.568013","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.09.24301069","pub_date":"2024-01-10","title":"Protection afforded by post-infection SARS-CoV-2 vaccine doses: a cohort study in Shanghai","abstract":"<jats:p>\n                  <jats:bold>ABSTRACT</jats:bold>\n                </jats:p>\n                <jats:p>\n                  <jats:bold>\n                    <jats:italic>Background</jats:italic>\n                  </jats:bold>\n                  <jats:italic>:</jats:italic>\n                  In many settings, a large fraction of the population has both been vaccinated against and infected by SARS-CoV-2. Hence, quantifying the protection provided by post-infection vaccination has become critical for policy. We aimed to estimate the protective effect against SARS-CoV-2 reinfection of an additional vaccine dose after an initial Omicron variant infection.\n                </jats:p>\n                <jats:p>\n                  <jats:bold>\n                    <jats:italic>Methods</jats:italic>\n                  </jats:bold>\n                  <jats:italic>:</jats:italic>\n                  We report a retrospective, population-based cohort study performed in Shanghai, China, using electronic databases with information on SARS-CoV-2 infections and vaccination history. We compared reinfection incidence by post-infection vaccination status in individuals initially infected during the April-May 2022 Omicron variant surge in Shanghai and who had been vaccinated before that period. Cox models were fit to estimate adjusted hazard ratios (aHR).\n                </jats:p>\n                <jats:p>\n                  <jats:bold>\n                    <jats:italic>Results</jats:italic>\n                  </jats:bold>\n                  <jats:italic>:</jats:italic>\n                  275,896 individuals were diagnosed with RT-PCR-confirmed SARS-CoV-2 infection in April-May 2022; 199,312/275,896 were included in analyses on the effect of a post-infection vaccine dose. Post-infection vaccination provided protection against reinfection (aHR 0.82; 95% CI 0.79-0.85). For patients who had received one, two or three vaccine doses before their first infection, hazard ratios for the post-infection vaccination effect were 0.84 (0.76-0.93), 0.87 (0.83-0.90) and 0.96 (0.74-1.23), respectively. Vaccination within 30 and 90 days before the second Omicron wave provided different degrees of protection (in aHR): 0.51 (0.44-0.58), and 0.67 (0.61-0.74), respectively. Moreover, for all vaccine types, but to different extents, a post-infection dose given to individuals who were fully vaccinated before first infection was protective.\n                </jats:p>\n                <jats:p>\n                  <jats:bold>\n                    <jats:italic>Conclusions:</jats:italic>\n                  </jats:bold>\n                  In previously vaccinated and infected individuals, an additional vaccine dose provided protection against Omicron variant reinfection. These observations will inform future policy decisions on COVID-19 vaccination in China and other countries.\n                </jats:p>","version":null,"doi":"10.1101/2024.01.09.24301069","journal":"medRxiv","score":null},{"id":"10.1101/2024.01.10.574981","pub_date":"2024-1-10","title":"Variant-specific interactions at the plasma membrane: Heparan sulfate\u2019s impact on SARS-CoV-2 binding kinetics","abstract":"The worldwide spread of SARS-CoV-2 has been characterised by the emergence of several variants of concern (VOCs) presenting an increasing number of mutations in the viral genome. The spike glycoprotein, responsible for engaging the viral receptor ACE2, exhibits the highest density of mutations, suggesting an ongoing evolution to optimize viral entry. However, previous studies focussed on isolated molecular interactions, neglecting the intricate composition of the plasma membrane and the interplay between viral attachment factors. Our study explores the role of avidity and of the complexity of the plasma membrane composition in modulating the virus-host binding kinetics during the early stages of viral entry for the original Wuhan strain and three VOCs: Omicron BA.1, Delta, and Alpha. We employ fluorescent liposomes decorated with spike from several VOCs as virion mimics in single-particle tracking studies on native supported lipid bilayers derived from pulmonary Calu-3 cells. Our findings reveal an increase in the affinity of the multivalent bond to the cell surface for Omicron driven by an increased association rate. We show that heparan sulfate (HS), a sulfated glycosaminoglycan commonly expressed on cells\u2019 plasma membrane, plays a central role in modulating the interaction with the cell surface and we observe a shift in its role from screening the interaction with ACE2 in early VOCs to an important binding factor for Omicron. This is caused by a \u223c10-fold increase in Omicron\u2019s affinity to HS compared to the original Wuhan strain, as shown using atomic force microscopy-based single-molecule force spectroscopy. Our results show the importance of coreceptors, particularly HS, and membrane complexity in the modulation of the attachment in SARS-CoV-2 VOCs. We highlight a transition in the variants\u2019 attachment strategy towards the use of HS as an initial docking site, which likely plays a role in shaping Omicron\u2019s tropism towards infection of the upper airways, milder symptoms, and higher transmissibility.","version":"1.1","doi":"10.1101/2024.01.10.574981","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.09.574819","pub_date":"2024-1-10","title":"Measuring concentration of nanoparticles in polydisperse mixtures using interferometric nanoparticle tracking analysis (iNTA)","abstract":"Quantitative measurements of nanoparticle concentration in liquid suspensions are in high demand, for example, in the medical and food industries. Conventional methods remain unsatisfactory, especially for polydisperse samples with overlapping size ranges. Recently, we introduced interferometric nanoparticle tracking analysis (iNTA) as a new method for high-precision measurement of nanoparticle size and refractive index. Here, we show that by counting the number of trajectories that cross the focal plane, iNTA can measure concentrations of subpopulations in a polydisperse mixture in a quantitative manner and without the need for a calibration sample. We evaluate our method on both monodisperse samples and mixtures of known concentrations. Furthermore, we assess the concentration of SARS-CoV-2 in supernatant samples obtained from infected cells.","version":"1.1","doi":"10.1101/2024.01.09.574819","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.08.574642","pub_date":"2024-1-09","title":"Preferential apical infection of intestinal cell monolayers by SARS-CoV-2 is associated with damage to cellular barrier integrity: Implications for the physiopathology of COVID-19","abstract":"SARS-CoV-2 can infect different organs, including the intestine. In Caco-2 intestinal cell line, SARS-CoV-2 modulates the ACE2 receptor expression and affects the expression of molecules involved in intercellular junctions. To further explore the possibility that the intestinal epithelium serves as an alternative infection route for SARS-CoV-2, we used a model of polarised intestinal cell monolayers grown on the polycarbonate membrane of Transwell inserts, inoculated with the virus either in the upper or lower chamber of culture. In both polarised Caco-2 cell monolayers and co-culture Caco-2/HT29 cell monolayer, apical SARS-CoV-2 inoculation was found to be much more effective in establishing infection than basolateral inoculation. In addition, apical SARS-CoV-2 infection triggers monolayer degeneration, as shown by histological examination, measurement of trans-epithelial electronic resistance, and cell adhesion molecule expression. During this process, the infectious viruses reach the lower chamber, suggesting either a transcytosis mechanism from the apical side to the basolateral side of cells, a paracellular trafficking of the virus after damage to intercellular junctions in the epithelial barrier, or both. Taken together, these data highlight a preferential tropism of SARS-CoV-2 for the apical side of the human intestinal tract and suggests that infection via the intestinal lumen leads to a systemic infection.","version":"1.1","doi":"10.1101/2024.01.08.574642","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.08.574531","pub_date":"2024-1-09","title":"Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli","abstract":"The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.","version":"1.1","doi":"10.1101/2024.01.08.574531","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.06.574466","pub_date":"2024-1-08","title":"The kinetics of SARS-CoV-2 nsp7-11 polyprotein processing and impact on complexation with nsp16","abstract":"In severe-acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, polyproteins (pp1a/pp1ab) are processed into non-structural proteins (nsps), which largely form the replication/transcription complex (RTC). The polyprotein processing and complex formation is critical and offers potential therapeutic targets. However, the interplay of polyprotein processing and RTC-assembly are poorly understood. Here, we studied two key aspects: The influence of the pp1a terminal nsp11 on the order of polyprotein processing by viral main protease Mpro and the influence of polyprotein processing on core enzyme complex formation. We established a method based on native MS to determine rate constants k considering the structural environment. This enabled us to quantify the multi-reaction kinetics of coronavirus polyprotein processing for the first time. Our results serve as a blueprint for other multi-cleavage reactions. Further, it offers a detailed and quantifiable perspective to the dynamic reactions of SARS-CoV-2 polyprotein processing, which is required for development of novel antivirals.","version":"1.1","doi":"10.1101/2024.01.06.574466","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.05.574280","pub_date":"2024-1-08","title":"Innate Immune Activation and Mitochondrial ROS Invoke Persistent Cardiac Conduction System Dysfunction after COVID-19","abstract":"Cardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. Radiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. COVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. The findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.","version":"1.1","doi":"10.1101/2024.01.05.574280","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.06.574128","pub_date":"2024-1-08","title":"GENOMIC PROFILING OF SARS-COV-2 STRAINS CIRCULATING IN SOUTH EASTERN REGION OF INDIA DURING THREE WAVES OF PANDEMIC","abstract":"Continuous bio-surveillance of SARS-CoV-2 is an ongoing task at local, national and global levels since the pandemic onset for understanding genetic evolution and vaccine efficacy. Present study was designed to track the emergence of new variants along the duration of three peaks of infection in the city of Puducherry, India. A total of 128 samples were subjected to Illumina deep RNA sequencing. The results showed predominance of uncommon, delta and omicron variants in first, second and third waves respectively. The most common pangolin lineage was B.1.560 and B.1.617.2. The study observed a total of 3133 common and 11 new mutations. The most common is in the Spike_D614G. A new set of mutations was observed in key viral factors such as NS16 that are implicated to be involved in immune evasion. This may have impact on enhanced disease virulence, vaccine efficiency and possible tolerance to current antivirals. This warrants further in vitro studies to understand the significance of the mutations. While the results presented would also augment the ongoing research on evolutionary and the genetic epidemiology of SARS-CoV-2, it also emphasizes the need for continuous genetic monitoring to predict the forthcoming threats due to the emergence of new or existing variants.","version":"1.1","doi":"10.1101/2024.01.06.574128","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.05.574431","pub_date":"2024-1-08","title":"Towards Real-Time Airborne Pathogen Sensing: Electrostatic Capture and On-Chip LAMP Based Detection of Airborne Viral Pathogens","abstract":"Considerable loss of life, economic slowdown, and public health risk associated with the transmission of airborne respiratory pathogens was underscored by the recent COVID-19 pandemic. Airborne transmission of zoonotic diseases such as the highly pathogenic avian influenza (HPAI) and porcine reproductive and respiratory syndrome virus (PRRSV) has caused major disruptions to domestic and global food security. Current ambient air pathogen monitoring systems involves the collection of air samples from indoor settings suspected of viral contamination, followed by subsequent processing of capture samples to determine the presence and species of airborne viral matter. Nucleic acid amplification techniques are considered the gold standard for pathogen diagnostics. Currently, the necessary extraction and purification of viral RNA from air collector systems prior to sample analysis is both time consuming and performed manually. A monitoring system with separate air sampling and biochemical detection procedures is prone to delay the response to emergent viral threats. In this paper, we present a pathogen monitoring system that overcomes these limitations related to extraction and purification of viral samples and lays the groundwork for a real-time monitor for airborne viral pathogens. We demonstrate a high flow electrostatic precipitator system, that uses small collection wells as counter electrodes for pathogen collection. Integrated reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is used for detection of captured viral matter within wells. On-chip heating of collection wells is enabled by integrated planar heaters and small volumes of reagent (30 \u03bcL) directly to the collection wells. We present the design of such a system and show experimental results that demonstrate the use of this device for detection of aerosolized SARS-CoV-2 virus like particles (VLPs), a model pathogen for SARV-CoV-2.","version":"1.1","doi":"10.1101/2024.01.05.574431","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.08.574715","pub_date":"2024-1-08","title":"Systemic Dosing of Virus-derived Serpin Improves Survival and Immunothrombotic Damage in Murine Colitis","abstract":"Inflammatory bowel disease (IBD) is potentially life-threatening, with risk of bleeding, clotting, infection, sepsis, cancer and toxic megacolon. Systemic and local immune and coagulation dysfunction increase IBD severity. Current treatments are partially effective, but there is no definitive cure. Serine protease cascades activate thrombotic, thrombolytic and complement pathways and are regulated by inhibitors, serpins. Viruses encode proteins evolved from endogenous central regulatory pathways. A purified secreted Myxomavirus-derived serpin, Serp-1, dosed as a systemic anti-inflammatory drug, has proven efficacy in vascular and inflammatory disorders. PEGylated Serp-1 protein (PEGSerp-1) has improved efficacy in lupus and SARS-CoV-2 models. We examined PEGSerp-1 treatment in a mouse Dextran Sodium Sulfate (DSS) colitis model. Prophylactic PEGSerp-1 significantly improved survival in acute severe 4-5% DSS colitis, reducing inflammation and crypt damage in acute 4-5% DSS induced colitis and when dosed as a chronic delayed treatment for recurrent 2% DSS colitis. PEGSerp-1 reduced iNOS+ M1 macrophage invasion, damage to crypt architecture and vascular inflammation with decreased uPAR, fXa, fibrinogen and complement activation. This work supports PEGSerp-1 as a tissue targeting serpin therapeutic.","version":"1.1","doi":"10.1101/2024.01.08.574715","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.05.574360","pub_date":"2024-1-06","title":"CGRP inhibits SARS-CoV-2 infection of bronchial epithelial cells and its pulmonary levels correlate with viral clearance in critical COVID-19 patients","abstract":"Upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), patients with critical coronavirus disease 2019 (COVID-19) present with life-threatening respiratory distress, pulmonary damage and cytokine storm. One unexplored hub in COVID-19 is the neuropeptide calcitonin gene-related peptide (CGRP), which is highly abundant in the airways and could converge in multiple aspects of COVID-19-related pulmonary pathophysiology. Whether CGRP affects SARS-CoV-2 infection directly remains elusive. We show that in critical COVID-19 patients, CGRP is increased in both plasma and lungs. Importantly, CGRP pulmonary levels are elevated in early SARS-CoV-2-positive patients, and restore to baseline upon subsequent viral clearance in SARS-CoV-2-negative patients. We further show that CGRP and its stable analogue SAX directly inhibit infection of bronchial Calu-3 epithelial cells with SARS-CoV-2 Omicron and Alpha variants in a dose-dependent manner. Both pre- and post-infection treatment with GRRP and/or SAX is enough to block SARS-CoV-2 productive infection of Calu3 cells. CGRP-mediated inhibition occurs via activation of the CGRP receptor and involves down-regulation of SARS-CoV-2 entry receptors at the surface of Calu-3 cells. Together, we propose that increased pulmonary CGRP mediates beneficial viral clearance in critical COVID-19 patients, by directly inhibiting SARS-CoV-2 infection. Hence, CGRP-based interventions could be harnessed for management of COVID-19. Pulmonary levels of the neuropeptide CGRP are increased in critical COVID-19 patients, and could clear virus by directly inhibiting SRAS-CoV-2 infection of bronchial epithelia cells.","version":"1.1","doi":"10.1101/2024.01.05.574360","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.05.574420","pub_date":"2024-1-06","title":"Rapid Emergence and Evolution of SARS-CoV-2 Variants in Advanced HIV Infection","abstract":"Previous studies have linked the evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic variants to persistent infections in people with immunocompromising conditions, but the evolutionary processes underlying these observations are incompletely understood. Here we used high-throughput, single-genome amplification and sequencing (HT-SGS) to obtain up to \u223c103 SARS-CoV-2 spike gene sequences in each of 184 respiratory samples from 22 people with HIV (PWH) and 25 people without HIV (PWOH). Twelve of 22 PWH had advanced HIV infection, defined by peripheral blood CD4 T cell counts (i.e., CD4 counts) <200 cells/\u03bcL. In PWOH and PWH with CD4 counts \u2265200 cells/\u03bcL, most single-genome spike sequences in each person matched one haplotype that predominated throughout the infection. By contrast, people with advanced HIV showed elevated intra-host spike diversity with a median of 46 haplotypes per person (IQR 14-114). Higher intra-host spike diversity immediately after COVID-19 symptom onset predicted longer SARS-CoV-2 RNA shedding among PWH, and intra-host spike diversity at this timepoint was significantly higher in people with advanced HIV than in PWOH. Composition of spike sequence populations in people with advanced HIV fluctuated rapidly over time, with founder sequences often replaced by groups of new haplotypes. These population-level changes were associated with a high total burden of intra-host mutations and positive selection at functionally important residues. In several cases, delayed emergence of detectable serum binding to spike was associated with positive selection for presumptive antibody-escape mutations. Taken together, our findings show remarkable intra-host genetic diversity of SARS-CoV-2 in advanced HIV infection and suggest that adaptive intra-host SARS-CoV-2 evolution in this setting may contribute to the emergence of new variants of concern (VOCs).","version":"1.1","doi":"10.1101/2024.01.05.574420","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.04.574272","pub_date":"2024-1-05","title":"Antigen-display exosomes provide adjuvant-free protection against SARS-CoV-2 disease at nanogram levels of spike protein","abstract":"As the only bionormal nanovesicle, exosomes have high potential as a nanovesicle for delivering vaccines and therapeutics. We show here that the loading of type-1 membrane proteins into the exosome membrane is induced by exosome membrane anchor domains, EMADs, that maximize protein delivery to the plasma membrane, minimize protein sorting to other compartments, and direct proteins into exosome membranes. Using SARS-CoV-2 spike as an example and EMAD13 as our most effective exosome membrane anchor, we show that cells expressing a spike-EMAD13 fusion protein produced exosomes that carry dense arrays of spike trimers on 50% of all exosomes. Moreover, we find that immunization with spike-EMAD13 exosomes induced strong neutralizing antibody responses and protected hamsters against SARS-CoV-2 disease at doses of just 0.5-5 ng of spike protein, without adjuvant, demonstrating that antigen-display exosomes are particularly immunogenic, with important implications for both structural and expression-dependent vaccines.","version":"1.1","doi":"10.1101/2024.01.04.574272","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.03.574064","pub_date":"2024-1-04","title":"A Murine Model of Post-acute Neurological Sequelae Following SARS-CoV-2 Variant Infection","abstract":"Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. Surviving mice showed no detectable viral RNA in the brain and minimal neuroinflammation post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and reduced levels of genes associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent T helper 1 prone cellular immune responses and high neutralizing antibodies against Delta and Omicron variants in the periphery for months post-acute infection. Overall, infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long COVID patients and may be useful for future assessment of the efficacy of vaccines and therapeutics against SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2024.01.03.574064","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.02.573408","pub_date":"2024-1-03","title":"BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population","abstract":"During the current COVID-19 pandemic, the rate of morbidity and mortality was considerably lower in BCG vaccinated countries like Pakistan. BCG has been shown to provide cross protection to both disseminated TB as well as non related viral infections in BCG vaccinated children which is consistent with COVID-19 morbidity in the younger age group. Recently, this cross protection was attributed to trained immunity (TI) associated with BCG recall responses in the innate arm of the immune system. Little is known about the longevity of BCG Trained Immunity (TI) beyond early childhood. To assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections. In this cross-sectional study, healthy young adults and adolescents (n=20) were recruited from 16\u201340 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition. BCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network {macrophages [M1 (TNF\u03b1, IFN\u03b3), M2 (IL10)], NK (IL2), Gamma delta (\u03b3\u03b4) T (IL17, IL4)} and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay. Significant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNF\u03b1 (9148 pg/ml) followed by IL10 (488 pg/ml) and IFN\u03b3 (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNF\u03b1, 40 fold for IL10, and 27 fold for IFN\u03b3. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG. The presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro-and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.","version":"1.1","doi":"10.1101/2024.01.02.573408","journal":"bioRxiv","score":null},{"id":"10.1101/2024.01.02.573675","pub_date":"2024-1-03","title":"Antigen non-specific CD8+ T cells accelerate cognitive decline in aged mice following respiratory coronavirus infection","abstract":"Primarily a respiratory infection, numerous patients infected with SARS-CoV-2 present with neurologic symptoms, some continuing long after viral clearance as a persistent symptomatic phase termed \u201clong COVID\u201d. Advanced age increases the risk of severe disease, as well as incidence of long COVID. We hypothesized that perturbations in the aged immune response predispose elderly individuals to severe coronavirus infection and post-infectious sequelae. Using a murine model of respiratory coronavirus, mouse hepatitis virus strain A59 (MHV-A59), we found that aging increased clinical illness and lethality to MHV infection, with aged animals harboring increased virus in the brain during acute infection. This was coupled with an unexpected increase in activated CD8+ T cells within the brains of aged animals but reduced antigen specificity of those CD8+ T cells. Aged animals demonstrated spatial learning impairment following MHV infection, which correlated with increased neuronal cell death and reduced neuronal regeneration in aged hippocampus. Using primary cell culture, we demonstrated that activated CD8+ T cells induce neuronal death, independent of antigen-specificity. Specifically, higher levels of CD8+ T cell-derived IFN-\u03b3 correlated with neuronal death. These results support the evidence that CD8+ T cells in the brain directly contribute to cognitive dysfunction following coronavirus infection in aged individuals. Using a murine model of respiratory coronavirus infection, we show that aging amplifies post-infectious cognitive dysfunction due to activated CD8+ T cells that secrete IFN-\u03b3 in the brain. These data provide evidence that CD8+ T cells in the brain negatively impact post-infectious cognitive function.","version":"1.1","doi":"10.1101/2024.01.02.573675","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.30.573713","pub_date":"2024-1-02","title":"Major role of S-glycoprotein in providing immunogenicity and protective immunity in mRNA lipid nanoparticle vaccines based on SARS-CoV-2 structural proteins","abstract":"Recently we have developed an mRNA lipid nanoparticle (mRNA-LNP) platform providing efficient long-term expression of an encoded gene in vivo after both intramuscular and intravenous application. Based on this platform, we have generated mRNA-LNP coding SARS-CoV-2 structural proteins M, N, S from different virus variants and studied their immunogenicity separately or in combinations in vivo. As a result, all candidate vaccine compositions coding S and N proteins induced excellent anti-RBD and N titers of binding antibodies. T cell responses mainly represented specific CD4+ T cell lymphocyte producing IL-2 and TNF-\u03b1. mRNA-LNP coding M protein did not show high immunogenicity. High neutralizing activity was detected in sera of mice vaccinated with mRNA-LNP coding S protein (alone or in combinations) against closely related strains but was not detectable or significantly lower against an evolutionarily distant variant. Our data showed that the addition of mRNAs encoding S and M antigens to the mRNA-N in the vaccine composition enhanced immunogenicity of mRNA-N inducing more robust immune response to the N protein. Based on our results, we suggested that the S protein plays a key role in enhancement of immune response to the N protein in the mRNA-LNP vaccine.","version":"1.1","doi":"10.1101/2023.12.30.573713","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.26.513886","pub_date":"2024-1-02","title":"Complex changes in serum protein levels in COVID-19 convalescents","abstract":"The COVID-19 pandemic, triggered by severe acute respiratory syndrome coronavirus 2, has affected millions of people worldwide. Much research has been dedicated to our understanding of COVID-19 disease heterogeneity and severity, but less is known about recovery associated changes. To address this gap in knowledge, we quantified the proteome from serum samples from 29 COVID-19 convalescents and 29 age-, race-, and sex-matched healthy controls. Samples were acquired within the first months of the pandemic. Many proteins from pathways known to change during acute COVID-19 illness, such as from the complement cascade, coagulation system, inflammation and adaptive immune system, had returned to levels seen in healthy controls. In comparison, we identified 22 and 15 proteins with significantly elevated and lowered levels, respectively, amongst COVID-19 convalescents compared to healthy controls. Some of the changes were similar to those observed for the acute phase of the disease, i.e. elevated levels of proteins from hemolysis, the adaptive immune systems, and inflammation. In contrast, some alterations opposed those in the acute phase, e.g. elevated levels of CETP and APOA1 which function in lipid/cholesterol metabolism, and decreased levels of proteins from the complement cascade (e.g. C1R, C1S, and VWF), the coagulation system (e.g. THBS1 and VWF), and the regulation of the actin cytoskeleton (e.g. PFN1 and CFL1) amongst COVID-19 convalescents. We speculate that some of these shifts might originate from a transient decrease in platelet counts upon recovery from the disease. Finally, we observed race-specific changes, e.g. with respect to immunoglobulins and proteins related to cholesterol metabolism.","version":"1.2","doi":"10.1101/2022.10.26.513886","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.31.573785","pub_date":"2024-1-02","title":"An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses","abstract":"Positive-strand RNA viruses and DNA viruses generate double-stranded RNA (dsRNA) during their replication processes and innate immune responses against viral infections are orchestrated by numerous interferon-stimulating genes, yet the detailed coordination of downstream signaling of anti-viral immune responses is not fully understood. Recent studies suggest 2\u2019-5\u2019- Oligoadenylate Synthetase 1 (OAS1) may have a protective role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections; however, the mechanism regulating OAS1 remains uninvestigated. Our aim is to understand the regulation of OAS1 and its modulation of RNaseL activity, as this has significant implications for responses to RNA viruses, including Vesicular stomatitis virus (VSV) and SARS-CoV-2. We explore the hypothesis that ABCF1 an ATP-binding cassette family member protein, a key regulator of innate immune responses and macrophage polarization and cytokine storm, play a role in regulating the antiviral responses and downstream dsRNA signaling revealed by measuring responses to the synthetic dsRNA analog termed poly (I:C). We utilize ABCF1 haplo-insufficient mice to discover that ABCF1 modulates the amplitude and frequency of VSV-specific Cytolytic T lymphocyte in anti-viral immune responses and suggests that innate immune responses underpin this process. To understand this mechanism, we describe that ABCF1 interacts with 2\u2019-5\u2019-oligoadenylate synthetase 1 (OAS1) which in turn modulates essential proteins that leads to the modulation of RNaseL activity via ABCE1. Furthermore, we find that ABCF1 also influences the production of interferon-\u03b1 (IFN-\u03b1) and interferon-\u03b2 (IFN-\u03b2) in bone marrow-derived macrophages. Overall,, we unexpectedly discovered that ABCF1 acts as a crucial link between innate and adaptive immunity, regulating the development of adaptive Cytolytic T lymphocyte responses and interacting with OAS1, a key regulator of innate immune responses against viral infections. Exploring pharmacological agents that target ABCE1 or ABCF1 may lead to the discovery of novel modalities for countering SARS CoV-2 and other viruses where OAS1 is a crucial innate immune response gene.","version":"1.1","doi":"10.1101/2023.12.31.573785","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.19.572480","pub_date":"2023-12-31","title":"Predicting the Trend of SARS-CoV-2 Mutation Frequencies Using Historical Data","abstract":"As the SARS-CoV-2 virus rapidly evolves, predicting the trajectory of viral variations has become a critical yet complex task. A deep understanding of future mutation patterns, in particular the mutations that will prevail in the near future, is vital in steering diagnostics, therapeutics, and vaccine strategies in the coming months. In this study, we developed a model to forecast future SARS-CoV-2 mutation surges in real-time, using historical mutation frequency data from the USA. To improve upon the accuracy of traditional time-series models, we transformed the prediction problem into a supervised learning framework using a sliding window approach. This involved breaking the time series of mutation frequencies into very short segments. Considering the time-dependent nature of the data, we focused on modeling the first-order derivative of the mutation frequency. We predicted the final derivative in each segment based on the preceding derivatives, employing various machine learning methods, including random forest, XGBoost, support vector machine, and neural network models, in this supervised learning setting. Empowered by the novel transformation strategy and the high capacity of machine learning models, we witnessed low prediction error that is confined within 0.1% and 1% when making predictions for future 30 and 80 days respectively. In addition, the method also led to a notable increase in prediction accuracy compared to traditional time-series models, as evidenced by lower MAE, and MSE for predictions made within different time horizons. To further assess the method\u2019s effectiveness and robustness in predicting mutation patterns for unforeseen mutations, we categorized all mutations into three major patterns. The model demonstrated its robustness by accurately predicting unseen mutation patterns when training on data from two pattern categories while testing on the third pattern category, showcasing its potential in forecasting a variety of mutation trajectories. To enhance accessibility and utility, we built our methodology into an R-shiny app (https://swdatpredicts.shinyapps.io/rshiny_predict/), a tool with potential applicability in studying other infectious diseases, thus extending its relevance beyond the current pandemic.","version":"1.3","doi":"10.1101/2023.12.19.572480","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.27.23300588","pub_date":"2023-12-30","title":"Hybrid immunity from SARS-CoV-2 infection and vaccination in Canadian adults: cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Few national-level studies have evaluated the impact of \u201chybrid\u201d immunity (vaccination coupled with recovery from infection) from the Omicron variants of SARS-CoV-2.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>From May 2020 to December 2022, we conducted serial assessments (each of \u223c4000-9000 adults) examining SARS-CoV-2 antibodies within a mostly representative Canadian cohort drawn from a national online polling platform. Adults, most of whom were vaccinated, reported viral test\u2013confirmed infections and mailed self-collected dried blood spots to a central lab. Samples underwent highly sensitive and specific antibody assays to spike and nucleocapsid protein antigens, the latter triggered only by infection. We estimated cumulative SARS-CoV-2 incidence prior to the Omicron period and during the BA.1/1.1 and BA.2/5 waves. We assessed changes in antibody levels and in age-specific active immunity levels.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Spike levels were higher in infected than in uninfected adults, regardless of vaccination doses. Among adults vaccinated at least thrice and infected more than six months earlier, spike levels fell notably and continuously for the nine months post-vaccination. By contrast, among adults infected within six months, spike levels declined gradually. Declines were similar by sex, age group, and ethnicity. Recent vaccination attenuated declines in spike levels from older infections. In a convenience sample, spike antibody and cellular responses were correlated. Near the end of 2022, about 35% of adults above age 60 had their last vaccine dose more than six months ago, and about 25% remained uninfected. The cumulative incidence of SARS-CoV-2 infection rose from 13% (95% CI 11-14%) before omicron to 78% (76-80%) by December 2022, equating to 25 million infected adults cumulatively. However, the COVID-19 weekly death rate during the BA.2/5 waves was less than half of that during the BA.1/1.1 wave, implying a protective role for hybrid immunity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Strategies to maintain population-level hybrid immunity require up-to-date vaccination coverage, including among those recovering from infection. Population-based, self-collected dried blood spots are a practicable biological surveillance platform.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Funding was provided by the COVID-19 Immunity Task Force, Canadian Institutes of Health Research, Pfizer Global Medical Grants, and St. Michael\u2019s Hospital Foundation. PJ and ACG are funded by the Canada Research Chairs Program.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.12.27.23300588","journal":"medRxiv","score":null},{"id":"10.1101/2023.12.28.573501","pub_date":"2023-12-28","title":"Tying the Knot: Unraveling the Intricacies of the Coronavirus Frameshift Pseudoknot","abstract":"Understanding and targeting functional RNA structures towards treatment of coronavirus infection can help us to prepare for novel variants of SARS-CoV-2 (the virus causing COVID-19), and any other coronaviruses that could emerge via human-to-human transmission or potential zoonotic (inter-species) events. Leveraging the fact that all coronaviruses use a mechanism known as \u22121 programmed ribosomal frameshifting (\u22121 PRF) to replicate, we apply algorithms to predict the most energetically favourable secondary structures (each nucleotide involved in at most one pairing) that may be involved in regulating the \u22121 PRF event in coronaviruses, especially SARS-CoV-2. We compute previously unknown most stable structure predictions for the frameshift site of coronaviruses via hierarchical folding, a biologically motivated framework where initial non-crossing structure folds first, followed by subsequent, possibly crossing (pseudoknotted), structures. Using mutual information from 181 coronavirus sequences, in conjunction with the algorithm KnotAli, we compute secondary structure predictions for the frameshift site of different coronaviruses. We then utilize the Shapify algorithm to obtain most stable SARS-CoV-2 secondary structure predictions guided by frameshift sequence-specific and genome-wide experimental data. We build on our previous secondary structure investigation of the singular SARS-CoV-2 68 nt frameshift element sequence, by using Shapify to obtain predictions for 132 extended sequences and including covariation information. Previous investigations have not applied hierarchical folding to extended length SARS-CoV-2 frameshift sequences. By doing so, we simulate the effects of ribosome interaction with the frameshift site, providing insight to biological function. We contribute in-depth discussion to contextualize secondary structure dual-graph motifs for SARS-CoV-2, highlighting the energetic stability of the previously identified 3 8 motif alongside the known dominant 3 3 and 3 6 (native-type) \u22121 PRF structures. Integrating experimental data within minimum free energy (MFE) hierarchical folding algorithms provides novel structure predictions to distill the relationship between RNA structure and function. In particular, fully categorizing most stable secondary structure predictions via hierarchical folding supports our identification of motif transitions and critical site targets for future therapeutic research. Finding evolutionary connections between coronaviruses frameshift element RNA structures is a worthwhile goal in contributing to treatment development for afflicted human and animal populations. Predicting the most energetically favourable RNA secondary structures, and how they may form via the hierarchical folding hypothesis, is an efficient use of computational resources to shed light on RNA structure-function. We used the KnotAli algorithm to obtain mutual information from 181 coronaviruses frameshift RNA sequences. Guided by this evolutionary information, we computed secondary structure predictions to allow comparison of marked similarities and subtle differences between SARS-CoV-2 and other coronaviruses frameshift element RNA structures. In addition, we applied the Shapify algorithm to predict secondary structures for extended SARS-CoV-2 frameshift element sequences informed by SHAPE reactivity data. Here we critically expand the known landscape of most stable \u22121 PRF secondary structure conformations, isolating the location of key secondary structure motif transitions that can improve site targeting of viral therapeutics. Our application of hierarchical folding algorithms contributes novel predictions of functional RNA structures, enhancing discussion of how secondary structures unfold or refold to regulate frameshifting in coronaviruses.","version":"1.1","doi":"10.1101/2023.12.28.573501","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.28.555062","pub_date":"2023-12-27","title":"Discovery of 2-amide-3-methylester thiophenes that target SARS-CoV-2 Mac1 and repress coronavirus replication, validating Mac1 as an anti-viral target","abstract":"The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 \u00b5M. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 \u00b5M and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.","version":"1.2","doi":"10.1101/2023.08.28.555062","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.20.549891","pub_date":"2023-12-27","title":"Associations between COVID-19 and putative markers of neuroinflammation: A diffusion basis spectrum imaging study","abstract":"COVID-19 remains a significant international public health concern. Yet, the mechanisms through which symptomatology emerges remain poorly understood. While SARS-CoV-2 infection may induce prolonged inflammation within the central nervous system, the evidence primarily stems from limited small-scale case investigations. To address this gap, our study capitalized on longitudinal UK Biobank neuroimaging data acquired prior to and following COVID-19 testing (N=416 including n=224 COVID-19 cases; Mage=58.6). Putative neuroinflammation was assessed in gray matter structures and white matter tracts using non-invasive Diffusion Basis Spectrum Imaging (DBSI), which estimates inflammation-related cellularity (DBSI-restricted fraction; DBSI-RF) and vasogenic edema (DBSI-hindered fraction; DBSI-HF).We hypothesized that COVID-19 case status would be associated with increases in DBSI markers after accounting for potential confound (age, sex, race, body mass index, smoking frequency, and data acquisition interval) and multiple testing. COVID-19 case status was not significantly associated with DBSI-RF (|\u03b2|\u2019s<0.28, pFDR >0.05), but with greater DBSI-HF in left pre- and post-central gyri and right middle frontal gyrus (\u03b2\u2019s>0.3, all pFDR=0.03). Intriguingly, the brain areas exhibiting increased putative vasogenic edema had previously been linked to COVID-19-related functional and structural alterations, whereas brain regions displaying subtle differences in cellularity between COVID-19 cases and controls included regions within or functionally connected to the olfactory network, which has been implicated in COVID-19 psychopathology. Nevertheless, our study might not have captured acute and transitory neuroinflammatory effects linked to SARS-CoV-2 infection, possibly due to symptom resolution before the imaging scan. Future research is warranted to explore the potential time- and symptom-dependent neuroinflammatory relationship with COVID-19.","version":"1.3","doi":"10.1101/2023.07.20.549891","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.21.572824","pub_date":"2023-12-22","title":"Virion morphology and on-virus spike protein structures of diverse SARS-CoV-2 variants","abstract":"The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins that are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S from SARS-CoV-2 variants has revealed this structural adaptation at high-resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, \u201cin-situ\u201d high-resolution S structures and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Considered together, our data is consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of S.","version":"1.1","doi":"10.1101/2023.12.21.572824","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.14.557558","pub_date":"2023-12-22","title":"MixOmics Integration of Biological Datasets Identifies Highly Correlated Key Variables of COVID-19 severity","abstract":"Despite several years since the COVID-19 pandemic was declared, challenges remain in understanding the factors that can predict the severity of COVID-19 disease and complications of SARS-CoV-2 infection. While many large-scale Multiomic datasets have been published, integration of these datasets has the potential to substantially increase the biological insight gained allowing a more complex comprehension of the disease pathogenesis. Such insight may improve our ability to predict disease progression, detect severe cases more rapidly and develop effective therapeutics. In this study we have applied an innovative machine learning algorithm to delineate COVID-severity based on integration of paired samples of proteomic and transcriptomic data from a small cohort of patients testing positive for SARS-CoV-2 infection with differential disease severity. Targeted plasma proteomics and an onco-immune targeted transcriptomic panel was performed on sequential samples from a cohort of 23 severe, 21 moderate and 10 mild COVID-19 patients. We applied DIABLO, a new integrative method, to identify multi-omics biomarker panels that can discriminate between multiple phenotypic groups, such as the varied severity of disease in COVID-19 patients. As COVID-19 severity is known among our sample group, we can train models using this as the outcome variable and calculate features that are important predictors of severe disease. In this study, we detect highly correlated key variables of severe COVID-19 using transcriptomic discriminant analysis and multi-omics integration methods. This approach highlights the power of data integration from a small cohort of patients offering a better biological understanding of the molecular mechanisms driving COVID-19 severity and an opportunity to improve prediction of disease trajectories and targeted therapeutics.","version":"1.2","doi":"10.1101/2023.09.14.557558","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.22.572975","pub_date":"2023-12-22","title":"Genomic analysis of Staphylococcus aureus isolates from bacteremia reveals genetic features associated with the COVID-19 pandemic","abstract":"Genomic analyses of bacterial isolates are necessary to monitor the prevalence of antibiotic resistance genes and virulence determinants. Herein, we provide a comprehensive genomic description of a collection of 339 Staphylococcus aureus strains isolated from patients with bacteremia between 2014 and 2022. Nosocomial acquisition accounted for 56.6% of episodes, with vascular catheters being the predominant source of infection (31.8%). Cases of fatality (27.4%), persistent bacteremia (19.5%) and diagnosis of septic emboli (24.2%) were documented. During the COVID-19 pandemic, we observed a 140% increase of the episodes of S. aureus bacteremia per year, with a concomitant increase of the cases from nosocomial origin. This prompted us to investigate the existence of genetic features associated with S. aureus isolates from the COVID-19 pandemic. While genes conferring resistance to \u03b2-lactams (blaI-blaR-blaZ), macrolides (ermA, ermC, ermT, mphC, msrA) and aminoglycosides (ant(4\u2019)-Ia, ant(9)-Ia, aph(3\u2019)-IIIa, aph(2\u2019\u2019)-Ih) were prevalent in our collection, detection of the msrA and mphC genes increased significantly in pandemic S. aureus isolates. Similarly, we observed a higher prevalence of isolates carrying the genes encoding the Clumping Factors A and B, involved in fibrinogen binding. Of note, macrolides were extensively used as accessory therapy for COVID-19 and fibrinogen levels were usually elevated upon SARS-CoV-2 infection. Therefore, our results reveal a remarkable adaptation of the S. aureus isolates to the COVID-19 pandemic context and demonstrates the potential of whole-genome sequencing to conduct molecular epidemiology studies.","version":"1.1","doi":"10.1101/2023.12.22.572975","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.20.572655","pub_date":"2023-12-21","title":"Persistence of an infectious form of SARS-CoV-2 post protease inhibitor treatment of permissive cells in vitro","abstract":"Reports have described SARS-CoV-2 rebound in COVID-19 patients treated with nirmatrelvir, a 3CL protease inhibitor. The cause remains a mystery, although drug resistance, re-infection, and lack of adequate immune responses have been excluded. We now present virologic findings that provide a clue to the cause of viral rebound, which occurs in \u223c20% of the treated cases. The persistence of an intermediary form of infectious SARS-CoV-2 was experimentally documented in vitro after treatment with nirmatrelvir or another 3CL protease inhibitor, but not with a polymerase inhibitor, remdesivir. This infectious intermediate decayed slowly with a half-life of \u223c1 day, suggesting that its persistence could outlive the treatment course to re-ignited SARS-CoV-2 infection as the drug is eliminated. Additional studies are needed to define the nature of this viral intermediate, but our findings point to a particular direction for future investigation and offer a specific treatment recommendation that should be tested clinically.","version":"1.1","doi":"10.1101/2023.12.20.572655","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.21.572736","pub_date":"2023-12-21","title":"Analysing the distribution of SARS-CoV-2 infections in schools: integrating model predictions with real world observations","abstract":"School closures were used as strategies to mitigate transmission in the COVID-19 pandemic. Understanding the nature of SARS-CoV-2 outbreaks and the distribution of infections in classrooms could help inform targeted or \u2018precision\u2019 preventive measures and outbreak management in schools, in response to future pandemics. In this work, we derive an analytical model of Probability Density Function (PDF) of SARS-CoV-2 secondary infections and compare the model with infection data from all public schools in Ontario, Canada between September-December, 2021. The model accounts for major sources of variability in airborne transmission like viral load and dose-response (i.e., the human body\u2019s response to pathogen exposure), air change rate, room dimension, and classroom occupancy. Comparisons between reported cases and the modeled PDF demonstrated the intrinsic overdispersed nature of the real-world and modeled distributions, but uncovered deviations stemming from an assumption of homogeneous spread within a classroom. The inclusion of near-field transmission effects resolved the discrepancy with improved quantitative agreement between the data and modeled distributions. This study provides a practical tool for predicting the size of outbreaks from one index infection, in closed spaces such as schools, and could be applied to inform more focused mitigation measures. At the start of the COVID-19 pandemic, there was huge uncertainty around the risks of SARS-CoV-2 spread in classrooms. In the absence of early predictions surrounding classroom risks, many jurisdictions across countries closed in-person education. There is great interest in adopting a more \u2018precision\u2019 approach to better inform future interventions in the context of airborne virus risks. For this purpose, we need tools that can predict the probability of the size of outbreaks within classrooms along with the impact of interventions including masks, better ventilation, and physical distancing by limiting the number of students per classroom. To this end, we have developed a robust but practical model that yields the probability of secondary infections stemming from index cases occurring within schools on a given day. During model development, the major underlying physical and biological factors that dictate the disease transmission process, both at long-range and close-range, have been accounted for. This enables our model to modify its predictions for different scenarios - and possibly allows its use beyond schools. Finally, the model\u2019s predictive capability has been verified by comparing its outputs with publicly available data on SARS-CoV-2 diagnoses in Ontario public schools. To our knowledge, this is the first time an analytical model derived from mostly first principles describes real-world infection distributions, satisfactorily. The quantitative match between the theoretical prediction and real-world data offers the proposed model as a possible powerful tool for better-informed precision pandemic mitigation strategies in indoor environments like schools.","version":"1.1","doi":"10.1101/2023.12.21.572736","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.20.572426","pub_date":"2023-12-21","title":"Tracking SARS-CoV-2 variants of concern in wastewater: an assessment of nine computational tools using simulated genomic data","abstract":"Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic \u201cnovel\u201d lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances, and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1% frequency, results were more reliable above a 5% threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of noise or bias in wastewater sequencing data and to appreciate the commonalities and differences across methods.","version":"1.1","doi":"10.1101/2023.12.20.572426","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.24.553565","pub_date":"2023-12-21","title":"Comparison of SARS-CoV-2 variants of concern in primary human nasal cultures demonstrates Delta as most cytopathic and Omicron as fastest replicating","abstract":"The SARS-CoV-2 pandemic was marked with emerging viral variants, some of which were designated as variants of concern (VOCs) due to selection and rapid circulation in the human population. Here we elucidate functional features of each VOC linked to variations in replication rate. Patient-derived primary nasal cultures grown at air-liquid-interface (ALI) were used to model upper-respiratory infection and human lung epithelial cell lines used to model lower-respiratory infection. All VOCs replicated to higher titers than the ancestral virus, suggesting a selection for replication efficiency. In primary nasal cultures, Omicron replicated to the highest titers at early time points, followed by Delta, paralleling comparative studies of population sampling. All SARS-CoV-2 viruses entered the cell primarily via a transmembrane serine protease 2 (TMPRSS2)-dependent pathway, and Omicron was more likely to use an endosomal route of entry. All VOCs activated and overcame dsRNA-induced cellular responses including interferon (IFN) signaling, oligoadenylate ribonuclease L degradation and protein kinase R activation. Among the VOCs, Omicron infection induced expression of the most IFN and IFN stimulated genes. Infections in nasal cultures resulted in cellular damage, including a compromise of cell-barrier integrity and loss of nasal cilia and ciliary beating function, especially during Delta infection. Overall, Omicron was optimized for replication in the upper-respiratory system and least-favorable in the lower-respiratory cell line; and Delta was the most cytopathic for both upper and lower respiratory cells. Our findings highlight the functional differences among VOCs at the cellular level and imply distinct mechanisms of pathogenesis in infected individuals. Comparative analysis of infections by SARS-CoV-2 ancestral virus and variants of concern including Alpha, Beta, Delta, and Omicron, indicated that variants were selected for efficiency in replication. In infections of patient-derived primary nasal cultures grown at air-liquid-interface to model upper-respiratory infection, Omicron reached highest titers at early time points, a finding that was confirmed by parallel population sampling studies. While all infections overcame dsRNA-mediated host responses, infections with Omicron induced the strongest interferon and interferon stimulated gene response. In both primary nasal cultures and lower-respiratory cell line infections by Delta were most damaging to the cells as indicated by syncytia formation, loss of cell barrier integrity and nasal ciliary function.","version":"1.2","doi":"10.1101/2023.08.24.553565","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.20.572660","pub_date":"2023-12-21","title":"Computational detection of antigen specific B cell receptors following immunization","abstract":"B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and Sars-CoV-2 infections. We show that different lineages converge to the same responding CDR3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.","version":"1.1","doi":"10.1101/2023.12.20.572660","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.21.572575","pub_date":"2023-12-21","title":"Humoral immune responses to the monovalent XBB.1.5-adapted BNT162b2 mRNA booster","abstract":"Continued SARS-CoV-2 evolution and immune escape necessitated the development of updated vaccines, and a monovalent vaccine incorporating the XBB.1.5 variant spike protein is currently being rolled out. Amidst the emergence of the highly mutated BA.2.86 lineage and against the backdrop of pronounced immune imprinting, it is important to characterize the antibody responses following vaccination, particularly in the elderly. Here, we show that the monovalent XBB.1.5-adapted booster vaccination substantially enhanced both binding and neutralising antibody responses against a panel of variants, including BA.2.86, in an older population with four or more previous vaccine doses. Furthermore, neutralizing antibody titers to XBB.1.5 and BA.2.86 were boosted more strongly than titers to historical variants were. Our findings thereby suggest increased vaccine induced protection against both antigenically matched variants, as well as the more distant BA.2.86 variant, and support current vaccine policies recommending a monovalent XBB.1.5 booster dose to older individuals.","version":"1.1","doi":"10.1101/2023.12.21.572575","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.19.572475","pub_date":"2023-12-21","title":"Inverse folding of protein complexes with a structure-informed language model enables unsupervised antibody evolution","abstract":"Large language models trained on sequence information alone are capable of learning high level principles of protein design. However, beyond sequence, the three-dimensional structures of proteins determine their specific function, activity, and evolvability. Here we show that a general protein language model augmented with protein structure backbone coordinates and trained on the inverse folding problem can guide evolution for diverse proteins without needing to explicitly model individual functional tasks. We demonstrate inverse folding to be an effective unsupervised, structure-based sequence optimization strategy that also generalizes to multimeric complexes by implicitly learning features of binding and amino acid epistasis. Using this approach, we screened \u223c30 variants of two therapeutic clinical antibodies used to treat SARS-CoV-2 infection and achieved up to 26-fold improvement in neutralization and 37-fold improvement in affinity against antibody-escaped viral variants-of-concern BQ.1.1 and XBB.1.5, respectively. In addition to substantial overall improvements in protein function, we find inverse folding performs with leading experimental success rates among other reported machine learning-guided directed evolution methods, without requiring any task-specific training data.","version":"1.2","doi":"10.1101/2023.12.19.572475","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.20.572501","pub_date":"2023-12-20","title":"Temperature impacts SARS-CoV-2 spike fusogenicity and evolution","abstract":"SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33\u00b0C and 37\u00b0C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that rising temperature from 33\u00b0C to 37\u00b0C or 39\u00b0C increased spike-mediated cell-cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell-cell fusion was maintained during evolution at 39\u00b0C, but was lost in a high proportion of viruses evolved at 33\u00b0C or 37\u00b0C. Consistently, sequencing of the spikes evolved at 33\u00b0C or 37\u00b0C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell-cell fusion, whereas this did not occur in spikes evolved at 39\u00b0C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell-cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike. When it infects humans, SARS-CoV-2 is exposed to different temperaures (e.g. replication site, fever\u2026). Temperature has been shown to strongly impact SARS-CoV-2 replication but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adapatation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.","version":"1.1","doi":"10.1101/2023.12.20.572501","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.20.572504","pub_date":"2023-12-20","title":"Cell type-specific adaptation of the SARS-CoV-2 spike","abstract":"SARS-CoV-2 can infect various human tissues and cell types, principally via interaction with its cognate receptor ACE2. However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After 20 passages, cell type-specific phenotypic changes were observed. Selected spike mutations were identified and functionally characterized in terms of entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, entry pathway and syncytia formation. We found that the effects of these mutations varied across cell types. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.","version":"1.1","doi":"10.1101/2023.12.20.572504","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.19.572469","pub_date":"2023-12-20","title":"Distinct SARS-CoV-2 populational immune backgrounds induce divergent RBD evolutionary preferences","abstract":"Immune evasion is a pivotal force shaping the evolution of viruses. Nonetheless, the extent to which virus evolution varies among populations with diverse immune backgrounds remains an unsolved mystery. Prior to the widespread SARS-CoV-2 infections in December 2022 and January 2023, the Chinese population possessed a markedly distinct (less potent) immune background due to its low infection rate, compared to countries experiencing multiple infection waves, presenting an unprecedented opportunity to investigate how the virus has evolved under different immune contexts. We compared the mutation spectrum and functional potential of BA.5.2.48, BF.7.14, and BA.5.2.49\u2014variants prevalent in China\u2014with their counterparts in other countries. We found that mutations in the RBD region in these lineages were more widely dispersed and evenly distributed across different epitopes. These mutations led to a higher ACE2 binding affinity and reduced potential for immune evasion compared to their counterparts in other countries. These findings suggest a milder immune pressure and less evident immune imprinting within the Chinese population. Despite the emergence of numerous immune-evading variants in China, none of them exhibited a transmission advantage. Instead, they were replaced by the imported XBB variant with stronger immune evasion since April 2023. Our findings demonstrated that the continuously changing immune background led to varying evolutionary pressures on SARS-CoV-2. Thus, in addition to the viral genome surveillance, immune background surveillance is also imperative for predicting forthcoming mutations and understanding how these variants spread in the population.","version":"1.1","doi":"10.1101/2023.12.19.572469","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.12.571160","pub_date":"2023-12-20","title":"A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines","abstract":"Continuous evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and monoclonal antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S2 subunit, which folds as a spring-loaded fusion machinery. Here, we describe a protein design strategy enabling prefusion-stabilization of the SARS-CoV-2 S2 subunit and high yield recombinant expression of trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to all sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S2 fusion machineries. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S2 trimer vaccine elicits broadly reactive sarbecovirus antibody responses and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with SARS-CoV-2 XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines motivating future development.","version":"1.2","doi":"10.1101/2023.12.12.571160","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.19.572339","pub_date":"2023-12-20","title":"Deep profiling of antigen-specific B cells from different pathogens identifies novel compartments in the IgG memory B cell and antibody-secreting cell lineages","abstract":"A better understanding of the bifurcation of human B cell differentiation into memory B cells (MBC) and antibody-secreting cells (ASC) and identification of MBC and ASC precursors is crucial to optimize vaccination strategies or block undesired antibody responses. To unravel the dynamics of antigen-induced B cell responses, we compared circulating B cells reactive to SARS-CoV-2 (Spike, RBD and Nucleocapsid) in COVID-19 convalescent individuals to B cells specific to Influenza-HA, RSV-F and TT, induced much longer ago. High-dimensional spectral flow cytometry indicated that the decision point between ASC- and MBC-formation lies in the CD43+CD71+IgG+ Activated B cell compartment, showing properties indicative of recent germinal center activity and recent antigen encounter. Within this Activated B cells compartment, CD86+ B cells exhibited close phenotypical similarity with ASC, while CD86\u2212 B cells were closely related to IgG+ MBCs. Additionally, different activation stages of the IgG+ MBC compartment could be further elucidated. The expression of CD73 and CD24, regulators of survival and cellular metabolic quiescence, discerned activated MBCs from resting MBCs. Activated MBCs (CD73-CD24lo) exhibited phenotypical similarities with CD86\u2212 IgG+ Activated B cells and were restricted to SARS-CoV-2 specificities, contrasting with the resting MBC compartment (CD73-/CD24hi) that exclusively encompassed antigen-specific B cells established long ago. Overall, these findings identify novel stages for IgG+ MBC and ASC formation and bring us closer in defining the decision point for MBC or ASC differentiation. In this study, researchers aimed to better understand human B cell differentiation and their role in establishing long-lived humoral immunity. Using high-dimensional flow cytometry, they studied B cells reactive to three SARS-CoV-2 antigens in individuals convalescent for COVID-19, and compared their phenotypes to B cells reactive to three distinct protein antigens derived from vaccines or viruses encountered months to decades before. Their findings showed that Activated B cells reflect recent germinal center graduates that may have diverse fates; with some feeding the pool of antibody-secreting cells and others fueling the resting memory B cell compartment. Activated B cells gradually differentiate into resting memory B cells through an activated MBC phase. Increased expression of the cellular metabolic regulators CD73 and CD24 in resting memory B cells distinguishes them from the activated memory B cells phase, and is likely involved in sustaining a durable memory of humoral immunity. These findings are crucial for the development of vaccines that provide lifelong protection and may show potential to define reactive B cells in diseases where the cognate-antigen is still unknown such as in autoimmunity, cancers, or novel viral outbreaks.","version":"1.1","doi":"10.1101/2023.12.19.572339","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.18.572126","pub_date":"2023-12-19","title":"Multi-target mode of action of Sulfodyne\u00ae, a stabilized Sulforaphane, against pathogenic effects of SARS-CoV-2 infection","abstract":"The coronavirus disease 2019 (COVID-19) due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown that, except vaccination, few therapeutics options for its treatment or prevention are available. Among the pathways that can be targeted for COVID-19 treatment, the Keap1/Nrf2 pathway seems of high interest as it regulates redox homeostasis and inflammation that are altered during SARS-CoV-2 infection. Here, we use three potent activators of the Keap1/Nrf2 pathway and showed that Sulfodyne\u00ae, a stabilized natural Sulforaphane preparation with optimal bioavailability, had the highest antiviral activity in pulmonary or colonic epithelial cell lines even when added late after SARS-CoV-2 infection. This antiviral activity was not dependent on NRF2 activity but associated with action on ER stress and mTOR signaling that are activated during SARS-CoV-2 infection. Sulfodyne\u00ae also decreased the inflammatory response of epithelial cell lines infected by SARS-CoV-2 independently of SARS-CoV-2 replication and reduced the activation of human monocytes that are recruited after infection of epithelial cells by SARS-CoV-2. Administration of Sulfodyne\u00ae had little effects on SARS-CoV-2 replication in mice and hamsters infected with SARS-CoV-2 but significantly reduced weight loss and disease severity. Altogether, these results pinpoint the natural compound Sulfodyne\u00ae as a potent therapeutic agent of COVID-19 symptomatology. Accumulating evidence shows that oxidative stress coupled with the systemic inflammation contribute to COVID-19 pathogenesis. As the Keap1/Nrf2 pathway is the major regulator of redox homeostasis and promotes resolution of inflammation and as lung biopsies from COVID-19 patients showed a decreased NRF2 target gene signature, pharmacological agents that are known to activate NRF2 are good candidates for COVID-19 treatment. We show herein that Sulfodyne\u00ae, an NRF2 activator that consists in a stabilized Sulforaphane preparation with optimal bioavailability, impairs SARS-CoV-2 replication in colonic or pulmonary epithelial cells. We show that this antiviral activity of Sulfodyne\u00ae is not dependent of NRF2 activation, characterize the pathways associated with the Sulfodyne\u00ae antiviral activity and show that Sulfodyne\u00ae displays multiple actions that result in a decrease of the inflammation associated with SARS-CoV-2 infection. Finally, we show that Sulfodyne\u00ae decreases the pathogenesis of mice or hamster infected with SARS-CoV-2. Overall, this study provides mechanistic explanations of the action of Sulfodyne\u00ae during SARS-CoV-2 infection and suggests that Sulfodyne\u00ae is a potential therapeutic agent of COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2023.12.18.572126","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.10.540124","pub_date":"2023-12-19","title":"PandoGen: Generating complete instances of future SARS-CoV-2 sequences using Deep Learning","abstract":"One of the challenges in a viral pandemic is the emergence of novel variants with different phenotypical characteristics. An ability to forecast future viral individuals at the sequence level enables advance preparation by characterizing the sequences and closing vulnerabilities in current preventative and therapeutic methods. In this article, we explore, in the context of a viral pandemic, the problem of generating complete instances of undiscovered viral protein sequences, which have a high likelihood of being discovered in the future using protein language models. Current approaches to training these models fit model parameters to a known sequence set, which does not suit pandemic forecasting as future sequences differ from known sequences in some respects. To address this, we develop a novel method, called PandoGen, to train protein language models towards the pandemic protein forecasting task. PandoGen combines techniques such as synthetic data generation, conditional sequence generation, and reward-based learning, enabling the model to forecast future sequences, with a high propensity to spread. Applying our method to modeling the SARS-CoV-2 Spike protein sequence, we find empirically that our model forecasts twice as many novel sequences with five times the case counts compared to a model that is thirty times larger. Our method forecasts unseen lineages months in advance, whereas models 4\u00d7 and 30\u00d7 larger forecast almost no new lineages. When trained on data available up to a month before the onset of important Variants of Concern, our method consistently forecasts sequences belonging to those variants within tight sequence budgets. PandoGen is available at: https://github.com/UIUC-ChenLab/PandoGen","version":"1.5","doi":"10.1101/2023.05.10.540124","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.18.572191","pub_date":"2023-12-19","title":"Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates","abstract":"Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs. Non-stabilized SARS-CoV-2 Spike mRNA vaccination activated B cells that target either conserved epitopes on SARS-CoV-2 Omicron variants of concern, or cross-neutralizing epitopes on pre-emergent Sarbecoviruses.","version":"1.1","doi":"10.1101/2023.12.18.572191","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.09.544327","pub_date":"2023-12-19","title":"CNP blocks mitochondrial depolarization and inhibits SARS-CoV-2 replication in vitro and in vivo","abstract":"The COVID-19 pandemic has claimed over 6.5 million lives worldwide and continues to have lasting impacts on the world\u2019s healthcare and economic systems. Several approved and emergency authorized therapeutics that inhibit early stages of the virus replication cycle have been developed however, effective late-stage therapeutical targets have yet to be identified. To that end, our lab identified that 2\u2019,3\u2019 cyclic-nucleotide 3\u2019-phosphodiesterase (CNP) inhibits SARS-CoV-2 virion assembly. We show that CNP inhibits the generation of new SARS-CoV-2 virions, reducing intracellular titers without inhibiting viral structural protein translation. Additionally, we show that targeting of CNP to mitochondria is necessary for inhibition, blocking mitochondrial depolarization and implicating CNP\u2019s proposed role as an inhibitor of the mitochondrial permeabilization transition pore (mPTP) as the mechanism of virion assembly inhibition. We also demonstrate that an adenovirus expressing virus expressing both human ACE2 and CNP inhibits SARS-CoV-2 titers to undetectable levels in lungs of mice. Collectively, this work shows the potential of CNP to be a new SARS-CoV-2 antiviral target. Upon entry into a cell, viruses manipulate the cellular environment for the benefit of their replication. We have found that upon infection, SARS-CoV-2 induces the mitochondria to release reactive oxygen species (ROS) into the cytoplasm which benefits the replication of the virus. We identified a phosphodiesterase, called CNP, that blocks this release via inhibition of an inducible pore called the mitochondrial permeability transition pore or mPTP. We also found that a drug targeting this pore inhibits SARS-CoV-2 replication. We test the function of CNP in vivo and find that if we overexpress CNP in mouse lungs, we inhibit SARS-CoV-2 replication. Together this demonstrates a key function of the mitochondria on SARS-CoV-2 replication and that antithetically ROS release enhances viral replication. We propose this is common across coronaviruses and potentially other viruses identifying a novel target for future therapies.","version":"1.2","doi":"10.1101/2023.06.09.544327","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.08.566227","pub_date":"2023-12-19","title":"Robust detection of SARS-CoV-2 exposure in the population using T-cell repertoire profiling","abstract":"The COVID-19 pandemic offers a powerful opportunity to develop methods for monitoring the spread of infectious diseases based on their signatures in population immunity. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become the method of choice for identifying T cell receptor (TCR) biomarkers encoding pathogen specificity and immunological memory. AIRR-seq can detect imprints of past and ongoing infections and facilitate the study of individual responses to SARS-CoV-2, as shown in many recent studies. Here, we have applied a machine learning approach to two large AIRR-seq datasets with more than 1,200 high-quality repertoires from healthy and COVID-19-convalescent donors to infer TCR repertoire features that were induced by SARS-CoV-2 exposure. The new batch effect correction method allowed us to use data from different batches together, as well as combine the analysis for data obtained using different protocols. Proper standardization of AIRR-seq batches, access to human leukocyte antigen (HLA) typing, and the use of both \u03b1- and \u03b2-chain sequences of TCRs resulted in a high-quality biomarker database and a robust and highly accurate classifier for COVID-19 exposure. This classifier is applicable to individual TCR repertoires obtained using different protocols, paving the way to AIRR-seq-based immune status assessment in large cohorts of donors.","version":"1.2","doi":"10.1101/2023.11.08.566227","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.19.572363","pub_date":"2023-12-19","title":"Comparison of mitochondrial response to SARS-CoV-2 spike protein receptor binding domain in human lung microvascular, coronary artery endothelial and bronchial epithelial cells","abstract":"Recent evidence indicate that SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVEC) and coronary artery endothelial (HCAEC) and bronchial epithelial cells (HBEC). Live Mito Orange staining, STED microscopy and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry, Agilent XFp analyser for mitochondrial/glycolytic activity, MitoSOX fluorescence for mitochondrial ROS, and qRT-PCR plus Luminex for cytokines. In HLMVEC, SARS-CoV-2 RBD fragmented the mitochondrial network, decreased cristae density, mitochondrial oxygen consumption and glycolysis and induced mitoROS-mediated GM-CSF and IL-1\u03b2 expression in all three investigated cell types and IL-8 - in both endothelial cell types. Mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVEC, HCAEC and HBEC, with the mitochondria of HLMVEC being more sensitive to SARS-CoV-2 RBD.","version":"1.1","doi":"10.1101/2023.12.19.572363","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.19.572347","pub_date":"2023-12-19","title":"Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera","abstract":"Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. As exposure histories for human sera become more and more complex, animal sera may provide an alternative for antigenic characterization of new variants. To bridge data obtained from animal sera with human sera, we here analyzed neutralizing antibody titers in human and hamster first infection sera in a highly controlled setting using the same live-virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity. Our results indicate that sera from infected hamsters are a good surrogate for the antigenic characterization of new variants.","version":"1.1","doi":"10.1101/2023.12.19.572347","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.18.572180","pub_date":"2023-12-19","title":"Neuropathological assessment of the olfactory bulb and tract in individuals with COVID-19","abstract":"The majority of patients with Parkinson disease (PD) experience a loss in their sense of smell and accumulate insoluble \u03b1-synuclein aggregates in their olfactory bulbs (OB). Subjects affected by a SARS-CoV-2-linked illness (COVID-19) frequently experience hyposmia. We previously hypothesized that \u03b1-synuclein and tau misprocessing could occur following host responses to microbial triggers. Using semiquantitative measurements of immunohistochemical signals, we examined OB and olfactory tract specimens collected serially at autopsies between 2020 and 2023. Deceased subjects comprised 50 adults, which included COVID19+ patients (n=22), individuals with Lewy body disease (e.g., PD and dementia with Lewy bodies (DLB; n=6)), Alzheimer disease (AD; n=3), other non-synucleinopathy-linked degenerative diseases (e.g., progressive supranuclear palsy (PSP; n=2) and multisystem atrophy (MSA; n=1)). Further, we included neurologically healthy controls (HCO; n=9) and those with an inflammation-rich brain disorder as neurological controls (NCO; n=7). When probing for inflammatory changes focusing on anterior olfactory nuclei (AON) using anti-CD68 immunostaining, scores were consistently elevated in NCO and AD cases. In contrast, inflammation on average was not significantly altered in COVID19+ patients relative to controls, although anti-CD68 reactivity in their OB and tracts declined with progression in age. Mild-to-moderate increases in phospho-\u03b1Syn and phospho-tau signals were detected in the AON of tauopathy-and synucleinopathy-afflicted brains, respectively, consistent with mixed pathology, as described by others. Lastly, when both sides were available for comparison in our case series, we saw no asymmetry in the degree of pathology of the left versus right OB and tracts. We concluded from our autopsy series that after a fatal course of COVID-19, microscopic changes -when present-in the rostral, intracranial portion of the olfactory circuitry generally reflected neurodegenerative processes seen elsewhere in the brain. In general, inflammation correlated best with the degree of Alzheimer\u2019s-linked tauopathy and declined with progression of age in COVID19+ patients.","version":"1.1","doi":"10.1101/2023.12.18.572180","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.18.571720","pub_date":"2023-12-19","title":"Interferon signaling in the nasal epithelium distinguishes among lethal and common cold respiratory viruses and is critical for viral clearance","abstract":"All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at air-liquid interface (ALI). HCoV-229E, HCoV-NL63 and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33\u00b0C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally-directed IFNs as potential therapeutics.","version":"1.1","doi":"10.1101/2023.12.18.571720","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.04.551687","pub_date":"2023-12-19","title":"Quantification of biases in predictions of protein-protein binding affinity changes upon mutations","abstract":"Understanding the impact of mutations on protein-protein binding affinity is a key objective for a wide range of biotechnological applications and for shedding light on disease-causing mutations, which are often located at protein-protein interfaces. Over the past decade, many computational methods using physics-based and/or machine learning approaches have been developed to predict how protein binding affinity changes upon mutations. They all claim to achieve astonishing accuracy on both training and test sets, with performances on standard benchmarks such as SKEMPI 2.0 that seem overly optimistic. Here we benchmarked eight well-known and well-used predictors and identified their biases and dataset dependencies, using not only SKEMPI 2.0 as a test set but also deep mutagenesis data on the SARS-CoV-2 spike protein in complex with the human angiotensin-converting enzyme 2. We showed that, even though most of the tested methods reach a significant degree of robustness and accuracy, they suffer from limited generalizability properties and struggle to predict unseen mutations. Interestingly, the generalizability problems are more severe for pure machine learning approaches while physics-based methods are less affected by this issue. Moreover, undesirable prediction biases towards specific mutation properties, the most marked being towards destabilizing mutations, are also observed and should be carefully considered by method developers. We conclude from our analyses that there is room for improvement in the prediction models and suggest ways to check, assess and improve their generalizability and robustness.","version":"1.2","doi":"10.1101/2023.08.04.551687","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.08.539897","pub_date":"2023-12-18","title":"Evolving spike-protein N-glycosylation in SARS-CoV-2 variants","abstract":"Since >3 years, SARS-CoV-2 has plunged humans into a colossal pandemic. Henceforth, multiple waves of infection have swept through the human population, led by variants that were able to partially evade acquired immunity. The co-evolution of SARS-CoV-2 variants with human immunity provides an excellent opportunity to study the interaction between viral pathogens and their human hosts. The heavily N-glycosylated spike-protein of SARS-CoV-2 plays a pivotal role in initiating infection and is the target for host immune-response, both of which are impacted by host-installed N-glycans. Using highly-sensitive DeGlyPHER approach, we compared the N-glycan landscape on spikes of the SARS-CoV-2 Wuhan-Hu-1 strain to seven WHO-defined variants of concern/interest, using recombinantly expressed, soluble spike-protein trimers, sharing same stabilizing-mutations. We found that N-glycan processing is conserved at most sites. However, in multiple variants, processing of N-glycans from high mannose- to complex-type is reduced at sites N165, N343 and N616, implicated in spike-protein function.","version":"1.2","doi":"10.1101/2023.05.08.539897","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.15.571826","pub_date":"2023-12-15","title":"SARS-CoV-2 and other coronaviruses in rats, Berlin, Germany, 2023","abstract":"We tested 130 rats trapped in Berlin for coronaviruses. Antibodies against SARS-CoV-2 were detected in a single animal only, but not in further 66 rats from the same location, speaking against virus circulation in the rat population. All animals tested negative for SARS-CoV-2 by RT-PCR. However, rodent-associated alphacoronaviruses were found.","version":"1.1","doi":"10.1101/2023.12.15.571826","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.14.571764","pub_date":"2023-12-15","title":"Structure of a SARS-CoV-2 spike S2 subunit in a pre-fusion, open conformation","abstract":"The 800 million human infections with SARS-CoV-2 and the likely emergence of new variants and additional coronaviruses necessitate a better understanding of the essential spike glycoprotein and the development of immunogens that foster broader and more durable immunity. The S2 fusion subunit is more conserved in sequence, is essential to function, and would be a desirable immunogen to boost broadly reactive antibodies. It is, however, unstable in structure and in its wild-type form, cannot be expressed alone without irreversible collapse into a six-helix bundle. In addition to the irreversible conformational changes of fusion, biophysical measurements indicate that spike also undergoes a reversible breathing action. However, spike in an open, \u201cbreathing\u201d conformation has not yet been visualized at high resolution. Here we describe an S2-only antigen, engineered to remain in its relevant, pre-fusion viral surface conformation in the absence of S1. We also describe a panel of natural human antibodies specific for S2 from vaccinated and convalescent individuals. One of these mAbs, from a convalescent individual, afforded a high-resolution cryo-EM structure of the prefusion S2. The structure reveals a complex captured in an \u201copen\u201d conformation with greater stabilizing intermolecular interactions at the base and a repositioned fusion peptide. Together, this work provides an antigen for advancement of next-generation \u201cbooster\u201d immunogens and illuminates the likely breathing adjustments of the coronavirus spike.","version":"1.1","doi":"10.1101/2023.12.14.571764","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.14.571774","pub_date":"2023-12-15","title":"Metacell-based differential expression analysis identifies cell type specific temporal gene response programs in COVID-19 patient PBMCs","abstract":"By resolving cellular heterogeneity in a biological sample, single cell RNA sequencing (scRNA-seq) can detect gene expression and its dynamics in different cell types. Its application to time-series samples can thus identify temporal genetic programs active in different cell types, for example, immune cells\u2019 responses to viral infection. However, current scRNA-seq analysis need improvement. Two issues are related to data generation. One is that the number of genes detected in each cell is relatively low especially when currently popular dropseq-based technology is used for analyzing thousands of cells or more. The other is the lack of sufficient replicates (often 1-2) due to high cost of library preparation and sequencing. The third issue lies in the data analysis \u2013-usage of individual cells as independent sampling data points leads to inflated statistics. To address these issues, we explore a new data analysis framework, specifically whether \u201cmetacells\u201d that are carefully constructed to maintain cellular heterogeneity within individual cell types (or clusters) can be used as \u201creplicates\u201d for statistical methods requiring multiple replicates. Toward this, we applied SEACells to a time-series scRNA-seq dataset from peripheral blood mononuclear cells (PBMCs) after SARS-Cov-2 infection to construct metacells, which were then used in maSigPro for quadratic regression to find significantly differentially expressed genes (DEGs) over time, followed by clustering analysis of the expression velocity trends. We found that metacells generated using the SEACells algorithm retained greater between-cell variance and produced more biologically meaningful results compared to metacells generated from random cells. Quadratic regression revealed significant DEGs through time that have been previously annotated in the SARS-CoV2 infection response pathway. It also identified significant genes that have not been annotated in this pathway, which were compared to baseline expression and showed unique expression patterns through time. The results demonstrated that this strategy could overcome the limitation of 1-2 replicates, as it correctly identified the known ISG15 interferon response program in almost all PBMC cell types. Its application further led to the uncovering of additional and more cell type-specific gene expression programs that potentially modulate different levels of host response after infection.","version":"1.1","doi":"10.1101/2023.12.14.571774","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.13.571479","pub_date":"2023-12-14","title":"A distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia","abstract":"Pathogen clearance and resolution of inflammation in patients with pneumonia require an effective local T cell response. Nevertheless, local T cell activation may drive lung injury, particularly during prolonged episodes of respiratory failure characteristic of severe SARS-CoV-2 pneumonia. While T cell responses in the peripheral blood are well described, the evolution of T cell phenotypes and molecular signatures in the distal lung of patients with severe pneumonia caused by SARS-CoV-2 or other pathogens is understudied. Accordingly, we serially obtained 432 bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia and respiratory failure, including 74 unvaccinated patients with COVID-19, and performed flow cytometry, transcriptional, and T cell receptor profiling on sorted CD8+ and CD4+ T cell subsets. In patients with COVID-19 but not pneumonia secondary to other pathogens, we found that early and persistent enrichment in CD8+ and CD4+ T cell subsets correlated with survival to hospital discharge. Activation of interferon signaling pathways early after intubation for COVID-19 was associated with favorable outcomes, while activation of NF-\u03baB-driven programs late in disease was associated with poor outcomes. Patients with SARS-CoV-2 pneumonia whose alveolar T cells preferentially targeted the Spike and Nucleocapsid proteins tended to experience more favorable outcomes than patients whose T cells predominantly targeted the ORF1ab polyprotein complex. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize patients who recover, yet these responses progress to NF-\u03baB activation against non-structural proteins in patients who go on to experience poor clinical outcomes.","version":"1.1","doi":"10.1101/2023.12.13.571479","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.13.571570","pub_date":"2023-12-14","title":"Inactivation of the Niemann Pick C1 cholesterol transporter 1 (NPC1) restricts SARS-CoV-2 infection","abstract":"The Niemann Pick C1 (NPC1) protein is an intracellular cholesterol transporter located in the late endosome/lysosome (LE/Ly) and is involved in cholesterol mobilization. Loss-of-function mutations of the NPC1 gene lead to accumulation of cholesterol and sphingolipids in LE/Ly, resulting in severe fatal NPC1 disease. Cellular alterations associated with NPC1 inactivation affect both the integrity of lipid rafts and the endocytic pathway. Because the angiotensin-converting enzyme 2 (ACE2) and type 2 serine transmembrane protease (TMPRSS2) of the SARS-CoV-2 Spike (S) protein also localize to lipid rafts, we sought to investigate the hypothesis that NPC1 inactivation would generate an intrinsically unfavorable barrier to SARS-CoV-2 entry. In this study, we demonstrate that NPC1 pharmacological inactivation or CRISP/R-Cas mediated ablation of NPC1 dramatically reduced SARS-CoV-2 infectivity. More specifically, our findings demonstrate that pharmacological inactivation of NPC1 results in massive accumulation of ACE2 in the autophagosomal/lysosomal compartment. A >40-fold decrease in virus titer indicates that this effectively prevents VSV-Spike-GFP infection by impeding virus binding and entry. A similarly marked decrease in viral infectivity is observed in cells that had NPC1 expression genetically abrogated. These observations were further confirmed in a de novo SARS-CoV-2 infection paradigm, where cells were infected with the naturally pathogenic SARS-CoV-2. Overall, this work offers strong evidence that NPC1 function is essential for successful SARS-CoV-2 infection, thus implicating NPC1 as a potential therapeutic target in COVID-19 management. A significant convergence exists between the cellular alterations associated with NPC1 inactivation and the mechanistic processes of SARS-CoV-2 infectivity. These alterations affect the integrity of lipid-enriched plasma membrane microdomains and the endocytic pathway. Furthermore, the cholesterol-regulated ACE2 receptor protein that facilitates SARS-Cov-2 viral binding and entry is targeted to the autophagolysosomal compartment upon NPC1 inactivation, thus hindering virus-host cell interaction. To our knowledge, this study provides the first evidence that NPC1 function represents a crucial factor for SARS-CoV-2 infection and suggests therapeutic opportunities.","version":"1.1","doi":"10.1101/2023.12.13.571570","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.08.556906","pub_date":"2023-12-14","title":"Differential outcomes of infection by wild-type SARS-CoV-2 and the B.1.617.2 and B.1.1.529 variants of concern in K18-hACE2 transgenic mice","abstract":"SARS-CoV-2 is a respiratory virus with neurological complications including loss of smell and taste, headache, and confusion that can persist for months or longer. Severe neuronal cell damage has also been reported in some cases. The objective of this study was to compare the infectivity of Wild-type virus, Delta and Omicron variants in transgenic mice that express the human angiotensin-converting enzyme 2 (hACE2) receptor under the control of the keratin 18 promoter (K18) and characterize the progression of infection and inflammatory response in the lung, brain medulla oblongata and olfactory bulbs of these animals. We hypothesized that Wild-type, Delta and Omicron differentially infect K18-hACE2 mice, thereby inducing distinct cellular responses. K18-hACE2 female mice were intranasally infected with Wild-type, Delta, or Omicron variants and euthanized either at 3 days post-infection (dpi) or at the humane endpoint. None of the animals infected with the Omicron variant reached the humane endpoint and were euthanized at day 8 dpi. Virological and immunological analyses were performed in the lungs, olfactory bulbs, medulla oblongata, and brains. Mice infected with Wild-type and Delta display higher levels of viral RNA in the lungs than mice infected with Omicron at 3dpi. Viral RNA levels in the brains of mice infected with the Wild-type virus were however significantly lower than those observed in mice infected with either Delta or Omicron at 3dpi. Viral RNA was also detected in the medulla oblongata of mice infected by all these virus strains at 3dpi. At this time point, mice infected with the Delta virus display a marked upregulation of inflammatory makers both in the lungs and brains. Upregulation of inflammatory markers was also observed in the brains of mice infected with Omicron but not in mice infected with the Wild-type virus, suggesting that during the initial phase of the infection only the Delta and Omicron variants induce strong inflammatory response in the brain. At the humane endpoint/8dpi, mice infected by any of these strains display elevated levels of viral RNA and upregulation of a subset of inflammatory markers in the lungs. There was also a significant increase in viral RNA in the brains of mice infected with Wild-type and Delta, as compared to 3dpi. This was accompanied by an increase in the expression of most cytokines and chemokines. In contrast, mice infected with the Omicron variant showed low levels of viral RNA and downregulation of cytokines and chemokines expression at 8dpi, suggesting that brain inflammation by this variant is attenuated. Reduced RNA levels and downregulation of inflammatory markers was also observed in the medulla oblongata and olfactory bulbs of mice infected with Omicron, while infection by Wild-type and Delta resulted in high levels of viral RNA and increased expression of inflammatory makers in these organs.","version":"1.2","doi":"10.1101/2023.09.08.556906","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.12.571262","pub_date":"2023-12-13","title":"Syncytia Formation Promotes Virus Resistance to Interferon and Neutralizing Antibodies","abstract":"SARS-CoV-2, like many viruses, generates syncytia. Using SARS-CoV-2 and S (S) expressing recombinant vesicular stomatitis and influenza A viruses, we show that S-mediated syncytia formation provides resistance to interferons in cultured cells, human small airway-derived air-liquid interface cultures and hACE2 transgenic mice. Amino acid substitutions that modulate fusogenicity in Delta- and Omicron-derived S have parallel effects on viral interferon resistance. Syncytia formation also decreases antibody virus neutralization activity in cultured cells. These findings explain the continued selection of fusogenic variants during SARS-CoV-2 evolution in humans and, more generally, the evolution of fusogenic viruses despite the adverse effects of syncytia formation on viral replication in the absence of innate or adaptive immune pressure.","version":"1.1","doi":"10.1101/2023.12.12.571262","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.20.524893","pub_date":"2023-12-12","title":"Whole transcriptome profiling of placental pathobiology in SARS-CoV-2 pregnancies identifies placental dysfunction signatures","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a \u201cpreeclampsia-like syndrome\u201d. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. We utilized whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n=7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n=9). Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified signatures associated with hypoxia and placental dysfunction during SARS-CoV-2 infection in pregnancy. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH), and preeclampsia (FLT1, EGFR, KISS1, PAPPA2), were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension, and inflammation, were also enriched in SARS-CoV-2 samples compared to uninfected controls. Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection. In this study, using spatial digital profiling transcriptomic approaches, we demonstrate that SARS-CoV-2 infection in pregnancy disrupts optimal placental function by altering the genomic architecture of trophoblasts and villous core stromal cells.","version":"1.2","doi":"10.1101/2023.01.20.524893","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.12.570971","pub_date":"2023-12-12","title":"G6PD deficiency mediated impairment of iNOS and lysosomal acidification affecting phagocytotic clearance in microglia in response to SARS-CoV-2","abstract":"The glucose-6-phosphate dehydrogenase (G6PD) deficiency is X-linked and is the most common enzymatic deficiency disorder globally. It is a crucial enzyme for the pentose phosphate pathway and produces NADPH, which plays a vital role in the regulation of oxidative stress of many cell types. The deficiency of G6PD causes hemolytic anemia, diabetes, cardiovascular and neurological disorders. Notably, the patient with G6PD deficiency was severely affected by SARS-CoV-2 and showed prolonged COVID-19 symptoms, neurological impacts, and high mortality. However, the mechanism of COVID-19 severity in G6PD deficient patients is still ambiguous. Here, using a CRISPR-edited G6PD deficient human microglia cell culture model, we observed a significant reduction in NADPH and an increase in basal reactive oxygen species (ROS) in microglia. Interestingly, the deficiency of the G6PD-NAPDH axis impairs induced nitric oxide synthase (iNOS) mediated nitric oxide (NO) production which plays a fundamental role in inhibiting viral replication. Surprisingly, we also observed that the deficiency of the G6PD-NADPH axis reduced lysosomal acidification, which further abrogates the lysosomal clearance of viral particles. Thus, impairment of NO production and lysosomal acidification as well as redox dysregulation in G6PD deficient microglia altered innate immune response, promoting the severity of SARS-CoV-2 pathogenesis.","version":"1.1","doi":"10.1101/2023.12.12.570971","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.11.571109","pub_date":"2023-12-12","title":"The complement pattern recognition molecule CL-11 promotes invasion and injury of respiratory epithelial cells by SARS-CoV-2","abstract":"Collectin-11 is a soluble C-type lectin produced at epithelial surfaces to initiate pathogen elimination by complement. Given the respiratory epithelium is a source of CL-11 and downstream complement-pathway components, we investigated the potential of CL-11 to impact the pathogenicity of SARS-CoV-2. While the SARS-CoV-2 spike trimer could bind CL-11 and trigger complement activation followed by MAC formation, the virus was resistant to lysis. Surprisingly, virus production by infected respiratory epithelial cells was enhanced by CL-11 opsonisation of virus but this effect was fully inhibited by sugar-blockade of CL-11. Moreover, SARS-CoV-2 spike protein expressed at the bronchial epithelial cell surface was associated with increased CL-11 binding and MAC formation. We propose that SARS-CoV-2 pathogenicity is exacerbated both by resistance to complement and CL-11 driven respiratory cell invasion and injury at the portal of entry. Contrary to expectation, CL-11 blockade could offer a novel approach to limit the pathogenicity of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.12.11.571109","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.11.571056","pub_date":"2023-12-12","title":"SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity","abstract":"It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved flexible pyrimidine nucleotides located in AU-rich areas and mismatch-containing areas in dsRNA via a nicking manner. The AU content and distribution in dsRNA along with the RNA length affected cleavage by SARS-CoV-2 nsp15. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.","version":"1.1","doi":"10.1101/2023.12.11.571056","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.11.571185","pub_date":"2023-12-12","title":"AlphaFold2-Enabled Atomistic Modeling of Epistatic Binding Mechanisms for the SARS-CoV-2 Spike Omicron XBB.1.5, EG.5 and FLip Variants: Convergent Evolution Hotspots Cooperate to Control Stability and Conformational Adaptability in Balancing ACE2 Binding and Antibody Resistance","abstract":"In this study, we combined AI-based atomistic structural modeling and microsecond molecular simulations of the SARS-CoV-2 Spike complexes with the host receptor ACE2 for XBB.1.5+L455F, XBB.1.5+F456L(EG.5) and XBB.1.5+L455F/F456L (FLip) lineages to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and physics-based rigorous computations of binding affinities, we identified binding energy hotspots and characterized molecular basis underlying epistatic couplings between convergent mutational hotspots. Consistent with the experiments, the results revealed the mediating role of Q493 hotspot in synchronization of epistatic couplings between L455F and F456L mutations providing a quantitative insight into the mechanism underlying differences between XBB lineages. Mutational profiling is combined with network-based model of epistatic couplings showing that the Q493, L455 and F456 sites mediate stable communities at the binding interface with ACE2 and can serve as stable mediators of non-additive couplings. Structure-based mutational analysis of Spike protein binding with the class 1 antibodies quantified the critical role of F456L and F486P mutations in eliciting strong immune evasion response. The results of this analysis support a mechanism in which the emergence of EG.5 and FLip variants may have been dictated by leveraging strong epistatic effects between several convergent revolutionary hotspots that provide synergy between the improved ACE2 binding and broad neutralization resistance. This interpretation is consistent with the notion that functionally balanced substitutions which simultaneously optimize immune evasion and high ACE2 affinity may continue to emerge through lineages with beneficial pair or triplet combinations of RBD mutations involving mediators of epistatic couplings and sites in highly adaptable RBD regions.","version":"1.1","doi":"10.1101/2023.12.11.571185","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.11.571101","pub_date":"2023-12-11","title":"Restoring Protein Glycosylation with GlycoShape","abstract":"During the past few years, we have been witnessing a revolution in structural biology. Leveraging on technological and computational advances, scientists can now resolve biomolecular structures at the atomistic level of detail by cryogenic electron microscopy (cryo-EM) and predict 3D structures from sequence alone by machine learning (ML). One technique often supports the other to provide the view of atoms in molecules required to capture the function of molecular machines. An example of the extraordinary impact of these advances on scientific discovery and on public health is given by how structural information supported the rapid development of COVID-19 vaccines based on the SARS-CoV-2 spike (S) glycoprotein. Yet, none of these new technologies can capture the details of the dense coat of glycans covering S, which is responsible for its natural, biologically active structure and function and ultimately for viral evasion. Indeed, glycosylation, the most abundant post-translational modification of proteins, is largely invisible through experimental structural biology and in turn it cannot be reproduced by ML, because of the lack of data to learn from. Molecular simulations through high-performance computing (HPC) can fill this crucial information gap, yet the computational resources, the users\u2019 skills and the long timescales involved limit applications of molecular modelling to single study cases. To broaden access to structural information on glycans, here we introduce GlycoShape (https://glycoshape.org) an open access (OA) glycan structure database and toolbox designed to restore glycoproteins to their native functional form by supplementing the structural information available on proteins in public repositories, such as the RCSB PDB (www.rcsb.org) and AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/), with the missing glycans derived from over 1 ms of cumulative sampling from molecular dynamics (MD) simulations. The GlycoShape Glycan Database (GDB) currently counts over 435 unique glycans principally covering the human glycome and with additional structures, fragments, and epitopes from other eukaryotic and prokaryotic organisms. The GDB feeds into Re-Glyco, a bespoke algorithm in GlycoShape designed to rapidly restore the natural glycosylation to protein 3D structures and to predict N-glycosylation occupancy, where unknown. Ultimately, integration of GlycoShape with other OA protein structure databases can provide a step-change in scientific discovery, from the structural and functional characterization of the active form of biomolecules, all the way down to pharmacological applications and drug discovery.","version":"1.1","doi":"10.1101/2023.12.11.571101","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.19.553950","pub_date":"2023-12-09","title":"Genome-wide Bioinformatics Analysis of Human Protease Specificity Identified Potential Cathepsin L Cleavage Site at K790 Position of the SARS-CoV-2 Spike Glycoprotein","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) primarily enters the cell by binding the virus\u2019s spike (S) glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface, followed by proteolytic cleavage by host proteases. Studies have identified furin and TMPRSS2 proteases in priming and triggering cleavages of the S glycoprotein, converting it into a fusion-competent form and initiating membrane fusion, respectively. Alternatively, SARS-CoV-2 can enter the cell through the endocytic pathway, where activation is triggered by lysosomal cathepsin L. However, other proteases are also suspected to be involved in both entry routes. In this study, we conducted a genome-wide bioinformatics analysis to explore the capacity of human proteases in hydrolyzing peptide bonds of the S glycoprotein. Predictive models of sequence specificity for 169 human proteases were constructed and applied to the S glycoprotein together with the method for predicting structural susceptibility to proteolysis of protein regions. After validating our approach on extensively studied S2\u2019 and S1/S2 cleavage sites, we applied our method to each peptide bond of the S glycoprotein across all 169 proteases. Our results indicate that various members of the PCSK, TTSP, and kallikrein families, as well as specific coagulation factors, are capable of cleaving S2\u2019 or S1/S2 sites. We have also identified a potential cleavage site of cathepsin L at the K790 position within the S2\u2019 loop. Structural analysis suggests that cleavage of this site induces conformational changes similar to the cleavage at the R815 (S2\u2019) position, leading to the exposure of the fusion peptide and subsequent fusion with the membrane. Other potential cleavage sites and the influence of mutations in common SARS-CoV-2 variants on proteolytic efficiency are discussed.","version":"1.3","doi":"10.1101/2023.08.19.553950","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.08.570782","pub_date":"2023-12-08","title":"Virological characteristics of the SARS-CoV-2 JN.1 variant","abstract":"The SARS-CoV-2 BA.2.86 lineage, first identified in August 2023, is phylogenetically distinct from the currently circulating SARS-CoV-2 Omicron XBB lineages, including EG.5.1 and HK.3. Comparing to XBB and BA.2, BA.2.86 carries more than 30 mutations in the spike (S) protein, indicating a high potential for immune evasion. BA.2.86 has evolved and its descendant, JN.1 (BA.2.86.1.1), emerged in late 2023. JN.1 harbors S:L455S and three mutations in non-S proteins. S:L455S is a hallmark mutation of JN.1: we have recently shown that HK.3 and other \u201cFLip\u201d variants carry S:L455F, which contributes to increased transmissibility and immune escape ability compared to the parental EG.5.1 variant. Here, we investigated the virological properties of JN.1.","version":"1.1","doi":"10.1101/2023.12.08.570782","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.07.570670","pub_date":"2023-12-08","title":"SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized COVID-19 patients","abstract":"Coronavirus disease 2019 (COVID-19) and associated severity has been linked to uncontrolled inflammation and may be associated with changes in the microbiome of mucosal sites including the gastrointestinal tract and oral cavity. These sites play an important role in host-microbe homeostasis and disruption of epithelial barrier integrity during COVID-19 may potentially lead to exacerbated inflammation and immune dysfunction. Outcomes in COVID-19 are highly disparate, ranging from asymptomatic to fatal, and the impact of microbial dysbiosis on disease severity is unclear. Here, we obtained plasma, rectal swabs, oropharyngeal swabs, and nasal swabs from 86 patients hospitalized with COVID-19 and 12 healthy volunteers. We performed 16S rRNA sequencing to characterize the microbial communities in the mucosal swabs and measured circulating cytokines, markers of gut barrier integrity, and fatty acids in the plasma samples. We compared these plasma concentrations and microbiomes between healthy volunteers and the COVID-19 patients who had survived or unfortunately died by the end of study enrollment, and between severe disease and healthy controls, as well as performed a correlation analysis between plasma variables and bacterial abundances. The rectal swabs of COVID-19 patients had reduced abundances of several commensal bacteria including Faecalibacterium prausnitsii, and an increased abundance of the opportunistic pathogens Eggerthella lenta and Hungatella hathewayi. Furthermore, the oral pathogen Scardovia wiggsiae was more abundant in the oropharyngeal swabs of COVID-19 patients who died. The abundance of both H. hathewayi and S. wiggsiae correlated with circulating inflammatory markers including IL-6, highlighting the possible role of the microbiome in COVID-19 severity, and providing potential therapeutic targets for managing COVID-19.","version":"1.1","doi":"10.1101/2023.12.07.570670","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.528032","pub_date":"2023-12-07","title":"SARS-COV-2 NSP5 Antagonizes MHC II Expresion by Subverting Histone Deacetylase 2","abstract":"SARS-CoV-2 interferes with antigen presentation by downregulating MHC II on antigen presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. This downregulation of CIITA is independent of NSP5\u2019s proteolytic activity, but rather, NSP5 delivers HDAC2 to IRF3 at an IRF binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response. SARS-CoV-2 alters the expression of many immunoregulatory proteins to limit and delay the host antiviral response, thereby producing a more severe and longer-lasting infection. Preventing and limiting the activation of helper T cells by reducing MHC II expression on antigen presenting cells is one of these strategies, but while this mechanism was identified early in the pandemic, the mechanism allowing SARS-CoV-2 to limit MHC II expression has remained unclear. Herein, we demonstrate that this occurs via a tripartite interaction between viral NSP5 and host HDAC2 and IRF3, where a complex of NSP5 and HDAC2 is recruited to IRF3 bound to the promoter of CIITA\u2014the master regulator of MHC II expression\u2014with the delivery of HDAC2 then mediating the deacetylation of the CIITA promoter and the suppression of MHC II expression.","version":"1.3","doi":"10.1101/2023.02.10.528032","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.05.570216","pub_date":"2023-12-07","title":"Algorithm for selecting potential SARS-CoV-2 dominant variants based on POS-NT frequency","abstract":"COVID-19, currently prevalent worldwide, is caused by a novel coronavirus, SARS-CoV-2. Similar to other RNA viruses, SARS-CoV-2 continues to evolve through random mutations, creating numerous variants, such as Alpha, Beta, and Delta. It is, therefore, necessary to predict the mutations constituting the dominant variant before they are generated. This can be achieved by continuously monitoring the mutation trends and patterns. Hence, in the current study, we sought to design a dominant variant candidate (DVC) selection algorithm. To this end, we obtained COVID-19 sequence data from GISAID and extracted position-nucleotide (POS-NT) frequency ratio data by country and date through data preprocessing. We then defined the dominant dates for each variant in the USA and developed a frequency ratio prediction model for each POS-NT. Based on this model, we applied DVC criteria to develop the selection algorithm, verified for Delta and Omicron. Using Condition 3 as the DVC criterion, 69 and 102 DVC POS-NTs were identified for Delta and Omicron an average of 47 and 82 days before the dominant dates, respectively. Moreover, 13 and 44 Delta- and Omicron-defining POS-NTs were recognized 18 and 25 days before the dominant dates, respectively. We identified all DVC POS-NTs before the dominant dates, including soaring and gently increasing POS-NTs. Considering that we successfully defined all POS-NT mutations for Delta and Omicron, the DVC algorithm may represent a valuable tool for providing early predictions regarding future variants, helping improve global health.","version":"1.1","doi":"10.1101/2023.12.05.570216","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.05.570280","pub_date":"2023-12-07","title":"In Silico Therapeutic Intervention on Cytokine Storm in COVID-19","abstract":"The recent global COVID-19 outbreak, attributed by the World Health Organization to the rapid spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), underscores the need for an extensive exploration of virological intricacies, fundamental pathophysiology, and immune responses. This investigation is vital to unearth potential therapeutic avenues and preventive strategies. Our study delves into the intricate interaction between SARS-CoV-2 and the immune system, coupled with exploring therapeutic interventions to counteract dysfunctional immune responses like the \u2018cytokine storm\u2019 (CS), a driver of disease progression. Understanding these immunological dimensions informs the design of precise multiepitopetargeted peptide vaccines using advanced immunoinformatics and equips us with tools to confront the cytokine storm. Employing a control theory-based approach, we scrutinize the perturbed behavior of key proteins associated with cytokine storm during COVID-19 infection. Our findings support ACE2 activation as a potential drug target for CS control and confirm AT1R inhibition as an alternative strategy. Leveraging deep learning, we identify potential drugs to individually target ACE2 and AT1R, with Lomefloxacin and Fostamatinib emerging as standout options due to their close interaction with ACE2. Their stability within the protein-drug complex suggests superior efficacy among many drugs from our deep-learning analysis. Moreover, there is a significant scope for optimization in fine-tuning protein-drug interactions. Strong binding alone may not be the sole determining factor for potential drugs; precise adjustments are essential. The application of advanced computational power offers novel solutions, circumventing time-consuming lab work. In scenarios necessitating both ACE2 and AT1R targeting, optimal drug combinations can be derived from our analysis of drug-drug interactions, as detailed in the manuscript.","version":"1.1","doi":"10.1101/2023.12.05.570280","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.05.570187","pub_date":"2023-12-07","title":"From Viral Infections to Alzheimer\u2019s Disease: Unveiling the Mechanistic Links Through Systems Bioinformatics","abstract":"Emerging evidence suggests that certain microorganisms, including viral infections, may contribute to the onset and/or progression of Alzheimer\u2019s Disease (AD), a neurodegenerative condition characterized by memory impairment and cognitive decline. However, the precise extent of their involvement and the underlying mechanisms through which specific viruses increase AD susceptibility risk remain elusive. We used an integrative systems bioinformatics approach to identity viral-mediated pathogenic mechanisms by which specific viral species, namely Herpes Simplex Virus 1 (HSV-1), Human Cytomegalovirus (HCMV), Epstein-Barr Virus (EBV), Kaposi Sarcoma-associated Herpesvirus (KSHV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Influenza A virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), could facilitate the pathogenesis of AD via virus-host protein-protein interactions (PPIs). We also sought to uncover potential synergistic pathogenic effects resulting from the reactivation of specific herpesviruses (HSV-1, HCMV and EBV) during acute SARS-CoV-2 infection, potentially increasing AD susceptibility. Our findings show that Herpesviridae Family members (HSV-1, EBV, KSHV, HCMV) impact AD-related processes like amyloid-beta formation, neuronal death, and autophagy. Hepatitis viruses (HBV, HCV) influence processes crucial for cellular homeostasis and dysfunction. Importantly, hepatitis viruses affect microglia activation via virus-host PPIs. Reactivation of HCMV during SARS-CoV-2 infection could potentially foster a lethal interplay of neurodegeneration, via synergistic pathogenic effects on AD-related processes like response to unfolded protein, regulation of autophagy, response to oxidative stress and amyloid-beta formation. Collectively, these findings underscore the complex link between viral infections and AD development. Perturbations in AD-related processes by viruses can arise from both shared and distinct mechanisms among viral species in different categories, potentially influencing variations in AD susceptibility.","version":"1.1","doi":"10.1101/2023.12.05.570187","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.01.569227","pub_date":"2023-12-06","title":"High-throughput ML-guided design of diverse single-domain antibodies against SARS-CoV-2","abstract":"Treating rapidly evolving pathogenic diseases such as COVID-19 requires a therapeutic approach that accommodates the emergence of viral variants over time. Our machine learning (ML)-guided sequence design platform combines high-throughput experiments with ML to generate highly diverse single-domain antibodies (VHHs) that bind and neutralize SARS-CoV-1 and SARS-CoV-2. Crucially, the model, trained using binding data against early SARS-CoV variants, accurately captures the relationship between VHH sequence and binding activity across a broad swathe of sequence space. We discover ML-designed VHHs that exhibit considerable cross-reactivity and successfully neutralize targets not seen during training, including the Delta and Omicron BA.1 variants of SARS-CoV-2. Our ML-designed VHHs include thousands of variants 4-15 mutations from the parent sequence with significantly improved activity, demonstrating that ML-guided sequence design can successfully navigate vast regions of sequence space to unlock and future-proof potential therapeutics against rapidly evolving pathogens.","version":"1.2","doi":"10.1101/2023.12.01.569227","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.27.568816","pub_date":"2023-12-06","title":"Modulation of human kinase activity through direct interaction with SARS-CoV-2 proteins","abstract":"The dysregulation of cellular signaling upon SARS-CoV-2 infection is mediated via direct protein interactions, with the human protein kinases constituting the major impact nodes in the signaling networks. Here, we employed a targeted yeast two-hybrid matrix approach to identify direct SARS-CoV-2 protein interactions with an extensive set of human kinases. We discovered 51 interactions involving 14 SARS-CoV-2 proteins and 29 human kinases, including many of the CAMK and CMGC kinase family members, as well as non-receptor tyrosine kinases. By integrating the interactions identified in our screen with transcriptomics and phospho-proteomics data, we revealed connections between SARS-CoV-2 protein interactions, kinase activity changes, and the cellular phospho-response to infection and identified altered activity patterns in infected cells for AURKB, CDK2, CDK4, CDK7, ABL2, PIM2, PLK1, NEK2, TRIB3, RIPK2, MAPK13, and MAPK14. Finally, we demonstrated direct inhibition of the FER human tyrosine kinase by the SARS-CoV-2 auxiliary protein ORF6, hinting at pressures underlying ORF6 changes observed in recent SARS-CoV-2 strains. Our study expands the SARS-CoV-2 \u2013 host interaction knowledge, illuminating the critical role of dysregulated kinase signaling during SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2023.11.27.568816","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.26.568730","pub_date":"2023-12-06","title":"XBB.1.5 monovalent mRNA vaccine booster elicits robust neutralizing antibodies against emerging SARS-CoV-2 variants","abstract":"COVID-19 vaccines have recently been updated with the spike protein of SARS-CoV-2 XBB.1.5 subvariant alone, but their immunogenicity in humans has yet to be fully evaluated and reported, particularly against emergent viruses that are rapidly expanding. We now report that administration of an updated monovalent mRNA vaccine (XBB.1.5 MV) to uninfected individuals boosted serum virus-neutralization antibodies significantly against not only XBB.1.5 (27.0-fold) and the currently dominant EG.5.1 (27.6-fold) but also key emergent viruses like HV.1, HK.3, JD.1.1, and JN.1 (13.3-to-27.4-fold). In individuals previously infected by an Omicron subvariant, serum neutralizing titers were boosted to highest levels (1,504-to-22,978) against all viral variants tested. While immunological imprinting was still evident with the updated vaccines, it was not nearly as severe as the previously authorized bivalent BA.5 vaccine. Our findings strongly support the official recommendation to widely apply the updated COVID-19 vaccines to further protect the public.","version":"1.2","doi":"10.1101/2023.11.26.568730","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.03.569831","pub_date":"2023-12-05","title":"Identification and Analysis of SARS-CoV-2 Mutation and Subtype using 2x tiled Primer Set with Oxford Nanopore Technologies Sequencing for Enhanced Variant Detection and Surveillance in Seoul, Korea","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a respiratory virus that contains RNA as its genetic material and has caused a global pandemic since its outbreak in 2020. This virus has many mutations, some of which can reduce the effectiveness of existing vaccines. Therefore, next-generation sequencing (NGS) is necessary to accurately identify new mutations. Current NGS analysis of SARS-CoV-2 uses the amplicon analysis method through a multiplex polymerase chain reaction. This study collected and validated RNA samples from patients who tested positive for SARS-CoV-2 from April to July 2022, and selected 613 samples for sequencing. The findings demonstrate the importance of long-read-based NGS analysis and 2x tiled primer set for identifying full SARS-CoV-2 genome sequence with new mutations and understanding the correlation between viral genotypes and patient characteristics for the effective management of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.12.03.569831","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.05.569965","pub_date":"2023-12-05","title":"Multi-variate statistical and machine learning reveals the interplay between sex and age in antibody responses to de novo SARS-CoV-2 infection and vaccination","abstract":"Prevention of negative COVID19 infection outcomes and infection/vaccine-acquired immunity is associated with the quality of antibody responses, whose variance by age and sex are poorly understood. Integrated, network approaches, identified sex and age effects in antibody responses and neutralization potential of de novo infection and vaccination throughout the Covid-19 pandemic. Cluster analysis found neutralization values followed SARS-CoV-2 specific receptor binding RIgG, spike SIgG and S and RIgA levels based on COVID19 status. Stochastic behavior tests and other analytical methods revealed sex differences only in persons <40y.o. Serum IgA antibody titers correlated with neutralization only in females 40-60y.o. Network analysis found males could improve IgA responses after vaccination dose 2, unlike >60y.o. females. Complex correlation analyses found vaccination induced less antibody isotype switching and neutralization in older persons, especially in females. Sex dependent antibody & neutralization behavior decayed fastest in older males and with vaccination. Such sex and age characterization by machine learning can direct studies integrating cell mediated responses to define yet elusive correlates of protection and inform age and sex precision-focused vaccine design.","version":"1.1","doi":"10.1101/2023.12.05.569965","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.04.569985","pub_date":"2023-12-05","title":"High throughput screening identifies broad-spectrum Coronavirus entry inhibitors","abstract":"The Covid-19 pandemic highlighted the pressing need for antiviral therapeutics capable of mitigating infection and spread of emerging coronaviruses (CoVs). A promising therapeutic strategy lies in inhibiting viral entry mediated by the Spike (S) glycoprotein. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified potential inhibitors by assessing their ability to inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Subsequent counter-screening against pseudoviruses with the Vesicular Stomatitis Virus glycoprotein (VSV-G), yielding sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds including the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested them against pseudoviruses bearing the S glycoprotein of the alpha, delta, and omicron variants as well as against bona fide SARS-CoV-2 in vitro. This rigorous approach revealed a novel inhibitor and its derivative as a potential broad-spectrum antiviral. These results validate the HTS platform and set the stage for lead optimization and future pre-clinical, in vivo studies.","version":"1.1","doi":"10.1101/2023.12.04.569985","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.13.532446","pub_date":"2023-12-04","title":"Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage","abstract":"The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-\u03b1 and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.","version":"1.2","doi":"10.1101/2023.03.13.532446","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.04.569917","pub_date":"2023-12-04","title":"Identification of key residues in MERS-CoV and SARS-CoV-2 main proteases for resistance against clinically applied inhibitors nirmatrelvir and ensitrelvir","abstract":"The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic, zoonotically emerging pathogen initially reported in Saudi Arabia in 2012. MERS-CoV has the potential to mutate or recombine with other coronaviruses, thus acquiring the ability to efficiently spread among humans and become pandemic. Its high mortality rate of up to 35 % and the absence of effective targeted therapies call for the development of antiviral drugs for this pathogen. Since the beginning of the SARS-CoV-2 pandemic, extensive research has focused on identifying protease inhibitors for the treatment of SARS-CoV-2. Our intention was therefore to assess whether these protease inhibitors are viable options for combating MERS-CoV. To that end, we used previously established protease assays to quantify inhibition of the SARS-CoV-2 and MERS-CoV main proteases. Furthermore, we selected MERS-CoV-Mpro mutants resistant against nirmatrelvir, the most effective inhibitor of this protease, with a safe, surrogate virus-based system, and suggest putative resistance mechanisms. Notably, nirmatrelvir demonstrated effectiveness against various viral proteases, illustrating its potential as a broad-spectrum coronavirus inhibitor. To adress the inherent resistance of MERS-CoV-Mpro to ensitrelvir, we applied directed mutagenesis to a key ensitrelvir-interacting residue and provided structural models. We investigate antivirals for MERS-CoV with a pool of SARS-CoV-2 antiviral drugs and study potential resistances developing against those drugs.","version":"1.1","doi":"10.1101/2023.12.04.569917","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.24.568532","pub_date":"2023-12-04","title":"Isogenic iPSC-derived proximal and distal lung-on-chip models: Tissue- and virus-specific immune responses in human lungs","abstract":"Micro-physiological systems (MPS) are set to play a vital role in preclinical studies, particularly in the context of future viral pandemics. Nonetheless, the development of MPS is often impeded by the scarcity of reliable cell sources, especially when seeking various organs or tissues from a single patient for comparative analysis of the host immune response. Herein, we developed human airway-on-chip and alveolus-on-chip models using induced pluripotent stem cell (iPSC)-derived isogenic lung progenitor cells. Both models demonstrated the replication of two different respiratory viruses, namely SARS-CoV-2 and Influenza, as well as related cellular damage and innate immune responses-on-chip. Our findings reveal distinct immune responses to SARS-CoV-2 in the proximal and distal lung-on-chip models. The airway chips exhibited a robust interferon (IFN)-dependent immune response, whereas the alveolus chips exhibited dysregulated IFN activation but a significantly upregulated chemokine pathway. In contrast, Influenza virus infection induced a more pronounced immune response and cellular damage in both chip models compared to SARS-CoV-2. Thus, iPSC-derived lung-on-chip models may aid in quickly gaining insights into viral pathology and screening potential drugs for future pandemics.","version":"1.2","doi":"10.1101/2023.11.24.568532","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.23.554506","pub_date":"2023-12-04","title":"Gut microbiome remains stable following COVID-19 vaccination in healthy and immuno-compromised individuals","abstract":"The bidirectional interaction between the immune system and the gut microbiota is a key contributor to various host physiological functions. Immune-associated diseases such as cancer and autoimmunity, as well as the efficacy of immunomodulatory therapies, have been linked to microbiome variation. While COVID-19 infection has been shown to cause microbial dysbiosis, it remains understudied whether the inflammatory response associated with vaccination also impacts the microbiota. Here, we investigate the temporal impact of COVID-19 vaccination on the gut microbiome in healthy and immuno-compromised individuals; the latter included patients with primary immunodeficiency and cancer patients on immunomodulating therapies. We find that the gut microbiome remained remarkably stable post-vaccination irrespective of diverse immune status, vaccine response, and microbial composition spanned by the cohort. The stability is evident at all evaluated levels including diversity, phylum, species, and functional capacity. Our results indicate the resilience of the gut microbiome to host immune changes triggered by COVID-19 vaccination and suggest minimal, if any, impact on microbiome-mediated processes. These findings encourage vaccine acceptance, particularly when contrasted with the significant microbiome shifts observed during COVID-19 infection.","version":"1.3","doi":"10.1101/2023.08.23.554506","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.01.569653","pub_date":"2023-12-03","title":"Design of SARS-CoV-2 papain-like protease inhibitor with antiviral efficacy in a mouse model","abstract":"The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PLpro) is a promising but challenging drug target. In this study, we designed and synthesized 85 noncovalent PLpro inhibitors that bind to the newly discovered Val70Ub site and the known BL2 groove pocket. Potent compounds inhibited PLpro with inhibitory constant Ki values from 13.2 to 88.2 nM. The co-crystal structures of PLpro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC50 from 0.44 to 2.02 \u00b5M. Oral treatment with Jun12682 significantly improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting PLpro inhibitors are promising oral SARS-CoV-2 antiviral candidates. Structure-guided design of SARS-CoV-2 PLpro inhibitors with in vivo antiviral efficacy in a mouse model.","version":"1.1","doi":"10.1101/2023.12.01.569653","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.01.569639","pub_date":"2023-12-03","title":"Memory B cells dominate the early antibody-secreting cell response to SARS-CoV-2 mRNA vaccination in na\u00efve individuals independently of their antibody affinity","abstract":"Memory B cells (MBCs) formed over the individual\u2019s lifetime constitute nearly half of the adult peripheral blood B cell repertoire in humans. To assess their response to novel antigens, we tracked the origin and followed the differentiation paths of MBCs in the early anti-S response to mRNA vaccination in SARS-CoV-2-na\u00efve individuals on single-cell and monoclonal antibody level. Newly generated and pre-existing MBCs differed in their differentiation paths despite similar levels of SARS-CoV-2 and common corona virus S-reactivity. Pre-existing highly mutated MBCs showed no signs of germinal center re-entry and rapidly developed into mature antibody secreting cells (ASCs). In contrast, newly generated MBCs derived from na\u00efve precursors showed strong signs of antibody affinity maturation before differentiating into ASCs. Thus, although pre-existing human MBCs have an intrinsic propensity to differentiate into ASCs, the quality of the anti-S antibody and MBC response improved through the clonal selection and affinity maturation of na\u00efve precursors. mRNA vaccination of SARS-CoV-2 na\u00efve individuals recruits na\u00efve and pre-existing MBCs with similar levels of S-reactivity into the response S-reactive na\u00efve but not pre-existing MBCs undergo affinity maturation S-reactive pre-existing MBCs dominate the early ASC response independent of their antigen affinity High-affinity S-reactive MBCs and ASCs develop over time and originate from affinity matured na\u00efve precursors","version":"1.1","doi":"10.1101/2023.12.01.569639","journal":"bioRxiv","score":null},{"id":"10.1101/2023.12.01.569046","pub_date":"2023-12-03","title":"Substrate recognition and selectivity in SARS-CoV-2 main protease: Unveiling the role of subsite interactions through dynamical nonequilibrium molecular dynamics simulations","abstract":"The main protease (Mpro) of the SARS-CoV-2 coronavirus employs a cysteine-histidine dyad in its active site to catalyse hydrolysis of the viral polyproteins. It is well established that binding of the substrate P1-Gln in the S1 subsite of Mpro active site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how Mpro dynamically recognises and responds to substrate binding remains difficult to probe by experimental methods. We thus employed the dynamical nonequilibrium molecular dynamics (D-NEMD) approach to probe the response of Mpro to systematic substrate variations. The results emphasise the importance of P1-Gln for initiating a productive enzymatic reaction. Specifically, substituting P1-Gln with alanine disrupts the conformations of the Cys145 and His41 dyad, causing Cys145 to transition from the productive gauche conformation to the non-productive trans conformation. Importantly, our findings indicate that Mpro exhibits dynamic responses to substrate binding and likely to substrate-mimicking inhibitors within each of the S4-S2\u2032 subsites. The results inform on the substrate selectivity requirements and shed light on the observed variations in hydrolytic efficiencies of Mpro towards different substrates. Some interactions between substrate residues and enzyme subsites involve more induced fit than others, implying that differences in functional group flexibility may optimise the binding of a substrate or inhibitor in a particular subsite.","version":"1.1","doi":"10.1101/2023.12.01.569046","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.08.566276","pub_date":"2023-12-02","title":"Generation and characterization of a multi-functional panel of monoclonal antibodies for SARS-CoV-2 research and treatment","abstract":"The Coronavirus disease 2019 (COVID19) pandemic caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron variants with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this study, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD, were generated and further characterized. These mAbs were shown by ELISA to be specific for each immunogen/antigen. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P2-20 is shown to be favorable for ELISA, immunoblotting, and immunohistochemistry (IHC), however, not live virus neutralization. In contrast, mAb CU-28-24 is most effective at live virus neutralization as well as ELISA and IHC. Moreover, mAb CU-28-24 was active against rRBD proteins from Omicron variants B.2 and B.4/B5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the immunoglobulin genes has been sequenced using Next Generation Sequencing, which allows the expression of respective recombinant proteins, thereby eliminating the need for long-term hybridoma maintenance. The synthetic peptides and hybridomas/mAbs are under the intellectual property management of the Clemson University Research Foundation, and the three CDRs have been submitted as an invention disclosure for further patenting and commercialization.","version":"1.3","doi":"10.1101/2023.11.08.566276","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.30.23299208","pub_date":"2023-12-01","title":"Safety and Immunogenicity of an Inactivated Recombinant Newcastle Disease Virus Vaccine Expressing SARS-CoV-2 Spike: A Randomised, Comparator-Controlled, Phase 2 Trial","abstract":"<jats:title>Summary</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Production of affordable coronavirus disease 2019 (COVID-19) vaccines in low- and lower-middle-income countries is needed. NDV-HXP-S is an inactivated egg-based recombinant Newcastle disease virus vaccine expressing the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A public sector manufacturer in Vietnam assessed the immunogenicity of NDV-HXP-S (COVIVAC) relative to an authorized vaccine.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    This phase 2 stage of a randomised, observer-blind, controlled, phase 1/2 trial was conducted at three community health centers in Thai Binh Province, Vietnam. Healthy males and non-pregnant females, 18 years of age and older, were eligible. Participants were randomised by age (18-59, \u226560 years) to receive one of three treatments by intramuscular injection twice, 28 days apart: COVIVAC at 3 \u00b5g or 6 \u00b5g, or AstraZeneca COVID-19 vaccine VAXZEVRIA. Participants and personnel assessing outcomes were masked to treatment. The main outcome was the induction of 50% neutralising antibody titers against vaccine-homologous pseudotyped virus 14 days (day 43) and 6 months (day 197) after the second vaccination by age group. The primary immunogenicity and safety analyses included all participants who received one dose of the vaccine.\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT05940194'>NCT05940194</jats:ext-link>\n                    .\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>During August 10-23, 2021, 737 individuals were screened, and 374 were randomised (124-125 per group); all received dose one, and three missed dose two. On day 43, the geometric mean fold rise of 50% neutralising antibody titers for subjects age 18-59 years was 31\u00b720 (COVIVAC 3 \u03bcg N=82, 95% CI 25\u00b714-38\u00b774), 35\u00b780 (COVIVAC 6 \u03bcg; N=83, 95% CI 29\u00b703-44\u00b715), 18\u00b785 (VAXZEVRIA; N=82, 95% CI 15\u00b710-23\u00b754), and for subjects age \u226560 years was 37\u00b727 (COVIVAC 3 \u03bcg; N=42, 95% CI 27\u00b743-50\u00b763), 50\u00b710 (COVIVAC 6 \u03bcg; N=40, 95% CI 35\u00b746-70\u00b776), 16\u00b711 (VAXZEVRIA; N=40, 95% CI 11\u00b773-22\u00b713). Among subjects seronegative for anti-S IgG at baseline, the day 43 geometric mean titer ratio of neutralising antibody (COVIVC 6 \u03bcg/VAXZEVRIA) was 1\u00b777 (95% CI 1\u00b730-2\u00b740) for subjects age 18-59 years and 3\u00b724 (95% CI 1\u00b798-5\u00b732) for subjects age \u226560 years. On day 197, the age-specific ratios were 1\u00b711 (95% CI 0\u00b751-2\u00b743) and 2\u00b732 (0\u00b769-7\u00b785). Vaccines were well tolerated; reactogenicity was predominantly mild and transient. The percentage of subjects with unsolicited adverse events (AEs) during 28 days after vaccinations was similar among treatments (COVIVAC 3 \u03bcg 29\u00b70%, COVIVAC 6 \u03bcg 23\u00b72%, VAXZEVRIA 31\u00b72%); no vaccine-related AE was reported.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Considering that induction of neutralising antibodies against SARS-CoV-2 has been correlated with the efficacy of COVID-19 vaccines, including VAXZEVRIA, our results suggest that vaccination with COVIVAC may afford clinical benefit matching or exceeding that of the VAXZEVRIA vaccine.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Vietnam\u2019s Institute of Vaccines and Medical Biologicals (including support from Vietnam\u2019s national COVID-19 vaccine fund and a charitable contribution from the Thien Tam fund of Vin group), Coalition for Epidemic Preparedness Innovations, a charitable contribution from Bayer AG, US National Institutes of Health.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.11.30.23299208","journal":"medRxiv","score":null},{"id":"10.1101/2023.11.29.568526","pub_date":"2023-12-01","title":"Gut Microbiome Dynamics and Predictive Value in Hospitalized COVID-19 Patients: A Comparative Analysis of Shallow and Deep Shotgun Sequencing","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has led to a wide range of clinical presentations, with respiratory symptoms being common. However, emerging evidence suggests that the gastrointestinal (GI) tract is also affected, with angiotensin-converting enzyme 2, a key receptor for SARS-CoV-2, abundantly expressed in the ileum and colon. The virus has been detected in GI tissues and fecal samples, even in cases with negative respiratory results. GI symptoms have been associated with an increased risk of ICU admission and mortality. The gut microbiome, a complex ecosystem of around 40 trillion bacteria, plays a crucial role in immunological and metabolic pathways. Dysbiosis of the gut microbiota, characterized by a loss of beneficial microbes and decreased microbial diversity, has been observed in COVID-19 patients, potentially contributing to disease severity. We conducted a comprehensive gut microbiome study in 204 hospitalized COVID-19 patients using both shallow and deep shotgun sequencing methods. We aimed to track microbiota composition changes induced by hospitalization, link these alterations to clinical procedures (antibiotics administration) and outcomes (ICU referral, survival), and assess the predictive potential of the gut microbiome for COVID-19 prognosis. Shallow shotgun sequencing was evaluated as a cost-effective diagnostic alternative for clinical settings.","version":"1.2","doi":"10.1101/2023.11.29.568526","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.29.569184","pub_date":"2023-11-30","title":"Mechanistic insights into ligand dissociation from the SARS-CoV-2 spike glycoprotein","abstract":"The COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an urgent need for effective therapeutic interventions. The spike glycoprotein of the SARS-CoV-2 is crucial for infiltrating host cells, rendering it a key candidate for drug development. By interacting with the human angiotensin-converting enzyme 2 (ACE2) receptor, the spike initiates the infection of SARS-CoV-2. Linoleate is known to bind the spike glycoprotein, subsequently reducing its interaction with ACE2. However, the detailed kinetics underlying the protein-ligand interaction remains unclear. In this study, we characterized the pathways of ligand dissociation and the conformational changes associated with the spike glycoprotein by using ligand Gaussian accelerated molecular dynamics (LiGaMD). Our simulations resulted in eight complete ligand dissociation trajectories, unveiling two distinct ligand unbinding pathways. The preference between these two pathways depends on the gate distance between two \u03b1-helices in the receptor binding domain (RBD) and the position of the N-linked glycan at N343. Our study also highlights the essential contributions of K417, N121 glycan, and N165 glycan in ligand unbinding, which are equally crucial in enhancing spike-ACE2 binding. We suggest that the presence of the ligand influences the motions of these residues and glycans, consequently reducing accessibility for spike-ACE2 binding. These findings enhance our understanding of ligand dissociation from the spike glycoprotein and offer significant implications for drug design strategies in the battle against COVID-19.","version":"1.1","doi":"10.1101/2023.11.29.569184","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.29.569330","pub_date":"2023-11-30","title":"Antiviral humoral immunity against SARS-CoV-2 Omicron subvariants induced by XBB.1.5 monovalent vaccine in infection-na\u00efve and XBB-infected individuals","abstract":"To control infection with SARS-CoV-2 Omicron XBB subvariants, the XBB.1.5 monovalent mRNA vaccine has been available since September 2023. However, we have found that natural infection with XBB subvariants, including XBB.1.5, does not efficiently induce humoral immunity against the infecting XBB subvariants. These observations raise the possibility that the XBB.1.5 monovalent vaccine may not be able to efficiently induce humoral immunity against emerging SARS-CoV-2 variants, including a variety of XBB subvariants (XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1 and HK.3) as well as BA.2.86. To address this possibility, we collected two types of sera from individuals vaccinated with the XBB.1.5 vaccine; those who had not been previously infected with SARS-CoV-2 and those who had been infected with XBB subvariants prior to XBB.1.5 vaccination. We collected sera before and 3-4 weeks after vaccination, and then performed a neutralization assay using these sera and pseudoviruses.","version":"1.1","doi":"10.1101/2023.11.29.569330","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.28.569129","pub_date":"2023-11-30","title":"Persistent immune imprinting after XBB.1.5 COVID vaccination in humans","abstract":"Immune imprinting - also known as \u2018original antigenic sin\u2019 - describes how the first exposure to a virus shapes the immunological outcome of subsequent exposures to antigenically related strains. SARS-CoV-2 Omicron breakthrough infections and bivalent COVID-19 vaccination were shown to primarily recall cross-reactive memory B cells and antibodies induced by prior mRNA vaccination with the Wuhan-Hu-1 spike rather than priming naive B cells that recognize Omicron-specific epitopes. These findings underscored a strong immune imprinting resulting from repeated Wuhan-Hu-1 spike exposures. To understand if immune imprinting can be overcome, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID mRNA vaccine booster. Our data show that the XBB.1.5 booster elicits neutralizing antibody responses against current variants that are dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. These results indicate that immune imprinting persists even after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 spike booster mRNA vaccination, which will need to be considered to guide the design of future vaccine boosters.","version":"1.1","doi":"10.1101/2023.11.28.569129","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.28.568639","pub_date":"2023-11-29","title":"Mechanism-based classification of SARS-CoV-2 Variants by Molecular Dynamics Resembles Phylogenetic Tree","abstract":"The COVID-19 pandemics has demonstrated the vulnerability of our societies to viral infectious disease. The mitigation of COVID-19 was complicated by the emergence of Variants of Concern (VOCs) with varying properties including increased transmissibility and immune evasion. Traditional population sequencing proved to be slow and not conducive for timely action. To tackle this challenge, we introduce the Persistence Score (PS) that assesses the pandemic potential of VOCs based on molecular dynamics of the interactions between the SARS-CoV-2 Receptor Binding Domain (RBD) and the ACE2 residues. Our mechanism-based classification approach successfully grouped VOCs into clinically relevant subgroups with higher sensitivity than classical affinity estimations and allows for risk assessment of hypothetical new VOCs. The PS-based interaction analysis across VOCs resembled the phylogenetic tree of SARS-Cov-2 demonstrating its predictive relevance for pandemic preparedness. Thus, PS allows for early detection of a variant\u2019s pandemic potential, and an early risk evaluation for data-driven policymaking.","version":"1.1","doi":"10.1101/2023.11.28.568639","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.28.569051","pub_date":"2023-11-29","title":"Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning","abstract":"The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we performed a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identified mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we showed that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.","version":"1.1","doi":"10.1101/2023.11.28.569051","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.28.568860","pub_date":"2023-11-28","title":"Expression and fusogenic activity of SARS CoV-2 Spike protein displayed in the HSV-1 Virion","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV) is a zoonotic pathogen that can cause severe respiratory disease in humans. The new SARS-CoV-2 is the cause of the current global pandemic termed coronavirus disease 2019 (COVID-19) that has resulted in many millions of deaths world-wide. The virus is a member of the Betacoronavirus family, its genome is a positive strand RNA molecule that encodes for many genes which are required for virus genome replication as well as for structural proteins that are required for virion assembly and maturation. A key determinant of this virus is the Spike (S) protein embedded in the virion membrane and mediates attachment of the virus to the receptor (ACE2). This protein also is required for cell-cell fusion (syncytia) that is an important pathogenic determinant. We have developed a pseudotyped herpes simplex virus type 1 (HSV-1) recombinant virus expressing S protein in the virion envelop. This virus has also been modified to express a Venus fluorescent protein fusion to VP16, a virion protein of HSV-1. The virus expressing Spike can enter cells and generates large multi-nucleated syncytia which are evident by the Venus fluorescence. The HSV-1 recombinant virus is genetically stable and virus amplification can be easily done by infecting cells. This recombinant virus provides a reproducible platform for Spike function analysis and thus adds to the repertoire of pseudotyped viruses expressing Spike. The isolation of a pseudotyped herpes simplex virus type 1 (HSV-1) virus using the Spike protein is new and innovative. This virus can be used to study entry and fusion events mediated by the S protein as well as test antibodies for their ability to neutralize this particle. In addition, these virions can be used for screening antibody specificity using the S protein displayed in its natural membrane bound conformation.","version":"1.1","doi":"10.1101/2023.11.28.568860","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.27.564378","pub_date":"2023-11-28","title":"Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls","abstract":"Understanding protein-protein interaction is essential when designing drugs or investigating biological processes. A variety of techniques can be employed in order to map the regions on proteins that are involved in binding eg., CryoEM, X-ray spectroscopy, linear epitope mapping, or mass spectrometry-based methods. The most commonly utilized mass spectrometry-based techniques are cross-linking and hydrogen-deuterium exchange (HDX). An alternative technique for identifying residues on the three-dimensional structure of proteins, that are involved in binding, can be hydroxyl radical protein footprinting (HRPF). However, this method is currently hampered by high initial cost and complex experimental setup. Here we set out to present a generally applicable method using Fenton chemistry for mapping of epitopes in a standard mass spectrometry laboratory. Furthermore, the described method illustrates the importance of controls on several levels when performing mass spectrometry-based epitope mapping. In particular, the inclusion of a negative antibody control has not previously been widely utilized in epitope mapping by HRPF analysis. In order to limit the number of false positives, we further introduced quantification by TMT labelling, thereby allowing for direct comparison between sample conditions and biological triplicates. Lastly, up to six technical replicates were incorporated in the experimental setup in order to achieve increased depth of the final analysis. Both binding and opening of regions on receptor-binding domain (RBD) from SARS-CoV-2 Spike Protein, Alpha, and Delta variants, were observed. The negative control antibody experiment combined with the high overlap between biological triplicates resulted in the exclusion of 40% of the significantly changed regions, including both binding and opening regions. The final identified binding region was mapped to a three-dimensional structure and agrees with the literature for neutralizing antibodies towards SARS-CoV-2 Spike Protein. The presented method is straightforward to implement for the analysis of HRPF in a generic MS-based laboratory. The high reliability of the data was achieved by increasing the number of technical and biological replicates combined with negative antibody controls.","version":"1.2","doi":"10.1101/2023.10.27.564378","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.24.568603","pub_date":"2023-11-27","title":"Variant- and Vaccination-Specific Alternative Splicing Profiles in SARS-CoV-2 Infections","abstract":"The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, and its subsequent variants has underscored the importance of understanding the host-viral molecular interactions to devise effective therapeutic strategies. A significant aspect of these interactions is the role of alternative splicing in modulating host responses and viral replication mechanisms. Our study sought to delineate the patterns of alternative splicing of RNAs from immune cells across different SARS-CoV-2 variants and vaccination statuses, utilizing a robust dataset of 190 RNA-seq samples from our previous studies, encompassing an average of 212 million reads per sample. We identified a dynamic alteration in alternative splicing and genes related to RNA splicing were highly deactivated in COVID-19 patients and showed variant- and vaccination-specific expression profiles. Overall, Omicron-infected patients exhibited a gene expression profile akin to healthy controls, unlike the Alpha or Beta variants. However, significantly, we found identified a subset of infected individuals, most pronounced in vaccinated patients infected with Omicron variant, that exhibited a specific dynamic in their alternative splicing patterns that was not widely shared amongst the other groups. Our findings underscore the complex interplay between SARS-CoV-2 variants, vaccination-induced immune responses, and alternative splicing, emphasizing the necessity for further investigations into these molecular cross-talks to foster deeper understanding and guide strategic therapeutic development.","version":"1.1","doi":"10.1101/2023.11.24.568603","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.24.568354","pub_date":"2023-11-27","title":"Antiviral innate immune memory in alveolar macrophages following SARS-CoV-2 infection","abstract":"Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors in airway-resident macrophages. Mechanistically, establishment of this innate immune memory required viral pattern recognition and canonical IFN-I signaling and augmented secondary antiviral responses. Past SARS-CoV-2 infection ameliorated disease caused by the heterologous respiratory pathogen influenza A virus. Insights into innate immune memory and how it affects subsequent infections with heterologous pathogens to influence disease pathology could facilitate the development of broadly effective therapeutic strategies.","version":"1.1","doi":"10.1101/2023.11.24.568354","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.18.567675","pub_date":"2023-11-27","title":"Development of a fast feature extraction method for SARS-CoV-2 spike sequences using amino acid physicochemical properties","abstract":"COVID-19 continues to spread today, leading to an accumulation of SARS-CoV-2 virus mutations in databases, and large amounts of genomic datasets are currently available. However, due to these large datasets, utilizing this amount of sequence data without random sampling is challenging. Major difficulties for downstream analyses include the increase in the dimension size along with the conversion of sequences into numerical values when using conventional amino acid representation methods, such as one-hot encoding and k-mer-based approaches that directly reflect sequences. Moreover, these sequences are deficient in physicochemical characteristics, such as structural information and hydrophilicity; hence, they fail to accurately represent the inherent function of the given sequences. In this study, we utilized the physicochemical properties of amino acids to develop a rapid and efficient approach for extracting feature parameters that are suitable for downstream processes of machine learning, such as clustering. A fixed-length feature vector representation of a spike sequence with reduced dimensionality was obtained by converting amino acid residues into physicochemical parameters. Next, t-distributed stochastic neighbor embedding (t- SNE), a method for dimensionality reduction and visualization of high-dimensional data, was performed, followed by density-based spatial clustering of applications with noise (DBSCAN). The results show that by using the physicochemical properties of amino acids rather than conventional methods that directly represent sequences into numerical values, SARS-CoV-2 spike sequences can be clustered with sufficient accuracy and a shorter runtime. Interestingly, the clusters obtained by using amino acid properties include subclusters that are distinct from those produced utilizing the method for the direct representation of amino acid sequences. A more detailed analysis indicated that the contributing parameters of this novel cluster identified exclusively when utilizing the physicochemical properties of amino acids significantly differ from one another. This suggests that representing amino acid sequences by physicochemical properties might enable the identification of clusters with enhanced sensitivity compared to conventional methods. One of the major causes of the global threat of SARS-CoV-2 is the rapid emergence of its variants. While analyzing these variants is crucial for understanding the mechanism of outbreaks, the expansion of database size is becoming a barrier for effective analysis. In this study, we provide an approach that allows researchers without vast computational resources to comprehensively analyze the variants of SARS-CoV-2 spike by representing the sequences using the physicochemical properties of amino acids. The result of clusters derived using this method demonstrates not only an accuracy comparable to the conventional approaches of directly converting sequences into numerical values but also indicates the potential for more detailed clustering outcomes. The results suggest that our approach is valuable for the rapid identification of characteristic residues in new variants of SARS-CoV-2 and other viruses that may arise in the future.","version":"1.2","doi":"10.1101/2023.11.18.567675","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.25.568642","pub_date":"2023-11-27","title":"Safe plant Hsp90 adjuvants elicit an effective immune response against SARS-CoV2 derived RBD antigen","abstract":"To better understand the role of pHsp90 adjuvant in immune response modulation, we proposed the use of the Receptor Binding Domain (RBD) of the Spike protein of SARS-CoV2, the principal candidate in the design of subunit vaccines. We evaluated the humoral and cellular immune responses against RBD through the strategy \u201cprotein mixture\u201d (Adjuvant + Antigen). The rRBD adjuvanted with rAtHsp81.2 group showed a higher increase of the anti-rRBD IgG1, while the rRBD adjuvanted with rNbHsp90.3 group showed a significant increase of anti-rRBD IgG2b/2a. These results were consistent with the cellular immune response analysis. Spleen cell cultures from rRBD+rNbHsp90.3-immunized mice showed significantly increased IFN-\u03b3 production. In contrast, spleen cell cultures from rRBD+rAtHsp81.2-immunized mice showed significant increased IL-4 levels. Finally, vaccines adjuvanted with rNbHsp90.3 induced higher neutralizing antibody responses compared to those adjuvanted with rAtHsp81.2. To know whether both chaperones must form complexes to generate an effective immune response, we performed co-immunoprecipitation (co-IP) assays. The results indicated that the greater neutralizing capacity observed in the rRBD adjuvanted with rNbHsp90.3 group would be given by the rRBD-rNbHsp90.3 interaction rather than by the quality of the immune response triggered by the adjuvants. These results, together with our previous results, provide a comparative benchmark of these two novel and safe vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV2 subunit vaccines. Furthermore, these results revealed differences in the ability to modulate the immune response between these two pHsp90s, highlighting the importance of adjuvant selection for future rational vaccine and adjuvant design.","version":"1.1","doi":"10.1101/2023.11.25.568642","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.567964","pub_date":"2023-11-27","title":"Tertiary folds of the SL5 RNA from the 5\u2032 proximal region of SARS-CoV-2 and related coronaviruses","abstract":"Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5\u2032 genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically-determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus SARS-CoV-2, resolved at 4.7 \u212b resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T\u2019s \u201carms.\u201d Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4-6.9 \u00c5 resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across the studied human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9-8.0 \u00c5 resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4-9.0 \u00c5 resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities, with implications for potential protein-binding modes and therapeutic targets. The three-dimensional structures of viral RNAs are of interest to the study of viral pathogenesis and therapeutic design, but the three-dimensional structures of viral RNAs remain poorly characterized. Here, we provide the first 3D structures of the SL5 domain (124-160 nt, 40.0-51.4 kDa) from the majority of human-infecting coronaviruses. All studied SL5s exhibit a similar 4-way junction, with their crossing angles grouped along phylogenetic boundaries. Further, across all species studied, conserved UUYYGU hexaloop pairs are located at opposing ends of a coaxial stack, suggesting that their three-dimensional arrangement is important for their as-of-yet defined function. These conserved tertiary features support the relevance of SL5 for pan-coronavirus fitness and highlight new routes in understanding its molecular and virological roles and in developing SL5-based antivirals. Classification: Biological Sciences, Biophysics and Computational Biology","version":"1.2","doi":"10.1101/2023.11.22.567964","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.568381","pub_date":"2023-11-23","title":"Enhanced surface accessibility of SARS-CoV-2 Omicron spike protein due to an altered glycosylation profile","abstract":"SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including ectodomain, receptor-binding domain, and N-terminal domain. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies. Through mass spectrometry and molecular dynamics simulations, SARS-CoV-2 Omicron spike is found to be less covered by glycans when compared to the D614G spike variant.","version":"1.1","doi":"10.1101/2023.11.22.568381","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.23.23298957","pub_date":"2023-11-23","title":"Improving SARS-CoV-2 variants monitoring in the absence of genomic surveillance capabilities: a serological study in Bolivian blood donors in October 2021 and June 2022","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Unlike genomic data, serological data have not been previously leveraged to evaluate the SARS-CoV-2 variants circulation. In Bolivia, sustained genomic surveillance capacities were lacking especially at the beginning of the pandemic. In 2021 and 2022 we estimated the prevalence of anti-SARS-CoV-2 antibodies in Bolivian blood donors and explored the feasibility of using virus serum neutralization data for variants thought to have circulated to map their circulation across all departments over a year-long follow-up period. Anti-S1 and anti-NCP SARS-CoV-2 IgGs were studied, along with virus neutralization tests for ancestral-D614G, Gamma, Delta, and Omicron BA.1 lineages of SARS-CoV-2. Between 2021 and 2022, the overall prevalence of anti-S1 and anti-NCP antibodies increased reaching values over 90%, demonstrating that a large proportion of the Bolivian population was no longer na\u00efve to the virus. Viral neutralization data, analyzed through multiple approaches, revealed the spread of the Gamma variant up to 2021, particularly impacting northern departments. In 2022, Gamma continued to circulate in southernmost departments of the country and the emergence of Omicron BA.1 was detected. These trends align with publicly available genomic data from neighboring countries. Our serological analyses successfully identified both new antigenic groups, such as Omicron BA.1, and individual variants related to previously circulating groups, such as Delta. The study contributes insights into overall population immunity to SARS-CoV-2 and variant-specific immunity levels across different regions of Bolivia. It also emphasizes the potency of seroprevalence studies in informing public health decisions and underscore their value in capturing the initial phases of emerging epidemics when variant diversity is limited, facilitating timely genomic surveillance setup.</jats:p>","version":null,"doi":"10.1101/2023.11.23.23298957","journal":"medRxiv","score":null},{"id":"10.1101/2023.11.22.568361","pub_date":"2023-11-23","title":"Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid","abstract":"The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38\u00b110 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.","version":"1.1","doi":"10.1101/2023.11.22.568361","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.568225","pub_date":"2023-11-23","title":"Development of attenuated coxsackievirus B3 vectored intranasal pre-emptive pan-coronavirus vaccine","abstract":"SARS-CoV-2 has the ability to evade immunity, resulting in breakthrough infections even after vaccination. Similarly, zoonotic coronaviruses pose a risk of spillover to humans. There is an urgent need to develop a pre-emptive pan-coronavirus vaccine that can induce systemic and mucosal immunity. Here, we employed a combination of immune-informatics approaches to identify shared immunodominant linear B- and T-cell epitopes from SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs), as well as zoonotic coronaviruses. Epitope-guided vaccine were designed and the attenuated coxsackievirus B3 vectored intranasal vaccines rCVB3-EPI and rCVB3-RBD-trimer were constructed. The immunogenicity of these candidate vaccines was evaluated using Balb/c mice. The results demonstrated effective immune responses, including the production of SARS-CoV-2-specific IgG and sIgA antibodies, as well as T cell-mediated responses. However, further verification is required to assess cross-reactivity with various variants. Our intranasal pre-emptive pan-coronavirus vaccine design framework offers an appealing candidate for future vaccine development.","version":"1.1","doi":"10.1101/2023.11.22.568225","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.21.568132","pub_date":"2023-11-22","title":"SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system","abstract":"SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, is associated with a range of neurological manifestations including haemorrhage, thrombosis and ischaemic necrosis and encephalitits. However, the mechanism by which this occurs is unclear. Neurological disease associated with SARS-CoV-2 infection has been proposed to occur following direct infection of the central nervous system and/or indirect sequelae as a result of peripheral inflammation. We profiled ACE2 and TMPRSS2 in brain tissue from five healthy human donors, and observed expression of these proteins in astrocytes, neurons and choroid plexus epithelium within frontal cortex and medulla. Primary human astrocytes, neurons and choroid plexus epithelial cells supported productive SARS-CoV-2 infection in an ACE2- dependent manner. Infected cells supported the full viral lifecycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells, pericytes and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 is neurotropic, and this may in part explain the neurological sequelae of infection. A subset of patients with COVID-19 develop neurological symptoms, but the underlying cause is poorly understood. We observed that cells within normal human brain express the SARS-CoV-2 entry factors ACE-2 and TMPRRS2, with expression mainly observed within astrocytes, neurons and choroid plexus epithelium. Primary human astrocytes, neurons and choroid plexus epithelial cells cultured in vitro supported the full SARS-CoV-2 life cycle with a range of SARS-CoV-2 variants. This study demonstrates that cells of the human central nervous system express SARS-CoV-2 entry factors in vivo and support viral infection in vitro, thus supporting a model where neurological symptoms seen in some COVID-19 patients may be as a result of direct viral infection of the central nervous system. Furthermore, these data highlight the importance of investigating the ability of therapeutics to clear virus from this potential reservoir of infection.","version":"1.1","doi":"10.1101/2023.11.21.568132","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.21.567575","pub_date":"2023-11-22","title":"SARS-CoV-2 monoclonal antibody treatment followed by vaccination shifts human memory B cell epitope recognition suggesting antibody feedback","abstract":"Therapeutic anti-SARS-CoV-2 monoclonal antibodies (mAbs) have been extensively studied in humans, but the impact on immune memory of mAb treatment during an ongoing immune response has remained unclear. Here, we evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2\u2013infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific defect in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating human memory B cell responses, both to infection and vaccination. These data indicate that mAb administration can promote alterations in the epitopes recognized by the B cell repertoire, and the single administration of mAb can continue to determine the fate of B cells in response to additional antigen exposures months later. Evaluating the therapeutic use of monoclonal antibodies during SARS-CoV-2 infection requires a comprehensive understanding of their impact on B cell responses at the cellular level and how these responses are shaped after vaccination. We report for the first time the effect of bamlanivimab on SARS-CoV-2 specific human memory B cells of COVID-19 infected humans receiving, or not, mRNA immunization.","version":"1.1","doi":"10.1101/2023.11.21.567575","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.567930","pub_date":"2023-11-22","title":"Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates","abstract":"SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there\u2019s a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.","version":"1.1","doi":"10.1101/2023.11.22.567930","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.22.568286","pub_date":"2023-11-22","title":"Transmission dynamics of MERS-CoV in a transgenic human DPP4 mouse model","abstract":"Since 2002, three novel coronavirus outbreaks have occurred: severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. A better understanding of the transmission potential of coronaviruses will result in adequate infection control precautions and an early halt of transmission within the human population. Experiments on the stability of coronaviruses in the environment, as well as transmission models, are thus pertinent. Here, we show that transgenic mice expressing human DPP4 can be infected with MERS-CoV via the aerosol route. Exposure to 5\u00d7106 TCID50 and 5\u00d7104 TCID50 MERS-CoV per cage via fomites resulted in transmission in 15 out of 20 and 11 out of 18 animals, respectively. Exposure of sentinel mice to donor mice one day post inoculation with 105 TCID50 MERS-CoV resulted in transmission in 1 out of 38 mice via direct contact and 4 out of 54 mice via airborne contact. Exposure to donor mice inoculated with 104 TCID50 MERS-CoV resulted in transmission in 0 out of 20 pairs via direct contact and 0 out of 5 pairs via the airborne route. Our model shows limited transmission of MERS-CoV via the fomite, direct contact, and airborne routes. The hDPP4 mouse model will allow assessment of the ongoing evolution of MERS-CoV in the context of acquiring enhanced human-to-human transmission kinetics and will inform the development of other transmission models.","version":"1.1","doi":"10.1101/2023.11.22.568286","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.15.23298565","pub_date":"2023-11-22","title":"Hematologic abnormalities after COVID-19 vaccination: A large Korean population-based cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Adverse hematologic events have been reported after COVID-19 vaccination. The objective of this study was to investigate whether hematologic abnormalities develop after COVID-19 vaccination. Retrospective cohort analyses of data from the Korean National Health Insurance Service (KNHIS) database were conducted from July 2022 to August 2023. We randomly selected data of half of those living in Seoul City as of January 1, 2021 with their diagnostic records up to December 31, 2021. The included participants were vaccinated and nonvaccinated persons aged 20 years or older (n= 4,203,887). Hematologic abnormalities after COVID-19 vaccination were identified as nutritional anemia, hemolytic anemia, aplastic anemia, coagulation defects, and neutropenia using International Classification of Diseases, Tenth Revision codes after index date. Incidence rates of hematologic abnormalities in the vaccination group 3 months after vaccination were significantly higher than those in the nonvaccinated group: 14.79 vs. 9.59 (P&lt;.001) for nutritional anemia, 7.83 vs. 5.00 (P&lt;.001) for aplastic anemia, and 4.85 vs. 1.85 (P&lt;.001) for coagulation defects. COVID-19 mRNA vaccine was associated with higher development of nutritional anemia (odds ratio [OR], 1.230 [95% CI, 1.129-1.339], P&lt;.001) and aplastic anemia (OR, 1.242 [95% CI, 1.110-1.390], P&lt;.001) than the viral vector vaccine. The risk of coagulation defects was increased (OR, 1.986 [95% CI, 1.523-2.589], P&lt;.001) after vaccination, and there was no risk difference between mRNA vaccine and viral vector vaccine (OR, 1.075 [95% CI, 0.936-1.233], P=.306). In conclusions, COVID-19 vaccination increased the risk of hematologic abnormalities. When administering the COVID-19 vaccine, careful observation will be necessary after vaccination.</jats:p>","version":null,"doi":"10.1101/2023.11.15.23298565","journal":"medRxiv","score":null},{"id":"10.1101/2023.11.19.567745","pub_date":"2023-11-21","title":"Microplastics dysregulate innate immunity in the SARS-CoV-2 infected lung","abstract":"Global microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis. Herein we described the effects of azide-free, 1 \u00b5m polystyrene MP beads co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum using a mouse model of mild COVID-19. Lung virus titres and viral RNA levels were not significantly affected by MPs, with overt clinical or histopathological changes also not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 days post infection (dpi) and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the \u2018cytokine release syndrome\u2019 signature seen in some severe COVID-19 patients. This study adds to a growing body of literature suggesting that MPs can dysregulate inflammation in specific disease settings. A single inoculation of microplastics dysregulated SARS-CoV-2 lung inflammation At the peak of SARS-CoV-2 infection microplastics decreased early innate responses Later post infection microplastics promoted a \u201ccytokine release syndrome\u201d signature A key mechanism may involve the inhibition of the phagocytosis of infected cells Azide-free microplastics were used, with no elevated ROS responses identified Postulated mechanisms whereby microplastics might decrease the proinflammatory responses 2 days after SARS-CoV-2 infection, yet promote the proinflammatory \u2018cytokine release syndrome\u2019 signature at 6 days post infection.","version":"1.1","doi":"10.1101/2023.11.19.567745","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.18.545507","pub_date":"2023-11-21","title":"Dysregulated Platelet Function in Patients with Post-Acute Sequelae of COVID-19","abstract":"Post-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. Our objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC. PASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. A mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets \u2013 a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. PASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2023.06.18.545507","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.18.567697","pub_date":"2023-11-21","title":"Accurate Characterization of Conformational Ensembles and Binding Mechanisms of the SARS-CoV-2 Omicron BA.2 and BA.2.86 Spike Protein with the Host Receptor and Distinct Classes of Antibodies Using AlphaFold2-Augmented Integrative Computational Modeling","abstract":"The latest wave SARS-CoV-2 Omicron variants displayed a growth advantage and the increased viral fitness through convergent evolution of functional hotspots that work synchronously to balance fitness requirements for productive receptor binding and efficient immune evasion. In this study, we combined AlphaFold2-based structural modeling approaches with all-atom MD simulations and mutational profiling of binding energetics and stability for prediction and comprehensive analysis of the structure, dynamics, and binding of the SARS-CoV-2 Omicron BA.2.86 spike variant with ACE2 host receptor and distinct classes of antibodies. We adapted several AlphaFold2 approaches to predict both structure and conformational ensembles of the Omicron BA.2.86 spike protein in the complex with the host receptor. The results showed that AlphaFold2-predicted conformational ensemble of the BA.2.86 spike protein complex can accurately capture the main dynamics signatures obtained from microscond molecular dynamics simulations. The ensemble-based dynamic mutational scanning of the receptor binding domain residues in the BA.2 and BA.2.86 spike complexes with ACE2 dissected the role of the BA.2 and BA.2.86 backgrounds in modulating binding free energy changes revealing a group of conserved hydrophobic hotspots and critical variant-specific contributions of the BA.2.86 mutational sites R403K, F486P and R493Q. To examine immune evasion properties of BA.2.86 in atomistic detail, we performed large scale structure-based mutational profiling of the S protein binding interfaces with distinct classes of antibodies that displayed significantly reduced neutralization against BA.2.86 variant. The results quantified specific function of the BA.2.86 mutations to ensure broad resistance against different classes of RBD antibodies. This study revealed the molecular basis of compensatory functional effects of the binding hotspots, showing that BA.2.86 lineage may have primarily evolved to improve immune escape while modulating binding affinity with ACE2 through cooperative effect of R403K, F486P and R493Q mutations. The study supports a hypothesis that the impact of the increased ACE2 binding affinity on viral fitness is more universal and is mediated through cross-talk between convergent mutational hotspots, while the effect of immune evasion could be more variant-dependent.","version":"1.1","doi":"10.1101/2023.11.18.567697","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.20.567927","pub_date":"2023-11-21","title":"Human coronavirus OC43 infection remodels Connexin 43 mediated gap junction intercellular communication in vitro","abstract":"\u03b2-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human \u03b2-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding \u03b2-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human \u03b2-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also showed a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post OC43 infection in the ERGIC region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human \u03b2-coronavirus infections. The enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human \u03b2-coronaviruses, advancing our preparedness for future coronavirus infections. Due to SARS-CoV-2 being highly infectious, another human \u03b2-coronavirus OC43 can be considered as an experimental model. One of the crucial pathways that can be considered is gap junction communication, as it is vital for cellular homeostasis. Our study seeks to understand the change in Cx43-mediated cell-to-cell communication during human \u03b2-coronavirus OC43 infection. In vitro studies showed the downregulation of the gap junction protein Cx43 and the upregulation of endoplasmic reticulum and oxidative stress markers post-OC43 infection. Furthermore, OC43 infection causes impairment of functional hemichannel and gap junction formation. Overall, this current study infers that OC43 infection reshapes intercellular communication, suggesting that this pathway may be a promising target for designing highly effective therapeutics against human coronaviruses by regulating Cx43 expression.","version":"1.1","doi":"10.1101/2023.11.20.567927","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.17.567570","pub_date":"2023-11-20","title":"Comprehensive contact tracing during an outbreak of alpha-variant SARS-CoV-2 in a rural community reveals less viral genomic diversity and higher household secondary attack rates than expected","abstract":"Sequencing of SARS-CoV-2 genomes throughout the COVID-19 pandemic has generated a wealth of data on viral evolution across populations, but only a few studies have so far explored SARS-CoV-2 evolution across transmission networks of tens to hundreds of persons. Here, we couple data from SARS-CoV-2 sequencing with contact tracing data from an outbreak with a single origin in a rural Norwegian community where samples from all exposed persons were collected prospectively. A total of 134 nasopharyngeal samples were positive by PCR. Among the 121 retrievable genomes, 81 were identical to the genome of the introductor, thus demonstrating that genomics offers limited additional value to manual contact-tracing. In the cases where mutations were discovered, five small genetic clusters were identified. We observed a household secondary attack rate of 67%, with 92% of household members infected among households with secondary transmission, suggesting that SARS-CoV-2 introduction into large families are likely to affect all household members. In outbreak investigations, obtaining a full overview of infected individuals within a population is seldom acheived. We here present an example of just that, where a single introduction of B1.1.7 SARS-CoV-2 within a rural community allowed for tracing of the virus, from an introductor via dissemination through larger gatherings, into households. The outbreak occurred before widespread vaccination, allowing for a \u201cnatural\u201d outbreak development with community lock-down. We show through sequencing that the virus can infect up to five consecutive persons without gaining mutations, thereby showing that contact tracing seems more important than sequencing for local outbreak investigations. We also show how families with small children are less likely to contain spread to all family members if SARS-CoV-2 enters the household either by a child or a caregiver, as isolation of the primary infected is difficult in such scenarios.","version":"1.1","doi":"10.1101/2023.11.17.567570","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.17.567583","pub_date":"2023-11-20","title":"Tropism of AAV.CPP.16 in the respiratory tract and its application for a CRISPR-based gene therapy against SARS-CoV-2","abstract":"Efficient gene delivery vectors are essential for developing gene therapies for respiratory diseases. Here, we report that AAV.CPP.16, a novel AAV9-derived adeno-associated virus vector, can efficiently transduce airway epithelium systems and lung parenchyma cells in both mice and non-human primates after intranasal administration. AAV.CPP.16 outperforms AAV6 and AAV9, two wild-type AAVs with demonstrated tropism to respiratory tract tissues, and can target major cell types in the respiratory tract and the lung. We also report an \u201call-in-one\u201d, CRISPR-Cas13d-based AAV gene therapy vector that targets the highly conserved RNA-dependent RNA polymerase (Rdrp) gene in SARS-CoV-2, and show the potential of such gene therapy against a broad range of circulating and emergent SARS-CoV-2 variants. Thus, AAV.CPP.16 could be a useful gene delivery vector for treating genetic respiratory diseases and airborne infections including for developing a potential prophilaxis to SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.11.17.567583","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.17.567629","pub_date":"2023-11-20","title":"Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15","abstract":"SARS-CoV-2 non-structural protein 15 (Nsp15) is critical for productive viral replication and evasion of host immunity. The uridine-specific endoribonuclease activity of Nsp15 mediates the cleavage of the polyuridine [poly(U)] tract of the negative-strand coronavirus genome to minimize the formation of dsRNA that activates the host antiviral interferon signaling. However, the molecular basis for the recognition and cleavage of the poly(U) tract by Nsp15 is incompletely understood. Here, we present cryogenic electron microscopy (cryoEM) structures of SARS-CoV-2 Nsp15 bound to viral replication intermediate dsRNA containing poly(U) tract at 2.7-3.3 \u00c5 resolution. The structures reveal one copy of dsRNA binds to the sidewall of an Nsp15 homohexamer, spanning three subunits in two distinct binding states. The target uracil is dislodged from the base-pairing of the dsRNA by amino acid residues W332 and M330 of Nsp15, and the dislodged base is entrapped at the endonuclease active site center. Up to 20 A/U base pairs are anchored on the Nsp15 hexamer, which explains the basis for a substantially shortened poly(U) sequence in the negative strand coronavirus genome compared to the long poly(A) tail in its positive strand. Our results provide mechanistic insights into the unique immune evasion strategy employed by coronavirus Nsp15.","version":"1.1","doi":"10.1101/2023.11.17.567629","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.15.567102","pub_date":"2023-11-20","title":"Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket","abstract":"To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate binding pocket. Of 631 fragments soaked, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (2 hits) or at crystal packing interfaces (3 hits). Notably, two fragments with a pose sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. Our structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. An X-ray crystallographic screen on SARS-CoV-2 3CL protease resulted in 29 fragment hits, including two isatin-based reversible covalent binders, and revealed a strong influence of the crystal form used for fragment soaking on the bound conformation of three additional reference fragments.","version":"1.2","doi":"10.1101/2023.11.15.567102","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.17.567633","pub_date":"2023-11-20","title":"Preclinical Characterization of the Omicron XBB.1.5-Adapted BNT162b2 COVID-19 Vaccine","abstract":"As SARS-CoV-2 continues to evolve, increasing in its potential for greater transmissibility and immune escape, updated vaccines are needed to boost adaptive immunity to protect against COVID-19 caused by circulating strains. Here, we report features of the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine, which contains the same mRNA backbone as the original BNT162b2 vaccine, modified by the incorporation of XBB.1.5-specific sequence changes in the encoded prefusion-stabilized SARS-CoV-2 spike protein (S(P2)). Biophysical characterization of Omicron XBB.1.5 S(P2) demonstrated that it maintains a prefusion conformation that adopts a flexible and predominantly open one-RBD-up state, with high affinity binding to the human ACE-2 receptor. When administered as a 4th dose in BNT162b2-experienced mice, the monovalent Omicron XBB.1.5 vaccine elicited substantially higher serum neutralizing titers against pseudotyped viruses of Omicron XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and HV.1 sublineages and the phylogenetically distant BA.2.86 lineage than the bivalent Wild Type + Omicron BA.4/5 vaccine. Similar trends were observed against Omicron XBB sublineage pseudoviruses when the vaccine was administered as a 2-dose primary series in na\u00efve mice. Strong S-specific Th1 CD4+ and IFN\u03b3+ CD8+ T cell responses were also observed. These findings, together with prior experience with variant-adapted vaccine responses in preclinical and clinical studies, suggest that the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine is anticipated to confer protective immunity against dominant SARS-CoV-2 strains. The monovalent Omicron XBB.1.5-adapted BNT162b2 mRNA vaccine encodes a prefusion-stabilized spike immunogen that elicits more potent neutralizing antibody responses against homologous XBB.1.5 and other circulating sublineage pseudoviruses compared to the bivalent Wild Type + Omicron BA.4/5 BNT162b2 vaccine, thus demonstrating the importance of annual strain changes to the COVID-19 vaccine.","version":"1.1","doi":"10.1101/2023.11.17.567633","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.16.567485","pub_date":"2023-11-17","title":"Human microbiota is a reservoir of SARS-CoV-2 advantageous mutations","abstract":"SARS-CoV-2 mutations are rapidly emerging, in particular advantageous mutations in the spike (S) protein, which either increase transmissibility or lead to immune escape, are posing a major challenge to pandemic prevention and treatment. However, how the virus acquires a high number of advantageous mutations in a short time remains a mystery. Here, we show that the human microbiota may contribute to mutations in variants of concern (VOCs). We identified a mutation and adjacent 6 amino acids (aa) in a viral mutation fragment (VMF) and searched for homologous fragments (HFs) in the National Center for Biotechnology Information (NCBI) database. Among the approximate 8000 HFs obtained, 61 mutations in S and other outer membrane proteins were found in bacteria, accounting for 62% of all mutation sources, which is a 12-fold higher than the natural variable proportion. Approximately 70% of these bacterial species belong to the human microbiota, are primarily distributed in the gut or lung and exhibit a composition pattern similar to that of COVID-19 patients. Importantly, SARS- CoV-2 RNA-dependent RNA polymerase (RdRp) replicates corresponding bacterial mRNAs harboring mutations, producing chimeric RNAs. Collectively, SARS-CoV-2 may acquire mutations from the human microbiota, resulting in alterations in the binding sites or antigenic determinants of the original virus. Our study sheds light on the evolving mutational mechanisms of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.11.16.567485","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.24.546363","pub_date":"2023-11-17","title":"Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50) values ranging from 1.42 to 32.7 \u00b5M. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half- maximum effective concentration (EC50) values of 21.73 \u00b5M and 31.19 \u00b5M, respectively. The anti-inflammatory role of azadirachtin and withanolide_A when assessed using HEK293T cells were found to significantly reduce the levels of CXCL10, TNF\u03b1, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-\u03b11). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2 specific enzymes and also host immune pathways involved in virus mediated inflammation.","version":"1.2","doi":"10.1101/2023.06.24.546363","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.16.566918","pub_date":"2023-11-17","title":"Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers","abstract":"Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFN\u03b3 ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 reliably immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.","version":"1.1","doi":"10.1101/2023.11.16.566918","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.16.567378","pub_date":"2023-11-17","title":"Dynamic label-free analysis of SARS-CoV-2 infection reveals virus-induced subcellular remodeling","abstract":"Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holo-tomographic microscopy (HTM) with Artificial Intelligence (AI) to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets (LD) and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs LD, changes mitochondrial shape and dry mass, and separates LD from mitochondria. We then used Bayesian statistics on organelle dry mass states to define organelle cross-regulation (OCR) networks and report modifications of OCR that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides a new AI-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.","version":"1.1","doi":"10.1101/2023.11.16.567378","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.13.566860","pub_date":"2023-11-17","title":"Fast evolution of SARS-CoV-2 BA.2.86 to JN.1 under heavy immune pressure","abstract":"While the BA.2.86 variant demonstrated significant antigenic drift and enhanced ACE2 binding affinity, its ability to evade humoral immunity was relatively moderate compared to dominant strains like EG.5 and HK.3. However, the emergence of a new subvariant, JN.1 (BA.2.86.1.1), which possesses an additional spike mutation, L455S, compared to BA.2.86, showed a markedly increased prevalence in Europe and North America, especially in France. Here, we found that L455S of JN.1 significantly enhances immune evasion capabilities at the expense of reduced ACE2 binding affinity. This mutation enables JN.1 to effectively evade Class 1 neutralizing antibodies, offsetting BA.2.86\u2019s susceptibility and thus allowing it to outcompete both its precursor BA.2.86 and the prevailing variants HV.1 (XBB.1.5+L452R+F456L) and JD.1.1 (XBB.1.5+L455F+F456L+A475V) in terms of humoral immune evasion. The rapid evolution from BA.2.86 to JN.1, similar to the earlier transition from BA.2.75 to CH.1.1, highlights the importance of closely monitoring strains with high ACE2 binding affinity and distinct antigenicity, despite their temporarily unremarkable immune evasion capabilities. Such strains could survive and transmit at low levels, since their large antigenic distance to dominant strains allow them to target distinct populations and accumulate immune-evasive mutations rapidly, often at the cost of receptor binding affinity.","version":"1.2","doi":"10.1101/2023.11.13.566860","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.03.543542","pub_date":"2023-11-17","title":"Species and habitat specific changes in bird activity in an urban environment during Covid 19 lockdown","abstract":"Covid-19 lockdowns provided ecologists with a rare opportunity to examine how animals behave when humans are absent. Indeed many, sometimes contradicting, studies reported various effects of lockdowns on animal activity, especially in urban areas and other human-dominated habitats. We explored how Covid-19 lockdowns in Israel have influenced bird activity in an urban environment by using continuous acoustic recordings to monitor three common bird species that differ in their level of adaptation to the urban ecosystem: (1) the hooded crow, an urban exploiter, which depends heavily on anthropogenic resources; (2) the rose-ringed parakeet, an invasive alien species that has adapted to exploit human resources; and (3) the graceful prinia, an urban adapter, which is relatively shy of humans and can be found urban habitats with shrubs and prairies. Acoustic recordings provided continuous monitoring of bird activity without an effect of the observer on the animal. We performed dense sampling of a 1.3 square km area in northern Tel-Aviv by placing 17 recorders for more than a month in different micro-habitats within this region including roads, residential areas and urban parks. We monitored both lockdown and no-lockdown periods. We portray a complex dynamic system where the activity of specific bird species depended on many environmental parameters and decreases or increases in a habitat-dependent manner during lockdown. Specifically, urban exploiter species decreased their activity in most urban habitats during lockdown, while human adapter species increased their activity during lockdown especially in parks where humans were absent. Our results also demonstrate the value of different habitats within urban environments for animal activity, specifically highlighting the importance of urban parks. These species- and habitat-specific changes in activity might explain the contradicting results reported by others who have not performed a habitat specific analysis.","version":"1.3","doi":"10.1101/2023.06.03.543542","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.15.567306","pub_date":"2023-11-17","title":"Unveiling the Emotional Turmoil: How Covid-19 impacted researchers and the pursuit of emotional well-being in academia","abstract":"The Covid-19 crisis unprecedentedly required researchers to adapt to significant changes in their work and personal lives. Our study aims to fill this gap analysing the Covid-19 emotional impact and confinement potential disruptions on researchers\u2019 activity (specifically, those related to working conditions, caring responsibilities, health, balance, and social support) considering the modulating role played by age, gender, and job position. An online survey was distributed during the first lockdown period of the Covid-19 pandemic, and answers from 1301 researchers (ECR %, senior researchers %) working in Sciences (28.1%), Social Sciences (25.9%), Humanities (16.2%), Health (16.2%) and in Engineering and Architecture (13.5%) were collected. The study highlights that the initial lockdown during the Covid-19 pandemic had a significant emotional impact on researchers, exacerbating pre-existing emotional distress and burnout within this group. Factors such as age, health, gender, and difficulties in balancing work and family life were associated with an increased risk of burnout and emotional distress. Lack of social support was identified as a significant risk factor, while the academic culture prioritizing productivity over well-being contributed to the issue. These findings underscore the need for greater support and cultural changes in academia to preserve researchers\u2019 mental health and prevent the chronicization of mental health issues in young academics.","version":"1.1","doi":"10.1101/2023.11.15.567306","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.15.567132","pub_date":"2023-11-16","title":"The Impact of SIV-Induced Immunodeficiency on Clinical Manifestation, Immune Response, and Viral Dynamics in SARS-CoV-2 Coinfection","abstract":"Persistent and uncontrolled SARS-CoV-2 replication in immunocompromised individuals has been observed and may be a contributing source of novel viral variants that continue to drive the pandemic. Importantly, the effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. Here we conducted a pilot study wherein two pigtail macaques (PTM) chronically infected with SIVmac239 were exposed to SARS-CoV-2 and monitored for six weeks for clinical disease, viral replication, and viral evolution, and compared to our previously published cohort of SIV-na\u00efve PTM infected with SARS-CoV-2. At the time of SARS-CoV-2 infection, one PTM had minimal to no detectable CD4+ T cells in gut, blood, or bronchoalveolar lavage (BAL), while the other PTM harbored a small population of CD4+ T cells in all compartments. Clinical signs were not observed in either PTM; however, the more immunocompromised PTM exhibited a progressive increase in pulmonary infiltrating monocytes throughout SARS-CoV-2 infection. Single-cell RNA sequencing (scRNAseq) of the infiltrating monocytes revealed a less activated/inert phenotype. Neither SIV-infected PTM mounted detectable anti-SARS-CoV-2 T cell responses in blood or BAL, nor anti-SARS-CoV-2 neutralizing antibodies. Interestingly, despite the diminished cellular and humoral immune responses, SARS-CoV-2 viral kinetics and evolution were indistinguishable from SIV-na\u00efve PTM in all sampled mucosal sites (nasal, oral, and rectal), with clearance of virus by 3-4 weeks post infection. SIV-induced immunodeficiency significantly impacted immune responses to SARS-CoV-2 but did not alter disease progression, viral kinetics or evolution in the PTM model. SIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants.","version":"1.1","doi":"10.1101/2023.11.15.567132","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.13.566859","pub_date":"2023-11-16","title":"Evolutionary arms race between SARS-CoV-2 and interferon signaling via dynamic interaction with autophagy","abstract":"SARS-CoV-2 emerged, and is evolving to efficiently infect humans worldwide. SARS-CoV-2 evades early innate recognition, interferon signaling activated only in bystander cells. This balance of innate activation and viral evasion has important consequences, but the pathways involved are incompletely understood. Here we find that autophagy genes regulate innate immune signaling, impacting the basal set point of interferons, and thus permissivity to infection. Mechanistically, autophagy genes negatively regulate MAVS, and this low basal level of MAVS is efficiently antagonized by SARS-CoV-2 ORF9b, blocking interferon activation in infected cells. However, upon loss of autophagy increased MAVS overcomes ORF9b-mediated antagonism suppressing infection. This has led to the evolution of SARS-CoV-2 variants to express higher levels of ORF9b, allowing SARS-CoV-2 to replicate under conditions of increased MAVS signaling. Altogether, we find a critical role of autophagy in the regulation of innate immunity and uncover an evolutionary trajectory of SARS-CoV-2 ORF9b to overcome host defenses.","version":"1.1","doi":"10.1101/2023.11.13.566859","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.507985","pub_date":"2023-11-16","title":"Unsupervised identification of significant lineages of SARS-CoV-2 through scalable machine learning methods","abstract":"Since its emergence in late 2019, SARS-CoV-2 has diversified into a large number of lineages and globally caused multiple waves of infection. Novel lineages have the potential to spread rapidly and internationally if they have higher intrinsic transmissibility and/or can evade host immune responses, as has been seen with the Alpha, Delta, and Omicron variants of concern (VoC). They can also cause increased mortality and morbidity if they have increased virulence, as was seen for Alpha and Delta, but not Omicron. Phylogenetic methods provide the gold standard for representing the global diversity of SARS-CoV-2 and to identify newly emerging lineages. However, these methods are computationally expensive, struggle when datasets get too large, and require manual curation to designate new lineages. These challenges together with the increasing volumes of genomic data available provide a motivation to develop complementary methods that can incorporate all of the genetic data available, without down-sampling, to extract meaningful information rapidly and with minimal curation. Here, we demonstrate the utility of using algorithmic approaches based on word-statistics to represent whole sequences, bringing speed, scalability, and interpretability to the construction of genetic topologies, and while not serving as a substitute for current phylogenetic analyses the proposed methods can be used as a complementary approach to identify and confirm new emerging variants.","version":"1.2","doi":"10.1101/2022.09.14.507985","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.15.566945","pub_date":"2023-11-15","title":"SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since emerging in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a mutant recombinant SARS-CoV-2 (nsp15mut) expressing a catalytically inactive nsp15. Infection with SARS-CoV-2 nsp15 mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type (WT) virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI culture. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a spectrum of respiratory disease ranging from asymptomatic infections to severe pneumonia and death. Innate immune responses during SARS-CoV-2 infection have been associated with clinical disease severity, with robust early interferon responses in the nasal epithelium reported to be protective. Thus, elucidating mechanisms through which SARS-CoV-2 induces and antagonizes host innate immune responses is crucial to understanding viral pathogenesis. CoVs encode various innate immune antagonists, including the conserved nonstructural protein 15 (nsp15) which contains an endoribonuclease (EndoU) domain. We demonstrate that SARS-CoV-2 EndoU is a crucial interferon antagonist, by providing further evidence for the role of the conserved CoV nsp15 in antagonizing innate immune activation, thereby optimizing CoV replication.","version":"1.1","doi":"10.1101/2023.11.15.566945","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.14.567145","pub_date":"2023-11-15","title":"CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in murine model of the SARS-CoV-2-related acute respiratory distress syndrome","abstract":"The acute respiratory distress syndrome (ARDS) secondary to viral pneumonitis is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019) \u2013 ongoing SARS-CoV-2 infection, reached more than 0.7 billion registered cases. Recently we elaborated non- surgical and reproducible method of unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice \u2013 a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data reads that two C-C chemokine receptor 5 (CCR5) ligands \u2013 macrophage inflammatory proteins (MIP) \u2013 (MIP-1\u03b1/CCL3) and (MIP-1\u03b2/CCL4) are upregulated in this DAD model up to three orders of magnitude compared to the background level. Here we showed that a nonpeptide compound TAK- 779, antagonist of CCR5/CXCR3, readily prevents DAD of the lung with a single injection of 2.5 mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung, and mononuclear infiltration of the wall and lumen of the alveoli in the TAK- 779-treated animals. Administration of the TAK-779 decreased 3-5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1\u03b1/\u03b2, MCP-1 and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779- treated animals. Our pre-clinical data suggest that TAK-779 is more effective than administration of dexamethasone or anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors recruited in ongoing clinical studies as a potential drugs for treatment of COVID19 and similar virus-induced inflammation syndromes. The pathogenesis of the SARS-CoV-2 infection is tightly linked with the cytokine storm resulting in the enormous release of cytokines and chemokines. Its clinical manifestation \u2013 the acute respiratory distress syndrome (ARDS), may be caused by self-sustaining hypersensitivity reactions leading to lung collapse even after virus clearance. Here we report that two macrophage inflammatory proteins, MIP-1\u03b1/CCL3 and MIP-1\u03b2/CCL4, seem to orchestrate mononuclear infiltration into the lungs during diffuse alveolar damage (DAD) in ICR mice \u2013 our murine model of ARDS caused by SARS-CoV-2. Inhibition of the C-C chemokine receptor 5 (CCR5) \u2013 parental receptor for MIP-1\u03b1 and MIP-1\u03b2, by nonpeptide antagonist TAK-779 results in significant amelioration of DAD in terms of reduced mononuclear infiltration into the lung, suppressed cytokine storm and restored physiology of affected lung according to computed tomography data. We suggest that targeted inhibition of CCR5 should be further elucidated as safe and effective approach to overcome severe viral pneumonia in humans.","version":"1.1","doi":"10.1101/2023.11.14.567145","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.14.566985","pub_date":"2023-11-15","title":"Virological characteristics of the SARS-CoV-2 Omicron HK.3 variant harboring the \u201cFLip\u201d substitution","abstract":"In November 2023, SARS-CoV-2 XBB descendants, including EG.5.1 (XBB.1.9.2.5.1), the currently predominant lineage, are circulating worldwide according to Nextstrain. EG.5.1 has a characteristic amino acid substitution in the spike protein (S), S:F456L, which contributes to its escape from humoral immunity. EG.5.1 has further evolved, and its descendant lineage harboring S:L455F (i.e., EG.5.1+S:L455F) emerged and was named HK.3 (XBB.1.9.2.5.1.1.3). HK.3 was initially discovered in East Asia and is rapidly spreading worldwide. Notably, the XBB subvariants bearing both S:L455F and S:F456L substitutions, including HK.3, are called the \u201cFLip\u201d variants. These FLip variants, such as JG.3 (XBB.1.9.2.5.1.3.3), JF.1 (XBB.1.16.6.1) and GK.3 (XBB.1.5.70.3), have emerged convergently, suggesting that the acquisition of these two substitutions confers a growth advantage to XBB in the human population. Here, we investigated the virological properties of HK.3 as a representative of the FLip variants.","version":"1.1","doi":"10.1101/2023.11.14.566985","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.13.566917","pub_date":"2023-11-14","title":"SARS-CoV-2 and its ORF3a, E and M viroporins activate inflammasome in human macrophages and induce of IL-1\u03b1 in pulmonary epithelial and endothelial cells","abstract":"Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger cell death and IL-1\u03b1 release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1\u03b1 and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1\u03b1 as a potential novel component driving the pulmonary immunothromobosis in COVID-19.","version":"1.1","doi":"10.1101/2023.11.13.566917","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.13.566961","pub_date":"2023-11-14","title":"Full-spike deep mutational scanning helps predict the evolutionary success of SARS-CoV-2 clades","abstract":"SARS-CoV-2 variants acquire mutations in spike that promote immune evasion and impact other properties that contribute to viral fitness such as ACE2 receptor binding and cell entry. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning to measure how >9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry, or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully impacted ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456, and 473\u2014however, the antigenic impacts of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.","version":"1.1","doi":"10.1101/2023.11.13.566961","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.13.566827","pub_date":"2023-11-14","title":"Synthesis of Dendritic Cell-Targeted Polymeric Nanoparticles for Selective Delivery of mRNA Vaccines to Elicit Enhanced Immune Responses","abstract":"Recent development of SARS-CoV-2 spike mRNA vaccines to control the pandemic is a breakthrough in the field of vaccine development. mRNA vaccines are generally formulated with lipid nanoparticles (LNPs) which are composed of several lipids with specific ratios; however, they generally lack selective delivery. To develop a simpler method selective delivery of mRNA, we reported here the synthesis of biodegradable copolymers decorated with guanidine and zwitterionic groups and an aryltrimannoside ligand as polymeric nanoparticles (PNPs) for encapsulation and selective delivery of an mRNA to dendritic cells (DCs). A representative DC-targeted SARS-CoV-2 spike mRNA-PNP vaccine was shown to elicit a stronger protective immune response in mice as compared to the mRNA-LNP and mRNA-PNP vaccines without the selective delivery design. It is anticipated that this technology will be generally applicable to development of DC-targeted mRNA vaccines with enhanced immune response. Dendritic cell-targeted mRNA-PNP vaccines","version":"1.1","doi":"10.1101/2023.11.13.566827","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.13.543274","pub_date":"2023-11-14","title":"Mycobiome analyses of critically ill COVID-19 patients","abstract":"COVID-19-associated pulmonary aspergillosis (CAPA) is a life-threatening complication in patients with severe COVID-19. Previously, acute respiratory distress syndrome in patients with COVID-19 has been associated with lung fungal dysbiosis, evidenced by reduced microbial diversity and Candida colonisation. Increased fungal burden in the lungs of critically ill COVID-19 patients is linked to prolonged mechanical ventilation and increased mortality. However, specific mycobiome signatures associated with severe COVID-19 in the context of survival and antifungal drug prophylaxis have not yet been determined and such knowledge could have an important impact on treatment. To understand the composition of the respiratory mycobiome in critically ill COVID-19 patients with and without CAPA and the impact of antifungal use in patient outcome. We performed a multi-national study of 39 COVID-19 patients in intensive care units (ICU) with and without CAPA. Respiratory mycobiome was profiled using ITS1 sequencing and Aspergillus fumigatus burden was further validated using qPCR. Fungal communities were investigated using alpha diversity, beta diversity, taxa predominance and taxa abundances. Respiratory mycobiomes of COVID-19 patients were dominated by Candida and Aspergillus. There was no significant association with corticosteroid use or CAPA diagnosis and respiratory fungal communities. Increased A. fumigatus burden was associated with mortality and, the use of azoles at ICU admission was linked with an absence of A. fumigatus. Our findings suggest that mould-active antifungal treatment at ICU admission may be linked with reduced A. fumigatus-associated mortality in severe COVID-19. However, further studies are warranted on this topic.","version":"1.3","doi":"10.1101/2023.06.13.543274","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.10.566587","pub_date":"2023-11-12","title":"Insights into B Cell and Antibody Kinetics Against SARS-CoV-2 Variants Using Mathematical Modelling","abstract":"B cells and antibodies are crucial in protecting against infections like SARS-CoV-2. However, antibody levels decline after infection or vaccination, reducing defences against future SARS-CoV-2 infections. To understand antibody production and decline, we developed a mathematical model that predicts germinal center B cell, long-lived plasma cell, memory B cell, and antibody dynamics. Our focus was on B cell activation and antibody generation following both primary and secondary SARS-CoV-2 infections. Aligning our model with clinical data, we adjusted antibody production rates for germinal center B cells and plasma B cells during primary and secondary infections. We also assessed antibody neutralization against Delta and Omicron variants post-primary and secondary exposure. Our findings showed reduced neutralization against Omicron due to its immune evasion. In primary and secondary exposures to Delta and Omicron, our predictions indicated enhanced antibody neutralization in the secondary response within a year of the primary response. We also explored waning immunity, demonstrating how B cell kinetics affect viral neutralization post-primary infection. This study enhances our understanding of humoral immunity to SARS-CoV-2 and can predict antibody dynamics post-infection or vaccination.","version":"1.1","doi":"10.1101/2023.11.10.566587","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.10.566576","pub_date":"2023-11-12","title":"The SARS-CoV-2 Omicron sub-variant BA.2.86 is attenuated in hamsters","abstract":"SARS-CoV-2 variants have emerged throughout the COVID-19 pandemic. There is a need to risk-assess newly emerged variants in near \u201creal-time\u201d to estimate their potential threat to public health. The recently emerged Omicron sub-variant BA.2.86 raised concerns as it carries a high number of mutations compared to its predecessors. Here, we assessed the virulence of BA.2.86 in hamsters. We compared the pathogenesis of BA.2.86 and BA.2.75, as the latter is one of the most virulent Omicron sub-variants in this animal model. Using digital pathology pipelines, we quantified the extent of pulmonary lesions measuring T cell and macrophage infiltrates, in addition to alveolar epithelial hyperplasia. We also assessed body weight loss, clinical symptoms, virus load in oropharyngeal swabs, and virus replication in the respiratory tract. Our data show that BA.2.86 displays an attenuated phenotype in hamsters, suggesting that it poses no greater risk to public health than its parental Omicron sub-variants. The newly emerged Omicron sub-variant BA.2.86 is attenuated in hamsters.","version":"1.1","doi":"10.1101/2023.11.10.566576","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.11.566634","pub_date":"2023-11-12","title":"Recreating the Biological Steps of Viral Infection on a Bioelectronic Platform to Profile Viral Variants of Concern","abstract":"Viral mutation rates frequently outpace the development of technologies used to detect and identify harmful variants; for SARS Coronavirus-2 (SARS-CoV-2), these are called variants of concern (VOC). Given the continual emergence of VOC, there is a critical need to develop platforms that can identify the presence of a virus and readily identify its propensity for infection. We present an electronic biomembrane sensing platform that recreates the multifaceted and sequential biological cues that give rise to distinct SARS-CoV-2 virus host cell entry pathways and reports the progression of entry steps of these pathways as electrical signals. Within these electrical signals, two necessary entry processes mediated by the viral Spike protein, virus binding and membrane fusion, can be distinguished. Remarkably, we find that closely related VOC exhibit distinct fusion signatures that correlate with trends reported in cell-based infectivity assays, allowing us to report quantitative differences in fusion characteristics among them that inform their infectivity potentials. This cell-free biomimetic infection platform also has a virus-free option that equally reports infectivity potential of the Spike proteins. We used SARS-CoV-2 as our prototype, but we anticipate that this platform will extend to other enveloped viruses and cell lines to quantifiably explore virus/host interactions. This advance should aid in faster determination of entry characteristics and fusogenicities of future VOC, necessary for rapid response.","version":"1.1","doi":"10.1101/2023.11.11.566634","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.29.560084","pub_date":"2023-11-10","title":"Protective non-neutralizing mAbs Ab94 and Ab81 retain high-affinity and potent Fc-mediated function against SARS-CoV-2 variants from Omicron to XBB1.5","abstract":"Antibodies play a central role in the immune defense against SARS-CoV-2. There is substantial evidence supporting that Fc-mediated effector functions of anti-spike antibodies contribute to anti-SARS-Cov-2 immunity. We have previously shown that two non-neutralizing but opsonic mAbs, Ab81 and Ab94, are protective against lethal Wuhan SARS-CoV-2 infection in mice. The protective effect was comparable to a potent neutralizing antibody, Ab59. Here, we hypothesized that, unlike the neutralizing antibodies, non-neutralizing opsonic antibodies would have a higher likelihood of retaining their function to the mutated variants, potentially functioning as broadly protective mAbs. Most of the mutations on the SARS-CoV-2 variants cluster on neutralizing epitopes, leaving other epitopes unaltered. We observed that neutralizing antibodies lost binding to Omicron. In contrast, seven non-neutralizing opsonic antibodies retained nanomolar affinity towards Omicron, BA.2, BA.4, and BA.5. Focusing on the two protective non-neutralizing antibodies Ab81 and Ab94, we showed that they maintain their strong reactivity even to XBB, XBB1.5, and BQ1.1. In the case of Ab94, interestingly, it even has increased affinity towards all variants except for XBB, which is comparable to WT. Finally, we show that Ab94 and Ab81 have potent Fc-mediated functions in vitro against the XBB and BQ1.1 and that combining the mAbs in a cocktail further enhances the effect. These results show that protective non-neutralizing mAbs such as Ab94 and Ab81 can be a viable strategy for anti-SARS-CoV-2 mAb therapies against current and possibly future SARS-CoV-2 variants and that opsonic epitopes could have implications for vaccine design.","version":"1.2","doi":"10.1101/2023.09.29.560084","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.13.557622","pub_date":"2023-11-10","title":"Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19","abstract":"The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-\u03b3 and TNF stimulated lung macrophages to chronically release IL-1\u03b2, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-\u03b3 and TNF, or IL-1\u03b2 after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.","version":"1.2","doi":"10.1101/2023.09.13.557622","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.07.565953","pub_date":"2023-11-09","title":"Attila and Aetius on the roof: The succession phenomenon in the roof greening area of a primary school in Beijing during the lockdown period of the COVID-19","abstract":"\u201cSuccession\u201d refers to the process of certain species replacing others over time. It is of great significance for understanding the characteristics and evolution of ecosystems. This article compares the changes in school rooftop green areas before and after the COVID-19 epidemic (2019, 2023), and studies the situation of wild grass invading roofs during the three years of the epidemic, as well as the response of original rooftop plants to the invasion. The results showed that: \u2460 invasive weeds were concentrated in 2 families and 4 species, forming a population advantage in most of the invaded planting boxes; \u2461 The succession has had a significant impact on the roof greening area; \u2462 Among the two cultivated plants (Phedimus aizoon (PA) and Sedum sarmentosum (SS)), SS was greatly affected, with the community almost disappearing, while PA was almost unaffected.","version":"1.1","doi":"10.1101/2023.11.07.565953","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.06.565781","pub_date":"2023-11-08","title":"The \u03b1-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses","abstract":"The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on \u03b1-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of \u03b1-dystroglycan (\u03b1-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of \u03b1-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant \u03b1-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant \u03b1-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.","version":"1.1","doi":"10.1101/2023.11.06.565781","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.07.566012","pub_date":"2023-11-08","title":"Predicting human and viral protein variants affecting COVID-19 susceptibility and repurposing therapeutics","abstract":"The COVID-19 disease is an ongoing global health concern. Although vaccination provides some protection, people are still susceptible to re-infection. Ostensibly, certain populations or clinical groups may be more vulnerable. Factors causing these differences are unclear and whilst socioeconomic and cultural differences are likely to be important, human genetic factors could influence susceptibility. Experimental studies indicate SARS-CoV-2 uses innate immune suppression as a strategy to speed-up entry and replication into the host cell. Therefore, it is necessary to understand the impact of variants in immunity-associated human proteins on susceptibility to COVID-19. In this work, we analysed missense coding variants in several SARS-CoV-2 proteins and its human protein interactors that could enhance binding affinity to SARS-CoV-2. We curated a dataset of 19 SARS-CoV-2: human protein 3D-complexes, from the experimentally determined structures in the Protein Data Bank and models built using AlphaFold2-multimer, and analysed impact of missense variants occurring in the protein-protein interface region. We analysed 468 missense variants from human proteins and 212 variants from SARS-CoV-2 proteins and computationally predicted their impacts on binding affinities to SARS-CoV-2 proteins, using 3D-complexes. We predicted a total of 26 affinity-enhancing variants from 14 human proteins implicated in increased binding affinity to SARS-CoV-2. These include key-immunity associated genes (TOMM70, ISG15, IFIH1, IFIT2, RPS3, PALS1, NUP98, RAE1, AXL, ARF6, TRIMM, TRIM25) as well as important spike receptors (KREMEN1, AXL and ACE2). We report both common (e.g., Y13N in IFIH1) and rare variants in these proteins and discuss their likely structural and functional impact, using information on known and predicted functional sites. Potential mechanisms associated with immune suppression implicated by these variants are discussed. Occurrence of certain predicted affinity-enhancing variants should be monitored as they could lead to increased susceptibility and reduced immune response to SARS-CoV-2 infection in individuals/populations carrying them. Our analyses aid in understanding the potential impact of genetic variation in immunity-associated proteins on COVID-19 susceptibility and help guide drug-repurposing strategies.","version":"1.1","doi":"10.1101/2023.11.07.566012","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.06.565765","pub_date":"2023-11-08","title":"Mucosal Adenoviral-vectored Vaccine Boosting Durably Prevents XBB.1.16 Infection in Nonhuman Primates","abstract":"Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens.","version":"1.1","doi":"10.1101/2023.11.06.565765","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.03.565419","pub_date":"2023-11-06","title":"Voltage-gated T-type calcium channel blockers reduce apoptotic body mediated SARS-CoV-2 cell-to-cell spread and subsequent cytokine storm","abstract":"SARS-CoV-2 typically utilises host angiotensin-converting enzyme 2 (ACE2) as a cellular surface receptor and host serine protease TMPRSS2 for the proteolytic activation of viral spike protein enabling viral entry. Although macrophages express low levels of ACE2, they are often found positive for SARS-CoV-2 in autopsied lungs from COVID-19 patients. As viral-induced macrophage inflammation and overwhelming cytokine release are key immunopathological events that drives exacerbated tissue damage in severe COVID-19 patients, insights into the entry of SARS-CoV-2 into macrophages are therefore critical to understand COVID-19 pathogenesis and devise novel COVID-19 therapies. Mounting evidence suggest that COVID-19 pathogenesis is associated with apoptosis, a type of programmed cell death that often leads to the release of numerous large extracellular vesicles (EVs) called apoptotic bodies (ApoBDs). Here, we showed that ApoBDs derived from SARS-CoV-2-infected cells carry viral antigens and infectious virions. Human monocyte-derived macrophages readily efferocytosed SARS-CoV-2-induced ApoBDs, resulting in SARS-CoV-2 entry and pro-inflammatory responses. To target this novel ApoBD-mediated viral entry process, we screened for ApoBD formation inhibitors and discovered that T-type voltage-gated calcium channel (T-channel) blockers can inhibit SARS-CoV-2-induced ApoBD formation. Mechanistically, T-channel blockers impaired the extracellular calcium influxes required for ApoBD biogenesis. Importantly, blockade of ApoBD formation by T-channel blockers were able to limit viral dissemination and virus-induced macrophage inflammation in vitro and in a pre-clinical mouse model of severe COVID-19. Our discovery of the ApoBD-efferocytosis-mediated viral entry reveals a novel route for SARS-CoV-2 infection and cytokine storm induction, expanding our understanding of COVID-19 pathogenesis and offering new therapeutic avenues for infectious diseases.","version":"1.1","doi":"10.1101/2023.11.03.565419","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.04.565660","pub_date":"2023-11-06","title":"Global Sensitivity Analysis of the Onset of Nasal Passage Infection by SARS-CoV-2 With Respect to Heterogeneity in Host Physiology and Host Cell-Virus Kinetic Interactions","abstract":"Throughout the COVID-19 pandemic, positive nasal swab tests have revealed dramatic population heterogeneity in viral titers spanning 6 orders-of-magnitude. Our goal here is to probe potential drivers of infection outcome sensitivity arising from (i) physiological heterogeneity between hosts and (ii) host-variant heterogeneity in the detailed kinetics of cell infection and viral replication. Toward this goal, we apply global sensitivity methods (Partial Rank Correlation Coefficient analysis and Latin Hypercube Sampling) to a physiologically faithful, stochastic, spatial model of inhaled SARS-CoV-2 exposure and infection in the human respiratory tract. We focus on the nasal passage as the primary origin of respiratory infection and site of clinical testing, and we simulate the spatial and dynamic progression of shed viral load and infected cells in the immediate 48 hours post infection. We impose immune evasion, i.e., suppressed immune protection, based on the preponderance of clinical evidence that nasal infections occur rapidly post exposure, largely independent of immune status. Global sensitivity methods provide the de-correlated outcome sensitivities to each source of within-host heterogeneity, including the dynamic progression of sensitivities at 12, 24, 36, and 48 hours post infection. The results reveal a dynamic rank-ordering of the drivers of outcome sensitivity in early infection, providing insights into the dramatic population-scale outcome diversity during the COVID-19 pandemic. While we focus on SARS-CoV-2, the model and methods are applicable to any inhaled virus in the immediate 48 hours post infection.","version":"1.1","doi":"10.1101/2023.11.04.565660","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.03.564190","pub_date":"2023-11-06","title":"Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2","abstract":"Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we argue that RNA-seq should be considered a routine diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers vital insights into a patient\u2019s immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 240 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that - combined with sequence alignments and pBLAST - they could be used to classify a patient\u2019s disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample. Computational deconvolution of transcriptomes can estimate immune cell abundances in SARS-CoV-2 patients, supplementing missing CBC data. 10 million RNA sequencing reads per sample suffice for analyzing immune responses and disease severity, including BCR/TCR identification.","version":"1.1","doi":"10.1101/2023.11.03.564190","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.04.565404","pub_date":"2023-11-06","title":"Dynamic gene expression analysis reveals distinct severity phases of immune and cellular dysregulation in COVID-19","abstract":"COVID-19 patients experience dynamic changes in immune and cellular function over time with potential clinical implications. However, there is insufficient research investigating, on a gene expression level, the mechanisms that become activated or suppressed over time as patients deteriorate or recover, which can inform use of repurposed and novel drugs as therapies. To investigate longitudinal changes in gene expression profiles throughout the COVID-19 disease timeline. Three-hundred whole blood samples from 128 adult patients were collected during hospitalization from COVID-19, with up to five samples per patient. Transcriptome sequencing (RNA-Seq), differential gene expression analysis and pathway enrichment was performed. Drug-gene set enrichment analysis was used to identify FDA-approved medications that could inhibit critical genes and proteins at each disease phase. Prognostic gene-expression signatures were generated using machine learning to distinguish 3 disease stages. Samples were longitudinally grouped by clinical criteria and gene expression into six disease phases: Mild, Moderate, Severe, Critical, Recovery, and Discharge. Distinct mechanisms with differing trajectories during COVID-19 hospitalization were apparent. Antiviral responses peaked early in COVID-19, while heme metabolism pathways became active much later during disease. Adaptive immune dysfunction, inflammation, and metabolic derangements were most pronounced during phases with higher disease severity, while hemostatic abnormalities were elevated early and persisted throughout the disease course. Drug-gene set enrichment analysis predicted repurposed medications for potential use, including platelet inhibitors in early disease, antidiabetic medications for patients with increased disease severity, and dasatinib throughout the disease course. Disease phases could be categorized using specific gene signatures for prognosis and treatment selection. Disease phases were also highly correlated to previously developed sepsis endotypes, indicating that severity and disease timing were significant contributors to heterogeneity observed in sepsis and COVID-19. Higher temporal resolution of longitudinal mechanisms in COVID-19 revealed multiple immune and cellular changes that were activated at different phases of COVID-19. Understanding how a patient\u2019s gene expression profile changes over time can permit more accurate risk stratification of patients and provide time-dependent personalized treatments with repurposed medications. This creates an opportunity for timely intervention before patients transition to a more severe phase, potentially accelerating patients to recovery.","version":"1.1","doi":"10.1101/2023.11.04.565404","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.03.23298032","pub_date":"2023-11-04","title":"Frequency and determinants of COVID-19 prevention behaviours: assessment of large-scale programmes in seven countries","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Pre-existing health and economic challenges mean residents of low- and middle-income countries (LMICs) are likely to be particularly vulnerable to infectious disease pandemics. Limited access to hygiene facilities, water, soap and masks, and dense living environments impeded effective practice of preventive behaviours \u2013 handwashing with soap (HWWS), mask wearing and physical distancing \u2013 a key line of primary defence against COVID-19. Here we describe a multi-country analysis of prevalence of key hygiene prevention behaviours and their determinants associated with an international non-governmental organisation (WaterAid) hygiene behaviour change programmes for COVID-19 prevention. The goal of this analysis is to inform future outbreak preparedness and pandemic response in LMICs. Cross-sectional household surveys were conducted in October-November 2020 in seven countries where WaterAid worked (Ethiopia, Ghana, Nepal, Nigeria, Rwanda, Tanzania and Zambia). Multivariable mixed-effects regression analyses were used to explore relationships between self-reported behavioural outcomes of interest (handwashing with soap, physical distancing, and mask use) and demographic characteristics, behavioural factors (knowledge, norms, barriers, motives), and exposure to COVID-19 communications. Most respondents (80%) reported increasing their handwashing behaviour after the pandemic, but practice of HWWS at COVID-19-specific prevention moments was low. Mask wearing (58%) and physical distancing (29%) varied substantially between countries. Determinants of key behaviours were identified, including age and socioeconomic status, perceived norms, self-regulation, and the motive of protecting others. These findings highlight that leveraging behaviour-specific emotional drivers and norms, reducing common barriers and promoting targeted messages about specific behaviours and actions individuals can take to reduce risk are necessary to support large-scale behaviour change. Learning from the COVID-19 response to more effectively integrate novel behaviours into existing health promotion will be vital for disease prevention and outbreak resilience.</jats:p>\n                <jats:sec id='s1'>\n                  <jats:title>Key messages</jats:title>\n                  <jats:sec id='s1a'>\n                    <jats:title>What is already known on this topic</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Facilitating COVID-19 prevention behaviours of hand hygiene, mask use and physical distancing in low- and-middle income countries comes with unique challenges</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Identifying effective strategies to promote adoption of key behaviours in diverse contexts over a period of rapid change will be key for future pandemic preparedness</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec id='s1b'>\n                    <jats:title>What this study adds</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>This multi-country analysis of areas where WaterAid implemented an initial mass media COVID-19 response in 2020 observed lower practice of handwashing at novel COVID-19 prevention moments compared to established moments and variable physical distancing behaviour, and examined behaviour-specific determinants and norms</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec id='s1c'>\n                    <jats:title>How this study might affect research, practice or policy</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Renewed focus on identified key drivers of behaviour: targeting critical age-groups and vulnerable populations, increasing descriptive norms and motives of protecting others and respect, and reducing common barriers, with targeted messaging for novel handwashing moments, may be key to ongoing COVID-19 response</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Learning from the rapid COVID-19 response on how well we are able to promote novel behaviours alongside established ones in a variety of contexts can inform future disease prevention and outbreak resilience.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2023.11.03.23298032","journal":"medRxiv","score":null},{"id":"10.1101/2023.11.01.564972","pub_date":"2023-11-03","title":"Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04","abstract":"Drug resistance poses a significant challenge in the development of effective therapies against SARS-CoV-2. Here, we identified two double mutations, M49K/M165V and M49K/S301P, in the 3C-like protease (3CLpro) that confer resistance to a novel non-covalent inhibitor, WU-04. Crystallographic analysis indicates that the M49K mutation destabilizes the WU-04 binding pocket, impacting the binding of WU-04 more significantly than the binding of 3CLpro substrates. The M165V mutation directly interferes with WU-04 binding. The S301P mutation, which is far from the WU-04 binding pocket, indirectly affects WU-04 binding by restricting the rotation of 3CLpro\u2019s C-terminal tail and impeding 3CLpro dimerization. We further explored 3CLpro mutations that confer resistance to two clinically used inhibitors: ensitrelvir and nirmatrelvir, and revealed a trade-off between the catalytic activity, thermostability, and drug resistance of 3CLpro. We found that mutations at the same residue (M49) can have distinct effects on the 3CLpro inhibitors, highlighting the importance of developing multiple antiviral agents with different skeletons for fighting SARS-CoV-2. These findings enhance our understanding of SARS-CoV-2 resistance mechanisms and inform the development of effective therapeutics.","version":"1.1","doi":"10.1101/2023.11.01.564972","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.02.565304","pub_date":"2023-11-03","title":"Virological characteristics of the SARS-CoV-2 BA.2.86 variant","abstract":"In late 2023, a lineage of SARS-CoV-2 emerged and was named the BA.2.86 variant. BA.2.86 is phylogenetically distinct from other Omicron sublineages identified so far, displaying an accumulation of over 30 amino acid mutations in its spike protein. Here, we performed multiscale investigations to reveal the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Experimental studies showed that four clinically-available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 is significantly lower than that of BA.2 in both in vitro cell cultures and in vivo, it is suggested that the attenuated pathogenicity of BA.2.86 is due to its decreased replication capacity.","version":"1.1","doi":"10.1101/2023.11.02.565304","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.01.565087","pub_date":"2023-11-02","title":"Phylogenetic meta-analysis of chronic SARS-CoV-2 infections in immunocompromised patients shows no evidence of elevated evolutionary rates","abstract":"Genomic sequences from rapidly evolving pathogens, sampled over time, hold information on disease origin, transmission, and evolution. Together with their sampling times, sequences can be used to estimate the rates of molecular evolution and date evolutionary events through molecular tip-dating. The validity of this approach, however, depends on whether detectable levels of genetic variation have accumulated over the given sampling interval, generating temporal signal. Moreover, different molecular dating methods have demonstrated varying degrees of systematic biases under different biologically realistic scenarios, such as the presence of phylo-temporal clustering. Chronic SARS-CoV-2 infection in immunocompromised patients has been linked to remarkably higher intra-host molecular rates than those of global lineages, facilitating the emergence of novel viral lineages. Yet, most studies reporting accelerated rates lack the evaluation of temporal signal or comparison of multiple methods of inference, both required to reliably estimate molecular rates. In this study, we use 26 previously published longitudinally sampled sequence series obtained from chronically infected immunocompromised patients to re-evaluate the rate of SARS-CoV-2 intrahost evolution. Using a range of methods, we analyse the strength of temporal signal and infer evolutionary rates from tip-calibrated phylogenies. Regardless of heterogeneity in rate estimates between sample series and methods, we find within-host rates to be in good agreement with rates derived from host-to-host transmission chains. Our findings suggest that when certain limitations of the methodology are disregarded, such as the underlying assumption of phylogenetic independence or the method\u2019s sensitivity to phylo- temporal grouping, evolutionary rates can be substantially overestimated. We demonstrate that estimating within-host rates is a challenging question necessitating careful interpretation of findings. While our results do not support faster evolution across the complete viral genome during chronic SARS-CoV-2 infection, prolonged viral shedding together with relapsing viral load dynamics may nevertheless promote the emergence of new viral variants in immunocompromised patients. The evolutionary origin of SARS-CoV-2 variants of concern (VOC) is a longstanding point of controversy, with multiple proposed explanations. Observations of immunocompromised individuals being at a greater risk of developing a prolonged SARS-CoV-2 infection have led to the \u2018Chronic infection hypothesis\u2019, suggesting that these cases may contribute to the emergence of VOCs. Correspondingly, many studies have reported accelerated viral evolution of SARS-CoV-2 within immunocompromised individuals with respect to the viral background population. However, many of these findings have not been validated with appropriate analytical methods. In this study we re-evaluate the rate of intrahost viral evolution of SARS- CoV-2 within immunocompromised patients utilising a range of methods. We assess the performance of different methodologies and compare our results to published estimates of SARS-CoV-2 evolutionary rates. Our systematic comparison showed no evidence supporting the previous claims of elevated levels of intrahost evolution in immunocompromised patients with chronic SARS-CoV-2. Instead, our findings exemplify the complexity of within-host viral dynamics, suggesting that a more comprehensive understanding of SARS-CoV-2 evolutionary processes would be derived from concurrent evaluation of viral genomic data together with patients\u2019 clinical information.","version":"1.1","doi":"10.1101/2023.11.01.565087","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.01.565098","pub_date":"2023-11-01","title":"SARS-CoV-2 spike protein induces the cytokine release syndrome by stimulating T cells to produce more IL-2","abstract":"Cytokine release syndrome (CRS) is one of the leading causes of mortality in COVID-19 patients caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more IL-2, which subsequently cooperates with spike protein to facilitate peripheral blood mononuclear cells to release IL-1\u03b2, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release TNF-\u03b1 and IFN-\u03b3, as well as T cells to release IFN-\u03b3. Mechanistically, IFN-\u03b3 and TNF-\u03b1 enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of TLR4 which serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of NF-\u03baB. Furthermore, TNF-\u03b1 also activates NF-\u03baB signaling in monocytes, which further cooperates with IFN-\u03b3 and spike protein to modulate NF-\u03baB-dependent transcription of CRS-related inflammatory cytokines. Targeting TNF-\u03b1/IFN-\u03b3 in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19.","version":"1.1","doi":"10.1101/2023.11.01.565098","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.31.565042","pub_date":"2023-11-01","title":"SARS-CoV-2 infection leads to sustained testicular injury and functional impairments in K18 hACE2 mice","abstract":"Compromised male reproductive health is one of the symptoms of long COVID with a decrease in male fertility markers including testosterone levels and sperm count for months in recovering patients. However, the long-term impact of SARS-CoV-2 infection on testicular injury and underlying mechanisms remains unknown. We previously demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis during the acute stage of the disease in K18-hACE2 mice. Here, we systematically delineate the consequences of SARS-CoV-2 infection on the testis injury and function both during the acute stage of the disease and up to 4 weeks after infection in survivor K18-hACE2 mice. The gross morphological defects included sloughing of healthy spermatids and spermatocytes into the lumen, lack of lumen, and increase in apoptotic cells that sustained for at least 2 weeks after infection. Testis injury correlated with systemic and testicular inflammation, and infiltration of immune cells in the interstitial space and seminiferous tubules. Transcriptomic analysis identified dysregulation of key pathways of testicular immune homeostasis, spermatogenesis, and cell death at the symptomatic and short-term recovery stages. Further, a significant reduction in testosterone levels was associated with transient reduction in sperm count and mouse fertility. Most of the testicular impairments except testosterone levels were resolved within 4 weeks, which is almost one spermatogenesis cycle in mice. These findings provide much-needed mechanistic insights beyond our current understanding of testicular pathogenesis, suggesting that recovering COVID-19 patients should be closely monitored to rescue the pathophysiological effects on male reproductive health.","version":"1.1","doi":"10.1101/2023.10.31.565042","journal":"bioRxiv","score":null},{"id":"10.1101/2023.11.01.565121","pub_date":"2023-11-01","title":"Assessment of The Broad-Spectrum Host Targeting Antiviral Efficacy of Halofuginone Hydrobromide in Human Airway, Intestinal and Brain Organoid Models","abstract":"Halofuginone hydrobromide has shown potent antiviral efficacy against a variety of viruses such as SARS-CoV-2, dengue, or chikungunya virus, and has, therefore, been hypothesized to have broad-spectrum antiviral activity. In this paper, we tested this broad-spectrum antiviral activity of Halofuginone hydrobomide against viruses from different families (Picornaviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae, and Flaviviridae). To this end, we used relevant human models of the airway and intestinal epithelium and regionalised neural organoids. Halofuginone hydrobomide showed antiviral activity against SARS-CoV-2 in the airway epithelium with no toxicity at equivalent concentrations used in human clinical trials but not against any of the other tested viruses. Halofuginone hydrobromide was identified as a possible broad-spectrum host targeting antiviral drug. Human organoid models offer a physiologically relevant and clinically translatable model for antiviral research. Halofuginone hydrobromide shows antiviral efficacy against SARS-CoV-2, but not against EV-A71, PeV-A1, IAV, RV-A16, HCMV or ZIKV in relevant organoid models. The efficacy of Halofuginone hydrobromide is concentration dependent as well as on proline content of the host receptor(s) or host factors for the specific virus in question.","version":"1.1","doi":"10.1101/2023.11.01.565121","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.30.564680","pub_date":"2023-10-30","title":"Characterization of Unique Pathological Features of COVID-Associated Coagulopathy: Studies with AC70 hACE2 Transgenic Mice Highly Permissive to SARS-CoV-2 Infection","abstract":"COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS-CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as \u223c 3 and \u223c 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and many inflammatory mediators could be readily detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio. Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy, including significantly elevated levels of D-dimer, t-PA, PAI-1, and circulating NETs, along with activated platelet/endothelium marker. Immunohistochemical staining with anti-PF4 antibody revealed profound platelet aggregates especially within blocked veins of the lungs. ANXA2 is known to interact with S100A10 to form heterotetrametric complexes, serving as coreceptors for t-PA to regulate membrane fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced AnxA2/S100A10 association in infected mice support an important role of this protein in the pathogenesis of acute COVID-19. In summary, we showed that acute SARS-CoV-2 infection of AC70 hACE2 Tg mice triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis, accompanied by dysregulation of ANXA2 system, which might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19. Accumulating evidence strongly suggests that COVID-associated coagulopathy characterized by dysregulation of the coagulation cascade, fibrinolysis system and pulmonary microvascular immune-thrombosis during different stages of SARS-CoV-2 infection may have a \u201cyet-to-be fully defined\u201d impact on the development of post-acute sequela of COVID-19. Herein we initially reported a comprehensively characterized AC70 hACE2 Tg mouse model for SARS-CoV-2 infection and disease. We next demonstrated the subsequent onset of imbalanced coagulation and fibrinolysis pathways in infected Tg mice, focusing on dysregulated formation of ANXA2/S100A10 complexes, key coreceptors for t-PA that regulates membrane fibrinolysis, in which elevated production of autoantibodies against ANXA2 induced by SARS-CoV-2 might play an intriguing role. Taken together, we demonstrated that AC70 hACE2 Tg mice lethally challenged with SARS-CoV-2 recapitulated several features of COVID-associated coagulopathy observed in patients and highlighted the potential role of ANXA2 in this phenomenon. Thus, ANXA2 might serve as a potentially novel druggable target to attenuate COVID-19-associated thrombotic events.","version":"1.1","doi":"10.1101/2023.10.30.564680","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.28.564096","pub_date":"2023-10-30","title":"Inhalation Delivered RNA Aptamer for Therapeutics against SARS-CoV2","abstract":"The continual emergence and re-emergence of infectious diseases has led to a pressing need for the development of swift and targeted therapeutic interventions. SARS-CoV-2, the causative agent of COVID-19, is a prime example of such a rapidly spreading virus[1]. The global crisis caused by this virus has propelled researchers to explore and adopt novel techniques in the hopes of effectively combating its spread[2]. One such innovative approach is the use of aptamers\u2014single-stranded RNA molecules that can bind targets with high specificity.","version":"1.1","doi":"10.1101/2023.10.28.564096","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.27.564440","pub_date":"2023-10-30","title":"NK cell-monocyte crosstalk underlies NK cell activation in severe COVID-19","abstract":"NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I interferon and TGF\u03b2, that underlie this dysregulation. However, the role of cell-cell interactions in mediating changes in NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell co-culture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro co-cultures in which NK cells from healthy donors were incubated with monocytes from COVID-19+ or healthy donors. Co-culture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from transwell co-cultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines as well as TGF\u03b2. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19. Natural killer (NK) cells are innate lymphocytes that are critical antiviral effectors. Because of their role in controlling acute viral infections, multiple studies have evaluated the role of NK cells in SARS-CoV-2 infection. Such studies revealed that NK cell phenotype and function are significantly altered by severe COVID-19; the peripheral NK cells of severe COVID-19 patients are highly activated and proliferative(1\u20135), with increased expression of cytotoxic molecules, Ki-67, and several surface markers of activation(3, 5\u20138). However, these NK cells also have dysfunctional cytotoxic responses to both tumor target cells(1, 2, 9, 10) and SARS-CoV-2-infected target cells(9, 10). Given that peripheral NK cells are thought to migrate to the lung during COVID-19(11\u201313), these results suggest that the NK cells of severe COVID-19 patients may be incapable of mounting a successful antiviral response to SARS-CoV-2 infection. Although the unique phenotype and dysfunctionality of NK cells in severe COVID-19 has been well-characterized, the processes underlying these phenomena have not. Only one study has conducted in vitro mechanistic experiments to identify a possible cause of NK cell dysfunction: Witkowski et al. identified serum-derived TGF\u03b2 as a suppressor of NK cell functionality in severe COVID-19 patients(9). However, this study did not identify the source of serum TGF\u03b2. Additionally, given the high degree of complexity within the immune system, there are likely other causes of NK cell dysfunction in COVID-19 that have thus far remain unexplored. One such mechanism may be the myriad of interactions between NK cells and other peripheral immune cells. NK cells are known to interact with CD4 and CD8 T cells, dendritic cells, neutrophils, and macrophages/monocytes(14), which can prime NK cell cytotoxicity or induce tolerance. Previous work by our lab suggested the potential for NK cell-monocyte crosstalk in severe COVID-19 through the expression of ligands for NK cell activating receptors on the monocytes of these patients(3). Crosstalk between NK cells and monocytes plays a role in regulating the NK cell response to other infections, including HIV-1(15, 16), mouse(17) and human cytomegalovirus(18), and malaria(19) through mechanisms including secretion of NK cell-regulating cytokines by monocytes. In this study, we used a combination of computational and in vitro methods to dissect the interactions between NK cells and monocytes in severe COVID-19. We utilized primary NK cells and monocytes from a large cohort of COVID-19 patients to demonstrate that co-culture of healthy NK cells with monocytes from severe COVID-19 donors can partially recapitulate the activated phenotype observed in the NK cells from COVID-19 patients. We then interrogated the mechanisms by which this activation occurs by performing NK cell-monocyte co-cultures in a transwell setting and using O-link to analyze the cytokines present in this system. Collectively, our work identifies monocytes as a driver of NK cell activation in severe COVID-19 and reveals interactions between NK cells and monocytes that may underlie this process.","version":"1.1","doi":"10.1101/2023.10.27.564440","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.27.564435","pub_date":"2023-10-30","title":"Colloidal aggregation confounds cell-based Covid-19 antiviral screens","abstract":"Colloidal aggregation is one of the largest contributors to false-positives in early drug discovery and chemical biology. Much work has focused on its impact on pure-protein screens; here we consider aggregations role in cell-based infectivity assays in Covid-19 drug repurposing. We began by investigating the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal-particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate antiviral efficacy of the drugs in cells we used spike pseudotyped lentivirus and pre-saturation of the colloids with BSA. The antiviral potency of the aggregators was diminished by at least 10-fold and often entirely eliminated in the presence of BSA, suggesting antiviral activity can be attributed to the non-specific nature of the colloids. In confocal microscopy, the aggregates induced fluorescent puncta of labeled spike protein, consistent with sequestration of the protein on the colloidal particles. Addition of either non-ionic detergent or of BSA disrupted these puncta. These observations suggest that colloidal aggregation is common among cell-based anti-viral drug repurposing, and perhaps cell-based assays more broadly, and offers rapid counter-screens to detect and eliminate these artifacts, allowing the community invest resources in compounds with true potential as a Covid-19 therapeutic.","version":"1.1","doi":"10.1101/2023.10.27.564435","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.28.564530","pub_date":"2023-10-30","title":"Deciphering the code of viral-host adaptation through maximum entropy models","abstract":"Understanding how the genome of a virus evolves depending on the host it infects is an important question that challenges our knowledge about several mechanisms of host-pathogen interactions, including mutational signatures, innate immunity, and codon optimization. A key facet of this general topic is the study of viral genome evolution after a host-jumping event, a topic which has experienced a surge in interest due to the fight against emerging pathogens such as SARS-CoV-2. In this work, we tackle this question by introducing a new method to learn Maximum Entropy Nucleotide Bias models (MENB) reflecting single, di- and tri-nucleotide usage, which can be trained from viral sequences that infect a given host. We show that both the viral family and the host leave a fingerprint in nucleotide usages which MENB models decode. When the task is to classify both the host and the viral family for a sequence of unknown viral origin MENB models outperform state of the art methods based on deep neural networks. We further demonstrate the generative properties of the proposed framework, presenting an example where we change the nucleotide composition of the 1918 H1N1 Influenza A sequence without changing its protein sequence, while manipulating the nucleotide usage, by diminishing its CpG content. Finally we consider two well-known cases of zoonotic jumps, for the H1N1 Influenza A and for the SARS-CoV-2 viruses, and show that our method can be used to track the adaptation to the new host and to shed light on the more relevant selective pressures which have acted on motif usage during this process. Our work has wide-ranging applications, including integration into metagenomic studies to identify hosts for diverse viruses, surveillance of emerging pathogens, prediction of synonymous mutations that effect immunogenicity during viral evolution in a new host, and the estimation of putative evolutionary ages for viral sequences in similar scenarios. Additionally, the computational frame-work introduced here can be used to assist vaccine design by tuning motif usage with fine-grained control. In our research, we delved into the fascinating world of viruses and their genetic changes when they jump from one host to another, a critical topic in the study of emerging pathogens. We developed a novel computational method to capture how viruses change the nucleotide usage of their genes when they infect different hosts. We found that viruses from various families have unique strategies for tuning their nucleotide usage when they infect the same host. Our model could accurately pinpoint which host a viral sequence came from, even when the sequence was vastly different from the ones we trained on. We demonstrated the power of our method by altering the nucleotide usage of an RNA sequence without affecting the protein it encodes, providing a proof-of-concept of a method that can be used to design better RNA vaccines or to fine-tune other nucleic acid-based therapies. Moreover the framework we introduce can help tracking emerging pathogens, predicting synonymous mutations in the adaptation to a new host and estimating how long viral sequences have been evolving in it. Overall, our work sheds light on the intricate interactions between viruses and their hosts.","version":"1.1","doi":"10.1101/2023.10.28.564530","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.26.23297597","pub_date":"2023-10-28","title":"Complement dysregulation is a predictive and therapeutically amenable feature of long COVID","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Long COVID encompasses a heterogeneous set of ongoing symptoms that affect many individuals after recovery from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The underlying biological mechanisms nonetheless remain obscure, precluding accurate diagnosis and effective intervention. Complement dysregulation is a hallmark of acute COVID-19 but has not been investigated as a potential determinant of long COVID.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We quantified a series of complement proteins, including markers of activation and regulation, in plasma samples from healthy convalescent individuals with a confirmed history of infection with SARS-CoV-2 and age/ethnicity/gender/infection/vaccine-matched patients with long COVID.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Markers of classical (C1s-C1INH complex), alternative (Ba, iC3b), and terminal pathway (C5a, TCC) activation were significantly elevated in patients with long COVID. These markers in combination had a receiver operating characteristic predictive power of 0.794. Other complement proteins and regulators were also quantitatively different between healthy convalescent individuals and patients with long COVID. Generalized linear modeling further revealed that a clinically tractable combination of just four of these markers, namely the activation fragments iC3b, TCC, Ba, and C5a, had a predictive power of 0.785.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>These findings suggest that complement biomarkers could facilitate the diagnosis of long COVID and further suggest that currently available inhibitors of complement activation could be used to treat long COVID.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This work was funded by the National Institute for Health Research (COV-LT2-0041), the PolyBio Research Foundation, and the UK Dementia Research Institute.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.10.26.23297597","journal":"medRxiv","score":null},{"id":"10.1101/2023.10.28.564520","pub_date":"2023-10-28","title":"Streamlined DNA template preparation and co-transcriptional 5\u2032 capped RNA synthesis enabled by solid-phase catalysis","abstract":"The success of SARS-CoV-2 mRNA vaccines demonstrated that rapid, large-scale manufacturing of synthetic mRNA is necessary for an effective and timely response to a pandemic. Innovations in areas such as template design and manufacturing processes are being implemented to facilitate more simple, cost-effective and scalable mRNA synthesis. In this study, for the first time, we demonstrate that the enzymatic steps in mRNA production (including DNA template linearization, RNA synthesis, 5\u2032 capping and methylation) can be carried out using enzymes immobilized to a solid support. Specifically, we demonstrate efficient IVT template DNA linearization using immobilized BspQI, where the linearized template DNA can be directly used in IVT without the need of purification. We also showed that immobilized T7 RNA polymerase, Faustovirus RNA capping enzyme (FCE), vaccinia cap 2\u2032-O-methyltransfease (2\u2032OMTase) and a novel FCE::T7RNAP fusion enable efficient enzymatic synthesis of Cap-1 RNA in a one-pot format. This solid-phase enzymatic platform may enable highly efficient, seamless and continuous mRNA synthesis workflows that minimizes sample loss and units of operation in biopharmaceutical manufacturing.","version":"1.1","doi":"10.1101/2023.10.28.564520","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.26.564184","pub_date":"2023-10-27","title":"Immune Epitopes of SARS-CoV-2 Spike Protein and Considerations for Universal Vaccine Development","abstract":"Despite the success of global vaccination programs in slowing the spread of COVID-19, these efforts have been hindered by the emergence of new SARS-CoV-2 strains capable of evading prior immunity. The mutation and evolution of SARS-CoV-2 have created a demand for persistent efforts in vaccine development. SARS-CoV-2 Spike protein has been the primary target for COVID-19 vaccine development, but it is also the hotspot of mutations directly involved in host susceptibility and immune evasion. Our ability to predict emerging mutants and select conserved epitopes is critical for the development of a broadly neutralizing therapy or a universal vaccine. In this article, we review the general paradigm of immune responses to COVID-19 vaccines, highlighting the immunological epitopes of Spike protein that are likely associated with eliciting protective immunity resulting from vaccination. Specifically, we analyze the structural and evolutionary characteristics of the SARS-CoV-2 Spike protein related to immune activation and function via the toll-like receptors (TLRs), B cells, and T cells. We aim to provide a comprehensive analysis of immune epitopes of Spike protein, thereby contributing to the development of new strategies for broad neutralization or universal vaccination.","version":"1.1","doi":"10.1101/2023.10.26.564184","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.26.564259","pub_date":"2023-10-27","title":"Impact of Memory T Cells on SARS-COV-2 Vaccine Response in Hematopoietic Stem Cell Transplant","abstract":"During the COVID-19 pandemic, hematopoietic stem cell transplant (HSCT) recipients faced an elevated mortality rate from SARS-CoV-2 infection, ranging between 10-40%. The SARS-CoV-2 mRNA vaccines are important tools in preventing severe disease, yet their efficacy in the post-transplant setting remains unclear, especially in patients subjected to myeloablative chemotherapy and immunosuppression. We evaluated the humoral and adaptive immune responses to the SARS-CoV-2 mRNA vaccination series in 42 HSCT recipients and 5 healthy controls. Peripheral blood mononuclear nuclear cells and serum were prospectively collected before and after each dose of the SARS-CoV-2 vaccine. Post-vaccination responses were assessed by measuring anti-spike IgG and nucleocapsid titers, and antigen specific T cell activity, before and after vaccination. In order to examine mechanisms behind a lack of response, pre-and post-vaccine samples were selected based on humoral and cellular responses for single-cell RNA sequencing with TCR and BCR sequencing. Our observations revealed that while all participants eventually mounted a humoral response, transplant recipients had defects in memory T cell populations that were associated with an absence of T cell response, some of which could be detected pre-vaccination.","version":"1.1","doi":"10.1101/2023.10.26.564259","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.08.561395","pub_date":"2023-10-27","title":"Lethal COVID-19 Associates With RAAS-Induced Inflammation For Multiple Organ Damage Including Mediastinal Lymph Nodes","abstract":"Lethal COVID-19 outcomes are most often attributed to classic cytokine storm and attendant excessive immune signaling. We re-visit this question using RNA sequencing in nasopharyngeal and 40 autopsy samples from COVID-19-positive and negative individuals. In nasal swabs, the top 100 genes which significantly correlated with COVID-19 viral load, include many canonical innate immune genes. However, 22 much less studied 'non-canonical' genes are found and despite the absence of viral transcripts, subsets of these are upregulated in heart, lung, kidney, and liver, but not mediastinal lymph nodes. An important regulatory potential emerges for the non-canonical genes for over-activating the renin-angiotensin-activation-system (RAAS) pathway, resembling this phenomenon in hereditary angioedema (HAE) and its overlapping multiple features with lethal COVID-19 infections. Specifically, RAAS overactivation links increased fibrin deposition, leaky vessels, thrombotic tendency, and initiating the PANoptosis death pathway, as suggested in heart, lung, and especially mediastinal lymph nodes, with a tightly associated mitochondrial dysfunction linked to immune responses. For mediastinal lymph nodes, immunohistochemistry studies validate the transcriptomic findings showing abnormal architecture, excess fibrin and collagen deposition, and pathogenic fibroblasts. Further, our findings overlap findings in SARS-CoV-2 infected hamsters, C57BL/6 and BALB/c mouse models, and importantly peripheral blood mononuclear cell (PBMC) and whole blood samples from COVID-19 patients infected with early variants and later SARS-CoV-2 strains. We thus present cytokine storm in lethal COVID-19 disease as an interplay between upstream immune gene signaling producing downstream RAAS overactivation with resultant severe organ damage, especially compromising mediastinal lymph node function.","version":"1.3","doi":"10.1101/2023.10.08.561395","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.26.564289","pub_date":"2023-10-27","title":"Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies","abstract":"A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.","version":"1.1","doi":"10.1101/2023.10.26.564289","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.24.563847","pub_date":"2023-10-26","title":"A deep learning framework for predicting the neutralizing activity of COVID-19 therapeutics and vaccines against evolving SARS-CoV-2 variants","abstract":"Understanding how viral variants evade neutralization is crucial for improving antibody-based treatments, especially with rapidly evolving viruses like SARS-CoV-2. Yet, conventional assays are limited in the face of rapid viral evolution, relying on a narrow set of viral isolates, and falling short in capturing the full spectrum of variants. To address this, we have developed a deep learning approach to predict changes in neutralizing antibody activity of COVID-19 therapeutics and vaccines against emerging viral variants. First, we trained a variational autoencoder (VAE) using all 67,885 unique SARS-CoV-2 spike protein sequences from the NCBI virus (up to October 31, 2022) database to encode spike protein variants into a latent space. Using this VAE and a curated dataset of 7,069 in vitro assay data points from the NCATS OpenData Portal, we trained a neural network regression model to predict fold changes in neutralizing activity of 40 COVID-19 therapeutics and vaccines against spike protein sequence variants, relative to their neutralizing activity against the ancestral strain (Wuhan-Hu-1). Our model also employs Bayesian inference to quantify prediction uncertainty, providing more nuanced and informative estimates. To validate the model\u2019s predictive capacity, we assessed its performance on a test set of in vitro assay data collected up to eight months after the data included in the model training (N = 980). The model accurately predicted fold changes in neutralizing activity for this prospective dataset, with an R2 of 0.77. Expanding our methodology to include all available data from NCBI virus and NCATS OpenData Portal up to date, we assessed predicted changes in activity for current COVID-19 monoclonal antibodies and vaccines against newly identified SARS-CoV-2 lineages. Our predictions suggest that current therapeutic and vaccine-induced antibodies will have significantly reduced activity against newer XBB descendants, notably EG.5, FL.1.5.1, and XBB.1.16. Using the model, we were able to primarily attribute the observed predicted loss in activity to the F456L spike mutation found in EG.5 and FL.1.5.1 sequences. Conversely, mRNA-bivalent vaccines are predicted to be less susceptible to the recent BA.2.86 variant compared to new XBB descendants. These findings align closely with recent research, underscoring the potential of deep learning in shaping therapeutic and vaccine strategies for emerging viral variants.","version":"1.1","doi":"10.1101/2023.10.24.563847","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.25.563806","pub_date":"2023-10-26","title":"Reduced Monocyte Proportions and Responsiveness in Convalescent COVID-19 Patients","abstract":"The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and COVID-19 suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We investigated the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and non-classical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients and uninfected control subjects. We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF-\u03b1 and IL-6 in response to LPS stimulation, than those from uninfected controls. In conclusion, SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.","version":"1.1","doi":"10.1101/2023.10.25.563806","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.24.563688","pub_date":"2023-10-25","title":"Discovery of CMX990: A Potent SARS-CoV-2 3CL Protease Inhibitor Bearing a Novel Covalent Warhead","abstract":"There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof towards human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CLpro inhibitor, CMX990, bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = PaxlovidTM), CMX990 has distinctly differentiated potency (\u223c5x more potent in primary cells) and human in vitro clearance (>4x better microsomal clearance and >10x better hepatocyte clearance), with good in vitro-in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.10.24.563688","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.24.561010","pub_date":"2023-10-25","title":"VIPERA: Viral Intra-Patient Evolution Reporting and Analysis","abstract":"Viral mutations within patients nurture the adaptive potential of SARS-CoV-2 during chronic infections, which are a potential source of variants of concern. However, there is no integrated framework for the evolutionary analysis of intra-patient SARS-CoV-2 serial samples. Herein we describe VIPERA (Viral Intra-Patient Evolution Reporting and Analysis), a new software that integrates the evaluation of the intra-patient ancestry of SARS-CoV-2 sequences with the analysis of evolutionary trajectories of serial sequences from the same viral infection. We have validated it using positive and negative control datasets and have successfully applied it to a new case, thus enabling an easy and automatic analysis of intra-patient SARS-CoV-2 sequences.","version":"1.1","doi":"10.1101/2023.10.24.561010","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488633","pub_date":"2023-10-25","title":"Benchmarking the accuracy of structure-based binding affinity predictors on Spike-ACE2 Deep Mutational Interaction Set","abstract":"Since the start of COVID-19 pandemic, a huge effort has been devoted to understanding the Spike (SARS-CoV-2)-ACE2 recognition mechanism. To this end, two deep mutational scanning studies traced the impact of all possible mutations across Receptor Binding Domain (RBD) of Spike and catalytic domain of human ACE2. By concentrating on the interface mutations of these experimental data, we benchmarked six commonly used structure-based binding affinity predictors (FoldX, EvoEF1, MutaBind2, SSIPe, HADDOCK, and UEP). These predictors were selected based on their user-friendliness, accessibility, and speed. As a result of our benchmarking efforts, we observed that none of the methods could generate a meaningful correlation with the experimental binding data. The best correlation is achieved by FoldX (R = -0.51). Also, when we simplified the prediction problem to a binary classification, i.e., whether a mutation is enriching or depleting the binding, we showed that the highest accuracy is achieved by FoldX with 64% success rate. Surprisingly, on this set, simple energetic scoring functions performed significantly better than the ones using extra evolutionary-based terms, as in Mutabind and SSIPe. Furthermore, we also demonstrated that recent AI approaches, mmCSM-PPI and TopNetTree, yielded comparable performances to the force field-based techniques. These observations suggest plenty of room to improve the binding affinity predictors in guessing the variant-induced binding profile changes of a host-pathogen system, such as Spike-ACE2. To aid such improvements we provide our benchmarking data at https://github.com/CSB-KaracaLab/RBD-ACE2-MutBench with the option to visualize our mutant models at https://rbd-ace2-mutbench.github.io/","version":"1.4","doi":"10.1101/2022.04.18.488633","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.23.563621","pub_date":"2023-10-24","title":"Heterotypic responses against nsp12/nsp13 from prior SARS-CoV-2 infection associates with lower subsequent endemic coronavirus incidence","abstract":"Immune responses from prior SARS-CoV-2 infection and COVID-19 vaccination do not prevent re-infections and may not protect against future novel coronaviruses (CoVs). We examined the incidence of and immune differences against human endemic CoVs (eCoV) as a proxy for response against future emerging CoVs. Assessment was among those with known SARS-CoV-2 infection, COVID-19 vaccination but no documented SARS-CoV-2 infection, or neither exposure. Retrospective cohort analyses suggest that prior SARS-CoV-2 infection, but not COVID-19 vaccination alone, protects against subsequent symptomatic eCoV infection. CD8+ T cell responses to the non-structural eCoV proteins, nsp12 and nsp13, were significantly higher in individuals with previous SARS-CoV-2 infection as compared to the other groups. The three groups had similar cellular responses against the eCoV spike and nucleocapsid, and those with prior spike exposure had lower eCoV-directed neutralizing antibodies. Incorporation of non-structural viral antigens in a future pan-CoV vaccine may improve protection against future heterologous CoV infections.","version":"1.1","doi":"10.1101/2023.10.23.563621","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.23.563088","pub_date":"2023-10-24","title":"Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity","abstract":"Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five-to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.","version":"1.1","doi":"10.1101/2023.10.23.563088","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.22.563481","pub_date":"2023-10-24","title":"Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD-20 therapy","abstract":"Patients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as Rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. We performed in depth antibody characterisation in sera collected four to six weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in Rituximab-treated primary vasculitis patients (n=14) using Luminex and pseudovirus neutralisation assays, whereas a novel microfluidic-based immunoassay was used to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. Comparative antibody profiling was performed at equivalent time points in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n=15) and in convalescent patients after WT SARS-CoV-2 infection (n=30). Rituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in Rituximab-treated patients and in healthy individuals. In the Rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT (median [range] KD: 21.6 [9.7-38.8] nM vs 4.6 [2.3-44.8] nM, p=0.0004). By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in Rituximab-treated patients (median [range] KD: 1.05 [0.45-1.84] nM vs 20.25 [13.2-38.8] nM, p=0.0002), underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection which was not observed in Rituximab-treated patients, despite repeated vaccination. Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in Rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.","version":"1.1","doi":"10.1101/2023.10.22.563481","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.30.555211","pub_date":"2023-10-24","title":"Convergent evolution of SARS-CoV-2 XBB lineages on receptor-binding domain 455-456 synergistically enhances antibody evasion and ACE2 binding","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) XBB lineages have achieved dominance worldwide and keep on evolving. Convergent evolution of XBB lineages on the receptor-binding domain (RBD) L455F and F456L is observed, resulting in variants like EG.5, FL.1.5.1, XBB.1.5.70, and HK.3. Here, we show that neutralizing antibody (NAb) evasion drives the convergent evolution of F456L, while the epistatic shift caused by F456L enables the subsequent convergence of L455F through ACE2 binding enhancement and further immune evasion. L455F and F456L evade Class 1 NAbs, reducing the neutralization efficacy of XBB breakthrough infection (BTI) and reinfection convalescent plasma. Importantly, L455F single substitution significantly dampens receptor binding; however, the combination of L455F and F456L forms an adjacent residue flipping, which leads to enhanced NAbs resistance and ACE2 binding affinity. The perturbed receptor-binding mode leads to the exceptional ACE2 binding and NAb evasion, as revealed by structural analyses. Our results indicate the evolution flexibility contributed by epistasis cannot be underestimated, and the evolution potential of SARS-CoV-2 RBD remains high.","version":"1.2","doi":"10.1101/2023.08.30.555211","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.22.563156","pub_date":"2023-10-24","title":"A 50-gene high-risk profile predictive of COVID-19 and Idiopathic Pulmonary Fibrosis mortality originates from a genomic imbalance in monocyte and T-cell subsets that reverses in survivors with post-COVID-19 Interstitial Lung Disease","abstract":"We aim to study the source of circulating immune cells expressing a 50-gene signature predictive of COVID-19 and IPF mortality. Whole blood and Peripheral Blood Mononuclear cells (PBMC) were obtained from 231 subjects with COVID-19, post-COVID-19-ILD, IPF and controls. We measured the 50-gene signature (nCounter, Nanostring), interleukin 6 (IL6), interferon \u03b3-induced protein (IP10), secreted phosphoprotein 1 (SPP1) and transforming growth factor beta (TGF-\u03b2) by Luminex. PCR was used to validate COVID-19 endotypes. For single-cell RNA sequencing (scRNA-seq) we used Chromium Controller (10X Genomics). For analysis we used the Scoring Algorithm of Molecular Subphenotypes (SAMS), Cell Ranger, Seurat, Propeller, Kaplan-Meier curves, CoxPH models, Two-way ANOVA, T-test, and Fisher\u2019s exact. We identified three genomic risk profiles based on the 50-gene signature, and a subset of seven genes, associated with low, intermediate, or high-risk of mortality in COVID-19 with significant differences in IL6, IP10, SPP1 and TGF\u03b2-1. scRNA-seq identified Monocytic-Myeloid-Derived Suppressive cells (M-MDSCs) expressing CD14+HLA DRlowCD163+ and high levels of the 7-gene signature (7Gene-M-MDSC) in COVID-19. These cells were not observed in post-COVID-19-ILD or IPF. The 43-gene signature was mostly expressed in CD4 T and CD8 T cell subsets. Increased expression of the 43 gene signature was seen in T cell subsets from survivors with post-COVID-19-ILD. The expression of these genes remained low in IPF. A 50-gene, high-risk profile in COVID-19 is characterized by a genomic imbalance in monocyte and T-cell subsets that reverses in survivors with post-COVID-19 Interstitial Lung Disease","version":"1.1","doi":"10.1101/2023.10.22.563156","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.23.563427","pub_date":"2023-10-23","title":"A bioactive peptide from the pearl has dual roles in resisting SARS-CoV-2 infection and its complications","abstract":"Angiotensin-converting enzyme 2 (ACE2) is a critical receptor for the entry of the SARS-CoV-2 virus into cells. Moreover, a decrease in ACE2 level and its activity due to SARS-CoV-2 infection is considered a crucial reason for the development of Covid-19-associated complications. Here, we report a bioactive peptide derived from the seawater pearl oyster Pinctada fucata, named SCOL polypeptide, which binds strongly to ACE2 and effectively inhibits 65% of the binding of the SARS-CoV-2 S protein to ACE2; thus, this peptide can be used as a blocker to enable cells to resist SARS-CoV-2 infection. The SCOL polypeptide also increases ACE2 enzyme activity by 3.76 times. Previous studies have shown that ACE2 deficiency is associated with inflammation, pain, cardiovascular diseases, insulin resistance, and nervous system injury. Therefore, the SCOL polypeptide can be used to treat or alleviate complications such as lung inflammation, pain, diabetes, cardiovascular diseases, and loss of taste or smell caused by SARS-CoV-2 infection. Thus, the SCOL polypeptide can play a dual role in resisting SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.10.23.563427","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.560010","pub_date":"2023-10-23","title":"Contributions of hyperactive mutations in Mpro from SARS-CoV-2 to drug resistance","abstract":"The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding and/or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but had not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a FRET-based yeast readout. We identified hundreds of mutations that significantly increased Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed three times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir-binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.","version":"1.3","doi":"10.1101/2023.09.28.560010","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.20.563308","pub_date":"2023-10-23","title":"Assessing nanobody interaction with SARS-CoV-2 Nsp9","abstract":"The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. However a different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.","version":"1.1","doi":"10.1101/2023.10.20.563308","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535453","pub_date":"2023-10-23","title":"Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5\u2019 untranslated region","abstract":"One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30 and C34, which bind to a four-way RNA helix called SL5 in the 5\u2019 UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2\u2019-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of C30 in the SARS-CoV-2 5\u2019 UTR, which was validated through mutagenesis and in vitro binding experiments. C30 was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5\u2019 UTR expressing cells. We further explored another RLR conjugation site on the E ring of C30/C34 and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells.","version":"1.2","doi":"10.1101/2023.04.03.535453","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.22.563490","pub_date":"2023-10-23","title":"Screening Peptide Drug Candidates to Neutralize Whole Viral Agents : A Case study with SARS-CoV-2 Virus","abstract":"Covid19 pandemic revealed the reality for the need of therapeutic and pharmaceutical molecule development in a short time with different approaches. Although the enhancement of immunological memory by vaccination was the quicker and robust strategy, still medication is required for immediate treatment for a patient. For this purpose, one of the approaches is developing new therapeutic molecule development like peptide-based drugs. Also, peptides can be used developing other molecules like nanobodies. Here, M13 phage display library was used for selecting SARS-CoV-2 interacting peptides for developing a neutralizing molecule for further use. Biopanning was applied with four iterative cycles to select phages displaying different 12-amino acid-long peptides. Then, the M13 phage genomic region where peptide sequences expressed were analyzed and sequences were obtained. Randomly selected peptide sequences were synthesized by solid-state peptide synthesis method. These peptides were analyzed by quartz crystal microbalance method in terms or peptide interaction capacity with specifically wild-type S protein. Next, QCM data was further validated by enzyme-linked immunosorbent assay (ELISA) in order to check peptides according to their neutralizing capacity rather than binding to S1 protein. The results showed that, phage display served an opportunity for selecting peptides which can be used and developed further as pharmaceutical molecules. More specifically, scpep3, scpep8 and scpep10 had both binding and neutralizing capacity for S1 protein as a candidate for therapeutic molecule.","version":"1.1","doi":"10.1101/2023.10.22.563490","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.23.563555","pub_date":"2023-10-23","title":"Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding","abstract":"Enveloped viruses carry one or multiple proteins with receptor binding functionalities. Functional receptors can either be glycans, proteinaceous or both, recombinant protein approaches are instrumental to gain more insight into these binding properties. Visualizing and measuring receptor binding normally entails antibody detection or direct labelling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here we report a suite of different fluorescent fusions, both N- and/or C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained a total of three or six fluorescent protein barrels and were applied directly to cells to determine receptor binding properties.","version":"1.1","doi":"10.1101/2023.10.23.563555","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.20.563154","pub_date":"2023-10-23","title":"In vivo affinity maturation of murine B cells reprogrammed to express human antibodies","abstract":"CRISPR-edited murine B cells engineered to express human antibody variable chains proliferate, class switch, and secrete these antibodies in vaccinated mice. However, current strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that recombined murine heavy- and kappa-variable genes can be directly and simultaneously overwritten, using Cas12a-mediated cuts at their 3\u2019-most J segments and 5\u2019 homology arms complementary to distal V segments. Cells edited in this way to express the HIV-1 broadly neutralizing antibodies 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated recipient mice. 10-1074 variants isolated from these mice bound and neutralized HIV-1 envelope glycoprotein more efficiently than wild-type 10-1074 while maintaining or improving its already low polyreactivity and long in vivo half-life. We further validated this approach by generating substantially broader and more potent variants of the anti-SARS-CoV-2 antibodies ZCB11 and S309. Thus, B cells edited at their native loci affinity mature, facilitating development of broad, potent, and bioavailable antibodies and expanding the potential applications of engineered B cells.","version":"1.1","doi":"10.1101/2023.10.20.563154","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.19.563184","pub_date":"2023-10-20","title":"Divergent spike mutations impact the activation of the fusion core in Delta and Omicron variants of SARS-CoV-2","abstract":"SARS-CoV-2 infects host cells by binding the receptor-binding domain (RBD) of its spike protein to the receptor, ACE2. A subset of highly effective spike mutations plays critical roles in altering the conformational dynamics of spike protein. Here, we use molecular dynamics simulations to investigate how spike mutations affect the conformational dynamics of spike/ACE2 complex in the D614G, Delta (B.1.617.2) and Omicron (B.1.1.529) SARS-CoV-2 variants. We observe that the increased positive-charged mutations in the Omicron spike amplify its structural rigidity and reduce its structural flexibility. The mutations (P681R in Delta and P681H in Omicron) at the S1/S2 junction facilitate S1/S2 cleavage and aid the activation of the fusion core. We report that high structural flexibility in Delta lowers the barrier for the activation of the S2 core; however, high structural rigidity in Omicron enhances the barrier for the same. Our results also explain why Omicron requires the presence of a higher number of ACE2 to activate its fusion core than Delta.","version":"1.1","doi":"10.1101/2023.10.19.563184","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.19.563209","pub_date":"2023-10-20","title":"Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant","abstract":"In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.","version":"1.1","doi":"10.1101/2023.10.19.563209","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.19.563117","pub_date":"2023-10-20","title":"Neuroinflammation in post-acute sequelae of COVID-19 (PASC) as assessed by [11C]PBR28 PET correlates with vascular disease measures","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has triggered a consequential public health crisis of post-acute sequelae of COVID-19 (PASC), sometimes referred to as long COVID. The mechanisms of the heterogeneous persistent symptoms and signs that comprise PASC are under investigation, and several studies have pointed to the central nervous and vascular systems as being potential sites of dysfunction. In the current study, we recruited individuals with PASC with diverse symptoms, and examined the relationship between neuroinflammation and circulating markers of vascular dysfunction. We used [11C]PBR28 PET neuroimaging, a marker of neuroinflammation, to compare 12 PASC individuals versus 43 normative healthy controls. We found significantly increased neuroinflammation in PASC versus controls across a wide swath of brain regions including midcingulate and anterior cingulate cortex, corpus callosum, thalamus, basal ganglia, and at the boundaries of ventricles. We also collected and analyzed peripheral blood plasma from the PASC individuals and found significant positive correlations between neuroinflammation and several circulating analytes related to vascular dysfunction. These results suggest that an interaction between neuroinflammation and vascular health may contribute to common symptoms of PASC.","version":"1.1","doi":"10.1101/2023.10.19.563117","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.19.563051","pub_date":"2023-10-20","title":"FAKHRAVAC and BBIBP-CorV vaccine seeds\u2019 binding to angiotensin-converting enzyme 2: A comparative molecular dynamics study","abstract":"Safety and efficacy of the SARS-CoV-2 inactivated vaccines have been question since the emergence of SARS-CoV-2 variants of concern (VOCs). Using residue fluctuations and statistically comparing RMSF values, have escalated the understanding of the binding dynamics of the viral proteins to their receptors and here in this study, we compared the interaction between inactivated spike proteins (representing FAKHRAVAC and BBIBP-CorV vaccines seed) and the human Angiotensin-Converting Enzyme 2 (hACE2) receptor. Through 100 set of accelerated 1 ns comparative molecular dynamics simulations, we analyze the binding dynamics and energy components of these interactions and compared residue backbone fluctuations using entropy and statistics including KL-Divergence and KS-test. Our results reveal that FAKHRAVAC and Sinopharm exhibit similar binding dynamics and affinity to hACE2. Further examination of residue-wise fluctuations highlights the common behavior of binding key residues and mutation sites between the two vaccines. However, subtle differences in residue fluctuations, especially at critical sites like Q24, Y435, L455, S477, Y505, and F486, raise the possibility of distinct efficacy profiles. These variations may influence vaccine immunogenicity and safety in response to evolving SARS-CoV-2 variants. The study underscores the importance of considering residue-wise fluctuations for understanding vaccine-pathogen interactions and their implications for vaccine design. It is fundamentally important to ensure the safety and efficacy of the FAKHRAVAC, as an inactivated vaccine candidate for SARS-CoV-2. Considering the previously published pre-clinical and clinical findings about the similarity of the FAKHRAVAC\u2019s safety and efficacy in comparison to the BBIBP-CorV vaccine seed (which is recalled as Sinopharm), it is necessary to gain more insights into structure and function of this vaccine at the molecular level, as well. Since the binding dynamics of the viral proteins to their receptor can imply the vaccine\u2019s immunogenicity and mechanism-of-action, binding dynamics of a vaccine candidate must be studied comprehensively. Hereby, we have compared binding dynamics of the FAKHRAVAC and Sinopharm vaccine seeds to the SARS-CoV-2 spike protein\u2019s receptor, the ACE2. We took advantage of a comparative molecular dynamics simulation approach to effectively compare binding dynamics using atom fluctuations and at the residue level to ensure the resolution of this study. We have found similar binding dynamics and binding mechanics between these two vaccines, validating the pre-clinical and clinical findings computationally, as well as highlighting residues with different fluctuations and discussed their potential roles.","version":"1.1","doi":"10.1101/2023.10.19.563051","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.18.563024","pub_date":"2023-10-19","title":"Caveolin-1 mediates neuroinflammation and cognitive impairment in SARS-CoV-2 infection","abstract":"Leukocyte infiltration of the CNS can contribute to neuroinflammation and cognitive impairment. Brain endothelial cells regulate adhesion, activation, and diapedesis of T cells across the blood-brain barrier (BBB) in inflammatory diseases. The integral membrane protein Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on T cell CNS infiltration in respiratory viral infections is unknown. In this study, we sought to determine the role of Cav-1 at the BBB in neuroinflammation in a COVID-19 mouse model. We used mice genetically deficient in Cav-1 to test the role of this protein in T cell infiltration and cognitive impairment. We found that SARS-CoV-2 infection upregulated brain endothelial Cav-1. Moreover, SARS-CoV-2 infection increased brain endothelial cell vascular cell adhesion molecule-1 (VCAM-1) and CD3+ T cell infiltration of the hippocampus, a region important for short term learning and memory. Concordantly, we observed learning and memory deficits. Importantly, genetic deficiency in Cav-1 attenuated brain endothelial VCAM-1 expression and T cell infiltration in the hippocampus of mice with SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results indicate the importance of BBB permeability in COVID-19 neuroinflammation and suggest potential therapeutic value of targeting Cav-1 to improve disease outcomes.","version":"1.1","doi":"10.1101/2023.10.18.563024","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.18.563016","pub_date":"2023-10-19","title":"Th2 and Th17-Associated Immunopathology Following SARS-CoV-2 Breakthrough Infection in Spike-Vaccinated ACE2-humanized Mice","abstract":"Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety. This research investigates the safety and efficacy of a Spike protein subunit vaccine adjuvanted with Alum and CpG in an ACE2-humanized mouse model, simulating SARS-CoV-2 breakthrough infections. The study reveals that despite robust protection against severe COVID-19, vaccinated mice exhibit substantial pulmonary immunopathology, including eosinophilia and enhanced Th2 effector immunity, following breakthrough infections. Surprisingly, the study also uncovers a significant systemic Th17 inflammatory response in vaccinated mice. This research sheds light on the potential risks associated with COVID-19 vaccine breakthrough infections and the need for a comprehensive understanding of vaccine-induced immune responses, emphasizing the importance of ongoing research, surveillance, and careful vaccine development for both protection and safety in the fight against the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2023.10.18.563016","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.17.562827","pub_date":"2023-10-18","title":"De novo generation of antibody CDRH3 with a pre-trained generative large language model","abstract":"Artificial Intelligence (AI) techniques have made great advances in assisting antibody design. However, antibody design still heavily relies on isolating antigen-specific antibodies from serum, which is a resource-intensive and time-consuming process. To address this issue, we propose a Pre-trained Antibody generative large Language Model (PALM) for the de novo generation of artificial antibodies heavy chain complementarity-determining region 3 (CDRH3) with desired antigen-binding specificity, reducing the reliance on natural antibodies. We also build a high-precision model antigen-antibody binder (A2binder) that pairs antigen epitope sequences with antibody sequences to predict binding specificity and affinity. PALM-generated antibodies exhibit binding ability to SARS-CoV-2 antigens, including the emerging XBB variant, as confirmed through in-silico analysis and in-vitro assays. The in-vitro assays validated that PALM-generated antibodies achieve high binding affinity and potent neutralization capability against both wild-type and XBB spike proteins of SARS-CoV-2. Meanwhile, A2binder demonstrated exceptional predictive performance on binding specificity for various epitopes and variants. Furthermore, by incorporating the attention mechanism into the PALM model, we have improved its interpretability, providing crucial insights into the fundamental principles of antibody design.","version":"1.1","doi":"10.1101/2023.10.17.562827","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.13.562139","pub_date":"2023-10-17","title":"Dual Ethanolamine Head Groups in Ionizable Lipids Facilitate Phospholipid-free Stable Nanoparticle Formulation for Augmented and Safer mRNA Delivery","abstract":"The accelerated advent of mRNA-based therapeutics and vaccines, highlighted by the battle against SARS-CoV-2, underscores the urgency to refine lipid nanoparticles (LNPs) for efficient mRNA delivery. In this work, we introduce a novel series of ionizable lipids characterized by double ethanolamine head groups, significantly amplifying mRNA binding affinity. A succinct three-component formulation is subsequently delineated, obviating the conventional dependency on phospholipids inherent in traditional four-component LNPs. Intriguingly, this formulation enables particle formation under neutral pH conditions, a notable departure from the acidic milieu traditionally required, attributable to the enhanced nonionic interactions predominating in mRNA encapsulation. The resultant particles exhibit exceptional stability, superior mRNA encapsulation efficiency, and maintain robust delivery efficacy. When deployed as a vaccine platform, the formulation elicited pronounced humoral and T-cell immune responses, concurrently exhibiting a favorable toxicity profile with a reduced induction of pro-inflammatory cytokines such as IL-6. Our exploration suggests that by fine-tuning the non-electrostatic interactions between the ionizable lipid and mRNA, the dynamics of particle formation can be considerably divergent from the prevailing paradigms of mRNA-LNP formation, hinting at a broader horizon for lipid optimization within the realm of mRNA delivery systems.","version":"1.1","doi":"10.1101/2023.10.13.562139","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.13.562198","pub_date":"2023-10-16","title":"Analysis the molecular similarity of least common amino acid sites in ACE2 receptor to predict the potential susceptible species for SARS-CoV-2","abstract":"This research offers a bioinformatics approach to forecasting both domestic and wild animals\u2019 likelihood of being susceptible to SARS-CoV-2 infection. Genomic sequencing can resolve phylogenetic relationships between the virus and the susceptible host. The genome sequence of SARS-CoV-2 is highly interactive with the specific sequence region of the ACE2 receptor of the host species. We further evaluate this concept to identify the most important SARS-CoV-2 binding amino acid sites in the ACE2 receptor sequence through the common similarity of the last common amino acid sites (LCAS) in known susceptible host species. Therefore, the SARS-CoV-2 viral genomic interacting key amino acid region in the ACE2 receptor sequence of known susceptible human host was summarized and compared with other reported known SARS-CoV-2 susceptible host species. We identified the 10 most significant amino acid sites for interaction with SARS-CoV-2 infection from the ACE2 receptor sequence region based on the LCAS similarity pattern in known sensitive SARS-CoV-2 hosts. The most significant 10 LCAS were further compared with ACE2 receptor sequences of unknown species to evaluate the similarity of the last common amino acid pattern (LCAP). We predicted the probability of SARS-CoV-2 infection risk in unknown species through the LCAS similarity pattern. This method can be used as a screening tool to assess the risk of SARS-CoV-2 infection in domestic and wild animals to prevent outbreaks of infection.","version":"1.1","doi":"10.1101/2023.10.13.562198","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.13.562192","pub_date":"2023-10-16","title":"Cross-sector collaboration is more effective than single sector actions at mitigating SARS-CoV-2 in white-tailed deer","abstract":"One Health helps achieve optimal health outcomes for people, animals, plants, and their shared environments. We describe a multidisciplinary effort to better understand and mitigate SARS-CoV-2 spread in white-tailed deer across One Health sectors. We first framed the risk problem with three governance sectors that manage captive and wild deer and human public health. The framing included the objectives for each sector, interactions that facilitate human-to-deer and deer-to-deer transmission, and alternatives intended to reduce risk. We then developed a dynamic compartmental model that linked wild and captive deer herds and humans and simulated SARS-CoV-2 dynamics. For baseline conditions, we estimated that median SARS-CoV-2 prevalence in wild and captive herds varied between 0.03 \u2013 0.07, incidence between 0.68 \u2013 1.46, and probability of persistence between 0.64 \u2013 0.97 across 120-day simulations. We then tested single-sector alternatives alone and in combination with other sector actions. We found that single sector alternatives varied in their ability to reduce transmission and that the best performing alternative required collaborative actions among wildlife management, agricultural management, and public health agencies.","version":"1.1","doi":"10.1101/2023.10.13.562192","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.15.23297013","pub_date":"2023-10-15","title":"Hyperglycemia-induced cathepsin L maturation: Linking to diabetic comorbidities and COVID-19 mortality","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  Diabetes, a prevalent chronic condition, significantly increases the risk of mortality from COVID-19, yet the underlying mechanisms remain elusive. Emerging evidence implicates Cathepsin L (CTSL) in diabetic complications, including nephropathy and retinopathy. Our previous research identified CTSL as a pivotal protease promoting SARS-CoV-2 infection. Here, we demonstrate elevated blood CTSL levels in individuals with diabetes, facilitating SARS-CoV-2 infection. Chronic hyperglycemia correlates positively with CTSL concentration and activity in diabetic patients, while acute hyperglycemia augments CTSL activity in healthy individuals.\n                  <jats:italic>In vitro</jats:italic>\n                  studies reveal high glucose, but not insulin, promotes SARS-CoV-2 infection in wild-type cells, with\n                  <jats:italic>CTSL</jats:italic>\n                  knockout cells displaying reduced susceptibility. Utilizing lung tissue samples from diabetic and non-diabetic patients, alongside db/db diabetic and control mice, we illustrate increased CTSL activity in both humans and mice under diabetic conditions. Mechanistically, high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum to the lysosome via the ER-Golgi-lysosome axis. Our findings underscore the pivotal role of hyperglycemia-induced CTSL maturation in diabetic comorbidities and complications.\n                </jats:p>","version":null,"doi":"10.1101/2023.10.15.23297013","journal":"medRxiv","score":null},{"id":"10.1101/2023.10.12.561995","pub_date":"2023-10-14","title":"Antibodies utilizing VL6-57 light chains target a convergent cryptic epitope on SARS-CoV-2 spike protein driving the genesis of Omicron variants","abstract":"Continued evolution of SARS-CoV-2 generates variants to challenge antibody immunity established by infection and vaccination. A connection between population immunity and genesis of virus variants has long been suggested but its molecular basis remains poorly understood. Here, we identify a class of SARS-CoV-2 neutralising public antibodies defined by their shared usage of VL6-57 light chains. Although heavy chains of diverse genotypes are utilized, convergent HCDR3 rearrangements have been observed among these public antibodies to cooperate with germline VL6-57 LCDRs to target a convergent epitope defined by RBD residues S371-S373-S375. Antibody repertoire analysis identifies that this class of VL6-57 antibodies is present in SARS-CoV-2-na\u00efve individuals and is clonally expanded in most COVID-19 patients. We confirm that Omicron specific substitutions at S371, S373 and S375 mediate escape of antibodies of the VL6-57 class. These findings support that this class of public antibodies constitutes immune pressure promoting the introduction of S371L/F-S373P-S375F in Omicron variants. The results provide further molecular evidences to support that antigenic evolution of SARS-CoV-2 is driven by antibody mediated population immunity.","version":"1.1","doi":"10.1101/2023.10.12.561995","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.12.561992","pub_date":"2023-10-13","title":"Nanobodies against SARS-CoV-2 non-structural protein Nsp9 inhibit viral replication by targeting innate immunity","abstract":"Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not target it to their full potential and as a consequence, inhibition of viral entry may not be efficient. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for viral genome replication. To find out if this strategy efficiently blocks viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA- 2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells with multiple SARS-CoV-2 variants and subjected them to LNP-mRNA-2NSP23 treatment. Analysis of total RNA isolated from infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.","version":"1.1","doi":"10.1101/2023.10.12.561992","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.12.561993","pub_date":"2023-10-13","title":"Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection","abstract":"Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. For positive-sense, single-stranded RNA viruses, several studies have attempted to monitor viral kinetics in vivo using reporter genomes. The application of such recombinant viruses can be limited by challenges in accommodating bioluminescent reporter genes in the viral genome. Conventional luminescence also exhibits relatively low tissue permeability and thus less sensitivity for visualization in vivo. Here we show that unlike NanoLuc bioluminescence, the improved method, termed AkaBLI, allows visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Syrian hamsters. By successfully incorporating a codon-optimized Akaluc luciferase gene into the SARS-CoV-2 genome, we visualized in vivo infection, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of neutralizing antibodies and mRNA vaccination by monitoring changes in Akaluc signals. Overall, AkaBLI is an effective technology for monitoring viral dynamics in live animals.","version":"1.1","doi":"10.1101/2023.10.12.561993","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.11.561987","pub_date":"2023-10-13","title":"Resolving the pharmacological redox-sensitivity of SARS-CoV-2 PLpro in drug repurposing screening enabled identification of the competitive GRL-0617 binding site inhibitor CPI-169","abstract":"The SARS CoV-2 Papain-Like protease has multiple roles in the viral replication cycle, related to both its polypeptide cleavage function and its capacity to antagonize host immune response. Targeting PLpro function is recognized as a promising mechanism to modulate viral replication whilst supporting host immune responses. However, development of PLpro specific inhibitors remains challenging. Upcoming studies revealed the limitation of reported inhibitors by profiling them through a pipeline of enzymatic, binding and cellular activity assays showing unspecific activity. GRL-0617 remained the only validated molecule with demonstrated anti-viral activity in cells. In this study we refer to the pitfalls of redox-sensitivity of PLpro. Using a screening-based approach to identify inhibitors of PLpro proteolytic activity, we made extensive efforts to validate the active compounds over a range of conditions and readouts, emphasising the need for comprehensive orthogonal data when profiling putative PLpro inhibitors. The remaining active compound CPI-169, showed to compete with GRL-0617 in NMR-based experiments, suggesting to share a similar binding mode, opening novel design opportunities for further developments as antiviral agents. The increasing knowledge about SARS-CoV-2 allowed the development of multiple strategies to contain the spread of COVID-19 infection. Nevertheless, effective antiviral pharmacological treatments are still rare and viral evolution allowed a fast adaptation and escape from available containment methods. The papain like protease (PLpro) has now become the next most promising SARS-CoV-2 therapeutic due to its multiple functions in virus replication cycle and antagonization of host immune response. However, due to inherent flexibility and sensitivity of this enzyme specific inhibitors are rare. Here we report on a screening strategy using repurposing of known drugs that takes into account PLpro characteristics to identify new inhibitors, showing the success of the approach by identifying CPI-169 that competitive targets the well described GRL-0617 inhibitor binding pocket of PLpro and helping to design further antiviral agents.","version":"1.1","doi":"10.1101/2023.10.11.561987","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.06.539698","pub_date":"2023-10-12","title":"Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue","abstract":"Omicron emerged following COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to lineages that continue to spread. Here, we show that Omicron exhibits increased infectivity in primary adult upper airway tissue relative to Delta. Using recombinant forms of SARS-CoV-2 and nasal epithelial cells cultured at the liquid-air interface, enhanced infectivity maps to the step of cellular entry and evolved recently through mutations unique to Omicron Spike. Unlike earlier variants of SARS-CoV-2, Omicron enters nasal cells independently of serine transmembrane proteases and instead relies upon metalloproteinases to catalyze membrane fusion. This entry pathway unlocked by Omicron Spike enables evasion of constitutive and interferon-induced antiviral factors that restrict SARS-CoV-2 entry following attachment. Therefore, the increased transmissibility exhibited by Omicron in humans may be attributed not only to its evasion of vaccine-elicited adaptive immunity, but also to its superior invasion of nasal epithelia and resistance to the cell-intrinsic barriers present therein.","version":"1.2","doi":"10.1101/2023.05.06.539698","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.559927","pub_date":"2023-10-12","title":"Plasma of COVID-19 patients does not alter electrical resistance of human endothelial blood-brain barrier in vitro","abstract":"The pandemic of Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) instigated the most serious global health crisis. Clinical presentation of COVID-19 frequently includes severe neurological and neuropsychiatric symptoms. However, it is presently unknown whether and to which extent pathological impairment of blood-brain barrier (BBB) contributes to the development of neuropathology during COVID-19 progression. In the present study we used human induced pluripotent stem cells-derived brain endothelial cells (iBECs) to study the effects of blood plasma derived from COVID-19 patients on the BBB integrity in vitro. We also performed a comprehensive analysis of the cytokine and chemokine profiles in the plasma of COVID-19 patients, healthy and recovered individuals. We found significantly increased levels of interferon \u03b3-induced protein 10 kDa (IP-10), hepatocyte growth factor (HGF), and interleukin-18 (IL-18) in the plasma of COVID-19 patients. However, blood plasma from COVID-19 patients did not affect transendothelial electrical resistance (TEER) in iBEC monolayers. Our results demonstrate that COVID-19-associated blood plasma inflammatory factors do not impair BBB integrity directly and suggest that pathological remodelling of BBB during COVID-19 may occur through indirect mechanisms.","version":"1.2","doi":"10.1101/2023.09.28.559927","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.11.561544","pub_date":"2023-10-12","title":"Sarbecovirus disease susceptibility is conserved across viral and host models","abstract":"Coronaviruses have caused three severe epidemics since the start of the 21st century: SARS, MERS and COVID-19. The severity of the ongoing COVID-19 pandemic and increasing likelihood of future coronavirus outbreaks motivates greater understanding of factors leading to severe coronavirus disease. We screened ten strains from the Collaborative Cross mouse genetic reference panel and identified strains CC006/TauUnc (CC006) and CC044/Unc (CC044) as coronavirus-susceptible and resistant, respectively, as indicated by variable weight loss and lung congestion scores four days post-infection. We generated a genetic mapping population of 755 CC006xCC044 F2 mice and exposed the mice to one of three genetically distinct mouse-adapted coronaviruses: clade 1a SARS-CoV MA15 (n=391), clade 1b SARS-CoV-2 MA10 (n=274), and clade 2 HKU3-CoV MA (n=90). Quantitative trait loci (QTL) mapping in SARS-CoV- and SARS-CoV-2-infected F2 mice identified genetic loci associated with disease severity. Specifically, we identified seven loci associated with variation in outcome following infection with either virus, including one, HrS45, that is present in both groups. Three of these QTL, including HrS45, were also associated with HKU3-CoV MA outcome. HrS45 overlaps with a QTL previously reported by our lab that is associated with SARS-CoV outcome in CC011xCC074 F2 mice and is also syntenic with a human chromosomal region associated with severe COVID-19 outcomes in humans GWAS. The results reported here provide: (a) additional support for the involvement of this locus in SARS-CoV MA15 infection, (b) the first conclusive evidence that this locus is associated with susceptibility across the Sarbecovirus subgenus, and (c) demonstration of the relevance of mouse models in the study of coronavirus disease susceptibility in humans.","version":"1.1","doi":"10.1101/2023.10.11.561544","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.03.510677","pub_date":"2023-10-12","title":"An agent-based modeling approach for lung fibrosis in response to COVID-19","abstract":"The severity of the COVID-19 pandemic has created an emerging need to investigate the long-term effects of infection on patients. Many individuals are at risk of suffering pulmonary fibrosis due to the pathogenesis of lung injury and impairment in the healing mechanism. Fibroblasts are the central mediators of extracellular matrix (ECM) deposition during tissue regeneration, regulated by anti-inflammatory cytokines including transforming growth factor beta (TGF-\u03b2). The TGF-\u03b2-dependent accumulation of fibroblasts at the damaged site and excess fibrillar collagen deposition lead to fibrosis. We developed an open-source, multiscale tissue simulator to investigate the role of TGF-\u03b2 sources in the progression of lung fibrosis after SARS-CoV-2 exposure, intracellular viral replication, infection of epithelial cells, and host immune response. Using the model, we predicted the dynamics of fibroblasts, TGF-\u03b2, and collagen deposition for 15 days post-infection in virtual lung tissue. Our results showed variation in collagen area fractions between 2% and 40% depending on the spatial behavior of the sources (stationary or mobile), the rate of activation of TGF-\u03b2, and the duration of TGF-\u03b2 sources. We identified M2 macrophages as primary contributors to higher collagen area fraction. Our simulation results also predicted fibrotic outcomes even with lower collagen area fraction when spatially-localized latent TGF-\u03b2 sources were active for longer times. We validated our model by comparing simulated dynamics for TGF-\u03b2, collagen area fraction, and macrophage cell population with independent experimental data from mouse models. Our results showed that partial removal of TGF-\u03b2 sources changed the fibrotic patterns; in the presence of persistent TGF-\u03b2 sources, partial removal of TGF-\u03b2 from the ECM significantly increased collagen area fraction due to maintenance of chemotactic gradients driving fibroblast movement. The computational findings are consistent with independent experimental and clinical observations of collagen area fractions and cell population dynamics not used in developing the model. These critical insights into the activity of TGF-\u03b2 sources may find applications in the current clinical trials targeting TGF-\u03b2 for the resolution of lung fibrosis. COVID-19 survivors are at risk of lung fibrosis as a long-term effect. Lung fibrosis is the excess deposition of tissue materials in the lung that hinder gas exchange and can collapse the whole organ. We identified TGF-\u03b2 as a critical regulator of fibrosis. We built a model to investigate the mechanisms of TGF-\u03b2 sources in the process of fibrosis. Our results showed spatial behavior of sources (stationary or mobile) and their activity (activation rate of TGF-\u03b2, longer activation of sources) could lead to lung fibrosis. Current clinical trials for fibrosis that target TGF-\u03b2 need to consider TGF-\u03b2 sources\u2019 spatial properties and activity to develop better treatment strategies.","version":"1.3","doi":"10.1101/2022.10.03.510677","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.02.522505","pub_date":"2023-10-12","title":"Nanoparticle-Conjugated TLR9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines","abstract":"Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and timescale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is critical need for new adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures, but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improve the magnitude and duration of antibody responses when compared to soluble CpG, and result in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhance the spatiotemporal control over co-delivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates comparable humoral responses as a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.","version":"1.3","doi":"10.1101/2023.01.02.522505","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.09.561492","pub_date":"2023-10-10","title":"Deep learning-guided selection of antibody therapies with enhanced resistance to current and prospective SARS-CoV-2 Omicron variants","abstract":"Most COVID-19 antibody therapies rely on binding the SARS-CoV-2 receptor binding domain (RBD). However, heavily mutated variants such as Omicron and its sublineages, which are characterized by an ever increasing number of mutations in the RBD, have rendered prior antibody therapies ineffective, leaving no clinically approved antibody treatments for SARS-CoV-2. Therefore, the capacity of therapeutic antibody candidates to bind and neutralize current and prospective SARS-CoV-2 variants is a critical factor for drug development. Here, we present a deep learning-guided approach to identify antibodies with enhanced resistance to SARS-CoV-2 evolution. We apply deep mutational learning (DML), a machine learning-guided protein engineering method to interrogate a massive sequence space of combinatorial RBD mutations and predict their impact on angiotensin-converting enzyme 2 (ACE2) binding and antibody escape. A high mutational distance library was constructed based on the full-length RBD of Omicron BA.1, which was experimentally screened for binding to the ACE2 receptor or neutralizing antibodies, followed by deep sequencing. The resulting data was used to train ensemble deep learning models that could accurately predict binding or escape for a panel of therapeutic antibody candidates targeting diverse RBD epitopes. Furthermore, antibody breadth was assessed by predicting binding or escape to synthetic lineages that represent millions of sequences generated using in silico evolution, revealing combinations with complementary and enhanced resistance to viral evolution. This deep learning approach may enable the design of next-generation antibody therapies that remain effective against future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.10.09.561492","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493714","pub_date":"2023-10-10","title":"The U.S. faculty job market survives the SARS-CoV-2 global pandemic","abstract":"This paper aims to identify the extent to which the COVID-19 pandemic disrupted the academic job market and the ways in which faculty job applicants altered their applications in response to a changing academia. The data presented here is the portion relevant to COVID-19 collected in a survey of faculty job applicants at the end of the 2019-2020 job cycle in North America (spring 2020). An additional \u201cmid-pandemic\u201d survey was used in fall 2020 for applicants participating in the following job search cycle to inquire about how they were adapting their application materials. A portion of data from the 2020-2022 job cycle surveys was used to represent the \u201clate-pandemic\u201d. Job posting data from the Higher Education Recruitment Consortium (HERC) is also used to study job availability. Examination of faculty job postings from 2018 through 2022 found that while they decreased in 2020, the market recovered in 2021 and beyond. While the market recovered, approximately 10% of the faculty job offers reported by 2019\u201320 survey respondents were rescinded. Respondents also reported altering their application documents in response to the pandemic as well as delaying or even abandoning their faculty job search. This paper provides a longitudinal perspective with quantitative data on how the academic job market changed through the major events of the COVID-19 pandemic in North America, a subject of intense discussion and stress, particularly amongst early career researchers.","version":"1.2","doi":"10.1101/2022.05.27.493714","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.09.561473","pub_date":"2023-10-09","title":"The SARS-CoV-2 Spike is a virulence determinant and plays a major role on the attenuated phenotype of Omicron virus in a feline model of infection","abstract":"To assess the role of the Omicron BA.1 Spike (S) protein in the pathogenesis of the severe acute respiratory coronavirus 2 (SARS-CoV-2), we generated recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 S gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 strain genome. The recombinant viruses were characterized in vitro and in vivo. Viral entry, cell-cell fusion, viral plaque size, and viral replication kinetics of the rWA1-Omi-S virus were markedly impaired when compared to the rWA1-D614G virus, demonstrating a lower fusogenicity and ability to spread cell-to-cell of rWA1-Omi-S. To assess the contribution of the Omicron BA.1 S protein to SARS-CoV-2 pathogenesis the pathogenicity of rWA1-D614G and rWA1-Omi-S viruses were compared using a feline model of infection. While the rWA1-D614G-inoculated cats became lethargic and showed increased body temperatures on days 2 and 3 post-infection (pi), rWA1-Omi-S-inoculated cats remained subclinical and gained weight throughout the 14-day experimental period. Animals inoculated with rWA1-D614G presented higher levels of infectious virus shedding in nasal secretions, when compared to rWA1-Omi-S-inoculated animals. In addition, tissue replication of the rWA1-Omi-S was markedly reduced compared to the rWA1-D614G, as evidenced by lower in situ viral RNA and lower viral load in tissues on days 3 and 5 pi. Histologic examination of the nasal turbinate and lungs revealed intense inflammatory infiltration in rWA1-D614G-inoculated animals, whereas rWA1-Omi-S-inoculated cats presented only mild to modest inflammation. Together, these results demonstrate that the S protein is a major virulence determinant for SARS-CoV-2 playing a major role for the attenuated phenotype of the Omicron virus. The SARS-CoV-2 Omicron sublineage BA.1 spread rapidly across the globe in late 2021/early 2022. Experimental studies have shown an overall lower pathogenicity of Omicron BA.1 when compared to the ancestral SARS-CoV-2 lineage B.1 (D614G). Recently, we have demonstrated that the Omicron BA.1.1 variant presents lower pathogenicity when compared to D614G (B.1) lineage in a feline model of SARS-CoV-2 infection. There are over 50 mutations in the Omicron genome, of which more than two thirds are present in the S gene. To assess the role of the Omicron BA.1 S on virus pathogenesis, recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 Spike gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 were characterized in vitro and in vivo. While the Omicron BA.1 S gene results in early entry into cells, the rWA1-Omi-S presents impaired cell-cell spread and fusogenic activity. Inoculation of cats with the recombinant viruses revealed an attenuated phenotype of rWA1-Omi-S, demonstrating a critical role for S protein on the pathogenicity of SARS-CoV-2 and indicating that the Omi-S is a major determinant of the attenuated disease phenotype of Omicron strains.","version":"1.1","doi":"10.1101/2023.10.09.561473","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.15.504010","pub_date":"2023-10-09","title":"Host and viral determinants of airborne transmission of SARS-CoV-2 in the Syrian hamster","abstract":"It remains poorly understood how SARS-CoV-2 infection influences the physiological host factors important for aerosol transmission. We assessed breathing pattern, exhaled droplets, and infectious virus after infection with Alpha and Delta variants of concern (VOC) in the Syrian hamster. Both VOCs displayed a confined window of detectable airborne virus (24-48 h), shorter than compared to oropharyngeal swabs. The loss of airborne shedding was linked to airway constriction resulting in a decrease of fine aerosols (1-10\u00b5m) produced, which are suspected to be the major driver of airborne transmission. Male sex was associated with increased viral replication and virus shedding in the air. Next, we compared the transmission efficiency of both variants and found no significant differences. Transmission efficiency varied mostly among donors, 0-100% (including a superspreading event), and aerosol transmission over multiple chain links was representative of natural heterogeneity of exposure dose and downstream viral kinetics. Co-infection with VOCs only occurred when both viruses were shed by the same donor during an increased exposure timeframe (24-48 h). This highlights that assessment of host and virus factors resulting in a differential exhaled particle profile is critical for understanding airborne transmission.","version":"1.3","doi":"10.1101/2022.08.15.504010","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.07.561170","pub_date":"2023-10-09","title":"Direct measures of liking and intensity of taste, smell, and chemesthetic stimuli are similar between young people reporting they did or did not have COVID-19","abstract":"The recovery period from post-COVID-19 smell and taste dysfunctions varies substantially, lasting from a few days to over a year. We aimed to assess the impact of COVID-19 on post-COVID-19 chemosensory sensitivity in a group of young convalescents of eastern/central European ancestry. We measured subjects\u2019 smell and taste capabilities with a standard testing kit, Monell Flavor Quiz (MFQ), and collected surveys on COVID-19 history. During testing, subjects rated liking and intensity of six odor samples (galaxolide, guaiacol, beta-ionone, trimethylamine, phenylethyl alcohol, 2-ethyl fenchol) and six taste samples (sucralose, sodium chloride, citric acid, phenylthiocarbamide, menthol, capsaicin) on a scale from 1 (dislike extremely, or no intensity) to 9 (like extremely, or extremely intense). There was no statistical difference in intensity ratings or liking of any sample between subjects who reported a history of COVID-19 (n = 34) and those reporting no history (n = 40), independent of presence/absence or severity of smell/taste impairments (P > 0.05). Additionally, neither vaccination status (full vaccination or no vaccination) nor time from the COVID-19 onset (2-27 months) correlated with liking or intensity. These results suggest that most young adults who had COVID-19 recovered their sense of smell and taste.","version":"1.1","doi":"10.1101/2023.10.07.561170","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.05.561101","pub_date":"2023-10-06","title":"Understanding SARS-CoV-2 Spike glycoprotein clusters and their impact on immunity of the population from Rio Grande do Norte, Brazil","abstract":"SARS-CoV-2 genome underwent mutations since it started circulating intensively within the human populations. The aim of this study was to understand the fluctuation of the spike clusters concomitant to high rate of population immunity either due to natural infection and/or vaccination in a state of Brazil that had high rate of infection and vaccination coverage. A total of 1715 SARS-CoV-2 sequences from the state of Rio Grande do Norte, Brazil, were retrieved from GISAID and subjected to cluster analysis. Immunoinformatics were used to predict T- and B-cell epitopes, followed by simulation to estimate either pro- or anti-inflammatory responses and correlate with circulating variants. From March 2020 to June 2022, Rio Grande do Norte reported 579,931 COVID-19 cases with a 1.4% fatality rate across three major waves: May-Sept 2020, Feb-Aug 2021, and Jan-Mar 2022. Cluster 0 variants (wild type strain, Zeta) were prevalent in the first wave and Delta in the latter half of 2021, featuring fewer unique epitopes. Cluster 1 (Gamma [P1]) dominated the first half of 2021. Late 2021 had Clusters 2 (Omicron) and 3 (Omicron sublineages) with the most unique epitopes, while Cluster 4 (Delta sublineages) emerged in the second half of 2021 with fewer unique epitopes. Cluster 1 epitopes showed a high pro-inflammatory propensity, while others exhibited a balanced cytokine induction. The clustering method effectively identified Spike groups that may contribute to immune evasion and clinical presentation, and explain in part the clinical outcome. Identification of epitopes of emerging or endemic pathogens is of importance to estimate population responses and predict clinical outcomes and contribute to vaccine improvement. In the case of SARS-CoV-2, the virus within 6 months of circulation transitioned from the wild-type to novel variants leading to distinct clinical outcomes. Immunoinformatics analysis of viral epitopes of isolates from the Brazilian state of Rio Grande do Norte was performed using a clustering method. This analysis aimed to clarify how the introduction of novel variants in a population characterized by high infection and/or vaccination rates resulted in immune evasion and distinct clinical disease. Our analysis showed that the epitope profiles of each variant explained the respective potential for cytokine production, including the variants that were more likely to cause cytokine storms. Finally, it serves as a mean to explain the multi-wave patterns observed during SARS-CoV-2 pandemics.","version":"1.1","doi":"10.1101/2023.10.05.561101","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.05.557343","pub_date":"2023-10-06","title":"Heterologous booster with a novel formulation containing glycosylated trimeric S protein is effective against Omicron","abstract":"In this study, we evaluated the efficacy of a heterologous three-dose vaccination schedule against the Omicron BA.1 SARS-CoV-2 variant infection using a mouse intranasal challenge model. The vaccination schedules tested in this study consisted of a primary series of 2 doses covered by two commercial vaccines: an mRNA-based vaccine (mRNA1273) or a non-replicative vector-based vaccine (AZD1222/ChAdOx1, hereafter referred to as AZD1222). These were followed by a heterologous booster dose using one of the two vaccine candidates previously designed by us: one containing the glycosylated and trimeric spike protein (S) from the ancestral virus (SW-Vac 2\u03bcg), and the other from the Delta variant of SARS-CoV-2 (SD-Vac 2\u03bcg), both formulated with Alhydrogel as an adjuvant. For comparison purposes, homologous three-dose schedules of the commercial vaccines were used. The mRNA-based vaccine, whether used in heterologous or homologous schedules, demonstrated the best performance, significantly increasing both humoral and cellular immune responses. In contrast, for the schedules that included the AZD1222 vaccine as the primary series, the heterologous schemes showed superior immunological outcomes compared to the homologous 3-dose AZD1222 regimen. For these schemes no differences were observed in the immune response obtained when SW-Vac 2\u03bcg or SD-Vac 2\u03bcg were used as a booster dose. Neutralizing antibody levels against Omicron BA.1 were low, especially for the schedules using AZD1222. However, a robust Th1 profile, known to be crucial for protection, was observed, particularly for the heterologous schemes that included AZD1222. All the tested schedules were capable of inducing populations of CD4 T effector, memory, and follicular helper T lymphocytes. It is important to highlight that all the evaluated schedules demonstrated a satisfactory safety profile and induced multiple immunological markers of protection. Although the levels of these markers were different among the tested schedules, they appear to complement each other in conferring protection against intranasal challenge with Omicron BA.1 in K18-hACE2 mice. In summary, the results highlight the potential of using the S protein (either ancestral Wuhan or Delta variant)-based vaccine formulation as heterologous boosters in the management of COVID-19, particularly for certain commercial vaccines currently in use.","version":"1.1","doi":"10.1101/2023.10.05.557343","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.26.559550","pub_date":"2023-10-05","title":"Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo","abstract":"The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spike from multiple SARS-CoV-2 variants, and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrate protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.","version":"1.2","doi":"10.1101/2023.09.26.559550","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.05.561047","pub_date":"2023-10-05","title":"Fourth dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-lasting Humoral Responses in mice","abstract":"The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skin\u2019s immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU+Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.","version":"1.1","doi":"10.1101/2023.10.05.561047","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.04.560875","pub_date":"2023-10-05","title":"Unveiling the antiviral capabilities of targeting Human Dihydroorotate Dehydrogenase against SARS-CoV-2","abstract":"The urgent need for effective treatments against emerging viral diseases, driven by drug-resistant strains and new viral variants, remains critical. We focus on inhibiting the human dihydroorotate dehydrogenase (HsDHODH), one of the enzymes in charge of pyrimidine nucleotide synthesis. This strategy could impede viral replication without provoking resistance. We evaluated quinone-based compounds, discovering potent HsDHODH inhibition (low nanomolar IC50) and promising in vitro anti-SARS-CoV-2 activity (low micromolar EC50). These compounds exhibited low toxicity, indicating potential for further development. Additionally, we employed computational tools like molecular docking and QSAR models to analyze protein-ligand interactions. These findings represent a significant step forward in the search for effective antiviral treatments and have great potential to impact the development of new broad-spectrum antiviral drugs.","version":"1.1","doi":"10.1101/2023.10.04.560875","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.04.23296554","pub_date":"2023-10-05","title":"Brain-targeted autoimmunity is strongly associated with Long COVID and its chronic fatigue syndrome as well as its affective symptoms","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Autoimmune responses contribute to the pathophysiology of Long COVID, affective symptoms and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>To examine whether Long COVID, and its accompanying affective symptoms and CFS are associated with immunoglobulin (Ig)A/IgM/IgG directed at neuronal proteins including myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), synapsin, \u03b1+\u03b2-tubulin, neurofilament protein (NFP), cerebellar protein-2 (CP2), and the blood-brain-barrier-brain-damage (BBD) proteins claudin-5 and S100B.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    IgA\n                    <jats:bold>/</jats:bold>\n                    IgM/IgG to the above neuronal proteins, human herpes virus-6 (HHV-6) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) were measured in 90 Long COVID patients and 90 healthy controls, while C-reactive protein (CRP), and advanced oxidation protein products (AOPP) in association with affective and CFS ratings were additionally assessed in a subgroup thereof.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Long COVID is associated with significant increases in IgG directed at tubulin (IgG-tubulin), MBP, MOG and synapsin; IgM-MBP, MOG, CP2, synapsin and BBD; and IgA-CP2 and synapsin. IgM-SARS-CoV-2 and IgM-HHV-6 antibody titers were significantly correlated with IgA/IgG/IgM-tubulin and -CP2, IgG/IgM-BBD, IgM-MOG, IgA/IgM-NFP, and IgG/IgM-synapsin. Binary logistic regression analysis shows that IgM-MBP and IgG-MBP are the best predictors of Long COVID. Multiple regression analysis shows that IgG-MOG, CRP and AOPP explain together 41.7% of the variance in the severity of CFS. Neural network analysis shows that IgM-synapsin, IgA-MBP, IgG-MOG, IgA-synapsin, IgA-CP2, IgG-MBP and CRP are the most important predictors of affective symptoms due to Long COVID with a predictive accuracy of r=0.801.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Brain-targeted autoimmunity contributes significantly to the pathogenesis of Long COVID and the severity of its physio-affective phenome.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.10.04.23296554","journal":"medRxiv","score":null},{"id":"10.1101/2023.09.22.558628","pub_date":"2023-10-04","title":"A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system","abstract":"Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions. Understanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment.","version":"1.2","doi":"10.1101/2023.09.22.558628","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.03.560426","pub_date":"2023-10-04","title":"SARS-CoV-2 N protein-induced Dicer, XPO5, SRSF3, and hnRNPA3 downregulation causes pneumonia","abstract":"Age is a major risk factor for coronavirus disease (COVID-19)-associated severe pneumonia and mortality; however, the underlying mechanism remains unclear. Herein, we investigated whether age-related deregulation of RNAi components and RNA splicing factors affects COVID-19 severity. Decreased expression of RNAi components (Dicer and XPO5) and splicing factors (SRSF3 and hnRNPA3) correlated with increased severity of COVID-19 and SARS-CoV-2 nucleocapsid (N) protein-induced pneumonia. N protein induced autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, repressing miRNA biogenesis and RNA splicing and inducing DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 were downregulated with age in mouse lung tissues. Older mice experienced more severe N protein-induced pneumonia than younger mice. However, treatment with a poly(ADP-ribose) polymerase inhibitor (PJ34) or aromatase inhibitor (anastrozole) relieved N protein-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.","version":"1.1","doi":"10.1101/2023.10.03.560426","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.03.560628","pub_date":"2023-10-04","title":"Virological characteristics correlating with SARS-CoV-2 spike protein fusogenicity","abstract":"The severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike (S) protein is essential in mediating membrane fusion of the virus with the target cells. Several reports demonstrated that SARS-CoV-2 S protein fusogenicity is reportedly closely associated with the intrinsic pathogenicity of the virus determined using hamster models. However, the association between S protein fusogenicity and other virological parameters remains elusive. In this study, we investigated the virological parameters of eleven previous variants of concern (VOCs) and variants of interest (VOIs) correlating with S protein fusogenicity. S protein fusogenicity was found to be strongly correlated with S1/S2 cleavage efficiency and plaque size formed by clinical isolates. However, S protein fusogenicity was less associated with pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics. Taken together, our results suggest that S1/S2 cleavage efficiency and plaque size could be potential indicators to predict the intrinsic pathogenicity of newly emerged SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2023.10.03.560628","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.04.560777","pub_date":"2023-10-04","title":"mRNA vaccines encoding membrane-anchored receptor-binding domains of SARS-CoV-2 mutants induce strong humoral responses and can overcome immune imprinting","abstract":"To address the limitations of whole-spike COVID vaccines, we explored mRNA vaccines encoding membrane-anchored receptor-binding domain (RBD-TMs), each a fusion of a variant RBD, the transmembrane (TM) and cytoplasmic tail (CT) fragments of the SARS-CoV-2 spike protein. In naive mice, RBD-TM mRNA vaccines against ancestral SARS-CoV-2, Beta, Delta, Delta-plus, Kappa, Omicron BA.1 or BA.5, all induced strong humoral responses against the target RBD. Multiplex surrogate viral neutralization (sVNT) assays indicated broad neutralizing activity against a range of variant RBDs. In the setting of a heterologous boost, against the background of exposure to ancestral whole spike vaccines, sVNT studies suggested that RBD-TM vaccines were able to overcome the detrimental effects of immune imprinting. Omicron BA.1 and BA.5 RBD-TM booster vaccines induced serum antibodies with 12 and 22-fold higher neutralizing activity against the target RBD than their equivalent whole spike variants. Boosting with BA.1 or BA.5 RBD-TM provided good protection against more recent variants including XBB and XBB.1.5. Each RBD-TM mRNA is 28% of the length of its whole-spike equivalent. This advantage will enable tetravalent mRNA vaccines to be developed at well-tolerated doses of formulated mRNA. mRNA vaccines encoding membrane-anchored RBDs of SARS-CoV-2 mutants are effective vaccines that can overcome immune imprinting in mice","version":"1.1","doi":"10.1101/2023.10.04.560777","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.03.560739","pub_date":"2023-10-04","title":"Comparative single-cell analysis reveals IFN-\u03b3 as a driver of respiratory sequelae post COVID-19","abstract":"Post-acute sequelae of SARS-CoV-2 infection (PASC) represents an urgent public health challenge, with its impact resonating in over 60 million individuals globally. While a growing body of evidence suggests that dysregulated immune reactions may be linked with PASC symptoms, most investigations have primarily centered around blood studies, with few focusing on samples derived from post-COVID affected tissues. Further, clinical studies alone often provide correlative insights rather than causal relationships. Thus, it is essential to compare clinical samples with relevant animal models and conduct functional experiments to truly understand the etiology of PASC. In this study, we have made comprehensive comparisons between bronchoalveolar lavage fluid (BAL) single-cell RNA sequencing (scRNAseq) data derived from clinical PASC samples and relevant PASC mouse models. This revealed a strong pro-fibrotic monocyte-derived macrophage response in respiratory PASC (R-PASC) in both humans and mice, and abnormal interactions between pulmonary macrophages and respiratory resident T cells. IFN-\u03b3 emerged as a key node mediating the immune anomalies in R-PASC. Strikingly, neutralizing IFN-\u03b3 post the resolution of acute infection reduced lung inflammation, tissue fibrosis, and improved pulmonary gas-exchange function in two mouse models of R-PASC. Our study underscores the importance of performing comparative analysis to understand the root cause of PASC for developing effective therapies.","version":"1.1","doi":"10.1101/2023.10.03.560739","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.05.543758","pub_date":"2023-10-04","title":"Post-acute immunological and behavioral sequelae in mice after Omicron infection","abstract":"Progress in understanding long COVID and developing effective therapeutics is hampered in part by the lack of suitable animal models. Here we used ACE2-transgenic mice recovered from Omicron (BA.1) infection to test for pulmonary and behavioral post-acute sequelae. Through in-depth phenotyping by CyTOF, we demonstrate that na\u00efve mice experiencing a first Omicron infection exhibit profound immune perturbations in the lung after resolving acute infection. This is not observed if mice were first vaccinated with spike-encoding mRNA. The protective effects of vaccination against post-acute sequelae were associated with a highly polyfunctional SARS-CoV-2-specific T cell response that was recalled upon BA.1 breakthrough infection but not seen with BA.1 infection alone. Without vaccination, the chemokine receptor CXCR4 was uniquely upregulated on multiple pulmonary immune subsets in the BA.1 convalescent mice, a process previously connected to severe COVID-19. Taking advantage of recent developments in machine learning and computer vision, we demonstrate that BA.1 convalescent mice exhibited spontaneous behavioral changes, emotional alterations, and cognitive-related deficits in context habituation. Collectively, our data identify immunological and behavioral post-acute sequelae after Omicron infection and uncover a protective effect of vaccination against post-acute pulmonary immune perturbations.","version":"1.2","doi":"10.1101/2023.06.05.543758","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.01.560357","pub_date":"2023-10-02","title":"The TMPRSS2 non-protease domains regulating SARS-CoV-2 Spike in mediated virus entry","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Through the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus infection, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2\u2019s cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.","version":"1.1","doi":"10.1101/2023.10.01.560357","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.23.521567","pub_date":"2023-10-02","title":"A large-scale serological survey in pets from October 2020 through June 2021 in France shows significantly higher exposure to SARS-CoV-2 in cats","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animals, including pets such as dogs and cats. Many studies have documented infection in companion animals by bio-molecular and serological methods. However, only a few have compared seroprevalence in cats and dogs from the general population, and these studies were limited by small sample sizes and collections over short periods. Our goal was to obtain a more accurate evaluation of seroprevalence in companion animals in France and to determine whether cats and dogs differ in their exposure to SARS-CoV-2. For this purpose, we conducted an extensive SARS-CoV-2 cross-sectional serological survey of 2036 cats and 3577 dogs sampled by veterinarians during medical examinations in clinics throughout France. Sampling was carried out from October 2020 through June 2021, a period encompassing the second and third waves of SARS-CoV-2 infections in humans in the country. Using a microsphere immunoassay targeting the receptor binding domain and trimeric spike protein, we found 7.1% seroprevalence in pets. In a subset of 308 seropositive samples, 26.3% had neutralizing antibodies. We found that cats were significantly more likely to test positive than dogs, with seropositivity rates of 9.3% and 5.9% in cats and dogs, respectively. Finally, data for both species showed that seroprevalence was lower in older animals and was not associated with the date of sampling or the sex of the animal. Our results show that cats are significantly more sensitive to SARS-CoV-2 than dogs, in line with experimental studies. Our large sample size provides for a reliable, statistically robust estimate of the frequency of infection of pets from their owners and offers strong support for the notion that cats are more sensitive to SARS-CoV-2 than dogs. Our findings emphasise the importance of a One-Health approach to the SARS-CoV-2 pandemic and raise the question of whether companion animals in close contact with humans should be vaccinated.","version":"1.4","doi":"10.1101/2022.12.23.521567","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.30.560318","pub_date":"2023-10-02","title":"Synthesis, Insertion, and Characterization of SARS-CoV-2 Membrane Protein Within Lipid Bilayers","abstract":"The membrane protein (M) is the most abundant structural protein in the SARS-CoV-2 virus and functions exclusively as a membrane-embedded homodimer. M protein is required for the formation of the SARS-CoV-2 virus particle and has been shown to interact with the Spike and Envelope proteins, as well as the RNA-packaging Nucleocapsid protein. Our knowledge of M protein is very limited due to its small size and challenges in expressing enough protein for use in structural and biophysical experiments. We report the successful development of a SUMO tag-based expression system to produce and purify significant quantities of M protein, and a method to insert the synthesized dimers into a suspended lipid membrane in a homogeneous orientation. We used AFM and Cryo-EM to image individual membrane-bound M protein dimers and characterize the configurations that they can assume. Our experimental results are in agreement with our molecular dynamics simulations which predict thinning of the membrane around the M protein and a propensity to induce local membrane curvature. Taken together, our results shed new light on M protein properties within the lipid bilayer and suggest mechanisms that could contribute to viral assembly and budding.","version":"1.1","doi":"10.1101/2023.09.30.560318","journal":"bioRxiv","score":null},{"id":"10.1101/2023.10.01.560365","pub_date":"2023-10-02","title":"Neutralisation of SARS-CoV-2 Omicron subvariants BA.2.86 and EG.5.1 by antibodies induced by earlier infection or vaccination","abstract":"Highly mutated SARS-CoV-2 Omicron subvariant BA.2.86 emerged in July 2023. We investigated the neutralisation of isolated virus by antibodies induced by earlier infection or vaccination. The neutralisation titres for BA.2.86 were comparable to those for XBB.1 and EG.5.1, by antibodies induced by XBB.1.5 or BA.4/5 breakthrough infection or BA.4/5 vaccination.","version":"1.1","doi":"10.1101/2023.10.01.560365","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.29.23296359","pub_date":"2023-10-02","title":"Dynamic Contact Networks of Residents of an Urban Jail in the Era of SARS-CoV-2","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>In custodial settings such as jails and prisons, infectious disease transmission is heightened by factors such as overcrowding and limited healthcare access. Specific features of social contact networks within these settings have not been sufficiently characterized, especially in the context of a large-scale respiratory infectious disease outbreak. The study aims to quantify contact network dynamics within the Fulton County Jail in Atlanta, Georgia, to improve our understanding respiratory disease spread to informs public health interventions.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>As part of the Surveillance by Wastewater and Nasal Self-collection of Specimens (SWANSS) study, jail roster data were utilized to construct social contact networks. Rosters included resident details, cell locations, and demographic information. This analysis involved 6,702 residents over 140,901 person days. Network statistics, including degree, mixing, and turnover rates, were assessed across age groups, race/ethnicities, and jail floors. We compared outcomes for two distinct periods (January 2022 and April 2022) to understand potential responses in network structures during and after the SARS-CoV-2 Omicron variant peak.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We found high cross-sectional network degree at both cell and block levels, indicative of substantial daily contacts. While mean degree increased with age, older residents exhibited lower degree during the Omicron peak, suggesting potential quarantine measures. Block-level networks demonstrated higher mean degrees than cell-level networks. Cumulative degree distributions for both levels increased from January to April, indicating heightened contacts after the outbreak. Assortative age mixing was strong, especially for residents aged 20\u201329. Dynamic network statistics illustrated increased degrees over time, emphasizing the potential for disease spread, albeit with a lower growth rate during the Omicron peak.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The contact networks within the Fulton County Jail presented ideal conditions for infectious disease transmission. Despite some reduction in network characteristics during the Omicron peak, the potential for disease spread remained high. Age-specific mixing patterns suggested unintentional age segregation, potentially limiting disease spread to older residents. The study underscores the need for ongoing monitoring of contact networks in carceral settings and provides valuable insights for epidemic modeling and intervention strategies, including quarantine, depopulation, and vaccination. This network analysis offers a foundation for understanding disease dynamics in carceral environments.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.09.29.23296359","journal":"medRxiv","score":null},{"id":"10.1101/2023.09.07.554570","pub_date":"2023-10-02","title":"Stereotypic persistent B cell receptor clonotypes in Alzheimer\u2019s Disease","abstract":"We constructed B cell receptor (BCR) repertoires in silico using peripheral blood (PB) samples collected from 44 Alzheimer\u2019s Disease (AD) patients at baseline and 37 patients at follow-up. For the control group (CG), we used BCR repertoire data from the chronologically collected PB samples of 55 healthy volunteers vaccinated with SARS-CoV-2 mRNA. The AD patients shared 3,983 stereotypic non-na\u00efve BCR clonotypes not found in CG, and their degree of overlap between patient pairs were significantly higher than that of CG pairs, even with the SARS-CoV-2 spike protein triggering a concerted BCR response. Twenty stereotypic non-na\u00efve AD patient-specific BCR clonotypes co-existed in more than four patients and persisted throughout two sampling points. One of these BCR clonotypes encoded an antibody reactive to the A\u03b242 peptide. Our findings strongly suggest that AD patients are exposed to common (auto)antigens associated with disease pathology, and their BCR repertoires show unique signatures with diagnostic potential.","version":"1.2","doi":"10.1101/2023.09.07.554570","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.11.528155","pub_date":"2023-9-30","title":"Olivar: automated variant aware primer design for multiplex tiled amplicon sequencing of pathogens","abstract":"Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 2 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (\u223c90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluated Olivar on real wastewater samples and found that Olivar had up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available as a web application at https://olivar.rice.edu. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https://github.com/treangenlab/Olivar.","version":"1.3","doi":"10.1101/2023.02.11.528155","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.559966","pub_date":"2023-9-29","title":"SARS-CoV-2 NSP14 governs mutational instability and assists in making new SARS-CoV-2 variants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the rapidly evolving RNA virus behind the COVID-19 pandemic, has spawned numerous variants since its 2019 emergence. The multifunctional NSP14 enzyme, possessing exonuclease and mRNA capping capabilities, serves as a key player. Notably, single and co-occurring mutations within NSP14 significantly influence replication fidelity and drive variant diversification. This study comprehensively examines 120 co-mutations, 68 unique mutations, and 160 conserved residues across NSP14 homologs, shedding light on their implications for phylogenetic patterns, pathogenicity, and residue interactions. Quantitative physicochemical analysis categorizes 3953 NSP14 variants into three clusters, revealing genetic diversity. This research underscores the dynamic nature of SARS-CoV-2 evolution, primarily governed by NSP14 mutations. Understanding these genetic dynamics provides valuable insights for therapeutic and vaccine development.","version":"1.1","doi":"10.1101/2023.09.28.559966","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.29.560163","pub_date":"2023-9-29","title":"Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease","abstract":"We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro) is a highly conserved and essential protease that plays key roles in viral replication and pathogenesis among various CoVs, representing one of the most attractive drug targets for antiviral drug development. Traditional antiviral drug development strategies focus on the pursuit of high-affinity binding inhibitors against MPro. However, this approach often suffers from issues such as toxicity, drug resistance, and a lack of broad-spectrum efficacy. Targeted protein degradation represents a promising strategy for developing next-generation antiviral drugs to combat infectious diseases. Here we leverage the proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Our previously developed MPro inhibitors MPI8 and MPI29 were used as MPro ligands to conjugate a CRBN E3 ligand, leading to compounds that can both inhibit and degrade SARS-CoV-2 MPro. Among them, MDP2 was demonstrated to effectively reduce MPro protein levels in 293T cells (DC50 = 296 nM), relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells, concurrently demonstrating potent anti-SARS-CoV-2 activity (EC50 = 492 nM). This proof-of-concept study highlights the potential of PROTAC-mediated targeted protein degradation of MPro as an innovative and promising approach for COVID-19 drug discovery.","version":"1.1","doi":"10.1101/2023.09.29.560163","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.560057","pub_date":"2023-9-29","title":"The Role of ATP Hydrolysis and Product Release in the Translocation Mechanism of SARS-CoV-2 NSP13","abstract":"In response to the emergence of COVID-19, caused by SARS-CoV-2, there has been a growing interest in understanding the functional mechanisms of the viral proteins to aid in the development of new therapeutics. Non-structural protein 13 (Nsp13) helicase is an attractive target for antivirals because it is essential for viral replication and has a low mutation rate; yet, the structural mechanisms by which this enzyme binds and hydrolyzes ATP to cause unidirectional RNA translocation remain elusive. Using Gaussian accelerated molecular dynamics (GaMD), we generated a comprehensive conformational ensemble of all substrate states along the ATP-dependent cycle. ShapeGMM clustering of the protein yields four protein conformations that describe an opening and closing of both the ATP pocket and RNA cleft. This opening and closing is achieved through a combination of conformational selection and induction along the ATP cycle. Furthermore, three protein-RNA conformations are observed that implicate motifs Ia, IV, and V as playing a pivotal role in an ATP-dependent inchworm translocation mechanism. Finally, based on a linear discriminant analysis of protein conformations, we identify L405 as a pivotal residue for the opening and closing mechanism and propose a L405D mutation as a way of testing our proposed mechanism. This research enhances our understanding of nsp13\u2019s role in viral replication and could contribute to the development of antiviral strategies.","version":"1.1","doi":"10.1101/2023.09.28.560057","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.560071","pub_date":"2023-9-29","title":"POP-UP TCR: Prediction of Previously Unseen Paired TCR-pMHC","abstract":"T lymphocytes (T-cells) major role in adaptive immunity drives efforts to elucidate the mechanisms behind T-cell epitope recognition. We analyzed solved structures of T-cell receptors (TCRs) and their cognate epitopes and used the data to train a set of machine learning models, POP-UP TCR, that predict the binding of any peptide to any TCR, including peptide and TCR sequences that were not included in the training set. We address biological issues that should be considered in the design of machine learning models for TCR-peptide binding and suggest that models trained only on beta chains give satisfactory predictions. Finally, we apply our models to large data set of TCR repertoires from COVID-19 patients and find that TCRs from patients in severe/critical condition have significantly lower scores for binding SARS-coV-2 epitopes compared to TCRs from moderate patients (p-value <0.001). POP-Up TCR is available at: https://github.com/NiliTicko/POP-UP-TCR nilibrac@bgu.ac.il","version":"1.1","doi":"10.1101/2023.09.28.560071","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.26.23296186","pub_date":"2023-09-29","title":"The more symptoms the better? Covid-19 vaccine side effects and long-term neutralizing antibody response","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Protection against SARS-CoV-2 wanes over time, and booster uptake has been low, in part because of concern about side effects. We examined the relationships between local and systemic symptoms, biometric changes, and neutralizing antibodies (nAB) after mRNA vaccination. Data were collected from adults (n = 364) who received two doses of either BNT162b2 or mRNA-1273. Serum nAB concentration was measured at 1 and 6 months post-vaccination. Daily symptom surveys were completed for six days starting on the day of each dose. Concurrently, objective biometric measurements, including skin temperature, heart rate, heart rate variability, and respiratory rate, were collected. We found that certain symptoms (chills, tiredness, feeling unwell, and headache) after the second dose were associated with increases in nAB at 1 and 6 months post-vaccination, to roughly 140-160% the level of individuals without each symptom. Each additional symptom predicted a 1.1-fold nAB increase. Greater increases in skin temperature and heart rate after the second dose predicted higher nAB levels at both time points, but skin temperature change was more predictive of durable (6 month) nAB response than of short-term (1 month) nAB response. In the context of low ongoing vaccine uptake, our convergent symptom and biometric findings suggest that public health messaging could seek to reframe systemic symptoms after vaccination as desirable.</jats:p>","version":null,"doi":"10.1101/2023.09.26.23296186","journal":"medRxiv","score":null},{"id":"10.1101/2023.09.29.560110","pub_date":"2023-9-29","title":"Network-based integrative multi-omics approach reveals biosignatures specific to COVID-19 disease phases","abstract":"COVID-19 disease is characterized by a spectrum of disease phases (mild, moderate, and severe). Each disease phase is marked by changes in omics profiles with corresponding changes in the expression of features (biosignatures). However, integrative analysis of multiple omics data from different experiments across studies to investigate biosignatures at various disease phases is limited. Exploring an integrative multi-omics profile analysis through a network approach could be used to determine biosignatures associated with specific disease phases and enable the examination of the relationships between the biosignatures. To identify and characterize biosignatures underlying various COVID-19 disease phases in an integrative multi-omics data analysis. We leveraged the correlation network approach to integrate transcriptomics, metabolomics, proteomics, and lipidomics data. The World Health Organization (WHO) Ordinal Scale (WOS) was used as a disease severity reference to harmonize COVID-19 patient metadata across two studies with independent data. A unified COVID-19 knowledge graph was constructed by assembling a disease-specific interactome from the literature and databases. Disease-state omics-specific graphs were constructed by integrating multi-omics data with the unified COVID-19 knowledge graph. We expanded on the network layers of multiXrank, a random walk with restart on multilayer network algorithm, to explore disease state omics-specific graphs and perform enrichment analysis. Network analysis revealed the biosignatures involved in inducing chemokines and inflammatory responses as hubs in the severe and moderate disease phases. We observed more shared biosignatures between severe and moderate disease phases as compared to mild-moderate and mild-severe disease phases. We further identified both biosignatures that discriminate between the disease states and interactions between biosignatures that are either common between or associated with COVID-19 disease phases. Interestingly, cross-layer interactions between different omics profiles increased with disease severity. This study identified both biosignatures of different omics types enriched in disease-related pathways and their associated interactions that are either common between or unique to mild, moderate, and severe COVID-19. These biosignatures include molecular features that underlie the observed clinical heterogeneity of COVID-19 and emphasize the need for disease-phase-specific treatment strategies. In addition, the approach implemented here can be used for other diseases. Integrative multi-omics analysis revealed biosignatures and biosignature interactions associated with COVID-19 disease states. Disease severity increases with biosignature interactions across different multi-omics data. The harmonization approach proposed and implemented here can be applied to other diseases","version":"1.1","doi":"10.1101/2023.09.29.560110","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.27.559799","pub_date":"2023-9-28","title":"Inhibition of SARS-CoV-2 Infection in Human Airway Epithelium with a Xeno-Nucleic Acid Aptamer","abstract":"SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.","version":"1.1","doi":"10.1101/2023.09.27.559799","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.27.559757","pub_date":"2023-9-28","title":"Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike","abstract":"Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be non-neutralizing, we show that they confer partial protection in vivo. We further demonstrate that such in vivo protection activity is diminished by the natural mutation D950N. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines.","version":"1.1","doi":"10.1101/2023.09.27.559757","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.28.559747","pub_date":"2023-9-28","title":"Efficient inhibition of fusion inhibitor HY3000 peptide to SARS-CoV-2 emerging EG.5, EG.5.1 and BA.2.86 variants","abstract":"SARS-CoV-2 continues to evolve and spread. Recently, the Omicron EG.5 lineage, bearing an additional F456L mutation in spike (S) protein compared to its ancestor XBB.1.9.2, and its sub-variant EG.5.1, which carries a further Q52H mutation, have raised concerns due to their increased prevalence and extended immune escape properties. Additionally, an alarming variant, BA.2.86, has also garnered global concern because it contains over 30 amino acid mutations in its S protein compared to BA.2, including more than 10 changes in receptor-binding domain (RBD), reminiscent of the appearance of the Omicron variant in late 2021. Therefore, there is an urgent need to assess the effectiveness of current vaccines and therapeutics against EG.5, EG.5.1 and BA.2.86. In our previous work, we reported the design and broad-spectrum antiviral activity of a peptide fusion inhibitor HY3000 against SARS-CoV-2 and its variants including XBB.1.5. Here, we continued to evaluate the inhibitory potency of the HY3000 peptide against the prevailing EG.5 and EG.5.1, as well as XBB.1.16, FL.1.5.1, FY.3 and BA.2.86. Our data indicated that the peptide retained its potent inhibitory activities against these variants, indicating its potential as a good virus fusion inhibitor with broad-spectrum therapeutic effect against current and future SARS-CoV-2 variants. Currently, the HY3000 has been finished in Phase II clinical trial in China and has also been approved to conduct clinical investigation by U.S. Food and Drug Administration (FDA), suggesting a good application prospect against the ongoing COVID-19.","version":"1.1","doi":"10.1101/2023.09.28.559747","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450614","pub_date":"2023-9-27","title":"ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication","abstract":"The \u03b2-coronavirus SARS-CoV-2 is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed an inert tagging strategy for SARS-CoV-2 E and find that it localises to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for ER-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify host-cell factors that interact with E and identify an ARFRP1/AP-1 dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across \u03b2-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing a trans-complementation assay, we show that both lysosomal trafficking of E and its viroporin activity are necessary for efficient viral replication and release.","version":"1.2","doi":"10.1101/2021.06.30.450614","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.27.559660","pub_date":"2023-9-27","title":"p38-MAPK is prerequisite for the synthesis of SARS-CoV-2 protein","abstract":"The inhibition of p38 mitogen-activated protein kinase (p38-MAPK) by small molecule chemical inhibitors was previously shown to impair severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) replication, however, mechanisms underlying antiviral activity remains unexplored. In this study, reduced growth of SARS-CoV-2 in p38-\u03b1 knockout Vero cells, together with enhanced viral yield in cells transfected with construct expressing p38\u03b1, suggested that p38-MAPK is essential for the propagation of SARS-CoV-2. The SARS-CoV-2 was also shown to induce phosphorylation (activation) of p38, at time when transcription/translational activities are considered to be at the peak levels. Further, we demonstrated that p38 supports viral RNA/protein synthesis without affecting viral attachment, entry, and budding in the target cells. In addition, we demonstrated that long-term culture of SARS-CoV-2 in the presence of p38 inhibitor SB203580 does not easily select resistant viral mutants. In conclusion, we provide mechanistic insights on the regulation of SARS-CoV-2 replication by p38 MAPK.","version":"1.1","doi":"10.1101/2023.09.27.559660","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.24.558921","pub_date":"2023-9-27","title":"Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine","abstract":"To explore viral evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) are as effective at neutralising the SARS-CoV-2 variant of concern (VOC) Delta (B 1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. Using a live-virus SARS-CoV-2 neutralisation assay in Vero E6 cells we determined neutralising antibody titres (nAbT) in 14 participants (vaccine-na\u00efve (n=2) and post-second dose of BNT162b2 vaccination (n=12), median age 45 years [IQR 29\u201365], median time after second dose = 21 days [IQR 19\u201328] against three SARS-CoV-2 strains: wild-type, Beta and Delta. The determination of nAbT was performed by visual inspection of cytopathic effect (CPE) and in-house quantitative reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 hrs, negative and positive controls underwent genome sequencing using the Respiratory Viral Oligo Panel version 2 (RVOP) (Illumina Inc. (San Diego, United States of America)) viral enrichment and short read sequencing using (Illumina Inc.San Diego, United States of America)(Figure 1). There was a significant reduction in nAbT observed against the Delta and Beta VOC compared with wild-type, 4.4-fold (p = >0.0006) and 2.3-fold (p = 0.0140), respectively (Figure 2). Neutralizing antibodies were not detected in one vaccinated immunosuppressed participant nor the vaccine-na\u00efve participants (n=2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARS-CoV-2 infection 12 months prior (Table S1). Limited consensus level mutations occurred in the SARS-CoV-2 genome of any lineage during in vitro neutralisation, however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S) and membrane protein. Significant reductions in nAbT post-vaccination were identified, with Delta demonstrating a 4.4-fold reduction. The reduction in nAbT for the VOC Beta has been previously documented, however, limited data is available on vaccine evasion for the Delta VOC, the predominant strain currently circulating worldwide at the time. Studies in high incidence countries may not be applicable to low incidence settings such as Australia as nAbT may be significantly higher in vaccine recipients previously infected with SARS-CoV-2, as seen in our cohort. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 variants on vaccine effectiveness as mutational profiles in the sub-consensus genome could indicate increases in transmissibility, virulence or allow the development of antiviral resistance.","version":"1.1","doi":"10.1101/2023.09.24.558921","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.24.559214","pub_date":"2023-9-27","title":"Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike","abstract":"Although the COVID-19 pandemic has officially ended, SARS-CoV-2 continues to spread and evolve. Recent infections have been dominated by XBB.1.5 and EG.5.1 subvariants. A new subvariant designated BA.2.86 has just emerged, spreading to 21 countries in 5 continents. This virus contains 34 spike mutations compared to its BA.2 predecessor, thereby raising concerns about its propensity to evade existing antibodies. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was not more resistant to human sera than XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from patients who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. The finding that the longer genetic distance of BA.2.86 did not yield a larger antigenic distance was partially explained by the mAb data. While BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the \u201cinner face\u201d of RBD that is exposed only when this domain is in the \u201cup\u201d position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.","version":"1.2","doi":"10.1101/2023.09.24.559214","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.22.559030","pub_date":"2023-9-27","title":"Survey of white-footed mice in Connecticut, USA reveals low SARS-CoV-2 seroprevalence and infection with divergent betacoronaviruses","abstract":"Diverse mammalian species display susceptibility to and infection with SARS-CoV-2. Potential SARS-CoV-2 spillback into rodents is understudied despite their host role for numerous zoonoses and human proximity. We assessed exposure and infection among white-footed mice (Peromyscus leucopus) in Connecticut, USA. We observed 1% (6/540) wild-type neutralizing antibody seroprevalence among 2020-2022 residential mice with no cross-neutralization of variants. We detected no SARS-CoV-2 infections via RT-qPCR, but identified non-SARS-CoV-2 betacoronavirus infections via pan-coronavirus PCR among 1% (5/468) of residential mice. Sequencing revealed two divergent betacoronaviruses, preliminarily named Peromyscus coronavirus-1 and -2. Both belong to the Betacoronavirus 1 species and are \u223c90% identical to the closest known relative, Porcine hemagglutinating encephalomyelitis virus. Low SARS-CoV-2 seroprevalence suggests white-footed mice may not be sufficiently susceptible or exposed to SARS-CoV-2 to present a long-term human health risk. However, the discovery of divergent, non-SARS-CoV-2 betacoronaviruses expands the diversity of known rodent coronaviruses and further investigation is required to understand their transmission extent.","version":"1.2","doi":"10.1101/2023.09.22.559030","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.26.559580","pub_date":"2023-9-27","title":"SARS-CoV-2 Omicron BA.2.86: less neutralization evasion compared to XBB sub-variants","abstract":"The continual emergence and circulation of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have caused a great challenge for the coronavirus disease 2019 (COVID-19) pandemic control. Recently, Omicron BA.2.86 was identified with more than 30 amino acid changes on the spike (S) protein, compared to Omicron BA.2 or XBB.1.5. The immune evasion potential of BA.2.86 is of great concern. In this study, we evaluated the neutralizing activities of sera collected from participants and mice. Participants were divided into five groups according to their vaccination (inactivated vaccine, protein subunit vaccine ZF2001 or ZF2202-A) and infection (Omicron BF.7/BA.5.2) status. ZF2202-A is ZF2001 vaccine\u2019s next-generation COVID-19 vaccine with updated bivalent Delta-BA.5 RBD-heterodimer immunogen. BALB/c mice were immunized with XBB.1.5 RBD-homodimer, BA.5-BA.2, Delta-XBB.1.5 or BQ.1.1-XBB.1.5 RBD-heterodimers protein vaccine candidates for evaluating the neutralizing responses. We found that Omicron BA.2.86 shows stronger immune evasion than BA.2 due to >30 additional mutations on S protein. Compared to XBB sub-variants, BA.2.86 does not display more resistance to the neutralizing responses induced by ZF2001-vaccination, BF.7/BA.5.2 breakthrough infection or a booster dose of ZF2202-A-vaccination. In addition, the mouse experiment results showed that BQ.1.1-XBB.1.5 RBD-heterodimer and XBB.1.5 RBD-homodimer induced high neutralizing responses against XBB sub-variants and BA.2.86, indicating that next-generation COVID-19 vaccine should be developed to enhance the protection efficacy against the circulating strains in the future.","version":"1.1","doi":"10.1101/2023.09.26.559580","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.01.526694","pub_date":"2023-9-27","title":"Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance","abstract":"The COVID-19 pandemic has necessitated the rapid development and implementation of whole genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of methodology on consensus genome concordance and interpretation. Data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) SARS-CoV-2 WGS proficiency testing program (PTP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimen were investigated, focusing on discordant sequence data, and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high Ct replicate which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture derived control specimen. While these sites were ultimately accurate after considering the participants\u2019 bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90% affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90% genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs, and minimum genome coverage of 90%. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Amidst the COVID-19 pandemic, a unique collaboration between a national multidisciplinary working group and a quality assurance program facilitated ongoing development of standardized quality control criteria and analysis methods for high-quality SARS-CoV-2 genomic approaches across Australia. With this article, we shed light on the robustness of amplicon sequencing and analysis methods to produce highly concordant genomes, while also presenting additional assessment criteria to guide laboratories in identifying areas for improvement. Insights from this nationwide collaboration underscore the need for real-time knowledge-sharing and iterative refinements to quality standards, particularly as situations and methods evolve during a pandemic. While the spotlight is on SARS-CoV-2, the analyses and findings have universal implications for genomic surveillance during infectious disease outbreaks. As WGS becomes increasingly central in outbreak surveillance, continuous evaluation and collaboration, like that described here, are vital to ensure data accuracy and inform future public health responses.","version":"1.2","doi":"10.1101/2023.02.01.526694","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.27.559689","pub_date":"2023-9-27","title":"Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera","abstract":"The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.","version":"1.1","doi":"10.1101/2023.09.27.559689","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.27.559677","pub_date":"2023-9-27","title":"Lessons from a multilaboratorial task force for diagnosis of a fatal toxoplasmosis outbreak in captive primates in Brazil","abstract":"As exemplified by the Coronavirus Disease 2019 (COVID-19) pandemic, infectious diseases may emerge and spread rapidly, often causing serious economic losses and public health concerns. In fact, disease outbreaks have become increasingly common, especially those of zoonotic origin. The Brazilian Ministry of Health is responsible for national epizootic surveillance. However, the system\u2019s focus primarily on diseases affecting humans has led to the neglect of other zoonotic diseases. In this report, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among captive neotropical primates housed at a primatology center in Brazil. After presenting a range of non-specific clinical signs, including fever, prostration, inappetence, and abdominal pain, ten primates from five different species died within approximately four days. Despite the state of health emergency due to the pandemic, a network of volunteer researchers was established to investigate the outbreak. A wide range of high-resolution techniques was used for different pathogens, including SARS-CoV-2 (RTq-PCR, ELISA and IHC), Toxoplasma gondii (IHC and IFA) and Escherichia coli (IFA), as well as a portable Metagenomic Sequencing utilizing Nanopore Technology. Within a span of four days after necropsies, we successfully identified T. gondii as the causative agent of this outbreak. This case highlights some of the obstacles faced with the current Brazilian surveillance system, which is still limited. A cross-platform interdisciplinary investigation could be a possible model for future epizootic investigations in non-human animals. The Brazilian epizootic surveillance system, under the regulation of the Ministry of Health, has been established to address a national list of compulsory notifiable diseases. However, focusing mainly on the risks to humans causes other zoonoses to be neglected. Here we present an outbreak that occurred during the first year of the COVID-19 pandemic that affected eleven neotropical primates (NP) belonging to six different species. Within four days of exhibiting a range of non-specific clinical signs, including fever, prostration, inappetence, and abdominal pain, ten NPs died. Despite testing negative for pathogens included in the national surveillance policy, a collaborative group of researchers investigated the outbreak in detail. Using integrated diagnostic techniques, we identified Toxoplasma gondii as the causative agent four days after necropsy. Toxoplasmosis causes devastating acute death outbreaks in neotropical primates and is currently absent in the national guidelines. This unified effort proved the effectiveness of a multidisciplinary collaborative surveillance network in facilitating precise diagnoses.","version":"1.1","doi":"10.1101/2023.09.27.559677","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.31.542721","pub_date":"2023-9-27","title":"Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A","abstract":"Furin is a mammalian serine protease with important roles in cellular homeostasis and disease. It cleaves and activates numerous endogenous and exogenous substrates, including the SARS-CoV-2 viral spike protein and protein toxins such as diphtheria toxin and Pseudomonas exotoxin A (PE). Recombinant immunotoxins (RITs) are toxin conjugates used as cancer therapeutics that connect tumor-directed antibodies with toxins for targeted cell killing. RITs based on PE have shown success in treating a variety of cancers, but often suffer from safety and efficacy concerns when used clinically. We have explored furin as a potential limiting factor in the intoxication pathway of PE-based RITs. Although the furin has widely recognized importance in RIT intoxication, its role is incompletely understood. Circumstantial evidence suggests that furin may act as a transporter for RITs in addition to its role of activation by cleavage. Here, we describe the creation of a CRISPR-engineered furin-deficient HEK293 cell line, \u0394Fur293. Using \u0394Fur293 and derivatives that express mutant forms of furin, we confirm the importance of furin in the PE RIT intoxication pathway and show that furin trafficking has a significant impact on RIT efficacy. Our data support the hypothesis that furin acts as a transporter during RIT intoxication, and suggest furin as a target to improve the effectiveness of RITs.","version":"1.2","doi":"10.1101/2023.05.31.542721","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.24.558358","pub_date":"2023-9-26","title":"RCoV19: A One-stop Hub for SARS-CoV-2 Genome Data Integration, Variants Monitoring, and Risk Pre-warning","abstract":"The Resource for Coronavirus 2019 (RCoV19, https://ngdc.cncb.ac.cn/ncov/) is an open-access information resource dedicated to providing valuable data on the genomes, mutations, and variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this updated implementation of RCoV19, we have made significant improvements and advancements over the previous version. Firstly, we have implemented a highly refined genome data curation model. This model now features an automated integration pipeline and optimized curation rules, enabling efficient daily updates of data in RCoV19. Secondly, we have developed a global and regional lineage evolution monitoring platform, alongside an outbreak risk pre-warning system. These additions provide a comprehensive understanding of SARS-CoV-2 evolution and transmission patterns, enabling better preparedness and response strategies. Thirdly, we have developed a powerful interactive mutation spectrum comparison module. This module allows users to compare and analyze mutation patterns, assisting in the detection of potential new lineages. Furthermore, we have incorporated a comprehensive knowledgebase on mutation effects. This knowledgebase serves as a valuable resource for retrieving information on the functional implications of specific mutations. In summary, RCoV19 serves as a vital scientific resource, providing access to valuable data, relevant information, and technical support in the global fight against COVID-19.","version":"1.1","doi":"10.1101/2023.09.24.558358","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.26.559506","pub_date":"2023-9-26","title":"Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10","abstract":"Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention. Mitochondria and metabolic alterations induced by SARS- CoV-2 accessory proteins ORF3a, ORF9b, ORF9c, ORF10 in pulmonary cells unravel new targets of intervention.","version":"1.1","doi":"10.1101/2023.09.26.559506","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.26.559465","pub_date":"2023-9-26","title":"In silico analyses identifies sequence contamination thresholds for Nanopore-generated SARS-CoV2 sequences","abstract":"The SARS-CoV-2 pandemic has brought molecular biology and genomic sequencing into the public consciousness and lexicon. With an emphasis on rapid turnaround, genomic data has been used to inform both diagnostic and surveillance decisions for the current pa ndemic at a previously unheard-of scale. The surge in the submission of genomic data to publicly-available databases has proved essential as comparing different genome sequences offers a wealth of knowledge, including phylogenetic links, modes of transmission, rates of evolution, and the impact of mutations on infection and disease severity. However, the scale of the pandemic has meant that once sequencing runs are performed, they are rarely repeated due to limited sample material and/or the availability of sequencing resources, resulting in some imperfect runs being uploaded to public repositories. As a result, it is crucial to investigate the data obtained from these imperfect runs to determine whether the results are reliable. Numerous studies have identified a variety of sources of contamination in public next-generation sequencing (NGS) data as the number of NGS studies increases along with the diversity of sequencing technologies and procedures [1\u20133]. For this study, we conducted an in silico experiment with known SARS-CoV-2 sequences produced from Oxford Nanopore Technologies sequencing to investigate the effect of contamination on lineage calls and single nucleotide variations (SNVs). Through a series of analyses, we identified a contamination threshold below which runs are expected to generate accurate lineage calls and maintain genomic sequence integrity. Together, these findings provide a benchmark below which imperfect runs may be considered robust for reporting results to both stakeholders and public repositories and reduce the need for repeat or wasted runs. Large-scale genomic comparisons provide a wealth of knowledge, including modes of transmission, rates of evolution, and the impact of mutations on infection, disease severity, and treatment effectiveness. As a result, the public release of genomic data has proven to be crucial. However, studies continue to show that some of the genomic data in public repositories are contaminated due to a variety of reasons. For instance, in the case of SARS-CoV-2 sequences, the pandemic prevented many sequencing runs from being repeated, resulting in some imperfect runs being uploaded to public repositories. It is of note that when genomic data is contaminated, both scientific decisions/studies and public health measures may be compromised. To identify genome contamination threshold(s) for SARS-CoV-2 sequences generated by Nanopore sequencing, computational biology techniques were utilized to generate artificially subsampled contaminated genomes. This is the first study of its kind and so our hope is that the results obtained provide a starting point for the investigation of reporting contamination of NGS data.","version":"1.1","doi":"10.1101/2023.09.26.559465","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.25.558837","pub_date":"2023-9-26","title":"Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain","abstract":"The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5\u2019-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.","version":"1.1","doi":"10.1101/2023.09.25.558837","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.25.559391","pub_date":"2023-9-26","title":"Using a function-first \u2018scout fragment\u2019-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes","abstract":"Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic \u2018scout fragments\u2019, which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.","version":"1.1","doi":"10.1101/2023.09.25.559391","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.28.538769","pub_date":"2023-9-25","title":"Lack of detection of SARS-CoV-2 in British wildlife 2020-21 and first description of a stoat (Mustela erminea) Minacovirus","abstract":"Repeat spillover of SARS-CoV-2 into new hosts has highlighted the critical role of cross species transmission of coronaviruses and establishment of new reservoirs of virus in pandemic and epizootic spread of coronaviruses. Species particularly susceptible to SARS-CoV-2 spill-over include Mustelidae (mink, ferrets and related animals), cricetid rodents (hamsters and related animals), felids domestic cats and related animals) and white tailed deer. These predispositions led us to screen British wildlife with sarbecovirus specific qPCR and pan coronavirus PCR assays for SARS-CoV-2 using samples collected during the human pandemic to establish if widespread spill-over was occurring. Fourteen wildlife species (n=402) were tested, including : 2 Red Foxes (Vulpes vulpes), 101 Badgers (Meles meles), 2 wild American Mink (Neogale vison), 41 Pine Marten (Martes martes), 2 Weasels (Mustela nivalis), 7 Stoats (Mustela erminea), 108 Water Voles (Arvicola amphibius), 39 Bank voles (Myodes glareolous), 10 Field Voles (Microtus agrestis), 15 Wood Mice (Apodemus sylvaticus), 1 Common Shrew (Sorex aranaeus), 2 Pygmy Shrews (Sorex minutus), 2 Hedgehogs (Erinaceus europaeus) and 75 Eurasian Otters (Lutra lutra). No cases of SARS-CoV-2 were detected in any animals, however a novel minacovirus related to mink and ferret alphacoronaviruses was detected in stoats recently introduced to the Orkney Islands. This group of viruses is of interest due to pathogenicity in ferrets. The impact of this virus on the health of stoat populations remains to be established.","version":"1.3","doi":"10.1101/2023.04.28.538769","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.22.559019","pub_date":"2023-9-25","title":"Immune Correlates of Hyperglycemia and Vaccination in a Non-human Primate Model of Long-COVID","abstract":"Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are major manifestations of the post-acute sequelae of SARS-CoV-2 (PASC). Our understanding of lasting glucometabolic disruptions after acute COVID-19 remains unclear due to the lack of animal models for metabolic PASC. Here, we report a non-human primate model of metabolic PASC using SARS-CoV-2 infected African green monkeys (AGMs). Using this model, we have identified a dysregulated chemokine signature and hypersensitive T cell population during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. This persistent hyperglycemia correlates with elevated hepatic glycogen, but there was no evidence of long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the AGM metabolic PASC model exhibits important similarities to human metabolic PASC and can be utilized to assess therapeutic candidates to combat this syndrome.","version":"1.1","doi":"10.1101/2023.09.22.559019","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.01.551474","pub_date":"2023-9-23","title":"Hierarchical Assembly of Single-Stranded RNA","abstract":"Single-stranded RNA (ssRNA) plays a major role in the flow of genetic information\u2013 most notably in the form of messenger RNA (mRNA)\u2013and in the regulation of biological processes. The highly dynamic nature of chains of unpaired nucleobases challenges structural characterizations of ssRNA by experiments or molecular dynamics (MD) simulations alike. Here we use hierarchical chain growth (HCG) to construct ensembles of ssRNA chains. HCG assembles the structures of protein and nucleic acid chains from fragment libraries created by MD simulations. Applied to homo- and heteropolymeric ssRNAs of different lengths, we find that HCG produces structural ensembles that overall are in good agreement with diverse experiments including nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and single-molecule F\u00f6rster resonance energy transfer (FRET). The agreement can be further improved by ensemble refinement using Bayesian inference of ensembles (BioEn). HCG can also be used to assemble RNA structures that combine base-paired and unpaired regions, as illustrated for the 5 untranslated region (UTR) of SARS-CoV-2 mRNA.","version":"1.2","doi":"10.1101/2023.08.01.551474","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.20.558551","pub_date":"2023-9-21","title":"COVID-19 ORF3a Viroporin Influenced Common and Unique Cellular Signalling Cascades in Lung, Heart and Brain Choroid Plexus Organoids with Additional Enriched MicroRNA Network Analyses for Lung and Brain Tissues","abstract":"Tissue specific implications of SARS-CoV-2 encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organs \u2013 the heart, lung, and brain. We analysed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Further we identified common and unique interacting host proteins, their targeting miRNAs (lung and brain), and delineated associated biological processes reanalysing RNA-seq data from the brain (COVID-19 infected/uninfected Choroid Plexus Organoids study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. Number of unique ORF3a interacting proteins in brain, lung and heart were 10, 7 and 1 respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with the history of hypertension taking calcium channel blockers (CCBs) or dihydorpyridine CCBs had elevated rate of intubation or increased rate of intubation/death respectively. Also likelihood of hospitalization of chronic CCB users with COVID-19 was more in comparison to long term Angiotensin Converting Enzyme inhibitors/Angiotensin Receptor Blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in brain and lung revealed, 2 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue specific COVID-19 drug/drug repurposing targets.","version":"1.1","doi":"10.1101/2023.09.20.558551","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.20.558606","pub_date":"2023-9-21","title":"Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells","abstract":"Respiratory syncytial virus (RSV) can cause severe disease especially in infants; however, mechanisms of age-associated disease severity remain elusive. Here, employing human bronchial epithelium models generated from tracheal aspirate-derived basal stem cells of neonates and adults, we investigated whether age regulates RSV-epithelium interaction to determine disease severity. We show that following RSV infection, only neonatal epithelium model exhibited cytopathy and mucus hyperplasia, and neonatal epithelium had more robust viral spread and inflammatory responses than adult epithelium. Mechanistically, RSV-infected neonatal ciliated cells displayed age-related impairment of STAT3 activation, rendering susceptibility to apoptosis, which facilitated viral spread. In contrast, SARS-CoV-2 infection of ciliated cells had no effect on STAT3 activation and was not affected by age. Taken together, our findings identify an age-related and RSV-specific interaction with neonatal bronchial epithelium that critically contributes to severity of infection, and STAT3 activation offers a potential strategy to battle severe RSV disease in infants.","version":"1.1","doi":"10.1101/2023.09.20.558606","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.19.558424","pub_date":"2023-9-20","title":"ACE2-Coated Virus-Like Particles Effectively Block SARS-CoV-2 Infection","abstract":"A large body of research accumulated over the past three years dedicated to our understanding and fighting COVID-19. Blocking the interaction between SARS-CoV-2 Spike and ACE2 receptor has been considered an effective strategy as anti-SARS-CoV-2 therapeutics. In this study, we developed ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus. These ACE2-VLPs exhibited high neutralization capacity even when applied at low doses, and displayed superior efficacy compared to extracellular vesicles carrying ACE2, in the in vitro pseudoviral assays. ACE2-VLPs were stable under different environmental temperatures, and they were effective in blocking all tested variants of concern in vitro. Finally, ACE2-VLPs displayed marked neutralization capacity against Omicron BA.1 in the Vero E6 cells. Based on their superior efficacy compared to extracellular vesicles, and their demonstrated success against live virus, ACE2-VLPs can be considered as vital candidates for treating SARS-CoV-2. This novel therapeutic approach of VLP coating with receptor particles can serve as proof-of-concept for designing effective neutralization strategies for other viral diseases in the future. In our study, we demonstrate the prevention of SARS-CoV-2 infection through the use of Ace2-coated VLPs.","version":"1.1","doi":"10.1101/2023.09.19.558424","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.19.558444","pub_date":"2023-9-20","title":"Characteristics and functions of infection-enhancing antibodies to the N-terminal domain of SARS-CoV-2","abstract":"Characterization of functional antibody responses to the N-terminal domain (NTD) of the SARS-CoV-2 spike (S) protein has included identification of both potent neutralizing activity and putative enhancement of infection. Fc\u03b3-receptor (Fc\u03b3R)-independent enhancement of SARS-CoV-2 infection mediated by NTD-binding monoclonal antibodies (mAbs) has been observed in vitro, but the functional significance of these antibodies in vivo is not clear. Here we studied 1,213 S-binding mAbs derived from longitudinal sampling of B-cells collected from eight COVID-19 convalescent patients and identified 72 (5.9%) mAbs that enhanced infection in a VSV-SARS-CoV-2-S-Wuhan pseudovirus (PV) assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least five months post-infection. Enhancement of PV infection by NTD-binding mAbs was not observed using intestinal (Caco-2) and respiratory (Calu-3) epithelial cells as infection targets and was diminished or lost against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire from two of the convalescent subjects identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum (i.e., functionally secreted antibody). Functional analysis of these mAbs demonstrated robust activation of Fc\u03b3RIIIa associated with antibody binding to recombinant S proteins. Taken together, these findings suggest functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display diverse attributes that include Fc\u03b3R activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.","version":"1.1","doi":"10.1101/2023.09.19.558444","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.19.558485","pub_date":"2023-9-20","title":"Immunogenicity and Efficacy of TNX-1800, A Live Virus Recombinant Poxvirus Vaccine Candidate, Against SARS-CoV-2 Challenge in Nonhuman Primates","abstract":"TNX-1800 is a synthetically derived live chimeric Horsepox Virus (rcHPXV) vaccine expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated, as indicated by the lack of serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated robust humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In Cynomolgus Macaques, a single dose of TNX-1800 induced a strong interferon-gamma (IFN-\u03b3) mediated T cell response, promoting both pathogen clearance in the upper and lower airways and generation of systemic neutralizing antibody response against WA strain SARS-CoV-2. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.","version":"1.1","doi":"10.1101/2023.09.19.558485","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.12.499603","pub_date":"2023-9-19","title":"Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants","abstract":"SARS-CoV-2 adaptation to humans is evidenced by the emergence of variants of concern (VOCs) with distinct genotypes and phenotypes that facilitate immune escape and enhance transmission frequency. Most recently Omicron subvariants have emerged with heavily mutated spike proteins which facilitate re-infection of immune populations through extensive antibody escape driving replacement of previously-dominant VOCs Alpha and Delta. Interestingly, Omicron is the first VOC to produce distinct subvariants. Here, we demonstrate that later Omicron subvariants, particularly BA.4 and BA.5, have evolved an enhanced capacity to suppress human innate immunity when compared to earliest subvariants BA.1 and BA.2. We find that, like previously dominant VOCs, later Omicron subvariants tend to increase expression of viral innate immune antagonists Orf6 and nucleocapsid. We show Orf6 to be a key contributor to enhanced innate immune suppression during epithelial replication by BA.5 and Alpha, reducing innate immune signaling through IRF3 and STAT1. Convergent VOC evolution of enhanced innate immune antagonist expression suggests common pathways of adaptation to humans and links VOC, and in particular Omicron subvariant, dominance to improved innate immune evasion.","version":"1.2","doi":"10.1101/2022.07.12.499603","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.15.557929","pub_date":"2023-9-19","title":"Third dose COVID-19 mRNA vaccine enhances IgG4 isotype switching and recognition of Omicron subvariants by memory B cells after mRNA but not adenovirus priming","abstract":"Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We here examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Healthy adults who received primary BNT162b2 (mRNA) (n=18) or ChAdOx1 (vector) (n=25) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1+. IgG4+ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4+ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4+ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.","version":"1.2","doi":"10.1101/2023.09.15.557929","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.17.558185","pub_date":"2023-9-18","title":"Evidence associating neutrophilia, lung damage, hyperlactatemia, blood acidosis, impaired oxygen transport, and mortality in critically ill COVID-19 patients","abstract":"COVID-19 severity and high in-hospital mortality are often associated with severe hypoxemia, hyperlactatemia, and acidosis. Since neutrophil numbers in severe COVID-19 can exceed 80% of the total circulating leukocytes and that they are massively recruited to infected lungs, we investigated whether metabolic acidosis mediated by the glycolytic neutrophils is associated with lung damage and impaired oxygen delivery in critically ill patients. Based on prospective mortality outcome, 102 critically ill-hospitalized COVID-19 patients were divided into two groups: ICU-Survivors (ICU-S, n=36) and ICU-Non-survivors (ICU-NS, n=66). Blood samples were collected from patients and control subjects to explore correlations between neutrophil counts, lung damage, glycolysis, blood lactate, blood pH, hemoglobin oxygen saturation, and mortality outcome. We also interrogated isolated neutrophils for glycolytic activities and for apoptosis using high-throughput fluorescence imaging complemented with transcriptomic analyses. Stratified survival analyses were conducted to estimate mortality risk associated with higher lactate among predefined subgroups. Neutrophil counts were consistently higher in critically ill patients while exhibiting remarkably lower apoptosis. Transcriptomic analysis revealed miRNAs associated with downregulation of genes involved in neutrophils apoptosis. Both CT lung damage scores and neutrophil counts predicted mortality. Severinghaus fitting of hemoglobin oxygen saturation curve revealed a right-shift indicating lower oxygen capacity in non-survivors, which is consistent with lower blood-pH observed in the same group. Levels of blood lactate were increased in patients but significantly more in the ICU-NS relative to the control group. ROC analysis followed by Kaplan-Meyer survival analysis stratified to the obtained cut-off values showed that CT damage scores, neutrophil counts, and lactate levels are predictors of mortality within 15 days following blood collection. The current results implicate neutrophilia as a potential player in metabolic acidosis and deranged oxygen delivery associating SARS-CoV-2 infection thus contributing to mortality outcome.","version":"1.1","doi":"10.1101/2023.09.17.558185","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.14.557679","pub_date":"2023-9-17","title":"Modeling the emergence of viral resistance for SARS-CoV-2 during treatment with an anti-spike monoclonal antibody","abstract":"The COVID-19 pandemic has led to over 760 million cases and 6.9 million deaths worldwide. To mitigate the loss of lives, emergency use authorization was given to several anti-SARS-CoV-2 monoclonal antibody (mAb) therapies for the treatment of mild-to-moderate COVID-19 in patients with a high risk of progressing to severe disease. Monoclonal antibodies used to treat SARS-CoV-2 target the spike protein of the virus and block its ability to enter and infect target cells. Monoclonal antibody therapy can thus accelerate the decline in viral load and lower hospitalization rates among high-risk patients with susceptible variants. However, viral resistance has been observed, in some cases leading to a transient viral rebound that can be as large as 3-4 orders of magnitude. As mAbs represent a proven treatment choice for SARS-CoV-2 and other viral infections, evaluation of treatment-emergent mAb resistance can help uncover underlying pathobiology of SARS-CoV-2 infection and may also help in the development of the next generation of mAb therapies. Although resistance can be expected, the large rebounds observed are much more difficult to explain. We hypothesize replenishment of target cells is necessary to generate the high transient viral rebound. Thus, we formulated two models with different mechanisms for target cell replenishment (homeostatic proliferation and return from an innate immune response anti-viral state) and fit them to data from persons with SARS-CoV-2 treated with a mAb. We showed that both models can explain the emergence of resistant virus associated with high transient viral rebounds. We found that variations in the target cell supply rate and adaptive immunity parameters have a strong impact on the magnitude or observability of the viral rebound associated with the emergence of resistant virus. Both variations in target cell supply rate and adaptive immunity parameters may explain why only some individuals develop observable transient resistant viral rebound. Our study highlights the conditions that can lead to resistance and subsequent viral rebound in mAb treatments during acute infection. Monoclonal antibodies have been used as a treatment for SARS-CoV-2. However, viral evolution and development of variants has compromised the use of all currently authorized monoclonal antibodies for SARS-CoV-2. In some individuals treated with one such monoclonal antibody, bamlanivimab, transient nasal viral rebounds of 3-4 logs associated with resistant viral strains occur. To better understand the mechanisms underlying resistance emergence with high viral load rebounds, we developed two different models that incorporate drug sensitive and drug resistant virus as well as target cell replenishment and fit them to data. The models accurately capture the observed viral dynamics as well as the proportion of resistant virus for each studied individual with little variation in model parameters. In the models with best-fit parameters, bamlanivimab selects for resistance mutants that can expand to high levels due to target cell replenishment. The ultimate clearance of virus however depends on the development of adaptive immunity.","version":"1.1","doi":"10.1101/2023.09.14.557679","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.15.557978","pub_date":"2023-9-17","title":"VirEvol platform : accurate prediction and visualization of SARS-CoV-2 evolutionary trajectory based on protein language model, structural information and immunological recognition mechanism","abstract":"Predicting the mutation direction of SARS-CoV-2 using exploratory computational methods presents a challenging, yet prospective, research avenue. However, existing research methods often ignore the effects of protein structure and multi-source viral information on mutation prediction, making it difficult to accurately predict the evolutionary trend of the SARS-CoV-2 S protein receptor-binding domain (RBD). To overcome this limitation, we proposed an interpretable language model combining structural, sequence and immune information. The dual utility of this model lies in its ability to predict SARS-CoV-2\u2019s affinity for the ACE2 receptor, and to assess its potential for immune evasion. Additionally, it explores the mutation trend of SARS-CoV-2 via a genetic algorithm-directed evolution. The model exhibits high accuracy in both regards and has displayed promising early warning capabilities, effectively identifying 13 out of 14 high-risk strains, marking a success rate of 93%.\u201d. This study provides a novel method for discerning the molecular evolutionary pattern, as well as predicting the evolutionary trend of SARS-CoV-2 which is of great significance for vaccine design and drug development of new coronaviruses. We further developed VirEvol, a unique platform designed to visualize the evolutionary trajectories of novel SARS-CoV-2 strains, thereby facilitating real-time predictive analysis for researchers. The methodologies adopted in this work may inspire new strategies and offer technical support for addressing challenges posed by other highly mutable viruses.","version":"1.1","doi":"10.1101/2023.09.15.557978","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.27.530277","pub_date":"2023-9-17","title":"Engineered Immunogens to Elicit Antibodies Against Conserved Coronavirus Epitopes","abstract":"Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employed computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant RBD. These engineered proteins bound with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interacted with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicited sera with broad betacoronavirus reactivity and protected as \u201cboosts\u201d against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.","version":"1.2","doi":"10.1101/2023.02.27.530277","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.17.545443","pub_date":"2023-9-17","title":"Detecting episodic evolution through Bayesian inference of molecular clock models","abstract":"Molecular evolutionary rate variation is a key aspect of the evolution of many organisms that can be modelled using molecular clock models. For example, fixed local clocks revealed the role of episodic evolution in the emergence of SARS-CoV-2 variants of concern. Like all statistical models, however, the reliability of such inferences is contingent on an assessment of statistical evidence. We present a novel Bayesian phylogenetic approach for detecting episodic evolution. It consists of computing Bayes factors, as the ratio of posterior and prior odds of evolutionary rate increases, effectively quantifying support for the effect size. We conducted an extensive simulation study to illustrate the power of this method and benchmarked it to formal model comparison of a range of molecular clock models using (log) marginal likelihood estimation, and to inference under a random local clock model. Quantifying support for the effect size has higher sensitivity than formal model testing and is straight-forward to compute, because it only needs samples from the posterior and prior distribution. However, formal model testing has the advantage of accommodating a wide range molecular clock models. We also assessed the ability of an automated approach, known as the random local clock, where branches under episodic evolution may be detected without their a priori definition. In an empirical analysis of a data set of SARS-CoV-2 genomes, we find \u2018very strong\u2019 evidence for episodic evolution. Our results provide guidelines and practical methods for Bayesian detection of episodic evolution, as well as avenues for further research into this phenomenon.","version":"1.2","doi":"10.1101/2023.06.17.545443","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.15.558006","pub_date":"2023-9-17","title":"Temporal profiling of human lymphoid tissues reveals coordinated defence to viral challenge","abstract":"Adaptive immunity is generated in lymphoid organs, but how these structures defend themselves during infection in humans is unknown. The nasal epithelium is a major site of viral entry, with adenoid nasal-associated lymphoid tissue (NALT) generating early adaptive responses. Here, using a nasopharyngeal biopsy, we examined longitudinal immune responses in NALT following viral challenge, using SARS-CoV-2 infection as a natural experimental model. In acute infection, infiltrating monocytes formed a subepithelial and peri-follicular shield, recruiting NET-forming neutrophils, whilst tissue macrophages expressed pro-repair molecules during convalescence to promote the restoration of tissue integrity. Germinal centre B cells expressed anti-viral transcripts that inversely correlated with fate-defining transcription factors. Among T cells, tissue-resident memory CD8 T cells alone showed clonal expansion and maintained cytotoxic transcriptional programmes into convalescence. Together our study provides a unique insight into how human nasal adaptive immune responses are generated and sustained in the face of viral challenge.","version":"1.1","doi":"10.1101/2023.09.15.558006","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.13.557561","pub_date":"2023-9-16","title":"Deep learning predictions of TCR-epitope interactions reveal epitope-specific chains in dual alpha T cells","abstract":"T cells have the ability to eliminate infected and cancer cells and play an essential role in cancer immunotherapy. T-cell activation is elicited by the binding of the T-cell receptor (TCR) to epitopes displayed on MHC molecules, and the TCR specificity is determined by the sequence of its \u03b1 and \u03b2 chains. Here, we collected and curated a dataset of 17,715 \u03b1\u03b2TCRs interacting with dozens of class I and class II epitopes. We used this curated data to develop MixTCRpred, a deep learning TCR-epitope interaction predictor. MixTCRpred accurately predicts TCRs recognizing several viral and cancer epitopes. MixTCRpred further provides a useful quality control tool for multiplexed single-cell TCR sequencing assays of epitope-specific T cells and pinpoints a substantial fraction of putative contaminants in public databases. Analysis of epitope-specific dual \u03b1 T cells demonstrates that MixTCRpred can identify \u03b1 chains mediating epitope recognition. Applying MixTCRpred to TCR repertoires from COVID-19 patients reveals enrichment of clonotypes predicted to bind an immunodominant SARS-CoV-2 epitope. Overall, MixTCRpred provides a robust tool to predict TCRs interacting with specific epitopes and interpret TCR-sequencing data from both bulk and epitope-specific T cells.","version":"1.1","doi":"10.1101/2023.09.13.557561","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.15.557899","pub_date":"2023-9-15","title":"The SARS-CoV-2 nucleoprotein associates with anionic lipid membranes","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N) and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we find the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, lipid binding is shown to occur in the N protein C-terminal domain, which is supported by extensive in silico analysis. Anionic lipid binding occurs for both the free and N oligomeric forms suggesting N can associate with membranes in the nucleocapsid form. Herein we present a lipid-dependent model based on in vitro, cellular and in silico data for the recruitment of N to M assembly sites in the lifecycle of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.09.15.557899","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.14.557682","pub_date":"2023-9-15","title":"Enhanced neutralization of SARS-CoV-2 XBB sub-lineages and BA.2.86 by a tetravalent COVID-19 vaccine booster","abstract":"As the SARS-CoV-2 virus continues to evolve, novel XBB sub-lineages such as XBB.1.5, XBB.1.16, EG.5, HK.3 (FLip), and XBB.2.3, as well as the most recent BA.2.86, have been identified and aroused global concern. Understanding the efficacy of current vaccines and the immune system\u2019s response to these emerging variants is critical for global public health. In this study, we evaluated the neutralization activities of sera from participants who received COVID-19 inactivated vaccines, or a booster vaccination of the recently approved tetravalent protein vaccine in China (SCTV01E), or had contracted a breakthrough infection with BA.5/BF.7/XBB virus. Comparative analysis of their neutralization profiles against a broad panel of 30 SARS-CoV-2 sub-lineage viruses revealed that strains such as BQ.1.1, CH.1.1, and all the XBB sub-lineages exhibited heightened resistance to neutralization than previous variants, however, despite the extra mutations carried by emerging XBB sub-lineages and BA.2.86, they did not demonstrate significantly increased resistance to neutralization compared to XBB.1.5. Encouragingly, the SCTV01E booster vaccination consistently induced robust and considerably higher neutralizing titers against all these variants than breakthrough infection did. Cellular immunity assays also showed that the SCTV01E booster vaccination elicited a higher frequency of virus-specific memory B cells but not IFN-\u03b3 secreting T cells. Our findings underline the importance of developing novel multivalent vaccines to more effectively combat future viral variants.","version":"1.1","doi":"10.1101/2023.09.14.557682","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.14.557399","pub_date":"2023-9-15","title":"Cooperativity and induced oligomerisation control the interaction of SARS-CoV-2 with its cellular receptor and patient-derived antibodies","abstract":"Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. Multivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.","version":"1.1","doi":"10.1101/2023.09.14.557399","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557241","pub_date":"2023-9-15","title":"Regulation of human interferon signaling by transposon exonization","abstract":"Innate immune signaling is essential for clearing pathogens and damaged cells, and must be tightly regulated to avoid excessive inflammation or autoimmunity. Here, we found that the alternative splicing of exons derived from transposable elements is a key mechanism controlling immune signaling in human cells. By analyzing long-read transcriptome datasets, we identified numerous transposon exonization events predicted to generate functional protein variants of immune genes, including the type I interferon receptor IFNAR2. We demonstrated that the transposon-derived isoform of IFNAR2 is more highly expressed than the canonical isoform in almost all tissues, and functions as a decoy receptor that potently inhibits interferon signaling including in cells infected with SARS-CoV-2. Our findings uncover a primate-specific axis controlling interferon signaling and show how a transposon exonization event can be co-opted for immune regulation.","version":"1.1","doi":"10.1101/2023.09.11.557241","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.13.557637","pub_date":"2023-9-14","title":"Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic","abstract":"Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Wholesale Seafood Market, the site with the most reported wildlife vendors in the city of Wuhan, China. Here, we analyze publicly available qPCR and sequencing data from environmental samples collected in the Huanan market in early 2020. We demonstrate that the SARS-CoV-2 genetic diversity linked to this market is consistent with market emergence, and find increased SARS-CoV-2 positivity near and within a particular wildlife stall. We identify wildlife DNA in all SARS-CoV-2 positive samples from this stall. This includes species such as civets, bamboo rats, porcupines, hedgehogs, and one species, raccoon dogs, known to be capable of SARS-CoV-2 transmission. We also detect other animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them to those from other markets. This analysis provides the genetic basis for a short list of potential intermediate hosts of SARS-CoV-2 to prioritize for retrospective serological testing and viral sampling.","version":"1.1","doi":"10.1101/2023.09.13.557637","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.12.557363","pub_date":"2023-9-13","title":"Identification of a Druggable Site on GRP78 at the GRP78-SARS-CoV-2 Interface and Compounds to Disrupt that Interface","abstract":"SARS-CoV-2, the virus that causes COVID-19, led to a global health emergency that claimed the lives of millions. Despite the widespread availability of vaccines, the virus continues to exist in the population in an endemic state which allows for the continued emergence of new variants. Most of the current vaccines target the spike glycoprotein interface of SARS-CoV-2, creating a selection pressure favoring viral immune evasion. Antivirals targeting other molecular interactions of SARS-CoV-2 can help slow viral evolution by providing orthogonal selection pressures on the virus. GRP78 is a host auxiliary factor that mediates binding of the SARS-CoV-2 spike protein to human cellular ACE2, the primary pathway of cell infection. As GRP78 forms a ternary complex with SARS-CoV-2 spike protein and ACE2, disrupting the formation of this complex is expected to hinder viral entry into host cells. Here, we developed a model of the GRP78-spike protein-ACE2 complex. We then used that model together with hot spot mapping of the GRP78 structure to identify the putative binding site for spike protein on GRP78. Next, we performed structure-based virtual screening of known drug/candidate drug libraries to identify binders to GRP78 that are expected to disrupt spike protein binding to the GRP78, and thereby preventing viral entry to the host cell. A subset of these compounds have previously been shown to have some activity against SARS-CoV-2. The identified hits are starting points for the further development of novel SARS-CoV-2 therapeutics, potentially serving as proof-of-concept for GRP78 as a potential drug target for other viruses.","version":"1.1","doi":"10.1101/2023.09.12.557363","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.12.557371","pub_date":"2023-9-13","title":"Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus","abstract":"Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad ACE2 usage and that RBD mutations further expand receptor promiscuity and enable human ACE2 utilization. We determined a cryoEM structure of the PRD-0038 RBD bound to R. alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryoEM and monoclonal antibody reactivity revealed its distinct antigenicity relative to SARS-CoV-2 and identified PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared to SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.","version":"1.1","doi":"10.1101/2023.09.12.557371","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.12.557330","pub_date":"2023-9-13","title":"Infectious touching: Has COVID-19 changed our perceptions of social touch? A neural and behavioral study","abstract":"Social touch is essential for reducing stress, improving mood and fostering a sense of social connectedness. Stimuli related to social touch are generally perceived as positive. Nevertheless, the social restrictions imposed by the COVID-19 pandemic may have changed the way human beings perceive and react to social touch. Indeed, the social distancing imposed by the pandemic may have had long term effects on human perceptions of social touch. In the current study, we examined how perceptions of interpersonal touch in social interactions were affected by the COVID-19 pandemic. Specifically, we compared behavioral and neural responses to observed social touch between two groups: pre- and post-COVID-19. Participants in both groups rated the pleasantness of photos of social touch between humans, nonsocial touch between inanimate objects or non-touch photos of either two humans or inanimate objects. We hypothesized that social touch in the post-COVID-19 group would induce hypervigilance due to the risk of infection. In line with our predictions, we found behavioral changes in perceptions of social touch among participants in this group, who rated photos with touch as less pleasant than did participants in the pre-COVID-19 group. Participants in the post-COVID-19 group also rated photos with humans as less pleasant than did participants in the pre-COVID-19 group. Additionally, EEG analysis revealed neural changes in the ERP components associated with hypervigilance: P1 and LPP. Contrary to pre-COVID-19 measures showing more positive P1 amplitudes for touch than for non-touch photos, after COVID-19 no differences in P1 amplitudes were found between touch and non-touch photos. Furthermore, after COVID-19 the P1 amplitudes for human and inanimate photos in the touch condition were similar, a pattern that did not emerge prior to COVID-19. These findings suggest that COVID-19 had a surprising impact on human perceptions of social touch, such that observing nonsocial touch evoked more positive emotions than observing human touch. Further, these findings may reflect shifts in attention or changes in the salience of touch-related information due to the altered circumstances brought about by the pandemic. Overall, our results indicate that COVID-19 has modified human perceptions of social touch, providing evidence that the pandemic has affected individuals\u2019 perceptual and evaluative processes and highlighting the importance of considering social and environmental factors in understanding subjective experiences.","version":"1.1","doi":"10.1101/2023.09.12.557330","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557206","pub_date":"2023-9-12","title":"Immune Evasion, Infectivity, and Fusogenicity of SARS-CoV-2 Omicron BA.2.86 and FLip Variants","abstract":"Evolution of SARS-CoV-2 requires the reassessment of current vaccine measures. Here, we characterized BA.2.86 and the XBB-lineage variant FLip by investigating their neutralization alongside D614G, BA.1, BA.2, BA.4/5, XBB.1.5, and EG.5.1 by sera from 3-dose vaccinated and bivalent vaccinated healthcare workers, XBB.1.5-wave infected first responders, and monoclonal antibody (mAb) S309. We assessed the biology of the variant Spikes by measuring viral infectivity and membrane fusogenicity. BA.2.86 is less immune evasive compared to FLip and other XBB variants, consistent with antigenic distances. Importantly, distinct from XBB variants, mAb S309 was unable to neutralize BA.2.86, likely due to a D339H mutation based on modeling. BA.2.86 had relatively high fusogenicity and infectivity in CaLu-3 cells but low fusion and infectivity in 293T-ACE2 cells compared to some XBB variants, suggesting a potentially differences conformational stability of BA.2.86 Spike. Overall, our study underscores the importance of SARS-CoV-2 variant surveillance and the need for updated COVID-19 vaccines.","version":"1.1","doi":"10.1101/2023.09.11.557206","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557190","pub_date":"2023-9-12","title":"Compartmentalized SARS-CoV-2 replication in upper versus lower respiratory tract after intranasal inoculation or aerosol exposure","abstract":"Non-human primate models are essential for the development of vaccines and antivirals against infectious diseases. Rhesus macaques are a widely utilized infection model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We compared cellular tropism and virus replication in rhesus macaques inoculated with SARS-CoV-2 via the intranasal route, or via exposure to aerosols. Intranasal inoculation results in replication in the upper respiratory tract and limited lower respiratory tract involvement, whereas exposure to aerosols results in infection throughout the respiratory tract. In comparison to multi-route inoculation, the intranasal and aerosol inoculation routes result in reduced SARS-CoV-2 replication in the respiratory tract.","version":"1.1","doi":"10.1101/2023.09.11.557190","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557279","pub_date":"2023-9-12","title":"Deep mutational scans of XBB.1.5 and BQ.1.1 reveal ongoing epistatic drift during SARS-CoV-2 evolution","abstract":"Substitutions that fix between SARS-CoV-2 variants can transform the mutational landscape of future evolution via epistasis. For example, large epistatic shifts in mutational effects caused by N501Y underlied the original emergence of Omicron variants, but whether such large epistatic saltations continue to define ongoing SARS-CoV-2 evolution remains unclear. We conducted deep mutational scans to measure the impacts of all single amino acid mutations and single-codon deletions in the spike receptor-binding domain (RBD) on ACE2-binding affinity and protein expression in the recent Omicron BQ.1.1 and XBB.1.5 variants, and we compared mutational patterns to earlier viral strains that we have previously profiled. As with previous RBD deep mutational scans, we find many mutations that are tolerated or even enhance binding to ACE2 receptor. The tolerance of sites to single-codon deletion largely conforms with tolerance to amino acid mutation. Though deletions in the RBD have not yet been seen in dominant lineages, we observe many tolerated deletions including at positions that exhibit indel variation across broader sarbecovirus evolution and in emerging SARS-CoV-2 variants of interest, most notably the well-tolerated \u0394483 deletion in BA.2.86. The substitutions that distinguish recent viral variants have not induced as dramatic of epistatic perturbations as N501Y, but we identify ongoing epistatic drift in SARS-CoV-2 variants, including interaction between R493Q reversions and mutations at positions 453, 455, and 456, including mutations like F456L that define the newly emerging EG.5 lineage. Our results highlight ongoing drift in the effects of mutations due to epistasis, which may continue to direct SARS-CoV-2 evolution into new regions of sequence space.","version":"1.1","doi":"10.1101/2023.09.11.557279","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.12.557347","pub_date":"2023-9-12","title":"Concurrent administration of COVID-19 and influenza vaccines enhances Spike-specific antibody responses","abstract":"The bivalent COVID-19 mRNA boosters became available in fall 2022 and were recommended alongside the seasonal influenza vaccine. However, the immunogenicity of concurrent versus separate administration of these vaccines remains unclear. Here, we analyzed antibody responses in healthcare workers who received the bivalent COVID-19 booster and the influenza vaccine on the same day or different days. IgG1 responses to SARS-CoV-2 Spike were higher at peak immunogenicity and 6 months following concurrent administration compared with separate administration of the COVID-19 and influenza vaccines. These data suggest that concurrent administration of these vaccines may yield higher and more durable SARS-CoV-2 antibody responses.","version":"1.1","doi":"10.1101/2023.09.12.557347","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557205","pub_date":"2023-9-12","title":"Examining Functional Linkages Between Conformational Dynamics, Protein Stability and Evolution of Cryptic Binding Pockets in the SARS-CoV-2 Omicron Spike Complexes with the ACE2 Host Receptor: Recombinant Omicron Variants Mediate Variability of Conserved Allosteric Sites and Binding Epitopes","abstract":"In the current study, we explore coarse-grained simulations and atomistic molecular dynamics together with binding energetics scanning and cryptic pocket detection in a comparative examination of conformational landscapes and systematic characterization of allosteric binding sites in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB.1 spike full-length trimer complexes with the host receptor ACE2. Microsecond simulations, Markov state models and mutational scanning of binding energies of the SARS-CoV-2 BA.2 and BA.2.75 receptor binding domain complexes revealed the increased thermodynamic stabilization of the BA.2.75 variant and significant dynamic differences between these Omicron variants. Molecular simulations of the SARS-CoV-2 Omicron spike full length trimer complexes with the ACE2 receptor complemented atomistic studies and enabled an in-depth analysis of mutational and binding effects on conformational dynamic and functional adaptability of the Omicron variants. Despite considerable structural similarities, Omicron variants BA.2, BA.2.75 and XBB.1 can induce unique conformational dynamic signatures and specific distributions of the conformational states. Using conformational ensembles of the SARS-CoV-2 Omicron spike trimer complexes with ACE2, we conducted a comprehensive cryptic pocket screening to examine the role of Omicron mutations and ACE2 binding on the distribution and functional mechanisms of the emerging allosteric binding sites. This analysis captured all experimentally known allosteric sites and discovered networks of inter-connected and functionally relevant allosteric sites that are governed by variant-sensitive conformational adaptability of the SARS-CoV-2 spike structures. The results detailed how ACE2 binding and Omicron mutations in the BA.2, BA.2.75 and XBB.1 spike complexes modulate the distribution of conserved and druggable allosteric pockets harboring functionally important regions. The results of are significant for understanding functional roles of druggable cryptic pockets that can be used for allostery-mediated therapeutic intervention targeting conformational states of the Omicron variants.","version":"1.1","doi":"10.1101/2023.09.11.557205","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557129","pub_date":"2023-9-12","title":"Expression Level Analysis of ACE2 Receptor Gene in African-American and Non-African-American COVID-19 Patients","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has spread rapidly across the continents. While the incidence of COVID-19 has been reported to be higher among African-American individuals, the rate of mortality has been lower compared to that of non-African-Americans. ACE2 is involved in COVID-19 as SARS-CoV-2 uses the ACE2 enzyme to enter host cells. Although the difference in COVID-19 incidence can be explained by many factors such as low accessibility of health insurance among the African-American community, little is known about ACE2 expression in African-American COVID-19 patients compared to non-African-American COVID-19 patients. The variable expression of genes can contribute to this observed phenomenon. In this study, transcriptomes from African-American and non-African-American COVID-19 patients were retrieved from the sequence read archive and analyzed for ACE2 gene expression. HISAT2 was used to align the reads to the human reference genome, and HTseq-count was used to get raw gene counts. EdgeR was utilized for differential gene expression analysis, and enrichR was employed for gene enrichment analysis. The datasets included 14 and 33 transcriptome sequences from COVID-19 patients of African-American and non-African-American descent, respectively. There were 24,092 differentially expressed genes, with 7,718 upregulated (log fold change > 1 and FDR 0.05) and 16,374 downregulated (log fold change \u22121 and FDR 0.05). The ACE2 mRNA level was found to be considerably downregulated in the African-American cohort (p-value = 0.0242, p-adjusted value = 0.038). The downregulation of ACE2 in the African-American cohort could indicate a correlation to the low COVID-19 severity observed among the African-American community.","version":"1.1","doi":"10.1101/2023.09.11.557129","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557219","pub_date":"2023-9-12","title":"Dual inhibition of coronavirus Mpro and PLpro enzymes by phenothiazines and their antiviral activity","abstract":"Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity in vitro was identified. In silico docking studies also predicted binding of the phenothiazine to the active sites of Mpro and PLpro from distantly related alphacoronavirus, HCoV-229E (229E) and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cell culture infection models. It was further demonstrated that the compound inhibited 229E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses. Coronavirus cysteine proteases Mpro and PLpro are targets for novel antiviral agents Phenothiazine ureas inhibit SARS-CoV-2 Mpro and PLpro protease activity Some phenothiazine ureas inhibit replication of diverse coronaviruses with minimal cytotoxicity Phenothiazine ureas inhibit early stages of coronavirus replication consistent with failure of viral polyprotein cleavage","version":"1.1","doi":"10.1101/2023.09.11.557219","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.12.557318","pub_date":"2023-9-12","title":"Digital holo-tomographic 3D maps of COVID-19 microclots in blood to assess disease severity","abstract":"The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders diagnosed in patients even after the recovery period. In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here we resolve individual microclot structures in platelet-rich plasma of donors with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 donors. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls when compared to COVID-19 donors. Our work highlights the utility of integrating DHTM in clinical settings that may allow the detection of individuals at risk of developing microvascular thrombotic disorders and for monitoring the efficiency of prescribed treatments by screening plasma samples.","version":"1.1","doi":"10.1101/2023.09.12.557318","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.10.557047","pub_date":"2023-9-11","title":"Less neutralization evasion of SARS-CoV-2 BA.2.86 than XBB sublineages and CH.1.1","abstract":"The highly mutated BA.2.86, with over 30 spike protein mutations in comparison to Omicron BA.2 and XBB.1.5 variants, has raised concerns about its potential to evade COVID-19 vaccination or prior SARS-CoV-2 infection-elicited immunity. In this study, we employ a live SARS-CoV-2 neutralization assay to compare the neutralization evasion ability of BA.2.86 with other emerged SARS-CoV-2 subvariants, including BA.2-derived CH.1.1, Delta-Omicron recombinant XBC.1.6, and XBB descendants XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1 and FL.1.5.1. Our results show that BA.2.86 is less neutralization evasive than XBB sublineages. Among all the tested variants, CH.1.1 exhibits the greatest neutralization evasion. In comparison to XBB.1.5, the more recent XBB descendants, particularly EG.5.1 and FL.1.5.1, display increased resistance to neutralization induced by parental COVID-19 mRNA vaccine and a BA.5-Bivalent-booster. In contrast, XBC.1.6 shows a slight reduction but remains comparable sensitivity to neutralization when compared to BA.5. Furthermore, a recent XBB.1.5-breakthrough infection significantly enhances the breadth and potency of cross-neutralization. These findings reinforce the expectation that the upcoming XBB.1.5 mRNA vaccine would likely boost the neutralization of currently circulating variants, while also underscoring the critical importance of ongoing surveillance to monitor the evolution and immune evasion potential of SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.09.10.557047","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.08.556349","pub_date":"2023-9-11","title":"Characterization of SARS-CoV-2 Convalescent Patients\u2019 Serological Repertoire Reveals High Prevalence of Iso\u2013RBD Antibodies","abstract":"While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that \u223c50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso\u2013RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso\u2013RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (\u223c8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso\u2013RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual\u2019s total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso\u2013RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso\u2013RBD serum antibodies which are unlikely to contribute to protection.","version":"1.1","doi":"10.1101/2023.09.08.556349","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.11.540343","pub_date":"2023-9-11","title":"Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform","abstract":"Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the COVID-19 pandemic, already established methods were challenged by the large genome of SARS-CoV-2. Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity, using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment \u2013 harboring all heterologous sequences \u2013 viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.","version":"1.4","doi":"10.1101/2023.05.11.540343","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.08.556901","pub_date":"2023-9-11","title":"The role of ion dissolution in metal and metal oxide surface inactivation of SARS-CoV-2","abstract":"Antiviral surface coatings are under development to prevent viral fomite transmission from high-traffic touch surfaces in public spaces. Copper\u2019s antiviral properties have been widely documented; but the antiviral mechanism of copper surfaces is not fully understood. We screened a series of metal and metal oxide surfaces for antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19). Copper and copper oxide surfaces exhibited superior anti-SARS-CoV-2 activity; however, level of antiviral activity was dependent upon the composition of the carrier solution used to deliver virus inoculum. We demonstrate that copper ions released into solution from test surfaces can mediate virus inactivation, indicating a copper ion dissolution-dependent antiviral mechanism. Level of antiviral activity is, however, not dependent on the amount of copper ions released into solution per se. Instead, our findings suggest that degree of virus inactivation is dependent upon copper ion complexation with other biomolecules (e.g., proteins/metabolites) in the virus carrier solution that compete with viral components. Although using tissue culture-derived virus inoculum is experimentally convenient to evaluate the antiviral activity of copper-derived test surfaces, we propose that the high organic content of tissue culture medium reduces the availability of \u201cuncomplexed\u201d copper ions to interact with the virus, negatively affecting virus inactivation and hence surface antiviral performance. We propose that laboratory antiviral surface testing should include virus delivered in a physiologically relevant carrier solution (saliva or nasal secretions when testing respiratory viruses) to accurately predict real-life surface antiviral performance when deployed in public spaces. The purpose of evaluating antiviral activity of test surfaces in the laboratory is to identify surfaces that will perform efficiently in preventing fomite transmission when deployed on high-traffic touch surfaces in public spaces. The conventional method in laboratory testing is to use tissue culture-derived virus inoculum, however this study demonstrates that antiviral performance of test copper-containing surfaces is dependent on the composition of the carrier solution in which the virus inoculum is delivered to test surfaces. Therefore, we recommend that laboratory surface testing should include virus delivered in a physiologically relevant carrier solution, to accurately predict real-life test surface performance in public spaces. Understanding the mechanism of virus inactivation is key to future rational design of improved antiviral surfaces. Here, we demonstrate that copper ions released from copper surfaces into small liquid droplets containing SARS-CoV-2, is a mechanism by which the virus that causes COVID-19 can be inactivated.","version":"1.1","doi":"10.1101/2023.09.08.556901","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.08.556912","pub_date":"2023-9-11","title":"Bayesian phylogenetics on globally emerging SARS-CoV-2 variant BA.2.86 suggest global distribution and rapid evolution","abstract":"Using bioinformatic pipelines and Bayseian phylogenetic analyses, we characterized a SARS-CoV-2 variant designated by the World Health Organization as a variant under monitoring in August 2023. Here we analyze the genomes of this SARS-CoV-2 variant, BA.2.86, deposited into GISAID within the two weeks of its emergence (2023-08-14 first submission to 2023-08-31), including the first BA.2.86 genome reported from a traveler originating from Japan. We present bioinformatics methods using publicly available tools to help analysts identify the lineage-defining 12 nucleotide insertion (S:Ins16MPLF), which is often masked by most bioinformatics pipelines. We also applied maximum-likelihood and Bayesian phylogenetics to demonstrate the high mutational rate of the tree branch leading to the emergence of BA.2.86, hinting at possible origins, and predict that BA.2.86 emerged around May 2023 and spread globally rapidly. Taken together, these results provide a framework for more rigorous bioinformatics approaches for teams performing genomic surveillance on viral respiratory pathogens.","version":"1.1","doi":"10.1101/2023.09.08.556912","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.08.556870","pub_date":"2023-9-11","title":"SARS-CoV-2 Omicron infection augments the magnitude and durability of systemic and mucosal immunity in triple-dose CoronaVac recipients","abstract":"The inactivated whole-virion vaccine, CoronaVac, is one of the most widely used coronavirus disease 2019 (COVID-19) vaccines worldwide. There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron breakthrough infection. In this prospective cohort study, 41 recipients of triple-dose CoronaVac and 14 unvaccinated individuals were recruited. We comprehensively profiled adaptive immune parameters in both groups, including spike-specific immunoglobulin (Ig) G and IgA titers, neutralizing activity, B cells, follicular helper T (Tfh) cells, CD4+ and CD8+ T cells, and their memory subpopulations at 12 months after the third booster dose and at 4 weeks and 20 weeks after Omicron BA.5 infection. Twelve months after the third CoronaVac vaccination, spike-specific antibody and cellular responses were detectable in most vaccinated individuals. BA.5 infection significantly augmented the magnitude, cross-reactivity and durability of serum neutralization activities, Fc-mediated phagocytosis, and nasal spike-specific IgA responses, memory B cells, activated Tfh cells memory CD4+ T cells, and memory CD8+ T cells for both the ancestral strain and Omicron subvariants, compared to unvaccinated individuals. Notably, the increase in BA.5-specific immunity after breakthrough infection was consistently higher than for the ancestral strain, suggesting no evidence of immune imprinting. Immune landscape analyses showed vaccinated individuals have better synchronization of multiple immune components than unvaccinated individuals upon heterologous SARS-CoV-2 infection. Our data provides detailed insight into the protective role of inactivated COVID-19 vaccine in shaping humoral and cellular immune responses to heterologous Omicron infection. ClinicalTrials.gov NCT05680896 This study was supported by the National Natural Science Foundation of China (92269118, 92269205), Nanjing Important Science & Technology Specific Projects (2021-11005), Scientific Research Project of Jiangsu Health Commission (M2022013), Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2021-LCYJ-PY-9), and Jiangsu graduate practice innovation project (JX22013929).","version":"1.1","doi":"10.1101/2023.09.08.556870","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.11.557161","pub_date":"2023-9-11","title":"The high infectivity of the SARS-CoV-2 Omicron variant is associated with an exclusive S477N spike receptor-binding domain mutation","abstract":"The spike glycoprotein receptor-binding domain (RBD) of SARS-CoV-2 facilitates viral binding to the ACE2 receptor and mediates viral infectivity. The Delta and Omicron variants of concern are the most infectious strains, presenting mutated amino acid residues in their spike RBD. The Omicron variant quickly dominated the COVID-19 pandemic, indicating its greater spreadability. Omicron\u2019s spreading might be associated with mutational substitutions at spike RBD residues. We employed in silico molecular dynamics (MD) simulation of the spike RBD-ACE2 interaction to compare the impact of specific mutations of the Delta and Omicron variants. The MD of the spike-ACE2 interaction showed the following: i) the amino acid profile involved in the spike-ACE2 interaction differs between Delta and Omicron; ii) the Omicron variant establishes several additional interactions, highlighting the spike RBD (S477), which is a flexible mutational residue. Since the S477N mutation is exclusive to Omicron, which may initiate binding with ACE2, the increased infectivity of Omicron might be associated not only with a mutated RBD but also with unmutated (e.g., G476 and L492) residues, initiating binding due to the influence of the N477 mutation. Compared to previous variants, Omicron\u2019s N477 residue represents a novelty within the spike-ACE2 interaction dynamics interface.","version":"1.1","doi":"10.1101/2023.09.11.557161","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.10.557067","pub_date":"2023-9-11","title":"Protocol for primary human lung organoid-derived air-liquid interface in vitro model to study response to SARS-CoV-2","abstract":"This article presents a comprehensive protocol for establishing primary human lung organoid-derived air-liquid interface (ALI) cultures from cryopreserved human lung tissue. These cultures serve as a physiologically relevant model to study human airway epithelium in vitro. The protocol encompasses lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. It also demonstrates SARS-CoV-2 infection in these cultures as an example of their utility. Quality control steps, ALI characterization, and technical readouts for monitoring virus response are included in the study. For additional details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al (https://doi.org/10.1016/j.isci.2023.107374). Human lung tissue dissection, embedding in OCT blocks, and tissue cryopreservation. Thawing & lung tissue dissociation for lung epithelium organoid generation. Organoid-derived air-liquid-interface cultures for the study of viral infection. Bulk RNA-Seq, flow cytometry, viral titer, and imaging to follow response to virus.","version":"1.1","doi":"10.1101/2023.09.10.557067","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.10.557088","pub_date":"2023-9-11","title":"Enhanced Omicron subvariant cross-neutralization efficacy of a monovalent SARS-CoV-2 BA.4/5 mRNA vaccine encoding a noncleaved, nonfusogenic spike antigen","abstract":"The rapid emergence of diverse SARS-CoV-2 variants, notably the Omicron variant, poses challenges to vaccine development. Here, we present a noncleaved, nonfusogenic spike (S) protein eliciting robust B- and T-cell immune responses against Omicron BA.5. The antigen incorporates the R685S and R815A mutations, effectively preventing the shedding of the S1 subunit and eliminating fusogenic activity of the resulting S antigen, termed S(SA). Through reverse genetic analysis, we found that the noncleaved form S protein with the R685S mutation enhances ACE2-dependent viral entry in vitro compared to the wild-type S protein, without increasing the virulence of the mutant virus in mice. The mRNA vaccine encoding the Omicron BA.4/5 S(SA) antigen conferred protective immunity in mice following two doses of 1 \u03bcg \u03a8-UTP- or UTP-incorporated mRNA vaccines. Despite a roughly 6-fold reduction in neutralizing potency, both mRNA vaccines exhibited broad neutralizing efficacy against Omicron subvariants, including the XBB lineage variants XBB.1.5 and XBB.1.16.","version":"1.1","doi":"10.1101/2023.09.10.557088","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.06.23295138","pub_date":"2023-09-08","title":"Quantity of SARS-CoV-2 RNA copies exhaled per minute during natural breathing over the course of COVID-19 infection","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>SARS-CoV-2 is spread through exhaled breath of infected individuals. A fundamental question in understanding transmission of SARS-CoV-2 is how much virus an individual is exhaling into the environment while they breathe, over the course of their infection. Research on viral load dynamics during COVID-19 infection has focused on internal swab specimens, which provide a measure of viral loads inside the respiratory tract, but not on breath. Therefore, the dynamics of viral shedding on exhaled breath over the course of infection are poorly understood. Here, we collected exhaled breath specimens from COVID-19 patients and used RTq-PCR to show that numbers of exhaled SARS-CoV-2 RNA copies during COVID-19 infection do not decrease significantly until day 8 from symptom-onset. COVID-19-positive participants exhaled an average of 80 SARS-CoV-2 viral RNA copies per minute during the first 8 days of infection, with significant variability both between and within individuals, including spikes over 800 copies a minute in some patients. After day 8, there was a steep drop to levels nearing the limit of detection, persisting for up to 20 days. We further found that levels of exhaled viral RNA increased with self-rated symptom-severity, though individual variation was high. Levels of exhaled viral RNA did not differ across age, sex, time of day, vaccination status or viral variant. Our data provide a fine-grained, direct measure of the number of SARS-CoV-2 viral copies exhaled per minute during natural breathing\u2014including 312 breath specimens collected multiple times daily over the course of infection\u2014in order to fill an important gap in our understanding of the time course of exhaled viral loads in COVID-19.</jats:p>","version":null,"doi":"10.1101/2023.09.06.23295138","journal":"medRxiv","score":null},{"id":"10.1101/2023.09.08.556788","pub_date":"2023-9-08","title":"SARS-CoV-2 ORF8 modulates lung inflammation and clinical disease progression","abstract":"The virus severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of the current COVID-19 pandemic. It possesses a large 30 kilobase (kb) genome that encodes structural, non-structural, and accessory proteins. Although not necessary to cause disease, these accessory proteins are known to influence viral replication and pathogenesis. Through the synthesis of novel infectious clones of SARS-CoV-2 that lack one or more of the accessory proteins of the virus, we have found that one of these accessory proteins, ORF8, is critical for the modulation of the host inflammatory response. Mice infected with a SARS-CoV-2 virus lacking ORF8 exhibit increased weight loss and exacerbated macrophage infiltration into the lungs. Additionally, infection of mice with recombinant SARS-CoV-2 viruses encoding ORF8 mutations found in variants of concern reveal that naturally occurring mutations in this protein influence disease severity. Our studies with a virus lacking this ORF8 protein and viruses possessing naturally occurring point mutations in this protein demonstrate that this protein impacts pathogenesis. Since its emergence in 2019, SARS-CoV-2 has accrued mutations throughout its 30kb genome. Of particular interest are the mutations present in the ORF8 protein, which occur in every major variant. The precise function and impact of this protein on disease severity and pathogenesis remains understudies. Our studies reveal that the ORF8 protein modulates the immune response by impacting macrophage infiltration into the lungs. Additionally, we have shown that the ORF8 protein of SARS-CoV-2 has accrued mutations throughout its evolution that lead to a loss of function phenotype in this protein. Our work reveals that the ORF8 protein of SARS-CoV-2 contributes significantly to disease progression through modulation of the inflammatory response.","version":"1.1","doi":"10.1101/2023.09.08.556788","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.13.536758","pub_date":"2023-9-08","title":"Resolving a Guanine-Quadruplex Structure in the SARS-CoV-2 Genome through Circular Dichroism and Multiscale Molecular Modeling","abstract":"The genome of SARS-CoV-2 coronavirus is made up of a single-stranded RNA fragment that can assume a specific secondary structure, whose stability can influence the virus ability to reproduce. Recent studies have identified putative guanine quadruplex sequences in SARS-CoV-2 genome fragments that are involved in coding for both structural and non-structural proteins. In this contribution, we focus on a specific G-rich sequence referred as RG-2, which codes for the non-structural protein 10 (Nsp10) and assumes a guanine-quadruplex (G4) arrangement. We provide the secondary structure of the RG-2 G4 at atomistic resolution by molecular modeling and simulation, validated by the superposition of experimental and calculated electronic circular dichroism spectrum. Through both experimental and simulation approaches, we have demonstrated that pyridostatin (PDS), a widely recognized G4 binder, can bind to and stabilize RG-2 G4 more strongly than RG-1, another G4 forming sequence that was previously proposed as a potential target for antiviral drug candidates. Overall, this study highlights RG-2 as a valuable target to inhibit the translation and replication of SARS-CoV-2 paving the way towards original therapeutic approaches against emerging RNA viruses.","version":"1.3","doi":"10.1101/2023.04.13.536758","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.07.23295190","pub_date":"2023-09-08","title":"The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The severity of COVID-19 is linked to excessive inflammation. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation, but their role in COVID-19 pathophysiology remains poorly understood. We conducted transcriptomic profiling of neutrophils obtained from patients with mild and severe COVID-19, as well as from SARS-CoV-2 infected mice, in comparison to non-infected healthy controls. In addition, we investigated the inflammasome formation potential in neutrophils from patients and mice upon SARS-CoV-2 infection. Transcriptomic analysis of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, revealed a striking type I interferon (IFN-I) gene signature in severe COVID-19 patients, contrasting with mild COVID-19 and healthy controls. Notably, low-density granulocytes (LDGs) from severe COVID-19 patients exhibited an immature neutrophil phenotype and lacked this IFN-I signature. Moreover, PMNs from severe COVID-19 patients showed heightened nigericin-induced caspase1 activation, but reduced responsiveness to exogenous inflammasome priming. Furthermore, IFN-I emerged as a priming stimulus for neutrophil inflammasomes, which was confirmed in a COVID-19 mouse model. These findings underscore the crucial role of neutrophil inflammasomes in driving inflammation during severe COVID-19. Altogether, these findings open promising avenues for targeted therapeutic interventions to mitigate the pathological processes associated with the disease.</jats:p>","version":null,"doi":"10.1101/2023.09.07.23295190","journal":"medRxiv","score":null},{"id":"10.1101/2023.09.07.556634","pub_date":"2023-9-07","title":"The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells","abstract":"The COVID-19 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, and has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. The aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-PCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of \u03b1-globin and \u03b3-globin mRNA accumulation. Inhibition of accumulation of \u03b6-globin and \u03b5-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.","version":"1.1","doi":"10.1101/2023.09.07.556634","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.06.556548","pub_date":"2023-9-07","title":"High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1","abstract":"SARS-CoV-2 variants with undetermined properties have emerged intermittently throughout the COVID-19 pandemic. Some variants possess unique phenotypes and mutations which allow further characterization of viral evolution and spike functions. Around 1100 cases of the B.1.640.1 variant were reported in Africa and Europe between 2021 and 2022, before the expansion of Omicron. Here, we analyzed the biological properties of a B.1.640.1 isolate and its spike. Compared to the ancestral spike, B.1.640.1 carried 14 amino acid substitutions and deletions. B.1.640.1 escaped binding by some anti-NTD and -RBD monoclonal antibodies, and neutralization by sera from convalescent and vaccinated individuals. In cell lines, infection generated large syncytia and a high cytopathic effect. In primary airway cells, B.1.640.1 replicated less than Omicron BA.1 and triggered more syncytia and cell death than other variants. The B.1.640.1 spike was highly fusogenic when expressed alone. This was mediated by two poorly characterized and infrequent mutations located in the spike S2 domain, T859N and D936H. Altogether, our results highlight the cytopathy of a hyper-fusogenic SARS-CoV-2 variant, supplanted upon the emergence of Omicron BA.1. Our results highlight the plasticity of SARS-CoV-2 spike to generate highly fusogenic and cytopathic strains with the causative mutations being uncharacterized in previous variants. We describe mechanisms regulating the formation of syncytia and the subsequent consequences in cell lines and a primary culture model, which are poorly understood.","version":"1.1","doi":"10.1101/2023.09.06.556548","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.06.556547","pub_date":"2023-9-07","title":"SARS-CoV-2 ORF7a mutation found in BF.5 and BF.7 sublineages impacts its functions","abstract":"A feature of the SARS-CoV-2 Omicron subvariants BF.5 and BF.7 that recently circulated mainly in China and Japan was the high prevalence of ORF7a: H47Y mutation. Here we evaluated the effect of this mutation on the three main functions ascribed to SARS-CoV-2 ORF7a protein. Our findings show that H47Y mutation impairs the ability of SARS-CoV-2 ORF7a to antagonize type-I interferon (IFN-I) response and to downregulate Major Histocompatibility Complex-I (MHC-I) cell surface levels, but had no effect in its anti-SERINC5 function. Overall, our results suggest that the H47Y mutation of ORF7a affects important functions of this protein resulting in changes in virus pathogenesis. In late 2021, the Omicron (B.1.1.529) VOC emerged and outcompeted the circulating VOC Delta (B.1.617.2). Soon afterwards, Omicron VOC has expanded and diversified in the world. Among the emerged subvariants of Omicron are BF.5 and BF.7 that are characterized by the presence of a number of mutations across their genome and spread quite effectively across China and other countries later on. One such mutation that was found in the vast majority of isolates of the BF.5 and BF.7 subvariants was ORF7a: H47Y, whose effect on ORF7a is unknown. In this report, we show that H47Y inhibits a number of ORF7a functions, which can potentially affect virus pathogenesis.","version":"1.1","doi":"10.1101/2023.09.06.556547","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508816","pub_date":"2023-9-07","title":"SARS-CoV-2 Sublingual Vaccine with RBD Antigen and Poly(I:C) Adjuvant: Preclinical Study in Cynomolgus Macaques","abstract":"Mucosal vaccine for sublingual route was prepared with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD) antigen and poly(I:C) adjuvant components. The efficacy of this sublingual vaccine was examined using Cynomolgus macaques. Nine of the macaque monkeys were divided into three groups of three animals; control (just 400 \u03bcg poly(I:C) per head); low dose (30 \u03bcg RBD and 400 \u03bcg poly(I:C) per head); and high dose (150 \u03bcg RBD and 400 \u03bcg poly(I:C) per head), respectively. N-acetylcysteine (NAC), a mild reducing agent losing mucin barrier, was used to enhance vaccine delivery to mucosal immune cells. RBD-specific IgA antibody secreted in pituita was detected in two of three monkeys of the high dose group and one of three animals of the low dose group. RBD-specific IgG and/or IgA antibodies in plasma were also detected in these monkeys. These indicated that the sublingual vaccine stimulated mucosal immune response to produce antigen-specific secretory IgA antibodies in pituita and/or saliva. This sublingual vaccine also affected systemic immune response to produce IgG (IgA) in plasma. Little RBD-specific IgE was detected in plasma, suggesting no allergic antigenicity of this sublingual vaccine. Thus, SARS-CoV-2 sublingual vaccine consisting of poly(I:C) adjuvant showed reasonable efficacy in a non-human primate model.","version":"1.3","doi":"10.1101/2022.09.21.508816","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.07.556636","pub_date":"2023-9-07","title":"Transmissibility, infectivity, and immune resistance of the SARS-CoV-2 BA.2.86 variant","abstract":"In September 2023, the SARS-CoV-2 XBB descendants, such as XBB.1.5 and EG.5.1 (originally XBB.1.9.2.5.1), are predominantly circulating worldwide. Unexpectedly, however, a lineage distinct from XBB was identified and named BA.2.86 on August 14, 2023. Notably, BA.2.86 bears more than 30 mutations in the spike (S) protein when compared to XBB and the parental BA.2, and many of them are assumed to be associated with immune evasion. Although the number of reported cases is low (68 sequences have been reported as of 7 September 2023), BA.2.86 has been detected in several continents (Europe, North America and Africa), suggesting that this variant may be spreading silently worldwide. On 17 August 2023, the WHO designated BA.2.86 as a variant under monitoring. Here we show evidence suggesting that BA.2.86 potentially has greater fitness than current circulating XBB variants including EG.5.1. The pseudovirus assay showed that the infectivity of BA.2.86 was significantly lower than that of B.1.1 and EG.5.1, suggesting that the increased fitness of BA.2.86 is not due to the increased infectivity. We then performed a neutralization assay using XBB breakthrough infection sera to address whether BA.2.86 evades the antiviral effect of the humoral immunity induced by XBB subvariants. The 50% neutralization titer of XBB BTI sera against BA.2.86 was significantly (1.6-fold) lower than those against EG.5.1. The sera obtained from individuals vaccinated with 3rd-dose monovalent, 4th-dose monovalent, BA.1 bivalent, and BA.5 bivalent mRNA vaccines exhibited very little or no antiviral effects against BA.2.86. Moreover, the three monoclonal antibodies (Bebtelovimab, Sotrovimab and Cilgavimab), which worked against the parental BA.2, did not exhibit antiviral effects against BA.2.86. These results suggest that BA.2.86 is one of the most highly immune evasive variants ever.","version":"1.1","doi":"10.1101/2023.09.07.556636","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.06.556620","pub_date":"2023-9-07","title":"Immunogenicity and tolerability of a SARS-CoV-2 TNX-1800, a live recombinant poxvirus vaccine candidate, in Syrian Hamsters and New Zealand White Rabbits","abstract":"TNX-1800 is a preclinical stage synthetic derived live chimeric horsepox virus vaccine that comprises an engineered SARS-CoV-2 spike (S) gene expression cassette. The objectives of this study were to assess the immunogenicity and tolerability of TNX-1800 administration in Syrian golden hamsters and New Zealand white rabbits. Animals were vaccinated via percutaneous inoculation and evaluated for dose tolerance and immunogenicity at three different dose levels. The 28-day study data showed that the single percutaneous administration of three TNX-1800 vaccine dose levels was well tolerated in both hamsters and rabbits. For all dose levels, rabbits had more dermal observations than hamsters at the same dose levels. Vaccine-induced viral load four weeks post-dosing was below the detection level for both species.","version":"1.1","doi":"10.1101/2023.09.06.556620","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.339317","pub_date":"2023-9-06","title":"Open Science Discovery of Potent Non-Covalent SARS-CoV-2 Main Protease Inhibitors","abstract":"We report the results of the COVID Moonshot, a fully open-science, crowd sourced, structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a non-covalent, non-peptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>840 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.","version":"1.5","doi":"10.1101/2020.10.29.339317","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.17.496544","pub_date":"2023-9-06","title":"COVFlow: phylodynamics analyses of viruses from selected SARS-CoV-2 genome sequences","abstract":"Phylodynamic analyses can generate important and timely data to optimise public health response to SARS-CoV-2 outbreaks and epidemics. However, their implementation is hampered by the massive amount of sequence data and the difficulty to parameterise dedicated software packages. We introduce the COVFlow pipeline, accessible at https://gitlab.in2p3.fr/ete/CoV-flow, which allows a user to select sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) database according to user-specified criteria, to perform basic phylogenetic analyses, and to produce an XML file to be run in the Beast2 software package. We illustrate the potential of this tool by studying two sets of sequences from the Delta variant in two French regions. This pipeline can facilitate the use of virus sequence data at the local level, for instance, to track the dynamics of a particular lineage or variant in a region of interest.","version":"1.7","doi":"10.1101/2022.06.17.496544","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.04.556164","pub_date":"2023-9-06","title":"Influenza A virus H1N1-derived circNP37 positively regulates viral replication by sponging host miR-361-5p","abstract":"RNA viruses, such as respiratory syncytial virus and SARS-CoV-2, can generate viral circular RNAs (circRNAs), which may play important roles during viral infection. However, whether influenza A viruses have this ability to generate viral circRNAs remains unknown. In this study, we discovered that the negative-strand RNA of the H1N1 nucleoprotein (NP) gene can generate a circRNA, designated circNP37. Furthermore, we demonstrated that circNP37 positively regulated viral replication by competitively sponging host miR-361-5p which inhibited polymerase basic protein 2 (PB2) expression. These results were confirmed using in vivo experiments. Compared with wild-type virus, infection with circNP37 knockout virus resulted in a reduced viral load in the lungs. This study demonstrates, for the first time, the existence and biological function of H1N1-derived circNP37. These findings help us better understand the mechanisms of influenza virus replication and pathogenicity. Negative-strand RNA of the H1N1 nucleoprotein gene can generate a circRNA CircNP37 plays important roles in viral replication and viral-host interactions CircNP37 positively regulates replication by competitively sponging host miR-361-5p In this study, we found that influenza A virus H1N1 infection can generate virus-derived circRNA, circNP37. We also demonstrated that circNP37 positively regulate viral PB2 gene expression and viral replication via sponge host miR-361-5p during viral infection.","version":"1.1","doi":"10.1101/2023.09.04.556164","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.05.556326","pub_date":"2023-9-05","title":"Deep unsupervised learning methods for the identification and characterization of TCR specificity to Sars-Cov-2","abstract":"The T-cell receptor (TCR) is one of the key players in the immune response to the Sars-Cov-2 virus. In this study, we used deep unsu-pervised learning methods to identify and characterize TCR speci-ficity. Our research focused on developing and applying state-of-the-art modelling techniques, including AutoEncoders, Variational Au-to Encoders and transfer learning with Transformers, to analyze TCR data. Through our experiments and analyses, we have achieved promis-ing results in identifying TCR patterns and understanding TCR speci-ficity for Sars-Cov-2. The insights gained from our research provide valuable tools and knowledge for interpreting the immunological re-sponse to the virus, ultimately contributing to the development of effective vaccines and treatments against the viral infection.","version":"1.1","doi":"10.1101/2023.09.05.556326","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.23.521761","pub_date":"2023-9-05","title":"Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16","abstract":"The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5\u2019 end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2\u2019-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the host\u2019s innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets. The SARS-CoV-2 proteins have constantly been evolving. These variants assist the virus to survive, adapt and evade the host immune responses. While the main focus has been on structural proteins like Spike, there is very limited structural and functional information on the effects of emerging mutations on other essential non-structural viral proteins. One such protein is NSP10, an essential cofactor for NSP14 and NSP16. This study demonstrates that NSP10 is more resistant to genetic variations than other SARS-CoV-2 non-structural proteins and that the presence of mutations conserve structural and dynamic changes in NSP10. The effects of naturally occurring mutations reflect the evolutionary relationship between structurally conserved essential cofactors, their function and the role they play in the survival of the virus.","version":"1.3","doi":"10.1101/2022.12.23.521761","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.19.533338","pub_date":"2023-9-05","title":"Enhanced protective efficacy of a novel, thermostable, RBD-S2 vaccine formulation against SARS-CoV-2 and its variants","abstract":"With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 \u00b0C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2023.03.19.533338","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.04.556272","pub_date":"2023-9-05","title":"Neutralization Escape by SARS-CoV-2 Omicron Subvariant BA.2.86","abstract":"The continued evolution of SARS-CoV-2 may lead to evasion of vaccine immunity and natural immunity. A highly mutated Omicron variant BA.2.86 has recently been identified with over 30 amino acid changes in Spike compared with BA.2 and XBB.1.5. As of September 4, 2023, BA.2.86 has been identified in 37 sequences from 10 countries, which is likely an underestimate due to limited surveillance. The ability of BA.2.86 to evade NAbs compared with other currently circulating Omicron variants remains unknown. Our data show that NAb responses to BA.2.86 were lower than to BA.2 but were comparable or slightly higher than to the current circulating recombinant variants XBB.1.5, XBB.1.16, EG.5, EG.5.1, and FL.1.5.1.","version":"1.1","doi":"10.1101/2023.09.04.556272","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.04.556192","pub_date":"2023-9-05","title":"Correlation of myeloid-derived suppressor cell expansion with upregulated transposable elements in severe COVID-19 unveiled in single-cell RNA sequencing reanalysis","abstract":"Some studies investigated the potential role of transposable elements (TEs) in COVID-19 pathogenesis and complications. However, to the best of our knowledge, there is no study to examine the possible association of TEs expression in cell functions and its potential role in COVID-19 immune response at the single-cell level. In this study, we reanalyzed single-cell RNA seq data of bronchoalveolar lavage (BAL) samples obtained from six severe COVID-19 patients and three healthy donors to assess the probable correlation of TE expression with the immune responses induced by the SARS-CoV-2 virus in COVID-19 patients. Our findings indicated that the expansion of myeloid-derived suppressor cells (MDSCs) may be a characteristic feature of COVID-19. Additionally, a significant increase in TEs expression in MDSCs was observed. This upregulation of TEs in COVID-19 may be linked to the adaptability of these cells in response to their microenvironments. Furthermore, it appears that the identification of overexpressed TEs by Pattern recognition receptors (PRRs) in MDSCs may enhance the suppressive capacity of these cells. Thus, this study emphasizes the crucial role of TEs in the functionality of MDSCs during COVID-19.","version":"1.1","doi":"10.1101/2023.09.04.556192","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.02.556038","pub_date":"2023-9-04","title":"Neutralization of SARS-CoV-2 EG.5/EG.5.1 by sera from ZF2001 RBD-dimer and its next-generation vaccines","abstract":"SARS-CoV-2 Omicron EG.5 and EG.5.1 are surging in several areas of the world, including China. Compared with XBB.1, EG.5 contains additional mutations of F456L and S486P in the spike protein receptor binding domain (RBD) and its subvariant EG.5.1 carries a further spike mutation Q52H. The immune escape potential of EG.5/EG.5.1 is of great concern. In this study, we evaluated the neutralization activities of sera from participants who received COVID-19 inactivated vaccines, protein subunit vaccine ZF2001 or a booster vaccination of Delta-BA.5 RBD-heterodimer protein vaccine, and participants who had a breakthrough infection during a wave of BF.7/BA.5.2 circulation in December 2022. Neutralization profiles elicited by bivalent RBD-heterodimer vaccine candidates containing XBB.1.5 antigen were evaluated in a murine model. We found that EG.5 and EG.5.1 displayed similar immune evasion potential to XBB.1 and XBB.1.5. The Delta-BA.5 RBD-heterodimer booster induced higher neutralizing titers against the tested XBB subvariants, including EG.5 and EG.5.1, than breakthrough infection by BF.7 or BA.5.2. In addition, Delta-XBB.1.5 and BQ.1.1-XBB.1.5 RBD-heterodimer vaccines induced high neutralizing activities against XBB sub-variants in a murine model, suggesting that next-generation COVID-19 vaccines with updated components must be developed to enhance the protection efficacy against the circulating SARS-CoV-2 strains.","version":"1.1","doi":"10.1101/2023.09.02.556038","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.01.555815","pub_date":"2023-9-04","title":"Antigenicity and infectivity characterization of SARS-CoV-2 BA.2.86","abstract":"The recently identified SARS-CoV-2 variant, BA.2.86, which carries a substantial number of Spike mutations, has raised a global alarm. An immediate assessment of its antigenic properties and infectivity is necessary. Here, we reveal the distinct antigenicity of BA.2.86 compared with previous variants including XBB.1.5. BA.2.86 significantly evades convalescent plasma from XBB breakthrough infection (BTI) and reinfections. Key mutations that mediate the enhanced resistance include N450D, K356T, L452W, A484K, V483del, and V445H on the RBD, while BA.2.86\u2019s NTD mutations and E554K on SD1 also largely contribute. However, we found that BA.2.86 pseudovirus exhibits compromised efficiency of infecting HEK293T-hACE2 cells compared to XBB.1.5 and EG.5, which may be caused by K356T, V483del, and E554K, and could potentially limit BA.2.86\u2019s transmissibility. In sum, it appears that BA.2.86 has traded its infectivity for higher immune evasion during long-term host-viral evolution. Close attention should be paid to monitoring additional mutations that could improve BA.2.86\u2019s infectivity.","version":"1.1","doi":"10.1101/2023.09.01.555815","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.01.555996","pub_date":"2023-9-04","title":"Trapping non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase","abstract":"The RNA dependent RNA polymerase (RdRp) in SARS-CoV-2 is a highly conserved enzyme responsible for viral genome replication/transcription. Here we investigate computationally natural non-cognate vs cognate nucleotide addition cycle (NAC) and intrinsic nucleotide selectivity during the viral RdRp elongation, focusing prechemically from initial nucleotide substrate binding (enzyme active site open) to insertion (active site closed) of RdRp in contrast with one-step only substrate binding process. Current studies have been first carried out using microsecond ensemble equilibrium all-atom molecular dynamics (MD) simulations. Due to slow conformational changes (from the open to closed) accompanying nucleotide insertion and selection, enhanced or umbrella sampling methods have been further employed to calculate free energy profiles of the non-cognate NTP insertion. Our studies show notable stability of noncognate dATP and GTP upon initial binding in the active-site open state. The results indicate that while natural cognate ATP and Remdesivir drug analogue (RDV-TP) are biased to be stabilized in the closed or insertion state, the natural non-cognate dATP and GTP can be well trapped in off-path initial binding configurations. Current work thus presents an intrinsic nucleotide selectivity mechanism of SARS-CoV-2 RdRp for natural substrate fidelity control in viral genome replication.","version":"1.1","doi":"10.1101/2023.09.01.555996","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.02.556033","pub_date":"2023-9-04","title":"Sensitivity of BA.2.86 to prevailing neutralising antibody responses","abstract":"A new SARS-CoV-2 variant, designated BA.2.86, has recently emerged with over 30 spike mutations relative to its parental BA.2, raising questions about its degree of resistance to neutralising antibodies. Using a spike-pseudotyped virus model we characterise neutralisation of BA.2.86 by clinically relevant monoclonal antibodies and by two cohorts of serum sampled from Stockholm, including both a recent cohort, and one sampled prior to the arrival of XBB in Sweden.","version":"1.1","doi":"10.1101/2023.09.02.556033","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.02.556069","pub_date":"2023-9-04","title":"Multi-omics profiling reveals phenotypic and functional heterogeneity of neutrophils in COVID-19","abstract":"Accumulating evidence has revealed unexpected phenotypic heterogeneity and diverse functions of neutrophils in several diseases. Coronavirus disease (COVID-19) can alter the leukocyte phenotype based on disease severity, including neutrophil activation in severe cases. However, the plasticity of neutrophil phenotypes and their relative impact on COVID-19 pathogenesis has not been well addressed. This study aimed to identify and validate the heterogeneity of neutrophils in COVID-19 and evaluate the phenotypic alterations for each subpopulation. We analyzed public single-cell RNA-seq, bulk RNA-seq, and human plasma proteome data from healthy donors and patients with COVID-19 to investigate neutrophil subpopulations and their response to disease pathogenesis. We identified eight neutrophil subtypes, namely C1\u2013C8, and found that they exhibited distinct features, including activation signatures and multiple enriched pathways. The neutrophil subtype C4 (DEFA1/1B/3+) associated with severe and fatal disease. Bulk RNA-seq and proteome dataset analyses using a cellular deconvolution approach validated the relative abundances of neutrophil subtypes and the expansion of C4 (DEFA1/1B/3+) in severe COVID-19 patients. Cell\u2013 cell communication analysis revealed representative ligand-receptor interactions among the identified neutrophil subtypes. Notably, the C4 (DEFA1/1B/3+) fraction showed transmembrane receptor expression of CD45 and CAP1 as well as the secretion of pro-platelet basic protein (PPBP). We further demonstrated the clinical potential of PPBP as a novel diagnostic biomarker for severe COVID-19. Our work has great value in terms of both clinical and public health as it furthers our understanding of the phenotypic and functional heterogeneity of neutrophils and other cell populations in multiple diseases.","version":"1.1","doi":"10.1101/2023.09.02.556069","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.01.555992","pub_date":"2023-9-02","title":"Therapeutic mitigation of measles-like immune amnesia and exacerbated disease after prior respiratory virus infections in ferrets","abstract":"After years of the COVID-19 pandemic, over 40 million children worldwide are at risk of measles due to delayed vaccination and temporary SARS-CoV-2 viral dominance. Acute measles has a case-fatality rate of \u223c1%, but most morbidity and mortality arise post-measles due to destruction of pre-existing immune memory by lymphotropic measles virus (MeV), a paramyxovirus of the Morbillivirus genus. MeV-induced immune amnesia is not mitigated by post-exposure vaccination and the impact of unrelated respiratory virus disease history on measles severity has not been defined. We used a lethal canine distemper virus (CDV)-ferret model as surrogate for human morbillivirus disease and employed the orally efficacious broad-spectrum paramyxovirus polymerase inhibitor GHP-88309 to establish measles treatment paradigms. Applying a receptor tropism-intact recombinant CDV with low lethality, we provide in vivo confirmation of the morbillivirus immune amnesia hypothesis and reveal an 8-day advantage of antiviral treatment versus therapeutic vaccination in preserving immune memory. Infection of ferrets with non-lethal influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks prior to CDV caused exacerbated CDV disease that rapidly advanced to fatal hemorrhagic pneumonia associated with lung onslaught by commensal bacteria. RNAseq of BAL samples and lung tissue identified CDV-induced expression of trefoil factor (TFF) peptides, which was absent in animals pre-infected with IAV, thus highlighting that immune priming by unrelated respiratory viruses influences morbillivirus infection outcome. Non-invasive pulmonary ferret MRI revealed that severe outcomes of consecutive IAV/CDV infections were prevented by oral GHP-88309 treatment even when initiated after peak clinical signs of CDV. These findings validate the morbillivirus immune amnesia hypothesis, define treatment paradigms for measles, identify prior disease history as risk factor for exacerbated morbillivirus disease, and demonstrate that treating morbillivirus infection with direct-acting oral antivirals provides therapeutic benefit regardless of whether the time window to mitigate primary clinical signs of infection has closed.","version":"1.1","doi":"10.1101/2023.09.01.555992","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.31.555625","pub_date":"2023-9-01","title":"Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53","abstract":"COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.08.31.555625","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.31.555800","pub_date":"2023-9-01","title":"Characterization of low copy number human angiotensin-converting enzyme 2 (hACE2)-transgenic mice as an improved model of SARS-CoV-2 infection","abstract":"Coronaviridae are significant human pathogens, as evidenced by several outbreaks of severe respiratory infections in the past 20 years and culminating with the COVID-19 pandemic. Mouse models of COVID-19 have included transgenic expression of the main SARS coronavirus entry receptor on human cells, human angiotensin-converting enzyme 2 (hACE2). However, the original hACE2-Tg mouse strain overexpresses many copies of the transgene, leading to neuropathology not representative of human infection. Aiming to improve physiological relevance, we generated two new lines of hACE2-Tg mice using the original transgene construct expressing hACE2 under the control of the keratin 18 promoter (K18-hACE2). We show that relative to the original strain, which expressed 8 copies of the transgene (8-hACE2-Tg), lines 1 and 2 expressed 1 and 2 copies of the transgene (1-hACE-2-Tg and 2-hACE-2-Tg, respectively). Upon intranasal (i.n.) infection with 103 plaque-forming units (pfu) SARS-CoV-2 WA-1/US, 8-hACE2-Tg mice succumbed to infection by d. 7. 2-hACE2-Tg mice exhibited 31% survival, with less viral replication in the lung and brain when compared to 8-hACE2-Tg mice. Furthermore, SARS-CoV-2 infection in 1-hACE2-Tg mice exhibited no mortality and had no detectable virus in the brain, although they did show clear virus replication in the lung. All three mouse strains analyzed showed SARS-CoV-2-related weight loss that tracked with the mortality rates. 1-hACE2-Tg mice mounted detectable primary and memory T effector cell and antibody responses. We conclude that these strains, particularly 1-hACE2-Tg mice, provide improved models to study hACE2-mediated viral infections.","version":"1.1","doi":"10.1101/2023.08.31.555800","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.01.555899","pub_date":"2023-9-01","title":"SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection","abstract":"Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.","version":"1.1","doi":"10.1101/2023.09.01.555899","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.31.555805","pub_date":"2023-9-01","title":"Immunogenicity and efficacy of a subcutaneously administered, adjuvanted vaccine containing modified S1 spike protein of SARS-CoV-2 variant C.1.2","abstract":"During the COVID-19 pandemic, vaccines have produced protective immunity sufficient enough to cause a decrease in hospitalizations and deaths; however, the pandemic continues due to mutational events, predominantly occurring in the S1 sequence of the spike protein of SARS-CoV-2. We have developed a baculovirus-expressed, modified S1 SARS-CoV-2 protein based on the C.1.2 variant, which was first identified in South Africa. This was encapsulated in a vitamin E containing, nonphospholipid liposome, which was then used to subcutaneously immunize Syrian hamsters. This vaccine, when administered at day 1 generates IgG responses that react to the modified C.1.2 S1 protein; full-length spike proteins from Wuhan-Hu-1, Delta, Omicron BA.1; and the Omicron recombinant variant XBB.1.5 in 100% of the animals. The second dose administered subcutaneously on day 28 demonstrated anamnestic response in the quantitative IgG assay to the Wuhan-Hu-1 spike Receptor Binding Domain (RBD). In addition, antibody IgA and IgM responses in sera were demonstrated. Serum IgG antibody responses to the spike proteins of the modified C.1.2 S1 and full-length spike proteins Wuhan-Hu-1, Delta, Omicron BA.1, and Omicron recombinant XBB.1.5 variants are elevated for over 120 days. Challenge of vaccinated and unvaccinated hamsters at day 126 of the study with an Omicron BA.1 resulted in a difference in weight change and viral load based on the qRT-PCR assay seven days after challenge.","version":"1.1","doi":"10.1101/2023.08.31.555805","journal":"bioRxiv","score":null},{"id":"10.1101/2023.09.01.555834","pub_date":"2023-9-01","title":"SARS-CoV-2 Spike amyloid fibrils specifically and selectively accelerates amyloid fibril formation of human prion protein and the amyloid \u03b2 peptide","abstract":"An increasing number of reports suggest an association between COVID-19 infection and initiation or acceleration of neurodegenerative diseases (NDs) including Alzheimer\u2019s disease (AD) and Creutzfeldt-Jakob disease (CJD). Both these diseases and several other NDs are caused by conversion of human proteins into a misfolded, aggregated amyloid fibril state. The fibril formation process is self-perpetuating by seeded conversion from preformed fibril seeds. We recently described a plausible mechanism for amyloid fibril formation of SARS-CoV-2 spike protein. Spike-protein formed amyloid fibrils upon cleavage by neutrophil elastase, abundant in the inflammatory response to COVID-19 infection. We here provide evidence of significant Spike-amyloid fibril seeded acceleration of amyloid formation of CJD associated human prion protein (HuPrP) using an in vitro conversion assay. By seeding the HuPrP conversion assay with other in vitro generated disease associated amyloid fibrils we demonstrate that this is not a general effect but a specific feature of spike-amyloid fibrils. We also showed that the amyloid fibril formation of AD associated A\u03b21-42 was accelerated by Spike-amyloid fibril seeds. Of seven different 20-amino acid long peptides, Spike532 (532NLVKNKCVNFNFNGLTGTGV551) was most efficient in seeding HuPrP and Spike601 (601GTNTSNQVAVLYQDVNCTEV620) was most effective in seeding A\u03b21-42, suggesting substrate dependent selectivity of the cross-seeding activity. Albeit purely in vitro, our data suggest that cross-seeding by Spike-amyloid fibrils can be implicated in the increasing number of reports of CJD, AD, and possibly other NDs in the wake of COVID-19.","version":"1.1","doi":"10.1101/2023.09.01.555834","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.31.555772","pub_date":"2023-9-01","title":"Sequential early-life viral infections modulate the microbiota and adaptive immune responses to systemic and mucosal vaccination","abstract":"Increasing evidence points to the microbial exposome as a critical factor in maturing and shaping the host immune system, thereby influencing responses to immune challenges such as infections or vaccines. To investigate the effect of early-life viral exposures on immune development and vaccine responses, we inoculated mice with six distinct viral pathogens in sequence beginning in the neonatal period, and then evaluated their immune signatures before and after intramuscular or intranasal vaccination against SARS-CoV-2. Sequential viral infection drove profound changes in all aspects of the immune system, including increasing circulating leukocytes, altering innate and adaptive immune cell lineages in tissues, and markedly influencing serum cytokine and total antibody levels. Beyond these immune responses changes, these exposures also modulated the composition of the endogenous intestinal microbiota. Although sequentially-infected mice exhibited increased systemic immune activation and T cell responses after intramuscular and intranasal SARS-CoV-2 immunization, we observed decreased vaccine-induced antibody responses in these animals. These results suggest that early-life viral exposures are sufficient to diminish antibody responses to vaccination in mice, and highlight their potential importance of considering prior microbial exposures when investigating vaccine responses.","version":"1.1","doi":"10.1101/2023.08.31.555772","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.30.555493","pub_date":"2023-8-31","title":"Epidemiological modeling of SARS-CoV-2 in white-tailed deer (Odocoileus virginianus) reveals conditions for introduction and widespread transmission","abstract":"Emerging infectious diseases with zoonotic potential often have complex socioecological dynamics and limited ecological data, requiring integration of epidemiological modeling with surveillance. Although our understanding of SARS-CoV-2 has advanced considerably since its detection in late 2019, the factors influencing its introduction and transmission in wildlife hosts, particularly white-tailed deer (Odocoileus virginianus), remain poorly understood. We use a Susceptible-Infected-Recovered-Susceptible epidemiological model to investigate the spillover risk and transmission dynamics of SARS-CoV-2 in wild and captive white-tailed deer populations across various simulated scenarios. We found that captive scenarios pose a higher risk of SARS-CoV-2 introduction from humans into deer herds and subsequent transmission among deer, compared to wild herds. However, even in wild herds, the transmission risk is often substantial enough to sustain infections. Furthermore, we demonstrate that the strength of introduction from humans influences outbreak characteristics only to a certain extent. Transmission among deer was frequently sufficient for widespread outbreaks in deer populations, regardless of the initial level of introduction. We also explore the potential for fence line interactions between captive and wild deer to elevate outbreak metrics in wild herds that have the lowest risk of introduction and sustained transmission. Our results indicate that SARS-CoV-2 could be introduced and maintained in deer herds across a range of circumstances based on testing a range of introduction and transmission risks in various captive and wild scenarios. Our approach and findings will aid One Health strategies that mitigate persistent SARS-CoV-2 outbreaks in white-tailed deer populations and potential spillback to humans.","version":"1.1","doi":"10.1101/2023.08.30.555493","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.30.554497","pub_date":"2023-8-31","title":"XBB.1.5 Spike Protein COVID-19 Vaccine Induces Broadly Neutralizing and Cellular Immune Responses Against EG.5.1 and Emerging XBB Variants","abstract":"Monovalent SARS-CoV-2 Prototype (Wuhan-Hu-1) and bivalent (Prototype + BA.4/5) COVID-19 vaccines have demonstrated a waning of vaccine-mediated immunity highlighted by lower neutralizing antibody responses against SARS-CoV-2 Omicron XBB sub-variants. The reduction of humoral immunity due to the rapid evolution of SARS-CoV-2 has signaled the need for an update to vaccine composition. A strain change for all authorized/approved vaccines to a monovalent composition with Omicron subvariant XBB.1.5 has been supported by the WHO, EMA, and FDA. Here, we demonstrate that immunization with a monovalent recombinant spike protein COVID-19 vaccine (Novavax, Inc.) based on the subvariant XBB.1.5 induces cross-neutralizing antibodies against XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1, and XBB.1.16.6 subvariants, promotes higher pseudovirus neutralizing antibody titers than bivalent (Prototype + XBB.1.5) vaccine, induces SARS-CoV-2 spike-specific Th1-biased CD4+ T-cell responses against XBB subvariants, and robustly boosts antibody responses in mice and nonhuman primates primed with a variety of monovalent and bivalent vaccines. Together, these data support updating the Novavax vaccine to a monovalent XBB.1.5 formulation for the 2023-2024 COVID-19 vaccination campaign.","version":"1.1","doi":"10.1101/2023.08.30.554497","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.29.555368","pub_date":"2023-8-30","title":"SARS-CoV-2 RNA Persists in the Central Nervous System of Non-Human Primates Despite Clinical Recovery","abstract":"Adverse neurological and psychiatric outcomes, collectively termed the post-acute sequelae of SARS-CoV-2 infection (PASC), persist in adults clinically recovered from COVID-19. Effective therapeutic interventions are fundamental to reducing the burden of PASC, necessitating an investigation of the pathophysiology underlying the debilitating neurological symptoms associated with the condition. Herein, eight non-human primates (Wild-Caught African Green Monkeys, n=4; Indian Rhesus Macaques, n=4) were inoculated with the SARS-CoV-2 isolate USA-WA1/2020 by either small particle aerosol or via multiple routes. At necropsy, tissue from the olfactory epithelium and pyriform cortex/amygdala of SARS-CoV-2 infected non-human primates were collected for ribonucleic acid in situ hybridization (i.e., RNAscope). First, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) mRNA are downregulated in the pyriform cortex/amygdala of non-human primates clinically recovered from SARS-CoV-2 inoculation relative to wildtype controls. Second, abundant SARS-CoV-2 mRNA was detected in clinically recovered non-human primates; mRNA which is predominantly harbored in pericytes. Collectively, examination of post-mortem pyriform cortex/amygdala brain tissue of non-human primates clinically recovered from SARS-CoV-2 infection revealed two early pathophysiological mechanisms potentially underlying PASC. Indeed, therapeutic interventions targeting the downregulation of ACE2, decreased expression of TMPRSS2, and/or persistent infection of pericytes in the central nervous system may effectively mitigate the debilitating symptoms of PASC.","version":"1.1","doi":"10.1101/2023.08.29.555368","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.30.555188","pub_date":"2023-8-30","title":"Immune Evasion and Membrane Fusion of SARS-CoV-2 XBB Subvariants EG.5.1 and XBB.2.3","abstract":"Immune evasion by SARS-CoV-2 paired with immune imprinting from monovalent mRNA vaccines has resulted in attenuated neutralizing antibody responses against Omicron subvariants. In this study, we characterized two new XBB variants rising in circulation \u2014 EG.5.1 and XBB.2.3, for their ability of neutralization and syncytia formation. We determined the neutralizing antibody in sera of individuals that received a bivalent mRNA vaccine booster, BA.4/5-wave infection, or XBB.1.5-wave infection. Bivalent vaccination-induced antibodies neutralized efficiently ancestral D614G, but to a much less extent, two new EG.5.1 and XBB.2.3 variants. In fact, the enhanced neutralization escape of EG.5.1 appeared to be driven by its key defining mutation XBB.1.5-F456L. Notably, infection by BA.4/5 or XBB.1.5 afforded little, if any, neutralization against EG.5.1, XBB.2.3 and previous XBB variants \u2014 especially in unvaccinated individuals, with average neutralizing antibody titers near the limit of detection. Additionally, we investigated the infectivity, fusion activity, and processing of variant spikes for EG.5.1 and XBB.2.3 in HEK293T-ACE2 and CaLu-3 cells but found no significant differences compared to earlier XBB variants. Overall, our findings highlight the continued immune evasion of new Omicron subvariants and, more importantly, the need to reformulate mRNA vaccines to include XBB spikes for better protection.","version":"1.1","doi":"10.1101/2023.08.30.555188","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.29.555437","pub_date":"2023-8-30","title":"Molecular Property Diagnostic Suite for COVID-19 (MPDSCOVID-19): An open access disease specific drug discovery portal","abstract":"Computational drug discovery is intrinsically interdisciplinary and has to deal with the multifarious factors which are often dependent on the type of disease. Molecular Property Diagnostic Suite (MPDS) is a Galaxy based web portal which was conceived and developed as a disease specific web portal, originally developed for tuberculosis (MPDSTB). As specific computational tools are often required for a given disease, developing a disease specific web portal is highly desirable. This paper emphasises on the development of the customised web portal for COVID-19 infection and is referred to as MPDSCOVID-19. Expectedly, the MPDS suites of programs have modules which are essentially independent of a given disease, whereas some modules are specific to a particular disease. In the MPDSCOVID-19 portal, there are modules which are specific to COVID-19, and these are clubbed in SARS-COV-2 disease library. Further, the new additions and/or significant improvements were made to the disease independent modules, besides the addition of tools from galaxy toolshed. This manuscript provides a latest update on the disease independent modules of MPDS after almost 6 years, as well as provide the contemporary information and tool-shed necessary to engage in the drug discovery research of COVID-19. The disease independent modules include file format converter and descriptor calculation under the data processing module; QSAR, pharmacophore, scaffold analysis, active site analysis, docking, screening, drug repurposing tool, virtual screening, visualisation, sequence alignment, phylogenetic analysis under the data analysis module; and various machine learning packages, algorithms and in-house developed machine learning antiviral prediction model are available. The MPDS suite of programs are expected to bring a paradigm shift in computational drug discovery, especially in the academic community, guided through a transparent and open innovation approach. The MPDSCOVID-19 can be accessed at http://mpds.neist.res.in:8085.","version":"1.1","doi":"10.1101/2023.08.29.555437","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.27.530188","pub_date":"2023-8-30","title":"Jet injection potentiates naked mRNA SARS-CoV-2 vaccine in mice and non-human primates by adding physical stress to the skin","abstract":"Naked mRNA-based vaccines may reduce the reactogenicity associated with delivery carriers, but their effectiveness has been suboptimal against infectious diseases. Herein, we aimed to enhance their efficacy by using a pyro-drive liquid jet injector that precisely controls pressure to widely disperse mRNA solution in the skin. The jet injection boosted naked mRNA delivery efficiency in the mouse skin. Mechanistic analyses indicate that dendritic cells, upon uptake of antigen mRNA in the skin, migrate to the draining lymph nodes for antigen presentation. Additionally, the jet injector activated innate immune responses in the skin, presumably by inducing physical stress, thus serving as a physical adjuvant. From a safety perspective, our approach, utilizing naked mRNA, restricted mRNA distribution solely to the injection site, preventing systemic pro-inflammatory reactions following vaccination. Ultimately, the jet injection of naked mRNA encoding SARS-CoV-2 spike protein elicited robust humoral and cellular immunity, providing protection against SARS-CoV-2 infection in mice. Furthermore, our approach induced plasma activity of neutralizing SARS-CoV-2 in non-human primates, comparable to that observed in mice, with no detectable systemic reactogenicity.","version":"1.2","doi":"10.1101/2023.02.27.530188","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.28.555120","pub_date":"2023-8-29","title":"Age-Associated Weaker Immunity to Coronaviruses is Characteristic of Children that Develop Multisystem Inflammatory Syndrome following SARS-CoV-2 Infection","abstract":"We analyzed the antibody and cytokine responses of twenty-three patients with multisystem inflammatory syndrome of children (MIS-C) that appeared with a three-to-six-week delay following a mild or asymptomatic SARS-CoV-2 infection. These responses were compared to healthy convalescent pediatric COVID-19 patients approximately twenty-eight days after the onset of symptoms. Both groups had strong IgG responses to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, but the MIS-C patients had weaker antibody responses to certain epitopes in the SARS-CoV-2 S and N proteins and to the S and N proteins of endemic human coronaviruses (HCoV) compared to pediatric convalescent COVID patients. HCoV antibody reactivity was correlated with age. In contrast, MIS-C patients had elevated serum levels of several proinflammatory cytokines compared to convalescent COVID patients, including interleukins IL-6, IL-8, IL-18 and chemokines CCL2, CCL8, CXCL5, CXCL9 and CXCL10 as well as tumor necrosis factor alpha and interferon gamma. Moreover, many cytokine responses of MIS-C patients were positively correlated with antibody responses to the SARS-CoV-2 S, N, membrane and ORF3a proteins while pediatric convalescent COVID patient cytokine responses were more often negatively correlated with antibody responses to the S, N and ORF3a proteins of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.08.28.555120","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.28.554806","pub_date":"2023-8-29","title":"Chlorpheniramine Maleate Displays Multiple Modes of Antiviral Action Against SARS-CoV-2: A Mechanistic Study","abstract":"Chlorpheniramine Maleate (CPM) has been identified as a potential antiviral compound against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In this study, we investigated the in vitro effects of CPM on key stages of the SARS-CoV-2 replication cycle, including viral adsorption, replication inhibition, and virucidal activity. Our findings demonstrate that CPM exhibits antiviral properties by interfering with viral adsorption, replication, and directly inactivating the virus. Molecular docking analysis revealed interactions between CPM and essential viral proteins, such as the main protease receptor, spike protein receptor, and RNA polymerase. CPM\u2019s interactions were primarily hydrophobic in nature, with an additional hydrogen bond formation in the RNA polymerase active site. These results suggest that CPM has the potential to serve as a multitarget antiviral agent against SARS-CoV-2 and potentially other respiratory viruses. Further investigations are warranted to explore its clinical implications and assess its efficacy in vivo.","version":"1.1","doi":"10.1101/2023.08.28.554806","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.29.555304","pub_date":"2023-8-29","title":"A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ Binding Motif to the cytoplasm","abstract":"The carboxy terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by E C-terminal specific antibodies neither by PDZ domain-containing protein binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.","version":"1.1","doi":"10.1101/2023.08.29.555304","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.24.550423","pub_date":"2023-8-29","title":"Predictive Systems Biology Modeling: Unraveling Host Metabolic Disruptions and Potential Drug Targets in Acute Viral Infections","abstract":"Host response is critical to the onset, progression, and outcome of viral infections. Since viruses hijack the host cellular metabolism for their replications, we hypothesized that restoring host cell metabolism can efficiently reduce viral production. Here, we present a viral-host Metabolic Modeling (vhMM) method to systematically evaluate the disturbances in host metabolism in viral infection and computationally identify targets for modulation by integrating genome-wide precision metabolic modeling and cheminformatics. We applied vhMM to SARS-CoV-2 infections and identified consistent changes in host metabolism and gene and endogenous metabolite targets between the original SARS-COV-2 and different variants (Alpha, Delta, and Omicron). Among six compounds predicted for repurposing, methotrexate, cinnamaldehyde, and deferiprone were tested in vitro and effective in inhibiting viral production with IC50 less than 4uM. Further, an analysis of real-world patient data showed that cinnamon usage significantly reduced the SARS-CoV-2 infection rate with an odds ratio of 0.65 [95%CI: 0.55\u223c0.75]. These results demonstrated that vhMM is an efficient method for predicting targets and drugs for viral infections.","version":"1.2","doi":"10.1101/2023.07.24.550423","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.25.554813","pub_date":"2023-8-28","title":"In Vivo Antiviral Efficacy of LCTG-002, a Pooled, Purified Human Milk Secretory IgA product, Against SARS-CoV-2 in a Murine Model of COVID-19","abstract":"Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosal compartments including the respiratory tract, with the secretory form of IgA (sIgA) being dominant and uniquely stable in these environments. sIgA is naturally found in human milk, which could be considered a global resource for this biologic, justifying the development of human milk sIgA as a dedicated airway therapeutic for respiratory infections such as SARS-CoV-2. In the present study, methods were therefore developed to efficiently extract human milk sIgA from donors who were either immunologically na\u00efve to SARS-CoV-2 (pooled as a control IgA) or had recovered from a PCR-confirmed SARS-CoV-2 infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA Abs in their milk (pooled together to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1.0% or greater were all associated with sIgA. None of the proteins exhibited statistically significant differences between batches. Western blot demonstrated all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher binding to Spike, and was also capable of blocking the Spike - ACE2 interaction in vitro with 6.3x greater potency compared to control IgA (58% inhibition at \u223c240ug/mL). LCTG-002 was then tested in vivo for its capacity to reduce viral burden in the lungs of K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 was demonstrated to significantly reduce SARS-CoV-2 titers in the lungs compared to control IgA when administered at either 250ug/day or 1 mg/day, as measured by TCID50, plaque forming units (PFU), and qRT-PCR, with a maximum reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure, efficacious, and well tolerated in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics against SARS-CoV-2 and other mucosal infections.","version":"1.1","doi":"10.1101/2023.08.25.554813","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.22.554373","pub_date":"2023-8-28","title":"Potent and broadly neutralizing antibodies against sarbecoviruses induced by sequential COVID-19 vaccination","abstract":"The current SARS-CoV-2 variants strikingly evade all authorized monoclonal antibodies and threaten the efficacy of serum-neutralizing activity elicited by vaccination or prior infection, urging the need to develop antivirals against SARS-CoV-2 and related sarbecoviruses. Here, we identified both potent and broadly neutralizing antibodies from a five-dose vaccinated donor who exhibited cross-reactive serum neutralizing activity against diverse coronaviruses. Through single B cell sorting and sequencing followed by a tailor-made computational pipeline, we successfully selected 86 antibodies with potential cross-neutralizing ability from 684 antibody sequences. Among them, one potently neutralized all SARS-CoV-2 variants that arose prior to Omicron BA.5, and the other three could broadly neutralize all current SARS-CoV-2 variants of concern, SARS-CoV and their related sarbecoviruses (Pangolin-GD, RaTG13, WIV-1, and SHC014). Cryo-EM analysis demonstrates that these antibodies have diverse neutralization mechanisms, such as disassembling spike trimers, or binding to RBM or SD1 to affect ACE2 binding. In addition, prophylactic administration of these antibodies significantly protects nasal turbinate and lung infections against BA.1, XBB.1 and SARS-CoV viral challenge in golden Syrian hamsters, respectively. This study reveals the potential utility of computational process to assist screening cross-reactive antibodies, as well as the potency of vaccine-induced broadly neutralizing antibodies against current SARS-CoV-2 variants and related sarbecoviruses, offering promising avenues for the development of broad therapeutic antibody drugs.","version":"1.2","doi":"10.1101/2023.08.22.554373","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.26.554935","pub_date":"2023-8-28","title":"Self-assembly vascularized human cardiac organoids model cardiac diseases in petri dishes and in mice","abstract":"In this study, we generated self-assembly cardiac organoids (COs) from human pluripotent stem cells by dual-phase modulation of Wnt/\u03b2-catenin pathway, utilizing CHIR99021 and IWR-1-endo. The resulting COs exhibited a diverse array of cardiac-specific cell lineages, cardiac cavity-like structures and demonstrated the capacity of spontaneous beating and vascularization in vitro. We further employed these complex and functional COs to replicate conditions akin to human myocardial infarction and SARS-CoV-2 induced fibrosis. These models accurately captured the pathological characteristics of these diseases, in both in vitro and in vivo settings. In addition, we transplanted the COs into NOD SCID mice and observed that they survived and exhibited ongoing expansion in vivo. Impressively, over a span of 75-day transplantation, these COs not only established blood vessel-like structures but also integrated with the host mice\u2019s vascular system. It is noteworthy that these COs developed to a size of approximately 8 mm in diameter, slightly surpassing the dimensions of the mouse heart. This innovative research highlighted the potential of our COs as a promising avenue for cardiovascular research and therapeutic exploration.","version":"1.1","doi":"10.1101/2023.08.26.554935","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.25.551434","pub_date":"2023-8-27","title":"Single-cell-resolved interspecies comparison identifies a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19","abstract":"Key issues for research of COVID-19 pathogenesis are the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leveraged the model to molecularly survey the disease progression from time-resolved single-cell RNA-sequencing data collected from healthy and SARS-CoV-2-infected Syrian and Roborovski hamster lungs. We compared our data to human COVID-19 studies, including BALF, nasal swab, and post-mortem lung tissue, and identified a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1 or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively. Activation of different immunological programs upon SARS-CoV-2 infection determines COVID-19 severity.","version":"1.1","doi":"10.1101/2023.08.25.551434","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.24.554732","pub_date":"2023-8-25","title":"Mucosal antibody responses to SARS-CoV-2 booster vaccination and breakthrough infection","abstract":"Coronavirus disease 2019 (COVID-19) vaccines have saved millions of lives. However, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged causing large numbers of breakthrough infections. These developments necessitated the rollout of COVID-19 vaccine booster doses. It has been reported that mucosal antibody levels in the upper respiratory tract, especially for secretory IgA (sIgA), correlate with protection from infection with SARS-CoV-2. However, it is still unclear how high levels of mucosal antibodies can be induced. In this study, we measured serum IgG, saliva IgG and saliva sIgA responses in individuals who received COVID-19 mRNA booster vaccinations or who experienced breakthrough infections. We found that mRNA booster doses could induce robust serum and saliva IgG responses, especially in individuals who had not experienced infections before, but saliva sIgA responses were weak. In contrast, breakthrough infections in individuals who had received the primary mRNA vaccination series induced robust serum and saliva IgG as well as saliva sIgA responses. Individuals who had received a booster dose and then had a breakthrough infection showed low IgG induction in serum and saliva but still responded with robust saliva sIgA induction. These data suggest that upper respiratory tract exposure to antigen is an efficient way of inducing mucosal sIgA while exposure via intramuscular injection is not. Antibodies on mucosal surfaces of the upper respiratory tract have been shown to be important for protection from infection with SARS-CoV-2. Here we investigate the induction of serum IgG, saliva IgG and saliva sIgA after COVID-19 mRNA booster vaccination or breakthrough infections.","version":"1.1","doi":"10.1101/2023.08.24.554732","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.24.554650","pub_date":"2023-8-25","title":"Efficient Sequence Embedding For SARS-CoV-2 Variants Classification","abstract":"Kernel-based methods, such as Support Vector Machines (SVM), have demonstrated their utility in various machine learning (ML) tasks, including sequence classification. However, these methods face two primary challenges:(i) the computational complexity associated with kernel computation, which involves an exponential time requirement for dot product calculation, and (ii) the scalability issue of storing the large n \u00d7 n matrix in memory when the number of data points(n) becomes too large. Although approximate methods can address the computational complexity problem, scalability remains a concern for conventional kernel methods. This paper presents a novel and efficient embedding method that overcomes both the computational and scalability challenges inherent in kernel methods. To address the computational challenge, our approach involves extracting the k-mers/nGrams (consecutive character substrings) from a given biological sequence, computing a sketch of the sequence, and performing dot product calculations using the sketch. By avoiding the need to compute the entire spectrum (frequency count) and operating with low-dimensional vectors (sketches) for sequences instead of the memory-intensive n \u00d7 n matrix or full-length spectrum, our method can be readily scaled to handle a large number of sequences, effectively resolving the scalability problem. Furthermore, conventional kernel methods often rely on limited algorithms (e.g., kernel SVM) for underlying ML tasks. In contrast, our proposed fast and alignment-free spectrum method can serve as input for various distance-based (e.g., k-nearest neighbors) and non-distance-based (e.g., decision tree) ML methods used in classification and clustering tasks. We achieve superior prediction for coronavirus spike/Peplomer using our method on real biological sequences excluding full genomes. Moreover, our proposed method outperforms several state-of-the-art embedding and kernel methods in terms of both predictive performance and computational runtime.","version":"1.1","doi":"10.1101/2023.08.24.554650","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.02.494552","pub_date":"2023-8-24","title":"Engineered Wnt7a ligands rescue blood brain barrier and neurobehavioral deficits in a mouse model of COVID-19","abstract":"Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating vascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we conduced unbiased transcriptional analysis to identify brain endothelial cell signaling pathways dysregulated by SARS-CoV-2 in vivo. This analysis revealed significant suppression of Wnt/\u03b2-catenin signaling, a critical regulator of blood brain barrier integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/\u03b2-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected blood brain barrier integrity, reduced T cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this therapeutic strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/\u03b2-catenin signaling or its downstream effectors could be potential interventional strategies for restoring cognitive health following acute viral infections.","version":"1.2","doi":"10.1101/2022.06.02.494552","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.24.554561","pub_date":"2023-8-24","title":"Rationally designed multimeric nanovaccines using icosahedral DNA origami for molecularly controlled display of SARS-CoV-2 receptor binding domain","abstract":"Multivalent antigen display on nanoparticles can enhance the immunogenicity of nanovaccines targeting viral moieties, such as the receptor binding domain (RBD) of SARS-CoV-2. However, particle morphology and size of current nanovaccines are significantly different from those of SARS-CoV-2. Additionally, surface antigen patterns are not controllable to enable the optimization of B cell activation. Herein, we employed an icosahedral DNA origami (ICO) as a display particle for SARS-CoV-2 RBD nanovaccines. The morphology and diameter of the particles were close to those of the virus (91 \u00b1 11 nm). The surface addressability of the DNA origami permitted facile modification of the ICO surface with numerous RBD antigen clusters (ICO-RBD) to form various antigen patterns. Using an in vitro screening system, we demonstrate that the antigen spacing, antigen copies within clusters and cluster number parameters of the surface antigen pattern all impact the ability of the nanovaccines to activate B cells. Importantly, the optimized ICO-RBD nanovaccines evoked stronger and more enduring humoral and T cell immune responses in mouse models compared to soluble RBD antigens. Our vaccines activated similar humoral immunity and slightly stronger cellular immunity compared to mRNA vaccines. These results provide reference principles for the rational design of nanovaccines and exemplify the utility of DNA origami as a display platform for vaccines against infectious disease.","version":"1.1","doi":"10.1101/2023.08.24.554561","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.23.554556","pub_date":"2023-8-24","title":"A fluorescence viewer for rapid molecular assay readout in space and low-resource terrestrial environments","abstract":"Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies \u2013 detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth.","version":"1.1","doi":"10.1101/2023.08.23.554556","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.05.494796","pub_date":"2023-8-24","title":"Development and application of an uncapped mRNA platform","abstract":"A novel uncapped mRNA platform was developed. Five lipid nanoparticle (LNP)-encapsulated mRNA constructs were made to evaluate several aspects of our platform, including transfection efficiency and durability in vitro and in vivo and the activation of humoral and cellular immunity in several animal models. The constructs were eGFP-mRNA-LNP (for enhanced green fluorescence mRNA), Fluc-mRNA-LNP (for firefly luciferase mRNA), S\u03b4T-mRNA-LNP (for Delta strain SARS-CoV-2 spike protein trimer mRNA), gDED-mRNA-LNP (for truncated glycoprotein D mRNA coding ectodomain from herpes simplex virus type 2 (HSV2)) and gDFR-mRNA-LNP (for truncated HSV2 glycoprotein D mRNA coding amino acids 1\u223c400). Quantifiable target protein expression was achieved in vitro and in vivo with eGFP-and Fluc-mRNA-LNP. S\u03b4T-mRNA-LNP, gDED-mRNA-LNP and gDFR-mRNA-LNP induced both humoral and cellular immune responses comparable to those obtained by previously reported capped mRNA-LNP constructs. Notably, S\u03b4T-mRNA-LNP elicited neutralizing antibodies in hamsters against the Omicron and Delta strains. Additionally, gDED-mRNA-LNP and gDFR-mRNA-LNP induced potent neutralizing antibodies in rabbits and mice. The mRNA constructs with uridine triphosphate (UTP) outperformed those with N1-methylpseudouridine triphosphate (N1m\u03c8TP) in the induction of antibodies via S\u03b4T-mRNA-LNP. Our uncapped, process-simplified, and economical mRNA platform may have broad utility in vaccines and protein replacement drugs.","version":"1.3","doi":"10.1101/2022.06.05.494796","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.22.553458","pub_date":"2023-8-23","title":"Administration of vaccine-boosted COVID-19 convalescent plasma to SARS-CoV-2 infected hamsters decreases virus replication in lungs and hastens resolution of the infection despite transiently enhancing disease and lung pathology","abstract":"The utility of COVID-19 convalescent plasma (CCP) for treatment of immunocompromised patients who are not able to mount a protective antibody response against SARS-CoV-2 and who have contraindications or adverse effects from currently available antivirals remains unclear. To better understand the mechanism of protection in CCP, we studied viral replication and disease progression in SARS-CoV-2 infected hamsters treated with CCP plasma obtained from recovered COVID patients that had also been vaccinated with an mRNA vaccine, hereafter referred to as Vaxplas. We found that Vaxplas dramatically reduced virus replication in the lungs and improved infection outcome in SARS-CoV-2 infected hamsters. However, we also found that Vaxplas transiently enhanced disease severity and lung pathology in treated animals likely due to the deposition of immune complexes, activation of complement and recruitment of increased numbers of macrophages with an M1 proinflammatory phenotype into the lung parenchyma.","version":"1.1","doi":"10.1101/2023.08.22.553458","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.22.554362","pub_date":"2023-8-23","title":"Recurrent SARS-CoV-2 mutations at Spike D796 evade antibodies from pre-Omicron convalescent and vaccinated subjects","abstract":"SARS-CoV-2 lineages of the Omicron variant rapidly became dominant in early 2022 and frequently cause human infections despite vaccination or prior infection with other variants. In addition to antibody-evading mutations in the Receptor Binding Domain, Omicron features amino acid mutations elsewhere in the Spike protein, however their effects generally remain ill-defined. The Spike D796Y substitution is present in all Omicron sub-variants and occurs at the same site as a mutation (D796H) selected during viral evolution in a chronically-infected patient. Here we map antibody reactivity to a linear epitope in the Spike protein overlapping position 796. We show that antibodies binding this region arise in pre-Omicron SARS-CoV-2 convalescent and vaccinated subjects, but that both D796Y and D796H abrogate their binding. These results suggest that D796Y contributes to the fitness of Omicron in hosts with pre-existing immunity to other variants of SARS-CoV-2 by evading antibodies targeting this site.","version":"1.1","doi":"10.1101/2023.08.22.554362","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.21.554193","pub_date":"2023-8-23","title":"Ensemble-Based Modeling of the SARS-CoV-2 Omicron BA.1 and BA.2 Spike Trimers and Systematic Characterization of Cryptic Binding Pockets in Distinct Functional States : Emergence of Conformation-Sensitive and Variant-Specific Allosteric Binding Sites","abstract":"A significant body of experimental structures of the SARS-CoV-2 spike trimers for the BA.1 and BA.2 variants revealed a considerable plasticity of the spike protein and emergence of druggable cryptic pockets. Understanding of the interplay of conformational dynamics changes induced by Omicron variants and identification of cryptic dynamic binding pockets in the S protein are of paramount importance as exploring broad-spectrum antiviral agents to combat the emerging variants is imperative. In the current study we explore conformational landscapes and characterize the universe of cryptic binding pockets in multiple open and closed functional spike states of the Omicron BA.1 and BA.2 variants. By using a combination of atomistic simulations, dynamics network analysis, and allostery-guided network screening of cryptic pockets in the conformational ensembles of BA.1 and BA.2 spike conformations, we identified all experimentally known allosteric sites and discovered significant variant-specific differences in the distribution of cryptic binding sites in the BA.1 and BA.2 trimers. This study provided in-depth structural analysis of the predicted allosteric site in the context of all available experimental information, revealing a critical role and effect of conformational plasticity on the distribution and function of allosteric binding sites. The results detailed how mutational and conformational changes in the BA.1 and BA.2 pike trimers can modulate the functional role of druggable allosteric pockets across different functional regions of the spike protein. The results of this study are particularly significant for understanding the universe of cryptic bindings sites and variant-specific preferences for druggable pockets. Exploring predicted druggable sites can present a new and previously underappreciated opportunity for therapeutic intervention of Omicron variants through conformation-selective and variant-specific targeting of functional sites involved in allosteric changes.","version":"1.1","doi":"10.1101/2023.08.21.554193","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.23.554463","pub_date":"2023-8-23","title":"Broad-spectrum antiviral activity of clinically approved CYP3A inhibitors against pathogenic human coronaviruses in vitro","abstract":"Coronaviruses pose a permanent risk of outbreaks, with three highly pathogenic species and strains (SARS-CoV, MERS-CoV, SARS-CoV-2) having emerged in the last twenty years. Limited antiviral therapies are currently available and their efficacy in randomized clinical trials enrolling SARS-CoV-2 patients has not been consistent, highlighting the need for more potent treatments. We previously showed that cobicistat, a clinically approved inhibitor of Cytochrome P450-3A (CYP3A), has direct antiviral activity against early circulating SARS-CoV-2 strains in vitro and in Syrian hamsters. Cobicistat is a derivative of ritonavir, which is co-administered as pharmacoenhancer with the SARS-CoV-2 protease inhibitor nirmatrelvir, to inhibit its metabolization by CPY3A and preserve its antiviral efficacy. Here, we used automated imaging and analysis for a screening and parallel comparison of the anti-coronavirus effects of cobicistat and ritonavir. Our data show that both drugs display antiviral activity at low micromolar concentrations against multiple SARS-CoV-2 variants in vitro, including epidemiologically relevant Omicron subvariants. Despite their close structural similarity, we found that cobicistat is more potent than ritonavir, as shown by significantly lower EC50 values in monotherapy and higher levels of viral suppression when used in combination with nirmatrelvir. Finally, we show that the antiviral activity of both cobicistat and ritonavir is maintained against other human coronaviruses, including HCoV-229E and the highly pathogenic MERS-CoV. Overall, our results demonstrate that cobicistat has more potent anti-coronavirus activity than ritonavir and suggest that dose adjustments could pave the way to the use of both drugs as broad-spectrum antivirals against highly pathogenic human coronaviruses.","version":"1.1","doi":"10.1101/2023.08.23.554463","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.21.554179","pub_date":"2023-8-22","title":"Heterologous sarbecovirus receptor binding domains as scaffolds for SARS-CoV-2 receptor binding motif presentation","abstract":"Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center upon targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potently neutralizing antibody response against diverse sarbecoviruses. Here, we used a \u201cresurfacing\u201d approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.","version":"1.1","doi":"10.1101/2023.08.21.554179","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.21.554197","pub_date":"2023-8-22","title":"AI-based antibody discovery platform identifies novel, diverse and pharmacologically active therapeutic antibodies against multiple SARS-CoV-2 strains","abstract":"A critical aspect of a successful pandemic response is expedient antibody discovery, manufacturing and deployment of effective lifesaving treatments to patients around the world. However, typical drug discovery and development is a lengthy multi-step process that must align drug efficacy with multiple developability criteria and can take years to complete. In this context, artificial intelligence (AI), and especially machine learning (ML), have great potential to accelerate and improve the optimization of therapeutics, increasing their activity and safety as well as decreasing their development time and manufacturing costs. Here we present a novel, cost-effective and accelerated approach to therapeutic antibody discovery, that couples AI-designed human antibody libraries, biased for improved developability attributes with high throughput and sensitive screening technologies. The applicability of our platform for effective therapeutic antibody discovery is demonstrated here with the identification of a panel of human monoclonal antibodies that are novel, diverse and pharmacologically active. These first-generation antibodies, without the need for affinity maturation, bind to the SARS-CoV-2 spike protein with therapeutically-relevant specificity and affinity and display neutralization of SARS-CoV-2 viral infectivity across multiple strains. Altogether, this platform is well suited for rapid response to infectious threats, such as pandemic response. Expedient discovery and manufacturing of lifesaving therapeutics is critical for pandemic response. The recent COVID pandemic has highlighted the current inefficiencies and the need for improvements. To this end, we present our therapeutic antibody discovery platform that couples artificial intelligence (AI) and innovative high throughput technologies, and we demonstrate its applicability to rapid response. This platform enabled the isolation, characterization, and rapid identification of effective broadly neutralizing SARS-CoV-2 antibodies with good developability attributes, anticipated to fit our current process development and manufacturing platform. As such, this would benefit cost-of-goods and improve therapeutic access to patients. The AI-derived antibodies represent an advantageous therapeutic modality that can be developed and deployed fast, thus well suited for rapid response to infectious threats, such as pandemic response.","version":"1.1","doi":"10.1101/2023.08.21.554197","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.21.553968","pub_date":"2023-8-22","title":"Antibody Neutralization of Emerging SARS-CoV-2: EG.5.1 and XBC.1.6","abstract":"SARS-CoV-2 variants EG.5.1 and XBC.1.6 have recently emerged, attracting increased attention due to their rapid expansion globally and in Australia, respectively. EG.5.1 evolved from Omicron subvariant XBB.1.9, harboring additional Q52H and F456L spike substitutions. The F456L mutation is located within the epitopes of many class-1 monoclonal antibodies (mAbs) directed to the receptor-binding domain (RBD), raising concerns about further antibody evasion. XBC.1.6, a descendant of a Delta-BA.2 recombinant, carries 15 additional spike mutations. The extent to which antibody evasion contributes to the growth advantage of XBC.1.6 in Australia remains to be determined. To assess the antibody evasion properties of the emergent variants, we conducted pseudovirus neutralization assays using sera from individuals who received three doses of COVID-19 mRNA monovalent vaccines plus one dose of a BA.5 bivalent vaccine, as well as from patients with BQ or XBB breakthrough infection. The assays were also performed using a panel of 14 mAbs that retained neutralizing activity against prior XBB subvariants. Our data suggested that EG.5.1 was slightly but significantly more resistant (< 2-fold) to neutralization by BQ and XBB breakthrough sera than XBB.1.16, which is known to be antigenically similar to XBB.1.5. Moreover, the F456L mutation in EG.5.1 conferred heightened resistance to certain RBD class-1 mAbs. In contrast, XBC.1.6 was more sensitive to neutralization by sera and mAbs than the XBB subvariants. Notably, XBB breakthrough sera retained only weak neutralization activity against XBB subvariants. In summary, EG.5.1 and XBC.1.6 exhibited distinct antibody evasion properties. The recent global expansion of EG.5.1 might be attributable, in part, to its enhanced neutralization resistance. That XBB breakthrough infections did not elicit a robust antibody neutralization response against XBB subvariants is indicative of immunological imprinting. The high prevalence of XBC.1.6 in Australia is not due to enhanced antibody evasion.","version":"1.1","doi":"10.1101/2023.08.21.553968","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.17.553767","pub_date":"2023-8-22","title":"Divergence of variant binding/neutralizing antibodies following SARS-CoV-2 booster vaccines in myeloma: Impact of hybrid immunity","abstract":"We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity.","version":"1.1","doi":"10.1101/2023.08.17.553767","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.22.554253","pub_date":"2023-8-22","title":"Real-time identification of epistatic interactions in SARS-CoV-2 from large genome collections","abstract":"The emergence and rapid spread of the SARS-CoV-2 virus has highlighted the importance of genomic epidemiology in understanding the evolution of pathogens and for guiding public health interventions. In particular, the Omicron variant underscored the role of epistasis in the evolution of lineages with both higher infectivity and immune escape, and therefore the necessity to update surveillance pipelines to detect them as soon as they emerge. In this study we applied a method based on mutual information (MI) between positions in a multiple sequence alignment (MSA), which is capable of scaling up to millions of samples. We showed how it could reliably predict known experimentally validated epistatic interactions, even when using as little as 10,000 sequences, which opens the possibility of making it a near real-time prediction system. We tested this possibility by modifying the method to account for sample collection date and applied it retrospectively to MSAs for each month between March 2020 and March 2023. We could detect a cornerstone epistatic interaction in the Spike protein between codons 498 and 501 as soon as 6 samples with a double mutation were present in the dataset, thus demonstrating the method\u2019s sensitivity. Lastly we provide examples of predicted interactions between genes, which are harder to test experimentally and therefore more likely to be overlooked. This method could become part of continuous surveillance systems tracking present and future pathogen outbreaks.","version":"1.1","doi":"10.1101/2023.08.22.554253","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.12.540230","pub_date":"2023-8-22","title":"Riding the Wave: Unveiling the Conformational Waves from RBD to ACE2","abstract":"The binding affinity between angiotensin-converting enzyme 2 (ACE2) and the receptor-binding domain (RBD) plays a crucial role in the transmission and re-infection of SARS-CoV2. Here, microsecond molecular dynamics simulations revealed that point mutations in the RBD domain induced conformational transitions that determined the binding affinity between ACE2 and RBD. These structural changes propagate through the RBD domain, altering the orientation of both ACE2 and RBD residues at the binding site. ACE2 receptor shows significant structural heterogeneity, whereas its binding to the RBD domain indicates a much greater degree of structural homogeneity. The receptor was more flexible in its unbound state, with the binding of RBD domains inducing structural transitions. The structural heterogeneity observed in the ACE2 unbound form plays a role in the promiscuity of viral entry as it may allow the receptor to interact with various related and unrelated ligands. Furthermore, rigidity may be important for stabilizing the complex and ensuring the proper orientation of the RBD-binding interface with ACE2. The greater structural homogeneity observed in the ACE2-RBD complex revealed the effectiveness of neutralizing antibodies and vaccines that are primarily directed towards the RBD-binding interface. The binding of the B38 monoclonal antibody revealed restricted conformational transitions in the RBD and ACE2 receptor, attributed to its potent binding interaction.","version":"1.2","doi":"10.1101/2023.05.12.540230","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.19.553970","pub_date":"2023-8-21","title":"No evidence for enhanced disease with polyclonal SARS-CoV-2 antibody in the ferret model","abstract":"Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-enhanced disease (AED) as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for AED, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.08.19.553970","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.18.553902","pub_date":"2023-8-20","title":"Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu","abstract":"Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.","version":"1.1","doi":"10.1101/2023.08.18.553902","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.17.553661","pub_date":"2023-8-18","title":"Purification, crystallization, and preliminary structural analysis of multivalent immunogenic effector protein-anchored SARS-CoV-2 RBD","abstract":"The continuous spread of highly transmissible variants of concern and the potential diminished effectiveness of existing vaccines necessitate ongoing research and development of new vaccines. Immunogenic molecule-anchored antigen has demonstrated superior efficacy in subunit vaccination, primarily due to enhanced cellular uptake facilitated by the affinity between the surface of Immunogenic molecule and the cell membrane. Based on the Immunogenic recombinase B. malayi RecA (BmRecA), we have overexpressed the construct of BmRecA with SARS-CoV-2 RBD (BmRecA-RBD) that exists as a stable helical filament formation; it was purified and crystallized to obtain X-ray diffraction data at 2.7 \u00c5, belonged to the hexagonal symmetry group P65 in the unit-cell parameters of a=b=122.12, c=75.55 and \u03b1=\u03b2=90\u00b0, \u03b3=120\u00b0. The Matthews coefficient was estimated to be 3.12 \u00c53 Da-1, corresponding to solvent contents of 52.65.","version":"1.1","doi":"10.1101/2023.08.17.553661","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.16.553581","pub_date":"2023-8-17","title":"Assembly of SARS-CoV-2 ribonucleosomes by truncated N* variant of the nucleocapsid protein","abstract":"The Nucleocapsid (N) protein of SARS-CoV-2 compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein SR region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N* or \u0394(1\u2013209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we show that \u0394(1\u2013209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of \u0394(1\u2013209) vRNPs requires the leucine-rich helix (LH) of the central disordered region, and that the LH promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to \u0394(1\u2013209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by SR phosphorylation.","version":"1.1","doi":"10.1101/2023.08.16.553581","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.16.553332","pub_date":"2023-8-17","title":"Virological characteristics of the SARS-CoV-2 XBB.1.5 variant","abstract":"Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the F486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determined the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. The intrinsic pathogenicity of XBB.1.5 in hamsters is lower than that of XBB.1. Importantly, we found that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC expression. In vivo experiments using recombinant viruses revealed that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, these data suggest that the mutations in ORF8 and S could enhance spreading of XBB.1.5 in humans.","version":"1.1","doi":"10.1101/2023.08.16.553332","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.16.553557","pub_date":"2023-8-17","title":"Molecular evolution and adaptation of SARS-CoV-2 omicron XBB sub-lineage Spike protein under African selection pressure","abstract":"The SARS-CoV-2 Omicron variant of concern (VOC) has multiple mutations in the spike (S) protein, which mediates viral infection and immunity. We analysed a sub-lineage of Omicron, designated XBB, that showed structural and functional changes in the S protein in response to the African selection pressures. We used molecular modelling to compare the S protein structures of Omicron and XBB and found that XBB had a reduced receptor-binding domain (RBD) due to the loss of some \u03b2-sheets, which may increase its affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. We also used Fast Unconstrained Bayesian AppRoximation (FUBAR) and Recombination Detection Program 4 (RDP 4) to perform selection and recombination analysis of the S protein sequences of Omicron and XBB and detected signals of positive selection and recombination in the N-terminal domain (NTD) of the S1 subunit, which contains antibody-binding epitopes, and the RBD, which is involved in viral entry. Our results reveal the structural and functional adaptation of the Omicron XBB variant in Africa and its potential implications for viral pathogenesis and immunity.","version":"1.1","doi":"10.1101/2023.08.16.553557","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.15.553430","pub_date":"2023-8-16","title":"Predicting host-based, synthetic lethal antiviral targets from omics data","abstract":"Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection. Thus, we hypothesized that genes depleted in CRISPR KO screens of virus-infected cells may be enriched in SL partners of proteins altered by infection. To investigate this, we established a computational pipeline predicting SL drug targets of viral infections. First, we identified SARS-CoV-2-induced changes in gene products via a large compendium of omics data. Second, we identified SL partners for each altered gene product. Last, we screened CRISPR KO data for SL partners required for cell viability in infected cells. Despite differences in virus-induced alterations detected by various omics data, they share many predicted SL targets, with significant enrichment in CRISPR KO-depleted datasets. Comparing data from SARS-CoV-2 and influenza infections, we found possible broad-spectrum, host-based antiviral SL targets. This suggests that CRISPR KO data are replete with common antiviral targets due to their SL relationship with virus-altered states and that such targets can be revealed from analysis of omics datasets and SL predictions.","version":"1.1","doi":"10.1101/2023.08.15.553430","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.16.553512","pub_date":"2023-8-16","title":"Coronaviruses Spike glycoprotein endodomains: the sequence and structure-based comprehensive study","abstract":"The flexibility of proteins makes them available to interact with many biomolecules in the cell. Specifically, such interactions in viruses help them to perform more functions despite having a smaller genome. Therefore, these flexible regions can be exciting and essential targets to be explored for their role in pathogenicity and therapeutic developments as they achieve essential interactions. In the continuation with our previous study on disordered analysis of SARS-CoV-2 spike cytoplasmic tail (CTR), or endodomain, here we have explored the disordered potential endodomains of six other coronaviruses using multiple bioinformatics approaches and molecular dynamics simulations. Based on the comprehensive analysis of its sequence and structural composition, we report the varying disorder propensity in endodomains of spike proteins of coronaviruses. The observations of this study may help to understand the importance of spike glycoprotein endodomain and creating therapeutic interventions against them.","version":"1.1","doi":"10.1101/2023.08.16.553512","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.14.553245","pub_date":"2023-8-15","title":"SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels","abstract":"COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome, and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA) is detectable and replicates in coronary atherosclerotic lesions taken at autopsy from patients with severe COVID-19. SARS-CoV-2 localizes to plaque macrophages and shows a stronger tropism for arterial lesions compared to corresponding perivascular fat, correlating with the degree of macrophage infiltration. In vitro infection of human primary macrophages highlights that SARS-CoV-2 entry is increased in cholesterol-loaded macrophages (foam cells) and is dependent, in part, on neuropilin-1 (NRP-1). Furthermore, although viral replication is abortive, SARS-CoV-2 induces a robust inflammatory response that includes interleukins IL-6 and IL-1\u03b2, key cytokines known to trigger ischemic cardiovascular events. SARS-CoV-2 infection of human atherosclerotic vascular explants recapitulates the immune response seen in cultured macrophages, including pro-atherogenic cytokine secretion. Collectively, our data establish that SARS-CoV-2 infects macrophages in coronary atherosclerotic lesions, resulting in plaque inflammation that may promote acute CV complications and long-term risk for CV events.","version":"1.1","doi":"10.1101/2023.08.14.553245","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.27.530232","pub_date":"2023-8-15","title":"ORF3c is expressed in SARS-CoV-2 infected cells and suppresses immune activation by inhibiting innate sensing","abstract":"SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While most of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. For example, the ORF3a sgRNA also encodes ORF3c, an enigmatic 4l-amino acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2 infected cells and suppresses RIG-I- and MDA5-mediated immune activation and IFN-\u03b2 induction. Mechanistic analyses revealed that ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c demonstrating that this reading frame is not essential for efficient viral replication in vivo and likely compensated by other viral proteins. In agreement with this, disruption of ORF3c did not significantly affect SARS-CoV-2 replication in CaCo-2 or CaLu-3 cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.","version":"1.2","doi":"10.1101/2023.02.27.530232","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.11.23293871","pub_date":"2023-08-15","title":"Novel risk loci for COVID-19 hospitalization among admixed American populations","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The genetic basis of severe COVID-19 has been thoroughly studied, and many genetic risk factors shared between populations have been identified. However, reduced sample sizes from non-European groups have limited the discovery of population-specific common risk loci. In this second study nested in the SCOURGE consortium, we conducted a GWAS for COVID-19 hospitalization in admixed Americans, comprising a total of 4,702 hospitalized cases recruited by SCOURGE and seven other participating studies in the COVID-19 Host Genetic Initiative. We identified four genome-wide significant associations, two of which constitute novel loci and were first discovered in Latin American populations (\n                  <jats:italic>BAZ2B</jats:italic>\n                  and\n                  <jats:italic>DDIAS</jats:italic>\n                  ). A trans-ethnic meta-analysis revealed another novel cross-population risk locus in\n                  <jats:italic>CREBBP</jats:italic>\n                  . Finally, we assessed the performance of a cross-ancestry polygenic risk score in the SCOURGE admixed American cohort. This study constitutes the largest GWAS for COVID-19 hospitalization in admixed Latin Americans conducted to date. This allowed to reveal novel risk loci and emphasize the need of considering the diversity of populations in genomic research.\n                </jats:p>","version":null,"doi":"10.1101/2023.08.11.23293871","journal":"medRxiv","score":null},{"id":"10.1101/2023.08.11.552998","pub_date":"2023-8-14","title":"Impact of age and sex on neuroinflammation following SARS-CoV-2 infection in a murine model","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent for the worldwide COVID-19 pandemic, is known to infect people of all ages and both sexes. Senior populations have the greatest risk of severe disease, and sexual dimorphism in clinical outcomes has been reported in COVID-19. SARS-CoV-2 infection in humans can cause damage to multiple organ systems, including the brain. Neurological symptoms are widely observed in patients with COVID-19, with many survivors suffering from persistent neurological and cognitive impairment, potentially accelerating Alzheimer\u2019s disease. The present study aims to investigate the impact of age and sex on the neuroinflammatory response to SARS-CoV-2 infection using a mouse model. Wild-type C57BL/6 mice were inoculated, by intranasal route, with SARS-CoV-2 lineage B.1.351 variant known to infect mice. Older animals and in particular males exhibited a significantly greater weight loss starting at 4 dpi. In addition, male animals exhibited higher viral RNA loads and higher titers of infectious virus in the lung, which was particularly evident in males at 16 months of age. Notably, no viral RNA was detected in the brains of infected mice, regardless of age or sex. Nevertheless, expression of IL-6, TNF-\u03b1, and CCL-2 in the lung and brain was increased with viral infection. An unbiased brain RNA-seq/transcriptomic analysis showed that SARS-CoV-2 infection caused significant changes in gene expression profiles in the brain, with innate immunity, defense response to virus, cerebravascular and neuronal functions, as the major molecular networks affected. The data presented in this study show that SARS-CoV-2 infection triggers a neuroinflammatory response despite the lack of detectable virus in the brain. Age and sex have a modifying effect on this pathogenic process. Aberrant activation of innate immune response, disruption of blood-brain barrier and endothelial cell integrity, and supression of neuronal activity and axonogenesis underlie the impact of SARS-CoV-2 infection on the brain. Understanding the role of these affected pathways in SARS-CoV-2 pathogenesis helps identify appropriate points of therapeutic interventions to alleviate neurological dysfunction observed during COVID-19.","version":"1.1","doi":"10.1101/2023.08.11.552998","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.12.553079","pub_date":"2023-8-14","title":"Pandemic preparedness through genomic surveillance: Overview of mutations in SARS-CoV-2 over the course of COVID-19 outbreak","abstract":"Genomic surveillance is a vital strategy for preparedness against the spread of infectious diseases and to aid in development of new treatments. In an unprecedented effort, millions of samples from COVID-19 patients have been sequenced worldwide for SARS-CoV-2. Using more than 8 million sequences that are currently available in GenBank\u2019s SARS-CoV-2 database, we report a comprehensive overview of mutations in all 26 proteins and open reading frames (ORFs) from the virus. The results indicate that the spike protein, NSP6, nucleocapsid protein, envelope protein and ORF7b have shown the highest mutational propensities so far (in that order). In particular, the spike protein has shown rapid acceleration in mutations in the post-vaccination period. Monitoring the rate of non-synonymous mutations (Ka) provides a fairly reliable signal for genomic surveillance, successfully predicting surges in 2022. Further, the external proteins (spike, membrane, envelope, and nucleocapsid proteins) show a significant number of mutations compared to the NSPs. Interestingly, these four proteins showed significant changes in Ka typically 2 to 4 weeks before the increase in number of human infections (\u201csurges\u201d). Therefore, our analysis provides real time surveillance of mutations of SARS-CoV-2, accessible through the project website http://pandemics.okstate.edu/covid19/. Based on ongoing mutation trends of the virus, predictions of what proteins are likely to mutate next are also made possible by our approach. The proposed framework is general and is thus applicable to other pathogens. The approach is fully automated and provides the needed genomic surveillance to address a fast-moving pandemic such as COVID-19.","version":"1.1","doi":"10.1101/2023.08.12.553079","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.13.553148","pub_date":"2023-8-14","title":"SARS-CoV-2 Neutralizing Antibodies Following a Second BA.5 Bivalent Booster","abstract":"Bivalent COVID-19 mRNA vaccines expressing both the ancestral D614G and Omicron BA.5 spike proteins were introduced in August 2022 with the goal of broadening immunity to emerging SARS-CoV-2 Omicron subvariants. Subsequent studies on bivalent boosters found neutralizing antibody responses similar to boosters with the original monovalent vaccine, likely the result of immunological imprinting. Guidelines allow for administration of a second bivalent booster in high-risk groups, but it remains unknown whether this would broaden antibody responses. To address this question, we assessed longitudinal serum SARS-CoV-2-neutralizing titers in 18 elderly immunocompetent individuals (mean age 69) following a fourth monovalent booster and two BA.5 bivalent booster vaccines using pseudovirus neutralization assays against D614G, Omicron BA.5, and Omicron XBB.1.5. There was a small but significant increase in peak neutralizing antibody responses against Omicron BA.5 and XBB.1.5 following the first bivalent booster, but no significant increase in peak titers following the second bivalent booster. Omicron-specific neutralizing titers remained low after both doses of the BA.5 bivalent booster. Our results suggest that a second dose of the BA.5 bivalent booster is not sufficient to broaden antibody responses and to overcome immunological imprinting. A monovalent vaccine targeting only the spike of the recently dominant SARS-CoV-2 may mitigate the \u201cback boosting\u201d associated with the \u201coriginal antigenic sin.\u201d","version":"1.1","doi":"10.1101/2023.08.13.553148","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.13.553144","pub_date":"2023-8-14","title":"Mutational basis of serum cross-neutralization profiles elicited by infection or vaccination with SARS-CoV-2 variants","abstract":"A series of SARS-CoV-2 variants emerged during the pandemic under selection for neutralization resistance. Convalescent and vaccinated sera show consistently different cross-neutralization profiles depending on infecting or vaccine variants. To understand the basis of this heterogeneity, we modeled serum cross-neutralization titers for 165 sera after infection or vaccination with historically prominent lineages tested against 18 variant pseudoviruses. Cross-neutralization profiles were well captured by models incorporating autologous neutralizing titers and combinations of specific shared and differing mutations between the infecting/vaccine variants and pseudoviruses. Infecting/vaccine variant-specific models identified mutations that significantly impacted cross-neutralization and quantified their relative contributions. Unified models that explained cross-neutralization profiles across all infecting and vaccine variants provided accurate predictions of holdout neutralization data comprising untested variants as infecting or vaccine variants, and as test pseudoviruses. Finally, comparative modeling of 2-dose versus 3-dose mRNA-1273 vaccine data revealed that the third dose overcame key resistance mutations to improve neutralization breadth. Modeled SARS-CoV-2 cross-neutralization using mutations at key sites Identified resistance mutations and quantified relative impact Accurately predicted holdout variant and convalescent/vaccine sera neutralization Showed that the third dose of mRNA-1273 vaccination overcomes resistance mutations","version":"1.1","doi":"10.1101/2023.08.13.553144","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.14.553212","pub_date":"2023-8-14","title":"Met58 and di-acidic motif located at C-terminal region of SARS-CoV-2 ORF6 plays a crucial role in its structural conformations","abstract":"Despite being mostly neglected in structural biology, the C-terminal Regions (CTRs) are studied to be multifunctional in humans as well as in viruses. Their role in cellular processes such as trafficking, protein-protein interactions, and protein-lipid interactions are known due to their structural properties. In our previous findings on SARS-CoV-2 Spike and NSP1 proteins, the C-terminal regions (CTRs) are observed to be disordered and experimental evidence showed a gain of structure properties in different physiological environments. In this line, we have investigated the structural dynamics of CTR (residues 38-61) of SARS-CoV-2 ORF6 protein, disrupting bidirectional transport between the nucleus and cytoplasm. Like Spike and NSP1-CTR, the ORF6-CTR is also disordered in nature but possesses gain of structure properties in minimal physiological conditions. As per studies, the residue such as Methionine at 58th position in ORF6 is critical for interaction with Rae1-Nup98. Therefore, along with M58, we have identified a few other mutations from the literature and performed extensive structure modelling and dynamics studies using computational simulations. The exciting revelations in CTR models provide evidence of its structural flexibility and possible capabilities to perform multifunctionality inside the host.","version":"1.1","doi":"10.1101/2023.08.14.553212","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.11.552988","pub_date":"2023-8-14","title":"PRIEST - Predicting viral mutations with immune escape capability of SARS-CoV-2 using temporal evolutionary information","abstract":"The dynamic evolution of the SARS-CoV-2 virus is largely driven by mutations in its genetic sequence, culminating in the emergence of variants with increased capability to evade host immune responses. Accurate prediction of such mutations is fundamental in mitigating pandemic spread and developing effective control measures. In this study, we introduce a robust and interpretable deep-learning approach called PRIEST. This innovative model leverages time-series viral sequences to foresee potential viral mutations. Our comprehensive experimental evaluations underscore PRIEST\u2019s proficiency in accurately predicting immune-evading mutations. Our work represents a substantial step forward in the utilization of deep-learning methodologies for anticipatory viral mutation analysis and pandemic response.","version":"1.1","doi":"10.1101/2023.08.11.552988","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.11.552671","pub_date":"2023-8-14","title":"Structural elucidation and antiviral activity of cathepsin L inhibitors with carbonyl and epoxide warheads","abstract":"Emerging RNA viruses including SARS-CoV-2 continue to be a major threat around the globe. The cell entry of SARS-CoV-2 particles via the endosomal pathway involves the cysteine protease cathepsin L (CatL) among other proteases. CatL is rendered as a promising drug target in the context of different viral and lysosome-related diseases. Hence, drug discovery and structure-based optimization of inhibitors is of high pharmaceutical interest. We herein verified and compared the anti-SARS-CoV-2 activity of a set of carbonyl and succinyl-epoxide-based inhibitors, which have previously been identified as cathepsin inhibitors. Calpain inhibitor XII (CI-XII), MG-101 and CatL inhibitor IV (CLI-IV) possess antiviral activity in the very low nanomolar IC50 range in Vero E6 cells. Experimental structural data on how these and related compounds bind to CatL are however notably lacking, despite their therapeutic potential. Consequently, we present and compare crystal structures of CatL in complex with 14 compounds, namely BOCA (N-BOC-2-aminoacetaldehyde), CLI-IV, CI-III, CI-VI, CI-XII, the main protease \u03b1-ketoamide inhibitor 13b, MG-101, MG-132 as well as E-64d (aloxistatin), E-64, CLIK148, CAA0225, TC-I (CID 16725315) and TPCK at resolutions better than 2 \u00c5. Overall, the presented data comprise a broad and solid basis for structure-guided understanding and optimization of CatL inhibitors towards protease drug development.","version":"1.1","doi":"10.1101/2023.08.11.552671","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.09.552643","pub_date":"2023-8-10","title":"Enhanced Deep Convolutional Neural Network for SARS-CoV-2 Variants Classification","abstract":"High-throughput sequencing techniques and sequence analysis have enabled the taxonomic classification of pathogens present in clinical samples. Sequencing provides an unbiased identification and systematic classification of pathogens and this is generally achieved by comparing novel sequences to pre-existing annotated reference databases. However, this approach is limited by large-scale reference databases which require considerable computational resources and skills to compare against. Alternative robust methods such as machine learning are currently employed in genome sequence analysis and classification, and it can be applied in classifying SARS-CoV-2 variants, whose continued evolution has resulted in the emergence of multiple variants. We developed a deep learning Convolutional Neural Networks-Long Short Term Memory (CNN-LSTM) model to classify dominant SARS-CoV-2 variants (omicron, delta, beta, gamma and alpha) based on gene sequences from the surface glycoprotein (spike gene). We trained and validated the model using > 26,000 SARS-CoV-2 sequences from the GISAID database. The model was evaluated using unseen 3,057 SARS-CoV-2 sequences. The model was compared to existing molecular epidemiology tool, nextclade. Our model achieved an accuracy of 98.55% on training, 99.19% on the validation and 98.41% on the test dataset. Comparing the proposed model to nextclade, the model achieved significant accuracy in classifying SARS-CoV-2 variants from unseen data. Nextclade identified the presence of recombinant strains in the evaluation data, a mechanism that the proposed model did not detect. This study provides an alternative approach to pre-existing methods employed in the classification of SARS-CoV-2 variants. Timely classification will enable effective monitoring and tracking of SARS-CoV-2 variants and inform public health policies in the control and management of the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2023.08.09.552643","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.08.552470","pub_date":"2023-8-10","title":"Development and Analytical Evaluation of a Point-of-Care Electrochemical Biosensor for Rapid and Accurate SARS-CoV-2 Detection","abstract":"The COVID-19 pandemic has underscored the critical need for rapid and accurate screening and diagnostic methods for potential respiratory viruses. Existing COVID-19 diagnostic approaches face limitations either in terms of turnaround time or accuracy. In this study, we present an electrochemical biosensor that offers nearly instantaneous and precise SARS-CoV-2 detection, suitable for point-of-care and environmental monitoring applications. The biosensor employs a stapled hACE-2 N-terminal alpha helix peptide to functionalize an in-situ grown polypyrrole conductive polymer on a nitrocellulose membrane backbone through a chemical process. We assessed the biosensor\u2019s analytical performance using heat-inactivated omicron and delta variants of the SARS-CoV-2 virus in artificial saliva (AS) and nasal swabs (NS) samples diluted in a strong ionic solution. Virus identification was achieved through electrochemical impedance spectroscopy (EIS) and frequency analyses. The assay demonstrated a limit of detection of 40 TCID50/mL, with 95% sensitivity and 100% specificity. Notably, the biosensor exhibited no cross-reactivity when tested against the influenza virus. The entire testing process using the biosensor takes less than a minute. In summary, our biosensor exhibits promising potential in the battle against pandemic respiratory viruses, offering a platform for the creation of rapid, compact, portable, and point-of-care devices capable of multiplexing various viruses. This groundbreaking development has the capacity to significantly bolster our readiness and response to future viral outbreaks.","version":"1.1","doi":"10.1101/2023.08.08.552470","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.09.552685","pub_date":"2023-8-10","title":"Open-source milligram-scale, four channel, automated protein purification system","abstract":"Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, \u201cone-protein-at-a-time\u201d purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.","version":"1.1","doi":"10.1101/2023.08.09.552685","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.08.552530","pub_date":"2023-8-09","title":"In silico thermodynamic evaluation of the effectiveness of RT-LAMP primers to SARS-CoV-2 variants detection","abstract":"Viral mutations are the primary cause of mismatches in primer-target hybridisation, affecting the sensibility of molecular techniques, potentially leading to detection dropouts. Despite its importance, little is known about the quantitative effect of mismatches in primer-target hybridisation. We use up-to-date and highly detailed thermodynamic model parameters of DNA mismatches to evaluate the sensibility to variants of SARS-CoV-2 RT-LAMP primers. We aligned 18 RT-LAMP primer sets, which were underwent clinical validation, to the genomes of Wuhan strain (ws), 7 variants and 4 subvariants, and calculated hybridisation temperatures allowing up to three consecutive mismatches. We calculate the coverage when the mismatched melting temperature falls by more than 5\u00b0C in comparison to the matched alignments. If no mismatches are considered, the average coverage found would be 94% for ws, falling the lowest value for Omicron: 84%. However, considering mismatches the coverage is much higher: 97% (ws) to 88% (Omicron). Stabilizing mismatches (higher melting temperatures), account for roughly 1/3 of this increase. The number of primer dropouts increases for new each variant, however the effect is much less severe if mismatches are considered. We suggest using melting temperature calculations to continuously assess the trend of primer dropouts.","version":"1.1","doi":"10.1101/2023.08.08.552530","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.09.552495","pub_date":"2023-8-09","title":"Heart-on-a-chip model of immune-induced cardiac dysfunction reveals the role of free mitochondrial DNA and therapeutic effects of endothelial exosomes","abstract":"Cardiovascular disease continues to take more human lives than all cancer combined, prompting the need for improved research models and treatment options. Despite a significant progress in development of mature heart-on-a-chip models of fibrosis and cardiomyopathies starting from induced pluripotent stem cells (iPSCs), human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip system with circulating immune cells to model SARS-CoV-2-induced acute myocarditis. Briefly, we observed hallmarks of COVID-19-induced myocardial inflammation in the heart-on-a-chip model, as the presence of immune cells augmented the expression levels of proinflammatory cytokines, triggered progressive impairment of contractile function and altered intracellular calcium transient activities. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the in vitro heart-on-a-chip model and then validated in COVID-19 patients with low left ventricular ejection fraction (LVEF), demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2 induced myocardial inflammation, we established that administration of human umbilical vein-derived EVs effectively rescued the contractile deficit, normalized intracellular calcium handling, elevated the contraction force and reduced the ccf- mtDNA and chemokine release via TLR-NF-kB signaling axis.","version":"1.1","doi":"10.1101/2023.08.09.552495","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.08.552503","pub_date":"2023-8-08","title":"Within-host evolution of SARS-CoV-2: how often are de novo mutations transmitted?","abstract":"Despite a relatively low mutation rate, the large number of SARS-CoV-2 infections has allowed for substantial genetic change, leading to a multitude of emerging variants. Using a recently determined mutation rate (per site replication), as well as parameter estimates for within-host SARS-CoV-2 infection, we apply a stochastic transmission-bottleneck model to describe the survival probability of de novo SARS-CoV-2 mutations. For narrow bottlenecks, we find mutations affecting pertarget-cell attachment rate (with phenotypes associated with fusogenicity and ACE2 binding), have similar transmission probabilities to mutations affecting viral load clearance (with phenotypes associated with humoral evasion). We further find that mutations affecting the eclipse rate (with phenotypes associated with reorganization of cellular metabolic processes and synthesis of viral budding precursor material) are highly favoured relative to all other traits examined. We find mutations leading to reduced removal rates of infected cells (with phenotypes associated with innate immune evasion) have limited transmission advantage relative to mutations leading to humoral evasion. Predicted transmission probabilities, however, for mutations affecting innate immune evasion are more consistent with the range of clinically-estimated household transmission probabilities for de novo mutations. This result suggests that although mutations affecting humoral evasion are more easily transmitted when they occur, mutations affecting innate immune evasion may occur more readily. We examine our predictions in the context of a number of previously characterized mutations in circulating strains of SARS-CoV-2. Our work offers both a null model for SARS-CoV-2 substitution rates and predicts which aspects of viral life history are most likely to successfully evolve, despite low mutation rates and repeated transmission bottlenecks.","version":"1.1","doi":"10.1101/2023.08.08.552503","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.14.520483","pub_date":"2023-8-08","title":"Crowdsourcing Temporal Transcriptomic Coronavirus Host Infection Data: resources, guide, and novel insights","abstract":"The emergence of SARS-CoV-2 reawakened the need to rapidly understand the molecular etiologies, pandemic potential, and prospective treatments of infectious agents. The lack of existing data on SARS-CoV-2 hampered early attempts to treat severe forms of COVID-19 during the pandemic. This study coupled existing transcriptomic data from SARS-CoV-1 lung infection animal studies with crowdsourcing statistical approaches to derive temporal meta-signatures of host responses during early viral accumulation and subsequent clearance stages. Unsupervised and supervised machine learning approaches identified top dysregulated genes and potential biomarkers (e.g., CXCL10, BEX2, and ADM). Temporal meta-signatures revealed distinct gene expression programs with biological implications to a series of host responses underlying sustained Cxcl10 expression and Stat signaling. Cell cycle switched from G1/G0 phase genes, early in infection, to a G2/M gene signature during late infection that correlated with the enrichment of DNA Damage Response and Repair genes. The SARS-CoV-1 meta-signatures were shown to closely emulate human SARS-CoV-2 host responses from emerging RNAseq, single cell and proteomics data with early monocyte-macrophage activation followed by lymphocyte proliferation. The circulatory hormone adrenomedullin was observed as maximally elevated in elderly patients that died from COVID-19. Stage-specific correlations to compounds with potential to treat COVID-19 and future coronavirus infections were in part validated by a subset of twenty-four that are in clinical trials to treat COVID-19. This study represents a roadmap to leverage existing data in the public domain to derive novel molecular and biological insights and potential treatments to emerging human pathogens. The data from this study is available in an interactive portal (http://18.222.95.219:8047).","version":"1.2","doi":"10.1101/2022.12.14.520483","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.07.552330","pub_date":"2023-8-08","title":"NVX-CoV2373 ancestral and NVX-CoV2540 BA.5 protein nanoparticle vaccines protect against Omicron BA.5 variant in Syrian hamsters","abstract":"The emergence of SARS-CoV-2 variants with greater transmissibility or immune evasion properties has jeopardized the existing vaccine and antibody-based countermeasures. Here, we evaluated the efficacy of boosting with the protein nanoparticle NVX-CoV2373 or NVX-CoV2540 vaccines containing ancestral or BA.5 S proteins, respectively, in mRNA-immunized pre-immune hamsters, against challenge with the Omicron BA.5 variant of SARS-CoV-2. Serum antibody binding and neutralization titers were quantified before challenge, and viral loads were measured 3 days after challenge. Compared to an mRNA vaccine boost, NVX-CoV2373 or NVX-CoV2540 induced higher serum antibody binding responses against ancestral Wuhan-1 or BA.5 spike proteins, and greater neutralization of Omicron BA.1 and BA.5 variants. One and three months after vaccine boosting, hamsters were challenged with the Omicron BA.5 variant. NVX-CoV2373 and NVX-CoV2540 boosted hamsters showed reduced viral infection in the nasal washes, nasal turbinates, and lungs compared to unvaccinated animals. Also, NVX-CoV2540 BA.5 boosted animals had fewer breakthrough infections than NVX-CoV2373 or mRNA-vaccinated hamsters. Thus, immunity induced by NVX-CoV2373 or NVX-CoV2540 boosting can protect against the Omicron BA.5 variant in the Syrian hamster model. As SARS-CoV-2 variants continue to emergence, the efficacy of prior and updated COVID-19 vaccines need to be tested. Here, we tested the efficacy of two nanoparticle protein-based vaccines in pre-immune hamsters against a challenge with the BA.5 Omicron variant of SARS-CoV-2. Compared to an mRNA vaccine boost, the nanoparticle vaccine NVX-CoV2373 and NVX-CoV2540 induced higher serum antibody binding and neutralization responses against ancestral Wuhan-1 or BA.5 variants. One and three months after the last immunization, hamsters were challenged with the Omicron BA.5 variant. NVX-CoV2373 and NVX-CoV2540 boosted hamsters showed reduced viral infection in the nasal washes, nasal turbinates, and lungs compared to unvaccinated animals. Animals that received the homologous vaccine, NVX-CoV2540, had fewer breakthrough infections than NVX-CoV2373 or mRNA-vaccinated hamsters. Together, our data shows that the BA.5 nanoparticle vaccine is effective and that it is important to update the COVID-19 vaccine to match currently circulating strains of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.08.07.552330","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.08.552415","pub_date":"2023-8-08","title":"Antiviral efficacy of the SARS-CoV-2 XBB breakthrough infection sera against Omicron subvariants including EG.5","abstract":"As of July 2023, EG.5.1 (a.k.a. XBB.1.9.2.5.1), a XBB subvariant bearing the S:Q52H and S:F456L substitutions, alongside the S:F486P substitution (Figure S1A), has rapidly spread in some countries. On July 19, 2023, the WHO classified EG.5 as a variant under monitoring. First, we showed that EG.5.1 exhibits a higher effective reproduction number compared with XBB.1.5, XBB.1.16, and its parental lineage (XBB.1.9.2), suggesting that EG.5.1 will spread globally and outcompete these XBB subvariants in the near future. We then addressed whether EG.5.1 evades from the antiviral effect of the humoral immunity induced by breakthrough infection (BTI) of XBB subvariants and performed a neutralization assay using XBB BTI sera. However, the 50% neutralization titer (NT50) of XBB BTI sera against EG.5.1 was comparable to those against XBB.1.5/1.9.2 and XBB.1.16. Moreover, the sensitivity of EG.5.1 to convalescent sera of XBB.1- and XBB.1.5-infected hamsters was similar to those of XBB.1.5/1.9 and XBB.1.16. These results suggest that the increased Re of EG.5.1 is attributed to neither increased infectivity nor immune evasion from XBB BTI, and the emergence and spread of EG.5 is driven by the other pressures. We previously demonstrated that Omicron BTI cannot efficiently induce antiviral humoral immunity against the variant infected. In fact, the NT50s of the BTI sera of Omicron BA.1, BA.2, and BA.5 against the variant infected were 3.0-, 2.2-, and 3.4-fold lower than that against the ancestral B.1.1 variant, respectively. However, strikingly, we found that the NT50 of the BTI sera of XBB1.5/1.9 and XBB.1.16 against the variant infected were 8.7- and 8.3-fold lower than that against the B.1.1 variant. These results suggest that XBB BTI cannot efficiently induce antiviral humoral immunity against XBB subvariants.","version":"1.1","doi":"10.1101/2023.08.08.552415","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.07.552269","pub_date":"2023-8-07","title":"Purifying selection and adaptive evolution proximate to the zoonosis of SARS-CoV-1 and SARS-CoV-2","abstract":"Over the past two decades the pace of spillovers from animal viruses to humans has accelerated, with COVID-19 becoming the most deadly zoonotic disease in living memory. Prior to zoonosis, it is conceivable that the virus might largely be subjected to purifying selection, requiring no additional selective changes for successful zoonotic transmission. Alternatively, selective changes occurring in the reservoir species may coincidentally preadapt the virus for human-to-human transmission, facilitating spread upon cross-species exposure. Here we quantify changes in the genomes of SARS-CoV-2 and SARS-CoV-1 proximate to zoonosis to evaluate the selection pressures acting on the viruses. Application of molecular-evolutionary and population-genetic approaches to quantify site-specific selection within both SARS-CoV genomes revealed strong purifying selection across many genes at the time of zoonosis. Even in the viral surface-protein Spike that has been fast-evolving in humans, there is little evidence of positive selection proximate to zoonosis. Nevertheless, in SARS-CoV-2, NSP12, a core protein for viral replication, exhibited a region under adaptive selection proximate to zoonosis. Furthermore, in both SARS-CoV-1 and SARS-CoV-2, regions of adaptive selection proximate to zoonosis were found in ORF7a, a putative Major Histocompatibility Complex modulatory gene. These findings suggest that these replication and immunomodulatory proteins have played a previously underappreciated role in the adaptation of SARS coronaviruses to human hosts.","version":"1.1","doi":"10.1101/2023.08.07.552269","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.04.551973","pub_date":"2023-8-07","title":"Recombinant SARS-CoV-2 lacking initiating and internal methionine codons within ORF10 is attenuated in vivo","abstract":"SARS-CoV-2 has been proposed to encode ORF10 as the 3\u2019 terminal gene in the viral genome. However, the potential role and even existence of a functional ORF10 product has been the subject of debate. There are significant structural features in the viral genomic RNA that could, by themselves, explain the retention of the ORF10 nucleotide sequences without the need for a functional protein product. To explore this question further we made two recombinant viruses, firstly a control virus (WT) based on the genome sequence of the original Wuhan isolate and with the inclusion of the early D614G mutation in the Spike protein. We also made a second virus, identical to WT except for two additional changes that replaced the initiating ORF10 start codon and an internal methionine codon for stop codons (ORF10KO). Here we show that the two viruses have apparently identical growth kinetics in a VeroE6 cell line that over expresses TMPRSS2 (VTN cells). However, in A549 cells over expressing ACE2 and TMPRSS2 (A549-AT cells) the ORF10KO virus appears to have a small growth rate advantage. Growth competition experiments were used whereby the two viruses were mixed, passaged in either VTN or A549-AT cells and the resulting output virus was sequenced. We found that in VTN cells the WT virus quickly dominated whereas in the A549-AT cells the ORF10KO virus dominated. We then used a hamster model of SARS-CoV-2 infection and determined that the ORF10KO virus has attenuated pathogenicity (as measured by weight loss). We found an almost 10-fold reduction in viral titre in the lower respiratory tract for ORF10KO vs WT. In contrast, the WT and ORF10KO viruses had similar titres in the upper respiratory tract. Sequencing of viral RNA in the lungs of hamsters infected with ORF10KO virus revealed that this virus frequently reverts to WT. Our data suggests that the retention of a functional ORF10 sequence is highly desirable for SARS-CoV-2 infection of hamsters and affects the virus\u2019s ability to propagate in the lower respiratory tract.","version":"1.1","doi":"10.1101/2023.08.04.551973","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.04.551867","pub_date":"2023-8-07","title":"Mucosal and systemic immune dynamics associated with COVID-19 outcomes: a longitudinal prospective clinical study","abstract":"COVID-19 severity varies widely; children and African Americans have low and high risk, respectively. Mechanistic data from these groups and the mucosa is lacking. To quantify mucosal and systemic viral and immune dynamics in a diverse cohort to identify mechanisms underpinning COVID-19 severity and outcome predictors. In this prospective study of unvaccinated children and adults COVID-19 outcome was based on an ordinal clinical severity scale. We quantified viral RNA, antigens, antibodies, and cytokines by PCR, ELISA, and Luminex from 579 longitudinally collected blood and nasal specimens from 78 subjects including 45 women and used modeling to determine functional relationships between these data. COVID-19 induced unique immune responses in African Americans (n=26) and children (n=20). Mild outcome was associated with more effective coordinated responses whereas moderate and severe outcomes had rapid seroconversion, significantly higher antigen, mucosal sCD40L, MCP-3, MCP-1, MIP-1\u03b1, and MIP-1\u03b2, and systemic IgA, IgM, IL-6, IL-8, IL-10, IL-15, IL-1RA, and IP-10, and uncoordinated early immune responses that went unresolved. Mucosal IL-8, IL-1\u03b2, and IFN-\u03b3 with systemic IL-1RA and IgA predicted COVID-19 outcomes. We present novel mucosal data, biomarkers, and therapeutic targets from a diverse cohort. Based on our findings, children and African Americans with COVID-19 have significantly lower IL-6 and IL-17 levels which may reduce responsiveness to drugs targeting IL-6 and IL-17. Unregulated immune responses persisted indicating moderate to severe COVID-19 cases may require prolonged treatments. Reliance on slower acting adaptive responses may cause immune crisis for some adults who encounter a novel virus. Despite the disparate outcomes for African Americans and children with COVID-19 and the vital role of mucosal immunity, the majority of mechanistic clinical studies lack these groups and mucosal assessments. To date, mucosal immune responses to SARS-CoV-2 has not been adequately described and we lack data from these understudied groups. This was a prospective cohort study of children and adults with confirmed COVID-19. Mortality was low (2.5%). Severity outcomes were associated with African American Race, shortness of breath, fever, respiratory disease, high blood pressure, and diabetes. We systematically characterized viral and immune factors in the mucosa and periphery and observed that moderate and severe COVID-19 were associated with longer duration, impaired clearance, early overexuberant antibody and cytokine production that was sustained. This study demonstrates that African Americans are at high risk of severe COVID-19 and display unique mucosal and peripheral immune responses. Children with COVID-19 also had distinct immune responses. This illustrates the importance of vaccination and careful clinical oversight of these populations (e.g., lower IL-6 and IL-17 levels may diminish tocilizumab, siltuximab, secukinumab, and brodalumab efficacy). This study identified generalizable outcomes predictors, systemic IL-1RA with mucosal IL-1\u03b2 and IL-8, and demonstrated the utility of mucosal sampling from diverse cohorts.","version":"1.1","doi":"10.1101/2023.08.04.551867","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.07.552249","pub_date":"2023-8-07","title":"phuEGO: A network-based method to reconstruct active signalling pathways from phosphoproteomics datasets","abstract":"Signalling networks are critical for virtually all cell functions. Our current knowledge of cell signalling has been summarised in signalling pathway databases, which, while useful, are highly biassed towards well-studied processes, and don\u2019t capture context specific network wiring or pathway cross-talk. Mass spectrometry-based phosphoproteomics data can provide a more unbiased view of active cell signalling processes in a given context, however, it suffers from low signal-to-noise ratio and poor reproducibility across experiments. Methods to extract active signalling signatures from such data struggle to produce unbiased and interpretable networks that can be used for hypothesis generation and designing downstream experiments. Here we present phuEGO, which combines three-layer network propagation with ego network decomposition to provide small networks comprising active functional signalling modules. PhuEGO boosts the signal-to-noise ratio from global phosphoproteomics datasets, enriches the resulting networks for functional phosphosites and allows the improved comparison and integration across datasets. We applied phuEGO to five phosphoproteomics data sets from cell lines collected upon infection with SARS CoV2. PhuEGO was better able to identify common active functions across datasets and to point to a subnetwork enriched for known COVID-19 targets. Overall, phuEGO provides a tool to the community for the improved functional interpretation of global phosphoproteomics datasets.","version":"1.1","doi":"10.1101/2023.08.07.552249","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.04.551565","pub_date":"2023-8-07","title":"CD4+ T cell senescence is associated with reduced reactogenicity in severe/critical COVID-19","abstract":"Aging is a critical risk factor for unfavorable clinical outcomes among COVID-19 patients and may affect vaccine efficacy. However, whether the senescence of T cells impact the progression to severe COVID-19 in the elderly individuals remains unclear. By using flow cytometry, we analyzed the frequency of senescent T cells (Tsens) in the peripheral blood from 100 elderly patients hospitalized for COVID-19 and compared the difference between mild/moderate and severe/critical illness. We also assessed correlations between the percentage of Tsens and the quantity and quality of spike-specific antibodies by ELISA, neutralizing antibody test kit and Elispot assay respectively, cytokine production profile of COVID-19 reactive T cells as well as plasma soluble factors by cytometric bead array (CBA). We found a significant elevated level of CD4+ Tsens in severe/critical disease compared to mild/moderate illness and patients with a higher level of CD4+ Tsens (>19.78%) showed a decreased survival rate as compared to those with a lower level (<19.78%), especially in the breakthrough infection. The percentage of CD4+ Tsens was negatively correlated with spike-specific antibody titers, neutralization ability and COVID-19 reactive IL-2+ CD4+ T cells. Additionally, IL-2 producing T cells and plasma levels of IL-2 were positively correlated with antibody levels. Our data illustrated that the percentage of CD4+ Tsens in the peripheral blood could act as an efficient biomarker for the capacity of spike-specific antibody production and the prognosis of severe COVID-19, especially in the breakthrough infection. Therefore, restoration of the immune response of CD4+ Tsens is one of the key factors to prevent severe illness and improve vaccine efficacy in older adults.","version":"1.1","doi":"10.1101/2023.08.04.551565","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.09.527892","pub_date":"2023-8-04","title":"Long COVID manifests with T cell dysregulation, inflammation, and an uncoordinated adaptive immune response to SARS-CoV-2","abstract":"Long COVID (LC), a type of post-acute sequelae of SARS-CoV-2 infection (PASC), occurs after at least 10% of SARS-CoV-2 infections, yet its etiology remains poorly understood. Here, we used multiple \u201comics\u201d assays (CyTOF, RNAseq/scRNAseq, Olink) and serology to deeply characterize both global and SARS-CoV-2-specific immunity from blood of individuals with clear LC and non-LC clinical trajectories, 8 months following infection and prior to receipt of any SARS-CoV-2 vaccine. Our analysis focused on deep phenotyping of T cells, which play important roles in immunity against SARS-CoV-2 yet may also contribute to COVID-19 pathogenesis. Our findings demonstrate that individuals with LC exhibit systemic inflammation and immune dysregulation. This is evidenced by global differences in T cell subset distribution in ways that imply ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. Individuals with LC harbored increased frequencies of CD4+ T cells poised to migrate to inflamed tissues, and exhausted SARS-CoV-2-specific CD8+ T cells. They also harbored significantly higher levels of SARS-CoV-2 antibodies, and in contrast to non-LC individuals, exhibited a mis-coordination between their SARS-CoV-2-specific T and B cell responses. RNAseq/scRNAseq and Olink analyses similarly revealed immune dysregulatory mechanisms, along with non-immune associated perturbations, in individuals with LC. Collectively, our data suggest that proper crosstalk between the humoral and cellular arms of adaptive immunity has broken down in LC, and that this, perhaps in the context of persistent virus, leads to the immune dysregulation, inflammation, and clinical symptoms associated with this debilitating condition.","version":"1.2","doi":"10.1101/2023.02.09.527892","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.03.551784","pub_date":"2023-8-04","title":"Comparing full variation profile analysis with the conventional consensus method in SARS-CoV-2 phylogeny","abstract":"This study proposes a novel approach to studying SARS-CoV-2 virus mutations through sequencing data comparison. Traditional consensus-based methods, which focus on the most common nucleotide at each position, might overlook or obscure the presence of low-frequency variants. Our method, in contrast, retains all sequenced nucleotides at each position, forming a genomic matrix. Utilizing simulated short reads from genomes with specified mutations, we contrasted our genomic matrix approach with the consensus sequence method. Our matrix methodology accurately reflected the known mutations and true compositions, demonstrating its efficacy in understanding the sample variability and their interconnections. Further tests using real data from GISAID and NCBI-SRA confirmed its reliability and robustness. As we see, the genomic matrix approach offers a more accurate representation of the viral genomic diversity, thereby providing superior insights into virus evolution and epidemiology. Future application recommendations are provided based on our observed results.","version":"1.1","doi":"10.1101/2023.08.03.551784","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.03.551813","pub_date":"2023-8-04","title":"Single-linkage molecular clustering of viral pathogens","abstract":"Public health faces the ongoing mission of safeguarding the population\u2019s health against various infectious diseases caused by a great number of pathogens. Epidemiology is an essential discipline in this field. With the rise of more advanced technologies, new tools are emerging to enhance the capability to intervene and control an epidemic. Among these approaches, molecular clustering comes forth as a promising option. However, appropriate genetic distance thresholds for defining clusters are poorly explored in contexts outside of Human Immunodeficiency Virus-1 (HIV-1). In this work, using the well-used pairwise Tamura-Nei 93 (TN93) distance threshold of 0.015 for HIV-1 as a point of reference for molecular cluster properties of interest, we perform molecular clustering on whole genome sequence datasets from HIV-1, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Zaire ebolavirus, and Mpox virus, to explore potential pairwise distances thresholds for these other viruses. We found the following pairwise TN93 distance thresholds as potential candidates for use in molecular clustering: 0.00016 (3 mutations) for Ebola, 0.00014 (4 mutations) for SARS-CoV-2, and 0.0000051 (1 mutation) for Mpox. This study provides valuable information for epidemic control strategies, and public health efforts in managing infectious diseases caused by these viruses. The identified pairwise distance thresholds for molecular clustering can serve as a foundation for future research and intervention to combat epidemics effectively. All relevant data and results can be found in the following repository: https://github.com/Niema-Lab/ENLACE-2023","version":"1.1","doi":"10.1101/2023.08.03.551813","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.02.551424","pub_date":"2023-8-03","title":"Neutralizing antibody responses and cellular responses against severe acute respiratory syndrome coronavirus 2 omicron subvariant BA.5 after an mRNA severe acute respiratory syndrome coronavirus 2 vaccine dose in kidney transplant recipients","abstract":"We examined the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein IgG antibody and neutralizing antibody titers and cellular immunity in 73 uninfected recipients and 17 uninfected healthy controls who received three doses of a coronavirus 2019 mRNA vaccine. Neutralizing antibody titers were evaluated using GFP-carrying recombinant SARS-CoV-2 with spike protein of B.1.1, omicron BA.1, or BA.5. For cellular immunity, peripheral blood mononuclear cells were stimulated with peptides corresponding to spike protein antigens of B.1.1, BA.1, and BA.5; spike-specific CD4/CD8 memory T cells were evaluated using intracellular cytokine staining. The median IgG antibody titers were 7.8 AU/mL in recipients and 143.0 AU/mL in healthy controls (p < 0.0001). Neutralizing antibody titers against all three viral variants were significantly lower in recipients (p < 0.0001). The number of spike-specific CD8 + memory T cells significantly decreased in recipients (p < 0.0001). Twenty recipients and seven healthy controls additionally received a bivalent omicron-containing booster vaccine, and IgG antibody and neutralizing antibody titers increased in both groups; however, the increase was significantly lower in recipients. Recipients did not gain sufficient immunity with a third dose of vaccine, suggesting a need to explore methods other than vaccines.","version":"1.1","doi":"10.1101/2023.08.02.551424","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.31.551349","pub_date":"2023-8-02","title":"Single-cell RNA sequencing reveals characteristics of myeloid cells in pulmonary post-acute sequelae of SARS-CoV-2","abstract":"Although our understanding of the immunopathology and subsequent risk and severity of COVID-19 disease is evolving, a detailed account of immune responses that contribute to the long-term consequences of pulmonary complication in COVID-19 infection remain unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequalae of SARS-CoV-2 infection with persistent pulmonary symptoms (PPASC). However, the dysregulation of the immune system that drives pulmonary sequelae in COVID-19 survivors and PASC sufferers remains largely unknown. To characterize the immunological features of pulmonary PASC (PPASC), we performed droplet-based single-cell RNA sequencing to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants na\u00efve to SARS-CoV-2 (Control) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC). We analyzed more than 34,139 PBMCs by integrating our dataset with previously reported control datasets (GSM4509024) cell distribution. In total, 11 distinct cell populations were identified based on the expression of canonical markers. The proportion of myeloid-lineage cells ([MLCs]; CD14+/CD16+monocytes and dendritic cells) was increased in PPASC compared to controls. MLCs from PPASC displayed up-regulation of genes associated with pulmonary symptoms/fibrosis, while glycolysis metabolism-related genes were downregulated. Similarly, pathway analysis showed that fibrosis- related (VEGF, WNT, and SMAD) and cell death pathways were up-regulated, but immune pathways were down-regulated in PPASC. In PPASC, we observed interactive VEGF ligand- receptor pairs among MLCs, and network modules in CD14+ (cluster 4) and CD16+ (Cluster 5) monocytes displayed a significant enrichment for biological pathways linked to adverse COVID- 19 outcomes, fibrosis, and angiogenesis. Further analysis revealed a distinct metabolic alteration in MLCs with a down-regulation of glycolysis/gluconeogenesis in PPASC compared to SARS- CoV-2 na\u00efve samples. This study offers valuable insights into the immune response and cellular landscape in PPASC. The presence of elevated MLC levels and their corresponding gene signatures associated with fibrosis, immune response suppression, and altered metabolic states suggests their potential role as a driver of PPASC.","version":"1.1","doi":"10.1101/2023.07.31.551349","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.31.551381","pub_date":"2023-8-02","title":"Growth media affects susceptibility of air-lifted human nasal epithelial cell cultures to SARS-CoV2, but not Influenza A, virus infection","abstract":"Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus-grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.","version":"1.1","doi":"10.1101/2023.07.31.551381","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.31.551037","pub_date":"2023-8-02","title":"Jointly modeling deep mutational scans identifies shifted mutational effects among SARS-CoV-2 spike homologs","abstract":"Deep mutational scanning (DMS) is a high-throughput experimental technique that measures the effects of thousands of mutations to a protein. These experiments can be performed on multiple homologs of a protein or on the same protein selected under multiple conditions. It is often of biological interest to identify mutations with shifted effects across homologs or conditions. However, it is challenging to determine if observed shifts arise from biological signal or experimental noise. Here, we describe a method for jointly inferring mutational effects across multiple DMS experiments while also identifying mutations that have shifted in their effects among experiments. A key aspect of our method is to regularize the inferred shifts, so that they are nonzero only when strongly supported by the data. We apply this method to DMS experiments that measure how mutations to spike proteins from SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.2) affect cell entry. Most mutational effects are conserved between these spike homologs, but a fraction have markedly shifted. We experimentally validate a subset of the mutations inferred to have shifted effects, and confirm differences of >1,000-fold in the impact of the same mutation on spike-mediated viral infection across spikes from different SARS-CoV-2 variants. Overall, our work establishes a general approach for comparing sets of DMS experiments to identify biologically important shifts in mutational effects. Amino-acid mutations to a protein have effects that can shift as the protein evolves or is put under new selective pressure. The effects of amino-acid mutations to a specific protein under a defined selective pressure can be measured by deep mutational scanning experiments. Here, we devise an approach to quantify shifts in mutational effects between experiments performed on different homologs (i.e. variants) of the same protein, or on the same protein selected under different conditions. We use this approach to compare experiments performed on three homologs of SARS-CoV-2 spike, identifying mutations that have shifted in their effect on spike-mediated viral infection by >1,000 fold across SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.07.31.551037","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.31.551354","pub_date":"2023-8-02","title":"Integrated Organ Immunity: Antigen-specific CD4-T cell-derived IFN-\u03b3 induced by BCG imprints prolonged lung innate resistance against respiratory viruses","abstract":"Bacille Calmette-Gu\u00e9rin (BCG) vaccination can confer non-specific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. Here, we show that mice immunized intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with SARS-CoV-2 and influenza. Protection was first evident between 14 - 21 days post vaccination, and lasted for at least 42 days. Remarkably, BCG induced a biphasic innate response in the lung, initially at day 1 and a subsequent prolonged phase starting at \u223c15 days post vaccination, and robust antigen-specific Th1 responses. MyD88-dependent TLR signaling was essential for the induction of the innate and Th1 responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or IFN-\u03b3 activity prior to infection obliterated innate activation and protection. Single cell and spatial transcriptomics revealed CD4-dependent expression of interferon-stimulated genes (ISGs) in myeloid, type II alveolar and lung epithelial cells. Thus, BCG elicits \u201cintegrated organ immunity\u201d where CD4+ T cells act on local myeloid and epithelial cells to imprint prolonged antiviral innate resistance.","version":"1.1","doi":"10.1101/2023.07.31.551354","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.01.551509","pub_date":"2023-8-02","title":"Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer","abstract":"DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90% along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.","version":"1.1","doi":"10.1101/2023.08.01.551509","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.01.551423","pub_date":"2023-8-02","title":"Gonadal androgens are associated with decreased type I interferon production by pDCs and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents","abstract":"mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Interferon type I (IFN-I) is an integral part of this early innate response and can prime several components of the adaptive immune response. Females are widely reported to respond better than males to seasonal tri- and quad-valent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production and female pDCs produce more IFN-I than male pDCs since the upstream receptor TLR7 is encoded by the X-chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains putative androgen response elements and androgens have been reported to suppress pDC IFN-I in-vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this cohort to determine the impact of IFN-I on anti-Spike and anti-receptor-binding domain titres to BNT162b2. Through LASSO modelling we determined that serum free testosterone was associated with reduced pDC IFN-I but, contrary to the well-described immunosuppressive role for androgens, the more potent androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live-attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together these data support a model where systemic IFN-I increased vaccine-mediated immune responses, but for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently vaccine-driven immunity. Type I interferons (IFN-I) are potent antiviral proteins which play a central role in activating the immune response and driving inflammation. IFN-I is predominantly produced by plasmacytoid dendritic cells (pDCs) and female pDCs produce more IFN-I than male pDCs. Consequently, females typically generate stronger antibody responses to vaccines such as seasonal influenza vaccines. In addition, females typically suffer more serious adverse events from vaccines. However, we recently reported in a study of adolescents that males generate stronger antibody responses to the SARS-CoV-2 mRNA vaccine BNT162b2 than females. Here we examine the IFN-I response of pDCs in adolescents co-/vaccinated with BNT162b2 and live-attenuated influenza vaccine (LAIV). We find that male sex hormones reduce pDC IFN-I but are associated with increased BNT162b2 antibody titres. We also observe that LAIV boosts BNT162b2 antibody titres through possible bystander activation of immune cells. These findings are consistent with a reportedly higher incidence of adverse events among males associated with this vaccine. Together these data suggest that IFN-I production typically enhances vaccine-specific immune responses but for new mRNA vaccines such as BNT162b2, that are modified to reduce innate immunogenicity, localised dampening of the IFN-I response in vaccinated tissue by male sex hormones may further delay the clearance of the vaccine, increasing vaccine antigen exposure and allowing time for a stronger antibody response.","version":"1.1","doi":"10.1101/2023.08.01.551423","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.25.542331","pub_date":"2023-8-02","title":"Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes","abstract":"RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consists of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the IBV genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.","version":"1.2","doi":"10.1101/2023.05.25.542331","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.28.550997","pub_date":"2023-8-01","title":"Tetherin restricts SARS-CoV-2 replication despite antagonistic effects of Spike and ORF7a","abstract":"SARS-CoV-2 infection induces interferon-stimulated genes, one of which encodes Tetherin, a transmembrane protein inhibiting the release of various enveloped viruses from infected cells. Previous studies revealed that SARS-CoV encodes two Tetherin antagonists: the Spike protein (S) inducing lysosomal degradation of Tetherin, and ORF7a altering its glycosylation. SARS-CoV-2 ORF7a has also been shown to antagonize Tetherin. Therefore, we here investigated whether SARS-CoV-2 S is also a Tetherin antagonist and compared the abilities and mechanisms of S and ORF7a in counteracting Tetherin. SARS-CoV and SARS-CoV-2 S reduced Tetherin cell surface levels in a cell type-dependent manner, possibly related to the basal protein levels of Tetherin. In HEK293T cells, under conditions of high exogenous Tetherin expression, SARS-CoV-2 S and ORF7a reduced total Tetherin levels much more efficiently than the respective counterparts derived from SARS-CoV. Nevertheless, ORF7a from both strains was able to alter Tetherin glycosylation. The ability to decrease total protein levels of Tetherin was conserved among S proteins from different SARS-CoV-2 variants (D614G, Cluster 5, \u03b1, \u03b3, \u03b4, \u03bf). While SARS-CoV-2 S and ORF7a both colocalized with Tetherin, only ORF7a directly interacted with the restriction factor. Despite the presence of two Tetherin antagonists, however, SARS-CoV-2 replication in Caco-2 cells was further enhanced upon Tetherin knockout. Altogether, our data show that endogenous Tetherin restricts SARS-CoV-2 replication, and that the antiviral activity of Tetherin is partially counteracted by two viral antagonists with differential and complementary modes of action, S and ORF7a. Viruses have adopted multiple strategies to cope with innate antiviral immunity. They blunt signaling and encode proteins that counteract antiviral host factors. One such factor is Tetherin, that tethers nascent virions to the cell membrane and interferes with virus release. For SARS-CoV, the viral glycoprotein Spike (S) and the accessory protein ORF7a are Tetherin antagonists. For pandemic SARS-CoV-2, such activity has only been shown for ORF7a. We therefore analyzed whether SARS-CoV-2 S is a Tetherin-counteracting protein and whether there are differences in the abilities of the viral proteins to antagonize Tetherin. Of note, the efficiency of Tetherin antagonism was more pronounced for S and ORF7a from SARS-CoV-2 compared to their SARS-CoV orthologs. Still, Tetherin was able to restrict SARS-CoV-2 replication. Our results highlight the fundamental importance of the innate immune response in the context of SARS-CoV-2 control and the evolutionary pressure on pathogenic viruses to withhold efficient Tetherin antagonism.","version":"1.1","doi":"10.1101/2023.07.28.550997","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.01.551417","pub_date":"2023-8-01","title":"Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning","abstract":"SARS-CoV-2 has continued to evolve throughout the COVID-19 pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2 and BA.2.75. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.","version":"1.1","doi":"10.1101/2023.08.01.551417","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.28.551035","pub_date":"2023-8-01","title":"Klebsiella pneumoniae Co-infection Leads to Fatal Pneumonia in SARS-CoV-2-infected Mice","abstract":"SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. Klebsiella pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a person\u2019s immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. We describe a mouse model to study disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae. Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae infection. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.","version":"1.1","doi":"10.1101/2023.07.28.551035","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.01.551500","pub_date":"2023-8-01","title":"Recombinant Rotavirus Expressing the Glycosylated S1 Protein of SARS-CoV-2","abstract":"Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment 7 (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment 7 RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the \u223c120-kDa S1 protein. The \u223c120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of \u223c80 kDa. Co-pulldown assays demonstrated that the \u223c120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibody, suggesting the rSA11/S1f virus expressed an authentic form of S1. Probably due to the presence of FLAG tags on their S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.08.01.551500","journal":"bioRxiv","score":null},{"id":"10.1101/2023.08.01.551467","pub_date":"2023-8-01","title":"Diversity of Short Linear Interaction Motifs in SARS-CoV-2 Nucleocapsid Protein","abstract":"Molecular mimicry of short linear interaction motifs has emerged as a key mechanism for viral proteins binding host domains and hijacking host cell processes. Here, we examine the role of RNA-virus sequence diversity in the dynamics of the virus-host interface, by analyzing the uniquely vast sequence record of viable SARS-CoV-2 species with focus on the multi-functional nucleocapsid protein. We observe the abundant presentation of motifs encoding several essential host protein interactions, alongside a majority of possibly non-functional and randomly occurring motif sequences absent in subsets of viable virus species. A large number of motifs emerge ex nihilo through transient mutations relative to the ancestral consensus sequence. The observed mutational landscape implies an accessible motif space that spans at least 25% of known eukaryotic motifs. This reveals motif mimicry as a highly dynamic process with the capacity to broadly explore host motifs, allowing the virus to rapidly evolve the virus-host interface.","version":"1.1","doi":"10.1101/2023.08.01.551467","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.28.550957","pub_date":"2023-8-01","title":"Epigenetic liquid biopsies reveal elevated vascular endothelial cell turnover and erythropoiesis in asymptomatic COVID-19 patients","abstract":"The full spectrum of tissues affected by SARS-CoV-2 infection is crucial for deciphering the heterogenous clinical course of COVID-19. Here, we analyzed DNA methylation and histone modification patterns in circulating chromatin to assess cell type-specific turnover in severe and asymptomatic COVID-19 patients, in relation to clinical outcome. Patients with severe COVID-19 had a massive elevation of circulating cell-free DNA (cfDNA) levels, which originated in lung epithelial cells, cardiomyocytes, vascular endothelial cells and erythroblasts, suggesting increased cell death or turnover in these tissues. The immune response to infection was reflected by elevated B cell and monocyte/macrophage cfDNA levels, and by evidence of an interferon response in cells prior to cfDNA release. Strikingly, monocyte/macrophage cfDNA levels (but not monocyte counts), as well as lung epithelium cfDNA and vascular endothelial cfDNA, predicted clinical deterioration and duration of hospitalization. Asymptomatic patients had elevated levels of immune-derived cfDNA but did not show evidence of pulmonary or cardiac damage. Surprisingly, these patients showed elevated levels of vascular endothelial cell and erythroblast cfDNA, suggesting that sub-clinical vascular and erythrocyte turnover are universal features of COVID-19, independent of disease severity. Epigenetic liquid biopsies provide non-invasive means of monitoring COVID-19 patients, and reveal sub-clinical vascular damage and red blood cell turnover.","version":"1.1","doi":"10.1101/2023.07.28.550957","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.28.551051","pub_date":"2023-8-01","title":"Targeted Amplification and Genetic Sequencing of the Severe Acute Respiratory Syndrome Coronavirus 2 Surface Glycoprotein","abstract":"The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.","version":"1.1","doi":"10.1101/2023.07.28.551051","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.31.551223","pub_date":"2023-7-31","title":"Immunities Specific to Both of the M Protein Ectodomain and RBD Synergize to Confer Cross-protection against SARS-CoV-2 Infections","abstract":"The effectiveness of the prototypic SARS-CoV-2 vaccine largely decreased overtime against the emerging virus strains, necessitating the universal vaccine development. The most abundant structural membrane (M) protein is highly conserved in amino acid sequence, which arouses our research interests in developing a universal immunogen based on it. Serological analysis showed that IgG responses specific to its N-terminal peptides can be strongly detected in many serum samples from both convalescent patients and vaccinees receiving inactivated vaccines, indicating the potential existence of human B-cell epitopes in reactive peptides. Microneutralization assays showed that the N-terminal peptide S2M2-30-specific hyperimmune serum was capable of cross-neutralizing the authentic viruses including wild-type HKU-001a, B.1.617.2/Delta, and Omicron subvariant BQ.1.1, and synergized with RBD-specific serum in reinforcing antiviral activities. Strong S2M2-30-specific immunities elicited in hACE2-transgenic mice could effectively inhibit B.1.1.7/Alpha (UK) infections. Our results suggest the potentiality of conserved M peptides as vaccine targets for conferring cross-protections against sarbecoviruses.","version":"1.1","doi":"10.1101/2023.07.31.551223","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.30.551145","pub_date":"2023-7-31","title":"Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19","abstract":"As many as 10\u201330% of the over 760 million survivors of COVID-19 develop persistent symptoms, of which respiratory symptoms are among the most common. To understand the cellular and molecular basis for respiratory PASC, we combined a machine learning based analysis of lung computed tomography (CT) with flow cytometry, single-cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples, and alveolar cytokine profiling in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed abnormalities involving 73% of the lung, which improved on subsequent imaging. Interstitial abnormalities suggestive of fibrosis on CT were associated with the increased numbers of neutrophils and presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid, reflecting unresolved epithelial injury. Persistent infection with SARS-CoV-2 was identified in six patients and secondary bacterial or viral infections in two others. These findings suggest that despite its heterogenous clinical presentations, respiratory PASC with radiographic abnormalities results from a common pathobiology characterized by the ongoing recruitment of neutrophils and profibrotic monocyte-derived alveolar macrophages driving lung fibrosis with implications for diagnosis and therapy.","version":"1.1","doi":"10.1101/2023.07.30.551145","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.19.549772","pub_date":"2023-7-31","title":"Structure, Dynamics and Free Energy Studies on the Effect of Spot Mutations on SARS-CoV-2 Spike Protein Binding with ACE2 Receptor","abstract":"The ongoing COVID-19 pandemic continues to infect people worldwide, and the virus continues to evolve in significant ways which can pose challenges to the efficiency of available vaccines and therapeutic drugs and cause future pandemic. Therefore, it is important to investigate the binding and interaction of ACE2 with different RBD variants. A comparative study using all-atom MD simulations was conducted on ACE2 binding with 8 different RBD variants, including N501Y, E484K, P479S, T478I, S477N, N439K, K417N and N501Y-E484K-K417N on RBD. Based on the RMSD, RMSF, and DSSP results, the overall binding of RBD variants with ACE2 is stable, and the secondary structures of RBD and ACE2 are consistent after the spot mutation. Besides that, a similar buried surface area, a consistent binding interface and a similar amount of hydrogen bonds formed between RBD with ACE2 although the exact residue pairs on the binding interface were modified. The change of binding free energy from spot mutation was predicted using the free energy perturbation (FEP) method. It is found that N501Y, N439K, and K417N can strengthen the binding of RBD with ACE2, while E484K and P479S weaken the binding, and S477N and T478I have negligible effect on the binding. Spot mutations modified the dynamic correlation of residues in RBD based on the dihedral angle covariance matrix calculation. Doing dynamic network analysis, a common intrinsic network community extending from the tail of RBD to central, then to the binding interface region was found, which could communicate the dynamics in the binding interface region to the tail thus to the other sections of S protein. The result can supply unique methodology and molecular insight on studying the molecular structure and dynamics of possible future pandemics and design novel drugs.","version":"1.2","doi":"10.1101/2023.07.19.549772","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.24.497555","pub_date":"2023-7-31","title":"SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery","abstract":"Today\u2019s genomics workflows typically require alignment to a reference sequence, which limits discovery. We introduce a new unifying paradigm, SPLASH (Statistically Primary aLignment Agnostic Sequence Homing), an approach that directly analyzes raw sequencing data to detect a signature of regulation: sample-specific sequence variation. The approach, which includes a new statistical test, is computationally efficient and can be run at scale. SPLASH unifies detection of myriad forms of sequence variation. We demonstrate that SPLASH identifies complex mutation patterns in SARS-CoV-2 strains, discovers regulated RNA isoforms at the single cell level, documents the vast sequence diversity of adaptive immune receptors, and uncovers biology in non-model organisms undocumented in their reference genomes: geographic and seasonal variation and diatom association in eelgrass, an oceanic plant impacted by climate change, and tissue-specific transcripts in octopus. SPLASH is a new unifying approach to genomic analysis that enables an expansive scope of discovery without metadata or references. SPLASH is a unifying, statistically driven approach to biological discovery from raw sequencing data, bypassing alignment.","version":"1.4","doi":"10.1101/2022.06.24.497555","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.27.550811","pub_date":"2023-7-28","title":"Butyrate Protects against SARS-CoV-2-induced Tissue Damage in Golden Hamsters","abstract":"Butyrate, produced by gut microbe during dietary fiber fermentation, plays anti-inflammatory and antioxidant effects in chronic inflammation diseases, yet it remains to be explored whether butyrate has protective effects against viral infections. Here, we demonstrated that butyrate alleviated tissue injury in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected golden hamsters with supplementation of butyrate before and during the infection. Butyrate-treated hamsters showed augmentation of type I interferon (IFN) response and activation of endothelial cells without exaggerated inflammation. In addition, butyrate regulated redox homeostasis by enhancing the activity of superoxide dismutase (SOD) to inhibit excessive apoptotic cell death. Therefore, butyrate exhibited an effective prevention against SARS-CoV-2 by upregulating antiviral immune responses and promoting cell survival. Since SARS-CoV-2 has caused severe disease characterized by acute respiratory distress syndrome (ARDS) in humans, it is essential to develop therapeutics based on relieving such severe clinical symptoms. Current therapy strategies mainly focus on individuals who have COVID-19, however, there is still a strong need for prevention and treatment of SARS-CoV-2 infection. This study showed that butyrate, a bacterial metabolite, improved the response of SARS-CoV-2-infected hamsters by reducing immunopathology caused by impaired antiviral defenses and inhibiting excessive apoptosis through reduction in oxidative stress.","version":"1.1","doi":"10.1101/2023.07.27.550811","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.18.499583","pub_date":"2023-7-28","title":"Discovering host protein interactions specific for SARS-CoV-2 RNA genome","abstract":"SARS-CoV-2, a positive single-stranded RNA virus, interacts with host cell proteins throughout its life cycle. These interactions are necessary for the host to recognize and hinder the replication of SARS-CoV-2. For the virus, to translate, transcribe and replicate its genetic material. However, many details of these interactions are still missing. We focused on the proteins binding to the highly structured 5\u2019 and 3\u2019 end regions of SARS-CoV-2 RNA that were predicted by the catRAPID algorithm to attract numerous proteins, exploiting RNA-Protein Interaction Detection coupled with Mass Spectrometry (RaPID-MS) technology. The validated interactors, which agreed with our predictions, include pseudouridine synthase PUS7 that binds to both ends of the viral RNA. Nanopore direct-RNA sequencing confirmed that the RNA virus is heavily modified, and PUS7 consensus regions were found in both SARS-CoV-2 RNA end regions. Notably, a modified site was detected in the viral Transcription Regulatory Sequence - Leader (TRS-L) and can influence the viral RNA structure and interaction propensity. Overall, our data map host protein interactions within SARS-CoV-2 UTR regions, pinpointing to a potential role of pseudouridine synthases and post-transcriptional modifications in the viral life cycle. These findings contribute to understanding virus-host dynamics and may guide the development of targeted therapies.","version":"1.3","doi":"10.1101/2022.07.18.499583","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.27.550709","pub_date":"2023-7-28","title":"Multiscale modelling of chromatin 4D organization in SARS-CoV-2 infected cells","abstract":"SARS-CoV-2 is able to re-structure chromatin organization and alters the epigenomic landscape of the host genome, though the mechanisms that produce such changes are still poorly understood. Here, we investigate with polymer physics chromatin re-organization of the host genome, in space and time upon SARS-CoV-2 viral infection. We show that re-structuring of A/B compartments is well explained by a re-modulation of intra-compartment homotypic affinities, which leads to the weakening of A-A interactions and enhances A-B mixing. At TAD level, re-arrangements are physically described by a general reduction of the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and spread in space of TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon (IFN) response, such as DDX58 or IFIT, results more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysis of time trajectories of pairwise gene-enhancer and higher-order contacts reveals that such variability derives from a more fluctuating dynamics in infected case, suggesting that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time. Overall, our study provides the first polymer-physics based 4D reconstruction of SARS-CoV-2 infected genome with mechanistic insights on the consequent gene mis-regulation.","version":"1.1","doi":"10.1101/2023.07.27.550709","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.27.550841","pub_date":"2023-7-28","title":"Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform","abstract":"The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525, was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming not just only that residues were linear but equally also the appropriate structural orientation of F6 upon the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV. Mapping epitopes within the receptor binding domains of viruses which are essential for viral tropism is critical for developing antiviral agents and subunit vaccines. In this study we have engineered the surface of Qubevirus to display a peptide library derived from the SARS-CoV S protein. In biopanning with S protein antibodies, we have identified three peptide fragments (EP1, EP2 and EP3) which reacted selectively with antibodies specific to the S protein. We demonstrated that all recombinant phage displayed peptide fragments both individually and as chimera exposed important immunological epitopes to their cognate antibodies. A peptide fragment F6 situated at S425-525, was found containing the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S437-492. The platform is rapidly to identify epitopes and receptor binding sites within viral receptors found in target host cell. Thus, this platform holds great significance.","version":"1.1","doi":"10.1101/2023.07.27.550841","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.28.550765","pub_date":"2023-7-28","title":"Prolonged exposure to lung-derived cytokines is associated with inflammatory activation of microglia in patients with COVID-19","abstract":"Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-\u0251 and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-19, was associated with lower levels of CXCL10, CCL8, and CCL2\u2014molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.","version":"1.1","doi":"10.1101/2023.07.28.550765","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.26.550755","pub_date":"2023-7-28","title":"New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity","abstract":"The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. Certain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches. Understanding Cas13a\u2019s allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants. These Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains. New strategies to improve Cas13a RNA-detection specificity developed via mismatch tolerance profiling, uncovering features that modulate specificity, and structure-guided engineering of LbuCas13a.","version":"1.1","doi":"10.1101/2023.07.26.550755","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.26.550688","pub_date":"2023-7-27","title":"Competitive fitness and homologous recombination of SARS-CoV-2 variants of concern","abstract":"SARS-CoV-2 variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22995 and 28866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination. SARS-CoV-2 variants or subvariants keep emerging and the epidemic strains keeps changing in humans and animals. The continued replacement of the epidemic strains was attributed to higher competitive fitness evolved by the newly appeared ones than the older ones, but which factors affect the final outcomes are still not entirely clear. In this study, we performed in vitro coinfection and serial passage with three VOCs and WT under different conditions. Our results showed that the competition outcomes of these viral strains varied in different cell lines or under different immune pressure, confirming the probable effects of these two factors for the competitive fitness of different SARS-CoV-2 viral strains. Meanwhile, strikingly, we found that coinfection and serial passage with different SARS-CoV-2 viral strains can mimic the recombination process of SARS-CoV-2 occurred in coinfection individual, indicating it is a novel model to investigate the SARS-CoV-2 recombination mechanism.","version":"1.1","doi":"10.1101/2023.07.26.550688","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.25.550460","pub_date":"2023-7-26","title":"Non-neutralizing SARS-CoV-2 N-terminal domain antibodies protect mice against severe disease using Fc-mediated effector functions","abstract":"Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of this non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb infusion did not suppress infectious viral titers in the lung as potently as NTD neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Finally, Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection in SARS-CoV-2 infection. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection. COVID-19 has claimed over 6.8 million lives worldwide and caused economic and social disruption globally. Preventing more deaths from COVID-19 is a principal goal of antibody biologic and vaccine developers. To guide design of such countermeasures, an understanding of how the immune system prevents severe COVID-19 disease is needed. We demonstrate here that antibody functions other than neutralization can contribute to protection from severe disease. Specifically, the functions of antibodies that rely on its Fc portion were shown to confer antibody-mediated protection of mice challenged with a mouse adapted version of SARS-CoV-2. Mice given an antibody that could not neutralize SARS-CoV-2 still showed a decrease in the amount of infectious virus in the lungs and less lung damage than mice given an irrelevant antibody. The decrease in infectious virus in the lungs was even larger when the non-neutralizing antibody was engineered to mediate non-neutralizing effector functions such as antibody-dependent cellular cytotoxicity more potently. Thus, in the absence of neutralization activity, non-neutralizing binding antibodies can contribute to the overall defense against SARS-CoV-2 infection and COVID-19 disease progression.","version":"1.1","doi":"10.1101/2023.07.25.550460","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.25.550568","pub_date":"2023-7-26","title":"Experimental infection of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) with SARS-CoV-2","abstract":"Elk (Cervus canadensis) and mule deer (Odocoileus hemionus) were experimentally evaluated for susceptibility to SARS-CoV-2. Elk did not shed infectious virus but produced low-level serological responses. Mule deer shed and transmitted virus in addition to mounting pronounced serological responses; they could therefore play a role in the epidemiology of SARS-CoV-2. Experimental infection of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) with SARS-CoV-2 revealed that while elk are minimally susceptible to infection, mule deer become infected, shed infectious virus, and can infect na\u00efve contacts.","version":"1.1","doi":"10.1101/2023.07.25.550568","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.24.550415","pub_date":"2023-7-25","title":"Mammalian cells-based platforms for the generation of SARS-CoV-2 virus-like particles","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Though many COVID-19 vaccines have been developed, most of them are delivered via intramuscular injection and thus confer relatively weak mucosal immunity against the natural infection. Virus-Like Particles (VLPs) are self-assembled nanostructures composed of key viral structural proteins, that mimic the wild-type virus structure but are non-infectious and non-replicating due to the lack of viral genetic material. In this study, we efficiently generated SARS-CoV-2 VLPs by co-expressing the four SARS-CoV-2 structural proteins, specifically the membrane (M), small envelope (E), spike (S) and nucleocapsid (N) proteins. We show that these proteins are essential and sufficient for the efficient formation and release of SARS-CoV-2 VLPs. Moreover, we used lentiviral vectors to generate human cell lines that stably produce VLPs. Because VLPs can bind to the virus natural receptors, hence leading to entry into cells and viral antigen presentation, this platform could be used to develop novel vaccine candidates that are delivered intranasally. Identification of protein requirements for SARS-CoV-2 VLP production by transient transfection Lentiviral transduction to create cells stably producing SARS-CoV-2 VLPs Isolation of cell clones for the production of SARS-CoV-2 VLPs New putative platforms for vaccine development","version":"1.1","doi":"10.1101/2023.07.24.550415","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.03.478930","pub_date":"2023-7-25","title":"Comprehensive analysis of next-generation sequencing data in COVID-19 and its secondary complications","abstract":"The ongoing pandemic of coronavirus disease 2019 (COVID-19) has made a serious public health threat globally. To discover key molecular changes in COVID-19 and its secondary complications, we analyzed next-generation sequencing (NGS) data of COVID-19. NGS data (GSE163151) was screened and downloaded from the Gene Expression Omnibus database (GEO). Differentially expressed genes (DEGs) were identified in the present study, using DESeq2 package in R programming software. Gene ontology (GO) and pathway enrichment analysis were performed, and the protein-protein interaction (PPI) network, module analysis, miRNA-hub gene regulatory network and TF-hub gene regulatory network were established. Subsequently, receiver operating characteristic curve (ROC) analysis was used to validate the diagonostics valuesof the hub genes. Firstly, 954 DEGs (477 up regulated and 477 down regulated) were identified from the four NGS dataset. GO enrichment analysis revealed enrichment of DEGs in genes related to the immune system process and multicellular organismal process, and REACTOME pathway enrichment analysis showed enrichment of DEGs in the immune system and formation of the cornified envelope. Hub genes were identified from the PPI network, module analysis, miRNA-hub gene regulatory network and TF-hub gene regulatory network. Furthermore, the ROC analysis indicate that COVID-19 and its secondary complications with following hub genes, namely, RPL10, FYN, FLNA, EEF1A1, UBA52, BMI1, ACTN2, CRMP1, TRIM42 and PTCH1, had good diagnostics values. This study identified several genes associated with COVID-19 and its secondary complications, which improves our knowledge of the disease mechanism.","version":"1.2","doi":"10.1101/2022.02.03.478930","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.24.550352","pub_date":"2023-7-24","title":"Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice","abstract":"Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.","version":"1.1","doi":"10.1101/2023.07.24.550352","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.24.550324","pub_date":"2023-7-24","title":"Revealing and evaluation of antivirals targeting multiple druggable sites of RdRp complex in SARS-CoV-2","abstract":"SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex consisting of nsp12, nsp7, and nsp8 as the key enzyme for viral genome replication and is a proven antiviral drug target. In this study, molecular interactions of nsp7 and nsp8 with nsp12 and the active site of nsp12 were coterminously targeted using in-silico screening of small molecule libraries to identify potential antivirals. Surface plasmon resonance (SPR) based assay using purified nsp7 and nsp8 proteins was developed, and the binding of identified molecules to targets was validated. The antiviral efficacy of identified small molecules was evaluated using cell-based assays, and potent antiviral effect with EC50 values of 0.56 \u03bcM, 0.73 \u03bcM, and 2.8 \u03bcM was demonstrated by fangchinoline, cepharanthine, and sennoside B, respectively. Further in vivo, investigation using hACE2 mice is being conducted. This is the first study that targets multiple sites in the RdRp complex of SARS-CoV-2 using a structure-based molecular repurposing approach and suggests potential therapeutic options for emerging variants of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.07.24.550324","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.12.484092","pub_date":"2023-7-24","title":"Mechanistic and thermodynamic characterization of antivirals targeting druggable pocket of SARS-CoV-2 nucleocapsid","abstract":"The N-terminal (NTD) and the C-terminal (CTD) domains comprises the structure of the SARS-CoV-2 Nucleocapsid (N) protein. Crystal structure of the SARS-CoV-2 N protein determined by Kang et al, 2020, reveals the N-terminal RNA binding domain as a unique drug binding site. The present study targets this unique pocket with identified antivirals using structure-based drug repurposing approach. The high-affinity binding of potential molecules was characterised thermodynamically using Isothermal titration calorimetry. The selected molecules showed an inhibitory RNA binding potential between 8.8 \u03bcM and 15.7 \u03bcM IC50 when evaluated with a fluorescent-based assay. Furthermore, in an in vitro cell-based antiviral assay, these ten antiviral molecules demonstrated high effectiveness in halting SARS-CoV-2 replication. Telmisartan and BMS-189453, the two highly potent antivirals, have \u223c0.98\u03bcM and 1.02 \u03bcM EC50 values with the selective index of >102, and >98, respectively. For the first time, this study presents drug molecules specifically targeting the NTD of SARS-CoV-2, offering essential insights for the development of therapeutic interventions against this virus, which is still a potential global threat to public health.","version":"1.4","doi":"10.1101/2022.03.12.484092","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.24.550379","pub_date":"2023-7-24","title":"The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 on Intracellular Trafficking, Autophagy, and Apoptosis","abstract":"The open reading frame 3a (ORF3a) is an accessory transmembrane protein that is important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (Yxx\u03a6; where x is any amino acid and \u03a6 is a hydrophobic amino acid with a bulky -R group). They have been implicated in the trafficking of membrane proteins to the cell plasma membrane and to intracellular organelles. Previous studies have indicated that mutation of the 160YSNV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. However, two additional canonical tyrosine-based sorting motifs (211YYQL213, 233YNKI236) exist in the cytoplasmic domain of ORF3a that have not been assessed. We removed all three potential tyrosine-based motifs and systematically restored them to assess the importance of each motif or combination of motifs that restored efficient trafficking to the cell surface and lysosomes. Our results indicate that the Yxx\u03a6 motif at position 160 was insufficient for the trafficking of ORF3a to the cell surface. Our studies also showed that ORF3a proteins with an intact Yxx\u03a6 at position 211 or at 160 and 211 were most important. We found that ORF3a cell surface expression correlated with the co-localization of ORF3a with LAMP-1 near the cell surface. These results suggest that Yxx\u03a6 motifs within the cytoplasmic domain may act cooperatively in ORF3a transport to the plasma membrane and endocytosis to lysosomes. Further, our results indicate that certain tyrosine mutants failed to activate caspase 3 and did not correlate with autophagy functions associated with this protein. Open reading frame 3a (ORF3a) encodes for the largest of the SARS-CoV-2 accessory proteins. While deletion of the ORF3a gene from SARS-CoV-2 results in a virus that replicates slightly less efficiently in cell culture, deletion also results in a virus that is less pathogenic in mouse models of SARS-CoV-2 infections. The ORF3a has been reported to be a viroporin, induces apoptosis and incomplete autophagy in cells. Thus, determining the domains involved in these functions will further our understanding of how this protein influences virus assembly and pathogenesis. Here, we investigated the role of the three potential tyrosine-based sorting signals in the cytoplasmic domain of the ORF3a on intracellular protein trafficking, apoptosis, and in the initiation of autophagy. Our results indicate that more than one Yxx\u03a6 motif is required for efficient transport of ORF3a, ORF3a expression resulted in minimal apoptosis, and cell surface expression was not required for autophagy.","version":"1.1","doi":"10.1101/2023.07.24.550379","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.23.549423","pub_date":"2023-7-24","title":"Phylogeny and evolution of the SARS-CoV-2 spike gene from December 2022 to February 2023","abstract":"By the end of 2022, new variants of SARS-CoV-2, such as BQ.1.1.10, BA.4.6.3, XBB, and CH.1.1, emerged with higher fitness than BA.5. The file (spikeprot0304), which contains spike protein sequences, isolates collected before March, 4, 2023, was downloaded from Global Initiative on Sharing All Influenza Data (GISAID). A total of 188 different spike protein sequences were chosen, of which their isolates were collected from December 2022 to February 2023. These sequences did not contain undetermined amino acid X, and each spike protein sequence had at least 100 identical isolate sequences in GISAID. Phylogenetic trees were reconstructed using IQ-TREE and MrBayes softwares. A median-join network was reconstructed using PopART software. Selection analyses were conducted using site model of PAML software. The phylogenetic tree of the spike DNA sequences revealed that the majority of variants belonged to three major lineages: BA.2 (BA.1.1.529.2), BA.5 (BA.1.1.529.5), and XBB. The median network showed that these lineages had at least six major diversifying centers. The spike DNA sequences of these diversifying centers had the representative accession IDs (EPI_ISL_) of 16040256 (BN.1.2), 15970311 (BA.5), 16028739 (BA.5.11), 16028774 (BQ.1), 16027638 (BQ.1.1.23), and 16044705 (XBB.1.5). Selection analyses revealed 26 amino-acid sites under positive selection. These sites included L5, V83, W152, G181, N185, V213, H245, Y248, D253, S255, S256, G257, R346, R408, K444, V445, G446, N450, L452, N460, F486, Q613, Q675, T883, P1162, and V1264. The spike proteins of SARS-CoV-2 from December 2022 to February 2023 were characterized by a swarm of variants that were evolved from three major lineages: BA.2 (BA.1.1.529.2), BA.5 (BA.1.1.529.5), and XBB. These lineages had at least six diversifying centers. Selection analysis identified 26 amino acid sites were under positive selection. Continued surveillance and research are necessary to monitor the evolution and potential impact of these variants on public health.","version":"1.1","doi":"10.1101/2023.07.23.549423","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.21.550109","pub_date":"2023-7-24","title":"Genomic epidemiology of European Aspergillus fumigatus causing COVID-19-associated pulmonary aspergillosis in Europe","abstract":"The opportunistic fungus Aspergillus fumigatus has been found to cause coinfections in patients with severe SARS-CoV-2 virus infection, leading to COVID-19-associated pulmonary aspergillosis (CAPA). The CAPA all-cause mortality rate is approximately 50% and may be complicated by azole-resistance. Genomic epidemiology can help shed light on the genetics of A. fumigatus causing CAPA including the prevalence of alleles that are associated with azole-resistance. Here, a population genomic analysis of 21 CAPA isolates from four European countries is presented. The CAPA isolates were compared with A. fumigatus from a wider population of 167 non-CAPA clinical isolates and 73 environmental isolates. Bioinformatic analysis and antifungal susceptibility testing were performed to quantify resistance and identify possible genetically-encoded azole-resistant mechanisms. Phylogenetic analysis of the 21 CAPA isolates showed a lack of genetic distinction from the wider A. fumigatus population, with isolates distributed within two distinct clades (A and B), with the majority of the CAPA isolates in clade B (71.4%). The prevalence of phenotypic azole-resistance in CAPA was 14.3% (n=3/21); all three CAPA isolates contained a known resistance-associated cyp51A polymorphism. CAPA isolates are drawn from the wider A. fumigatus population rather than forming a unique genetic background showing that COVID-19 patients are susceptible to the entire A. fumigatus population. However, the relatively high prevalence of azole-resistance alleles that we document poses a threat to treatment success rates, warranting enhanced detection and surveillance of A. fumigatus genotypes in these patients. Furthermore, potential changes to antifungal first-line treatment guidelines may be needed to improve patient outcomes.","version":"1.1","doi":"10.1101/2023.07.21.550109","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.19.549739","pub_date":"2023-7-20","title":"Design of SARS-CoV-2 protease inhibitors with improved affinity and reduced sensitivity to mutations","abstract":"Inhibitors of the SARS-CoV-2 main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function on these mutants are thus urgently needed. We hypothesized that the covalent HCV protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more tightly than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to NTV. A crucial feature of ML2006a4 is a novel derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Finally, ML2006a4 is less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory drug design can preemptively address potential resistance mechanisms.","version":"1.1","doi":"10.1101/2023.07.19.549739","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.549113","pub_date":"2023-7-20","title":"Megakaryocyte infection by SARS-CoV-2 drives the formation of pathogenic afucosylated IgG antibodies in mice","abstract":"More than 90% of total human plasma immunoglobulin G (IgG) is found in a fucosylated form, but specific IgGs with low core fucosylation (afucosylated IgGs) are found in response to infections with enveloped viruses and to alloantigens on blood cells. Afucosylated IgGs mediate immunopathology in severe COVID-19 and dengue fever in humans. In COVID-19, the early formation of non-neutralizing afucosylated IgG against the spike protein predicts and directly mediates disease progression to severe form. IgG lacking core fucosylation causes dramatically increased antibody-dependent cellular toxicity mediated by intense Fc\u03b3R-mediated stimulation of macrophages, monocytes, natural killer cells, and platelets. The mechanism and the context within which afucosylated IgG formation occurs in response to enveloped virus antigens have remained elusive thus far in COVID-19, dengue fever, and other infections. This study demonstrates that administration of human bone marrow megakaryocytes infected by SARS-CoV-2 into the circulation of K18-hACE2 transgenic mice drives the formation of pathogenic afucosylated anti-spike IgG antibodies, and is sufficient to reproduce severe COVID-19 manifestations of pulmonary vascular thrombosis, acute lung injury, and death in mice.","version":"1.2","doi":"10.1101/2023.07.14.549113","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.19.549731","pub_date":"2023-7-20","title":"Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors","abstract":"Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.","version":"1.1","doi":"10.1101/2023.07.19.549731","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.01.538516","pub_date":"2023-7-20","title":"Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting","abstract":"The continuous emergence of highly immune evasive SARS-CoV-2 variants, like XBB.1.51,2 and XBB.1.163,4, highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by wildtype (WT)-based vaccination would compromise the antibody response to Omicron-based boosters5-9. Vaccination strategies that can counter immune imprinting are critically needed. In this study, we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. Our results show that in mice, the efficacy of single Omicron-boosting is heavily limited by immune imprinting, especially when using variants antigenically distinct from WT, like XBB, while the concerning situation could be largely mitigated by a second Omicron booster. Similarly, in humans, we found that repeated Omicron infections could also alleviate WT-vaccination-induced immune imprinting and generate high neutralizing titers against XBB.1.5 and XBB.1.16 in both plasma and nasal mucosa. By isolating 781 RBD-targeting mAbs from repeated Omicron infection cohorts, we revealed that double Omicron exposure alleviates immune imprinting by generating a large proportion of highly matured and potent Omicron-specific antibodies. Importantly, epitope characterization using deep mutational scanning (DMS) showed that these Omicron-specific antibodies target distinct RBD epitopes compared to WT-induced antibodies, and the bias towards non-neutralizing epitopes observed in single Omicron exposures due to imprinting was largely restored after repeated Omicron stimulation, together leading to a substantial neutralizing epitope shift. Based on the DMS profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated the combinations of these mutations could further boost XBB.1.5\u2019s immune-evasion capability while maintaining high ACE2 binding affinity. Our findings suggest the WT component should be abandoned when updating COVID-19 vaccine antigen compositions to XBB lineages, and those who haven\u2019t been exposed to Omicron yet should receive two updated vaccine boosters.","version":"1.4","doi":"10.1101/2023.05.01.538516","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.19.549800","pub_date":"2023-7-20","title":"Protective role of N-acetylcysteine and Sulodexide on endothelial cells after SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus-2 causes hyperinflammation and activation of coagulation cascade and in the result aggravates endothelial cell dysfunction. N-acetylcysteine and Sulodexide have been found to mitigate endothelial damage. The influence on coronary artery endothelial cells of serum collected after 4+/-1 months from coronavirus infection was studied. The concentrations of serum samples of interleukin 6, von Willebrand Factor, tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 were studied. The cultures with serum of patients after coronavirus infection were incubated with N-acetylocysteine and Sulodexide to estimate their potential protective role. The blood inflammatory parameters were increased in the group of cultures incubated with serum from patients after coronavirus infection. Supplementation of the serum from patients after coronavirus infection with N-acetylcysteine or Sulodexide reduced the synthesis of interleukin 6, von Willebrand Factor. No changes in the synthesis of tissue Plasminogen Activator were observed. N-acetylcysteine reduced the synthesis of Plasminogen Activator Inhibitor-1. N-acetylcysteine and Sulodexide increased the tPA/PAI-1 ratio. N-acetylcysteine may have a role in reducing the myocardial injury occurring in the post-COVID-19 syndrome. Sulodexide can also play a protective role in post-COVID-19 patients.","version":"1.1","doi":"10.1101/2023.07.19.549800","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.491266","pub_date":"2023-7-19","title":"Activated interstitial macrophages are a predominant target of viral takeover and focus of inflammation in COVID-19 initiation in human lung","abstract":"Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices cultured ex vivo can be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of \u201cinfection pseudotime\u201d for individual lung cell types. This revealed that the prominent SARS-CoV-2 target is a population of activated interstitial macrophages (IMs), which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover of IMs, there is cell-autonomous induction of a pro-fibrotic program (TGFB1, SPP1), and an inflammatory program characterized by the early interferon response, chemokines (CCL2, 7, 8, 13, CXCL10) and cytokines (IL6, IL10), along with destruction of cellular architecture and formation of dense viral genomic RNA bodies revealed by super-resolution microscopy. In contrast, alveolar macrophages (AMs) showed neither viral takeover nor induction of a substantial inflammatory response, although both purified AMs and IMs supported production of infectious virions. Spike-dependent viral entry into AMs was neutralized by blockade of ACE2 or Sialoadhesin/CD169, whereas IM entry was neutralized only by DC-SIGN/CD209 blockade. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated IMs as a prominent site of viral takeover and focus of inflammation and fibrosis, and suggest therapeutic targeting of the DC-SIGN/CD209 entry mechanism to prevent IM infection, destruction and early pathology in COVID-19 pneumonia. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.","version":"1.2","doi":"10.1101/2022.05.10.491266","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.18.549478","pub_date":"2023-7-19","title":"Epigenetic Age Acceleration in Surviving versus Deceased COVID-19 Patients with Acute Respiratory Distress Syndrome following Hospitalization","abstract":"Aging has been reported as a major risk factor for severe symptoms and higher mortality rates in COVID-19 patients. Molecular hallmarks such as epigenetic alterations and telomere attenuation reflect the biological process of aging. Epigenetic clocks have been shown to be valuable tools for measuring biological age in a variety of tissues and samples. As such, these epigenetic clocks can determine accelerated biological aging and time-to-mortality across various tissues. Previous reports have shown accelerated biological aging and telomere attrition acceleration following SARS-CoV-2 infection. However, the effect of accelerated epigenetic aging on outcome (death/recovery) in COVID-19 patients with Acute Respiratory Distress Syndrome (ARDS) has not been well investigated. In this study, we measured DNA methylation age and telomere attrition in 87 severe COVID-19 cases with ARDS under mechanical ventilation. Furthermore, we compared dynamic changes in epigenetic aging across multiples time-points until recovery or death. Epigenetic age was measured using the Horvath, Hannum, DNAm skin and blood, GrimAge, and PhenoAge clocks, whereas telomere length was calculated using the surrogate marker DNAmTL. Our analysis revealed significant accelerated epigenetic aging but no telomere attrition acceleration in severe COVID-19 cases. In addition, we observed epigenetic age deceleration at inclusion vs end of follow-up in recovered but not in deceased COVID-19 cases using certain clocks. When comparing dynamic changes in epigenetic age acceleration (EAA), we detected higher EAA using both the Horvath and PhenoAge clocks in deceased vs recovered patients. The DNAmTL measurements revealed telomere attrition acceleration in deceased COVID19 patients between inclusion and end of follow-up as well as a significant change in dynamic telomere attrition acceleration when comparing patients who recovered vs those who died. In conclusion, EAA and telomere attrition acceleration was associated with treatment outcome in hospitalized COVID-19 Patients with ARDS. A better understanding of the long-term effects of EAA in COVID19 patients and how they might contribute to Long COVID symptoms in recovered individuals is urgently needed.","version":"1.1","doi":"10.1101/2023.07.18.549478","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.18.549530","pub_date":"2023-7-18","title":"ACE2 mimetic antibody potently neutralizes all SARS-CoV-2 variants and fully protects in XBB.1.5 challenged monkeys","abstract":"The rapid evolution of SARS-CoV-2 to variants with improved transmission efficiency and reduced sensitivity to vaccine-induced humoral immunity has abolished the protective effect of licensed therapeutic human monoclonal antibodies (mAbs). To fill this unmet medical need and protect vulnerable patient populations, we isolated the P4J15 mAb from a previously infected, vaccinated donor, with <20 ng/ml neutralizing activity against all Omicron variants including the latest XBB.2.3 and EG.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares \u223c93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. Although SARS-CoV-2 mutants escaping neutralization by P4J15 were selected in vitro, these displayed lower infectivity, poor binding to ACE2, and the corresponding \u2018escape\u2019 mutations are accordingly rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, we show that P4J15 confers complete prophylactic protection. We conclude that the P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug.","version":"1.1","doi":"10.1101/2023.07.18.549530","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.549044","pub_date":"2023-7-18","title":"Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies","abstract":"Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays.","version":"1.2","doi":"10.1101/2023.07.14.549044","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.17.549430","pub_date":"2023-7-18","title":"Deep mutational scanning of whole SARS-CoV-2 spike in an inverted infection system","abstract":"In order to investigate SARS-CoV-2 mutations and their impact on immune evasion and infectivity, we developed a Deep Mutational Scanning (DMS) platform utilizing an inverted infection assay to measure spike expression, ACE2 affinity, and viral infectivity in human cells. Surprisingly, our analysis reveals that spike protein expression, rather than ACE2 affinity, is the primary factor affecting viral infectivity and correlated with SARS-CoV-2 evolution. Notably, within the N-terminal domain (NTD), spike expression and infectivity-enhancing mutations are concentrated in flexible loops. We also observed that Omicron variants BA.1 and BA.2 exhibit immune evasion through receptor binding domain (RBD) mutations, although these mutations reduce structural stability. Interestingly, the NTD has evolved to increase stability, compensating for the RBD instability and resulting in heightened overall infectivity. Our findings, available in SpikeScanDB, emphasize the importance of spike expression levels and compensatory mutations in both the NTD and RBD domains for shaping Omicron variant infectivity.","version":"1.1","doi":"10.1101/2023.07.17.549430","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.18.549524","pub_date":"2023-7-18","title":"Breakthrough infection elicits hypermutated IGHV3-53/3-66 public antibodies with broad and potent neutralizing activity against SARS-CoV-2 variants including BQ and XBB lineages","abstract":"The rapid emergence of SARS-CoV-2 variants of concern (VOCs) calls for efforts to study broadly neutralizing antibodies elicited by infection or vaccination so as to inform the development of vaccines and antibody therapeutics with broad protection. Here, we identified two convalescents of breakthrough infection with relatively high neutralizing titers against all tested viruses including BQ and XBB lineages. Among 50 spike-specific monoclonal antibodies (mAbs) cloned from their B cells, the top 6 neutralizing mAbs (KXD01-06) belong to previously defined IGHV3-53/3-66 public antibodies. Although most antibodies in this class are dramatically escaped by VOCs, KXD01-06 exhibit broad neutralizing capacity with the IC50s of KXD01 ranging from 0.011\u223c0.059\u03bcg/ml. Deep mutational scanning reveals that KXD01-06 target highly conserved sites on RBD including D420, Y421, L455, F456, A475 and N487. Genetic and functional analysis further indicates that the extent of somatic hypermutation is critical for the breadth of IGHV3-53/3-66 public antibodies. Overall, we discovered and characterized IGHV3-53/3-66 public antibodies with broad and potent neutralizing activity against SARS-CoV-2, which provides rationale for novel vaccines and antibody therapeutics based on this class of antibodies.","version":"1.1","doi":"10.1101/2023.07.18.549524","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.17.549425","pub_date":"2023-7-18","title":"A laboratory framework for ongoing optimisation of amplification based genomic surveillance programs","abstract":"Constantly evolving viral populations affect the specificity of primers and quality of genomic surveillance. This study presents a framework for continuous optimisation of sequencing efficiency for public health surveillance based on the ongoing evolution of the COVID-19 pandemic. SARS-CoV-2 genomic clustering capacity based on three amplification based whole genome sequencing schemes was assessed using decreasing thresholds of genome coverage and measured against epidemiologically linked cases. Overall genome coverage depth and individual amplicon depth were used to calculate an amplification efficiency metric. Significant loss of genome coverage over time was documented which was recovered by optimisation of primer pooling or implementation of new primer sets. A minimum of 95% genome coverage was required to cluster 94% of epidemiologically defined SARS-CoV-2 transmission events. Clustering resolution fell to 70% when only 85% of genome coverage was achieved. The framework presented in this study can provide public health genomic surveillance programs a systematic process to ensure an agile and effective laboratory response during rapidly evolving viral outbreaks.","version":"1.1","doi":"10.1101/2023.07.17.549425","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.13.548462","pub_date":"2023-7-17","title":"A simulation framework for modeling the within-patient evolutionary dynamics of SARS-CoV-2","abstract":"The global impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to considerable interest in detecting novel beneficial mutations and other genomic changes that may signal the development of variants of concern (VOCs). The ability to accurately detect these changes within individual patient samples is important in enabling early detection of VOCs. Such genomic scans for positive selection are best performed via comparison of empirical data to simulated data wherein evolutionary factors, including mutation and recombination rates, reproductive and infection dynamics, and purifying and background selection, can be carefully accounted for and parameterized. While there has been work to quantify these factors in SARS-CoV-2, they have yet to be integrated into a baseline model describing intra-host evolutionary dynamics. To construct such a baseline model, we develop a simulation framework that enables one to establish expectations for underlying levels and patterns of patient-level variation. By varying eight key parameters, we evaluated 12,096 different model-parameter combinations and compared them to existing empirical data. Of these, 592 models (\u223c5%) were plausible based on the resulting mean expected number of segregating variants. These plausible models shared several commonalities shedding light on intra-host SARS-CoV-2 evolutionary dynamics: severe infection bottlenecks, low levels of reproductive skew, and a distribution of fitness effects skewed towards strongly deleterious mutations. We also describe important areas of model uncertainty and highlight additional sequence data that may help to further refine a baseline model. This study lays the groundwork for the improved analysis of existing and future SARS-CoV-2 within-patient data. Despite its tremendous impact on human health, a comprehensive evolutionary baseline model has yet to be developed for studying the within-host population genomics of SARS-CoV-2. Importantly, such modeling would enable improved analysis and provide insights into the key evolutionary dynamics governing SARS-CoV-2 evolution. Given this need, we have here quantified a set of plausible baseline models via large-scale simulation. The commonly shared features of these relevant models - including severe infection bottlenecks, low levels of progeny skew, and a high rate of strongly deleterious mutations - lay the foundation for sophisticated analyses of SARS-CoV-2 evolution within patients using these baseline models.","version":"1.2","doi":"10.1101/2023.07.13.548462","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.15.549135","pub_date":"2023-7-17","title":"Pre-existing interferon gamma conditions the lung to mediate early control of SARS-CoV-2","abstract":"Interferons (IFNs) are critical for anti-viral host defence. Type-1 and type-3 IFNs are typically associated with early control of viral replication and promotion of inflammatory immune responses; however, less is known about the role of IFN\u03b3 in anti-viral immunity, particularly in the context of SARS-CoV-2. We have previously observed that lung infection with attenuated bacteria Mycobacterium bovis BCG achieved though intravenous (iv) administration provides strong protection against SARS-CoV-2 (SCV2) infection and disease in two mouse models. Assessment of the pulmonary cytokine milieu revealed that iv BCG induces a robust IFN\u03b3 response and low levels of IFN\u03b2. Here we examined the role of ongoing IFN\u03b3 responses due to pre-established bacterial infection on SCV2 disease outcomes in two murine models. We report that IFN\u03b3 is required for iv BCG induced reduction in pulmonary viral loads and that this outcome is dependent on IFN\u03b3 receptor expression by non-hematopoietic cells. Further analysis revealed that BCG infection promotes the upregulation of interferon-stimulated genes (ISGs) with reported anti-viral activity by pneumocytes and bronchial epithelial cells in an IFN\u03b3-dependent manner, suggesting a possible mechanism for the observed protection. Finally, we confirmed the importance of IFN\u03b3 in these anti-viral effects by demonstrating that the recombinant cytokine itself provides strong protection against SCV2 challenge when administered intranasally. Together, our data show that a pre-established IFN\u03b3 response within the lung is protective against SCV2 infection, suggesting that concurrent or recent infections that drive IFN\u03b3 may limit the pathogenesis of SCV2 and supporting possible prophylactic uses of IFN\u03b3 in COVID-19 management.","version":"1.1","doi":"10.1101/2023.07.15.549135","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.16.549184","pub_date":"2023-7-17","title":"Using big sequencing data to identify chronic SARS-Coronavirus-2 infections","abstract":"The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants, many of which may have arisen during chronic infections of immunocompromised individuals. Here, we harness a global phylogeny of \u223c11.7 million SARS-CoV-2 genomes and search for clades composed of sequences with identical metadata (location, age, and sex) spanning more than 21 days. We postulate that such clades represent repeated sampling from the same chronically infected individual. A set of 271 such chronic-like clades was inferred, and displayed signatures of an elevated rate of adaptive evolution, in line with validated chronic infections. More than 70% of adaptive mutations present in currently circulating variants are found in BA.1 chronic-like clades that predate the circulating variants by months, demonstrating the predictive nature of such clades. We find that in chronic-like clades the probability of observing adaptive mutations is approximately 10-20 higher than that in global transmission chains. We next employ language models to find mutations most predictive of chronic infections and use them to infer hundreds of additional chronic-like clades in the absence of metadata and phylogenetic information. Our proposed approach presents an innovative method for mining extensive sequencing data and providing valuable insights into future evolutionary patterns.","version":"1.1","doi":"10.1101/2023.07.16.549184","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.12.503821","pub_date":"2023-7-17","title":"SIRT-1 is required for release of enveloped enteroviruses","abstract":"Enterovirus D68 is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1\u2019s proviral activity is, we demonstrate, mediated through the repression of ER stress. Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.","version":"1.3","doi":"10.1101/2022.08.12.503821","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.549076","pub_date":"2023-7-15","title":"Oral immunization with rVSV bivalent vaccine elicits protective immune responses, including ADCC, against both SARS-CoV-2 and Influenza A viruses","abstract":"COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SP\u0394C1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral cavity delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significant low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.","version":"1.1","doi":"10.1101/2023.07.14.549076","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.549077","pub_date":"2023-7-15","title":"Absence of SARS-CoV-2 in Wildlife of Northeastern Minnesota and Isle Royale National Park","abstract":"We investigated the presence of SARS-CoV-2 in free-ranging wildlife populations in Northeastern Minnesota on the Grand Portage Indian Reservation and Isle Royale National Park. 120 nasal samples were collected from white-tailed deer, moose, gray wolves, and black bears monitored for conservation efforts during 2022-2023. Samples were tested for viral RNA by RT-qPCR using the CDC N1/N2 primer set. Our data indicate that no wildlife samples were positive for SARS-CoV-2 RNA. Continued surveillance is therefore crucial to better understand the changing landscape of zoonotic SARS-CoV-2 in the Upper Midwest.","version":"1.1","doi":"10.1101/2023.07.14.549077","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.10.519890","pub_date":"2023-7-14","title":"SWAMPy: Simulating SARS-CoV-2 Wastewater Amplicon Metagenomes with Python","abstract":"Tracking SARS-CoV-2 variants through genomic sequencing has been an important part of the global response to the pandemic. As well as whole-genome sequencing of clinical samples, this surveillance effort has been aided by amplicon sequencing of wastewater samples, which proved effective in real case studies. Because of its relevance to public healthcare decisions, testing and benchmarking wastewater sequencing analysis methods is also crucial, which necessitates a simulator. Although metagenomic simulators exist, none are fit for the purpose of simulating the metagenomes produced through amplicon sequencing of wastewater. Our new simulation tool, SWAMPy (Simulating SARS-CoV-2 Wastewater Amplicon Metagenomes with Python), is intended to provide realistic simulated SARS-CoV-2 wastewater sequencing datasets with which other programs that rely on this type of data can be evaluated and improved. The code for this project is available at https://github.com/goldman-gp-ebi/SWAMPy It can be installed on any Unix-based operating system and is available under the GPL-v3 license.","version":"1.2","doi":"10.1101/2022.12.10.519890","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.549026","pub_date":"2023-7-14","title":"Performance of amplicon and capture based next-generation sequencing approaches for the epidemiological surveillance of Omicron SARS-CoV-2 and other variants of concern","abstract":"To control the SARS-CoV-2 pandemic, healthcare systems have focused on ramping up their capacity for epidemiological surveillance through viral whole genome sequencing. In this paper, we tested the performance of two protocols of SARS-CoV-2 nucleic acid enrichment, an amplicon enrichment using different versions of the ARTIC primer panel and a hybrid-capture method using KAPA RNA Hypercap. We focused on the challenge of the Omicron variant sequencing, the advantages of automated library preparation and the influence of the bioinformatic analysis in the final consensus sequence. All 94 samples were sequenced using Illumina iSeq 100 and analysed with two bioinformatic pipelines: a custom-made pipeline and an Illumina-owned pipeline. We were unsuccessful in sequencing six samples using the capture enrichment due to low reads. On the other hand, amplicon dropout and mispriming caused the loss of mutation G21987A and the erroneous addition of mutation T15521A respectively using amplicon enrichment. Overall, we found high sequence agreement regardless of method of enrichment, bioinformatic pipeline or the use of automation for library preparation in eight different SARS-CoV-2 variants. Automation and the use of a simple app for bioinformatic analysis can simplify the genotyping process, making it available for more diagnostic facilities and increasing global vigilance.","version":"1.1","doi":"10.1101/2023.07.14.549026","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.14.548971","pub_date":"2023-7-14","title":"Deep spatial proteomic exploration of severe COVID-19-related pulmonary injury in post-mortem specimens","abstract":"The lung, as a primary target of SARS-CoV-2, exhibits heterogeneous microenvironment accompanied by various histopathological changes following virus infection. However, comprehensive insight into the protein basis of COVID-19-related pulmonary injury with spatial resolution is currently deficient. Here, we generated a region-resolved quantitative proteomic atlas of seven major pathological structures within the lungs of COVID-19 victims by integrating histological examination, laser microdissection, and ultrasensitive proteomic technologies. Over 10,000 proteins were quantified across 71 dissected FFPE post-mortem specimens. By comparison with control samples, we identified a spectrum of COVID-19-induced protein and pathway dysregulations in alveolar epithelium, bronchial epithelium, and pulmonary blood vessels, providing evidence for the proliferation of transitional-state pneumocytes. Additionally, we profiled the region-specific proteomes of hallmark COVID-19 pulmonary injuries, including bronchiole mucus plug, pulmonary fibrosis, airspace inflammation, and hyperplastic alveolar type 2 cells. Bioinformatic analysis revealed the enrichment of cell-type and functional markers in these regions (e.g. enriched TGFBI in fibrotic region). Furthermore, we identified the up-regulation of proteins associated with viral entry, host restriction, and inflammatory response in COVID-19 lungs, such as FURIN and HGF. Collectively, this study provides spatial proteomic insights for understanding COVID-19-caused pulmonary injury, and may serve as a valuable reference for improving therapeutic intervention for severe pneumonia.","version":"1.1","doi":"10.1101/2023.07.14.548971","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.13.548895","pub_date":"2023-7-14","title":"Multivalent Exosome based protein vaccine: a \u201cmix and match\u201d approach to epidemic viruses\u2019 challenges","abstract":"Endemic viruses are becoming increasingly the norm, and the development of a rapid and effective vaccine is emergent. Here, we used our StealthX \u2122 exosome platform to express either Influenza H3 (Stealth\u2122 X-Hemagglutinin, STX-H3) or SARS-CoV-2 Delta spike (Stealth\u2122 X-Spike, STX-S) protein on the surface and facilitate their trafficking to the exosomes. When administered as single product, both STX-H3 and STX-S induced a strong immunization with the production of a potent humoral and cellular immune response in mice. Interestingly, these effects were obtained with administration of nanograms of protein and without adjuvant. Therefore, we tested the possibility of a multivalent vaccine: STX-H3 and STX-S exosomes were formulated together in a \u201cmix and match\u201d approach and the immune response was further evaluated. We showed that our STX-H3+S cocktail vaccine is as effective as the single components administered separately, resulting in a strong antibody and T-cell response. Our data show that our exosome platform has an enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation, and for therapeutics by enabling cell and tissue specific targeting.","version":"1.1","doi":"10.1101/2023.07.13.548895","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.12.548630","pub_date":"2023-7-13","title":"Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2","abstract":"SARS-CoV-2 has caused millions of infections worldwide since its emergence in 2019. Understanding how infection and vaccination induce mucosal immune responses and how they fluctuate over time is important, especially since they are key in preventing infection and reducing disease severity. We established a novel methodology for assessing SARS-CoV-2 cytokine and antibody responses at the nasal epithelium by using nasopharyngeal swabs collected longitudinally before and after either SARS-CoV-2 infection or vaccination. We then compared responses between mucosal and systemic compartments. We demonstrate that cytokine and antibody profiles differ markedly between compartments. Nasal cytokines show a wound healing phenotype while plasma cytokines are consistent with pro-inflammatory pathways. We found that nasal IgA and IgG have different kinetics after infection, with IgA peaking first. Although vaccination results in low nasal IgA, IgG induction persists for up to 180 days post-vaccination. This research highlights the importance of studying mucosal responses in addition to systemic responses to respiratory infections to understand the correlates of disease severity and immune memory. The methods described herein can be used to further mucosal vaccine development by giving us a better understanding of immunity at the nasal epithelium providing a simpler, alternative clinical practice to studying mucosal responses to infection. A nasopharyngeal swab can be used to study the intranasal immune response and yields much more information than a simple viral diagnosis.","version":"1.1","doi":"10.1101/2023.07.12.548630","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.12.548617","pub_date":"2023-7-12","title":"The recency and geographical origins of the bat viruses ancestral to SARS-CoV and SARS-CoV-2","abstract":"The emergence of SARS-CoV in 2002 and SARS-CoV-2 in 2019 has led to increased sampling of related sarbecoviruses circulating primarily in horseshoe bats. These viruses undergo frequent recombination and exhibit spatial structuring across Asia. Employing recombination-aware phylogenetic inference on bat sarbecoviruses, we find that the closest-inferred bat virus ancestors of SARS-CoV and SARS-CoV-2 existed just \u223c1\u20133 years prior to their emergence in humans. Phylogeographic analyses examining the movement of related sarbecoviruses demonstrate that they traveled at similar rates to their horseshoe bat hosts and have been circulating for thousands of years in Asia. The closest-inferred bat virus ancestor of SARS-CoV likely circulated in western China, and that of SARS-CoV-2 likely circulated in a region comprising southwest China and northern Laos, both a substantial distance from where they emerged. This distance and recency indicate that the direct ancestors of SARS-CoV and SARS-CoV-2 could not have reached their respective sites of emergence via the bat reservoir alone. Our recombination-aware dating and phylogeographic analyses reveal a more accurate inference of evolutionary history than performing only whole-genome or single gene analyses. These results can guide future sampling efforts and demonstrate that viral genomic fragments extremely closely related to SARS-CoV and SARS-CoV-2 were circulating in horseshoe bats, confirming their importance as the reservoir species for SARS viruses.","version":"1.1","doi":"10.1101/2023.07.12.548617","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.12.548725","pub_date":"2023-7-12","title":"Endocytosis Inhibitors Block SARS-CoV-2 Pseudoparticle Infection of Mink Lung Epithelium","abstract":"Both spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback. The purpose of our study was to determine the SARS-CoV-2 entry mechanism using mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors. Mv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. Dyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, while TMPRSS2 inhibitors had minimal impact, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans.","version":"1.1","doi":"10.1101/2023.07.12.548725","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.01.538902","pub_date":"2023-7-12","title":"Predicting Virus Fitness: Towards a structure-based computational model","abstract":"Predicting the impact of new emerging virus mutations is of major interest in surveillance and for understanding the evolutionary forces of the pathogen. The SARS-CoV-2 surface spike-protein (S-protein) binds to human ACE2 receptors as a critical step in host cell infection. At the same time, S-protein binding to human antibodies neutralizes the virus and prevents interaction with ACE2. Here we combine these two binding properties in a simple virus fitness model, using structure-based computation of all possible mutation effects averaged over 10 ACE2 complexes and 10 antibody complexes of the S-protein (\u223c3,80,000 computed mutations), and validated the approach against diverse experimental binding/escape data of ACE2 and antibodies. The ACE2-antibody selectivity change caused by mutation (i.e., the differential change in binding to ACE2 vs. immunity-inducing antibodies) is proposed to be a key metric of fitness model, enabling systematic error cancelation when evaluated. In this model, new mutations become fixated if they increase the selective binding to ACE2 relative to circulating antibodies, assuming that both are present in the host in a competitive binding situation. We use this model to categorize viral mutations that may best reach ACE2 before being captured by antibodies. Our model may aid the understanding of variant-specific vaccines and molecular mechanisms of viral evolution in the context of a human host.","version":"1.2","doi":"10.1101/2023.05.01.538902","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.10.548464","pub_date":"2023-7-11","title":"Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers","abstract":"Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence the \u223c30,000 nucleotide SARS-CoV-2 genome that use a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with \u2018Ns\u2019 inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). This study uses seven long-range PCR primers with an amplicon size of \u223c4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.","version":"1.1","doi":"10.1101/2023.07.10.548464","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.02.547368","pub_date":"2023-7-11","title":"Alternative cell entry mechanisms for SARS-CoV-2 and multiple animal viruses","abstract":"The cell entry mechanism of SARS-CoV-2, the causative agent of the COVID-19 pandemic, is not fully understood. Most animal viruses hijack cellular endocytic pathways as an entry route into the cell. Here, we show that in cells that do not express serine proteases such as TMPRSS2, genetic depletion of all dynamin isoforms blocked the uptake and strongly reduced infection with SARS-CoV-2 and its variant Delta. However, increasing the viral loads partially and dose-dependently restored infection via a thus far uncharacterized entry mechanism. Ultrastructural analysis by electron microscopy showed that this dynamin-independent endocytic processes appeared as 150-200 nm non-coated invaginations and was efficiently used by numerous mammalian viruses, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, and rhinovirus. Both the dynamin-dependent and dynamin-independent infection of SARS-CoV-2 required a functional actin cytoskeleton. In contrast, the alphavirus Semliki Forest virus, which is smaller in diameter, required actin only for the dynamin-independent entry. The presence of TMPRSS2 protease rescued SARS-CoV-2 infection in the absence of dynamins. Collectively, these results indicate that some viruses such as canine parvovirus and SARS-CoV-2 mainly rely on dynamin for endocytosis-dependent infection, while other viruses can efficiently bypass this requirement harnessing an alternative infection entry route dependent on actin.","version":"1.3","doi":"10.1101/2023.07.02.547368","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.10.548424","pub_date":"2023-7-11","title":"An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV","abstract":"The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.","version":"1.1","doi":"10.1101/2023.07.10.548424","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.11.548309","pub_date":"2023-7-11","title":"Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2\u2032 proline of the viral polyprotein","abstract":"The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG\u2193X motif found in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2\u2032 position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2\u2032 contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.","version":"1.1","doi":"10.1101/2023.07.11.548309","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.10.548406","pub_date":"2023-7-11","title":"Effects of aluminum-salt, CpG and emulsion adjuvants on the stability and immunogenicity of a virus-like particle displaying the SARS-CoV-2 receptor-binding domain (RBD)","abstract":"Second-generation COVID-19 vaccines with improved immunogenicity (e.g., breadth, duration) and availability (e.g., lower costs, refrigerator stable) are needed to enhance global coverage. In this work, we formulated a clinical-stage SARS-CoV-2 receptor binding domain (RBD) virus-like particle (VLP) vaccine candidate (IVX-411) with widely available adjuvants. Specifically, we assessed the in vitro storage stability and in vivo mouse immunogenicity of IVX-411 formulated with aluminum-salt adjuvants (Alhydrogel\u2122, AH and Adjuphos\u2122, AP), without or with the TLR-9 agonist CpG-1018\u2122 (CpG), and compared these profiles to IVX-411 adjuvanted with an oil-in-water nano-emulsion (AddaVax\u2122, AV). Although IVX-411 bound both AH and AP, lower binding strength of antigen to AP was observed by Langmuir binding isotherms. Interestingly, AH- and AP-adsorbed IVX-411 had similar storage stability profiles as measured by antigen binding assays (competitive ELISAs), but the latter displayed higher pseudovirus neutralizing titers (pNT) in mice, at levels comparable to titers elicited by AV-adjuvanted IVX-411. CpG addition to alum (AP or AH) resulted in a marginal trend of improved pNTs in stressed samples only, yet did not impact the storage stability profiles of IVX-411. In contrast, previous work with AH-formulations of a monomeric RBD antigen showed greatly improved immunogenicity and decreased stability upon CpG addition to alum. At elevated temperatures (25, 37\u00b0C), IVX-411 formulated with AH or AP displayed decreased in vitro stability compared to AV-formulated IVX-411and this rank-ordering correlated with in vivo performance (mouse pNT values). This case study highlights the importance of optimizing antigen-adjuvant interactions to develop low cost, aluminum-salt adjuvanted recombinant subunit vaccine candidates.","version":"1.1","doi":"10.1101/2023.07.10.548406","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.09.548285","pub_date":"2023-7-10","title":"Proteome profiling of nasopharynx reveals pathophysiological signature of COVID-19 disease severity","abstract":"An aberrant innate immune system caused by the beta coronavirus SARS-CoV-2 is a characteristic manifestation of severe coronavirus disease 2019 (COVID-19). Here, we performed proteome profiling of nasopharyngeal (NP) swabs from 273 hospitalized patients with mild and severe COVID-19 symptoms, including non-survivors. We identified depletion in STAT1-mediated type I interferon response, retinol metabolism and NRF2 antioxidant system that are associated with disease severity in our patient demography. We found that the dysregulation of glucocorticoid signaling and renin-angiotensin-aldosterone system (RAAS) contribute to the pathophysiology of COVID-19 fatality. Hyperactivation of host innate immune system was observed in severe patients, marked by elevated proteins involved in neutrophil degranulation and platelet aggregation. Our study using high-throughput proteomics on the nasopharynx of COVID-19 patients provides additional evidence on the SARS-CoV-2-induced pathophysiological signatures of disease severity and fatality.","version":"1.1","doi":"10.1101/2023.07.09.548285","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.17.536926","pub_date":"2023-7-10","title":"Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection","abstract":"SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.","version":"1.2","doi":"10.1101/2023.04.17.536926","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.10.548360","pub_date":"2023-7-10","title":"High-resolution map of the Fc-functions mediated by COVID-19 neutralizing antibodies","abstract":"A growing body of evidence shows that Fc-dependent antibody effector functions play an important role in protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To unravel the mechanisms that drive these responses, we analyzed the phagocytosis and complement deposition mediated by a panel of 482 human monoclonal antibodies (nAbs) neutralizing the original Wuhan virus, expressed as recombinant IgG1. Our study confirmed that nAbs no longer neutralizing SARS-CoV-2 Omicron variants can retain their Fc-functions. Surprisingly, we found that nAbs with the most potent Fc-function recognize the N- terminal domain, followed by those targeting Class 3 epitopes in the receptor binding domain. Interestingly, nAbs direct against the Class 1/2 epitopes in the receptor binding motif, which are the most potent in neutralizing the virus, were the weakest in Fc-functions. The divergent properties of the neutralizing and Fc- function mediating antibodies were confirmed by the use of different B cell germlines and by the observation that Fc-functions of polyclonal sera differ from the profile observed with nAbs, suggesting that not-neutralizing antibodies also contribute to Fc-functions. These data provide a high-resolution picture of the Fc-antibody response to SARS-CoV-2 and suggest that the Fc contribution should be considered for the design of improved vaccines, the selection of therapeutic antibodies and the evaluation of correlates of protection.","version":"1.1","doi":"10.1101/2023.07.10.548360","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.07.548083","pub_date":"2023-7-08","title":"Human complement Factor H and Properdin act as soluble pattern recognition receptors and differentially modulate SARS-CoV-2 Infection","abstract":"Severe cases of SARS-CoV-2 infection are characterised by an imbalanced immune response, excessive inflammation, and the development of acute respiratory distress syndrome, which can lead to multiorgan failure and death. Several studies have demonstrated dysregulated complement activity as an indicator of immunopathogenesis in the SARS-CoV-2 infection. Notably, the complement alternative pathway has been implicated in driving the excessive inflammation during severe SARS-CoV-2 infection. Reduced levels of factor H (FH), a down-regulator of the alternative pathway, and increased levels of properdin (Factor P/FP), the only known up-regulator of the alternative pathway, have been observed in individuals with severe COVID-19 infection. The present study investigated the complement activation-independent, and a more direct role of FH and FP against SARS-CoV-2 infection. Using direct ELISA, the interactions of FH and FP with the SARS-CoV-2 spike (S) and receptor binding domain (RBD) were assessed. Using S protein expressing lentiviral pseudotypes, the cell binding and luciferase-based virus entry assays were employed to assess the potential modulatory effects of FH, FP, and recombinant thrombospondin repeats 4 and 5 (TSR4+5) on SARS-CoV-2 cell entry. We also evaluated the immunomodulatory functions of FH and FP in the cytokine response triggered by SARS-CoV-2 pseudotypes via RT-qPCR. SARS-CoV-2 S and RBD proteins were found to bind both FH and FP. Treatment of A549 cells expressing human ACE2 and TMPRSS2 with FP or TSR4+5 resulted in increased cell entry and binding of SARS-CoV-2 pseudotypes. In silico studies revealed that FP increases affinity between SARS-CoV-2 and host ACE2. The impact of FP on viral cell entry and binding was reversed by anti-FP antibody treatment in A549-hACE2+TMPRSS2 cells. However, FH treatment reduced the cell entry and binding of SARS-CoV-2 lentiviral pseudotypes. Furthermore, the A549-hACE2+TMPRSS2 cells challenged with SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes pre-treated with FP or TSR4+5, exhibited upregulation of the transcripts of pro-inflammatory cytokines, such as IL-1\u03b2, IL-8, IL-6, TNF-\u03b1, IFN-\u03b1 and RANTES (as well as NF-\u03baB). Conversely, FH pre-treatment downregulated the expression of these pro-inflammatory cytokines. Treatment of A549-hACE2+TMPRSS2 cells with FP increased S protein-mediated NF-\u03baB activation, while FH treatment reduced it. These findings suggest that FH may act as an inhibitor of SARS-CoV-2 cell entry and binding, thereby attenuating the infection-associated inflammatory response in a complement activation-independent manner. FP may contribute to viral cell entry, binding, and exacerbating the immune response. That may result in potentially influencing the severity of the infection.","version":"1.1","doi":"10.1101/2023.07.07.548083","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.07.548087","pub_date":"2023-7-07","title":"Temporal Pattern of Mutation Accumulation in SARS-CoV-2 Proteins: Insights from Whole Genome Sequences Pan-India Using Data Mining Approach","abstract":"Mutation is a fundamental factor that affects host-pathogen biology and consequently viral survival and spread. Close monitoring and observation of such mutation help decipher essential changes in the SARS Cov2 genome. A plethora of mutations have been documented owing to increased whole genomic sequencing. Understanding how conserved the specific mutations are and the temporal pattern of mutation accumulation is of paramount interest. Using an in-house data mining approach, pan-India data was mined and analysed for 26 proteins expressed by SARS-CoV-2 to understand the spread of mutations over 28 months (January 2021-April 2023). It was observed that proteins such as Nsp3, Nsp4, ORF9b, among others, acquired mutations over the period. In contrast, proteins such as Nsp6-10 were highly stable, with no detectable conserved mutations. Further, it was observed that many of the mutations that were highly prevalent in the delta variants were not observed in the omicron variants, which probably influenced the host-pathogen relationship. The study attempts to catalogue and focus on well-conserved mutations across all the SARS-CoV-2 proteins, highlighting the importance of understanding non-spike mutations.","version":"1.1","doi":"10.1101/2023.07.07.548087","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.07.548077","pub_date":"2023-7-07","title":"Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant","abstract":"Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, the S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production of the recombinant S trimer and, more importantly, its immunogenicity, suggesting that these two parameters are related. However, S-2P still shows some molecular instability and it is produced with low yield. Thus, S-2P production can be further optimized. Here we described a novel set of mutations identified by molecular modelling and located in the S2 region of the Spike that increase S-2P production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 spike mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development. The rapid development of SARS-CoV-2 vaccines have been pivotal in the control of the COVID-19 pandemic worldwide. Most of these vaccines include the S glycoprotein as the main immunogen since this protein, and particularly its receptor binding domain (RBD), is the major target of neutralizing antibodies. SARS-CoV-2 have been evolving from the beginning of the pandemic and several variants with increased transmissibility, pathogenicity or resistance to infection\u2013 or vaccine-induced immunity have emerged. Different strategies have been adopted to improve vaccine protection including additional booster doses or the adaptation of the S immunogens to the novel SARS-CoV-2 variants. As a complementary strategy we have identified a combination of non-proline mutations that increase S production by 5-fold (S-29 protein). Despite the sequence of this novel S-29 immunogen is based on the ancestral SARS-CoV-2 WH1 variant, it effectively protects animal model from the highly pathogenic and neutralization resistant SARS-CoV-2 Beta variant. Thus, we describe a novel set of mutations that can increase the production and efficacy of S-based COVID-19 vaccines.","version":"1.1","doi":"10.1101/2023.07.07.548077","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.07.547941","pub_date":"2023-7-07","title":"First Eurasian cases of SARS-CoV-2 seropositivity in a free-ranging urban population of wild fallow deer","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects wildlife. Recent studies highlighted that variants of concern (VOC) may expand into novel animal reservoirs with the potential for reverse zoonosis. North American white-tailed deer are the only deer species in which SARS-CoV-2 has been documented, raising the question whether further reservoir species exist as new VOC emerge. Here, we report the first cases of deer SARS-CoV-2 seropositivity in Eurasia, in a city population of fallow deer in Dublin, Ireland. Deer were seronegative in 2020 (circulating variant in humans: Alpha), one animal was seropositive in 2021 (Delta variant), and 57% of animals tested in 2022 were seropositive (Omicron variant). Ex vivo, a clinical isolate of Omicron BA.1 infected fallow deer precision cut lung slice type-2 pneumocytes, also a major target of infection in human lungs. Our findings suggest a change in host tropism as new variants emerged in the human reservoir, highlighting the importance of continued wildlife disease monitoring and limiting human-wildlife contacts. Teaser: This study is the first report of SARS-CoV-2 seropositivity in fallow deer, highlighting expansion of viral variants into new host reservoirs.","version":"1.1","doi":"10.1101/2023.07.07.547941","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.06.547955","pub_date":"2023-7-07","title":"Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states","abstract":"The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.07.06.547955","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.06.547945","pub_date":"2023-7-07","title":"In vitro broad-spectrum antiviral activity of MIT-001, a mitochondria-targeted reactive oxygen species scavenger, against severe acute respiratory syndrome coronavirus 2 and multiple zoonotic viruses","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 becomes a serious threat to global health and requires the development of effective antiviral therapies. Current therapies that target viral proteins have limited efficacy with side effects. In this study, we investigated the antiviral activity of MIT-001, a small molecule reactive oxygen species (ROS) scavenger targeting mitochondria, against SARS-CoV-2 and other zoonotic viruses in vitro. The antiviral activity of MIT-001 was quantified by RT-qPCR and plaque assay. We also evaluated the functional analysis of MIT-001 by JC-1 staining to measure mitochondrial depolarization, total RNA sequencing to investigate gene expression changes, and immunoblot to quantify protein expression levels. The results showed that MIT-001 effectively inhibited the replication of B.1.617.2 and BA.1 strains, Zika virus, Seoul virus, and Vaccinia virus. Treatment with MIT-001 restored the expression of heme oxygenase-1 (HMOX1) and NAD(P)H: quinone oxidoreductase 1 (NqO1) genes, anti-oxidant enzymes reduced by SARS-CoV-2, to normal levels. The presence of MIT-001 also alleviated mitochondrial depolarization caused by SARS-CoV-2 infection. These findings highlight the potential of MIT-001 as a broad-spectrum antiviral compound that targets for zoonotic RNA and DNA viruses, providing a promising therapeutic approach to combat viral infection.","version":"1.1","doi":"10.1101/2023.07.06.547945","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.07.548130","pub_date":"2023-7-07","title":"Cytokine storm mitigation for exogenous immune agonists","abstract":"Cytokine storm is a life-threatening inflammatory response characterized by hyperactivation of the immune system. It can be caused by various therapies, auto-immune conditions, or pathogens, such as respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes coronavirus disease COVID-19. Here we propose a conceptual mathematical model describing the phenomenology of cytokine-immune interactions when a tumor is treated by an exogenous immune cell agonist which has the potential to cause a cytokine storm, such as CAR T cell therapy. Numerical simulations reveal that as a function of just two model parameters, the same drug dose and regimen could result in one of four outcomes: treatment success without a storm, treatment success with a storm, treatment failure without a storm, and treatment failure with a storm. We then explore a scenario in which tumor control is accompanied by a storm and ask if it is possible to modulate the duration and frequency of drug administration (without changing the cumulative dose) in order to preserve efficacy while preventing the storm. Simulations reveal existence of a \u201csweet spot\u201d in protocol space (number versus spacing of doses) for which tumor control is achieved without inducing a cytokine storm. This theoretical model, which contains a number of parameters that can be estimated experimentally, contributes to our understanding of what triggers a cytokine storm, and how the likelihood of its occurrence can be mitigated.","version":"1.1","doi":"10.1101/2023.07.07.548130","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.06.548022","pub_date":"2023-7-07","title":"Establishing thresholds for cytokine storm and defining their relationship to disease severity in respiratory viral infections","abstract":"Previous studies have identified cytokines associated with respiratory virus infection illness outcome. However, few studies have included comprehensive cytokine panels, longitudinal analyses, and/or simultaneous assessment across the severity spectrum. This, coupled with subjective definitions of cytokine storm syndrome (CSS), have contributed to inconsistent findings of cytokine signatures, particularly with COVID severity. Here, we measured 38 plasma cytokines and compared profiles in healthy, SARS-CoV-2 infected, and multisystem inflammatory syndrome in children (MIS-C) patients (n = 169). Infected patients spanned the severity spectrum and were classified as Asymptomatic, Mild, Moderate or Severe. Our results showed acute cytokine profiles and longitudinal dynamics of IL1Ra, IL10, MIP1b, and IP10 can differentiate COVID severity groups. Only 4% of acutely infected patients exhibited hypercytokinemia. Of these subjects, 3 were Mild, 3 Moderate, and 1 Severe, highlighting the lack of association between CSS and COVID severity. Additionally, we identified IL1Ra and TNFa as potential biomarkers for patients at high risk for long COVID. Lastly, we compare hypercytokinemia profiles across COVID and influenza patients and show distinct elevated cytokine signatures, wherein influenza induces the most elevated cytokine profile. Together, these results identify key analytes that, if obtained at early time points, can predict COVID illness outcome and/or risk of complications, and provide novel insight for improving the conceptual framework of hypercytokinemia, wherein CSS is a subgroup that requires concomitant severe clinical manifestations, and including a list of cytokines that can distinguish between subtypes of hypercytokinemia.","version":"1.1","doi":"10.1101/2023.07.06.548022","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.05.547902","pub_date":"2023-7-06","title":"SARS-CoV-2 Nsp1 regulates translation start site fidelity to promote infection","abstract":"A better mechanistic understanding of virus-host interactions can help reveal vulnerabilities and identify opportunities for therapeutic interventions. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in polysome composition and protein synthesis during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones whose inhibition impairs infectious particle production without major toxicity to the host. We find that CoV2 non-structural protein Nsp1 selectively enhances virus translation through functional interactions with initiation factor EIF1A. When EIF1A is depleted, more ribosomes initiate translation from an upstream CUG start codon, inhibiting translation of non-structural genes and reducing viral titers. Together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and identifies druggable targets for potential antiviral development.","version":"1.1","doi":"10.1101/2023.07.05.547902","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.05.547781","pub_date":"2023-7-05","title":"The receptor binding domain of SARS-CoV-2 spike protein fused with the type IIb E. coli heat-labile enterotoxin A subunit as an intranasal booster after mRNA vaccination","abstract":"The outbreak of SARS-CoV-2 infections had led to the COVID-19 pandemic which has a significant impact on global public health and the economy. The spike (S) protein of SARS-CoV-2 contains the receptor binding domain (RBD) which binds to human angiotensin-converting enzyme 2 receptor. Numerous RBD-based vaccines have been developed and recently focused on the induction of neutralizing antibodies against the immune evasive Omicron BQ.1.1 and XBB.1.5 subvariants. In this preclinical study, we reported the use of a direct fusion of the type IIb Escherichia coli heat-labile enterotoxin A subunit with SARS CoV-2 RBD protein (RBD-LTA) as an intranasal vaccine candidate. The results showed that intranasal immunization with the RBD-LTA fusion protein in BALB/c mice elicited potent neutralizing antibodies against the Wuhan-Hu-1 and several SARS-CoV-2 variants as well as the production of IgA antibodies in bronchoalveolar lavage fluids (BALFs). Furthermore, the RBD-LTA fusion protein was used as a second-dose booster after bivalent mRNA vaccination. The results showed that the neutralizing antibody titers elicited by the intranasal RBD-LTA booster were similar to the bivalent mRNA booster, but the RBD-specific IgA titers in sera and BALFs significantly increased. Overall, this preclinical study suggests that the RBD-LTA fusion protein could be a promising candidate as a mucosal booster COVID-19 vaccine.","version":"1.1","doi":"10.1101/2023.07.05.547781","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.03.547244","pub_date":"2023-7-03","title":"Lack of detection of SARS-CoV-2 in Wildlife from Kerala, India in 2020-21","abstract":"Spill over of SARs-CoV-2 into a variety of wild and domestic animals has been an ongoing feature of the human pandemic. The establishment of a new reservoir in white tailed deer in North America and increasing divergence of the viruses circulating in them from those circulating in the human population has highlighted the ongoing risk this poses for global health. Some parts of the world have seen more intensive monitoring of wildlife species for SARS-CoV-2 and related coronaviruses but there are still very large gaps in geographical and species-specific information. This paper reports negative results for SARS-CoV-2 PCR based testing using a pan coronavirus end point RDRP PCR and a Sarbecovirus specific E gene qPCR on lung and or gut tissue from wildlife from the Indian State of Kerala. These animals included: 121 Rhinolophus rouxii (Rufous Horsehoe Bat), 6 Rhinolophus bedommei (Lesser Woolly Horseshoe Bat), 15 Rossettus leschenaultii (Fulvous Fruit Bat), 47 Macaca radiata (Bonnet macaques), 35 Paradoxurus hermaphroditus (Common Palm Civet), 5 Viverricula indica (Small Indian Civet), 4 Herpestes edwardsii (Common Mongoose), 10 Panthera tigris (Bengal Tiger), 8 Panthera pardus fusca (Indian Leopard), 4 Prionailurus bengalensis (Leopard cats), 2 Felis chaus (Jungle cats), 2 Cuon alpinus (Wild dogs) and 1 Melursus ursinus (sloth bear).","version":"1.1","doi":"10.1101/2023.07.03.547244","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.10.531533","pub_date":"2023-7-03","title":"Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike","abstract":"Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.","version":"1.3","doi":"10.1101/2023.03.10.531533","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.02.547440","pub_date":"2023-7-03","title":"RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid","abstract":"The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1000nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with different RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD\u2019s secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N. The nucleocapsid protein of the SARS-CoV-2 coronavirus binds to viral RNA, both to protect and condense it inside the viral particle and to facilitate viral transcription inside infected host cells. Evidence suggests that variations in RNA structure impact how and where it binds to the nucleocapsid, but these differences are not well understood at a structural level. Using nuclear magnetic resonance spectroscopy, we examine the interactions between each folded domain of the nucleocapsid and different RNA structures. Binding affinities and NMR chemical shift profiles demonstrate that binding between the N-terminal domain and single stranded RNA is driven by strong interactions at a specific site, while multiple weak nonspecific interactions at newly discovered sites lead to phase separation and RNA condensation.","version":"1.1","doi":"10.1101/2023.07.02.547440","journal":"bioRxiv","score":null},{"id":"10.1101/2023.07.02.547076","pub_date":"2023-7-03","title":"Antigenic cartography using variant-specific hamster sera reveals substantial antigenic variation among Omicron subvariants","abstract":"SARS-CoV-2 has developed substantial antigenic variability. As the majority of the population now has pre-existing immunity due to infection or vaccination, the use of experimentally generated animal immune sera can be valuable for measuring antigenic differences between virus variants. Here, we immunized Syrian hamsters by two successive infections with one of eight SARS-CoV-2 variants. Their sera were titrated against 14 SARS-CoV-2 variants and the resulting titers visualized using antigenic cartography. The antigenic map shows a condensed cluster containing all pre-Omicron variants (D614G, Alpha, Delta, Beta, Mu, and an engineered B.1+E484K variant), and a considerably more distributed positioning among a selected panel of Omicron subvariants (BA.1, BA.2, BA.4/5, the BA.5 descendants BF.7 and BQ.1.18; the BA.2.75 descendant BN.1.3.1; and the BA.2-derived recombinant XBB.2). Some Omicron subvariants were as antigenically distinct from each other as the wildtype is from the Omicron BA.1 variant. The results highlight the potential of using variant-specifically infected hamster sera for the continued antigenic characterisation of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.07.02.547076","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.30.547241","pub_date":"2023-6-30","title":"SARS-CoV-2 spike glycosylation affects function and neutralization sensitivity","abstract":"The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. Here, we investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain (NTD) and the receptor binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated spike protein. Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies (mAbs) cloned from convalescent individuals. The N343 glycan reduced overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 spike glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan.","version":"1.1","doi":"10.1101/2023.06.30.547241","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.29.546885","pub_date":"2023-6-30","title":"Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication","abstract":"G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.","version":"1.1","doi":"10.1101/2023.06.29.546885","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.29.547094","pub_date":"2023-6-30","title":"Identification of side effects of COVID-19 drug candidates on embryogenesis using an integrated zebrafish screening platform","abstract":"Drug repurposing is an important strategy in COVID-19 treatment, but many clinically approved compounds have not been extensively studied in the context of embryogenesis, thus limiting their administration during pregnancy. Here we used the zebrafish embryo model organism to test the effects of 162 marketed drugs on cardiovascular development. Among the compounds used in the clinic for COVD-19 treatment, we found that Remdesivir led to reduced body size and heart functionality at clinically relevant doses. Ritonavir and Baricitinib showed reduced heart functionality and Molnupiravir and Baricitinib showed effects on embryo activity. Sabizabulin was highly toxic at concentrations only 5 times higher than Cmax and led to a mean mortality of 20% at Cmax. Furthermore, we tested if zebrafish could be used as a model to study inflammatory response in response to spike protein treatment and found that Remdesivir, Ritonavir, Molnupiravir, Baricitinib as well as Sabizabulin counteracted the inflammatory response related gene expression upon SARS-CoV-2 spike protein treatment. Our results show that the zebrafish allows to study immune-modulating properties of COVID-19 compounds and highlights the need to rule out secondary defects of compound treatment on embryogenesis. All results are available on a user friendly web-interface https://share.streamlit.io/alernst/covasc_dataapp/main/CoVasc_DataApp.py that provides a comprehensive overview of all observed phenotypic effects and allows personalized search on specific compounds or group of compounds. Furthermore, the presented platform can be expanded for rapid detection of developmental side effects of new compounds for treatment of COVID-19 and further viral infectious diseases. A zebrafish screening platform assesses side effects on cardiovascular development and behavior of FDA approved drugs used in clinical practice to treat COVID-19 and their immune modulatory effect upon spike protein treatment.","version":"1.1","doi":"10.1101/2023.06.29.547094","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.27.546805","pub_date":"2023-6-29","title":"Identification of cross-reacting IgG hotspots to prevent immune evasion of SARS-CoV-2 variants","abstract":"The major factor that shapes the global perspective for increase or diminution of successive pandemic waves of COVID-19 is the immunological protection. The SARS-CoV-2 virus constantly develops new variants, and capability of immune evasion is among the major factors that promote variant spreading in the human population. After two years of the pandemic and virus evolution, it is almost impossible to explain effects of all possible combinations different viral strains, a few types of vaccinations or new variants infecting an individual patient. Instead of variant-to-variant comparisons, identification of key protein regions linked to immune evasion could be efficient. Here we report an approach for experimental identification of SARS-CoV-2 protein regions that (i) have characteristics of cross-reacting IgG hot-spots, and (ii) are highly immunogenic. Cross-reacting IgG hot spots are regions of protein frequently recognized in many variants by cross-reacting antibodies. Immunogenic regions efficiently induce specific IgG production in SARS-CoV-2 infected patients. We determined four regions that demonstrate both significant immunogenicity and the activity of a cross-reacting IgG hot-spot in protein S, and two such regions in protein N. Their distribution within the proteins suggests that they may be useful in vaccine design and in serological diagnostics of COVID-19.","version":"1.1","doi":"10.1101/2023.06.27.546805","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.27.546764","pub_date":"2023-6-29","title":"Decreased efficacy of a COVID-19 vaccine due to mutations present in early SARS-CoV-2 variants of concern","abstract":"With the SARS-CoV-2 virus still circulating and evolving, there remains an outstanding question if variant-specific vaccines represent the optimal path forward, or if other strategies might be more efficacious towards providing broad protection against emerging variants. Here, we examine the efficacy of strain-specific variants of our previously reported, pan-sarbecovirus vaccine candidate, DCFHP-alum, a ferritin nanoparticle functionalized with an engineered form of the SARS-CoV-2 spike protein. In non-human primates, DCFHP-alum elicits neutralizing antibodies against all known VOCs that have emerged to date and SARS-CoV-1. During development of the DCFHP antigen, we investigated the incorporation of strain-specific mutations from the major VOCs that had emerged to date: D614G, Epsilon, Alpha, Beta, and Gamma. Here, we report the biochemical and immunological characterizations that led us to choose the ancestral Wuhan-1 sequence as the basis for the final DCFHP antigen design. Specifically, we show by size exclusion chromatography and differential scanning fluorimetry that mutations in the VOCs adversely alter the antigen\u2019s structure and stability. More importantly, we determined that DCFHP without strain-specific mutations elicits the most robust, cross-reactive response in both pseudovirus and live virus neutralization assays. Our data suggest potential limitations to the variant-chasing approach in the development of protein nanoparticle vaccines, but also have implications for other approaches including mRNA-based vaccines.","version":"1.2","doi":"10.1101/2023.06.27.546764","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.29.547038","pub_date":"2023-6-29","title":"Nanoparticle endocytosis is driven by monocyte phenotype rather than nanoparticle size under high shear flow conditions","abstract":"Monocytes are members of the mononuclear phagocyte system involved in pathogen clearance and nanoparticle pharmacokinetics. Monocytes play a critical role in the development and progression of cardiovascular disease and, recently, in SARS-CoV-2 pathogenesis. While studies have investigated the effect of nanoparticle modulation on monocyte uptake, their capacity for nanoparticle clearance is poorly studied. In this study, we investigated the impact of ACE2 deficiency, frequently observed in individuals with cardiovascular complications, on monocyte nanoparticle endocytosis. Moreover, we investigated nanoparticle uptake as a function of nanoparticle size, physiological shear stress, and monocyte phenotype. Our Design of Experiment (DOE) analysis found that the THP-1 ACE2- cells showed a greater preference for 100nm particles under atherosclerotic conditions than THP-1 wild-type cells. Observing how nanoparticles can modulate monocytes in the context of disease can inform precision dosing.","version":"1.1","doi":"10.1101/2023.06.29.547038","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.27.546784","pub_date":"2023-6-28","title":"Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential","abstract":"Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.","version":"1.1","doi":"10.1101/2023.06.27.546784","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.27.546790","pub_date":"2023-6-28","title":"A human primary airway microphysiological system infected with SARS-CoV-2 distinguishes the treatment efficacy between nirmatrelvir and repurposed compounds fluvoxamine and amodiaquine","abstract":"The COVID-19 pandemic necessitated a rapid mobilization of resources toward the development of safe and efficacious vaccines and therapeutics. Finding effective treatments to stem the wave of infected individuals needing hospitalization and reduce the risk of adverse events was paramount. For scientists and healthcare professionals addressing this challenge, the need to rapidly identify medical countermeasures became urgent, and many compounds in clinical use for other indications were repurposed for COVID-19 clinical trials after preliminary preclinical data demonstrated antiviral activity against SARS-CoV-2. Two repurposed compounds, fluvoxamine and amodiaquine, showed efficacy in reducing SARS-CoV-2 viral loads in preclinical experiments, but ultimately failed in clinical trials, highlighting the need for improved predictive preclinical tools that can be rapidly deployed for events such as pandemic emerging infectious diseases. The PREDICT96-ALI platform is a high-throughput, high-fidelity microphysiological system (MPS) that recapitulates primary human tracheobronchial tissue and supports highly robust and reproducible viral titers of SARS-CoV-2 variants Delta and Omicron. When amodiaquine and fluvoxamine were tested in PREDICT96-ALI, neither compound demonstrated an antiviral response, consistent with clinical outcomes and in contrast with prior reports assessing the efficacy of these compounds in other human cell-based in vitro platforms. These results highlight the unique prognostic capability of the PREDICT96-ALI proximal airway MPS to assess the potential antiviral response of lead compounds.","version":"1.1","doi":"10.1101/2023.06.27.546790","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.15.500120","pub_date":"2023-6-28","title":"Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq","abstract":"Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-\u03baB inhibitor I\u03baB\u03b1 (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.","version":"1.2","doi":"10.1101/2022.07.15.500120","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.27.545921","pub_date":"2023-6-27","title":"Paridiprubart inhibits TLR4-dependant NF-\u03baB activation by multiple pathogens","abstract":"Respiratory pathogens such as SARS-CoV-2 and influenza can activate an exaggerated inflammatory response (cytokine storm) in the lungs that may result in acute respiratory distress syndrome (ARDS), hospitalization, and death. Therapies that target a specific pathogen (i.e. anti-virals) must, by nature, be selected after a specific diagnosis and may become ineffective due to pathogen evolution. An alternate strategy is to counter the exaggerated innate immune response present in ARDS patients using host-directed drug therapies that are agnostic to the infectious agent to overcome both of these challenges. Originally described as the innate immune receptor for lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) is now understood to be an important mediator of inflammation caused by a variety of pathogen-associated molecular patterns (PAMPs) and host-derived damage-associated molecular patterns (DAMPs). Here we show that paridiprubart, a monoclonal antibody that prevents TLR4 dimer formation, inhibits the response to TLR4 agonists including LPS, the SARS-CoV-2 spike protein, the DAMP high mobility group box 1 (HMGB1), as well as the NF-\u03baB response to infection by both viral and bacterial pathogens. Notable in this regard, we demonstrate that SARS-CoV-2 increases HMGB1 levels, and that paridiprubart inhibits both the SARS-CoV-2 and HMGB1-triggered NF-\u03baB response, illustrating its potential to suppress this self-amplifying inflammatory signal. We also observed that the inhibitory effect of paridiprubart is apparent when cells are exposed to the SARS-CoV-2 spike protein, which is itself a direct TLR4 agonist. In the context of active infection, paridiprubart suppressed the NF-\u03baB-dependent response elicited by infection with SARS-CoV-2, the seasonal coronavirus 229E, influenza A virus or Haemophilus influenzae, a gram-negative bacterial pathogen. Combined, these findings reinforce the central role played by TLR4 in the inflammatory response to infection by diverse pathogens, and demonstrates the protective potential of paridiprubart-dependent inhibition of pathogenic TLR4 responses.","version":"1.1","doi":"10.1101/2023.06.27.545921","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.26.546514","pub_date":"2023-6-26","title":"A pseudovirus-based method to dynamically mimic SARS-CoV-2-associated cell-to-cell fusion and transmission","abstract":"SARS-CoV-2 has caused the global tremendous loss and continues to evolve to generate variants. Entry of SARS-CoV-2 into the host cells is primarily mediated by Spike (S), which binds to the host receptor hACE2 and initiates virus-cell membrane fusion. Cell fusion contributes to viral entry, cell-to-cell transmission and tissue damage in COVID-19 patients. Many reporter assays have been developed to study S-mediated cell fusion by equally coculturing S-expressing cells and hACE2-positive cells. However, these strategies cannot fully simulate cell-to-cell fusion and transmission of SARS-CoV-2 infection, in which virions from a single target cell transmit to the neighbor cells and induce syncytia formation. Here, we design a pseudovirus-based method to dynamically mimic cell-to-cell fusion and transmission of SARS-CoV-2. We coculture a small number of pseudovirus-producing 293FT cells and a large number of hACE2-expressing 293T cells, and demonstrate that a single cell producing S-pseudotyped virions can induce significant syncytia of hACE2-positive cells. This pseudovirus-based method is a powerful tool to screen and estimate potential inhibitors of S-driven syncytia. Moreover, this strategy can also be utilized to explore fusogenic ability of SARS-CoV-2 variants. Together, the pseudovirus-based method we report here will be beneficial to drug screening and scientific research against SARS-CoV-2 or future emerging coronavirus.","version":"1.1","doi":"10.1101/2023.06.26.546514","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.26.546492","pub_date":"2023-6-26","title":"Oligomeric state of \u03b2-coronavirus non-structural protein 10 stimulators studied by OmniSEC and Small Angle X-ray Scattering","abstract":"Members of the \u03b2-coronavirus family such as SARS-CoV-2, SARS, and MERS have caused pandemics over the last 20 years. Future pandemics are likely and studying the coronavirus family members is necessary for their understanding and treatment. Coronaviruses possess 16 non-structural proteins, many of which are involved in viral replication and other vital functions. Non-structural protein 10 (nsp10) is an essential stimulator of nsp14 and nsp16, modulating RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to understanding its multifunctional role. We report the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. Proteins were subjected to a combination of OmniSEC and SEC-MALS to characterise their oligomeric state. Full-length nsp10s were predominantly monomeric in solution, while truncated versions of nsp10 have a higher tendency to oligomerise. Small angle X-ray scattering (SAXS) experiments reveal a globular shape of nsp10 which is conserved in all three coronaviruses, including MERS nsp10, which diverges most from SARS and SARS-CoV-2 nsp10s. In conclusion, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 are globular and predominantly monomeric in solution. Additionally, we describe for the first time a functional role of the C-terminus of nsp10 for tight binding to nsp14.","version":"1.1","doi":"10.1101/2023.06.26.546492","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.22.546100","pub_date":"2023-6-26","title":"Classification of patients with COVID-19 by blood RNA endotype: A prospective cohort study","abstract":"Although the development of vaccines has considerably reduced the severity of COVID-19, its incidence is still high. Hence, a targeted approach based on RNA endotypes of a population should be developed to help design biomarker-based therapies for COVID-19. We evaluated the major RNAs transcribed in blood cells during COVID-19 using PCR to further elucidate its pathogenesis and determine predictive phenotypes in COVID-19 patients. In a discovery cohort of 40 patients with COVID-19, 26,354 RNAs were measured on day 1 and day 7. Five RNAs associated with disease severity and prognosis were derived. In a validation cohort of 153 patients with COVID-19 treated in the intensive care unit, we focused on prolactin (PRL), and toll-like receptor 3 (TLR3) among RNAs, which have a strong association with prognosis, and evaluated the accuracy for predicting survival of PRL-to-TL3 ratios (PRL/TLR3) with the areas under the ROC curves (AUC). The validation cohort was divided into two groups based on the cut-off value in the ROC curve with the maximum AUC. The two groups were defined by high PRL/TLR3 (n=47) and low PRL/TLR3 groups (n=106) and the clinical outcomes were compared. In the validation cohort, the AUC for PRL/TLR3 was 0.79, showing superior prognostic ability compared to severity scores such as APACHE II and SOFA. The high PRL/TLR3 group had a significantly higher 28-day mortality than the low PRL/TLR3 group (17.0% vs 0.9%, P<0.01). A new RNA endotype classified using high PRL/TLR3 was associated with mortality in COVID-19 patients.","version":"1.1","doi":"10.1101/2023.06.22.546100","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.23.533961","pub_date":"2023-6-25","title":"Adverse outcomes in SARS-CoV-2 infected pregnant mice are gestational age-dependent and resolve with antiviral treatment","abstract":"SARS-CoV-2 infection during pregnancy is associated with severe COVID-19 and adverse fetal outcomes, but the underlying mechanisms remain poorly understood. Moreover, clinical studies assessing therapeutics against SARS-CoV-2 in pregnancy are limited. To address these gaps, we developed a mouse model of SARS-CoV-2 infection during pregnancy. Outbred CD1 mice were infected at embryonic day (E) 6, E10, or E16 with a mouse adapted SARS-CoV-2 (maSCV2) virus. Outcomes were gestational age-dependent, with greater morbidity, reduced anti-viral immunity, greater viral titers, and more adverse fetal outcomes occurring with infection at E16 (3rd trimester-equivalent) than with infection at either E6 (1st trimester-equivalent) or E10 (2nd trimester-equivalent). To assess the efficacy of ritonavir-boosted nirmatrelvir (recommended for pregnant individuals with COVID-19), we treated E16-infected dams with mouse equivalent doses of nirmatrelvir and ritonavir. Treatment reduced pulmonary viral titers, decreased maternal morbidity, and prevented adverse offspring outcomes. Our results highlight that severe COVID-19 during pregnancy and adverse fetal outcomes are associated with heightened virus replication in maternal lungs. Ritonavir-boosted nirmatrelvir mitigated adverse maternal and fetal outcomes of SARS-CoV-2 infection. These findings prompt the need for further consideration of pregnancy in preclinical and clinical studies of therapeutics against viral infections.","version":"1.2","doi":"10.1101/2023.03.23.533961","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.24.546374","pub_date":"2023-6-24","title":"PARP14 is a writer, reader and eraser of mono-ADP-ribosylation","abstract":"PARP14/BAL2 is a large multidomain enzyme involved in signaling pathways with relevance to cancer, inflammation, and infection. Inhibition of its mono-ADP-ribosylating PARP homology domain and its three ADP-ribosyl binding macro domains has been regarded as a potential means of therapeutic intervention. Macrodomains-2 and -3 are known to stably bind to ADP-ribosylated target proteins; but the function of macrodomain-1 has remained some-what elusive. Here, we used biochemical assays of ADP-ribosylation levels to characterize PARP14 macrodomain-1 and the homologous macrodomain-1 of PARP9. Our results show that both macrodomains display an ADP-ribosyl glycohydrolase activity that is not directed toward specific protein side chains. PARP14 macrodomain-1 is unable to degrade poly(ADP-ribose), the enzymatic product of PARP1. The F926A mutation of PARP14 and the F244A mutation of PARP9 strongly reduced ADP-ribosyl glycohydrolase activity of the respective macrodomains, suggesting mech-anistic homology to the Mac1 domain of the SARS-CoV-2 Nsp3 protein. This study adds two new enzymes to the previously known six human ADP-ribosyl glycohydrolases. Our results have key implications for how PARP14 and PARP9 will be studied and how their functions will be understood.","version":"1.1","doi":"10.1101/2023.06.24.546374","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.23.546214","pub_date":"2023-6-23","title":"Infection-induced vascular inflammation in COVID-19 links focal microglial dysfunction with neuropathologies through IL-1/IL-6-related systemic inflammatory states","abstract":"COVID-19 is associated with diverse neurological abnormalities, which predict poor outcome in patients. However, the mechanisms whereby infection-induced inflammation could affect complex neuropathologies in COVID-19 are unclear. We hypothesized that microglia, the resident immune cells of brain, are centrally involved in this process. To study this, we developed an autopsy platform allowing the integration of molecular anatomy-, protein- and mRNA data sets in post-mortem mirror blocks of brain and peripheral organ samples from COVID-19 cases. Nanoscale microscopy, single-cell RNA sequencing and analysis of inflammatory and metabolic signatures revealed distinct mechanisms of microglial dysfunction associated with cerebral SARS-CoV-2 infection. We observed focal loss of microglial P2Y12R at sites of virus-associated vascular inflammation together with dysregulated microglia-vascular-astrocyte interactions, Cx3Cr1-fractalkine axis deficits and mitochondrial failure in severely affected medullary autonomic nuclei and other brain areas. Microglial dysfunction occurs at sites of excessive synapse- and myelin phagocytosis and loss of glutamatergic terminals. While central and systemic viral load is strongly linked in individual patients, the regionally heterogenous microglial reactivity in the brain correlated with the extent of central and systemic inflammation related to IL-1 / IL-6 via virus-sensing pattern recognition receptors (PRRs) and inflammasome activation pathways. Thus, SARS-CoV-2-induced central and systemic inflammation might lead to a primarily glio-vascular failure in the brain, which could be a common contributor to diverse COVID-19-related neuropathologies.","version":"1.1","doi":"10.1101/2023.06.23.546214","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.23.546114","pub_date":"2023-6-23","title":"PCR data accurately predict infectious virus: a characterization of SARS-CoV-2 in non-human primates","abstract":"Researchers and clinicians often rely on molecular assays like PCR to identify and monitor viral infections instead of the resource-prohibitive gold standard of viral culture. However, it remains unclear when (if ever) PCR measurements of viral load are reliable indicators of replicating or infectious virus. Here, we compare total RNA, subgenomic RNA, and viral culture results from 24 studies of SARS-CoV-2 in non-human primates using bespoke statistical models. On out-of-sample data, our best models predict subgenomic RNA from total RNA with 91% accuracy, and they predict culture positivity with 85% accuracy. Total RNA and subgenomic RNA showed equivalent performance as predictors of culture positivity. Multiple cofactors, including exposure conditions and host traits, influence culture predictions for total RNA quantities spanning twelve orders of magnitude. Our model framework can be adapted to compare any assays, in any host species, and for any virus, to support laboratory analyses, medical decisions, and public health guidelines.","version":"1.1","doi":"10.1101/2023.06.23.546114","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.22.546079","pub_date":"2023-6-22","title":"The impact of non-lineage defining mutations in the structural stability for variants of concern of SARS-CoV-2","abstract":"The identification of the most important mutations, that lead to a structural and functional change in a highly transmissible virus variants, is essential to understand the impacts and the possible chances of vaccine and antibody escape. Strategies to rapidly associate mutations to functional and conformational properties are needed to rapidly analyze mutations in proteins and their impacts in antibodies and human binding proteins. Comparative analysis showed the main structural characteristics of the essential mutations found for each variant of concern in relation to the reference proteins. The paper presented a series of methodologies to track and associate conformational changes and the impacts promoted by the mutations. yasmmin.c.martins@gmail.com.br Supplementary data are available at Bioinformatics online.","version":"1.1","doi":"10.1101/2023.06.22.546079","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.21.545910","pub_date":"2023-6-21","title":"Spatial and Temporal Analysis of SARS-CoV-2 Genome Evolutionary Patterns","abstract":"The spread of SARS-CoV-2 virus accompanied by availability of abundant sequence data publicly, provides a window for determining the spatio-temporal patterns of viral evolution in response to vaccination. In this study, SARS-CoV-2 genome sequences were collected from seven countries in the period January 2020-December 2022. The sequences were classified into three phases, namely: pre-vaccination, post-vaccination, and recent period. Comparison was performed between these phases based on parameters like mutation rates, selection pressure (dN/dS ratio), and transition to transversion ratios (Ti/Tv). Similar comparisons were performed among SARS-CoV-2 variants. Statistical significance was tested using Graphpad unpaired t-test. The comparative analysis showed an increase in the percent genomic mutation rates post-vaccination and in recent periods across different countries from the pre-vaccination phase. The dN/dS ratios showed positive selection that increased after vaccination, and the Ti/Tv ratios decreased after vaccination. C\u2192U and G\u2192U were the most frequent transitions and transversions in all the countries. However, U\u2192G was the most frequent transversion in recent period. The Omicron variant had the highest genomic mutation rates, while Delta showed the highest dN/dS ratio. Mutation rates were highest in NSP3, S, N and NSP12b before and increased further after vaccination. NSP4 showed the largest change in mutation rates after vaccination. N, ORF8, ORF3a and ORF10 were under highest positive selection before vaccination. They were overtaken by E, S and NSP1 in the after vaccination as well as recent sequences, with the largest change observed in NSP1. Protein-wise dN/dS ratio was also seen to vary across the different variants. Irrespective of the different vaccine technologies used, geographical regions and host genetics, variations in the SARS-CoV-2 genome have maintained similar patterns worldwide. To the best of our knowledge, there exists no other large-scale study of the genomic and protein-wise mutation patterns during the time course of evolution in different countries. Analysing the SARS-CoV-2 evolution patterns in response to spatial, temporal, and biological signals is important for diagnostics, therapeutics, and pharmacovigilance of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.06.21.545910","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.20.545832","pub_date":"2023-6-21","title":"Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in severe COVID-19","abstract":"Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, the treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. Here, we show that the oral antiviral ensitrelvir combined with the anti-inflammatory corticosteroid methylprednisolone has higher therapeutic effects and better outcomes in a delayed dosing model of SARS-CoV-2 infected hamsters compared to the monotherapy with ensitrelvir or methylprednisolone alone. Combination therapy with these drugs improved respiratory conditions and the development of pneumonia in hamsters even when the treatment was started after 2 days post infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulated expressions of genes involved in inflammatory response. Furthermore, we found that the combination treatment is effective in infection with both highly pathogenic delta and circulating omicron variants. Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment. Since both drugs are available as oral medications, this combination therapy could provide a clinical and potent therapeutic option for COVID-19.","version":"1.1","doi":"10.1101/2023.06.20.545832","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.12.540592","pub_date":"2023-6-21","title":"Towards Computing Attributions for Dimensionality Reduction Techniques","abstract":"We describe the problem of computing local feature attributions for dimensionality reduction methods. We use one such method that is well established within the context of supervised classification \u2013 using the gradients of target outputs with respect to the inputs \u2013 on the popular dimensionality reduction technique t-SNE, widely used in analyses of biological data. We provide an efficient implementation for the gradient computation for this dimensionality reduction technique. We show that our explanations identify significant features using novel validation methodology; using synthetic datasets and the popular MNIST benchmark dataset. We then demonstrate the practical utility of our algorithm by showing that it can produce explanations that agree with domain knowledge on a SARS-CoV-2 sequence dataset. Throughout, we provide a road map so that similar explanation methods could be applied to other dimensionality reduction techniques to rigorously analyze biological datasets.","version":"1.2","doi":"10.1101/2023.05.12.540592","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479343","pub_date":"2023-6-20","title":"Machine learning-based approach KEVOLVE efficiently identifies SARS-CoV-2 variant-specific genomic signatures","abstract":"Machine learning was shown to be effective at identifying distinctive genomic signatures among viral sequences. These signatures are defined as pervasive motifs in the viral genome that allow discrimination between species or variants. In the context of SARS-CoV-2, the identification of these signatures can assist in taxonomic and phylogenetic studies, improve in the recognition and definition of emerging variants, and aid in the characterization of functional properties of polymorphic gene products. In this paper, we assess KEVOLVE, an approach based on a genetic algorithm with a machine-learning kernel, to identify multiple genomic signatures based on minimal sets of k-mers. In a comparative study, in which we analyzed large SARS-CoV-2 genome dataset, KEVOLVE was more effective at identifying variant-discriminative signatures than several gold-standard statistical tools. Subsequently, these signatures were characterized using a new extension of KEVOLVE (KANALYZER) to highlight variations of the discriminative signatures among different classes of variants, their genomic location, and the mutations involved. The majority of identified signatures were associated with known mutations among the different variants, in terms of functional and pathological impact based on available literature. Here we showed that KEVOLVE is a robust machine learning approach to identify discriminative signatures among SARS-CoV-2 variants, which are frequently also biologically relevant, while bypassing multiple sequence alignments. The source code of the method and additional resources are available at: https://github.com/bioinfoUQAM/KEVOLVE.","version":"1.2","doi":"10.1101/2022.02.07.479343","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481499","pub_date":"2023-6-20","title":"A SARS-CoV-2 peptide vaccine which elicits T-cell responses in mice but does not protect against infection or disease","abstract":"We vaccinated BALB/c mice with peptides derived from the SARS-CoV-2 proteome selected in silico to elicit T-cell responses and/or B-cell responses against linear epitopes. These peptides were administered in combination with either of two adjuvants, poly(I:C) and the STING agonist BI-1387466. Antibody responses against predicted linear epitopes were not observed but both adjuvants consistently elicited T-cell responses to the same peptides, which were primarily from the set chosen for predicted T-cell immunogenicity. The magnitude of T-cell responses was significantly higher with BI-1387466 compared with poly(I:C). Neither adjuvant group, however, provided any protection against infection with the murine adapted virus SARS-CoV-2-MA10 or from disease following infection. In light of more recent evidence for protection from severe disease mediated by CD8+ T-cells, we suspect that the epitopes selected for vaccination were not presented by infected murine cells.","version":"1.2","doi":"10.1101/2022.02.22.481499","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.16.504128","pub_date":"2023-6-19","title":"Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds","abstract":"Multivalent antigen display is a well-established principle to enhance humoral immunity. Protein-based virus-like particles (VLPs) are commonly used to spatially organize antigens. However, protein-based VLPs are limited in their ability to control valency on fixed scaffold geometries and are thymus-dependent antigens that elicit neutralizing B cell memory themselves, which can distract immune responses. Here, we investigated DNA origami as an alternative material for multivalent antigen display in vivo, applied to the receptor binding domain (RBD) of SARS-CoV-2 that is the primary antigenic target of neutralizing antibody responses. Icosahedral DNA-VLPs elicited neutralizing antibodies to SARS-CoV-2 in a valency-dependent manner following sequential immunization in mice, quantified by pseudo-and live-virus neutralization assays. Further, induction of B cell memory against the RBD required T cell help, but the immune sera did not contain boosted, class-switched antibodies against the DNA scaffold. This contrasted with protein-based VLP display of the RBD that elicited B cell memory against both the target antigen and the scaffold. Thus, DNA-based VLPs enhance target antigen immunogenicity without generating off-target, scaffold-directed immune memory, thereby offering a potentially important alternative material for particulate vaccine design.","version":"1.3","doi":"10.1101/2022.08.16.504128","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.15.545172","pub_date":"2023-6-16","title":"SARS-CoV-2 Delta Variant Remains Viable in Environmental Biofilms found in Meat Packaging Plants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus that directly infects human airway epithelial cells and caused the COVID-19 pandemic. At the start of the pandemic in 2020, meat-packaging plants saw a surge in SARS-CoV-2 cases, which forced many to temporarily close. To determine why SARS-CoV-2 appears to thrive specifically well in meat packaging plants, we used SARS-CoV-2 Delta variant and meat packaging plant drain samples to develop mixed-species biofilms on materials commonly found within meat packaging plants (stainless steel (SS), PVC, and ceramic tile). Our data provides evidence that SARS-CoV-2 Delta variant remained viable on all the surfaces tested with and without an environmental biofilm. We observed that SARS-CoV-2 Delta variant was able to remain infectious with each of the environmental biofilms, however, we detected a significant reduction in viability post-exposure to Plant B biofilm on SS, PVC, and on ceramic tile chips, and to Plant C biofilm on SS and PVC chips. The numbers of viable SARS-CoV-2 Delta viral particles was 1.81 \u2013 4.57-fold high than the viral inoculum incubated with the Plant B and Plant C environmental biofilm on SS, and PVC chips. We did not detect a significant difference in viability when SARS-CoV-2 Delta variant was incubated with the biofilm obtained from Plant A on any of the materials tested and SARS-CoV-2 Delta variant had higher plaque numbers when inoculated with Plant C biofilm on tile chips, with a 2.75-fold difference compared to SARS-CoV-2 Delta variant on tile chips by itself. In addition, we detected an increase in the biofilm biovolume in response to SARS-CoV-2 Delta variant which is also a concern for food safety due to the potential for foodborne pathogens to respond likewise when they come into contact with the virus. These results indicate a complex virus-environmental biofilm interaction which correlates to the different bacteria found in each biofilm. Our results also indicate that there is the potential for biofilms to protect SARS-CoV-2 from disinfecting agents and remaining prevalent in meat packaging plants. With the highly infectious nature of some SARS-CoV-2 variants such as Delta, and more so with the Omicron variant, even a minimal amount of virus could have serious health implications for the spread and reoccurrence of SARS-CoV-2 outbreaks in meat packaging plants.","version":"1.1","doi":"10.1101/2023.06.15.545172","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.15.545129","pub_date":"2023-6-16","title":"Repurposing Remdesivir for COVID-19: Computational Drug Design Targeting SARS-CoV-2 RNA Polymerase and Main Protease using Molecular Dynamics Approach","abstract":"The coronavirus disease of 2019 (COVID-19) is a highly contagious respiratory illness that has become a global health crisis with new variants, an unprecedented number of infections, and deaths and demands urgent manufacturing of potent therapeutics. Despite the success of vaccination campaigns around the globe, there is no particular therapeutics approved to date for efficiently treating infected individuals. Repositioning or repurposing previously effective antivirals against RNA viruses to treat COVID-19 patients is a feasible option. Remdesivir is a broad-spectrum antiviral drug that the Food and Drug Administration (FDA) licenses for treating COVID-19 patients who are critically ill patients. Remdesivir\u2019s low efficacy, which has been shown in some clinical trials, possible adverse effects, and dose-related toxicities are issues with its use in clinical use. Our study aimed to design potent derivatives of remdesivir through the functional group modification of the parent drug targeting RNA-dependent RNA polymerase (RdRp) and main protease (MPro) of SARS-CoV-2. The efficacy and stability of the proposed derivatives were assessed by molecular docking and extended molecular dynamics simulation analyses. Furthermore, the pharmacokinetic activity was measured to ensure the safety and drug potential of the designed derivatives. The derivatives were non-carcinogenic, chemically reactive, highly interactive, and stable with the target proteins. D-CF3 is one of the designed derivatives that finally showed stronger interaction than the parent drug, according to the docking and dynamics simulation analyses, with both target proteins. However, in vitro and in vivo investigations are guaranteed to validate the findings in the future.","version":"1.1","doi":"10.1101/2023.06.15.545129","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.28.477987","pub_date":"2023-6-16","title":"Mapping SARS-CoV-2 antigenic relationships and serological responses","abstract":"During the SARS-CoV-2 pandemic, multiple variants escaping pre-existing immunity emerged, causing concerns about continued protection. Here, we use antigenic cartography to analyze patterns of cross-reactivity among a panel of 21 variants and 15 groups of human sera obtained following primary infection with 10 different variants or after mRNA-1273 or mRNA-1273.351 vaccination. We find antigenic differences among pre-Omicron variants caused by substitutions at spike protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months post-2nd dose. We find changes in immunodominance of different spike regions depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine strain selection. Antigenic Cartography of SARS-CoV-2 variants reveals amino acid substitutions governing immune escape and immunodominance patterns.","version":"1.3","doi":"10.1101/2022.01.28.477987","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.16.545251","pub_date":"2023-6-16","title":"High-confidence placement of difficult-to-fit fragments into electron density by using anomalous signals - a case study using hits targeting SARS-CoV-2 non-structural protein 1","abstract":"The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (< 300 Da) although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tuneable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to advance the rational optimisation of SARS-CoV-2 nsp1 targeting fragment hits.","version":"1.1","doi":"10.1101/2023.06.16.545251","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.14.544834","pub_date":"2023-6-15","title":"Immunogenicity of COVID-19 vaccines and their effect on the HIV reservoir in older people with HIV","abstract":"Older individuals and people with HIV (PWH) were prioritized for COVID-19 vaccination, yet comprehensive studies of the immunogenicity of these vaccines and their effects on HIV reservoirs are not available. We followed 68 PWH aged 55 and older and 23 age-matched HIV-negative individuals for 48 weeks from the first vaccine dose, after the total of three doses. All PWH were on antiretroviral therapy (cART) and had different immune status, including immune responders (IR), immune non-responders (INR), and PWH with low-level viremia (LLV). We measured total and neutralizing Ab responses to SARS-CoV-2 spike and RBD in sera, total anti-spike Abs in saliva, frequency of anti-RBD/NTD B cells, changes in frequency of anti-spike, HIV gag/nef-specific T cells, and HIV reservoirs in peripheral CD4+ T cells. The resulting datasets were used to create a mathematical model for within-host immunization. Various regimens of BNT162b2, mRNA-1273, and ChAdOx1 vaccines elicited equally strong anti-spike IgG responses in PWH and HIV- participants in serum and saliva at all timepoints. These responses had similar kinetics in both cohorts and peaked at 4 weeks post-booster (third dose), while half-lives of plasma IgG also dramatically increased post-booster in both groups. Salivary spike IgA responses were low, especially in INRs. PWH had diminished live virus neutralizing titers after two vaccine doses which were \u2018rescued\u2019 after a booster. Anti-spike T cell immunity was enhanced in IRs even in comparison to HIV- participants, suggesting Th1 imprinting from HIV, while in INRs it was the lowest. Increased frequency of viral \u2018blips\u2019 in PWH were seen post-vaccination, but vaccines did not affect the size of the intact HIV reservoir in CD4+ T cells in most PWH, except in LLVs. Thus, older PWH require three doses of COVID-19 vaccine to maximize neutralizing responses against SARS-CoV-2, although vaccines may increase HIV reservoirs in PWH with persistent viremia.","version":"1.1","doi":"10.1101/2023.06.14.544834","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.14.544985","pub_date":"2023-6-15","title":"Inclusion of glycopeptides in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike ectodomain provides in-creased sequence coverage","abstract":"Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein\u2019s conformational dynamics across varied states, such as heat-denatured vs. native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of glycopeptides has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their isotopic mass shifts from deuteration. Inclusion of the deu-terated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.","version":"1.1","doi":"10.1101/2023.06.14.544985","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.13.544630","pub_date":"2023-6-14","title":"Biophysical evolution of the receptor binding domains of SARS-CoVs","abstract":"With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening. This study holds significant relevance in the context of the COVID-19 pandemic and the broader understanding of coronaviruses. A comparison of the receptor binding domains (RBDs) of SARS-CoV and SARS-CoV-2 reveals significant differences in their structure, stability, aggregation, and function. Despite divergent sequences, the RBDs share a similar fold and ACE2 receptor binding capability, likely through convergent evolution. These findings are crucial for understanding coronavirus evolution, interactions with human receptors, and the spillover of coronaviruses from animals to humans. The study also has implications for vaccine design strategies for SARS-CoVs, where the RBD is used as an antigen in protein subunit vaccines. By anticipating future outbreaks and enhancing our understanding of zoonotic spillover, this research contributes to safeguarding human health.","version":"1.1","doi":"10.1101/2023.06.13.544630","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.13.544519","pub_date":"2023-6-14","title":"Partially hydrolyzed guar gum attenuates the symptoms of SARS-CoV-2 infection through gut microbiota modulation in an animal model","abstract":"The coronavirus disease 2019 (COVID-19) pandemic has caused worldwide health issues. Although several vaccines have been developed, it is still difficult to prevent and reduce the inflammation caused by the infection. Studies have shown that there are correlations between the gut environment and severity of symptoms caused by SARS-CoV-2 infection. Several gut metabolites produced by the gut microbiota such as SCFAs and the secondary bile acid UDCA are reported to improve the survival rate of the host after viral infection in an animal model through modulation of the host immune system. Therefore, in this study, we attempted to use the prebiotic dietary fiber PHGG to modulate the gut microbiome and intestinal metabolites for improvement of host survival rate after SARS-CoV-2 infection in a Syrian hamster model. We were able to show that PHGG significantly improved the host survival rate and body weight reduction. Analysis of the gut microbiome, serum, and intestinal metabolites revealed that PHGG significantly increased the concentrations of several intestinal SCFAs, fecal secondary bile acids, and serum secondary bile acids. Furthermore, several microbial species and metabolites identified in this study are consistent with reports in humans. Taken together, our data suggest that PHGG is a candidate prebiotic food for reducing the morbidity of COVID-19.","version":"1.1","doi":"10.1101/2023.06.13.544519","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.14.544560","pub_date":"2023-6-14","title":"AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor","abstract":"Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.","version":"1.1","doi":"10.1101/2023.06.14.544560","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.12.540483","pub_date":"2023-6-14","title":"Regulation of coronavirus nsp15 cleavage specificity by RNA structure","abstract":"SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had an enduring impact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (NSPS) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established function of nsp15 as a uridine-specific endonuclease, little is known about other determinants of its cleavage specificity. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS 3\u2019UTR as a model for our structural studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus high probably targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the GNRNA pentaloop of the RNA. Further investigation revealed that the position of uridine within the pentaloop also impacted nsp15 cleavage efficiency, suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of recognition of RNA by nsp15.","version":"1.2","doi":"10.1101/2023.05.12.540483","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.13.544745","pub_date":"2023-6-13","title":"SARS-CoV-2 Envelope protein alters calcium signaling via SERCA interactions","abstract":"The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor, however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirmed that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling and molecular dynamics of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.06.13.544745","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.20.513049","pub_date":"2023-6-13","title":"De novo design of anti-variant COVID-19 Vaccine","abstract":"Recent studies have shown the efficacy of hybrid SARS-COV-2 vaccines using wild-type nucleocapsid (N) and Spike (S) protein. We upgraded this strategy one step further using clinically proven spike protein by considering the delta and post-delta omicron variant of concern (VOC) mutations and nucleocapsid peptides conferring T-cell immunity. Nucleocapsid peptides are considered much better immunological replacement of nucleocapsid proteins. Hence, peptide linking strategy is applied which is more economic for cellular biosynthesis than whole proteins. An envelope peptide with potent T-cell immune response is also selected. All these peptides are clustered in this hybrid spike\u2019s designed cytoplasmic region separated by non-immunogenic helical linkers. The resulting domain is more folded in the construct devoid of transmembrane domain after AlphaFold analysis. Alongside, we also propose the idea of introduction of any T-cell peptide like other Human Corona Viruses (HuCoV) in these linker regions whenever required. In addition to SARS-COV-2, the same approach can be applied for any emergency or even long-term unsolved outbreaks of Influenza, Dengue and West Nile Virus etc. In this era of novelty as presented by subunit and nucleic acid vaccines, multiepitope strategies like this can help to combat multiple diseases successfully in real time to give hope for better future.","version":"1.2","doi":"10.1101/2022.10.20.513049","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.12.544648","pub_date":"2023-6-13","title":"C1q Enables Influenza HA Stem Binding Antibodies to Block Viral Attachment and Broadens the Antibody Escape Repertoire","abstract":"Broadly neutralizing, anti-hemagglutinin stem antibodies (Abs) are a promising universal influenza vaccine target. While anti-stem Abs are not believed to block viral attachment, we show that C1q confers attachment inhibition and boosts fusion and neuraminidase inhibition, greatly enhancing virus neutralization activity in vitro and in mice challenged with influenza virus via the respiratory route. These effects reflect increased steric interference and not increased Ab avidity. Remarkably, C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the hemagglutinin. Some substitutions cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-RBD anti-SARS-CoV-2 Spike Abs, an effect dependent on Spike density on the virion surface. Together, our findings show that first, Ab function must be considered in a physiological context and second, inferring the exact selection pressure for Ab-driven viral evolution is risky business, at best.","version":"1.1","doi":"10.1101/2023.06.12.544648","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.12.544498","pub_date":"2023-6-13","title":"Involvement of a serotonin/GLP-1 circuit in adolescent isolation-induced diabetes","abstract":"In 2020, stay-at-home orders were implemented to stem the spread of SARS-CoV-2 worldwide. Social isolation can be particularly harmful to children and adolescents\u2014during the pandemic, the prevalence of obesity increased by \u223c37% in persons aged 2-19. Obesity is often comorbid with type 2 diabetes, which was not assessed in this human pandemic cohort. Here, we investigated whether male mice isolated throughout adolescence develop type 2 diabetes in a manner consistent with human obesity-induced diabetes, and explored neural changes that may underlie such an interaction. We find that isolating C57BL/6J mice throughout adolescence is sufficient to induce type 2 diabetes. We observed fasted hyperglycemia, diminished glucose clearance in response to an insulin tolerance test, decreased insulin signaling in skeletal muscle, decreased insulin staining of pancreatic islets, increased nociception, and diminished plasma cortisol levels compared to group-housed control mice. Using Promethion metabolic phenotyping chambers, we observed dysregulation of sleep and eating behaviors, as well as a time-dependent shift in respiratory exchange ratio of the adolescent-isolation mice. We profiled changes in neural gene transcription from several brain areas and found that a neural circuit between serotonin-producing and GLP-1-producing neurons is affected by this isolation paradigm. Overall, spatial transcription data suggest decreased serotonin neuron activity (via decreased GLP-1-mediated excitation) and increased GLP-1 neuron activity (via decreased serotonin-mediated inhibition). This circuit may represent an intersectional target to further investigate the relationship between social isolation and type 2 diabetes, as well as a pharmacologically-relevant circuit to explore the effects of serotonin and GLP-1 receptor agonists. Isolating C57BL/6J mice throughout adolescence is sufficient to induce type 2 diabetes, presenting with fasted hyperglycemia. Adolescent-isolation mice have deficits in insulin responsiveness, impaired peripheral insulin signaling, and decreased pancreatic insulin production. Transcriptional changes across the brain include the endocannabinoid, serotonin, and GLP-1 neurotransmitters and associated receptors. The neural serotonin/GLP-1 circuit may represent an intersectional target to further investigate the relationship between social isolation and type 2 diabetes. Serotonin-producing neurons of adolescent-isolation mice produce fewer transcripts for the GLP-1 receptor, and GLP-1 neurons produce fewer transcripts for the 5-HT1A serotonin receptor.","version":"1.1","doi":"10.1101/2023.06.12.544498","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.12.544552","pub_date":"2023-6-12","title":"Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2","abstract":"Why individuals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.","version":"1.1","doi":"10.1101/2023.06.12.544552","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.22.537917","pub_date":"2023-6-12","title":"SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response","abstract":"Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, a key driver of replication, on the UPR using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their co-expression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Re-analysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.","version":"1.2","doi":"10.1101/2023.04.22.537917","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.12.532219","pub_date":"2023-6-12","title":"Evolutionary changes in the number of dissociable amino acids on spike proteins and nucleoproteins of SARS-CoV-2 variants","abstract":"The spike protein of SARS-CoV-2 is responsible for target recognition, cellular entry, and endosomal escape of the virus. At the same time, it is the part of the virus which exhibits the greatest sequence variation across the many variants which have emerged during its evolution. Recent studies have indicated that with progressive lineage emergence, the positive charge on the spike protein has been increasing, with certain positively charged amino acids improving the binding of the spike protein to cell receptors. We have performed a detailed analysis of dissociable amino acids of more than 1400 different SARS-CoV-2 lineages which confirms these observations while suggesting that this progression has reached a plateau with omicron and its subvariants and that the positive charge is not increasing further. Analysis of the nucleocapsid protein shows no similar increase of positive charge with novel variants, which further indicates that positive charge of the spike protein is being evolutionarily selected for. Furthermore, comparison with the spike proteins of known coronaviruses shows that already the wild-type SARS-CoV-2 spike protein carries an unusually large amount of positively charged amino acids when compared to most other betacoronaviruses. Our study sheds a light on the evolutionary changes in the number of dissociable amino acids on the spike protein of SARS-CoV-2, complementing existing studies and providing a stepping stone towards a better understanding of the relationship between the spike protein charge and viral infectivity and transmissibility.","version":"1.2","doi":"10.1101/2023.03.12.532219","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.09.544432","pub_date":"2023-6-12","title":"RBD-based high affinity ACE2 antagonist limits SARS-CoV-2 replication in upper and lower airways","abstract":"SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.","version":"1.1","doi":"10.1101/2023.06.09.544432","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.12.544536","pub_date":"2023-6-12","title":"Defining the single base importance of human mRNAs and lncRNAs","abstract":"As the fundamental unit of a gene and its transcripts, nucleotides have enormous impacts on molecular function and evolution, and thus on phenotypes and diseases. Given that different nucleotides on one gene often exhibit diverse levels of effects, it is quite crucial to comprehensively and quantitatively measure the importance of each base on a gene transcript, however, tools are still not available. Here we proposed Base Importance Calculator (BIC), an algorithm to calculate the importance score of single bases based on sequence information of human mRNAs and long noncoding RNAs (lncRNAs). We then confirmed its power by applying BIC to three different tasks. Firstly, we revealed that BIC can effectively evaluate the pathogenicity of both genes and single bases by analyzing the BIC scores and the pathogenicity of single nucleotide variations (SNVs). Moreover, the BIC score in the Cancer Genome Atlas (TCGA) somatic mutations is able to predict the prognosis of some cancers. Finally, we show that BIC can also precisely predict the transmissibility of SARS-CoV-2. The above results indicate that BIC is a useful tool for evaluating the single base important of human mRNAs and lncRNAs. BIC could measure the single base importance of human mRNAs and lncRNAs. BIC could be applied to many aspects including measuring the pathogenicity of SNVs and enhancing the ability of predicting cancer survival. BIC could predict the transmissibility of SARS-CoV-2","version":"1.1","doi":"10.1101/2023.06.12.544536","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.07.544133","pub_date":"2023-6-08","title":"Anti-Viral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein Against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models","abstract":"SARS-CoV-2 Variants of Concern (VOCs) continue to evolve and re-emerge with chronic inflammatory long-COVID sequelae necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the Receptor for Advanced Glycation End products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of the RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an anti-viral and anti-inflammatory therapeutic effect in the COVID-19 system. The protective therapeutic effect of RAGE-Ig was determined in vitro in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six various VOCs of SARS-CoV-2. The underlying anti-viral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated: (i) significant dose-dependent protection (i.e. greater survival, less weight loss, lower virus replication in the lungs); (ii) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (iii) a RAGE-Ig dose-dependent increase in the expression of type I interferons (IFN-\u03b1, and IFN-\u03b2) and type III interferon (IFN\u03bb2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (iv) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Our pre-clinical findings revealed type I and III interferons-mediated anti-viral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2023.06.07.544133","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.06.515327","pub_date":"2023-6-08","title":"Temporal Vascular Endothelial Growth Factor Sub-type Gene Switching in SARS-CoV-related Inflammation - Basis for a Dual Gene Biomarker Approach","abstract":"This study examines temporal gene expression (GE) patterns in a murine model of SARS-CoV infection. We focused on a Temporal Gene Set (TGS) comprising pro-inflammatory genes (TNF, NFKB1, VEGF-A) and VEGF-B. A systematic search of the NCBI Geo database for MA15 (SARS-CoV) pulmonary studies using C57BL Wild (WT) mice and filtering according to TGS GE patterns eluded seven datasets for further analysis. Encompassing the GE profiles from these datasets alluded to a rising and falling pattern in TNF and NFKB1 GE. Also, our findings reveal a temporal decrease in VEGF-A GE coinciding with an increase in VEGF-B GE post-immunogenic stimulation. Notably, differential responses were observed with the MA15 dosage and in comparison, to other antigens (dORF6 and NSP16). Further, the human SARS-CoV-2 gene enrichment in this murine study confirms the MA15 murine model\u2019s relevance for SARS research. Our study also suggests potential interactions between SARS-CoV-2 Spike protein and VEGF-related receptors, hinting at other pathophysiological mechanisms. Our results indicate severe inflammation may lead to a flattened VEGF-B GE response, influencing VEGF-B\u2019s cell survival role. We underline the significance of considering VEGF-A/B interactions, particularly temporal differences, in manipulating angiogenic processes. Future research needs to consider temporal changes in VEGF-A and VEGF-B GE, in terms of time-associated gene-switching, in line with changing host inflammation.","version":"1.2","doi":"10.1101/2022.11.06.515327","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.07.544062","pub_date":"2023-6-08","title":"Single-cell RNA sequencing reveals HIF1A as a severity-sensitive immunological scar in circulating monocytes of convalescent comorbidity-free COVID-19 patients","abstract":"COVID-19, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is characterized by a wide range of clinical symptoms and a poorly predictable disease course. Although in-depth transcriptomic investigations of peripheral blood samples from COVID-19 patients have been performed, the detailed molecular mechanisms underlying an asymptomatic, mild or severe disease course, particularly in patients without relevant comorbidities, remain poorly understood. While previous studies have mainly focused on the cellular and molecular dissection of ongoing COVID-19, we set out to characterize transcriptomic immune cell dysregulation at the single-cell level at different time points in patients without comorbidities after disease resolution to identify signatures of different disease severities in convalescence. With single-cell RNA sequencing we reveal a role for hypoxia-inducible factor 1-alpha (HIF1A) as a severity-sensitive long-term immunological scar in circulating monocytes of convalescent COVID-19 patients. Additionally, circulating complexes formed by monocytes with either T cells or NK cells represent a characteristic cellular marker in convalescent COVID-19 patients irrespective of their preceding symptom severity. Together, these results provide cellular and molecular correlates of recovery from COVID-19 and could help in immune monitoring and in the design of new treatment strategies.","version":"1.1","doi":"10.1101/2023.06.07.544062","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.07.544141","pub_date":"2023-6-08","title":"An evolutionarily conserved strategy for ribosome binding and inhibition by \u03b2-coronavirus non-structural protein 1","abstract":"An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Nsp1, which suppresses host gene expression and stunts antiviral signaling. SARS-CoV-2 Nsp1 binds the ribosome to inhibit translation through mRNA displacement and induces degradation of host mRNAs through an unknown mechanism. Here we show that Nsp1-dependent host shutoff is conserved in diverse coronaviruses, but only Nsp1 from \u03b2-CoV inhibits translation through ribosome binding. The C-terminal domain of all \u03b2-CoV Nsp1s confers high-affinity ribosome-binding despite low sequence conservation. Modeling of interactions of four Nsp1s to the ribosome identified few absolutely conserved amino acids that, together with an overall conservation in surface charge, form the \u03b2-CoV Nsp1 ribosome-binding domain. Contrary to previous models, the Nsp1 ribosome-binding domain is an inefficient translation inhibitor. Instead, the Nsp1-CTD likely functions by recruiting Nsp1\u2019s N-terminal \u201ceffector\u201d domain. Finally, we show that a viral cis-acting RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. Together, our work provides new insight into the diversity and conservation of ribosome-dependent host-shutoff functions of Nsp1, knowledge that could aide future efforts in pharmacological targeting of Nsp1 from SARS-CoV-2, but also related human-pathogenic \u03b2-coronaviruses. Our study also exemplifies how comparing highly divergent Nsp1 variants can help to dissect the different modalities of this multi-functional viral protein.","version":"1.1","doi":"10.1101/2023.06.07.544141","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.05.543759","pub_date":"2023-6-07","title":"Atypical Chemokine Receptor 1 (Ackr1)-deficient Mice Resist Lethal SARS-CoV-2 Challenge","abstract":"High pro-inflammatory chemokine levels have been reported in blood and lung in patients with COVID-19. To investigate specific roles in pathogenesis, we studied the regulation of chemokine ligands and receptors in the lungs of 4-6-month-old wild type C57BL/6 mice infected with the MA10 mouse-adapted strain of SARS-CoV-2. We found that atypical chemokine receptor 1 (Ackr1, also known as Duffy antigen receptor for chemokines/DARC) was the most highly upregulated chemokine receptor in infected lung, where it localized to endothelial cells of veins and arterioles. In a screen of 7 leukocyte chemoattractant or chemoattractant receptor knockout mouse lines, Ackr1-/- mice were unique in having lower mortality after SARS-CoV-2 infection, particularly in males. ACKR1 is a non-signaling chemokine receptor that in addition to endothelium is also expressed on erythrocytes and Purkinje cells of the cerebellum. It binds promiscuously to both inflammatory CC and CXC chemokines and has been reported to control chemokine availability which may influence the shape of chemotactic gradients and the ability of leukocytes to extravasate and produce immunopathology. Of note, erythrocyte ACKR1 deficiency is fixed in sub-Saharan African populations where COVID-19 has been reported to result in low mortality compared to worldwide data. Our data suggest the possibility of a causal contribution of ACKR1 deficiency to low sub-Saharan COVID-19 mortality and identify ACKR1 as a possible drug target in the disease.","version":"1.1","doi":"10.1101/2023.06.05.543759","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.06.543969","pub_date":"2023-6-07","title":"Widespread impact of immunoglobulin V gene allelic polymorphisms on antibody reactivity","abstract":"The ability of human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V gene (IGV) allelic polymorphisms. However, previous studies have provided only a limited number of examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many IGV allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiment further demonstrates that paratope allelic mutations on both heavy and light chain often abolish antibody binding. We also illustrate the importance of minor IGV allelic variants with low frequency in several broadly neutralizing antibodies to SARS-CoV-2 and influenza virus. Overall, this study not only highlights the pervasive impact of IGV allelic polymorphisms on antibody binding, but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.","version":"1.1","doi":"10.1101/2023.06.06.543969","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.30.526314","pub_date":"2023-6-06","title":"Fitness effects of mutations to SARS-CoV-2 proteins","abstract":"Knowledge of the fitness effects of mutations to SARS-CoV-2 can inform assessment of new variants, design of therapeutics resistant to escape, and understanding of the functions of viral proteins. However, experimentally measuring effects of mutations is challenging: we lack tractable lab assays for many SARS-CoV-2 proteins, and comprehensive deep mutational scanning has been applied to only two SARS-CoV-2 proteins. Here we develop an approach that leverages millions of publicly available SARS-CoV-2 sequences to estimate effects of mutations. We first calculate how many independent occurrences of each mutation are expected to be observed along the SARS-CoV-2 phylogeny in the absence of selection. We then compare these expected observations to the actual observations to estimate the effect of each mutation. These estimates correlate well with deep mutational scanning measurements. For most genes, synonymous mutations are nearly neutral, stop-codon mutations are deleterious, and amino-acid mutations have a range of effects. However, some viral accessory proteins are under little to no selection. We provide interactive visualizations of effects of mutations to all SARS-CoV-2 proteins (https://jbloomlab.github.io/SARS2-mut-fitness/). The framework we describe is applicable to any virus for which the number of available sequences is sufficiently large that many independent occurrences of each neutral mutation are observed.","version":"1.2","doi":"10.1101/2023.01.30.526314","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.27.504955","pub_date":"2023-6-06","title":"CD8+ T-cell memory induced by successive SARS-CoV-2 mRNA vaccinations is characterized by clonal replacement","abstract":"mRNA vaccines against the Spike glycoprotein of severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) elicit strong T-cell responses. However, it\u2019s not known whether T cell clonotypes responding to the first vaccination repeatedly expand with booster vaccinations. Here, we temporally tracked the CD8+ T-cell repertoire in individuals who received three shots of the BNT162b2 mRNA vaccine. By analyzing the kinetic profile of CD8+ T-cell clonotypes responding to the first, second, or third shot, we demonstrated that newly expanded clonotypes elicited by the second shot replaced many of those that responded to the first shot. Although these 2nd responder clonotypes expanded after the third shot, their clonal diversity was skewed, and they were partially replaced by newly elicited the 3rd responders. Furthermore, this replacement of vaccine-responding clonotypes occurred within the same Spike epitope. These results suggest that CD8+ T-cell memory induced by repetitive mRNA vaccination is characterized by the emergence of new dominant clones.","version":"1.2","doi":"10.1101/2022.08.27.504955","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.03.482731","pub_date":"2023-6-06","title":"A mechanistic understanding of the modes of Ca ion binding to the SARS-CoV-1 fusion peptide and their role in the dynamics of host membrane penetration","abstract":"The SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP) segment that mediates fusion of the viral and host cell membranes. Calcium ions are thought to position the FP optimally for membrane insertion by interacting with negatively charged residues in this segment (E801, D802, D812, E821, D825, and D830); however, which residues bind to calcium and in what combinations supportive of membrane insertion are unknown. Using biological assays and molecular dynamics studies, we have determined the functional configurations of FP-Ca+2 binding which promote membrane insertion. We first mutated the negatively charged residues in the SARS CoV-1 FP to assay their role in cell entry and syncytia formation, finding that charge loss in the D802A or D830A mutants reduced syncytia formation and pseudoparticle transduction. Interestingly, the D812A mutation led to increased pseudoparticle transduction, indicating the Ca2+ effect depends on binding at specific FP sites. To interpret mechanistically these results and learn how specific modes of FP-Ca2+ binding modulate membrane insertion, we performed molecular dynamics simulations. Preferred residue pairs for Ca2+ binding were identified (E801/D802; E801/D830; D812/E821) which promote FP membrane insertion. In contrast, binding to residues E821/D825 inhibited FP membrane insertion, which is also supported by our biological assays. Our findings show that Ca2+ binding to SARS-CoV-1 FP residue pairs E801/D802 and D812/E821 facilitates membrane insertion, whereas binding to the E801/D802 and D821/D825 pairs is detrimental. These conclusions provide an improved and nuanced mechanistic understanding of calcium binding modes to FP residues and their dynamic effects on host cell entry.","version":"1.3","doi":"10.1101/2022.03.03.482731","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.03.543589","pub_date":"2023-6-05","title":"A deep learning-based drug repurposing screening and validation for anti-SARS-CoV-2 compounds by targeting the cell entry mechanism","abstract":"The recent outbreak of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a severe threat to the global public health and economy, however, effective drugs to treat COVID-19 are still lacking. Here, we employ a deep learning-based drug repositioning strategy to systematically screen potential anti-SARS-CoV-2 drug candidates that target the cell entry mechanism of SARS-CoV-2 virus from 2,635 FDA-approved drugs and 1,062 active ingredients from Traditional Chinese Medicine herbs. In silico molecular docking analysis validates the interactions between the top compounds and host receptors or viral spike proteins. Using a SARS-CoV-2 pseudovirus system, we further identify several drug candidates including Fostamatinib, Linagliptin, Lysergol and Sophoridine that can effectively block the cell entry of SARS-CoV-2 variants into human lung cells even at a nanomolar scale. These efforts not only illuminate the feasibility of applying deep learning-based drug repositioning for antiviral agents by targeting a specified mechanism, but also provide a valuable resource of promising drug candidates or lead compounds to treat COVID-19.","version":"1.1","doi":"10.1101/2023.06.03.543589","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.02.543487","pub_date":"2023-6-05","title":"Potential involvement of protein phosphatase PP2CA on protein synthesis and cell cycle during SARS-CoV-2 infection. A meta analysis investigation","abstract":"Coronavirus disease 2019 is a multi-systemic syndrome that caused a pandemic. Proteomic studies demonstrate changes in protein expression and interaction involved in signaling pathways related to SARS-CoV-2 infections. Protein phosphatases are important for cell signaling regulation. Here we aimed to understand the involvement of protein phosphatases and the signaling pathways that may be involved during SARS-CoV-2 infection. Then, we carried out a metanalysis of protein phosphatase interaction directly or indirectly with viral proteins. Additionally, we analyzed the expression degree of protein phosphatases, and phosphorylation degree of intermediate proteins. Our analyses revealed that PP2CA and PTEN were the key protein involved in the cell cycle and apoptosis regulation, during SARS-CoV-2 infection. Showing it as potential target for COVID-19 control.","version":"1.1","doi":"10.1101/2023.06.02.543487","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.02.543458","pub_date":"2023-6-05","title":"Machine Learning-Guided Antibody Engineering That Leverages Domain Knowledge To Overcome The Small Data Problem","abstract":"The application of Machine Learning (ML) tools to engineer novel antibodies having predictable functional properties is gaining prominence. Herein, we present a platform that employs an ML-guided optimization of the complementarity-determining region (CDR) together with a CDR framework (FR) shuffling method to engineer affinity-enhanced and clinically developable monoclonal antibodies (mAbs) from a limited experimental screen space (order of 10^2 designs) using only two experimental iterations. Although high-complexity deep learning models like graph neural networks (GNNs) and large language models (LLMs) have shown success on protein folding with large dataset sizes, the small and biased nature of the publicly available antibody-antigen interaction datasets is not sufficient to capture the diversity of mutations virtually screened using these models in an affinity enhancement campaign. To address this key gap, we introduced inductive biases learned from extensive domain knowledge on protein-protein interactions through feature engineering and selected model hyper parameters to reduce overfitting of the limited interaction datasets. Notably we show that this platform performs better than GNNs and LLMs on an in-house validation dataset that is enriched in diverse CDR mutations that go beyond alanine-scanning. To illustrate the broad applicability of this platform, we successfully solved a challenging problem of redesigning two different anti-SARS-COV-2 mAbs to enhance affinity (up to 2 orders of magnitude) and neutralizing potency against the dynamically evolving SARS-COV-2 Omicron variants.","version":"1.1","doi":"10.1101/2023.06.02.543458","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.02.543047","pub_date":"2023-6-02","title":"Impact of reference design on estimating SARS-CoV-2 lineage abundances from wastewater sequencing data","abstract":"Sequencing of SARS-CoV-2 RNA from wastewater samples has emerged as a valuable tool for detecting the presence and relative abundances of SARS-CoV-2 variants in a community. By analyzing the viral genetic material present in wastewater, public health officials can gain early insights into the spread of the virus and inform timely intervention measures. The construction of reference datasets from known SARS-CoV-2 lineages and their mutation profies has become state-of-the-art for assigning viral lineages and their relative abundances from wastewater sequencing data. However, the selection of reference sequences or mutations directly affects the predictive power. Here, we show the impact of a mutation- and sequence-based reference reconstruction for SARS-CoV-2 abundance estimation. We benchmark three data sets: 1) synthetic \u201cspike-in\u201d mixtures, 2) German samples from early 2021, mainly comprising Alpha, and 3) samples obtained from wastewater at an international airport in Germany from the end of 2021, including 1rst signals of Omicron. The two approaches differ in sub-lineage detection, with the marker-mutation-based method, in particular, being challenged by the increasing number of mutations and lineages. However, the estimations of both approaches depend on selecting representative references and optimized parameter settings. By performing parameter escalation experiments, we demonstrate the effects of reference size and alternative allele frequency cutoffs for abundance estimation. We show how different parameter settings can lead to different results for our test data sets, and illustrate the effects of virus lineage composition of wastewater samples and references. Here, we compare a mutation- and sequence-based reference construction and assignment for SARS-CoV-2 abundance estimation from wastewater samples. Our study highlights current computational challenges, focusing on the general reference design, which significantly and directly impacts abundance allocations. We illustrate advantages and disadvantages that may be relevant for further developments in the wastewater community and in the context of higher standardization.","version":"1.1","doi":"10.1101/2023.06.02.543047","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.02.543298","pub_date":"2023-6-02","title":"Deep Phenotyping of the Lipidomic Response in COVID and non-COVID Sepsis","abstract":"Lipids may influence cellular penetrance by pathogens and the immune response that they evoke. Here we find a broad based lipidomic storm driven predominantly by secretory (s) phospholipase A2 (sPLA2) dependent eicosanoid production occurs in patients with sepsis of viral and bacterial origin and relates to disease severity in COVID-19. Elevations in the cyclooxygenase (COX) products of arachidonic acid (AA), PGD2 and PGI2, and the AA lipoxygenase (LOX) product, 12-HETE, and a reduction in the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients, correlate with the inflammatory response and link to disease severity. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflect disease severity in COVID-19. AA and LA metabolites and LPC-O-16:0 linked variably to the immune response. These studies yield prognostic biomarkers and therapeutic targets for patients with sepsis, including COVID-19. An interactive purpose built interactive network analysis tool was developed, allowing the community to interrogate connections across these multiomic data and generate novel hypotheses.","version":"1.1","doi":"10.1101/2023.06.02.543298","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.01.543345","pub_date":"2023-6-02","title":"AGILE Platform: A Deep Learning-Powered Approach to Accelerate LNP Development for mRNA Delivery","abstract":"Ionizable lipid nanoparticles (LNPs) have seen widespread use in mRNA delivery for clinical applications, notably in SARS-CoV-2 mRNA vaccines. Despite their successful use, expansion of mRNA therapies beyond COVID-19 is impeded by the absence of LNPs tailored to different target cell types. The traditional process of LNP development remains labor-intensive and cost-inefficient, relying heavily on trial and error. In this study, we present the AI-Guided Ionizable Lipid Engineering (AGILE) platform, a synergistic combination of deep learning and combinatorial chemistry. AGILE streamlines the iterative development of ionizable lipids, crucial components for LNP-mediated mRNA delivery. This approach brings forth three significant features: efficient design and synthesis of combinatorial lipid libraries, comprehensive in silico lipid screening employing deep neural networks, and adaptability to diverse cell lines. Using AGILE, we were able to rapidly design, synthesize, and evaluate new ionizable lipids for mRNA delivery in muscle and immune cells, selecting from a library of over 10,000 candidates. Importantly, AGILE has revealed cell-specific preferences for ionizable lipids, indicating the need for different tail lengths and head groups for optimal delivery to varying cell types. These results underscore the potential of AGILE in expediting the development of customized LNPs. This could significantly contribute to addressing the complex needs of mRNA delivery in clinical practice, thereby broadening the scope and efficacy of mRNA therapies. AI and combinatorial chemistry expedite ionizable lipid creation for mRNA delivery.","version":"1.1","doi":"10.1101/2023.06.01.543345","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.31.543159","pub_date":"2023-6-01","title":"Combinatorial Regimens Augment Drug Monotherapy for SARS-CoV-2 Clearance in Mice","abstract":"Direct acting antivirals (DAAs) represent critical tools for combating SARS-CoV-2 variants of concern (VOCs) that evolve to escape spike-based immunity and future coronaviruses with pandemic potential. Here, we used bioluminescence imaging to evaluate therapeutic efficacy of DAAs that target SARS-CoV-2 RNA-dependent RNA polymerase (favipiravir, molnupiravir) or Main protease (nirmatrelvir) against Delta or Omicron VOCs in K18-hACE2 mice. Nirmatrelvir displayed the best efficacy followed by molnupiravir and favipiravir in suppressing viral loads in the lung. Unlike neutralizing antibody treatment, DAA monotherapy did not eliminate SARS-CoV-2 in mice. However, targeting two viral enzymes by combining molnupiravir with nirmatrelvir resulted in superior efficacy and virus clearance. Furthermore, combining molnupiravir with Caspase-1/4 inhibitor mitigated inflammation and lung pathology whereas combining molnupiravir with COVID-19 convalescent plasma yielded rapid virus clearance and 100% survival. Thus, our study provides insights into treatment efficacies of DAAs and other effective combinations to bolster COVID-19 therapeutic arsenal.","version":"1.1","doi":"10.1101/2023.05.31.543159","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.31.543129","pub_date":"2023-6-01","title":"CD4+ T-cell immunity of SARS-CoV-2 patients determine pneumonia development","abstract":"Most humans infected with SARS-CoV-2 will recover without developing pneumonia. A few SARS-CoV-2 infected patients, however, develop pneumonia, and occasionally develop cytokine storms. In such cases, it is assumed that there is an inadequate immune response to eliminate viral infected cells and an excessive inappropriate immune response causing organ damage, but little is known about this mechanism. In this study, we used single cell RNA sequencing and mass cytometry to analyze peripheral blood T cells from patients hospitalized with proven COVID-19 infection in order to clarify the differences in host immune status among COVID-19 pneumonia cases, non-pneumonia cases, and healthy controls. The results showed that a specific CD4+ T cell cluster with chemokine receptor expression patterns, CXCR3+CCR4-CCR6+ (Th1/17), was less abundant in COVID-19 pneumonia patients. Interestingly, these CD4+ T-cell clusters were identical to those we have reported to correlate with antitumor immunity and predict programmed cell death (PD)-1 blockade treatment response in lung cancer. The Th1/17 cell percentages had biomarker performance in diagnosing pneumonia cases. In addition, CTLA-4 expression of type17 helper T cells (Th17) and regulatory T cells (Treg) was found to be significantly lower. This indicates that functional suppression of Th17 was less effective and Treg function was impaired in pneumonia cases. These results suggest that imbalance of CD4+ T-cell immunity generates excessive immunity that does not lead to viral eradication. This might be a potential therapeutic target mechanism to prevent severe viral infections. In this observational study, 49 consecutive patients with SARS-CoV-2 infection confirmed by PCR testing and admitted to Saitama Medical University Hospital and Saitama Medical University International Medical Centre between December 4, 2020 and January 17, 2022 were included. Of these 49 patients, 29 were diagnosed with COVID-19 pneumonia by computed tomography (CT) scan (Table 1). The unique CD4+ T-cell immunity with less abundant Th1/17 CD4+ T-cell cluster and low expression of CTLA-4 in Th17 and Treg was consistently found in SARS-CoV-2 pneumonia patients on admission and 1-week of admission. The imbalance of CD4+ T-cell immunity may contribute to develop pneumonia in SARS-CoV-2 virus infected patients by delaying viral clearance and resulting in an excessive immune response.","version":"1.1","doi":"10.1101/2023.05.31.543129","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.26.542482","pub_date":"2023-6-01","title":"The HLA-II immunopeptidome of SARS-CoV-2","abstract":"Targeted synthetic vaccines have the potential to transform our response to viral outbreaks; yet the design of these vaccines requires a comprehensive knowledge of viral immunogens, including T-cell epitopes. Having previously mapped the SARS-CoV-2 HLA-I landscape, here we report viral peptides that are naturally processed and loaded onto HLA-II complexes in infected cells. We identified over 500 unique viral peptides from canonical proteins, as well as from overlapping internal open reading frames (ORFs), revealing, for the first time, the contribution of internal ORFs to the HLA-II peptide repertoire. Most HLA-II peptides co-localized with the known CD4+ T cell epitopes in COVID-19 patients. We also observed that two reported immunodominant regions in the SARS-CoV-2 membrane protein are formed at the level of HLA-II presentation. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and non-structural and non-canonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize the vaccine effectiveness.","version":"1.1","doi":"10.1101/2023.05.26.542482","journal":"bioRxiv","score":null},{"id":"10.1101/2023.06.01.543234","pub_date":"2023-6-01","title":"Prenatal SARS-CoV-2 infection alters postpartum human milk-derived extracellular vesicles","abstract":"Human milk-derived extracellular vesicles (HMEVs) are crucial functional components in breast milk, contributing to infant health and development. Maternal conditions could affect HMEV cargos; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study evaluated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. Milk samples (9 prenatal SARS-CoV-2 vs. 9 controls) were retrieved from the IMPRINT birth cohort. After defatting and casein micelle disaggregation, 1 mL milk was subjected to a sequential process of centrifugation, ultrafiltration, and qEV-size exclusion chromatography. Particle and protein characterizations were performed following the MISEV2018 guidelines. EV lysates were analyzed through proteomics and miRNA sequencing, while the intact EVs were biotinylated for surfaceomic analysis. Multi-Omics was employed to predict HMEV functions associated with prenatal SARS-CoV-2 infection. Demographic data between the prenatal SARS-CoV-2 and control groups were similar. The median duration from maternal SARS-CoV-2 test positivity to milk collection was 3 months (range: 1-6 months). Transmission electron microscopy showed the cup-shaped nanoparticles. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1e11 particles from 1 mL milk. Western immunoblots detected ALIX, CD9 and HSP70, supporting the presence of HMEVs in the isolates. Thousands of HMEV cargos and hundreds of surface proteins were identified and compared. Multi-Omics predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming and mucosal tissue development, while mitigating inflammation and lower EV transmigration potential. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era.","version":"1.1","doi":"10.1101/2023.06.01.543234","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.01.526736","pub_date":"2023-6-01","title":"Omicron\u2019s Intrinsic Gene-Gene Interactions Jumped Away from Earlier SARS-CoV-2 Variants and Gene Homologs between Humans and Animals","abstract":"Omicron and its subvariants have become the predominant SARS-CoV-2 variants worldwide. The Omicron\u2019s basic reproduction number (R0) has been close to 20 or higher. However, it is not known what caused such an extremely high R0. This work aims to find an explanation for such high R0 Omicron infection. We found that Omicron\u2019s intrinsic gene-gene interactions jumped away from earlier SARS-CoV-2 variants which can be fully described by a miniature set of genes reported in our earlier work. We found that the gene PTAFR (Platelet Activating Factor Receptor) is highly correlated with Omicron variants, and so is the gene CCNI (Cyclin I), which is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. The combination of PTAFR and CCNI can lead to a 100% accuracy of differentiating Omicron COVID-19 infection and COVID-19 negative. We hypothesize that Omicron variants were potentially jumped from COVID-19-infected animals back to humans. In addition, there are also several other two-gene interactions that lead to 100% accuracy. Such observations can explain Omicron\u2019s fast-spread reproduction capability as either of those two-gene interactions can lead to COVID-19 infection, i.e., multiplication of R0s leads to a much higher R0. At the genomic level, PTAFR, CCNI, and several other genes identified in this work rise to Omicron druggable targets and antiviral drugs besides the existing antiviral drugs.","version":"1.2","doi":"10.1101/2023.02.01.526736","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.30.542314","pub_date":"2023-6-01","title":"A semi-quantitative, rapid, point of care SARS-CoV-2 serologic assay predicts neutralizing antibody levels","abstract":"The ongoing COVID-19 pandemic has caused millions of deaths and the continued emergence of new variants suggests continued circulation in the human population. In the current time of vaccine availability and new therapeutic development, including antibody-based therapies, many questions about long-term immunity and protection remain uncertain. Identification of protective antibodies in individuals is often done using highly specialized and challenging assays such as functional neutralizing assays, which are not available in the clinical setting. Therefore, there is a great need for the development of rapid, clinically available assays that correlate with neutralizing antibody assays to identify individuals who may benefit from additional vaccination or specific COVID-19 therapies. In this report, we apply a novel semi-quantitative method to an established lateral flow assay (sqLFA) and analyze its ability to detect the presence functional neutralizing antibodies from the serum of COVID-19 recovered individuals. We found that the sqLFA has a strong positive correlation with neutralizing antibody levels. At lower assay cutoffs, the sqLFA is a highly sensitive assay to identify the presence of a range of neutralizing antibody levels. At higher cutoffs, it can detect higher levels of neutralizing antibody with high specificity. This sqLFA can be used both as a screening tool to identify individuals with any level of neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or as a more specific tool to identify those with high neutralizing antibody levels who may not benefit from antibody-based therapies or further vaccination.","version":"1.1","doi":"10.1101/2023.05.30.542314","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.31.543022","pub_date":"2023-6-01","title":"Universal features of Nsp1-mediated translational shutdown by coronaviruses","abstract":"Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses \u2013 SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV \u2013 using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.","version":"1.1","doi":"10.1101/2023.05.31.543022","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.29.542735","pub_date":"2023-5-30","title":"Antibodies against SARS-CoV-2 control complement-induced inflammatory responses to SARS-CoV-2","abstract":"Dysregulated immune responses contribute to pathogenesis of COVID-19 leading to uncontrolled and exaggerated inflammation observed during severe COVID-19. However, it remains unclear how immunity to SARS-CoV-2 is induced and subsequently controlled. Notably, here we have uncovered an important role for complement in the induction of innate and adaptive immunity to SARS-CoV-2. Complement rapidly opsonized SARS-CoV-2 via the lectin pathway. Complement-opsonized SARS-CoV-2 efficiently interacted with dendritic cells (DCs), inducing type I IFN and pro-inflammatory cytokine responses, which were inhibited by antibodies against the complement receptors (CR)3 and CR4. These data suggest that complement is important in inducing immunity via DCs in the acute phase against SARS-CoV-2. Strikingly, serum from COVID-19 patients as well as monoclonal antibodies against SARS-CoV-2 attenuated innate and adaptive immunity induced by complement-opsonized SARS-CoV-2. Blocking the FcyRII, CD32, restored complement-induced immunity. These data strongly suggest that complement opsonization of SARS-CoV-2 is important for inducing innate and adaptive immunity to SARS-CoV-2. Subsequent induction of antibody responses is important to limit the immune responses and restore immune homeostasis. These data suggest that dysregulation in complement and FcyRII signalling might underlie mechanisms causing severe COVID-19.","version":"1.1","doi":"10.1101/2023.05.29.542735","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.481978","pub_date":"2023-5-30","title":"Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-\u03baB pathway towards SARS-CoV-2","abstract":"Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of SARS-CoV-2 infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral factors, such as interferons (IFN). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-\u03baB pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-\u03baB pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.","version":"1.2","doi":"10.1101/2022.02.25.481978","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.08.518776","pub_date":"2023-5-30","title":"Using drivers and transmission pathways to identify SARS-like coronavirus spillover risk hotspots","abstract":"The emergence of SARS-like coronaviruses is a multi-stage process from wildlife reservoirs to people. Here we characterize multiple drivers\u2014landscape change, host distribution, and human exposure\u2014associated with the risk of spillover of SARS-like coronaviruses to help inform surveillance and mitigation activities. We consider direct and indirect transmission pathways by modeling four scenarios with livestock and mammalian wildlife as potential and known reservoirs before examining how access to healthcare varies within clusters and scenarios. We found 19 clusters with differing risk factor contributions within a single country (N=9) or transboundary (N=10). High-risk areas were mainly closer (11-20%) rather than far (<1%) from healthcare. Areas far from healthcare reveal healthcare access inequalities, especially Scenario 3, which includes wild mammals as secondary hosts. China (N=2) and Indonesia (N=1) had clusters with the highest risk. Our findings can help stakeholders in land use planning integrating healthcare implementation and One Health actions.","version":"1.2","doi":"10.1101/2022.12.08.518776","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.29.542720","pub_date":"2023-5-29","title":"Development of a SARS-CoV-2 monoclonal antibody panel and its applicability as a reagent in high-throughput fluorescence reduction neutralization and immunohistochemistry assays","abstract":"Since its emergence in late 2019, infection by SARS-CoV-2 (COVID-19 disease) has quickly spread worldwide, leading to a pandemic that has caused millions of deaths and huge socio-economic losses. Although vaccination against COVID-19 has significantly reduced disease mortality, it has been shown that protection wanes over time, and that circulating SARS-CoV-2 variants may escape vaccine-derived immunity. Therefore, serological studies are still necessary to assess protection in the population and better guide vaccine booster programs. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard method for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. In this study, we developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we also developed and characterized in-house monoclonal antibodies (mAbs) to lower assay costs and reduce the vulnerability of the test to reagent shortages. Using samples collected before the pandemic and from individuals vaccinated against COVID-19, we showed that the results of the FRNA we developed using commercial and in-house mAbs strongly correlated with those of the standard PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high-throughput alternative for measuring nAbs, the FRNA can be easily customized to assess other SARS-CoV-2 variants of concern (VOCs).","version":"1.1","doi":"10.1101/2023.05.29.542720","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.26.542489","pub_date":"2023-5-27","title":"SARS-CoV-2 lineage assignments using phylogenetic placement/UShER are superior to pangoLEARN machine learning method","abstract":"With the rapid spread and evolution of SARS-CoV-2, the ability to monitor its transmission and distinguish among viral lineages is critical for pandemic response efforts. The most commonly used software for the lineage assignment of newly isolated SARS-CoV-2 genomes is pangolin, which offers two methods of assignment, pangoLEARN and pUShER. PangoLEARN rapidly assigns lineages using a machine learning algorithm, while pUShER performs a phylogenetic placement to identify the lineage corresponding to a newly sequenced genome. In a preliminary study, we observed that pangoLEARN (decision tree model), while substantially faster than pUShER, offered less consistency across different versions of pangolin v3. Here, we expand upon this analysis to include v3 and v4 of pangolin, which moved the default algorithm for lineage assignment from pangoLEARN in v3 to pUShER in v4, and perform a thorough analysis confirming that pUShER is not only more stable across versions but also more accurate. Our findings suggest that future lineage assignment algorithms for various pathogens should consider the value of phylogenetic placement.","version":"1.1","doi":"10.1101/2023.05.26.542489","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.26.542392","pub_date":"2023-5-26","title":"Modifications to the SR-Rich Region of the SARS-CoV-2 Nucleocapsid Regulate Self-Association and Attenuate RNA Interactions","abstract":"The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, and maturation. N is comprised of two folded domains that are separated by a highly conserved, disordered, Ser/Arg-rich linker, and flanked by disordered tails. Using NMR spectroscopy and analytical ultracentrifugation we identify an alpha-helical region in the linker that undergoes concentration dependent self-association. NMR and gel shift assays show that the linker binds viral RNA but this binding is dampened by both phosphorylation and a naturally occurring mutation, whereas in contrast, RNA binding to the full-length protein is not affected. Interestingly, phase separation with RNA is significantly reduced upon phosphorylation but enhanced with the mutation. We attribute these differences to changes in the linker helix self-association which dissociates upon phosphorylation but forms more stable higher order oligomers in the variant. These data provide a structural mechanism for how the linker region contributes to protein-protein interactions, RNA-protein interactions, liquid-liquid phase separation and N protein regulation.","version":"1.1","doi":"10.1101/2023.05.26.542392","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.25.542297","pub_date":"2023-5-26","title":"Multi-omic Profiling Reveals Early Immunological Indicators for Identifying COVID-19 Progressors","abstract":"The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a rapid response by the scientific community to further understand and combat its associated pathologic etiology. A focal point has been on the immune responses mounted during the acute and post-acute phases of infection, but the immediate post-diagnosis phase remains relatively understudied. We sought to better understand the immediate post-diagnosis phase by collecting blood from study participants soon after a positive test and identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14\u2212CD16+ and intermediate CD14+CD16+ monocytes. Additionally, in the lymphocyte compartment, CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. Importantly, the identification of these cellular and molecular immune changes occurred at the early stages of COVID-19 disease. These observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19. Immunological changes associated with COVID-19 progression can be detected during the early stages of infection.","version":"1.1","doi":"10.1101/2023.05.25.542297","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.25.542379","pub_date":"2023-5-26","title":"In vitro reconstitution of SARS CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g","abstract":"SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on \u03b2-globin, EMCV IRES and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g\u2019s RRM domain. The cleavage site was located in the coding region 18 nucleotides downstream from the mRNA entrance indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1\u2019s N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g\u2019s RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of Nsp1-NTD and eIF3g\u2019s RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.","version":"1.1","doi":"10.1101/2023.05.25.542379","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.24.542181","pub_date":"2023-5-25","title":"Longitudinal host transcriptional responses to SARS-CoV-2 infection in adults with extremely high viral load","abstract":"Current understanding of viral dynamics of SARS-CoV-2 and host responses driving the pathogenic mechanisms in COVID-19 is rapidly evolving. Here, we conducted a longitudinal study to investigate gene expression patterns during acute SARS-CoV-2 illness. Cases included SARS-CoV-2 infected individuals with extremely high viral loads early in their illness, individuals having low SARS-CoV-2 viral loads early in their infection, and individuals testing negative for SARS-CoV-2. We could identify widespread transcriptional host responses to SARS-CoV-2 infection that were initially most strongly manifested in patients with extremely high initial viral loads, then attenuating within the patient over time as viral loads decreased. Genes correlated with SARS-CoV-2 viral load over time were similarly differentially expressed across independent datasets of SARS-CoV-2 infected lung and upper airway cells, from both in vitro systems and patient samples. We also generated expression data on the human nose organoid model during SARS-CoV-2 infection. The human nose organoid-generated host transcriptional response captured many aspects of responses observed in the above patient samples, while suggesting the existence of distinct host responses to SARS-CoV-2 depending on the cellular context, involving both epithelial and cellular immune responses. Our findings provide a catalog of SARS-CoV-2 host response genes changing over time.","version":"1.1","doi":"10.1101/2023.05.24.542181","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.24.541920","pub_date":"2023-5-25","title":"Dichotomy of neutralizing antibody, B cell and T cell responses to SARS-CoV-2 vaccination and protection in healthy adults","abstract":"Heterogeneity in SARS-CoV-2 vaccine responses is not understood. Here, we identify four patterns of live-virus neutralizing antibody responses: individuals with hybrid immunity (with confirmed prior infection); rare individuals with low responses (paucity of S1-binding antibodies); and surprisingly, two further groups with distinct serological repertoires. One group \u2013 broad responders \u2013 neutralize a range of SARS-CoV-2 variants, whereas the other \u2013 narrow responders \u2013 neutralize fewer, less divergent variants. This heterogeneity does not correlate with Ancestral S1-binding antibody, rather the quality of the serological response. Furthermore, IgDlowCD27-CD137+ B cells and CCR6+ CD4+ T cells are enriched in broad responders before dose 3. Notably, broad responders have significantly longer infection-free time after their third dose. Understanding the control and persistence of these serological profiles could allow personalized approaches to enhance serological breadth after vaccination.","version":"1.1","doi":"10.1101/2023.05.24.541920","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.476803","pub_date":"2023-5-24","title":"Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 Spike protein","abstract":"SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper- proinflammatory state involves activation of signaling by NF\u03baB and ENaC, and expression of high levels of cytokines and chemokines. Post-infection inflammation may contribute to \u201cLong COVID\u201d, and there are long term consequences for acute severe COVID-19, which double or triple the chances of dying from any cause within a year. Enhanced signaling by NF\u03baB and ENaC also marks the airway of patients suffering from cystic fibrosis, a lethal proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might be due to inhibition of CFTR signaling by SARS- CoV-2 Spike protein. This hypothesis was tested using the hTERT-transformed BCi-NS1.1 basal stem cell, previously derived from small airway epithelia, which were differentiated into a model of small airway epithelia on an air-liquid-interface (ALI). CyclicAMP-activated CFTR chloride channel activity was measured using an Ussing Chamber. Cell surface-CFTR was labeled with the impermeant biotin method. Exposure of differentiated airway epithelia to SARS-CoV-2 Spike protein resulted in loss of CFTR protein expression. As hypothesized, TNF\u03b1/NF\u03baB signaling was activated, based on increased protein expression of TNFR1, the TNF\u03b1 receptor; TRADD, the first intracellular adaptor for the TNF\u03b1/TNFR1 complex; phosphorylated I\u03baB\u03b1, and the chemokine IL8. ENaC activity was also activated, based on specific changes in molecular weights for \u03b1 and \u03b3 ENaC. Exposure of the epithelia to viral Spike protein suppressed cAMP-activated CFTR chloride channel activity. However, 30 nM concentrations of cardiac glycoside drugs ouabain, digitoxin and digoxin, prevented loss of channel activity. ACE2 and CFTR were found to co- immunoprecipitate (co-IP) in both basal cells and epithelia, suggesting that the mechanism for Spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, Spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane, suggesting that failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Based on experiments with this model of small airway epithelia, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by SARS-CoV-2 Spike protein, thus inducing a CFTR-null, cystic fibrosis-like clinical phenotype.","version":"1.3","doi":"10.1101/2022.01.18.476803","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.23.542024","pub_date":"2023-5-24","title":"A Multi-Epitope/CXCL11 Prime/Pull Coronavirus Mucosal Vaccine Boosts the Frequency and the Function of Lung-Resident CD4+ and CD8+ Memory T Cells and Protects Against COVID-19-like Symptoms and Death Caused by SARS-CoV-2 infection","abstract":"The pandemic of the coronavirus disease 2019 (COVID-19) has created the largest global health crisis in almost a century. Following exposure to SARS-CoV-2, the virus particles replicate in the lungs, induce a \u201ccytokine storm\u201d and potentially cause life-threatening inflammatory disease. Low frequencies of function SARS-CoV-2-specific CD4+ and CD8+ T cells in the lungs of COVID-19 patients were associated with severe cases of COVID-19. The apparent low level of T cell-attracting CXCL9, CXCL10, and CXCL11 chemokines in infected lungs may not be sufficient enough to assure the sequestration and/or homing of CD4+ and CD8+ T cells from the circulation into infected lungs. We hypothesize that a Coronavirus vaccine strategy that boosts the frequencies of functional SARS-CoV-2-specific CD4+ and CD8+ T cells in the lungs would lead to better protection against SARS-CoV-2 infection, COVID19-like symptoms, and death. In the present study, we designed and pre-clinically tested the safety, immunogenicity, and protective efficacy of a novel multi-epitope//CXCL11 prime/pull mucosal Coronavirus vaccine. This prime/pull vaccine strategy consists of intranasal delivery of a lung-tropic adeno-associated virus type 9 (AAV-9) vector that incorporates highly conserved human B, CD4+ CD8+ cell epitopes of SARS-CoV-2 (prime) and pulling the primed B and T cells into the lungs using the T cell attracting chemokine, CXCL-11 (pull). We demonstrated that immunization of HLA-DR*0101/HLA-A*0201/hACE2 triple transgenic mice with this multi-epitope//CXCL11 prime/pull Coronavirus mucosal vaccine: (i) Increased the frequencies of CD4+ and CD8+ TEM, TCM, and TRM cells in the lungs; and (ii) reduced COVID19-like symptoms, lowered virus replication, and prevented deaths following challenge with SARS-CoV-2. These findings discuss the importance of bolstering the number and function of lung-resident memory CD4+ and CD8+ T cells for better protection against SARS-CoV-2 infection, COVID-19-like symptoms, and death.","version":"1.1","doi":"10.1101/2023.05.23.542024","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.24.541850","pub_date":"2023-5-24","title":"Cross-Protection Induced by Highly Conserved Human B, CD4+, and CD8+ T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern","abstract":"The Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of SARS-CoV-2 infections has decreased significantly; the long-term outlook of COVID-19 remains a serious cause of high death worldwide; with the mortality rate still surpassing even the worst mortality rates recorded for the influenza viruses. The continuous emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, have prolonged the COVID-19 pandemic and outlines the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. In the present study, we designed a multi-epitope-based Coronavirus vaccine that incorporated B, CD4+, and CD8+ T cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-Coronavirus vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. The Pan-Coronavirus vaccine: (i) is safe; (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells; and (iii) provides robust protection against virus replication and COVID-19-related lung pathology and death caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2) and Omicron (B.1.1.529). Conclusions: A multi-epitope pan-Coronavirus vaccine bearing conserved human B and T cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that cleared the virus, and reduced COVID-19-related lung pathology and death caused by multiple SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2023.05.24.541850","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.24.542061","pub_date":"2023-5-24","title":"Computational insights on the destabilizing mutations in the binding site of 3CL-protease SARS-CoV-2 Omicron (VOC)","abstract":"The COVID-19 (Corona Virus Disease 19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is causing enormous difficulties in the world\u2019s economies and there is uncertainty as to whether the current prophylactic measures will offer adequate protection globally after the appearance of virus variants that like that indicated as Omicron emerged in the presence of global vaccine-based immunization. While several studies are available describing the main differences in the spike protein of Omicron compared to the other variants previously emerged, there was no structural insights into the 3CL-protease (3CLpro) associated to the new variant. Herein, we performed a computational study based on genomic data and amino acid sequences available in the most updated COVID-19-related databases that allowed us to build up in silico the 3D structure of Omicron 3CLpro. Moreover, by molecular dynamics simulation we demonstrated that currently available drugs acting as inhibitors of the SARS-CoV-2 main protease could be less effective in the case of Omicron variant due to the different chemical interactions in the binding site occurred after the recent amino acid mutations. Ultimately, our study highlights the need of exploiting in silico and in vitro methods to discover novel 3CLpro inhibitors starting from the computationally based structure we presented herein, and more in general to direct the major efforts to targeting the most conserved 3CLpro regions that appeared unchanged in the context of the Omicron variant.","version":"1.1","doi":"10.1101/2023.05.24.542061","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.24.542043","pub_date":"2023-5-24","title":"The impact of RSV/SARS-CoV-2 co-infection on clinical disease and viral replication: insights from a BALB/c mouse model","abstract":"RSV and SARS-CoV-2 are prone to co-infection with other respiratory viruses. In this study, we use RSV/SARS-CoV-2 co-infection to evaluate changes to clinical disease and viral replication in vivo. To consider the severity of RSV infection, effect of sequential infection, and the impact of infection timing, mice were co-infected with varying doses and timing. Compared with a single infection of RSV or SARS-CoV-2, the co-infection of RSV/SARS-CoV-2 and the primary infection of RSV followed by SARS-CoV-2 results in protection from SARS-CoV-2-induced clinical disease and reduces SARS-CoV-2 replication. Co-infection also augmented RSV replication at early timepoints with only the low dose. Additionally, the sequential infection of RSV followed by SARS-CoV-2 led to improved RSV clearance regardless of viral load. However, SARS-CoV-2 infection followed by RSV results in enhanced SARS-CoV-2-induced disease while protecting from RSV-induced disease. SARS-CoV-2/RSV sequential infection also reduced RSV replication in the lung tissue, regardless of viral load. Collectively, these data suggest that RSV and SARS-CoV-2 co-infection may afford protection from or enhancement of disease based on variation in infection timing, viral infection order, and/or viral dose. In the pediatric population, understanding these infection dynamics will be critical to treat patients and mitigate disease outcomes. Infants and young children are commonly affected by respiratory viral co-infections. While RSV and SARS-CoV-2 are two of the most prevalent respiratory viruses, their co-infection rate in children remains surprisingly low. In this study, we investigate the impact of RSV/SARS-CoV-2 co-infection on clinical disease and viral replication using an animal model. The findings indicate that RSV infection either simultaneously or prior to SARS-CoV-2 infection in mice protect against SARS-CoV-2-induced clinical disease and viral replication. On the other hand, infection with SARS-CoV-2 followed by RSV results in worsening of SARS-CoV-2-induced clinical disease, but also protection from RSV-induced clinical disease. These results highlight a protective role for RSV exposure, given this occurs before infection with SARS-CoV-2. This knowledge could help guide vaccine recommendations in children and sets a basis for future mechanistic studies.","version":"1.1","doi":"10.1101/2023.05.24.542043","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.29.474427","pub_date":"2023-5-23","title":"Dynamic Expedition of Leading Mutations in SARS-CoV-2 Spike Glycoproteins","abstract":"Throughout the coronavirus disease 2019 (COVID-19) pandemic, the continuous genomic evolution of its etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has generated many new variants with enhanced transmissibility and immune escape capabilities. Being an essential mediator of infections and a key target of antibodies, mutations of its spike glycoprotein play a vital role in modulating its evolutionary trajectory. Here, we present a time-resolved statistical method, Dynamic Expedition of Leading Mutations (deLemus), to analyze the evolutionary dynamics of the SARS-CoV-2 spike. Together with analysis of its single amino acid polymorphism (SAP), we propose the use of L-index in quantifying the mutation strength of each amino acid site, such that the evolutionary mutation pattern of the spike glycoprotein can be unravelled.","version":"1.2","doi":"10.1101/2021.12.29.474427","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.22.540829","pub_date":"2023-5-23","title":"Vaccine-mediated protection against merbecovirus and sarbecovirus challenge in mice","abstract":"The emergence of three distinct highly pathogenic human coronaviruses \u2013 SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019 \u2013 underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines are highly protective against severe COVID-19 disease, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor binding domains (RBDs), which elicited live-virus neutralizing antibody responses and broad protection. Specifically, a monovalent SARS-CoV-2 RBD scNP vaccine only protected against sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both merbecovirus and sarbecovirus challenge in highly pathogenic and lethal mouse models. Moreover, the trivalent RBD scNP elicited serum neutralizing antibodies against SARS-CoV, MERS-CoV and SARS-CoV-2 BA.1 live viruses. Our findings show that a trivalent RBD nanoparticle vaccine displaying merbecovirus and sarbecovirus immunogens elicits immunity that broadly protects mice against disease. This study demonstrates proof-of-concept for a single pan-betacoronavirus vaccine to protect against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.","version":"1.1","doi":"10.1101/2023.05.22.540829","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.22.541294","pub_date":"2023-5-23","title":"Breakthrough infections by SARS-CoV-2 variants boost cross-reactive hybrid immune responses in mRNA-vaccinated Golden Syrian Hamsters","abstract":"Hybrid immunity to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B and T-cell responses.","version":"1.2","doi":"10.1101/2023.05.22.541294","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.18.541286","pub_date":"2023-5-23","title":"Contrasting the open access dissemination of COVID-19 and SDG research","abstract":"This paper examines the extent to which research has been published open access in response to two global threats: COVID-19 and the Sustainable Development Goals (SDGs), including climate change. We compare the accessibility of COVID-19 content versus SDG literature using the Dimensions database between 2000 and 2021, classifying each publication as gold open access, green, bronze, hybrid, or closed. We found that 79.9% of COVID-19 research papers published between January 2020 and December 2021 was open access, with 39.0% published with gold open access licenses. In contrast, just 55.7% of SDG papers were open access in the same time period, with only 36.0% published with gold open access licenses. Papers related to the climate emergency overall had the second-lowest level of open access at just 55.5%. Papers published by the largest for-profit publishers that committed to both the SDG Publishers Compact and climate actions were not predominantly published open access. The paper highlights the need for continued efforts to promote open access publishing to facilitate scientific research and technological development to address global challenges. In contrast to COVID-19 papers, research on UN Sustainable Development Goals including the climate emergency have not been made open access by leading global science publishers despite their corporate commitments to sustainability and climate action.","version":"1.2","doi":"10.1101/2023.05.18.541286","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.22.541685","pub_date":"2023-5-22","title":"ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface","abstract":"Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.","version":"1.1","doi":"10.1101/2023.05.22.541685","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.10.519730","pub_date":"2023-5-22","title":"Dynamical nonequilibrium molecular dynamics simulations identify allosteric sites and positions associated with drug resistance in the SARS-CoV-2 main protease","abstract":"The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalysing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behaviour of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.","version":"1.3","doi":"10.1101/2022.12.10.519730","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.20.541592","pub_date":"2023-5-22","title":"Markov State Models and Perturbation-Based Approaches Reveal Distinct Dynamic Signatures and Hidden Allosteric Pockets in the Emerging SARS-Cov-2 Spike Omicron Variants Complexes with the Host Receptor: The Interplay of Dynamics and Convergent Evolution Modulates Allostery and Functional Mechanisms","abstract":"The new generation of SARS-CoV-2 Omicron variants displayed a significant growth advantage and the increased viral fitness by acquiring convergent mutations, suggesting that the immune pressure can promote convergent evolution leading to the sudden acceleration of SARS-CoV-2 evolution. In the current study, we combined structural modeling, extensive microsecond MD simulations and Markov state models to characterize conformational landscapes and identify specific dynamic signatures of the SARS-CoV-2 spike complexes with the host receptor ACE2 for the recently emerged highly transmissible XBB.1, XBB.1.5, BQ.1, and BQ.1.1 Omicron variants. Microsecond simulations and Markovian modeling provided a detailed characterization of the conformational landscapes and revealed the increased thermodynamic stabilization of the XBB.1.5 subvariant which is contrasted to more dynamic BQ.1 and BQ.1.1 subvariants. Despite considerable structural similarities, Omicron mutations can induce unique dynamic signatures and specific distributions of conformational states. The results suggested that variant-specific changes of conformational mobility in the functional interfacial loops of the spike receptor binding domain can be fine-tuned through cross-talk between convergent mutations thereby providing an evolutionary path for modulation of immune escape. By combining atomistic simulations and Markovian modeling analysis with perturbation-based approaches, we determined important complementary roles of convergent mutation sites as effectors and receivers of allosteric signaling involved in modulating conformational plasticity at the binding interface and regulating allosteric responses. This study also characterized the dynamics-induced evolution of allosteric pockets in the Omicron complexes that revealed hidden allosteric pockets and suggested that convergent mutation sites could control evolution and distribution of allosteric pockets through modulation of conformational plasticity in the flexible adaptable regions. Through integrative computational approaches, this investigation provides a systematic analysis and comparison of the effects of Omicron subvariants on conformational dynamics and allosteric signaling in the complexes with the ACE2 receptor.","version":"1.1","doi":"10.1101/2023.05.20.541592","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.19.541367","pub_date":"2023-5-22","title":"An Atlas of Adaptive Evolution in Endemic Human Viruses","abstract":"Through antigenic evolution, viruses like seasonal influenza evade recognition by neutralizing antibodies elicited by previous infection or vaccination. This means that a person with antibodies well-tuned to an initial infection will not be protected against the same virus years later and that vaccine-mediated protection will decay. It is not fully understood which of the many endemic human viruses evolve in this fashion. To expand that knowledge, we assess adaptive evolution across the viral genome in 28 endemic viruses, spanning a wide range of viral families and transmission modes. We find that surface proteins consistently show the highest rates of adaptation, and estimate that ten viruses in this panel undergo antigenic evolution to selectively fix mutations that enable the virus to escape recognition by prior immunity. We compare overall rates of amino acid substitution between these antigenically-evolving viruses and SARS-CoV-2, showing that SARS-CoV-2 viruses are accumulating protein-coding changes at substantially faster rates than these endemic viruses.","version":"1.1","doi":"10.1101/2023.05.19.541367","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.15.516351","pub_date":"2023-5-22","title":"Inflammation durably imprints memory CD4+ T cells","abstract":"Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs when primed in these two contexts. Notably, viral infection is generally associated with higher levels of systemic inflammation than is vaccination. To assess whether the inflammatory milieu at the time of CD4+ T cell priming has long-term effects on memory, we compared Spike-specific memory CD4+ T cells in 22 individuals around the time of the participants\u2019 third SARS-CoV-2 mRNA vaccination, with stratification by whether the participants\u2019 first exposure to Spike was via virus or mRNA vaccine. Multimodal single-cell profiling of Spike-specific CD4+ T cells revealed 755 differentially expressed genes that distinguished infection- and vaccine-primed memory CD4+ T cells. Spike-specific CD4+ T cells from infection-primed individuals had strong enrichment for cytotoxicity and interferon signaling genes, whereas Spike-specific CD4+ T cells from vaccine-primed individuals were enriched for proliferative pathways by gene set enrichment analysis. Moreover, Spike-specific memory CD4+ T cells established by infection had distinct epigenetic landscapes driven by enrichment of IRF-family transcription factors, relative to T cells established by mRNA vaccination. This transcriptional imprint was minimally altered following subsequent mRNA vaccination or breakthrough infection, reflecting the strong bias induced by the inflammatory environment during initial memory differentiation. Together, these data suggest that the inflammatory context during CD4+ T cell priming is durably imprinted in the memory state at transcriptional and epigenetic levels, which has implications for personalization of vaccination based on prior infection history. SARS-CoV-2 infection versus SARS-CoV-2 mRNA vaccination prime durable transcriptionally and epigenetically distinct Spike-specific CD4+ T cell memory landscapes.","version":"1.2","doi":"10.1101/2022.11.15.516351","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.18.541401","pub_date":"2023-5-19","title":"Host microRNA interactions with the SARS-CoV-2 viral genome 3\u2019-untranslated region","abstract":"The 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has marked the spread of a novel human coronavirus. While the viral life cycle is well understood, most of the interactions at the virus-host interface remain elusive. Furthermore, the molecular mechanisms behind disease severity and immune evasion are still largely unknown. Conserved elements of the viral genome such as secondary structures within the 5\u2019- and 3\u2019-untranslated regions (UTRs) serve as attractive targets of interest and could prove crucial in furthering our understanding of virus-host interactions. It has been proposed that microRNA (miR) interactions with viral components could be used by both the virus and host for their own benefit. Analysis of the SARS-CoV-2 viral genome 3\u2019-UTR has revealed the potential for host cellular miR binding sites, providing sites for specific interactions with the virus. In this study, we demonstrate that the SARS-CoV-2 genome 3\u2019-UTR binds the host cellular miRNAs miR-760-3p, miR-34a-5p, and miR-34b-5p, which have been shown to influence translation of interleukin-6 (IL-6), the IL-6 receptor (IL-6R), as well as progranulin (PGRN), respectively, proteins that have roles in the host immune response and inflammatory pathways. Furthermore, recent work suggests the potential of miR-34a-5p and miR-34b-5p to target and inhibit translation of viral proteins. Native gel electrophoresis and steady-state fluorescence spectroscopy were utilized to characterize the binding of these miRs to their predicted sites within the SARS-CoV-2 genome 3\u2019-UTR. Additionally, we investigated 2\u2019-fluoro-D-arabinonucleic acid (FANA) analogs of these miRNAs as competitive binding inhibitors for these miR binding interactions. The mechanisms detailed in this study have the potential to drive the development of antiviral treatments for SARS-CoV-2 infection, and provide a potential molecular basis for cytokine release syndrome and immune evasion which could implicate the host-virus interface. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now plagued the world for over three years. In this time, scientific advancements have allowed for the development of mRNA vaccines and targeted antiviral drugs. However, many mechanisms of the viral life cycle, as well as the interactions at the host-virus interface, remain unknown. The host immune response is of particular interest in combating SARS-CoV-2 infection, with observed dysregulation in both severe and mild cases of infection. To uncover the link between SARS-CoV-2 infection and observed immune dysregulation, we investigated host microRNAs associated with the immune response, particularly miR-760-3p, miR-34a-5p, and miR-34b-5p and emphasize them as targets of binding by the viral genome 3\u2019-UTR. We utilized biophysical methods to characterize the interactions between these miRs and the SARS-CoV-2 viral genome 3\u2019-UTR. Lastly, we introduce 2\u2019-fluoro-D-arabinonucleic acid analogs of these microRNAs as disruptors of the binding interactions, with intent of therapeutic intervention.","version":"1.1","doi":"10.1101/2023.05.18.541401","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.04.531075","pub_date":"2023-5-19","title":"Uncovering strain- and age-dependent differences in innate immune response to SARS-CoV-2 infection in nasal epithelia using 10X single-cell sequencing","abstract":"Assessing the impact of SARS-CoV-2 variants on the host is crucial with continuous emergence of new variants. We employed single-cell sequencing to investigate host transcriptomic response to ancestral and Alpha-strain SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from adults and adolescents. Strong innate immune responses were observed across lowly-infected and bystander cell-types, and heightened in Alpha-infection. Contrastingly, the innate immune response of highly-infected cells was like mock-control cells. Alpha highly-infected cells showed increased expression of protein refolding genes compared with ancestral-strain-infected adolescent cells. Oxidative phosphorylation- and translation-related genes were down-regulated in bystander cells versus infected and mock-control cells, suggesting that the down-regulation is protective and up-regulation supports viral activity. Infected adult cells revealed up-regulation of these pathways compared with infected adolescents, implying enhanced pro-viral states in infected adults. Overall, this highlights the complexity of cell-type-, age- and viral-strain-dependent host epithelial responses to SARS-CoV-2 and the value of air-liquid-interface cultures.","version":"1.2","doi":"10.1101/2023.03.04.531075","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.15.540684","pub_date":"2023-5-18","title":"Mechanism Underlying the Immune Responses of a Sublingual Vaccine for SARS-CoV-2 with RBD Antigen and Adjuvant, Poly(I:C) or AddaS03, in Non-human Primates","abstract":"A sublingual vaccine formulated with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD) antigen and Poly(I:C)) adjuvant was assessed for its safety in non-human primates. This Poly(I:C)-adjuvanted sublingual vaccine was safe compared to the AddaS03-adjuvanted vaccine in blood tests and plasma CRP. The safety of the vaccine was also confirmed through quantitative reverse transcription PCR of six genes and ELISA of four cytokines associated with inflammation and related reactions. The Poly(I:C)- or AddaS03-adjuvanted sublingual vaccine produced RBD-specific IgA antibodies in nasal washings, saliva, and plasma. SARS-CoV-2 neutralizing antibodies were detected in plasma, suggesting that adjuvanted-sublingual vaccines protect against SARS-CoV-2 infection. \u201cYin and Yang\u201d-like unique transcriptional regulation was observed through DNA microarray analyses of white blood cell RNAs from both vaccines, suppressing and enhancing immune responses and up- or downregulating genes associated with these immune responses. Poly(I:C) adjuvanted sublingual vaccination induced atypical up- or downregulation of genes related to immune suppression or tolerance; Treg differentiation; and T-cell exhaustion. Therefore, Poly(I:C) adjuvant is safe and favorable for sublingual vaccination and can induce a balanced \u201cYin/Yang\u201d -like effect on immune responses.","version":"1.2","doi":"10.1101/2023.05.15.540684","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.17.541127","pub_date":"2023-5-17","title":"Single-cycle SARS-CoV-2 vaccine elicits high protection and sterilizing immunity in hamsters","abstract":"Vaccines have been central in ending the COVID-19 pandemic, but newly emerging SARS-CoV-2 variants increasingly escape first-generation vaccine protection. To fill this gap, live particle-based vaccines mimicking natural infection aim at protecting against a broader spectrum of virus variants. We designed \u201csingle-cycle SARS-CoV-2 viruses\u201d (SCVs) that lack essential viral genes, possess superior immune-modulatory features and provide an excellent safety profile in the Syrian hamster model. Full protection of all intranasally vaccinated animals was achieved against an autologous challenge with SARS-CoV-2 virus using an Envelope-gene-deleted vaccine candidate. By deleting key immune-downregulating genes, sterilizing immunity was achieved with an advanced candidate without virus spread to contact animals. Hence, SCVs have the potential to induce a broad and durable protection against COVID-19 superior to a natural infection.","version":"1.1","doi":"10.1101/2023.05.17.541127","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.17.541098","pub_date":"2023-5-17","title":"SARS-CoV-2 infection leads to Tau pathological signature in neurons","abstract":"The coronavirus disease 19 (COVID-19) has represented an issue for global health since its outbreak in March 2020. It is now evident that the SARS-CoV-2 infection results in a wide range of long-term neurological symptoms and is worryingly associated with the aggravation of Alzheimer\u2019s disease. Little is known about the molecular basis of these manifestations. Several SARS-CoV-2 strain variants were used to infect SH-SY5Y neuroblastoma cells and K18-hACE C57BL/6J mice. The Tau phosphorylation profile and aggregation propensity upon infection were investigated using immunoblot and immunofluorescence on cellular extracts, subcellular fractions, and brain tissue. The viral proteins Spike, Nucleocapsid, and Membrane were overexpressed in SH-SY5Y cells and the direct effect on Tau phosphorylation was checked using immunoblot experiments. Upon infection, Tau is phosphorylated at several pathological epitopes associated with Alzheimer\u2019s disease and other tauopathies. Moreover, this event increases Tau\u2019s propensity to form insoluble aggregates and alters its subcellular localization. Our data support the evidence that SARS-CoV-2 infection in the Central Nervous System triggers downstream effects altering Tau function, eventually leading to the impairment of neuronal function.","version":"1.1","doi":"10.1101/2023.05.17.541098","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.17.541103","pub_date":"2023-5-17","title":"Enhanced Airway Epithelial Response to SARS-CoV-2 Infection in Children is Critically Tuned by the Cross-Talk Between Immune and Epithelial Cells","abstract":"To cope with novel virus infections to which no prior adaptive immunity exists, the body strongly relies on the innate immune system. In such cases, including infections with SARS-CoV-2, children tend to fair better than adults. In the context of COVID-19, it became evident that a rapid interferon response at the site of primary infection is key for successful control of the virus and prevention of severe disease. The airway epithelium of children was shown to exhibit a primed state already at homeostasis and to respond particularly well to SARS-CoV-2 infection. However, the underlying mechanism for this priming remained elusive. Here we show that interactions between airway mucosal immune cells and epithelial cells are stronger in children, and via cytokine-mediated signaling lead to IRF-1-dependent upregulation of the viral sensors RIG-I and MDA5. Based on a cellular in vitro model we show that stimulated human peripheral blood mononuclear cells (PBMC) can induce a robust interferon-beta response towards SARS-CoV-2 in a lung epithelial cell line otherwise unresponsive to this virus. This is mediated by type I interferon, interferon-gamma and TNF, and requires induction of both, RIG-I and MDA5. In single cell-analysis of nasal swab samples the same cytokines are found to be elevated in mucosal immune cells of children, correlating with elevated epithelial expression of viral sensors. In vitro analysis of PBMC derived from healthy adolescents and adults confirm that immune cells of younger individuals show increased cytokine production and potential to prime epithelial cells. In co-culture with SARS-CoV-2-infected A549 cells, PBMC from adolescents significantly enhance the antiviral response. Taken together, our study suggests that higher numbers and a more vigorous activity of innate immune cells in the airway mucosa of children tune the set-point of the epithelial antiviral system. This likely is a major contributor to the robust immune response to SARS-CoV-2 in children. Our findings shed light on the molecular underpinnings of the stunning resilience of children towards severe COVID-19, and may propose a novel concept for immunoprophylactic treatments.","version":"1.1","doi":"10.1101/2023.05.17.541103","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.16.523994","pub_date":"2023-5-16","title":"SARS-CoV-2 evolution in the absence of selective immune pressures, results in antibody resistance, interferon suppression and phenotypic differences by lineage","abstract":"The persistence of COVID-19 is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of Wuhan-like SARS-CoV-2 in Balb/c mice yielded mouse-adapted strains with greater infectivity and mortality. We investigated if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing human ACE2 receptor, in a BSL-3 laboratory without selective pressures, would drive human health-relevant evolution and if evolution was lineage-dependent. Late-passage virus caused more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurred with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, was co-inherited at times with spike E1182G per Nanopore sequencing, existing in different quasi-species at others. Both are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2\u2019s tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribute.","version":"1.4","doi":"10.1101/2023.01.16.523994","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.15.540806","pub_date":"2023-5-16","title":"Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs","abstract":"Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.","version":"1.1","doi":"10.1101/2023.05.15.540806","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.15.540756","pub_date":"2023-5-16","title":"A synthetic delivery vector for mucosal vaccination","abstract":"The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+ T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and its in vitro folding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+ T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.","version":"1.1","doi":"10.1101/2023.05.15.540756","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.14.540726","pub_date":"2023-5-15","title":"Metformin an anti-diabetic drug, possess ACE-2 receptor-SARS-Cov-2 RBD binding antagonist activity, anti-inflammatory and cytokine inhibitory properties suitable for treatment of COVID-19","abstract":"Metformin is a widely used and is a safe anti-diabetic drug. It has also been shown to have anti-inflammatory and anti-viral activities in humans and animal models. Specifically we explored its activity in SARS-CoV-2 initiated COVID19 disease. Here we show that metformin 1. blocks the binding of SARS-CoV-2 spike protein receptor binding domain RBD to human ACE2 receptor 2. We also show that it has anti-inflammatory effects and reduces cytokine secretion as well as blocks the recruitment of monocytes to endothelial cells 3. Finally we show its activity in a hamster in vivo model of SARS-CoV-2 infection as a nasal formulation. Based on the safety and the therapeutic properties relevant to COVID-19 it is feasible to propose a nasal spray of metformin that can be used in treatment of this disease. A nasal spray would deliver the drug to the target organ lung and spare other organs which get exposed upon oral dosing.","version":"1.1","doi":"10.1101/2023.05.14.540726","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.13.540576","pub_date":"2023-5-15","title":"A peptide derived from the SARS-CoV-2 S2-protein heptad-repeat-2 inhibits pseudoviral fusion at micromolar concentrations: Role of palmitic acid conjugation","abstract":"SARS-CoV-2 S protein-mediated fusion is thought to involve the interaction of the membrane-distal, or N-terminal heptad repeat (NHR) (\u201cHR1\u201d) of the cleaved S2 segment of the protein, and the membrane-proximal, or C-terminal heptad repeat (CHR) (\u201cHR2\u201d) regions of the protein. Following the observations of Xia et al (Xia S, Liu M, Wang C, Xu W, Lan Q, Feng S, Qi F, Bao L, Du L, Liu S, Qin C, Sun F, Shi Z, Zhu Y, Jiang S, Lu L. Cell Res. 2020b Apr;30(4):343-355), we examined the fusion inhibitory activity of a PEGylated HR2-derived peptide and its palmitoylated derivative, using a pseudovirus infection assay. The latter peptide caused a 76% reduction in fusion activity at 10 \u03bcM. Our results suggest that small variations in peptide derivatization and differences in the membrane composition of pseudovirus preparations may affect the inhibitory potency of HR2-derived peptides.","version":"1.1","doi":"10.1101/2023.05.13.540576","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.29.533758","pub_date":"2023-5-13","title":"Large-scale template-based structural modeling of T-cell receptors with known antigen specificity reveals complementarity features","abstract":"T-cell receptor (TCR) recognition of foreign peptides presented by the major histocompatibility complex (MHC) initiates the adaptive immune response against pathogens. A large number of TCR sequences specific to different antigens are known to date, however, the structural data describing the conformation and contacting residues for TCR:antigen:MHC complexes is relatively limited. In the present study we aim to extend and analyze the set of available structures by performing highly accurate template-based modeling of TCR:antigen:MHC complexes using TCR sequences with known specificity. Using the set of 29 complex templates (including a template with SARS-CoV-2 antigen) and 732 specificity records, we built a database of 1585 model structures carrying substitutions in either TCR\u03b1 or TCR\u03b2 chains with some models representing the result of different mutation pathways for the same final structure. This database allowed us to analyze features of amino acid contacts in TCR:antigen interfaces that govern antigen recognition preferences and interpret these interactions in terms of physicochemical properties of interacting residues. Our results provide a methodology for creating high-quality TCR:antigen:MHC models for antigens of interest that can be utilized to predict TCR specificity.","version":"1.3","doi":"10.1101/2023.03.29.533758","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520166","pub_date":"2023-5-12","title":"Broad and Durable Humoral Responses Following Single Hydrogel Immunization of SARS-CoV-2 Subunit Vaccine","abstract":"Most vaccines require several immunizations to induce robust immunity, and indeed, most SARS-CoV-2 vaccines require an initial two-shot regimen followed by several boosters to maintain efficacy. Such a complex series of immunizations unfortunately increases the cost and complexity of populations-scale vaccination and reduces overall compliance and vaccination rate. In a rapidly evolving pandemic affected by the spread of immune-escaping variants, there is an urgent need to develop vaccines capable of providing robust and durable immunity. In this work, we developed a single immunization SARS-CoV-2 subunit vaccine that could rapidly generate potent, broad, and durable humoral immunity. We leveraged injectable polymer-nanoparticle (PNP) hydrogels as a depot technology for the sustained delivery of a nanoparticle COVID antigen displaying multiple copies of the SARS-CoV-2 receptor-binding-domain (RBD-NP), and potent adjuvants including CpG and 3M-052. Compared to a clinically relevant prime-boost regimen with soluble vaccines formulated with CpG/Alum or 3M-052/Alum adjuvants, PNP hydrogel vaccines more rapidly generated higher, broader, and more durable antibody responses. Additionally, these single-immunization hydrogel-based vaccines elicited potent and consistent neutralizing responses. Overall, we show that PNP hydrogels elicit improved anti-COVID immune responses with only a single administration, demonstrating their potential as critical technologies to enhance our overall pandemic readiness.","version":"1.3","doi":"10.1101/2022.12.12.520166","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.10.540101","pub_date":"2023-5-11","title":"Metabolic alterations unravel the materno\u2013fetal immune responses with disease severity in pregnant women infected with SARS-CoV-2","abstract":"Pregnancy being immune compromised state, COVID-19 disease poses high risk of premature delivery and threat to fetus. Plasma metabolome regulates immune cellular responses and we aimed to analyze the plasma secretome, metabolome and immune cells in COVID-19 positive pregnant mother and cord blood. COVID-19 RT-PCR positive pregnant females (n=112) asymptomatic (n=82), or with mild (n=21) or moderate (n=9) disease and control healthy pregnant (n=10) females were included. Mother\u2019s blood and cord blood (n=80) was analysed for untargeted metabolome profiling and plasma cytokines by high-resolution mass spectrometry (MS) and multiplex cytokine bead array. Immune scan in mothers was done using flow cytometry. In asymptomatic SARS-CoV-2 infection, --the amino acid metabolic pathways such as glycine, serine, L-lactate and threonine metabolism was upregulated, riboflavin and tyrosine metabolism, downregulated. In mild to moderate disease, the pyruvate and NAD+ metabolism (energy metabolic pathways) were mostly altered. In addition to raised TNF-\u03b1, IFN-\u03b1, IFN-\u03b3, IL-6 cytokine storm, IL-9 was increased in both mothers and neonates. Pyruvate and NAD+ metabolic pathways along with IL-9 and IFN-\u03b3 had impact on non-classical monocytes, increased CD4 T cells and B cells but depleted CD8+ T cells. Cord blood mimicked the mother\u2019s metabolomic profiles by showing altered valine, leucine, isoleucine, glycine, serine, threonine in asymptomatic and NAD+ and riboflavin metabolism in mild and moderate disease subjects. Our results demonstrate a graduated immune-metabolomic interplay in mother and fetus in pregnant females with different degrees of severity of COVID-19 disease. IL-9 and IFN- \u03b3 regulated pyruvate, lactate TCA metabolism and riboflavin metabolism with context to disease severity are hall marks of this materno-fetal metabolome. SARS-CoV-2 infection alters energy consumption metabolic pathways during pregnancy. Pregnant women with mild to moderate COVID-19 show increased energy demands, and consume stored glucose by upregulating pyruvate and NAD+ metabolism. Increased TNF-\u03b1 and IL-9 in mild COVID-19 disease involve TCA cycle to produce lactate and consume stored glucose by up regulating pyruvate and nicotinamide and nicotinate metabolism. With mild to moderate disease, raised IL-9 and TNF-\u03b1, decreased riboflavin pathway, exhaustion of T and B cells cause pathogenesis. Cord blood mimics the metabolic profile of mother\u2019s peripheral blood, SARS- CoV-2 infection reshapes immune-metabolic profiles of mother-infant dyad.","version":"1.1","doi":"10.1101/2023.05.10.540101","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.10.540228","pub_date":"2023-5-11","title":"D614G and Omicron SARS-CoV-2 variant spike proteins differ in the effects of N-glycan modifications on spike expression, virus infectivity, and neutralization by some therapeutic antibodies","abstract":"The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, authentic SARS-CoV-2 grown in the presence of kifunensine decreased titers more for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between spike glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization. The SARS-CoV-2 spike surface protein is covered in glycans that may affect its function and ability to evade antibodies. Omicron variants have over 30 mutations compared to the D614G variant, yet all 22 potential N-glycosylation sites are highly conserved. Here we compared the impact of glycan changes in the spikes of the Omicron and D614G variants on virus infectivity and neutralization. We found that loss of specific glycans in the transmembrane subunit of spike greatly reduced Omicron, but not D614G, spike expression and incorporation into pseudoviruses. Changes in the overall glycan composition also reduced the infectivity of Omicron pseudovirus and authentic virus more than D614G pseudoviruses and authentic viruses. We further show that changes in specific glycans directly or indirectly affected susceptibility of pseudoviruses to therapeutic antibodies, but the effects differed among the variants. These findings highlight differences in the interplay between spike glycans and amino acid residues among SARS-CoV-2 variants that can contribute to spike plasticity and modify spike expression, function, and immune evasion properties.","version":"1.1","doi":"10.1101/2023.05.10.540228","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.10.540233","pub_date":"2023-5-11","title":"Mild/Asymptomatic Maternal SARS-CoV-2 Infection Leads to Immune Paralysis in Fetal Circulation and Immune Dysregulation in Fetal-Placental Tissues","abstract":"Few studies have addressed the impact of maternal mild/asymptomatic SARS-CoV-2 infection on the developing neonatal immune system. In this study, we analyzed umbilical cord blood and placental chorionic villi from newborns of unvaccinated mothers with mild/asymptomatic SARS-CoV-2 infection during pregnancy using flow cytometry, single-cell transcriptomics, and functional assays. Despite the lack of vertical transmission, levels of inflammatory mediators were altered in cord blood. Maternal infection was also associated with increased memory T, B cells, and non-classical monocytes as well as increased activation. However, ex vivo responses to stimulation were attenuated. Finally, within the placental villi, we report an expansion of fetal Hofbauer cells and infiltrating maternal macrophages and rewiring towards a heightened inflammatory state. In contrast to cord blood monocytes, placental myeloid cells were primed for heightened antiviral responses. Taken together, this study highlights dysregulated fetal immune cell responses in response to mild maternal SARS-CoV-2 infection during pregnancy.","version":"1.1","doi":"10.1101/2023.05.10.540233","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.10.511541","pub_date":"2023-5-10","title":"SARS-CoV-2 spike-induced syncytia are senescent and contribute to exacerbated heart failure","abstract":"Patients with pre-existing heart failure are at a particularly high risk of morbidity and mortality resulting from SARS-CoV-2 infection. Direct acute cardiac injury or cytokine storms have been proposed to contribute to depressed cardiac function. However, the pathogenic mechanisms underlying the increased vulnerability to heart failure in SARS-CoV-2 infected patients are still largely unknown. Here, we found that the senescent outcome of SARS-CoV-2 spike protein (SARS-2-S)-induced syncytia exacerbated heart failure progression. We first demonstrated that syncytium formation in cells expressing SARS-2-S delivered by DNA plasmid or LNP-mRNA exhibits a senescence-like phenotype. Extracellular vesicles containing SARS-2-S (S-EVs) also confer a potent ability to form senescent syncytia without denovosynthesis of SARS-2-S. Mechanistically, SARS-2-S syncytia provoke the formation of functional MAVS aggregates, which regulate the senescence fate of SARS-2-S syncytia by TNF \u03b1 . We further demonstrate that senescent SARS-2-S syncytia exhibit shrinked morphology, leading to the activation of WNK1 and impaired cardiac metabolism. In pre-existing heart failure mice, the WNK1 inhibitor WNK463, anti-syncytial drug niclosamide, and senolytic dasatinib protect the heart from exacerbated heart failure triggered by pseudovirus expressing SARS-2-S (SARS-2-Spp). Signs of senescent multinucleated cells are identified in ascending aorta from SARS-CoV-2 omicron variant-infected patient. Our findings thus suggest a potential mechanism for COVID-19-mediated cardiac pathology and recommend the application of WNK1 inhibitor for therapy. In this paper, we directly linked SARS-2-S-triggered syncytium formation with the ensuing induction of cellular senescence and its pathophysiological contribution to heart failure. We propose that both SARS-2-S expression and SARS-2-S protein internalization were sufficient to induce senescence in nonsenescent ACE2-expressing cells. This is important because of the persistent existence of SARS-2-S or extracellular vesicles containing SARS-2-S during the acute and post-acute stages of SARS-CoV-2 infection in human subjects. In searching for the underlying molecular mechanisms determining syncytial fate, the formation of functional MAVS aggregates dependent on RIG-I was observed at an early stage during fusion and regulated the anti-death to senescence fate of SARS-2-S syncytia through the TNF\u03b1-TNFR2 axis. We also found impaired cardiac metabolism in SARS-2-S syncytia induced by condensed WNK1. Importantly, SARS-2-Spp-exacerbated heart failure could be largely rescued by WNK1 inhibitor, anti-syncytial drug or senolytic agent. Together, we suggest that rescuing metabolism dysfunction in senescent SARS-2-S syncytia should be taken into consideration in individuals during the acute or post-acute stage of SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2022.10.10.511541","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.18.537104","pub_date":"2023-5-10","title":"A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo","abstract":"Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by \u223c10-fold, mutations to aspartic acid (N40D) reduced activity by \u223c100-fold relative to wildtype. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.","version":"1.2","doi":"10.1101/2023.04.18.537104","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.444128","pub_date":"2023-5-10","title":"Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects","abstract":"Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.","version":"1.3","doi":"10.1101/2021.05.15.444128","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.08.539929","pub_date":"2023-5-09","title":"Toll-like receptor 7 (TLR7)-mediated antiviral response protects mice from lethal SARS-CoV-2 infection","abstract":"SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses cause fatal pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and lethal inflammatory responses in-vivo are not well defined. CoVs possess single-stranded RNA (ssRNA) genome that is abundantly produced during infection and stimulates both antiviral interferon (IFN) and inflammatory cytokine/ chemokine responses. Therefore, in this study, using wild-type control and TLR7 deficient BALB/c mice infected with a mouse-adapted SARS-COV-2 (MA-CoV-2), we evaluated the role of TLR7 signaling in MA-CoV-2-induced antiviral and inflammatory responses and disease outcome. We show that TLR7-deficient mice are more susceptible to MA-CoV-2 infection as compared to infected control mice. Further evaluation of MA-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral type I (IFN\u03b1/\u03b2) and type III (IFN\u03bb) IFNs, IFN stimulated genes (ISGs, ISG15 and CXCL10), and several pro-inflammatory cytokines/chemokines in TLR7 deficient compared to control mice. Reduced lung IFN/ISG levels and increased morbidity/mortality in TLR7 deficient mice correlated with high lung viral titer. Detailed examination of total cells from MA-CoV-2 infected lungs showed high neutrophil count in TLR7 deficient mice compared to control mice. Additionally, blocking TLR7 activity post-MA-CoV-2 infection using a specific inhibitor also enhanced disease severity. In summary, our results conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite robust inflammatory response, TLR7-mediated IFN/ISG responses likely protect the host from lethal disease. Given similar outcomes in control and TLR7 deficient humans and mice, these results show that MA-CoV-2 infected mice serve as excellent model to study COVID-19.","version":"1.1","doi":"10.1101/2023.05.08.539929","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.08.519651","pub_date":"2023-5-09","title":"Biochemistry-informed design selects potent siRNAs against SARS-CoV-2","abstract":"RNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel COVID-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, and ex vivo assessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September 2022). Our approach is easily generalizable to other viruses as long as a variant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.","version":"1.2","doi":"10.1101/2022.12.08.519651","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.09.539943","pub_date":"2023-5-09","title":"Dissecting the impact of somatic hypermutation on SARS-CoV-2 neutralization and viral escape","abstract":"Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2 neutralizing antibodies. Yet, several potent SARS-CoV-2 antibodies carry no or only few mutations, leaving the question of how ongoing SHM affects neutralization. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, some antibodies, including the public clonotype VH1-58, remained unaffected for Wu01 activity. Moreover, while mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical to neutralize Omicron BA.1/BA.2. Notably, we exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2-neutralizer. These findings substantially broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation, but also counteracts antigenic imprinting through antibody diversification and thus increases the chances of neutralizing viral escape variants.","version":"1.1","doi":"10.1101/2023.05.09.539943","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.476458","pub_date":"2023-5-09","title":"Genomic and transcriptomic characterization of Delta SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus)","abstract":"White-tailed deer are susceptible to SARS-CoV-2 and represent a highly important species for surveillance. Nasal swabs and retropharyngeal lymph nodes from white-tailed deer (n=258) collected in November 2021 from Qu\u00e9bec, Canada were analyzed for SARS-CoV-2 RNA. We employed viral genomics and transcriptomics to further characterize infection and investigate host response to infection. We detected Delta SARS-CoV-2 (AY.44) in deer from the Estrie region; sequences clustered with human sequences from GISAID collected in October 2021 from Vermont, USA, which borders this region. Mutations in the S-gene and a deletion in ORF8 encoding a truncated protein were detected. Host expression patterns in SARS-CoV-2 infected deer were associated with the innate immune response, including signalling pathways related to anti-viral, pro- and anti-inflammatory signalling, and host damage. Our findings provide preliminary insights of host response to SARS-CoV-2 infection in deer and underscores the importance of ongoing surveillance of key wildlife species for SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.01.20.476458","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.06.539715","pub_date":"2023-5-08","title":"SARS-COV-2 Spike Protein Fragment eases Amyloidogenesis of \u03b1-Synuclein","abstract":"Parkinson\u2019s Disease is accompanied by presence of amyloids in the brain formed of \u03b1-synuclein chains. Correlation between COVID-19 and the onset of Parkinson\u2019s disease let to the idea that amyloidogenic segments in SARS-COV-2 proteins can induce aggregation of \u03b1-synuclein. Using molecular dynamic simulations, we show that the fragment FKNIDGYFKI of the spike protein, which is unique for SARS-COV-2, shifts preferentially the ensemble of \u03b1-synuclein monomer towards rod-like fibril seeding conformations, and at the same time stabilizes differentially this polymorph over the competing twister-like structure. Our results are compared with earlier work relying on a different protein fragment that is not specific for SARS-COV-2.","version":"1.1","doi":"10.1101/2023.05.06.539715","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.05.539661","pub_date":"2023-5-08","title":"Host inducible-HSP70A1A is an irresistible drug target to combat SARS-CoV2 infection and pathogenesis","abstract":"One of the fundamental mechanisms developed by the host to contain the highly infectious and rapidly proliferating SARS coronavirus is elevation of body temperature, a natural fallout of which is Heat Shock Protein (HSP) over-expression. Here, for the first time, we demonstrate that the SARS-CoV-2 virus exploits the host Hsp70 chaperone for its entry and propagation and blocking it can combat the infection. SARS-CoV-2 infection as well as febrile temperature enhanced Hsp70 overexpression in host Vero E6 cells. In turn, Hsp70 overexpression elevated the host cell autophagic response that is a prerequisite for viral propagation. Suppressive and prophylactic treatment of Vero E6 cells with HSP70 inhibitor PES-Cl, a small molecule derivative of Pifithrin \u03bc, abrogated viral infection more potently than the currently used drug Remdesivir by suppressing host HSP70 and autophagic response. In conclusion, our study not only provides a fundamental insight into the role of host Hsp70 in SARS-CoV-2 pathogenesis, it paves the way for the development of potent and irresistible anti-viral therapeutics.","version":"1.1","doi":"10.1101/2023.05.05.539661","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.05.539395","pub_date":"2023-5-08","title":"A multiadjuvant polysaccharide-amino acid-lipid (PAL) subunit nanovaccine generates robust systemic and lung-specific mucosal immune responses against SARS-CoV-2 in mice","abstract":"Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, which are essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters could overcome the shortcomings of parenteral vaccines and enhance pre- existing systemic immunity. Here we present a new protein subunit nanovaccine using multiadjuvanted (e.g. RIG-I: PUUC, TLR9: CpG) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL- NPs, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lung, and showed robust systemic humoral immunity. Interestingly, as a purely intranasal vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. Our data suggest that PUUC+CpG PAL-NP subunit vaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.","version":"1.1","doi":"10.1101/2023.05.05.539395","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.07.539752","pub_date":"2023-5-08","title":"Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein","abstract":"The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein has been found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identified only a symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6\u00b0, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.","version":"1.1","doi":"10.1101/2023.05.07.539752","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.248880","pub_date":"2023-5-08","title":"Alpha-1-antitrypsin and its variant-dependent role in COVID-19 pathogenesis","abstract":"SARS-CoV-2 entry into host cells is facilitated by endogenous and exogenous proteases that proteolytically activate the spike glycoprotein and antiproteases inhibiting this process. Understanding the key actors in viral entry is crucial for advancing knowledge of virus tropism, pathogenesis, and potential therapeutic targets. We aimed to investigate the role of na\u00efve serum and alpha-1-antitrypsin (AAT) in inhibiting protease-mediated SARS-CoV-2 entry and explore the implications of AAT deficiency on susceptibility to different SARS-CoV-2 variants. Our study demonstrates that na\u00efve serum exhibits significant inhibition of SARS-CoV-2 entry, with AAT identified as the major serum protease inhibitor potently restricting entry. Using pseudoparticles, replication-competent pseudoviruses, and authentic SARS-CoV-2, we show that AAT inhibition occurs at low concentrations compared with those in serum and bronchoalveolar tissues, suggesting physiological relevance. Furthermore, sera from subjects with an AAT-deficient genotype show reduced ability to inhibit entry of both Wuhan-Hu-1 (WT) and B.1.617.2 (Delta) but exhibit no difference in inhibiting B.1.1.529 (Omicron) entry. AAT may have a variant-dependent therapeutic potential against SARS-CoV-2. Our findings highlight the importance of further investigating the complex interplay between proteases, antiproteases, and spike glycoprotein activation in SARS-CoV-2 and other respiratory viruses to identify potential therapeutic targets and improve understanding of disease pathogenesis.","version":"1.2","doi":"10.1101/2020.08.14.248880","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.15.512291","pub_date":"2023-5-05","title":"Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. As of October 2022, there have been over 625 million confirmed cases of COVID-19, including over 6.5 million deaths. Epidemiological studies have indicated that comorbidities of obesity and diabetes mellitus are associated with increased morbidity and mortality following SARS-CoV-2 infection. We determined how the comorbidities of obesity and diabetes affect morbidity and mortality following SARS-CoV-2 infection in unvaccinated and adjuvanted spike nanoparticle (NVX-CoV2373) vaccinated mice. We find that obese/diabetic mice infected with SARS-CoV-2 have increased morbidity and mortality compared to age matched normal mice. Mice fed a high-fat diet (HFD) then vaccinated with NVX-CoV2373 produce equivalent neutralizing antibody titers to those fed a normal diet (ND). However, the HFD mice have reduced viral clearance early in infection. Analysis of the inflammatory immune response in HFD mice demonstrates a recruitment of neutrophils that was correlated with increased mortality and reduced clearance of the virus. Depletion of neutrophils in diabetic/obese vaccinated mice reduced disease severity and protected mice from lethality. This model recapitulates the increased disease severity associated with obesity and diabetes in humans with COVID-19 and is an important comorbidity to study with increasing obesity and diabetes across the world. SARS-CoV-2 has caused a wide spectrum of disease in the human population, from asymptomatic infections to death. It is important to study the host differences that may alter the pathogenesis of this virus. One clinical finding in COVID19 patients, is that people with obesity or diabetes are at increased risk of severe illness from SARS-CoV-2 infection. We used a high fat diet model in mice to study the effects of obesity and Type 2 diabetes on SARS-CoV-2 infection as well as how these comorbidities alter the response to vaccination. We find that diabetic/obese mice have increased disease after SARS-CoV-2 infection and they have slower clearance of virus. We find that the lungs of these mice have increased neutrophils and that removing these neutrophils protect diabetic/obese mice from disease. This demonstrates why these diseases have increased risk of severe disease and suggests specific interventions upon infection.","version":"1.2","doi":"10.1101/2022.10.15.512291","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.04.539453","pub_date":"2023-5-05","title":"An inimitable proprotein convertase subtilisin kexin-9 (PCSK9) cleavage site VFAQ on Spike protein along with furin cleavage site makes SARS-CoV-2 unique","abstract":"A novel coronavirus (2019-nCoV) or Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) that affects humans has been discovered in Wuhan, China, in 2019. Its genome has been sequenced, and the genetic data was quickly made public. We discovered a novel proprotein convertase subtilisin kexin-9 (PCSK9) cleavage site in the Spike protein of the 2019-nCoV. The recent research also demonstrates that the previously found proprotein convertase 3 (PC3) or furin cleavage site, which was assumed to be unique, is already present in animal corona viruses. In this article, we suggest that the combination of the both proprotein convertase PC3 cleavage site and the PCSK9 site renders SARS-CoV-2 unique in terms of the pathogenicity, potential functional effects, and implications for the development of antiviral drugs.","version":"1.1","doi":"10.1101/2023.05.04.539453","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.04.539462","pub_date":"2023-5-05","title":"Paired associated SARS-CoV-2 spike variable positions: a network analysis approach to emerging variants","abstract":"Amino acids in variable positions of proteins may be correlated, with potential structural and functional implications. Here, we apply exact tests of independence in R \u00d7 C contingency tables to examine noise-free associations between variable positions of the SARS-CoV-2 spike protein, using as a paradigm sequences from Greece deposited in GISAID (N=6,683/1,078 full-length) for the period February 29, 2020 to April 26, 2021 that essentially covers the first three pandemic waves. We examine the fate and complexity of these associations by network analysis, using associated positions (exact p\u22640.001 and Average Product Correction \u22652) as links and the corresponding positions as nodes . We found a temporal linear increase of positional differences and a gradual expansion of the number of position associations over time, represented by a temporally evolving intricate web, resulting in a non-random complex network of 69 nodes and 252 links. Overconnected nodes corresponded to the most adapted variant positions in the population, suggesting a direct relation between network degree and position functional importance. Modular analysis revealed 25 k-cliques comprising three to 11 nodes. At different k- clique resolutions, one to four communities were formed, capturing epistatic associations of circulating variants (Alpha, Beta, B.1.1.318), but also Delta, which dominated the evolutionary landscape later in the pandemic. Cliques of aminoacidic positional associations tended to occur in single sequences, enabling the recognition of epistatic positions in real-world virus populations. Our findings provide a novel way of understanding epistatic relationships in viral proteins with potential applications in the design of virus control procedures. Paired positional associations of adapted amino acids in virus proteins may provide new insights for understanding virus evolution and variant formation. We investigated potential intramolecular relationships between variable SARS-CoV-2 spike positions by exact tests of independence in R \u00d7 C contingency tables, having applied Average Product Correction (APC) to eliminate background noise. Associated positions (exact p\u22640.001 and APC\u22652) formed a non- random, epistatic network of 25 cliques and 1-4 communities at different clique resolutions, revealing evolutionary ties between variable positions of circulating variants, and a predictive potential of previously unknown network positions. Cliques of different sizes represented theoretical combinations of changing residues in sequence space, allowing the identification of significant aminoacidic combinations in single sequences of real-world populations. Our analytic approach that links network structural aspects to mutational aminoacidic combinations in the spike sequence population offers a novel way to understand virus epidemiology and evolution.","version":"1.1","doi":"10.1101/2023.05.04.539462","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.04.539429","pub_date":"2023-5-05","title":"Adsorption of respiratory syncytial virus (RSV), rhinovirus, SARS-CoV-2, and F+ bacteriophage MS2 RNA onto wastewater solids from raw wastewater","abstract":"Despite the wide adoption of wastewater surveillance, more research is needed to understand the fate and transport of viral genetic markers in wastewater. This information is essential for the interpretation of wastewater surveillance data and the development of mechanistic models that link wastewater measurements to the number of individuals shedding virus. In this study, we examined the solid-liquid partitioning behavior of four viruses in wastewater: SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus (RV), and F+ coliphage/MS2. We used two approaches to achieve this: we (1) conducted laboratory partitioning experiments using lab-grown viruses and (2) examined the distribution of endogenous viruses in wastewater. Partition experiments were conducted at 4\u00b0C and 22\u00b0C; wastewater samples were spiked with varying concentrations of each virus and stored for three hours to allow the system to equilibrate. Solids and liquids were separated via centrifugation and viral RNA concentrations were quantified using reverse-transcription-digital droplet PCR (RT-ddPCR). For the distribution experiment, wastewater samples were collected from six wastewater treatment plants and processed without spiking exogenous viruses; viral RNA concentrations were measured in wastewater solids and liquid. Overall, RNA concentrations were higher in solids than the liquid fraction of wastewater by approximately 3\u20134 orders of magnitude. Partition coefficients (KF) from laboratory experiments were determined using the Freundlich model and ranged from 2,000\u2013270,000 ml\u00b7g-1 across viruses and temperature conditions. Distribution coefficients (Kd) determined from endogenous wastewater viruses were consistent with results from laboratory experiments.Further research is needed to understand how virus and wastewater characteristics might influence the partition of viral genetic markers in wastewater. We examined the solid-liquid partitioning behavior of SARS-CoV-2, RSV, RV, and F+coliphage/MS2 RNA in wastewater influent. Overall, partition/distribution coefficients were similar across viruses and temperature conditions.","version":"1.1","doi":"10.1101/2023.05.04.539429","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.04.539267","pub_date":"2023-5-04","title":"\u201cBroad-Spectrum Heavily Mutated Monoclonal Antibody Isolated from COVID-19 Convalescent Vaccinee with Capacity to Neutralize SARS-CoV2 Variants Ranging from B.1 to BQ.1.1.\u201d","abstract":"Choudhary et al. have isolated and characterized Acovimab, a broadly neutralizing RBM-specific human monoclonal antibody with a relatively high level of somatic hypermutation, which potently neutralizes SARS-CoV2 variants ranging from WuhanB.1 to OmicronBQ.1.1, but not the XBB.1.5 variant. Acovimab also possesses strong synergistic neutralizing activity against some Omicron variants when combined with Sotrovimab. Polyclonal plasma antibodies from COVID-19 vaccinees who had recovered from SARS-CoV2 infection were shown to possess low neutralizing titers of antibodies against conserved RBD targets of CoV2 variants including XBB.1.5, which also synergistically neutralize with Sotrovimab against this variant. The increasing prevalence of the highly antibody-evasive Omicron sublineages increases the risk of breakthrough infections and leaves high-risk and vulnerable immunocompromised individuals with no effective options for prophylactic or therapeutic antibody treatments. Here, we report a heavily mutated anti-RBD monoclonal antibody, Acovimab, directed against a site in the receptor-binding motif (RBM) region of the CoV2 receptor-binding domain (RBD), that possesses very broad and highly potent neutralizing activity against CoV2 variants, including many Omicron variants. This antibody is derived from the IGHV1-58*01 germline sequence and possesses a relatively high level of mutation (15.5% of the VH aa sequence), which is unusual for anti-RBD antibodies. Neutralizing activity was very potent (IC50s range of 1-9 ng/ml) for early Omicron subvariants that possess an unmutated F486 residue and is retained but less potent (IC50s of 200-650 ng/ml) for more resistant Omicron subvariants which contain the F486V mutation (BA4/5, BA4.6, and BQ1.1), but is lost for the later ultra-resistant variants that contain F486S (XBB) or F486P (XBB.1.5) mutations. Based on these specificities, it is predicted that Acovimab by itself should protect against CoV2 infections other than those caused by the XBB/XBB.1.5 family. Acovimab also shows strong synergy in neutralization when combined with Sotrovimab, which neutralizes all Omicron variants, including XBB.1.5. Plasma from subjects with hybrid immunity (induced by vaccination + infection) possessed low levels of XBB.1.5 RBM-targeting plasma-neutralizing antibodies, and these also neutralized synergistically when combined with Sotrovimab. These results suggest potentially novel immunotherapeutic options for treating most of the CoV2 variants responsible for current infections.","version":"1.1","doi":"10.1101/2023.05.04.539267","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.02.522449","pub_date":"2023-5-04","title":"Composition of nasopharyngeal microbiota in individuals with SARS-CoV-2 infection across three COVID-19 waves in India","abstract":"Multiple variants of the SARS-CoV-2 virus have been plaguing the world through successive waves of infection over the past three years. Studies by independent research groups across geographies have shown that the microbiome composition in COVID-19 patients (CP) differ from that of healthy individuals (CN). However, such observations were based on limited-sized sample-sets collected primarily from the early days of the pandemic. Here, we study the nasopharyngeal microbiota in COVID-19 patients, wherein the samples have been collected across the three COVID-19 waves witnessed in India, which were driven by different variants of concern. We also present the variations in microbiota of symptomatic vs asymptomatic COVID-19 patients. The nasopharyngeal swabs were collected from 589 subjects providing samples for diagnostics purposes at Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India. CP showed a marked shift in the microbial diversity and composition compared to CN, in a wave-dependent manner. Rickettsiaceae was the only family that was noted to be consistently depleted in CP samples across the waves. The genera Staphylococcus, Anhydrobacter, Thermus, and Aerococcus were observed to be highly abundant in the symptomatic CP patients when compared to the asymptomatic group. In general, we observed a decrease in the burden of opportunistic pathogens in the host microbiota during the later waves of infection. To our knowledge, this is the first longitudinal study which was designed to understand the relation between the evolving nature of the virus and the changes in the human nasopharyngeal microbiota. Such studies not only pave way for better understanding of the disease pathophysiology but also help gather preliminary evidence on whether interventions to the host microbiota can help in better protection or faster recovery.","version":"1.4","doi":"10.1101/2023.01.02.522449","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.01.538506","pub_date":"2023-5-04","title":"Role of SARS-CoV-2 mutations in the evolution of the COVID-19 pandemic","abstract":"RNA viruses, including SARS-CoV-2, evolve by mutation acquisition, or by hybridization between viral genomes. The SARS-CoV-2 pandemic provided an exceptional opportunity to analyze the mutations that appeared over a three-year period. In this study, we analysed the type of mutations and their epidemic consequences on the thousands of genomes produced in our laboratory. These were obtained by next-generation sequencing from respiratory samples performed for genomic surveillance. The frequencies of mutations were calculated using Nextclade, Microsoft Excel, and an in-house Python script. In total, 61,397 genomes matching 483 Pangolin lineages were analyzed; 22,225 nucleotide mutations were identified, and of them 220 (1.0%) were each at the root of at least 836 genomes, a frequency threshold classifying mutations as \u201chyperfertile\u201d. Two of these seeded the pandemic in Europe, namely a mutation in the RNA-dependent RNA polymerase associated with an increased mutation rate (P323L) and one in the spike protein (D614G), which plays a particular role in virus fitness. Most of these 220 \u201chyperfertile\u201d mutations occurred in areas not predicted to be associated with increased virulence. Their number was 8\u00b16 (0-22) per 1,000 nucleotides on average per gene. They were 3.7 times more frequent in accessory than informational genes (14 versus 4; p= 0.0037). Particularly, they were 4.1 times more frequent in ORF8 than in the gene encoding RNA polymerase. Interestingly, stop codons were present in 97 positions, almost only in six accessory genes including ORF7a (25 per 100 codons) and ORF8 (21). Furthermore, 1,661 mutations (16.3%) were associated with a lower number of \u201coffspring\u201d (50-835) and classified as \u201cfertile\u201d. In conclusion, except for two initial mutations that could predict a change in the dynamics of the epidemic (mutation rate and change in the virus attachment site), most of the \u201chyperfertile\u201d mutations did not predict the emergence of a new epidemic form. Significantly, some mutations were in non-coding areas and some consisted of stop codons, indicating that some genes (particularly ORF7a and ORF8) were rather \u201cnon-virulence genes\u201d at a given stage of the epidemic, which is an unusual concept for viruses.","version":"1.1","doi":"10.1101/2023.05.01.538506","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.04.539332","pub_date":"2023-5-04","title":"A bivalent ChAd nasal vaccine protects against SARS-CoV-2 BQ.1.1 and XBB.1.5 infection and disease in mice and hamsters","abstract":"We previously described a nasally delivered monovalent adenoviral-vectored SARS- CoV-2 vaccine (ChAd-SARS-CoV-2-S, targeting Wuhan-1 spike [S]; iNCOVACC\u00ae) that is currently used in India as a primary or booster immunization. Here, we updated the mucosal vaccine for Omicron variants by creating ChAd-SARS-CoV-2-BA.5-S, which encodes for a pre- fusion and surface-stabilized S protein of the BA.5 strain, and then tested monovalent and bivalent vaccines for efficacy against circulating variants including BQ.1.1 and XBB.1.5. Whereas monovalent ChAd-vectored vaccines effectively induced systemic and mucosal antibody responses against matched strains, the bivalent ChAd-vectored vaccine elicited greater breadth. However, serum neutralizing antibody responses induced by both monovalent and bivalent vaccines were poor against the antigenically distant XBB.1.5 Omicron strain and did not protect in passive transfer experiments. Nonetheless, nasally delivered bivalent ChAd- vectored vaccines induced robust antibody and spike-specific memory T cell responses in the respiratory mucosa, and conferred protection against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in the upper and lower respiratory tracts of both mice and hamsters. Our data suggest that a nasally delivered bivalent adenoviral-vectored vaccine induces protective mucosal and systemic immunity against historical and emerging SARS-CoV-2 strains without requiring high levels of serum neutralizing antibody.","version":"1.1","doi":"10.1101/2023.05.04.539332","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.03.539268","pub_date":"2023-5-04","title":"Deep immunological imprinting due to the ancestral spike in the current bivalent COVID-19 vaccine","abstract":"With the aim of broadening immune responses against the evolving SARS-CoV-2 Omicron variants, bivalent COVID-19 mRNA vaccines that encode the ancestral and Omicron BA.5 spike proteins have been authorized for clinical use, supplanting the original monovalent counterpart in numerous countries. However, recent studies have demonstrated that administering either a monovalent or bivalent vaccine as a fourth vaccine dose results in similar neutralizing antibody titers against the latest Omicron subvariants, raising the possibility of immunological imprinting. Utilizing binding immunoassays, pseudotyped virus neutralization assays, and antigenic mapping, we investigated antibody responses from 72 participants who received three monovalent mRNA vaccine doses followed by either a bivalent or monovalent booster, or who experienced breakthrough infections with the BA.5 or BQ subvariant after vaccinations with an original monovalent vaccine. Compared to a monovalent booster, the bivalent booster did not yield noticeably higher binding titers to D614G, BA.5, and BQ.1.1 spike proteins, nor higher virus-neutralizing titers against SARS-CoV-2 variants including the predominant XBB.1.5 and the emergent XBB.1.16. However, sera from breakthrough infection cohorts neutralized Omicron subvariants significantly better. Multiple analyses of these results, including antigenic mapping, made clear that inclusion of the ancestral spike prevents the broadening of antibodies to the BA.5 component in the bivalent vaccine, thereby defeating its intended goal. Our findings suggest that the ancestral spike in the current bivalent COVID-19 vaccine is the cause of deep immunological imprinting. Its removal from future vaccine compositions is therefore strongly recommended.","version":"1.1","doi":"10.1101/2023.05.03.539268","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.17.524183","pub_date":"2023-5-04","title":"Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential","abstract":"There has been limited characterisation of bat-borne coronaviruses in Europe. Here, we screened for coronaviruses in 48 faecal samples from 16 of the 17 bat species breeding in the UK, collected through a bat rehabilitation and conservationist network. We recovered nine (two novel) complete genomes across six bat species: four alphacoronaviruses, a MERS-related betacoronavirus, and four closely related sarbecoviruses. We demonstrate that at least one of these sarbecoviruses can bind and use the human ACE2 receptor for infecting human cells, albeit suboptimally. Additionally, the spike proteins of these sarbecoviruses possess an R-A-K-Q motif, which lies only one nucleotide mutation away from a furin cleavage site (FCS) that enhances infectivity in other coronaviruses, including SARS-CoV-2. However, mutating this motif to an FCS does not enable spike cleavage. Overall, while UK sarbecoviruses would require further molecular adaptations to infect humans, their zoonotic risk is unknown and warrants closer surveillance.","version":"1.5","doi":"10.1101/2023.01.17.524183","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.02.539139","pub_date":"2023-5-03","title":"Chronic alcohol consumption dysregulates innate immune response to SARS-CoV-2 in the lung","abstract":"Alcohol consumption is widespread with over half of the individuals over 18 years of age in the U.S. reporting alcohol use in the last 30 days. Moreover, 9 million Americans engaged in binge or chronic heavy drinking (CHD) in 2019. CHD negatively impacts pathogen clearance and tissue repair, including in the respiratory tract, thereby increasing susceptibility to infection. Although, it has been hypothesized that chronic alcohol consumption negatively impacts COVID-19 outcomes; the interplay between chronic alcohol use and SARS-CoV-2 infection outcomes has yet to be elucidated. Therefore, in this study we investigated the impact of chronic alcohol consumption on SARS-CoV-2 anti-viral responses in bronchoalveolar lavage cell samples from humans with alcohol use disorder and rhesus macaques that engaged in chronic drinking. Our data show that in both humans and macaques, the induction of key antiviral cytokines and growth factors was decreased with chronic ethanol consumption. Moreover, in macaques fewer differentially expressed genes mapped to Gene Ontology terms associated with antiviral immunity following 6 month of ethanol consumption while TLR signaling pathways were upregulated. These data are indicative of aberrant inflammation and reduced antiviral responses in the lung with chronic alcohol drinking.","version":"1.1","doi":"10.1101/2023.05.02.539139","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.13.520255","pub_date":"2023-5-03","title":"Preclinical evaluation of PHH-1V vaccine candidate against SARS-CoV-2 in non-human primates","abstract":"SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programmes, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralising antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.","version":"1.2","doi":"10.1101/2022.12.13.520255","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.25.538336","pub_date":"2023-5-03","title":"Association between SARS-CoV-2 and metagenomic content of samples from the Huanan Seafood Market","abstract":"The role of the Huanan Seafood Market in the early SARS-CoV-2 outbreak remains unclear. Recently the Chinese CDC released data from deep sequencing of environmental samples collected from the market after it was closed on January-1-2020 (Liu et al. 2023a). Prior to this release, Crits-Christoph et al. (2023) analyzed data from a subset of the samples. Both studies concurred that the samples contained genetic material from a variety of species, including some like raccoon dogs that are susceptible to SARS-CoV-2. However, neither study systematically analyzed the relationship between the amount of genetic material from SARS-CoV-2 and different animal species. Here I implement a fully reproducible computational pipeline that jointly analyzes the number of reads mapping to SARS-CoV-2 and the mitochondrial genomes of chordate species across the full set of samples. I validate the presence of genetic material from numerous species, and calculate mammalian mitochondrial compositions similar to those reported by Crits-Christoph et al. (2023). However, the number of SARS-CoV-2 reads is not consistently correlated with reads mapping to non-human susceptible species. For instance, 14 samples have >20% of their chordate mitochondrial material from raccoon dogs, but only one of these samples contains any SARS-CoV-2 reads, and that sample only has 1 of \u223c200,000,000 reads mapping to SARS-CoV-2. Instead, SARS-CoV-2 reads are most correlated with reads mapping to various fish, such as catfish and largemouth bass. These results suggest that while metagenomic analysis of the environmental samples is useful for identifying animals or animal products sold at the market, co-mingling of animal and viral genetic material is unlikely to reliably indicate whether any animals were infected by SARS-CoV-2.","version":"1.2","doi":"10.1101/2023.04.25.538336","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.02.539155","pub_date":"2023-5-03","title":"Host-Microbiome Associations in Saliva Predict COVID-19 Severity","abstract":"Established evidence indicates that oral microbiota plays a crucial role in modulating host immune responses to viral infection. Following Severe Acute Respiratory Syndrome Coronavirus 2 \u2013 SARS-CoV-2 \u2013 there are coordinated microbiome and inflammatory responses within the mucosal and systemic compartments that are unknown. The specific roles that the oral microbiota and inflammatory cytokines play in the pathogenesis of COVID-19 are yet to be explored. We evaluated the relationships between the salivary microbiome and host parameters in different groups of COVID-19 severity based on their Oxygen requirement. Saliva and blood samples (n = 80) were collected from COVID-19 and from non-infected individuals. We characterized the oral microbiomes using 16S ribosomal RNA gene sequencing and evaluated saliva and serum cytokines using Luminex multiplex analysis. Alpha diversity of the salivary microbial community was negatively associated with COVID-19 severity. Integrated cytokine evaluations of saliva and serum showed that the oral host response was distinct from the systemic response. The hierarchical classification of COVID-19 status and respiratory severity using multiple modalities separately (i.e., microbiome, salivary cytokines, and systemic cytokines) and simultaneously (i.e., multi-modal perturbation analyses) revealed that the microbiome perturbation analysis was the most informative for predicting COVID-19 status and severity, followed by the multi-modal. Our findings suggest that oral microbiome and salivary cytokines may be predictive of COVID-19 status and severity, whereas atypical local mucosal immune suppression and systemic hyperinflammation provide new cues to understand the pathogenesis in immunologically na\u00efve populations. The oral mucosa is one of the first sites encountered by bacterial and viral infections, including SARS-CoV-2. It consists of a primary barrier occupied by a commensal oral microbiome. The primary function of this barrier is to modulate immunity and provide protection against invading infection. The occupying commensal microbiome is an essential component that influences the immune system\u2019s function and homeostasis. The present study showed that the host oral immune response performs unique functions in response to SARS-CoV-2 when compared to systemic responses during the acute phase. We also demonstrated that there is a link between oral microbiome diversity and COVID-19 severity. Additionally, the salivary microbiome was predictive of not only disease status but also severity.","version":"1.1","doi":"10.1101/2023.05.02.539155","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.01.538955","pub_date":"2023-5-02","title":"Diverging maternal and infant cord antibody functions from SARS-CoV-2 infection and vaccination in pregnancy","abstract":"Immunization in pregnancy is a critical tool that can be leveraged to protect the infant with an immature immune system but how vaccine-induced antibodies transfer to the placenta and protect the maternal-fetal dyad remains unclear. Here, we compare matched maternal-infant cord blood from individuals who in pregnancy received mRNA COVID-19 vaccine, were infected by SARS-CoV-2, or had the combination of these two immune exposures. We find that some but not all antibody neutralizing activities and Fc effector functions are enriched with vaccination compared to infection. Preferential transport to the fetus of Fc functions and not neutralization is observed. Immunization compared to infection enriches IgG1-mediated antibody functions with changes in antibody post-translational sialylation and fucosylation that impact fetal more than maternal antibody functional potency. Thus, vaccine enhanced antibody functional magnitude, potency and breadth in the fetus are driven more by antibody glycosylation and Fc effector functions compared to maternal responses, highlighting prenatal opportunities to safeguard newborns as SARS-CoV-2 becomes endemic. SARS-CoV-2 vaccination in pregnancy induces diverging maternal and infant cord antibody functions","version":"1.1","doi":"10.1101/2023.05.01.538955","journal":"bioRxiv","score":null},{"id":"10.1101/2023.05.02.539082","pub_date":"2023-5-02","title":"Discovery and characterization of highly potent and selective covalent inhibitors of SARS-CoV-2 PLpro","abstract":"Coronavirus infections, such as the global COVID-19 pandemic, have had a profound impact on many aspects of our daily life including working style, economy, and the healthcare system. To prevent the rapid viral transmission and speed up recovery from the infection, many academic organizations and industry research labs have conducted extensive research on discovering new therapeutic options for SARS-CoV-2. Among those efforts, RNA-dependent RNA polymerase (RdRp) inhibitors such as Remdesivir, Molnupiravir and 3CLpro inhibitor such as Nirmatrelvir (Paxlovid\u2122) have been widely used as the therapeutic options. Given the recent emergence of several new variants that caused a resurgence of the virus, it would be beneficial to discover more diverse therapeutic options with novel anti-viral mechanisms. In this regard, PLpro has been highlighted since it, along with 3CLpro, is one of the two most important proteases that are required for SARS-CoV-2 viral processing. While 3CLpro inhibitors were extensively investigated in the light of Emergency Use Authorizations of Nirmatrelvir, PLpro inhibitors have not been thoroughly investigated even preclinically. Thus, discovery efforts on antivirals acting against PLpro will be valuable. PLpro inhibitors may exert their activity by inhibiting viral replication and enhancing the host defense system through blocking virus-induced cell signaling events for evading host immune response. In this study, we report the discovery and development of two covalent irreversible PLpro inhibitors, HUP0109 and its deuterated analog DX-027, out of our quest for novel anti-COVID 19 therapeutic agents for the past two and half years. HUP0109 selectively targets the viral catalytic cleft of PLpro and covalently modifies its active site cysteine residue (C111). Promising results from preclinical evaluation suggest that DX-027 can be developed as a potential COVID-19 treatment.","version":"1.1","doi":"10.1101/2023.05.02.539082","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.07.506979","pub_date":"2023-5-02","title":"A novel biopolymer for mucosal adjuvant against respiratory pathogens","abstract":"Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a novel biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, using COVID as an example, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.","version":"1.2","doi":"10.1101/2022.09.07.506979","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.28.538747","pub_date":"2023-5-01","title":"Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron Variants in Mice and Hamsters","abstract":"The continued evolution and emergence of novel SARS-CoV-2 variants has resulted in challenges to vaccine and antibody efficacy. The emergence of each new variant necessitates the need to re-evaluate and refine animal models used for countermeasure testing. Here, we tested a currently circulating SARS-CoV-2 Omicron lineage variant, BQ.1.1, in multiple rodent models including K18-hACE2 transgenic, C57BL/6J, and 129S2 mice, and Syrian golden hamsters. In contrast to a previously dominant BA.5.5 Omicron variant, inoculation of K18-hACE2 mice with BQ.1.1 resulted in a substantial weight loss, a characteristic seen in pre-Omicron variants. BQ.1.1 also replicated to higher levels in the lungs of K18-hACE2 mice and caused greater lung pathology than the BA.5.5 variant. However, C57BL/6J mice, 129S2 mice, and Syrian hamsters inoculated with BQ.1.1 showed no differences in respiratory tract infection or disease compared to animals administered BA.5.5. Airborne or direct contact transmission in hamsters was observed more frequently after BQ.1.1 than BA.5.5 infection. Together, these data suggest that the BQ.1.1 Omicron variant has increased virulence in some rodent species, possibly due to the acquisition of unique spike mutations relative to other Omicron variants. As SARS-CoV-2 continues to evolve, there is a need to rapidly assess the efficacy of vaccines and antiviral therapeutics against newly emergent variants. To do so, the commonly used animal models must also be reevaluated. Here, we determined the pathogenicity of the circulating BQ.1.1 SARS-CoV-2 variant in multiple SARS-CoV-2 animal models including transgenic mice expressing human ACE2, two strains of conventional laboratory mice, and Syrian hamsters. While BQ.1.1 infection resulted in similar levels of viral burden and clinical disease in the conventional laboratory mice tested, increases in lung infection were detected in human ACE2-expressing transgenic mice, which corresponded with greater levels of pro-inflammatory cytokines and lung pathology. Moreover, we observed a trend towards greater animal-to-animal transmission of BQ.1.1 than BA.5.5 in Syrian hamsters. Together, our data highlight important differences in two closely related Omicron SARS-CoV-2 variant strains and provide a foundation for evaluating countermeasures.","version":"1.1","doi":"10.1101/2023.04.28.538747","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.19.524684","pub_date":"2023-4-28","title":"Immunogenicity and safety in pigs of PHH-1V, a SARS-CoV-2 RBD fusion heterodimer vaccine candidate","abstract":"The continuing high global incidence of COVID-19 and the undervaccinated status of billions of persons strongly motivate the development of a new generation of efficacious vaccines. We have developed an adjuvanted vaccine candidate, PHH-1V, based on a protein comprising the receptor binding domain (RBD) of the Beta variant of SARS-CoV-2 fused in tandem with the equivalent domain of the Alpha variant, with its immunogenicity, safety and efficacy previously demonstrated in mouse models. In the present study, we immunized pigs with different doses of PHH-1V in a prime-and-boost scheme showing PHH-1V to exhibit an excellent safety profile in pigs and to produce a solid RBD-specific humoral response with neutralising antibodies to 7 distinct SARS-CoV-2 variants of concern, with the induction of a significant IFN\u03b3+ T-cell response. We conclude that PHH-1V is safe and elicits a robust immune response to SARS-CoV-2 in pigs, a large animal preclinical model.","version":"1.2","doi":"10.1101/2023.01.19.524684","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.28.538473","pub_date":"2023-4-28","title":"Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 that Exhibits Antiviral Efficacy in SARS-CoV-2 Infected African Green Monkeys","abstract":"Remdesivir 1 is an amidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here we describe the discovery of a 5\u2019-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and three to seven-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved pre-systemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice-daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2 which supports development of 3 as a promising COVID-19 treatment.","version":"1.1","doi":"10.1101/2023.04.28.538473","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.09.531948","pub_date":"2023-4-28","title":"Impaired potency of neutralizing antibodies against cell-cell fusion mediated by SARS-CoV-2","abstract":"The SARS-CoV-2 Omicron subvariants have dominated the pandemic due to their high transmissibility and immune evasion conferred by the spike mutations. The Omicron subvariants can spread by cell-free virus infection and cell-cell fusion, the latter of which is more effective but has not been extensively investigated. In this study, we developed a simple and high-throughput assay that provides a rapid readout to quantify cell-cell fusion mediated by the SARS-CoV-2 spike proteins without using live or pseudotyped virus. This assay can be used to identify variants of concern and to screen for prophylactic and therapeutic agents. We further evaluated a panel of monoclonal antibodies (mAbs) and vaccinee sera against D614G and Omicron subvariants, finding that cell-cell fusion is substantially more resistant to mAb and serum inhibition than cell-free virus infection. Such results have important implications for the development of vaccines and antiviral antibody drugs against cell-cell fusion induced by SARS-CoV-2 spikes.","version":"1.2","doi":"10.1101/2023.03.09.531948","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.26.538488","pub_date":"2023-4-27","title":"Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift","abstract":"Developing broad coronavirus vaccines requires identifying and understanding the molecular basis of broadly neutralizing antibody (bnAb) spike sites. In our previous work, we identified sarbecovirus spike RBD group 1 and 2 bnAbs. We have now shown that many of these bnAbs can still neutralize highly mutated SARS-CoV-2 variants, including the XBB.1.5. Structural studies revealed that group 1 bnAbs use recurrent germline encoded CDRH3 features to interact with a conserved RBD region that overlaps with class 4 bnAb site. Group 2 bnAbs recognize a less well-characterized 'site V' on the RBD and destabilize spike trimer. The site V has remained largely unchanged in SARS-CoV- 2 variants and is highly conserved across diverse sarbecoviruses, making it a promising target for broad coronavirus vaccine development. Our findings suggest that targeted vaccine strategies may be needed to induce effective B cell responses to escape resistant subdominant spike RBD bnAb sites.","version":"1.1","doi":"10.1101/2023.04.26.538488","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.26.538490","pub_date":"2023-4-27","title":"Designer DNA NanoGripper","abstract":"DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the \u201cNanoGripper\u201d, that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of \u223c 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza. Design, synthesis, characterization, and functional showcase of a human-hand like designer DNA nanobot","version":"1.1","doi":"10.1101/2023.04.26.538490","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.19.537514","pub_date":"2023-4-26","title":"Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses","abstract":"The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and variants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learned. As a component of the Platform, the SARS-CoV-2 Data Hubs enabled the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.","version":"1.2","doi":"10.1101/2023.04.19.537514","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.25.538264","pub_date":"2023-4-26","title":"Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA","abstract":"Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Our results indicate that no sampled wildlife were positive for SARS-CoV-2. This finding is surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American\u2019s wildlife populations.","version":"1.1","doi":"10.1101/2023.04.25.538264","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.25.538294","pub_date":"2023-4-26","title":"Intranasal VLP-RBD vaccine adjuvanted with BECC470 confers immunity against Delta SARS-CoV-2 challenge in K18-hACE2-mice","abstract":"As the COVID-19 pandemic transitions to endemic, seasonal boosters are a plausible reality across the globe. We hypothesize that intranasal vaccines can provide better protection against asymptomatic infections and more transmissible variants of SARS-CoV-2. To formulate a protective intranasal vaccine, we utilized a VLP-based platform. Hepatitis B surface antigen- based virus like particles (VLP) linked with receptor binding domain (RBD) antigen were paired with the TLR4-based agonist adjuvant, BECC 470. K18-hACE2 mice were primed and boosted at four-week intervals with either VLP-RBD-BECC or mRNA-1273. Both VLP-RBD-BECC and mRNA-1273 vaccination resulted in production of RBD-specific IgA antibodies in serum. RBD- specific IgA was also detected in the nasal wash and lung supernatants and were highest in VLP-RBD-BECC vaccinated mice. Interestingly, VLP-RBD-BECC vaccinated mice showed slightly lower levels of pre-challenge IgG responses, decreased RBD-ACE2 binding inhibition, and lower neutralizing activity in vitro than mRNA-1273 vaccinated mice. Both VLP-RBD-BECC and mRNA-1273 vaccinated mice were protected against challenge with a lethal dose of Delta variant SARS-CoV-2. Both vaccines limited viral replication and viral RNA burden in the lungs of mice. CXCL10 is a biomarker of severe SARS-CoV-2 infection and we observed both vaccines limited expression of serum and lung CXCL10. Strikingly, VLP-RBD-BECC when administered intranasally, limited lung inflammation at early timepoints that mRNA-1273 vaccination did not. VLP-RBD-BECC immunization elicited antibodies that do recognize SARS-CoV-2 Omicron variant. However, VLP-RBD-BECC immunized mice were protected from Omicron challenge with low viral burden. Conversely, mRNA-1273 immunized mice had low to no detectable virus in the lungs at day 2. Together, these data suggest that VLP-based vaccines paired with BECC adjuvant can be used to induce protective mucosal and systemic responses against SARS- CoV-2.","version":"1.1","doi":"10.1101/2023.04.25.538294","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.24.538130","pub_date":"2023-4-25","title":"How Do Deer Respiratory Epithelial Cells Weather The Initial Storm of SARS-CoV-2?","abstract":"The potential infectivity of SARS-CoV-2 in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with SARS-CoV-2 was assessed throughout 48 hours post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to SARS-COV-2, with significantly (P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via IL-17 and NF-\u03baB signaling pathways. Inhibition of the NF-\u03baB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans. White-tailed deer primary respiratory epithelial cells are susceptible to SARS- CoV-2 without causing hyper cytokine gene expression. Downregulation of IL-17 and NF-\u03baB signaling pathways after SARS-CoV-2 infection could be key to the regulated cytokine response in deer cells. Deer innate immune system could play a critical role in early antiviral and tissue repair response following SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.04.24.538130","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.24.538161","pub_date":"2023-4-25","title":"SARS-CoV-2 Spike Protein Reduces Burst Activities in Neurons Measured by Micro-Electrode Arrays","abstract":"SARS-CoV-2 caused a large-scale global pandemic between 2020 and 2022. Despite efforts to understand its biology and mechanisms of pathogenicity, the viral impact on the neurological systems remains unclear. The main goal of this study was to quantify the neurological phenotypes induced by SARS-CoV-2 spike protein in neurons, measured by in-vitro multi-well micro-electrode arrays (MEAs). We extracted the whole-brain neurons from the newborn P1 mice and plated them on multi-well micro-electrode arrays and administered purified recombinant spike proteins (S1 and S2 subunits respectively) from the SARS-CoV-2 virus. The signals from the MEAs were transmitted from an amplifier to a high-performance computer for recording and analysis. We used an in-house developed algorithm to quantify neuronal phenotypes. Among all the phenotypic features analyzed, we discovered that the S1 protein of SARS-CoV-2 decreased the mean burst numbers observed on each electrode; This effect was not observed for the spike 2 protein (S2) and could be rescued by an anti-S1 antibody. Finally, our data strongly suggest that the receptor binding domain (RBD) of S1 is responsible for the reduction of burst activities in neurons. Overall, our results strongly indicate that spike proteins may play an important role in altering neuronal phenotypes, specifically the burst patterns, when neurons are exposed during early development.","version":"1.1","doi":"10.1101/2023.04.24.538161","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.20.492834","pub_date":"2023-4-24","title":"SARS-CoV-2 Infects Peripheral and Central Neurons Before Viremia, Facilitated by Neuropilin-1","abstract":"Neurological symptoms associated with COVID-19, acute and long-term, suggest SARS-CoV-2 affects both central and peripheral nervous systems. Although studies have shown olfactory and hematogenous entry into the brain and neuroinflammation, little attention has been paid to the susceptibility of the peripheral nervous system to infection or to alternative routes of CNS invasion. We show that neurons in the central and peripheral nervous system are susceptible to productive infection with SARS-CoV-2. Infection of K18-hACE2 mice, wild-type mice, golden Syrian hamsters, and primary neuronal cultures demonstrate viral RNA, protein, and infectious virus in peripheral nervous system neurons and satellite glial cells, spinal cord, and specific brain regions. Moreover, neuropilin-1 facilitates SARS-CoV-2 neuronal infection. Our data show that SARS-CoV-2 rapidly invades and establishes a productive infection in the peripheral and central nervous system via direct invasion of neurons prior to viremia, which may underlie some cognitive and sensory symptoms associated with COVID-19.","version":"1.2","doi":"10.1101/2022.05.20.492834","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.24.538100","pub_date":"2023-4-24","title":"The enigma of the SARS-CoV-2 microcirculation dysfunction: evidence for modified endothelial junctions","abstract":"Published evidence indicates that Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection causes endothelial cell (EC) injury in the Coronavirus Disease 2019 (COVID-19). Endothelial junctions (EJ) are crucial to maintain EC integrity and normal microvascular functions due to the adhesive properties of Vascular endothelial (VE)-cadherin to glue EC together. Here we report studies in vitro and in vivo that indicate VE-cadherin to be a target for cleavage by ACE2. We have identified that the extracellular domain of VE-cadherin contains these two amino acid sequences at the positions 256P-F257 and 321PMKP-325L for ACE2 substrate recognition. Incubation of purified sVE with ACE2 revealed a dose-dependent loss of immunoreactivity detected with an antibody directed against the Extracellular domain 1 (EC1) domain of sVE. We confirmed the presence of ACE2 on ECs using immunofluorescence studies, and by western blotting on ECs extracts. We also present evidence from patients with severe COVID-19 disease for a circulating form of ACE2. Its apparent molecular weight of 70 kDa is in agreement with a previously described extracellular form of ACE2 bearing the catalytic site of the ectopeptidase. Consistent with the experimental evidence for our hypothesis, the level of circulating soluble VE-cadherin fragments was increased in the blood of patients with severe COVID-19 disease. Further studies are needed to determine if increased circulating fragments of ACE2 and VE-cadherin may contribute to the future development of post-acute COVID-19 syndrome characterized by vascular endothelial injury, hypoxia, and inflammatory state. SARS-CoV-2 infection promotes vascular dysfunction but the processes are not completely understood. The vascular endothelium is composed of a monolayer of endothelial cells (ECs) that exclusively express VE-cadherin at adherens junctions (AJs). The published structure of VE-cadherin has revealed crucial residues in the domains EC1-2 for ECs adhesiveness. In this report, we demonstrate for the first time that VE-cadherin is a target for ACE2 ectoenzyme in the domains EC2-3. In addition, in COVID-19 patients\u2019 blood, we identify truncated forms of ACE2 and VE-cadherin that are correlated with severe SARS-CoV-2 infection. Because the turnover rate of ECs is very low, this could provide part of the explanation for Long CoVID-19 disease. These exciting results highlight the role of proteases and AJs, and the need for continuing efforts to elucidate whether these circulating proteins might be of prime significance for clinicians to facilitate personalized medicine.","version":"1.1","doi":"10.1101/2023.04.24.538100","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.21.513237","pub_date":"2023-4-24","title":"Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants","abstract":"The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs, but also revealed how quickly viral escape can curtail effective options. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including EvusheldTM and its constituent, cilgavimab. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.","version":"1.2","doi":"10.1101/2022.10.21.513237","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.30.535005","pub_date":"2023-4-24","title":"S:D614G and S:H655Y are gateway mutations that act epistatically to promote SARS-CoV-2 variant fitness","abstract":"SARS-CoV-2 variants bearing complex combinations of mutations that confer increased transmissibility, COVID-19 severity, and immune escape, were first detected after S:D614G had gone to fixation, and likely originated during persistent infection of immunocompromised hosts. To test the hypothesis that S:D614G facilitated emergence of such variants, S:D614G was reverted to the ancestral sequence in the context of sequential Spike sequences from an immunocompromised individual, and within each of the major SARS-CoV-2 variants of concern. In all cases, infectivity of the S:D614G revertants was severely compromised. The infectivity of atypical SARS-CoV-2 lineages that propagated in the absence of S:D614G was found to be dependent upon either S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y are only detected in SARS-CoV-2, which is also distinguished by a polybasic S1/S2 cleavage site. Genetic and biochemical experiments here showed that S:Q613H, S:D614G, and S:H655Y each stabilize Spike on virions, and that they are dispensable in the absence of S1/S2 cleavage, consistent with selection of these mutations by the S1/S2 cleavage site. CryoEM revealed that either S:D614G or S:H655Y shift the Spike receptor binding domain (RBD) towards the open conformation required for ACE2-binding and therefore on pathway for infection. Consistent with this, an smFRET reporter for RBD conformation showed that both S:D614G and S:H655Y spontaneously adopt the conformation that ACE2 induces in the parental Spike. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y are convergent adaptations to the polybasic S1/S2 cleavage site which stabilize S1 on the virion in the open RBD conformation and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations. S:D614G is ubiquitous among SARS-CoV-2 B-lineage Spikes and is required for infectivity of the main Variants of Concern In an example of convergent evolution, SARS-CoV-2 A lineage viruses maintained transmission chains in the absence of S:D614G, but were instead dependent upon S:Q613H or S:H655Y S:D614G and S:H655Y are both adaptations to the polybasic S1/S2 cleavage site Increased infectivity of S:D614G and S:H655Y is associated with a more open RBD conformation and increased steady-state levels of virion-associated S1","version":"1.2","doi":"10.1101/2023.03.30.535005","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.23.537985","pub_date":"2023-4-24","title":"Mechanism of the Covalent Inhibition of Human Transmembrane Protease Serine 2 as an Original Antiviral Strategy","abstract":"The Transmembrane Protease Serine 2 (TMPRSS2) is a human enzyme which is involved in the maturation and post-translation of different proteins. In addition of being overexpressed in cancer cells, TMPRSS2 plays a further fundamental role in favoring viral infections by allowing the fusion of the virus envelope and the cellular membrane, notably in SARS-CoV-2. In this contribution we resort to multiscale molecular modeling to unravel the structural and dynamical features of TMPRSS2 and its interaction with a model lipid bilayer. Furthermore, we shed light into the mechanism of action of a potential inhibitor (Nafamostat), determining the free-energy profile associated with the inhibition reaction, and showing the facile poisoning of the enzyme. Our study, while providing the first atomistically resolved mechanism of TMPRSS2 inhibition, is also fundamental in furnishing a solid framework for further rational design targeting transmembrane proteases in a host-directed antiviral strategy.","version":"1.1","doi":"10.1101/2023.04.23.537985","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460664","pub_date":"2023-4-21","title":"Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19","abstract":"Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity. Genetically diverse mice model a spectrum of clinically relevant innate immune responses to SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2021.09.17.460664","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.20.537738","pub_date":"2023-4-21","title":"An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.","version":"1.1","doi":"10.1101/2023.04.20.537738","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.19.537460","pub_date":"2023-4-20","title":"Machine learning detection of SARS-CoV-2 high-risk variants","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved many high-risk variants, resulting in repeated COVID-19 waves of pandemic during the past years. Therefore, accurate early-warning of high-risk variants is vital for epidemic prevention and control. Here we construct a machine learning model to predict high-risk variants of SARS-CoV-2 by LightGBM algorithm based on several important haplotype network features. As demonstrated on a series of different retrospective testing datasets, our model achieves accurate prediction of all variants of concern (VOC) and most variants of interest (AUC=0.96). Prediction based on the latest sequences shows that the newly emerging lineage BA.5 has the highest risk score and spreads rapidly to become a major epidemic lineage in multiple countries, suggesting that BA.5 bears great potential to be a VOC. In sum, our machine learning model is capable to early predict high-risk variants soon after their emergence, thus greatly improving public health preparedness against the evolving virus.","version":"1.1","doi":"10.1101/2023.04.19.537460","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.19.537521","pub_date":"2023-4-20","title":"SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replication in vitro","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is believed to have a zoonotic origin. Bats are a suspected natural host of SARS-CoV-2 because of sequence homology with other bat coronaviruses. Understanding the origin of the virus and determining species susceptibility is essential for managing the transmission potential during a pandemic. In a previous study, we established an in vitro animal model of SARS-CoV-2 susceptibility and replication in a non-permissive avian fibroblast cell line (DF1) based on expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) from different animal species. In this work, we express the ACE2 of seven bat species in DF1 cells and determine their ability to support attachment and replication of the original SARS-CoV-2 Wuhan lineage virus, as well as two variants, Delta and Lambda. We demonstrate that the ACE2 receptor of all seven species: little brown bat (Myotis lucifugus), great roundleaf bat (Hipposideros armiger), Pearson\u2019s horseshoe bat (Rhinolophus pearsonii), greater horseshoe bat (Rhinolophus ferrumequinum), Brazilian free-tailed bat (Tadarida brasiliensis), Egyptian rousette (Rousettus aegyptiacus), and Chinese rufous horseshoe bat (Rhinolophus sinicus), made the DF1 cells permissible to the three isolates of SARS-CoV-2. However, the level of virus replication differed between bat species and variant tested. In addition, the Wuhan lineage SARS-CoV-2 virus replicated to higher titers (104.5-105.5 TCID50) than either variant virus (103.5-104.5 TCID50) on pass 1. Interestingly, all viruses tested grew to higher titers (approximately 106 TCID50) when cells expressed the human ACE2 gene compared to bat ACE2. This study provides a practical in vitro method for further testing of animal species for potential susceptibility to current and emerging SARS-CoV-2 viruses.","version":"1.1","doi":"10.1101/2023.04.19.537521","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.20.537680","pub_date":"2023-4-20","title":"Establishment of a screening platform based on human coronavirus OC43 for the identification of microbial natural products with antiviral activity","abstract":"Human coronaviruses (HCoVs) cause respiratory tract infections and are of great importance due to the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Human betacoronavirus OC43 (HCoV-OC43) is an adequate surrogate for SARS-CoV-2 because it infects the human respiratory system, presents a comparable biology, and is transmitted in a similar way. Its use is advantageous since it only requires biosafety level (BSL)-2 infrastructure which minimizes costs and biosafety associated limitations. In this report, we describe a high-throughput screening (HTS) platform to identify compounds that inhibit the propagation of HCoV-OC43. Optimization of assays based on inhibition of the cytopathic effect and virus immunodetection with a specific antibody, has provided a robust methodology for the screening of a selection of microbial natural product extracts from the Fundaci\u00f3n MEDINA collection. Using this approach, a subset of 1280 extracts has been explored. Of these, upon hit confirmation and early LC-MS dereplication, 10 extracts were identified that contain potential new compounds. In addition, we report on the novel antiviral activity of some previously described natural products whose presence in bioactive extracts was confirmed by LC/MS analysis. The COVID-19 pandemic has revealed the lack of effective treatments against betacoronaviruses and the urgent need for new broad-spectrum antivirals. Natural products are a valuable source of bioactive compounds with pharmaceutical potential that may lead to the discovery of new antiviral agents. Specifically, compared to conventional synthetic molecules, microbial natural extracts possess a unique and vast chemical diversity and are amenable to large-scale production. The implementation of a high-throughput screening platform using the betacoronavirus OC43 in a human cell line infection model has provided proof of concept of the approach and has allowed for the rapid and efficient evaluation of 1280 microbial extracts. The identification of several active compounds validates the potential of the platform for the search for new compounds with antiviral capacity.","version":"1.1","doi":"10.1101/2023.04.20.537680","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.20.536837","pub_date":"2023-4-20","title":"Sialoglycan binding triggers spike opening in a human coronavirus","abstract":"Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.","version":"1.1","doi":"10.1101/2023.04.20.536837","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.31.505985","pub_date":"2023-4-19","title":"Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants","abstract":"Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals developed a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS- CoV-2 variants were found to be neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validated the olfactory pathway as a major entry point into the brain in vivo and demonstrated in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks. \n","version":"1.2","doi":"10.1101/2022.08.31.505985","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.18.537373","pub_date":"2023-4-19","title":"An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy","abstract":"COVID-19 has claimed millions of lives since the emergence of SARS-CoV-2, and lung disease appears the primary cause of the death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where the human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors, reflecting the heterogeneity of human populations. In particular, two cytokines (IP-10 and IL-8) were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei, not only inhibited virus replication but also production of pro-inflammatory cytokines, and ameliorated the histopathological changes of the lungs caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs. Here we established an ex vivo human precision-cut lung slice platform for assessing SARS-CoV-2 infection, viral replication kinetics, innate immune response, disease progression, and antiviral drugs. Using this platform, we identified early induction of specific cytokines, especially IP-10 and IL-8, as potential predictors for severe COVID-19, and uncovered a hitherto unrecognized phenomenon that while infectious virus disappears at late times of infection, viral RNA persists and lung histopathology commences. This finding may have important clinical implications for both acute and post-acute sequelae of COVID-19. This platform recapitulates some of the characteristics of lung disease observed in severe COVID-19 patients and is therefore a useful platform for understanding mechanisms of SARS-CoV-2 pathogenesis and for evaluating the efficacy of antiviral drugs.","version":"1.1","doi":"10.1101/2023.04.18.537373","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.25.493484","pub_date":"2023-4-19","title":"Molecular Investigations of Selected Spike Protein Mutations in SARS-CoV-2: Delta and Omicron Variants and Omicron Subvariants","abstract":"Among the multiple SARS-CoV-2 variants recently reported, the Delta variant has generated most perilous and widespread effects. Another variant, Omicron, has been identified specifically for its high transmissibility. Omicron contains numerous spike (S) protein mutations and in numbers much larger than those of its predecessor variants. In this report we discuss some essential structural aspects and time-based structure changes of a selected set of spike protein mutations within the Delta and Omicron variants. The expected impact of multiple-point mutations within the spike protein\u2019s receptor-binding domain (RBD) and S1 of these variants are examined. Additionally, RBD of the more recently emerged subvariants BA.4, BA.5 and BA.2.12.1 are discussed. Within the latter group, BA.5 represents globally, the most prevalent form of SARS-CoV-2 at the present time. Temporal mutation profile for the subvariant BF.7 and currently circulating variants of interest (VOI) and variants under monitoring (VUMs) including XBB.1.5, BQ.1, BA.2.75, CH.1.1, XBB and XBF are computationally explored here briefly with the expectation that these structural data will be helpful to identify drug targets and to neutralize antibodies for the evolving variants/subvariants of SARS-CoV-2.","version":"1.5","doi":"10.1101/2022.05.25.493484","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.17.537235","pub_date":"2023-4-18","title":"A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity","abstract":"An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (\u22063678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of \u22063678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the \u22063678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest \u22063678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.04.17.537235","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.17.537220","pub_date":"2023-4-18","title":"A novel simian adenovirus vectored COVID-19 vaccine elicits effective mucosal and systemic immunity in mice by intranasal and intramuscular vaccination regimens","abstract":"The failure of COVID-19 vaccines to prevent SARS-CoV-2 infection and transmission, a possibly critical reason was the lack of protective mucosal immunity in respiratory tract. Here, we evaluated the effects of mucosal and systemic immunity from a novel simian adenovirus vectored COVID-19 vaccine (Sad23L- nCoV-S) in mice in comparison with Ad5-nCoV-S by intranasal (IN) drip and intramuscular (IM) injection vaccinations. As good as the well-known Ad5-nCoV-S vaccine, a single-dose IN inoculation of 1\u00d7109 PFU Sad23L-nCoV-S vaccine induced a similar level of IgG S-binding antibody (S-BAb) and neutralizing antibody (NAb) and higher IgA in serum, while IN route raised significantly higher IgG and IgA S- BAb and NAb in bronchoalveolar lavage (BAL), and specific IFN-\u03b3 secreting T cell response in lung compared with IM route, but lower T cell response in spleen. By prime-boost vaccination regimens with different combination of IN and IM inoculations of Sad23L-nCoV-S vaccine, the IN involved vaccinations stimulated higher protective mucosal or local immunity in BAL and lung, while the IM involved immunizations induced higher systemic immunity in serum and spleen. A long-term sustained systemic and mucosal NAb and T cell immunity to SARS-CoV-2 was maintained at high levels over 32 weeks by prime-boost vaccination regimens with IN and IM routes. In conclusion, priming or boosting immunization with IN inoculation of Sad23L-nCoV-S vaccine could induced effective mucosal immunity and in combination of IM route could additionally achieve systemic immunity, which provided an important reference for vaccination regimens against respiratory virus infection. The essential goal of vaccination is to generate potent and long-term protection against diseases. Several factors including type of vector, delivery route, boosting regimen influence the outcome of prime-boost immunization approaches. The immunization regimen by constructing a novel simian adenovirus vectored COVID-19 vaccine and employing combination of intranasal and intramuscular inoculations, could elicit mucosal neutralizing antibodies against five mutant strains in the respiratory tract, and strong systemic immunity. Immune protection could last for more than 32 weeks. Vectored vaccine construction and immunization regimens have positively impacted respiratory disease prevention.","version":"1.1","doi":"10.1101/2023.04.17.537220","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458196","pub_date":"2023-4-18","title":"The beneficial role of Candida intermedia and Saccharomyces boulardii yeasts on the immune response of mice vaccinated with a SARS-CoV-2 experimental vaccine","abstract":"Non-Saccharomyces yeasts emerge as possible new probiotics with a beneficial effect equal to or greater than the reference probiotic yeast, Saccharomyces boulardii. In this work, we evaluated the immunomodulation effect caused by Candida intermedia in mice vaccinated with inactivated SARS-CoV-2. We conducted preliminary tests using murine macrophages (RAW 264.7) stimulated with viable and heat-killed yeast cells, culture supernatant, and DNA, using qPCR to detect the mRNA transcription. Next, mice were supplemented with C. intermedia before each dose of the SARS-CoV-2 vaccine, and then antibody production was measured by ELISA. The probiotic strain S. boulardii CNCM I-745 was used as a control. We also explored the differences in fecal microbiomes between the non-supplemented and supplemented groups. Live cells of C. intermedia increased the transcription of IL-4, IL-13, and STAT3 by macrophages RAW 264.7, while heat-killed cells up-regulated TNF\u03b1 and Bcl6, and the culture supernatant positively impacted TLR2 transcription. Concanavalin, zymosan, and lipopolysaccharide were used to stimulate splenocytes from C. intermedia-supplemented animals, which showed increased transcription of TNF\u03b1, IFN\u03b3, IL-4, Bcl6, and STAT3. Sera from these animals showed enhanced levels of anti-SARS-CoV-2 IgG, as well as IgG1 and IgM isotypes, and sIgA in fecal samples. The microbiome of the C. intermedia-supplemented group showed a higher abundance of Bacteroides spp. and Clostridium spp., impacting the Bacteroidetes/Firmicutes balance. We concluded that C. intermedia and S. boulardii could stimulate and impact the gene expression of cells important for innate immunity, influence the composition of the gastrointestinal microbiome, and primarily boost the humoral response after vaccination. The present work was carried out with the support of Conselho Nacional de Desenvolvimento Cient\u00edfico (CNPq, Brazil), grant number 150538/2021-9.","version":"1.2","doi":"10.1101/2021.08.30.458196","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.15.536998","pub_date":"2023-4-17","title":"Human airway ex vivo models: new tools to study the airway epithelial cell response to SARS-CoV-2 infection","abstract":"Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models) infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using publicly available and our own bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) at day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) also were altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins also was deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to understand the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.","version":"1.1","doi":"10.1101/2023.04.15.536998","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.14.533542","pub_date":"2023-4-17","title":"SARS-CoV-2 occurrence in white-tailed deer throughout their range in the conterminous United States","abstract":"Broad-scale data show SARS-CoV-2 occurrence in white-tailed deer throughout much of their range in the conterminous United States and reinforce findings of considerable SARS-CoV-2 infection and exposure. Results shed light on both current infections and prior exposure, with prevalence decreasing over time and seroprevalence increasing. White-tailed deer are infected with, and have been exposed to, SARS-CoV-2 throughout their range in the conterminous US.","version":"1.1","doi":"10.1101/2023.04.14.533542","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.15.537011","pub_date":"2023-4-17","title":"SARS-CoV-2 shifts transcription of host gene to increase Spike acylation and boost infectivity","abstract":"SARS-CoV-2 infection requires Spike protein mediating fusion between the viral and cellular membranes. The fusogenic activity of Spike requires its post-translational lipid modification by host S-acyltransferases, predominantly ZDHHC20. Previous observations indicate that SARS-CoV-2 infection augments the S-acylation of Spike when compared to transfection. Here, we find that SARS-CoV-2 infection triggers a change in the transcriptional start site of the zddhc20 gene, both in cells and in an in vivo infection model, resulting in a 67-amino\u2013acid-long N-terminally extended protein with 37-times higher Spike acylating activity, leading to enhanced viral infectivity. Furthermore, we observed the same induced transcriptional change in response to other challenges, such as chemically induced colitis, indicating that SARS-CoV-2 hijacks an existing cell damage response pathway to generate more infectious viruses.","version":"1.1","doi":"10.1101/2023.04.15.537011","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.12.534029","pub_date":"2023-4-15","title":"Analysis of SARS-CoV-2 Recombinant Lineages XBC and XBC.1 in the Philippines and Evidence for Delta-Omicron Co-infection as a Potential Origin","abstract":"We report the sequencing and analysis of 60 XBC and 114 XBC.1 SARS-CoV-2 lineages detected in the Philippines from August to September 2022, which are regarded as recombinant lineages of the BA.2 Omicron and B.1.617.2 Delta (21I Clade) variants. The sequences described here place the Philippines as the country with the earliest and highest number of XBC and XBC.1 cases within the included period. Majority of the detected cases were sampled from the adjacent Davao and Soccskargen regions in southern Philippines, but have also been observed at lower proportions in other regions of the country. Time-scaled phylogenetic analysis with global samples from GISAID reaffirms the supposed root of XBC-like cases from the Philippines. Furthermore, the apparent clustering of some foreign cases separate from those collected in the country suggests several occurrences of cross-border transmissions resulting in the spread of XBC-like lineages within and among those countries. The consensus mutation profile shows regions harboring mutations specific to either the Omicron BA.2 or Delta B.1.617.2 lineages, supporting the recombinant nature of XBC. Finally, alternative allele fraction pattern and intrahost mutation analysis revealed that a relatively early case of XBC collected in March 2022 is likely to be an active co-infection event. This suggests that co-infection of Omicron and Delta was already occurring in the Philippines early in 2022, facilitating the generation of recombinants that may have further evolved and gained additional mutations enabling its spread across certain local populations at a later time. More recently, various lineages of the SARS-CoV-2 virus, the causative agent COVID-19 pandemic, have been observed to form recombinant lineages, further expanding the ways by which the virus can evolve and adapt to human interventions. Therefore, a large part of biosurveillance efforts is dedicated to detecting and observing new lineages, including recombinants, for early and effective control. In this paper, we present an analysis of 174 XBC and XBC.1 cases detected in the Philippines between August and September of 2022 which contextualize these cases as some of the earliest reported cases of this hybrid lineage. We show that when compared to cases from other countries collected at a similar time, the earliest cases of the XBC lineage are from the Philippines. Additionally, when samples were reclassified following an update of Pangolin, a tool for assigning SARS-CoV-2 lineages to samples, we found two samples of interest reclassified as XBC pointing to a potential origin via co-infection events occurring as early as March of 2022.","version":"1.3","doi":"10.1101/2023.04.12.534029","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.13.536832","pub_date":"2023-4-14","title":"Determination of the factors responsible for host tropism of SARS-CoV-2-related bat coronaviruses","abstract":"Differences in host ACE2 genes may affect the host range of SARS-CoV-2-related coronaviruses (SC2r-CoVs) and further determine the tropism of host ACE2 for the infection receptor. However, the factor(s) responsible for determining the host tropism of SC2r-CoVs, which may in part be determined by the tropism of host ACE2 usage, remains unclear. Here, we use the pseudoviruses with the spike proteins of two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and the cells expressing ACE2 proteins of eight different Rhinolophus bat species, and show that these two spikes have different tropisms for Rhinolophus bat ACE2. Through structural analysis and cell culture experiments, we demonstrate that this tropism is determined by residue 493 of the spike and residues 31 and 35 of ACE2. Our results suggest that SC2r-CoVs exhibit differential ACE2 tropism, which may be driven by adaptation to different Rhinolophus bat ACE2 proteins.","version":"1.1","doi":"10.1101/2023.04.13.536832","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.14.536885","pub_date":"2023-4-14","title":"Signal-amplification for cell-free biosensors, an analog-to-digital converter","abstract":"Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and Sars-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficientl contained leakage, boosted the expression fold-change from OFF to ON, and decreased the detection limit of the switches by three to four orders of magnitude. Ultimately, the integrase circuit converted the analog switches\u2019 signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.","version":"1.1","doi":"10.1101/2023.04.14.536885","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.14.536864","pub_date":"2023-4-14","title":"The airborne transmission of viruses causes tight transmission bottlenecks","abstract":"The transmission bottleneck describes the number of viral particles that found an infection in a new host. Previous studies have used genome sequence data to suggest that transmission bottlenecks for influenza and SARS-CoV-2 involve few viral particles, but the general principles underlying these bottlenecks are not fully understood. Here we show that, across a broad range of circumstances, tight transmission bottlenecks arise as a consequence of the physical process underlying airborne viral transmission. We use a mathematical model to describe the process of infectious particles being emitted by an infected individual and inhaled by others nearby. The extent to which exposure to particles translates into infection is determined by an effective viral load, which is calculated as a function of the epidemiological parameter R0. Across multiple scenarios, including those present at a superspreading event, our model suggests that the great majority of transmission bottlenecks involve few viral particles, with a high proportion of infections being caused by a single viral particle. Our results provide a physical explanation for previous inferences of bottleneck size and predict that tight transmission bottlenecks prevail more generally in respiratory virus transmission.","version":"1.1","doi":"10.1101/2023.04.14.536864","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.13.535896","pub_date":"2023-4-13","title":"SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition","abstract":"Following SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic. The study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA. The frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups. The antibody responses to the spike protein, but not the RBD-specific B cell responses diverge between convalescent males and females, and those who develop PCC or not. Our findings suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens. Post-COVID Condition (PCC) is lingering illness that afflicts a significant proportion of COVID-19 patients from three months after clearing SARS-CoV-2 infection. Therapy for PCC is only palliative and the underlying disease mechanisms are unclear. The wide spectrum of PCC symptoms that can affect different organs and the detection of viral components in tissues distant from lungs raise the possibility that PCC may be associated with aberrant immune response due to presence of viral antigens. Therefore, we studied B cell and antibody responses to the spike and nucleoprotein antigens in PCC patients who cleared mild SARS-CoV-2 infection during the pre-vaccination COVID-19 pandemic period. We observed divergent patterns of immune reactivity to the spike protein in PCC males and females at different times post-infection, suggesting that the immune responses in PCC may also be influenced by sex-related factors.","version":"1.1","doi":"10.1101/2023.04.13.535896","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.12.536671","pub_date":"2023-4-13","title":"SARS-CoV-2 selectively induces the expression of unproductive splicing isoforms of interferon, class I MHC and splicing machinery genes","abstract":"Splicing is a highly conserved, intricate mechanism intimately linked to transcription elongation, serving as a pivotal regulator of gene expression. Alternative splicing may generate specific transcripts incapable of undergoing translation into proteins, designated as unproductive. A plethora of respiratory viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), strategically manipulate the host\u2019s splicing machinery to circumvent antiviral responses. During the infection, SARS-CoV-2 effectively suppresses interferon (IFN) expression, leading to B cell and CD8+ T cell leukopenia, while simultaneously increasing the presence of macrophages and neutrophils in patients with severe COVID-19. In this study, we integrated publicly available omics datasets to systematically analyze transcripts at the isoform level and delineate the nascent-peptide translatome landscapes of SARS-CoV-2-infected human cells. Our findings reveal a hitherto uncharacterized mechanism whereby SARS-CoV-2 infection induces the predominant expression of unproductive splicing isoforms in key IFN signaling genes, interferon-stimulated genes (ISGs), class I MHC genes, and splicing machinery genes, including IRF7, OAS3, HLA-B, and HNRNPH1. In stark contrast, cytokine and chemokine genes, such as IL6, CXCL8, and TNF, predominantly express productive (protein-coding) splicing isoforms in response to SARS-CoV-2 infection. We postulate that SARS-CoV-2 employs a previously unreported tactic of exploiting the host splicing machinery to bolster viral replication and subvert the immune response by selectively upregulating unproductive splicing isoforms from antigen presentation and antiviral response genes. Our study sheds new light on the molecular interplay between SARS-CoV-2 and the host immune system, offering a foundation for the development of novel therapeutic strategies to combat COVID-19.","version":"1.1","doi":"10.1101/2023.04.12.536671","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.12.536590","pub_date":"2023-4-13","title":"Evaluation of mRNA-LNP and adjuvanted protein SARS-CoV-2 vaccines in a maternal antibody mouse model","abstract":"Maternal antibodies (matAbs) protect against a myriad of pathogens early in life; however, these antibodies can also inhibit de novo immune responses against some vaccine platforms. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) matAbs are efficiently transferred during pregnancy and protect infants against subsequent SARS-CoV-2 infections. It is unknown if matAbs inhibit immune responses elicited by different types of SARS-CoV-2 vaccines. Here, we established a mouse model to determine if SARS-CoV-2 spike-specific matAbs inhibit immune responses elicited by recombinant protein and nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccines. We found that SARS-CoV-2 mRNA-LNP vaccines elicited robust de novo antibody responses in mouse pups in the presence of matAbs. Recombinant protein vaccines were also able to circumvent the inhibitory effects of matAbs when adjuvants were co-administered. While additional studies need to be completed in humans, our studies raise the possibility that mRNA-LNP-based and adjuvanted protein-based SARS-CoV-2 vaccines have the potential to be effective when delivered very early in life.","version":"1.1","doi":"10.1101/2023.04.12.536590","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.11.536467","pub_date":"2023-4-12","title":"An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease","abstract":"Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 C\u03b1 atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.","version":"1.1","doi":"10.1101/2023.04.11.536467","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.21.501023","pub_date":"2023-4-12","title":"Learning from pre-pandemic data to forecast viral escape","abstract":"Effective pandemic preparedness relies on anticipating viral mutations that are able to evade host immune responses in order to facilitate vaccine and therapeutic design. However, current strategies for viral evolution prediction are not available early in a pandemic \u2013 experimental approaches require host polyclonal antibodies to test against and existing computational methods draw heavily from current strain prevalence to make reliable predictions of variants of concern. To address this, we developed EVEscape, a generalizable, modular framework that combines fitness predictions from a deep learning model of historical sequences with biophysical structural information. EVEscape quantifies the viral escape potential of mutations at scale and has the advantage of being applicable before surveillance sequencing, experimental scans, or 3D structures of antibody complexes are available. We demonstrate that EVEscape, trained on sequences available prior to 2020, is as accurate as high-throughput experimental scans at anticipating pandemic variation for SARS-CoV-2 and is generalizable to other viruses including Influenza, HIV, and understudied viruses with pandemic potential such as Lassa and Nipah. We provide continually updated escape scores for all current strains of SARS-CoV-2 and predict likely additional mutations to forecast emerging strains as a tool for ongoing vaccine development (evescape.org).","version":"1.2","doi":"10.1101/2022.07.21.501023","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.18.512756","pub_date":"2023-4-11","title":"Endonuclease fingerprint indicates a synthetic origin of SARS-CoV-2","abstract":"To prevent future pandemics, it is important that we understand whether SARS-CoV-2 spilled over directly from animals to people, or indirectly in a laboratory accident. The genome of SARS-COV-2 contains a peculiar pattern of unique restriction endonuclease recognition sites allowing efficient dis- and re-assembly of the viral genome characteristic of synthetic viruses. Here, we report the likelihood of observing such a pattern in coronaviruses with no history of bioengineering. We find that SARS-CoV-2 is an anomaly, more likely a product of synthetic genome assembly than natural evolution. The restriction map of SARS-CoV-2 is consistent with many previously reported synthetic coronavirus genomes, meets all the criteria required for an efficient reverse genetic system, differs from closest relatives by a significantly higher rate of synonymous mutations in these synthetic-looking recognitions sites, and has a synthetic fingerprint unlikely to have evolved from its close relatives. We report a high likelihood that SARS-CoV-2 may have originated as an infectious clone assembled in vitro. To construct synthetic variants of natural coronaviruses in the lab, researchers often use a method called in vitro genome assembly. This method utilizes special enzymes called restriction enzymes to generate DNA building blocks that then can be \u201cstitched\u201d together in the correct order of the viral genome. To make a virus in the lab, researchers usually engineer the viral genome to add and remove stitching sites, called restriction sites. The ways researchers modify these sites can serve as fingerprints of in vitro genome assembly. We found that SARS-CoV has the restriction site fingerprint that is typical for synthetic viruses. The synthetic fingerprint of SARS-CoV-2 is anomalous in wild coronaviruses, and common in lab-assembled viruses. The type of mutations (synonymous or silent mutations) that differentiate the restriction sites in SARS-CoV-2 are characteristic of engineering, and the concentration of these silent mutations in the restriction sites is extremely unlikely to have arisen by random evolution. Both the restriction site fingerprint and the pattern of mutations generating them are extremely unlikely in wild coronaviruses and nearly universal in synthetic viruses. Our findings strongly suggest a synthetic origin of SARS-CoV2.","version":"1.2","doi":"10.1101/2022.10.18.512756","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.08.536123","pub_date":"2023-4-10","title":"Antibodies that neutralize all current SARS-CoV-2 variants of concern by conformational locking","abstract":"SARS-CoV-2 continues to evolve and evade most existing neutralizing antibodies, including all clinically authorized antibodies. We have isolated and characterized two human monoclonal antibodies, 12-16 and 12-19, which exhibited neutralizing activities against all SARS-CoV-2 variants tested, including BQ.1.1 and XBB.1.5. They also blocked infection in hamsters challenged with Omicron BA.1 intranasally. Structural analyses revealed both antibodies targeted a conserved quaternary epitope located at the interface between the N-terminal domain and subdomain 1, revealing a previously unrecognized site of vulnerability on SARS-CoV-2 spike. These antibodies prevent viral receptor engagement by locking the receptor-binding domain of spike in the down conformation, revealing a novel mechanism of virus neutralization for non-RBD antibodies. Deep mutational scanning showed that SARS-CoV-2 could mutate to escape 12-19, but the responsible mutations are rarely found in circulating viruses. Antibodies 12-16 and 12-19 hold promise as prophylactic agents for immunocompromised persons who do not respond robustly to COVID-19 vaccines.","version":"1.1","doi":"10.1101/2023.04.08.536123","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.07.536037","pub_date":"2023-4-10","title":"SARS-CoV-2\u2019s evolutionary capacity is mostly driven by host antiviral molecules","abstract":"The COVID-19 pandemic has been characterised by sequential variant-specific waves shaped by viral, individual human and population factors. SARS-CoV-2 variants are defined by their unique combinations of mutations and there has been a clear adaptation to human infection since its emergence in 2019. Here we use machine learning models to identify shared signatures, i.e., common underlying mutational processes, and link these to the subset of mutations that define the variants of concern (VOCs). First, we examined the global SARS-CoV-2 genomes and associated metadata to determine how viral properties and public health measures have influenced the magnitude of waves, as measured by the number of infection cases, in different geographic locations using regression models. This analysis showed that, as expected, both public health measures and not virus properties alone are associated with the rise and fall of regional SARS-CoV-2 reported infection numbers. This impact varies geographically. We attribute this to intrinsic differences such as vaccine coverage, testing and sequencing capacity, and the effectiveness of government stringency. In terms of underlying evolutionary change, we used non-negative matrix factorisation to observe three distinct mutational signatures, unique in their substitution patterns and exposures from the SARS-CoV-2 genomes. Signatures 0, 1 and 3 were biased to C\u2192T, T\u2192C/A\u2192G and G\u2192T point mutations as would be expected of host antiviral molecules APOBEC, ADAR and ROS effects, respectively. We also observe a shift amidst the pandemic in relative mutational signature activity from predominantly APOBEC-like changes to an increasingly high proportion of changes consistent with ADAR editing. This could represent changes in how the virus and the host immune response interact, and indicates how SARS-CoV-2 may continue to accumulate mutations in the future. Linkage of the detected mutational signatures to the VOC defining amino acids substitutions indicates the majority of SARS-CoV-2\u2019s evolutionary capacity is likely to be associated with the action of host antiviral molecules rather than virus replication errors.","version":"1.1","doi":"10.1101/2023.04.07.536037","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.04.498645","pub_date":"2023-4-09","title":"Spatial and Temporal Origin of The Third SARS-Cov-2 Outbreak in Taiwan","abstract":"Since the first report of SARS-CoV-2 in December 2019, Taiwan has gone through three local outbreaks. Unlike the first two outbreaks, the spatial and temporal origin of the third outbreak (April 20 to November 5, 2021) is still unclear. We assembled and analyzed a data set of more than 6,000 SARS-CoV-2 genomes, including 300 from Taiwan and 5812 related sequences downloaded from GISAID as of 2021/12/08. We found that the third outbreak in Taiwan was caused by a single virus lineage belonging to Alpha (B.1.1.7) strain. This lineage, T-III (the third outbreak in Taiwan), carries a distinct genetic fingerprint, consisting of spike M1237I (S-M1237I) and three silent mutations, C5812T, C15895T, and T27869C. The T-III is closest to the sequences derived from Turkey on February 8, 2021. The estimated age of the most recent common ancestor (TMRCA) of T-III is March 23, 2021 (95% highest posterior density [HPD] February 24 - April 13, 2021), almost one month before the first three confirmed cases on April 20, 2021. The effective population size of the T-III showed approximately 20-fold increase after the onset of the outbreak and reached a plateau in early June 2021. Our results reconcile several unresolved observations, including the occurrence of two infection clusters at the same time without traceable connection and several airline pilots who were PCR negative but serum IgM-/IgG+ for SARS-CoV-2 in late April. Therefore, in contrast to the general notion that the third SARS-CoV-2 outbreak in Taiwan was sparked by two imported cases from USA on April 20, 2021, which, in turn, was caused by the partial relaxation of entry quarantine measures in early April 2021, our comprehensive analyses demonstrated that the outbreak was most likely originated from Europe in February 2021.","version":"1.3","doi":"10.1101/2022.07.04.498645","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.06.535883","pub_date":"2023-4-09","title":"Virological characteristics of the SARS-CoV-2 Omicron XBB.1.16 variant","abstract":"At the end of March 2023, XBB.1.16, a SARS-CoV-2 omicron XBB subvariant, emerged and was detected in various countries. Compared to XBB.1.5, XBB.1.16 has two substitutions in the S protein: E180V is in the N-terminal domain, and T478R in the receptor-binding domain (RBD). We first show that XBB.1.16 had an effective reproductive number (Re) that was 1.27- and 1.17-fold higher than the parental XBB.1 and XBB.1.5, respectively, suggesting that XBB.1.16 will spread worldwide in the near future. In fact, the WHO classified XBB.1.16 as a variant under monitoring on March 30, 2023. Neutralization assays demonstrated the robust resistance of XBB.1.16 to breakthrough infection sera of BA.2 (18-fold versus B.1.1) and BA.5 (37-fold versus B.1.1). We then used six clinically-available monoclonal antibodies and showed that only sotrovimab exhibits antiviral activity against XBB subvariants, including XBB.1.16. Our results suggest that, similar to XBB.1 and XBB.1.5, XBB.1.16 is robustly resistant to a variety of anti-SARS-CoV-2 antibodies. Our multiscale investigations suggest that XBB.1.16 that XBB.1.16 has a greater growth advantage in the human population compared to XBB.1 and XBB.1.5, while the ability of XBB.1.16 to exhibit profound immune evasion is comparable to XBB.1 and XBB.1.5. The increased fitness of XBB.1.16 may be due to (1) different antigenicity than XBB.1.5; and/or (2) the mutations in the non-S viral protein(s) that may contribute to increased viral growth efficiency.","version":"1.3","doi":"10.1101/2023.04.06.535883","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.25.061499","pub_date":"2023-4-07","title":"A rapid, low-cost, and highly sensitive SARS-CoV-2 diagnostic based on whole-genome sequencing","abstract":"Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosing and studying SARS-CoV-2 infection. Dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), this method amplifies the entirety of the SARS-CoV-2 genome. This contrasts with typical RT-PCR-based diagnostic tests, which amplify only a few loci. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. Using this method, we can reliably (>95% accuracy) detect SARS-CoV-2 at a concentration of 84 genome equivalents per milliliter (GE/mL). Almost all diagnostic methods currently authorized for use by the United States Food and Drug Administration with the Coronavirus Disease 2019 (COVID-19) Emergency Use Authorization require larger concentrations of the virus to achieve this degree of accuracy. In addition, we can reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. The genotypic data contained in these genome assemblies enable the more effective analysis of disease spread than is possible with an ordinary binary diagnostic. These data can also help identify vaccine and drug targets. Finally, we show that the diagnoses obtained using POLAR of both positive and negative clinical nasopharyngeal swab samples 100% match the diagnoses obtained in a clinical diagnostic lab using the Center for Disease Control\u2019s 2019-Novel Coronavirus test. Using POLAR, a single person can manually process 192 samples over an 8- hour experiment at the cost of \u223c$36 per patient (as of December 7th, 2022), enabling a 24-hour turnaround with sequencing and data analysis time. We anticipate that further testing and refinement will allow greater sensitivity in this approach.","version":"1.4","doi":"10.1101/2020.04.25.061499","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.07.535766","pub_date":"2023-4-07","title":"Silymarin Inhibits In Vitro SARS-CoV-2 Infection In Vero E6 Cells","abstract":"The study evaluated the invitro ability of Silymarin to inhibit SARS-COV-2 infection on Vero Cells. We set out to evaluate the hypothesis that Silymarin has both preventive and curative against SARS-COV-2. To study this, we first evaluated the safety profile of Silymarin using the Drosophila melanogaster(Harwich strain) model. Silymarin tablet film coated 140mg(Silybon-140) was used for the study. We evaluated the fly for acute toxicity, Locomotor performance, estimation of total thiol level, determination of Acetylcholinesterases (AchE) activity, Catalase activity, Glutathion-S-transferase(GST) activity and fecundity assay. To evaluate the invitro activity of Silymarin against SARS-COV-2, SARS-COV-2 isolates from oropharyngeal swabs and confirmed using qRT-PCR were cultured in Vero E6 monolayer cells. Different concentrations of silymarin concentration were used to determine pre- or post-exposure activity. The result showed that daily exposure to silymarin dose between 50% to 2000% adult dose showed no adverse effect after 28 days. Treatment of Vero cells with silymarin at the concentration 250-500ug/ml all revealed a pre-treatment effect to SARS-COV-2 in vitro and no inhibition effect was observed when the virus was first added before the addition of Silymarin. Silymarin had no adverse effect on D. melanogaster and can be used a preventive drug against SARS-COV-2","version":"1.1","doi":"10.1101/2023.04.07.535766","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.13.499346","pub_date":"2023-4-07","title":"The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein","abstract":"Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections, as well as enteric and neurological diseases. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 \u03bcg/ml and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.","version":"1.2","doi":"10.1101/2022.07.13.499346","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.06.535927","pub_date":"2023-4-06","title":"SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in mice","abstract":"Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (\u0394Mac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV \u0394Mac1 BACs, SARS-CoV-2 \u0394Mac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 \u0394Mac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, \u0394Mac1 was quickly cleared causing minimal pathology without any morbidity. \u0394Mac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. \u0394Mac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target. All CoVs, including SARS-CoV-2, encode for a conserved macrodomain (Mac1) that counters host ADP-ribosylation. Prior studies with SARS-CoV-1 and MHV found that Mac1 blocks IFN production and promotes CoV pathogenesis, which has prompted the development of SARS-CoV-2 Mac1 inhibitors. However, development of these compounds into antivirals requires that we understand how SARS-CoV-2 lacking Mac1 replicates and causes disease in vitro and in vivo. Here we found that SARS-CoV-2 containing a complete Mac1 deletion replicates normally in cell culture but induces an elevated IFN response, has reduced viral loads in vivo, and does not cause significant disease in mice. These results will provide a roadmap for testing Mac1 inhibitors, help identify Mac1 functions, and open additional avenues for coronavirus therapies.","version":"1.1","doi":"10.1101/2023.04.06.535927","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.05.535806","pub_date":"2023-4-06","title":"A new tractable method for generating Human Alveolar Macrophage Like cells in vitro to study lung inflammatory processes and diseases","abstract":"Alveolar macrophages (AMs) are unique lung resident cells that contact airborne pathogens and environmental particulates. The contribution of human AMs (HAM) to pulmonary diseases remains poorly understood due to difficulty in accessing them from human donors and their rapid phenotypic change during in vitro culture. Thus, there remains an unmet need for cost-effective methods for generating and/or differentiating primary cells into a HAM phenotype, particularly important for translational and clinical studies. We developed cell culture conditions that mimic the lung alveolar environment in humans using lung lipids, i.e., Infasurf (calfactant, natural bovine surfactant) and lung-associated cytokines (GM-CSF, TGF-\u03b2, and IL-10) that facilitate the conversion of blood-obtained monocytes to an AM-Like (AML) phenotype and function in tissue culture. Similar to HAM, AML cells are particularly susceptible to both Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. This study reveals the importance of alveolar space components in the development and maintenance of HAM phenotype and function, and provides a readily accessible model to study HAM in infectious and inflammatory disease processes, as well as therapies and vaccines. Millions die annually from respiratory disorders. Lower respiratory track gas-exchanging alveoli maintain a precarious balance between fighting invaders and minimizing tissue damage. Key players herein are resident AMs. However, there are no easily accessible in vitro models of HAMs, presenting a huge scientific challenge. Here we present a novel model for generating AML cells based on differentiating blood monocytes in a defined lung component cocktail. This model is non-invasive, significantly less costly than performing a bronchoalveolar lavage, yields more AML cells than HAMs per donor and retains their phenotype in culture. We have applied this model to early studies of M. tuberculosis and SARS-CoV-2. This model will significantly advance respiratory biology research.","version":"1.1","doi":"10.1101/2023.04.05.535806","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.06.535837","pub_date":"2023-4-06","title":"Insights into the ISG15 transfer cascade by the UBE1L activating enzyme","abstract":"The attachment of the ubiquitin-like protein ISG15 to substrates is a well-established antiviral signalling mechanism of the innate immune response. However, despite the identification of thousands of substrates and clear roles in antiviral immunity, a molecular understanding of ISG15 selection and transfer through its cognate E1-E2- E3 enzyme cascade is largely unknown. Here, we present a 3.45 \u00c5 cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L. Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validated the structure and confirmed the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enabled the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study provides much needed insight into the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.","version":"1.1","doi":"10.1101/2023.04.06.535837","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.04.535604","pub_date":"2023-4-05","title":"SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-19","abstract":"Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus\u2019s acute and potential chronic impact on the central nervous system. In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spike\u2019s persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities. The accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis presents potential molecular mechanisms and therapeutic targets for neurological complications in long-COVID-19 patients.","version":"1.1","doi":"10.1101/2023.04.04.535604","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.05.531513","pub_date":"2023-4-05","title":"The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection","abstract":"Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.","version":"1.1","doi":"10.1101/2023.04.05.531513","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.05.535700","pub_date":"2023-4-05","title":"AI-Guided Discovery of Novel SARS-CoV-2 PLpro Inhibitors: Accelerating Antiviral Drug Development in the Fight Against COVID-19","abstract":"The global pandemic caused by SARS-CoV-2 has highlighted the urgent need for effective antiviral drugs. The papain-like protease (PLpro) is a key viral enzyme involved in the replication and immune evasion of SARS-CoV-2, making it a promising target for antiviral drug development. In this study, we employed an artificial intelligence (AI)-driven drug discovery platform, LIME, to generate novel inhibitors of the SARS-CoV-2 PLpro. LIME is based on generative language models that can generate diverse, valid, and synthetically accessible compounds. The LIME software was used to identify potential inhibitors with strong binding affinity and specificity to the target protein. The top 13 hit compounds were tested in vitro, and the top 5 inhibitors with strong binding affinity and specificity were selected for further analysis. A top candidate molecule, CSEMRS-1376, exhibited similar binding energies and structural similarities to the known SARS-CoV-2 PLpro inhibitor, XR8-89. Computational analysis of the absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles of the hit compounds and XR8-89 showed that both CSEMRS-1376 and XR8-89 demonstrated favorable ADMET profiles. Overall, the LIME software successfully identified several novel molecules, including CSEMRS-1376, with strong potential as a therapeutic agent against SARS-CoV-2. The study highlights the potential of AI-driven drug discovery platforms, such as LIME, to accelerate the drug development process and pave the way for more efficient and effective therapeutics.","version":"1.1","doi":"10.1101/2023.04.05.535700","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.05.535744","pub_date":"2023-4-05","title":"How Interactions During Viral-Viral Coinfection Can Shape Infection Kinetics","abstract":"Respiratory virus infections are a leading cause of disease worldwide with multiple viruses detected in 20-30% of cases and several viruses simultaneously circulating. Some infections with viral copathogens have been shown to result in reduced pathogenicity while other virus pairings can worsen disease. The mechanisms driving these dichotomous outcomes are likely variable and have only begun to be examined in the laboratory and clinic. To better understand viral-viral coinfections and predict potential mechanisms that result in distinct disease outcomes, we first systematically fit mathematical models to viral load data from ferrets infected with respiratory syncytial virus (RSV) followed by influenza A virus (IAV) after 3 days. The results suggested that IAV reduced the rate of RSV production while RSV reduced the rate of IAV infected cell clearance. We then explored the realm of possible dynamics for scenarios not examined experimentally, including different infection order, coinfection timing, interaction mechanisms, and viral pairings. IAV coinfection with rhinovirus (RV) or SARS-CoV-2 (CoV2) was examined by using human viral load data from single infections together with murine weight loss data from IAV-RV, RV-IAV, and IAV-CoV2 coinfections to guide the interpretation of the model results. Similar to the results with RSV-IAV coinfection, this analysis showed that the increased disease severity observed during murine IAV-RV or IAV-CoV2 coinfection was likely due to slower clearance of IAV infected cells by the other viruses. On the contrary, the improved outcome when IAV followed RV could be replicated when the rate of RV infected cell clearance was reduced by IAV. Simulating viral-viral coinfections in this way provides new insights about how viral-viral interactions can regulate disease severity during coinfection and yields testable hypotheses ripe for experimental evaluation.","version":"1.1","doi":"10.1101/2023.04.05.535744","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535424","pub_date":"2023-4-04","title":"The Functional RNA Identification (FRID) Pipeline: Identification of Potential Pseudoknot-Containing RNA Elements as Therapeutic Targets for SARS-CoV-2","abstract":"The COVID-19 pandemic persists despite the development of effective vaccines. As such, it remains crucial to identify new targets for antiviral therapies. The causative virus of COVID-19, SARS-CoV-2, is a positive-sense RNA virus with RNA structures that could serve as therapeutic targets. One such RNA with established function is the frameshift stimulatory element (FSE), which promotes programmed ribosomal frameshifting. To accelerate identification of additional functional RNA elements, we introduce a novel computational approach termed the Functional RNA Identification (FRID) pipeline. The guiding principle of our pipeline, which uses established component programs as well as customized component programs, is that functional RNA elements have conserved secondary and pseudoknot structures that facilitate function. To assess the presence and conservation of putative functional RNA elements in SARS-CoV-2, we compared over 6,000 SARS-CoV-2 genomic isolates. We identified 22 functional RNA elements from the SARS-CoV-2 genome, 14 of which have conserved pseudoknots and serve as potential targets for small molecule or antisense oligonucleotide therapeutics. The FRID pipeline is general and can be applied to identify pseudoknotted RNAs for targeted therapeutics in genomes or transcriptomes from any virus or organism.","version":"1.1","doi":"10.1101/2023.04.03.535424","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535004","pub_date":"2023-4-04","title":"The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 Gamma variant","abstract":"The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N-glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N-glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody binding. Glycan site-occupancy analyses revealed that the mutation resulted in N-glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.","version":"1.1","doi":"10.1101/2023.04.03.535004","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535504","pub_date":"2023-4-04","title":"Longitudinal study of humoral immunity against SARS-CoV-2 of health professionals in Brazil: the impact of booster dose and reinfection on antibody dynamics","abstract":"The pandemic caused by SARS-CoV-2 has had a major impact on health systems. Vaccines have been shown to be effective in improving the clinical outcome of COVID-19, but they are not able to fully prevent infection and reinfection, especially that caused by new variants. Here, we tracked for 450 days the humoral immune response and reinfection in 52 healthcare workers from Brazil. Infection and reinfection were confirmed by RT-qPCR, while IgM and IgG antibody levels were monitored by rapid test. Of the 52 participants, 19 (36%) got reinfected during the follow-up period, all presenting mild symptoms. For all participants, IgM levels dropped sharply, with over 47% of them becoming seronegative by the 60th day. For IgG, 90% of the participants became seropositive within the first 30 days of follow-up. IgG antibodies also dropped after this period reaching the lowest level on day 270 (68.5\u00b172.3, p<0.0001). Booster dose and reinfection increased the levels of both antibodies, with the interaction between them resulting in an increase in IgG levels of 130.3 units. Overall, our data indicate that acquired humoral immunity declines over time and suggests that IgM and IgG antibody levels are not associated with the prevention of reinfection. This prospective observational study monitored the kinetics of humoral response and the occurrence of reinfection in a population of healthcare workers (HCW) who got COVID-19 over a period of 450 days. During the study period, HCW was a prioritized in COVID-19 vaccination campaign, several SARS-CoV-2 variants of concern circulated in the country, and nineteen participants of the study got reinfected. So, we were able to investigate the duration of humoral response against COVID-19, the impact of vaccination boost and reinfection in the production of anti-SARS-CoV-2 antibodies, and the associating of this antibodies with protection against reinfection. These information are important to support health managers in defining COVID19 surveillance and control actions.","version":"1.1","doi":"10.1101/2023.04.03.535504","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.15.529513","pub_date":"2023-4-04","title":"Modelling the viral dynamics of the SARS-CoV-2 Delta and Omicron variants in different cell types","abstract":"We use viral kinetic models fitted to viral load data from in vitro studies to explain why the SARS-CoV-2 Omicron variant replicates faster than the Delta variant in nasal cells, but slower than Delta in lung cells, which could explain Omicron\u2019s higher transmission potential and lower severity. We find that in both nasal and lung cells, viral infectivity is higher for Omicron but the virus production rate is higher for Delta. However, the differences are unequal between cell types, and ultimately leads to the basic reproduction number and growth rate being higher for Omicron in nasal cells, and higher for Delta in lung cells. In nasal cells, Omicron alone can enter via a TMPRSS2-independent pathway, but it is primarily increased efficiency of TMPRSS2-dependent entry which accounts for Omicron\u2019s increased activity. This work paves the way for using within-host mathematical models to understand the transmission potential and severity of future variants.","version":"1.3","doi":"10.1101/2023.03.15.529513","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535447","pub_date":"2023-4-04","title":"Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 Spike ferritin nanoparticle vaccine antigen produced from two different cell lines","abstract":"The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of \u223c100%, \u223c40% or \u223c10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4\u00b0C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.","version":"1.1","doi":"10.1101/2023.04.03.535447","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.528026","pub_date":"2023-4-04","title":"GILEA: GAN Inversion-enabled latent eigenvalue analysis for phenome profiling and editing","abstract":"Modeling heterogeneous disease states by data-driven methods has great potential to advance biomedical research. However, a comprehensive analysis of phenotypic heterogeneity is often challenged by the complex nature of biomedical datasets and emerging imaging methodologies. Here, we propose a novel GAN Inversion-enabled Latent Eigenvalue Analysis (GILEA) framework and apply it to phenome profiling and editing. As key use cases for fluorescence and natural imaging, we demonstrate the power of GILEA using publicly available SARS-CoV-2 datasets stained with the multiplexed fluorescence cell-painting protocol as well as real-world medical images of common skin lesions captured by dermoscopy. The quantitative results of GILEA can be biologically supported by editing latent representations and simulating dynamic phenotype transitions between physiological and pathological states. In conclusion, GILEA represents a new and broadly applicable approach to the quantitative and interpretable analysis of biomedical image data. The GILEA code and video demos are publicly available at https://github.com/CTPLab/GILEA.","version":"1.3","doi":"10.1101/2023.02.10.528026","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535215","pub_date":"2023-4-03","title":"Human Surfactant Protein A Alleviates SARS-CoV-2 Infectivity in Human Lung Epithelial Cells","abstract":"SARS coronavirus 2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing lung epithelial cells through its spike (S) protein. The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collagen-containing C-type lectin, expressed by mucosal epithelial cells and mediates its antiviral activities by binding to viral glycoproteins. This study examined the mechanistic role of human SP-A in SARS-CoV-2 infectivity. The interactions between human SP-A and SARS-CoV-2 S protein and hACE2 receptor, and SP-A level in COVID-19 patients were assessed by ELISA. The effect of SP-A on SARS-CoV-2 infectivity was analyzed by infecting human lung epithelial cells (A549-ACE2) with pseudoviral particles and infectious SARS-CoV-2 (Delta variant) pre-incubated with SP-A. Virus binding, entry, and infectivity were assessed by RT-qPCR, immunoblotting, and plaque assay. The results showed that human SP-A can bind SARS-CoV-2 S protein/RBD and hACE2 in a dose-dependent manner (p<0.01). Human SP-A inhibited virus binding and entry, and reduce viral load in lung epithelial cells, evidenced by the dose-dependent decrease in viral RNA, nucleocapsid protein, and titer (p<0.01). Increased SP-A level was observed in the saliva of COVID-19 patients compared to healthy controls (p<0.05), but severe COVID-19 patients had relatively lower SP-A levels than moderate COVID-19 patients (p<0.05). Therefore, SP-A plays an important role in mucosal innate immunity against SARS-CoV-2 infectivity by directly binding to the S protein and inhibiting its infectivity in host cells. SP-A level in the saliva of COVID-19 patients might serve as a biomarker for COVID-19 severity.","version":"1.1","doi":"10.1101/2023.04.03.535215","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.534161","pub_date":"2023-4-03","title":"A broadly protective CHO cell expressed recombinant spike protein subunit vaccine (IMT-CVAX) against SARS-CoV-2","abstract":"Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S). The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response. In pseudovirus-based assays, IMT-CVAX\u2013 immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus. The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.","version":"1.1","doi":"10.1101/2023.04.03.534161","journal":"bioRxiv","score":null},{"id":"10.1101/2023.04.03.535401","pub_date":"2023-4-03","title":"nanoCLAMP potently neutralizes SARS-CoV-2 and protects K18-hACE2 mice from infection","abstract":"Intranasal treatments, combined with vaccination, has the potential to slow mutational evolution of virusues by reducing transmission and replication. Here we illustrate the development of a SARS-CoV-2 receptor binding domain (RBD) nanoCLAMP and demonstrate its potential as an intranasally administered therapeutic. A multi-epitope nanoCLAMP was made by fusing a pM affinity single-domain nanoCLAMP (P2710) to alternate epitope binding nanoCLAMP, P2609. The resulting multimerised nanoCLAMP P2712 had sub-pM affinity for the Wuhan and South African (B.1.351) RBD (KD < 1 pM), and decreasing affinity for the Delta (B.1.617.2) and Omicron (B.1.1.529) variants (86 pM and 19.7 nM, respectively). P2712 potently inhibited ACE2:RBD interaction, suggesting its utility as a therapeutic. With an IC50 = 0.4 \u00b1 0.1 nM obtained from neutralization experiments using pseudoviral particles as well as patient cultured SARS-CoV-2 samples, nanoCLAMP P2712 protected K18-hACE2 mice from SARS-CoV-2 infection, reduced viral loads in the lungs and brains, and reduced associated upregulation of inflammatory cytokines and chemokines. Together, our findings warrant further investigation into the development of nanoCLAMPs as effective intranasally delivered COVID19 therapeutics.","version":"1.1","doi":"10.1101/2023.04.03.535401","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.14.532590","pub_date":"2023-3-31","title":"Persistence of SARS-CoV-2 and its surrogate, bacteriophage Phi6, on surfaces and in water","abstract":"The COVID-19 pandemic has motivated research on the persistence of infectious SARS-CoV-2 in environmental reservoirs such as surfaces and water. Viral persistence data has been collected for SARS-CoV-2 and its surrogates, including bacteriophage Phi6. Despite its wide use, no side-by-side comparisons between Phi6 and SARS-CoV-2 exist. Here, we quantified the persistence of SARS-CoV-2 and Phi6 on surfaces (plastic and metal) and in water and evaluated the influence that the deposition solution has on viral persistence by using four commonly used deposition solutions: two culture media (DMEM and Tryptone Soya Broth (TSB)), Phosphate Buffered Saline (PBS), and human saliva. Phi6 remained infectious in water significantly longer than SARS-CoV-2, having a half-life of 27 hours as compared with 15 hours for SARS-CoV-2. The persistence of viruses on surfaces was significantly influenced by the virus used and the deposition solution, but not by the surface material. Phi6 remained infectious significantly longer than SARS-CoV-2 when the inoculation solution was culture media (DMEM, TSB) and saliva. Using culture media and saliva led to half-lives between 9 hours and 2 weeks for Phi6, as compared to 0.5 to 2 hours for SARS-CoV-2. Using PBS as a deposition solution led to half-lives shorter than 4 hours for both viruses on all surfaces. Our results showed that, although it has been frequently used as a surrogate for coronaviruses, bacteriophage Phi6 is not an adequate surrogate for studies quantifying SARS-CoV-2 persistence, as it over-estimates infectiousness. Additionally, our findings reveal the need of using adequate deposition solutions when evaluating viral persistence on surfaces.","version":"1.2","doi":"10.1101/2023.03.14.532590","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.30.534980","pub_date":"2023-3-31","title":"Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia","abstract":"The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression towards severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an \u201cearly\u201d inflammatory/immune signature preceding a \u201clate\u201d type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.","version":"1.1","doi":"10.1101/2023.03.30.534980","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.31.535057","pub_date":"2023-3-31","title":"miRNA binding pressure channels evolution of SARS-CoV-2 genomes","abstract":"In somatic cells, microRNAs (miRNAs) bind to the genomes of RNA viruses and influence their translation and replication. Here we demonstrate that a significant number of miRNA binding sites locate in the NSP4 region of the SARS-CoV-2 genome, and the intestinal human miRNAs exert evolutionary pressure on this region. Notably, in infected cells, NSP4 promotes the formation of double-membrane vesicles, which serve as the scaffolds for replication-transcriptional complexes and protect viral RNA from intracellular destruction. In three years of selection, the loss of many miRNA binding sites, in particular, those within the NSP4, has shaped the SARS-CoV-2 genomes to promote the descendants of the BA.2 variants as the dominant strains and define current momentum of the pandemics.","version":"1.1","doi":"10.1101/2023.03.31.535057","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.31.535072","pub_date":"2023-3-31","title":"Explicit Modelling of Antibody Levels for Infectious Disease Simulations in the Context of SARS-CoV-2","abstract":"Measurable levels of immunoglobulin G antibodies develop after infections with and vaccinations against SARS-CoV-2. These antibodies are temporarily dynamic; due to waning, antibody levels will drop below detection thresholds over time. As a result, epidemiological studies could underestimate population protection, given that antibodies are a marker for protective immunity. During the COVID-19 pandemic, multiple models predicting infection dynamics were used by policymakers to plan public health policies. Explicitly integrating antibody and waning effects into the models is crucial for reliable calculations of individual infection risk. However, only few approaches have been suggested that explicitly treat these effects. This paper presents a methodology that explicitly models antibody levels and the resulting protection against infection for individuals within an agent-based model. This approach can be integrated in general frameworks, allowing complex population studies with explicit antibody and waning effects. We demonstrate the usefulness of our model in two use cases.","version":"1.1","doi":"10.1101/2023.03.31.535072","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470748","pub_date":"2023-3-31","title":"Mechanistic investigation of SARS-CoV-2 Omicron variant spike mutants via full quantum mechanical modeling","abstract":"Ab initio quantum mechanical models can characterize and predict intermolecular binding, but only recently have models including more than a few hundred atoms gained traction. Here, we simulate \u223c13,000 atoms to predict and characterize binding of SARS-CoV-2 spike variants to the human receptor ACE2 (hACE2). We compare four spike variants in our analysis: Wuhan, Omicron, and two Omicron-based variants. To assess binding, we mechanistically characterize the energetic contribution of each amino acid involved, and predict the effect of select single point mutations. We validate our computational predictions experimentally by comparing binding efficacy of spike variants to cells expressing hACE2. We argue that this computational model, QM-CR, can identify mutations critical for intermolecular interactions and inform the engineering of high-specificity interactors. Ab initio modeling can predict the strength of SARS-CoV-2 variants\u2019 binding to human cell receptor.","version":"1.2","doi":"10.1101/2021.12.01.470748","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.28.534588","pub_date":"2023-3-31","title":"Real time analysis of SARS-CoV-2 induced cytolysis reveals distinct variant-specific replication profiles","abstract":"The continuous evolution of new SARS-CoV-2 variants with enhanced immune evasion capacity suggests the entire population is and will continue to be potentially vulnerable to infection despite pre-existing immunity. The ability of each new variant to evade host humoral immunity is the focus of intense research across the globe. Each variant may also harbor unique replication capabilities relevant for disease and transmission. Here we demonstrate the utility of a new approach to assessing viral replication kinetics using Real Time Cell Analysis (RTCA). Virus induced cell death is measured in real time by the detection of electrical impedance through cell monolayers. Using this system, we quantified replication kinetics of five clinically important viral variants; USA WA1/2020 (an A1 ancestral lineage isolate), Delta, and Omicron subvariants BA.1, BA.4, and BA.5. We identified multiple kinetic measures that proved useful in variant replication comparisons including time (in hours) to the maximum rate of cell death at each log10 viral dilution and the slope at the maximum rate of cell death. We found that WA1/2020 and Delta were the most rapid but in distinct ways. While WA1/2020 induced cell death most rapidly after inoculation, Delta was slightly slower to reach cell death, it appeared to kill cells faster once cytotoxic effects began. Interestingly, BA.1, showed substantially reduced replication kinetics relative to all other variants. Together, these data show that real time analysis of cell death is a robust method to assess replicative capacity of any given SARS-CoV-2 variant rapidly and quantitatively, which may be useful in assessment of newly emerging variants.","version":"1.2","doi":"10.1101/2023.03.28.534588","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.31.535059","pub_date":"2023-3-31","title":"Determinants of species-specific utilization of ACE2 by human and animal coronaviruses","abstract":"Utilization of human ACE2 allowed several bat coronaviruses (CoVs), including the causative agent of COVID-19, to infect humans either directly or via intermediate hosts. Here, we analyzed the ability of Spike proteins from 24 human or animal CoVs to use ACE2 receptors across nine reservoir, potential intermediate and human hosts. We show that overall SARS-CoV-2 Omicron variants evolved more efficient ACE2 usage but mutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats. Spikes from most CoVs showed species-specific differences in ACE2 usage, partly due to variations in ACE2 residues 31, 41 or 354. Mutation of T403R allowed the RaTG13 bat CoV Spike to use all ACE2 orthologs analysed for viral entry. Sera from COVID-19 vaccinated individuals neutralized the Spike proteins of a range of bat Sarbecoviruses. Our results define determinants of ACE2 receptor usage of diverse CoVs and suggest that COVID-19 vaccination may protect against future zoonoses of SARS-CoV-related bat viruses. Mutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats Variations in ACE2 residues 31, 41 or 354 affect utilization by coronavirus Spike proteins Residue R403 in the Spike protein of bat coronavirus allow broad and effective ACE2 usage Sera from COVID-19 vaccinated individuals neutralize Spike proteins of bat Sarbecoviruses","version":"1.1","doi":"10.1101/2023.03.31.535059","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.28.534602","pub_date":"2023-3-31","title":"Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes","abstract":"Development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. Current strategies for developing pan-coronavirus countermeasures have largely focused on the receptor binding domain (RBD) and S2 regions of the coronavirus Spike protein; it has been unclear whether the N-terminal domain (NTD) is a viable target for universal vaccines and broadly neutralizing antibodies (Abs). Additionally, many RBD-targeting Abs have proven susceptible to viral escape. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees using multiplexed panels of uniquely barcoded antigens in a high-throughput single cell workflow to isolate over 9,000 SARS-CoV-2-specific monoclonal Abs (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. We observed many instances of clonal coalescence between individuals, suggesting that Ab responses frequently converge independently on similar genetic solutions. Among the recovered antibodies was TXG-0078, a public neutralizing mAb that binds the NTD supersite region of the coronavirus Spike protein and recognizes a diverse collection of alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy chain-dominant binding pattern seen in other NTD supersite-specific neutralizing Abs with much narrower specificity. We also report the discovery of CC24.2, a pan-sarbecovirus neutralizing mAb that targets a novel RBD epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 provides protection against in vivo challenge with SARS-CoV-2, suggesting potential future use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.","version":"1.2","doi":"10.1101/2023.03.28.534602","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.29.534854","pub_date":"2023-3-30","title":"Post-infection treatment with the E protein inhibitor BIT225 reduces disease severity and increases survival of k18-hACE2 transgenic mice infected with a lethal dose of SARS-CoV-2","abstract":"The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E-protein but did not inhibit endogenous calcium-induced ion channel activity or TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 h before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). When treatment started at 24 h after infection, body weight loss, and mortality were also prevented (100% survival, n=5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection. Antiviral agents are highly important for the management of COVID-19. We need new antivirals, because available drugs have major drawbacks that limit their use and are threatened by drug resistance. This study demonstrates that the small molecule drug BIT225 is an inhibitor of an important viral ion channel (E-protein). E-protein is required for virus replication and is involved in eliciting inflammatory response to infection. Exacerbated inflammation is a hallmark of severe COVID-19 in mice and in humans. In a mouse model of severe SARS-CoV-2 infection, BIT225 treatment starting before or 24 hours after infection could protect all treated mice from developing disease, from experiencing weight loss and from death (100%, n=17), while all untreated mice developed severe disease, started to lose body weight from day 3 onwards and died within 9 days after infection. BIT225 treatment was associated with potent suppression of virus load, and reduced inflammation markers, consistent with effective clearance of the virus. These results are remarkable for the exceptionally high efficacy achieved with a new mechanism of action. BIT225 is a clinical stage drug candidate with an established human safety profile. These results support clinical evaluation of BIT225 for the treatment of human SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.03.29.534854","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.25.513760","pub_date":"2023-3-30","title":"One for all \u2013 Human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses","abstract":"In vitro investigations of host-pathogen interactions of viruses are reliant on suitable cell and tissue culture models. Results are only as good as the model they have been generated in. However, choosing cell models for in vitro work often depends on what is available in labs or what has previously been used. Despite the vast increase in coronavirus research activity over the past few years, researchers are still heavily reliant on: non-human cells, for example Vero E6, highly heterogeneous or not fully differentiated cells, such as Calu-3, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors, such as ACE2 and TMPRSS2. Complex cell models, such as primary cell-derived air-liquid interface epithelial models are highly representative of human tissues but are expensive and time-consuming to develop and maintain. They have limited suitability for large-scale and high-throughput analysis. Using tissue-specific expression pattern as a selection criteria, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the highly characterized human kidney cell line Caki-1 for infection with three human coronaviruses: Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and Alphacoronavirus human coronavirus 229E (hCoV-229E). Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. Using a panel of 21 antibodies in infected Caki-1 cells using immunocytochemistry shows the location of viral proteins during replication. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison of not only a broad range of coronaviruses but respiratory viruses in general. Investigating how viruses interact with their host relies models used for laboratory research. The closer a model matches the host, the more conclusive results are. Complex cell systems based on primary epithelial or stem cells are the gold standard of in vitro research. However, they are expensive, time consuming, and laborious to establish. Therefore, cell lines remain the backbone of virus research. Despite vastly increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models, for example ells of non-human origin like the VeroE6 African Green Monkey cell line. Using known expression patterns of the entry factors of the COVID-19 causative agent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) we identified the Caki-1 cell line as a target for SARS-CoV-2. This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show that SARS-CoV-2 proteins can be stained for and localized in Caki-1 cells and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.","version":"1.2","doi":"10.1101/2022.10.25.513760","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.30.534872","pub_date":"2023-3-30","title":"Broad and potent neutralizing mAbs are elicited in vaccinated individuals following Delta/BA.1 breakthrough infection","abstract":"Despite the success of COVID-19 vaccines in preventing infection and/or severe disease, with the emergence of SARS-CoV-2 variants of concern (VOC) which encode mutations in Spike, and the waning of vaccine induced immunity, there has been an increase in SARS-CoV-2 infections in vaccinated individuals which leads to increased serum neutralization breadth. However, how exposure to a heterologous Spike broadens the neutralizing response at the monoclonal antibody (mAb) level is not fully understood. Through isolation of 119 mAbs from three individuals receiving two-doses of BNT162b2 vaccine before becoming delta or omicron/BA.1-infected, we show that breadth arises from re-activation and maturation of B cells generated through previous COVID-19 vaccination rather than a de novo response specific to the VOC Spike. Isolated mAbs frequently show reduced neutralization of current circulating variants including BA.2.75.2, XBB, XBB.1.5 and BQ.1.1 confirming continuous selective pressure on Spike to evolve and evade neutralization. However, isolation of mAbs that display effective cross-neutralization against all variants indicate the presence of conserved epitopes on RBD and a lesser extent NTD. These findings have implications for selection of Spike antigens for next-generation COVID-19 vaccines.","version":"1.1","doi":"10.1101/2023.03.30.534872","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.29.534838","pub_date":"2023-3-30","title":"Improving mRNA vaccine safety and efficiency with cationized lipid nanoparticle formula","abstract":"The widespread use of Covid-19 mRNA vaccines has highlighted the need to address rare but concerning side effects. Systemic off-target gene expression has been identified as a primary cause of acute adverse reactions and side effects associated with nucleoside-modified mRNA vaccines. In this study, we incorporated the permanent cationic lipid Dotap component into the mRNA-LNP formula associated with the FDA-approved mRNA vaccine Comirnaty to create a novel positively charged LNP carrier for mRNA vaccine delivery. Using the optimized LNP formula to prepare SARS-Cov-2 Spike mRNA vaccines for immunogenicity testing, Balb/c mice exhibited improved immunogenicity kinetics with initial antibody titers being lower but showing a continuous upward trend, ultimately reaching levels comparable to those of control mRNA vaccines 8 weeks after boost immunization. The mRNA vaccines encapsulated in the modified LNPs have demonstrated a superior safety profile in respect to systemic delivery of LNP constituents, off-target gene expression, and the systemic pro-inflammatory stimulation. Consequently, it may represent a safer alternative of conventional mRNA-LNP vaccines.","version":"1.1","doi":"10.1101/2023.03.29.534838","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.15.520606","pub_date":"2023-3-29","title":"Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection","abstract":"The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are novel in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1. SARS-CoV-2 infections can result in diverse clinical outcomes, including severe disease. Monoclonal antibodies (mAbs) have been used therapeutically to treat infection, but the emergence of variants has compromised their efficacy. Thus, identifying mAbs that are more durable in the face of SARS-CoV-2 evolution is a pressing need. Here, we describe four new mAbs isolated from a Delta-breakthrough infection, that can potently neutralize diverse variants, including multiple Omicron variants. In addition, one mAb shows broader activity against coronaviruses. The breadth of these mAbs is due to their focus on highly conserved regions of the viral protein antigen, including regions that are required for the virus to enter the cell. These properties make them promising candidates for therapeutic use.","version":"1.2","doi":"10.1101/2022.12.15.520606","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.26.533897","pub_date":"2023-3-27","title":"Evaluation of antiviral drugs against newly emerged SARS-CoV-2 Omicron subvariants","abstract":"The ongoing emergence of SARS-CoV-2 Omicron subvariants and their rapid worldwide spread pose a threat to public health. From November 2022 to February 2023, newly emerged Omicron subvariants, including BQ.1.1, BF.7, BA.5.2, XBB.1, XBB.1.5, and BN.1.9, became prevalent global strains (>5% global prevalence). These Omicron subvariants are resistant to several therapeutic antibodies. Thus, the antiviral activities of current drugs such as remdesivir, molnupiravir, and nirmatrelvir, which target highly conserved regions of SARS-CoV-2, against newly emerged Omicron subvariants need to be evaluated. We assessed the antiviral efficacy of the drugs using half maximal inhibitory concentration (IC50) against human isolated 23 Omicron subvariants and four former SARS-CoV-2 variants of concern (VOC) and compared them with the antiviral efficacy of these drugs against the SARS-CoV-2 reference strain (hCoV/Korea/KCDC03/2020). Maximal IC50 fold changes of remdesivir, molnupiravir, and nirmatrelvir were 1.9- (BA.2.75.2), 1.2-(B.1.627.2), and 1.4-fold (BA.2.3), respectively, compared to median IC50 values of the reference strain. Moreover, median IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir against the Omicron variants were 0.96, 0.4, and 0.62, similar to 1.02, 0.88, and 0.67, respectively, of median IC50-fold changes for previous VOC. Although K90R and P132H in Nsp 5, and P323L, A529V, G671S, V405F, and ins823D in Nsp 12 mutations were identified, these amino acid substitutions did not affect drug antiviral activity. Altogether, these results indicated that the current antivirals retain antiviral efficacy against newly emerged Omicron subvariants, and provide comprehensive information on the antiviral efficacy of these drugs.","version":"1.1","doi":"10.1101/2023.03.26.533897","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.27.534381","pub_date":"2023-3-27","title":"SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling","abstract":"SARS-CoV-2, the cause of the COVID19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. Transcriptomic analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease. Functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID19 evidenced altered gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID19 disease. Viral diseases, COVID19 insights","version":"1.1","doi":"10.1101/2023.03.27.534381","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.25.534209","pub_date":"2023-3-27","title":"AI-Designed, Mutation-Resistant Broad Neutralizing Antibodies Against Multiple SARS-CoV-2 Strains","abstract":"In this study, we generated a Digital Twin for SARS-CoV-2 by integrating data and meta-data with multiple data types and processing strategies, including machine learning, natural language processing, protein structural modeling, and protein sequence language modeling. This approach enabled the computational design of broadly neutralizing antibodies against over 1300 different historical strains of SARS-COV-2 containing 64 mutations in the receptor binding domain (RBD) region. The AI-designed antibodies were experimentally validated in real-virus neutralization assays against multiple strains including the newer Omicron strains that were not included in the initial design base. Many of these antibodies demonstrate strong binding capability in ELISA assays against the RBD of multiple strains. These results could help shape future therapeutic design for existing strains, as well as predicting hidden patterns in viral evolution that can be learned by AI for developing future antiviral treatments.","version":"1.1","doi":"10.1101/2023.03.25.534209","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.27.23287795","pub_date":"2023-03-27","title":"Effect of an enhanced public health contact tracing intervention on the secondary transmission of SARS-CoV-2 in educational settings: the four-way decomposition analysis","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>We assessed the impact of testing contacts immediately instead of at the end of quarantine on the transmission of SARS-CoV-2 in schools in Reggio Emilia Province.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We analysed surveillance data on notification of COVID-19 cases in schools between 1 September 2020 and 4 April 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Median tracing delay decreased from 7 to 3.1 days and the percentage of the known infection source increased from 34% to 54.8% (IRR 1.61 1.40-1.86). Implementation of prompt contact tracing was associated with a 10% decrease in the number of secondary cases (excess relative risk, EER -0.1 95%CI -0.35 to 0.15). Knowing the source of infection of the index case led to a decrease in secondary transmission (IRR 0.75 95% CI 0.63-0.91) while the decrease in tracing delay was associated with decreased risk of secondary cases (1/IRR 0.97 95%CI 0.94-1.01 per one day of delay). The direct effect of the intervention accounted for the 29% decrease in the number of secondary cases (EER -0.29 95% -0.61 to 0.03).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Prompt contact testing in the community seems to reduce the time of contact tracing and increases the ability to identify the source of infection in school outbreaks. Yet, observed differences can be also due to differences in the force of infection and to other control measures put in place.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This project was carried out with the technical and financial support of the Italian Ministry of Health \u2013 CCM 2020 and Ricerca Corrente Annual Program 2023.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.03.27.23287795","journal":"medRxiv","score":null},{"id":"10.1101/2022.10.04.510658","pub_date":"2023-3-24","title":"A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8","abstract":"Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein in this context.","version":"1.3","doi":"10.1101/2022.10.04.510658","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.24.534062","pub_date":"2023-3-24","title":"Intra-Host Mutation Rate of Acute SARS-CoV-2 Infection During the Initial Pandemic Wave","abstract":"Our understanding of SARS-CoV-2 evolution and mutation rate is limited. The rate of SARS-CoV-2 evolution is minimized through a proofreading function encoded by NSP-14 and may be affected by patient comorbidity. Current understanding of SARS-CoV-2 mutational rate is through population based analysis while intra-host mutation rate remains poorly studied. Viral genome analysis was performed between paired samples and mutations quantified at allele frequencies (AF) \u22650.25, \u22650.5 and \u22650.75. Mutation rate was determined employing F81 and JC69 evolution models and compared between isolates with (\u0394NSP-14) and without (wtNSP-14) non-synonymous mutations in NSP-14 and by patient comorbidity. Forty paired samples with median interval of 13 days [IQR 8.5-20] were analyzed. The estimated mutation rate by F81 modeling was 93.6 (95%CI:90.8-96.4], 40.7 (95%CI:38.9-42.6) and 34.7 (95%CI:33.0-36.4) substitutions/genome/year at AF \u22650.25, \u22650.5, \u22650.75 respectively. Mutation rate in \u0394NSP-14 were significantly elevated at AF>0.25 vs wtNSP-14. Patients with immune comorbidities had higher mutation rate at all allele frequencies. Intra-host SARS-CoV-2 mutation rates are substantially higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated mutation rate at low AF. Immunosuppressed patients have elevated mutation rate at all AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.","version":"1.1","doi":"10.1101/2023.03.24.534062","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.22.533759","pub_date":"2023-3-24","title":"Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs","abstract":"Factors of the innate immune response to SARS-CoV-2 in the lungs are pivotal for the ability of the host to deal with the infection. In humans, excessive macrophage infiltration is associated with disease severity. Using 3D spatiotemporal analysis of optically cleared hamster lung slices in combination with virological, immunohistochemical and RNA sequence analyses, we visualized the spread of SARS-CoV-2 through the lungs and the rapid anti-viral response in infected lung epithelial cells, followed by a wave of monocyte-derived macrophage (MDM) infiltration and virus elimination from the tissue. These SARS-CoV-2 induced innate immune processes are closely related to the onset of necrotizing inflammatory and consecutive remodelling responses in the lungs, which manifests as extensive cell death, vascular damage, thrombosis, and cell proliferation. Here we show that MDM are directly linked to virus clearance, and appear in connection with tissue injury and blood vessel damage. Rapid initiation of prothrombotic factor upregulation, tissue repair and alveolar cell proliferation results in tissue remodelling, which is followed by fibrosis development despite a decrease in inflammatory and anti-viral activities. Thus, although the hamsters are able to resolve the infection following the MDM influx and repair lung tissue integrity, longer-term alterations of the lung tissues arise as a result of concurrent tissue damage and regeneration processes.","version":"1.2","doi":"10.1101/2023.03.22.533759","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.21.533720","pub_date":"2023-3-22","title":"BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine","abstract":"Despite medical interventions and several approved vaccines, the COVID-19 pandemic is continuing into its third year. Recent publications have explored single-dose intranasal (i.n.) adenovirus-based vaccines as an effective strategy for curbing SARS-CoV-2 in na\u00efve animal models. However, the effects of prior immunizations and infections have yet to be considered within these models. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization on a subsequent S-protein expressing i.n. Ad vaccination, termed Ad(Spike). We found that Ad(Spike) alone conferred long-term protection from severe SARS-CoV-2 pathology within a mouse model, yet it was unable to limit initial infection 6 months post-vaccination. While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) vaccination potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish had no effect on the ability of Ad(Spike) to generate and maintain humoral immunity, it promoted the generation of cytotoxic and Th1 responses over suppressive FoxP3+ TREG cells in the lungs of infected mice. These data demonstrate a novel vaccination strategy that may prove useful in limiting future viral pandemics by potentiating the long-term efficacy of next generation mucosal vaccines within the context of the safe and widely distributed BCG vaccine. BCG enhances anti-SARS-CoV-2 immunity and protection afforded by a novel adenovirus-vectored vaccine.","version":"1.1","doi":"10.1101/2023.03.21.533720","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.22.533805","pub_date":"2023-3-22","title":"Evolving antibody evasion and receptor affinity of the Omicron BA.2.75 sublineage of SARS-CoV-2","abstract":"SARS-CoV-2 Omicron BA.2.75 has diversified into multiple subvariants with additional spike mutations, and several are expanding in prevalence, particularly CH.1.1 and BN.1. Here, we investigated the viral receptor affinities and neutralization evasion properties of major BA.2.75 subvariants actively circulating in different regions worldwide. We found two distinct evolutionary pathways and three newly identified mutations that shaped the virological features of these subvariants. One phenotypic group exhibited a discernible decrease in viral receptor affinities, but a noteworthy increase in resistance to antibody neutralization, as exemplified by CH.1.1, which is apparently as resistant as XBB.1.5. In contrast, a second group demonstrated a substantial increase in viral receptor affinity but only a moderate increase in antibody evasion, as exemplified by BN.1. We also observed that all prevalent SARS-CoV-2 variants in the circulation presently, except for BN.1, exhibit profound levels of antibody evasion, suggesting this is the dominant determinant of virus transmissibility today.","version":"1.1","doi":"10.1101/2023.03.22.533805","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.21.533701","pub_date":"2023-3-22","title":"Balancing Functional Tradeoffs between Protein Stability and ACE2 Binding in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB Lineages : Dynamics-Based Network Models Reveal Epistatic Effects Modulating Compensatory Dynamic and Energetic Changes","abstract":"The evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity for ACE2, conformational plasticity, protein stability and allosteric modulation. In this study, we systematically characterize conformational dynamics, protein stability and binding affinities of the SARS-CoV-2 Spike Omicron complexes with the host receptor ACE2 for BA.2, BA.2.75, XBB.1 and XBB.1.5 variants. We combined multiscale molecular simulations and dynamic analysis of allosteric interactions together with the ensemble-based mutational scanning of the protein residues and network modeling of epistatic interactions. This multifaceted computational study characterized molecular mechanisms and identified energetic hotspots that can mediate the predicted increased stability and the enhanced binding affinity of the BA.2.75 and XBB.1.5 complexes. The results suggested a mechanism driven by the stability hotspots and a spatially localized group of the Omicron binding affinity centers, while allowing for functionally beneficial neutral Omicron mutations in other binding interface positions. A network-based community model for the analysis of non-additive epistatic contributions in the Omicron complexes is proposed revealing the key role of the binding hotspots R498 and Y501 in mediating community-based epistatic couplings with other Omicron sites and allowing for compensatory dynamics and binding energetic changes. The results also showed that mutations in the convergent evolutionary hotspot F486 can modulate not only local interactions but also rewire the global network of local communities in this region allowing the F486P mutation to restore both the stability and binding affinity of the XBB.1.5 variant which may explain the growth advantages over the XBB.1 variant. The results of this study are consistent with a broad range of functional studies rationalizing functional roles of the Omicron mutation sites that form a coordinated network of hotspots enabling balance of multiple fitness tradeoffs and shaping up a complex functional landscape of virus transmissibility.","version":"1.1","doi":"10.1101/2023.03.21.533701","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.20.533485","pub_date":"2023-3-21","title":"SARS-CoV-2 infection induces dopaminergic neuronal loss in midbrain organoids during short and prolonged cultures","abstract":"COVID-19 is mainly associated with respiratory symptoms, although several reports showed that SARS-CoV-2 affects the nervous system. We evaluated the effects of infection in prolonged culture of midbrain organoids, showing that the virus induces changes in gene expression, and fragmentation and loss of dopaminergic neurons. Our findings highlight the direct viral-induced damage to midbrain organoids indicating the relevance of assessing the neurological long-term evolution of COVID-19 patients.","version":"1.1","doi":"10.1101/2023.03.20.533485","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.21.533610","pub_date":"2023-3-21","title":"SARS-CoV-2 infection activates endogenous retroviruses of the LTR69 subfamily","abstract":"Accumulating evidence suggests that endogenous retroviruses (ERVs) play an important role in the host response to infection and the development of disease. By combining RNA- and ChIP-sequencing analyses with RT-qPCR, we show that SARS-CoV-2 infection induces the LTR69 subfamily of ERVs, both in vitro and in vivo. Using functional assays, we identified one SARS-CoV-2-activated LTR69 locus, termed Dup69, which exhibits enhancer activity and is responsive to the transcription factors IRF3 and p65/RelA. LTR69-Dup69 is located about 500 bp upstream of a long non-coding RNA gene (ENSG00000289418) and within the PTPRN2 gene encoding a diabetes-associated autoantigen. Both ENSG00000289418 and PTPRN2 showed a significant increase in expression upon SARS-CoV-2 infection. Thus, our study sheds light on the interplay of exogenous with endogenous viruses and helps to understand how ERVs regulate gene expression during infection.","version":"1.1","doi":"10.1101/2023.03.21.533610","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.20.533560","pub_date":"2023-3-21","title":"Nanograms of SARS-CoV-2 Spike Protein Delivered by Exosomes Induce Potent Neutralization of Both Delta and Omicron Variants","abstract":"Exosomes are emerging as potent and safe delivery carriers for use in vaccinology and therapeutics. A better vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to provide improved, broader, longer lasting neutralization of SARS-CoV-2, a more robust T cell response, enable widespread global usage, and further enhance the safety profile of vaccines given the likelihood of repeated booster vaccinations. Here, we use Capricor\u2019s StealthX\u2122 platform to engineer exosomes to express native SARS-CoV-2 spike Delta variant (STX-S) protein on the surface for the delivery of a protein-based vaccine for immunization against SARS-CoV-2 infection. The STX-S vaccine induced a strong immunization with the production of a potent humoral immune response as demonstrated by high levels of neutralizing antibody not only against the delta SARS-CoV-2 virus but also two Omicron variants (BA.1 and BA.5), providing broader protection than current mRNA vaccines. Additionally, both CD4+ and CD8+ T cell responses were increased significantly after treatment. Quantification of spike protein by ELISA showed that only nanograms of protein were needed to induce a potent immune response. This is a significantly lower dose than traditional recombinant protein vaccines with no adjuvant required, which makes the StealthX\u2122 exosome platform ideal for the development of multivalent vaccines with a better safety profile. Importantly, our exosome platform allows novel proteins, or variants in the case of SARS-CoV-2, to be engineered onto the surface of exosomes in a matter of weeks, comparable with mRNA vaccine technology, but without the cold storage requirements. The ability to utilize exosomes for cellular delivery of proteins, as demonstrated by STX-S, has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation at an extremely low dose resulting in a potent, broad antibody response.","version":"1.1","doi":"10.1101/2023.03.20.533560","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.21.533609","pub_date":"2023-3-21","title":"The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication","abstract":"Recent technical advances have significantly improved our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells, with a particular focus on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, notably SARS-CoV-2, revealing both similarities and differences between the RBP profiles of viral and cellular mRNAs. Herein, we comprehensively identified the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, which associates with the viral mRNAs, results in lower levels of viral mRNAs within infected cells, and a decreased yield of infectious viral particles. Overall, our results uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs, and point to a role of the viral polymerase in orchestrating the assembly of viral mRNPs.","version":"1.1","doi":"10.1101/2023.03.21.533609","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.18.533204","pub_date":"2023-3-20","title":"Mouse Adapted SARS-CoV-2 Model Induces \u201cLong-COVID\u201d Neuropathology in BALB/c Mice","abstract":"The novel coronavirus SARS-CoV-2 has caused significant global morbidity and mortality and continues to burden patients with persisting neurological dysfunction. COVID-19 survivors develop debilitating symptoms to include neuro-psychological dysfunction, termed \u201cLong COVID\u201d, which can cause significant reduction of quality of life. Despite vigorous model development, the possible cause of these symptoms and the underlying pathophysiology of this devastating disease remains elusive. Mouse adapted (MA10) SARS-CoV-2 is a novel mouse-based model of COVID-19 which simulates the clinical symptoms of respiratory distress associated with SARS-CoV-2 infection in mice. In this study, we evaluated the long-term effects of MA10 infection on brain pathology and neuroinflammation. 10-week and 1-year old female BALB/cAnNHsd mice were infected intranasally with 104 plaque-forming units (PFU) and 103 PFU of SARS-CoV-2 MA10, respectively, and the brain was examined 60 days post-infection (dpi). Immunohistochemical analysis showed a decrease in the neuronal nuclear protein NeuN and an increase in Iba-1 positive amoeboid microglia in the hippocampus after MA10 infection, indicating long-term neurological changes in a brain area which is critical for long-term memory consolidation and processing. Importantly, these changes were seen in 40-50% of infected mice, which correlates to prevalence of LC seen clinically. Our data shows for the first time that MA10 infection induces neuropathological outcomes several weeks after infection at similar rates of observed clinical prevalence of \u201cLong COVID\u201d. These observations strengthen the MA10 model as a viable model for study of the long-term effects of SARS-CoV-2 in humans. Establishing the viability of this model is a key step towards the rapid development of novel therapeutic strategies to ameliorate neuroinflammation and restore brain function in those suffering from the persistent cognitive dysfunction of \u201cLong-COVID\u201d.","version":"1.1","doi":"10.1101/2023.03.18.533204","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.18.533280","pub_date":"2023-3-20","title":"SARS-CoV-2 NSP12 associates with the TRiC complex and the P323L substitution is a host adaption","abstract":"SARS-CoV-2 emerged into the human population in late 2019 and human to human transmission has dominated the evolutionary landscape and driven the selection of different lineages. The first major change that resulted in increased transmission was the D614G substitution in the spike protein. This was accompanied by the P323L substitution in the viral RNA dependent RNA polymerase (RdRp) (NSP12). Together, with D614G these changes are the root of the predominant global SARS-CoV-2 landscape. Here, we found that NSP12 formed an interactome with cellular proteins. The functioning of NSP12 was dependent on the T-complex protein Ring Complex, a molecular chaperone. In contrast, there was differential association between NSP12 variants and components of a phosphatase complex (PP2/PP2A and STRN3). Virus expressing NSP12L323 was less sensitive to perturbations in PP2A and supports the paradigm that ongoing genotype to phenotype adaptation of SARS- CoV-2 in humans is not exclusively restricted to the spike protein.","version":"1.1","doi":"10.1101/2023.03.18.533280","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.02.494559","pub_date":"2023-3-20","title":"Zooanthroponotic transmission of SARS-CoV-2 and host-specific viral mutations revealed by genome-wide phylogenetic analysis","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including selection of adaptive mutations and emergence of new variant lineages. Here we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, \u223c\u2154 were plausibly introduced into these animal species from local human populations, while the remaining \u223c\u2153 were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health. SARS-CoV-2, the causative agent of COVID-19, can infect many animal species, making eradication difficult because it can be reseeded from different reservoirs. When viruses replicate in different species, they may be faced with different evolutionary pressures and acquire new mutations, with unknown consequences for transmission and virulence in humans. Here we analyzed SARS-CoV-2 genome sequences from cats, dogs, deer, and mink to estimate transmission between each of these species and humans. We found several transmission events from humans to each animal, but relatively few detectable transmissions from animals back to humans, with the exception of mink. We also identified three mutations more likely to be found in mink than humans, and 26 in deer. These mutations could help the virus adapt to life in these different species. Ongoing surveillance of SARS-CoV-2 from animals will be important to understand their potential impacts on both human and animal health.","version":"1.3","doi":"10.1101/2022.06.02.494559","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.19.533317","pub_date":"2023-3-20","title":"Analysis of spike protein variants evolved in a novel mouse model of persistent SARS-CoV-2 infection","abstract":"SARS-CoV-2 mutation rates have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants; however, there is currently no animal model to recapitulate persistent infection. We established a novel model of persistent infection using xenografts of Calu-3 human lung cancer cells in immunocompromised mice. After infection with wild-type SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three isolated viral clones with defined mutations produced higher virus titers than the wild-type virus in Calu-3 cells without cytotoxic effects. In K18-hACE2 mice, the variants were less lethal than the wild-type virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of wild-type S protein and provided protective immunity against subsequent challenge with wild-type virus. These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study persistent SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.03.19.533317","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.528025","pub_date":"2023-3-20","title":"Predicting changes in neutralizing antibody activity for SARS-CoV-2 XBB.1.5 using in silico protein modeling","abstract":"The SARS-CoV-2 variant XBB.1.5 is of concern as it has high transmissibility. XBB.1.5 currently accounts for upwards of 30% of new infections in the United States. One year after our group published the predicted structure of the Omicron (B.1.1.529) variant\u2019s receptor binding domain (RBD) and antibody binding affinity, we return to investigate the new mutations seen in XBB.1.5 which is a descendant of Omicron. Using in silico ico modeling approaches against newer neutralizing antibodies that are shown effective against B.1.1.529, we predict the immune consequences of XBB.1.5\u2019s mutations and show that there is no statistically significant difference in overall antibody evasion when comparing to the B.1.1.529 and other related variants (e.g., BJ.1 and BM.1.1.1). However, noticeable changes in antibody binding affinity were seen due to specific amino acid changes of interest in the newer variants.","version":"1.4","doi":"10.1101/2023.02.10.528025","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.04.522629","pub_date":"2023-3-20","title":"Activity of Sotrovimab against BQ.1.1 and XBB.1 Omicron sublineages in a hamster model","abstract":"The successive emergence of SARS-CoV-2 Omicron variants has completely changed the modalities of use of therapeutic monoclonal antibodies. Recent in vitro studies indicated that only Sotrovimab has maintained partial activity against BQ.1.1 and XBB.1. In the present study, we used the hamster model to determine whether Sotrovimab retains antiviral activity against these Omicron variants in vivo. Our results show that at exposures consistent with those observed in humans, Sotrovimab remains active against BQ.1.1 and XBB.1, although for BQ.1.1 the efficacy is lower than that observed against the first globally dominant Omicron sublineages BA.1 and BA.2.","version":"1.2","doi":"10.1101/2023.01.04.522629","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.17.533092","pub_date":"2023-3-19","title":"Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine","abstract":"COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.","version":"1.2","doi":"10.1101/2023.03.17.533092","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.17.533105","pub_date":"2023-3-17","title":"Magnipore: Prediction of differential single nucleotide changes in the Oxford Nanopore Technologies sequencing signal of SARS-CoV-2 samples","abstract":"Oxford Nanopore Technologies (ONT) allows direct sequencing of ribonucleic acids (RNA) and, in addition, detection of possible RNA modifications due to deviations from the expected ONT signal. The software available so far for this purpose can only detect a small number of modifications. Alternatively, two samples can be compared for different RNA modifications. We present Magnipore, a novel tool to search for significant signal shifts between samples of Oxford Nanopore data from similar or related species. Magnipore classifies them into mutations and potential modifications. We use Magnipore to compare SARS-CoV-2 samples. Included were representatives of the early 2020s Pango lineages (n=6), samples from Pango lineages B.1.1.7 (n=2, Alpha), B.1.617.2 (n=1, Delta), and B.1.529 (n=7, Omicron). Magnipore utilizes position-wise Gaussian distribution models and a comprehensible significance threshold to find differential signals. In the case of Alpha and Delta, Magnipore identifies 55 detected mutations and 15 sites that hint at differential modifications. We predicted potential virus-variant and variant-group-specific differential modifications. Magnipore contributes to advancing RNA modification analysis in the context of viruses and virus variants.","version":"1.1","doi":"10.1101/2023.03.17.533105","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.15.532878","pub_date":"2023-3-16","title":"The highly conserved stem-loop II motif is dispensable for SARS-CoV-2","abstract":"The stem-loop II motif (s2m) is a RNA structural element that is found in the 3\u2019 untranslated region (UTR) of many RNA viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over twenty-five years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro, or growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3\u2019 UTR of wild type and s2m deletion viruses using SHAPE-MaP and DMS-MaPseq. These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3\u2019UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2. RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contain functional structures to support virus replication, translation and evasion of the host antiviral immune response. The 3\u2019 untranslated region of early isolates of SARS-CoV-2 contained a stem-loop II motif (s2m), which is a RNA structural element that is found in many RNA viruses. This motif was discovered over twenty-five years ago, but its functional significance is unknown. We created SARS-CoV-2 with deletions or mutations of the s2m and determined the effect of these changes on viral growth in tissue culture and in rodent models of infection. Deletion or mutation of the s2m element had no effect on growth in vitro, or growth and viral fitness in Syrian hamsters in vivo. We also observed no impact of the deletion on other known RNA structures in the same region of the genome. These experiments demonstrate that the s2m is dispensable for SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.03.15.532878","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.15.532808","pub_date":"2023-3-16","title":"Tetravalent SARS-CoV-2 S1 Subunit Protein Vaccination Elicits Robust Humoral and Cellular Immune Responses in SIV-Infected Rhesus Macaque Controllers","abstract":"The COVID-19 pandemic has highlighted the need for safe and effective vaccines to be rapidly developed and distributed worldwide, especially considering the emergence of new SARS-CoV-2 variants. Protein subunit vaccines have emerged as a promising approach due to their proven safety record and ability to elicit robust immune responses. In this study, we evaluated the immunogenicity and efficacy of an adjuvanted tetravalent S1 subunit protein COVID-19 vaccine candidate composed of the Wuhan, B.1.1.7 variant, B.1.351 variant, and P.1 variant spike proteins in a nonhuman primate model with controlled SIVsab infection. The vaccine candidate induced both humoral and cellular immune responses, with T- and B cell responses mainly peaking post-boost immunization. The vaccine also elicited neutralizing and cross-reactive antibodies, ACE2 blocking antibodies, and T-cell responses, including spike specific CD4+ T cells. Importantly, the vaccine candidate was able to generate Omicron variant spike binding and ACE2 blocking antibodies without specifically vaccinating with Omicron, suggesting potential broad protection against emerging variants. The tetravalent composition of the vaccine candidate has significant implications for COVID-19 vaccine development and implementation, providing broad antibody responses against numerous SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.03.15.532808","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.15.532841","pub_date":"2023-3-16","title":"An alphacoronavirus polymerase structure reveals conserved co-factor functions","abstract":"Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryoelectron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs. Coronaviruses are important human and animal pathogens with a history of crossing over from animal reservoirs into humans leading to epidemics or pandemics. Betacoronaviruses, such as SARS-CoV and SARS-CoV-2, have been the focus of research efforts in the field of coronaviruses, leaving other genera (alpha, gamma, and delta) understudied. To broaden our understanding, we studied an alphacoronavirus polymerase complex. We solved the first structure of a non-betacoronavirus replication complex, and in doing so identified previously unknown, and conserved, aspects of polymerase cofactor interactions. Our work displays the importance of studying coronaviruses from all genera and provides important insight into coronavirus replication that can be used for antiviral drug development.","version":"1.1","doi":"10.1101/2023.03.15.532841","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.09.503414","pub_date":"2023-3-15","title":"Function and Cryo-EM structures of broadly potent bispecific antibodies against multiple SARS-CoV-2 Omicron sublineages","abstract":"The SARS-CoV-2 variant, Omicron (B.1.1.529), rapidly swept the world since its emergence. Compared with previous variants, Omicron has a high number of mutations, especially those in its spike glycoprotein that drastically dampen or abolish the efficacy of currently available vaccines and therapeutic antibodies. Several major sublineages of Omicron evolved, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75, which rapidly changing the global and regional landscape of the pandemic. Although vaccines are available, therapeutic antibodies remain critical for infected and especially hospitalized patients. To address this, we have designed and generated a panel of human/humanized therapeutic bispecific antibodies against Omicron and its sub-lineage variants, with activity spectrum against other lineages. Among these, the top clone CoV2-0213 has broadly potent activities against multiple SARS-CoV-2 ancestral and Omicron lineages, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75. We have solved the cryo-EM structure of the lead bi-specific antibody CoV-0213 and its major Fab arm MB.02. Three-dimensional structural analysis shows distinct epitope of antibody - spike receptor binding domain (RBD) interactions and reveals that both Fab fragments of CoV2-0213 can simultaneously target one single spike RBD or two adjacent ones in the same spike trimer, further corroborating its mechanism of action. CoV2-0213 represents a unique and potent broad-spectrum SARS-CoV-2 neutralizing bispecific antibody (nbsAb) against the currently circulating major Omicron variants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5). CoV2-0213 is primarily human and ready for translational testing as a countermeasure against the ever-evolving pathogen.","version":"1.2","doi":"10.1101/2022.08.09.503414","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.14.532609","pub_date":"2023-3-15","title":"Epithelial galectin-3 induces mitochondrial complex inhibition and cell cycle arrest of CD8+ T Cells in severe/critical ill COVID-19","abstract":"Several studies have identified the presence of functionally depleted CD8+ T cells in COVID-19 patients, and particularly abnormally reduced CD8+ T cells in severe/critical patients, which may be a major cause of disease progression and poor prognosis. In this study, a proliferating-depleted CD8+ T cell phenotype was observed in severe/critical COVID-19 patients through scRNA-seq and scTCR-seq analysis. These CD8+ T cells were subsequently found to be characterized by cell cycle arrest and downregulation of mitochondrial biogenesis and respiratory chain complex genes. Cellchat analysis revealed that the Galectin signaling pathways between infected lung epithelial cells and CD8+ T cells play the key role in inducing CD8+ T cell reduction and dysfunction in severe/critical COVID-19. We used SARS-COV-2 ORF3a to transfect A549 epithelial cells, and co-cultured with CD8+ T cells. The ex vivo experiments confirmed that galectin-3 inhibited the transcription of mitochondrial respiratory chain complex III/IV genes in CD8+ T cells by suppressing the nuclear translocation of nuclear respiratory factor 1 (NRF1). In addition, the regulatory effect of galectin-3 was correlated with the activation of ERK signaling and/or the inhibition of Akt signaling. Galectin-3 inhibitor, TD-139, promoted nuclear translocation of NRF1, and enhanced mitochondrial respiratory chain complex III/IV gene expression and mitochondrial biogenesis, then restore the expansion ability of CD8+ T cells. Our study improved the understanding the immunopathogenesis and provided new target for the prevention and treatment of severe/critical COVID-19.","version":"1.1","doi":"10.1101/2023.03.14.532609","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.12.532269","pub_date":"2023-3-15","title":"Urine proteomic characterization of active and recovered COVID-19 patients","abstract":"The molecular changes in COVID-19 patients have been reported in many studies. However, there were limited attention has been given to the disease sequelae in the recovered COVID-19 patients. Here, we profiled the urine proteome of a cohort of 29 COVID-19 patients in their disease onset and recovery period, including mild, severe, and fatal patients and survivors who recovered from mild or severe symptoms. The molecular changes in the COVID-19 onset period suggest that viral infections, immune response changes, multiple organ damage, cell injury, coagulation system changes and metabolic changes are associated with COVID-19 progression. The patients who recovered from COVID-19 still exhibited an innate immune response, coagulation system changes and central nervous system changes. We also proposed four potential biomarkers to monitor the whole progression period of COVID-19. Our findings provide valuable knowledge about the potential molecular pathological changes and biomarkers that can be used to monitor the whole period of COVID-19.","version":"1.2","doi":"10.1101/2023.03.12.532269","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.14.532352","pub_date":"2023-3-14","title":"Genomic surveillance reveals circulation of multiple variants and lineages of SARS-CoV-2 during COVID-19 pandemic in Indian city of Bengaluru","abstract":"Genomic surveillance in response to coronavirus disease (COVID-19) pandemic is crucial for tracking spread, identify variants of concern (VoCs) and understand the evolution of its etiological agent, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). India has experienced three waves of COVID-19 cases, which includes a deadly wave of COVID-19 that was driven by the Delta lineages (second/Delta wave) followed by another wave driven by the Omicron lineages (third/Omicron wave). These waves were particularly dramatic in the metropolitan cities due to high population density. We evaluated the prevalence, and mutational spectrum of SARS-CoV-2 variants/lineages in one such megapolis, Bengaluru city, across these three waves between October 2020 and June 2022. 15,134 SARS-CoV-2 samples were subjected to whole genome sequencing (WGS). Phylogenetic analysis revealed, SARS-CoV-2 variants in Bengaluru city belonged to 18 clades and 196 distinct lineages. As expected, the Delta lineages were the most dominant lineages during the second wave of COVID-19. The Omicron lineage BA.2 and its sublineages accounted for most of the COVID-19 cases in the third wave. Most number of amino acid changes were observed in spike protein. Among the 18 clades, majority of the mutations and least similarity at nucleotide sequence level with the reference genome were observed in Omicron clades.","version":"1.1","doi":"10.1101/2023.03.14.532352","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.14.532528","pub_date":"2023-3-14","title":"Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants","abstract":"In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two VHH-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S- 2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.","version":"1.1","doi":"10.1101/2023.03.14.532528","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.13.532347","pub_date":"2023-3-13","title":"Resolving the developmental mechanisms of coagulation abnormalities characteristic of SARS-CoV2 based on single-cell transcriptome analysis","abstract":"The COVID-19 outbreak caused by the SARS-CoV-2 virus has developed into a global health emergency. In addition to causing respiratory symptoms following SARS-CoV-2 infection, COVID-19-associated coagulopathy (CAC) is the main cause of death in patients with severe COVID-19. In this study, we performed single-cell sequencing analysis of the right ventricular free wall tissue from healthy donors, patients who died in the hypercoagulable phase of CAC, and patients in the fibrinolytic phase of CAC. Among these, we collected 61,187 cells, which were enriched in 24 immune cell subsets and 13 cardiac-resident cell subsets. We found that in response to SARS-CoV-2 infection, CD9highCCR2highmonocyte-derived m\u00f8 promoted hyperactivation of the immune system and initiated the extrinsic coagulation pathway by activating CXCR-GNB/G-PI3K-AKT. This sequence of events is the main process contributing the development of coagulation disorders subsequent to SARS-CoV-2 infection. In the characteristic coagulation disorder caused by SARS-CoV-2, excessive immune activation is accompanied by an increase in cellular iron content, which in turn promotes oxidative stress and intensifies intercellular competition. This induces cells to alter their metabolic environment, resulting in an increase in sugar uptake, such as that via the glycosaminoglycan synthesis pathway, in CAC coagulation disorders. In addition, high levels of reactive oxygen species generated in response elevated iron levels promote the activation of unsaturated fatty acid metabolic pathways in endothelial cell subgroups, including vascular endothelial cells. This in turn promotes the excessive production of the toxic peroxidation by-product malondialdehyde, which exacerbates both the damage caused to endothelial cells and coagulation disorders.","version":"1.1","doi":"10.1101/2023.03.13.532347","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.11.532204","pub_date":"2023-3-13","title":"Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals","abstract":"The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid (N) protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, a mAb-based bELISA was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition (PI) cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 6.95%, and 5.15%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with COVID-19-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.","version":"1.1","doi":"10.1101/2023.03.11.532204","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.11.532212","pub_date":"2023-3-13","title":"In vitro comparison of SARS-CoV-2 variants","abstract":"The Coronaviridae family hosts various coronaviruses responsible for many diseases, from the common cold, severe lung infections to pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic, and numerous basic and applied laboratory techniques were utilized in virus culture and examination of the disease. Understanding the replication kinetics and characterizing the virus\u2019 effect on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will give us a clear understanding of the characteristic differences in pathogenicity. In this study, we compared the cytopathic effect (CPE) and replication of Wild Type (WT), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on 5 different cell lines; VeroE6, VeroE6 expressing high endogenous ACE2, VeroE6 highly expressing human ACE2 (VeroE6/ACE2) and TMPRSS2 (VeroE6/hACE2/ TMPRSS2), Calu3 cells highly expressing human ACE2 and A549 cells. All 3 VeroE6 cell lines were susceptible to WT strain, where CPE and replication were observed. Along with being susceptible to Wild type, VeroE6/hACE2/TMPRSS2 cells were susceptible to both omicron and delta strains, whereas VeroE6/ACE2 cells were only susceptible to omicron in a dose-dependent manner. No CPE was observed in both human lung cell lines, A549 and Calu3/hACE2, but Wild type and omicron replication was observed. As SAR-CoV-2 continues to evolve, this data will benefit researchers in experimental planning, viral pathogenicity analysis, and providing a baseline for testing future variants.","version":"1.1","doi":"10.1101/2023.03.11.532212","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.13.532357","pub_date":"2023-3-13","title":"Inhibition of SARS-CoV-2 3CLpro in vitro by chemically modified tyrosinase from Agaricus bisporus","abstract":"Antiviral compounds are crucial to controlling the SARS-CoV-2 pandemic. Approved drugs have been tested for their efficacy against COVID-19, and new pharmaceuticals are being developed as a complementary tool to vaccines However, there are not any effective treatment against this disease yet. In this work, a cheap and fast purification method of natural tyrosinase from Agaricus bisporus fresh mushrooms was developed in order to evaluate the potential of this enzyme as a therapeutic protein by the inhibition of SARS-CoV-2 3CLpro protease activity in vitro. Tyrosinase showed a mild inhibition of 3CLpro of around 15%. Thus, different variants of this protein were synthesized through chemical modifications, covalently binding different tailor-made glycans and peptides to the amino terminal groups of the protein. These new tyrosinase conjugates were purified and characterized by circular dichroism and fluorescence spectroscopy analyses, and their stability under different conditions. Then all these tyrosinase conjugates were tested in 3CLpro protease inhibition. From them, the conjugate between tyrosinase and dextran-aspartic acid (6kDa) polymer showed the highest inhibition, with an IC50 of 2.5 \u03bcg/ml and IC90 of 5 \u03bcg/ml, results that highlight the potential use of modified tyrosinase as a therapeutic protein and opens the possibility of developing this and other enzymes as pharmaceutical drugs against diseases.","version":"1.1","doi":"10.1101/2023.03.13.532357","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.13.532385","pub_date":"2023-3-13","title":"SARS-CoV-2 N-protein induces the formation of composite \u03b1-synuclein/N-protein fibrils that transform into a strain of \u03b1-synuclein fibrils","abstract":"The presence of deposits of alpha-synuclein fibrils in cells of the brain are a hallmark of several \u03b1-synucleinopathies, including Parkinson\u2019s disease. As most disease cases are not familial, it is likely that external factors play a role in disease onset. One of the external factors that may influence disease onset are viral infections. It has recently been shown that in the presence of SARS-Cov-2 N-protein, \u03b1S fibril formation is faster and proceeds in an unusual two-step aggregation process. Here, we show that faster fibril formation is not due to a SARS-CoV-2 N-protein-catalysed formation of an aggregation-prone nucleus. Instead, aggregation starts with the formation of a population of mixed \u03b1S/N-protein fibrils with low affinity for \u03b1S. After the depletion of N-protein, fibril formation comes to a halt, until a slow transformation to fibrils with characteristics of pure \u03b1S fibril strains occurs. This transformation into a strain of \u03b1S fibrils subsequently results in a second phase of fibril growth until a new equilibrium is reached. Our findings point at the possible relevance of fibril strain transformation in the cell-to-cell spread of the \u03b1S pathology and disease onset.","version":"1.1","doi":"10.1101/2023.03.13.532385","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.12.532265","pub_date":"2023-3-13","title":"Monoclonal antibodies against human coronavirus NL63 spike","abstract":"The COVID-19 pandemic has illustrated the potential for monoclonal antibody therapeutics as prophylactic and therapeutic agents against pandemic viruses. No such therapeutics currently exist for other human coronaviruses. NL63 is a human alphacoronavirus that typically causes the common cold and uses the same receptor, ACE2, as the highly pathogenic SARS-CoV and SARS-CoV-2 pandemic viruses. In a cohort of healthy adults, we characterised humoral responses against the NL63 spike protein. While NL63 spike and receptor binding domain-specific binding antibodies and neutralisation activity could be detected in plasma of all subjects, memory B cells against NL63 spike were variable and relatively low in frequency compared to that against SARS-CoV-2 spike. From these donors, we isolated a panel of antibodies against NL63 spike and characterised their neutralising potential. We identified potent neutralising antibodies that recognised the receptor binding domain (RBD) and other non-RBD epitopes within spike.","version":"1.1","doi":"10.1101/2023.03.12.532265","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.12.532292","pub_date":"2023-3-13","title":"Pump-free and high-throughput generation of monodisperse hydrogel beads by microfluidic step emulsification for dLAMP-on-a-chip","abstract":"Step emulsification (SE), which generates droplets by a sharp change in confinement, has emerged as a potential alternative to flow-focusing technology. Water/dispersed phase is continuously pumped through a shallow inlet channel into a deep chamber pre-filled with the oil/continuous phase. The need for one or more pumps to maintain a continuous flow for droplet generation, and the consequent use of high sample volumes, limit this technique to research labs. Here, we report a pumpfree SE technique for rapid and high-throughput generation of monodisperse hydrogel (agarose) beads using <40 \u00b5l sample volume. Instead of using syringe pumps, we sequentially pipetted oil and liquid agarose into a microfluidic SE device to generate between 20000 and 80000 agarose beads in \u223c 2 min. We also demonstrated the encapsulation of loop-mediated isothermal amplification mixture inside these beads at the time of their formation. Finally, using these beads as reaction chambers, we amplified nucleic acids from P. falciparum and SARS-CoV-2 inside them. The pump-free operation, tiny sample volume, and high-throughput generation of droplets by SE make our technique suitable for point-of-care diagnostics.","version":"1.1","doi":"10.1101/2023.03.12.532292","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.10.532014","pub_date":"2023-3-12","title":"Improving the sensitivity of fluorescence-based immunoassays by time-resolved and spatial-resolved measurements","abstract":"Detection of target molecules, such as proteins, antibodies, or specific DNA sequences, is critical in medical laboratory science. Commonly used assays rely on tagging the target molecules with fluorescent probes. These are then fed to high-sensitivity detection systems. Such systems typically consist of a photodetector or camera and use time-resolved measurements that require sophisticated and expensive optics. Magnetic modulation biosensing (MMB) is a novel, fast, and sensitive detection technology that has been used successfully to detect viruses such as Zika and SARS-CoV-2. While this powerful tool is known for its high analytical and clinical sensitivity, the current signal-processing method for detecting the target molecule and estimating its dose is based on time-resolved measurements only. To improve the MMB-system performance, we propose here a novel signal processing algorithm that uses both temporally and spatially resolved measurements. We show that this combination significantly improves the sensitivity of the MMB-based assay. To evaluate the new method statistically, we performed multiple dose responses of Human Interleukin 9 (IL \u22128) on different days. Compared to standard time-resolved methods, the new algorithm provides a 2-3 fold improvement in detection limit and a 25% improvement in quantitative resolution.","version":"1.1","doi":"10.1101/2023.03.10.532014","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.09.531992","pub_date":"2023-3-10","title":"A single C-terminal residue controls SARS-CoV-2 spike trafficking and virion assembly","abstract":"The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virions and viral fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.","version":"1.1","doi":"10.1101/2023.03.09.531992","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.09.531961","pub_date":"2023-3-10","title":"SARS-CoV-2 protein structure and sequence mutations: evolutionary analysis and effects on virus variants","abstract":"Proteins sequence, structure, and function are related, so that any changes in the protein sequence may cause modifications in its structure and function. Thanks to the exponential growth of data availability, many studies have addressed different questions such as: (i) how structure evolves based on the sequence changes, (ii) how structure and function change over time. Computational experiments have contributed to the study of viral protein structures. For instance the Spike (S) protein has been investigated for its role in binding receptors and infection activity in COVID-19, hence the interest of scientific researchers in studying the effects of virus mutations due to sequence, structure and vaccination effects. Protein Contact Networks (PCNs) can be used for investigating protein structures to detect biological properties thorough network topology. We apply topological studies based on graph theory of the PCNs to compare the structural changes with sequence changes, and find that both node centrality and community extraction analysis play a relevant role in changes in protein stability and functionality caused by mutations. We compare the structural evolution to sequence changes and study mutations from a temporal perspective focusing on virus variants. We finally highlight a timeline correlation between Omicron variant identification and the vaccination campaign.","version":"1.1","doi":"10.1101/2023.03.09.531961","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.10.455872","pub_date":"2023-3-09","title":"N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2","abstract":"The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N- dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of \u03b2- 0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse. The ongoing COVID-19 pandemic necessitates new approaches to reduce disease caused by SARS-CoV-2. We tested the immunoadjuvant N-dihydrogalactochitosan (GC), used previously as an immunostimulant for tumor therapy and adjuvant for viral vaccines, as a potential COVID-19 countermeasure. When GC was administered before and after inoculation of a lethal dose of SARS-CoV-2 into the nose of humanized mice expressing an entry receptor for the virus, fewer mice showed weight loss and died compared to mice that received only the vehicle but no GC. GC-treated mice also had lower levels of infectious SARS-CoV-2 in their upper airway. These results suggest that GC may be a candidate to prevent or treat COVID-19. The immunoadjuvant N-dihydrogalactochitosan diminishes SARS-CoV-2 disease in humanized ACE2 mice, representing a new countermeasure against COVID-19.","version":"1.3","doi":"10.1101/2021.08.10.455872","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.26.501570","pub_date":"2023-3-09","title":"Immunological imprinting of humoral immunity to SARS-CoV-2 in children","abstract":"Omicron variants of SARS-CoV-2 are globally dominant and infection rates are very high in children. We determined immune responses following Omicron BA.1/2 infection in children aged 6-14 years and related this to prior and subsequent SARS-CoV-2 infection or vaccination. Primary Omicron infection elicited a weak antibody response with poor functional neutralizing antibodies. Subsequent Omicron reinfection or COVID-19 vaccination elicited increased antibody titres with broad neutralisation of Omicron subvariants. Prior pre-Omicron SARS-CoV-2 virus infection or vaccination primed for robust antibody responses following Omicron infection but these remained primarily focussed against ancestral variants. Primary Omicron infection thus elicits a weak antibody response in children which is boosted after reinfection or vaccination. Cellular responses were robust and broadly equivalent in all groups, providing protection against severe disease irrespective of SARS-CoV-2 variant. Immunological imprinting is likely to act as an important determinant of long-term humoral immunity, the future clinical importance of which is unknown.","version":"1.2","doi":"10.1101/2022.07.26.501570","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.09.531862","pub_date":"2023-3-09","title":"Inhibition mechanism and antiviral activity of an \u03b1-ketoamide based SARS-CoV-2 main protease inhibitor","abstract":"SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an \u03b1-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the \u03b1-ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.","version":"1.1","doi":"10.1101/2023.03.09.531862","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.08.531833","pub_date":"2023-3-09","title":"Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology","abstract":"The global COVID-19 pandemic continues with an increasing number of cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology and titers were comparable between rescued molecular clones and a clinical isolate (VIDO-01 strain), thus providing confidence that the rescued viruses can be used as effective replication tools. Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells, were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death and a novel lung cell line NCI-H23ACE2 A3 showed about \u223c99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection and are currently being used in CRISPR knockout and activation screens in our lab. We used a reverse genetics system to generate a wild type as well as a nanoluciferase-expressing reporter clone of SARS-CoV-2. The reporter virus allows for rapid transient replication assays and high throughput screens by detection of virus replication using luciferase assays. In addition, the reverse genetic system can be used to generate mutant viruses to study phenotypes of variant mutations. Additionally, unique human lung cell lines supporting SARS-CoV-2 replication will aid in studying the virus in a lung-relevant environment and based on high cytopathology induced in some cell lines, will be useful for screens that rely on virus-induced cell death for selection. Our study aims to enhance and contribute to the current replication tools available to study SARS-CoV-2 by providing rapid methods, virus clones and novel lung cell lines.","version":"1.1","doi":"10.1101/2023.03.08.531833","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.09.531709","pub_date":"2023-3-09","title":"Hybrid immunity elicits potent cross-variant ADCC against SARS-CoV-2 through a combination of anti-S1 and S2 antibodies","abstract":"Antibodies capable of neutralising SARS-CoV-2 have been well studied, but the Fc receptor-dependent antibody activities that also significantly impact the course of infection have not been studied in such depth. SARS-CoV-2 infection induces antibody-dependent NK cell responses targeting multiple antigens, however, as most vaccines induce only anti-spike antibodies, we investigated spike-specific antibody-dependent cellular cytotoxicity (ADCC). Vaccination produced antibodies that only weakly induced ADCC, however, antibodies from individuals who were infected prior to vaccination (\u2018hybrid\u2019 immunity) elicited much stronger anti-spike ADCC. Quantitative and qualitative aspects of humoral immunity contributed to this capability, with infection skewing IgG antibody production towards S2, vaccination skewing towards S1 and hybrid immunity evoking strong responses against both domains. The capacity for hybrid immunity to provide superior spike-directed ADCC was associated with selectively increased antibody responses against epitopes within both S1 and S2. Antibodies targeting both spike domains were important for strong antibody-dependent NK cell activation, with three regions of antibody reactivity outside the receptor-binding domain (RBD) corresponding with potent anti-spike ADCC. Consequently, ADCC induced by hybrid immunity with ancestral antigen was conserved against variants containing neutralisation escape mutations in the RBD [Delta and Omicron (BA.1)]. Induction of antibodies recognizing a broad range of spike epitopes and eliciting strong and durable ADCC may partially explain why hybrid immunity provides superior protection against infection and disease than vaccination alone, and demonstrates that spike-only subunit vaccines would benefit from strategies to induce a combination of S1- and S2-specific antibody responses. Neutralising antibodies prevent the entry of cell-free virus, however, antibodies that promote Fc-dependent activities such as ADCC are critical to control cell-associated virus. Although current SARS-CoV-2 vaccines induce potent neutralising antibodies, they fail to induce robust ADCC. Our demonstration that hybrid immunity induces superior ADCC with pan-variant activity may partially explain why hybrid immunity offers enhanced protection against reinfection. It also highlights that vaccine strategies based on expression of the spike subunit alone should not focus solely on inducing antibody responses targeting the receptor binding domain.","version":"1.1","doi":"10.1101/2023.03.09.531709","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.23.477397","pub_date":"2023-3-09","title":"Ronapreve (REGN-CoV; casirivimab and imdevimab) reduces the viral burden and alters the pulmonary response to the SARS-CoV-2 Delta variant (B.1.617.2) in K18-hACE2 mice using an experimental design reflective of a treatment use case","abstract":"Ronapreve demonstrated clinical application in post-exposure prophylaxis, mild/moderate disease and in the treatment of seronegative patients with severe COVID19 prior to the emergence of the Omicron variant in late 2021. Numerous reports have described loss of in vitro neutralisation activity of Ronapreve and other monoclonal antibodies for BA.1 Omicron and subsequent sub-lineages of the Omicron variant. With some exceptions, global policy makers have recommended against the use of existing monoclonal antibodies in COVID19. Gaps in knowledge regarding the mechanism of action of monoclonal antibodies are noted, and further preclinical study will help understand positioning of new monoclonal antibodies under development. The purpose of this study was to investigate the impact of Ronapreve on compartmental viral replication as a paradigm for a monoclonal antibody combination. The study also sought to confirm absence of in vivo activity against BA.1 Omicron (B.1.1.529) relative to the Delta (B.1.617.2) variant. Virological efficacy of Ronapreve was assessed in K18-hACE2 mice inoculated with either the SARS-CoV-2 Delta or Omicron variants. Viral replication in tissues was quantified using qRT-PCR to measure sub-genomic viral RNA to the E gene (sgE) as a proxy. A histological examination in combination with staining for viral antigen served to determine viral spread and associated damage. Ronapreve reduced sub-genomic viral RNA levels in lung and nasal turbinate, 4 and 6 days post infection, for the Delta variant but not the Omicron variant of SARS-CoV-2 at doses 2-fold higher than those shown to be active against previous variants of the virus. It also appeared to block brain infection which is seen with high frequency in K18-hACE2 mice after Delta variant infection. At day 6, the inflammatory response to lung infection with the Delta variant was altered to a mild multifocal granulomatous inflammation in which the virus appeared to be confined. A similar tendency was also observed in Omicron infected, Ronapreve-treated animals. The current study provides evidence of an altered tissue response to the SARS-CoV-2 after treatment with a monoclonal antibody combination that retains neutralization activity. These data also demonstrate that experimental designs that reflect the treatment use case are achievable in animal models for monoclonal antibodies deployed against susceptible variants. Extreme caution should be taken when interpreting prophylactic experimental designs when assessing plausibility of monoclonal antibodies for treatment use cases.","version":"1.2","doi":"10.1101/2022.01.23.477397","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.07.531609","pub_date":"2023-3-09","title":"Early transcriptional responses of human nasal epithelial cells to infection with Influenza A and SARS-CoV-2 virus differ and are influenced by physiological temperature","abstract":"Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an un-derstudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insights into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affects SCV2, but not IAV, viral replicative fitness and that SCV2 infected cultures are slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changes the basal transcriptomic landscape of epithelial cells, but that it also impacts the response to infection. The induction of interferon and other innate immune responses were not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remains consistent, there may be metabolic or signaling changes that affect how well the cultures are able to adapt to new pressures such as infection. Finally, we show that hNECs respond differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.","version":"1.1","doi":"10.1101/2023.03.07.531609","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.08.531718","pub_date":"2023-3-09","title":"COVID-19 as a continuous-time stochastic process","abstract":"In this article a mathematical treatment of Covid-19 as a stochastic process is discussed. The chance of extinction and the consequences of introducing new Covid-19 infectives into the population are evaluated by using certain approximate arguments. It is shown, in general terms, that the stochastic formulation of a recurrent epidemic like Covid-19 leads to the prediction of a permanent succession of undamped outbreaks of disease. It is also shown that one is able to derive certain useful conclusions about Covid-19 without consideration of immune individuals in a population.","version":"1.1","doi":"10.1101/2023.03.08.531718","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.01.530717","pub_date":"2023-3-08","title":"Assessment of neutralization susceptibility of Omicron subvariants XBB.1.5 and BQ.1.1 against broad-spectrum neutralizing antibodies through epitopes mapping","abstract":"The emergence of new variants of the SARS-CoV-2 virus has posed a significant challenge in developing broadly neutralizing antibodies (nAbs) with guaranteed therapeutic potential. Some nAbs, such as Sotrovimab, have exhibited varying levels of efficacy against different variants, while others, such as Bebtelovimab and Bamlanivimab-etesevimab are ineffective against specific variants, including BQ.1.1 and XBB. This highlights the urgent need for developing broadly active mAbs providing prophylactic and therapeutic benefits to high-risk patients, especially in the face of the risk of reinfection from new variants. Here, we aimed to investigate the feasibility of redirecting existing mAbs against new variants of SARS-CoV-2, as well as to understand how BQ.1.1 and XBB.1.5 can evade broadly neutralizing mAbs. By mapping epitopes and escape sites, we discovered that the new variants evade multiple mAbs, including FDA-approved Bebtelovimab, which showed resilience against other Omicron variants. Our approach, which included simulations, free energy perturbations, and shape complementarity analysis, revealed the possibility of identifying mAbs that are effective against both BQ.1.1 and XBB.1.5. We identified two broad-spectrum mAbs, R200-1F9 and R207-2F11, as potential candidates with increased binding affinity to XBB.1.5 and BQ.1.1 compared to the wild-type virus. Additionally, we propose that these mAbs do not interfere with ACE2 and bind to conserved epitopes on the RBD that are not-overlapping, potentially providing a solution to neutralize these new variants either independently or as part of a combination (cocktail) treatment.","version":"1.2","doi":"10.1101/2023.03.01.530717","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.06.531252","pub_date":"2023-3-07","title":"Unraveling the Interactions between Human DPP4 Receptor, SARS-CoV-2 Variants, and MERS-CoV, converged for Pulmonary Disorders Integrating through Immunoinformatics and Molecular Dynamics","abstract":"Human coronaviruses like MERS CoV are known to utilize dipeptidyl peptidase 4 (DPP4), apart from angiotensin-converting enzyme 2(ACE2) as potential co-receptor for viral cell entry. DPP4, ubiquitous membrane-bound aminopeptidase is closely associated with elevation of disease severity in comorbidities. In SARS-CoV-2, there is inadequate evidence for combination of spike protein variants with DPP4, and underlying adversity in COVID19. To elucidate this mechanistic basis, we have investigated interaction of spike protein variants with DPP4 through molecular docking and simulation studies. The possible binding interactions between receptor binding domain (RBD) of different spike variants of SARS-CoV-2 and DPP4 have been compared with interactions observed in experimentally determined structure of complex of MERS-CoV with DPP4. Comparative binding affinity confers that Delta-CoV-2:DPP4 shows close proximity with MERS-CoV:DPP4, as depicted from accessible surface area, radius of gyration, number of hydrogen bonding and energy of interactions. Mutation in delta variant, L452R and T478K, directly participate in DPP4 interaction enhancing DPP4 binding. E484K in alpha and gamma variant of spike protein is also found to interact with DPP4. Hence, DPP4 interaction with spike protein gets more suitable due to mutation especially due to L452R, T478K and E484K. Furthermore, perturbation in the nearby residues Y495, Q474 and Y489 is evident due to L452R, T478K and E484K respectively. Virulent strains of spike protein are more susceptible to DPP4 interaction and are prone to be victimized in patients due to comorbidities. Our results will aid the rational optimization of DPP4 as a potential therapeutic target to manage COVID-19 disease severity.","version":"1.1","doi":"10.1101/2023.03.06.531252","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.06.531431","pub_date":"2023-3-07","title":"Genome-scale CRISPR\u2012Cas9 screen identifies novel host factors as potential therapeutic targets for SARS-CoV-2 infection","abstract":"Although many host factors important for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported, the mechanisms by which the virus interacts with host cells remain elusive. Here, we identified tripartite motif containing (TRIM) 28, TRIM33, euchromatic histone lysine methyltransferase (EHMT) 1, and EHMT2 as novel proviral factors involved in SARS-CoV-2 infection by CRISPR\u2012Cas9 screening. We demonstrated that TRIM28 plays a role(s) in viral particle formation and that TRIM33, EHMT1, and EHMT2 are involved in viral transcription and replication using cells with suppressed gene expression. UNC0642, a compound that specifically inhibits the methyltransferase activity of EHMT1/2, strikingly suppressed SARS-CoV-2 growth in cultured cells and reduced disease severity in a hamster infection model. This study suggests that EHMT1/2 may be a novel therapeutic target for SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.03.06.531431","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.07.531527","pub_date":"2023-3-07","title":"Development of Fully Human, Bispecific Antibodies that Effectively Block Omicron Variant Pseudovirus Infections","abstract":"The emergence of highly immune invasive and transmissible variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has decreased the effectiveness of existing vaccines. It is, therefore, critical to develop effective and safe therapeutics for SARS-CoV-2 infections, especially for the most vulnerable and immunocompromised patients. Neutralizing antibodies have been shown to be successful at preventing severe disease from early SARS-CoV-2 strains, although their efficacy has diminished with the emergence of new variants. Here, we aim to develop fully human and broadly neutralizing monoclonal (mAb) and bispecific (BsAb) antibodies against SARS-CoV-2 and its variants. Specifically, we first identified two antibodies from human transgenic mice that bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and are capable of neutralizing SARS-CoV-2 and variants of concern with high to moderate affinity. Two non-competing clones with the highest affinity and functional blocking of ACE2 binding were then selected to be engineered into two BsAbs, which were then demonstrated to have relatively improved affinity, ACE2 blocking ability, and pseudovirus inhibition against several variants, including Omicron (B.1.1.529). Our findings provide one mAb candidate and two bsAb candidates for consideration of further clinical development and suggest that the bispecific format may be more effective than mAbs for SARS-CoV-2 treatment.","version":"1.1","doi":"10.1101/2023.03.07.531527","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.06.531335","pub_date":"2023-3-07","title":"Cooperative and structural relationships of the trimeric Spike with infectivity and antibody escape of the strains Delta (B.1.617.2) and Omicron (BA.2, BA.5, and BQ.1)","abstract":"Herein, we simulated the trimeric Spike of the variants B.1.617.2, BA.2, BA.5 and BQ.1 for 300 ns. We derived mechanisms by which the substitutions K417N, L452R, N444T and N460K may favor resistance to neutralizing antibodies. The K417N and L452R contribute to the expansion of the networks of hydrogen bonding interactions with neighboring residues, decreasing their capacity to interact with neutralizing antibodies. The SpikeBQ.1 possesses two unique K444T and N460K mutations that expand the network of hydrogen bonding interactions. This lysine also contributes one novel strong saline interaction and both substitutions may favor resistance to neutralizing antibodies. We also investigated how the substitutions D614G, P681R, and P681H impact Spike structural conformations and discuss the impact of these changes to infectivity and lethality. The prevalent D614G substitution plays a key role in the communication between the glycine and the residues of a \u03b2-strand located between the NTD and the RBD, impacting the transition between up- and down-RBD states. The P681R mutation, found in the Delta variant, favors intra- and inter-protomer correlations between the subunits S1 and S2. Conversely, in Omicron sub-variants, P681H decreases the intra- and inter-protomer long-range interactions within the trimeric Spike, providing an explanation for the reduced fusogenicity of this variant. Taken together, our results enhance the knowledge on how novel mutations lead to changes in infectivity and reveal mechanisms by which SARS-CoV-2 may evade the immune system.","version":"1.2","doi":"10.1101/2023.03.06.531335","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.04.531078","pub_date":"2023-3-06","title":"Tau protein aggregation associated with SARS-CoV-2 main protease","abstract":"The primary function of virus proteases is the proteolytic processing of the viral polyprotein. These enzymes can also cleave host cell proteins, which is important for viral pathogenicity, modulation of cellular processes, viral replication, the defeat of antiviral responses and modulation of the immune response. It is known that COVID-19 can influence multiple tissues or organs and that infection can damage the functionality of the brain in multiple ways. After COVID-19 infections, amyloid-\u03b2, neurogranin, tau and phosphorylated tau were detected extracellularly, implicating possible neurodegenerative processes. The present study describes the possible induction of protein aggregation by the SARS-CoV-2 3CL protease (3CLpro) possibly relevant in neuropathology, such as aggregation of tau, alpha-synuclein and TPD-43. Further investigations demonstrated that tau was proteolytically cleaved by the viral protease 3CL and, consequently, generated aggregates. However, more evidence is needed to confirm that COVID-19 is able to trigger neurodegenerative diseases.","version":"1.1","doi":"10.1101/2023.03.04.531078","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.31.502215","pub_date":"2023-3-06","title":"An Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease","abstract":"Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir\u2019s inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds up in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here we investigated the H172Y Mpro\u2019s conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggests that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weakens the nirma-trelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intel-ligence approaches, and together with biochemical experiments they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the discovery of new antiviral drugs. The presented workflow can be applicable to characterize mutation effects on any protein drug targets.","version":"1.2","doi":"10.1101/2022.07.31.502215","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.03.531067","pub_date":"2023-3-06","title":"Convergent Evolution of A-Lineage (Clade 19B) SARS-CoV-2 Spike Sequences with B-Lineage Variants of Concern Affects Virus Replication in a Temperature-Dependent Manner on Human Nasal Epithelial Cell Cultures","abstract":"The first three months of the COVID-19 pandemic was dominated by two SARS-CoV-2 lineages: A-lineages (Clade 19B) and B-lineages (Clade 19A). However, with the emergence of the Spike D614G substitution in B.1 lineages (Clade 20A), both early lineages were outcompeted and remained near-extinction from mid-2020 onwards. In early-2021, there was a re-emergence and persistence of novel A-lineage variants with substitutions in the Spike gene resembling those found in Variants of Concern (VOCs). An early A.3 variant (MD-HP00076/2020) and three A.2.5 variants (MD-HP02153/2021, MD-HP05922/2021 and CA-VRLC091/2021) were isolated and characterized for their genomic sequences, antibody neutralization, and in vitro replication. All A.2.5 isolates had five Spike mutations relative to the A.3 variant sequence: D614G, L452R, \u0394141-143, D215A, and ins215AGY. Plaque reduction neutralization assays demonstrated that A.2.5 isolates had a 2.5 to 5-fold reduction in neutralization using contemporaneous COVID-19 convalescent plasma when compared to A.3. In vitro viral characterization in VeroE6 cell lines revealed that the A.3 isolate grew faster and spread more than A.2.5. On VeroE6-TMPRSS2 cells, significant syncytia formation was also observed with the A.2.5 isolates, however Spike cleavage efficiency did not explain these differences. In human nasal epithelial cell (hNEC) cultures, the A.2.5 isolates grew significantly faster and to higher total infectious virus titers than A.3. All A.2.5 lineage isolates grew significantly faster at 37\u00b0C than at 33\u00b0C irrespective of cell type, and to higher peak titers except compared to A.3. This suggests A.2.5\u2019s adapted to improve replication using similar mutations found in the B-lineage SARS-CoV-2 variants. While both A- and B-lineage SARS-CoV-2 variants emerged and circulated together during the early months of the pandemic, the B-lineages that acquired Spike D614G eventually outcompeted all other variants. We show that the A-lineage variants eventually evolved mutations including Spike D614G and Spike L452R that improved their in vitro replication in human nasal epithelial cells in a temperature dependent manner, suggesting there are some highly selectable mutation landscapes that SARS-CoV-2 can acquire to adapt to replication and transmission in humans.","version":"1.1","doi":"10.1101/2023.03.03.531067","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.05.531227","pub_date":"2023-3-06","title":"Predicting the feasibility of targeting a conserved region on the S2 domain of the SARS-CoV-2 spike protein","abstract":"The efficacy of vaccines against the SARS-CoV-2 virus significantly declines with the emergence of mutant strains, prompting investigation into the feasibility of targeting highly conserved but often cryptic regions on the S2 domain of spike protein. Using tools from molecular dynamics, we find that this conserved S2 epitope located in the central helices below the receptor binding domains is unlikely to be exposed by dynamic fluctuations without any external facilitating factors, in spite of previous computational evidence suggesting transient exposure of this region. Furthermore, glycans inhibit opening dynamics, and thus stabilize spike in addition to immunologically shielding the protein surface, again in contrast to previous computational findings. Though the S2 epitope region examined here is central to large scale conformational changes during viral entry, free energy landscape analysis obtained using the path coordinate formalism reveals no inherent \u201cloaded spring\u201d effect, suggesting that a vaccine immunogen would tend to present the epitope in a pre-fusion-like conformation and may be effective in neutralization. These findings contribute to a deeper understanding of the dynamic origins of the function of the spike protein, as well as further characterizing the feasibility of the S2 epitope as a therapeutic target.","version":"1.1","doi":"10.1101/2023.03.05.531227","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.05.531143","pub_date":"2023-3-06","title":"Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity and neutralization sensitivity","abstract":"SARS-CoV-2 has remarkable ability to respond to and evolve against the selection pressure by host immunity exemplified by emergence of Omicron lineage. Here, we characterized the functional significance of mutations in Omicron spike. By systematic transfer of mutations in WT spike we assessed neutralization sensitivity, fusogenicity, and TMPRSS2-dependence for entry. The data revealed that the mutations in both S1 and S2 complement to make Omicron highly resistant. Strikingly, the mutations in Omicron S2 modulated the neutralization sensitivity to NTD- and RBD-antibodies, but not to S2 specific neutralizing antibodies, suggesting that the mutations in S2 were primarily acquired to gain resistance to S1-antibodies. Although all six mutations in S2 appeared to act in concert, D796Y showed greatest impact on neutralization sensitivity and rendered WT virus >100-fold resistant to S309, COVA2-17, and 4A8. S2 mutations greatly reduced the antigenicity for NAbs due to reduced exposure of epitopes. In terms of the entry pathway, S1 or S2 mutations only partially altered the entry phenotype of WT and required both sets of mutations for complete switch to endosomal route and loss of syncytia formation. In particular, N856K and L981F in Omicron reduced fusion capacity and explain why subsequent Omicron variants lost them to regain fusogenicity.","version":"1.1","doi":"10.1101/2023.03.05.531143","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.14.520411","pub_date":"2023-3-06","title":"COVID-19db linkage maps of cell surface proteins and transcription factors in immune cells","abstract":"The highly contagious SARS-CoV-2 and its associated disease (COVID-19) are a threat to global public health and economies. To develop effective treatments for COVID-19, we must understand the host cell types, cell states and regulators associated with infection and pathogenesis such as dysregulated transcription factors (TFs) and surface proteins, including signalling receptors. To link cell surface proteins with TFs, we recently developed SPaRTAN (Single-cell Proteomic and RNA-based Transcription factor Activity Network) by integrating parallel single-cell proteomic and transcriptomic data based on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and gene cis-regulatory information. We apply SPaRTAN to CITE-seq datasets from patients with varying degrees of COVID-19 severity and healthy controls to identify the associations between surface proteins and TFs in host immune cells. Here, we present COVID-19db of Immune Cell States (https://covid19db.streamlit.app/), a web server containing cell surface protein expression, SPaRTAN-inferred TF activities, and their associations with major host immune cell types. The data include four high-quality COVID-19 CITE-seq datasets with a toolset for userfriendly data analysis and visualization. We provide interactive surface protein and TF visualizations across major immune cell types for each dataset, allowing comparison between various patient severity groups for the discovery of potential therapeutic targets and diagnostic biomarkers.","version":"1.2","doi":"10.1101/2022.12.14.520411","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448868","pub_date":"2023-3-06","title":"Visual and Quantitative Analyses of Virus Genomic Sequences using a Metric-based Algorithm","abstract":"This work aims to study the virus RNAs using a novel algorithm for accelerated exploring any-length genomic fragments in sequences using Hamming distance between the binary-expressed characters of an RNA and query patterns. The found repetitive genomic sub-sequences of different lengths were placed on one plot as genomic trajectories (walks) to increase the effectiveness of geometrical multi-scale genomic studies. Primary attention was paid to the building and analysis of the atg-triplet walks composing the schemes or skeletons of the viral RNAs. The 1-D distributions of these codon-starting atg-triplets were built with the single-symbol walks for full-scale analyses. The visual examination was followed by calculating statistical parameters of genomic sequences, including the estimation of geometry deviation and fractal properties of inter-atg distances. This approach was applied to the SARS CoV-2, MERS CoV, Dengue and Ebola viruses, whose complete genomic sequences are taken from GenBank and GISAID databases. The relative stability of these distributions for SARS CoV-2 and MERS CoV viruses was found, unlike the Dengue and Ebola distributions that showed an increased deviation of their geometrical and fractal characteristics of atg-distributions. The results of this work can found in classification of the virus families and in the study of their mutation.","version":"1.3","doi":"10.1101/2021.06.17.448868","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.31.526458","pub_date":"2023-3-04","title":"Scalable neighbour search and alignment with uvaia","abstract":"Despite millions of SARS-CoV-2 genomes being sequenced and shared globally, manipulating such data sets is still challenging, especially selecting sequences for focused phylogenetic analysis. We present a novel method, uvaia, which is based on partial and exact sequence similarity for quickly extracting database sequences similar to query sequences of interest. Many SARS-CoV-2 phylogenetic analyses rely on very low numbers of ambiguous sites as a measure of quality since ambiguous sites do not contribute to single nucleotide polymorphism (SNP) differences, which uvaia alleviates by using measures of sequence similarity that consider partially ambiguous sites. Such fine-grained definition of similarity allows not only for better phylogenetic analyses, but also for improved classification and biogeographical inferences. Uvaia works natively with compressed files, can use multiple cores and efficiently utilises memory, being able to analyse large data sets on a standard desktop.","version":"1.2","doi":"10.1101/2023.01.31.526458","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.01.530733","pub_date":"2023-3-03","title":"Frequent emergence of resistance mutations following complex intra-host genomic dynamics in SARS-CoV-2 patients receiving Sotrovimab","abstract":"The emergence of the Omicron variant of SARS-CoV-2 represented a challenge to the treatment of COVID-19 with monoclonal antibodies. Only Sotrovimab maintained partial activity, allowing it to be used in high-risk patients infected with the Omicron variant. However, the reports of resistance mutations to Sotrovimab call for efforts to better understand the intra-patient emergence of this resistance. A retrospective genomic analysis was conducted on respiratory samples from immunocompromised patients infected with SARS-CoV-2 who received Sotrovimab at our hospital between December 2021 and August 2022. The study involved 95 sequential specimens from 22 patients (1-12 samples/patient; 3-107 days post-infusion (Ct \u2264 32)). Resistance mutations (in P337, E340, K356, and R346) were detected in 68% of cases; the shortest time to detection of a resistance mutation was 5 days after Sotrovimab infusion. The dynamics of resistance acquisition were highly complex, with up to 11 distinct amino acid changes in specimens from the same patient. In two patients, the mutation distribution was compartmentalized in respiratory samples from different sources. This is the first study to examine the acquisition of resistance to Sotrovimab in the BA.5 lineage, enabling us to determine the lack of genomic or clinical differences between Sotrovimab resistance in BA.5 relative to BA.1/2. Across all Omicron lineages, the acquisition of resistance delayed SARS-CoV-2 clearance (40.67 vs 19.5 days). Close, real-time genomic surveillance of patients receiving Sotrovimab should be mandatory to facilitate early therapeutic interventions.","version":"1.1","doi":"10.1101/2023.03.01.530733","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.02.530883","pub_date":"2023-3-03","title":"Residual Sanitization of Three Human Respiratory Viruses on a Hard, Non-Porous Surface","abstract":"Human pathogenic viruses that are present in bioaerosols released by coughing, sneezing, or breathing can contaminate fomites and other inanimate environmental surfaces. Most are enveloped respiratory viruses that are vulnerable to inactivation by a broad spectrum of antimicrobial actives. Quaternary ammonium compounds are highly diverse in structure and are among the most widely utilized antimicrobial agents. The objective of this study was to evaluate two commercially available, ready-to-use quaternary ammonium compound-based disinfectants (one of which also contains a surface binding agent) for antiviral activity against Influenza A (H1N1), human coronavirus 229E, and SARS-CoV-2 (Washington) following a rigorous procedure of wear and abrasions with regular re-inoculations of virus in the presence of a 6% organic soil load. Formulation TF-A demonstrated variable residual efficacy against the three viruses, achieving log10reductions of 1.62, 3.33, and 0.92, respectively. Formulation TF-B lowered each test virus by greater than 3-log10to non-detectable levels on all carriers in demonstration of residual antiviral activity.","version":"1.1","doi":"10.1101/2023.03.02.530883","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.02.530652","pub_date":"2023-3-02","title":"Systematic analyses of the resistance potential of drugs targeting SARS-CoV-2 main protease","abstract":"Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance in a yeast screen to nirmatrelvir (contained in the drug Paxlovid), and ensitrelvir (Xocova) that is currently in phase III trials. The most impactful resistance mutation (E166V) recently reported in multiple viral passaging studies with nirmatrelvir showed the strongest drug resistance score for nirmatrelvir, while P168R had the strongest resistance score for ensitrelvir. Using a systematic approach to assess potential drug resistance, we identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir. Among these mutations, 99 caused apparent resistance to both inhibitors, suggesting a strong likelihood for the evolution of cross-resistance. Many mutations that exhibited inhibitor-specific resistance were consistent with distinct ways that each inhibitor protrudes beyond the substrate envelope. In addition, mutations with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and secondary mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations.","version":"1.1","doi":"10.1101/2023.03.02.530652","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.02.530738","pub_date":"2023-3-02","title":"Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats","abstract":"While the spike proteins from SARS-CoV and SARS-CoV-2 bind to host ACE2 to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol, and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016, as well as an ACE2-independent virus, RsHuB2019. Although our stocks of RsHuB2019 rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light onto the intricacies of coronavirus isolation and propagation in vitro. Several coronaviruses have transmitted from animals to people and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.","version":"1.1","doi":"10.1101/2023.03.02.530738","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.28.530557","pub_date":"2023-3-01","title":"Type I interferon signaling induces a delayed antiproliferative response in Calu-3 cells during SARS-CoV-2 infection","abstract":"Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex \u2013 IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.","version":"1.1","doi":"10.1101/2023.02.28.530557","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.28.530547","pub_date":"2023-3-01","title":"COVID-19 adenoviral vector vaccination elicits a robust memory B cell response with the capacity to recognize Omicron BA.2 and BA.5 variants","abstract":"Following the COVID-19 pandemic caused by SARS-CoV-2, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the Spike receptor binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response elicited by the mRNA vaccine BNT162b2 (Pfizer-BioNTech). Whole blood was sampled from 31 healthy adults pre-vaccination, and four weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each timepoint. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1, and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.","version":"1.1","doi":"10.1101/2023.02.28.530547","journal":"bioRxiv","score":null},{"id":"10.1101/2023.03.01.530454","pub_date":"2023-3-01","title":"IgG4 serum levels are not elevated in cases of Post-COVID syndrome","abstract":"Recently, unexpectedly high virus-specific IgG4 levels were reported after more than two mRNA vaccinations. Class switch towards IgG4 occurs after long-term antigen exposure, downregulates immune responses and is associated with several autoimmune diseases. Here, we examined differences in antigen-specific IgG subtypes in serum samples from 64 Post-COVID patients and an equally sized cohort of convalescent controls. In both cohorts, the relative amounts of spike protein-specific IgG subtypes were comparable. IgG1 was the most frequent, followed by IgG3, IgG2, and IgG4. A difference between cohorts was observed only for IgG2, which was significantly lower in the Post-COVID cohort. Further analysis of the reactive IgG4 revealed a small but significant difference for the spike protein receptor-binding domain but not for the spike ectodomain. Since the total IgG4 levels are very low, we do not expect a biologically relevant role in Post-COVID syndrome. However, reduced virus-specific IgG2 levels could contribute to the persistence of SARS-CoV-2, causing chronic inflammation in the setting of Post-COVID syndrome.","version":"1.1","doi":"10.1101/2023.03.01.530454","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.28.530489","pub_date":"2023-3-01","title":"Coarse-Grained Molecular Simulations and Ensemble-Based Mutational Profiling of Protein Stability in the Different Functional Forms of the SARS-CoV-2 Spike Trimers : Balancing Stability and Adaptability in BA.1, BA.2 and BA.2.75 Variants","abstract":"The evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity, conformational plasticity, protein stability and allosteric modulation. In this study, we embarked on a systematic comparative analysis of the conformational dynamics, electrostatics, protein stability and allostery in the different functional states of spike trimers for BA.1, BA.2, and BA.2.75 variants. Using efficient and accurate coarse-grained simulations and atomistic reconstruction of the ensembles, we examined conformational dynamics of the spike trimers that agrees with the recent functional studies, suggesting that BA.2.75 trimers are the most stable among these variants. A systematic mutational scanning of the inter-protomer interfaces in the spike trimers revealed a group of conserved structural stability hotspots that play a key role in modulation of functional dynamics and are also involved in the inter-protomer couplings through local contacts and interaction networks with the Omicron mutational sites. The results of mutational scanning provided evidence that BA.2.75 trimers are more stable than BA.2 and comparable in stability to BA.1 variant. Using dynamic network modeling of the S Omicron BA.1, BA.2 and BA.2.75 trimers we showed that the key network positions driving long-range signaling are associated with the major stability hotspots that are inter-connected along potential communication pathways, while sites of Omicron mutations may often correspond to weak spots of stability and allostery but are coupled to the major stability hotspots through interaction networks. The presented analysis of the BA.1, BA.2 and BA.2.75 trimers suggested that thermodynamic stability of BA.1 and BA.2.75 variants may be intimately linked with the residue interaction network organization that allows for a broad ensemble of allosteric communications in which signaling between structural stability hotspots may be modulated by the Omicron mutational sites. The findings provided plausible rationale for mechanisms in which Omicron mutations can evolve to balance thermodynamic stability and conformational adaptability in order to ensure proper tradeoff between stability, binding and immune escape.","version":"1.1","doi":"10.1101/2023.02.28.530489","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.28.530444","pub_date":"2023-2-28","title":"SARS-CoV-2 surveillance between 2020 and 2021 of all mammalian species in two Flemish zoos (Antwerp Zoo and Planckendael Zoo)","abstract":"The COVID-19 pandemic has led to millions of human infections and deaths worldwide. Several other mammal species are also susceptible to SARS-CoV-2, and multiple instances of transmission from humans to pets, farmed mink, wildlife and zoo animals have been recorded. We conducted a systematic surveillance of SARS-CoV-2 in all mammal species in two zoos in Belgium between September and December 2020 and July 2021 in four sessions, and a targeted surveillance of selected mammal enclosures following SARS-CoV-2 infection in hippos in December 2021. A total of 1523 faecal samples were tested for SARS-CoV-2 via real-time PCR. None of the samples tested positive for SARS-CoV-2. Additional surrogate virus neutralization tests conducted on 50 routinely collected serum samples during the same period were all negative. This study is a first to our knowledge to conduct active SARS-CoV-2 surveillance for several months in all mammal species of a zoo. We conclude that at the time of our investigation, none of the screened animals were excreting SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.02.28.530444","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516114","pub_date":"2023-2-28","title":"A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein","abstract":"The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target. This study reports on the discovery of a macrocyclic peptide that is able to inhibit SARS-CoV-2 infection by exploiting a new vulnerable site in the spike glycoprotein. This region is highly conserved across SARS-CoV-2 variants and the subgenus sarbecovirus. Due to the inaccessability and mutational contraint of this site, it is anticipated to be resistant to the development of resistance through antibody selective pressure. In addition to the discovery of a new molecule for development of potential new peptide or biomolecule therapeutics, the discovery of this broadly active conserved site can also stimulate a new direction of drug development, which together may prevent future outbreaks of related viruses.","version":"1.3","doi":"10.1101/2022.11.11.516114","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.27.530346","pub_date":"2023-2-28","title":"Neutralizing antibody levels and epidemiological information of patients with breakthrough COVID-19 infection in Toyama, Japan","abstract":"Breakthrough infection (BI) after coronavirus disease 2019 (COVID-19) vaccination has exploded owing to the emergence of various SARS-CoV-2 variants and has become a major problem at present. In this study, we analyzed the epidemiological information and possession status of neutralizing antibodies in patients with BI using SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Analysis of 44 specimens diagnosed with COVID-19 after two or more vaccinations showed high inhibition of infection by 90% or more against the Wuhan strain and the Alpha and Delta variants of pseudotyped viruses in 40 specimens. In contrast, almost no neutralizing activity was observed against the Omicron BA.1 variant. Many cases without neutralizing activity or BI were immunosuppressed individuals. The results of this study show that BI occurs even when there are sufficient neutralizing antibodies in the blood due to exposure to close contacts at the time of infection. Thus, even after vaccination, sufficient precautions must be taken to prevent infection.","version":"1.1","doi":"10.1101/2023.02.27.530346","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.27.530294","pub_date":"2023-2-28","title":"Rolosense: Mechanical detection of SARS-CoV-2 using a DNA-based motor","abstract":"Assays detecting viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here we present Rolosense, a virus sensing platform that transduces the motion of synthetic DNA-based motors transporting 5-micron particles on RNA fuel chips. Motors and chips are modified with virus-binding aptamers that lead to stalling of motion. Therefore, motors perform a \u201cmechanical test\u201d of viral target and stall in the presence of whole virions which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 103 copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as we demonstrated one-pot detection of influenza A and SARS-CoV-2. As a proof-of-concept, we show readout can be achieved using a smartphone camera in as little as 15 mins without any sample preparation steps. Taken together, mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost, point-of-care screening of circulating viruses.","version":"1.1","doi":"10.1101/2023.02.27.530294","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.26.530067","pub_date":"2023-2-27","title":"Genomic perspectives of SARS CoV-2 in liver disease patients with its clinical correlation: A single centre retrospective study","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2), is a causative agent of current global pandemic of Coronavirus disease-19 (COVID-19). Due to propagated outbreak and global vaccination drive an immense immunological selection pressure has been exerted on SARS CoV-2 leading to evolution of new variants. This study was performed to compare the mutational and clinical profile of liver disease patients infected with different variants of SARS CoV-2. This was a single-centre, retrospective, cohort study in which clinicogenomic analysis of liver disease (LD) patients infected with SARS CoV-2 was performed. Complete demographic and clinical details were retrieved from Hospital Information System (HIS). QC-threshold passed FASTA files containing sequences from COVID-19 patients (n=174) were compared with a reference genome of SARS-CoV-2 isolate named Wuhan-Hu-1 (NCBI Reference Sequence: NC_045512.2) for mutational analysis. Out of 232 finally analysed patients 137 (59.1%) were LD-CoV (+) and 95 (40.9%) were LD-CoV(-). LD patients with comorbidities were affected more with COVID-19 (p=0.002). On comparing the outcome in the terms of mortality, LD-CoV (+) had 2.29 times (OR 2.29, CI 95%, 1.25-4.29) higher of odds of succumbing to COVID-19 (p=0.006). Multivariate regression analysis revealed, abdominal distention (p=0.05), severe COVID-19 pneumonia (p=0.046) and the change in serum bilirubin levels (p=0.005) as well as Alkaline phosphatase (ALP) levels (p=0.003) to have an association with adverse outcome in LD patients with COVID-19. In Delta (22%) and Omicron (48%) groups, Spike gene harboured maximum mutations. On comparing the mutations between LD-CoV(+/D) and LD-CoV(+/O) a total of nine genes had more mutations in LD-CoV(+/O) whereas three genes had more mutations in LD-CoV(+/D). We concluded that LD patients are more susceptible to COVID-19 as compared to a healthy adult with associated adverse clinical outcomes in terms of mortality and morbidity. Therefore this special group should be given priority while devising and introducing new vaccination and vaccination policies. The infection with different variants did not result in different outcome in our group of patients.","version":"1.1","doi":"10.1101/2023.02.26.530067","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.25.530000","pub_date":"2023-2-27","title":"Rapid resistance profiling of SARS-CoV-2 protease inhibitors","abstract":"Resistance to nirmatrelvir (Paxlovid) has been shown by multiple groups and may already exist in clinical SARS-CoV-2 isolates. Here a panel of SARS-CoV-2 main protease (Mpro) variants and a robust cell-based assay are used to compare the resistance profiles of nirmatrelvir, ensitrelvir, and FB2001. The results reveal distinct resistance mechanisms (\u201cfingerprints\u201d) and indicate that these next-generation drugs have the potential to be effective against nirmatrelvir-resistant variants and vice versa.","version":"1.1","doi":"10.1101/2023.02.25.530000","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.24.529933","pub_date":"2023-2-27","title":"Binding of SARS-CoV-2 non-structured protein 1 to 40S ribosome inhibits mRNA translation","abstract":"Experiments have shown that non-structural protein 1 (NSP1) of SARS-CoV-2 is a factor that restricts cellular gene expression and prevents mRNA translation in the ribosome 40S subunit. However, the molecular mechanism of this phenomenon remains unclear. To clarify this issue, all-atom steered molecular dynamics and coarse-grained alchemical simulations were used to compare the binding affinity of mRNA to 40S ribosome in the absence and presence of NSP1. We found that NSP1 binding to the 40S ribosome dramatically increases the binding affinity of mRNA, which, in agreement with experiment, suggests that NSP1 can stall mRNA translation. The mRNA translation has been found to be driven by electrostatic mRNA-40S ribosome interactions. Water molecules have been demonstrated to play an important role in stabilizing the mRNA-40S ribosome complex. The NSP1 residues that are critical in triggering a translation arrest have been identified.","version":"1.1","doi":"10.1101/2023.02.24.529933","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.24.529952","pub_date":"2023-2-27","title":"Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy","abstract":"We recently reported that SARS-CoV-2 Nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the seasonal human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and non-infected cells by binding heparan-sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a non-overlapping set of 6 cytokines (CKs). As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12\u03b2-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and endemic HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionary conserved roles in manipulating host innate immunity and as a target for adaptive immunity.","version":"1.1","doi":"10.1101/2023.02.24.529952","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.25.529934","pub_date":"2023-2-27","title":"MDTOMO: Continuous conformational variability analysis in cryo electron subtomogram data using flexible fitting based on Molecular Dynamics simulations","abstract":"Cryo electron tomography (cryo-ET) allows observing macromolecular complexes in their native environment. The common routine of subtomogram averaging (STA) allows obtaining the three-dimensional (3D) structure of abundant macromolecular complexes, and can be coupled with discrete classification to reveal conformational heterogeneity of the sample. However, the number of complexes extracted from cryo-ET data is usually small, which restricts the discrete-classification results to a small number of enough populated states and, thus, results in a largely incomplete conformational landscape. Alternative approaches are currently being investigated to explore the continuity of the conformational landscapes that in situ cryo-ET studies could provide. In this article, we present MDTOMO, a method for analyzing continuous conformational variability in cryo-ET subtomograms based on Molecular Dynamics (MD) simulations. MDTOMO allows obtaining an atomic-scale model of conformational variability and the corresponding free-energy landscape, from a given set of cryo-ET subtomograms. The article presents the performance of MDTOMO on a synthetic ABC exporter dataset and an in situ SARS-CoV-2 spike dataset. MDTOMO allows analyzing dynamic properties of molecular complexes to understand their biological functions, which could also be useful for structure-based drug discovery.","version":"1.1","doi":"10.1101/2023.02.25.529934","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.26.530085","pub_date":"2023-2-26","title":"2019-20 H1N1 clade A5a.1 viruses have better in vitro replication compared with the co-circulating A5a.2 clade","abstract":"Surveillance for emerging human influenza virus clades is important for identifying changes in viral fitness and assessing antigenic similarity to vaccine strains. While fitness and antigenic structure are both important aspects of virus success, they are distinct characteristics and do not always change in a complementary manner. The 2019-20 Northern Hemisphere influenza season saw the emergence of two H1N1 clades: A5a.1 and A5a.2. While several studies indicated that A5a.2 showed similar or even increased antigenic drift compared with A5a.1, the A5a.1 clade was still the predominant circulating clade that season. Clinical isolates of representative viruses from these clades were collected in Baltimore, Maryland during the 2019-20 season and multiple assays were performed to compare both antigenic drift and viral fitness between clades. Neutralization assays performed on serum from healthcare workers pre- and post-vaccination during the 2019-20 season show a comparable drop in neutralizing titers against both A5a.1 and A5a.2 viruses compared with the vaccine strain, indicating that A5a.1 did not have antigenic advantages over A5a.2 that would explain its predominance in this population. Plaque assays were performed to investigate fitness differences, and the A5a.2 virus produced significantly smaller plaques compared with viruses from A5a.1 or the parental A5a clade. To assess viral replication, low MOI growth curves were performed on both MDCK-SIAT and primary differentiated human nasal epithelial cell cultures. In both cell cultures, A5a.2 yielded significantly reduced viral titers at multiple timepoints post-infection compared with A5a.1 or A5a. Receptor binding was then investigated through glycan array experiments which showed a reduction in receptor binding diversity for A5a.2, with fewer glycans bound and a higher percentage of total binding attributable to the top three highest bound glycans. Together these data indicate that the A5a.2 clade had a reduction in viral fitness, including reductions in receptor binding, that may have contributed to the limited prevalence observed after emergence.","version":"1.1","doi":"10.1101/2023.02.26.530085","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.29.526074","pub_date":"2023-2-25","title":"Chimeric chikungunya virus-like particles with surface exposed SARS-CoV-2 RBD elicits potent immunogenic responses in mice","abstract":"The SARS-CoV-2 pandemic has reinforced efforts for developing effective vaccination strategy for existing and emerging viruses. Currently there are various vaccine technology available for treating viral diseases, however it is imperative to develop and investigate second-generation vaccines such as chimeric virus-like particles (chi-VLPs) vaccine for increased immunogenicity, ease of production and scalability to supplement the worldwide vaccine supply. Intriguingly, chi-VLPs expresses more than one antigenic epitope on its surface, hence it is expected to be a more effective vaccine candidate. Hereby, this study reports, a novel bivalent vaccine design of chimeric alphavirus coronavirus virus-like particles (ChAC-VLPs), displaying fusion glycoproteins of CHIKV and receptor binding domain (RBD) of SARS-CoV-2 on its surface. Uniqueness and versatility of ChAC-VLPs has been demonstrated via a various techniques including Western blot, Immunofluorescence, cryoEM, and dynamic light scattering (DLS). The multimeric epitope display of immunogenic antigens, i.e CHIKV envelop glycoprotein and SARS-CoV-2 RBD was validated by cell-based assays. ChAC-VLP immunized mice has shown substantial neutralization titres for CHIKV (PRNT50 of 1:25) from the serum collected after 2nd booster doses. Similarly, serum antibodies were detected for SARS-CoV2 RBD as observed by antigen specific ELISA and validated using surface plasmon resonance (SPR). SPR binding response was detected to be >200 RU for anti-RBD antibody in post-immunized mice sera. In conclusion, present study proposes ChAC-VLPs as a potential hybrid vaccine candidate for CHIKV and SARS-CoV-2 infection and contributes valuable insights in chi-VLPs domain.","version":"1.2","doi":"10.1101/2023.01.29.526074","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.23.529742","pub_date":"2023-2-24","title":"Isolation may select for earlier and higher peak viral load but shorter duration in SARS-CoV-2 evolution","abstract":"During the COVID-19 pandemic, human behavior change as a result of nonpharmaceutical interventions such as isolation may have induced directional selection for viral evolution. By combining previously published empirical clinical data analysis and multi-level mathematical modeling, we found that the SARS-CoV-2 variants selected for as the virus evolved from the pre-Alpha to the Delta variant had earlier and higher infectious periods but a shorter duration of infection. Selection for increased transmissibility shapes the viral load dynamics, and the isolation measure is likely to be a driver of these evolutionary transitions. In addition, we showed that a decreased incubation period and an increased proportion of asymptomatic infection were also positively selected for as SARS-CoV-2 mutated to the extent that people did not isolate. We demonstrated that the Omicron variants evolved in these ways to adapt to human behavior. The quantitative information and predictions we present here can guide future responses in the potential arms race between pandemic interventions and viral evolution.","version":"1.1","doi":"10.1101/2023.02.23.529742","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.23.529833","pub_date":"2023-2-24","title":"Machine learning on large scale perturbation screens for SARS-CoV-2 host factors identifies \u03b2-catenin/CBP inhibitor PRI-724 as a potent antiviral","abstract":"Expanding antiviral treatment options against SARS-CoV-2 remains crucial as the virus evolves rapidly and drug resistant strains have emerged. Broad spectrum host-directed antivirals (HDA) are promising therapeutic options, however the robust identification of relevant host factors by CRISPR/Cas9 or RNA interference screens remains challenging due to low consistency in the resulting hits. To address this issue, we employed machine learning based on experimental data from knockout screens and a drug screen. As gold standard, we assembled perturbed genes reducing virus replication or protecting the host cells. The machines based their predictions on features describing cellular localization, protein domains, annotated gene sets from Gene Ontology, gene and protein sequences, and experimental data from proteomics, phospho-proteomics, protein interaction and transcriptomic profiles of SARS-CoV-2 infected cells. The models reached a remarkable performance with a balanced accuracy of 0.82 (knockout based classifier) and 0.71 (drugs screen based classifier), suggesting patterns of intrinsic data consistency. The predicted host dependency factors were enriched in sets of genes particularly coding for development, morphogenesis, and neural related processes. Focusing on development and morphogenesis-associated gene sets, we found \u03b2-catenin to be central and selected PRI-724, a canonical \u03b2-catenin/CBP disruptor, as a potential HDA. PRI-724 limited infection with SARS-CoV-2 variants, SARS-CoV-1, MERS-CoV and IAV in different cell line models. We detected a concentration-dependent reduction in CPE development, viral RNA replication, and infectious virus production in SARS-CoV-2 and SARS-CoV-1-infected cells. Independent of virus infection, PRI-724 treatment caused cell cycle deregulation which substantiates its potential as a broad spectrum antiviral. Our proposed machine learning concept may support focusing and accelerating the discovery of host dependency factors and the design of antiviral therapies. Drug resistance to pathogens is a well-known phenomenon which was also observed for SARS-CoV-2. Given the gradually increasing evolutionary pressure on the virus by herd immunity, we attempted to enlarge the available antiviral repertoire by focusing on host proteins that are usurped by viruses. The identification of such proteins was followed within several high throughput screens in which genes are knocked out individually. But, so far, these efforts led to very different results. Machine learning helps to identify common patterns and normalizes independent studies to their individual designs. With such an approach, we identified genes that are indispensable during embryonic development, i.e., when cells are programmed for their specific destiny. Shortlisting the hits revealed \u03b2-catenin, a central player during development, and PRI-724, which inhibits the interaction of \u03b2-catenin with cAMP responsive element binding (CREB) binding protein (CBP). In our work, we confirmed that the disruption of this interaction impedes virus replication and production. In A549-AT cells treated with PRI-724, we observed cell cycle deregulation which might contribute to the inhibition of virus infection, however the exact underlying mechanisms needs further investigation.","version":"1.1","doi":"10.1101/2023.02.23.529833","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.24.529520","pub_date":"2023-2-24","title":"Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology","abstract":"Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection. A vaccine candidate with polyprotein antigens displayed on nanocages induces neutralizing antibodies to multiple SARS-like coronaviruses.","version":"1.1","doi":"10.1101/2023.02.24.529520","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.22.529625","pub_date":"2023-2-23","title":"Broadly effective ACE2 decoy proteins protect mice from lethal SARS-CoV-2 infection","abstract":"As SARS-CoV-2 variants have been causing increasingly serious drug resistance problem, development of broadly effective and hard-to-escape anti-SARS-CoV-2 agents is in urgent need. Here we describe further development and characterization of two SARS-CoV-2 receptor decoy proteins, ACE2-Ig-95 and ACE2-Ig-105/106. We found that both proteins had potent and robust in vitro neutralization activities against diverse SARS-CoV-2 variants including Omicron, with an average IC50 of up to 37 pM. In a stringent lethal SARS-CoV-2 infection mouse model, both proteins lowered lung viral load by up to \u223c1000 fold, prevented the emergence of clinical signs in >75% animals, and increased animal survival rate from 0% (untreated) to >87.5% (treated). These results demonstrate that both proteins are good drug candidates for protecting animals from severe COVID-19. In a head-to-head comparison of these two proteins with five previously-described ACE2-Ig constructs, we found that two of these constructs, each carrying five surface mutations in the ACE2 region, had partial loss of neutralization potency against three SARS-CoV-2 variants. These data suggest that extensively mutating ACE2 residues near the RBD-binding interface should be avoided or performed with extra caution. Further, we found that both ACE2-Ig-95 and ACE2-Ig-105/106 could be produced to gram/liter level, demonstrating the developability of them as biologic drug candidates. Stress-condition stability test of them further suggests that more studies are required in the future to improve the stability of these proteins. These studies provide useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. Engineering soluble ACE2 proteins that function as a receptor decoy to block SARS-CoV-2 infection is a very attractive approach to broadly effective and hard-to-escape anti-SARS-CoV-2 agents. This study here describes development of two antibody-like soluble ACE2 proteins that broadly block diverse SARS-CoV-2 variants including Omicron. In a stringent COVID-19 mouse model, both proteins successfully protected >87.5% animals from lethal SARS-CoV-2 infection. In addition, a head-to-head comparison of the two constructs developed in this study with five previously-described ACE2 decoy constructs were performed here. Two previously-described constructs with relatively more ACE2-surface mutations were found with less robust neutralization activities against diverse SARS-CoV-2 variants. Further, the developability of the two proteins as biologic drug candidates was also assessed here. This study provides two broadly anti-SARS-CoV-2 drug candidates and useful insight into critical factors for engineering and preclinical development of ACE2 decoy as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. Two antibody-like ACE2 decoy proteins could block diverse SARS-CoV-2 variants and prevent animals from severe COVID-19.","version":"1.1","doi":"10.1101/2023.02.22.529625","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.22.469642","pub_date":"2023-2-23","title":"Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing","abstract":"Single cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNA-Seq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3\u2032 (10X 3\u2032) and Chromium Next GEM Single Cell V(D)J (10X 5\u2032) sequencing, we find that 10X 5\u2032 with an extended read 1 (R1) sequencing strategy maximizes the detection of sgmRNAs by increasing the number of unambiguous reads spanning leader-sgmRNA junction sites. Using this method, we show that viral gene expression is highly correlated across cells suggesting a relatively consistent proportion of viral sgmRNA production throughout infection. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.","version":"1.2","doi":"10.1101/2021.11.22.469642","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.23.529497","pub_date":"2023-2-23","title":"Analysis of the SARS-CoV-2 spike protein revealed that blocked receptor-binding domain antigenicity decreases the production of neutralizing antibodies in vivo","abstract":"The identification of SARS-CoV-2 spike protein distribution and function in target cells has raised concerns about its possible impacts on vaccine efficacy and pathogenic effect in host cells. Thus, a better understanding of such consequences is necessary. In this study, we studied the biological characteristics of six variants of SARS-CoV-2 in A549 and HEK293 cells using four different technologies. The results showed that compared to the other fragments, the full-length spike protein exhibited the highest expression on the cell surface and was detectable in the cell supernatant, cytoplasm, and nucleus. Except for the cell surface, the S1 subunit generally expressed higher than the full-length spike protein. RBD and S2 subunits were expressed in the cytoskeleton. The SS-RBD peptide, which consists of a 19-amino acid signal peptide sequence (SS)-linked RBD, exhibited the highest expression in the cell supernatant among all other studied peptides. The SS positively enhanced the expression, migration, and secretion of SS-RBD from the cytoskeleton to the supernatant. Importantly, the FACS assay results showed that neutralizing antibodies (NAbs) could recognize SS-RBD but not RBD in the transfected cells, suggesting that RBD was tightly bound by ACE2 in HEK293 cells. In contrast, the antigenicity of the RBD in the spike protein was revealed and efficiently monitored only by 6-His-tag mAbs. Thus, our findings demonstrated that ACE2 blocks crucial immunogenic epitopes of the RBD, and the full-length spike protein mainly induces non-neutralizing antibodies in vivo. Therefore, we suggest that reducing ACE2 binding affinity and exposing the immunogenicity of the RBD on the spike protein is imperative for improving vaccine efficacy and generating new SARS-CoV-2 mRNA vaccines.","version":"1.1","doi":"10.1101/2023.02.23.529497","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.19.23286150","pub_date":"2023-02-23","title":"The Impact of COVID-19 on Cancer Screening and Treatment in Older Adults: The Multiethnic Cohort Study","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The Coronavirus Disease of 2019 (COVID-19) has impacted the health and day-to-day life of individuals, especially the elderly and people with certain pre-existing medical conditions, including cancer. The purpose of this study was to investigate how COVID-19 impacted access to cancer screenings and treatment, by studying the participants in the Multiethnic Cohort (MEC) study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>The MEC has been following over 215,000 residents of Hawai\u2019i and Los Angeles for the development of cancer and other chronic diseases since 1993-1996. It includes men and women of five racial and ethnic groups: African American, Japanese American, Latino, Native Hawaiian, and White. In 2020, surviving participants were sent an invitation to complete an online survey on the impact of COVID-19 on their daily life activities, including adherence to cancer screening and treatment. Approximately 7,000 MEC participants responded. A cross-sectional analysis was performed to investigate the relationships between the postponement of regular health care visits and cancer screening procedures or treatment with race and ethnicity, age, education, and comorbidity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Women with more education, women with lung disease, COPD, or asthma, and women and men diagnosed with cancer in the past 5 years were more likely to postpone any cancer screening test/procedure due to the COVID-19 pandemic. Groups less likely to postpone cancer screening included older women compared to younger women and Japanese American men and women compared to White men and women.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>This study revealed specific associations of race/ethnicity, age, education level, and comorbidities with the cancer-related screening and healthcare of MEC participants during the COVID-19 pandemic. Increased monitoring of patients in high-risk groups for cancer and other diseases is of the utmost importance as the chance of undiagnosed cases or poor prognosis is increased as a result of delayed screening and treatment.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This research was partially supported by the Omidyar \u2018Ohana Foundation and grant U01 CA164973 from the National Cancer Institute.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.02.19.23286150","journal":"medRxiv","score":null},{"id":"10.1101/2022.10.27.514096","pub_date":"2023-2-22","title":"Tocilizumab treatment leads to early resolution of lymphopenia and myeloid dysregulation in patients hospitalized with COVID-19","abstract":"High interleukin (IL)-6 levels are associated with more severe clinical manifestations in patients hospitalized with COVID-19, but the complex role of IL-6 in antiviral and inflammatory processes has made it difficult to decipher its involvement in the disease. IL-6 receptor blockade by tocilizumab (anti-IL6R; Actemra) is used globally for the treatment of severe COVID-19, yet a molecular understanding of the therapeutic benefit remains unclear. We characterized the immune profile and identified cellular and molecular pathways directly modified by tocilizumab in peripheral blood samples collected from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial that assessed the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19 pneumonia. We identified factors predicting disease severity and clinical outcomes, including markers of inflammation, lymphopenia, myeloid dysregulation, and organ injury. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab in addition to identifying novel pharmacodynamic biomarkers. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysregulation associated with severe COVID-19, indicating greater anti-inflammatory activity relative to standard of care and potentially leading to faster recovery in patients hospitalized with COVID-19. Interleukin-6 receptor blockade with tocilizumab accelerated resolution of myeloid dysfunction and lymphopenia in patients hospitalized with COVID-19","version":"1.2","doi":"10.1101/2022.10.27.514096","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.21.529344","pub_date":"2023-2-21","title":"Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad and durable systemic and mucosal immune responses","abstract":"Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8+ cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (TH1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8+ T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.","version":"1.1","doi":"10.1101/2023.02.21.529344","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.14.528476","pub_date":"2023-2-21","title":"Sarbecoviruses of British Horseshoe Bats; Sequence Variation and Epidemiology","abstract":"Horseshoe bats are the natural hosts of the Sarbecovirus subgenus that includes SARS-CoV-1 and 2. Despite the devastating impacts of the COVID-19 pandemic, there is still little known about the underlying epidemiology and virology of sarbecoviruses in their natural hosts, leaving large gaps in our pandemic preparedness. Here we describe the results of PCR testing for sarbecoviruses in the two horseshoe bat species (Rhinolophus hipposideros and R. ferrumequinum) present in Great Britain, collected in 2021-22 during the peak of COVID-19 pandemic. One hundred and ninety seven R. hipposideros samples from 33 roost sites and 277 R. ferremequinum samples from 20 roost sites were tested. No coronaviruses were detected in any samples from R. ferrumequinum whereas 44% and 56% of individual and pooled (respectively) faecal samples from R. hipposideros across multiple roost sites tested positive in a sarbecovirus-specific qPCR. Full genome sequences were generated from three of the positive samples (and partial genomes from two more) using Illumina RNAseq on unenriched samples. Phylogenetic analyses showed that the obtained sequences belong to the same monophyletic clade, with >95% similarity, as previously reported European isolates from R. hipposideros. The sequences differed in the presence or absence of accessory genes ORF 7b, 9b and 10. All lacked the furin cleavage site of SARS-CoV-2 spike gene and are therefore unlikely to be infective for humans. These results demonstrate a lack, or at least low incidence, of SARS-CoV-2 spill over from humans to susceptible GB bats, and confirm that sarbecovirus infection is widespread in R. hipposideros. Despite frequently sharing roost sites with R. ferrumequinum, no evidence of cross-species transmission was found.","version":"1.2","doi":"10.1101/2023.02.14.528476","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.19.529105","pub_date":"2023-2-21","title":"Efficient Viral Capture and Inactivation from Bioaerosols Using Electrostatic Precipitation","abstract":"The presence of infectious viral particles in bioaerosols generated during laparoscopic surgery places surgical staff at significant risk of infection and represents a major cause of nosocomial infection. These factors contributed to the postponement and cancellation of countless surgical procedures during the early stages of the ongoing COVID-19 pandemic, causing backlogs, increased waiting times for surgical procedures and excess deaths indirectly related to the pandemic. The development and implementation of devices that effectively inactivate viral particles from bioaerosols would be beneficial in limiting or preventing the spread of infections from such bioaerosols. Here, we sought to evaluate whether electrostatic precipitation (EP) is a viable means to capture and inactivate both non-enveloped (Adenovirus) and enveloped (SARS-CoV-2 Pseudotyped Lentivirus) viral particles present in bioaerosols. We developed a closed-system model to mimic the release of bioaerosols during laparoscopic surgery. Known concentrations of each virus were aerosolised into the model system, exposed to EP using a commercially available system (UltravisionTM, Alesi Surgical Limited, UK) and collected in a BioSampler for analysis. Using qPCR to quantify viral genomes and transduction assays to quantify biological activity, we show that both enveloped and non-enveloped viral particles were efficiently captured and inactivated by EP. Both capture and inactivation could be further enhanced when increasing the voltage to 10kV, or when using two Ultravision\u2122 discharge electrodes together at 8kV. This study highlights EP as an efficient means for capturing and inactivating viral particles present in bioaerosols. The use of EP may limit the spread of diseases, reducing nosocomial infections and potentially enable the continuation of surgical procedures during periods of viral pandemics. Bioaerosols released from patients during surgery have the potential to facilitate viral spread. Ultravision\u2122 technology works via the process of electrostatic precipitation. Electrostatic precipitation can be manipulated to capture and inactivate aerosolised viral particles, preventing viral spread. Electrostatic precipitation is effective against both enveloped and non-enveloped viral particles. Electrostatic precipitation represents a viable means to reduce nosocomial infections.","version":"1.1","doi":"10.1101/2023.02.19.529105","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.20.529249","pub_date":"2023-2-20","title":"Antibody-mediated cell entry of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. We report here that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interactions with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may expand the viral tropism to otherwise nonpermissive cell types; they have important implications for viral transmission and pathogenesis.","version":"1.1","doi":"10.1101/2023.02.20.529249","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.13.528205","pub_date":"2023-2-20","title":"Regulatory T Cell-like Response to SARS-CoV-2 in Jamaican Fruit Bats (Artibeus jamaicensis) Transduced with Human ACE2","abstract":"Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer\u2019s patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-\u03b2, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptibility to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease. Bats are reservoir hosts of many viruses that infect humans, yet little is known about how they host these viruses, principally because of a lack of relevant and susceptible bat experimental infection models. Although SARS-CoV-2 originated in bats, no robust infection models of bats have been established. We determined that Jamaican fruit bats are poorly susceptible to SARS-CoV-2; however, their lungs can be transduced with human ACE2, which renders them susceptible to SARS-CoV-2. Despite robust infection of the lungs and diminishment of pulmonary cellularity, the bats showed no overt signs of disease and cleared the infection after two weeks. Despite clearance of infection, only low-titer antibody responses occurred and only a single bat made neutralizing antibody. Assessment of the CD4+ helper T cell response showed that activated cells expressed the regulatory T cell cytokines IL-10 and TGF\u03b2 that may have tempered pulmonary inflammation.","version":"1.2","doi":"10.1101/2023.02.13.528205","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.19.529128","pub_date":"2023-2-20","title":"A robust Platform for Integrative Spatial Multi-omics Analysis to Map Immune Responses to SARS-CoV-2 infection in Lung Tissues","abstract":"The SARS-CoV-2 (COVID-19) virus has caused a devastating global pandemic of respiratory illness. To understand viral pathogenesis, methods are available for studying dissociated cells in blood, nasal samples, bronchoalveolar lavage fluid, and similar, but a robust platform for deep tissue characterisation of molecular and cellular responses to virus infection in the lungs is still lacking. We developed an innovative spatial multi-omics platform to investigate COVID-19-infected lung tissues. Five tissue-profiling technologies were combined by a novel computational mapping methodology to comprehensively characterise and compare the transcriptome and targeted proteome of virus infected and uninfected tissues. By integrating spatial transcriptomics data (Visium, GeoMx and RNAScope) and proteomics data (CODEX and PhenoImager HT) at different cellular resolutions across lung tissues, we found strong evidence for macrophage infiltration and defined the broader microenvironment surrounding these cells. By comparing infected and uninfected samples, we found an increase in cytokine signalling and interferon responses at different sites in the lung and showed spatial heterogeneity in the expression level of these pathways. These data demonstrate that integrative spatial multi-omics platforms can be broadly applied to gain a deeper understanding of viral effects on cellular environments at the site of infection and to increase our understanding of the impact of SARS-CoV-2 on the lungs.","version":"1.1","doi":"10.1101/2023.02.19.529128","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.20.529234","pub_date":"2023-2-20","title":"Bovine milk glycoproteins inhibit SARS-CoV-2 and influenza virus co-infection","abstract":"The attachment of S1 subunit of spike (S) protein to angiotensin-converting enzyme 2 (ACE2) is the first and crucial step of SARS-CoV-2 infection. Although S protein and ACE2 are heavily glycosylated, the precise roles of glycans in their interactions are still unclear. Here, we profiled the glycopatterns of S1 subunit of SARS-CoV-2 and ACE2, and found that the galactosylated glycoforms were dominant in both S1 subunit and ACE2. Interestingly, S1 subunit exhibited the property of glycan-binding protein (GBP) and adhered to the ACE2 via binding to the galactosylated glycans on the ACE2. Our earlier findings demonstrated that the sialylated glycoproteins isolated from bovine milk potently inhibit and neutralize viral activity against influenza A virus (IAV). Importantly, we proved further that the galactosylated glycans on isolated glycoproteins bind to the glycan recognition domains of S1 subunit and competitively inhibit binding of S1 subunit to ACE2 and ultimately impede the entry of SARS-CoV-2 pseudovirus into host cells. We provided a potential protein drug that could be multiple simultaneous inhibitor for coronavirus and IAV co-infection.","version":"1.1","doi":"10.1101/2023.02.20.529234","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.13.507829","pub_date":"2023-2-20","title":"SARS-CoV-2 Protein Nsp2 Stimulates Translation Under Normal and Hypoxic Conditions","abstract":"When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.","version":"1.4","doi":"10.1101/2022.09.13.507829","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.20.529243","pub_date":"2023-2-20","title":"Genetic consequences of effective and suboptimal dosing with mutagenic drugs in a hamster model of SARS-CoV-2 infection","abstract":"Mutagenic antiviral drugs have shown promising results against multiple viruses, yet concerns have been raised about whether their use might promote the emergence of new and harmful viral variants. Here, we examine the genetic consequences of effective and suboptimal dosing of favipiravir and molnupiravir in the treatment of SARS-CoV-2 infection in a hamster model. We identify a dose-dependent effect upon the mutational load in a viral population, with molnupiravir having a greater potency than favipiravir per mg/kg of treatment. The emergence of de novo variants was largely driven by stochastic processes, with evidence of compensatory adaptation but not of the emergence of drug resistance or novel immune phenotypes. Effective doses for favipiravir and molunpiravir correspond to similar levels of mutational load. Combining both drugs had an increased impact on both efficacy and mutational load. Our results suggest the potential for mutational load to provide a marker for clinical efficacy.","version":"1.1","doi":"10.1101/2023.02.20.529243","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.17.529036","pub_date":"2023-2-19","title":"Tracking and curating putative SARS-CoV-2 recombinants with RIVET","abstract":"Identifying and tracking recombinant strains of SARS-CoV-2 is critical to understanding the evolution of the virus and controlling its spread. But confidently identifying SARS-CoV-2 recombinants from thousands of new genome sequences that are being shared online every day is quite challenging, causing many recombinants to be missed or suffer from weeks of delay in being formally identified while undergoing expert curation. We present RIVET \u2013 a software pipeline and visual platform that takes advantage of recent algorithmic advances in recombination inference to comprehensively and sensitively search for potential SARS-CoV-2 recombinants, and organizes the relevant information in a web interface that would help greatly accelerate the process identifying and tracking recombinants. RIVET-based web interface displaying the most updated analysis of potential SARS-CoV-2 recombinants is available at https://rivet.ucsd.edu/. RIVET\u2019s frontend and backend code is freely available under MIT license at https://github.com/TurakhiaLab/rivet. All inputs necessary for running the RIVET\u2019s backend workflow for SARS-CoV-2 are available through a public database maintained by UCSC (https://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/). yturakhia@ucsd.edu","version":"1.1","doi":"10.1101/2023.02.17.529036","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.14.527605","pub_date":"2023-2-19","title":"Protective effect of plasma neutralization from prior SARS-CoV-2 Omicron infection against BA.5 subvariant symptomatic reinfection","abstract":"From December 2022 to January 2023, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) swept across mainland China. It is crucial to estimate the protective effect of the neutralizing antibodies generated by such mass infections against the next potential SARS-CoV-2 reinfection wave, especially if driven by CH.1.1 or XBB.1.5. Previously, we recruited and continuously followed a cohort of individuals that experienced Omicron BA.1, BA.2, and BA.5 breakthrough infections, as well as a control cohort with no history of SARS-CoV-2 infection. In the previously uninfected cohort, the total symptomatic infection rate surveyed during the outbreak was 91.6%, while the symptomatic reinfection rate was 32.9%, 10.5%, and 2.8% among individuals with prior Omicron BA.1, BA.2 and BA.5 infection, respectively, with median intervals between infections of 335, 225 and 94 days. Pseudovirus neutralization assays were performed in plasma samples collected from previously Omicron BA.1-infected individuals approximately 3 months before the outbreak. Results indicate a robust correlation between the plasma neutralizing antibody titers and the protective effect against symptomatic reinfection. The geometric mean of the 50% neutralizing titers (NT50) against D614G, BA.5, and BF.7 were 2.0, 2.5, and 2.3-fold higher in individuals without symptomatic reinfection than in those with symptomatic reinfection (p < 0.01). Low plasma neutralizing antibody titer (below the geometric mean of NT50) was associated with an enhanced cumulative risk of symptomatic reinfection, with a hazard ratio (HR) of 23.55 (95% CI: 9.23-60.06) against BF.7 subvariant. Importantly, neutralizing antibodies titers post one month after BF.7/BA.5 breakthrough infections against CH.1.1 and XBB.1.5 are similar to that against BF.7 from individuals with prior BA.1 infection while not experiencing a symptomatic BF.7/BA.5 reinfection (plasma collected 3 months before the outbreak), suggesting that the humoral immunity generated by the current BF.7/BA.5 breakthrough infection may provide protection against CH.1.1 and XBB.1.5 symptomatic reinfection wave for 4 months. Of note, the higher hACE2 binding of XBB.1.5 may reduce the protection period since the potential increase of infectivity.","version":"1.1","doi":"10.1101/2023.02.14.527605","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.07.527406","pub_date":"2023-2-18","title":"Neutralization of SARS-CoV-2 BQ.1.1 and XBB.1.5 by Breakthrough Infection Sera from Previous and Current Waves in China","abstract":"SARS-CoV-2 is continuing to evolve and diversify, with an array of various Omicron sub-lineages, including BA.5, BA.2.75, BN.1, BF.7, BQ.1, BQ.1.1, XBB and XBB.1.5, now circulating globally at recent time. In this study, we evaluated the neutralization sensitivity of a comprehensive panel of Omicron subvariants to sera from different clinical cohorts, including individuals who received homologous or heterologous booster vaccinations, vaccinated people who had Delta or BA.2 breakthrough infection in previous waves, and patients who had BA.5 or BF.7 breakthrough infection in the current wave in China. All the Omicron subvariants exhibited substantial neutralization evasion, with BQ.1, BQ.1.1, XBB.1, and XBB.1.5 being the strongest escaped subvariants. Sera from Omicron breakthrough infection, especially the recent BA.5 or BF.7 breakthrough infection, exhibited higher neutralizing activity against all Omicron sub-lineages, indicating the chance of BA.5 and BF.7 being entirely replaced by BQ or XBB subvariants in China in a short-term might be low. We also demonstrated that the BQ and XBB subvariants were the most resistant viruses to monoclonal antibodies. Continuing to monitor the immune escape of SARS-CoV-2 emerging variants and developing novel broad-spectrum vaccines and antibodies are still crucial.","version":"1.2","doi":"10.1101/2023.02.07.527406","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.16.23286017","pub_date":"2023-02-18","title":"Long-term outdoor air pollution and COVID-19 mortality in London: an individual-level analysis","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The risk of COVID-19 severity and mortality differs markedly by age, socio-demographic characteristics and pre-existing health status. Various studies have suggested that higher air pollution exposures also increase the likelihood of dying from COVID-19.</jats:p>\n                  <jats:p>\n                    Objectives: To assess the association between long-term outdoor air pollution (NO\n                    <jats:sub>2</jats:sub>\n                    , NOx, PM\n                    <jats:sub>10</jats:sub>\n                    and PM\n                    <jats:sub>2.5</jats:sub>\n                    ) concentrations and the risk of death involving COVID-19, using a large individual-level dataset.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We used comprehensive individual-level data from the Office for National Statistics\u2019 Public Health Data Asset for September 2020 to January 2022 and London Air Quality Network modelled air pollution concentrations available for 2016. Using Cox proportional hazard regression models, we adjusted for potential confounders including age, sex, vaccination status, dominant virus variants, geographical factors (such as population density), ethnicity, area and household-level deprivation, and health comorbidities.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    There were 737,356 confirmed COVID-19 cases including 9,315 COVID-related deaths. When only adjusting for age, sex, and vaccination status, there was an increased risk of dying from COVID-19 with increased exposure to all air pollutants studied (NO\n                    <jats:sub>2</jats:sub>\n                    : HR 1.07 [95% confidence interval: 1.04-1.12] per 10 \u03bcg/m\n                    <jats:sup>3</jats:sup>\n                    ; NOx: 1.05[1.02-1.09] per 20 \u03bcg/m\n                    <jats:sup>3</jats:sup>\n                    ; PM\n                    <jats:sub>10</jats:sub>\n                    : 1.32[1.15-1.51] per 10 \u03bcg/m\n                    <jats:sup>3</jats:sup>\n                    ; PM\n                    <jats:sub>2.5</jats:sub>\n                    : 1.29[1.12-1.49] per 5 \u03bcg/m\n                    <jats:sup>3</jats:sup>\n                    ). However, after adjustment including ethnicity and socio-economic factors the HRs were close to unity (NO\n                    <jats:sub>2</jats:sub>\n                    : 0.98[0.90-1.06]; NOx: 0.99[0.94-1.04]; PM\n                    <jats:sub>10</jats:sub>\n                    : 0.95[0.74-1.22]; PM\n                    <jats:sub>2.5</jats:sub>\n                    : 0.90[0.67-1.20]). Additional adjustment for dominant variant or pre-existing health comorbidities did not alter the results.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Observed associations between long-term outdoor air pollution exposure and COVID-19 mortality in London are strongly confounded by geography, ethnicity and deprivation.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Summary</jats:title>\n                  <jats:p>Using a large individual-level dataset, we found that a positive association between long-term outdoor air pollution and COVID-19 mortality in London did not persist after adjusting for confounders including population density, ethnicity and deprivation.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2023.02.16.23286017","journal":"medRxiv","score":null},{"id":"10.1101/2023.02.16.528881","pub_date":"2023-2-17","title":"Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19","abstract":"It has been revealed that SARS-CoV-2 can be efficiently isolated from clinical specimens such as nasal/nasopharyngeal swabs or saliva in cultured cells. In this study, we examined the efficiency of viral isolation including SARS-CoV-2 mutant strains between nasal/nasopharyngeal swab or saliva specimens. Furthermore, we also examined the comparison of viral isolation rates by sample species using simulated specimens for COVID- 19. As a result, it was found that the isolation efficiency of SARS-CoV-2 in the saliva specimens was significantly lower than that in the nasal/nasopharyngeal swab specimens. In order to determine which component of saliva is responsible for the lower isolation rate of saliva specimens, we tested the abilities of lactoferrin, amylase, cathelicidin, and mucin, which are considered to be abundant in saliva, to inhibit the infection of SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Lactoferrin and amylase were found to inhibit SARS-CoV-2pv infection. In conclusion, even if the same number of viral genome copies was detected by the real-time RT-PCR test, infection of SARS-CoV-2 present in saliva is thought to be inhibited by inhibitory factors such as lactoferrin and amylase, compared to nasal/nasopharyngeal swab specimens.","version":"1.1","doi":"10.1101/2023.02.16.528881","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.17.528914","pub_date":"2023-2-17","title":"A burns and COVID-19 shared stress responding gene network deciphers CD1C-CD141-DCs as the key cellular components in septic prognosis","abstract":"Differential body responses to various stresses, infectious or noninfectious, govern clinical outcomes ranging from asymptoma to death. However, the common molecular and cellular nature of the stress responsome across different stimuli is not described. In this study, we compared the expression behaviors between burns and COVID-19 infection by choosing the transcriptome of peripheral blood from related patients as the analytic target since the blood cells reflect the systemic landscape of immune homeostasis. We identified an immune co-stimulator (CD86)-centered network, named stress-response core (SRC), which coordinated multiple immune processes and was robust in membership and highly related to the clinical traits in both burns and COVID-19. An independent whole blood single-cell RNA sequencing of COVID-19 patients demonstrated that the monocyte-dendritic cell (Mono-DC) wing was the major cellular source of the SRC, among which the higher expression of the SRC in the monocyte was associated with the asymptomatic COVID-19 patients, while the quantity-restricted and function-defected CD1C-CD141-DCs were recognized as the key signature which linked to bad consequences in COVID-19. Specifically, the proportion of the CD1C-CD141-DCs and their SRC expression levels were step-wise reduced along with worse clinic conditions while the sub-cluster of CD1C-CD141-DCs of the critical COVID-19 patients was characterized of IFN signaling quiescence, high mitochondrial metabolism and immune-communication inactivation. Thus, our study identified an expression-synchronized and function-focused gene network which was decreased under burns and COVID-19 stress and argued the CD1C-CD141-DC as the prognosis-related cell population which might serve as a new target of diagnosis and therapy.","version":"1.1","doi":"10.1101/2023.02.17.528914","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.15.528742","pub_date":"2023-2-16","title":"Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the \u0394orf2-8, \u0394orf2.4, and \u0394orf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the \u0394orf2-8 and \u0394orf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the \u0394orf2.4-puro replicon by inserting a puromycin-resistant gene into the \u0394orf2.4 replicon. We observed the more sustained replication of the \u0394orf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.","version":"1.1","doi":"10.1101/2023.02.15.528742","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.15.528538","pub_date":"2023-2-15","title":"Sotrovimab retains activity against SARS-CoV-2 Omicron variant BQ.1.1 in a non-human primate model","abstract":"The SARS-CoV2 Omicron variants have acquired new Spike mutations leading to escape from the most of the currently available monoclonal antibody treatments reducing the options for patients suffering from severe Covid-19. Recently, both in vitro and in vivo data have suggested that Sotrovimab could retain partial activity against recent omicron sub-lineage such as BA.5 variants, including BQ.1.1. Here we report full efficacy of Sotrovimab against BQ.1.1 viral replication as measure by RT-qPCR in a non-human primate challenge model.","version":"1.1","doi":"10.1101/2023.02.15.528538","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.15.528632","pub_date":"2023-2-15","title":"Development and characterization of a multimeric recombinant protein based on the spike protein receptor binding domain of SARS-CoV-2 that can neutralize virus infection","abstract":"The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, has four structural proteins and sixteen non-structural proteins. The S-protein is one of the structural proteins exposed on the surface of the virus and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry. We stably expressed in a constitutive manner in HEK293 cells a new recombinant protein containing a signal sequence of immunoglobulin to produce an extended C-terminal portion of the RBD followed by a region responsible for the trimerization inducer of the bacteriophage T4, and a sequence of 6 histidines. The protein was produced and released in the culture supernatant of cells and was purified by Ni-agarose column and exclusion chromatography. It was then characterized by SDS-polyacrylamide gel and used as antigen to generate protective antibodies to inhibit ACE2 receptor interaction and virus entry into Vero cells. The purified protein displayed a molecular mass of 135 kDa and with a secondary structure like the monomeric RBD. Electrophoresis analysis in SDS-polyacrylamide gel with and without reducing agents, and in the presence of crosslinkers indicated that it forms a multimeric structure composed of trimers and hexamers. The purified protein was able to bind the ACE2 receptor and generated high antibody titers in mice (1:10000), capable of inhibiting the binding of biotin labeled ACE2 to the virus S1 subunit, and to neutralize the entry of the SARS-CoV-2 Wuhan strain into cells. Our results characterize a new multimeric protein based on S1 subunit to combat COVID-19, as a possible immunogen or antigen for diagnosis.","version":"1.1","doi":"10.1101/2023.02.15.528632","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.13.528411","pub_date":"2023-2-15","title":"Vulture: Cloud-enabled scalable mining of microbial reads in public scRNA-seq data","abstract":"The rapidly growing collection of public single-cell sequencing data have become a valuable resource for molecular, cellular and microbial discovery. Previous studies mostly overlooked detecting pathogens in human single-cell sequencing data. Moreover, existing bioinformatics tools lack the scalability to deal with big public data. We introduce Vulture, a scalable cloud-based pipeline that performs microbial calling for single-cell RNA sequencing (scRNA-seq) data, enabling meta-analysis of host-microbial studies from the public domain. In our scalability benchmarking experiments, Vulture can outperform the state-of-the-art cloud-based pipeline Cumulus with a 40% and 80% reduction of runtime and cost, respectively. Furthermore, Vulture is 2-10 times faster than PathogenTrack and Venus, while generating comparable results. We applied Vulture to two COVID-19, three hepatocellular carcinoma (HCC), and two gastric cancer human patient cohorts with public sequencing reads data from scRNA-seq experiments and discovered cell-type specific enrichment of SARS-CoV2, hepatitis B virus (HBV), and H. pylori positive cells, respectively. In the HCC analysis, all cohorts showed hepatocyte-only enrichment of HBV, with cell subtype-associated HBV enrichment based on inferred copy number variations. In summary, Vulture presents a scalable and economical framework to mine unknown host-microbial interactions from large-scale public scRNA-seq data. Vulture is available via an open-source license at https://github.com/holab-hku/Vulture.","version":"1.1","doi":"10.1101/2023.02.13.528411","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.13.528235","pub_date":"2023-2-14","title":"P-Selectin promotes SARS-CoV-2 interactions with platelets and the endothelium","abstract":"COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo, authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.","version":"1.1","doi":"10.1101/2023.02.13.528235","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.14.528496","pub_date":"2023-2-14","title":"Transcription regulation of SARS-CoV-2 receptor ACE2 by Sp1: a potential therapeutic target","abstract":"Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Induction of ACE2 expression may represent an effective tactic employed by SARS-CoV-2 to facilitate its own propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. By employing an array of 45 different luciferase reporters, we identify that the transcription factor Sp1 positively and HNF4\u03b1 negatively regulate the expression of ACE2 at the transcriptional levels in HPAEpiC cells, a human lung epithelial cell line. SARS-CoV-2 infection promotes and inhibits the transcription activity of Sp1 and HNF4\u03b1, respectively. The PI3K/AKT signaling pathway, which is activated by SARS-CoV-2 infection, is a crucial node for induction of ACE2 expression by increasing Sp1 phosphorylation, an indicator of its activity, and reducing HNF4\u03b1 nuclear location. Furthermore, we show that colchicine could inhibit the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. Inhibition of Sp1 by either its inhibitor mithramycin A or colchicine reduces viral replication and tissue injury in Syrian hamsters infected with SARS-CoV-2. In summary, our study uncovers a novel function of Sp1 in regulating ACE2 expression and suggests that Sp1 is a potential target to reduce SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.02.14.528496","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.23.497375","pub_date":"2023-2-14","title":"Predicting the antigenic evolution of SARS-COV-2 with deep learning","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) antigenic profile evolves in response to the vaccine and natural infection-derived immune pressure, resulting in immune escape and threatening public health. Exploring the possible antigenic evolutionary potentials improves public health preparedness, but it is limited by the lack of experimental assays as the sequence space is exponentially large. Here we introduce the Machine Learning-guided Antigenic Evolution Prediction (MLAEP), which combines structure modeling, multi-task learning, and genetic algorithm to model the viral fitness landscape and explore the antigenic evolution via in silico directed evolution. As demonstrated by existing SARS-COV-2 variants, MLAEP can infer the order of variants along antigenic evolutionary trajectories, which is also strongly correlated with their sampling time. The novel mutations predicted by MLAEP are also found in immunocompromised covid patients and newly emerging variants, like XBB1.5. The predictions of MLAEP were validated by conducting in vitro neutralizing antibody binding assay, which demonstrated that the model-generated variants displayed significantly increased immune evasion ability compared with the controls. In sum, our approach enables profiling existing variants and forecasting prospective antigenic variants, thus may help guide the development of vaccines and increase preparedness against future variants. Our model is available at https://mlaep.cbrc.kaust.edu.sa.","version":"1.3","doi":"10.1101/2022.06.23.497375","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.13.528341","pub_date":"2023-2-14","title":"SARS-CoV-2 Neutralizing Antibodies After Bivalent vs. Monovalent Booster","abstract":"Bivalent mRNA vaccine boosters expressing Omicron BA.5 spike and ancestral D614G spike were introduced to attempt to boost waning antibody titers and broaden coverage against emerging SARS-CoV-2 lineages. Previous reports showed that peak serum neutralizing antibody (NAb) titers against SARS-CoV-2 variants following bivalent booster were similar to peak titers following monovalent booster. It remains unknown whether these antibody responses would diverge over time. We assessed serum virus-neutralizing titers in 41 participants who received three monovalent mRNA vaccine doses followed by bivalent booster, monovalent booster, or BA.5 breakthrough infection at one month and three months after the last vaccine dose or breakthrough infection using pseudovirus neutralization assays against D614G and Omicron subvariants (BA.2, BA.5, BQ.1.1, and XBB.1.5). There was no significant difference at one month and three months post-booster for the two booster cohorts. BA.5 breakthrough patients exhibited significantly higher NAb titers at three months against all Omicron subvariants tested compared against monovalent and bivalent booster cohorts. There was a 2-fold drop in mean NAb titers in the booster cohorts between one and three month time points, but no discernible waning of titers in the BA.5 breakthrough cohort over the same period. Our results suggest that NAb titers after boosting with one dose of bivalent mRNA vaccine are not higher than boosting with monovalent vaccine. Perhaps inclusion of D614G spike in the bivalent booster exacerbates the challenge posed by immunological imprinting. Hope remains that a second bivalent booster could induce superior NAb responses against emerging variants.","version":"1.1","doi":"10.1101/2023.02.13.528341","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.13.528349","pub_date":"2023-2-14","title":"Propylene glycol inactivates respiratory viruses and prevents airborne transmission","abstract":"Viruses are vulnerable as they transmit between hosts and we aimed to exploit this critical window. We found that the ubiquitous, safe, inexpensive and biodegradable small molecule propylene glycol (PG) has robust virucidal activity. Propylene glycol rapidly inactivates influenza, SARS-CoV-2 and a broad range of other enveloped viruses, and reduces disease burden in mice when administered intranasally at concentrations commonly found in nasal sprays. Most critically, aerosolized PG efficiently abolishes influenza and SARS-CoV-2 infectivity within airborne droplets, potently preventing infection at levels significantly below those well-tolerated by mammals. We present PG vapor as a first-in-class non-toxic airborne virucide, to prevent transmission of existing and emergent viral pathogens, with clear and immediate implications for public health. Propylene glycol is a potent and safe virucidal compound that could be used to limit and control infections.","version":"1.1","doi":"10.1101/2023.02.13.528349","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442634","pub_date":"2023-2-13","title":"Single-virus fusion measurements reveal multiple mechanistically equivalent pathways for SARS-CoV-2 entry","abstract":"SARS-CoV-2 binds to cell-surface receptors and is activated for membrane fusion and cell entry via proteolytic cleavage. Phenomenological data have shown that SARS-CoV-2 can be activated for entry at either the cell surface or in endosomes, but the relative roles in different cell types and mechanisms of entry have been debated. Here we use single-virus fusion experiments and exogenously controlled proteases to probe activation directly. We find that plasma membrane and an appropriate protease are sufficient to support SARS-CoV-2 pseudovirus fusion. Furthermore, fusion kinetics of SARS-CoV-2 pseudoviruses are indistinguishable no matter which of a broad range of proteases was used to activate the virus. This suggests that fusion mechanism is insensitive to protease identity or even whether activation occurs before or after receptor binding. These data support a model for opportunistic fusion by SARS-CoV-2, where subcellular location of entry likely depends on the differential activity of airway, cell-surface, and endosomal proteases, but all support infection. Inhibiting any single host protease may thus reduce infection in some cells but may be less clinically robust. SARS-CoV-2 can use multiple pathways to infect cells, as demonstrated recently when new viral variants switched dominant infection pathways. Here, we use single-virus fusion experiments together with biochemical reconstitution to show that these multiple pathways coexist simultaneously and specifically that the virus can be activated by different proteases in different cellular compartments with mechanistically identical effect. The consequences of this are that the virus is evolutionarily plastic and that therapies targeting viral entry should address multiple pathways at once to achieve optimal clinical effects.","version":"1.3","doi":"10.1101/2021.05.04.442634","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.527906","pub_date":"2023-2-13","title":"LINE1-mediated reverse transcription and genomic integration of SARS-CoV-2 mRNA detected in virus-infected but not in viral mRNA-transfected cells","abstract":"SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1,000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches for the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10 \u2013 20-fold more sensitive per tested cell, TagMap can interrogate 1,000 \u2013 2,000-fold more cells and therefore can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected in contrast to transfected cells may be facilitated because virus infection in contrast to viral RNA transfection results in significantly higher viral RNA levels and stimulates LINE1-expression which causes cellular stress.","version":"1.1","doi":"10.1101/2023.02.10.527906","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.527914","pub_date":"2023-2-13","title":"The disordered N-terminal tail of SARS CoV-2 Nucleocapsid protein forms a dynamic complex with RNA","abstract":"The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA Binding Domain (RBD) and its flanking disordered N-Terminal Domain (NTD) tail, using single-molecule F\u00f6rster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.","version":"1.1","doi":"10.1101/2023.02.10.527914","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.29.526145","pub_date":"2023-2-13","title":"Quantitative Annotations of T-Cell Repertoire Specificity","abstract":"The specificity of a T-cell receptor (TCR) repertoire determines personalized immune capacity. Existing methods have modelled the qualitative aspects of TCR specificity, while the quantitative aspects remained unaddressed. We developed a package, TCRanno, to quantify the specificity of TCR repertoires. Applying TCRanno to 4,195 TCR repertoires revealed quantitative changes in repertoire specificity upon infections, autoimmunity and cancers. Specifically, TCRanno found cytomegalovirus-specific TCRs in seronegative healthy individuals, supporting the possibility of abortive infections. TCRanno discovered age-accumulated fraction of SARS-CoV2-specific TCRs in pre-pandemic samples, which may explain the aggressive symptoms and age-related severity of COVID-19. TCRanno also identified the encounter of Hepatitis B antigens as a potential trigger of systemic lupus erythematosus. TCRanno annotations showed capability in distinguishing TCR repertoires of healthy and cancers including melanoma, lung and breast cancers. TCRanno may also facilitate single-cell TCRseq+gene expression data analyses by isolating T-cells with the specificity of interest.","version":"1.2","doi":"10.1101/2023.01.29.526145","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.528014","pub_date":"2023-2-10","title":"Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p","abstract":"The stem loop 2 motif (s2m), a highly conserved 41-nucleotide hairpin structure in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a greater than 99% correlation of a single nucleotide mutation at the 15th position (G15U) in Delta SARS-CoV-2. Based on 1H NMR assignments comparing the imino proton resonance region of s2m and the G15U at 19\u00b0C, we find that the U15-A29 base pair closes resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we find that the s2m G15U mutation drastically reduces the binding affinity of the host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on the immune response.","version":"1.1","doi":"10.1101/2023.02.10.528014","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.10.527437","pub_date":"2023-2-10","title":"Antagonistic pleiotropy plays an important role in governing the evolution and genetic diversity of SARS-CoV-2","abstract":"Analyses of the genomic diversity of SARS-CoV-2 found that some sites across the genome appear to have mutated independently multiple times with frequency significantly higher than four-fold sites, which can be either due to mutational bias, i.e., elevated mutation rate in some sites of the genome, or selection of the variants due to antagonistic pleiotropy, a condition where mutations increase some components of fitness at a cost to others. To examine how different forces shaped evolution of SARS-CoV-2 in 2020\u20132021, we analyzed a large set of genome sequences (~ 2 million). Here we show that while evolution of SARS-CoV-2 during the pandemic was largely mutation-driven, a group of nonsynonymous changes is probably maintained by antagonistic pleiotropy. To test this hypothesis, we studied the function of one such mutation, spike M1237I. Spike I1237 increases viral assembly and secretion, but decreases efficiency of transmission in vitro. Therefore, while the frequency of spike M1237I may increase within hosts, viruses carrying this mutation would be outcompeted at the population level. We also discuss how the antagonistic pleiotropy might facilitate positive epistasis to promote virus adaptation and reconcile discordant estimates of SARS-CoV-2 transmission bottleneck sizes in previous studies.","version":"1.1","doi":"10.1101/2023.02.10.527437","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.09.527920","pub_date":"2023-2-10","title":"Population genetics in the early emergence of the Omicron SARS-CoV-2 variant in the provinces of South Africa","abstract":"Population genetic analyses of viral genome populations provide insight into the emergence and evolution of new variants of SARS-CoV-2. In this study, we use a population genetic approach to examine the evolution of the Omicron variant of SARS-CoV-2 in four provinces of South Africa (Eastern Cape, Gauteng, KwaZulu-Natal, and Mpumalanga) during the first months before emergence and after early spread. Our results show that Omicron polymorphisms increase sharply from September to November. We found differences between SARS-CoV-2 populations from Gauteng and Kwazulu-Natal and viruses from the Eastern Cape, where allele frequencies were higher, suggesting that natural selection may have contributed to the increase in frequency or that this was the site of origin. We found that the frequency of variants N501Y, T478K, and D614G increased in the spike in November compared with other mutations, some of which are also present in other animal hosts. Gauteng province was the most isolated, and most genetic variation was found within populations. Our population genomic approach is useful for small-scale genomic surveillance and identification of novel allele-level variants that can help us understand how SARS-CoV-2 will continue to adapt to humans and other hosts.","version":"1.1","doi":"10.1101/2023.02.09.527920","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.05.498834","pub_date":"2023-2-10","title":"Comparing the evolutionary dynamics of predominant SARS-CoV-2 virus lineages co-circulating in Mexico","abstract":"Over 200 different SARS-CoV-2 lineages have been observed in Mexico by November 2021. To investigate lineage replacement dynamics, we applied a phylodynamic approach and explored the evolutionary trajectories of five dominant lineages that circulated during the first year of local transmission. For most lineages, peaks in sampling frequencies coincided with different epidemiological waves of infection in Mexico. Lineages B.1.1.222 and B.1.1.519 exhibited similar dynamics, constituting clades that likely originated in Mexico and persisted for >12 months. Lineages B.1.1.7, P.1 and B.1.617.2 also displayed similar dynamics, characterized by multiple introduction events leading to a few successful extended local transmission chains that persisted for several months. For the largest B.1.617.2 clades, we further explored viral lineage movements across Mexico. Many clades were located within the south region of the country, suggesting that this area played a key role in the spread of SARS-CoV-2 in Mexico.","version":"1.3","doi":"10.1101/2022.07.05.498834","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.09.527802","pub_date":"2023-2-09","title":"Seroprevalence of IgG Antibody against SARS-CoV-2 Nucleocapsid protein and Associated Risk Factors","abstract":"Estimation of antibody development against SARS-CoV-2 is essential means for understanding the immune response against the virus. We reported IgG antibody development status against Nucleocapsid protein of the virus and compared with lifestyle (health and food habits), co-existing diseases, vaccination and COVID-19 infection status. ELISA (Enzyme Linked Immunosorbent Assay) was performed to assess IgG antibodies targeted against the Nucleocapsid protein of SARS-CoV-2 in participants (n=500). In this seroprevalence study, serological data were estimated for a period of 10 months in the participants who were aged 10 years and above. Sociodemographic and risk factors related data were collected through a written questionnaire and chi-square test was performed to determine the association with seropositivity. The overall seroprevalence of anti-SARS-CoV-2 antibodies among the study subjects was 47.8%. Estimates were highest among the participants of 21-40 years old (55.1%), and lowest in older aged (>60 years) participants (39.5%). Among the Sinopharm vaccinated individuals 81.8% had developed anti-Nucleocapsid antibody. Physical exercise and existence of comorbidities like hypertension and diabetes were the distinguishing factors between seropositive and seronegative individuals. Seropositivity rate largely varied among symptomatic (67%) and asymptomatic (33.1%) COVID-19 infected participants. The findings suggest that residents of Dhaka city had a higher prevalence of anti-nucleocapsid antibody in the second year of the pandemic. This indicates the improvement of immunological status among the population. Finally, the study emphasizes on maintaining active and healthy lifestyle to improve immunity. However, the absence of IgG antibodies in many cases of COVID-19 infected individuals suggests that antibodies wane with time. The overall seroprevalence of anti-Nucleocapsid IgG among the study subjects was determined to be 47.8%. Age, regular physical exercise, existence of comorbidities were the identified parameters associated with seroprevalence. This study observed lower prevalence of Anti-Nucleocapsid antibody among asymptomatic cases of COVID-19 infected individuals compared to symptomatic cases.","version":"1.1","doi":"10.1101/2023.02.09.527802","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.31.525914","pub_date":"2023-2-09","title":"Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6","abstract":"Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (30kb). Here, we designed a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.","version":"1.1","doi":"10.1101/2023.01.31.525914","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.08.527785","pub_date":"2023-2-09","title":"Functional comparisons of the virus sensor RIG-I from humans, the microbat Myotis daubentonii, and the megabat Rousettus aegyptiacus, and their response to SARS-CoV-2 infection","abstract":"Bats (order Chiroptera) are a major reservoir for emerging and re-emerging zoonotic viruses. Their tolerance towards highly pathogenic human viruses led to the hypothesis that bats may possess an especially active antiviral interferon (IFN) system. Here, we cloned and functionally characterized the virus RNA sensor, Retinoic Acid-Inducible Gene-I (RIG-I), from the \u201cmicrobat\u201d Myotis daubentonii (suborder Yangochiroptera) and the \u201cmegabat\u201d Rousettus aegyptiacus (suborder Yinpterochiroptera), and compared them to the human ortholog. Our data show that the overall sequence and domain organization is highly conserved and that all three RIG-I orthologs can mediate a similar IFN induction in response to viral RNA at 37\u00b0 and 39\u00b0C, but not at 30\u00b0C. Like human RIG-I, bat RIG-Is were optimally activated by double stranded RNA containing a 5\u2019-triphosphate end and required Mitochondrial Antiviral-Signalling Protein (MAVS) for antiviral signalling. Moreover, the RIG-I orthologs of humans and of R. aegyptiacus, but not of M. daubentonii, enable innate immune sensing of SARS-CoV-2 infection. Our results thus show that microbats and megabats express a RIG-I that is not substantially different from the human counterpart with respect to function, temperature dependency, antiviral signaling, and RNA ligand properties, and that human and megabat RIG-I are able to sense SARS-CoV-2 infection. A common hypothesis holds that bats (order Chiroptera) are outstanding reservoirs for zoonotic viruses because of a special antiviral interferon (IFN) system. However, functional studies about key components of the bat IFN system are rare. RIG-I is a cellular sensor for viral RNA signatures that activates the antiviral signalling chain to induce IFN. We cloned and functionally characterized RIG-I genes from representatives of the suborders Yangochiroptera and Yinpterochiroptera. The bat RIG-Is were conserved in their sequence and domain organization, and similar to human RIG-I in (i) mediating virus- and IFN-activated gene expression, (ii) antiviral signalling, (iii) temperature dependence, and (iv) recognition of RNA ligands. Moreover, RIG-I of Rousettus aegyptiacus (suborder Yinpterochiroptera) and of humans were found to recognize SARS-CoV-2 infection. Thus, members of both bat suborders encode RIG-Is that are comparable to their human counterpart. The ability of bats to harbour zoonotic viruses therefore seems due to other features.","version":"1.1","doi":"10.1101/2023.02.08.527785","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.29.466408","pub_date":"2023-2-08","title":"Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface","abstract":"Mucins play an essential role in protecting the respiratory tract against microbial infections but can also serve as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on SARS-CoV-2 epithelial entry. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. Both MUC1 and MUC16 are expressed on the surface of human air-liquid interface (ALI) differentiated airway organoids and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 entry and replication. On the surface of Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are often co-expressed and StcE treatment results in enhanced binding of purified spike protein and SARS-CoV-2 pseudovirus. This study points at an important role for glycosylated mucin domains as components of the host defense that can restrict SARS-CoV-2 infection. SARS-CoV-2, the virus that has caused the devastating COVID-19 pandemic, causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A fundamental understanding of host factors influencing viral entry is critical to elucidate SARS-CoV-2\u2013host interactions and identify novel therapeutic targets. In this study, we investigated the role of host mucins and mucin glycans on SARS-CoV-2 entry into the airway epithelial cells. Mucins are a family of high molecular weight O-glycosylated proteins that play an essential role in protecting the respiratory tract against viral and bacterial infections. The gel-forming mucins MUC5AC and MUC5B clear pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict or facilitate microbial invasion at the apical surface of the epithelium. The mucin-selective protease StcE specifically cleaves the glycosylated extracellular part of the mucins without perturbing the underlying domains. We show that removal of mucins from the surface of Calu-3 cells and primary airway epithelial cultures with StcE mucinase increases binding of the SARS-CoV-2 spike protein to the respiratory surface and greatly enhances infection. This study demonstrates the important role of glycosylated extracellular mucin domains as a host defense mechanism during SARS-CoV-2 entry. Future efforts should be focused on characterizing the role of specific soluble and transmembrane mucins during the different stages of SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2021.10.29.466408","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.07.527372","pub_date":"2023-2-08","title":"Counterintuitive effect of antiviral therapy on influenza A-SARS-CoV-2 coinfection due to viral interference","abstract":"The resurgence of influenza and continued circulation of SARS-CoV-2 raise the question of how these viruses interact in a co-exposed host. Here we studied virus-virus and host-virus interactions during influenza A virus (IAV) -SARS-CoV-2 coinfection using differentiated cultures of the human airway epithelium. Coexposure to IAV enhanced the tissue antiviral response during SARS-CoV-2 infection and suppressed SARS-CoV-2 replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the antiviral response and paradoxically restored SARS-CoV-2 replication. These results highlight the importance of diagnosing coinfections and compel further study of how coinfections impact the outcome of antiviral therapy.","version":"1.1","doi":"10.1101/2023.02.07.527372","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.07.23285574","pub_date":"2023-02-08","title":"Quantification of impact of COVID-19 pandemic on cancer screening programmes -a case study from Argentina, Bangladesh, Colombia, Morocco, Sri Lanka and Thailand","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>It is quite well-documented that the COVID-19 pandemic disrupted cancer screening services in all countries, irrespective of their resources and healthcare settings. While quantitative estimates on reduction in volume of screening tests or diagnostic evaluation are readily available from the high-income countries, very little data is available from the low- and middle-income countries (LMICs). From the CanScreen5 global cancer screening data repository we identified six LMICs through purposive sampling based on the availability of cancer screening data at least for the years 2019 and 2020. These countries represented different human development index (HDI) categories and were from Asia (Bangladesh and Thailand), Africa (Morocco) and Latin America (Argentina and Colombia). The reduction in the volume of tests in 2020 compared to the previous years ranged from 14.1% in Bangladesh to 72.9% in Argentina (regional programme) for cervical screening, from 14.1% in Bangladesh to 52.2% in Morocco for breast cancer screening and 30.7% in Thailand for colorectal cancer screening. Number of colposcopies was reduced in 2020 compared to previous year by 88.9% in Argentina, 38.2% in Colombia, 27.4% in Bangladesh and 52.3% in Morocco. The reduction in detection rates of CIN 2 or worse lesions ranged from 20.7% in Morocco to 45.4% in Argentina. Reduction of breast cancer detection by 19.2% was reported from Morocco. No association of the impact of pandemic could be seen with HDI categories. Quantifying the impact of service disruptions in screening and diagnostic tests will allow the programmes to strategize how to ramp up services to clear the backlogs in screening and more crucially in further evaluation of screen-positives. The data can be used to estimate the impact on stage distribution and avoidable mortality from these common cancers.</jats:p>","version":null,"doi":"10.1101/2023.02.07.23285574","journal":"medRxiv","score":null},{"id":"10.1101/2023.02.06.527330","pub_date":"2023-2-07","title":"Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers","abstract":"Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. We characterized antibody binding and functionality in convalescent plasma from postpartum women and their infants infected with SARS-CoV-2 from a vaccine-na\u00efve prospective cohort in Nairobi, Kenya. Antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels were significantly higher in infants than in mothers. Plasma antibodies from mothers and infants bound to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants displayed higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants had significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, neutralization titers between infants and mothers were similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody repertoires and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.","version":"1.1","doi":"10.1101/2023.02.06.527330","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.06.527382","pub_date":"2023-2-07","title":"A COVID-19 DNA Vaccine Candidate Elicits Broadly Neutralizing Antibodies Against Multiple SARS-CoV-2 Variants Including the Currently Circulating Omicron BF.5, BF.7, BQ.1 and XBB","abstract":"Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants pose a global challenge to pandemic control today. We have previously reported a pVAX1-based DNA vaccine candidate, pAD1002, which encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous Sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 prototype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-\u03b3+ cellular responses in BALB/c and C57BL/6 mice. More importantly, pAD1016 vaccination in mice and rabbits generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in C57BL/6 mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy further translational study as a COVID-19 vaccine candidate.","version":"1.1","doi":"10.1101/2023.02.06.527382","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.07.527501","pub_date":"2023-2-07","title":"Macrodomain Mac1 of SARS-CoV-2 Nonstructural Protein 3 Hydrolyzes Diverse ADP-ribosylated Substrates","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD+ to various acceptors, e.g., proteins, DNA, RNA, contributing on a cell\u2019s biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved \u03b1-NAD+, but not \u03b2-NAD+, consistent with stereospecificity at the C-1\u201d bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1\u201d-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave \u03b1-ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. SARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. \u03b1-NAD+ and O-acetyl-ADP-ribose. However, Mac1 did not hydrolyze \u03b2-NAD+, ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a \u03b1-NAD+ consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1\u2019s mechanisms of action is important to provide possible therapeutic targets for COVID-19.","version":"1.1","doi":"10.1101/2023.02.07.527501","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.06.527376","pub_date":"2023-2-07","title":"A SARS-CoV-2 vaccine designed for manufacturability results in unexpected potency and non-waning humoral response","abstract":"The rapid development of several highly efficacious SARS-CoV-2 vaccines was an unprecedented scientific achievement that saved millions of lives. However, now that SARS-CoV-2 is transitioning to the endemic stage, there exists an unmet need for new vaccines that provide durable immunity, protection against variants, and can be more easily manufactured and distributed. Here we describe a novel protein component vaccine candidate, MT-001, based on a fragment of the SARS-CoV-2 spike protein that encompasses the receptor binding domain (RBD). Mice and hamsters immunized with a prime-boost regimen of MT-001 demonstrated extremely high anti-spike IgG titers, and remarkably this humoral response did not appreciably wane for up to 12 months following vaccination. Further, virus neutralization titers, including titers against variants such as Delta and Omicron BA.1, remained high without the requirement for subsequent boosting. MT-001 was designed for manufacturability and ease of distribution, and we demonstrate that these attributes are not inconsistent with a highly immunogenic vaccine that confers durable and broad immunity to SARS-CoV-2 and its emerging variants. These properties suggest MT-001 could be a valuable new addition to the toolbox of SARS-CoV-2 vaccines and other interventions to prevent infection and curtail additional morbidity and mortality from the ongoing worldwide pandemic.","version":"1.1","doi":"10.1101/2023.02.06.527376","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.06.527236","pub_date":"2023-2-07","title":"Underlying driving forces of the SARS-CoV-2 evolution: immune evasion and ACE2 binding affinity","abstract":"The evolution of SARS-CoV-2 is characterized by the emergence of new variants with a sheer number of mutations compared to their predecessors, which conferred resistance to pre-existing antibodies and/or increased transmissibility. The recently emerged Omicron subvariants also exhibit a strong tendency for immune evasion, suggesting adaptive evolution. However, previous studies have been limited to specific lineages or subsets of mutations, the overall evolutionary trajectory of SARS-CoV-2 and the underlying driving forces are still not fully understood. In this study, we analyzed the mutations present in all open-access SARS-CoV-2 genomes (until November 2022) and correlated the mutation\u2019s incidence and fitness change with its impact on immune evasion and ACE2 binding affinity. Our results showed that the Omicron lineage had an accelerated mutation rate in the RBD region, while the mutation incidence in other genomic regions did not change dramatically over time. Moreover, mutations in the RBD region (but not in any other genomic regions) exhibited a lineage-specific pattern and tended to become more aggregated over time, and the mutation incidence was positively correlated with the strength of antibody pressure on the specific position. Additionally, the incidence of mutation was also positively correlated with changes in ACE2 binding affinity, but with a lower correlation coefficient than with immune evasion. In contrast, the mutation\u2019s effect on fitness was more closely correlated with changes in ACE2 binding affinity than immune evasion. In conclusion, our results suggest that immune evasion and ACE2 binding affinity play significant and diverse roles in the evolution of SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.02.06.527236","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.07.527419","pub_date":"2023-2-07","title":"Dogs and cats are less susceptible to the omicron variant of concern of SARS-CoV-2 \u2013 a field study","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of unprecedented extent. Beside humans, a number of animal species can be infected, however, in some species differing susceptibilities were observed depending on the virus variant. Here, we serologically investigated cats and dogs living in households with human COVID-19 patients. The study was conducted during the transition period from delta as the dominating variant of concern (VOC) to omicron (BA.1/BA.2) to investigate the frequency of virus transmission of both VOCs from infected owners to their pets. The animal sera were tested by surrogate virus neutralization tests (sVNT) using either the original receptor-binding domain (RBD), enabling the detection of antibodies against the delta variant, or an omicron-specific RBD. Of the 290 canine samples, 20 tested positive by sVNT, but there were marked differences between the sampling time, and, related thereto, the virus variants, the dogs had contact to. While in November 2021 infected owners led to 50% seropositive dogs (18/36), only 0.8% (2/254) of animals with household contacts to SARS-CoV-2 between December 2021 and April 2022 tested positive. In all cases, the positive reaction was recorded against the original RBD. For cats, a similar pattern was seen, as in November 2021 38.1% (16/42) tested positive and between December 2021 and March 2022 only 5.0% (10/199). The markedly reduced ratio of seropositive animals during the period of omicron circulation suggests a considerably lower susceptibility of dogs and cats to this VOC. To examine the effect of BA.2, BA.4 and BA.5 omicron subvariants, sera taken in the second and third quarter of 2022 from randomly selected cats were investigated. 2.3% (11/372) tested seropositive and all of them showed a stronger reaction against the original RBD, further supporting the assumption of a lower susceptibility of companion animals to the omicron VOC.","version":"1.1","doi":"10.1101/2023.02.07.527419","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.07.527429","pub_date":"2023-2-07","title":"A Bayesian inference method to estimate transmission trees with multiple introductions; applied to SARS-CoV-2 in Dutch mink farms","abstract":"Knowledge of who infected whom during an outbreak of an infectious disease is important to determine risk factors for transmission and to design effective control measures. Both whole-genome sequencing of pathogens and epidemiological data provide useful information about the transmission events and underlying processes. Existing models to infer transmission trees usually assume that the pathogen is introduced only once from outside into the population of interest. However, this is not always true. For instance, SARS-CoV-2 is suggested to be introduced multiple times in mink farms in the Netherlands from the SARS-CoV-2 pandemic among humans. Here, we developed a Bayesian inference method combining whole-genome sequencing data and epidemiological data, allowing for multiple introductions of the pathogen in the population. Our method does not a priori split the outbreak into multiple phylogenetic clusters, nor does it break the dependency between the processes of mutation, within-host dynamics, transmission, and observation. We implemented our method as an additional feature in the R-package phybreak. On simulated data, our method identifies the number of introductions with high accuracy. Moreover, when a single introduction was simulated, our method produces similar estimates of parameters and transmission trees as the existing package. When applied to data from a SARS-CoV-2 outbreak in Dutch mink farms, the method provides strong evidence for 13 introductions, which is 20 percent of all infected farms. Using the new feature of the phybreak package, transmission routes of a more complex class of infectious disease outbreaks can be inferred which will aid infection control in future outbreaks.","version":"1.1","doi":"10.1101/2023.02.07.527429","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445313","pub_date":"2023-2-07","title":"Identification of evolutionary trajectories shared across human betacoronaviruses","abstract":"Comparing the evolution of distantly related viruses can provide insights into common adaptive processes related to shared ecological niches. Phylogenetic approaches, coupled with other molecular evolution tools, can help identify mutations informative on adaptation, whilst the structural contextualization of these to functional sites of proteins may help gain insight into their biological properties. Two zoonotic betacoronaviruses capable of sustained human-to-human transmission have caused pandemics in recent times (SARS-CoV-1 and SARS-CoV-2), whilst a third virus (MERS-CoV) is responsible for sporadic outbreaks linked to animal infections. Moreover, two other betacoronaviruses have circulated endemically in humans for decades (HKU1 and OC43). To search for evidence of adaptive convergence between established and emerging betacoronaviruses capable of sustained human-to-human transmission (HKU1, OC43, SARS-CoV-1 and SARS-CoV-2), we developed a methodological pipeline to classify shared non-synonymous mutations as putatively denoting homoplasy (repeated mutations that do not share direct common ancestry) or stepwise evolution (sequential mutations leading towards a novel genotype). In parallel, we look for evidence of positive selection, and draw upon protein structure data to identify potential biological implications. We find 30 mutations, with four of these [codon sites 18121 (nsp14/residue 28), 21623 (spike/21), 21635 (spike/25) and 23948 (spike/796); SARS-CoV-2 genome numbering] displaying evolution under positive selection and proximity to functional protein regions. Our findings shed light on potential mechanisms underlying betacoronavirus adaptation to the human host and pinpoint common mutational pathways that may occur during establishment of human endemicity.","version":"1.2","doi":"10.1101/2021.05.24.445313","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.05.527215","pub_date":"2023-2-06","title":"Prior vaccination enhances immune responses during SARS-CoV-2 breakthrough infection with early activation of memory T cells followed by production of potent neutralizing antibodies","abstract":"SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened Spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific CD4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production, and primary responses to non-Spike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.02.05.527215","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.05.527173","pub_date":"2023-2-06","title":"Taxonomical and ontological analysis of verified natural and laboratory human coronavirus hosts","abstract":"To fully understand COVID-19, it is critical to identify and analyze all the possible hosts of SARS-CoV-2 (the pathogen of COVID-19) and compare them with the hosts of other human coronaviruses. In this study, we collected, annotated, and performed taxonomical and ontological analysis of all the reported and verified hosts for all human coronaviruses including SARS-CoV, MERS-CoV, SARS-CoV-2, and four others that cause the common cold. A total of 37 natural hosts and 19 laboratory animal hosts of host human coronaviruses were identified based on experimental or clinical evidence. Our taxonomical ontology-based analysis found that all the verified susceptible natural and laboratory animals belong to therian mammals. Specifically, these 37 natural therian hosts include one wildlife marsupial mammal (i.e., Didelphis virginiana) and 36 Eutheria mammals (a.k.a. placental mammals). The 19 laboratory animal hosts are also classified as placental mammals. While several non-therian animals (including snake, housefly, zebrafish) were reported to be likely SARS-CoV-2 hosts, our analysis excluded them due to the lack of convincing evidence. Genetically modified mouse models with human Angiotensin-converting enzyme 2 (ACE2) or dipeptidyl peptidase-4 (DPP4) protein were more susceptible to virulent human coronaviruses with clear symptoms. Coronaviruses often became more virulent and adaptive in the mouse hosts after a series of viral passages in the mice. To support knowledge standardization and analysis, we have also represented the annotated host knowledge in the Coronavirus Infectious Disease Ontology (CIDO) and provided ways to automatically query the knowledge.","version":"1.1","doi":"10.1101/2023.02.05.527173","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.480904","pub_date":"2023-2-06","title":"Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) as an auxiliary host factor for RNA viruses including SARS-CoV-2","abstract":"Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the highly LRP1-positive human cell line HuH-7, LRP1 also promoted the early infection stages of Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), La Crosse virus (LACV; family Peribunyaviridae, order Bunyavirales), had a minor effect on RNA levels during the late infection stages by vesicular stomatitis virus (VSV; family Rhabdoviridae, order Mononegavirales), whereas infection by Encephalomyocarditis virus (EMCV, family Picornaviridae) was entirely LRP1-independent. Moreover, siRNA experoments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.","version":"1.2","doi":"10.1101/2022.02.17.480904","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.22.497114","pub_date":"2023-2-06","title":"S373P Mutation Stabilizes the Receptor-binding Domain of Spike Protein in Omicron and Promotes Binding","abstract":"A cluster of several newly occurring mutations on Omicron are found at the \u03b2-core region of spike protein\u2019s receptor-binding domain (RBD), where mutation rarely happened before. Notably, the binding of SARS-CoV-2 to human receptor ACE2 via RBD happens in a dynamic airway environment, where mechanical force caused by coughing or sneezing occurs and applies to the proteins. Thus, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of RBDs and found that the mechanical stability of Omicron RBD increased by ~20% compared with the wild-type. Molecular dynamics simulations revealed that Omicron RBD showed more hydrogen bonds in the \u03b2-core region due to the closing of the \u03b1-helical motif caused primarily by mutation S373P, which was further confirmed experimentally. Moreover, the binding ability of Omicron to ACE2 is promoted with a stabilized RBD. This work reveals the effect of the highly conserved mutation S373P which is present in most Omicron subvariants, including BA.1-5, BQ. 1, XBB, and CH.1.1.","version":"1.3","doi":"10.1101/2022.06.22.497114","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.03.527052","pub_date":"2023-2-05","title":"Automated Agnostic Designation of Pathogen Lineages","abstract":"Pathogen nomenclature systems are a key component of effective communication and collaboration for researchers and public health workers. Since February 2021, the Pango nomenclature for SARS-CoV-2 has been sustained by crowdsourced lineage proposals as new isolates were added to a growing global dataset. This approach to dynamic lineage designation is dependent on a large and active epidemiological community identifying and curating each new lineage. This is vulnerable to time-critical delays as well as regional and personal bias. To address these issues, we developed a simple heuristic approach that divides a phylogenetic tree into lineages based on shared ancestral genotypes. We additionally provide a framework that automatically prioritizes the lineages by growth rate and association with key mutations or locations, extensible to any pathogen. Our implementation is efficient on extremely large phylogenetic trees and produces similar results to existing Pango lineage designations when applied to SARS-CoV-2. This method offers a simple, automated and consistent approach to pathogen nomenclature that can assist researchers in developing and maintaining phylogeny-based classifications in the face of ever increasing genomic datasets.","version":"1.1","doi":"10.1101/2023.02.03.527052","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.03.526944","pub_date":"2023-2-03","title":"Mechanisms of SARS-CoV-2 Inactivation using UVC Laser Radiation","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50mJ/cm2 using a UVC laser at 266nm achieved an inactivation efficiency of 99.89%, whilst infectious virions were undetectable at 75mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterisation of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV-2 spike protein\u2019s ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.","version":"1.1","doi":"10.1101/2023.02.03.526944","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.02.526749","pub_date":"2023-2-02","title":"Investigations on SARS-CoV-2 and other coronaviruses in mink farms in France at the end of the first year of COVID-19 pandemic","abstract":"Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks (Neovison vison) for fur was detected in several countries of Europe. The risk of a new reservoir formation and of a reverse zoonosis from minks was then a major concern. The aim of this study was to investigate the four French mink farms for the circulation of SARS-CoV-2 at the end of 2020. The investigations took place during the slaughtering period thus facilitating different types of sampling (swabs and blood). In one of the four mink farms, 96.6% of serum samples were positive in SARS-CoV-2 ELISA coated with purified N protein recombinant antigen and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced in this farm indicated the co-circulation of several lineages at the time of sampling. All SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled at the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.5 and 1.2% in the three other farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus highly similar to a mink coronavirus sequence observed in Danish farms in 2015. In addition, a mink Caliciviridae was identified in one of the two positive farms for Alphacoronavirus. The clinical impact of these unapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses in mink farms could contribute to explain the diversity of clinical symptoms noted in different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 within a mink farm would increase potentially the risk of viral recombination between alpha and betacoronaviruses already suggested in wild and domestic animals, as well as in humans. France is not a country of major mink fur production. Following the SARS-CoV-2 contamination of mink farms in Denmark and the Netherlands, the question arose for the four French farms. The investigation conducted at the same time in the four farms revealed the contamination of one of them by a variant different from the one circulating at the same time in Denmark and the Netherlands mink farms. Investigation of three other farms free of SARS-CoV-2 contamination revealed the circulation of other viruses including a mink Alphacoronavirus and Caliciviridae, which could modify the symptomatology of SARS-CoV-2 infection in minks.","version":"1.1","doi":"10.1101/2023.02.02.526749","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469695","pub_date":"2023-2-02","title":"Antiviral activity of Molnupiravir precursor NHC against SARS-CoV-2 Variants of Concern (VOCs) and implications for the therapeutic window and resistance","abstract":"Several regulatory agencies have either licensed or given emergency use approval for treatment of patients at risk of developing severe COVID-19 with the anti-viral drug, Molnupiravir. Recent trials involving Molnupiravir suggested the drug was not as efficacious as earlier studies suggested. This study aimed to: (i) determine the effectiveness of the Molnupiravir active metabolite (NHC) against different SARS-CoV-2 Variants of Concern (VoCs), (ii) establish the therapeutic window of NHC in a human lung cell model, and (iii) and evaluate the genetic barrier to resistance. Dose response assays were performed in parallel to determine the IC50 (the concentration required to inhibit virus titre by 50%) of NHC against different variants. Human ACE-2 A549 cells were treated with NHC at different time points either before, during or after infection with SARS-CoV-2. Multiple passaging in the presence or absence of drug was used to evaluate whether resistance occurred. To obtain genomic information, virus was sequenced at regular intervals. After 20 passages in the presence of the drug, dose response assays and sequencing showed the virus did not appear to have developed resistance. The drug had equivalent activity against four VOCs ranging from 0.04 to 0.16\u03bcM IC50. The efficacy of the drug diminished when applied after 24 hours post-infection. Our results suggest that earlier administration in patients, perhaps pre- or post-exposure rather than symptom onset, would be a more effective treatment option.","version":"1.3","doi":"10.1101/2021.11.23.469695","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.02.526761","pub_date":"2023-2-02","title":"Discovering SARS-CoV-2 neoepitopes and the associated TCR-pMHC recognition mechanisms by combining single-cell sequencing, deep learning, and molecular dynamics simulation techniques","abstract":"The molecular mechanisms underlying the recognition of epitopes by T cell receptors (TCRs) are critical for activating T cell immune responses and rationally designing TCR-based therapeutics. Single-cell sequencing techniques vastly boost the accumulation of TCR sequences, while the limitation of available TCR-pMHC structures hampers further investigations. In this study, we proposed a comprehensive strategy that incorporates structural information and single-cell sequencing data to investigate the epitope-recognition mechanisms of TCRs. By antigen specificity clustering, we mapped the epitope sequences between epitope-known and epitope-unknown TCRs from COVID-19 patients. One reported SARS-CoV-2 epitope, NQKLIANQF (S919-927), was identified for a TCR expressed by 614 T cells (TCR-614). Epitope screening also identified a potential cross-reactive epitope, KLKTLVATA (NSP31790-1798), for a TCR expressed by 204 T cells (TCR-204). According to the molecular dynamics (MD) simulations, we revealed the detailed epitope-recognition mechanisms for both TCRs. The structural motifs responsible for epitope recognition revealed by the MD simulations are consistent with the sequential features recognized by the sequence-based clustering method. This strategy will facilitate the discovery and optimization of TCR-based therapeutics. In addition, the comprehensive strategy can also promote the development of cancer vaccines in virtue of the ability to discover neoepitopes and epitope-recognition mechanisms.","version":"1.1","doi":"10.1101/2023.02.02.526761","journal":"bioRxiv","score":null},{"id":"10.1101/2023.02.01.526623","pub_date":"2023-2-02","title":"Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses","abstract":"With the spread of SARS-CoV-2 throughout the globe to cause the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from beluga whale. Comparison with the apo structure of SW1 Mpro, which we also present here, enabled identifying structural arrangements upon inhibitor binding at the active site. The binding modes and interactions of two covalent inhibitors, PF-00835231 (lufotrelvir) bound to HKU15 and GC376 bound to SW1 Mpro, reveal features that may be leveraged to target diverse coronaviruses and toward structure-based design of pan-CoV inhibitors.","version":"1.1","doi":"10.1101/2023.02.01.526623","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.30.526308","pub_date":"2023-2-01","title":"Mucociliary Clearance Augmenting Drugs Block SARS-Cov-2 Replication in Human Airway Epithelial Cells","abstract":"The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve barrier function of the airway epithelium, reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury, thus, it was further studied using biochemical, genetic and biophysical methods to ascertain mechanism of action via improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression and motion was required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that Intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.","version":"1.1","doi":"10.1101/2023.01.30.526308","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.19.504450","pub_date":"2023-2-01","title":"Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range","abstract":"Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. The SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.","version":"1.2","doi":"10.1101/2022.08.19.504450","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.31.525328","pub_date":"2023-2-01","title":"Metaproteomic analysis of nasopharyngeal swab samples to identify microbial peptides and potential co-infection status in COVID-19 patients","abstract":"Respiratory infections disrupt the microbiota in the upper respiratory tract (URT), putting patients at a risk for subsequent infections. During the pandemic, cases of COVID-19 were aggravated by secondary infections because of impaired immunity and medical interventions, which was clearly evident in the second wave of COVID-19 in India. The potential dangers and clinical difficulties of bacterial and fungal secondary infections in COVID-19 patients necessitate microbial exploration of the URT. In this regard, mass spectrometry (MS)-based proteome data of nasopharyngeal swab samples from COVID-19 patients was used to investigate the metaproteome. The MS datasets were searched against a comprehensive protein sequence database of common URT pathogens using multiple search platforms (MaxQuant, MSFragger, and Search GUI/PeptideShaker). The detected microbial peptides were verified using PepQuery, which analyses peptide-spectrum pairs to give statistical output for determining confident microbial peptides. Finally, a protein sequence database was generated using the list of verified microbial peptides for identification and quantitation of microbial peptides and proteins, respectively. The taxonomic analysis of the detected peptides revealed several opportunistic pathogens like Streptococcus pneumoniae, Rhizopus microsporus, Clavispora lusitaniae, and Syncephalastrum racemosum among others. Using parallel reaction monitoring (PRM), we validated a few identified microbial peptides in clinical samples. The analysis also revealed proteins belonging to species like Pseudomonas fluorescens, Enterobacter, and Clostridium to be up-regulated in severe COVID-19 samples. Thus, MS can serve as a powerful tool for untargeted detection of a wide range of microorganisms. Metaproteomic analysis in COVID-19 patients for early identification and characterisation of co-infecting microorganisms can significantly impact the diagnosis and treatment of patients.","version":"1.1","doi":"10.1101/2023.01.31.525328","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.27.474250","pub_date":"2023-1-31","title":"Structural analysis of the Spike of the Omicron SARS-COV-2 variant by cryo-EM and implications for immune evasion","abstract":"The Omicron (B.1.1.529) SARS-COV-2 was reported on November 24, 2021 and declared a variant of concern a couple of days later. With its constellation of mutations acquired by this variant on its Spike glycoprotein and the speed at which this new variant has replaced the previously dominant variant Delta in South Africa and the United Kingdom, it is crucial to have atomic structural insights to reveal the mechanism of its rapid proliferation. Here we present a high-resolution cryo-EM structure of the Spike protein of the Omicron variant.","version":"1.3","doi":"10.1101/2021.12.27.474250","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.18.524660","pub_date":"2023-1-31","title":"SARS-CoV-2 Omicron XBB.1.5 May Be a Variant That Spreads More Widely and Faster Than Other Variants","abstract":"In this research, we aimed to predict the relative risk of the recent new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the basis of our previous research. We first performed molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to determine the binding affinities to human cells of three new variants of SARS-CoV-2: Omicron BQ.1, XBB, and XBB.1.5 We then investigated the three variants to discover the evolutionary distance of the spike protein gene (S gene) from the Wuhan, Omicron BA.1, and Omicron BA.4/5 variants, to understand the changes in the S gene. The results indicated that the XBB.1.5 variant had the highest binding affinity of the spike protein with ACE2 and the longest evolutionary distance of the S gene. This in silico evidence suggested that the XBB.1.5 variant may produce infections that spread more widely and faster than can infections of preexisting variants.","version":"1.4","doi":"10.1101/2023.01.18.524660","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.448932","pub_date":"2023-1-31","title":"SARS-CoV-2 spike RBD and nucleocapsid encoding DNA vaccine elicits T cell and neutralising antibody responses that cross react with variants","abstract":"Although the efficacy of vaccines targeting SARS-CoV-2 is apparent now that the approved mRNA and adenovirus vector vaccines are in widespread use, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. We have therefore designed a DNA vaccine encoding both the SARS-CoV-2 spike receptorbinding domain (\u2018RBD\u2019) and nucleocapsid proteins, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4+ Th1 and CD8+ T-cell responses to both proteins in mice and rats, with responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD in mice that efficiently neutralise in pseudotype and live virus neutralisation assays and show cross reactivity with spike proteins from the variants B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). The vaccine also showed good protection in a viral challenge model in ACE2 receptor transgenic mice. This DNA platform can be easily adapted to target variant proteins and we show that a vaccine variant encoding the Beta variant sequence stimulates cross-reactive humoral and T cell responses. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for rapidly targeting emerging SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.06.18.448932","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.22.469117","pub_date":"2023-1-30","title":"Preclinical evaluation of a COVID-19 vaccine candidate based on a recombinant RBD fusion heterodimer of SARS-CoV-2","abstract":"Current C0VID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease and a decrease in mortality rates. However, SARS-CoV-2 variants are continuously evolving, and development of new accessible COVID-19 vaccines is essential to mitigate the pandemic. Here, we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine (PHH-1V) consisting of an RBD fusion heterodimer comprising the B.1.351 and B.1.1.7 SARS-CoV-2 variants formulated in SQBA adjuvant, an oil-in-water emulsion. A prime-boost immunisation with PHH-1V in BALB/c and K18-hACE2 mice induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralising activity against several variants, and also showed a good tolerability profile. Significantly, RBD fusion heterodimer vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice, but also reducing Beta, Delta and Omicron infection in lower respiratory airways. These findings demonstrate the feasibility of this recombinant vaccine strategy.","version":"1.7","doi":"10.1101/2021.11.22.469117","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.30.526275","pub_date":"2023-1-30","title":"Human Amylin in the Presence of SARS-COV-2 Protein Fragments","abstract":"Covid-19 can lead to the onset of type-II diabetes which is associated with aggregation of islet amyloid polypeptides, also called amylin. Using molecular dynamics simulations, we investigate how the equilibrium, between amylin monomers in its functional form and fibrils associated with diabetes, is altered in presence of SARS-COV-2 protein fragments. For this purpose, we study the interaction between the fragment SFYVYSRVK of the Envelope protein or the fragment FKNIDGYFKI of the Spike protein with the monomer and two amylin fibril models. Our results are compared with earlier work studying such interactions for two different proteins.","version":"1.1","doi":"10.1101/2023.01.30.526275","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.27.525936","pub_date":"2023-1-29","title":"MOTIFS IN SARS-COV-2 EVOLUTION","abstract":"We present a novel framework enhancing the prediction of whether novel lineage poses the threat of eventually dominating the viral population. The framework is based purely on genomic sequence data, without requiring prior established biological analysis. Its building blocks are sets of co-evolving sites in the alignment (motifs), identified via co-evolutionary signals. The collection of such motifs forms a relational structure over the polymorphic sites. Motifs are constructed using distances quantifying the co-evolutionary coupling of pairs and manifest as co-evolving clusters of sites. We present an approach to genomic surveillance based on this notion of relational structure. Our system will issue an alert regarding a lineage, based on its contribution to drastic changes in the relational structure. We then conduct a comprehensive retrospective analysis of the COVID-19 pandemic based on SARS-CoV-2 genomic sequence data in GISAID from October 2020 to September 2022, across 21 lineages and 27 countries with weekly resolution. We investigate the performance of this surveillance system in terms of its accuracy, timeliness and robustness. Lastly, we study how well each lineage is classified by such a system.","version":"1.1","doi":"10.1101/2023.01.27.525936","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.28.525917","pub_date":"2023-1-29","title":"SARS-CoV-2 Mpro protease variants of concern display altered viral and host target processing but retain potency towards antivirals","abstract":"Main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOC) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and subsequent significant decrease in cytokine secretion, providing evidence for alterations in escape of host-antiviral mechanisms. Accordingly, mutations associated with the highly virulent Delta VOC resulted in significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.","version":"1.1","doi":"10.1101/2023.01.28.525917","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.27.525575","pub_date":"2023-1-29","title":"Omicron BA.1 breakthrough infection drives long-term remodeling of the memory B cell repertoire in vaccinated individuals","abstract":"How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reaction, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies.","version":"1.1","doi":"10.1101/2023.01.27.525575","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.23.525251","pub_date":"2023-1-28","title":"Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry","abstract":"Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding. Next generation protein-adaptive DSF (paDSF) enables rapid and general measurements of protein stability and dynamics.","version":"1.3","doi":"10.1101/2023.01.23.525251","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.26.525770","pub_date":"2023-1-27","title":"Infection of equine bronchial epithelial cells with a SARS-CoV-2 pseudovirus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, can infect animals by binding to the angiotensin-converting enzyme 2 (ACE2). Equine infection appears possible due to high homology (\u224897%) between human and equine ACE2, evidence of in vitro infection in cell lines expressing equine ACE2, and evidence of seroconversion in horses after exposure to persons infected with SARS-CoV-2. Our objective was to examine susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a SARS-CoV-2 pseudovirus relative to human bronchial epithelial cells (HBECs; positive control). ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were co-cultivated with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean \u0394 Ct values from quantitative PCR were significantly (P < 0.0001) higher in HBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. Equine respiratory tract cells were susceptible to infection with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infectious to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted. This study provides the first published evidence for SARS-CoV-2 pseudovirus infection in equine airway epithelial cells, which were less susceptible to infection than cells of human origin. This was presumably due to lower ACE2 expression in equine cells, lower viral affinity for equine ACE2, or both. Our results are important considering recent evidence for asymptomatic seroconversion in horses following exposure to COVID-19 positive humans, despite this lower susceptibility, and increased affinity of viral variants of concern for equine ACE2 compared to ancestral strains. Thus, there is great need to better characterize SARS-CoV-2 susceptibility in horses for the benefit of veterinary and human health.","version":"1.1","doi":"10.1101/2023.01.26.525770","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.21.524927","pub_date":"2023-1-27","title":"CompCorona: A Web Portal for Comparative Analysis of the Host Transcriptome of PBMC and Lung SARS-CoV-2, SARS-CoV, and MERS-CoV","abstract":"Understanding the host response to SARS-CoV-2 infection is crucial for deciding on the correct treatment of this epidemic disease. Although several recent studies reported the comparative transcriptome analyses of the three coronaviridae (CoV) members; namely SARS-CoV, MERS-CoV, and SARS-CoV-2, there is yet to exist a web-tool to compare increasing number of host transcriptome response datasets against the pre-processed CoV member datasets. Therefore, we developed a web application called CompCorona, which allows users to compare their own transcriptome data of infected host cells with our pre-built datasets of the three epidemic CoVs, as well as perform functional enrichment and principal component analyses (PCA). Comparative analyses of the transcriptome profiles of the three CoVs revealed that numerous differentially regulated genes directly or indirectly related to several diseases (e.g., hypertension, male fertility, ALS, and epithelial dysfunction) are altered in response to CoV infections. Transcriptome similarities and differences between the host PBMC and lung tissue infected by SARS-CoV-2 are presented. Most of our findings are congruent with the clinical cases recorded in the literature. Hence, we anticipate that our results will significantly contribute to ongoing studies investigating the pre-and/or post-implications of SARS-CoV-2 infection. In addition, we implemented a user-friendly public website, CompCorona for biomedical researchers to compare users own CoV-infected host transcriptome data against the built-in CoV datasets and visualize their results via interactive PCA, UpSet and Pathway plots. CompCorona is freely available on the web at http://compcorona.mu.edu.tr tugbasuzek@mu.edu.tr","version":"1.2","doi":"10.1101/2023.01.21.524927","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.26.525759","pub_date":"2023-1-27","title":"The Omicron variant BQ.1* with mutations at positions 28,311 and 28,312 in the SARS-CoV-2 N gene have minimal impact on CDC N1 target detection","abstract":"Ensuring COVID-19 testing remains accurate and reliable is of critical importance as the SARS-CoV-2 virus continues to evolve. Currently, a number of Omicron variants are dominating infection across the globe in including BQ.1 and XBB. Both variants and their sublineages (BQ.1* and XBB*) contain a 28,311 C/U mutation inherited from the original Omicron variant (BA.1). This mutation overlaps with a commonly used fluorescent probe for N gene detection in many Emergency Use Authorization (EUA) assays, as this target was originally established by the U.S. Centers for Disease Control and Prevention (CDC) in their EUA test for COVID-19 (2019-nCoV_N1). This C to U mutation was previously shown to have no impact on CDC N1 target detection. The rise of Omicron sublineages has increased the likelihood of additional point mutations occurring within the same assay target. A subpopulation of BQ.1* has an additional 28,312 C/U mutation within the CDC 2019_nCoV_N1 fluorescent probe in addition to the 28,311 C/U mutation. The double mutation could adversely affect the ability of diagnostic assays to detect the virus in patient samples and therefore it is important to verify the impacts of this additional mutation. Using in vitro transcribed (IVT) N gene RNA representing the wildtype (GenBank/GISAID ID MN908947.3) and Omicron BQ.1.1 variant (BQ.1, GISAID ID EPI_ISL_ 15155651), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays successfully detected the mutant N gene sequence efficiently even at 10 copies of input, although the double mutation caused a 0.5\u223c1 Cq delay on average when compared to the wild-type sequence. These data suggest that circulating BQ.1* lineage viruses with this double mutation likely have minimal impact on diagnostic assays that use the 2019-nCoV-N1 primer-probe.","version":"1.1","doi":"10.1101/2023.01.26.525759","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.26.525578","pub_date":"2023-1-27","title":"The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.","version":"1.1","doi":"10.1101/2023.01.26.525578","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.25.525551","pub_date":"2023-1-26","title":"LY6E protects mice from pathogenic effects of murine coronavirus and SARS-CoV-2","abstract":"LY6E is an antiviral protein that inhibits coronavirus entry. Its expression in immune cells allows mice to control murine coronavirus infection. However, it is not known which immune cell subsets mediate this control or whether LY6E protects mice from SARS-CoV-2. In this study, we used tissue-specific Cre recombinase expression to ablate Ly6e in distinct immune compartments or in all epiblast-derived cells, and bone marrow chimeras to target Ly6e in a subset of radioresistant cells. Mice lacking Ly6e in Lyz2-expressing cells and radioresistant Vav1-expressing cells were more susceptible to lethal murine coronavirus infection. Mice lacking Ly6e globally developed clinical disease when challenged with the Gamma (P.1) variant of SARS-CoV-2. By contrast, wildtype mice and mice lacking type I and type III interferon signaling had no clinical symptoms after SARS-CoV-2 infection. Transcriptomic profiling of lungs from SARS-CoV-2-infected wildtype and Ly6e knockout mice revealed a striking reduction of secretory cell-associated genes in infected knockout mice, including Muc5b, an airway mucin-encoding gene that may protect against SARS-CoV-2-inflicted respiratory disease. Collectively, our study reveals distinct cellular compartments in which Ly6e confers cell intrinsic antiviral effects, thereby conferring resistance to disease caused by murine coronavirus and SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.01.25.525551","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.25.525589","pub_date":"2023-1-26","title":"Rapid engineering of SARS-CoV-2 therapeutic antibodies to increase breadth of neutralization including XBB.1.5 and BQ.1.1","abstract":"An antibody panel that broadly neutralizes currently circulating Omicron variants was obtained by in vitro affinity maturation using phage display. Starting from a single parent clone, antibody engineering was performed in iterative stages in real time as variants emerged using a proprietary technology called STage-Enhanced Maturation (STEM). Humanized from a rabbit antibody, the parent clone showed undetectable neutralization of later Omicron variants, while an early stage IgG possessing only an engineered light chain potently neutralizes some BA.2 but not BA.4/BA.5 lineage variants. However, the final heavy and light chain engineered mAbs show potent neutralization of XBB.1.5 and BQ.1.1 by surrogate virus neutralization test, and biolayer interferometry shows pM KD affinity for both variants. Our work not only details novel therapeutic candidates but also validates a unique general strategy to create broadly neutralizing mAbs to current and future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.01.25.525589","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.25.524586","pub_date":"2023-1-25","title":"A role for Toll-like receptor 3 in lung vascular remodeling associated with SARS-CoV-2 infection","abstract":"Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs). In vitro, SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.","version":"1.1","doi":"10.1101/2023.01.25.524586","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.25.525485","pub_date":"2023-1-25","title":"Long-term respiratory mucosal immune memory to SARS-CoV-2 after infection and vaccination","abstract":"Respiratory mucosal immunity induced by vaccination is vital for protection from coronavirus infection in animal models. In humans, SARS-CoV-2 immunity has been studied extensively in blood. However, the capacity of peripheral vaccination to generate sustained humoral and cellular immunity in the lung mucosa, and how this is influenced by prior SARS-CoV-2 infection, is unknown. Bronchoalveolar lavage samples obtained from vaccinated donors with or without prior infection revealed enrichment of spike-specific antibodies, class-switched memory B cells and T cells in the lung mucosa compared to the periphery in the setting of hybrid immunity, whereas in the context of vaccination alone, local anti-viral immunity was limited to antibody responses. Spike-specific T cells persisted in the lung mucosa for up to 5 months post-vaccination and multi-specific T cell responses were detected at least up to 11 months post-infection. Thus, durable lung mucosal immunity against SARS-CoV-2 seen after hybrid exposure cannot be achieved by peripheral vaccination alone, supporting the need for vaccines targeting the airways.","version":"1.1","doi":"10.1101/2023.01.25.525485","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.06.467547","pub_date":"2023-1-25","title":"Adaptive trends of sequence compositional complexity over pandemic time in the SARS-CoV-2 coronavirus","abstract":"During the spread of the COVID-19 pandemic, the SARS-CoV-2 coronavirus underwent mutation and recombination events that altered its genome compositional structure, thus providing an unprecedented opportunity to search for adaptive evolutionary trends in real-time. The mutation rate in coronavirus is known to be lower than expected for neutral evolution, thus suggesting a role for natural selection. We summarize the compositional heterogeneity of each viral genome by computing its Sequence Compositional Complexity (SCC). To study the full range of SCC diversity, random samples of high-quality coronavirus genomes covering pandemic time span were analyzed. We then search for evolutionary trends that could inform on the adaptive process of the virus to its human host by computing the phylogenetic ridge regression of SCC against time (i.e., the collection date of each viral isolate). In early samples, we find no statistical support for any trend in SCC, although the viral genome appears to evolve faster than Brownian Motion (BM) expectation. However, in samples taken after the emergence of high fitness variants, and despite the brief time span elapsed, a driven decreasing trend for SCC, and an increasing one for its absolute evolutionary rate, are detected, pointing to a role for selection in the evolution of SCC in coronavirus genomes. We conclude that the higher fitness of variant genomes leads to adaptive trends of SCC over pandemic time in the coronavirus.","version":"1.6","doi":"10.1101/2021.11.06.467547","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.25.525479","pub_date":"2023-1-25","title":"Coronaviruses use ACE2 monomers as entry receptors","abstract":"The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of preferably multimeric ACE2 receptors to achieve a higher binding and infection efficiency. However, our current knowledge about the influence of ACE2 expression and organization in the plasma membrane on SARS-CoV-2 infection efficiency remains elusive. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors \u03bcm-2. In addition, binding of trimeric S proteins does not induce clustering of ACE2 receptors in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection which attests SARS-CoV-2 a high infectivity.","version":"1.1","doi":"10.1101/2023.01.25.525479","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.25.525527","pub_date":"2023-1-25","title":"The Evolution of Local Energetic Frustration in Protein Families","abstract":"Energetic local frustration offers a biophysical perspective to interpret the effects of sequence variability on protein families. Here we present a methodology to analyze local frustration patterns within protein families that allows us to uncover constraints related to stability and function, and identify differential frustration patterns in families with a common ancestry. We have analyzed these signals in very well studied cases such as PDZ, SH3, \u03b1 and \u03b2 globins and RAS families. Recent advances in protein structure prediction make it possible to analyze a vast majority of the protein space. An automatic and unsupervised proteome-wide analysis on the SARS-CoV-2 virus demonstrates the potential of our approach to enhance our understanding of the natural phenotypic diversity of protein families beyond single protein instances. We have applied our method to modify biophysical properties of natural proteins based on their family properties, as well as perform unsupervised analysis of large datasets to shed light on the physicochemical signatures of poorly characterized proteins such as emergent pathogens.","version":"1.1","doi":"10.1101/2023.01.25.525527","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.24.525203","pub_date":"2023-1-24","title":"Multimodal characterization of antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection","abstract":"The human immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we utilize multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after BNT162b2 immunization. Our data reveal distinct subpopulations of CD8+ T cells which reliably appear 28 days after prime vaccination (7 days post boost). Using a suite of cross-modality integration tools, we define their transcriptome, accessible chromatin landscape, and immunophenotype, and identify unique biomarkers within each modality. By leveraging DNA-oligo-tagged peptide-MHC multimers and T cell receptor sequencing, we demonstrate that this vaccine-induced population is SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we also identify these CD8+ populations in scRNA-seq datasets from COVID-19 patients and find that their relative frequency and differentiation outcomes are predictive of subsequent clinical outcomes. Our work contributes to our understanding of T cell immunity, and highlights the potential for integrative and multimodal analysis to characterize rare cell populations.","version":"1.1","doi":"10.1101/2023.01.24.525203","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.23.525275","pub_date":"2023-1-24","title":"Genome-wide CRISPR screens identify noncanonical translation factor eIF2A as an enhancer of SARS-CoV-2 programmed \u20131 ribosomal frameshifting","abstract":"Many positive-strand RNA viruses, including all known coronaviruses, employ programmed \u20131 ribosomal frameshifting (\u20131 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate \u20131 PRF. Through a reporter-based genome-wide CRISPR/Cas9 knockout screen, we identified several host factors that either suppressed or enhanced \u20131 PRF of SARS-CoV-2. One of these factors is eukaryotic translation initiation factor 2A (eIF2A), which specifically and directly enhanced \u20131 PRF in vitro and in cells. Consistent with the crucial role of efficient \u20131 PRF in transcriptase/replicase expression, loss of eIF2A reduced SARS-CoV-2 replication in cells. Transcriptome-wide analysis of eIF2A-interacting RNAs showed that eIF2A primarily interacted with 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results revealed an unexpected role for eIF2A in modulating the translation of specific RNAs independent of its previously described role during initiation.","version":"1.1","doi":"10.1101/2023.01.23.525275","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.22.525048","pub_date":"2023-1-23","title":"In-silico Analysis of SARS-Cov2 Spike Proteins of Different Field Variants","abstract":"Coronaviruses belong to the group of RNA family of viruses which trigger diseases in birds, humans, and mammals, which can cause respiratory tract infections. The COVID-19 pandemic has badly affected every part of the world, and the situation in the world is getting worse with the emergence of novel variants. Our study aims to explore the genome of SARS-,CoV2 followed by in silico analysis of its proteins. Different nucleotide and protein variants of SARS-Cov2 were retrieved from NCBI. Contigs & consensus sequences were developed to identify variations in these variants by using SnapGene. Data of variants that significantly differ from each other was run through Predict Protein software to understand changes produced in protein structure The SOPMA web server was used to predict the secondary structure of proteins. Tertiary structure details of selected proteins were analyzed using the online web server SWISS-MODEL. Sequencing results shows numerous single nucleotide polymorphisms in surface glycoprotein, nucleocapsid, ORF1a, and ORF1ab polyprotein. While envelope, membrane, ORF3a, ORF6, ORF7a, ORF8, and ORF10 genes have no or few SNPs. Contigs were mto identifyn of variations in Alpha & Delta Variant of SARs-CoV-2 with reference strain (Wuhan). The secondary structures of SARs-CoV-2 proteins were predicted by using sopma software & were further compared with reference strain of SARS-CoV-2 (Wuhan) proteins. The tertiary structure details of only spike proteins were analyzed through the SWISS-MODEL and Ramachandran plot. By Swiss-model, a comparison of the tertiary structure model of SARS-COV-2 spike protein of Alpha & Delta Variant was made with reference strain (Wuhan). Alpha & Delta Variant of SARs-CoV-2 isolates submitted in GISAID from Pakistan with changes in structural and nonstructural proteins were compared with reference strain & 3D structure mapping of spike glycoprotein and mutations in amino acid were seen. The surprising increased rate of SARS-CoV-2 transmission has forced numerous countries to impose a total lockdown due to an unusual occurrence. In this research, we employed in silico computational tools to analyze SARS-CoV-2 genomes worldwide to detect vital variations in structural proteins and dynamic changes in all SARS-CoV-2 proteins, mainly spike proteins, produced due to many mutations. Our analysis revealed substantial differences in functional, immunological, physicochemical, & structural variations in SARS-CoV-2 isolates. However real impact of these SNPs can only be determined further by experiments. Our results can aid in vivo and in vitro experiments in the future.","version":"1.1","doi":"10.1101/2023.01.22.525048","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.20.524748","pub_date":"2023-1-23","title":"A Novel Monoclonal Antibody Targeting a Large Surface of the Receptor Binding Motif Shows Pan-neutralizing SARS-CoV-2 Activity Including BQ.1.1 Variant","abstract":"In the present study we report the functional and structural characterization of 17T2, a new highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (mAb) isolated from a convalescent COVID-19 individual infected during the first wave of the COVID-19 pandemic. 17T2 is a class 1 VH1-58/\u03ba3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA memory B cell and developed as a human recombinant IgG1. Functional characterization revealed that 17T2 mAb has a high and exceptionally broad neutralizing activity against all SARS-CoV-2 spike variants tested, including BQ.1.1. Moreover, 17T2 mAb has in vivo prophylactic activity against Omicron BA.1.1 infection in K18-hACE2 transgenic mice. 3D reconstruction from cryogenic-electron microscopy (cryo-EM) showed that 17T2 binds the Omicron BA.1 spike protein with the RBD domains in \u201cup\u201d position and recognizes an epitope overlapping with the receptor binding motif, as it is the case for other structurally similar neutralizing mAbs, including S2E12. Yet, unlike S2E12, 17T2 retained its high neutralizing activity against all Omicron sublineages tested, probably due to a larger contact area with the RBD, which could confer a higher resilience to spike mutations. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 mAb as a potential candidate for future therapeutic and prophylactic interventions.","version":"1.1","doi":"10.1101/2023.01.20.524748","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.23.525130","pub_date":"2023-1-23","title":"Prediction of SARS-CoV-2 spike protein mutations using Sequence-to-Sequence and Transformer models","abstract":"In the study of viral epidemics, having information about the structural evolution of the virus can be very helpful in controlling the disease and making vaccines. Various deep learning and natural language processing techniques (NLP) can be used to analyze genetic structure of viruses, namely to predict their mutations. In this paper, by using Sequence-to-Sequence (Seq2Seq) model with Long Short-Term Memory (LSTM) cell and Transformer model with the attention mechanism, we investigate the spike protein mutations of SARS-CoV-2 virus. We make time-series datasets of the spike protein sequences of this virus and generate upcoming spike protein sequences. We also determine the mutations of the generated spike protein sequences, by comparing these sequences with the Wuhan spike protein sequence. We train the models to make predictions in December 2021, February 2022, and October 2022. Furthermore, we find that some of our generated spike protein sequences have been reported in December 2021 and February 2022, which belong to Delta and Omicron variants. The results obtained in the present study could be useful for prediction of future mutations of SARS-CoV-2 and other viruses.","version":"1.1","doi":"10.1101/2023.01.23.525130","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.22.525117","pub_date":"2023-1-23","title":"A New Approach for Active Coronavirus Infection Identification by Targeting the Negative RNA Strand- A Replacement for the Current Positive RNA-based qPCR Detection Method","abstract":"This manuscript describes the development of an alternative method to detect active coronavirus infection, in light of the current COVID-19 pandemic caused by the SARS-CoV-2 virus. The pandemic, which was first identified in Wuhan, China in December 2019, has had a significant impact on global health as well as on the economy and daily life in the world. The current positive RNA-based detection systems are unable to discriminate between replicating and non-replicating viruses, complicating decisions related to quarantine and therapeutic interventions. The proposed method targets the negative strand of the virus and has the potential to effectively distinguish between active and inactive infections, which could provide a more accurate means of determining the spread of the virus and guide more effective public health measures during the current pandemic.","version":"1.1","doi":"10.1101/2023.01.22.525117","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.22.525079","pub_date":"2023-1-23","title":"Waning Immunity Against XBB.1.5 Following Bivalent mRNA Boosters","abstract":"The SARS-CoV-2 Omicron variant has continued to evolve. XBB is a recombinant between two BA.2 sublineages, XBB.1 includes the G252V mutation, and XBB.1.5 includes the G252V and F486P mutations. XBB.1.5 has rapidly increased in frequency and has become the dominant virus in New England. The bivalent mRNA vaccine boosters have been shown to increase neutralizing antibody (NAb) titers to multiple variants, but the durability of these responses remains to be determined. We assessed humoral and cellular immune responses in 30 participants who received the bivalent mRNA boosters and performed assays at baseline prior to boosting, at week 3 after boosting, and at month 3 after boosting. Our data demonstrate that XBB.1.5 substantially escapes NAb responses but not T cell responses after bivalent mRNA boosting. NAb titers to XBB.1 and XBB.1.5 were similar, suggesting that the F486P mutation confers greater transmissibility but not increased immune escape. By month 3, NAb titers to XBB.1 and XBB.1.5 declined essentially to baseline levels prior to boosting, while NAb titers to other variants declined less strikingly.","version":"1.1","doi":"10.1101/2023.01.22.525079","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.19.524784","pub_date":"2023-1-20","title":"Extremely potent pan-sarbecovirus neutralizing antibodies generated by immunization of macaques with an AS03-adjuvanted monovalent subunit vaccine against SARS-CoV-2","abstract":"The rapid emergence of SARS-CoV-2 variants that evade immunity to vaccination has placed a global health imperative on the development of therapeutic countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent pan-sarbecovirus antibodies from non-human primates vaccinated with an AS03 adjuvanted subunit vaccine against SARS-CoV-2 that recognize conserved epitopes in the receptor binding domain (RBD) with femtomolar affinities. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells for at least one year following primary vaccination. 514 monoclonal antibodies (mAbs) were generated from antigen-specific memory B cells. Antibodies isolated at 5 to 12 months following vaccination displayed greater potency and breadth, relative to those identified at 1.4 months. Notably, 15 out of 338 (\u223c4.4%) antibodies isolated at 1.4\u223c6 months after the primary vaccination showed extraordinary neutralization potency against SARS-CoV-2 omicron BA.1, despite the absence of BA.1 neutralization in serum. Two of them, 25F9 and 20A7, neutralized authentic clade Ia sarbecoviruses (SARS-CoV, WIV-1, SHC014) and clade Ib sarbecoviruses (SARS-CoV-2 D614G, SARS-CoV-2 BA.1, Pangolin-GD) with half-maximal inhibition concentrations of (0.85 ng/ml, 3 ng/ml, 6 ng/ml, 6 ng/ml, 42 ng/ml, 6 ng/ml) and (13 ng/ml, 2 ng/ml, 18 ng/ml, 9 ng/ml, 6 ng/ml, 345 ng/ml), respectively. Furthermore, 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants of concern and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1 and XBB variants. X-ray crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved RBD sites. In vivo prophylactic protection of 25F9, 20A7 and 27A12 was confirmed in aged Balb/c mice. Notably, administration of 25F9 provided complete protection against SARS-CoV-2, SARS-CoV-2 BA.1, SARS-CoV, and SHC014 challenge, underscoring that these mAbs are promising pan-sarbecovirus therapeutic antibodies. Extremely potent pan-sarbecovirus neutralizing antibodies","version":"1.1","doi":"10.1101/2023.01.19.524784","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.19.524762","pub_date":"2023-1-20","title":"Induction of SARS-CoV-2 N-specific CD8+ T cell immunity in lungs by engineered extracellular vesicles associates with strongly impaired viral replication","abstract":"Induction of effective immunity in lungs should be a pre-requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. In the case of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection, vaccination is expected to associate with significant inhibition of viral replication in lungs. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of SARS-CoV-2 Nucleocapsid (N) protein can protect K18-hACE2 transgenic mice from the lethal intranasal infection with the ancestral Wuhan isolate. Actually, it was widely demonstrated that these transgenic mice succumb to SARS-CoV-2 intranasal infection mainly as a consequence of the viral invasiveness of central nervous system, a pathogenetic mechanism almost absent in humans. On the other hand, K18-hACE2 transgenic mice support viral replication in lungs, an event strictly mirroring the major pathogenic signature linked to the severe disease in humans. However, nothing is known about the ability of N-specific CD8+ T cell immunity induced by engineered EVs in controlling viral replication in lungs. To fill the gap, we investigated the immunity generated in lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8+ T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in lungs were evaluated. We found that three weeks after second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8+ T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting. This inhibitory effect associated with the persistence of a N-specific CD8+ T-resident memory lymphocytes in lungs of N-immunized mice. In view of the quite conserved sequence of the N protein among SARS-CoV-2 variants, these results support the idea that a vaccine strategy focused on the induction of anti-N CD8+ T cell immunity in lungs has the potential to control the replication of emerging variants.","version":"1.1","doi":"10.1101/2023.01.19.524762","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.19.524824","pub_date":"2023-1-20","title":"Broad-spectrum antiviral inhibitors targeting pandemic potential RNA viruses","abstract":"RNA viruses continue to remain a clear and present threat for potential pandemics due to their rapid evolution. To mitigate their impact, we urgently require antiviral agents that can inhibit multiple families of disease-causing viruses, such as arthropod-borne and respiratory pathogens. Potentiating host antiviral pathways can prevent or limit viral infections before escalating into a major outbreak. Therefore, it is critical to identify broad-spectrum antiviral agents. We have tested a small library of innate immune agonists targeting pathogen recognition receptors, including TLRs, STING, NOD, Dectin and cytosolic DNA or RNA sensors. We observed that TLR3, STING, TLR8 and Dectin-1 ligands inhibited arboviruses, Chikungunya virus (CHIKV), West Nile virus (WNV) and Zika virus, to varying degrees. Cyclic dinucleotide (CDN) STING agonists, such as cAIMP, diABZI, and 2\u2019,3\u2019-cGAMP, and Dectin-1 agonist scleroglucan, demonstrated the most potent, broad-spectrum antiviral function. Comparative transcriptome analysis revealed that CHIKV-infected cells had larger number of differentially expressed genes than of WNV and ZIKV. Furthermore, gene expression analysis showed that cAIMP treatment rescued cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provided protection against CHIKV in a CHIKV-arthritis mouse model. Cardioprotective effects of synthetic STING ligands against CHIKV, WNV, SARS-CoV-2 and enterovirus D68 (EV-D68) infections were demonstrated using human cardiomyocytes. Interestingly, the direct-acting antiviral drug remdesivir, a nucleoside analogue, was not effective against CHIKV and WNV, but exhibited potent antiviral effects against SARS-CoV-2, RSV (respiratory syncytial virus), and EV-D68. Our study identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses, which can be rapidly deployed to prevent or mitigate future pandemics.","version":"1.1","doi":"10.1101/2023.01.19.524824","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.22.517339","pub_date":"2023-1-19","title":"Host response of Syrian hamster to SARS-CoV-2 infection, including differences with humans and between sexes","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the importance of having proper tools and models to study the pathophysiology of emerging infectious diseases to test therapeutic protocols, assess changes in viral phenotype and evaluate the effect of viral evolution. This study provides a comprehensive characterization of the Syrian hamster (Mesocricetus auratus) as an animal model for SARS-CoV-2 infection, using different approaches (description of clinical signs, viral load, receptor profiling and host immune response) and targeting four different organs (lungs, intestine, brain and PBMCs). Our data showed that both male and female hamsters are susceptible to the infection and develop a disease similar to the one observed in patients with COVID-19, including moderate to severe pulmonary lesions, inflammation and recruitment of the immune system in lungs and at systemic level. However, all animals recovered within 14 days without developing the severe pathology seen in humans, and none of them died. We found faint evidence for intestinal and neurological tropism associated with the absence of lesions and a minimal host response in intestines and brains, highlighting another crucial difference with the multi-organ impairment of severe COVID-19. When comparing male and female hamsters, it was observed that males sustained higher viral RNA shedding and replication in the lungs, suffered from more severe symptoms and histopathological lesions and triggered higher pulmonary inflammation. Overall, these data confirm the Syrian hamster as a suitable model for mildmoderate COVID-19 and reflect sex-related differences in the response against the virus observed in humans.","version":"1.2","doi":"10.1101/2022.11.22.517339","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.15.460543","pub_date":"2023-1-19","title":"Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin","abstract":"The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.","version":"1.3","doi":"10.1101/2021.09.15.460543","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.10.523356","pub_date":"2023-1-19","title":"Exosome based multivalent vaccine: achieving potent immunization, broadened reactivity, and strong T cell responses with nanograms of proteins","abstract":"Current approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused solely on the spike protein to provide immunity. The first vaccines were developed rapidly using spike mRNA delivered by lipid nanoparticles but required ultra-low storage and have had limited immunity against variations in spike. Subsequently, protein-based vaccines were developed which offer broader immunity but require significant time for development and use of an adjuvant to boost immune response. Here, exosomes were used to deliver a bi-valent protein-based vaccine, in which two independent viral proteins were used. Exosomes were engineered to express either SARS-CoV-2 Delta spike (Stealth X-Spike, STX-S) or the more conserved nucleocapsid (Stealth X-Nucleocapsid, STX-N) protein on the surface. When administered as single product (STX-S or STX-N) or in combination (STX-S+N), both STX-S and STX-N induced a strong immunization with the production of a potent humoral and cellular immune response. Interestingly, these results were obtained with administration of only nanograms of protein and without adjuvant. In two independent animal models (mouse and rabbit), administration of nanograms of the STX-S+N vaccine resulted in increased antibody production, potent neutralizing antibodies with cross-reactivity to other variants of spike and strong T-cell responses. Importantly, no competition in immune response was observed, allowing for delivery of nucleocapsid with spike to offer improved SARS-CoV-2 immunity. These data show that the StealthXTM exosome platform has an enormous potential to revolutionize vaccinology by combining the advantages of mRNA and recombinant protein vaccines into a superior, rapidly generated, low dose vaccine resulting in potent, broader immunity.","version":"1.2","doi":"10.1101/2023.01.10.523356","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.334532","pub_date":"2023-1-18","title":"Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19","abstract":"COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that rapidly caused a pandemic. Coalescence of this virus with seasonal respiratory viruses, particularly influenza virus is a global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, they exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by infection with IAV is responsible for the observed inhibition of SARS-CoV-2, however, infection with attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest that the concept of \u2018twinfection\u2019 is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.","version":"1.3","doi":"10.1101/2020.10.13.334532","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424231","pub_date":"2023-1-18","title":"Genomic diversity of SARS-CoV-2 can be accelerated by mutations in the nsp14 gene","abstract":"Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a proofreading exonuclease, nonstructural protein 14 (nsp14), that helps ensure replication competence at a low evolutionary rate compared with other RNA viruses. In the current pandemic, SARS-CoV-2 has accumulated diverse genomic mutations including in nsp14. Here, to clarify whether amino acid substitutions in nsp14 affect the genomic diversity and evolution of SARS-CoV-2, we searched for amino acid substitutions in nature that may interfere with nsp14 function. We found that viruses carrying a proline-to-leucine change at position 203 (P203L) have a high evolutionary rate and that a recombinant SARS-CoV-2 virus with the P203L mutation acquired more diverse genomic mutations than wild-type virus during its replication in hamsters. Our findings suggest that substitutions, such as P203L, in nsp14 may accelerate the genomic diversity of SARS-CoV-2, contributing to virus evolution during the pandemic.","version":"1.4","doi":"10.1101/2020.12.23.424231","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.17.524469","pub_date":"2023-1-18","title":"A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease","abstract":"SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (MPro) for replication and pathogenesis. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MPro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 reversibly covalent dipeptidyl MPro inhibitors and characterized them on in vitro enzymatic inhibition potency, structures of their complexes with MPro, cellular MPro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that MPro has a flexible S2 pocket that accommodates dipeptidyl inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro[4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have optimal characteristics. One compound MPI60 containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability and can be potentially advanced to further preclinical tests.","version":"1.1","doi":"10.1101/2023.01.17.524469","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.17.524329","pub_date":"2023-1-18","title":"Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology","abstract":"Aging is the primary risk factor for most neurodegenerative diseases, and recently coronavirus disease 2019 (COVID-19) has been associated with severe neurological manifestations that can eventually impact neurodegenerative conditions in the long-term. The progressive accumulation of senescent cells in vivo strongly contributes to brain aging and neurodegenerative co-morbidities but the impact of virus-induced senescence in the aetiology of neuropathologies is unknown. Here, we show that senescent cells accumulate in physiologically aged brain organoids of human origin and that senolytic treatment reduces inflammation and cellular senescence; for which we found that combined treatment with the senolytic drugs dasatinib and quercetin rejuvenates transcriptomic human brain aging clocks. We further interrogated brain frontal cortex regions in postmortem patients who succumbed to severe COVID-19 and observed increased accumulation of senescent cells as compared to age-matched control brains from non-COVID-affected individuals. Moreover, we show that exposure of human brain organoids to SARS-CoV-2 evoked cellular senescence, and that spatial transcriptomic sequencing of virus-induced senescent cells identified a unique SARS-CoV-2 variant-specific inflammatory signature that is different from endogenous naturally-emerging senescent cells. Importantly, following SARS-CoV-2 infection of human brain organoids, treatment with senolytics blocked viral retention and prevented the emergence of senescent corticothalamic and GABAergic neurons. Furthermore, we demonstrate in human ACE2 overexpressing mice that senolytic treatment ameliorates COVID-19 brain pathology following infection with SARS-CoV-2. In vivo treatment with senolytics improved SARS-CoV-2 clinical phenotype and survival, alleviated brain senescence and reactive astrogliosis, promoted survival of dopaminergic neurons, and reduced viral and senescence-associated secretory phenotype gene expression in the brain. Collectively, our findings demonstrate SARS-CoV-2 can trigger cellular senescence in the brain, and that senolytic therapy mitigates senescence-driven brain aging and multiple neuropathological sequelae caused by neurotropic viruses, including SARS-CoV-2.","version":"1.1","doi":"10.1101/2023.01.17.524329","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.09.523209","pub_date":"2023-1-18","title":"Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern","abstract":"The high mutation rate of SARS-CoV-2 leads to emergence of several variants, some of which are resistant to vaccines and drugs targeting viral elements. Targeting host dependency factors \u2013 cell proteins required for viral replication - would help avoid resistance. However, whether different SARS-CoV-2 variants induce conserved cell responses and exploit the same core host factors is still unclear. We compared three variants of concern and observed that the host transcriptional response was conserved, differing only in kinetics and magnitude. By CRISPR screening we identified the host genes required for infection by each variant: most of the identified genes were shared by multiple variants, both in lung and colon cells. We validated our hits with small molecules and repurposed FDA-approved drugs. All drugs were highly effective against all tested variants, including delta and omicron, new variants that emerged during the study. Mechanistically, we identified ROS production as a pivotal step in early virus propagation. Antioxidant drugs, such as N-acetyl cysteine (NAC), were effective against all variants both in human lung cells, and in a humanised mouse model. Our study supports the use of available antioxidant drugs, such as NAC, as a general and effective anti-COVID-19 approach.","version":"1.2","doi":"10.1101/2023.01.09.523209","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.17.524472","pub_date":"2023-1-18","title":"A deep generative model of the SARS-CoV-2 spike protein predicts future variants","abstract":"SARS-CoV-2 has demonstrated a robust ability to adapt in response to environmental pressures\u2014increasing viral transmission and evading immune surveillance by mutating its molecular machinery. While viral sequencing has allowed for the early detection of emerging variants, methods to predict mutations before they occur remain limited. This work presents SpikeGPT2, a deep generative model based on ProtGPT2 and fine-tuned on SARS-CoV-2 spike (S) protein sequences deposited in the NIH Data Hub before May 2021. SpikeGPT2 achieved 88.8% next-residue prediction accuracy and successfully predicted amino acid substitutions found only in a held-out set of spike sequences deposited on or after May 2021, to which SpikeGPT2 was never exposed. When compared to several other methods, SpikeGPT2 achieved the best performance in predicting such future mutations. SpikeGPT2 also predicted several novel variants not present in the NIH SARS-CoV-2 Data Hub. A binding affinity analysis of all 54 generated substitutions identified 5 (N439A, N440G, K458T, L492I, and N501Y) as predicted to simultaneously increase S/ACE2 affinity, and decrease S/tixagevimab+cilgavimab affinity. Of these, N501Y has already been well-described to increase transmissibility of SARS-CoV-2. These findings indicate that SpikeGPT2 and other similar models may be employed to identify high-risk future variants before viral spread has occurred.","version":"1.1","doi":"10.1101/2023.01.17.524472","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.16.524211","pub_date":"2023-1-18","title":"The emergence of goblet inflammatory or ITGB6hi nasal progenitor cells determines age-associated SARS-CoV-2 pathogenesis","abstract":"Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (<11y), adult (30- 50y) and elderly (>70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hi progenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways.","version":"1.2","doi":"10.1101/2023.01.16.524211","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.17.524254","pub_date":"2023-1-18","title":"Higher Angiotensin I Converting Enzyme 2 (ACE2) levels in the brain of individuals with Alzheimer\u2019s disease","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of death in the elderly. Cognitive decline due to Alzheimer\u2019s disease (AD) is frequent in the geriatric population disproportionately affected by the COVID-19 pandemic. Interestingly, central nervous system (CNS) manifestations have been reported in SARS-CoV-2-infected patients. In this study, we investigated the levels of Angiotensin I Converting Enzyme 2 (ACE2), the main entry receptor of SARS-COV-2 in cells, in postmortem parietal cortex samples from two independent AD cohorts, totalling 142 persons. Higher concentrations of ACE2 protein and mRNA were found in individuals with a neuropathological diagnosis of AD compared to age-matched healthy control subjects. Brain levels of soluble ACE2 were inversely associated with cognitive scores (p = 0.02), markers of pericytes (PDGFR\u03b2, p=0.02 and ANPEP, p = 0.007) and caveolin1 (p = 0.03), but positively correlated with soluble amyloid-\u03b2 peptides (A\u03b2) concentrations (p = 0.01) and insoluble phospho- tau (S396/404, p = 0.002). No significant differences in ACE2 were observed in the 3xTgAD mouse model of tau and A\u03b2 neuropathology. Results from immunofluorescence and Western blots showed that ACE2 protein is mainly localized in neurons in the human brain but predominantly in microvessels in the mouse brain. The present data show that an AD diagnosis is associated with higher levels of soluble ACE2 in the human brain, which might contribute to a higher risk of CNS SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.01.17.524254","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.17.524492","pub_date":"2023-1-18","title":"Discovery of highly potent small molecule pan-coronavirus fusion inhibitors","abstract":"The unprecedented pandemic of COVID-19, caused by a novel coronavirus, SARS-CoV-2, has led to massive human suffering, death, and economic devastation worldwide. The virus is mutating fast to more transmissible and infectious variants. The Delta variant (B.1.617.2), initially identified in India, and the omicron variant (BA.4 and BA.5) have spread worldwide. In addition, recently alarming antibody evasive SARS-CoV-2 subvariants, BQ and XBB, have been reported. These new variants may pose a substantial challenge to controlling the spread of this virus. Therefore, the continued development of novel drugs having pan-coronavirus inhibition to treat and prevent infection of COVID-19 is urgently needed. These drugs will be critically important in dealing with new pandemics that will emerge in the future. We report the discovery of several highly potent small molecule pan-coronavirus inhibitors. One of which, NBCoV63, showed low nM potency against SARS-CoV-2 (IC50: 55 nM), SARS-CoV (IC50: 59 nM), and MERS-CoV (IC50: 75 nM) in pseudovirus-based assays with excellent selectivity indices (SI: as high as > 900) demonstrating its pan-coronavirus inhibition. NBCoV63 showed equally effective antiviral potency against SARS-CoV-2 mutant (D614G) and several variants of concerns (VOCs) such as B.1.617.2 (Delta), B.1.1.529/BA.1 and BA.4/BA.5 (Omicron) and K417T/E484K/N501Y (Gamma). NBCoV63 also showed similar efficacy profiles to Remdesivir against authentic SARS-CoV-2 (Hong Kong strain) and two of its variants (Delta and Omicron) by plaque reduction in Calu3 cells. Additionally, we show that NBCoV63 inhibits virus-mediated cell-to-cell fusion in a dose-dependent manner. Furthermore, the Absorption, distribution, metabolism, and excretion (ADME) data of NBCoV63 demonstrated drug-like properties.","version":"1.1","doi":"10.1101/2023.01.17.524492","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.11.523616","pub_date":"2023-1-17","title":"Intranasal mRNA-LNP vaccination protects hamsters from SARS-CoV-2 infection","abstract":"Intranasal vaccination represents a promising approach for preventing disease caused by respiratory pathogens by eliciting a mucosal immune response in the respiratory tract that may act as an early barrier to infection and transmission. This study investigated immunogenicity and protective efficacy of intranasally administered messenger RNA (mRNA)\u2013lipid nanoparticle (LNP) encapsulated vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian golden hamsters. Intranasal mRNA-LNP vaccination systemically induced spike-specific binding (IgG and IgA) and neutralizing antibodies with similar robustness to intramuscular controls. Additionally, intranasal vaccination decreased viral loads in the respiratory tract, reduced lung pathology, and prevented weight loss after SARS-CoV-2 challenge. This is the first study to demonstrate successful immunogenicity and protection against respiratory viral infection by an intranasally administered mRNA-LNP vaccine.","version":"1.2","doi":"10.1101/2023.01.11.523616","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.15.524090","pub_date":"2023-1-17","title":"T492I mutation alters SARS-CoV-2 properties via modulating viral non-structural proteins","abstract":"The historically dominant SARS-CoV-2 Delta variants and the currently dominant Omicron variants carry a T492I substitution within the non-structural protein 4 (NSP4). Based on a combination of in silico analyses, we predicted that the T492I mutation increases the transmissibility and adaptability of the virus. We confirmed this hypothesis by performing competition experiments in hamsters and in human airway tissue culture models. Furthermore, we show that the T492I mutation also increases the replication capacity and infectiveness of the virus, and improves its ability to evade antibody neutralization induced by previous variants. Mechanistically, the T492I mutation increases cleavage efficiency of the viral main protease NSP5 by enhancing enzyme-substrate binding, resulting in increased production of nearly all non-structural proteins processed by NSP5. Importantly, T492I mutation suppresses the viral RNA associated chemokines in monocytic macrophages, which may contribute to the attenuated pathogenicity of Omicron variants. Our results highlight the importance of the NSP4 mutation in the evolutionary dynamics of SARS-CoV-2 and identify a novel target for the development of broad-spectrum antiviral agents.","version":"1.1","doi":"10.1101/2023.01.15.524090","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.15.524078","pub_date":"2023-1-17","title":"SARS-CoV-2 Envelope protein triggers depression and dysosmia via TLR2 mediated neuroinflammation","abstract":"Depression and dysosmia have been regarded as the main neurological symptoms in COVID-19 patients, the mechanism of which remains unclear. Current studies have demonstrated that SARS-CoV-2 envelope protein served as a pro-inflammatory factor as sensed by Toll like receptor 2 (TLR2), suggesting the viral infection independent pathological feature of E protein. In this study, we aim to determine the role of E protein in depression, dysosmia and associated neuroinflammation in central nervous system (CNS). Depression and olfactory function were observed in both female and male mice as receiving intracisternal injection of envelope protein. Immunohistochemistry was applied in conjunction with RT-PCR to assess the glial activation, blood-brain barrier status and mediators synthesis in cortex, hippocampus and olfactory bulb. TLR2 was pharmacologically blocked to determine its role in E protein related depression and dysosmia. Intracisternal injection of envelope protein evoked depression and dysosmia in both female and male mice. Immunohistochemistry suggested that envelope protein upregulated IBA1 and GFAP in cortex, hippocampus and olfactory bulb, while ZO-1 was downregulated. Moreover, IL-1\u03b2, TNF-\u03b1, IL-6, CCL2, MMP2 and CSF1 were upregulated in both cortex and hippocampus, whereas IL-1\u03b2, IL-6 and CCL2 were upregulated in olfactory bulb. Furtherly, inhibiting microglia, but not astrocyte, alleviated depression and dysosmia induced by envelope protein. Finally, RT-PCR and immunohistochemistry suggested that TLR2 was upregulated in cortex, hippocampus and olfactory bulb, the blocking of which mitigated depression and dysosmia induced by envelope protein. Our study demonstrates that envelope protein could directly induce depression and dysosmia together with obvious neuroinflammation in CNS. TLR2 mediated depression and dysosmia induced by envelope protein, which could serve as a promising therapeutic target for neurological manifestation in COVID-19 patients.","version":"1.1","doi":"10.1101/2023.01.15.524078","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.14.524034","pub_date":"2023-1-17","title":"Exploration of the Link Between COVID-19 and Alcoholic Hepatitis from the Perspective of Bioinformatics and Systems Biology","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been suggested to purpose threats to health of mankind. Alcoholic hepatitis (AH) is a life-threatening acute and chronic liver failure that takes place in sufferers who drink excessively. During the epidemic, AH has an increasing incidence of severe illness and mortality. However, for these two diseases, the intrinsic relationship of molecular pathogenesis, as well as common therapeutic strategies are still poorly understood. The transcriptome of the COVID-19 and AH has been compared to obtain the altered genes and hub genes were screened out through protein-protein interaction (PPI) network analysis. Via gene ontology (GO), pathway enrichment and transcription regulator analysis, a deeper appreciation of the interplay mechanism between hub genes were established. With 181 common differentially expressed genes (DEGs) of AH and COVID-19 were obtained, 10 hub genes were captured. Follow-up studies located that these 10 genes typically mediated the diseases occurrence by regulating the activities of the immune system. Other results suggest that the common pathways of the two ailments are enriched in regulating the function of immune cells and the release of immune molecules. This study reveals the common pathogenesis of COVID-19 and AH and assist to discover necessary therapeutic targets to combat the ongoing pandemic induced via SARS-CoV-2 infection and acquire promising remedy strategies for the two diseases.","version":"1.1","doi":"10.1101/2023.01.14.524034","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.13.523998","pub_date":"2023-1-17","title":"Maintained imbalance of triglycerides, apolipoproteins, energy metabolites and cytokines in long-term COVID-19 syndrome (LTCS) patients","abstract":"Deep metabolomic, proteomic and immunologic phenotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients have matched a wide diversity of clinical symptoms with potential biomarkers for coronavirus disease 2019 (COVID-19). Within here, several studies described the role of metabolites, lipoproteins and inflammation markers during infection and in recovered patients. In fact, after SARS-CoV-2 viral infection almost 20-30% of patients experience persistent symptoms even after 12 weeks of recovery which has been defined as long-term COVID-19 syndrome (LTCS). Emerging evidence revealed that a dysregulated immune system and persisting inflammation could be one of the key drivers of LTCS. However, how these small biomolecules such as metabolites, lipoprotein, cytokines and chemokines altogether govern pathophysiology is largely underexplored. Thus, a clear understanding how these parameters into an integrated fashion could predict the disease course may help to stratify LTCS patients from acute COVID-19 or recovered specimen and would help to elucidate a potential mechanistic role of these biomolecules during the disease course. Here, we report an integrated analysis of blood serum and plasma by in vitro diagnostics research NMR spectroscopy and flow cytometry-based cytokine quantification in a total of 125 individuals (healthy controls (HC; n=73), recovered (n=12), acute (n=7) and LTCS (n=33)). We identified that in LTCS patients lactate and pyruvate were significantly different from either healthy controls or acute COVID-19 patients. Further correlational analysis of cytokines and metabolites indicated that creatine, glutamine, and high-density lipoprotein (HDL) phospholipids were distributed differentially amongst patients or individuals. Of note, triglycerides and several lipoproteins (apolipoproteins Apo-A1 and A2) in LTCS patients demonstrate COVID-19-like alterations compared to HC. Interestingly, LTCS and acute COVID-19 samples were distinguished mostly by their creatinine, phenylalanine, succinate, 3-hydroxybutyrate (3-HB) and glucose concentrations, illustrating an imbalanced energy metabolism. Most of the cytokines and chemokines were present at low levels in LTCS patients compared with HC except IL-18 chemokine, which tended to be higher in LTCS patients and correlated positively with several amino acids (creatine, histidine, leucine, and valine), metabolites (lactate and 3-HB) and lipoproteins. The identification of these persisting plasma metabolites, lipoprotein and inflammation alterations will help to better stratify LTCS patients from other diseases and could help to predict ongoing severity of LTCS patients. Almost 20-30% of individuals infected with the SARS-CoV-2 virus regardless of hospitalization status experience long-term COVID-19 syndrome (LTCS). It is devasting for millions of individuals worldwide and hardly anything is known about why some people experience these symptoms even after 3 to 12 months after the acute phase. In this, we attempted to understand whether dysregulated metabolism and inflammation could be contributing factors to the ongoing symptoms in LTCS patients. Total blood triglycerides and the Cory cycle metabolites (lactate and pyruvate) were significantly higher, lipoproteins (Apo-A1 and A2) were drastically lower in LTCS patients compared to healthy controls. Correlation analysis revealed that either age or gender are positively correlated with several metabolites (citrate, glutamate, 3-hydroxybutyrate, glucose) and lipoproteins (Apo-A1, HDL Apo-A1, LDL triglycerides) in LTCS patients. Several cytokines and chemokines were also positively correlated with metabolites and lipoproteins thus, dysregulation in metabolism and inflammation could be a potential contributory factor for LTCS symptoms.","version":"1.1","doi":"10.1101/2023.01.13.523998","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.16.524178","pub_date":"2023-1-17","title":"Enhanced transmissibility, infectivity and immune resistance of the SARS-CoV-2 Omicron XBB.1.5 variant","abstract":"In 2022, we have elucidated the characteristics of a variety of newly emerging SARS-CoV-2 Omicron subvariants. At the end of 2022, the XBB.1.5 variant, an descendant of XBB.1 that acquired the S:F486P substitution, emerged and is rapidly spreading in the USA and is the latest variant of concern. Although the features of XBB.1.5 was already reported by another group as a preprint, we think multiple and independent evaluations important, and these reports are crucial for sustained global health. In this study, our epidemic dynamics analysis revealed that the relative effective reproduction number (Re) of XBB.1.5 is more than 1.2-fold greater than that of the parental XBB.1, and XBB.1.5 is outcompeting BQ.1.1, the predominant lineage in the USA as of December 2022. Our data suggest that XBB.1.5 will rapidly spread worldwide in the near future. Yeast surface display assay and pseudovirus assay respectively showed that the ACE2 binding affinity and infectivity of XBB.1.5 is 4.3-fold and 3.3-fold higher than those of XBB.1, respectively. Moreover, neutralization assay revealed that XBB.1.5 is robustly resistant to BA.2 breakthrough infection sera (41-fold versus B.1.1, 20-fold versus BA.2) and BA.5 breakthrough infection sera (32-fold versus B.1.1, 9.5-fold versus BA.5), respectively. Because the immune resistance of XBB.1.5 is comparable to that of XBB.1, our results suggest that XBB.1.5 is the most successful XBB lineage as of January 2023 by acquiring the S:F486P substitution to augment ACE2 binding affinity without losing remarkable immune resistance, which leads to greater transmissibility.","version":"1.1","doi":"10.1101/2023.01.16.524178","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.15.524170","pub_date":"2023-1-17","title":"Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2","abstract":"SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to Serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus\u2019 evolution. The Spike strain of SARS-CoV-2 has accumulated many mutations during its time in circulation, most of which have occurred in the S1 region, and more specifically in the RBD, in an effort to either improve the virus\u2019s affinity for the receptor ACE2 or to enhance its ability to evade the immune system. Mutations in the Spike S2 region have more far-reaching effects than those in the S1 region because it is more conserved across sarbecoviruses. By comparing SARS and SARS2, we found that an important substitution at amino acid position 813 in the S2 region (T813S) disrupts the utilization of TMPRSS2 and can significantly influence viral entry into cells. This discovery deepens our knowledge of S proteins and provides new prospects for tracing the evolution of Sarbecoviruses.","version":"1.1","doi":"10.1101/2023.01.15.524170","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.13.524025","pub_date":"2023-1-17","title":"Redistribution and activation of CD16brightCD56dim NK cell subset to fight against Omicron subvariant BA.2 after COVID-19 vaccination","abstract":"With the alarming surge in COVID-19 cases globally, vaccination must be prioritised to achieve herd immunity. Immune dysfunction is detected in the majority of patients with COVID-19; however, it remains unclear whether the immune responses elicited by COVID-19 vaccination function against the Omicron subvariant BA.2. Of the 508 Omicron BA.2-infected patients enrolled, 102 were unvaccinated controls and 406 were vaccinated. Despite the presence of clinical symptoms in both groups, vaccination led to a significant decline in nausea or vomiting, abdominal pain, headache, pulmonary infection, overall clinical symptoms, and a moderate rise in body temperature. Omicron BA.2-infected individuals were also characterised by a mild increase in both serum pro- and anti-inflammatory cytokine levels after vaccination. There were no significant differences or trend changes between T and B lymphocyte subsets; however, a significant expansion of NK lymphocytes in COVID-19-vaccinated patients was observed. Moreover, the most effective CD16brightCD56dim subsets of NK cells showed increased functional capacities, as evidenced by a significantly greater IFN-\u03b3 secretion and stronger cytotoxic potential in Omicron BA.2-infected patients after vaccination. Collectively, these results suggest that COVID-19 vaccination interventions promote the redistribution and activation of CD16brightCD56dim NK cell subsets against viral infections, and could facilitate the clinical management of Omicron BA.2-infected patients.","version":"1.1","doi":"10.1101/2023.01.13.524025","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.06.511203","pub_date":"2023-1-14","title":"Incipient parallel evolution of SARS-CoV-2 Deltacron variant in South Brazil","abstract":"With the coexistence of multiple lineages and increased international travel, recombination and gene flow are likely to become increasingly important in the adaptive evolution of SARS-CoV-2. This could result in the incipient parallel evolution of multiple recombinant lineages. However, identifying recombinant lineages is challenging, and the true extent of recombinant evolution in SARS-CoV-2 may be underestimated. This study describes the first SARS-CoV-2 Deltacron recombinant case identified in Brazil. We demonstrate that the recombination breakpoint is at the beginning of Spike gene (S). The 5\u2032 genome portion (circa 22 kb) resembles the AY.101 lineage (VOC Delta), and the 3\u2032 genome portion (circa 8 kb nucleotides) is most similar to the BA.1.1 lineage (VOC Omicron). Furthermore, evolutionary genomic analyses indicate that the new strain emerged after a single recombination event between lineages of diverse geographical locations in December 2021 in South Brazil. This Deltacron, named AYBA-RS, is one out of almost 30 recombinants described this year. The submission of only four sequences in the GISAID database suggests that this Brazilian lineage had a minor epidemiological impact. On the other hand, the recent emergence of this and various other Deltacron recombinant lineages (i.e., XD, XF, and XS) suggests that gene flow and recombination may play an increasingly important role in the COVID-19 pandemic. We explain the evolutionary and population genetic theory that support this assertion, and we conclude that this stresses the need for continued genomic and epidemiological surveillance. This is particularly important for countries where multiple variants are present, as well as for countries that receive significant inbound international travel.","version":"1.2","doi":"10.1101/2022.10.06.511203","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.12.523876","pub_date":"2023-1-13","title":"Pathogenesis of Breakthrough Infections with SARS-CoV-2 Variants in Syrian Hamsters","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has evolved into multiple variants. Animal models are important to understand variant pathogenesis, particularly for those with mutations that have significant phenotypic or epidemiological effects. Here, cohorts of na\u00efve or previously infected Syrian hamsters (Mesocricetus auratus) were infected with variants to investigate viral pathogenesis and disease protection. Na\u00efve hamsters infected with SARS-CoV-2 variants had consistent clinical outcomes, tissue viral titers, and pathology, while hamsters that recovered from initial infection and were reinfected demonstrated less severe clinical disease and lung pathology than their na\u00efve counterparts. Males had more frequent clinical signs than females in most variant groups, but few sex variations in tissue viral titers and lung pathology were observed. These findings support the use of Syrian hamsters as a SARS-CoV-2 model and highlight the importance of considering sex differences when using this species. With the continued circulation and emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, understanding differences between the initial and a subsequent reinfection on disease pathogenesis is critical and highly relevant. This study characterizes Syrian hamsters as an animal model to study reinfection with SARS-CoV-2. Previous infection reduced the disease severity of reinfection with different SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2023.01.12.523876","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.12.523677","pub_date":"2023-1-13","title":"Structure-based discovery of inhibitors of the SARS-CoV-2 Nsp14 N7-methyltransferase","abstract":"An under-explored target for SARS-CoV-2 is non-structural protein 14 (Nsp14), a crucial enzyme for viral replication that catalyzes the methylation of N7-guanosine of the viral RNA at 5\u2032-end; this enables the virus to evade the host immune response by mimicking the eukaryotic post-transcriptional modification mechanism. We sought new inhibitors of the S-adenosyl methionine (SAM)-dependent methyltransferase (MTase) activity of Nsp14 with three large library docking strategies. First, up to 1.1 billion make-on-demand (\u201ctangible\u201d) lead-like molecules were docked against the enzyme\u2019s SAM site, seeking reversible inhibitors. On de novo synthesis and testing, three inhibitors emerged with IC50 values ranging from 6 to 43 \u03bcM, each with novel chemotypes. Structure-guided optimization and in vitro characterization supported their non-covalent mechanism. In a second strategy, docking a library of 16 million tangible fragments revealed nine new inhibitors with IC50 values ranging from 12 to 341 \u03bcM and ligand efficiencies from 0.29 to 0.42. In a third strategy, a newly created library of 25 million tangible, virtual electrophiles were docked to covalently modify Cys387 in the SAM binding site. Seven inhibitors emerged with IC50 values ranging from 3.2 to 39 \u03bcM, the most potent being a reversible aldehyde. Initial optimization of a second series yielded a 7 \u03bcM acrylamide inhibitor. Three inhibitors characteristic of the new series were tested for selectivity against 30 human protein and RNA MTases, with one showing partial selectivity and one showing high selectivity. Overall, 32 inhibitors encompassing eleven chemotypes had IC50 values <50 \u03bcM and 5 inhibitors in four chemotypes had IC50 values <10 \u03bcM. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing multiple starting points for optimizing towards antiviral activity.","version":"1.1","doi":"10.1101/2023.01.12.523677","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.17.516989","pub_date":"2023-1-12","title":"Running ahead of evolution - AI based simulation for predicting future high-risk SARS-CoV-2 variants","abstract":"The never-ending emergence of SARS-CoV-2 variations of concern (VOCs) has challenged the whole world for pandemic control. In order to develop effective drugs and vaccines, one needs to efficiently simulate SARS-CoV-2 spike receptor binding domain (RBD) mutations and identify high-risk variants. We pretrain a large protein language model with approximately 408 million protein sequences and construct a high-throughput screening for the prediction of binding affinity and antibody escape. As the first work on SARS-CoV-2 RBD mutation simulation, we successfully identify mutations in the RBD regions of 5 VOCs and can screen millions of potential variants in seconds. Our workflow scales to 4096 NPUs with 96.5% scalability and 493.9\u00d7 speedup in mixed precision computing, while achieving a peak performance of 366.8 PFLOPS (reaching 34.9% theoretical peak) on Pengcheng Cloudbrain-II. Our method paves the way for simulating coronavirus evolution in order to prepare for a future pandemic that will inevitably take place. Our models are released at https://github.com/ZhiweiNiepku/SARS-CoV-2_mutation_simulation to facilitate future related work. We develop a novel multi-constraint variation prediction framework to simulate SARS-CoV-2 RBD mutations, reaching a peak performance of 366.8 PFLOPS with 96.5% scalability and achieving 493.9\u00d7 speedup. Our method facilitates the prediction and prioritization of future high-risk variants for the early deployment of drugs and vaccines. Coronavirus Disease 2019 (COVID-19) has spread rapidly to more than 200 countries or regions since December 2019. Due to its high infectivity, there have been over 645 million confirmed cases, including approximately 6.6 million deaths, reported by the World Health Organization (WHO) as of December 20221. In addition to being a serious threat to human health, COVID-19 has had a catastrophic impact on the global economy.","version":"1.4","doi":"10.1101/2022.11.17.516989","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.12.523465","pub_date":"2023-1-12","title":"The natural tannins oligomeric proanthocyanidins and punicalagin are potent inhibitors of infection by SARS-CoV-2 in vitro","abstract":"The COVID-19 pandemic continues to infect people worldwide. While the vaccinated population has been increasing, the rising breakthrough infection persists in the vaccinated population. For living with the virus, the dietary guidelines to prevent virus infection are worthy of and timely to develop further. Tannic acid has been demonstrated to be an effective inhibitor of coronavirus and is under clinical trial. Here we found that two other members of the tannins family, oligomeric proanthocyanidins (OPCs) and punicalagin, are also potent inhibitors against SARS-CoV-2 infection with different mechanisms. OPCs and punicalagin showed inhibitory activity against omicron variants of SARS-CoV-2 infection. The water extractant of the grape seed was rich in OPCs and also exhibited the strongest inhibitory activities for viral entry of wild-type and other variants in vitro. Moreover, we evaluated the inhibitory activity of grape seed extractants (GSE) supplementation against SARS-CoV-2 viral entry in vivo and observed that serum samples from the healthy human subjects had suppressive activity against different variants of SARS-CoV-2 vpp infection after taking GSE capsules. Our results suggest that natural tannins acted as potent inhibitors against SARS-CoV-2 infection, and GSE supplementation could serve as healthy food for infection prevention. OPCs and Punicalagin had inhibitory activity against omicron variants of SARS-CoV-2 infection. OPCs serve as a dual inhibitor of the viral Mpro and the cellular TMPRSS2 protease. Punicalagin possesses the most potent activity to suppress the Mpro and block the interaction of the viral spike protein and human ACE2. OPCs-enriched grape seed extractant exhibited inhibitory activities for viral entry of wild-type and other variants of SARS-CoV-2. The daily intake of grape seed extractants may be able to prevent SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2023.01.12.523465","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.31.522401","pub_date":"2023-1-12","title":"Prophylaxis and Treatment of SARS-CoV-2 infection by an ACE2 Receptor Decoy","abstract":"The emergence of SARS-CoV-2 variants with highly mutated spike proteins has presented an obstacle to the use of monoclonal antibodies for the prevention and treatment of SARS-CoV-2 infection. We show that a high affinity receptor decoy protein in which a modified ACE2 ectodomain is fused to a single domain of an immunoglobulin heavy chain Fc region dramatically suppressed virus loads in mice upon challenge with a high dose of parental SARS-CoV-2 or Omicron variants. The decoy also potently suppressed virus replication when administered shortly post-infection. The decoy approach offers protection against the current viral variants and, potentially, against SARS-CoV-2 variants that may emerge with the continued evolution of the spike protein or novel viruses that use ACE2 for virus entry.","version":"1.2","doi":"10.1101/2022.12.31.522401","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.11.523649","pub_date":"2023-1-12","title":"Vectored Immunoprophylaxis and Treatment of SARS-CoV-2 Infection","abstract":"Vectored immunoprophylaxis was first developed as a means to establish engineered immunity to HIV through the use of an adeno-associated viral vector expressing a broadly neutralizing antibody. We have applied this concept to establish long-term prophylaxis against SARS-CoV-2 by adeno-associated and lentiviral vectors expressing a high affinity ACE2 decoy receptor. Administration of decoy-expressing AAV vectors based on AAV2.retro and AAV6.2 by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and active against recent SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective when administered up to 24 hours post-infection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.","version":"1.1","doi":"10.1101/2023.01.11.523649","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.10.523494","pub_date":"2023-1-12","title":"Microfluidics-enabled fluorescence-activated cell sorting of single pathogen-specific antibody secreting cells for the rapid discovery of monoclonal antibodies","abstract":"Monoclonal antibodies are increasingly used to prevent and treat viral infections, playing a pivotal role in pandemic response efforts. Antibody secreting cells (ASCs, plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methodologies to study antigen-specific ASCs either have low throughput, require expensive and labour-intensive screening or are technically demanding and therefore not accessible to the wider research community. Here, we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs: we combine microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. With our technology, we screened millions of mouse and human ASCs and obtained anti-SARS-CoV-2 monoclonal antibodies with high affinity (pM) and neutralising capacity (<100 ng/mL) in two weeks with a high hit rate (>85%). By facilitating access into the underexplored ASC compartment, we enable fast and efficient antibody discovery as well as immunological studies into the generation of protective antibodies.","version":"1.1","doi":"10.1101/2023.01.10.523494","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.10.523518","pub_date":"2023-1-11","title":"Anthracyclines inhibit SARS-CoV-2 infection","abstract":"Vaccines and drugs are two effective medical interventions to mitigate SARS-CoV-2 infection. Three SARS-CoV-2 inhibitors, remdesivir, paxlovid, and molnupiravir, have been approved for treating COVID-19 patients, but more are needed, because each drug has its limitation of usage and SARS-CoV-2 constantly develops drug resistance mutations. In addition, SARS-CoV-2 drugs have the potential to be repurposed to inhibit new human coronaviruses, thus help to prepare for future coronavirus outbreaks. We have screened a library of microbial metabolites to discover new SARS-CoV-2 inhibitors. To facilitate this screening effort, we generated a recombinant SARS-CoV-2 Delta variant carrying the nano luciferase as a reporter for measuring viral infection. Six compounds were found to inhibit SARS-CoV-2 at the half maximal inhibitory concentration (IC50) below 1 \u03bcM, including the anthracycline drug aclarubicin that markedly reduced viral RNA-dependent RNA polymerase (RdRp)-mediated gene expression, whereas other anthracyclines inhibited SARS-CoV-2 by activating the expression of interferon and antiviral genes. As the most commonly prescribed anti-cancer drugs, anthracyclines hold the promise of becoming new SARS-CoV-2 inhibitors. Microbial metabolites are a rich source of bioactive molecules. The best examples are antibiotics and immunosuppressants that have transformed the practice of modern medicine and saved millions of lives. Recently, some microbial metabolites were reported to have antiviral activity, including the inhibition of Zika virus and Ebola virus. In this study, we discovered several microbial metabolites that effectively inhibit SARS-CoV-2 infection, including anthracyclines that have also been shown to inhibit other viruses including Ebola virus through enhancing interferon responses, which indicates potentially broad antiviral properties of these microbial metabolites and can lead to the discovery of pan-antiviral molecules.","version":"1.1","doi":"10.1101/2023.01.10.523518","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.27.522023","pub_date":"2023-1-11","title":"Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs","abstract":"RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs (svRNAs) generated by SARS-CoV-1 and SARS-CoV-2 remained largely unexplored, mainly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA-seq datasets, we observed that human cells infected by SARS-CoV-1 or SARS-CoV-2 produce multiple short viral RNAs (svRNAs), ranging in size from 15 to 26 nt and deriving predominantly from (+) RNA strands. In addition, we verified the presence of the five most abundant SARS-CoV-2 svRNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by qPCR. Interestingly, the copy number of the observed SARS-CoV-2 svRNAs dramatically exceeded the expression of previously reported viral miRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 svRNAs could serve as biomarkers for early infection stages due to their high abundance. Finally, we found that both SARS-CoV-1 and SARS-CoV-2 infection induced up- and down-regulation of multiple endogenous human short RNAs that align predominantly to protein-coding and lncRNA transcripts. Interestingly, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various hg38 sequences, suggesting opportunities to investigate regulatory roles of svRNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.","version":"1.2","doi":"10.1101/2022.12.27.522023","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.11.23284424","pub_date":"2023-01-11","title":"Country Learning on Maintaining Quality Essential Health Services (EHS) during COVID-19 in Timor-Leste: A mixed methods qualitative analysis","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>This research study examines the enabling factors, strengths, and challenges experienced by the Timor-Leste health system as it sought to maintain quality essential health services (EHS) during the COVID-19 pandemic.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>A mixed methods qualitative analysis</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>National, municipal, facility levels in Baucau, Dili and Ermera Municipalities in TLS</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>Key informant interviews (n=40) and focus group discussions (n=6) working to maintain quality EHS in TLS.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>A reduction in people accessing general health services was observed in 2020, reportedly due to fears of contracting COVID-19 in healthcare settings, limited resources (eg. human resources, personal protective equipment, clinical facilities, etc) and closure of health services. However, improvements in maternal child health services simultaneously improved in the areas of skilled birth attendants, prenatal coverage, and vitamin A distribution, for example. Five themes emerged as enabling factors for maintaining quality EHS including 1) high level strategy for maintaining quality EHS, 2) implementation of quality activities across the three levels of the health system, 3) measurement for quality and factors affecting service utilization 4) the positive impact of quality improvement leadership in health facilities during COVID-19, and 5) learning from each other for maintaining quality EHS now and for the future. Other countries may benefit from the challenges, strengths and enablers found on planning for quality.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>The maintenance of quality essential health services (EHS) is critical to mitigate adverse health effects from the COVID-19 pandemic. When quality health services are delivered prior to and maintained during public health emergencies, they build trust within the health system and promote healthcare seeking behavior. Planning for quality as part of emergency preparedness can facilitate a high standard of care by ensuring health services continue to provide a safe environment, reduce harm, improve clinical care, and engage patients, facilities, and communities.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>DATA SHARING</jats:title>\n                  <jats:p>All data is kept with MBK and GR and is available upon request. The dataset analysis is available from the corresponding author upon reasonable request.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>QUALITATIVE CHECKLIST</jats:title>\n                  <jats:p>The Standards for Reporting Qualitative Research (SRQR) checklist was used for this original research.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>STRENGTHS AND LIMITATIONS OF THIS STUDY</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>The qualitative data gave detailed insights to the operationalization of key strategic COVID-19 emergency documents and the national quality implementation strategy.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Data collection was performed in three out of thirteen municipalities, including the largest metropolitan city of Dili.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>The qualitative research was conducted in the participants native language (Tetum).</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Not all pre-identified national level KII participants were available to provide feedback.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2023.01.11.23284424","journal":"medRxiv","score":null},{"id":"10.1101/2022.11.17.515635","pub_date":"2023-1-10","title":"Hamsters are a model for post-COVID-19 alveolar regeneration mechanisms: an opportunity to understand post-acute sequelae of SARS-CoV-2","abstract":"A relevant number of coronavirus disease 2019 (COVID-19) survivors suffers from post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (PASC). Current evidence suggests a dysregulated alveolar regeneration in COVID-19 as a possible explanation for respiratory PASC symptoms, a phenomenon which deserves further investigation in a suitable animal model. This study investigates morphological, phenotypical and transcriptomic features of alveolar regeneration in SARS-CoV-2 infected Syrian golden hamsters. We demonstrate that CK8+ alveolar differentiation intermediate (ADI) cells occur following SARS-CoV-2-induced diffuse alveolar damage. A subset of ADI cells shows nuclear accumulation of TP53 at 6- and 14-days post infection (dpi), indicating a prolonged arrest in the ADI state. Transcriptome data show the expression of gene signatures driving ADI cell senescence, epithelial-mesenchymal transition, and angiogenesis. Moreover, we show that multipotent CK14+ airway basal cell progenitors migrate out of terminal bronchioles, aiding alveolar regeneration. At 14 dpi, presence of ADI cells, peribronchiolar proliferates, M2-type macrophages, and sub-pleural fibrosis is observed, indicating incomplete alveolar restoration. The results demonstrate that the hamster model reliably phenocopies indicators of a dysregulated alveolar regeneration of COVID-19 patients. The results provide important information on a translational COVID-19 model, which is crucial for its application in future research addressing pathomechanisms of PASC and in testing of prophylactic and therapeutic approaches for this syndrome.","version":"1.2","doi":"10.1101/2022.11.17.515635","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.10.523422","pub_date":"2023-1-10","title":"Human iPS cell-derived sensory neurons can be infected by SARS-CoV-2 strain WA1/2020 as well as variants delta and omicron","abstract":"COVID-19 has impacted billions of people in the world since 2019 and unfolded a major healthcare crisis. With an increasing number of deaths and the emergence of more transmissible variants, it is crucial to better understand the biology of the disease-causing virus, the SARS-CoV-2. Peripheral neuropathies appeared as a specific COVID-19 symptom occurring at later stages of the disease. In order to understand the impact of SARS-CoV-2 on the peripheral nervous system, we generated human sensory neurons from induced pluripotent stem cells that we infected with the SARS-CoV-2 strain WA1/2020 and the variants delta and omicron. Using single cell RNA sequencing, we found that human sensory neurons can be infected by SARS-CoV-2 but are unable to produce new viruses. Our data suggests that sensory neurons can be infected by the original WA1/2020 strain of SARS-CoV-2 as well as the delta and omicron variants.","version":"1.1","doi":"10.1101/2023.01.10.523422","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.13.512134","pub_date":"2023-1-10","title":"Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron","abstract":"The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.","version":"1.2","doi":"10.1101/2022.10.13.512134","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.02.506332","pub_date":"2023-1-10","title":"SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b","abstract":"The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding - prevented PKR activation, inhibited \u03b2-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera. SARS-CoV-2 nucleocapsid protein N is an antagonist of innate immunity but how it averts virus detection by intracellular sensors remains subject to debate. We provide evidence that SARS-CoV-2 N, by sequestering dsRNA through domain N2b, prevents PKR-mediated activation of the integrated stress response as well as detection by RIG-I-like receptors and ensuing type I interferon expression. This function, conserved in human coronavirus 229E, is not affected by mutations that prevent posttranslational modifications, previously implicated in immune evasion, or that target its binding to stress granule scaffold proteins. Our findings further our understanding of how SARS-CoV-2 evades innate immunity, how this may drive viral evolution and why increased N expression may have been a selective advantage to SARS-CoV-2 variants of concern.","version":"1.2","doi":"10.1101/2022.09.02.506332","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.09.523338","pub_date":"2023-1-10","title":"Inspiration of SARS-CoV-2 envelope protein mutations on pathogenicity of Omicron XBB","abstract":"Predicting pathogenicity of Omicron sub-variants is critical for assessing disease dynamics and developing public health strategies. As an important virulence factor, SARS-CoV-2 envelope protein (2-E) causes cell death and acute respiratory distress syndrome (ARDS)-like pathological damages. Evaluation of 2-E mutations might offer clues to pathogenicity forecast. Here, the frequency and cell lethality of 92 mutations of 2-E in five early \u201cvariants of concern\u201d (VOCs, Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.2, BA.3, BA.4, and BA.5) were analyzed, which could be divided into three classes. Most (87) mutations belong to Class I, no obvious frequency changes. Class II consists of 2 mutations, exhibiting enhanced cell lethality but decreased frequency. The rest 3 mutations in Class III were characterized by attenuated cell lethality and increased frequency. Remarkably, the Class II mutations are always observed in the VOCs with high disease severity while the Class III mutations are highly conserved in the VOCs with weakened pathogenicity. For example, P71L, the most lethal mutation, dropped to nearly 0.00% in the milder Omicrons from 99.12% in Beta, while the less lethal mutation T9I, sharply increased to 99.70% in BA.1 and is highly conserved in BA.1-5. Accordingly, we proposed that some key 2-E mutations are pathogenicity markers of the virus. Notably, the highly contagious Omicron XBB retained T9I also. In addition, XBB gained a new dominant-negative mutation T11A with frequency 90.52%, exhibiting reduced cell lethality, cytokine induction and viral production capabilities in vitro, and particularly weakened lung damages in mice. No mutations with enhanced cell lethality were observed in XBB. These clues imply a further weakened pathogenicity of XBB among Omicron sub-variants.","version":"1.1","doi":"10.1101/2023.01.09.523338","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.09.523288","pub_date":"2023-1-10","title":"HOGVAX: Exploiting Peptide Overlaps to Maximize Population Coverage in Vaccine Design with Application to SARS-CoV-2","abstract":"Peptide vaccines present a safe and cost-efficient alternative to traditional vaccines. Their efficacy depends on the peptides included in the vaccine and the ability of major histocompatibility complex (MHC) molecules to bind and present these peptides. Due to the high diversity of MHC alleles, their diverging peptide binding specificities, and physical constraints on the maximum length of peptide vaccine constructs, choosing a set of peptides that effectively achieve immunization across a large proportion of the population is challenging. Here, we present HOGVAX, a combinatorial optimization approach to select peptides that maximize population coverage. The key idea behind HOGVAX is to exploit overlaps between peptide sequences to include a large number of peptides in limited space and thereby also cover rare MHC alleles. We formalize the vaccine design task as a theoretical problem, which we call the Maximum Scoring k-Superstring Problem (MSKS). We show that MSKS is NP-hard, reformulate it into a graph problem using the hierarchical overlap graph (HOG), and present a haplotype-aware variant of MSKS to take linkage disequilibrium between MHC loci into account. We give an integer linear programming formulation for the graph problem and provide an open source implementation. We demonstrate on a SARS-CoV-2 case study that HOGVAX-designed vaccine formulations contain significantly more peptides than vaccine sequences built from concatenated peptides. We predict over 98% population coverage and high numbers of per-individual presented peptides, leading to robust immunity against new pathogens or viral variants.","version":"1.1","doi":"10.1101/2023.01.09.523288","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.07.523115","pub_date":"2023-1-09","title":"The Ecology of Viruses in Urban Rodents with a Focus on SARS-CoV-2","abstract":"Wild animals are naturally infected with a range of viruses, some of which may be zoonotic for humans. During the human COIVD pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this we have sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralisation ability, that are consistent with rats being exposed to SARS-CoV-2 and / or exposed to other viruses that result in cross-reactive antibodies.","version":"1.1","doi":"10.1101/2023.01.07.523115","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.289355","pub_date":"2023-1-09","title":"Leveraging T-cell receptor \u2013 epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution","abstract":"Despite the general agreement on the importance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain, while this knowledge may indicate how to adjust vaccines and maintain robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to epitopes unique to SARS-CoV-2 (SC-unique) or shared with other coronaviruses (CoV-common), we trained a large number of TCR-epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. Applying those models to longitudinal COVID-19 TCR repertoires of critical and non-critical COVID-19 patients, we discovered that notwithstanding comparable CD8+ T-cell depletion and the sizes of putative CoV-common CD8+ TCR repertoires in all symptomatic patients at the initial stage of the disease, the temporal dynamics of putative SC2-unique TCRs differed depending on the disease severity. Only non-critical patients had developed large and diverse SC2-unique CD8+ T-cell response by the second week of the disease. Additionally, only this patient group demonstrated redundancy in CD8+ TCRs putatively recognizing unique and common SARS-CoV-2 epitopes. Our findings thus emphasize the role of the de novo CD8+ T-cell response and support the argument against the clinical benefit of pre-existing cross-reactive CD8+ T cells. Now, the analytical framework of this study can not only be employed to track specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire but also be generalized to more epitopes and be employed for adaptive immune response assessment and monitoring to inform public health decisions.","version":"1.2","doi":"10.1101/2020.09.09.289355","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485248","pub_date":"2023-1-09","title":"Macrophages only sense infectious SARS-CoV-2 when they express sufficient ACE2 to permit viral entry, where rapid cytokine responses then limit viral replication","abstract":"Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication. Lack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.","version":"1.2","doi":"10.1101/2022.03.22.485248","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.09.523246","pub_date":"2023-1-09","title":"Unique amino acid substitution in RBD region of SARS-CoV-2 Omicron XAY.2","abstract":"We attempted to explain the rare mutation at the receptor binding domain of the spike protein in the XAY.2 variant of SARS-CoV-2 from the perspective of hydrophobic interactions. We propose that decreasing hydrophobicity at position 446 and 486 of the RBD region of the spike protein might affect the infectivity of SARS-CoV-2. We also estimated the probable mutations at the 446 and 486 position the virus may acquire, leading to a decreased hydrophobicity.","version":"1.1","doi":"10.1101/2023.01.09.523246","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.03.522213","pub_date":"2023-1-09","title":"A Gamma-adapted recombinant subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from infection","abstract":"The COVID-19 pandemic continues with the emergence of successive new variants of concern (VOC). One strategy to prevent breakthrough infections is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD recombinant vaccine candidate derived from the Gamma SARS-CoV-2 variant adjuvanted with alum. Gamma RBD-derived antigen elicited better neutralizing antibody and T cell responses than formulation containing ancestral RBD antigen. The Gamma-adapted subunit vaccine elicited a long-lasting antibody response with cross-neutralizing activity against different VOC including the Omicron variant. Additionally, Gamma variant RBD-adapted vaccine elicited robust T cells responses with induction of Th1 and CD8+ T cell responses in spleen and lung. Vaccine-induced immunity protected K18-hACE2 mice from intranasal challenge with SARS-CoV-2 increasing survival, reducing body weight loss and viral burden in the lungs and brain. Importantly, the subunit vaccine demonstrated a potent effect as heterologous booster of different vaccine platforms including the non-replicating adenovirus vaccine ChAdOx1-S, the mRNA vaccine BNT162b2 and the inactivated SARS-CoV-2 vaccine BBIBP-CorV, increasing cross-reactive antibody responses. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate to combat SARS-CoV-2 variants including Omicron.","version":"1.2","doi":"10.1101/2023.01.03.522213","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.05.522964","pub_date":"2023-1-09","title":"Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides","abstract":"With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors can be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped VSV vectors and viral plaque assays. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed for the Spike proteins from Beta, Gamma and Omicron. The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor or engineered peptides that bind to the receptor binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in mouse models, confirming their potential as an alternative to antibody therapies.","version":"1.2","doi":"10.1101/2023.01.05.522964","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.06.522349","pub_date":"2023-1-09","title":"Effects of Variants of Concern Mutations on the Force-Stability of the SARS-CoV-2:ACE2 Interface and Virus Transmissibility","abstract":"Viruses mutate under a variety of selection pressures, allowing them to continuously adapt to their hosts. Mutations in SARS-CoV-2 have shown effective evasion of population immunity and increased affinity to host factors, in particular to the cellular receptor ACE2. However, in the dynamic environment of the respiratory tract forces act on the binding partners, which raises the question whether not only affinity, but also force-stability of the SARS-CoV-2:ACE2 bond, might be a selection factor for mutations. Here, we use magnetic tweezers (MT) to study the effect of amino acid substitutions in variants of concern (VOCs) on RBD:ACE2 bond kinetics with and without external load. We find higher affinity for all VOCs compared to wt, in good agreement with previous affinity measurements in bulk. In contrast, Alpha is the only VOC that shows significantly higher force stability compared to wt. Investigating the RBD:ACE2 interactions with molecular dynamics simulations, we are able to rationalize the mechanistic molecular origins of this increase in force-stability. Our study emphasizes the diversity of contributions to the assertiveness of variants and establishes force-stability as one of several factors for fitness. Understanding fitness-advantages opens the possibility for prediction of likely mutations allowing rapid adjustment of therapeutics, vaccination, and intervention measures.","version":"1.1","doi":"10.1101/2023.01.06.522349","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.08.523127","pub_date":"2023-1-09","title":"Bivalent mRNA vaccine improves antibody-mediated neutralization of many SARS-CoV-2 Omicron lineage variants","abstract":"The early Omicron lineage variants evolved and gave rise to diverging lineages that fueled the COVID-19 pandemic in 2022. Bivalent mRNA vaccines, designed to broaden protection against circulating and future variants, were authorized by the U.S. Food and Drug Administration (FDA) in August 2022 and recommended by the U.S. Centers for Disease Control and Prevention (CDC) in September 2022. The impact of bivalent vaccination on eliciting neutralizing antibodies against homologous BA.4/BA.5 viruses as well as emerging heterologous viruses needs to be analyzed. In this study, we analyze the neutralizing activity of sera collected after a third dose of vaccination (2-6 weeks post monovalent booster) or a fourth dose of vaccination (2-7 weeks post bivalent booster) against 10 predominant/recent Omicron lineage viruses including BA.1, BA.2, BA.5, BA.2.75, BA.2.75.2, BN.1, BQ.1, BQ.1.1, XBB, and XBB.1. The bivalent booster vaccination enhanced neutralizing antibody titers against all Omicron lineage viruses tested, including a 10-fold increase in neutralization of BQ.1 and BQ.1.1 viruses that predominated in the U.S. during the last two months of 2022. Overall, the data indicate the bivalent vaccine booster strengthens protection against Omicron lineage variants that evolved from BA.5 and BA.2 progenitors.","version":"1.1","doi":"10.1101/2023.01.08.523127","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.06.519322","pub_date":"2023-1-09","title":"A Binary RNA and DNA Self-Amplifying Platform for Next Generation Vaccines and Therapeutics","abstract":"Conventional mRNA-based vaccines were instrumental in lowering the burden of the pandemic on healthcare systems and in reducing mortality. However, such first-generation vaccines have significant weaknesses. Here, we describe a high-performance binary recombinant vectoral platform offering the flexibility to be used as a self-amplifying mRNA or a self-amplifying DNA. Both formats drive long-lasting expression and actuate robust antibody responses against SAR-CoV-2 spike, and neither format require encapsulation with lipid nanoparticles (LNP) in the generation immune responses. The platform combines the power of conventional mRNA with the low-dosage of self-amplifying vectors together with the simplicity, rapid creation, ease of storage, and convenience of distribution of plasmid DNA vectors. This platform promises to pave the way for more effective, less expensive, and truly democratized vaccines and therapeutics. Gemini: a versatile platform that improves on existing vaccine formats in terms of effectiveness, manufacturing, distribution, and cost.","version":"1.2","doi":"10.1101/2022.12.06.519322","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.06.522977","pub_date":"2023-1-06","title":"Omicron Spike Protein Is Vulnerable to Reduction","abstract":"SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.","version":"1.1","doi":"10.1101/2023.01.06.522977","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.05.522845","pub_date":"2023-1-05","title":"Poor neutralizing antibody responses against SARS-CoV-2 Omicron BQ.1.1 and XBB in Norway in October 2022","abstract":"New sub-lineages of the SARS-CoV-2 omicron variants with enhanced ability to evade existing antibody responses continue to evolve. A better understanding how susceptible emerging virus variants are to immunity induced by vaccination or infection could help predict which strains will become dominant going forward. Here we evaluate neutralizing antibodies against several clinical isolates of omicron variants including BQ.1.1 and XBB in sera from 3x mRNA vaccinated individuals and individuals with breakthrough infections with early (BA.1 or 2) or late (BA.5) omicron variants. In addition, we evaluate neutralizing antibodies in serum samples harvested from 32 individuals from the middle of October 2022, to provide a more recent estimate of immunity. As expected, serum samples harvested after breakthrough infections were more efficient at neutralizing all the omicron variants, compared to sera from non-infected individuals. While neutralization remained high against variants such as BA.2.75.2, BR.1 and BF.7, there was a marked reduction in neutralizing titers against BQ.1.1 and XBB. Similarly, most serum samples harvested in October 2022 had very low neutralizing antibodies against BQ.1.1 and XBB, suggesting that these variants and their descendants will dominate infection waves in Norway this winter season.","version":"1.1","doi":"10.1101/2023.01.05.522845","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.05.522853","pub_date":"2023-1-05","title":"The SARS-CoV-2 Spike protein induces long-term transcriptional perturbations of mitochondrial metabolic genes, causes cardiac fibrosis, and reduces myocardial contractile in obese mice","abstract":"As the pandemic evolves, post-acute sequelae of CoV-2 (PACS) including cardiovascular manifestations have emerged as a new health threat. This study aims to study whether the Spike protein plus obesity can exacerbate PACS-related cardiomyopathy. A Spike protein-pseudotyped (Spp) virus with the proper surface tropism of SARS-CoV-2 was developed for viral entry assay in vitro and administration into high fat diet (HFD)-fed mice. The systemic viral loads and cardiac transcriptomes were analyzed at 2 and 24 hrs, 3, 6, and 24 weeks post introducing (wpi) Spp using RNA-seq or real time RT-PCR. Echocardiography was used to monitor cardiac functions. Low-density lipoprotein cholesterol enhanced viral uptake in endothelial cells, macrophages, and cardiomyocyte-like H9C2 cells. Selective cardiac and adipose viral depositions were observed in HFD mice but not in normal-chow-fed mice. The cardiac transcriptional signatures in HFD mice at 3, 6, and 24 wpi showed systemic suppression of mitochondria respiratory chain genes including ATP synthases and nicotinamide adenine dinucleotide:ubiquinone oxidoreductase gene members, upregulation of stress pathway-related crucial factors such as nuclear factor-erythroid 2-related factor 1 and signal transducer and activator of transcription 5A, and increases in expression of glucose metabolism-associated genes. As compared with the age-matched HFD control mice, cardiac ejection fraction and fractional shortening were significantly decreased, while left ventricular end-systolic diameter and volume were significantly elevated, and cardiac fibrosis was increased in HFD mice at 24 wpi. Our data demonstrated that the Spike protein could induce long-term transcriptional suppression of mitochondria metabolic genes and cause cardiac fibrosis and myocardial contractile impairment, providing mechanistic insights to PACS-related cardiomyopathy.","version":"1.1","doi":"10.1101/2023.01.05.522853","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.04.522794","pub_date":"2023-1-05","title":"Targets and cross-reactivity of human T cell recognition of Common Cold Coronaviruses","abstract":"The Coronavirus (CoV) family includes a variety of viruses able to infect humans. Endemic CoVs that can cause common cold belong to the alphaCoV and betaCoV genera, with the betaCoV genus also containing subgenera with zoonotic and pandemic concern, including sarbecoCoV (SARS-CoV and SARS-CoV-2) and merbecoCoV (MERS-CoV). It is therefore warranted to explore pan-CoV vaccine concepts, to provide adaptive immune protection against new potential CoV outbreaks, particularly in the context of betaCoV sub lineages. To explore the feasibility of eliciting CD4+ T cell responses widely cross-recognizing different CoVs, we utilized samples collected pre-pandemic to systematically analyze T cell reactivity against representative alpha (NL63) and beta (OC43) common cold CoVs (CCC). Similar to previous findings on SARS-CoV-2, the S, N, M, and nsp3 antigens were immunodominant for both viruses while nsp2 and nsp12 were immunodominant for NL63 and OC43, respectively. We next performed a comprehensive T cell epitope screen, identifying 78 OC43 and 87 NL63-specific epitopes. For a selected subset of 18 epitopes, we experimentally assessed the T cell capability to cross-recognize sequences from representative viruses belonging to alphaCoV, sarbecoCoV, and beta-non-sarbecoCoV groups. We found general conservation within the alpha and beta groups, with cross-reactivity experimentally detected in 89% of the instances associated with sequence conservation of >67%. However, despite sequence conservation, limited cross-reactivity was observed in the case of sarbecoCoV (50% of instances), indicating that previous CoV exposure to viruses phylogenetically closer to this subgenera is a contributing factor in determining cross-reactivity. Overall, these results provided critical insights in the development of future pan-CoV vaccines.","version":"1.1","doi":"10.1101/2023.01.04.522794","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.03.522427","pub_date":"2023-1-05","title":"Enhanced transmissibility of XBB.1.5 is contributed by both strong ACE2 binding and antibody evasion","abstract":"SARS-CoV-2 recombinant subvariant XBB.1.5 is growing rapidly in the United States, carrying an additional Ser486Pro substitution compared to XBB.1 and outcompeting BQ.1.1 and other XBB sublineages. The underlying mechanism for such high transmissibility remains unclear. Here we show that XBB.1.5 exhibits a substantially higher hACE2-binding affinity compared to BQ.1.1 and XBB/XBB.1. Convalescent plasma samples from BA.1, BA.5, and BF.7 breakthrough infection are significantly evaded by both XBB.1 and XBB.1.5, with XBB.1.5 displaying slightly weaker immune evasion capability than XBB.1. Evusheld and Bebtelovimab could not neutralize XBB.1/XBB.1.5, while Sotrovimab remains weakly reactive and notably, SA55 is still highly effective. The fact that XBB.1 and XBB.1.5 showed comparable antibody evasion but distinct transmissibility suggests enhanced receptor-binding affinity would indeed lead to higher growth advantages. The strong hACE2 binding of XBB.1.5 could also enable its tolerance of further immune escape mutations, which should be closely monitored.","version":"1.2","doi":"10.1101/2023.01.03.522427","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.03.519511","pub_date":"2023-1-04","title":"The prospect of universal coronavirus immunity: a characterization of reciprocal and non-reciprocal T cell responses against SARS-CoV2 and common human coronaviruses","abstract":"T cell immunity plays a central role in clinical outcomes of Coronavirus Infectious Disease 2019 (COVID-19). Therefore, T cell-focused vaccination or cellular immunotherapy might provide enhanced protection for immunocompromised patients. Pre-existing T cell memory recognizing SARS-CoV2 antigens antedating COVID-19 infection or vaccination, may have developed as an imprint of prior infections with endemic non-SARS human coronaviruses (hCoVs) OC43, HKU1, 229E, NL63, pathogens of \u201ccommon cold\u201d. In turn, SARS-CoV2-primed T cells may recognize emerging variants or other hCoV viruses and modulate the course of subsequent hCoV infections. Cross-immunity between hCoVs and SARS-CoV2 has not been well characterized. Here, we systematically investigated T cell responses against the immunodominant SARS-CoV2 spike, nucleocapsid and membrane proteins and corresponding antigens from \u03b1- and \u03b2-hCoVs among vaccinated, convalescent, and unexposed subjects. Broad T cell immunity against all tested SARS-CoV2 antigens emerged in COVID-19 survivors. In convalescent and in vaccinated individuals, SARS-CoV2 spike-specific T cells reliably recognized most SARS-CoV2 variants, however cross-reactivity against the omicron variant was reduced by approximately 50%. Responses against spike, nucleocapsid and membrane antigens from endemic hCoVs were more extensive in COVID-19 survivors than in unexposed subjects and displayed cross-reactivity between \u03b1- and \u03b2-hCoVs. In some, non-SARS hCoV-specific T cells demonstrated a prominent non-reciprocal cross-reactivity with SARS-CoV2 antigens, whereas a distinct anti-SARS-CoV2 immunological repertoire emerged post-COVID-19, with relatively limited cross-recognition of non-SARS hCoVs. Based on this cross-reactivity pattern, we established a strategy for in-vitro expansion of universal anti-hCoV T cells for adoptive immunotherapy. Overall, these results have implications for the future design of universal vaccines and cell-based immune therapies against SARS- and non-SARS-CoVs.","version":"1.1","doi":"10.1101/2023.01.03.519511","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.04.522709","pub_date":"2023-1-04","title":"Bioinformatics Approaches to Determine the Effect of SARS-CoV-2 Infection on Patients with Intrahepatic Cholangiocarcinoma","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), has infected millions of individuals throughout the world, which poses a serious threat to human health. COVID-19 is a systemic disease that affects tissues and organs, including the lung and liver. Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common liver cancer, and cancer patients are particularly at high risk of SARS-CoV-2 infection. The relationship between HCC and COVID-19 has been reported in previous studies, but ICC has been rare. With the methods of systems biology and bioinformatics, this study explored the link between COVID-19 and ICC. Transcriptional profiling of COVID-19 and ICC were obtained from the GEO database. A total of 70 common differentially expressed gene (DEGs) of both diseases were identified to investigate shared pathways. Then top-ranked 10 key DEGs (SCD, ACSL5, ACAT2, HSD17B4, ALDOA, ACSS1, ACADSB, CYP51A1, PSAT1, and HKDC1) were identified as hub genes by protein-protein interaction (PPI) network analysis. In addition, transcriptional regulatory networks regulating hub genes were revealed by hub Gene-transcription factor (TF) interaction analysis and hub gene-microRNA (miRNAs) interaction analysis. This study is expected to provide new references for future research and treatment of COVID-19 and ICC.","version":"1.1","doi":"10.1101/2023.01.04.522709","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.04.521629","pub_date":"2023-1-04","title":"Assessment of Immunogenicity and Efficacy of CV0501 mRNA-based Omicron COVID-19 Vaccination in Small Animal Models","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.5) represent the most commonly circulating variants of concern (VOC) in the coronavirus disease 2019 (COVID-19) pandemic in 2022. Despite high vaccination rates with approved SARS-CoV-2 vaccines encoding the ancestral spike (S) protein, these Omicron subvariants have collectively resulted in increased viral transmission and disease incidence. This necessitates the development and characterization of vaccines incorporating later emerging S proteins to enhance protection against VOC. In this context, bivalent vaccine formulations may induce broad protection against VOC and potential future SARS-CoV-2 variants. Here, we report preclinical data for a lipid nanoparticle (LNP)-formulated RNActive\u00ae N1-methylpseudouridine (N1m\u03a8) modified mRNA vaccine (CV0501) based on our second-generation SARS-CoV-2 vaccine CV2CoV, encoding the S protein of Omicron BA.1. The immunogenicity of CV0501, alone or in combination with a corresponding vaccine encoding the ancestral S protein (ancestral N1m\u03a8), was first measured in dose-response and booster immunization studies performed in Wistar rats. Both monovalent CV0501 and bivalent CV0501/ancestral N1m\u03a8 immunization induced robust neutralizing antibody titers against the BA.1, BA.2 and BA.5 Omicron subvariants, in addition to other SARS-CoV-2 variants in a booster immunization study. The protective efficacy of monovalent CV0501 against live SARS-CoV-2 BA.2 infection was then assessed in hamsters. Monovalent CV0501 significantly reduced SARS-CoV-2 BA.2 viral loads in the airways, demonstrating protection induced by CV0501 vaccination. CV0501 has now advanced into human Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT05477186).","version":"1.1","doi":"10.1101/2023.01.04.521629","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.18.500514","pub_date":"2023-1-04","title":"COVID-19-associated pulmonary aspergillosis in immunocompetent patients: A virtual patient cohort study","abstract":"The opportunistic fungus Aspergillus fumigatus infects the lungs of immunocompromised hosts, including patients undergoing chemotherapy or organ transplantation. More recently however, immunocompetent patients with severe SARS-CoV2 have been reported to be affected by COVID-19 Associated Pulmonary Aspergillosis (CAPA), in the absence of the conventional risk factors for invasive aspergillosis. This paper explores the hypothesis that contributing causes are the destruction of the lung epithelium permitting colonization by opportunistic pathogens. At the same time, the exhaustion of the immune system, characterized by cytokine storms, apoptosis, and depletion of leukocytes may hinder the response to A. fumigatus infection. The combination of these factors may explain the onset of invasive aspergillosis in immunocompetent patients. We used a previously published computational model of the innate immune response to infection with Aspergillus fumigatus. Variation of model parameters was used to create a virtual patient population. A simulation study of this virtual patient population to test potential causes for co-infection in immunocompetent patients. The two most important factors determining the likelihood of CAPA were the inherent virulence of the fungus and the effectiveness of the neutrophil population, as measured by granule half-life and ability to kill fungal cells. Varying these parameters across the virtual patient population generated a realistic distribution of CAPA phenotypes observed in the literature. Computational models are an effective tool for hypothesis generation. Varying model parameters can be used to create a virtual patient population for identifying candidate mechanisms for phenomena observed in actual patient populations.","version":"1.2","doi":"10.1101/2022.07.18.500514","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495205","pub_date":"2023-1-04","title":"Decoding the fundamental drivers of phylodynamic inference","abstract":"Despite its increasing role in the understanding of infectious disease transmission at the applied and theoretical levels, phylodynamics lacks a well-defined notion of ideal data and optimal sampling. We introduce a formal method to visualise and quantify the relative impact of pathogen genome sequence and sampling times\u2014two fundamental sources of data for phylodynamics under birth-death-sampling models\u2014to understand how each drive phylodynamic inference. Applying our method to simulations and outbreaks of SARS-CoV-2 and H1N1 Influenza data, we use this insight to elucidate fundamental trade-offs and guidelines for phylodynamic analyses to draw the most from sequence data. Phylodynamics promises to be a staple of future responses to infectious disease threats globally. Continuing research into the inherent requirements and trade-offs of phylodynamic data and inference will help ensure phylodynamic tools are wielded in ever more targeted and efficient ways.","version":"1.2","doi":"10.1101/2022.06.07.495205","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.01.522328","pub_date":"2023-1-03","title":"SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression","abstract":"microRNAs (miRNAs) inhibit mRNA translation initiation by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5\u2019 cap structure. Viruses utilise miRNAs to impair the host antiviral immune system and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-\u00df, and thereby impairs the host antiviral immune response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) and enhances the translational repression mediated by natural miRNA binding sites in the 3\u2019 UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery.","version":"1.1","doi":"10.1101/2023.01.01.522328","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.31.522389","pub_date":"2023-1-03","title":"Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies","abstract":"The antiviral component of Paxlovid, nirmatrelvir (NIR), forms a covalent bond with Cys145 of SARS-CoV-2 nsp5. To explore NIR resistance we designed mutations to impair binding of NIR over substrate. Using 12 Omicron (BA.1) and WA.1 SARS-CoV-2 replicons, cell-based complementation and enzymatic assays, we showed that in both strains, E166V imparted high NIR resistance (\u223c55-fold), with major decrease in WA1 replicon fitness (\u223c20-fold), but not BA.1 (\u223c2-fold). WA1 replicon fitness was restored by L50F. These differences may contribute to a potentially lower barrier to resistance in Omicron than WA1. E166V is rare in untreated patients, albeit more prevalent in paxlovid-treated EPIC-HR clinical trial patients. Importantly, NIR-resistant replicons with E166V or E166V/L50F remained susceptible to a) the flexible GC376, and b) PF-00835231, which forms additional interactions. Molecular dynamics simulations show steric clashes between the rigid and bulky NIR t-butyl and \u03b2-branched V166 distancing the NIR warhead from its Cys145 target. In contrast, GC376, through \u201cwiggling and jiggling\u201d accommodates V166 and still covalently binds Cys145. PF-00835231 uses its strategically positioned methoxy-indole to form a \u03b2-sheet and overcome E166V. Drug design based on strategic flexibility and main chain-targeting may help develop second-generation nsp5-targeting antivirals efficient against NIR-resistant viruses.","version":"1.1","doi":"10.1101/2022.12.31.522389","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.01.522064","pub_date":"2023-1-03","title":"Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques","abstract":"Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection. No evidence of association between CH and COVID-19 clinical severity in macaques. The presence of CH is associated with prolonged local inflammatory responses in COVID-19. SARS-CoV-2 persists longer in respiratory tracts of macaques with CH following infection.","version":"1.1","doi":"10.1101/2023.01.01.522064","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.02.522466","pub_date":"2023-1-03","title":"Time Dependent Dihedral Angle Oscillations of the Spike Protein of SARS-CoV-2 Reveal Favored Frequencies of Dihedral Angle Rotations","abstract":"The spike protein of SARS-CoV-2 is critical to viral infection of host cells which ultimately results in COVID-19. In this study we analyze the behavior of dihedral (phi and psi) angles of the spike protein over time from molecular dynamics and identify that the oscillations of these dihedral angles are dominated by a few discrete, relatively low frequencies in the 23-63 MHz range with 42.96875 MHz being the most prevalent frequency sampled by the oscillations. We further observe that upon tallying the populations of each individual frequency for all residues along the frequency spectrum, there is a regular alternation between high and low population counts along the increasing frequency values in the spectrum. This alternation of the counts becomes less pronounced and ultimately stabilizes as the frequency values increase. These observations thus suggest a regularity and propensity in the spike protein\u2019s dihedral angles to avoid similar oscillation population counts between vicinal frequencies. We also observe that for amino acids that are least abundant in the S protein, there are certain frequencies at which the dihedral angles never oscillate, in contrast to relatively abundant amino acids that ultimately cover the entire spectrum. This suggests that the frequency components of dihedral angle oscillations may also be a function of position in the primary structure: the more positions an amino acid is found in, the more frequencies it can sample. Lastly, certain residues identified in the literature as constituting the inside of a druggable pocket of the spike protein, as well as other residues identified as allosteric sites, are observed in our data to have distinctive time domain profiles. This motivates us to propose residues from our dynamic data, with similar time domain profiles, which may be of potential interest to the vaccine and drug design communities, for further investigation. Thus our findings indicate that there is a particular frequency domain profile for the spike protein, hidden within the time domain data, and this information, perhaps with the suggested residues, might provide additional insight into therapeutic development strategies for COVID-19 and beyond.","version":"1.1","doi":"10.1101/2023.01.02.522466","journal":"bioRxiv","score":null},{"id":"10.1101/2023.01.01.522435","pub_date":"2023-1-03","title":"L-shape distribution of the relative substitution rate (c/\u03bc) observed for SARS-COV-2\u2019s genome, inconsistent with the selectionist theory, the neutral theory and the nearly neutral theory but a near-neutral balanced selection theory: implication on \u201cneutralist-selectionist\u201d debate","abstract":"The genomic substitution rate (GSR) of SARS-CoV-2 exhibits a molecular clock feature and does not change under fluctuating environmental factors such as the infected human population (100-107), vaccination etc.. The molecular clock feature is believed to be inconsistent with the selectionist theory (ST). The GSR shows lack of dependence on the effective population size, suggesting Ohta\u2019s nearly neutral theory (ONNT) is not applicable to this virus. Big variation of the substitution rate within its genome is also inconsistent with Kimura\u2019s neutral theory (KNT). Thus, all three existing evolution theories fail to explain the evolutionary nature of this virus. In this paper, we proposed a Segment Substitution Rate Model (SSRM) under non-neutral selections and pointed out that a balanced mechanism between negative and positive selection of some segments that could also lead to the molecular clock feature. We named this hybrid mechanism as near-neutral balanced selection theory (NNBST) and examined if it was followed by SARS-CoV-2 using the three independent sets of SARS-CoV-2 genomes selected by the Nextstrain team. Intriguingly, the relative substitution rate of this virus exhibited an L-shaped probability distribution consisting with NNBST rather than Poisson distribution predicted by KNT or an asymmetric distribution predicted by ONNT in which nearly neutral sites are believed to be slightly deleterious only, or the distribution that is lack of nearly neutral sites predicted by ST. The time-dependence of the substitution rates for some segments and their correlation with the vaccination were observed, supporting NNBST. Our relative substitution rate method provides a tool to resolve the long standing \u201cneutralist-selectionist\u201d controversy. Implications of NNBST in resolving Lewontin\u2019s Paradox is also discussed.","version":"1.1","doi":"10.1101/2023.01.01.522435","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.29.522275","pub_date":"2023-1-02","title":"An engineered ACE2 decoy broadly neutralizes Omicron subvariants and shows therapeutic effect in SARS-CoV-2-infected cynomolgus macaques","abstract":"The Omicron variant continuously evolves under the humoral immune pressure obtained by vaccination and SARS-CoV-2 infection and the resultant Omicron subvariants exhibit further immune evasion and antibody escape. Engineered ACE2 decoy composed of high-affinity ACE2 and IgG1 Fc domain is an alternative modality to neutralize SARS-CoV-2 and we previously reported its broad spectrum and therapeutic potential in rodent models. Here, we show that engineered ACE2 decoy retains the neutralization activity against Omicron subvariants including the currently emerging XBB and BQ.1 which completely evade antibodies in clinical use. The culture of SARS-CoV-2 under suboptimal concentration of neutralizing drugs generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against engineered ACE2 decoy. As the efficient drug delivery to respiratory tract infection of SARS-CoV-2, inhalation of aerosolized decoy treated mice infected with SARS-CoV-2 at a 20-fold lower dose than the intravenous administration. Finally, engineered ACE2 decoy exhibited the therapeutic efficacy for COVID-19 in cynomolgus macaques. Collectively, these results indicate that engineered ACE2 decoy is the promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation can be considered as a non-invasive approach to enhance efficacy in the treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.12.29.522275","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.16.496402","pub_date":"2023-1-02","title":"De novo design of site-specific protein interactions with learned surface fingerprints","abstract":"Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic, and structural data grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof-of-principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1, and CTLA-4. Several designs were experimentally optimized while others were purely generated in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.","version":"1.2","doi":"10.1101/2022.06.16.496402","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.30.522311","pub_date":"2022-12-30","title":"Surveillance for SARS-CoV-2 in Ohio\u2019s wildlife, companion, and agricultural animals","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly to become a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g. shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g. pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n=76), mink (n=57), multiple species of bats (n=65), and other wildlife in addition to domestic cats (n=275) and pigs (n= 184). In total, we tested 800 animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, due to lack of validated tests for animals, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing re-emergence of SARS-CoV-2 in humans or animals.","version":"1.1","doi":"10.1101/2022.12.30.522311","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.28.474326","pub_date":"2022-12-29","title":"Intranasal inhibitor broadly blocks SARS-CoV-2 including recent highly immunoevasive Omicron subvariants","abstract":"The recent emergence of novel SARS-CoV-2 variants capable of efficiently escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models. However, there remains a dire need for SARS-CoV-2 blocking agents that are less vulnerable to mutational variation in the neutralization epitopes of the viral spike glycoprotein and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and extremely stable trimeric human SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Omicron BA.5 as well as the latest and most immunoevasive variants like BF.7, XBB, and BQ.1.1. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before a challenge with SARS-CoV-2 efficiently protected mice from infection, and was still effective even when given 4 h after the viral challenge. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. The high potency and robust biochemical properties of TriSb92 together with the remarkable resistance of its inhibitory action against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2021.12.28.474326","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.28.522082","pub_date":"2022-12-29","title":"Erythromycin, Retapamulin, Pyridoxine, Folic acid and Ivermectin dose dependently inhibit cytopathic effect, Papain-like Protease and MPRO of SARS-CoV-2","abstract":"We previously showed that Erythromycin, Retapamulin, Pyridoxine, Folic acid and Ivermectin inhibit SARS-COV-2 induced cytopathic effect (CPE) in Vero cells. In this study and using validated quantitative neutral red assay, we show that the inhibition of CPE is concentration dependent with Inhibitory Concentration-50(IC50) of 3.27 \u03bcM, 4.23 \u03bcM, 9.29 \u03bcM, 3.19 \u03bcM and 84.31 \u03bcM respectively. Furthermore, Erythromycin, Retapamulin, Pyridoxine, Folic acid and Ivermectin dose dependently inhibit SARS-CoV-2 Papain-like Protease with IC50 of 0.94 \u03bcM, 0.88 \u03bcM, 1.14 \u03bcM, 1.07 \u03bcM, 1.51 \u03bcM respectively and the main protease(MPRO) with IC50 of 1.35 \u03bcM, 1.25 \u03bcM, 7.36 \u03bcM, 1.15 \u03bcM and 2.44 \u03bcM respectively. The IC50 for all the drugs, except ivermectin, are at the clinically achievable plasma concentration in human, which supports a possible role for the drugs in the management of COVID-19. The lack of inhibition of CPE by Ivermectin at clinical concentrations could be part of the explanation for its lack of effectiveness in clinical trials.","version":"1.1","doi":"10.1101/2022.12.28.522082","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.29.522217","pub_date":"2022-12-29","title":"SARS-CoV-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast","abstract":"Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The SARS-CoV-2 small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small ORFs. ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a specifically localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.","version":"1.1","doi":"10.1101/2022.12.29.522217","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.29.522202","pub_date":"2022-12-29","title":"Immunogenicity of COVID-eVax Is Moderately Impacted by Temperature and Molecular Isoforms","abstract":"DNA integrity is a key issue in gene therapy and genetic vaccine approaches based on plasmid DNA. In contrast to messenger RNA that requires a controlled cold chain for efficacy, DNA molecules are considered to be more stable. In this study, we challenged this concept by characterizing the immunological response induced by a plasmid DNA vaccine delivered using electroporation. As a model, we used COVID-eVax, which is a plasmid DNA vaccine that targets the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Increased nicked DNA was produced by using either an accelerated stability protocol or a lyophilization protocol. Surprisingly, the immune response induced in vivo was only minimally affected by the percentage of open circular DNA. This result suggests that plasmid DNA vaccines, such as COVID-eVax that has completed a phase I clinical trial, retain their efficacy upon storage at higher temperatures and this feature may facilitate their use in low-/middle-income countries.","version":"1.1","doi":"10.1101/2022.12.29.522202","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.27.521990","pub_date":"2022-12-27","title":"An alpaca-derived nanobody recognizes a unique conserved epitope and retains potent activity against the SARS-CoV-2 omicron variant","abstract":"The SARS-CoV2 Omicron variant sub-lineages spread rapidly through the world, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for anti-SARS-CoV-2 agents that are effective against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production and potential for delivery via inhalation. Here, we characterize the RBD-specific nanobody W25, which we previously isolated from an alpaca, and show superior neutralization activity towards Omicron lineage BA.1 in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike surface glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. Furthermore, we show that W25 also binds the spike protein from the emerging, more infectious Omicron BA.2 lineage with picomolar affinity. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse prioritization of W25 for further clinical development.","version":"1.1","doi":"10.1101/2022.12.27.521990","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.27.521979","pub_date":"2022-12-27","title":"Tissue protective role of Ganetespib in SARS-CoV-2-infected Syrian golden hamsters","abstract":"The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are therefore urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyper-inflammatory state generated upon infection, at reducing lung tissue pathology and endothelial damages, along with viral replication. Previous research has pointed a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. In this study, we analyzed the effects of the potent HSP90 inhibitor Ganetespib in vitro on alveolar epithelial cells and alveolar macrophages to characterize its effects on cell activation and viral replication. Additionally, to evaluate its efficacy in controlling systemic inflammation and the viral burden after infection in vivo, a Syrian hamster model was used. In vitro, Ganetespib reduced viral replication on AECs in a dose-dependent manner and lowered significantly the expression of pro-inflammatory genes, in both AECs and alveolar macrophages. In vivo, administration of Ganetespib led to an overall improvement of the clinical condition of infected animals, with decreased systemic inflammation, reduced edema formation and lung tissue pathology. Altogether, we show that Ganetespib could be a potential medicine to treat moderate and severe cases of COVID-19.","version":"1.1","doi":"10.1101/2022.12.27.521979","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.26.521940","pub_date":"2022-12-27","title":"ESCRT recruitment to mRNA-encoded SARS-CoV-2 spike induces virus-like particles and enhanced antibody responses","abstract":"Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely-arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T-cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared to conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for three months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.","version":"1.1","doi":"10.1101/2022.12.26.521940","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.27.521986","pub_date":"2022-12-27","title":"Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants","abstract":"In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.","version":"1.1","doi":"10.1101/2022.12.27.521986","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.23.521847","pub_date":"2022-12-27","title":"Evaluation of the molecular diversity of Brazilian strains of the B.1.1 variant of SARS-CoV-2 used in vaccines","abstract":"In this work, 28 sequences with 57,570 sites of the B.1.1 variant of SARS-CoV-2, from Brazilian states, were used. All sequences (publicly available on the National Center for Biotechnology Information platform (NCBI)) were aligned with Mega X software and all gaps, ambiguous sites and lost data were extracted, resulting in a region in a segment with 8,799 polymorphic (15.2% of the total) that were analyzed for their molecular diversity, FST, demographic and spatial expansion. Phylogenetic relationships of ancestry revealed the absence of genetically distinct subgroups, which was corroborated by the low value of FST found (15.38%). The low degree of polymorphism found among these samples, corroborated by the almost non-existent genetic distance, helped or established the absence of a genetic structuring pattern, demonstrating a satisfactory pattern of response to vaccines, since all the sequences analyzed were part of the Brazilian strains of variant B.1.1 of SARS-CoV-2 used in vaccines.","version":"1.1","doi":"10.1101/2022.12.23.521847","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.23.521817","pub_date":"2022-12-26","title":"In Vitro Inhibition of SARS-CoV-2 Infection by Bromhexine hydrochloride","abstract":"The world is enduring the SARS CoV-2 pandemic, and although extensive research has been conducted on the issue, only a few antivirals have been approved to treat patients with COVID-19. Bromhexine hydrochloride was previously identified as a potent inhibitor of TMPRSS2, an essential protease for ACE-2 virus receptor interactions. In the present study, we investigated whether bromhexine treatment could reduce SARS CoV-2 replication in vitro. To evaluate bromhexine\u2019s effectiveness against SARS COV-2 infection, viral load was measured using Caco-2 cell lines expressing TMPRSS2. Our molecular docking results indicate that bromhexine displays an affinity with the active site of TMPRSS2. The drug significantly inhibited SARS CoV-2, both parental and P1 variant strains, infection in the Caco-2 cell line, reducing about 40% of SARS-CoV-2 entrance and about 90% of viral progeny in the supernatant 48h post-infection. Furthermore, bromhexine did not exhibit any direct virucidal activity on SARS CoV-2. In conclusion, bromhexine hydrochloride efficiently disrupts SARS CoV-2 infection in vitro and has the potential to become an effective antiviral agent in COVID-19 treatment.","version":"1.1","doi":"10.1101/2022.12.23.521817","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.25.521651","pub_date":"2022-12-26","title":"Innovative, rapid, high-throughput method for drug repurposing in a pandemic \u2013 a case study of SARS-CoV-2 and COVID-19","abstract":"Several efforts to repurpose drugs for COVID-19 treatment have largely either failed to identify a suitable agent or agents identified did not translate to clinical use; either because of demonstrated lack of clinical efficacy in trials, inappropriate dose requirements and probably use of inappropriate pre-clinical laboratory surrogates of effectiveness. In this study, we used an innovative algorithm, that incorporates dissemination and implementation considerations, to identify potential drugs for COVID-19 using iterative computational and wet laboratory methods that highlight inhibition of viral induced cytopathic effect (CPE) as a laboratory surrogate of effectiveness. Erythromycin, pyridoxine, folic acid and retapamulin were found to inhibit SARS-CoV-2 induced CPE in Vero cells at concentrations that are clinically achievable. Additional studies may be required to further characterize the inhibitions of CPE and the possible mechanisms. TETFund Covid-19 Special Intervention Research grant(grant number TETFund/DR&D/CE/ SI/COVID-19/UDUS/VOL 1)","version":"1.1","doi":"10.1101/2022.12.25.521651","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.07.503099","pub_date":"2022-12-26","title":"Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors","abstract":"Vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease (Mpro/3CLpro) of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy. Resistance to protease inhibitor drugs, nirmatrelvir (Paxlovid) and ensitrelvir (Xocova), exists in SARS-CoV-2 variants in the human population.","version":"1.2","doi":"10.1101/2022.08.07.503099","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.25.521903","pub_date":"2022-12-26","title":"Impact of BA.1, BA.2, and BA.4/BA.5 Omicron Mutations on Therapeutic Monoclonal Antibodies","abstract":"The emergence of Omicron SARS-CoV-2 subvariants (BA.1, BA.2, BA.4, and BA.5) with an unprecedented number of mutations in their receptor-binding domain (RBD) of the spike-protein has fueled a new surge of COVID-19 infections, posing a major challenge to the efficacy of existing vaccines and monoclonal antibody (mAb) therapeutics. Here, a thorough and systematic molecular dynamics (MD) simulation study is conducted to investigate how the RBD mutations on these subvariants affect the interactions with broad mAbs including AstraZeneca (COV2-2196 and COV2-2130), Brii Biosciences (BRII-196), Celltrion (CT-P59), Eli Lilly (LY-CoV555 and LY-CoV016), Regeneron (REGN10933 and REGN10987), Vir Biotechnology (S309), and S2X259. Our results show a complete loss of binding for COV2-2196, BRII-196, CT-P59, and LY-CoV555 with all Omicron RBDs. REGN10987 also loses its binding against BA.1 but partially retains against BA.2 and BA.4/5. The reduction in binding is either significant for LY-CoV016 and REGN10933 or moderate for COV2-2130. S309 and S2X259 retain their binding strength against BA.1 but decrease against others. We introduce a mutational escape map for each mAb to identify the key RBD sites and critical mutation. Overall, our findings suggest that majority of therapeutic mAbs have diminished or lost their activity against Omicron subvariants, indicating the urgent need for a new therapeutic mAb, modifying current ones with a better mAb design, or seeking an alternative approach.","version":"1.1","doi":"10.1101/2022.12.25.521903","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.25.521784","pub_date":"2022-12-26","title":"A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates","abstract":"While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster \u223cone year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly booster vaccine, and as a primary vaccine for pediatric use including in infants.","version":"1.1","doi":"10.1101/2022.12.25.521784","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.24.521858","pub_date":"2022-12-26","title":"Amplification and extraction free quantitative detection of viral nucleic acids and single-base mismatches using magnetic signal amplification circuit","abstract":"Established nucleic acid detection assays require extraction and purification before sequence amplification and/or enzymatic reactions, hampering their widespread applications in point-of-care (POC) formats. Magnetic immunoassays based on magnetic particle spectroscopy and magnetic nanoparticles (MNPs) are isothermal, extraction- and purification-free, and can be quantitative and benchtop, making them suitable for POC settings. Here, we demonstrate a Magnetic signal Amplification Circuit (MAC) that combines specificity of toehold-mediated DNA strand displacement with magnetic response of MNPs to a clustering/declustering process. Our MAC assays require neither amplification nor extraction of target nucleic acids, and reveal four times better sensitivity than that of a magnetic circuit without signal amplification. Using MAC, we detect a highly specific 43 nucleotides sequence of SARS-CoV-2 virus. The MAC enables sensing both DNA and RNA targets with varying lengths and resolving single-base mismatches. Our MAC can be a powerful tool for translating research of nucleic acids detection to the clinic.","version":"1.1","doi":"10.1101/2022.12.24.521858","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507370","pub_date":"2022-12-25","title":"Clomipramine inhibits dynamin GTPase activity by L-\u03b1-phosphatidyl-L-serine stimulation","abstract":"Three dynamin isoforms play critical roles in clathrin-dependent endocytosis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells via clathrin-dependent endocytosis. We previously reported that 3-(3-chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (clomipramine) inhibits the GTPase activity of dynamin 1, which is in mainly neuron. Therefore, we investigated whether clomipramine inhibits the activity of other dynamin isoforms in this study. We found that, similar to its inhibitory effect on dynamin 1, clomipramine inhibited the L-\u03b1-phosphatidyl-L-serine-stimulated GTPase activity of dynamin 2, which is expressed ubiquitously, and dynamin 3, which is expressed in the lung. Inhibition of GTPase activity raises the possibility that clomipramine can suppress SARS-CoV-2 entry into host cells.","version":"1.4","doi":"10.1101/2022.09.09.507370","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.04.490614","pub_date":"2022-12-23","title":"SARS-CoV-2 Omicron subvariants evolved to promote further escape from MHC-I recognition","abstract":"SARS-CoV-2 variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress MHC I expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC I, Omicron subvariants exhibit a greater ability to suppress surface MHC-I expressions. Collectively, our data suggest that, in addition to escape from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition. Numerous pathogenic viruses have developed strategies to evade host CD8+ T cell-mediated clearance. Here, we demonstrated that SARS-CoV-2 encodes multiple viral factors that can modulate MHC-I expression in the host cells. We found that MHC-I upregulation was strongly suppressed during SARS-CoV-2 infection in vivo. Notably, the Omicron subvariants showed an enhanced ability to suppress MHC-I compared to the original strain and the earlier SARS-CoV-2 variants of concern (VOCs). Our results point to the inherently strong ability of SARS-CoV-2 to hinder MHC-I expression and demonstrated that Omicron subvariants have evolved an even more optimized capacity to evade CD8 T cell recognition.","version":"1.3","doi":"10.1101/2022.05.04.490614","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.22.521201","pub_date":"2022-12-23","title":"Enhanced neutralization escape to therapeutic monoclonal antibodies by SARS-CoV-2 Omicron sub-lineages","abstract":"The landscape of SARS-CoV-2 variants dramatically diversified with the simultaneous appearance of multiple sub-variants originating from BA.2, BA.4 and BA.5 Omicron sub-lineages. They harbor a specific set of mutations in the spike that can make them more evasive to therapeutic monoclonal antibodies. In this study, we compared the neutralizing potential of monoclonal antibodies against the Omicron BA.2.75.2, BQ.1, BQ.1.1 and XBB variants, with a pre-Omicron Delta variant as a reference. Sotrovimab retains some activity against BA.2.75.2, BQ.1 and XBB as it did against BA.2/BA.5, but is less active against BQ.1.1. Within the Evusheld/AZD7442 cocktail, Cilgavimab lost all activity against all subvariants studied, resulting in loss of Evusheld activity. Finally, Bebtelovimab, while still active against BA.2.75, also lost all neutralizing activity against BQ.1, BQ.1.1 and XBB variants.","version":"1.1","doi":"10.1101/2022.12.22.521201","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.22.521662","pub_date":"2022-12-23","title":"Contact-number-driven virus evolution: a multi-level modeling framework for the evolution of acute or persistent RNA virus infection","abstract":"Viruses evolve in infected host populations, and host population dynamics affect viral evolution. RNA viruses with a short duration of infection and a high peak viral load, such as and SARS-CoV-2, are maintained in human populations. By contrast, RNA viruses characterized by a long infection duration and a low peak viral load (e.g., borna disease virus) can be maintained in nonhuman populations, and why the persistent viruses evolved has been rarely explored. Here, using a multi-level modeling approach including both individual-level virus infection dynamics and population-scale transmission, we consider virus evolution based on the host environment, specifically, the effect of the contact history of infected hosts. We found that, with a highly dense contact history, viruses with a high virus production rate but low accuracy are likely to be optimal, resulting in a short infectious period with a high peak viral load. In contrast, with a low-density contact history, viral evolution is toward low virus production but high accuracy, resulting in long infection durations with low peak viral load. Our study sheds light on the origin of persistent viruses and why acute viral infections but not persistent virus infection tends to prevail in human society.","version":"1.1","doi":"10.1101/2022.12.22.521662","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.15.520561","pub_date":"2022-12-22","title":"SARS-CoV-2 causes periodontal fibrosis by deregulating mitochondrial \u03b2-oxidation","abstract":"The global high prevalence of COVID-19 is a major challenge for health professionals and patients. SARS-CoV-2 virus mutate predominantly in the spike proteins, whilst the other key viral components remain stable. Previous studies have shown that the human oral cavity can potentially act as reservoir of the SARS-CoV-2 virus. COVID-19 can cause severe oral mucosa lesions and is likely to be connected with poor periodontal conditions. However, the consequence of SARS-CoV-2 viral infection on human oral health has not been systematically examined. In this research, we aimed to study the pathogenicity of SARS-CoV-2 viral components on human periodontal tissues and cells. We found that by exposing to SARS-CoV-2, especially to the viral envelope and membrane proteins, the human periodontal fibroblasts could develop fibrotic pathogenic phenotypes, including hyperproliferation that was concomitant induced together with increased apoptosis and senescence. The fibrotic degeneration was mediated by a down-regulation of mitochondrial \u03b2-oxidation in the fibroblasts. Fatty acid \u03b2-oxidation inhibitor, etomoxir treatment could mirror the same pathological consequence on the cells, similar to SARS- CoV-2 infection. Our results therefore provide novel mechanistic insights into how SARS- CoV-2 infection can affect human periodontal health at the cell and molecular level with potential new therapeutic targets for COVID-19 induced fibrosis.","version":"1.2","doi":"10.1101/2022.12.15.520561","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.22.521642","pub_date":"2022-12-22","title":"Targeting Spike Glycans to Inhibit SARS-CoV2 Viral Entry","abstract":"SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.","version":"1.1","doi":"10.1101/2022.12.22.521642","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.22.521558","pub_date":"2022-12-22","title":"Common dandelion (Taraxacum officinale) leaf extract efficiently inhibits SARS-CoV-2 Omicron infection in vitro","abstract":"As the COVID-19 pandemic continues to pose a health risk concern to humans, despite a significant increase in vaccination rates, an effective prevention and treatment of SARS-CoV-2 infection is being sought worldwide. Herbal medicines have been used for years and played a tremendous role in several epidemics of respiratory viral infections. Thus, they are considered as a promising platform to combat SARS-CoV-2. Previously, we reported that common dandelion (Taraxacum officinale) leaf extract and its high molecular weight compounds strongly suppressed in vitro lung cell infection by SARS-CoV-2 Spike D614 and Delta variant pseudotyped lentivirus. We now here demonstrate that T. officinale extract protects against the most prominent Omicron variant using hACE2-TMPRSS2 overexpressing A549 cells as in vitro model system. Notably, compared to the original D614, and the Delta variant, we could confirm a higher efficacy. Short-term interval treatment of only 30 min was then sufficient to block the infection by 80% at 10 mg/mL extract. Further subfractionation of the extract identified compounds larger than 50 kDa as effective ACE2-Spike binding inhibitors. In summary, the evolution of SARS-CoV-2 virus to the highly transmissible Omicron variant did not lead to resistance, but rather increased sensitivity to the preventive effect of the extract.","version":"1.1","doi":"10.1101/2022.12.22.521558","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.425396","pub_date":"2022-12-22","title":"Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic","abstract":"The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS- CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function. Since it was identified in 2019, SARS-CoV-2 has displayed voracious transmissibility which has resulted in rapid spread of the virus and a global pandemic. SARS-CoV-2 is a member of the Coronaviridae family whose members are encapsulated by a host-derived protective membrane shell. Whilst the viral envelope may provide protection for the virus, it also provides an opportunity for the host cell to restrict the virus and stop it spreading. The anti-viral restriction factor, tetherin, acts to crosslink viruses to the surface of infected cells and prevent their spread to uninfected cells. Here, we demonstrate that SARS-CoV-2 undergoes viral restriction by tetherin, and that SARS-CoV-2 moves tetherin away from the site of Coronavirus budding to enhance its ability to escape and infect na\u00efve cells. Tetherin depletion from cells enhanced SARS-CoV-2 viral release and increased propagation of the virus. We found that the SARS-CoV-2 protein, ORF3a, redirects tetherin away from the biosynthetic organelles where tetherin would become incorporated to newly forming SARS-CoV-2 virions \u2013 and instead relocalises tetherin to late endocytic organelles. We also found that SARS-CoV-2 Spike downregulates tetherin. These two mechanisms, in addition to the well described antagonism of interferon and subsequent ISGs highlight the multiple mechanisms by which SARS-CoV-2 abrogates tetherin function. Our study provides new insights into how SARS-CoV-2 subverts human antiviral responses and escapes from infected cells.","version":"1.2","doi":"10.1101/2021.01.06.425396","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.22.22283843","pub_date":"2022-12-22","title":"Association of close-range contact patterns with SARS-CoV-2: a household transmission study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Households are an important location for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, especially during periods where travel and work was restricted to essential services. We aimed to assess the association of close-range contact patterns with SARS-CoV-2 transmission.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We deployed proximity sensors for two weeks to measure face-to-face interactions between household members after SARS-CoV-2 was identified in the household, in South Africa, 2020 - 2021. We calculated duration, frequency and average duration of close range proximity events with SARS-CoV-2 index cases. We assessed the association of contact parameters with SARS-CoV-2 transmission using mixed effects logistic regression accounting for index and household member characteristics.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    We included 340 individuals (88 SARS-CoV-2 index cases and 252 household members). On multivariable analysis, factors associated with SARS-CoV-2 acquisition were index cases with minimum C\n                    <jats:sub>t</jats:sub>\n                    value &lt;30 (aOR 10.2 95%CI 1.4-77.4) vs &gt;35, contacts aged 13-17 years (aOR 7.7 95%CI 1.0-58.2) vs &lt;5 years and female contacts (aOR 2.3 95%CI 1.1-4.8). No contact parameters were associated with acquisition (aOR 1.0 95%CI 1.0-1.0) for all three of duration, frequency and average duration.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>We did not find an association between close-range proximity events and SARS-CoV-2 household transmission. It may be that droplet-mediated transmission during close-proximity contacts play a smaller role than airborne transmission of SARS-CoV-2 in the household, due to high contact rates in households or study limitations.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Wellcome Trust (Grant number 221003/Z/20/Z) in collaboration with the Foreign, Commonwealth and Development Office, United Kingdom.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.12.22.22283843","journal":"medRxiv","score":null},{"id":"10.1101/2022.12.22.521646","pub_date":"2022-12-22","title":"Glyco-engineered pentameric SARS-CoV-2 IgMs show superior activities compared to IgG1 orthologues","abstract":"Immunoglobulin M (IgM) is the largest antibody isotype with unique features like extensive glycosylation and oligomerization. Major hurdles in characterizing its properties are difficulties in the production of well-defined multimers. Here we report the expression of two SARS-CoV-2 neutralizing monoclonal antibodies in glycoengineered plants. Isotype switch from IgG1 to IgM resulted in the production of pentameric IgMs, comprising of correctly assembled 21 human protein subunits. All four recombinant monoclonal antibodies carried a highly reproducible human-type N-glycosylation profile, with a single dominant N-glycan species at each glycosite. Both pentameric IgMs exhibited increased antigen binding and virus neutralization potency, up to 390-fold, compared to the parental IgG1. Collectively, the results may impact on the future design of vaccines, diagnostics and antibody-based therapies and emphasize the versatile use of plants for the expression of highly complex human proteins with targeted posttranslational modifications.","version":"1.1","doi":"10.1101/2022.12.22.521646","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.21.521463","pub_date":"2022-12-22","title":"T-cell cellular stress and reticulocyte signatures, but not loss of na\u00efve T lymphocytes, characterize severe COVID-19 in older adults","abstract":"In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19 we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of na\u00efve T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.","version":"1.1","doi":"10.1101/2022.12.21.521463","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.21.521431","pub_date":"2022-12-21","title":"Mechanism of LLPS of SARS-CoV-2 N protein","abstract":"SARS-CoV-2 nucleocapsid (N) protein with low mutation rate is the only structural protein not only functioning to package viral genomic RNA, but also manipulating the host-cell machineries, thus representing a key target for drug development. Recent discovery of its liquid-liquid phase separation (LLPS) not only sheds light on previously-unknown mechanisms underlying the host-SARS-CoV-2 interaction and viral life cycle, but most importantly opens up a new direction for developing anti-SARS-CoV-2 strategies/drugs. However, so far the high-resolution mechanism of LLPS of N protein still remains unknown because it is not amenable for high-resolution biophysical investigations. Here we systematically dissected N protein into differential combinations of domains followed by DIC and NMR characterization. We successfully identified N (1-249), which not only gives high-quality NMR spectra, but phase separates as the full-length N protein. The results together decode for the first time: 1) nucleic acid modulates LLPS by dynamic but specific interactions multivalently over both folded NTD/CTD and Arg/Lys residues within IDRs. 2) ATP, mysteriously with concentrations >mM in all living cells but absent in viruses, not only specifically binds NTD/CTD, but also Arg residues within IDRs with Kd of 2.8 mM. 3) ATP dissolves LLPS by competitively displacing nucleic acid from binding the protein. Therefore, ATP and nucleic acid interplay in modulating LLPS by specific competitions for binding over the highly overlapped binding sites. Our study deciphers the mechanism of LLPS of N protein, which is targetable by small molecules. ATP is not only emerging as a cellular factor controlling the host-SARS-CoV-2 interaction, but also provides a lead for developing anti-SARS-CoV-2 drugs efficient for different variants of SARS-CoV-2. Fundamentally, our results imply that the mechanisms of LLPS of IDR-containing proteins mediated by ATP and nucleic acids appear to be highly conserved from human to virus.","version":"1.1","doi":"10.1101/2022.12.21.521431","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.21.521388","pub_date":"2022-12-21","title":"SARS-CoV-2 Variant-Specific Differences in Inhibiting the Effects of the PKR-Activated Integrated Stress Response","abstract":"The integrated stress response (ISR) is a eukaryotic cell pathway that triggers translational arrest and the formation of stress granules (SGs) in response to various stress signals, including those caused by viral infections. The SARS-CoV-2 nucleocapsid protein has been shown to disrupt SGs, but SARS-CoV-2 interactions with other components of the pathway remains poorly characterized. Here, we show that SARS-CoV-2 infection triggers the ISR through activation of the eIF2\u03b1-kinase PKR while inhibiting a variety of downstream effects. In line with previous studies, SG formation was efficiently inhibited and the induced eIF2\u03b1 phosphorylation only minimally contributed to the translational arrest observed in infected cells. Despite ISR activation and translational arrest, expression of the stress-responsive transcripts ATF4 and CHOP was not induced in SARS-CoV-2 infected cells. Finally, we found variant-specific differences in the activation of the ISR between ancestral SARS-CoV-2 and the Delta and Omicron BA.1 variants in that Delta infection induced weaker PKR activation while Omicron infection induced higher levels of p-eIF2\u03b1 and greatly increased SG formation compared to the other variants. Our results suggest that different SARS-CoV-2 variants can affect normal cell functions differently, which can have an impact on pathogenesis and treatment strategies.","version":"1.1","doi":"10.1101/2022.12.21.521388","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.20.521139","pub_date":"2022-12-21","title":"Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity","abstract":"The success of the human body in fighting SARS-CoV-2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance. We report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV-2 compared with uninfected controls. In contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients. These features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges.","version":"1.1","doi":"10.1101/2022.12.20.521139","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.20.521247","pub_date":"2022-12-21","title":"Identification of new drugs to counteract anti-spike IgG-induced hyperinflammation in severe COVID-19","abstract":"Previously, we and others have shown that SARS-CoV-2 spike-specific IgG antibodies play a major role in disease severity in COVID-19 by triggering macrophage hyperactivation, disrupting endothelial barrier integrity, and inducing thrombus formation. This hyper-inflammation is dependent on high levels of anti-spike IgG with aberrant Fc tail glycosylation, leading to Fc\u03b3 receptor hyper-activation. For development of immune-regulatory therapeutics, drug specificity is crucial to counteract excessive inflammation while simultaneously minimizing inhibition of antiviral immunity. We here developed an in vitro activation assay to screen for small molecule drugs that specifically counteract antibody-induced pathology. We identified that anti-spike induced inflammation is specifically blocked by small molecule inhibitors against SYK and PI3K. We identified SYK inhibitor entospletinib as the most promising candidate drug, which also counteracted anti-spike-induced endothelial dysfunction and thrombus formation. Moreover, entospletinib blocked inflammation by different SARS-CoV-2 variants of concern. Combined, these data identify entospletinib as a promising treatment for severe COVID-19.","version":"1.1","doi":"10.1101/2022.12.20.521247","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.20.521221","pub_date":"2022-12-21","title":"Structure adaptation in Omicron SARS-CoV-2/hACE2: Biophysical origins of evolutionary driving forces","abstract":"Since its emergence, the Covid19 pandemic has been sustained by a series of transmission waves initiated by new variants of the SARS-CoV-2 virus. Some of these arise with higher transmissivity and/or increased disease severity. Here we use molecular dynamics simulations to examine the modulation of the fundamental interactions between the receptor binding domain (RBD) of the spike glycoprotein and the host cell receptor (human angiotensin-converting enzyme 2: hACE2) arising from Omicron variant mutations (BA.1 and BA.2) relative to the original wild type strain. We find significant structural differences in the complexes which overall bring the spike protein and its receptor into closer proximity. These are consistent with and attributed to the higher positive charge on the RBD conferred by BA.1 and BA.2 mutations relative to the wild type. However, further differences between sub-variants BA.1 and BA.2 (which have equivalent RBD charges) are also evident: Mutations affect interdomain interactions between the up-chain and its clockwise neighbor chain, resulting in enhanced flexibility for BA.2. Consequently, additional close contacts arise in BA.2 which include binding to hACE2 by a second spike protein monomer, in addition to the up-chain - a motif not found in BA.1. Finally, the mechanism by which the glycans stabilize the up state of the Spike protein differs for the wild type and the Omicrons. We also found the glycan on N90 of hACE2 turns from inhibiting, to facilitating the binding to Omicron spike protein. These structural and electrostatic differences offer further insight into the mechanisms by which viral mutations modulate host cell binding and provide a biophysical basis for evolutionary driving forces.","version":"1.1","doi":"10.1101/2022.12.20.521221","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.19.517879","pub_date":"2022-12-20","title":"An oral vaccine for SARS-CoV-2 RBD mRNA-bovine milk-derived exosomes induces a neutralizing antibody response in vivo","abstract":"The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has presented numerous challenges to global health. The vaccines, including lipid-based nanoparticle mRNA, inactivated virus and recombined protein, have been used to prevent SARS-CoV-2 infections in clinics and are immensely helpful against the epidemic. Here, we first present an oral mRNA vaccine based on bovine milk-derived exosomes (milk-exos), which encodes the SARS-CoV-2 receptor binding domain (RBD) as an immunogen. The results indicated that RBD mRNA delivered by milk-derived exosomes can produce secreted RBD peptide in 293 cells in vitro and stimulated neutralizing antibodies against RBD in mice. These results indicated that bovine milk-derived exosome-based mRNA vaccine could serve as a new strategy for preventing SARS-CoV-2 infection. Meanwhile, it also can work as a new oral delivery system for mRNA. Oral SARS-CoV-2 mRNA vaccine based on bovine milk-derived exosomes can stimulate neutralizing antibodies in mice.","version":"1.1","doi":"10.1101/2022.12.19.517879","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.19.521129","pub_date":"2022-12-20","title":"Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection","abstract":"While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2022.12.19.521129","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.19.521127","pub_date":"2022-12-20","title":"Adenoviral-Based Vaccine Elicits Robust Systemic and Mucosal Cross-Reactive Responses in African Green Monkeys and Reduces Shedding after SARS-CoV-2 Challenge","abstract":"As new SARS-CoV-2 variants continue to emerge and impact communities worldwide, efforts to develop next generation vaccines that enhance mucosal immunity would be beneficial for protecting individuals and reducing community transmission. We have developed a non-replicating recombinant adenovirus vector (rAd5) vaccine delivered by mucosal administration engineered to express both a protein antigen and a novel molecular adjuvant in the same cell. Here we describe the immunogenicity of three unique SARS-CoV-2 rAd5 vaccine preclinical candidates and their efficacy following viral challenge in African green monkeys. Animals were prime and boost immunized intranasally twenty-nine days apart with rAd5 vaccine candidates containing viral SARS-CoV-2 spike protein alone or in combination with viral nucleocapsid. Mucosal immunization elicited significant increases in antigen-specific serum antibody responses and functional neutralizing activity against multiple variants of concern. Robust antigen specific mucosal IgA responses were observed after a single administration of rAd5 and generated strong cross-reactive neutralizing antibodies against multiple variants including delta. Importantly, all vaccinated animals exhibited a significant reduction in viral loads and infectious particle shedding in both the nasal passages and lower airways compared to unvaccinated controls following challenge with SARS-CoV-2. These findings demonstrate that mucosal immunization using rAd5 is highly immunogenic, confers protective cross-reactive humoral responses in both the circulation and mucosa, and reduces viral loads and shedding upon challenge with multiple SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.12.19.521127","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.20.521197","pub_date":"2022-12-20","title":"Genome-based comparison between the recombinant SARS-CoV-2 XBB and its parental lineages","abstract":"Recombination is the main contributor to RNA virus evolution, and SARS-CoV-2 during the pandemic produced several recombinants. The most recent SARS-CoV-2 recombinant is the lineage labeled XBB, also known as Gryphon, which arose from BJ.1 and BM. 1.1.1. Here we performed a genome-based survey aimed to compare the new recombinant with its parental lineages that never became dominant. Genetic analyses indicated that the recombinant XBB and its first descendant XBB.1 show an evolutionary condition typical of an evolutionary blind background with no further epidemiologically relevant descendant. Genetic variability and expansion capabilities are slightly higher than parental lineages. Bayesian Skyline Plot indicates that XBB reached its plateau around October 6, 2022 and after an initial rapid growth the viral population size did not further expand, and around November 10, 2022 its levels of genetic variability decreased. Simultaneously with the reduction of the XBB population size, an increase of the genetic variability of its first sub-lineage XBB.1 occurred, that in turn reached the plateau around November 9, 2022 showing a kind of vicariance with its direct progenitors. Structure analysis indicates that the affinity for ACE2 surface in XBB/XBB.1 RBDs is weaker than for BA.2 RBD. In conclusion, nowadays XBB and XBB.1 do not show evidence about a particular danger or high expansion capability. Genome-based monitoring must continue uninterrupted in order to individuate if further mutations can make XBB more dangerous or generate new subvariants with different expansion capability.","version":"1.1","doi":"10.1101/2022.12.20.521197","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.19.521064","pub_date":"2022-12-20","title":"Different B cell activation patterns in asymptomatic and symptomatic COVID-19 patients","abstract":"Early and persistent defects in B cell subsets such as memory B cells were shown to be correlated with poor outcomes in COVID-19 patients. This research aimed to develop a molecular pathway model to understand the B cell development in COVID-19. A B cell transcriptomics dataset, obtained from COVID-19 patients, was analyzed on the resulting pathway model to study B cell activation. The pathway showed two distinct gene expression profiles between asymptomatic and symptomatic patients. In asymptomatic patients, there is an increase in transcript levels of antiviral interferon-stimulated genes such as ISG15, IFITM1, and NEAT1 and a driving gene for the extrafollicular pathway CXCR4 indicating a formation of plasmablast. In symptomatic patients, the results suggest an inhibition occurring at the germinal center hinting at a reduction in memory B cell production. Transcripts of driver gene CXCR5 involved in germinal center development is one of the most downregulated genes. This could contribute to the shortage in the formation of memory B cells in COVID-19. Concluding, in SARS-CoV-2 infection, B cells follow different activation routes in asymptomatic and symptomatic patients. In this study, we constructed a pathway that allowed us to analyze and interpret activation patterns of B cells in COVID-19 patients and their link to disease severity. Importantly, the pathway and approach can be reused for further research in COVID-19 or other diseases.","version":"1.1","doi":"10.1101/2022.12.19.521064","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.19.521044","pub_date":"2022-12-20","title":"An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics","abstract":"This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced with consistent purity and potency in less than 24 hours. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed anywhere in the world where a viral pathogen might emerge. The current emergence of viral variants has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.","version":"1.1","doi":"10.1101/2022.12.19.521044","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.30.498338","pub_date":"2022-12-20","title":"Transcriptional Profiles Analysis of COVID-19 and Malaria Patients Reveals Potential Biomarkers in Children","abstract":"The clinical presentation overlap between malaria and COVID-19 poses special challenges for rapid diagnosis in febrile children. In this study, we collected RNA-seq data of children with malaria and COVID-19 infection from the public databases as raw data in fastq format paired end files. A group of six, five and two biological replicates of malaria, COVID-19 and healthy donors respectively were used for the study. We conducted differential gene expression analysis to visualize differences in the expression profiles. Using edgeR, we explored particularly gene expression levels in different phenotype groups and found that 1084 genes and 2495 genes were differentially expressed in the malaria samples and COVID-19 samples respectively when compared to healthy controls. The highly expressed gene in the COVID-19 group we found CD151 gene which is facilitates in T cell proliferation, while in the malaria group, among the highly expressed gene we identified GBP5 gene which involved in inflammatory response and response to bacterium. By comparing both malaria and COVID-19 infections, the overlap of 62 differentially expressed genes patterns were identified. Among them, three genes (ENSG00000234998, H2AC19 and TXNDC5) were highly upregulated in both infections. Strikingly, we observed 13 genes such as HBQ1, HBM, SLC7A5, SERINC2, ATP6V0C, ST6GALNAC4, RAD23A, PNPLA2, GAS2L1, TMEM86B, SLC6A8, UBALD1, RNF187 were downregulated in children with malaria and uniquely upregulated in children with COVID-19, thus may be further validated as potential biomarkers to delineate COVID-19 from malaria-related febrile infection. The hemoglobin complexes and lipid metabolism biological pathways are highly expressed in both infections. Our study provided new insights for further investigation of the biological pattern in hosts with malaria and COVID-19 coinfection.","version":"1.3","doi":"10.1101/2022.06.30.498338","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.16.520829","pub_date":"2022-12-19","title":"Prewhitening and Normalization Help Detect a Strong Cross-Correlation Between Daily Wastewater SARS-CoV-2 RNA Abundance and COVID-19 Cases in a Community","abstract":"Wastewater surveillance is a promising technology for real-time tracking and even early detection of COVID-19 infections in communities. Although correlation analysis between wastewater surveillance data and the daily clinical COVID-19 case numbers has been frequently conducted, the importance of stationarity of the time-series data has not been well addressed. In this study, we demonstrated that strong yet spurious correlation could arise from non-stationary time-series data in wastewater surveillance, and data prewhitening to remove trends helped to reveal distinct cross-correlation patterns between daily clinical case numbers and daily wastewater SARS-CoV-2 concentration during a lockdown period in 2020 in Honolulu, Hawaii. Normalization of wastewater SARS-CoV-2 concentration by the endogenous fecal viral markers in the same samples significantly improved the cross-correlation, and the best correlation was detected at a two-day lag of the daily clinical case numbers. The detection of a significant correlation between daily wastewater SARS-CoV-2 RNA abundance and clinical case numbers also suggests that disease burden fluctuation in the community should not be excluded as a contributor to the often observed weekly cyclic patterns of clinical cases. Wastewater surveillance represents an emerging water technology with significant human health benefits. The study demonstrated that non-stationary time-series data could lead to spurious correlation, highlighting the need for prewhitening. Normalization strategies could alleviate variations in sample collection and analyses, which is useful for detecting actual underlying relationships between wastewater surveillance data and clinical data.","version":"1.1","doi":"10.1101/2022.12.16.520829","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.17.520843","pub_date":"2022-12-19","title":"Transgenic Mouse Models Establish a Protective Role of Type 1 IFN Response in SARS-CoV-2 infection-related Immunopathology","abstract":"Type 1 interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infections remained to be perplexing. Here, we developed two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1\u2212/\u2212) and the other with dampened IFN-I response (hACE2; Ifnar1\u2212/\u2212) to comprehend the role of IFN-I response during SARS-CoV-2 invasion. We found that hACE2; Irgm1\u2212/\u2212 mice were resistant to lethal SARS-CoV-2 infection with substantially reduced cytokine storm and immunopathology. In striking contrast, a severe SARS-CoV-2 infection along with immune cells infiltration, inflammatory response, and enhanced pathology was observed in the lungs of hACE2; Ifnar1\u2212/\u2212 mice. Additionally, hACE2; Ifnar1\u2212/\u2212 mice were highly susceptible to SARS-CoV-2 neuroinvasion in the brain accompanied by immune cell infiltration, microglia/astrocytes activation, cytokine response, and demyelination of neurons. The hACE2; Irgm1\u2212/\u2212 Ifnar1\u2212/\u2212 double knockout mice or hACE2; Irgm1\u2212/\u2212 mice treated with STING or RIPK2 pharmacological inhibitors displayed loss of the protective phenotypes observed in hACE2; Irgm1\u2212/\u2212 mice suggesting that heightened IFN-I response accounts for the observed immunity. Taken together, we explicitly demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target.","version":"1.1","doi":"10.1101/2022.12.17.520843","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.18.520908","pub_date":"2022-12-19","title":"How SARS-CoV-2 alters the regulation of gene expression in infected cells\u2020","abstract":"Non-structural accessory proteins in viruses play a key role in hijacking the basic cellular mechanisms, which is essential to promote the virus survival and evasion of the immune system. The immonuglobulin-like open reading frame 8 (ORF8) protein expressed by SARS-CoV-2 accumulates in the nucleus and may influence the regulation of the gene expression in infected cells. In this contribution, by using micro-second time-scale all-atom molecular dynamics simulations, we unravel the structural bases behind the epigenetic action of ORF8. In particular, we highlight how the protein is able to form stable aggregates with DNA through a histone tail-like motif, and how this interaction is influenced by post-translational modifications, such as acetylation and methylation, which are known epigenetic markers in histones. Our work not only clarifies the molecular mechanisms behind the perturbation of the epigenetic regulation caused by the viral infection, but also offers an unusual perspective which may foster the development of original antivirals.","version":"1.1","doi":"10.1101/2022.12.18.520908","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.17.520865","pub_date":"2022-12-19","title":"A versatile and interoperable computational framework for the analysis and modeling of COVID-19 disease mechanisms","abstract":"The COVID-19 Disease Map project is a large-scale community effort uniting 277 scientists from 130 Institutions around the globe. We use high-quality, mechanistic content describing SARS-CoV-2-host interactions and develop interoperable bioinformatic pipelines for novel target identification and drug repurposing. Community-driven and highly interdisciplinary, the project is collaborative and supports community standards, open access, and the FAIR data principles. The coordination of community work allowed for an impressive step forward in building interfaces between Systems Biology tools and platforms. Our framework links key molecules highlighted from broad omics data analysis and computational modeling to dysregulated pathways in a cell-, tissue- or patient-specific manner. We also employ text mining and AI-assisted analysis to identify potential drugs and drug targets and use topological analysis to reveal interesting structural features of the map. The proposed framework is versatile and expandable, offering a significant upgrade in the arsenal used to understand virus-host interactions and other complex pathologies.","version":"1.1","doi":"10.1101/2022.12.17.520865","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.18.22283642","pub_date":"2022-12-19","title":"Changes in cancer prevention and management and patient needs during the COVID-19 pandemic: An umbrella review of systematic reviews","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>The COVID-19 pandemic led to relocation and reconstruction of health care resources and systems, and to a decrease in healthcare utilization, and this may have affected the treatment, diagnosis, prognosis, and psychosocial well-being of cancer patients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To summarize and quantify the evidence on the impact of the COVID-19 pandemic on the full spectrum of cancer care.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We performed an umbrella review to summarize and quantify the findings from systematic reviews on impact of the COVID-19 pandemic on cancer treatment modification, delays, and cancellations; delays or cancellations in screening and diagnosis; psychosocial well-being, financial distress, and use of telemedicine as well as on other aspects of cancer care. PubMed was searched for relevant systematic reviews with or without meta-analysis published before November 29\n                    <jats:sup>th</jats:sup>\n                    , 2022. Abstract, full text screening and data extraction were performed by two independent reviewers. AMSTAR-2 was used for critical appraisal of included systematic reviews.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>45 systematic reviews evaluating different aspects of cancer care were included in our analysis. Most reviews were based on observational studies judged to be at medium and high risk of bias. Only 2 of the included reviews had high or moderate scores based on AMSTAR-2. Findings suggest treatment modifications in cancer care during the pandemic versus the pre-pandemic period were based on low level of evidence. Different degrees of delays and cancellations in cancer treatment, screening and diagnosis were observed, with low-and-middle income countries and countries that implemented lockdowns being disproportionally affected. A shift from in-person appointments to telemedicine use was observed, but utility of telemedicine, challenges in implementation and cost-effectiveness in different areas of cancer care were little explored. Evidence was consistent in suggesting psychosocial well-being (e.g., depression, anxiety, and social activities) of cancer patients deteriorated, and cancer patients experienced financial distress, albeit results were in general not compared to pre-pandemic levels. Impact of cancer care disruption during the pandemic on cancer prognosis was little explored.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Substantial but heterogenous impact of COVID-19 pandemic on cancer care has been observed. Evidence gaps exist on this topic, with mid- and long-term impact on cancer care being most uncertain.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.12.18.22283642","journal":"medRxiv","score":null},{"id":"10.1101/2022.12.16.520800","pub_date":"2022-12-19","title":"Comprehensive profiling of wastewater viromes by genomic sequencing","abstract":"Genomic material in wastewater provides a rich source of data for detection and surveillance of microbes. Used for decades to monitor poliovirus and other pathogens, the SARS-CoV-2 pandemic and the falling costs of high-throughput sequencing have substantially boosted the interest in and the usage of wastewater monitoring. We have longitudinally collected over 100 samples from a wastewater treatment plant in Berlin/Germany, from March 2021 to July 2022, in order to investigate three aspects. First, we conducted a full metagenomic analysis and exemplified the depth of the data by temporal tracking strains and to a certain extent also variants of human astroviruses and enteroviruses. Second, targeting respiratory pathogens, a broad enrichment panel enabled us to detect waves of RSV, influenza, or common cold coronaviruses in high agreement with clinical data. Third, by applying a profile Hidden Markov Model-based search for novel viruses, we identified more than 100 thousand novel transcript assemblies likely not belonging to known virus species, thus substantially expanding our knowledge of virus diversity. Taken together, we present a longitudinal and deep investigation of the viral genomic information in wastewater that underlines the value of sewage surveillance for both public health purposes and planetary virome research.","version":"1.1","doi":"10.1101/2022.12.16.520800","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.25.517977","pub_date":"2022-12-16","title":"Plasma after both SARS-CoV-2 boosted vaccination and COVID-19 potently neutralizes BQ.1.1 and XBB.1","abstract":"Recent 2022 SARS-CoV-2 Omicron variants, have acquired resistance to most neutralizing anti-Spike monoclonal antibodies authorized, and the BQ.1.* sublineages are notably resistant to all authorized monoclonal antibodies. Polyclonal antibodies from individuals both vaccinated and recently recovered from Omicron COVID-19 (VaxCCP) could retain new Omicron neutralizing activity. Here we reviewed BQ.1.* virus neutralization data from 920 individual patient samples from 43 separate cohorts defined by boosted vaccinations with or without recent Omicron COVID-19, as well as infection without vaccination. More than 90% of the plasma samples from individuals in the recently (within 6 months) boosted VaxCCP study cohorts neutralized BQ.1.1, and BF.7 with 100% neutralization of WA-1, BA.4/5, BA.4.6 and BA.2.75. The geometric mean of the geometric mean 50% neutralizing titers (GM (GMT50) were 314, 78 and 204 for BQ.1.1, XBB.1 and BF.7, respectively. Compared to VaxCCP, plasma sampled from COVID-19 na\u00efve subjects who also recently within 6 months received at least a third vaccine dose had about half of the GM (GMT50) for all viral variants. Boosted VaxCCP characterized by either recent vaccine dose or infection event within 6 months represents a robust, variant-resilient, passive immunotherapy against the new Omicron BQ.1.1, XBB.1 and BF.7 variants.","version":"1.3","doi":"10.1101/2022.11.25.517977","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.16.520799","pub_date":"2022-12-16","title":"Surface-modified measles vaccines encoding oligomeric, fusion-stabilized SARS-CoV-2 spike glycoproteins bypass measles seropositivity, boosting neutralizing antibody responses to omicron and historical variants","abstract":"Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.","version":"1.1","doi":"10.1101/2022.12.16.520799","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.16.520599","pub_date":"2022-12-16","title":"Cholesterol and ceramide facilitate SARS-CoV-2 Spike protein-mediated membrane fusion","abstract":"SARS-CoV-2 entry into host cells is mediated by the Spike (S) protein of the viral envelope. The S protein is composed of two subunits: S1 that induces binding to the host cell via its interaction with the ACE2 receptor of the cell surface and S2 that triggers fusion between viral and cellular membranes. Fusion by S2 depends on its heptad repeat domains that bring membranes close together, and its fusion peptide (FP) that interacts with and perturb the membrane structure to trigger fusion. Recent studies suggest that cholesterol and ceramide lipids from the cell surface may facilitate SARS-CoV-2 entry into host cells, but their exact mode of action remains unknown. We have used a combination of in vitro liposome-liposome and in situ cell-cell fusion assays to study the lipid determinants of S-mediated membrane fusion. We found that cholesterol and ceramide both facilitated fusion, suggesting that targeting lipids could be effective against SARS-CoV-2. As proof of concept, we examined the effect of chlorpromazine (CPZ), an antipsychotic drug known to perturb membrane structure. We found that CPZ inhibited S-mediated membrane fusion and thus potentially SARS-CoV-2 entry.","version":"1.1","doi":"10.1101/2022.12.16.520599","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.16.520794","pub_date":"2022-12-16","title":"The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design","abstract":"The main protease of SARS-CoV-2 (Mpro) is an important target for developing COVID-19 therapeutics. Recent work has highlighted Mpro\u2019s susceptibility to undergo redox-associated conformational changes in response to cellular and immune-system-induced oxidation. Despite structural evidence indicating large-scale rearrangements upon oxidation, the mechanisms of conformational change and its functional consequences are poorly understood. Here, we present the crystal structure of a new Mpro point mutant (H163A) that shows an oxidized conformation with the catalytic cysteine in a disulfide bond. We hypothesize that Mpro adopts this conformation under oxidative stress to protect against over-oxidation. Our metadynamics simulations illustrated a potential mechanism by which H163 modulates this transition and suggest that this equilibrium exists in the wild-type enzyme. We show that other point mutations can also significantly shift the equilibrium towards this state by altering conformational free energies. New therapeutic strategies against SARS-CoV-2 can be explored by understanding how H163 modulates this equilibrium.","version":"1.1","doi":"10.1101/2022.12.16.520794","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.01.518149","pub_date":"2022-12-16","title":"SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response","abstract":"Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Moderna\u2019s mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (m\u03a8), is terminated by a long poly(A) tail (~100 nucleotides) followed by an m\u03a8Cm\u03a8AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of m\u03a8Cm\u03a8AG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after m\u03a8Cm\u03a8AG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after m\u03a8Cm\u03a8AG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.","version":"1.2","doi":"10.1101/2022.12.01.518149","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.15.520569","pub_date":"2022-12-15","title":"Progressive loss of conserved spike protein neutralizing antibody sites in Omicron sublineages is balanced by preserved T-cell recognition epitopes","abstract":"The continued evolution of the SARS-CoV-2 Omicron variant has led to the emergence of numerous sublineages with different patterns of evasion from neutralizing antibodies. We investigated neutralizing activity in immune sera from individuals vaccinated with SARS-CoV-2 wild-type spike (S) glycoprotein-based COVID-19 mRNA vaccines after subsequent breakthrough infection with Omicron BA.1, BA.2, or BA.4/BA.5 to study antibody responses against sublineages of high relevance. We report that exposure of vaccinated individuals to infections with Omicron sublineages, and especially with BA.4/BA.5, results in a boost of Omicron BA.4.6, BF.7, BQ.1.1, and BA.2.75 neutralization, but does not efficiently boost neutralization of sublineages BA.2.75.2 and XBB. Accordingly, we found in in silico analyses that with occurrence of the Omicron lineage a large portion of neutralizing B-cell epitopes were lost, and that in Omicron BA.2.75.2 and XBB less than 12% of the wild-type strain epitopes are conserved. In contrast, HLA class I and class II presented T-cell epitopes in the S glycoprotein were highly conserved across the entire evolution of SARS-CoV-2 including Alpha, Beta, and Delta and Omicron sublineages, suggesting that CD8+ and CD4+ T-cell recognition of Omicron BQ.1.1, BA.2.75.2, and XBB may be largely intact. Our study suggests that while some Omicron sublineages effectively evade B-cell immunity by altering neutralizing antibody epitopes, S protein-specific T-cell immunity, due to the very nature of the polymorphic cell-mediated immune, response is likely to remain unimpacted and may continue to contribute to prevention or limitation of severe COVID-19 manifestation.","version":"1.1","doi":"10.1101/2022.12.15.520569","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.15.520197","pub_date":"2022-12-15","title":"Understanding the mechanism of the SARS CoV-2 coinfection with other respiratory viruses","abstract":"Coinfections have a potential role in increased morbidity and mortality rates during pandemics. Our investigation is aimed at evaluating the viral coinfection prevalence in COVID-19 patients. Rapid diagnostic tests are tools with a paramount impact both on improving patient care. Particularly in the case of respiratory infections, it is of great importance to quickly confirm/exclude the involvement of pathogens. The COVID-19 pandemic has been associated with changes in respiratory virus infections worldwide, which have differed between virus types. In this paper, we systematically searched the percentage of coinfection of various respiratory viruses in COVID-19-positive samples. We included patients of all ages, in all settings. The main outcome was the proportion of patients with viral coinfection. By describing the differences in changes between viral species across different geographies over the course of the COVID-19 pandemic, we may better understand the complex factors involved in the community cocirculation of respiratory viruses.","version":"1.1","doi":"10.1101/2022.12.15.520197","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.13.520303","pub_date":"2022-12-15","title":"Effect of field strength on RF power deposition near conductive leads: A simulation study of SAR in DBS lead models during MRI at 1.5 T - 10.5 T","abstract":"Since the advent of magnetic resonance imaging (MRI) nearly four decades ago, there has been a quest for ever-higher magnetic field strengths. Strong incentives exist to do so, as increasing the magnetic field strength increases the signal-to-noise ratio of images. However, ensuring patient safety becomes more challenging at high and ultrahigh field MRI (i.e., \u22653 T) compared to lower fields. The problem is exacerbated for patients with conductive implants, such as those with deep brain stimulation (DBS) devices, as excessive local heating can occur around implanted lead tips. Despite extensive effort to assess radio frequency (RF) heating of implants during MRI at 1.5 T, a comparative study that systematically examines the effects of field strength and various exposure limits on RF heating is missing. This study aims to perform numerical simulations that systematically compare RF power deposition near DBS lead models during MRI at common clinical and ultra-high field strengths, namely 1.5, 3, 7, and 10.5 T. Furthermore, we assess the effects of different exposure constraints on RF power deposition by imposing limits on either the B1+ or global head specific absorption rate (SAR) as these two exposure limits commonly appear in MRI guidelines. We created 33 unique DBS lead models based on postoperative computed tomography (CT) images of patients with implanted DBS devices and performed electromagnetic simulations to evaluate the SAR of RF energy in the tissue surrounding lead tips during RF exposure at frequencies ranging from 64 MHz (1.5 T) to 447 MHz (10.5 T). The RF exposure was implemented via realistic MRI RF coil models created based on physical prototypes built in our institutions. We systematically examined the distribution of local SAR at different frequencies with the input coil power adjusted to either limit the B1+ or the global head SAR. The MRI RF coils at higher resonant frequencies generated lower SARs around the lead tips when the global head SAR was constrained. The trend was reversed when the constraint was imposed on B1+. At higher static fields, MRI is not necessarily more dangerous than at lower fields for patients with conductive leads. Specifically, when a conservative safety criterion, such as constraints on the global SAR, is imposed, coils at a higher resonant frequency tend to generate a lower local SAR around implanted leads due to the decreased B1+ and, by proxy, E field levels.","version":"1.1","doi":"10.1101/2022.12.13.520303","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520124","pub_date":"2022-12-14","title":"Trivalent SARS-CoV-2 S1 Subunit Protein Vaccination Induces Broad Humoral Responses in BALB/c Mice","abstract":"This paper presents a novel approach for improving the efficacy of COVID-19 vaccines against emergent SARS-CoV-2 variants. We have evaluated the immunogenicity of unadjuvanted wild-type (WU S1-RS09cg) and variant-specific (Delta S1-RS09cg and OM S1-RS09cg) S1 subunit protein vaccines delivered either as a monovalent or a trivalent antigen in BALB/c mice. Our results show that a trivalent approach induced a broader humoral response with more coverage against antigenically distinct variants, especially when compared to monovalent Omicron-specific S1. This trivalent approach was also found to have increased or equivalent ACE2 binding inhibition, and increased S1 IgG endpoint titer at early timepoints, against SARS-CoV-2 spike variants when compared monovalent Wuhan, Delta, or Omicron S1. Our results demonstrate the utility of protein subunit vaccines against COVID-19 and provide insights into the impact of variant-specific COVID-19 vaccine approaches on the immune response in the current SARS-CoV-2 variant landscape. Particularly, our study provides insight into effects of further increasing valency of currently approved SARS-CoV-2 vaccines, a promising approach for improving protection to curtail emerging viral variants.","version":"1.1","doi":"10.1101/2022.12.12.520124","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.27.514054","pub_date":"2022-12-14","title":"Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model","abstract":"Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the \u201cgold-standard\u201d preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens. Despite the availability of efficacious COVID vaccines that reduce disease severity, SARS-CoV-2 continues to spread. To limit SARS-CoV-2 transmission, the next generation of vaccines must induce immunity in the mucosa of the upper respiratory tract. Therefore, we performed proof-of-principle, unadjuvanted intranasal vaccination studies with a recombinant protein nanoparticle scaffold, SpyCage, decorated with the receptor-binding domain (RBD) of the S protein (SpyCage+RBD). We show that SpyCage+RBD was immunogenic and enhanced SARS-CoV-2 clearance from the nose and lungs of Syrian hamsters. Moreover, covalent grafting of the RBD to the scaffold was required to induce an immune response when given via the intranasal route. These proof-of-concept findings indicate that with further enhancements to immunogenicity (e.g., adjuvant incorporation, antigen optimization), the SpyCage scaffold has potential as a versatile, intranasal vaccine platform for respiratory pathogens.","version":"1.2","doi":"10.1101/2022.10.27.514054","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.22.504819","pub_date":"2022-12-14","title":"Direction mutation pressure of SARS-CoV-2 helps to understand the past and predict the future evolution: C>U and G>U biased mutagenesis forces the majority of amino-acid substitutions to be from CG-rich losers to U-rich gainers","abstract":"Evolution is a function of mutagenesis and selection. To analyse the role of mutagenesis on the structure of the SARS-CoV-2 genome, we reconstructed the mutational spectrum, which was highly C>U and G>U biased. This bias forces the SARS-CoV-2 genome to become increasingly U-rich unless selection cancels it. We analysed the consequences of this bias on the composition of the most neutral (four-fold degenerate synonymous substitutions) and the least neutral positions (nonsynonymous substitutions). The neutral nucleotide composition is already highly saturated by U and, according to our model, it is at equilibrium, suggesting that in the future, we don\u2019t expect any more increase in U. However, nonsynonymous changes continue slowly evolve towards equilibrium substituting CG-rich amino-acids (\u201closers\u201d) with U-rich ones (\u201cgainers\u201d). This process is universal for all genes of SARS-CoV-2 as well as for other coronaviridae species. In line with the direction mutation pressure hypothesis, we show that viral-specific amino acid content is associated with the viral-specific mutational spectrum due to the accumulation of effectively neutral slightly deleterious variants (losers to gainers) during the molecular evolution. The tuning of a protein space by the mutational process is expected to be typical for species with relaxed purifying selection, suggesting that the purging of slightly-deleterious variants in the SARS-CoV-2 population is not very effective, probably due to the fast expansion of the viral population during the pandemic. Understanding the mutational process can help to design more robust vaccines, based on gainer-rich motifs, close to the mutation-selection equilibrium.","version":"1.2","doi":"10.1101/2022.08.22.504819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.14.520006","pub_date":"2022-12-14","title":"2-Thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes significant morbidity and mortality worldwide, seriously impacting not only human health but also the global economy. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these and many other viruses have been approved, they continue to be responsible for large-scale epidemics and global pandemics. Thus, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral nucleoside analogue that exhibited antiviral activity against SARS-CoV-2 and its variants of concern, including the Delta and Omicron variants, as well as a number of other positive-sense single-stranded RNA (ssRNA+) viruses, including DENV. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with SARS-CoV-2 or DENV in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against SARS-CoV-2 and DENV but other ssRNA+ viruses.","version":"1.1","doi":"10.1101/2022.12.14.520006","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.14.520265","pub_date":"2022-12-14","title":"COVID-19 Associated Pulmonary Aspergillosis isolates are genomically diverse but similar to each other in their responses to infection-relevant stresses","abstract":"Secondary infections caused by the pulmonary fungal pathogen Aspergillus fumigatus are a significant cause of mortality in patients with severe Coronavirus Disease 19 (COVID-19). Even though epithelial cell damage and aberrant cytokine responses have been linked with susceptibility to COVID-19 associated pulmonary aspergillosis (CAPA), little is known about the mechanisms underpinning co-pathogenicity. Here, we analysed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centres from different European countries. CAPA isolates did not cluster based on geographic origin in a genome-scale phylogeny of representative A. fumigatus isolates. Phenotypically, CAPA isolates were more similar to the A. fumigatus A1160 reference strain than to the Af293 strain when grown in infection-relevant stresses; except for interactions with human immune cells wherein macrophage responses were similar to those induced by the Af293 reference strain. Collectively, our data indicates that CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses. A larger number of isolates from CAPA patients should be studied to identify genetic drivers of co-pathogenicity in patients with COVID-19. Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) has been globally reported as a life-threatening complication in some patients with severe COVID-19. Most of these infections are caused by the environmental mould Aspergillus fumigatus which ranks third in the fungal pathogen priority list of the WHO. However, little is known about the molecular epidemiology of Aspergillus fumigatus CAPA strains. Here, we analysed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centres from different European countries and, carried out phenotypic analyses with a view to understand the pathophysiology of the disease. Our data indicates that A. fumigatus CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses.","version":"1.1","doi":"10.1101/2022.12.14.520265","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520032","pub_date":"2022-12-13","title":"Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters","abstract":"The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2. SARS-CoV-2 infects and causes disease in all age groups. While injectable SARS-CoV-2 vaccines are effective against severe COVID-19, they do not fully prevent SARS-CoV-2 replication and transmission. This study describes the preclinical comparison in hamsters of B/HPIV3/S-2P and B/HPIV3/S-6P, live-attenuated pediatric vector vaccine candidates expressing the \u201c2P\u201d prefusion stabilized version of the SARS-CoV-2 spike protein, or the further-stabilized \u201c6P\u201d version. B/HPIV3/S-6P induced significantly stronger anti-S serum IgA and IgG responses than B/HPIV3/S-2P. A single intranasal immunization with B/HPIV3/S-6P elicited broad systemic antibody responses in hamsters that efficiently neutralized the vaccine-matched isolate as well as variants of concern, including Omicron. B/HPIV3/S-6P immunization induced near-complete airway protection against the vaccine-matched SARS-CoV-2 isolate as well as two variants. Furthermore, following SARS-CoV-2 challenge, immunized hamsters exhibited strong anamnestic serum antibody responses. Based on these data, B/HPIV3/S-6P will be further evaluated in a phase I study.","version":"1.1","doi":"10.1101/2022.12.12.520032","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.13.520307","pub_date":"2022-12-13","title":"Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants","abstract":"Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of enveloped viruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 \u03bcM). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 \u03bcM), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner. Despite the success of the vaccines against SARS-CoV-2, new variants of the virus are still emerging and spreading, underlining the need for additional effective antiviral countermeasures. An interesting antiviral target for enveloped viruses is the fusion of the viral envelope with host cell membranes, a crucial early step in the life cycle of coronaviruses like SARS-CoV-2. Here, we present a sensitive impedance-based method to monitor in real-time cell-cell fusion elicited by the SARS-CoV-2 spike protein. With this technique we can profile entry inhibitors and determine the inhibitory potential of fusion inhibitors for SARS-CoV-2. In addition, with cellular electrical impedance we can evaluate the fusogenic properties of new emerging SARS-CoV-2 variants. Overall, the impedance technology adds valuable information on the fusion process of circulating coronaviruses and helps unravel the mode of action of new antivirals, opening new avenues for the development of next generation fusion inhibitors with improved antiviral activity.","version":"1.1","doi":"10.1101/2022.12.13.520307","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520172","pub_date":"2022-12-13","title":"Fortuitous Somatic Mutations during Antibody Evolution Endow Broad Neutralization against SARS-CoV-2 Omicron Variants","abstract":"Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.","version":"1.1","doi":"10.1101/2022.12.12.520172","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520021","pub_date":"2022-12-13","title":"SARS-CoV-2 vaccine-breakthrough infections (VBIs) by Omicron (B.1.1.529) variant and consequences in structural and functional impact","abstract":"This study investigated the efficacy of existing vaccinations against hospitalization and infection due to the Omicron variant of COVID-19, particularly for those who received two doses of Moderna or Pfizer vaccines and one dose of a vaccine by Johnson & Johnson or who were vaccinated more than five months previously. A total of 36 variants in Omicron\u2019s spike protein, targeted by all three vaccinations, have made antibodies less effective at neutralizing the virus. Genotyping of SARS-CoV-2 viral sequencing revealed clinically significant variants such as E484K in three genetic mutations (T95I, D614G, and del142-144). One woman displayed two of these mutations, indicating a potential risk of infection following successful immunization, as recently reported by Hacisuleyman (2021). We examined the effects of mutations on domains (NID, RBM, and SD2) found at the interfaces of spike domains Omicron B.1.1529, Delta/B.1.1529, Alpha/B.1.1.7, VUM B.1.526, B.1.575.2, and B.1.1214 (formerly VOI Iota). We tested the affinity of Omicron for hACE2 and found that the wild and mutant spike proteins were using atomistic molecular dynamics simulations. According to binding free energies calculated during mutagenesis, hACE2 bound Omicron spike more strongly than SARS-CoV-2 wild strain. T95I, D614G, and E484K are three substitutions that significantly contribute to the RBD, corresponding to hACE2 binding energies and a doubling of Omicron spike proteins\u2019 electrostatic potential. Omicron appears to bind hACE2 with greater affinity, increasing its infectivity and transmissibility. The spike virus was designed to strengthen antibody immune evasion through binding while boosting receptor binding by enhancing IgG and IgM antibodies that stimulate human \u03b2-cell, as opposed to the wild strain, which has more vital stimulation of both antibodies.","version":"1.1","doi":"10.1101/2022.12.12.520021","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.12.520110","pub_date":"2022-12-13","title":"Long COVID: G Protein-Coupled Receptors (GPCRs) responsible for persistent post-COVID symptoms","abstract":"As of early December 2022, COVID-19 had a significant impact on the lives of people all around the world, with over 630 million documented cases and over 6 million deaths. A recent clinical analysis revealed that under certain conditions, a patient\u2019s disease symptoms are more likely to persist. Long COVID is characterised by many symptoms that continue long after the SARS-CoV-2 infection has resolved. This work utilised computational methods to analyse the persistence of COVID symptoms after recovery and to identify the relevant genes. Based on functional similarity, differentially expressed genes (DEGs) of SARS-CoV-2 infection and 255 symptoms of long covid were examined, and potential genes were identified based on the rank of functional similarity. Then, hub genes were identified by analysing the interactions between proteins. Using the identified key genes and the drug-gene interaction score, FDA drugs with potential for possible alternatives were identified. Also discovered were the gene ontology and pathways for 255 distinct symptoms. A website (https://longcovid.omicstutorials.com/) with a list of significant genes identified as biomarkers and potential treatments for each symptom was created. All of the hub genes associated with the symptoms, GNGT1, GNG12, GNB3, GNB4, GNG13, GNG8, GNG3, GNG7, GNG10, and GNAI1, were discovered to be associated with G-protein coupled receptors. This demonstrates that persistent COVID infection affects various organ systems and promotes chronic inflammation following infection. CTLA4, PTPN22, KIT, KRAS, NF1, RET, and CTNNB1 were identified as the common genes that regulate T-cell immunity via GPCR and cause a variety of symptoms, including autoimmunity, cardiovascular, dermatological, general symptoms, gastrointestinal, pulmonary, reproductive, genitourinary, and endocrine symptoms (RGEM). Among other functions, they were found to be involved in the positive regulation of protein localization to the cell cortex, the regulation of triglyceride metabolism, the binding of G protein-coupled receptors, the binding of G protein-coupled serotonin receptors, the heterotrimeric G-protein complex, and the cell cortex region. These biomarker data, together with the gene ontology and pathway information that accompanies them, are intended to aid in determining the cause and improving the efficacy of treatment.","version":"1.1","doi":"10.1101/2022.12.12.520110","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488678","pub_date":"2022-12-13","title":"Read2Tree: scalable and accurate phylogenetic trees from raw reads","abstract":"The inference of phylogenetic trees is foundational to biology. However, state-of-the-art phylogenomics requires running complex pipelines, at significant computational and labour costs, with additional constraints in sequencing coverage, assembly and annotation quality. To overcome these challenges, we present Read2Tree, which directly processes raw sequencing reads into groups of corresponding genes. In a benchmark encompassing a broad variety of datasets, our assembly-free approach was 10-100x faster than conventional approaches, and in most cases more accurate\u2014the exception being when sequencing coverage was high and reference species very distant. To illustrate the broad applicability of the tool, we reconstructed a yeast tree of life of 435 species spanning 590 million years of evolution. Applied to Coronaviridae samples, Read2Tree accurately classified highly diverse animal samples and near-identical SARS-CoV-2 sequences on a single tree\u2014thereby exhibiting remarkable breadth and depth. The speed, accuracy, and versatility of Read2Tree enables comparative genomics at scale.","version":"1.2","doi":"10.1101/2022.04.18.488678","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.11.519990","pub_date":"2022-12-12","title":"Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep","abstract":"Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases such as COVID-19. Enhancing the efficacy of vaccine antigens to induce robust and specific antibody responses remains central to developing highly effective formulations. The direct fusion of immunoglobulin (IgG) Fc domains or other immune-stimulating proteins to antigens has shown promise in several mammalian species but has not yet been tested and optimized in commercially-relevant ruminant species. Here we show that the immunization of sheep with fusions of the receptor binding domain (RBD) of SARS-CoV-2 to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population was shown to contain elevated levels of neutralizing antibodies that suppress binding between the RBD and soluble hACE2 receptors in vitro. The parallel evaluation of a second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance at the group level. Furthermore, we demonstrate that the antibodies induced by these fusion antigens are transferred from maternal serum into colostrum/milk. These antibodies also demonstrate cross-neutralizing activity against diverse SARS-CoV-2 variants including delta and omicron. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.","version":"1.1","doi":"10.1101/2022.12.11.519990","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.10.517707","pub_date":"2022-12-12","title":"Visualization of early RNA replication kinetics of SARS-CoV-2 by using single molecule RNA-FISH","abstract":"SARS-CoV-2 infection has caused a major global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of plus stranded genomic and subgenomic RNAs (gRNA and sgRNA) starting from the RNA from the first virus that enters the cell. We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA in infected cells and carried out a time course analysis to determine the kinetics of their replication. We visualized the single molecules of gRNA within the cytoplasm of infected cells 30 minutes post-infection and detected the co-expression of gRNA and sgRNA within two hours post-infection. Furthermore, we observed the formation of a replication organelle (RO) from a single RNA, which led to the formation of multiple ROs within the same cells. Single molecule analysis indicated that while gRNA resided in the center of these ROs, the sgRNAs were found to radiate and migrate out of these structures. Our results also indicated that after the initial delay, there was a rapid but asynchronous replication, and the gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post-infection forming multiple ROs that filled the entire cytoplasm. These results provide insight into the kinetics of early post-entry events of SARS-CoV-2 and the formation of RO, which will help to understand the molecular events associated with viral infection and facilitate the identification of new therapeutic targets that can curb the virus at a very early stage of replication to combat COVID-19. SARS-CoV-2 infection continues to be a global burden. Soon after the entry, SARS-CoV-2 replicates by an elaborate process, producing genomic and subgenomic RNAs (gRNA and sgRNAs) within specialized structures called replication organelles (RO). Many questions including the timing of multiplication of gRNA and sgRNA, the generation, subcellular localization, and function of the ROs, and the mechanism of vRNA synthesis within ROs is not completely understood. Here, we have developed probes and methods to simultaneously detect the viral gRNA and a sgRNA at single cell single molecule resolution and have employed a method to scan thousands of cells to visualize the early kinetics of gRNA and sgRNA synthesis soon after the viral entry into the cell. Our results reveal that the replication is asynchronous and ROs are rapidly formed from a single RNA that enters the cell within 2 hours, which multiply to fill the entire cell cytoplasm within ~4 hours after infection. Furthermore, our studies provide a first glimpse of the gRNA and sgRNA synthesis within ROs at single molecule resolution. Our studies may facilitate the development of drugs that inhibit the virus at the earliest possible stages of replication to minimize the pathogenic impact of viral infection.","version":"1.1","doi":"10.1101/2022.12.10.517707","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.10.518819","pub_date":"2022-12-12","title":"Identification of genomic signatures and multiple lineage markers from the second and third wave samples of COVID-19 in Western Rajasthan, India","abstract":"Most of the mutations occurred in SARS-CoV-2 are either relatively neutral or swiftly purged. However, some mutations have altered the functional aspects in terms of infectivity and transmission, host-viral interactions, disease severity and immune or vaccine escape. There are emerging evidence that certain mutations are jeopardizing the immune based therapies. The present research report is focused on the identification of genomic signatures of SARS-CoV-2 variant that caused mortality during second and third wave of COVID-19 in Western Rajasthan, India. We identified that Delta clade of SARS-CoV-2 is the predominant cause of mortality during second wave and even third wave in Western Rajasthan, India. Importantly, this study also revealed the unique and common substitution mutations within the spike domain, those are present in mortality and survived persons during the second and third wave of COVID-19 in India. In addition, this study also revealed the multiple lineage markers (Delta and Omicron), that would update with insightful understanding in the clade development of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.12.10.518819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.11.520008","pub_date":"2022-12-12","title":"Using machine learning to detect coronaviruses potentially infectious to humans","abstract":"Establishing the host range for novel viruses remains a challenge. Here, we address the challenge of identifying non-human animal coronaviruses that may infect humans by creating an artificial neural network model that learns from the binding of the spike protein of alpha and beta coronaviruses to their host receptor. The proposed method produces a human-Binding Potential (h-BiP) score that distinguishes, with high accuracy, the binding potential among human coronaviruses. Two viruses, previously unknown to bind human receptors, were identified: Bat coronavirus BtCoV/133/2005 (a MERS related virus) and Rhinolophus affinis coronavirus isolate LYRa3 a SARS related virus. We further analyze the binding properties of these viruses using molecular dynamics. To test whether this model can be used for surveillance of novel coronaviruses, we re-trained the model on a set that excludes SARS-COV-2 viral sequences. The results predict the binding of SARS-CoV-2 with a human receptor, indicating that machine learning methods are an excellent tool for the prediction of host expansion events.","version":"1.1","doi":"10.1101/2022.12.11.520008","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.18.517156","pub_date":"2022-12-10","title":"SARS-CoV-2 exposure in Norway rats (Rattus norvegicus) from New York City","abstract":"Millions of Norway rats (Rattus norvegicus)inhabit New York City (NYC), presenting the potential for transmission of SARS-CoV-2 from humans to rats and other wildlife. We evaluated SARS-CoV-2 exposure among 79 rats captured from NYC during the fall of 2021. Results showed that 13 of 79 rats (16.5%) tested IgG or IgM positive, and partial genomes of SARS-CoV-2 were recovered from four rats that were qRT-PCR positive. Using a virus challenge study, we also showed that Alpha, Delta, and Omicron variants can cause robust infections in wild-type Sprague Dawley (SD) rats, including high level replications in the upper and lower respiratory tracts and induction of both innate and adaptive immune responses. Additionally, the Delta variant resulted in the highest infectivity. In summary, our results indicated that rats are susceptible to infection with Alpha, Delta, and Omicron variants, and rats in the NYC municipal sewer systems have been exposed to SARS-CoV-2. Our findings highlight the potential risk of secondary zoonotic transmission from urban rats and the need for further monitoring of SARS-CoV-2 in those populations. Since its emergence causing the COVID-19 pandemic, the host tropism expansion of SARS-CoV-2 raises a potential risk for reverse-zoonotic transmission of emerging variants into rodent species, including wild rat species. In this study, we presented both genetic and serological evidence for SARS-CoV-2 exposure in wild rat population from New York City, and these viruses are potentially linked to the viruses during the early stages of the pandemic. We also demonstrated that rats are susceptible to additional variants (i.e., Alpha, Delta, and Omicron) predominant in humans and that the susceptibility to different variants vary. Our findings highlight the potential risk of secondary zoonotic transmission from urban rats and the need for further monitoring of SARS-CoV-2 in those populations.","version":"1.2","doi":"10.1101/2022.11.18.517156","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.09.519765","pub_date":"2022-12-09","title":"TMPRSS2 is essential for SARS-CoV-2 Beta and Omicron infection","abstract":"The COVID-19 pandemic remains a global health threat and novel antiviral strategies are urgently needed. SARS-CoV-2 employs the cellular serine protease TMPRSS2 for entry into lung cells and TMPRSS2 inhibitors are being developed for COVID-19 therapy. However, the SARS-CoV-2 Omicron variant, which currently dominates the pandemic, prefers the endo/lysosomal cysteine protease cathepsin L over TMPRSS2 for cell entry, raising doubts whether TMPRSS2 inhibitors would be suitable for treatment of patients infected with the Omicron variant. Nevertheless, the contribution of TMPRSS2 to spread of SARS-CoV-2 in the infected host is largely unclear. Here, we show that loss of TMPRSS2 strongly reduced the replication of the Beta variant in nose, trachea and lung of C57BL mice and protected the animals from weight loss and disease. Infection of mice with the Omicron variant did not cause disease, as expected, but again TMPRSS2 was essential for efficient viral spread in the upper and lower respiratory tract. These results identify a key role of TMPRSS2 in SARS-CoV-2 Beta and Omicron infection and highlight TMPRSS2 as an attractive target for antiviral intervention.","version":"1.1","doi":"10.1101/2022.12.09.519765","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.08.519593","pub_date":"2022-12-09","title":"Global loss of cellular m6A RNA methylation following infection with different SARS-CoV-2 variants","abstract":"Host-viral interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N6-methyladenosine modification (m6A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during a stress response. Gene expression profiles observed post-infection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m6A readers and erasers. We found that infection with SARS-CoV-2 variants caused a loss of m6A in cellular RNAs, whereas m6A was detected abundantly in viral RNA. METTL3, the m6A methyltransferase, showed an unusual cytoplasmic localization post-infection. The B.1.351 variant had a less pronounced effect on METTL3 localization and loss of m6A than the B.1 and B.1.1.7 variants. We also observed a loss of m6A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m6A loss is characteristic of SARS-CoV-2 infected cells. Further, transcripts with m6A modification were preferentially down-regulated post-infection. Inhibition of the export protein XPO1 resulted in the restoration of METTL3 localization, recovery of m6A on cellular RNA, and increased mRNA expression. Stress granule formation, which was compromised by SARS-CoV-2 infection, was restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m6A-dependent manner.","version":"1.1","doi":"10.1101/2022.12.08.519593","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.08.519672","pub_date":"2022-12-09","title":"wastewaterSPAdes: SARS-CoV-2 strain deconvolution using SPAdes toolkit","abstract":"SARS-CoV-2 wastewater samples are extensively collected and studied because it allows quantitatively assess a viral load in surrounding populations. Additionally, SARS-CoV-2 strain deconvolution gives more insights into pandemic dynamics, and the uprising of new strains. Usually, the solution to the strain deconvolution problem starts with read alignment of wastewater short read sequencing data to the SARS-CoV-2 reference genome. After variants are called and their abundances are estimated, a reference database is used to assign variants to strains, select a subset of strains, and infer relative abundance of these strains based on some mathematical model. Assembly-based methods have its own strengths, but currently reside in the shadow of alignment-based methods. In this paper we propose a new assembly-based approach based on SPAdes toolkit codebase \u2013\u2013\u2013 wastewaterSPAdes, that is able to deconvolve wastewater without a need of read alignment. Our results show that watewaterSPAdes is able to accurately identify strains presented in a sample, and correctly estimate abundances for most of the samples. https://cab.spbu.ru/software/wastewaterspades/ a.korobeynikov@spbu.ru Supplementary data are available at Bioinformatics","version":"1.1","doi":"10.1101/2022.12.08.519672","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.07.519508","pub_date":"2022-12-08","title":"Mild SARS-CoV-2 infection results in long-lasting microbiota instability","abstract":"Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of SARS-CoV-2 infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2 positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the United States), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. Taken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2 it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.","version":"1.1","doi":"10.1101/2022.12.07.519508","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.11.507506","pub_date":"2022-12-08","title":"Broad host tropism of ACE2-using MERS-related coronaviruses and determinants restricting viral recognition","abstract":"Phylogenetically distant coronaviruses have evolved to use ACE2 as their common receptors, including NL63 and many Severe acute respiratory syndrome (SARS) coronavirus-related viruses. We recently reported two Middle East respiratory syndrome coronavirus (MERS-CoV) closely related bat merbecoviruses, NeoCoV and PDF-2180, use Angiotensin-converting enzyme 2 (ACE2) for entry. However, their host range and cross-species transmissibility remain unknown. Here, we characterized their species-specific receptor preference by testing ACE2 orthologs from 49 bats and 53 non-bat mammals. Both viruses exhibited broad receptor recognition spectra and are unable to use ACE2 orthologs from 24 species, mainly Yinpterochiropteran bats. Comparative analyses of bat ACE2 orthologs underscored four crucial host range determinants, all confirmed by subsequent functional assays in human and bat cells. Among them, residue 305, participating in a critical interaction, plays a crucial role in host tropism determination. NeoCoV-T510F, a mutation that enhances human ACE2 recognition, further expanded the potential host range via tighter interaction with an evolutionary conserved hydrophobic pocket. Our results elucidated the molecular basis for the species-specific ACE2 usage of MERS-related viruses across mammals and shed light on their zoonotic risks.","version":"1.2","doi":"10.1101/2022.09.11.507506","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.07.519460","pub_date":"2022-12-07","title":"Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance","abstract":"Obesity and Type 2 Diabetes Mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including COVID-19. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used SARS-CoV-2 mRNA vaccines. To establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. We utilized a murine model of diet-induced obesity and insulin resistance to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet (ND), HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in ND mice but not in HFD mice. We demonstrate impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases. Obesity and type 2 diabetes impair SARS-CoV-2 mRNA vaccine efficacy in a murine model.","version":"1.1","doi":"10.1101/2022.12.07.519460","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477133","pub_date":"2022-12-07","title":"Outbreak.info Research Library: A standardized, searchable platform to discover and explore COVID-19 resources","abstract":"To combat the ongoing COVID-19 pandemic, scientists have been conducting research at breakneck speeds, producing over 52,000 peer-reviewed articles within the first year. To address the challenge in tracking the vast amount of new research located in separate repositories, we developed outbreak.info Research Library, a standardized, searchable interface of COVID-19 and SARS-CoV-2 resources. Unifying metadata from sixteen repositories, we assembled a collection of over 350,000 publications, clinical trials, datasets, protocols, and other resources as of October 2022. We used a rigorous schema to enforce consistency across different sources and resource types and linked related resources. Researchers can quickly search the latest research across data repositories, regardless of resource type or repository location, via a search interface, public API, and R package. Finally, we discuss the challenges inherent in combining metadata from scattered and heterogeneous resources and provide recommendations to streamline this process to aid scientific research.","version":"1.5","doi":"10.1101/2022.01.20.477133","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.04.519037","pub_date":"2022-12-06","title":"Genome charaterization based on the Spike-614 and NS8-84 loci of SARS-CoV-2 reveals two major onsets of the COVID-19 pandemic","abstract":"The global COVID-19 pandemic has lasted for three years since its outbreak, however its origin is still unknown. Here, we analyzed the genotypes of 3.14 million SARS-CoV-2 genomes based on the amino acid 614 of the Spke (S) and the amino acid 48 of NS8 (nonstructural protein 8), and identified 16 linkage haplotypes. The GL haplotype (S_614G and NS8_48L) was the major haplotype driving the global pandemic and accounted for 99.2% of the sequenced genomes, while the DL haplotype (S_614D and NS8_48L) caused the pandemic in China in the spring of 2020 and accounted for approximately 60% of the genomes in China and 0.45% of the global genomes. The GS (S_614G and NS8_48S), DS (S_614D and NS8_48S) and NS (S_614N and NS8_48S) haplotypes accounted for 0.26%, 0.06%, and 0.0067% of the genomes, respectively. The main evolutionary trajectory of SARS-CoV-2 is DS\u2192DL\u2192GL, whereas the other haplotypes are minor byproducts in the evolution. Surprisingly, the newest haplotype GL had the oldest time of most recent common ancestor (tMRCA), which was May 1 2019 by mean, while the oldest haplotype had the newest tMRCA with a mean of October 17, indicating that the ancestral strains that gave birth to GL had been extinct and replaced by the more adapted newcomer at the place of its origin, just like the sequential rise and fall of the delta and omicron variants. However, they arrived and evolved into toxic strains and ignited a pandemic in China where the GL strains did not exist at the end of 2019. The GL strains had spread all over the world before they were discovered, and ignited the global pandemic, which had not been noticed until the pandemic was declared in China. However, the GL haplotype had little influence in China during the early phase of the pandemic due to its late arrival as well as the strict transmission controls in China. Therefore, we propose two major onsets of the COVID-19 pandemic, one was mainly driven by the haplotype DL in China, the other was driven by the haplotype GL globally.","version":"1.1","doi":"10.1101/2022.12.04.519037","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.05.519032","pub_date":"2022-12-06","title":"Superiority of intranasal over systemic administration of bioengineered soluble ACE2 for survival and brain protection against SARS-CoV-2 infection","abstract":"The present study was designed to investigate the effects of a soluble ACE2 protein termed ACE2 618-DDC-ABD, bioengineered to have long duration of action and high binding affinity to SARS-CoV-2, when administered either intranasally (IN) or intraperitoneally (IP) and before or after SARS-CoV-2 inoculation. K18hACE2 mice permissive for SARS-CoV-2 infection were inoculated with 2\u00d7104 PFU wildtype SARS-CoV-2. In one protocol, ACE2 618-DDC-ABD was given either IN or IP, pre- and post-viral inoculation. In a second protocol, ACE2 618-DDC-ABD was given either IN, IP or IN+IP but only post-viral inoculation. In addition, A549 and Vero E6 cells were used to test neutralization of SARS-CoV-2 variants by ACE2 618-DDC-ABD at different concentrations. Survival by day 5 was 0% in infected untreated mice, and 40% in mice from the ACE2 618-DDC-ABD IP-pre treated group. By contrast, in the IN-pre group survival was 90%, histopathology of brain and kidney was essentially normal and markedly improved in the lungs. When ACE2 618-DDC-ABD was administered only post viral inoculation, survival was 30% in the IN+IP group, 20% in the IN and 0% in the IP group. Brain SARS-CoV-2 titers were high in all groups except for the IN-pre group where titers were undetectable in all mice. In cells permissive for SARS-CoV-2 infection, ACE2 618-DDC-ABD neutralized wildtype SARS-CoV-2 at high concentrations, whereas much lower concentrations neutralized omicron BA. 1. We conclude that ACE2 618-DDC-ABD provides much better survival and organ protection when administered intranasally than when given systemically or after viral inoculation and that lowering brain titers is a critical determinant of survival and organ protection.","version":"1.1","doi":"10.1101/2022.12.05.519032","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.05.519191","pub_date":"2022-12-06","title":"A screen for modulation of nucleocapsid protein condensation identifies small molecules with anti-coronavirus activity","abstract":"Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase separated condensates that are hypothesized to play critical roles in viral replication, transcription and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43 and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.","version":"1.1","doi":"10.1101/2022.12.05.519191","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.03.519007","pub_date":"2022-12-05","title":"Increased migratory/activated CD8+ T cell and low avidity SARS-CoV-2 reactive cellular response in post-acute COVID-19 syndrome","abstract":"The role of autoimmunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although clinicians observe a growing population of convalescent COVID-19 patients with manifestation of post-acute sequelae of COVID-19. We analyzed the immune response in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. The phenotyping of lymphocytes showed a significantly higher number of CD8+ T cells expressing the Epstein-Barr virus induced G protein coupled receptor 2, chemokine receptor CXCR3 and C-C chemokine receptor type 5 playing an important role in inflammation and migration in PASC patients compared to controls. Additionally, a stronger, SARS-CoV-2 reactive CD8+ T cell response, characterized by IFN\u03b3 production and predominant TEMRA phenotype but low SARS-CoV-2 avidity was detected in PASC patients compared to controls. Furthermore, higher titers of several autoantibodies were detected among PASC patients. Our data suggest that a persistent inflammatory response triggered by SARS-CoV-2 might be responsible for the observed sequelae in PASC patients. These results may have implications on future therapeutic strategies.","version":"1.1","doi":"10.1101/2022.12.03.519007","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.05.518843","pub_date":"2022-12-05","title":"Convergent evolution in SARS-CoV-2 Spike creates a variant soup that causes new COVID-19 waves","abstract":"The first 2 years of the COVID-19 pandemic were mainly characterized by convergent evolution of mutations of SARS-CoV-2 Spike protein at residues K417, L452, E484, N501 and P681 across different variants of concern (Alpha, Beta, Gamma, and Delta). Since Spring 2022 and the third year of the pandemic, with the advent of Omicron and its sublineages, convergent evolution has led to the observation of different lineages acquiring an additional group of mutations at different amino acid residues, namely R346, K444, N450, N460, F486, F490, Q493, and S494. Mutations at these residues have become increasingly prevalent during Summer and Autumn 2022, with combinations showing increased fitness. The most likely reason for this convergence is the selective pressure exerted by previous infection- or vaccine-elicited immunity. Such accelerated evolution has caused failure of all anti-Spike monoclonal antibodies, including bebtelovimab and cilgavimab. While we are learning how fast coronaviruses can mutate and recombine, we should reconsider opportunities for economically sustainable escape-proof combination therapies, and refocus antibody-mediated therapeutic efforts on polyclonal preparations that are less likely to allow for viral immune escape.","version":"1.1","doi":"10.1101/2022.12.05.518843","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.02.518937","pub_date":"2022-12-05","title":"Deep mutational scanning to predict antibody escape in SARS-CoV-2 Omicron subvariants","abstract":"The major concern of COVID-19 therapeutic monoclonal antibodies is the loss of efficacy to continuously emerging SARS-CoV-2 variants. To predict the antibodies efficacy to the future Omicron subvariants, we conducted deep mutational scanning (DMS) encompassing all single mutations in the receptor binding domain of BA.2 strain. In case of bebtelovimab that preserves neutralization activity against BA.2 and BA.5, broad range of amino acid substitutions at K444, V445 and G446 and some substitutions at P499 and T500 were indicated to achieve the antibody escape. Among currently increasing subvariants, BA2.75 carrying G446S partly and XBB with V445P and BQ.1 with K444T completely evade the neutralization of bebtelovimab, consistent with the DMS results. DMS can comprehensively characterize the antibody escape for efficient and effective management of future variants.","version":"1.1","doi":"10.1101/2022.12.02.518937","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.05.519140","pub_date":"2022-12-05","title":"Cyclic lipopeptides as membrane fusion inhibitors against SARS-CoV-2: new tricks for old dogs","abstract":"With the resurgence of the coronavirus pandemic, the repositioning of FDA-approved drugs against coronovirus and finding alternative strategies for antiviral therapy are both important. We previously identified the viral lipid envelope as a potential target for the prevention and treatment of SARS-CoV-2 infection with plant alkaloids [1]. Here, we investigated the effects of eleven cyclic lipopeptides (CLPs), including well-known antifungal and antibacterial compounds, on the liposome fusion triggered by calcium, polyethylene glycol 8000, and a fragment of SARS-CoV-2 fusion peptide (816-827) by calcein release assays. Differential scanning microcalorimetry of the gel-to-liquid-crystalline and lamellar-to-inverted hexagonal phase transitions and confocal fluorescence microscopy demonstrated the relation of the fusion inhibitory effects of CLPs to alterations in lipid packing, membrane curvature stress and domain organization. The effects of the compounds were evaluated in an in vitro Vero-based cell model, and aculeacin A, anidulafugin, iturin A, and mycosubtilin attenuated the cytopathogenicity of SARS-CoV-2 without specific toxicity.","version":"1.1","doi":"10.1101/2022.12.05.519140","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.03.518963","pub_date":"2022-12-05","title":"Enhanced Protection from SARS-CoV-2 Variants by MVA-Based Vaccines Expressing Matched or Mismatched S Proteins Administered Intranasally to hACE2 Mice","abstract":"The continuous evolution of SARS-CoV-2 strains is contributing to the prolongation of the global pandemic. We previously reported the prevention or more rapid clearance of SARS-CoV-2 from the nasal turbinates and lungs of susceptible K18-hACE2 mice that had been vaccinated intranasally (IN) rather than intramuscularly (IM) with a recombinant MVA (rMVA) expressing a modified S protein of the ancestor SARS-CoV-2 strain. Here, we constructed additional rMVAs and pseudoviruses expressing modified S protein of SARS-CoV-2 variants and compared the ability of vaccines with S proteins that were matched or mismatched to neutralize variants, bind to S proteins and protect K18-hACE2 mice against SARS-CoV-2 challenge. Although vaccines with matched S proteins induced higher neutralizing antibodies, vaccines with mismatched S proteins still protected against severe disease and reduced virus and mRNAs in the lungs and nasal turbinates, though not as well as vaccines with matched S proteins. In mice earlier primed and boosted with rMVA expressing ancestral S, antibodies to the latter increased after one immunization with rMVA expressing Omicron S, but neutralizing antibody to Omicron required a second immunization. Passive transfer of Wuhan immune serum with Omicron S binding but undetectable neutralizing activities reduced infection of the lungs by the variant. Notably, the reduction in infection of the nasal turbinates and lungs was significantly greater when the rMVAs were administered IN rather than IM and this held true for vaccines that were matched or mismatched to the challenge SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.12.03.518963","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.05.519151","pub_date":"2022-12-05","title":"Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane","abstract":"Entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells depends on refolding of the virus-encoded spike protein from a prefusion conformation, metastable after cleavage, to a lower energy, stable postfusion conformation. This transition overcomes kinetic barriers for fusion of viral and target cell membranes. We report here a cryo-EM structure of the intact postfusion spike in a lipid bilayer that represents single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membraneinteracting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.","version":"1.1","doi":"10.1101/2022.12.05.519151","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.03.518949","pub_date":"2022-12-05","title":"High resolution cryo-EM structures of two potently SARS-CoV-2 neutralizing monoclonal antibodies of same donor origin that vary in neutralizing Omicron variants","abstract":"While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency. Two potent human mAbs obtained from a single donor differ binding to Omicron spikes Pattern of binding and conformation of these mAbs bound to full length spike differs Antibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms. Cryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs.","version":"1.1","doi":"10.1101/2022.12.03.518949","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.05.519085","pub_date":"2022-12-05","title":"Convergent evolution of the SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant","abstract":"In late 2022, although the SARS-CoV-2 Omicron subvariants have highly diversified, some lineages have convergently acquired amino acid substitutions at five critical residues in the spike protein. Here, we illuminated the evolutionary rules underlying the convergent evolution of Omicron subvariants and the properties of one of the latest lineages of concern, BQ.1.1. Our phylogenetic and epidemic dynamics analyses suggest that Omicron subvariants independently increased their viral fitness by acquiring the convergent substitutions. Particularly, BQ.1.1, which harbors all five convergent substitutions, shows the highest fitness among the viruses investigated. Neutralization assays show that BQ.1.1 is more resistant to breakthrough BA.2/5 infection sera than BA.5. The BQ.1.1 spike exhibits enhanced binding affinity to human ACE2 receptor and greater fusogenicity than the BA.5 spike. However, the pathogenicity of BQ.1.1 in hamsters is comparable to or even lower than that of BA.5. Our multiscale investigations provide insights into the evolutionary trajectory of Omicron subvariants.","version":"1.1","doi":"10.1101/2022.12.05.519085","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.03.518956","pub_date":"2022-12-05","title":"Bioinformatics techniques for efficient structure prediction of SARS-CoV-2 protein ORF7a via structure prediction approaches","abstract":"Protein is the building block for all organisms. Protein structure prediction is always a complicated task in the field of proteomics. DNA and protein databases can find the primary sequence of the peptide chain and even similar sequences in different proteins. Mainly, there are two methodologies based on the presence or absence of a template for Protein structure prediction. Template-based structure prediction (threading and homology modeling) and Template-free structure prediction (ab initio). Numerous web-based servers that either use templates or do not can help us forecast the structure of proteins. In this current study, ORF7a, a transmembrane protein of the SARS-coronavirus, is predicted using Phyre2, IntFOLD, and Robetta. The protein sequence is straightforwardly entered into the sequence bar on all three web servers. Their findings provided information on the domain, the region with the disorder, the global and local quality score, the predicted structure, and the estimated error plot. Our study presents the structural details of the SARS-CoV protein ORF7a. This immunomodulatory component binds to immune cells and induces severe inflammatory reactions.","version":"1.1","doi":"10.1101/2022.12.03.518956","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.02.22283026","pub_date":"2022-12-05","title":"Healthcare in England was affected by the COVID-19 pandemic across the pancreatic cancer pathway: a cohort study using OpenSAFELY-TPP","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Healthcare across all sectors, in the UK and globally, was negatively affected by the COVID-19 pandemic. We analysed healthcare services delivered to people with pancreatic cancer from January 2015 to March 2023 to investigate the effect of the COVID-19 pandemic.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>With the approval of NHS England, and drawing from a nationally representative OpenSAFELY-TPP dataset of 24 million patients (over 40% of the English population), we undertook a cohort study of people diagnosed with pancreatic cancer. We queried electronic healthcare records for information on the provision of healthcare services across the pancreatic cancer pathway. To estimate the effect of the COVID-19 pandemic, we predicted the rates of healthcare services if the pandemic had not happened. We used generalised linear models (GLM) and the pre-pandemic data from January 2015 to February 2020 to predict rates in March 2020 to March 2023. The 95% confidence intervals of the predicted values were used to estimate the significance of the difference between the predicted and observed rates.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The rate of pancreatic cancer and diabetes diagnoses in the cohort was not affected by the pandemic. There were 26,840 people diagnosed with pancreatic cancer from January 2015 to March 2023. The mean age at diagnosis was 72 (\u00b111 SD), 48% of people were female, 95% were of White ethnicity and 40% were diagnosed with diabetes. We found a reduction in surgical resections by 25% to 28% during the pandemic. In addition, 20%, 10% and 4% fewer people received BMI, HbA1c and liver function tests respectively before they were diagnosed with pancreatic cancer. There was no impact of the pandemic on the number of people making contact with primary care, but the number of contacts increased on average by 1 to 2 per person amongst those who made contact. Reporting of jaundice decreased by 28%, but recovered within twelve months into the pandemic. Emergency department visits, hospital admissions and deaths were not affected.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The pandemic affected healthcare in England across the pancreatic cancer pathway. Positive lessons could be learnt from the services that were resilient and those that recovered quickly. The reductions in healthcare experienced by people with cancer have the potential to lead to worse outcomes. Current efforts should focus on addressing the unmet needs of people with cancer.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This work was jointly funded by the Wellcome Trust (222097/Z/20/Z); MRC (MR/V015757/1, MC_PC-20059, MR/W016729/1); NIHR (NIHR135559, COV-LT2-0073), and Health Data Research UK (HDRUK2021.000, 2021.0157). This work was funded by Medical Research Council (MRC) grant reference MR/W021390/1 as part of the postdoctoral fellowship awarded to AL and undertaken at the Bennett Institute, University of Oxford. The views expressed are those of the authors and not necessarily those of the NIHR, NHS England, UK Health Security Agency (UKHSA) or the Department of Health and Social Care. Funders had no role in the study design, collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.12.02.22283026","journal":"medRxiv","score":null},{"id":"10.1101/2022.11.05.515296","pub_date":"2022-12-03","title":"4\u2019-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses","abstract":"Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4\u2019-Fluorouridine (4\u2019-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus, we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium organoids, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4\u2019-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar potency in organoids. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4\u2019-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4\u2019-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented direct-contact transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (Ca09) with 2 mg/kg 4\u2019-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating an unusually broad window for effective intervention. Therapeutic oral 4\u2019-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pandemic Ca09. Recoverees were fully protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4\u2019-FlU and supports 4\u2019-FlU as developmental candidate for the treatment of seasonal and pandemic influenza. Next-generation antiviral therapeutics are needed to better mitigate seasonal influenza and prepare against zoonotic virus spillover from animal reservoirs. At greatest risk are the immunocompromised and patients infected with highly pathogenic influenza viruses. In this study, we have demonstrated efficacy of a broad-spectrum nucleoside analog, 4\u2019-fluorouridine, against a representative panel of influenza viruses in cell culture, human organoids, and two animal models, ferrets and mice. Acting as an immediate chain terminator of the influenza virus polymerase, once-daily oral treatment protected against lethal infection with seasonal and highly pathogenic avian influenza viruses, prevented direct-contact transmission to untreated sentinels, and mitigated lethal infection of immunocompromised hosts. These results support the developmental potential of 4\u2019-fluorouridine for treatment of vulnerable patient groups and mitigation of pandemic influenza, providing a much-needed additional therapeutic option for improved disease management.","version":"1.2","doi":"10.1101/2022.11.05.515296","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.01.518127","pub_date":"2022-12-02","title":"Potent Immunogenicity and Broad-Spectrum Protection Potential of Microneedle Array Patch-Based COVID-19 DNA Vaccine Candidates Encoding Dimeric RBD Chimera of SARS-CoV and SARS-CoV-2 Variants","abstract":"Breakthrough infections by SARS-CoV-2 variants pose a global challenge to pandemic control, and the development of more effective vaccines of broadspectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding heterodimeric receptor-binding domain (RBD) chimera of SARS-CoV and SARS-CoV-2 Omicron BA.1 (RBDSARS/BA1), SARS-CoV and SARS-CoV-2 Beta (RBDSARS/Beta), or Omicron BA.1 and Beta (RBDBA1/Beta) in secreted form. When i.m. injected in mice, RBDSARS/BA1 and RBDSARS/Beta encoding plasmids (pAD1002 and pAD131, respectively) were by far more immunogenic than RBDBA1/Beta plasmid (pAD1003). Dissolvable microneedle array patches (MAP) laden with these DNA plasmids were fabricated. All 3 resulting MAP-based vaccine candidates, namely MAP-1002, MAP1003 and MAP-131, were comparable to i.m. inoculated plasmids with electroporation assistance in eliciting strong and durable IgG responses in BALB/c and C57BL/6 mice as well as rabbits, while MAP-1002 was comparatively the most immunogenic. More importantly, MAP-1002 significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-\u03b3+ T cells. Moreover, MAP-1002 antisera effectively neutralized pseudoviruses displaying spike proteins of SARS-CoV, prototype SARS-CoV-2 or Beta, Delta, Omicron BA1, BA2 and BA4/5 variants. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.","version":"1.1","doi":"10.1101/2022.12.01.518127","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.01.518541","pub_date":"2022-12-02","title":"Mice Humanized for Major Histocompatibility Complex and Angiotensin-Converting Enzyme 2 with High Permissiveness to SARS-CoV-2 Omicron Replication","abstract":"Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike glycoprotein. The murine ACE2 ortholog does not interact efficiently with SARS-CoV-2 Spike and therefore the conventional laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in C57BL/6 \u201cHHD-DR1\u201d background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. Compared to the previously available B6.K18-ACE22Prlmn/JAX mice, which have limited permissiveness to SARS-CoV-2 Omicron replication, these three new hACE2 transgenic strains display higher levels of hACE2 mRNA expression, associated with high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral vector-based COVID-19 vaccine candidate.","version":"1.1","doi":"10.1101/2022.12.01.518541","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.02.518847","pub_date":"2022-12-02","title":"Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias","abstract":"In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.","version":"1.1","doi":"10.1101/2022.12.02.518847","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.02.518860","pub_date":"2022-12-02","title":"Gut microbiota dysbiosis is associated with altered tryptophan metabolism and dysregulated inflammatory response in severe COVID-19","abstract":"The clinical course of the 2019 coronavirus disease (COVID-19) is variable and to a substantial degree still unpredictable, especially in persons who have neither been vaccinated nor recovered from previous infection. We hypothesized that disease progression and inflammatory responses were associated with alterations in the microbiome and metabolome. To test this, we integrated metagenome, metabolome, cytokine, and transcriptome profiles of longitudinally collected samples from hospitalized COVID-19 patients at the beginning of the pandemic (before vaccines or variants of concern) and non-infected controls, and leveraged detailed clinical information and post-hoc confounder analysis to identify robust within- and cross-omics associations. Severe COVID-19 was directly associated with a depletion of potentially beneficial intestinal microbes mainly belonging to Clostridiales, whereas oropharyngeal microbiota disturbance appeared to be mainly driven by antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine, and reduced levels of various other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Decreased abundance of Clostridiales potentially mediated the observed reduction in 5-hydroxytryptophan levels. Moreover, altered plasma levels of various tryptophan metabolites and lower abundances of Clostridiales explained significant increases in the production of IL-6, IFN\u03b3 and/or TNF\u03b1. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19.","version":"1.1","doi":"10.1101/2022.12.02.518860","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.22.517465","pub_date":"2022-12-01","title":"Nirmatrelvir treatment blunts the development of antiviral adaptive immune responses in SARS-CoV-2 infected mice","abstract":"Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir \u2013 an orally available inhibitor of the 3-chymotrypsin-like cysteine protease \u2013 has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using a mouse model of SARS-CoV-2 infection, we show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.","version":"1.2","doi":"10.1101/2022.11.22.517465","journal":"bioRxiv","score":null},{"id":"10.1101/2022.12.01.518643","pub_date":"2022-12-01","title":"Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2","abstract":"Seasonal \u201ccommon-cold\u201d human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but identification and characterization of the T cell response to seasonal human coronaviruses remain largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal human coronavirus OC43. We identified MHC-I and MHC-II bound peptides derived from the viral spike, nucleocapsid, hemagglutinin-esterase, 3C-like proteinase, and envelope proteins. Only three MHC-I bound OC43-derived peptides were observed, possibly due to the potent MHC-I downregulation induced by OC43 infection. By contrast, 80 MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. These peptides elicited low-abundance recall T cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T cell lines. Among the validated epitopes, S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. N54-68 and HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow T cell cross-reactivity after infection or vaccination and could aid in the selection of epitopes for inclusion in pan-coronavirus vaccines. There is much current interest in cellular immune responses to seasonal common-cold coronaviruses because of their possible role in mediating protection against SARS-CoV-2 infection or pathology. However, identification of relevant T cell epitopes and systematic studies of the T cell responses responding to these viruses are scarce. We conducted a study to identify naturally processed and presented MHC-I and MHC-II epitopes from human cells infected with the seasonal coronavirus HCoV-OC43, and to characterize the T cell responses associated with these epitopes. We found epitopes specific to the seasonal coronaviruses, as well as epitopes cross-reactive between HCoV-OC43 and SARS-CoV-2. These epitopes should be useful in following immune responses to seasonal coronaviruses and identifying their roles in COVID-19 vaccination, infection, and pathogenesis.","version":"1.1","doi":"10.1101/2022.12.01.518643","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.30.518633","pub_date":"2022-12-01","title":"BBIBP-CorV (Sinopharm) vaccination- induced immunity is affected by age, gender and prior COVID-19 and activates responses to spike and other antigens","abstract":"Long-term solutions against SARS-CoV-2 infections require understanding of immune protection induced by different vaccine COVID-19 formulations. We investigated humoral and cellular immunity induced by Sinopharm (BBIBP-CorV) in a region of high SARS-CoV-2 seroprevalence. Levels of IgG antibodies to SARS-CoV-2 spike protein and its receptor-binding domain (RBD) were determined 24-weeks. Cellular immunity was investigated using a commercially available IFN-\u03b3 release assay to SARS-CoV-2 spike (Ag1 and 2) and extended genome antigens (Ag3). Increasing IgG seropositivity to Spike protein and RBD was observed post-vaccination. Seropositivity was reduced in those over 50 years and raised in females and those with prior COVID-19. After 20 weeks post-vaccination, only one third of participants had positive T cell responses to SARS-CoV-2 antigens. Prior COVID-19 impacted IFN\u03b3 responses, with reactivity enhanced in those infected earlier. The frequency of IFN\u03b3 responses was highest to extended genome antigen set. Overall, BBIBP-CorV- induced antibody responses were impacted by age, gender and prior COVID-19. Cellular immunity was present in a limited number of individuals after 20 weeks but was enhanced by prior infection. This suggests the need for booster vaccinations in older individuals. BBIBP-CorV-induced cellular activation is broader than to spike, requiring further study to understand how to monitor vaccine effectiveness.","version":"1.1","doi":"10.1101/2022.11.30.518633","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518438","pub_date":"2022-11-30","title":"Targeted photodynamic neutralization of SARS-CoV-2 mediated by singlet oxygen","abstract":"The SARS-CoV-2 virus has been on a rampage for more than two years. Vaccines in combination with neutralizing antibodies (NAbs) against SARS-CoV-2 carry great hope in the treatment and final elimination of COVID-19. However, the relentless emergence of variants of concern (VOC), including the most recent Omicron variants, presses for novel measures to counter these variants that often show immune evasion. Hereby we developed a targeted photodynamic approach to neutralize SARS-CoV-2 by engineering a genetically encoded photosensitizer (SOPP3) to a diverse list of antibodies targeting the WT spike protein, including human antibodies isolated from a 2003 SARS patient, potent monomeric and multimeric nanobodies targeting RBD, and non-neutralizing antibodies (non-NAbs) targeting the more conserved NTD region. As confirmed by pseudovirus neutralization assay, this targeted photodynamic approach significantly increased the efficacy of these antibodies, especially that of non-NAbs, against not only the WT but also the Delta strain and the heavily immune escape Omicron strain (BA.1). Subsequent measurement of infrared phosphorescence at 1270 nm confirmed the generation of singlet oxygen (1O2) in the photodynamic process. Mass spectroscopy assay uncovered amino acids in the spike protein targeted by 1O2. Impressively, Y145 and H146 form an oxidization \u201chotspot\u201d, which overlaps with the antigenic \u201csupersite\u201d in NTD. Taken together, our study established a targeted photodynamic approach against the SARS-CoV-2 virus and provided mechanistic insights into the photodynamic modification of protein molecules mediated by 1O2.","version":"1.1","doi":"10.1101/2022.11.29.518438","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.26.518005","pub_date":"2022-11-30","title":"An Optimized Circular Polymerase Extension Reaction-based Method for Functional Analysis of SARS-CoV-2","abstract":"Reverse genetics systems have been crucial for studying specific viral genes and their relevance in the virus lifecycle, and become important tools for the rational attenuation of viruses and thereby for vaccine design. Recent rapid progress has been made in the establishment of reverse genetics systems for functional analysis of SARS-CoV-2, a coronavirus that causes the ongoing COVID-19 pandemic that has resulted in detrimental public health and economic burden. Among the different reverse genetics approaches, CPER (circular polymerase extension reaction) has become one of the leading methodologies to generate recombinant SARS-CoV-2 infectious clones due to its accuracy, efficiency, and flexibility. Here, we report an optimized CPER methodology which, through the use of a modified linker plasmid and by performing DNA nick ligation and direct transfection of permissive cells, overcomes certain intrinsic limitations of the \u2018traditional\u2019 CPER approaches for SARS-CoV-2, allowing for efficient virus rescue. This optimized CPER system may facilitate research studies to assess the contribution of SARS-CoV-2 genes and individual motifs or residues to virus replication, pathogenesis and immune escape, and may also be adapted to other viruses.","version":"1.1","doi":"10.1101/2022.11.26.518005","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.18.512708","pub_date":"2022-11-30","title":"Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis","abstract":"We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.","version":"1.2","doi":"10.1101/2022.10.18.512708","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.23.517619","pub_date":"2022-11-30","title":"Feline Coronavirus Infection of Domestic Cats Causes Development of Cross-Reactive Antibodies to SARS-CoV-2 Receptor Binding Domain","abstract":"The current study was initiated when our specific pathogen-free laboratory toms developed unexpectedly high levels of cross-reactive antibodies to human SARS-CoV-2 (SCoV2) receptor binding domain (RBD) upon mating with feline coronavirus (FCoV)-positive queens. Multi-sequence alignment analyses of SCoV2 Wuhan RBD and four strains each from FCoV serotypes 1 and 2 (FCoV1, FCoV2) demonstrated amino acid sequence identity of 11.5% and similarity of 31.8% with FCoV1 RBD, as well as 12.2% identity and 36.5% similarity for FCoV2 RBD. The sera from all three toms and three mated queens cross-reacted with SCoV2 RBD and reacted with FCoV1 RBD and FCoV2 spike-2, nucleocapsid, and membrane proteins of FCoV2 whole-virus, but not with FCoV2 RBD. Additionally, the plasma from all six FCoV2-inoculated laboratory cats reacted with FCoV2 and SCoV2 RBDs, but not with FCoV1 RBD. In another study, eight group-housed laboratory cats from a different lineage had a range of serum cross-reactivity to SCoV2 RBD even 15 months later. Such cross-reactivity was also observed in FCoV1-positive group-housed pet cats. The SCoV2 RBD at a high non-toxic dose and FCoV2 RBD at a 60-400-fold lower dose blocked the in vitro FCoV2 infection of the feline cells, demonstrating their close structural conformations essential as vaccine immunogens. Furthermore, such cross-reactivity to SCoV2 RBD was also detected by the peripheral blood mononuclear cells of both transient and chronically FCoV1-infected cats. Overall, the cross-reactivity with SCoV2 RBD by the sera from both serotypes of FCoV-infected cats also suggests that the cross-reactive epitope(s) on FCoV1 and FCoV2 RBDs may be similar to those of SCoV2 RBD and provides essential insights to developing a pan-CoV vaccine. To date, there are no reports on the sera from feline coronavirus (FCoV)-infected cats cross-reacting with either SARS-CoV-1 or SARS-CoV2 (SCoV2) receptor binding domains (RBDs). This report describes the presence of cross-reactive antibodies to SCoV2 RBD in the sera of FCoV-infected laboratory cats, even though SCoV2 RBD and each FCoV serotype (FCoV1, FCoV2) RBD had minimal sequence similarity. However, this observation of serum cross-reactivity to SCoV2 RBD was confirmed by more stringent antibody-based assays and viral assays. Furthermore, both serotypes of FCoV-infected cats, including FCoV1-infected pet cats, produced the cross-reactive antibodies, and such cross-reactivity to SCoV2 RBD was also detected, most likely, by the T cells in peripheral blood mononuclear cells of both transient and chronically FCoV1-infected cats. Since SCoV2 RBD is essential component for current vaccines against COVID-19 disease, our findings should provide essential insights to developing a pan-coronavirus vaccine that induces full-scale immunity to completely prevent SCoV2 infection in humans and pet animals.","version":"1.2","doi":"10.1101/2022.11.23.517619","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518411","pub_date":"2022-11-30","title":"Proteomic profiling identifies biomarkers of COVID-19 severity","abstract":"SARS-CoV-2 infection remains a major public health concern, particularly for the aged and those individuals with co-morbidities at risk for developing severe COVID-19. Understanding the pathogenesis and biomarkers associated with responses to SARS-CoV-2 infection remain critical components in developing effective therapeutic approaches, especially in cases of severe and long-COVID-19. In this study blood plasma protein expression was compared in subjects with mild, moderate, and severe COVID-19 disease. Evaluation of an inflammatory protein panel confirms upregulation of proteins including TNF\u03b2, IL-6, IL-8, IL-12, already associated with severe cytokine storm and progression to severe COVID-19. Importantly, we identify several proteins not yet associated with COVID-19 disease, including mesothelin (MSLN), that are expressed at significantly higher levels in severe COVID-19 subjects. In addition, we find a subset of markers associated with T-cell and dendritic cell responses to viral infection that are significantly higher in mild cases and decrease in expression as severity of COVID-19 increases, suggesting that an immediate and effective activation of T-cells is critical in modulating disease progression. Together, our findings identify new targets for further investigation as therapeutic approaches for the treatment of SARS-CoV-2 infection and prevention of complications of severe COVID-19.","version":"1.1","doi":"10.1101/2022.11.29.518411","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518257","pub_date":"2022-11-30","title":"SARS-CoV-2 Envelope-mediated Golgi pH dysregulation interferes with ERAAP retention in cells","abstract":"Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) trims peptide precursors in the ER for presentation by major histocompatibility (MHC)-I molecules to surveying CD8+ T-cells. This function allows ERAAP to regulate the nature and quality of the peptide repertoire and, accordingly, the resulting immune responses. We recently showed that infection with murine cytomegalovirus leads to a dramatic loss of ERAAP levels in infected cells. In mice, this loss is associated with the activation of QFL T-cells, a subset of T-cells that monitor ERAAP integrity and eliminate cells experiencing ERAAP dysfunction. In this study, we aimed to identify host factors that regulate ERAAP expression level and determine whether these could be manipulated during viral infections. We performed a CRISPR knockout screen and identified ERp44 as a factor promoting ERAAP retention in the ER. ERp44\u2019s interaction with ERAAP is dependent on the pH gradient between the ER and Golgi. We hypothesized that viruses that disrupt the pH of the secretory pathway interfere with ERAAP retention. Here, we demonstrate that expression of the Envelope (E) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) leads to Golgi pH neutralization and consequently decrease of ERAAP intracellular levels. Furthermore, SARS-CoV-2-induced ERAAP loss correlates with its release into the extracellular environment. ERAAP\u2019s reliance on ERp44 and a functioning ER/Golgi pH gradient for proper localization and function led us to propose that ERAAP serves as a sensor of disturbances in the secretory pathway during infection and disease.","version":"1.1","doi":"10.1101/2022.11.29.518257","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.30.518473","pub_date":"2022-11-30","title":"DAPTEV: Deep aptamer evolutionary modelling for COVID-19 drug design","abstract":"Typical drug discovery and development processes are costly, time consuming and often biased by expert opinion. Aptamers are short, single-stranded oligonucleotides (RNA/DNA) that bind to target proteins and other types of biomolecules. Compared with small-molecule drugs, aptamers can bind to their targets with high affinity (binding strength) and specificity (uniquely interacting with the target only). The conventional development process for aptamers utilizes a manual process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which is costly, slow, dependent on library choice and often produces aptamers that are not optimized. To address these challenges, in this research, we create an intelligent approach, named DAPTEV, for generating and evolving aptamer sequences to support aptamer-based drug discovery and development. Using the COVID-19 spike protein as a target, our computational results suggest that DAPTEV is able to produce structurally complex aptamers with strong binding affinities. Compared with small-molecule drugs, aptamer drugs are short RNAs/DNAs that can specifically bind to targets with high strength. With the interest of discovering novel aptamer drugs as an alternative to address the long-lasting COVID-19 pandemic, in this research, we developed an artificial intelligence (AI) framework for the in silico design of novel aptamer drugs that can prevent the SARS-CoV-2 virus from entering human cells. Our research is valuable as we explore a novel approach for the treatment of SARS-CoV-2 infection and the AI framework could be applied to address future health crises.","version":"1.1","doi":"10.1101/2022.11.30.518473","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518418","pub_date":"2022-11-29","title":"Calpain-2 mediates SARS-CoV-2 entry and represents a therapeutic target","abstract":"Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, much effort has been dedicated to identifying effective antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of calpain inhibitors show excellent antiviral activities against SARS-CoV-2 by targeting the viral main protease (Mpro), which plays an essential role in processing viral polyproteins. In this study, we found that calpain inhibitors potently inhibited the infection of a chimeric vesicular stomatitis virus (VSV) encoding the SARS-CoV-2 spike protein, but not Mpro. In contrast, calpain inhibitors did not exhibit antiviral activities towards the wild-type VSV with its native glycoprotein. Genetic knockout of calpain-2 by CRISPR/Cas9 conferred resistance of the host cells to the chimeric VSV-SARS-CoV-2 virus and a clinical isolate of wild-type SARS-CoV-2. Mechanistically, calpain-2 facilitates SARS-CoV-2 spike protein-mediated cell attachment by positively regulating the cell surface levels of ACE2. These results highlight an Mpro-independent pathway targeted by calpain inhibitors for efficient viral inhibition. We also identify calpain-2 as a novel host factor and a potential therapeutic target responsible for SARS-CoV-2 infection at the entry step.","version":"1.1","doi":"10.1101/2022.11.29.518418","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518406","pub_date":"2022-11-29","title":"Atomic-level characterization of the conformational transition pathways in SARS-CoV-1 and SARS-CoV-2 spike proteins","abstract":"Severe acute respiratory syndrome (SARS) coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) derive transmissibility from spike protein activation in the receptor binding domain (RBD) and binding to the host cell angiotensin converting enzyme 2 (ACE2). However, the mechanistic details that describe the large-scale conformational changes associated with spike protein activation or deactivation are still somewhat unknown. Here, we have employed an extensive set of nonequilibrium all-atom molecular dynamics (MD) simulations, utilizing a novel protocol, for the SARS-CoV-1 (CoV-1) and SARS-CoV-2 (CoV-2) prefusion spike proteins in order to characterize the conformational pathways associated with the active-to-inactive transition. Our results indicate that both CoV-1 and CoV-2 spike proteins undergo conformational transitions along pathways unique to each protein. We have identified a number of key residues that form various inter-domain saltbridges, suggesting a multi-stage conformational change along the pathways. We have also constructed the free energy profiles along the transition pathways for both CoV-1 and CoV-2 spike proteins. The CoV-2 spike protein must overcome larger free energy barriers to undergo conformational changes towards protein activation or deactivation, when compared to CoV-1.","version":"1.1","doi":"10.1101/2022.11.29.518406","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478415","pub_date":"2022-11-29","title":"Toward Atomistic Models of Intact SARS-CoV-2 via Martini Coarse-Grained Molecular Dynamics Simulations","abstract":"The causative pathogen of Coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an enveloped virus assembled by a lipid envelope and multiple structural proteins. In this study, by integrating experimental data, structural modeling, and coarse-grained molecular dynamics simulations, we constructed multiscale models of SARS-CoV-2. Our 500-ns coarse-grained simulation of the intact virion allowed us to investigate the dynamic behavior of the membrane-embedded proteins and the surrounding lipid molecules in situ. Our results indicated that the membrane-embedded proteins are highly dynamic, and certain types of lipids exhibit various binding preferences to specific sites of the membrane-embedded proteins. The equilibrated virion model was transformed into atomic resolution, which provided a 3D structure for scientific demonstration and can serve as a framework for future exascale all-atom MD simulations.","version":"1.2","doi":"10.1101/2022.01.31.478415","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518385","pub_date":"2022-11-29","title":"SARS-CoV-2 Vaccine Booster Elicits Robust Prolonged Maternal Antibody Responses and Passive Transfer Via The Placenta And Breastmilk","abstract":"Infection during pregnancy can result in adverse outcomes for both pregnant persons and offspring. Maternal vaccination is an effective mechanism to protect both mother and neonate into post-partum. However, our understanding of passive transfer of antibodies elicited by maternal SARS-CoV-2 mRNA vaccination during pregnancy remains incomplete. We aimed to evaluate the antibody responses engendered by maternal SARS-CoV-2 vaccination following initial and booster doses in maternal circulation and breastmilk to better understand passive immunization of the newborn. We collected longitudinal blood samples from 121 pregnant women who received SARS-CoV-2 mRNA vaccines spanning from early gestation to delivery followed by collection of blood samples and breastmilk between delivery and 12 months post-partum. During the study, 70% of the participants also received a booster post-partum. Paired maternal plasma, breastmilk, umbilical cord plasma, and newborn plasma samples were tested via enzyme-linked immunosorbent assays (ELISA) to evaluate SARS-CoV-2 specific IgG antibody levels. Vaccine-elicited maternal antibodies were detected in both cord blood and newborn blood, albeit at lower levels than maternal circulation, demonstrating transplacental passive immunization. Booster vaccination significantly increased spike specific IgG antibody titers in maternal plasma and breastmilk. Finally, SARS-CoV-2 specific IgG antibodies in newborn blood correlated negatively with days post initial maternal vaccine dose. Vaccine-induced maternal SARS-CoV-2 antibodies were passively transferred to the offspring in utero via the placenta and after birth via breastfeeding. Maternal booster vaccination, regardless of gestational age at maternal vaccination, significantly increased antibody levels in breastmilk and maternal plasma, indicating the importance of this additional dose to maximize passive protection against SARS-CoV-2 infection for neonates and infants until vaccination eligibility.","version":"1.1","doi":"10.1101/2022.11.29.518385","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512954","pub_date":"2022-11-29","title":"Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients","abstract":"Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.","version":"1.2","doi":"10.1101/2022.10.19.512954","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518404","pub_date":"2022-11-29","title":"Virus-like particle displaying SARS-CoV-2 receptor binding domain elicits neutralizing antibodies and is protective in a challenge model","abstract":"While the effort to vaccinate people against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has largely been successful, particularly in the developed world, the rise of new variants as well as waning immunity illustrate the need for a new generation of vaccines that provide broader and/or more durable protection against infection and severe disease. Here we describe the generation and characterization of IVX-411, a computationally designed, two-component virus-like particle (VLP) displaying the ancestral SARS-CoV-2 receptor binding domain (RBD) on its surface. Immunization of mice with IVX-411 generates neutralizing antibodies against the ancestral strain as well as three variants of concern. Neutralizing antibody titers elicited by IVX-411 are durable and significantly higher than those elicited by immunization with soluble RBD and spike antigens. Furthermore, immunization with IVX-411 is shown to be protective in a Syrian Golden hamster challenge model using two different strains of SARS-CoV-2. Overall, these studies demonstrate that IVX-411 is highly immunogenic and capable of eliciting broad, protective immunity.","version":"1.1","doi":"10.1101/2022.11.29.518404","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.29.518231","pub_date":"2022-11-29","title":"Structural and Computational Design of a SARS-2 Spike Antigen with Increased Receptor Binding Domain Exposure and Improved Immunogenicity","abstract":"Emerging SARS-CoV-2 variants of concern challenge the efficacy of approved vaccines and emphasize the need for improved antigens. Using an evolutionary-based design approach starting from the widely used engineered Spike antigen, S-2P, we sought to increase antigen production levels and the exposure of highly conserved and neutralization sensitive receptor-binding domain (RBD) epitopes. Thirty-six prototypes were generated in silico, of which fifteen were produced and tested in biochemical assays. Design S2D14, which contains 20 mutations within the Spike S2 domain, showed a 6-fold increase in expression while preserving similar thermal stability and antigenicity as S-2P. Cryo-EM structures indicate that the dominant populations of S2D14 particles have RBDs in exposed states, and analysis of these structures revealed how modifications within the S2 domain balance trimer stability and RBD accessibility through formation and removal of hydrogen bonds and surface charge alterations. Importantly, vaccination of mice with adjuvanted S2D14 resulted in higher levels of neutralizing antibodies than adjuvanted S-2P against SARS-CoV-2 Wuhan strain and four variants of concern. These results can guide the design of next generation vaccines to combat current, and future coronaviruses and the approaches used may be broadly applicable to streamline the successful design of vaccine antigens.","version":"1.1","doi":"10.1101/2022.11.29.518231","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.28.518301","pub_date":"2022-11-29","title":"Brewpitopes: a pipeline to refine B-cell epitope predictions during public health emergencies","abstract":"The application of B-cell epitope identification for the development of therapeutic antibodies is well established but consuming in terms of time and resources. For this reason, in the last few years, the immunoinformatic community has developed several computational predictive tools. While relatively successful, most of these tools only use a few properties of the candidate region to determine their likelihood of being a true B-cell epitope. However, this likelihood is influenced by a wide variety of protein features, including the presence of glycosylated residues in the neighbourhood of the candidate epitope, the subcellular location of the protein region or the three-dimensional information about their surface accessibility in the parental protein. In this study we created Brewpitopes, an integrative pipeline to curate computational predictions of B-cell epitopes by accounting for all the aforementioned features. To this end, we implemented a set of rational filters to mimic the conditions for the in vivo antibody recognition to enrich the B-cell epitope predictions in actionable candidates. To validate Brewpitopes, we analyzed the SARS-CoV-2 proteome. In the S protein, Brewpitopes enriched the initial predictions in 5-fold on epitopes with neutralizing potential (p-value < 2e-4). Other than S protein, 4 out of 16 proteins in the proteome contain curated B-cell epitopes and hence, have also potential interest for viral neutralization, since mutational escape mainly affects the S protein. Our results demonstrate that Brewpitopes is a powerful pipeline for the rapid prediction of refined B-cell epitopes during public health emergencies. We have created Brewpitopes, a new pipeline that integrates additional important features such as glycosylation or structural accessibility, to curate B-cell epitope more likely to be functional in vivo. We have also validated Brewpitopes against SARS-CoV-2 not only for S protein but also for the entire viral proteome demonstrating that is a rapid and reliable epitope predictive tool to be implemented in present or future public health emergencies. Brewpitopes has identified 7 SARS-CoV-2 epitopes in S and epitopes allocated in 4 other proteins. Overall, offering an accurate selection of epitopes that might be scaled up to the production of new antibodies.","version":"1.1","doi":"10.1101/2022.11.28.518301","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.24.517882","pub_date":"2022-11-28","title":"SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects","abstract":"Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than for the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterwards. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines which use spike protein as the generated antigen.","version":"1.1","doi":"10.1101/2022.11.24.517882","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.28.518195","pub_date":"2022-11-28","title":"Highly potent antisense oligonucleotides (ASOs) targeting the SARS-CoV-2 RNA genome","abstract":"Currently the world is dealing with the third outbreak of the human-infecting coronavirus with potential lethal outcome, cause by a member of the Nidovirus family, the SARS-CoV-2. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused the last worldwide pandemic. Successful development of vaccines highly contributed to reduce the severeness of the COVID-19 disease. To establish a control over the current and newly emerging coronaviruses of epidemic concern requires development of substances able to cure severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA using antisense oligonucleotides (ASOs) and identify locked nucleic acid gapmers (LNA gapmers) potent to reduce by up to 96% the intracellular viral load in vitro. Our results strongly suggest promise of our preselected ASOs for further development as therapeutic or prophylactic anti-viral agents. ASOs (LNA gapmers) targeting the SARS-CoV-2 RNA genome have been effective in viral RNA (load) reduction in vitro.","version":"1.1","doi":"10.1101/2022.11.28.518195","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.27.518117","pub_date":"2022-11-28","title":"Fc\u03b3 receptor-dependent antibody effector functions are required for vaccine protection against infection by antigenic variants of SARS-CoV-2","abstract":"Emerging SARS-CoV-2 variants with antigenic changes in the spike protein are neutralized less efficiently by serum antibodies elicited by legacy vaccines against the ancestral Wuhan-1 virus. Nonetheless, these vaccines, including mRNA-1273 and BNT162b2, retained their ability to protect against severe disease and death, suggesting that other aspects of immunity control infection in the lung. Although vaccine-elicited antibodies can bind Fc gamma receptors (Fc\u03b3Rs) and mediate effector functions against SARS-CoV-2 variants, and this property correlates with improved clinical COVID-19 outcome, a causal relationship between Fc effector functions and vaccine-mediated protection against infection has not been established. Here, using passive and active immunization approaches in wild-type and Fc-gamma receptor (Fc\u03b3R) KO mice, we determined the requirement for Fc effector functions to protect against SARS-CoV-2 infection. The antiviral activity of passively transferred immune serum was lost against multiple SARS-CoV-2 strains in mice lacking expression of activating Fc\u03b3Rs, especially murine Fc\u03b3R III (CD16), or depleted of alveolar macrophages. After immunization with the preclinical mRNA-1273 vaccine, protection against Omicron BA.5 infection in the respiratory tract also was lost in mice lacking Fc\u03b3R III. Our passive and active immunization studies in mice suggest that Fc-Fc\u03b3R engagement and alveolar macrophages are required for vaccine-induced antibody-mediated protection against infection by antigenically changed SARS-CoV-2 variants, including Omicron strains.","version":"1.1","doi":"10.1101/2022.11.27.518117","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.24.515932","pub_date":"2022-11-28","title":"Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2","abstract":"Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2\u2019 site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants. Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.","version":"1.1","doi":"10.1101/2022.11.24.515932","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.23.517532","pub_date":"2022-11-28","title":"Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants","abstract":"The SARS-CoV-2 Omicron variant continues to evolve, with new BQ and XBB subvariants now rapidly expanding in Europe/US and Asia, respectively. As these new subvariants have additional spike mutations, they may possess altered antibody evasion properties. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals who were boosted with a WA1/BA.5 bivalent mRNA vaccine. Compared to the ancestral strain D614G, serum neutralizing titers against BQ and XBB subvariants were lower by 13-81-fold and 66-155-fold, respectively, far beyond what had been observed to date. A panel of monoclonal antibodies capable of neutralizing the original Omicron variant, including those with Emergency Use Authorization, were largely inactive against these new subvariants. The spike mutations that conferred antibody resistance were individually studied and structurally explained. Finally, the ACE2-binding affinities of the spike proteins of these novel subvariants were found to be similar to those of their predecessors. Taken together, our findings indicate that BQ and XBB subvariants present serious threats to the efficacy of current COVID-19 vaccines, render inactive all authorized monoclonal antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.","version":"1.1","doi":"10.1101/2022.11.23.517532","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.24.517008","pub_date":"2022-11-28","title":"Extracellular disintegration of viral proteins as an innovative strategy for developing broad-spectrum antivirals against coronavirus","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives worldwide, not to mention innumerable losses in the global economy and disruptions in social relationships. Unfortunately, state-of-the-art treatments still lag behind the fast emergence of new variants of concern. The key to resolve this issue is to develop broad-spectrum antivirals with innovative antiviral mechanisms in which coronaviruses are deactivated regardless of their variant development. Herein, we report a new antiviral strategy involving extracellular disintegration of viral proteins that are indispensable for viral infection with hyperanion-grafted enediyne molecules. The sulfate groups ensure low cellular permeability and rather low cytotoxicity of the molecules, while the core enediyne generates reactive radical species and causes significant damage to the spike (S) protein of coronavirus. The enediyne compounds exhibit antiviral activity at micromolar to nanomolar concentrations, and the selectivity index of up to 20,000 against four kinds of human coronaviruses, including the SARS-CoV-2 omicron variant, suggesting the high potential of this new strategy in combating the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2022.11.24.517008","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.28.518175","pub_date":"2022-11-28","title":"Fc mediated pan-sarbecovirus protection after alphavirus vector vaccination","abstract":"Two group 2B \u03b2-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. The mechanisms of cross protection driven by the sarbecovirus spike, a dominant immunogen, are less clear yet critically important for pan-sarbecovirus vaccine development. We evaluated the mechanisms of cross-sarbecovirus protective immunity using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination did not prevent virus replication, it protected against lethal heterologous disease outcomes in both SARS-CoV-2 and clade 2 bat sarbecovirus HKU3-SRBD challenge models. The spike vaccines tested primarily elicited a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. We found non-neutralizing antibody functions that mediated cross protection in wild-type mice were mechanistically linked to FcgR4 and spike S2-binding antibodies. Protection was lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.","version":"1.1","doi":"10.1101/2022.11.28.518175","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.23.493121","pub_date":"2022-11-27","title":"Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course","abstract":"Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.","version":"1.2","doi":"10.1101/2022.05.23.493121","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.10.511545","pub_date":"2022-11-27","title":"A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens","abstract":"Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to the pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here we describe a multi-parametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform was validated by blocking SARS-CoV-2 spike particles with nanobodies and IgGs from human serum samples. A fast screening platform tackling host-pathogen interactions.","version":"1.2","doi":"10.1101/2022.10.10.511545","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.25.22282759","pub_date":"2022-11-27","title":"Prolonged T-cell activation and long COVID symptoms independently associate with severe disease at 3 months in a UK cohort of hospitalized COVID-19 patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  COVID-19 causes immune perturbations which may persist long-term, and patients frequently report ongoing symptoms for months after recovery. We assessed the extent and nature of immune activation at 3 months post hospital admission in patients with mild, moderate or severe COVID-19 and investigated whether immune activation associates with disease severity and long COVID. Patients with severe disease displayed persistent activation of CD4\n                  <jats:sup>+</jats:sup>\n                  and CD8\n                  <jats:sup>+</jats:sup>\n                  T-cells, based on expression of HLA-DR, CD38, Ki67 and granzyme B, but they lacked activation of other immune subsets. Elevated plasma levels of IL-4, IL-7, IL-17 and TNF-\u03b1 were present in patients with severe compared to mild and/or moderate disease. Plasma from severe patients caused T-cells from healthy donors to upregulate IL-15R\u03b1, suggesting that factors in the plasma of severe patients may increase T-cell responsiveness to IL-15-driven \u2018bystander\u201d activation, which may drive persistent T-cell activation after severe COVID-19. Patients with severe disease reported a higher number of long COVID symptoms which correlated with the frequency of two subsets of activated CD4\n                  <jats:sup>+</jats:sup>\n                  and CD8\n                  <jats:sup>+</jats:sup>\n                  T cells (CD4\n                  <jats:sup>+</jats:sup>\n                  T-cell population 2 and CD8\n                  <jats:sup>+</jats:sup>\n                  T-cell population 4; FDR p&lt;0.05), however these associations were lost after adjusting for age, sex and disease severity. Our data suggests that persistent immune activation and long COVID correlate independently with severe disease.\n                </jats:p>","version":null,"doi":"10.1101/2022.11.25.22282759","journal":"medRxiv","score":null},{"id":"10.1101/2022.11.23.517678","pub_date":"2022-11-24","title":"An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission","abstract":"SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and airborne aerosols. To prevent viral infection and reduce viral spread, vaccine strategies must elicit protective immunity in the airways. FcRn transfers IgG across epithelial barriers; we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized S protein; the resulting S-Fc bound to S-specific antibodies (Ab) and FcRn. A significant increase in Ab responses was observed following the intranasal immunization of mice with S-Fc formulated in CpG as compared to the immunization with S alone or PBS. Furthermore, we intranasally immunize adult or aged mice and hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2, including Delta and Omicron variants. Intranasal immunization also significantly reduced viral transmission between immunized and naive hamsters. Protection was mediated by nasal IgA, serum-neutralizing Abs, tissue-resident memory T cells, and bone marrow S-specific plasma cells. Hence FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission. Based on these findings, FcRn-targeted non-invasive respiratory immunizations are superior strategies for preventing highly contagious respiratory viruses from spreading.","version":"1.1","doi":"10.1101/2022.11.23.517678","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.21.517375","pub_date":"2022-11-24","title":"Fragment-Based Hit Discovery via Unsupervised Learning of Fragment-Protein Complexes","abstract":"The process of finding molecules that bind to a target protein is a challenging first step in drug discovery. Crystallographic fragment screening is a strategy based on elucidating binding modes of small polar compounds and then building potency by expanding or merging them. Recent advances in high-throughput crystallography enable screening of large fragment libraries, reading out dense ensembles of fragments spanning the binding site. However, fragments typically have low affinity thus the road to potency is often long and fraught with false starts. Here, we take advantage of high-throughput crystallography to reframe fragment-based hit discovery as a denoising problem \u2013 identifying significant pharmacophore distributions from a fragment ensemble amid noise due to weak binders \u2013 and employ an unsupervised machine learning method to tackle this problem. Our method screens potential molecules by evaluating whether they recapitulate those fragment-derived pharmacophore distributions. We retrospectively validated our approach on an open science campaign against SARS-CoV-2 main protease (Mpro), showing that our method can distinguish active compounds from inactive ones using only structural data of fragment-protein complexes, without any activity data. Further, we prospectively found novel hits for Mpro and the Mac1 domain of SARS-CoV-2 non-structural protein 3. More broadly, our results demonstrate how unsupervised machine learning helps interpret high throughput crystallography data to rapidly discover of potent chemical modulators of protein function.","version":"1.1","doi":"10.1101/2022.11.21.517375","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.23.517706","pub_date":"2022-11-23","title":"Molecular and cellular similarities in the brain of SARS-CoV-2 and Alzheimer\u2019s disease individuals","abstract":"Infection with the etiological agent of COVID-19, SARS-CoV-2, appears capable of impacting cognition, which some patients with Post-acute Sequelae of SARS-CoV-2 (PASC). To evaluate neuro-pathophysiological consequences of SARS-CoV-2 infection, we examine transcriptional and cellular signatures in the Broadman area 9 (BA9) of the frontal cortex and the hippocampal formation (HF) in SARS-CoV-2, Alzheimer\u2019s disease (AD) and SARS-CoV-2 infected AD individuals, compared to age- and gender-matched neurological cases. Here we show similar alterations of neuroinflammation and blood-brain barrier integrity in SARS-CoV-2, AD, and SARS-CoV-2 infected AD individuals. Distribution of microglial changes reflected by the increase of Iba-1 reveal nodular morphological alterations in SARS-CoV-2 infected AD individuals. Similarly, HIF-1\u03b1 is significantly upregulated in the context of SARS-CoV-2 infection in the same brain regions regardless of AD status. The finding may help to inform decision-making regarding therapeutic treatments in patients with neuro-PASC, especially those at increased risk of developing AD. SARS-CoV-2 and Alzheimer\u2019s disease share similar neuroinflammatory processes, which may help explain neuro-PASC.","version":"1.1","doi":"10.1101/2022.11.23.517706","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.22.517574","pub_date":"2022-11-23","title":"Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 Spike protein","abstract":"SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin \u03b1v\u03b23. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.","version":"1.1","doi":"10.1101/2022.11.22.517574","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.10.511571","pub_date":"2022-11-23","title":"GenSLMs: Genome-scale language models reveal SARS-CoV-2 evolutionary dynamics","abstract":"We seek to transform how new and emergent variants of pandemiccausing viruses, specifically SARS-CoV-2, are identified and classified. By adapting large language models (LLMs) for genomic data, we build genome-scale language models (GenSLMs) which can learn the evolutionary landscape of SARS-CoV-2 genomes. By pretraining on over 110 million prokaryotic gene sequences and finetuning a SARS-CoV-2-specific model on 1.5 million genomes, we show that GenSLMs can accurately and rapidly identify variants of concern. Thus, to our knowledge, GenSLMs represents one of the first whole genome scale foundation models which can generalize to other prediction tasks. We demonstrate scaling of GenSLMs on GPU-based supercomputers and AI-hardware accelerators utilizing 1.63 Zettaflops in training runs with a sustained performance of 121 PFLOPS in mixed precision and peak of 850 PFLOPS. We present initial scientific insights from examining GenSLMs in tracking evolutionary dynamics of SARS-CoV-2, paving the path to realizing this on large biological data.","version":"1.2","doi":"10.1101/2022.10.10.511571","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.23.517609","pub_date":"2022-11-23","title":"Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses","abstract":"Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.","version":"1.1","doi":"10.1101/2022.11.23.517609","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.22.517073","pub_date":"2022-11-22","title":"Environmental and genetic drivers of population differences in SARS-CoV-2 immune responses","abstract":"Humans display vast clinical variability upon SARS-CoV-2 infection, partly due to genetic and immunological factors. However, the magnitude of population differences in immune responses to SARS-CoV-2 and the mechanisms underlying such variation remain unknown. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells from 222 healthy donors of various ancestries stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces a weaker, but more heterogeneous interferon-stimulated gene activity than influenza A virus, and a unique pro-inflammatory signature in myeloid cells. We observe marked population differences in transcriptional responses to viral exposure that reflect environmentally induced cellular heterogeneity, as illustrated by higher rates of cytomegalovirus infection, affecting lymphoid cells, in African-descent individuals. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell proportions on population differences in immune responses, with genetic variants having a narrower but stronger effect on specific loci. Additionally, natural selection has increased immune response differentiation across populations, particularly for variants associated with SARS-CoV-2 responses in East Asians. We document the cellular and molecular mechanisms through which Neanderthal introgression has altered immune functions, such as its impact on the myeloid response in Europeans. Finally, colocalization analyses reveal an overlap between the genetic architecture of immune responses to SARS-CoV-2 and COVID-19 severity. Collectively, these findings suggest that adaptive evolution targeting immunity has also contributed to current disparities in COVID-19 risk.","version":"1.1","doi":"10.1101/2022.11.22.517073","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.19.517190","pub_date":"2022-11-22","title":"Novel mRNA vaccines encoding Monkeypox virus M1R and A35R protect mice from a lethal virus challenge","abstract":"The outbreak of Monkeypox virus infection urgently need effective vaccines. However, the vaccines so far approved are all based on whole-virus, which raises safety concerns. MRNA vaccines has demonstrated its high efficacy and safety against SARS-Cov-2 infection. Here, we developed three mRNA vaccines encoding Monkeypox proteins M1R and A35R, including A35R-M1R fusions (VGPox1 and VGPox 2) and a combination of encapsulated full-length mRNAs for A35R and M1R (VGPox 3). All three vaccines induced anti-A35R total IgGs as early as day 7 following a single vaccination. However, only VGPox 1 and 2 produced anti-M1R total IgGs at early dates following vaccination while VGPox 3 did not show significant anti-M1R antibody till day 35. Similar results were also found in neutralizing antibodies and T cell immune response. However, all mRNA vaccine groups completely protected mice from a lethal dose virus challenge and effectively cleared virus in lungs. Collectively, our results indicate that the novel mRNA vaccines coding for a fusion protein of A35R and M1R had a better anti-virus immunity than co-expression of the two individual proteins. The mRNA vaccines are highly effective and can be an alternative to the current whole-virus vaccines to defend Monkeypox virus infection.","version":"1.2","doi":"10.1101/2022.11.19.517190","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.19.517207","pub_date":"2022-11-21","title":"Evolution of the SARS-CoV-2 mutational spectrum","abstract":"SARS-CoV-2 evolves rapidly in part because of its high mutation rate. Here we examine whether this mutational process itself has changed during viral evolution. To do this, we quantify the relative rates of different types of single nucleotide mutations at four-fold degenerate sites in the viral genome across millions of human SARS-CoV-2 sequences. We find clear shifts in the relative rates of several types of mutations during SARS-CoV-2 evolution. The most striking trend is a roughly two-fold decrease in the relative rate of G\u2192T mutations in Omicron versus early clades, as was recently noted by Ruis et al (2022). There is also a decrease in the relative rate of C\u2192T mutations in Delta, and other subtle changes in the mutation spectrum along the phylogeny. We speculate that these changes in the mutation spectrum could arise from viral mutations that affect genome replication, packaging, and antagonization of host innate-immune factors\u2014although environmental factors could also play a role. Interestingly, the mutation spectrum of Omicron is more similar than that of earlier SARS-CoV-2 clades to the spectrum that shaped the long-term evolution of sarbecoviruses. Overall, our work shows that the mutation process is itself a dynamic variable during SARS-CoV-2 evolution, and suggests that human SARS-CoV-2 may be trending towards a mutation spectrum more similar to that of other animal sarbecoviruses.","version":"1.1","doi":"10.1101/2022.11.19.517207","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.20.517271","pub_date":"2022-11-21","title":"Paxlovid-like nirmatrelvir/ritonavir fails to block SARS-CoV-2 transmission in ferrets","abstract":"Despite the continued spread of SARS-CoV-2 and emergence of variants of concern (VOC) that are capable of escaping preexisting immunity, therapeutic options are underutilized. In addition to preventing severe disease in high-risk patients, antivirals may contribute to interrupting transmission chains. The FDA has granted emergency use authorizations for two oral drugs, molnupiravir and paxlovid. Initial clinical trials suggested an efficacy advantage of paxlovid, giving it a standard-of-care-like status in the United States. However, recent retrospective clinical studies suggested a more comparable efficacy of both drugs in preventing complicated disease and case-fatalities in older adults. For a direct efficacy comparison under controlled conditions, we assessed potency of both drugs against SARS-CoV-2 in two relevant animal models; the Roborovski dwarf hamster model for severe COVID-19 in high-risk patients and the ferret model of upper respiratory tract disease and transmission. After infection of dwarf hamsters with VOC omicron, paxlovid and molnupiravir were efficacious in mitigating severe disease and preventing death. However, a pharmacokinetics-confirmed human equivalent dose of paxlovid did not significantly reduce shed SARS-CoV-2 titers in ferrets and failed to block virus transmission to untreated direct-contact ferrets, whereas transmission was fully suppressed in a group of animals treated with a human-equivalent dose of molnupiravir. Prophylactic administration of molnupiravir to uninfected ferrets in direct contact with infected animals blocked productive SARS-CoV-2 transmission, whereas all contacts treated with prophylactic paxlovid became infected. These data confirm retrospective reports of similar therapeutic benefit of both drugs for older adults, and reveal that treatment with molnupiravir, but not paxlovid, may be suitable to reduce the risk of SARS-CoV-2 transmission.","version":"1.1","doi":"10.1101/2022.11.20.517271","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.20.517193","pub_date":"2022-11-21","title":"Differential haplotype expression in class I MHC genes during SARS-CoV-2 infection of human lung cell lines","abstract":"Cell entry of SARS-CoV-2 causes genome-wide disruption of the transcriptional profiles of genes and biological pathways involved in the pathogenesis of COVID-19. Expression allelic imbalance is characterized by a deviation from the Mendelian expected 1:1 expression ratio and is an important source of allele-specific heterogeneity. Expression allelic imbalance can be measured by allele-specific expression analysis (ASE) across heterozygous informative expressed single nucleotide variants (eSNVs). ASE reflects many regulatory biological phenomena that can be assessed by combining genome and transcriptome information. ASE contributes to the interindividual variability associated with disease. We aim to estimate the transcriptome-wide impact of SARS-CoV-2 infection by analyzing eSNVs. We compared ASE profiles in the human lung cell lines Calu-3, A459, and H522 before and after infection with SARS-CoV-2 using RNA-Seq experiments. We identified 34 differential ASE (DASE) sites in 13 genes (HLA-A, HLA-B, HLA-C, BRD2, EHD2, GFM2, GSPT1, HAVCR1, MAT2A, NQO2, SUPT6H, TNFRSF11A, UMPS), all of which are enriched in protein binding functions and play a role in COVID-19. Most DASE sites were assigned to the MHC class I locus and were predominantly upregulated upon infection. DASE sites in the MHC class I locus also occur in iPSC-derived airway epithelium basal cells infected with SARS-CoV-2. Using an RNA-Seq haplotype reconstruction approach, we found DASE sites and adjacent eSNVs in phase (i.e., predicted on the same DNA strand), demonstrating differential haplotype expression upon infection. We found a bias towards the expression of the HLA alleles with a higher binding affinity to SARS-CoV-2 epitopes. Independent of gene expression compensation, SARS-CoV-2 infection of human lung cell lines induces transcriptional allelic switching at the MHC loci. This suggests a response mechanism to SARS-CoV-2 infection that swaps HLA alleles with poor epitope binding affinity, an expectation supported by publicly available proteome data.","version":"1.1","doi":"10.1101/2022.11.20.517193","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.18.517139","pub_date":"2022-11-21","title":"SARS-CoV-2 variant transition dynamics are associated with vaccination rates, number of co-circulating variants, and natural immunity","abstract":"Throughout the COVID-19 pandemic, the SARS-CoV-2 virus has continued to evolve, with new variants outcompeting existing variants and often leading to different dynamics of disease spread. In this paper, we performed a retrospective analysis using longitudinal sequencing data to characterize differences in the speed, calendar timing, and magnitude of 13 SARS-CoV-2 variant waves/transitions for 215 countries and sub-country regions, between October 2020 and October 2022. We then clustered geographic locations in terms of their variant behavior across all Omicron variants, allowing us to identify groups of locations exhibiting similar variant transitions. Finally, we explored relationships between heterogeneity in these variant waves and time-varying factors, including vaccination status of the population, governmental policy, and the number of variants in simultaneous competition. This work demonstrates associations between the behavior of an emerging variant and the number of co-circulating variants as well as the demographic context of the population. We also observed an association between high vaccination rates and variant transition dynamics prior to the Mu and Delta variant transitions. These results suggest the behavior of an emergent variant may be sensitive to the immunologic and demographic context of its location. Additionally, this work represents the most comprehensive characterization of variant transitions globally to date. Laboratory Directed Research and Development (LDRD), Los Alamos National Laboratory SARS-CoV-2 variants with a selective advantage are continuing to emerge, resulting in variant transitions that can give rise to new waves in global COVID-19 cases and changing dynamics of disease spread. While variant transitions have been well studied individually, more work is needed to better understand how variant transitions have occurred in the past and how properties of these transitions may relate to vaccination rates, natural immunity, and population demographics. Our retrospective study integrates metadata based on 12.8 million SARS-CoV-2 sequences available through the Global Initiative on Sharing All Influenza Data (GISAID) with clinical and demographic data to characterize heterogeneity in variant waves/transitions across the globe throughout the COVID-19 pandemic. We demonstrate that properties of the variant transitions (e.g., speed, timing, and magnitude of the transition) are associated with vaccination rates, prior COVID-19 cases, and the number of co-circulating variants in competition. Our results indicate that there is substantial heterogeneity in how an emerging variant may compete with other viral variants across locations, and suggest that each location\u2019s contemporaneous immunologic landscape may play a role in these interactions.","version":"1.1","doi":"10.1101/2022.11.18.517139","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.29.502029","pub_date":"2022-11-21","title":"A multispecific antibody prevents immune escape and confers pan-SARS-CoV-2 neutralization","abstract":"Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies . To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.","version":"1.3","doi":"10.1101/2022.07.29.502029","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.21.517352","pub_date":"2022-11-21","title":"Comparative aerosol and surface stability of SARS-CoV-2 Variants of Concern","abstract":"SARS-CoV-2 is transmitted principally via air; contact and fomite transmission may also occur. Variants-of-concern (VOCs) are more transmissible than ancestral SARS-CoV-2. We find that early VOCs show greater aerosol and surface stability than the early WA1 strain, but Delta and Omicron do not. Stability changes do not explain increased transmissibility.","version":"1.1","doi":"10.1101/2022.11.21.517352","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.20.517236","pub_date":"2022-11-21","title":"Polymorphic regions in BA.2.12.1, BA.4 and BA.5 likely implicated in immunological evasion of Omicron subvariant BQ.1.1","abstract":"In this work, 45 Spike glycoprotein Chain B polypeptides were used in the subvariants BA.2.12.1, BA.4 and BA.5 were recovered from GENBANK. All sequences were publicly available on the National Biotechnology Information Center (NCBI) platform. The results indicate the existence of informative polymorphic and parsimony sites that may be implicated in the level of diversity of the studied strains, as well as reflect the immunological evasion potential of the subvariant BQ1.1. of the variant \u00d4micron d and SARS-CoV-2. The results also suggest the formation of ancestral polymorphism with slight retention, and the probable is responsible the diversity of the whole studied set.","version":"1.1","doi":"10.1101/2022.11.20.517236","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.17.516888","pub_date":"2022-11-21","title":"Resistance of Omicron subvariants BA.2.75.2, BA.4.6 and BQ.1.1 to neutralizing antibodies","abstract":"Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.","version":"1.2","doi":"10.1101/2022.11.17.516888","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.17.516978","pub_date":"2022-11-18","title":"Human Early Syncytiotrophoblasts Are Highly Susceptible to SARS-CoV-2 Infection","abstract":"The ongoing and devastating pandemic of coronavirus disease 2019 (COVID-19) has led to a global public health crisis. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and can potentially pose a serious risk to maternal and neonatal health. Cases of abnormal pregnancy and vertical transmission of SARS-CoV-2 from mother to foetus have been reported but no firm conclusions are drawn. Trophoblasts are the major constituents of the placenta to protect and nourish the developing foetus. However, direct in vivo investigation of trophoblast\u2019s susceptibility to SARS-CoV-2 and of COVID-19 and pregnancy is challenging. Here we report that human early syncytiotrophoblasts (eSTBs) are highly susceptible to SARS-CoV-2 infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. From human expanded potential stem cells (hEPSCs), we derived bona fide trophoblast stem cells (TSCs) that resembled those originated from the blastocyst and the placenta in generating functional syncytiotrophoblasts (STBs) and extravillus trophoblasts (EVTs) and in low expression of HLA-A/B and amniotic epithelial (AME) cell signature. The EPSC-TSCs and their derivative trophoblasts including trophoblast organoids could be infected by SARS-CoV-2. Remarkably, eSTBs expressed high levels of ACE2 and produced substantially higher amounts of virion than Vero E6 cells which are widely used in SARS-CoV-2 research and vaccine production. These findings provide experimental evidence for the clinical observations that opportunistic SARS-CoV-2 infection during pregnancy can occur. At low concentrations, two well characterized antivirals, remdesivir and GC376, effectively eliminated infection of eSTBs by SARS-CoV-2 and middle east respiratory syndrome-related coronavirus (MERS-CoV), and rescued their developmental arrest caused by the virus infection. Several human cell lines have been used in coronavirus research. However, they suffer from genetic and/or innate immune defects and have some of the long-standing technical challenges such as cell transfection and genetic manipulation. In contrast, hEPSCs are normal human stem cells that are robust in culture, genetically stable and permit efficient gene-editing. They can produce and supply large amounts of physiologically relevant normal and genome-edited human cells such as eSTBs for isolation, propagation and production of coronaviruses for basic research, antivirus drug tests and safety evaluation.","version":"1.1","doi":"10.1101/2022.11.17.516978","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.18.517035","pub_date":"2022-11-18","title":"Design and validation of an exposure system for efficient inter-animal SARS-CoV-2 airborne transmission in Syrian hamsters","abstract":"SARS-CoV-2 is a highly transmissible respiratory pathogen whose main transmission route is airborne. Development of an animal model and exposure system that recapitulates airborne transmission of SARS-CoV-2 is integral for understanding the dynamics of SARS-CoV-2 spread in individuals and populations. Here we designed, built, and characterized a hamster transmission caging and exposure system that allows for efficient SARS-CoV-2 airborne transmission from an infected index animal to na\u00efve recipients under unidirectional airflow, without contribution from fomite or direct contact transmission. To validate our system, we assessed a 1:1 or 1:4 ratio of infected index to naive recipient hamsters and compared their virological and clinical measurements after eight hours of airborne exposure. Airborne exposure concentrations and pulmonary deposited dose of SARS-CoV-2 in index and na\u00efve hamsters, respectively, were similar in both groups. Daily nasal viral RNA levels, and terminal (day 5) lung viral RNA and infectious virus, and fecal viral RNA levels were statistically similar among 1:1 and 1:4 naive animals. However, virological measurements in the 1:4 na\u00efve animals were more variable than the 1:1 na\u00efve animals, likely due to hamster piling behavior creating uneven SARS-CoV-2 exposure during the grouped 1:4 airborne exposure. This resulted in slight, but not statistically significant, changes in daily body weights between the 1:1 and 1:4 naive groups. Our report describes a multi-chamber caging and exposure system that allowed for efficient SARS-CoV-2 airborne transmission in single and grouped hamsters. This system can be used to better define airborne transmission dynamics and test transmission-blocking therapeutic strategies against SARS-CoV-2. The main route of SARS-CoV-2 transmission is airborne. However, there are few experimental systems that can assess airborne transmission dynamics of SARS-CoV-2 in vivo. Here, we designed, built, and characterized a hamster transmission caging and exposure system that allows for efficient SARS-CoV-2 airborne transmission in Syrian hamsters, without contributions from fomite or direct contact transmission. We successfully measured SARS-CoV-2 viral RNA in aerosols and demonstrated that SARS-CoV-2 is transmitted efficiently at either a 1:1 or 1:4 infected index to na\u00efve recipient hamster ratio. This is meaningful as a 1:4 infected index to na\u00efve hamster ratio would allow for simultaneous comparisons of various interventions in na\u00efve animals to determine their susceptibility of infection by aerosol transmission of SARS-CoV-2. Our SARS-CoV-2 exposure system allows for testing viral airborne transmission dynamics and transmission-blocking therapeutic strategies against SARS-CoV-2 in Syrian hamsters.","version":"1.1","doi":"10.1101/2022.11.18.517035","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.18.517047","pub_date":"2022-11-18","title":"Innate immune response to SARS-CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons","abstract":"Severe acute respiratory coronavirus 2 (SARS-CoV-2) infection causes neurological disease in some patients suggesting that infection can affect both the peripheral and central nervous system (PNS and CNS, respectively). It is not clear whether the outcome of SARS-CoV-2 infection of PNS and CNS neurons is similar, and which are the key factors that cause neurological disease: SARS-CoV-2 infection or the subsequent immune response. Here, we addressed these questions by infecting human induced-pluripotent stem cell-derived CNS and PNS neurons with the \u03b2 strain of SARS-CoV-2. Our results show that SARS-CoV-2 infects PNS neurons more efficiently than CNS neurons, despite lower expression levels of angiotensin converting enzyme 2. Infected PNS neurons produced interferon \u03bb1, several interferon stimulated genes and proinflammatory cytokines. They also displayed neurodegenerative-like alterations, as indicated by increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and \u03b1-synuclein and lower levels of nicotinamide mononucleotide adenylyltransferase 2 and \u03b2-III-tubulin. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neurodegeneration, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS.","version":"1.1","doi":"10.1101/2022.11.18.517047","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.18.517046","pub_date":"2022-11-18","title":"Rising SARS-CoV-2 Seroprevalence and Patterns of Cross-Variant Antibody Neutralization in UK Domestic Cats","abstract":"Recent evidence confirming cat-to-human SARS-CoV-2 transmission has highlighted the importance of monitoring infection in domestic cats. Although the effects of SARS-CoV-2 infection on feline health are poorly characterized, cats have close contact with humans, and with both domesticated and wild animals. Accordingly, they could act as a reservoir of infection, an intermediate host and a source of novel variants. To investigate the spread of the virus in the cat population, serum samples were tested for SARS-CoV-2 antibodies by ELISA and a pseudotype-based virus neutralization assay, designed to detect exposure to variants known to be circulating in the human population. Overall seroprevalence was 3.2%, peaking at 5.3% in autumn 2021. Variant-specific neutralizing antibody responses were detected with titers waning over time. The variant-specific response in the feline population correlated with and trailed the variants circulating in the human population, indicating multiple ongoing human-to-cat spill-over events.","version":"1.1","doi":"10.1101/2022.11.18.517046","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.15.516323","pub_date":"2022-11-18","title":"The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity","abstract":"The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria during infection, where it inhibits innate immunity by restricting IFN-\u03b2 production, but not NF-\u03baB activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c acts after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen.","version":"1.2","doi":"10.1101/2022.11.15.516323","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.17.516905","pub_date":"2022-11-17","title":"Effect of surfactants on SARS-CoV-2: Molecular Dynamics Simulations","abstract":"Surfactants are commonly used as disinfection agents in personal care products against bacteria and viruses, including SARS-CoV-2. However, there is a lack of understanding of the molecular mechanisms of the inactivation of viruses by surfactants. Here, we employ coarse grain (CG) and all-atom (AA) molecular dynamics simulations to investigate the interaction between general families of surfactants and the SARS-CoV-2 virus. To this end, we considered a CG model of a full virion. Overall, we found that surfactants have only a small impact over the virus envelope, being inserted into the envelope without dissolving it or generating pores, at the conditions considered here. However, we found that surfactants may induce a deep impact on the spike protein of the virus (responsible for its infectivity), easily covering it and inducing its collapse over the envelope surface of the virus. AA simulations confirmed that both negatively and positively charged surfactants are able to extensively adsorb over the spike protein and get inserted into the virus envelope. Our results suggest that the best strategy for the design of surfactants as virucidal agents will be to focus on those strongly interacting with the spike protein.","version":"1.1","doi":"10.1101/2022.11.17.516905","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.01.502301","pub_date":"2022-11-17","title":"Free energy perturbation calculations of mutation effects on SARS-CoV-2 RBD::ACE2 binding affinity","abstract":"The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein-protein binding affinities and in practical applications such as the optimization of neutralizing antibodies.","version":"1.2","doi":"10.1101/2022.08.01.502301","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.17.516898","pub_date":"2022-11-17","title":"Improved Neutralization of Omicron BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 with Bivalent BA.4/5 Vaccine","abstract":"The BNT162b2 bivalent BA.4/5 COVID-19 vaccine has been authorized to mitigate COVID-19 due to current Omicron and potentially future variants. New sublineages of SARS-CoV-2 Omicron continue to emerge and have acquired additional mutations, particularly in the spike protein, that may lead to improved viral fitness and immune evasion. The present study characterized neutralization activities against new Omicron sublineages BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 after a 4th dose (following three doses of BNT162b2) of either the original monovalent BNT162b2 or the bivalent BA.4/5 booster in individuals >55 years of age. For all participants, the 4th dose of monovalent BNT162b2 vaccine induced a 3.0\u00d7, 2.9\u00d7, 2.3\u00d7, 2.1\u00d7, 1.8\u00d7, and 1.5\u00d7 geometric mean neutralizing titer fold rise (GMFR) against USA/WA1-2020 (a strain isolated in January 2020), BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1, respectively; the bivalent vaccine induced 5.8\u00d7, 13.0\u00d7, 11.1\u00d7, 6.7\u00d7, 8.7\u00d7, and 4.8\u00d7 GMFRs. For individuals without SARS-CoV-2 infection history, BNT162b2 monovalent induced 4.4\u00d7, 3.0\u00d7, 2.5\u00d7, 2.0\u00d7, 1.5\u00d7, and 1.3\u00d7 GMFRs, respectively; the bivalent vaccine induced 9.9\u00d7, 26.4\u00d7, 22.2\u00d7, 8.4\u00d7, 12.6\u00d7, and 4.7\u00d7 GMFRs. These data suggest the bivalent BA.4/5 vaccine is more immunogenic than the original BNT162b2 monovalent vaccine against circulating Omicron sublineages, including BQ.1.1 that is becoming prevalent globally.","version":"1.1","doi":"10.1101/2022.11.17.516898","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.15.516662","pub_date":"2022-11-16","title":"SpikeScape: A Tool for Analyzing Structural Diversity in Experimental Structures of the SARS-CoV-2 Spike Glycoprotein","abstract":"In this application note we describe a tool which we developed to help structural biologists who study the SARS-CoV-2 spike glycoprotein. There are more than 500 structures of this protein available in the Protein Data Bank. These structures are available in different flavors: wild type spike, different variants, 2P substitutions, structures with bound antibodies, structures with Receptor Binding Domains in closed or open conformation, etc. Understanding differences between these structures could provide insight to how the spike structure changes in different variants or upon interaction with different molecules such as receptors or antibodies. However, inconsistencies among deposited structures, such as different chain or sequence numbering, hamper a straightforward comparison of all structures. The tool described in this note fixes those inconsistencies and calculates the distribution of the requested distance between any two atoms across all SARS-CoV-2 spike structures available in the Protein Data Bank, with the option to filter by various selections. The tool provides a histogram and cumulative frequency of the calculated distribution, as the ability to download the results and corresponding PDB IDs.","version":"1.1","doi":"10.1101/2022.11.15.516662","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.16.516726","pub_date":"2022-11-16","title":"\u03b2-Cyclodextrins as affordable antivirals to treat coronavirus infection","abstract":"The SARS-CoV-2 pandemic made evident that we count with few coronavirus-fighting drugs. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety and tolerability profiles. We began elaborating a list of 116 drugs previously used to treat other pathologies or characterized in pre-clinical studies with potential to treat coronavirus infections. We next employed molecular modelling tools to rank the 44 most promising inhibitors and tested their efficacy as antivirals against a panel of \u03b1 and \u03b2 coronavirus, e.g., the HCoV-229E and SARS-CoV-2 viruses. Four drugs, OSW-1, U18666A, hydroxypropyl-\u03b2-cyclodextrin (H\u03b2CD) and phytol, showed antiviral activity against both HCoV-229E (in MRC5 cells) and SARS-CoV-2 (in Vero E6 cells). The mechanism of action of these compounds was studied by transmission electron microscopy (TEM) and by testing their capacity to inhibit the entry of SARS-CoV-2 pseudoviruses in ACE2-expressing HEK-293T cells. The entry was inhibited by H\u03b2CD and U18666A, yet only H\u03b2CD could inhibit SARS-CoV-2 replication in the pulmonary cells Calu-3. With these results and given that cyclodextrins are widely used for drug encapsulation and can be safely administered to humans, we further tested 6 native and modified cyclodextrins, which confirmed \u03b2-cyclodextrins as the most potent inhibitors of SARS-CoV-2 replication in Calu-3 cells. All accumulated data points to \u03b2-cyclodextrins as promising candidates to be used in the therapeutic treatments for SARS-CoV-2 and possibly other respiratory viruses.","version":"1.1","doi":"10.1101/2022.11.16.516726","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.09.467981","pub_date":"2022-11-15","title":"Fibroblast-expressed LRRC15 suppresses SARS-CoV-2 infection and controls antiviral and antifibrotic transcriptional programs","abstract":"Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here we use whole genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe COVID-19 infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.","version":"1.2","doi":"10.1101/2021.11.09.467981","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516206","pub_date":"2022-11-14","title":"SARS-CoV-2 vaccination of laboratory rhesus monkeys (Macaca mulatta): Monitoring and efficacy","abstract":"The availability of effective vaccines and a high vaccination rate allowed the recent mitigation, or even withdrawal, of many protective measures for containing the SARS CoV-2 pandemic. At the same time, new and highly mutated variants of the virus are found to have significantly higher transmissibility and reduced vaccine efficacy, thus causing high infection rates during the third year of the pandemic. The combination of reduced measures and increased infectivity poses a particular risk for unvaccinated individuals, including animals susceptible to the virus. Among the latter, non-human primates (NHPs) are particularly vulnerable. They serve as important models in various fields of biomedical research and because of their cognitive capabilities, they receive particular attention in animal welfare regulations around the world. Yet, although they played an extraordinarily important role for developing and testing vaccines against SARS-CoV-2, the protection of captive rhesus monkeys against Covid-19 has rarely been discussed. We here report upon twofold mRNA vaccination of a cohort of 19 rhesus monkeys (Macaca mulatta) against infection by SARS-CoV-2. All animals were closely monitored on possible side effects of vaccination, and were tested for neutralising antibodies against the virus. The data show that vaccination of rhesus monkeys is a safe and reliable measure to protect these animals against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.11.11.516206","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516239","pub_date":"2022-11-14","title":"Efficient SARS-CoV-2 detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis","abstract":"The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to a pandemic threat in the form of vaccines and antigen tests. The causative agent of COVID-19 is SARS-CoV-2. The spike protein on the virus surface interacts with the human angiotensin-converting enzyme (ACE2) via the so-called receptor binding domain (RBD), facilitating virus entry. The RBD thus represents a prime target for vaccines, therapeutic antibodies, and antigen test systems. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. In this study, we produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the viral RBD as Cts1 fusions and screened their RBD binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of an inexpensive antigen test utilizing unconventionally secreted nanobodies as RBD trap and a matching ubiquitous and biogenic surface for immobilization.","version":"1.1","doi":"10.1101/2022.11.11.516239","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.14.516398","pub_date":"2022-11-14","title":"Multi-omic spatial profiling reveals the unique virus-driven immune landscape of COVID-19 placentitis","abstract":"COVID-19 placentitis, a rare complication of maternal SARS-CoV-2 infection, only shows detectable virus in the placenta of a subset of cases. We provide a deep multi-omic spatial characterisation of placentitis from obstetrically complicated maternal COVID-19 infection. We found that SARS-CoV-2 infected placentas have a distinct transcriptional and immunopathological signature. This signature overlaps with virus-negative cases supporting a common viral aetiology. An inverse correlation between viral load and disease duration suggests viral clearance over time. Quantitative spatial analyses revealed a unique microenvironment surrounding virus-infected trophoblasts characterised by PDL1-expressing macrophages, T-cell exclusion, and interferon blunting. In contrast to uninfected mothers, ACE2 was localised to the maternal side of the placental trophoblast layer of almost all mothers with placental SARS-CoV-2 infection, which may explain variable susceptibility to placental infection. Our results demonstrate a pivotal role for direct placental SARS-CoV-2 infection in driving the unique immunopathology of COVID-19 placentitis.","version":"1.1","doi":"10.1101/2022.11.14.516398","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516052","pub_date":"2022-11-14","title":"Genetic and structural data on the SARS-CoV-2 Omicron BQ.1 variant reveal its low potential for epidemiological expansion","abstract":"The BQ.1 SARS-CoV-2 variant, also known as Cerberus, is one of the most recent Omicron descendant lineages. Compared to its direct progenitor BA.5, BQ.1 carries out some additional spike mutations in some key antigenic site which confer it further immune escape ability over other circulating lineage. In such a context, here we performed a genome-based survey aimed to obtain an as complete as possible nuance of this rapidly evolving Omicron subvariant. Genetic data suggests that BQ.1 represents an evolutionary blind background, lacking of the rapid diversification which is typical of a dangerous lineage. Indeed, the evolutionary rate of BQ.1 is very similar to that of BA.5 (7.6 \u00d7 10\u22124 and 7 \u00d7 10\u22124 subs/site/year, respectively), which is circulating by several months. Bayesian Skyline Plot reconstruction, indicates low level of genetic variability, suggesting that the peak has been reached around September 3, 2022. Structure analyses performed by comparing the properties of BQ.1 and BA.5 RBD indicated that the impact of the BQ.1 mutations on the affinity for ACE2 may be modest. Likewise, immunoinformatic analyses showed modest differences between the BQ.1 and the BA5 potential B-cells epitope. In conclusion, genetic and structural analysis on SARS-CoV-2 BQ.1 suggest that, it does not show evidence about its particular dangerous or high expansion capability. The monitoring genome-based must continue uninterrupted for a better understanding of its descendant and all other lineages.","version":"1.1","doi":"10.1101/2022.11.11.516052","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516125","pub_date":"2022-11-14","title":"Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses","abstract":"SARS-CoV-2 mutational variants evade humoral immune responses elicited by vaccines and current monoclonal antibody (mAb) therapies. Novel antibody-based treatments will thus need to exhibit broad neutralization against different variants. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies into one antibody taking advantage of the avidity, synergy and cooperativity provided by targeting two different epitopes. Here we used controlled Fab-arm exchange (cFAE), a versatile and straightforward method, to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader but less potent antibodies that also neutralize SARS-CoV. We demonstrate that the parental IgG\u2019s rely on avidity for their neutralizing activity by comparing their potency to bsAbs containing one irrelevant \u201cdead\u201d Fab arm. We used single particle mass photometry to measure formation of antibody:spike complexes, and determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike (S), observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent or multivalent agents to provide a robust activity against circulating variants, as well as future SARS-like coronaviruses.","version":"1.1","doi":"10.1101/2022.11.11.516125","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.10.516025","pub_date":"2022-11-11","title":"Cotranslational formation of disulfides guides folding of the SARS COV-2 receptor binding domain","abstract":"Many secreted proteins contain multiple disulfide bonds. How disulfide formation is coupled to protein folding in the cell remains poorly understood at the molecular level. Here, we combine experiment and simulation to address this question as it pertains to the SARS-CoV-2 receptor binding domain (RBD). We show that, whereas RBD can refold reversibly when its disulfides are intact, their disruption causes misfolding into a nonnative molten-globule state that is highly prone to aggregation and disulfide scrambling. Thus, non-equilibrium mechanisms are needed to ensure disulfides form prior to folding in vivo. Our simulations suggest that co-translational folding may accomplish this, as native disulfide pairs are predicted to form with high probability at intermediate lengths, ultimately committing the RBD to its metastable native state and circumventing nonnative intermediates. This detailed molecular picture of the RBD folding landscape may shed light on SARS-CoV-2 pathology and molecular constraints governing SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2022.11.10.516025","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.23.509206","pub_date":"2022-11-11","title":"A pan-variant mRNA-LNP T cell vaccine protects HLA transgenic mice from mortality after infection with SARS-CoV-2 Beta","abstract":"Clinically licensed COVID-19 vaccines ameliorate viral infection by inducing vaccinee production of neutralizing antibodies that bind to the SARS-CoV-2 Spike protein to inhibit viral cellular entry (Walsh et al., 2020; Baden et al., 2021), however the clinical effectiveness of these vaccines is transitory as viral variants arise that escape antibody neutralization (Tregoning et al., 2021; Willett et al., 2022). Vaccines that solely rely upon a T cell response to combat viral infection could be transformational because they can be based on highly conserved short peptide epitopes that hold the potential for pan-variant immunity, but a mRNA-LNP T cell vaccine has not been shown to be sufficient for effective antiviral prophylaxis. Here we show that a mRNA-LNP vaccine based on highly conserved short peptide epitopes activates a CD8+ and CD4+ T cell response that prevents mortality in HLA-A*02:01 transgenic mice infected with the SARS-CoV-2 Beta variant of concern (B.1.351). In mice vaccinated with the T cell vaccine, 24% of the nucleated cells in lung were CD8+ T cells on day 7 post infection. This was 5.5 times more CD8+ T cell infiltration of the lungs in response to infection compared to the Pfizer-BioNTech Comirnaty\u00ae vaccine. Between days 2 and 7 post infection, the number of CD8+ T cells in the lung increased in mice vaccinated with the T cell vaccine and decreased in mice vaccinated with Comirnaty\u00ae. The T cell vaccine did not produce neutralizing antibodies, and thus our results demonstrate that SARS-CoV-2 viral infection can be controlled by a T cell response alone. Our results suggest that further study is merited for pan-variant T cell vaccines, and that T cell vaccines may be relevant for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.","version":"1.3","doi":"10.1101/2022.09.23.509206","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.11.516111","pub_date":"2022-11-11","title":"Attenuated humoral responses in HIV infection after SARS-CoV-2 vaccination are linked to global B cell defects and cellular immune profiles","abstract":"People living with HIV (PLWH) on suppressive antiretroviral therapy (ART) can have residual immune dysfunction and often display poorer responses to vaccination. We assessed in a cohort of PLWH (n=110) and HIV negative controls (n=64) the humoral and spike-specific B-cell responses following 1, 2 or 3 SARS-CoV-2 vaccine doses. PLWH had significantly lower neutralizing antibody (nAb) titers than HIV-negative controls at all studied timepoints. Moreover, their neutralization breadth was reduced with fewer individuals developing a neutralizing response against the Omicron variant (BA.1) relative to controls. We also observed a delayed development of neutralization in PLWH that was underpinned by a reduced frequency of spike-specific memory B cells (MBCs) and pronounced B cell dysfunction. Improved neutralization breadth was seen after the third vaccine dose in PLWH but lower nAb responses persisted and were associated with global, but not spike-specific, MBC dysfunction. In contrast to the inferior antibody responses, SARS-CoV-2 vaccination induced robust T cell responses that cross-recognized variants in PLWH. Strikingly, a subset of PLWH with low or absent neutralization had detectable functional T cell responses. These individuals had reduced numbers of circulating T follicular helper cells and an enriched population of CXCR3+CD127+CD8+ T cells after two doses of SARS-CoV-2 vaccination, which may compensate for sub-optimal serological responses in the event of infection. Therefore, normalisation of B cell homeostasis could improve serological responses to vaccines in PLWH and evaluating T cell immunity could provide a more comprehensive immune status profile in these individuals and others with B cell imbalances.","version":"1.1","doi":"10.1101/2022.11.11.516111","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.07.515557","pub_date":"2022-11-11","title":"Normalized Semi-Covariance Co-Efficiency Analysis of Spike Proteins from SARS-CoV-2 variant Omicron and Other Coronaviruses for their Infectivity and Virulence","abstract":"Spectrum-based Mass-Charge modeling is increasingly used in biological analysis. To explain statistical phenomenon with positive and negative fluctuations of amino acid charges in spike protein sequences from Omicron and other coronaviruses, we propose calculation-based Mass-Charge modeling, a normalized derivation algorithm with exact Excel and MATLAB tool involving separate quadrant extension to normalized covariance, which is still compatible with Pearson covariance co-efficiency. The number of amino acids, molecular weight, isoelectric point, amino acid composition, charged residues, mass-charge ratio, hydropathicity of the proteins were taken into consideration in the analyses, and the relative peak and dip of the average with spike protein sequences based on hydrophobic mass to isoelectric charges of amino acids were also examined. The analyses with the algorithm provide more clear insights leading to revealing underline evolving trends of the viral proteins. Spike proteins from SARS-CoV-2 variants, seasonal and murine coronaviruses were taken as representative examples in this study. The analyses demonstrate that the Mass-Charge covariance co-efficiency can distinguish subtle differences between biological properties of spike proteins and correlate well with viral infectivity and virulence.","version":"1.2","doi":"10.1101/2022.11.07.515557","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.04.515139","pub_date":"2022-11-10","title":"Evasive spike variants elucidate the preservation of T cell immune response to the SARS-CoV-2 omicron variant","abstract":"The Omicron variants boast the highest infectivity rates among all SARS-CoV-2 variants. Despite their lower disease severity, they can reinfect COVID-19 patients and infect vaccinated individuals as well. The high number of mutations in these variants render them resistant to antibodies that otherwise neutralize the spike protein of the original SARS-CoV-2 spike protein. Recent research has shown that despite its strong immune evasion, Omicron still induces strong T Cell responses similar to the original variant. This work investigates the molecular basis for this observation using the neural network tools NetMHCpan-4.1 and NetMHCiipan-4.0. The antigens presented through the MHC Class I and Class II pathways from all the notable SARS-CoV-2 variants were compared across numerous high frequency HLAs. All variants were observed to have equivalent T cell antigenicity. A novel positive control system was engineered in the form of spike variants that did evade T Cell responses, unlike Omicron. These evasive spike proteins were used to statistically confirm that the Omicron variants did not exhibit lower antigenicity in the MHC pathways. These results suggest that T Cell immunity mounts a strong defense against COVID-19 which is difficult for SARS-CoV-2 to overcome through mere evolution.","version":"1.1","doi":"10.1101/2022.11.04.515139","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.09.515832","pub_date":"2022-11-10","title":"Helminth exposure protects against murine SARS-CoV-2 infection through macrophage dependent T cell activation","abstract":"Helminth endemic regions report lower COVID-19 morbidity and mortality. Here, we show that lung remodeling from a prior infection with a lung migrating helminth, Nippostrongylus brasiliensis, enhances viral clearance and survival of human-ACE2 transgenic mice challenged with SARS-CoV-2 (SCV2). This protection is associated with a lymphocytic infiltrate including an increased accumulation of pulmonary SCV2-specific CD8+ T cells and anti-CD8 antibody depletion abrogated the N. brasiliensis-mediated reduction in viral loads. Pulmonary macrophages with a type-2 transcriptional signature persist in the lungs of N. brasiliensis exposed mice after clearance of the parasite and establish a primed environment for increased antigen presentation. Accordingly, depletion of macrophages ablated the augmented viral clearance and accumulation of CD8+ T cells driven by prior N. brasiliensis infection. Together, these findings support the concept that lung migrating helminths can limit disease severity during SCV2 infection through macrophage-dependent enhancement of anti-viral CD8+ T cell responses.","version":"1.1","doi":"10.1101/2022.11.09.515832","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.08.515589","pub_date":"2022-11-10","title":"Identification of small molecules with virus growth enhancement properties","abstract":"The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the pandemic disease known as coronavirus disease 2019 (COVID-19). COVID-19 vaccines were developed at record speed and were authorized approximately a year after the original outbreak. This fast response saved the lives of countless individuals and reduced the disease burden of many more. The experience has served as a reminder of the necessity to implement solid vaccine development platforms and fast production pipelines. Manufacturing vaccines for enveloped viruses, including some SARS-CoV-2 vaccines, often relies on the production of large quantities of viruses in vitro. Thus, speeding up or increasing virus production would expedite vaccine development. With this objective in mind, we established a high throughput screening (HTS) to identify small molecules that enhance or speed up host-virus membrane fusion. Among the HTS hits, we identified that ethynylestradiol augments SARS-CoV-2 fusion activity in both the absence and presence of TMPRSS2. Furthermore, we confirmed that ethynylestradiol can boost the growth of not only SARS-CoV-2 but also Influenza A virus in vitro. A small molecule with these characteristics could be implemented to improve vaccines production. The (COVID-19) pandemic had a tremendous impact on our healthcare systems and the global economy. The rapid development of effective vaccines saved the lives of countless individuals and reduced the disease burden of many more. Intending to increase vaccine production, we developed and performed a high-throughput screening (HTS) to identify small molecules that enhance viral and cellular membrane fusion. Among the HTS hits, we confirmed that Ethynylestradiol can boost the growth of SARS-CoV-2 and Influenza A virus in vitro.","version":"1.1","doi":"10.1101/2022.11.08.515589","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.08.22282050","pub_date":"2022-11-10","title":"Characterizing responsiveness to the COVID-19 pandemic in the United States and Canada using mobility data","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Mobile phone-derived human mobility data are a proxy for disease transmission risk and have proven useful during the COVID-19 pandemic for forecasting cases and evaluating interventions. We propose a novel metric using mobility data to characterize responsiveness to rising case rates.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We examined weekly reported COVID-19 incidence and retail and recreation mobility from Google Community Mobility Reports for 50 U.S. states and nine Canadian provinces from December 2020 to November 2021. For each jurisdiction, we calculated the responsiveness of mobility to COVID-19 incidence when cases were rising. Responsiveness across countries was summarized using subgroup meta-analysis. We also calculated the correlation between the responsiveness metric and the reported COVID-19 death rate during the study period.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>\n                    Responsiveness in Canadian provinces (\n                    <jats:italic>\u03b2</jats:italic>\n                    = -1\u00b745; 95% CI: -2\u00b745, -0\u00b744) was approximately five times greater than in U.S. states (\n                    <jats:italic>\u03b2</jats:italic>\n                    = -0\u00b730; 95% CI: -0\u00b738, -0\u00b721). Greater responsiveness was moderately correlated with a lower reported COVID-19 death rate during the study period (Spearman\u2019s\n                    <jats:italic>\u03c1</jats:italic>\n                    = 0\u00b751), whereas average mobility was only weakly correlated the COVID-19 death rate (Spearman\u2019s\n                    <jats:italic>\u03c1</jats:italic>\n                    = 0\u00b720).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Our study used a novel mobility-derived metric to reveal a near-universal phenomenon of reductions in mobility subsequent to rising COVID-19 incidence across 59 states and provinces of the U.S. and Canada, while also highlighting the different public health approaches taken by the two countries.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This study received no funding.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before the study</jats:title>\n                    <jats:p>There exists a wide body of literature establishing the usefulness of mobile phone-derived human mobility data for forecasting cases and other metrics during the COVID-19 pandemic. We performed a literature search to identify studies examining the opposite relationship, attempting to quantify the responsiveness of human mobility to changes in COVID-19 incidence. We searched PubMed on October 21, 2022 using the keywords \u201cCOVID-19\u201d, \u201c2019-nCoV\u201d, or \u201cSARS-CoV-2\u201d in combination with \u201cresponsiveness\u201d and one or more of \u201cmobility\u201d, \u201cdistancing\u201d, \u201clockdown\u201d, and \u201cnon-pharmaceutical interventions\u201d. We scanned 46 published studies and found one that used a mobile phone data-derived index to measure the intensity of social distancing in U.S. counties from January 2020 to January 2021. The authors of this study found that an increase in cases in the last 7 days was associated with an increase in the intensity of social distancing, and that this effect was larger during periods of lockdown/shop closures.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of the study</jats:title>\n                    <jats:p>Our study developed a metric of the responsiveness of mobility to rising case rates for COVID-19 and calculated it for 59 subnational jurisdictions in the United States and Canada. While nearly all jurisdictions displayed some degree of responsiveness, average responsiveness in Canada was nearly five times greater than in the United States. Responsiveness was moderately associated with the reported COVID-19 death rate during the study period, such that jurisdictions with greater responsiveness had lower death rates, and was more strongly associated with death rates than average mobility in a jurisdiction.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>Mobile phone-derived human mobility data has proven useful in the context of infectious disease surveillance during the COVID-19 pandemic, such as for forecasting cases and evaluating non-pharmaceutical interventions. In our study, we derived a metric of responsiveness to show that mobility data may be used to track the efficiency of public health responses as the pandemic evolves. This responsiveness metric was also correlated with reported COVID-19 death rates during the study period. Together, these results demonstrate the usefulness of mobility data for making broad characterizations of public health responses across jurisdictions during the COVID-19 pandemic and reinforce the value of mobility data as an infectious disease surveillance tool for answering present and future threats.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2022.11.08.22282050","journal":"medRxiv","score":null},{"id":"10.1101/2022.11.09.515752","pub_date":"2022-11-09","title":"SARS-CoV-2 ORF8 limits expression levels of Spike antigen","abstract":"Survival from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of SARS-CoV-2 mechanisms counteracting antibody-mediated immunity is needed. Here, we report that ORF8 is a SARS-CoV-2 factor that controls cellular Spike antigen levels. ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. With limited Spike availability, ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Our studies propose an ORF8-dependent SARS-CoV-2 strategy that allows immune evasion of infected cells for extended viral production.","version":"1.1","doi":"10.1101/2022.11.09.515752","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.12.246389","pub_date":"2022-11-09","title":"Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture","abstract":"In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drugtreatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.","version":"1.2","doi":"10.1101/2020.08.12.246389","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.08.515725","pub_date":"2022-11-09","title":"Cross-neutralization and viral fitness of SARS-CoV-2 Omicron sublineages","abstract":"The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 \u2265 BA.2 \u2265 BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguisable cross-neutralizations against Omicron sublineages, but exhibited little neutralizations against the index virus (i.e., USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titers did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.","version":"1.1","doi":"10.1101/2022.11.08.515725","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.09.515748","pub_date":"2022-11-09","title":"Impact of variants of concern on SARS-CoV-2 viral dynamics in non-human primates","abstract":"The impact of variants of concern (VoC) on SARS-CoV-2 viral dynamics remains poorly understood and essentially relies on observational studies subject to various sorts of biases. In contrast, experimental models of infection constitute a powerful model to perform controlled comparisons of the viral dynamics observed with VoC and better quantify how VoC escape from the immune response. Here we used molecular and infectious viral load of 78 cynomolgus macaques to characterize in detail the effects of VoC on viral dynamics. We first developed a mathematical model that recapitulate the observed dynamics, and we found that the best model describing the data assumed a rapid antigen-dependent stimulation of the immune response leading to a rapid reduction of viral infectivity. When compared with the historical variant, all VoC except beta were associated with an escape from this immune response, and this effect was particularly sensitive for delta and omicron variant (p<10\u22126 for both). Interestingly, delta variant was associated with a 1.8-fold increased viral production rate (p=0.046), while conversely omicron variant was associated with a 14-fold reduction in viral production rate (p<10\u22126). During a natural infection, our models predict that delta variant is associated with a higher peak viral RNA than omicron variant (7.6 log10 copies/mL 95% CI 6.8 \u2013 8 for delta; 5.6 log10 copies/mL 95% CI 4.8 \u2013 6.3 for omicron) while having similar peak infectious titers (3.7 log10 PFU/mL 95% CI 2.4 \u2013 4.6 for delta; 2.8 log10 PFU/mL 95% CI 1.9 \u2013 3.8 for omicron). These results provide a detailed picture of the effects of VoC on total and infectious viral load and may help understand some differences observed in the patterns of viral transmission of these viruses.","version":"1.1","doi":"10.1101/2022.11.09.515748","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.08.515436","pub_date":"2022-11-08","title":"Large clones of pre-existing T cells drive early immunity against SARS-COV-2 and LCMV infection","abstract":"We analyzed the dynamics of the earliest T cell response to SARS-COV-2. A wave of TCRs strongly but transiently expand during infection, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Most epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains, but not with circulating human coronaviruses. Many expanding CDR3s were also present at high precursor frequency in pre-pandemic TCR repertoires. A similar set of early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the pre-infection na\u00efve repertoire. High frequency na\u00efve precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection. High frequency na\u00efve precursors underly the rapid T cell response during the crucial early phases of acute viral infection.","version":"1.1","doi":"10.1101/2022.11.08.515436","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.28.501856","pub_date":"2022-11-08","title":"Computer Simulation of the interaction between SARS-CoV-2 Spike Protein and the Surface of Coinage Metals","abstract":"A prominent feature of the SARS-CoV-2 virus is the presence of a large glycoprotein spike protruding from the virus envelope. The spike determines the interaction of the virus with the environment and the host. Here, we used an all-atom molecular dynamics simulation method to investigate the interaction of up and down conformations of the S1 subunit of the SARS-CoV-2 spike with the (100) surface of Au, Ag and Cu. Our results revealed that the spike protein is adsorbed onto the surface of these metals, being Cu the metal with the highest interaction with the spike. In our simulations, we considered the spike protein in both its up conformation Sup (one receptor binding domain exposed) and down conformation Sdown (no exposed receptor binding domain). We found that the affinity of the metals for the up conformation was higher than their affinity for the down conformation. The structural changes in the Spike in the up conformation were also larger than the changes in the down conformation. Comparing the present results for metals with those obtained in our previous MD simulations of Sup with other materials (celulose, graphite, and human skin models), we see that Au induces the highest structural change in Sup, larger than those obtained in our previous studies.","version":"1.2","doi":"10.1101/2022.07.28.501856","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.08.515567","pub_date":"2022-11-08","title":"Enhanced stability of the SARS CoV-2 spike glycoprotein trimer following modification of an alanine cavity in the protein core","abstract":"The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity formed by Ala1016 and Ala1020 that form part of the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. The trimer stabilizing effects of filling the Ala1016/Ala1020 cavity was linked to improved S glycoprotein membrane fusion function. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited very high titers of neutralizing antibodies to ancestral and Delta-derived viruses (1/2,700-1/5,110), while neutralization titer was somewhat reduced with Omicron BA.1 (1/210-1,1744). The antigens elicited antibody specificities that could compete with ACE2-Fc for binding to the receptor-binding motif (RBM) and NAbs directed to key neutralization epitopes within the receptor-binding domain (RBD), N-terminal domain (NTD) and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S ectodomain trimers in the absence of an external trimerization motif (T4 foldon). The VI mutation represents a method for producing an intrinsically stable trimeric S ectodomain glycoprotein vaccine in the absence of a foreign trimerization tag. First-generation SARS CoV-2 vaccines that generate immune responses to ancestral Spike glycoprotein sequences have averted at least 14.4 million deaths, but their effectiveness against the recently emerged Omicron lineages is reduced. The updating of booster vaccines with variant Spike sequences are therefore likely required to maintain immunity as the pandemic continues to evolve. The Spike is a trimeric integral membrane protein with a membrane spanning sequence at its C-terminus. The Spike protein-based vaccine that is currently licensed for human use is produced by a complex process that reconstitutes the Spike in an artificial membrane. Alternatively, production of the Spike trimer as a soluble protein generally requires replacement of the membrane spanning sequence with a foreign often highly immunogenic trimerization motif that can complicate clinical advancement. We used systematic structure-directed mutagenesis coupled with functional studies to identify an alternative stabilization approach that negates the requirement for an external trimerization motif or membrane-spanning sequence. The replacement of 2 alanine residues that form a cavity in the core of the Spike trimer with bulkier hydrophobic residues resulted in increased Spike thermal stability. Thermostable Spike mutants retained major conserved neutralizing antibody epitopes and the ability to elicit broad and potent neutralizing antibody responses. One such mutation, referred to as VI, enabled the production of intrinsically stable Omicron variant Spike ectodomain trimers in the absence of an external trimerization motif. The VI mutation potentially enables a simplified method for producing a stable trimeric S ectodomain glycoprotein vaccine.","version":"1.1","doi":"10.1101/2022.11.08.515567","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.08.515673","pub_date":"2022-11-08","title":"Prediction of Transport, Deposition, and Resultant Immune Response of Nasal Spray Vaccine Droplets using a CFPD-HCD Model in a 6-Year-Old Upper Airway Geometry to Potentially Prevent COVID-19","abstract":"This study focuses on the transport, deposition, and triggered immune response of intranasal vaccine droplets to the Angiotensin-converting enzyme 2-rich region (i.e., the olfactory region (OR)) in the nasal cavity of a 6-year-old female to possibly prevent COVID-19. To investigate how administration strategy can influence nasal vaccine efficiency, a validated multiscale model (i.e., computational fluid-particle dynamics (CFPD) and host-cell dynamics (HCD) model) was employed. Droplet deposition fraction, size change, residence time, and the area percentage of OR covered by the vaccine droplets and triggered immune system response were predicted with different spray cone angles, initial droplet velocities, and compositions. Numerical results indicate that droplet initial velocity and composition have negligible influences on the vaccine delivery efficiency to OR. In contrast, the spray cone angle can significantly impact the vaccine delivery efficiency. The triggered immunity was not significantly influenced by the administration investigated in this study, due to the low percentage of OR area covered by the droplets. To enhance the effectiveness of the intranasal vaccine to prevent COVID-19 infection, it is necessary to optimize the vaccine formulation and administration strategy so that the vaccine droplets can cover more epithelial cells in OR to minimize the available receptors for SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.11.08.515673","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.07.515545","pub_date":"2022-11-08","title":"Optimization of the Illumina COVIDSeq\u2122 protocol for decentralized, cost-effective genomic surveillance","abstract":"A decentralized surveillance system to identify local outbreaks and monitor SARS-CoV-2 Variants of Concern is one of the primary strategies for the pandemic\u2019s containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the technology is still cost-prohibitive for decentralized sequencing, particularly in small independent labs with limited resources. We have optimized the Illumina COVID-seq protocol to reduce cost without compromising accuracy. 90% of genomic coverage was achieved for 142/153 samples analyzed in this study. The lineage was correctly assigned to all samples (152/153) except for one. This modified protocol can help laboratories with constrained resources contribute to decentralized SARS-CoV-2 surveillance in the post-vaccination era.","version":"1.1","doi":"10.1101/2022.11.07.515545","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.09.495482","pub_date":"2022-11-07","title":"ARF6 is a host factor for SARS-CoV-2 infection in vitro","abstract":"SARS-CoV-2 is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors, and the mechanism of fusion of the virus at the plasma membrane have been extensively investigated, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynamin but dependent on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV-2 replication and it is involved in the entry and infection of several pathogenic viruses. Using CRISPR-Cas9 genetic deletion, we observed that ARF6 is important for SARS-CoV-2 uptake and infection in Huh-7. This finding was corroborated using a pharmacologic inhibitor, whereby the ARF6 inhibitor NAV-2729 showed a dose-dependent inhibition of viral infection. Importantly, NAV-2729 reduced SARS-CoV-2 viral loads also in more physiologic models of infection: Calu-3 and kidney organoids. This highlighted the importance of ARF6 in multiple cell contexts. Together, these experiments points to ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.06.09.495482","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.05.515197","pub_date":"2022-11-07","title":"Pharmacological modulators of epithelial immunity uncovered by synthetic genetic tracing of SARS-CoV-2 infection responses","abstract":"Epithelial immune responses govern tissue homeostasis and offer drug targets against maladaptation. Here, we report a framework to generate drug discovery-ready reporters of cellular responses to viral infection. We reverse engineered epithelial cell responses to SARS-CoV-2, the viral agent fueling the ongoing COVID-19 pandemic and designed synthetic transcriptional reporters whose molecular logic comprises interferon-\u03b1/\u03b2/\u03b3-, and NF-\u03baB pathways. Such regulatory potential reflected single-cell data from experimental models to severe COVID-19 patient epithelial cells infected by SARS-CoV-2. SARS-CoV-2, type-I interferons, and RIG-I drive reporter activation. Live-cell-image-based phenotypic drug screens identified JAK inhibitors and DNA damage inducers as antagonistic modulators of epithelial cell response to interferons, RIG-I stimulation, and SARS-CoV-2. Synergistic or antagonistic modulation of the reporter by drugs underscored their similar mechanism of action. Thus, this study describes a tool for dissecting antiviral responses to infection and sterile cues, and a rapid approach to other emerging viruses of public health concern in order to discover rational drug combinations.","version":"1.1","doi":"10.1101/2022.11.05.515197","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.05.515305","pub_date":"2022-11-07","title":"Novel inhibitors against COVID-19 main protease suppressed viral infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, can cause severe disease with high mortality rates, especially among older and vulnerable populations. Despite the recent success of vaccines and approval of first-generation anti-viral inhibitor against SARS-CoV-2, an expanded arsenal of anti-viral compounds that limit viral replication and ameliorate disease severity is still urgently needed in light of the continued emergence of viral variants of concern (VOC). The main protease (Mpro) of SARS-CoV-2 is the major non-structural protein required for the processing of viral polypeptides encoded by the open reading frame 1 (ORF1) and ultimately replication. Structural conservation of Mpro among SARS-CoV-2 variants make this protein an attractive target for the anti-viral inhibition by small molecules. Here, we developed a structure-based in-silico screening of approximately 11 million compounds in ZINC15 database inhibiting Mpro, which prioritized 9 lead compounds for the subsequent in vitro validation in SARS-CoV-2 replication assays using both Vero and Calu-3 cells. We validated three of these compounds significantly inhibited SARS-CoV-2 replication in the micromolar range. In summary, our study identified novel small-molecules significantly suppressed infection and replication of SARS-CoV-2 in human cells.","version":"1.1","doi":"10.1101/2022.11.05.515305","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.07.515399","pub_date":"2022-11-07","title":"Long-range Hill-Robertson effect in adapting populations with recombination and standing variation","abstract":"In sexual populations, closely-situated genes have linked evolutionary fates, while genes spaced far in genome are commonly thought to evolve independently due to recombination. In the case where evolution depends essentially on supply of new mutations, this assumption has been confirmed by mathematical modeling. Here I examine it in the case of pre-existing genetic variation, where mutation is not important. A haploid population with N genomes, L loci, a fixed selection coefficient, and a small initial frequency of beneficial alleles f0 is simulated by a Monte-Carlo algorithm. The results demonstrate the existence of extremely strong linkage effects, including clonal interference and genetic background effects, that depend neither on the distance between loci nor on the average number of recombination crossovers. When the number of loci, L, is larger than 4log2(Nf0), beneficial alleles become extinct at most loci. The substitution rate varies broadly between loci, with the fastest rate exceeding the one-locus model prediction. All observables and the transition to the independent-locus limit are controlled by single composite parameter log2(Nf0)/L. The potential link between these findings and the emergence of new Variants of Concern of SARS CoV-2 is discussed.","version":"1.1","doi":"10.1101/2022.11.07.515399","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.22.504731","pub_date":"2022-11-06","title":"Contributions of adaptation and purifying selection to SARS-CoV-2 evolution","abstract":"Continued evolution and adaptation of SARS-CoV-2 has lead to more transmissible and immune-evasive variants with profound impact on the course of the pandemic. Here I analyze the evolution of the virus over 2.5 years since its emergence and estimate rates of evolution for synonymous and non-synonymous changes separately for evolution within clades \u2013 well defined mono-phyletic groups with gradual evolution \u2013 and for the pandemic overall. The rate of synonymous mutations is found to be around 6 changes per year. Synonymous rates within variants vary little from variant to variant and are compatible with the overall rate of 7 changes per year (or 7.5 \u00d7 10\u22124 per year and codon). In contrast, the rate at which variants accumulate amino acid changes (non-synonymous mutation) was initially around 12-16 changes per year, but in 2021 and 2022 dropped to 6-9 changes per year. The overall rate of non-synonymous evolution, that is across variants, is estimated to be about 26 amino acid changes per year (or 2.7 \u00d7 10\u22123 per year and codon). This strong acceleration of the overall rate compared to within clade evolution indicates that the evolutionary process that gave rise to the different variants is qualitatively different from that in typical transmission chains and likely dominated by adaptive evolution. I further quantify the spectrum of mutations and purifying selection in different SARS-CoV-2 proteins and show that the massive global sampling of SARS-CoV-2 is sufficient to estimate site specific fitness costs across the entire genome. Many accessory proteins evolve under limited evolutionary constraint with little short term purifying selection. About half of the mutations in other proteins are strongly deleterious.","version":"1.2","doi":"10.1101/2022.08.22.504731","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.31.513793","pub_date":"2022-11-04","title":"Antiviral effect of candies containing persimmon-derived tannin against SARS-CoV-2 delta strain","abstract":"Inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the mouth has the potential to reduce the spread of coronavirus disease 2019 (COVID-19) because the virus is readily transmitted by dispersed saliva. Persimmon-derived tannin has strong antioxidant and antimicrobial activity owing to its strong adhesiveness to proteins, and it also exhibited antiviral effects against non-variant and alpha variant SARS-CoV-2 in our previous study. In this report, we first demonstrated the antiviral effects of persimmon-derived tannin against the delta variant of SARS-CoV-2 in vitro via the plaque assay method. We then examined the effects of candy containing persimmon-derived tannin. Our plaque assay results show that saliva samples provided by healthy volunteers while they were eating tannin-containing candy remarkably suppressed the virus titers of the SARS-CoV-2 delta variant. In addition, we found that the SARS-CoV-2 viral load in saliva from patients with COVID-19 that was collected immediately after they had eaten the tannin-containing candy was below the level of detection by PCR for SARS-CoV-2. These data suggest that adding persimmon-derived tannin to candy and holding such candy in the mouth is an effective method by which to inactivate the SARS-CoV-2 in saliva, and the application of this approach has potential for inhibiting the transmission of COVID-19.","version":"1.1","doi":"10.1101/2022.10.31.513793","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510352","pub_date":"2022-11-04","title":"Distinct phenotype of SARS-CoV-2 Omicron BA.1 in human primary cells but no increased host range in cell lines of putative mammalian reservoir species","abstract":"SARS-CoV-2\u2019s genetic plasticity has led to several variants of concern (VOCs). Here we studied replicative capacity for seven SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, and Omicron BA.1) in primary reconstituted airway epithelia (HAE) and lung-derived cell lines. Furthermore, to investigate the host range of Delta and Omicron compared to ancestral SARS-CoV-2, we assessed replication in 17 cell lines from 11 non-primate mammalian species, including bats, rodents, insectivores and carnivores. Only Omicron\u2019s phenotype differed in vitro, with rapid but short replication and efficient production of infectious virus in nasal HAEs, in contrast to other VOCs, but not in lung cell lines. No increased infection efficiency for other species was observed, but Delta and Omicron infection efficiency was increased in A549 cells. Notably replication in A549 and Calu3 cells was lower than in nasal HAE. Our results suggest better adaptation of VOCs towards humans, without an extended host range.","version":"1.2","doi":"10.1101/2022.10.04.510352","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.31.514580","pub_date":"2022-11-04","title":"Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1, and XBB.1 by 4 doses of parental mRNA vaccine or a BA.5-bivalent booster","abstract":"The newly emerged SARS-CoV-2 Omicron BQ.1.1, XBB.1, and other sublineages have accumulated additional spike mutations that may affect vaccine effectiveness. Here we report neutralizing activities of three human serum panels collected from individuals 1-3 months after dose 4 of parental mRNA vaccine (post-dose-4), 1 month after a BA.5-bivalent-booster (BA.5-bivalent-booster), or 1 month after a BA.5-bivalent-booster with previous SARS-CoV-2 infection (BA.5-bivalent-booster-infection). Post-dose-4 sera neutralized USA-WA1/2020, BA.5, BF.7, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 SARS-CoV-2 with geometric mean titers (GMTs) of 1533, 95, 69, 62, 26, 22, and 15, respectively; BA.5-bivalent-booster sera improved the GMTs to 3620, 298, 305, 183, 98, 73, and 35; BA.5-bivalent-booster-infection sera further increased the GMTs to 5776, 1558,1223, 744, 367, 267, and 103. Thus, although BA.5-bivalent-booster elicits better neutralization than parental vaccine, it does not produce robust neutralization against the newly emerged Omicron BA.2.75.2, BQ.1.1, and XBB.1. Previous infection enhances the magnitude and breadth of BA.5-bivalent-booster-elicited neutralization.","version":"1.2","doi":"10.1101/2022.10.31.514580","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.03.515011","pub_date":"2022-11-04","title":"Evaluation of antibody kinetics and durability in health subjects vaccinated with inactivated COVID-19 vaccine (CoronaVac): A cross-sectional and cohort study in Zhejiang, China","abstract":"Although inactivated COVID-19 vaccines are proven to be safe and effective in the general population, the dynamic response and duration of antibodies after vaccination in the real world should be further assessed. We enrolled 1067 volunteers who had been vaccinated with one or two doses of CoronaVac in Zhejiang Province, China. Another 90 healthy adults without previous vaccinations were recruited and vaccinated with three doses of CoronaVac, 28 days and 6 months apart. Serum samples were collected from multiple timepoints and analyzed for specific IgM/IgG and neutralizing antibodies (NAbs) for immunogenicity evaluation. Antibody responses to the Delta and Omicron variants were measured by pseudovirus-based neutralization tests. Our results revealed that binding antibody IgM peaked 14\u201328 days after one dose of CoronaVac, while IgG and NAbs peaked approximately 1 month after the second dose then declined slightly over time. Antibody responses had waned by month 6 after vaccination and became undetectable in the majority of individuals at 12 months. Levels of NAbs to live SARS-CoV-2 were correlated with anti-SARS-CoV-2 IgG and NAbs to pseudovirus, but not IgM. Homologous booster around 6 months after primary vaccination activated anamnestic immunity and raised NAbs 25.5-fold. The NAb inhibition rate subsequently rose to 36.0% for Delta (p=0.03) and 4.3% for Omicron (p=0.004), and the response rate for Omicron rose from 7.9% (7/89) to 17.8% (16/90). Two doses of CoronaVac vaccine resulted in limited protection over a short duration. The homologous booster slightly increased antibody responses to the Delta and Omicron variants; therefore, the optimization of booster procedures is vital. Key Research and Development Program of Zhejiang Province; Key Program of Health Commission of Zhejiang Province/ Science Foundation of National Health Commission; Major Program of Zhejiang Municipal Natural Science Foundation.","version":"1.1","doi":"10.1101/2022.11.03.515011","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.03.515010","pub_date":"2022-11-03","title":"Towards increased accuracy and reproducibility in SARS-CoV-2 next generation sequence analysis for public health surveillance","abstract":"During the COVID-19 pandemic, SARS-CoV-2 surveillance efforts integrated genome sequencing of clinical samples to identify emergent viral variants and to support rapid experimental examination of genome-informed vaccine and therapeutic designs. Given the broad range of methods applied to generate new viral genomes, it is critical that consensus and variant calling tools yield consistent results across disparate pipelines. Here we examine the impact of sequencing technologies (Illumina and Oxford Nanopore) and 7 different downstream bioinformatic protocols on SARS-CoV-2 variant calling as part of the NIH Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Tracking Resistance and Coronavirus Evolution (TRACE) initiative, a public-private partnership established to address the COVID-19 outbreak. Our results indicate that bioinformatic workflows can yield consensus genomes with different single nucleotide polymorphisms, insertions, and/or deletions even when using the same raw sequence input datasets. We introduce the use of a specific suite of parameters and protocols that greatly improves the agreement among pipelines developed by diverse organizations. Such consistency among bioinformatic pipelines is fundamental to SARS-CoV-2 and future pathogen surveillance efforts. The application of analysis standards is necessary to more accurately document phylogenomic trends and support data-driven public health responses.","version":"1.1","doi":"10.1101/2022.11.03.515010","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.02.514944","pub_date":"2022-11-03","title":"The Longitudinal Analysis of Convergent Antibody VDJ Regions in SARS-CoV-2 Positive Patients Using RNA-seq","abstract":"The severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has infected over 600 million individuals and caused over 6.5 million deaths. To understand the immune response individuals have from the SARS-CoV-2 infection, we studied the immunoglobulins against the virus\u2019s antigens. The diversified complementarity determining region 3 (CDR3) can be used to characterize an antibody. We downloaded four public RNA-seq data sets that were collected be-tween March 2020 and March 2022 from the Gene Expression Omnibus (GEO) in our longitudinal analysis. In total, there were 269 SARS-CoV-2 positive patients and 26 negative patients who served as a control group. Samples were grouped based on their SARS-CoV-2 variant type and/or the time they were collected. Among 629,137 immunoglobulin V(D)J sequences identified by reconstructing the V(D)J sequences, we found 1011 common V(D)Js (same V gene, J gene and CDR3 sequences in each SARS-CoV-2 positive group) shared by more than one patient in each group and no common V(D)Js were from the negative control group. In our clustering analysis, we identified 129 convergent clusters from the SARS-CoV-2 positive groups. One of these convergent clusters matched the protein sequence of crystal 3D structures of the antibodies against SARS-CoV-2 in the Protein Data Bank (PDB). In our longitudinal analysis between the Alpha and Omicron variant, we found 2.7% of common CDR3s were shared although the longitudinal profiling of common V(D)Js was variant specific. Although diverse immunoglobulin profiles were observed, the convergence of common V(D)Js suggests that there exists antibodies with similar antigenic specificities across patients in different groups over various stages of the pandemic.","version":"1.1","doi":"10.1101/2022.11.02.514944","journal":"bioRxiv","score":null},{"id":"10.1101/2022.11.01.514722","pub_date":"2022-11-02","title":"Substantial Neutralization Escape by the SARS-CoV-2 Omicron Variant BQ.1.1","abstract":"Omicron BA.5 has been the globally dominant SARS-CoV-2 variant and has demonstrated substantial neutralization escape compared with prior variants. Additional Omicron variants have recently emerged, including BA.4.6, BF.7, BA.2.75.2, and BQ.1.1, all of which have the Spike R346T mutation. In particular, BQ.1.1 has rapidly increased in frequency, and BA.5 has recently declined to less than half of viruses in the United States. Our data demonstrate that BA.2.75.2 and BQ.1.1 escape NAbs induced by infection and vaccination more effectively than BA.5. BQ.1.1 NAb titers were lower than BA.5 NAb titers by a factor of 7 in two cohorts of individuals who received the monovalent or bivalent mRNA vaccine boosters. These findings provide the immunologic context for the rapid increase in BQ.1.1 prevalence in regions where BA.5 is dominant and have implications for both vaccine immunity and natural immunity.","version":"1.1","doi":"10.1101/2022.11.01.514722","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.31.514592","pub_date":"2022-11-01","title":"Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody to antigenically distinct omicron SARS-CoV-2 subvariants","abstract":"The rapid evolution of SARS-CoV-2 Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identify S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) and derived from an individual previously infected with SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrates broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1). Furthermore, it protected hamsters against in vivo challenges with wildtype, Delta, and BA.1 viruses. Structural analysis reveals that this antibody targets a class 1 epitope via multiple hydrophobic and polar interactions with its CDR-H3, in addition to common class 1 motifs in CDR-H1/CDR-H2. Importantly, this epitope is more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.10.31.514592","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.31.513750","pub_date":"2022-11-01","title":"Negligible peptidome diversity of SARS-CoV-2 and its higher taxonomic ranks","abstract":"The unprecedented increase in SARS-CoV-2 sequence data limits the application of alignment-dependent approaches to study viral diversity. Herein, we applied our recently published UNIQmin, an alignment-free tool to study the protein sequence diversity of SARS-CoV-2 (sub-species) and its higher taxonomic lineage ranks (species, genus, and family). Only less than 0.5% of the reported SARS-CoV-2 protein sequences are required to represent the inherent viral peptidome diversity, which only increases to a mere \u223c2% at the family rank. This is expected to remain relatively the same even with further increases in the sequence data. The findings have important implications in the design of vaccines, drugs, and diagnostics, whereby the number of sequences required for consideration of such studies is drastically reduced, short-circuiting the discovery process, while still providing for a systematic evaluation and coverage of the pathogen diversity.","version":"1.1","doi":"10.1101/2022.10.31.513750","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.31.514483","pub_date":"2022-11-01","title":"An optimised method for the recovery of laboratory generated SARS-CoV-2 aerosols by plaque assay","abstract":"We present an optimised method for the recovery of laboratory generated SARS-CoV-2 virus by plaque assay. This method allows easy incorporation into existing standard operating procedures of biological containment level 3 (BCL3) laboratories.","version":"1.1","doi":"10.1101/2022.10.31.514483","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.31.514636","pub_date":"2022-11-01","title":"mRNA bivalent booster enhances neutralization against BA.2.75.2 and BQ.1.1","abstract":"The emergence of the highly divergent SARS-CoV-2 Omicron variant has jeopardized the efficacy of vaccines based on the ancestral spike. The bivalent COVID-19 mRNA booster vaccine within the United States is comprised of the ancestral and the Omicron BA.5 spike. Since its approval and distribution, additional Omicron subvariants have been identified with key mutations within the spike protein receptor binding domain that are predicted to escape vaccine sera. Of particular concern is the R346T mutation which has arisen in multiple subvariants, including BA.2.75.2 and BQ.1.1. Using a live virus neutralization assay, we evaluated serum samples from individuals who had received either one or two monovalent boosters or the bivalent booster to determine neutralizing activity against wild-type (WA1/2020) virus and Omicron subvariants BA.1, BA.5, BA.2.75.2, and BQ.1.1. In the one monovalent booster cohort, relative to WA1/2020, we observed a reduction in neutralization titers of 9-15-fold against BA.1 and BA.5 and 28-39-fold against BA.2.75.2 and BQ.1.1. In the BA.5-containing bivalent booster cohort, the neutralizing activity improved against all the Omicron subvariants. Relative to WA1/2020, we observed a reduction in neutralization titers of 3.7- and 4-fold against BA.1 and BA.5, respectively, and 11.5- and 21-fold against BA.2.75.2 and BQ.1.1, respectively. These data suggest that the bivalent mRNA booster vaccine broadens humoral immunity against the Omicron subvariants.","version":"1.1","doi":"10.1101/2022.10.31.514636","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.28.513849","pub_date":"2022-10-31","title":"Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations","abstract":"Mutations carried by SARS-CoV-2 spike protein variants may promote viral escape from immune protection. Humoral immunity is sensitive to evasion by SARS-CoV-2 mutants, but the impact of viral evolution on the interplay between virus and host CD8 T cell reactivity remains uncertain. By a systematic functional analysis of 30 spike variant mutations, we show that in vaccinated as well as convalescent subjects, mutated epitopes can have not only a neutral or abrogating effect on the recognition by CD8 T cells but can also enhance or even generate de novo CD8 T cell responses. Large pools of peptides spanning the entire spike sequence and comprising previously identified CD8 T cell epitopes were then used in parallel with variant peptides to define strength and multispecificity of total anti-spike CD8 responses. In some individuals, CD8 cells were narrowly focused on a few epitopes indicating that in this context of weak and oligospecific responses the overall antiviral protection can likely benefit of the function enhancing effect of heteroclitic-like mutations. In conclusion, appearance of mutated stimulatory epitopes likely reflects an epiphenomenon of SARS-CoV-2 evolution driven by antibody evasion and increased transmissibility, that might bear clinical relevance in a subset of individuals with weak and oligospecific CD8 T cell responses.","version":"1.2","doi":"10.1101/2022.10.28.513849","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.27.514070","pub_date":"2022-10-28","title":"A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients","abstract":"The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.","version":"1.1","doi":"10.1101/2022.10.27.514070","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.06.490962","pub_date":"2022-10-28","title":"The SARS-CoV-2 cellular receptor ACE2 is expressed in oropharyngeal cells and is modulated in vitro by the bacterial lysate Lantigen B","abstract":"Angiotensin-converting enzyme2 (ACE2) is the main cell surface receptor of the SARS-CoV-2 spike protein and is expressed in a variety of cell types, including cells of the respiratory tract. A bacterial lysate used for the prophylaxis of respiratory infections (OM-85), was recently shown to downregulate the expression of ACE2 in epithelial cells, suggesting its possible role as a prophylaxis of the onset of COVID19. Another bacterial lysate (Lantigen B, administered sublingually) is used in the prophylaxis of recurrent respiratory tract infections. It contains antigens obtained by chemical lysis from the most representative microbes of the respiratory tract. In this in vitro study, the capacity of Lantigen B to decrease ACE2 in human oropharyngeal cells was evaluated. The study was carried out in 40 healthy donors undergoing oropharyngeal swab for routine SARS-CoV-2 detection. Cells were treated in vitro with a 1:2 of Lantigen B. ACE2 expression was evaluated using a fluorescent anti-ACE2 monoclonal antibody and flow cytometry. A reduction in the number of positive cells was observed in 72% of the patients, while a modulation of ACE2 expression was observed in 62% of the samples. As a control, the expression of the CD54 rhinovirus receptor in the same cells was unaffected. To evaluate the functional effects of down regulation, in a subset of samples, the same oropharynx cells were incubated with Lantigen B and infected with wild-type SARS-CoV-2. After 24 hours, viral RNA, as assessed by rt-PCR, was significantly lower in samples treated with Lantigen B. In conclusion, this study demonstrates that Lantigen B, at a pharmacological dose, modulates the expression of the main SARS-CoV-2 receptor in oropharyngeal cells, and reduces viral yield. This activity could be synergistic with other approaches (vaccination and therapy) by reducing the number of potentially infected cells and thus reducing the effects of SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2022.05.06.490962","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.27.514012","pub_date":"2022-10-27","title":"Vitamin D deficiency and SARS\u2011CoV\u20112 infection: Big-data analysis from March 2020 to March 2021. D-COVID study","abstract":"Vitamin D has been proposed to have immunomodulatory functions and therefore play a role in coronavirus infection (COVID-19). However, there is no conclusive evidence on its impact on COVID-19 infection and evolution. To study the association between COVID-19 infection and vitamin D deficiency in patients of a terciary university hospital. To investigate the clinical evolution and prognosis of patients with COVID-19 and vitamin D deficiency. Using big-data analytics and artificial intelligence through the SAVANA Manager clinical platform, we analysed clinical data from patients with COVID-19 atended in a terciary university hospital from March 2020 to March 2021. Of the 143.157 analysed patients, 36.261 subjects had COVID-19 infection (25.33%); during this period; of these 2588 had vitamin D deficiency (7.14%). Among subjects with COVID-19 and vitamin D deficiency, there was a higher proportion of women OR 1.45 [95% CI 1.33-1.57], adults older than 80 years OR 2.63 [95%CI 2.38-2.91], people living in nursing homes OR 2.88 [95%CI 2.95-3.45] and walking dependence OR 3.45 [95%CI 2.85-4.26]. Regarding clinical course, a higher number of subjects with COVID-19 and vitamin D deficiency required hospitalitation OR 2.41 [95%CI 2.22-2-61], intensive unit care (ICU) OR 2.22 [95% CI 1.64-3.02], had a longer mean hospital stay 3.94 (2.29) p=0.02 and higher mortality OR 1.82 [95%CI 1.66-2.01].) Low serum 25 (OH) Vitamin-D level was significantly associated with a worse clinical evolution and prognosis of COVID-19 infection. We found a higher proportion of institutionalised and dependent people over 80 years of age among patients with COVID-19 and vitamin D deficiency.","version":"1.1","doi":"10.1101/2022.10.27.514012","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.25.513804","pub_date":"2022-10-26","title":"Immunity to seasonal coronavirus spike proteins does not protect from SARS-CoV-2 challenge in a mouse model but has no detrimental effect on protection mediated by COVID-19 mRNA vaccination","abstract":"Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection as compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. The impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.10.25.513804","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.25.513090","pub_date":"2022-10-26","title":"SARS-CoV-2 S1 Subunit Booster Vaccination Elicits Robust Humoral Immune Responses in Aged Mice","abstract":"Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death of the disease. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants raises concerns of reduced vaccine effectiveness and increased risk of infection. Repeated homologous booster in elderly individuals and immunocompromised patients is considered to solve severe form of disease caused by new SARS-CoV-2 variants but cannot protect completely against breakthrough infection. In our previous study we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) in mice, resulting in that a single immunization with Ad5.S1, via subcutaneously injection or intranasal delivery, induced robust humoral and cellular immune responses [1]. As a follow up study, here we showed that vaccinated mice had high titers of anti-S1 antibodies at one year after vaccination compared to PBS immunized mice. Furthermore, one booster dose of non-adjuvanted recombinant S1Beta (rS1Beta) subunit vaccine was effective in stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Beta, and Delta strain with 3.6- to 19.5-fold change increases. Importantly, the booster dose elicits cross-reactive antibody responses resulting in ACE2 binding inhibition against spike of SARS-CoV-2 variants (Wuhan, Alpha, Beta, Gamma, Delta, Zeta, Kappa, New York, India) as early as two-week post-boost injection, persisting over 28 weeks after a booster vaccination. Interestingly, levels of neutralizing antibodies were correlated with not only level of S1-binding IgG but also level of ACE2 inhibition in the before- and after-booster serum samples. Our findings show that S1 recombinant protein subunit vaccine candidate as a booster has potential to offer cross-neutralization against broad variants, and has important implications for vaccine control of new emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccine like AZD1222 and Ad26.COV2.S.","version":"1.1","doi":"10.1101/2022.10.25.513090","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.25.513701","pub_date":"2022-10-26","title":"Assessing SARS-CoV-2 evolution through the analysis of emerging mutations","abstract":"The number of studies on SARS-CoV-2 published on a daily basis is constantly increasing, in an attempt to understand and address the challenges posed by the pandemic in a better way. Most of these studies also include a phylogeny of SARS-CoV-2 as background context, always taking into consideration the latest data in order to construct an updated tree. However, some of these studies have also revealed the difficulties of inferring a reliable phylogeny. [13] have shown that reliable phylogeny is an inherently complex task due to the large number of highly similar sequences, given the relatively low number of mutations evident in each sequence. From this viewpoint, there is indeed a challenge and an opportunity in identifying the evolutionary history of the SARS-CoV-2 virus, in order to assist the phylogenetic analysis process as well as support researchers in keeping track of the virus and the course of its characteristic mutations, and in finding patterns of the emerging mutations themselves and the interactions between them. The research question is formulated as follows: Detecting new patterns of co-occurring mutations beyond the strain-specific / strain-defining ones, in SARS-CoV-2 data, through the application of ML methods. Going beyond the traditional phylogenetic approaches, we will be designing and implementing a clustering method that will effectively create a dendrogram of the involved sequences, based on a feature space defined on the present mutations, rather than the entire sequence. Ultimately, this ML method is tested out in sequences retrieved from public databases and validated using the available metadata as labels. The main goal of the project is to design, implement and evaluate a software that will automatically detect and cluster relevant mutations, that could potentially be used to identify trends in emerging variants. tasos1109@gmail.com","version":"1.1","doi":"10.1101/2022.10.25.513701","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.24.513632","pub_date":"2022-10-25","title":"Characterization of Three Variants of SARS-CoV-2 in vivo Shows Host-Dependent Pathogenicity in Hamsters","abstract":"Animal models are used in preclinical trials to test vaccines, antivirals, monoclonal antibodies, and immunomodulatory drug therapies against SARS-CoV-2. However, these drugs often do not produce equivalent results in human clinical trials. Here, we show how different animal models infected with some of the most clinically relevant SARS-CoV-2 variants, WA1/2020, B.1.617.2/Delta, B.1.1.529/Omicron and BA5.2/Omicron, have independent outcomes. We show that in mice, B.1.617.2 is more pathogenic, followed by WA1, while B.1.1.529 showed an absence of clinical signs. Only B.1.1.529 was able to infect C57BL/6J mice, which lack the human ACE2 receptor. B.1.1.529-infected ACE2 mice had different T cell profiles compared to infected K18-hACE2 mice, while viral shedding profiles and viral titers in lungs were similar between the ACE2 and the C57BL/6J mice. These data suggest B.1.1.529 virus adaptation to a new host and shows that asymptomatic carriers can accumulate and shed virus. Next, we show how B.1.617.2, WA1 and BA5.2/Omicron have similar viral replication kinetics, pathogenicity, and viral shedding profiles in hamsters, demonstrating that the increased pathogenicity of B.1.617.2 observed in mice is host-dependent. Overall, these findings suggest that small animal models are useful to parallel human clinical data, but the experimental design places an important role in interpreting the data. There is a need to investigate SARS-CoV-2 variants phenotypes in different animal models due to the lack of reproducible outcomes when translating experiments to the human population. Our findings highlight the correlation of clinically relevant SARS-CoV-2 variants in animal models with human infections. Experimental design and understanding of correct animal models are essential to interpreting data to develop antivirals, vaccines, and other therapeutic compounds against COVID-19.","version":"1.1","doi":"10.1101/2022.10.24.513632","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.24.513610","pub_date":"2022-10-25","title":"Fever temperatures modulate intraprotein dynamics and enhance the binding affinity between monoclonal antibodies and the Spike protein from SARS-CoV-2","abstract":"Fever is a typical symptom of most infectious diseases. While prolonged fever may be clinically undesirable, mild reversible fever (< 39\u00b0C, 312K) can potentiate the immune responses against pathogens. Here, using molecular dynamics, we investigated the effect of febrile temperatures (38\u00b0C to 40\u00b0C, 311K to 313K) on the immune complexes formed by the SARS-CoV-2 spike protein with two neutralizing antibodies. We found that, at mild fever temperatures (311-312K), the binding affinities of the two antibodies improve when compared to the physiological body temperature (37\u00b0C, 310K). Furthermore, only at 312K, antibodies exert distinct mechanical effects on the receptor binding domains of the spike protein that may hinder SARS-CoV-2 infectivity. Enhanced antibody binding affinity may thus be obtained using appropriate temperature conditions.","version":"1.1","doi":"10.1101/2022.10.24.513610","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.24.513619","pub_date":"2022-10-25","title":"Immunogenicity of the BA.5 Bivalent mRNA Vaccine Boosters","abstract":"Waning immunity following mRNA vaccination and the emergence of SARS-CoV-2 variants has led to reduced mRNA vaccine efficacy against both symptomatic infection and severe disease. Bivalent mRNA boosters expressing the Omicron BA.5 and ancestral WA1/2020 Spike proteins have been developed and approved, because BA.5 is currently the dominant SARS-CoV-2 variant and substantially evades neutralizing antibodies (NAbs). Our data show that BA.5 NAb titers were comparable following monovalent and bivalent mRNA boosters.","version":"1.1","doi":"10.1101/2022.10.24.513619","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.24.449733","pub_date":"2022-10-25","title":"Integrating T-cell receptor and transcriptome for large-scale single-cell immune profiling analysis","abstract":"Recent advancements in single-cell immune profiling that enable the measurement of the transcriptome and T-cell receptor (TCR) sequences simultaneously have emerged as a promising approach to study immune responses at cellular resolution. Yet, combining these different types of information from multiple datasets into a joint representation is complicated by the unique characteristics of each modality and the technical effects between datasets. Here, we present mvTCR, a multimodal generative model to learn a unified representation across modalities and datasets for joint analysis of single-cell immune profiling data. We show that mvTCR allows the construction of large-scale and multimodal T-cell atlases by distilling modality-specific properties into a shared view, enabling unique and improved data analysis. Specifically, we demonstrated mvTCR\u2019s potential by revealing and separating SARS-CoV-2-specific T-cell clusters from bystanders that would have been missed in individual unimodal data analysis. Finally, mvTCR can enable automated analysis of new datasets when combined with transfer-learning approaches. Overall, mvTCR provides a principled solution for standard analysis tasks such as multimodal integration, clustering, specificity analysis, and batch correction for single-cell immune profiling data.","version":"1.2","doi":"10.1101/2021.06.24.449733","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.21.512606","pub_date":"2022-10-24","title":"Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques","abstract":"Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFN\u03b12 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN-\u03b1 and IFN-\u03b2 pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.","version":"1.1","doi":"10.1101/2022.10.21.512606","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.22.513351","pub_date":"2022-10-24","title":"Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequalae of SARS-CoV-2 infection","abstract":"Low-density granulocytes (LDGs) are a distinct subset of neutrophils whose increased abundance is associated with the severity of COVID-19. However, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on LDG levels and phenotypic alteration remain unexplored. Using participants na\u00efve to SARS-CoV-2 (NP), infected with SARS-CoV-2 with no residual symptoms (NRS), and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC), we compared LDG levels and their phenotype by measuring the expression of markers for activation, maturation, and neutrophil extracellular trap (NET) formation using flow cytometry. The number of LDGs was significantly elevated in PPASC compared to NP. Individuals infected with SARS-CoV-2 (NRS and PPASC) demonstrated increased CD10+ and CD16HI subset counts of LDGs compared to NP group. Further characterization of LDGs demonstrated that LDGs from PPASC displayed higher NET forming ability and aggregation with platelets compared to LDGs from NP and NRS. Our data demonstrates that mature neutrophils with a heightened activation phenotype remain in circulation long after initial SARS-CoV-2 infection. Persistent elevation of markers for neutrophil activation and NET formation on LDGs, as well as an enhanced proclivity for platelet-neutrophil aggregation (PNA) formation in individuals with PPASC may be associated with the development of long-term pulmonary sequelae.","version":"1.1","doi":"10.1101/2022.10.22.513351","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.22.513347","pub_date":"2022-10-24","title":"The ACE-2 receptor accelerates but is not biochemically required for SARS-CoV-2 membrane fusion","abstract":"The SARS-CoV-2 coronavirus infects human cells via the ACE-2 receptor. Circumstantial evidence suggests that ACE-2 may not just serve as an attachment factor but also help activate the SARS-CoV-2 spike protein for membrane fusion. Here, we test that hypothesis directly, using DNA-lipid tethering as a synthetic attachment factor in the place of ACE-2. We find that SARS-CoV-2 pseudovirus and viruslike particles are both capable of membrane fusion if attached in the absence of ACE-2 and activated with an appropriate protease. However, addition of soluble ACE-2 speeds the fusion reaction. This is observed for both the Wuhan strain and the B.1.1.529 Omicron variant. Kinetic analysis suggests that there are at least two rate-limiting steps for SARS-CoV-2 membrane fusion, one of which is ACE-2 dependent and one of which is not. These data establish that, in the presence of an alternative attachment factor, ACE-2 is not biochemically required for SARS-CoV-2 membrane fusion. Since ACE-2 serves as the high-affinity attachment factor on human cells, the possibility to replace it with other factors has implications for the evolvability of SARS-CoV-2 and the fitness landscape for future related coronaviruses.","version":"1.1","doi":"10.1101/2022.10.22.513347","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.23.513379","pub_date":"2022-10-24","title":"Antibody avidity and multi-specificity combined to confer protection against SARS-CoV-2 and resilience against viral escape","abstract":"SARS-CoV-2, the causative agent of COVID-19, has been responsible for a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs), and in deployment by the need for high doses. In this study, we leverage the MULTI-specific, multi-Affinity antiBODY (Multabody, MB) platform, derived from the human apoferritin protomer, to drive the multimerization of antibody fragments and generate exceptionally potent and broad SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity for the core of these engineered antibody-like molecules at 2.1 \u00c5 resolution. We demonstrate that neutralization potency improvements of the MB over corresponding IgGs translates into superior in vivo protection: in the SARS-CoV-2 mouse challenge model, comparable in vivo protection was achieved for the MB delivered at 30x lower dose compared to the corresponding IgGs. Furthermore, we show how MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding IgGs lose their ability to neutralize potently. Multiple mAb specificities could also be combined into a single MB molecule to expand the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.","version":"1.1","doi":"10.1101/2022.10.23.513379","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.24.513517","pub_date":"2022-10-24","title":"Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response","abstract":"A detailed understanding of the molecular features of the neutralizing epitopes developed by viral escape mutants is important for predicting and developing vaccines or therapeutic antibodies against continuously emerging SARS-CoV-2 variants. Here, we report three human monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during first wave of pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, but poorly neutralized Beta and completely failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these three mAbs in complex with trimeric spike protein showed that all three mAbs are involved in bivalent spike binding with two mAbs targeting class-1 and one targeting class-4 Receptor Binding Domain (RBD) epitope. Comparison of immunogenetic makeup, structure, and function of these three mAbs with our recently reported class-3 RBD binding mAb that potently neutralized all SARS-CoV-2 variants revealed precise antibody footprint, specific molecular interactions associated with the most potent multi-variant binding / neutralization efficacy. This knowledge has timely significance for understanding how a combination of certain mutations affect the binding or neutralization of an antibody and thus have implications for predicting structural features of emerging SARS-CoV-2 escape variants and to develop vaccines or therapeutic antibodies against these.","version":"1.1","doi":"10.1101/2022.10.24.513517","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.22.513349","pub_date":"2022-10-24","title":"Antibody responses to Omicron BA.4/BA.5 bivalent mRNA vaccine booster shot","abstract":"The SARS-CoV-2 Omicron variant and its numerous sub-lineages have exhibited a striking ability to evade humoral immune responses induced by prior vaccination or infection. The Food and Drug Administration (FDA) has recently granted Emergency Use Authorizations (EUAs) to new bivalent formulations of the original Moderna and Pfizer mRNA SARS-CoV-2 vaccines that target both the ancestral strain as well as the Omicron BA.4/BA.5 variant. Despite their widespread use as a vaccine boost, little is known about the antibody responses induced in humans. Here, we collected sera from several clinical cohorts: individuals after three or four doses of the original monovalent mRNA vaccines, individuals receiving the new bivalent vaccines as a fourth dose, and individuals with BA.4/BA.5 breakthrough infection following mRNA vaccination. Using pseudovirus neutralization assays, these sera were tested for neutralization against an ancestral SARS-CoV-2 strain, several Omicron sub-lineages, and several related sarbecoviruses. At ~3-5 weeks post booster shot, individuals who received a fourth vaccine dose with a bivalent mRNA vaccine targeting BA.4/BA.5 had similar neutralizing antibody titers as those receiving a fourth monovalent mRNA vaccine against all SARS-CoV-2 variants tested, including BA.4/BA.5. Those who received a fourth monovalent vaccine dose had a slightly higher neutralizing antibody titers than those who received the bivalent vaccine against three related sarbecoviruses: SARS-CoV, GD-Pangolin, and WIV1. When given as a fourth dose, a bivalent mRNA vaccine targeting Omicron BA.4/BA.5 and an ancestral SARS-CoV-2 strain did not induce superior neutralizing antibody responses in humans, at the time period tested, compared to the original monovalent vaccine formulation.","version":"1.1","doi":"10.1101/2022.10.22.513349","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.24.513415","pub_date":"2022-10-24","title":"Vaccine- and BTI-elicited pre-Omicron immunity more effectively neutralizes Omicron sublineages BA.1, BA.2, BA.4 and BA.5 than pre-Omicron infection alone","abstract":"Since the emergence of SARS-CoV-2 Omicron BA.1 and BA.2, several Omicron sublineages have emerged, supplanting their predecessors. BA.5 is the current dominant sublineage. Here we compared the neutralization of Omicron sublineages BA.1, BA.2, BA.4 and BA.5 by human sera collected from individuals who were infected with the ancestral B.1 (D614G) strain, vaccinated (3 doses), or with hybrid immunity from vaccination (2 doses) followed by pre-Omicron breakthrough infection (BTI) with Gamma or Delta. All Omicron sublineages exhibited extensive escape from all sera compared to the ancestral B.1 strain and to Delta, albeit to different levels depending on the origin of the sera. Convalescent sera were unable to neutralize BA.1, and partly neutralized BA.2, BA.4 and BA.5. Vaccinee sera partly neutralized BA.2, but BA.1, BA.4 and BA.5 evaded neutralizing antibodies. BTI sera were either non-neutralizing or partially neutralizing. In this case, they had similar neutralizing ability against all Omicron sublineages. Despite similar levels of anti-Spike and anti-Receptor Binding Domain (RBD) antibody in all groups, BTI sera had the highest cross-neutralizing ability against all Omicron sublineages and convalescent sera were the least neutralizing. The NT50:antibody titer ratio, which reflects antibody avidity, was significantly higher in sera from BTI patients compared to convalescent sera, underscoring qualitative differences in antibodies elicited by infection alone and by vaccination. Together these findings highlight the importance of vaccination to trigger highly cross-reactive antibodies that neutralize phylogenetically and antigenically distant strains, and suggest that immune imprinting by first generation vaccines may restrict, but not abolish cross-neutralization.","version":"1.1","doi":"10.1101/2022.10.24.513415","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.21.513318","pub_date":"2022-10-24","title":"ViReaDB: A user-friendly database for compactly storing viral sequence data and rapidly computing consensus genome sequences","abstract":"In viral molecular epidemiology, reconstruction of consensus genomes from sequence data is critical for tracking mutations and variants of concern. However, storage of the raw sequence data can become prohibitively large, and computing consensus genome from sequence data can be slow and requires bioinformatics expertise. ViReaDB is a user-friendly database system for compactly storing viral sequence data and rapidly computing consensus genome sequences. From a dataset of 1 million trimmed mapped SARS-CoV-2 reads, it is able to compute the base counts and the consensus genome in 16 minutes, store the reads alongside the base counts and consensus in 50 MB, and optionally store just the base counts and consensus (without the reads) in 300 KB. ViReaDB is freely available on PyPI (https://pypi.org/project/vireadb) and on GitHub (https://github.com/niemasd/ViReaDB) as an open-source Python software project. niema@ucsd.edu","version":"1.1","doi":"10.1101/2022.10.21.513318","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.21.513196","pub_date":"2022-10-21","title":"SARS-CoV-2 nsp3-4 suffice to form a pore shaping replication organelles","abstract":"Coronavirus replication is associated with the remodeling of cellular membranes resulting in the formation of double-membrane vesicles (DMVs). Recently, a pore spanning DMV was identified as a putative portal for viral RNA transcription and replication products providing a novel target for antiviral intervention. However, the exact components and the structure of the SARS-CoV-2 pore remain to be determined. Here, we investigate the structure of DMV pores by in situ cryo-electron tomography combined with subtomogram averaging. We reveal non-structural proteins (nsp) 3 and 4 as minimal components forming a DMV spanning pore and show that nsp3 Ubl1-Ubl2 domains are critical for inducing membrane curvature and DMV formation. Altogether, SARS-CoV-2 nsp3-4 has a dual role by driving the biogenesis of replication organelles and forming DMV-spanning replicopores. Biogenesis of SARS-CoV-2 replication organelles is driven by nsp3-4 constituting the double-membrane vesicle spanning pore.","version":"1.1","doi":"10.1101/2022.10.21.513196","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.20.513002","pub_date":"2022-10-21","title":"SARS-CoV-2 E and 3a proteins are inducers of pannexin currents","abstract":"Controversial reports have suggested that SARS-CoV E and 3a proteins may be viroporins that conduct currents through the plasma membrane of the infected cells. If true, these proteins would represent accessible targets for the development of new antiviral drugs by using high-throughput patch-clamp techniques. Here we aimed at better characterizing the cell responses induced by E or 3a protein with a particular focus on the ion conductances measured at the cell surface. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly-acquired round shape, tending to detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this hypothesis by using flow cytometry, in agreement with earlier reports on other cell types. In adhering cells expressing E or 3a protein, whole-cell currents were in fact not different from the control condition indicating that E and 3a proteins are not plasma membrane viroporins. In contrast, recording currents on detached cells uncovered outwardly-rectifying currents, much larger than those observed in control. The current characteristics are reminiscent of what was previously observed in cells expressing SARS-CoV-1 E or 3a proteins. Herein, we illustrate for the first time that carbenoxolone blocks these outward currents suggesting that they are conducted by pannexin channels, mostly likely activated by cell morphology change and/or cell death. Alongside we also demonstrate that truncation of the C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent pannexin currents suggesting distinct pathways for cell death and pannexin currents induced by E and 3a proteins. We conclude that SARS-CoV-2 E and 3a proteins are not acting as viroporins expressed at the plasma membrane. A viroporin (or viral porin) is a class of proteins that is encoded by a virus genome. It is named porin because its biological role is to conduct ions through a pore that it created in a lipid membrane such as the one surrounding a human cell. if such viroporin is present at the external membrane of a human cell infected by a virus, it can be an easy target of an antiviral agent which thus does not have to enter the cell to be active. One example of viroporin is the flu M2 protein that is the target of amantadine, an antiviral agent used against flu. In previous studies, two proteins of SARS-CoV viruses, named E protein and 3a protein, have been suggested to be viroporins at the surface of infected human cells, potentially opening a new research avenue against SARS. Here we demonstrate that both proteins are not viroporins at the external membrane but they rather trigger changes in the cell shape and promote cell death. They only indirectly induce the activity of a porin that is encoded by the cell genome, named pannexin.","version":"1.2","doi":"10.1101/2022.10.20.513002","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512927","pub_date":"2022-10-21","title":"Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories","abstract":"A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic\u2014Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. Systematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.","version":"1.1","doi":"10.1101/2022.10.19.512927","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.21.513200","pub_date":"2022-10-21","title":"A systems approach evaluating the impact of SARS-CoV-2 variant of concern mutations on CD8+ T cell responses","abstract":"T cell recognition of SARS-CoV-2 antigens after vaccination and/or natural infection has played a central role in resolving SARS-CoV-2 infections and generating adaptive immune memory. However, the clinical impact of SARS-CoV-2-specific T cell responses is variable and the mechanisms underlying T cell interaction with target antigens are not fully understood. This is especially true given the virus\u2019 rapid evolution, which leads to new variants with immune escape capacity. In this study, we used the Omicron variant as a model organism and took a systems approach to evaluate the impact of mutations on CD8+ T cell immunogenicity. We computed an \u2018immunogenicity potential\u2019 score for each SARS-CoV-2 peptide antigen from the ancestral strain and Omicron, capturing both antigen presentation and T cell recognition probabilities. By comparing ancestral vs. Omicron immunogenicity scores, we reveal a divergent and heterogeneous landscape of impact for CD8+ T cell recognition of mutated targets in Omicron variants. While T cell recognition of Omicron peptides is broadly preserved, we observed mutated peptides with deteriorated immunogenicity that may assist breakthrough infection in some individuals. We then combined our scoring scheme with an in-silico mutagenesis, to characterise the position- and residue-specific theoretical mutational impact on immunogenicity. While we predict many escape trajectories from the theoretical landscape of substitutions, our study suggests that Omicron mutations in T cell epitopes did not develop under cell-mediated pressure. Our study provides a generalisable platform for fostering a deeper understanding of existing and novel variant impact on antigen-specific vaccine- and/or infection-induced T cell immunity.","version":"1.1","doi":"10.1101/2022.10.21.513200","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.20.513136","pub_date":"2022-10-21","title":"Diversity, composition, and networking of saliva microbiota distinguish the severity of COVID-19 episodes as revealed by an analysis of 16S rRNA variable V1-V3 regions sequences","abstract":"Studies on the role of the oral microbiome in SARS-CoV-2 infection and severity of the disease are limited. We aimed to characterize the bacterial communities present in the saliva of patients with varied COVID-19 severity to learn if there are differences in the characteristics of the microbiome among the clinical groups. We included asymptomatic subjects with no previous COVID-19 infection or vaccination; patients with mild respiratory symptoms, positive or negative for SARS-CoV-2 infection; patients that required hospitalization because of severe COVID-19 with oxygen saturation below 92%, and fatal cases of COVID-19. Saliva samples collected before any treatment were tested for SARS-CoV-2 by PCR. Oral microbiota in saliva was studied by amplification and sequencing of the V1-V3 variable regions of 16S gene using a Illumina MiSeq platform. We found significant changes in diversity, composition, and networking in saliva microbiota of patients with COVID-19, as well as patterns associated with severity of disease. The presence or abundance of several commensal species and opportunistic pathogens were associated with each clinical stage. Patterns of networking were also found associated with severity of disease: a highly regulated bacterial community (normonetting) was found in healthy people whereas poorly regulated populations (disnetting) were characteristic of severe cases. Characterization of microbiota in saliva may offer important clues in the pathogenesis of COVID-19 and may also identify potential markers for prognosis in the severity of the disease. SARS-CoV-2 infection is the most severe pandemic of humankind in the last hundred years. The outcome of the infection ranges from asymptomatic or mild to severe and even fatal cases, but reasons for this remain unknown. Microbes normally colonizing the respiratory tract form communities that may mitigate the transmission, symptoms, and severity of viral infections, but very little is known on the role of these microbial communities in the severity of COVID-19. We aimed to characterize the bacterial communities in saliva of patients with different severity of COVID-19 disease, from mild to fatal cases. Our results revealed clear differences in the composition and in the nature of interactions (networking) of the bacterial species present in the different clinical groups and show community-patterns associated with disease severity. Characterization of the microbial communities in saliva may offer important clues to learn ways COVID-19 patients may suffer from different disease severities.","version":"1.1","doi":"10.1101/2022.10.20.513136","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512957","pub_date":"2022-10-20","title":"SARS-CoV-2 infected cells sprout actin-rich filopodia that facilitate viral invasion","abstract":"Emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a great threat to human health and economics. Although SARS-CoV-2 entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the host cell remodeling to facilitate virus invasion process. Here we unveil that SARS-CoV-2 boosts and repurposes filopodia for entry to the target cells. Using SARS-CoV-2 virus-like particle (VLP), real-time live-cell imaging and simulation of active gel model, we reveal that VLP-induced Cdc42 activation leads to the formation of filopodia, which reinforce the viral entry to host cells. By single-particle tracking and sparse deconvolution algorithm, we uncover that VLP particles utilize filopodia to reach the entry site in two patterns, \u2018surfing\u2019 and \u2018grabbing\u2019, which are more efficient and faster than entry via flat plasma membrane regions. Furthermore, the entry process via filopodia is dependent on the actin cytoskeleton and actin-associated proteins fascin, formin, and Arp2/3. Importantly, either inhibition the actin cross-linking protein fascin or the active level of Cdc42 could significantly hinders both the VLP and the authentic SARS-CoV-2 entry. Together, our results highlight that the spatial-temporal regulation of the actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a \u2018highway\u2019 for virus entry, which emerges as an antiviral target. Revealing the mechanism of SARS-CoV-2 invasion is of great significance to explain its high pathogenic and rapid transmission in the world. We discovered a previously unknown route of SARS-CoV-2 entry. SARS-CoV-2 virus-like particles boost cellular filopodia formation by activating Cdc42. Using state-of-art-technology, we spatial-temporally described how virus utilize filopodia to enter the target cell in two modes: \u2018surfing\u2019 and \u2018grabbing\u2019. Filopodia can directly transport the virus to endocytic hot spots to avoid the virus from disorderly searching on the plasma membrane. Our study complements current knowledge of SARS-CoV-2 that filopodia and its components not only play an important role in virus release and cell-cell transmission, but also in the entry process, and provides several potential therapeutic targets for SARS-CoV-2. SARS-CoV-2 VLP infection promotes filopodia formation by activating Cdc42 SARS-CoV-2 VLP utilizes filopodia to enter target cell via two modes, \u2018surfing\u2019 and \u2018grabbing\u2019 Filopodia disruption compromises the invasion of both VLP and authentic SARS-CoV-2","version":"1.1","doi":"10.1101/2022.10.19.512957","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.31.502203","pub_date":"2022-10-20","title":"SARS-CoV-2-specific CD4+ and CD8+ T cell responses can originate from cross-reactive CMV-specific T cells","abstract":"Detection of SARS-coronavirus-2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relative high frequency and prevalence of cross-reactive T cells, we hypothesized CMV may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved PBMCs with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.07.31.502203","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512979","pub_date":"2022-10-20","title":"Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth","abstract":"Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. Development of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.10.19.512979","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512884","pub_date":"2022-10-20","title":"Intra-host viral populations of SARS-CoV-2 in immunosuppressed patients with hematologic cancers","abstract":"Throughout the SARS-CoV-2 pandemic, several variants of concern (VOC) have been identified, many of which share recurrent mutations in the spike protein\u2019s receptor binding domain (RBD). This region coincides with known epitopes and can therefore have an impact on immune escape. Protracted infections in immunosuppressed patients have been hypothesized to lead to an enrichment of such mutations and therefore drive evolution towards VOCs. Here, we show that immunosuppressed patients with hematologic cancers develop distinct populations with immune escape mutations throughout the course of their infection. Notably, by investigating the co-occurrence of substitutions on individual sequencing reads in the RBD, we found quasispecies harboring mutations that confer resistance to known monoclonal antibodies (mAbs) such as S:E484K and S:E484A. Furthermore, we provide the first evidence for a viral reservoir based on intra-host phylogenetics. Our results on viral reservoirs can shed light on protracted infections interspersed with periods where the virus is undetectable as well as an alternative explanation for some long-COVID cases. Our findings also highlight that protracted infections should be treated with combination therapies rather than by a single mAbs to clear pre-existing resistant mutations.","version":"1.1","doi":"10.1101/2022.10.19.512884","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512891","pub_date":"2022-10-20","title":"Distinct Neutralizing Antibody Escape of SARS-CoV-2 Omicron Subvariants BQ.1, BQ.1.1, BA.4.6, BF.7 and BA.2.75.2","abstract":"Continued evolution of SARS-CoV-2 has led to the emergence of several new Omicron subvariants, including BQ.1, BQ. 1.1, BA.4.6, BF.7 and BA.2.75.2. Here we examine the neutralization resistance of these subvariants, as well as their ancestral BA.4/5, BA.2.75 and D614G variants, against sera from 3-dose vaccinated health care workers, hospitalized BA.1-wave patients, and BA.5-wave patients. We found enhanced neutralization resistance in all new subvariants, especially the BQ.1 and BQ.1.1 subvariants driven by a key N460K mutation, and to a lesser extent, R346T and K444T mutations, as well as the BA.2.75.2 subvariant driven largely by its F486S mutation. The BQ.1 and BQ.1.1 subvariants also exhibited enhanced fusogenicity and S processing dictated by the N460K mutation. Interestingly, the BA.2.75.2 subvariant saw an enhancement by the F486S mutation and a reduction by the D1199N mutation to its fusogenicity and S processing, resulting in minimal overall change. Molecular modelling revealed the mechanisms of receptor-binding and non-receptor binding monoclonal antibody-mediated immune evasion by R346T, K444T, F486S and D1199N mutations. Altogether, these findings shed light on the concerning evolution of newly emerging SARS-CoV-2 Omicron subvariants.","version":"1.1","doi":"10.1101/2022.10.19.512891","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.20.512999","pub_date":"2022-10-20","title":"Altered infective competence of the human gut microbiome in COVID-19","abstract":"Infections with SARS-CoV-2 have a pronounced impact on the gastrointestinal tract and its resident microbiome. Clear differences between severe cases of infection and healthy individuals have been reported, including the loss of commensal taxa. We aimed to understand if microbiome alterations including functional shifts are unique to severe cases or a common effect of COVID-19. We used high-resolution systematic multi-omic analyses to profile the gut microbiome in asymptomatic-to-moderate COVID-19 individuals compared to a control group. We found a striking increase in the overall abundance and expression of both virulence factors and antimicrobial resistance genes in COVID-19. Importantly, these genes are encoded and expressed by commensal taxa from families such as Acidaminococcaceae and Erysipelatoclostridiaceae, which we found to be enriched in COVID-19 positive individuals. We also found an enrichment in the expression of a betaherpesvirus and rotavirus C genes in COVID-19 positive individuals compared to healthy controls. Our analyses identified an altered and increased infective competence of the gut microbiome in COVID-19 patients.","version":"1.1","doi":"10.1101/2022.10.20.512999","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512816","pub_date":"2022-10-20","title":"A collaborative approach to improve representation in viral genomic surveillance","abstract":"The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building microbial genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness. Genomic surveillance involves decoding a pathogen\u2019s genetic code to track its spread and evolution. During the pandemic, genomic surveillance programs around the world provided valuable data to scientists, doctors, and public health officials. Knowing the complete SARS-CoV-2 genome has helped detect the emergence of new variants, including ones that are more transmissible or cause more severe disease, and has supported the development of diagnostics, vaccines, and therapeutics. The impact of genomic surveillance on public health depends on representative sampling that accurately reflects the diversity and distribution of populations, as well as rapid turnaround time from sampling to data sharing. After a slow start, SARS-CoV-2 genomic surveillance in the United States grew exponentially. Despite this, many rural regions and ethnic minorities remain poorly represented, leaving significant gaps in the data that informs public health responses. To address this problem, we formed a network of universities and clinics in Louisiana, Georgia, and Mississippi with the goal of increasing SARS-CoV-2 sequencing volume, representation, and equity. Our results demonstrate the advantages of rapidly sequencing pathogens in the same communities where the cases occur and present a model that leverages existing academic and clinical infrastructure for a powerful decentralized genomic surveillance system.","version":"1.2","doi":"10.1101/2022.10.19.512816","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.19.512980","pub_date":"2022-10-20","title":"Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner","abstract":"Mucosal vaccines and vaccines that block pathogen transmission are under-appreciated in vaccine development. However, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has shown that blocking viral transmission is an important attribute of efficient vaccines. Here, we investigated if recombinant influenza virus neuraminidase (NA) vaccines delivered at a mucosal site could protect from onward transmission of influenza B viruses in the guinea pig model. We tested four different scenarios in which sequential transmission was investigated in chains of four guinea pigs. The variables tested included a low and a high viral inoculum (104 vs 105 plaque forming units) in the initial donor guinea pig and variation of exposure/cohousing time (1 day vs 6 days). In three out of four scenarios \u2013 low inoculum-long exposure, low inoculum-short exposure and high inoculum-short exposure \u2013 transmission chains were efficiently blocked. Based on this data we believe an intranasal recombinant NA vaccine could be used to efficiently curtail influenza virus spread in the human population during influenza epidemics. Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here we describe how a recombinant neuraminidase-based influenza virus vaccines reduces transmission in vaccinated guinea pigs in an exposure-intensity based manner.","version":"1.1","doi":"10.1101/2022.10.19.512980","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.18.512323","pub_date":"2022-10-19","title":"The humoral and cellular immune response of a third dose adenoviral-based vectored vaccine after a single or 2 shots of inactivated COVID-19 vaccine in healthy adults","abstract":"The coronavirus pandemic is a severe infectious respiratory disease which caused massive loss worldwide. Thailand was affected by the wild-type, and the Delta variant started in mid-2021. Due to the shortage of effective vaccines, the ministry of public health of Thailand suggested the heterologous vaccine scheme, which comprises inactivated COVID-19 vaccine (CoronaVac) and an adenoviral-based vectored vaccine (ChAdOx1). However, data on the humoral and cellular immune responses of the single or 2 shots of inactivated vaccine followed by the adenoviral-based vaccine are very limited. In this current study, the sera from participants who received either single or 2 shots of CoronaVac followed by the ChAdOx1 vaccine were evaluated for SARS-CoV-2 spike receptor-binding-domain (RBD) IgG. The cytokine level was also assessed using Luminex immunoassay. The PBMC were collected to evaluate spike-specific T-cell and B-cell responses. Participants who received 2 shots of CoronaVac followed by ChAdOx1 possessed significantly (P<0.0001) higher levels of spike RBD-specific IgG. They also exhibited a higher level of CD4+T-cell and IFN-gamma than those who received only 1 shot of CoronaVac followed by the ChAdOx1 vaccine. The volunteers who received two shots of CoronaVac followed by ChAdOx1 had a significantly (p<0.01) higher marginal B-cell response against wild-type SARS-CoV-2 S peptides than those who received only one shot of CoronaVac followed by ChAdOx1. Surprisingly, the class switch B-cell response to Delta variant SARS-CoV-2 S peptides of the volunteers who received 1 shot of CoronaVac followed by ChAdOx1 was significantly (p<0.01) higher than those who received 2 shots of CoronaVac. However, participants who received only a single shot of inactivated vaccine followed by an adenoviral-based vectored vaccine possessed a higher level of TNF-alpha and IL-6. This study indicated that boosting the ChAdOx1 as a third dose after completing 2 shots of CoronaVac induced strong humoral and cellular immune responses.","version":"1.1","doi":"10.1101/2022.10.18.512323","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.03.494608","pub_date":"2022-10-19","title":"Taxonium, a web-based tool for exploring large phylogenetic trees","abstract":"The COVID-19 pandemic has resulted in a step change in the scale of sequencing data, with more genomes of SARS-CoV-2 having been sequenced than any other organism on earth. These sequences reveal key insights when represented as a phylogenetic tree, which captures the evolutionary history of the virus, and allows the identification of transmission events and the emergence of new variants. However, existing web-based tools for exploring phylogenies do not scale to the size of datasets now available for SARS-CoV-2. We have developed Taxonium, a new tool that uses WebGL to allow the exploration of trees with tens of millions of nodes in the browser for the first time. Taxonium links each node to associated metadata and supports mutation-annotated trees, which are able to capture all known genetic variation in a dataset. It can either be run entirely locally in the browser, from a server-based backend, or as a desktop application. We describe insights that analysing a tree of five million sequences can provide into SARS-CoV-2 evolution, and provide a tool at cov2tree.org for exploring a public tree of more than five million SARS-CoV-2 sequences. Taxonium can be applied to any tree, and is available at taxonium.org, with source code at github.com/theosanderson/taxonium.","version":"1.4","doi":"10.1101/2022.06.03.494608","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.18.512746","pub_date":"2022-10-19","title":"Regional epidemic dynamics and Delta variant diversity resulted in varying rates of spread of Omicron-BA.1 in Mexico","abstract":"The Omicron subvariant BA.1 of SARS-CoV-2 was first detected in November 2021 and quickly spread worldwide, displacing the Delta variant. In Mexico, this subvariant began spreading during the first week of December 2021 and became dominant in the next three weeks, causing the fourth COVID-19 epidemiological surge in the country. Unlike previous SARS-CoV-2 variants, BA.1 did not acquire local substitutions nor exhibited a geographically distinct circulation pattern in Mexico. However, a regional difference in the speed of the replacement of the Delta variant was observed, as some northern states showed persistence of Delta lineages well into February 2022. Mexican states were divided into four regions (North, Central North, Central South, and Southeast) based on the lineage circulation before the dominance of BA.1 to study possible causes for this difference. For each region, the time to fixation of BA.1, the diversity of Delta sublineages in the weeks preceding BA.1 entry, the population density, and the level of virus circulation during the inter-wave interval were determined. An association between a faster Omicron spread and lower Delta diversity, as well as fewer COVID-19 cases during the Delta-BA.1.x inter-wave period, was observed. For example, the North region exhibited the slowest spread but had the highest diversity of Delta sublineages and the greatest number of inter-wave cases relative to the maximum amount of the virus circulating in the region, whereas the Southeast region showed the opposite. Viral diversity and the relative abundance of the virus in a particular area around the time of the introduction of a new lineage seem to have influenced the spread dynamics. Nonetheless, if there is a significant difference in the fitness of the variants or the time allowed for the competition is sufficient, it seems the fitter virus will eventually become dominant, as observed in the eventual dominance of the BA.1.x variant in Mexico. The surveillance of lineage circulation of SARS-CoV-2 has helped identify variants that have a transmission advantage and are of concern to public health and to track the virus dispersion accurately. However, many factors contributing to differences in lineage spread dynamics beyond the acquisition of specific mutations remain poorly understood. In this work, a description of BA.1 entry and dispersion within Mexico is presented, and which factors potentially affected the spread rates of the Omicron variant BA.1 among geographical regions in the country are analyzed, underlining the importance of population density, the proportion of active cases, and viral lineage diversity and identity before the entry of BA.1. This work was carried out using data shared through the GISAID initiative. All sequences and metadate are available through GISAID with the accession EPI_SET_220927gw, accession numbers and metadata are also reported in the supplemental material of this article. Epidemiological data was obtained though the Secretar\u00eda de Salud website (https://www.gob.mx/salud/documentos/datos-abiertos-152127),","version":"1.1","doi":"10.1101/2022.10.18.512746","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.17.512637","pub_date":"2022-10-18","title":"Deep learning-based Drug discovery of Mac domain of SARS-CoV-2 (WT) Spike inhibitors: using experimental ACE2 Inhibition TR-FRET Assay Screening and Molecular Dynamic Simulations","abstract":"SARS-CoV-2 exploits the homotrimer transmembrane Spike glycoproteins (S protein) during host cell invasion. Omicron, delta, and prototype SARS-CoV-2 receptor-binding domain show similar binding strength to hACE2 (Angiotensin-Converting Enzyme 2). Here we utilized multi-ligand virtual screening to identify small molecule inhibitors for their efficacy against SARS-CoV-2 virus using quantum Docking, pseudovirus ACE2 Inhibition TR-FRET Assay Screening, and Molecular Dynamic simulations (MDS). 350-thousand compounds were screened against the macrodomain of non-structural protein 3 of SARS-CoV-2. Using TR-FRET Assay, we filtered out two of 10 compounds that had no reported activity in in-vitro screen against Spike S1: ACE2 binding assay. Percentage Inhibition at 30 \u00b5M was found to be 79% for \u201cCompound F1877-0839\u201d and 69% for \u201cCompound F0470-0003\u201d. This first of its kind study identified \u201cFILLY\u201d pocket in macrodomains. Our 200 ns MDS revealed stable binding poses of both leads. They can be used for further development of preclinical candidates. Iqbal et al., described a deep learning guided drug discovery, efficacy against SARS-CoV-2 Spike inhibitors: using experimental pseudovirus ACE2 Inhibition TR-FRET Assay. Our molecular dynamic simulation results were next validated a posteriori against the corresponding experimental data of identified leads with 80 percent inhibition. Moreover, this study is first of kind to identify \u201cFILLY\u201d pocket in macrodomains. Experimental pseudovirus ACE2 Inhibition TR-FRET Assay and HTS lead to identification of two potential clinical leads. Conformational Dynamics analysis reveal the structural stability of complexes throughout 200 ns molecular dynamic simulations. Unveiling of the impact surface charge on the Variant of Concerns Detection of conformational changes within ACE2/RBD complex We identified the FILLY pocket in the SARS viruses.","version":"1.1","doi":"10.1101/2022.10.17.512637","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.17.512617","pub_date":"2022-10-18","title":"Infection of primary nasal epithelial cells differentiates among lethal and seasonal human coronaviruses","abstract":"The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal (SARS-CoV-2 and MERS-CoV) and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures but diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV replication, enhancing MERS-CoV replication but reducing that of SARS-CoV-2 and HCoV-NL63. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.","version":"1.1","doi":"10.1101/2022.10.17.512617","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.15.512351","pub_date":"2022-10-17","title":"Investigating the role of binding free energy, binding affinity and antibody escape in the evolution of SARS-CoV-2 spike protein","abstract":"SARS-CoV-2 is considered a pandemic virus and presents a major strain on public health globally. SARS-CoV-2 infects mammalian cells by binding to its receptor, ACE2 which is mediated by the viral spike glycoprotein, specifically the receptor binding domain (RBD) within the spike protein. Recent development of vaccines against SARS-CoV-2 spike protein are currently the best strategy to reduce morbidity and mortality from infection. Like all viruses, SARS-CoV-2 evolves which may result in mutations which are benign or alter its viral fitness. The evolution of SARS-CoV-2 may increase the virulence, possibly by increasing the infectivity of the virus through strengthening the binding of the RBD to ACE2 or enabling the virus to evade naturally or vaccine induced immune responses. To address the need to characterise the evolution of SARS-CoV-2, this study has compared SARS-CoV2 sequences globally to the Wuhan reference strain at different time points. Additionally, by assigning scores to sequence data, which quantify each sequences binding strength to ACE2 and ability to evade patient derived antibodies, we have demonstrated that over time SARS-CoV-2 has evolved in less than one year to increase its ability to evade antibodies and increase the binding free energy between the RBD and ACE2.","version":"1.1","doi":"10.1101/2022.10.15.512351","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.16.512395","pub_date":"2022-10-17","title":"Host independent deletion hotspots in the SARS-CoV-2 genome","abstract":"SARS-CoV-2 infects a wide range of hosts in varying degrees. The RNA genome of SARS-CoV-2 makes it prone to mutations. Advantageous mutations help the virus to evolve and the virus maintains such mutations across species. Here in this study, all non-human hosts-derived SARS-CoV-2 genomic sequences from the GISAID database were analyzed, and identified several deletion hotspots, which are maintained by the virus, across various host species, indicating their important role in the virus evolution. Several of these deletion hotspots are also found in human-derived SARS-CoV-2 genomic sequences. These deletion hotspots have the potential to affect the pathogenicity and virulence of the virus and have a role in molecular and serological diagnostics. Potentially, they can lead to immune escape, resulting in vaccine failure and drug-resistant variants.","version":"1.1","doi":"10.1101/2022.10.16.512395","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.15.512346","pub_date":"2022-10-17","title":"Comparative Mutagenesis of SARS-CoV-2 Nonstructural Proteins (NSPs) Across Variants: The Case for RdRp as a Therapeutic Target","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenicity has been studied extensively from the perspective of structural (S, E, M, N) proteins for purposes in vaccine development. The virus\u2019 nonstructural protein (nsp) components are less characterized, and demonstrate significant potential in efforts to develop novel therapeutic agents. NSP 7, 8, and 12, formed from the cleavage of pp1a and pp1ab polyproteins, comprise the viral replicase (RdRp) complex, the site for the mechanism of action of Remdesivir. Presented herein is a phylogenetic analysis for the evolution of SARS-CoV-2 replicase components between variant and related coronaviruses with the aim to delineate its current and long-term efficacy as a drug target.","version":"1.1","doi":"10.1101/2022.10.15.512346","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.14.512325","pub_date":"2022-10-17","title":"The impact of the ABO/Rh blood group on susceptibility and severity among COVID-19 patients in Luanda, Angola","abstract":"SARS-CoV-2 is a public health concern worldwide. Identification of biological factors that could influence transmission and worsen the disease has been the subject of extensive investigation. Herein, we investigate the impact of the ABO/Rh blood group on susceptibility and severity among COVID-19 patients in Luanda, Angola. This was a multicentric cohort study conducted with 101 COVID-19 patients. Chi-square and logistic regression were calculated to check factors related to the worsening of the disease and deemed significant when p<0.05. Blood type O (51.5%) and Rh-positive (93.1%) were the most frequent. Patients from blood type O had a high risk to severe disease [OR: 1.33 (95% CI: 0.42 - 4.18), p=0.630] and hospitalization [OR: 2.59 (95% CI: 0.84 - 8.00), p=0.099]. Also, Rh-positive blood type presented a high risk for severe disease (OR: 10.6, p=0.007) and hospitalization (OR: 6.04, p=0.026). We find a high susceptibility, severity, hospitalization, and mortality, respectively, among blood group O and Rh-positive patients, while blood group AB presented a low susceptibility, severity, hospitalization, and mortality, respectively. Our findings add to the body of evidence suggesting that ABO/Rh blood groups play an important role in the course of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.10.14.512325","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.15.512322","pub_date":"2022-10-17","title":"Determinants and Mechanisms of the Low Fusogenicity and Endosomal Entry of Omicron Subvariants","abstract":"The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation, as shown by increased molecular interactions in structural modeling as well as reduced S1 shedding. Importantly, the H655Y mutation also determines the low fusogenicity and high dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5 and BA.2.75. These results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis.","version":"1.1","doi":"10.1101/2022.10.15.512322","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.16.512436","pub_date":"2022-10-17","title":"The Impact of Protein Dynamics on Residue-Residue Coevolution and Contact Prediction","abstract":"The need to maintain protein structure constrains evolution at the sequence level, and patterns of coevolution in homologous protein sequences can be used to predict their 3D structures with high accuracy. Our understanding of the relationship between protein structure and evolution has traditionally been benchmarked by computational models\u2019 ability to predict contacts from a single representative, experimentally determined structure per protein family. However, proteins in vivo are highly dynamic and can adopt multiple functionally relevant conformations. Here we demonstrate that interactions that stabilize alternate conformations, as well those that mediate conformational changes, impose an underappreciated but significant set of evolutionary constraints. We analyze the extent of these constraints over 56 paralogous G protein coupled receptors (GPCRs), \u03b2-arrestin and the human SARS-CoV2 receptor ACE2. Specifically, we observe that contacts uniquely found in molecular dynamics (MD) simulation data and alternate-conformation crystal structures are successfully predicted by unsupervised language models. In GPCRs, adding these contacts as positives increases the percentage of top contacts classified as true positives, as predicted by a state-of-the-art language model, from 69% to 87%. Our results show that protein dynamics impose constraints on molecular evolution and demonstrate the ability of unsupervised language models to measure these constraints.","version":"1.1","doi":"10.1101/2022.10.16.512436","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.17.22281058","pub_date":"2022-10-17","title":"Eleven key measures for monitoring general practice clinical activity during COVID-19 using federated analytics on 48 million adults\u2019 primary care records through OpenSAFELY","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The COVID-19 pandemic has had a significant impact on delivery of NHS care. We have developed the OpenSAFELY Service Restoration Observatory (SRO) to describe this impact on primary care activity and monitor its recovery.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>To develop key measures of primary care activity and describe the trends in these measures throughout the COVID-19 pandemic.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>With the approval of NHS England we developed an open source software framework for data management and analysis to describe trends and variation in clinical activity across primary care electronic health record (EHR) data on 48 million adults.</jats:p>\n                  <jats:p>We developed SNOMED-CT codelists for key measures of primary care clinical activity selected by a expert clinical advisory group and conducted a population cohort-based study to describe trends and variation in these measures January 2019-December 2021, and pragmatically classified their level of recovery one year into the pandemic using the percentage change in the median practice level rate.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We produced 11 measures reflective of clinical activity in general practice. A substantial drop in activity was observed in all measures at the outset of the COVID-19 pandemic. By April 2021, the median rate had recovered to within 15% of the median rate in April 2019 in six measures. The remaining measures showed a sustained drop, ranging from a 18.5% reduction in medication reviews to a 42.0% reduction in blood pressure monitoring. Three measures continued to show a sustained drop by December 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The COVID-19 pandemic was associated with a substantial change in primary care activity across the measures we developed, with recovery in most measures. We delivered an open source software framework to describe trends and variation in clinical activity across an unprecedented scale of primary care data. We will continue to expand the set of key measures to be routinely monitored using our publicly available NHS OpenSAFELY SRO dashboards with near real-time data.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.10.17.22281058","journal":"medRxiv","score":null},{"id":"10.1101/2022.10.14.22281103","pub_date":"2022-10-17","title":"No evidence that analgesic use after COVID-19 vaccination negatively impacts antibody responses","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Uptake of mRNA vaccines, especially booster immunizations, against COVID-19 has been lower than hoped, perhaps in part due to their reactogenicity. Analgesics might alleviate symptoms associated with vaccination, but studies to measure their impact on immune responses have been limited to relatively small cohorts. We semi-quantitatively measured antibody responses following COVID-19 vaccination in 2354 human participants surveyed about analgesic use. Participants who used non-steroidal anti-inflammatory drugs (NSAIDs) or acetaminophen after vaccination showed elevated antibody levels against the receptor binding domain of Spike protein relative to those who did not use analgesics. This pattern was observed for both mRNA-1273 and BNT162b2 and across age groups. Participants who used analgesics more frequently reported fatigue, muscle aches, and headaches than those who did not use painkillers. Amongst participants who reported these symptoms, we observed no statistically significant differences in antibody levels irrespective of analgesic use. These data suggest that antibody levels are elevated as a function of symptoms and inflammatory processes rather than painkiller use per se. Taken together, we find no evidence that analgesic use reduces antibody responses after COVID-19 vaccination. Recommendation of their use to alleviate symptoms might improve uptake of booster immunizations.</jats:p>","version":null,"doi":"10.1101/2022.10.14.22281103","journal":"medRxiv","score":null},{"id":"10.1101/2022.10.14.512324","pub_date":"2022-10-16","title":"SARS-CoV-2 multi-antigen protein microarray for detailed characterization of antibody responses in COVID-19 patients","abstract":"Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target multiple epitopes on different domains of the spike protein, and other SARS-CoV-2 proteins. We developed a SARS-CoV-2 multi-antigen protein microarray with the nucleocapsid, spike and its domains (S1, S2), and variants with single (D614G, E484K, N501Y) or double substitutions (N501Y/Deletion69/70), allowing a more detailed high-throughput analysis of the antibody repertoire following infection. The assay was demonstrated to be reliable and comparable to ELISA. We analyzed antibodies from 18 COVID-19 patients and 12 recovered convalescent donors. S IgG level was higher than N IgG in most of the COVID-19 patients, receptor-binding domain of S1 showed high reactivity, but no antibodies were detected against heptad repeat domain 2 of S2. Furthermore, antibodies were detected against S variants with single and double substitutions in COVID-19 patients who were infected with SARS-CoV-2 early in the pandemic. Here we demonstrated that SARS-CoV-2 multi-antigen protein microarray is a powerful tool for detailed characterization of antibody responses, with potential utility in understanding the disease progress and assessing current vaccines and therapies against evolving SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.10.14.512324","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.14.512296","pub_date":"2022-10-16","title":"Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes","abstract":"SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in \u201cup\u201d position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.","version":"1.1","doi":"10.1101/2022.10.14.512296","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.507904","pub_date":"2022-10-15","title":"The SARS-CoV-2 spike N-terminal domain engages 9-O-acetylated \u03b12-8-linked sialic acids","abstract":"SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, Alpha, Beta, Delta, and Omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 Beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells. Coronaviruses utilize their N-terminal domain (NTD) for initial reversible low-affinity interaction to (sialylated) glycans. This initial low-affinity/high-avidity engagement enables viral surfing on the target membrane, potentially followed by a stronger secondary receptor interaction. Several coronaviruses, such as HKU1 and OC43, possess a hemagglutinin-esterase for viral release after sialic acid interaction, thus allowing viral dissemination. Other coronaviruses, such as MERS-CoV, do not possess a hemagglutinin-esterase, but interact reversibly to sialic acids allowing for viral surfing and dissemination. The early 501Y.V2-1 subvariant of the Beta SARS-CoV-2 Variant of Concern has attained a receptor-binding functionality towards 9-O-acetylated sialic acid using its NTD. This binding functionality was selected against rapidly, most likely due to poor dissemination. Ablation of sialic acid binding in more recent SARS-CoV-2 Variants of Concern suggests a fine balance of sialic acid interaction of SARS-CoV-2 is required for infection and/or transmission.","version":"1.2","doi":"10.1101/2022.09.14.507904","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.13.512127","pub_date":"2022-10-14","title":"SARS-CoV-2 immune complex triggers human monocyte necroptosis","abstract":"We analyzed the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) itself and SARS-CoV-2-IgG immune complexes to trigger human monocyte necroptosis. SARS-CoV-2 was able to induce monocyte necroptosis dependently of MLKL activation. Necroptosis-associated proteins (RIPK1, RIPK3 and MLKL) were involved in SARS-CoV-2 N1 gene expression in monocytes. SARS-CoV-2 immune complexes promoted monocyte necroptosis in a RIPK3- and MLKL-dependent manner, and Syk tyrosine kinase was necessary for SARS-CoV-2 immune complex-induced monocyte necroptosis, indicating the involvement of Fc\u03b3 receptors on necroptosis. Finally, we provide evidence that elevated LDH levels as a marker of lytic cell death are associated with COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2022.10.13.512127","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.01.450475","pub_date":"2022-10-14","title":"CRISPRa screening with real world evidence identifies potassium channels as neuronal entry factors and druggable targets for SARS-CoV-2","abstract":"Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been successful, there are no good treatments for those who are actively infected. While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from sensory organs and the brain are also susceptible to infection. While many patients suffer from diverse neurological symptoms, the virus\u2019s neuronal entry remains mysterious. To discover host factors involved in SARS-CoV-2 viral entry, we performed CRISPR activation (CRISPRa) screens targeting all 6000+ human membrane proteins in cells with and without overexpression of ACE2 using Spike-pseudotyped lentiviruses. This unbiased gain-of-function screening identified both novel and previously validated host factors. Notably, newly found host factors have high expression in neuronal and immune cells, including potassium channel KCNA6, protease LGMN, and MHC-II component HLA-DPB1. We validated these factors using replication-competent SARS-CoV-2 infection assays. Notably, the overexpression of KCNA6 led to a marked increase in infection even in cells with undetectable levels of ACE2 expression. Analysis of human olfactory epithelium scRNA-seq data revealed that OLIG2+/TUJ1+ cells--previously identified as sites of infection in COVID-19 autopsy studies-- have high KCNA6 expression and minimal levels of ACE2. The presence of KCNA6 may thus explain sensory/neuronal aspects of COVID-19 symptoms. Further, we demonstrate that FDA-approved compound dalfampridine, an inhibitor of KCNA-family potassium channels, suppresses viral entry in a dosage-dependent manner. Finally, we identified common prescription drugs likely to modulate the top identified host factors, and performed a retrospective analysis of insurance claims of ~8 million patients. This large cohort study revealed a statistically significant association between top drug classes, particularly those targeting potassium channels, and COVID-19 severity. Taken together, the potassium channel KCNA6 facilitates neuronal entry of SARS-CoV-2 and is a promising target for drug repurposing and development.","version":"1.2","doi":"10.1101/2021.07.01.450475","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.14.512203","pub_date":"2022-10-14","title":"Computational Prediction of Binding Affinities of Human Angiotensin Converting Enzyme-2 with SARS-CoV-2 Spike Protein Variants: Omicron Variants and Potentially Deleterious Mutations","abstract":"The Omicron variant (BA.1) and its sub-variants of the SARS-CoV-2 virus which causes the COVID-19 disease continues to spread across the United States and the World at large. As new sub-variants of SARS-CoV-2 continue to proliferate, a reliable computational method of quickly determining the potential infectivity of these new variants is needed to assess their potential threat. In the present study, we have tested and validated an efficient computational protocol, which includes an efficient energy minimization and subsequent molecular mechanics/Poisson Boltzmann surface area (MM-PBSA) calculation of the binding free energy between the SARS-CoV-2 spike protein and human angiotensin converting enzyme-2 (ACE2), to predict the binding affinities of these spike/ACE2 complexes based upon the calculated binding free energies and a previously calibrated linear correlation relationship. The predicted binding affinities are in good agreement with available experimental data including those for Omicron variants, suggesting that the predictions based on this protocol should be reasonable. Further, we have investigated several hundred potential mutations of both the wildtype and Omicron variants of the SARS-CoV-2 spike protein. Based on the predicted binding affinity data, we have identified several mutations that have the potential to vastly increase the binding affinity of the spike protein to ACE2 within both the wildtype and Omicron variants. As well known, the coronavirus responsible for COVID-19 disease enters human cells through its spike protein binding with a human receptor protein known as angiotensin converting enzyme-2. So, the binding affinity between the spike protein and angiotensin converting enzyme-2 contributes to the infectivity of the coronavirus and its variants. In this study, we demonstrated that a generally applicable, fast and easy-to-use computational protocol was able to accurately predict the binding affinity of angiotensin converting enzyme-2 with spike protein of the currently known variants of the coronavirus. Hence, we believe that this computational protocol may be used to reliably predict the binding affinity of angiotensin converting enzyme-2 with spike protein of new variants to be identified in the future. Using this computational protocol, we have further examined a number of possible single mutations on the spike protein of both the wildtype and Omicron variants and predicted their binding affinity with angiotensin converting enzyme-2, demonstrating that several mutations have the potential to vastly increase the binding affinity of the spike protein to angiotensin converting enzyme-2.","version":"1.1","doi":"10.1101/2022.10.14.512203","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495116","pub_date":"2022-10-14","title":"The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir","abstract":"The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors.","version":"1.2","doi":"10.1101/2022.06.07.495116","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.14.512216","pub_date":"2022-10-14","title":"Broad SARS-CoV-2 Neutralization by Monoclonal and Bispecific Antibodies Derived from a Gamma-infected Individual","abstract":"The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.","version":"1.1","doi":"10.1101/2022.10.14.512216","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.12.511991","pub_date":"2022-10-14","title":"Rapid transmission and tight bottlenecks constrain the evolution of highly transmissible SARS-CoV-2 variants","abstract":"Transmission bottlenecks limit the spread of novel mutations and reduce the efficiency of natural selection along a transmission chain. Many viruses exhibit tight bottlenecks, and studies of early SARS-CoV-2 lineages identified a bottleneck of 1-3 infectious virions. While increased force of infection, host receptor binding, or immune evasion may influence bottleneck size, the relationship between transmissibility and the transmission bottleneck is unclear. Here, we compare the transmission bottleneck of non-variant-of-concern (non-VOC) SARS-CoV-2 lineages to those of the Alpha, Delta, and Omicron variants. We sequenced viruses from 168 individuals in 65 multiply infected households in duplicate to high depth of coverage. In 110 specimens collected close to the time of transmission, within-host diversity was extremely low. At a 2% frequency threshold, 51% had no intrahost single nucleotide variants (iSNV), and 42% had 1-2 iSNV. In 64 possible transmission pairs with detectable iSNV, we identified a bottleneck of 1 infectious virion (95% CI 1-1) for Alpha, Delta, and Omicron lineages and 2 (95% CI 2-2) in non-VOC lineages. The latter was driven by a single iSNV shared in one non-VOC household. The tight transmission bottleneck in SARS-CoV-2 is due to low genetic diversity at the time of transmission, a relationship that may be more pronounced in rapidly transmissible variants. The tight bottlenecks identified here will limit the development of highly mutated VOC in typical transmission chains, adding to the evidence that selection over prolonged infections in immunocompromised patients may drive their evolution.","version":"1.1","doi":"10.1101/2022.10.12.511991","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.13.512105","pub_date":"2022-10-14","title":"TCID50 Measurements of anti-viral efficacy on metal printed masks and plastic surfaces","abstract":"The SARS-CoV-2 pandemic has created a need for effective personal protective equipment (PPE) to prevent viral spread. PPE like face masks contain the spread of virus-filled droplets and thus reduce infection rates, has been a critical tool in stopping the spread of SARS-CoV-2. PET plastic barriers have also been used in public settings to reduce face to face viral transmission. However, in some cases, they have provided additional contact with the virus due to contamination. In order study, we evaluated the effectiveness of face masks and PET plastics coated in different metals in reducing viral load. We compared PPE printed with silver, copper, or zinc for their ability to inactivate live human coronavirus HCoV 229E. Our results show that silver and copper have significant anti-viral efficacy when printed on nonwoven fabric compared to the controls. The metal-printed PET showed around 70% anti-viral efficacy with any formulations, with copper performing the best. This work builds more data to support the development of metal printed materials for enhanced protection against coronaviruses.","version":"1.1","doi":"10.1101/2022.10.13.512105","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.379115","pub_date":"2022-10-14","title":"Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19","abstract":"Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections. In a severe systemic inflammatory response, such as sepsis and COVID-19, neutrophils play a central role in organ damage. Thus, finding new ways to inhibit the exacerbated response of these cells is greatly needed. Here, we demonstrate that in vitro treatment of whole blood with the viral neuraminidase inhibitors Oseltamivir or Zanamivir, inhibits the activity of human neuraminidases as well as the exacerbated response of neutrophils. In experimental models of severe sepsis, oseltamivir decreased neutrophil activation and increased the survival rate of mice. Moreover, Oseltamivir or Zanamivir ex vivo treatment of whole blood cells from severe COVID-19 patients rewire neutrophil function.","version":"1.5","doi":"10.1101/2020.11.12.379115","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.10.511625","pub_date":"2022-10-13","title":"Statistical challenges for inferring multiple SARS-CoV-2 spillovers with early outbreak phylodynamics","abstract":"Understanding how SARS-CoV-2 entered the human population, thereby causing the COVID-19 pandemic, is one of the most urgent questions in science today. Two hypotheses are widely acknowledged as being most likely to explain the pandemic\u2019s origin in late 2019: (i) the \u201cnatural origin\u201d hypothesis that one or more cross-species transmissions from animals into humans occurred, most likely at the Huanan Seafood Market in Wuhan, China; (ii) the \u201claboratory origin\u201d hypothesis, that scientific research activities led to the unintentional leak of SARS-CoV-2 from a laboratory into the general population. A recent analysis of SARS-CoV-2 genomes by Pekar et al. [Science 377:960-966 (2022)] claims to establish at least two separate spillover events from animals into humans, thus claiming to provide strong evidence for the natural origin hypothesis. However, here we use outbreak simulations to show that the findings of Pekar et al. are heavily impacted by two methodological artifacts: the dubious exclusion of informative SARS-CoV-2 genomes, and their reliance on unrealistic phylodynamic models of SARS-CoV-2. Absent models that incorporate these effects, one cannot conclude multiple SARS-CoV-2 spillovers into humans. Our results cast doubt on a primary point of evidence in favor of the natural origin hypothesis. It is not known if SARS-CoV-2 spilled over from animals into humans at the Huanan Seafood Market, or arose as a result of research activities studying bat coronaviruses. Two recent papers had claimed to answer this question, but here we show those papers are both inconclusive as they fail to account for biases in how medical managers became alerted to SARS-CoV-2 and how public health authorities sampled early cases. Additionally, key data points conflicting with the authors\u2019 conclusions were improperly excluded from the analysis. The papers\u2019 methods do not justify their conclusions, and the origin of SARS-CoV-2 remains an urgent, open question for science.","version":"1.1","doi":"10.1101/2022.10.10.511625","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.13.512053","pub_date":"2022-10-13","title":"SARS-CoV-2 infection in domestic rats after transmission from their infected owner","abstract":"We report the transmission of SARS-CoV-2 Omicron variant from a COVID-19 symptomatic individual to two domestic rats, one of which developed severe symptoms. Omicron carries several mutations which permit rodent infection. This report demonstrates that pet, and likely wild, rodents could therefore contribute to SARS-CoV-2 spread and evolution.","version":"1.1","doi":"10.1101/2022.10.13.512053","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.13.512054","pub_date":"2022-10-13","title":"Combination of the parent analogue of Remdesivir (GS-441524) and Molnupiravir results in a markedly potent antiviral effect in SARS-CoV-2 infected Syrian hamsters","abstract":"Remdesivir was the first drug to be approved for the treatment of severe COVID-19; followed by molnupiravir (another prodrug of a nucleoside analogue) and the protease inhibitor nirmatrelvir. Combination of antiviral drugs may result in improved potency and help to avoid or delay the development of resistant variants. We set out to explore the combined antiviral potency of GS-441524 (the parent nucleoside of remdesivir) and molnupiravir against SARS-CoV-2. In SARS-CoV-2 (BA.5) infected A549-Dual\u2122 hACE2-TMPRSS2 cells, the combination resulted in an overall additive antiviral effect with a synergism at certain concentrations. Next, the combined effect was explored in Syrian hamsters infected with SARS-CoV-2 (Beta, B.1.351); treatment was started at the time of infection and continued twice daily for four consecutive days. At 4 day 4 post-infection, GS-441524 (50 mg/kg, oral BID) and molnupiravir (150 mg/kg, oral BID) as monotherapy reduced infectious viral loads by 0.5 and 1.6 log10, respectively, compared to the vehicle control. When GS-441524 (50 mg/kg, BID) and molnupiravir (150 mg/kg, BID) were combined, infectious virus was no longer detectable in the lungs of 7 out of 10 of the treated hamsters (4.0 log10 reduction) and titers in the other animals were reduced by ~2 log10. The combined antiviral activity of molnupiravir which acts by inducing lethal mutagenesis and GS-441524, which acts as a chain termination appears to be highly effective in reducing SARS-CoV-2 replication/infectivity. The unexpected potent antiviral effect of the combination warrants further exploration as a potential treatment for COVID-19.","version":"1.1","doi":"10.1101/2022.10.13.512054","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492068","pub_date":"2022-10-13","title":"Variation in the ACE2 receptor has limited utility for SARS-CoV-2 host prediction","abstract":"Transmission of SARS-CoV-2 from humans to other species threatens wildlife conservation and may create novel sources of viral diversity for future zoonotic transmission. A variety of computational heuristics have been developed to pre-emptively identify susceptible host species based on variation in the ACE2 receptor used for viral entry. However, the predictive performance of these heuristics remains unknown. Using a newly-compiled database of 96 species we show that, while variation in ACE2 can be used by machine learning models to accurately predict animal susceptibility to sarbecoviruses (accuracy = 80.2%, binomial confidence interval [CI]: 70.8 \u2013 87.6%), the sites informing predictions have no known involvement in virus binding and instead recapitulate host phylogeny. Models trained on host phylogeny alone performed equally well (accuracy = 84.4%, CI: 75.5 \u2013 91.0%) and at a level equivalent to retrospective assessments of accuracy for previously published models. These results suggest that the predictive power of ACE2-based models derives from strong correlations with host phylogeny rather than processes which can be mechanistically linked to infection biology. Further, biased availability of ACE2 sequences misleads projections of the number and geographic distribution of at-risk species. Models based on host phylogeny reduce this bias, but identify a very large number of susceptible species, implying that model predictions must be combined with local knowledge of exposure risk to practically guide surveillance. Identifying barriers to viral infection or onward transmission beyond receptor binding and incorporating data which are independent of host phylogeny will be necessary to manage the ongoing risk of establishment of novel animal reservoirs of SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.05.16.492068","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.13.512056","pub_date":"2022-10-13","title":"A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike","abstract":"A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.","version":"1.1","doi":"10.1101/2022.10.13.512056","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.12.511994","pub_date":"2022-10-13","title":"Reduction in RBD Binding Affinity to Glycosylated ACE2 is Entropic in Origin","abstract":"The spike protein in the virus SARS-CoV-2 (the causative agent of COVID-19) recognizes the host cell by binding to the peptidase domain (PD) of the extracellular enzyme Angiotensin-converting Enzyme 2 (ACE2). A variety of carbohydrates could be attached to the six asparagines in the PD, resulting in a heterogeneous population of ACE2 glycoforms. Experiments have shown that the binding affinity of glycosylated and deglycosylated ACE2 to the virus is virtually identical. In most cases, the reduction in glycan size correlates with stronger binding, which suggests that volume exclusion, and hence entropic forces, determine the binding affinity. Here, we quantitatively test the entropy-based hypothesis by developing a lattice model for the complex between ACE2 and the SARS-CoV-2 spike protein Receptor-binding Domain (RBD). Glycans are treated as branched polymers with only volume exclusion, which we justify using all atom molecular dynamics simulations in explicit water. We show that the experimentally measured changes in the ACE2-RBD dissociation constants for a variety of engineered ACE2 glycoforms are well accounted for by our theory, thus affirming that ACE2 glycans have only a weak, entropic effect on RBD binding.","version":"1.1","doi":"10.1101/2022.10.12.511994","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.12.512011","pub_date":"2022-10-13","title":"Laboratory evaluation of a quaternary ammonium compound (QAC)-based antimicrobial coating used in public transport during the COVID-19 pandemic","abstract":"The virucidal activity of a quaternary ammonium compound (QAC)-based antimicrobial coating used by the UK rail industry during the COVID-19 pandemic was evaluated using the bacteriophage \u03d56 as a surrogate for SARS-CoV-2. Immediately after application and in the absence of interfering substance, the product showed efficacy (>3 log10 reduction) on some materials typically used in rail carriages (stainless steel, high pressure laminate and plastic), variable efficacy on glass and no efficacy (<3 log10 reduction) on a train armrest made of Terluran 22. If, after application of the product, the surfaces remained undisturbed, the antimicrobial coating retained its efficacy for at least 28 days on all materials where it was effective immediately after application. However, regardless of the material coated or time since application, the presence of organic debris (fetal bovine serum) significantly reduced the viricidal activity of the coating. Wiping the surface with a wetted cloth after organic debris deposition was not sufficient to restore efficacy. We conclude that the product is likely to be of limited effectiveness in a busy multi-user environment such as public transport. This study evaluated the performance of a commercially available antimicrobial coating used by the transport industry in the UK during the COVID-19 pandemic. While the product initially showed efficacy against \u03d56 when applied to some materials, when organic debris was subsequently deposited, the efficacy was severely diminished and could not be recovered through wiping (cleaning) the surface. This highlights the importance of including relevant materials and conditions when evaluating antimicrobial coatings in the laboratory. Further efforts are required to identify suitable infection prevention and control practices for the transport industry.","version":"1.1","doi":"10.1101/2022.10.12.512011","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.11.511804","pub_date":"2022-10-12","title":"Development of SARS-CoV-2 replicons for the ancestral virus and variant of concern Delta for antiviral screening","abstract":"SARS-CoV-2 is the aetiologic agent of COVID-19 and the associated ongoing pandemic. As the pandemic has progressed, Variants of Concern (VOC) have emerged with lineage defining mutations. Using a SARS-CoV-2 reverse genetic system, based on transformation associated recombination in yeast, a series of replicons were produced for the ancestral Wuhan virus and the SARS-CoV-2 VOC Delta in which different combinations of the Spike, membrane, ORF6 and ORF7a coding sequences were replaced with sequences encoding the selectable marker puromycin N-acetyl transferase and reporter proteins (Renilla luciferase, mNeonGreen and mScarlet). Replicon RNAs were replication competent in African green monkey kidney (Vero E6) derived cells and a range of human cell lines, with a Vero E6 cell line expressing ACE2 and TMPRSS2 showing much higher transfection efficiency and overall levels of Renilla luciferase activity. The replicons could be used for transient gene expression studies, but cell populations that stably maintained the replicons could not be propagated. Replication of the transiently expressed replicon RNA genomes was sensitive to remedesivir, providing a system to dissect the mechanism of action of antiviral compounds.","version":"1.1","doi":"10.1101/2022.10.11.511804","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.11.511764","pub_date":"2022-10-12","title":"SARS-CoV2 associated secretion of nanoLuciferase reports on virus and Virus-Like Particle production","abstract":"SARS-CoV2 is a positive-strand RNA virus in the Coronaviridae family that has caused world-wide morbidity and mortality. While much progress has been made we still need expanded rapid anti-virals. The top advanced antiviral candidates all target stages of RNA replication, leaving virus assembly an unexplored avenue of antiviral research. To address this gap, and explore the biochemical and cell biological features of viral assembly, we have employed an improved virus-like particle (VLP) system. We exploited the small nanoLuciferase protein for enhanced signal and surprisingly found that the protein itself appears to be packaged into both SARS CoV2 VLPs and virions and secreted from cells. Interestingly, nLuc is not co-secreted with dengue or Zika infection, suggesting the large virion of Coronavirus can encaspidate and secrete a cellularly expressed reporter protein. Our findings open the way for powerful new approaches to measure viral particle production, egress and viral entry mechanisms.","version":"1.1","doi":"10.1101/2022.10.11.511764","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.11.511826","pub_date":"2022-10-12","title":"Discovering hub genes involved in the pathophysiological impact of COVID-19 on diabetes kidney disease by differential gene expression and interactome analysis","abstract":"Diabetic kidney disease (DKD) is a frequently chronic kidney pathology derived from diabetes comorbidity. This condition has irreversible damage, and its risk factor increases with SARS-CoV-2 infection. The prognostic outcome for diabetic patients with COVID-19 is dismal, even with intensive medical treatment. However, there is still scarce information on critical genes involved in the pathophysiological impact of COVID-19 on DKD. Herein, we characterize differential expression gene (DEG) profiles and determine hub genes undergoing transcriptional reprogramming in both disease conditions. Out of 995 DEGs, we identified 42 DEGs shared with COVID-19 pathways. Enrichment analysis elucidated that they are significantly induced with implications for immune and inflammatory responses. By performing a protein-protein interaction (PPI) network and applying topological methods, we determine the following five hub genes STAT1, IRF7, ISG15, MX1, and OAS1. Then, by network deconvolution, we determine their co-expressed gene modules. Moreover, we validate the conservancy of their upregulation using the Coronascape database (DB). Finally, tissue-specific regulation of the five predictive hub genes indicates that OAS1 and MX1 expression levels are lower in healthy kidney tissue. Altogether, our results suggest that these genes could play an essential role in developing severe outcomes of COVID-19 in DKD patients.","version":"1.1","doi":"10.1101/2022.10.11.511826","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.12.511145","pub_date":"2022-10-12","title":"Alterations in platelet proteome signature and impaired platelet integrin \u03b1IIb\u03b23 activation in patients with COVID-19","abstract":"Patients with coronavirus disease-19 (COVID-19) are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. We aimed to characterise the mechanism of altered platelet function in COVID-19 patients. The platelet proteome, platelet functional responses and platelet-neutrophil aggregates were compared between patients hospitalised with COVID-19 and healthy control subjects using Tandem Mass Tag (TMT) proteomic analysis, Western blotting and flow cytometry. COVID-19 patients showed a different profile of platelet protein expression (858 altered out of 5773 quantified). Levels of COVID-19 plasma markers were enhanced in COVID-19 platelets. Gene ontology (GO) pathway analysis demonstrated that levels of granule secretory proteins were raised, whereas some platelet activation proteins, such as the thrombopoietin receptor and PKC\u03b1, were lowered. Basally, COVID-19 platelets showed enhanced phosphatidylserine (PS) exposure, with unaltered integrin \u03b1IIb\u03b23 activation and P-selectin expression. Agonist-stimulated integrin \u03b1IIb\u03b23 activation and PS exposure, but not P-selectin expression, were significantly decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This interaction was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. Overall, our data suggests the presence of two platelet populations in patients with COVID-19: one with circulating platelets with an altered proteome and reduced functional responses and another with P-selectin expressing neutrophil-associated platelets. Platelet driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients. COVID-19 patient platelet function and platelet proteins were compared with healthy controls Proteomic analysis of platelets indicated that COVID-19 decreased platelet activation proteins Agonist induced PS exposure and integrin \u03b1IIb\u03b23 activation were impaired in COVID-19 COVID-19 led to maximal levels of P-selectin dependent platelet-neutrophil aggregates","version":"1.1","doi":"10.1101/2022.10.12.511145","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.11.511775","pub_date":"2022-10-11","title":"The SARS-CoV-2 envelope (E) protein forms a calcium- and voltage-activated calcium channel","abstract":"The function of ion channels is essential in the infectious cycle of many viruses. To facilitate viral uptake, maturation and export, viruses must modify the ionic balance of their host cells, in particular of calcium ions (Ca2+). Viroporins encoded in the viral genome play a key part in altering the cell\u2019s ionic homeostasis. In SARS-Coronavirus-2 (SARS-CoV-2) \u2013 the causative agent of Covid-19 \u2013 the envelope (E) protein is considered to form ion channels in ERGIC organellar membranes, whose function is closely linked to disease progression and lethality. Deletion, blockade, or loss-of-function mutation of coronaviral E proteins results in propagation-deficient or attenuated virus variants. The exact physiological function of the E protein, however, is not sufficiently understood. Since one of the key features of the ER is its function as a Ca2+ storage compartment, we investigated the activity of E in the context of this cation. Molecular dynamics simulations and voltage-clamp electrophysiological measurements show that E exhibits ion channel activity that is regulated by increased luminal Ca2+ concentration, membrane voltage, post-translational protein modification, and negatively charged ERGIC lipids. Particularly, calcium ions bind to a distinct region at the ER-luminal channel entrance, where they activate the channel and maintain the pore in an open state. Also, alongside monovalent ions, the E protein is highly permeable to Ca2+. Our results suggest that the physiological role of the E protein is the release of Ca2+ from the ER, and that the distinct Ca2+ activation site may serve as a promising target for channel blockers, potentially inhibiting the infectious cycle of coronaviruses.","version":"1.1","doi":"10.1101/2022.10.11.511775","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.10.511623","pub_date":"2022-10-11","title":"Genomic tracking of SARS-COV-2 variants in Myanmar","abstract":"In December 2019, the COVID-19 disease started in Wuhan, China. WHO declared a pandemic on March 12, 2020, and the disease started in Myanmar on March 23, 2020. December brought variants around the world, threatening the healthcare systems. To counter those threats, Myanmar started the COVID-19 variant surveillance program in late 2020. Whole genome sequencing was done six times between January 2021 and March 2022. We chose 83 samples with a PCR threshold cycle of less than 25. Then, we used MiSeq FGx for sequencing and Illumina DRAGEN COVIDSeq pipeline, command line interface, GISAID, and MEGA version 7 for data analysis. January 2021 results showed no variant. The second run during the rise of cases in June 2021 showed multiple variants like Alpha, Delta, and Kappa. There is only Delta in the third run at the height of mortality in August, and Delta alone continued until the fourth run in December. After the world reported the Omicron variant in November, Myanmar started a surveillance program. The fifth run in January 2022 showed both Omicron and Delta variants. The sixth run in March 2022 showed only Omicron BA.2. Amino acid mutation at receptor binding domain (RBD) of Spike glycoprotein started since the second run coupling with high transmission, recurrence, and vaccine escape. We also found the mutation at the primer targets used in current RT-PCR platforms. The occurrence of multiple variants and mutations claimed vigilance at ports of entry and preparedness for effective control measures. Genomic surveillance with the observation of evolutionary data is required to predict imminent threats of the current disease and diagnose emerging infectious diseases.","version":"1.1","doi":"10.1101/2022.10.10.511623","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481247","pub_date":"2022-10-11","title":"A novel intranasal administration adenoviral vector-based platform for rapid COVID-19 vaccine development","abstract":"The coronavirus SARS-CoV-2 has a severe impact on global public health, and the emerging variants threaten the efficacy of the circulating vaccines. Here, we report that a single vaccination with a non-replicating chimpanzee adenovirus-based vaccine against the SARS-CoV-2 Delta variant (JS1-delta) elicits potent humoral, cellular and mucosal immunity in mice. Additionally, a single intranasal administration of JS1-delta provides effective protection against the Delta (B.1.617.2) variant challenge in mice. This study indicates that chimpanzee adenovirus type 3 (ChAd3) derived vector represents a promising platform for antiviral vaccine development against respiratory infections and JS1-delta is worth further investigation in human clinical trials. A new chimpanzee adenoviral vaccine against the SARS-CoV-2 Delta variant was developed. The vaccine elicited potent humoral, cellular and mucosal immunity in mice. The vaccine protected mice from the Delta variant challenge.","version":"1.2","doi":"10.1101/2022.02.21.481247","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.08.511408","pub_date":"2022-10-10","title":"Gut microbial disruption in critically ill patients with COVID-19 associated pulmonary aspergillosis","abstract":"COVID-19 disease can be exacerbated by Aspergillus superinfection (CAPA). The causes of CAPA are not yet fully understood. Recently, alterations in the gut microbiome have been associated with a complicating course and increasing severity of COVID-19 disease, most likely via immunological mechanisms. Aim of this study was to investigate a potential association between severe CAPA and alterations in the gut and bronchial microbiota. We performed 16S rRNA gene amplicon sequencing of stool and bronchial samples from a total of 16 COVID-19 patients with CAPA and 26 patients without CAPA. All patients were admitted to the intensive care unit. Results were carefully tested for potential influences on the microbiome during hospitalization. We found that late in COVID-19 disease, CAPA patients exhibited a trend towards reduced gut microbial diversity. Furthermore, late stage CAPA disease showed an increased presence of Staphylococcus epidermidis in the gut. This is not found in late non-CAPA cases or early disease. The analysis of bronchial samples did not show significant results. This is the first study showing alterations in the gut microbiome accompany severe CAPA and possibly influence the host\u2019s immunological response. In particular, an increase of Staphylococcus epidermidis in the intestine could be of importance. The composition of intestinal bacteria in severe CAPA disease is altered with an increase in Staphylococcus epidermidis in the gut. Alterations in the composition of intestinal bacteria in severe CAPA may indicate immunologic involvement of the gut in the disease.","version":"1.1","doi":"10.1101/2022.10.08.511408","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.08.511397","pub_date":"2022-10-08","title":"SiRNA Molecules as Potential RNAi Therapeutics to Silence RdRP Region and N-Gene of SARS-CoV-2: An In Silico Approach","abstract":"COVID-19 pandemic keeps pressing onward and effective treatment option against it is still far-off. Since the onslaught in 2020, 13 different variants of SARS-CoV-2 have been surfaced including 05 different variants of concern. Success in faster pandemic handling in the future largely depends on reinforcing therapeutics along with vaccines. As a part of RNAi therapeutics, here we developed a computational approach for predicting siRNAs, which are presumed to be intrinsically active against two crucial mRNAs of SARS-CoV-2, the RNA-dependent RNA polymerase (RdRp), and the nucleocapsid phosphoprotein gene (N gene). Sequence conservancy among the alpha, beta, gamma, and delta variants of SARS-CoV-2 was integrated in the analyses that warrants the potential of these siRNAs against multiple variants. We preliminary found 13 RdRP-targeting and 7 N gene-targeting siRNAs using the siDirect V.2.0. These siRNAs were subsequently filtered through different parameters at optimum condition including macromolecular docking studies. As a result, we selected 4 siRNAs against the RdRP and 3 siRNAs against the N-gene as RNAi candidates. Development of these potential siRNA therapeutics can significantly synergize COVID-19 mitigation by lessening the efforts, furthermore, can lay a rudimentary base for the in silico design of RNAi therapeutics for future emergencies.","version":"1.1","doi":"10.1101/2022.10.08.511397","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.511351","pub_date":"2022-10-08","title":"Inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution","abstract":"The genome of SARS-CoV-2 encodes for a helicase called nsp13 that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds short DNA duplexes. Our data confirm that nsp13 uses the inchworm mechanism to move along the DNA in single-nucleotide steps, translocating at ~1000 nt/s or unwinding at ~100 bp/s. Nanopore tweezers\u2019 high spatio-temporal resolution enables observation of the fundamental physical steps taken by nsp13 even as it translocates at speeds in excess of 1000 nucleotides per second enabling detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13\u2019s motion in the presence of the ATPase inhibitor ATP\u03b3S. Our data reveals that ATP\u03b3S interferes with nsp13\u2019s action by affecting several different kinetic processes. The dominant mechanism of inhibition differs depending on the application of assisting force. These advances demonstrate that nanopore tweezers are a powerful method for studying viral helicase mechanism and inhibition.","version":"1.1","doi":"10.1101/2022.10.07.511351","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.511252","pub_date":"2022-10-07","title":"Baculoviral COVID-19 Delta DNA vaccine cross-protects against SARS-CoV2 variants in K18-ACE2 transgenic mice","abstract":"After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronavirus\u2019s intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50% survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100% survival upon challenge with Delta and Omicron variants and 71.4% survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants. After the SARS-CoV2 pandemic, it is known that the existing vaccine has diminished efficacy against the emerging variants. We developed a baculoviral COVID19 DNA vaccine for the Delta variant (AcHERV-COVIS19D). Compared to AcHERV-COVID19S, designed to protect from the prototype of SARS-CoV2, AcHERV-COVID19D elicited higher humoral and cellular immunity and showed perfect protection against SARS-CoV2 delta strain and Omicron challenge. The broad and robust cellular immunity of the AcHERV-COVID19D vaccine appears to have played a significant role in the cross-protection of the Omicron variant. Our AcHERV-COVID19D can be a potential vaccine against emerging SARS-CoV2 variants.","version":"1.1","doi":"10.1101/2022.10.07.511252","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.511336","pub_date":"2022-10-07","title":"Fate and plasticity of SARS-CoV-2-specific B cells during memory and recall response in humans","abstract":"B cell responses to different pathogens recruit tailored effector mechanisms, resulting in functionally specialized subsets. For human memory B cells (MBCs), these include CD21+ resting, CD21\u2212CD27+ activated, and CD21\u2212CD27\u2212 atypical cells. Whether these subsets follow deterministic or interconnected fates is unknown. We demonstrate in COVID-19 patients that single clones of SARS-CoV-2-specific MBCs followed multiple fates with distinctive phenotypic and functional characteristics. 6\u201312 months after infection, most circulating MBCs were CD21+ resting cells, which also accumulated in peripheral lymphoid organs where they acquired markers of tissue residency. Conversely, at acute infection and following SARS-CoV-2-specific immunization, CD21\u2212 MBCs became the predominant subsets, with atypical MBCs expressing high T-bet, inhibitory molecules, and distinct chemokine receptors. B cell receptor sequencing allowed tracking of individual MBC clones differentiating into CD21+, CD21\u2212CD27+, and CD21\u2212CD27\u2212 cell fates. Collectively, single MBC clones can adopt functionally different trajectories, thus contributing to immunity to infection.","version":"1.1","doi":"10.1101/2022.10.07.511336","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.511319","pub_date":"2022-10-07","title":"Development of SARS-CoV-2 mRNA vaccines encoding spike N-terminal and receptor binding domains","abstract":"With the success of mRNA vaccines against coronavirus disease 2019 (COVID-19), strategies can now focus on improving vaccine potency, breadth, and stability. We present the design and preclinical evaluation of domain-based mRNA vaccines encoding the wild-type spike-protein receptor-binding (RBD) and/or N-terminal domains (NTD). An NTD-RBD linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2-8\u00b0C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses and protection from viral challenge were observed against wild-type, beta, delta, or omicron (BA. 1) compared with mRNA-1273 immunized mice, especially at lower vaccine dosages. These results support clinical assessment of mRNA-1283 (NCT05137236). A domain-based mRNA vaccine, mRNA-1283, is immunogenic and protective against SARS-CoV-2 and emerging variants in mice.","version":"1.1","doi":"10.1101/2022.10.07.511319","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.511324","pub_date":"2022-10-07","title":"Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets","abstract":"The infection and replication cycle of all viruses depend on interactions between viral and host proteins. Each of these protein-protein interactions is therefore a potential drug target. These host-virus interactions often involve a disordered protein region on one side of the interface and a folded protein domain on the other. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind peptides from human proteins. Of 281 high/medium confidence peptides, 23 interactions involving eight SARS-CoV-2 protein domains were tested by fluorescence polarization, and binding was observed with affinities spanning the whole micromolar range. The key specificity determinants were established for six of these domains, two based on ProP-PD and four by alanine scanning SPOT arrays. Finally, two cell-penetrating peptides, targeting Nsp9 and Nsp16, respectively, were shown to function as inhibitors of viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously provide information on potential host-virus interactions and identify ligands with antiviral properties.","version":"1.1","doi":"10.1101/2022.10.07.511324","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.511263","pub_date":"2022-10-07","title":"Neutralization of SARS-CoV-2 Omicron BA.4/BA.5 subvariant by a booster dose of bivalent adjuvanted subunit vaccine containing Omicron BA.4/BA.5 and BA.1 subvariants","abstract":"The dominance of SARS-CoV-2 variants of concern (VoC), such as the Omicron subvariants, is a threat to the current vaccination scheme due to increased resistance to immune neutralization and greater transmissibility. To develop the next generation of prefusion SARS-CoV-2 spike protein (S-2P) subunit vaccine adjuvanted with CpG1018 and aluminum hydroxide, mice immunized with two doses of the adjuvanted ancestral Wuhan strain (W) followed by the third dose of the W or Omicron variants (BA.1 or BA.4/BA.5) S-2P, or a combination of the above bivalent S-2Ps. Antisera from mice were tested against pseudovirus neutralization assay of ancestral SARS-CoV-2 (WT) and Omicron BA.4/BA.5 subvariant. Boosting with bivalent mixture of Omicron BA.4/BA.5 and W S-2P achieved the highest neutralizing antibody titers against BA.4/BA.5 subvariant pseudovirus compared to other types of S-2P as boosters.","version":"1.1","doi":"10.1101/2022.10.07.511263","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510837","pub_date":"2022-10-07","title":"Community engagement through \u201cstudent-led science\u201d for dengue prevention during the COVID-19 pandemic in C\u00f3rdoba, Argentina","abstract":"During 2019-2020 while COVID-19 pandemic, the Americas were facing the biggest dengue fever epidemic in recent history. Traditional vector control programs, based on insecticide application have been insufficient to control the spread of dengue fever. Several studies suggest refocusing on education with the aim of an integrated vector management strategy within the local ecological-community context. We aim to assess community perceptions, knowledge, attitude, preventive practice, and action through student-led science assignments regarding dengue fever, prevention, and socio-ecological factors in temperate C\u00f3rdoba, Argentina. The study was conducted during the COVID-19 quarantine when schools switched to online education for the first time. Several activities through Google Classroom platform included a survey to one student\u2019s family member, and an outdoor activity to assess their attitudes and to clean the backyard and gardens. Significant number of respondents developed good preventive practices and increased their knowledge about the vector and disease highlighting that 75% of responders knew that dengue fever was transmitted by a mosquito, 81.96% declared having obtained knowledge regarding dengue and vector through television, 56% affirm that dengue is a severe illness, 67% of respondents admitted that individuals play an important role in the prevention of dengue. Regarding mosquito control activities, 90% of respondents reported turning containers. This highlights the need for school programs with curricula to address vector biology and the prevention of vector-borne diseases not only during activity periods when mosquitoes batter people but all year long to do real prevention.","version":"1.1","doi":"10.1101/2022.10.04.510837","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.07.510750","pub_date":"2022-10-07","title":"Upregulation of CD55 complement regulator in distinct PBMC subpopulations of COVID-19 patients is associated with suppression of interferon responses","abstract":"Complement activation has been verified in COVID-19 patients by both increased serum levels of complement factors C3a and C5b-9 and increased complement deposition at the tissue levels. Complement regulatory proteins (CRPs) CD55, CD46, CD59 and CR1 act to control complement overactivation and eliminate complement deposition and cell lysis. The aim of the study was to investigate the expression of CRPs in COVID-19 in order to identify potential dysregulated expression patterns of CRPs and address whether these may contribute to disease pathogenesis. Single cell RNA-sequencing (scRNA-seq) analysis performed on isolated PBMCs revealed an increase of CD55 expression in severe and critical COVID-19 patients compared to healthy controls. This increase was also detected upon integrated subclustering analysis of the monocyte, T cell and B cell populations. Flow cytometric analysis verified the distinct pattern of upregulated CD55 expression in monocyte and T cell sub populations of severe COVID-19 patients. This upregulation was associated with decreased expression of interferon stimulated genes (ISGs) in patients with severe COVID-19 suggesting a potential suppressor effect of CD55 on interferon responses. The present study identifies a COVID-19 specific CD55 expression pattern in PBMC subpopulations that coincides with reduced interferon responses thus indicating that the complement regulator CD55 may contribute to COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2022.10.07.510750","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510830","pub_date":"2022-10-06","title":"SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein","abstract":"The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication, and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin effect is not associated with acute cellular toxicity but is prevented by mutations within the channel pore of E. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.10.04.510830","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.06.511069","pub_date":"2022-10-06","title":"Is the Stalk of the SARS-CoV-2 Spike Protein Druggable?","abstract":"The SARS-CoV-2 virus spike protein (SP) is the vector of the virus infectivity. The high propensity to mutate in key regions responsible for the recognition of the human angiotensinconverting enzyme 2 (hACE2) or the antibodies produced by the immune system following infection or vaccination makes subunit 1 of the SP a difficult to target and, to date, efforts have not delivered any ACE2 binding inhibitor yet. The inherent flexibility of the stalk region, within subunit S2, is key to SARS-CoV-2 high infectivity because it facilitates the receptor binding domain encounter with ACE2. Thus, it could be a valuable therapeutic target. By employing a fragment-based strategy, we computationally studied the druggability of the conserved part of the SP stalk by means of an integrated approach that combines molecular docking with high-throughput molecular dynamics simulations. Our results suggest that the druggability of the stalk is challenging and provide the structural basis for such difficulty.","version":"1.1","doi":"10.1101/2022.10.06.511069","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.05.511057","pub_date":"2022-10-06","title":"Long-term passaging of replication competent pseudo-typed SARS-CoV-2 reveals the antiviral breadth of monoclonal and bispecific antibody cocktails","abstract":"The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12+10D4+2G1 and 7B9-9D11+2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12+10D4+2G1 and 7B9-9D11+2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.10.05.511057","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.06.511104","pub_date":"2022-10-06","title":"Design and Immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein","abstract":"The potential of immune evasive mutations accumulation of the SARS-CoV-2 virus has led to its rapid spread causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. Huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants renews interest in DNA vaccine technology. Here we report a rapid generation and immunological evaluation of novel DNA vaccine candidates against Wuhan-Hu-1 and Omicron variants, based on the RBD protein fused with the Potato virus X coat protein (PVXCP). Delivery of DNA vaccines using electroporation in a two-doses regimen induced high antibody titers and profound cellular response in mice. Antibody titers induced against Omicron variant of the vaccine were sufficient for the effective protection against both the Omicron and Wuhan-Hu-1 virus infections. PVXCP protein in the vaccine construct shifted immune response to the favorable Th1-like type and provided oligomerization of RBD-PVXCP protein. A naked DNA delivery by the needle-free injection device allowed us to achieve antibody titers comparable with the mRNA-LNP delivery in rabbits. This data identifies the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study.","version":"1.1","doi":"10.1101/2022.10.06.511104","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.09.491254","pub_date":"2022-10-06","title":"Three-dose vaccination-induced immune responses protect against SARS-CoV-2 Omicron BA.2","abstract":"The ongoing outbreak of SARS-CoV-2 Omicron BA.2 infections in Hong Kong, the model city of universal masking of the world, has resulted in a major public health crisis. Although the third vaccination resulted in strong boosting of neutralization antibody, vaccine efficacy and corelates of immune protection against the major circulating Omicron BA.2 remains to be investigated. We investigated the vaccine efficacy against the Omicron BA.2 breakthrough infection among 470 public servants who had received different SARS-CoV-2 vaccine regimens including two-dose BNT162b2 (2\u00d7BNT, n=169), three-dose BNT162b2 (3\u00d7BNT, n=170), two-dose CoronaVac (2\u00d7CorV, n=34), three-dose CoronaVac (3\u00d7CorV, n=67) and third-dose BNT162b2 following 2\u00d7CorV (2\u00d7CorV+1BNT, n=32). Humoral and cellular immune responses after three-dose vaccination were further characterized and correlated with clinical characteristics of BA.2 infection. During the BA.2 outbreak, 27.7% vaccinees were infected. The timely third-dose vaccination provided significant protection with lower incidence rates of breakthrough infections (2\u00d7BNT 49.2% vs 3\u00d7BNT 13.1%, p <0.0001; 2\u00d7CorV 44.1% vs 3\u00d7CoV 19.4%, p=0.003). Investigation of immune response on blood samples derived from 92 subjects in three-dose vaccination cohorts collected before the BA.2 outbreak revealed that the third-dose vaccination activated spike (S)-specific memory B cells and Omicron cross-reactive T cell responses, which correlated with reduced frequencies of breakthrough infections and disease severity rather than with types of vaccines. Moreover, the frequency of S-specific activated memory B cells was significantly lower in infected vaccinees than uninfected vaccinees before vaccine-breakthrough infection whereas IFN-\u03b3+ CD4 T cells were negatively associated with age and viral clearance time. Critically, BA.2 breakthrough infection boosted cross-reactive memory B cells with enhanced cross-neutralizing antibodies to Omicron sublineages, including BA.2.12.1 and BA.4/5, in all vaccinees tested. Our results imply that the timely third vaccination and immune responses are likely required for vaccine-mediated protection against Omicron BA.2 pandemic. Although BA.2 conferred the highest neutralization resistance compared with variants of concern tested before the emergence of BA.2.12.1 and BA.4/5, the third dose vaccination-activated S-specific memory B cells and Omicron cross-reactive T cell responses contributed to reduced frequencies of breakthrough infection and disease severity. Neutralizing antibody potency enhanced by BA. 2 breakthrough infection with previous 3 doses of vaccines (CoronaVac or BNT162b2) may reduce the risk for infection of ongoing BA.2.12.1 and BA.4/5. Hong Kong Research Grants Council Collaborative Research Fund, Health and Medical Research Fund, Wellcome Trust, Shenzhen Science and Technology Program, the Health@InnoHK, Innovation and Technology Commission of Hong Kong, China, National Program on Key Research Project, Emergency Key Program of Guangzhou Laboratory, donations from the Friends of Hope Education Fund and the Hong Kong Theme-Based Research Scheme.","version":"1.2","doi":"10.1101/2022.05.09.491254","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510846","pub_date":"2022-10-06","title":"Discovery of novel papillomaviruses in the critically-endangered Malayan and Chinese pangolins","abstract":"Pangolins are scaly and toothless mammals which are distributed across Africa and Asia. Currently, the Malayan, Chinese and Philippine pangolins are all designated as critically-endangered species. Although few pangolin viruses have been described, their viromes have received more attention following the discovery that they harbour sarbecoviruses related to SARS-CoV-2. Using a large-scale genome mining strategy, we discovered novel lineages of papillomaviruses infecting the Malayan and Chinese pangolins. We were able to assemble 3 complete circular papillomavirus genomes with an intact coding capacity, and 5 additional L1 genes encoding the major capsid protein. Phylogenetic analysis revealed that 7 out of 8 L1 sequences formed a monophyletic group which is the sister lineage to the Tree shrew papillomavirus 1, isolated from Yunnan province in China. Additionally, a single L1 sequence assembled from a Chinese pangolin was placed in a clade closer to alpha- and omegapapillomaviruses. Examination of the SRA data from 95 re-sequenced genomes revealed that 49.3% Malayan pangolins and 50% Chinese pangolins, were positive for papillomavirus reads. Our results indicate that pangolins in South East Asia are the hosts to diverse and highly prevalent papillomaviruses, which may have implications for pangolin health and conservation.","version":"1.2","doi":"10.1101/2022.10.04.510846","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.30.502143","pub_date":"2022-10-05","title":"Evolution to increased positive charge on the viral spike protein may be part of the adaptation of SARS-CoV-2 to human transmission","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, continues to evolve and infect individuals. The exterior surface of the SARS-CoV-2 virion is dominated by the spike protein and the current work examined spike protein biochemical features that have changed during the 2 years that SARS-CoV-2 has infected humans. These biochemical properties may influence virion survival and promote movement through the environment and within the human airway to reach target cells to bind, enter and establish the next round of infection. In addition to selective pressure to avoid immune recognition of viral proteins, we hypothesised that SARS-CoV-2 emerged from an animal reservoir capable of human infection and transmission but in a sub-optimum state and a second level of selective pressure is acting on these biochemical features. Our analysis identified a striking change in spike protein charge, from \u22128.3 in the original Lineage A and B viruses to \u22121.26 in the current Omicron viruses. In summary, we conclude that in addition to immune selection pressure, the evolution of SARS-CoV-2 has also altered viral spike protein biochemical properties. Future vaccine and therapeutic development should also exploit and target these biochemical properties.","version":"1.2","doi":"10.1101/2022.07.30.502143","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.507920","pub_date":"2022-10-05","title":"Nanobody engineering for SARS-CoV-2 neutralization and detection","abstract":"In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, \u2018nanobodies\u2019. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for viral evolution in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel detection assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.","version":"1.2","doi":"10.1101/2022.09.14.507920","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510919","pub_date":"2022-10-05","title":"Real-time inactivation of airborne SARS-CoV-2 using ultraviolet-C","abstract":"COVID-19 is a life-threatening respiratory infection that has had a profound impact on indoor air quality awareness. Ultraviolet-C (UV-C) is a physical disinfection process that triggers microbial inactivation through creating irreversible genetic material damage. An upper room device equipped with germicidal UV-C (UR GUV) was evaluated against airborne SARS-CoV-2 for antimicrobial efficacy using a robust aerosol testing protocol. In 30 minutes, it led to a virucidal efficacy of 99.994 % in a large, room-sized chamber. UR GUV is a promising mitigation strategy for airborne pathogens.","version":"1.1","doi":"10.1101/2022.10.04.510919","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.05.490805","pub_date":"2022-10-05","title":"Novel ACE2 nanoparticles universally block SARS-CoV-2 variants in the human respiratory tract","abstract":"The continual evolution of SARS-CoV-2 has challenged the efficacy of many COVID19 vaccines and treatment options. One strategy that evades viral escape is using the entry receptor, human Angiotensin-Converting Enzyme 2 (hACE2). Soluble hACE2 receptor domains show potential as decoys but genetic modifications are necessary to provide sufficient efficacy. However, these engineered constructs are potentially susceptible to viral escape. We combined native hACE2 with viral vectors to form nanoparticles presenting hACE2 analogous to human cells. Cell-based viral infection assays and cryogenic in-situ tomography show that hACE2 nanoparticles sequester viruses through aggregation, efficiently blocking entry of SARS-CoV-2 and its variants in model cell systems and human respiratory tract explants using native hACE2. Thus, we show that hACE2 nanoparticles have high potential as pan-variant COVID19 therapeutics.","version":"1.2","doi":"10.1101/2022.05.05.490805","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.05.510928","pub_date":"2022-10-05","title":"A novel antiviral formulation inhibits SARS-CoV-2 infection of human bronchial epithelium","abstract":"A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of in vitro cellular toxicity was detected in ViruSAL treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.","version":"1.1","doi":"10.1101/2022.10.05.510928","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510657","pub_date":"2022-10-04","title":"Relevance of the viral Spike protein/cellular Estrogen Receptor-\u03b1 interaction for endothelial-based coagulopathy induced by SARS-CoV-2","abstract":"Severe coagulopathy has been observed at the level of the microcirculation in several organs including lungs, heart and kidneys in patients with COVID-19, and in a minority of subjects receiving the SARS-CoV-2 vaccine. Various mechanisms have been implicated in these effects, including increases in circulating neutrophil extracellular traps, excessive inflammation, and endothelial dysfunction. Even if a correlation between infection by SARS-CoV-2 and upregulation of coagulation cascade components has been established in the lung, no direct proofs have been yet provided about the transcriptional machinery controlling the expression of these factors. Recent results obtained by us reported a novel transcriptional function of the SARS-CoV-2 Spike (S) viral protein involving a direct protein-protein interaction with the human Estrogen Receptor-\u03b1 (ER\u03b1). Given the implications of ER\u03b1 in the control of key effectors in the coagulation cascade, we hypothesized that S-protein might increase the pro-coagulation activity of endothelial cells via the transcriptional activity of the ER\u03b1, thus justifying the enhanced risk of thrombosis. To assess this, we tested the effects of S-protein on the expression of Tissue Factor (TF) and the overall procoagulation activity in a human endothelial cell line and confirmed this finding by overexpressing S-protein by gene transfer in mice. We then designed and tested two-point mutations in the S2 S-protein sequence that abolished the pro-coagulation function of S-protein in vitro and in vivo, without compromising its immunogenicity. In addition to reveal a new potential transcriptional function of S-protein, these results inspire the design of new vaccines with lower risk of thrombogenesis. Indeed, while the benefit/risk ratio remains overwhelming in favor of COVID-19 vaccination, our results shed light on the causal mechanisms of some rare anti-SARS-CoV-2 vaccine adverse events, and are thus essential for current and future vaccination and booster campaigns.","version":"1.1","doi":"10.1101/2022.10.04.510657","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.04.510754","pub_date":"2022-10-04","title":"SARS-CoV-2 ORF3c impairs mitochondrial respiratory metabolism, oxidative stress and autophagic flow","abstract":"Coronaviruses encode a variable number of accessory proteins that play a role in host-virus interactions, in the suppression of immune responses, or in immune evasion. Accessory proteins in SARS-CoV-2 consist of at least twelve viral proteins whose roles during infection have been extensively studied. Nevertheless, the role of the ORF3c accessory protein, an alternative open reading frame of ORF3a, has remained elusive. Herein, we characterized ORF3c in terms of cellular localization, host\u2019s antiviral response modulation, and effects on mitochondrial metabolism. We show that ORF3c has a mitochondrial localization and alters mitochondrial metabolism, resulting in increased ROS production, block of the autophagic flux, and accumulation of autophagosomes/autolysosomes. Notably, we also found that ORF3c induces a shift from glucose to fatty acids oxidation and enhanced oxidative phosphorylation. This is similar to the condition observed in the chronic degenerative phase of COVID-19. Altogether these data suggest that ORF3c could be a key protein for SARS-CoV-2 pathogenesis and that it may play a role in disease progression.","version":"1.1","doi":"10.1101/2022.10.04.510754","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.15.508093","pub_date":"2022-10-04","title":"O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern","abstract":"The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern.","version":"1.3","doi":"10.1101/2022.09.15.508093","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.15.507787","pub_date":"2022-10-04","title":"Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution","abstract":"Continuous evolution of Omicron has led to numerous subvariants that exhibit growth advantage over BA.5. Such rapid and simultaneous emergence of variants with enormous advantages is unprecedented. Despite their rapidly divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots, including R346, K356, K444, L452, N460K and F486. The driving force and destination of such convergent evolution and its impact on humoral immunity established by vaccination and infection remain unclear. Here, we demonstrate that these convergent mutations can cause striking evasion of convalescent plasma, including those from BA.5 breakthrough infection, and existing antibody drugs, including Evusheld and Bebtelovimab. BR.2, CA.1, BQ.1.1, BM.1.1.1, and especially XBB, are the most antibody-evasive strain tested, far exceeding BA.5 and approaching SARS-CoV-1 level. To delineate the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies (mAbs) isolated from BA.2 and BA.5 breakthrough-infection convalescents. Importantly, due to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection caused significant reductions in the epitope diversity of neutralizing antibodies and increased proportion of non-neutralizing mAbs, which in turn concentrated humoral immune pressure and promoted the convergent RBD evolution. Additionally, the precise convergent RBD mutations and evolution trends of BA.2.75/BA.5 subvariants could be inferred by integrating the neutralization-weighted DMS profiles of mAbs from various immune histories (3051 mAbs in total). Moreover, we demonstrated that as few as five additional convergent mutations based on BA.5 or BA.2.75 could completely evade most plasma samples, including those from BA.5 breakthrough infection, while retaining sufficient hACE2-binding affinity. These results suggest that current herd immunity and BA.5 vaccine boosters may not provide sufficiently broad protection against infection. Broad-spectrum SARS-CoV-2 vaccines and NAb drugs development should be of high priority, and the constructed convergent mutants could serve to examine their effectiveness in advance.","version":"1.3","doi":"10.1101/2022.09.15.507787","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.30.510331","pub_date":"2022-10-03","title":"CiDRE+ M2c macrophages hijacked by SARS-CoV-2 cause COVID-19 severity","abstract":"Infection of the lungs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin I converting enzyme 2 (ACE2) receptor induces a type of systemic inflammation known as a cytokine storm. However, the precise mechanisms involved in severe coronavirus disease 2019 (COVID-19) pneumonia are unknown. Here, we show that interleukin-10 (IL-10) changed normal alveolar macrophages into ACE2-expressing M2c-type macrophages that functioned as spreading vectors for SARS-CoV-2 infection. The depletion of alveolar macrophages and blockade of IL-10 attenuated SARS-CoV-2 pathogenicity. Furthermore, genome-wide association and quantitative trait locus analyses identified novel mRNA transcripts in human patients, COVID-19 infectivity enhancing dual receptor (CiDRE), which has unique synergistic effects within the IL-10-ACE2 system in M2c-type macrophages. Our results demonstrate that alveolar macrophages stimulated by IL-10 are key players in severe COVID-19. Collectively, CiDRE expression levels are potential risk factors that predict COVID-19 severity, and CiDRE inhibitors might be useful as COVID-19 therapies.","version":"1.1","doi":"10.1101/2022.09.30.510331","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.03.510566","pub_date":"2022-10-03","title":"Intranasal delivery of NS1-deleted influenza virus vectored COVID-19 vaccine restrains the SARS-CoV-2 inflammatory response","abstract":"The emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) variants and \u201canatomical escape\u201d characteristics threaten the effectiveness of current coronavirus disease (COVID-19) vaccines. There is an urgent need to understand the immunological mechanism of broad-spectrum respiratory tract protection to guide broader vaccines development. In this study, we investigated immune responses induced by an NS1-deleted influenza virus vectored intranasal COVID-19 vaccine (dNS1-RBD) which provides broad-spectrum protection against SARS-CoV-2 variants. Intranasal delivery of dNS1-RBD induced innate immunity, trained immunity and tissue-resident memory T cells covering the upper and lower respiratory tract. It restrained the inflammatory response by suppressing early phase viral load post SARS-CoV-2 challenge and attenuating pro-inflammatory cytokine (IL-6, IL-1B, and IFN-\u03b3) levels, thereby reducing excess immune-induced tissue injury compared with the control group. By inducing local cellular immunity and trained immunity, intranasal delivery of NS1-deleted influenza virus vectored vaccine represents a broad-spectrum COVID-19 vaccine strategy to reduce disease burden.","version":"1.1","doi":"10.1101/2022.10.03.510566","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.01.510442","pub_date":"2022-10-03","title":"SARS-CoV-2 spike protein induces endothelial dysfunction in 3D engineered vascular networks","abstract":"With new daily discoveries about the long-term impacts of COVID-19 there is a clear need to develop in vitro models that can be used to better understand the pathogenicity and impact of COVID-19. Here we demonstrate the utility of developing a model of endothelial dysfunction that utilizes induced pluripotent stem cell-derived endothelial progenitors encapsulated in collagen hydrogels to study the effects of COVID-19 on the endothelium. We found that treating these cell-laden hydrogels with SARS-CoV-2 spike protein resulted in a significant decrease in the number of vessel-forming cells as well as vessel network connectivity. Following treatment with the anti-inflammatory drug dexamethasone, we were able to prevent SARS-CoV-2 spike protein-induced endothelial dysfunction. In addition, we confirmed release of inflammatory cytokines associated with the COVID-19 cytokine storm. In conclusion, we have demonstrated that even in the absence of immune cells, we are able to use this 3D in vitro model for angiogenesis to reproduce COVID-19 induced endothelial dysfunction seen in clinical settings.","version":"1.1","doi":"10.1101/2022.10.01.510442","journal":"bioRxiv","score":null},{"id":"10.1101/2022.10.03.507132","pub_date":"2022-10-03","title":"Biophysical characterisation of the structure of a SARS-CoV-2 self-amplifying - RNA (saRNA) vaccine","abstract":"The current SARS-Covid-2 pandemic has led to an acceleration of messenger \u2013 ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA) has required concomitant development of analytical characterisation techniques. Characterising the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This paper describes the biophysical characterisation of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterise the IMP-1 RNA molecule. In this paper we use UV spectroscopy, dynamic light scattering (DLS), size-exclusion chromatography small angle scattering (SEC-SAXS) and circular dichroism (CD) to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule. This paper is highly significant as it provides a prescient biophysical characterisation of an efficacious Sars-Cov-2 vaccine self-amplifying (sa)RNA molecule. RNA vaccines have been a major scientific breakthrough of the Covid-19 pandemic. saRNA is a further development of conventional mRNA vaccines, amplifying the RNA of interest in the cell, allowing the vaccine to be administered at lower dosages. These new biologics are distinct from previous biologics and have required distinct analytical characterisation. The analytics described herein provide detailed information on the size, shape, and structure of the RNA molecule. This paper is therefore an important step in characterising large saRNA biological relevant molecules.","version":"1.1","doi":"10.1101/2022.10.03.507132","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.30.510319","pub_date":"2022-10-03","title":"Minor intron containing genes: Achilles\u2019 heel of viruses?","abstract":"The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the world\u2019s unpreparedness to deal with the emergence of novel pathogenic viruses, pointing to the urgent need to identify targets for broad-spectrum antiviral strategies. Here, we report that proteins encoded by Minor Intron-containing Genes (MIGs) are significantly enriched in datasets of cellular proteins that are leveraged by SARS-CoV-2 and other viruses. Pointing to a general gateway for viruses to tap cellular machinery, MIG-encoded proteins (MIG-Ps) that react to the disruption of the minor spliceosome are most important points of viral attack, suggesting that MIG-Ps may pan-viral drug targets. While contemporary anti-viral drugs shun MIG-Ps, we surprisingly found that anti-cancer drugs that have been repurposed to combat SARS-CoV-2, indeed target MIG-Ps, suggesting that such genes can potentially be tapped to efficiently fight viruses.","version":"1.1","doi":"10.1101/2022.09.30.510319","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.472151","pub_date":"2022-10-01","title":"Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline","abstract":"SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest. Using this method, we found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting bidirectional nuclear transport, due to differences in the NUP98-binding C-terminal region that is required for the inhibition. The N-terminal region promotes membrane binding and was required for activity, but could be replaced by constructs which forced oligomerization in solution. Based on these data, we propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the FG domains of NUP98 at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.","version":"1.2","doi":"10.1101/2021.12.10.472151","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.30.509852","pub_date":"2022-9-30","title":"Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape","abstract":"Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.09.30.509852","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.29.510149","pub_date":"2022-9-30","title":"The SARS-CoV-2 mutation landscape is shaped before replication starts","abstract":"Mutation landscapes and signatures have been thoroughly studied in SARS-CoV-2. Here, we analyse those patterns and link their changes to the viral replication niche. Surprisingly, those patterns look to be also modified after vaccination. Hence, by deductive reasoning we identify the steps of the coronavirus infection cycle in which those mutations initiate.","version":"1.1","doi":"10.1101/2022.09.29.510149","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.30.510287","pub_date":"2022-9-30","title":"Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor","abstract":"Spike protein of SARS-CoV-2 variants play critical role in the infection and transmission through its interaction with hACE2 receptor. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with hACE2 receptor. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike proteins of Wild (Wuhan Hu-1), Delta, C.1.2 and Omicron variants. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptor expressing HEK-293T cell line and evaluated binding efficiencies of the variants and competitive binding of spike variants with D614G spike pseudotyped virus. In lines with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. From the analysis in receptor binding domain (RBD) revealed that a single common modification, N501Y, present in both Omicron and C.1.2 is driving the enhanced spike binding to the receptor and showed two-fold superior competitive binding than Delta. Our study using cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors and signifies the role of single common modification N501Y in RBD towards imparting superior binding efficiencies. Our approach would be instrumental in understanding the disease progression and developing therapeutics. Spike proteins of evolving SARS-CoV2 variants demonstrated their signature binding to hACE2 receptor, in turn contributed to driving the infection and transmission. Prior studies to scale the binding efficiencies between the spike variant and the receptor had consensus in distinct variants, but discrepancies in the closely related ones. To this end, we compared spike variants-receptor interactions with ELISA, from cells expressing hACE2 receptor. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. More importantly, competitive binding studies in presence of pseudovirus, demonstrated that a single common modification, N501Y, present in both Omicron and C.1.2 showed two fold superior competitive binding than Delta. Collectively, our study suggests a precise approach to evaluate the binding interactions between spike sub-lineages to hACE2 receptor. This would be instrumental in understanding the disease progression and developing therapeutics.","version":"1.1","doi":"10.1101/2022.09.30.510287","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509819","pub_date":"2022-9-30","title":"COVIDpro: Database for mining protein dysregulation in patients with COVID-19","abstract":"The ongoing pandemic of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has limited treatment options partially due to our incomplete understanding of the molecular dysregulations of the COVID-19 patients. We aimed to generate a repository and data analysis tools to examine the modulated proteins underlying COVID-19 patients for the discovery of potential therapeutic targets and diagnostic biomarkers. We built a web server containing proteomic expression data from COVID-19 patients with a toolset for user-friendly data analysis and visualization. The web resource covers expert-curated proteomic data from COVID-19 patients published before May 2022. The data were collected from ProteomeXchange and from select publications via PubMed searches and aggregated into a comprehensive dataset. Protein expression by disease subgroups across projects was compared by examining differentially expressed proteins. We also visualize differentially expressed pathways and proteins. Moreover, circulating proteins that differentiated severe cases were nominated as predictive biomarkers. We built and maintain a web server COVIDpro (https://www.guomics.com/covidPro/) containing proteomics data generated by 41 original studies from 32 hospitals worldwide, with data from 3077 patients covering 19 types of clinical specimens, the majority from plasma and sera. 53 protein expression matrices were collected, for a total of 5434 samples and 14,403 unique proteins. Our analyses showed that the lipopolysaccharide-binding protein, as identified in the majority of the studies, was highly expressed in the blood samples of patients with severe disease. A panel of significantly dysregulated proteins was identified to separate patients with severe disease from non-severe disease. Classification of severe disease based on these proteomic signatures on five test sets reached a mean AUC of 0.87 and ACC of 0.80. COVIDpro is an online database with an integrated analysis toolkit. It is a unique and valuable resource for testing hypotheses and identifying proteins or pathways that could be targeted by new treatments of COVID-19 patients. National Key R&D Program of China: Key PDPM technologies (2021YFA1301602, 2021YFA1301601, 2021YFA1301603), Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars (LR19C050001), Hangzhou Agriculture and Society Advancement Program (20190101A04), National Natural Science Foundation of China (81972492) and National Science Fund for Young Scholars (21904107), National Resource for Network Biology (NRNB) from the National Institute of General Medical Sciences (NIGMS-P41 GM103504) Although an increasing number of therapies against COVID-19 are being developed, they are still insufficient, especially with the rise of new variants of concern. This is partially due to our incomplete understanding of the disease\u2019s mechanisms. As data have been collected worldwide, several questions are now worth addressing via meta-analyses. Most COVID-19 drugs function by targeting or affecting proteins. Effectiveness and resistance to therapeutics can be effectively assessed via protein measurements. Empowered by mass spectrometry-based proteomics, protein expression has been characterized in a variety of patient specimens, including body fluids (e.g., serum, plasma, urea) and tissue (i.e., formalin-fixed and paraffin-embedded (FFPE)). We expert-curated proteomic expression data from COVID-19 patients published before May 2022, from the largest proteomic data repository ProteomeXhange as well as from literature search engines. Using this resource, a COVID-19 proteome meta-analysis could provide useful insights into the mechanisms of the disease and identify new potential drug targets. We integrated many published datasets from patients with COVID-19 from 11 nations, with over 3000 patients and more than 5434 proteome measurements. We collected these datasets in an online database, and generated a toolbox to easily explore, analyze, and visualize the data. Next, we used the database and its associated toolbox to identify new proteins of diagnostic and therapeutic value for COVID-19 treatment. In particular, we identified a set of significantly dysregulated proteins for distinguishing severe from non-severe patients using serum samples. COVIDpro will support the navigation and analysis of patterns of dysregulated proteins in various COVID-19 clinical specimens for identification and verification of protein biomarkers and potential therapeutic targets.","version":"1.2","doi":"10.1101/2022.09.27.509819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.28.509985","pub_date":"2022-9-30","title":"Treenome Browser: co-visualization of enormous phylogenies and millions of genomes","abstract":"Treenome Browser is a web browser tool to interactively visualize millions of genomes alongside huge phylogenetic trees. Treenome Browser for SARS-CoV-2 can be accessed at cov2tree.org, or at taxonium.org for user-provided trees. Source code and documentation are available at github.com/theosanderson/taxonium and docs.taxonium.org/en/latest/treenome.html. alex.kramer@ucsc.edu, rucorbet@ucsc.edu","version":"1.1","doi":"10.1101/2022.09.28.509985","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.30.510283","pub_date":"2022-9-30","title":"Novel and simple simulation method to design and development of antisense template","abstract":"Antisense technology is emerging as potential therapeutics against lethal infections. Basically, Antisense-mRNA complex inhibits the protein translation of pathogens and thus it is used for treatment. Based on previous online tools and literatures and difficulties for designing antisense template, finding high conserved regions from large number of long sequences, by taking all those factors in consideration, we proposed new innovative offline target simulation methods i.e. Deletion of unwanted region from viral sequence alignment (DURVA) and Most frequent region (MFR) for designing and developing antisense template from large number of long sequence or genomic data. Based on current pandemic crisis and long genomic sequence of SARS-CoV-2, we chose coronavirus for simulation. Initially, we hypothesized that DURVA-MFR would find stable region from large annotated sequencing data. As per Chan et.al. guidelines for antisense designing and development, we designed couple of algorithms and python scripts to process the data of approximately 30kbp sequence length and 1Gb file size in short turnaround time. The steps involved were as: 1) Simplifying whole genome sequence in single line; 2) Deletion of unwanted region from Virus sequence alignment(DURVA); 3)Most frequent antisense target region(MFR) and 4)Designing and development of antisense template. This simulation method is identifying most frequent regions between 20-30bp long, GC count\u226510. Our study concluded that targets were highly identical with large population and similar with high number of remaining sequences. In addition, designed antisense sequences were stable and each sequence is having tighter binding with targets. After studying each parameter, here we suggested that our proposed method would be helpful for finding best antisense against all present and upcoming lethal infection.The initial design of this logic was published in Indian Patent Office Journal No.08/2021withApplication number202121005964A. The antisense development is state of the art for modern therapeutics. There are number of online soft-wares and open sources for designing of antisense template. But all other tools did not consider frequency as major factor for designing antisense. Also; all sources excepting our simulation approach does not process large file or long sequences. Therefore; we designed an offline innovative simulation method which deletes the unwanted region from sequences and stores the data which are fulfilled antisense criteria. Further; the calculation of frequency from these short listed target regions; the most frequent region is desire antisense target and further antisense template will be designed according to Watson-Crick model. This article explained all information about how our new approach is best for designing antisense template against SARS-CoV-2 and many lethal infectious viruses etc.","version":"1.1","doi":"10.1101/2022.09.30.510283","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.29.510112","pub_date":"2022-9-29","title":"A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 600 million cases and over 6 million deaths worldwide. Vaccination has been the main strategy used to contain the spread of the virus, and to avoid hospitalizations and deaths. Currently, there are two mRNA-based and one adenovirus vectored vaccines approved and available for use in the U.S. population. The versatility, low cost and rapid-to-manufacture attributes of DNA vaccines are important advantages over other platforms. However, DNA vaccination must meet higher efficiency levels for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been successfully used in the veterinary field. Here we evaluated the safety, immunogenicity and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate for delivered by intramuscular injection followed by electroporation (Vet-ePorator\u2122) in ferrets. The results demonstrated that the linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects, and was able to elicit neutralizing antibodies and T cell responses using a low dose of the linear DNA construct in prime-boost regimen, and significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.","version":"1.1","doi":"10.1101/2022.09.29.510112","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.29.510004","pub_date":"2022-9-29","title":"Sensitivity of diffusion-tensor and correlated diffusion imaging to white-matter microstructural abnormalities: application in COVID-19","abstract":"There has been growing attention on the effect of COVID-19 on white-matter microstructure, especially among those that self-isolated after being infected. There is also immense scientific interest and potential clinical utility to evaluate the sensitivity of single-shell diffusion MRI methods for detecting such effects. In this work, the sensitivities of three single-shell-compatible diffusion MRI modeling methods are compared for detecting the effect of COVID-19, including diffusion-tensor imaging, diffusion-tensor decomposition of orthogonal moments and correlated diffusion imaging. Imaging was performed on self-isolated patients at baseline and 3-month follow-up, along with age- and sex-matched controls. We demonstrate through simulations and experimental data that correlated diffusion imaging is associated with far greater sensitivity, being the only one of the three single-shell methods to demonstrate COVID-19-related brain effects. Results suggest less restricted diffusion in the frontal lobe in COVID-19 patients, but also more restricted diffusion in the cerebellar white matter, in agreement with several existing studies highlighting the vulnerability of the cerebellum to COVID-19 infection. These results, taken together with the simulation results, suggest that a significant proportion of COVID-19 related white-matter microstructural pathology manifests as a change in water diffusivity. Interestingly, different b-values also confer different sensitivities to the effects. No significant difference was observed in patients at the 3-month follow-up, likely due to the limited size of the follow-up cohort. To summarize, correlated diffusion imaging is shown to be a sensitive single-shell diffusion analysis approach that allows us to uncover opposing patterns of diffusion changes in the frontal and cerebellar regions of COVID-19 patients, suggesting the two regions react differently to viral infection.","version":"1.1","doi":"10.1101/2022.09.29.510004","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.28.509903","pub_date":"2022-9-28","title":"Nirmatrelvir-resistant SARS-CoV-2 is efficiently transmitted in Syrian hamsters","abstract":"The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic target for the treatment of COVID-19. Nirmatrelvir is the only the 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization; other 3Lpro inhibitors are in development. We recently repored on the in vitro selection of a SARS-CoV2 3CLpro (L50F-E166A-L167F; short 3CLprores) virus that is cross-resistant with nirmatrelvir and yet other 3CLpro inhibitors. Here, we demonstrate that the resistant virus replicates efficiently in the lungs of intranassaly infected hamsters and that it causes a lung pathology that is comparable to that caused by the WT virus. Moreover, 3CLprores infected hamsters transmit the virus efficiently to co-housed non-infected contact hamsters. Fortunately, resistance to Nirmatrelvir does not readily develop (in the clinical setting) since the drug has a relatively high barrier to resistance. Yet, as we demonstrate, in case resistant viruses emerge, they may easily spread and impact therapeutic options for others. Therefore, the use of SARS-CoV-2 3CLpro protease inhibitors in combinations with drugs that have a different mechanism of action, may be considered to avoid the development of drug-resistant viruses in the future.","version":"1.1","doi":"10.1101/2022.09.28.509903","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509803","pub_date":"2022-9-28","title":"Activation of SARS-CoV-2 by trypsin-like proteases in the clinical specimens of patients with COVID-19","abstract":"SARS-CoV-2 enters host cells through the angiotensin converting enzyme 2 (ACE2) receptor and/or transmembrane protease, serine 2 (TMPRSS2). Serine proteases, such as TMPRSS2 and trypsin, promote viral entry. In this study, we investigated whether proteases increased SARS-CoV-2 infectivity using pseudotyped viruses and clinical specimens from patients with COVID-19. First, we investigated how trypsin increased infectivity using the pseudotyped virus. Our findings revealed that trypsin increased infectivity after the virus was adsorbed on the cells, but no increase in infectivity was observed when the virus was treated with trypsin. We examined the effect of trypsin on SARS-CoV-2 infection in clinical specimens and found that the infectivity of the SARS-CoV-2 delta variant increased 36,000-fold after trypsin treatment. By contrast, the infectivity of SARS-CoV-2 omicron variant increased to less than 20-fold in the clinical specimens. Finally, infectivity of clinical specimens containing culture supernatants of Fusobacterium necrophorum was increased from several- to 10-fold. Because SARS-CoV-2 infectivity increases in the oral cavity, which may contain anaerobic bacteria, keeping the oral cavities clean may help prevent SARS-CoV-2 infection. In this study, we examined whether trypsin-like proteases increased the infectivity of SARS-CoV-2. We found that trypsin-like proteases increased the infectivity of both the pseudotyped viruses and the live virus in the clinical specimens. The increase in infectivity was significantly higher for the delta than the omicron variant. A large amount of protease in the oral cavity during SARS-CoV-2 infection is expected to increase infectivity. Therefore, keeping the oral cavity clean is important for preventing infection.","version":"1.1","doi":"10.1101/2022.09.27.509803","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509689","pub_date":"2022-9-28","title":"SARS-CoV-2 variants show temperature-dependent enhanced polymerase activity in the upper respiratory tract and high transmissibility","abstract":"With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33\u00b0C) and lower (37\u00b0C) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33\u00b0C compared to 37\u00b0C and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33\u00b0C, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.","version":"1.2","doi":"10.1101/2022.09.27.509689","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509738","pub_date":"2022-9-28","title":"IgG3 subclass antibodies recognize antigenically drifted influenza viruses and SARS-CoV-2 variants through efficient bivalent binding","abstract":"The constant domains of antibodies are important for effector functions, but less is known about how they can affect binding and neutralization of viruses. Here we evaluated a panel of human influenza virus monoclonal antibodies (mAbs) expressed as IgG1, IgG2 or IgG3. We found that many influenza virus-specific mAbs have altered binding and neutralization capacity depending on the IgG subclass encoded, and that these differences result from unique bivalency capacities of the subclasses. Importantly, subclass differences in antibody binding and neutralization were greatest when the affinity for the target antigen was reduced through antigenic mismatch. We found that antibodies expressed as IgG3 bound and neutralized antigenically drifted influenza viruses more effectively. We obtained similar results using a panel of SARS-CoV-2-specific mAbs and the antigenically advanced B.1.351 strain of SARS-CoV-2. We found that a licensed therapeutic mAb retained neutralization breadth against SARS-CoV-2 variants when expressed as IgG3, but not IgG1. These data highlight that IgG subclasses are not only important for fine-tuning effector functionality, but also for binding and neutralization of antigenically drifted viruses. Influenza viruses and coronaviruses undergo continuous change, successfully evading human antibodies elicited from prior infections or vaccinations. It is important to identify features that allow antibodies to bind with increased breadth. Here we examined the effect that different IgG subclasses have on monoclonal antibody binding and neutralization. We show that IgG subclass is a determinant of antibody breadth, with IgG3 affording increased neutralization of antigenically drifted variants of influenza virus and SARS-CoV-2. Future studies should evaluate IgG3 therapeutic antibodies and vaccination strategies or adjuvants that may skew antibody responses toward broadly reactive isotypes.","version":"1.1","doi":"10.1101/2022.09.27.509738","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509649","pub_date":"2022-9-28","title":"Mutational spectra distinguish SARS-CoV-2 replication niches","abstract":"Exposure to different mutagens leaves distinct mutational patterns that can allow prediction of pathogen replication niches (Ruis 2022). We therefore hypothesised that analysis of SARS-CoV-2 mutational spectra might show lineage-specific differences, dependant on the dominant site(s) of replication and onwards transmission, and could therefore rapidly infer virulence of emergent variants of concern (VOC; Konings 2021). Through mutational spectrum analysis, we found a significant reduction in G>T mutations in Omicron, which replicates in the upper respiratory tract (URT), compared to other lineages, which replicate in both upper and lower respiratory tracts (LRT). Mutational analysis of other viruses and bacteria indicates a robust, generalisable association of high G>T mutations with replication within the LRT. Monitoring G>T mutation rates over time, we found early separation of Omicron from Beta, Gamma and Delta, while the mutational burden in Alpha varied consistent with changes in transmission source as social restrictions were lifted. This supports the use of mutational spectra to infer niches of established and emergent pathogens.","version":"1.1","doi":"10.1101/2022.09.27.509649","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.06.506714","pub_date":"2022-9-28","title":"Heterologous boost with mRNA vaccines against SARS-CoV-2 Delta/Omicron variants following an inactivated whole-virus vaccine","abstract":"The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants following an order of BA.1>BA.2>BA.4/5. In na\u00efve animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccine\u2019s targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection across all VOCs. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by \u201crecall\u201d and \u201creshape\u201d the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine\u2013 primed populations.","version":"1.3","doi":"10.1101/2022.09.06.506714","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509702","pub_date":"2022-9-28","title":"Cell type-independent profiling of interactions between intracellular pathogens and the human phosphoproteome","abstract":"Interactions between proteins from intracellular pathogens and host proteins in an infected cell are often mediated by post-translational modifications encoded in the host proteome. Identifying protein modifications, such as phosphorylation, that dictate these interactions remains a defining challenge in unraveling the molecular mechanisms of pathogenesis. We have developed a platform in engineered bacteria that displays over 110,000 phosphorylated human proteins coupled to a fluorescent reporter system capable of identifying the host-pathogen interactome of phosphoproteins (H-PIP). This resource broadly enables cell-type independent interrogation and discovery of proteins from intracellular pathogens capable of binding phosphorylated human proteins. As an example of the H-PIP platform, we generated a unique, high-resolution SARS-CoV-2 interaction network which expanded our knowledge of viral protein function and identified understudied areas of host pathology.","version":"1.1","doi":"10.1101/2022.09.27.509702","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509633","pub_date":"2022-9-27","title":"Nuclear translocation of spike mRNA and protein is a novel pathogenic feature of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe pathophysiology in vulnerable older populations and appears to be highly pathogenic and more transmissible than SARS-CoV or MERS-CoV [1, 2]. The spike (S) protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. Although the S protein is a surface transmembrane type 1 glycoprotein, it has been predicted to be translocated into the nucleus due to the novel nuclear localization signal (NLS) \u201cPRRARSV\u201d, which is absent from the S protein of other coronaviruses. Indeed, S proteins translocate into the nucleus in SARS-CoV-2-infected cells. To our surprise, S mRNAs also translocate into the nucleus. S mRNA colocalizes with S protein, aiding the nuclear translocation of S mRNA. While nuclear translocation of nucleoprotein (N) has been shown in many coronaviruses, the nuclear translocation of both S mRNA and S protein reveals a novel pathogenic feature of SARS-CoV-2. One of the novel sequence insertions resides at the S1/S2 boundary of Spike (S) protein and constitutes a functional nuclear localization signal (NLS) motif \u201cPRRARSV\u201d, which may supersede the importance of previously proposed polybasic furin cleavage site \u201cRRAR\u201d. Indeed, S protein\u2019s NLS-driven nuclear translocation and its possible role in S mRNA\u2019s nuclear translocation reveal a novel pathogenic feature of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.09.27.509633","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.26.22280387","pub_date":"2022-09-27","title":"Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Guidelines for SARS-CoV-2 have relied on limited data on duration of viral infectiousness and correlation with COVID-19 symptoms and diagnostic testing.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We enrolled ambulatory adults with acute SARS-CoV-2 infection and performed serial measurements of COVID-19 symptoms, nasal swab viral RNA, nucleocapsid (N) and spike (S) antigens, and replication-competent SARS-CoV-2 by culture. We determined average time from symptom onset to a first negative test result and estimated risk of infectiousness, as defined by a positive viral culture.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Among 95 adults, median [interquartile range] time from symptom onset to first negative test result was 9 [5] days, 13 [6] days, 11 [4] days, and &gt;19 days for S antigen, N antigen, viral culture growth, and viral RNA by RT-PCR, respectively. Beyond two weeks, viral cultures and N antigen titers were rarely positive, while viral RNA remained detectable among half (26/51) of participants tested 21-30 days after symptom onset. Between 6-10 days from symptom onset, N antigen was strongly associated with viral culture positivity (relative risk=7.61, 95% CI: 3.01-19.2), whereas neither viral RNA nor symptoms were associated with culture positivity. During the 14 days following symptom onset, presence of N antigen (adjusted relative risk=7.66, 95% CI: 3.96-14.82), remained strongly associated with viral culture positivity, regardless of COVID-19 symptoms.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Most adults have replication-competent SARS-CoV-2 for 10-14 after symptom onset, and N antigen testing is a strong predictor of viral infectiousness. Within two weeks from symptom onset, N antigen testing, rather than absence of symptoms or viral RNA, should be used to safely discontinue isolation.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Bill and Melinda Gates Foundation</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.09.26.22280387","journal":"medRxiv","score":null},{"id":"10.1101/2022.09.27.509682","pub_date":"2022-9-27","title":"Wild red deer (Cervus elaphus) do not play a role as vectors or reservoirs of SARS-CoV-2 in northeastern Poland","abstract":"Several studies reported a high prevalence of SARS-CoV-2 among white-tailed deer in North America. Monitoring cervids in all regions to better understand SARS-CoV-2 infection and circulation in other deer populations has been urged. To evaluate deer exposure to SARS-CoV-2 in Poland, we sampled 90 reed deer individuals shot by hunters in five hunting districts in northeastern Poland. Serum and nasopharyngeal swabs were collected, and then the Immunofluorescent Assay (IFA) to detect anti-SARS-CoV-2 antibodies was performed as well as real-time PCR with reverse transcription for direct virus detection. No positive samples were detected. There is no evidence of spillover of SARS-CoV-2 from the human to deer population in Poland.","version":"1.1","doi":"10.1101/2022.09.27.509682","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.03.506425","pub_date":"2022-9-27","title":"Plant production of high affinity nanobodies that block SARS-CoV-2 spike protein binding with its receptor, human angiotensin converting enzyme","abstract":"Nanobodies\u00ae (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb) derived from camelids. Here, we demonstrate production of VHH nanobodies against the SARS-CoV-2 spike proteins in the solanaceous plant Nicotiana benthamiana through transient expression and their subsequent detection verified through western blot. We demonstrate that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2 (ACE2). We present plant production of nanobodies as an economical and scalable alternative to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture.","version":"1.2","doi":"10.1101/2022.09.03.506425","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.27.509668","pub_date":"2022-9-27","title":"The structure of monkeypox virus 2\u2019-O-ribose methyltransferase VP39 in complex with sinefungin provides the foundation for inhibitor design","abstract":"Monkeypox is an emerging, rapidly spreading disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a dsDNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present the crystal structure of its 2\u2019-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin. A comparison of this 2\u2019-O RNA MTase with enzymes from unrelated ssRNA viruses (SARS-CoV-2 and Zika) reveals a surprisingly conserved sinefungin binding mode implicating that a single inhibitor could be used against unrelated viral families.","version":"1.1","doi":"10.1101/2022.09.27.509668","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.22.509123","pub_date":"2022-9-23","title":"Generation and functional analysis of defective viral genomes during SARS-CoV-2 infection","abstract":"Defective viral genomes (DVGs) have been identified in many RNA viruses as a major factor influencing antiviral immune response and viral pathogenesis. However, the generation and function of DVGs in SARS-CoV-2 infection are less known. In this study, we elucidated DVG generation in SARS-CoV-2 and its relationship with host antiviral immune response. We observed DVGs ubiquitously from RNA-seq datasets of in vitro infections and autopsy lung tissues of COVID-19 patients. Four genomic hotspots were identified for DVG recombination and RNA secondary structures were suggested to mediate DVG formation. Functionally, bulk and single cell RNA-seq analysis indicated the IFN stimulation of SARS-CoV-2 DVGs. We further applied our criteria to the NGS dataset from a published cohort study and observed significantly higher DVG amount and frequency in symptomatic patients than that in asymptomatic patients. Finally, we observed unusually high DVG frequency in one immunosuppressive patient up to 140 days after admitted to hospital due to COVID-19, first-time suggesting an association between DVGs and persistent viral infections in SARS-CoV-2. Together, our findings strongly suggest a critical role of DVGs in modulating host IFN responses and symptom development, calling for further inquiry into the mechanisms of DVG generation and how DVGs modulate host responses and infection outcome during SARS-CoV-2 infection. Defective viral genomes (DVGs) are ubiquitously generated in many RNA viruses, including SARS-CoV-2. Their interference activity to full-length viruses and IFN stimulation provide them the potential for novel antiviral therapies and vaccine development. SARS-CoV-2 DVGs are generated through the recombination of two discontinuous genomic fragments by viral polymerase complex and the recombination is also one of the major mechanisms for the emergence of new coronaviruses. Focusing on the generation and function of SARS-CoV-2 DVGs, these studies identify new hotspots for non-homologous recombination and strongly suggest that the secondary structures within viral genomes mediate the recombination. Furthermore, these studies provide the first evidence for IFN stimulation activity of de novo DVGs during natural SARS-CoV-2 infection. These findings set up the foundation for further mechanism studies of SARS-CoV-2 recombination and provide the evidence to harness DVGs\u2019 immunostimulatory potential in the development of vaccine and antivirals for SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.09.22.509123","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.22.508962","pub_date":"2022-9-23","title":"Pharmacological inhibition of bromodomain and extra-terminal proteins induces NRF-2-mediated inhibition of SARS-CoV-2 replication and is subject to viral antagonism","abstract":"Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential therapeutics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-induced antiviral activity and its susceptibility to viral antagonism remain incompletely understood. iBET treatment transiently inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. Our functional assays confirmed JQ-1-mediated downregulation of ACE2 expression and multi-omics analysis uncovered induction of an antiviral NRF-2-mediated cytoprotective response as an additional antiviral component of JQ-1 treatment. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variants. JQ-1 antiviral activity was transient in human bronchial airway epithelial cells (hBAECs) treated prior to infection and absent when administered therapeutically. We propose that JQ-1 exerts pleiotropic effects that collectively induce a transient antiviral state that is ultimately nullified by an established SARS-CoV-2 infection, raising questions on their clinical suitability in the context of COVID-19.","version":"1.1","doi":"10.1101/2022.09.22.508962","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.23.22280264","pub_date":"2022-09-23","title":"Post-COVID-19 syndrome: retinal microcirculation as a potential marker for chronic fatigue","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Post-COVID-19 syndrome (PCS) summarizes persisting sequelae after infection with the severe-acute-respiratory-syndrome-Coronavirus-2 (SARS-CoV-2). PCS can affect patients of all covid-19 disease severities. As previous studies revealed impaired blood flow as a provoking factor for triggering PCS, it was the aim of the present study to investigate a potential association of self-reported chronic fatigue and retinal microcirculation in patients with PCS, potentially indicating an objective biomarker.</jats:p>\n                <jats:p>A prospective study was performed, including 201 subjects: 173 patients with PCS and 28 controls. Retinal microcirculation was visualized by OCT-Angiography (OCT-A) and quantified by the Erlangen-Angio-Tool as macula and peripapillary vessel density (VD). Chronic Fatigue (CF) was assessed with the variables \u2018Bell score\u2019, age and gender. The VD in the superficial vascular plexus (SVP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP) were analyzed considering the repetitions (12 times). Taking in account of such repetitions a mixed model was performed to detect possible differences in the least square means between different groups of analysis.</jats:p>\n                <jats:p>An age effect on VD was observed between patients and controls (p&lt;0.0001). Gender analysis yielded that women with PCS showed lower VD levels in SVP compared to male patients (p=0.0015). The PCS patients showed significantly lower VD of ICP as compared to the controls (p=0.0001, [CI: 0.32; 1]). Moreover, considering PCS patients, the mixed model reveals a significant difference between chronic fatigue (CF) and without CF in VD of SVP (p=0.0033, [CI: -4.5; -0.92]). The model included age, gender and the variable \u2018Bell score\u2019, representing a subjective marker for CF. Consequently, the retinal microcirculation might be an objective biomarker in subjective-reported chronic fatigue of patients with PCS.</jats:p>","version":null,"doi":"10.1101/2022.09.23.22280264","journal":"medRxiv","score":null},{"id":"10.1101/2022.09.22.508999","pub_date":"2022-9-22","title":"Triple COVID-19 vaccination induces humoral and cellular immunity to SARS-CoV-2 with cross-recognition of the Omicron variant and IgA secretion","abstract":"COVID-19 vaccination is the leading strategy to prevent severe courses after SARS-CoV-2 infection. In our study, we analyzed humoral and cellular immune responses in detail to three consecutive homologous or heterologous COVID-19 vaccinations. All individuals (n=20) responded to vaccination with increasing S1- /RBD-/S2-specific IgG levels, whereas specific plasma IgA displayed individual variability. The third dose increased antibody inhibitory capacity (AIC) against immune-escape variants Beta and Omicron independently from age. The mRNA-primed vaccination induced IgG and IgA immunity more efficiently, whereas vector-primed individuals displayed higher levels of memory T and B cells. Vaccinees showed a SARS-CoV-2-specific T cell responses, which were further improved and specified after Omicron breakthrough infections in parallel to appearance of new variant-specific antibodies. In conclusion, the third vaccination was essential to increase IgG levels, mandatory to boost AIC against immune-escape variants and induced SARS-CoV-2-specific T cells. Breakthrough infection with Omicron generates additional spike specificities covering all known variants.","version":"1.1","doi":"10.1101/2022.09.22.508999","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508904","pub_date":"2022-9-22","title":"In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury","abstract":"The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.","version":"1.1","doi":"10.1101/2022.09.21.508904","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.22.509040","pub_date":"2022-9-22","title":"SARS-CoV-2 Omicron boosting induces de novo B cell response in humans","abstract":"The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs). It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS- CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a na\u00efve B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.","version":"1.1","doi":"10.1101/2022.09.22.509040","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.11.495733","pub_date":"2022-9-22","title":"Evolutionary trajectory of receptor binding specificity and promiscuity of the spike protein of SARS-CoV-2","abstract":"SARS-CoV-2 infects cells by attachment to its receptor \u2013 the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation- and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affect the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism, and capacity to evade immune responses during evolution.","version":"1.2","doi":"10.1101/2022.06.11.495733","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.22.509013","pub_date":"2022-9-22","title":"Evolution of new variants of SARS-COV-2 during the pandemic: mutation limited or selection limited?","abstract":"The recent pandemic caused by SARS-Cov-2 has witnessed an evolving succession of variants of the virus. While the phenomenon of invasion by immunity evading variants is known for other viruses such as influenza, the dynamics of the ecological and evolutionary process in the succession is little known. Since during the Covid-19 pandemic, large scale epidemiological data were collected and made available in the public domain, it is possible to seek answers to a number of evolutionary questions, which will also have public health implications. We list multiple alternative hypotheses about the origin and invasion of the variants and evaluate them in the light of epidemiological data. Our analysis shows that invasion by novel variants is selection limited and not mutation limited. Further novel variants are not the necessary and sufficient causes of the repeated waves during the pandemic. Rather there is substantial overlap between the conditions leading to a wave and those favoring selection of a partial immune evading variant. This is likely to lead to an association between invasion by new variant and the rise of a new wave. But the association is not sufficiently strong and does not support a causal role of the new variant. The dynamics of interaction between epidemiological processes and selection on viral variants have many public health implications that can guide future policies for effective control of infectious epidemics.","version":"1.1","doi":"10.1101/2022.09.22.509013","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508922","pub_date":"2022-9-22","title":"Evolution of antibody immunity following Omicron BA.1 breakthrough infection","abstract":"Understanding the evolution of antibody immunity following heterologous SAR-CoV-2 breakthrough infection will inform the development of next-generation vaccines. Here, we tracked SARS-CoV-2 receptor binding domain (RBD)-specific antibody responses up to six months following Omicron BA.1 breakthrough infection in mRNA-vaccinated individuals. Cross-reactive serum neutralizing antibody and memory B cell (MBC) responses declined by two- to four-fold through the study period. Breakthrough infection elicited minimal de novo Omicron-specific B cell responses but drove affinity maturation of pre-existing cross-reactive MBCs toward BA.1. Public clones dominated the neutralizing antibody response at both early and late time points, and their escape mutation profiles predicted newly emergent Omicron sublineages. The results demonstrate that heterologous SARS-CoV-2 variant exposure drives the evolution of B cell memory and suggest that convergent neutralizing antibody responses continue to shape viral evolution.","version":"1.1","doi":"10.1101/2022.09.21.508922","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508960","pub_date":"2022-9-22","title":"Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome","abstract":"Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8\u2019s ability to sense coronavirus 3CLpros, and instead enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.","version":"1.1","doi":"10.1101/2022.09.21.508960","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508823","pub_date":"2022-9-22","title":"FDA-approved drug screening identified micafungin as an antiviral agent against bat-borne emerging zoonotic Pteropine orthoreovirus","abstract":"Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, severe acute respiratory syndrome (SARS) coronavirus, and SARS-CoV-2. Pteropine orthoreovirus (PRV), which spillover event occurred from fruit bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is a critical tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. A library of 2,943 FDA-approved drugs was screened to find potential antiviral drugs of Pteropine orthoreovirus. Six hit compounds dramatically inhibited viral replication in vitro. Micafungin possessed antiviral activity to multiple strains of PRV.","version":"1.1","doi":"10.1101/2022.09.21.508823","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.20.508614","pub_date":"2022-9-21","title":"Parallel use of pluripotent human stem cell lung and heart models provide new insights for treatment of SARS-CoV-2","abstract":"SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems. Rational treatment strategies for SARS-CoV-2 derived from human PSC models","version":"1.1","doi":"10.1101/2022.09.20.508614","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.20.508790","pub_date":"2022-9-21","title":"Prediction of RNA secondary structures in SARS-CoV-2 and comparison with contemporary predictions","abstract":"SARS-CoV-2, the causative agent of covid-19, is known to exhibit secondary structure in its 5\u2019 and 3\u2019 untranslated regions, along with the frameshifting stimulatory element situated between ORF1a and 1b. To identify further regions containing conserved structure, multiple sequence alignment with related coronaviruses was used as a starting point from which to apply a modified computational pipeline developed to identify non-coding RNA elements in vertebrate eukaryotes. Three different RNA structural prediction approaches were employed in this modified pipeline. Forty genomic regions deemed likely to harbour structure were identified, ten of which exhibited three-way consensus substructure predictions amongst our predictive utilities. Intracomparison of the pipeline\u2019s predictive utilities, along with intercomparison with three previously published SARS-CoV-2 structural datasets, were performed. Limited agreement as to precise structure was observed, although different approaches appear to agree upon regions likely to contain structure in the viral genome.","version":"1.1","doi":"10.1101/2022.09.20.508790","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508818","pub_date":"2022-9-21","title":"Exposure to BA.4/BA.5 Spike glycoprotein drives pan-Omicron neutralization in vaccine-experienced humans and mice","abstract":"The SARS-CoV-2 Omicron variant and its sublineages show pronounced viral escape from neutralizing antibodies elicited by vaccination or prior SARS-CoV-2 variant infection owing to over 30 amino acid alterations within the spike (S) glycoprotein. We and others have recently reported that breakthrough infection of vaccinated individuals with Omicron sublineages BA.1 and BA.2 are associated with distinct patterns of cross-neutralizing activity against SARS-CoV-2 variants of concern (VOCs). BA.2 breakthrough infection mediated overall stronger cross-neutralization of BA.2 and its descendants (BA.2.12.1, BA.4, and BA.5) compared to BA.1 breakthrough infection. Here we characterized the effect of Omicron BA.4/BA.5 S glycoprotein exposure on the magnitude and breadth of the neutralizing antibody response upon breakthrough infection in vaccinated individuals and in mice upon booster vaccination. We show that immune sera from triple mRNA-vaccinated individuals with subsequent Omicron BA.4/BA.5 breakthrough infection display broad and robust neutralizing activity against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5. Administration of a prototypic BA.4/BA.5-adapted mRNA booster vaccine to mice following SARS-CoV-2 wild-type strain-based primary immunization is associated with similarly broad neutralizing activity. Immunization of na\u00efve mice with a bivalent mRNA vaccine (wild-type + Omicron BA.4/BA.5) induces strong and broad neutralizing activity against Omicron VOCs and previous variants. These findings suggest that when administered as boosters, mono- and bivalent Omicron BA.4/BA.5-adapted vaccines may enhance neutralization breadth, and in a bivalent format may also have the potential to confer protection to individuals with no pre-existing immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.09.21.508818","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.21.508870","pub_date":"2022-9-21","title":"Association of polymorphisms of IL-6 pathway genes (IL6, IL6R and IL6ST) with COVID-19 severity in an Amazonian population","abstract":"Interleukin-6 have been recognized as a major role player in COVID-19 severity, being an important regulator of cytokine storm. Hence, the evaluation of the influence of polymorphisms in key genes of IL-6 pathway, namely IL6, IL6R and IL6ST, may provide valuable prognostic/predictive biomarkers on COVID-19. The present cross-sectional study genotyped three Single Nucleotide Polymorphisms - SNPs (rs1800795, rs2228145 and rs7730934) at IL6, IL6R and IL6ST genes, respectively, in 227 COVID-19 patients (132 hospitalized and 95 non-hospitalized). Genotype frequencies were compared between these groups. As control group, published data on gene and genotype frequencies was gathered from published studies from before the pandemic started. Our major results point to an association of IL6 C allele with COVID-19 severity. Moreover, IL-6 plasmatic levels were higher among IL6 CC genotype carriers. Additionally, the frequency of symptoms was higher at IL6 CC and IL6R CC genotypes. In conclusion the data suggest an important role of IL6 C allele and IL6R CC genotype on COVID-19 severity, in agreement with indirect evidences from literature about association of these genotypes with mortality rates, pneumonia, heightening of protein plasmatic levels proinflammatory driven effects.","version":"1.1","doi":"10.1101/2022.09.21.508870","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474095","pub_date":"2022-9-20","title":"Early Computational Detection of Potential High Risk SARS-CoV-2 Variants","abstract":"The ongoing COVID-19 pandemic is leading to the discovery of hundreds of novel SARS-CoV-2 variants on a daily basis. While most variants do not impact the course of the pandemic, some variants pose a significantly increased risk when the acquired mutations allow better evasion of antibody neutralisation in previously infected or vaccinated subjects or increased transmissibility. Early detection of such high risk variants (HRVs) is paramount for the proper management of the pandemic. However, experimental assays to determine immune evasion and transmissibility characteristics of new variants are resource-intensive and time-consuming, potentially leading to delays in appropriate responses by decision makers. Here we present a novel in silico approach combining spike (S) protein structure modelling and large protein transformer language models on S protein sequences to accurately rank SARS-CoV-2 variants for immune escape and fitness potential. These metrics can be combined into an automated Early Warning System (EWS) capable of evaluating new variants in minutes and risk-monitoring variant lineages in near real-time. The system accurately pinpoints the putatively dangerous variants by selecting on average less than 0.3% of the novel variants each week. With only the S protein nucleotide sequence as input, the EWS detects HRVs earlier and with better precision than baseline metrics such as the growth metric (which requires real-world observations) or random sampling. Notably, Omicron BA.1 was flagged by the EWS on the day its sequence was made available. Additionally, our immune escape and fitness metrics were experimentally validated using in vitro pseudovirus-based virus neutralisation test (pVNT) assays and binding assays. The EWS flagged as potentially dangerous all 16 variants (Alpha-Omicron BA.1/2/4/5) designated by the World Health Organisation (WHO) with an average lead time of more than one and a half months ahead of them being designated as such. A COVID-19 Early Warning System combining structural modelling with machine learning to detect and monitor high risk SARS-CoV-2 variants, identifying all 16 WHO designated variants on average more than one and a half months in advance by selecting on average less than 0.3% of the weekly novel variants.","version":"1.2","doi":"10.1101/2021.12.24.474095","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.20.508745","pub_date":"2022-9-20","title":"Deep mutational scans for ACE2 binding, RBD expression, and antibody escape in the SARS-CoV-2 Omicron BA.1 and BA.2 receptor-binding domains","abstract":"SARS-CoV-2 continues to acquire mutations in the spike receptor-binding domain (RBD) that impact ACE2 receptor binding, folding stability, and antibody recognition. Deep mutational scanning prospectively characterizes the impacts of mutations on these biochemical properties, enabling rapid assessment of new mutations seen during viral surveillance. However, the effects of mutations can change as the virus evolves, requiring updated deep mutational scans. We determined the impacts of all amino acid mutations in the Omicron BA.1 and BA.2 RBDs on ACE2-binding affinity, RBD folding, and escape from binding by the LY-CoV1404 (bebtelovimab) monoclonal antibody. The effects of some mutations in Omicron RBDs differ from those measured in the ancestral Wuhan-Hu-1 background. These epistatic shifts largely resemble those previously seen in the Beta variant due to the convergent epistatically modifying N501Y substitution. However, Omicron variants show additional lineage-specific shifts, including examples of the epistatic phenomenon of entrenchment that causes the Q498R and N501Y substitutions present in Omicron to be more favorable in that background than in earlier viral strains. In contrast, the Omicron substitution Q493R exhibits no sign of entrenchment, with the derived state, R493, being as unfavorable for ACE2 binding in Omicron RBDs as in Wuhan-Hu-1. Likely for this reason, the R493Q reversion has occurred in Omicron sub-variants including BA.4/BA.5 and BA.2.75, where the affinity buffer from R493Q reversion may potentiate concurrent antigenic change. Consistent with prior studies, we find that Omicron RBDs have reduced expression, and identify candidate stabilizing mutations that ameliorate this deficit. Last, our maps highlight a broadening of the sites of escape from LY-CoV1404 antibody binding in BA.1 and BA.2 compared to the ancestral Wuhan-Hu-1 background. These BA.1 and BA.2 deep mutational scanning datasets identify shifts in the RBD mutational landscape and inform ongoing efforts in viral surveillance. SARS-CoV-2 evolves in part through mutations in its spike receptor-binding domain. As these mutations accumulate in evolved variants, they shape the future evolutionary potential of the virus through the phenomenon of epistasis. We characterized the functional impacts of mutations in the Omicron BA.1 and BA.2 receptor-binding domains on ACE2 receptor binding, protein folding, and recognition by the clinical LY-CoV1404 antibody. We then compared the measurements to prior data for earlier variants. These comparisons identify patterns of epistasis that may alter future patterns of Omicron evolution, such as turnover in the availability of specific affinity-enhancing mutations and an expansion in the number of paths of antibody escape from a key monoclonal antibody used for therapeutic treatment of COVID-19. This work informs continued efforts in viral surveillance and forecasting.","version":"1.1","doi":"10.1101/2022.09.20.508745","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.507948","pub_date":"2022-9-19","title":"HLA variants and TCR diversity against SARS-CoV-2 in the pre-COVID-19 era","abstract":"HLA antigen presentation and T-cell immunity are critical to control viral infection such as SARS-CoV-2. This study performed on samples collected in the pre-COVID-19 era demonstrates that individuals are fully equiped at the genetic level in terms of TCR repertoire and HLA variants to recognize and kill SARS-CoV-2 infected cells. HLA diversity, heterologous immunity and random somatic TCR recombination could explain these observations.","version":"1.2","doi":"10.1101/2022.09.14.507948","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.18.508442","pub_date":"2022-9-19","title":"HCR Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing","abstract":"The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/\u03bcL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/\u03bcL, 1000 copies/\u03bcL, 2000 copies/\u03bcL, 2000 copies/\u03bcL, and 20,000 copies/\u03bcL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious disease. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection. Amplified Instrument-free At-home 60 min Naked eye SARS-CoV-2 200 copies/\u03bcL","version":"1.1","doi":"10.1101/2022.09.18.508442","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.11.483930","pub_date":"2022-9-19","title":"Proinflammatory innate cytokines and metabolomic signatures shape the T cell response in active COVID-19","abstract":"The underlying factors contributing to the evolution of SARS-CoV-2-specific T cell responses during COVID-19 infection remain unidentified. To address this, we characterized innate and adaptive immune responses with metabolomic profiling longitudinally at three different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young mildly symptomatic active COVID-19 patients infected during the first wave in mid-2020. We observed that anti-RBD IgG and viral neutralization are significantly reduced against the Delta variant compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell responses remain preserved against the delta and omicron variants. We determined innate immune responses during the early stage of active infection in response to TLR 3/7/8 mediated activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-derived proinflammatory cytokines, IL-18, IL-1\u03b2, and IL-23, and the abundance of plasma metabolites involved in arginine biosynthesis were predictive of a robust SARS-CoV-2-specific Th1 response at a later stage (two weeks after PCR positivity). These observations may contribute to designing effective vaccines and adjuvants that promote innate immune responses and metabolites to induce long-lasting anti-SARS-CoV-2 specific T cells response.","version":"1.3","doi":"10.1101/2022.03.11.483930","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.18.508418","pub_date":"2022-9-19","title":"Guild-level microbiome signature associated with COVID-19 severity and prognosis","abstract":"COVID-19 severity has been associated with alterations of the gut microbiota. However, the relationship between gut microbiome alterations and COVID-19 prognosis remains elusive. Here, we performed a genome-resolved metagenomic analysis on fecal samples collected from 300 in-hospital COVID-19 patients at time of admission. Among the 2,568 high quality metagenome-assembled genomes (HQMAGs), Redundancy Analysis identified 33 HQMAGs which showed differential distribution among mild, moderate, and severe/critical severity groups. Random Forest model based on these 33 HQMAGs classified patients from different severity groups (average AUC = 0.79). Co-abundance network analysis found that the 33 HQMAGs were organized as two competing guilds. Guild 1 harbored more genes for short-chain fatty acid biosynthesis, and fewer genes for virulence and antibiotic resistance, compared with Guild 2. Random Forest regression showed that these 33 HQMAGs at admission had the capacity to predict 8 clinical parameters, which are predictors for COVID-19 prognosis, at Day 7 in hospital. Moreover, the dominance of Guild 1 over Guild 2 at admission predicted the death/discharge outcome of the critical patients (AUC = 0.92). Random Forest models based on these 33 HQMAGs classified patients with different COVID-19 symptom severity, and differentiated COVID-19 patients from healthy subjects, non-COVID-19, and pneumonia controls in three independent datasets. Thus, this genome-based guild-level signature may facilitate early identification of hospitalized COVID-19 patients with high risk of more severe outcomes at time of admission.","version":"1.1","doi":"10.1101/2022.09.18.508418","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.442029","pub_date":"2022-9-19","title":"A non-coding A-to-T Kozak site change related to the transmissibility of Alpha, Delta and Omicron VOCs","abstract":"Three prevalent SARS-CoV-2 Variants of Concern (VOCs) were emerged and caused epidemic waves. It is essential to uncover the key genetic changes that cause the high transmissibility of VOCs. However, different viral mutations are generally tightly linked so traditional population genetic methods may not reliably detect beneficial mutation. In this study, we proposed a new pandemic-scale phylogenomic approach to detect mutations crucial to transmissibility. We analyzed 3,646,973 high-quality SARS-CoV-2 genomic sequences and the epidemiology metadata. Based on the sequential occurrence order of mutations and the instantaneously accelerated furcation rate, the analysis revealed that two non-coding mutations at the position of 28271 (g.a28271-/t) might be crucial for the high transmissibility of Alpha, Delta and Omicron VOCs. Both two mutations cause an A-to-T change at the core Kozak site of the N gene. The analysis also revealed that the non-coding mutations (g.a28271-/t) alone are unlikely to cause high viral transmissibility, indicating epistasis or multilocus interaction in viral transmissibility. A convergent evolutionary analysis revealed that g.a28271-/t, S:P681H/R and N:R203K/M occur independently in the three-VOC lineages, suggesting a potential interaction among these mutations. Therefore, this study unveils that non-synonymous and non-coding mutations could affect the transmissibility synergistically.","version":"1.3","doi":"10.1101/2021.04.30.442029","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.16.508278","pub_date":"2022-9-16","title":"Semantic and population analysis of the genetic targets related to COVID-19 and its association with genes and diseases","abstract":"SARS-CoV-2 is a coronavirus responsible for one of the most serious, modern worldwide pandemics, with lasting and multi-faceted effects. By late 2021, SARS-CoV-2 has infected more than 180 million people and has killed more than 3 million. The virus gains entrance to human cells through binding to ACE2 via its surface spike protein and causes a complex disease of the respiratory system, termed COVID-19. Vaccination efforts are being made to hinder the viral spread and therapeutics are currently under development. Towards this goal, scientific attention is shifting towards variants and SNPs that affect factors of the disease such as susceptibility and severity. This genomic grammar, tightly related to the dark part of our genome, can be explored through the use of modern methods such as natural language processing. We present a semantic analysis of SARS-CoV-2 related publications, which yielded a repertoire of SNPs, genes and disease ontologies. Population data from the 100Genomes Project were subsequently integrated into the pipeline. Data mining approaches of this scale have the potential to elucidate the complex interaction between COVID-19 pathogenesis and host genetic variation; the resulting knowledge can facilitate the management of high-risk groups and aid the efforts towards precision medicine.","version":"1.1","doi":"10.1101/2022.09.16.508278","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.10.483652","pub_date":"2022-9-15","title":"Open modification searching of SARS-CoV-2\u2013human protein interaction data reveals novel viral modification sites","abstract":"The outbreak of the SARS-CoV-2 coronavirus, the causative agent of the COVID-19 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus\u2013host protein\u2013protein interactions (PPIs) through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications (PTMs). We have reanalyzed public affinity purification mass spectrometry data using open modification searching to investigate the presence of PTMs in the context of the SARS-CoV-2 virus\u2013host PPI network. Based on an over two-fold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus\u2013host interactions for alternative viruses, as well as previously unknown protein interactions. Additionally, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.03.10.483652","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.508057","pub_date":"2022-9-15","title":"SARS-CoV-2 infection of human neurons requires endosomal cell entry and can be blocked by inhibitors of host phosphoinositol-5 kinase","abstract":"COVID-19 is a disease caused by coronavirus SARS-CoV-2. In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms that can last from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to infection of brain cells. We found that a small fraction of cortical neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), which was expressed at very low levels. Although only a limited number of neurons was infectable, the infection was productive, as demonstrated by the presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout the neuronal cell, and an increase in viral RNA in the culture medium within the first 48 h of infection (viral release). The productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface protein spike, which can occur at the plasma membrane or from endo/lysosomes. Using specific combinations of small-molecule inhibitors, we found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases found on the cell surface, including TMPRSS2. In contrast, the infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K) that regulates endosomal maturation. COVID-19 is a disease caused by coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures and encephalopathy, as well as anatomical abnormalities such as changes in brain morphology. Whether these symptoms are linked to brain infection is not clear. The mechanism of the virus entry into neurons has also not been characterized. Here we investigated SARS-CoV-2 infection using a human iPSC-derived neural cell model and found that a small fraction of cortical neurons was naturally susceptible to infection. The infection depended on the ACE2 receptor and was productive. We also found that the virus used the late endosomal/lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of the cellular phosphatidyl-inositol 5 kinase.","version":"1.1","doi":"10.1101/2022.09.14.508057","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.13.507852","pub_date":"2022-9-15","title":"IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques","abstract":"The pro- and anti-inflammatory pathways that determine the balance of inflammation and viral control during SARS-CoV-2 infection are not well understood. Here we examine the roles of IFN\u03b3 and IL-10 in regulating inflammation, immune cell responses and viral replication during SARS-CoV-2 infection of rhesus macaques. IFN\u03b3 blockade tended to decrease lung inflammation based on 18FDG-PET/CT imaging but had no major impact on innate lymphocytes, neutralizing antibodies, or antigen-specific T cells. In contrast, IL-10 blockade transiently increased lung inflammation and enhanced accumulation of virus-specific T cells in the lower airways. However, IL-10 blockade also inhibited the differentiation of virus-specific T cells into airway CD69+CD103+ TRM cells. While virus-specific T cells were undetectable in the nasal mucosa of all groups, IL-10 blockade similarly reduced the frequency of total TRM cells in the nasal mucosa. Neither cytokine blockade substantially affected viral load and infection ultimately resolved. Thus, in the macaque model of mild COVID-19, the pro- and anti-inflammatory effects of IFN\u03b3 and IL-10 have no major role in control of viral replication. However, IL-10 has a key role in suppressing the accumulation of SARS-CoV-2-specific T cells in the lower airways, while also promoting TRM at respiratory mucosal surfaces.","version":"1.2","doi":"10.1101/2022.09.13.507852","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.15.507991","pub_date":"2022-9-15","title":"A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2","abstract":"The ongoing pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further drug development.","version":"1.1","doi":"10.1101/2022.09.15.507991","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.507659","pub_date":"2022-9-15","title":"Fast bioluminescent nucleic acid detection using one-pot isothermal amplification and dCas9-based split luciferase complementation","abstract":"Nucleic acid detection methods based on isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or post-amplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. Whereas LUNAS itself features a detection limit of \u223c1 pM for dsDNA targets, the LUNAS platform is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a single-pot assay. We designed a one-pot RT-RPA-LUNAS assay for detecting SARS-CoV-2 RNA without the need for RNA isolation and demonstrated the diagnostic performance for COVID-19 patient nasopharyngeal swab samples using a digital camera to record the ratiometric signal. Detection of SARS-CoV-2 from samples with viral RNA loads of \u223c200 cp/\u03bcL was achieved within \u223c20 minutes, showing that RPA-LUNAS is attractive for point-of-care diagnostic applications.","version":"1.1","doi":"10.1101/2022.09.12.507659","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.20.457146","pub_date":"2022-9-14","title":"SARS-CoV-2 hijacks p38\u03b2/MAPK11 to promote virus replication","abstract":"SARS-CoV-2, the causative agent of the COVID-19 pandemic, drastically modifies infected cells in an effort to optimize virus replication. Included is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammation and is a central driver of COVID-19 clinical presentations. Inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduces both cytokine production and viral replication. Here, we combined genetic screening with quantitative phosphoproteomics to better understand interactions between the p38/MAPK pathway and SARS-CoV-2. We found that several components of the p38/MAPK pathway impacted SARS-CoV-2 replication and that p38\u03b2 is a critical host factor for virus replication, and it prevents activation of the type-I interferon pathway. Quantitative phosphoproteomics uncovered several SARS-CoV-2 nucleocapsid phosphorylation sites near the N-terminus that were sensitive to p38 inhibition. Similar to p38\u03b2 depletion, mutation of these nucleocapsid residues was associated with reduced virus replication and increased activation of type-I interferon signaling. Taken together, this study reveals a unique proviral function for p38\u03b2 that is not shared with p38\u03b1 and supports exploring p38\u03b2 inhibitor development as a strategy towards developing a new class of COVID-19 therapies. SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 mitogen-activated protein kinase (MAPK) pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.","version":"1.2","doi":"10.1101/2021.08.20.457146","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.13.507484","pub_date":"2022-9-14","title":"Trans-synaptic dwelling of SARS-CoV-2 particles perturbs neural synapse organization and function","abstract":"SARS-CoV-2 infection is associated with short- and long-term neurological and psychiatric complications, referred to as neuroCOVID. These symptoms are relatively heterogenous and fluctuating, hampering the discovery of molecular mechanisms underlying viro-induced brain perturbations. Here, we show that the human cerebral cortex poorly supports SARS-CoV-2 dissemination using post-mortem COVID-19 patient samples, ex vivo organotypic cultures of human brain explants and stem cell-derived cortical organoids. Despite restricted infection, the sole exposure of neural cells to SARS-CoV-2 particles is sufficient to induce significant perturbations on neural synapse organization associated to electrical activity dysfunction. Single-organoid proteomics revealed that exposure to SARS-CoV-2 is associated to trans-synaptic proteins upregulation and unveiled that incoming virions dwell at LPHN3/FLRT3-containing synapses. Our study provides new mechanistic insights on the origin of SARS-CoV-2-induced neurological disorders. SARS-CoV-2 modulates neural plasticity and electrical activity as viral particles lodge at the trans-synaptic interface.","version":"1.1","doi":"10.1101/2022.09.13.507484","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.507842","pub_date":"2022-9-14","title":"Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants","abstract":"There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.","version":"1.1","doi":"10.1101/2022.09.14.507842","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.13.507781","pub_date":"2022-9-14","title":"The landscape of antibody binding affinity in SARS-CoV-2 Omicron BA.1 evolution","abstract":"The Omicron BA.1 variant of SARS-CoV-2 escapes convalescent sera and monoclonal antibodies that are effective against earlier strains of the virus. This immune evasion is largely a consequence of mutations in the BA.1 receptor binding domain (RBD), the major antigenic target of SARS-CoV-2. Previous studies have identified several key RBD mutations leading to escape from most antibodies. However, little is known about how these escape mutations interact with each other and with other mutations in the RBD. Here, we systematically map these interactions by measuring the binding affinity of all possible combinations of these 15 RBD mutations (215 = 32,768 genotypes) to four monoclonal antibodies (LY-CoV016, LY-CoV555, REGN10987, and S309) with distinct epitopes. We find that BA.1 can lose affinity to diverse antibodies by acquiring a few large-effect mutations and can reduce affinity to others through several small-effect mutations. However, our results also reveal alternative pathways to antibody escape that do not include every large-effect mutation. Moreover, epistatic interactions are shown to constrain affinity decline in S309 but only modestly shape the affinity landscapes of other antibodies. Together with previous work on the ACE2 affinity landscape, our results suggest that escape of each antibody is mediated by distinct groups of mutations, whose deleterious effects on ACE2 affinity are compensated by another distinct group of mutations (most notably Q498R and N501Y).","version":"1.1","doi":"10.1101/2022.09.13.507781","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.13.507876","pub_date":"2022-9-14","title":"Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test","abstract":"Neutralization assays are important in understanding and quantifying neutralizing antibody responses towards SARS-CoV-2. The SARS-CoV-2 Lentivirus Surrogate Neutralization Assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable, alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and pre-screening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity produced acceptable results with 100% (95% CI: 94-100) specificity and 100% (95% CI: 94-100) sensitivity against ancestral Wuhan spike pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike pseudotyped lentivirus resulted in 88.3% (95% CI: 77.8 to 94.2) and 100% (95% CI: 94-100), respectively. Assay precision measuring intra-assay variability produced acceptable results for High (1:\u2265 640 PRNT50), Mid (1:160 PRNT50) and Low (1:40 PRNT50) antibody titer concentration ranges based on the PRNT50, with %CV of 14.21, 12.47, and 13.28 respectively. Intermediate precision indicated acceptable ranges for the High and Mid concentrations, with %CV of 15.52 and 16.09, respectively. However, the Low concentration did not meet the acceptance criteria with a %CV of 26.42. Acceptable ranges were found in the robustness evaluation for both intra-assay and inter-assay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT.","version":"1.1","doi":"10.1101/2022.09.13.507876","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.13.507833","pub_date":"2022-9-14","title":"Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses","abstract":"Identifying host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of coronavirus disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify pro-viral host factors for highly pathogenic human coronaviruses. Very few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was completely unknown, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and regulates cell proliferation, and neuronal development, among other cellular processes. Interestingly, individuals with Down syndrome overexpress DYRK1A 1.5-fold and exhibit 5-10x higher hospitalization and mortality rates from COVID-19 infection. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and MERS-CoV entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the pro-viral activity of DYRK1A is conserved across species using cells of monkey and human origin and an in vivo mouse model. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses. Whether DYRK1A overexpression contributes to heightened COVID-19 severity in individuals with Down syndrome through ACE2 regulation warrants further future investigation.","version":"1.1","doi":"10.1101/2022.09.13.507833","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.14.507947","pub_date":"2022-9-14","title":"Structure and Epitope of a Neutralizing Monoclonal Antibody that Targets the Stem Helix of \u03b2 Coronaviruses","abstract":"Monoclonal antibodies (MAbs) that retain neutralizing activity against distinct coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1\u00c5 resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743-749, which interact with the C-terminal end of the SH helix in the S postfusion conformation. The crystal structure shows that BNMAb 1249A8 disrupts SH secondary structure and packing rearrangements required for CoV S to adopt its prefusion conformation that mediates membrane fusion and ultimately infection. The mechanisms regulating BNMAb 1249A8 CoV S specificity are also defined. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.","version":"1.1","doi":"10.1101/2022.09.14.507947","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.507647","pub_date":"2022-9-13","title":"Humoral immunogenicity of a Coronavirus Disease 2019 (COVID-19) DNA Vaccine in Rhesus Macaques (Macaca mulatta) Delivered using Needle-free Jet Injection","abstract":"A SARS-CoV-2 DNA vaccine targeting the spike protein and delivered by jet injection, nCOV-S(JET), previously shown to protect wild-type and immunosuppressed Syrian hamsters (Mesocricetus auratus), was evaluated via two needle-free delivery methods in rhesus macaques (Macaca mulatta). The methods included intramuscular delivery of 2 mg per vaccination with the PharmaJet Stratis device and intradermal delivery of 0.4 mg per vaccination with the PharmaJet Tropis device. We hypothesized that the nCOV-S(JET) vaccine would mount detectable neutralizing antibody responses when delivered by needle-free jet injection by either the intradermal or intramuscular route. When delivered intramuscularly, the vaccines elicited neutralizing and variant (Beta, Gamma, and Delta) cross-neutralizing antibodies against SARS-CoV-2 in all six animals after three vaccinations. When delivered at a lower dose by the intradermal route, strong neutralizing antibody responses were only detected in two of six animals. This study confirms that a vaccine previously shown to protect in a hamster model can elicit neutralizing and cross-neutralizing antibodies against SARS-CoV-2 in nonhuman primates. We posit that nCOV-S(JET) has the potential for use as booster vaccine in heterologous vaccination strategies against COVID-19.","version":"1.1","doi":"10.1101/2022.09.12.507647","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.507671","pub_date":"2022-9-13","title":"Evaluation of endogenous and therapeutic 25-hydroxycholesterols in murine models of pulmonary SARS-CoV-2 infection","abstract":"Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7\u03b1,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo. 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h\u2212/\u2013 and Gpr183\u2212/\u2013 mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.","version":"1.1","doi":"10.1101/2022.09.12.507671","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507373","pub_date":"2022-9-13","title":"Does Vaping Increase the Likelihood of SARS-CoV-2 Infection? Paradoxically Yes and No","abstract":"Data on the relationship between electronic cigarettes (ECs) and SARS-CoV-2 infection are limited and contradictory. Evidence indicates that EC aerosols or nicotine increase ACE2, SARS-CoV-2 virus receptors, which increase virus binding and susceptibility. Our objectives were to determine if EC aerosols increased SARS-CoV-2 infection of human bronchial epithelial cells and to identify the causative chemical(s). A 3D organotypic model (EpiAirway\u2122) in conjunction with air liquid interface (ALI) exposure was used to test the effects of aerosols produced from JUUL\u2122 \u201cVirginia Tobacco\u201d and BLU\u2122 ECs, or individual chemicals (nicotine, propylene glycol, vegetable glycerin (PG/VG), and benzoic acid) on infection using SARS-CoV-2 pseudoparticles. Exposure of EpiAirway\u2122 to JUUL\u2122 aerosols increased ACE2, while BLU\u2122 and lab-made EC aerosols containing nicotine increased ACE2 levels and TMPRSS2 activity, a spike protease that enables viral-cell fusion. Pseudoparticle infection of EpiAirway\u2122 increased with aerosols produced from PG/VG, PG/VG plus nicotine, or BLU\u2122 ECs. JUUL\u2122 EC aerosols did not increase infection above controls. The baseline level of infection in JUUL\u2122 treated aerosol groups was attributed to benzoic acid, which mitigated the enhanced infection caused by PG/VG or nicotine. The benzoic acid protection from enhanced infection continued at least 48 hours after exposure. TMPRSS2 activity was significantly correlated with e-liquid pH, which in turn was significantly correlated with infection, with lower pH blocking PG/VG and nicotine-induced-enhanced infection. While ACE2 levels increased in EpiAirway\u2122 tissues exposed to EC aerosols, infection depended on the ingredients of the e-liquids. PG/VG and nicotine enhanced infection, an effect that was mitigated by benzoic acid.","version":"1.1","doi":"10.1101/2022.09.09.507373","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.505486","pub_date":"2022-9-13","title":"High throughput Bioluminescent assay to characterize and monitor the activity of SARS-CoV-2 Methyltransferases","abstract":"The fast rate of viral mutations of SARS CoV-2 result in decrease in the efficacy of the vaccines that have been developed before the emergence of these mutations. Thus, it is believed that using additional measures to combat the virus is not only advisable but also beneficial. Two antiviral drugs were authorized for emergency use by the FDA, namely Pfizer\u2019s two-drug regimen sold under the brand name Paxlovid, and Merck\u2019s drug Lagevrio. Pfizer\u2019s two-drug combination consists of nirmatrelvir, a protease inhibitor that blocks coronavirus ability to multiply and another antiviral, ritonavir, that lowers the rate of drug clearance to boost the longevity and activity of the protease inhibitor. Merck\u2019s drug Lagevrio (molnupiravir) is a nucleoside analogue with a mechanism of action that aims to introduce errors into the genetic code of the virus. We believe the armament against the virus can be augmented by the addition of another class of enzyme inhibitors that are required for viral survival and its ability to replicate. Enzymes like nsp14 and nsp10/16 methyltransferases represent another class of drug targets since they are required for viral RNA translation and evading the host immune system. In this communication, we have successfully verified that the Methyltransferase Glo, which is universal and homogeneous methyltransferase assay can be used to screen for inhibitors of the two pivotal enzymes nsp14 and nsp16 of SARS CoV-2. Furthermore, we have carried out extensive studies on those enzymes using different RNA substrates and tested their activity using various inhibitors and verified the utility of this assay for use in drug screening programs. We anticipate our work will be pursued further to screen for large libraries to discover new and selective inhibitors for the viral enzymes particularly that these enzymes are structurally different from their mammalian counterparts.","version":"1.1","doi":"10.1101/2022.09.12.505486","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.507614","pub_date":"2022-9-13","title":"Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant","abstract":"The emergence of SARS-CoV-2 variants in the Omicron lineage with large numbers of substitutions in the spike protein that can evade antibody neutralization has resulted in diminished vaccine efficacy and persistent transmission. One strategy to broaden vaccine-induced immunity is to administer bivalent vaccines that encode for spike proteins from both historical and newly-emerged variant strains. Here, we evaluated the immunogenicity and protective efficacy of two bivalent vaccines that recently were authorized for use in Europe and the United States and contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary immunization series in BALB/c mice, both bivalent vaccines induced broader neutralizing antibody responses than the constituent monovalent vaccines (mRNA-1273 [Wuhan-1], mRNA-1273.529 [BA.1], and mRNA-1273-045 [BA.4/5]). When administered to K18-hACE2 transgenic mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced greater breadth and magnitude of neutralizing antibodies compared to an mRNA-1273 booster. Moreover, the response in bivalent vaccine-boosted mice was associated with increased protection against BA.5 infection and inflammation in the lung. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and protection against currently circulating SARS-CoV-2 strains.","version":"1.1","doi":"10.1101/2022.09.12.507614","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.506773","pub_date":"2022-9-13","title":"Monitoring correlates of SARS-CoV-2 infection in cell culture using two-photon microscopy and a novel fluorescent calcium-sensitive dye","abstract":"The organism-wide effects of viral infection SARS-CoV-2 are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. Two-photon (2P) imaging combined with a proper subcellular staining technique has been an effective tool for studying mechanisms at such resolutions and organelle levels. Herein, we report the development of a novel calcium sensor molecule along with a 2P-technique for identifying imaging patterns associated with cellular correlates of infection damage within the cells. The method works as a cell viability assay and also provides valuable information on how the calcium level and intracellular distribution are perturbed by the virus. Moreover, it allows the quantitative analysis of infection dynamics. This novel approach facilitates the study of the infection progression and the quantification of the effects caused by viral variants and viral load.","version":"1.1","doi":"10.1101/2022.09.12.506773","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.12.507666","pub_date":"2022-9-13","title":"Comparative multi-OMICS single cell atlas of five COVID-19 (rAdVV and mRNA) vaccines describe unique and distinct mechanisms of action","abstract":"COVID-19 vaccines based on a range of expression platforms have shown considerable protective efficacy, generating antibody and T cell immune responses. However, molecular pathways underpinning COVID-19 vaccine priming of immunity against the SARS-CoV-2 virus have not yet been explored extensively. This analysis is critical to optimization of future vaccination strategies, schedules, and combinations. Thus, we investigated a cohort of individuals pre- and post-vaccination to understand the humoral and cellular immune response against different COVID-19 vaccines, including recombinant adenoviral vector (rAdVV) and mRNA-based vaccines. Single-cell RNA sequencing allowed characterization of monocytes, T, NK and B cell activation at the transcriptomics/proteomic level, in response to different COVID-19 vaccines. Our data revealed that different COVID-19 vaccines elicit a unique and distinct mechanism of action. Specifically, we revealed that rAdVV vaccines negatively regulate CD4+ T cell activation, leukocytes chemotaxis, IL-18 signalling and antigen presentation by monocytes whilst mRNA vaccines positively regulate NKT cell activation, platelets activation and chemokine signalling pathways. An antigen-specific T cell response was already observed following the 1st vaccine dose and was not further augmented after the subsequent 2nd dose of the same vaccine and it was dependent on the type of vaccination used. Our integrated three layered-analyses highlights that COVID-19 vaccines evoke a strong but divergent immune response at the RNA, protein, and cellular levels. Our approach is able to pinpoint efficacy and mechanisms controlling immunity to vaccination and open the door for better vaccination which could induce innate and adaptive immunity equally in the long term. Decrease in major three cell types classical and non-classical monocytes and NK type III cells after COVID-19 vaccination Individual vaccination (AZ, JJ, MD, PB) has differential effect on various immune cell subsets and regulates unique cell populations, whilst no change was observed for CV vaccination rAdVV and mRNA vaccines have different mechanism of action for activation of lymphocytes and monocytes, respectively rAdVV vaccines negatively regulates CD4+ T cell activation, leukocytes chemotaxis, IL-18 signalling and antigen presentation whilst mRNA vaccines positively regulate NKT cell activation, platelets activation and chemokine signalling pathways. An antigen-specific T cell response was prompted after the 1st vaccine dose and not augmented after the subsequent 2nd dose of the same vaccine.","version":"1.1","doi":"10.1101/2022.09.12.507666","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507349","pub_date":"2022-9-12","title":"The SARS-CoV-2 Spike Protein Mutation Explorer: Using an Interactive Application to Improve the Public Understanding of SARS-CoV-2 Variants of Concern","abstract":"SARS-CoV-2 is the virus responsible for the COVID-19 pandemic, which began in late 2019 and has resulted in millions of death globally. The need to understand the pandemic means that detailed descriptions of features of this virus are now of interest to non-expert audiences. In particular, there has been much public interest in the spike protein that protrudes from the surface of the SARS-CoV-2 virus particle. The spike is the major determinant of viral infectivity and the main target for protective immune responses, and included in vaccines, and so its properties influence the impact of the pandemic on people\u2019s lives. This protein is rapidly evolving, with mutations that enhance transmissibility or weaken vaccine protection creating new variants of concern (VOCs) and associated sub-lineages. The spread of SARS-CoV-2 VOCs has been tracked by groups such as the COVID-19 Genomics UK consortium (COG-UK). Their online mutation explorer (COG-UK/ME), which analyses and shares SARS-CoV-2 sequence data, contains information about VOCs that is designed primarily for an expert audience but is potentially of general interest during a pandemic. We wished to make this detailed information about SARS-CoV-2 VOCs more widely accessible. Previously work has shown that visualisations and interactivity can facilitate active learning and boost engagement with molecular biology topics, while animations of these topics can boost understanding on protein structure, function, and dynamics. We therefore set out to develop an educational graphical resource, the SARS-CoV-2 Spike Protein Mutation Explorer (SSPME), which contains interactive 3D molecular models and animations explaining SARS-CoV-2 spike protein variants and VOCs. We performed user-testing of the original COG-UK/ME website and of the SSPME, using a within-groups design to measure knowledge acquisition and a between-groups design to contrast the effectiveness and usability. Statistical analysis demonstrated that, when compared to the COG-UK/ME, the SSPME had higher usability and significantly improved participant knowledge confidence and knowledge acquisition. The SSPME therefore provides an example of how 3D interactive visualisations can be used for effective science communication and education on complex biomedical topics, as well as being a resource to improve the public understanding of SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2022.09.09.507349","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.13.476267","pub_date":"2022-9-12","title":"Modelling how the altered usage of cell entry pathways by the SARS-CoV-2 Omicron variant may affect the efficacy and synergy of TMPRSS2 and Cathepsin B/L inhibitors","abstract":"The SARS-CoV-2 Omicron variant harbours mutations in its spike protein, which may affect its cell entry, tropism, and response to interventions. To elucidate these effects, we developed a mathematical model of SARS-CoV-2 entry into cells and applied it to analyse recent in vitro data. SARS-CoV-2 enters cells using host proteases, either Cathepsin B/L or TMPRSS2. We estimated >4-fold increase and >3-fold decrease in entry efficiency using Cathepsin B/L and TMPRSS2, respectively, of the Omicron variant relative to the original or other strains in a cell type-dependent manner. Our model predicted that Cathepsin B/L inhibitors would be more and TMPRSS2 inhibitors less efficacious against the Omicron than the original strain. Furthermore, the two inhibitor classes would exhibit synergy, although the drug concentrations maximizing synergy would have to be tailored to the Omicron variant. These findings provide insights into the cell entry mechanisms of the Omicron variant and have implications for interventions.","version":"1.3","doi":"10.1101/2022.01.13.476267","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507363","pub_date":"2022-9-11","title":"Treatment with anti-inflammatory viral serpin modulates immuno-thrombotic responses and improves outcomes in SARS-CoV-2 infected mice","abstract":"Severe acute respiratory distress syndrome (ARDS) during SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infection, manifests as uncontrolled lung inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies can reduce COVID-19 severity if administered in the early viremic phase, but treatments for later stage immuno-thrombotic syndrome and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases during thrombotic, thrombolytic and immune responses. The myxoma poxvirus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated coagulation and complement protease pathways as part of a self-defense strategy to combat viral clearance by the innate immune system. When purified and utilized as an anti-immune therapeutic, Serp-1 is effective as an anti-inflammatory drug in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 (PEGSerp-1) as a therapy for immuno-thrombotic complications during ARDS. Treatment with PEGSerp-1 in two distinct mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved clinical outcomes. PEGSerp-1 significantly reduced M1 macrophage invasion in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR) and complement membrane attack complex (MAC). Sequential changes in urokinase-type plasminogen activator receptor (uPAR) and serpin gene expression were observed in lung and heart with PEGSerp-1 treatment. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for treatment of severe viral ARDS with additional potential to reduce late SARS-CoV-2 complications related to immune-thrombotic events that persist during long COVID. Severe acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection manifests as uncontrolled tissue inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies reduce COVID-19 severity if administered early, but treatments for later stage immuno-thrombosis are limited. Serine protease inhibitors (SERPINS) regulate thrombotic, thrombolytic and complement pathways. We investigate here systemic treatment with purified poxvirus-derived PEGSerp-1 as a therapeutic for immuno-thrombotic complications in viral ARDS. PEGSerp-1 treatment in two mouse-adapted SARS-CoV-2 models (C57Bl/6 and BALB/c) significantly reduced lung and heart inflammation and improved clinical outcomes, with sequential changes in thrombolytic (uPAR) and complement expression. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for immune-thrombotic complications in severe viral ARDS and has potential benefit for long COVID.","version":"1.1","doi":"10.1101/2022.09.09.507363","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507342","pub_date":"2022-9-11","title":"Targeting intracellular Neu1 for Coronavirus Infection Treatment","abstract":"There are no effective therapies for COVID-19 or antivirals against SARS-CoV-2. Furthermore, current vaccines appear less efficacious for new SARS-CoV-2 variants. Thus, there is an urgent need to better understand the virulence mechanisms of SARS-CoV-2 and the host response to develop therapeutic agents. Here, we show host Neu1 regulates coronavirus replication by controlling sialylation on coronavirus nucleocapsid protein. Coronavirus nucleocapsid proteins in COVID-19 patients and in coronavirus HCoV-OC43-infected cells were heavily sialylated; this sialylation controlled the RNA binding activity and replication of coronavirus. Neu1 overexpression increased HCoV-OC43 replication, whereas Neu1 knockdown reduced HCoV-OC43 replication. Moreover, a newly developed Neu1 inhibitor, Neu5Ac2en-OAcOMe, selectively targeted intracellular sialidase, which dramatically reduced HCoV-OC43 and SARS-CoV-2 replication in vitro and rescued mice from HCoV-OC43 infection-induced death. Our findings suggest that Neu1 inhibitors could be used to limit SARS-CoV-2 replication in patients with COVID-19, making Neu1 a potential therapeutic target for COVID-19 and future coronavirus pandemics.","version":"1.1","doi":"10.1101/2022.09.09.507342","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.08.507221","pub_date":"2022-9-09","title":"Use of the particle agglutination/particle agglutination-inhibition test for antigenic analysis of SARS-CoV-2","abstract":"The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination-inhibition (HA/HI) tests are well-known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 is unlikely to agglutinate to human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated enzyme-linked immunosorbent assay (ELISA) or cell-based assays such as the microneutralization assay. In this study, we developed the particle agglutination/particle agglutination-inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The PA titer was positively correlated with the plaque-forming units. The PAI titer using post-infection Syrian hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The results show the PAI test is useful for easy and rapid antigenic analysis of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.09.08.507221","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507250","pub_date":"2022-9-09","title":"Mucosal vaccination for SARS-CoV-2 elicits superior systemic T central memory function and cross-neutralizing antibodies against variants of concern","abstract":"COVID-19 vaccines used in humans are highly effective in limiting disease and death caused by the SARS-CoV-2 virus, yet improved vaccines that provide greater protection at mucosal surfaces, which could reduce break-through infections and subsequent transmission, are still needed. Here we show that intranasal (I.N.) vaccination with the receptor binding domain of Spike antigen of SARS-CoV-2 (S-RBD) in combination with the mucosal adjuvant mastoparan-7 improved systemic T cell responses compared to an equivalent dose of antigen delivered by the sub-cutaneous (S.C.) route, adjuvanted by either M7 or the gold-standard adjuvant, alum. T cell phenotypes induced by I.N. vaccine administration included enhanced polyfunctionality (combined IFN-\u03b3 and TNF expression) and greater numbers of T central memory (TCM) cells. These phenotypes were T cell-intrinsic and could be recalled in the lungs and/or brachial LNs upon antigen challenge after adoptive T cell transfer to na\u00efve recipients. Furthermore, mucosal vaccination induced antibody responses that were similarly effective in neutralizing the binding of the parental strain of S-RBD to its ACE2 receptor, but showed greater cross-neutralizing capacity against multiple variants of concern (VOC), compared to S.C. vaccination. These results highlight the role of nasal vaccine administration in imprinting an immune profile associated with long-term T cell retention and diversified neutralizing antibody responses, which could be applied to improve vaccines for COVID-19 and other infectious diseases.","version":"1.1","doi":"10.1101/2022.09.09.507250","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507307","pub_date":"2022-9-09","title":"A new and highly effective method for predicting T-cell response targets implemented on SARS-CoV-2 data","abstract":"Computational T-cell epitope prediction is essential in many immunological projects, including the development of vaccines. T-cells of immunocompetent vertebrate hosts can recognize as non-self only peptides which are present in the parasite\u2019s proteins and absent in the host\u2019s proteins. This basic principle allows us to predict which peptides can elicit T-cells\u2019 response. We built on the fact that the specificity of T-cells reacting to SARS-CoV-2 antigens has been recently mapped in detail. Using Monte Carlo tests, we found that empirically confirmed peptides that stimulate T-cells contain an increased fraction of pentapeptides, hexapeptides, and heptapeptides which are not found in the human proteome (p < 0.0001). Similarly, hexapeptides absent in human proteins were overrepresented in peptides that elicited T-cell response in a published empirical study (p = 0.027). The new theory-based method predicted T-cell immunogenicity of SARS-CoV-2 peptides four times more effectively than current empirically based methods.","version":"1.1","doi":"10.1101/2022.09.09.507307","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507133","pub_date":"2022-9-09","title":"SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction","abstract":"Processing of polyproteins pp1a and pp1ab in Coronaviruses by main protease Mpro is a crucial event in virus replication and a promising target for antiviral drug development. Mpro recognizes multiple recognition sites within polyproteins in a defined order, but its mechanism remains enigmatic due to lack of structural information of the polyprotein substrate bound Mpro complex. Here, we present the cryo-EM structures of the SARS-CoV-2 Mpro in an apo-form and in complex with the nsp7-10 region of pp1a polyprotein. The structure shows that the interaction of Mpro with polyproteins is limited to the recognition site connecting nsp9 and nsp10 proteins without any tight association with the rest of polyprotein structure or sequence. Comparison between the apo-form and the polyprotein bound structures of Mpro highlights the flexible nature of active site region allowing the Mpro to accommodate various recognition sites connecting series of nsp proteins. These observations suggest that the role of Mpro for selecting a preferred cleavage site within the polyprotein is limited and underscore the structure, conformation and/or dynamics of polyprotein determining the sequential polyprotein cleavage by Mpro.","version":"1.1","doi":"10.1101/2022.09.09.507133","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507257","pub_date":"2022-9-09","title":"Cellular stress modulates severity of the acute respiratory distress syndrome in COVID-19","abstract":"Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we demonstrate that cell surface BiP (cs-BiP) responds by increasing its levels in leukocytes. Neutrophiles show the highest levels of cs-BiP and respond by increasing their population, whereas alveolar macrophages increase their levels of cs-BiP. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.","version":"1.1","doi":"10.1101/2022.09.09.507257","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.09.507303","pub_date":"2022-9-09","title":"Expression profiling of immune-response genes common in SARS-CoV-1 and influenza A virus disease, viz-a-viz neutropenia disorder, using integrated bioinformatics tools","abstract":"Viral diseases have proven to be an existential threat to man due to its fatality and its consistent emergence and re-emergence, so the need for novel ideas in combating this menace. Advances in genetic studies has proven to be indispensable in this fight as knowledge of organismal genetic variability has been useful in therapy development, as well as in mounting defense against the treacherous infectious agents. However, there are still fallow grounds needing to be explored in this war which informed this study, with focus on expression profiling of similar immune-responsegenes in SARS-COV-1 and influenza A virus (AIV) in respect to neutropenia (NP), using integrated bioinformatics techniques such as extraction of microarray dataset in the gene expression omnibus (GEO) database, generation of DEGs using GEO2R tool, construction of protein-protein interaction (PPI) network using string and Cytoscape software, Venn diagram analysis and then gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) for enrichment and pathway analysis respectively. Ten genes which includes ELANE, ITGA2B, CXCR1, CSF3R, SPI1, MS4A3, MMP8, CEACAM8, RNASE3, and DEFA4 were identified, with ELANE gene moreover identified as a key gene, which might be responsible in the regulation of immune response during viral infection, based on its characteristic feature in the PPI network.","version":"1.1","doi":"10.1101/2022.09.09.507303","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.07.506919","pub_date":"2022-9-08","title":"A clinical stage LMW-DS drug inhibits cell infection by coronaviruses and modulates reactive cytokine release from microglia","abstract":"Most coronaviruses infect animals including bats, birds and mammals, which act as hosts and reservoirs for the viruses, but the viruses can sometimes move host species and infect humans. Coronoviruses were first identified as human pathogens in the 1960s and now there are seven types known to infect humans. Whilst four of these types cause mild-to-moderate respiratory disease, the other three may cause more severe and possibly even fatal disease in vulnerable individuals particularly, with the most recent SARS-CoV-2 pandemic being associated with severe acute respiratory syndrome (SARS) in many infected people. The aim of the present study was to evaluate the potential of a unique low molecular weight dextran sulphate (LMW-DS) clinical stage drug, ILB\u00ae, to inhibit infection of human cells by the NL63 coronavirus assessed by immunofluorescence of viral particles, and also to see if the drug directly blocked the interaction of the SARS-CoV-2 viral spike protein with the ACE2 receptor. Furthermore, we evaluated if ILB\u00ae could modulate the downstream consequences of viral infection including the reactive cytokine release from human microglia induced by various SARS-CoV-2 variant spike proteins. We demonstrated that ILB\u00ae blocked ACE2:spike protein interaction and inhibited coronaviral infection. ILB\u00ae also attenuated the omicron-induced release of pro-inflammatory cytokines, including TNF\u03b1, from human microglia, indicating control of post-viral neuroinflammation. In conclusion, given the safety profile of ILB\u00ae established in a number of Phase I and Phase II clinical trials, these results highlight the potential of ILB\u00ae to treat patients infected with coronaviruses to both limit infectivity and attenuate the progression to severe disease. There is now an opportunity to translate these findings quickly by the clinical investigation of drug efficacy.","version":"1.1","doi":"10.1101/2022.09.07.506919","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.06.506768","pub_date":"2022-9-07","title":"MultiOMICs landscape of SARS-CoV-2-induced host responses in human lung epithelial cells","abstract":"Despite the availability of vaccines and approved therapeutics, the COVID-19 pandemic continues to rise owing to the emergence of newer variants. Several multi-omics studies have made available extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the biochemical and cellular dynamics is critical to expanding the current knowledge on the host response to SARS-CoV-2 infections. Here, employing unbiased global transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We provide evidence for the interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies.","version":"1.1","doi":"10.1101/2022.09.06.506768","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.05.506640","pub_date":"2022-9-07","title":"Future COVID19 surges prediction based on SARS-CoV-2 mutations surveillance","abstract":"COVID19 has aptly revealed that airborne viruses such as SARS-CoV-2 with the ability to rapidly mutate, combined with high rates of transmission and fatality can cause a deadly world-wide pandemic in a matter of weeks. Apart from vaccines and post-infection treatment options, strategies for preparedness will be vital in responding to the current and future pandemics. Therefore, there is wide interest in approaches that allow predictions of increase in infections (\u201csurges\u201d) before they occur. We describe here real time genomic surveillance particularly based on mutation analysis, of viral proteins as a methodology for a priori determination of surge in number of infection cases. The full results are available for SARS-CoV-2 at http://pandemics.okstate.edu/covid19/, and are updated daily as new virus sequences become available. This approach is generic and will also be applicable to other pathogens.","version":"1.1","doi":"10.1101/2022.09.05.506640","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.07.506878","pub_date":"2022-9-07","title":"Immunogenicity and Protective Efficacy of a SARS-CoV-2 mRNA Vaccine Encoding Secreted Non-Stabilized Spike Protein in Mice","abstract":"Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP) \u201cChulaCov19\u201d. In BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 \u03bcg given 2 doses, 21 days apart, elicited robust neutralizing antibody (NAb) and T cells responses in a dose-dependent relationship. The geometric mean titer (GMT) of micro-virus neutralizing (micro-VNT) antibody against wild-type virus was 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induced better cross-neutralizing antibody against Delta and Omicron variants. This elicited specific immunogenicity was significantly higher than those induced by homologous prime-boost with inactivated (CoronaVac) or viral vector (AZD1222) vaccine. In heterologous prime-boost study, mice primed with either CoronaVac or AZD1222 vaccine and boosted with 5 \u03bcg ChulaCov19 generated NAb 7-fold higher against wild-type virus (WT) and was also significantly higher against Omicron (BA.1 and BA.4/5) than homologous CoronaVac or AZD1222 vaccination. AZD1222-prime/mRNA-boost had mean spike-specific IFN\u03b3 positive T cells of 3,725 SFC/106 splenocytes, which was significantly higher than all groups except homologous ChulaCov19. Challenge study in human-ACE-2-expressing transgenic mice showed that ChulaCov19 at 1 \u03bcg or 10 \u03bcg protected mice from COVID-19 symptoms, prevented SARS-CoV-2 viremia, significantly reduced tissue viral load in nasal turbinate, brain, and lung tissues 99.9-100%, and without anamnestic of Ab response which indicated its protective efficacy. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or a boost vaccination and has entered clinical development.","version":"1.1","doi":"10.1101/2022.09.07.506878","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.05.506622","pub_date":"2022-9-07","title":"A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile","abstract":"Cellular immune defects associated with suboptimal responses to SARS-CoV-2 mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyzed antibody, B cell, CD4+ and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CI). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses and enhances comparatively more Thelper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (TNF\u03b1/IL-2 skewing), while others (CCR6, CXCR6, PD-1 and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieve robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.","version":"1.1","doi":"10.1101/2022.09.05.506622","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.05.506628","pub_date":"2022-9-06","title":"Resistance of SARS-CoV-2 Omicron Subvariant BA.4.6 to Antibody Neutralization","abstract":"SARS-CoV-2 Omicron subvariants BA.4.6, BA.4.7, and BA.5.9 have recently emerged, and BA.4.6 appears to be expanding even in the presence of BA.5 that is globally dominant. Compared to BA.5, these new subvariants harbor a mutation at R346 residue in the spike glycoprotein, raising concerns for further antibody evasion. We compared the viral receptor binding affinity of the new Omicron subvariants with BA.5 by surface plasmon resonance. We also performed VSV-based pseudovirus neutralization assays to evaluate their antigenic properties using sera from individuals who received three doses of a COVID-19 mRNA vaccine (boosted) and patients with BA.1 or BA.2 breakthrough infection, as well as using a panel of 23 monoclonal antibodies (mAbs). Compared to the BA.5 subvariant, BA.4.6, BA.4.7, and BA.5.9 showed similar binding affinities to hACE2 and exhibited similar resistance profiles to boosted and BA.1 breakthrough sera, but BA.4.6 was slightly but significantly more resistant than BA.5 to BA.2 breakthrough sera. Moreover, BA.4.6, BA.4.7, and BA.5.9 showed heightened resistance over to a class of mAbs due to R346T/S/I mutation. Notably, the authorized combination of tixagevimab and cilgavimab completely lost neutralizing activity against these three subvariants. The loss of activity of tixagevimab and cilgavimab against BA.4.6 leaves us with bebtelovimab as the only therapeutic mAb that has retained potent activity against all circulating forms of SARS-CoV-2. As the virus continues to evolve, our arsenal of authorized mAbs may soon be depleted, thereby jeopardizing the wellbeing of millions of immunocompromised persons who cannot robustly respond to COVID-19 vaccines.","version":"1.1","doi":"10.1101/2022.09.05.506628","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.05.22279572","pub_date":"2022-09-06","title":"Distinct immune signatures discriminate SARS-CoV-2 vaccine combinations","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Several vaccines have been found effective against COVID-19, usually administered in homologous regimens, with the same vaccine used for the prime and boost doses. However, recent studies have demonstrated improved protection via heterologous mix-and-match COVID-19 vaccine combinations, and a direct comparison among these regimens is needed to identify the best employment strategies. Here, we show a single-cohort comparison of changes to the humoral and cellular immune compartments following five different COVID-19 vaccines spanning three technologies (adenoviral, mRNA and inactivated vaccines). These vaccines were administered in a combinatorial fashion, resulting in sixteen different homologous and heterologous regimens. SARS-CoV-2-targeting antibody titres were highest when the boost dose consisted of mRNA-1273, independent of the vaccine used for priming. Priming with BBIBP-CorV induced less class-switching among spike-binding memory B cells and the highest antigen-specific T cell responses in heterologous combinations. These were generally more immunogenic in terms of specific antibodies and cellular responses compared to homologous regimens. Finally, single-cell analysis of 754 samples revealed specific B and T cell signatures of the vaccination regimens, indicating distinctive differences in the immune responses. These data provide new insights on the immunological effects of COVID-19 vaccine combinations and a framework for the design of improved vaccination strategies for other pathogens and cancer.</jats:p>","version":null,"doi":"10.1101/2022.09.05.22279572","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.28.497978","pub_date":"2022-9-06","title":"Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir","abstract":"The SARS-CoV-2 main protease (Mpro) is the drug target of Pfizer\u2019s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in Mpro raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring Mpro mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (kcat/Km <10-fold change) and resistance to nirmatrelvir (Ki >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that Mpro mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance. Paxlovid resistant SARS-CoV-2 viruses with mutations in the main protease have been identified from clinical isolates.","version":"1.2","doi":"10.1101/2022.06.28.497978","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.05.506626","pub_date":"2022-9-06","title":"Immune dynamics at single cell protein level after delta/omicron infection in COVID-19 vaccinated convalescent individuals","abstract":"Both COVID-19 mRNA or recombinant Adenovirus vector (rAdVV) based vaccines have shown a great efficacy in generating humoral and cellular immune responses. Two doses of the COVID-19 vaccines generate enough antibodies and generate spike-specific T cell responses. However, after 6-8 months there is a decline in antibody production and T cell responses. Due to the rise of new SARS-CoV-2 variants of concern, a third or even fourth dose of vaccine was recommended for the elderly, immune comprised and frontline medical health care workers. However, despite additional booster doses given, those who were infected with either delta or omicron (during December 2021 \u2013 March 2022) had symptoms of illness. By what means these COVID-19 vaccines provide immunity against the SARS-CoV-2 virus at the molecular level is not explored extensively yet and, it is an emerging research field as to how the SARS-CoV-2 virus is able to evade the host immunity. Most of the infected people had mild symptoms whilst some were asymptomatic. Many of the people had developed nucleocapsid antibodies against the SARS-CoV-2 delta/omicron variants confirming a humoral immune response against viral infection. Furthermore, cellular analysis shows that post-vaccinated recovered COVID-19 individuals have significantly reduced NK cells and increased T na\u00efve CD4+, TEM CD8+ and B cells. This decrease in cellular immunity corresponds to individuals who recovered from alpha variants infection and had mild symptoms. Our results highlight that booster doses clearly reduce the severity of infection against delta/omicron infection. Furthermore, our cellular and humoral immune system is trained by vaccines and ready to deal with breakthrough infections in the future.","version":"1.1","doi":"10.1101/2022.09.05.506626","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.06.506814","pub_date":"2022-9-06","title":"Climate change has affected the spillover risk of bat-borne pathogens","abstract":"Bat-borne viruses are a threat to global health and have in recent history had major impacts to human morbidity and mortality. Examples include diseases such as rabies, Ebola, SARS-Cov-1, and SARS-Cov-2 (COVID-19). Climate change could exacerbate the emergence of bat-borne pathogens by affecting the distribution and abundance of bats in tropical ecosystems. Here we report an assessment of historical climate and vampire bat occurrence data for the last century, which revealed a relationship between climatic variation and risk of disease spillover triggered by changes in bat distributions. This report represents one of the first examples of empirical evidence of global change effects on continental patterns of bat-borne pathogen transmission risk. We therefore recommend that more research is necessary on the impacts of climate change on bat-borne pathogen spillover risk, and that climate change impacts on bat-borne disease should be considered in global security initiatives. Bat-borne viruses are a threat to global health and include diseases such as rabies, Ebola, SARS-Cov-1, and SARS-Cov-2 (COVID-19). Climate change could exacerbate the emergence of bat-borne pathogens by affecting the distribution and abundance of bats. Here we report an assessment of historical climate and vampire-bat occurrence data for the last century, which reveals a relationship between climatic variation and risk of disease spillover triggered by changes in bat distributions.","version":"1.1","doi":"10.1101/2022.09.06.506814","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.05.506686","pub_date":"2022-9-06","title":"Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity","abstract":"Intradermal delivery of self-replicating RNA (srRNA) is a promising vaccine platform. Considering that human skin temperature is around 33\u00b0C, lower than core body temperature of 37\u00b0C, we have developed an srRNA that functions optimally at skin temperature and is inactivated at or above 37\u00b0C as a safety switch. This temperature-controllable srRNA (c-srRNA), when tested as an intradermal vaccine against SARS-CoV-2, functions when injected naked without lipid nanoparticles. Unlike most currently available vaccines, c-srRNA vaccines predominantly elicit cellular immunity with little or no antibody production. Interestingly, c-srRNA-vaccinated mice produced antigen-specific antibodies upon subsequent stimulation with antigen protein. Antigen-specific antibodies were also produced when B-cell stimulation using antigen protein was followed by c-srRNA booster vaccination. Using c-srRNA, we have designed a pan-coronavirus booster vaccine that incorporates both spike receptor binding domains as viral surface proteins and evolutionarily conserved nucleoproteins as viral non-surface proteins, from both SARS-CoV-2 and MERS-CoV. It can thereby potentially immunize against SARS-CoV-2, SARS-CoV, MERS-CoV, and their variants. c-srRNA may provide a route to activate cellular immunity against a wide variety of pathogens.","version":"1.1","doi":"10.1101/2022.09.05.506686","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.29.505743","pub_date":"2022-9-06","title":"Molecular fate-mapping of serum antibodies reveals the effects of antigenic imprinting on repeated immunization","abstract":"The ability of serum antibody to protect against pathogens arises from the interplay of antigen-specific B cell clones of different affinities and fine specificities. These cellular dynamics are ultimately responsible for serum-level phenomena such as antibody imprinting or \u201cOriginal Antigenic Sin\u201d (OAS), a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells that responded to a stimulus upon exposure to related antigens. Imprinting/OAS is thought to pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2. Precise measurement of the extent to which imprinting/OAS inhibits the recruitment of new B cell clones by boosting is challenging because cellular and temporal origins cannot readily be assigned to antibodies in circulation. Thus, the extent to which imprinting/OAS impacts the induction of new responses in various settings remains unclear. To address this, we developed a \u201cmolecular fate-mapping\u201d approach in which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that, upon sequential homologous boosting, the serum antibody response strongly favors reuse of the first cohort of B cell clones over the recruitment of new, na\u00cfve-derived B cells. This \u201cprimary addiction\u201d decreases as a function of antigenic distance, allowing secondary immunization with divergent influenza virus or SARS-CoV-2 glycoproteins to overcome imprinting/OAS by targeting novel epitopes absent from the priming variant. Our findings have implications for the understanding of imprinting/OAS, and for the design and testing of vaccines aimed at eliciting antibodies to evolving antigens.","version":"1.2","doi":"10.1101/2022.08.29.505743","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.06.506799","pub_date":"2022-9-06","title":"Omicron-induced interferon signalling prevents influenza A virus infection","abstract":"Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active type I (\u03b1/\u03b2) and III (\u03bb) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signalling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knock-out prevented it. The JAK/ STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/ STAT signalling. In conclusion, our study 1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, 2) shows that Omicron infection protects cells from influenza A virus super-infection, and 3) indicates that BA.1 and BA.5 induce comparable antiviral states.","version":"1.1","doi":"10.1101/2022.09.06.506799","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.03.506470","pub_date":"2022-9-05","title":"Optimization and deoptimization of codons in SARS-CoV-2 and the implications for vaccine development","abstract":"The spread of Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 coronavirus, has progressed into a global pandemic. To date, thousands of genetic variants have been identified across SARS-CoV-2 isolates from patients. Sequence analysis reveals that the codon usage of viral sequences decreased over time but fluctuated from time to time. In this study, through evolution modeling, we found that this phenomenon might result from the virus\u2019 preference for mutations during transmission. Using dual luciferase assays, we further discovered that the deoptimization of codons on viruses might weaken protein expression during the virus evolution, indicating that the choice of codon usage might play important role in virus fitness. Finally, given the importance of codon usage in protein expression and particularly for mRNA vaccine, we designed several omicron BA.2.12.1 and BA.4/5 spike mRNA vaccine candidates based on codon optimization, and experimentally validated their high levels of expression. Our study highlights the importance of codon usage in virus evolution and mRNA vaccine development.","version":"1.1","doi":"10.1101/2022.09.03.506470","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.03.506479","pub_date":"2022-9-05","title":"Combined Molnupiravir and Nirmatrelvir Treatment Improves the Inhibitory Effect on SARS-CoV-2 in Rhesus Macaques","abstract":"The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have each recently been approved as monotherapy for use in high risk COVID-19 patients. As preclinical data are only available for rodent and ferret models, we originally assessed the efficacy of MK-4482 and PF-07321332 alone and then in combination Against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs. Combined treatment resulted in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated here in the closest COVID-19 surrogate model. The combination of molnupiravir and nirmatrelvir inhibits SARS-CoV-2 replication and shedding more effectively than individual treatments in the rhesus macaque model.","version":"1.1","doi":"10.1101/2022.09.03.506479","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.03.506499","pub_date":"2022-9-05","title":"Delayed SARS-CoV-2 Spread and Olfactory Cell Lineage Impairment in Close-Contact Infection Syrian Hamster Models","abstract":"Close contact with patients with COVID-19 is speculated to be the most common cause of viral transmission, but the pathogenesis of COVID-19 by close contact remains to be elucidated. In addition, despite olfactory impairment being a unique complication of COVID-19, the impact of SARS-CoV-2 on the olfactory cell lineage has not been fully validated. This study aimed to elucidate close-contact viral transmission to the nose and lungs and to investigate the temporal damage in the olfactory receptor neuron (ORN) lineage caused by SARS-CoV-2. Syrian hamsters were orally administered SARS-CoV-2 as direct-infection models. On day 7 after inoculation, infected and uninfected hamsters were housed in the same cage for 30 minutes. These uninfected hamsters were subsequently assigned to a close-contact group. First, viral presence in the nose and lungs was verified in the infection and close-contact groups at several time points. Next, the impacts on the olfactory epithelium, including olfactory progenitors, immature ORNs, and mature ORNs, were examined histologically. Then, the viral transmission status and chronological changes in tissue damage were compared between the direct-infection and close-contact groups. In the close-contact group, viral presence could not be detected in both the nose and lungs on day 3, and the virus was identified in both tissues on day 7. In the direct-infection group, the viral load was highest in the nose and lungs on day 3, decreased on day 7, and was no longer detectable on day 14. Histologically, in the direct-infection group, mature ORNs were most depleted on day 3 (p < 0.001) and showed a recovery trend on day 14, with similar trends for olfactory progenitors and immature ORNs. In the close-contact group, there was no obvious tissue damage on day 3, but on day 7, the number of all ORN lineage cells significantly decreased (p < 0.001). SARS-CoV-2 was transmitted even after brief contact and subsequent olfactory epithelium and lung damage occurred more than 3 days after the trigger of infection. The present study also indicated that SARS-CoV-2 damages all ORN lineage cells, but this damage can begin to recover approximately 14 days post infection.","version":"1.1","doi":"10.1101/2022.09.03.506499","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.04.506474","pub_date":"2022-9-05","title":"ApoE4 causes severe COVID-19 outcomes via downregulation of ACE2","abstract":"The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); host cell entry by this virus relies on the interaction between the receptor-binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor on cell membranes. In addition to serving as a receptor for SARS-CoV-2, ACE2 was originally discovered as a protective factor in the renin\u2013angiotensin system (RAS) that catalyses the degradation of angiotensin II (Ang II) to Ang 1-7, which is involved in multiple organ pathology. Recent genetic and clinical studies reported that ApoE4 expression is associated with increased susceptibility to SARS-CoV-2 infection and the development of severe COVID-19, but the underlying mechanism is currently unclear. In the present study, by using immunofluorescence staining, molecular dynamics simulations, proximity ligation assay (PLA) and coimmunoprecipitation (Co-IP) combined with a biolayer interferometry (BLI) assay, we found that ApoE interacts with both the spike protein and ACE2 but does not show obvious isoform-dependent binding effects. These data suggest that ApoE4 increases SARS-CoV-2 infectivity in a manner that may not depend on differential interactions with the spike protein or ACE2. Importantly, further immunoblotting and immunofluorescence staining results showed that ApoE4 significantly downregulates ACE2 protein expression in vitro and in vivo and subsequently decreases the conversion of Ang II to Ang 1-7, which could worsen tissue lesions; these findings provide a possible explain by which ApoE4 exacerbates COVID-19 disease.","version":"1.1","doi":"10.1101/2022.09.04.506474","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.02.506438","pub_date":"2022-9-03","title":"High resolution photocatalytic mapping of SARS-CoV-2 Spike protein-host cell membrane interactions","abstract":"Identifying protein environments at the virus-host cell interface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the virus behind the ongoing COVID-19 pandemic, uses the cell surface ACE2 protein as a major receptor, but the contribution of other cellular proteins in the entry process is unknown. To probe the microenvironment of SARS-CoV-2 Spike-ACE2 protein interactomes on human cells, we developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) employing iridium photocatalysts conjugated to Spike for visible-light driven proximity labelling on host cells. Application of ViraMap on ACE2-expressing cells captured ACE2, the established co-receptor NRP1, as well as other proteins implicated in host cell entry and immunomodulation. We further investigated these enriched proteins via loss-of-function and over-expression in pseudotype and authentic infection models and observed that the Ig receptor PTGFRN and tyrosine kinase ligand EFNB1 can serve as SARS-CoV-2 entry factors. Our results highlight additional host targets that participate infection and showcase ViraMap for interrogating virus-host cell surface interactomes.","version":"1.1","doi":"10.1101/2022.09.02.506438","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.02.506428","pub_date":"2022-9-03","title":"The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins","abstract":"The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.","version":"1.1","doi":"10.1101/2022.09.02.506428","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.02.506368","pub_date":"2022-9-02","title":"White-tailed deer (Odocoileus virginianus) may serve as a wildlife reservoir for nearly extinct SARS-CoV-2 variants of concern","abstract":"The spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans into white-tailed deer (WTD) and its ability to transmit from deer-to-deer raised concerns about the role of WTD in the epidemiology and ecology of the virus. In the present study, we conducted a comprehensive investigation to assess the prevalence, genetic diversity, and evolution of SARS-CoV-2 in WTD in the State of New York (NY). A total of 5,462 retropharyngeal lymph node (RPLN) samples collected from free-ranging hunter-harvested WTD during the hunting seasons of 2020 (Season 1, September-December 2020, n=2,700) and 2021 (Season 2, September-December 2021, n=2,762) were tested by SARS-CoV-2 real-time RT-PCR. SARS-CoV-2 RNA was detected in 17 samples (0.6%) from Season 1 and in 583 (21.1%) samples from Season 2. Hotspots of infection were identified in multiple confined geographic areas of NY. Sequence analysis of SARS-CoV-2 genomes from 164 samples demonstrated the presence multipls SARS-CoV-2 lineages as well as the co-circulation of three major variants of concern (VOCs) (Alpha, Gamma, and Delta) in WTD. Our analysis suggests the occurrence of multiple spillover events (human-to-deer) of the Alpha and Delta lineages with subsequent deer-to-deer transmission of the viruses. Detection of Alpha and Gamma variants in WTD long after their broad circulation in humans in NY suggests that WTD may serve as a wildlife reservoir for VOCs no longer circulating in humans. Thus, implementation of continuous surveillance programs to monitor SARS-CoV-2 dynamics in WTD are warranted, and measures to minimize virus transmission between humans and animals are urgently needed. White-tailed deer (WTD) are highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and are known to efficiently transmit the virus to other susceptible animals. Evidence of natural exposure or infection of wild WTD in North America raised significant concerns about their role on the ecology of the virus and its impact on the control of the coronavirus disease 2019 (COVID-19) pandemic. This comprehensive study demonstrates widespread infection of SARS-CoV-2 in the WTD populations across the State of New York. Additionally, we showed co-circulation of three major SARS-CoV-2 variants of concern (VOCs) in this wildlife population, long after their broad circulation in humans. These findings indicate that WTD \u2013 the most abundant large mammal in North America \u2013 may serve as a reservoir for variant SARS-CoV-2 strains that no longer circulate in the human population.","version":"1.1","doi":"10.1101/2022.09.02.506368","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.02.506305","pub_date":"2022-9-02","title":"Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75","abstract":"We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.","version":"1.1","doi":"10.1101/2022.09.02.506305","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.31.506117","pub_date":"2022-9-02","title":"Microfluidic immuno-serology assay revealed a limited diversity of protection against COVID-19 in patients with altered immunity","abstract":"The immune response to SARS-CoV-2 for patients with altered immunity such as hematologic malignancies and autoimmune disease may differ substantially from that in general population. These patients remain at high risk despite wide-spread adoption of vaccination. It is critical to examine the differences at the systems level between the general population and the patients with altered immunity in terms of immunologic and serological responses to COVID-19 infection and vaccination. Here, we developed a novel microfluidic chip for high-plex immuno-serological assay to simultaneously measure up to 50 plasma or serum samples for up to 50 soluble markers including 35 plasma proteins, 11 anti-spike/RBD IgG antibodies spanning all major variants, and controls. Our assay demonstrated the quintuplicate test in a single run with high throughput, low sample volume input, high reproducibility and high accuracy. It was applied to the measurement of 1,012 blood samples including in-depth analysis of sera from 127 patients and 21 healthy donors over multiple time points, either with acute COVID infection or vaccination. The protein association matrix analysis revealed distinct immune mediator protein modules that exhibited a reduced degree of diversity in protein-protein cooperation in patients with hematologic malignancies and patients with autoimmune disorders receiving B cell depletion therapy. Serological analysis identified that COVID infected patients with hematologic malignancies display impaired anti-RBD antibody response despite high level of anti-spike IgG, which could be associated with limited clonotype diversity and functional deficiency in B cells and was further confirmed by single-cell BCR and transcriptome sequencing. These findings underscore the importance to individualize immunization strategy for these high-risk patients and provide an informative tool to monitor their responses at the systems level.","version":"1.1","doi":"10.1101/2022.08.31.506117","journal":"bioRxiv","score":null},{"id":"10.1101/2022.09.01.506264","pub_date":"2022-9-02","title":"An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis","abstract":"Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5\u2019 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.","version":"1.1","doi":"10.1101/2022.09.01.506264","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.30.505966","pub_date":"2022-9-01","title":"Impact of reinfection with SARS-CoV-2 Omicron variants in previously infected hamsters","abstract":"The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. The emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2022.08.30.505966","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.25.505217","pub_date":"2022-9-01","title":"SARS-CoV-2 Omicron BA.2.75 variant may be much more infective than preexisting variants","abstract":"In our previous research, we developed a mathematical model via molecular simulation analysis to predict the infectivity of seven SARS-CoV-2 variants. In this report, we aimed to predict the relative risk of the recent new variants of SARS-CoV-2 as based on our previous research. We subjected Omicron BA.4/5 and BA.2.75 variants of SARS-CoV-2 to the analysis to determine the absolute evolutionary distance of the spike protein gene (S gene) of the variants from the Wuhan variant so as to appreciate the changes in the spike protein. We performed the molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to understand the docking affinities of these variants. We then compared the evolutionary distances and the docking affinities of these variants with those of the seven variants that we had analyzed in our previous research. The evolutionary distances of the S gene in BA.4/5 and BA.2.75 from the Wuhan variant were longer than those of other variants. BA.2.75 had the highest docking affinity of the spike protein with ACE2 (ratio per Wuhan variant). The important results from this analysis are the following: BA.2.75 has both the highest docking affinity and the longest evolutionary distance of the S gene. These results suggest that BA.2.75 infection can spread farther than can infections of preexisting variants.","version":"1.4","doi":"10.1101/2022.08.25.505217","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.31.506107","pub_date":"2022-9-01","title":"Structural basis of nirmatrelvir and ensitrelvir resistance profiles against SARS-CoV-2 Main Protease naturally occurring polymorphisms","abstract":"SARS-CoV-2 is the causative agent of COVID-19. Mpro is the main viral protease, with a critical role in replication and, therefore, an attractive target for antiviral drug discovery. The clinically approved drug nirmatrelvir from Pfizer, and the clinical candidate ensitrelvir from Shionogi Pharmaceuticals had so far showed great potential for treatment of viral infections. Despite the importance of new therapeutics, the broad use of antivirals is often associated with mutation selection and resistance generation. Herein, we characterized 14 naturally occurring polymorphisms that are already in circulation and are within the radius of action of these two antivirals. Nirmatrelvir retained most of its in vitro activity against most polymorphism tested, while mutants G143S and Q189K were associated with higher resistance. For ensitrelvir, higher resistance was observed for polymorphisms M49I, G143S and R188S, but not for Q189K, suggesting a distinct resistance profile difference between the two inhibitors. The crystal structures of selected polymorphism reveal the structural basis for resistance generation. Our data will assist the monitoring of potential resistant strains, support the design of combined therapy to avoid resistance, as well as assist the development of a next generation of Mpro inhibitors","version":"1.1","doi":"10.1101/2022.08.31.506107","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.31.506023","pub_date":"2022-8-31","title":"The SARS-CoV-2 Spike S1 Protein Induces Global Proteomic Changes in ATII-Like Rat L2 Cells that are Attenuated by Hyaluronan","abstract":"The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants that have been characterized to date, COVID-19 has led to 571,198,904 confirmed cases, and 6,387,863 deaths worldwide (as of July 15th, 2022). Despite tremendous advances in our understanding of COVID19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics applications combined with systems biology analysis identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in an ATII-like Rat L2 cells that include three significant network hubs: E2F1, CREB1/ RelA, and ROCK2/ RhoA. Separately, we found that pre-treatment with High Molecular Weight Hyaluronan (HMW-HA), greatly attenuated the S1 effects. Immuno-targeted studies carried out on E2F1 and Rock2/ RhoA induction and kinase-mediated activation, in addition to cell cycle measurements, validated these observations. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with standard immuno-targeted and cell cycle measurements revealed profound and novel biological changes that contribute to our current understanding of both Spike S1 and Hyaluronan biology. This data shows that the Spike S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV2 induced cell injury.","version":"1.1","doi":"10.1101/2022.08.31.506023","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.30.505885","pub_date":"2022-08-30","title":"Evidence for coordinated evolution at amino acid sites of SARS-CoV-2 spike","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>It is currently unclear why SARS-Cov-2 has adapted in a stepwise manner, with multiple beneficial mutations accumulating in a rapid succession at the origins of VOCs. Here, we searched for coordinated evolution of amino acid sites in the spike protein of SARS-Cov-2. We searched for concordantly evolving site pairs (CSP) for which changes at one site were rapidly followed by changes at the other site in the same lineage. We detected 46 sites which formed 45 CSP. Sites in CSP were closer to each other in the protein structure than random pairs, indicating that concordant evolution has a functional basis. Notably, site pairs carrying lineage defining mutations of the four VOCs that circulated before May 2021 are enriched in CSP, indicating that the origin of these VOCs could have been facilitated by positive epistasis. Additionally, we detected four discordantly evolving pairs of sites where mutations at one site unexpectedly rarely occurred on the background of a specific allele at another site, namely on the wild-type D at site 614 (for two pairs) or at derived Y in the site 501 (for two other pairs). Our findings hint that positive epistasis between accumulating mutations could have delayed the assembly of advantageous combinations of mutations comprising at least some of the VOCs.</jats:p>","version":null,"doi":"10.1101/2022.08.30.505885","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.26.505369","pub_date":"2022-08-29","title":"Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk and enhanced NTD dynamics","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>SARS-CoV-2 emergent variants are characterized by increased transmissibility and each show multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track correlative changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in Omicron BA.1 S traced through Alpha S and Delta S variants. Stabilization preceded progressive enhancement in dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from specific S mutations detail the evolutionary trajectory of S protein in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.</jats:p>","version":null,"doi":"10.1101/2022.08.26.505369","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.26.505399","pub_date":"2022-8-26","title":"Niacinamide enhances cathelicidin mediated SARS-CoV-2 membrane disruption","abstract":"The continual emergence of SARS-CoV-2 variants threatens to compromise the effectiveness of worldwide vaccination programs, and highlights the need for complementary strategies for a sustainable containment plan. An effective approach is to mobilize the body\u2019s own antimicrobial peptides (AMPs), to combat SARS-CoV-2 infection and propagation. We have found that human cathelicidin (LL37), an AMP found at epithelial barriers as well as in various bodily fluids, has the capacity to neutralise multiple strains of SARS-CoV-2. Biophysical and computational studies indicate that LL37\u2019s mechanism of action is through the disruption of the viral membrane. This antiviral activity of LL37 is enhanced by the hydrotropic action of niacinamide, which may increase the bioavailability of the AMP. Interestingly, we observed inverse correlation between LL37 levels and disease severity of COVID-19 positive patients, suggesting enhancement of AMP response as an interesting therapeutic avenue to mitigate disease severity. The combination of niacinamide and LL37 is a potent antiviral formulation that targets viral membranes of various variants and can potentially overcome vaccine escape.","version":"1.1","doi":"10.1101/2022.08.26.505399","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.25.505365","pub_date":"2022-8-26","title":"Antimicrobial copper as an effective and practical deterrent to surface transmission of SARS-CoV-2","abstract":"The aerosols are critical for SARS-CoV-2 transmission, however in areas with high confluence of people the contaminated surfaces take an important role that we could attack using antimicrobial surfaces including copper. In this study, we wanted to challenge infectious SARS-CoV-2 with two samples of copper surfaces and one plastic surface as control at different direct times contact. To evaluate and quantify virucidal activity of copper against SARS-CoV-2, two methods of experimental infection were performed, TCID50 and plaque assays on VeroE6 cells, showing significant inactivation of high titer of SARS-CoV-2 within minutes reaching 99.9 % of inactivation of infectivity on both copper surfaces. Daily high demand surfaces contamination is an issue that we have to worry about not only during the actual pandemic time but also for future, where copper or its alloys will have a pivotal role. Quantitative data obtained of TCID50 and plaque assay with infectious SARS-CoV-2 virus showed that after direct contact with copper or copper alloys, viruses were inactivated within minutes. Notably, the SARS-CoV-2 virus used in these assays was in high titer (106 PFU/mL) showing strong copper inactivation of the infectious SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.08.25.505365","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.26.505425","pub_date":"2022-8-26","title":"Correlated substitutions reveal SARS-like coronaviruses recombine frequently with a diverse set of structured gene pools","abstract":"Quantifying SARS-like coronavirus (SL-CoV) evolution is critical to understanding the origins of SARS-CoV-2 and the molecular processes that could underlie future epidemic viruses. While genomic evidence implicates recombination as a factor in the emergence of SARS-CoV-2, few studies have quantified recombination rates among SL-CoVs. Here, we infer recombination rates of SL-CoVs from correlated substitutions in sequencing data using a coalescent model with recombination. Our computationally-efficient, non-phylogenetic method infers recombination parameters of both sampled sequences and the unsampled gene pools with which they recombine. We apply this approach to infer recombination parameters for a range of positive-sense RNA viruses. We then analyze a set of 191 SL-CoV sequences (including SARS-CoV-2) and find that ORF1ab and S genes frequently undergo recombination. We identify which SL-CoV sequence clusters have recombined with shared gene pools, and show that these pools have distinct structures and high recombination rates, with multiple recombination events occurring per synonymous substitution. We find that individual genes have recombined with different viral reservoirs. By decoupling contributions from mutation and recombination, we recover the phylogeny of non-recombined portions for many of these SL-CoVs, including the position of SARS-CoV-2 in this clonal phylogeny. Lastly, by analyzing 444,145 SARS-CoV-2 whole genome sequences, we show current diversity levels are insufficient to infer the within-population recombination rate of the virus since the pandemic began. Our work offers new methods for inferring recombination rates in RNA viruses with implications for understanding recombination in SARS-CoV-2 evolution and the structure of clonal relationships and gene pools shaping its origins. Quantifying the population genetics of SARS-like coronavirus (SL-CoV) evolution is vital to deciphering the origins of SARS-CoV-2 and pinpointing viruses with epidemic potential. While some Bayesian approaches can quantify recombination for these pathogens, the required simulations of recombination networks do not scale well with the massive amounts of sequences available in the genomics era. Our approach circumvents this by measuring correlated substitutions in sequences and fitting these data to a coalescent model with recombination. This allows us to analyze hundreds of thousands of sample sequences, and infer recombination rates for unsampled viral reservoirs. Our results provide insights into both the clonal relationships of sampled SL-CoV sequence clusters and the evolutionary dynamics of the gene pools with which they recombine.","version":"1.1","doi":"10.1101/2022.08.26.505425","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.26.505450","pub_date":"2022-8-26","title":"Characterization of SARS-CoV-2 Omicron BA.2.75 clinical isolates","abstract":"The prevalence of the Omicron subvariant BA.2.75 is rapidly increasing in India and Nepal. In addition, BA.2.75 has been detected in at least 34 other countries and is spreading globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs was higher than that of BA.2 and BA.5. Of note, BA.2.75 caused focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which was not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicated better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 and should be closely monitored.","version":"1.1","doi":"10.1101/2022.08.26.505450","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.24.505118","pub_date":"2022-8-25","title":"The diversity of the glycan shield of sarbecoviruses closely related to SARS-CoV-2","abstract":"The animal reservoirs of sarbecoviruses represent a significant risk of emergent pandemics, as evidenced by the impact of SARS-CoV-2. Vaccines remain successful at limiting severe disease and death, however the continued emergence of SARS-CoV-2 variants, together with the potential for further coronavirus zoonosis, motivates the search for pan-coronavirus vaccines that induce broadly neutralizing antibodies. This necessitates a better understanding of the glycan shields of coronaviruses, which can occlude potential antibody epitopes on spike glycoproteins. Here, we compare the structure of several sarbecovirus glycan shields. Many N-linked glycan attachment sites are shared by all sarbecoviruses, and the processing state of certain sites is highly conserved. However, there are significant differences in the processing state at several glycan sites that surround the receptor binding domain. Our studies reveal similarities and differences in the glycosylation of sarbecoviruses and show how subtle changes in the protein sequence can have pronounced impacts on the glycan shield.","version":"1.1","doi":"10.1101/2022.08.24.505118","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.25.505249","pub_date":"2022-8-25","title":"Modelling SARS-CoV-2 spike-protein mutation effects on ACE2 binding","abstract":"The binding affinity of the SARS-CoV-2 spike (S)-protein \u0394\u0394Gbind to the human membrane protein ACE2 is critical for virus function and evolution. Computational structure-based screening of new S-protein mutations for ACE2 binding lends promise to rationalize virus function directly from protein structure and ideally aid early detection of potentially concerning variants. We used a computational protocol based on cryo-electron microscopy structures of the S-protein to estimate the ACE2-binding that gave good trend agreement with experimental ACE2 affinities. We then expanded predictions to all possible S-protein mutations in 21 different S-protein-ACE2 complexes (400,000 \u0394\u0394Gbind data points in total), using mutation group comparisons to reduce systematic errors. We show that mutations that have arisen in major variants as a group maintain ACE2 affinity significantly more than random mutations in the total protein, at the interface, and at evolvable sites, with differences between variant mutations being small relative to these effects. Omicron mutations as a group had a modest change in binding affinity compared to mutations in other major variants. The single-mutation effects are consistent with ACE2 binding being optimized and maintained in omicron, despite increased importance of other selection pressures (antigenic drift). As epistasis, glycosylation and in vivo conditions will modulate these effects, computational predictive SARS-CoV-2 evolution remains far from achieved, but the feasibility of large-scale computation is substantially aided by using many structures and comparison of mutation groups rather than single mutation effects, which are very uncertain. Our results demonstrate substantial challenges but indicate ways to improve the quality of computer models for assessing SARS-CoV-2 mutation effects.","version":"1.1","doi":"10.1101/2022.08.25.505249","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.24.505127","pub_date":"2022-8-25","title":"Antigen presentation dynamics shape the response to emergent variants like SARS-CoV-2 Omicron strain after multiple vaccinations with wild type strain","abstract":"The Omicron variant of SARS-CoV-2 evades neutralization by most serum antibodies elicited by two doses of mRNA vaccines, but a third dose of the same vaccine increases anti-Omicron neutralizing antibodies. By combining computational modeling with data from vaccinated humans we reveal mechanisms underlying this observation. After the first dose, limited antigen availability in germinal centers results in a response dominated by B cells with high germline affinities for immunodominant epitopes that are significantly mutated in an Omicron-like variant. After the second dose, expansion of these memory cells and differentiation into plasma cells shape antibody responses that are thus ineffective for such variants. However, in secondary germinal centers, pre-existing higher affinity antibodies mediate enhanced antigen presentation and they can also partially mask dominant epitopes. These effects generate memory B cells that target subdominant epitopes that are less mutated in Omicron. The third dose expands these cells and boosts anti-variant neutralizing antibodies.","version":"1.1","doi":"10.1101/2022.08.24.505127","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.06.499075","pub_date":"2022-8-25","title":"Maast: genotyping thousands of microbial strains efficiently","abstract":"Genotyping single nucleotide polymorphisms (SNPs) of intraspecific genomes is a prerequisite to performing population genetic analysis and microbial epidemiology. However, existing algorithms fail to scale for species with thousands of sequenced strains, nor do they account for the biased sampling of strains that has produced considerable redundancy in genome databases. Here we present Maast, a tool that reduces the computational burden of SNP genotyping by leveraging this genomic redundancy. Maast implements a novel algorithm to dynamically identify a minimum set of phylogenetically diverse conspecific genomes that contains the maximum number of SNPs above a user-specified allele frequency. Then it uses these genomes to construct a SNP panel for each species. A species\u2019 SNP panel enables Maast to rapidly genotype thousands of strains using a hybrid of whole-genome alignment and k-mer exact matching. Maast works with both genome assemblies and unassembled sequencing reads. Compared to existing genotyping methods, Maast is more accurate and up to two orders of magnitude faster. We demonstrate Maast\u2019s utility on species with thousands of genomes by reconstructing the genetic structure of Helicobacter pylori across the globe and tracking SARS-CoV-2 diversification during the COVID-19 outbreak. Maast is a fast, reliable SNP genotyping tool that empowers population genetic meta-analysis of microbes at an unrivaled scale. source code of Maast is available at https://github.com/zjshi/Maast. kpollard@gladstone.ucsf.edu","version":"1.2","doi":"10.1101/2022.07.06.499075","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.24.505060","pub_date":"2022-8-24","title":"Predicting antiviral resistance mutations in SARS-CoV-2 main protease with computational and experimental screening","abstract":"The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 Mpro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of Mpro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of Mpro. The E166M mutation reduced the half-maximal inhibitory concentration (IC50) of NTV 24-fold, and 118-fold for the non-covalent peptide inhibitor. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages.","version":"1.1","doi":"10.1101/2022.08.24.505060","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.23.504798","pub_date":"2022-8-24","title":"Integrated Immunopeptidomics and Proteomics Study Reveals Imbalanced Innate and Adaptive Immune Responses to SARS-Cov-2 Infection","abstract":"We present an integrated immunopeptidomics and proteomics study of SARS-Cov-2 infection to comprehensively decipher the changes in host cells in response to viral infection. Our results indicated that innate immune response in Calu-3 cells was initiated by TLR3, followed by activation of interferon signaling pathway. Host cells also present viral antigens to the cell surface through both Class I and Class II MHC system for recognition by adaptive immune system. SARS-Cov-2 infection led to the disruption of antigen presentation as demonstrated by higher level of HLA proteins from the flow-through of MHC immunoprecipitation. Glycosylation analysis of HLA proteins from the elution and flow-through of immunoprecipitation revealed that the synthesis and degradation of HLA protein was affected by SARS-Cov-2 infection. This study provided many useful information to study the host response to SARS-Cov-2 infection and would be helpful for the development of therapeutics and vaccine for Covid-19 and future pandemic.","version":"1.1","doi":"10.1101/2022.08.23.504798","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.23.504936","pub_date":"2022-8-24","title":"Enhanced Recombination Among SARS-CoV-2 Omicron Variants Contributes to Viral Immune Escape","abstract":"SARS-CoV-2 virus evolution occurs as a result of antigenic drift and shift. Although antigenic drift has been extensively studied, antigenic shift, which for SARS-CoV-2 occurs through genetic recombination, has been examined scarcely. To gain a better understanding of the emergence and prevalence of recombinant SARS-CoV-2 lineages through time and space, we analyzed SARS-CoV-2 genome sequences from public databases. Our study revealed an extraordinary increase in the emergence of SARS-CoV-2 recombinant lineages during the Omicron wave, particularly in Northern America and Europe. This phenomenon was independent of sequencing density or genetic diversity of circulating SARS-CoV-2 strains. In SARS-CoV-2 genomes, recombination breakpoints were found to be more concentrated in the 3\u2019 UTR followed by ORF1a. Additionally, we noted enrichment of certain amino acids in the spike protein of recombinant lineages, which have been reported to confer immune escape from neutralizing antibodies, increase ACE2 receptor binding, and enhance viral transmission in some cases. Overall, we report an important and timely observation of accelerated recombination in the currently circulating Omicron variants and explore their potential contribution to viral fitness, particularly immune escape.","version":"1.1","doi":"10.1101/2022.08.23.504936","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.23.505031","pub_date":"2022-8-24","title":"New Insights into How JUUL\u2122 Electronic Cigarette Aerosols and Aerosol Constituents Affect SARS-CoV-2 Infection of Human Bronchial Epithelial Cells","abstract":"The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Most studies have been done with tobacco cigarettes, while few have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with high concentrations of nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Responses of BEAS-2B cells to authentic JUUL\u2122 aerosols or their individual constituents (propylene glycol (PG)/vegetable glycerin (VG) and nicotine) were compared using three exposure platforms: submerged culture, air-liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex\u00ae system, which produces authentic heated EC aerosols. SARS-CoV-2 infection machinery was assessed using immunohistochemistry and Western blotting. Specifically, the levels of the SARS-CoV-2 receptor ACE2 (angiotensin converting enzyme 2) and a spike modifying enzyme, TMPRSS2 (transmembrane serine protease 2), were evaluated. Following each exposure, lentivirus pseudoparticles with spike protein and a green-fluorescent reporter were used to test viral penetration and the susceptibility of BEAS-2B cells to infection. Nicotine, EC fluids, and authentic JUUL\u2122 aerosols increased both ACE2 levels and TMPRSS2 activity, which in turn increased viral particle entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. In the Cultex system, PG/VG, PG/VG/nicotine, and JUUL\u2122 aerosols significantly increased infection above clean air controls. However, both the PG/VG and JUUL\u2122 treatments were significantly lower than nicotine/PG/VG. PG/VG increased infection only in the Cultex\u00ae system, which produces heated aerosol. Our data are consistent with the conclusion that authentic JUUL\u2122 aerosols or their individual constituents (nicotine or PG/VG) increase SARS-CoV-2 infection. The strong effect produced by nicotine was modulated in authentic JUUL aerosols, demonstrating the importance of studying mixtures and aerosols from actual EC products. These data support the idea that vaping increases the likelihood of contracting COVID-19.","version":"1.1","doi":"10.1101/2022.08.23.505031","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.24.505169","pub_date":"2022-8-24","title":"Human anti-ACE2 monoclonal antibodies as pan-sarbecovirus prophylactic agents","abstract":"Human monoclonal antibodies from convalescent individuals that target the SARS-CoV-2 spike protein have been deployed as therapeutics against SARS-CoV-2. However, nearly all of these antibodies have been rendered obsolete by SARS-CoV-2 variants that evolved to resist similar, naturally occurring antibodies. Here, we describe the development of human monoclonal antibodies that bind the ACE2 receptor rather than the viral spike protein. These antibodies block infection by all ACE2 binding sarbecoviruses, including emergent SARS-CoV-2 variants. Structural and biochemical analyses revealed that the antibodies target an ACE2 epitope that engages SARS-CoV-2 spike. Importantly, the antibodies do not inhibit ACE2 enzymatic activity, nor do they induce ACE depletion from cell surfaces. The antibodies exhibit favorable pharmacology and protect human ACE2 knock-in mice against SARS-CoV-2 infection. Such antibodies should be useful prophylactic and treatment agents against any current and future SARS-CoV-2 variants, as well as ACE2-binding sarbecoviruses that might emerge as future pandemic threats.","version":"1.1","doi":"10.1101/2022.08.24.505169","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500716","pub_date":"2022-8-24","title":"Evasion of neutralizing antibodies by Omicron sublineage BA.2.75","abstract":"An emerging SARS-CoV-2 Omicron sublineage, BA.2.75, is increasing in frequency in India and has been detected in at least 15 countries as of 19 July 2022. Relative to BA.2, BA.2.75 carries nine additional mutations in spike. Here we report the sensitivity of the BA.2.75 spike to neutralization by a panel of clinically-relevant and pre-clinical monoclonal antibodies, as well as by serum from blood donated in Stockholm, Sweden, before and after the BA.1/BA.2 infection wave. BA.2.75 largely maintains sensitivity to bebtelovimab, despite a slight reduction in potency, and exhibits moderate susceptibility to tixagevimab and cilgavimab. For sera sampled both before and after the BA.1/BA.2 infection wave, BA.2.75 does not show significantly greater antibody evasion than the currently-dominating BA.5.","version":"1.2","doi":"10.1101/2022.07.19.500716","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.23.504908","pub_date":"2022-8-23","title":"Mucosal gene expression in response to SARS-CoV-2 is associated with early viral load","abstract":"Little is known about the relationships between symptomatic early-time SARS-CoV-2 viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate COVID-19. We measured SARS-CoV-2 viral load using qRT-PCR. We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 85% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited co-detection of common respiratory viruses i.e., only the human Rhinovirus (HRV) being identified in 6% of the samples. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusted for age, sex and race. Interestingly, the expression levels of most of these genes plateaued at a CT value of ~25. Overall, our data shows that early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, which potentially could modify COVID-19 outcomes. Several prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load and airway mucosal gene expression and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during Spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load with interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load-dependent and may modify COVID-19 outcomes.","version":"1.1","doi":"10.1101/2022.08.23.504908","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.22.504904","pub_date":"2022-8-23","title":"Isolation and characterization of SARS-CoV-2 in Kenya","abstract":"The emergence of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) from Wuhan, China, in December 2019 raised a global health concern that eventually became a pandemic affecting almost all countries worldwide. The respiratory disease has infected over 530 million people worldwide, with over 950,000 deaths recorded. This has led scientists to focus their efforts on understanding the virus to develop effective means to diagnose, treat, prevent, and control this pandemic. One of the areas of focus is isolation of this virus, which plays a crucial role in understanding the viral dynamics in the laboratory. In this study, we report the isolation and detection of locally circulating SARS-CoV-2 in Kenya. The isolates were cultured on Vero Cercopithecus cell line (CCL-81) cells, RNA extraction conducted from the supernatants, and reverse transcriptase-polymerase chain reaction (RT-PCR). Genome sequencing was done to profile the strains phylogenetically and identify novel and previously reported mutations. Vero CCL-81 cells were able to support the growth of SARS-CoV-2 in vitro, and mutations were detected from the two isolates sequenced (001 and 002). These virus isolates will be expanded and made available to the Kenya Ministry of Health and other research institutions to advance SARS-CoV-2 research in Kenya and the region. The Coronavirus disease 2019 (COVID-19) pandemic is caused by a type of coronavirus that emerged in Wuhan, China in December 2019 and later spread to almost all countries. Many countries are still finding ways to contain it. The virus has been studied in many ways to investigate its origin, infectivity, and evolution. Different variants of the virus have emerged and spread, causing a lot of concern as to whether the pandemic will end soon. Significant studies have proven the ability of the virus to grow in the laboratory using cell lines that offer the necessary conditions. Therefore, this study sought to find out the growth of the virus in specific monkey cell line and the variant circulating within the Kenyan population. We found that the selected cell lines supported viral growth outside a human host system. In addition, the circulating virus was found to have evolved to enhance its survival mechanism. This is the first study in Kenya to report this virus\u2019s isolation, culture, and identification in monkey kidney cells. These cells supported the growth of the virus in the laboratory and analysing the genome of the growth products showed the virus was related to previously reported strains with multiple changes in its whole DNA sequence.","version":"1.1","doi":"10.1101/2022.08.22.504904","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.22.504888","pub_date":"2022-8-23","title":"SARS-CoV-2 infects multiple species of North American deer mice and causes clinical disease in the California mouse","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes coronavirus disease-19 (COVID-19), emerged in late 2019 in Wuhan, China and its rapid global spread has resulted in millions of deaths. An important public health consideration is the potential for SARS-CoV-2 to establish endemicity in a secondary animal reservoir outside of Asia or acquire adaptations that result in new variants with the ability to evade the immune response and reinfect the human population. Previous work has shown that North American deer mice (Peromyscus maniculatus) are susceptible and can transmit SARS-CoV-2 to na\u00efve conspecifics, indicating its potential to serve as a wildlife reservoir for SARS-CoV-2 in North America. In this study, we report experimental SARS-CoV-2 susceptibility of two additional subspecies of the North American deer mouse and two additional deer mouse species, with infectious virus and viral RNA present in oral swabs and lung tissue of infected deer mice and neutralizing antibodies present at 15 days post-challenge. Moreover, some of one species, the California mouse (P. californicus) developed clinical disease, including one that required humane euthanasia. California mice often develop spontaneous liver disease, which may serve as a comorbidity for SARS-CoV-2 severity. The results of this study suggest broad susceptibility of rodents in the genus Peromyscus and further emphasize the potential of SARS-CoV-2 to infect a wide array of North American rodents. A significant concern is the spillback of SARS-CoV-2 into North American wildlife species. We have determined that several species of peromyscine rodents, the most abundant mammals in North America, are susceptible to SARS-CoV-2 and that infection is likely long enough that the virus may be able to establish persistence in local rodent populations. Strikingly, some California mice developed clinical disease that suggests this species may be useful for the study of human co-morbidities often associated with severe and fatal COVID-19 disease.","version":"1.1","doi":"10.1101/2022.08.22.504888","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.23.503528","pub_date":"2022-8-23","title":"An outbreak of SARS-CoV-2 in big hairy armadillos (Chaetophractus villosus) associated with Gamma variant in Argentina three months after being undetectable in humans","abstract":"The present pandemic produced by SARS-CoV-2 and its variants represents an example of the one health concept in which humans and animals are components of the same epidemiologic chain. Animal reservoirs of these viruses are thus the focus of surveillance programs to monitor their circulation and evolution in potentially new hosts and reservoirs. In this work, we report the detection of SARS-CoV-2 Gamma variant infection in four specimens of Chaetophractus villosus (big hairy armadillo/armadillo peludo) in Argentina. In addition to the finding of a new wildlife species susceptible to SARS-CoV-2 infection, the identification of the Gamma variant three months after its last detection in humans is a noteworthy result, raising the question of potential unidentified viral reservoirs.","version":"1.1","doi":"10.1101/2022.08.23.503528","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.19.504307","pub_date":"2022-8-23","title":"The efficiency of Grignard Pure\u2122 to inactivate airborne SARS-CoV-2 surrogate","abstract":"Grignard Pure\u2122 (GP) is a unique and proprietary blend of Triethylene Glycol (TEG) and inert ingredients designed for continuous antimicrobial treatment of air. GP received approval from the US EPA under its Section 18 Public Health Emergency Exemption program for use in seven states. This study characterizes the efficacy of GP for inactivating MS2 bacteriophage \u2013 a non-enveloped virus widely used as a surrogate for SARs-CoV-2. Experiments measured the decrease in the airborne viable MS2 concentration in the presence of different concentrations of GP from 60 to 90 minutes, accounting for both natural die-off and settling of MS2. Experiments were conducted both by introducing GP aerosol into air containing MS2 and by introducing airborne MS2 into air containing GP aerosol. GP is consistently able to rapidly reduce viable MS2 bacteriophage concentration by 2-3 logs at GP concentrations of 0.02 mg/m3 to 0.5 mg/m3 (corresponding to TEG concentrations of 0.012 mg/m3 to 0.287 mg/m3). Related GP efficacy experiments by the US EPA, as well as GP (TEG) safety and toxicology, are also discussed. Limited research on the germicidal properties of triethylene glycol against airborne pathogens was conducted during the 1940s and 50s. This paper investigates the inactivation rate of airborne bacteriophage MS2 by Grignard Pure\u2122 product, containing a unique and proprietary blend of Triethylene Glycol (TEG) and inert ingredients.","version":"1.1","doi":"10.1101/2022.08.19.504307","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.22.504823","pub_date":"2022-8-23","title":"Effective Matrix Designs for COVID-19 Group Testing","abstract":"Grouping samples with low prevalence of positives into pools and testing these pools can achieve considerable savings in testing resources compared with individual testing in the context of COVID-19. We review published pooling matrices, which encode the assignment of samples into pools and describe decoding algorithms, which decode individual samples from pools. Based on the findings we propose new one-round pooling designs with high compression that can efficiently be decoded by combinatorial algorithms. This expands the admissible parameter space for the construction of pooling matrices compared to current methods. By arranging samples in a grid and using polynomials to construct pools, we develop direct formulas for an Algorithm (Polynomial Pools (PP)) to generate assignments of samples into pools. Designs from PP guarantee to correctly decode all samples with up to a specified number of positive samples. PP includes recent combinatorial methods for COVID-19, and enables new constructions that can result in more effective designs. For low prevalences of COVID-19, group tests can save resources when compared to individual testing. Constructions from the recent literature on combinatorial methods have gaps with respect to the designs that are available. We develop a method (PP), which generalizes previous constructions and enables new designs that can be advantageous in various situations.","version":"1.1","doi":"10.1101/2022.08.22.504823","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.19.504579","pub_date":"2022-8-22","title":"Overview of the SARS-CoV-2 genotypes circulating in Latin America during 2021","abstract":"Latin America is one of the regions in which the COVID-19 pandemic has had a stronger impact, with more than 72 million reported infections and 1.6 million deaths until June 2022. Since this region is ecologically diverse and is affected by enormous social inequalities, efforts to identify genomic patterns of the circulating SARS-CoV-2 genotypes are necessary for the suitable management of the pandemic. To contribute to the genomic surveillance of the SARS-CoV-2 in Latin America, we extended the number of SARS-CoV-2 genomes available from the region by sequencing and analyzing the viral genome from COVID-19 patients from seven countries (Argentina, Brazil, Costa Rica, Colombia, Mexico, Bolivia and Peru). Subsequently, we analyzed the genomes circulating mainly during 2021 including records from GISAID database from Latin America. A total of 1534 genome sequences were generated from seven countries, demonstrating the laboratory and bioinformatics capabilities for genomic surveillance of pathogens that have been developed locally. For Latin America, patterns regarding several variants associated with multiple re-introductions, a relatively low percentage of sequenced samples, as well as an increment in the mutation frequency since the beginning of the pandemic, are in line with worldwide data. Besides, some variants of concern (VOC) and variants of interest (VOI) such as Gamma, Mu and Lambda, and at least 83 other lineages have predominated locally with a country-specific enrichments. This work has contributed to the understanding of the dynamics of the pandemic in Latin America as part of the local and international efforts to achieve timely genomic surveillance of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.08.19.504579","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.22.504760","pub_date":"2022-8-22","title":"Airway epithelial cells and macrophages trigger IL-6-CD95/CD95L axis and mediate initial immunopathology of COVID-19","abstract":"Airway epithelial cells (AEC) are the first in contact with SARS-CoV-2 and drive the interface with macrophage to generate inflammation. To elucidate how those initial events contribute to the immunopathology or to dysregulate the immune response observed in severe and critical COVID-19, we determined the direct and indirect interactions of these cells. AEC lineage (Calu-3) infected with SARS-CoV-2 and epithelial cells (CD45-EpCAM+) from intubated COVID-19 patients showed high expression of CD95L. Infected-Calu-3 cells secreted IL-6, and expressed annexin V and caspase-3, apoptosis markers. The direct interaction of macrophages with sorted apoptotic Calu-3 cells, driven by SARS-CoV-2 infection, resulted in macrophage death and increased expression of CD95, CD95L and CD163. Macrophages exposed to tracheal aspirate supernatants from intubated COVID-19 patients or to recombinant human IL-6 exhibited decreased HLA-DR and increased CD95 and CD163 expression. IL-6 effects on macrophages were prevented by tocilizumab (anti-IL-6 receptor mAb) and Kp7-6 (CD95/CD95L antagonist). Similarly, lung inflammation and death of AEC were decreased in CD95 and IL-6 knockout mice infected with SARS-CoV-2. Our results show that the AEC-macrophage interaction via CD95/CD95L signaling is an initial key step of immunopathology of severe COVID-19 and should be considered as a therapeutic target. - SARS-CoV-2-infected airway epithelial cells (AEC) secrete IL-6, express Fas/FasL and undergo apoptosis; - SARS-CoV-2-infected apoptotic AEC induces Fas/FasL expression and death in macrophages; - IL-6 induces IL-1\u03b2 secretion, reduction of HLA-DR and increase of Fas and CD163 expression in macrophages; - Blockade of IL-6 signaling and Fas/FasL restores the expression of HLA-DR and reduces the expression of Fas and CD163, and secretion of IL-1\u03b2 on isolated macrophages; in vivo, the deficiency of Fas and IL-6 decreases acute pulmonary inflammation in SARS-CoV-2-infected mice.","version":"1.1","doi":"10.1101/2022.08.22.504760","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.08.491108","pub_date":"2022-8-22","title":"Imprinted antibody responses against SARS-CoV-2 Omicron sublineages","abstract":"SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.","version":"1.4","doi":"10.1101/2022.05.08.491108","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.24.493068","pub_date":"2022-8-22","title":"Host-directed therapy with 2-Deoxy-D-glucose inhibits human rhinoviruses, endemic coronaviruses, and SARS-CoV-2","abstract":"Rhinoviruses (RVs) and coronaviruses (CoVs) upregulate host cell metabolic pathways such as glycolysis to meet their bioenergetic demands for rapid multiplication. Using the glycolysis inhibitor 2-deoxy-D-glucose (2-DG), we assessed the dose-dependent inhibition of viral replication of minor- and major-receptor group RVs in epithelial cells. 2-DG disrupted RV infection cycle by inhibiting template negative-strand as well as genomic positive-strand RNA synthesis, resulting in less progeny virus and RV-mediated cell death. Assessment of 2-DG\u2019s intracellular kinetics revealed that after a short-exposure to 2-DG, the active intermediate, 2-DG6P, is stored intracellularly for several hours. Finally, we confirmed the antiviral effect of 2-DG on pandemic SARS-CoV-2 and showed for the first time that 2-DG also reduces replication of endemic human coronaviruses (HCoVs). These results provide further evidence that 2-DG could be utilized as a broad-spectrum antiviral. 2-DG inhibits replication of minor- and major-group rhinoviruses in epithelial cells including human nasal epithelial cell. 2-DG disrupts rhinovirus infection cycle and reduces rhinovirus-mediated cell death in vitro. 2-DG treatment attenuates viral load of endemic coronaviruses in vitro.","version":"1.2","doi":"10.1101/2022.05.24.493068","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.470978","pub_date":"2022-8-22","title":"Secreted ORF8 is a pathogenic cause of severe COVID-19 and is potentially targetable with select NLRP3 inhibitors","abstract":"COVID-19 is a significant cause of morbidity and mortality in blood cancer patients, especially those on immunosuppressive therapy. Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the life-threatening inflammatory cytokine response in patients with severe COVID-19 remains unidentified. Herein we demonstrate that the virus-encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro and in symptomatic patients with COVID-19. ORF8 specifically binds to the NOD-like receptor family pyrin domain-containing 3 (NLRP3) in CD14+ monocytes to induce a non-canonical inflammasomal response, and a canonical response when the second activation signal is present. Levels of ORF8 protein in the blood correlate with severity and disease-specific mortality in patients with acute SARS-CoV-2 infection. Furthermore, the ORF8-induced inflammasome response was readily inhibited by the NLRP3 inhibitor MCC950 in vitro. Our study identifies a dominant cause of pathogenesis, its underlying mechanism, and a potential new treatment for severe COVID-19. Secreted glycoprotein ORF8 induces monocytic pro-inflammatory cytokines involving the activation of the NLPR3 inflammasome pathway. ORF8 is prognostically present in the blood of symptomatic patients with covid-19 and is targetable with NLRP3 inhibitor MCC-950.","version":"1.2","doi":"10.1101/2021.12.02.470978","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.21.22279047","pub_date":"2022-08-22","title":"Associations of proton pump inhibitors with susceptibility to influenza, pneumonia, and COVID-19: Evidence from a large population based cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Adverse effects of proton pump inhibitors (PPIs) have raised wide concerns. The association of PPIs with influenza is unexplored, while that with pneumonia or COVID-19 remains controversial. Our study aims to evaluate whether PPI use increases the risks of these respiratory infections. The current study included 160,923 eligible participants at baseline who completed questionnaires on medication use, which included PPI or histamine-2 receptor antagonist (H2RA), from the UK Biobank. Cox proportional hazards regression and propensity score-matching analyses were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs). Comparisons with H2RA users were tested. PPI use was associated with increased risks of developing influenza (HR 1.32, 95%CI 1.12-1.56) and pneumonia (hazard ratio [HR] 1.42, 95% confidence interval [CI] 1.26-1.59). In contrast, the risk of COVID-19 infection was not significant with regular PPI use (HR 1.08, 95%CI 0.99-1.17), while the risks of severe COVID-19 (HR 1.19. 95%CI 1.11-1.27) and mortality (HR 1.37. 95%CI 1.29-1.46) were increased. However, when compared with H2RA users, PPI users were associated with a higher risk of influenza (HR 1.74, 95%CI 1.19-2.54), but the risks with pneumonia or COVID-19-related outcomes were not evident. In conclusion, PPI users are associated with increased risks of influenza, pneumonia, as well as COVID-19 severity and mortality compared to non-users, while the effects on pneumonia or COVID-19-related outcomes under PPI use were attenuated when compared to the use of H2RAs. Appropriate use of PPIs based on comprehensive evaluation is required.</jats:p>","version":null,"doi":"10.1101/2022.08.21.22279047","journal":"medRxiv","score":null},{"id":"10.1101/2022.08.20.504642","pub_date":"2022-8-22","title":"Development of Metal organic Framework Based Biosensor to Detect the Coronavirus (Covid-19)","abstract":"Recent outbreak of novel coronavirus (COVID-19) caused around 7 million deaths people worldwide and still afflicting on the global health, economy and social setup. Timely detection and diagnosis are crucial steps to reduce the spread and prevention of any pandemic. Different types of diagnosis methos has been used. In last decade nanomaterials and metal organic frameworks (MOFs) based biosensors has been developed to detect the other viruses. We have designed the Zeolitic imidazolate framework-8 (ZIF-8) based biosensor to detect the COVID-19. ZIF-8 work as fluorescence quenching and re-emergence platform to detect the COVID-19 RNA sequences. ZIF-8 platform is highly sensitive which can distinguish the highly conserved single strand RNA and with 200 pM concentrations. It can distinguish down to the single mismatch nucleotide in RNA sequences.","version":"1.1","doi":"10.1101/2022.08.20.504642","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.16.484616","pub_date":"2022-8-20","title":"Pre-exposure to mRNA-LNP inhibits adaptive immune responses and alters innate immune fitness in an inheritable fashion","abstract":"Hundreds of millions of SARS-CoV-2 mRNA-LNP vaccine doses have already been administered to humans. However, we lack a comprehensive understanding of the immune effects of this platform. The mRNA-LNP-based SARS-CoV-2 vaccine is highly inflammatory, and its synthetic ionizable lipid component responsible for the induction of inflammation has a long in vivo half-life. Since chronic inflammation can lead to immune exhaustion and non-responsiveness, we sought to determine the effects of pre-exposure to the mRNA-LNP on adaptive immune responses and innate immune fitness. We found that pre-exposure to mRNA-LNPs or LNP alone led to long-term inhibition of the adaptive immune responses, which could be overcome using standard adjuvants. On the other hand, we report that after pre-exposure to mRNA-LNPs, the resistance of mice to heterologous infections with influenza virus increased while Candida albicans decreased. The diminished resistance to Candida albicans correlated with a general decrease in blood neutrophil percentages. Interestingly, mice pre-exposed to the mRNA-LNP platform can pass down the acquired immune traits to their offspring, providing better protection against influenza. In summary, the mRNA-LNP vaccine platform induces long-term unexpected immunological changes affecting both adaptive immune responses and heterologous protection against infections. Thus, our studies highlight the need for more research to determine this platform\u2019s true impact on human health. We bring experimental evidence that pre-exposure to mRNA-LNPs or its LNP component affects innate and adaptive immune responses. Pre-exposure to mRNA-LNPs led to long-term inhibition of the adaptive immune responses, which the use of adjuvants could overcome. On the other hand, we report that after pre-exposure to mRNA-LNPs, the resistance of mice to heterologous infections with influenza virus increased while Candida albicans decreased. We also detected a general neutropenia in the mRNA-LNP exposed mice. Interestingly, mice pre-exposed to mRNA-LNPs can pass down the acquired immune traits to their offspring. In summary, the mRNA-LNP vaccine platform induces long-term immunological changes that can affect both adaptive immune responses and heterologous protection against infections, some of which can be inherited by the offspring. More studies are needed to understand the mechanisms responsible for these effects and determine this platform\u2019s impact on human health.","version":"1.2","doi":"10.1101/2022.03.16.484616","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.17.504362","pub_date":"2022-8-19","title":"Ancestral lineage of SARS-CoV-2 is more stable in human biological fluids than Alpha, Beta and Omicron variants of concern","abstract":"SARS-CoV-2 is a zoonotic virus which was first identified in 2019, and has quickly spread worldwide. The virus is primarily transmitted through respiratory droplets from infected persons; however, the virus-laden excretions can contaminate surfaces which can serve as a potential source of infection. Since the beginning of the pandemic, SARS-CoV-2 has continued to evolve and accumulate mutations throughout its genome leading to the emergence of variants of concern (VOCs) which exhibit increased fitness, transmissibility, and/or virulence. However, the stability of SARS-CoV-2 VOCs in biological fluids has not been thoroughly investigated so far. The aim of this study was to determine and compare the stability of different SARS-CoV-2 strains in human biological fluids. Here, we demonstrate that the ancestral strain of Wuhan-like lineage A was more stable than the Alpha VOC B.1.1.7, and the Beta VOC B.1.351 strains in human liquid nasal mucus and sputum. In contrast, there was no difference in stability among the three strains in dried biological fluids. Furthermore, we also show that the Omicron VOC B.1.1.529 strain was less stable than the ancestral Wuhan-like strain in liquid nasal mucus. These studies provide insight into the effect of the molecular evolution of SARS-CoV-2 on environmental virus stability, which is important information for the development of countermeasures against SARS-CoV-2. Genetic evolution of SARS-CoV-2 leads to the continuous emergence of novel variants, posing a significant concern to global public health. Five of these variants have been classified so far into variants of concern (VOCs); Alpha, Beta, Gamma, Delta, and Omicron. Previous studies investigated the stability of SARS-CoV-2 under various conditions, but there is a gap of knowledge on the survival of SARS-CoV-2 VOCs in human biological fluids which are clinically relevant. Here, we present evidence that Alpha, Beta, and Omicron VOCs were less stable than the ancestral Wuhan-like strain in human biological fluids. Our findings highlight the potential risk of contaminated human biological fluids in SARS-CoV-2 transmission and contribute to the development of countermeasures against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.08.17.504362","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.19.504551","pub_date":"2022-8-19","title":"SARS-CoV-2 Airway Infection Results in Time-dependent Sensory Abnormalities in a Hamster Model","abstract":"Despite being largely confined to the airways, SARS-CoV-2 infection has been associated with sensory abnormalities that manifest in both acute and long-lasting phenotypes. To gain insight on the molecular basis of these sensory abnormalities, we used the golden hamster infection model to characterize the effects of SARS-CoV-2 versus Influenza A virus (IAV) infection on the sensory nervous system. Efforts to detect the presence of virus in the cervical/thoracic spinal cord and dorsal root ganglia (DRGs) demonstrated detectable levels of SARS-CoV-2 by quantitative PCR and RNAscope uniquely within the first 24 hours of infection. SARS-CoV-2-infected hamsters demonstrated mechanical hypersensitivity during acute infection; intriguingly, this hypersensitivity was milder, but prolonged when compared to IAV-infected hamsters. RNA sequencing (RNA-seq) of thoracic DRGs from acute infection revealed predominantly neuron-biased signaling perturbations in SARS-CoV-2-infected animals as opposed to type I interferon signaling in tissue derived from IAV-infected animals. RNA-seq of 31dpi thoracic DRGs from SARS-CoV-2-infected animals highlighted a uniquely neuropathic transcriptomic landscape, which was consistent with substantial SARS-CoV-2-specific mechanical hypersensitivity at 28dpi. Ontology analysis of 1, 4, and 30dpi RNA-seq revealed novel targets for pain management, such as ILF3. Meta-analysis of all SARS-CoV-2 RNA-seq timepoints against preclinical pain model datasets highlighted both conserved and unique pro-nociceptive gene expression changes following infection. Overall, this work elucidates novel transcriptomic signatures triggered by SARS-CoV-2 that may underlie both short- and long-term sensory abnormalities while also highlighting several therapeutic targets for alleviation of infection-induced hypersensitivity. SARS-CoV-2 infection results in an interferon-associated transcriptional response in sensory tissues underlying time-dependent hypersensitivity.","version":"1.1","doi":"10.1101/2022.08.19.504551","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.17.504313","pub_date":"2022-8-19","title":"Epistasis lowers the genetic barrier to SARS-CoV-2 neutralizing antibody escape","abstract":"Consecutive waves of SARS-CoV-2 infection have been driven in part by the repeated emergence of variants with mutations that confer resistance to neutralizing antibodies Nevertheless, prolonged or repeated antigen exposure generates diverse memory B-cells that can produce affinity matured receptor binding domain (RBD)-specific antibodies that likely contribute to ongoing protection against severe disease. To determine how SARS-CoV-2 omicron variants might escape these broadly neutralizing antibodies, we subjected chimeric viruses encoding spike proteins from ancestral, BA.1 or BA.2 variants to selection pressure by a collection of 40 broadly neutralizing antibodies from individuals with various SARS-CoV-2 antigen exposures. Notably, pre-existing substitutions in the BA.1 and BA.2 spikes facilitated acquisition of resistance to many broadly neutralizing antibodies. Specifically, selection experiments identified numerous RBD substitutions that did not confer resistance to broadly neutralizing antibodies in the context of the ancestral Wuhan-Hu-1 spike sequence, but did so in the context of BA.1 and BA.2. A subset of these substitutions corresponds to those that have appeared in several BA.2 daughter lineages that have recently emerged, such as BA.5. By including as few as 2 or 3 of these additional changes in the context of BA.5, we generated spike proteins that were resistant to nearly all of the 40 broadly neutralizing antibodies and were poorly neutralized by plasma from most individuals. The emergence of omicron variants has therefore not only allowed SARS-CoV-2 escape from previously elicited neutralizing antibodies but also lowered the genetic barrier to the acquisition of resistance to the subset of antibodies that remained effective against early omicron variants.","version":"1.1","doi":"10.1101/2022.08.17.504313","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.18.504053","pub_date":"2022-8-19","title":"Single-cell RNA sequencing highlights a reduced function of natural killer and cytotoxic T cell in recovered COVID-19 pregnant women","abstract":"Pregnancy is a complex phenomenon during which women undergo immense immunological change throughout this period. Having an infection with the SARS-CoV-2 virus leads to an additional burden on the highly stretched immune response. Some studies suggest that age-matched pregnant women are more prone to SARS-CoV-2 infection compared with normal healthy (non-pregnant) women, while alternative evidence proposed that pregnant women are neither susceptible nor develop severe symptoms. This discrepancy in different findings regarding the immune responses of pregnant women infected with SARS-CoV-2 virus is not well understood. In this study, we investigated how SARS-CoV-2 viral infection could modulate the immune landscape during the active infection phase and recovery in pregnant females. Using flow cytometry, we identified that intermediate effector CD8+ T cells were increased in pregnant women who had recovered from COVID-19 as opposed to those currently infected. Similarly, an increase in CD4+ T helper cells (early or late) during the recovered phase was observed during the recovery phase compared with infected pregnant women or healthy pregnant women, whilst infected pregnant women had a reduced number of late effector CD4+ T cells. CD3+CD4- CD8-NKT cells that diminished during active infection in contrast to healthy pregnant women were significant increase in recovered COVID-19 recovered pregnant women. Further, our single-cell RNA sequencing data revealed that infection of SARS-CoV-2 had changed the gene expression profile of monocytes, CD4+ effector cells and antibody producing B cells in convalescent as opposed to healthy pregnant women. Additionally, several genes with cytotoxic function, interferon signalling type I & II, and pro- and anti-inflammatory functions in natural killer cells and CD8+ cytotoxic T cells were compromised in recovered patients compared with healthy pregnant women. Overall, our study highlights that SARS-CoV-2 infection deranged the adaptive immune response in pregnant women and could be implicated in pregnancy complications in ongoing pregnancies.","version":"1.1","doi":"10.1101/2022.08.18.504053","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.18.504268","pub_date":"2022-8-19","title":"A syntenin inhibitor blocks endosomal entry of SARS-CoV-2 and a panel of RNA viruses","abstract":"Viruses are dependent on interactions with host factors in order to efficiently establish an infection and replicate. Targeting such interactions provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking, and has previously been shown to be important for HPV infection. Here we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits SARS-CoV-2 infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection, and strongly reduced flavivirus infection, which are completely dependent on receptor mediated endocytosis for their entry. In conclusion, we have identified a novel pan-viral inhibitor that efficiently target a broad range of RNA viruses.","version":"1.1","doi":"10.1101/2022.08.18.504268","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.19.504545","pub_date":"2022-8-19","title":"Disrupted chromatin architecture in olfactory sensory neurons: A missing link from COVID-19 infection to anosmia","abstract":"We tackle here genomic mechanisms of a rapid onset and recovery from anosmia - a useful diagnostic indicator for early-stage COVID-19 infection. On the basis of earlier observed specifics of olfactory receptors (ORs) regulation in the mice chromatin structures, we hypothesized that the disruption of OR function can be caused by chromatin reorganization taking place upon SARS-CoV-2 infection. We reconstructed the chromatin ensembles of ORs obtained from COVID-19 patients and control samples using our original computational framework for the whole-genome chromatin ensemble 3D reconstruction. We have also developed here a new procedure for the analysis of fine structural hierarchy in local, megabase scale, parts of chromosomes containing the OR genes and corresponding epigenetic factors. We observed structural modifications in COVID-19 patients on different levels of chromatin organization, from alteration of the whole genome structure and chromosomal intermingling to reorganization of contacts between the chromatin loops at the level of topologically associating domains. While complementary data on known regulatory elements point to pathology-associated changes within the overall picture of chromatin alterations, further investigation using additional epigenetic factors mapped on 3D reconstructions with improved resolution will be required for better understanding of anosmia caused by SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.08.19.504545","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.07.499047","pub_date":"2022-8-18","title":"Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir","abstract":"Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-19. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.","version":"1.2","doi":"10.1101/2022.08.07.499047","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.08.503157","pub_date":"2022-8-18","title":"Protein Geometry, Function and Mutation","abstract":"This survey for mathematicians summarizes several works by the author on protein geometry and protein function with applications to viral glycoproteins in general and the spike glycoprotein of the SARS-CoV-2 virus in particular. Background biology and biophysics are sketched. This body of work culminates in a postulate that protein secondary structure regulates mutation, with backbone hydrogen bonds materializing in critical regions to avoid mutation, and disappearing from other regions to enable it.","version":"1.2","doi":"10.1101/2022.08.08.503157","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.17.504290","pub_date":"2022-8-17","title":"Genetic diversity and spread dynamics of SARS-CoV-2 variants present in African populations","abstract":"The dynamics of COVID-19 disease have been extensively researched in many settings around the world, but little is known about these patterns in Africa. 6139 complete nucleotide genomes from 51 African nations were obtained and analyzed from the National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Influenza Data (GISAID) databases to examine genetic diversity and spread dynamics of SARS-CoV-2 lineages circulating in Africa. We investigated their diversity using several clade and lineage nomenclature systems, and used maximum parsimony inference methods to recreate their evolutionary divergence and history. According to this study, only 193 of the 2050 Pango lineages discovered worldwide circulated in Africa after two years of the COVID-19 pandemic outbreak, with five different lineages dominating at various points during the outbreak. We identified South Africa, Kenya, and Nigeria as key sources of viral transmissions between Sub-Saharan African nations because they had the most SARS-CoV-2 genomes sampled and sequenced. These results shed light on the evolutionary dynamics of the circulating viral strains in Africa. Genomic surveillance is one of the important techniques in the pandemic preparedness toolbox and to better understand the molecular, evolutionary, epidemiological, and spatiotemporal dynamics of the COVID-19 pandemic in Africa, genomic surveillance activities across the continent must be expanded. The effectiveness of molecular surveillance as a method for tracking pandemics strongly depends on continuous and reliable sampling, speedy virus genome sequencing, and prompt reporting and we have to improve in all these aspects in Africa. Additionally, the pandemic breakout revealed that current land-border regulations aimed at limiting virus\u2019s international transmission are ineffective and a lot needs to be done to implement and improve our African land-borders as far as epidemiology is concerned in order to contain such outbreaks in the future.","version":"1.1","doi":"10.1101/2022.08.17.504290","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.15.22278603","pub_date":"2022-08-17","title":"Risk Factors Associated with Post-Acute Sequelae of SARS-CoV-2 in an EHR Cohort: A National COVID Cohort Collaborative (N3C) Analysis as part of the NIH RECOVER program","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>More than one-third of individuals experience post-acute sequelae of SARS-CoV-2 infection (PASC, which includes long-COVID).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To identify risk factors associated with PASC/long-COVID.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Retrospective case-control study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>31 health systems in the United States from the National COVID Cohort Collaborative (N3C).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Patients</jats:title>\n                  <jats:p>8,325 individuals with PASC (defined by the presence of the International Classification of Diseases, version 10 code U09.9 or a long-COVID clinic visit) matched to 41,625 controls within the same health system.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Measurements</jats:title>\n                  <jats:p>Risk factors included demographics, comorbidities, and treatment and acute characteristics related to COVID-19. Multivariable logistic regression, random forest, and XGBoost were used to determine the associations between risk factors and PASC.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Among 8,325 individuals with PASC, the majority were &gt;50 years of age (56.6%), female (62.8%), and non-Hispanic White (68.6%). In logistic regression, middle-age categories (40 to 69 years; OR ranging from 2.32 to 2.58), female sex (OR 1.4, 95% CI 1.33-1.48), hospitalization associated with COVID-19 (OR 3.8, 95% CI 3.05-4.73), long (8-30 days, OR 1.69, 95% CI 1.31-2.17) or extended hospital stay (30+ days, OR 3.38, 95% CI 2.45-4.67), receipt of mechanical ventilation (OR 1.44, 95% CI 1.18-1.74), and several comorbidities including depression (OR 1.50, 95% CI 1.40-1.60), chronic lung disease (OR 1.63, 95% CI 1.53-1.74), and obesity (OR 1.23, 95% CI 1.16-1.3) were associated with increased likelihood of PASC diagnosis or care at a long-COVID clinic. Characteristics associated with a lower likelihood of PASC diagnosis or care at a long-COVID clinic included younger age (18 to 29 years), male sex, non-Hispanic Black race, and comorbidities such as substance abuse, cardiomyopathy, psychosis, and dementia. More doctors per capita in the county of residence was associated with an increased likelihood of PASC diagnosis or care at a long-COVID clinic. Our findings were consistent in sensitivity analyses using a variety of analytic techniques and approaches to select controls.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>This national study identified important risk factors for PASC such as middle age, severe COVID-19 disease, and specific comorbidities. Further clinical and epidemiological research is needed to better understand underlying mechanisms and the potential role of vaccines and therapeutics in altering PASC course.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>KEY POINTS</jats:title>\n                  <jats:sec>\n                    <jats:title>Question</jats:title>\n                    <jats:p>What risk factors are associated with post-acute sequelae of SARS-CoV-2 (PASC) in the National COVID Cohort Collaborative (N3C) EHR Cohort?</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Findings</jats:title>\n                    <jats:p>This national study identified important risk factors for PASC such as middle age, severe COVID-19 disease, specific comorbidities, and the number of physicians per capita.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Meaning</jats:title>\n                    <jats:p>Clinicians can use these risk factors to identify patients at high risk for PASC while they are still in the acute phase of their infection and also to support targeted enrollment in clinical trials for preventing or treating PASC.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2022.08.15.22278603","journal":"medRxiv","score":null},{"id":"10.1101/2022.08.17.504157","pub_date":"2022-8-17","title":"Natural variations within the glycan shield of SARS-CoV-2 impact viral spike dynamics","abstract":"The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the glycosylation of a recombinant viral spike mimetic of the P.1 (Gamma) strain, which exhibits two additional N-linked glycan sites compared to the equivalent mimetic of the Wuhan strain. We determine the site-specific glycosylation of these variants and investigate the impact of these glycans by molecular dynamics. The N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the antigenic surface.","version":"1.1","doi":"10.1101/2022.08.17.504157","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.15.22278787","pub_date":"2022-08-17","title":"The impact of the Covid-19 pandemic on Italian population-based cancer screening activities and test coverage: results from national cross-sectional repeated surveys","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>In Italy, population-based screening programs for breast, cervical and colorectal cancers are mandatory, and Regions are in charge of their delivery. From March to May 2020, a severe lockdown was imposed due to the Covid-19 pandemic by the Italian Ministry of Health, with the suspension of screening programs. This paper describes the impact of the pandemic on Italian screening activities and test coverage in 2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>The regional number of subjects invited and of screening tests performed in 2020 were compared with those in 2019. Invitation and examination coverage were also calculated. PASSI surveillance system, through telephone interviews, investigated the population screening test coverage, before and during the pandemic, accordingly to educational attainment, perceived economic difficulties and citizenship.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>A reduction of subjects invited and tests performed, with differences among periods and geographic macro areas, was observed in 2020 vs. 2019. The reduction in examination coverage was larger than that in invitation coverage for all screening campaigns. From the second half of 2020, the trend for test coverage showed a decrease in all the macro areas for all the screening campaigns. Compared with the pre-pandemic period, there was a greater difference according to level of education in the odds of having had a test last year vs. never having been screened or not being up to date with screening tests. In addition, foreigners had less access to screening than Italians did.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The lockdown and the ongoing Covid-19 emergency caused an important delay in screening activities. This increased the pre-existing individual and geographical inequalities in access. The opportunistic screening did not mitigate the pandemic impact.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This study was partially supported by Italian Ministry of Health \u2013 Ricerca Corrente Annual Program 2023.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.08.15.22278787","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.13.476223","pub_date":"2022-8-16","title":"Genomic Biomarker Heterogeneities Between SARS-CoV-2 and COVID-19","abstract":"Genes functionally associated with SARS-CoV-2 infection and genes functionally related to COVID-19 disease can be different, whose distinction will become the first essential step for successfully fighting against the COVID-19 pandemic. Unfortunately, this first step has not been completed in all biological and medical research. Using a newly developed maxcompeting logistic classifier, two genes, ATP6V1B2 and IFI27, stand out to be critical in transcriptional response to SARS-CoV-2 infection with differential expressions derived from NP/OP swab PCR. This finding is evidenced by combining these two genes with one another gene in predicting disease status to achieve better-indicating accuracy than existing classifiers with the same number of genes. In addition, combining these two genes with three other genes to form a five-gene classifier outperforms existing classifiers with ten or more genes. These two genes can be critical in fighting against the COVID-19 pandemic as a new focus and direction with their exceptional predicting accuracy. Comparing the functional effects of these genes with a five-gene classifier with 100% accuracy identified and tested from blood samples in our earlier work, genes and their transcriptional response and functional effects to SARS-CoV-2 infection and genes and their functional signature patterns to COVID-19 antibody are significantly different, which can be interpreted as the former is the point of a phenomenon, and the latter is the essence of the disease. We will use a total of fourteen cohort studies (including breakthrough infections and omicron variants) with 1481 samples to justify our results. Such significant findings can help explore the causal and pathological clue between SARS-CoV-2 infection and COVID-19 disease and fight against the disease with more targeted genes, vaccines, antiviral drugs, and therapies.","version":"1.2","doi":"10.1101/2022.01.13.476223","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.15.503946","pub_date":"2022-8-16","title":"Collateral impacts of pandemic COVID-19 drive the nosocomial spread of antibiotic resistance","abstract":"Circulation of multidrug-resistant bacteria (MRB) in healthcare facilities is a major public health problem. These settings have been greatly impacted by the COVID-19 pandemic, notably due to surges in COVID-19 caseloads and the implementation of infection control measures. Yet collateral impacts of pandemic COVID-19 on MRB epidemiology remain poorly understood. Here, we present a dynamic transmission model in which SARS-CoV-2 and MRB co-circulate among patients and staff in a hospital population in an early pandemic context. Responses to SARS-CoV-2 outbreaks are captured mechanistically, reflecting impacts on factors relevant for MRB transmission, including contact behaviour, hand hygiene compliance, antibiotic prescribing and population structure. In a first set of simulations, broad parameter ranges are accounted for, representative of diverse bacterial species and hospital settings. On average, COVID-19 control measures coincide with MRB prevention, including fewer incident cases and fewer cumulative person-days of patient MRB colonization. However, surges in COVID-19 caseloads favour MRB transmission and lead to increased rates of antibiotic resistance, especially in the absence of concomitant control measures. In a second set of simulations, methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamase-producing Escherichia coli are simulated in specific hospital wards and pandemic response scenarios. Antibiotic resistance dynamics are highly context-specific in these cases, and SARS-CoV-2 outbreaks significantly impact bacterial epidemiology only in facilities with high underlying risk of bacterial transmission. Crucially, antibiotic resistance burden is reduced in facilities with timelier, more effective implementation of COVID-19 control measures. This highlights the control of antibiotic resistance as an important collateral benefit of robust pandemic preparedness. Impacts of COVID-19 on the spread of antibiotic resistance are poorly understood. Here, an epidemiological model accounting for the simultaneous spread of SARS-CoV-2 and antibiotic-resistant bacteria is presented. The model is tailored to healthcare settings during the first wave of the COVID-19 pandemic, and accounts for hand hygiene, inter-individual contact behaviour, and other factors relevant for pathogen spread. Simulations demonstrate that public health policies enacted to slow the spread of COVID-19 also tend to limit bacterial transmission. However, surges in COVID-19 cases simultaneously select for higher rates of antibiotic resistance. Selection for resistance is thus mitigated by prompt implementation of effective COVID-19 prevention policies. This highlights the control of antibiotic resistance as an important collateral benefit of pandemic preparedness.","version":"1.1","doi":"10.1101/2022.08.15.503946","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.16.504117","pub_date":"2022-8-16","title":"Intrahost SARS-CoV-2 k-mer identification method (iSKIM) for rapid detection of mutations of concern reveals emergence of global mutation patterns","abstract":"Despite unprecedented global sequencing and surveillance of SARS-CoV-2, timely identification of the emergence and spread of novel variants of concern (VoCs) remains a challenge. Several million raw genome sequencing runs are now publicly available. We sought to survey these datasets for intrahost variation to study emerging mutations of concern. We developed iSKIM (\u201cintrahost SARS-CoV-2 k-mer identification method\u201d) to relatively quickly and efficiently screen the many SARS-CoV-2 datasets to identify intrahost mutations belonging to lineages of concern. Certain mutations surged in frequency as intrahost minor variants just prior to, or while lineages of concern arose. The Spike N501Y change common to several VoCs was found as a minor variant in 834 samples as early as October 2020. This coincides with the timing of the first detected samples with this mutation in the Alpha/B.1.1.7 and Beta/B.1.351 lineages. Using iSKIM, we also found that Spike L452R was detected as an intrahost minor variant as early as September 2020, prior to the observed rise of the Epsilon/B.1.429/B.1.427 lineages in late 2020. iSKIM rapidly screens for mutations of interest in raw data, prior to genome assembly, and can be used to detect increases in intrahost variants, potentially providing an early indication of novel variant spread.","version":"1.1","doi":"10.1101/2022.08.16.504117","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442873","pub_date":"2022-8-16","title":"Optimized Quantification of Intrahost Viral Diversity in SARS-CoV-2 and Influenza Virus Sequence Data","abstract":"High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller, and use of replicate sequencing have the most significant impact on single nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false negative rates. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intrahost viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intrahost variation, viral diversity, and viral evolution. When viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in inclusion of false positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant calling tools. We used simulated and synthetic data to test their performance against a true set of variants, and then used these studies to inform variant identification in data from clinical SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.","version":"1.2","doi":"10.1101/2021.05.05.442873","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.06.487325","pub_date":"2022-8-16","title":"Delta-Omicron recombinant escapes therapeutic antibody neutralization","abstract":"The emergence of recombinant viruses is a threat to public health. Recombination of viral variants may combine variant-specific features that together catalyze viral escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. Multi-method amplicon and metagenomic sequencing of a clinical swab and the in vitro grown virus allowed for high-confidence detection of a novel recombinant variant. Mutational, phylogeographic, and structural analyses determined features of the recombinant genome and spike protein. Neutralization assays using infectious as well as pseudotyped viruses and point mutants thereof defined the recombinant\u2019s sensitivity to a panel of monoclonal antibodies and sera from vaccinated and/or convalescent individuals. A novel Delta-Omicron SARS-CoV-2 recombinant was identified in an unvaccinated, immunosuppressed kidney transplant recipient treated with monoclonal antibody Sotrovimab. The recombination breakpoint is located in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site, and results in a 5\u2019-Delta AY.45 and a 3\u2019-Omicron BA.1 mosaic spike protein. Delta and BA.1 are sensitive to Sotrovimab neutralization, whereas the Delta-Omicron recombinant is highly resistant to Sotrovimab, both with and without the RBD resistance mutation E340D. Recombination between circulating SARS-CoV-2 variants can functionally contribute to immune escape. It is critical to validate phenotypes of mosaic viruses and monitor immunosuppressed COVID-19 patients treated with monoclonal antibodies for the selection of recombinant and immune escape variants. (Funded by NYU, the National Institutes of Health, and others)","version":"1.2","doi":"10.1101/2022.04.06.487325","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.26.493529","pub_date":"2022-8-16","title":"The emergence of variants with increased fitness accelerates the slowdown of genome sequence heterogeneity in the SARS-CoV-2 coronavirus","abstract":"The author has withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2021/467547. Therefore, the author does not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.2","doi":"10.1101/2022.05.26.493529","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.13.503857","pub_date":"2022-8-15","title":"Antisense oligonucleotide to SARS-Cov-2 trs gene: antiviral activity on an in vitro model and possibilities of vital and postmortem diagnosis of COVID-19","abstract":"The data on the relevance of the 5 \u2018-AGC CGA GTG ACA GCC ACA CAG antisense oligonucleotide for binding to the trs-gene of the SARS-CoV-2 virus, which causes the new coronavirus infection COVID-19, are presented. The high stability and conservatism of this section of the SARS-CoV-2 genome is shown, which allows it to be used as an application point for antisense oligonucleotides. By evaluating plaque inhibition, the ability of this antisense oligonucleotide with phosphorothioate and 2\u2019-oxymethyl modification to suppress viral replication was found. The effective dosage reducing the virus titer by 50% is 3.84 mcg/ml. No toxicity was shown up to a dosage of 100 \u03bcg/mL, which is more than 28.8 chemotherapeutic index. The ability of this oligonucleotide conjugated to the fluorescent dye TAMRA to detect the SARS-CoV-2 virus in the fluorescent hybridization reaction in situ in cytological preparations of nasopharyngeal smears and blood smears, as well as in histological preparations of internal tissues is shown.","version":"1.1","doi":"10.1101/2022.08.13.503857","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.12.503822","pub_date":"2022-8-15","title":"The Spike protein of SARS-coV2 19B (S) clade mirrors critical features of viral adaptation and coevolution","abstract":"Pathogens including viruses evolve in tandem with diversity in their animal and human hosts. For SARS-coV2, the focus is generally for understanding such coevolution on the virus spike protein since it demonstrates high mutation rates compared to other genome regions, particularly in the receptor-binding domain (RBD). Viral sequences of the SARS-coV2 19B (S) clade and variants of concern from different continents, were investigated, with a focus on the A.29 lineage which presented with different mutational patterns within the 19B (S) lineages in order to learn more about how SARS-coV2 may have evolved and adapted to widely diverse populations globally. Results indicated that SARS-coV2 went through evolutionary constrains and intense selective pressure, particularly in Africa. This was manifested in a departure from neutrality with excess nonsynonymous mutations and a negative Tajima D consistent with rapid expansion and directional selection as well as deletion and deletion-frameshifts in the N-terminal domain (NTD region) of the spike protein. In conclusion, viral transmission during epidemics through population of diverse genomic structure and marked complexity may be a significant factor for the virus to acquire distinct patterns of mutations within these populations in order to ensure its survival and fitness, hence in the emergence of novel variants and strains. In this study, we justify the fact that the virus\u2019s evolution varies across continents, with each continent showing different amounts and patterns of mutations and deletions, which was manifested in the 19B (S) clade of SARS-coV2, particularly in areas with high population complexity, such as Africa, despite the low rate of sampling and data sharing. The findings show that SARS-coV2 was subject to evolutionary constraints and intense selective pressure. This study will contribute to the scanty amount of research on the SARS-coV2 coevolution and adaptation, in which the host variation is of great significance in understanding the intricacies of viral host coevolution.","version":"1.1","doi":"10.1101/2022.08.12.503822","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.14.503921","pub_date":"2022-8-15","title":"Evasion of Neutralizing Antibody Response by the SARS-CoV-2 BA.2.75 Variant","abstract":"The newly emerged BA.2.75 SARS-CoV-2 variant exhibits an alarming 9 additional mutations in its spike (S) protein compared to the ancestral BA.2 variant. Here we examine the neutralizing antibody escape of BA.2.75 in mRNA-vaccinated and BA.1-infected individuals, as well as the molecular basis underlying functional changes in the S protein. Notably, BA.2.75 exhibits enhanced neutralization resistance over BA.2, but less than the BA.4/5 variant. The G446S and N460K mutations of BA.2.75 are primarily responsible for its enhanced resistance to neutralizing antibodies. The R493Q mutation, a reversion to the prototype sequence, reduces BA.2.75 neutralization resistance. The mutational impact is consistent with their locations in common neutralizing antibody epitopes. Further, the BA.2.75 variant shows enhanced cell-cell fusion over BA.2, driven largely by the N460K mutation, which enhances S processing. Structural modeling revealed a new receptor contact introduced by N460K, supporting a mechanism of potentiated receptor utilization and syncytia formation.","version":"1.1","doi":"10.1101/2022.08.14.503921","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.12.503154","pub_date":"2022-8-15","title":"CD98 is critical for a conserved inflammatory response to diverse injury stimuli relevant to IPF exacerbations and COVID pneumonitis","abstract":"Progressive fibrosing interstitial lung diseases (PFILDs) cause substantial morbidity and mortality. Antifibrotic agents slow progression, but most of the clinical need remains unmet. The archetypal PFILD is idiopathic pulmonary fibrosis (IPF). Chronic progression is driven by transforming growth factor (TGF-)\u03b21 signalling. It is punctuated by inflammatory flares known as acute exacerbations (AE-IPF), which are associated with accelerated decline and high mortality. We hypothesized that acute injury responses underlying exacerbations and the mechanisms of chronic fibrosis overlap at the molecular level, via a cell surface assembly nucleated by galectin-3 that we term the \u2018gal-3-fibrosome\u2019. We focused upon a putative pro-inflammatory galectin-3 ligand, the CD98:integrin complex. Our data indicate CD98 and \u03b21-integrin co-localise with galectin-3 within epithelial cells in IPF lung tissue, and within 40 nm in human lung tissue treated with TGF-\u03b21 compared to controls. CD98 is required for interleukin (IL-)6 and IL-8 responses to biochemical and biophysical conditions mimicking stimuli of AE-IPF in vivo, ex vivo and in cells, and for an interstitial neutrophilic response in a mouse model. We demonstrate this pathway progresses via intracellular influx of Ca2+ mediated by TRPV4, and NF-\u03baB activation, operating in positive feedback. Lastly we show the CD98- and galectin-3-dependence of IL-6 and IL-8 responses to the SARS-CoV-2 spike protein receptor binding domain and the conservation of this response pattern between lung epithelial cells and monocyte-derived macrophages. Taken together our findings identify CD98 as a key mediator of both pro-fibrotic and acute inflammatory responses in the lung with relevance to AE- and chronic progression of IPF, and the priming of fibrotic lungs for acute inflammatory responses. They similarly implicate CD98 and galectin-3 as mediators of COVID pneumonitis and worse outcomes in ILD patients with COVID.","version":"1.1","doi":"10.1101/2022.08.12.503154","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.11.503574","pub_date":"2022-8-12","title":"SARS-CoV-2 specific plasma cells acquire the phenotype of long-lived plasma cells in the human bone marrow","abstract":"Establishment of long-lived plasma cells (PC) in the bone marrow (BM) is important for the development of long-term specific humoral immunity. While SARS-CoV-2-specific, resting, affinity-matured, IgG-secreting plasma cells were described in human bone marrow approx. 6-7 months after infection or vaccination, the long-term durability of these PC remains unclear. We here show that approximately 20% of SARS-CoV-2-specific human BM plasma cells, including RBD-specific PC accommodate the phenotype of long-lived plasma cells, characterized by the lack of CD19 and/or CD45. This result provides evidence in support of the emergence of persistent SARS-CoV-2 specific plasma cells in humans sustaining the durable production of specific serum IgG protecting against severe courses of COVID-19.","version":"1.1","doi":"10.1101/2022.08.11.503574","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.11.503706","pub_date":"2022-8-12","title":"The P681H mutation in the Spike glycoprotein escapes IFITM restriction and is necessary for type I interferon resistance in the SARS-CoV-2 alpha variant","abstract":"The appearance of new dominant variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the COVID-19 pandemic. Of these, the alpha variant (also known as B.1.1.7) that appeared initially in the UK became the dominant variant in much of Europe and North America in the first half of 2021. The Spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation in the polybasic cleavage site that has been suggested to enhance S cleavage. Here, we show that the alpha S protein confers a level of resistance to the effects of interferon-\u03b2 (IFN\u03b2) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN\u03b2 and context-dependent resistance to IFITMs in the alpha S. However, while this appears to confer changes in sensitivity to endosomal protease inhibition consistent with enhanced cell-surface entry, its reversion does not reduce cleaved S incorporation into particles, indicating a role downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOCs may well also confer replication and/or transmission advantage through adaptation to resist innate immune mechanisms. The emergence of Variants of Concern of SARS-CoV-2 has been a key challenge in the global response to the COVID-19 pandemic. Accumulating evidence suggests VOCs are being selected to evade the human immune response, with much interest focussed on mutations in the Spike protein that escape from neutralizing antibody responses. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type-1 interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2, and enhancement by its paralogue IFITM3, that block virus entry into target cells. The key determinant of this is a proline to histidine change at position 681 in S adjacent to the furin-cleavage site that we have shown previously modulates IFITM2 sensitivity. Unlike other VOCs, in the context of the alpha spike, P681H modulates cell entry pathways of SARS-CoV-2, further reducing its dependence one endosomal proteases. Reversion of position 681 to a proline in viruses bearing the alpha spike is sufficient to restore interferon and IFITM2 sensitivity without reducing furin-mediated spike cleavage, suggesting post cleavage conformational changes in S are changing the viral entry pathway and therefore sensitivity to interferon. These data highlight the dynamic nature of the SARS CoV-2 S as it adapts to both innate and adaptive immunity in the human population.","version":"1.1","doi":"10.1101/2022.08.11.503706","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.11.503601","pub_date":"2022-8-12","title":"Memory B cell responses to Omicron subvariants after SARS-CoV-2 mRNA breakthrough infection","abstract":"Individuals that receive a 3rd mRNA vaccine dose show enhanced protection against severe COVID19 but little is known about the impact of breakthrough infections on memory responses. Here, we examine the memory antibodies that develop after a 3rd or 4th antigenic exposure by Delta or Omicron BA.1 infection, respectively. A 3rd exposure to antigen by Delta breakthrough increases the number of memory B cells that produce antibodies with comparable potency and breadth to a 3rd mRNA vaccine dose. A 4th antigenic exposure with Omicron BA.1 infection increased variant specific plasma antibody and memory B cell responses. However, the 4th exposure did not increase the overall frequency of memory B cells or their general potency or breadth compared to a 3rd mRNA vaccine dose. In conclusion, a 3rd antigenic exposure by Delta infection elicits strain-specific memory responses and increases in the overall potency and breadth of the memory B cells. In contrast, the effects of a 4th antigenic exposure with Omicron BA.1 is limited to increased strain specific memory with little effect on the potency or breadth of memory B cell antibodies. The results suggest that the effect of strain-specific boosting on memory B cell compartment may be limited.","version":"1.1","doi":"10.1101/2022.08.11.503601","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.10.503531","pub_date":"2022-8-11","title":"Development of highly potent non-covalent inhibitors of SARS-CoV-2 3CLpro","abstract":"The SARS-CoV-2 virus is the causal agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19). There is an urgent need for potent, specific antiviral compounds against SARS-CoV-2. The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses, and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, non-covalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment. A oral non-covalent inhibitor of 3C-like protease effectively inhibits SARS-CoV-2 replication.","version":"1.1","doi":"10.1101/2022.08.10.503531","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.10.503534","pub_date":"2022-8-11","title":"A single-administration therapeutic interfering particle reduces SARS-CoV-2 viral shedding and pathogenesis in hamsters","abstract":"The high transmissibility of SARS-CoV-2 is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, post-exposure TIP dose lowers SARS-CoV-2 nasal shedding and at 5 days post-infection infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Co-housed \u2018contact\u2019 animals exposed to infected, TIP-treated, animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals co-housed with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission. COVID-19 vaccines are exceptionally effective in preventing severe disease and death, but they have mixed efficacy in preventing virus transmission, consistent with established literature that parenteral vaccines for other viruses fail to prevent mucosal virus shedding or transmission. Likewise, small-molecule antivirals, while effective in reducing viral-disease pathogenesis, also appear to have inconsistent efficacy in preventing respiratory virus transmission including for SARS-CoV-2. Recently, we reported the discovery of a single-administration antiviral Therapeutic Interfering Particle (TIP) against SARS-CoV-2 that prevents severe disease in hamsters and exhibits a high genetic barrier to the evolution of resistance. Here, we report that TIP intervention also reduces SARS-CoV-2 transmission between hamsters.","version":"1.1","doi":"10.1101/2022.08.10.503534","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.11.503553","pub_date":"2022-8-11","title":"Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein","abstract":"Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.","version":"1.1","doi":"10.1101/2022.08.11.503553","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.10.22278636","pub_date":"2022-08-11","title":"Phylodynamics of SARS-CoV-2 transmissions in France, Europe and the world during 2020","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Although France was one of the most affected European countries by the COVID-19 pandemic in 2020, the dynamics of SARS-CoV-2 transmissions within France, Europe and worldwide remain only partially characterized during the first year of the pandemic.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Here, we analyzed GISAID deposited sequences from January to December 2020 (n = 638,706 sequences). To tackle the huge number of sequences without the bias of analyzing a single sequence subset, we produced 100 independent and randomly selected sequence datasets and related phylogenetic trees for different geographic scales (worldwide, European countries and French administrative regions) and time periods (first and second half of 2020). We applied a maximum likelihood discrete trait phylogeographic method to date transmission events and to estimate the geographic spread of SARS-CoV-2 to, from and within France, Europe and worldwide.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The results unraveled two different patterns of inter- and intra-territory transmission events between the first and second half of 2020. Throughout the year, Europe was systematically associated with most of the intercontinental transmissions, for which France has played a pivotal role. SARS-CoV-2 transmissions with France were concentrated with North America and Europe (mainly Italy, Spain, United Kingdom, Belgium and Germany) during the first wave, and were limited to neighboring countries without strong intercontinental transmission during the second one. Regarding French administrative regions, the Paris area was the main source of transmissions during the first wave. But, for the second epidemic wave, it equally contributed to virus spread with Lyon and Marseille area, the two other most densely populated cities in France.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>By enabling the inclusion of tens of thousands of viral sequences, this original phylogenetic strategy enabled us to robustly depict SARS-CoV-2 transmissions through France, Europe and worldwide in 2020.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.08.10.22278636","journal":"medRxiv","score":null},{"id":"10.1101/2022.08.11.503614","pub_date":"2022-8-11","title":"Interfering with nucleotide excision by the coronavirus 3\u2019-to-5\u2019 exoribonuclease","abstract":"Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3\u2019-to-5\u2019 proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3\u2019-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3\u2019-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.","version":"1.1","doi":"10.1101/2022.08.11.503614","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.09.503429","pub_date":"2022-8-10","title":"Stability of SARS-CoV-2 in cold-chain transportation environments and the efficacy of disinfection measures","abstract":"Cold-chain environment could extend the survival duration of SARS-CoV-2 and increases the risk of transmission. However, the effect of clod-chain environmental factors and packaging materials on SARS-CoV-2 stability and the efficacy of intervention measures to inactivate SARS-CoV-2 under cold-chain environment remains uncertain. This study aimed to unravel cold-chain environmental factors that preserved the stability of SARS-CoV-2 and disinfection measures against SARS-CoV-2 under the cold-chain environment. The spike gene of SARS-CoV-2 isolated from Wuhan hu-1 was used to construct the SARS-CoV-2 pseudovirus and used as model of the SARS-CoV-2 virus. The decay rate of SARS-CoV-2 pseudovirus in the cold-chain environment, various types of packaging material surfaces i.e., PE plastic, stainless steel, Teflon and cardboard, and in frozen seawater was investigated. The influence of LED visible light(wavelength 450 nm-780 nm) and airflow movement on the stability of SARS-CoV-2 pseudovirus at -18\u00b0 C were subsequently assessed. The results show that SARS-CoV-2 pseudovirus decayed more rapidly on porous cardboard surface compared with the non-porous surfaces including PE plastic, stainless steel and Teflon. Compared with 25\u00b0 C, the decay rate of SARS-CoV-2 pseudovirus was significantly lower at low temperature. Seawater preserved viral stability both at -18\u00b0 C and repeated freeze-thawing cycles compared with deionized water. LED visible light illumination and airflow movement environment at -18\u00b0 C reduced the SARS-CoV-2 pseudovirus stability. In conclusion, our results indicate cold-chain temperature and seawater as risk factors for SARS-CoV-2 transmission and LED visible light illumination and airflow movement as possible disinfection measures of SARS-CoV-2 under the cold-chain environment. It is widely recognized that low temperature is a condition for maintaining virus vitality, and cold-chain transportation spreads the events of the SARS-CoV-2 were reported. This study provides that the decay rate of the SARS-CoV-2 pseudovirus at low temperatures varies on different packaging materials, and salt ions present in frozen foods such as seafood may protect virus survival. These results provide evidence for the possibility of SARS-CoV-2 transmission through cold-chain transport and also suggest the importance for disinfection of items. However, the commonly used disinfection methods of ultraviolet radiation and chemical reagents are generally not suitable for the disinfection of frozen food. Our study shows LED visible light illumination and airflow movement as possible disinfection measures of SARS-CoV-2 under the cold-chain environment. This has implications for reducing the long-distance transmission of the virus through cold-chain transportation.","version":"1.1","doi":"10.1101/2022.08.09.503429","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.27.478063","pub_date":"2022-8-10","title":"Ultraviolet Dosage and Decontamination Efficacy was Widely Variable across 14 UV Devices after Testing a Dried Enveloped Ribonucleic Acid Virus Surrogate for SARS-CoV-2","abstract":"The dosages and efficacy of 14 ultraviolet (UV) decontamination technologies were measured against a SARS-CoV-2 surrogate virus that was dried on to different materials for lab and field testing. A live enveloped, ribonucleic acid (RNA) virus surrogate for SARS- CoV-2 was dried on stainless steel 304 (SS304), Navy Top Coat-painted SS304 (NTC), cardboard, polyurethane, polymethyl methacrylate (PMMA), and acrylonitrile butadiene styrene (ABS) at > 8.0 log10 plaque-forming units (PFU) per test coupon. The coupons were then exposed to UV radiation during both lab and field testing. Commercial and prototype UV- emitting devices were measured for efficacy: 4 handheld devices, 3 room/surface-disinfecting machines, 5 air-disinfection devices, and 2 larger custom-made machines. UV device dosages ranged from 0.01-729 mJ cm-2. Anti-viral efficacy among the different UV devices ranged from no decontamination up to nearly achieving sterilization. Importantly, cardboard required far more dosage than SS304. Enormous variability in dosage and efficacy was measured among the different UV devices. Porous materials limit the utility of UV decontamination. UV devices have wide variability in dosages, efficacy, hazards, and UV output over time indicating that each UV device needs independent technical measurement and assessment for product development, prior to, and during use.","version":"1.2","doi":"10.1101/2022.01.27.478063","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.09.503400","pub_date":"2022-8-10","title":"Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps","abstract":"The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells (\u201creceptor traps\u201d). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.","version":"1.1","doi":"10.1101/2022.08.09.503400","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.09.503384","pub_date":"2022-8-10","title":"Further humoral immunity evasion of emerging SARS-CoV-2 BA.4 and BA.5 subvariants","abstract":"Multiple BA.4 and BA.5 subvariants with R346 mutations on the spike glycoprotein have been identified in various countries, such as BA.4.6/BF.7 harboring R346T, BA.4.7 harboring R346S, and BA.5.9 harboring R346I. These subvariants, especially BA.4.6, exhibit substantial growth advantages compared to BA.4/BA.5. In this study, we showed that BA.4.6, BA.4.7, and BA.5.9 displayed higher humoral immunity evasion capability than BA.4/BA.5, causing 1.5 to 1.9-fold decrease in NT50 of the plasma from BA.1 and BA.2 breakthrough-infection convalescents compared to BA.4/BA.5. Importantly, plasma from BA.5 breakthrough-infection convalescents also exhibits significant neutralization activity decrease against BA.4.6, BA.4.7, and BA.5.9 than BA.4/BA.5, showing on average 2.4 to 2.6-fold decrease in NT50. For neutralizing antibody drugs, Bebtelovimab remains potent, while Evusheld is completely escaped by these subvariants. Together, our results rationalize the prevailing advantages of the R346 mutated BA.4/BA.5 subvariants and urge the close monitoring of these mutants, which could lead to the next wave of the pandemic.","version":"1.1","doi":"10.1101/2022.08.09.503384","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.18.500332","pub_date":"2022-8-10","title":"Characterizations of enhanced infectivity and antibody evasion of Omicron BA.2.75","abstract":"Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a local growth advantage over BA.2.38, BA.2.76 and BA.5 in India. The underlying mechanism of BA.2.75\u2019s enhanced infectivity, especially compared to BA.5, remains unclear. Here, we show that BA.2.75 exhibits substantially higher ACE2-binding affinity than BA.5. Also, BA.2.75 spike shows decreased thermostability and increased \u201cup\u201d RBD conformation in acidic conditions, suggesting enhanced low-pH-endosomal cell-entry pathway utilization. BA.2.75 is less humoral immune evasive than BA.4/BA.5 in BA.1/BA.2 breakthrough-infection convalescents; however, BA.2.75 shows heavier neutralization evasion in Delta breakthrough-infection convalescents. Importantly, plasma from BA.5 breakthrough infection exhibit significantly weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75\u2019s distinct RBD and NTD-targeting antibody escaping pattern from BA.4/BA.5. Additionally, Evusheld and Bebtelovimab remain effective against BA.2.75, and Sotrovimab recovered RBD-binding affinity. Together, our results suggest BA.2.75 may prevail after the global BA.4/BA.5 wave, and its increased receptor-binding capability could allow further incorporation of immune-evasive mutations.","version":"1.2","doi":"10.1101/2022.07.18.500332","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.08.503239","pub_date":"2022-8-09","title":"A replicon RNA vaccine induces durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned","abstract":"The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.","version":"1.1","doi":"10.1101/2022.08.08.503239","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.09.503302","pub_date":"2022-8-09","title":"Spike-specific CXCR3+ TFH cells play a dominant functional role in supporting antibody responses in SARS-CoV-2 infection and vaccination","abstract":"CD4+ T follicular helper (TFH) cells are required for high-quality antibody generation and maintenance. However, the longevity and functional role of these cells are poorly defined in COVID-19 convalescents and vaccine recipients. Here, we longitudinally investigated the dynamics and functional roles of spike-specific circulating TFH cells and their subsets in convalescents at the 2nd, 5th, 8th, 12th and 24th months after COVID-19 symptom onset and in vaccinees after two and three doses of inactivated vaccine. SARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, of which spike-specific CXCR3+ TFH cells but not spike-specific CXCR3\u2212 TFH cells and neutralizing antibodies were persistent for at least two years in more than 80% of convalescents who experienced symptomatic COVID-19, which was well coordinated between spike-specific TFH cell and antibody responses at the 5th month after infection. Inactivated vaccine immunization also induced spike-specific TFH cell and antibody responses; however, these responses rapidly declined after six months with a two-dose standard administration, and a third dose significantly promoted antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells exhibited better responsiveness than spike-specific CXCR3\u2212 TFH cells upon spike protein stimulation in vitro and showed superior capacity in supporting spike-specific antibody secreting cell (ASC) differentiation and antibody production than spike-specific CXCR3\u2212 TFH cells cocultured with autologous memory B cells. In conclusion, spike-specific CXCR3+ TFH cells played a dominant functional role in antibody elicitation and maintenance in SARS-CoV-2 infection and vaccination, suggesting that induction of CXCR3-biased spike-specific TFH cell differentiation will benefit SARS-CoV-2 vaccine development aiming to induce long-term protective immune memory. SARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, which persisted for at least two years in the majority of symptomatic COVID-19 convalescent patients. Inactivated vaccine immunization also elicited spike-specific TFH cell and antibody responses, which rapidly declined over time, and a third dose significantly promoted antibody maturation and potency. Spike-specific CXCR3+ TFH cells exhibited more durable responses than spike-specific CXCR3\u2212 TFH cells, correlated with antibody responses and showed superior capacity in supporting ASC differentiation and antibody production than spike-specific CXCR3\u2212 TFH cells.","version":"1.1","doi":"10.1101/2022.08.09.503302","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.09.503270","pub_date":"2022-8-09","title":"Acetylsalicylic acid and Salicylic acid inhibit SARS-CoV-2 replication in precision-cut lung slices","abstract":"Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses and at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analyzed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and in a patient-near replication system using human precision-cut lung slices by two orders of magnitude. The compounds did not interfere with viral entry but led to lower viral RNA expression after 24 h.","version":"1.1","doi":"10.1101/2022.08.09.503270","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.08.503231","pub_date":"2022-8-09","title":"Vitamin B12 attenuates leukocyte inflammatory signature in COVID-19 via methyl-dependent changes in epigenetic marks","abstract":"COVID-19 induces chromatin remodeling in host immune cells, and it had previously been shown that vitamin B12 downregulates some inflammatory genes via methyl-dependent epigenetic mechanisms. In this work, whole blood cultures from moderate or severe COVID-19 patients were used to assess the potential of B12 as adjuvant drug. The vitamin normalized the expression of a panel of inflammatory genes still dysregulated in the leukocytes despite glucocorticoid therapy during hospitalization. B12 also increased the flux of the sulfur amino acid pathway, raising the bioavailability of methyl. Accordingly, B12-induced downregulation of CCL3 strongly and negatively correlated with the hypermethylation of CpGs in its regulatory regions. Transcriptome analysis revealed that B12 attenuates the effects of COVID-19 on most inflammation-related pathways affected by the disease. As far as we are aware, this is the first study to demonstrate that pharmacological modulation of epigenetic marks in leukocytes favorably regulates central components of COVID-19 physiopathology. B12 has great potential as an adjuvant drug for alleviating inflammation in COVID-19.","version":"1.1","doi":"10.1101/2022.08.08.503231","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.06.503050","pub_date":"2022-8-08","title":"The Defenders of the Alveolus Succumb in COVID-19 Pneumonia to SARS-CoV-2, Necroptosis, Pyroptosis and Panoptosis","abstract":"The alveolar type II (ATII) pneumocyte has been called the defender of the alveolus because, amongst the cell\u2019s many important roles, repair of lung injury is particularly critical. We investigated the extent to which SARS-CoV-2 infection incapacitates the ATII reparative response in fatal COVID-19 pneumonia, and describe massive infection and destruction of ATI and ATII cells. We show that both type I interferon-negative infected ATII and type I-interferon-positive uninfected ATII cells succumb to TNF-induced necroptosis, BTK-induced pyroptosis and a new PANoptotic hybrid form of inflammatory cell death that combines apoptosis, necroptosis and pyroptosis in the same cell. We locate pathway components of these cell death pathways in a PANoptosomal latticework that mediates emptying and disruption of ATII cells and destruction of cells in blood vessels associated with microthrombi. Early antiviral treatment combined with inhibitors of TNF and BTK could preserve ATII cell populations to restore lung function and reduce hyperinflammation from necroptosis, pyroptosis and panoptosis. In fatal COVID-19 pneumonia, the initial destruction of Type II alveolar cells by SARS-CoV-2 infection is amplified by infection of the large numbers of spatially contiguous Type II cells supplied by the proliferative reparative response. Interferon-negative infected cells and interferon-positive uninfected cells succumb to inflammatory forms of cell death, TNF-induced necroptosis, BTK-induced pyroptosis, and PANoptosis. All of the cell death pathway components, including a recently identified NINJ1 component, are localized in a PANoptosome latticework that empties in distinctive patterns to generate morphologically distinguishable cell remnants. Early combination treatment with inhibitors of SARS-CoV-2 replication, TNF and BTK could reduce the losses of Type II cells and preserve a reparative response to regenerate functional alveoli.","version":"1.1","doi":"10.1101/2022.08.06.503050","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472252","pub_date":"2022-8-08","title":"Improved neutralisation of the SARS-CoV-2 Omicron variant after Pfizer-BioNTech (BNT162b2) COVID-19 vaccine boosting with a third dose","abstract":"In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired 15 mutations in the receptor binding domain of the spike protein, raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three and six months post-two doses of Pfizer-BioNTech BNT162b2 has a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres are boosted. Despite this increase, neutralising antibody titres are reduced four-fold for Omicron compared to lineage A.2.2 SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.12.12.472252","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.07.503115","pub_date":"2022-8-08","title":"Virological characteristics of the SARS-CoV-2 Omicron BA.2.75","abstract":"SARS-CoV-2 Omicron BA.2.75 emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically different from BA.5, the currently predominant BA.2 descendant. Here, we showed that the effective reproduction number of BA.2.75 is greater than that of BA.5. While the sensitivity of BA.2.75 to vaccination- and BA.1/2 breakthrough infection-induced humoral immunity was comparable to that of BA.2, the immunogenicity of BA.2.75 was different from that of BA.2 and BA.5. Three clinically-available antiviral drugs were effective against BA.2.75. BA.2.75 spike exhibited a profound higher affinity to human ACE2 than BA.2 and BA.5 spikes. The fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were comparable to those of BA.5 but were greater than those of BA.2. Our multiscale investigations suggest that BA.2.75 acquired virological properties independently of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.","version":"1.1","doi":"10.1101/2022.08.07.503115","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.06.503039","pub_date":"2022-8-08","title":"A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations","abstract":"The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.","version":"1.1","doi":"10.1101/2022.08.06.503039","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.05.22278464","pub_date":"2022-08-08","title":"Differential immune response induced by two immunization schedules with an inactivated SARS-CoV-2 vaccine in a randomized phase 3 clinical trial","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>\n                    The development of vaccines to control the COVID-19 pandemic progression is a worldwide priority. CoronaVac\n                    <jats:sup>\u00ae</jats:sup>\n                    is an inactivated SARS-CoV-2 vaccine approved for emergency use with robust efficacy and immunogenicity data reported in trials in China, Brazil, Indonesia, Turkey, and Chile.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    This study is a randomized, multicenter, and controlled phase 3 trial in healthy Chilean adults aged \u226518 years. Volunteers received two doses of CoronaVac\n                    <jats:sup>\u00ae</jats:sup>\n                    separated by two (0-14 schedule) or four weeks (0-28 schedule). 2,302 volunteers were enrolled, 440 were part of the immunogenicity arm, and blood samples were obtained at different times. Samples from a single center are reported. Humoral immune responses were evaluated by measuring the neutralizing capacities of circulating antibodies. Cellular immune responses were assessed by ELISPOT and flow cytometry. Correlation matrixes were performed to evaluate correlations in the data measured.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Both schedules exhibited robust neutralizing capacities with the response induced by the 0-28 schedule being better. No differences were found in the concentration of antibodies against the virus and different variants of concern between schedules. Stimulation of PBMCs with MPs induced the secretion of IFN-\u03b3 and the expression of activation induced markers for both schedules. Correlation matrixes showed strong correlations between neutralizing antibodies and IFN-\u03b3 secretion.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    Immunization with CoronaVac\n                    <jats:sup>\u00ae</jats:sup>\n                    in Chilean adults promotes robust cellular and humoral immune responses. The 0-28 schedule induced a stronger humoral immune response than the 0-14 schedule.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Ministry of Health, Government of Chile, Confederation of Production and Commerce &amp; Millennium Institute on Immunology and Immunotherapy, Chile.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Clinical trial number</jats:title>\n                  <jats:p>NCT04651790.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>summary</jats:title>\n                  <jats:p>\n                    Two immunization schedules were evaluated for the inactivated SARS-CoV-2 vaccine, Coronavac\n                    <jats:sup>\u00ae</jats:sup>\n                    , with two doses of the vaccine separated by two or four weeks. We compared humoral and cellular immune responses, showing they are mostly similar, with differences in neutralization capacities.\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.08.05.22278464","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.29.502055","pub_date":"2022-8-08","title":"Neutralization of SARS-CoV-2 Omicron sublineages by 4 doses of mRNA vaccine","abstract":"Since the initial emergence of SARS-CoV-2 Omicron BA.1, several Omicron sublineages have emerged, leading to BA.5 as the current dominant sublineage. Here we report the neutralization of different Omicron sublineages by human sera collected from individuals who had distinct mRNA vaccination and/or BA.1 infection. Four-dose-vaccine sera neutralize the original USA-WA1/2020, Omicron BA.1, BA.2, BA.212.1, BA.3, and BA.4/5 viruses with geometric mean titers (GMTs) of 1554, 357, 236, 236, 165, and 95, respectively; 2-dose-vaccine-plus-BA.1-infection sera exhibit GMTs of 2114, 1705, 730, 961, 813, and 274, respectively; and 3-dose-vaccine-plus-BA.1-infection sera show GMTs of 2962, 2038, 983, 1190, 1019, and 297, respectively. Thus, 4-dose-vaccine elicits the lowest neutralization against BA.5; 2-dose-vaccine-plus-BA.1-infection elicits significantly higher GMTs against Omicron sublineages than 4-dose-vaccine; and 3-dose-vaccine-plus-BA.1-infection elicits slightly higher GMTs (statistically insignificant) than the 2-dose-vaccine-plus-BA.1-infection. Finally, compared with BA.5, the newly emerged BA.2.75 is equally evasive of 4-dose-vaccine-elicited neutralization, but more susceptible to 3-dose-vaccine-plus-BA.1-infection-elicited neutralization.","version":"1.2","doi":"10.1101/2022.07.29.502055","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.479924","pub_date":"2022-8-08","title":"SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff","abstract":"In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. Nevertheless, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains elusive. We set out to characterize the interactome of full-length Nsp1 and its topology by crosslinking mass spectrometry in order to investigate the role of the N-terminal domain and linker regions in host translational shutoff. We find that these regions are in contact with 40S proteins lining the mRNA entry channel and detect a novel interaction with the G subunit of the eIF3 complex. The crosslink-derived distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit, reporting on the dynamic interface between Nsp1, the ribosome and the eIF3 complex. The significance of the Nsp1-eIF3G interaction is supported by further evidence that Nsp1 predominantly binds to 40-43S complexes. Our results point towards a mechanism by which Nsp1 is preferentially recruited to canonical initiation complexes, leading to subsequent mRNA degradation.","version":"1.2","doi":"10.1101/2022.02.10.479924","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.08.503075","pub_date":"2022-8-08","title":"Human immune and gut microbial parameters associated with inter-individual variations in COVID-19 mRNA vaccine-induced immunity","abstract":"COVID-19 mRNA vaccines induce protective adaptive immunity against SARS-CoV-2 in most individuals, but there is wide variation in levels of vaccine-induced antibody and T-cell responses. However, factors associated with this inter-individual variation remain unclear. Here, using a systems biology approach based on multi-omics analyses of human blood and stool samples, we find that baseline expression of AP-1 transcription factors, FOS and ATF3, is inversely correlated with BNT162b2 mRNA vaccine-induced T-cell responses. FOS expression is associated with transcription modules related to baseline immunity, but it is negatively associated with those related to T-cell activation upon BNT162b2 mRNA stimulation. Interestingly, the gut microbial fucose/rhamnose degradation pathway is positively correlated with FOS and ATF3 expression and inversely correlated with BNT162b2-induced T-cell responses. Taken together, these results demonstrate that baseline expression of AP-1 genes, which is associated with the gut microbial fucose/rhamnose degradation pathway, is a key negative correlate of BNT162b2-induced T-cell responses.","version":"1.1","doi":"10.1101/2022.08.08.503075","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.06.503019","pub_date":"2022-8-08","title":"Evolutionary progression of collective mutations in Omicron sub-lineages towards efficient RBD-hACE2: allosteric communications between and within viral and human proteins","abstract":"The interaction between the Spike (S) protein of SARS-CoV-2 and the human angiotensin converting enzyme 2 (hACE2) is essential for infection, and is a target for neutralizing antibodies. Consequently, selection of mutations in the S protein is expected to be driven by the impact on the interaction with hACE2 and antibody escape. Here, for the first time, we systematically characterized the collective effects of mutations in each of the Omicron sub-lineages (BA.1, BA.2, BA.3 and BA.4) on both the viral S protein receptor binding domain (RBD) and the hACE2 protein using post molecular dynamics studies and dynamic residue network (DRN) analysis. Our analysis suggested that Omicron sub-lineage mutations result in altered physicochemical properties that change conformational flexibility compared to the reference structure, and may contribute to antibody escape. We also observed changes in the hACE2 substrate binding groove in some sub-lineages. Notably, we identified unique allosteric communication paths in the reference protein complex formed by the DRN metrics betweenness centrality and eigencentrality hubs, originating from the RBD core traversing the receptor binding motif of the S protein and the N-terminal domain of the hACE2 to the active site. We showed allosteric changes in residue network paths in both the RBD and hACE2 proteins due to Omicron sub-lineage mutations. Taken together, these data suggest progressive evolution of the Omicron S protein RBD in sub-lineages towards a more efficient interaction with the hACE2 receptor which may account for the increased transmissibility of Omicron variants.","version":"1.1","doi":"10.1101/2022.08.06.503019","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.05.22278466","pub_date":"2022-08-06","title":"Performance of Rapid Antigen Tests to Detect Symptomatic and Asymptomatic SARS-CoV-2 Infection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Performance of rapid antigen tests for SARS-CoV-2 (Ag-RDT) varies over the course of an infection, and their performance in screening for SARS-CoV-2 is not well established. We aimed to evaluate performance of Ag-RDT for detection of SARS-CoV-2 for symptomatic and asymptomatic participants.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Participants &gt;2 years old across the United States enrolled in the study between October 2021 and February 2022. Participants completed Ag-RDT and molecular testing (RT-PCR) for SARS-CoV-2 every 48 hours for 15 days. This analysis was limited to participants who were asymptomatic and tested negative on their first day of study participation. Onset of infection was defined as the day of first positive RT-PCR result. Sensitivity of Ag-RDT was measured based on testing once, twice (after 48-hours), and thrice (after 96 hours). Analysis was repeated for different Days Post Index PCR Positivity (DPIPP) and stratified based on symptom-status.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>In total, 5,609 of 7,361 participants were eligible for this analysis. Among 154 participants who tested positive for SARS-CoV-2, 97 were asymptomatic and 57 had symptoms at infection onset. Serial testing with Ag-RDT twice 48-hours apart resulted in an aggregated sensitivity of 93.4% (95% CI: 89.1-96.1%) among symptomatic participants on DPIPP 0-6. Excluding singleton positives, aggregated sensitivity on DPIPP 0-6 for two-time serial-testing among asymptomatic participants was lower at 62.7% (54.7-70.0%) but improved to 79.0% (71.0-85.3%) with testing three times at 48-hour intervals.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Discussion</jats:title>\n                  <jats:p>Performance of Ag-RDT was optimized when asymptomatic participants tested three-times at 48-hour intervals and when symptomatic participants tested two-times separated by 48-hours.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.08.05.22278466","journal":"medRxiv","score":null},{"id":"10.1101/2022.08.05.502940","pub_date":"2022-8-05","title":"Human endogenous retrovirus-R envelope is a host restriction factor against severe acute respiratory syndrome-coronavirus-2","abstract":"Coronavirus induced disease-19 (COVID-19), caused by the SARS-CoV-2 remains a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that got integrated into the ancestral human genome. HERVs are important in development and diseases, including cancer, inflammation and viral infections. Here, we analyzed the expression of several HERVs in SARS-CoV-2 infected cells and observed increased activity of HERV-E, HERV-V, HERV-FRD, HERV-MER34, HERV-W and HERV-KHML2. In contrast, HERV-R-envelope was downregulated in cell-based models and COVID-19 patient PBMCs. HERV-R overexpression inhibited SARS-CoV-2 replication, suggesting its antiviral action. Further studies demonstrated the role of extracellular signal-regulated kinase (ERK) in regulating HERV-R antiviral activity. Cross-talk between the ERK and p38 MAPK controls HERV-R envelope synthesis, which in turn modulates the replication of SARS-CoV-2. These findings establish the importance of HERV-R envelope as a host restriction factor against SARS-CoV-2 and illustrate the advantage of integration and evolutionary maintenance of retroviral-elements in the human genome.","version":"1.1","doi":"10.1101/2022.08.05.502940","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.05.502936","pub_date":"2022-8-05","title":"SARS-CoV-2 entry route impacts a range of downstream viral and cellular processes","abstract":"SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.","version":"1.1","doi":"10.1101/2022.08.05.502936","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.05.502758","pub_date":"2022-8-05","title":"Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5","abstract":"Unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants imposes us to continuous control measurement. Given the rapid spread, new Omicron subvariant named BA.5 is urgently required for characterization. Here we analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1 comprehensively. Although in vitro growth kinetics of BA.5 is comparable among the Omicron subvariants, BA.5 become much more fusogenic than BA.1 and BA.2. The airway-on-a-chip analysis showed that the ability of BA.5 to disrupt the respiratory epithelial and endothelial barriers is enhanced among Omicron subvariants. Furthermore, in our hamster model, in vivo replication of BA.5 is comparable with that of the other Omicrons and less than that of the ancestral B.1.1. Importantly, inflammatory response against BA.5 is strong compared with BA.1 and BA.2. Our data suggest that BA.5 is still low pathogenic compared to ancestral strain but evolved to induce enhanced inflammation when compared to prior Omicron subvariants.","version":"1.1","doi":"10.1101/2022.08.05.502758","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.04.502828","pub_date":"2022-8-05","title":"mRNA vaccines and hybrid immunity use different B cell germlines to neutralize Omicron BA.4 and BA.5","abstract":"SARS-CoV-2 omicron BA.4 and BA.5, characterized by high transmissibility and ability to escape natural and vaccine induced immunity, are rampaging worldwide. To understand the escape mechanisms, we tested the neutralizing activity against omicron BA.4 and BA.5 of a panel of 482 human monoclonal antibodies that had been isolated from people who received two or three mRNA vaccine doses or from people that had been vaccinated after infection. None of the antibodies isolated after two vaccine doses neutralized omicron BA.4 and BA.5, while these variants were neutralized by approximately 15% of antibodies obtained from people that received three doses or had been vaccinated after infection. Remarkably, the antibodies isolated after three vaccine doses targeted mainly the receptor binding domain (RBD) Class 1/2 epitope region and were encoded by the IGHV1-69 and IGHV3-66 B cell germlines, while the antibodies isolated after infection recognized mostly the RBD Class 3 epitope region and the NTD, and were encoded by the IGHV2-5;IGHJ4-1 and IGHV1-24;IGHJ4-1 germlines. The observation that mRNA vaccination and hybrid immunity elicit a different immunity against the same antigen is intriguing and its understanding may help to design the next generation of therapeutics and vaccines against COVID-19.","version":"1.1","doi":"10.1101/2022.08.04.502828","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.04.502768","pub_date":"2022-8-04","title":"A specific anti-IFITM2 antibody bars the way to SARS-CoV-2 entry into host cells","abstract":"The early steps of viral infection involve protein complexes and structural lipid rearrangements, which mark the characteristic strategies of each virus in entering permissive host cells. Human IFITM proteins have been described as inhibitors of a broad range of viruses. Despite their homology and functional redundancy, recently it has been surprisingly shown that SARS-CoV-2 is able to specifically hijack the IFITM2 protein. Here has been reported the characterization of a newly generated specific anti-IFITM2 mAb able to impair SARS-CoV-2 Spike protein internalization and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Importantly, as evidence of the more general involvement of IFITM2 in virus entry, the anti-IFITM2 mAb was able to efficiently reduce HSVs- and RSV-dependent cytopathic effects. Hence, IFITM proteins could be promising targets that can foster the development of biological antiviral molecules, or suggest additional therapeutic strategies for the treatment of viral infections.","version":"1.1","doi":"10.1101/2022.08.04.502768","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.04.502609","pub_date":"2022-8-04","title":"Neutralization sensitivity of the SARS-CoV-2 Omicron BA.2.75 sublineage","abstract":"The recently emerged BA.2.75 Omicron sublineage of SARS-CoV-2 identified in numerous countries is rapidly increasing in prevalence in regions of India. Compared with BA.2, the spike protein of BA.2.75 differs in nine amino acid residues. To determine the impact of the spike mutations on polyclonal and monoclonal antibody activity, we investigated the neutralization sensitivity of BA.2.75 in comparison with B.1, BA.2, BA.2.12.1, and BA.4/5. Analysis of post-boost samples from 30 vaccinated individuals revealed significantly lower serum neutralizing activity against BA.2.75 than against BA.2. However, BA.2.75 was more sensitive to serum neutralization than the widely circulating BA.4/5 sublineages. Moreover, evaluation of 17 clinical-stage monoclonal antibodies demonstrated individual differences in Omicron sublineage activity. Notably, some authorized antibodies with low activity against other Omicron sublineages demonstrated high BA.2.75 neutralizing potency. Our results indicate a less pronounced degree of antibody evasion of BA.2.75 compared with BA.4/5 and suggest that factors beyond immune evasion may be required for an expansion of BA.2.75 over BA.4/5.","version":"1.1","doi":"10.1101/2022.08.04.502609","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.03.502672","pub_date":"2022-8-04","title":"A long interval between priming and boosting SARS-CoV-2 mRNA vaccine doses enhances B cell responses with limited impact on T cell immunity","abstract":"Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare in SARS-CoV-2 na\u00efve donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype and function of CD4+ and CD8+ T cell responses at post-boost memory timepoints. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.","version":"1.1","doi":"10.1101/2022.08.03.502672","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.03.502703","pub_date":"2022-8-04","title":"Double-dose mRNA vaccination to SARS-CoV-2 progressively increases recognition of variants-of-concern by Spike RBD-specific memory B cells","abstract":"SARS-CoV-2 vaccination with BNT162b2 (Pfizer BioNTech) has been shown to be 95% effective. Double-dose vaccination generates high levels of spike-specific antibodies, memory B cells (Bmem) and T cells. However, variants of concern (VoC) with mutations in the spike Receptor Binding Domain (RBD) can evade antibody responses. Booster vaccinations improve antibody recognition of VoC, but it is unclear if this is due to higher total antibodies or their capacity to bind VoC. We here addressed the capacity of surface Ig on single Wuhan-specific Bmem after first and second dose BNT162b2 vaccination to recognize variant RBD. Samples were collected from 30 healthy COVID-19 naive individuals pre-BNT162b2 vaccination, 3 weeks post-dose 1 and 4-weeks post-dose 2. Plasma antibodies and Bmem were evaluated using recombinant RBD proteins of the Wuhan, Gamma and Delta strains. All individuals generated a robust antibody response to BNT162b2 vaccination with all participants producing neutralizing antibodies following dose 2. IgM+ and IgG+ RBD-specific Bmem were generated after one vaccine dose, and those expressing IgG1 increased in absolute number after dose 2. The majority of RBD-specific Bmem bound the Gamma and/or Delta variants, and this proportion significantly increased after the second dose. The second dose of BNT162b2 increases the number of circulating Ig-class switched RBD-specific Bmem. Importantly, the second dose of vaccination is required for a high frequency of RBD-specific Bmem to recognize Gamma and Delta variants. This suggests that dose 2 not only increases the number of RBD-specific Bmem but also the affinity of the Bmem to overcome the point mutations in VoC.","version":"1.1","doi":"10.1101/2022.08.03.502703","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.03.499114","pub_date":"2022-8-04","title":"Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents","abstract":"SARS-CoV-2 Omicron sublineages have escaped most RBD-targeting therapeutic neutralizing antibodies (NAbs), which proves the previous NAb drug screening strategies deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following the criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection. Our screening strategy can also be applied to identify broad-spectrum NAb drugs against other fast-evolving viruses, such as influenza viruses.","version":"1.1","doi":"10.1101/2022.08.03.499114","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.03.502673","pub_date":"2022-8-04","title":"Phenotype-driven identification of drug targets for post-COVID-19 anosmia","abstract":"Anosmia (loss of sense of smell) is one symptom of COVID-19 which can linger long after acute infection has passed, with major impact on quality of life. Given the number of people impacted by COVID-19-related anosmia, there is an urgent need to identify effective therapeutics in a faster fashion than using traditional drug discovery and development methods. We used our knowledge graph, the Phenograph, to navigate from phenotypes to genes to drug targets, to rapidly find druggable targets associated with anosmia. This process shortlisted six targets: NRP1, SCN9A, EGR1, VEGFB, PRKCE, and FGFR1. Neuropilin-1 (NRP1) is under active study for its involvement in SARS-CoV-2 infection. Importantly, there is no direct link between anosmia and NRP1 in our knowledge graph; the relationship was inferred through the graph structure. Based on this external validation, we derived hypotheses for the involvement of the remaining five targets in COVID-19-related anosmia, and the mechanism of action desired in a drug candidate to correct the hypothesized dysregulation.","version":"1.1","doi":"10.1101/2022.08.03.502673","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.15.452549","pub_date":"2022-8-04","title":"Visualizing Amino Acid Substitutions in a Physicochemical Vector Space","abstract":"A three-dimensional representation of the twenty proteinogenic amino acids in a physicochemical space is presented. Vectors corresponding to amino acid substitutions are classified based on whether they are accessible via a single-nucleotide mutation. It is shown that the standard genetic code establishes a \u201cchoice architecture\u201d that permits nearly independent tuning of the properties related with size and those related with hydrophobicity. This work sheds light on the non-arbitrary benefits of evolvability that may have shaped the development standard genetic code to increase the probability that adaptive point mutations will be generated. Illustrations of the usefulness of visualizing amino acid substitutions in a 3D physicochemical space are shown using recent datasets collected regarding the SARS-CoV-2 receptor binding domain. First, the substitutions most responsible for antibody escape are almost always inaccessible via single nucleotide mutation, and change multiple properties concurrently. Second, it is shown that assays of ACE2 binding by sarbecovirus variants, including the viruses responsible for SARS and COVID-19, are more easily understood when plotted with this method. The results of this research can extend our understanding of certain hereditary disorders caused by point mutations, as well as guide the development of rational protein and vaccine design.","version":"1.3","doi":"10.1101/2021.07.15.452549","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.02.502430","pub_date":"2022-8-03","title":"Prediction, scanning and designing of TNF-\u03b1 inducing epitopes for human and mouse","abstract":"Tumor Necrosis Factor alpha (TNF-\u03b1) is a pleiotropic pro-inflammatory cytokine that plays a crucial role in controlling signaling pathways within the immune cells. Recent studies reported that the higher expression levels of TNF-\u03b1 is associated with the progression of several diseases including cancers, cytokine release syndrome in COVID-19 and autoimmune disorders. Thus, it is the need of the hour to develop immunotherapies or subunit vaccines to manage TNF-\u03b1 progression in various disease conditions. In the pilot study, we have proposed a host-specific in-silico tool for the prediction, designing and scanning of TNF-\u03b1 inducing epitopes. The prediction models were trained and validated on the experimentally validated TNF-\u03b1 inducing/non-inducing for human and mouse hosts. Firstly, we developed alignment free (machine learning based models using composition of peptides) methods for predicting TNF-\u03b1 inducing peptides and achieved maximum AUROC of 0.79 and 0.74 for human and mouse hosts, respectively. Secondly, alignment based (using BLAST) method has been used for predicting TNF-\u03b1 inducing epitopes. Finally, a hybrid method (combination of alignment free and alignment-based method) has been developed for predicting epitopes. Our hybrid method achieved maximum AUROC of 0.83 and 0.77 on an independent dataset for human and mouse hosts, respectively. We have also identified the potential TNF-\u03b1 inducing peptides in different proteins of HIV-1, HIV-2, SARS-CoV-2 and human insulin. Best models developed in this study has been incorporated in a webserver TNFepitope (https://webs.iiitd.edu.in/raghava/tnfepitope/), standalone package and GitLab (https://gitlab.com/raghavalab/tnfepitope). TNF-\u03b1 is a multifunctional pleiotropic pro-inflammatory cytokine. Anti-TNF-\u03b1 therapy used as an effective treatment in several autoimmune disorders. Composition-based features generated using Pfeature for each peptide sequence. Alignment-based and alignment-free models developed. Prediction and scanning of TNF-\u03b1 inducing regions in antigens. TNFepitope is available as a web-server, standalone package and GitLab.","version":"1.1","doi":"10.1101/2022.08.02.502430","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.15.500170","pub_date":"2022-8-02","title":"Pangenome analysis of SARS-CoV2 strains to Identify Potential vaccine targets by Reverse Vaccinology","abstract":"Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) leads to respiratory failure and obstructive alveolar damage, which may be fatal in immunocompromised individuals. COVID-19 pandemic has severe global implications badly, and the situation in the world is depreciating with the emergence of novel variants. The aim of our study is to explore the genome of SARS-CoV2 followed by in silico reverse vaccinology analysis. This will help to identify the most putative vaccine candidate against the virus in a robust manner and enables cost-effective development of vaccines compared with traditional strategies. The genomic sequencing data is retrieved from NCBI (Reference Sequence Number NC_045512.2). The sequences are explored through comparative genomics approaches by GENOMICS to find out the core genome. The comprehensive set of proteins obtained was employed in computational vaccinology approaches for the prediction of the best possible B and T cell epitopes through ABCpred and IEDB Analysis Resource, respectively. The multi-epitopes were further tested against human toll-like receptor and cloned in E. coli plasmid vector. The designed Multiepitope Subunit Vaccine was non-allergenic, antigenic (0.6543), & non-toxic, with significant connections with the human leukocyte antigen (HLA) binding alleles, and collective global population coverage of 84.38%. It has 276 amino acids, consisting of an adjuvant with the aid of EAAAK linker, AAY linkers used to join the 4 CTL epitopes, GPGPG linkers used to join the 3 HTL epitopes and KK linkers used to join the 7 B-cell epitopes. MESV docking with human pathogenic toll-like receptors-3 (TLR3) exhibited a stable & high binding affinity. An in-silico codon optimization approach was used in the codon system of E. coli (strain K12) to obtain the GC-Content of Escherichia coli (strain K12): 50.7340272413779 and CAI-Value of the improved sequence: 0.9542834278823386. The multi-epitope vaccine\u2019s optimized gene sequence was cloned in-silico in E. coli plasmid vector pET-30a (+), BamHI and HindIII restriction sites were added to the N and C-terminals of the sequence, respectively. There is a pressing need to combat COVID-19 and we need quick and reliable approaches against Covid-19. By using In-silico approaches, we acquire an effective vaccine that could trigger adequate immune responses at the cellular and humoral level. The suggested sequences can be further validated through in vivo and in vitro experimentation. Current developments in the immunological bioinformatics areas has resulted in different servers and tools that are cost and time efficient for the traditional vaccine development. Though for designing a multiple epitope vaccine the antigenic epitopes prediction of a relevant protein by immunoinformatic methods are very helpful.","version":"1.2","doi":"10.1101/2022.07.15.500170","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.02.502186","pub_date":"2022-8-02","title":"DNA Origami Presenting the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Response","abstract":"Effective and safe vaccines are invaluable tools in the arsenal to fight infectious diseases. The rapid spreading of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible of the coronavirus disease 2019 pandemic has highlighted the need to develop methods for rapid and efficient vaccine development. DNA origami nanoparticles (DNA-NPs) presenting multiple antigens in prescribed nanoscale patterns have recently emerged as a safe, efficient, and easily scalable alternative for rational design of vaccines. Here, we are leveraging the unique properties of these DNA-NPs and demonstrate that precisely patterning ten copies of a reconstituted trimer of the receptor binding domain (RBD) of SARS-CoV-2 along with CpG adjuvants on the DNA-NPs is able to elicit a robust protective immunity against SARS-CoV-2 in a mouse model. Our results demonstrate the potential of our DNA-NP-based approach for developing safe and effective nanovaccines against infectious diseases.","version":"1.1","doi":"10.1101/2022.08.02.502186","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.02.502439","pub_date":"2022-8-02","title":"Serological surveillance for wild rodent infection with SARS-CoV-2 in Europe","abstract":"We report serological surveillance for exposure to SARS-CoV-2 in 1,237 wild rodents and other small mammals across Europe. All samples were negative with the possible exception of one. Given the ongoing circulation of this virus in humans and potential host jumps, we suggest such surveillance be continued.","version":"1.1","doi":"10.1101/2022.08.02.502439","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.02.502427","pub_date":"2022-8-02","title":"UVC-based air disinfection system for rapid inactivation of SARS-CoV-2 present in the air","abstract":"The novel coronavirus disease 2019 (COVID-19) infections have rapidly spread throughout the world, and the virus has acquired an ability to spread via aerosols even at long distances. Hand washing, face-masking, and social distancing are the primary preventive measures against infections. With mounting scientific evidence, World Health Organisation (WHO) declared COVID-19 an air-borne disease. This ensued the need to disinfect air to reduce the transmission. Ultraviolet C (UVC) comprising the light radiation of 200-280 nm range is a commonly used method for inactivation of pathogens. The heating, ventilation, and air conditioning (HVAC) systems are not beneficial in closed spaces due to poor or no ability to damage circulating viruses. Therefore, standard infection-prevention practices coupled with a strategy to reduce infectious viral load in air substantially might be helpful in reducing virus transmissibility. In this study, we implemented UV light-based strategies to combat COVID-19 and future pandemics. We tested various disinfection protocols by using UVC-based air purification systems and currently installed such a system in workspaces, rushed out places, hospitals and healthcare facilities for surface, air, and water disinfection. In this study, we designed a prototype device to test the dose of UVC required to inactivate SARS-CoV-2 in aerosols and demonstrate that the radiation rapidly destroys the virus in aerosols. The UVC treatment renders the virus non-infectious due to chemical modification of nucleic acid. We also demonstrate that UVC treatment alters the Spike protein conformation that may further affect the infectivity of the virus. We show by using a mathematical model based on the experimental data that UVC-based air disinfection strategy can substantially reduce the risk of virus transmission. The systematic treatment by UVC of air in the closed spaces via ventilation systems could be helpful in reducing the active viral load in the air.","version":"1.1","doi":"10.1101/2022.08.02.502427","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.02.502100","pub_date":"2022-8-02","title":"NF-\u03baB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids","abstract":"As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-\u03baB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more I\u03baB\u03b1 expression in infected than bystander cells, but found concurrent nuclear translocation of NF-\u03baB that I\u03baB\u03b1 usually prevents. Overexpressing a nondegradable I\u03baB\u03b1 mutant reduced NF-\u03baB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-\u03baB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.","version":"1.1","doi":"10.1101/2022.08.02.502100","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.01.502390","pub_date":"2022-8-02","title":"SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta","abstract":"Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Due to confounding factors in the human population, such as pre-existing immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were upregulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggests that in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.","version":"1.1","doi":"10.1101/2022.08.01.502390","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.02.502461","pub_date":"2022-8-02","title":"Omicron BA.2 breakthrough infection enhances cross-neutralization of BA.2.12.1 and BA.4/BA.5","abstract":"Recently, we reported that BNT162b2-vaccinated individuals after Omicron BA.1 breakthrough infection have strong serum neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 variants of concern (VOCs), yet less against the highly contagious Omicron sublineages BA.4 and BA.5 that have displaced previous variants. As the latter sublineages are derived from Omicron BA.2, we characterized serum neutralizing activity of COVID-19 mRNA vaccine triple-immunized individuals who experienced BA.2 breakthrough infection. We demonstrate that sera of these individuals have broadly neutralizing activity against previous VOCs as well as all tested Omicron sublineages, including BA.2 derived variants BA.2.12.1, BA.4/BA.5. Furthermore, applying antibody depletion we showed that neutralization of BA.2 and BA.4/BA.5 sublineages by BA.2 convalescent sera is driven to a significant extent by antibodies targeting the N-terminal domain (NTD) of the spike glycoprotein, whereas their neutralization by Omicron BA.1 convalescent sera depends exclusively on antibodies targeting the receptor binding domain (RBD). These findings suggest that exposure to Omicron BA.2, in contrast to BA.1 spike glycoprotein, triggers significant NTD specific recall responses in vaccinated individuals and thereby enhances the neutralization of BA.4/BA.5 sublineages. Given the current epidemiology with a predominance of BA.2 derived sublineages like BA.4/BA.5 and rapidly ongoing evolution, these findings are of high relevance for the development of Omicron adapted vaccines.","version":"1.1","doi":"10.1101/2022.08.02.502461","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.01.502275","pub_date":"2022-8-01","title":"SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses","abstract":"Many patients with severe COVID-19 suffer from pneumonia, and thus elucidation of the mechanisms underlying the development of such severe pneumonia is important. The ORF8 protein is a secreted protein of SARS-CoV-2, whose in vivo function is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2. We found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrate that the serum ORF8 protein levels are correlated well with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a viral cytokine of SARS-CoV-2 involved in the in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.","version":"1.1","doi":"10.1101/2022.08.01.502275","journal":"bioRxiv","score":null},{"id":"10.1101/2022.08.01.502311","pub_date":"2022-8-01","title":"Epitranscriptomic N6-methyladenosine profile of SARS-CoV-2-infected human lung epithelial cells","abstract":"N6-methyladenosine (m6A) is a dynamic post-transcriptional RNA modification that plays an important role in determining transcript fate. Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused the global pandemic of coronavirus disease 2019 (COVID-19) and the virus has been extensively studied. However, how m6A modification of host cell RNAs change during SARS-CoV-2 infection has not been reported. Here we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. Biological pathway analyses revealed that differentially methylated transcripts were significantly associated with cancer-related pathways, protein processing in the endoplasmic reticulum, cell death and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include proteins involved in the type I interferon response, inflammation, and cytokine signaling. These data suggest that m6A modification of cellular RNA is an important mechanism of regulating host gene expression during SARS-CoV-2 infection of lung epithelial cells.","version":"1.1","doi":"10.1101/2022.08.01.502311","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.30.501940","pub_date":"2022-8-01","title":"Recombinant Human ACE2-Fc : A promising therapy for SARS-CoV2 infection","abstract":"SARS-CoV2 entry is mediated by binding of viral spike-protein(S) to the transmembrane Angiotensin-Converting Enzyme-2 (ACE2) of the host cell. Thus, to prevent transmission of disease, strategies to abrogate the interaction are important. However, ACE2 cannot be blocked since its normal function is to convert the Angiotensin II peptide to Angiotensin(1-7) to reduce hypertension. This work reports a recombinant cell line secreting soluble ACE2-ectopic domain (MFcS2), modified to increase binding and production efficacy and fused to human immunoglobulin-Fc. While maintaining its enzymatic activity, the molecule trapped and neutralized SARS CoV2 virus in vitro with an IC50 of 64 nM. In vivo, with no pathology in the vital organs, it inhibited the viral load in lungs in SARS-CoV2 infected Golden-Syrian-hamster. The Intravenous pharmacokinetic profiling of MFcS2 in hamster at a dose of 5 mg/Kg presented a maximum serum concentration of 23.45 \u00b5g/mL with a half-life of 29.5 hrs. These results suggest that MFcS2 could be used as an effective decoy based therapeutic strategy to treat COVID19. This work also reports usage of a novel oral-cancer cell line as in vitro model of SARS-Cov2 infection, validated by over expressing viral-defence pathways upon RNA-seq analysis and over-expression of ACE2 and TMPRSS upon growth in hyperglycaemic condition.","version":"1.1","doi":"10.1101/2022.07.30.501940","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.31.502235","pub_date":"2022-8-01","title":"Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75","abstract":"The SARS-CoV-2 Omicron subvariant BA.2.75 emerged recently and appears to be spreading rapidly. It has nine mutations in its spike compared to BA.2, raising concerns it may further evade vaccine-elicited and therapeutic antibodies. Here, we found BA.2.75 to be moderately more neutralization resistant to sera from vaccinated/boosted individuals than BA.2 (1.8-fold), similar to BA.2.12.1 (1.1-fold), but more neutralization sensitive than BA.4/5 (0.6-fold). Relative to BA.2, BA.2.75 showed heightened resistance to class 1 and class 3 monoclonal antibodies to the receptor-binding domain, while gaining sensitivity to class 2 antibodies. The resistance was largely conferred by the G446S and R460K mutations. Of note, BA.2.75 was slightly resistant (3.7-fold) to bebtelovimab, the only therapeutic antibody with potent activity against all Omicron subvariants. BA.2.75 also exhibited higher receptor binding affinity than other Omicron subvariants. BA.2.75 provides yet another example of the ongoing evolution of SARS-CoV-2 as it gains transmissibility while incrementally evading antibody neutralization.","version":"1.1","doi":"10.1101/2022.07.31.502235","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.31.500554","pub_date":"2022-8-01","title":"Memory B cells and memory T cells induced by SARS-CoV-2 booster vaccination or infection show different dynamics and efficacy to the Omicron variant","abstract":"Although BNT162b2 vaccination was shown to prevent infection and reduce COVID-19 severity, and the persistence of immunological memory generated by the vaccination has not been well elucidated. We evaluated memory B and T cell responses to the SARS-CoV-2 spike protein before and after the third BNT162b2 booster. Although the antibody titer against the spike receptor-binding domain (RBD) decreased significantly 8 months after the second vaccination, the number of memory B cells continued to increase, while the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the antibody titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, while memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- cTfh1 was positively correlated with RBD-specific antibody-secreting B cells. Furthermore, T cell-dependent antibody production from reactivated memory B cells in vitro was correlated to the Tfh-like cytokine levels. For the response to variant RBDs, although 60%-80% of memory B cells could bind to the Omicron RBD, their binding affinity was low, while memory T cells show an equal response to the Omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate antibody production and T cell responses after Omicron strain infection, which prevents severe illness and death due to COVID-19.","version":"1.1","doi":"10.1101/2022.07.31.500554","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.31.501809","pub_date":"2022-8-01","title":"InterClone: Store, Search and Cluster Adaptive Immune Receptor Repertoires","abstract":"B and T cell receptor repertoire data has the potential to fundamentally change the way we diagnose and treat a wide range of diseases. However, there are few resources for storing or analyzing repertoire data. InterClone provides tools for storing, searching, and clustering repertoire datasets. Efficiency is achieved by encoding the complementarity-determining regions of sequences as mmseqs2 databases. Single chain search or cluster results can be merged into paired (alpha-beta or heavy-light) results for analysis of single-cell sequencing data. We illustrate the use of InterClone with two recently reported examples: 1) searching for SARS-CoV-2 infection-enhancing antibodies in bulk COVID-19 and healthy donor repertoires; 2) identification of SARS-CoV-2 specific TCRs by clustering paired and bulk sequences from COVID-19, BNT162b2 vaccinated and healthy unvaccinated donors. The core functions of InterClone have been implemented as a web server and integrated database (https://sysimm.org/interclone). All source code is available upon request.","version":"1.1","doi":"10.1101/2022.07.31.501809","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.18.469078","pub_date":"2022-7-29","title":"Metformin Suppresses SARS-CoV-2 in Cell Culture","abstract":"Comorbidities such as diabetes worsen COVID-19 severity and recovery. Metformin, a first-line medication for type 2 diabetes, has antiviral properties and certain studies have also indicated its prognostic potential in COVID-19. Here, we report that metformin significantly inhibits SARS-CoV-2 growth in cell culture models. First, a steady increase in AMPK phosphorylation was detected as infection progressed, suggesting its important role during viral infection. Activation of AMPK in Calu3 and Caco2 cell lines using metformin revealed that metformin suppresses SARS-CoV-2 infectious titers up to 99%, in both na\u00efve as well as infected cells. TCID50 values from dose-variation studies in infected cells were found to be 0.8 and 3.5 mM in Calu3 and Caco2 cells, respectively. Role of AMPK in metformin\u2019s antiviral suppression was further confirmed using other pharmacological compounds, AICAR and Compound C. Collectively, our study demonstrates that metformin is effective in limiting the replication of SARS-CoV-2 in cell culture and thus possibly could offer double benefits s diabetic COVID-19 patients by lowering both blood glucose levels and viral load.","version":"1.3","doi":"10.1101/2021.11.18.469078","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.29.502045","pub_date":"2022-7-29","title":"OM-85 reduces SARS-COV-2 viral RNA expression in nasopharyngeal cells from COVID-19 patients","abstract":"OM-85 is a bacterial lysate from common respiratory tract pathogens, with an excellent safety profile, widely used to prevent recurrent respiratory tract infections. Several studies have been reporting the antiviral roles of OM-85. Here we demonstrated the effect of ex-vivo OM-85 exposure in nasopharyngeal cells collected from COVID-19 patients. OM-85 decreased the SARS-CoV-2 N1 gene expression and increased RIG-I (DDX58) gene expression in these cells. These data support the antiviral effect of OM-85 against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.07.29.502045","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.27.501771","pub_date":"2022-7-29","title":"MEPSi: A tool for simulating tomograms of membrane-embedded proteins","abstract":"The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question. Tools for a-priory assessment of the feasibility of samples to answer biological questions and how to optimize imaging parameters to do so would be a major advantage. Here we describe MEPSi (Membrane Embedded Protein Simulator), a simulation tool aimed at rapid and convenient evaluation and optimization of cryo-ET data acquisition parameters for studies of transmembrane proteins in their native environment. We demonstrate the utility of MEPSi by showing how to detangle the influence of different data collection parameters and different orientations in respect to tilt axis and electron beam for two examples: (1) simulated plasma membranes with embedded single-pass transmembrane \u03b1IIb\u03b23 integrin receptors and (2) simulated virus membranes with embedded SARS-CoV-2 spike proteins. Tool to simulate tomograms of membrane-embedded proteins Detangles influence of data acquisition parameters from sample quality issues Rapid evaluation and optimization of cryo-ET data acquisition parameters Proof-of-concept provided with integrins and SARS-CoV-2 spike simulations","version":"1.1","doi":"10.1101/2022.07.27.501771","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.29.502072","pub_date":"2022-7-29","title":"Infection- or vaccine mediated immunity reduces SARS-CoV-2 transmission, but increases competitiveness of Omicron in hamsters","abstract":"Omicron has demonstrated a competitive advantage over Delta in vaccinated people. To understand this, we designed a transmission chain experiment using na\u00efve, intranasally (IN) or intramuscularly (IM) vaccinated, and previously infected (PI) hamsters. Vaccination and previous infection protected animals from disease and virus replication after Delta and Omicron dual challenge. A gradient in transmission blockage was observed: IM vaccination displayed moderate transmission blockage potential over three airborne chains (approx. 70%), whereas, IN vaccination and PI blocked airborne transmission in >90%. In na\u00efve hamsters, Delta completely outcompeted Omicron within and between hosts after dual infection in onward transmission. Although Delta also outcompeted Omicron in the vaccinated and PI transmission chains, an increase in Omicron competitiveness was observed in these groups. This correlated with the increase in the strength of the humoral response against Delta, with the strongest response seen in PI animals. These data highlight the continuous need to assess the emergence and spread of novel variants in populations with pre-existing immunity and address the additional evolutionary pressure this may exert on the virus.","version":"1.1","doi":"10.1101/2022.07.29.502072","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.27.501719","pub_date":"2022-7-28","title":"Development of Equine Polyclonal Antibodies as a Broad-Spectrum Therapy Against SARS-CoV-2 Variants","abstract":"The Coronavirus disease 19 (COVID-19) pandemic has accumulated over 550 million confirmed cases and more than 6.34 million deaths worldwide. Although vaccinations has largely protected the population through the last two years, the effect of vaccination has been increasingly challenged by the emerging SARS-CoV-2 variants. Although several therapeutics including both monoclonal antibodies and small molecule drugs have been used clinically, high cost, viral escape mutations, and potential side effects have reduced their efficacy. There is an urgent need to develop a low cost treatment with wide-spectrum effect against the novel variants of SARS-CoV-2. Here we report a product of equine polyclonal antibodies that showed potential broad spectrum neutralization effect against the major variants of SARS-CoV-2. The equine polyclonal antibodies were generated by horse immunization with the receptor binding domain (RBD) of SARS-CoV-2 spike protein and purified from equine serum. A high binding affinity between the generated equine antibodies and the RBD was observed. Although designed against the RBD of the early wild type strain sequenced in 2020, the equine antibodies also showed a highly efficient neutralization capacity against the major variants of SARS-CoV-2, including the recent BA.2 Omicron variant (IC50 =1.867\u03bcg/ml) in viral neutralization assay in Vero E6 cells using live virus cultured. The broad-spectrum neutralization capacity of the equine antibodies was further confirmed using pseudovirus neutralization assay covering the major SARS-CoV-2 variants including wild type, alpha, beta, delta, and omicron, showing effective neutralization against all the tested strains. Ex vivo reconstructed human respiratory organoids representing nasal, bronchial, and lung epitheliums were employed to test the treatment efficacy of the equine antibodies. Antibody treatment protected the human nasal, bronchial, and lung epithelial organoids against infection of the novel SARS-CoV-2 variants challenging public health, the Delta and Omicron BA.2 isolates, by reducing >95% of the viral load. The equine antibodies were further tested for potential side effects in a mouse model by inhalation and no significant pathological feature was observed. Equine antibodies, as a mature medical product, have been widely applied in the treatment of infectious diseases for more than a century, which limits the potential side effects and are capable of large scale production at a low cost. A cost-effective, wide-spectrum equine antibody therapy effective against the major SARS-CoV-2 variants can contribute as an affordable therapy to cover a large portion of the world population, and thus potentially reduce the transmission and mutation of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.07.27.501719","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.28.501852","pub_date":"2022-7-28","title":"P2G3 human monoclonal antibody neutralizes SARS-CoV-2 Omicron subvariants including BA.4 and BA.5 and Bebtelovimab escape mutants","abstract":"The rapid evolution of SARS-CoV-2 has led to a severe attrition of the pool of monoclonal antibodies still available for COVID-19 prophylaxis or treatment. Omicron subvariants notably escape most antibodies developed so far, with Bebtelovimab last amongst clinically approved therapeutic antibodies to display still good activity against all of them including the currently dominant BA.4/BA.5. We recently described P2G3, a broadly active SARS-CoV-2 monoclonal antibody, which targets a region of Spike partly overlapping with the site recognized by Bebtelovimab. Here, we reveal that P2G3 efficiently neutralizes SARS-CoV-2 omicron subvariants including BA.4/BA.5. We further demonstrate that P2G3 neutralizes Omicron BA.2 and BA.4 mutants escaping Bebtelovimab blockade, whereas the converse is not true. EU COVICIS program; private foundation advised by CARIGEST SA.","version":"1.1","doi":"10.1101/2022.07.28.501852","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.27.497816","pub_date":"2022-7-28","title":"Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 Macrodomain of SARS-CoV-2","abstract":"The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 \u00c5 resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1. SARS-CoV-2 encodes a viral macrodomain protein (Mac1) that hydrolyzes ribo-adenylate marks on viral proteins, disrupting the innate immune response to the virus. Catalytic mutations in the enzyme make the related SARS-1 virus less pathogenic and non-lethal in animals, suggesting that Mac1 will be a good antiviral target. However, no potent inhibitors of this protein class have been described, and pharmacologically the enzyme remains an orphan. Here, we computationally designed potent inhibitors of Mac1, determining 150 inhibitor-enzyme structures to ultra-high resolution by crystallography. In silico fragment linking and molecular docking of > 450 million virtual compounds led to inhibitors with submicromolar activity. These molecules may template future drug discovery efforts against this crucial but understudied viral target.","version":"1.2","doi":"10.1101/2022.06.27.497816","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.27.501708","pub_date":"2022-7-28","title":"Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain","abstract":"Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.","version":"1.1","doi":"10.1101/2022.07.27.501708","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.28.501901","pub_date":"2022-7-28","title":"Effect of Delta and Omicron mutations on the RBD-SD1 domain of the Spike protein in SARS-CoV-2 and the Omicron mutations on RBD-ACE2 interface complex","abstract":"The receptor-binding domain (RBD) is the essential part in the Spike-protein (S-protein) of SARS-CoV-2 virus that directly binds to the human ACE2 receptor, making it a key target for many vaccines and therapies. Therefore, any mutations at this domain could affect the efficacy of these treatments as well as the viral-cell entry mechanism. We introduce ab initio DFT-based computational study that mainly focuses on two parts: (1) Mutations effects of both Delta and Omicron variants in the RBD-SD1 domain. (2) Impact of Omicron RBD mutations on the structure and properties of the RBD-ACE2 interface system. The in-depth analysis is based on the novel concept of amino acid-amino acid bond pair units (AABPU) that reveal the differences between the Delta and/or Omicron mutations and its corresponding wild-type strain in terms of the role played by non-local amino acid interactions, their 3D shapes and sizes, as well as contribution to hydrogen bonding and partial charge distributions. Our results also show that the interaction of Omicron RBD with ACE2 significantly increased its bonding between amino acids at the interface providing information on the implications of penetration of S-protein into ACE2, and thus offering a possible explanation for its high infectivity. Our findings enable us to present in more conspicuous atomic level detail the effect of specific mutations that may help in predicting and/or mitigating the next variant of concern.","version":"1.1","doi":"10.1101/2022.07.28.501901","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.26.501656","pub_date":"2022-7-27","title":"Honokiol inhibits SARS-CoV-2 replication in cell culture","abstract":"SARS-CoV-2 emerged in 2019 and since its global spread has caused the death of over 6 million people. There are currently few antiviral options for treatment of COVID-19. Repurposing of known drugs can be a fast route to obtain molecules that inhibit viral infection and/or modulate pathogenic host responses. Honokiol is a small molecule from Magnolia trees, for which several biological effects have been reported,, including anticancer and anti-inflammatory activity. Honokiol has also been shown to inhibit several viruses in cell culture. In this study, we show that honokiol protected Vero E6 cells from SARS-CoV-2-mediated cytopathic effect with an EC50 of 7.8 \u00b5M. In viral load reduction assays we observed that honokiol decreased viral RNA copies as well as viral infectious progeny titers. The compound also inhibited SARS-CoV-2 replication in the more relevant A549 cells, expressing ACE2 and TMPRSS2. A time-of-addition assay showed that honokiol inhibited virus replication even when added post infection, suggesting it acts at a post-entry step of the replication cycle. Honokiol was also effective against more recent variants of SARS-CoV-2, including omicron and it inhibited other human coronaviruses as well. Our study suggests that honokiol is an interesting molecule to evaluate in animal studies and clinical trials to investigate its effect on virus replication and pathogenic (inflammatory) host responses.","version":"1.1","doi":"10.1101/2022.07.26.501656","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.26.501655","pub_date":"2022-7-27","title":"High Frequencies of Phenotypically and Functionally Senescent and Exhausted CD56+CD57+PD-1+ Natural Killer Cells, SARS-CoV-2-Specific Memory CD4+ and CD8+ T cells Associated with Severe Disease in Unvaccinated COVID-19 Patients","abstract":"Unvaccinated COVID-19 patients display a large spectrum of symptoms, ranging from asymptomatic to severe symptoms, the latter even causing death. Distinct Natural killer (NK) and CD4+ and CD8+ T cells immune responses are generated in COVID-19 patients. However, the phenotype and functional characteristics of NK cells and T-cells associated with COVID-19 pathogenesis versus protection remain to be elucidated. In this study, we compared the phenotype and function of NK cells SARS-CoV-2-specific CD4+ and CD8+ T cells in unvaccinated symptomatic (SYMP) and unvaccinated asymptomatic (ASYMP) COVID-19 patients. The expression of senescent CD57 marker, CD45RA/CCR7differentiation status, exhaustion PD-1 marker, activation of HLA-DR, and CD38 markers were assessed on NK and T cells from SARS-CoV-2 positive SYMP patients, ASYMP patients, and Healthy Donors (HD) using multicolor flow cytometry. We detected significant increases in the expression levels of both exhaustion and senescence markers on NK and T cells from SYMP patients compared to ASYMP patients and HD controls. In SYMP COVID-19 patients, the T cell compartment displays several alterations involving naive, central memory, effector memory, and terminally differentiated T cells. The senescence CD57 marker was highly expressed on CD8+ TEM cells and CD8+ TEMRA cells. Moreover, we detected significant increases in the levels of proinflammatory TNF-\u03b1, IFN-\u03b3, IL-6, IL-8, and IL-17 cytokines from SYMP COVID-19 patients, compared to ASYMP COVID-19 patients and HD controls. The findings suggest exhaustion and senescence in both NK and T cell compartment is associated with severe disease in critically ill COVID-19 patients. Unvaccinated COVID-19 patients display a large spectrum of symptoms, ranging from asymptomatic to severe symptoms, the latter even causing death. Distinct Natural killer (NK) and CD4+ and CD8+ T cells immune responses are generated in COVID-19 patients. In this study, we detected significant increases in the expression levels of both exhaustion and senescence markers on NK and T cells from unvaccinated symptomatic (SYMP) compared to unvaccinated asymptomatic (ASYMP) COVID-19 patients. Moreover, we detected significant increases in the levels of proinflammatory TNF-\u03b1, IFN-\u03b3, IL-6, IL-8, and IL-17 cytokines from SYMP COVID-19 patients, compared to ASYMP COVID-19 patients. The findings suggest exhaustion and senescence in both NK and T cell compartment is associated with severe disease in critically ill COVID-19 patients. Significant exhaustion and senescence in both NK and T cells were detected in unvaccinated symptomatic COVID-19 patients, suggesting a weakness in both innate and adaptive immune systems leads to severe disease in critically ill COVID-19 patients.","version":"1.1","doi":"10.1101/2022.07.26.501655","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.26.501658","pub_date":"2022-7-27","title":"Environmental Stability of Enveloped Viruses is Impacted by the Initial Volume and Evaporation Kinetics of Droplets","abstract":"Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 \u00b5L) and relative humidity (RH: 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage, Phi6, a common surrogate for enveloped viruses, and SARS-CoV-2 under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e. a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with initial droplet volume. We observed more rapid virus decay when the droplets were still wet and undergoing evaporation, and slower decay after the droplets had dried. Initial droplet volume had a major effect on virus viability over the first few hours; whereby the decay rate of influenza virus was faster in smaller droplets. In general, influenza virus and SARS-CoV-2 decayed similarly. Overall, this study suggests that virus decay in media is closely correlated with the extent of droplet evaporation, which is controlled by RH. Taken together, these data suggest that decay of different viruses is more similar at higher RH and in smaller droplets and is distinct at lower RH and in larger droplets. Importantly, accurate assessment of transmission risk requires use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. National Institute of Allergy and Infectious Diseases, National Institute of Neurological Disorders and Stroke, National Institutes of Health; Department of Health and Human Services; Flu Lab. During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments involving SARS-CoV-2 applied to surfaces in large droplets at various humidity conditions. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets likely over-estimates transmission risk for contaminated surfaces, as occurred during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.","version":"1.1","doi":"10.1101/2022.07.26.501658","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.488969","pub_date":"2022-7-26","title":"Intrinsic furin-mediated cleavability of the spike S1/S2 site from SARS-CoV-2 variant B.1.1.529 (Omicron)","abstract":"The ability of SARS-CoV-2 to be primed for viral entry by the host cell protease furin has become one of the most investigated of the numerous transmission and pathogenicity features of the virus. SARS-CoV-2 The variant B.1.1.529 (Omicron) emerged in late 2020 and has continued to evolve and is now present in several distinct sub-variants. Here, we analyzed the \u201cfurin cleavage site\u201d of the spike protein of SARS-CoV-2 B.1.1.529 (Omicron variant) in vitro, to assess the role of two key mutations (spike, N679K and P681H) that are common across all subvariants compared to the ancestral B.1 virus and other notable lineages. We observed significantly increased intrinsic cleavability with furin compared to an original B lineage virus (Wuhan-Hu1), as well as to two variants, B.1.1.7 (Alpha) and B.1.617 (Delta) that subsequently had wide circulation. Increased furin-mediated cleavage was attributed to the N679K mutation, which lies outside the conventional furin binding pocket. Our findings suggest that B.1.1.529 (Omicron variant) has gained genetic features linked to intrinsic furin cleavability, in line with its evolution within the population as the COVID-19 pandemic has proceeded.","version":"1.2","doi":"10.1101/2022.04.20.488969","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.25.501479","pub_date":"2022-7-26","title":"Intranasal delivery of lipid nanoparticle encapsulated SARS-CoV-2 and RSV-targeting siRNAs reduces lung infection","abstract":"RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) in infected mouse lungs. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The studies presented here demonstrates an attractive alternate therapeutic delivery strategy for the treatment of both future and emerging respiratory viral diseases.","version":"1.1","doi":"10.1101/2022.07.25.501479","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.26.501505","pub_date":"2022-7-26","title":"The highly conserved RNA-binding specificity of nucleocapsid protein facilitates the identification of drugs with broad anti-coronavirus activity","abstract":"The binding of SARS-CoV-2 nucleocapsid (N) protein to both the 5\u2032- and 3\u2032-ends of genomic RNA has different implications arising from its binding to the central region during virion assembly. However, the mechanism underlying selective binding remains unknown. Herein, we performed the high-throughput RNA-SELEX (HTR-SELEX) to determine the RNA-binding specificity of the N proteins of various SARS-CoV-2 variants as well as other \u03b2-coronaviruses and showed that N proteins could bind two unrelated sequences, both of which were highly conserved across all variants and species. Interestingly, both these sequence motifs are virtually absent from the human transcriptome; however, they exhibit a highly enriched, mutually complementary distribution in the coronavirus genome, highlighting their varied functions in genome packaging. Our results provide mechanistic insights into viral genome packaging, thereby increasing the feasibility of developing drugs with broad-spectrum anti-coronavirus activity by targeting RNA binding by N proteins.","version":"1.1","doi":"10.1101/2022.07.26.501505","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.25.501370","pub_date":"2022-7-25","title":"Poor sensitivity of iPSC-derived neural progenitors and glutamatergic neurons to SARS-CoV-2","abstract":"COVID-19 is a respiratory disease affecting multiple organs including the central nervous system (CNS), with a characteristic loss of smell and taste. Although frequently reported, the neurological symptoms remain enigmatic. There is no consensus on the extent of CNS infection. Here, we derived human induced pluripotent stem cells (hiPSC) into neural progenitor cells (NPCs) and cortical excitatory neurons to study their permissiveness to SARS-CoV-2 infection. Flow cytometry and western blot analysis indicated that NPCs and neurons do not express detectable levels of the SARS-CoV-2 receptor ACE2. We thus generated cells expressing ACE2 by lentiviral transduction to analyze in a controlled manner the properties of SARS-CoV-2 infection relative to ACE2 expression. Sensitivity of parental and ACE2 expressing cells was assessed with GFP- or luciferase-carrying pseudoviruses and with authentic SARS-CoV-2 Wuhan, D614G, Alpha or Delta variants. SARS-CoV-2 replication was assessed by microscopy, RT-qPCR and infectivity assays. Pseudoviruses infected only cells overexpressing ACE2. Neurons and NPCs were unable to efficiently replicate SARS-CoV-2, whereas ACE2 overexpressing neurons were highly sensitive to productive infection. Altogether, our results indicate that primary NPCs and cortical neurons remain poorly permissive to SARS-CoV-2 across the variants\u2019 spectrum, in the absence of ACE2 expression.","version":"1.1","doi":"10.1101/2022.07.25.501370","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.22.501212","pub_date":"2022-7-25","title":"SARS-CoV-2 Non-Structural Protein 1(NSP1) Mutation Virulence and Natural Selection: Evolutionary Trends in the Six Continents","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an unsegmented positivesense single-stranded RNA virus that belongs to the \u03b2-coronavirus. This virus was the cause of a novel severe acute respiratory syndrome in 2019 (COVID-19) that emerged in Wuhan, China at the early stage of the pandemic and rapidly spread around the world. Rapid transmission and reproduction of SARS-CoV-2 threaten worldwide health with a high mortality rate from the virus. According to the significant role of non-structural protein 1 (NSP1) in inhibiting host mRNA translation, this study focuses on the link between amino acid sequences of NSP1 and alterations of them spreading around the world. The SARS-CoV-2 NSP1 protein sequences were analyzed and FASTA files were processed by Python language programming libraries. Reference sequences compared with each NSP1 sample to identify every mutation and categorize them were based on continents and frequencies. NSP1 mutations rate divided into continents were different. Based on continental studies, E87D in global vision and also in Europe notably increased. The E87D mutation has significantly risen especially in the last months of the study as the first frequent mutation observed. The remarkable mutations, H110Y and R24C, have the second and third frequencies, respectively. Based on this mutational information, despite NSP1 being a conserved sequence occurrence, these mutations change the rate of flexibility and stability of the NSP1 protein, which can eventually affect inhibiting the host translation. In this study, we analyzed 6,510,947 sequences of non-structural protein 1 as a conserved region of SARS-CoV-2. According to the obtained results, 93.4819% of samples had no mutant regions on their amino acid sequences. Heat map data of mutational samples demonstrated high percentages of mutations that occurred in the region of 72 to 126 amino acids indicating a hot spot region of the protein. Increased rates of E87D, H110Y, and R24C mutations in the timeline of our study were reported as significant compared to available mutant samples. Analyzing the details of replacing amino acids in the most frequent E87D mutation reveals the role of this alteration in increasing molecule flexibility and destabilizing the structure of the protein.","version":"1.1","doi":"10.1101/2022.07.22.501212","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.22.501141","pub_date":"2022-7-25","title":"SARS-CoV-2 omicron variants succumb in vitro to Artemisia annua hot water extracts","abstract":"The SARS-CoV-2 (COVID-19) global pandemic continuous to infect and kill millions while rapidly evolving new variants that are more transmissible and evading vaccine-elicited antibodies. Artemisia annua L. extracts have shown potency against all previously tested variants. Here we further queried extract efficacy against omicron and its recent subvariants. Using Vero E6 cells, we measured the in vitro efficacy (IC50) of stored (frozen) dried-leaf hot-water A. annua L. extracts of four cultivars (A3, BUR, MED, and SAM) against SARS-CoV-2 variants: original WA1 (WT), BA.1.1.529+R346K (omicron), BA.2, BA.2.12.1, and BA.4. IC50 values normalized to the extract artemisinin (ART) content ranged from 0.5-16.5 \u00b5M ART. When normalized to dry mass of the extracted A. annua leaves, values ranged from 20-106 \u00b5g. Although IC50 values for these new variants are slightly higher than those reported for previously tested variants, they were within limits of assay variation. There was no measurable loss of cell viability at leaf dry weights \u226450 \u00b5g of any cultivar extract. Results continue to indicate that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective approach to help stave off this pandemic virus and its rapidly evolving variants.","version":"1.1","doi":"10.1101/2022.07.22.501141","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.22.501163","pub_date":"2022-7-25","title":"Waning and boosting of functional humoral immunity to SARS-CoV-2","abstract":"Since the emergence of the SARS-CoV-2 virus, we have witnessed a revolution in vaccine development with the rapid emergence and deployment of both traditional and novel vaccine platforms. The inactivated CoronaVac vaccine and the mRNA-based Pfizer/BNT162b2 vaccine are among the most widely distributed vaccines, both demonstrating high, albeit variable, vaccine effectiveness against severe COVID-19 over time. Beyond the ability of the vaccines to generate neutralizing antibodies, antibodies can attenuate disease via their ability to recruit the cytotoxic and opsinophagocytic functions of the immune response. However, whether Fc-effector functions are induced differentially, wane with different kinetics, and are boostable, remains unknown. Here, using systems serology, we profiled the Fc-effector profiles induced by the CoronaVac and BNT162b2 vaccines, over time. Despite the significantly higher antibody functional responses induced by the BNT162b2 vaccine, CoronaVac responses waned more slowly, albeit still found at levels below those present in the systemic circulation of BNT162b2 immunized individuals. However, mRNA boosting of the CoronaVac vaccine responses resulted in the induction of significantly higher peak antibody functional responses with increased humoral breadth, including to Omicron. Collectively, the data presented here point to striking differences in vaccine platform-induced functional humoral immune responses, that wane with different kinetics, and can be functionally rescued and expanded with boosting.","version":"1.1","doi":"10.1101/2022.07.22.501163","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.23.501242","pub_date":"2022-7-25","title":"Determining the antiviral activity of two polyene macrolide antibiotics following treatment in Kidney Cells infected with SARS-CoV-2","abstract":"There has been much speculation that polyene macrolide antibiotics, such as amphotericin B (AmB) and Nystatin (NYS) may have antiviral activity against several viruses including SARS-CoV-2. The objective of this short communication was to determine the antiviral activity of two polyene macrolides, AmB and NYS, following treatment in kidney cells infected with SARS-CoV-2. A serial dilution of AmB, NYS, and irbesartan (a drug known to bind to the ACE-2 receptor as a positive control) were then added (n=4 at each concentration) to the infected Vero\u201976 kidney cells in 100 \u00b5L media. Cells were also examined for contamination at 24 hours, and for cytopathic effect (CPE) and cytotoxicity (if noticeable) under a microscope at 48 hours. In a second study, AmB and Remdesivir were incubated in kidney cells infected with the virus and inhibition of the virus was determined by an immunoassay. Amphotericin B (AmB) showed a significant reduction in the TCID50 titer, with the 50% effective concentration (EC50) of 1.24 \u00b5M, which was 2.5 times lower than the cytotoxicity concentration. NYS and Irbesartan both exhibited substantially less active and would not be considered a suitable choice for further investigations. In addition, when measuring viral inhibition by immunoassay, AmB was significantly more potent than remdesivir (EC50 31.8 nM vs. 1.15 \u00b5M). Taken together, these preliminary findings suggest that AmB may have significant activity against SARS-CoV-2. However, further cell and animal studies are warranted.","version":"1.1","doi":"10.1101/2022.07.23.501242","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.479867","pub_date":"2022-7-25","title":"Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2","abstract":"Advanced mRNA vaccines play vital roles against SARS-CoV-2. However, due to their poor stability, most current mRNA delivery platforms need to be stored at -20\u00b0C or -70\u00b0C, which severely limits their distribution. Herein, we present lyophilized SARS-CoV-2 mRNA-lipid nanoparticle vaccines, which can be stored at room temperature with long-term thermostability. In the in vivo Delta virus challenge experiment, lyophilized Delta variant mRNA vaccine successfully protected mice from infection and cleared the virus. Lyophilized omicron mRNA vaccine enabled to elicit both potent humoral and cellular immunity. In booster immunization experiments in mice and old monkeys, lyophilized omicron mRNA vaccine could effectively increase the titers of neutralizing antibodies against wild-type coronavirus and omicron variants. In humans, lyophilized omicron mRNA vaccine as a booster shot could also engender excellent immunity and had less severe adverse events. This lyophilization platform overcomes the instability of mRNA vaccines without affecting their bioactivity, and significantly improved their accessibility, particularly in remote regions.","version":"1.3","doi":"10.1101/2022.02.10.479867","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.23.501111","pub_date":"2022-7-25","title":"McAN: an ultrafast haplotype network construction algorithm","abstract":"Haplotype network is becoming popular due to its increasing use in analyzing genealogical relationships of closely related genomes. We newly proposed McAN, a minimum-cost arborescence based haplotype network construction algorithm, by considering mutation spectrum history (mutations in ancestry haplotype should be contained in descendant haplotype), node size (corresponding to sample count for a given node) and sampling time. McAN is two orders of magnitude faster than the state-of-the-art algorithms, making it suitable for analyzation of massive sequences. Source code is written in C/C++ and available at https://github.com/Theory-Lun/McAN and https://ngdc.cncb.ac.cn/biocode/tools/BT007301 under the MIT license. The online web service of McAN is available at https://ngdc.cncb.ac.cn/ncov/online/tool/haplotype. SARS-CoV-2 dataset are available at https://ngdc.cncb.ac.cn/ncov/.","version":"1.1","doi":"10.1101/2022.07.23.501111","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.23.501235","pub_date":"2022-7-25","title":"Multi-omics integrated analysis reveals a specific phenotype of CD8+ T cell may contribute to immunothromosis via Th17 response in severe and critical COVID-19","abstract":"T lymphocyte reduction and immunosenescence frequently occur in severe and critical coronavirus disease 2019 (COVID-19) patients, which may cause immunothrombosis and numerous sequelae. This study integrated analyzed multi-omics data from healthy donors, pneumonia, COVID-19 patients (mild & moderate, severe, and critical), and convalescences, including clinical, laboratory test, PBMC bulk RNA-seq, PBMC scRNA-seq and TCR-seq, BAL scRNA-seq, and lung proteome. We revealed that there are certain associations among T lymphocyte reduction, CD8+ T cell senescence, Th17 immune activation, and immunothrombosis. A specific phenotype (S. P.) CD8+ T cells were identified in severe and critical COVID-19 patients in both PBMC and BAL scRNA-seq, which showed highly TCR homology with terminal effector CD8+ T cells and senescent CD8+ T cells. Pseudotime analysis showed that the S. P. CD8+ T cells were located in the transition trajectory from mild to severe disease. Which may be activated by terminal effector CD8+ T cells or senescent CD8+ T cells, thereby promoting Th17 cell differentiation. This phenomenon was absent in healthy donors, mild and moderate COVID-19 patients, or convalescences. Our findings are an important reference for avoiding the conversion of patients with mild to severe diseases and provide insight into the future prevention and control of COVID-19 and its variants.","version":"1.1","doi":"10.1101/2022.07.23.501235","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.24.501275","pub_date":"2022-7-24","title":"Structural basis of a high-affinity antibody binding to glycoprotein region with consecutive glycosylation sites","abstract":"Consecutive glycosylation sites occur in both self and viral proteins. Glycan-shielding of underneath peptide region is a double-edged sword, that avoids immune attack to self-proteins, but helps viruses including HIV-1 and SARS-CoV2 to escape antibody binding. Here we report a high-affinity antibody, 16A, binding to linear peptide containing consecutive glycosylation sites. Co-crystallization of 16A Fab and glycopeptides with GalNAc modifications at different sites showed that STAPPAHG is the sequence recognized by 16A antibody. GalNAc modification at Threonine site on STAPPAHG sequence significantly increased the affinity of Fab binding by 30.6 fold (KD=6.7nM). The increased affinity is conferred by hydrophilic and pi-stacking interactions between the GalNAc residue on Threonine site and a Trp residue from the CDR1 region of the heavy chain. Furthermore, molecular modeling suggested that GalNAc on T site causes more favorable conformation for antibody binding. These results showed that glycan modification most proximal to linear peptide core epitope significantly increases antigenicity of a glycopeptide epitope. The antibody recognition mode by peptide-binding CDR groove with a glycan-binding edge, may shed light on designing of linear glycopeptide-based vaccines for cancer and viral diseases. A high-affinity antibody was found to bind densely glycosylated glycoprotein region by a peptide binding groove of the antibody\u2019s variant region, with a glycan-binding edge specific to glycosylation site most proximal to core peptide epitope.","version":"1.1","doi":"10.1101/2022.07.24.501275","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.20.500860","pub_date":"2022-7-22","title":"A linear DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain elicits protective immunity in domestic cats","abstract":"Since its first detection in China in late 2019, SARS-CoV-2, the etiologic agent of COVID-19 pandemic, has infected a wide range of animal species, especially mammals, all over the world. Indeed, as reported by the American Veterinary Medical Association, besides human-to-human transmission, human-to-animal transmission has been observed in some wild animals and pets, especially in cats. With animal models as an invaluable tool in the study of infectious diseases combined with the fact that the intermediate animal source of SARS-CoV-2 is still unknown, researchers have demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a randomized Phase I/II clinical study conducted in domestic cats to assess safety and immunogenicity of a linear DNA (\u201clinDNA\u201d) vaccine encoding the RBD domain of SARS-CoV-2. No significant adverse events occurred and both RBD-specific binding/neutralizing antibodies and T cells were detected. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.","version":"1.1","doi":"10.1101/2022.07.20.500860","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.21.501010","pub_date":"2022-7-22","title":"Durability of the Neutralizing Antibody Response to mRNA Booster Vaccination Against SARS-CoV-2 BA.2.12.1 and BA.4/5 Variants","abstract":"The recent emergence of the SARS-CoV-2 BA.4/5 and BA.2.12.1 variants has led to rising COVID-19 case numbers and concerns over the continued efficacy of mRNA booster vaccination. Here we examine the durability of neutralizing antibody (nAb) responses against these SARS-CoV-2 Omicron subvariants in a cohort of health care workers 1-40 weeks after mRNA booster dose administration. Neutralizing antibody titers fell by \u223c1.5-fold 4-6 months and by \u223c2.5-fold 7-9 months after booster dose, with average nAb titers falling by 11-15% every 30 days, far more stable than two dose induced immunity. Notably, nAb titers from booster recipients against SARS-CoV-2 BA.1, BA.2.12.1, and BA.4/5 variants were \u223c4.7-, 7.6-, and 13.4-fold lower than against the ancestral D614G spike. However, the rate of waning of booster dose immunity was comparable across variants. Importantly, individuals reporting prior infection with SARS-CoV-2 exhibited significantly higher nAb titers compared to those without breakthrough infection. Collectively, these results highlight the broad and stable neutralizing antibody response induced by mRNA booster dose administration, implicating a significant role of virus evolution to evade nAb specificity, versus waning humoral immunity, in increasing rates of breakthrough infection.","version":"1.1","doi":"10.1101/2022.07.21.501010","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.22.501169","pub_date":"2022-7-22","title":"Synergism of interferon-beta with antiviral drugs against SARS-CoV-2 variants","abstract":"Omicron BA.1 variant isolates were previously shown to replicate less effectively in interferon-competent cells and to be more sensitive to interferon treatment than a Delta isolate. Here, an Omicron BA.2 isolate displayed intermediate replication patterns in interferon-competent Caco-2-F03 cells when compared to BA.1 and Delta isolates. Moreover, BA.2 was less sensitive than BA.1 and similarly sensitive as Delta to betaferon treatment. Delta and BA.1 displayed similar sensitivity to the approved anti-SARS-CoV-2 drugs remdesivir, nirmatrelvir, EIDD-1931 (the active metabolite of molnupiravir) and the protease inhibitor aprotinin, whereas BA.2 was less sensitive than Delta and BA.1 to EIDD-1931, nirmatrelvir and aprotinin. Nirmatrelvir, EIDD-1931, and aprotinin (but not remdesivir) exerted synergistic antiviral activity in combination with betaferon, with some differences in the extent of synergism detected between the different SARS-CoV-2 variants. In conclusion, even closely related SARS-CoV-2 (sub)variants can differ in their biology and in their response to antiviral treatments. Betaferon combinations with nirmatrelvir and, in particular, with EIDD-1931 and aprotinin displayed high levels of synergism, which makes them strong candidates for clinical testing. Notably, effective antiviral combination therapies are desirable, as a higher efficacy is expected to reduce resistance formation.","version":"1.1","doi":"10.1101/2022.07.22.501169","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.28.497981","pub_date":"2022-7-21","title":"Crystal structure of the membrane (M) protein from a SARS-COV-2-related coronavirus","abstract":"The membrane (M) protein is the most abundant structural protein of coronaviruses including SARS-COV-2 and plays a central role in virus assembly through its interaction with various partner proteins. However, mechanistic details about how M protein interacts with others remain elusive due to lack of high-resolution structures. Here, we present the first crystal structure of a coronavirus M protein from Pipistrellus bat coronavirus HKU5 (batCOV5-M), which is closely related to SARS-COV-2 M protein. Furthermore, an interaction analysis indicates that the carboxy-terminus of the batCOV5 nucleocapsid (N) protein mediates its interaction with batCOV5-M. Combined with a computational docking analysis an M-N interaction model is proposed, providing insight into the mechanism of M protein-mediated protein interactions.","version":"1.2","doi":"10.1101/2022.06.28.497981","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.21.500897","pub_date":"2022-7-21","title":"Buying time: detecting Vocs in SARS-CoV-2 via co-evolutionary signals","abstract":"We present a novel framework facilitating the rapid detection of variants of interest (VOI) and concern (VOC) in a viral multiple sequence alignment (MSA). The framework is purely based on the genomic sequence data, without requiring prior established biological analysis. The framework\u2019s building blocks are sets of co-evolving sites (motifs), identified via co-evolutionary signals within the MSA. Motifs form a weighted simplicial complex, whose vertices are sites that satisfy a certain nucleotide diversity. Higher dimensional simplices are constructed using distances quantifying the co-evolutionary coupling of pairs and in the context of our method maximal motifs manifest as clusters. The framework triggers an alert via a cluster with a significant fraction of newly emerging polymorphic sites. We apply our method to SARS-CoV-2, analyzing all alerts issued from November 2020 through August 2021 with weekly resolution for England, USA, India and South America. Within a week at most a handful of alerts, each of which involving on the order of 10 sites are triggered. Cross referencing alerts with a posteriori knowledge of VOI/VOC-designations and lineages, motif-induced alerts detect VOIs/VOCs rapidly, typically weeks earlier than current methods. We show how motifs provide insight into the organization of the characteristic mutations of a VOI/VOC, organizing them as co-evolving blocks. Finally we study the dependency of the motif reconstruction on metric and clustering method and provide the receiver operating characteristic (ROC) of our alert criterion.","version":"1.1","doi":"10.1101/2022.07.21.500897","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.21.500987","pub_date":"2022-7-21","title":"Temporal profiling of CD4 T-cell activation and differentiation upon SARS-CoV-2 spike protein immunisation","abstract":"CD4 T-cells require T-cell receptor (TCR) signalling for their activation and differentiation. Foxp3+ regulatory T-cells (Treg) are dependent on TCR signals for their differentiation and suppressive function. However, it is not fully known how TCR signalling controls the differentiation of polyclonal CD4 T-cells upon antigen recognition at the single-cell level in vivo. In this study, using Nr4a3-Tocky (Timer-of-cell-kinetics-and-activity), which analyses temporal changes of antigen-reactive T-cells following TCR signalling, we investigated T-cell response to Spike protein fragments (S1a, S1b, S2a, and S2b) upon immunisation. We show that S1a and S2a induced the differentiation of PD1hiCXCR5+ T follicular helper (Tfh) cells, which is related to CD4 T-cell immunogenicity. In contrast, S1b induced CD25hiGITRhiPD-1int Treg, which intermittently received TCR signalling. Using Foxp3-Tocky, which analyses Foxp3 transcriptional dynamics, the S1b-reactive Treg sustained Foxp3 transcription over time, which is a hallmark of activated Treg. Foxp3 fate-mapping showed that the S1b-reactive Treg were derived not from pre-existing thymic Treg, suggesting Foxp3 induction in non-Treg cells. Thus, the current study reveals temporally dynamic differentiation of CD4 T-cells and Treg upon immunisation in the polyclonal TCR repertoire.","version":"1.1","doi":"10.1101/2022.07.21.500987","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500688","pub_date":"2022-7-20","title":"Safety and biodistribution of Nanoligomers\u2122 targeting SARS-CoV-2 genome for treatment of COVID-19","abstract":"As the world braces to enter its third year in the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer,\u2122 a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed after three separate administration methods, namely intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.","version":"1.1","doi":"10.1101/2022.07.19.500688","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.08.483429","pub_date":"2022-7-20","title":"Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting","abstract":"SARS-CoV-2 is currently causing an unprecedented pandemic. While vaccines are massively deployed, we still lack effective large-scale antiviral therapies. In the quest for antivirals targeting conserved structures, we focused on molecules able to bind viral RNA secondary structures. Aminoglycosides are a class of antibiotics known to interact with the ribosomal RNA of both prokaryotes and eukaryotes and have previously been shown to exert antiviral activities by interacting with viral RNA. Here we show that the aminoglycoside geneticin is endowed with antiviral activity against all tested variants of SARS-CoV-2, in different cell lines and in a respiratory tissue model at non-toxic concentrations. The mechanism of action is an early inhibition of RNA replication and protein expression related to a decrease in the efficiency of the -1 programmed ribosomal frameshift (PRF) signal of SARS-CoV-2. Using in silico modelling, we have identified a potential binding site of geneticin in the pseudoknot of frameshift RNA motif. Moreover, we have selected, through virtual screening, additional RNA binding compounds, interacting with the same site with increased potency.","version":"1.2","doi":"10.1101/2022.03.08.483429","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500631","pub_date":"2022-7-20","title":"Regulation of mRNA transcripts, protein isoforms, glycosylation and spatial localization of ACE2 and other SARS-CoV-2-associated molecules in human airway epithelium upon viral infection and type 2 inflammation","abstract":"SARS-CoV-2 infection continues to pose a significant life threat, especially in patients with comorbidities. It remains unknown, if asthma or allergen- and virus-induced airway inflammation are risk factors or can constitute some forms of protection against COVID-19. ACE2 and other SARS-CoV-2-related host proteins are limiting factors of an infection, expression of which is regulated in a more complex way than previously anticipated. Hence, we studied the expression of ACE2 mRNA and protein isoforms, together with its glycosylation and spatial localization in house dust mite (HDM)-, interleukin-13 (IL-13)- and human rhinovirus (RV)-induced inflammation in the primary human bronchial airway epithelium of healthy subjects and patients with asthma. IL-13 decreased the expression of long ACE2 mRNA and glycosylation of full-length ACE2 protein via alteration of the N-linked glycosylation process, limiting its availability on the apical side of ciliated cells. RV infection increased short ACE2 mRNA, but it did not influence its protein expression. HDM exposure did not affect ACE2 mRNA or protein. IL-13 and RV significantly regulated mRNA, but not protein expression of TMPRSS2 and NRP1. Regulation of ACE2 and other host proteins was similar in healthy and asthmatic epithelium, underlining the lack of intrinsic differences, but rather the dependence on the inflammatory milieu in the airways.","version":"1.1","doi":"10.1101/2022.07.19.500631","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500639","pub_date":"2022-7-19","title":"Orally administered niclosamide-based organic/inorganic hybrid suppresses SARS-CoV-2 infection","abstract":"The COVID-19 pandemic is a serious global health threat mainly due to the surging cases along with new variants of COVID-19. Though global vaccinations have indeed some effects on the virus spread, its longevity is still unknown. Therefore an orally administrable anti-viral agent against SARS-CoV-2 would be of substantial benefit in controlling the COVID-19 pandemic. Herein, we repurposed niclosamide (NIC), an FDA approved anthelmintic drug in to MgO, which was further coated with hydroxyl propyl methyl cellulose (HPMC) to get the de-sired product called NIC-MgO-HPMC, which has improved anti-SARS-CoV-2 replication in the Syrian hamster model. The inhibitory effect of NIC-MgO-HPMC on SARS-CoV-2 replication leads to the prevention of inflammation as well as lung injury. These data strongly support that repurposed NIC-MgO-HPMC could be highly beneficial for controlling the ongoing pandemic thereby achieving an endemic phase.","version":"1.1","doi":"10.1101/2022.07.19.500639","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500626","pub_date":"2022-7-19","title":"Monovalent and trivalent VSV-based COVID-19 vaccines elicit potent neutralizing antibodies and immunodominant CD8+ T cells against diverse SARS-CoV-2 variants","abstract":"Recombinant vesicular stomatitis virus (rVSV) vaccines expressing Spike proteins of Wuhan, Beta and/or Delta variants of SARS-CoV-2 were generated and tested for induction of antibody and T cell immune responses in mice. rVSV-Wuhan and rVSV-Delta vaccines and a rVSV-Trivalent (mixed rVSV-Wuhan, -Beta, -Delta) vaccine elicited potent neutralizing antibodies (nAbs) against live SARS-CoV-2 Wuhan (USAWA1), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) viruses. Prime-boost vaccination with rVSV-Beta was less effective in this capacity. Heterologous boosting of rVSV-Wuhan with rVSV-Delta induced strong nAb responses against Delta and Omicron viruses, with rVSV-Trivalent vaccine consistently effective in inducing nAbs against all the SARS-CoV-2 variants tested. All vaccines, including rVSV-Beta, elicited a spike-specific immunodominant CD8+ T cell response. Collectively, rVSV vaccines targeting SARS-CoV-2 variants of concern may be considered in the global fight against COVID-19.","version":"1.1","doi":"10.1101/2022.07.19.500626","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.18.500565","pub_date":"2022-7-19","title":"The First Geographic Identification by Country of Sustainable Mutations of SARS-COV2 Sequence Samples: Worldwide Natural Selection Trends","abstract":"The high mutation rates of RNA viruses, coupled with short generation times and large population sizes, allow viruses to evolve rapidly and adapt to the host environment. The rapidity of viral mutation also causes problems in developing successful vaccines and antiviral drugs. With the spread of SARS-CoV-2 worldwide, thousands of mutations have been identified, some of which have relatively high incidences, but their potential impacts on virus characteristics remain unknown. The present study analyzed mutation patterns, SARS-CoV-2 AASs retrieved from the GISAID database containing 10,500,000 samples. Python 3.8.0 programming language was utilized to pre-process FASTA data, align to the reference sequence, and analyze the sequences. Upon completion, all mutations discovered were categorized based on geographical regions and dates. The most stable mutations were found in nsp1(8% S135R), nsp12(99.3% P323L), nsp16 (1.2% R216C), envelope (30.6% T9I), spike (97.6% D614G), and Orf8 (3.5% S24L), and were identified in the United States on April 3, 2020, and England, Gibraltar, and, New Zealand, on January 1, 2020, respectively. The study of mutations is the key to improving understanding of the function of the SARS-CoV-2, and recent information on mutations helps provide strategic planning for the prevention and treatment of this disease. Viral mutation studies could improve the development of vaccines, antiviral drugs, and diagnostic assays designed with high accuracy, specifically useful during pandemics. This knowledge helps to be one step ahead of new emergence variants. More than two years into the global COVID-19 pandemic, the focus of attention is shifted to the emergence and spread of the SARS-CoV-2 variants that cause the evolutionary trend. Here, we analyzed and compared about 10.5 million sequences of SARS-CoV-2 to extract the stable mutations, frequencies and the substitute amino acid that changed with the wild-type one in the evolutionary trend. Also, developing and designing accurate vaccines could prepare long-term immunization against different local variants. In addition, according to the false negative results of the COVID-19 PCR test report in the diagnosis of new strains, investigating local mutation patterns could help to design local primer and vaccine.","version":"1.1","doi":"10.1101/2022.07.18.500565","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500637","pub_date":"2022-7-19","title":"Vaccination shapes evolutionary trajectories of SARS-CoV-2","abstract":"The large-scale evolution of the SARS-CoV-2 virus has been marked by rapid turnover of genetic clades. New variants show intrinsic changes, notably increased transmissibility, as well as anti-genic changes that reduce the cross-immunity induced by previous infections or vaccinations. How this functional variation shapes the global evolutionary dynamics has remained unclear. Here we show that selection induced by vaccination impacts on the recent antigenic evolution of SARS-CoV-2; other relevant forces include intrinsic selection and antigenic selection induced by previous infections. We obtain these results from a fitness model with intrinsic and antigenic fitness components. To infer model parameters, we combine time-resolved sequence data, epidemiological records, and cross-neutralisation assays. This model accurately captures the large-scale evolutionary dynamics of SARS-CoV-2 in multiple geographical regions. In particular, it quantifies how recent vaccinations and infections affect the speed of frequency shifts between viral variants. Our results show that timely neutralisation data can be harvested to identify hotspots of antigenic selection and to predict the impact of vaccination on viral evolution.","version":"1.1","doi":"10.1101/2022.07.19.500637","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.18.500430","pub_date":"2022-7-19","title":"Experimental infection of Mexican free-tailed bats (Tadarida brasiliensis) with SARS-CoV-2","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats (Tadarida brasiliensis: TABR) live in large colonies in the southern United States, often in urban areas, and as such, could be exposed to the virus from infected humans. We experimentally challenged wild TABR with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of nine bats oronasally inoculated with SARS-CoV-2, five became infected and orally excreted moderate amounts of virus for up to 18 days post inoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while TABR are susceptible to SARS-CoV-2 infection, infection of wild populations of TABR would not likely cause mortality. However, the transmission of SARS-CoV-2 from TABR to or from humans, or to other animal species, is a distinct possibility requiring further investigation to better define.","version":"1.2","doi":"10.1101/2022.07.18.500430","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.19.500662","pub_date":"2022-7-19","title":"Persistence of SARS-CoV-2 neutralizing antibodies longer than 13 months in naturally-infected, captive white-tailed deer (Odocoileus virginianus), Texas","abstract":"After identifying a captive herd of white-tailed deer (WTD) in central Texas with >94% seroprevalence with SARS-CoV-2 neutralizing antibodies in September 2021, we worked retrospectively through archived serum samples of 21 deer and detected seroconversion of all animals between December 2020 and January 2021. We then collected prospective samples to conclude that the duration of persistence of neutralizing antibodies is at least 13 months for 19 (90.5%) of the animals, with two animals converting to seronegative after six and eight months. Antibody titers generally waned over this time frame, but three deer had a temporary 4 to 8-fold increases in PRNT titers over a month after seroconversion; anamnestic response cannot be ruled out.","version":"1.1","doi":"10.1101/2022.07.19.500662","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.15.22277570","pub_date":"2022-07-19","title":"Pharmacometric assessment of the\n                  <i>in vivo</i>\n                  antiviral activity of ivermectin in early symptomatic COVID-19","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>\n                    There is no generally accepted methodology for\n                    <jats:italic>in vivo</jats:italic>\n                    assessment of antiviral activity in SARS-CoV-2 infection. Ivermectin has been recommended widely as a treatment of COVID-19, but whether it has significant antiviral activity\n                    <jats:italic>in vivo</jats:italic>\n                    is uncertain.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    In a multicentre open label, randomized, controlled adaptive platform trial, adult patients with early symptomatic COVID-19 were randomized to one of six treatment arms including high dose ivermectin (600\u00b5g/kg daily for seven days), the monoclonal antibodies casirivimab and imdevimab (600mg/600mg), and no study drug. Viral clearance rates were derived from daily duplicate oropharyngeal quantitative PCR measurements. This ongoing trial is registered at\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    (\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT05041907'>NCT05041907</jats:ext-link>\n                    ).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Randomization to the ivermectin arm was stopped after enrolling 205 patients into all arms, as the prespecified futility threshold was reached. Compared with the no study drug arm, the mean estimated SARS-CoV-2 viral clearance following ivermectin was 9.1% slower [95%CI -27.2% to +11.8%; n=45 versus n=41], whereas in a preliminary analysis of the casirivimab/imdevimab arm it was 52.3% faster [95%CI +7.0% to +115.1%; n=10 (Delta variant) versus n=41].</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    High dose ivermectin did not have measurable antiviral activity in early symptomatic COVID-19. Measured in this way viral clearance rate is a valuable pharmacodynamic measure in assessing antiviral COVID-19 therapeutics\n                    <jats:italic>in vivo</jats:italic>\n                    .\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>\u201cFinding treatments for COVID-19: A phase 2 multi-centre adaptive platform trial to assess antiviral pharmacodynamics in early symptomatic COVID-19 (PLAT-COV)\u201d is funded by the Wellcome Therapeutics Accelerator (223195/Z/21/Z).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Impact</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>Rate of viral clearance determined from daily duplicate oropharyngeal swabs over one week is an efficient measure of antiviral efficacy in early COVID-19 infection.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>High dose ivermectin did not demonstrate measurable antiviral activity in early symptomatic COVID-19 infection.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.15.22277570","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.18.500363","pub_date":"2022-7-18","title":"Identification of a Papain-Like Protease Inhibitor with Potential for Repurposing in Combination with an Mpro Protease Inhibitor for Treatment of SARS-CoV-2","abstract":"SARS-CoV-2 requires two cysteine proteases for viral polypeptide processing to allow maturation and replication: the 3C-like protease also known as the Main protease (Mpro) and the papain-like protease (PLpro). In addition to its critical role in viral replication, PLpro removes post-translational modifications like ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins through its deubiquitinase domain, leading to host immunosuppression and increased ability of the virus to evade the host antiviral immune response. Through screening of a custom clinical compound library, we identified eltrombopag (DDL-701), a thrombopoietin receptor agonist, as having PLpro inhibitory activity that is sustained in the presence of the Mpro inhibitor nirmatrelvir. DDL-701 also suppressed both the deubiquitinase and ISG15 cleavage activities of PLpro. In addition, DDL-701 partially restored interferon-\u03b2 induction \u2013 an element of the host immune response - in an in vitro model system. Further, modeling and docking studies suggest DDL-701 interacts with the active site region of the PLpro enzyme and pilot pharmacokinetic studies indicate it is brain permeable. DDL-701 is already approved for treatment of thrombocytopenia and has previously been shown to achieve human plasma levels after oral dosing that is above the IC50 needed for it to exert its PLpro inhibitory activity in vivo. In addition, it has also been reported to have antiviral efficacy against SARS-CoV-2. DDL-701 thus represents a drug that can immediately be repurposed and undergo clinical evaluation as a PLpro inhibitor that may be most effectively used in a protease inhibitor cocktail with an Mpro inhibitor such as nirmatrelvir (Paxlovid) for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.07.18.500363","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.28.470269","pub_date":"2022-7-18","title":"COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis","abstract":"In the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options. Using an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR. COVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric), and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2. Like IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung diseases. This work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780). Severe COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease In its aftermath, the COVID-19 pandemic has left many survivors, almost a third of those who recovered, with a mysterious long-haul form of the disease which culminates in a fibrotic form of interstitial lung disease (post-COVID-19 ILD). Post-COVID-19 ILD remains a largely unknown entity. Currently, we lack insights into the core cytopathic features that drive this condition. Using an AI-guided approach, which involves the use of sets of gene signatures, protein-protein network analysis, and a hamster model of COVID-19, we have revealed here that COVID-19 -lung fibrosis resembles IPF, the most common form of ILD, at a fundamental level\u2014showing similar gene expression patterns in the lungs and blood, and dysfunctional AT2 processes (ER stress, telomere instability, progenitor cell arrest, and senescence). These findings are insightful because AT2 cells are known to contain an elegant quality control network to respond to intrinsic or extrinsic stress; a failure of such quality control results in diverse cellular phenotypes, of which ER stress appears to be a point of convergence, which appears to be sufficient to drive downstream fibrotic remodeling in the lung. Because unbiased computational methods identified the shared fundamental aspects of gene expression and cellular processes between COVID-19 and IPF, the impact of our findings is likely to go beyond COVID-19 or any viral pandemic. The insights, tools (disease models, gene signatures, and biomarkers), and mechanisms identified here are likely to spur the development of therapies for patients with IPF and, other fibrotic interstitial lung diseases, all of whom have limited or no treatment options. To dissect the validated prognostic biomarkers to assess and track the risk of pulmonary fibrosis and develop therapeutics to halt fibrogenic progression.","version":"1.4","doi":"10.1101/2021.11.28.470269","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485312","pub_date":"2022-7-18","title":"Maximum likelihood pandemic-scale phylogenetics","abstract":"Phylogenetics plays a crucial role in the interpretation of genomic data. Phylogenetic analyses of SARS-CoV-2 genomes have allowed the detailed study of the virus\u2019s origins, of its international and local spread, and of the emergence and reproductive success of new variants, among many applications. These analyses have been enabled by the unparalleled volumes of genome sequence data generated and employed to study and help contain the pandemic. However, preferred model-based phylogenetic approaches including maximum likelihood and Bayesian methods, mostly based on Felsenstein\u2019s \u2018pruning\u2019 algorithm, cannot scale to the size of the datasets from the current pandemic, hampering our understanding of the virus\u2019s evolution and transmission. We present new approaches, based on reworking Felsenstein\u2019s algorithm, for likelihood-based phylogenetic analysis of epidemiological genomic datasets at unprecedented scales. We exploit near-certainty regarding ancestral genomes, and the similarities between closely related and densely sampled genomes, to greatly reduce computational demands for memory and time. Combined with new methods for searching amongst candidate evolutionary trees, this results in our MAPLE (\u2018MAximum Parsimonious Likelihood Estimation\u2019) software giving better results than popular approaches such as FastTree 2, IQ-TREE 2, RAxML-NG and UShER. Our approach therefore allows complex and accurate proba-bilistic phylogenetic analyses of millions of microbial genomes, extending the reach of genomic epidemiology. Future epidemiological datasets are likely to be even larger than those currently associated with COVID-19, and other disciplines such as metagenomics and biodiversity science are also generating huge numbers of genome sequences. Our methods will permit continued use of preferred likelihood-based phylogenetic analyses.","version":"1.2","doi":"10.1101/2022.03.22.485312","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.15.22277678","pub_date":"2022-07-17","title":"Influence of vitamin D supplementation on SARS-CoV-2 vaccine efficacy and immunogenicity","abstract":"<jats:sec>\n                  <jats:title>SUMMARY</jats:title>\n                  <jats:sec>\n                    <jats:title>Background &amp; Aims</jats:title>\n                    <jats:p>Vitamin D deficiency has been reported to associate with impaired development of antigen-specific responses following vaccination. We aimed to determine whether vitamin D supplements might boost immunogenicity and efficacy of SARS-CoV-2 vaccination.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods</jats:title>\n                    <jats:p>We conducted three sub-studies nested within the CORONAVIT randomised controlled trial, which investigated effects of offering vitamin D supplements at a dose of 800 IU/day or 3200 IU/day vs. no offer on risk of acute respiratory infections, including COVID-19, in UK adults with circulating 25-hydroxyvitamin D concentrations &lt;75 nmol/L. Sub-study 1 (n=2808) investigated effects of vitamin D supplementation on risk of breakthrough SARS-CoV-2 infection following two doses of SARS-CoV-2 vaccine. Sub-study 2 (n=1853) investigated effects of vitamin D supplementation on titres of combined IgG, IgA and IgM (IgGAM) anti-Spike antibodies in eluates of dried blood spots collected after SARS-CoV-2 vaccination. Sub-study 3 (n=100) investigated effects of vitamin D supplementation on neutralising antibody and cellular responses in venous blood samples collected after SARS-CoV-2 vaccination.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Results</jats:title>\n                    <jats:p>1945/2808 (69.3%) sub-study 1 participants received two doses of ChAdOx1 nCoV-19 (Oxford\u2013AstraZeneca); the remainder received two doses of BNT162b2 (Pfizer). Vitamin D supplementation did not influence risk of breakthrough SARS-CoV-2 infection (800 IU/day vs. no offer: adjusted hazard ratio 1.28, 95% CI 0.89 to 1.84; 3200 IU/day vs. no offer: 1.17, 0.81 to 1.70). Neither did it influence IgGAM anti-Spike titres, neutralising antibody titres or IFN-\u03b3 concentrations in supernatants of S peptide-stimulated whole blood.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions</jats:title>\n                    <jats:p>Among adults with sub-optimal baseline vitamin D status, vitamin D replacement at a dose of 800 or 3200 IU/day did not influence protective efficacy or immunogenicity of SARS-CoV-2 vaccination.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Clinical Trial Registration</jats:title>\n                    <jats:p>\n                      <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                      <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04579640'>NCT04579640</jats:ext-link>\n                      .\n                    </jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.15.22277678","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.17.500346","pub_date":"2022-7-17","title":"Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform","abstract":"Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the HK2 inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.","version":"1.1","doi":"10.1101/2022.07.17.500346","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.500148","pub_date":"2022-7-17","title":"Novavax NVX-COV2373 triggers potent neutralization of Omicron sub-lineages","abstract":"The SARS-CoV-2 Omicron (B.1.1.529) Variant of Concern (VOC) and its sub-lineages (including BA.2, BA.4/5, BA.2.12.1) contain spike mutations that confer high level resistance to neutralizing antibodies. The NVX-CoV2373 vaccine, a protein nanoparticle vaccine, has value in countries with constrained cold-chain requirements. Here we report neutralizing titers following two or three doses of NVX-CoV2373. We show that after two doses, Omicron sub-lineages BA.1 and BA.4 were resistant to neutralization by 72% (21/29) and 59% (17/29) of samples. However, after a third dose of NVX-CoV2373, we observed high titers against Omicron BA.1 (GMT: 1,197) and BA.4 (GMT: 582), with responses similar in magnitude to those triggered by three doses of an mRNA vaccine. These data are of particular relevance as BA.4 is emerging to become the dominant strain in many locations, and highlight the potential utility of the NVX-CoV2373 vaccine as a booster in resource-limited environments.","version":"1.1","doi":"10.1101/2022.07.14.500148","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.13.499991","pub_date":"2022-7-15","title":"SARS-CoV-2 causes brain inflammation via impaired neuro-immune interactions","abstract":"The brain inflammation that frequently occurs in SARS-CoV-2 is the cause of neurological complications and long COVID . However, many aspects of its pathogenesis mechanism remain unknown  and no method of treatment has been established . By administering a non-proliferating adenovirus vector expressing SARS-CoV-2 S1 protein into the nasal cavity of mice, we developed a mouse model (S1 mouse) reproducing brain inflammation, fatigue, depressive symptoms, and lung inflammation. Having intracellular calcium elevating activity, S1 protein increased olfactory bulb apoptosis, and reduced the number of acetylcholine producing cells in the medial septal and the diagonal band of Broca as well as the amount of acetylcholine in the brain. This resulted in disrupting the cholinergic anti-inflammatory pathway (CAP)  and enhancing inflammation in the brain. Previously, nothing was known about anti-inflammatory factors in the CAP but we discovered that, in the inflammation occurring in the S1 mouse brain, the action of the RNA binding protein ZFP36  in degrading inflammatory cytokine mRNA was impaired. The symptoms exhibited by the S1 mouse were improved by administering donepezil, a drug with a cholinergic action used in the treatment of dementia. These findings clarify the mechanism of brain inflammation in COVID-19 and indicate the possibility of applying donepezil in the treatment of neurological complications in COVID-19 and long COVID.","version":"1.1","doi":"10.1101/2022.07.13.499991","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.499651","pub_date":"2022-7-15","title":"Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models","abstract":"ACE2 is a major receptor for cell entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2\u2019s binding, how to efficiently and flexibly control ACE2 levels for prevention of SARS-CoV-2 infection has not been explored. Here, we revealed Vitamin C (VitC) administration as an effective strategy to prevent SARS-CoV-2 infection. VitC reduced ACE2 protein levels in a dose-dependent manner, while partial reduction of ACE2 can greatly restrict SARS-CoV-2 infection. Further studies uncovered that USP50 is a crucial regulator of ACE2 protein levels, and VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination at Lys788 and degradation of ACE2, without disrupting ACE2 transcriptional expression. Importantly, VitC administration reduced host ACE2 and largely blocked SARS-CoV-2 infection in mice. This study identified an in vivo ACE2 balance controlled by both USP50 and an essential nutrient VitC, and revealed a critical role and application of VitC in daily protection from SARS-CoV-2 infection. VitC reduces ACE2 protein levels in a dose-dependent manner VitC and USP50 regulate K48-linked ubiquitination at Lys788 of ACE2 VitC blocks the interaction between USP50 and ACE2 VitC administration lowers host ACE2 and prevents SARS-CoV-2 infection in vivo The deubiquitinase USP50 controls ACE2 protein stability and levels, while Vitamin C blocks the USP50-ACE2 interaction and therefore results in ACE2 degradation, offering a flexible and efficient approach to protection of the host from SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.07.14.499651","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.500063","pub_date":"2022-7-15","title":"Genome-first detection of emerging resistance to novel therapeutic agents for SARS-CoV-2","abstract":"Some COVID-19 patients are unable to clear their infection or are at risk of severe disease, requiring treatment with neutralising monoclonal antibodies (nmAb) and/or antivirals. The rapid roll-out of novel therapeutics means there is limited understanding of the likely genetic barrier to drug resistance. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to the detection of emerging drug resistance. Here we report the accrual of mutations in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the epitopes of the respective nmAbs. For casirivimab+imdevimab these are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.","version":"1.1","doi":"10.1101/2022.07.14.500063","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.500039","pub_date":"2022-7-15","title":"SARS-CoV-2 BA.4 infection triggers more cross-reactive neutralizing antibodies than BA.1","abstract":"SARS-CoV-2 variants of concern (VOCs) differentially trigger neutralizing antibodies with variable cross-neutralizing capacity. Here we show that unlike SARS-CoV-2 Omicron BA.1, which triggered neutralizing antibodies with limited cross-reactivity, BA.4/5 infection triggers highly cross-reactive neutralizing antibodies. Cross-reactivity was observed both in the absence of prior vaccination and also in breakthrough infections following vaccination. This suggests that next-generation vaccines incorporating BA.4, which is spreading globally, might result in enhanced neutralization breadth.","version":"1.1","doi":"10.1101/2022.07.14.500039","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.500068","pub_date":"2022-7-15","title":"Ending transmission of SARS-CoV-2: sterilizing immunity using an intranasal subunit vaccine","abstract":"Immunization programs against SARS-CoV-2 with commercial intramuscular (IM) vaccines prevent disease but not infections. The continued evolution of variants of concern (VOC) like Delta and Omicron has increased infections even in countries with high vaccination coverage. This is due to commercial vaccines being unable to prevent viral infection in the upper airways and exclusively targeting the spike (S) protein that is subject to continuous evolution facilitating immune escape. Here we report a multi-antigen, intranasal vaccine, NanoSTING-NS that yields sterilizing immunity and leads to the rapid and complete elimination of viral loads in both the lungs and the nostrils upon viral challenge with SARS-CoV-2 VOC. We formulated vaccines with the S and nucleocapsid (N) proteins individually to demonstrate that immune responses against S are sufficient to prevent disease whereas combination immune responses against both proteins prevents viral replication in the nasal compartment. Studies with the highly infectious Omicron VOC showed that even in vaccine-na\u00efve animals, a single dose of NanoSTING-NS significantly reduced transmission. These observations have two implications: (1) mucosal multi-antigen vaccines present a pathway to preventing transmission and ending the pandemic, and (2) an explanation for why hybrid immunity in humans is superior to vaccine-mediated immunity by current IM vaccines.","version":"1.1","doi":"10.1101/2022.07.14.500068","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.15.500185","pub_date":"2022-7-15","title":"Lasting alterations in monocyte and dendritic cell subsets in individuals after hospitalization for COVID-19","abstract":"After more than two years the COVID-19 pandemic continues to burden healthcare systems and economies worldwide, and it is evident that long-term effects of the disease can persist for months post-recovery in some individuals. The activity of myeloid cells such as monocytes and dendritic cells (DC) is essential for correct mobilization of the innate and adaptive responses to a pathogen. Impaired levels and responses of monocytes and DC to SARS-CoV-2 is likely to be a driving force behind the immune dysregulation that characterizes severe COVID-19. Here, we followed, for 6-7 months, a cohort of COVID-19 patients hospitalized during the early waves of the pandemic. The levels and phenotypes of circulating monocyte and DC subsets were assessed to determine both the early and long-term effects of the SARS-CoV-2 infection. We found increased monocyte levels that persisted for 6-7 months, mostly attributed to elevated levels of classical monocytes. While most DC subsets recovered from an initial decrease, we found elevated levels of cDC2/cDC3 at the 6-7 month timepoint. Analysis of functional markers on monocytes and DC revealed sustained reduction in PD-L1 expression but increased CD86 expression across almost all cell types examined. Finally, viral load and CRP correlated to the appearance of circulating antibodies and levels of circulating DC and monocyte subsets, respectively. By elucidating some of the long-term effects that SARS-CoV-2 infection has on these key innate myeloid cells, we have shed more light on how the immune landscape remains affected in the months following severe COVID-19.","version":"1.1","doi":"10.1101/2022.07.15.500185","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.500042","pub_date":"2022-7-15","title":"Fc effector activity and neutralization against SARS-CoV-2 BA.4 is compromised in convalescent sera, regardless of the infecting variant","abstract":"The SARS-CoV-2 Omicron BA.1 variant, which exhibits high level neutralization resistance, has since evolved into several sub-lineages including BA.4 and BA.5, which have dominated the fifth wave of infection in South Africa. Here we assessed the sensitivity of BA.4 to neutralization and antibody dependent cellular cytotoxicity (ADCC) in convalescent donors infected with four previous variants of SARS-CoV-2, as well as in post-vaccination breakthrough infections (BTIs) caused by Delta or BA.1. We confirm that BA.4 shows high level resistance to neutralization, regardless of the infecting variant. However, breakthrough infections, which trigger potent neutralization, retained activity against BA.4, albeit at reduced titers. Fold reduction of neutralization in BTIs was lower than that seen in unvaccinated convalescent donors, suggesting maturation of neutralizing responses to become more resilient against VOCs in hybrid immunity. BA.4 sensitivity to ADCC was reduced but remained detectable in both convalescent donors and in BTIs. Overall, the high neutralization resistance of BA.4, even to antibodies from BA.1 infections, provides an immunological mechanism for the rapid spread of BA.4 immediately after a BA.1-dominated wave. Furthermore, although ADCC activity against BA.4 was reduced, residual activity may nonetheless contribute to the protection from disease.","version":"1.1","doi":"10.1101/2022.07.14.500042","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.14.500031","pub_date":"2022-7-15","title":"A zebrafish model of COVID-19-associated cytokine storm syndrome reveals that the Spike protein signals via TLR2","abstract":"Understanding the mechanism of virulence of SARS-CoV-2 and host innate immune responses are essential to develop novel therapies. One of the most studied defense mechanisms against invading pathogens, including viruses, are Toll-like receptors (TLRs). Among them, TLR3, TLR7, TLR8 and TLR9 detect different forms of viral nucleic acids in endosomal compartments, whereas TLR2 and TLR4 recognize viral structural and nonstructural proteins outside the cell. Although many different TLRs have been shown to be involved in SARS-CoV-2 infection and detection of different structural proteins, most studies have been performed in vitro and the results obtained are rather contradictory. In this study, we report using the unique advantages of the zebrafish model for in vivo imaging and gene editing that the S1 domain of the Spike protein from the Wuhan strain (S1WT) induced hyperinflammation in zebrafish larvae via a Tlr2/Myd88 signaling pathway and independently of interleukin-1\u03b2 production. In addition, S1WT also triggered emergency myelopoiesis, but in this case through a Tlr2/Myd88-independent signaling pathway. These results shed light on the mechanisms involved in the COVID-19-associated cytokine storm syndrome.","version":"1.1","doi":"10.1101/2022.07.14.500031","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.13.22277579","pub_date":"2022-07-15","title":"Long COVID in Cancer patients: Preponderance of Symptoms in Majority of Patients over Long Time Period","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>An increasing number of observational studies have reported the persistence of symptoms following recovery from acute COVID-19 disease in non-cancer patients. The long-term consequences of COVID-19 are not fully understood particularly in the cancer patient population. The purpose of this study is to assess post-acute sequelae of SARS-CoV-2 infection (PASC) in cancer patients following acute COVID-19 recovery.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We identified cancer patients at MD Anderson Cancer Center who were diagnosed with COVID-19 disease between March 1, 2020 and Sept 1, 2020 and followed them till May 2021. To assess PASC, we collected patients reported outcomes through questionnaires that were sent to patients daily for 14 days after COVID-19 diagnosis then weekly for 3 months, and then monthly thereafter. We also reviewed patients\u2019 electronic medical records to capture the the persistence or emergence of new COVID19-related symptoms reported during any clinic or hospital encounter beyond 30 days of the acute illness and up to 14 months.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We included 312 cancer patients with a median age of 57 years (18-86). The majority of patients had solid tumors (75%). Of the 312 patients, 188 (60%) reported long COVID-19 symptoms with a median duration of 7 months and up to 14 months after COVID-19 diagnosis. The most common symptoms reported included fatigue (82%), sleep disturbances (78%), myalgias (67%) and gastrointestinal symptoms (61%), followed by headache, altered smell or taste, dyspnea (47%) and cough (46%). A higher number of females reported a persistence of symptoms compared to males (63% vs 37%; p=0.036). Cancer type, neutropenia, lymphocytopenia, and hospital admission during acute COVID-19 disease were comparable in both groups. Among the 188 patients with PASC, only 16 (8.5%) were readmitted for COVID-related reasons.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>More than one out of two cancer patients, and more likely females, report PASC that may persist beyond 6 months and even one year. The most common symptoms are non-respiratory and consist of fatigue, sleep disturbance, myalgia and gastro-intestinal symptoms. Most of the cancer patients with PASC were managed on outpatient basis with only 8,5% requiring a COVID-19 related re-admission.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.13.22277579","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.14.500041","pub_date":"2022-7-15","title":"Neutralization sensitivity of Omicron BA.2.75 to therapeutic monoclonal antibodies","abstract":"Since the end of 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant outcompeted other variants and took over the world. After the emergence of original Omicron BA.1, Omicron BA.2 subvariant emerged and outcompeted BA.1. As of July 2022, some BA.2 subvariants, including BA.2.12.1, BA.4 and BA.5, emerged in multiple countries and begun outcompeting original BA.2. Moreover, a novel BA.2 subvariant, BA.2.75, was detected in eight countries including India at the end of June 2022, and preliminary investigations suggest that BA.2.75 is more transmissible over the other BA.2 subvariants. On July 7, 2022, the WHO classified BA.2.75 as a variant-of-concern lineage under monitoring. We have recently demonstrated that BA.4/5 is highly resistant to a therapeutic monoclonal antibody, cilgavimab, than BA.2. The resistance of SARS-CoV-2 variants to therapeutic antibodies can be attributed to the mutations in the viral spike protein. Compared to the BA.2 spike, BA.2.12.1 and BA.4/5 respectively bear two and four mutations in their spike proteins. On the other hand, the majority of BA.2.75 spike bears nine substitutions. The fact that the mutation number in the BA.2.75 spike is larger than those in the BA.4/5 spike raises the possibility that the BA.2.75 spike significantly reduces sensitivity towards therapeutic monoclonal antibodies than BA.2 and BA.4/5. In this study, we generated pseudoviruses harboring the spike proteins of BA.2.75, BA.4/5 and BA.2 and evaluated the efficacy of ten therapeutic monoclonal antibodies and three antibody cocktails against BA.2.75.","version":"1.1","doi":"10.1101/2022.07.14.500041","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.13.22277580","pub_date":"2022-07-15","title":"Procalcitonin for Antimicrobial Stewardship Among Cancer Patients Admitted with COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Procalcitonin (PCT) has been used to guide antibiotic therapy in bacterial infections. We aimed to determine the role of PCT in decreasing the duration of empiric antibiotic therapy among cancer patients admitted with COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This retrospective study included cancer patients admitted to our institution for COVID-19 between March 1, 2020, and June 28, 2021, with a PCT test done within 72 hours after admission. Patients were divided into 2 groups: PCT &lt;0.25 ng/ml and PCT \u22650.25 ng/ml. We assessed pertinent cultures, antibacterial use, and duration of empiric antibacterial therapy.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The study included 530 patients (median age, 62 years [range, 13-91]). All the patients had \u22651 culture test within 7 days following admission. Patients with PCT &lt;0.25 ng/ml were less likely to have a positive culture than were those with PCT \u22650.25 ng/ml (6% [20/358] vs 17% [30/172]; p&lt;0.0001). PCT &lt;0.25 ng/ml had a high negative predictive value for bacteremia and 30-day mortality. Patients with PCT &lt;0.25 ng/ml were less likely to receive intravenous (IV) antibiotics for &gt;72 hours than were patients with PCT \u22650.25 ng/ml (45% [162/358] vs 69% [119/172]; p&lt;0.0001). Among patients with PCT &lt;0.25 ng/ml and negative cultures, 30-day mortality was similar between those who received IV antibiotics for \u226572 hours and those who received IV antibiotics for shorter durations (2% [2/111] vs 3% [5/176], p=0.71).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Among cancer patients with COVID-19, PCT level &lt;0.25 ng/ml is associated with lower likelihood of bacterial co-infection and greater likelihood of a shorter antibiotic course. In patients with PCT level &lt;0.25 ng/ml and negative cultures, an antibiotic course of &gt; 72 hours is unnecessary. PCT could be useful in enhancing antimicrobial stewardship in cancer patients with COVID-19.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.13.22277580","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.13.443734","pub_date":"2022-7-14","title":"Optimised non-coding regions of mRNA SARS-CoV-2 vaccine CV2CoV improves homogeneous and heterogenous neutralising antibody responses","abstract":"More than two years after the emergence of SARS-CoV-2, 33 COVID-19 vaccines, based on different platforms, have been approved in 197 countries. Novel variants that are less efficiently neutralised by antibodies raised against ancestral SARS-CoV-2 are circulating, highlighting the need to adapt vaccination strategies. Here, we compare the immunogenicity of a first-generation mRNA vaccine candidate, CVnCoV, with a second-generation mRNA vaccine candidate, CV2CoV, in rats. Higher levels of spike (S) protein expression were observed in cell culture with CV2CoV mRNA than with CVnCoV mRNA. Vaccination with CV2CoV also induced higher titres of virus neutralising antibodies with accelerated kinetics in rats compared with CVnCoV. Significant cross-neutralization of the SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), and the \u2018mink\u2019 variant (B1.1.298) that were circulating at the time in early 2021 was also demonstrated. In addition, CV2CoV induced higher levels of antibodies at lower doses than CVnCoV, suggesting that dose-sparing could be possible with the next generation SARS-CoV-2 vaccine which could improve worldwide vaccine supply.","version":"1.2","doi":"10.1101/2021.05.13.443734","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.12.499813","pub_date":"2022-7-13","title":"Inducible Bronchus-Associated Lymphoid Tissue in SARS-CoV-2 Infected Rhesus Macaques","abstract":"Pulmonary immunity against SARS-CoV-2 infection has not been well studied. This study investigated the distribution of immune cells int the lungs of 8 rhesus macaques experimentally infected with SARS-CoV-2, and euthanized 11-14 days later. Using immunohistochemistry, inducible bronchus-associated lymphoid tissue was found in all animals. The inducible bronchus-associated lymphoid tissues were composed of B cells, T cells, and follicular dendritic cells with evidence of lymphocyte priming and differentiation. This suggests local immunity plays an important role in the SARS-CoV-2 infection. Further study of local immunity in the lung would benefit our understanding of SARS-CoV-2 pathogenesis and could lead to new interventions to control the SARS-CoV-2 infection and disease.","version":"1.1","doi":"10.1101/2022.07.12.499813","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.24.497526","pub_date":"2022-7-13","title":"In Vitro Evaluation and Mitigation of Niclosamide\u2019s Liabilities as a COVID-19 Treatment","abstract":"Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future. There is still an urgent need for effective anti-SARS-CoV-2 therapeutics due to waning vaccine efficacy, the emergence of variants of concern, and limited efficacy of existing antivirals. One potential therapeutic option is niclosamide, an FDA approved anthelmintic compound that has shown promising anti-SARS-CoV-2 activity in cell-based assays. Unfortunately, there are significant barriers for the clinical utility of niclosamide as a COVID-19 therapeutic. Our work emphasizes these limitations by showing that niclosamide has high cytotoxicity at antiviral concentrations, variable potency against variants of concern, and significant polypharmacology as a result of its activity as a nonspecific protonophore. Some of these clinical limitations can be mitigated, however, through structural modifications to the niclosamide scaffold, which we demonstrate through a preliminary structure activity relationship analysis. Overall, we show that niclosamide is not a suitable candidate for the treatment of COVID-19, but that structural analogs with improved drug properties may have higher clinical-translational potential.","version":"1.2","doi":"10.1101/2022.06.24.497526","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.13.499851","pub_date":"2022-7-13","title":"Pathobiology and dysbiosis of the respiratory and intestinal microbiota in 14 months old Golden Syrian hamsters infected with SARS-CoV-2","abstract":"The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS2) affected the geriatric population. Among research models, Golden Syrian hamsters (GSH) are one of the most representative to study SARS2 pathogenesis and host responses. However, animal studies that recapitulate the effects of SARS2 in the human geriatric population are lacking. To address this gap, we inoculated 14 months old GSH (resembling people over 60 years old) with a prototypic ancestral strain of SARS2 and studied the effects on virus pathogenesis, virus shedding, and respiratory and gastrointestinal microbiome changes. SARS2 infection led to high vRNA loads in the nasal turbinates (NT), lungs, and trachea as well as higher pulmonary lesions scores later in infection. Dysbiosis throughout SARS2 disease progression was observed in the pulmonary microbial dynamics with the enrichment of opportunistic pathogens (Haemophilus, Fusobacterium, Streptococcus, Campylobacter, and Johnsonella) and microbes associated with inflammation (Prevotella). Changes in the gut microbial community also reflected an increase in multiple genera previously associated with intestinal inflammation and disease (Helicobacter, Mucispirillum, Streptococcus, unclassified Erysipelotrichaceae, and Spirochaetaceae). Influenza A virus (FLUAV) pre-exposure resulted in slightly more pronounced pathology in the NT and lungs early on (3 dpc), and more notable changes in lungs compared to the gut microbiome dynamics. Similarities among aged GSH and the microbiome in critically ill COVID-19 patients, particularly in the lower respiratory tract, suggest that GSHs are a representative model to investigate microbial changes during SARS2 infection. The relationship between the residential microbiome and other confounding factors, such as SARS2 infection, in a widely used animal model, contributes to a better understanding of the complexities associated with the host responses during viral infections. The SARS-CoV-2 pandemic led to millions of human losses, notably affecting the geriatric population, who are at greater risk of developing acute respiratory distress infection leading to prolonged hospitalization and death. However, the mechanism of age-related pathogenicity is not fully understood. Here, we utilized an aged Syrian hamster model resembling ~60-year-old humans to analyze the pathobiology, host response, and effects of SARS2 on the respiratory and intestinal microbiome. We identified specific microbial markers observed in severe COVID-19 patients within the lungs of aged hamsters infected with SARS-CoV-2. Prior influenza A virus (H1N1) exposure amplified these changes. Similarities among aged GSH and critically ill COVID-19 patients suggest that GSHs are a valuable model for investigating microbial changes during SARS2 infection. The relationship between the age, residential microbiome and viral pathogens contributes to a better understanding of the complexities associated with the host responses during viral infection while limiting potential environmental factors that may contribute to inter-individual variation.","version":"1.1","doi":"10.1101/2022.07.13.499851","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.13.499586","pub_date":"2022-7-13","title":"Structural basis for the enhanced infectivity and immune evasion of Omicron subvariants","abstract":"The Omicron variants of SARS-CoV-2 have recently become the globally dominant variants of concern in the COVID-19 pandemic. At least five major Omicron sub-lineages have been characterized: BA.1, BA.2, BA.3, BA.4 and BA.5. They all possess over 30 mutations on the Spike (S) protein. Here we report the cryo-EM structures of the trimeric S proteins from the five subvariants, of which BA.4 and BA.5 share the same mutations of S protein, each in complex with the surface receptor ACE2. All three receptor binding domains of S protein from BA.2 and BA.4/BA.5 are \u201cup\u201d, while the BA.1 S protein has two \u201cup\u201d and one \u201cdown\u201d. The BA.3 S protein displays increased heterogeneity, with the majority in the all \u201cup\u201d RBD state. The differentially preferred conformations of the S protein are consistent with their varied transmissibilities. Analysis of the well defined S309 and S2K146 epitopes reveals the underlie immune evasion mechanism of Omicron subvariants.","version":"1.1","doi":"10.1101/2022.07.13.499586","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.23.497404","pub_date":"2022-7-12","title":"The Functional Landscape of SARS-CoV-2 3CL Protease","abstract":"SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CLpro), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CLpro, and validate our results both in yeast and in authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CLpro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CLpro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.","version":"1.2","doi":"10.1101/2022.06.23.497404","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.12.499687","pub_date":"2022-7-12","title":"Hotspot residues and resistance mutations in the nirmatrelvir-binding site of SARS-CoV-2 main protease: Design, identification, and correlation with globally circulating viral genomes","abstract":"Since the onset of the COVID-19 pandemic, SARS-CoV-2 has acquired numerous variations in its intracellular proteins to quickly adapt, become more infectious, and ultimately develop drug resistance by mutating certain hotspot residues. To keep the emerging variants at bay, including Omicron and subvariants, FDA has approved the antiviral nirmatrelvir for mild-to-moderate and high-risk COVID-19 cases. Like other viruses, SARS-CoV-2 could acquire mutations in its main protease (Mpro) to adapt and develop resistance against nirmatrelvir. Employing a unique high-throughput protein design technique, the hotspot residues and signatures of adaptation of Mpro having the highest probability of mutating and rendering nirmatrelvir ineffective were identified. Our results show that \u223c40% of the designed mutations in Mpro already exist in the globally circulating SARS-CoV-2 lineages. The work provides a first-hand explanation of the resistance mutations in Mpro and is crucial in comprehending viral adaptation, robust antiviral design, and surveillance of evolving Mpro variations.","version":"1.1","doi":"10.1101/2022.07.12.499687","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.11.22277368","pub_date":"2022-07-12","title":"Structural epitope profiling identifies antibodies associated with critical COVID-19 and long COVID","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Even within a single protein, antibody binding can have beneficial, neutral, or harmful effects during the response to infection. Resolving a polyclonal antibody repertoire across a pathogen\u2019s proteome to specific epitopes may therefore explain much of the heterogeneity in susceptibility to infectious disease. However, the three-dimensional nature of antibody-epitope interactions makes the discovery of non-obvious targets challenging. We implemented a novel computational method and synthetic biology pipeline for identifying epitopes that are functionally important in the SARS-CoV-2 proteome and identified an IgM-dominant response to an exposed Membrane protein epitope which to our knowledge is the strongest correlate of severe disease identified to date (adjusted OR 72.14, 95% CI: 9.71 \u2013 1300.15), stronger even than the exponential association of severe disease with age. We also identify persistence (&gt; 2 years) of this IgM response in individuals with longCOVID, and a correlation with fatigue and depression symptom burden. The repetitive arrangement of this epitope and the pattern of isotype class switching is consistent with this being a previously unrecognized T independent antigen. These findings point to a coronavirus host-pathogen interaction characteristic of severe virus driven immune pathology. This epitope is a promising vaccine and therapeutic target as it is highly conserved through SARS-CoV-2 variant evolution in humans to date and in related coronaviruses (e.g. SARS-CoV), showing far less evolutionary plasticity than targets on the Spike protein. This provides a promising biomarker for longCOVID and a target to complement Spike-directed vaccination which could broaden humoral protection from severe or persistent disease or novel coronavirus spillovers.</jats:p>\n                <jats:sec>\n                  <jats:title>One-Sentence Summary</jats:title>\n                  <jats:p>Using a novel protein-structure-based B cell epitope discovery method with a wide range of possible applications, we have identified a simple to measure host-pathogen antibody signature associated with severe COVID-19 and longCOVID and suggest the viral Membrane protein contains an epitope that acts as a T independent antigen during infection triggering extrafollicular B cell activation.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.11.22277368","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.11.499644","pub_date":"2022-7-12","title":"Insights from Incorporating Quantum Computing into Drug Design Workflows","abstract":"While many quantum computing (QC) methods promise theoretical advantages over classical counterparts, quantum hardware remains limited. Exploiting near-term QC in computer-aided drug design (CADD) thus requires judicious partitioning between classical and quantum calculations. We present HypaCADD, a hybrid classical-quantum workflow for finding ligands binding to proteins, while accounting for genetic mutations. We explicitly identify modules of our drug design workflow currently amenable to replacement by QC: non-intuitively, we identify the mutation-impact predictor as the best candidate. HypaCADD thus combines classical docking and molecular dynamics with quantum machine learning (QML) to infer the impact of mutations. We present a case study with the SARS-CoV-2 protease and associated mutants. We map a classical machine-learning module onto QC, using a neural network constructed from qubit-rotation gates. We have implemented this in simulation and on two commercial quantum computers. We find that the QML models can perform on par with, if not better than, classical baselines. In summary, HypaCADD offers a successful strategy for leveraging QC for CADD.","version":"1.1","doi":"10.1101/2022.07.11.499644","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.08.499374","pub_date":"2022-7-11","title":"Evolution of SARS-CoV-2 during the first year of the COVID-19 pandemic in Northwestern Argentina","abstract":"Studies about the evolution of SARS-CoV-2 lineages in different backgrounds such as naive populations, are still scarce, especially from South America. The aim of this work was to study the introduction and diversification pattern of SARS-CoV-2 during the first year of the COVID-19 pandemic in the Northwestern Argentina (NWA) region and to analyze the evolutionary dynamics of the main lineages found. In this study, we analyzed a total of 260 SARS-CoV-2 whole-genome sequences from Argentina, belonging to the Provinces of Jujuy, Salta and Tucum\u00e1n, from March 31st, 2020, to May 22nd, 2021, which covered the full first wave and the early second wave of the COVID-19 pandemic in Argentina. In the first wave, eight lineages were identified: B.1.499 (76.9%), followed by N.5 (10.2%), B.1.1.274 (3.7%), B.1.1.348 (3.7%), B.1 (2.8%), B.1.600 (0.9%), B.1.1.33 (0.9%) and N.3 (0.9%). During the early second wave, the first-wave lineages were displaced by the introduction of variants of concern (VOC) (Alpha, Gamma), or variants of interest (VOI) (Lambda, Zeta, Epsilon) and other lineages with more limited distribution. Phylodynamic analyses of the B.1.499 and N.5, the two most prevalent lineages in NWA, revealed that the substitution rate of lineage N.5 (7.9 \u00d7 10\u22124 substitutions per site per year, s/s/y) was a \u223c40% faster than that of lineage B.1.499 (5.9 \u00d7 10\u22124 s/s/y), although both are in the same order of magnitude than other non-VOC lineages. No mutations associated with a biological characteristic of importance were observed as signatures markers of the phylogenetic groups established in Northwestern Argentina, however, single sequences in non-VOC lineages did present mutations of biological importance or associated with VOCs as sporadic events, showing that many of these mutations could emerge from circulation in the general population. This study contributed to the knowledge about the evolution of SARS-CoV-2 in a pre-vaccination and without post-exposure immunization period.","version":"1.1","doi":"10.1101/2022.07.08.499374","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.08.499336","pub_date":"2022-7-11","title":"COVID-19 Causes Ciliary Dysfunction as Demonstrated by Human Intranasal Micro-Optical Coherence Tomography Imaging","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV-2), causative agent of coronavirus disease 2019 (COVID-19), binds via ACE2 receptors, highly expressed in ciliated cells of the nasal epithelium. Micro-optical coherence tomography (\u03bcOCT) is a minimally invasive intranasal imaging technique that can determine cellular and functional dynamics of respiratory epithelia at 1-\u03bcm resolution, enabling real time visualization and quantification of epithelial anatomy, ciliary motion, and mucus transport. We hypothesized that respiratory epithelial cell dysfunction in COVID-19 will manifest as reduced ciliated cell function and mucociliary abnormalities, features readily visualized by \u03bcOCT. Symptomatic outpatients with SARS-CoV-2 aged \u2265 18 years were recruited within 14 days of symptom onset. Data was interpreted for subjects with COVID-19 (n=13) in comparison to healthy controls (n=8). Significant reduction in functional cilia, diminished ciliary beat frequency, and abnormal ciliary activity were evident. Other abnormalities included denuded epithelium, presence of mucus rafts, and increased inflammatory cells. Our results indicate that subjects with mild but symptomatic COVID-19 exhibit functional abnormalities of the respiratory mucosa underscoring the importance of mucociliary health in viral illness and disease transmission. Ciliary imaging enables investigation of early pathogenic mechanisms of COVID-19 and may be useful for evaluating disease progression and therapeutic response.","version":"1.1","doi":"10.1101/2022.07.08.499336","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.09.499414","pub_date":"2022-7-11","title":"Landscape of infection enhancing antibodies in COVID-19 and healthy donors","abstract":"To assess the frequency of SARS-CoV-2 infection enhancing antibodies in the general population, we searched over 64 million heavy chain antibody sequences from healthy and COVID-19 patient repertoires for sequences similar to 11 previously reported enhancing antibodies. Although the distribution of sequence identities was similar in COVID-19 and healthy repertoires, the COVID-19 hits were significantly more clonally expanded than healthy hits. Furthermore, among the tested hits, 17 out of 94 from COVID-19, compared with 2 out of 96 from healthy, bound to the enhancing epitope. A total of 6 of the 19 epitope-binding antibodies enhanced ACE2 receptor binding to the spike protein. Together, this study revealed that enhancing antibodies are far more frequent in COVID-19 patients than in healthy donors, but a reservoir of potential enhancing antibodies exists in healthy donors that could potentially mature to actual enhancing antibodies upon infection.","version":"1.1","doi":"10.1101/2022.07.09.499414","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.11.499512","pub_date":"2022-7-11","title":"Re-emergence of Severe Acute Diarrhea Syndrome Coronavirus (SADS-CoV) in Guangxi, China, 2021","abstract":"Severe acute diarrhea syndrome coronavirus (SADS-CoV) has had a major impact on the swine industry in China, but has not been detected since 2019. Using real-time qPCR and metagenomic surveillance we identified SADS-CoV in a pig farm experiencing diarrheal disease. Genomic analysis supported the undetected circulation of SADS-CoV since 2019.","version":"1.1","doi":"10.1101/2022.07.11.499512","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495186","pub_date":"2022-7-11","title":"Efficient reconciliation of genomic datasets of high similarity","abstract":"We apply Invertible Bloom Lookup Tables (IBLTs) to the comparison of k-mer sets originated from large DNA sequence datasets. We show that for similar datasets, IBLTs provide a more space-efficient and, at the same time, more accurate method for estimating Jaccard similarity of underlying k-mer sets, compared to MinHash which is a go-to sketching technique for efficient pairwise similarity estimation. This is achieved by combining IBLTs with k-mer sampling based on syncmers, which constitute a context-independent alternative to minimizers and provide an unbiased estimator of Jaccard similarity. A key property of our method is that involved data structures require space proportional to the difference of k-mer sets and are independent of the size of sets themselves. As another application, we show how our ideas can be applied in order to efficiently compute (an approximation of) k-mers that differ between two datasets, still using space only proportional to their number. We experimentally illustrate our results on both simulated and real data (SARS-CoV-2 and Streptococcus Pneumoniae genomes). Available at: https://github.com/yhhshb/km-peeler.git","version":"1.2","doi":"10.1101/2022.06.07.495186","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.10.499455","pub_date":"2022-7-10","title":"Remdesivir does not affect mitochondrial DNA copy number or deletion mutation frequency in aged male rats","abstract":"Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivir\u2019s effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. For the 33-month-old control rats, the average mtDNA copy number per nucleus was 2567, 1100, and 1869 for heart, kidney, and quadriceps, respectively. MtDNA deletion mutation frequency was 2.6\u00d710\u22124, 1.6\u00d710\u22124 and 4.7\u00d710\u22123for heart, kidney, and quadriceps, respectively. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples.","version":"1.1","doi":"10.1101/2022.07.10.499455","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.07.22277371","pub_date":"2022-07-10","title":"Covid-19 vaccination and menstrual cycle length in the Apple Women\u2019s Health Study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>COVID-19 vaccination may be associated with change in menstrual cycle length following vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a longitudinal analysis within a subgroup of 14,915 participants in the Apple Women\u2019s Health Study (AWHS) who enrolled between November 2019 and December 2021 and met the following eligibility criteria: were living in the U.S., met minimum age requirements for consent, were English speaking, actively tracked their menstrual cycles, and responded to the COVID-19 Vaccine Update survey. In the main analysis, we included tracked cycles recorded when premenopausal participants were not pregnant, lactating, or using hormonal contraceptives. We used conditional linear regression and multivariable linear mixed-effects models with random intercepts to estimate the covariate-adjusted difference in mean cycle length, measured in days, between pre-vaccination cycles, cycles in which a vaccine was administered, and post-vaccination cycles within vaccinated participants, and between vaccinated and unvaccinated participants. We further compared associations between vaccination and menstrual cycle length by the timing of vaccine dose within a menstrual cycle (i.e., in follicular or luteal phase). We present Bonferroni-adjusted 95% confidence intervals to account for multiple comparisons.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>A total of 128,094 cycles (median = 10 cycles per participant; interquartile range: 4-22) from 9,652 participants (8,486 vaccinated; 1,166 unvaccinated) were included. The average within-individual standard deviation in cycle length was 4.2 days. Fifty-five percent of vaccinated participants received Pfizer-BioNTech\u2019s mRNA vaccine, 37% received Moderna\u2019s mRNA vaccine, and 7% received the Johnson &amp; Johnson/Janssen vaccine (J&amp;J). We found no evidence of a difference between mean menstrual cycle length in the unvaccinated and vaccinated participants prior to vaccination (0.24 days, 95% CI: \u22120.34, 0.82).</jats:p>\n                  <jats:p>Among vaccinated participants, COVID-19 vaccination was associated with a small increase in mean cycle length (MCL) for cycles in which participants received the first dose (0.50 days, 95% CI: 0.22, 0.78) and cycles in which participants received the second dose (0.39 days, 95% CI: 0.11, 0.67) of mRNA vaccines compared with pre-vaccination cycles. Cycles in which the single dose of J&amp;J was administered were, on average, 1.26 days longer (95% CI: 0.45, 2.07) than pre-vaccination cycles. Post-vaccination cycles returned to average pre-vaccination length. Estimates for pre vs post cycle lengths were 0.14 days (95% CI: \u22120.13, 0.40) in the first cycle following vaccination, 0.13 days (95% CI: \u22120.14, 0.40) in the second, \u22120.17 days (95% CI: \u22120.43, 0.10) in the third, and \u22120.25 days (95% CI: \u22120.52, 0.01) in the fourth cycle post-vaccination. Follicular phase vaccination was associated with an increase in MCL in cycles in which participants received the first dose (0.97 days, 95% CI: 0.53, 1.42) or the second dose (1.43 days, 95% CI: 1.06, 1.80) of mRNA vaccines or the J&amp;J dose (2.27 days, 95% CI: 1.04, 3.50), compared with pre-vaccination cycles.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>COVID-19 vaccination was associated with an immediate short-term increase in menstrual cycle length overall, which appeared to be driven by doses received in the follicular phase. However, the magnitude of this increase was small and diminished in each cycle following vaccination. No association with cycle length persisted over time. The magnitude of change associated with vaccination was well within the natural variability in the study population. Menstrual cycle change following COVID-19 vaccination appears small and temporary and should not discourage individuals from becoming vaccinated.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.07.22277371","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.07.22277391","pub_date":"2022-07-10","title":"Modeling the impact of the Omicron infection wave in Germany","abstract":"<jats:sec>\n                  <jats:title>BACKGROUND</jats:title>\n                  <jats:p>In November 2021, the first case of SARS-CoV-2 \u201cvariant of concern\u201d (VOC) B.1.1.529 (\u201cOmicron\u201d) was reported in Germany, alongside global reports of reduced vaccine efficacy against infections with this variant. The potential threat posed by the rapid spread of this variant in Germany remained, at the time, elusive.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>We developed a variant-dependent population-averaged susceptible-exposed-infected-recovered (SEIR) infectious disease model. The model was calibrated on the observed fixation dynamics of the Omicron variant in December 2021, and allowed us to estimate potential courses of upcoming infection waves in Germany, focusing on the corresponding burden on intensive care units (ICUs) and the efficacy of contact reduction strategies.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>A maximum median incidence of approximately 300 000 (50% PI in 1000: [181,454], 95% PI in 1000: [55,804]) reported cases per day was expected with the median peak occurring in the mid of February 2022, reaching a cumulative Omicron case count of 16.5 million (50% PI in mio: [11.4, 21.3], 95% PI in mio: [4.1, 27.9]) until Apr 1, 2022. These figures were in line with the actual Omicron waves that were subsequently observed in Germany with respective peaks occurring in mid February (peak: 191k daily new cases) and mid March (peak: 230k daily new cases), cumulatively infecting 14.8 million individuals during the study period. The model peak incidence was observed to be highly sensitive to variations in the assumed generation time and decreased with shorter generation time. Low contact reductions were expected to lead to containment. Early, strict, and short contact reductions could have led to a strong \u201crebound\u201d effect with high incidences after the end of the respective non-pharmaceutical interventions. Higher vaccine uptake would have led to a lower outbreak size. To ensure that ICU occupancy remained below maximum capacity, a relative risk of requiring ICU care of 10%\u201320% was necessary (after infection with Omicron vs. infection with Delta).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>We expected a large cumulative number of infections with the VOC Omicron in Germany with ICU occupancy likely remaining below capacity nevertheless, even without additional non-pharmaceutical interventions. Our estimates were in line with the retrospectively observed waves. The results presented here informed legislation in Germany. The methodology developed in this study might be used to estimate the impact of future waves of COVID-19 or other infectious diseases.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.07.22277391","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.08.22277420","pub_date":"2022-07-09","title":"Brain imaging and neuropsychological assessment of individuals recovered from a mild to moderate SARS-CoV-2 infection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  As SARS-CoV-2 infections have been shown to affect the central nervous system, the investigation of associated alterations of brain structure and neuropsychological sequelae is crucial to help address future health care needs. Therefore, we performed a comprehensive neuroimaging and neuropsychological assessment of 223 non-vaccinated individuals recovered from a mild to moderate SARS-CoV-2 infection (100 female/123 male, age [years], mean \u00b1 SD, 55.54 \u00b1 7.07; median 9.7 months after infection) in comparison with 223 matched controls (93 female/130 male, 55.74 \u00b1 6.60) within the framework of the Hamburg City Health Study. Primary study outcomes were advanced diffusion magnetic resonance imaging (MRI) measures of white matter microstructure, cortical thickness, white matter hyperintensity load and neuropsychological test scores. Among all 11 MRI markers tested, significant differences were found in global measures of mean diffusivity and extracellular free-water which were elevated in the white matter of post-SARS-CoV-2 individuals comparing to matched controls (free-water: 0.148 \u00b1 0.018 vs. 0.142 \u00b1 0.017,\n                  <jats:italic>P</jats:italic>\n                  &lt;.001; mean diffusivity [10\n                  <jats:sup>\u22123</jats:sup>\n                  mm\n                  <jats:sup>2</jats:sup>\n                  /s]: 0.747 \u00b1 0.021 vs. 0.740 \u00b1 0.020,\n                  <jats:italic>P</jats:italic>\n                  &lt;.001). Group classification accuracy based on diffusion imaging markers was up to 80%. Neuropsychological test scores did not significantly differ between groups. Collectively, our findings suggest that subtle changes in white matter extracellular water content last beyond the acute infection with SARS-CoV-2. However, in our sample, a mild to moderate SARS-CoV-2 infection was not associated with neuropsychological deficits, significant changes in cortical structure or vascular lesions several months after recovery. External validation of our findings and longitudinal follow-up investigations are needed.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Significance statement</jats:title>\n                  <jats:p>In this case-control study, we demonstrate that non-vaccinated individuals recovered from a mild to moderate SARS-CoV-2 infection show significant alterations of the cerebral white matter identified by diffusion weighted imaging, such as global increases in extracellular free-water and mean diffusivity. Despite the observed brain white matter alterations in this sample, a mild to moderate SARS-CoV-2 infection was not associated with worse cognitive functions within the first year after recovery. Collectively, our findings indicate the presence of a prolonged neuroinflammatory response to the initial viral infection. Further longitudinal research is necessary to elucidate the link between brain alterations and clinical features of post-SARS-CoV-2 individuals.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.08.22277420","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.07.499204","pub_date":"2022-7-08","title":"Correlation of Alpha-1 Antitrypsin Levels and Exosome Associated Neutrophil Elastase Endothelial Injury in Subjects with SARS-CoV2 Infection","abstract":"Severe acute respiratory syndrome caused by a novel coronavirus 2 (SARS-CoV-2) has infected more than 18 million people worldwide. The activation of endothelial cells is a hallmark of signs of SARS-CoV-2 infection that includes altered integrity of vessel barrier and endothelial inflammation. Pulmonary endothelial activation is suggested to be related to the profound neutrophil elastase (NE) activity, which is necessary for sterilization of phagocytosed bacterial pathogens. However, unopposed activity of NE increases alveolocapillary permeability and extracellular matrix degradation. The uncontrolled protease activity of NE during the inflammatory phase of lung diseases might be due to the resistance of exosome associated NE to inhibition by alpha-1 antitrypsin. 31 subjects with a diagnosis of SARS-CoV2 infection were recruited in the disease group and samples from 30 voluntaries matched for age and sex were also collected for control. We measured the plasma levels of exosome-associated NE in SARS-CoV-2 patients which, was positively correlated with the endothelial damage in those patients. Notably, we also found strong correlation with plasma levels of alpha-1 antitrypsin and exosome-associated NE in SARS-CoV-2 patients. Using macrovascular endothelial cells, we also observed that purified NE activity is inhibited by purified alpha-1 antitrypsin while, NE associated with exosomes are resistant to inhibition and show less sensitivity to alpha-1 antitrypsin inhibitory activity, in vitro. Our results point out the role of exosome-associated NE in exacerbation of endothelial injury in SARS-CoV-2 infection. We have demonstrated that exosome-associated NE could be served as a new potential therapeutic target of severe systemic manifestations of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.07.07.499204","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.08.499297","pub_date":"2022-7-08","title":"Carbohydrate-Binding Protein from Stinging Nettle as Fusion Inhibitor for SARS-CoV-2 Variants of Concern","abstract":"Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks entry of pseudotyped SARS-CoV-2 in A549.ACE2+-TMPRSS2 cells, with IC50 values ranging from 0.32 to 1.22 \u00b5M. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2+ cells (IC50 values are 1.6 and 0.9 \u00b5M, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent and broad-spectrum fusion inhibitor for SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.07.08.499297","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.15.459697","pub_date":"2022-7-06","title":"Molecular architecture and dynamics of SARS-CoV-2 envelope by integrative modeling","abstract":"Despite tremendous efforts by the research community during the COVID-19 pandemic, the exact structure of SARS-CoV-2 and related betacoronaviruses remains elusive. Being a key structural component of the SARS-CoV-2 virion, the envelope encapsulates viral RNA and is composed of three structural proteins, spike (S), membrane (M), and envelope (E), which interact with each other and with the lipids acquired from the host membranes. Here, we developed and applied an integrative multiscale computational approach to model the envelope structure of SARS-CoV-2 with near atomistic detail, focusing on studying the dynamic nature and molecular interactions of its most abundant, but largely understudied, M protein. The molecular dynamics simulations allowed us to test the envelope stability under different configurations and revealed that the M dimers agglomerated into large, filament-like, macromolecular assemblies with distinct molecular patterns formed by M\u2019s transmembrane and intravirion (endo) domains. These results are in good agreement with current experimental data, demonstrating a generic and versatile integrative approach to model the structure of a virus de novo. We anticipate our work to provide insights into critical roles of structural proteins in the viral assembly and integration, proposing new targets for the antiviral therapies.","version":"1.2","doi":"10.1101/2021.09.15.459697","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.05.498807","pub_date":"2022-7-06","title":"Understanding The Role of Heparinoids on the SARS-CoV-2 Spike Protein through Molecular Dynamics Simulations","abstract":"The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a threat, with an estimated number of deaths exceeding 5 million. SARS-CoV-2 entry into the cell is mediated by its transmembrane spike glycoprotein (S protein), and the angiotensin-converting enzyme 2 (ACE2) receptor on the human cell surface. The extracellular heparan sulphate (EcHS) enhances the S protein binding through a mechanism that is still unknown. Surprisingly, low molecular weight heparin (LMWH) and HS in the disaccharide form (dHS) hinder the S protein binding to ACE2, despite the similarity with EcHS. We investigated the molecular mechanism behind this inhibition through molecular dynamics (MD) simulations to understand the interaction pattern of the heparinoids with S protein and ACE2 receptor.","version":"1.1","doi":"10.1101/2022.07.05.498807","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.06.498864","pub_date":"2022-7-06","title":"A Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2","abstract":"Using the regioselective cyanobenzothiazole condensation reaction with the N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and ACE2, the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 \u03bcM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 \u03bcM. The current study demonstrates that two kinetically-controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals.","version":"1.1","doi":"10.1101/2022.07.06.498864","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.05.498881","pub_date":"2022-7-06","title":"Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors","abstract":"Antiviral therapeutics to treat SARS-CoV-2 are much desired for the on-going pandemic. A well-precedented viral enzyme is the main protease (MPro), which is now targeted by an approved drug and by several investigational drugs. With the inevitable liabilities of these new drugs, and facing viral resistance, there remains a call for new chemical scaffolds against MPro. We virtually docked 1.2 billion non-covalent and a new library of 6.5 million electrophilic molecules against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 \u03bcM and 20 \u03bcM, respectively. Several series were optimized, resulting in inhibitors active in the low micromolar range. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. Together, these compounds reveal new chemotypes to aid in further discovery of MPro inhibitors for SARS-CoV-2 and other future coronaviruses.","version":"1.1","doi":"10.1101/2022.07.05.498881","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.03.498624","pub_date":"2022-7-06","title":"Targeting C5aR1 signaling reduced neutrophil extracellular traps and ameliorates COVID-19 pathology","abstract":"Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment.","version":"1.2","doi":"10.1101/2022.07.03.498624","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.05.498883","pub_date":"2022-7-06","title":"Post-vaccination Omicron infections induce broader immunity across antigenic space than prototype mRNA COVID-19 booster vaccination or primary infection","abstract":"The rapid emergence of new SARS-CoV-2 variants challenges vaccination strategies. Here, we measured antigenic diversity among variants and interpreted neutralizing antibody responses following single and multiple exposures in longitudinal infection and vaccine cohorts. Antigenic cartography using primary infection antisera showed that BA.2, BA.4/BA.5, and BA.2.12.1 are distinct from BA.1 and closer to the Beta cluster. Three doses of an mRNA COVID-19 vaccine increased breadth to BA.1 more than to BA.4/BA.5 or BA.2.12.1. Omicron BA.1 post-vaccination infection elicited antibody landscapes characterized by broader immunity across antigenic space than three doses alone, although with less breadth than expected to BA.2.12.1 and BA.4/BA.5. Those with Omicron BA.1 infection after two or three vaccinations had similar neutralizing titer magnitude and antigenic breadth. Accounting for antigenic differences among variants of concern when interpreting neutralizing antibody titers aids understanding of complex patterns in humoral immunity and informs selection of future COVID-19 vaccine strains.","version":"1.1","doi":"10.1101/2022.07.05.498883","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.05.22277281","pub_date":"2022-07-06","title":"Efficacy and longevity of immune response to 3\n                  <sup>rd</sup>\n                  COVID-19 vaccine and effectiveness of a 4\n                  <sup>th</sup>\n                  dose in severely immunocompromised patients with cancer","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Cancer patients show increased morbidity with COVID-19 and need effective immunization strategies. We demonstrate that a 3\n                  <jats:sup>rd</jats:sup>\n                  dose of COVID-19 vaccine leads to seroconversion in 57% of patients that were seronegative after primary vaccination. The immune response is durable as assessed by anti-S antibody titers, T-cell activity and neutralization activity against wild-type SARS-CoV2 and BA1.1.529 at 6 months of follow up. A subset of severely immunocompromised hematologic malignancy patients were unable to mount adequate immune response after the 3\n                  <jats:sup>rd</jats:sup>\n                  dose and were treated with a 4\n                  <jats:sup>th</jats:sup>\n                  dose in a prospective clinical trial which led to adequate immune-boost in 67% of patients. Low baseline IgM levels and CD19 counts were associated with inadequate seroconversion. Booster doses induced limited neutralization activity against the Omicron variant. These results indicate that vaccine booster-induced immunity is durable in cancer patients and additional doses can further stimulate immunity in a subset of hematologic malignancy patients.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Statement of significance</jats:title>\n                  <jats:p>\n                    We demonstrate that a 3\n                    <jats:sup>rd</jats:sup>\n                    dose of vaccine leads to seroconversion in 57% of negative patients with durable immune responses at 6 months. A 4\n                    <jats:sup>th</jats:sup>\n                    dose of vaccine can seroconvert hematologic malignancy patients with higher baseline IgM and CD19 levels.\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.05.22277281","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.29.498117","pub_date":"2022-7-05","title":"COVID-19 Neuropathology: evidence for SARS-CoV-2 invasion of Human Brainstem Nuclei","abstract":"Neurological manifestations are common in COVID-19, the disease caused by SARS-CoV-2. Despite reports of SARS-CoV-2 detection in the brain and cerebrospinal fluid of COVID-19 patients, it\u2019s still unclear whether the virus can infect the central nervous system, and which neuropathological alterations can be ascribed to viral tropism, rather than immune-mediated mechanisms. Here, we assess neuropathological alterations in 24 COVID-19 patients and 18 matched controls who died due to pneumonia / respiratory failure. Aside from a wide spectrum of neuropathological alterations, SARS-CoV-2-immunoreactive neurons were detected in specific brainstem nuclei of 5 COVID-19 subjects. Viral RNA was also detected by real-time RT-PCR. Quantification of reactive microglia revealed an anatomically segregated pattern of inflammation within affected brainstem regions, and was higher when compared to controls. While the results of this study support the neuroinvasive potential of SARS-CoV-2, the role of SARS-CoV-2 neurotropism in COVID-19 and its long-term sequelae require further investigation.","version":"1.1","doi":"10.1101/2022.06.29.498117","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.04.498661","pub_date":"2022-7-05","title":"Proteome dynamics of COVID-19 severity learnt by a graph convolutional network of multi-scale topology","abstract":"Many efforts have been recently done to characterise the molecular mechanisms of COVID-19 disease. These efforts resulted in a full structural identification of ACE2 as principal receptor of the Sars-CoV-2 spike protein in the cell. However, there are still important open questions related to other proteins involved in the progression of the disease. To this end, we have modelled the plasma proteome of 384 COVID patients. The model calibrated proteins measures at three time tags and make also use of the detailed clinical evaluation outcome of each patient after their hospital stay at day 28. Our analysis is able to discriminate severity of the disease by means of a metric based on available WHO scores of disease progression. Then, we identify by topological vectorisation those proteins shifting the most in their expression depending on that severity classification. Finally, the extracted topological invariants respect the protein expression at different times were used as base of a graph convolutional network. This model enabled the dynamical learning of the molecular interactions produced between the identified proteins.","version":"1.1","doi":"10.1101/2022.07.04.498661","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.03.498630","pub_date":"2022-7-05","title":"Cross-attention PHV: Prediction of human and virus protein-protein interactions using cross-attention\u2013based neural networks","abstract":"Viral infections represent a major health concern worldwide. The alarming rate at which SARS-CoV-2 spreads, for example, led to a worldwide pandemic. Viruses incorporate genetic material into the host genome to hijack host cell functions such as the cell cycle and apoptosis. In these viral processes, protein-protein interactions (PPIs) play critical roles. Therefore, the identification of PPIs between humans and viruses is crucial for understanding the infection mechanism and host immune responses to viral infections and for discovering effective drugs. Experimental methods such as yeast two-hybrid assays and mass spectrometry are widely used to identify human-virus PPIs, but these experimental methods are time-consuming, expensive, and laborious. To overcome this problem, we developed a novel computational predictor, named cross-attention PHV, by implementing two key technologies of the cross-attention mechanism and a one- dimensional convolutional neural network (1D-CNN). The cross-attention mechanisms were very effective in enhancing prediction and generalization abilities. Application of 1D-CNN to the word2vec-generated feature matrices reduced computational costs, thus extending the allowable length of protein sequences to 9000 amino acid residues. Cross- attention PHV outperformed existing state-of-the-art models using a benchmark dataset and accurately predicted PPIs for unknown viruses. Cross-attention PHV also predicted human\u2013SARS-CoV-2 PPIs with area under the curve values >0.95.","version":"1.1","doi":"10.1101/2022.07.03.498630","journal":"bioRxiv","score":null},{"id":"10.1101/2022.07.01.22277143","pub_date":"2022-07-03","title":"Multiple effects of TNF\u03b1 inhibitors on the development of the adaptive immune response after SARS-CoV-2 vaccination","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>The humoral immune response to SARS-CoV-2 vaccination in patients with chronic inflammatory disease (CID) declines more rapidly with TNF\u03b1 inhibition. Furthermore, the efficacy of current vaccines against Omicron variants of concern (VOC) including BA.2 is limited. Alterations within immune cell populations, changes in IgG affinity and the ability to neutralise a pre-VOC strain and the BA.2 virus were investigated in these at-risk patients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Serum levels of anti-SARS-CoV-2 IgG, IgG avidity and neutralising antibodies (NA) were determined in anti-TNF\u03b1 patients (n=10) and controls (n=24 healthy individuals; n=12 patients under other disease-modifying anti-rheumatic drugs, oDMARD) before and after the second and third vaccination by ELISA, immunoblot and live virus neutralisation assay. SARS-CoV-2-specific B-and T cell subsets were analysed by multicolour flow cytometry.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>IgG avidity and anti-pre-VOC NA titres decreased faster in anti-TNF\u03b1 recipients than in controls 6 months after the second vaccination (healthy individuals: avidity: p\u22640.0001; NA: p=0.0347; oDMARDs: avidity: p=0.0012; NA: p=0.0293). Total plasma cell counts were increased in anti-TNF\u03b1 patients (Healthy individuals: p=0.0344; oDMARDs: p=0.0254), whereas absolute numbers of SARS-CoV-2-specific cells were comparable 7 days after vaccination. These patients had lower BA.2 NA titres compared to both other groups, even after the third vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>We show a reduced SARS-CoV-2 neutralising capacity in patients under TNF\u03b1 blockade. In this cohort, the plasma cell response appears to be less specific and show stronger bystander activation. While these effects were observable after the first two vaccinations and with older VOC, the differences in responses to BA.2 were magnified.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>What is already known on this topic</jats:title>\n                  <jats:p>Patients with chronic inflammatory diseases treated with TNF\u03b1 inhibitors show a greater decrease in SARS-CoV-2 IgG 6 months after the second vaccination than patients taking oDMARDs and healthy individuals.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>What this study adds</jats:title>\n                  <jats:p>Antibodies from patients taking TNF\u03b1 blockers have a lower SARS-CoV-2 neutralising capacity and maturity. Plasma cells from these patients exhibit less specific immune reaction. SARS-CoV-2-specific T cells are less activated. Neutralisation against BA.2 is drastically reduced even after the third vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>How this study might affect research, practice or policy</jats:title>\n                  <jats:p>This study emphasizes the need to protect vulnerable groups such as patients using TNF inhibitors. They could benefit from Omicron-adapted vaccination, but most likely they need to be protected by additional means other than vaccination.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.07.01.22277143","journal":"medRxiv","score":null},{"id":"10.1101/2022.07.01.496571","pub_date":"2022-7-01","title":"CovidOutcome2: a tool for SARS-CoV2 mutation identification and for disease severity prediction","abstract":"Our goal was to develop a platform, CovidOutcome2, capable of predicting disease severity from viral mutation profiles using automated machine learning (autoML) and deep neural networks applied to the available large corpus of sequenced SARS-CoV2 genomes. CovidOutcome2 accepts either user-submitted genomes or user defined mutation combinations as the input. The output is a predicted severity score plus a list of identified, annotated mutations and their functional effects in VCF format. The best model performance is a ROC-AUC 0.899 for the model including patient age and ROC-AUC 0.83 for the model without patient age. CovidOutcome is freely available online under the URL https://www.covidoutcome.bio-ml.com as well as in a standalone version https://github.com/bio-apps/covid-outcome.","version":"1.1","doi":"10.1101/2022.07.01.496571","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.30.498305","pub_date":"2022-6-30","title":"Inactivated SARS-CoV-2 Reprograms the Tumor Immune Microenvironment and Improves Murine Cancer Outcomes","abstract":"Following the breakthrough of immune check point inhibitors (ICIs), a new era of immuno-oncology agents has emerged and established immunotherapy as a part of cancer treatment. Despite the improving outcomes of ICIs, many patients with initial response are known to develop acquired resistance later. There is increasing interest in utilizing other stimulatory means, such as anti-pathogen immune responses to induce anti-tumor immune responses. The immunostimulatory effects of anti-pathogen-treated tumors in combinations with ICI are known to potentially amplify anti-tumor immunity resulting in increased tumor responses and improved outcomes. Anti-pathogen-treated tumors can become immune-infiltrated \u201chot\u201d tumors and demonstrate higher treatment response rates and improved survival. Our research group has previously demonstrated that tumors can be converted from \u201ccold\u201d to \u201chot\u201d by intratumoral injection of a commercially available seasonal influenza vaccine. In continuation with our work, in deciphering the role of anti-viral immunity in the context of tumor immunology, we studied the role of inactivated SARS-CoV-2 virus as anti-tumor agent. Here we report that intratumoral injections of inactivated SARS-CoV-2 convert the immunologically cold tumors to hot by generating anti-tumor-mediated CD8+ T cells. Our findings suggest that inactivated SARS-CoV-2 can be used as an immune modulator in immunotherapy for melanoma and triple-negative breast cancer.","version":"1.1","doi":"10.1101/2022.06.30.498305","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.29.498206","pub_date":"2022-6-30","title":"Identification and Mechanistic Basis of non-ACE2 Blocking Neutralizing Antibodies from COVID-19 Patients with Deep RNA Sequencing and Molecular Dynamics Simulations","abstract":"Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (16,315 reads vs 1,412 reads, p=0.02). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.","version":"1.1","doi":"10.1101/2022.06.29.498206","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.29.498191","pub_date":"2022-6-30","title":"Inhibitory effects of GT0918 on acute lung injury and the molecular mechanisms of anti-inflammatory response","abstract":"Coronavirus disease 2019 (COVID-19) has caused the public health crisis in the whole world. Anti-androgens block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and protect against severe clinical COVID-19 outcomes. GT0918, a novel androgen receptor antagonist, accelerated viral clearance and increased recovery rate in outpatients by blocking SARS-CoV-2 infection though down-regulating ACE2 and TMPRSS2 expression. Further clinical study showed that GT0918 reduced mortality rate and shortened hospital stay in hospitalized COVID-19 patients. GT0918 also exhibits protective efficacy in severe COVID-19 patient in critical care. However, the mechanism of GT0918 treatment for severe COVID-19 disease is unknown. Here, we found GT0918 decreased the expression and secretion of proinflammatory cytokines through NF-\u03baB signaling pathway. The acute lung injury induced by LPS or Poly(I:C) was also attenuated in GT0918-treated mice, compared with vehicle control group. Moreover, GT0918 elevated the NRF2 protein level but not mRNA transcription activity. GT0918 induced proinflammatory cytokines downregulation was partially dependent on NRF2. In conclusion, our data demonstrate that GT0918 reduced cytokine release and suppressed inflammatory responses through inhibiting NF-\u03baB signaling and activating NRF2. GT0918 is not only effective for treatment of mild to moderate COVID-19 patients, but also a potential therapeutic drug for severe COVID-19 patients by reducing the risk of cytokine storm and acute respiratory distress syndrome.","version":"1.1","doi":"10.1101/2022.06.29.498191","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.29.498158","pub_date":"2022-6-30","title":"Vitamin D and the ability to produce 1,25(OH)2D are critical for protection from viral infection of the lungs","abstract":"Vitamin D supplementation has been linked to improved outcomes from respiratory virus infection, and the COVID19 pandemic has renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of Vitamin D using animal models of pandemic H1N1 influenza and SARS-CoV-2 infection. In mice, dietary induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D-Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficient/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.06.29.498158","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.29.498082","pub_date":"2022-6-30","title":"Trapping virus-loaded aerosols using granular protein nanofibrils and iron oxyhydroxides nanoparticles","abstract":"The ongoing COVID-19 pandemic has revealed that developing effective therapeutics against viruses might be outpaced by emerging variants, waning immunity, vaccine skepticism/hesitancy, lack of resources, and the time needed to develop virus-specific therapeutics, emphasizing the importance of non-pharmaceutical interventions as the first line of defense against virus outbreaks and pandemics. However, fighting the spread of airborne viruses has proven extremely challenging, much more if this needs to be achieved on a global scale and in an environmentally-friendly manner. Here, we introduce an aerosol filter made of granular material based on whey protein nanofibrils and iron oxyhydroxides nanoparticles. The material is environmentally-friendly, biodegradable, and composed mainly of a dairy industry byproduct. It features remarkable filtration efficiencies between 95.91% and 99.99% for both enveloped and non-enveloped viruses, including SARS-CoV-2, the influenza A virus strain H1N1, enterovirus 71, bacteriophage \u03a66, and bacteriophage MS2. The developed material is safe to handle and recycle, with a simple baking step sufficient to inactivate trapped viruses. The high filtration efficiency, virtually-zero environmental impact, and low cost of the material illuminate a viable role in fighting current and future pandemics on a global scale.","version":"1.1","doi":"10.1101/2022.06.29.498082","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.27.497883","pub_date":"2022-6-28","title":"The Staphylococcus aureus iron-regulated surface determinant A (IsdA) increases SARS CoV-2 replication by modulating JAK-STAT signaling","abstract":"The emergence and spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) and the associated Coronavirus disease (COVID-19) pandemic have affected millions globally. Like other respiratory viruses, a significant complication of COVID-19 infection is secondary bacterial co-infection, which is seen in approximately 25% of severe cases. The most common organism isolated from co-infection is the Gram-positive bacterium Staphylococcus aureus. Here, we developed an in vitro co-infection model where both CoV-2 and S. aureus replication kinetics can be examined. We demonstrate CoV-2 infection does not alter how S. aureus attaches to or grows in host epithelial cells. In contrast, the presence of replicating S. aureus enhances the replication of CoV-2 by 10-15-fold. We identify this pro-viral activity is due to the S. aureus iron-regulated surface determinant A (IsdA) and this effect is mimicked across different SARS CoV-2 permissive cell lines infected with multiple viral variants. Analysis of co-infected cells demonstrated an IsdA dependent modification of host transcription. Using chemical inhibition, we determined S. aureus IsdA modifies host Janus Kinase \u2013 Signal Transducer and Activator of Transcription (JAK-STAT) signalling, ultimately leading to increased viral replication. These findings provide key insight into the molecular interactions that occur between host cells, CoV-2 and S. aureus during co-infection. Bacterial co-infection is a common and significant complication of respiratory viral infection, including in patients with COVID-19, and leads to increased morbidity and mortality. The relationship between virus, bacteria and host is largely unknown, which makes it difficult to design effective treatment strategies. In the present study we created a model of co-infection between SARS CoV-2 and Staphylococcus aureus, the most common species identified in COVID-19 patients with co-infection. We demonstrate that the S. aureus protein IsdA enhances the replication of SARS CoV-2 in vitro by modulating host cell signal transduction pathways. The significance of this finding is in identifying a bacterial component that enhances CoV-2 pathogenesis, which could be a target for the development of co-infection specific therapy in the future. In addition, this protein can be used as a tool to decipher the mechanisms by which CoV-2 manipulates the host cell, providing a better understanding of COVID-19 virulence.","version":"1.1","doi":"10.1101/2022.06.27.497883","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.27.497875","pub_date":"2022-6-28","title":"Combination therapy with nirmatrelvir and molnupiravir improves the survival of SARS-CoV-2 infected mice","abstract":"As the SARS-CoV-2 pandemic remains uncontrolled owing to the continuous emergence of variants of concern, there is an immediate need to implement the most effective antiviral treatment strategies, especially for risk groups. Here, we evaluated the therapeutic potency of nirmatrelvir, remdesivir, and molnupiravir and their combinations in SARS-CoV-2-infected K18-hACE2 transgenic mice. Systemic treatment of mice with each drug (20 mg/kg) resulted in slightly enhanced antiviral efficacy and yielded an increased life expectancy of only about 20\u201340% survival. However, combination therapy with nirmatrelvir (20 mg/kg) and molnupiravir (20 mg/kg) in lethally infected mice showed profound inhibition of SARS-CoV-2 replication in both the lung and brain and synergistically improved survival times up to 80% compared to those with nirmatrelvir (P= 0.0001) and molnupiravir (P= 0.0001) administered alone. This combination therapy effectively reduced clinical severity score, virus-induced tissue damage, and viral distribution compared to those in animals treated with these monotherapies. Furthermore, all these assessments associated with this combination were also significantly higher than that of mice receiving remdesivir monotherapy (P= 0.0001) and the nirmatrelvir (20 mg/kg) and remdesivir (20 mg/kg) combination (P= 0.0001), underscored the clinical significance of this combination. By contrast, the nirmatrelvir and remdesivir combination showed less antiviral efficacy, with lower survival compared to nirmatrelvir monotherapy, demonstrating the inefficient therapeutic effect of this combination. The combination therapy with nirmatrelvir and molnupiravir contributes to alleviated morbidity and mortality, which can serve as a basis for the design of clinical studies of this combination in the treatment of COVID-19 patients. Since SARS-CoV-2 spread rapidly with the emergence of new variants of concerns, it is necessary to develop effective treatment strategies to treat elderly individuals and those with comorbidities. Antiviral therapy using a combination of drugs is more effective in eradicating viruses and will undoubtedly improve the clinical outcome and survival probability of hospitalized SARS-CoV-2 patients. In the current study, we observed three FDA-approved antivirals nirmatrelvir, remdesivir, and molnupiravir have therapeutic significance with moderate survival for their monotherapies against SARS-CoV-2 infected K18-hACE2 mouse model. The combination of nirmatrelvir and molnupiravir showed significant antiviral activity and a higher survival rate of approximately 80%, providing in vivo evidence of the potential utility of this combination. In contrast, nirmatrelvir and remdesivir combination showed less antiviral potency and emphasized the ineffective significance with less survival. The current study suggests that the nirmatrelvir and molnupiravir combination is an effective drug regimen strategy in treating SARS-CoV-2 patients.","version":"1.1","doi":"10.1101/2022.06.27.497875","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.08.495396","pub_date":"2022-6-28","title":"Binding and unbinding pathways of peptide substrate on SARS-CoV-2 3CL protease","abstract":"Based on many crystal structures of ligand complexes, much study has been devoted to understanding the molecular recognition of SARS-CoV-2 3C-like protease (3CLpro), a potent drug target for COVID-19. In this research, to extend this present static view, we examined the kinetic process of binding/unbinding of an eight-residue substrate peptide to/from 3CLpro by evaluating the path ensemble with the weighted ensemble simulation. The path ensemble showed the mechanism of how a highly flexible peptide folded into the bound form. At the early stage, the dominant motion was the diffusion on the protein surface showing a broad distribution, whose center was led into the cleft of the Chymotrypsin fold. We observed a definite sequential formation of the hydrogen bonds at the later stage occurring in the cleft, initiated between Glu166 (3CLpro) and P3_Val (peptide), followed by binding to the oxyanion hole and completed by the sequencespecific recognition at P1_Gln.","version":"1.2","doi":"10.1101/2022.06.08.495396","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.15.430863","pub_date":"2022-6-28","title":"Versatile live attenuated SARS-CoV-2 vaccine platform applicable to variants induces protective immunity","abstract":"Live attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37-39\u00b0C, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.","version":"1.2","doi":"10.1101/2021.02.15.430863","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.27.497248","pub_date":"2022-6-28","title":"Pre-existing immunity modulates responses to mRNA boosters","abstract":"mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower antibody levels before boost were associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the boosting capacity of mRNA vaccines. Mechanistic studies in mice show that pre-existing antibodies significantly limit antigen expression and priming of B cell responses after mRNA vaccination. Furthermore, we demonstrate that the relative superiority of an updated Omicron vaccine over the original vaccine is critically dependent on the serostatus of the host. These data demonstrate that pre-existing immunity dictates responses to mRNA vaccination, elucidating specific circumstances when updated SARS-CoV-2 vaccines confer superior protection to original vaccines.","version":"1.1","doi":"10.1101/2022.06.27.497248","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.28.497989","pub_date":"2022-6-28","title":"Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes","abstract":"Protection from SARS-related coronaviruses with spillover potential and SARS-CoV-2 variants could prevent and/or end pandemics. We show that mice immunized with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicit cross-reactive anti-sarbecovirus antibodies against conserved class 1/4 and class 3 RBD epitopes. Monoclonal antibodies (mAbs) identified from initial screening of <10,000 single B-cells secreting IgGs binding two or more sarbecovirus RBDs showed cross-reactive binding and neutralization of SARS-CoV-2 variants and animal sarbecoviruses. Single-particle cryo-EM structures of antibody\u2013spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes and revealed neutralization mechanisms, potentials for intra-spike trimer crosslinking by single IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticles to identify therapeutic pan-sarbecovirus and pan-variant mAbs and to elicit them by vaccination.","version":"1.1","doi":"10.1101/2022.06.28.497989","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.22.492693","pub_date":"2022-6-27","title":"RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity","abstract":"The spread of SARS-CoV-2 has fueled the COVID-19 pandemic with its enduring medical and socioeconomic challenges due to subsequent waves and long-term consequences of great concern. Here we charted the molecular basis of COVID-19 pathogenesis, by analysing patients\u2019 immune response at single-cell resolution across disease course and severity. This approach uncovered cell subpopulation-specific dysregulation in COVID-19 across disease course and severity and identified a severity-associated activation of the receptor for advanced glycation endproduct (RAGE) pathway in monocytes. In vitro experiments confirmed that monocytes bind the SARS-CoV-2 S1-RBD via RAGE and that RAGE-Spike interactions drive monocyte infection. Our results demonstrate that RAGE is a novel functional receptor of SARS-CoV-2 contributing to COVID-19 severity. Monocyte SARS-CoV-2 infection via the receptor for advanced glycation endproduct triggers severe COVID-19.","version":"1.2","doi":"10.1101/2022.05.22.492693","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.27.497749","pub_date":"2022-6-27","title":"The RNA demethylase FTO controls m6A marking on SARS-CoV-2 and classifies COVID-19 severity in patients","abstract":"The RNA modification N6-methyladenosine (m6A) plays a key role in the life cycles of several RNA viruses. Whether this applies to SARS-CoV-2 and whether m6A affects the outcome of COVID-19 disease is still poorly explored. Here we report that the RNA demethylase FTO strongly affects both m6A marking of SARS-CoV-2 and COVID-19 severity. By m6A profiling of SARS-CoV-2, we confirmed in infected cultured cells and showed for the first time in vivo in hamsters that the regions encoding TRS_L and the nucleocapsid protein are multiply marked by m6A, preferentially within RRACH motifs that are specific to \u03b2-coronaviruses and well conserved across SARS-CoV-2 variants. In cells, downregulation of the m6A demethylase FTO, occurring upon SARS-CoV-2 infection, increased m6A marking of SARS-CoV-2 RNA and slightly promoted viral replication. In COVID-19 patients, a negative correlation was found between FTO expression and both SARS-CoV-2 expression and disease severity. FTO emerged as a classifier of disease severity and hence a potential stratifier of COVID-19 patients.","version":"1.1","doi":"10.1101/2022.06.27.497749","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.26.497669","pub_date":"2022-6-27","title":"Antiviral T-cell Biofactory platform for SARS-CoV-2","abstract":"Vaccines help reduce new infections, but interventions that can prevent the disease from transitioning to a severe stage are rather limited. Dysregulated IFN kinetics are mostly exploited by pathogenic viruses, including SARS-CoV-2. The clinical benefits of systemically infused IFN are, unfortunately, mired by undesired side effects. To address this situation, we engineered a T cell to synthesize interferons (IFNs) as antiviral proteins upon recognizing the virus envelop protein of SARS-CoV-2, i.e., anti-SARS T-cell Biofactory. The T-cell Biofactory, capable of regulating the IFN expression with spatiotemporal resolution within the infected tissues, can mitigate these concerns. In this work, we determined the prophylactic and therapeutic effects of the type-I and type-III IFNs produced from the T-cell Biofactory against SARS-CoV-2 infection in host cells and investigated the expression profiles of ensuing IFN-stimulated genes (ISGs). To enable the translation of T-cell Biofactory as an effective antiviral countermeasure, we also investigated an irradiation dose that renders the T-cell Biofactory non-proliferative and thus non-oncogenic. The ongoing public health crisis motivated us to direct the T-cell Biofactory technology to target SARS-CoV-2. The T-cell Biofactory, based on T cells engineered with chimeric antigen receptors (CAR T cells), is a platform technology that can be rapidly re-engineered and become available for targeting any new pathogen.","version":"1.1","doi":"10.1101/2022.06.26.497669","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.26.493517","pub_date":"2022-6-27","title":"Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, and BA.5","abstract":"SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 have surged dramatically to become dominant in the United States and South Africa, respectively. These novel subvariants carrying additional mutations in their spike proteins raise concerns that they may further evade neutralizing antibodies, thereby further compromising the efficacy of COVID-19 vaccines and therapeutic monoclonals. We now report findings from a systematic antigenic analysis of these surging Omicron subvariants. BA.2.12.1 is only modestly (1.8-fold) more resistant to sera from vaccinated and boosted individuals than BA.2. However, BA.4/5 is substantially (4.2-fold) more resistant and thus more likely to lead to vaccine breakthrough infections. Mutation at spike residue L452 found in both BA.2.12.1 and BA.4/5 facilitates escape from some antibodies directed to the so-called class 2 and 3 regions of the receptor-binding domain. The F486V mutation found in BA.4/5 facilitates escape from certain class 1 and 2 antibodies but compromises the spike affinity for the viral receptor. The R493Q reversion mutation, however, restores receptor affinity and consequently the fitness of BA.4/5. Among therapeutic antibodies authorized for clinical use, only bebtelovimab retains full potency against both BA.2.12.1 and BA.4/5. The Omicron lineage of SARS-CoV-2 continues to evolve, successively yielding subvariants that are not only more transmissible but also more evasive to antibodies.","version":"1.2","doi":"10.1101/2022.05.26.493517","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.29.474469","pub_date":"2022-6-27","title":"Unsupervised genome-wide cluster analysis: nucleotide sequences of the omicron variant of SARS-CoV-2 are similar to sequences from early 2020","abstract":"The GISAID database contains more than 1,000,000 SARS-CoV-2 genomes, including sequences of the recently discovered SARS-CoV-2 omicron variant and of prior SARS-CoV-2 strains that have been collected from patients around the world since the beginning of the pandemic. We applied unsupervised cluster analysis to the SARS-CoV-2 genomes, assessing their similarity at a genome-wide level based on the Jaccard index and principal component analysis. Our analysis results show that the omicron variant sequences are most similar to sequences that have been submitted early in the pandemic around January 2020. Furthermore, the omicron variants in GISAID are spread across the entire range of the first principal component, suggesting that the strain has been in circulation for some time. This observation supports a long-term infection hypothesis as the omicron strain origin.","version":"1.3","doi":"10.1101/2021.12.29.474469","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492178","pub_date":"2022-6-27","title":"Unsupervised outlier detection applied to SARS-CoV-2 nucleotide sequences can identify sequences of common variants and other variants of interest","abstract":"As of June 2022, the GISAID database contains more than one million SARS-CoV-2 genomes, including several thousand nucleotide sequences for the most common variants such as delta or omicron. These SARS-CoV-2 strains have been collected from patients around the world since the beginning of the pandemic. We start by assessing the similarity of all pairs of nucleotide sequences using the Jaccard index and principal component analysis. As shown previously in the literature, an unsupervised cluster analysis applied to the SARS-CoV-2 genomes results in clusters of sequences according to certain characteristics such as their strain or their clade. Importantly, we observe that nucleotide sequences of common variants are often outliers in clusters of sequences stemming from variants identified earlier on during the pandemic. Motivated by this finding, we are interested in applying outlier detection to nucleotide sequences. We demonstrate that nucleotide sequences of common variants (such as alpha, delta, or omicron) can be identified solely based on a statistical outlier criterion. We argue that outlier detection might be a useful surveillance tool to identify emerging variants in real time as the pandemic progresses.","version":"1.2","doi":"10.1101/2022.05.16.492178","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.26.497634","pub_date":"2022-6-26","title":"Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism","abstract":"SARS-CoV-2 Omicron variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of antibodies induced by vaccination. Here, we describe the SARS-CoV-2 neutralizing SP1-77 antibody that was generated from a humanized mouse model with a single human VH1-2 and V\u03ba1-33-associated with immensely diverse complementarity-determining-region-3 (CDR3) sequences. SP1-77 potently and broadly neutralizes SARS-CoV-2 variants of concern and binds the SARS-CoV-2 spike protein receptor-binding-domain (RBD) via a novel-CDR3-based mode. SP1-77 does not block RBD-binding to the ACE2-receptor or endocytosis step of viral entry, but rather blocks membrane fusion. Our findings provide the first mechanistic insight into how a non-ACE2 blocking antibody potently neutralizes SARS-CoV-2, which may inform strategies for designing vaccines that robustly neutralize current and future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.06.26.497634","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.23.497376","pub_date":"2022-6-24","title":"Monitoring SARS-CoV-2 infection using a double reporter-expressing virus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 fundamental biology and the impact of anti-viral therapeutics are robust methods to detect for the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals are limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively), or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes, or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bireporter-expressing rSARS-CoV-2 for the study SARS-CoV-2 in vitro and in vivo. Despite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use to specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualize SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS).","version":"1.1","doi":"10.1101/2022.06.23.497376","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.23.497326","pub_date":"2022-6-24","title":"DESIGN OF A CHIMERIC ACE-2/Fc-SILENT FUSION PROTEIN WITH ULTRAHIGH AFFINITY AND NEUTRALIZING CAPACITY FOR SARS-CoV-2 VARIANTS","abstract":"As the coronavirus SARS-CoV-2 continues to mutate into Variants of Concern (VOC), there is a growing and urgent need to develop effective antivirals to combat the newly emerged infectious disease COVID-19. Recent data indicate that monoclonal antibodies developed early in the pandemic are no longer capable of effectively neutralizing currently active VOCs. This report describes the design of a class of variant-agnostic chimeric molecules consisting of an Angiotensin Converting Enzyme-2 (ACE-2) domain mutated to retain ultrahigh affinity binding to a wide variety of SARS-CoV-2 variants, coupled to an Fc-silent immunoglobulin domain that eliminates antibody-dependent enhancement (ADE) and simultaneously extends biological half-life compared to existing mABs. Molecular modeling revealed that ACE-2 mutations L27, V34 and E90 resulted in ultrahigh affinity binding of the LVE-ACE-2 domain to the widest variety of VOCs, with KDs of 93 pM, 507 pM and 73 pM for binding to the Alpha B1.1.7, Delta B.1.617.2 and Omicron B.1.1.529 variants, and notably, 78fM affinity to the Omicron BA.2 variant, respectively. Surrogate viral neutralization assays (sVNT) revealed titers of \u22654.9ng/ml, for neutralization of recombinant viral proteins corresponding to the Alpha, Delta and Omicron variants. The values above were obtained with LVE-ACE-2/mAB chimeras containing the Y-T-E sequence that enhances binding to the FcRn receptor, which in turn is expected to extend biological half-life 3-4-fold. It is proposed that this new class of chimeric ACE-2/mABs will constitute variant-agnostic and cost-effective prophylactics against SARS-CoV-2, particularly when administered by nasal delivery systems.","version":"1.1","doi":"10.1101/2022.06.23.497326","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.23.497239","pub_date":"2022-6-24","title":"Investigating the mutations in the SARS-CoV-2 proteins among European countries","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new member of the Coronaviridae family, triggering more than 190 million cases and more than two million deaths in European societies. Emerging the new variants due to mutations in genomic regions is foremost responsible for influencing the infectivity and mortality potential of such a virus. In the current study, we considered mutations among spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 in the Europe continent by exploring the frequencies of mutations and the timeline of emerging them. For this purpose, Amino-acid sequences (AASs) were gathered from the GISAID database, and Mutation tracking was performed by detecting any difference between samples and a reference sequence; Wuhan-2019. In the next step, we compared the achieved results with worldwide sequences. 8.6%, 63.6%, 24.7%, and 1.7% of S, E, M, and N samples did not demonstrate any mutation among European countries. Also, the regions of 508 to 635 AA, 7 to 14 AA, 66 to 88 AA, and 164 to 205 AA in S, E, M, and N samples contained the most mutations relative to the total AASs in both Europe AASs and worldwide samples. D614G, A222V, S477N, and L18F were the first to fifth frequent mutations in S AASs among European samples, and T9I, I82T, and R203M were the first frequent mutations among E, M, and S AASs of the Europe continent. Investigating the mutations among structural proteins of SARS-CoV-2 can improve the strength of therapeutic and diagnostic strategies to efficient combat the virus and even maybe efficient in predicting new emerging variants of concern.","version":"1.1","doi":"10.1101/2022.06.23.497239","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.24.496409","pub_date":"2022-6-24","title":"A cell-based, spike protein binding assay highlights differences in antibody neutralising capacity for SARS-CoV-2 variants","abstract":"The engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells and accordingly blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine-elicited serum antibodies. The emergence of SARS-CoV-2 variants necessitates the development of adaptable assays that can be applied to assess the effectiveness of therapeutics. Through testing of a range of recombinant spike proteins, we have developed a cell based, ACE2/spike protein binding assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell bound spike protein fluorescence. Using spike proteins from the original \u2018Wuhan\u2019 SARS-CoV-2 virus, as well as the delta and omicron variants, we identify differential blocking activity of three monoclonal antibodies directed against the spike receptor binding domain. Importantly, biological activity in the spike binding assay translated to efficacy in a SARS-CoV-2 infection assay. Hence, the spike binding assay has utility to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable to quantify impact of antibodies against new and emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.06.24.496409","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478697","pub_date":"2022-6-24","title":"Mutability Patterns Across the Spike Glycoprotein Reveal the Diverging and Lineage-specific Evolutionary Space of SARS-CoV-2","abstract":"Mutations in the spike glycoprotein of SARS-CoV-2 allow the virus to probe the sequence space in search of higher-fitness states. New sublineages of SARS-CoV-2 variants-of-concern (VOCs) continuously emerge with such mutations. Interestingly, the sites of mutation in these sublineages vary between the VOCs. Whether such differences reflect the random nature of mutation appearance or distinct evolutionary spaces of spike in the VOCs is unclear. Here we show that each position of spike has a lineage-specific likelihood for mutations to appear and dominate descendent sublineages. This likelihood can be accurately estimated from the lineage-specific mutational profile of spike at a protein-wide level. The mutability environment of each position, including adjacent sites on the protein structure and neighboring sites on the network of comutability, accurately forecast changes in descendent sublineages. Mapping of imminent changes within the VOCs can contribute to the design of immunogens and therapeutics that address future forms of SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.02.01.478697","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.22.497189","pub_date":"2022-6-24","title":"Increased levels of circulating neurotoxic metabolites in patients with mild Covid19","abstract":"SARS-CoV-2 corona virus causes a multi-faceted and poorly defined clinical and pathological phenotype involving hyperinflammation, cytokine release, and long-term cognitive deficits, with an undefined neuropathological mechanism. Inflammation increases the activity of the kynurenine pathway, which is linked to neurodegenerative and psychiatric disorders. We sought to determine whether the kynurenine pathway is impacted in patients with mild COVID-19, leading to elevated neurotoxic metabolites in blood, and whether such changes are associated with pro-inflammatory cytokines. Serum samples were taken from 150 patients and analyzed by ELISA and ultra-high performance liquid chromatography (UHPLC). The data were analyzed using multiple linear regression models adjusted for age and sex. We found increased levels of kynurenine, quinolinic acid and 3-hydroxykynurenine in serum from patients with mild COVID-19, together with increased levels of IL-6, ICAM-1, VCAM-1 and neopterin. The levels of neurotoxic metabolites were significantly associated with key inflammatory cytokines including IL-6 and TNF\u03b1. The COVID-19 risk-factor hypertension was associated with the highest levels of neurotoxic metabolites in plasma. These neuroactive metabolites could be part of the pathological mechanisms underlying cognitive impairment during and post-COVID and should be explored as potential biomarkers for long-COVID symptoms.","version":"1.2","doi":"10.1101/2022.06.22.497189","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481100","pub_date":"2022-6-23","title":"Mitoquinone mesylate targets SARS-CoV-2 infection in preclinical models","abstract":"To date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo. Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy. Mitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2022.02.22.481100","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.21.497047","pub_date":"2022-6-23","title":"Within-host evolutionary dynamics and tissue compartmentalization during acute SARS-CoV-2 infection","abstract":"The global evolution of SARS-CoV-2 depends in part upon the evolutionary dynamics within individual hosts with varying immune histories. To characterize the within-host evolution of acute SARS-CoV-2 infection, we deep sequenced saliva and nasal samples collected daily from immune and unvaccinated individuals early during infection. We show that longitudinal sampling facilitates high-confidence genetic variant detection and reveals evolutionary dynamics missed by less-frequent sampling strategies. Within-host dynamics in both na\u00efve and immune individuals appeared largely stochastic; however, we identified clear mutational hotspots within the viral genome, consistent with selection and differing between na\u00efve and immune individuals. In rare cases, minor genetic variants emerged to frequencies sufficient for forward transmission. Finally, we detected significant genetic compartmentalization of virus between saliva and nasal swab sample sites in many individuals. Altogether, these data provide a high-resolution profile of within-host SARS-CoV-2 evolutionary dynamics.","version":"1.2","doi":"10.1101/2022.06.21.497047","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.22.495814","pub_date":"2022-6-23","title":"Mouse genomic rewriting and tailoring: synthetic Trp53 and humanized ACE2","abstract":"Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection.","version":"1.1","doi":"10.1101/2022.06.22.495814","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.22.497134","pub_date":"2022-6-22","title":"Mutational insights among the structural proteins of SARS-CoV-2: frequencies and evolutionary trends in American countries","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a role in the mortality of more than 6 million people worldwide. This virus owns the genome, which contains four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N). The occurrence of structural mutations can induce the emergence of new variants. Depending on the mutations, the variants may display different patterns of infectivity, mortality, and sensitivity toward drugs and vaccines. In this study, we analyzed samples of amino-acid sequences (AASs) for structural proteins from the coronavirus 2019 (COVID-19) declaration as a pandemic to April 2022 among American countries. The analysis process included considering mutations\u2019 frequencies, locations, and evolutionary trends utilizing sequence alignment to the reference sequence. In the following, the results were compared with the same analyses among the samples of the entire world. Results displayed that despite samples of North America and international countries that own the region of 508 to 635 with the highest mutation frequency among S AASs, the region with the same characteristic was concluded as 1 to 127 in South America. Besides, the most frequent mutations in S, E, M, and N proteins from North America and worldwide samples were concluded as D614G, T9I, I82T, and R203M. In comparison, R203K was the first frequent mutation in N samples in South America. Widely comparing mutations between North America and South America and between the Americas and the world can help scientists introduce better drug and vaccine development strategies.","version":"1.1","doi":"10.1101/2022.06.22.497134","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.21.496751","pub_date":"2022-6-22","title":"Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models","abstract":"The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19\u2013convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.","version":"1.1","doi":"10.1101/2022.06.21.496751","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.22.22276764","pub_date":"2022-06-22","title":"An international observational study to assess the impact of the Omicron variant emergence on the clinical epidemiology of COVID-19 in hospitalised patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Whilst timely clinical characterisation of infections caused by novel SARS-CoV-2 variants is necessary for evidence-based policy response, individual-level data on infecting variants are typically only available for a minority of patients and settings.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Here, we propose an innovative approach to study changes in COVID-19 hospital presentation and outcomes after the Omicron variant emergence using publicly available population-level data on variant relative frequency to infer SARS-CoV-2 variants likely responsible for clinical cases. We apply this method to data collected by a large international clinical consortium before and after the emergence of the Omicron variant in different countries.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Our analysis, that includes more than 100,000 patients from 28 countries, suggests that in many settings patients hospitalised with Omicron variant infection less often presented with commonly reported symptoms compared to patients infected with pre-Omicron variants. Patients with COVID-19 admitted to hospital after Omicron variant emergence had lower mortality compared to patients admitted during the period when Omicron variant was responsible for only a minority of infections (odds ratio in a mixed-effects logistic regression adjusted for likely confounders, 0.67 [95% confidence interval 0.61 \u2013 0.75]). Qualitatively similar findings were observed in sensitivity analyses with different assumptions on population-level Omicron variant relative frequencies, and in analyses using available individual-level data on infecting variant for a subset of the study population.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Although clinical studies with matching viral genomic information should remain a priority, our approach combining publicly available data on variant frequency and a multi-country clinical characterisation dataset with more than 100,000 records allowed analysis of data from a wide range of settings and novel insights on real-world heterogeneity of COVID-19 presentation and clinical outcome.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.06.22.22276764","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.21.22276660","pub_date":"2022-06-22","title":"Impact of Pre-Existing Chronic Viral Infection and Reactivation on the Development of Long COVID","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>The presence and reactivation of chronic viral infections such as Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) have been proposed as potential contributors to Long COVID (LC), but studies in well-characterized post-acute cohorts of individuals with COVID-19 over a longer time course consistent with current case definitions of LC are limited. In a cohort of 280 adults with prior SARS-CoV-2 infection, we observed that LC symptoms such as fatigue and neurocognitive dysfunction at a median of 4 months following initial diagnosis were independently associated with serological evidence of recent EBV reactivation (early antigen-D [EA-D] IgG positivity) or high nuclear antigen IgG levels, but not with ongoing EBV viremia. Evidence of EBV reactivation (EA-D IgG) was most strongly associated with fatigue (OR 2.12). Underlying HIV infection was also independently associated with neurocognitive LC (OR 2.5). Interestingly, participants who had serologic evidence of prior CMV infection were less likely to develop neurocognitive LC (OR 0.52) and tended to have less severe (&gt;5 symptoms reported) LC (OR 0.44). Overall, these findings suggest differential effects of chronic viral co-infections on the likelihood of developing LC and predicted distinct syndromic patterns. Further assessment during the acute phase of COVID-19 is warranted.</jats:p>\n                <jats:sec>\n                  <jats:title>SUMMARY</jats:title>\n                  <jats:p>The authors found that Long COVID symptoms in a post-acute cohort were associated with serological evidence of recent EBV reactivation and pre-existing HIV infection when adjusted for participant factors, sample timing, comorbid conditions and prior hospitalization, whereas underlying CMV infection was associated with a decreased risk of Long COVID.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.06.21.22276660","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.21.497040","pub_date":"2022-6-22","title":"Enhancement of mitochondrial function fosters B cell immune memory","abstract":"Differentiation of T and B cells to effector and memory cell fates are associated with extensive metabolic changes which are accompanied by altered mitochondrial dynamics. However, whether alterations in mitochondrial structure and function plays an active role in regulating effector versus memory cell fate decisions during immune responses remains unclear. Our studies here characterize changes in mitochondrial dynamics in activated B cells and show that increased mitochondrial mass and activity is a distinct feature of memory B cell lineage commitment in vivo. Using a directed screen of mitochondrial modulators, we identify mitochondrial fission inhibitor, Mdivi-1 as an agent that could enhance mitochondrial mass and function leading to augmented memory B cell differentiation. The enhanced memory B cell responses mediated by Mdivi-1, translated to more robust recall responses upon secondary antigen exposures. Moreover, Mdivi-1 when used in combination with subunit (SARS-CoV2) and inactivated (H1N1 influenza) vaccines led to remarkably improved vaccine efficacies and protection from lethal viral (H1N1) challenge. Single-cell transcriptomics revealed enhanced commitment to memory lineage differentiation in B cells following Mdivi-1 treatment. We propose that mitochondrial modulators such as Mdivi-1 are a novel class of \u201cimmune enhancers\u201d that specifically reinforces immunological memory and could be broadly applied to improve the fidelity of immune responses and vaccine efficacies.","version":"1.1","doi":"10.1101/2022.06.21.497040","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.20.496929","pub_date":"2022-6-21","title":"ct2vl: Converting Ct Values to Viral Loads for SARS-CoV-2 RT-qPCR Test Results","abstract":"RT-qPCR is the de facto reference method for detecting the presence of SARS-CoV-2 genomic material in infected individuals (1). Although RT-qPCR is inherently quantitative and despite SARS-CoV-2 viral loads varying by 10 orders of magnitude and therefore being potentially highly clinically informative, in practice SARS-CoV-2 RT-qPCR results are usually reported qualitatively as simply positive or negative. This is both because of the mathematical complexity of converting from Ct values to viral loads and because the same Ct value can correspond to orders-of-magnitude differences in viral load depending on the testing platform (2, 3, 4). To address this problem, here we present ct2vl, a Python package designed to help individual clinical laboratories, investigators, and test developers convert from Ct values to viral loads on their own platforms, using only the data generated during validation of those platforms. It allows any user to convert Ct values to viral loads and is readily applicable to other RT-qPCR tests. ct2vl is open source, has 100% code coverage, and is freely available via the Python Package Index (PyPI). Up to now, COVID-19 test results have been reported as positive vs. negative, even though \u201cpositive\u201d can mean anywhere from 1 copy of SARS-CoV-2 virus per milliliter of transport media to over 1 billion copies/mL, with attendant clinical consequences. Democratizing access to this quantitative data is the first step toward its eventual incorporation into test development, the research literature, and clinical care.","version":"1.1","doi":"10.1101/2022.06.20.496929","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.14.22276397","pub_date":"2022-06-21","title":"Association between Bisphosphonate use and COVID-19 related outcomes: a retrospective cohort study","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Although there are several efficacious vaccines against COVID-19, vaccination rates in many regions around the world remain insufficient to prevent continued high disease burden and emergence of viral variants. Repurposing of existing therapeutics that prevent or mitigate severe COVID-19 could help to address these challenges. The objective of this study was to determine whether prior use of bisphosphonates is associated with reduced incidence and/or severity of COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>A retrospective cohort study utilizing payer-complete health insurance claims data from 8,239,790 patients with continuous medical and prescription insurance from 1-1-2019 to 6-30-2020 was performed. The primary exposure of interest was use of any bisphosphonate from 1-1-2019 to 2-29-2020. Outcomes of interest included: (a) testing for SARS-CoV-2 infection; (b) COVID-19 diagnosis; and (c) hospitalization with COVID-19 diagnosis between 3-1-2020 and 6-30-2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>7,906,603 patients for whom continuous medical and prescription insurance information was available were selected. 450,366 bisphosphonate users were identified and 1:1 propensity score-matched to bisphosphonate non-users by age, gender, insurance type, primary-care-provider visit in 2019, and comorbidity burden. Bisphosphonate users had lower odds ratios (OR) of testing for SARS-CoV-2 infection (OR=0.22; 95%CI:0.21-0.23; p&lt;0.001), COVID-19 diagnosis (OR=0.23; 95%CI:0.22-0.24; p&lt;0.001), and COVID-19-related hospitalization (OR=0.26; 95%CI:0.24-0.29; p&lt;0.001). Sensitivity analyses yielded results consistent with the primary analysis. Bisphosphonate-use was also associated with decreased odds of acute bronchitis (OR=0.23; 95%CI:0.22-0.23; p&lt;0.001) or pneumonia (OR=0.32; 95%CI:0.31-0.34; p&lt;0.001) in 2019, suggesting that bisphosphonates may protect against respiratory infections by a variety of pathogens, including but not limited to SARS-CoV-2.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Prior bisphosphonate-use was associated with dramatically reduced odds of SARS-CoV-2 testing, COVID-19 diagnosis, and COVID-19-related hospitalizations. Prospective clinical trials will be required to establish a causal role for bisphosphonate-use in COVID-19-related outcomes.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.06.14.22276397","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.19.496718","pub_date":"2022-6-21","title":"Anamnestic Humoral Correlates of Immunity Across SARS-CoV-2 Variants of Concern","abstract":"While immune correlates against SARS-CoV-2 are typically defined at peak immunogenicity following vaccination, immunologic responses that expand selectively during the anamnestic response following infection can provide mechanistic and detailed insights into the immune mechanisms of protection. Moreover, whether anamnestic correlates are conserved across VoCs, including the Delta and more distant Omicron variant of concern (VoC), remains unclear. To define the anamnestic correlates of immunity, across VOCs, we deeply profiled the humoral immune response in individuals recently infected with either the Delta or Omicron VoC. While limited acute N-terminal domain and RBD-specific immune expansion was observed following breakthrough, a significant immunodominant expansion of opsinophagocytic Spike-specific antibody responses focused largely on the conserved S2-domain of SARS-CoV-2 was observed 1 week after breakthrough infection. This S2-specific functional humoral response continued to evolve over 2-3 weeks following both Delta and Omicron breakthrough infection, targeting multiple VoCs and common coronaviruses. These responses were focused largely on the fusion peptide 2 and heptad repeat 1, both associated with enhanced rates of viral clearance. Taken together, our results point to a critical role of highly conserved, functional S2-specific responses in the control of SARS-CoV-2 infection, across VOCs, and thus humoral response linked to virus attenuation can guide next-generation generation vaccine boosting approaches to confer broad protection against future SARS-CoV-2 VoCs.","version":"1.1","doi":"10.1101/2022.06.19.496718","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.21.496991","pub_date":"2022-6-21","title":"Identification of a guanine-specific pocket in the protein N of SARS-CoV-2","abstract":"The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveals a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.","version":"1.1","doi":"10.1101/2022.06.21.496991","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.20.496341","pub_date":"2022-6-21","title":"SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D","abstract":"Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally-infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogated the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We found that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (\u201cNKG2D-L\u201d). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we found that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work reveals that SARS-CoV-2 evades NK cell cytotoxicity and describes a mechanism by which this occurs.","version":"1.1","doi":"10.1101/2022.06.20.496341","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.20.496903","pub_date":"2022-6-21","title":"Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning","abstract":"Increasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the N-terminal domain (NTD) has been long overlooked due to the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD, but also provides invaluable insights into S-based immunogen design.","version":"1.1","doi":"10.1101/2022.06.20.496903","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.20.496780","pub_date":"2022-6-21","title":"SEMA: Antigen B-cell conformational epitope prediction using deep transfer learning","abstract":"One of the primary tasks in vaccine design and development of immunotherapeutic drugs is to predict conformational B-cell epitopes corresponding to primary antibody binding sites within the antigen tertiary structure. To date, multiple approaches have been developed to address this issue. However, for a wide range of antigens their accuracy is limited. In this paper, we applied the transfer learning approach using pretrained deep learning models to develop a model that predicts conformational B-cell epitopes based on the primary antigen sequence and tertiary structure. A pretrained protein language model, ESM-1b, and an inverse folding model, ESM-IF1, were fine-tuned to quantitatively predict antibody-antigen interaction features and distinguish between epitope and non-epitope residues. The resulting model called SEMA demonstrated the best performance on an independent test set with ROC AUC of 0.76 compared to peer-reviewed tools. We show that SEMA can quantitatively rank the immunodominant regions within the RBD domain of SARS-CoV-2. SEMA is available at https://github.com/AIRI-Institute/SEMAi and the web-interface http://sema.airi.net.","version":"1.1","doi":"10.1101/2022.06.20.496780","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.20.496826","pub_date":"2022-6-20","title":"Trim69 is a microtubule regulator that acts as a pantropic viral inhibitor","abstract":"To identify novel cellular modulators of HIV-1 infection in IFN-stimulated myeloid cells, we have carried out a screen that combines functional and evolutionary analyses in THP-1-PMA cells that led us to the Tripartite Motif Protein 69 (Trim69), a poorly studied member of the Trim family of innate immunity regulators. Trim69 inhibits HIV-1, primate lentiviruses and the negative and positive-strand RNA viruses VSV and SARS-CoV2, overall indicating it is a broad-spectrum antiviral factor. Trim69 binds directly to microtubules and its antiviral activity is intimately linked to its ability to promote the accumulation of stable MTs, a specialized subset of microtubules. By analyzing the behavior of primary blood cells, we provide evidence that a program of MT stabilization is commonly observed in response to IFN-I in cells of the myeloid lineage and Trim69 is the key factor behind this program. Overall, our study identifies Trim69 as the first antiviral innate defense factor that regulates the properties of microtubules to limit viral spread, highlighting the possibility that the cytoskeleton may be a novel unappreciated fighting ground in the host-pathogen interactions that underlie viral infections.","version":"1.1","doi":"10.1101/2022.06.20.496826","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.17.496600","pub_date":"2022-6-19","title":"Efficient direct and limited environmental transmission of SARS-CoV-2 lineage B.1.22 in domestic cats","abstract":"Susceptibility of domestic cats for infection with SARS-CoV-2 has been demonstrated by several experimental studies and field observations. We performed an extensive study to further characterize transmission of SARS-CoV-2 between cats, both by direct contact as well as by indirect contact. To that end, we estimated the transmission rate parameter and the decay parameter for infectivity in the environment. Using four groups of pair-transmission experiment, all donor (inoculated) cats became infected, shed virus and seroconverted, while three out of four direct contact cats got infected, shed virus and two of those seroconverted. One out of eight cats exposed to a SARS-CoV-2-contaminated environment became infected but did not seroconvert. Statistical analysis of the transmission data gives a reproduction number R0 of 2.18 (95% CI: (0.92-4.08), a transmission rate parameter \u03b2 of 0.23 day-1 (95% CI: 0.06-0.54), and a virus decay rate parameter \u03bc of 2.73 day-1 (95% CI: 0.77-15.82). These data indicate that transmission between cats can be sustained (R0>1), however, infectiousness of a contaminated environment decays rapidly (mean duration of infectiousness 1/2.73 days). Infections of cats via exposure to a SARS-CoV-2-contaminated environment cannot be excluded if cats are exposed shortly after contamination.","version":"1.1","doi":"10.1101/2022.06.17.496600","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.17.496635","pub_date":"2022-6-19","title":"Compensatory epistasis maintains ACE2 affinity in SARS-CoV-2 Omicron BA.1","abstract":"The Omicron BA.1 variant emerged in late 2021 and quickly spread across the world. Compared to the ancestral Wuhan Hu-1 strain and other pre-Omicron SARS-CoV-2 variants, BA.1 has many mutations, a number of which are known to enable antibody escape . Many of these antibody-escape mutations individually decrease the spike receptor-binding domain (RBD) affinity for ACE2 in the background of early SARS-CoV-2 variants , but BA.1 still binds ACE2 with high affinity . The fitness and evolution of the BA.1 lineage is therefore driven by the combined effects of numerous mutations. Here, we systematically map the epistatic interactions between the 15 mutations in the RBD of BA.1 relative to the Wuhan Hu-1 strain. Specifically, we measure the ACE2 affinity of all possible combinations of these 15 mutations (2  = 32,768 genotypes), spanning all possible evolutionary intermediates from the ancestral Wuhan Hu-1 strain to BA.1. We find that immune escape mutations in BA.1 individually reduce ACE2 affinity but are compensated by epistatic interactions with other affinity-enhancing mutations, including Q498R and N501Y. Thus, the ability of BA.1 to evade immunity while maintaining ACE2 affinity is contingent on acquiring multiple interacting mutations. Our results implicate compensatory epistasis as a key factor driving substantial evolutionary change for SARS-CoV-2 and are consistent with Omicron BA.1 arising from a chronic infection.","version":"1.1","doi":"10.1101/2022.06.17.496635","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.17.496646","pub_date":"2022-6-19","title":"Integrating Conformational Dynamics and Perturbation-Based Network Modeling for Mutational Profiling of Binding and Allostery in the SARS-CoV-2 Spike Variant Complexes with Antibodies: Balancing Local and Global Determinants of Mutational Escape Mechanisms","abstract":"In this study, we combined all-atom MD simulations, the ensemble-based mutational scanning of protein stability and binding, and perturbation-based network profiling of allosteric interactions in the SARS-Cov-2 Spike complexes with a panel of cross-reactive and ultra-potent single antibodies (B1-182.1 and A23-58.1) as well as antibody combinations (A19-61.1/B1-182.1 and A19-46.1/B1-182.1). Using this approach, we quantify local and global effects of mutations in the complexes, identify structural stability centers, characterize binding energy hotspots and predict the allosteric control points of long-range interactions and communications. Conformational dynamics and distance fluctuation analysis revealed the antibody-specific structural stability signatures of the spike complexes that can dictate the pattern of mutational escape. By employing an integrated analysis of conformational dynamics and binding energetics, we found that the potent antibodies that efficiently neutralize Omicron spike variant can form the dominant binding energy hotpots with the conserved stability centers in which mutations may be restricted by the requirements of the folding stability and binding to the host receptor. The results show that protein stability and binding energetics of the SARS-CoV-2 spike complexes with the panel of cross-reactive ultrapotent antibodies are tolerant to the constellation of Omicron mutations. A network-based perturbation approach for mutational profiling of allosteric residues potentials revealed how antibody binding can modulate allosteric interactions and identified allosteric control points that can form vulnerable sites for mutational escape. This study suggested a mechanism in which the pattern of specific escape mutants for ultrapotent antibodies may not be solely determined by the binding interaction changes but are driven by a complex balance and tradeoffs between different local and global factors including the impact of mutations on structural stability, binding strength, long-range interactions and fidelity of allosteric signaling.","version":"1.1","doi":"10.1101/2022.06.17.496646","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.18.496304","pub_date":"2022-6-19","title":"Deciphering inhibitory mechanism of coronavirus replication through host miRNAs-RNA-dependent RNA polymerase (RdRp) interactome","abstract":"Despite what we know so far, Covid-19, caused by SARS-CoV-2 virus, remains a pandemic that still require urgent healthcare intervention. The frequent mutations of the SARS-CoV-2 virus has rendered disease control with vaccines and antiviral drugs quite difficult and challenging, with newer variants surfacing constantly. There is therefore the need for newer, effective and efficacious drugs against coronaviruses. Considering the role of RNA dependent, RNA polymerase (RdRp) as an important enzyme necessary for the virus life cycle and its conservation among coronaviruses, we investigated potential host miRNAs that can be employed as broad-range antiviral drugs averse to coronaviruses, with particular emphasis on BCoV, MERS-CoV, SARS-CoV and SARS-CoV-2. miRNAs are small molecules capable of binding mRNA and regulate expression at transcriptional or translational levels. Our hypothesis is that host miRNAs have the potential of blocking coronavirus replication through miRNA-RdRp mRNA interaction. To investigate this, we downloaded the open reading frame (ORF 1ab) nucleotide sequences and used them to interrogate miRNA databases for miRNAs that can bind them. We employed various bioinformatics tools to predict and identify the most effective host miRNAs. In all, we found 27 miRNAs that target RdRp mRNA of multiple coronaviruses, of which three - hsa-miR-1283, hsa-miR-579-3p, and hsa-miR-664b-3p target BCoV, SARS-CoV and SARS-CoV-2. Additionally, hsa-miR-374a-5p has three bovine miRNAs homologs viz bta-miR-374a, bta-miR-374b, and bta-miR-374c. Inhibiting the expression of RdRp enzyme via non-coding RNA is novel and of great therapeutic importance in the control of coronavirus replication, and could serve as a broad-spectrum antiviral, with hsa-miR-1283, hsa-miR-579-3p, and hsa-miR-664b-3p highly promising.","version":"1.1","doi":"10.1101/2022.06.18.496304","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.16.496458","pub_date":"2022-6-17","title":"Electrostatic features for the Receptor binding domain of SARS-COV-2 wildtype and its variants. Compass to the severity of the future variants with the charge-rule","abstract":"Electrostatic intermolecular interactions are important in many aspects of biology. We have studied the main electrostatic features involved in the interaction of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with the human receptor Angiotensin-converting enzyme 2 (ACE2). As the principal computational tool, we have used the FORTE approach, capable to model proton fluctuations and computing free energies for a very large number of protein-protein systems under different physical-chemical conditions, here focusing on the RBD-ACE2 interactions. Both the wild-type and all critical variants are included in this study. From our large ensemble of extensive simulations, we obtain, as a function of pH, the binding affinities, charges of the proteins, their charge regulation capacities, and their dipole moments. In addition, we have calculated the pKas for all ionizable residues and mapped the electrostatic coupling between them. We are able to present a simple predictor for the RBD-ACE2 binding based on the data obtained for Alpha, Beta, Gamma, Delta, and Omicron variants, as a linear correlation between the total charge of the RBD and the corresponding binding affinity. This \u201cRBD charge rule\u201d should work as a quick test of the degree of severity of the coming SARS-CoV-2 variants in the future. Categories and Subject Descriptors:","version":"1.1","doi":"10.1101/2022.06.16.496458","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.16.496383","pub_date":"2022-6-16","title":"Development and Evaluation of RT-LAMP Assays to Identify Variants of SARS-CoV-2","abstract":"Emergence of new variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) during current pandemic of Coronavirus Disease 2019 (COVID-19) and several waves of infections by some of variants emphasized the importance of continuous surveillance. While genomic surveillance through whole genome sequencing is performed as a standard method, identification of known variants through mutation-targeting molecular diagnosis such as qRT-PCR is also useful for timely investigation. However, there are limited studies regarding the concurrent detection and identification of SARS-CoV-2 variants through a LAMP-based method. In this study, we developed and evaluated RT-LAMP assays to detect characteristic deletions of SARS-CoV-2 variants. In addition, we evaluated a fluorescent probe mediated method for identification of single nucleotide substitution by RT-LAMP. Finally, we discussed restrictions and perspectives regarding pathogen screening and surveillance of variants by RT-LAMP based on our observations.","version":"1.1","doi":"10.1101/2022.06.16.496383","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.16.496324","pub_date":"2022-6-16","title":"SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-\u03baB non-canonical pathway and mitochondrial remodeling","abstract":"Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non-productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-\u03baB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.","version":"1.1","doi":"10.1101/2022.06.16.496324","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.14.496214","pub_date":"2022-6-16","title":"Oxysterols drive inflammation via GPR183 during influenza virus and SARS-CoV-2 infection","abstract":"Severe viral respiratory infections are often characterized by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain elusive. To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. Preclinical murine models of influenza virus and SARS-CoV-2 infection. Oxidized cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte-macrophage infiltration to the lung during influenza virus (IAV) and SARS-CoV-2 infections. Both IAV and SARS-CoV-2 infections upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidized cholesterols 25-hydroxycholesterol and 7\u03b1,25-dihydroxycholesterol (7\u03b1,25-OHC). Loss-of-function mutation of GPR183, or treatment with a GPR183 antagonist, reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist also significantly attenuated the severity of SARS-CoV-2 infection by reducing weight loss and viral loads. This study demonstrates that oxysterols drive inflammation in the lung and provides the first preclinical evidence for therapeutic benefit of targeting GPR183 during severe viral respiratory infections. Viral infections trigger oxysterol production in the lung, attracting macrophages via GPR183. Blocking GPR183 reduced inflammation and disease severity in SARS-CoV-2 infection, making GPR183 a putative target for therapeutic intervention.","version":"1.2","doi":"10.1101/2022.06.14.496214","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.16.496428","pub_date":"2022-6-16","title":"Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses","abstract":"Previously, we showed that coagulation factors directly cleave SARS-CoV-2 spike and promote viral entry (Kastenhuber et al., 2022). Here, we show that substitutions in the S1/S2 cleavage site observed in SARS-CoV-2 variants of concern (VOCs) exhibit divergent interactions with host proteases, including factor Xa and furin. Nafamostat remains effective to block coagulation factor-mediated cleavage of variant spike sequences. Furthermore, host protease usage has likely been a selection pressure throughout coronavirus evolution, and we observe convergence of distantly related coronaviruses to attain common host protease interactions, including coagulation factors. Interpretation of genomic surveillance of emerging SARS-CoV-2 variants and future zoonotic spillover is supported by functional characterization of recurrent emerging features.","version":"1.1","doi":"10.1101/2022.06.16.496428","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.16.496375","pub_date":"2022-6-16","title":"An S1 subunit vaccine and combination adjuvant (COVAC-1) elicits robust protection against SARS-CoV-2 challenge in African green monkeys","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent responsible for the ongoing global pandemic. With over 500 million cases and more than 6 million deaths reported globally, the need for access to effective vaccines is clear. An ideal SARS-CoV-2 vaccine will prevent pathology in the lungs and prevent virus replication in the upper respiratory tract, thus reducing transmission. Here, we assessed the efficacy of an adjuvanted SARS-CoV-2 S1 subunit vaccine, called COVAC-1, in an African green monkey (AGM) model. AGMs immunized and boosted with COVAC-1 were protected from SARS-CoV-2 challenge compared to unvaccinated controls based on reduced pathology and reduced viral RNA levels and infectious virus in the respiratory tract. Both neutralizing antibodies and antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) were observed in vaccinated animals prior to the challenge. COVAC-1 induced effective protection, including in the upper respiratory tract, thus supporting further development and utility for determining the mechanism that confers this protection. Vaccines that can prevent the onward transmission of SARS-CoV-2 and prevent disease are highly desirable. Whether this can be accomplished without mucosal immunization by a parenterally administered subunit vaccine is not well established. Here we demonstrate that following two vaccinations, a protein subunit vaccine containing the S1 portion of the SARS-CoV-2 spike glycoprotein and the novel adjuvant TriAdj significantly reduces the amount of virus in the lungs and also mediates rapid clearance of the virus from the upper respiratory tract. Further support of the effectiveness of COVAC-1 was the observation of reduced pathology in the lungs and viral RNA being largely absent from tissues, blood, and rectal swabs. Thus COVAC-1 appears promising at mediating protection in both the upper and lower respiratory tract and may be capable of reducing subsequent transmission of SARS-CoV-2. Further investigation into the mechanism of protection in the upper respiratory tract and the initial immune response that supports this would be warranted.","version":"1.1","doi":"10.1101/2022.06.16.496375","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.15.496220","pub_date":"2022-6-16","title":"The Omicron variant BA.1.1 presents a lower pathogenicity than B.1 D614G and Delta variants in a feline model of SARS-CoV-2 infection","abstract":"Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern (VOC), which emerged in late 2021 and rapidly achieved global predominance in early 2022. In this study, we compared the infection dynamics, tissue tropism and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2) and Omicron BA.1.1 sublineage (B.1.1.529) variants in a highly susceptible feline model of infection. While D614G- and Delta-inoculated cats became lethargic, and showed increased body temperatures between days 1 and 3 post-infection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log10 TCID50.ml-1), whereas strikingly lower level of viruses shedding (<3.1 log10 TCID50.ml-1) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by in situ viral RNA detection, in situ immunofluorescence, and quantification of viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main - but not the only - sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats had severe pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model. The SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VOC) emerged in South Africa late in 2021 and rapidly spread across the world causing a significant increase in the number of infections. Importantly, this variant was also associated with an increased risk of reinfections. However, the number of hospitalizations and deaths due to COVID-19 did not follow the same trends. These early observations, suggested effective protection conferred by immunizations and/or overall lower virulence of the highly mutated variant virus. In this study we present novel evidence demonstrating that the Omicron BA.1.1 variant of concern (VOC) presents a lower pathogenicity when compared to D614G- or Delta variants in cats. Clinical, virological and pathological evaluations revealed lower disease severity, viral replication and lung pathology in Omicron-infected cats when compared to D614G and Delta variant inoculated animals, confirming that Omicron BA.1.1 is less pathogenic in a highly susceptible feline model of infection.","version":"1.2","doi":"10.1101/2022.06.15.496220","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.15.496006","pub_date":"2022-6-15","title":"A single-component luminescent biosensor for the SARS-CoV-2 spike protein","abstract":"Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of E. coli and S. cerevisiae. This biosensor is deployed in multiple homogenous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an un-augmented cell phone camera. Binding Activated Tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.","version":"1.1","doi":"10.1101/2022.06.15.496006","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.09.22276196","pub_date":"2022-06-14","title":"Accelerated waning of the humoral response to SARS-CoV-2 vaccines in obesity","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  Obesity is associated with an increased risk of severe Covid-19. However, the effectiveness of SARS-CoV-2 vaccines in people with obesity is unknown. Here we studied the relationship between body mass index (BMI), hospitalization and mortality due to Covid-19 amongst 3.5 million people in Scotland. Vaccinated people with severe obesity (BMI&gt;40 kg/m\n                  <jats:sup>2</jats:sup>\n                  ) were significantly more likely to experience hospitalization or death from Covid-19. Excess risk increased with time since vaccination. To investigate the underlying mechanisms, we conducted a prospective longitudinal study of the immune response in a clinical cohort of vaccinated people with severe obesity. Compared with normal weight people, six months after their second vaccine dose, significantly more people with severe obesity had unquantifiable titres of neutralizing antibody against authentic SARS-CoV-2 virus, reduced frequencies of antigen-experienced SARS-CoV-2 Spike-binding B cells, and a dissociation between anti-Spike antibody levels and neutralizing capacity. Neutralizing capacity was restored by a third dose of vaccine, but again declined more rapidly in people with severe obesity. We demonstrate that waning of SARS-CoV-2 vaccine-induced humoral immunity is accelerated in people with severe obesity and associated with increased hospitalization and mortality from breakthrough infections. Given the prevalence of obesity, our findings have significant implications for global public health.\n                </jats:p>","version":null,"doi":"10.1101/2022.06.09.22276196","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.14.495413","pub_date":"2022-6-14","title":"Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior protective humoral immune response to SARS-CoV-2","abstract":"Immunization with vesicular stomatitis virus (VSV)-vectored COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein in place of the VSV glycoprotein relies implicitly on expression of the ACE2 receptor at the muscular injection site. Here, we report that such a viral vector vaccine did not induce protective immunity following intramuscular immunization of K18-hACE2 transgenic mice. However, when the viral vector was trans-complemented with the VSV glycoprotein, intramuscular immunization resulted in high titers of spike-specific neutralizing antibodies. The vaccinated animals were fully protected following infection with a lethal dose of SARS-CoV-2-SD614G via the nasal route, and partially protected if challenged with the SARS-CoV-2Delta variant. While dissemination of the challenge virus to the brain was completely inhibited, replication in the lung with consequent lung pathology was not entirely controlled. Thus, intramuscular immunization was clearly enhanced by trans-complementation of the VSV-vectored vaccines by the VSV glycoprotein and led to protection from COVID-19, although not achieving sterilizing immunity.","version":"1.1","doi":"10.1101/2022.06.14.495413","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.14.496062","pub_date":"2022-6-14","title":"HIV skews the SARS-CoV-2 B cell response toward an extrafollicular maturation pathway","abstract":"HIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. We compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing and regulatory features. This revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal center (GC) activity, homing capacity and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated na\u00efve B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2 specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. Despite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge. This work was supported by a grant from the Wellcome Trust to the Africa Health Research Institute (Wellcome Trust Strategic Core Award [grant number 201433/Z/16/Z]). Additional funding was received from the South African Department of Science and Innovation through the National Research Foundation (South African Research Chairs Initiative, [grant number 64809]), and the Victor Daitz Foundation.","version":"1.1","doi":"10.1101/2022.06.14.496062","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.09.495472","pub_date":"2022-6-14","title":"SARS-CoV-2 requires acidic pH to infect cells","abstract":"SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity. Infection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement. Detailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells","version":"1.2","doi":"10.1101/2022.06.09.495472","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.12.495816","pub_date":"2022-6-14","title":"Two ligand-binding sites on SARS-CoV-2 non-structural protein 1 revealed by fragment-based x-ray screening","abstract":"The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 through suppressing its replication and or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.95 \u00c5). Binding affinities of hits against nsp1 were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal sift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medially relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.","version":"1.2","doi":"10.1101/2022.06.12.495816","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.14.496021","pub_date":"2022-6-14","title":"Protective efficacy of COVAXIN\u00ae against Delta and Omicron variants in hamster model","abstract":"The immunity acquired after natural infection or vaccinations against SARS-CoV-2 tend to wane with time. Vaccine effectiveness also varies with the variant of infection. Here, we compared the protective efficacy of COVAXIN\u00ae following 2 and 3 dose immunizations against the Delta variant and also studied the efficacy of COVAXIN\u00ae against Omicron variants in a Syrian hamster model. The antibody response, clinical observations, viral load reduction and lung disease severity after virus challenge were studied. Protective response in terms of the reduction in lung viral load and lung lesions were observed in both the 2 dose as well as 3 doses COVAXIN\u00ae immunized group when compared to placebo group following the Delta variant challenge. In spite of the comparable neutralizing antibody response against the homologous vaccine strain in both the 2 dose and 3 dose immunized groups, considerable reduction in the lung disease severity was observed in the 3 dose immunized group post Delta variant challenge indicating the involvement of cell mediated immune response also in protection. In the vaccine efficacy study against the Omicron variants i.e., BA.1 and BA.2, lesser virus shedding, lung viral load and lung disease severity were observed in the immunized groups in comparison to the placebo groups. The present study shows that administration of COVAXIN\u00ae booster dose will enhance the vaccine effectiveness against the Delta variant infection and give protection against the Omicron variants BA.1.1 and BA.2.","version":"1.1","doi":"10.1101/2022.06.14.496021","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.13.495792","pub_date":"2022-6-14","title":"Antigenic escape accelerated by the presence of immunocompromised hosts","abstract":"The repeated emergence of SARS-CoV-2 escape mutants from host immunity has obstructed the containment of the current pandemic and poses a serious threat to humanity. Prolonged infection in immunocompromised patients has received increasing attention as a driver of immune escape, and accumulating evidence suggests that viral genomic diversity and emergence of immune-escape mutants are promoted in immunocompromised patients. However, because immunocompromised patients comprise a small proportion of the host population, whether they have a significant impact on antigenic evolution at the population level is unknown. We used an evolutionary epidemiological model combining antigenic evolution and epidemiological dynamics in host populations with heterogeneity in immune competency to determine the impact of immunocompromised patients on the pathogen evolutionary dynamics of antigenic escape from host immunity. We derived analytical formulae of the speed of antigenic evolution in heterogeneous host populations and found that even a small number of immunocompromised hosts in the population significantly accelerates antigenic evolution. Our results demonstrate that immunocompromised hosts play a key role in viral adaptation at the population level and emphasize the importance of critical care and surveillance of immunocompromised hosts.","version":"1.1","doi":"10.1101/2022.06.13.495792","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.30.489997","pub_date":"2022-6-14","title":"BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection","abstract":"SARS-CoV-2 Omicron sublineages BA.2.12.1, BA.4 and BA.5 exhibit higher transmissibility over BA.2. The new variants\u2019 receptor binding and immune evasion capability require immediate investigation. Here, coupled with Spike structural comparisons, we show that BA.2.12.1 and BA.4/BA.5 exhibit comparable ACE2-binding affinities to BA.2. Importantly, BA.2.12.1 and BA.4/BA.5 display stronger neutralization evasion than BA.2 against the plasma from 3-dose vaccination and, most strikingly, from post-vaccination BA.1 infections. To delineate the underlying antibody evasion mechanism, we determined the escaping mutation profiles, epitope distribution and Omicron neutralization efficacy of 1640 RBD-directed neutralizing antibodies (NAbs), including 614 isolated from BA.1 convalescents. Interestingly, post-vaccination BA.1 infection mainly recalls wildtype-induced humoral memory. The resulting elicited antibodies could neutralize both wildtype and BA.1 and are enriched on non-ACE2-competing epitopes. However, most of these cross-reactive NAbs are heavily escaped by L452Q, L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1; nevertheless, these NAbs are largely escaped by BA.2/BA.4/BA.5 due to D405N and F486V, and react weakly to pre-Omicron variants, exhibiting poor neutralization breadths. As for therapeutic NAbs, Bebtelovimab and Cilgavimab can effectively neutralize BA.2.12.1 and BA.4/BA.5, while the S371F, D405N and R408S mutations would undermine most broad sarbecovirus NAbs. Together, our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection, suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants.","version":"1.2","doi":"10.1101/2022.04.30.489997","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.12.495779","pub_date":"2022-6-13","title":"An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants","abstract":"As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA. 1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.","version":"1.1","doi":"10.1101/2022.06.12.495779","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.12.495856","pub_date":"2022-6-13","title":"SARS2Mutant: SARS-CoV-2 Amino-Acid Mutation Atlas Database","abstract":"The coronavirus disease 19 (COVID-19) is a highly pathogenic viral infection of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the global pandemic of 2020.A lack of therapeutic and preventive approaches including drugs and vaccines, has quickly posed significant threats to world health. A comprehensive understanding of the evolution and natural selection of SARS-CoV-2 against the host interaction and symptoms at the phenotype level could impact the candidate\u2019s strategies for the fight against this virus. SARS-CoV-2 Mutation (SARS2Mutant, http://sars2mutant.com/) is a database thatprovides comprehensive analysis results based on tens of thousands of high-coverage and high-quality SARS-CoV-2 complete protein sequences. The structure of this database is designed to allow the users to search for the three different strategies among amino acid substitution mutations based on gene name, geographical zone or comparative analysis. Based on each strategy, five data types are available to the user: mutated sample frequencies, heat map of the mutated amino acid positions, timeline trend for mutation survivals and natural selections, and charts of changed amino acids and their frequencies. Due to the increase of virus protein sequence samples published daily showing the latest trends of current results, all sequences in the database are reanalyzed and updated monthly. The SARS-2Mutant database providescurrent analysis and updated data of mutation patterns and conserved regions, helpful in developing and designing targeted vaccines, primers and drug discoveries.","version":"1.1","doi":"10.1101/2022.06.12.495856","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.12.495841","pub_date":"2022-6-13","title":"Structure of SARS-CoV-2 M protein in lipid nanodiscs","abstract":"SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 \u00c5 resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.","version":"1.1","doi":"10.1101/2022.06.12.495841","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.13.491759","pub_date":"2022-6-13","title":"In situ architecture and membrane fusion of SARS-CoV-2 Delta variant","abstract":"Among the current five Variants of Concern, infections caused by the SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, architecture of intact Delta virions remains veiled. Moreover, molecular evidences for the detailed mechanism of S-mediated membrane fusion are missing. Here we reported the in situ structure and distribution of S on the authentic Delta variant, and discovered invagination in the distinctive Delta architecture. We also captured fusion snapshots from the virus-virus fusion events, provided structural evidences for Delta\u2019s attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S over that of the Wuhan-Hu-1 S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant. Cryo-ET of intact SARS-CoV-2 Delta variant revealed its distinctive architecture and captured snapshots of its membrane fusion in action.","version":"1.2","doi":"10.1101/2022.05.13.491759","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.13.495895","pub_date":"2022-6-13","title":"Dynamics of SARS-CoV-2 genetic mutations and their information entropy","abstract":"We report an investigation of the mutations dynamics of the SARS-CoV-2 virus using Shannon\u2019s information theory. Our study includes seventeen RNA genetic sequences collected at different geographic locations and timeframes ranging from Dec. 2019 to Oct. 2021. The data shows a previously unobserved relationship between the information entropy of genomes and their mutation dynamics. The information entropy of the mutated variants decreases linearly with the number of genetic mutations with a negative slope of 1.52 \u00d7 10-5 bits / mutations, pointing to a possible deterministic approach to the dynamics of genetic mutations. The method proposed here could be used to develop a predictive algorithm of genetic mutations.","version":"1.1","doi":"10.1101/2022.06.13.495895","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.30.474519","pub_date":"2022-6-13","title":"SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1\u03b1/XBP1 ER stress pathway in human lung-derived cells","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available, or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the master UPR sensor IRE1\u03b1 kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1\u03b1-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1\u03b1 as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1\u03b1 through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1\u03b1 was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1\u03b1, perhaps as a strategy to eliminate detection by the host immune system. SARS-CoV-2 is the third lethal respiratory coronavirus after MERS-CoV and SARS-CoV to emerge this century, causing millions of deaths world-wide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1\u03b1 (IRE1\u03b1) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1\u03b1 kinase and RNase activities, SARS-CoV-2 only partially activates IRE1\u03b1, promoting its kinase activity but not RNase activity. Based on IRE1\u03b1-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1\u03b1 RNase activation as a strategy to limit detection by the host immune system.","version":"1.2","doi":"10.1101/2021.12.30.474519","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.13.495912","pub_date":"2022-6-13","title":"Accurate and Fast Clade Assignment via Deep Learning and Frequency Chaos Game Representation","abstract":"Since the beginning of the COVID-19 pandemic there has been an explosion of sequencing of the SARS-CoV-2 virus, making it the most widely sequenced virus in the history. Several databases and tools have been created to keep track of genome sequences and variants of the virus, most notably the GISAID platform hosts millions of complete genome sequences, and it is continuously expanding every day. A challenging task is the development of fast and accurate tools that are able to distinguish between the different SARS-CoV-2 variants and assign them to a clade. In this paper, we leverage the Frequency Chaos Game Representation (FCGR) and Convolutional Neural Networks (CNNs) to develop an original method that learns how to classify genome sequences that we implement into CouGaR-g, a tool for the clade assignment problem on SARS-CoV-2 sequences. On a testing subset of the GISAID, CouGaR-g achieves an 96.29% overall accuracy, while a similar tool, Covidex, obtained a 77, 12% overall accuracy. As far as we know, our method is the first using Deep Learning and FCGR for intra-species classification. Furthermore, by using some feature importance methods CouGaR-g allows to identify k-mers that matches SARS-CoV-2 marker variants. By combining FCGR and CNNs, we develop a method that achieves a better accuracy than Covidex (which is based on Random Forest) for clade assignment of SARS-CoV-2 genome sequences, also thanks to our training on a much larger dataset, with comparable running times. Our method implemented in CouGaR-g is able to detect k-mers that capture relevant biological information that distinguishes the clades, known as marker variants. The trained models can be tested online providing a FASTA file (with one or multiple sequences) at https://huggingface.co/spaces/BIASLab/sars-cov-2-classification-fcgr. CouGaR-g is also available at https://github.com/AlgoLab/CouGaR-g under the GPL.","version":"1.1","doi":"10.1101/2022.06.13.495912","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.10.495677","pub_date":"2022-6-12","title":"The Fc-effector function of COVID-19 convalescent plasma contributes to SARS-CoV-2 treatment efficacy in mice","abstract":"COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, specific CCP characteristics that promote SARS-CoV-2 control in recipients are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing and high Fc-effector activity, in contrast to those with poor Fc-function, afford effective prophylaxis and therapy in K18-hACE2 mice lethally challenged with SARS-CoV-2-nLuc. Macrophages and neutrophils significantly contributed to CCP effects during therapy but to a reduced extent under prophylaxis. Both IgG and Ig(M+A) were required during therapy, but the IgG fraction alone was sufficient during prophylaxis. Finally, despite neutralizing poorly, SARS-CoV-2 Wuhan-elicited CCPs delayed Delta and Beta variants of concern (VOC)-induced mortality in mice illustrating the contribution of polyclonal Fc-effector functions in immunity against VOCs. Thus, in addition to neutralization, Fc-effector activity is a significant criterion for CCP selection for therapeutic applications.","version":"1.1","doi":"10.1101/2022.06.10.495677","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.10.22276252","pub_date":"2022-06-12","title":"Ivermectin for Treatment of Mild-to-Moderate COVID-19 in the Outpatient Setting: A Decentralized, Placebo-controlled, Randomized, Platform Clinical Trial","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The effectiveness of ivermectin to shorten symptom duration or prevent hospitalization among outpatients in the United States with mild-to-moderate symptomatic coronavirus disease 2019 (COVID-19) is unknown.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>We evaluated the efficacy of ivermectin 400 \u00b5g/kg daily for 3 days compared with placebo for the treatment of early mild-to-moderate COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>ACTIV-6 is an ongoing, decentralized, double-blind, randomized, placebo-controlled platform trial to evaluate repurposed therapies in outpatients with mild-to-moderate COVID-19. Non-hospitalized adults age \u226530 years with confirmed COVID-19, experiencing \u22652 symptoms of acute infection for \u22647 days were randomized to receive ivermectin 400 \u00b5g/kg daily for 3 days or placebo. The main outcome measure was time to sustained recovery, defined as achieving at least 3 consecutive days without symptoms. Secondary outcomes included a composite of hospitalization or death by day 28.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Of the 3457 participants who consented to be evaluated for inclusion in the ivermectin arm, 1591 were eligible for this study arm, randomized to receive ivermectin 400 \u00b5g/kg (n=817) or placebo (n=774), and received study drug. Of those enrolled, 47% reported receiving at least 2 doses of SARS-CoV-2 vaccination. The posterior probability for any improvement in time to recovery was 0.91 (hazard ratio 1.07, 95% credible interval 0.96\u20131.17). The posterior probability of this benefit exceeding 24 hours was less than 0.01, as measured by the difference in mean time unwell. Hospitalizations or deaths were uncommon (ivermectin [n=10]; placebo [n=9]). Ivermectin at 400 \u00b5g/kg was safe and without serious adverse events as compared with placebo (ivermectin [n=10]; placebo [n=9]).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Ivermectin dosed at 400 \u00b5g/kg daily for 3 days resulted in less than one day of shortening of symptoms and did not lower incidence of hospitalization or death among outpatients with COVID-19 in the United States during the delta and omicron variant time periods.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Trial registration</jats:title>\n                  <jats:p>\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    Identifier:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04885530'>NCT04885530</jats:ext-link>\n                    .\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.06.10.22276252","journal":"medRxiv","score":null},{"id":"10.1101/2022.06.09.495586","pub_date":"2022-6-11","title":"SARS-CoV-2 type I Interferon modulation by nonstructural proteins 1 and 2","abstract":"Since the beginning of the COVID-19 pandemic, enormous efforts were devoted to understanding how SARS-CoV-2 escapes the antiviral response. Yet, modulation of type I interferons (IFNs) by this virus is not completely understood. Using in vitro and in vivo approaches, we have characterized the type I IFN response during SARS-CoV-2 infection as well as immune evasion mechanisms. The transcriptional and translational expression of IFNs, cytokines and chemokines were measured in lung homogenates of Wuhan-like, Beta, and Delta SARS-CoV-2 K18-ACE2 transgenic mice. Using in vitro experiments, we measured SARS-CoV-2 and its non-structural proteins 1 and 2 (Nsp1-2) to modulate expression of IFN\u03b2 and interferon-stimulated genes (ISG). Our data show that infection of mice with Wuhan-like virus induces robust expression of Ifna and Ifnb1 mRNA and limited type I production. In contrast, Beta and Delta variant infected mice failed to activate and produce IFN\u03b1. Using in vitro systems, Ifn\u03b2 gene translation inhibition was observed using an Nsp1 expression vector. Conversely, SARS-CoV-2 and its variants induce robust expression of NF-\u03baB-driven genes such as those encoding CCL2 ans CXCL10 chemokines. We also identified Nsp2 as an activator of NF-\u03baB that partially counteracts the inhibitory actions of Nsp1. In summary, our work indicates that SARS-CoV-2 skews the antiviral response in favor of an NF-\u03baB-driven inflammatory response, a hallmark of acute COVID-19, and that Nsp2 is partly responsible for this effect. Several studies suggest that SARS-CoV-2 possess multiple mechanisms aimed shunting the type I interferon response. However, few studies have studied type I IFN modulation in the context of infection. Our work indicates that mice and human cells infected with SARS-CoV-2 produce sufficient type I IFN to activate an antiviral response, despite Nsp1 translational blockade of IFN\u03921 mRNA. In contrast to Wuhan-like virus, Beta and Delta variants failed to induce Ifna gene expression. Our work also showcases the importance of studying protein functions in the context of infection, as demonstrated by the partial antagonizing properties of the Nsp2 protein on the activities of Nsp1. Our studies also highlight that the innate immune response triggered by SARS-CoV-2 is chiefly driven by NF-\u03baB responsive genes for which Nsp2 is partially responsible.","version":"1.1","doi":"10.1101/2022.06.09.495586","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.10.495727","pub_date":"2022-6-11","title":"Hybrid immunity shifts the Fc-effector quality of SARS-CoV-2 mRNA vaccine-induced immunity","abstract":"Despite the robust immunogenicity of SARS-CoV-2 mRNA vaccines, emerging data reveal enhanced neutralizing antibody and T cell cross-reactivity among individuals that previously experienced COVID-19, pointing to a hybrid immune advantage with infection-associated immune priming. Beyond neutralizing antibodies and T cell immunity, mounting data point to a potential role for additional antibody effector functions, including opsinophagocytic activity, in the resolution of symptomatic COVID-19. Whether hybrid immunity modifies the Fc-effector profile of the mRNA vaccine-induced immune response remains incompletely understood. Thus, here we profiled the SARS-CoV-2 specific humoral immune response in a group of individuals with and without prior COVID-19. As expected, hybrid Spike-specific antibody titers were enhanced following the primary dose of the mRNA vaccine, but were similar to those achieved by na\u00efve vaccinees after the second mRNA vaccine dose. Conversely, Spike-specific vaccine-induced Fc-receptor binding antibody levels were higher after the primary immunization in individuals with prior COVID-19, and remained higher following the second dose compared to na\u00efve individuals, suggestive of a selective improvement in the quality, rather than the quantity, of the hybrid humoral immune response. Thus, while the magnitude of antibody titers alone may suggest that any two antigen exposures \u2013 either hybrid immunity or two doses of vaccine alone - represent a comparable prime/boost immunologic education, we find that hybrid immunity offers a qualitatively improved antibody response able to better leverage Fc effector functions against conserved regions of the virus.","version":"1.1","doi":"10.1101/2022.06.10.495727","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495149","pub_date":"2022-6-10","title":"SARS-CoV-2 spike protein induces TLR-4-mediated long-term cognitive dysfunction recapitulating post-COVID syndrome","abstract":"Cognitive dysfunction is often reported in post-COVID patients, but its underlying mechanisms remain unknown. While some evidence indicate that SARS-CoV-2 can reach and directly impact the brain, others suggest viral neuroinvasion as a rare event. Independently of brain viral infection, the ability of SARS-CoV-2 spike (S) protein to cross the BBB and reach memory-related brain regions has already been shown. Here, we demonstrate that brain infusion of S protein in mice induces late cognitive impairment and increases serum levels of neurofilament light chain (NFL), which recapitulates post-COVID features. Neuroinflammation, hippocampal microgliosis and synapse loss are induced by S protein. Increased engulfment of hippocampal presynaptic terminals late after S protein brain infusion were found to temporally correlate with cognitive deficit in mice. Blockage of TLR4 signaling prevented S-associated detrimental effects on synapse and memory loss. In a cohort of 86 patients recovered from mild COVID-19, genotype GG TLR4 -2604G>A (rs10759931) was associated with poor cognitive outcome. Collectively, these findings indicate that S protein directly impacts the brain and suggest that TLR4 is a potential target to prevent post-COVID cognitive dysfunction. TLR4 mediates long-term cognitive impairment in mice and its genetic variant increases the risk of poor cognitive outcome in post-COVID patients.","version":"1.2","doi":"10.1101/2022.06.07.495149","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.07.490748","pub_date":"2022-6-10","title":"Inferring selection effects in SARS-CoV-2 with Bayesian Viral Allele Selection","abstract":"The global effort to sequence millions of SARS-CoV-2 genomes has provided an unprecedented view of viral evolution. Characterizing how selection acts on SARS-CoV-2 is critical to developing effective, long-lasting vaccines and other treatments, but the scale and complexity of genomic surveillance data make rigorous analysis challenging. To meet this challenge, we develop Bayesian Viral Allele Selection (BVAS), a principled and scalable probabilistic method for inferring the genetic determinants of differential viral fitness and the relative growth rates of viral lineages, including newly emergent lineages. After demonstrating the accuracy and efficacy of our method through simulation, we apply BVAS to 6.9 million SARS-CoV-2 genomes. We identify numerous mutations that increase fitness, including previously identified mutations in the SARS-CoV-2 Spike and Nucleocapsid proteins, as well as mutations in non-structural proteins whose contribution to fitness is less well characterized. In addition, we extend our baseline model to identify mutations whose fitness exhibits strong dependence on vaccination status as well as pairwise interaction effects, i.e. epistasis. Strikingly, both these analyses point to the pivotal role played by the N501 residue in the Spike protein. Our method, which couples Bayesian variable selection with a diffusion approximation in allele frequency space, lays a foundation for identifying fitness-associated mutations under the assumption that most alleles are neutral.","version":"1.2","doi":"10.1101/2022.05.07.490748","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.493843","pub_date":"2022-6-10","title":"Anti-COVID-19 Activity of FDA Approved Drugs through RNA G-quadruplex Binding","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ, PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ/PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against fast mutating viruses like SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.05.31.493843","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.10.495670","pub_date":"2022-6-10","title":"SARS-CoV-2 minor variant genomes at the start of the pandemic contained markers of VoCs","abstract":"SARS-CoV-2 emerged through limited zoonotic spillovers and was predicted to have constrained sequence diversity. The dominant consensus and minor variant genomes were determined from the earliest samples associated with the Huanan market and the start of the pandemic. The sequence data confirmed that the dominant consensus genomes shared very close homology. However, there were minor variant genomes present in each sample, which encompassed synonymous and non-synonymous changes. Fusion sequences characteristic of defective RNAs were identified that could be linked between patients. Several substitutions (but not deletions) associated with much later variants of concern (VoCs) were already present as minor variant genomes. This suggests it may be possible to predict futures variants at the start of a pandemic by examining where variability in sequence occurs.","version":"1.1","doi":"10.1101/2022.06.10.495670","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.09.495422","pub_date":"2022-6-09","title":"SARS-CoV-2-neutralizing humoral IgA response occurs earlier but modest and diminishes faster compared to IgG response","abstract":"Secretory immunoglobulin A (IgA) plays a crucial role in the mucosal immunity for preventing the invasion of the exogenous antigens, however, little has been understood about the neutralizing activity of serum IgA. Here, to examine the role of IgA antibodies against COVID-19 illnesses, we determined the neutralizing activity of serum/plasma IgG and IgA purified from previously SARS-CoV-2-infected and COVID-19 mRNA-vaccine-receiving individuals. We found that serum/plasma IgA possesses substantial but rather modest neutralizing activity against SARS-CoV-2 compared to IgG with no significant correlation with the disease severity. Neutralizing IgA and IgG antibodies achieved the greatest activity at approximately 25 and 35 days after symptom onset, respectively. However, neutralizing IgA activity quickly diminished and went down below the detection limit approximately 70 days after onset, while substantial IgG activity was observed till 200 days after onset. The total neutralizing activity in sera/plasmas of those with COVID-19 largely correlated with that in purified-IgG and purified-IgA and levels of anti-SARS-CoV-2-S1-binding IgG and anti-SARS-CoV-2-S1-binding IgA. In individuals who were previously infected with SARS-CoV-2 but had no detectable neutralizing IgA activity, a single dose of BNT162b2 or mRNA-1273 elicited potent serum/plasma neutralizing IgA activity but the second dose did not further strengthen the neutralization antibody response. The present data show that the systemic immune stimulation with natural infection and COVID-19 mRNA-vaccines elicit both SARS-CoV-2-specific neutralizing IgG and IgA response in serum, but the IgA response is modest and diminishes faster compared to IgG response. Immunoglobulin A (IgA) is the most abundant type of antibody in the body mostly located on mucosal surfaces as a dimeric secretory IgA. Such secretory IgA plays an important role in preventing the adherence and invasions of foreign objects by its neutralizing activity, while monomeric serum IgA is thought to relate to the phagocytic immune system activation. Here, we report that individuals with the novel coronavirus disease (COVID-19) developed both systemic neutralizing IgG and IgA active against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the neutralizing IgA response was quick and reached the highest activity 25 days post-symptom-onset, compared to 35 days for IgG response, neutralizing IgA activity was modest and diminished faster than neutralizing IgG response. In individuals, who recovered from COVID-19 but had no detectable neutralizing IgA activity, a single dose of COVID-19 mRNA-vaccine elicited potent neutralizing IgA activity but the second dose did not further strengthen the antibody response. Our study provides novel insights into the role and the kinetics of serum IgA against the viral pathogen both in naturally-infected and COVID-19 mRNA-vaccine-receiving COVID-19-convalescent individuals.","version":"1.1","doi":"10.1101/2022.06.09.495422","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.09.495433","pub_date":"2022-6-09","title":"A cellular assay for spike/ACE2 fusion: quantification of fusion-inhibitory antibodies after COVID-19 and vaccination","abstract":"Not all antibodies against SARS-CoV-2 inhibit viral entry and hence infection. Neutralizing antibodies are more likely to reflect real immunity, however certain of these tests investigate protein/protein interaction rather than the fusion event. Viral and pseudoviral entry assays detect functionally active antibodies, however they are cumbersome and burdened by biosafety and standardization issues. We have developed a Spike/ACE2-dependant cell-to-cell fusion assay, based on a split luciferase. Hela cells stably transduced with Spike and a large fragment of luciferase were co-cultured with Hela cells transduced with ACE2 and the complementary small fragment of luciferase. Within 24h, cell fusion occured allowing the measurement of luminescence. Light emission was abolished in the absence of Spike and reduced in the presence of an inhibitor of Spike-processing proteases. Serum samples from COVID-19-negative, non-vaccinated individuals, or sera from patients at the moment of first symptoms did not lead to a significant reduction of fusion. In contrast, sera from COVID-19-positive patients as well as sera from vaccinated individuals reduced the fusion. In conclusion, we report a new method measuring fusion-inhibitory antibodies in serum, combining the advantage of a functional full Spike/ACE2 interaction with a high degree of standardization, easily allowing automation in a standard bio-safety environment.","version":"1.1","doi":"10.1101/2022.06.09.495433","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495215","pub_date":"2022-6-08","title":"Efficacy of Parainfluenza Virus 5 (PIV5)-vectored Intranasal COVID-19 Vaccine as a Single Dose Vaccine and as a Booster against SARS-CoV-2 Variants","abstract":"Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has greatly reduced coronavirus disease 2019 (COVID-19)-related deaths and hospitalizations, but waning immunity and the emergence of variants capable of immune escape indicate the need for novel SARS-CoV-2 vaccines. An intranasal parainfluenza virus 5 (PIV5)-vectored COVID-19 vaccine CVXGA1 has been proven efficacious in animal models and blocks contact transmission of SARS-CoV-2 in ferrets. CVXGA1 vaccine is currently in human clinical trials in the United States. This work investigates the immunogenicity and efficacy of CVXGA1 and other PIV5-vectored vaccines expressing additional antigen SARS-CoV-2 nucleoprotein (N) or SARS-CoV-2 variant spike (S) proteins of beta, delta, gamma, and omicron variants against homologous and heterologous challenges in hamsters. A single intranasal dose of CVXGA1 induces neutralizing antibodies against SARS-CoV-2 WA1 (ancestral), delta variant, and omicron variant and protects against both homologous and heterologous virus challenges. Compared to mRNA COVID-19 vaccine, neutralizing antibody titers induced by CVXGA1 were well-maintained over time. When administered as a boost following two doses of a mRNA COVID-19 vaccine, PIV5-vectored vaccines expressing the S protein from WA1 (CVXGA1), delta, or omicron variants generate higher levels of cross-reactive neutralizing antibodies compared to three doses of a mRNA vaccine. In addition to the S protein, the N protein provides added protection as assessed by the highest body weight gain post-challenge infection. Our data indicates that PIV5-vectored COVID-19 vaccines, such as CVXGA1, can serve as booster vaccines against emerging variants. With emerging new variants of concern (VOC), SARS-CoV 2 continues to be a major threat to human health. Approved COVID-19 vaccines have been less effective against these emerging VOCs. This work demonstrates the protective efficacy, and strong boosting effect, of a new intranasal viral-vectored vaccine against SARS-CoV-2 variants in hamsters.","version":"1.1","doi":"10.1101/2022.06.07.495215","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.493653","pub_date":"2022-6-08","title":"COVID-MVP: an interactive visualization for tracking SARS-CoV-2 mutations, variants, and prevalence, enabled by curated functional annotations and portable genomics workflow","abstract":"The SARS-CoV-2 pandemic has reemphasized the importance of genomic epidemiology to track the evolution of the virus, dynamics of epidemics, geographic origins, and the emerging variants. It is vital in understanding the epidemiological spread of the virus on global, national, and local scales. Several analytical (bioinformatics) resources have been developed for molecular surveillance. However, a resource that combines genetic mutations and functional annotations on the impact of these mutations has been lacking in SARS-CoV-2 genomics surveillance. COVID-MVP provides an interactive visualization application that summarizes the mutations and their prevalence in SARS-CoV-2 viral lineages and provides functional annotations from the literature curated in an ongoing effort, Pokay. COVID-MVP is a tool that can be used for routine surveillance including spatio-temporal analyses. We have powered the visualization through a scalable and reproducible genomic analysis workflow nf-ncov-voc wrapped in Nextflow. COVID-MVP allows users to interactively explore data and download summarized surveillance reports. COVID-MVP, Pokay, and nf-ncov-voc are open-source tools available under the Massachusetts Institute of Technology (MIT) and GPL-3.0 licenses. COVID-MVP source code is available at https://github.com/cidgoh/COVID-MVP and an instance is hosted at https://covidmvp.cidgoh.ca.","version":"1.1","doi":"10.1101/2022.06.07.493653","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.05.494889","pub_date":"2022-6-08","title":"Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by 3 doses of BNT162b2 vaccine or BA.1 infection","abstract":"Distinct SARS-CoV-2 Omicron sublineages have evolved showing increased fitness and immune evasion than the original Omicron variant BA.1. Here we report the neutralization activity of sera from BNT162b2 vaccinated individuals or unimmunized Omicron BA.1-infected individuals against Omicron sublineages and \u201cDeltacron\u201d variant (XD). BNT162b2 post-dose 3 immune sera neutralized USA-WA1/2020, Omicron BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and XD-spike SARS-CoV-2s with geometric mean titers (GMTs) of 1335, 393, 298, 315, 216, 103, and 301, respectively; thus, BA.4/5 SARS-CoV-2 spike variant showed the highest propensity to evade vaccine neutralization compared to the original Omicron variants BA.1. BA.1-convalescent sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron-spike SARS-CoV-2s with GMTs of 15, 430, 110, 109, 102, 25, and 284, respectively. The low neutralization titers of vaccinated sera or convalescent sera from BA. 1 infected individuals against the emerging and rapidly spreading Omicron BA.4/5 variants provide important results for consideration in the selection of an updated vaccine in the current Omicron wave.","version":"1.2","doi":"10.1101/2022.06.05.494889","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487660","pub_date":"2022-6-08","title":"A potent synthetic nanobody with broad-spectrum activity neutralizes SARS-Cov-2 virus and Omicron variant through a unique binding mode","abstract":"The major challenge to control COVID pandemic is the rapid mutation rate of the SARS-Cov-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-Cov-2 variants. Here, we reported a synthetic nanobody (named C5G2) obtianed by phage display and subsequent antibody engineering. C5G2 has a single digit nanomolar binding affinity to RBD domain and inhibits its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assay indicated that the monovalent C5G2 could protect the cells from the infection of SARS-Cov-2 wild type virus and most of the virus of concern, i.e. Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron among all the variants with the IC50 of 4.9ng/mL. The Cryo-EM structure of C5G2 in complex with the Spike trimer showed that C5G2 bind to RBD mainly through its CDR3 at a conserved region that not overlapping with the ACE2 binding surface. Additionally, C5G2 bind simultaneously to the neighboring NTD domain of spike trimer through the same CDR3 loop, which may further increase its potency against the virus infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may be served as an effective drug for the prophylaxis and therapy against Omicron as well as future variants.","version":"1.2","doi":"10.1101/2022.04.11.487660","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.06.494921","pub_date":"2022-6-07","title":"Nirmatrelvir Resistant SARS-CoV-2 Variants with High Fitness in Vitro","abstract":"The oral protease inhibitor nirmatrelvir is expected to play a pivotal role for prevention of severe cases of coronavirus disease 2019 (COVID-19). To facilitate monitoring of potentially emerging resistance, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir. Resistant variants selected in cell culture harbored different combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetic studies in a homologous infectious cell culture system revealed up to 80-fold resistance conferred by the combination of substitutions L50F and E166V. Resistant variants had high fitness increasing the likelihood of occurrence and spread of resistance. Molecular dynamics simulations revealed that E166V and L50F+E166V weakened nirmatrelvir-Mpro binding. The SARS-CoV-2 polymerase inhibitor remdesivir retained activity against nirmatrelvir resistant variants and combination of remdesivir and nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatment programs with high efficacy against SARS-CoV-2 and potentially emerging coronaviruses.","version":"1.1","doi":"10.1101/2022.06.06.494921","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495065","pub_date":"2022-6-07","title":"Development of an Escape-resistant SARS CoV-2 Neutralizing Synthetic Nanobody","abstract":"An emerging COVID-19 pandemic resulted in a global crisis, but also accelerated vaccine development and antibody discovery. In this work, we identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD), by screening synthetic humanized antibody library with more than 1011 diversity. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12\u00d73-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, their affinities are weakly influenced by SARS-CoV-2 VoC mutations. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\\ml and 9.6 ng\\ml for H7-Fc and G12\u00d73-Fc antibodies respectively for emerging Omicron variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.06.07.495065","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.494579","pub_date":"2022-6-07","title":"GABA-receptors are a new druggable target for limiting disease severity, lung viral load, and death in SARS-CoV-2 infected mice","abstract":"GABA-receptors (GABA-Rs) are well-known neurotransmitter receptors in the central nervous system. GABA-Rs are also expressed by immune cells and lung epithelial cells and GABA-R agonists/potentiators reduce inflammatory immune cell activities and limit acute lung injuries. Notably, plasma GABA levels are reduced in hospitalized COVID-19 patients. Hence, GABA-R agonists may have therapeutic potential for treating COVID-19. Here, we show that oral GABA treatment initiated just after SARS-CoV-2 infection, or 2 days later near the peak of lung viral load, reduced disease severity, lung coefficient index, and death rates in K18-hACE2 mice. GABA-treated mice had a reduced viral load in their lungs and displayed shifts in their serum cytokine and chemokine levels that are associated with better outcomes in COVID-19 patients. Thus, GABA-R activation had multiple beneficial effects in this mouse model which are also desirable for the treatment of COVID-19. A number of GABA-R agonists are safe for human use and can be readily tested in clinical trials with COVID-19 patients. We also discuss their potential for limiting COVID-19-associated neuroinflammation.","version":"1.1","doi":"10.1101/2022.06.07.494579","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495142","pub_date":"2022-6-07","title":"Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks","abstract":"Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.","version":"1.1","doi":"10.1101/2022.06.07.495142","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.07.495101","pub_date":"2022-6-07","title":"Predicting clinical outcomes of SARS-CoV-2 drug treatments with a high throughput human airway on chip platform","abstract":"Despite the relatively common observation of therapeutic efficacy in discovery screens with immortalized cell lines, the vast majority of drug candidates do not reach clinical development. Candidates that do move forward often fail to demonstrate efficacy when progressed from animal models to humans. This dilemma highlights the need for new drug screening technologies that can parse drug candidates early in development with regard to predicted relevance for clinical use. PREDICT96-ALI is a high-throughput organ-on-chip platform incorporating human primary airway epithelial cells in a dynamic tissue microenvironment. Here we demonstrate the utility of PREDICT96-ALI as an antiviral screening tool for SARS-CoV-2, combining the high-throughput functionality of a 96-well plate format in a high containment laboratory with the relevant biology of primary human tissue. PREDICT96-ALI resolved differential efficacy in five antiviral compounds over a range of drug doses. Complementary viral genome quantification and immunofluorescence microscopy readouts achieved high repeatability between devices and replicate plates. Importantly, results from testing the three antiviral drugs currently available to patients (nirmatrelvir, molnupiravir, and remdesivir) tracked with clinical outcomes, demonstrating the value of this technology as a prognostic drug discovery tool.","version":"1.1","doi":"10.1101/2022.06.07.495101","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.05.494897","pub_date":"2022-6-06","title":"Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for SARS-CoV-2 3CL Protease","abstract":"The pandemic of coronavirus disease 2019 (COVID-19) has urgently necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report the new class of covalent inhibitors for 3CLpro possessing chlorofluoroacetamide (CFA) as a cysteine reactive warhead. Based on the aza-peptide scaffold, we synthesized the series of CFA derivatives in enantiopure form and evaluated their biochemical efficiencies. The data revealed that 8a (YH-6) with R configuration at the CFA unit strongly blocks the SARS-CoV-2 replication in the infected cells and this potency is comparable to that of nirmatrelvir. The X-ray structural analysis shows that 8a (YH-6) forms a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and sufficient pharmacokinetics property of 8a (YH-6) suggest its potential as a lead compound for treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.06.05.494897","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.06.494965","pub_date":"2022-6-06","title":"Genomic Surveillance of SARS CoV2 in COVID-19 vaccinated healthcare workers in Lebanon","abstract":"The emergence of SARS-CoV-2 variants including the Delta and Omicron along with waning of vaccine-induced immunity over time contributed to increased rates of breakthrough infection specifically among healthcare workers (HCWs). SARS-CoV-2 genomic surveillance is an important tool for timely detection and characterization of circulating variants as well as monitoring the emergence of new strains. Our study is the first national SARS-CoV-2 genomic surveillance among HCWs in Lebanon. We collected 250 samples from five hospitals across Lebanon between December 2021 and January 2022. We extracted viral RNA and performed whole genome sequencing using the Illumina NextSeq 500 platform. A total of 133 (57.1%) samples belonging to the Omicron (BA.1.1) sub-lineage were identified, as well as 44 (18.9%) samples belonging to the BA.1 sub-lineage, 28 (12%) belonging to the BA.2 sub-lineage, and only 15 (6.6%) samples belonging to the Delta variant sub-lineage B.1.617.2. These results show that Lebanon followed the global trend in terms of circulating SARS-CoV-2 variants with Delta rapidly replaced by the Omicron variant. This study underscores the importance of continuous genomic surveillance programs in Lebanon for the timely detection and characterization of circulating variants. The latter is critical to guide public health policy making and to timely implement public health interventions.","version":"1.1","doi":"10.1101/2022.06.06.494965","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.09.455472","pub_date":"2022-6-06","title":"Neutrophilic inflammation promotes SARS-CoV-2 infectivity and augments the inflammatory responses in airway epithelial cells","abstract":"In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co- morbidities correlating to incidence of severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS- CoV-2 infection. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. SARS-CoV-2 infection of the airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented pro-inflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory response is polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity in response to SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2021.08.09.455472","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.06.491344","pub_date":"2022-6-06","title":"The T cell epitope landscape of SARS-CoV-2 variants of concern","abstract":"During the COVID-19 pandemic, several SARS-CoV-2 variants of concern (VOC) emerged, bringing with them varying degrees of health and socioeconomic burdens. In particular, the Omicron VOC displayed distinct features of increased transmissibility accompanied by anti-genic drift in the spike protein that partially circumvented the ability of pre-existing anti-body responses in the global population to neutralize the virus. However, T cell immunity has remained robust throughout all the different VOC transmission waves and has emerged as a critically important correlate of protection against SARS-CoV-2 and it\u2019s VOCs, in both vaccinated and infected individuals. Therefore, as SARS-CoV-2 VOCs continue to evolve, it is crucial that we characterize the correlates of protection and the potential for immune escape for both B cell and T cell human immunity in the population. Generating the insights necessary to understand T cell immunity, experimentally, for the global human population is at present critical but a time consuming, expensive, and laborious process. Further, it is not feasible to generate global or universal insights into T cell immunity in an actionable time frame for potential future emerging VOCs. However, using computational means we can expedite and provide early insights into the correlates of T cell protection. In this study, we generated and reveal insights on the T cell epitope landscape for the five main SARS-CoV-2 VOCs observed to date. We demonstrated here using a unique AI prediction platform, a strong concordance in global T cell protection across all mutated peptides for each VOC. This was modeled using the most frequent HLA alleles in the human population and covers the most common HLA haplotypes in the human population. The AI resource generated through this computational study and associated insights may guide the development of T cell vaccines and diagnostics that are even more robust against current and future VOCs, and their emerging subvariants.","version":"1.1","doi":"10.1101/2022.06.06.491344","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.06.494969","pub_date":"2022-6-06","title":"The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm","abstract":"The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1\u03b2, IL-6, IL-12p70, TNF-\u03b1, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases.","version":"1.1","doi":"10.1101/2022.06.06.494969","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456305","pub_date":"2022-6-06","title":"ImputeCoVNet: 2D ResNet Autoencoder for Imputation of SARS-CoV-2 Sequences","abstract":"We describe a new deep learning approach for the imputation of SARS-CoV-2 variants. Our model, ImputeCoVNet, consists of a 2D ResNet Autoencoder that aims at imputing missing genetic variants in SARS-CoV-2 sequences in an efficient manner. We show that ImputeCoVNet leads to accurate results at minor allele frequencies as low as 0.0001. When compared with an approach based on Hamming distance, ImputeCoVNet achieved comparable results with significantly less computation time. We also present the provision of geographical metadata (e.g., exposed country) to decoder increases the imputation accuracy. Additionally, by visualizing the embedding results of SARS-CoV-2 variants, we show that the trained encoder of ImputeCoVNet, or the embedded results from it, recapitulates viral clade\u2019s information, which means it could be used for predictive tasks using virus sequence analysis.","version":"1.3","doi":"10.1101/2021.08.13.456305","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.06.494494","pub_date":"2022-6-06","title":"SARS-CoV-2 S protein antagonizes type I interferon downstream signal pathway through interacting and attenuating phosphorylation of STAT1/STAT2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may keep patients in a clinically asymptomatic state by blocking cellular innate antiviral immunity, but the molecular mechanism remains unclear. Here, we screened the viral proteins of SARS-CoV-2 and found that the spike (S) protein inhibits the activation of interferon-stimulated genes (ISGs) and even reduces the expression of these genes to below background values. Mechanistically, the S protein interacted with STAT1, STAT2, and IRF9 and impedes the phosphorylation of STAT1/STAT2, thus preventing the formation of the interferon-stimulating gene factor 3 (ISGF3) complex and inhibiting the downstream production of Interferon-stimulated genes (ISGs). Remarkably, we also have found that the inhibitory mechanism of the S protein was conservative among SARS-CoV-2 variants and other human coronaviruses, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Truncation studies indicated that the most conserved S2 domain played a major inhibitory role. Altogether, our findings unveil a new mechanism by which SARS-CoV-2 S protein attenuated the host\u2019s antiviral immune response and provide new insights into the pathogenic mechanism of coronavirus.","version":"1.1","doi":"10.1101/2022.06.06.494494","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.20.492819","pub_date":"2022-6-06","title":"Revealing druggable cryptic pockets in the Nsp-1 of SARS-CoV-2 and other \u03b2-coronaviruses by simulations and crystallography","abstract":"Non-structural protein 1 (Nsp1) is a main pathogenicity factor of \u03b1- and \u03b2-coronaviruses. Nsp1 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suppresses the host gene expression by sterically blocking 40S host ribosomal subunits and promoting host mRNA degradation. This mechanism leads to the downregulation of the translation-mediated innate immune response in host cells, ultimately mediating the observed immune evasion capabilities of SARS-CoV-2. Here, by combining extensive Molecular Dynamics simulations, fragment screening and crystallography, we reveal druggable pockets in Nsp1. Structural and computational solvent mapping analyses indicate the partial crypticity of these newly discovered and druggable binding sites. The results of fragment-based screening via X-ray crystallography confirm the druggability of the major pocket of Nsp1. Finally, we show how the targeting of this pocket could disrupt the Nsp1-mRNA complex and open a novel avenue to design new inhibitors for other Nsp1s present in homologous \u03b2-coronaviruses.","version":"1.3","doi":"10.1101/2022.05.20.492819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.05.493249","pub_date":"2022-6-06","title":"Pathogen-Host Adhesion between SARS-CoV-2 Spike Proteins from Different Variants and Human ACE2 Probed at Single-Molecule and Single-Cell Levels","abstract":"Pathogen-Host adhesion is considered the first step of infection for many pathogens such as bacteria and virus. The binding of the receptor binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) onto human angiotensin-converting enzyme 2 (ACE2) is considered as the first step for the SARS-CoV-2 to adhere onto the host cells during the infection. Within three years, a number of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been found all around the world. Here, we investigated the adhesion of S Proteins from different variants and ACE2 using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and single-cell force spectroscopy (SCFS). We found that the unbinding force and binding probability of the S protein from Delta variant to the ACE2 was the highest among the variants tested in our study at both single-molecule and single-cell levels. Molecular dynamics simulation showed that ACE2-RBD (Omicron) complex is destabilized by the E484A and Y505H mutations and stabilized by S477N and N501Y mutations, when compared with Delta variant. In addition, a neutralizing antibody, produced by immunization with wild type RBD of S protein, could effectively inhibit the binding of S proteins from wild type, Delta and Omicron variants onto ACE2. Our results provide new insight for the molecular mechanism of the adhesive interactions between S protein and ACE2 and suggest that effective monoclonal antibody can be prepared using wild type S protein against the different variants.","version":"1.1","doi":"10.1101/2022.06.05.493249","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.05.494856","pub_date":"2022-6-06","title":"A uniquely stable trimeric model of SARS-CoV-2 spike transmembrane domain","abstract":"The spike (S) protein of SARS-CoV-2 effectuates membrane fusion and virus entry into target cells. Its transmembrane domain (TMD) represents a homotrimer of \u03b1-helices anchoring the spike in the viral envelope. Although S-protein models available to date include the TMD, its precise configuration was given brief consideration. Understanding viral fusion entails realistic TMD models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD (S-TMD) based solely on its primary structure. First, we performed amino acid sequence pattern matching and compared molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and thus selected the TMD of the tumour necrosis factor receptor 1 (TNFR-1) for subsequent template-based modelling. We then iteratively built an all-atom homotrimer model of S-TMD based on \u201cdynamic MHP portraits\u201d and residue variability motifs. In this model each helix possessed two overlapping interfaces interacting with either of the remaining helices, which include conservative residues I1216, F1220, I1227, M1229, and M1233. Finally, the stability of this and several alternative models (including a recent NMR structure) and a set of mutant forms was tested in all-atom molecular dynamics (MD) simulations in a POPC bilayer mimicking the viral envelope membrane. Unlike other configurations, our model trimer remained extraordinarily tightly packed over a microsecond-range MD and retained its stability when palmitoylated in accordance with experimental data. Palmitoylation had no significant impact on the TMD conformation nor the way in which the lipid bilayer was perturbed in the presence of the trimer. Overall, the resulting model of S-TMD conforms to known basic principles of TM helix packing and will be further used to explore the complex machinery of membrane fusion from a broader perspective beyond the TMD.","version":"1.1","doi":"10.1101/2022.06.05.494856","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.05.494906","pub_date":"2022-6-06","title":"Implications of Spike Protein Interactions with Zn-bound form of ACE2: A Computational Structural Study","abstract":"The COVID-19 pandemic has generated a major interest in designing inhibitors to prevent SARS-CoV-2 binding on host cells to protect against infection. One promising approach to such research utilizes molecular dynamics (MD) to identify potential inhibitors that can prevent the interaction between spike (S) protein on the virus and angiotensin converting enzyme 2 (ACE2) receptor on the host cells. In these studies, many groups have chosen to exclude a zinc (Zn) ion bound to the ACE2 molecule which is critical for enzymatic activity. While the relatively distant location of Zn ion from the S protein binding site (S1 domain), combined with the difficulties in modeling this ion have motivated the decision of exclusion, Zn can potentially contribute to the structural stability of the entire protein, and thus, may have implications on spike protein interaction. In this study, we explored the effects of excluding Zn on the structural stability and binding free energy of the ACE2-S1 protein complex. We generated two versions of an experimentally-derived structure of the ACE2-S1 protein complex: one with Zn and one without. Examining the differences between these two complexes during MD simulation, we found that the Zn-bound complex exhibited greater instability at nearly all residues except for the interacting residues, which were more stable in the Zn-bound complex. Additionally, the Zn-bound complex had a stronger binding free energy at all internal dielectric constants greater than one. Since binding free energy is often used to score inhibitors\u2019 performances, excluding Zn could potentially have implications on inhibitor selection and performance, both in the ACE2-S1 protein system and other protein complexes that include the Zn ion.","version":"1.1","doi":"10.1101/2022.06.05.494906","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.03.494640","pub_date":"2022-6-03","title":"Discovery of host-directed modulators of virus infection by probing the SARS-CoV-2-host protein-protein interaction network","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based algorithm that expands the SARS-CoV-2-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19. We experimentally tested seven chemical inhibitors of the identified host proteins for modulation of SARS-CoV-2 infection in human cells that express ACE2 and TMPRSS2. Inhibition of the epigenetic regulators bromodomain-containing protein 4 (BRD4) and histone deacetylase 2 (HDAC2), along with ubiquitin specific peptidase (USP10), enhanced SARS-CoV-2 infection. Such proviral effect was observed upon treatment with compounds JQ1, vorinostat, romidepsin, and spautin-1, when measured by cytopathic effect and validated by viral RNA assays, suggesting that HDAC2, BRD4 and USP10 host proteins have antiviral functions. Mycophenolic acid and merimepodib, two inhibitors of inosine monophosphate dehydrogenase (IMPDH 1 and IMPDH 2), showed modest antiviral effects with no toxicity in mock-infected control cells. The network-based approach enables systematic identification of host-targets that selectively modulate the SARS-CoV-2 interactome, as well as reveal novel chemical tools to probe virus-host interactions that regulate virus infection. Viruses exploit host machinery and therefore it is important to understand the virus-host dependencies to gain better insight of the key regulators of viral infection. Using a context-specific SARS-COV-2 PPI network, a computational framework was developed to identify host modulators of viral infection. Chromatin modifying host proteins HDAC2 and BRD4, along with deubiquitinating enzyme USP10, act as antiviral proteins. IMPDH inhibitors mycophenolic acid and merimipodib showed modest antiviral response to SARS-COV-2 infection, and no toxic effects. Cell context specificity is a critical factor when identifying selective modulators of viral infection and potential antiviral therapeutics. Topology-based network models cannot distinguish between host-proteins, the inhibition of which leads to either virus suppressive or enhancing effects.","version":"1.1","doi":"10.1101/2022.06.03.494640","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.02.493651","pub_date":"2022-6-03","title":"Distinct antibody responses to endemic coronaviruses pre- and post-SARS-CoV-2 infection in Kenyan infants and mothers","abstract":"Pre-existing antibodies that bind endemic human coronaviruses (eHCoVs) can cross-react with SARS-CoV-2, the betacoronavirus that causes COVID-19, but whether these responses influence SARS-CoV-2 infection is still under investigation and is particularly understudied in infants. In this study, we measured eHCoV and SARS-CoV-1 IgG antibody titers before and after SARS-CoV-2 seroconversion in a cohort of Kenyan women and their infants. Pre-existing eHCoV antibody binding titers were not consistently associated with SARS-CoV-2 seroconversion in infants or mothers, though we observed a very modest association between pre-existing HCoV-229E antibody levels and lack of SARS-CoV-2 seroconversion in infants. After seroconversion to SARS-CoV-2, antibody binding titers to endemic betacoronaviruses HCoV-OC43 and HCoV-HKU1, and the highly pathogenic betacoronavirus SARS-CoV-1, but not endemic alphacoronaviruses HCoV-229E and HCoV-NL63, increased in mothers. However, eHCoV antibody levels did not increase following SARS-CoV-2 seroconversion in infants, suggesting the increase seen in mothers was not simply due to cross-reactivity to naively generated SARS-CoV-2 antibodies. In contrast, the levels of antibodies that could bind SARS-CoV-1 increased after SARS-CoV-2 seroconversion in both mothers and infants, both of whom are unlikely to have had a prior SARS-CoV-1 infection, supporting prior findings that SARS-CoV-2 responses cross-react with SARS-CoV-1. In summary, we find evidence for increased eHCoV antibody levels following SARS-CoV-2 seroconversion in mothers but not infants, suggesting eHCoV responses can be boosted by SARS-CoV-2 infection when a prior memory response has been established, and that pre-existing cross-reactive antibodies are not strongly associated with SARS-CoV-2 infection risk in mothers or infants.","version":"1.1","doi":"10.1101/2022.06.02.493651","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.03.494642","pub_date":"2022-6-03","title":"Single-cell RNA-sequencing data analysis reveals a highly correlated triphasic transcriptional response to SARS-CoV-2 infection","abstract":"Single-cells RNA sequencing (scRNA-seq) is currently one of the most powerful techniques available to study the transcriptional response of cells to external perturbations. However, the use of conventional bulked RNA-seq analysis methods can miss important patterns underlying in the scRNA-seq data. Here, we present a reanalysis of scRNA-seq data from human bronchial epithelial cells and colon and ileum organoids using pseudo-time profiles based on the degree of virus accumulation which reflect the progress of infection. Our analysis revealed a transcriptional response to infection characterized by three distinct up- and down-regulatory phases, that cannot be detected using classical two-group comparisons. Interrogation of results, focused on genes involved in interferon-response, transcription factors and RNA-binding proteins, suggests a highly correlated transcriptional response for most genes. In addition, correlation network analysis revealed a distinct response of genes involved in translation and mitochondrially-encoded genes. Based on our data, we propose a model where modulation of nucleocytoplasmic traffic by the viral protein nsp1 explains the triphasic transcriptional response to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.06.03.494642","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.01.494451","pub_date":"2022-6-03","title":"A Big Data COVID-19 literature pattern discovery using NLP","abstract":"As our collective knowledge about COVID-19 continues to grow at an exponential rate, it becomes more difficult to organize and observe emerging trends. In this work, we built an open source methodology that uses topic modeling and a pretrained BERT model to organize large corpora of COVID-19 publications into topics over time and over location. Additionally, it assesses the association of medical keywords against COVID-19 over time. These analyses are then automatically pushed into an open source web application that allows a user to obtain actionable insights from across the globe.","version":"1.1","doi":"10.1101/2022.06.01.494451","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.03.494698","pub_date":"2022-6-03","title":"Atomic-scale Quantum Chemical Calculation of Omicron Mutations Near Cleavage Sites of the Spike Protein","abstract":"The attachment of the Spike-protein in SARS-CoV-2 to host cells and the initiation of viral invasion are two critical processes in the viral infection and transmission processes in which the presence of unique furin (S1/S2) and TMPRSS2 (S2\u2019) cleavage sites play a pivotal role. In this study, we provide detailed analysis of the impact of the BA.1 Omicron variant mutations, vicinal to these two cleavage sites using a novel computational method based on Amino acid \u2013 amino acid bond pair unit (AABPU), a specific protein structural unit in 3D as a proxy for quantifying the atomic interaction. We have identified several key features related to the electronic structure as well as bonding of the Omicron mutations near the cleavage sites that significantly increase the size of the relevant AABPUs and the fraction of the positive partial charge. These results of the ultra-large-scale quantum calculations enable us to conjecture on the biological role of Omicron mutations and their specific effects on cleavage sites, as well as identify the principles that can be of some value in analyzing other new variants or subvariants.","version":"1.1","doi":"10.1101/2022.06.03.494698","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.01.494373","pub_date":"2022-6-02","title":"Selection for immune evasion in SARS-CoV-2 revealed by high-resolution epitope mapping combined with genome sequence analysis","abstract":"A deeper understanding of the molecular determinants that drive humoral responses to coronaviruses, and in particular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is critical for improving and developing diagnostics, therapies and vaccines. Moreover, viral mutations can change key antigens in a manner that alters the ability of the immune system to detect and clear infections. In this study, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses of asymptomatic or recovered COVID-19-positive patients relative to COVID-19-negative patients. We made use of a novel high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses to rapidly identify B cell epitopes recognized by distinct antibody isotypes in patients\u2019 blood sera. Using our integrated computational pipeline, we then evaluated the fine immunological properties of detected SARS-CoV-2 epitopes and relate them to their evolutionary and structural properties. While some epitopes are common across all CoVs, others are private to specific hCoVs. We also highlight the existence of hotspots of pre-existing immunity and identify a subset of cross-reactive epitopes that contributes to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves and SARS-CoV-2 variants, which have important implications for genomic surveillance and vaccine design. Lastly, we show that mutations in Spike and Nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts.","version":"1.1","doi":"10.1101/2022.06.01.494373","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.02.494502","pub_date":"2022-6-02","title":"The Nucleocapsid Protein Of SARS-CoV-2, Combined With ODN-39M, Is A Potential Component For An Intranasal Bivalent Pancorona Vaccine","abstract":"Despite the rapid development of vaccines and their reported efficacy for controlling the COVID-19 waves, two key challenges remain: the scope of the immunity against upcoming variants and zoonosis events, and the induction of mucosal immunity able to clear the virus in the upper respiratory tract for halting the transmission. The present study is aiming at assessing a potential component for a new generation of vaccines so as to overcome such limitations. The recombinant nucleocapsid (N) protein from SARS-CoV-2 Delta variant was combined with a phosphodiester backbone CpG ODN (ODN-39M), forming high molecular weight aggregates. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive Cell-Mediated-Immunity (CMI). In turn, this combination was able to induce anti-N IgA in lungs, which along with the specific IgG in sera and CMI in spleen, resulted cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N+ODN-39M preparation combined with the RBD Delta protein, as inductor of neutralizing Abs, enhanced the local and systemic immune response against RBD with a modulation toward a Th1 pattern. Taken together, these results make the N+ODN-39M preparation a suitable component for a future intranasal pancorona vaccine against Sarbecoviruses. Particularly, the bivalent vaccine formulation N+ODN-39M+RBD could be used as an effective nasal booster in previously vaccinated population.","version":"1.1","doi":"10.1101/2022.06.02.494502","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.01.494461","pub_date":"2022-6-02","title":"A Multitrait Locus Regulates Sarbecovirus Pathogenesis","abstract":"Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.","version":"1.1","doi":"10.1101/2022.06.01.494461","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.01.494385","pub_date":"2022-6-02","title":"Characterization of entry pathways, species-specific ACE2 residues determining entry, and antibody neutralization evasion of Omicron BA.1, BA.1.1, BA.2, and BA.3 variants","abstract":"The SARS-CoV-2 Omicron variants were first detected in November 2021, and several Omicron lineages (BA.1, BA.2, BA.3, BA.4, and BA.5) have since rapidly emerged. Studies characterizing the mechanisms of Omicron variant infection and sensitivity to neutralizing antibodies induced upon vaccination are ongoing by several groups. In the present study, we used pseudoviruses to show that the transmembrane serine protease 2 (TMPRSS2) enhances infection of BA.1, BA.1.1, BA.2, and BA.3 Omicron variants to lesser extent compared to ancestral D614G. We further show that Omicron variants have higher sensitivity to inhibition by soluble angiotensin converting enzyme 2 (ACE2) and the endosomal inhibitor chloroquine compared to D614G. The Omicron variants also more efficiently used ACE2 receptors from nine out of ten animal species tested, and unlike the D614G variant, used mouse ACE2 due to the Q493R and Q498R spike substitutions. Finally, neutralization of the Omicron variants by antibodies induced by three doses of Pfizer/BNT162b2 mRNA vaccine was 7-8-fold less potent than the D614G, and the Omicron variants still evade neutralization more efficiently.","version":"1.1","doi":"10.1101/2022.06.01.494385","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.26.461873","pub_date":"2022-6-01","title":"SARS- CoV-2 viroporins: A multi-omics insight from nucleotides to amino acids","abstract":"COVID-19 is caused by SARS-CoV-2 which has so far affected more than 500 million people worldwide and killed over 6 million as of 1st May, 2022. The approved emergency-use vaccines were lifesaving to such a devastating pandemic. Viroporins are important players of the life cycle of SARS-CoV-2 and are primary to its pathogenesis. We studied the two prominent viroporins of SARS-CoV-2 (i) Orf3a and (ii) Envelope (E) protein from a sequential and structural point of view. Orf3a is a cation selective viral ion channel which has been shown to disrupt the endosomal pathways. E protein is one of the most conserved proteins among the SARS-CoV proteome which affects the ERGIC related pathways. The aqueous medium through the viroporins mediates the non-selective translocation of cations, affecting ionic homeostasis in the host cellular compartments. This ionic imbalance could potentially lead to increased inflammatory response in the host cell. Our results shed light into the mechanism of viroporin action, which can be potentially leveraged for the development of antiviral therapeutics. Our results corroborate with previously published transcriptomic data from COVID-19 infected lung alveolar cells where inflammatory responses and molecular regulators directly impacted by ion channelling were upregulated.","version":"1.2","doi":"10.1101/2021.09.26.461873","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.494211","pub_date":"2022-6-01","title":"SARS-CoV-2 Variant Spike and accessory gene mutations alter pathogenesis","abstract":"The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against SARS-CoV-2, the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the Spike protein. Although these differences in Spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome which may affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis, we synthesized viruses where the WA1 Spike is replaced by each variant spike genes in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to the full variant viruses. Our work has revealed that non-spike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host and can lead to attenuating phenotypes in circulating variants of concern. This work suggests that while Spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may lead to less clinical disease, extended time toward knowing an infection exists in a person and thus increased time for transmission to occur. A hallmark of the COVID19 pandemic has been the emergence of SARS-CoV-2 variants that have increased transmission and immune evasion. Each variant has a set of mutations that can be tracked by sequencing but little is known about their affect on pathogenesis. In this work we first identify accessory genes that are responsible for pathogenesis in vivo as well as identify the role of variant spike genes on replication and disease in mice. Isolating the role of Spike mutations in variants identifies the non-Spike mutations as key drivers of disease for each variant leading to the hypothesis that viral fitness depends on balancing increased Spike binding and immuno-evasion with attenuating phenotypes in other genes in the SARS-CoV-2 genome.","version":"1.1","doi":"10.1101/2022.05.31.494211","journal":"bioRxiv","score":null},{"id":"10.1101/2022.06.01.494101","pub_date":"2022-6-01","title":"Antiviral immune responses, cellular metabolism and adhesion are differentially modulated by SARS-CoV-2 ORF7a or ORF7b","abstract":"SARS-CoV-2, the causative agent of the present COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome, and some have been implicated in facilitating infection and pathogenesis through their interaction with cellular components. Among these proteins, accessory protein ORF7a and ORF7b functions are poorly understood. In this study, A549 cells were transduced to express ORF7a and ORF7b, respectively, to explore more in depth the role of each accessory protein in the pathological manifestation leading to COVID-19. Bioinformatic analysis and integration of transcriptome results identified defined canonical pathways and functional groupings revealing that after expression of ORF7a or ORF7b, the lung cells are potentially altered to create conditions more favorable for SARS-CoV-2, by inhibiting the IFN-I response, increasing proinflammatory cytokines release, and altering cell metabolic activity and adhesion. Based on these results, it is reasonable to suggest that ORF7a and ORF7b could be targeted by new therapies or used as future biomarkers during this pandemic.","version":"1.1","doi":"10.1101/2022.06.01.494101","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.494262","pub_date":"2022-6-01","title":"SUMOylation of SARS-CoV-2 Nucleocapsid protein enhances its interaction affinity and plays a critical role for its nuclear translocation","abstract":"Viruses, such as SARS-CoV-2, infect hosts and take advantages of host cellular machinery for their genome replication and new virion production. Identification and elucidation of host pathways for viral infection are critical for understanding the viral life cycle and novel therapeutics development. SARS-CoV-2 N protein is critical for viral RNA(vRNA) genome packaging in new virion formation, Here, we report that identification of SUMOylation sites of SARS-CoV-2 N protein and role of SUMO modification in N protein interaction affinity with itself using our qFRET/MS coupled method. We found, for the first time, that the SUMO modification of N protein can significantly increase its interaction affinity with itself and may support its oligomer formation. One of the identified Lys residues, K65 was critical for N protein translocation to nucleus, where the vRNA replication and packaging take place. The in vitro assessment of the affinity of N protein to N protein with SUMO mutants provides insight of the oligomerized N protein formation after SUMO modification. These results suggest that the host human SUMOylation pathway may be very critical for N protein functions in viral replication. The host SUMOylation pathway may be a critical host factor for the SARS-CoV-2 virus life cycle. Identification and inhibition of critical host SUMOyaltion could provide a novel strategy for future anti-viral therapeutics development, such as SARS-CoV-2 and other viruses. The SARS-CoV-2 virus N protein plays a critical role critical for viral RNA(vRNA) genome packaging in host cell nucleus for new virion formation. Therefore, deciphering the molecular mechanisms modulating N activity could be a strategy to identify potential targets amenable to therapeutics. Here, we identify a comprehensive SUMOylation sites of N proteins using an in vitro reconstitute SUMOyaltion assay containing SUMO E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. We find that SUMOylation modification of N protein can significantly enhance it interaction affinity with itself, indicating an increased oligomerization capability, which is critical for N protein activity for vRNA genome packaging. In addition, we find one of SUMOylation sites of N protein is critical for its nucleus translocation, which is a critical for viral genome packaging. The SUMOylation modification may represent novel potential approach to design new antivirals with the ability to modulate SARS-CoV-2 virus replication.","version":"1.1","doi":"10.1101/2022.05.31.494262","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493682","pub_date":"2022-6-01","title":"Structural basis of a two-antibody cocktail exhibiting highly potent and broadly neutralizing activities against SARS-CoV-2 variants including diverse Omicron sublineages","abstract":"SARS-CoV-2 variants of concern (VOCs), especially the latest Omicron, have exhibited severe antibody evasion. Broadly neutralizing antibodies with high potency against Omicron are urgently needed for understanding working mechanisms and developing therapeutic agents. In this study, we characterized previously reported F61, which was isolated from convalescent patients infected with prototype SARS-CoV-2, as a broadly neutralizing antibody against all VOCs including Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 sublineages by utilizing antigen binding and cell infection assays. We also identified and characterized another broadly neutralizing antibody D2 with epitope distinct from that of F61. More importantly, we showed that a combination of F61 with D2 exhibited synergy in neutralization and protecting mice from SARS-CoV-2 Delta and Omicron BA.1 variants. Cryo-EM structures of the spike-F61 and spike-D2 binary complexes revealed the distinct epitopes of F61 and D2 at atomic level and the structural basis for neutralization. Cryo-EM structure of the Omicron-spike-F61-D2 ternary complex provides further structural insights into the synergy between F61 and D2. These results collectively indicated F61 and F61-D2 cocktail as promising therapeutic antibodies for combating SARS-CoV-2 variants including diverse Omicron sublineages.","version":"1.3","doi":"10.1101/2022.05.27.493682","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.494147","pub_date":"2022-6-01","title":"Plasmacytoid dendritic cells regulate megakaryocyte and platelet homeostasis","abstract":"Platelet homeostasis is essential for vascular integrity and immune defense. While the process of platelet formation by fragmenting megakaryocytes (thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of megakaryocytes by their progenitor cells (megakaryopoiesis) remains unclear. Here we use intravital 2 photon microscopy to track individual megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as crucial bone marrow niche cells that regulate megakaryopoiesis. pDCs monitor the bone marrow for platelet-producing megakaryocytes and deliver IFN-\u03b1 to the megakaryocytic niche to trigger local on-demand proliferation of megakaryocyte progenitors. This fine-tuned coordination between thrombopoiesis and megakaryopoiesis is crucial for megakaryocyte and platelet homeostasis in steady state and stress. However, uncontrolled pDC function within the megakaryocytic niche is detrimental. Accordingly, we show that pDCs activated by SARS-CoV2 drive inappropriate megakaryopoiesis associated with thrombotic complications. Together, we uncover a hitherto unknown megakaryocytic bone marrow niche maintained by the constitutive delivery of pDC-derived IFN-\u03b1.","version":"1.1","doi":"10.1101/2022.05.31.494147","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.30.494036","pub_date":"2022-5-31","title":"SARS-CoV-2 Helicase might interfere with cellular nonsense-mediated RNA decay, insights from a bioinformatics study","abstract":"Unraveling molecular interactions between viral proteins and host cells is key to understanding the pathogenesis of viral diseases. We hypothesized that potential sequence and structural similarities between SARS-CoV2 proteins and proteins of infected cells might influence host cell biology and antiviral defense. Comparing the proteins of SARS-CoV-2 with human and mammalian proteins revealed sequence and structural similarities between viral helicase with human UPF1. The latter is a protein that is involved in nonsense mediated RNA decay (NMD), an mRNA surveillance pathway which also acts as a cellular defense mechanism against viruses. Protein sequence similarities were also observed between viral nsp3 and human Poly ADP-ribose polymerase (PARP) family of proteins. Gene set enrichment analysis on transcriptomic data derived from SARS-CoV-2 positive samples illustrated the enrichment of genes belonging to the NMD pathway compared with control samples. Moreover, comparing transcriptomic data from SARS-CoV2-infected samples with transcriptomic data derived from UPF1 knockout cells demonstrated a significant overlap between datasets. These findings suggest that helicase/UPF1 sequence and structural similarity might have the ability to interfere with the NMD pathway with pathogenic and immunological implications.","version":"1.1","doi":"10.1101/2022.05.30.494036","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.494153","pub_date":"2022-5-31","title":"SARS-CoV-2 impacts the transcriptome and epigenome at the maternal-fetal interface in pregnancy","abstract":"During pregnancy, the maternal-fetal interface plays vital roles in fetal development. Its disruption is frequently found in pregnancy complications. Recent works show increased incidences of adverse pregnancy outcomes in COVID-19 patients; however, the mechanism remains unclear. Here, we analyzed the molecular impacts of SARS-CoV-2 infection on the maternal-fetal interface. Generating bulk and single-nucleus transcriptomic and epigenomic profiles from COVID-19 patients and control samples, we discovered aberrant immune activation and angiogenesis patterns in patients. Surprisingly, retrotransposons were dysregulated in specific cell types. Notably, reduced enhancer activities of LTR8B elements were functionally linked to the downregulation of Pregnancy-Specific Glycoprotein genes in syncytiotrophoblasts. Our findings revealed that SARS-CoV-2 infection induced significant changes to the epigenome and transcriptome at the maternal-fetal interface, which may be associated with pregnancy complications. Pregnant COVID-19 patients show placental epigenetic and transcriptional changes, associated with adverse pregnancy outcomes.","version":"1.1","doi":"10.1101/2022.05.31.494153","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.494162","pub_date":"2022-5-31","title":"The salivary and nasopharyngeal microbiomes are associated with SARS-CoV-2 infection and disease severity","abstract":"Oral and upper respiratory microbiota play important roles in modulating host immune responses to viral infection. As emerging evidence suggests the host microbiome may be involved in the pathophysiology of COVID-19, we aimed to investigate associations between the oral and nasopharyngeal microbiome and COVID-19 severity. We collected saliva (n = 78) and nasopharyngeal swab (n = 66) samples from a COVID-19 cohort and characterized the microbiomes using 16S ribosomal RNA gene sequencing. We also examined associations between the salivary and nasopharyngeal microbiome and age, COVID-19 symptoms, and blood cytokines. SARS-CoV-2 infection status, but not COVID-19 severity, was associated with community-level differences in the oral and nasopharyngeal microbiomes. Salivary and nasopharyngeal microbiome alpha diversity negatively correlated with age and were associated with fever and diarrhea. Several bacterial genera were differentially abundant by COVID-19 severity, including oral Bifidobacterium, Lactobacillus, and Solobacterium, all of which were depleted in patients with severe COVID-19. Nasopharyngeal Paracoccus was depleted while nasopharyngeal Proteus, Cupravidus, and Lactobacillus were increased in patients with severe COVID-19. Further analysis revealed that the abundance of oral Bifidobacterium was negatively associated with plasma concentrations of known COVID-19 biomarkers interleukin 17F (IL-17F) and monocyte chemoattractant protein-1 (MCP-1). In conclusion, our results suggest COVID-19 disease severity is associated with the relative abundance of certain bacterial taxa.","version":"1.1","doi":"10.1101/2022.05.31.494162","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.30.493765","pub_date":"2022-5-31","title":"Potent and pan-neutralization of SARS-CoV-2 variants of concern by DARPins","abstract":"We report the engineering and selection of two synthetic proteins \u2013 FSR16m and FSR22 \u2013 for possible treatment of SARS-CoV-2 infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon and exhibit broad spectrum neutralization of SARS-Cov-2 strains. The IC50 values of FSR16m against authentic B.1.351, B.1.617.2 and BA.1.1 variants are 3.4 ng/mL, 2.2 ng/mL and 7.4 ng/mL, respectively, comparable to currently used therapeutic antibodies. Despite the use of the spike protein from a now historical wild-type virus for design, FSR16m and FSR22 both exhibit increased neutralization against newly-emerged variants of concern (39- to 296-fold) in pseudovirus assays. Cryo-EM structures revealed that these DARPins recognize a region of the receptor binding domain (RBD, residues 455-456, 486-489) overlapping a critical portion of the ACE2-binding surface. K18-hACE2 transgenic mice inoculated with a B.1.617.2 variant and receiving intranasally-administered FSR16m were protected as judged by less weight loss and 10-100-fold reductions in viral burden in the upper and lower respiratory tracts. The strong and broad neutralization potency make FSR16m and FSR22 promising candidates for prevention and treatment of infection by current and potential future strains of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.05.30.493765","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.493925","pub_date":"2022-5-31","title":"Comprehensive characterization of the transcriptional response to COVID-19 in multiple organs reveals shared signatures across tissues","abstract":"Infection by Coronavirus SARS-CoV2 is a severe and often deadly disease that has implications for the respiratory system and multiple organs across the human body. While the effects in the lung have been extensively studied, less is known about COVID-19\u2019s cellular impact across other organs. Here we contribute a single-nuclei RNA sequencing atlas comprising six human organs across 20 autopsies where we analyzed the transcriptional changes due to COVID-19 in multiple cell types. Computational cross-organ analysis for endothelial cells and macrophages identified systemic transcriptional changes in these cell types in COVID-19 samples. In addition, analysis of signaling pathways from multiple datasets showed several systemic dysregulations of signaling interaction in different cell types. Altogether, the COVID Tissue Atlas enables the investigation of both cell type-specific and cross-organ transcriptional responses to COVID-19, providing insights into the molecular networks affected by the disease and highlighting novel potential targets for therapies and drug development.","version":"1.1","doi":"10.1101/2022.05.31.493925","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.06.487257","pub_date":"2022-5-31","title":"Delineating antibody escape from Omicron sublineages","abstract":"SARS-CoV-2 neutralizing antibodies play a critical role in prevention and treatment of COVID-19 but are challenged by viral evolution and antibody evasion, exemplified by the highly resistant Omicron BA.1 sublineage. Importantly, the recently identified Omicron sublineages BA.2.12.1 and BA.4/5 with differing spike mutations are rapidly emerging in various countries. By determining polyclonal serum activity of 50 convalescent or vaccinated individuals against BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4/5, we reveal a further reduction of BA.4/5 susceptibility to vaccinee sera. Most notably, delineation of the sensitivity to an extended panel of 163 antibodies demonstrates pronounced antigenic differences of individual sublineages with distinct escape patterns and increased antibody resistance of BA.4/5 compared to the most prevalent BA.2 sublineage. These results suggest that the antigenic distance from BA.1 and the increased resistance compared to BA.2 may favor immune escape-mediated expansion of BA.4/5 after the first Omicron wave. Finally, while most monoclonal antibodies in clinical stages are inactive against all Omicron sublineages, we identify promising novel antibodies with high pan-Omicron neutralizing potency. Our study provides a detailed understanding of the antibody escape from the most recently emerging Omicron sublineages that can inform on effective strategies to prevent and treat COVID-19.","version":"1.2","doi":"10.1101/2022.04.06.487257","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.31.494170","pub_date":"2022-5-31","title":"Accurate Prediction of Virus-Host Protein-Protein Interactions via a Siamese Neural Network Using Deep Protein Sequence Embeddings","abstract":"Prediction and understanding of tissue-specific virus-host interactions have relevance for the development of novel therapeutic interventions strategies. In addition, virus-like particles (VLPs) open novel opportunities to deliver therapeutic compounds to targeted cell types and tissues. Given our incomplete knowledge of virus-host interactions on one hand and the cost and time associated with experimental procedures on the other, we here propose a novel deep learning approach to predict virus-host protein-protein interactions (PPIs). Our method (Siamese Tailored deep sequence Embedding of Proteins - STEP) is based on recent deep protein sequence embedding techniques, which we integrate into a Siamese neural network architecture. After evaluating the high prediction performance of STEP in comparison to an existing method, we apply it to two use cases, SARS-CoV-2 and John Cunningham polyomavirus (JCV), to predict virus protein to human host interactions. For the SARS-CoV-2 spike protein our method predicts an interaction with the sigma 2 receptor, which has been suggested as a drug target. As a second use case, we apply STEP to predict interactions of the JCV VP1 protein showing an enrichment of PPIs with neurotransmitters, which are known to function as an entry point of the virus into glial brain cells. In both cases we demonstrate how recent techniques from the field of Explainable AI (XAI) can be employed to identify those parts of a pair of sequences, which most likely contribute to the protein-protein interaction. Altogether our work highlights the potential of deep sequence embedding techniques originating from the field of natural language processing as well as XAI methods for the analysis of biological sequences. We have made our method publicly available via GitHub. Development of novel cell and tissue specific therapies requires a profound knowledge about protein-protein interactions (PPIs). Identifying these PPIs with experimental approaches such as biochemical assays or yeast two-hybrid screens is cumbersome, costly, and at the same time difficult to scale. Computational approaches can help to prioritize huge amounts of possible PPIs by learning from biological sequences plus already-known PPIs. In this work, we developed a novel approach (Siamese Tailored deep sequence Embedding of Proteins - STEP) that is based on recent deep protein sequence embedding techniques, which we integrate into a Siamese neural network architecture. We use this approach to train models by utilizing protein sequence information and known PPIs. After evaluating the high prediction performance of STEP in comparison to an existing method, we apply it to two use cases, SARS-CoV-2 and John Cunningham polyomavirus (JCV), to predict virus protein to human host interactions. Altogether our work highlights the potential of deep sequence embedding techniques originating from the field of natural language processing as well as Explainable AI methods for the analysis of biological sequence data. A novel deep learning approach (STEP) predicts virus protein to human host protein interactions based on recent deep protein sequence embedding and a Siamese neural network architecture Prediction of protein-protein interactions of the JCV VP1 protein and of the SARS-CoV-2 spike protein Identification of parts of sequences that most likely contribute to the protein-protein interaction using Explainable AI (XAI) techniques DSML 3: Development/Pre-production: Data science output has been rolled out/validated across multiple domains/problems","version":"1.1","doi":"10.1101/2022.05.31.494170","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.29.493850","pub_date":"2022-5-30","title":"The SARS-CoV-2 accessory factor ORF7a downregulates MHC class I surface expression","abstract":"The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.","version":"1.1","doi":"10.1101/2022.05.29.493850","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493798","pub_date":"2022-5-30","title":"Monitoring for SARS-CoV-2 drug resistance mutations in broad viral populations","abstract":"The search for drugs against COVID-19 and other diseases caused by coronaviruses focuses on the most conserved and essential proteins, mainly the main (Mpro) and the papain-like (PLpro) proteases and the RNA-dependent RNA polymerase (RdRp). Nirmatrelvir, an inhibitor for Mpro, was recently approved by FDA as a part of a two-drug combination, Paxlovid, and many more drugs are in various stages of development. Multiple candidates for the PLpro inhibitors are being studied, but none have yet progressed to clinical trials. Several repurposed inhibitors of RdRp are already in use. We can expect that once anti-COVID-19 drugs become widely used, resistant variants of SARS-CoV-2 will emerge, and we already see that for the drugs targeting SARS-CoV-2 RdRp. We hypothesize that emergence of such variants can be anticipated by identifying possible escape mutations already present in the existing populations of viruses. Our group previously developed the coronavirus3D server (https://coronavirus3d.org), tracking the evolution of SARS-CoV-2 in the context of the three-dimensional structures of its proteins. Here we introduce dedicated pages tracking the emergence of potential drug resistant mutations to Mpro and PLpro, showing that such mutations are already circulating in the SARS-CoV-2 viral population. With regular updates, the drug resistance tracker provides an easy way to monitor and potentially predict the emergence of drug resistance-conferring mutations in the SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2022.05.27.493798","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.29.493866","pub_date":"2022-5-30","title":"Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge","abstract":"The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to the insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). Not only displays the pSpike/PP-sNp superior gene-transfection and favorable biocompatibility in the mouse airway, but pSpike/PP-sNp promotes a tripartite immunity consisting of systemic, cellular and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp might be a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.05.29.493866","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493767","pub_date":"2022-5-30","title":"Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro","abstract":"SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its \u223c30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein substrate. We elucidated the structural basis of order of cleavage of SARS-CoV-2 nsp7-11 polyprotein, with implications for Mpro inhibition.","version":"1.1","doi":"10.1101/2022.05.27.493767","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.29.493871","pub_date":"2022-5-30","title":"Genome similarities between human-derived and mink-derived SARS-CoV-2 make mink a potential reservoir of the virus","abstract":"The SARS-CoV-2 has RNA as the genome, which makes the virus more prone to mutations. Occasionally, mutations help a virus to cross the species barrier. The SARS-CoV-2 infection to humans and minks (Neovison vison) are examples of zoonotic spillover. Many studies have been published on the analysis of human-derived SARS-CoV-2, here we performed mutation analysis on the minks-derived SARS-CoV-2 genome sequences. We analyzed all available full-length mink derived SARS-CoV-2 genome sequences on GISAID (214 from Netherlands and 133 from Denmark). We found that the mutation pattern in the Netherlands and Denmark derived samples were different. Out of a total of 201 mutations, we found in this study, only 13 mutations were common in the Netherlands and Denmark derived samples. We found 4 mutations prevailed in the Netherlands and Denmark mink derived samples and these 4 mutations are also reported to prevail in human-derived SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.05.29.493871","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.29.493923","pub_date":"2022-5-30","title":"Potentiating the cross-reactive IFN-\u03b3 T cell and polyfunctional T cell responses by heterologous GX-19N DNA booster in mice primed with either a COVID-19 mRNA vaccine or inactivated vaccine","abstract":"Waning vaccine-induced immunity, coupled with the emergence of SARS-CoV-2 variants, has inspired the widespread implementation of COVID-19 booster vaccinations. Here, we evaluated the potentials of the GX-19N DNA vaccine as a heterologous booster to enhance the protective immune response to SARS-CoV-2 in mice primed with either an inactivated virus particle (VP) or mRNA vaccine. We found that in the VP-primed condition, GX-19N enhanced the response of both vaccine-specific antibodies and cross-reactive T-cells to the SARS-CoV-2 variant of concern (VOC) compared to the homologous VP vaccine prime-boost. Under the mRNA-primed condition, GX-19N induced higher vaccine-induced T-cell responses but lower antibody responses than the homologous mRNA vaccine prime-boost. Furthermore, heterologous GX-19N boost induced higher S-specific polyfunctional CD4+ and CD8+ T cell responses than the homologous VP or mRNA prime-boost vaccinations. Our results provide new insights into booster vaccination strategies for the management of novel COVID-19 variants.","version":"1.1","doi":"10.1101/2022.05.29.493923","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.10.463724","pub_date":"2022-5-30","title":"Recoverability of Ancestral Recombination Graph Topologies","abstract":"Recombination is a powerful evolutionary process that shapes the genetic diversity observed in the populations of many species. Reconstructing genealogies in the presence of recombination from sequencing data is a very challenging problem, as this relies on mutations having occurred on the correct lineages in order to detect the recombination and resolve the ordering of coalescence events in the local trees. We investigate the probability of reconstructing the true topology of ancestral recombination graphs (ARGs) under the coalescent with recombination and gene conversion. We explore how sample size and mutation rate affect the inherent uncertainty in reconstructed ARGs, which sheds light on the theoretical limitations of ARG reconstruction methods. We illustrate our results using estimates of evolutionary rates for several organisms; in particular, we find that for parameter values that are realistic for SARS-CoV-2, the probability of reconstructing genealogies that are close to the truth is low.","version":"1.2","doi":"10.1101/2021.10.10.463724","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.26.493537","pub_date":"2022-5-28","title":"Specific Pupylation as IDEntity Reporter (SPIDER) for the identification of Protein-Biomolecule interactions","abstract":"Protein-biomolecule interactions play pivotal roles in almost all biological processes, the identification of the interacting protein is essential. By combining a substrate-based proximity labelling activity from the pupylation pathway of Mycobacterium tuberculosis, and the streptavidin (SA)-biotin system, we developed Specific Pupylation as IDEntity Reporter (SPIDER) for identifying protein-biomolecular interactions. As a proof of principle, SPIDER was successfully applied for global identification of interacting proteins, including substrates for enzyme (CobB), the readers of m6A, the protein interactome of mRNA, and the target proteins of drug (lenalidomide). In addition, by SPIDER, we identified SARS-CoV-2 Omicron variant specific receptors on cell membrane and performed in-depth analysis for one candidate, Protein-g. These potential receptors could explain the differences between the Omicron variant and the Prototype strain, and further serve as target for combating the Omicron variant. Overall, we provide a robust technology which is applicable for a wide-range of protein-biomolecular interaction studies.","version":"1.2","doi":"10.1101/2022.05.26.493537","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493569","pub_date":"2022-5-27","title":"Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery","abstract":"Uneven worldwide vaccination coverage against SARS-CoV-2 and emergence of variants escaping immunity call for broadly-effective and easily-deployable therapeutics. We previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alfa, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes infectivity and fusogenic activity of Omicron BA.1 and BA.2 variants. Cryo-EM analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. Moreover, we demonstrate that nebulized scFv76 exhibits therapeutic efficacy in a severe hACE2 transgenic mouse model of COVID-19 pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with the inhibition of lung inflammation observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. Altogether the results support the use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants emerged so far.","version":"1.1","doi":"10.1101/2022.05.27.493569","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.24.493348","pub_date":"2022-5-27","title":"Intranasal vaccination induced cross-protective secretory IgA antibodies against SARS-CoV-2 variants with reducing the potential risk of lung eosinophilic immunopathology","abstract":"To control the coronavirus disease 2019 (COVID-19) pandemic, there is a need to develop vaccines to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. One candidate is a nasal vaccine capable of inducing secretory IgA antibodies in the mucosa of the upper respiratory tract, the initial site of infection. However, regarding the development of COVID-19 vaccines, there is concern about the potential risk of inducing lung eosinophilic immunopathology as a vaccine-associated enhanced respiratory disease as a result of the T helper 2 (Th2)-dominant adaptive immune response. In this study, we investigated the protective effect against virus infection induced by intranasal vaccination of recombinant trimeric spike protein derived from SARS-CoV-2 adjuvanted with CpG oligonucleotides, ODN2006, in mouse model. The intranasal vaccine combined with ODN2006 successfully induced not only systemic spike-specific IgG antibodies, but also secretory IgA antibodies in the nasal mucosa. Secretory IgA antibodies showed high protective ability against SARS-CoV-2 variants (Alpha, Beta and Gamma variants) compared to IgG antibodies in the serum. The nasal vaccine of this formulation induced a high number of IFN-\u03b3-secreting cells in the draining cervical lymph nodes and a lower spike-specific IgG1/IgG2a ratio compared to that of subcutaneous vaccination with alum as a typical Th2 adjuvant. These features are consistent with the induction of the Th1 adaptive immune response. In addition, mice intranasally vaccinated with ODN2006 showed less lung eosinophilic immunopathology after viral challenge than mice subcutaneously vaccinated with alum adjuvant. Our findings indicate that intranasal vaccine adjuvanted with ODN2006 could be a candidate that can prevent the infection of antigenically different variant viruses, reducing the risk of vaccine-associated enhanced respiratory disease.","version":"1.2","doi":"10.1101/2022.05.24.493348","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493693","pub_date":"2022-5-27","title":"Development of a Novel SARS-CoV-2 Immune Complex Vaccine Candidate (CRCx) with Broad Immune Responses: A Preclinical Trial in Animal Model","abstract":"The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a serious threat to global public health and imposes a severe burden on the entire human population. Faced with a virus that can mutate its structure while immunity is incapacitated, a need to develop a universal vaccine that can boost immunity to coronaviruses is highly needed. Five formulations of two types (CRCx2 and CRCx3) of immune complexes with an immunogen adjuvant were evaluated in a mouse model as candidate SARS CoV-2 vaccines in a pretrial prior to clinical trials in humans. CRCx3 comprises 3 different formulas and CRCx2 comprises 2. Balb/c mice were vaccinated intraperitoneally on days 0/7 with a high or low dose of CRCx2 or on days 0/7/14 with a high, medium, or low dose of CRCx3 series, and their blood was sampled for serum antibody measurements. Mice were challenged with live virus after immunization with either vaccine to evaluate prophylaxis ability or treated with them after challenge to evaluate therapeutic ability on day 15. Immunological markers and histopathological studies as well as titration of neutralizing antibodies to the vaccines were evaluated and analyzed. CRCx 3 and CRCx 2 vaccine candidates induced elevated levels of positive neutralizing antibodies as well as a cellular immune response with safety, efficient productivity, and good genetic stability for vaccine manufacturing to provide protection against SARS-CoV-2 with relatively higher levels with the high dose CRCx2 candidate combination. Highly efficient protection and therapeutic effect against SARS-CoV-2 were obtained with a double-dose immunization schedule spaced at 7-day intervals using injections 0.25 of or 0.40 ml of CRCx2 vaccine formulations with a 25-mm needle. These results support further evaluation of CRCx in a clinical trial on humans.","version":"1.1","doi":"10.1101/2022.05.27.493693","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.27.493400","pub_date":"2022-5-27","title":"The Glycan-Specificity of the Pineapple Lectin AcmJRL and its Carbohydrate-Dependent Binding of the SARS-CoV-2 Spike Protein","abstract":"The current SARS-CoV-2 pandemic has become one of the most challenging global health threats, with over 530 million reported infections by May 2022. In addition to vaccines, research and development have also been directed towards novel drugs. Since the highly glycosylated spike protein of SARS-CoV-2 is essential for infection, it constitutes a prime target for antiviral agents. The pineapple-derived jacalin-related lectin (AcmJRL) is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we elucidated its ligand specificity by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to recombinantly produced spike protein. We could demonstrate that AcmJRL binds the spike protein with a low micromolar KD in a carbohydrate-dependent fashion, suggesting its use as a potential SARS-CoV-2 neutralising agent.","version":"1.1","doi":"10.1101/2022.05.27.493400","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.14.464320","pub_date":"2022-5-27","title":"Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids","abstract":"While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.","version":"1.2","doi":"10.1101/2021.10.14.464320","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.25.22275603","pub_date":"2022-05-26","title":"The prevalence of SARS-CoV-2 infection and other public health outcomes during the BA.2/BA.2.12.1 surge, New York City, April-May 2022","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Routine case surveillance data for SARS-CoV-2 are incomplete, unrepresentative, missing key variables of interest, and may be increasingly unreliable for both timely surge detection and understanding the burden of infection and access to treatment.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a cross-sectional survey of a representative sample of 1,030 New York City (NYC) adult residents \u226518 years on May 7-8, 2022, when BA.2.12.1 comprised 47% of reported cases per genomic surveillance. We estimated the prevalence of SARS-CoV-2 infection during the preceding 14-day period (April 23-May 8), weighted to represent the 2020 NYC adult population. Respondents were asked about SARS-CoV-2 testing (including at-home rapid antigen tests), testing outcomes, COVID-like symptoms, and contact with SARS-CoV-2 cases. Based on responses, we classified individuals into three mutually exclusive categories of SARS-CoV-2 infection according to a hierarchical case definition as follows: confirmed (positive test with a provider), probable (positive at home rapid test), and possible (COVID-like symptoms and close contact with a confirmed/probable case). SARS-CoV-2 prevalence estimates were age- and sex-adjusted to the 2020 US population. Individuals with SARS-CoV-2 were asked about awareness/use of antiviral medications. We triangulated survey-based prevalence estimates with NYC\u2019s official SARS-CoV-2 metrics on cases, hospitalizations, and deaths, as well as SARS-CoV-2 concentrations in wastewater for the same time period.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>An estimated 22.1% (95%CI 17.9%-26.2%) of respondents had SARS-CoV-2 infection during the two-week study period, corresponding to \u223c1.5 million adults (95%CI 1.3-1.8 million). The official SARS-CoV-2 case count during the study period was 51,218. This 22.1% prevalence estimate included 11.4%, 6.5%, and 4.3% who met the confirmed, probable, and possible criteria of our case definition, respectively. Prevalence was estimated at 34.9% (95%CI 26.9%-42.8%) among individuals with co-morbidities, 14.9% (95% CI 11.0%-18.8%) among those 65+ years, and 18.9% (95%CI 10.2%-27.5%) among unvaccinated persons. Hybrid immunity (i.e., history of both vaccination and prior infection) was 66.2% (95%CI 55.7%-76.7%) among those with COVID and 46.3% (95%CI 40.2-52.2) among those without. Among individuals with COVID, 44.1% (95%CI 33.0%-55.1%) were aware of the antiviral nirmatrelvir/ritonavir (Paxlovid\u2122), and 15.1% (95%CI 7.1%-23.1%) reported receiving it. Deaths and hospitalizations increased, but remained well below the levels of the BA.1 surge. SARS-CoV-2 virus concentrations in wastewater surveillance showed only a modest signal in comparison to that of the BA.1 surge.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions and Relevance</jats:title>\n                  <jats:p>The true magnitude of NYC\u2019s BA.2/BA.2.12.1 surge may have been vastly underestimated by routine SARS-CoV-2 case counts and wastewater surveillance. Hybrid immunity, bolstered by the recent BA.1 surge, likely limited the impact of the BA.2/BA.2.12.1 surge on severe outcomes. Representative surveys are needed as part of routine surveillance for timely surge detection, and to estimate the true burden of infection, hybrid immunity, and uptake of time-sensitive treatments among those most vulnerable to severe COVID.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Short abstract</jats:title>\n                  <jats:p>Changes in testing practices and behaviors, including increasing at-home rapid testing and decreasing provider-based testing make it challenging to assess the true prevalence of SARS-CoV-2. We conducted a population-representative survey of adults in New York City to estimate the prevalence of SARS-CoV-2 infection during the BA.2./BA.2.12.1 surge in late April/early May 2022. We triangulated survey-based SARS-CoV-2 prevalence estimates with contemporaneous city-wide SARS-CoV-2 metrics on diagnosed cases, hospitalizations, deaths, and SARS-CoV-2 concentration in wastewater. Survey-based prevalence estimates were nearly 30 times higher than official case counts, and estimates of recently acquired hybrid immunity among those with active infection were high. We conclude that no single data source provides a complete or accurate assessment of the epidemiologic situation. Taken together, however, our results suggest that the magnitude of the BA.2/BA.2.12.1 surge was likely significantly underestimated, and high levels of hybrid immunity likely prevented a major surge in BA.2/BA.2.12.1-associated hospitalizations/deaths.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.05.25.22275603","journal":"medRxiv","score":null},{"id":"10.1101/2022.05.26.493539","pub_date":"2022-5-26","title":"Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5","abstract":"After the global spread of SARS-CoV-2 Omicron BA.2 lineage, some BA.2-related variants that acquire mutations in the L452 residue of spike protein, such as BA.2.9.1 and BA.2.13 (L452M), BA.2.12.1 (L452Q), and BA.2.11, BA.4 and BA.5 (L452R), emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these L452R/M/Q-bearing BA.2-related Omicron variants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1 and BA.2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. Furthermore, infection experiments using hamsters indicated that BA.4/5 is more pathogenic than BA.2. Altogether, our multiscale investigations suggest that the risk of L452R/M/Q-bearing BA.2-related Omicron variants, particularly BA.4 and BA.5, to global health is potentially greater than that of original BA.2. Spike L452R/Q/M mutations increase the effective reproduction number of BA.2 BA.4/5 is resistant to the immunity induced by BA.1 and BA.2 infections BA.2.12.1 and BA.4/5 more efficiently spread in human lung cells than BA.2 BA.4/5 is more pathogenic than BA.2 in hamsters","version":"1.1","doi":"10.1101/2022.05.26.493539","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.25.493467","pub_date":"2022-5-26","title":"Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins","abstract":"Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual SARS-CoV-2 viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately 5-fold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I downregulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I downregulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single-amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T-cells. Speficially, ORF7a prevented the assembly of the MHC-I peptide loading complex and causing retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.","version":"1.1","doi":"10.1101/2022.05.25.493467","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.24.493347","pub_date":"2022-5-25","title":"Subcutaneous delivery of an antibody against SARS-CoV-2 from a supramolecular hydrogel depot","abstract":"Prolonged maintenance of therapeutically-relevant levels of broadly neutralizing antibodies (bnAbs) is necessary to enable passive immunization against infectious disease. Unfortunately, protection only lasts for as long as these bnAbs remain present at a sufficiently high concentration in the body. Poor pharmacokinetics and burdensome administration are two challenges that need to be addressed in order to make pre- and post-exposure prophylaxis with bnAbs feasible and effective. In this work, we develop a supramolecular hydrogel as an injectable, subcutaneous depot to encapsulate and deliver antibody drug cargo. This polymer-nanoparticle (PNP) hydrogel exhibits shear-thinning and self-healing properties that are required for an injectable drug delivery vehicle. In vitro drug release assays and diffusion measurements indicate that the PNP hydrogels prevent burst release and slow the release of encapsulated antibodies. Delivery of bnAbs against SARS-CoV-2 from PNP hydrogels is compared to standard routes of administration in a preclinical mouse model. We develop a multi-compartment model to understand the ability of these subcutaneous depot materials to modulate the pharmacokinetics of released antibodies; the model is extrapolated to explore the requirements needed for novel materials to successfully deliver relevant antibody therapeutics with different pharmacokinetic characteristics.","version":"1.1","doi":"10.1101/2022.05.24.493347","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.24.493187","pub_date":"2022-5-25","title":"Hemin shows antiviral activity in vitro, possibly through suppression of viral entry mediators","abstract":"Heme oxygenase-1 (HO-1) is a stress-induced enzyme that catalyzes the breakdown of heme into biliverdin, carbon monoxide, and iron. Targeting HO-1 to treat severe COVID-19 has been suggested by several groups, yet the role of HO-1 in SARS-CoV-2 infection remains unclear. Based on this, we aimed to investigate the antiviral activity of Hemin, an activator of HO-1. Infectivity of SARS-CoV-2 was decreased in Vero E6 cells treated with Hemin. Hemin also decreased TMPRSS2 and ACE2 mRNA levels in non-infected cells, possibly explaining the observed decrease in infectivity. TMPRSS2 protein expression and proteolytic activity were decreased in Vero E6 cells treated with Hemin. Besides that, experimental studies supported with in silico calculations. Overall, our study supports further exploration of Hemin as a potential antiviral and inflammatory drug for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.05.24.493187","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.25.493397","pub_date":"2022-5-25","title":"Distinct antigenic properties of the SARS-CoV-2 Omicron lineages BA.4 and BA.5","abstract":"Over the course of the pandemic variants have arisen at a steady rate. The most recent variants to emerge, BA.4 and BA.5, form part of the Omicron lineage and were first found in Southern Africa where they are driving the current wave of infection. In this report, we perform an in-depth characterisation of the antigenicity of the BA.4/BA.5 Spike protein by comparing sera collected post-vaccination, post-BA.1 or BA.2 infection, or post breakthrough infection of vaccinated individuals with the Omicron variant. In addition, we assess sensitivity to neutralisation by commonly used therapeutic monoclonal antibodies. We find sera collected post-vaccination have a similar ability to neutralise BA.1, BA.2 and BA.4/BA.5. In contrast, in the absence of vaccination, prior infection with BA.2 or, in particular, BA.1 results in an antibody response that neutralises BA.4/BA.5 poorly. Breakthrough infection with Omicron in vaccinees leads to a broad neutralising response against the new variants. The sensitivity of BA.4/BA.5 to neutralisation by therapeutic monoclonal antibodies was similar to that of BA.2. These data suggest BA.4/BA.5 are antigenically distinct from BA.1 and, to a lesser extent, BA.2. The enhanced breadth of neutralisation observed following breakthrough infection with Omicron suggests that vaccination with heterologous or multivalent antigens may represent viable strategies for the development of cross-neutralising antibody responses.","version":"1.1","doi":"10.1101/2022.05.25.493397","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481551","pub_date":"2022-5-24","title":"Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission","abstract":"Wildlife reservoirs of SARS-CoV-2 may enable viral adaptation and spillback from animals to humans. In North America, there is evidence of unsustained spillover of SARS-CoV-2 from humans to white-tailed deer (Odocoileus virginianus), but no evidence of transmission from deer to humans. Through a biosurveillance program in Ontario, Canada we identified a new and highly divergent lineage of SARS-CoV-2 in white-tailed deer. This lineage is the most divergent SARS-CoV-2 lineage identified to date, with 76 consensus mutations (including 37 previously associated with non-human animal hosts) and signatures of considerable evolution and transmission within wildlife. Phylogenetic analysis also revealed an epidemiologically linked human case. Together, our findings represent the first clear evidence of sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.","version":"1.3","doi":"10.1101/2022.02.22.481551","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.23.493138","pub_date":"2022-5-24","title":"Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation","abstract":"The nucleocapsid (N) protein of coronaviruses is responsible for compaction of the \u223c30-kb RNA genome in the \u223c100-nm virion. Cryo-electron tomography suggests that each virion contains 35-40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined with viral RNA fragments in vitro, forms cylindrical 15-nm particles similar to the vRNP structures observed within coronavirus virions. These vRNPs form in the presence of stem-loop-containing RNA and depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine (SR) region weakens these interactions and disrupts vRNP assembly. We propose that unmodified N binds stem-loop-rich regions in genomic RNA to form compact vRNP complexes within the nucleocapsid, while phosphorylated N maintains uncompacted viral RNA to promote the protein\u2019s transcriptional function.","version":"1.1","doi":"10.1101/2022.05.23.493138","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.22.492976","pub_date":"2022-5-24","title":"Bayesian Inference of Dependent Population Dynamics in Coalescent Models","abstract":"The coalescent is a powerful statistical framework that allows us to infer past population dynamics leveraging the ancestral relationships reconstructed from sampled molecular sequence data. In many biomedical applications, such as in the study of infectious diseases, cell development, and tumorgenesis, several distinct populations share evolutionary history and therefore become dependent. The inference of such dependence is a highly important, yet a challenging problem. With advances in sequencing technologies, we are well positioned to exploit the wealth of high-resolution biological data for tackling this problem. Here, we present a novel probabilistic model that relies on jointly distributed Markov random fields. We use this model to estimate past population dynamics of dependent populations and to quantify their degree of dependence. An essential feature of our approach is the ability to track the time-varying association between the populations while making minimal assumptions on their functional shapes via Markov random field priors. We provide nonparametric estimators, extensions of our base model that integrate multiple data sources, and fast scalable inference algorithms. We test our method using simulated data under various dependent population histories and demonstrate the utility of our model in shedding light on evolutionary histories of different variants of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.05.22.492976","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.21.492920","pub_date":"2022-5-23","title":"The SARS-CoV-2 spike protein binds and modulates estrogen receptors","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ER\u03b1). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ER\u03b1 binding mode. In cultured cells, S DNA transfection increased ER\u03b1 cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [18F]fluoroestradiol (FES) localized lung pathology with increased ER\u03b1 lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ER\u03b1 expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ER\u03b1 interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.","version":"1.1","doi":"10.1101/2022.05.21.492920","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.21.492922","pub_date":"2022-5-23","title":"Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes","abstract":"Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the still ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have also engineered reporter-expressing recombinant viruses in which we place the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression that facilitates the study of SARS-CoV-2 in vitro and in vivo. Although successful, the genetic manipulation of the BAC containing the entire SARS-CoV-2 genome of \u223c30,000 nucleotides, is challenging. Herein, we depict the technical details to engineer rSARS-CoV-2 expressing reporter genes using the BAC-based RG approach. We describe i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, ii) verification of the pBeloBAC-FL, iii) cloning of a Venus reporter gene into the pBeloBAC-FL, and iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with basic molecular biology and gene engineering techniques knowledge will be able to generate wild-type and reporter-expressing rSARS-CoV-2.","version":"1.1","doi":"10.1101/2022.05.21.492922","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.21.492923","pub_date":"2022-5-23","title":"Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine candidate is protective in macaques","abstract":"Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways, as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal IgA and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T-cell responses, including tissue-resident memory cells in lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine. Intranasal parainfluenza virus-vectored COVID-19 vaccine induces anti-S antibodies, T-cell memory and protection in macaques.","version":"1.1","doi":"10.1101/2022.05.21.492923","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.491922","pub_date":"2022-5-23","title":"SARS-CoV-2 ORF1abA1061S potentiate autoreactive T cell responses via epitope mimicry: an explanation to hepatitis of unknown cause","abstract":"The World Health Organization have recently announced outbreak news of acute, severe hepatitis of unknown cause in children under a Covid-19 pandemic. Whether it is associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still under debating. Here, we performed genomic sequence alignment analysis of the genome of SARS-Cov-2 (Wuhan-hu-1) to the human genome reference. Sequence analysis revealed that the SARS-CoV-2 ORF1ab1056-1173 presented high identities with the human protein PAPR1453-176(3Q6Z_A). After searching the fully sequenced SARS-CoV-2 genomes deposited in GISAID (https://www.gisaid.org/), we detected 170 SARS-CoV-2 variants with mutation in ORF1ab1061, where alanine (A) was substituted by serine (S). This alteration made a 7-amino acid peptide (VVVNASN) in ORF1ab1056-1062 identical to its counterpart in PARP1453-59(3Q6Z_A). HLA prediction suggested that the peptides with high identities in PARP14 and ORF1ab could be presented by a same globally prevalent HLA-A*11:01 molecule. And in consistent with the first reported case of hepatitis of unknown, SARS-CoV-2 ORF1abVVVNASN variants were mostly identified as Delta lineages in UK by the late 2021, with an overall frequency of 0.00161%. Thus, our preliminary results raised a possibility that infection by SARS-CoV-2 ORF1abVVVNASN variant might elicit an autoimmune T cell response via epitope mimicry and is associated with the outbreak of unknown hepatitis. We anticipated that these findings will alert the human societies to pay more attention to rare mutations beyond the spike proteins.","version":"1.3","doi":"10.1101/2022.05.16.491922","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.21.492903","pub_date":"2022-5-23","title":"Reduced Neutralization of SARS-CoV-2 Omicron Variant in Sera from SARS-CoV-1 Survivors after 3-dose of Vaccination","abstract":"Recent studies found that Omicron variant escapes vaccine-elicited immunity. Interestingly, potent cross-clade pan-sarbecovirus neutralizing antibodies were found in survivors of the infection by SARS-CoV-1 after BNT162b2 mRNA vaccination (N Engl J Med. 2021 Oct 7;385(15):1401-1406). These pan-sarbecovirus neutralizing antibodies were observed to efficiently neutralize the infection driven by the S protein from both SARS-CoV and multiple SARS-CoV-2 variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta). However, whether these cross-reactive antibodies could neutralize the Omicron variant is still unknown. Based on the data collected from a cohort of SARS-CoV-1 survivors received 3-dose of immunization, our studies reported herein showed that a high level of neutralizing antibodies against both SARS-CoV-1 and SARS-CoV-2 were elicited by a 3rd-dose of booster vaccination of protein subunit vaccine ZF2001. However, a dramatically reduced neutralization of SARS-CoV-2 Omicron Variant (B.1.1.529) is observed in sera from these SARS-CoV-1 survivors received 3-dose of Vaccination. Our results indicates that the rapid development of pan-variant adapted vaccines is warranted.","version":"1.1","doi":"10.1101/2022.05.21.492903","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.23.492800","pub_date":"2022-5-23","title":"Predictions of immunogenicity reveal potent SARS-CoV-2 CD8+ T-cell epitopes","abstract":"The recognition of pathogen or cancer-specific epitopes by CD8+ T cells is crucial for the clearance of infections and the response to cancer immunotherapy. This process requires epitopes to be presented on class I Human Leukocyte Antigen (HLA-I) molecules and recognized by the T-Cell Receptor (TCR). Machine learning models capturing these two aspects of immune recognition are key to improve epitope predictions. Here we assembled a high-quality dataset of naturally presented HLA-I ligands and experimentally verified neo-epitopes. We then integrated these data with new algorithmic developments to improve predictions of both antigen presentation and TCR recognition. Applying our tool to SARS-CoV-2 proteins enabled us to uncover several epitopes. TCR sequencing identified a monoclonal response in effector/memory CD8+ T cells against one of these epitopes and cross-reactivity with the homologous SARS-CoV-1 peptide.","version":"1.1","doi":"10.1101/2022.05.23.492800","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.21.492928","pub_date":"2022-5-23","title":"Modeling suggests that multiple immunizations or infections will reveal the benefits of updating SARS-CoV-2 vaccines","abstract":"When should vaccines to evolving pathogens such as SARS-CoV-2 be updated? Our computational models address this focusing on updating SARS-CoV-2 vaccines to the currently circulating Omicron variant. Current studies typically compare the antibody titers to the new variant following a single dose of the original-vaccine versus the updated-vaccine in previously immunized individuals. These studies find that the updated-vaccine does not induce higher titers to the vaccine-variant compared with the original-vaccine, suggesting that updating may not be needed. Our models recapitulate this observation but suggest that vaccination with the updated-vaccine generates qualitatively different humoral immunity, a small fraction of which is specific for unique epitopes to the new variant. Our simulations suggest that these new variant-specific responses could dominate following subsequent vaccination or infection with either the currently circulating or future variants. We suggest a two-dose strategy for determining if the vaccine needs updating and for vaccinating high-risk individuals.","version":"1.1","doi":"10.1101/2022.05.21.492928","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.21.492554","pub_date":"2022-5-23","title":"Further antibody escape by Omicron BA.4 and BA.5 from vaccine and BA.1 serum","abstract":"The Omicron lineage of SARS-CoV-2, first described in November 2021, spread rapidly to become globally dominant and has split into a number of sub-lineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa\u2019s Gauteng region uncovered two new sub-lineages, BA.4 and BA.5 which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences and, although closely related to BA.2, contain further mutations in the receptor binding domain of spike. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by serum from triple AstraZeneca or Pfizer vaccinated individuals compared to BA.1 and BA.2. Furthermore, using serum from BA.1 vaccine breakthrough infections there are likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.","version":"1.1","doi":"10.1101/2022.05.21.492554","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.19.22275214","pub_date":"2022-05-22","title":"Antibody levels following vaccination against SARS-CoV-2: associations with post-vaccination infection and risk factors","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>SARS-CoV-2 antibody levels can be used to assess humoral immune responses following SARS-CoV-2 infection or vaccination, and may predict risk of future infection. From cross-sectional antibody testing of 9,361 individuals from TwinsUK and ALSPAC UK population-based longitudinal studies (jointly in April-May 2021, and TwinsUK only in November 2021-January 2022), we tested associations between antibody levels following vaccination and: (1) SARS-CoV-2 infection following vaccination(s); (2) health, socio-demographic, SARS-CoV-2 infection and SARS-CoV-2 vaccination variables.</jats:p>\n                <jats:p>Within TwinsUK, single-vaccinated individuals with the lowest 20% of anti-Spike antibody levels at initial testing had 3-fold greater odds of SARS-CoV-2 infection over the next six to nine months, compared to the top 20%. In TwinsUK and ALSPAC, individuals identified as at increased risk of COVID-19 complication through the UK \u201cShielded Patient List\u201d had consistently greater odds (2 to 4-fold) of having antibody levels in the lowest 10%. Third vaccination increased absolute antibody levels for almost all individuals, and reduced relative disparities compared with earlier vaccinations.</jats:p>\n                <jats:p>These findings quantify the association between antibody level and risk of subsequent infection, and support a policy of triple vaccination for the generation of protective antibodies.</jats:p>\n                <jats:sec>\n                  <jats:title>Lay summary</jats:title>\n                  <jats:p>In this study, we analysed blood samples from 9,361 participants from two studies in the UK: an adult twin registry, TwinsUK (4,739 individuals); and the Avon Longitudinal Study of Parents and Children, ALSPAC (4,622 individuals). We did this work as part of the UK Government National Core Studies initiative researching COVID-19. We measured blood antibodies which are specific to SARS-CoV-2 (which causes COVID-19). Having a third COVID-19 vaccination boosted antibody levels. More than 90% of people from TwinsUK had levels after third vaccination that were greater than the average level after second vaccination. Importantly, this was the case even in individuals on the UK \u201cShielded Patient List\u201d. We found that people with lower antibody levels after first vaccination were more likely to report having COVID-19 later on, compared to people with higher antibody levels. People on the UK \u201cShielded Patient List\u201d, and individuals who reported that they had poorer general health, were more likely to have lower antibody levels after vaccination. In contrast, people who had had a previous COVID-19 infection were more likely to have higher antibody levels following vaccination compared to people without infection. People receiving the Oxford/AstraZeneca rather than the Pfizer BioNTech vaccine had lower antibody levels after one or two vaccinations. However, after a third vaccination, there was no difference in antibody levels between those who had Oxford/AstraZeneca and Pfizer BioNTech vaccines for their first two doses. These findings support having a third COVID-19 vaccination to boost antibodies.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.05.19.22275214","journal":"medRxiv","score":null},{"id":"10.1101/2022.05.19.492641","pub_date":"2022-5-20","title":"Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests","abstract":"Widespread and frequent testing is critical to prevent the spread of COVID-19, and rapid antigen tests are the diagnostic tool of choice in many settings. With new viral variants continuously emerging and spreading rapidly, the effect of mutations on antigen test performance is a major concern. In response to the spread of variants the National Institutes of Health\u2019s Rapid Acceleration of Diagnostics (RADx\u00ae) initiative created a Variant Task Force to assess the impact of emerging SARS-CoV-2 variants on in vitro diagnostic testing. To evaluate the impact of mutations on rapid antigen tests we developed a lentivirus-mediated mammalian surface-display platform for the SARS-CoV-2 Nucleocapsid protein, the target of the majority of rapid antigen tests. We employed deep mutational scanning (DMS) to directly measure the effect of all possible Nucleocapsid point mutations on antibody binding by 17 diagnostic antibodies used in 11 commercially available antigen tests with FDA emergency use authorization (EUA). The results provide a complete map of the antibodies\u2019 epitopes and their susceptibility to mutational escape. This approach identifies linear epitopes, conformational epitopes, as well as allosteric escape mutations in any region of the Nucleocapsid protein. All 17 antibodies tested exhibit distinct escape mutation profiles, even among antibodies recognizing the same folded domain. Our data predict no vulnerabilities of rapid antigen tests for detection of mutations found in currently and previously dominant variants of concern and interest. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutation profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system. Further, our mammalian surface-display platform combined with DMS is a generalizable platform for complete mapping of protein-protein interactions.","version":"1.1","doi":"10.1101/2022.05.19.492641","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.20.492764","pub_date":"2022-5-20","title":"SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly","abstract":"CTL-mediated killing of virally infected or malignant cells is orchestrated at a specialized intercellular junction, the immune synapse (IS). We hypothesized that SARS-CoV-2 may target IS assembly in CTLs to escape killing. We show that primary human CD8+ T cells strongly upregulate the expression of ACE2, the Spike protein receptor, during differentiation to CTLs. CTL pre-incubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarisation to the IS, resulting in impaired target cell killing. These defects were reversed by anti-Spike antibodies that interfere with ACE2 binding and were reproduced by ACE2 engagement with Angiotensin-II or an anti-ACE2 antibody, but not by the ACE2 product Ang (1-7). These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent the elimination of infected cells. We report a new mechanism of immune evasion by SARS-CoV-2 based on direct disabling CTLs to form immune synapses through Spike protein binding to ACE2. This mechanism could contribute to the failure of the immune system to control SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.05.20.492764","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.20.492832","pub_date":"2022-5-20","title":"The spike-stabilizing D614G mutation interacts with S1/S2 cleavage site mutations to promote the infectious potential of SARS-CoV-2 variants","abstract":"SARS-CoV-2 remained genetically stable during the first three months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide, and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. The successive emergence of SARS-CoV-2 variants is fueling the COVID pandemic, thus causing a major and persistent public health issue. The parameters involved in the emergence of variants with higher pathogenic potential remain incompletely understood. The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions, and resulting in viral particles with higher infectious capacity. The Alpha and the Delta variants that spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic when compared to the original SARS-CoV-2 strain. Interestingly, Alpha and Delta both carried mutations in a spike cleavage site that needs to be processed by cellular proteases prior to viral entry. The cleavage site mutations P681H/R made the Alpha and Delta spikes more efficient at viral fusion, by generating a higher fraction of cleaved spikes subunits S1 and S2. We show here that the early D614G mutation and the late P681H/R mutations act synergistically to increase the fusion capacity of SARS-CoV-2 variants. Specifically, viruses with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite to the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.","version":"1.1","doi":"10.1101/2022.05.20.492832","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.20.492815","pub_date":"2022-5-20","title":"Structural basis for substrate selection by the SARS-CoV-2 replicase","abstract":"The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC). Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP. Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation. The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5\u2019 RNA cap.","version":"1.1","doi":"10.1101/2022.05.20.492815","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.20.492779","pub_date":"2022-5-20","title":"Omicron breakthrough infections in vaccinated or previously infected hamsters","abstract":"The second and third years of the SARS-CoV-2 pandemic have been marked by the repeated emergence and replacement of \u2018variants\u2019 with genetic and phenotypic distance from the ancestral strains, the most recent examples being Delta and Omicron. Here we describe a hamster contact exposure challenge model to assess protection conferred by vaccination or prior infection against re-infection. We found that 2-doses of self-amplifying RNA vaccine based on the ancestral spike ameliorated weight loss following Delta infection and decreased viral loads, but had minimal effect on Omicron/BA.1 infection. Prior infection with ancestral or Alpha variant was partially protective against Omicron/BA.1 infection, whereas all animals previously infected with Delta and exposed to Omicron became infected, although shed less virus. We further tested whether prior infection with Omicron/BA.1 protected from re-infection with Delta or Omicron/BA.2. Omicron/BA.1 was protective against Omicron/BA.2, but not Delta reinfection, again showing Delta and Omicron have a very large antigenic distance. Indeed, cross-neutralisation assays with human antisera from otherwise immunona\u00efve individuals (unvaccinated and no known prior infection), confirmed a large antigenic distance between Delta and Omicron. Prior vaccination followed by Omicron or Delta breakthrough infection led to a higher degree of cross-reactivity to all tested variants. To conclude, cohorts whose only immune experience of COVID is Omicron/BA.1 infection may be particularly vulnerable to future circulation of Delta or Delta-like derivatives. In contrast, repeated exposure to antigenically distinct spikes, via infection and or vaccination drives a more cross-reactive immune response, both in hamsters and people. Infection with the Delta and Omicron SARS-CoV-2 variants do not provide cross-protective immunity against reinfection with one another in hamsters.","version":"1.1","doi":"10.1101/2022.05.20.492779","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476672","pub_date":"2022-5-19","title":"High activity of an affinity-matured ACE2 decoy against Omicron SARS-CoV-2 and pre-emergent coronaviruses","abstract":"The viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly its cell-binding spike protein gene, has undergone rapid evolution during the coronavirus disease 2019 (COVID-19) pandemic. Variants including Omicron BA.1 and Omicron BA.2 now seriously threaten the efficacy of therapeutic monoclonal antibodies and vaccines that target the spike protein. Viral evolution over a much longer timescale has generated a wide range of genetically distinct sarbecoviruses in animal populations, including the pandemic viruses SARS-CoV-2 and SARS-CoV-1. The genetic diversity and widespread zoonotic potential of this group complicates current attempts to develop drugs in preparation for the next sarbecovirus pandemic. Receptor-based decoy inhibitors can target a wide range of viral strains with a common receptor and may have intrinsic resistance to escape mutant generation and antigenic drift. We previously generated an affinity-matured decoy inhibitor based on the receptor target of the SARS-CoV-2 spike protein, angiotensin-converting enzyme 2 (ACE2), and deployed it in a recombinant adeno-associated virus vector (rAAV) for intranasal delivery and passive prophylaxis against COVID-19. Here, we demonstrate the exceptional binding and neutralizing potency of this ACE2 decoy against SARS-CoV-2 variants including Omicron BA.1 and Omicron BA.2. Tight decoy binding tracks with human ACE2 binding of viral spike receptor-binding domains across diverse clades of coronaviruses. Furthermore, in a coronavirus that cannot bind human ACE2, a variant that acquired human ACE2 binding was bound by the decoy with nanomolar affinity. Considering these results, we discuss a strategy of decoy-based treatment and passive protection to mitigate the ongoing COVID-19 pandemic and future airway virus threats. Viral sequences can change dramatically during pandemics lasting multiple years. Likewise, evolution over centuries has generated genetically diverse virus families posing similar threats to humans. This variation presents a challenge to drug development, in both the breadth of achievable protection against related groups of viruses and the durability of therapeutic agents or vaccines during extended outbreaks. This phenomenon has played out dramatically during the coronavirus disease 2019 (COVID-19) pandemic. The highly divergent Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have upended previous gains won by vaccine and monoclonal antibody development. Moreover, ecological surveys have increasingly revealed a broad class of SARS-CoV-2-like viruses in animals, each poised to cause a future human pandemic. Here, we evaluate an alternative to antibody-based protection and prevention\u2014a decoy molecule based on the SARS-CoV-2 receptor. Our engineered decoy has proven resistant to SARS-CoV-2 evolution during the ongoing COVID-19 pandemic and can neutralize all variants of concern, including Omicron BA.1 and Omicron BA.2. Furthermore, the decoy binds tightly to a broad class of sarbecoviruses related to pandemic SARS-CoV-2 and SARS-CoV-1, indicating that receptor decoys offer advantages over monoclonal antibodies and may be deployed during the COVID-19 pandemic and future coronavirus outbreaks to prevent and treat severe illness.","version":"1.2","doi":"10.1101/2022.01.17.476672","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.18.492546","pub_date":"2022-5-19","title":"mRNA-based vaccines against SARS-CoV-2 do not stimulate interferon stimulatory gene expression in individuals affected by Aicardi Gouti\u00e8res Syndrome","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses threats to individuals with rare disease, in part because so little is known about the impact of COVID-19 infection and vaccination safety in rare disease populations. Of particular concern, given the overlap in disease manifestations and interferon dysregulation, are a group of heritable autoinflammatory conditions called type I interferonopathies. The most common of these, Aicardi Gouti\u00e8res Syndrome (AGS), is caused by altered nucleic acid metabolism and sensing, resulting in additional concerns surrounding the use of mRNA vaccination approaches. To determine whether mRNA vaccines induce an interferon response in AGS, we applied mRNA SARS-CoV-2 vaccines to whole blood samples and assessed internalization and interferon signaling gene expression responses to the mRNA. In all cases (11 AGS and 11 control samples), interferon signatures did not significantly increase from baseline, regardless of baricitinib treatment status in the AGS subjects, and were even decreased, when using codon optimized SARS-CoV-2 di-proline modified spike sequence (S2P). Internalization of S2P in human dendritic cells was verified by Western Blot, and in control and AGS blood cells was verified by Luciferase activity. Although numbers of tested samples in this rare disease are small, based on these findings, we suggest that COVID vaccination is unlikely to directly stimulate the interferon signaling gene expression in AGS patients via response to mRNA internalization. The in vitro nature of this study cannot exclude an exaggerated interferon response to spike protein production at a systemic level in individuals with a primary heritable interferonopathy. In the context of continued SARS-CoV-2 spread in the community, we do not recommend withholding vaccination in this rare disease group. However, we recommend that vaccinations for AGS patients are provided in a controlled setting with appropriate observation and used with caution in individuals with prior vaccine associated adverse events.","version":"1.1","doi":"10.1101/2022.05.18.492546","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.18.492441","pub_date":"2022-5-19","title":"Comprehensive analysis of pathways in Coronavirus 2019 (COVID-19) using an unsupervised machine learning method","abstract":"The World Health Organization (WHO) introduced \u201cCoronavirus disease 19\u201d or \u201cCOVID-19\u201d as a novel coronavirus in March 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires the fast discovery of effective treatments to fight this worldwide crisis. Artificial intelligence and bioinformatics analysis pipelines can assist with finding biomarkers, explanations, and cures. Artificial intelligence and machine learning methods provide powerful infrastructures for interpreting and understanding the available data. On the other hand, pathway enrichment analysis, as a dominant tool, could help researchers discover potential key targets present in biological pathways of host cells that are targeted by SARS-CoV-2. In this work, we propose a two-stage machine learning approach for pathway analysis. During the first stage, four informative gene sets that can represent important COVID-19 related pathways are selected. These \u201crepresentative genes\u201d are associated with the COVID-19 pathology. Then, two distinctive networks were constructed for COVID-19 related signaling and disease pathways. In the second stage, the pathways of each network are ranked with respect to some unsupervised scorning method based on our defined informative features. Finally, we present a comprehensive analysis of the top important pathways in both networks. Materials and implementations are available at: https://github.com/MahnazHabibi/Pathway.","version":"1.1","doi":"10.1101/2022.05.18.492441","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.487697","pub_date":"2022-5-19","title":"Epitope Mapping of SARS-CoV-2 Spike Protein Reveals Distinct Antibody Binding Activity of Vaccinated and Infected Individuals","abstract":"Previous studies have attempted to characterize the antibody response of individuals to the SARS-CoV-2 virus on a linear peptide level by utilizing peptide microarrays. These studies have helped to identify epitopes that have potential to be used for diagnostic tests to identify infected individuals, however, the immunological responses of individuals who have received the currently available Moderna mRNA-1273 or Pfizer BNT162b2 mRNA vaccines have not been characterized. We aimed to identify linear peptides of the SARS-CoV-2 spike protein that elicited high IgG or IgA binding activity and to compare the immunoreactivity of infected individuals to those who received both doses of either vaccines by utilizing peptide microarrays. Our results revealed peptide epitopes of significant IgG binding among recently infected individuals. Some of these peptides are located near functional domains implicated in the high infectivity of SARS-CoV-2. Vaccinated individuals lacked these distinct markers despite overall binding activity being similar.","version":"1.3","doi":"10.1101/2022.04.13.487697","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.19.492649","pub_date":"2022-5-19","title":"Programming the lymph node immune response with Amphiphile-CpG induces potent cellular and humoral immunity following COVID-19 subunit vaccination in mice and non-human primates","abstract":"Despite the success of currently authorized vaccines for the reduction of severe COVID-19 disease risk, rapidly emerging viral variants continue to drive pandemic waves of infection, resulting in numerous global public health challenges. Progress will depend on future advances in prophylactic vaccine activity, including advancement of candidates capable of generating more potent induction of cross-reactive T cells and durable cross-reactive antibody responses. Here we evaluated an Amphiphile (AMP) adjuvant, AMP-CpG, admixed with SARS-CoV-2 Spike receptor binding domain (RBD) immunogen, as a lymph node-targeted protein subunit vaccine (ELI-005) in mice and non-human primates (NHPs). AMP-mediated targeting of CpG DNA to draining lymph nodes resulted in comprehensive local immune activation characterized by extensive transcriptional reprogramming, inflammatory proteomic milieu, and activation of innate immune cells as key orchestrators of antigen-directed adaptive immunity. Prime-boost immunization with AMP-CpG in mice induced potent and durable T cell responses in multiple anatomical sites critical for prophylactic efficacy and prevention of severe disease. Long-lived memory responses were rapidly expanded upon re-exposure to antigen. In parallel, RBD-specific antibodies were long-lived, and exhibited cross-reactive recognition of variant RBD. AMP-CpG-adjuvanted prime-boost immunization in NHPs was safe and well tolerated, while promoting multi-cytokine-producing circulating T cell responses cross-reactive across variants of concern (VOC). Expansion of RBD-specific germinal center (GC) B cells in lymph nodes correlated to rapid seroconversion with variant-specific neutralizing antibody responses exceeding those measured in convalescent human plasma. These results demonstrate the promise of lymph-node adjuvant-targeting to coordinate innate immunity and generate robust adaptive responses critical for vaccine efficacy.","version":"1.1","doi":"10.1101/2022.05.19.492649","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.18.492443","pub_date":"2022-5-18","title":"Using unsupervised learning algorithms to identify essential genes associated with SARS-CoV-2 as potential therapeutic targets for COVID-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires the fast discovery of effective treatments to fight this worldwide concern. Several genes associated with the SARS-CoV-2, which are essential for its functionality, pathogenesis, and survival, have been identified. These genes, which play crucial roles in SARS-CoV-2 infection, are considered potential therapeutic targets. Developing drugs against these essential genes to inhibit their regular functions could be a good approach for COVID-19 treatment. Artificial intelligence and machine learning methods provide powerful infrastructures for interpreting and understanding the available data and can assist in finding fast explanations and cures. We propose a method to highlight the essential genes that play crucial roles in SARS-CoV-2 pathogenesis. For this purpose, we define eleven informative topological and biological features for the biological and PPI networks constructed on gene sets that correspond to COVID-19. Then, we use three different unsupervised learning algorithms with different approaches to rank the important genes with respect to our defined informative features. Finally, we present a set of 18 important genes related to COVID-19. Materials and implementations are available at: https://github.com/MahnazHabibi/Gene_analysis. m_habibi@qiau.ac.ir Supplementary data are available at Bioinformatics online.","version":"1.1","doi":"10.1101/2022.05.18.492443","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.17.492220","pub_date":"2022-5-18","title":"Molecular analysis of a public cross-neutralizing antibody response to SARS-CoV-2","abstract":"As SARS-CoV-2 variants of concerns (VOCs) continue to emerge, cross-neutralizing antibody responses become key towards next-generation design of a more universal COVID-19 vaccine. By analyzing published data from the literature, we report here that the combination of germline genes IGHV2-5/IGLV2-14 represents a public antibody response to the receptor-binding domain (RBD) that potently cross-neutralizes all VOCs to date, including Omicron and its sub-lineages. Detailed molecular analysis shows that the complementarity-determining region H3 sequences of IGHV2-5/IGLV2-14-encoded RBD antibodies have a preferred length of 11 amino acids and a conserved HxIxxI motif. In addition, these antibodies have a strong allelic preference due to an allelic polymorphism at amino-acid residue 54 of IGHV2-5, which locates at the paratope. These findings have important implications for understanding cross-neutralizing antibody responses to SARS-CoV-2 and its heterogenicity at the population level as well as the development of a universal COVID-19 vaccine.","version":"1.1","doi":"10.1101/2022.05.17.492220","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.18.464900","pub_date":"2022-5-18","title":"Preclinical Efficacy of IMM-BCP-01, a Highly Active Patient-Derived Anti-SARS-CoV-2 Antibody Cocktail","abstract":"Using an unbiased interrogation of the memory B cell repertoire of convalescent COVID-19 patients, we identified human antibodies that demonstrated robust antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants. Here, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant neutralizing antibodies directed at non-overlapping surfaces on the SARS-CoV-2 spike protein. Two antibodies, IMM20184 and IMM20190 directly block spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its unique epitope on the outer surface of RBD, alters the conformation of the spike trimer, promoting release of spike monomers. These antibodies decreased SARS-CoV-2 infection in the lungs of Syrian golden hamsters, and efficacy in vivo efficacy was associated with broad antiviral neutralizing activity against multiple SARS-CoV-2 variants and robust antiviral effector function response, including phagocytosis, ADCC, and complement pathway activation. Our pre-clinical data demonstrate that the three antibody cocktail IMM-BCP-01 shows promising potential for preventing or treating SARS-CoV-2 infection in susceptible individuals. IMM-BCP-01 cocktail triggers Spike Trimer dissociation, neutralizes all tested variants in vitro, activates a robust effector response and dose-dependently inhibits virus in vivo.","version":"1.2","doi":"10.1101/2021.10.18.464900","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.18.492452","pub_date":"2022-5-18","title":"SARS-CoV-2 Subunit Virus-Like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study","abstract":"Public health threat coming from a rapidly developing COVID-19 pandemic calls for developing safe and effective vaccines with innovative designs. This paper presents preclinical trial results of \u201cBetuvax-CoV-2\u201d, a vaccine developed as a subunit vaccine containing a recombinant RBD-Fc fusion protein and betulin-based spherical virus-like nanoparticles as an adjuvant (\u201cBetuspheres\u201d). The aim of the study was to demonstrate vaccine safety in mice, rats, and Chinchilla rabbits through acute, subchronic, and reproductive toxicity studies. Along with safety, the vaccine demonstrated protective efficacy through SARS-CoV-2-neutralizing antibody production in mice, rats, hamsters, rabbits, and primates (rhesus macaque), and lung damage and infection protection in hamsters and rhesus macaque model. Eventually, \u201cBetuvax-CoV-2\u201d was proved to confer superior efficacy and protection against the SARS-CoV-2 in preclinical studies. Based on the above results, the vaccine was enabled to enter clinical trials that are currently underway.","version":"1.1","doi":"10.1101/2022.05.18.492452","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.17.492310","pub_date":"2022-5-18","title":"Adaptation-proof SARS-CoV-2 vaccine design","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through \u2018epitope grafting,\u2019 in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of \u2018epitope grafting\u2019 in rational vaccine design.","version":"1.1","doi":"10.1101/2022.05.17.492310","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.471004","pub_date":"2022-5-18","title":"Online Phylogenetics using Parsimony Produces Slightly Better Trees and is Dramatically More Efficient for Large SARS-CoV-2 Phylogenies than de novo and Maximum-Likelihood Approaches","abstract":"Phylogenetics has been foundational to SARS-CoV-2 research and public health policy, assisting in genomic surveillance, contact tracing, and assessing emergence and spread of new variants. However, phylogenetic analyses of SARS-CoV-2 have often relied on tools designed for de novo phylogenetic inference, in which all data are collected before any analysis is performed and the phylogeny is inferred once from scratch. SARS-CoV-2 datasets do not fit this mould. There are currently over 10 million sequenced SARS-CoV-2 genomes in online databases, with tens of thousands of new genomes added every day. Continuous data collection, combined with the public health relevance of SARS-CoV-2, invites an \u201conline\u201d approach to phylogenetics, in which new samples are added to existing phylogenetic trees every day. The extremely dense sampling of SARS-CoV-2 genomes also invites a comparison between likelihood and parsimony approaches to phylogenetic inference. Maximum likelihood (ML) methods are more accurate when there are multiple changes at a single site on a single branch, but this accuracy comes at a large computational cost, and the dense sampling of SARS-CoV-2 genomes means that these instances will be extremely rare because each internal branch is expected to be extremely short. Therefore, it may be that approaches based on maximum parsimony (MP) are sufficiently accurate for reconstructing phylogenies of SARS-CoV-2, and their simplicity means that they can be applied to much larger datasets. Here, we evaluate the performance of de novo and online phylogenetic approaches, and ML and MP frameworks, for inferring large and dense SARS-CoV-2 phylogenies. Overall, we find that online phylogenetics produces similar phylogenetic trees to de novo analyses for SARS-CoV-2, and that MP optimizations produce more accurate SARS-CoV-2 phylogenies than do ML optimizations. Since MP is thousands of times faster than presently available implementations of ML and online phylogenetics is faster than de novo, we therefore propose that, in the context of comprehensive genomic epidemiology of SARS-CoV-2, MP online phylogenetics approaches should be favored.","version":"1.2","doi":"10.1101/2021.12.02.471004","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.07.481785","pub_date":"2022-5-17","title":"SARS-CoV-2 Spike evolution influences GBP and IFITM sensitivity","abstract":"SARS-CoV-2 spike requires proteolytic processing for viral entry. The presence of a polybasic furin-cleavage site (FCS) in spike, and evolution towards an optimised FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show that interferon-inducible antiviral restriction factors Guanylate binding proteins (GBP) 2 and 5 interfere with furin-mediated cleavage of SARS-CoV-2 spike and inhibit the infectivity of early-lineage Wuhan-Hu-1, while VOCs Alpha and Delta have evolved to escape restriction. Strikingly, we find Omicron is unique amongst VOCs, being restricted by GBP2/5, and also IFITM1, 2 and 3. Replacing the spike S2 domain in Omicron with Delta shows S2 is the determinant of entry route and IFITM sensitivity. We conclude that VOC evolution under different selective pressures has influenced sensitivity to spike-targeting restriction factors, with Omicron selecting spike changes that not only mediate antibody escape, and altered tropism, but also sensitivity to innate immunity.","version":"1.2","doi":"10.1101/2022.03.07.481785","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.17.492198","pub_date":"2022-5-17","title":"SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression","abstract":"Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein \u03b22-microglobulin (\u03b22m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8+ T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of \u03b22m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2. Viruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with \u03b22m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of \u03b22m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.","version":"1.1","doi":"10.1101/2022.05.17.492198","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492158","pub_date":"2022-5-17","title":"Differential Evasion of Delta and Omicron Immunity and Enhanced Fusogenicity of SARS-CoV-2 Omicron BA.4/5 and BA.2.12.1 Subvariants","abstract":"The rising case numbers of the SARS-CoV-2 Omicron BA.4, BA.5, and BA.2.12.1 subvariants has generated serious concern about the course of the pandemic. Here we examine the neutralization resistance, infectivity, processing, and fusogenicity of spike from the BA.4/5 and BA.2.12.1 SARS-CoV-2 variants compared with other Omicron subvariants and Delta. Critically, we found that the new Omicron subvariants BA.4/5 and BA.2.12.1 were more resistant to neutralization by mRNA-vaccinated and boosted health care worker sera and Omicron-BA.1-wave patient sera than were the BA.1 and BA.2 variants. Interestingly, Delta-wave patient sera neutralized more efficiently against not only Delta but also BA.4/5 and BA.2.12.1 variants that also contain substitutions at position L452, similar to Delta. The BA.4/5 and BA.2.12.1 variants also exhibited higher fusogenicity, and increased spike processing, dependent on the L452 substitution. These results highlight the key role of the L452R and L452Q mutations in BA.4/5 and BA.2.12.1 subvariants.","version":"1.1","doi":"10.1101/2022.05.16.492158","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.17.491668","pub_date":"2022-5-17","title":"The ATLAS\u2122 screening assay reveals distinct CD4+ and CD8+ SARS-CoV-2 antigen response profiles which have implications to Omicron cellular immunity","abstract":"The emergence of SARS-CoV-2 variants are a persistent threat to the efficacy of currently developed prophylactic vaccines and therapeutic antibodies. These variants accumulate mutations in the spike protein which encodes the epitopes necessary for neutralizing antibody binding. Moreover, emerging evidence suggest that robust antibody responses are insufficient to prevent severe disease and long-lasting viral immunity requires T cells. Thus, understanding how the T cell antigen landscape evolves in the context of these emerging variants remains crucial. T cells responses are durable and recognize a wider breadth of epitopes reducing the possibility of immune escape through mutation. Here, we deploy the ATLAS\u2122 assay which identifies CD4+ and CD8+ T cell antigens by utilizing the endogenous HLA class-I and class-II peptide processing pathways. Profiling of T cells from exposed and unexposed donors revealed rich and complex patterns which highlighted the breadth of antigenic potential encoded in SARS-CoV-2. ATLAS revealed several common or frequent antigenic regions as well as an abundance of responses in the unexposed cohort potentially the result of pre-exposure to related coronaviruses. ORF10 was a common CD4+ response in the unexposed cohort while spike was identified as a common and frequent target in both cohorts. Moreover, the spike response profiles allowed us to accurately predict the impact of Omicron spike mutations. This analysis could thus be applied to study the impact of future emerging VOCs.","version":"1.1","doi":"10.1101/2022.05.17.491668","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492112","pub_date":"2022-5-17","title":"Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis","abstract":"Severe coronavirus disease (COVID-19) is accompanied with acute respiratory distress syndrome & pulmonary pathology, and is presented mostly with inflammatory cytokine release, dysregulated immune response, skewed neutrophil/ lymphocyte ratio, and hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19 related mortality, the limitation of use of vaccine against immunocompromised, comorbidity, and emerging variants remains a concern. In the current study we investigate for the first-time the efficacy of Glycyrrhiza glabra (GG) extract, a potent immunomodulator, against SARS-CoV-2 infection in hamsters. Prophylactic treatment with GG showed protection against loss in body weight and 35-40% decrease in lung viral load along with reduced lung pathology in the hamster model. Remarkably, GG reduced the mRNA expression of pro-inflammatory cytokines and Plasminogen activator inhibito-1 (PAI-1). In-vitro, GG acted as potent immunomodulator by reducing Th2 and Th17 differentiation and IL-4 and IL-17A cytokine production. In addition, GG also showed robust potential to suppress ROS, mtROS and NETs generation in a concentration dependent manner in both human polymorphonuclear neutrophils (PMNs) and murine bone marrow derived neutrophils (BMDNs). Taken together, we provide evidence for the protective efficacy of GG against COVID-19 and its putative mechanistic insight, which might be developed as a future immunomodulatory approach against various pathologies with high cytokine production, aberrant neutrophil activation including coronavirus infection.","version":"1.1","doi":"10.1101/2022.05.16.492112","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484956","pub_date":"2022-5-16","title":"Human Galectin-9 Potently Enhances SARS-CoV-2 Replication and Inflammation in Airway Epithelial Cells","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a \u03b2-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. Here, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including primary AECs in air-liquid interface (ALI) culture. Gal-9-glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an ACE2-dependent manner, enhancing the binding affinity of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induce the expression of key pro-inflammatory programs in AECs including the IL-6, IL-8, IL-17, EIF2, and TNF\u03b1 signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection. COVID-19 continues to have a major global health and economic impact. Identifying host molecular determinants that modulate SARS-CoV-2 infectivity and pathology is a key step in discovering novel therapeutic approaches for COVID-19. Several recent studies have revealed that plasma concentrations of the human \u03b2-galactoside-binding protein galectin-9 (Gal-9) are highly elevated in COVID-19 patients. In this study, we investigated the impact of Gal-9 on SARS-CoV-2 pathogenesis ex vivo in airway epithelial cells (AECs), the critical initial targets of SARS-CoV-2 infection. Our findings reveal that Gal-9 potently enhances SARS-CoV-2 replication in AECs, interacting with glycans to enhance the binding between viral particles and entry receptors on the target cell surface. Moreover, we determined that Gal-9 accelerates and exacerbates several virus-induced pro-inflammatory programs in AECs that are established signature characteristics of COVID-19 disease and SARS-CoV-2-induced acute respiratory distress syndrome (ARDS). Our findings suggest that Gal-9 is a promising pharmacological target for COVID-19 therapies.","version":"1.3","doi":"10.1101/2022.03.18.484956","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492062","pub_date":"2022-5-16","title":"Functional evolution of SARS-COV-2 Spike protein: adaptation on translation and infection via surface charge of spike protein","abstract":"The SARS-COV-2 virus, which causes the COVID-19, is rapidly accumulating mutations to adapt to the hosts. We collected SARS-COV-2 sequence data from the end of 2019 to April 2022 to analyze for their evolutionary features during the pandemic. We found that most of the SARS-COV-2 genes are undergoing negative purifying selection, while the spike protein gene (S-gene) is undergoing rapid positive selection. From the original strain to the alpha, delta and omicron variant types, the Ka/Ks of the S-gene increases, while the Ka/Ks within one variant type decreases over time. During the evolution, the codon usage did not evolve towards optimal translation and protein expression. In contrast, only S-gene mutations showed a remarkable trend on accumulating more positive charges. This facilitates the infection via binding human ACE2 for cell entry and binding furin for cleavage. Such a functional evolution emphasizes the survival strategy of SARS-COV-2, and indicated new druggable target to contain the viral infection. The nearly fully positively-charged interaction surfaces indicated that the infectivity of SARS-COV-2 virus may approach a limit.","version":"1.1","doi":"10.1101/2022.05.16.492062","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492045","pub_date":"2022-5-16","title":"Isolation of bat sarbecoviruses of SARS-CoV-2 clade, Japan","abstract":"Betacoronaviruses have caused 3 outbreaks in the past 2 decades. SARS-CoV-2, in particular, has caused a serious pandemic. As the betacoronaviruses are considered to originate from bats, surveillance of bat betacoronaviruses is crucial for understanding the mechanism of cross-species transition and potential for future outbreaks. We previously detected and characterized a SARS-CoV-2-related sarbecovirus, Rc-o319, from Rhinolophus cornutus in Japan. Here, we detected several bat sarbecoviruses of the SARS-CoV-2 clade from R. cornutus in multiple locations in Japan, and successfully isolated them using Vero/TMPRSS2 cells stably expressing R. cornutus ACE2 (Vero-RcACE2). The coding sequences of S1 region varied among isolates, whereas other genetic regions were highly conserved. Isolates were efficiently grown in Vero-RcACE2 cells, but did not replicate in Vero/TMPRSS2 cells stably expressing human ACE2, suggesting a narrow host range. Further long-term epidemiological studies of sarbecoviruses in wildlife are expected to facilitate the assessment of the risk of their spillover potential.","version":"1.1","doi":"10.1101/2022.05.16.492045","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.13.491916","pub_date":"2022-5-16","title":"A SCID mouse model to evaluate the efficacy of antivirals against SARS-CoV-2 infection","abstract":"Ancestral SARS-CoV-2 lacks the intrinsic ability to bind to the mouse ACE2 receptor and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different Wild type (WT) mice species. Here, we desribe the establishment of SARS-CoV-2 beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small molecule inhibitors. Intranasal infection of male SCID mice with 105 TCID50 of the beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir (200 mg/kg, BID) or Nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 1.9 and 3.8 log10 TCID50/mg tissue, respectively and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mice/beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2. Unlike the ancestral SARS-CoV-2 strain, the beta (B.1.351) VoC has been reported to replicate to some extent in WT mice (species C57BL/6 and BALB/c). We here demonstrate that infection of SCID mice with SARS-CoV-2 beta variant results in high viral loads in the lungs on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir or Nirmatrelvir for 3 consecutive days markedly reduced the infectious virus titers in the lungs and improved lung pathology. The advantages of using this mouse model over the standard hamster infection models to assess the in vivo efficacy of small molecule antiviral drugs are (i) the use of a clinical isolate without the need to use mouse-adapted strains or genetically modified animals (ii) lower amount of the test drug is needed and (ii) more convenient housing conditions compared to bigger rodents such as hamsters.","version":"1.1","doi":"10.1101/2022.05.13.491916","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.491949","pub_date":"2022-5-16","title":"Human Surfactant Protein D Facilitates SARS-CoV-2 Pseudotype Binding and Entry in DC-SIGN Expressing Cells, and Downregulates Spike protein Induced Inflammation","abstract":"Pattern recognition receptors are crucial for innate anti-viral immunity, including C-type lectin receptors. Two such examples are Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) which are soluble and membrane-bound C-type lectin receptors, respectively. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction as an antigen-presenting cell with na\u00efve T cells to mount an anti-viral immune response. Both SP-D and DC-SIGN have been shown to interact with various viruses, including HIV-1, Influenza A virus and SARS-CoV-2. SARS-CoV-2 is an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of \u03b1-helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP-1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. The RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-\u03b1, IFN-\u03b1, IL-1\u03b2, IL-6, IL-8, and RANTES (as well as NF-\u03baB) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction of SARS-CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.05.16.491949","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.16.492138","pub_date":"2022-5-16","title":"A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants","abstract":"Vaccines are a cornerstone in COVID-19 pandemic management. Here, we compare immune responses to and preclinical efficacy of the mRNA vaccine BNT162b2, an adenovirus-vectored spike vaccine, and the live-attenuated-virus vaccine candidate sCPD9 after single and double vaccination in Syrian hamsters. All regimens containing sCPD9 showed superior efficacy. The robust immunity elicited by sCPD9 was evident in a wide range of immune parameters after challenge with heterologous SARS-CoV-2 including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue. Our results demonstrate that use of live-attenuated vaccines may offer advantages over available COVID-19 vaccines, specifically when applied as booster, and may provide a solution for containment of the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2022.05.16.492138","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.14.491911","pub_date":"2022-5-16","title":"SARS-CoV-2 Omicron Variant Wave in India: Advent, Phylogeny and Evolution","abstract":"SARS-CoV-2 evolution has continued to generate variants, responsible for new pandemic waves locally and globally. Varying disease presentation and severity has been ascribed to inherent variant characteristics and vaccine immunity. This study analyzed genomic data from 305 whole genome sequences from SARS-CoV-2 patients before and through the third wave in India. Delta variant was responsible for disease in patients without comorbidity(97%), while Omicron BA.2 caused disease primarily in those with comorbidity(77%). Tissue adaptation studies brought forth higher propensity of Omicron variants to bronchial tissue than lung, contrary to observation in Delta variants from Delhi. Study of codon usage pattern distinguished the prevalent variants, clustering them separately, Omicron BA.2 isolated in February grouped away from December strains, and all BA.2 after December acquired a new mutation S959P in ORF1b (44.3% of BA.2 in the study) indicating ongoing evolution. Loss of critical spike mutations in Omicron BA.2 and gain of immune evasion mutations including G142D, reported in Delta but absent in BA.1, and S371F instead of S371L in BA.1 could possibly be due to evolutionary trade-off and explain very brief period of BA.1 in December 2021, followed by complete replacement by BA.2.","version":"1.1","doi":"10.1101/2022.05.14.491911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.423603","pub_date":"2022-5-16","title":"Fatty Acid Synthase inhibitor TVB-3166 prevents S-acylation of the Spike protein of human coronaviruses","abstract":"The Spike protein of SARS-CoV2 and other coronaviruses mediate host cell entry and are S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes of the ZDHHC family have been reported to modify Spike proteins post-translationally. Using resin-assisted capture mass spectrometry, we demonstrate that the Spike protein is S-acylated in SARS-CoV2 infected human and monkey cells. We further show that increased abundance of the human acyltransferase ZDHHC5 results in increased S-acylation of the SARS-CoV2 Spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also find that the S-acylation of the Spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also found on the immunocaptured protein. Yet, ZDHHC5 was highly selective for palmitate, suggesting that other ZDHHC enzymes mediated the incorporation of other fatty acyl chains. Thus, since multiple ZDHHC isoforms may modify the Spike protein, we examined the ability of the fatty acid synthase inhibitor TVB-3166 to prevent the S-acylation of the Spike proteins of SARS-CoV-2 and human CoV-229E. Treating cells with TVB-3166 inhibited S-acylation of ectopically expressed SARS-CoV2 Spike and attenuated the ability of SARS-CoV2 and human CoV-229E to spread in vitro. Additionally, treatment of mice with a comparatively low dose of TVB-3166 promoted survival from an otherwise fatal murine coronavirus infection. Our findings further substantiate the necessity of CoV Spike protein S-acylation and the potential use of fatty acid synthase inhibitors.","version":"1.2","doi":"10.1101/2020.12.20.423603","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.13.491763","pub_date":"2022-5-16","title":"AI-based search for convergently expanding, advantageous mutations in SARS-CoV-2 by focusing on oligonucleotide frequencies","abstract":"Among mutations that occur in SARS-CoV-2, efficient identification of mutations advantageous for viral replication and transmission is important to characterize and defeat this rampant virus. Mutations rapidly expanding frequency in a viral population are candidates for advantageous mutations, but neutral mutations hitchhiking with advantageous mutations are also likely to be included. To distinguish these, we focus on mutations that appear to occur independently in different lineages and expand in frequency in a convergent evolutionary manner. Batch-learning SOM (BLSOM) can separate SARS-CoV-2 genome sequences according by lineage from only providing the oligonucleotide composition. Focusing on remarkably expanding 20-mers, each of which is only represented by one copy in the viral genome, allows us to correlate the expanding 20-mers to mutations. Using visualization functions in BLSOM, we can efficiently identify mutations that have expanded remarkably both in the Omicron lineage, which is phylogenetically distinct from other lineages, and in other lineages. Most of these mutations involved changes in amino acids, but there were a few that did not, such as an intergenic mutation.","version":"1.1","doi":"10.1101/2022.05.13.491763","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.14.488372","pub_date":"2022-5-16","title":"Hijacking of Cellular Functions by Severe Acute Respiratory Syndrome Coronavirus-2. Permeabilization and Polarization of the Host Lipid Membrane by Viroporins","abstract":"As all viral infections, SARS-CoV-2 acts at multiple levels hijacking fundamental cellular functions and assuring its replication and immune system evasion. In particular, it has been observed that the viral 3\u2019 Open Reading Frame (ORF3a) codes for a hydrophobic protein which embeds in the cellular membrane, where it acts as an ion viroporin and is related to strong inflammatory response. Here we report equilibrium and enhanced sampling molecular dynamic simulation of the SARS-CoV-2 ORF3a in a model lipid bilayer, showing how the protein permeabilizes the lipid membrane, via the formation of a water channel, which in turn assures ion transport. We report the free energy profile for both K+ and Cl- transfer from the cytosol to the extracellular domain. The important role of ORF3a in the viral cycle, and its highly conservation among coronaviruses, may also make it a target of choice for future antiviral development, further justifying the elucidation of its mechanism at the atomistic level.","version":"1.2","doi":"10.1101/2022.04.14.488372","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.22274866","pub_date":"2022-05-14","title":"Elevated liver enzymes and bilirubin following SARS-CoV-2 infection in children under 10","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Recently, the Centers for Diseases and Control released a nationwide health alert about an increase in hepatitis cases of unknown origin in children, raising concern about potential sequelae of COVID-19 infection. In this study, we test whether there was increased risk of elevated serum liver enzymes and bilirubin following COVID-19 infection in children. We performed a retrospective cohort study on a nation-wide database of patient electronic health records (EHRs) in the US. The study population comprise 796,369 children between the ages of 1-10 years including 245,675 who had contracted COVID-19 during March 11, 2020 - March 11, 2022 and 550,694 who contracted non-COVID other respiratory infection (ORI) during the same timeframe.</jats:p>\n                <jats:p>Compared to children infected with other respiratory infections, children infected with COVID-19 infection were at significantly increased risk for elevated AST or ALT (hazard ratio or HR: 2.52, 95% confidence interval or CI: 2.03-3.12) and total bilirubin (HR: 3.35, 95% CI: 2.16-5.18). These results suggest acute and long-term hepatic sequelae of COVID-19 in pediatric patients. Further investigation is needed to clarify if post-COVID-19 related hepatic injury described in this study is related to the current increase in pediatric hepatitis cases of unknown origin.</jats:p>","version":null,"doi":"10.1101/2022.05.10.22274866","journal":"medRxiv","score":null},{"id":"10.1101/2022.05.13.491823","pub_date":"2022-5-13","title":"Humoral immunity to SARS-CoV-2 elicited by combination COVID-19 vaccination regimens","abstract":"The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID- 19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV2.S and two-dose ChAdOx1 or combination ChAdOx1/mRNA vaccination. Plasma neutralizing activity as well as the magnitude, clonal composition and antibody maturation of the RBD-specific memory B cell compartment showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV2.S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.","version":"1.1","doi":"10.1101/2022.05.13.491823","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.13.491770","pub_date":"2022-5-13","title":"Structural insights for neutralization of BA.1 and BA.2 Omicron variants by a broadly neutralizing SARS-CoV-2 antibody","abstract":"The SARS-CoV-2 BA.1 and BA.2 (Omicron) variants contain more than 30 mutations within the spike protein and evade therapeutic monoclonal antibodies (mAbs). Here, we report a receptor-binding domain (RBD) targeting human antibody (002-S21F2) that effectively neutralizes live viral isolates of SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Gamma, Delta, and Omicron (BA.1 and BA.2) with IC50 ranging from 0.02 \u2013 0.05 \u03bcg/ml. This near germline antibody 002-S21F2 has unique genetic features that are distinct from any reported SARS-CoV-2 mAbs. Structural studies of the full-length IgG in complex with spike trimers (Omicron and WA.1) reveal that 002-S21F2 recognizes an epitope on the outer face of RBD (class-3 surface), outside the ACE2 binding motif and its unique molecular features enable it to overcome mutations found in the Omicron variants. The discovery and comprehensive structural analysis of 002-S21F2 provide valuable insight for broad and potent neutralization of SARS-CoV-2 Omicron variants BA.1 and BA.2.","version":"1.1","doi":"10.1101/2022.05.13.491770","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.12.491597","pub_date":"2022-5-13","title":"Delivery of Circular mRNA via Degradable Lipid Nanoparticles against SARS-CoV-2 Delta Variant","abstract":"mRNA vaccines have emerged as a most promising and potent platform in the fight against various diseases including the COVID-19 pandemic. However, the intrinsic instability, varying side effects associated with the delivery systems, and continuous emergence of virus variants highlight the urgent need for the development of stable, safe and efficacious mRNA vaccines. In this study, by screening a panel of proprietary biodegradable ionizable lipidoids, we reported on a novel mRNA vaccine (cmRNA-1130) formed from a biodegradable lipidoid with eight ester bonds in the branched tail (AX4) and synthetic circular mRNA (cmRNA) encoding the trimeric Delta receptor binding domain (RBD) of SARS-CoV-2 spike protein for the induction of robust immune activation. The AX4-based lipid nanoparticles (AX4-LNP) revealed much faster elimination rate from liver and spleen in comparison with commercialized MC3-based LNP (MC3-LNP) and afforded normal level of alanine transferase (ALT), aspartate aminotransferase (AST), and creatinine (CRE) in BALB/c mice. Following intramuscular (IM) administration in BALB/c mice, cmRNA-1130 elicited potent and sustained neutralizing antibodies, RBD-specific CD4+ and CD8+ T effector memory cells (Tem), and Th1-biased T cell activations. cmRNA-1130 vaccine showed excellent stability against 6-month storage at 4 \u00b0C and freezing-thawing cycles. In brief, our study highlights mRNA vaccines based on cmRNA and biodegradable AX4 lipids hold great potential as superb therapeutic platforms for the treatment of varying diseases.","version":"1.2","doi":"10.1101/2022.05.12.491597","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.13.491706","pub_date":"2022-5-13","title":"Three-doses of BNT162b2 COVID-19 mRNA vaccine establishes long-lasting CD8+ T cell immunity in CLL and MDS patients","abstract":"Patients with hematological malignancies are prioritized for COVID-19 vaccine due to their high risk for severe SARS-CoV-2 infection related disease and mortality. To understand T cell immunity, its long-term persistence, and correlation with antibody response, we evaluated the BNT162b2 COVID-19 mRNA vaccine-specific immune response in chronic lymphocytic leukemia (CLL) and myeloid dysplastic syndrome (MDS) patients. Longitudinal analysis of CD8+ T cells using DNA-barcoded peptide-MHC multimers covering the full SARS-CoV-2 Spike-protein (415 peptides) showed vaccine-specific T cell activation and persistence of memory T cells up to six months post-vaccination. Surprisingly, a higher frequency of vaccine-induced antigen-specific CD8+ T cell was observed in the patient group compared to a healthy donor group. Furthermore, and importantly, immunization with the second booster dose significantly increased the frequency of antigen-specific CD8+ T cells as well as the total number of T cell specificities. Altogether 59 BNT162b2 vaccine-derived immunogenic epitopes were identified, of which 23 established long-term CD8+ T cell memory response with a strong immunodominance for NYNYLYRLF (HLA-A24:02) and YLQPRTFLL (HLA-A02:01) epitopes. In summary, we mapped the vaccine-induced antigen-specific CD8+ T cells and showed a booster-specific activation and enrichment of memory T cells that could be important for long-term disease protection in this patient group. COVID-19 mRNA vaccine induced an early and persistent activation of antigen-specific CD8+ T cells in this patient group. Vaccination with a booster dose is required to maintain vaccine-specific CD8+ T cells.","version":"1.1","doi":"10.1101/2022.05.13.491706","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.31.474653","pub_date":"2022-5-13","title":"The altered entry pathway and antigenic distance of the SARS-CoV-2 Omicron variant map to separate domains of spike protein","abstract":"The SARS-CoV-2 Omicron/BA.1 lineage emerged in late 2021 and rapidly displaced the Delta variant before being overtaken itself globally by, the Omicron/BA.2 lineage in early 2022. Here, we describe how Omicron BA.1 and BA.2 show a lower severity phenotype in a hamster model of pathogenicity which maps specifically to the spike gene. We further show that Omicron is attenuated in a lung cell line but replicates more rapidly, albeit to lower peak titres, in human primary nasal cells. This replication phenotype also maps to the spike gene. Omicron spike (including the emerging Omicron lineage BA.4) shows attenuated fusogenicity and a preference for cell entry via the endosomal route. We map the altered Omicron spike entry route and partially map the lower fusogenicity to the S2 domain, particularly the substitution N969K. Finally, we show that pseudovirus with Omicron spike, engineered in the S2 domain to confer a more Delta-like cell entry route retains the antigenic properties of Omicron. This shows a distinct separation between the genetic determinants of these two key Omicron phenotypes, raising the concerning possibility that future variants with large antigenic distance from currently circulating and vaccine strains will not necessarily display the lower intrinsic severity seen during Omicron infection.","version":"1.2","doi":"10.1101/2021.12.31.474653","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.13.491757","pub_date":"2022-5-13","title":"Optimising Elastic Network Models for Protein Dynamics and Allostery: Spatial and Modal Cut-offs and Backbone Stiffness","abstract":"The family of coarse-grained models for protein dynamics known as Elastic Network Models (ENMs) require a careful choice of parameters to represent well experimental measurements or fully-atomistic simulations. The most basic ENM that represents each protein residue by a node at the position of its C-alpha atom, all connected by springs of equal stiffness, up to a cut-off in distance. Even at this level, a choice is required of the optimum cut-off distance and the upper limit of elastic normal modes taken in any sum for physical properties, such as dynamic correlation or allosteric effects on binding. Additionally, backbone-enhanced ENM (BENM) may improve the model by allocating a higher stiffness to springs that connect along with the protein backbone. This work reports on the effect of varying these three parameters (distance and mode cutoffs, backbone stiffness) on the dynamical structure of three proteins, Catabolite Activator Protein (CAP), Glutathione S-transferase (GST), and the SARS-CoV- 2 Main Protease (Mpro). Our main results are: (1) balancing B-factor and dispersion-relation predictions, a near-universal optimal value of 8.5 angstroms is advisable for ENMs; (2) inhomogeneity in elasticity brings the first mode containing spatial structure not well-resolved by the ENM typically within the first 20; (3) the BENM only affects modes in the upper third of the distribution, and, additionally to the ENM, is only able to model the dispersion curve better in this vicinity; (4) BENM does not typically affect fluctuation-allostery, which also requires careful treatment of the effector binding to the host protein to capture.","version":"1.1","doi":"10.1101/2022.05.13.491757","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.491351","pub_date":"2022-5-12","title":"The SARS-CoV-2 Spike Protein Activates the Epidermal Growth Factor Receptor-Mediated Signaling","abstract":"The coronavirus disease-19 (COVID-19) pandemic is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the molecular and cellular levels, the SARS-Cov-2 uses its envelope glycoprotein, the spike S protein, to infect the target cells in the lungs via binding with their transmembrane receptor, the angiotensin-converting enzyme 2 (ACE2). Here, we wanted to invesitgate if other molecular targets and pathways may be used by SARS-Cov-2. We investigated the possibility for the spike 1 S protein and its receptor-binding domain (RBD) to target the epidermal growth factor receptor (EGFR) and its downstream signaling pathway in vitro using the lung cancer cell line (A549 cells). Protein expression and phosphorylation was examined upon cell treatment with the recombinant full spike 1 S protein or RBD. We demonstrate for the first time the activation of EGFR by the Spike 1 protein associated with the phosphorylation of the canonical ERK1/2 and AKT kinases and an increase of survivin expression controlling the survival pathway. Our study suggests the putative implication of EGFR and its related signaling pathways in SARS-CoV-2 infectivity and Covid-19 pathology. This may open new perspectives in the treatment of Covid-19 patients by targeting EGFR.","version":"1.1","doi":"10.1101/2022.05.10.491351","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.12.491584","pub_date":"2022-5-12","title":"Low immune response after 1.5 years of primary SARS-CoV-2 infection and Covishield vaccination lead to SARS-CoV-2 reinfection","abstract":"We have investigated six COVID recovered cases with two doses of Covishield vaccination followed by reinfection. The primary SARS-CoV-2 infection found to occur with B.1 and reinfection with Omicron BA.1 and BA.2 variants. The genomic characterization and duration between two infections confirms these cases as SARS-CoV-2 reinfection. The mutation analysis of the reinfection cases correlated with immune evasion potential of BA.1 and BA.2 sub lineages. The immune response determined at different time intervals demonstrated boost post two dose vaccination, decline in pre-reinfection sera post 7 months and rise post reinfection. Apparently, these cases suffered from SARS-CoV-2 reinfection with the declined hybrid immunity acquired from primary infection and two dose covishield vaccination. This suggests the need for booster dose of vaccination. Besides this, multiple non-pharmaceutical interventions should be used to cope up with SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.05.12.491584","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.11.491588","pub_date":"2022-5-12","title":"A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern","abstract":"Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a \u201csingle\u201d agent to simultaneously target multiple distinct epitopes on the spike is desirable to overcome the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained the specificity and simultaneously bound to the two distinct epitopes on RBD and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs) and variants being monitored (VBMs) compared with its parental mAbs. Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs including Alpha (B.1.1.7), Beta (B.1.351) and Kappa (B.1.617.1) and VOCs including Delta (B.1.617.2), Omicron BA.1 and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50= 27.3 ng/mL) and Omicron BA.2 (IC50= 121.1 ng/mL) compared with their parental mAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. The new highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, that simultaneously targets SARS-CoV-2 RBD and S2 and improved neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy to treat and prevent COVID-19.","version":"1.1","doi":"10.1101/2022.05.11.491588","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.491349","pub_date":"2022-5-12","title":"Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants","abstract":"Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9+ million SARS-CoV-2 sequences revealed mutations across Nsp15\u2019s three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.","version":"1.1","doi":"10.1101/2022.05.10.491349","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.11.491583","pub_date":"2022-5-12","title":"Effect on the conformations of spike protein of SARS-CoV-2 due to mutation","abstract":"The spike protein of SARS CoV-2 mediates receptor binding and cell entry and is the key immunogenic target for virus neutralization and the present attention of many vaccine layouts. It exhibits significant conformational flexibility. We study the structural fluctuations of spike protein among the most common mutations appeared in variant of concerns (VOC). We report the thermodynamics of conformational changes in mutant spike protein with respect to the wildtype from the distributions of the dihedral angles obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. We find that the mutation causes the increase in distance between N-terminal domain and receptor binding domain leading to an obtuse angle cosine \u03b8 distribution in the trimeric structure in spike protein. Thus, increase in open-state is conferred to the more infectious variants of SARS-CoV-2. The thermodynamically destabilized and disordered residues of receptor binding motif among the mutant variants of spike protein are proposed to serve as better binding sites for host factor. We identify a short stretch of region connecting the N-terminal domain and receptor binding domain forming linker loop where many residues undergo stabilization in the open state compared to the closed one.","version":"1.1","doi":"10.1101/2022.05.11.491583","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.11.491557","pub_date":"2022-5-12","title":"A Mathematical Model of the Within-Host Kinetics of SARS-CoV-2 Neutralizing Antibodies Following COVID-19 Vaccination","abstract":"Compelling evidence continues to build to support the idea that SARS-CoV-2 Neutralizing Antibody (NAb) levels in an individual can serve as an important indicator of the strength of protective immunity against infection. It is not well understood why NAb levels in some individuals remain high over time, while in others levels decline rapidly. In this work, we present a two-population mathematical model of within-host NAb dynamics in response to vaccination. By fitting only four host-specific parameters, the model is able to capture individual-specific NAb levels over time as measured by the AditxtScore\u2122 for NAbs. The model can serve as a foundation for predicting NAb levels in the long-term, understanding connections between NAb levels, protective immunity, and break-through infections, and potentially guiding decisions about whether and when a booster vaccination may be warranted.","version":"1.1","doi":"10.1101/2022.05.11.491557","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489942","pub_date":"2022-5-11","title":"Immediate myeloid depot for SARS-CoV-2 in the human lung","abstract":"In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.","version":"1.2","doi":"10.1101/2022.04.28.489942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.09.467911","pub_date":"2022-5-11","title":"Evidence for a long-range RNA-RNA interaction between ORF8 and Spike of SARS-CoV-2","abstract":"SARS-CoV-2 has affected people worldwide as the causative agent of COVID-19. The virus is related to the highly lethal SARS-CoV responsible for the 2002-2003 SARS outbreak in Asia. Research is ongoing to understand why both viruses have different spreading capacities and mortality rates. Like other beta coronaviruses, RNA-RNA interactions occur between different parts of the viral genomic RNA, resulting in discontinuous transcription and production of various sub-genomic RNAs. These sub-genomic RNAs are then translated into other viral proteins. In this work, we performed a comparative analysis for novel long-range RNA-RNA interactions that may involve the Spike region. Comparing predictions between reference sequences of SARS-CoV-1 and SARS-CoV-2 revealed several predictions amongst which a thermodynamically stable long-range RNA-RNA interaction between (23660-23703 Spike) and (28025-28060 ORF8) unique to SARS-CoV-2 was observed. Using data gathered worldwide, sequence variation patterns observed in the population support the in-silico RNA-RNA base-pairing predictions within these regions, suggesting further evidence for the interaction. The predicted interactions can potentially be related to the regulation of sub-genomic RNA production rates in SARS-CoV-2 and their subsequent accessibility to the host transcriptome.","version":"1.2","doi":"10.1101/2021.11.09.467911","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.491295","pub_date":"2022-5-11","title":"Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals","abstract":"The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named \u03b1Reps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent \u03b1Rep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, \u03b1Reps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment. The entry of SARS-CoV-2 in permissive cells is mediated by the binding of its spike to angiotensin-converting enzyme 2 (ACE2) on the cell surface. To select ligands able to block this interaction, we screened a library of phages encoding artificial proteins (named \u03b1Reps) for binding to its receptor binding domain (RBD). Two of them were able to bind the RBD with high affinity and block efficiently the virus entry in cultured cells. Assembled \u03b1Reps through covalent or non-covalent linkages blocked virus entry at lower concentration than their precursors (with around 20-fold activity increase for a trimeric \u03b1Rep). These \u03b1Reps derivates neutralize efficiently SARS-CoV-2 \u03b2, \u03b3, \u03b4 and Omicron virus variants. Instillation of an \u03b1Rep dimer in the nasal cavity effectively reduced virus replication in the hamster model of SARS-CoV-2 and pathogenicity.","version":"1.1","doi":"10.1101/2022.05.10.491295","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446179","pub_date":"2022-5-11","title":"Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium","abstract":"SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins \u2013 the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.","version":"1.4","doi":"10.1101/2021.05.28.446179","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.09.491227","pub_date":"2022-5-11","title":"Investigating the evolutionary origins of the first three SARS-CoV-2 variants of concern","abstract":"The emergence of Variants of Concern (VOCs) of SARS-CoV-2 with increased transmissibility, immune evasion properties, and virulence poses a great challenge to public health. Despite unprecedented efforts to increase genomic surveillance, fundamental facts about the evolutionary origins of VOCs remain largely unknown. One major uncertainty is whether the VOCs evolved during transmission chains of many acute infections or during long-term infections within single individuals. We test the consistency of these two possible paths with the observed dynamics, focusing on the clustered emergence of the first three VOCs, Alpha, Beta, and Gamma, in late 2020, following a period of relative evolutionary stasis. We consider a range of possible fitness landscapes, in which the VOC phenotypes could be the result of single mutations, multiple mutations that each contribute additively to increasing viral fitness, or epistatic interactions among multiple mutations that do not individually increase viral fitness\u2014a \u201cfitness plateau\u201d. Our results suggest that the timing and dynamics of the VOC emergence, together with the observed number of mutations in VOC lineages, are in best agreement with the VOC phenotype requiring multiple mutations and VOCs having evolved within single individuals with long-term infections.","version":"1.1","doi":"10.1101/2022.05.09.491227","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.490700","pub_date":"2022-5-11","title":"Delayed antigen-specific CD4+ T-cell induction correlates with impaired immune responses to SARS-COV-2 mRNA vaccination in the elderly","abstract":"Despite the clinical efficacy of coronavirus disease 2019 mRNA vaccines, the elderly demonstrate lower IgG levels and neutralizing titers and a higher risk of severe diseases. CD4+ T cells play a central role in regulating antigen-specific antibody and CD8+ T-cell responses; however, because their composition and functionality change significantly with age, relationships between age-associated defects in T cells and the immunogenicity of or reactogenicity to mRNA vaccines are unclear. Using a vaccine cohort (n = 216), we found that the elderly (aged \u226565 years) showed delayed induction and early contraction of vaccine-specific CD4+ T cells, and that the compromised C\u2013X\u2013C motif chemokine receptor 3+ circulating T follicular helper cell response after the first dose was associated with the lower IgG levels. Additionally, the elderly experienced significantly fewer systemic adverse effects (AEs) after the second dose, with those exhibiting few AEs showing lower cytokine+ CD4+ T cells after the first dose and lower antibody levels after the second dose. Furthermore, T helper 1 cells in the elderly expressed higher levels of programmed cell death protein-1, a negative regulator of the T-cell response, which was associated with less production of vaccine-specific CD4+ T cells and impaired CD8+ T-cell expansion. Thus, efficient induction of vaccine-specific effector/memory CD4+ T cells after the first dose may trigger robust cytokine production after the second dose, leading to effective vaccine responses and higher systemic reactogenicity. These results suggested that an enhanced CD4+ T-cell response after the first dose is key to improved vaccination efficacy in the elderly. We compared immunogenicity and reactogenicity to COVID-19 mRNA vaccine in 107 adults (aged <65 years) and 109 elderly (aged \u226565) individuals.","version":"1.1","doi":"10.1101/2022.05.10.490700","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.09.491179","pub_date":"2022-5-10","title":"Adenovirus-Vectored SARS-CoV-2 Vaccine Expressing S1-N Fusion Protein","abstract":"Additional COVID-19 vaccines that are safe, easy to manufacture, and immunogenic are needed for global vaccine equity. Here, we developed a recombinant type 5 adenovirus vector encoding for the SARS-CoV-2-S1 subunit antigen and nucleocapsid as a fusion protein (Ad5.SARS-CoV-2-S1N) delivered to BALB/c mice through multiple vaccine administration routes. A single subcutaneous (S.C.) immunization with Ad5.SARS-CoV-2-S1N induced a similar humoral response, along with a significantly higher S1-specific cellular response, as a recombinant type 5 adenovirus vector encoding for S1 alone (Ad5.SARS-CoV-2-S1). Immunogenicity was improved by homologous prime boost strategies, using either S.C. or intranasal (I.N.) delivery of Ad5.SARS-CoV-2-S1N, and further improved through heterologous prime boost, with traditional intramuscular (I.M.) injection, using subunit recombinant S1 protein. Priming with low dose (1\u00d71010 v.p.) of Ad5.SARS-CoV-2-S1N and boosting with either wildtype recombinant rS1 or B.1.351 recombinant rS1 induced a robust neutralizing response, that was sustained against immune evasive Beta and Gamma SARS-CoV-2 variants, along with a long-lived plasma cell response in the bone marrow 29 weeks post vaccination. This novel Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity in mice and supports the further development of COVID-19 based vaccines incorporating the nucleoprotein as a target antigen.","version":"1.2","doi":"10.1101/2022.05.09.491179","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.10.491301","pub_date":"2022-5-10","title":"Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants","abstract":"The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike(S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the Wild-type, B.1.1.7, B.1.351, B.1.617.2, and the omicron B.1.1.529 variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. We also proved the safety of RQ3013 in NHP models. Our results provided key support for the evaluation of RQ3013 in clinical trials.","version":"1.1","doi":"10.1101/2022.05.10.491301","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.07.491004","pub_date":"2022-5-10","title":"SARS-CoV-2 Spike N-Terminal Domain modulates TMPRSS2-dependent viral entry and fusogenicity","abstract":"Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. We have previously shown Delta spike confers rapid cell-cell fusion kinetics; here we show that increased fusogenicity can be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of successful SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.","version":"1.2","doi":"10.1101/2022.05.07.491004","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.09.491201","pub_date":"2022-5-10","title":"COVID-19 mRNA third dose induces a unique hybrid immunity-like antibody response","abstract":"The continuous evolution of SARS-CoV-2 generated highly mutated variants, like omicron BA.1 and BA.2, able to escape natural and vaccine-induced primary immunity. The administration of a third dose of mRNA vaccines induces a secondary response with increased protection. We investigated, at single-cell level, the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses. A total of 4,100 spike protein specific memory B cells were single cell sorted and 350 neutralizing antibodies were identified. The third dose increased the antibody neutralization potency and breadth against all SARS-CoV-2 variants of concern as previously observed with hybrid immunity. However, the B cell repertoire that stands behind the response is dramatically different. The increased neutralizing response was largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization such as IGHV3-53;IGHJ6-1 and IGHV3-66;IGHJ4-1. Divergently to hybrid immunity, cross-protection after a third dose was mainly guided by Class 1/2 antibodies encoded by IGHV1-58;IGHJ3-1 and IGHV1-69;IGHJ4-1 germlines. The IGHV2-5;IGHJ3-1 germline, which induced broadly cross-reactive Class 3 antibodies after infection or viral vector vaccination, was not induced by a third mRNA dose. Our data show that while neutralizing breadth and potency can be improved by different immunization regimens, each of them has a unique molecular signature which should be considered while designing novel vaccines and immunization strategies.","version":"1.1","doi":"10.1101/2022.05.09.491201","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444881","pub_date":"2022-5-09","title":"Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques","abstract":"Coronavirus disease 2019 (COVID-19), which is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, continues to threaten global public health. Developing a vaccine that only requires single immunization but provides long-term protection for the prevention and control of COVID-19 is important. Here, we developed an adeno-associated virus (AAV)-based vaccine expressing a stable receptor-binding domain (SRBD) protein. The vaccine requires only a single shot but provides effective neutralizing antibodies (NAbs) over 598 days in rhesus macaques (Macaca mulatta). Importantly, our results showed that the NAbs were kept in high level and long lasting against authentic wild-type SARS-CoV-2, Beta, Delta and Omicron variants using plaque reduction neutralization test. Of note, although we detected pre-existing AAV2/9 antibodies before immunization, the vaccine still induced high and effective NAbs against COVID-19 in rhesus macaques. AAV-SRBD immune serum also efficiently inhibited the binding of ACE2 with RBD in the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1/P.2 (Gamma), B.1.617.2 (Delta), B.1.617.1/3(Kappa), and C.37 (Lambda) variants. Thus, these data suggest that the vaccine has great potential to prevent the spread of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.05.19.444881","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484436","pub_date":"2022-5-09","title":"High neutralizing antibody levels against SARS-CoV-2 Omicron BA.1 and BA.2 after UB-612 vaccine booster","abstract":"The highly transmissible Omicron variant has caused high rates of breakthrough infections among vaccinated and convalescent individuals. Here, we demonstrate that a booster dose of UB-612 vaccine candidate delivered 7-9 months after primary vaccination increases neutralizing antibody levels by 131-, 61- and 49-fold against ancestral SARS-CoV-2, Omicron BA.1 and BA.2 variants, respectively. Based on the RBD protein binding antibody responses, we estimated a \u223c95% efficacy against symptomatic COVID-19 caused by the ancestral strain after a UB-612 booster. Our results support UB-612 vaccine as a potent booster against current and emerging SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2022.03.18.484436","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.06.22274613","pub_date":"2022-05-09","title":"Exploratory data on the clinical efficacy of monoclonal antibodies against SARS-CoV-2 Omicron Variant of Concern","abstract":"<jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Recent in-vitro data have shown that the activity of monoclonal antibodies (mAbs) targeting SARS-CoV-2 varies according to the Variant of Concern (VOC). No studies have compared the clinical efficacy of different mAbs against Omicron VOC.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>The MANTICO trial is a non-inferiority randomised controlled trial comparing the clinical efficacy of bamlanivimab/etesevimab, casirivimab/imdevimab, and sotrovimab in outpatients aged 50 or older with early COVID-19. As the patient enrolment was interrupted for possible futility after the onset of the Omicron wave, the analysis was performed according to the SARS-CoV-2 VOC. The primary outcome was COVID-19 progression (hospitalisation, need of supplemental oxygen therapy, or death through day 14). Secondary outcomes included the time to symptom resolution, assessed using the product-limit method. Kaplan-Meier estimator and Cox proportional hazard model were used to assess the association with predictors. Log rank test was used to compare survival functions.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Overall, 319 patients were included. Among 141 patients infected with Delta, no disease progression was recorded and the time to symptom resolution did not differ significantly between treatment groups (Log-rank Chi-square 0.22,\n                    <jats:italic>p</jats:italic>\n                    0.895). Among 170 patients infected with Omicron (80.6% BA.1, 19.4% BA.1.1), two disease progressions were recorded in the bamlanivimab/etesevimab group and the median time to symptom resolution was 5 days shorter in the sotrovimab group compared to bamlanivimab/etesevimab and casirivimab/imdevimab (HR 0.526 and HR 0.451, 95% CI 0.359 - 0.77 and 95% CI 0.303 - 0.669,\n                    <jats:italic>p</jats:italic>\n                    0.001 and 0.0001, respectively).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>These results confirm the in-vitro data of superiority of sotrovimab versus casirivimab/imdevimab and bamlanivimab/etesivamab in reducing the time to recovery in patients infected with Omicron BA.1 and BA.1.1, while no difference was detected in Delta infections. Casirivimab/imdevimab seems to maintain a role in preventing severe COVID-19 in the Omicron population. Adaptive clinical trials comparing mAbs by VOC should be pursued to promptly inform clinical recommendations.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This trial was funded by the Italian Medicines Agency (Agenzia Italiana del Farmaco, AIFA). The VOC identification was funded by the ORCHESTRA (Connecting European Cohorts to Increase Common and Effective Response to SARS-CoV-2 Pandemic) project, which has received funding from the European Union\u2019s Horizon 2020 research and innovation programme under grant agreement No 101016167.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Clinical trial number</jats:title>\n                  <jats:p>NCT05205759</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.05.06.22274613","journal":"medRxiv","score":null},{"id":"10.1101/2022.05.09.491196","pub_date":"2022-5-09","title":"Persistent serum protein signatures define an inflammatory subset of long COVID","abstract":"Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months after acute SARS-CoV-2 infection. The etiologies are unknown but may include persistent inflammation, unresolved tissue damage, or delayed clearance of viral protein or RNA. Attempts to classify subsets of PASC by symptoms alone have been unsuccessful. To molecularly define PASC, we evaluated the serum proteome in longitudinal samples from 55 PASC individuals with symptoms lasting \u226560 days after onset of acute infection and compared this to symptomatically recovered SARS-CoV-2 infected and uninfected individuals. We identified subsets of PASC with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-\u03baB signaling (particularly associated with TNF), were the most differentially enriched pathways. These findings help to resolve the heterogeneity of PASC, identify patients with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance. Serum proteome profiling identifies subsets of long COVID patients with evidence of persistent inflammation including key immune signaling pathways that may be amenable to therapeutic intervention.","version":"1.1","doi":"10.1101/2022.05.09.491196","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.07.491022","pub_date":"2022-5-08","title":"Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection","abstract":"The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host-virus interactions is essential for development of new COVID-19 treatment strategies. Here we show that SARS-CoV-2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. No changes to other cyclins or cyclin dependent kinases were observed. Further, cyclin D depletion was independent from SARS-CoV-2 mediated cell cycle arrest in early S phase or S/G2/M phase. Cyclin D3 knockdown by small interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co-immunoprecipitated with SARS-CoV-2 Envelope and Membrane proteins. We propose that cyclin D3 inhibits virion assembly and is depleted during SARS-CoV-2 infection to restore efficient assembly and release of newly produced virions.","version":"1.1","doi":"10.1101/2022.05.07.491022","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.07.491038","pub_date":"2022-5-08","title":"Simultaneous and sequential multi-species coronavirus vaccination","abstract":"Although successful COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging for development of multi-species coronavirus vaccines. Here we developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 (Delta variant), SARS-CoV and MERS-CoV, and test how multiplexing of these LNP-mRNAs can induce effective immune responses in animal models. A triplex scheme of LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV, with a relatively weaker MERS-CoV response in this setting. Single cell RNA-seq profiled the global systemic immune repertoires and the respective transcriptome signatures of multiplexed vaccinated animals, which revealed a systemic increase in activated B cells, as well as differential gene expression signatures across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated the feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species. Multiplexed mRNA vaccination in simultaneous and sequential modes provide broad and potent immunity against pathogenic coronavirus species.","version":"1.1","doi":"10.1101/2022.05.07.491038","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.06.490915","pub_date":"2022-5-06","title":"Reaction Conditions Promoting the Specific Detection of SARS-CoV-2 NendoU Enzymatic Activity","abstract":"Methods that enable rapid detection of SARS-CoV-2 provide valuable tools for detecting and controlling Covid-19 outbreaks and also facilitate more effective treatment of infected individuals. The predominant approaches developed use PCR to detect viral nucleic acids or immunoassays to detect viral proteins. Each approach has distinct advantages and disadvantages, but alternatives that do not share the same limitations could enable substantial improvements in outbreak detection and management. For instance, methods that have comparable sensitivity to PCR, but that are not prone to the false-positive results that stem from the tendency of PCR to detect molecular degradation products could improve accurate identification of infected individuals. An alternative approach with potential to achieve this entails harnessing the unique enzymatic properties of SARS-CoV-2 enzymes to generate SARS-Cov-2-specific signals that indicate the presence of the virus. This route benefits from the high sensitivity provided by enzymatic signal amplification and also the fact that signal is generated only by intact viral enzymes, not degradation products. Here, we demonstrate enzymatic reaction conditions that enable the preferential detection of NendoU of SARS-CoV-2, versus several of its orthologues, with a fluorogenic oligonucleotide substrate. These compositions provide a possible technical foundation for a novel approach for detecting SARS-CoV-2 that has distinct advantages from current approaches.","version":"1.1","doi":"10.1101/2022.05.06.490915","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.06.490867","pub_date":"2022-5-06","title":"SARS-CoV-2 evolution and patient immunological history shape the breadth and potency of antibody-mediated immunity","abstract":"Since the emergence of SARS-CoV-2, humans have been exposed to distinct SARS-CoV-2 antigens, either by infection with different variants, and/or vaccination. Population immunity is thus highly heterogeneous, but the impact of such heterogeneity on the effectiveness and breadth of the antibody-mediated response is unclear. We measured antibody-mediated neutralisation responses against SARS-CoV-2Wuhan, SARS-CoV-2\u03b1, SARS-CoV-2\u03b4 and SARS-CoV-2\u03bf pseudoviruses using sera from patients with distinct immunological histories, including naive, vaccinated, infected with SARS-CoV-2Wuhan, SARS-CoV-2\u03b1 or SARS-CoV-2\u03b4, and vaccinated/infected individuals. We show that the breadth and potency of the antibody-mediated response is influenced by the number, the variant, and the nature (infection or vaccination) of exposures, and that individuals with mixed immunity acquired by vaccination and natural exposure exhibit the broadest and most potent responses. Our results suggest that the interplay between host immunity and SARS-CoV-2 evolution will shape the antigenicity and subsequent transmission dynamics of SARS-CoV-2, with important implications for future vaccine design. Neutralising antibodies provide protection against viruses and are generated because of vaccination or prior infections. The main target of anti-SARS-CoV-2 neutralising antibodies is a protein called Spike, which decorates the viral particle and mediates viral entry into cells. As SARS-CoV-2 evolves, mutations accumulate in the spike protein, allowing the virus to escape antibody-mediated immunity and decreasing vaccine effectiveness. Multiple SARS-CoV-2 variants have appeared since the start of the COVID-19 pandemic, causing various waves of infection through the population and infecting-in some cases-people that had been previously infected or vaccinated. Since the antibody response is highly specific, individuals infected with different variants are likely to have different repertoires of neutralising antibodies. We studied the breadth and potency of the antibody-mediated response against different SARS-CoV-2 variants using sera from vaccinated people as well as from people infected with different variants. We show that potency of the antibody response against different SARS-CoV-2 variants depends on the particular variant that infected each person, the exposure type (infection or vaccination) and the number and order of exposures. Our study provides insight into the interplay between virus evolution and immunity, as well as important information for the development of better vaccination strategies.","version":"1.1","doi":"10.1101/2022.05.06.490867","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.05.490815","pub_date":"2022-5-06","title":"Immunological findings in a group of individuals who were non-responders to standard two-dose SARS-CoV-2 vaccines","abstract":"Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic. The virus has infected more than 505 million people and caused more than 6 million deaths. However, data on non-responders to SARS-CoV-2 vaccines in the general population are limited. The objective of the study is to comprehensively compare the immunological characteristics of non-responders to SARS-CoV-2 vaccines in the 18-59 years with that in the 60 years and older using internationally recognized cutoff values. Participants included 627 individuals who received physical examinations and volunteered to participate in COVID-19 vaccination from the general population. The main outcome was an effective seroconversion characterized by anti-SARS-CoV-2 spike IgG level of at least 4-fold increase from baseline. Profiling of naive immune cells was analyzed prior to vaccination to demonstrate baseline immunity. Outcomes of effective seroconversion in the 18-59 years with that in the 60 years and older were compared. The quantitative level of the anti-spike IgG was significantly lower in the 60 years and older and in men among the 18-59 years. There were 7.5% of non-responders among the 18-59 years and 11.7% of non-responders in the 60 years and older using the 4-fold increase parameter. The effective seroconversion rate was significantly related to the level of certain immune cells before vaccination, such as CD4 cells, CD8 cells and B cells and the age. An individual with a titer of anti-SARS-CoV-2 spike IgG that is below 50 BAU/mL might be considered a non-responder between 14-90 days after the last vaccine dose. Booster vaccination or additional protective measures should be recommended for non-responders as soon as possible to reduce disease severity and mortality.","version":"1.1","doi":"10.1101/2022.05.05.490815","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.05.490850","pub_date":"2022-5-06","title":"ORF6 protein of SARS-CoV-2 inhibits TRIM25 mediated RIG-I ubiquitination to mitigate type I IFN induction","abstract":"Evasion and antagonism of host cellular immunity upon SARS-CoV-2 infection confers a profound replication advantage on the virus and contributes to COVID-19 pathogenesis. We explored the ability of different SARS-CoV-2 proteins to antagonize the host innate immune system and found that the ORF6 protein mitigated type-I IFN (interferon) induction and downstream IFN signaling. Our findings also corroborated previous reports that ORF6 blocks the nuclear import of IRF3 and STAT1 to inhibit IFN induction and signaling. Here we show that ORF6 directly interacts with RIG-I and blocks downstream type-I IFN induction and signaling by inhibiting K-63 linked ubiquitination of RIG-I by the E3 Ligase TRIM25. This involves ORF6-mediated targeting of TRIM25 for degradation, also observed during SARS-CoV-2 infection. The type-I IFN antagonistic activity of ORF6 was mapped to its C-terminal cytoplasmic tail, specifically to amino acid residues 52-61. Overall, we provide new insights into how the SARS-CoV-2 ORF6 protein inhibits type I-IFN induction and signaling through distinct mechanisms.","version":"1.1","doi":"10.1101/2022.05.05.490850","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.06.490883","pub_date":"2022-5-06","title":"Modelling the within-host spread of SARS-CoV-2 infection, and the subsequent immune response, using a hybrid, multiscale, individual-based model. Part I: Macrophages","abstract":"Individual responses to SARS-CoV-2 infection vary significantly, ranging from mild courses of infection that do not require hospitalisation to the development of disease which not only requires hospitalisation but can be fatal. Whilst many immunological studies have revealed fundamental insights into SARS-CoV-2 infection and COVID-19, mathematical and computational modelling can offer an additional perspective and enhance understanding. The majority of mathematical models for the within-host spread of SARS-CoV-2 infection are ordinary differential equations, which neglect spatial variation. In this article, we present a hybrid, multiscale, individual-based model to study the within-host spread of SARS-CoV-2 infection. The model incorporates epithelial cells (each containing a dynamical model for viral entry and replication), macrophages and a subset of cytokines. We investigate the role of increasing initial viral deposition, increasing delay in type I interferon secretion from epithelial cells (as well as the magnitude of secretion), increasing macrophage virus internalisation rate and macrophage activation, on the spread of infection.","version":"1.1","doi":"10.1101/2022.05.06.490883","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.06.490927","pub_date":"2022-5-06","title":"Molecular dynamics of spike variants in the locked conformation: RBD interfaces, fatty acid binding and furin cleavage sites","abstract":"Since December 2019 the SARS-CoV-2 virus has infected billions of people around the world and caused millions of deaths. The ability for this RNA virus to mutate has produced variants that have been responsible for waves of infections across the globe. The spike protein on the surface of the SARS-CoV-2 virion is responsible for cell entry in the infection process. Here we have studied the spike proteins from the Original, Alpha (B.1.1.7), Delta (B1.617.2), Delta-plus (B1.617.2-AY1), Omicron BA.1 and Omicron BA.2 variants. Using models built from cryo-EM structures with linoleate bound (6BZ5.pdb) and the N-terminal domain from 7JJI.pdb, each is built from the first residue, with missing loops modelled and 45 disulphides per trimer. Each spike variant was modified from the same Original model framework to maximise comparability. Three replicate, 200 ns atomistic molecular dynamics simulations were performed for each case. (These data also provide the basis for further, non-equilibrium molecular dynamics simulations, published elsewhere.) The analysis of our equilibrium molecular dynamics reveals that sequence variation at the closed receptor binding domain interface particularly for Omicron BA.2 has implications for the avidity of the locked conformation, with potential effects on Omicron BA.1 and Delta-plus. Linoleate binding has a mildly stabilizing effect on furin cleavage site motions in the Original and Alpha variants, but has no effect in Delta, Delta-plus and slightly increases motions at this site for Omicron BA.1, but not BA.2, under these simulation conditions.","version":"1.1","doi":"10.1101/2022.05.06.490927","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.04.490692","pub_date":"2022-5-05","title":"Net-shaped DNA nanostructure designed for rapid/sensitive detection and potential inhibition of SARS-CoV-2 virus","abstract":"We present a net-shaped DNA nanostructure (called \u201cDNA Net\u201d herein) design strategy for selective recognition and high-affinity capture of the intact SARS-CoV-2 virions through spatial pattern-matching and multivalent interactions between the aptamers (targeting wild type spike-RBD) positioned on the DNA Net and the trimeric spike glycoproteins displayed on the viral outer surface. Carrying a designer nanoswitch, the DNA Net-aptamers releases fluorescent signal upon virus binding that is easily read by a hand-held fluorimeter for a rapid (in 10 mins), simple (mix- and-read), sensitive (PCR equivalent), room temperature compatible, and inexpensive (\u223c $1.26/test) COVID-19 test assay. The DNA Net-aptamers also impede authentic wild-type SARS-CoV-2 infection in cell culture with a near 1\u00d7103-fold enhancement of the monomeric aptamer. Furthermore, our DNA Net design principle and strategy can be customized to tackle other life-threatening and economically influential viruses like influenza and HIV, whose surfaces carry class-I viral envelope glycoproteins like the SARS-CoV-2 spikes in trimeric forms.","version":"1.1","doi":"10.1101/2022.05.04.490692","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.04.490631","pub_date":"2022-5-04","title":"Adsorption of Pulmonary and Exogeneous Surfactants on SARS-CoV-2 Spike Protein","abstract":"COVID-19 is transmitted by inhaling SARS-CoV-2 virions, which are enveloped by a lipid bilayer decorated by a \u201ccrown\u201d of Spike protein protrusions. In the respiratory tract, virions interact with surfactant films composed of phospholipids and cholesterol that coat lung airways. Here, we explore by using coarse-grained molecular dynamics simulations the physico-chemical mechanisms of surfactant adsorption on Spike proteins. With examples of zwitterionic dipalmitoyl phosphatidyl choline, cholesterol, and anionic sodium dodecyl sulphate, we show that surfactants form micellar aggregates that selectively adhere to the specific regions of S1 domain of the Spike protein that are responsible for binding with ACE2 receptors and virus transmission into the cells. We find high cholesterol adsorption and preferential affinity of anionic surfactants to Arginine and Lysine residues within S1 receptor binding motif. These findings have important implications for informing the search for extraneous therapeutic surfactants for curing and preventing COVID-19 by SARS-CoV-2 and its variants.","version":"1.1","doi":"10.1101/2022.05.04.490631","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.21.469172","pub_date":"2022-5-04","title":"Identification of a promiscuous conserved CTL epitope within the SARS-CoV-2 spike protein","abstract":"The COVID-19 disease caused by infection with SARS-CoV-2 and its variants is devastating to the global public health and economy. To date, over a hundred COVID-19 vaccines are known to be under development and the few that have been approved to fight the disease are using the spike protein as the primary target antigen. Although virus neutralizing epitopes are mainly located within the RBD of the spike protein, the presence of T cell epitopes, particularly the CTL epitopes that are likely to be needed for killing infected cells, has received comparatively little attention. In this study, we predicted several potential T cell epitopes with web-based analytic tools, and narrowed them down from several potential MHC-I and MHC-II epitopes by ELIspot and cytolytic assays to a conserved MHC-I epitope. The epitope is highly conserved in current viral variants including the most recent Omicron and compatible with presentation by most HLA alleles worldwide. In conclusion, we identified a CTL epitope suitable for evaluating the CD8+ T cell-mediated cellular response and potentially for addition into future COVID-19 vaccine candidates to maximize CTL responses against SARS-CoV-2.","version":"1.3","doi":"10.1101/2021.11.21.469172","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.472458","pub_date":"2022-5-04","title":"Computational Mapping of the Human-SARS-CoV-2 Protein-RNA Interactome","abstract":"Strong evidence suggests that human human RNA-binding proteins (RBPs) are critical factors for viral infection, yet there is no feasible experimental approach to map exact binding sites of RBPs across the SARS-CoV-2 genome systematically at a large scale. We investigated the role of RBPs in the context of SARS-CoV-2 by constructing the first in silico map of human RBP / viral RNA interactions at nucleotide-resolution using two deep learning methods (pysster and DeepRiPe) trained on data from CLIP-seq experiments. We evaluated conservation of RBP binding between 6 other human pathogenic coronaviruses and identified sites of conserved and differential binding in the UTRs of SARS-CoV-1, SARS-CoV-2 and MERS. We scored the impact of variants from 11 viral strains on protein-RNA interaction, identifying a set of gain-and loss of binding events. Lastly, we linked RBPs to functional data and OMICs from other studies, and identified MBNL1, FTO and FXR2 as potential clinical biomarkers. Our results contribute towards a deeper understanding of how viruses hijack host cellular pathways and are available through a comprehensive online resource (https://sc2rbpmap.helmholtz-muenchen.de).","version":"1.3","doi":"10.1101/2021.12.22.472458","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.03.490381","pub_date":"2022-5-03","title":"Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9+Spp1+ macrophages promoting viral clearance and inflammation resolution","abstract":"While SARS-CoV-2 pathogenesis has been intensively investigated, the host mechanisms of viral clearance and inflammation resolution are still elusive because of the ethical limitation of human studies based on COVID-19 convalescents. Here we infected Syrian hamsters by authentic SARS-CoV-2 and built an ideal model to simulate the natural recovery process of SARS-CoV-2 infection from severe pneumonia. We developed and applied a spatial transcriptomic sequencing technique with subcellular resolution and tissue-scale extensibility, i.e., Stereo-seq, together with single-cell RNA sequencing (scRNA-seq), to the entire lung lobes of 45 hamsters and obtained an elaborate map of the pulmonary spatiotemporal changes from acute infection, severe pneumonia to the late viral clearance and inflammation resolution. While SARS-CoV-2 infection caused massive damages to the hamster lungs, including na\u00efve T cell infection and deaths related to lymphopenia, we identified a group of monocyte-derived proliferating Slamf9+Spp1+ macrophages, which were SARS-CoV-2 infection-inducible and cell death-resistant, recruiting neutrophils to clear viruses together. After viral clearance, the Slamf9+Spp1+ macrophages differentiated into Trem2+ and Fbp1+ macrophages, both responsible for inflammation resolution and replenishment of alveolar macrophages. The existence of this specific macrophage subpopulation and its descendants were validated by RNAscope in hamsters, immunofluorescence in hACE2 mice, and public human autopsy scRNA-seq data of COVID-19 patients. The spatiotemporal landscape of SARS-CoV-2 infection in hamster lungs and the identification of Slamf9+Spp1+ macrophages that is pivotal to viral clearance and inflammation resolution are important to better understand the critical molecular and cellular players of COVID-19 host defense and also develop potential interventions of COVID-19 immunopathology.","version":"1.1","doi":"10.1101/2022.05.03.490381","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.03.490409","pub_date":"2022-5-03","title":"Sensitivity of novel SARS-CoV-2 Omicron subvariants, BA.2.11, BA.2.12.1, BA.4 and BA.5 to therapeutic monoclonal antibodies","abstract":"As of May 2022, Omicron BA.2 variant is the most dominant variant in the world. Thereafter, Omicron subvariants have emerged and some of them began outcompeting BA.2 in multiple countries. For instance, Omicron BA.2.11, BA.2.12.1 and BA.4/5 subvariants are becoming dominant in France, the USA and South Africa, respectively. In this study, we evaluated the sensitivity of these new Omicron subvariants (BA.2.11, BA.2.12.1 and BA.4/5) to eight therapeutic monoclonal antibodies (bamlanivimab, bebtelovimab, casirivimab, cilgavimab, etesevimab, imdevimab, sotrovimab and tixagevimab). Notably, we showed that although cilgavimab is antiviral against BA.2, BA.4/5 exhibits higher resistance to this antibody compared to BA.2. Since mutations are accumulated in the spike proteins of newly emerging SARS-CoV-2 variants, we suggest the importance of rapid evaluation of the efficiency of therapeutic monoclonal antibodies against novel SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.05.03.490409","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.03.490428","pub_date":"2022-5-03","title":"Protection from Omicron and other VOCs by Bivalent S-Trimer COVID-19 Vaccine","abstract":"The Omicron variant of SARS-COV-2 (GISAID GRA clade [B.1.1.529, BA.1 and BA.2]) is now the single dominant Variant of Concern (VOC). The high number of mutations in the Omicron Spike (S) protein promotes humoral immunological escape. Although a third homologous boost with S, derived from the ancestral strain, was able to increase neutralizing antibody titers and breadth including to Omicron, the magnitude of virus neutralization could benefit from further optimization. Moreover, combining SARS-COV-2 strains as additional valences may address the current antigenicity range occupied by VOCs. Using Trimer-Tag\u2122 platform we have previously demonstrated phase 3 efficacy and safety of a prototypic vaccine SCB-2019 in the SPECTRA trial and have submitted applications for licensure. Here, we successfully generated a bivalent vaccine candidate including both Ancestor and Omicron variant S-proteins. Preclinical studies demonstrate this SARS-CoV-2 bivalent S-Trimer subunit vaccine elicits high titers of neutralizing antibodies against all VOCs, with markedly enhanced Omicron specific neutralizing antibody responses.","version":"1.1","doi":"10.1101/2022.05.03.490428","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.02.490272","pub_date":"2022-5-02","title":"Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation","abstract":"Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus- infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the highly pathogenic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2\u03b1 (eIF2\u03b1) phosphorylation and SG formation induced by exogenous stress (e.g. sodium arsenite treatment). Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV-2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2\u03b1 phosphorylation, and the Nsp1 of SARS- CoV2 alone was sufficient to cause decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear retention of the SG- nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in infection compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication. Host cells possess many mechanisms that can detect viral infections and trigger defense programs to suppress viral replication and spread. One of such antiviral mechanisms is the formation of stress granules \u2013 large aggregates of RNA and proteins that sequester viral components and cellular factors needed by the virus to replicate. Because of this threat, viruses evolved specific mechanisms that prevent stress granule formation. Understanding these mechanisms can reveal potential targets for therapies that would disable viral inhibition of stress granules and render cells resistant to infection. In this study we analyzed inhibition of stress granules by two human coronaviruses: the common cold coronavirus OC43 and the pandemic SARS-CoV2. We have demonstrated that these viruses employ at least two proteins \u2013 nucleocapsid protein (N) and the non-structural protein 1 (Nsp1) to suppress stress granules. These proteins act through distinct complementary mechanisms to ensure successful virus replication. Because both OC43 and SARS-CoV2 each dedicate more than one gene product to inhibit stress granule formation, our work suggests that viral disarming of stress granule responses is central for a productive infection.","version":"1.1","doi":"10.1101/2022.05.02.490272","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.30.486882","pub_date":"2022-5-02","title":"The highly conserved stem-loop II motif is important for the lifecycle of astroviruses but dispensable for SARS-CoV-2","abstract":"The stem-loop II motif (s2m) is an RNA element present in viruses from divergent viral families, including astroviruses and coronaviruses, but its functional significance is unknown. We created deletions or substitutions of the s2m in astrovirus VA1 (VA1), classic human astrovirus 1 (HAstV1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For VA1, recombinant virus could not be rescued upon partial deletion of the s2m or substitutions of G-C base pairs. Compensatory substitutions that restored the G-C base-pair enabled recovery of VA1. For HAstV1, a partial deletion of the s2m resulted in decreased viral titers compared to wild-type virus, and reduced activity in a replicon system. In contrast, deletion or mutation of the SARS-CoV-2 s2m had no effect on the ability to rescue the virus, growth in vitro, or growth in Syrian hamsters. Our study demonstrates the importance of the s2m is virus-dependent.","version":"1.1","doi":"10.1101/2022.04.30.486882","journal":"bioRxiv","score":null},{"id":"10.1101/2022.05.01.490203","pub_date":"2022-5-02","title":"SARS-CoV-2 Main Protease: a Kinetic Approach","abstract":"In this article, I present a new model of the interaction of the main protease (Mpro) from SARS-CoV-2 virus with its substrate. The reaction scheme used to describe this mechanism is an extension of the well-known Michaelis-Menten model proposed in 1913 by Leonor Michaelis and Maud Menten [1]. The model I present here takes into account that one Mpro enzyme monomer interacts with another Mpro monomer in the presence of the substrate, leading to the formation of an enzyme dimer bound to one substrate molecule. Indeed, this dimer is formed by the sequentially binding of one Mpro enzyme monomer to one molecule of substrate, followed by another Mpro enzyme monomer binding to this Mpro-substrate complex. This reaction mechanism is also known in the literature as substrate-induced dimerization [3]. Starting from this new reaction scheme established for this catalytic mechanism, I derived a mathematical expression describing the catalytic rate of the active Mpro enzyme dimer as a function of the substrate concentration [S]. The plot corresponding to this substrate-induced dimerization reaction shows a function f ([S]) that is not monotonic, i.e. not strictly increasing or decreasing, but with a second derivative initially negative and then becoming positive after having passed the Vmax point. This is typically a type of curve showing a phenomenon like the one of substrate inhibition (for instance, inhibition by excess-substrate [7]). The graphical representation of this process shows an interesting behaviour: from zero \u03bcM/s, the reaction rate increases progressively, similar to the kind of curve described by the Michaelis-Menten model. However, after having reached its maximum catalytic rate, Vmax, the reaction rate decreases progressively as we continue to increase the substrate concentration. I propose an explanation to this interesting behavior. At the moment where Vcat is maximum, we can assume that, in theory, every single substrate molecule in solution is bound to two enzyme monomers (i.e. to one active dimer). The catalytic rate is thus theoretically maximized. At the time where the reaction rate begins to decrease, we observe a new phenomenon that appears: the enzyme monomers begin to be \u201cdiluted\u201d in the solution containing the excess substrate. The dimers begin to dissociate and to bind increasingly to the substrate as inactive monomers instead of active dimers. Hence, it is more and more unlikely for the enzyme monomers to sequentially bind twice to the same substrate molecule (here, [E] \u226a [S]). Thus, at this stage, the substrate-induced dimerization occurs less often. At the limit, when the substrate is in high excess, there is virtually no more dimerization which occurs. This is one example of excess-substrate inhibition. Furthermore, after having established this fact, I wanted to see if this catalytic behavior was also observed in vitro. Therefore, I conducted an experiment where I measured the catalytic rate of the Mpro dimer for different substrate concentrations. The properties of my substrate construct were such, that I could determine the catalytic rate of the enzyme dimer by directly measuring the spectrophotometric absorbance of the cleaved substrate at \u03bb = 405 nm. The results show explicitly \u2014 within a margin of error \u2014 that the overall shape of the experimental curve looks like the one of the theoretical curve. I thus conclude that the biochemical behavior of the Mpro in vitro follows a new path when it is in contact with its substrate: an excess substrate concentration decreases the activity of the enzyme by the phenomenon of a type of excess-substrate inhibition. This finding could open a new door in the discovery of drugs directed against the Mpro enzyme of the SARS-CoV-2 virus, acting on the inhibition by excess-substrate of the Mpro enzyme, this protein being a key component in the metabolism of the virus. Furthermore, I have established that the maximum of the fitted curve, Vmax, depends only on [E]T and not on [S].  exhibits the same dependence pattern. Therefore, if I keep [E]T close to zero, the catalytic rate of the enzyme will also be greatly reduced, which can be understood intuitively. Finally, if we dilute the enzyme sufficiently in the host cell by injecting a suitably high concentration of the octapeptide substrate AVLQSGFR (an inhibitor of the original substrate), this artificial substrate will bind to the \u201cintermediate\u201d dimer from the polypeptide and prevent the precursor Mpro from auto-cleaving and dimerizing due to the \u201cdistorted key\u201d effect of the octapeptide on the \u201cintermediate\u201d dimer. The precursor peptide Mpro will auto-cleave to a lesser extent than in the absence of the artificial octapeptide and thus the concentration of the total enzyme [E]T will be lowered in the cell. It would therefore be possible to control the virulence of the virus by adjusting the concentration of the artificial inhibitory octapeptide. However, this is only speculation and has yet to be verified in practice.","version":"1.1","doi":"10.1101/2022.05.01.490203","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.29.490117","pub_date":"2022-4-30","title":"Long Term Virucidal Activity of Rosin Soap on Surfaces","abstract":"Microbiocidal products for decontaminating both animate and inanimate surfaces that may act as vectors for disease transmission are a well-established method for inactivating viruses of clinical significance. There are a wide variety of such microbiocidal products that can be purchased commercially, many with different active ingredients. We have recently shown that Rosin soap (derived from Tall Oil that has been produced by trees) is a highly effective virucidal product against enveloped viruses like influenza A virus and SARS-CoV-2 when tested in solution. Here we further demonstrate that Rosin soap retains its virucidal activity against influenza A virus and SARS-CoV-2 when applied to surfaces, such as plastic, glass and steel, either before or after virus inoculation. The virucidal activity extended out to seven days post administration. Together, our results show the potential for Rosin soap-based disinfectants to be used as a surface coating to protect against clinically relevant enveloped viruses, such as influenza viruses and coronaviruses.","version":"1.1","doi":"10.1101/2022.04.29.490117","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.07.483258","pub_date":"2022-4-29","title":"Intragenomic rearrangements in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses","abstract":"Variation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses. Specifically, we report numerous genomic insertions of 5\u2019-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain 5\u2019-UTR-derived inserted sequences. In limited instances, there is mounting evidence that these insertions alter the fundamental biological properties of mutant viruses. Intragenomic rearrangements add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise. Understanding mechanisms of variation in coronaviruses is vital to control of their associated diseases. Beyond point mutations, insertions, deletions and recombination, we here describe for the first time intragenomic rearrangements and their relevance to changes in transmissibility, immune escape and/or virulence documented during the SARS-CoV-2 pandemic.","version":"1.2","doi":"10.1101/2022.03.07.483258","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.29.490044","pub_date":"2022-4-29","title":"A Novel Y-Shaped, S-O-N-O-S-Bridged Crosslink between Three Residues C22, C44, and K61 Is a Redox Switch of the SARS-CoV-2 Main Protease","abstract":"As the COVID-19 pathogen, SARS-CoV-2 relies on its main protease (MPro) for pathogenesis and replication. During the crystallographic analyses of MPro crystals that were exposed to the air, a uniquely Y-shaped, S-O-N-O-S-bridged posttranslational crosslink that connects three residues C22, C44, and K61 at their side chains was frequently observed. As a novel posttranslational modification, this crosslink serves as a redox switch to regulate the catalytic activity of MPro, a demonstrated drug target of COVID-19. The formation of this linkage leads to a much more opened active site that can be potentially targeted for the development of novel SARS-CoV-2 antivirals. The inactivation of MPro by this crosslink indicates that small molecules that lock MPro in the crosslinked form can be potentially used with other active site-targeting molecules such as paxlovid for synergistic effects in inhibiting the SARS-CoV-2 viral replication. Therefore, this new finding reveals a unique aspect of the SARS-CoV-2 pathogenesis and is potentially paradigm-shifting in our current understanding of the function of MPro and the development of its inhibitors as COVID-19 antivirals.","version":"1.1","doi":"10.1101/2022.04.29.490044","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489809","pub_date":"2022-4-29","title":"A bacteriophage-based, highly efficacious, needle and adjuvant-free, mucosal COVID-19 vaccine","abstract":"The authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected and effective in preventing COVID-19, but do not induce efficient mucosal immunity, or prevent viral transmission. We developed a bacteriophage T4-based, multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of T4-COVID vaccine induced higher virus neutralization antibody titers against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger CD4+ and CD8+ T cell immunity, and higher secretory IgA titers in sera and bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain and its Delta variant with minimal lung histopathology. This mucosal vaccine is an excellent candidate for boosting immunity of immunized and/or as a second-generation vaccine for the unimmunized population.","version":"1.1","doi":"10.1101/2022.04.28.489809","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.06.413682","pub_date":"2022-4-29","title":"Scientific publications and COVID-19 \u201cresearch pivots\u201d during the pandemic: An initial bibliometric analysis","abstract":"An examination is presented of scientific research publication trends during the global coronavirus (COVID-19) pandemic in 2020. After reviewing the timing of the emergence of the pandemic in 2020 and the growth of governmental responses, available secondary sources are used to highlight impacts of COVID-19 on scientific research. A bibliometric analysis is then undertaken to analyze developments in COVID-19 related scientific publications through to October of 2020 by broad trends, fields, countries, and organizations. Two publication data sources are used: PubMed and the Web of Science. While there has been a massive absolute increase in PubMed and Web of Science papers directly focused on COVID-19 topics, especially in medical, biological science, and public health fields, this is still a relatively small proportion of publication outputs across all fields of science. Using Web of Science publication data, the paper examines the extent to which researchers across all fields of science have pivoted their research outputs to focus on topics related to COVID-19. A COVID-19 research pivot is defined as the extent to which the proportion of output in a particular research field has shifted to a focus on COVID-19 topics in 2020 (to date) compared with 2019. Significant variations are found by specific fields (identified by Web of Science Subject Categories). In a top quintile of fields, not only in medical specialties, biomedical sciences, and public health but also in subjects in social sciences and arts and humanities, there are relatively high to medium research pivots. In lower quintiles, including other subjects in science, social science, and arts and humanities, low to zero COVID-19 research pivoting is identified. In a new Appendix to the paper, an updated analysis is provided through to mid-April 2022. Shapira, P. \u201cScientific publications and COVID-19 \u201cresearch pivots\u201d during the pandemic: An initial bibliometric analysis,\u201d bioRxiv 2020.12.06.413682; doi: https://doi.org/10.1101/2020.12.06.413682 Version 1: Original paper, completed on December 6, 2020; posted at bioRxiv on December 7, 2020. Version 2: Minor grammar items corrected. Version 3: Updated bibliometric analysis through to mid-April 2022 added on April 29, 2022, as new Appendix 2.","version":"1.2","doi":"10.1101/2020.12.06.413682","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489857","pub_date":"2022-4-29","title":"Systematic analysis of alternative splicing in time course data using Spycone","abstract":"During disease progression or organism development, alternative splicing (AS) may lead to isoform switches (IS) that demonstrate similar temporal patterns and reflect the AS co-regulation of such genes. Tools for dynamic process analysis usually neglect AS. Here we propose Spycone (https://github.com/yollct/spycone), a splicing-aware framework for time course data analysis. Spycone exploits a novel IS detection algorithm and offers downstream analysis such as network and gene set enrichment. We demonstrate the performance of Spycone using simulated and real-world data of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.04.28.489857","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.26.489580","pub_date":"2022-4-28","title":"Diet induced obesity and type 2 diabetes drives exacerbated sex-associated disease profiles in K18-hACE2-mice challenged with SARS-CoV-2","abstract":"SARS-CoV-2 infection results in wide-ranging disease manifestation from asymptomatic to potentially lethal. Infection poses an increased threat of severity to at-risk populations including those with hypertension, diabetes, and obesity. Type 2 Diabetes (T2DM), is characterized, in part, by insulin insensitivity and impaired glucose regulation. T2DM patients have increased disease severity and poorer outcomes with COVID-19. We utilized the diet-induced obesity (DIO) model of Type 2 Diabetes in SARS-CoV-2-susceptible K18-hACE2 transgenic mice to better understand the obesity co-morbidity. Female DIO, but not male DIO mice challenged with SARS-CoV-2 were observed to have shortened time to morbidity compared to normal diet mice. Increase in susceptibility to SARS-CoV2 in female DIO was associated with increased total viral RNA burden compared to male mice. RNAseq analysis was performed on the lungs of non-challenged, challenged, females, males, of either normal diet or DIO cohorts to determine the disease specific transcriptional profiles. DIO female mice had more total activated genes than normal diet mice after challenge; however, male mice experienced a decrease. GO term analysis revealed the DIO condition increased interferon response signatures and interferon gamma production following challenge. Male challenged mice had robust expression of antibody-related genes suggesting antibody producing cell localization in the lung. DIO reduced antibody gene expression in challenged males. Collectively this study establishes a preclinical T2DM/obesity co-morbidity model of COVID-19 in mice where we observed sex and diet specific responses that begin to explain the effects of obesity and diabetes on COVID-19 disease.","version":"1.1","doi":"10.1101/2022.04.26.489580","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474799","pub_date":"2022-4-28","title":"Putative host-derived insertions in the genomes of circulating SARS-CoV-2 variants","abstract":"Insertions in the SARS-CoV-2 genome have the potential to drive viral evolution, but the source of the insertions is often unknown. Recent proposals have suggested that human RNAs could be a source of some insertions, but the small size of many insertions makes this difficult to confirm. Through an analysis of available direct RNA sequencing data from SARS-CoV-2 infected cells, we show that viral-host chimeric RNAs are formed through what are likely stochastic RNA-dependent RNA polymerase template switching events. Through an analysis of the publicly available GISAID SARS-CoV-2 genome collection, we identified two genomic insertions in circulating SARS-CoV-2 variants that are identical to regions of the human 18S and 28S rRNAs. These results provide direct evidence of the formation of viral-host chimeric sequences and the integration of host genetic material into the SARS-CoV-2 genome, highlighting the potential importance of host-derived insertions in viral evolution. Throughout the COVID-19 pandemic, the sequencing of SARS-CoV-2 genomes has revealed the presence of insertions in multiple globally circulating lineages of SARS-CoV-2, including the Omicron variant. The human genome has been suggested to be the source of some of the larger insertions, but evidence for this kind of event occurring is still lacking. Here, we leverage direct RNA sequencing data and SARS-CoV-2 genomes to show host-viral chimeric RNAs are generated in infected cells and two large genomic insertions have likely been formed through the incorporation of host rRNA fragments into the SARS-CoV-2 genome. These host-derived insertions may increase the genetic diversity of SARS-CoV-2 and expand its strategies to acquire genetic materials, potentially enhancing its adaptability, virulence, and spread.","version":"1.2","doi":"10.1101/2022.01.04.474799","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489834","pub_date":"2022-4-28","title":"Induction of neutralizing antibodies against SARS-CoV-2 variants by a multivalent mRNA-lipid nanoparticle vaccine encoding SARS-CoV-2/SARS-CoV Spike protein receptor-binding domains","abstract":"To address the need for multivalent vaccines against Coronaviridae that can be rapidly developed and manufactured, we compared antibody responses against SARS-CoV, SARS-CoV-2, and several variants of concern in mice immunized with mRNA-lipid nanoparticle vaccines encoding homodimers or heterodimers of SARS-CoV/SARS-CoV-2 receptor-binding domains. All vaccine constructs induced robust anti-viral antibody responses, and the heterodimeric vaccine elicited an IgG response capable of cross-neutralizing SARS-CoV, SARS-CoV-2 Wuhan-Hu-1, B.1.351 (beta), and B.1.617.2 (delta) variants.","version":"1.1","doi":"10.1101/2022.04.28.489834","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.27.489436","pub_date":"2022-4-28","title":"Removal of Clinically Relevant SARS-CoV-2 Variants by An Affinity Resin Containing Galanthus nivalis Agglutinin","abstract":"The Coronavirus-19 (COVID-19) pandemic due to the SARS-CoV-2 virus has now exceeded two years in duration. The pandemic has been characterized by the development of a succession of variants containing mutations in the spike protein affecting infectiousness, virulence and efficacy of vaccines and monoclonal antibodies. Resistance to vaccination and limitations in the current treatments available require the ongoing development of therapies especially for those with severe disease. The plant lectin Galanthus nivalis binds to mannose structures in the viral envelope. We hypothesized that viral binding should be unaffected by spike protein mutations. Known concentrations of seven clinically relevant SARS-CoV-2 variants were spiked in medium and passed three times over columns containing 1 gm of GNA affinity resin. Percent decrease in viral titer was compared with a control sample. Viral capture efficiency was found to range from 53 to 89% for all variants. Extrapolation indicated that an adult Aethlon Hemopurifier\u00ae would have more than sufficient binding capacity for viral loads observed in adult patients with severe COVID-19 infection.","version":"1.1","doi":"10.1101/2022.04.27.489436","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489772","pub_date":"2022-4-28","title":"Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike","abstract":"The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.","version":"1.1","doi":"10.1101/2022.04.28.489772","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489618","pub_date":"2022-4-28","title":"A Pan-Coronavirus Vaccine Candidate: Nine Amino Acid Substitutions in the ORF1ab Gene Attenuate 99% of 365 Unique Coronaviruses: A Comparative Effectiveness Research Study","abstract":"The COVID-19 pandemic has been a watershed event. Industry and governments have reacted, investing over US$105 billion in vaccine research. The \u2018Holy Grail\u2019 is a universal, pan-coronavirus, vaccine to protect humankind from future SARS-CoV-2 variants and the thousands of similar coronaviruses with pandemic potential. This paper proposes a new vaccine candidate that appears to attenuate the SARS-Cov-2 coronavirus variants to render it safe to use as a vaccine. Moreover, these results indicate it may be efficacious against 99% of 365 coronaviruses. This research model is wet-dry-wet; it originated in genomic sequencing laboratories, evolved to computational modeling, and the candidate result now require validation back in a wet lab. This study\u2019s purpose was to test the hypothesis that machine learning applied to sequenced coronaviruses\u2019 genomes could identify which amino acid substitutions likely attenuate the viruses to produce a safe and effective pan-coronavirus vaccine candidate. This candidate is now eligible to be pre-clinically then clinically tested and proven. If validated, it would constitute a traditional attenuated virus vaccine to protect against hundreds of coronaviruses, including the many future variants of SARS-CoV-2 predicted from continuously recombining in unvaccinated populations and spreading by modern mass travel. Using machine learning, this was an in silico comparative effectiveness research study on trinucleotide functions in nonstructural proteins of 365 novel coronavirus genomes. Sequences of 7,097 codons in the ORF1ab gene were collected from 65 global locations infecting 68 species and reported to the US National Institute of Health. The data were proprietarily transformed twice to enable machine learning ingestion, mapping, and interpretation. The set of 2,590,405 data points was randomly divided into three cohorts: 255 (70%) observations for training; and two cohorts of 55 (15%) observations each for testing. Machine learning models were trained in the statistical programming language R and compared to identify which mixture of the 7.097 \u00d7 1023 possible amino-acid-location combinations would attenuate SARS-CoV-2 and other coronaviruses that have infected humans. Contests of machine-learning algorithms identified nine amino-acid point substitutions in the ORF1ab gene that likely attenuate 98.98% of 365 (361) novel coronaviruses. Notably, seven substitutions are for the amino acid alanine. Most of the locations (5 of 9) are in nonstructural proteins (NSPs) 2 and 3. The substitutions are alanine to (1) valine at codon 4273; (2) leucine at codon 5077; (3) phenylalanine at codon 2001; (4) leucine at codon 372; (5) proline at codon 354; (6) phenylalanine at codon 2811; (7) phenylalanine at codon 4703; (8) leucine to serine at codon 2333; and, (9) threonine to alanine at codon 5131. The primary outcome is a new, highly promising, pan-coronavirus vaccine candidate based on nine amino-acid substitutions in the ORF1ab gene. The secondary outcome was evidence that sequences of wet-dry lab collaborations \u2013 here machine learning analysis of viral genomes informing codon functions -- may discover new broader and more stable vaccines candidates more quickly and inexpensively than traditional methods.","version":"1.1","doi":"10.1101/2022.04.28.489618","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.27.489746","pub_date":"2022-4-28","title":"Equilibrative Nucleoside Transporter 3 is an IFN-stimulated Gene that Facilitates Viral Genome Release","abstract":"An increasing body of evidence emphasizes the role of metabolic reprogramming in immune cells to fight off infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that equilibrative nucleoside transporter 3 (ENT3) expression is part of the innate immune response, and is rapidly upregulated upon bacterial and viral infection. The transcription of ENT3 is directly under the regulation of IFN-induced signaling, positioning this metabolite transporter as an Interferon-stimulated gene (ISG). Moreover, we unveil that several viruses, including SARS-CoV2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of ENT3 expression is sufficient to significantly decrease viral replication in vitro and in vivo.","version":"1.1","doi":"10.1101/2022.04.27.489746","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.27.489747","pub_date":"2022-4-28","title":"Two types of human TCR differentially regulate reactivity to self and non-self antigens","abstract":"Based on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are polyreactive to self and microbial antigens. Thus, >50% of cord blood TCRs are responsive to SARS-CoV2 and other common pathogens. TDT- dependent TCRs present distinct structural features and are less shared among subjects. TDT- dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes.","version":"1.1","doi":"10.1101/2022.04.27.489747","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.28.489537","pub_date":"2022-4-28","title":"The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype","abstract":"Variant of concern (VOC) Omicron-BA1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and multiple animal models is urgently needed. Here, we characterized Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in na\u00efve hamsters, ferrets and hACE2-expressing mice, and in immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In Syrian hamsters, Delta showed dominance over Omicron-BA.1 and in ferrets, Omicron-BA.1 infection was abortive. In mice expressing the authentic hACE2-receptor, Delta and a Delta spike clone also showed dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in na\u00efve K18-hACE2 mice, we observed Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of both Delta and Omicron-BA.1 replication and pathogenicity. Finally, the Omicron-BA.1 spike clone was less well controlled by mRNA-vaccination in K18-hACE2-mice and became more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.","version":"1.1","doi":"10.1101/2022.04.28.489537","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.27.489750","pub_date":"2022-4-28","title":"Ligand Binding Prediction using Protein Structure Graphs and Residual Graph Attention Networks","abstract":"Computational prediction of ligand-target interactions is a crucial part of modern drug discovery as it helps to bypass high costs and labor demands of in vitro and in vivo screening. As the wealth of bioactivity data accumulates, it provides opportunities for the development of deep learning (DL) models with increasing predictive powers. Conventionally, such models were either limited to the use of very simplified representations of proteins or ineffective voxelization of their 3D structures. Herein, we present the development of the PSG-BAR (Protein Structure Graph \u2013Binding Affinity Regression) approach that utilizes 3D structural information of the proteins along with 2D graph representations of ligands. The method also introduces attention scores to selectively weight protein regions that are most important for ligand binding. The developed approach demonstrates the state-of-the-art performance on several binding affinity benchmarking datasets. The attention-based pooling of protein graphs enables identification of surface residues as critical residues for protein-ligand binding. Finally, we validate our model predictions against an experimental assay on a viral main protease (Mpro)\u2013 the hallmark target of SARS-CoV-2 coronavirus. The code for PSG-BAR is made available at https://github.com/diamondspark/PSG-BAR acherkasov@prostatecentre.com","version":"1.1","doi":"10.1101/2022.04.27.489750","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.16.440173","pub_date":"2022-4-27","title":"Neuroinvasion and neurotropism by SARS-CoV-2 variants in the K18-hACE2 mouse","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, also after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally orientated regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates, and broadly support investigation of agents with adequate penetration into relevant regions of the CNS.","version":"1.2","doi":"10.1101/2021.04.16.440173","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.25.489427","pub_date":"2022-4-27","title":"Faster SARS-CoV-2 sequence validation and annotation for GenBank using VADR","abstract":"In 2020 and 2021, more than 1.5 million SARS-CoV-2 sequences were submitted to GenBank. The initial version (v1.0) of the VADR (Viral Annotation DefineR) software package that GenBank uses to automatically validate and annotate incoming viral sequences is too slow and memory intensive to process many thousands of SARS-CoV-2 sequences in a reasonable amount of time. Additionally, long stretches of ambiguous N nucleotides, which are common in many SARS-CoV-2 sequences, prevent VADR from accurate validation and annotation. VADR has been updated to more accurately and rapidly annotate SARS-CoV-2 sequences. Stretches of consecutive Ns are now identified and temporarily replaced with expected nucleotides to facilitate processing, and the slowest steps have been overhauled using blastn and glsearch, increasing speed, reducing the memory requirement from 64Gb to 2Gb per thread, and allowing simple, coarse-grained parallelization on multiple processors per host. VADR is now nearly 1000 times faster than it was in early 2020 for processing SARS-CoV-2 sequences submitted to GenBank. It has been used to screen and annotate more than 1.5 million SARS-CoV-2 sequences since June 2020, and it is now efficient enough to cope with the current rate of hundreds of thousands of submitted sequences per month. Version 1.4.1 is freely available (https://github.com/ncbi/vadr) for local installation and use.","version":"1.1","doi":"10.1101/2022.04.25.489427","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.26.489630","pub_date":"2022-4-27","title":"P681 mutations within the polybasic motif of spike dictate fusogenicity and syncytia formation of SARS CoV-2 variants","abstract":"The rapid spread and dominance of the Omicron SARS-CoV-2 over its Delta variant has posed severe global challenges. While extensive research on the role of the Receptor Binding Domain on viral infectivity and vaccine sensitivity has been documented, the role of the spike 681PRRAR/SV687 polybasic motif is less clear. Here we monitored infectivity and vaccine sensitivity of Omicron SARS-CoV-2 pseudovirus against sera samples that were drawn four months post administration of the third dose of BNT162b2 mRNA vaccine. Our findings show that relative to Wuhan-Hu and Delta SARS-CoV-2, Omicron displayed enhanced infectivity and a sharp decline in its sensitivity to vaccine-induced neutralizing antibodies. Furthermore, while the spike proteins form Wuhan-Hu (P681), Omicron (H681) and BA.2 (H681) pseudoviruses modestly promoted cell fusion and syncytia formation, Delta spike (P681R) displayed enhanced fusogenic activity and syncytia formation capability. Live-viruses plaque formation assays confirmed these findings and demonstrated that relatively to the Wuhan-Hu and Omicron SARS-CoV-2, Delta formed more plaques that were smaller in size. Introducing a single P681R point mutation within the Wuhan-Hu spike, or H681R within Omicron spike, restored fusion potential to similar levels observed for Delta spike. Conversely, a R681P point mutation within Delta spike efficiency abolished fusion potential. We conclude that over time, the efficiency of the third dose of the Pfizer vaccine against SARS CoV-2 is waned, and cannot neutralize Omicron. We further verify that the P681 position of the viral spike dictates fusogenicity and syncytia formation.","version":"1.1","doi":"10.1101/2022.04.26.489630","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.27.489676","pub_date":"2022-4-27","title":"Targeting Neutrophils Extracellular Traps (NETs) reduces multiple organ injury in a COVID-19 mouse model","abstract":"COVID-19 is characterized by severe acute lung injury, which is associated with neutrophils infiltration and release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potential deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.","version":"1.1","doi":"10.1101/2022.04.27.489676","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.26.489505","pub_date":"2022-4-27","title":"Discovery of new senolytics using machine learning","abstract":"Cellular senescence is a stress response characterised by a permanent cell cycle arrest and a proinflammatory secretome. In addition to its tumour suppressor role, senescence is involved in ageing and promotes many disease processes such as cancer, type 2 diabetes, osteoarthritis, and SARS-CoV-2 infection. There is a growing interest in therapies based on targeted elimination of senescent cells, yet so far only a few such senolytics are known, partly due to the poor grasp of the molecular mechanisms that control the senescence survival programme. Here we report a highly effective machine learning pipeline for the discovery of senolytic compounds. Using solely published data, we trained machine learning algorithms to classify compounds according to their senolytic action. Models were trained on as few as 58 known senolytics against a background of FDA-approved compounds or in late-stage clinical development (2,523 in total). We computationally screened various chemical libraries and singled out top candidates for validation in human lung fibroblasts (IMR90) and lung adenocarcinoma (A549) cell lines. This led to the discovery of three novel senolytics: ginkgetin, oleandrin and periplocin, with potency comparable to current senolytics and a several hundred-fold reduction in experimental screening costs. Our work demonstrates that machine learning can take maximum advantage of existing drug screening data, paving the way for new open science approaches to drug discovery for senescence-associated diseases.","version":"1.1","doi":"10.1101/2022.04.26.489505","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.26.489529","pub_date":"2022-4-26","title":"A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2","abstract":"Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 \u03bcg of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.","version":"1.1","doi":"10.1101/2022.04.26.489529","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.24.489298","pub_date":"2022-4-26","title":"Anti-SARS-Cov-2 S-RBD IgG formed after BNT162b2 vaccination can bind C1q and activate complement","abstract":"Activation of the classical complement pathway through C1q binding to immunoglobulins (Ig) contributes to pathogen neutralization, thus, the ability of Ig produced after vaccination to bind C1q could affect vaccine efficacy. In this study, we investigated C1q binding and subsequent complement activation by anti-spike (S) protein receptor-binding domain (RBD) specific antibodies produced following vaccination with either the mRNA vaccine BNT162b2 or the inactivated vaccine BBIBP-CorV. Serum samples were collected in the period July 2021-March 2022. Participants\u2019 demographic data, type of vaccine, date of vaccination, as well as adverse effects of the vaccine were recorded. The serum samples were incubated with S protein RBD-coated plates. Levels of human IgG, IgM, and C1q, that were bound to the plate, as well as formed C5b-9, were compared between different groups of participants. A total of 151 samples were collected from vaccinated (n=116) and non-vaccinated (n=35) participants. Participants who received either one or two doses of BNT162b2 formed higher levels of anti-RBD IgG than participants who received BBIBP-CorV. The anti-RBD IgG formed following either vaccine bound C1q, but significantly more C1q binding was observed in participants who received BNT162b2. Subsequently, C5b-9 formation was significantly higher in participants who received BNT162b2, while no significant difference in C5b-9 formation was found between the non-vaccinated and BBIBP-CorV groups. Formation of C5b-9 was strongly correlated to C1q binding, additionally, the ratio of formed C5b-9/ bound C1q was significantly higher in the BNT162b2 group. Anti-RBD IgG formed following vaccination can bind C1q with subsequent complement activation, the degree of terminal complement pathway activation differed between vaccines, which could play a role in in the protection offered by COVID-19 vaccines. Further investigation into the correlation between vaccine protection and the ability of vaccine generated antibodies to activate complement is required.","version":"1.1","doi":"10.1101/2022.04.24.489298","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456317","pub_date":"2022-4-22","title":"Point Mutations in SARS-CoV-2 Variants Induce Long-Range Dynamical Perturbations in Neutralizing Antibodies","abstract":"Monoclonal antibodies are emerging as a viable treatment for the coronavirus disease 19 (COVID-19). However, newly evolved variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can reduce the efficacy of currently available antibodies and can diminish vaccine-induced immunity. Here, we demonstrate that the microscopic dynamics of neutralizing monoclonal antibodies can be profoundly modified by the mutations present in the spike proteins of the SARS-COV-2 variants currently circulating in the world population. The dynamical perturbations within the antibody structure, which alter the thermodynamics of antigen recognition, are diverse and can depend both on the nature of the antibody and on the spatial location of the spike mutation. The correlation between the motion of the antibody and that of the spike receptor binding domain (RBD) can also be changed, modulating binding affinity. Using protein-graph-connectivity networks, we delineated the mutant-induced modifications in the information-flow along allosteric pathway throughout the antibody. Changes in the collective dynamics were spatially distributed both locally and across long-range distances within the antibody. On the receptor side, we identified an anchor-like structural element that prevents the detachment of the antibodies; individual mutations there can significantly affect the antibody binding propensity. Our study provides insight into how virus neutralization by monoclonal antibodies can be impacted by local mutations in the epitope via a change in dynamics. This realization adds a new layer of sophistication to the efforts for rational design of monoclonal antibodies against new variants of SARS-CoV2, taking the allostery in the antibody into consideration.","version":"1.3","doi":"10.1101/2021.08.13.456317","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.488933","pub_date":"2022-4-22","title":"A Quantum Dot Biomimetic for SARS-CoV-2 to Interrogate Dysregulation of the Neurovascular Unit Relevant to Brain Inflammation","abstract":"Despite limited evidence for competent infection and viral replication of SARS-CoV-2 in the central nervous system (CNS), neurologic dysfunction is a common post-acute medical condition reported in \u201crecovered\u201d COVID-19 patients. To identify a potential noninfectious route for SARS-CoV-2-mediated neurological damage, we constructed colloidal nanocrystal quantum dots linked to micelles decorated with spike protein (COVID-QDs) as a biomimetic to interrogate how blood-brain barrier (BBB) dysregulation may subsequently induce neuroinflammation in the absence of infection. In transwell co-culture of endothelial bEnd.3 monolayers and primary neuroglia, we exposed only the bEnd.3 monolayers to COVID-QDs and examined by fluorescence microscopy whether such treatment led to (i) increased inflammation and leakage across the bEnd.3 monolayers, (ii) permeability of the COVID-QDs across the monolayers, and (iii) induction of neuroinflammation in neuroglial cultures. The results of our study provide evidence of neuroinflammatory hallmarks in cultured neurons and astrocytes without direct exposure to SARS-CoV-2-like nanoparticles. Additionally, we found that pre-treatment of our co-cultures with a small-molecule, broad-spectrum inhibitor of mixed lineage and leucine rich repeat kinases led to reversal of the observed dysregulation in endothelial monolayers and resulted in neuroglial protection. The results reported here may serve to guide future studies into the potential mechanisms by which SARS-CoV-2 mediates neurologic dysfunction.","version":"1.1","doi":"10.1101/2022.04.20.488933","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.488878","pub_date":"2022-4-22","title":"An Efficient Approach for SARS-CoV-2 Monoclonal Antibody Production via Modified mRNA-LNP Immunization","abstract":"Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult to express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies. In addition, the mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.","version":"1.1","doi":"10.1101/2022.04.20.488878","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.22.489083","pub_date":"2022-4-22","title":"The Free Fatty Acid-Binding Pocket is a Conserved Hallmark in Pathogenic \u03b2-Coronavirus Spike Proteins from SARS-CoV to Omicron","abstract":"As COVID-19 persists, severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor-binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA-binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic \u03b2-coronaviruses (\u03b2-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2 and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation while the open, infectious conformation is LA-free. Electron tomography of SARS-CoV-2 infected cells reveals that LA-treatment inhibits viral replication, resulting in fewer, deformed virions. Our results establish FFA-binding as a hallmark of pathogenic \u03b2-CoV infection and replication, highlighting potential antiviral strategies. Free fatty acid-binding is conserved in pathogenic \u03b2-coronavirus S proteins and suppresses viral infection and replication.","version":"1.1","doi":"10.1101/2022.04.22.489083","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.21.489022","pub_date":"2022-4-22","title":"SARS-CoV-2 spike variants differ in their allosteric response to linoleic acid","abstract":"The SARS-CoV-2 spike protein contains a fatty acid binding site, also found in some other coronaviruses (e.g. SARS-CoV), which binds linoleic acid and is functionally important. When occupied by linoleic acid, it reduces infectivity, by \u2018locking\u2019 the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. These simulations show that the fatty acid site is coupled to functional regions of the protein, some of them far from the site (e.g. in the receptor-binding motif, N-terminal domain, the furin cleavage site located in position 679-685 and the fusion peptide-surrounding regions) and identify the allosteric networks involved in these connections. Comparison of the response of the original (\u2018Wuhan\u2019) spike with four variants: Alpha, Delta, Delta plus and Omicron BA.1 show that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the fatty acid site on Alpha are generally similar to the original protein, except for the receptor-binding motif and S71-R78 region which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629 and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, e.g. in differences in transmissibility and virulence. Experimental comparison of the effects of linoleic acid on different variants is warranted.","version":"1.1","doi":"10.1101/2022.04.21.489022","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.21.489072","pub_date":"2022-4-22","title":"Impaired immune response drives age-dependent severity of COVID-19","abstract":"SARS-CoV-2 is a highly contagious respiratory virus and the causative agent for COVID-19. The severity of disease varies from mildly symptomatic to lethal and shows an extraordinary correlation with increasing age, which represents the major risk factor for severe COVID-19. However, the precise pathomechanisms leading to aggravated disease in the elderly are currently unknown. Delayed and insufficient antiviral immune responses early after infection as well as dysregulated and overshooting immunopathological processes late during disease were suggested as possible mechanisms. Here we show that the age-dependent increase of COVID-19 severity is caused by the disruption of a timely and well-coordinated innate and adaptive immune response due to impaired interferon (IFN) responses. To overcome the limitations of mechanistic studies in humans, we generated a mouse model for severe COVID-19 and compared the kinetics of the immune responses in adult and aged mice at different time points after infection. Aggravated disease in aged mice was characterized by a diminished IFN-\u03b3 response and excessive virus replication. Accordingly, adult IFN-\u03b3 receptor-deficient mice phenocopied the age-related disease severity and supplementation of IFN-\u03b3 reversed the increased disease susceptibility of aged mice. Mimicking impaired type I IFN immunity in adult and aged mice, a second major risk factor for severe COVID-19, we found that therapeutic treatment with IFN-\u03bb in adult and a combinatorial treatment with IFN-\u03b3 and IFN-\u03bb in aged Ifnar1-/-mice was highly efficient in protecting against severe disease. Our findings provide an explanation for the age-dependent disease severity of COVID-19 and clarify the nonredundant antiviral functions of type I, II and III IFNs during SARS-CoV-2 infection in an age-dependent manner. Based on our data, we suggest that highly vulnerable individuals combining both risk factors, advanced age and an impaired type I IFN immunity, may greatly benefit from immunotherapy combining IFN-\u03b3 and IFN-\u03bb.","version":"1.1","doi":"10.1101/2022.04.21.489072","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.21.22274150","pub_date":"2022-04-22","title":"Epidemiological impact and cost-effectiveness analysis of COVID-19 vaccination in Kenya","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Few studies have assessed the benefits of COVID-19 vaccines in settings where most of the population had been exposed to SARS-CoV-2 infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a cost-effectiveness analysis of COVID-19 vaccine in Kenya from a societal perspective over a 1.5-year time frame. An age-structured transmission model assumed at least 80% of the population to have prior natural immunity when an immune escape variant was introduced. We examine the effect of slow (18 months) or rapid (6 months) vaccine roll-out with vaccine coverage of 30%, 50% or 70% of the adult (&gt; 18 years) population prioritizing roll-out in over 50-year olds (80% uptake in all scenarios). Cost data were obtained from primary analyses. We assumed vaccine procurement at $7 per dose and vaccine delivery costs of $3.90-$6.11 per dose. The cost-effectiveness threshold was USD 919.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Slow roll-out at 30% coverage largely targets over 50-year-olds and resulted in 54% fewer deaths (8,132(7,914 to 8,373)) than no vaccination and was cost-saving (ICER=US$-1,343 (-1,345 to - 1,341) per DALY averted). Increasing coverage to 50% and 70%, further reduced deaths by 12% (810 (757 to 872) and 5% (282 (251 to 317) but was not cost-effective, using Kenya\u2019s cost-effectiveness threshold ($ 919.11). Rapid roll-out with 30% coverage averted 63% more deaths and was more cost-saving (ICER=$-1,607 (-1,609 to -1,604) per DALY averted) compared to slow roll-out at the same coverage level, but 50% and 70% coverage scenarios were not cost-effective.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>With prior exposure partially protecting much of the Kenyan population, vaccination of young adults may no longer be cost-effective.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>KEY QUESTIONS</jats:title>\n                  <jats:sec>\n                    <jats:title>What is already known?</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>The COVID-19 pandemic has led to a substantial number of cases and deaths in low-and middle-income countries.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>COVID-19 vaccines are considered the main strategy of curtailing the pandemic. However, many African nations are still at the early phase of vaccination.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Evidence on the cost-effectiveness of COVID-19 vaccines are useful in estimating value for money and illustrate opportunity costs. However, there is a need to balance these economic outcomes against the potential impact of vaccination.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>What are the new findings?</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>In Kenya, a targeted vaccination strategy that prioritizes those of an older age and is deployed at a rapid rollout speed achieves greater marginal health impacts and is better value for money.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Given the existing high-level population protection to COVID-19 due to prior exposure, vaccination of younger adults is less cost-effective in Kenya.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>What do the new findings imply?</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Rapid deployment of vaccines during a pandemic averts more cases, hospitalisations, and deaths and is more cost-effective.</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Against a context of constrained fiscal space for health, it is likely more prudent for Kenya to target those at severe risk of disease and possibly other vulnerable populations rather than to the whole population.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2022.04.21.22274150","journal":"medRxiv","score":null},{"id":"10.1101/2022.04.22.489229","pub_date":"2022-4-22","title":"Detection and discrimination of single nucleotide polymorphisms by quantification of CRISPR-Cas catalytic efficiency","abstract":"The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., SARS-CoV-2 variants. Such assays often employ endpoint measurements of SNP or wild type (WT) activated Cas12 trans-cleavage activity; however, the fundamental kinetic effects of SNP versus WT activation remain unknown. We here show that endpoint-based assays are limited by arbitrary experimental choices (like used reporter concentration and assay duration) and work best for known target concentrations. More importantly, we show that SNP (versus WT) activation results in measurable shifts in the Cas12 trans-cleavage substrate affinity (KM) and apparent catalytic efficiency . To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michalis-Menten parameters and apply this assay to a 20-base pair WT target of the SARS-CoV-2 E gene. We find that the  measured for WT is 130-fold greater than the lowest  among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). KM also offers strong ability to distinguish SNPs, varies 27-fold over all the cases, and is insensitive to target concentration. Lastly, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.","version":"1.1","doi":"10.1101/2022.04.22.489229","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.488968","pub_date":"2022-4-21","title":"Lung spatial profiling reveals a T cell signature in COPD patients with fatal SARS-CoV-2 infection","abstract":"People with pre-existing lung diseases like chronic obstructive pulmonary disease (COPD) are more likely to get very sick from SARS-CoV-2 disease 2019 (COVID-19), but an interrogation of the immune response to COVID-19 infection, spatial throughout the lung structure is lacking in patients with COPD. To profile the immune microenvironment of lung parenchyma, airways, and vessels of never- and ever-smokers with or without COPD, whom all died of COVID-19, using spatial transcriptomic and proteomic profiling. The parenchyma, airways, and vessels of COPD patients, compared to control lungs had: 1) significant enrichment for lung resident CD45RO+ memory T cells; 2) downregulation of genes associated with T cell antigen-priming and memory T cell differentiation; 3) higher expression of proteins associated with SARS-CoV-2 entry and major receptor ubiquitously across the ROIs and in particular the lung parenchyma, despite similar SARS-CoV-2 structural gene expression levels. The lung parenchyma, airways, and vessels of COPD patients have increased T-lymphocytes with a blunted memory T cell response and a more invasive SARS-CoV-2 infection pattern, and may underlie the higher death toll observed with COVID-19.","version":"1.1","doi":"10.1101/2022.04.20.488968","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434300","pub_date":"2022-4-21","title":"Pediatric nasal epithelial cells are less permissive to SARS-CoV-2 replication compared to adult cells","abstract":"Children typically experience more mild symptoms of COVID-19 when compared to adults. There is a strong body of evidence that children are also less susceptible to SARS-CoV-2 infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remains to be determined. Here, we use primary nasal epithelial cells from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the nasal epithelial cells of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the nasal epithelial cells of children. Importantly, the Delta variant also replicated to significantly lower titres in the nasal epithelial cells of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.","version":"1.3","doi":"10.1101/2021.03.08.434300","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488682","pub_date":"2022-4-21","title":"Elucidating host cell response pathways and repurposing therapeutics for SARS-CoV-2 and other coronaviruses using gene expression profiles of chemical and genetic perturbations","abstract":"COVID-19 is an ongoing pandemic that has been causing devastation across the globe for over 2 years. Although there are multiple vaccines that can prevent severe symptoms, effective COVID-19 therapeutics are still of importance. Using our proprietary in silico SMarTR\u2122 engine, we screened more than 22,000 unique compounds represented by over half a million gene expression profiles to uncover compounds that can be repurposed for SARS-CoV-2 and other coronaviruses in a timely and cost-efficient manner. We then tested 13 compounds in vitro and found three with potency against SARS-CoV-2 with reasonable cytotoxicity. Bortezomib and homoharringtonine are some of the most promising hits with IC50 of 1.39 \u03bcM and 0.16 \u03bcM, respectively for SARS-CoV-2. Tanespimycin and homoharringtonine were effective against the common cold coronaviruses. In-depth analysis highlighted proteasome, ribosome, and heat shock pathways as key targets in modulating host responses during viral infection. Further studies of these pathways and compounds have provided novel and impactful insights into SARS-CoV-2 biology and host responses that could be further leveraged for COVID-19 therapeutics development.","version":"1.2","doi":"10.1101/2022.04.18.488682","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.21.489021","pub_date":"2022-4-21","title":"Peptide derived nanobody inhibits entry of SARS-CoV-2 variants","abstract":"Emergence of the new escape mutants of the SARS-CoV-2 virus has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS variants and other viruses in a short period of time becomes essential. Blocking the SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-ACE2 interaction has been already exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a 9 amino acid peptide from the receptor-binding motif (RBM) of Spike (S) protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the nanobody. In summary, our study presents a faster and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. This approach can also be widely extended to target other viruses and pathogens in the future.","version":"1.1","doi":"10.1101/2022.04.21.489021","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433360","pub_date":"2022-4-21","title":"Fragment-based computational design of antibodies targeting structured epitopes","abstract":"De novo design methods hold the promise of reducing the time and cost of antibody discovery, while enabling the facile and precise targeting of predetermined epitopes. Here we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterisation showed that all designs are highly stable, and bind their intended targets with affinities in the nanomolar range without any in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as our method yields similar predictions when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to pre-selected epitopes. A combinatorial method can rapidly design nanobodies for predetermined epitopes, which bind with KDs in the nanomolar range.","version":"1.2","doi":"10.1101/2021.03.02.433360","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.488873","pub_date":"2022-4-20","title":"Uncovering the structural flexibility of SARS-CoV-2 glycoprotein spike variants","abstract":"The severe acute respiratory syndrome CoV-2 rapidly spread worldwide, causing a pandemic. After a period of evolutionary stasis, a set of SARS-CoV-2 mutations has arisen in the spike, the leading glycoprotein at the viral envelope and the primary antigenic candidate for vaccines against the 2019 CoV disease (COVID-19). Here, we present comparative biochemical data of the glycosylated full-length ancestral and D614G spike together with three other highly transmissible strains classified by the World Health Organization as variants of concern (VOC): beta, gamma, and delta. By showing that only D614G early variant has less hydrophobic surface exposure and trimer persistence at mid-temperatures, we place D614G with features that support a model of temporary fitness advantage for virus spillover worldwide. Further, during the SARS-CoV-2 adaptation, the spike accumulates alterations leading to less structural rigidity. The decreased trimer stability observed for the ancestral and the gamma strain and the presence of D614G uncoupled conformations mean higher ACE-2 affinities when compared to the beta and delta strains. Mapping the energetic landscape and flexibility of spike variants is necessary to improve vaccine development.","version":"1.1","doi":"10.1101/2022.04.20.488873","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.488895","pub_date":"2022-4-20","title":"Emergence of new subgenomic mRNAs in SARS-CoV-2","abstract":"Two mutations occurred in SARS-CoV-2 early during the COVID-19 pandemic that have come to define circulating virus lineages: first a change in the spike protein (D614G) that defines the B.1 lineage and second, a double substitution in the nucleocapsid protein (R203K, G204R) that defines the B.1.1 lineage, which has subsequently given rise to three Variants of Concern: Alpha, Gamma and Omicron. While the latter mutations appear unremarkable at the protein level, there are dramatic implications at the nucleotide level: the GGG\u2192AAC substitution generates a new Transcription Regulatory Sequence (TRS) motif, driving SARS-CoV-2 to express a novel subgenomic mRNA (sgmRNA) encoding a truncated C-terminal portion of nucleocapsid (N.iORF3), which is an inhibitor of type I interferon production. We find that N.iORF3 also emerged independently within the Iota variant, and further show that additional TRS motifs have convergently evolved to express novel sgmRNAs; notably upstream of Spike within the nsp16 coding region of ORF1b, which is expressed during human infection. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level, reminiscent of eukaryotic evolution. Greater attention to this aspect in the assessment of emerging strains of SARS-CoV-2 is warranted.","version":"1.1","doi":"10.1101/2022.04.20.488895","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.476940","pub_date":"2022-4-20","title":"Targeting SARS-CoV-2 infection through CAR-T like bispecific T cell engagers incorporating ACE2","abstract":"Despite advances in antibody treatments and vaccines, COVID-19 caused by SARS-CoV-2 infection remains a major health problem resulting in excessive morbidity and mortality and the emergence of new variants has reduced the effectiveness of current vaccines. Here, as a proof-of-concept we engineered primary CD8 T cells to express SARS-CoV-2 Spike protein-specific CARs, using extracellular region of ACE2, and demonstrated their highly specific and potent cytotoxicity towards Spike-expressing target cells. To improve on this concept as a potential therapeutic, we developed a bispecific T cell engager combining ACE2 with an anti-CD3 scFv (ACE2-Bite) to target infected cells and the virus. Similar to CAR-T cell approach, ACE2-Bite endowed cytotoxic cells to selectively kill Spike-expressing targets. Furthermore, ACE2-Bite neutralized the pseudoviruses of SARS-CoV, SARS-CoV-2 wild-type and variants including Delta and Omicron, as a decoy protein. Remarkably, ACE2-Bite molecule showed a higher binding and neutralization affinity to Delta and Omicron variants compared to SARS-CoV-2 wild-type Spike proteins, suggesting the potential of this approach as a variant-proof, therapeutic strategy for future SARS-CoV-2 variants, employing both humoral and cellular arms of the adaptive immune response.","version":"1.2","doi":"10.1101/2022.01.19.476940","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.20.485440","pub_date":"2022-4-20","title":"Low-dose bivalent mRNA vaccine is highly effective against different SARS-CoV-2 variants in a transgenic mouse model","abstract":"Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve COVID-19 control. We compared monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein, primarily in a transgenic mouse model and a Wistar rat model. The low-dose bivalent mRNA vaccine contained half the mRNA of each respective monovalent vaccine, but induced comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, specific CD4+ and CD8+ responses, and fully protected transgenic mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduced viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized Wistar rats also contained neutralizing antibodies against the B.1.1.529 (Omicron BA.1) variant. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins is a feasible approach for increasing the potency of vaccines against emerging and co-circulating SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.04.20.485440","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.19.488843","pub_date":"2022-4-20","title":"Broadly neutralizing antibodies against Omicron variants of SARS-CoV-2 derived from mRNA-lipid nanoparticle-immunized mice","abstract":"The COVID-19 pandemic continues to threaten human health worldwide, as new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged. Currently, the predominant circulating strains around the world are Omicron variants, which can evade many therapeutic antibodies. Thus, the development of new broadly neutralizing antibodies remains an urgent need. In this work, we address this need by using the mRNA-lipid nanoparticle immunization method to generate a set of Omicron-targeting monoclonal antibodies. Five of our novel K-RBD-mAbs show strong binding and neutralizing activities toward all SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron). Notably, the epitopes of these five K-RBD-mAbs are overlapping and localized around K417 and F486 of the spike protein receptor binding domain (RBD). Chimeric derivatives of the five antibodies (K-RBD-chAbs) neutralize Omicron sublineages BA.1 and BA.2 with low IC50 values that range from 5.7 to 12.9 ng/mL. Additionally, we performed antibody humanization on a broadly neutralizing chimeric antibody to create K-RBD-hAb-62, which still retains excellent neutralizing activity against Omicron. Our results collectively suggest that these five therapeutic antibodies may effectively combat current and emerging SARS-CoV-2 variants, including Omicron BA.1 and BA.2. Therefore, the antibodies can potentially be used as universal neutralizing antibodies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.04.19.488843","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.19.488826","pub_date":"2022-4-20","title":"Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic","abstract":"Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1*105 to 1*10\u22124 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1*10\u22123 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1*102 TCID50 as the optimal dose for experimental infections. This compellingly high susceptibility resulting in productive infections in Golden Syrian hamsters needs to be considered also as a source of SARS-CoV-2 infections in humans.","version":"1.1","doi":"10.1101/2022.04.19.488826","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.05.475107","pub_date":"2022-4-20","title":"Autophagy and evasion of immune system by SARS-CoV-2. Structural features of the Non-structural protein 6 from Wild Type and Omicron viral strains interacting with a model lipid bilayer. \u2020","abstract":"The viral cycle of SARS-CoV-2 is based on a complex interplay with the cellular machinery, which is mediated by specific proteins eluding or hijacking the cellular defense mechanisms. Among the complex pathways called by the viral infection autophagy is particularly crucial and is strongly influenced by the action of the non-structural protein 6 (Nsp6) interacting with the endoplasmic reticulum membrane. Importantly, differently from other non-structural proteins Nsp6 is mutated in the recently emerged Omicron variant, suggesting a possible different role of autophagy. In this contribution we explore, for the first time, the structural property of Nsp6 thanks to long-time scale molecular dynamic simulations and machine learning analysis, identifying the interaction patterns with the lipid membrane. We also show how the mutation brought by the Omicron variant may indeed modify some of the specific interactions, and more particularly help anchoring the viral protein to the lipid bilayer interface. Analysis protein of the secondary structure and of the specific lipid/amino acid interactions. RMSF per amino acid. Distribution of the distance between the center of mass of the 89 to 99 \u03b1-helix and the center of the lipid bilayer. Analysis of the behavior of the 195 to 207 \u03b1-helix. See DOI: 10.1039/x0xx00000x","version":"1.2","doi":"10.1101/2022.01.05.475107","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.19.488722","pub_date":"2022-4-19","title":"Tracking the circulating SARS-CoV-2 variants in Turkey: Complete genome sequencing and molecular characterization of 1000 SARS-CoV-2 samples","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus and has caused a pandemic of acute respiratory disease, named \u2018coronavirus disease 2019\u2019 (COVID-19). COVID-19 has a deep impact on public health as one of the most serious pandemics in the last century. Tracking SARS-CoV-2 is important for monitoring and assessing its evolution. This is only possible by detecting all mutations in the viral genome through genomic sequencing. Moreover, accurate detection of SARS-CoV-2 and tracking its mutations is also required for its correct diagnosis. Potential effects of mutations on the prognosis of the disease can be observed. Assignment of epidemiological lineages in an emerging pandemic requires efforts. To address this, we collected 1000 SARS-CoV-2 samples from different geographical regions in Turkey and analyze their genome comprehensively. To track the virus across Turkey we focus on 10 distinct cities in different geographic regions. Each SARS-CoV-2 genome was analyzed and named according to the nomenclature system of Nextclade and Pangolin Lineage. Furthermore, the frequency of the variations observed in 10 months was also determined by region. In this way, we have observed how the virus mutations and what kind of transmission mechanism it has. The effects of age and disease severity on lineage distribution were other considered parameters. The temporal rates of SARS-CoV-2 variants by time in Turkey were close to the global trend. This study is one of the most comprehensive whole genome analyses of SARS-CoV-2 that represents a general picture of the distribution of SARS-CoV-2 variations in Turkey in 2021. Since the outbreak of the COVID-19 pandemic in 2019, the viral genome of SARS-CoV-2 was analysed intensively all over the world both to detect its zoonotic origin and the emerging variants worldwide together with the variants\u2019 effect on the prognosis and treatment, respectively, of the infection. Remarkable COVID-19 studies were also made in Turkey as it was in the rest of the world. To date, indeed, almost all studies on COVID-19 in Turkey either sequenced only a small number of the viral genome or analysed the viral genome which was obtained from online databases. In respect thereof, our study constitutes a milestone regarding both the huge sample size consisting of 1000 viral genomes and the widespread geographic origin of the viral genome samples. Our study provides new insights both into the SARS-CoV-2 landscape of Turkey and the transmission of the emerging viral pathogen and its interaction with its vertebrate host.","version":"1.1","doi":"10.1101/2022.04.19.488722","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.15.487518","pub_date":"2022-4-19","title":"V\u03b39V\u03b42 T cells are potent inhibitors of SARS-CoV-2 replication and exert effector phenotypes in COVID-19 patients","abstract":"V\u03b39V\u03b42 T cells play a key role in the innate immune response to viral infections, including SARS-CoV-1 and 2, and are activated through butyrophilin (BTN)-3A. Here, the objectives were to: 1) characterize the effects of SARS-CoV-2 infection on the number, phenotype, and activation of V\u03b39V\u03b42 T cells in infected patients, and 2) assess the effects of in vitro SARS-CoV-2 infection on the expression of BTN3A and its impact on the activation and response of V\u03b39V\u03b42 T cells to an anti-BTN3A antibody. Blood V\u03b39V\u03b42 T cells decreased in clinically mild SARS-CoV-2 infections compared to healthy volunteers (HV). This decrease was maintained up to 28 days and in the recovery period. Terminally differentiated V\u03b39V\u03b42 T cells tend to be enriched on the day of diagnosis, 28 days after and during the recovery period compared to HV. Furthermore, these cells showed cytotoxic and inflammatory activities as shown by TNF\u03b1, IFN\u03b3 and CD107a/b increase following anti-BTN3A activation. Moreover, BTN3A upregulation and V\u03b39V\u03b42 T cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection, as compared to HV. In vitro, SARS-CoV-2 infection significantly increased BTN3A expression in macrophages and lung cell lines. The activation via BTN3A enhanced the anti-SARS-CoV-2 V\u03b39V\u03b42 T cells cytotoxicity and IFN-\u03b3 and TNF\u03b1 in SARS-CoV-2 infected patient. Increasing concentrations of anti-BTN3A were accompanied by an inhibition of viral replication. Altogether, these data suggest that V\u03b39V\u03b42 T cells are important in the immune response against SARS-CoV-2 infection and that activation by an anti-BTN3A antibody may enhance their response. SARS-CoV-2 mediates upregulation of the key receptor of V\u03b39V\u03b42 T cells BTN3A on lung tissues and cell lines as well as monocytes During SARS-CoV-2 infection, V\u03b39V\u03b42 are differentiated and efficiently degranulate and secrete cytokines upon activation with BTN3A mAb","version":"1.1","doi":"10.1101/2022.04.15.487518","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.487732","pub_date":"2022-4-19","title":"Redox regulation of the SARS-CoV-2 main protease provides new opportunities for drug design","abstract":"Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for stopping the current COVID-19 pandemic and preventing future outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include sulfenylation, disulfide formation between the catalytic cysteine and a proximal cysteine, and generation of an allosteric lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and possess antiviral activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS and SARS-CoV, indicating their potential as common druggable sites.","version":"1.1","doi":"10.1101/2022.04.18.487732","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488717","pub_date":"2022-4-19","title":"COVID-19 Infection and Transmission Includes Complex Sequence Diversity","abstract":"SARS-CoV-2 whole genome sequencing has played an important role in documenting the emergence of polymorphisms in the viral genome and its continuing evolution during the COVID-19 pandemic. Here we present data from over 360 patients to characterize the complex sequence diversity of individual infections identified during multiple variant surges (e.g., Alpha and Delta; requiring \u2265 80% genome coverage and \u2265100X read depth). Across our survey, we observed significantly increasing SARS-CoV-2 sequence diversity during the pandemic and frequent occurrence of multiple biallelic sequence polymorphisms in all infections. This sequence polymorphism shows that SARS-CoV-2 infections are heterogeneous mixtures. Convention for reporting microbial pathogens guides investigators to report a majority consensus sequence. In our study, we found that this approach would under-report at least 79% of the observed sequence variation. As we find that this sequence heterogeneity is efficiently transmitted from donors to recipients, our findings illustrate that infection complexity must be monitored and reported more completely to understand SARS-CoV-2 infection and transmission dynamics involving both immunocompetent and immunocompromised patients. Many of the nucleotide changes that would not be reported in a majority consensus sequence have now been observed as lineage defining SNPs in Omicron BA.1 and/or BA.2 variants. This suggests that minority alleles in earlier SARS-CoV-2 infections may play an important role in the continuing evolution of new variants of concern. Evolution of the virus causing COVID-19 (SARS-CoV-2) has been associated with significant transmission surges. With evolution of SARS-CoV-2, evidence has accumulated regarding increased transmissibility of lineages, varying severity of illness, evasion of vaccines and diagnostic tests. Continuous tracking of SARS-CoV-2 lineage evolution distills very large and complex viral sequence data sets down to consensus sequences that report the majority nucleotide at each of over 29,000 positions in the SARS-CoV-2 genome. We observe that this eliminates considerable sequence variation and leads to a significant underestimation of SARS-CoV-2 infection diversity and transmission complexity. Additionally, concentration on the majority consensus sequence diverts attention from genetic variation that may contribute significantly to the continuing evolution of the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2022.04.18.488717","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488640","pub_date":"2022-4-19","title":"BCG vaccination of Diversity Outbred mice induces cross-reactive antibodies to SARS-CoV-2 spike protein","abstract":"The Bacillus Calmette-Gu\u00e9rin (BCG) vaccine, the only vaccine against tuberculosis, induces cross-protection against pathogens unrelated to Mycobacterium, including viruses. Epidemiological studies have identified potential benefits of BCG vaccination against SARS-CoV-2 infection. While BCG\u2019s heterologous effects have been widely attributable to trained immunity, we hypothesized BCG vaccination could induce cross-reactive antibodies against the spike protein of SARS-CoV-2 Wuhan-Hu-1. The concentration of IgG reactive to SARS-CoV-2 spike protein from the sera of BCG-vaccinated, Diversity Outbred (DO) mice and C57BL/6J inbred mice was measured using ELISA. Sera from 10/15 BCG-vaccinated DO mice possessed more IgG reactive to recombinant spike protein than sera from BCG-vaccinated C57BL/6J mice and unvaccinated DO mice. Amino acid sequences common to BCG cell wall/membrane proteins and SARS-CoV-2 spike protein were identified as potential antigen candidates for future study. These results imply a humoral mechanism, influenced by genotype, by which BCG vaccination could confer immunity to SARS-CoV-2. \n\n","version":"1.1","doi":"10.1101/2022.04.18.488640","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488629","pub_date":"2022-4-19","title":"Persistent cross-species SARS-CoV-2 variant infectivity predicted via comparative molecular dynamics simulation","abstract":"Widespread human transmission of SARS-CoV-2 highlights the substantial public health, economic, and societal consequences of virus spillover from wildlife and also presents a repeated risk of reverse spillovers back to na\u00efve wildlife populations. We employ comparative statistical analyses of a large set of short-term molecular dynamic (MD) simulations to investigate potential human to bat (Rhinolophus macrotis) cross-species infectivity allowed by the binding of SARS-CoV-2 receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) across the bat progenitor strain and emerging human strain variants of concern (VOC). We statistically compare the dampening of atom motion during binding across protein sites upon the formation of the RBD/ACE2 binding interface using bat vs. human target receptors (i.e. bACE2 and hACE2). We report that while the bat progenitor viral strain RaTG13 shows some pre-adaption to binding hACE2, it also exhibits stronger overall affinity to bACE2. However, while the early emergent human strains and later VOC\u2019s exhibit robust binding to both hACE2 and bACE2, the delta and omicron variants exhibit evolutionary adaption of binding to hACE2. However, we conclude there is a still significant risk of mammalian cross-species infectivity of human VOC\u2019s during upcoming waves of infection as COVID-19 transitions from a pandemic to endemic status.","version":"1.1","doi":"10.1101/2022.04.18.488629","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.19.488806","pub_date":"2022-4-19","title":"The Spike protein of SARS-CoV-2 impairs lipid metabolism and increases susceptibility to lipotoxicity: implication for a role of Nrf2","abstract":"Coronavirus disease 2019 (COVID-19) patients exhibit lipid metabolic alterations, but the mechanism remains unknown. In this study, we aimed to investigate whether the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs lipid metabolism in host cells. A Spike cell line in HEK293 was generated using the pcDNA vector carrying the Spike gene expression cassette. A control cell line was generated using the empty pcDNA vector. Gene expression profiles related to lipid metabolic, autophagic, and ferroptotic pathways were investigated. Palmitic acid (PA)-overload was used to assess lipotoxicity-induced necrosis. As compared with controls, the Spike cells showed a significant increase in lipid depositions on cell membranes as well as dysregulation of expression of a panel of molecules involved lipid metabolism, autophagy, and ferroptosis. The Spike cells showed an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a multifunctional transcriptional factor, in response to PA. Furthermore, the Spike cells exhibited increased necrosis in response to PA-induced lipotoxicity compared to control cells in a time- and dose-dependent manner via ferroptosis, which could be attenuated by the Nrf2 inhibitor trigonelline. The Spike protein impairs lipid metabolic and autophagic pathways in host cells, leading to increased susceptibility to lipotoxicity via ferroptosis which can be suppressed by a Nrf2 inhibitor. This data also suggests a central role of Nrf2 in Spike-induced lipid metabolic impairments. The Spike protein increases lipid deposition in host cell membranes The Spike protein impairs lipid metabolic and autophagic pathways The Spike protein exaggerates PA-induced lipotoxicity in host cells via ferroptosis Nrf2 inhibitor Trigonelline can mitigate the Spike protein-induced necrosis","version":"1.1","doi":"10.1101/2022.04.19.488806","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488695","pub_date":"2022-4-19","title":"An intranasal nanoparticle STING agonist has broad protective immunity against respiratory viruses and variants","abstract":"Respiratory viral infections, especially Influenza (endemic) or SARS-CoV-2 (pandemic since 2020), cause morbidity and mortality worldwide. Despite remarkable progress in the development and deployment of vaccines, they are clearly impacted by the rapid emergence of viral variants. The development of an off-the-shelf, effective, safe, and low-cost drug for prophylaxis against respiratory viral infections is a major unmet medical need. Here, we developed NanoSTING, a liposomally encapsulated formulation of the endogenous STING agonist, 2\u2019-3\u2019 cGAMP, to function as an immunoantiviral. NanoSTING rapidly activates the body\u2019s innate immune system to facilitate a broad-spectrum antiviral response against SARS-CoV-2 and influenza variants in hamsters and mice. We demonstrate that a single intranasal dose of NanoSTING can: (1) treat infections throughout the respiratory system and minimize clinical symptoms, (2) protect against highly pathogenic strains of SARS-CoV-2 (alpha and delta), (3) provide durable protection against reinfection from the same strains without the need for retreatment, (4) prevent transmission of the highly infectious SARS-CoV-2 Omicron strain, and (5) provide protection against both oseltamivir-sensitive and resistant strains of influenza. Mechanistically, administration of NanoSTING rapidly upregulated interferon-stimulated and antiviral pathways in both the nasal turbinates and lung. Our results support using NanoSTING as a thermostable, immunoantiviral with broad-spectrum antiviral properties making it appealing as a therapeutic for prophylactic or early post-exposure treatment.","version":"1.1","doi":"10.1101/2022.04.18.488695","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.19.488067","pub_date":"2022-4-19","title":"Phylogeny and Metadata Network Database for Epidemiologic Surveillance","abstract":"The ongoing SARS-CoV-2 pandemic has highlighted the difficulty in integrating disparate data sources for epidemiologic surveillance. To address this challenge, we have created a graph database to integrate phylogenetic trees, associated metadata, and community surveillance data for phylodynamic inference. As an example use case, we divided 22,713 SARS-CoV-2 samples into 5 groups, generated maximum likelihood trees, and inferred a potential transmission network from a forest of minimum spanning trees built on patristic distances between samples. We then used Cytoscape to visualize the resultant graphs.","version":"1.1","doi":"10.1101/2022.04.19.488067","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.19.488803","pub_date":"2022-4-19","title":"Nonstructural protein 1 (nsp1) widespread RNA decay phenotype varies among Coronaviruses","abstract":"Extensive remodeling of the host gene expression environment by coronaviruses nsp1 proteins is a well-documented and conserved piece of the coronavirus-host takeover battle. However, whether and how the underlying mechanism of regulation or the transcriptional target landscape differ amongst coronaviruses remains mostly uncharacterized. In this study we use comparative transcriptomics to investigate the diversity of transcriptional targets between four different coronavirus nsp1 proteins (from MERS, SARS1, SARS2 and 229E). In parallel, we performed Affinity Purification followed by Mass-Spectrometry to identify common and divergent interactors between these different nsp1. For all four nsp1 tested, we detected widespread RNA destabilization, confirming that both \u03b1- and \u03b2-Coronavirus nsp1 broadly affect the host transcriptome. Surprisingly, we observed that even closely related nsp1 showed little similarities in the clustering of genes targeted. Additionally, we show that the RNA targeted by nsp1 from the \u03b1-CoV 229E partially overlapped with MERS nsp1 targets. Given MERS nsp1 preferential targeting of nuclear transcripts, these results may indicate that these nsp1 proteins share a similar targeting mechanism. Finally, we show that the interactome of these nsp1 proteins differ widely. Intriguingly, our data indicate that the 229E nsp1, which is the smallest of the nsp1 proteins tested here, interacts with the most host proteins, while MERS nsp1 only engaged with a few host proteins. Collectively, our work highlights that while nsp1 is a rather well-conserved protein with conserved functions across different coronaviruses, its precise effects on the host cell is virus specific. Coronaviruses extensively co-opt their host gene expression machinery in order to quicky benefit from the host resources. The viral protein nsp1 plays a major role in this takeover as nsp1 is known to induce a widespread shutdown of the host gene expression, both at the RNA and the translational level. Previous work characterized the molecular basis for nsp1-mediated host shutdown. However, this was mostly conducted in the context of \u03b2-coronaviruses and in particular SARS-CoV1, CoV2 and MERS due to the important public health burden that these viruses represent. Here instead, we explored the impact of nsp1 on the host using a comparative approach, defining the influence of 4 nsp1 protein from \u03b1- and \u03b2-coronaviruses. We delineated the impact of these 4 nsp1 on the host transcriptome and mapped their interactome. We revealed that host target range and interactomes vary widely among different nsp1, suggesting a viral-specific targeting. Understanding how these differences shape infection will be important to better inform antiviral drug development.","version":"1.1","doi":"10.1101/2022.04.19.488803","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.17.488607","pub_date":"2022-4-18","title":"Ocular tropism of SARS-CoV-2 with retinal inflammation through neuronal invasion in animal models","abstract":"Although ocular manifestations are commonly reported in patients with coronavirus disease 2019 (COVID-19), there is currently no consensus on ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To investigate this, we infected K18-hACE2 mice with SARS-CoV-2 using various routes. We observed ocular manifestation and retinal inflammation with cytokine production in the eyes of intranasally (IN) infected mice. An intratracheal (IT) injection resulted in virus spread from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasion were confirmed by an intracerebral (IC) infection. Notably, eye-dropped (ED) virus did not infect the lungs and was undetectable with time. Using infectious SARS-CoV-2-mCherry clones, we demonstrated the ocular and neurotropic distribution of the virus in vivo by a fluorescence-imaging system. Evidence for the ocular tropic and neuroinvasive characteristics of SARS-CoV-2 was confirmed in wild-type Syrian hamsters. Our data provides further understanding of the viral transmission; SARS-CoV-2 clinical characteristics; and COVID-19 control procedures. SARS-CoV-2 can spread from the respiratory tract to the brain and eyes via trigeminal and optic nerves in animal models. This ocular tropism of SARS-CoV-2 through neuronal invasion likely causes ocular manifestation and retinal inflammation.","version":"1.1","doi":"10.1101/2022.04.17.488607","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.25.485826","pub_date":"2022-4-18","title":"Identification of consensus hairpin loop structure among the negative sense sub-genomic RNAs of SARS-CoV-2","abstract":"SARS-CoV-2 is the causative agent of worldwide pandemic disease COVID-19. SARS-CoV-2 bears positive sense RNA genome, that have organized and complex pattern of replication/transcription process including the generation of subgenomic RNAs. Transcription regulatory sequences (TRS) have important role in the pausing of replication/transcription and generation of subgenomic RNAs. In the present bioinformatics analysis a consensus secondary structure was identified among negative sense subgenomic RNAs at the adjacent of initiation codon. This study proposed that consensus structured domain could involve in mediating the long pausing of replication/transcription complex and responsible for subgenomic RNA production.","version":"1.2","doi":"10.1101/2022.03.25.485826","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471528","pub_date":"2022-4-18","title":"The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies","abstract":"A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. The SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.","version":"1.2","doi":"10.1101/2021.12.06.471528","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488660","pub_date":"2022-4-18","title":"Modeling COVID-19 disease biology to identify drug treatment candidates","abstract":"Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, there are a limited number of effective treatments. A variety of drugs that have been approved for other diseases are being tested for the treatment of COVID-19, and thus far only remdesevir, dexamethasone, baricitinib, tofacitinib, tocilizumab, and sarilumab have been recommended by the National Institutes of Health (NIH) COVID-19 Treatment Guidelines Panel for the therapeutic management of hospitalized adults with COVID-19. Using a disease biology modeling approach, we constructed a protein-protein interactome network based on COVID-19- associated genes/proteins described in research literature together with known protein-protein interactions in epithelial cells. Phenotype and disease enrichment analysis of the COVID-19 disease biology model demonstrated strong statistical enrichments consistent with patients\u2019 clinical presentation. The model was used to interrogate host biological response induced by SARS-CoV-2 and identify COVID-19 drug treatment candidates that may inform on drugs currently being evaluated or provide insight into possible targets for potential new therapeutic agents. We focused on cancer drugs as they are often used to control inflammation, inhibit cell division, and modulate the host microenvironment to control the disease. From the top 30 COVID-19 drug candidates, twelve have a role as an antineoplastic agent, seven of which are approved for human use. Altogether, nearly 40% of the drugs identified by our model have been identified by others for COVID-19 clinical trials. Disease biology modeling incorporating disease-associated genes/proteins discussed in the research literature together with known molecular interactions in relevant cell types is a useful method to better understand disease biology and identify potentially effective therapeutic interventions.","version":"1.1","doi":"10.1101/2022.04.18.488660","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487528","pub_date":"2022-4-18","title":"Cryo-EM structures of SARS-CoV-2 Omicron BA.2 spike","abstract":"The BA.2 sub-lineage of the SARS-CoV-2 Omicron variant has gained in proportion relative to BA.1. As differences in spike (S) proteins may underlie differences in their pathobiology, here we determine cryo-EM structures of a BA.2 S ectodomain and compare these to previously determined BA.1 S structures. BA.2 Receptor Binding Domain (RBD) mutations induced remodeling of the internal RBD structure resulting in its improved thermostability and tighter packing within the 3-RBD-down spike. In the S2 subunit, the fusion peptide in BA.2 was less accessible to antibodies than in BA.1. Pseudovirus neutralization and spike binding assays revealed extensive immune evasion while defining epitopes of two RBD-directed antibodies, DH1044 and DH1193, that bound the outer RBD face to neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the 3-RBD-down state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant, and reveal differences in key functional regions in the BA.1 and BA.2 S proteins.","version":"1.2","doi":"10.1101/2022.04.07.487528","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.17.488095","pub_date":"2022-4-18","title":"The SARS-CoV-2 Omicron BA.1 spike G446S potentiates HLA-A*24:02-restricted T cell immunity","abstract":"Although the Omicron variant of the SARS-CoV-2 virus is resistant to neutralizing antibodies, it retains susceptibility to cellular immunity. Here, we characterized vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppressed Omicron BA.1 replication. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication was observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation was lost when target cells were treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell immunity towards emerging variants.","version":"1.1","doi":"10.1101/2022.04.17.488095","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481345","pub_date":"2022-4-18","title":"Mucosal Immunization of Cynomolgus Macaques with Adenoviral Vector Vaccine Elicits Neutralizing Nasal and Serum Antibody to Several SARS-CoV-2 Variants","abstract":"The emergence of SARS-CoV-2 variants continues to be a major obstacle for controlling the global pandemic. Despite the currently authorized SARS-CoV-2 vaccines ability to reduce severe disease and hospitalization, new immunization strategies are needed that enhance mucosal immune responses, inhibit community transmission, and provide protection against emerging variants. We have developed a mucosally delivered, non-replicating recombinant adenovirus vector (rAd5) vaccine, that has proven efficacy in the clinic against other respiratory viruses [1]. Here we evaluated the immunogenicity of three candidate SARS-CoV-2 vaccines in cynomolgus macaques that contained spike (S) and/or nucleocapsid (N) from either the Wuhan or the beta variant to select a candidate for future clinical development. Mucosal immunization with the Wuhan specific S vaccine (ED90) induced significant cross-reactive serum IgG responses against to Wuhan, beta, gamma and delta lineages, and generated substantial serum neutralizing activity. In nasal samples, ED90 immunization induced 1000-fold increases in IgA to all variants of concern tested and had neutralizing activity against Wuhan and delta. While immunization with the beta specific vaccine (ED94) enhanced IgG and IgA responses to homologous beta variant S and RBD, this approach resulted in less cross-reactive responses to other variants in the serum and nasal passages compared to ED90. As ED90 immunization induced the most robust cross-reactive systemic and mucosal antibody responses, this candidate was chosen for future clinical development.","version":"1.2","doi":"10.1101/2022.02.21.481345","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.16.488556","pub_date":"2022-4-18","title":"In Silico transcriptional analysis of asymptomatic and severe COVID-19 patients reveals the susceptibility of severe patients to other comorbidities and non-viral pathological conditions","abstract":"COVID-19 is a severe respiratory disease caused by SARS-CoV-2, a novel human coronavirus. The host response to SARS-CoV-2 infection is not clearly understood. Patients infected with SARS-CoV-2 exhibit heterogeneous intensity of symptoms, i.e., asymptomatic, mild, and severe. Moreover, effects on organs also vary from person to person. These heterogeneous responses pose pragmatic hurdles for implementing appropriate therapy and management of COVID-19 patients. Post-COVID complications pose another major challenge in managing the health of these patients. Thus, understanding the impact of disease severity at the molecular level is vital to delineate the precise host response and management. In the current study, we performed a comprehensive transcriptomics analysis of publicly available seven asymptomatic and eight severe COVID-19 patients. Exploratory data analysis using Principal Component Analysis (PCA) showed the distinct clusters of asymptomatic and severe patients. Subsequently, the differential gene expression analysis using DESeq2 identified 1,224 significantly upregulated genes (logFC>= 1.5, p-adjusted value <0.05) and 268 significantly downregulated genes (logFC<= -1.5, p-adjusted value <0.05) in severe samples in comparison to asymptomatic samples. Eventually, Gene Set Enrichment Analysis (GSEA) of upregulated genes revealed significant enrichment of terms, i.e., anti-viral and anti-inflammatory pathways, secondary infections, Iron homeostasis, anemia, cardiac-related, etc. Gene set enrichment analysis of downregulated genes indicates lipid metabolism, adaptive immune response, translation, recurrent respiratory infections, heme-biosynthetic pathways, etc. In summary, severe COVID-19 patients are more susceptible to other health issues/concerns, non-viral pathogenic infections, atherosclerosis, autoinflammatory diseases, anemia, male infertility, etc. And eventually, these findings provide insight into the precise therapeutic management of severe COVID-19 patients and efficient disease management.","version":"1.1","doi":"10.1101/2022.04.16.488556","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.18.488614","pub_date":"2022-4-18","title":"Antibody escape and cryptic cross-domain stabilization in the SARS-CoV-2 Omicron spike protein","abstract":"The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.","version":"1.1","doi":"10.1101/2022.04.18.488614","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.17.488474","pub_date":"2022-4-18","title":"Persistent post-COVID-19 smell loss is associated with inflammatory infiltration and altered olfactory epithelial gene expression","abstract":"Most human subjects infected by SARS-CoV-2 report an acute alteration in their sense of smell, and more than 25% of COVID patients report lasting olfactory dysfunction. While animal studies and human autopsy tissues have suggested mechanisms underlying acute loss of smell, the pathophysiology that underlies persistent smell loss remains unclear. Here we combine objective measurements of smell loss in patients suffering from post-acute sequelae of SARS-CoV-2 infection (PASC) with single cell sequencing and histology of the olfactory epithelium (OE). This approach reveals that the OE of patients with persistent smell loss harbors a diffuse infiltrate of T cells expressing interferon-gamma; gene expression in sustentacular cells appears to reflect a response to inflammatory signaling, which is accompanied by a reduction in the number of olfactory sensory neurons relative to support cells. These data identify a persistent epithelial inflammatory process associated with PASC, and suggests mechanisms through which this T cell-mediated inflammation alters the sense of smell.","version":"1.1","doi":"10.1101/2022.04.17.488474","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.25.465798","pub_date":"2022-4-18","title":"Statistics of antibody binding to the spike protein explain the dependence of COVID 19 infection risk on antibody concentration and affinity","abstract":"The increase of COVID-19 breakthrough infection risk with time since vaccination has a clear relationship to the decrease of antibody concentration with time. The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface, leading to blocking of binding to the receptor: namely that the probability of infection is the probability that a critical number of the spike proteins protruding from the virus are unblocked. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of infection given measured antibody concentration. It can be used to relate population breakthrough infection risk to the distribution across the population of antibody concentration, and its variation with time.","version":"1.3","doi":"10.1101/2021.10.25.465798","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479776","pub_date":"2022-4-18","title":"Distinct Core Glycan and O-Glycoform Utilization of SARS-CoV-2 Omicron Variant Spike Protein RBD Revealed by Top-Down Mass Spectrometry","abstract":"The SARS-CoV-2 Omicron (B.1.1.529) variant possesses numerous spike (S) mutations particularly in the S receptor-binding domain (S-RBD) that significantly improve transmissibility and evasion of neutralizing antibodies. But exactly how the mutations in the Omicron variant enhance viral escape from immunological protection remains to be understood. The S-RBD remains the principal target for neutralizing antibodies and therapeutics, thus new structural insights into the Omicron S-RBD and characterization of the post-translational glycosylation changes can inform rational design of vaccines and therapeutics. Here we report the molecular variations and O-glycoform changes of the Omicron S-RBD variant as compared to wild-type (WA1/2020) and Delta (B.1.617.2) variants using high-resolution top-down mass spectrometry (MS). A novel O-glycosite (Thr376) unique to the Omicron variant is identified. Moreover, we have directly quantified the Core 1 and Core 2 O-glycan structures and characterized the O-glycoform structural heterogeneity of the three variants. Our findings reveal high resolution detail of Omicron O-glycoforms and their utilization to provide direct molecular evidence of proteoform alterations in the Omicron variant which could shed light on how this variant escapes immunological protection.","version":"1.2","doi":"10.1101/2022.02.09.479776","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484814","pub_date":"2022-4-18","title":"Persistent Immune and Clotting Dysfunction Detected in Saliva and Blood Plasma after COVID-19","abstract":"A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal vs. pathologic convalescence process have not been well-delineated. We characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 4-6 weeks after initial clinical symptoms resolved. Convalescent subjects showed robust IgA and IgG responses and positive antibody correlations between matched saliva and plasma samples. However, global shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells and clotting factors in plasma (e.g., fibrinogen and antithrombin), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered pathways that correlated positively with IgA levels. Our study positions saliva as a viable fluid to monitor immunity beyond plasma to document COVID-19 immune, inflammatory, and coagulation-related sequelae.","version":"1.2","doi":"10.1101/2022.03.18.484814","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.15.488536","pub_date":"2022-4-16","title":"Crystal structure of the Rubella virus protease reveals a unique papain-like protease fold","abstract":"Rubella is well-controlled due to an effective vaccine, but outbreaks are still occurring without any available antiviral treatments. There is still much to learn about the rubella virus (RUBV) papain-like protease (RubPro) that could be a potential drug target. This protease is crucial to RUBV replication, cleaving the non-structural polyprotein p200 into 2 multi-functional proteins, p150 and p90. Here we report a novel crystal structure of RubPro at 1.64 \u212b resolution. It has a similar catalytic core structure to that of SARS-CoV-2 and foot-mouth-disease virus (FMDV) proteases. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. The RubPro construct was shown to have protease activity in trans against a construct of RUBV protease-helicase and fluorogenic peptide. A protease-helicase construct was also cleaved in E. coli expression. RubPro was demonstrated to possess deubiquitylation activity, suggesting a potential role of RubPro in modulating the host\u2019s innate immune responses. The structural and functional insights of the RubPro will advance our current understanding of its function and point to more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics in the future.","version":"1.1","doi":"10.1101/2022.04.15.488536","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.488249","pub_date":"2022-4-14","title":"Whole-body metabolic modelling predicts isoleucine dependency of SARS-CoV-2 replication","abstract":"We aimed at investigating host-virus co-metabolism during SARS-CoV-2 infection. Therefore, we extended comprehensive sex-specific, whole-body organ resolved models of human metabolism with the necessary reactions to replicate SARS-CoV-2 in the lung as well as selected peripheral organs. Using this comprehensive host-virus model, we obtained the following key results: 1. The predicted maximal possible virus shedding rate was limited by isoleucine availability. 2. The supported initial viral load depended on the increase in CD4+ T-cells, consistent with the literature. 3. During viral infection, the whole-body metabolism changed including the blood metabolome, which agreed well with metabolomic studies from COVID-19 patients and healthy controls. 4. The virus shedding rate could be reduced by either inhibition of the guanylate kinase 1 or availability of amino acids, e.g., in the diet. 5. The virus variants achieved differed in their maximal possible virus shedding rates, which could be inversely linked to isoleucine occurrences in the sequences. Taken together, this study presents the metabolic crosstalk between host and virus and emphasis the role of amino acid metabolism during SARS-CoV-2 infection, in particular of isoleucine. As such, it provides an example of how computational modelling can complement more canonical approaches to gain insight into host-virus crosstalk and to identify potential therapeutic strategies.","version":"1.1","doi":"10.1101/2022.04.13.488249","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.487939","pub_date":"2022-4-14","title":"SARS-CoV-2 Omicron BA.1 variant infection of human colon epithelial cells","abstract":"Omicron B.1.1.529 became the predominant SARS-CoV-2 variant in early 2022, causing a new wave of public anxiety. Compared to the ancestral strain, Omicron has 50 mutations, with over 30 mutations in the spike protein. These differences likely underlie the changes in Omicron biology noted in other studies, including an attenuation in the lung parenchyma, compared to the ancestral SARS-CoV-2 strain and other variants, as well as a preference for endosomal entry, in place of the TMPRSS2-mediated membrane fusion pathway. This raises questions on Omicron tropism and infectivity in various target organ systems, including the gastrointestinal (GI) tract. Up to 70% of COVID-19 patients report GI symptoms, including nausea, vomiting, and diarrhea. Here, we show that in the context of donor intrinsic genetic heterogeneity, the SARS-CoV-2 Omicron variant infects human colonoids similarly, if not less effectively, than the ancestral WT (WA1) strain or the Delta variant. Additionally, we note a higher ratio of viral RNA to infectious virus titer, which may suggest that Omicron is potentially less infectious in the intestine. This study lays the foundation for further work defining mechanisms mediating intestinal infection and pathogenesis by Omicron.","version":"1.1","doi":"10.1101/2022.04.13.487939","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.488221","pub_date":"2022-4-14","title":"Determinants of Spike Infectivity, Processing and Neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2","abstract":"The SARS-CoV-2 Omicron variant rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission, immune evasion and pathogenicity is thought to be driven by numerous mutations in the Omicron Spike protein. Here, we examined the impact of amino acid changes that are characteristic for the BA.1 and/or BA.2 Omicron lineages on Spike function, processing and susceptibility to neutralization. Individual mutations of S371F/L, S375F and T376A in the ACE2 receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes of G339D, D614G, N764K and L981F moderately enhanced it. Most mutations in the N-terminal region and the receptor binding domain reduced sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine or therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that allowed this SARS-CoV-2 variant to overtake the current pandemic. S371F/L, S373P and S375F impair Spike function and revert in some BA. 1 isolates Changes of Q954H and N969K in HR1 reduce while L981F enhances S-mediated infection Omicron-specific mutations in the NTD and RBD of Spike reduce neutralization N440K, G446S, E484A and Q493K confer resistance to bamlanivimab or imdevimab","version":"1.1","doi":"10.1101/2022.04.13.488221","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.488264","pub_date":"2022-4-14","title":"Genomic surveillance unfolds the dynamics of SARS-CoV-2 transmission and divergence in Bangladesh over the past two years","abstract":"The highly pathogenic virus SARS-CoV-2 has shattered the healthcare system of the world causing the COVID-19 pandemic since first detected in Wuhan, China. Therefore, scrutinizing the genome structure and tracing the transmission of the virus has gained enormous interest in designing appropriate intervention strategies to control the pandemic. In this report, we examined 4622 sequences from Bangladesh and found that they belonged to thirty-five major PANGO lineages, while Delta alone accounted for 39%, and 78% were from just four primary lineages. Our research has also shown Dhaka to be the hub of viral transmission and observed the virus spreading back and forth across the country at different times by building a transmission network. The analysis resulted in 7659 unique mutations, with an average of 24.61 missense mutations per sequence. Moreover, our analysis of genetic diversity and mutation patterns revealed that eight genes were under negative selection pressure to purify deleterious mutations, while three genes were under positive selection pressure. With 29,122 deaths, 1.95 million infections and a shattered healthcare system from SARS-CoV-2 in Bangladesh, the only way to avoid further complications is to break the transmission network of the virus. Therefore, it is vital to shedding light on the transmission, divergence, mutations, and emergence of new variants using genomic data analyses and surveillance. Here, we present the geographic and temporal distribution of different SARS-CoV-2 variants throughout Bangladesh over the past two years, and their current prevalence. Further, we have developed a transmission network of viral spreads, which in turn will help take intervention measures. Then we analyzed all the mutations that occurred and their effect on evolution as well as the currently present mutations that could trigger a new variant of concern. In short, together with an ongoing genomic surveillance program, these data will help to better understand SARS-CoV-2, its evolution, and pandemic characteristics in Bangladesh.","version":"1.1","doi":"10.1101/2022.04.13.488264","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.486321","pub_date":"2022-4-14","title":"Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells","abstract":"With the emergence of multiple highly transmissible SARS-CoV-2 variants during the recent pandemic, the comparison of their infectivity has become a substantially critical issue for public health. However, a direct assessment of these viral characteristics has been challenging due to the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing techniques to facilitate the evaluation. In a proof-of-concept study using the assay with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our results demonstrate that the Omicron (BA.1) variant is 3-5-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the early phase of infection. To our knowledge, this study provides the first direct measurement of the infectivity of the Omicron variant and new experimental procedures that can be applied for monitoring newly emerging viral variants.","version":"1.1","doi":"10.1101/2022.04.13.486321","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.04.483019","pub_date":"2022-4-14","title":"Soluble P2X7 receptor is elevated in the plasma of COVID-19 patients and correlates with disease severity","abstract":"Inflammation is a tightly coordinated response against bacterial and viral infections, triggered by the production of pro-inflammatory cytokines. SARS-CoV-2 infection induces COVID-19 disease, characterized by an inflammatory response mediated through the activation of the NLRP3 inflammasome, which results in the production of IL-1\u03b2 and IL-18 along with pyroptotic cell death. The NLRP3 inflammasome could be also triggered by sterile danger signals such as extracellular ATP triggering the purinergic P2X7 receptor. Severe inflammation in the lungs of SARS-CoV-2 infected individuals is associated with pneumonia, hypoxia and acute respiratory distress syndrome, these being the causes of death associated with COVID-19. Both the P2X7 receptor and NLRP3 have been considered as potential pharmacological targets for treating inflammation in COVID-19. However, there is no experimental evidence of the involvement of the P2X7 receptor during COVID-19 disease. In the present study we determined the concentration of different cytokines and the P2X7 receptor in the plasma of COVID-19 patients and found that along with the increase in IL-6, IL-18 and the IL-1 receptor antagonist in the plasma of COVID-19 patients, there was also an increase in the purinergic P2X7 receptor. Increase in COVID-19 severity and C-reactive protein concentration positively correlated with increased concentration of the P2X7 receptor in the plasma, but not with IL-18 cytokine. The P2X7 receptor was found in the supernatant of human peripheral blood mononuclear cells after inflammasome activation. Therefore, our data suggest that determining levels of the P2X7 receptor in the plasma could be a novel biomarker of COVID-19 severity.","version":"1.3","doi":"10.1101/2022.03.04.483019","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.488236","pub_date":"2022-4-14","title":"Mutual inhibition of airway epithelial responses supports viral and fungal co-pathogenesis during coinfection","abstract":"Awareness that fungal coinfection complicates viral respiratory infections causing worse disease outcome has recently emerged. The environmental fungus Aspergillus fumigatus (Af) has been reported as the main driver of fungal coinfection in patients suffering from viral infections caused by Cytomegalovirus, Influenza or more recently SARS-CoV2. The airway epithelium is the first common point of contact between inhaled pathogens and the host. Aberrant airway epithelial cell (AEC) responses against fungal challenge have been described in patients susceptible to aspergillosis. Therefore, it is likely that a dysregulation of AEC responses during fungal-viral coinfection represents a potent driver for the development of fungal disease. Here we used an in vitro model of Af-viral infection of AECs to determine outcomes of spore internalisation, killing and viral replication during coinfection. Our data indicate that viral stimulation, while boosting Af uptake by AECs, limits Af spore killing by those cells, favouring fungal persistence and growth. Type I viral-induced interferon release was significantly decreased in the presence of Af hyphal forms suggesting a possible role of Af secreted factors in modulating viral pathogenicity. We next explored the impact of Af challenge in SARS-CoV2 replication within airway epithelial cells using nano-luciferase as a measure of viral replication. We found that Af increased SARS-CoV2 pathogenicity in a strain-dependent manner. Collectively, our findings demonstrate a mutual inhibition of antifungal and antiviral AEC responses during Af-viral coinfection and also suggest that some fungal factors might be key regulators of co-pathogenicity during in lung infection.","version":"1.1","doi":"10.1101/2022.04.13.488236","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.14.488348","pub_date":"2022-4-14","title":"This is GlycoQL","abstract":"We have previously designed and implemented a tree-based ontology to represent glycan structures with the aim of searching these structures with a glyco-driven syntax. This resulted in creating the GlySTreeM knowledge-base as a linchpin of the matching procedure and we now introduce a query language, called GlycoQL, for the actual implementation of a glycan structure search. The methodology is described and illustrated with a use-case focused on SARS-CoV-2 spike protein glycosylation. We show how to enhance site annotation with federated queries involving UniProt and GlyConnect, our glycoprotein database. currently only available for reviewers at: https://beta.glyconnect.expasy.org/glycoql/ catherine.hayes@unige.ch; frederique.lisacek@sib.swiss Supplementary data are available at https://glyconnect.expasy.org/glystreem/wiki.","version":"1.1","doi":"10.1101/2022.04.14.488348","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.488051","pub_date":"2022-4-13","title":"Markers of Fungal Translocation Are Elevated During Post-Acute Sequelae of SARS-CoV-2 Infection and Induce NF-\u03baB Triggered Inflammation","abstract":"Long COVID, a type of Post-Acute Sequelae of SARS CoV-2 infection (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the pathophysiological mechanisms that drive this inflammation remain unknown. Inflammation during acute Coronavirus Disease 2019 (COVID-19) could be exacerbated by microbial translocation (from the gut and/or lung) to the blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We found higher levels of fungal translocation \u2013 measured as \u03b2-glucan, a fungal cell wall polysaccharide \u2013 in the plasma of individuals experiencing PASC compared to those without PASC or SARS-CoV-2 negative controls. The higher \u03b2-glucan correlated with higher levels of markers of inflammation and elevated levels of host metabolites involved in activating N-Methyl-D-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neuro-toxic properties. Mechanistically, \u03b2-glucan can directly induce inflammation by binding to myeloid cells (via the Dectin-1 receptor) and activating Syk/NF-\u03baB signaling. Using an in vitro Dectin-1/NF-\u03baB reporter model, we found that plasma from individuals experiencing PASC induced higher NF-\u03baB signaling compared to plasma from SARS-CoV-2 negative controls. This higher NF-\u03baB signaling was abrogated by the Syk inhibitor Piceatannol. These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.","version":"1.1","doi":"10.1101/2022.04.12.488051","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.488092","pub_date":"2022-4-13","title":"SARS-CoV-2 Delta breakthrough infections in vaccinated patients","abstract":"The continuous emergence of SARS-CoV-2 variants with increased transmission and immune evasion has caused breakthrough infections in vaccinated population. It is important to determine the threshold of neutralizing antibody titers that permit breakthrough infections. Here we tested the neutralization titers of vaccinated patients who contracted Delta variant. All 75 patients with Delta breakthrough infections exhibited neutralization titers (NT50) of less than 70. Among the breakthrough patients, 76%, 18.7%, and 5.3% of them had the NT50 ranges of <20, 20-50, and 50-69, respectively. These clinical laboratory results have implications in vaccine strategy and public health policy.","version":"1.1","doi":"10.1101/2022.04.12.488092","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.26.484261","pub_date":"2022-4-13","title":"Titers of antibodies the receptor-binding domain (RBD) of ancestral SARS-CoV-2 are predictive for levels of neutralizing antibodies to multiple variants","abstract":"Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.","version":"1.2","doi":"10.1101/2022.03.26.484261","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.13.488132","pub_date":"2022-4-13","title":"The Omicron (B.1.1.529) SARS-CoV-2 variant of concern also affects companion animals","abstract":"The recent emergence of the Omicron variant (B.1.1.529) has brought with it a large increase in the incidence of SARS-CoV-2 disease worldwide. However, there is hardly any data on the incidence of this new variant in companion animals. In this study, we have detected the presence of this new variant in domestic animals such as dogs and cats living with owners with COVID19 in Spain that have been sampled at the most optimal time for the detection of the disease. None of the RT-qPCR positive animals (10.13%) presented any clinical signs and the viral loads detected were very low. In addition, the shedding of viral RNA lasted a short period of time in the positive animals. Infection with the Omicron variant of concern (VOC) was confirmed by a specific RT-qPCR for the detection of this variant and by sequencing. These outcomes suggest a lower virulence of this variant in infected cats and dogs. This study demonstrates the transmission of this new variant from infected humans to domestic animals and highlights the importance of doing active surveillance as well as genomic research to detect the presence of VOCs or mutations associated with animal hosts.","version":"1.1","doi":"10.1101/2022.04.13.488132","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.488087","pub_date":"2022-4-13","title":"Broadly neutralizing and protective nanobodies against diverse sarbecoviruses","abstract":"As SARS-CoV-2 Omicron and other variants of concern continue spreading around the world, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with exceptional breadth and potency against diverse sarbecoviruses including SARS-CoV-1, Omicron BA.1, and BA.2. Crystal structure analysis of one representative nanobody, 3-2A2-4, revealed a highly conserved epitope between the cryptic and the outer face of the receptor binding domain (RBD). The epitope is readily accessible regardless of RBD in \u201cup\u201d or \u201cdown\u201d conformation and distinctive from the receptor ACE2 binding site. Passive delivery of 3-2A2-4 protected K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. This group of nanobodies and the epitope identified should provide invaluable reference for the development of next generation antibody therapies and vaccines against wide varieties of SARS-CoV-2 infection and beyond.","version":"1.1","doi":"10.1101/2022.04.12.488087","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.09.487739","pub_date":"2022-4-12","title":"Disrupting ACE2 Dimerization Mitigates the Infection by SARS-COV-2","abstract":"The coronavirus disease 2019 (COVID-19) pandemic has caused over 6 million death and 460 million reported cases globally. More effective antiviral medications are needed to curb the continued spread of this disease. The infection by SARS-COV-2 virus is initiated via the interaction between the receptor binding domain (RBD) of the viral glycoprotein Spike (S protein) and the N-term peptidase domain (PD) of the angiotensin-converting enzyme 2 (ACE2) expressed on host cell membrane. ACE2 forms protein homodimer primarily through its ferredoxin-like fold domain (aka. Neck-domain). We investigated whether the dimerization of ACE2 receptor plays a role in SARS-COV-2 virus infection. We report here that the ACE2 receptor dimerization enhances the recognition of SARS-COV-2 S protein. A 43 amino acid peptide based on the N-term of Neck-domain could block the ACE2 dimerization and the interaction between RBD and ACE2, and mitigate the SARS-COV-2/host cell interaction. Our study illustrated a new route to develop potential therapeutics for the prevention and treatment of SARS-COV-2 viral infection.","version":"1.2","doi":"10.1101/2022.04.09.487739","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.487988","pub_date":"2022-4-12","title":"An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity","abstract":"The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.","version":"1.1","doi":"10.1101/2022.04.12.487988","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431652","pub_date":"2022-4-12","title":"How the replication and transcription complex functions in jumping transcription of SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although unprecedented efforts are underway to develop therapeutic strategies against this disease, scientists have acquired only a little knowledge regarding the structures and functions of the CoV replication and transcription complex (RTC) and 16 non-structural proteins, named NSP1-16. In the present study, we proposed a two-route model to answer how the RTC functions in the jumping transcription of CoVs. The key step leading to this model was that the motif AAACH for METTL3 recognition flanking the transcription regulatory sequence (TRS) motif was discovered to determine the m6A methylation of SARS-CoV-2 RNAs, by reanalyzing public Nanopore RNA-seq data. As the most important finding, TRS hairpins were reported for the first time to interpret NSP15 cleavage, RNA methylation of CoVs and their association at the molecular level. In addition, we reported canonical TRS motifs of all CoVs to prove the importance of our findings. The main conclusions are: (1) TRS hairpins can be used to identify recombination regions in CoV genomes; (2) RNA methylation of CoVs participates in the determination of the RNA secondary structures by affecting the formation of base pairing; and (3) The eventual determination of the CoV RTC global structure needs to consider METTL3 in the experimental design. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, providing a crucial basis for future studies.","version":"1.2","doi":"10.1101/2021.02.17.431652","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.487379","pub_date":"2022-4-12","title":"Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants","abstract":"SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variants revealed that SARS-CoV-2 WA1 or Delta infects a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possesses broader cellular invasion capacity into the submucosa, while Omicron displays longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon is more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa is accompanied by a decline of phagocytosis related genes. Furthermore, robust basal stem cell activation contributes to neuroepithelial regeneration and restores ACE2 expression post-infection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration post infection. The shifting characteristics of viral infection at the airway portal provides insight into the variability of COVID-19 clinical features and may suggest differing strategies for early local intervention.","version":"1.1","doi":"10.1101/2022.04.12.487379","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.488042","pub_date":"2022-4-12","title":"The SARS-CoV-2 receptor-binding domain facilitates neutrophil transepithelial migration and nanoparticle uptake in the mice airways","abstract":"SARS-CoV-2-induced infection is still dangerous. Mouse models are convenient to the investigation of virus-activated immune response mechanisms. However, mice are not proper model organisms to study COVID-19 due to decreased interaction affinity between the SARS-CoV-2 receptor-binding domain (RBD) and mouse angiotensin-converting enzyme 2 (ACE2) compared with human ACE2. In the present study, we propose a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system. To mimic the effects of RBD\u2013 ACE2 high-affinity interaction, mice received the ACE2 inhibitor MLN-4760. To simulate virus loading, we applied 100 nm particles suspended in the solution of RBD via the oropharyngeal route to mice. In this model, MLN-4760 application enhanced neutrophil egress from the bone marrow to the bloodstream and RBD attracted neutrophils to the luminal side of the conducting airway epithelium. By contrast, inert 100 nm particles were not potent to stimulate neutrophil recruitment to the conducting airway mucosa. Using this model, and by altering the dosage of the ACE2 inhibitor, nanoparticles, and RBD, one can adapt it to investigate different COVID-19 states characterized with mild or severe airway inflammation. This study presents a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system and investigates immune cell-model virus particle interactions in the conducting airway mucosa.","version":"1.1","doi":"10.1101/2022.04.12.488042","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486373","pub_date":"2022-4-12","title":"Prediction of infectivity of SARS-CoV2: Mathematical model with analysis of docking simulation for spike proteins and angiotensin-converting enzyme 2","abstract":"Variants of a coronavirus (SARS-CoV-2) have been spreading in a global pandemic. Improved understanding of the infectivity of future new variants is important so that effective countermeasures against them can be quickly undertaken. In our research reported here, we aimed to predict the infectivity of SARS-CoV-2 by using a mathematical model with molecular simulation analysis, and we used phylogenetic analysis to determine the evolutionary distance of the spike protein gene (S gene) of SARS-CoV-2. We subjected the six variants and the wild type of spike protein and human angiotensin-converting enzyme 2 (ACE2) to molecular docking simulation analyses to understand the binding affinity of spike protein and ACE2. We then utilized regression analysis of the correlation coefficient of the mathematical model and the infectivity of SARS-CoV-2 to predict infectivity. The evolutionary distance of the S gene correlated with the infectivity of SARS-CoV-2 variants. The coefficient of the mathematical model obtained with results of molecular docking simulation also correlated with the infectivity of SARS-CoV-2 variants. These results suggest that the data from the docking simulation for the receptor binding domain of variant spike proteins and human ACE2 were valuable for prediction of SARS-CoV-2 infectivity. In addition, we developed a mathematical model for prediction of SARS-CoV-2 variant infectivity by using binding affinity obtained via molecular docking and the evolutionary distance of the S gene.","version":"1.3","doi":"10.1101/2022.03.30.486373","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.487999","pub_date":"2022-4-12","title":"Model evolution in SARS-CoV-2 spike protein sequences using a generative neural network","abstract":"Modelling evolutionary elements inherent in protein sequences, emerging from one clade into another of the SARS-CoV-2 virus, would provide insights to augment our understanding of its impact on public health and may help in formulating better strategies to contain its spread. Deep learning methods have been used to model protein sequences for SARS-CoV-2 viruses. A few significant drawbacks in these studies include being deficient in modelling end-to-end protein sequences, modelling only those genomic positions that show high activity and upsampling the number of sequences at each genomic position for balancing the frequency of mutations. To mitigate such drawbacks, the current approach uses a generative model, an encoder-decoder neural network, to learn the natural progression of spike protein sequences through adjacent clades of the phylogenetic tree of Nextstrain clades. Encoder transforms a set of spike protein sequences from the source clade (20A) into its latent representation. Decoder uses the latent representation, along with Gaussian distributed noise, to generate a different set of protein sequences that are closer to the target clade (20B). The source and target clades are adjacent nodes in the phylogenetic tree of different evolving clades of the SARS-CoV-2 virus. Sequences of amino acids are generated, for the entire length, at each genomic position using the latent representation of the amino acid generated at a previous step. Using trained models, protein sequences from the source clade are used to generate sequences that form a collection of evolved sequences belonging to all children clades of the source clade. A comparison of this predicted evolution (between source and generated sequences) of proteins with the true evolution (between source and target sequences) shows a high pearson correlation (> 0.7). Moreover, the distribution of the frequencies of substitutions per genomic position, including high- and low-frequency positions, in source-target sequences and source-generated sequences exhibit a high resemblance (pearson correlation > 0.7). In addition, the model partially predicts a few substitutions at specific genomic positions for the sequences of unseen clades (20J (Gamma)) where they show little activity during training. These outcomes show the potential of this approach in learning the latent mechanism of evolution of SARS-CoV-2 viral sequences. https://github.com/anuprulez/clade_prediction","version":"1.1","doi":"10.1101/2022.04.12.487999","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.12.488010","pub_date":"2022-4-12","title":"Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis","abstract":"Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.","version":"1.1","doi":"10.1101/2022.04.12.488010","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.08.467705","pub_date":"2022-4-12","title":"Molecular signature of postmortem lung tissue from COVID-19 patients suggests distinct trajectories driving mortality","abstract":"The precise molecular mechanisms behind life-threatening lung abnormalities during severe SARS-CoV-2 infections are still unclear. To address this challenge, we performed whole transcriptome sequencing of lung autopsies from 31 patients suffering from severe COVID-19 related complications and 10 uninfected controls. Using a metatranscriptome analysis of lung tissue samples we identified the existence of two distinct molecular signatures of lethal COVID-19. The dominant \u201cclassical\u201d signature (n=23) showed upregulation of unfolded protein response, steroid biosynthesis and complement activation supported by massive metabolic reprogramming leading to characteristic lung damage. The rarer signature (n=8) potentially representing \u201cCytokine Release Syndrome\u201d (CRS) showed upregulation of cytokines such IL1 and CCL19 but absence of complement activation and muted inflammation. Further, dissecting expression of individual genes within enriched pathways for patient signature suggests heterogeneity in host response to the primary infection. We found that the majority of patients cleared the SARS-CoV-2 infection, but all suffered from acute dysbiosis with characteristic enrichment of opportunistic pathogens such as Staphylococcus cohnii in \u201cclassical\u201d patients and Pasteurella multocida in CRS patients. Our results suggest two distinct models of lung pathology in severe COVID-19 patients that can be identified through the status of the complement activation, presence of specific cytokines and characteristic microbiome. This information can be used to design personalized therapy to treat COVID-19 related complications corresponding to patient signature such as using the identified drug molecules or mitigating specific secondary infections.","version":"1.2","doi":"10.1101/2021.11.08.467705","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487970","pub_date":"2022-4-12","title":"Discrimination of SARS-CoV-2 Omicron sub-lineages BA.1 and BA.2 using a high-resolution melting-based assay: A pilot study","abstract":"The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting peaks of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variants BA.1/BA.1.1, and 8 Omicron BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2.","version":"1.1","doi":"10.1101/2022.04.11.487970","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487920","pub_date":"2022-4-12","title":"Hierarchical Computational Modeling and Dynamic Network Analysis of Allosteric Regulation in the SARS-CoV-2 Spike Omicron Trimer Structures: Omicron Mutations Cooperate to Allosterically Control Balance of Protein Stability and Conformational Adaptability","abstract":"Structural and computational studies of the Omicron spike protein in various functional states and complexes provided important insights into molecular mechanisms underlying binding, high transmissibility, and escaping immune defense. However, the regulatory roles and functional coordination of the Omicron mutations are poorly understood and often ignored in the proposed mechanisms. In this work, we explored the hypothesis that the SARS-CoV-2 spike protein can function as a robust allosterically regulated machinery in which Omicron mutational sites are dynamically coupled and form a central engine of the allosteric network that regulates the balance between conformational plasticity, protein stability, and functional adaptability. In this study, we employed coarse-grained dynamics simulations of multiple full-length SARS-CoV-2 spike Omicron trimers structures in the closed and open states with the local energetic frustration analysis and collective dynamics mapping to understand the determinants and key hotspots driving the balance of protein stability and conformational adaptability. We have found that the Omicron mutational sites at the inter-protomer regions form regulatory clusters that control functional transitions between the closed and open states. Through perturbation-based modeling of allosteric interaction networks and diffusion analysis of communications in the closed and open spike states, we quantify the allosterically regulated activation mechanism and uncover specific regulatory roles of the Omicron mutations. The network modeling demonstrated that Omicron mutations form the inter-protomer electrostatic bridges that connect local stable communities and function as allosteric switches of signal transmission. The results of this study are consistent with the experiments, revealing distinct and yet complementary role of the Omicron mutational sites as a network of hotspots that enable allosteric modulation of structural stability and conformational changes which are central for spike activation and virus transmissibility.","version":"1.1","doi":"10.1101/2022.04.11.487920","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487879","pub_date":"2022-4-12","title":"Broadly neutralizing antibodies target the coronavirus fusion peptide","abstract":"The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2\u2019 cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2\u2019 cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. Rare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.","version":"1.1","doi":"10.1101/2022.04.11.487879","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.375626","pub_date":"2022-4-12","title":"DeepDrug: A general graph-based deep learning framework for drug-drug interactions and drug-target interactions prediction","abstract":"Computational approaches for accurate prediction of drug interactions, such as drug-drug interactions (DDIs) and drug-target interactions (DTIs), are highly demanded for biochemical researchers due to the efficiency and cost-effectiveness. Despite the fact that many methods have been proposed and developed to predict DDIs and DTIs respectively, their success is still limited due to a lack of systematic evaluation of the intrinsic properties embedded in the corresponding chemical structure. In this paper, we develop a deep learning framework, named DeepDrug, to overcome the above limitation by using residual graph convolutional networks (RGCNs) and convolutional networks (CNNs) to learn the comprehensive structural and sequential representations of drugs and proteins in order to boost the DDIs and DTIs prediction accuracy. We benchmark our methods in a series of systematic experiments, including binary-class DDIs, multi-class/multi-label DDIs, binary-class DTIs classification and DTIs regression tasks using several datasets. We then demonstrate that DeepDrug outperforms state-of-the-art methods in terms of both accuracy and robustness in predicting DDIs and DTIs with multiple experimental settings. Furthermore, we visualize the structural features learned by DeepDrug RGCN module, which displays compatible and accordant patterns in chemical properties and drug categories, providing additional evidence to support the strong predictive power of DeepDrug. Ultimately, we apply DeepDrug to perform drug repositioning on the whole DrugBank database to discover the potential drug candidates against SARS-CoV-2, where 3 out of 5 top-ranked drugs are reported to be repurposed to potentially treat COVID-19. To sum up, we believe that DeepDrug is an efficient tool in accurate prediction of DDIs and DTIs and provides a promising insight in understanding the underlying mechanism of these biochemical relations. The source code of the DeepDrug can be freely downloaded from https://github.com/wanwenzeng/deepdrug.","version":"1.2","doi":"10.1101/2020.11.09.375626","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460265","pub_date":"2022-4-12","title":"The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets","abstract":"Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2\u2019-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA-RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function.","version":"1.3","doi":"10.1101/2021.09.14.460265","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487836","pub_date":"2022-4-11","title":"SARS-ANI: A Global Open Access Dataset of Reported SARS-CoV-2 Events in Animals","abstract":"The zoonotic origin of SARS-CoV-2, the etiological agent of COVID-19, is not yet fully resolved. Although natural infections in animals are reported in a wide range of species, large knowledge and data gaps remain regarding SARS-CoV-2 animal hosts. We used two major health databases to extract unstructured data and generated a comprehensive global dataset of thoroughly documented SARS-CoV-2 events in animals. The dataset integrates relevant epidemiological and clinical data on each event and is readily usable for analytical purposes. We also share the code for technical and visual validation of the data and created a user-friendly dashboard for data exploration. Data on SARS-CoV-2 occurrence in animals is critical to adapt monitoring strategy, prevent the formation of animal reservoirs, and tailor future human and animal vaccination programs. The FAIRness and analytical flexibility of the data will support research efforts on SARS-CoV-2 at the human-animal-environment interface. We intend to update this dataset weekly for at least one year and, through collaborative processes, to develop the dataset further and expand its use.","version":"1.1","doi":"10.1101/2022.04.11.487836","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487828","pub_date":"2022-4-11","title":"Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants","abstract":"Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from human to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, in order to evaluate the impact of S mutations, we constructed 20 Hela cell lines stably expressing ACE2 orthologs from different animals, and prepared 27 pseudotyped SARS-CoV-2 carrying different spike mutants, among which 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.135), Lambda (B.1.429) and Mu (B.1.525). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammals ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.","version":"1.1","doi":"10.1101/2022.04.11.487828","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.15.468737","pub_date":"2022-4-11","title":"SARS-CoV-2 RdRp uses NDPs as a substrate and is able to incorporate NHC into RNA from diphosphate form molnupiravir","abstract":"The coronavirus disease 2019 (COVID-19) has been ravaging throughout the world for more than two years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) employs the viral RNA-dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Here, we reveal that RdRp can recognize and utilize nucleoside diphosphates (NDPs) as a substrate to synthesize RNAs with an efficiency of about two thirds of using nucleoside triphosphates (NTPs) as a substrate. NDPs incorporation is also template-specific and has high fidelity. Moreover, RdRp can incorporate \u03b2-d-N4-hydroxycytidine (NHC) into RNA while using diphosphate form molnupiravir (MDP) as a substrate. We also observed that MDP is a better substrate for RdRp than the triphosphate form molnupiravir (MTP).","version":"1.2","doi":"10.1101/2021.11.15.468737","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487924","pub_date":"2022-4-11","title":"Independent acquisition of short insertions at the RIR1 site in the spike N-terminal domain of the SARS-CoV-2 BA.2 lineage","abstract":"Although the SARS-CoV-2 variants BA.1 and BA.2 share over 30 non-synonymous substitutions in the spike glycoprotein, they show several unique mutations that were likely acquired after the split between these two major omicron lineages. One of the most intriguing mutations associated with BA.1 is the presence of the inserted tripeptide Glu-Pro-Glu within the N-terminal domain. While the functional implications of this insertion are still unclear, several other SARS-CoV-2 lineages had previously independently acquired similarly short insertions at the very same site, named RIR1. We have previously identified this site, located approximately between codon 212 and codon 216, as a hotspot of insertions, which usually involve small nucleotide sequences including three or four codons. Here we show that similar insertion events have independently occurred at least 13 times in early 2022 within the BA.2 lineage, being occasionally associated with significant community transmission. One of these omicron sublineages, characterized by a Ser-Gly-Arg insertion in position 212, is responsible of over 2% of all SARS-CoV-2 cases recorded in Denmark, as of early April 2022. Molecular surveillance data highlight a slow but steady growth compared with the parental BA.2 lineage in all Danish regions, suggesting that the RIR1 insertion may confer a selective advantage. We report the identification of other currently circulating BA.2 sublineages showing similar insertions, whose spread should be therefore carefully monitored in the upcoming months.","version":"1.1","doi":"10.1101/2022.04.11.487924","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.11.487882","pub_date":"2022-4-11","title":"Receptor binding domain of SARS-CoV-2 is a functional \u03b1v-integrin agonist","abstract":"Among the novel mutations distinguishing SARS-CoV-2 from similar respiratory coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2 (ACE2)-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. In the present study, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared to RGD-containing, integrin-binding fragments of fibronectin. S1-RBD supported adhesion of both fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells. Cell adhesion to S1-RBD was cation- and RGD-dependent, and was inhibited by blocking antibodies against \u03b1v and \u03b23, but not \u03b15 or \u03b21, integrins. Similarly, direct binding of S1-RBD to recombinant human \u03b1v\u03b23 and \u03b1v\u03b26 integrins, but not \u03b15\u03b21 integrins, was observed by surface plasmon resonance. Adhesion to S1-RBD initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin, Akt activation, and supported cell proliferation. Together, these data demonstrate that the RGD sequence within S1-RBD can function as an \u03b1v-selective integrin agonist. This study provides evidence that cell surface \u03b1v-containing integrins can respond functionally to spike protein and raise the possibility that S1-mediated dysregulation of ECM dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.04.11.487882","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.10.486823","pub_date":"2022-4-11","title":"Subtyping of major SARS-CoV-2 variants reveals different transmission dynamics","abstract":"SARS-CoV-2 continues to evolve, causing waves of the pandemic. Up to March 2022, eight million genome sequences have accumulated, which are classified into five major variants of concern. With the growing number of sequenced genomes, analysis of the big dataset has become increasingly challenging. Here we developed systematic approaches for comprehensive subtyping and pattern recognition for transmission dynamics. By analyzing the first two million viral genomes as of July 2021, we found that different subtypes of the same variant exhibited distinct temporal trajectories. For example, some Delta subtypes did not spread rapidly, while others did. We identified sets of characteristic single nucleotide variations (SNVs) that appeared to enhance transmission or decrease efficacy of antibodies for some subtypes of the Delta and Alpha variants. We also identified a set of SNVs that appeared to suppress transmission or increase viral sensitivity to antibodies. These findings are later confirmed in an analysis of six million genomes as of December 2021. For the Omicron variant, the dominant type in the world, we identified the subtypes with enhanced and suppressed transmission in an analysis of seven million genomes as of January 2022 and further confirmed the findings in a later analysis of eight million genomes as of March 2022. While the \u201cenhancer\u201d SNVs exhibited an enriched presence on the spike protein, the \u201csuppressor\u201d SNVs are mainly elsewhere. Disruption of the SNV correlation largely destroyed the enhancer-suppressor phenomena. These results suggest the importance of fine subtyping of variants, and point to potential complex interactions among SNVs.","version":"1.1","doi":"10.1101/2022.04.10.486823","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.10.487815","pub_date":"2022-4-11","title":"Single-cell colocalization analysis using a deep generative model","abstract":"Analyzing colocalization of single cells with heterogeneous molecular phenotypes is essential for understanding cell-cell interactions, cellular responses to external stimuli, and their biological functions in diseases and tissues. However, high-throughput methods for identifying spatial proximity at single-cell resolution are practically unavailable. Here, we introduce DeepCOLOR, a computational framework based on a deep generative model that recovers inter-cellular colocalization networks with single cell resolution by the integration of single cell and spatial transcriptomes. It segregates cell populations defined by the colocalization relationships and predicts cell-cell interactions between colocalized single cells. DeepCOLOR could identify plausible cell-cell interaction candidates in mouse brain tissues, human squamous cell carcinoma samples, and human lung tissues infected with SARS-CoV-2 by reconstructing spatial colocalization maps at single-cell resolution. DeepCOLOR is typically applicable to studying cell-cell interactions in any spatial niche. Our newly developed computational framework could help uncover molecular pathways across single cells connected with colocalization networks.","version":"1.1","doi":"10.1101/2022.04.10.487815","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.10.487761","pub_date":"2022-4-11","title":"Candidate genes associated with neurological manifestations of COVID-19: Meta-analysis using multiple computational approaches","abstract":"COVID-19 develops certain neurological symptoms, the molecular pathophysiology of which is obscure. In the present study, two networks were constructed and their hub-bottleneck and driver nodes were evaluated to consider them as \u2018target genes\u2019 followed by identifying \u2018candidate genes\u2019 and their associations with neurological phenotypes of COVID-19. A tripartite network was first constructed using literature-based neurological symptoms of COVID-19 as input. The target genes evaluated therefrom were then used as query genes to identify the co-expressed genes from the RNA-sequence data of the frontal cortex of COVID-19 patients using pair-wise mutual information to genes. A \u2018combined gene network\u2019 (CGN) was constructed with 189 genes selected from TN and 225 genes co-expressed in COVID-19. Total 44 \u2018target genes\u2019 evaluated from both networks and their connecting genes in respective networks were analyzed functionally by measuring pair-wise \u2018semantic similarity scores\u2019 (SSS) and finding Enrichr annotation terms against a set of genes. A new integrated \u2018weighted harmonic mean score\u2019 was formulated using SSS and STRING-based \u2018combined score\u2019 to select 21 gene-pairs among \u2018target genes\u2019 that provided 21 \u2018candidate genes\u2019 with their properties as \u2018indispensable driver nodes\u2019 of CGN. Finally, six pairs providing seven prevalent candidate genes (ADAM10, ADAM17, AKT1, CTNNB1, ESR1, PIK3CA, FGFR1) exhibited direct linkage with the neurological phenotypes under tumour/cancer, cellular signalling, neurodegeneration and neurodevelopmental diseases. The other phenotypes under behaviour/cognitive and motor dysfunctions showed indirect associations with the former genes through other candidate genes. The pathophysiology of \u2018prevalent candidate genes\u2019 has been discussed for better interpretation of neurological manifestation in COVID-19.","version":"1.1","doi":"10.1101/2022.04.10.487761","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487556","pub_date":"2022-4-08","title":"SARS-CoV-2 and its variants, but not Omicron, induces thymic atrophy and impaired T cell development","abstract":"Pathogenic infections cause thymic atrophy, perturb thymic-T cell development and alter immunological response. Previous studies reported dysregulated T cell function and lymphopenia in coronavirus disease-19 (COVID-19) patients. However, immune-pathological changes, in the thymus, post severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report SARS-CoV-2 infects thymocytes, depletes CD4+CD8+ (double positive; DP) T cell population associated with an increased apoptosis of thymocytes, which leads to severe thymic atrophy in K18-hACE2-Tg mice. CD44+CD25-T cells were found to be enriched in infected thymus, indicating an early arrest in the T cell developmental pathway. Further, Interferon gamma (IFN-\u03b3) was crucial for thymic atrophy, as anti-IFN-\u03b3 antibody neutralization rescued the loss of thymic involution. Therapeutic use of remdesivir (prototype anti-viral drug) was also able to rescue thymic atrophy. While Omicron variant of SARS-CoV2 caused marginal thymic atrophy, delta variant of SARS-CoV-2 exhibited most profound thymic atrophy characterized by severely depleted DP T cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore thymic developmental pathway of T cells. Together, we provide the first report of SARS-CoV-2 associated thymic atrophy resulting from impaired T cell developmental pathway and also explains dysregulated T cell function in COVID-19.","version":"1.1","doi":"10.1101/2022.04.07.487556","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487520","pub_date":"2022-4-08","title":"Hippo Signaling Pathway Activation during SARS-CoV-2 Infection Contributes to Host Antiviral Response","abstract":"SARS-CoV-2, responsible for the COVID-19 pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19 associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples, and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.","version":"1.1","doi":"10.1101/2022.04.07.487520","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487347","pub_date":"2022-4-08","title":"Plant-produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants","abstract":"SARS-CoV-2 is a novel and highly pathogenic coronavirus, which has caused an outbreak in Wuhan City, China, in 2019 and then spread rapidly throughout the world. Although several COVID-19 vaccines are currently available for mass immunization, they are less effective against emerging SARS-CoV-2 variants, especially the Omicron (B.1.1.529). Recently, we successfully produced receptor-binding domain (RBD) variants of spike (S) protein of SARC-CoV-2 and an antigen cocktail in Nicotiana benthamiana, which are highly produced in plants and elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. In this study, we demonstrate that these protein-based vaccine candidates are highly effective against Delta and Omicron variants. These data support that plant produced RBD and cocktail-based antigens are most promising vaccine candidates and may protect against Delta and Omicron-mediated COVID-19. Based on the neutralization ability, plant produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants.","version":"1.1","doi":"10.1101/2022.04.07.487347","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.08.487674","pub_date":"2022-4-08","title":"NVX-CoV2373 vaccination induces functional SARS-CoV-2\u2013specific CD4+ and CD8+ T cell responses","abstract":"NVX-CoV2373 is an adjuvanted recombinant full-length SARS-CoV-2 spike trimer protein vaccine demonstrated to be protective against COVID-19 in efficacy trials. Here we demonstrate that vaccinated subjects made CD4+ T cell responses after one and two doses of NVX-CoV2373, and a subset of individuals made CD8+ T cell responses. Characterization of the vaccine-elicited CD8+T cells demonstrated IFN\u03b3 production. Characterization of the vaccine-elicited CD4+ T cells revealed both circulating T follicular helper cells (cTFH) and TH1 cells (IFN\u03b3, TNF\u03b1, and IL-2) were detectable within 7 days of the primary immunization. Spike-specific CD4+ T cells were correlated with the magnitude of the later SARS-CoV-2 neutralizing antibody titers, indicating that robust generation of CD4+ T cells, capable of supporting humoral immune responses, may be a key characteristic of NVX-CoV2373 which utilizes Matrix-M\u2122 adjuvant.","version":"1.1","doi":"10.1101/2022.04.08.487674","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487415","pub_date":"2022-4-08","title":"The Delta variant SARS-CoV-2 spike protein uniquely promotes aggregation of pseudotyped viral particles","abstract":"Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike\u2019s unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants.","version":"1.1","doi":"10.1101/2022.04.07.487415","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.08.487623","pub_date":"2022-4-08","title":"SARS-CoV-2 mutations affect proteasome processing to alter CD8+ T cell responses","abstract":"Viral CD8+ epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8+ epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs. Natural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8+ T cell responses leading to partial escape.","version":"1.1","doi":"10.1101/2022.04.08.487623","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.08.487615","pub_date":"2022-4-08","title":"Recycling of Polymerase Chain Reaction (PCR) kits","abstract":"During the outbreak of the SARS-CoV-2 pandemic, PCR (polymerase chain reaction) kits have been used as a common diagnosing method, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic there was a shortage of PCR kits. So far, the ecosystem of PCR kit is linear use, that is kits are produced, used one-time, and disposed in biolab wastes. Here we show that, to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits, based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotides monophosphates that are then phosphorylated in triphosphates using a method that is a development of a recently published one. We used oligonucleotides with 3\u2019-terminal phosphorothioate (PS) backbone modification as non-hydrolysable PCR primers, so to undergo the recycling process unchanged. We have successfully recycled both PCR primers (\u223c65% yield for 4-PS modification, and \u223c40% yield for 2-PS modification) and nucleotides (\u223c75% yield). We demonstrate that the method allows for direct re-use of the PCR kits. We also show that the recycled primers can be isolated and then added to end point or quantitative PCR. This recycling approach provides a new path for circularly reusing PCR nucleic acids.","version":"1.1","doi":"10.1101/2022.04.08.487615","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487460","pub_date":"2022-4-07","title":"Distinct evolutionary trajectories of SARS-CoV-2 interacting proteins in bats and primates identify important host determinants of COVID-19","abstract":"The COVID-19 pandemic is caused by SARS-CoV-2, a novel coronavirus that spilled from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional-genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. Here, we performed high-throughput evolutionary analyses of 334 SARS- CoV-2 interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms-race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo important interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified functional adaptations involved in SARS- CoV-2 infection in bats and primates, critically enlightening modern genetic determinants of virus susceptibility and severity. Evolutionary history of 334 SARS-CoV-2 interacting proteins (VIPs) in bats and primates identifying how the past has shaped modern viral reservoirs and humans \u2013 results publicly-available in an online resource. Identification of 81 primate and 38 bat VIPs with signatures of adaptive evolution. The common ones among species delineate a core adaptive interactome, while the ones displaying distinct evolutionary trajectories enlighten host lineage-specific determinants. Evidence of primate specific adaptation of the entry factor TMPRSS2 pointing to its host- specific in vivo importance and predicting molecular interfaces. FYCO1 sites associated with severe COVID-19 in human (GWAS) display hallmarks of ancient adaptive evolution in primates, highlighting its importance in SARS-CoV-2 replication or pathogenesis and differences with the bat reservoir. Identification of adaptive evolution in the bat\u2019s multifunctional RIPK1 at residues that may differentially regulate inflammation.","version":"1.1","doi":"10.1101/2022.04.07.487460","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.18.473309","pub_date":"2022-4-07","title":"The Roles of APOBEC-mediated RNA Editing in SARS-CoV-2 Mutations, Replication and Fitness","abstract":"During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure. Efficient Editing of SARS-CoV-2 genomic RNA by Host APOBEC deaminases and Its Potential Impacts on the Viral Replication and Emergence of New Strains in COVID-19 Pandemic","version":"1.2","doi":"10.1101/2021.12.18.473309","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.06.487394","pub_date":"2022-4-07","title":"Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology","abstract":"Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease. There remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.","version":"1.1","doi":"10.1101/2022.04.06.487394","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.482049","pub_date":"2022-4-07","title":"Efficient Neutralization of SARS-CoV-2 Omicron and Other VOCs by a Broad Spectrum Antibody 8G3","abstract":"Numerous mutations in the spike protein of SARS-CoV-2 B.1.1.529 Omicron variant pose a crisis for antibody-based immunotherapies. The efficacy of emergency use authorized (EUA) antibodies that developed in early SARS-CoV-2 pandemic seems to be in flounder. We tested the Omicron neutralization efficacy of an early B cell antibody repertoire as well as several EUA antibodies in pseudovirus and authentic virus systems. More than half of the antibodies in the repertoire that showed good activity against WA1/2020 previously had completely lost neutralizing activity against Omicron, while antibody 8G3 displayed non-regressive activity. EUA antibodies Etesevimab, Casirivimab, Imdevimab and Bamlanivimab were entirely desensitized by Omicron. Only Sotrovimab targeting the non-ACE2 overlap epitope showed a dramatic decrease activity. Antibody 8G3 efficiently neutralized Omicron in pseudovirus and authentic virus systems. The in vivo results showed that Omicron virus was less virulent than the WA1/2020 strain, but still caused deterioration of health and even death in mice. Treatment with 8G3 quickly cleared virus load of mice. Antibody 8G3 also showed excellent activity against other variants of concern (VOCs), especially more efficient against authentic Delta plus virus. Collectively, our results suggest that neutralizing antibodies with breadth remains broad neutralizing activity in tackling SARS-CoV-2 infection despite the universal evasion from EUA antibodies by Omicron variant.","version":"1.3","doi":"10.1101/2022.02.25.482049","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.07.487489","pub_date":"2022-4-07","title":"Antibody Resistance of SARS-CoV-2 Omicron BA.1, BA.1.1, BA.2 and BA.3 Sub-lineages","abstract":"The SARS-CoV-2 Omicron variant has been partitioned into four sub-lineages designated BA.1, BA.1.1, BA.2 and BA.3, with BA.2 becoming dominant worldwide recently by outcompeting BA.1 and BA.1.1. We and others have reported the striking antibody evasion of BA.1 and BA.2, but side-by-side comparison of susceptibility of all the major Omicron sub-lineages to vaccine-elicited or monoclonal antibody (mAb)-mediated neutralization are urgently needed. Using VSV-based pseudovirus, we found that sera from individuals vaccinated by two doses of inactivated whole-virion vaccines (BBIBP-CorV) showed very weak to no neutralization activity, while a homologous inactivated vaccine booster or a heterologous booster with protein subunit vaccine (ZF2001) markedly improved the neutralization titers against all Omicron variants. The comparison between sub-lineages indicated that BA.1.1, BA.2 and BA.3 had comparable or even greater antibody resistance than BA.1. We further evaluated the neutralization profile of a panel of 20 mAbs, including 10 already authorized or approved, against these Omicron sub-lineages as well as viruses with different Omicron spike single or combined mutations. Most mAbs lost their neutralizing activity completely or substantially, while some demonstrated distinct neutralization patterns among Omicron sub-lineages, reflecting their antigenic difference. Taken together, our results suggest all four Omicron sub-lineages threaten the efficacies of current vaccines and antibody therapeutics, highlighting the importance of vaccine boosters to combat the emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.04.07.487489","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.06.22273202","pub_date":"2022-04-07","title":"Prediction of deterioration from COVID-19 in patients in skilled nursing facilities using wearable and contact-free devices: a feasibility study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background and Rationale</jats:title>\n                  <jats:p>Approximately 35% of all COVID-19 deaths occurred in Skilled Nursing Facilities (SNFs). In a healthy general population, wearables have shown promise in providing early alerts for actionable interventions during the pandemic. We tested this promise in a cohort of SNFs patients diagnosed with COVID-19 and admitted for post-acute care under quarantine. We tested if 1) deployment of wearables and contact-free biosensors is feasible in the setting of SNFs and 2) they can provide early and actionable insights into deterioration.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    This prospective clinical trial has been IRB-approved (\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04548895'>NCT04548895</jats:ext-link>\n                    ). We deployed two commercially available devices detecting continuously every 2-3 minutes heart rate (HR), respiratory rate (RR) and uniquely providing the following biometrics: 1) the wrist-worn bracelet by Biostrap yielded continuous oxygen saturation (O2Sat), 2) the under-mattress ballistocardiography sensor by Emfit tracked in-bed activity, tossing, and sleep disturbances. Patients also underwent routine monitoring by staff every 2-4 h. For death outcomes, cases are reported due to the small sample size. For palliative care versus at-home discharges, we report mean\u00b1SD at p&lt;0.05.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>From 12/2020 - 03/2021, we approached 26 PCR-confirmed SarsCoV2-positive patients at two SNFs: 5 declined, 21 were enrolled into monitoring by both sensors (female=13, male=8; age 77.2\u00b19.1). We recorded outcomes as discharged to home (8, 38%), palliative care (9, 43%) or death (4, 19%). The O2Sat threshold of 91% alerted for intervention. Biostrap captured hypoxic events below 91% nine times as often as the routine intermittent pulse oximetry. In the patient deceased, two weeks prior we observed a wide range of O2Sat values (65-95%) captured by the Biostrap device and not noticeable with the routine vital sign spot checks. In this patient, the Emfit sensor yielded a markedly reduced RR (7/min) in contrast to 18/min from two routine spot checks performed in the same period of observation as well as compared to the seven patients discharged home over a total of 86 days of monitoring (RR 19\u00b1 4). Among the patients discharged to palliative care, a total of 76 days were monitored, HR did not differ compared to the patients discharged home (68\u00b18 vs 70\u00b17 bpm). However, we observed a statistically significant reduction of RR at 16\u00b14/min as well as the variances in RR (10\u00b16 vs 19\u00b14/min vs16\u00b113) and activity of palliative care patients vs. patients discharged home.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion/Discussion</jats:title>\n                  <jats:p>We demonstrate that wearables and under-mattress sensors can be integrated successfully into the SNF workflows and are well tolerated by the patients. Moreover, specific early changes of oxygen saturation fluctuations and other biometrics herald deterioration from COVID-19 two weeks in advance and evaded detection without the devices. Wearable devices and under-mattress sensors in SNFs hold significant potential for early disease detection.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.04.06.22273202","journal":"medRxiv","score":null},{"id":"10.1101/2022.04.05.22273453","pub_date":"2022-04-06","title":"Unsuppressed HIV infection impairs T cell responses to SARS-CoV-2 infection and abrogates T cell cross-recognition","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  HIV infection has been identified as one of the major risk factors for severe COVID-19 disease, but the mechanisms underpinning this susceptability are still unclear. Here, we assessed the impact of HIV infection on the quality and epitope specificity of SARS-CoV-2 T cell responses in the first wave and second wave of the COVID-19 epidemic in South Africa. Flow cytometry was used to measure T cell responses following PBMC stimulation with SARS-CoV-2 peptide pools. Culture expansion was used to determine T cell immunodominance hierarchies and to assess potential SARS-CoV-2 escape from T cell recognition. HIV-seronegative individuals had significantly greater CD4\n                  <jats:sup>+</jats:sup>\n                  and CD8\n                  <jats:sup>+</jats:sup>\n                  T cell responses against the Spike protein compared to the viremic PLWH. Absolute CD4 count correlated positively with SARS-CoV-2 specific CD4\n                  <jats:sup>+</jats:sup>\n                  and CD8\n                  <jats:sup>+</jats:sup>\n                  T cell responses (CD4 r= 0.5, p=0.03; CD8 r=0.5, p=0.001), whereas T cell activation was negatively correlated with CD4\n                  <jats:sup>+</jats:sup>\n                  T cell responses (CD4 r= \u22120.7, p=0.04). There was diminished T cell cross-recognition between the two waves, which was more pronounced in individuals with unsuppressed HIV infection. Importantly, we identify four mutations in the Beta variant that resulted in abrogation of T cell recognition. Together, we show that unsuppressed HIV infection markedly impairs T cell responses to SARS-Cov-2 infection and diminishes T cell cross-recognition. These findings may partly explain the increased susceptibility of PLWH to severe COVID-19 and also highlights their vulnerability to emerging SARS-CoV-2 variants of concern.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>One sentence summary</jats:title>\n                  <jats:p>Unsuppressed HIV infection is associated with muted SARS-CoV-2 T cell responses and poorer recognition of the Beta variant.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.04.05.22273453","journal":"medRxiv","score":null},{"id":"10.1101/2022.04.05.487240","pub_date":"2022-4-06","title":"Vascular inflammation exposes perivascular cells to SARS-CoV-2 infection","abstract":"Pericytes stabilize blood vessels and promote vascular barrier function. However, vessels subjected to pro-inflammatory conditions have impaired barrier function, which has been suggested to potentially expose perivascular cells to SARS-CoV-2. To test this hypothesis, we engineered pericyte-supported vascular capillaries on-a-chip, and determined that the extravasation and binding of spike protein (S1) on perivascular cells of inflamed vessels to be significantly higher that in healthy controls, indicating a potential target to understand COVID-19 vascular complications.","version":"1.1","doi":"10.1101/2022.04.05.487240","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.06.487306","pub_date":"2022-4-06","title":"An Ultralong Bovine CDRH3 that Targets a Conserved, Cryptic Epitope on SARS-CoV and SARS-CoV-2","abstract":"The ability of broadly neutralising antibodies to target conserved epitopes gives them huge potential as antibody-based therapeutics, particularly in the face of constant viral antigen evolution. Certain bovine antibodies are highly adept at binding conserved, glycosylated epitopes, courtesy of their ultralong complementarity determining region (CDR)H3. Here, we used a SARS-na\u00efve, bovine ultralong CDRH3 library and mammalian cell display, to isolate a bovine paratope that engages the SARS-CoV and SARS-CoV-2 receptor-binding domain (RBD). This neutralises viruses pseudo-typed with SARS-CoV Spike protein but not by competition with RBD binding to ACE2. Instead, using differential hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis, we demonstrate that this ultralong CDRH3 recognises a rarely identified, conserved, cryptic epitope that overlaps the target of pan-sarbecovirus antibodies (7D6/6D6). The epitope is glycan-shielded and becomes accessible only transiently via inter-domain movements. This represents the first bovine anti-sarbecovirus paratope and highlights the power of this approach in identifying novel tools to combat emerging pathogens.","version":"1.1","doi":"10.1101/2022.04.06.487306","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.15.440067","pub_date":"2022-4-06","title":"Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2","abstract":"SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and the evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors in clinical settings such as cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically na\u00efve rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less active in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of IFITM2 and IFITM3 via an endolysosomal remodeling program known as microautophagy. Rapalogs that promote virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating a lysosome-mediated suppression of intrinsic immunity. Rapamycin is an immunosuppressant used in humans to treat cancer, autoimmunity, and other disease states. Here, we show that rapamycin and related compounds promote the first step of the SARS-CoV-2 infection cycle\u2014entry into cells\u2014by disarming cell-intrinsic immune defenses. We outline the molecular basis for this effect by identifying a rapamycin derivative that is inactive, laying the foundation for improved mTOR inhibitors that do not suppress intrinsic immunity. We find that rapamycin analogs that promote SARS-CoV-2 entry are those that activate TFEB, a transcription factor that triggers the degradation of antiviral membrane proteins inside of cells. Finally, rapamycin administration to rodents prior to SARS-CoV-2 challenge results in enhanced viral disease, revealing that its use in humans may increase susceptibility to infection.","version":"1.4","doi":"10.1101/2021.04.15.440067","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.05.487186","pub_date":"2022-4-06","title":"SARS-CoV-2 Omicron (BA.1 and BA.2) specific novel CD8+ and CD4+ T cell epitopes targeting spike protein","abstract":"The Omicron (BA.1/B.1.1.529) variant of SARS-CoV-2 harbors an alarming 37 mutations on its spike protein, reducing the efficacy of current COVID-19 vaccines. This study identified CD8+ and CD4+ T cell epitopes from SARS-CoV-2 S protein mutants. To identify the highest quality CD8 and CD4 epitopes from the Omicron variant, we selected epitopes with a high binding affinity towards both MHC I and MHC II molecules and applied other clinical checkpoint predictors including immunogenicity, antigenicity, allergenicity, instability, and toxicity. Subsequently, we found eight Omicron (BA.1/B.1.1.529) specific CD8+ and eleven CD4+ T cell epitopes with a world population coverage of 76.16% and 97.46%, respectively. Additionally, we identified common epitopes across Omicron BA.1 and BA.2 lineages that target mutations critical to SARS-CoV-2 virulence. Further, we identified common epitopes across B.1.1.529 and other circulating SARS-CoV-2 variants, such as B.1.617.2 (Delta). We predicted CD8 epitopes\u2019 binding affinity to murine MHC alleles to test the vaccine candidates in preclinical models. The CD8 epitopes were further validated using our previously developed software tool PCOptim. We then modeled the three-dimensional structures of our top CD8 epitopes to investigate the binding interaction between peptide-MHC and peptide-MHC-TCR complexes. Importantly, our identified epitopes are targeting the mutations on the RNA-binding domain and the fusion sites of S protein. This could potentially eliminate viral infections and form long-term immune responses compared to rather short-lived mRNA vaccines and maximize the efficacy of vaccine candidates against the current pandemic and potential future variants.","version":"1.1","doi":"10.1101/2022.04.05.487186","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.03.481940","pub_date":"2022-4-06","title":"Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies Against Delta and Omicron in Mice","abstract":"As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We have previously developed highly thermo-tolerant monomeric and trimeric receptor binding domain derivatives that can withstand 100\u00b0C for 90 minutes and 37\u00b0C for four weeks, and help eliminate cold chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations, and 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for the upcoming Phase I human clinical trials, and that there is potential for increasing efficacy with vaccine matching to improve responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions which show that while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible, and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.","version":"1.3","doi":"10.1101/2022.03.03.481940","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.25.461785","pub_date":"2022-4-06","title":"LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19","abstract":"One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs for given therapeutic targets. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified novel therapeutics for cancer, lifestyle-related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. We envision that LIGHTHOUSE will bring about a paradigm shift in translational medicine, providing a bridge from bench side to bedside.","version":"1.3","doi":"10.1101/2021.09.25.461785","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.03.482810","pub_date":"2022-4-06","title":"Z-RNA and the flipside of the SARS Nsp13 helicase","abstract":"We present evidence that the severe acute respiratory syndrome coronavirus (SARS) non-structural protein 13 (Nsp13) modulates the Z-RNA dependent regulated cell death pathways [1]. We show that Z-prone sequences (called flipons [2]) exist in coronavirus and provide a signature (Z-sig) that enables identification of the animal viruses from which the human pathogens arose. We also identify a potential RIP Homology Interaction Motif (RHIM) in the helicase Nsp13 that resembles those present in proteins that initiate Z-RNA-dependent cell death through interactions with the Z-RNA sensor protein ZBP1. These two observations allow us to suggest a model in which Nsp13 down regulates Z-RNA activated innate immunity by two distinct mechanisms. The first involves a novel ATP-independent Z-flipon helicase (flipase) activity in Nsp13 that differs from that of canonical A-RNA helicases. This flipase prevents formation of Z-RNAs that would otherwise activate cell death pathways. The second mechanism likely inhibits the interactions between ZBP1 and the Receptor Interacting Proteins Kinases RIPK1 and RIPK3 by targeting their RHIM domains. Together the described Nsp13 RHIM and flipase activities have the potential to alter the host response to coronaviruses and impact the design of drugs targeting the Nsp13 protein. The Z-sig and RHIM domains may provide a way of identifying previously uncharacterized viruses that are potentially pathogenic for humans.","version":"1.2","doi":"10.1101/2022.03.03.482810","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.04.487020","pub_date":"2022-4-05","title":"The network of SARS-CoV-2\u2014cancer molecular interactions and pathways","abstract":"Relatively little is known about the long-term impacts of SARS-CoV-2 biology, including whether it increases the risk of cancer. This study aims to identify the molecular interactions between COVID-19 infections and cancer processes. We integrated recent data on SARS-CoV-2 \u2013 host protein interactions, risk factors for critical illness, known oncogenes, tumor suppressor genes and cancer drivers in EpiGraphDB, a database of disease biology and epidemiology. We used these data to reconstruct the network of molecular links between SARS-CoV-2 infections and cancer processes in various tissues expressing the angiotensin-converting enzyme 2 (ACE2) receptor. We applied community detection algorithms and Gene Set Enrichment Analysis (GSEA) to identify cancer-relevant pathways that may be perturbed by SARS-CoV-2 infection. In lung tissue, the results showed that 4 oncogenes are potentially targeted by SARS-CoV-2, and 92 oncogenes interact with other human genes targeted by SARS-CoV-2. We found evidence of potential SARS-CoV-2 interactions with Wnt and hippo signaling pathways, telomere maintenance, DNA replication, protein ubiquitination and mRNA splicing. Some of these pathways were potentially affected in multiple tissues. The long-term implications of SARS-CoV-2 infection are still unknown, but our results point to the potential impact of infection on pathways relevant to cancer affecting cell proliferation, development and survival, favoring DNA degradation, preventing the repair of damaging events and impeding the translation of RNA into working proteins. This highlights the need for further research to investigate whether such effects are transient or longer lasting. Our results are openly available in the EpiGraphDB platform at https://epigraphdb.org/covid-cancer and the repository https://github.com/MRCIEU/covid-cancer (https://doi.org/10.5281/zenodo.6391588).","version":"1.1","doi":"10.1101/2022.04.04.487020","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.04.486994","pub_date":"2022-4-05","title":"Screening of SARS-CoV-2 Antivirals Through a Cell-Based RNA-Dependent RNA Polymerase (RdRp) Reporter Assay","abstract":"COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose international public health threat and thus far, has resulted in greater than 5.6 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast spreading COVID-19 variants that may elude vaccines efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using a known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5\u2019CT, and dasabuvir. Among these inhibitors, dasabuvir (FDA-approved drug) exhibited promising RdRp inhibitory activity. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with IC50 values 9.47 \u03bcM and 10.48 \u03bcM, for USA-WA1/2020 and B.1.617.2 variants, respectively). Our results suggests that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. In addition, our assays provide robust, target-specific, and high-throughput screening compatible (z- and z\u2019-factors of > 0.5) platforms that will be a valuable tool for the screening SARS-CoV-2 RdRp inhibitors. SARS-CoV-2 has caused a major public crisis world has seen in recent history. Development of vaccines and emergency use authorization of anti-virals are helping in reducing the burden of SARS-CoV-2 caused hospitalization and deaths. However, there is still need for optimal anti-viral(s) that can efficiently block viral propagation, and targeting viral polymerase (RdRp) is an among the most suitable targets for clamping viral replication. In this study, we developed a cell-based assay to screen potential compounds capable of blocking RdRp activity. The efficacy of our assay was validated by using already approved anti-virals, which reduced RdRp activity and slowed the replication of two SARS-CoV-2 variants (WA1 USA-WA1/2020 and B.1.617.2) in a cell culture model. This confirmed that our system can be used for identifying potential anti-SARS-CoV-2 anti-virals.","version":"1.1","doi":"10.1101/2022.04.04.486994","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.05.487060","pub_date":"2022-4-05","title":"Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection","abstract":"Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.","version":"1.1","doi":"10.1101/2022.04.05.487060","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.04.487083","pub_date":"2022-4-05","title":"Impact of prior infection on SARS-CoV-2 antibody responses in vaccinated long-term care facility staff","abstract":"SARS-CoV-2 emerged in 2019 and has resulted in millions of deaths worldwide. Certain populations are at higher risk for infection, especially staff and residents at long term care facilities (LTCF), due to the congregant living setting, and residents with many comorbidities. Prior to vaccine availability, these populations represented a large fraction of total COVID-19 cases and deaths in the U.S. Due to the high-risk setting and outbreak potential, staff and residents were among the first groups to be vaccinated. To define the impact of prior infection on response to vaccination, we measured antibody responses in a cohort of staff members at a LTCF, many of whom were previously infected by SARS-CoV-2. We found that neutralizing, receptor-binding-domain (RBD) and nucleoprotein (NP) binding antibody levels were significantly higher post-full vaccination course in individuals that were previously infected, and NP antibody levels could discriminate individuals with prior infection from vaccinated individuals. While an anticipated antibody titer increase was observed after vaccine booster dose in na\u00efve individuals, boost response was not observed in individuals with previous COVID-19 infection. We observed a strong relationship between neutralizing antibodies and RBD-binding antibodies post-vaccination across all groups, suggesting RBD-binding antibodies may be used as a correlate of neutralization. One individual with high levels of neutralizing and binding antibodies experienced a breakthrough infection (prior to the introduction of Omicron), demonstrating that the presence of antibodies is not always sufficient for complete protection against infection. These results highlight that history of COVID-19 exposure significantly increases SARS-CoV-2 antibody responses following vaccination. Long-term care facilities (LTCFs) have been disproportionately impacted by COVID-19, due to their communal nature, high-risk profile of residents and vulnerability to respiratory pathogens. In this study, we analyzed the role of prior natural immunity to SARS-CoV-2 on post-vaccination antibody responses. The LTCF in our cohort experienced a large outbreak with almost 40% of staff becoming infected. We found that individuals that were infected prior to vaccination, had higher levels of neutralizing and binding antibodies post-vaccination. Importantly, the second vaccine dose significantly boosted antibody levels in those that were immunologically na\u00efve prior to vaccination, but not those that had prior immunity. Regardless of pre-vaccination immune status, levels of binding and neutralizing antibodies were highly correlated. The presence of NP-binding antibodies can be used to identify individuals that were previously infected when pre-vaccination immune status is not known. Our results reveal that vaccination antibody responses differ depending on prior natural immunity.","version":"1.1","doi":"10.1101/2022.04.04.487083","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.04.486920","pub_date":"2022-4-05","title":"Immunopharmacological evaluation of adjuvant efficacy of Monophosphoryl lipid-A and CpG ODN with SARS-CoV-2 RBD antigen","abstract":"SARS-CoV-2 infection has made the mankind to witness most sever and serious pandemic situation in the history. Millions of people have suffered and are still suffering with this infection which has caused a mass mortality in the past three years. Development of an effective vaccine to control the spread of infection and to prevent this viral infection is need of the hour. Adjuvanted vaccines have proven their efficacy in controlling many other viral infections like flu, keeping this context in view we have evaluated the immunopharmacological efficacy of two adjuvants MPL-A and CpG ODN in combination with MF59 emulsion against SARS-CoV-2 antigen. From the data obtained we can infer that both the adjuvants were capable of eliciting a potent antibody response against antigen alone and MF59 groups. Comparatively MPL-A was eliciting a Th1 polarized response in terms of IgG2a and cytokine production. Both the adjuvants were capable of enhancing the CD 4, 8 and 19 cell populations. Overall the pre clinical evaluation has given a clue of the effectiveness of MPL-A and CpG adjuvants against SARS-CoV-2 antigen.","version":"1.1","doi":"10.1101/2022.04.04.486920","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.05.487114","pub_date":"2022-4-05","title":"Time series analysis of SARS-CoV-2 genomes and correlations among highly prevalent mutations","abstract":"The efforts of the scientific community to tame the recent SARS-CoV-2 pandemic seems to have been diluted by the emergence of new viral strains. Therefore, it becomes imperative to study and understand the effect of mutations on viral evolution, fitness and pathogenesis. In this regard, we performed a time-series analysis on 59541 SARS-CoV-2 genomic sequences from around the world. These 59541 genomes were grouped according to the months (January 2020-March 2021) based on the collection date. Meta-analysis of this data led us to identify highly significant mutations in viral genomes. Correlation and Hierarchical Clustering of the highly significant mutations led us to the identification of sixteen mutation pairs that were correlated with each other and were present in >30% of the genomes under study. Among these mutation pairs, some of the mutations have been shown to contribute towards the viral replication and fitness suggesting the possible role of other unexplored mutations in viral evolution and pathogenesis. Additionally, we employed various computational tools to investigate the effects of T85I, P323L, and Q57H mutations in Non-structural protein 2 (Nsp2), RNA-dependent RNA polymerase (RdRp) and Open reading frame 3a (ORF3a) respectively. Results show that T85I in Nsp2 and Q57H in ORF3a mutations are deleterious and destabilize the parent protein whereas P323L in RdRp is neutral and has a stabilizing effect. The normalized linear mutual information (nLMI) calculations revealed the significant residue correlation in Nsp2 and ORF3a in contrast to reduce correlation in RdRp protein.","version":"1.1","doi":"10.1101/2022.04.05.487114","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.05.487103","pub_date":"2022-4-05","title":"Evolution of Delta variant by non-Spike signature co-appearing mutations: trailblazer of COVID-19 disease outcome","abstract":"The high transmissibility and infectivity of a SARS-CoV-2 variant is usually ascribed to the Spike mutations, while emerging non-spike mutations might be a serious threat to the current Spike-recombinant vaccines. In addition to mutations in structural Spike glycoprotein, rapid accumulation of mutations across non-structural genes is leading to continuous virus evolution, altering its pathogenicity. We performed whole genome sequencing of SARS-CoV-2 positive samples collected from different clinical groups from eastern India, during the second pandemic wave (April-May, 2021). In addition to the several common spike mutations in Delta variant, two mutually explicit signature constellations of non-spike co-appearing mutations were identified, driving symptomatic and asymptomatic infections. We attempted to correlate these unique signatures of non-Spike co-appearing mutations to COVID-19 disease outcome. Results revealed that the Delta strains harboring a unique constellation of 9 non-spike co-appearing mutations could be the wheeler and dealer of symptomatic infection, even post vaccination. The strains predominantly driving asymptomatic infection possessed 7 non-spike co-appearing mutations, which were mutually exclusive in contrast to the set of mutations causing symptomatic disease. Phylodynamic analysis depicted high probability of emergence of these unique sub-clusters within India, with subsequent spread worldwide. Interestingly, some mutations of this signature were selected in Omicron and IHU variants, which suggest that gradual accumulation of such co-existing mutations may lead to emergence of more \u201cvaccine-evading variants\u201d in future. Hence, unfaltering genome sequencing and tracking of non-Spike mutations might be significant in formulation of any future vaccines against emerging SARS-CoV-2 variants that might evade the current vaccine-induced immunity.","version":"1.1","doi":"10.1101/2022.04.05.487103","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.02.462862","pub_date":"2022-4-04","title":"In Vitro Activity of Cysteamine Against SARS-CoV-2 Variants","abstract":"Global COVID-19 pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Continuous emergence of new variants and their rapid spread are jeopardizing vaccine countermeasures to a significant extent. While currently available vaccines are effective at preventing illness associated with SARS-CoV-2 infection, these have been shown to be less effective at preventing breakthrough infection and transmission from a vaccinated individual to others. Here we demonstrate broad antiviral activity of cysteamine HCl in vitro against major emergent infectious variants of SARS-CoV-2 in a highly permissible Vero cell line. Cysteamine HCl inhibited infection of wild type, alpha, beta, gamma, delta, lambda, and omicron variants effectively. Cysteamine is a very well-tolerated US FDA-approved drug used chronically as a topical ophthalmic solution to treat ocular cystinosis in patients who receive it hourly or QID lifelong at concentrations 6 to 10 times higher than that required to completely inhibit SARS CoV-2 in tissue culture. Application of cysteamine as a topical nasal treatment can potentially1) mitigate existing infection 2) prevent infection in exposed individuals, and 3) limit the contagion in vulnerable populations.","version":"1.2","doi":"10.1101/2021.10.02.462862","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.289488","pub_date":"2022-4-04","title":"Screening of candidate host cell membrane proteins involved in SARS-CoV-2 entry","abstract":"Coronavirus disease (COVID-19) represents a real threat to the global population, and understanding the biological features of the causative virus, i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and co-receptors (co-factors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. DPP4, Cadherin-17, and CD133 were found to co-localize with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. The experimental infection with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these co-receptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses.","version":"1.2","doi":"10.1101/2020.09.09.289488","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486499","pub_date":"2022-4-04","title":"Brequinar and Dipyridamole in Combination Exhibits Synergistic Antiviral Activity Against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19","abstract":"The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of currently available vaccines and monoclonal antibody (mAb)-based treatment options for COVID-19. The limited number of authorized small-molecule direct-acting antivirals present challenges with pill burden, the necessity for intravenous administration or potential drug interactions. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.","version":"1.2","doi":"10.1101/2022.03.30.486499","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.04.486975","pub_date":"2022-4-04","title":"Differential pathogenesis of SARS-CoV-2 variants of concern in human ACE2-expressing mice","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in millions of deaths worldwide. Increasingly contagious variants of concern (VoC) have fueled recurring global infection waves. A major question is the relative severity of disease caused by the previous and currently circulating variants of SARS-CoV-2. In this study, we evaluated the pathogenesis of SARS-CoV-2 variants in human ACE-2-expressing (K18-hACE2) mice. Eight-week-old K18-hACE2 mice were inoculated intranasally with a representative virus from the original B.1 lineage, or the emerging B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta) or B.1.1.529 (omicron) lineages. We also infected a group of mice with the mouse-adapted SARS-CoV-2 (MA10). Our results demonstrate that B.1.1.7, B.1.351 and B.1.617.2 viruses are significantly more lethal than B.1 strain in K18-hACE2 mice. Infection with B.1.1.7, B.1.351 and B.1.617.2 variants resulted in significantly higher virus titers in the lungs and brain of mice compared to the B.1 virus. Interestingly, mice infected with the B.1.1.529 variant exhibited less severe clinical signs and high survival rate. We found that B.1.1.529 replication was significantly lower in the lungs and brain of infected mice in comparison to other VoC. Transcription levels of cytokines and chemokines in the lungs of the B.1.1.529-infected mice were significantly less when compared to those challenged with the B.1.1.7 virus. Together, our data provide insights into the pathogenesis of the previous and circulating SARS-CoV-2 VoC in mice.","version":"1.1","doi":"10.1101/2022.04.04.486975","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477469","pub_date":"2022-4-04","title":"Design of immunogens for eliciting antibody responses that may protect against SARS-CoV-2 variants","abstract":"The rise of SARS-CoV-2 variants and the history of outbreaks caused by zoonotic coronaviruses point to the need for next-generation vaccines that confer protection against variant strains. Here, we combined analyses of diverse sequences and structures of coronavirus spikes with data from deep mutational scanning to design SARS-CoV-2 variant antigens containing the most significant mutations that may emerge. We trained a neural network to predict RBD expression and ACE2 binding from sequence, which allowed us to determine that these antigens are stable and bind to ACE2. Thus, they represent viable variants. We then used a computational model of affinity maturation (AM) to study the antibody response to immunization with different combinations of the designed antigens. The results suggest that immunization with a cocktail of the antigens is likely to promote evolution of higher titers of antibodies that target SARS-CoV-2 variants than immunization or infection with the wildtype virus alone. Finally, our analysis of 12 coronaviruses from different genera identified the S2\u2019 cleavage site and fusion peptide as potential pan-coronavirus vaccine targets. SARS-CoV-2 variants have already emerged and future variants may pose greater threats to the efficacy of current vaccines. Rather than using a reactive approach to vaccine development that would lag behind the evolution of the virus, such as updating the sequence in the vaccine with a current variant, we sought to use a proactive approach that predicts some of the mutations that could arise that could evade current immune responses. Then, by including these mutations in a new vaccine antigen, we might be able to protect against those potential variants before they appear. Toward this end, we used various computational methods including sequence analysis and machine learning to design such antigens. We then used simulations of antibody development, and the results suggest that immunization with our designed antigens is likely to result in an antibody response that is better able to target SARS-CoV-2 variants than current vaccines. We also leveraged our sequence analysis to suggest that a particular site on the spike protein could serve as a useful target for a pan-coronavirus vaccine.","version":"1.2","doi":"10.1101/2022.01.24.477469","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.25.445601","pub_date":"2022-4-04","title":"Genomic Surveillance of COVID-19 Variants with Language Models and Machine Learning","abstract":"The global efforts to control COVID-19 are threatened by the rapid emergence of novel SARS-CoV-2 variants that may display undesirable characteristics such as immune escape, increased transmissibility or pathogenicity. Early prediction for emergence of new strains with these features is critical for pandemic preparedness. We present Strainflow, a supervised and causally predictive model using unsupervised latent space features of SARS-CoV-2 genome sequences. Strainflow was trained and validated on 0.9 million sequences for the period December, 2019 to June, 2021 and the frozen model was prospectively validated from July, 2021 to December, 2021. Strainflow captured the rise in cases two months ahead of the Delta and Omicron surges in most countries including the prediction of a surge in India as early as beginning of November, 2021. Entropy analysis of Strainflow unsupervised embeddings clearly reveals the explore-exploit cycles in genomic feature-space, thus adding interpretability to the deep learning based model. We also conducted codon-level analysis of our model for interpretability and biological validity of our unsupervised features. Strainflow application is openly available as an interactive web-application for prospective genomic surveillance of COVID-19 across the globe.","version":"1.4","doi":"10.1101/2021.05.25.445601","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.04.487067","pub_date":"2022-4-04","title":"Surface detection of SARS-CoV-2 by lateral flow LAMP","abstract":"Slowing the transmission of SARS-CoV-2 requires rapid and accurate diagnostic testing. Toward this end, loop-mediated isothermal amplification (LAMP), an isothermal genomic detection method, offers great promise but the readout tends to be difficult because it does not generate linear DNA products. Rapid antigen tests are coupled to lateral flow strips, with one (negative) or two (positive) bands providing simple rapid readout, but are less sensitive than genomic amplification methods. To address the need for a genomic amplification method that can be visualized on a lateral flow strip, we developed a novel strand-displacement probe. In this work we validate this pipeline for purified RNA, intact virus, and even virus deposited onto a surface. We demonstrate robust sensitivity (100 genomic copies) and and we demonstrate the utility of our assay as a surveillance system, with the capability to detect viral particles from surfaces, even after a week of complete dry-down. Our innovation couples the diagnostic advantages of a nucleic acid amplification test (NAAT) with the simplicity of lateral-flow readouts.","version":"1.1","doi":"10.1101/2022.04.04.487067","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.03.486854","pub_date":"2022-4-03","title":"Conformational Flexibility in Neutralization of SARS-CoV-2 by Naturally Elicited Anti-SARS-CoV-2 Antibodies","abstract":"As new variants of SARS-CoV-2 continue to emerge, it is important to assess the neutralizing capabilities of naturally elicited antibodies against SARS-CoV-2. In the present study, we evaluated the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviuses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are far more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which kept their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solved the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 A\u030a and 2.42 A\u030a, respectively, revealing a similar mode of binding to that between the RBD and the ACE2 receptor. Furthermore, we provide five additional structures (at resolutions of 5.54 A\u030a, 7.76 \u00c5, 6.47 A\u030a, 3.45 A\u030a, and 7.32 A\u030a) of a second antibody, non-ACE2bs mAb TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides new mechanistic insights about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insight about the likelihood of reinfections.","version":"1.1","doi":"10.1101/2022.04.03.486854","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.02.486853","pub_date":"2022-4-03","title":"Dietary \u03b1KG inhibits SARS CoV-2 infection and rescues inflamed lungs to restore normal O2 saturation in animals","abstract":"Our recent works described the rescue effect of \u03b1-ketoglutarate (\u03b1KG, a metabolite of Krebs cycle) on thrombosis and inflammation in animals. \u03b1KG augments activity of prolyl hydroxylase 2 (PHD2), which in turn degrades proline residues of substrates like phosphorylated Akt (pAkt) and hypoxia inducible factor (HIF)\u03b1. Here we describe the inhibitory effect of octyl \u03b1KG on pAkt as well as on HIF1\u03b1/HIF2\u03b1, and in turn decreasing SARS CoV-2 replication in Vero E6 cells. \u03b1KG failed to inhibit the viral replication and Akt phosphorylation in PHD2-knockdown U937 cells transiently expressing ACE2. Contrastingly, triciribine (TCN, an Akt-inhibitor) inhibited viral replication alongside a downmodulation of pAkt in PHD2-KD cells. Dietary \u03b1KG significantly inhibited viral infection and rescued hamsters from thrombus formation and inflammation in lungs, the known causes of acute respiratory distress syndrome (ARDS) in COVID-19. \u03b1KG supplementation also reduced the apoptotic death of lung tissues in infected animals, alongside a downmodulation of pAkt and HIF2\u03b1. \u03b1KG supplementation neither affected IgG levels against SARS CoV-2 RBD protein nor altered the neutralization antibody response against SARS CoV-2. It did not interfere with the percentage of interferon-\u03b3 positive (IFN\u03b3+) CD4+ and IFN\u03b3+CD8+ T cells in infected animals. The extended work in balb/c mice transiently expressing ACE2 showed a similar effect of \u03b1KG in reducing accumulation of inflammatory immune cells and cytokines, including IL6, IL1\u03b2 and TNF\u03b1, in lungs as well as in circulation of infected animals. Pro-thrombotic markers like platelet microparticles and platelet-leukocyte aggregates were reduced significantly in infected mice after \u03b1KG supplementation. Importantly, \u03b1KG supplementation restored the O2 saturation (SpO2) in circulation of SARS CoV-2 infected hamsters and mice, suggesting a potential therapeutic role of this metabolite in COVID-19 treatment.","version":"1.1","doi":"10.1101/2022.04.02.486853","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.03.486830","pub_date":"2022-4-03","title":"Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature upon SARS-CoV-2 sensing by monocytes in COVID-19","abstract":"Alterations in the myeloid immune compartment have been observed in COVID-19, but the specific mechanisms underlying these impairments are not completely understood. Here we examined the functionality of classical CD14+ monocytes as a main myeloid cell component in well-defined cohorts of patients with mild and moderate COVID-19 during the acute phase of infection and compared them to that of healthy individuals. We found that ex vivo isolated CD14+ monocytes from mild and moderate COVID-19 patients display specific patterns of costimulatory and inhibitory receptors that clearly distinguish them from healthy monocytes, as well as altered expression of histone marks and a dysfunctional metabolic profile. Decreased NF\u03baB activation in COVID-19 monocytes ex vivo is accompanied by an intact type I IFN antiviral response. Subsequent pathogen sensing ex vivo led to a state of functional unresponsiveness characterized by a defect in pro-inflammatory cytokine expression, NF\u03baB-driven cytokine responses and defective type I IFN response in moderate COVID-19 monocytes. Transcriptionally, COVID-19 monocytes switched their gene expression signature from canonical innate immune functions to a pro-thrombotic phenotype characterized by increased expression of pathways involved in hemostasis and immunothrombosis. In response to SARS-CoV-2 or other viral or bacterial components, monocytes displayed defects in the epigenetic remodelling and metabolic reprogramming that usually occurs upon pathogen sensing in innate immune cells. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 may contribute to disease pathology.","version":"1.1","doi":"10.1101/2022.04.03.486830","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.03.486864","pub_date":"2022-4-03","title":"SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant","abstract":"Recent studies have revealed the unique virological characteristics of Omicron, the newest SARS-CoV-2 variant of concern, such as pronounced resistance to vaccine-induced neutralizing antibodies, less efficient cleavage of the spike protein, and poor fusogenicity. However, it remains unclear which mutation(s) in the spike protein determine the virological characteristics of Omicron. Here, we show that the representative characteristics of the Omicron spike are determined by its receptor-binding domain. Interestingly, the molecular phylogenetic analysis revealed that the acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidate that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue in another protomer in the spike trimer, which confers the attenuated spike cleavage efficiency and fusogenicity of Omicron. Our data shed light on the evolutionary events underlying Omicron emergence at the molecular level. Omicron spike receptor binding domain determines virological characteristics Spike S375F mutation results in the poor spike cleavage and fusogenicity in Omicron Acquisition of the spike S375F mutation triggered the explosive spread of Omicron F375-H505-mediated \u03c0-\u03c0 interaction in the spike determines the phenotype of Omicron","version":"1.1","doi":"10.1101/2022.04.03.486864","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.05.471310","pub_date":"2022-4-01","title":"An ACE2-dependent Sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines","abstract":"Spillover of sarbecoviruses from animals to humans has resulted in outbreaks of severe acute respiratory syndrome SARS-CoVs and the ongoing COVID-19 pandemic. Efforts to identify the origins of SARS-CoV-1 and \u22122 has resulted in the discovery of numerous animal sarbecoviruses \u2013 the majority of which are only distantly related to known human pathogens and do not infect human cells. The receptor binding domain (RBD) on sarbecoviruses engages receptor molecules on the host cell and mediates cell invasion. Here, we tested the receptor tropism and serological cross reactivity for RBDs from two sarbecoviruses found in Russian horseshoe bats. While these two viruses are in a viral lineage distinct from SARS-CoV-1 and \u22122, one virus, Khosta-2, was capable of using human ACE2 to facilitate cell entry. Viral pseudotypes with a recombinant, SARS-CoV-2 spike encoding for the Khosta 2 RBD were resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Our findings further demonstrate that sarbecoviruses circulating in wildlife outside of Asia also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2 European bat coronaviruses that are only distantly related to SARS-CoV-2 but use the same cell entry route, escape the immune response against SARS-CoV-2 vaccines, driving the need for broader vaccines.","version":"1.2","doi":"10.1101/2021.12.05.471310","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.01.486719","pub_date":"2022-4-01","title":"Potent Human Broadly SARS-CoV-2 Neutralizing IgA and IgG Antibodies Effective Against Omicron BA.1 and BA.2","abstract":"Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.","version":"1.1","doi":"10.1101/2022.04.01.486719","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.31.486548","pub_date":"2022-4-01","title":"Antibody evolution to SARS-CoV-2 after single-dose Ad26.COV2.S vaccine","abstract":"The single dose Ad.26.COV.2 (Janssen) vaccine elicits lower levels of neutralizing antibodies and shows more limited efficacy in protection against infection than either of the available mRNA vaccines. In addition, the Ad.26.COV.2 has been less effective in protection against severe disease during the Omicron surge. Here, we examined the memory B cell response to single dose Ad.26.COV.2 vaccination. Compared to mRNA vaccines, Ad.26.COV.2 recipients had significantly lower numbers of RBD-specific memory B cells 1.5 or 6 months after vaccination. Memory antibodies elicited by both vaccine types show comparable neutralizing potency against SARS-CoV-2 and Delta. However, the number of memory cells producing Omicron neutralizing antibodies was somewhat lower after Ad.26.COV.2 than mRNA vaccination. The data help explain why boosting Ad.26.COV.2 vaccine recipients with mRNA vaccines is effective, and why the Janssen vaccine appears to have been less protective against severe disease during the Omicron surge than the mRNA vaccine. Ad.26.COV.2 vaccine results in lower quantity but comparable quality of protective memory B cells compared to mRNA vaccines.","version":"1.1","doi":"10.1101/2022.03.31.486548","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.31.486561","pub_date":"2022-4-01","title":"Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike","abstract":"The SARS-CoV-2 spike protein is the target of neutralizing antibodies and the immunogen used in all currently approved vaccines. The global spread of the virus has resulted in emergence of lineages which are of concern for the effectiveness of immunotherapies and vaccines based on the early Wuhan isolate. Here we describe two SARS-CoV-2 isolates with large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis showed that the deletions result in complete reshaping of the antigenic surface of the NTD supersite. The remodeling of the NTD affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite for both spike variants. A unique escape mechanism with high antigenic impact observed in the \u0394N135 variant was based on the loss of the Cys15-Cys136 disulfide due to the P9L-mediated shift of the signal peptide cleavage site and deletion of residues 136-144. Although the observed large loop and disulfide deletions are rare, similar modifications became independently established in several other lineages, highlighting the possibility of a general escape mechanism via the NTD supersite. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.03.31.486561","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.461948","pub_date":"2022-4-01","title":"Inflammasome activation in infected macrophages drives COVID-19 pathology","abstract":"Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system. Blocking either viral replication with Remdesivir or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.","version":"1.2","doi":"10.1101/2021.09.27.461948","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471377","pub_date":"2022-4-01","title":"Omicron BA.1 and BA.2 Variants Increase the Interactions of SARS-CoV-2 Spike Glycoprotein with ACE2","abstract":"SARS-CoV-2 infection is initiated by binding of the receptor-binding domain (RBD) of its spike glycoprotein to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. Recently detected Omicron variant of SARS-CoV-2 (B.1.1.529) is heavily mutated on RBD. Currently, the most common Omicron variants are the original BA.1 Omicron strain and the BA.2 variant, which became more prevalent since it first appeared. To investigate how these mutations affect RBD-PD interactions, we performed all-atom molecular dynamics simulations of the BA.1 and BA.2 RBD-PD in the presence of full-length glycans, explicit water and ions. Simulations revealed that RBDs of BA.1 and BA.2 variants exhibit a more dispersed interaction network and make an increased number of salt bridges and hydrophobic interactions with PD compared to wild-type RBD. Although BA.1 and BA.2 differ in two residues at the RBD-ACE2 interface, no major difference in RBD-PD interactions and binding strengths were observed between these variants. Using the conformations sampled in each trajectory, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method estimated ~34% and ~51% stronger binding free energies for BA.1 and BA.2 RBD, respectively, than wild-type RBD, which may result in higher binding efficiency of the Omicron variant to infect host cells.","version":"1.3","doi":"10.1101/2021.12.06.471377","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.01.486726","pub_date":"2022-4-01","title":"Recall of pre-existing cross-reactive B cell memory following Omicron breakthrough infection","abstract":"Understanding immune responses following SARS-CoV-2 breakthrough infection will facilitate the development of next-generation vaccines. Here, we profiled spike (S)-specific B cell responses following Omicron/BA.1 infection in mRNA-vaccinated donors. The acute antibody response was characterized by high levels of somatic hypermutation (SHM) and a bias toward recognition of ancestral SARS-CoV-2 strains, suggesting the early activation of vaccine-induced memory B cells (MBCs). BA.1 breakthrough infection induced a shift in B cell immunodominance hierarchy from the S2 subunit toward the receptor binding domain (RBD). A large proportion of RBD-directed neutralizing antibodies isolated from BA.1 breakthrough infection donors displayed convergent sequence features and broadly recognized SARS-CoV-2 variants of concern (VOCs). Together, these findings provide fundamental insights into the role of pre-existing immunity in shaping the B cell response to heterologous SARS-CoV-2 variant exposure. BA.1 breakthrough infection activates pre-existing memory B cells with broad activity against SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.04.01.486726","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.10.483772","pub_date":"2022-4-01","title":"Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions","abstract":"There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2, and to a lesser extent, the alphacoronavirus HCoV-229E. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting synthesis of full-length and subgenomic HCoV RNAs. Furthermore, consistent with our previous report on the effects of thiopurines on influenza A virus glycoproteins, we observed that 6-TG inhibited accumulation of Spike glycoproteins from diverse HCoVs. Specifically, 6-TG treatment decreased the accumulation of Spike proteins and increased their electrophoretic mobility, consistent with Spike migration following the enzymatic removal of N-linked oligosaccharides with Peptide:N-glycosidase F (PNGaseF). SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on lentiviruses pseudotyped with SARS-CoV-2 Spike; lentiviruses could be harvested from cell supernatants but were deficient in Spike and unable to infect human cells bearing ACE2 receptors. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. At low micromolar doses, the primary known mode of action of 6-TG is selective inhibition of the small GTPase Rac1. However, we observed that selective chemical inhibitors of the small GTPases Rac1, CDC42 and Rho had no effect on Spike processing and accumulation. The GTPase agonist ML099 countered the effects of 6-TG, suggesting that an unknown GTPase could be the relevant 6-TG-target protein involved in regulating Spike processing and accumulation. Overall, these findings provide important clues about the mechanism of action of a candidate antiviral that can broadly target HCoVs and suggest that small GTPases are promising targets for host-targeted antivirals. The COVID-19 pandemic has ignited efforts to repurpose existing drugs as safe and effective antivirals. Rather than directly inhibiting viral enzymes, host-targeted antivirals inhibit host cell processes to indirectly impede viral replication and/or stimulate antiviral responses. Here, we describe a new antiviral mechanism of action for the FDA-approved thiopurine 6-thioguanine. We demonstrate that this thiopurine is a pro-drug that must be metabolized by host enzymes to gain antiviral activity. We show that it can inhibit the replication of several human coronaviruses, including SARS-CoV-2, at least in part by interfering with the processing and accumulation of Spike glycoproteins, thereby impeding assembly of infectious progeny viruses. We provide evidence implicating host cell GTPase enzymes in the antiviral mechanism of action.","version":"1.3","doi":"10.1101/2022.03.10.483772","journal":"bioRxiv","score":null},{"id":"10.1101/2022.04.01.486695","pub_date":"2022-4-01","title":"Omicron breakthrough infection drives cross-variant neutralization and memory B cell formation","abstract":"Omicron is the evolutionarily most distinct SARS-CoV-2 variant (VOC) to date and displays multiple amino acid alterations located in neutralizing antibody sites of the spike (S) protein. We report here that Omicron breakthrough infection in BNT162b2 vaccinated individuals results in strong neutralizing activity not only against Omicron, but also broadly against previous SARS-CoV-2 VOCs and against SARS-CoV-1. We found that Omicron breakthrough infection mediates a robust B cell recall response, and primarily expands preformed memory B cells that recognize epitopes shared broadly by different variants, rather than inducing new B cells against strictly Omicron-specific epitopes. Our data suggest that, despite imprinting of the immune response by previous vaccination, the preformed B cell memory pool has sufficient plasticity for being refocused and quantitatively remodeled by exposure to heterologous S protein, thus allowing effective neutralization of variants that evade a previously established neutralizing antibody response. Breakthrough infection in individuals double- and triple-vaccinated with BNT162b2 drives cross-variant neutralization and memory B cell formation.","version":"1.1","doi":"10.1101/2022.04.01.486695","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486409","pub_date":"2022-3-31","title":"Cross-neutralization of Omicron BA.1 against BA.2 and BA.3 SARS-CoV-2","abstract":"The Omicron SARS-CoV-2 has three distinct sublineages, among which sublineage BA.1 is responsible for the initial Omicron surge and is now being replaced by BA.2 world-wide, whereas BA.3 is currently at a low frequency. The ongoing BA.1-to-BA.2 replacement underscores the importance to understand the cross-neutralization among the three Omicron sublineages. Here we tested the neutralization of BA.1-infected human sera against BA.2, BA.3, and USA/WA1-2020 (a strain isolated in late January 2020). The BA.1-infected sera neutralized BA.1, BA.2, BA.3, and USA/WA1-2020 SARS-CoV-2s with geometric mean titers (GMTs) of 445, 107, 102, and 16, respectively. Thus, the neutralizing GMTs against heterologous BA.2, BA.3, and USA/WA1-2020 were 4.2-, 4.4-, and 28.4-fold lower than the GMT against homologous BA.1, respectively. These findings have implications in COVID-19 vaccine strategy.","version":"1.1","doi":"10.1101/2022.03.30.486409","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.473063","pub_date":"2022-3-31","title":"ACE2-independent SARS-CoV-2 infection and mouse adaption emerge after passage in cells expressing human and mouse ACE2","abstract":"Human ACE2 (hACE2) is the key cell attachment and entry receptor for SARS-CoV-2, with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe a new system for generating mouse-adapted SARS-CoV-2 in vitro by serial passaging virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses emerged with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused severe lung histopathology. One mouse-adapted virus was also able to replicate efficiently in ACE2-negative cell lines, with ACE2-independent entry by SARS-CoV-2 representing a new biology for SARS-CoV-2 that has potential widespread implications for disease and intervention development.","version":"1.2","doi":"10.1101/2021.12.16.473063","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486418","pub_date":"2022-3-31","title":"Contributions of the N-terminal intrinsically disordered region of the SARS-CoV-2 nucleocapsid protein to RNA-induced phase separation","abstract":"SARS-CoV-2 nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein\u2019s ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by disease-relevant mutations. Here we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2. In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE\u2019s structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein\u2019s RNA-mediated liquid-liquid phase separation.","version":"1.1","doi":"10.1101/2022.03.30.486418","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.31.22273222","pub_date":"2022-03-31","title":"Reducing societal impacts of SARS-CoV-2 interventions through subnational implementation","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>To curb the initial spread of SARS-CoV-2, many countries relied on nation-wide implementation of non-pharmaceutical intervention measures, resulting in substantial socio-economic impacts. Potentially, subnational implementations might have had less of a societal impact, but comparable epidemiological impact. Here, using the first COVID-19 wave in the Netherlands as a case in point, we address this issue by developing a high-resolution analysis framework that uses a demographically-stratified population and a spatially-explicit, dynamic, individual contact-pattern based epidemiology, calibrated to hospital admissions data and mobility trends extracted from mobile phone signals and Google. We demonstrate how a subnational approach could achieve similar level of epidemiological control in terms of hospital admissions, while some parts of the country could stay open for a longer period. Our framework is exportable to other countries and settings, and may be used to develop policies on subnational approach as a better strategic choice for controlling future epidemics.</jats:p>","version":null,"doi":"10.1101/2022.03.31.22273222","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.31.486531","pub_date":"2022-3-31","title":"SARS-CoV-2 Omicron is specifically restricted in its replication in human lung tissue, compared to other variants of concern","abstract":"SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron, compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC - a difference which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.","version":"1.1","doi":"10.1101/2022.03.31.486531","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486313","pub_date":"2022-3-31","title":"Identification of C270 as a novel site for allosteric modulators of SARS-CoV-2 papain-like protease","abstract":"The papain-like protease (PLpro) in coronavirus is one of key cysteine proteases responsible for the proteolytic processing of viral polyproteins, and plays an important role in dysregulation of host immune response. PLpro is a promising therapeutic target with a major challenge in inhibitor design due to the restricted S1/S2 sites for two consecutive glycine of substrates. Here we reported the discovery of two activators of the SARS-CoV-2 PLpro from a biochemical screening, and the identification of the unique residue, C270, as an allosteric and covalent regulation site for the activators. This site was also specifically modified by glutathione oxidized, resulting in the S-glutathionylation and activation of the protease. Furthermore, one compound was found to allosterically inhibit the protease by covalent binding to this crucial site. Together, these results elucidated an unrevealed molecular mechanism for allosteric modulation of the protease\u2019s activity, and provided a new strategy for discovery of allosteric inhibitors of the SARS-CoV-2 PLpro.","version":"1.1","doi":"10.1101/2022.03.30.486313","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486461","pub_date":"2022-3-31","title":"Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2Omicron variant BA.1 infection of human airway epithelial explant cultures","abstract":"Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (1, 2). Our results not only show broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 days, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data underscore the broad effects of MB, MPA, POS, and Niclo against SARS-CoV-2 and the currently circulating VoC, and reinforce the concept of repurposing drugs in clinical trials against COVID-19.","version":"1.1","doi":"10.1101/2022.03.30.486461","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.31.22273226","pub_date":"2022-03-31","title":"Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using\n                  <jats:italic>in vitro</jats:italic>\n                  microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs immediately resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.\n                </jats:p>","version":null,"doi":"10.1101/2022.03.31.22273226","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.29.486190","pub_date":"2022-3-30","title":"CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses","abstract":"Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNF\u03b1, IL-6 and IL-1\u03b2) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response. Over-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.","version":"1.2","doi":"10.1101/2022.03.29.486190","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.29.486331","pub_date":"2022-3-30","title":"Genetic surveillance of SARS-CoV-2 Mpro reveals high sequence and structural conservation prior to the introduction of protease inhibitor Paxlovid","abstract":"SARS-CoV-2 continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (Mpro). Nirmatrelvir is a potent Mpro inhibitor and the antiviral component of Paxlovid\u2122. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of Mpro prior to the introduction of Mpro inhibitors, Mpro sequences and its cleavage junction regions were retrieved from \u223c4,892,000 high-quality SARS-CoV-2 genomes in GISAID. Any mutations identified from comparison to the reference sequence (Wuhan-hu-1) were cataloged and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of Mpro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (alpha-, beta-, and gamma-coronaviruses). Additionally, we showed that over time the SARS-CoV-2 Mpro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the EUA approval of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 Mpro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals. The recent authorization of oral SARS-CoV-2 antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. Emergence of new variants, as well as selective pressure imposed by antiviral drugs themselves, raise concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of Mpro evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and Mpro inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 Mpro mutational landscape.","version":"1.1","doi":"10.1101/2022.03.29.486331","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486356","pub_date":"2022-3-30","title":"Differential gene expression profiling reveals potential biomarkers and pharmacological compounds against SARS-CoV-2: insights from machine learning and bioinformatics approaches","abstract":"SARS-CoV-2 continues to spread and evolve worldwide, despite intense efforts to develop multiple vaccines and therapeutic options against COVID-19. Moreover, the precise role of SARS-CoV-2 in the pathophysiology of the nasopharyngeal tract (NT) is still unfathomable. Therefore, we used the machine learning methods to analyze 22 RNA-seq datasets from COVID-19 patients (n=8), recovered individuals (n=7), and healthy individuals (n=7) to find disease-related differentially expressed genes (DEGs). In comparison to healthy controls, we found 1960 and 153 DEG signatures in COVID-19 patients and recovered individuals, respectively. We compared dysregulated DEGs to detect critical pathways and gene ontology (GO) connected to COVID-19 comorbidities. In COVID-19 patients, the DEG\u2013 miRNA and DEG\u2013transcription factors (TFs) interactions network analysis revealed that E2F1, MAX, EGR1, YY1, and SRF were the most highly expressed TFs, whereas hsa-miR-19b, hsa-miR-495, hsa-miR-340, hsa-miR-101, and hsa-miR-19a were the overexpressed miRNAs. Three chemical agents (Valproic Acid, Alfatoxin B1, and Cyclosporine) were abundant in COVID-19 patients and recovered individuals. Mental retardation, mental deficit, intellectual disability, muscle hypotonia, micrognathism, and cleft palate were the significant diseases associated with COVID-19 by sharing DEGs. Finally, we detected DEGs impacted by SARS-CoV-2 infection and mediated by TFs and miRNA expression, indicating that SARS-CoV-2 infection may contribute to various comorbidities. These pathogenetic findings can provide some crucial insights into the complex interplay between COVID-19 and the recovery stage and support its importance in the therapeutic development strategy to combat against COVID-19 pandemic. Despite it has now been over two years since the beginning of the COVID-19 pandemic, many crucial questions about SARS-CoV-2 infection and the different COVID-19 symptoms it causes remain unresolved. An intriguing question about COVID-19 is how SARS-CoV-2 interplays with the host during infection and how SARS-CoV-2 infection can cause so many disease symptoms. Our analysis of three different datasets (COVID-19, recovered, and healthy) revealed significantly higher DEGs in COVID-19 patients than recovered humans and healthy controls. Some of these DEGs were found to be co-expressed in both COVID-19 patients. They recovered humans supporting the notion that DEGs level is directly correlated with the viral load, disease progression, and different comorbidities. The protein-protein interaction consisting of 24 nodes and 72 edges recognized eight hub-nodes as potential hub-proteins (i.e., RPL4, RPS4X, RPL19, RPS12, RPL19, EIF3E, MT-CYB, and MT-ATP6). Protein\u2013chemical interaction analysis identified three chemical agents (e.g., Valproic Acid, Alfatoxin B1, and Cyclosporine) enriched in COVID-19 patients and recovered individuals. Mental retardation, mental deficiency, intellectual disability, muscle hypotonia, micrognathism, and cleft palate were the significant diseases associated with COVID-19 by sharing DEGs.","version":"1.1","doi":"10.1101/2022.03.30.486356","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486403","pub_date":"2022-3-30","title":"SARS-CoV-2 accessory protein ORF8 decreases antibody-dependent cellular cytotoxicity","abstract":"SARS-CoV-2 Spike glycoprotein is the major target of host neutralizing antibodies and the most changing viral protein in the continuously emerging SARS-CoV-2 variants as a result of frequent viral evasion from host antibody responses. In addition, SARS-CoV-2 encodes multiple accessory proteins that modulate host antiviral immunity by different mechanisms. Among all SARS-CoV-2 accessory proteins, ORF8 is rapidly evolving and a deletion in this protein has been linked to milder disease. Here, we studied the effect of ORF8 on peripheral blood mononuclear cells (PBMC). Specifically, we found that ORF8 can bind monocytes as well as NK cells. Strikingly, ORF8 binds CD16a (Fc\u03b3RIIIA) with nanomolar affinity and decreases the overall level of CD16 at the surface of monocytes and, to a lesser extent, NK cells. Strikingly, this decrease significantly reduces the capacity of PBMCs and particularly monocytes to mediate antibody-dependent cellular cytotoxicity (ADCC). Overall, our data identifies a new immune-evasion activity used by SARS-CoV-2 to escape humoral responses.","version":"1.1","doi":"10.1101/2022.03.30.486403","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486345","pub_date":"2022-3-30","title":"Ancestral origins are associated with SARS-CoV-2 susceptibility and protection in a Florida patient population","abstract":"COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The severity of COVID-19 is highly variable and related to known (e.g., age, obesity, immune deficiency) and unknown risk factors. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, e.g., whether individual differences in immunity play a role in patient symptomatology and how much human leukocyte antigen (HLA) contributes to this. To reveal genetic determinants of susceptibility to COVID-19 severity in the population and further explore potential immune-related factors, we performed a genome-wide association study on 284 confirmed COVID-19 patients (cases) and 95 healthy individuals (controls). We compared cases and controls of European (EUR) ancestry and African American (AFR) ancestry separately. We identified two loci on chromosomes 5q32 and 11p12, which reach the significance threshold of suggestive association (p<1\u00d710-5 threshold adjusted for multiple trait testing) and are associated with the COVID-19 susceptibility in the European ancestry (index rs17448496: odds ratio [OR] = 0.173; 95% confidence interval [CI], 0.08\u20130.36 for G allele; p=5.15\u00d7 10-5 and index rs768632395: OR = 0.166; 95% CI, 0.07\u20130.35 for A allele; p= 4.25\u00d710-6, respectively), which were associated with two genes, PPP2R2B at 5q32, and LRRC4C at 11p12, respectively. To explore the linkage between HLA and COVID-19 severity, we applied fine-mapping analysis to dissect the HLA association with mild and severe cases. Using In-silico binding predictions to map the binding of risk/protective HLA to the viral structural proteins, we found the differential presentation of viral peptides in both ancestries. Lastly, extrapolation of the identified HLA from the cohort to the worldwide population revealed notable correlations. The study uncovers possible differences in susceptibility to COVID-19 in different ancestral origins in the genetic background, which may provide new insights into the pathogenesis and clinical treatment of the disease.","version":"1.1","doi":"10.1101/2022.03.30.486345","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.30.486377","pub_date":"2022-3-30","title":"ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies","abstract":"Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing \u20182P\u2019 mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.","version":"1.1","doi":"10.1101/2022.03.30.486377","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.29.486282","pub_date":"2022-3-29","title":"Repeated ethanol exposure and withdrawal alters ACE2 expression in discrete brain regions: Implications for SARS-CoV-2 infection","abstract":"Emerging evidence suggests that people with alcohol use disorders are at higher risk for SARS-CoV-2. SARS-CoV-2 engages angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) receptors for cellular entry. While ACE2 and TMPRSS2 genes are upregulated in the cortex of alcohol-dependent individuals, information on expression in specific brain regions and neural populations implicated in SARS-CoV-2 neuroinvasion, particularly monoaminergic neurons, is limited. We sought to clarify how chronic alcohol exposure affects ACE2 and TMPRSS2 expression in monoaminergic brainstem circuits and other putative SARS-CoV-2 entry points. C57BL/6J mice were exposed to chronic intermittent ethanol (CIE) vapor for 4 weeks and brains were examined using immunofluorescence. We observed increased ACE2 levels in the olfactory bulb and hypothalamus following CIE, which are known to mediate SARS-CoV-2 neuroinvasion. Total ACE2 immunoreactivity was also elevated in the raphe magnus (RMG), raphe obscurus (ROB), and locus coeruleus (LC), while in the dorsal raphe nucleus (DRN), ROB, and LC we observed increased colocalization of ACE2 with monoaminergic neurons. ACE2 also increased in the periaqueductal gray (PAG) and decreased in the amygdala. Whereas ACE2 was detected in most brain regions, TMPRSS2 was only detected in the olfactory bulb and DRN but was not significantly altered after CIE. Our results suggest that previous alcohol exposure may increase the risk of SARS-CoV-2 neuroinvasion and render brain circuits involved in cardiovascular and respiratory function as well as emotional processing more vulnerable to infection, making adverse outcomes more likely. Additional studies are needed to define a direct link between alcohol use and COVID-19 infection.","version":"1.1","doi":"10.1101/2022.03.29.486282","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.29.486173","pub_date":"2022-3-29","title":"A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants","abstract":"Large-scale populations in the world have been vaccinated with COVID-19 vaccines, however, breakthrough infections of SARS-CoV-2 are still growing rapidly due to the emergence of immune-evasive variants, especially Omicron. It is urgent to develop effective broad-spectrum vaccines to better control the pandemic of these variants. Here, we present a mosaic-type trimeric form of spike receptor-binding domain (mos-tri-RBD) as a broad-spectrum vaccine candidate, which carries the key mutations from Omicron and other circulating variants. Tests in rats showed that the designed mos-tri-RBD, whether used alone or as a booster shot, elicited potent cross-neutralizing antibodies against not only Omicron but also other immune-evasive variants. Neutralizing antibody titers induced by mos-tri-RBD were substantially higher than those elicited by homo-tri-RBD (containing homologous RBDs from prototype strain) or the inactivated vaccine BBIBP-CorV. Our study indicates that mos-tri-RBD is highly immunogenic, which may serve as a broad-spectrum vaccine candidate in combating SARS-CoV-2 variants including Omicron.","version":"1.1","doi":"10.1101/2022.03.29.486173","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.29.482838","pub_date":"2022-3-29","title":"Subcellular mapping of the protein landscape of SARS-CoV-2 infected cells for target-centric drug repurposing","abstract":"The COVID-19 pandemic has resulted in millions of deaths and affected socioeconomic structure worldwide and the search for new antivirals and treatments are still ongoing. In the search for new drug target and to increase our understanding of the disease, we used large scale immunofluorescence to explore the host cell response to SARS-CoV-2 infection. Among the 602 host proteins studied in this host response screen, changes in abundance and subcellular localization were observed for 97 proteins, with 45 proteins showing increased abundance and 10 reduced abundances. 20 proteins displayed changed localization upon infection and an additional 22 proteins displayed altered abundance and localization, together contributing to diverse reshuffling of the host cell protein landscape. We then selected existing and approved small-molecule drugs (n =123) against our identified host response proteins and identified 3 compounds - elesclomol, crizotinib and rimcazole, that significantly reduced antiviral activity. Our study introduces a novel, targeted and systematic approach based on host protein profiling, to identify new targets for drug repurposing. The dataset of \u223c75,000 immunofluorescence images from this study are published as a resource available for further studies.","version":"1.1","doi":"10.1101/2022.03.29.482838","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.28.486152","pub_date":"2022-3-29","title":"Convergent epitope specificities, V gene usage and public clones elicited by primary exposure to SARS-CoV-2 variants","abstract":"While humoral immune responses to infection or vaccination with ancestral SARS-CoV-2 have been well-characterized, responses elicited by infection with variants are less understood. Here we characterized the repertoire, epitope specificity, and cross-reactivity of antibodies elicited by Beta and Gamma variant infection compared to ancestral virus. We developed a high-throughput approach to obtain single-cell immunoglobulin sequences and isolate monoclonal antibodies for functional assessment. Spike-, RBD- and NTD-specific antibodies elicited by Beta- or Gamma-infection exhibited a remarkably similar hierarchy of epitope immunodominance for RBD and convergent V gene usage when compared to ancestral virus infection. Additionally, similar public B cell clones were elicited regardless of infecting variant. These convergent responses may account for the broad cross-reactivity and continued efficacy of vaccines based on a single ancestral variant. WA1, Beta and Gamma variants of SARS-CoV-2 all elicit antibody responses targeting similar RBD epitopes; public and cross-reactive clones are common.","version":"1.1","doi":"10.1101/2022.03.28.486152","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.29.486253","pub_date":"2022-3-29","title":"Discovery of a druggable copper-signaling pathway that drives cell plasticity and inflammation","abstract":"Inflammation is a complex physiological process triggered in response to harmful stimuli. It involves specialized cells of the immune system able to clear sources of cell injury and damaged tissues to promote repair. Excessive inflammation can occur as a result of infections and is a hallmark of several diseases. The molecular basis underlying inflammatory responses are not fully understood. Here, we show that the cell surface marker CD44, which characterizes activated immune cells, acts as a metal transporter that promotes copper uptake. We identified a chemically reactive pool of copper(II) in mitochondria of inflammatory macrophages that catalyzes NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with a rationally-designed dimer of metformin triggers distinct metabolic and epigenetic states that oppose macrophage activation. This drug reduces inflammation in mouse models of bacterial and viral (SARS-CoV-2) infections, improves well-being and increases survival. Identifying mechanisms that regulate the plasticity of immune cells provides the means to develop next-generation medicine. Our work illuminates the central role of copper as a regulator of cell plasticity and unveils a new therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.","version":"1.1","doi":"10.1101/2022.03.29.486253","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.25.485832","pub_date":"2022-3-28","title":"A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model","abstract":"The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery, which improve vaccination compliance and demonstrate efficacy against emerging variants. Here we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprised of stabilized, pre-fusion Spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction of lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.","version":"1.1","doi":"10.1101/2022.03.25.485832","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.28.486075","pub_date":"2022-3-28","title":"An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2","abstract":"Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22.v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE22.v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoy\u2019s therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE22.v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE22.v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.03.28.486075","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485633","pub_date":"2022-3-28","title":"Neutralization of Omicron BA.1, BA.2, and BA.3 SARS-CoV-2 by 3 doses of BNT162b2 vaccine","abstract":"The newly emerged Omicron SARS-CoV-2 has 3 distinct sublineages: BA.1, BA.2, and BA.3. BA.1 accounts for the initial surge and is being replaced by BA.2, whereas BA.3 is at a low prevalence at this time. Here we report the neutralization of BNT162b2-vaccinated sera (collected at 1 month after dose 3) against the three Omicron sublineages. To facilitate the neutralization testing, we engineered the complete BA.1, BA.2, or BA.3 spike into an mNeonGreen USA-WA1/2020 SRAS-CoV-2. All BNT162b2-vaccinated sera neutralized USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s with titers of >20; the neutralization geometric mean titers (GMTs) against the four viruses were 1211, 336, 300, and 190, respectively. Thus, the BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s were 3.6-, 4.0-, and 6.4-fold less efficiently neutralized than the USA-WA1/2020, respectively. Our data have implications in vaccine strategy and understanding the biology of Omicron sublineages.","version":"1.1","doi":"10.1101/2022.03.24.485633","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.14.464390","pub_date":"2022-3-28","title":"Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis","abstract":"While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral \u2018RG\u2019 motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2\u2019s continued adaptation to human infection. Since its emergence, SARS-CoV-2 has continued to adapt for human infection resulting in the emergence of variants with unique genetic profiles. Most studies of genetic variation have focused on spike, the target of currently available vaccines, leaving the importance of variation elsewhere understudied. Here, we characterize a highly variable motif at residues 203-205 in nucleocapsid. Recreating the prominent nucleocapsid R203K+G204R mutation in an early pandemic background, we show that this mutation is alone sufficient to enhance SARS-CoV-2 replication and pathogenesis. We also link augmentation of SARS-CoV-2 infection by the R203K+G204R mutation to its modulation of nucleocapsid phosphorylation. Finally, we characterize an analogous alanine double substitution at positions 203-204. This mutant was found to mimic R203K+G204R, suggesting augmentation of infection occurs by disrupting the ancestral sequence. Together, our findings illustrate that mutations outside of spike are key components of SARS-CoV-2\u2019s adaptation to human infection.","version":"1.2","doi":"10.1101/2021.10.14.464390","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.484759","pub_date":"2022-3-28","title":"Broad neutralization of SARS-CoV-2 variants by circular mRNA producing VFLIP-X spike in mice","abstract":"Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X elicited neutralizing antibodies for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in TH1 and TH2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA confers humoral and cellular immune responses, as well as neutralizing activity against broad SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2022.03.17.484759","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.26.485903","pub_date":"2022-3-28","title":"The interplay between lncRNAs, RNA-binding proteins and viral genome during SARS-CoV-2 infection reveals strong connections with regulatory events involved in RNA metabolism and immune response","abstract":"Viral infections are complex processes based on an intricate network of molecular interactions. The infectious agent hijacks components of the cellular machinery for its profit, circumventing the natural defense mechanisms triggered by the infected cell. The successful completion of the replicative viral cycle within a cell depends on the function of viral components versus the cellular defenses. Non-coding RNAs (ncRNAs) are important cellular modulators, either promoting or preventing the progression of viral infections. Among these ncRNAs, the long non-coding RNA (lncRNA) family is especially relevant due to their intrinsic functional properties and ubiquitous biological roles. Specific lncRNAs have been recently characterized as modulators of the cellular response during infection of human host cells by single stranded RNA viruses. However, the role of host lncRNAs in the infection by human RNA coronaviruses such as SARS-CoV-2 remains uncharacterized. In the present work, we have performed a transcriptomic study of a cohort of patients with different SARS-CoV-2 viral load. Our results revealed the existence of a SARS-CoV-2 infection-dependent pattern of transcriptional up-regulation in which specific lncRNAs are an integral component. To determine the role of these lncRNAs, we performed a functional correlation analysis complemented with the study of the validated interactions between lncRNAs and RNA-binding proteins (RBPs). This combination of in silico functional association studies and experimental evidence allowed us to identify a lncRNA signature composed of six elements - NRIR, BISPR, MIR155HG, FMR1-IT1, USP30-AS1, and U62317.2 - associated with the regulation of SARS-CoV-2 infection. We propose a competition mechanism between the viral RNA genome and the regulatory lncRNAs in the sequestering of specific RBPs that modulates the interferon response and the regulation of RNA surveillance by nonsense-mediated decay (NMD). Model of interactions among lncRNA and cognate RNA-binding proteins in SARS-CoV-2 infection. According to our model, the viral genome can establish direct interactions with three core proteins (DDX3X, UPF1 and IGF2BP2) involved in mRNA metabolism and regulation of the interferon response, which are also components of a SARS-CoV-2 lncRNA-centered regulatory network. The competition between viral RNA and lncRNAs could act as a counteracting factor for the normal function of homeostatic lncRNA-centered regulatory networks, contributing to viral progression and replication. Black arrows depict physical interactions between network components; red arrows represent functional relationships.","version":"1.1","doi":"10.1101/2022.03.26.485903","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.27.485958","pub_date":"2022-3-28","title":"Genetically engineered MRI-trackable extracellular vesicles as SARS-CoV-2 mimetics for mapping ACE2 binding in vivo","abstract":"The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup, call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible, and thus, allows mapping of the binding of RBD to ACE2 receptors non-invasively in live subjects. Importantly, the proposed mimetics can be easily modified to display the RBD of SARS-CoV-2mutants, namely Delta and Omicron, allowing rapid screening of newly raised variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner.","version":"1.1","doi":"10.1101/2022.03.27.485958","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.26.485922","pub_date":"2022-3-28","title":"Unique molecular signatures sustained in circulating monocytes and regulatory T cells in Convalescent COVID-19 patients","abstract":"Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-\u03b2 high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.","version":"1.1","doi":"10.1101/2022.03.26.485922","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450632","pub_date":"2022-3-28","title":"Spike protein cleavage-activation in the context of the SARS-CoV-2 P681R mutation: an analysis from its first appearance in lineage A.23.1 identified in Uganda","abstract":"Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences.","version":"1.6","doi":"10.1101/2021.06.30.450632","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.25.485875","pub_date":"2022-3-28","title":"Mosaic RBD nanoparticles protect against multiple sarbecovirus challenges in animal models","abstract":"To combat future SARS-CoV-2 variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles presenting randomly-arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against conserved/relatively-occluded, rather than variable/immunodominant/exposed, epitopes. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD-nanoparticles in mice and macaques, observing stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants including Omicron and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest mosaic-8 RBD-nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.","version":"1.1","doi":"10.1101/2022.03.25.485875","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.23.485575","pub_date":"2022-3-27","title":"Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry","abstract":"SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemia that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the virus RNA ratio allowing SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and S. Furthermore, monochrome, dual-color fluorescent or photoconvertible VLPs were produced. Thanks to live fluorescence and super-resolution microscopy, we quantified VLPs size and concentration. It shows a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a minimum concentration of 10e12 VLP/ml. SARS-CoV-2 VLPs could tolerate the integration of fluorescent N and M tagged proteins without impairing particle assembly. In this condition, we were able to establish incorporation of the mature Spike in fluorescent VLPs. The Spike functionality was then shown by monitoring fluorescent MNES-VLPs docking and endocytosis in human pulmonary cells expressing the receptor hACE2. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.","version":"1.1","doi":"10.1101/2022.03.23.485575","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.23.485570","pub_date":"2022-3-27","title":"Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes","abstract":"Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously. We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.","version":"1.1","doi":"10.1101/2022.03.23.485570","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.23.485576","pub_date":"2022-3-27","title":"The Envelope Protein of SARS-CoV-2 Inhibits Viral Protein Synthesis and Infectivity of Human Immunodeficiency Virus type 1 (HIV-1)","abstract":"The human coronavirus SARS-CoV-2 encodes for a small 75 amino acid transmembrane protein known as the envelope (E) protein. The E protein forms an ion channel, like the viroporins from human immunodeficiency virus type 1 (HIV-1) (Vpu) and influenza A virus (M2). Here, we analyzed HIV-1 virus infectivity in the presence of four different \u03b2-coronavirus E proteins. We observed that the SARS-CoV-2 and SARS-CoV E proteins reduced HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. This was also reflected in the levels of HIV-1 protein synthesis in cells. Mechanistically, we show that that the E protein neither affected reverse transcription nor genome integration. However, SARS-CoV-2 E protein activated the ER-stress pathway associated with the phosphorylation of eIF-2\u03b1, which is known to attenuate protein synthesis in cells. Finally, we show that these four E proteins and the SARS-CoV-2 N protein did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2) while the spike (S) proteins of SARS-CoV and SARS-CoV-2, and HIV-1 Vpu efficiently down-regulated cell surface BST-2 expression. The results of this study show for the first time that viroporins from a heterologous virus can suppress HIV-1 infection. The E protein of coronaviruses is a viroporin that is required for efficient release of infectious virus and for viral pathogenicity. We determined if the E protein from four \u03b2-coronaviruses could restrict virus particle infectivity of HIV-1 infection. Our results indicate that the E proteins from SARS-CoV-2 and SARS-CoV potently restricted HIV-1 while those from MERS-CoV and HCoV-OC43 were less restrictive. Substitution of the highly conserved proline in the cytoplasmic domain of SARS-CoV-2 E abrogated the restriction on HIV-1 infection. Mechanistically, the SARS-CoV-2 E protein did not interfere with viral integration or RNA synthesis but rather reduced viral protein synthesis. We show that the E protein-initiated ER stress causing phosphorylation of eIF-2\u03b1, which is known to attenuate protein synthesis. Companion studies suggest that the E protein also triggers autophagy. These results show for the first time that a viroporin from a coronavirus can restrict infection of another virus.","version":"1.1","doi":"10.1101/2022.03.23.485576","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.26.22272984","pub_date":"2022-03-27","title":"Monitoring of the SARS-CoV-2 Omicron BA.1/BA.2 variant transition in the Swedish population reveals higher viral quantity in BA.2 cases","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Throughout the SARS-CoV-2 pandemic, multiple waves of variants of concern have swept across populations, leading to a chain of new and yet more contagious lineages dominating COVID-19 cases. Here, we tracked the remarkably rapid shift from Omicron BA.1 to BA.2 sub-variant dominance in the Swedish population during January\u2013March 2022. By analysis of 174,933 clinical nasopharyngeal swab samples using a custom variant-typing RT-PCR assay, we uncover nearly two-fold higher levels of viral RNA in cases with Omicron BA.2. Importantly, increased viral load in the upper pharynx upon BA.2 infection may provide part of the explanation why Omicron BA.2 is more transmissible and currently outcompetes the BA.1 variant across populations.</jats:p>","version":null,"doi":"10.1101/2022.03.26.22272984","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.24.485591","pub_date":"2022-3-27","title":"Antiviral roles of interferon regulatory factor (IRF)-1, 3 and 7 against human coronavirus infection","abstract":"Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). As many human coronaviruses are known to be sensitive to IFN, antiviral roles of IRFs are yet to be fully understood. TypeI or II IFN treatment protected MRC5 cells from infection of human coronavirus 229E, but not human coronavirus OC43. Infection of 229E or OC43 efficiently upregulated ISGs, indicating that antiviral transcription is not suppressed during their infection. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of the IRFs demonstrated that IRF1 and IRF3 have antiviral property against OC43 while only IRF3 and IRF7 are effective to restrict 229E infection. Our study demonstrates that IRF3 plays critical roles against infection of human coronavirus 229E and OC43, which may be an anti-human coronavirus therapeutic target.","version":"1.1","doi":"10.1101/2022.03.24.485591","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485734","pub_date":"2022-3-25","title":"SARS-CoV-2 harnesses host translational shutoff and autophagy to optimize virus yields: The role of the envelope (E) protein","abstract":"The SARS-CoV-2 virion is composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). E spans the membrane a single time and is the smallest, yet most enigmatic of the structural proteins. E is conserved among coronaviruses and has an essential role in virus-mediated pathogenesis. We found that ectopic expression of E had deleterious effects on the host cell as it activated stress responses, leading to phosphorylation of the translation initiation factor eIF2\u03b1 and LC3 lipidation that resulted in host translational shutoff. During infection E is highly expressed although only a small fraction is incorporated into virions, suggesting that E activity is regulated and harnessed by the virus to its benefit. In support of this, we found that the \u03b31 34.5 protein of herpes simplex virus 1 (HSV-1) prevented deleterious effects of E on the host cell and allowed for E protein accumulation. This observation prompted us to investigate whether other SARS-CoV-2 structural proteins regulate E. We found that the N and M proteins enabled E protein accumulation, whereas S prevented E accumulation. While \u03b31 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. This negative effect of \u03b31 34.5 was most likely associated with failure of SARS-CoV-2 to divert the translational machinery and with deregulation of autophagy pathways. Overall, our data suggest that SARS-CoV-2 causes stress responses and subjugates these pathways, including host protein synthesis (phosphorylated eIF2\u03b1) and autophagy, to support optimal virus production. In 2020, a new \u03b2-coronavirus, SARS-CoV-2, entered the human population that has caused a pandemic resulting in 6 million deaths worldwide. Although closely related to SARS-CoV, the mechanisms of SARS-CoV-2 pathogenesis are not fully understood. We found that ectopic expression of the SARS-CoV-2 E protein had detrimental effects on the host cell, causing metabolic alterations including shutoff of protein synthesis and mobilization of cellular resources through autophagy activation. Co-expression of E with viral proteins known to subvert host antiviral responses such as autophagy and translational inhibition, either from SARS-CoV-2 or from heterologous viruses increased cell survival and E protein accumulation. However, such factors were found to negatively impact SARS-CoV-2 infection, as autophagy contributes to formation of viral membrane factories, and translational control offers an advantage for viral gene expression. Overall, SARS-CoV-2 has evolved mechanisms to harness host functions that are essential for virus replication.","version":"1.1","doi":"10.1101/2022.03.24.485734","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388710","pub_date":"2022-3-25","title":"Universally available herbal teas based on sage and perilla elicit potent antiviral activity against SARS-CoV-2 variants of concern by HMOX-1 upregulation in human cells","abstract":"The current SARS-CoV-2/COVID-19 pandemic wreaks medical and socioeconomic havoc. Despite the availability of vaccines, cost-effective acute treatment options preventing morbidity and mortality are urgently needed. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 in therapeutic as well as prophylactic regimens. The herbal infusions exerted antiviral effects comparable to interferon-\u03b2 and remdesivir but outperformed convalescent sera and interferon-\u03b12 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron.","version":"1.3","doi":"10.1101/2020.11.18.388710","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485222","pub_date":"2022-3-25","title":"A Computational Pipeline to Identify Potential Drug Targets and Interacting Chemotypes in SARS-CoV-2","abstract":"Minimizing the human and economic costs of the COVID-19 pandemic and of future pandemics requires the ability to develop and deploy effective treatments for novel pathogens as soon as possible after they emerge. To this end, we introduce a unique, computational pipeline for the rapid identification and characterization of binding sites in the proteins of novel viruses as well as the core chemical components with which these sites interact. We combine molecular-level structural modeling of proteins with clustering and cheminformatic techniques in a computationally efficient manner. Similarities between our results, experimental data, and other computational studies provide support for the effectiveness of our predictive framework. While we present here a demonstration of our tool on SARS-CoV-2, our process is generalizable and can be applied to any new virus, as long as either experimentally solved structures for its proteins are available or sufficiently accurate homology models can be constructed.","version":"1.1","doi":"10.1101/2022.03.24.485222","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.25.485815","pub_date":"2022-3-25","title":"Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system","abstract":"During the SARS-CoV-2 intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-COVID-19 drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the Receptor Binding Domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. When used to immunise mice, the 3CLpro-RBD chimer elicited antibodies mainly to the 3CL-pro portion of the molecule indicating that a different chimeric composition (i.e., RBD/full Spike-3CLpro) or expression system (i.e., mammalian cells), might be required to produce and deliver a RBD with immunogenicity similar to the native protein. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.","version":"1.1","doi":"10.1101/2022.03.25.485815","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485649","pub_date":"2022-3-25","title":"B cell receptor repertoire analysis unveils dynamic antibody response and severity markers in COVID-19 patients","abstract":"Humoral and cell mediated immunity are critical against viral infections. The knowledge of composition, diversity, gene usage of the B cell repertoires helps in determining the immune response to SARS-CoV-2 infection. Examining B cell response provides insights on therapeutic antibodies, disease severity markers and aids in predicting vaccine response. We have analyzed public domain immunoglobulin sequencing data from PBMCs of SARS-CoV-2 infected individuals to gain a better understanding of B cell repertoire in patients. Public clonotypes showed increased usage of IGHV3, IGHV4, IGKV1, IGKV3, IGLV3 and IGLV2 family genes during the acute phase infection. Identical CDR3 sequences were identified for heavy (H), kappa (K) and lambda (L) chains across individuals, indicating the convergence of B cell selection during SARS-CoV-2 infection. While the immune repertoire dynamically changed over the course of convalescence, there were persistent clones across early and late timepoints. The diversity of antibody repertoire, measured by Shannon-Weiner diversity index for H and K chains, reduced during the acute phase of infection. In addition, the repertoire diversity was low in severe patients compared to patients with mild or moderate symptoms. Increased usage of IGHV4-59 gene was observed in COVID-19 patients with severe symptoms requiring ventilator support at 2 weeks and 3 weeks post symptom onset. IGHV4-59 is reported to have rheumatoid factor (RF) activity with high affinity for IgG and the elevated level of IGHV4-59 provides a potential mechanism for the increased autoimmune responses in severe patients. Correlation of the clinical features with the B cell receptor repertoire dynamics elucidated public antibody clonotypes and disease severity markers for COVID-19.","version":"1.1","doi":"10.1101/2022.03.24.485649","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.25.485748","pub_date":"2022-3-25","title":"Changes of urinary proteomic before and after QIV and COVID-19 vaccination","abstract":"We first collected a young people\u2019s urine samples cohort of quadrivalent influenza vaccine. Urine protein at 24 hours after vaccination was enriched in immune-related pathways, though the specific pathways varied. Perhaps because different people may be in a previous life encountered some of the viruses in the vaccine, the second immunization was triggered. Or everyone has a different constitution, exposure to the same virus triggering different immunity. We then collected urine samples from several uninfected SARS-CoV-2 young people before and after the first, second, and third doses of the COVID-19 vaccine. We found that the differential protein compared between after the second dose (24h) and before the second dose enriched pathways were involved in regulated exocytosis and immune-related pathways, indicating not first exposure to antigen. Surprisingly, the urine differential protein-enriched pathways before and after the first dose were similar to those before and after the second dose. We assume that although the volunteers have not been infected with SARS-CoV-2, they might have been exposed to other coimmunogenic coronaviruses. 2~4h after the third vaccination, the differentially expressed protein also enriched regulated exocytosis and immune-related pathways, indicating that the body has triggered the immune response in a very short time after vaccination, and urine proteome is a good window to monitor the changes of human immune function.","version":"1.1","doi":"10.1101/2022.03.25.485748","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.442182","pub_date":"2022-3-24","title":"LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants","abstract":"SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variants No loss of potency against currently circulating variants Binding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database Breadth of neutralizing activity and potency supports clinical development","version":"1.6","doi":"10.1101/2021.04.30.442182","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485614","pub_date":"2022-3-24","title":"SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications","abstract":"The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Five antibody candidates were selected out of a na\u00efve camelid library by phage display and expressed as full-length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.","version":"1.1","doi":"10.1101/2022.03.24.485614","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485596","pub_date":"2022-3-24","title":"Differences in neuroinflammation in the olfactory bulb between D614G, Delta and Omicron BA.1 SARS-CoV-2 variants in the hamster model","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. SARS-CoV-2 infection induces neuroinflammation in the central nervous system (CNS), whereat the olfactory bulb seems to be involved most frequently. Here we show differences in the neuroinvasiveness and neurovirulence among SARS-CoV-2 variants in the hamster model five days post inoculation. Replication in the olfactory mucosa was observed in all hamsters, but most prominent in D614 inoculated hamsters. We observed neuroinvasion into the CNS via the olfactory nerve in D614G-, but not Delta (B.1.617.2)- or Omicron BA.1 (B.1.1.529) inoculated hamsters. Neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G but hardly in Delta or Omicron BA.1. Altogether, this indicates that there are differences in the neuroinvasive and neurovirulent potential among SARS-CoV-2 variants in the acute phase of the infection in the hamster model.","version":"1.1","doi":"10.1101/2022.03.24.485596","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485618","pub_date":"2022-3-24","title":"Improved And Optimized Drug Repurposing For The SARS-CoV-2 Pandemic","abstract":"The active global SARS-CoV-2 pandemic caused more than 426 million cases and 5.8 million deaths worldwide. The development of completely new drugs for such a novel disease is a challenging, time intensive process. Despite researchers around the world working on this task, no effective treatments have been developed yet. This emphasizes the importance of drug repurposing, where treatments are found among existing drugs that are meant for different diseases. A common approach to this is based on knowledge graphs, that condense relationships between entities like drugs, diseases and genes. Graph neural networks (GNNs) can then be used for the task at hand by predicting links in such knowledge graphs. Expanding on state-of-the-art GNN research, Doshi et al. recently developed the Dr-COVID model. We further extend their work using additional output interpretation strategies. The best aggregation strategy derives a top-100 ranking of 8,070 candidate drugs, 32 of which are currently being tested in COVID-19-related clinical trials. Moreover, we present an alternative application for the model, the generation of additional candidates based on a given pre-selection of drug candidates using collaborative filtering. In addition, we improved the implementation of the Dr-COVID model by significantly shortening the inference and pre-processing time by exploiting data-parallelism. As drug repurposing is a task that requires high computation and memory resources, we further accelerate the post-processing phase using a new emerging hardware \u2014 we propose a new approach to leverage the use of high-capacity Non-Volatile Memory for aggregate drug ranking.","version":"1.1","doi":"10.1101/2022.03.24.485618","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.428685","pub_date":"2022-3-24","title":"Self-assembling SARS-CoV-2 nanoparticle vaccines targeting the S protein induces protective immunity in mice","abstract":"The spike (S), a homotrimer glycoprotein, is the most important antigen target in the research and development of SARS-CoV-2 vaccine. There is no doubt that fully simulating the advanced structure of this homotrimer in the subunit vaccine development strategy is the most likely way to improve the immune protective effect of the vaccine. In this study, the preparation strategies of S protein receptor-binding domain (RBD) trimer, S1 region trimer, and ectodomain (ECD) trimer nanoparticles were designed based on ferritin nanoparticle self-assembly technology. The Bombyx mori baculovirus expression system was used to prepare these three nanoparticle vaccines with high expression levels in the silkworm. The immune results of mice show that the nanoparticle vaccine prepared by this strategy can not only induce an immune response by subcutaneous administration but also effective by oral administration. Given the stability of these ferritin-based nanoparticles vaccine, easy-to-use and low-cost oral immunization strategy can make up for the vaccination blind areas caused by the shortage of ultralow-temperature equipment and medical resources in underdeveloped areas. And the oral vaccine is also a very potential candidate to cut off the spread of SARS-CoV-2 in domestic and farmed animals, especially in stray and wild animals.","version":"1.2","doi":"10.1101/2021.02.05.428685","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.24.485560","pub_date":"2022-3-24","title":"Structural insights into the viral proteins binding by TRIM7 reveal a general C-terminal glutamine recognition mechanism","abstract":"The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. A recent study found that TRIM7 inhibits human enteroviruses through ubiquitination and proteasomal degradation of viral 2BC protein by targeting the 2C moiety of 2BC protein. Here, we report the crystal structures of TRIM7 in complex with 2C, where the C-terminal region of 2C is inserted into a positively charged groove of the TRIM7 PRY-SPRY domain. Structure-guided biochemical studies revealed the C-terminus glutamine residue of 2C as the primary determinant for TRIM7 binding. Such a glutamine-end motif binding mechanism can be successfully extended to other substrates of TRIM7. More importantly, leveraged by this finding, we were able to identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. We further show that TRIM7 may function as a restriction factor to promote the degradation of the viral proteins of norovirus and SARS-CoV-2, thereby restoring the Type I interferon immune response and inhibiting viral infection. Several crystal structures of TRIM7 in complex with SARS-CoV-2 proteins are also determined, and a conserved C-terminus glutamine-specific interaction is observed. These findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.","version":"1.1","doi":"10.1101/2022.03.24.485560","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.23.22272828","pub_date":"2022-03-24","title":"Hyperactive immature state and differential CXCR2 expression of neutrophils in severe COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Neutrophils are vital in defence against pathogens but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome (ARDS). COVID-19 is associated with systemic expansion of immature neutrophils but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10\n                  <jats:sup>\u2212</jats:sup>\n                  subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable three months post symptom onset, indication long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients downregulate the chemokine receptor CXCR2, while neutrophils from severely ill individuals failed to do so, suggesting altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10\n                  <jats:sup>\u2212</jats:sup>\n                  and CXCR2\n                  <jats:sup>hi</jats:sup>\n                  neutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for identification of individuals at high risk of progressing to severe COVID-19.\n                </jats:p>","version":null,"doi":"10.1101/2022.03.23.22272828","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.22.485425","pub_date":"2022-3-23","title":"Influenza infection in ferrets with SARS-CoV-2 infection history","abstract":"Non-pharmaceutical interventions (NPIs) to contain the SARS-CoV-2 pandemic drastically reduced human-to-human interactions, decreasing the circulation of other respiratory viruses as well. As a consequence, influenza virus circulation \u2013 normally responsible for 3-5 million hospitalizations per year globally \u2013 was significantly reduced. With downscaling the NPI countermeasures, there is a concern for increased influenza disease, particularly in individuals suffering from post-acute effects of SARS-CoV-2 infection. To investigate this possibility, we performed a sequential influenza H1N1 infection 4 weeks after an initial SARS-CoV-2 infection in the ferret model. Upon H1N1 infection, ferrets that were previously infected with SARS-CoV-2 showed an increased tendency to develop clinical symptoms compared to the control H1N1 infected animals. Histopathological analysis indicated only a slight increase for type II pneumocyte hyperplasia and bronchitis. The effects of the sequential infection thus appeared minor. However, ferrets were infected with B.1.351-SARS-CoV-2, the beta variant of concern, which replicated poorly in our model. The histopathology of the respiratory organs was mostly resolved 4 weeks after SARS-CoV-2 infection, with only reminiscent histopathological features in the upper respiratory tract. Nevertheless, SARS-CoV-2 specific cellular and humoral responses were observed, confirming an established infection. Thus, there may likely be a SARS-CoV-2 variant-dependent effect on the severity of disease upon a sequential influenza infection as we observed mild effects upon a mild infection. It, however, remains to be determined what the impact is of more virulent SARS-CoV-2 variants. During the COVID-19 pandemic, the use of face masks, social distancing and isolation were not only effective in decreasing the circulation of SARS-CoV-2, but also in reducing other respiratory viruses such as influenza. With less restrictions, influenza is slowly returning. In the meantime, people still suffering from long-COVID, could be more vulnerable to an influenza virus infection and develop more severe influenza disease. This study provides directions to the effect of a previous SARS-CoV-2 exposure on influenza disease severity in the ferret model. This model is highly valuable to test sequential infections under controlled settings for translation to humans. We could not induce clear long-term COVID-19 effects as SARS-CoV-2 infection in ferrets was mild. However, we still observed a slight increase in influenza disease severity compared to ferrets that had not encountered SARS-CoV-2 before. It may therefore be advisable to include long-COVID patients as a risk group for influenza vaccination.","version":"1.1","doi":"10.1101/2022.03.22.485425","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.23.485487","pub_date":"2022-3-23","title":"In silico analysis predicts a limited impact of SARS-CoV-2 variants on CD8 T cell recognition","abstract":"Since the start of the COVID-19 pandemic, mutations have led to the emergence of new SARS-CoV-2 variants, and some of these have become prominent or dominant variants of concern. This natural course of development can have an impact on how protective the previously naturally or vaccine induced immunity is. Therefore, it is crucial to understand whether and how variant specific mutations influence host immunity. To address this, we have investigated how mutations in the recent SARS-CoV-2 variants of interest and concern influence epitope sequence similarity, predicted binding affinity to HLA, and immunogenicity of previously reported SARS-CoV-2 CD8 T cell epitopes. Our data suggests that the vast majority of SARS-CoV-2 CD8 T cell recognized epitopes are not altered by variant specific mutations. Interestingly, for the CD8 T cell epitopes that are altered due to variant specific mutations, our analyses show there is a high degree of sequence similarity between mutated and reference SARS-CoV-2 CD8 T cell epitopes. However, mutated epitopes, primarily derived from the spike protein, in SARS-CoV-2 variants Delta, AY.4.2 and Mu display reduced predicted binding affinity to their restriction element. These findings indicate that the recent SARS-CoV-2 variants of interest and concern have limited ability to escape memory CD8 T cell responses raised by vaccination or prior infection with SARS-CoV-2 early in the pandemic. The overall low impact of the mutations on CD8 T cell cross-recognition is in accordance with the notion that mutations in SARS-CoV-2 are primarily the result of receptor binding affinity and antibody selection pressures exerted on the spike protein, unrelated to T cell immunity.","version":"1.1","doi":"10.1101/2022.03.23.485487","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.481974","pub_date":"2022-3-23","title":"An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques","abstract":"The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab\u2019)2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites. PET/CT detected SARS-CoV-2 infection of 4 different tissues in the male genital tract illuminates the cause of COVID-19 clinical sequalae of male sexual health and fertility\n\nFigure 1.\nDiagram shows schematic illustration of the male genital tract of the rhesus macaque. Virus icon shows sites of SARS-CoV-2 PET signal. Text highlighting the clinical sequalae associated with each sight of infection is shown in text adjacent to each infection site.\n\n Diagram shows schematic illustration of the male genital tract of the rhesus macaque. Virus icon shows sites of SARS-CoV-2 PET signal. Text highlighting the clinical sequalae associated with each sight of infection is shown in text adjacent to each infection site.","version":"1.2","doi":"10.1101/2022.02.25.481974","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485373","pub_date":"2022-3-23","title":"Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant protect Vero E6 cells against infection by viral pseudotyped particles expressing SARS-CoV-2, SARS-CoV or MERS-CoV Spike protein","abstract":"Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses (HuCoV) that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surface and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with \u223c97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S- pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:16 dilution, their infectivity was dramatically reduced by \u223c98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.","version":"1.1","doi":"10.1101/2022.03.22.485373","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485418","pub_date":"2022-3-23","title":"Heterotypic vaccination responses against SARS-CoV-2 Omicron BA.2","abstract":"The Omicron sub-lineage BA.2 of SARS-CoV-2 has recently become dominant across many areas in the world in the on-going waves of COVID-19. Compared to the ancestral/wild-type (WT) virus, Omicron lineage variants, both BA.1 and BA.2, contain high number of mutations, especially in the spike protein, causing significant immune escape that leads to substantial reduction of vaccine and antibody efficacy. Because of this antigenic drift, BA.2 exhibited differential resistance profile to monoclonal antibodies than BA.1. Thus, it is important to understand whether the immunity elicited by currently available vaccines are effective against the BA.2 subvariant. We directly tested the heterotypic vaccination responses against Omicron BA.2, using vaccinated serum from animals receiving WT- and variant-specific mRNA vaccine in lipid nanoparticle (LNP) formulations. Omicron BA.1 and BA.2 antigen showed similar reactivity to serum antibodies elicited by two doses of WT, B.1.351 and B.1.617 LNP-mRNAs. Neutralizing antibody titers of B.1.351 and B.1.617 LNP-mRNA were ~2-fold higher than that of WT LNP-mRNA. Both homologous boosting with WT LNP-mRNA and heterologous boosting with BA.1 LNP-mRNA substantially increased waning immunity of WT vaccinated mice against both BA.1 and BA.2 subvariants. The BA.1 LNP-mRNA booster was ~3-fold more efficient than WT LNP-mRNA at elevating neutralizing antibody titers of BA.2. Together, these data provided a direct preclinical evaluation of WT and variant-specific LNP-mRNAs in standard two-dose and as boosters against BA.1 and BA.2 subvariants.","version":"1.1","doi":"10.1101/2022.03.22.485418","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.23.485397","pub_date":"2022-3-23","title":"Topologically engineered antibodies and Fc-fusion proteins: a new class of multifunctional therapeutic candidates for SARS-CoV-2, cancer, and other disease","abstract":"The ability of antibodies and Fc-fusion proteins to bind multiple targets cooperatively is limited by their topology. Here we describe our discovery that ACE2 Fc-fusion proteins spontaneously cross-dimerize, forming topologically distinct \u201csuperdimers\u201d that demonstrate extraordinary SARS-CoV-2 intra-spike cooperative binding and potently neutralize Omicron B.1.1.529 at least 100-fold better than eight clinically authorized antibodies. We also exploited cross- dimerization to topologically engineer novel superdimeric antibodies and Fc-fusion proteins with antibody-like plasma half-lives to address cancer and infectious disease therapy. These include bispecific ACE2-antibody superdimers that potently neutralize all major SARS-CoV-2 variants, and bispecific anti-cancer and anti-viral antibody superdimers that are more potent than two-antibody cocktails. Superdimers are efficiently produced from single cells, providing a new therapeutic approach to many disease indications.","version":"1.1","doi":"10.1101/2022.03.23.485397","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485230","pub_date":"2022-3-23","title":"A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability","abstract":"mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing enhanced stability, improved manufacturability, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of long-lived bone marrow-resident antibody secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability at room temperature for at least 6 months when lyophilized. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.","version":"1.1","doi":"10.1101/2022.03.22.485230","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485323","pub_date":"2022-3-23","title":"Intranasal Nanoemulsion Adjuvanted S-2P Vaccine Demonstrates Protection in Hamsters and Induces Systemic, Cell-Mediated and Mucosal Immunity in Mice","abstract":"With the rapid progress made in the development of vaccines to fight the SARS-CoV-2 pandemic, almost >90% of vaccine candidates under development and a 100% of the licensed vaccines are delivered intramuscularly (IM). While these vaccines are highly efficacious against COVID-19 disease, their efficacy against SARS-CoV-2 infection of upper respiratory tract and transmission is at best temporary. Development of safe and efficacious vaccines that are able to induce robust mucosal and systemic immune responses are needed to control new variants. In this study, we have used our nanoemulsion adjuvant (NE01) to intranasally (IN) deliver stabilized spike protein (S-2P) to induce immunogenicity in mouse and hamster models. Data presented demonstrate the induction of robust immunity in mice resulting in 100% seroconversion and protection against SARS-CoV-2 in a hamster challenge model. There was a significant induction of mucosal immune responses as demonstrated by IgA- and IgG-producing memory B cells in the lungs of animals that received intranasal immunizations compared to an alum adjuvanted intramuscular vaccine. The efficacy of the S-2P/NE01 vaccine was also demonstrated in an intranasal hamster challenge model with SARS-CoV-2 and conferred significant protection against weight loss, lung pathology, and viral clearance from both upper and lower respiratory tract. Our findings demonstrate that intranasal NE01-adjuvanted vaccine promotes protective immunity against SARS-CoV-2 infection and disease through activation of three arms of immune system: humoral, cellular, and mucosal, suggesting that an intranasal SARS-CoV-2 vaccine may play a role in addressing a unique public health problem and unmet medical need.","version":"1.1","doi":"10.1101/2022.03.22.485323","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485413","pub_date":"2022-3-23","title":"A CNN model for predicting binding affinity changes between SARS-CoV-2 spike RBD variants and ACE2 homologues","abstract":"The cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the association of its receptor binding domain (RBD) with human angiotensin converting enzyme 2 (hACE2) as the first crucial step. Efficient and reliable prediction of RBD-hACE2 binding affinity changes upon amino acid substitutions can be valuable for public health surveillance and monitoring potential spillover and adaptation into non-human species. Here, we introduce a convolutional neural network (CNN) model trained on protein sequence and structural features to predict experimental RBD-hACE2 binding affinities of 8,440 variants upon single and multiple amino acid substitutions in the RBD or ACE2. The model achieves a classification accuracy of 83.28% and a Pearson correlation coefficient of 0.85 between predicted and experimentally calculated binding affinities in five-fold cross-validation tests and predicts improved binding affinity for most circulating variants. We pro-actively used the CNN model to exhaustively screen for novel RBD variants with combinations of up to four single amino acid substitutions and suggested candidates with the highest improvements in RBD-ACE2 binding affinity for human and animal ACE2 receptors. We found that the binding affinity of RBD variants against animal ACE2s follows similar trends as those against human ACE2. White-tailed deer ACE2 binds to RBD almost as tightly as human ACE2 while cattle, pig, and chicken ACE2s bind weakly. The model allows testing whether adaptation of the virus for increased binding with other animals would cause concomitant increases in binding with hACE2 or decreased fitness due to adaptation to other hosts.","version":"1.1","doi":"10.1101/2022.03.22.485413","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.484725","pub_date":"2022-3-23","title":"Jupytope: Computational extraction of structural properties of viral epitopes","abstract":"Epitope residues located on viral surface proteins are of immense interest in immunology and related applications such as vaccine development, disease diagnosis and drug design. Most tools rely on sequence based statistical comparisons, such as information entropy of residue positions in aligned columns to infer location and properties of epitope sites. To facilitate cross-structural comparisons of epitopes on viral surface proteins, a python-based extraction tool implemented with Jupyter notebook is presented (Jupytope). Given a viral antigen structure of interest, a list of known epitope sites and a reference structure, the corresponding epitope structural properties can quickly be obtained. The tool integrates biopython modules for commonly used software such as NACCESS, DSSP as well as residue depth and outputs a list of structure derived properties such as dihedral angles, solvent accessibility, residue depth and secondary structure that can be saved in several convenient data formats. To ensure correct spatial alignment, Jupytope takes a list of given epitope sites and their corresponding reference structure and aligns them before extracting the desired properties. Examples are demonstrated for epitopes of Influenza and SARS-CoV2 viral strains. The extracted properties assist detection of two Influenza subtypes and show potential in distinguishing between four major clades of SARS-CoV2, as compared with randomized labels. The tool will facilitate analytical and predictive works on viral epitopes through the extracted structural information. Jupytope combines existing 3D-structural software to extract the properties of viral epitopes into a convenient text or csv file format The structural properties serve as parameters or features that quantitatively capture viral epitopes Association of structural properties to viral subtypes (for Influenza) or clades (SARS-CoV2) is demonstrated with a simple XGBoost model Structure datasets mapped to SARS-CoV2 WHO clades and Pango lineages, as well as chain annotations are available for download","version":"1.1","doi":"10.1101/2022.03.22.484725","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485401","pub_date":"2022-3-23","title":"Inhalable polymer nanoparticles for versatile mRNA delivery and mucosal vaccination","abstract":"An inhalable platform for mRNA therapeutics would enable minimally invasive and lung targeted delivery for a host of pulmonary diseases. Development of lung targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) polyplexes for mRNA delivery using end group modifications and polyethylene glycol. Our polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for SARS-CoV-2. Intranasal vaccination with spike protein mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected K18-hACE2 mice from lethal viral challenge. Inhaled polymer nanoparticles (NPs) achieve high mRNA expression in the lung and induce protective immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.03.22.485401","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.20.22272549","pub_date":"2022-03-23","title":"Viral load dynamics of SARS-CoV-2 Delta and Omicron variants following multiple vaccine doses and previous infection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>An important, and often neglected, aspect of vaccine effectiveness is its impact on pathogen transmissibility, harboring major implications for public health policies. As viral load is a prominent factor affecting infectivity, its laboratory surrogate, qRT-PCR cycle threshold (Ct), can be used to investigate the infectivity-related component of vaccine effectiveness. While vaccine waning has previously been observed for viral load, during the Delta wave, it is yet unknown how Omicron viral load is affected by vaccination status, and whether vaccine-derived and natural infection protection are sustainable. By analyzing results of more than 460,000 individuals we show that while recent vaccination reduces Omicron viral load, its effect wanes rapidly. In contrast, a significantly slower waning rate is demonstrated for recovered COVID-19 individuals. Thus, while the vaccine is effective in decreasing morbidity and mortality, their relative minute effect on transmissibility and rapid waning call for reassessment of the scientific justification for \u201cvaccine certificate\u201d, as it may promote false reassurance and promiscuous behavior.</jats:p>","version":null,"doi":"10.1101/2022.03.20.22272549","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.21.485243","pub_date":"2022-3-22","title":"A potent SARS-CoV-2 antibody neutralizes Omicron variant by disassembling the spike trimer","abstract":"The continuous emergence of novel SARS-CoV-2 variants poses new challenges to the fight against the COVID-19 pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of antibody targeting conserved and universal epitope is urgently needed. A subset neutralizing antibody(nAbs) against COVID-19 from convalescent patients were isolated in our previous study. Here in this study, we investigated the accommodation of these nAbs to SARS-CoV-2 variants of concerns (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of SARS-CoV-2 Omicron variant. In addition, we determined the cryo-EM structure of SARS-CoV-2 spike complexed with three antibodies targeting different epitopes, including 553-49, 553-15 and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes virus by disassembling spike trimers. 553-15, an antibody that neutralizes all the other VOCs except omicron, cross-links two spike trimers to form trimer dimer, demonstrating that 553-15 neutralizes virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 49 and other antibody targeting this highly conserved epitope as promising cocktail therapeutics reagent for COVID-19. The newly emergence of Omicron strain caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 Omicron neutralizing antibody 553-49, which neutralizes Omicron variant by targeting a completely conserved novel epitope. Besides, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by crosslinking virions and 553-60 functions by blocking receptor binding. Comparison of different RBD epitopes revealed that the epitope of 553-49 is hidden in the S trimer and keeps high conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutics reagent to fight against the emerging Omicron and future variant of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.03.21.485243","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.21.485247","pub_date":"2022-3-22","title":"Trivalent NDV-HXP-S vaccine protects against phylogenetically distant SARS-CoV-2 variants of concern in mice","abstract":"Equitable access to vaccines is necessary to limit the global impact of the coronavirus disease 2019 (COVID-19) pandemic and the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In previous studies, we described the development of a low-cost vaccine based on a Newcastle Disease virus (NDV) expressing the prefusion stabilized spike protein from SARS-CoV-2, named NDV-HXP-S. Here, we present the development of next-generation NDV-HXP-S variant vaccines, which express the stabilized spike protein of the Beta, Gamma and Delta variants of concerns (VOC). Combinations of variant vaccines in bivalent, trivalent and tetravalent formulations were tested for immunogenicity and protection in mice. We show that the trivalent preparation, composed of the ancestral Wuhan, Beta and Delta vaccines, substantially increases the levels of protection and of cross-neutralizing antibodies against mismatched, phylogenetically distant variants, including the currently circulating Omicron variant.","version":"1.1","doi":"10.1101/2022.03.21.485247","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.21.485224","pub_date":"2022-3-22","title":"A mRNA vaccine encoding for a RBD 60-mer nanoparticle elicits neutralizing antibodies and protective immunity against the SARS-CoV-2 delta variant in transgenic K18-hACE2 mice","abstract":"Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the delta variant elicits robust neutralizing antibody response with neutralizing titers an order of magnitude above currently approved mRNA vaccines. The construct also provides protective immunity against the delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2022.03.21.485224","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.21.22272611","pub_date":"2022-03-22","title":"Regional importation and asymmetric within-country spread of SARS-CoV-2 variants of concern in the Netherlands","abstract":"<jats:p>Variants of concern (VOCs) of SARS-CoV-2 have caused resurging waves of infections worldwide. In the Netherlands, Alpha, Beta, Gamma and Delta variants circulated widely between September 2020 and August 2021. To understand how various control measures had impacted the spread of these VOCs, we analyzed 39,844 SARS-CoV-2 genomes collected under the Dutch national surveillance program. We found that all four VOCs were introduced before targeted flight restrictions were imposed on countries where the VOCs first emerged. Importantly, foreign introductions, predominantly from other European countries, continued during these restrictions. Our findings show that flight restrictions had limited effectiveness in deterring VOC introductions due to the strength of regional land travel importation risks. We also found that the Alpha and Delta variants largely circulated more populous regions \nwith international connections after their respective introduction before asymmetric bidirectional transmissions occurred with the rest of the country and the variant dominated infections in the Netherlands. As countries consider scaling down SARS-CoV-2 surveillance efforts in the post-crisis phase of the pandemic, our results highlight that robust surveillance in regions of early spread is important for providing timely information for variant detection and outbreak control.</jats:p>","version":null,"doi":"10.1101/2022.03.21.22272611","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.06.451353","pub_date":"2022-3-22","title":"Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern","abstract":"We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax\u2122 vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.","version":"1.2","doi":"10.1101/2021.07.06.451353","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.22.485299","pub_date":"2022-3-22","title":"Methylene Blue Is a Nonspecific Protein-Protein Interaction Inhibitor with Potential for Repurposing as an Antiviral for COVID-19","abstract":"We have previously identified methylene blue, a tricyclic phenothiazine dye approved for clinical use for the treatment of methemoglobinemia and used for other medical applications, as a small-molecule inhibitor of the protein-protein interaction (PPI) between the spike protein of the SARS-CoV-2 coronavirus and ACE2, the first critical step of the attachment and entry of this coronavirus responsible for the COVID-19 pandemic. Here, we show that methylene blue concentration-dependently inhibits this PPI for the spike protein of the original strain as well as for those of variants of concerns such as the D614G mutant and delta (B.1.617.2) with IC50 in the low micromolar range (1-5 \u03bcM). Methylene blue also showed promiscuous activity and inhibited several other PPIs of viral proteins (e.g., HCoV-NL63 \u2013 ACE2, hepatitis C virus E \u2013 CD81) as well as others (e.g., IL-2 \u2013 IL-2R\u03b1) with similar potency. This non-specificity notwithstanding, methylene blue inhibited the entry of pseudoviruses bearing the spike protein of SARS-CoV-2 in hACE2-expressing host cells both for the original strain and the delta variant. It also blocked SARS-CoV-2 (B.1.5) virus replication in Vero E6 cells with an IC50 in the low micromolar range (1.7 \u03bcM) when assayed using quantitative PCR of the viral RNA. Thus, while it seems to be a promiscuous PPI inhibitor with low micromolar activity and it has a relatively narrow therapeutic index, methylene blue inhibits entry and replication of SARS-CoV-2, including several of its mutant variants, and has potential as a possible inexpensive, broad-spectrum, orally bioactive small-molecule antiviral for the prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.03.22.485299","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473140","pub_date":"2022-3-22","title":"Human genetic variants associated with COVID-19 severity are enriched in immune and epithelium regulatory networks","abstract":"Human genetic variants can influence the severity of symptoms being infected with SARS-COV-2. Several genome-wide association studies have identified human genomic risk SNPs associated with COVID-19 severity. However, the causal tissues or cell types of COVID-19 severity are uncertain and candidate genes associated with these human risk SNPs were investigated in genomic proximity instead of their functional cellular contexts. Here, we compiled regulatory networks of 77 human contexts and revealed those risk SNPs\u2019 enriched cellular contexts and associated transcript factors, regulatory elements, and target genes. Twenty-one human contexts were identified and grouped into two categories: immune cells and epithelium cells. We further aggregated the regulatory networks of immune cells, epithelium cells, and immune-epithelium crosstalk and investigated their association with risk SNPs\u2019 regulation. Two genomic clusters, chemokine receptors cluster and OAS cluster, showed the strongest association with COVID-19 severity and different regulations in immune and epithelium contexts. Our findings were supported by analysis on both microarray and whole genome sequencing based GWAS summary statistics.","version":"1.2","doi":"10.1101/2021.12.17.473140","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.08.483451","pub_date":"2022-3-21","title":"Reorganization of F-actin nanostructures is required for the late phases of SARS-CoV-2 replication in pulmonary cells","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is worldwide the main cause of the COVID-19 pandemic. After infection of human pulmonary cells, intracellular viral replication take place in different cellular compartments resulting in the destruction of the host cells and causing severe respiratory diseases. Although cellular trafficking of SARS-CoV-2 have been explored, little is known about the role of the cytoskeleton during viral replication in pulmonary cells. Here we show that SARS-CoV-2 infection induces dramatic changes of F-actin nanostructures overtime. Ring-like actin nanostructures are surrounding viral intracellular organelles, suggesting a functional interplay between F-actin and viral M clusters during particle assembly. Filopodia-like structures loaded with viruses to neighbour cells suggest these structures as mechanism for cell-to-cell virus transmission. Strikingly, gene expression profile analysis and PKN inhibitor treatments of infected pulmonary cells reveal a major role of alpha-actinins superfamily proteins in SARS-CoV-2 replication. Overall, our results highlight cell actors required for SARS-CoV2 replication that are promises for antiviral targets. Impairing regulation of actin filaments inhibits SARS-CoV-2 particle production in human pulmonary cells.","version":"1.2","doi":"10.1101/2022.03.08.483451","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.14.484288","pub_date":"2022-3-21","title":"Dual spatially resolved transcriptomics for SARS-CoV-2 host-pathogen colocalization studies in humans","abstract":"To advance our understanding of cellular host-pathogen interactions, technologies that facilitate the co-capture of both host and pathogen spatial transcriptome information are needed. Here, we present an approach to simultaneously capture host and pathogen spatial gene expression information from the same formalin-fixed paraffin embedded (FFPE) tissue section using the spatial transcriptomics technology. We applied the method to COVID-19 patient lung samples and enabled the dual detection of human and SARS-CoV-2 transcriptomes at 55 \u03bcm resolution. We validated our spatial detection of SARS-CoV-2 and identified an average specificity of 94.92% in comparison to RNAScope and 82.20% in comparison to in situ sequencing (ISS). COVID-19 tissues showed an upregulation of host immune response, such as increased expression of inflammatory cytokines, lymphocyte and fibroblast markers. Our colocalization analysis revealed that SARS-CoV-2+ spots presented shifts in host RNA metabolism, autophagy, NF\u03baB, and interferon response pathways. Future applications of our approach will enable new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes.","version":"1.2","doi":"10.1101/2022.03.14.484288","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484953","pub_date":"2022-3-21","title":"Humoral and cellular immune memory to four COVID-19 vaccines","abstract":"Multiple COVID-19 vaccines, representing diverse vaccine platforms, successfully protect against symptomatic COVID-19 cases and deaths. Head-to-head comparisons of T cell, B cell, and antibody responses to diverse vaccines in humans are likely to be informative for understanding protective immunity against COVID-19, with particular interest in immune memory. Here, SARS-CoV-2-spike\u2014specific immune responses to Moderna mRNA-1273, Pfizer/BioNTech BNT162b2, Janssen Ad26.COV2.S and Novavax NVX-CoV2373 were examined longitudinally for 6 months. 100% of individuals made memory CD4+ T cells, with cTfh and CD4-CTL highly represented after mRNA or NVX-CoV2373 vaccination. mRNA vaccines and Ad26.COV2.S induced comparable CD8+ T cell frequencies, though memory CD8+ T cells were only detectable in 60-67% of subjects at 6 months. Ad26.COV2.S was not the strongest immunogen by any measurement, though the Ad26.COV2.S T cell, B cell, and antibody responses were relatively stable over 6 months. A differentiating feature of Ad26.COV2.S immunization was a high frequency of CXCR3+ memory B cells. mRNA vaccinees had substantial declines in neutralizing antibodies, while memory T cells and B cells were comparatively stable over 6 months. These results of these detailed immunological evaluations may also be relevant for vaccine design insights against other pathogens.","version":"1.1","doi":"10.1101/2022.03.18.484953","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.21.485157","pub_date":"2022-3-21","title":"Stability and expression of SARS-CoV-2 spike-protein mutations","abstract":"Protein fold stability likely plays a role in SARS-CoV-2 S-protein evolution, together with ACE2 binding and antibody evasion. While few thermodynamic stability data are available for S-protein mutants, many systematic experimental data exist for their expression. In this paper, we explore whether such expression levels relate to the thermodynamic stability of the mutants. We studied mutation-induced SARS-CoV-2 S-protein fold stability, as computed by three very distinct methods and eight different protein structures to account for method- and structure-dependencies. For all methods and structures used (24 comparisons), computed stability changes correlate significantly (99% confidence level) with experimental yeast expression from the literature, such that higher expression is associated with relatively higher fold stability. Also significant, albeit weaker, correlations were seen for ACE2 binding. The effect of thermodynamic fold stability may be direct or a correlate of amino acid or site properties, notably the solvent exposure of the site. Correlation between computed stability and experimental expression and ACE2 binding suggests that functional properties of the SARS-CoV-2 S-protein mutant space are largely determined by a few simple features, due to underlying correlations. Our study lends promise to the development of computational tools that may ideally aid in understanding and predicting SARS-CoV-2 S-protein evolution.","version":"1.1","doi":"10.1101/2022.03.21.485157","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.20.485050","pub_date":"2022-3-21","title":"SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells","abstract":"While there is clinical evidence of neurological manifestation in coronavirus disease-19, it\u2019s unclear whether this is due to differential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake from blood by cells of the cerebrovasculature. SARS-CoV-2 and its spike protein (SP) interact with the endothelium but the roles of extracellular peptidase domain on angiotensin converting enzyme 2 receptors (ACE2) and ACE2 independent pathways (such as glycans) are not fully elucidated. In addition, for SARS-CoV-2 to enter the brain parenchyma from blood it has to cross several cell types, including the endothelium, pericytes and vascular smooth muscle. Since SARS-CoV-2 interacts with host cells via it SP at the entry point of it life cycle, we used fluorescently labelled SP (SP-555) (wild type and mutants) to model viral behaviour, in vitro, for these cell types (endothelial, pericytes and vascular smooth muscle) to explore pathways of viral entry into brain from blood. There was differential SP uptake by these cell types. The endothelial cells had the least uptake, which may limit SP uptake into brain from blood. Uptake was mediated by ACE2, but it was dependent on SP interaction with ganglioside GM1 in the lipid raft. Mutation sites, N501Yand E484K and D614G, as seen in variants of interest, were differentially taken up by these cell types. There was greater uptake but neutralization with anti-ACE2 and anti-GM1antibodies was less effective. Our data suggested that GM1/lipid raft is an important entry point of SARS-CoV-2 into these cells since inhibition of SP uptake with both anti-ACE2 and anti-GM1 together was similar to that with only anti-GM1, and both ACE2 and GM1 are within the lipid raft region of plasma membrane. Thus, GM1 is a potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.","version":"1.1","doi":"10.1101/2022.03.20.485050","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.20.485044","pub_date":"2022-3-21","title":"Robust and durable prophylactic protection conferred by RNA interference in preclinical models of SARS-CoV-2","abstract":"RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral genome. We screened lipophilic small-interfering RNA (siRNA) conjugates targeting highly conserved regions of the SARS-CoV-2 genome and identified leads targeting outside of the spike-encoding region capable of achieving \u22653-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single-siRNA approach. A two-siRNA combination delivered intranasally protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach to limit SARS-CoV-2 infection that may help combat emergent variants, complement existing interventions, or protect populations where vaccines are less effective. Most importantly, this strategy has implications for developing medicines that may be valuable in protecting against future coronavirus pandemics.","version":"1.1","doi":"10.1101/2022.03.20.485044","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.21.485084","pub_date":"2022-3-21","title":"SARS-CoV-2 Omicron spike H655Y mutation is responsible for enhancement of the endosomal entry pathway and reduction of cell surface entry pathways","abstract":"The SARS-CoV-2 Omicron variant reportedly displays decreased usage of the cell surface entry pathway mediated by the host transmembrane protease, serine 2 (TMPRSS2) and increased usage of the endosomal entry pathway mediated by cathepsin B/L. These differences result in different cell tropisms and low fusogenicity from other SARS-CoV-2 variants. Recent studies have revealed that host metalloproteases are also involved in cell surface entry and fusogenic activity of SARS-CoV-2, independent of TMPRSS2. However, the involvement of metalloproteinase-mediated cell entry and fusogenicity in Omicron infections has not been investigated. Here, we report that Omicron infection is less sensitive to the metalloproteinase inhibitor marimastat, like the TMPRSS2 inhibitor nafamostat, and is more sensitive to the cathepsin B/L inhibitor E-64d than infections with wild-type SARS-CoV-2 and other variants. The findings indicate that Omicron preferentially utilizes the endosomal pathway rather than cell surface pathways for entry. Moreover, the Omicron variant also displays poor syncytia formation mediated by metalloproteinases, even when the S cleavage status mediated by fusion-like proteases is unchanged. Intriguingly, the pseudovirus assay showed that a single mutation, H655Y, of the Omicron spike (S) is responsible for the preferential entry pathway usage without affecting the S cleavage status. These findings suggest that the Omicron variant has altered entry properties and fusogenicity, probably through the H655Y mutation in its S protein, leading to modulations of tissue and cell tropism, and reduced pathogenicity. Recent studies have suggested that the SARS-CoV-2 Omicron variant displays altered cell tropism and fusogenicity, in addition to immune escape. However, comprehensive analyses of the usage of viral entry pathways in Omicron variant have not been performed. Here, we used protease inhibitors to block each viral entry pathway mediated by the three host proteases (cathepsin B/L, TMPRSS2, and metalloproteinases) in various cell types. The results clearly indicated that Omicron exhibits enhanced cathepsin B/L-dependent endosome entry and reduced metalloproteinase-dependent and TMPRSS2-dependent cell surface entry. Furthermore, the H655Y mutation of Omicron S determines the relative usage of the three entry pathways without affecting S cleavage by the host furin-like proteases. Comparative data among SARS-CoV-2 variants, including Omicron, may clarify the biological and pathological phenotypes of Omicron but increase the understanding of disease progression in infections with other SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.03.21.485084","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.19.484981","pub_date":"2022-3-21","title":"Identification of a Novel SARS-CoV-2 Delta-Omicron Recombinant Virus in the United States","abstract":"Recombination between SARS-CoV-2 virus variants can result in different viral properties (e.g., infectiousness or pathogenicity). In this report, we describe viruses with recombinant genomes containing signature mutations from Delta and Omicron variants. These genomes are the first evidence for a Delta-Omicron hybrid Spike protein in the United States.","version":"1.1","doi":"10.1101/2022.03.19.484981","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484404","pub_date":"2022-3-21","title":"Omicron variant of SARS-COV-2 gains new mutations in New Zealand and Hong Kong","abstract":"The authors have withdrawn this manuscript because an issue was raised about the SARS- COV-2 genome sequence data used in the study. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.2","doi":"10.1101/2022.03.15.484404","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.16.484554","pub_date":"2022-3-21","title":"Targeting an evolutionarily conserved \u201cE-L-L\u201d motif in the spike protein to develop a small molecule fusion inhibitor against SARS-CoV-2","abstract":"As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved \u201cE-L-L\u201d motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing the post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this \u201cE-L-L\u201d motif resulting in effective inhibition of SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy towards blocking S protein-mediated viral entry, mutations within the \u201cE-L-L\u201d motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential \u201cE-L-L\u201d motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.","version":"1.2","doi":"10.1101/2022.03.16.484554","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484178","pub_date":"2022-3-20","title":"Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for therapy","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.","version":"1.1","doi":"10.1101/2022.03.18.484178","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.22272574","pub_date":"2022-03-20","title":"Maternal Antibody Response and Transplacental Transfer Following SARS-CoV-2 Infection or Vaccination in Pregnancy","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Pregnant persons are at increased risk of severe COVID-19 and adverse obstetric outcomes. Understanding maternal antibody response and transplacental transfer after SARS-CoV-2 infection and COVID-19 vaccination is important to inform public health recommendations.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This prospective observational cohort study included 351 birthing individuals who had SARS-CoV-2 infection or COVID-19 vaccination during pregnancy. IgG and IgM to SARS-CoV-2 S1 receptor binding domain were measured in maternal and cord blood. Antibody levels and transplacental transfer ratios were compared across 1) disease severity for those with SARS-CoV-2 infection and 2) infection versus vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>There were 252 individuals with SARS-CoV-2 infection and 99 who received COVID-19 vaccination during pregnancy. Birthing people with more severe SARS-CoV-2 infection category had higher maternal and cord blood IgG levels (p=0.0001, p=0.0001). Median IgG transfer ratio was 0.87-1.2. Maternal and cord blood IgG were higher after vaccination than infection (p=0.001, p=0.001). Transfer ratio was higher after 90 days in the vaccinated group (p&lt;0.001). Modeling showed higher amplitude and half-life of maternal IgG following vaccination (p&lt;0.0001). There were no significant differences by fetal sex.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>COVID-19 vaccination in pregnancy leads to higher and longer lasting maternal IgG levels, higher cord blood IgG, and higher transfer ratio after 90 days compared to SARS-CoV-2 infection. Greater infection severity leads to higher maternal and cord blood antibodies. Maternal IgG decreases over time following both vaccination and infection, reinforcing the importance of vaccination, even after infection, and vaccine boosters for pregnant patients.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.03.17.22272574","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.20.485024","pub_date":"2022-3-20","title":"A Novel High-Throughput Single B-Cell Cloning Platform for Isolation and Characterization of High-Affinity and potent SARS-CoV-2 Neutralizing Antibodies","abstract":"Monoclonal antibodies (mAbs) that are specific to SARS-CoV-2 can be useful in diagnosing, preventing, and treating the coronavirus (COVID-19) illness. Strategies for the high-throughput and rapid isolation of these potent neutralizing antibodies are critical toward the development of therapeutically targeting COVID-19 as well as other infectious diseases. In the present study, a single B-cell cloning method was used to screen SARS-CoV-2 receptor-binding domain (RBD) specific, high affinity, and neutralizing mAbs from patients\u2019 blood samples. An RBD-specific antibody, SAR03, was discovered that showed high binding (ELISA and SPR) and neutralizing activity (competitive ELISA and pseudovirus-based reporter assay) against Sars-CoV-2. Mechanistic studies on human cells revealed that SAR03 competes with the ACE-2 receptor for binding with the RBD domain (S1 subunit) present in the spike protein of Sars-CoV-2. This study highlights the potential of the single B cell cloning method for the rapid and efficient screening of high-affinity and effective neutralizing antibodies for Sars-CoV-2 and other emerging infectious diseases. Single B-cell cloning is a high-throughput and efficient method of generating high affinity neutralizing antibodies Single B-cell cloning method was used to screen SARS-CoV-2 receptor-binding domain (RBD) specific, high affinity, and neutralizing monoclonal antibodies from patient\u2019s blood samples. An RBD-specific antibody, SAR03, was discovered that showed high binding and neutralizing activity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.03.20.485024","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484950","pub_date":"2022-3-20","title":"Durable protection against SARS-CoV-2 Omicron induced by an adjuvanted subunit vaccine","abstract":"Despite the remarkable efficacy of COVID-19 vaccines, waning immunity, and the emergence of SARS-CoV-2 variants such as Omicron represents a major global health challenge. Here we present data from a study in non-human primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine, consisting of RBD (receptor binding domain) on the I53-50 nanoparticle, adjuvanted with AS03, currently in Phase 3 clinical trial (NCT05007951). Vaccination induced robust neutralizing antibody (nAb) titers that were maintained at high levels for at least one year after two doses (Pseudovirus nAb GMT: 2207, Live-virus nAb GMT: 1964) against the ancestral strain, but not against Omicron. However, a booster dose at 6-12 months with RBD-Wu or RBD-\u03b2 (RBD from the Beta variant) displayed on I53-50 elicited equivalent and remarkably high neutralizing titers against the ancestral as well as the Omicron variant. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Importantly, vaccination resulted in protection against Omicron infection in the lung (no detectable virus in any animal) and profound suppression of viral burden in the nares (median peak viral load of 7567 as opposed to 1.3\u00d7107 copies in unvaccinated animals) at 6 weeks post final booster. Even at 6 months post vaccination, there was significant protection in the lung (with 7 out of 11 animals showing no viral load, 3 out of 11 animals showing ~20-fold lower viral load than unvaccinated controls) and rapid control of virus in the nares. These results highlight the durable cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine platform.","version":"1.1","doi":"10.1101/2022.03.18.484950","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484954","pub_date":"2022-3-20","title":"Peptidome Surveillance Across Evolving SARS-CoV-2 Lineages Reveals HLA Binding Conservation in Nucleocapsid Among Variants With Most Potential for T-Cell Epitope Loss In Spike","abstract":"To provide a unique global view of the relative potential for evasion of CD8+ and CD4+ T cells by SARS-CoV-2 lineages as they evolve over time, we performed a comprehensive analysis of predicted HLA-I and HLA-II binding peptides in spike (S) and nucleocapsid (N) protein sequences of all available SARS-CoV-2 genomes as provided by NIH NCBI at a bi-monthly interval between March and December of 2021. A data supplement of all B.1.1.529 (Omicron) genomes from GISAID in early December was also used to capture the rapidly spreading variant. A key finding is that throughout continued viral evolution and increasing rates of mutations occurring at T-cell epitope hotspots, protein instances with worst case binding loss did not become the most frequent for any Variant of Concern (VOC) or Variant of Interest (VOI) lineage; suggesting T-cell evasion is not likely to be a dominant evolutionary pressure on SARS-CoV-2. We also determined that throughout the course of the pandemic in 2021, there remained a relatively steady ratio of viral variants that exhibit conservation of epitopes in the N protein, despite significant potential for epitope loss in S relative to other lineages. We further localized conserved regions in N with high epitope yield potential, and illustrated HLA-I binding heterogeneity across the S protein consistent with empirical observations. Although Omicron\u2019s high volume of mutations caused it to exhibit more epitope loss potential than most frequently observed versions of proteins in almost all other VOCs, epitope candidates across its most frequent N proteins were still largely conserved. This analysis adds to the body of evidence suggesting that N may have merit as an additional antigen to elicit immune responses to vaccination with increased potential to provide sustained protection against COVID-19 disease in the face of emerging variants.","version":"1.1","doi":"10.1101/2022.03.18.484954","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427501","pub_date":"2022-3-18","title":"SARS-CoV-2 Can Infect Human Embryos","abstract":"The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. We note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. Using fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, we observe robust infection of trophectoderm cells, and this permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.","version":"1.2","doi":"10.1101/2021.01.21.427501","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484873","pub_date":"2022-3-18","title":"SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties","abstract":"The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one another\u2019s escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.","version":"1.1","doi":"10.1101/2022.03.18.484873","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.484786","pub_date":"2022-3-18","title":"Prime-boost vaccinations with two serologically distinct chimpanzee adenovirus vectors expressing SARS-CoV-2 spike or nucleocapsid tested in a hamster COVID-19 model","abstract":"Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing the spike (S) or nucleocapsid (N) proteins of an early SARS-CoV-2 isolate were tested individually or as a mixture in a hamster COVID-19 challenge model. The N protein, which was expressed as a fusion protein within herpes simplex virus glycoprotein D (gD) stimulated antibodies and CD8+ T cells. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gDN fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable that the S protein may potentiate and prolong protection achieved by the currently used genetic COVID-19 vaccines.","version":"1.1","doi":"10.1101/2022.03.17.484786","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441151","pub_date":"2022-3-18","title":"Large-scale analysis of SARS-CoV-2 synonymous mutations reveals the adaptation to the human codon usage during the virus evolution","abstract":"Many large national and transnational studies have been dedicated to the analysis of SARS-CoV-2 genome, most of which focused on missense and nonsense mutations. However, approximately 30% of the SARS-CoV-2 variants are synonymous, therefore changing the target codon without affecting the corresponding protein sequence. By performing a large-scale analysis of sequencing data generated from almost 400,000 SARS-CoV-2 samples, we show that silent mutations increasing the similarity of viral codons to the human ones tend to fixate in the viral genome over-time. This indicates that SARS-CoV-2 codon usage is adapting to the human host, likely improving its effectiveness in using the human aminoacyl-tRNA set through the accumulation of deceitfully neutral silent mutations. Synonymous SARS-CoV-2 mutations related to the activity of different mutational processes may positively impact viral evolution by increasing its adaptation to human codon usage.","version":"1.2","doi":"10.1101/2021.04.23.441151","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.484787","pub_date":"2022-3-18","title":"Resilience of S309 and AZD7442 monoclonal antibody treatments against infection by SARS-CoV-2 Omicron lineage strains","abstract":"Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2). Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human Fc\u03b3 R transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo.","version":"1.1","doi":"10.1101/2022.03.17.484787","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.484837","pub_date":"2022-3-18","title":"Cytokine and chemokine profile in patients hospitalized with COVID-19: A comparative study","abstract":"Abnormal cytokine and chemokine concentrations during SARS-CoV-2 infection may represent disease severity. We aimed to assess plasma cytokine and chemokine concentrations in patients with SARS-CoV-2 in Addis Ababa, Ethiopia. In this study, 260 adults: 126 hospitalized patients with confirmed COVID-19 sorted into severity groups: severe (n=68) and mild or moderate (n=58), and 134 healthy controls were enrolled. We quantified 39 plasma cytokines and chemokines using multiplex ELISA. Spearman rank correlation and Mann-Whitney U test were used to identify mechanistically coupled cytokines/chemokines and compare disease severity. Compared to healthy controls, patients with COVID-19 had significantly higher levels of interleukins 1\u03b1, 2, 6, 7, 8, 10 and 15, C-reactive protein (CRP), serum amyloid A (SAA), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), IFN-\u03b3-inducible protein-10 (IP-10), macrophage inflammatory protein-1 alpha (MIP-1\u03b1), eotaxin-3, interferon-gamma (IFN-\u03d2), tumor necrosis factor-\u03b1 (TNF-\u03b1), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and fms-like tyrosine kinase 1 (Flt-1). Patients with severe COVID-19 had higher IL-10 and lower macrophage-derived chemokine (MDC) compared to the mild or moderate group (P<0.05). In the receiver operating characteristic curve, SAA, IL-6 and CRP showed strong sensitivity and specificity predicting the severity and prognosis of COVID-19. Greater age and higher CRP had a significant association with disease severity (P<0.05). Our findings reveal that CRP, SAA, VCAM-1, IP-10, MDC and IL-10 levels are promising biomarkers for COVID-19 disease severity, suggesting that plasma cytokines/chemokines could be used as warning indicators of COVID-19 severity, aid in COVID-19 prognosis and treatment. SARS-CoV-2 triggers inflammatory reaction resulting in respiratory discomfort and in critical case may result in death. Cytokines and chemokines are inflammatory biomarkers that regulate and determine the nature of immune responses. Measuring cytokine and chemokine levels is useful in stratification, management and treatment of COVID-19 patients as well as guide resource allocations and therapeutic options. Here, we examined ctytokine and chemokine profiles in COVID-19 patients. Understanding how distinct cytokines and chemokines change over time as COVID-19 disease progresses might aid clinicians in detecting severe illness earlier and thereby improve patient prognosis.","version":"1.1","doi":"10.1101/2022.03.17.484837","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.18.484843","pub_date":"2022-3-18","title":"COVID-19 patients have increased levels of membrane-associated and soluble CD48","abstract":"COVID-19 is a respiratory-centered systemic disorder caused by SARS-CoV-2. The disease can progress into a severe form causing acute lung injury. CD48 is a co-signaling receptor, existing as both membrane-bound and soluble forms reported to be dysregulated in several inflammatory conditions. Therefore, we reasoned that CD48 could be deregulated in COVID-19 as well. Here we analyzed CD48 expression in autoptic sections and peripheral blood leukocytes and sera of COVID-19 patients by gene expression profiling (HTG\u00ae autoimmune panel), immunohistochemistry, flow cytometry and ELISA. Lung tissue of COVID-19 patients showed increased CD48 mRNA expression and infiltration of CD48+ lymphocytes. In the peripheral blood, mCD48 was considerably increased on all evaluated cells, and additionally, sCD48 levels were significantly higher in COVID-19 patients independently of disease severity. Considering the alterations of mCD48 and sCD48, a specific role for CD48 in COVID-19 can be assumed, suggesting it as a potential target for therapy.","version":"1.1","doi":"10.1101/2022.03.18.484843","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.484817","pub_date":"2022-3-18","title":"Potent and specific human monoclonal antibodies against SARS-CoV-2 Omicron variant by rapid mRNA immunization of humanized mice","abstract":"The Omicron variant (B.1.1.529) of SARS-CoV-2 rapidly becomes dominant globally. Its extensive mutations confer severe efficacy reduction to most of existing antibodies or vaccines. Here, we developed RAMIHM, a highly efficient strategy to generate fully human monoclonal antibodies (mAbs), directly applied it with Omicron-mRNA immunization, and isolated three potent and specific clones against Omicron. Rapid mRNA immunization elicited strong anti-Omicron antibody response in humanized mice, along with broader anti-coronavirus activity. Customized single cell BCR sequencing mapped the clonal repertoires. Top-ranked clones collectively from peripheral blood, plasma B and memory B cell populations showed high rate of Omicron-specificity (93.3%) from RAMIHM-scBCRseq. Clone-screening identified three highly potent neutralizing antibodies that have low nanomolar affinity for Omicron RBD, and low ng/mL level IC50 in neutralization, more potent than majority of currently approved or authorized clinical RBD-targeting mAbs. These lead mAbs are fully human and ready for downstream IND-enabling and/or translational studies.","version":"1.1","doi":"10.1101/2022.03.17.484817","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.16.468802","pub_date":"2022-3-18","title":"NUDT18 catalyzes the hydrolysis of active metabolites of the antivirals Remdesivir, Ribavirin and Molnupiravir","abstract":"Remdesivir and Molnupiravir have gained considerable interest due to their activity against SARS-CoV-2. Cellular hydrolysis of their active triphosphate forms, Remdesivir-TP and Molnupiravir-TP, would decrease drug efficiency. We therefore tested Remdesivir-TP as a substrate against a panel of human hydrolases and found that NUDT18 catalyzes the hydrolysis of Remdesivir-TP. The kcat value of NUDT18 for Remdesivir-TP was determined to 2.6 s-1 and the Km value was 156 \u03bcM, suggesting that NUDT18 catalyzed hydrolysis of Remdesivir-TP occurs in cells. We demonstrate that the triphosphates of the antivirals Ribavirin and Molnupiravir are hydrolyzed by NUDT18, albeit with a lower efficiency compared to Remdesivir-TP. NUDT18 also hydrolyses the triphosphates of Sofosbuvir and Aciclovir although with significantly lower activity. These results suggest that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of Remdesivir, Molnupiravir and Ribavirin. NUDT18 is expressed in respiratory epithelial cells and may limit the antiviral efficacy of Remdesivir and Molnupiravir against SARS-CoV2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action.","version":"1.2","doi":"10.1101/2021.11.16.468802","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.04.483032","pub_date":"2022-3-18","title":"Chimeric mRNA based COVID-19 vaccine induces protective immunity against Omicron and Delta","abstract":"The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future.","version":"1.2","doi":"10.1101/2022.03.04.483032","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.07.483373","pub_date":"2022-3-17","title":"Extensive neutralization against SARS-CoV-2 variants elicited by Omicron-specific subunit vaccine booster","abstract":"The currently dominant variant of SARS-CoV-2 Omicron, carrying a great number of mutations, has been verified its strong capacity of immune escape in COVID-19 convalescents and vaccinated individuals. An increased risk of SARS-CoV-2 reinfection or breakthrough infection should be concerned. Here we reported higher humoral immune response elicited by Delta and Omicron variants after breaking through previous infection and cross-neutralization against VOCs, compared to the ancestral wild-type (WT) virus infection. To overcome the immune escape of Omicron, Omicron-specific vaccine was considered as a novel and potential strategy. Mouse models were used to verify whether Omicron-specific RBD subunit boost immune response by immunizing Omicron-RBD recombinant proteins. Three doses of Omicron-RBD immunization elicit comparable neutralizing antibody (NAb) titers with three doses of WT-RBD immunization, but the neutralizing activity was not cross-active. By contrast, two doses of WT-RBD with an Omicron-RBD booster increased the NAb geometric mean titers against Omicron by 9 folds. Moreover, an additional boost vaccination with Omicron-RBD protein could increase humoral immune response against both WT and current VOCs. These results suggest that the Omicron-specific subunit booster shows its advantages in the immune protection from both WT and current VOCs, and that SARS-CoV-2 vaccines administration using two or more virus lineages as antigens might improve the NAb response.","version":"1.2","doi":"10.1101/2022.03.07.483373","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.16.484099","pub_date":"2022-3-17","title":"The effect of waning on antibody levels and memory B cell recall following SARS-CoV-2 infection or vaccination","abstract":"As of March 2022, there have been over 450 million reported SARS-CoV-2 cases worldwide, and more than 4 billion people have received their primary series of a COVID-19 vaccine. In order to longitudinally track SARS-CoV-2 antibody levels in people after vaccination or infection, a large-scale COVID-19 sero-surveillance progam entitled SPARTA (SeroPrevalence and Respiratory Tract Assessment) was established early in the pandemic. Anti-RBD antibody levels were tracked in more than 1,000 people. There was no significant decrease in antibody levels during the first 14 months after infection in unvaccinated participants, however, significant waning of antibody levels was observed following vaccination, regardless of previous infection status. Moreover, participants who were pre-immune to SARS-CoV-2 prior to vaccination seroconverted to significantly higher antibody levels, and antibodies were maintained at significantly higher levels than in previously infected, unvaccinated participants. This pattern was entirely due to differences in the magnitude of the initial seroconversion event, and the rate of antibody waning was not significantly different based on the pre-immune status. Participants who received a third (booster) dose of an mRNA vaccine not only increased their anti-RBD antibody levels \u223c14-fold, but they also had \u223c3 times more anti-RBD antibodies compared to the peak of their antibody levels after receiving their primary vaccine series. In order to ascertain whether the presence of serum antibodies is important for long-term seroprotection, PBMCs from 13 participants who lost all detectable circulating antibodies after vaccination or infection were differentiated into memory cells in vitro. There was a significant recall of memory B cells in the absence of serum antibodies in 70% of the vaccinated participants, but not in any of the infected participants. Therefore, there is a strong connection between anti-RBD antibody levels and the effectiveness of memory B cell recall.","version":"1.1","doi":"10.1101/2022.03.16.484099","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.17.484640","pub_date":"2022-3-17","title":"Long-term T cell perturbations and waning antibody levels in individuals needing hospitalization for COVID-19","abstract":"COVID-19 is being extensively studied, and much remains unknown regarding the long-term consequences of the disease on immune cells. The different arms of the immune system are interlinked, with humoral responses and the production of high-affinity antibodies being largely dependent on T cell immunity. Here, we longitudinally explored the effect COVID-19 has on T cell populations and the virus-specific T cells, as well as neutralizing antibody responses, for 6-7 months following hospitalization. The CD8+ TEMRA and exhausted CD57+CD8+ T cells were markedly affected with elevated levels that lasted long into convalescence. Further, markers associated with T-cell activation were upregulated at the inclusion, and in the case of CD69+CD4+ T cells this lasted all through the study duration. The levels of T cells expressing negative immune checkpoint molecules were increased in COVID-19 patients and sustained for a prolonged duration following recovery. Within 2-3 weeks after symptom onset, all COVID-19 patients developed anti-nucleocapsid IgG and spike-neutralizing IgG as well as SARS-CoV-2-specific T cell responses. In addition, we found alterations in follicular T helper (TFH) cell populations, such as enhanced TFH-TH2 following recovery from COVID-19. Our study revealed significant and long-term alterations in T cell populations and key events associated with COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2022.03.17.484640","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.14.484208","pub_date":"2022-3-16","title":"Parsing the role of NSP1 in SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.","version":"1.2","doi":"10.1101/2022.03.14.484208","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484484","pub_date":"2022-3-16","title":"Allosteric binders of ACE2 are promising anti-SARS-CoV-2 agents","abstract":"The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for an acute treatment for the disease. We investigate whether compounds that bind the human ACE2 protein can interrupt SARS-CoV-2 replication without damaging ACE2\u2019s natural enzymatic function. Initial compounds were screened for binding to ACE2 but little interruption of ACE2 enzymatic activity. This set of compounds was extended by application of quantitative structure-activity analysis, which resulted in 512 virtual hits for further confirmatory screening. A subsequent SARS-CoV-2 replication assay revealed that five of these compounds inhibit SARS-CoV-2 replication in human cells. Further effort is required to completely determine the antiviral mechanism of these compounds, but they serve as a strong starting point for both development of acute treatments for COVID-19 and research into the mechanism of infection. TOC Graphic: Overall study design.","version":"1.1","doi":"10.1101/2022.03.15.484484","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429146","pub_date":"2022-3-16","title":"CovRadar: Continuously tracking and filtering SARS-CoV-2 mutations for molecular surveillance","abstract":"The SARS-CoV-2 pandemic underlined the importance of molecular surveillance to track the evolution of the virus and inform public health interventions. Fast analysis, easy visualization and convenient filtering of the latest virus sequences are essential for this purpose. However, access to computational resources, the lack of bioinformatics expertise, and the sheer volume of sequences in public databases complicate surveillance efforts. CovRadar combines an analytical pipeline and a web application designed for the molecular surveillance of the spike gene of SARS-CoV-2, an important vaccine target. The intuitive web front-end focuses on mutations rather than viral lineages and provides easy access to frequencies and spatio-temporal distributions from global sample collections. The data is regularly updated based on a scalable and reproducible analytical back-end. With this platform, we aim to give users, those with or without bioinformatics skills or sufficient computational resources, the possibility to track and explore mutational changes in the SARS-CoV-2 spike gene and to filter, download, and further analyze data that meet their questions and needs. Advanced computational users have the ability to apply the analytical pipeline and data visualization methods locally on their own data. CovRadar is freely accessible at https://covradar.net, source code is available at https://gitlab.com/dacs-hpi/covradar.","version":"1.3","doi":"10.1101/2021.02.03.429146","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.479519","pub_date":"2022-3-16","title":"Aerosol Formation During Processing of Potentially Infectious Samples on Roche Immunochemistry Analyzers (cobas e analyzers) and in an End-to-End Laboratory Workflow to Model SARS-CoV-2 Infection Risk for Laboratory Operators","abstract":"This study assessed formation of potentially infectious aerosols during processing of infectious samples in a real-world laboratory setting, which could then be applied in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This two-part study assessed aerosol formation when using cobas e analyzers only and in an end-to-end laboratory workflow. To estimate aerosol formation, recombinant hepatitis B surface antigen (HBsAg) was used as a surrogate marker for infectious virus particles to evaluate the potential risk of SARS-CoV-2 infection to laboratory operators. Using the HBsAg model, air sampling was performed at different positions around the cobas e analyzers and in four scenarios reflecting critical handling and/or transport locations in an end-to-end laboratory workflow. Aerosol formation of HBsAg was quantified using the Elecsys\u00ae HBsAg II quant II assay. The model was then applied to a SARS-CoV-2 context using SARS-CoV-2 infection-specific parameters to calculate viral RNA copies. Following application to SARS-CoV-2, the mean HBsAg uptake per hour when recalculated into viral RNA copies was 1.9 viral RNA copies across the cobas e analyzers and 0.87 viral RNA copies across all tested scenarios in an end-to-end laboratory workflow. This corresponds to a maximum aspiration rate of <16 viral RNA copies during an 8-hour shift when using cobas e analyzers and/or in an end-to-end laboratory workflow. The low production of marker-containing aerosol when using cobas e analyzers and in an end-to-end laboratory workflow is consistent with a remote risk of laboratory-acquired SARS-CoV-2 infection for laboratory operators. This study investigated the formation of potentially infectious aerosols during processing of infectious samples in a model using hepatitis B surface antigen (HBsAg) as a marker for infectious virus particles. The risk to laboratory operators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was then inferred. Air sampling was performed around cobas e analyzers and in an end-to-end laboratory workflow, after which HBsAg was quantified and applied to SARS-CoV-2 using SARS-CoV-2 infection-specific parameters. The maximum aspiration rate of <16 viral RNA copies/8-hour shift, when applied to a SARS-CoV-2 context, poses a remote risk of SARS-CoV-2 infection to laboratory operators.","version":"1.2","doi":"10.1101/2022.02.08.479519","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484542","pub_date":"2022-3-16","title":"Omicron BA.1 and BA.2 neutralizing activity elicited by a comprehensive panel of human vaccines","abstract":"The SARS-CoV-2 Omicron variant of concern comprises three sublineages designated BA.1, BA.2, and BA.3, with BA.2 steadily replacing the globally dominant BA.1. We show that the large number of BA.1 and BA.2 spike mutations severely dampen plasma neutralizing activity elicited by infection or seven clinical vaccines, with cross-neutralization of BA.2 being consistently more potent than that of BA.1, independent of the vaccine platform and number of doses. Although mRNA vaccines induced the greatest magnitude of Omicron BA.1 and BA.2 plasma neutralizing activity, administration of a booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1 and BA.2 across all vaccines evaluated. Our data suggest that although BA.1 and BA.2 evade polyclonal neutralizing antibody responses, current vaccine boosting regimens may provide sufficient protection against Omicron-induced disease.","version":"1.1","doi":"10.1101/2022.03.15.484542","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484467","pub_date":"2022-3-16","title":"A conserved immune trajectory of recovery in hospitalized COVID-19 patients","abstract":"Many studies have provided insights into the immune response to COVID-19; however, little is known about the immunological changes and immune signaling occurring during COVID-19 resolution. Individual heterogeneity and variable disease resolution timelines obscure unifying immune characteristics. Here, we collected and profiled >200 longitudinal peripheral blood samples from patients hospitalized with COVID-19, with other respiratory infections, and healthy individuals, using mass cytometry to measure immune cells and signaling states at single cell resolution. COVID-19 patients showed a unique immune composition and an early, coordinated and elevated immune cell signaling profile, which correlated with early hospital discharge. Intra-patient time course analysis tied to clinically relevant events of recovery revealed a conserved set of immunological processes that accompany, and are unique to, disease resolution and discharge. This immunological process, together with additional changes in CD4 regulatory T cells and basophils, accompanies recovery from respiratory failure and is associated with better clinical outcomes at the time of admission. Our work elucidates the biological timeline of immune recovery from COVID-19 and provides insights into the fundamental processes of COVID-19 resolution in hospitalized patients.","version":"1.1","doi":"10.1101/2022.03.15.484467","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484439","pub_date":"2022-3-15","title":"Neutrophils initiate the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters","abstract":"The loss of smell related to SARS-CoV-2 infection is one of the most prevalent symptoms of COVID-19. It is now clear that this symptom is related to the massive infection by SARS-CoV-2 of the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamster, we show here that while apoptosis remains at a low level in damaged infected epithelium, the latter is invaded by innate immunity cells. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we reduced the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease of SARS-CoV-2 infection levels in the nasal cavity. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus.","version":"1.1","doi":"10.1101/2022.03.15.484439","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484274","pub_date":"2022-3-15","title":"Vascular dysregulation following SARS-CoV-2 infection involves integrin signalling through a VE-Cadherin mediated pathway","abstract":"The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins have emerged as possible receptor candidates for SARS-CoV-2, and their complex intracellular signalling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation through hijacking integrin \u03b1V\u03b23. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin \u03b1V\u03b23 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein \u2013 through its RGD motif \u2013 binds to \u03b1V\u03b23 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.","version":"1.1","doi":"10.1101/2022.03.15.484274","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484379","pub_date":"2022-3-15","title":"Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model","abstract":"Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.","version":"1.1","doi":"10.1101/2022.03.15.484379","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484448","pub_date":"2022-3-15","title":"Pulmonary lesions following inoculation with the SARS-CoV-2 Omicron BA.1 (B.1.1.529) variant in Syrian golden hamsters","abstract":"The Omicron BA.1 (B.1.1.529) SARS-CoV-2 variant is characterized by a high number of mutations in the viral genome, associated with immune-escape and increased viral spread. It remains unclear whether milder COVID-19 disease progression observed after infection with Omicron BA.1 in humans is due to reduced pathogenicity of the virus or due to pre-existing immunity from vaccination or previous infection. Here, we inoculated hamsters with Omicron BA.1 to evaluate pathogenicity and kinetics of viral shedding, compared to Delta (B.1.617.2) and to animals re-challenged with Omicron BA.1 after previous SARS-CoV-2 614G infection. Omicron BA.1 infected animals showed reduced clinical signs, pathological changes, and viral shedding, compared to Delta-infected animals, but still showed gross- and histopathological evidence of pneumonia. Pre-existing immunity reduced viral shedding and protected against pneumonia. Our data indicate that the observed decrease of disease severity is in part due to intrinsic properties of the Omicron BA.1 variant.","version":"1.1","doi":"10.1101/2022.03.15.484448","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484018","pub_date":"2022-3-15","title":"Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation","abstract":"COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Gu\u00e9rin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.","version":"1.1","doi":"10.1101/2022.03.15.484018","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.15.484421","pub_date":"2022-3-15","title":"Structural basis of Omicron immune evasion: A comparative computational study of Spike protein-Antibody interaction","abstract":"The COVID-19 pandemic has caused more than 424 million infections and 5.9 million deaths so far. The vaccines used against SARS-COV-2 by now have been able to develop some neutralising antibodies in the vaccinated human population and slow down the infection rate. The effectiveness of the vaccines has been challenged by the emergence of the new strains with numerous mutations in the spike (S) protein of SARS-CoV-2. Since S protein is the major immunogenic protein of the virus and also contains Receptor Binding Domain (RBD) that interacts with the human Angiotensin-Converting Enzyme 2 (ACE2) receptors, any mutations in this region should affect the neutralisation potential of the antibodies leading to the immune evasion. Several variants of concern (VOC) of the virus have emerged so far. Among them, the most critical are Delta (B.1.617.2), and recently reported Omicron (B. 1.1.529) which have acquired a lot of mutations in the spike protein. We have mapped those mutations on the modelled RBD and evaluated the binding affinities of various human antibodies with it. Docking and molecular dynamics simulation studies have been used to explore the effect of the mutations on the structure of the RBD and the RBD-antibody interaction. The analysis shows that the mutations mostly at the interface of a nearby region lower the binding affinity of the antibody by ten to forty per cent, with a downfall in the number of interactions formed as a whole and therefore, it implies the generation of immune escape variants. Notable mutations and their effect was characterised by performing various analyses that explain the structural basis of antibody efficacy in Delta and a compromised neutralisation effect for the Omicron variant. Our results pave the way for robust vaccine design that can be effective for many variants. The research study utilises comparative docking and MD simulations analyses to illustrate how mutations in delta and omicron variants affect the binding of antibodies to the spike receptor binding domain (RBD) of SARS CoV-2.","version":"1.1","doi":"10.1101/2022.03.15.484421","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.13.484172","pub_date":"2022-3-14","title":"Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide Coronavirus Disease 2019 (COVID-19) pandemic. Despite high efficacy of the authorized vaccines, protection against the surging variants of concern (VoC) was less robust. Live-attenuated vaccines (LAV) have been shown to elicit robust and long-term protection by induction of host innate and adaptive immune responses. We sought to develop a COVID-19 LAV by generating 3 double open reading frame (ORF)-deficient recombinant (r)SARS-CoV-2 simultaneously lacking two accessory open reading frame (ORF) proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). Here, we report that these double ORF-deficient rSARS-CoV-2 have slower replication kinetics and reduced fitness in cultured cells as compared to their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2 showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against different SARS-CoV-2 VoC, and also activated viral component-specific T-cell responses. Notably, the double ORF-deficient rSARS-CoV-2 were able to protect, as determined by inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2. Collectively, our results demonstrate the feasibility to implement these double ORF-deficient rSARS-CoV-2 as safe, stable, immunogenic and protective LAV for the prevention of SARS-CoV-2 infection and associated COVID-19 disease.","version":"1.1","doi":"10.1101/2022.03.13.484172","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.13.484123","pub_date":"2022-3-14","title":"Development of a ferritin-based nanoparticle vaccine against the SARS-CoV-2 Omicron variant","abstract":"A new SARS-CoV-2 variant named Omicron (B.1.1.529) discovered initially in South Africa has recently been proposed as a variant of concern (VOC) by the World Health Organization, because of its high transmissibility and resistance to current vaccines and therapeutic antibodies. Therefore, rapid development of vaccines against prevalent variants including Omicron is urgently needed for COVID-19 prevention. Here, we designed a self-assembling ferritin-based nanoparticle (FNP) vaccine against the SARS-CoV-2 Omicron variant. The purified Fc-RBDOmicron automatically formed a dimer depending on the nature of the Fc tag, thus assembling onto the nanoparticles by the Fc-protein A tag interaction (FNP-Fc-RBDOmicron). The results of hACE2-transgenic mice immunization showed that SARS-CoV-2 Omicron RBD-specific IgG titer induced by FNP-Fc-RBDOmicron was much higher than that by Fc-RBDOmicron. Consistently, the sera showed a higher neutralizing activity against SARS-CoV-2 Omicron BA.1 and BA.2 in the FNP-Fc-RBDOmicron immunized mice, indicating that immunization of a self-assembling ferritin-based nanoparticle vaccine offers a robust humoral immune response against Omicron variants. This study offers a great potential for the quick response of the emerging SARS-CoV-2 variants and affords versatility to develop universal vaccines against other emerging and reemerging coronaviruses in the future.","version":"1.1","doi":"10.1101/2022.03.13.484123","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.11.483867","pub_date":"2022-3-14","title":"Accelerating PERx Reaction Enables Covalent Nanobodies for Potent Neutralization of SARS-Cov-2 and Variants","abstract":"The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here we developed covalent nanobodies capable of binding with SARS-CoV-2 spike protein irreversibly via proximity-enabled reactive therapeutic (PERx) mechanism. A novel latent bioreactive amino acid FFY was designed and genetically encoded into nanobodies to accelerate PERx reaction rate. After covalent engineering, nanobodies binding with the Spike in the down state, but not in the up state, were discovered to possess striking enhancement in inhibiting viral infection. In comparison with the noncovalent wildtype nanobody, the FFY-incorporated covalent nanobody neutralized both authentic SARS-CoV-2 and its Alpha and Delta variants with potency drastically increased over tens of folds. This PERx-enabled covalent nanobody strategy and uncovered insights on potency increase can be valuable to developing effective therapeutics for various viral infections.","version":"1.1","doi":"10.1101/2022.03.11.483867","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.13.484129","pub_date":"2022-3-14","title":"Tracking SARS-CoV-2 Omicron diverse spike gene mutations identifies multiple inter-variant recombination events","abstract":"The current pandemic of COVID-19 is fueled by more infectious emergent Omicron variants. Ongoing concerns of emergent variants include possible recombinants, as genome recombination is an important evolutionary mechanism for the emergence and re-emergence of human viral pathogens. Although recombination events among SARS-CoV-1 and MERS-CoV were well-documented, it has been difficult to detect the recombination signatures in SARS-CoV-2 variants due to their high degree of sequence similarity. In this study, we identified diverse recombination events between two Omicron major subvariants (BA.1 and BA.2) and other variants of concern (VOCs) and variants of interest (VOIs), suggesting that co-infection and subsequent genome recombination play important roles in the ongoing evolution of SARS-CoV-2. Through scanning high-quality completed Omicron spike gene sequences, eighteen core mutations of BA.1 variants (frequency >99%) were identified (eight in NTD, five near the S1/S2 cleavage site, and five in S2). BA.2 variants share three additional amino acid deletions with the Alpha variants. BA.1 subvariants share nine common amino acid mutations (three more than BA.2) in the spike protein with most VOCs, suggesting a possible recombination origin of Omicron from these VOCs. There are three more Alpha-related mutations (del69-70, del144) in BA.1 than BA.2, and therefore BA.1 may be phylogenetically closer to the Alpha variant. Revertant mutations are found in some dominant mutations (frequency >95%) in the BA.1 subvariant. Most notably, multiple additional amino acid mutations in the Delta spike protein were also identified in the recently emerged Omicron isolates, which implied possible recombination events occurred between the Omicron and Delta variants during the on-going pandemic. Monitoring the evolving SARS-CoV-2 genomes especially for recombination is critically important for recognition of abrupt changes to viral attributes including its epitopes which may call for vaccine modifications.","version":"1.1","doi":"10.1101/2022.03.13.484129","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.11.484006","pub_date":"2022-3-14","title":"Dual inhibition of vacuolar ATPase and TMPRSS2 is required for complete blockade of SARS-CoV-2 entry into cells","abstract":"An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using non-specific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.","version":"1.1","doi":"10.1101/2022.03.11.484006","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.12.484088","pub_date":"2022-3-14","title":"The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes","abstract":"Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD\u2019s class 1 and class 2 epitopes, including sites 417, 478, and 484\u2013486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.","version":"1.1","doi":"10.1101/2022.03.12.484088","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.13.484037","pub_date":"2022-3-14","title":"A broad and potent neutralization epitope in SARS-related coronaviruses","abstract":"Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.","version":"1.1","doi":"10.1101/2022.03.13.484037","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.13.484191","pub_date":"2022-3-14","title":"Molecular docking between human TMPRSS2 and the serine protease Kunitz-type inhibitor rBmTI-A","abstract":"SARS-CoV-2 entrance into host cells is dependent of ACE2 receptor and viral protein S initiation by serine protease TMPRSS2. Cleavage of coronavirus protein S at the junctions Arg685/Ser686 and Arg815/Ser816 leads to the production of the S1/S2 and S2\u2019 fragments needed for the fusion of viral and cell membranes. Studying and identifying serine protease inhibitors is an important step towards the development of candidate drugs to prevent SARS-CoV-2 infection. It has already been stablished that camostat mesylate, a serine protease inhibitor, is capable of blocking TMPRSS2 activity and prevent SARS-CoV-2 entrance into host cells. In this work, the interaction between the two domains of Kunitz-type serine protease inhibitor rBmTI-A and TMPRSS2 was studied through molecular docking. rBmTI-A domain 2 (P1 site Leu84) had the best complex results with predicted binding affinity of -12 Kcal.mol-1 and predicted dissociation constant at 25\u00b0C of 1.6 nM. The results suggest that rBmTI-A is capable of binding TMPRSS2 cleavage site at the junction Arg815/Ser816 using essentially the same residues that camostat mesylate.","version":"1.1","doi":"10.1101/2022.03.13.484191","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.13.484180","pub_date":"2022-3-14","title":"Peritoneal M2 macrophage-derived extracellular vesicles as natural multi-target nanotherapeutics to attenuate cytokine storm after severe infections","abstract":"Cytokine storm is a primary cause for multiple organ damage and death after severe infections, such as SARS-CoV-2. However, current single cytokine-targeted strategies display limited therapeutic efficacy. Here, we report that peritoneal M2 macrophages-derived extracellular vesicles (M2-EVs) are multi-target nanotherapeutics to resolve cytokine storm. In detail, primary peritoneal M2 macrophages exhibited superior anti-inflammatory potential than immobilized cell lines. Systemically administrated M2-EVs entered major organs and were taken up by phagocytes (e.g., macrophages). M2-EVs treatment effectively reduced excessive cytokine (e.g., TNF-\u03b1 and IL-6) release in vitro and in vivo, thereby attenuated oxidative stress and multiple organ (lung, liver, spleen and kidney) damage in endotoxin-induced cytokine storm. Moreover, M2-EVs simultaneously inhibited multiple key proinflammatory pathways (e.g., NF-\u03baB, JAK-STAT and p38 MAPK) by regulating complex miRNA-gene and gene-gene networks, and this effect was collectively mediated by many functional cargos (miRNAs and proteins) in EVs. In addition to the direct anti-inflammatory role, human peritoneal M2-EVs expressed angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2 spike protein, and thus could serve as nanodecoys to prevent SARS-CoV-2 pseudovirus infection in vitro. As cell-derived nanomaterials, the therapeutic index of M2-EVs can be further improved by genetic/chemical modification or loading with specific drugs. This study highlights that peritoneal M2-EVs are promising multifunctional nanotherapeutics to attenuate infectious diseases-related cytokine storm.","version":"1.1","doi":"10.1101/2022.03.13.484180","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477105","pub_date":"2022-3-14","title":"In Silico Analysis Of The Effects Of Omicron Spike Amino Acid Changes On The Interactions With Human ACE2 Receptor And Structurally Characterized Complexes With Human Antibodies","abstract":"The new SARS-CoV-2 variant Omicron is characterised, among others, by more than 30 amino acid changes (including 4 deletions and 1 insertion) occurring on the spike glycoprotein. We report a comprehensive analysis of the effects of the Omicron spike amino acid changes in the interaction with human ACE2 receptor or with human antibodies, obtained by analysing the publicly available resolved 3D structures. Our analysis predicts that amino acid changes occurring on amino acids interacting with the ACE2 receptor may increase Omicron transmissibility. The interactions of Omicron spike with human antibodies can be both negatively and positively affected by amino acid changes, with a predicted total loss of interactions only in few complexes. We believe that such an approach can be used to better understand SARS-CoV-2 transmissibility, detectability, and epidemiology, especially when extended to other than spike proteins.","version":"1.2","doi":"10.1101/2022.01.20.477105","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.09.22272113","pub_date":"2022-03-12","title":"Evidence for SARS-CoV-2 Delta and Omicron co-infections and recombination","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Between November 2021 and February 2022, SARS-CoV-2 Delta and Omicron variants co-circulated in the United States, allowing for co-infections and possible recombination events. We sequenced 29,719 positive samples during this period and analyzed the presence and fraction of reads supporting mutations specific to either the Delta or Omicron variant. We identified 18 co-infections, one of which displayed evidence of a low Delta-Omicron recombinant viral population. We also identified two independent cases of infection by a Delta-Omicron recombinant virus, where 100% of the viral RNA came from one clonal recombinant. In the three cases, the 5\u2019-end of the viral genome was from the Delta genome, and the 3\u2019-end from Omicron including the majority of the spike protein gene, though the breakpoints were different. Delta-Omicron recombinant viruses were rare, and there is currently no evidence that Delta-Omicron recombinant viruses are more transmissible between hosts compared to the circulating Omicron lineages.</jats:p>","version":null,"doi":"10.1101/2022.03.09.22272113","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.11.483836","pub_date":"2022-3-11","title":"Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development","abstract":"The SARS-CoV-2 non-structural protein 14 (NSP14) is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both enzymatic activities appear to be essential for the viral life cycle and thus may be targeted for anti-viral therapeutics. NSP14 forms a stable complex with the SARS-CoV-2 zinc binding protein NSP10, and this interaction greatly enhances the nuclease but not the methyltransferase activity. In this study, we have determined the crystal structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 \u00c5 resolution. Comparisons of this structure with the structure of NSP14/NSP10 complexes solved to date reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including significant movements and helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. Conformational changes are also seen in the MTase active site within a SAM/SAH interacting loop that plays a key role in viral mRNA capping. We have also determined the structure of NSP14 in complex with cap analogue 7MeGpppG, offering new insights into MTase enzymatic activity. We have used our high resolution crystals to perform X-ray fragment screening of NSP14, revealing 72 hits bound to potential sites of inhibition of the ExoN and MTase domains. These structures serve as excellent starting point tools for structure guided development and optimization of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.","version":"1.1","doi":"10.1101/2022.03.11.483836","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.02.482662","pub_date":"2022-3-11","title":"Comparative infection and pathogenesis of SARS-CoV-2 Omicron and Delta variants in aged and young Syrian hamsters","abstract":"Coronavirus disease 2019 continues to batter the world with the unceasing introduction of new variants of the causative virus, SARS-CoV-2. In order to understand differences in disease caused by variants of concern and to develop variant-specific vaccines, suitable small animal models are required that mimic disease progression in humans at various stages of life. In this study, we compared the dynamics of infection with two SARS-CoV-2 variants of concern (Delta and Omicron) in aged (>1 year 3 months old) and young (<5 weeks old) Syrian hamsters (Mesocricetus auratus). We show that no weight loss occurred in Omicron infected groups regardless of age, while infection with the Delta variant caused weight loss of up to 10% by day 7 post-infection with slower and incomplete recovery in the aged group. Omicron replicated to similar levels as Delta in the lungs, trachea and nasal turbinates, with no significant differences in the tissue viral loads of aged versus young animals for either variant. In contrast to rare necrosis observed in Omicron-infected animals regardless of age, severe necrosis was observed in the olfactory epithelium in Delta-infected animals. Omicron infection also resulted in mild pulmonary disease in both young and aged animals compared to the moderate acute necrotizing bronchointerstitial pneumonia seen in Delta-infected animals. These results suggest that Omicron infection results in an attenuated clinical disease outlook in Syrian hamsters compared to infection with the Delta variant irrespective of age.","version":"1.2","doi":"10.1101/2022.03.02.482662","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470440","pub_date":"2022-3-11","title":"Heteologous saRNA-Prime, DNA Dual-Antigen-Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies","abstract":"We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nano-lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AdS+N boost of an SASA S prime particularly enhanced both CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strain. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against emerging SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.11.29.470440","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.11.483934","pub_date":"2022-3-11","title":"Increased receptor affinity and reduced recognition by specific antibodies contribute to immune escape of SARS-CoV-2 variant Omicron","abstract":"In this report, we mechanistically reveal how the Variant of Concern (VOC) SARS-CoV-2 Omicron (B.1.1.529) escapes neutralizing antibody responses, by characterization of this variant, and wildtype Wuhan and Delta variant (B.1.617.2). Convalescent sera as well as sera obtained from participants who received two or three doses of mRNA vaccines (Moderna-mRNA-1273\u00ae or Pfizer-BNT162b2\u00ae) were used for comparison in this study. Our data demonstrate that both the Delta as well as Omicron variants exhibit higher affinity for the receptor ACE2, facilitating infection and causing antibody escape by receptor affinity (affinity escape), due to reduced ability of antibodies to compete with RBD-receptor interaction and virus neutralization. In contrast, only Omicron but not Delta variant escaped antibody recognition, most likely because only Omicron exhibit the mutation at position E484 associated with reduced recognition, resulting in further reduced neutralization (specificity escape). Nevertheless, the immunizations with RNA based vaccines resulted in marked viral neutralization in vitro for all strains, compatible with the fact that Omicron is still largely susceptible to vaccination-induced antibodies, despite affinity- and specificity escape.","version":"1.1","doi":"10.1101/2022.03.11.483934","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.11.483948","pub_date":"2022-3-11","title":"Alveolar regeneration following viral infection is independent of tuft cells","abstract":"Severe injuries following viral infection cause lung epithelial destruction with the presence of ectopic basal progenitor cells (EBCs), although the exact function of EBCs remains controversial. We and others previously showed the presence of ectopic tuft cells in the disrupted alveolar region following severe influenza infection. Here, we further revealed that the ectopic tuft cells are derived from EBCs. This process is amplified by Wnt signaling inhibition but suppressed by Notch inhibition. Further analysis revealed that p63-CreER labeled population de novo arising during regeneration includes alveolar epithelial cells when Tamoxifen was administrated after viral infection. The generation of the p63-CreER labeled alveolar cells is independent of tuft cells, demonstrating segregated differentiation paths of EBCs in lung repair. EBCs and ectopic tuft cells can also be found in the lung parenchyma post SARS-CoV-2 infection, suggesting a similar response to severe injuries in humans.","version":"1.1","doi":"10.1101/2022.03.11.483948","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.480950","pub_date":"2022-3-11","title":"A high-throughput yeast display approach to profile pathogen proteomes for MHC-II binding","abstract":"T cells play a critical role in the adaptive immune response, recognizing peptide antigens presented on the cell surface by Major Histocompatibility Complex (MHC) proteins. While assessing peptides for MHC binding is an important component of probing these interactions, traditional assays for testing peptides of interest for MHC binding are limited in throughput. Here we present a yeast display-based platform for assessing the binding of tens of thousands of user-defined peptides in a high throughput manner. We apply this approach to assess a tiled library covering the SARS-CoV-2 proteome and four dengue virus serotypes for binding to human class II MHCs, including HLA-DR401, -DR402, and -DR404. This approach identifies binders missed by computational prediction, highlighting the potential for systemic computational errors given even state-of-the-art training data, and underlines design considerations for epitope identification experiments. This platform serves as a framework for examining relationships between viral conservation and MHC binding, and can be used to identify potentially high-interest peptide binders from viral proteins. These results demonstrate the utility of this approach for determining high-confidence peptide-MHC binding.","version":"1.2","doi":"10.1101/2022.02.22.480950","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.09.483704","pub_date":"2022-3-10","title":"Dissecting the Role of the Human Microbiome in COVID-19 via Metagenome-assembled Genomes","abstract":"Coronavirus disease 2019 (COVID-19), primarily a respiratory disease caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is often accompanied by gastrointestinal symptoms. However, little is known about the relation between the human microbiome and COVID-19, largely due to the fact that previous studies fail to provide high taxonomic resolution to identify microbes that likely interact with SARS-CoV-2 infection. Here we used whole-metagenome shotgun sequencing data together with assembly and binning strategies to reconstruct metagenome-assembled genomes (MAGs) from a total of 514 nasopharyngeal and fecal samples of patients with COVID-19 and controls. We reconstructed a total of 11,584 medium-and high-quality microbial MAGs and obtained 5,403 non-redundant MAGs (nrMAGs) with strain-level resolution. We found that, thanks to the high taxonomic resolution of nrMAGs, the gut microbiome signatures can accurately distinguish COVID-19 cases from healthy controls and predict the progression of COVID-19. Moreover, we identified a set of nrMAGs with a putative causal role in the clinical manifestations of COVID-19 and revealed their functional pathways that potentially interact with SARS-CoV-2 infection. The presented results highlight the importance of incorporating the human gut microbiome in our understanding of SARS-CoV-2 infection and disease progression. The genomic content of nrMAGs presented here has the potential to inform microbiome-based therapeutic developments for COVID-19 progression and post-COVID conditions.","version":"1.1","doi":"10.1101/2022.03.09.483704","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.10.483726","pub_date":"2022-3-10","title":"More or less deadly? A mathematical model that predicts SARS-CoV-2 evolutionary direction","abstract":"SARS-CoV-2 has caused tremendous deaths world wild. It is of great value to predict the evolutionary direction of SARS-CoV-2. In this paper, we proposed a novel mathematical model that could predict the evolutionary trend of SARS-CoV-2. We focus on the mutational effects on viral assembly capacity. A robust coarse-grained mathematical model is constructed to simulate the virus dynamics in the host body. Both virulence and transmissibility can be quantified in this model. The relationship between virulence and transmissibility can be simulated. A delicate equilibrium point that optimizing the transmissibility can be numerically obtained. Based on this model, we predict the virulence of SARS-CoV-2 might further decrease, accompanied by an enhancement of transmissibility. However, this trend is not continuous; its virulence will not disappear but remains at a relatively stable range. We can also explain the cross-species transmission phenomenon of certain RNA virus based on this model. A small-scale model which simulates the virus packing process is also proposed. It can be explained why a small number of mutations would lead to a significant divergence in clinical performance, both in the overall particle formation quantity and virulence. This research provides a mathematical attempt to elucidate the evolutionary driving force in RNA virus evolution.","version":"1.1","doi":"10.1101/2022.03.10.483726","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.09.483703","pub_date":"2022-3-10","title":"SARS-CoV-2 Omicron variant is more stable than the ancestral strain on various surfaces","abstract":"The Omicron BA.1 SARS-CoV-2 variant of concern spreads quickly around the world and outcompetes other circulating strains. We examined the stability of this SARS-CoV-2 variant on various surfaces and revealed that the Omicron variant is more stable than its ancestral strain on smooth and porous surfaces.","version":"1.1","doi":"10.1101/2022.03.09.483703","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479349","pub_date":"2022-3-10","title":"Omicron BA.2 specifically evades broad sarbecovirus neutralizing antibodies","abstract":"Omicron sub-lineage BA.2 has rapidly surged globally, accounting for over 60% of recent SARS-CoV-2 infections. Newly acquired RBD mutations and high transmission advantage over BA.1 urge the investigation of BA.2\u2019s immune evasion capability. Here, we show that BA.2 causes strong neutralization resistance, comparable to BA.1, in vaccinated individuals\u2019 plasma. However, BA.2 displays more severe antibody evasion in BA.1 convalescents, and most prominently, in vaccinated SARS convalescents\u2019 plasma, suggesting a substantial antigenicity difference between BA.2 and BA.1. To specify, we determined the escaping mutation profiles of 714 SARS-CoV-2 RBD neutralizing antibodies, including 241 broad sarbecovirus neutralizing antibodies isolated from SARS convalescents, and measured their neutralization efficacy against BA.1, BA.1.1, BA.2. Importantly, BA.2 specifically induces large-scale escape of BA.1/BA.1.1-effective broad sarbecovirus neutralizing antibodies via novel mutations T376A, D405N, and R408S. These sites were highly conserved across sarbecoviruses, suggesting that Omicron BA.2 arose from immune pressure selection instead of zoonotic spillover. Moreover, BA.2 reduces the efficacy of S309 (Sotrovimab) and broad sarbecovirus neutralizing antibodies targeting the similar epitope region, including BD55-5840. Structural comparisons of BD55-5840 in complexes with BA.1 and BA.2 spike suggest that BA.2 could hinder antibody binding through S371F-induced N343-glycan displacement. Intriguingly, the absence of G446S mutation in BA.2 enabled a proportion of 440-449 linear epitope targeting antibodies to retain neutralizing efficacy, including COV2-2130 (Cilgavimab). Together, we showed that BA.2 exhibits distinct antigenicity compared to BA.1 and provided a comprehensive profile of SARS-CoV-2 antibody escaping mutations. Our study offers critical insights into the humoral immune evading mechanism of current and future variants.","version":"1.3","doi":"10.1101/2022.02.07.479349","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.09.483635","pub_date":"2022-3-09","title":"Nucleocapsid-specific humoral responses improve the control of SARS-CoV-2","abstract":"The spike protein of SARS-CoV-2 is a critical antigen present in all approved SARS-CoV-2 vaccines. This surface viral protein is also the target for all monoclonal antibody therapies, but it is unclear whether antibodies targeting other viral proteins can also improve protection against COVID-19. Here, we interrogate whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine, and then transferred sera from these mice into na\u00efve mice. On the next day, the recipient mice were challenged intranasally with SARS-CoV-2 to evaluate whether nucleocapsid-specific humoral responses affect viral control. Interestingly, mice that received nucleocapsid-specific sera exhibited enhanced control of a SARS-CoV-2 infection. These findings provide the first demonstration that humoral responses specific to an internal coronavirus protein can help clear infection, warranting the inclusion of other viral antigens in next-generation SARS-CoV-2 vaccines and providing a rationale for the clinical evaluation of nucleocapsid-specific monoclonals to treat COVID-19. A SARS-CoV-2 nucleocapsid vaccine elicits robust nucleocapsid-specific antibody responses. This nucleocapsid vaccine generates memory B cells (MBC). Nucleocapsid-specific humoral responses do not prevent SARS-CoV-2 infection. Nucleocapsid-specific humoral responses help control a SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.03.09.483635","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.08.483569","pub_date":"2022-3-09","title":"Modeling within-host and aerosol dynamics of SARS-CoV-2: the relationship with infectiousness","abstract":"The relationship between transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the amount of virus present in the proximity of a susceptible host is not understood. Here, we developed a within-host and aerosol mathematical model and used it to determine the relationship between viral kinetics in the upper respiratory track, viral kinetics in the aerosols, and new transmissions in golden hamsters challenged with SARS-CoV-2. We determined that infectious virus shedding early in infection correlates with transmission events, shedding of infectious virus diminishes late in the infection, and high viral RNA levels late in the infection is a poor indicator of transmission. We further showed that viral infectiousness increases in a density dependent manner with viral RNA and that their relative ratio is time-dependent. Such information is useful for designing interventions. Quantifying the relationship between SARS-CoV-2 dynamics in upper respiratory tract and in aerosols is key to understanding SARS-CoV-2 transmission and evaluating intervention strategies. Of particular interest is the link between the viral RNA measured by PCR and a subject\u2019s infectiousness. Here, we developed a mechanistic model of viral transmission in golden hamsters and used data in upper respiratory tract and aerosols to evaluate key within-host and environment based viral parameters. The significance of our research is in identifying the timing and duration of viral shedding, how long it stays infectious, and the link between infectious virus and total viral RNA. Such knowledge enhances our understanding of the SARS-CoV-2 transmission window.","version":"1.1","doi":"10.1101/2022.03.08.483569","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.03.467186","pub_date":"2022-3-09","title":"Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants","abstract":"We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wt (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 \u03bcM, 8.5 \u03bcM, 24.1 \u03bcM, 8.2 \u03bcM and 13.6 \u03bcM, respectively. A separate experiment focusing on a combination of 10 \u03bcM stenoparib and 0.5 \u03bcM remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha (B.1.1.7) variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.","version":"1.4","doi":"10.1101/2021.11.03.467186","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.18.481028","pub_date":"2022-3-08","title":"Furin-cleavage site is present in an antiparallel \u03b2-strand in SARS-CoV2 Spike protein","abstract":"Furin cleavage-site (CS) present between the S1/S2 junction in SARS-CoV2 spike (S) protein is critical to drive the fusion of SARS-CoV2 with the host cell. SARS-CoV2 falls in the sarbecovirus lineage that doesn\u2019t comprise of furin CS and therefore makes its origin enigmatic. The available wild-type (Wt) SARS-CoV2 S protein with PDB ID: 6yvb lacks a stretch of amino acid including furin CS as well. All investigators till date have shown this stretch existing in the form of a loop. We are for the first time reporting that this stretch comprises of 14 amino acid residues (677QTNSPRRARSVASQ689), forming an antiparallel \u03b2-sheet comprising of PRRAR furin CS. We observed the presence of this antiparallel \u03b2-sheet in MERS spike protein as well. While switching over from Wt. SARS-CoV2 with PRRAR furin CS to B.1.1.7 variant with HRRAR furin CS, we found 3% increase in the percentage content of \u03b2 stands. Interestingly, we found that the change of B.1.1.7 to B.1.617 variant comprising of RRRAR furin CS shifted the percentage secondary structure back to that found in Wt. SARS-CoV2. We anticipate that this \u03b2-sheet is used as a docking site by host cell proteases to act on furin-CS. Additionally, we studied the interaction of modeled SARS-CoV2 S protein with transmembrane protease, serine 2 (TMPRSS2), and furin proteases, which clearly highlighted that these proteases exclusively uses furin CS located in \u03b2-sheet to cleave the SARS-CoV2 S protein at its S1/S2 junction.","version":"1.3","doi":"10.1101/2022.02.18.481028","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.08.483381","pub_date":"2022-3-08","title":"Hetero-bivalent Nanobodies Provide Broad-spectrum Protection against SARS-CoV-2 Variants of Concern including Omicron","abstract":"Following Delta, Omicron variant triggered a new wave of SARS-CoV-2 infection globally, adaptive evolution of the virus may not stop, the development of broad-spectrum antivirals is still urgent. We previously developed two hetero-bivalent nanobodies with potent neutralization against original WT SARS-CoV-2, termed aRBD-2-5 and aRBD-2-7, by fusing aRBD-2 with aRBD-5 or aRBD-7, respectively. Here, we resolved crystal structures of these nanobodies in complex with RBD, and found the epitope of aRBD-2 differs from that of aRBD-5, aRBD-7. aRBD-2 binds to a conserved epitope which renders its binding activity to all variants of concern (VOCs) including Omicron. Interestingly, although monovalent aRBD-5 and aRBD-7 lost binding to some variants, they effectively improved the overall affinity when transformed into the hetero-bivalent form after being fused with aRBD-2. Consistent with the high binding affinities, aRBD-2-5-Fc and aRBD-2-7-Fc exhibited ultra-potent neutralization to all five VOCs; particularly, aRBD-2-5-Fc neutralized authentic virus of Beta, Delta and Omicron with the IC50of 5.98\u223c9.65 ng/mL or 54.3\u223c87.6 pM. Importantly, aRBD-2-5-Fc provided in vivo prophylactic protection for mice against WT and mouse-adapted SARS-CoV-2, and provided full protection against Omicron in hamster model when administrated either prophylactically or therapeutically. Taken together, we found a conserved epitope on RBD, and hetero-bivalent nanobodies had increased affinity for VOCs over its monovalent form, and provided potent and broad-spectrum protection both in vitro and in vivo against all tested major variants, and potentially future emerging variants. Our strategy provides a new solution in the development of therapeutic antibodies for COVID-19 caused by newly emergent VOCs.","version":"1.1","doi":"10.1101/2022.03.08.483381","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.08.481609","pub_date":"2022-3-08","title":"The origins and molecular evolution of SARS-CoV-2 lineage B.1.1.7 in the UK","abstract":"The first SARS-CoV-2 variant of concern (VOC) to be designated was lineage B.1.1.7, later labelled by the World Health Organisation (WHO) as Alpha. Originating in early Autumn but discovered in December 2020, it spread rapidly and caused large waves of infections worldwide. The Alpha variant is notable for being defined by a long ancestral phylogenetic branch with an increased evolutionary rate, along which only two sequences have been sampled. Alpha genomes comprise a well-supported monophyletic clade within which the evolutionary rate is more typical of SARS-CoV-2. The Alpha epidemic continued to grow despite the continued restrictions on social mixing across the UK, and the imposition of new restrictions, in particular the English national lockdown in November 2020. While these interventions succeeded in reducing the absolute number of cases, the impact of these non-pharmaceutical interventions was predominantly to drive the decline of the SARS-CoV-2 lineages which preceded Alpha. We investigate the only two sampled sequences that fall on the branch ancestral to Alpha. We find that one is likely to be a true intermediate sequence, providing information about the order of mutational events that led to Alpha. We explore alternate hypotheses that can explain how Alpha acquired a large number of mutations yet remained largely unobserved in a region of high genomic surveillance: an under-sampled geographical location, a non-human animal population, or a chronically-infected individual. We conclude that the last hypothesis provides the best explanation of the observed behaviour and dynamics of the variant, although we find that the individual need not be immunocompromised, as persistently-infected immunocompetent hosts also display a higher within-host rate of evolution. Finally, we compare the ancestral branches and mutation profiles of other VOCs to each other, and identify that Delta appears to be an outlier both in terms of the genomic locations of its defining mutations, and its lack of rapid evolutionary rate on the ancestral branch. As new variants, such as Omicron, continue to evolve (potentially through similar mechanisms) it remains important to investigate the origins of other variants to identify ways to potentially disrupt their evolution and emergence.","version":"1.1","doi":"10.1101/2022.03.08.481609","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.07.483402","pub_date":"2022-3-08","title":"High-throughput molecular dynamics-based alchemical free energy calculations for predicting the binding free energy change associated with the common mutations in the spike receptor-binding domain of SARS-CoV-2","abstract":"The ongoing pandemic caused by SARS-CoV-2 has gone through various phases. From the initial outbreak the virus has mutated several times, with some lineages showing even stronger infectivity and faster spread than the original virus. Among all the variants, beta, gamma, delta and the latest (omicron) are currently classified as variants of concern (VOC) while the remaining are labelled either as variants of interest (VOI) or variants under monitoring (VUM). In this work, we have focused on the mutations observed in important variants, particularly at the receptor-binding domain (RBD) of the spike protein that is responsible for the interactions with the host ACE2 receptor and binding of antibodies. Studying these mutations is particularly important for understanding the viral infectivity, spread of the disease and for tracking the escape routes of this virus from antibodies. Molecular dynamics (MD) based alchemical free energy calculations have been shown to be very accurate in predicting the free energy change due to a mutation that could have a deleterious or a stabilising effect on the protein itself or its binding affinity to another protein. Here, we investigated the significance of six commonly observed spike RBD mutations on the stability of the spike protein binding to ACE2 by free energy calculations using high throughput MD simulations. For comparison, we also used other (rigorous and non-rigorous) binding free energy prediction methods and compared our results with the experimental data if available. The alchemical free energy-based method consistently predicted the free-energy changes with an accuracy close to \u00b11.0 kcal/mol when compared with the available experimental values. As per our simulation data the most significant mutations responsible for stabilising the spike RBD interactions with human ACE2 are N501Y and L452R.","version":"1.1","doi":"10.1101/2022.03.07.483402","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.06.483172","pub_date":"2022-3-07","title":"Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication","abstract":"New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase (OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.","version":"1.1","doi":"10.1101/2022.03.06.483172","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.05.483025","pub_date":"2022-3-07","title":"Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors","abstract":"While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly \u201cdruggable\u201d or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.","version":"1.1","doi":"10.1101/2022.03.05.483025","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.05.483104","pub_date":"2022-3-07","title":"Characterization of Hydrophobic Interactions of SARS-CoV-2 and MERS-CoV Spike Protein Fusion Peptides Using Single Molecule Force Measurements","abstract":"We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide sequences within coronavirus (CoV) spike proteins. Within the fusion peptides of SARS-CoV-2 and MERS-CoV, a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. While a non-polar triad (LLF) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore if single molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask if sequence variations between FP1 from SARS and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 \u00b1 0.03 nN versus 0.68 \u00b1 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single residue diversity in viral fusion peptides, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts. Fusion of coronaviruses (CoVs) and host cells is mediated by the insertion of the fusion peptide (FP) of the viral spike protein into the host cell membrane. Hydrophobic interactions between FPs with their host cell membranes regulate the viral membrane fusion process and are key to determining infection ability. However, it is not fully understood how the amino acid sequences in FPs mediate hydrophobic interactions. We use single-molecule force measurements to characterize hydrophobic interactions of FPs from SARS-CoV-2 and MERS-CoV. Our findings provide insight into the mechanisms by which the amino acid composition of FPs encodes hydrophobic interactions and their implications for fusion activity critical to the spread of infection.","version":"1.1","doi":"10.1101/2022.03.05.483104","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.07.481737","pub_date":"2022-3-07","title":"Anti-spike antibody response to natural infection with SARS-CoV-2 and its activity against emerging variants","abstract":"The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has substantially impacted human health globally. Spike-specific antibody response plays a major role in protection against SARS-CoV-2. Here, we demonstrated that acute SARS-CoV-2 infection elicits rapid and robust spike-binding and ACE2-blocking antibody responses, which wane approximately 11 months after infection. Serological responses were found to be correlated with the frequency of spike-specific memory B cell responses to natural infections. Further, significantly higher spike-binding, ACE2-blocking, and memory B cell responses were detected in patients with fever and pneumonia. Spike-specific antibody responses were found to be greatly affected by spike mutations in emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization. As spike protein-specific antibody responses play a major role in protection against SARS-CoV-2, we examined the spike-binding and ACE2-blocking antibody responses in SARS-CoV-2 infection at different time points. We found robust responses following acute infection, which waned approximately 11 months after infection. Further, the serological responses were correlated with the frequency of spike-specific memory B cell responses to natural infections. Patients with fever and pneumonia showed significantly stronger spike-binding, ACE2-blocking antibody, and memory B cell responses. Moreover, the spike-specific antibody responses were substantially affected by the emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization.","version":"1.1","doi":"10.1101/2022.03.07.481737","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.06.483197","pub_date":"2022-3-07","title":"Angiotensin converting enzyme 2 (ACE2): Virus accomplice or host defender","abstract":"The current coronavirus disease-19 (COVID-19) caused by the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has seriously disrupted the daily life of human, mainly attributed to the fact that we know too little about SARS-CoV-2. Increasing studies show that viral infection alters host cells glucose metabolism, which is crucial for viral nucleic acid replication. Here, we integrated RNA-sequencing results and found that SARS-CoV-2 infection alters the aerobic glycolysis, pentose phosphate pathway (oxiPPP), and DNA replication in lung tissues and cells. However, the direction of metabolic flux and DNA replication were dominated by angiotensin-converting enzyme 2 (ACE2), a host cell-expressed viral receptor protein. More interesting, although hosts with high expression of ACE2 are more likely to be infected with SARS-CoV-2, the invading virus cannot perform nucleic acid replication well due to the restriction of glucose metabolism, and eventually resulting prolonged infection-cycle or infection failure. Our findings, after a typical epidemiological investigation and modeling analysis, preliminarily explain the reasons for the emergence of asymptomatic infections or lower copy virus at early stage in host with higher ACE2 levels, which will provide important help for the development of more accurate and effective detection methods for diagnosing COVID-19.","version":"1.1","doi":"10.1101/2022.03.06.483197","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.05.483145","pub_date":"2022-3-07","title":"Predicted binding interface between coronavirus nsp3 and nsp4","abstract":"Double membrane vesicles (DMVs) in coronavirus-infected cells feature pores that span both membranes. DMV pores were observed to have six-fold symmetry and include the nsp3 protein. Co-expression of SARS-CoV nsp3 and nsp4 induces DMV formation, and elements of nsp3 and nsp4 have been identified that are essential for membrane disruption. I describe a predicted luminal binding interface between nsp3 and nsp4 that is membrane-associated, conserved in SARS-CoV-2 during the COVID-19 pandemic and in diverse coronaviruses, and stable in molecular dynamics simulation. Combined with structure predictions for the full-length nsp4 monomer and cryo-EM data, this suggests a DMV pore model in which nsp4 spans both membranes with nsp3 and nsp4 inserted into the same bilayer. This approach may be able to identify additional protein-protein interactions between coronavirus proteins.","version":"1.1","doi":"10.1101/2022.03.05.483145","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.05.483092","pub_date":"2022-3-07","title":"mRNA-based vaccine candidate COReNAPCIN\u00ae induces robust humoral and cellular immunity in mice and non-human primates","abstract":"At the forefront of biopharmaceutical industry, the messenger RNA (mRNA) technology offers a flexible and scalable platform to address the urgent need for world-wide immunization in pandemic situations. This strategic powerful platform has recently been used to immunize millions of people proving both of safety and highest level of clinical efficacy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we provide preclinical report of COReNAPCIN\u00ae; a vaccine candidate against SARS-CoV-2 infection. COReNAPCIN\u00ae is a nucleoside modified mRNA-based vaccine formulated in lipid nanoparticles (LNPs) for encoding the full-length prefusion stabilized SARS-CoV-2 spike glycoprotein on the cell surface. Vaccination of C57BL/6 and BALB/c mice and rhesus macaque with COReNAPCIN\u00ae induced strong humoral responses with high titers of virus-binding and neutralizing antibodies. Upon vaccination, a robust SARS-CoV-2 specific cellular immunity was also observed in both mice and non-human primate models. Additionally, vaccination protected rhesus macaques from symptomatic SARS-CoV-2 infection and pathological damage to the lung upon challenging the animals with high viral loads of up to 2\u00d7108 live viral particles. Overall, our data provide supporting evidence for COReNAPCIN\u00ae as a potent vaccine candidate against SARS-CoV-2 infection for clinical studies.","version":"1.1","doi":"10.1101/2022.03.05.483092","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.04.479488","pub_date":"2022-3-07","title":"Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease","abstract":"Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.","version":"1.1","doi":"10.1101/2022.03.04.479488","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.05.483133","pub_date":"2022-3-07","title":"Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel \u03b2-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human \u03b2-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human \u03b2-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.","version":"1.1","doi":"10.1101/2022.03.05.483133","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.07.483324","pub_date":"2022-3-07","title":"In silico screening and testing of FDA approved small molecules to block SARS-CoV-2 entry to the host cell by inhibiting Spike protein cleavage","abstract":"The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting Spike (S) protein cleavage by several proteases. We develop a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.","version":"1.1","doi":"10.1101/2022.03.07.483324","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.04.22271911","pub_date":"2022-03-07","title":"Impact of the COVID-19 pandemic on breast cancer screening indicators in a Spanish population-based program: a cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>To assess the effect of the COVID-19 pandemic on performance indicators in the population-based breast cancer screening program of Parc de Salut Mar (PSMAR), Barcelona, Spain.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a before-and-after, quasi-experimental study to evaluate participation, recall, false-positives, cancer detection rate, and cancer characteristics in our screening population from March 2020 to March 2021 compared with the four previous rounds (2012-2019). Using independent logistic regression models, we estimated the adjusted odds ratios (aOR) of each of the performance indicators for the COVID-19 period, controlling by type of screening (prevalent or incident), socioeconomic index, family history of breast cancer, and menopausal status. We analyzed 144,779 observations from 47,571 women.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>During the COVID-19 period, the odds of participation were 11% lower in first-time invitees (aOR=0.89[95%CI=0.84-0.96]) and in those who had previously participated regularly and irregularly (aOR=0.65 [95%CI=0.61-0.69] and aOR=0.93 [95%CI=0.85-1.03], respectively). Participation showed a modest increase in women not attending any of the previous rounds (aOR=1.07 [95%CI=0.99-1.17]). The recall rate slightly decreased in both prevalent and incident screening (aOR=0.89 [95%CI=0.78-1.01] and aOR=0.89 [95%CI=0.79-1.00], respectively). No significant differences were observed in false-positives (prevalent - aOR=1.07 [95%CI=0.92-1.24] and incident screening -aOR=0.94 [95%CI=0.82-1.08]), cancer detection rate (aOR=0.91 [95%CI=0.69-1.18]), or cancer stages.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The COVID-19 pandemic negatively affected screening attendance, especially in previous participants and newcomers. We found no marked differences in recall, false-positives, or cancer detection, indicating the program\u2019s resilience. There is a need for further evaluations of interval cancers and potential diagnostic delays.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.03.04.22271911","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.04.482636","pub_date":"2022-3-04","title":"Immunogenicity of an Ad26-based SARS-CoV-2 Omicron Vaccine in Na\u00efve Mice and SARS-CoV-2 Spike Pre-immune Hamsters","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant sparked concern due to its fast spread and the unprecedented number of mutations in the spike protein that enables it to partially evade spike-based COVID-19 vaccine-induced humoral immunity. In anticipation of a potential need for an Omicron spike-based vaccine, we generated an Ad26 vector encoding an Omicron (BA.1) spike protein (Ad26.COV2.S.529). Ad26.COV2.S.529 encodes for a prefusion stabilized spike protein, similar to the current COVID-19 vaccine Ad26.COV2.S encoding the Wuhan-Hu-1 spike protein. We verified that spike expression by Ad26.COV2.S.529 was comparable to Ad26.COV2.S. Immunogenicity of Ad26.COV2.S.529 was then evaluated in na\u00efve mice and SARS-CoV-2 Wuhan-Hu-1 spike pre-immunized hamsters. In na\u00efve mice, Ad26.COV2.S.529 elicited robust neutralizing antibodies against SARS-CoV-2 Omicron (BA.1) but not to SARS-CoV-2 Delta (B.1.617.2), while the opposite was observed for Ad26.COV2.S. In pre-immune hamsters, Ad26.COV2.S.529 vaccination resulted in robust increases in neutralizing antibody titers against both SARS-CoV-2 Omicron (BA.1) and Delta (B.1.617.2), while Ad26.COV2.S vaccination only increased neutralizing antibody titers against the Delta variant. Our data imply that Ad26.COV2.S.529 can both expand and boost a Wuhan-Hu-1 spike-primed humoral immune response to protect against distant SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.03.04.482636","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.03.482819","pub_date":"2022-3-04","title":"Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication","abstract":"As SARS-CoV-2 triggered a global health crisis, there is an urgent need to provide patients with safe, effective, accessible, and preferably oral therapeutics for COVID-19 that complement mRNA vaccines. Zinc compounds are widely known for their antiviral properties. Therefore, we have prepared a library of zinc complexes with pyrithione (1-hydroxy-2(1H)-pyridinethione) and its analogues, all of which showed promising in vitro inhibition of cathepsin L, an enzyme involved in SARS-CoV-2 entry, and PLPro, an enzyme involved in SARS-CoV-2 replication both in (sub)micromolar range. Zinc pyrithione 1a is a well-established, commercially available antimicrobial agent and was therefore selected for further evaluation of its SARS-CoV-2 entry and replication inhibition in an ex vivo system derived from primary human lung tissue. Our results suggest that zinc pyrithione complex 1a provides a multitarget approach to combat SARS-CoV-2 and should be considered for repurposing as a potential therapeutic against the insidious COVID-19 disease. In our study, we show that zinc pyrithione holds immense potential for the development of a possible out-patient treatment for SARS-CoV-2 due to its inhibition of viral entry and replication.","version":"1.1","doi":"10.1101/2022.03.03.482819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.24.481778","pub_date":"2022-3-04","title":"IgG1 responses following SARS-CoV-2 infection are polyclonal and highly personalized, whereby each donor and each clone displays a distinct pattern of cross-reactivity against SARS-CoV-2 variants","abstract":"Using a recently introduced efficient mass spectrometry-based approach we monitored individual donors\u2019 IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.","version":"1.2","doi":"10.1101/2022.02.24.481778","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.01.482592","pub_date":"2022-3-03","title":"Determining the optimal SARS-CoV-2 mRNA vaccine dosing interval for maximum immunogenicity","abstract":"Emerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear. This study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples 6 months (170 to 190 days) after the first vaccine dose. The main exposure variable was vaccine dosing interval (days), categorized as \u201cshort\u201d (first quartile), \u201cmoderate\u201d (second quartile), \u201clong\u201d (third quartile), and \u201clongest\u201d interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included: spike and RBD IgG antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations. A total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to \u201cshort interval\u201d (\u226430 days), higher dosing interval quartiles (moderate: 31-38 days; long: 39-73 days and longest: \u226574 days) were all associated with increased Elescys spike total antibody concentration. Compared to the short interval, \u201clong\u201d and \u201clongest\u201d interval quartiles were associated with higher spike and RBD IgG antibody concentrations. Similarly, increasing dosing intervals increased inhibition of ACE-2 binding to viral spike protein, regardless of the vaccine type. Increased mRNA vaccine dosing intervals longer than 30 days result in higher levels of circulating antibodies and viral neutralization when assessed at 6 months.","version":"1.1","doi":"10.1101/2022.03.01.482592","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.03.482788","pub_date":"2022-3-03","title":"Lack of antiviral activity of probenecid in Vero E6 cells and Syrian golden hamsters: a need for better understanding of inter-lab differences in preclinical assays","abstract":"Antiviral interventions are urgently required to support vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data in support of candidate plausibility are required. The speed at which preclinical models have been developed during the pandemic are unprecedented but there is a vital need for standardisation and assessment of the Critical Quality Attributes. This work provides cross-validation for the recent report demonstrating potent antiviral activity of probenecid against SARS-CoV-2 in preclinical models (1). Vero E6 cells were pre-incubated with probenecid, across a 7-point concentration range, or control media for 2 hours before infection with SARS-CoV-2 (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020, Pango B; MOI 0.05). Probenecid or control media was then reapplied and plates incubated for 48 hours. Cells were fixed with 4% v/v paraformaldehyde, stained with crystal violet and cytopathic activity quantified by spectrophotometry at 590 nm. Syrian golden hamsters (n=5 per group) were intranasally inoculated with virus (SARS-CoV-2 Delta variant B.1.617.2; 103 PFU/hamster) for 24 hours prior to treatment. Hamsters were treated with probenecid or vehicle for 4 doses. Hamsters were ethically euthanised before quantification of total and sub-genomic pulmonary viral RNAs. No inhibition of cytopathic activity was observed for probenecid at any concentration in Vero E6 cells. Furthermore, no reduction in either total or sub-genomic RNA was observed in terminal lung samples from hamsters on day 3 (P > 0.05). Body weight of uninfected hamsters remained stable throughout the course of the experiment whereas both probenecid- (6 - 9% over 3 days) and vehicle-treated (5 - 10% over 3 days) infected hamsters lost body weight which was comparable in magnitude (P > 0.5). The presented data do not support probenecid as a SARS-CoV-2 antiviral. These data do not support use of probenecid in COVID-19 and further analysis is required prior to initiation of clinical trials to investigate the potential utility of this drug.","version":"1.1","doi":"10.1101/2022.03.03.482788","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.481957","pub_date":"2022-3-02","title":"A structural dynamic explanation for observed escape of SARS-CoV-2 BA.2 variant mutation S371L/F","abstract":"The SARS-CoV-2 Omicron sub-variants BA.1 and BA.2 have become the dominant variants worldwide due to enhanced transmissibility and immune evasion. In response to the rise of BA.1 and BA.2, two recent studies by Liu et al. and Iketani et al. provide a detailed analysis of loss of therapeutic antibody potency through evaluation of escape by pseudotyped viruses harboring BA.1 and BA.2 receptor binding domain (RBD) point mutations. Surprisingly, Liu et al. and Iketani et al. observed a profoundly broad escape effect for the individual mutations S371L and S371F. This result cannot be explained by known escape mechanisms of the SARS-CoV-2 RBD, and conflicts with existing computational and experimental escape measurements for S371 mutations performed on monomeric RBD. Through an examination of these conflicting datasets and a structural analysis of the antibodies assayed by Liu et al. and Iketani et al., we propose a mechanism to explain S371L/F escape according to a perturbation of spike trimer conformational dynamics that has not yet been described for any SARS-CoV-2 escape mutation. The proposed mechanism is relevant to Omicron and future variant surveillance as well as therapeutic antibody design.","version":"1.2","doi":"10.1101/2022.02.25.481957","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.01.22271582","pub_date":"2022-03-02","title":"Duration of viable virus shedding in SARS-CoV-2 omicron variant infection","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>Clinical features of SARS-CoV-2 Omicron variant infection, including incubation period and transmission rates, distinguish this variant from preceding variants. However, whether the duration of shedding of viable virus differs between omicron and previous variants is not well understood. To characterize how variant and vaccination status impact shedding of viable virus, we serially sampled symptomatic outpatients newly diagnosed with COVID-19. Anterior nasal swabs were tested for viral load, sequencing, and viral culture. Time to PCR conversion was similar between individuals infected with the Delta and the Omicron variant. Time to culture conversion was also similar, with a median time to culture conversion of 6 days (interquartile range 4-8 days) in both groups. There were also no differences in time to PCR or culture conversion by vaccination status.</jats:p>","version":null,"doi":"10.1101/2022.03.01.22271582","journal":"medRxiv","score":null},{"id":"10.1101/2022.03.01.482536","pub_date":"2022-3-02","title":"Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening","abstract":"The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen \u223c38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100\u00b5M, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.","version":"1.1","doi":"10.1101/2022.03.01.482536","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.02.482651","pub_date":"2022-3-02","title":"Intranasal Immunization with a Proteosome-Adjuvanted SARS-CoV2 Spike Protein-Based Vaccine is Immunogenic and Efficacious in Mice & Hamsters","abstract":"With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.","version":"1.1","doi":"10.1101/2022.03.02.482651","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.01.482462","pub_date":"2022-3-02","title":"Rapid hypermutation B cell trajectory recruits previously primed B cells upon third SARS-CoV-2 mRNA vaccination","abstract":"High antibody affinity against the ancestral SARS-CoV-2 strain seems to be necessary (but not always sufficient) for the control of emerging immune-escape variants. Therefore, aiming at strong B cell somatic hypermutation - not only at high antibody titers - is a priority when utilizing vaccines that are not targeted at individual variants. Here, we developed a next-generation sequencing based SARS-CoV-2 B cell tracking protocol to rapidly determine the level of immunoglobulin somatic hypermutation at distinct points during the immunization period. The percentage of somatically hypermutated B cells in the SARS-CoV-2 specific repertoire was low after the primary vaccination series, evolved further over months and increased steeply after boosting. The third vaccination mobilized not only na\u00efve, but also antigen-experienced B cell clones into further rapid somatic hypermutation trajectories indicating increased affinity. Together, the strongly mutated post-booster repertoires and antibodies deriving from this may explain why the booster, but not the primary vaccination series, offers some protection against immune-escape variants such as Omicron B.1.1.529. Priming SARS-CoV-2 vaccinations generate antibodies from low-level matured B cells while the third vaccination strongly boosts somatic hypermutation potentially explaining different protection from immune-escape variants.","version":"1.1","doi":"10.1101/2022.03.01.482462","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.02.480688","pub_date":"2022-3-02","title":"Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease","abstract":"Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.","version":"1.1","doi":"10.1101/2022.03.02.480688","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.15.452246","pub_date":"2022-3-02","title":"Gut microbiome dysbiosis during COVID-19 is associated with increased risk for bacteremia and microbial translocation","abstract":"The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate in a mouse model that SARS-CoV-2 infection can induce gut microbiome dysbiosis, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Comparison with stool samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.","version":"1.2","doi":"10.1101/2021.07.15.452246","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.01.482548","pub_date":"2022-3-02","title":"Immunological memory to Common Cold Coronaviruses assessed longitudinally over a three-year period","abstract":"Understanding immune memory to Common Cold Coronaviruses (CCCs) is relevant for assessing its potential impact on the outcomes of SARS-CoV-2 infection, and for the prospects of pan-corona vaccines development. We performed a longitudinal analysis, of pre-pandemic samples collected from 2016-2019. CD4+ T cells and antibody responses specific for CCC and to other respiratory viruses, and chronic or ubiquitous pathogens were assessed. CCC-specific memory CD4+ T cells were detected in most subjects, and their frequencies were comparable to those for other common antigens. Notably, responses to CCC and other antigens such as influenza and Tetanus Toxoid (TT) were sustained over time. CCC-specific CD4+ T cell responses were also associated with low numbers of HLA-DR+CD38+ cells and their magnitude did not correlate with yearly changes in the prevalence of CCC infections. Similarly, spike RBD-specific IgG responses for CCC were stable throughout the sampling period. Finally, high CD4+ T cell reactivity to CCC, but not antibody responses, was associated with high pre-existing SARS-CoV-2 immunity. Overall, these results suggest that the steady and sustained CCC responses observed in the study cohort are likely due to a relatively stable pool of CCC-specific memory CD4+ T cells instead of fast decaying responses and frequent reinfections.","version":"1.1","doi":"10.1101/2022.03.01.482548","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432821","pub_date":"2022-3-01","title":"Impaired activation of Transposable Elements in SARS-CoV-2 infection","abstract":"Transposable element (TE) transcription is induced in response to viral infections. TE induction triggers a robust and durable interferon (IFN) response, providing a host defense mechanism. Still, the connection between SARS-CoV-2 IFN response and TEs remains largely unknown. Here, we analyzed TE expression changes in response to SARS-CoV-2 infection in different human cellular models. We find that compared to other viruses, which cause global upregulation of TEs, SARS-CoV-2 infection results in a significantly milder TE response in both primary lung epithelial cells and in iPSC-derived lung alveolar type 2 cells. TE activation precedes, and correlates with, the induction of IFN-related genes, suggesting that the limited activation of TEs following SARS-CoV-2 infection may be the reason for the weak IFN response. Diminished TE activation was not observed in lung cancer cell lines with very high viral load. Moreover, we identify two variables which explain most of the observed diverseness in immune responses: basal expression levels of TEs in the pre-infected cells, and the viral load. Finally, analyzing the SARS-CoV-2 interactome, as well as the epigenetic landscape around the TEs that are activated following infection, we identify SARS-CoV-2 interacting proteins, which may regulate chromatin structure and TE transcription in response to a high viral load. This work provides a functional explanation for SARS-CoV-2\u2019s success in its fight against the host immune system, and suggests that TEs could be used as sensors and serve as potential drug targets for COVID-19. \n\nUnlike other viruses, SARS-CoV-2 invokes a weak and inefficient transposable element (TE) response\nTE induction precedes and predicts IFN response\nBasal TE expression and viral load explain immune responses\nDistinct chromatin and enhancer binding factors occupancy on TEs induced by SARS-CoV-2\n\n Unlike other viruses, SARS-CoV-2 invokes a weak and inefficient transposable element (TE) response TE induction precedes and predicts IFN response Basal TE expression and viral load explain immune responses Distinct chromatin and enhancer binding factors occupancy on TEs induced by SARS-CoV-2","version":"1.3","doi":"10.1101/2021.02.25.432821","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.28.482305","pub_date":"2022-3-01","title":"Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection","abstract":"Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.","version":"1.1","doi":"10.1101/2022.02.28.482305","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.28.482334","pub_date":"2022-3-01","title":"RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2","abstract":"SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.","version":"1.1","doi":"10.1101/2022.02.28.482334","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465476","pub_date":"2022-3-01","title":"A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike","abstract":"Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.10.22.465476","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.28.482283","pub_date":"2022-3-01","title":"Mutation patterns in SARS-COV-2 Alpha and Beta variants indicate non-neutral evolution","abstract":"Due to the emergence of new variants of the SARS-CoV-2 coronavirus, the question of how the viral genomes evolved, leading to the formation of highly infectious strains, becomes particularly important. Two early emergent strains, Alpha and Beta, characterized by a significant number of missense mutations, provide natural testing samples. In this study we are exploring the history of each of the segregating sites present in Alpha and Beta variants of concern, to address the question whether defining mutations were accumulating gradually leading to the formation of sequence characteristic of these variants. Our analysis exposes data features that suggest other than neutral evolution of SARS-CoV-2 genomes, leading to emergence of variants of concern. We observe only small number of possible combinations of mutations indicating rapid evolution of genomes. In addtion, mutation patterns observed in whole genome samples of Alpha and Beta variants also indicate presence of stronger selection than in remaining genome samples.","version":"1.1","doi":"10.1101/2022.02.28.482283","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.27.482162","pub_date":"2022-3-01","title":"Stable nebulization and muco-trapping properties of Regdanvimab/IN-006 supports its development as a potent, dose-saving inhaled therapy for COVID-19","abstract":"The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries around the world, can effectively trap SARS-CoV-2 virus-like-particles in fresh human airway mucus. IN-006, a reformulation of Regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.","version":"1.1","doi":"10.1101/2022.02.27.482162","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.28.482377","pub_date":"2022-3-01","title":"Sleep and circadian rhythm disruption alters the lung transcriptome to predispose to viral infection","abstract":"Sleep and circadian rhythm disruption (SCRD), as encountered during shift work, increases the risk of respiratory viral infection including SARS-CoV-2. However, the mechanism(s) underpinning higher rates of respiratory viral infection following SCRD remain poorly characterised. To address this, we investigated the effects of acute sleep deprivation on the mouse lung transcriptome. Here we show that sleep deprivation profoundly alters the transcriptional landscape of the lung, causing the suppression of both innate and adaptive immune systems, disrupting the circadian clock, and activating genes implicated in SARS-CoV-2 replication, thereby generating a lung environment that promotes viral infection and associated disease pathogenesis. Our study provides a mechanistic explanation of how SCRD increases the risk of respiratory viral infections including SARS-CoV-2 and highlights therapeutic avenues for the prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.02.28.482377","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.28.482287","pub_date":"2022-3-01","title":"Evolutionary analysis of genomes of SARS-CoV-2-related bat viruses suggests old roots, constant effective population size, and possible increase of fitness","abstract":"It is of vital practical interest to understand the co-evolution of bat \u03b2-coronaviruses with their hosts, since a number of these most likely crossed the species boundaries and infected humans. Complete sequences of 47 consensus genomes are available for bat \u03b2-coronaviruses related to the SARS-CoV-2 human virus. We carried out several types of evolutionary analyses using these data. First, using the publicly available BEAST 2 software, we generated phylogenetic trees and skyline plots. The roots of the trees, both for the entire sequences and subsequences coding for the E and S proteins as well as the 5\u2019 and 3\u2019 UTR regions, are estimated to be located from several decades to more than a thousand years ago, while the effective population sizes remained largely constant. Motivated by this, we developed a simple estimator of the effective population size in a Moran model with constant population, which, under the model is equal to the expected age of the MRCA measured in generations. Comparisons of these estimates to those produced by BEAST 2 shows qualitative agreement. We also compared the site frequency spectra (SFS) of the bat genomes to those provided by the Moran Tug-of-War model. Comparison does not exclude the possibility that overall fitness of the bat \u03b2-coronaviruses was increasing over time as a result of directional selection. Stability of interactions of bats and their viruses was considered likely on the basis of specific manner in which bat immunity is tuned, and it seems consistent with our analysis.","version":"1.1","doi":"10.1101/2022.02.28.482287","journal":"bioRxiv","score":null},{"id":"10.1101/2022.03.01.481391","pub_date":"2022-3-01","title":"Omicron-specific mRNA vaccine elicits potent immune responses in mice, hamsters, and nonhuman primates","abstract":"SARS-CoV-2 has infected more than 400 million people around the globe and caused millions of deaths. Since its identification in November 2021, Omicron, a highly transmissible variant, has become the dominant variant in most countries. Omicron\u2019s highly mutated spike protein, the main target of vaccine development, significantly compromises the immune protection from current vaccination. We develop an mRNA vaccine (SOmicron-6P) based on an Omicron-specific sequence. In mice, SOmicron-6P shows superior neutralizing antibodies inducing abilities to a clinically approved inactivated virus vaccine, a clinically approved protein subunit vaccine, and an mRNA vaccine (SWT-2P) with the same sequence of BNT162b2 RNA. Significantly, SOmicron-6P induces a 14.4\u223c27.7-fold and a 28.3\u223c50.3-fold increase of neutralizing activity against the pseudovirus of Omicron and authentic Omicron compared to SWT-2P, respectively. In addition, two doses SOmicron-6P significantly protects Syrian hamsters against challenge with SARS-CoV-2 Omicron variant and elicits high titers of nAbs in a dose-dependent manner in macaques. Our results suggest that SOmicron-6P offers advantages over current vaccines, and it will be helpful for those with weak immunity.","version":"1.1","doi":"10.1101/2022.03.01.481391","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477673","pub_date":"2022-3-01","title":"Despite the odds: formation of the SARS-CoV-2 methylation complex","abstract":"Coronaviruses protect their single-stranded RNA genome with a methylated cap during replication. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16 where nsp10 acts as a co-factor to both. Aditionally, nsp14 carries an exonuclease domain, which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins should be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex. This interaction is expected to encourage formation of mature capped viral mRNA, modulating the nsp14\u2019s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.","version":"1.2","doi":"10.1101/2022.01.25.477673","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.481997","pub_date":"2022-2-28","title":"Adenosine A2A Receptor (A2AR) agonists improve survival in K28-hACE2 mice following SARS CoV-2 infection","abstract":"Effective and available therapies for the treatment of COVID-19 disease are limited. Apadenoson is a highly potent selective anti-inflammatory adenosine A2A receptor (A2AR) agonist and potential treatment option for COVID-19 patients. Apadenoson, when administered after infection with SARS CoV-2, was found to decrease weight loss, improve clinical symptoms, reduce levels of a several proinflammatory cytokines and chemokines in bronchial lavage (BAL) fluid, and promote increased survival in K18hACE2 transgenic mice. Of note, administering apadenoson after, but not prior to Covid-19 infection, caused a rapid decrease in lung viral burden. The work presented provides the foundation for further examination of these drugs as a therapy option for COVID-19. Apadenoson therapy improves COVID-19 outcome","version":"1.2","doi":"10.1101/2022.02.25.481997","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.27.482153","pub_date":"2022-2-28","title":"A strategy to optimize the peptide-based inhibitors against different mutants of the spike protein of SARS-CoV-2","abstract":"SARS-CoV-2 virus has caused high-priority health concerns at a global level. Vaccines have stalled the proliferation of viruses to some extent. Yet, the emergence of newer, potentially more infectious, and dangerous mutants such as delta and omicron are among the major challenges in finding a more permanent solution for this pandemic. The effectiveness of antivirals Molnupiravir and Paxlovid, authorized for emergency use by the FDA, are yet to be assessed at larger populations. Patients with a high risk of disease progression or hospitalization have received treatment with a combination of antibodies (antibody-cocktail). Most of the mutations leading to the new lineage of SARS-CoV-2 are found in the spike protein of this virus that plays a key role in facilitating host entry. The current study has investigated how to modify a promising peptide-based inhibitor of spike protein, LCB3, against common mutations in the target protein so that it retains its efficacy against the spike protein. LCB3 being a prototype for protein-based inhibitors is an ideal testing system to learn about protein-based inhibitors. Two common mutations N501Y and K417N are considered in this work. Using a structure-based approach that considers free energy decomposition of residues, distance, and the interactions between amino acids, we propose the substitutions of amino acid residues of LCB3 inhibitors. Our binding free energy calculations suggest a possible improvement in the binding affinity of existing inhibitor LCB3 to the mutant forms of the S-protein using simple substitutions at specific positions of the inhibitor. This approach, being general, can be used in different inhibitors and other mutations and help in fighting against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.02.27.482153","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480449","pub_date":"2022-2-28","title":"Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2","abstract":"The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-na\u00efve mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.","version":"1.2","doi":"10.1101/2022.02.14.480449","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.27.482176","pub_date":"2022-2-28","title":"Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV2 nsp3 Macrodomain","abstract":"A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low \u03bcM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.","version":"1.1","doi":"10.1101/2022.02.27.482176","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.27.482147","pub_date":"2022-2-28","title":"Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergence\u2019s impact on choosing optimal drug treatment.","version":"1.1","doi":"10.1101/2022.02.27.482147","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.08.372995","pub_date":"2022-2-27","title":"Human coronaviruses disassemble processing bodies","abstract":"A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this report, no information was known about how human coronaviruses (hu CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold hu CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that N protein-mediated PB loss correlated with elevated RNA for PB-localized transcripts encoding TNF and IL-6. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV-1) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to enhanced proinflammatory cytokine production observed during severe SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2020.11.08.372995","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429164","pub_date":"2022-2-26","title":"Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants","abstract":"SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.","version":"1.4","doi":"10.1101/2021.02.03.429164","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477274","pub_date":"2022-2-26","title":"Host Chitinase 3-like-1 is a Universal Therapeutic Target for SARS-CoV-2 Viral Variants in COVID 19","abstract":"COVID 19 is the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2; SC2) which has caused a world-wide pandemic with striking morbidity and mortality. Evaluation of SC2 strains demonstrated impressive genetic variability and many of these viral variants are now defined as variants of concern (VOC) that cause enhanced transmissibility, decreased susceptibility to antibody neutralization or therapeutics and or the ability to induce severe disease. Currently, the delta (\u03b4) and omicron (o) variants are particularly problematic based on their impressive and unprecedented transmissibility and ability to cause break through infections. The delta variant also accumulates at high concentrations in host tissues and has caused waves of lethal disease. Because studies from our laboratory have demonstrated that chitinase 3-like-1 (CHI3L1) stimulates ACE2 and Spike (S) priming proteases that mediate SC2 infection, studies were undertaken to determine if interventions that target CHI3L1 are effective inhibitors of SC2 viral variant infection. Here we demonstrate that CHI3L1 augments epithelial cell infection by pseudoviruses that express the alpha, beta, gamma, delta or omicron S proteins and that the CHI3L1 inhibitors anti-CHI3L1 and kasugamycin inhibit epithelial cell infection by these VOC pseudovirus moieties. Thus, CHI3L1 is a universal, VOC-independent therapeutic target in COVID 19.","version":"1.2","doi":"10.1101/2022.01.21.477274","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.24.481901","pub_date":"2022-2-26","title":"Protection of Hamsters Challenged with SARS-CoV-2 after Two Doses of MVC-COV1901 Vaccine Followed by a Single Intranasal Booster with Nanoemulsion Adjuvanted S-2P Vaccine","abstract":"Intramuscular vaccines have greatly reduced hospitalization and death due to severe COVID-19. However, most countries are experiencing a resurgence of infection driven predominantly by the Delta and Omicron variants of SARS-CoV-2. In response, booster dosing of COVID-19 vaccines has been implemented in many countries to address waning immunity and reduced protection against the variants. However, intramuscular boosting fails to elicit mucosal immunity and therefore does not solve the problem of persistent viral carriage and transmission, even in patients protected from severe disease. In this study, two doses of stabilized prefusion SARS-CoV-2 spike (S-2P)-based intramuscular vaccine adjuvanted with Alum/CpG1018, MVC-COV1901, were used as a primary vaccination series, followed by an intranasal booster vaccination with nanoemulsion (NE01)-adjuvanted S-2P vaccine in a hamster model to demonstrate immunogenicity and protection from viral challenge. Here we report that this vaccination regimen resulted not only in the induction of robust immunity and protection against weight loss and lung pathology following challenge with SARS-CoV-2, but also led to increased viral clearance from both upper and lower respiratory tracts. Our findings showed that intramuscular MVC-COV1901 vaccine followed by a booster with intranasal NE01-adjuvanted vaccine promotes protective immunity against both viral infection and disease, suggesting that this immunization protocol may offer a solution in addressing a significant, unmet medical need for both the COVID-19 and future pandemics.","version":"1.1","doi":"10.1101/2022.02.24.481901","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480347","pub_date":"2022-2-26","title":"Molecular dynamics simulations of the Spike trimeric ectodomain of the SARS-CoV-2 Omicron variant: structural relationships with infectivity, evasion to immune system and transmissibility","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is replacing Delta, the most prevalent variant worldwide from the beginning of 2021 until early 2022. The Omicron variant is highly transmissible and responsible for a new worldwide COVID-19 wave. Herein, we calculated molecular dynamics simulations of the SARS-CoV-2 trimeric spike protein of Wuhan-Hu-1 strain (wild type, WT) and the Omicron variant of concern. Structural analyses reveal that the SpikeOmicron presents more conformational flexibility than SpikeWT, mainly in the N-terminal domain (NTD) and receptor-binding domain (RBD). Such flexibility results in a broader spectrum of different conformations for SpikeOmicron, whereby the RBD can more easily visit an up-conformational state. We reported how the mutations in this variant may influence the intra- and inter-protomer contacts caused by conformational flexibility of the NTD. Based on our analysis, we suggest that the differences in conformational flexibility between SpikeOmicron and SpikeWT may explain the observed gains in infectivity, immune system evasion and transmissibility in this novel variant.","version":"1.2","doi":"10.1101/2022.02.14.480347","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.481941","pub_date":"2022-2-26","title":"Follow-up investigation and detailed mutational characterization of the SARS-CoV-2 Omicron variant lineages (BA.1, BA.2, BA.3 and BA.1.1)","abstract":"Aided by extensive protein mutations, the SARS-CoV-2 Omicron (B.1.1.529) variant overtook the previously dominant Delta variant and rapidly spread around the world. It was shown to exhibit significant resistance to current vaccines and evasion from neutralizing antibodies. It is therefore critical to investigate the Omicron mutations\u2019 trajectories. In this study, a literature search of published articles and SARS-CoV-2 databases was conducted, We explored the full list of mutations in Omicron BA.1, BA.1.1, BA.2, and BA.3 lineages. We described in detail the prevalence and occurrence of the mutations across variants, and how Omicron differs from them. We used GISAID as our primary data source, which provides open-access to genomics data of the SARS-CoV-2 virus, in addition to epidemiological and geographical data. We examined how these mutations interact with each other, their co-occurrence and clustering. Our study offers for the first time a comprehensive description of all mutations with a focus on non-spike mutations and demonstrated that mutations in regions other than the Spike (S) genes are worth investigating further. Our research established that the Omicron variant has retained some mutations reported in other SARS-CoV-2 variants, yet many of its mutations are extremely rare in other variants and unique to Omicron. Some of these mutations have been linked to the transmissibility and immune escape of the virus, and indicate a significant shift in SARS-CoV-2 evolution. The most likely theories for the evolution of the Omicron variant were also discussed.","version":"1.1","doi":"10.1101/2022.02.25.481941","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.24.481899","pub_date":"2022-2-26","title":"Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution","abstract":"SARS-CoV-2 has evolved variants with substitutions in the spike receptor-binding domain (RBD) that impact its affinity for ACE2 receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites\u2014a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single amino-acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently N501Y, cause epistatic shifts in the effects of mutations at other sites, thereby shaping subsequent evolutionary change. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2022.02.24.481899","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.25.481966","pub_date":"2022-2-26","title":"Proteomic analysis of human milk reveals nutritional and immune benefits in the colostrum from mothers with COVID-19","abstract":"The range of benefits breastfeeding provides neonates and infants include nutrition, improved neonatal survival, and reduced morbidity from certain diseases. It also aids maternal health by speeding postpartum recovery. However, due to concern about the risk of SARS-CoV-2 transmission and the lack of evidence of breastmilk\u2019s protective effects against the virus, whether mothers with COVID-19 should be encouraged to breastfeed is under debate. Here, we present the results of proteomic and glycoproteomic studies of breast milk (colostrum and mature milk) from mothers with confirmed COVID-19. All colostrum samples exhibited significantly upregulated immune-related proteins, especially whey proteins with antiviral properties against SARS-CoV-2, and increased glycosylation levels and heterogeneity at those proteins. Such adaptive differences in milk from COVID-19 mothers tend to fade in mature milk from the same mothers one month postpartum. These results suggest the immune benefits of colostrum from mothers with COVID-19 and provide molecular-level insights that aid breastmilk feeding decisions in cases of active infection.","version":"1.1","doi":"10.1101/2022.02.25.481966","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.24.481866","pub_date":"2022-2-26","title":"Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression","abstract":"How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. The K18-hACE2 mouse model has been widely used for evaluation of new interventions for COVID-19. Herein we use RNA-Seq data and bioinformatics approaches to compare the transcriptional responses in the SARS-CoV-2 infected lungs of K18-hACE2 mice with those seen in humans. Overlap in differentially expressed genes was generally poor (\u224820-30%), even when multiple studies were combined. The overlap was not substantially improved when a second mouse model was examined wherein hACE was expressed from the mouse ACE2 promoter. In contrast, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between the species. As immunity and immunopathology are the focus of most studies, these hACE2 transgenic mouse models can thus be viewed as representative and relevant models of COVID-19.","version":"1.1","doi":"10.1101/2022.02.24.481866","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.474050","pub_date":"2022-2-26","title":"Convergent evolution of multiple mutations improves the viral fitness of SARS-CoV-2 variants by balancing positive and negative selection","abstract":"Multiple mutations have been seen to undergo convergent evolution in SARS-CoV-2 variants of concern. One such evolution occurs in Beta, Gamma, and Omicron variants at three amino acid positions K417, E484, and N501 in the receptor binding domain of the spike protein. We examined the physical mechanisms underlying the convergent evolution of three mutations K417T/E484K/N501Y by delineating the individual and collective effects of mutations on binding to angiotensin converting enzyme 2 receptor, immune escape from neutralizing antibodies, protein stability and expression. Our results show that each mutation serves a distinct function that improves virus fitness supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. Compared to the wild-type, K417T escapes Class 1 antibodies, has increased stability and expression; however, it has decreased receptor binding. E484K escapes Class 2 antibodies; however, it has decreased receptor binding, stability and expression. N501Y increases receptor binding; however, has decreased stability and expression. When these mutations come together, the deleterious effects are mitigated due to the presence of compensatory effects. Triple mutant K417T/E484K/N501Y has increased receptor binding, escapes both Class 1 and Class 2 antibodies, and has similar stability and expression as that of the wild-type. These results show the implications of presence of multiple mutations on virus evolution that enhance viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together.","version":"1.2","doi":"10.1101/2021.12.23.474050","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481341","pub_date":"2022-2-24","title":"Potent Neutralizing Activity of Polyclonal Equine Antibodies against Omicron SARS-CoV-2","abstract":"Using a polyclonal approach of equine anti-SARS-CoV-2 F(ab\u2019)2 antibodies we have achieved a high level of neutralizing potency against all SARS-CoV-2 variants tested. Neutralization titers were in the range of 105-106 IU/mL including Omicron: 111,403 UI/mL, which is 2-3 orders of magnitude what is normally achieved in response to SARS-CoV-2 infection and/or vaccination. The presence of high titers of a repertoire of antibodies targeting conserved epitopes in different regions of the spike protein could plausibly account for this remarkable breadth of neutralization. These results warrant the clinical investigation of anti-SARS-CoV-2 equine polyclonal F(ab\u2019)2 antibodies as a novel therapeutic strategy against COVID-19","version":"1.2","doi":"10.1101/2022.02.21.481341","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476556","pub_date":"2022-2-24","title":"Structural basis for Nirmatrelvir in vitro efficacy against SARS-CoV-2 variants","abstract":"The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID\u2122 (nirmatrelvir tablets and ritonavir tablets). We evaluated the in vitro catalytic activity and in vitro potency of nirmatrelvir against the main protease (Mpro) of prevalent variants of concern (VOC) or variants of interest (VOI): Alpha (\u03b1, B.1.1.7), Beta (\u03b2, B.1.351), Delta (\u03b4, B1.617.2), Gamma (\u03b3, P.1), Lambda (\u03bb, B.1.1.1.37/C37), Omicron (o, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the Mpro specifically for: \u03b1, \u03b2, \u03b3 (K90R), \u03bb (G15S) and o (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrate that they are catalytically comparable to wildtype. Nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the o, \u03bb and \u03b2 Mpro at 1.63 - 2.09 \u00c5 resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells (1).","version":"1.2","doi":"10.1101/2022.01.17.476556","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.23.481644","pub_date":"2022-2-24","title":"Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants","abstract":"The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly. The Omicron variant termed BA.2, which differs genetically substantially from BA.1, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed. Here, we used antigenic cartography to quantify and visualize antigenic differences between SARS-CoV-2 variants using hamster sera obtained after primary infection. Whereas early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape (vaccine-induced) antibody responses as a result of different antigenic characteristics. Close monitoring of the antigenic changes of SARS-CoV-2 using antigenic cartography can be helpful in the selection of future vaccine strains.","version":"1.1","doi":"10.1101/2022.02.23.481644","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.23.481492","pub_date":"2022-2-24","title":"Modeling predicts mechanisms altered by mutations of the SARS-CoV-2 delta and omicron variants","abstract":"We apply our mechanistic, within-host, pre-immunity, respiratory tract infection model for unvaccinated, previously uninfected, and immune-compromised individuals. Starting from published cell infection and viral replication data for the SARS-CoV-2 alpha variant, we explore variability in outcomes of viral load and cell infection due to three plausible mechanisms altered by SARS-CoV-2 mutations of delta and omicron. We seek a mechanistic explanation of clinical test results: delta nasal infections express \u223c3 orders-of-magnitude higher viral load than alpha, while omicron infections express an additional 1 to 2 orders-of-magnitude rise over delta. Model simulations reveal shortening of the eclipse phase (the time between cellular uptake of the virus and onset of infectious viral replication and shedding) alone can generate 3-5 orders-of-magnitude higher viral load within 2 days post initial infection. Higher viral replication rates by an infected cell can generate at most one order-of-magnitude rise in viral load, whereas higher cell infectability has minimal impact and lowers the viral load.","version":"1.1","doi":"10.1101/2022.02.23.481492","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.15.480166","pub_date":"2022-2-24","title":"Neutralization of SARS-CoV-2 Omicron BA.2 by Therapeutic Monoclonal Antibodies","abstract":"Monoclonal antibody therapy for the treatment of SARS-CoV-2 infection has been highly successful in decreasing disease severity; however, the recent emergence of the heavily mutated Omicron variant has posed a challenge to this treatment strategy. The Omicron variant BA.1 has been found to evade neutralization by several of the therapeutic monoclonal antibodies authorized for emergency use, while Vir-7831 and a cocktail consisting of monoclonal antibodies AZD8895+AZD1061 retain significant neutralizing activity. A newly emerged variant, Omicron BA.2, containing some of the BA.1 mutations plus an additional 6 mutations and 3 deletions, 3 of which lie in the receptor binding domain, has been found to be spreading with increased transmissibility. We report here, using spike protein-pseudotyped lentiviruses, decreased neutralization of BA.2 by several therapeutic monoclonal antibodies but that the mixture of AZD8895+AZD1061 retained substantial neutralizing activity against BA.2.","version":"1.2","doi":"10.1101/2022.02.15.480166","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.18.481058","pub_date":"2022-2-24","title":"Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains","abstract":"The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.","version":"1.2","doi":"10.1101/2022.02.18.481058","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.23.481620","pub_date":"2022-2-24","title":"Pre-clinical testing of two serologically distinct chimpanzee-origin adenovirus vectors expressing spike of SARS-CoV-2","abstract":"Two serologically distinct chimpanzee-origin, replication-defective adenovirus (AdC) vectors expressing the spike (S) protein of an early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate were generated and tested for induction of antibodies in young and aged mice. Both vectors induced S protein-specific antibodies including neutralizing antibodies. Levels of antibodies increased after a boost. The effectiveness of the boost depended on vector dose and timing between the two immunizations. Using two heterologous AdC vectors was more effective than vaccinating with the same vector repeatedly. Antibodies partially crossreacted between different S protein variants. Cross-reactivity increased after booster immunization with vectors carrying the same S gene, expression of two different S proteins by the AdC vectors used for the prime and the boost did not selectively increase responses against the variants.","version":"1.1","doi":"10.1101/2022.02.23.481620","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.23.481658","pub_date":"2022-2-24","title":"Bacterial metatranscriptomes in wastewater can differentiate virally infected human populations","abstract":"Monitoring wastewater samples at building-level resolution screens large populations for SARS-CoV-2, prioritizing testing and isolation efforts. Here we perform untargeted metatranscriptomics on virally-enriched wastewater samples from 10 locations on the UC San Diego campus, demonstrating that resulting bacterial taxonomic and functional profiles discriminate SARS-CoV-2 status even without direct detection of viral transcripts. Our proof-of-principle reveals emergent threats through changes in the human microbiome, suggesting new approaches for untargeted wastewater-based epidemiology.","version":"1.1","doi":"10.1101/2022.02.23.481658","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481491","pub_date":"2022-2-23","title":"Molnupiravir (MK-4482) is efficacious against Omicron and other SARS-CoV-2 variants in the Syrian hamster COVID-19 model","abstract":"The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the Syrian hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants. MK-4482 inhibits replication of multiple SARS-CoV-2 variants of concern, including Omicron, in the Syrian hamster COVID-19 model","version":"1.1","doi":"10.1101/2022.02.22.481491","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481223","pub_date":"2022-2-23","title":"Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions","abstract":"The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1\u03b1/\u03b2. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.","version":"1.1","doi":"10.1101/2022.02.21.481223","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481472","pub_date":"2022-2-23","title":"Ivermectin does not protect against SARS-CoV-2 infection in the Syrian hamster model","abstract":"Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio\u2122) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2022.02.22.481472","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.479559","pub_date":"2022-2-23","title":"Computational study and design of effective siRNAs to silence structural proteins associated genes of Indian SARS-CoV-2 strains","abstract":"SARS-CoV-2 is a highly transmissible and pathogenic coronavirus that first emerged in late 2019 and has since triggered a pandemic of acute respiratory disease named COVID-19 which poses a significant threat to all public health institutions in the absence of specific antiviral treatment. Since the outbreak began in March 2020, India has reported 4.77 lakh Coronavirus deaths, according to the World Health Organization (WHO). The innate RNA interference (RNAi) pathway, on the other hand, allows for the development of nucleic acid-based antiviral drugs in which complementary small interfering RNAs (siRNAs) mediate the post-transcriptional gene silencing (PTGS) of target mRNA. Therefore, in this current study, the potential of RNAi was harnessed to construct siRNA molecules that target the consensus regions of specific structural proteins associated genes of SARS-CoV-2, such as the envelope protein gene (E), membrane protein gene (M), nucleocapsid phosphoprotein gene (N), and surface glycoprotein gene (S) which are important for the viral pathogenesis. Conserved sequences of 811 SARS-CoV-2 strains from around India were collected to design 21 nucleotides long siRNA duplex based on various computational algorithms and parameters targeting E, M, N and S genes. The proposed siRNA molecules possessed sufficient nucleotide-based and other features for effective gene silencing and BLAST results revealed that siRNAs\u2019 targets have no significant matches across the whole human genome and hence, siRNAs were found to have no off-target effects on the genome, ruling out the possibility of off-target silencing. Finally, out of 157 computationally identified siRNAs, only 4 effective siRNA molecules were selected for each target gene which is proposed to exert the best action based on GC content, free energy of folding, free energy of binding, melting temperature, heat capacity and molecular docking analysis with Human AGO2 protein. Our engineered siRNA candidates could be used as a genome-level therapeutic treatment against various sequenced SARS-CoV-2 strains in India. However, future applications will necessitate additional validations in vitro and in vivo animal models.","version":"1.2","doi":"10.1101/2022.02.08.479559","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481436","pub_date":"2022-2-23","title":"Characterization of various remdesivir-resistant mutations of SARS-CoV-2 by mathematical modeling and molecular dynamics simulation","abstract":"Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection. Considering the emerging Omicron strain, quick characterization of SARS-CoV-2 mutations is important. However, owing to the difficulties in genetically modifying SARS-CoV-2, limited groups have produced multiple mutant viruses. Our cutting-edge reverse genetics technique enabled construction of eight reporter-carrying mutant SARS-CoV-2 in this study. We developed a mathematical model taking into account sequential changes and identified antiviral effects against mutant viruses with differing propagation capacities and lethal effects on cells. In addition to identifying the positions of mutations, we analyzed the structural changes in SARS-CoV-2 NSP12 by computer simulation to understand the mechanism of resistance. This multidisciplinary approach promotes the evaluation of future resistance mutations.","version":"1.1","doi":"10.1101/2022.02.22.481436","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481262","pub_date":"2022-2-23","title":"Serological screening in wild ruminants in Germany, 2021/22: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus","abstract":"Wildlife animals may be susceptible for multiple infectious agents of public health or veterinary relevance, thereby potentially forming a reservoir that bears the constant risk of re-introduction into the human or livestock population. Here, we serologically investigated 493 wild ruminant samples collected in the 2021/22 hunting season in Germany for the presence of antibodies against the severe acute respiratory coronavirus 2 (SARS-CoV-2) and four viruses pathogenic for domestic ruminants, namely the orthobunyavirus Schmallenberg virus (SBV), the reovirus bluetongue virus (BTV) and ruminant pestiviruses like bovine viral diarrhoea virus or border disease virus. The animal species comprised fallow deer, red deer, roe deer, mouflon and wisent. For coronavirus serology, additional 307 fallow, roe and red deer samples collected between 2017 and 2020 at three military training areas were included. While antibodies against SBV could be detected in about 13.6% of the samples collected in 2021/22, only one fallow deer of unknown age tested positive for anti-BTV antibodies and all samples reacted negative for antibodies against ruminant pestiviruses. In an ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2, 25 out of 493 (5.1%) samples collected in autumn and winter 2021/22 scored positive. This sero-reactivity could not be confirmed by the highly specific virus neutralization test, occurred also in 2017, 2018 and 2019, i.e. prior to the human SARS-CoV-2 pandemic, and was likewise observed against the RBD of the related SARS-CoV-1. Therefore, the SARS-CoV-2-seroreactivity was most likely induced by another, hitherto unknown deer virus belonging to the subgenus Sarbecovirus of betacoronaviruses.","version":"1.1","doi":"10.1101/2022.02.21.481262","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481485","pub_date":"2022-2-23","title":"Confirming Multiplex Q-PCR Use in COVID-19 with Next Generation Sequencing: Strategies for Epidemiological Advantage","abstract":"Rapid classification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the Variants of Concern and tracking this pandemic. However, NGS remains expensive and time-consuming for large-scale monitoring of COVID-19. This study analyzed a total of 78 de-identified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole genome sequencing then compared with two commercially available Q-PCR assays for spike protein mutation(s). The data showed good concordance with Q-PCR and NGS analysis for specific SARS-COV-2 lineages and characteristic mutations. Deployment of Q-PCR testing to detect known SARS-COV-2 variants may be extremely beneficial. These assays are quick and cost-effective, thus can be implemented as an alternative to sequencing for screening known mutations of SARS-COV-2 for clinical and epidemiological interest. The findings support the great potential for Q-PCR to be an effective strategy offering several COVID-19 epidemiological advantages.","version":"1.1","doi":"10.1101/2022.02.22.481485","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.22.481430","pub_date":"2022-2-23","title":"SARS-CoV-2 Variant Delta Potently Suppresses Innate Immune Response and Evades Interferon-Activated Antiviral Responses","abstract":"Delta variant of SARS-CoV-2 has caused more severe infections than its previous variants. We studied the host innate immune response to Delta, Alpha and two earlier variants to map the evolution of the recent ones. Our biochemical and transcriptomic studies reveal that Alpha and Delta have progressively evolved over the ancestral variants by silencing innate immune response, thereby limiting cytokine and chemokine production. Though Alpha silenced RLR pathway just as Delta, it failed to persistently silence the innate immune response unlike Delta. Both Alpha and Delta have evolved to resist IFN treatment while they are still susceptible to RLR activation, further highlighting the importance of RLR-mediated, IFN-independent mechanisms in restricting SARS-CoV-2. Our studies reveal that SARS-CoV-2 Delta has integrated multiple mechanisms to silence host innate immune response and evade IFN response. Delta\u2019s silent replication and sustained suppression of host innate immune response, possibly resulting in delayed or reduced intervention by the adaptive immune response, could potentially contribute to the severe symptoms and poor recovery index associated with it.","version":"1.1","doi":"10.1101/2022.02.22.481430","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.16.480524","pub_date":"2022-2-23","title":"Experimental infection of mink with SARS-COV-2 Omicron (BA.1) variant leads to symptomatic disease with lung pathology and transmission","abstract":"We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that minks remain virus RNA positive for days, develop clinical signs and histopathological changes, and transmit the virus to uninfected recipients warranting further studies and preparedness.","version":"1.2","doi":"10.1101/2022.02.16.480524","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481360","pub_date":"2022-2-23","title":"Effect of an amyloidogenic SARS-COV-2 protein fragment on \u03b1-synuclein monomers and fibrils","abstract":"Using molecular dynamic simulations we study whether amyloidogenic regions in viral proteins can initiate and modulate formation of \u03b1-synuclein aggregates, thought to be the disease-causing agent in Parkinson\u2019s Disease. As an example we choose the nine-residue fragment SFYVYSRVK (SK9), located on the C-terminal of the Envelope protein of SARS-COV-2. We probe how the presence of SK9 affects the conformational ensemble of \u03b1-synuclein monomers and the stability of two resolved fibril polymorphs. We find that the viral protein fragment SK9 may alter \u03b1-synuclein amyloid formation by shifting the ensemble toward aggregation-prone and preferentially rod-like fibril seeding conformations. However, SK9 has only little effect of the stability of pre-existing or newly-formed fibrils.","version":"1.1","doi":"10.1101/2022.02.21.481360","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481324","pub_date":"2022-2-23","title":"A single-dose of the deactivated rabies virus vectored COVID-19 vaccine, CORAVAX, is highly efficacious and alleviates lung inflammation in the hamster model","abstract":"Without sufficient herd immunity through either vaccination or natural infection, the Coronavirus Disease 2019 pandemic is unlikely to be controlled. Waning immunity with the currently approved vaccines suggests the need to evaluate vaccines causing the induction of long-term responses. Here we report the immunogenicity and efficacy of our adjuvanted single-dose Rabies vectored SARS CoV-2 S1 vaccine, CORAVAX, in hamsters. CORAVAX induces high SARS CoV-2 S1 specific and virus-neutralizing antibodies (VNA) that prevent weight loss, viral loads, disease, lung inflammation, and the cytokine storm in hamsters. We also observed high Rabies VNA titers. In summary, CORAVAX is a promising dual antigen vaccine candidate for clinical evaluation against SARS CoV-2 and Rabies virus.","version":"1.1","doi":"10.1101/2022.02.21.481324","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481311","pub_date":"2022-2-23","title":"Biophysical fitness landscape of the SARS-CoV-2 Delta variant receptor binding domain","abstract":"Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased number of deaths. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single mutations as well as the double mutation on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than receptor binding, is the main biophysical parameter determining the fitness landscape of the Delta variant RBD and is determined by the L452R mutation.","version":"1.1","doi":"10.1101/2022.02.21.481311","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.18.481096","pub_date":"2022-2-23","title":"Omicron and Alpha P680H block SARS-CoV2 spike protein from accessing cholinergic inflammatory pathway via \u03b19-nAChR mitigating the risk of MIS-C","abstract":"Sequence homology between neurotoxins and the site encompassing the furin cleavage site 680SPRRAR685 in the spike protein (S) of CoV2 suggested that this site could interact with nicotinic acetylcholine receptors (nAChRs). Molecular dynamics simulations confirm robust structural similarity between wild-type (WT) CoV2 and the binding motif of \u03b1-conotoxin to \u03b19 nAChR, which is known to modulate IL-1\u03b2 in immune cells. We show that the structural integrity of this binding motif is eliminated by Alpha P681H mutation, reemerged in Delta variant P681R, and disappeared again with Omicron N679H/P681H. Interactions between the toxin-mimic CoV2 motif and \u03b19-nAChR are expected to trigger the release of pro-inflammatory cytokines an effect that is mollified by Alpha and Omicron. Clinical features of this interaction site are relevant because, contrary to most regions in the S protein, the furin binding site does not appear to trigger an immune response prior to cleavage, indicating that the cholinergic pathway should be activated in the respiratory tract and nasal mucosa where \u03b19-nAChR co-localizes with the virus. The correlation of changes on this motif by the different variants closely matches the reported cases of Multisystem Inflammatory Syndrome in Children by the CDC, and predicts significant mitigation of MIS-C with the Omicron variant. Our findings strongly motivate further study of this cholinergic pathway as one source of the cytokine storm triggered by CoV2.","version":"1.1","doi":"10.1101/2022.02.18.481096","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481269","pub_date":"2022-2-23","title":"An in-silico study of the mutation-associated effects on the spike protein of SARS-CoV-2, Omicron variant","abstract":"The emergence of Omicron (B.1.1.529), a new Variant of Concern in the COVID-19 outbreak, while accompanied by the ongoing Delta variant infection, has once again fueled fears of a new infection wave and global health concern. In the Omicron variant, the receptor-binding domain (RBD) of its spike glycoprotein is heavily mutated, a feature critical for the transmission rate of the virus by interacting with hACE2. In this study, we used a combination of conventional and advanced neural network-based in silico approaches to predict how these mutations would affect the spike protein. The results demonstrated a decrease in the electrostatic potentials of residues corresponding to receptor recognition sites, an increase in the alkalinity of the protein, a change in hydrophobicity, variations in functional residues, and an increase in the percentage of alpha-helix structure. Our next step was to predict the structural changes of the spike protein using the AI-based tool Alphafold2 and dock it with hACE2. The results revealed that the RBD of the Omicron variant had a higher affinity than the reference. Moreover, all-atom molecular dynamics simulations concluded that the RBD of the Omicron variant exhibits a more dispersed interaction network since mutations resulted in an increased number of hydrophobic interactions and hydrogen bonds with hACE2 compared to the reference RBD. In summary, our current study highlighted the potential structural basis for the enhanced transmissibility and pathogenicity of the Omicron variant, although further research is needed to investigate its epidemiological and biological implications.","version":"1.1","doi":"10.1101/2022.02.21.481269","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.19.481110","pub_date":"2022-2-23","title":"Omicron booster in ancestral strain vaccinated mice augments protective immunities against both the Delta and Omicron variants","abstract":"A booster vaccination is called for constraining the evolving epidemic of SARS-CoV-2. However, the necessity of a new COVID-19 vaccine is currently unclear. To compare the effect of an Omicron-matched S DNA vaccine and an ancestral S DNA vaccine in boosting cross-reactive immunities, we firstly immunized mice with two-dose of a DNA vaccine encoding the spike protein of the ancestral Wuhan strain. Then the mice were boosted with DNA vaccines encoding spike proteins of either the Wuhan strain or the Omicron variant. Specific antibody and T cell responses were measured at 4 weeks post boost. Our data showed that the Omicron-matched vaccine efficiently boosted RBD binding antibody and neutralizing antibody responses against both the Delta and the Omicron variants. Of note, antibody responses against the Omicron variant elicited by the Omicron-matched vaccine were much stronger than those induced by the ancestral S DNA vaccine. Meanwhile, CD8+ T cell responses against both the ancestral Wuhan strain and the Omicron strain also tended to be higher in mice boosted by the Omicron-matched vaccine than those in mice boosted with the ancestral S DNA vaccine, albeit no significant difference was observed. Our findings suggest that an Omicron-matched vaccine is preferred for boosting cross-reactive immunities.","version":"1.2","doi":"10.1101/2022.02.19.481110","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.19.481107","pub_date":"2022-2-22","title":"Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron","abstract":"To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.","version":"1.1","doi":"10.1101/2022.02.19.481107","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.03.479007","pub_date":"2022-2-22","title":"VE607 Stabilizes SARS-CoV-2 Spike In the \u201cRBD-up\u201d Conformation and Inhibits Viral Entry","abstract":"SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its \u201cup\u201d conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.","version":"1.2","doi":"10.1101/2022.02.03.479007","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.10.455737","pub_date":"2022-2-22","title":"Potential autoimmunity resulting from molecular mimicry between SARS-CoV-2 Spike and human proteins","abstract":"SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. We computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins such as PRKG1 and tropomyosin. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.","version":"1.3","doi":"10.1101/2021.08.10.455737","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.24.440952","pub_date":"2022-2-22","title":"Household transmission of SARS-CoV-2 from humans to pets in Washington and Idaho: burden and risk factors","abstract":"SARS-CoV-2 is believed to have emerged from an animal reservoir; however, the frequency of and risk factors for inter-species transmission remain unclear. We carried out a community-based study of pets in households with one or more confirmed SARS-CoV-2 infection in humans. Among 119 dogs and 57 cats with completed surveys, clinical signs consistent with SARS-CoV-2 were reported in 20 dogs (21%) and 19 cats (39%). Out of 81 dogs and 32 cats sampled for testing, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive; this discordance may be due to delays in sampling. Respondents commonly reported close human-animal contact and willingness to take measures to prevent transmission to their pets. Reported preventative measures showed a slightly protective trend for both illness and seropositivity in pets, while sharing of beds and bowls had slight harmful effects.","version":"1.2","doi":"10.1101/2021.04.24.440952","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481175","pub_date":"2022-2-22","title":"Alterations in SARS-CoV-2 Omicron and Delta peptides presentation by HLA molecules","abstract":"The T-cell immune response is a major determinant of effective SARS-CoV-2 clearance. Here, using the recently developed T-CoV bioinformatics pipeline (https://t-cov.hse.ru) we analyzed the peculiarities of the viral peptide presentation for the Omicron, Delta and Wuhan variants of SARS-CoV-2. First, we showed the absence of significant differences in the presentation of SARS-CoV-2-derived peptides by the most frequent HLA class I/II alleles and the corresponding HLA haplotypes. Then, the analysis was limited to the set of peptides originating from the Spike proteins of the considered SARS-CoV-2 variants. The major finding was the destructive effect of the Omicron mutations on PINLVRDLPQGFSAL peptide, which was the only tight binder from the Spike protein for HLA-DRB1*03:01 allele and some associated haplotypes. Specifically, we predicted a dramatical decline in binding affinity of HLA-DRB1*03:01 and this peptide after N211 deletion, L212I substitution and EPE 212-214 insertion. The computational prediction was experimentally validated by ELISA with the use of corresponding thioredoxin-fused peptides and recombinant HLA-DR molecules. Another finding was the significant reduction in the number of tightly binding Spike peptides for HLA-B*07:02 HLA class I allele (both for Omicron and Delta variants). Overall, the majority of HLA alleles and haplotypes was not significantly affected by the mutations, suggesting the maintenance of effective T-cell immunity against the Omicron and Delta variants. Finally, we introduced the Omicron variant to T-CoV portal and added the functionality of haplotype-level analysis to it.","version":"1.1","doi":"10.1101/2022.02.21.481175","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.20.480711","pub_date":"2022-2-22","title":"Modular capsid decoration boosts adenovirus vaccine-induced humoral and cellular immunity against SARS-CoV-2","abstract":"Adenovirus vector vaccines have been widely and successfully deployed in response to COVID-19. However, despite inducing potent T cell immunity, improvement of vaccine-specific antibody responses upon homologous boosting is modest compared to other technologies. Here, we describe a system to enable modular decoration of adenovirus capsid surfaces with protein antigens and demonstrate induction of potent humoral immunity against these displayed antigens. Ligand attachment via a covalent isopeptide bond was achieved in a rapid and spontaneous reaction, requiring simple co-incubation of ligand and vector components. We used a recently described protein superglue, DogTag/DogCatcher, which is similar to the widely used SpyTag/SpyCatcher ligation system but performs better in loop structures. DogTag was inserted into surface-exposed loops in the adenovirus hexon protein to allow attachment of DogCatcher-fused ligands on virus particles. Efficient coverage of the capsid surface was achieved using a variety of ligands and vector infectivity was retained in each case. Capsid decoration shielded particles from anti-adenovirus neutralizing antibodies. In prime-boost regimens, proof-of-concept COVID-19 adenovirus vaccines decorated with the receptor-binding domain (RBD) of SARS-CoV-2 spike induced >10-fold higher SARS-CoV-2 neutralization titers compared to an undecorated adenovirus vector encoding spike. Importantly, decorated vectors retained robust T cell immunogenicity to encoded antigens, a key hallmark of adenovirus vector vaccines. We propose capsid decoration via protein superglue-mediated covalent ligation as a novel strategy to improve the efficacy and boostability of adenovirus-based vaccines and therapeutics. Decorating the capsid surface of adenovirus vaccine vectors using a spontaneous protein superglue induces potent pathogen-specific immunity","version":"1.1","doi":"10.1101/2022.02.20.480711","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.474009","pub_date":"2022-2-22","title":"Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation","abstract":"Phosphorylated within its Ser/Arg-rich region, the SARS-CoV-2 nucleoprotein (N) recruits the phosphopeptide-binding human 14-3-3 proteins that play a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that phosphorylation of SARS-CoV-2 N at either of two pseudo-repeats centered at Ser197 and Thr205 is sufficient for 14-3-3 binding. According to fluorescence anisotropy, the pT205-motif, present in SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which display unforeseen pT205/pS197 binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data indicate 14-3-3 binding occludes the Ser/Arg-rich region, inhibiting its dephosphorylation. This Ser/Arg-rich region of N is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting how the strength of its 14-3-3 binding can be linked with the replicative fitness of the virus.","version":"1.3","doi":"10.1101/2021.12.23.474009","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472725","pub_date":"2022-2-22","title":"Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection","abstract":"Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.","version":"1.2","doi":"10.1101/2021.12.14.472725","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473804","pub_date":"2022-2-22","title":"Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant","abstract":"The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge. Omicron, carrying \u223c30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.","version":"1.2","doi":"10.1101/2021.12.22.473804","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.19.481139","pub_date":"2022-2-22","title":"SARS-CoV-2 Viroporins Activate The NLRP3-Inflammasome Via The Mitochondrial Permeability Transition Pore","abstract":"Cytokine storm precipitated by activation of the host innate immune defenses is a major cause of COVID19 death. To elucidate how SARS-CoV-2 initiates this inflammatory process, we studied viroporin proteins E and Orf3a (2-E+2-3a). Expression of 2-E+2-3a in human 293T cells resulted in increased cytosolic Ca++ and then elevated mitochondrial Ca++, taken up through the MUCi11-sensitive mitochondrial calcium uniporter (MCU). Increased mitochondrial Ca++ resulted in stimulation of mitochondrial reactive oxygen species (mROS) production, which was blocked by mitochondrially-targeted catalase or MnTBAP. To determined how mROS activates the inflammasome, we transformed 293T cells with NLRP3, ASC, pro-caspase-1 and pro-IL-1\u03b2 plus used THP1 derived macrophages to monitor the secretion of mature IL-1\u03b2. This revealed that mROS activates a factor that is released via the NIM811-sensitive mitochondrial permeability pore (mtPTP) to activate the inflammasome. Hence, interventions targeting mROS and the mtPTP may mitigate the severity of COVID19 cytokine storms.","version":"1.1","doi":"10.1101/2022.02.19.481139","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.05.471263","pub_date":"2022-2-22","title":"The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation","abstract":"The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.","version":"1.3","doi":"10.1101/2021.12.05.471263","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.19.481089","pub_date":"2022-2-22","title":"Targeted Down Regulation Of Core Mitochondrial Genes During SARS-CoV-2 Infection","abstract":"Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19. Covid-19 is associated with targeted inhibition of mitochondrial gene transcription.","version":"1.1","doi":"10.1101/2022.02.19.481089","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.20.481163","pub_date":"2022-2-22","title":"Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine","abstract":"Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for \u223c9-10 months after the primary 2-dose mRNA vaccine series, as well as for \u223c3 months after a 3rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and \u223c40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3rd dose. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.","version":"1.1","doi":"10.1101/2022.02.20.481163","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.21.481356","pub_date":"2022-2-22","title":"Supervised Rank aggregation (SRA): A novel rank aggregation approach for ensemble-based feature selection","abstract":"Feature selection (FS) is critical for high dimensional data analysis. Ensemble based feature selection (EFS) is a commonly used approach to develop FS techniques. Rank aggregation (RA) is an essential step of EFS where results from multiple models are pooled to estimate feature importance. However, the literature primarily relies on rule-based methods to perform this step which may not always provide an optimal feature set. This study proposes a novel Supervised Rank Aggregation (SRA) approach to allow RA step to dynamically learn and adapt the model aggregation rules to obtain feature importance. The approach creates a performance matrix containing feature and model performance value from all models and prepares a supervised learning model to get the feature importance. Then, unsupervised learning is performed to select the features using their importance. We evaluate the performance of the algorithm using simulation studies and implement it into real research studies, and compare its performance with various existing RA methods. The proposed SRA method provides better or at par performance in terms of feature selection and predictive performance of the model compared to existing methods. SRA method provides an alternative to the existing approaches of RA for EFS. While the current study is limited to the continuous cross-sectional outcome, other endpoints such as longitudinal, categorical, and time-to-event medical data could also be used.","version":"1.1","doi":"10.1101/2022.02.21.481356","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.22271030","pub_date":"2022-02-19","title":"Clinical severity of Omicron sub-lineage BA.2 compared to BA.1 in South Africa","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>Early data indicated that infection with Omicron BA.1 sub-lineage was associated with a lower risk of hospitalisation and severe illness, compared to Delta infection. Recently, the BA.2 sub-lineage has increased in many areas globally. We aimed to assess the severity of BA.2 infections compared to BA.1 in South Africa. We performed data linkages for (i) national COVID-19 case data, (ii) SARS-CoV-2 laboratory test data, and (iii) COVID-19 hospitalisations data, nationally. For cases identified using TaqPath COVID-19 PCR, infections were designated as S-gene target failure (SGTF, proxy for BA.1) or S-gene positive (proxy for BA.2). Disease severity was assessed using multivariable logistic regression models comparing individuals with S-gene positive infection to SGTF-infected individuals diagnosed between 1 December 2021 to 20 January 2022. From week 49 (starting 5 December 2021) through week 4 (ending 29 January 2022), the proportion of S-gene positive infections increased from 3% (931/31,271) to 80% (2,425/3,031). The odds of being admitted to hospital did not differ between individuals with S-gene positive (BA.2 proxy) infection compared to SGTF (BA.1 proxy) infection (adjusted odds ratio (aOR) 0.96, 95% confidence interval (CI) 0.85-1.09). Among hospitalised individuals, after controlling for factors associated with severe disease, the odds of severe disease did not differ for individuals with S-gene positive infection compared to SGTF infection (aOR 0.91, 95%CI 0.68-1.22). These data suggest that while BA.2 may have a competitive advantage over BA.1 in some settings, the clinical profile of illness remains similar.</jats:p>","version":null,"doi":"10.1101/2022.02.17.22271030","journal":"medRxiv","score":null},{"id":"10.1101/2022.02.17.480826","pub_date":"2022-2-18","title":"Genomic characterization of SARS-CoV-2 from Islamabad, Pakistan by Rapid Nanopore sequencing","abstract":"Since the start of COVID-19 pandemic, Pakistan has experienced four waves of pandemic. The fourth wave ended in October, 2021 while the fifth wave of pandemic starts in January, 2022. The data regarding the circulating strains after the fourth wave of pandemic from Pakistan is not available. The current study explore the genomic diversity of SARS-CoV-2 after fourth wave and before fifth wave of pandemic through whole genome sequencing. The results showed the circulation of different strains of SARS-CoV-2 during November-December, 2021. We have Omicron BA.1 (n=4), Lineage A (n=2) and delta AY.27 (n=1) variants of SARS-CoV-2 in the population of Islamabad. All the isolates harbors characteristics mutations of omicron and delta variant in the genome. The lineage A isolate harbors a nine amino acid (68-76) and a ten amino acid (679-688) deletion in the genome. The circulation of omicron in the population before the fifth wave of pandemic and subsequent upsurges of COVID-19 positive cases in Pakistan highlights the importance of genomic surveillance.","version":"1.1","doi":"10.1101/2022.02.17.480826","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.18.480872","pub_date":"2022-2-18","title":"SARS-CoV-2 has not emerged in roe, red or fallow deer in Germany or Austria during the COVID 19 pandemic","abstract":"Spillover of SARS-CoV-2 to North American white tailed deer (Odocoileus virginianus) has been documented. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white tailed deer, we found no evidence of SARS-CoV-2 exposure. Using a sensitive serological assay, 433 pre and pandemic deer samples from Germany and Austria tested negative for SARS-CoV-2-specific antibodies highlighting a major difference between central European and North American deer exposure and in their epidemiologic roles.","version":"1.1","doi":"10.1101/2022.02.18.480872","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.18.480994","pub_date":"2022-2-18","title":"Mosaic receptor-binding domain nanoparticles induce protective immunity against SARS-CoV-2 challenges","abstract":"Recurrent spillovers of \u03b1- and \u03b2-coronaviruses (CoV) such as acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, SARS-CoV-2, and possibly human CoV (NL63, 229E, OC43, and HKU1) have caused serious morbidity and mortality worldwide. Six receptor binding domains (RBDs) derived from \u03b1- and \u03b2-CoV that are considered to have originated from animals and cross-infected humans were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric subunits, PCNA1, PCNA2, and PCNA3. These were used to form a scaffold-based mosaic multivalent antigen, 6RBD-np. Electron microscopic and atomic force microscopic images show a ring-shaped disk with six protruding RBDs, like jewels in a crown, with a size of 40 nm. Prime-boost immunizations with 6RBD-np in BALB/c mice elicited strong, dose-dependent antibody responses. In human angiotensin converting enzyme 2-transgenic mice, the same immunization induced full-protection against SARS-CoV-2 wild type and Delta challenges, resulting in a 100% survival rate. The mosaic 6RBD-np provides a potential platform for developing a pan-CoV vaccine against newly emerging SARS-CoV-2 variants and future CoV spillovers. Despite the arsenal of COVID-19 vaccines, hospitalization and mortality associated with SARS-CoV-2 (acute respiratory syndrome coronavirus 2) variants remain high. There is an urgent need to develop next-generation COVID vaccines that provide broad protection against diseases by current and newly emerging SARS-CoV-2 variants. In this study, six receptor binding domains (RBDs) derived from \u03b1- and \u03b2-CoV were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric scaffolds. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens. The prime-boost immunization in BALB/c and human angiotensin converting enzyme 2-transgenic mice with the 6RBD-np elicited strong, dose-dependent antibody responses and induced full-protection against both the SARS-CoV-2 wild type (WT) and Delta challenges. This study provides proof-of-concept that the mosaic 6RBD-np induces 100% protection against SARS-CoV-2 WT and Delta. It provides the potential of co-displaying heterologous antigens for novel vaccine designs, which can be deployed countering future pandemics.","version":"1.1","doi":"10.1101/2022.02.18.480994","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.480819","pub_date":"2022-2-18","title":"Metagenomic analysis reveals the abundance and diversity of opportunistic fungal pathogens in the nasopharyngeal tract of COVID-19 patients","abstract":"The nasopharyngeal tract (NT) of human is a habitat of a diverse microbial community that work together with other gut microbes to maintain the host immunity. In our previous study, we reported that SARS-CoV-2 infection reduces human nasopharyngeal commensal microbiome (bacteria, archaea and commensal respiratory viruses) but increases the abundance of pathobionts. This study aimed to assess the possible changes in the resident fungal diversity by the inclusion of opportunistic fungi due to the infection of SARS-CoV-2 in the NT of humans. Twenty-two (n = 22) nasopharyngeal swab samples (including COVID-19 = 8, Recovered = 7, and Healthy = 7) were collected for RNAseq-based metagenomics analyses. Our results indicate that SARS-CoV-2 infection significantly increased (p < 0.05, Wilcoxon test) the population and diversity of NT fungi with a high inclusion of opportunistic pathogens. We detected 863 fungal species including 533, 445, and 188 species in COVID-19, Recovered, and Healthy individuals, respectively that indicate a distinct microbiome dysbiosis due to the SARS-CoV-2 infection. Remarkably, 37% of the fungal species were exclusively associated with SARS-CoV-2 infection, where S. cerevisiae (88.62%) and Phaffia rhodozyma (10.30%) were two top abundant species in the NT of COVID-19 patients. Importantly, 16% commensal fungal species found in the Healthy control were not detected in either COVID-19 patients or when they were recovered from the COVID-19. Pairwise Spearman\u2019s correlation test showed that several altered metabolic pathways had significant positive correlations (r > 0.5, p < 0.01) with dominant fungal species detected in three metagenomes. Taken together, our results indicate that SARS-CoV-2 infection causes significant dysbiosis of fungal microbiome and alters some metabolic pathways and expression of genes in the NT of human. Findings of our study might be helpful for developing microbiome-based diagnostics, and also devising appropriate therapeutic regimens including antifungal drugs for prevention and control of concurrent fungal coinfections in COVID-19 patients. The SARS-CoV-2 is a highly transmissible and pathogenic betacoronavirus that primarily enters into the human body through NT to cause fearsome COVID-19 disease. Recent high throughput sequencing and downstream bioinformatic analyses revealed that microbiome dysbiosis associated with SARS-CoV-2 infection are not limited to bacteria, and fungi are also implicated in COVID-19 development in susceptible individuals. This study demonstrates that SARS-CoV-2 infection results in remarkable depletion of NT commensal fungal microbiomes with inclusion of various opportunistic fungal pathogens. We discussed the role of these altered fungal microbiomes in the pathophysiology of the SARS-CoV-2 infection. Our results suggest that dysbiosis in fungal microbiomes and associated altered metabolic functional pathways (or genes) possibly play a determining role in the progression of SARS-CoV-2 pathogenesis. Thus, the identifiable changes in the diversity and composition of the NT fungal population and their related genomic features demonstrated in this study might lay a foundation for better understanding of the underlying mechanism of co-pathogenesis, and the ongoing development of therapeutic agents including antifungal drugs for the resolution of COVID-19 pandemic.","version":"1.1","doi":"10.1101/2022.02.17.480819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.15.480592","pub_date":"2022-2-18","title":"SARS-CoV-2 variants show a gradual declining pathogenicity and pro-inflammatory cytokine spur, an increasing antigenic and antiinflammatory cytokine induction, and rising structural protein instability","abstract":"Hyper-transmissibility with decreased disease severity are typical characteristics of Omicron variant. To understand this phenomenon, we used various bioinformatics approaches to analyze randomly selected genome sequences (one each) of the Gamma, Delta, and Omicron variants submitted to NCBI from 15 to 31 December 2021. We show that: (i) Pathogenicity of SARS-CoV-2 variants decreases in the order: Wuhan > Gamma > Delta > Omicron; however, the antigenic property follows the order: Omicron > Gamma > Wuhan > Delta. (ii) Omicron Spike RBD has lower pathogenicity but higher antigenicity than other variants. (iii) Decreased disease severity by Omicron variant may be due to its decreased pro-inflammatory and IL-6 stimulation and increased IFN-\u03b3 and IL-4 induction efficacy. (iv) Mutations in N protein are associated with decreased IL-6 induction and human DDX21-mediated increased IL-4 production in Omicron. (v) Due to mutations, the stability of S, M, N, and E proteins decreases in the order: Omicron > Gamma > Delta > Wuhan. (vi) Stronger Spike RBD-hACE2 binding in Omicron is associated with increased transmissibility. However, the lowest stability of the Omicron Spike protein makes Spike RBD-hACE2 interaction weak for systemic infection and for causing severe disease. Finally (vii), the highest instability of Omicron E protein may also be associated with decreased viral maturation and low viral load leading to less severe disease and faster recovery. Our method may be used for other similar viruses, and these findings will contribute to the understanding of the dynamics of SARS-CoV-2 variants and the management of emerging variants.","version":"1.2","doi":"10.1101/2022.02.15.480592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453908","pub_date":"2022-2-18","title":"The Achilles\u2019 heel of coronaviruses: targeting the 5\u2019 Polyuridines tract of the antigenome to inhibit Mouse coronavirus-induced cell death","abstract":"The current coronavirus pandemic situation is worsened by the rapidly-spreading SARS-CoV-2 virus variants. Identification of viral targets that are indispensable for the virus can be targeted to inhibit mutation-based new escape variant development. The 5\u2019-polyU tract of the antigenome offers such a target. Host cells do not harbor 5\u2019-polyU tracts on any of their transcripts, making the tract an attractive, virus-specific target. Inhibiting the 5\u2019-polyU can limit the use of the tract as template to generate 3\u2019 polyA tails of +RNAs of coronaviruses. Here, a modified DNA oligo with 3\u2019 polyAs is used to target the 5-polyU tract in mouse coronavirus (MHV-A59). The oligo treatment in mouse 17CL-1 cells infected with MHV-A59 significantly prevented virus-induced cell deaths. This proof-of-concept result shows a unique mode of action against mouse coronavirus without affecting host cells, and can be used for the development of novel classes of drugs that inhibit coronavirus infection in host cells, specifically by the COVID-19-causing virus SARS-CoV-2. In addition, as the 5\u2019-polyU tract is immediately generated upon infection, the tag can also be targeted for reliable early detection of viral infection.","version":"1.3","doi":"10.1101/2021.07.26.453908","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.480845","pub_date":"2022-2-18","title":"Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity","abstract":"During the RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10 mediated modulation remains unclear in the absence of nsp14 structure. Here we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10 bound structures explain the modulatory role of the co-factor protein. Further, the structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.","version":"1.1","doi":"10.1101/2022.02.17.480845","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.480939","pub_date":"2022-2-18","title":"On Correlation between Structural Properties and Viral Escape Measurements from Deep Mutational Scanning","abstract":"Encouraged by recent efforts to map responses of SARS-CoV-2 mutations to various antibody treatments with deep mutational scanning, we explored the possibility of tying measurable structural contact information from the binding complexes of antibodies and their targets to experimentally determined viral escape responses. With just a single crystal structure for each binding complex, we find that the average correlation coefficient R is surprisingly high at 0.76. Our two methods for calculating contact information use binary contacts measured between all residues of two proteins. By varying the parameters to obtain binary contacts, we find that 3.6 \u00c5 and 7 \u00c5 are pivotal distances to toggle the binary step function when tallying the contacts for each method. The correlations are improved by short simulations (\u223c25 ns), which increase average R to 0.78. With blind tests using the random forest model, we can further improve average R to 0.84. These easy-to-implement measurements can be utilized in computational screening of viral mutations that escape antibody treatments and potentially other protein-protein interaction problems.","version":"1.1","doi":"10.1101/2022.02.17.480939","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.480751","pub_date":"2022-2-17","title":"An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants","abstract":"The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity. A human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants","version":"1.1","doi":"10.1101/2022.02.17.480751","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.479764","pub_date":"2022-2-17","title":"Biomechanical dependence of SARS-CoV-2 infections","abstract":"Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome, including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, i.e., the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly.","version":"1.1","doi":"10.1101/2022.02.17.479764","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.16.480801","pub_date":"2022-2-17","title":"A natural broad-spectrum inhibitor of enveloped virus entry, effective against SARS-CoV-2 and Influenza A Virus in preclinical animal models","abstract":"The COVID-19 pandemic has highlighted the need for novel antivirals for pandemic management and preparedness. Targeting host processes that are co-opted by viruses is an attractive strategy for developing antivirals with a high resistance barrier. Picolinic acid (PA) is a byproduct of tryptophan metabolism, endogenously produced in humans and other mammals. Here we report broad-spectrum antiviral effects of PA against enveloped viruses, including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), Influenza A virus (IAV), Flaviviruses, Herpes Simplex Virus, and Human Parainfluenza Virus. We further demonstrate using animal models that PA is effective against SARS-CoV-2 and IAV, especially as an oral prophylactic. The mode of action studies revealed that PA inhibits viral entry of enveloped viruses, primarily by interfering with viral-cellular membrane fusion, inhibiting virus-mediated syncytia formation, and dysregulating cellular endocytosis. Overall, our data establish PA as a broad-spectrum antiviral agent, with promising preclinical efficacy against pandemic viruses SARS-CoV-2 and IAV.","version":"1.1","doi":"10.1101/2022.02.16.480801","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.16.480759","pub_date":"2022-2-17","title":"Bioinformatic analysis of B and T cell epitopes from SARS-CoV-2 Spike, Membrane and Nucleocapsid proteins as a strategy to assess possible cross-reactivity between emerging variants, including Omicron, and other human coronaviruses","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 produced a global health emergency since December 2019, that up to the end of January 2022 had caused the death of more than 5.6 million people worldwide. Despite emergence of new variants of concern, vaccination remains one of the most important tools to control the pandemic. All approved vaccines and most of the vaccine candidates use the spike protein of the virus as a target antigen to induce protective immune responses. Several variants of the virus present key mutations in this protein which render the virus, at different rates, to evade the neutralizing antibody response. Although experimental evidence suggests that cross-reactive responses between coronaviruses are present in the population, it is unknown which potential antigens shared between different coronaviruses could be responsible for these responses. This study provides predictions of new potential B and T cell epitopes within SARS-CoV-2 Spike (S), Membrane (M) and Nucleocapsid (N) proteins together with a review of the reported B epitopes of these proteins. We also analyse amino acid changes present in the epitopes of variants of concern (VOC) and variants being monitored (VBM), and how these might affect the immune response, as these changes may alter the peptides\u2019 immunogenicity index and the antigen presentation by related HLA alleles. Finally, given these observations, we performed an identity analysis between the repertoire of potential epitopes of SARS-CoV-2 and other human coronaviruses to identify which are conserved among them. The results shown here together with the published experimental evidence, allow us to support the hypothesis that antibody and T cell cross-reactive responses to common coronaviruses epitopes, could contribute to broaden the protective response to SARS-CoV-2 and its variants. This evidence could help not only to understand cross-reactive responses among coronaviruses but also contribute to elucidate their role in immunity to SARS-CoV-2 induced by infection and/or vaccination. Finally, these findings could promote targeted analysis of antigen-specific immune responses and might orient and drive the rational development of new SARS-CoV-2 vaccines including candidates that ideally provide \u201cuniversal\u201d protection against other coronaviruses relevant to human health.","version":"1.1","doi":"10.1101/2022.02.16.480759","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.461434","pub_date":"2022-2-17","title":"Immunogenicity of SARS-CoV-2 trimetric spike protein associated to Poly(I:C) plus Alum","abstract":"The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (spike protein with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.","version":"1.3","doi":"10.1101/2021.10.05.461434","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.475291","pub_date":"2022-2-17","title":"Comprehensive Evaluation of ACE2-Fc Combination with Neutralization Antibody on Broad Protection against SARS-CoV-2 and Its Variants","abstract":"Emerging SARS-CoV-2 variants are threatening the efficacy of antibody therapies. Combination treatments including ACE2-Fc have been developed to overcome the evasion of neutralizing antibodies (NAbs) in individual cases. Here we conducted a comprehensive evaluation of this strategy by combining ACE2-Fc with NAbs of diverse epitopes on the RBD. NAb+ACE2-Fc combinations efficiently neutralized HIV-based pseudovirus carrying the spike protein of the Delta or Omicron variants, achieving a balance between efficacy and breadth. In an antibody escape assay using replication-competent VSV-SARS-CoV-2-S, all the combinations had no escape after fifteen passages. By comparison, all the NAbs without combo with ACE2-Fc had escaped within six passages. Further, the VSV-S variants escaped from NAbs were neutralized by ACE2-Fc, revealing the mechanism of NAb+ACE2-Fc combinations survived after fifteen passages. We finally examined ACE2-Fc neutralization against pseudovirus variants that were resistant to the therapeutic antibodies currently in clinic. Our results suggest ACE2-Fc is a universal combination partner to combat SARS-CoV-2 variants including Delta and Omicron.","version":"1.2","doi":"10.1101/2022.01.17.475291","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.480851","pub_date":"2022-2-17","title":"Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA","abstract":"Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable \u2018sterilizing immunity\u2019 at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 \u2264 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting. We unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.","version":"1.1","doi":"10.1101/2022.02.17.480851","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.17.479653","pub_date":"2022-2-17","title":"CriSNPr: a single interface for the curated and de-novo design of gRNAs for CRISPR diagnostics using diverse Cas systems","abstract":"Nucleic acid detection and variant calling through CRISPR-based diagnostics (CRISPRDx) has facilitated clinical decision-making, particularly during the COVID-19 pandemic. This has been further accelerated through the discovery of newer and engineered CRISPR effectors, expanding the portfolio of such diagnostic applications to a wide variety of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline requires customized detection schemes originating from fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is particularly relevant for variant detection, an attractive low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver CriSNPr (CRISPR based SNP recognition), which provides the user the opportunity to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. In addition, we provide a database of curated pre-designed gRNAs and target/off-target for all human and SARS-CoV-2 variants reported so far. CriSNPr has been validated on multiple Cas proteins and highlights its broad and immediate scope of utilization across multiple detection platforms. CriSNPr is available at URL http://crisnpr.igib.res.in/.","version":"1.1","doi":"10.1101/2022.02.17.479653","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.479891","pub_date":"2022-2-17","title":"Variational phylodynamic inference using pandemic-scale data","abstract":"The ongoing global pandemic has sharply increased the amount of data available to researchers in epidemiology and public health. Unfortunately, few existing analysis tools are capable of exploiting all of the information contained in a pandemic-scale data set, resulting in missed opportunities for improved surveillance and contact tracing. In this paper, we develop the variational Bayesian skyline (VBSKY), a method for fitting Bayesian phylodynamic models to very large pathogen genetic data sets. By combining recent advances in phylodynamic modeling, scalable Bayesian inference and differentiable programming, along with a few tailored heuristics, VBSKY is capable of analyzing thousands of genomes in a few minutes, providing accurate estimates of epidemiologically relevant quantities such as the effective reproduction number and overall sampling effort through time. We illustrate the utility of our method by performing a rapid analysis of a large number of SARS-CoV-2 genomes, and demonstrate that the resulting estimates closely track those derived from alternative sources of public health data.","version":"1.1","doi":"10.1101/2022.02.10.479891","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.15.480603","pub_date":"2022-2-16","title":"A possible way to relate the effects of SARS-CoV-2 induced changes in transferrin to severe COVID-19 associated diseases","abstract":"The potentially life-threatening Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is responsible for the coronavirus pandemic in 2019 (COVID-19). The transferrin as an essential component of iron-metabolism was suggested to be a link between iron transport associated diseases and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis and deconvolution of the thermal transition curves showed that the Tm of transferrin related second peak decreased by 5.16 \u00b0C (6.4%) in the presence of SARS-CoV-2 virus. The ratio of the under-curve area of the two main peaks was greatly affected while the total enthalpy of the heat denaturation was nearly unchanged in the presence of the virus. Based on the results it is possible to conclude that SARS-CoV-2 through binding to transferrin can influence it\u2019s Fe3+ uptake by inducing thermodynamic changes. Transferrin may stay in iron-free apo-conformational state, which probably depends on the SARS-CoV-2 concentration. SARS-CoV-2 might induce disturbance in the erythropoiesis due to the free iron overload generated iron toxicity. As a late consequence iron toxicity related hepatocellular carcinoma can even develop. Our work can support the basic role of transferrin in COVID-19 related severe diseases.","version":"1.1","doi":"10.1101/2022.02.15.480603","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.12.468374","pub_date":"2022-2-16","title":"Updated vaccine protects from infection with SARS-CoV-2 variants, prevents transmission and is immunogenic against Omicron in hamsters","abstract":"Current first-generation COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) that threaten to escape vaccine-mediated protection. Here we show in a stringent hamster model that immunization using prototypic spike expressed from a potent YF17D viral vector (1) provides vigorous protection against infection with ancestral virus (B lineage) and VOC Alpha (B.1.1.7), however, is insufficient to provide maximum protection against the Beta (B.1.351) variant. To improve vaccine efficacy, we created a revised vaccine candidate that carries an evolved spike antigen. Vaccination of hamsters with this updated vaccine candidate provides full protection against intranasal challenge with all four VOCs Alpha, Beta, Gamma (P.1) and Delta (B.1.617.2) resulting in complete elimination of infectious virus from the lungs and a marked improvement in lung pathology. Vaccinated hamsters did also no longer transmit the Delta variant to non-vaccinated sentinels. Hamsters immunized with our modified vaccine candidate also mounted marked neutralizing antibody responses against the recently emerged Omicron (B.1.1.529) variant, whereas the old vaccine employing prototypic spike failed to induce immunity to this antigenically distant virus. Overall, our data indicate that current first-generation COVID-19 vaccines need to be urgently updated to cover newly emerging VOCs to maintain vaccine efficacy and to impede virus spread at the community level. SARS-CoV-2 keeps mutating rapidly, and the ongoing COVID-19 pandemic is fueled by new variants escaping immunity induced by current first-generation vaccines. There is hence an urgent need for universal vaccines that cover variants of concern (VOC). In this paper we show that an adapted version of our vaccine candidate YF-S0* provides full protection from infection, virus transmission and disease by VOCs Alpha, Beta, Gamma and Delta, and also results in markedly increased levels of neutralizing antibodies against recently emerged Omicron VOC in a stringent hamster model. Our findings underline the necessity to update COVID-19 vaccines to curb the pandemic, providing experimental proof on how to maintain vaccine efficacy in view of an evolving SARS-CoV-2 diversity.","version":"1.3","doi":"10.1101/2021.11.12.468374","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.02.450964","pub_date":"2022-2-16","title":"SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome","abstract":"Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1,000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.","version":"1.3","doi":"10.1101/2021.07.02.450964","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.15.480585","pub_date":"2022-2-16","title":"Mathematical model of a cytokine storm","abstract":"Cytokine storm is a life-threatening inflammatory response that is characterized by hyperactivation of the immune system, and which can be caused by various therapies, auto-immune conditions, or pathogens, such as respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease COVID-19. While initial causes of cytokine storms can vary, late-stage clinical manifestations of cytokine storm converge and often overlap, and therefore a better understanding of how normal immune response turns pathological is warranted. Here we propose a theoretical framework, where cytokine storm phenomenology is captured using a conceptual mathematical model, where cytokines can both activate and regulate the immune system. We simulate normal immune response to infection, and through variation of system parameters identify conditions where, within the frameworks of this model, cytokine storm can arise. We demonstrate that cytokine storm is a transitional regime, and identify three main factors that must converge to result in storm-like dynamics, two of which represent individual-specific characteristics, thereby providing a possible explanation for why some people develop CRS, while others may not. We also discuss possible ecological insights into cytokine-immune interactions and provide mathematical analysis for the underlying regimes. We conclude with a discussion of how results of this analysis can be used in future research.","version":"1.1","doi":"10.1101/2022.02.15.480585","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480338","pub_date":"2022-2-15","title":"Oral administration of S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and accelerates recovery from clinical aspects of COVID-19","abstract":"In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622, a novel inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates the disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated eminent pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron variant. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase II/III clinical trial, possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.","version":"1.1","doi":"10.1101/2022.02.14.480338","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480317","pub_date":"2022-2-15","title":"SARS-CoV-2-specific T cells associate with reduced lung function and inflammation in pulmonary post-acute sequalae of SARS-CoV-2","abstract":"As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had up to 34-fold higher frequencies of IFN-\u03b3- and TNF-\u03b1-producing SARS-CoV-2-specific CD4+ and CD8+ T cells in peripheral blood and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-\u03b1-producing SARS-CoV-2-specific CD4+ and CD8+ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV1), while increased frequencies of IFN-\u03b3-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden. Long COVID-19 or post-acute sequelae of SARS-CoV-2 (PASC) impacts 20-30% of those infected with SARS-CoV-2 and is characterized by COVID-19 symptoms exceeding 4 weeks from symptom onset. While those with PASC experience a wide variety of persistent symptoms including shortness of breath, cough, chest pain, irregular heartbeat, brain fog, fatigue, and intermittent fever, lung-related conditions are the most common. Although, infection with SARS-CoV-2 is clearly the inciting factor for PASC, the mechanisms responsible for long-term lung dysfunction are unclear and current treatments are ineffective at resolving pulmonary symptoms. Generalized PASC has been associated with SARS-CoV-2-specific T cells, a component of adaptive immunity, suggesting that residual virus may persist. Here, we investigated the frequency and function of virus-specific T cells in the blood of individuals with pulmonary PASC and correlated their presence with systemic inflammation and lung function. Our findings demonstrated that T cells specific for SARS-CoV-2 are elevated in the blood of those with pulmonary PASC and are associated with increased IL-6, a cytokine strongly associated with COVID-19 severity, and decreased lung function. These findings provide mechanistic insight into the pathophysiology of pulmonary PASC needed for the development of new treatments to improve quality of life for those affected.","version":"1.1","doi":"10.1101/2022.02.14.480317","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.15.480527","pub_date":"2022-2-15","title":"Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immunity responses","abstract":"Cell-intrinsic responses mounted in vivo in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or are, at least partially, resulting from physical interaction with virus particles, remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. Bulk and single cell RNA-sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes, but not pro-inflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG base-line profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, non-productive physical interaction of PBMCs with SARS-CoV-2-but not SARS-CoV particles stimulates JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.","version":"1.1","doi":"10.1101/2022.02.15.480527","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.15.480515","pub_date":"2022-2-15","title":"A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies","abstract":"COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.","version":"1.1","doi":"10.1101/2022.02.15.480515","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480353","pub_date":"2022-2-15","title":"The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylated IgG1 in naive but not antigen-experienced vaccinees","abstract":"The onset of severe SARS-CoV-2 infection is characterized by the presence of afucosylated IgG1 responses against the viral spike (S) protein, which can trigger exacerbated inflammatory responses. Here, we studied IgG glycosylation after BNT162b2 SARS-CoV-2 mRNA vaccination to explore whether vaccine-induced S protein expression on host cells also generates afucosylated IgG1 responses. SARS-CoV-2 naive individuals initially showed a transient afucosylated anti-S IgG1 response after the first dose, albeit to a lower extent than severely ill COVID-19 patients. In contrast, previously infected, antigen-experienced individuals had low afucosylation levels, which slightly increased after immunization. Afucosylation levels after the first dose correlated with low fucosyltransferase 8 (FUT8) expression levels in a defined plasma cell subset. Remarkably, IgG afucosylation levels after primary vaccination correlated significantly with IgG levels after the second dose. Further studies are needed to assess efficacy, inflammatory potential, and protective capacity of afucosylated IgG responses. A transient afucosylated IgG response to the BNT162b2 mRNA vaccine was observed in naive but not in antigen-experienced individuals, which predicted antibody titers upon the second dose.","version":"1.1","doi":"10.1101/2022.02.14.480353","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480460","pub_date":"2022-2-15","title":"A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions","abstract":"We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcriptional regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (\u03943678). The \u03943678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The \u03943678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the \u03943678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the \u03943678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter \u03943678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that \u03943678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.","version":"1.1","doi":"10.1101/2022.02.14.480460","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480430","pub_date":"2022-2-15","title":"A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants","abstract":"A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. We mapped alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We found that SARS-CoV-2 rewires host lipid metabolism, altering 409 lipid species up to 64-fold relative to controls. We correlated these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We found that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We found that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.","version":"1.1","doi":"10.1101/2022.02.14.480430","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480335","pub_date":"2022-2-15","title":"Virological characteristics of SARS-CoV-2 BA.2 variant","abstract":"Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.","version":"1.1","doi":"10.1101/2022.02.14.480335","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.11.480177","pub_date":"2022-2-15","title":"A novel consensus-based computational pipeline for rapid screening of antibody therapeutics for efficacy against SARS-CoV-2 variants of concern including omicron variant","abstract":"Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to evolve carrying flexible amino acid substitutions in the spike protein\u2019s receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with three-fold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero-dimeric and \u2013trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variant\u2019s RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow_3D11, and C118) \u2014 that may likely neutralize the recently emerged omicron variant \u2014 facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.02.11.480177","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.479919","pub_date":"2022-2-15","title":"Preclinical establishment of a divalent vaccine against SARS-CoV-2","abstract":"First-generation vaccines against SARS-CoV-2 have been administered to more than 60% of the population in developed countries. However, the monovalent vaccines currently available in Europe do not confer adequate and durable immune protection. To satisfy the need for a novel vaccine, we engineered a divalent gene construct consisting of the receptor binding domain (RBD, 300-685 aa) of the spike protein and the immunodominant region of the nucleocapsid (100-300 aa). This fusion protein was cloned into a pET-30a plasmid and expressed either in Escherichia coli or in a recombinant baculovirus in insect cells. Following purification via its His-tag, the fusion protein was mixed with adjuvant, and administered to mice in a prime-booster-mode. Upon testing for IgG antibody response against nucleocapsid and RBD, a titer of 10\u22124 - 10\u22125 was demonstrated 14 days after the first booster injection in 72% of the animals, which could be increased to 100% by a second booster. Notably, comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed the presence of IgG beyond 90 days. In addition, granzyme A and perforin mRNA expression (cytolytic effector cell molecules) was increased in cytotoxic lymphocytes isolated from peripheral blood. Ex vivo stimulation of T-cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 in vaccinated mice among their CD4+ and CD8+ T-cells. No side-effect was documented in the central nervous system, be it either endothelial inflammation or neuronal damage. Cumulatively, the combined induction of B-cell and T-cell response by a bivalent protein-based vaccine directed against two structural SARS-CoV-2 proteins represents a proof-of-principle approach alternative to existing mRNA vaccination strategies, which could confer long-lasting immunity against all known viral strains.","version":"1.3","doi":"10.1101/2022.02.10.479919","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.14.480298","pub_date":"2022-2-15","title":"Milk of cow and goat, immunized by recombinant protein vaccine ZF-UZ-VAC2001(Zifivax), contains neutralizing antibodies against SARS-CoV-2 and remains active after standard milk pasteurization","abstract":"Here we report the first experimental validation of the possibility for obtaining immune milk with neutralizing antibodies against SARS-CoV-2 from vaccinated cow and goat using recombinant protein human vaccine, ZF-UZ-VAC2001. In the period of two weeks after first vaccination, we detected the neutralizing antibodies against coronavirus in the blood serum of vaccinated animals. The neutralizing activity, in its peak on the 21st days after receiving the third dose (77th day from first dose), was effective in Neutralization Test using a live SARS-CoV-2 in Vero E6 cells, even after 120-fold serum titration. Colostrum of the first day after 3rd dose vaccinated cow after calving had a greater activity to neutralize the SARS-CoV-2 compared to colostrum of subsequent three days (4.080 \u00b5g/ml vs 2.106, 1.960 and 1.126 \u00b5g/ml), goat milk (1,486 \u00b5g/ml), and cow milk (0.222 \u00b5g/ml) in MAGLUMI\u00ae SARS-CoV-2 neutralizing antibody competitive chemiluminescence immunoassay. We observed a positive correlation of receptor-binding domain (RBD)-specific IgG antibodies between the serum of actively immunized cow and milk-feeding calf during the entire course of vaccination (r = 0.95, p = 0.027). We showed an optimal regime for immune milk pasteurization at 62.5\u00b0C for 30 min, which retained specific neutralizing activity to SARS-CoV-2, potentially useful for passive immunization against coronavirus infection threats.","version":"1.1","doi":"10.1101/2022.02.14.480298","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.22270721","pub_date":"2022-02-15","title":"Direct and indirect mortality impacts of the COVID-19 pandemic in the US, March 2020-April 2021","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Excess mortality studies provide crucial information regarding the health burden of pandemics and other large-scale events. Here, we used time series approaches to separate the direct contribution of SARS-CoV-2 infections on mortality from the indirect consequences of pandemic interventions and behavior changes in the United States. We estimated deaths occurring in excess of seasonal baselines stratified by state, age, week and cause (all causes, COVID-19 and respiratory diseases, Alzheimer\u2019s disease, cancer, cerebrovascular disease, diabetes, heart disease, and external causes, including suicides, opioids, accidents) from March 1, 2020 to April 30, 2021. Our estimates of COVID-19 excess deaths were highly correlated with SARS-CoV-2 serology, lending support to our approach. Over the study period, we estimate an excess of 666,000 (95% Confidence Interval (CI) 556000, 774000) all-cause deaths, of which 90% could be attributed to the direct impact of SARS-CoV-2 infection, and 78% were reflected in official COVID-19 statistics. Mortality from all disease conditions rose during the pandemic, except for cancer. The largest direct impacts of the pandemic were seen in mortality from diabetes, Alzheimer\u2019s, and heart diseases, and in age groups over 65 years. In contrast, the largest indirect consequences of the pandemic were seen in deaths from external causes, which increased by 45,300 (95% CI 30,800, 59,500) and were statistically linked to the intensity of non-pharmaceutical interventions. Within this category, increases were most pronounced in mortality from accidents and injuries, drug overdoses, and assaults and homicides, while the rate of death from suicides remained stable. Younger age groups suffered the brunt of these indirect effects. Overall, on a national scale, the largest consequences of the COVID-19 pandemic are attributable to the direct impact of SARS-CoV-2 infections; yet, the secondary impacts dominate among younger age groups, in periods of stricter interventions, and in mortality from external causes. Further research on the drivers of indirect mortality is warranted to optimize interventions in future pandemics.</jats:p>","version":null,"doi":"10.1101/2022.02.10.22270721","journal":"medRxiv","score":null},{"id":"10.1101/2022.02.14.480394","pub_date":"2022-2-15","title":"Increased Potency and Breadth of SARS-CoV-2 Neutralizing Antibodies After a Third mRNA Vaccine Dose","abstract":"The omicron variant of SARS-CoV-2 infected very large numbers of SARS-CoV-2 vaccinated and convalescent individuals. The penetrance of this variant in the antigen experienced human population can be explained in part by the relatively low levels of plasma neutralizing activity against Omicron in people who were infected or vaccinated with the original Wuhan-Hu-1 strain. The 3rd mRNA vaccine dose produces an initial increase in circulating anti-Omicron neutralizing antibodies, but titers remain 10-20-fold lower than against Wuhan-Hu-1 and are, in many cases, insufficient to prevent infection. Despite the reduced protection from infection, individuals that received 3 doses of an mRNA vaccine were highly protected from the more serious consequences of infection. Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving 3 mRNA vaccine doses. We find that the 3rd dose is accompanied by an increase in, and evolution of, anti-receptor binding domain specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the 2nd vaccine dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared to antibodies obtained after the 2nd vaccine dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells that differed from the persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analyzed neutralizing antibodies in the memory compartment obtained from individuals receiving a 3rd mRNA vaccine dose neutralized Omicron. Thus, individuals receiving 3 doses of an mRNA vaccine encoding Wuhan-Hu-1, have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help explain why a 3rd dose of an mRNA vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.","version":"1.1","doi":"10.1101/2022.02.14.480394","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.11.22270775","pub_date":"2022-02-15","title":"Favipiravir, lopinavir-ritonavir or combination therapy (FLARE): a randomised, double blind, 2x2 factorial placebo-controlled trial of early antiviral therapy in COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Early antiviral treatment is effective for COVID-19 but currently available agents are expensive. Favipiravir is routinely used in many countries, but efficacy is unproven. Antiviral combinations have not been systematically studied. We aimed to evaluate the effect of favipiravir, lopinavir-ritonavir or the combination of both agents on SARS-CoV-2 viral load trajectory when administered early.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We conducted a Phase 2, proof of principle, randomised, placebo-controlled, 2\u00d72 factorial, double-blind trial of outpatients with early COVID-19 (within 7 days of symptom onset) at two sites in the United Kingdom. Participants were randomised using a centralised online process to receive: favipiravir (1800mg twice daily on Day 1 followed by 400mg four times daily on Days 2-7) plus lopinavir-ritonavir (400mg/100mg twice daily on Day 1, followed by 200mg/50mg four times daily on Days 2-7); favipiravir plus lopinavir-ritonavir placebo; lopinavir-ritonavir plus favipiravir placebo; or both placebos. The primary outcome was SARS-CoV-2 viral load at Day 5, accounting for baseline viral load. ClinicalTrials\u00b7gov:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04499677'>NCT04499677</jats:ext-link>\n                    .\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>\n                    Between 6 October 2020 and 4 November 2021, we recruited 240 participants. For the favipiravir+lopinavir-ritonavir, favipiravir+placebo, lopinavir-ritonavir+placebo and placebo-only arms, we recruited 61, 59, 60 and 60 participants and analysed 55, 56, 55 and 58 participants respectively who provided viral load measures at Day 1 and Day 5. In the primary analysis, the mean viral load in the favipiravir+placebo arm had decreased by 0.57 log\n                    <jats:sub>10</jats:sub>\n                    (95% CI -1.21 to 0.07, p=0.08) and in the lopinavir-ritonavir+placebo arm by 0.18 log\n                    <jats:sub>10</jats:sub>\n                    (95% CI -0.82 to 0.46, p=0.58) more than in the placebo arm at Day 5. There was no significant interaction between favipiravir and lopinavir-ritonavir (interaction coefficient term: 0.59 log\n                    <jats:sub>10</jats:sub>\n                    , 95% CI -0.32 to 1.50, p=0.20). More participants had undetectable virus at Day 5 in the favipiravir+placebo arm compared to placebo only (46.3% vs 26.9%, odds ratio (OR): 2.47, 95% CI 1.08 to 5.65; p=0.03). Adverse events were observed more frequently with lopinavir-ritonavir, mainly gastrointestinal disturbance. Favipiravir drug levels were lower in the combination arm than the favipiravir monotherapy arm.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>At the current doses, no treatment significantly reduced viral load in the primary analysis. Favipiravir requires further evaluation with consideration of dose escalation. Lopinavir-ritonavir administration was associated with lower plasma favipiravir concentrations.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>LifeArc, UK.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.02.11.22270775","journal":"medRxiv","score":null},{"id":"10.1101/2022.02.14.480356","pub_date":"2022-2-15","title":"Characterisation and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry","abstract":"Large RNA including messenger RNA (mRNA) has emerged as an important new class of therapeutic. Recently this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study we have developed an automated, high throughput workflow for the rapid characterisation and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilised on magnetic particles was performed in conjunction with high resolution liquid chromatography mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS Co-V2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation can be completed within 90 minutes. The ability to rapidly identify, characterise and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation and impurity analysis.","version":"1.2","doi":"10.1101/2022.02.14.480356","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.04.455134","pub_date":"2022-2-15","title":"GlycoSHIELD: a versatile pipeline to assess glycan impact on protein structures","abstract":"More than 75% of surface and secreted proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, influencing protein dynamics and interactions with other molecules. Glycan conformational freedom impairs complete structural elucidation of glycoproteins. Computer simulations may be used to model glycan structure and dynamics. However, such simulations typically require thousands of computing hours on specialized supercomputers, thus limiting routine use. Here, we describe a reductionist method that can be implemented on personal computers to graft ensembles of realistic glycan conformers onto static protein structures in a matter of minutes. Using this open-source pipeline, we reconstructed the full glycan cover of SARS-CoV-2 Spike protein (S-protein) and a human GABAA receptor. Focusing on S-protein, we show that GlycoSHIELD recapitulates key features of extended simulations of the glycosylated protein, including epitope masking, and provides new mechanistic insights on N-glycan impact on protein structural dynamics.","version":"1.3","doi":"10.1101/2021.08.04.455134","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.05.459013","pub_date":"2022-2-15","title":"ScanNet: An interpretable geometric deep learning model for structure-based protein binding site prediction","abstract":"Predicting the functional sites of a protein from its structure, such as the binding sites of small molecules, other proteins or antibodies sheds light on its function in vivo. Currently, two classes of methods prevail: Machine Learning (ML) models built on top of handcrafted features and comparative modeling. They are respectively limited by the expressivity of the handcrafted features and the availability of similar proteins. Here, we introduce ScanNet, an end-to-end, interpretable geometric deep learning model that learns features directly from 3D structures. ScanNet builds representations of atoms and amino acids based on the spatio-chemical arrangement of their neighbors. We train ScanNet for detecting protein-protein and protein-antibody binding sites, demonstrate its accuracy - including for unseen protein folds - and interpret the filters learned. Finally, we predict epitopes of the SARS-CoV-2 spike protein, validating known antigenic regions and predicting previously uncharacterized ones. Overall, ScanNet is a versatile, powerful, and interpretable model suitable for functional site prediction tasks. A webserver for ScanNet is available from http://bioinfo3d.cs.tau.ac.il/ScanNet/","version":"1.2","doi":"10.1101/2021.09.05.459013","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.22270635","pub_date":"2022-02-15","title":"Surveillance of Myopericarditis following COVID-19 Booster Dose Vaccination in a Large Integrated Health System","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Purpose</jats:title>\n                  <jats:p>The risk of myopericarditis following COVID-19 booster vaccination has not been extensively evaluated. We provide a timely case ascertainment of myocarditis following COVID-19 booster vaccine in individuals age 18-39 years from an integrated health system.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We studied a cohort of 65,785 Kaiser Permanente (KP) Northwest Health Plan members aged 18-39 years who received a COVID-19 vaccine booster at least 5 months following completion of the primary series. We identified cases of myopericarditis by searching the electronic health record for the National Center for Health Statistics (NCHS) text label for \u2018myocarditis\u2019 or \u2018pericarditis\u2019 diagnosis codes in all inpatient and outpatient encounters through January 18\n                    <jats:sup>th</jats:sup>\n                    2022. The cohort was followed for 21 days after their booster. We excluded anyone with a documented diagnosis of myocarditis or pericarditis before their first COVID-19 vaccination. Two physicians independently reviewed the identified patient records and applied the CDC myocarditis and pericarditis surveillance case definition to classify records as confirmed, probable or excluded based on the prior published definition.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Our method identified 6 patients who met the confirmed or probable CDC case definition for acute myocarditis or pericarditis within 21 days of COVID-19 booster dose among 65,785 eligible members. Four cases occurred in 27,253 men. Overall, we estimated 9.1 cases (exact 95% CI 3.4 to 19.9) of post-booster myopericarditis per 100,000 booster doses given. In men, we estimated 14.7 cases (exact 95% CI 4.0 to 37.6) per 100,000 booster doses given.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>We identified a rate of 9.1 cases of myopericarditis per 100,000 COVID-19 booster doses which is higher than prior estimates reported by the Vaccine Adverse Event Reporting System (VAERS). Myopericarditis occurs following COVID-19 booster vaccine and may be underreported by current surveillance methods. High sensitivity of these case estimates is essential when modeling risk and benefit for sequential COVID-19 vaccinations for the general population.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.02.08.22270635","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.26.477915","pub_date":"2022-2-14","title":"Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine","abstract":"Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies in non-human primates (NHPs) against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants. The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 10.6-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.","version":"1.2","doi":"10.1101/2022.01.26.477915","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473265","pub_date":"2022-2-14","title":"Possible Cross Reactivity of Feline and White-tailed Deer Antibodies Against the SARS-CoV-2 Receptor Binding Domain","abstract":"In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2\u2019s RBD is antigenically distinct from common human and animal coronaviruses allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect ELISA for SARS-CoV-2\u2019s RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2\u2019s RBD. Surprisingly pre-pandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were \u223c50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing as cross-reactive antibodies towards SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. We report cross-reactive antibodies from pre-pandemic cats and post-pandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2\u2019s RBD region. However, we lack conclusive evidence of pre-pandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.","version":"1.2","doi":"10.1101/2021.12.17.473265","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.13.480238","pub_date":"2022-2-14","title":"SARS-CoV-2 Permissive Glioblastoma Cell Line for High Throughput Antiviral Screening","abstract":"Despite the great success of the administered vaccines against SARS-CoV-2, the virus can still spread, as evidenced by the current circulation of the highly contagious Omicron variant. This emphasizes the additional need to develop effective antiviral countermeasures. In the context of early preclinical studies for antiviral assessment, robust cellular infection systems are required to screen drug libraries. In this study, we reported the implementation of a human glioblastoma cell line, stably expressing ACE2, in a SARS-CoV-2 cytopathic effect (CPE) reduction assay. These glioblastoma cells, designated as U87.ACE2+, expressed ACE2 and cathepsin B abundantly, but had low cellular levels of TMPRSS2 and cathepsin L. The U87.ACE2+ cells fused highly efficiently and quickly with SARS-CoV-2 spike expressing cells. Furthermore, upon infection with SARS-CoV-2 wild-type virus, the U87.ACE2+ cells displayed rapidly a clear CPE that resulted in complete cell lysis and destruction of the cell monolayer. By means of several readouts we showed that the U87.ACE2+ cells actively replicate SARS-CoV-2. Interestingly, the U87.ACE2+ cells could be successfully implemented in an MTS-based colorimetric CPE reduction assay, providing IC50 values for Remdesivir in the low nanomolar range. Lastly, the U87.ACE2+ cells were consistently permissive to all tested SARS-CoV-2 variants of concern, including the current Omicron variant. Thus, ACE2 expressing glioblastoma cells are highly permissive to SARS-CoV-2 with productive viral replication and with the induction of a strong CPE that can be utilized in high-throughput screening platforms.","version":"1.1","doi":"10.1101/2022.02.13.480238","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.12.480218","pub_date":"2022-2-14","title":"Distinct upper airway epithelium interferon-stimulated and profibrotic gene expression between adult and infant rhesus macaques infected with SARS-CoV-2","abstract":"The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for respiratory viral diseases. To elucidate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analysis of the respiratory tract and evaluated systemic cytokine and antibody responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum while CXCL10 was induced in all animals. Both groups mounted neutralizing antibody (nAb) responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal tissue isolated at day 14 post-infection revealed significant upregulation of multiple interferon-stimulated genes in infants compared to adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix (ECM) composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared to infants. Our observations suggest age-dependent differential airway responses to SARS-CoV-2 infection that could explain the distinction in pathogenesis between infants and adults.","version":"1.1","doi":"10.1101/2022.02.12.480218","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.12.480188","pub_date":"2022-2-14","title":"Next-generation intranasal Covid-19 vaccine: a polymersome-based protein subunit formulation that provides robust protection against multiple variants of concern and early reduction in viral load of the upper airway in the golden Syrian hamster model","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (Covid-19), an ongoing global public health emergency. Despite the availability of safe and efficacious vaccines, achieving herd immunity remains a challenge due in part to rapid viral evolution. Multiple variants of concern (VOCs) have emerged, the latest being the heavily mutated Omicron, which exhibits the highest resistance to neutralizing antibodies from past vaccination or infection. Currently approved vaccines generate robust systemic immunity, yet poor immunity at the respiratory tract. We have demonstrated that a polymersome-based protein subunit vaccine with wild type (WT) spike protein and CpG adjuvant induces robust systemic immunity (humoral and T cell responses) in mice. Both antigen and adjuvant are encapsulated in artificial cell membrane (ACM) polymersomes \u2013 synthetic, nanoscale vesicles that substantially enhance the immune response through efficient delivery to dendritic cells. In the present study, we have formulated a vaccine candidate with the spike protein from Beta variant and assessed its immunogenicity in golden Syrian hamsters. Two doses of ACM-Beta spike vaccine administered via intramuscular (IM) injection evoke modest serum neutralizing titers that are equally efficacious towards WT and Beta viruses. In contrast, the ACM-WT spike vaccine induces a predominantly WT-specific serum neutralizing response with pronounced reduction in potency towards the Beta variant. Remarkably, immunogenicity of the ACM-Beta spike vaccine is greatly enhanced through intranasal (IN) administration. Following IN challenge with the Beta variant, IM-immunized hamsters are fully protected from disease but not infection, displaying similar peak viral RNA loads in oral swabs as non-vaccinated controls. In contrast, hamsters IN vaccinated with ACM-Beta spike vaccine are protected from disease and infection, exhibiting a \u223c100-fold drop in total and subgenomic RNA load as early as day 2 post challenge. We further demonstrate that nasal washes from IN-but not IM-immunized animals possess virus neutralizing activity that is broadly efficacious towards Delta and Omicron variants. Altogether, our results show IN administration of ACM-Beta spike vaccine to evoke systemic and mucosal antibodies that cross-neutralize multiple SARS-CoV-2 VOCs. Our work supports IN administration of ACM-Beta spike vaccine for a next-generation vaccination strategy that not only protects against disease but also an infection of the respiratory tract, thus potentially preventing asymptomatic transmission.","version":"1.1","doi":"10.1101/2022.02.12.480188","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.13.480261","pub_date":"2022-2-14","title":"Omicron Spike protein has a positive electrostatic surface that promotes ACE2 recognition and antibody escape","abstract":"High transmissibility is a hallmark of the Omicron variant of SARS-CoV-2. Understanding the molecular determinants of Omicron\u2019s transmissibility will impact development of intervention strategies. Here we map the electrostatic potential surface of the Spike protein to show that major SARS-CoV-2 variants have accumulated positive charges in solvent-exposed regions of the Spike protein, especially its ACE2-binding interface. Significantly, the Omicron Spike-ACE2 complex has complementary electrostatic surfaces. In contrast, interfaces between Omicron and neutralizing antibodies tend to have similar positively charged surfaces. Structural modeling demonstrates that the electrostatic property of Omicron\u2019s Spike receptor binding domain (S RBD) plays a role in enhancing ACE2 recognition and destabilizing Spike-antibody complexes. Collectively, our structural analysis implies that Omicron S RBD interaction interfaces have been optimized to simultaneously promote access to human ACE2 receptors and evade antibodies. These findings suggest that electrostatic interactions are a major contributing factor for increased Omicron transmissibility relative to other variants.","version":"1.1","doi":"10.1101/2022.02.13.480261","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454529","pub_date":"2022-2-14","title":"Severe COVID-19 shares a common neutrophil activation signature with other acute inflammatory states","abstract":"Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaky disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflamatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.","version":"1.2","doi":"10.1101/2021.07.30.454529","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.22270799","pub_date":"2022-02-13","title":"Evaluating the effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control teams: the COG-UK hospital-onset COVID-19 infection study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data-collection period, followed by intervention periods comprising 8 weeks of \u2018rapid\u2019 (&lt;48h) and 4 weeks of \u2018longer-turnaround\u2019 (5-10 day) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital onset COVID-19 infections (HOCIs; detected \u226548h from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on incidence of probable/definite hospital-acquired infections (HAIs) was evaluated.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    A total of 2170 HOCI cases were recorded from October 2020-April 2021, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (IRR 1.60, 95%CI 0.85-3.01;\n                    <jats:italic>P=</jats:italic>\n                    0.14) or rapid (0.85, 0.48-1.50;\n                    <jats:italic>P=</jats:italic>\n                    0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8% and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2% and 11.6% of cases where the report was returned. In a per-protocol sensitivity analysis there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.02.10.22270799","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.07.242347","pub_date":"2022-2-12","title":"Ig-VAE: Generative Modeling of Protein Structure by Direct 3D Coordinate Generation","abstract":"While deep learning models have seen increasing applications in protein science, few have been implemented for protein backbone generation\u2014an important task in structure-based problems such as active site and interface design. We present a new approach to building class-specific backbones, using a variational auto-encoder to directly generate the 3D coordinates of immunoglobulins. Our model is torsion- and distance-aware, learns a high-resolution embedding of the dataset, and generates novel, high-quality structures compatible with existing design tools. We show that the Ig-VAE can be used to create a computational model of a SARS-CoV2-RBD binder via latent space sampling. We further demonstrate that the model\u2019s generative prior is a powerful tool for guiding computational protein design, motivating a new paradigm under which backbone design is solved as constrained optimization problem in the latent space of a generative model.","version":"1.2","doi":"10.1101/2020.08.07.242347","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.22.477073","pub_date":"2022-2-11","title":"Bacteriophage-derived dsRNA exerts anti-SARS-CoV-2 activity in vitro and in Golden Syrian hamsters in vivo","abstract":"Bacteriophage-derived dsRNA, also known as Larifan, is nationally well-known broad-spectrum antiviral medication. The goal of this study was to ascertain the antiviral activity of Larifan against the novel SARS-CoV-2. The antiviral activity of Larifan against SARS-CoV-2 in vitro was measured in human lung adenocarcinoma (Calu3) and primary human small airway epithelial cells (HSAEC) using cytopathic effect assay, viral RNA copy number detection by digital droplet PCR (ddPCR) and infectious virus titration in cells supernatants in Vero E6 cells by end-point titration method. The antiviral effect of Larifan in vivo was detected in SARS-CoV-2 infection model in Golden Syrian hamsters. Larifan (5 mg/kg) was administered either subcutaneously or intranasally twice before and after virus infection with a 24-hour interval between doses. The viral RNA copies and infectious virus titre were detected in animal lungs at day three and five post-infection using ddPCR and end-point titration in Vero E6 cells, respectively. Histopathology of lungs was analysed as well. Larifan inhibited SARS-CoV-2 replication in Calu3 cells both after the drug addition pre- and post-infection with a substantial drop in the supernatant viral RNA copy numbers from eight (p = 0.0013) to twenty (p = 0.0042) times, respectively. Similarly, infectious virus titre in Vero E6 cells dropped by 3.6log10 TCID50 and 2.8log10 TCID50 after the drug addition pre- and post-infection, respectively. In HSAEC, Larifan inhibited SARS-CoV-2 replication at the similar level. Larifan also markedly reduced virus numbers in the lungs of infected hamsters (p = 0.0032) both at day three and five post-infection with a more pronounced effect after intranasal administration reaching a drop by 2.7log10 at day three and 2.0log10 at day five. The administration of Larifan also reduced the amount of infections virus titer in lungs (p = 0.0039) by 4.3log10 TCID50 and 2.8log10 TCID50 at day three and five post-infection, respectively. Improvements in the infection-induced pathological lesion severity in the lungs of animals treated with Larifan were also demonstrated by histological analyses. The inhibition of SARS-CoV-2 replication in vitro and the reduction of the viral load in the lungs of infected hamsters treated with Larifan alongside the improved lung histopathology, suggests a potential use of Larifan in controlling the COVID-19 disease in humans.","version":"1.2","doi":"10.1101/2022.01.22.477073","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.479714","pub_date":"2022-2-11","title":"Profiling of the most reliable mutations from sequenced SARS-CoV-2 genomes scattered in Uzbekistan","abstract":"Due to rapid mutations in the coronavirus genome over time and re-emergence of multiple novel variants of concerns (VOC), there is a continuous need for a periodic genome sequencing of SARS-CoV-2 genotypes of particular region. This is for on-time development of diagnostics, monitoring and therapeutic tools against virus in the global pandemics condition. Toward this goal, we have generated 18 high-quality whole-genome sequence data from 32 SARS-CoV-2 genotypes of PCR-positive COVID-19 patients, sampled from the Tashkent region of Uzbekistan. The nucleotide polymorphisms in the sequenced sample genomes were determined, including nonsynonymous (missense) and synonymous mutations in coding regions of coronavirus genome. Phylogenetic analysis grouped fourteen whole genome sample sequences (1, 2, 4, 5, 8, 10-15, 17, 32) into the G clade (or GR sub-clade) and four whole genome sample sequences (3, 6, 25, 27) into the S clade. A total of 128 mutations were identified, consisting of 45 shared and 83 unique mutations. Collectively, nucleotide changes represented one unique frameshift mutation, four upstream region mutations, six downstream region mutations, 50 synonymous mutations, and 67 missense mutations. The sequence data, presented herein, is the first coronavirus genomic sequence data from the Republic of Uzbekistan, which should contribute to enrich the global coronavirus sequence database, helping in future comparative studies. More importantly, the sequenced genomic data of coronavirus genotypes of this study should be useful for comparisons, diagnostics, monitoring, and therapeutics of COVID-19 disease in local and regional levels.","version":"1.1","doi":"10.1101/2022.02.10.479714","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479493","pub_date":"2022-2-11","title":"In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance","abstract":"In vitro selection of remdesivir-resistant SARS-CoV-2 revealed the emergence of a V166L substitution, located outside of the polymerase active site of the nsp12 protein, after 9 passages. V166L remained the only nsp12 substitution after 17 passages at a final concentration of 10 \u00b5M RDV, conferring a 2.3-fold increase in EC50. When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to RDV resistance.","version":"1.1","doi":"10.1101/2022.02.07.479493","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.11.480029","pub_date":"2022-2-11","title":"Omicron (BA.1) and Sub-Variants (BA.1, BA.2 and BA.3) of SARS-CoV-2 Spike Infectivity and Pathogenicity: A Comparative Sequence and Structural-based Computational Assessment","abstract":"The Omicron variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread throughout the world. We used computational tools to assess the spike infectivity, transmission, and pathogenicity of Omicron (BA.1) and sub-variants (BA.1.1, BA.2, and BA.3) in this study. BA.1 has 39 mutations, BA.1.1 has 40 mutations, BA.2 has 31 mutations, and BA.3 has 34 mutations, with 21 shared mutations between all. We observed 11 common mutations in Omicron\u2019s receptor-binding domain and sub-variants. In pathogenicity analysis, the Y505H, N786K, T95I, N211I, N856K, and V213R mutations in omicron and sub-variants are predicted to be deleterious. Due to the major effect of the mutations characterising, in the receptor-binding domain (RBD), we found that Omicron and sub-variants had a higher positive electrostatic surface potential. This could increase interaction between RBD and electronegative human angiotensin-converting enzyme 2 (hACE2). Omicron and sub-variants had a higher affinity for hACE2 and the potential for increased transmission when compared to the wild type. Among Omicron sub-lineages, BA.2 and BA.3 have a higher transmission potential than BA.1 and BA.1.1. We predicted that mutated residues in BA.1.1 (K478), BA.2 (R400, R490, R495), and BA.3 (R397 and H499) formation of new salt bridges and hydrogen bonds. Omicron and sub-variant mutations at Receptor-binding Motif (RBM) residues such as Q493R, N501Y, Q498, T478K, and Y505H all contribute significantly to binding affinity with human ACE2. Interactions with Omicron variant mutations at residues 493, 496, 498, and 501 seem to restore ACE2 binding effectiveness lost due to other mutations like K417N and Y505H.","version":"1.1","doi":"10.1101/2022.02.11.480029","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.11.480063","pub_date":"2022-2-11","title":"Molecular Dynamics Simulations Studies On The Effects Of Mutations On The Binding Affinities Between SARS-CoV-2 Spike RBD And Human ACE2","abstract":"The SARS-CoV-2 viruses had made a great impact on humankind and the world economy. Phylogenetic analysis revealed the newly identified B.1.617.1 and B.1.617.2 lineages possessed with few key mutations predominantly circulating. The signature mutations possessed by these lineages are situated in the RBD motif of S protein. Reports revealed variants L452R, T478K, and E484Q harbours in enhancement with hACE2 binding while P681R situated in furin cleavage site resulting in better transmissibility. To gain a deeper understanding of the impact of these variants (L452R, T478K and E484Q) binding with hACE2, structural dynamics at the interface between S-RBD protein and hACE2 were studied. We performed our dynamics studies with both single mutant complex (L452R, T478K and E484Q) and in the combination of triple mutants (L452R + T478K + E484Q) at 100ns in contrast with the wild type. Interfacial docking interactions and Molecular Mechanics approach exhibited that the spike mutants \u2212L452R, T478K and E484Q harbour with higher binding affinity on hACE2 in contrast with its native spike protein. The presence of interfacial residue, intermolecular contacts such as hydrogen bonding, salt bridge and non-hydrogen bonded interactions might be the reason for its higher binding affinity. Hence the findings from our study unravelled plausible mechanism for the increase in affinities of mutants to hACE2 thus leading to higher transmissibility and infection of emerging variants. Further, the conformational alterations in the course of dynamics at the RBD motif led to enhancement of hACE2 binding and immune escape. These results suggest that the structural changes introduced by these variants enhance the binding affinities of the S protein with the hACE2 that could form the basis to further aid in designing therapeutics that could inhibit at the interface of S protein and hACE2 receptor.","version":"1.1","doi":"10.1101/2022.02.11.480063","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.10.480009","pub_date":"2022-2-11","title":"Binding Interactions between RBD of Spike-Protein and Human ACE2 in Omicron variant","abstract":"Emergence of new SARS-CoV-2 Omicron VOC (OV) has exacerbated the COVID-19 pandemic due to a large number of mutations in the spike-protein, particularly in the receptor-binding domain (RBD), resulting in highly contagious and/or vaccine-resistant strain. Herein, we present a systematic analysis based on detailed molecular dynamics (MD) simulations in order to understand how the OV RBD mutations affect the ACE2 binding. We show that the OV RBD binds to ACE2 more efficiently and tightly due predominantly to strong electrostatic interactions, thereby promoting increased infectivity and transmissibility compared to other strains. Some of OV RBD mutations are predicted to affect the antibody neutralization either through their role in the S-protein conformational changes, such as S371L, S373P, and S375F, or through changing its surface charge distribution, such as G339D, N440K, T478K, and E484A. Other mutations, such as K417N, G446S, and Y505H, decrease the ACE2 binding, whereas S447N, Q493R, G496S, Q498R, and N501Y tend to increase it.","version":"1.1","doi":"10.1101/2022.02.10.480009","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.11.480072","pub_date":"2022-2-11","title":"Extensive in vitro and in vivo protein translation via in situ circularized RNAs","abstract":"RNAs are a powerful therapeutic class. However their inherent transience impacts their activity both as an interacting moiety as well as a template. Circularization of RNA has been demonstrated as a means to improve persistence, however simple and scalable approaches to achieve this are lacking. Utilizing autocatalytic RNA circularization, here we engineer in situ circularized RNAs (icRNAs). This approach enables icRNA delivery as simple linear RNA that is circularized upon delivery into the cell, thus making them compatible with routine synthesis, purification, and delivery formulations. We confirmed extensive protein translation from icRNAs both in vitro and in vivo and explored their utility in three contexts: first, we delivered the SARS-CoV-2 Omicron spike protein in vivo as icRNAs and showed corresponding induction of humoral immune responses; second, we demonstrated robust genome targeting via zinc finger nucleases delivered as icRNAs; and third, to enable compatibility between persistence of expression and immunogenicity, we developed a novel long range multiplexed (LORAX) protein engineering methodology to screen progressively deimmunized Cas9 proteins, and demonstrated efficient genome and epigenome targeting via their delivery as icRNAs. We anticipate this highly simple and scalable icRNA methodology could have broad utility in basic science and therapeutic applications.","version":"1.1","doi":"10.1101/2022.02.11.480072","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479842","pub_date":"2022-2-10","title":"The rise and fall of SARS-CoV-2 variants and the emergence of competing Omicron lineages","abstract":"In late December of 2019, high throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we analyze a subset (n=83,204) of all publicly available SARS-CoV-2 genomes (n=~5.6 million) that were randomly selected, but equally distributed over the course of the pandemic. We plot the emergence and extinction of new variants of concern (VOCs) over time and show how this corresponds to the ongoing accumulation of mutations in SARS-CoV-2 genomes and individual proteins. While the accumulation of mutations generally follows a linear regression, non-synonymous mutations are significantly greater in Omicron viruses than in previous variants\u2013especially in the spike and nucleoproteins\u2013and these differences are more pronounced in a recently identified sub-lineage (BA.2) of Omicron. Omicron is the fifth SARS-CoV-2 variant to be designated a Variant of Concern (VOC) by the World Health Organization (WHO). Here we provide a retrospective analysis of SARS-CoV-2 variants and explain how the Omicron variant is distinct. Our work shows that the spike and nucleoproteins have accumulated the most mutations in Omicron variants, but that the accessory proteins of SARS-CoV-2 sequences are changing most rapidly relative to their size. Collectively, this \u201cObservation\u201d provides a concise overview of SARS-CoV-2 evolution, reveals mutational differences between two Omicron lineages, and highlights changes in the SARS-CoV-2 proteome that have been under reported.","version":"1.1","doi":"10.1101/2022.02.09.479842","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479786","pub_date":"2022-2-10","title":"Predicting Epitope Candidates for SARS-CoV-2","abstract":"Epitopes are short amino acid sequences that define the antigen signature to which an antibody binds. In light of the current pandemic, epitope analysis and prediction is paramount to improving serological testing and developing vaccines. In this paper, we leverage known epitope sequences from SARS-CoV, SARS-CoV-2 and other Coronaviridae and use those known epitopes to identify additional antigen regions in 62k SARS-CoV-2 genomes. Additionally, we present epitope distribution across SARS-CoV-2 genomes, locate the most commonly found epitopes, discuss where epitopes are located on proteins, and how epitopes can be grouped into classes. We also discuss the mutation density of different regions on proteins using a big data approach. We find that there are many conserved epitopes between SARS-CoV-2 and SARS-CoV, with more diverse sequences found in Nucleoprotein and Spike Glycoprotein.","version":"1.1","doi":"10.1101/2022.02.09.479786","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479840","pub_date":"2022-2-10","title":"Remdesivir and GS-441524 retain antiviral activity against Delta, Omicron, and other emergent SARS-CoV-2 variants","abstract":"Genetic variation of SARS-CoV-2 has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern or interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV, VEKLURY\u00ae) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein ELISA and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with EC50 values 0.31 to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.15 to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.","version":"1.1","doi":"10.1101/2022.02.09.479840","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479835","pub_date":"2022-2-10","title":"Dual Inhibition of Cathepsin L and 3CL-Pro by GC-376 Constrains SARS Cov2 Infection Including Omicron Variant","abstract":"Recurrent waves of SARS CoV2 infections remain a major global health concern. Emergence of highly infectious variants with reduced sensitivity to neutralization by vaccines and monoclonal antibodies (mAb) necessitates a deeper understanding of factors involved in SARS CoV2 infections and identification of drug candidates to halt infection. Here, we determined the primacy of endosomal protease cathepsin-L in mediating SARS CoV2 entry and screened a library of well-annotated bioactive compounds for potent cathepsin-L inhibitory activity. Whilst the potent cathepsin-L inhibitors were capable of inhibiting SARS CoV2 entry and cytopathic effect (CPE) in less susceptible cell lines such as human ACE2 expressing 293T cells, these drugs failed to inhibit SARS CoV2 in highly susceptible cell lines such as human TMPRSS2 or human-ACE2-TMPRSS2 overexpressing Vero E6 cells. Only drugs with dual inhibitory effect on both host cathepsin-L and virus 3CL-Protease enzymes such as Z-FA-FMK and GC-376 were capable of inhibiting prototypic (USA-WA1/2020, Lineage A) SARS CoV2 induced CPE in highly susceptible cell lines. Moreover, these drugs inhibited delta (Lineage-B.1.617.2) and omicron (Lineage-B.1.1.529) infection with equal potency showing that the newer mutations harbored in these variants did not affect the mechanism of action of these drugs such as cathepsin-L or 3CL-Pro inhibition. Moreover, our early evidence that 3CL-Pro inhibition can effectively inhibit omicron-induced CPE in highly susceptible cell lines suggests that the recently FDA-approved oral drug, a 3CL-Pro inhibitor which is a combination of nirmatrelvir/ritonavir (Paxlovid) could be effective against omicron variant which shows reduced sensitivity to vaccines and mAb. We report that cathepsin-L and 3CL-Pro as major targets for designing antivirals against SARS CoV2. Dual inhibition of cathepsin-L and 3CL-Pro by GC-376 renders it effective in inhibiting SARS CoV2-induced cytopathic effect in highly susceptible cell lines. Moreover, this candidate drug is equally effective against prototypic SARS CoV2 lineage A and emerging variants such as delta and omicron which show reduced sensitivity to vaccines and monoclonal antibodies. Given the recent wave of SARS CoV2 omicron variant infection around the world, and 3CL-Pro inhibitor nirmatrelvir is one of the components of the FDA-approved Paxlovid, our findings are timely, important and should be of broad interest.","version":"1.1","doi":"10.1101/2022.02.09.479835","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479781","pub_date":"2022-2-10","title":"Engineering Defensin \u03b1-helix to produce high-affinity SARS-CoV-2 Spike protein binding ligands","abstract":"The binding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike protein to the Angiotensin-Converting Enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 peptidomimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small and highly stable proteins, and engineered the amino acid residues on its conformationally constrained alpha-helices to mimic the critical residues on the ACE2 helix 1 that interacts with the Spike-protein. The engineered proteins were soluble and purified to homogeneity with high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability, high-affinity binding to the Spike protein with dissociation constants in the low nanomolar range, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical peptidomimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing high-affinity ligands. The engineered proteins developed in this study are cost-effective and highly stable reagents for SARS-CoV-2 detection. These features may allow large-scale and cost-effective production of diagnostic tests to assist COVID-19 diagnostic and prevention.","version":"1.1","doi":"10.1101/2022.02.09.479781","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479755","pub_date":"2022-2-10","title":"Cell delivery peptides for small interfering RNAs targeting SARS-CoV-2 new variants through a bioinformatics and deep learning design","abstract":"Nucleic acid technologies with designed delivery systems have surged as one the most promising therapies of the future, due to their contribution in combating SARS-CoV-2 severe disease. Nevertheless, the emergence of new variants of concern still represents a real threat in the years to come. It is here that the use of small interfering RNA sequences to inhibit gene expression and, thus, protein synthesis, may complement the already developed vaccines, with faster design and production. Here, we have designed new sequences targeting COVID-19 variants and other related viral diseases through bioinformatics, while also addressing the limited number of delivery peptides by a deep learning approach. Two sequences databases were produced, from which 62 were able to target the virus mRNA, and ten displayed properties present in delivery peptides, which we compared to the broad use TAT delivery peptide.","version":"1.1","doi":"10.1101/2022.02.09.479755","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479588","pub_date":"2022-2-10","title":"Epigenetic Memory of COVID-19 in Innate Immune Cells and Their Progenitors","abstract":"Severe coronavirus disease 2019 (COVID-19) is characterized by systemic inflammation and can result in protracted symptoms. Robust systemic inflammation may trigger persistent changes in hematopoietic cells and innate immune memory through epigenetic mechanisms. We reveal that rare circulating hematopoietic stem and progenitor cells (HSPC), enriched from human blood, match the diversity of HSPC in bone marrow, enabling investigation of hematopoiesis and HSPC epigenomics. Following COVID-19, HSPC retain epigenomic alterations that are conveyed, through differentiation, to progeny innate immune cells. Epigenomic changes vary with disease severity, persist for months to a year, and are associated with increased myeloid cell differentiation and inflammatory or antiviral programs. Epigenetic reprogramming of HSPC may underly altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors. Transcriptomic and epigenomic analysis of blood reveal sustained changes in hematopoiesis and innate immunity after COVID-19.","version":"1.1","doi":"10.1101/2022.02.09.479588","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.27.478053","pub_date":"2022-2-09","title":"Neutralization Of SARS-CoV-2 Variants By A Human Polyclonal Antibody Therapeutic (COVID-HIG, NP-028) With High Neutralizing Titers To SARS-CoV-2","abstract":"Since the start of the COVID-19 outbreak the World Health Organization (WHO) has classified multiple SARS-CoV-2 Variants-of-Concern and Variants-of-Interest (VOC/VOI) with mutations in their Spike protein that increase transmissibility and/or reduce the effectiveness of vaccines and monoclonal antibody therapeutics. The emergence of these variants represents a significant health risk and highlights the need for additional COVID-19 therapeutics that maintain the ability to neutralize current, as well as future variants. COVID-HIG (NP-028) is a polyclonal Anti-SARS-CoV-2 human Immunoglobulin purified from source human plasma screened for high antibody titers to SARS-CoV-2 antigens. COVID-HIG was previously evaluated in INSIGHT 013 clinical trial [NCT04546581] which was an international, multi-center, adaptive, randomized, double-blind, placebo-controlled trial of the safety, tolerability and efficacy of a single dose infusion (up to 400 mL) of Anti-Coronavirus Hyperimmune Intravenous Immunoglobulin (hIVIG) for the treatment of adult recently hospitalized COVID-19 patients (N=593). COVID-HIG is currently being evaluated for clinical efficacy in a Phase 3 placebo-controlled study INSIGHT 012 (NCT04910269) to compare the safety and efficacy of a single infusion of anti-COVID-19 hyperimmune immunoglobulin (hIVIG) versus placebo among adults with recently diagnosed SARS-CoV-2 infection who do not require hospitalization. In the present study, in-vitro pseudovirus and live virus neutralization assays were used to assess the impact of SARS-CoV-2 variant spike mutations on neutralizing potency of COVID-HIG. These assays are valuable tools for monitoring the potential impact of variant mutations on efficacy of antibody therapeutics as well as vaccines/natural immunity. To date, COVID-HIG (NP-028) has been shown to retain neutralizing potency against 20 full spike protein sequence SARS-CoV-2 pseudovirus variants including all currently classified VOC/VOI (Alpha, Beta, Gamma, Delta/Delta+, Eta, Iota, Kappa, Lambda, Mu as of Sept 2021) as well as 4 live virus variants (Alpha, Beta, Gamma, and Iota).","version":"1.1","doi":"10.1101/2022.01.27.478053","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479471","pub_date":"2022-2-09","title":"The mechanism of RNA capping by SARS-CoV-2","abstract":"The SARS-CoV-2 RNA genome contains a 5\u2019-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2\u2019-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.","version":"1.1","doi":"10.1101/2022.02.07.479471","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.479664","pub_date":"2022-2-09","title":"SARS-CoV-2 ORF6 disrupts innate immune signalling by inhibiting cellular mRNA export","abstract":"SARS-CoV-2 is a betacoronavirus and the etiological agent of COVID-19, a devastating infectious disease. Due to its far-reaching effect on human health, there is an urgent and growing need to understand the viral molecular biology of SARS-CoV-2 and its interaction with the host cell. SARS-CoV-2 encodes 9 predicted accessory proteins, which are presumed to be dispensable for in vitro replication, most likely having a role in modulating the host cell environment to aid viral replication. Here we show that the ORF6 accessory protein interacts with cellular Rae1 to inhibit cellular protein production by blocking mRNA export. We utilised cell fractionation coupled with mRNAseq to explore which cellular mRNA species are affected by ORF6 expression and show that ORF6 can inhibit the export of many mRNA including those encoding antiviral factors such as IRF1 and RIG-I. We also show that export of these mRNA is blocked in the context of SARS-CoV-2 infection. Together, our studies identify a novel mechanism by which SARS-CoV-2 can manipulate the host cell environment to supress antiviral responses, providing further understanding to the replication strategies of a virus that has caused an unprecedented global health crisis. SARS-CoV-2 is the virus responsible for the current COVID-19 pandemic. Coronaviruses, like SARS-CoV-2, replicate their genome in the cytoplasm of the host cell by hijacking the cellular machinery. In addition to structural proteins and viral enzymes, SARS-CoV-2 encodes 9 accessory proteins. Although these are not required for in vitro replication, they are thought to modulate the host cell environment to favour viral replication. In this work, we show that the ORF6 accessory protein can supress cellular protein production by blocking mRNA nuclear export through interacting with the cellular protein Rae1, a known mRNA export factor. We also investigated which cellular mRNAs were retained in the nucleus when ORF6 was overexpressed. Interestingly, we found that ORF6 inhibited the export of many different mRNAs, including those encoding antiviral factors, like IRF1 and RIG-I, even in the absence of stimulation by interferon. Importantly, we found that the export of these mRNAs was similarly affected in the context of SARS-CoV-2 infection. Therefore, we believe we have identified a novel mechanism that SARS-CoV-2 uses to suppress antiviral responses in order to make the cell more permissive to infection.","version":"1.1","doi":"10.1101/2022.02.08.479664","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.479634","pub_date":"2022-2-09","title":"The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters","abstract":"A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.","version":"1.1","doi":"10.1101/2022.02.08.479634","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479468","pub_date":"2022-2-09","title":"Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOC) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.","version":"1.1","doi":"10.1101/2022.02.07.479468","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.479661","pub_date":"2022-2-09","title":"N-acylethanolamine acid amide hydrolase is a novel target for drugs against SARS-CoV-2 and Zika virus","abstract":"Several compounds have been tested against SARS-CoV-2; at present, COVID-19 treatments decrease the deleterious inflammatory response and acute lung injury. However, the best therapeutic response would be expected by combining anti-inflammatory properties, while concomitantly blocking viral replication. These combined effects should drastically reduce both infection rate and severe complications induced by novel SARS-CoV-2 variants. Therefore, we explored the antiviral potency of a class of anti-inflammatory compounds that inhibit the N-Acylethanolamine acid amidase (NAAA). This enzyme catalyzes the hydrolysis of palmitoylethanolamide (PEA), a bioactive lipid that mediates anti-inflammatory and analgesic activity through the activation of peroxisome proliferator receptor-\u03b1 (PPAR-\u03b1). Similarly, this pathway is likely to be a significant target to impede viral replication since PPAR-\u03b1 activation leads to dismantling of lipid droplets, where viral replication of Flaviviruses and Coronaviruses occurs. Here, we show that either genetic or pharmacological inhibition of the NAAA enzyme leads to five-fold reduction in the replication of both SARS-CoV-2 and ZIKV in various cell lines. Once NAAA enzyme is blocked, both ZIKV and SARS CoV-2 replication decrease, which parallels a sudden five-fold decrease in virion release. These effects induced by NAAA inhibition occurs concomitantly with stimulation of autophagy during infection. Remarkably, parallel antiviral and anti-inflammatory effects of NAAA antagonism were confirmed in ex-vivo experiments, within SARS-CoV-2 infected human PBMC cells, in which both viral genomes and TNF-\u03b1 production drop by ~60%. It is known that macrophages contribute to viral spread, excessive inflammation and macrophage activation syndrome that NAAA inhibitors might prevent, reducing the macrophage-induced acute respiratory distress syndrome and subsequent death of COVID-19 patients.","version":"1.1","doi":"10.1101/2022.02.08.479661","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479443","pub_date":"2022-2-09","title":"Olverembatinib inhibits SARS-CoV-2-Omicron variant-mediated cytokine release","abstract":"The Omicron variant has become dominant in the U.S. and around the world. This variant is found to be 2-fold more infectious than the Delta variant, posing a significant threat of severe cases and death. We and others have recently reported that the N-terminus domain (NTD) of the SARS-CoV-2 of various variants is responsible for inducing cytokine release in human PBMCs. Here, we demonstrate that the NTD of the Omicron variant remains highly effective at inducing cytokine release in human PBMCs. Furthermore, we show that Ponatinib and a novel compound, Olverembatinib, are potent Omicron NTD-mediated cytokine release inhibitors. Target profiling revealed that Olverembatinib blocks most of the previously identified kinases responsible for cytokine release. Together, we propose that Ponatinib and Olverembatinib may represent an attractive therapeutic option for treating moderate to severe COVID-19 cases. The N-terminus domain (NTD) of the SARS-CoV-2 Omicron variant strongly induces multiple inflammatory molecules in PBMCs, unaffected by the mutations observed in the NTD. The cytokine release mediated by the Omicron variant is comparable to the Delta variant. Olverembatinib, a clinical-stage multi-kinase inhibitor, potently inhibits Omicron NTD-mediated cytokine release. Olverembatinib could relieve severe symptoms associated with COVID-19 Omicron and Delta variants.","version":"1.1","doi":"10.1101/2022.02.07.479443","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479306","pub_date":"2022-2-09","title":"Antibody Evasion Properties of SARS-CoV-2 Omicron Sublineages","abstract":"The identification of the Omicron variant (B.1.1.529.1 or BA.1) of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in Botswana in November 2021 immediately raised alarms due to the sheer number of mutations in the spike glycoprotein that could lead to striking antibody evasion. We and others recently reported results in this Journal confirming such a concern. Continuing surveillance of Omicron evolution has since revealed the rise in prevalence of two sublineages, BA.1 with an R346K mutation (BA.1+R346K) and B.1.1.529.2 (BA.2), with the latter containing 8 unique spike mutations while lacking 13 spike mutations found in BA.1. We therefore extended our studies to include antigenic characterization of these new sublineages. Polyclonal sera from patients infected by wild-type SARS-CoV-2 or recipients of current mRNA vaccines showed a substantial loss in neutralizing activity against both BA.1+R346K and BA.2, with drops comparable to that already reported for BA.1. These findings indicate that these three sublineages of Omicron are antigenically equidistant from the wild-type SARS-CoV-2 and thus similarly threaten the efficacies of current vaccines. BA.2 also exhibited marked resistance to 17 of 19 neutralizing monoclonal antibodies tested, including S309 (sotrovimab), which had retained appreciable activity against BA.1 and BA.1+R346K. This new finding shows that no presently approved or authorized monoclonal antibody therapy could adequately cover all sublineages of the Omicron variant.","version":"1.1","doi":"10.1101/2022.02.07.479306","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479477","pub_date":"2022-2-09","title":"The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature","abstract":"The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.","version":"1.1","doi":"10.1101/2022.02.07.479477","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.08.479556","pub_date":"2022-2-09","title":"Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein","abstract":"Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.","version":"1.1","doi":"10.1101/2022.02.08.479556","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479546","pub_date":"2022-2-09","title":"Narrow transmission bottlenecks and limited within-host viral diversity during a SARS-CoV-2 outbreak on a fishing boat","abstract":"The long-term evolution of viruses is ultimately due to viral mutants that arise within infected individuals and transmit to other individuals. Here we use deep sequencing to investigate the transmission of viral genetic variation among individuals during a SARS-CoV-2 outbreak that infected the vast majority of crew members on a fishing boat. We deep-sequenced nasal swabs to characterize the within-host viral population of infected crew members, using experimental duplicates and strict computational filters to ensure accurate variant calling. We find that within-host viral diversity is low in infected crew members. The mutations that did fix in some crew members during the outbreak are not observed at detectable frequencies in any of the sampled crew members in which they are not fixed, suggesting viral evolution involves occasional fixation of low-frequency mutations during transmission rather than persistent maintenance of within-host viral diversity. Overall, our results show that strong transmission bottlenecks dominate viral evolution even during a superspreading event with a very high attack rate.","version":"1.1","doi":"10.1101/2022.02.09.479546","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.09.479669","pub_date":"2022-2-09","title":"Auto-Immunoproteomics Analysis of COVID-19 ICU Patients Revealed Increased Levels of Autoantibodies Related to Male Reproductive System","abstract":"The role of autoantibodies in coronavirus disease (COVID-19) complications is not yet fully understood. The current investigation screened two independent cohorts of 97 COVID-19 patients (Discovery (Disc) cohort from Qatar (n = 49) and Replication (Rep) cohort from New York (n = 48)) utilizing high-throughput KoRectly Expressed (KREX) immunome protein-array technology. Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls (P \u2264 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients that served as controls. Both cohorts showed substantial similarities (r2 = 0.73) and exhibited higher autoantibodies responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. KREX analysis of the specific IgG autoantibody responses indicates that the targeted host proteins are supposedly increased in COVID-19 patients. The autoantigen-autoantibody response was cross-validated for SPANXN4 and STK25 proteins using Uniprot BLASTP and sequence alignment tools. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19 associated male reproductive tract complications and warrants further research. Coronavirus disease (COVID-19), caused by the SARS-CoV-2 virus, has emerged as a global pandemic with a high morbidity rate and multiorgan complications. It is observed that the host immune system contributes to the varied responses to COVID-19 pathogenesis. Autoantibodies, immune system proteins that mistakenly target the body\u2019s own tissue, may underlie some of this variation. We screened total IgG autoantibody responses against 1,318 human proteins in two COVID-19 patient cohorts. We observed several novel markers in COVID-19 patients that are associated with male fertility, such as sperm protein SPANXN4, STK25, and the apoptotic factor ATF4. Particularly, elevated levels of autoantibodies against the testicular tissue-specific protein SPANXN4 offer significant evidence of anticipating the protein role in COVID-19 associated male reproductive complications.","version":"1.1","doi":"10.1101/2022.02.09.479669","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479419","pub_date":"2022-2-09","title":"Boosting with Omicron-matched or historical mRNA vaccines increases neutralizing antibody responses and protection against B.1.1.529 infection in mice","abstract":"The B.1.1.529 Omicron variant jeopardizes vaccines designed with early pandemic spike antigens. Here, we evaluated in mice the protective activity of the Moderna mRNA-1273 vaccine against B.1.1.529 before or after boosting with preclinical mRNA-1273 or mRNA-1273.529, an Omicron-matched vaccine. Whereas two doses of mRNA-1273 vaccine induced high levels of serum neutralizing antibodies against historical WA1/2020 strains, levels were lower against B.1.1.529 and associated with infection and inflammation in the lung. A primary vaccination series with mRNA-1273.529 potently neutralized B.1.1.529 but showed limited inhibition of historical or other SARS-CoV-2 variants. However, boosting with mRNA-1273 or mRNA-1273.529 vaccines increased serum neutralizing titers and protection against B.1.1.529 infection. Nonetheless, the levels of inhibitory antibodies were higher, and viral burden and cytokines in the lung were slightly lower in mice given the Omicron-matched mRNA booster. Thus, in mice, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against B.1.1.529 infection with limited differences in efficacy measured.","version":"1.1","doi":"10.1101/2022.02.07.479419","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479352","pub_date":"2022-2-08","title":"A highly attenuated SARS-CoV-2 related pangolin coronavirus variant has a 104nt deletion at the 3\u2032-terminus untranslated region","abstract":"SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3\u2019-terminus untranslated region (3\u2019-UTR). We further report the characterization of the GX_P2V variant in in vitro and in vivo infection models. In cultured Vero and BGM cells, the GX_P2V variant produced minimal cell damage and small plaques. The GX_P2V variant infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V variant is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely due to the 104-nt deletion in the HVR in the 3\u2019-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.02.07.479352","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459480","pub_date":"2022-2-08","title":"Targeted isolation of panels of diverse human protective broadly neutralizing antibodies against SARS-like viruses","abstract":"The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy . Development of broadly effective coronavirus vaccines that can mitigate these threats is needed . Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone . Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination . Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.","version":"1.2","doi":"10.1101/2021.09.08.459480","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.05.479274","pub_date":"2022-2-08","title":"EMoMiS: A Pipeline for Epitope-based Molecular Mimicry Search in Protein Structures with Applications to SARS-CoV-2","abstract":"Epitope-based molecular mimicry occurs when an antibody cross-reacts with two different antigens due to structural and chemical similarities. Molecular mimicry between proteins from two viruses can lead to beneficial cross-protection when the antibodies produced by exposure to one also react with the other. On the other hand, mimicry between a protein from a pathogen and a human protein can lead to auto-immune disorders if the antibodies resulting from exposure to the virus end up interacting with host proteins. While cross-protection can suggest the possible reuse of vaccines developed for other pathogens, cross-reaction with host proteins may explain side effects. There are no computational tools available to date for a large-scale search of antibody cross-reactivity. We present a comprehensive Epitope-based Molecular Mimicry Search (EMoMiS) pipeline for computational molecular mimicry searches. EMoMiS, when applied to the SARS-CoV-2 Spike protein, identified eight examples of molecular mimicry with viral and human proteins. These findings provide possible explanations for (a) differential severity of COVID-19 caused by cross-protection due to prior vaccinations and/or exposure to other viruses, and (b) commonly seen COVID-19 side effects such as thrombocytopenia and thrombophilia. Our findings are supported by previously reported research but need validation with laboratory experiments. The developed pipeline is generic and can be applied to find mimicry for novel pathogens. It has applications in improving vaccine design. The developed Epitope-based Molecular Mimicry Search Pipeline (EMoMiS) is available from https://biorg.cs.fiu.edu/emomis/. giri@cs.fiu.edu","version":"1.2","doi":"10.1101/2022.02.05.479274","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.05.22269279","pub_date":"2022-02-08","title":"Spatial and temporal epidemiology of SARS-CoV-2 virus lineages in Teesside, UK, in 2020: effects of socio-economic deprivation, weather, and lockdown on lineage dynamics","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>SARS-CoV-2 emerged in the UK in January 2020. The UK government introduced control measures including national \u2018lockdowns\u2019 and local \u2018tiers\u2019 in England to control virus transmission. As the outbreak continued, new variants were detected through two national monitoring programmes that conducted genomic sequencing. This study aimed to determine the effects of weather, demographic features, and national and local COVID-19 restrictions on positive PCR tests at a sub-regional scale.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We examined the spatial and temporal patterns of COVID-19 in the Teesside sub-region of the UK, from January to December 2020, capturing the first two waves of the epidemic. We used a combination of disease mapping and mixed-effect modelling to analyse the total positive tests, and those of the eight most common virus lineages, in response to potential infection risk factors: socio-economic deprivation, population size, temperature, rainfall, government interventions, and a government restaurant subsidy (\u201cEat Out to Help Out\u201d).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Total positive tests of SARS-CoV-2 were decreased by temperature and the first national lockdown (the only one to include school closures), while deprivation, population, the second national lockdown, and the local tiered interventions were associated with increased cases. The restaurant subsidy and rainfall had no apparent effect. The relationships between positive tests and covariates varied greatly between lineages, likely due to the strong heterogeneity in their spatial and temporal distributions. Cases during the second wave appeared to be higher in areas that recorded fewer first-wave cases, however, an additional model showed the number of first-wave cases was not predictive of second-wave cases.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Discussion</jats:title>\n                  <jats:p>National and local government interventions appeared to be ineffective at the sub-regional level if they did not include school closures. Examination of viral lineages at the sub-regional scale was less useful in terms of investigating covariate associations but may be more useful for tracking spread within communities. Our study highlights the importance of understanding the effects of government interventions in local and regional contexts, and the importance of applying local restrictions appropriately within such settings.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.02.05.22269279","journal":"medRxiv","score":null},{"id":"10.1101/2022.02.08.479543","pub_date":"2022-2-08","title":"Spike Protein-independent Attenuation of SARS-CoV-2 Omicron Variant in Laboratory Mice","abstract":"Despite being more transmissible, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant was found to cause milder diseases in laboratory animals, often accompanied by a lower viral load compared to previous variants of concern. This study revealed the structural basis for a robust interaction between the receptor binding domain of the Omicron spike protein and mouse ACE2. Pseudovirus bearing the Omicron spike protein efficiently utilized mouse ACE2 for entry. By comparing viral load and disease severity among laboratory mice infected by a natural Omicron variant or recombinant ancestral viruses bearing either the entire Omicron Spike or only the N501Y/Q493R mutations in its spike, we found that mutations outside the spike protein in the Omicron variant may be responsible for the observed lower viral load. Together, our results indicated that a post-entry block to the Omicron variant exists in laboratory mice.","version":"1.1","doi":"10.1101/2022.02.08.479543","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441806","pub_date":"2022-2-08","title":"Recombination patterns in coronaviruses","abstract":"As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.","version":"1.2","doi":"10.1101/2021.04.28.441806","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450225","pub_date":"2022-2-08","title":"An Efficient Coalescent Epoch Model for Bayesian Phylogenetic Inference","abstract":"We present a two headed approach called Bayesian Integrated Coalescent Epoch PlotS (BICEPS) for efficient inference of coalescent epoch models. Firstly, we integrate out population size parameters and secondly we introduce a set of more powerful Markov chain Monte Carlo (MCMC) proposals for flexing and stretching trees. Even though population sizes are integrated out and not explicitly sampled through MCMC, we are still able to generate samples from the population size posteriors. This allows demographic reconstruction through time and estimating the timing and magnitude of population bottlenecks and full population histories. Altogether, BICEPS can be considered a more muscular version of the popular Bayesian skyline model. We demonstrate its power and correctness by a well calibrated simulation study. Furthermore, we demonstrate with an application to SARS-CoV-2 genomic data that some analyses that have trouble converging with the traditional Bayesian skyline prior and standard MCMC proposals can do well with the BICEPS approach. BICEPS is available as open source package for BEAST 2 under GPL license and has a user friendly graphical user interface. Bayesian phylogenetics, coalescent model, BEAST 2, BICEPS","version":"1.2","doi":"10.1101/2021.06.28.450225","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.06.479285","pub_date":"2022-2-07","title":"Vaccine Protection Against the SARS-CoV-2 Omicron Variant in Macaques","abstract":"The rapid spread of the SARS-CoV-2 Omicron (B.1.1.529) variant, including in highly vaccinated populations, has raised important questions about the efficacy of current vaccines. Immune correlates of vaccine protection against Omicron are not known. 30 cynomolgus macaques were immunized with homologous and heterologous prime-boost regimens with the mRNA-based BNT162b2 vaccine and the adenovirus vector-based Ad26.COV2.S vaccine. Following vaccination, animals were challenged with the SARS-CoV-2 Omicron variant by the intranasal and intratracheal routes. Omicron neutralizing antibodies were observed following the boost immunization and were higher in animals that received BNT162b2, whereas Omicron CD8+ T cell responses were higher in animals that received Ad26.COV2.S. Following Omicron challenge, sham controls showed more prolonged virus in nasal swabs than in bronchoalveolar lavage. Vaccinated macaques demonstrated rapid control of virus in bronchoalveolar lavage, and most vaccinated animals also controlled virus in nasal swabs, showing that current vaccines provide substantial protection against Omicron in this model. However, vaccinated animals that had moderate levels of Omicron neutralizing antibodies but negligible Omicron CD8+ T cell responses failed to control virus in the upper respiratory tract. Virologic control correlated with both antibody and T cell responses. BNT162b2 and Ad26.COV2.S provided robust protection against high-dose challenge with the SARS-CoV-2 Omicron variant in macaques. Protection against this highly mutated SARS-CoV-2 variant correlated with both humoral and cellular immune responses.","version":"1.1","doi":"10.1101/2022.02.06.479285","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.07.451463","pub_date":"2022-2-07","title":"SARS-CoV-2 promotes microglial synapse elimination in human brain organoids","abstract":"Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection causes an extensive cell death and loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synaptic stripping. To a large extent, SARS-CoV-2 exposed microglia display a transcriptomic profile previously observed in neurodegenerative disorders characterized by early a synapse loss and an increased incident risk after a Covid-19 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from Covid-19.","version":"1.2","doi":"10.1101/2021.07.07.451463","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.06.479332","pub_date":"2022-2-07","title":"A potent SARS-CoV-2 neutralizing antibody recognizing a conserved epitope with broad mutant variant and SARS-CoV activity","abstract":"COVID-19 is the deadliest respiratory virus pandemic since 1918 and the latest of several coronavirus epidemics and pandemics in recent years. Despite the unprecedented response by both the government and private sectors to develop vaccines and therapies, the evolution of SARS-CoV-2 variants resistant to these interventions reveals a crucial need for therapeutics that maintain their efficacy against current and future mutant variants. Here we describe a SARS-CoV-2 neutralizing antibody, ABP-310, with potent activity against all variants tested including the Omicron variant. ABP-310 also displays potent neutralizing activity against SARS-CoV, highlighting the conserved nature of the ABP-310 epitope. By targeting a conserved epitope, we believe that ABP-310 has therapeutic promise not only against the current SARS-CoV-2 variants but would be expected to maintain efficacy against future variants and possibly even novel coronaviruses.","version":"1.1","doi":"10.1101/2022.02.06.479332","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.04.479171","pub_date":"2022-2-07","title":"SARS-CoV-2 variant of concern type and biological sex affect efficacy of molnupiravir in dwarf hamster model of severe COVID-19","abstract":"SARS-CoV-2 variants of concern (VOC) have triggered distinct infection waves in the coronavirus disease 2019 (COVID-19) pandemic, culminating in currently all-time high incidence rates of VOC omicron. Orally available direct-acting antivirals such as molnupiravir promise to improve disease management and limit SARS-CoV-2 spread. However, molnupiravir efficacy against VOC delta was questioned based on clinical trial results and its potency against omicron is unknown. This study evaluates molnupiravir against a panel of relevant VOC in three efficacy models: primary human airway epithelium organoids, the ferret model of upper respiratory disease, and a lethal Roborovski dwarf hamster efficacy model of severe COVID-19-like acute lung injury. All VOC were equally efficiently inhibited by molnupiravir in cultured cells and organoids. Treatment consistently reduced upper respiratory VOC shedding in ferrets and prevented viral transmission. Pathogenicity in the dwarf hamsters was VOC-dependent and highest for gamma, omicron, and delta with fulminant lung histopathology. Oral molnupiravir started 12 hours after infection resulted in complete survival of treated dwarf hamsters independent of challenge VOC. However, reduction in lung virus differed VOC-dependently, ranging from one (delta) to four (gamma) orders of magnitude compared to vehicle-treated animals. Dwarf hamsters infected with VOC omicron showed significant individual variation in response to treatment. Virus load reduction was significant in treated males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir seen in human trials and alerts that therapeutic benefit of approved antivirals must be continuously reassessed in vivo as new VOC emerge.","version":"1.1","doi":"10.1101/2022.02.04.479171","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478647","pub_date":"2022-2-07","title":"Why SARS-CoV-2 Omicron variant is milder? A single high-frequency mutation of structural envelope protein matters","abstract":"SARS-CoV-2 Omicron variant is highly transmissible and extensive morbidity, which has raised concerns for antiviral therapy. In addition, the molecular basis for the attenuated pathogenicity and replication capacity of Omicron remains elusive. Here, we report for the first time that a high-frequency mutation T9I on 2-E of SARS-CoV-2 variant Omicron forms a non-selective ion channel with abolished calcium permeability and reduced acid sensitivity compared to the WT channel. In addition, T9I caused less cell death and a weaker cytokine production. The channel property changes might be responsible for the Omicron variant releases less efficiently and induces a comparatively lower level of cell damage in the infected cells. Our study gives valuable insights into key features of the Omicron variant, further supporting 2-E is a promising drug target against SARS-CoV-2 and providing critical information for the COVID-19 treatment.","version":"1.2","doi":"10.1101/2022.02.01.478647","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.04.479189","pub_date":"2022-2-07","title":"Detection of SARS-CoV-2 Omicron variant (B.1.1.529) infection of white-tailed deer","abstract":"White-tailed deer (Odocoileus virginianus) are highly susceptible to infection by SARS-CoV-2, with multiple reports of widespread spillover of virus from humans to free-living deer. While the recently emerged SARS-CoV-2 B.1.1.529 Omicron variant of concern (VoC) has been shown to be notably more transmissible amongst humans, its ability to cause infection and spillover to non-human animals remains a challenge of concern. We found that 19 of the 131 (14.5%; 95% CI: 0.10\u20130.22) white-tailed deer opportunistically sampled on Staten Island, New York, between December 12, 2021, and January 31, 2022, were positive for SARS-CoV-2 specific serum antibodies using a surrogate virus neutralization assay, indicating prior exposure. The results also revealed strong evidence of age-dependence in antibody prevalence. A significantly (\u03c7, p < 0.001) greater proportion of yearling deer possessed neutralizing antibodies as compared with fawns (OR=12.7; 95% CI 4\u201337.5). Importantly, SARS-CoV-2 nucleic acid was detected in nasal swabs from seven of 68 (10.29%; 95% CI: 0.0\u20130.20) of the sampled deer, and whole-genome sequencing identified the SARS-CoV-2 Omicron VoC (B.1.1.529) is circulating amongst the white-tailed deer on Staten Island. Phylogenetic analyses revealed the deer Omicron sequences clustered closely with other, recently reported Omicron sequences recovered from infected humans in New York City and elsewhere, consistent with human to deer spillover. Interestingly, one individual deer was positive for viral RNA and had a high level of neutralizing antibodies, suggesting either rapid serological conversion during an ongoing infection or a \u201cbreakthrough\u201d infection in a previously exposed animal. Together, our findings show that the SARS-CoV-2 B.1.1.529 Omicron VoC can infect white-tailed deer and highlights an urgent need for comprehensive surveillance of susceptible animal species to identify ecological transmission networks and better assess the potential risks of spillback to humans. These studies provide strong evidence of infection of free-living white-tailed deer with the SARS-CoV-2 B.1.1.529 Omicron variant of concern on Staten Island, New York, and highlight an urgent need for investigations on human-to-animal-to-human spillovers/spillbacks as well as on better defining the expanding host-range of SARS-CoV-2 in non-human animals and the environment.","version":"1.1","doi":"10.1101/2022.02.04.479189","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.05.479221","pub_date":"2022-2-07","title":"Mechanistic origin of different binding affinities of SARS-CoV and SARS-CoV-2 spike RBDs to human ACE2","abstract":"The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein mediates viral entry into host cells through binding to the cell-surface receptor angiotensin-converting enzyme 2 (ACE2). It has been shown that SARS-CoV-2 RBD (RBDCoV2) has a higher binding affinity to human ACE2 than its highly homologous SARS-CoV RBD (RBDCoV), for which the mechanistic reasons still remain to be elucidated. Here, we used the multiple-replica molecular dynamics (MD) simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations, and interface residue contact network (IRCN) analysis approach to explore the mechanistic origin of different ACE2 binding affinities of these two RBDs. The results demonstrate that, when compared to the RBDCoV2-ACE2 complex, the RBDCoV-ACE2 complex features the enhanced overall structural fluctuations and inter-protein positional movements and increased conformational entropy and diversity. The inter-protein electrostatic attractive interactions are a dominant force in determining the high ACE2 affinities of both RBDs, while the significantly strengthened electrostatic forces of attraction of ACE2 to RBDCoV2 determine the higher ACE2 binding affinity of RBDCoV2 than of RBDCoV. Comprehensive comparative analyses of the residue binding free energy components and IRCNs reveal that, although any RBD residue substitution involved in the charge change can significantly impact the inter-protein electrostatic interaction strength, it is the substitutions at the RBD interface that lead to the overall stronger electrostatic attractive force of RBDCoV2-ACE2, which in turn not only tightens the interface packing and suppresses the dynamics of RBDCoV2-ACE2, but also enhances the ACE2 binding affinity of RBDCoV2 compared to that of RBDCoV. Since the RBD residue substitutions involving gain/loss of the positively/negatively charged residues, in particular those near/at the binding interfaces with the potential to form hydrogen bonds and/or salt bridges with ACE2, can greatly enhance the ACE2 binding affinity, the SARS-CoV-2 variants carrying such mutations should be paid special attention to.","version":"1.1","doi":"10.1101/2022.02.05.479221","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.03.479080","pub_date":"2022-2-07","title":"In vitro effect of a non-immunosuppressive FKBP ligand, FK1706, on SARS-CoV-2 replication in combination with antivirals","abstract":"FKBP, a naturally occurring ubiquitous intracellular protein, has been proposed as a potential target for coronavirus replication. A non-immunosuppressive FKBP ligand, FK1706, was studied in vitro in a Vero cell model to assess potential activity alone and in combination with antivirals against SARS-CoV-2 replication. When combined with remdesivir, synergistic activity was seen (summary synergy score 24.7+9.56). FK1706 warrants in vivo testing as a potential new combination therapeutic for the treatment of COVID-19 infections.","version":"1.2","doi":"10.1101/2022.02.03.479080","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.07.479348","pub_date":"2022-2-07","title":"An mRNA vaccine candidate for the SARS-CoV-2 Omicron variant","abstract":"The newly emerged Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains more than 30 mutations on the spike protein, 15 of which are located within the receptor binding domain (RBD). Consequently, Omicron is able to extensively escape existing neutralizing antibodies and may therefore compromise the efficacy of current vaccines based on the original strain, highlighting the importance and urgency of developing effective vaccines against Omicron. Here we report the rapid generation and evaluation of an mRNA vaccine candidate specific to Omicron. This mRNA vaccine encodes the RBD of Omicron (designated RBD-O) and is formulated with lipid nanoparticle. Two doses of the RBD-O mRNA vaccine efficiently induce neutralizing antibodies in mice; however, the antisera are effective only on the Omicron variant but not on the wildtype and Delta strains, indicating a narrow neutralization spectrum. It is noted that the neutralization profile of the RBD-O mRNA vaccine is opposite to that observed for the mRNA vaccine expressing the wildtype RBD (RBD-WT). Our work demonstrates the feasibility and potency of an RBD-based mRNA vaccine specific to Omicron, providing important information for further development of bivalent or multivalent SARS-CoV-2 vaccines with broad-spectrum efficacy.","version":"1.1","doi":"10.1101/2022.02.07.479348","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.13.476204","pub_date":"2022-2-07","title":"Covariance predicts conserved protein residue interactions important to the emergence and continued evolution of SARS-CoV-2 as a human pathogen","abstract":"SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21st century and that likely emerged from animal reservoirs. Differences in nucleotide and protein sequence composition within related \u03b2-coronaviruses are often used to better understand CoV evolution, host adaptation, and their emergence as human pathogens. Here we report the comprehensive analysis of amino acid residue changes that have occurred in lineage B \u03b2-coronaviruses that show covariance with each other. This analysis revealed patterns of covariance within conserved viral proteins that potentially define conserved interactions within and between core proteins encoded by SARS-CoV-2 related \u03b2-coranaviruses. We identified not only individual pairs but also networks of amino acid residues that exhibited statistically high frequencies of covariance with each other using an independent pair model followed by a tandem model approach. Using 149 different CoV genomes that vary in their relatedness, we identified networks of unique combinations of alleles that can be incrementally traced genome by genome within different phylogenic lineages. Remarkably, covariant residues and their respective regions most abundantly represented are implicated in the emergence of SARS-CoV-2 are also enriched in dominant SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2022.01.13.476204","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.04.479134","pub_date":"2022-2-07","title":"A Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19","abstract":"SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allowed for rapid movement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.","version":"1.1","doi":"10.1101/2022.02.04.479134","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.04.479136","pub_date":"2022-2-07","title":"Mutations of Omicron variant at the interface of the receptor domain motif and human angiotensin-converting enzyme-2","abstract":"The most recent Omicron variant of SARS-CoV-2 has caused global concern and anxiety. The only thing certain about this strain, with large number of mutations in the spike protein, is that it spreads quickly, seems to evade immune defense and mitigates the benefits of existing vaccines. Based on the ultra-large-scale ab initio computational modeling of the receptor binding motif (RBM) and human angiotensin-converting enzyme-2 (ACE2) interface we provide the details of the effect of Omicron mutations at the fundamental atomic scale level. In-depth analysis anchored in the novel concept of amino acid-amino acid bond pair units (AABPU), indicates that mutations in the Omicron variant are connected with (i) significant changes in the shape and structure of AABPU components, together with (ii) significant increase in the positive partial charge which facilitates the interaction with ACE2. The calculated bond order, based on AABPU, reveals that the Omicron mutations increase the binding strength of RBM to ACE2. Our findings correlate with and are instrumental to explain the current observations and can contribute to the prediction of next potential new variant of concern.","version":"1.1","doi":"10.1101/2022.02.04.479136","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.05.478644","pub_date":"2022-2-05","title":"A scalable pipeline for SARS-CoV-2 replicon construction based on de-novo synthesis","abstract":"Replicons are synthetic viral RNA molecules that recapitulate the self-replicating activities of the virus but are missing its infectivity potential. Here, we report on a scalable pipeline to generate a replicon of any SARS-CoV-2 strain using de-novo synthesis. Our pipeline relies only on publicly available sequencing data without requiring access to any material, simplifying logistical and bureaucratic issues of sample acquisition. In addition, our system retains the nucleotide sequence of most of the SARS-CoV-2 full genome and therefore better captures its underlying genomic and biological functions as compared to the popular pseudotypes or any replicon system published to-date. We utilized our system to synthesize a SARS-CoV-2 non-infectious version of the Beta strain. We then confirmed that the resulting RNA molecules are non-infectious and safe to handle in a BSL2/CL2 facility. Finally, we show that our replicon can be specifically inhibited by molnupiravir and RNAi treatments, demonstrating its utility for drug research and development.","version":"1.1","doi":"10.1101/2022.02.05.478644","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.02.478897","pub_date":"2022-2-04","title":"Primary macrophages exhibit a modest inflammatory response early in SARS-CoV-2 infection","abstract":"Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-\u03b1 and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 infected MDM with notable absence of IFN-\u03b2 induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-\u03b1 enhanced proinflammatory cytokine expression upon infection. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.","version":"1.1","doi":"10.1101/2022.02.02.478897","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.03.479081","pub_date":"2022-2-04","title":"Circulatory exosomes from COVID-19 patients trigger NLRP3 inflammasome in endothelial cells","abstract":"SARS-CoV-2 infection induces inflammatory response, cytokine storm, venous thromboembolism, coagulopathy, and multiple organ damage. Resting endothelial cells prevent coagulation, control blood flow and inhibit inflammation. However, it remains unknown how SARS-CoV-2 induces strong molecular signals in distant cells for immunopathogenesis. In this study, we examined the consequence of human endothelial cells (microvascular endothelial cells (HMEC-1) and liver endothelial cells (TMNK-1)) to exosomes from plasma of severe COVID-19 patients. We observed a significant induction of NLRP3, caspase-1 and IL-1\u03b2 mRNA expression in the endothelial cells following exposure to exosomes from plasma of COVID-19 patients as compared to that of healthy donors. Activation of caspase-1 was noted in the endothelial cell culture medium following exposure to the COVID-19 exosomes. Further, COVID-19 exosomes significantly induced mature IL-1\u03b2 secretion in the endothelial cell culture medium. Thus, our results demonstrated for the first time that exosomes from COVID-19 plasma trigger NLRP3 inflammasome in endothelial cells of distant organs.","version":"1.1","doi":"10.1101/2022.02.03.479081","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.03.479037","pub_date":"2022-2-04","title":"mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron","abstract":"SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to antibody neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-specific vaccines would enhance immunity and protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing antibody titers against D614G were 4760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (pre-boost), respectively, and 320 and 110 for Omicron. Two weeks after boost, titers against D614G and Omicron increased to 5360 and 2980, respectively, for mRNA-1273 and 2670 and 1930 for mRNA-Omicron. Following either boost, 70-80% of spike-specific B cells were cross-reactive against both WA1 and Omicron. Significant and equivalent control of virus replication in lower airways was observed following either boost. Therefore, an Omicron boost may not provide greater immunity or protection compared to a boost with the current mRNA-1273 vaccine.","version":"1.1","doi":"10.1101/2022.02.03.479037","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.02.478671","pub_date":"2022-2-03","title":"Development and optimisation of a high-throughput screening assay for in vitro anti\u2013SARS-CoV-2 activity: evaluation of 5676 phase 1 passed structures","abstract":"Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging\u2013based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti\u2013SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.","version":"1.1","doi":"10.1101/2022.02.02.478671","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.02.478873","pub_date":"2022-2-03","title":"Unraveling the Enzymatic Mechanism of the SARS-CoV-2 RNA-Dependent-RNA-Polymerase. An Unusual Active Site Leading to High Replication Rates","abstract":"Viral infection relies on the hijacking of cellular machineries to enforce the reproduction of the infecting virus and its subsequent diffusion. In this context the replication of the viral genome is a key step performed by specific enzymes, i.e. polymerases. The replication of SARS-CoV-2, the causative agent of the COVID-19 pandemics, is based on the duplication of its RNA genome, an action performed by the viral RNA-dependent-RNA polymerase. In this contribution, for the first time and by using two-dimensional enhanced sampling quantum mechanics/ molecular mechanics, we have determined the chemical mechanisms leading to the inclusion of a nucleotide in the nascent viral RNA strand. We prove the high efficiency of the polymerase, which lowers the activation free energy to less than 10 kcal/mol. Furthermore, the SARS-CoV-2 polymerase active site is slightly different from those found usually found in other similar enzymes, and particularly it lacks the possibility to enforce a proton shuttle via a nearby histidine. Our simulations show that this absence is partially compensate by lysine, whose proton assist the reaction opening up an alternative, but highly efficient, reactive channel. Our results present the first mechanistic resolution of SARS-CoV-2 genome replication and shed light on unusual enzymatic reactivity paving the way for future rational design of antivirals targeting emerging RNA viruses.","version":"1.1","doi":"10.1101/2022.02.02.478873","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.02.478719","pub_date":"2022-2-03","title":"COVID-19 infection enhances susceptibility to oxidative-stress induced parkinsonism","abstract":"Viral induction of neurological syndromes has been a concern since parkinsonian-like features were observed in patients diagnosed with encephalitis lethargica subsequent to the 1918 influenza pandemic. Given the similarities in the systemic responses following SARS-CoV-2 infection with those observed after pandemic influenza, there is a question if a similar syndrome of post-encephalic parkinsonism could follow COVID-19 infection. To determine if prior infection with SARS-CoV-2 increased sensitivity to a mitochondrial toxin known to induce parkinsonism. hACE2 mice were infected with SARS-CoV-2 to induce mild to moderate disease. After 31 days recovery, mice were administered a non-lesion inducing dose of the parkinsonian toxin MPTP. Subsequent neuroinflammation and SNpc dopaminergic neuron loss was determined and compared to SARS-CoV-2 or MPTP alone. hACE2 mice infected with SARS-CoV-2 or MPTP showed no SNpc DA neuron loss following MPTP. In mice infected and recovered from SARS-CoV-2 infection, MPTP induced a 23% or 19% greater loss of SNpc dopaminergic neurons than SARS-CoV-2 or MPTP, respectively (p\u25a1<\u25a10.05). Examination of microglial activation showed a significant increase in the number of activated microglia in the SARS-CoV-2 + MPTP group compared to SARS-CoV-2 or MPTP alone. Our observations have important implications for long-term public health, given the number of people that have survived SARS-CoV-2 infection as well as for future public policy regarding infection mitigation. However, it will be critical to determine if other agents known to increase risk of PD also have synergistic effects with SARS-CoV-2 and if are abrogated by vaccination. This work was supported by grant from the State of North Carolina (PS, JE, DOR, RJS) and R21 NS122280 (RJS).","version":"1.1","doi":"10.1101/2022.02.02.478719","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.28.478180","pub_date":"2022-2-03","title":"RNase L-mediated RNA decay alters 3\u2019 end formation and splicing of host mRNAs","abstract":"The antiviral endoribonuclease, RNase L, is a vital component of the mammalian innate immune response that destroys host and viral RNA to reduce viral gene expression. Herein, we show that a consequence of RNase L-mediated decay of cytoplasmic host RNAs is the widespread re-localization of RNA-binding proteins (RBPs) from the cytoplasm to the nucleus, due to the presence of nuclear RNA. Concurrently, we observe global alterations to host RNA processing in the nucleus, including alterations of splicing and 3\u2019 end formation, with the latter leading to downstream of gene (DoG) transcripts. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and coincide with the retention of their mRNAs in the nucleus. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is fundamentally dictated by the availability of RNA in each compartment and thus viral infections that trigger cytoplasmic RNA degradation alter RNA processing due to the nuclear influx of RNA binding proteins.","version":"1.2","doi":"10.1101/2022.01.28.478180","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478701","pub_date":"2022-2-02","title":"CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation","abstract":"Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Autopsy demonstrates the presence of thrombosis and microangiopathy in the small vessels and capillaries. Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS. It has been shown that sera from individuals with COVID-19 triggered NET release in vitro, and spleen tyrosine kinase (Syk) inhibitor R406 inhibited NETosis caused by COVID-19 plasma. However, the serum components responsible for NET formation are still unknown. In this study, we found that virus-free extracellular vesicles (EVs) from COVID-19 patients (COVID-19 EVs) induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A). Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation and lung fibrosis were attenuated dramatically in clec5a-/-/tlr2-/- mice. These results suggest that COVID-19 EVs play critical roles in SARS-CoV-2-induced immunothrombosis, and blockade of CLEC5A and TLR2 is a promising strategy to inhibit SARS-CoV-2-induced intravascular coagulopathy and reduce the risk of ARDS in COVID-19 patients.","version":"1.1","doi":"10.1101/2022.02.01.478701","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478724","pub_date":"2022-2-02","title":"Metabolic dyshomeostasis induced by SARS-CoV-2 structural proteins reveals immunological insights into viral olfactory interactions","abstract":"One of the most common symptoms in COVID-19 is a sudden loss of smell. SARS-CoV-2 has been detected in the olfactory bulb (OB) from animal models and sporadically in COVID-19 patients. To decipher the specific role over the SARS-CoV-2 proteome at olfactory level, we characterized the in-depth molecular imbalance induced by the expression of GFP-tagged SARS-CoV-2 structural proteins (M, N, E, S) on mouse OB cells. Transcriptomic and proteomic trajectories uncovered a widespread metabolic remodeling commonly converging in extracellular matrix organization, lipid metabolism and signaling by receptor tyrosine kinases. The molecular singularities and specific interactome expression modules were also characterized for each viral structural factor. The intracellular molecular imbalance induced by each SARS-CoV-2 structural protein was accompanied by differential activation dynamics in survival and immunological routes in parallel with a differentiated secretion profile of chemokines in OB cells. Machine learning through a proteotranscriptomic data integration uncovered TGF-beta signaling as a confluent activation node by the SARS-CoV-2 structural proteome. Taken together, these data provide important avenues for understanding the multifunctional immunomodulatory properties of SARS-CoV-2 M, N, S and E proteins beyond their intrinsic role in virion formation, deciphering mechanistic clues to the olfactory inflammation observed in COVID-19 patients.","version":"1.1","doi":"10.1101/2022.02.01.478724","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478685","pub_date":"2022-2-02","title":"Chronic alcohol intake regulates expression of SARS-CoV2 infection-relevant genes in an organ-specific manner","abstract":"Chronic alcohol consumption and alcohol use disorder (AUD) have a tremendous impact on the patient\u2019s psychological and physiological health. There is some evidence that chronic alcohol consumption influences SARS-CoV2 infection risk, but the molecular mechanism is unknown. Here, we generated expression data of SARS-CoV2 infection relevant genes (Ace2, Tmprss2 and Mas) in different organs in rat models of chronic alcohol exposure and alcohol dependence. ACE2 and TMPRSS2 represent the virus entry point whereas Mas is activating the anti-inflammatory response once the cells are infected. Across three different chronic alcohol test conditions, we found a consistent upregulation of Ace2 in the lung, which is the most affected organ in Covid-19 patients. Other organs such as liver, ileum, kidney, heart, and the brain showed also up-regulation of Ace2 and Mas but in a less consistent manner across the different animal models, while Tmprss2 was unaffected in all conditions. We suggest that alcohol-induced up-regulation of Ace2 can lead to an elevated stochastic probability of cellular virus entry and may thus confer a molecular risk factor for a SARS-CoV2 infection.","version":"1.1","doi":"10.1101/2022.02.01.478685","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478759","pub_date":"2022-2-02","title":"Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings","abstract":"Accurate, high-resolution environmental monitoring of SARS-CoV-2 traces indoors through sentinel cards is a promising approach to help students safely return to in-person learning. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface types, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution, but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, wet wipes, RNase Away) at three different viral loads: \u201chigh\u201d (4 x 104 GE/mL), \u201cmedium\u201d (1 x 104 GE/mL), and \u201clow\u201d (2.5 x 103 GE/mL). RNAse Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. Because SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are persistent signal or new infectious cases, and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning \u201csentinel cards\u201d to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2022.02.01.478759","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478506","pub_date":"2022-2-02","title":"Detection and Interspecies Comparison of SARS-CoV-2 Delta Variant (AY.3) in Feces from a Domestic Cat and Human Samples","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have spilled over from humans to companion and wild animals since the inception of the global COVID-19 pandemic. However, whole genome sequencing data of the viral genomes that infect non-human animal species has been scant. Here, we detected and sequenced a SARS-CoV-2 delta variant (AY.3) in fecal samples from an 11-year-old domestic house cat previously exposed to an owner who tested positive for SARS-CoV-2. Molecular testing of two fecal samples collected 7 days apart yielded relatively high levels of viral RNA. Sequencing of the feline-derived viral genomes showed the two to be identical, and differing by between 4 and 14 single nucleotide polymorphisms in pairwise comparisons to human-derived lineage AY.3 sequences collected in the same geographic area and time period. However, several mutations unique to the feline samples reveal their divergence from this cohort on phylogenetic analysis. These results demonstrate continued spillover infections of emerging SARS-CoV-2 variants that threaten human and animal health, as well as highlight the importance of collecting fecal samples when testing for SARS-CoV-2 in animals. To the authors\u2019 knowledge, this is the first published case of a SARS-CoV-2 delta variant in a domestic cat in the United States.","version":"1.1","doi":"10.1101/2022.01.31.478506","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478628","pub_date":"2022-2-02","title":"USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING","abstract":"Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFN (IFN-\u03bb) exhibit cell-type specific and long-lasting functions in autoinflammation, tumorigenesis and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-\u03bb secretion and SARS-CoV-2 infections in native human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-\u03bb and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2\u20193\u2019-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-\u03bb expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.","version":"1.1","doi":"10.1101/2022.02.01.478628","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478425","pub_date":"2022-2-02","title":"HOW CONCERNING IS A SARS-COV-2 VARIANT OF CONCERN? COMPUTATIONAL PREDICTIONS AND THE VARIANTS LABELING SYSTEM","abstract":"We herein report a study to evaluate the use of computational prediction of SARS-CoV-2 genetic variations in improving the current variants labeling system. First, we reviewed the basis of the system developed by the World Health Organization (WHO) for the labeling of SARS-CoV-2 genetic variants and the adaptations made to it by the United States Center of Diseases Control (CDC). We observed that the labeling system is based upon the virus\u2019 major attributes. However, we found that the labeling criteria of the SARS-CoV-2 variants derived from these attributes are not accurately defined and are used differently by the two health management agencies. Consequently, discrepancies exist between the labels given by WHO and CDC to same variants. Our observations suggest that giving the VOC label to a new variant is premature and might not be appropriate. Therefore, we carried out a comparative computational study to predict the effects of the mutations on the virus structure and functions of five VOCs. By linking these data to the criteria used by WHO and the CDC for variant labeling, we ascertained that comparative computational predictions of the impact of genetic variations are a better ground for rapid and more accurate labelling of SARS-CoV-2 variants. We propose to label all emergent variants VUM or VBM and to carry out computational predictive studies and thorough variants comparison, upon which more appropriate and informative labels can be attributed. Furthermore, harmonization of the variants labeling system would be globally beneficial to communicate about and fight COVID-19 pandemic.","version":"1.3","doi":"10.1101/2022.01.31.478425","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.02.478775","pub_date":"2022-2-02","title":"Intracellular flow cytometry complements RT-qPCR detection of circulating SARS-CoV-2 variants of concern","abstract":"Despite the efficacy of current vaccines against SARS-CoV-2, the spread of the virus is still not under control, as evidenced by the ongoing circulation of the highly contagious SARS-CoV-2 Omicron variant. Basic and antiviral research on SARS-CoV-2 relies on cellular assays of virus replication in vitro. In addition, accurate detection of virus-infected cells and released virus particles is needed to study virus replication and to profile new candidate antiviral drugs. Here, by flow cytometry, we detect SARS-CoV-2 infection at single cell level and distinguish infected Vero E6 cells from uninfected bystander cells. Furthermore, based on the viral nucleocapsid expression, subpopulations of infected cells that are in an early or late phase of viral replication can be differentiated. Importantly, this flow cytometric technique complements RT-qPCR detection and can be applied to all current SARS-CoV-2 variants of concern, including the highly mutated Omicron variant. This study describes the characterization of SARS-CoV-2 infected cells using intracellular flow cytometric viral nucleocapsid staining that complements RT-qPCR quantification of viral RNA. The technique makes it possible to distinguish between infected cells in the early (low N) or late phase (high N) of viral replication. It can also be applied to the different variants of concern of SARS-CoV-2, including the Omicron variant.","version":"1.1","doi":"10.1101/2022.02.02.478775","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478520","pub_date":"2022-2-02","title":"Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in na\u00efve hamsters compared to ancestral vaccine","abstract":"In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Initial studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. We report here the rapid development of a replicating RNA vaccine expressing the B.1.1.529 spike and show that this B.1.1.529-targeted vaccine is immunogenic in mice and hamsters. Interestingly, we found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. Rapidly developed RNA vaccine protects against SARS-CoV-2 Omicron variant","version":"1.1","doi":"10.1101/2022.01.31.478520","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478632","pub_date":"2022-2-02","title":"Design and development of potent h-ACE2 derived peptide mimetics in SARS-CoV-2 Omicron variant therapeutics","abstract":"The pandemic of COVID-19 has become the global health challenge due to the emergence of new variants. The Receptor binding domain (RBD) of spike protein that makes direct interaction with ACE-2 has shown unique mutated residues in most of the variants of concern (VOC). Recently WHO declared the Omicron (B.1.1.529) as VOC considering it as a highly mutated variant which includes a total of 60 mutations out of which 15 mutations occurred in RBD region of SARS-CoV-2. Inhibition of Protein-protein (Omicron RBD-h-ACE2) interaction was already proved to inhibit the viral infection. In this study, by using molecular dynamic simulations efforts are made to explore the atomistic details of Omicron RBD-h-ACE2 interaction. Based on MD simulations, h-ACE2 motif is found to be interacting with omicron RBD domain. Interaction analysis had provided key residues interacting with Omicron-RBD that helped to extract h-ACE2 peptide. Here, rational design of the peptides that have resemblance with h-ACE2 is done and the peptide library is subjected for inhibition studies against Omicron-RBD. The current study helped to identify the significant peptides that can inhibit Omicron-RBD. Altogether the performed studies will provide an opportunity to develop potential therapeutic peptidomimetics effective against Omicron variant of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.02.01.478632","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478507","pub_date":"2022-2-02","title":"Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine","abstract":"Despite recent success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability, remain. Although mRNA, pDNA, and viral-vector based vaccines are being administered, no protein subunit-based SARS-CoV-2 vaccine is approved. Molecular adjuvants targeting pathogen-recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulate various PRRs, including toll-like receptors (TLRs) and retinoic-acid-inducible gene I-like receptors (RIG-I). We hypothesized that targeting the same PRRs using adjuvants on nanoparticles along with a stabilized spike (S) protein antigen could provide broad and efficient immune responses. Formulations targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer-nanoparticles (NPs) were combined with the S1 subunit of S protein and assessed in vitro with isogeneic mixed lymphocyte reactions (iso-MLRs). For in vivo studies, the adjuvanted nanoparticles were combined with stabilized S protein and assessed using intranasal and intramuscular prime-boost vaccination models in mice. Combination NP-adjuvants targeting both TLR and RIG-I (MPLA+PUUC, CpG+PUUC, or R848+PUUC) differentially increased proinflammatory cytokine secretion (IL-1\u03b2, IL-12p70, IL-27, IFN-\u03b2) by APCs cultured in vitro, and induced differential T cell proliferation. When delivered intranasally, MPLA+PUUC NPs enhanced local CD4+CD44+ activated memory T cell responses while MPLA NPs increased anti-S-protein-specific IgG and IgA in the lung. Following intramuscular delivery, PUUC-carrying NPs induced strong humoral immune responses, characterized by increases in anti-S-protein IgG and neutralizing antibody titers and germinal center B cell populations (GL7+ and BCL6+ B cells). MPLA+PUUC NPs further boosted S-protein-neutralizing antibody titers and T follicular helper cell populations in draining lymph nodes. These results suggest that SARS-CoV-2-mimicking adjuvants and subunit vaccines could lead to robust and unique route-specific adaptive immune responses and may provide additional tools against the pandemic.","version":"1.1","doi":"10.1101/2022.01.31.478507","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.476607","pub_date":"2022-2-02","title":"Differences in environmental stability among SARS-CoV-2 variants of concern: Omicron has higher stability","abstract":"SARS-CoV-2 variants of concern (VOCs) could cause significant human and economic damage owing to increased infectivity and transmissibility, and understanding their characteristics is crucial for infection control. Here, we analyzed differences in viral stability and disinfection efficacy between the Wuhan strain and all VOCs. On plastic and skin surfaces, Alpha, Beta, Delta, and Omicron variants exhibited more than two-fold longer survival than the Wuhan strain, and the Omicron variant had the longest survival time. Specifically, survival times of the Wuhan strain, Alpha variant, Beta variant, Gamma variant, Delta variant, and Omicron variant on skin surfaces were 8.6 h (95% CI, 6.5\u201310.9 h), 19.6 h (95% CI, 14.8\u201325.3 h), 19.1 h (95% CI, 13.9\u2013 25.3 h), 11.0 h (95% CI, 8.1\u201314.7 h), 16.8 h (95% CI, 13.1\u201321.1 h), and 21.1 h (95% CI, 15.8\u2013 27.6 h), respectively. In vitro, disinfectant effectiveness evaluations showed that Alpha, Beta, Delta, and Omicron were slightly more resistant to ethanol than the Wuhan strain. However, ex vivo evaluation showed that on human skin, all viruses were completely inactivated by exposure to 35 w/w % ethanol for 15 s. The high environmental stability of these VOCs could increase transmission risk and contribute to spread. Additionally, the Omicron variant might have been replaced by the Delta variant due to its increased environmental stability and rapid spread. To prevent VOC spread, it is highly recommended that current infection control practices use disinfectants with appropriate ethanol concentrations.","version":"1.2","doi":"10.1101/2022.01.18.476607","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.30.478380","pub_date":"2022-2-02","title":"Using Unassigned NMR Chemical Shifts to Model RNA Secondary Structure","abstract":"NMR-derived chemical shifts are sensitive probes of RNA structure. However, the need to assign NMR spectra hampers their utility as a direct source of structural information. In this report, we describe a simple method that uses unassigned 2D NMR spectra to model the secondary structure of RNAs. Similar to assigned chemical shifts, we could use unassigned chemical shift data to reweight conformational libraries such that the highest weighted structure closely resembles their reference NMR structure. Furthermore, the application of our approach to the 3\u2019- and 5\u2019-UTR of the SARS-CoV-2 genome yields structures that are, for the most part, consistent with the secondary structure models derived from chemical probing data. Therefore, we expect the framework we describe here will be useful as a general strategy for rapidly generating preliminary structural RNA models directly from unassigned 2D NMR spectra. As we demonstrated for the 337-nt and 472-nt UTRs of SARS-CoV-2, our approach could be especially valuable for modeling the secondary structures of large RNA.","version":"1.2","doi":"10.1101/2022.01.30.478380","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.30.478400","pub_date":"2022-2-01","title":"Clonal dynamics of SARS-CoV-2-specific T cells in children and adults with COVID-19","abstract":"Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV- 2-specific na\u00efve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. More na\u00efve interferon-activated CD4+ T cells were recruited into the memory compartment and recovery was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection. Children have diverse polyclonal SARS-CoV-2-specific na\u00efve T cells Adults have clonally expanded exhausted SARS-CoV-2-specific memory T cells Interferon-activated na\u00efve T cells differentiate into memory T cells in adults but not children Adults but not children develop robust memory T cell responses to SARS-CoV-2","version":"1.1","doi":"10.1101/2022.01.30.478400","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450181","pub_date":"2022-2-01","title":"A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type and Delta variants","abstract":"Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.","version":"1.4","doi":"10.1101/2021.06.28.450181","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.07.467640","pub_date":"2022-2-01","title":"An interactome landscape of SARS-CoV-2 virus-human protein-protein interactions by protein sequence-based multi-label classifiers","abstract":"The new coronavirus species, SARS-CoV-2, caused an unprecedented global pandemic of COVID-19 disease since late December 2019. A comprehensive characterization of protein-protein interactions (PPIs) between SARS-CoV-2 and human cells is a key to understanding the infection and preventing the disease. Here we present a novel approach to predict virus-host PPIs by multi-label machine learning classifiers of random forests and XGBoost using amino acid composition profiles of virus and human proteins. Our models harness a large-scale database of Viruses.STRING with >80,000 virus-host PPIs along with evidence scores for multi-level evidence prediction, which is distinct from predicting binary interactions in previous studies. Our multi-label classifiers are based on 5 evidence levels binned from evidence scores. Our best model of XGBoost achieves 74% AUC and 68% accuracy on average in 10-fold cross validation. The most important amino acids are cysteine and histidine. In addition, our model predicts experimental PPIs with higher accuracy than text mining-based PPIs by 4% despite their smaller data size by more than 6-fold. We then predict evidence levels of \u223c2,000 SARS-CoV-2 virus-human PPIs from public experimental proteomics data. Interactions with SARS-CoV-2 Nsp7b show high evidence. We also predict evidence levels of all pairwise PPIs of \u223c550,000 between the SARS-CoV-2 and human proteomes to provide a draft virus-host interactome landscape for SARS-CoV-2 infection in humans in a comprehensive and unbiased way in silico. Most human proteins from 140 highest evidence predictions interact with SARS-CoV-2 Nsp7, Nsp1, and ORF14, with significant enrichment in the top 2 pathways of vascular smooth muscle contraction (CALD1, NPR2, CALML3) and Myc targets (CBX3, PES1). Our prediction also suggests that histone H2A components are targeted by multiple SARS-CoV-2 proteins.","version":"1.4","doi":"10.1101/2021.11.07.467640","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437194","pub_date":"2022-2-01","title":"D155Y Substitution of SARS-CoV-2 ORF3a Weakens Binding with Caveolin-1","abstract":"The clinical manifestation of the recent pandemic COVID-19, caused by the novel SARS-CoV-2 virus, varies from mild to severe respiratory illness. Although environmental, demographic and co-morbidity factors have an impact on the severity of the disease, contribution of the mutations in each of the viral genes towards the degree of severity needs a deeper understanding for designing a better therapeutic approach against COVID-19. Open Reading Frame-3a (ORF3a) protein has been found to be mutated at several positions. In this work, we have studied the effect of one of the most frequently occurring mutants, D155Y of ORF3a protein, found in Indian COVID-19 patients. Using computational simulations we demonstrated that the substitution at 155th changed the amino acids involved in salt bridge formation, hydrogen-bond occupancy, interactome clusters, and the stability of the protein compared with the other substitutions found in Indian patients. Protein-protein docking using HADDOCK analysis revealed that substitution D155Y weakened the binding affinity of ORF3a with caveolin-1 compared with the other substitutions, suggesting its importance in the overall stability of ORF3a-caveolin-1 complex, which may modulate the virulence property of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.03.26.437194","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478504","pub_date":"2022-2-01","title":"Engineering SARS-CoV-2 cocktail antibodies into a bispecific format improves neutralizing potency and breadth","abstract":"One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce antibody resistance. We engineered two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) increases antigen-binding and virus-neutralizing activities and spectrum against multiple SARS-CoV-2 variants including the Omicron, than the cocktail. X-ray crystallography and computational simulations reveal distinct neutralizing mechanisms for individual cocktail antibodies and suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and the Beta, Gamma, and Delta variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.","version":"1.1","doi":"10.1101/2022.02.01.478504","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.29.477140","pub_date":"2022-2-01","title":"Significant Broad Spectrum Antiviral activity of Bi121 Against Different Variants of SARS-CoV-2","abstract":"The SARS-CoV-2 pandemic infected 343 million people with over 5.59 million deaths. New mutated lineages of SARS-CoV-2 such as Omicron are evolving faster. Broad-spectrum viral inhibitors that block the initial stage of infection by reducing virus proliferation and disease severity is an unmet global medical need. We studied Bi121, a standardized polyphenolic-rich compound isolated from Pelargonium sidoides, against recombinant Vesicular Stomatitis Virus (rVSV)-pseudotyped SARS-CoV-2S (spike) that represent mutations in the spike protein of six different variants of SARS-CoV-2. Bi121 was effective in neutralizing all six rVSV-\u0394G-SARS-CoV-2S variants expressing different mutations. The antiviral activity of Bi121 was then assessed against three variants of SARS-CoV-2 (USA WA1/2020, Hongkong/VM20001061/2020, B.1.167.2 (Delta)) using RT-qPCR and plaque assays in two different cell lines (Vero cells and HEK-ACE2). Bi121 showed significant activity toward all the three variants tested, suggesting a broad-spectrum activity.","version":"1.2","doi":"10.1101/2022.01.29.477140","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478657","pub_date":"2022-2-01","title":"Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses","abstract":"Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the influence of immune responses induced by other vaccinations on the durability and efficacy of the immune response to SARS-CoV-2 vaccine is still unknown. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 S1 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that the hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2022.02.01.478657","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478460","pub_date":"2022-2-01","title":"A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor","abstract":"The SARS-CoV-2 main protease, Mpro, is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after Mpro-mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensor developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.","version":"1.1","doi":"10.1101/2022.01.31.478460","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.465025","pub_date":"2022-2-01","title":"Oral Lisinopril Raises Tissue Levels of ACE2, the SARS-CoV-2 Receptor, in Healthy Male and Female Mice","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the established cellular receptor for SARS-CoV-2. However, it is unclear whether ACE1 inhibitors (e.g., lisinopril) or angiotensin receptor blockers (e.g., losartan) alter tissue ACE2 expression. This study sought to determine whether lisinopril or losartan, as monotherapies or in combination, change tissue levels of ACE2 in healthy male and female mice. Mice received lisinopril (10 mg/kg/day), losartan (10 mg/kg/day), or both for 21 days via drinking water. A control group received water without drug. ACE2 protein index (ACE2 protein / total protein) was determined on small intestine, lung, kidney, and brain. Oral lisinopril increased ACE2 protein index across all tissues (p < 0.0001 vs control). In contrast, the combination of lisinopril plus losartan did not increase ACE2 levels in any tissue (p = 0.89 vs control) and even decreased tissue expression of the Ace2 gene (p < 0.001 vs control). Tissue ACE2 remained elevated in mice 21 days after cessation of lisinopril (p = 0.02). Across both cohorts, plasma ACE2 did not correlate with ACE2 protein index in any tissue. A sex difference was observed: kidney ACE2 levels were higher in males than females (p < 0.0001). Oral lisinopril increases ACE2, the cellular receptor for SARS-CoV-2, in tissues that are relevant to the transmission and pathogenesis of COVID-19. Remarkably, the addition of losartan prevented lisinopril-induced increases in ACE2 across tissues. These results suggest that ACE inhibitors and angiotensin receptor blockers interact to determine tissue levels of ACE2.","version":"1.2","doi":"10.1101/2021.10.19.465025","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440626","pub_date":"2022-2-01","title":"Identification of druggable host targets needed for SARS-CoV-2 infection by combined pharmacological evaluation and cellular network directed prioritization both in vitro and in vivo","abstract":"Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.","version":"1.3","doi":"10.1101/2021.04.20.440626","journal":"bioRxiv","score":null},{"id":"10.1101/2022.02.01.478695","pub_date":"2022-2-01","title":"Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins","abstract":"SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for the antibody system. Neutralizing antibodies targeting the RBD bind to several different sites on this domain. In contrast, most neutralizing antibodies to NTD characterized to date bind to a single supersite, however these antibodies were obtained by methods that were not NTD specific. Here we use NTD specific probes to focus on anti-NTD memory B cells in a cohort of pre-omicron infected individuals some of which were also vaccinated. Of 275 NTD binding antibodies tested 103 neutralized at least one of three tested strains: Wuhan-Hu-1, Gamma, or PMS20, a synthetic variant which is extensively mutated in the NTD supersite. Among the 43 neutralizing antibodies that were further characterized, we found 6 complementation groups based on competition binding experiments. 58% targeted epitopes outside the NTD supersite, and 58% neutralized either Gamma or Omicron, but only 14% were broad neutralizers. Three of the broad neutralizers were characterized structurally. C1520 and C1791 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to previously described antibodies allowing for greater potency and cross-reactivity with 7 different variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously uncharacterized class of NTD-directed antibodies that recognizes the viral membrane proximal side of the NTD and SD2 domain, leading to cross-neutralization of Beta, Gamma and Omicron. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-NTD antibodies some of which can neutralize variants of concern. Rapid recruitment of these cells into the antibody secreting plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.","version":"1.1","doi":"10.1101/2022.02.01.478695","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478497","pub_date":"2022-2-01","title":"Human Cardiac Organoids to Model COVID-19 Cytokine Storm Induced Cardiac Injuries","abstract":"Acute cardiac injuries occur in 20-25% of hospitalized COVID-19 patients. Despite urgent needs, there is a lack of 3D organotypic models of COVID-19 hearts for mechanistic studies and drug testing. Herein, we demonstrate that human cardiac organoids (hCOs) are a viable platform to model the cardiac injuries caused by COVID-19 hyperinflammation. As IL-1\u03b2is an upstream cytokine and a core COVID-19 signature cytokine, it was used to stimulate hCOs to induce the release of a milieu of proinflammatory cytokines that mirror the profile of COVID-19 cytokine storm. The IL-1 \u03b2 treated hCOs recapitulated transcriptomic, structural, and functional signatures of COVID-19 hearts. The comparison of IL-1\u03b2 treated hCOs with cardiac tissue from COVID-19 autopsies illustrated the critical roles of hyper-inflammation in COVID-19 cardiac insults and indicated the cardioprotective effects of endothelium. The IL-1\u03b2 treated hCOs also provide a viable model to assess the efficacy and potential side effects of immunomodulatory drugs, as well as the reversibility of COVID-19 cardiac injuries at baseline and simulated exercise conditions.","version":"1.1","doi":"10.1101/2022.01.31.478497","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.27.477964","pub_date":"2022-1-31","title":"Virucidal activity and mechanism of action of cetylpyridinium chloride against SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. Recent studies have shown the importance of the throat and salivary glands as sites of virus replication and transmission. The viral host receptor, angiotensin-converting enzyme 2 (ACE2), is broadly enriched in epithelial cells of the salivary glands and oral mucosae. Oral care products containing cetylpyridinium chloride (CPC) as a bactericidal ingredient are known to exhibit antiviral activity against SARS-CoV-2 in vitro. However, the exact mechanism of action remains unknown. This study examined the antiviral activity of CPC against SARS-CoV-2 and its inhibitory effect on the interaction between the viral spike (S) protein and ACE2 using an enzyme-linked immunosorbent assay. CPC (0.05%, 0.1% and 0.3%) effectively inactivated SARS-CoV-2 within the contact times (20 and 60 s) in directions for use of oral care products in vitro. The binding ability of both the S protein and ACE2 were reduced by CPC. Our results suggest that CPC inhibits the interaction between S protein and ACE2, and thus, reduces infectivity of SARS-CoV-2 and suppresses viral adsorption.","version":"1.1","doi":"10.1101/2022.01.27.477964","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.30.478343","pub_date":"2022-1-31","title":"High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients","abstract":"SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with \u03b1-CCCs and \u03b2-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-\u03b3-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with \u03b2-CCCs strains but significantly fewer co-infections with \u03b1-CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with \u03b1-CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with \u03b1-CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines. A broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells were associated with severe disease onset in COVID-19 patients. High frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients. Compared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4+ and CD8+ T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens. The cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients. Compared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the \u03b1-CCCs strains. Compared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4+ and CD8+ T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the \u03b2-CCCs strains.","version":"1.1","doi":"10.1101/2022.01.30.478343","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.27.478128","pub_date":"2022-1-31","title":"A pseudotyped lentivirus-based assay to titer SARS-CoV-2 neutralizing antibodies in Mexico","abstract":"Measuring the neutralizing potential of SARS-CoV-2 antigens-exposed sera informs on effective humoral immunity. This is relevant to 1-monitor levels of protection within an asymptomatic population, 2-evaluate the efficacy of existing and novel vaccines against emerging variants, 3-test prospective therapeutic monoclonal neutralizing antibodies (NAbs) and, overall, to contribute to understand SARS-CoV-2 immunity. However, the gold-standard method to titer NAbs is a functional assay of virus-mediated infection, which requires biosafety level 3 (BSL-3) facilities. As these facilities are insufficient in Latin American countries, including Mexico, scant information has been obtained about NAb in these countries during the COVID-19 pandemic. An alternative solution to acquire NAb information locally is to use non-replicative viral particles that display the SARS-CoV-2 Spike (S) protein on their surface, and deliver a reporter gene into target cells upon transduction. Here we present the development of a NAb-measuring assay based on Nanoluc-mediated luminescence measurements from SARS-CoV-2 S-pseudotyped lentiviral particle-infected cells. The successive steps of development are presented, including lentiviral particles production, target cell selection, and TCID50 determination. We applied the optimized assay in a BSL-2 facility to measure NAbs in 15 pre-pandemic, 18 COVID-19 convalescent and 32 BNT162b2 vaccinated serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. The assay highlighted heterogeneity in neutralization curves which are relevant in discussing neutralization potency dynamics. Overall, this is the first report of a BSL-2 safe functional assay to measure SARS-CoV-2 in Mexico and a cornerstone methodology necessary to measure NAb with a functional assay in the context of limited resources settings. Evaluating effective humoral immunity against SARS-CoV-2 requires a functional assay with infectious virus. Handling the authentic SARS-CoV-2 virus requires specialized facilities that are not readily available in Latin America, including Mexico. Here we produce non-replicative viral particles pseudotyped with the SARS-CoV-2 S protein that are used as safe surrogate viral particles in an optimized BSL-2 ready neutralization assay. The establishment of this assay is critical to allow the evaluation of effective humoral immunity to SARS-CoV-2 post-infection and to monitor the efficacy of existing or novel vaccines against emerging variants in the Mexican population.","version":"1.1","doi":"10.1101/2022.01.27.478128","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477616","pub_date":"2022-1-31","title":"Visible blue light inactivates SARS-CoV-2 variants and inhibits Delta replication in differentiated human airway epithelia","abstract":"The emergence of SARS-CoV-2 variants that evade host immune responses has prolonged the COVID-19 pandemic. Thus, the development of an efficacious, variant-agnostic therapeutic for the treatment of early SARS-CoV-2 infection would help reduce global health and economic burdens. Visible light therapy has the potential to fill these gaps. In this study, visible blue light centered around 425 nm efficiently inactivated SARS-CoV-2 variants in cell-free suspensions and in a translationally relevant well-differentiated tissue model of the human large airway. Specifically, 425 nm light inactivated cell-free SARS-CoV-2 variants Alpha, Beta, Delta, Gamma, Lambda, and Omicron by up to 99.99% in a dose-dependent manner, while the monoclonal antibody bamlanivimab did not neutralize the Beta, Delta, and Gamma variants. Further, we observed that 425 nm light reduced virus binding to host ACE-2 receptor and limited viral entry to host cells in vitro. Further, the twice daily administration of 32 J/cm2 of 425 nm light for three days reduced infectious SARS-CoV-2 Beta and Delta variants by >99.99% in human airway models when dosing began during the early stages of infection. In more established infections, logarithmic reductions of infectious Beta and Delta titers were observed using the same dosing regimen. Finally, we demonstrated that the 425 nm dosing regimen was well-tolerated by the large airway tissue model. Our results indicate that blue light therapy has the potential to lead to a well-tolerated and variant-agnostic countermeasure against COVID-19.","version":"1.1","doi":"10.1101/2022.01.25.477616","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.30.478159","pub_date":"2022-1-31","title":"An intranasal lentiviral booster broadens immune recognition of SARS-CoV-2 variants and reinforces the waning mRNA vaccine-induced immunity that it targets to lung mucosa","abstract":"As the COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines wains and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal humoral and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization because it is non-cytopathic, non-replicative and scarcely inflammatory. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine\u2013primed and -boosted mice, with waning primary humoral immunity at 4 months post-vaccination, were boosted intranasally with LV::SBeta-2P. Strong boost effect was detected on cross-sero-neutralizing activity and systemic T-cell immunity. In addition, mucosal anti-Spike IgG and IgA, lung resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19.","version":"1.1","doi":"10.1101/2022.01.30.478159","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.27.478082","pub_date":"2022-1-31","title":"Susceptibility of wild canids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a promiscuous virus, capable of infecting a variety of different animal species, but much work remains in determining the susceptibility of common wildlife species to the virus. Here, we demonstrate that following experimental inoculation with SARS-CoV-2, red fox (Vulpes vulpes) become infected and can shed virus in oral and respiratory secretions. Conversely, experimentally challenged coyotes (Canis latrans) did not become infected or shed virus. Our results add red fox to the animal species known to be susceptible to SARS-CoV-2 and suggest that they may contribute to continued maintenance and transmission of the virus. Experimental infection of red fox (Vulpes vulpes) and coyotes (Canis latrans) with SARS-CoV-2 revealed that red fox are susceptible to infection and can shed virus, while coyotes do not become infected.","version":"1.1","doi":"10.1101/2022.01.27.478082","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478406","pub_date":"2022-1-31","title":"Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants","abstract":"The emerging SARS-CoV-2 variants of concern (VOC) harbor mutations associated with increasing transmission and immune escape, hence undermine the effectiveness of current COVID-19 vaccines. In late November of 2021, the Omicron (B.1.1.529) variant was identified in South Africa and rapidly spread across the globe. It was shown to exhibit significant resistance to neutralization by serum not only from convalescent patients, but also from individuals receiving currently used COVID-19 vaccines with multiple booster shots. Therefore, there is an urgent need to develop next generation vaccines against VOCs like Omicron. In this study, we develop a panel of mRNA-LNP-based vaccines using the receptor binding domain (RBD) of Omicron and Delta variants, which are dominant in the current wave of COVID-19. In addition to the Omicron- and Delta-specific vaccines, the panel also includes a \u201cHybrid\u201d vaccine that uses the RBD containing all 16 point-mutations shown in Omicron and Delta RBD, as well as a bivalent vaccine composed of both Omicron and Delta RBD-LNP in half dose. Interestingly, both Omicron-specific and Hybrid RBD-LNP elicited extremely high titer of neutralizing antibody against Omicron itself, but few to none neutralizing antibody against other SARS-CoV-2 variants. The bivalent RBD-LNP, on the other hand, generated antibody with broadly neutralizing activity against the wild-type virus and all variants. Surprisingly, similar cross-protection was also shown by the Delta-specific RBD-LNP. Taken together, our data demonstrated that Omicron-specific mRNA vaccine can induce potent neutralizing antibody response against Omicron, but the inclusion of epitopes from other variants may be required for eliciting cross-protection. This study would lay a foundation for rational development of the next generation vaccines against SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2022.01.31.478406","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.29.478316","pub_date":"2022-1-31","title":"Differences between Omicron SARS-CoV-2 RBD and other variants in their ability to interact with cell receptors and monoclonal antibodies","abstract":"SARS-CoV-2 has caused immeasurable damage worldwide and available treatments with high efficacy are still scarce. With the continuous emergence of new variants of the virus, such as Omicron, Alpha, Beta, Gamma, and Delta - the so-called variants of concern, the available therapeutic and prevention strategies had to return to the experimental trial to verify their effectiveness against them. This work aims to expand the knowledge about the SARS-CoV-2 receptor-binding domain (RBD) interactions with cell receptors and monoclonal antibodies (mAbs). Special attention is given to the Omicron variant and its comparison with the others, including its sublineage BA.2 and two new ones (B.1.640.1 and B.1.640.2/IHU) recently found in France. By using constant-pH Monte Carlo simulations, the free energy of interactions between the SARS-CoV-2 receptor-binding domain (RBD) from different variants and several partners (Angiotensin-Converting Enzyme-2 (ACE2) polymorphisms and several mAbs) were calculated. It was evaluated both the impact of mutations for the RBD-ACE2 and how strongly each of mAb can bind to the virus RBD, which can indicate their therapeutic potential for neutralization. RBD-ACE2-binding affinities were higher for two ACE2 polymorphisms typically found in Europeans (rs142984500 and rs4646116), indicating that these types of polymorphisms may be related to genetic susceptibility to COVID-19. The antibody landscape was computationally investigated with the largest set of mAbs so far in the literature. From the 33 studied binders, groups of mAbs were identified with weak (e.g. S110 and Ab3b4), medium (e.g. CR3022), and strong binding affinities (e.g. P01\u2019\u2019\u2019, S2K146 and S230). All the mAbs with strong binding capacity could also bind to the RBD from SARS-CoV-1, SARS-CoV-2 wt, and all studied variants. These mAbs and especially their combination are amenable to experimentation and clinical trials because of their high binding affinities and neutralization potential for current known virus mutations and a universal coronavirus.","version":"1.1","doi":"10.1101/2022.01.29.478316","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.30.478305","pub_date":"2022-1-31","title":"Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies","abstract":"The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant.","version":"1.1","doi":"10.1101/2022.01.30.478305","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.29.478335","pub_date":"2022-1-31","title":"SARS-CoV-2 leverages airway epithelial protective mechanism for viral infection","abstract":"Despite much concerted effort to better understand SARS-CoV-2 viral infection, relatively little is known about the dynamics of early viral entry and infection in the airway. Here we analyzed a single-cell RNA sequencing dataset of early SARS-CoV-2 infection in a humanized in vitro model, to elucidate key mechanisms by which the virus triggers a cell-systems-level response in the bronchial epithelium. We find that SARS-CoV-2 virus preferentially enters the tissue via ciliated cell precursors, giving rise to a population of infected mature ciliated cells, which signal to basal cells, inducing further rapid differentiation. This feed-forward loop of infection is mitigated by further cell-cell communication, before interferon signaling begins at three days post-infection. These findings suggest hijacking by the virus of potentially beneficial tissue repair mechanisms, possibly exacerbating the outcome. This work both elucidates the interplay between barrier tissues and viral infections, and may suggest alternative therapeutic approaches targeting non-immune response mechanisms.","version":"1.1","doi":"10.1101/2022.01.29.478335","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.31.478157","pub_date":"2022-1-31","title":"In silico analysis of predicted differential MHC binding and CD8+ T-cell immune escape of SARS-CoV-2 B.1.1.529 variant mutant epitopes","abstract":"The B.1.1.529 (Omicron) SARS-CoV-2 variant has raised global concerns due to its high number of mutations and its rapid spread. It is of major importance to understand the impact of this variant on the acquired and induced immunity. Several preliminary studies have reported the impact of antibody binding and to this date, there are few studies on Omicron\u2019s CD8+ T-cell immune escape. We first assessed the impact of Omicron and B.1.617.2 (Delta) variant mutations on the SARS-CoV-2 spike epitopes submitted to the Immune Epitope Database (IEDB) with positive out-come on MHC ligand or T-cell assays (n=411). From those epitopes modified by a mutation, we found the corresponding homologous epitopes in Omicron and Delta. We then ran the netMHCpan computational MHC binding prediction on the pairs of IEDB epitopes and matching homologous epitopes over top 5 MHC I alleles on some selected populations. Lastly, we applied a Fisher test to find mutations enriched for homologous epitopes with decreased predicted binding affinity. We found 31 and 78 IEDB epitopes modified by Delta and Omicron mutations, respectively. The IEDB spike protein epitopes redundantly cover the protein sequence. The WT pMHC with a strong predicted binding tend to have homologous mutated pMHC with decreased binding. A similar trend is observed in Delta over all HLA genes, while in Omicron only for HLA-B and HLA-C. Finally, we obtained one and seven mutations enriched for homologous mutated pMHC with decreased MHC binding affinity in Delta and Omicron, respectively. Three of the Omicron mutations, VYY143-145del, K417N and Y505H, are replacing an aromatic or large amino acid, which are reported to be enriched in immunogenic epitopes. K417N is common with Beta variants, while Y505H and VYY143-145del are novel Omicron mutations. In summary, pMHC with Delta and Omicron mutations show decreased MHC binding affinity, which results in a trend specific to SARS-CoV-2 variants. Such epitopes may decrease overall presentation on different HLA alleles suggesting evasion from CD8+ T-cell responses in specific HLA alleles. However, our results show B.1.1.529 (Omicron) will not totally evade the immune system through a CD8+ immune escape mechanism. Yet, we identified mutations in B.1.1.529 (Omicron) introducing amino acids associated with increased immunogenicity. All the code and results from this study are available at https://github.com/TRON-bioinformatics/omicron-analysis.","version":"1.1","doi":"10.1101/2022.01.31.478157","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477819","pub_date":"2022-1-31","title":"Analysis of receptors responsible for the dysfunction of the human immune system by different viral infections","abstract":"There are difficulties in creating direct anti-viral drugs for all viruses, including new, suddenly arising infections, such as COVID-19. Therefore, pathogenetic therapy is often used to treat severe viral infections. Despite significant distinctions in the etiopathogenesis of viral diseases, they are often associated with the substantial dysfunction of the immune system. To identify shared mechanisms of immune dysfunction during infection by nine different viruses (cytomegalovirus, Ebstein-Barr virus, human T-cell leukemia virus type 1, Hepatitis B and C viruses, human immunodeficiency virus, Dengue virus, SARS-CoV, and SARS-CoV-2), we applied analysis of corresponding transcription profiles from peripheral blood mononuclear cells (PBMC). As a result, we revealed common pathways, cellular processes, and master regulators for studied viral infections. We found that all nine viral infections cause immune activation, exhaustion, cell proliferation disruption, and increased susceptibility to apoptosis. An application of network analysis allowed us to identify receptors of PBMC that are the proteins at the top of signaling pathways, which may be responsible for the observed transcription changes. The identified relationships between some of them and virus-induced immune disfunction are new, with little or no information in the literature, e.g., receptors for autocrine motility factor, insulin, prolactin, angiotensin II, and immunoglobulin epsilon.","version":"1.1","doi":"10.1101/2022.01.26.477819","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477860","pub_date":"2022-1-30","title":"Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms","abstract":"With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.","version":"1.2","doi":"10.1101/2022.01.26.477860","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.28.22270044","pub_date":"2022-01-30","title":"Transmission of SARS-CoV-2 Omicron VOC subvariants BA.1 and BA.2: Evidence from Danish Households","abstract":"<jats:label>1</jats:label>\n                <jats:title>Abstract</jats:title>\n                <jats:p>The Omicron SARS-CoV-2 variant of concern (VOC lineage B.1.1.529), which became dominant in many countries during early 2022, includes several subvariants with strikingly different genetic characteristics. Several countries, including Denmark, have observed the two Omicron subvariants: BA.1 and BA.2. In Denmark the latter has rapidly replaced the former as the dominant subvariant.</jats:p>\n                <jats:p>Based on nationwide Danish data, we estimate the transmission dynamics of BA.1 and BA.2 following the spread of Omicron VOC within Danish households in late December 2021 and early January 2022.</jats:p>\n                <jats:p>Among 8,541 primary household cases, of which 2,122 were BA.2, we identified a total of 5,702 secondary infections among 17,945 potential secondary cases during a 1-7 day follow-up period. The secondary attack rate (SAR) was estimated as 29% and 39% in households infected with Omicron BA.1 and BA.2, respectively.</jats:p>\n                <jats:p>We found BA.2 to be associated with an increased susceptibility of infection for unvaccinated individuals (Odds Ratio (OR) 2.19; 95%-CI 1.58-3.04), fully vaccinated individuals (OR 2.45; 95%-CI 1.77-3.40) and booster-vaccinated individuals (OR 2.99; 95%-CI 2.11-4.24), compared to BA.1. We also found an increased transmissibility from unvaccinated primary cases in BA.2 households when compared to BA.1 households, with an OR of 2.62 (95%-CI 1.96-3.52). The pattern of increased transmissibility in BA.2 households was not observed for fully vaccinated and booster-vaccinated primary cases, where the OR of transmission was below 1 for BA.2 compared to BA.1.</jats:p>\n                <jats:p>We conclude that Omicron BA.2 is inherently substantially more transmissible than BA.1, and that it also possesses immune-evasive properties that further reduce the protective effect of vaccination against infection, but do not increase its transmissibility from vaccinated individuals with breakthrough infections.</jats:p>","version":null,"doi":"10.1101/2022.01.28.22270044","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.25.477724","pub_date":"2022-1-29","title":"Distinct genetic determinants and mechanisms of SARS-CoV-2 resistance to remdesivir","abstract":"The nucleoside analog remdesivir (RDV) is an FDA-approved antiviral for the treatment of SARS- CoV-2 infections, and as such it is critical to understand potential genetic determinants and barriers to RDV resistance. In this study, SARS-CoV-2 was subjected to 13 passages in cell culture with increasing concentrations of GS-441524, the parent nucleoside of RDV. At passage 13 the RDV resistance of the lineages ranged from 2.7-to 10.4-fold increase in EC50. Sequence analysis of the three lineage populations identified non-synonymous mutations in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two of the three lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first then combined with S759A. Introduction of the S759A and V792I substitutions at homologous nsp12 positions in viable isogenic clones of the betacoronavirus murine hepatitis virus (MHV) demonstrated their transferability across CoVs, up to 38-fold RDV resistance in combination, and a significant replication defect associated with their introduction. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a \u223c10- fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, while nsp12-V792I diminished the UTP concentration needed to overcome the template-dependent inhibition associated with RDV. The in vitro selected substitutions here identified were rare or not detected in the >6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in various clinical settings. SARS-CoV-2 develops in vitro resistance to remdesivir by distinct and complementary mutations and mechanisms in the viral polymerase","version":"1.1","doi":"10.1101/2022.01.25.477724","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.28.22269990","pub_date":"2022-01-29","title":"BA.1 and BA.2 sub-lineages of Omicron variant have comparable replication kinetics and susceptibility to neutralization by antibodies","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  The Omicron variant of SARS-CoV-2 is capable of infecting unvaccinated, vaccinated and previously-infected individuals due to its ability to evade neutralization by antibodies. With three sub-lineages of Omicron emerging in the last four months, there is inadequate information on the quantitative antibody response generated upon natural infection with Omicron variant and whether these antibodies offer cross-protection against other sub-lineages of Omicron variant. In this study, we characterized the growth kinetics of Kappa, Delta and Omicron variants of SARS-CoV-2 in Calu-3 cells. Relatively higher amounts infectious virus titers, cytopathic effect and disruption of epithelial barrier functions was observed with Delta variant whereas infection with Omicron variant led to a more robust induction of interferon pathway, lower level of virus replication and mild effect on epithelial barrier. The replication kinetics of BA.1 and BA.2 sub-lineages of the Omicron variant were comparable in cell culture and natural Omicron infection in a subset of individuals led to a significant increase in binding and neutralizing antibodies to both BA.1 and BA.2 sub-lineages but these levels were lower than that produced against the Delta variant. Finally, we show that Cu\n                  <jats:sup>2+</jats:sup>\n                  , Zn\n                  <jats:sup>2+</jats:sup>\n                  and Fe\n                  <jats:sup>2+</jats:sup>\n                  salts inhibited\n                  <jats:italic>in vitro</jats:italic>\n                  RdRp activity but only Cu\n                  <jats:sup>2+</jats:sup>\n                  and Fe\n                  <jats:sup>2+</jats:sup>\n                  inhibited both the Delta and Omicron variants in cell culture. Thus, our results suggest that high levels of interferons induced upon infection with Omicron variant may counter virus replication and spread. Waning neutralizing antibody titers rendered subjects susceptible to infection by Omicron variant and natural Omicron infection elicits neutralizing antibodies that can cross-react with other sub-lineages of Omicron and other variants of concern.\n                </jats:p>","version":null,"doi":"10.1101/2022.01.28.22269990","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.22.477323","pub_date":"2022-1-28","title":"Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion","abstract":"Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S protein\u2019s autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.","version":"1.3","doi":"10.1101/2022.01.22.477323","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477612","pub_date":"2022-1-28","title":"Endogenous pancreatic microRNAs differentially target the Delta, Omicron, and Wuhan SARS-CoV-2 genomes to upregulate the Diabetes-associated genes","abstract":"The SARS-CoV-2 viral genome is mutating and evolving into new variations, including the recently discovered Delta and Omicron. In this study, we used and evaluated our notion of differential targeting of SARS-CoV-2 variant genomes by microRNAs (miRNAs) of the infected human pancreas. We found that even with UTR mutations, the Delta, Omicron, and original Wuhan variations\u2019 genomes would be differentially targeted by the host pancreas cell\u2019s native miRNAs in the same way. The miRNAs show a difference in Minimal Free Energy (MFE) with the different viral variants\u2019 genomes; however, they would still be responsible for the upregulation of the diabetes-associated genes.","version":"1.1","doi":"10.1101/2022.01.26.477612","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477298","pub_date":"2022-1-28","title":"The N764K and N856K mutations in SARS-CoV-2 Omicron BA.1 S protein generate potential cleavage sites for SKI-1/S1P protease","abstract":"Spike (S) protein is a key protein in coronaviruses life cycle. SARS-CoV-2 Omicron BA.1 variant of concern (VoC) presents an exceptionally high number of 30 substitutions, 6 deletions and 3 insertions in the S protein. Recent works revealed major changes in the SARS-CoV-2 Omicron biological properties compared to earlier variants of concern (VoCs). Here, these major changes could be explained, at least in part, by the mutations N764K and/or N856K in S2 subunit. These mutations were not previously detected in other VoCs. N764K and N856K generate two potential cleavage sites for SKI-1/S1P serine protease, known to cleave viral envelope glycoproteins. The new sites where SKI-1/S1P could cleave S protein might impede the exposition of the internal fusion peptide for membrane fusion and syncytia formation. Based on the human protein atlas, SKI-1/S1P protease is not found in lung tissues (alveolar cells type I/II and endothelial cells), but present in bronchus and nasopharynx. This may explain why Omicron has change of tissue tropism. Viruses have evolved to use several host proteases for cleavage/activation of envelope glycoproteins. Mutations that allow viruses to change of protease may have a strong impact in host range, cell and tissue tropism, and pathogenesis.","version":"1.2","doi":"10.1101/2022.01.21.477298","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.384743","pub_date":"2022-1-27","title":"Transmission of SARS-CoV-2 from humans to animals and potential host adaptation","abstract":"SARS-CoV-2, the agent of the COVID-19 pandemic, can infect a wide range of mammals. Since its spread in humans, secondary host jumps of SARS-CoV-2 from humans to a variety of domestic and wild populations of mammals have been documented. The evolution of SARS-CoV-2 in different host species is of fundamental interest while also providing indication of how SARS-CoV-2 may have adapted to human hosts soon after the initial host jump, a time window for which there are no genome sequences available. Moreover, the study of SARS-CoV-2 circulating in animals is critical to assess the risk that the transmission of animal-adapted viral lineages back into humans (i.e., spillback) may pose. Here, we compared the genomic landscapes of SARS-CoV-2 isolated from animal species relative to that in humans, profiling the mutational biases indicative of potentially different selective pressures in animals. We focused on viral genomes collected in infected mink (Neovison vison) and white-tailed deer (Odocoileus virginianus) for which reports of multiple independent spillover events and subsequent animal-to-animal transmission are available. We identified six candidate mutations for animal-specific adaptation in mink (NSP9_G37E, Spike_F486L, Spike_N501T, Spike_Y453F, ORF3a_T229I, ORF3a_L219V), and one in deer (NSP3a_L1035F), though these mutations appear to confer minimal advantage for circulation in humans. Additionally, circulation of SARS-CoV-2 in mink and deer has not caused considerable changes to the evolutionary trajectory of SARS-CoV-2 thus far. Finally, our results suggest that minimal adaptation was required for human-to-animal spillover and subsequent onward transmission in mink and deer, highlighting the \u2018generalist\u2019 nature of SARS-CoV-2 as a pathogen of mammalian hosts.","version":"1.2","doi":"10.1101/2020.11.16.384743","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477969","pub_date":"2022-1-27","title":"Effect of SARS-CoV-2 spike mutations on its activation by TMPRSS2 and TMPRSS13","abstract":"The continuous emergence of new SARS-CoV-2 variants urges better understanding of the functional motifs in the spike (S) protein and their tolerance towards mutations. We here focus on the S2\u2019 motif which, during virus entry, requires cleavage by a cell surface protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2\u2019 motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant S-pseudotyped viruses, we show that K814, which precedes the scissile R815 residue, is dispensable for SARS-CoV-2 spike activation by TMPRSS2 but not TMPRSS13. The latter protease lost its activity towards SARS-CoV-2 S when the S2\u2019 motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689) and also the reverse effect was seen. This swap had no impact on TMPRSS2 activation. Also in the MERS-CoV spike, introducing a dibasic scissile motif was fully accepted by TMPRSS13 but less so by TMPRSS2. Our findings are the first to demonstrate which S2\u2019 residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS13 exhibiting higher preference for K/R rich motifs than TMPRSS2. This preemptive insight can help to estimate the impact of S2\u2019 motif changes as they may appear in new SARS-CoV-2 variants. Since the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning in a direct manner. Remarkably, though located in a strong immunogenic region, the S2\u2019 cleavage motif has, thus far, remained highly conserved. This suggests that its amino acid sequence is critical for spike activation by airway proteases. To investigate this, we assessed which S2\u2019 site mutations affect processing by TMPRSS2 and TMPRSS13, two main activators of the SARS-CoV-2 spike. Being the first in its kind, our study will help to assess the biological impact of S2\u2019 site variations as soon as they are detected during variant surveillance.","version":"1.1","doi":"10.1101/2022.01.26.477969","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477757","pub_date":"2022-1-27","title":"Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance","abstract":"Coronaviruses, as exemplified by SARS-CoV-2, can evolve and spread rapidly to cause severe disease morbidity and mortality. Direct acting antivirals (DAAs) are highly effective in decreasing disease burden especially when they target essential viral enzymes, such as proteases and polymerases, as demonstrated in HIV-1 and HCV and most recently SARS-CoV-2. Optimization of these DAAs through iterative structure-based drug design has been shown to be critical. Particularly, the evolutionarily conserved molecular mechanisms underlying viral replication can be leveraged to develop robust antivirals against rapidly evolving viral targets. The main protease (Mpro) of SARS-CoV-2, which is evolutionarily constrained to recognize and cleave 11 specific sites to promote viral maturation, exemplifies one such target. In this study we define the substrate envelope of Mpro by determining the molecular basis of substrate recognition, through nine high-resolution cocrystal structures of SARS-CoV-2 Mpro with the viral cleavage sites. These structures enable identification of evolutionarily vulnerable sites beyond the substrate envelope that may be susceptible to drug resistance and compromise binding of the newly developed Mpro inhibitors.","version":"1.1","doi":"10.1101/2022.01.25.477757","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477774","pub_date":"2022-1-27","title":"The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition","abstract":"The ongoing SARS-CoV-2 pandemic continues to be a significant threat to global health. First reported in November 2021, the Omicron variant (B.1.1.529) is more transmissible and can evade immunity better than previous SARS-CoV-2 variants, fueling an unprecedented surge in cases. To produce functional proteins from this polyprotein, SARS-CoV-2 relies on the cysteine proteases Nsp3/papain-like protease (PLpro) and Nsp5/Main Protease (Mpro)/3C-like protease to cleave at three and more than 11 sites, respectively. Therefore, Mpro and PLpro inhibitors are considered to be some of the most promising SARS-CoV-2 antivirals. On December 22, 2021, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for PAXLOVID, a ritonavir-boosted formulation of nirmatrelvir. Nirmatrelvir is a first-in-class orally bioavailable SARS-CoV-2 Mpro inhibitor. Thus, the scientific community must vigilantly monitor potential mechanisms of drug resistance, especially because SARS-CoV-2 is na\u00efve to Mpro inhibitors. Mutations have been well identified in variants to this point. Notably, Omicron Mpro (OMpro) harbors a single mutation\u2013 P132H. In this study we characterize the enzymatic activity, drug inhibition, and structure of OMpro while evaluating the past and future implications of Mpro mutations.","version":"1.1","doi":"10.1101/2022.01.26.477774","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477937","pub_date":"2022-1-27","title":"Germinal center-derived broadly neutralizing antibodies adapt to SARS-CoV-2 antigenic drift","abstract":"The outbreak of SARS-CoV-2 variant Omicron which harbors a striking number of mutations in the spike protein has been raising concerns about the effectiveness of vaccines and antibody treatment. Here, we confirmed a substantial reduction in neutralizing potency against Omicron in all convalescent and vaccinated sera. However, we found that some people infected by the early strain show relatively higher neutralization to Omicron. From those B cells, we developed neutralizing antibodies inhibiting broad variants including Delta and Omicron. Unlike reported antibodies, one had an extremely large interface and widely covered receptor binding motif of spike, thereby interfering with diversified variants. Somatic mutations introduced by long-term germinal center reaction contributed to the key structure of antibodies and the universal interaction with spike variants. Recalling such rare B cells may confer sustainable protection against SARS-CoV-2 variants emerging one after another.","version":"1.1","doi":"10.1101/2022.01.26.477937","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477545","pub_date":"2022-1-27","title":"Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19","abstract":"While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.","version":"1.1","doi":"10.1101/2022.01.24.477545","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.476850","pub_date":"2022-1-27","title":"Impact of various vaccine boosters on neutralization against Omicron following prime vaccinations with inactivated or adenovirus-vectored vaccine","abstract":"Since the first report on November 24, 2021, the Omicron SARS-CoV-2 variant is now overwhelmingly spreading across the world. Two SARS-CoV-2 inactivated vaccines (IAVs), one recombinant protein subunit vaccine (PRV), and one adenovirus-vectored vaccine (AdV) have been widely administrated in many countries including China to pursue herd immunity. Here we investigated cross-neutralizing activities in 341 human serum specimens elicited by full-course vaccinations with IAV, PRV and AdV, and by various vaccine boosters following prime IAV and AdV vaccinations. We found that all types of vaccines induced significantly lower neutralizing antibody titers against the Omicron variant than against the prototype strain. For prime vaccinations with IAV and AdV, heterologous boosters with AdV and PRV, respectively, elevated serum Omicron-neutralizing activities to the highest degrees. In a mouse model, we further demonstrated that among a series of variant-derived RBD-encoding mRNA vaccine boosters, it is only the Omicron booster that significantly enhanced Omicron neutralizing antibody titers compared with the prototype booster following a prime immunization with a prototype S-encoding mRNA vaccine candidate. In summary, our systematical investigations of various vaccine boosters inform potential booster administrations in the future to combat the Omicron variant.","version":"1.1","doi":"10.1101/2022.01.25.476850","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477782","pub_date":"2022-1-26","title":"Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral non-covalent, non-peptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug-design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel non-covalent inhibitor could be a potential oral agent for treating COVID-19.","version":"1.1","doi":"10.1101/2022.01.26.477782","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477753","pub_date":"2022-1-26","title":"The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells","abstract":"The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses.","version":"1.1","doi":"10.1101/2022.01.25.477753","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.26.477856","pub_date":"2022-1-26","title":"Anti-SARS-CoV-2 swine glyco-humanized polyclonal antibody XAV-19 retains neutralizing activity against SARS-CoV-2 B.1.1.529 (Omicron)","abstract":"B.1.1.529 is the SARS-CoV-2 variant designated Omicron by the WHO in November 2021. It is a highly divergent variant with a high number of mutations, including 26-32 mutations in the spike protein among which 15 in the Receptor Binding Domain (RBD) including at the human angiotensin converting enzyme 2 (ACE-2) receptor interacting interface. Because of a decreased affinity for the ACE-2 receptor and a geometric reorganization of the S1-S2 cleavage site, the Omicron variant is predicted to not have a significant infectivity advantage over the delta variant and to be less pathogenic than Delta. However, in Omicron, neutralizing epitopes are greatly affected, suggesting that current vaccines and neutralizing monoclonal antibodies might confer reduced protection against this variant. In contrast, we and others previously demonstrated that polyclonal antibodies against SARS-CoV-2 RBD obtained from hyperimmunized animal hosts do maintain their neutralizing properties against Alpha to Delta. Here, we confirmed these findings by showing that XAV-19, a swine glyco-humanized polyclonal antibody retains full neutralizing activity against Omicron.","version":"1.1","doi":"10.1101/2022.01.26.477856","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477784","pub_date":"2022-1-26","title":"Structural diversity of the SARS-CoV-2 Omicron spike","abstract":"Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.","version":"1.1","doi":"10.1101/2022.01.25.477784","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.475953","pub_date":"2022-1-26","title":"SARS-CoV-2 Omicron Variant AI-based Primers","abstract":"As the COVID-19 pandemic continues to affect the world, a new variant of concern, B.1.1.529 (Omicron), has been recently identified by the World Health Organization. At the time of writing, there are still no available primer sets specific to the Omicron variant, and its identification is only possible by using multiple targets, checking for specific failures, amplifying the suspect samples, and sequencing the results. This procedure is considerably time-consuming, in a situation where time might be of the essence. In this paper we use an Artificial Intelligence (AI) technique to identify a candidate primer set for the Omicron variant. The technique, based on Evolutionary Algorithms (EAs), has been already exploited in the recent past to develop primers for the B.1.1.7/Alpha variant, that have later been successfully tested in the lab. Starting from available virus samples, the technique explores the space of all possible subsequences of viral RNA, evaluating them as candidate primers. The criteria used to establish the suitability of a sequence as primer includes its frequency of appearance in samples labeled as Omicron, its absence from samples labeled as other variants, a specific range of melting temperature, and its CG content. The resulting primer set has been validated in silico and proves successful in preliminary laboratory tests. Thus, these results prove further that our technique could be established as a working template for a quick response to the appearance of new SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.01.21.475953","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477535","pub_date":"2022-1-26","title":"Cytotoxic T lymphocytes targeting a conserved SARS-CoV-2 spike epitope are efficient serial killers","abstract":"Understanding the cellular immune response to infections, cancers and vaccines lags behind the investigation of humoral responses. While neutralizing antibody responses wane over time, the ability of T cells to recognize viruses including SARS-CoV-2 is instrumental to providing long-term immunity. Although T-cell receptor (TCR) repertoire screening can provide insights into the skewing of a T-cell response elicited upon vaccination or infection, they unfortunately provide no assessment into the functional capacity of T cells or their ability to eliminate virally infected targets. We have used time-lapse imaging microscopy in nanowell grids (TIMING) to integrate the migration of individual T cells with analysis of effector functions including cytokine secretion and cytotoxicity. Machine learning is then applied to study thousands of videos of dynamic interactions as T cells with specificity for SARS-CoV-2 eliminate targets bearing spike protein as a surrogate for viral infection. Our data provide the first direct evidence that cytotoxic T lymphocytes from a convalescent patient targeting an epitope conserved across all known variants of concern (VoC) are serial killers capable of eliminating multiple infected targets. These data have implications for development of vaccines to provide broad and sustained cellular immunity and for the recovery and monitoring of individuals who have been exposed to SARS-CoV-2. We present an imaging platform that uses artificial intelligence (AI) to track thousands of individual cell-cell interactions within nanowell arrays. We apply this platform to quantify how the T cell component of adaptive immunity responds to infections. Our results show that T cells specific for a conserved epitope within the SARS-CoV-2 spike protein are serial killers that can rapidly eliminate virally infected targets. The ability to map the functional capacity of T cells and their ability to kill infected cells provides fundamental insights into the immunology of vaccines and recovery from infections.","version":"1.1","doi":"10.1101/2022.01.24.477535","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477789","pub_date":"2022-1-26","title":"Lineage-mosaic and mutation-patched spike proteins for broad-spectrum COVID-19 vaccine","abstract":"The widespread SARS-CoV-2 in humans results in the continuous emergence of new variants. Recently emerged Omicron variant with multiple spike mutations sharply increases the risk of breakthrough infection or reinfection, highlighting the urgent need for new vaccines with broad-spectrum antigenic coverage. Using inter-lineage chimera and mutation patch strategies, we engineered a recombinant monomeric spike variant (STFK1628x), which showed high immunogenicity and mutually complementary antigenicity to its prototypic form (STFK). In hamsters, a bivalent vaccine comprised of STFK and STFK1628x elicited high titers of broad-spectrum antibodies to neutralize all 14 circulating SARS-CoV-2 variants, including Omicron; and fully protected vaccinees from intranasal SARS-CoV-2 challenges of either the ancestral strain or immune-evasive Beta variant. Strikingly, the vaccination of hamsters with the bivalent vaccine completely blocked the within-cage virus transmission to unvaccinated sentinels, for either the ancestral SARS-CoV-2 or Beta variant. Thus, our study provides new insights and antigen candidates for developing next-generation COVID-19 vaccines.","version":"1.1","doi":"10.1101/2022.01.25.477789","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472257","pub_date":"2022-1-26","title":"High temporal resolution systems profiling reveals distinct patterns of interferon response after Covid-19 mRNA vaccination and SARS-CoV2 infection","abstract":"Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here we investigated responses to COVID-19 mRNA vaccination via ultra-low-volume sampling and high-temporal-resolution transcriptome profiling (23 subjects across 22 timepoints, and with 117 COVID-19 patients used as comparators). There were marked differences in the timing and amplitude of the responses to the priming and booster doses. Notably, we identified two distinct interferon signatures. The first signature (A28/S1) was robustly induced both post-prime and post-boost and in both cases correlated with the subsequent development of antibody responses. In contrast, the second interferon signature (A28/S2) was robustly induced only post-boost, where it coincided with a transient inflammation peak. In COVID19 patients, a distinct phenotype dominated by A28/S2 was associated with longer duration of intensive care. In summary, high-temporal-resolution transcriptomic permitted the identification of post- vaccination phenotypes that are determinants of the course of COVID-19 disease.","version":"1.2","doi":"10.1101/2021.12.12.472257","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477770","pub_date":"2022-1-26","title":"Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability","abstract":"Immunization with SARS-CoV-2 spike elicits diverse antibodies, but can any of these neutralize broadly? Here, we report the isolation and characterization of antibody WS6, from a mouse immunized with mRNA encoding the SARS-CoV-2 spike. WS6 bound diverse beta-coronavirus spikes and neutralized SARS-CoV-2 variants, SARS-CoV, and related sarbecoviruses. Epitope mapping revealed WS6 to target a region in the S2 subunit, which was conserved among SARS-CoV-2, MERS-CoV, and hCoV-OC43. The crystal structure at 2-\u00c5 resolution of WS6 with its S2 epitope revealed recognition to center on a conserved helix, which was occluded in both prefusion and post-fusion spike conformations. Structural and neutralization analyses indicated WS6 to neutralize by inhibiting fusion, post-viral attachment. Comparison of WS6 to other antibodies recently identified from convalescent donors or mice immunized with diverse spikes indicated a stem-helical supersite \u2013 centered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156 \u2013 to be a promising target for vaccine design. SARS-CoV-2 spike mRNA-immunized mouse elicited an antibody, WS6, that cross reacts with spikes of diverse human and bat beta-coronaviruses WS6 neutralizes SARS-CoV-2 variants, SARS-CoV, and related viruses Crystal structure at 2-\u00c5 resolution of WS6 in complex with a conserved S2 peptide reveals recognition of a helical epitope WS6 neutralizes by inhibition of fusion, post-viral attachment WS6 recognizes a supersite of vulnerability also recognized by other recently identified antibodies Helical supersite of vulnerability comprises a hydrophobic cluster spanning three helical turns, with acid residues framing the center turn Genetic and structural analysis indicate supersite recognition to be compatible with diverse antibody ontogenies","version":"1.1","doi":"10.1101/2022.01.25.477770","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477505","pub_date":"2022-1-26","title":"Biodistribution and Environmental Safety of a Live-attenuated YF17D-vectored SARS-CoV-2 Vaccine Candidate","abstract":"New platforms are urgently needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as vector for several licensed vaccines and platform for novel vaccine candidates. Based on YF17D, we developed YF-S0 as exceptionally potent COVID-19 vaccine candidate. However, use of such live RNA virus vaccines raises safety concerns, i.e., adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic; YEL-AND, and viscerotropic disease; YEL-AVD). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in STAT2 signaling as new preclinical model of YEL-AND/AVD. Compared to parental YF17D, YF-S0 showed an improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence evaluated in comparison to readily transmitting YFV-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of vector mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other novel vaccines that are based on the same YF17D platform.","version":"1.1","doi":"10.1101/2022.01.24.477505","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477597","pub_date":"2022-1-26","title":"Unadjuvanted intranasal spike vaccine booster elicits robust protective mucosal immunity against sarbecoviruses","abstract":"As the SARS-CoV-2 pandemic enters its third year, vaccines that not only prevent disease, but also prevent transmission are needed to help reduce global disease burden. Currently approved parenteral vaccines induce robust systemic immunity, but poor immunity at the respiratory mucosa. Here we describe the development of a novel vaccine strategy, Prime and Spike, based on unadjuvanted intranasal spike boosting that leverages existing immunity generated by primary vaccination to elicit mucosal immune memory within the respiratory tract. We show that Prime and Spike induces robust T resident memory cells, B resident memory cells and IgA at the respiratory mucosa, boosts systemic immunity, and completely protects mice with partial immunity from lethal SARS-CoV-2 infection. Using divergent spike proteins, Prime and Spike enables induction of cross-reactive immunity against sarbecoviruses without invoking original antigenic sin. Broad sarbecovirus protective mucosal immunity is generated by unadjuvanted intranasal spike boost in preclinical model.","version":"1.1","doi":"10.1101/2022.01.24.477597","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.03.474779","pub_date":"2022-1-26","title":"Host kinase CSNK2 is a target for inhibition of pathogenic \u03b2-coronaviruses including SARS-CoV-2","abstract":"Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine \u03b2-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in \u03b2-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-\u03b2-coronavirus drugs.","version":"1.3","doi":"10.1101/2022.01.03.474779","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477043","pub_date":"2022-1-25","title":"Substantial immune response in Omicron infected breakthrough and unvaccinated individuals against SARS-CoV-2 variants of concerns","abstract":"The recent emergence of highly mutated SARS-CoV-2 Omicron variant has debilitating effect on public health system of the affected countries worldwide. Currently India is facing third wave of COVID-19 pandemic and going through a severe crisis. Within short span of time, the variant has shown high transmissibility and capability of evading the immune response generated against natural infection and vaccination. The immune escape potential of Omicron is a serious concern and further needs to be explored. In the present study, we have assessed the IgG and neutralizing antibody (NAb) response in breakthrough individuals vaccinated with two doses ChAdOx1 nCoV-19 vaccine (n=25), breakthrough individuals vaccinated with two doses of BNT162b2 mRNA vaccine (n=8) and unvaccinated individuals (n=6). All these individuals were infected with Omicron variant. The IgG antibody activity in the sera of the ChAdOx1 nCoV-19 and BNT162b2 mRNA breakthrough individuals was comparable with S1-RBD, while it was lesser in BNT162b2 mRNA breakthrough individuals with N protein and inactivated whole antigen IgG ELISA. BNT162b2 mRNA breakthrough individuals showed moderate reduction in NAb GMTs compared to ChAdOx1 nCoV-19 against Alpha, Beta and Delta. However, 3-fold higher reduction was observed with omicron variant in BNT162b2 mRNA than ChAdOx1 nCoV-19. Apparently, Alpha variant was modestly resistant to the sera of unvaccinated individuals than Beta, Delta and Omicron. Our study demonstrated substantial immune response in the individuals infected with Omicron. The neutralizing antibodies could effectively neutralize the Omicron and other VOCs including the most prevalent Delta variant.","version":"1.1","doi":"10.1101/2022.01.24.477043","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477288","pub_date":"2022-1-25","title":"Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail","abstract":"The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-CoV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the cilgavimab (AZD1061) mAb. Here, we show that this residue substitution remodels the ACE2-binding site allosterically, thereby dampening receptor recognition severely and altering the epitopes recognized by these three mAbs. Although vaccine-elicited neutralizing antibody titers are decreased similarly against the E406 mutant and the Delta or Epsilon variants, broadly neutralizing sarbecovirus mAbs, including a clinical mAb, inhibit the E406W spike mutant.","version":"1.1","doi":"10.1101/2022.01.21.477288","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477625","pub_date":"2022-1-25","title":"Converting non-neutralizing SARS-CoV-2 antibodies targeting conserved epitopes into broad-spectrum inhibitors through receptor blockade","abstract":"All but one of the authorized monoclonal antibody-based treatments for SARS-CoV-2 are largely ineffective against Omicron, highlighting the critical need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective therapeutic antibodies target epitopes that are not highly conserved. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to antibodies that are known to be non-neutralizing, but which target highly conserved epitopes in the viral spike protein. These inhibitors, called Receptor-blocking conserved non-neutralizing Antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOC), including Omicron. Neutralization potency is dependent on both the binding and inhibitory ReconnAb components as activity is lost when the linker joining the two is severed. In addition, a bifunctional ReconnAb, made by linking ACE2 to a bispecific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad- spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.","version":"1.1","doi":"10.1101/2022.01.24.477625","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.30.474580","pub_date":"2022-1-25","title":"SARS-CoV-2 entry sites are present in all structural elements of the human glossopharyngeal and vagal nerves: clinical implications","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections result in the temporary loss of smell and taste (anosmia and dysgeusia) in about one third of confirmed cases. Several investigators have reported that the viral spike protein receptor is present in olfactory neurons. However, no study has been published to date showing the presence of viral entry sites angiotensin-converting enzyme 2 (ACE2), neuropilin1 (NRP1), and TMPRSS2, the serine protease necessary for priming the viral proteins, in human nerves that are responsible for taste sensation (cranial nerves: VII, IX and X). We used immunocytochemistry to examine three postmortem donor samples of the IXth (glossopharyngeal) and Xth (vagal) cranial nerves where they leave/join the medulla from three donors to confirm the presence of ACE2, NRP1 and TMPRSS2. Two samples were paraffin embedded; one was a frozen sample. In addition to staining sections from the latter, we isolated RNA from it, made cDNA, and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. All three of the proteins required for SARS-CoV-2 infections appear to be present in the human IXth and Xth nerves near the medulla. Direct infection of these nerves by the COVID-19 virus is likely to cause the loss of taste experienced by many patients. In addition, potential viral spread through these nerves into the adjacent brainstem respiratory centers might also aggravate the respiratory problems patients are experiencing.","version":"1.3","doi":"10.1101/2021.12.30.474580","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.473679","pub_date":"2022-1-25","title":"SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-weeks interval between doses","abstract":"Continuous emergence of SARS-CoV-2 variants of concern (VOC) is fueling the COVID-19 pandemic. Omicron (B.1.1.529), is rapidly spreading worldwide. The large number of mutations in its Spike raised concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses was shown to elicit antibodies that efficiently recognize Spikes from different VOCs. Here we evaluated the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 na\u00efve and previously-infected individuals that received their BNT162b2 mRNA vaccine 16-weeks apart. Omicron Spike was recognized less efficiently than D614G, Alpha, Beta, Gamma and Delta Spikes. We compared to plasma activity from participants receiving a short (4-weeks) interval regimen. Plasma from individuals of the long interval cohort recognized and neutralized better the Omicron Spike compared to those that received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.","version":"1.2","doi":"10.1101/2021.12.21.473679","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477163","pub_date":"2022-1-25","title":"Mutations of SARS-CoV-2 variants of concern escaping Spike-specific T cells","abstract":"The amino acid (AA) mutations that characterise the different variants of concern (VOCs), which replaced the ancestral SARS-CoV-2 Wuhan-Hu-1 isolate worldwide, provide biological advantages such as increased infectivity and partial escape from humoral immunity. Here we analysed the impact of these mutations on vaccination- and infection-induced Spike-specific T cells. We confirmed that, in the majority of infected or vaccinated individuals, different mutations present in a single VOC (Delta) or a combined mosaic of more than 30 AA substitutions and deletions found in Alpha, Beta, Gamma, Delta and Omicron VOCs cause modest alteration in the global Spike-specific T cell response. However, distinct numerically dominant Spike-specific CD4 and CD8 T cells preferentially targeted regions affected by AA mutations and do not recognise the mutated peptides. Importantly, some of these mutations, such as N501Y (present in Alpha, Beta, Gamma, and Omicron) and L452R (present in Delta), known to provide biological advantage to SARS-CoV-2 in terms of infectivity also abolished CD8 T cell recognition. Taken together, our data show that while global mRNA vaccine- and infection-induced Spike-specific T cells largely tolerate the diverse mutations present in VOCs, single Spike-specific T cells might contribute to the natural selection of SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.01.20.477163","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477633","pub_date":"2022-1-25","title":"Binding of Human ACE2 and RBD of Omicron Enhanced by Unique Interaction Patterns Among SARS-CoV-2 Variants of Concern","abstract":"The 2019 coronavirus disease (COVID-19) pandemic has had devastating impacts on our global health. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has now overtaken the Delta variant and become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor-binding domain (RBD) of the spike protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD-ACE2 interface through N501Y, Q493K/R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Our MST data confirmed that the Omicron mutations in RBD are associated with a five-fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our result could help explain the Omicron variant\u2019s prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity.","version":"1.1","doi":"10.1101/2022.01.24.477633","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.25.477671","pub_date":"2022-1-25","title":"Structural insight into antibody evasion of SARS-CoV-2 omicron variant","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to mutate and evolve with the emergence of omicron (B.1.1.529) as the new variant of concern. The rapid spread of this variant regionally and globally could be an allusion to increased infectivity, transmissibility, and antibody resistance. The omicron variant has a large set of mutations in its spike protein, specifically in the receptor binding domain (RBD), reflecting their significance in ACE2 interaction and antibody recognition. We have carried out the present study to understand how these mutations structurally impact the binding of the antibodies to their target epitope. We have computationally evaluated the binding of different classes of RBD targeted antibodies, namely, CB6 (etesevimab), REGN10933 (casirivimab), S309 (sotrovimab), and S2X259 to the omicron mutation-induced RBD. Molecular dynamics simulations and binding free energy calculations unveil the binding affinity and stability of the antibody-RBD complexes. All the four antibodies show reduced binding affinity towards the omicron RBD. The therapeutic antibody CB6 aka etesevimab was substantially affected due to numerous omicron mutations occurring in its target epitope. This study provides a structural insight into the reduced efficacy of RBD targeting antibodies against the SARS-CoV-2 omicron variant.","version":"1.1","doi":"10.1101/2022.01.25.477671","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.09.454215","pub_date":"2022-1-25","title":"Fully Human Antibody Immunoglobulin from Transchromosomic Bovines is Potent Against SARS-CoV-2 Variant Pseudoviruses","abstract":"SAB-185 is a fully human polyclonal anti-SARS-CoV-2 immunoglobulin produced from the plasma of transchromosomic bovines that are hyperimmunized with recombinant SARS-CoV-2 Wuhan-Hu-1 Spike protein. SAB-185 is being evaluated for efficacy in aphase 3 clinical trial. The World Health Organization (WHO) has identified multiple Variants-of-Concern and Variants-of-Interest (VOC/VOI) that have mutations in their Spike protein that appear to increase transmissibility and/or reduce the effectiveness of therapeutics and vaccines, among other parameters of concern. SAB-185 was evaluated using lentiviral-based pseudovirus assays performed in a BSL2 environment that incorporates stable or transient cell lines that express human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). The results indicate that SAB-185 retained neutralization potency against multiple SARS-CoV-2 pseudovirus variants, including the Delta, Kappa, Lambda and Omicron variants, that have or are supplanting other VOC/VOI in many countries and regions around the world.","version":"1.2","doi":"10.1101/2021.08.09.454215","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477037","pub_date":"2022-1-25","title":"Emergence of glycogen synthase kinase-3 interaction domain enhances phosphorylation of SARS-CoV-2 nucleocapsid protein","abstract":"A structural protein of SARS-CoV-2, nucleocapsid (N) protein is abundantly expressed during viral replication. The N protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3\u03b2 constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), commonly found in many endogenous GSK-3\u03b2 binding proteins, such as Axins, FRATs, WWOX and GSKIP. Indeed, N interacts with GSK-3\u03b2 similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. Unlike with endogenous GID proteins, the N interaction neither disturbs endogenous GSK-3 activity nor regulates subsequent canonical Wnt activity and the Snail-EMT program. Notably, N abundance in SARS-CoV-2 is incomparably high compared to other coronaviruses, such as 229E, OC43 and HKU1. Compared to other coronaviruses, N harbors a CDK1 primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3\u03b2. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allows increased abundance and hyper-phosphorylation of N. Our observations suggest that the emergence of GID and mutations for increased phosphorylation in N may have contributed to the emergence of SARS-CoV-2 and evolution of variants, respectively. Further study, especially in a BSL3-equipped facility, is required to elucidate the functional importance of GID and N phosphorylation in SARS-CoV-2 and variants.","version":"1.1","doi":"10.1101/2022.01.24.477037","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477651","pub_date":"2022-1-25","title":"Structural Ramifications of Spike Protein D614G Mutation in SARS-CoV-2","abstract":"A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). D614G mutation induces structural changes in the Spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on wild-type structure. The introduction of mutation with ordered 630-loop induces structural changes toward S-G614 Cryo-EM structure. An ordered 630-loop weakens the stabilizing interactions of the anionic D614, suggesting its disorder in wild-type. The mutation allosterically alters the receptor binding domain (RBD) forming an asymmetric and mobile Down conformation, which facilitate Up transition. The loss of D614_K854 salt-bridge upon mutation, generally stabilize S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding of the molecular basis of D614G is crucial as it dominates in all variants of concern including Delta and Omicron.","version":"1.1","doi":"10.1101/2022.01.24.477651","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477502","pub_date":"2022-1-25","title":"Template-based design of peptides to inhibit SARS-CoV-2 RNA-dependent RNA polymerase complexation","abstract":"The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between the subunits of the RdRp complex are highly conserved, facilitating the design of inhibitors with high affinity for the interaction hotspots of the complex. Here, we report development and application of a structural bioinformatics protocol to design peptides that can inhibit RdRp complex formation by targeting the interface of its core subunit nonstructural protein (nsp) 12 with accesory factor nsp7. We adopt a top-down approach for protein design by using interaction hotspots of the nsp7-nsp12 complex obtained from a long molecular dynamics trajectory as template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in silico to determine peptide sequences with highest shape and interaction complementarity for the nsp7-nsp12 interface. Two lead designed peptide are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation and anti-viral activity. Their binding affinity to nsp7 (target), as determined from surface plasmon resonance (SPR) assay, is found to be comparable to that of the nsp7-nsp12 complex. Further, one of the designed peptides gives 46 % inhibition of nsp7-nsp12 complex at 10:1 peptide:nsp7 molar concentration (from ELISA assay). Further optimization of cell penetrability and target affinity of these designed peptides is expected to provide lead candidates with high anti-viral activity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.24.477502","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477490","pub_date":"2022-1-25","title":"Close relatives of MERS-CoV in bats use ACE2 as their functional receptors","abstract":"Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes\u2019 S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using \u201cMERS-CoV-2\u201d with both high fatality and transmission rate.","version":"1.1","doi":"10.1101/2022.01.24.477490","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.24.477579","pub_date":"2022-1-25","title":"The Existence of At Least Three Genomic Signature Patterns and At Least Seven Subtypes of COVID-19 and the End of the Disease","abstract":"Hoping to find genomic clues linked to COVID-19 and end the pandemic has driven scientists\u2019 tremendous efforts to try all kinds of research. Signs of progress have been achieved but are still limited. This paper intends to prove the existence of at least three genomic signature patterns and at least seven subtypes of COVID-19 driven by five critical genes (the smallest subset of genes). These signatures and subtypes provide crucial genomic information in COVID-19 diagnosis (including ICU patients), research focuses, and treatment methods. Unlike existing approaches focused on gene fold-changes and pathways, gene-gene nonlinear and competing interactions are the driving forces in finding the signature patterns and subtypes. Furthermore, the method leads to 100% accuracy, which shows biological and mathematical equivalences between COVID-19 status and the signature patterns and a methodological advantage over other existing methods that cannot lead to 100% accuracy. As a result, as new biomarkers, the new findings can be much more informative than other findings for interpreting biological mechanisms, developing the second (third) generation of vaccines, antiviral drugs, and treatment methods, and eventually bringing new hopes to an end of the pandemic.","version":"1.1","doi":"10.1101/2022.01.24.477579","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477164","pub_date":"2022-1-24","title":"Severe acute respiratory disease in American mink (Neovison vison) experimentally infected with SARS-CoV-2","abstract":"An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, a non-transgenic model that develops severe acute respiratory disease has not been described. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding for nonstructural protein 6 in open reading frame 1a/1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID19 and as such, are uniquely suited to test viral countermeasures. SARS-CoV-2 infected mink develop severe respiratory disease that recapitulates some components of severe acute respiratory disease, including ARDS.","version":"1.1","doi":"10.1101/2022.01.20.477164","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477296","pub_date":"2022-1-24","title":"Nasally-delivered interferon-\u03bb protects mice against upper and lower respiratory tract infection of SARS-CoV-2 variants including Omicron","abstract":"Although vaccines and monoclonal antibody countermeasures have reduced the morbidity and mortality associated with SARS-CoV-2 infection, variants with constellations of mutations in the spike gene threaten their efficacy. Accordingly, antiviral interventions that are resistant to further virus evolution are needed. The host-derived cytokine IFN-\u03bb has been proposed as a possible treatment based on correlative studies in human COVID-19 patients. Here, we show IFN-\u03bb protects against SARS-CoV-2 B.1.351 (Beta) and B.1.1.529 (Omicron)variants in three strains of conventional and human ACE2 transgenic mice. Prophylaxis or therapy with nasally-delivered IFN-\u03bb2 limited infection of historical or variant (B.1.351 and B.1.1.529) SARS-CoV-2 strains in the upper and lower respiratory tracts without causing excessive inflammation. In the lung, IFN-\u03bb was produced preferentially in epithelial cells and acted on radio-resistant cells to protect against of SARS-CoV-2 infection. Thus, inhaled IFN-\u03bb may have promise as a treatment for evolving SARS-CoV-2 variants that develop resistance to antibody-based countermeasures.","version":"1.1","doi":"10.1101/2022.01.21.477296","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477067","pub_date":"2022-1-24","title":"SARS-CoV-2 Omicron variant virus isolates are highly sensitive to interferon treatment","abstract":"Recently, we have shown that SARS-CoV-2 Omicron virus isolates are less effective at inhibiting the host cell interferon response than Delta viruses. Here, we present further evidence that reduced interferon-antagonising activity explains at least in part why Omicron variant infections are inherently less severe than infections with other SARS-CoV-2 variants. Most importantly, we here also show that Omicron variant viruses display enhanced sensitivity to interferon treatment, which makes interferons promising therapy candidates for Omicron patients, in particular in combination with other antiviral agents.","version":"1.1","doi":"10.1101/2022.01.20.477067","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477244","pub_date":"2022-1-24","title":"Significance of the RBD mutations in the SARS-CoV-2 Omicron: from spike opening to antibody escape and cell attachment","abstract":"We computationally investigated the role of the Omicron RBD mutations on its structure and interactions with ACE2. Our results suggest that, compared to the WT and Delta, the mutations in the Omicron RBD facilitate a more efficient RBD opening and ACE2 attachment. These effects, combined with antibody evasion, may contribute to its dominance over Delta. While the Omicron RBD escapes most antibodies from prior infections, epitope analysis shows that it harbors sequences with significantly improved antigenicity compared to other variants, suggesting more potent Omicron-specific neutralizing antibodies.","version":"1.1","doi":"10.1101/2022.01.21.477244","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477147","pub_date":"2022-1-23","title":"SARS-CoV-2 Viral Genes Compromise Survival and Functions of Human Pluripotent Stem Cell-derived Cardiomyocytes via Reducing Cellular ATP Level","abstract":"Cardiac manifestations are commonly observed in COVID-19 patients and prominently contributed to overall mortality. Human myocardium could be infected by SARS-CoV-2, and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are susceptible to SARS-CoV-2 infection. However, molecular mechanisms of SARS-CoV-2 gene-induced injury and dysfunction of human CMs remain elusive. Here, we find overexpression of three SARS-CoV-2 coding genes, Nsp6, Nsp8 and M, could globally compromise transcriptome of hPSC-CMs. Integrated transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with hPSC-CMs of Nsp6, Nsp8 or M overexpression identified concordantly activated genes enriched into apoptosis and immune/inflammation responses, whereas reduced genes related to heart contraction and functions. Further, Nsp6, Nsp8 or M overexpression induce prominent apoptosis and electrical dysfunctions of hPSC-CMs. Global interactome analysis find Nsp6, Nsp8 and M all interact with ATPase subunits, leading to significantly reduced cellular ATP level of hPSC-CMs. Finally, we find two FDA-approved drugs, ivermectin and meclizine, could enhance the ATP level, and ameliorate cell death and dysfunctions of hPSC-CMs overexpressing Nsp6, Nsp8 or M. Overall, we uncover the global detrimental impacts of SARS-CoV-2 genes Nsp6, Nsp8 and M on the whole transcriptome and interactome of hPSC-CMs, define the crucial role of ATP level reduced by SARS-CoV-2 genes in CM death and functional abnormalities, and explore the potentially pharmaceutical approaches to ameliorate SARS-CoV-2 genes-induced CM injury and abnormalities.","version":"1.1","doi":"10.1101/2022.01.20.477147","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.22269605","pub_date":"2022-01-23","title":"Outcomes of SARS-CoV-2 Omicron infection in residents of Long-Term Care","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Recently there has been a rapid, global increase in SARS-CoV-2 infections associated with the Omicron variant (B.1.1.529). Although severity of Omicron cases may be reduced, the scale of infection suggests hospital admissions and deaths may be substantial. Definitive conclusions about disease severity require evidence from populations with the greatest risk of severe outcomes, such as residents of Long-Term Care Facilities (LTCFs).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We used a cohort study to compare the risk of hospital admission or death in LTCF residents in England who had tested positive for SARS-CoV-2 in the period shortly before Omicron emerged (Delta dominant) and the Omicron-dominant period, adjusting for age, sex, vaccine type, and booster vaccination. Variants were confirmed by sequencing or spike-gene status in a subset.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Risk of hospital admission was markedly lower in 1241 residents infected in the Omicron-period (4.01% hospitalised, 95% CI: 2.87-5.59) compared to 398 residents infected in the pre-Omicron period (10.8% hospitalised, 95% CI: 8.13-14.29, adjusted Hazard Ratio 0.50, 95% CI: 0.29-0.87, p=0.014); findings were similar in residents with confirmed variant. No residents with previous infection were hospitalised in either period. Mortality was lower in the Omicron versus the pre-Omicron period, (p&lt;0.0001).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Risk of severe outcomes in LTCF residents with the SARS-CoV-2 Omicron variant was substantially lower than that seen for previous variants. This suggests the current wave of Omicron infections is unlikely to lead to a major surge in severe disease in LTCF populations with high levels of vaccine coverage and/or natural immunity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Trial Registration Number</jats:title>\n                  <jats:p>ISRCTN 14447421</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.01.21.22269605","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.19.22269391","pub_date":"2022-01-23","title":"COVID-19 and its clinical severity are associated with alterations of plasma sphingolipids and enzyme activities of sphingomyelinase and ceramidase","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>In the current pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19), a better understanding of the underlying mechanisms is essential to reduce morbidity and mortality and treat post-COVID-19 disease. Here, we analyzed alterations of sphingolipids and their metabolizing enzymes in 125 men and 74 women tested positive for SARS-CoV-2 and hospitalized with mild, moderate or severe symptoms or after convalescence.</jats:p>\n                <jats:p>The activities of acid and neutral sphingomyelinases (ASM, NSM), which hydrolyze sphingomyelin to ceramide, were significantly increased in COVID-19 patients, while the activity of neutral ceramidase (NC), which hydrolyzes ceramide to sphingosine, was reduced. These alterations could each contribute to elevated ceramide levels in patients. Accordingly, liquid chromatography tandem-mass spectrometry (LC-MS/MS) yielded increased levels of ceramides 16:0 and 18:0 with highest levels in severely affected patients and similar effects for dihydroceramides 16:0 and 18:0, whereas levels of (dihydro-)ceramides 24:0 were reduced. Furthermore, sphingomyelin 20:0; 22:0 and 24:0 as substrates of ASM and NSM as well as their dihydrosphingomyelin counterparts were reduced in patients as well as sphingosine-1-phosphate further downstream of NC activity. Effects of NSM, NC, ceramides and sphingomyelins remained significant after Bonferroni correction. SARS-CoV-2 antibody levels in convalescent patients were associated with age but none of the sphingolipid parameters. Based on our data, COVID-19 is associated with a dysregulation of sphingolipid homeostasis in a severity-dependent manner, particularly focused around a reduction of sphingomyelins and an accumulation of ceramides by increased enzyme activities leading to ceramide elevation (ASM, NSM) combined with a decreased activity of enzymes (NC) reducing ceramide levels. The potential of a combined sphingolipid/enzyme pattern as a diagnostic and prognostic marker and therapeutic target deserves further exploration.</jats:p>","version":null,"doi":"10.1101/2022.01.19.22269391","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.20.477107","pub_date":"2022-1-22","title":"Broadly-recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles","abstract":"Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can impact the T cell response in COVID-19. We studied T cells recognizing SARS-CoV-2 and HCoVs in convalescent COVID-19 donors, and identified a highly conserved SARS-CoV-2 sequence S811-831, with two overlapping epitopes presented by common MHC-II proteins HLA-DQ5 and HLA-DP4. These epitopes were recognized by CD4+ T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and by low-abundance CD4+ T cells in uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. CD4+ T cell cross-reactivity was driven by the remarkably strong conservation of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC-II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly-conserved CD4+ T cell epitope broadly recognized across human populations.","version":"1.1","doi":"10.1101/2022.01.20.477107","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.476344","pub_date":"2022-1-22","title":"Neutralization of Omicron SARS-CoV-2 by 2 or 3 doses of BNT162b2 vaccine","abstract":"We report the antibody neutralization against Omicron SARS-CoV-2 after 2 and 3 doses of BNT162b2 mRNA vaccine. Vaccinated individuals were serially tested for their neutralization against wild-type SARS-CoV-2 (strain USA-WA1/2020) and an engineered USA-WA1/2020 bearing the Omicron spike glycoprotein. Plaque reduction neutralization results showed that at 2 or 4 weeks post-dose-2, the neutralization geometric mean titers (GMTs) were 511 and 20 against the wild-type and Omicron-spike viruses, respectively, suggesting that two doses of BNT162b2 were not sufficient to elicit robust neutralization against Omicron; at 1 month post-dose-3, the neutralization GMTs increased to 1342 and 336, respectively, indicating that three doses of vaccine increased the magnitude and breadth of neutralization against Omicron; at 4 months post-dose-3, the neutralization GMTs decreased to 820 and 171, respectively, suggesting similar neutralization decay kinetics for both variants. The data support a three-dose vaccine strategy and provide the first glimpse of the neutralization durability against Omicron.","version":"1.1","doi":"10.1101/2022.01.21.476344","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477115","pub_date":"2022-1-22","title":"ESCPE-1 Mediates Retrograde Endosomal Sorting of the SARS-CoV-2 Host Factor Neuropilin-1","abstract":"Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed \u2018cargoes\u2019) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.20.477115","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437769","pub_date":"2022-1-21","title":"A human antibody reveals a conserved site on beta-coronavirus spike proteins and confers protection against SARS-CoV-2 infection","abstract":"Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses (\u03b2-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 \u00c5 resolution and found that the peptide adopted a mainly helical structure. Conserved residues in \u03b2-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on \u03b2-CoV spike proteins for protective antibodies that may facilitate the development of pan-\u03b2-CoV vaccines. A human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-\u03b2-CoV vaccine design strategies","version":"1.2","doi":"10.1101/2021.03.30.437769","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477194","pub_date":"2022-1-21","title":"ReadItAndKeep: rapid decontamination of SARS-CoV-2 sequencing reads","abstract":"Viral sequence data from clinical samples frequently contain human contamination, which must be removed prior to sharing for legal and ethical reasons. To enable host read removal for SARS-CoV-2 sequencing data on low-specification laptops, we developed ReadItAndKeep, a fast lightweight tool for Illumina and nanopore data that only keeps reads matching the SARS-CoV-2 genome. Peak RAM usage is typically below 10MB, and runtime less than one minute. We show that by excluding the polyA tail from the viral reference, ReadItAndKeep prevents bleed-through of human reads, whereas mapping to the human genome lets some reads escape. We believe our test approach (including all possible reads from the human genome, human samples from each of the 26 populations in the 1000 genomes data, and a diverse set of SARS-CoV-2 genomes) will also be useful for others. ReadItAndKeep is implemented in C++, released under the MIT license, and available from https://github.com/GenomePathogenAnalysisService/read-it-and-keep.","version":"1.1","doi":"10.1101/2022.01.21.477194","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.21.477258","pub_date":"2022-1-21","title":"Nanoformulated Remdesivir with Extremely Low Content of Poly(2-oxazoline) - Based Stabilizer for Aerosol Treatment of COVID-19","abstract":"The rise of the novel virus SARS-CoV2 which causes the disease known as COVID-19 has led to a global pandemic claiming millions of lives. With no clinically approved treatment for COVID-19, physicians initially struggled to treat the disease and there is still need for improved anti-viral therapies in this area. We conceived early in the pandemic that an inhalable formulation of the drug Remdesivir which directly targets the virus at the initial site of infection could improve therapeutic outcomes in COVID-19. We developed a set of requirements that would be conducive to rapid drug approval: 1) try to use GRAS or GRAS similar reagents 2) minimize excipient concentration and 3) achieve a working concentration of 5 mg/mL Remdesivir to achieve a deliverable dose which is 5-10% of the IV dose. In this work, we discovered that Poly(2-oxazoline) block copolymers can stabilize drug nanocrystal suspensions and provide suitable formulation characteristics for aerosol delivery while maintaining anti-viral efficacy. We believe POx block copolymers can be used as a semi-ubiquitous stabilizer for the rapid development of nanocrystal formulations for new and existing diseases.","version":"1.1","doi":"10.1101/2022.01.21.477258","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.22269426","pub_date":"2022-01-21","title":"Investigating sensitivity of nasal or throat (ISNOT): A combination of both swabs increases sensitivity of SARS-CoV-2 rapid antigen tests","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The COVID-19 pandemic has been hallmarked by several waves of variants of concern (VoCs), each with novel challenges. Currently, the highly transmissible Omicron VOC is predominant worldwide, and sore throat is common among other cold-like symptoms. Anecdotes on social media suggested sampling one\u2019s throat can increase sensitivity for Omicron detection by antigen-based rapid testing devices (Ag-RDTs). This work determines whether the sensitivity of Ag-RDTs designed for nasal sampling is altered with use of self-administered throat swabs in self-perceived asymptomatic individuals. This investigation compared results of a common Ag-RDT (i.e. Abbott Panbio COVID-19 Ag Rapid Test Device) using three sampling sites: nasal swab; throat swab and; combined nasal/throat. All Ag-RDT results were confirmed with molecular testing. Compared to RT-PCR, samples from nasal or throat swabs each detected 64.5% of SARS-CoV-2 cases; however, combining the contributions of each swab increased sensitivity to 88.7%. This trend was also evident with the Rapid Response Ag-RDT (BTNX), which uses a more flexible swabs than Panbio. When nasal swab collection was compared to paired sampling of the nasal/throat using a single swab with the Panbio Ag-RDT, the sensitivity of each was 68.4% and 81.6%, respectively. No false-positive results were observed with nasal, throat, or combined nasal/throat sampling. Self-administered throat and nasal/throat swabs both had &gt;90% acceptability. These findings support the use of self-collected combined nasal/throat sampling for Ag-RDT based SARS-CoV-2 detection in self perceived asymptomatic individuals.</jats:p>","version":null,"doi":"10.1101/2022.01.18.22269426","journal":"medRxiv","score":null},{"id":"10.1101/2021.12.14.472554","pub_date":"2022-1-21","title":"Relationship between monomer packing, receptor binding domain pocket status, and pH, in the spike trimer of SARS-CoV-2 variants","abstract":"Existence of a SARS-CoV-2 spike protein trimer form with closer packing between monomers when receptor binding domains (RBDs) are all down, locked as opposed to closed, has been associated with linoleic acid (LA) binding at neutral pH, or can occur at acidic pH in the absence of LA binding. The relationship between degree of closure of the LA binding pocket of the RBD, and monomer burial in the trimer, is examined for a range of spike protein structures, including those with D614G mutation, and that of the Delta variant (which also carries D614G). Some spike protein structures with this aspartic acid mutation show monomer packing approaching that of the locked form (at neutral pH, without LA binding) for two segments, a third (around the RBD) remains less closely packed. Mutations in the RBD are a focus for the Omicron variant spike protein. Structure reports suggest that these mutations are involved in increased RBD-RBD interactions, and also that they could lead to a closing of the LA pocket, both of which could impact on pH-dependence. One potential outcome is that the extent of pH-dependent conformational transitions of the pre-fusion SARS-CoV-2 spike trimer are reduced in the Omicron variant.","version":"1.2","doi":"10.1101/2021.12.14.472554","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.22269586","pub_date":"2022-01-21","title":"Immunogenicity of a third dose of BNT162b2 to ancestral SARS-CoV-2 &amp; Omicron variant in adults who received two doses of inactivated vaccine","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Limited data exist on antibody responses to mixed vaccination strategies involving inactivated COVID-19 vaccines, particularly in the context of emerging variants.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted an open label trial of a third vaccine dose of an mRNA vaccine (BNT162b2, Fosun Pharma/BioNTech) in adults aged \u226530 years who had previously received two doses of inactivated COVID-19 vaccine. We collected blood samples before administering the third dose and 28 days later, and tested for antibodies to the ancestral virus using a binding assay (ELISA), a surrogate virus neutralization test (sVNT) and a live virus plaque reduction neutralization test (PRNT). We also tested for antibodies against the Omicron variant using live-virus PRNT.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    In 315 participants, a third dose of BNT162b2 substantially increased antibody titers on each assay. Mean ELISA levels increased from an optical density (OD) of 0.3 to 2.2 (p&lt;0. 001), and mean sVNT levels increased from an inhibition of 17% to 96% (p&lt;0.001). In a random subset of 20 participants, the geometric mean PRNT\n                    <jats:sub>50</jats:sub>\n                    titers rose very substantially by at least 24 fold from Day 0 to Day 28 against the ancestral virus (p&lt;0.001) and rose by at least 11 fold against the Omicron variant (p&lt;0.001). In daily monitoring, post-vaccination reactions subsided within 7 days for over 99% of participants.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>A third dose of COVID-19 vaccination with an mRNA vaccine substantially improved antibody levels against the ancestral virus and the Omicron variant with well-tolerated safety profile, in adults who had received two doses of inactivated vaccine 6 months earlier.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Summary</jats:title>\n                  <jats:p>In this open label trial of Chinese adults aged \u226530 years who received two doses of inactivated COVID-19 vaccine 6 months earlier, third-dose mRNA vaccine substantially improved antibody levels against the ancestral virus and Omicron variant with well-tolerated safety profile.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.01.20.22269586","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.19.476893","pub_date":"2022-1-20","title":"NRP1 and furin as putative mediators of SARS-CoV-2 entry into human brain cells","abstract":"COVID-19 has prominent neurological manifestations including psychiatric symptoms, indicating significant synaptic pathology. Surprisingly, existing evidence suggests negligible expression of the key SARS-CoV-2 host cell entry mediators ACE2 and TMPRSS2 in human brain, which complicates understanding of the pathomechanisms of the neuropsychiatric manifestations in COVID-19. Recent studies suggested that an alternative host-cell entry receptor, NRP1, can mediate entry of furin cleaved SARS-CoV-2 spike proteins into the host cells. However, the role of NRP1 and furin in mediating SARS-CoV-2 entry in human brain cells has been least explored and remains a lacuna in the literature. We performed an in silico analysis of the transcriptomic and proteomic expressions of SARS-CoV-2 host-cell entry receptors and associated tissue proteases in human brain tissue, using the publically available databases. Based on the expression analysis, SARS-CoV-2 entry in human brain cells is likely to be mediated through NRP1 and furin.","version":"1.1","doi":"10.1101/2022.01.19.476893","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.476786","pub_date":"2022-1-20","title":"SARS-CoV-2 infection results in lasting and systemic perturbations post recovery","abstract":"SARS-CoV-2 has been found capable of inducing prolonged pathologies collectively referred to as Long-COVID. To better understand this biology, we compared the short- and long-term systemic responses in the golden hamster following either SARS-CoV-2 or influenza A virus (IAV) infection. While SARS-CoV-2 exceeded IAV in its capacity to cause injury to the lung and kidney, the most significant changes were observed in the olfactory bulb (OB) and olfactory epithelium (OE) where inflammation was visible beyond one month post SARS-CoV-2 infection. Despite a lack of detectable virus, OB/OE demonstrated microglial and T cell activation, proinflammatory cytokine production, and interferon responses that correlated with behavioral changes. These findings could be corroborated through sequencing of individuals who recovered from COVID-19, as sustained inflammation in OB/OE tissue remained evident months beyond disease resolution. These data highlight a molecular mechanism for persistent COVID-19 symptomology and characterize a small animal model to develop future therapeutics.","version":"1.1","doi":"10.1101/2022.01.18.476786","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477038","pub_date":"2022-1-20","title":"Evidence of antibodies against SARS-CoV-2 in wild mustelids from Brittany (France)","abstract":"In the French region of Brittany, mainly in the department of the C\u00f4tes d\u2019Armor, during the first semester of 2021, seropositivity for SARS-CoV-2 was detected in five wild mustelids out of 32 animals tested. Anti-SARS-CoV-2 IgG against at least four out of five recombinant viral proteins (S1 receptor binding domain, nucleocapsid, S1 subunit, S2 subunit and spike) were detected using automated western blot technique in three martens (Martes martes) and two badgers (Meles meles). An ELISA test also objectified seropositivities. Although the 171 qPCRs carried out on samples from the 33 mustelids were all negative, these preliminary results (observational study) nevertheless bear witness to infections of unknown origin. The epidemiological surveillance of Covid-19 in wildlife must continue, in particular with the tools of efficient serology.","version":"1.1","doi":"10.1101/2022.01.20.477038","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.476998","pub_date":"2022-1-20","title":"Discovery of a SARS-CoV-2 Broadly-Acting Neutralizing Antibody with Activity against Omicron and Omicron + R346K Variants","abstract":"The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.","version":"1.1","doi":"10.1101/2022.01.19.476998","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.476497","pub_date":"2022-1-20","title":"T cell response following anti COVID-19 BNT162b2 vaccination is maintained against the SARS-CoV-2 Omicron B.1.1.529 variant of concern","abstract":"The progression of the COVID-19 pandemic leads to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about a reduced protective T cell immunity and consequently to more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type, Wuhan-1 SARS-CoV-2 ancestral spike protein and Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from 8 healthy volunteers 4-5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or Omicron SARS-CoV- 2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4) secreting cells. For all the examined individuals, comparable level of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg secreting cells and only limited numbers of IL-10 and IL-4 secreting cells. The data demonstrates a stable T cell activity to the emerging Omicron variant in the tested individuals, therefore the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.","version":"1.1","doi":"10.1101/2022.01.19.476497","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.476801","pub_date":"2022-1-20","title":"A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants","abstract":"The spike glycoprotein of SARS-CoV-2 engages with human angiotensin-converting enzyme 2 (ACE2) to facilitate infection. Here, we describe an alpaca-derived heavy chain antibody fragment (VHH), saRBD-1, that disrupts this interaction by competitively binding to the spike protein receptor-binding domain. We further generated an engineered bivalent nanobody construct engineered by a flexible linker, and a dimeric Fc conjugated nanobody construct. Both multivalent nanobodies blocked infection at picomolar concentrations and demonstrated no loss of potency against emerging variants of concern including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427/429), and Delta (B.1.617.2). saRBD-1 tolerates elevated temperature, freeze-drying, and nebulization, making it an excellent candidate for further development into a therapeutic approach for COVID-19.","version":"1.1","doi":"10.1101/2022.01.18.476801","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476608","pub_date":"2022-1-20","title":"Serological screening suggests single SARS-CoV-2 spillover events to cattle","abstract":"Widespread human SARS-CoV-2 infections pose a constant risk for virus transmission to animals. Here, we serologically investigated 1000 cattle samples collected in late 2021 in Germany. Eleven sera tested antibody-positive, indicating that cattle may be occasionally infected by contact to SARS-CoV-2-positive keepers, but there is no indication of further spreading.","version":"1.1","doi":"10.1101/2022.01.17.476608","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.477009","pub_date":"2022-1-20","title":"Computation of Antigenicity Predicts SARS-CoV-2 Vaccine Breakthrough Variants","abstract":"It has been reported that multiple SARS-CoV-2 variants of concerns (VOCs) including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) can reduce neutralisation by antibodies, resulting in vaccine breakthrough infections. Virus-antiserum neutralisation assays are typically performed to monitor potential vaccine breakthrough strains. However, such experimental-based methods are slow and cannot instantly validate whether newly emerging variants can break through current vaccines or therapeutic antibodies. To address this, we sought to establish a computational model to predict the antigenicity of SARS-CoV-2 variants by sequence alone and in real time. In this study, we firstly identified the relationship between the antigenic difference transformed from the amino acid sequence and the antigenic distance from the neutralisation titres. Based on this correlation, we obtained a computational model for the receptor binding domain (RBD) of the spike protein to predict the fold decrease in virus-antiserum neutralisation titres with high accuracy (~0.79). Our predicted results were comparable with experimental neutralisation titres of variants, including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), B.1.429 (Epsilon), P.1 (Gamma), B.1.526 (Iota), B.1.617.1 (Kappa), and C.37 (Lambda), as well as SARS-CoV. Here, we firstly predicted the fold of decrease of B.1.1.529 (Omicron) as 17.4-fold less susceptible to neutralisation. We visualised all 1521 SARS-CoV-2 lineages to indicate variants including B.1.621 (Mu), B.1.630, B.1.633, B.1.649, and C.1.2, which can induce vaccine breakthrough infections in addition to reported VOCs B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Our study offers a quick approach to predict the antigenicity of SARS-CoV-2 variants as soon as they emerge. Furthermore, this approach can facilitate future vaccine updates to cover all major variants. An online version can be accessed at http://jdlab.online.","version":"1.1","doi":"10.1101/2022.01.19.477009","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.476693","pub_date":"2022-1-20","title":"SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation","abstract":"Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a novel mechanism by which the SARS-CoV-2 virus co-opts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-\u03b2. We reveal that the SARS-CoV-2 encoded Non-Structural Protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-\u03b2 production, leading to reduced viral infection. Our findings reveal a new target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.","version":"1.1","doi":"10.1101/2022.01.19.476693","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.476892","pub_date":"2022-1-20","title":"Molecular basis of broad neutralization against SARS-CoV-2 variants including Omicron by a human antibody","abstract":"Omicron, a newly emerging SARS-CoV-2 variant, carried a large number of mutations in the spike protein leading to an unprecedented evasion from many neutralizing antibodies (nAbs). Here, we performed a head-to-head comparison of Omicron with other existing highly evasive variants in terms of their reduced sensitivities to antibodies, and found that Omicron variant is significantly more evasive than Beta and Mu variants. Of note, some key mutations occur in the conserved epitopes identified previously, especially in the binding sites of Class 4 nAbs, contributing to the increased Ab evasion. We also reported a broadly nAb (bnAb), VacW-209, which effectively neutralized all tested SARS-CoV-2 variants and even SARS-CoV. Finally, we determined six cryo-electron microscopy structures of VacW-209 complexed with the spike ectodomains of wild-type, Delta, Mu, C.1.2, Omicron, and SARS-CoV, and revealed the molecular basis of the broadly neutralizing activities of VacW-209 against SARS-CoV-2 variants. Overall, Omicron has once again raised the alarm over virus variation with significantly compromised neutralization. BnAbs targeting more conserved epitopes among variants will continue to play a key role in pandemic control and prevention. Structural and functional analyses reveal that a human antibody named VacW-209 confers broad neutralization against SARS-CoV-2 variants including Omicron by recognizing a highly conserved epitope.","version":"1.1","doi":"10.1101/2022.01.19.476892","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.477013","pub_date":"2022-1-20","title":"Pathogenicity of SARS-CoV-2 Omicron in Syrian hamsters and its neutralization with different Variants of Concern","abstract":"SARS-CoV-2 Omicron variant is rampantly spreading across the globe. Animal models are useful in understanding the disease characteristics as well as properties of emerging SARS-CoV-2 variants. We assessed the pathogenicity and immune response generated by BA.1 sub-lineage of SARS-CoV-2 Omicron variant with R346K mutation in 5 to 6-week old Syrian hamsters. Virus shedding, organ viral load, lung disease and immune response generated were sequentially assessed. The disease characteristics of Omicron were found to be similar to that of other SARS-CoV-2 variants of concerns in hamsters like high viral replication in the respiratory tract and interstitial pneumonia. The infected hamsters demonstrated lesser body weight gain in comparison to the uninfected control hamsters. Viral RNA could be detected in nasal washes and respiratory organs (nasal turbinate, trachea, bronchi and lungs) till 10 and 14 days respectively. The clearance of the virus was observed from nasal washes and lungs by day 7. Neutralizing antibody response against Omicron variant was detected from day 5 with rising antibody titers till 14 days. However, the cross-neutralization titre of the sera against other variants showed severe reduction ie., 7 fold reduction against Alpha and no titers against B.1, Beta and Delta. This preliminary data shows that Omicron variant infection can produce moderate to severe lung disease and the neutralizing antibodies produced in response to Omicron variant infection shows poor neutralizing ability against other co-circulating SARS-CoV-2 variants like Delta which necessitates caution as it may lead to increased cases of reinfection.","version":"1.1","doi":"10.1101/2022.01.19.477013","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.473990","pub_date":"2022-1-20","title":"Designing multi-epitope based peptide vaccine targeting spike protein SARS-CoV-2 B1.1.529 (Omicron) variant using computational approaches","abstract":"Since the SARS-CoV-2 outbreak in 2019, millions of people have been infected with the virus, and due to its high human-to-human transmission rate, there is a need for a vaccine to protect people. Although some vaccines are in use, due to the high mutation rate in the SARS-CoV-2 multiple variants, the current vaccines may not be sufficient to immunize people against new variant threats. One of the emerging variants of concern is B1.1.529 (Omicron), which carries\u223c30 mutations in the Spike protein of SARS-CoV-2 is predicted to evade antibodies recognition even from vaccinated people. We used a structure-based approach along with an epitope prediction server to develop a Multi-Epitope based Subunit Vaccine (MESV) involving SARS-CoV-2 B1.1.529 variant spike glycoprotein. The predicted epitope with better antigenicity and non-toxicity were used for designing and predicting vaccine construct features and structure models. The MESV construct In-silico cloning in pET28a expression vector predicted the construct to be highly translational. The proposed MESV vaccine construct was also subjected to immune simulation prediction and was found to be highly antigenic and elicit a cell-mediated immune response. The proposed MESV in the present study has the potential to be evaluated further for vaccine production against the newly identified B1.1.529 (Omicron) variant of concern.","version":"1.2","doi":"10.1101/2021.12.23.473990","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.19.476898","pub_date":"2022-1-20","title":"SARS-CoV-2 Omicron efficiently infects human airway, but not alveolar epithelium","abstract":"In late 2021, the highly mutated SARS-CoV-2 Omicron variant emerged, raising concerns about its potential extensive immune evasion, increased transmissibility and pathogenicity. Here, we used organoids of the human airways and alveoli to investigate Omicron\u2019s fitness and replicative potential in comparison with earlier SARS-CoV-2 variants. We report that Omicron replicates more rapidly in the airways and has an increased fitness compared to the early 614G variant and Delta. In contrast, Omicron did not replicate productively in human alveolar type 2 cells. Mechanistically, we show that Omicron does not efficiently use TMPRSS2 for entry or spread through cell-cell fusion. Altogether, our data show that Omicron has an altered tropism and protease usage, potentially explaining its higher transmissibility and decreased pathogenicity.","version":"1.1","doi":"10.1101/2022.01.19.476898","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.476864","pub_date":"2022-1-20","title":"Longitudinal Assessment of SARS-CoV-2 Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multi-Cytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity","abstract":"Cellular-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 SARS-CoV-2-infected survivors over a one year post symptom onset (PSO) interval by ex vivo cytokine ELISpot assay. All subjects demonstrated SARS-CoV-2-specific IFN-\u03b3, IL-2, and Granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (Nucleocapsid, Membrane, Spike) except Envelope, with GzmB > IL-2 > IFN-\u03b3. Mathematical modeling predicted that: 1) cytokine responses peaked at 6 days for IFN-\u03b3, 36 days for IL-2, and 7 days for GzmB, 2) severe illness was associated with reduced IFN-\u03b3 and GzmB, but increased IL-2 production rates, 3) males displayed greater production of IFN-\u03b3, whereas females produced more GzmB. Ex vivo responses declined over time with persistence of IL-2 in 86% and of IFN-\u03b3 and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modelled to be 139 days (\u223c4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the Nucleocapsid and Membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent. Our findings highlight the relative importance of SARS-CoV-2-specific GzmB-producing T cell responses in SARS-CoV-2 control, shared CD4 and CD8 immunodominant epitopes in seasonal coronaviruses or SARS-CoV-1, and indicate robust persistence of T cell memory at least one year after infection. Our findings should inform future strategies to induce T cell vaccines against SARS-CoV-2 and other coronaviruses.","version":"1.1","doi":"10.1101/2022.01.18.476864","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.477056","pub_date":"2022-1-20","title":"Sialic acid and fucose residues on the SARS-CoV-2 receptor binding domain modulate IgG reactivity","abstract":"The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.","version":"1.1","doi":"10.1101/2022.01.20.477056","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.20.476754","pub_date":"2022-1-20","title":"Omicron variant of SARS-CoV-2 exhibits an increased resilience to the antiviral type I interferon response","abstract":"The new variant of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Omicron (B.1.1.529), is genetically very different from other VOCs. We compared Omicron with the preceding VOC Delta (B.1.617.2) and the wildtype strain (B.1) with respect to their interactions with the antiviral type I interferon (IFN-alpha/beta) response in infected cells. Our data indicate that Omicron has gained an elevated capability to suppress IFN-beta induction upon infection and to better withstand the antiviral state imposed by exogenously added IFN-alpha.","version":"1.1","doi":"10.1101/2022.01.20.476754","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476560","pub_date":"2022-1-20","title":"Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF signaling","abstract":"The lung\u2019s gas exchange surface comprises thin alveolar type 1 (AT1) cells and cuboidal surfactant-secreting AT2 cells that are corrupted in some of the most common and deadly diseases including adenocarcinoma, emphysema, and SARS/Covid-19. These cells arise from an embryonic progenitor whose development into an AT1 or AT2 cell is thought to be dictated by differential mechanical forces. Here we show the critical determinant is FGF signaling. FGF Receptor 2 (Fgfr2) is expressed in mouse progenitors then restricts to nascent AT2 cells and remains on throughout life. Its ligands are expressed in surrounding mesenchyme and can, in the absence of differential mechanical cues, induce purified, uncommitted E16.5 progenitors to form alveolus-like structures with intermingled AT2 and AT1 cells. FGF signaling directly and cell autonomously specifies AT2 fate; progenitors lacking Fgfr2 in vitro and in vivo exclusively acquire AT1 fate. Fgfr2 loss in AT2 cells perinatally results in reprogramming to AT1 fate, whereas loss or inhibition later in life immediately triggers AT2 apoptosis followed by a compensatory regenerative response. We propose Fgfr2 signaling directly selects AT2 fate during development, induces a cell non-autonomous secondary signal for AT1 fate, and stays on throughout life to continuously maintain healthy AT2 cells. FGF signaling induces and distinguishes the two cell types of the lung\u2019s gas exchange surface, and the pathway remains on throughout life to maintain one that can be transformed into lung cancer or targeted in the deadly form of SARS/Covid-19.","version":"1.1","doi":"10.1101/2022.01.17.476560","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.14.476298","pub_date":"2022-1-20","title":"Long dsRNA mediated RNA interference (dsRNAi) is antiviral in interferon competent mammalian cells","abstract":"In invertebrate cells, RNA interference (RNAi) acts as a powerful defense against virus infection by cleaving virally produced long dsRNA into siRNA by Dicer and loaded into RISC which can then destroy/disrupt complementary viral mRNA sequences. Comparatively in mammalian cells, the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. Although the cellular machinery for RNAi functions in mammalian cells, its role in the antiviral response remains controversial. Here we show that IFN competent mammalian cells engage in dsRNA-mediated RNAi. We found that pre-soaking mammalian cells with concentrations of sequence-specific dsRNA too low to induce IFN production could significantly inhibit viral replication, including SARS-CoV-2. This phenomenon was dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, Dicer-knockout cell lines were incapable of this inhibition, confirming use of RNAi. This represents the first evidence that soaking with gene-specific dsRNA can generate viral knockdown in mammalian cells. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform.","version":"1.1","doi":"10.1101/2022.01.14.476298","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476677","pub_date":"2022-1-19","title":"Prediction and validation of host cleavage targets of SARS-CoV-2 3C-like protease","abstract":"How SARS-CoV-2 causes the observed range of clinical manifestations and disease severity remains poorly understood. SARS-CoV-2 encodes for two proteases (3CLPro and PLPro), vital for viral production, but also promiscuous with respect to host protein targets, likely contributing to the range of disease. Pharmacological inhibition of the 3C-like3 protease has revealed remarkable reduction in hospitalization and death in phase 2/3 clinical studies. However, the mechanisms responsible for the pathology mediated by those proteases are still unclear. In this study, we develop a bioinformatic algorithm, leveraging experimental data from SARS-CoV, to predict host cleavage targets of the SARS-CoV-2 3C-like protease, or 3CLPro. We capture targets of the 3CL protease described previously for SARS-CoV, and we identify hundreds of new putative targets. We experimentally validate a number of these predicted targets, including the giant sarcomeric protein Obscurin, and show that expression of 3CL protease alone recapitulates the sarcomeric disorganization seen by SARS-CoV-2 infection of hiPSC-derived cardiomyocytes. Our data provide a resource to identify putative host cleavage targets of 3CL protease that contribute to mechanisms and heterogeneity of disease in COVID-19 and future coronavirus outbreaks.","version":"1.1","doi":"10.1101/2022.01.17.476677","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476644","pub_date":"2022-1-19","title":"Nirmatrelvir, an orally active Mpro inhibitor, is a potent inhibitor of SARS-CoV-2 Variants of Concern","abstract":"New variants of SARS-CoV-2 with potential for enhanced transmission, replication, and immune evasion capabilities continue to emerge causing reduced vaccine efficacy and/or treatment failure. As of January 2021, the WHO has defined five \u2018variants of concern\u2019 (VOC): B.1.1.7 (Alpha, \u03b1), B.1.351 (Beta, \u03b2), P.1 (Gamma, \u03b3), B.1.617.2 (Delta, \u03b4), and B.1.1.529 (Omicron, o). To provide a therapeutic option for the treatment of COVID-19 and variants, Nirmatrelvir, the antiviral component of PAXLOVID\u2122, an oral outpatient treatment recently authorized for conditional or emergency use treatment of COVID-19, was developed to inhibit SARS-CoV-2 replication. Nirmatrelvir (PF-07321332) is a specific inhibitor of coronavirus main protease (Mpro, also referred to as 3CLpro), with potent antiviral activity against several human coronaviruses, including SARS-CoV-2, SARS-CoV, and MERS (Owen et al, Science 2021. doi: 10.1126/science.abl4784). Here, we evaluated PF-07321332 against the five SARS-CoV-2 VOC (\u03b1, \u03b2, \u03b3, \u03b4,, o) and two Variants of Interest or VOI, C.37 (\u03bb) and B.1.621 (\u03bc), using qRT-PCR in VeroE6 cells lacking the P-glycoprotein (Pgp) multidrug transporter gene (VeroE6 P-gp knockout cells). Nirmatrelvir potently inhibited USA-WA1/2020 strain, and \u03b1, \u03b2, \u03b3, \u03bb, \u03b4, \u03bc, and o variants in VeroE6 P-gp knockout cells with mean EC50 values 38.0 nM, 41.0 nM, 127.2 nM, 24.9 nM, 21.2 nM, 15.9 nM, 25.7 nM and 16.2 nM, respectively. Sequence analysis of the Mpro encoded by the variants showed ~100% identity of active site amino acid sequences, reflecting the essential role of Mpro during viral replication leading to ability of Nirmatrelvir to exhibit potent activity across all the variants.","version":"1.1","doi":"10.1101/2022.01.17.476644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.11.459844","pub_date":"2022-1-19","title":"Rapid and parallel adaptive mutations in spike S1 drive clade success in SARS-CoV-2","abstract":"Given the importance of variant SARS-CoV-2 viruses with altered receptor-binding or antigenic phenotypes, we sought to quantify the degree to which adaptive evolution is driving accumulation of mutations in the SARS-CoV-2 genome. Here we assessed adaptive evolution across genes in the SARS-CoV-2 genome by correlating clade growth with mutation accumulation as well as by comparing rates of nonsynonymous to synonymous divergence, clustering of mutations across the SARS-CoV-2 phylogeny and degree of convergent evolution of individual mutations. We find that spike S1 is the focus of adaptive evolution, but also identify positively-selected mutations in other genes that are sculpting the evolutionary trajectory of SARS-CoV-2. Adaptive changes in S1 accumulated rapidly, resulting in a remarkably high ratio of nonsynonymous to synonymous divergence that is 2.5X greater than that observed in HA1 at the beginning of the 2009 H1N1 pandemic.","version":"1.4","doi":"10.1101/2021.09.11.459844","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.17.476685","pub_date":"2022-1-19","title":"Nirmatrelvir, Molnupiravir, and Remdesivir maintain potent in vitro activity against the SARS-CoV-2 Omicron variant","abstract":"Variants of SARS-CoV-2 have become a major public health concern due to increased transmissibility, and escape from natural immunity, vaccine protection, and monoclonal antibody therapeutics. The highly transmissible Omicron variant has up to 32 mutations within the spike protein, many more than previous variants, heightening these concerns of immune escape. There are now multiple antiviral therapeutics that have received approval for emergency use by the FDA and target both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and the main protease (Mpro), which have accumulated fewer mutations in known SARS-CoV-2 variants. Here we test nirmatrelvir (PF-07321332), and other clinically relevant SARS-CoV-2 antivirals, against a panel of SARS-CoV-2 variants, including the novel Omicron variant, in live-virus antiviral assays. We confirm that nirmatrelvir and other clinically relevant antivirals all maintain activity against all variants tested, including Omicron.","version":"1.1","doi":"10.1101/2022.01.17.476685","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.22.453287","pub_date":"2022-1-19","title":"Competent immune responses to SARS-CoV-2 variants in older adults following mRNA vaccination","abstract":"Aging is associated with a reduced magnitude of primary immune responses to vaccination and constriction of immune receptor repertoire diversity. Clinical trials demonstrated high efficacy of mRNA based SARS-CoV-2 vaccines in older adults but concerns about virus variant escape have not been well addressed. We have conducted an in-depth analysis of humoral and cellular immunity against an early-pandemic viral isolate and compared that to the P.1. (Gamma) and B.1.617.2 (Delta) variants in <50 and >55 age cohorts of mRNA vaccine recipients. We have further measured neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595; a SARS-CoV-2 isolate bearing Spike mutation E484Q. As reported, robust immunity required the second dose of vaccine. Older vaccinees manifested robust cellular immunity against early-pandemic SARS-CoV-2 and more recent variants, which remained statistically comparable to the adult group. The older cohort had lower neutralizing capacity at the first time point following the second dose, but at later time points immunity was indistinguishable between them. While the duration of these immune responses remains to be determined over longer periods of time, these results provide reasons for optimism regarding vaccine protection of older adults against SARS-CoV-2 variants and inform thinking about boost vaccination with variant vaccines. Vaccine responses are often diminished with aging, but we found strong responses to SARS-CoV-2 in older adults following mRNA vaccination. T cell responses were not diminished when confronted by SARS-CoV-2 variants. Neutralizing Ab were reduced but not more than those in adults. Created with BioRender.com","version":"1.2","doi":"10.1101/2021.07.22.453287","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.18.476863","pub_date":"2022-1-19","title":"SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice","abstract":"The COVID-19 pandemic has been fueled by novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strain\u2019s pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta- challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing infected to uninfected mice revealed that Alpha-challenged mice have more total genes differentially activated, conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-\u03b3 production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines. The Delta variant of SARS-CoV-2 is known to be more transmissible and cause severe disease in human hosts due to mutations in its genome that are divergent from previous variants of concern (VOC). Our study evaluates the pathogenesis of Delta in the K18-hACE2 mouse model compared to the Alpha VOC. We observed that relative to Alpha, Delta challenge results in enhanced inflammation and tissue damage with stronger antiviral responses. These observations provide insight into Delta\u2019s unique pathogenesis.","version":"1.1","doi":"10.1101/2022.01.18.476863","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.15.476426","pub_date":"2022-1-18","title":"ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells","abstract":"Currently, the COVID-19 pandemic, caused by SARS-CoV-2 infection, represents a serious public health problem worldwide. Although it has been shown that ACE2 serves as the main receptor for SARS-CoV-2 entry into host cells, studies have shown that ACE2 is expressed at extremely low levels in various tissues, especially in some organs where virus particles have been found, such as the heart and liver. Therefore, these organs potentially express additional SARS-CoV-2 receptors that have not yet been discovered. Here, by a genome-wide CRISPR-Cas9 activation library screening, we found that ASGR1 promoted SARS-CoV-2 infection of 293T cells. In Huh-7 and HepG2 cell lines, simultaneous knock out of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary liver parenchymal cells, both of which hardly express ACE2, SARS-CoV-2 could successfully establish an infection. After treatment with ASGR1 antibody, the infection rate significantly reduced. This suggests that SARS-CoV-2 infects liver cells mainly through an ASGR1-dependent mechanism. Finally, we also found that the soluble ASGR1 could not only prevent the SARS-CoV-2 pseudovirus, which binds to the ASGR1 receptors, from infecting host liver cells, but also had a protective effect on those expressing ACE2, indicating that administration of soluble ASGR1 protein may represent a new treatment approach. Colletively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells. We show that ASGR1 is a candidate receptor for SARS-CoV-2 to infect liver cells.","version":"1.1","doi":"10.1101/2022.01.15.476426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.19.452809","pub_date":"2022-1-18","title":"CRISPR screens for host factors critical for infection by SARS-CoV-2 variants of concern identify GATA6 as a central modulator of ACE2","abstract":"The global spread of SARS-CoV-2 led to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SASR-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.","version":"1.2","doi":"10.1101/2021.07.19.452809","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.459055","pub_date":"2022-1-18","title":"Short-term instantaneous prophylaxis and efficient treatment against SARS-CoV-2 in hACE2 mice conferred by an intranasal nanobody (Nb22)","abstract":"Current COVID-19 vaccines need to take at least one month to complete inoculation and then become effective. Around 51% global population are still not fully vaccinated. Instantaneous protection is an unmet need among those who are not fully vaccinated. In addition, breakthrough infections caused by SARS-CoV-2 are widely reported. All these highlight the unmet needing for short-term instantaneous prophylaxis (STIP) in the communities where SARS-CoV-2 is circulating. Previously, we reported nanobodies isolated from an alpaca immunized with the spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its variants. Herein, we found that Nb22, among our previously reported nanobodies, exhibited ultrapotent neutralization against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM). Furthermore, the crystal structural analysis revealed that the binding of Nb22 to WH01 and Delta RBDs both effectively blocked the binding of RBD to hACE2. Additionally, intranasal Nb22 exhibited protection against SARS-CoV-2 Delta variant in the post-exposure prophylaxis (PEP) and pre-exposure prophylaxis (PrEP). Of note, intranasal Nb22 also demonstrated high efficacy against SARS-CoV-2 Delta variant in STIP for seven days administered by single dose and exhibited long-lasting retention in the respiratory system for at least one month administered by four doses, providing a means of instantaneous short-term prophylaxis against SARS-CoV-2. Thus, ultrahigh potency, long-lasting retention in the respiratory system as well as stability at room-temperature make the intranasal or inhaled Nb22 to be a potential therapeutic or STIP agent against SARS-CoV-2. Nb22 exhibits ultrahigh potency against Delta variant in vitro and is exploited by crystal structural analysis; furthermore, animal study demonstrates high effectiveness in the treatment and short-term instantaneous prophylaxis in hACE2 mice via intranasal administration. Nb22 exhibits ultrapotent neutralization against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM). Structural analysis elucidates the ultrapotent neutralization of Nb22 against Delta variant. Nb22 demonstrates complete protection in the treatment of Delta variant infection in hACE2 transgenic mice. We complete the proof of concept of STIP against SARS-CoV-2 using intranasal Nb22 with ultrahigh potency and long-lasting retention in respiratory system.","version":"1.2","doi":"10.1101/2021.09.06.459055","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.16.476016","pub_date":"2022-1-18","title":"Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection","abstract":"Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.","version":"1.1","doi":"10.1101/2022.01.16.476016","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.14.475727","pub_date":"2022-1-18","title":"Obesity associated with attenuated tissue immune cell responses in COVID-19","abstract":"Obesity is common and associated with more severe COVID-19, proposed to be in part related to an adipokine-driven pro-inflammatory state. Here we analysed single cell transcriptomes from bronchiolar lavage in three adult cohorts, comparing obese (Ob, body mass index (BMI) >30m2) and non-obese (N-Ob, BMI <30m2). Surprisingly, we found that Ob subjects had attenuated lung immune/inflammatory responses in SARS-CoV-2 infection, with decreased expression of interferon (IFN)\u03b1, IFN\u03b3 and tumour necrosis factor (TNF) alpha response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Analysis of peripheral blood immune cells in an independent adult cohort showed a similar, but less marked, reduction in type I IFN and IFN\u03b3 response genes, as well as decreased serum IFN\u03b1, in Ob patients with SARS-CoV-2. Nasal immune cells from Ob children with COVID-19 also showed reduced enrichment of IFN\u03b1 and IFN\u03b3 response genes. Altogether, these findings show blunted tissue immune responses in Ob COVID-19 patients, with clinical implications.","version":"1.1","doi":"10.1101/2022.01.14.475727","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.13.476252","pub_date":"2022-1-18","title":"Immunokinetic Model for COVID-19 Patients","abstract":"In this paper, we develop a fractional-order differential model for the dynamics of immune responses to SARS-CoV-2 viral load in one host. In the model, a fractional-order derivative is incorporated to represent the effects of temporal long-run memory on immune cells and tissues for any age group of patients. The population of cytotoxic T-cells (CD8+), natural killer (NK) cells and infected viruses are unknown in this model. Some interesting sufficient conditions that ensure the asymptotic stability of the steady states are obtained. This model indicates some complex phenomena in COVID-19 such as \u201cimmune exhaustion\u201d and \u201cLong COVID\u201d. Sensitivity analysis is also investigated for model parameters to determine the parameters that are effective in determining of the long COVID duration, disease control and future treatment as well as vaccine design. The model is verified with clinical and experimental data of 5 patients with COVID-19.","version":"1.1","doi":"10.1101/2022.01.13.476252","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.15.476448","pub_date":"2022-1-18","title":"RASCL: Rapid Assessment Of SARS-CoV-2 Clades Through Molecular Sequence Analysis","abstract":"An important component of efforts to manage the ongoing COVID19 pandemic is the Rapid Assessment of how natural selection contributes to the emergence and proliferation of potentially dangerous SARS-CoV-2 lineages and CLades (RASCL). The RASCL pipeline enables continuous comparative phylogenetics-based selection analyses of rapidly growing clade-focused genome surveillance datasets, such as those produced following the initial detection of potentially dangerous variants. From such datasets RASCL automatically generates down-sampled codon alignments of individual genes/ORFs containing contextualizing background reference sequences, analyzes these with a battery of selection tests, and outputs results as both machine readable JSON files, and interactive notebook-based visualizations. RASCL is available from a dedicated repository at https://github.com/veg/RASCL and as a Galaxy workflow https://usegalaxy.eu/u/hyphy/w/rascl. Existing clade/variant analysis results are available here: https://observablehq.com/@aglucaci/rascl. Dr. Sergei L Kosakovsky Pond (spond@temple.edu). N/A","version":"1.1","doi":"10.1101/2022.01.15.476448","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.14.476382","pub_date":"2022-1-18","title":"Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function","abstract":"Among the 30 non-synonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any genomes within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron over all previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.","version":"1.1","doi":"10.1101/2022.01.14.476382","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475746","pub_date":"2022-1-17","title":"Tuftsin: a natural molecule against SARS-CoV-2 infection","abstract":"Coronavirus disease 2019 (COVID-19) continuously proceeds despite the application of a variety of vaccines. It is still urgent to find effective ways to treat COVID-19. Recent studies indicate that NRP1, an important receptor of the natural peptide tuftsin, facilitates SARS-CoV-2 infection. Importantly, tuftsin is a natural human molecule released from IgG. Here, we found 91 overlapping genes between tuftsin targets and COVID-19-associated genes. Bioinformatics analyses indicated that tuftsin could also target ACE2 and exert some immune-related functions to treat COVID-19. Using surface plasmon resonance (SPR) analysis, we confirmed that tuftsin can bind ACE2 and NRP1 directly. Moreover, tuftsin effectively impairs the binding of SARS-CoV-2 S1 to ACE2. Thus, tuftsin is an attractive drug against COVID-19. And tuftsin as natural immunostimulating peptide in human, we speculate that tuftsin may has crucial roles in asymptomatic carriers or mild cases of COVID-19.","version":"1.1","doi":"10.1101/2022.01.10.475746","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457378","pub_date":"2022-1-17","title":"SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19","abstract":"Coagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis. To understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including PT, APTT, D-Dimer and TFPI. The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue-plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer. The investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease. In summary, induction of soluble P-selectin, sCD40L, fibrinogen and PAI-1 in moderate COVID-19 cases suggests the activation of platelets and coagulation system before patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity.","version":"1.2","doi":"10.1101/2021.08.23.457378","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452680","pub_date":"2022-1-16","title":"Variable susceptibility of intestinal organoid-derived monolayers to SARS-CoV-2 infection","abstract":"Gastrointestinal effects associated with COVID-19 are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures differentiated from primary human specimens as a model to examine inter-individual variability. Infection of intestinal organoids derived from different donors with SARS-CoV-2 resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with ACE2 expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue indicating this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.","version":"1.2","doi":"10.1101/2021.07.16.452680","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.27.474275","pub_date":"2022-1-15","title":"Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern","abstract":"We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved.","version":"1.2","doi":"10.1101/2021.12.27.474275","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.29.474402","pub_date":"2022-1-15","title":"Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant","abstract":"The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an \u201cactive\u201d conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.","version":"1.2","doi":"10.1101/2021.12.29.474402","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.13.22269257","pub_date":"2022-01-14","title":"Quantifying the impact of immune history and variant on SARS-CoV-2 viral kinetics and infection rebound: a retrospective cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The combined impact of immunity and SARS-CoV-2 variants on viral kinetics during infections has been unclear.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We characterized 2,875 infections from the National Basketball Association occupational health cohort identified between June 2020 and January 2022 using serial RT-qPCR testing. Logistic regression and semi-mechanistic viral RNA kinetics models were used to quantify the effect of variant, symptom status, age, infection history, vaccination and antibody titer to founder SARS-CoV-2 strain on the duration of potential infectiousness and overall viral kinetics. The frequency of viral rebounds was quantified under multiple cycle threshold (Ct) value-based definitions.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Among individuals detected partway through their infection, 51.0% (95% credible interval [CrI]: 48.2-53.6%) remained potentially infectious (Ct&lt;30) five days post detection, with small differences across variants and vaccination history. Only seven viral rebounds (0.7%; N=999) were observed, with rebound defined as 3+ days with Ct&lt;30 following an initial clearance of 3+ days with Ct\u226530. High antibody titers against the founder SARS-CoV-2 strain predicted lower peak viral loads and shorter durations of infection. Among Omicron BA.1 infections, boosted individuals had lower pre-booster antibody titers and longer clearance times than non-boosted individuals.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>SARS-CoV-2 viral kinetics are partly determined by immunity and variant but dominated by individual-level variation. Since booster vaccination protects against infection, longer clearance times for BA.1-infected, boosted individuals may reflect a less effective immune response, more common in older individuals, that increases infection risk and reduces viral RNA clearance rate. The shifting landscape of viral kinetics underscores the need for continued monitoring to optimize isolation policies and to contextualize the health impacts of therapeutics and vaccines.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Supported in part by CDC contract 200-2016-91779, Emergent Ventures at the Mercatus Center, the Huffman Family Donor Advised Fund, the MorrisSinger Fund, the National Basketball Association, and the National Basketball Players Association.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.01.13.22269257","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.14.22268980","pub_date":"2022-01-14","title":"HAT-field: a cheap, robust and quantitative point-of-care serological test for Covid-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>We have recently described a very simple and cheap serological test called HAT to detect antibodies directed against the RBD of the SARS-Cov-2 virus. HAT is based on hemagglutination, triggered by a single reagent (IH4-RBD) comprised of the viral RBD domain fused to a nanobody specific for glycophorin, which is expressed at very high levels at the surface of human red blood cells (RBCs).</jats:p>\n                <jats:p>One of the main initial goals of this study was to devise a test protocol that would be sensitive and reliable, yet require no specialized laboratory equipment such as adjustable pipets, so that it could be performed in the most remote corners of the world by people with minimal levels of training. Because antibody levels against the viral RBD have been found to correlate closely with sero-neutralisation titers, and thus with protection against reinfection, it has become obvious during the course of this study that making this test reliably quantitative would be a further significant advantage.</jats:p>\n                <jats:p>Using IH4-RBD based on the original Wuhan sequence, we have found that, in PBN, a buffer which contains BSA and sodium azide, the reagent is stable for over 6 months at room temperature, and that PBN also improves HAT performance compared to using straight PBS. We also show that performing HAT at either 4\u00b0C, room temperature or 37\u00b0C has minimal influence on the results, and that quantitative evaluation of the levels of antibodies directed against the SARS-CoV-2 RBD can be achieved in a single step using titration of the IH4-RBD reagent.</jats:p>\n                <jats:p>The HAT-field protocol described here requires only very simple disposable equipment and a few microliters of whole blood, such as can be obtained by finger prick. Because it is based on a single soluble reagent, the test can be adapted very simply and rapidly to detect antibodies against variants of the SARS-CoV-2, or conceivably against different pathogens. HAT-field appears well suited to provide quantitative assessments of the serological protection of populations as well as individuals, and given its very low cost, the stability of the IH4-RBD reagent in the adapted buffer, and the simplicity of the procedure, could be deployed pretty much anywhere, including in the poorest countries and the most remote corners of the globe.</jats:p>\n                <jats:p>\n                  <jats:bold>Note: This manuscript has been refereed</jats:bold>\n                  by Review Commons, and modified thanks to the comments and suggestions from three referees. Those comments, and our replies, are provided at the end of the manuscript\u2019s pdf, and can also be accessed by clicking on the blue tab found to the right of the MedRXiv window.\n                </jats:p>","version":null,"doi":"10.1101/2022.01.14.22268980","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.13.476159","pub_date":"2022-1-14","title":"In vitro Kinase-to-Phosphosite database (iKiP-DB) predicts kinase activity in phosphoproteomic datasets","abstract":"Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. While databases like PhosphoSitePlus contain information about many phosphorylation sites, the vast majority of known sites are not assigned to any protein kinase. Assigning changes in the phosphoproteome to the activity of individual kinases therefore remains a key challenge.. A recent large-scale study systematically identified in vitro substrates for most human protein kinases. Here, we reprocessed and filtered these data to generate an in vitro Kinase-to-Phosphosite database (iKiP-DB). We show that iKiP-DB can accurately predict changes in kinase activity in published phosphoproteomic datasets for both well-studied and poorly characterized kinases. We apply iKiP-DB to a newly generated phosphoproteomic analysis of SARS-CoV-2 infected human lung epithelial cells and provide evidence for coronavirus-induced changes in host cell kinase activity. In summary, we show that iKiP-DB is widely applicable to facilitate the functional analysis of phosphoproteomic datasets.","version":"1.1","doi":"10.1101/2022.01.13.476159","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.12.476120","pub_date":"2022-1-13","title":"An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer","abstract":"The protective human antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus focuses on the spike (S) protein which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor- binding domain or a single dominant epitope (\u2018supersite\u2019) on the N terminal domain (NTD). Here, using the single B cell technology LIBRA-seq, we isolated a large panel of NTD-reactive and SARS-CoV-2 neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies to the NTD supersite commonly are encoded by the IGHV1-24 gene, forming a genetic cluster that represents a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited cell-to-cell spread of virus in culture, and conferred protection in human ACE2 transgenic mice against SARS-CoV-2 challenge. This study provides insight about antibody targeting of the S protein trimer interface region, suggesting this region may be a site of virus vulnerability.","version":"1.1","doi":"10.1101/2022.01.12.476120","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.12.475688","pub_date":"2022-1-13","title":"matOptimize: A parallel tree optimization method enables online phylogenetics for SARS-CoV-2","abstract":"Phylogenetic tree optimization is necessary for precise analysis of evolutionary and transmission dynamics, but existing tools are inadequate for handling the scale and pace of data produced during the COVID-19 pandemic. One transformative approach, online phylogenetics, aims to incrementally add samples to an ever-growing phylogeny, but there are no previously-existing approaches that can efficiently optimize this vast phylogeny under the time constraints of the pandemic. Here, we present matOptimize, a fast and memory-efficient phylogenetic tree optimization tool based on parsimony that can be parallelized across multiple CPU threads and nodes, and provides orders of magnitude improvement in runtime and peak memory usage compared to existing state-of-the-art methods. We have developed this method particularly to address the pressing need during the COVID-19 pandemic for daily maintenance and optimization of a comprehensive SARS-CoV-2 phylogeny. Thus, our approach addresses an important need for daily maintenance and refinement of a comprehensive SARS-CoV-2 phylogeny. Phylogenetic trees have been central to genomic surveillance, epidemiology, and contact tracing efforts during the COVD-19 pandemic. With over 6 million SARS-CoV-2 genome sequences now available, maintaining an accurate, comprehensive phylogenetic tree of all available SARS-CoV-2 sequences is becoming computationally infeasible with existing software, but is essential for getting a detailed picture of the virus\u2019 evolution and transmission. Our novel phylogenetic software, matOptimize, is helping refine possibly the largest-ever phylogenetic tree, containing millions of SARS-CoV-2 sequences, thus providing an unprecedented resolution for studying the pathogen\u2019s evolutionary and transmission dynamics.","version":"1.1","doi":"10.1101/2022.01.12.475688","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.13.475409","pub_date":"2022-1-13","title":"Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants","abstract":"The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies, and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.","version":"1.1","doi":"10.1101/2022.01.13.475409","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470356","pub_date":"2022-1-13","title":"A potent human monoclonal antibody with pan-neutralizing activities directly dislocates S trimer of SARS-CoV-2 through binding both up and down forms of RBD","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.","version":"1.4","doi":"10.1101/2021.11.29.470356","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.12.476031","pub_date":"2022-1-13","title":"The SARS-CoV-2 Omicron (B.1.1.529) variant exhibits altered pathogenicity, transmissibility, and fitness in the golden Syrian hamster model","abstract":"The newly emerging SARS-CoV-2 Omicron (B.1.1.529) variant first identified in South Africa in November 2021 is characterized by an unusual number of amino acid mutations in its spike that renders existing vaccines and therapeutic monoclonal antibodies dramatically less effective. The in vivo pathogenicity, transmissibility, and fitness of this new Variant of Concerns are unknown. We investigated these virological attributes of the Omicron variant in comparison with those of the currently dominant Delta (B.1.617.2) variant in the golden Syrian hamster COVID-19 model. Omicron-infected hamsters developed significantly less body weight losses, clinical scores, respiratory tract viral burdens, cytokine/chemokine dysregulation, and tissue damages than Delta-infected hamsters. The Omicron and Delta variant were both highly transmissible (100% vs 100%) via contact transmission. Importantly, the Omicron variant consistently demonstrated about 10-20% higher transmissibility than the already-highly transmissible Delta variant in repeated non-contact transmission studies (overall: 30/36 vs 24/36, 83.3% vs 66.7%). The Delta variant displayed higher fitness advantage than the Omicron variant without selection pressure in both in vitro and in vivo competition models. However, this scenario drastically changed once immune selection pressure with neutralizing antibodies active against the Delta variant but poorly active against the Omicron variant were introduced, with the Omicron variant significantly outcompeting the Delta variant. Taken together, our findings demonstrated that while the Omicron variant is less pathogenic than the Delta variant, it is highly transmissible and can outcompete the Delta variant under immune selection pressure. Next-generation vaccines and antivirals effective against this new VOC are urgently needed. The novel SARS-CoV-2 Omicron variant, though less pathogenic, is highly transmissible and outcompetes the Delta variant under immune selection pressure in the golden Syrian hamster COVID-19 model.","version":"1.1","doi":"10.1101/2022.01.12.476031","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.12.473243","pub_date":"2022-1-13","title":"COVID-19 vaccine booster induces a strong CD8+ T cell response against Omicron variant epitopes in HLA-A*02:01+ individuals","abstract":"The >30 mutated residues in the Omicron spike protein have led to its rapid classification as a new SARS-CoV-2 variant of concern. As a result, Omicron may escape from the immune system, decreasing the protection provided by COVID-19 vaccines. Preliminary data shows a weaker neutralizing antibody response to Omicron compared to the ancestral SARS-CoV-2 virus, which can be increased after a booster vaccine. Here, we report that CD8+ T cells can recognize Omicron variant epitopes presented by HLA-A*02:01 in both COVID-19 recovered and vaccinated individuals, even 6 months after infection or vaccination. Additionally, the T cell response was stronger for Omicron variant epitopes after the vaccine booster. Altogether, T cells can recognize Omicron variants, especially in vaccinated individuals after the vaccine booster. CD8+ T cells response against Omicron variant epitopes is stronger after the vaccine booster.","version":"1.1","doi":"10.1101/2022.01.12.473243","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473248","pub_date":"2022-1-13","title":"SARS-CoV-2 Omicron spike mediated immune escape and tropism shift","abstract":"The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.","version":"1.3","doi":"10.1101/2021.12.17.473248","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.12.475264","pub_date":"2022-1-13","title":"Hypercapnia limits \u03b2-catenin-mediated alveolar type 2 cell progenitor function by altering Wnt production from adjacent fibroblasts","abstract":"Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, we show that hypercapnia limits \u03b2-catenin signaling in alveolar type 2 (AT2) cells, leading to reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRa+-fibroblasts from those maintaining AT2 progenitor activity and towards those that antagonize \u03b2-catenin signaling and limit progenitor function. Activation of \u03b2-catenin signaling in AT2 cells, rescues the inhibition AT2 proliferation induced by hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality.","version":"1.1","doi":"10.1101/2022.01.12.475264","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475889","pub_date":"2022-1-12","title":"Favipiravir, umifenovir and camostat mesylate: a comparative study against SARS-CoV-2","abstract":"Since the first cases the coronavirus disease caused by SARS-CoV-2 (COVID-19) reported in December 2019, worldwide continuous efforts have been placed both for the prevention and treatment of this infectious disease. As new variants of the virus emerge, the need for an effective antiviral treatment continues. The concept of preventing SARS-CoV-2 on both pre-entry and post-entry stages has not been much studied. Therefore, we compared the antiviral activities of three antiviral drugs which have been currently used in the clinic. In silico docking analyses and in vitro viral infection in Vero E6 cells were performed to delineate their antiviral effectivity when used alone or in combination. Both in silico and in vitro results suggest that the combinatorial treatment by favipiravir and umifenovir or camostat mesylate has more antiviral activity against SARS-CoV-2 rather than single drug treatment. These results suggest that inhibiting both viral entry and viral replication at the same time is much more effective for the antiviral treatment of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.11.475889","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475947","pub_date":"2022-1-12","title":"SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction","abstract":"Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson\u2019s disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation utilising a model of human monocyte-derived microglia. We identified that SARS-CoV-2 isolates can bind and enter microglia, triggering inflammasome activation in the absence of viral replication. Mechanistically, microglial NLRP3 could be both primed and activated with SARS-CoV-2 spike glycoprotein in a NF-\u03baB and ACE2-dependent manner. Notably, virus- and spike protein-mediated inflammasome activation in microglia was significantly enhanced in the presence of \u03b1-synuclein fibrils, which was entirely ablated by NLRP3-inhibition. These results support a possible mechanism of microglia activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson\u2019s disease in certain COVID-19 infected individuals, and a potential therapeutic avenue for intervention. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) principally affects the lungs, however there is evidence that the virus can also reach the brain and lead to chronic neurological symptoms. In this study, we examined the interaction SARS-CoV-2 with brain immune cells, by using an ex-vivo model of human monocyte-derived microglia. We identified robust activation of the innate immune sensor complex, NLRP3 inflammasome, in cells exposed to SARS-CoV-2. This was dependent on spike protein-ACE2 receptor interaction and was potentiated in the presence of \u03b1-synuclein. We therefore identify a possible mechanism for SARS-CoV-2 and increased vulnerability to developing neurological dysfunction. These findings support a potential therapeutic avenue for treatment of SARS-CoV-2 driven neurological manifestations, through use of NLRP3 inflammasome or ACE2 inhibitors.","version":"1.1","doi":"10.1101/2022.01.11.475947","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475327","pub_date":"2022-1-12","title":"Systemic infection of SARS-CoV-2 in free ranging Leopard (Panthera pardus fusca) in India","abstract":"We report patho-morphological and virological characterization of SARS-CoV-2 in naturally infected, free ranging Indian Leopard (Panthera pardus fusca). Whole genome sequence analysis confirmed infection of Delta variant of SARS-CoV-2, possibly spill over from humans, but the case was detected when infection level had dropped significantly in human population. This report underlines the need for intensive screening of wild animals for keeping track of the virus evolution and development of carrier status of SARS-CoV-2 among wildlife species.","version":"1.1","doi":"10.1101/2022.01.11.475327","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475752","pub_date":"2022-1-12","title":"A novel structure-based approach for identification of vertebrate susceptibility to SARS-CoV-2: implications for future surveillance programmes","abstract":"Understanding the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a highly debatable and unsolved challenge for the scientific communities across the world. A key to dissect the susceptibility profiles of animal species to SARS-CoV-2 is to understand how virus enters into the cells. The interaction of SARS-CoV-2 ligands (RBD on spike protein) with its host cell receptor, angiotensin-converting enzyme 2 (ACE2), is a critical determinant of host range and cross-species transmission. In this study, we developed and implemented a rigorous computational approach for predicting binding affinity between 299 ACE2 orthologs from diverse vertebrate species and the SARS-CoV-2 spike protein. The findings show that the spike protein of SARS-CoV-2 can bind to many vertebrate species carrying evolutionary divergent ACE2, implying a broad host range at the virus entry level, which may contribute to cross-species transmission and further viral evolution. Additionally, the present study facilitated the identification of genetic determinants that may differentiate susceptible from the resistant host species based on the conservation of ACE2-spike protein interacting residues in vertebrate host species known to facilitate SARS-CoV-2 infection; however, these genetic determinants warrant in vivo experimental confirmation. The molecular interactions associated with varied binding affinity of distinct ACE2 isoforms in a specific bat species were identified using protein structure analysis, implying the existence of diversified susceptibility of bat species to SARS-CoV-2. The findings from current study highlight the importance of intensive surveillance programs aimed at identifying susceptible hosts, particularly those with the potential to transmit zoonotic pathogens, in order to prevent future outbreaks.","version":"1.1","doi":"10.1101/2022.01.10.475752","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475901","pub_date":"2022-1-12","title":"Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein","abstract":"To study effects on cellular innate immune responses to novel genes ORF8 and ORF10, and the more conserved Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, either alone, or in combination with cannabidiol (CBD). HEK293 cells were transfected with a control plasmid, or plasmids expressing ORF8, ORF10, or M protein, and assayed for cell number and markers of apoptosis at 24 h, and expression of interferon and interferon-stimulated genes at 14 h. A significant reduction in cell number, and increase in early and late apoptosis, was found after 24 h in cells where expression of viral genes was combined with 1-2 \u03bcM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. CBD (2 \u03bcM) augmented expression of IFN\u03b3, IFN\u03bb1 and IFN\u03bb2/3, as well as the 2\u2019-5\u2019-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL, in cells expressing ORF8, ORF10, and M protein. CBD also augmented expression of these genes in control cells not expressing viral genes, without enhancing apoptosis. Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but suggest an augmented innate anti-viral response to these genes in the presence of CBD. Furthermore, our results indicate that CBD may prime components of the innate immune system, increasing readiness to respond to viral infection without activating apoptosis, and therefore could be studied for potential in prophylaxis.","version":"1.1","doi":"10.1101/2022.01.11.475901","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475922","pub_date":"2022-1-12","title":"Structural and functional impact by SARS-CoV-2 Omicron spike mutations","abstract":"The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bearing an unusually high number of mutations, has become a dominant strain in many countries within several weeks. We report here structural, functional and antigenic properties of its full-length spike (S) protein with a native sequence in comparison with those of previously prevalent variants. Omicron S requires a substantially higher level of host receptor ACE2 for efficient membrane fusion than other variants, possibly explaining its unexpected cellular tropism. Mutations not only remodel the antigenic structure of the N-terminal domain of the S protein, but also alter the surface of the receptor-binding domain in a way not seen in other variants, consistent with its remarkable resistance to neutralizing antibodies. These results suggest that Omicron S has acquired an extraordinary ability to evade host immunity by excessive mutations, which also compromise its fusogenic capability.","version":"1.1","doi":"10.1101/2022.01.11.475922","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475722","pub_date":"2022-1-12","title":"UV-C light completely blocks highly contagious Delta SARS-CoV-2 aerosol transmission in hamsters","abstract":"Behavioral and medical control measures are not effective in containing the spread of SARS-CoV-2. Here we report on the effectiveness of a preemptive environmental strategy using UV-C light to prevent airborne transmission of the virus in a hamster model and show that UV-C exposure completely prevents airborne transmission between individuals","version":"1.1","doi":"10.1101/2022.01.10.475722","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475397","pub_date":"2022-1-12","title":"Investigation of the Effects of N-Linked Glycans on the Stability of the Spike Protein in SARS-CoV-2 by Molecular Dynamics Simulations","abstract":"We perform all-atom molecular dynamics simulations to study the effects of the N-linked glycans on the stability of the spike glycoprotein in SARS-CoV-2. After a 100 ns of simulation on the spike proteins without and with the N-linked glycans, we found that the presence of glycans increases the local stability in their vicinity; even though their effect on the full structure is negligible.","version":"1.1","doi":"10.1101/2022.01.07.475397","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475918","pub_date":"2022-1-12","title":"Shift of lung macrophage composition is associated with COVID-19 disease severity and recovery","abstract":"Though it has been 2 years since the start of the Coronavirus Disease 19 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive vaccines, very little progress has been made to identify curative therapies to treat COVID-19 and other inflammatory diseases which remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and death to develop tailored immunotherapy strategies to halt disease progression. Here we assembled the Mount Sinai COVID-19 Biobank which was comprised of ~600 hospitalized patients followed longitudinally during the peak of the pandemic. Moderate disease and survival were associated with a stronger antigen (Ag) presentation and effector T cell signature, while severe disease and death were associated with an altered Ag presentation signature, increased numbers of circulating inflammatory, immature myeloid cells, and extrafollicular activated B cells associated with autoantibody formation. Strikingly, we found that in severe COVID-19 patients, lung tissue resident alveolar macrophages (AM) were not only severely depleted, but also had an altered Ag presentation signature, and were replaced by inflammatory monocytes and monocyte-derived macrophages (MoM\u03a6). Notably, the size of the AM pool correlated with recovery or death, while AM loss and functionality were restored in patients that recovered. These data therefore suggest that local and systemic myeloid cell dysregulation is a driver of COVID-19 severity and that modulation of AM numbers and functionality in the lung may be a viable therapeutic strategy for the treatment of critical lung inflammatory illnesses.","version":"1.1","doi":"10.1101/2022.01.11.475918","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447760","pub_date":"2022-1-12","title":"Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.","version":"1.2","doi":"10.1101/2021.06.09.447760","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475768","pub_date":"2022-1-11","title":"SARS-CoV-2 Point Mutation and Deletion Spectra, and Their Association with Different Disease Outcome","abstract":"Mutant spectra of RNA viruses are important to understand viral pathogenesis, and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultra-deep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of thirty nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity, and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.10.475768","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475725","pub_date":"2022-1-11","title":"A single-cell atlas reveals shared and distinct immune responses and metabolism during SARS-CoV-2 and HIV-1 infections","abstract":"SARS-CoV-2 and HIV-1 are RNA viruses that have killed millions of people worldwide. Understanding the similarities and differences between these two infections is critical for understanding disease progression and for developing effective vaccines and therapies, particularly for 38 million HIV-1+ individuals who are vulnerable to SARS-CoV-2 co-infection. Here, we utilized single-cell transcriptomics to perform a systematic comparison of 94,442 PBMCs from 7 COVID-19 and 9 HIV-1+ patients in an integrated immune atlas, in which 27 different cell types were identified using an accurate consensus single-cell annotation method. While immune cells in both cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activities, and downregulated mitophagy. Our results elucidate transcriptional signatures associated with COVID-19 and HIV-1 that may reveal insights into fundamental disease biology and potential therapeutic targets to treat these viral infections. COVID-19 and HIV-1+ patients show disease-specific inflammatory immune signatures COVID-19 patients show more productive humoral responses than HIV-1+ patients SARS-CoV-2 elicits more enriched IFN-I signaling relative to HIV-I Divergent, impaired metabolic programs distinguish SARS-CoV-2 and HIV-1 infections","version":"1.1","doi":"10.1101/2022.01.10.475725","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475820","pub_date":"2022-1-11","title":"SARS-CoV-2 Triggers Complement Activation through Interactions with Heparan Sulfate","abstract":"The complement system has been heavily implicated in severe COVID-19 with clinical studies revealing widespread gene induction, deposition, and activation. However, the mechanism by which complement is activated in this disease remains incompletely understood. Herein we examined the relationship between SARS-CoV-2 and complement by inoculating the virus in lepirudin-anticoagulated human blood. This caused progressive C5a production after 30 minutes and 24 hours, which was blocked entirely by inhibitors for factor B, C3, C5, and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface deposition of complement and interactions with heparan sulfate. Additional functional analysis revealed that complement-dependent granulocyte and monocyte activation was delayed. Indeed, C5aR1 internalisation and CD11b upregulation on these cells only occurred after 24 hours. Thus, SARS-CoV-2 is a non-canonical complement activator that triggers the alternative pathway through interactions with heparan sulfate.","version":"1.1","doi":"10.1101/2022.01.11.475820","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.435594","pub_date":"2022-1-11","title":"Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants of concern (VOC), such as Delta (B.1.617.2) and Omicron (B.1.1.529), has continued to drive the worldwide pandemic. Therefore, there is a high demand for vaccines with enhanced efficacy, high thermostability, superior design flexibility, and fast manufacturing speed. Here, we report a circular RNA (circRNA) vaccine that encodes the trimeric RBD of SARS-CoV-2 Spike protein. Without the need of nucleotide modification, 5\u2019-capping or 3\u2019-polyadenylation, circRNA could be rapidly produced via in vitro transcription and is highly thermostable whether stored in naked or lipid-nanoparticle (LNP)-encapsulated format. LNP-encapsulated circRNARBD elicited potent neutralizing antibodies and T cell responses, providing robust protection against Beta (B.1.351) and native viruses in mice and rhesus macaques, respectively. Notably, circRNA vaccine enabled higher and more durable antigen production than 1m\u03a8-modified mRNA vaccine, eliciting a higher proportion of neutralizing antibodies and stronger Th1-biased immune responses. Importantly, we found that circRNARBD-Omicron vaccine induced effective neutralizing antibodies against only Omicron but not Delta variant. By contrast, circRNARBD-Delta could elicit high level of neutralizing antibodies against both Delta and Omicron. Following two doses of either native- or Delta-specific vaccination, circRNARBD-Delta, but not Omicron or Beta vaccines, could effectively boost the neutralizing antibodies against both Delta and Omicron variants. These results suggest that circRNARBD-Delta is a favorable choice for vaccination to provide a broad-spectrum protection against the current variants of concern of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.03.16.435594","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.472066","pub_date":"2022-1-11","title":"Structure of the 5\u2019 untranslated region in SARS-CoV-2 genome and its specific recognition by innate immune system via the human oligoadenylate synthase 1\u2020","abstract":"2\u2019-5\u2019-Oligoadenylate synthetase 1 (OAS1) is one of the key enzymes driving the innate immune system response to SARS-CoV-2 infection whose activity has been related to COVID-19 severity. In particular, OAS1 is a sensor of endogenous RNA that triggers the 2\u2019-5\u2019 oligoadenylate/RNase L pathway in response to viral infections, ultimately activating the RNA-Lyase which cleaves endogenous and exogenous RNA impeding the viral maturation. Upon SARS-CoV-2 infection, OAS1 is responsible for the recognition of viral RNA and has been shown to possess a particularly high sensitivity for the 5\u2019-untranslated (5\u2019-UTR) RNA region, which is organized in a double-strand stem loop motif (SLA). Since the structure of the nucleic acid/protein complex has not been resolved, here we report its structure obtained by molecular modeling, including enhanced sampling approaches. We also pinpoint that the SL1 region enhances the interaction network with the enzyme, promoting specific hydrogen bonds, absent in normal double strand RNA fragments, hence rationalizing the high affinity for OAS1.","version":"1.2","doi":"10.1101/2021.12.10.472066","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.11.475898","pub_date":"2022-1-11","title":"Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis","abstract":"Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.","version":"1.1","doi":"10.1101/2022.01.11.475898","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475443","pub_date":"2022-1-10","title":"Evaluation of an optimized protocol and Illumina ARTIC V4 primer pool for sequencing of SARS-CoV-2 using COVIDSeq\u2122 and DRAGEN\u2122 COVID Lineage App workflow","abstract":"Next-Generation Sequencing based genomic surveillance has been widely implemented for identification and tracking of emerging SARS-CoV-2 variants to guide the Public Health response to the COVID-19 pandemic. Amplicon-based assays, such as the Illumina\u00ae COVIDSeq\u2122 Test (RUO) and COVIDSeq Assay (RUO), enable scalable sequencing of SARS-CoV-2, leveraging V3 and V4 primer designs from the ARTIC community and DRAGEN\u2122 COVID Lineage App analysis available on Illumina BaseSpace\u2122. We report here a comparison of COVIDSeq performance for SARS-CoV-2 genome reporting using the ARTIC V3 based primer pool (including primers for human control genes) that is provided with the COVIDSeq kit versus the ARTIC V4 based Illumina COVIDSeq V4 primer pool, using an optimized protocol and DRAGEN COVID Lineage App analysis. The data indicates that both primer pools enable robust reporting of SARS-CoV-2 variants. The Illumina COVIDSeq V4 primer pool has superior performance for SARS-CoV-2 genome reporting, particularly in samples with low virus load, and is therefore the recommended primer pool for genomic surveillance of SARS-CoV-2 for research use using COVIDSeq.","version":"1.1","doi":"10.1101/2022.01.07.475443","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475453","pub_date":"2022-1-10","title":"Mild respiratory SARS-CoV-2 infection can cause multi-lineage cellular dysregulation and myelin loss in the brain","abstract":"Survivors of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection frequently experience lingering neurological symptoms, including impairment in attention, concentration, speed of information processing and memory. This long-COVID cognitive syndrome shares many features with the syndrome of cancer therapy-related cognitive impairment (CRCI). Neuroinflammation, particularly microglial reactivity and consequent dysregulation of hippocampal neurogenesis and oligodendrocyte lineage cells, is central to CRCI. We hypothesized that similar cellular mechanisms may contribute to the persistent neurological symptoms associated with even mild SARS-CoV-2 respiratory infection. Here, we explored neuroinflammation caused by mild respiratory SARS-CoV-2 infection \u2013 without neuroinvasion - and effects on hippocampal neurogenesis and the oligodendroglial lineage. Using a mouse model of mild respiratory SARS-CoV-2 infection induced by intranasal SARS-CoV-2 delivery, we found white matter-selective microglial reactivity, a pattern observed in CRCI. Human brain tissue from 9 individuals with COVID-19 or SARS-CoV-2 infection exhibits the same pattern of prominent white matter-selective microglial reactivity. In mice, pro-inflammatory CSF cytokines/chemokines were elevated for at least 7-weeks post-infection; among the chemokines demonstrating persistent elevation is CCL11, which is associated with impairments in neurogenesis and cognitive function. Humans experiencing long-COVID with cognitive symptoms (48 subjects) similarly demonstrate elevated CCL11 levels compared to those with long-COVID who lack cognitive symptoms (15 subjects). Impaired hippocampal neurogenesis, decreased oligodendrocytes and myelin loss in subcortical white matter were evident at 1 week, and persisted until at least 7 weeks, following mild respiratory SARS-CoV-2 infection in mice. Taken together, the findings presented here illustrate striking similarities between neuropathophysiology after cancer therapy and after SARS-CoV-2 infection, and elucidate cellular deficits that may contribute to lasting neurological symptoms following even mild SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2022.01.07.475453","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.22268729","pub_date":"2022-01-10","title":"Reconstructing transmission chains of SARS-CoV-2 amid multiple outbreaks in a geriatric acute-care hospital","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>There is ongoing uncertainty regarding transmission chains and the respective roles of healthcare workers (HCWs) and elderly patients in nosocomial outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in geriatric settings.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We performed a retrospective cohort study including patients with nosocomial coronavirus disease 2019 (COVID-19) in four outbreak-affected wards, and all SARS-CoV-2 RT-PCR positive HCWs from a Swiss university-affiliated geriatric acute-care hospital that admitted both Covid-19 and non-Covid-19 patients during the first pandemic wave in Spring 2020. We combined epidemiological and genetic sequencing data using a Bayesian modelling framework, and reconstructed transmission dynamics of SARS-CoV-2 involving patients and HCWs, in order to determine who infected whom. We evaluated general transmission patterns according to type of case (HCWs working in dedicated Covid-19 cohorting wards: HCW\n                    <jats:sub>covid</jats:sub>\n                    ; HCWs working in non-Covid-19 wards where outbreaks occurred: HCW\n                    <jats:sub>outbreak</jats:sub>\n                    ; patients with nosocomial Covid-19: patient\n                    <jats:sub>noso</jats:sub>\n                    ) by deriving the proportion of infections attributed to each type of case across all posterior trees and comparing them to random expectations.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    During the study period (March 1 to May 7, 2020) we included 180 SARS-CoV-2 positive cases: 127 HCWs (91 HCW\n                    <jats:sub>covid</jats:sub>\n                    , 36 HCW\n                    <jats:sub>outbreak</jats:sub>\n                    ) and 53 patients. The attack rates ranged from 10-19% for patients, and 21% for HCWs. We estimated that there were 16 importation events (3 patients, 13 HCWs) that jointly led to 16 secondary cases. Most patient-to-patient transmission events involved patients having shared a ward (97.6%, 95% credible interval [CrI] 90.4-100%), in contrast to those having shared a room (44.4%, 95%CrI 27.8-62.5%). Transmission events tended to cluster by type of case: patient\n                    <jats:sub>noso</jats:sub>\n                    were almost twice as likely to be infected by other patient\n                    <jats:sub>noso</jats:sub>\n                    than expected (observed:expected ratio 1.91, 95%CrI 1.08 \u2013 4.00,\n                    <jats:bold>p</jats:bold>\n                    = 0.02); similarly, HCW\n                    <jats:sub>outbreak</jats:sub>\n                    were more than twice as likely to be infected by other HCW\n                    <jats:sub>outbreak</jats:sub>\n                    than expected (2.25, 95%CrI 1.00-8.00,\n                    <jats:bold>p</jats:bold>\n                    = 0.04). The proportion of infectors of HCW\n                    <jats:sub>covid</jats:sub>\n                    were as expected as random. The proportions of high transmitters (\u22652 secondary cases) were significantly higher among HCW\n                    <jats:sub>outbreak</jats:sub>\n                    than patient\n                    <jats:sub>noso</jats:sub>\n                    in the late phases (26.2% vs. 13.4%, p&lt;2.2e-16) of the outbreak.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    Most importation events were linked to HCW. Unexpectedly, transmission between HCW\n                    <jats:sub>covid</jats:sub>\n                    was more limited than transmission between patients and HCW\n                    <jats:sub>outbreak</jats:sub>\n                    . This highlights gaps in infection control and suggests possible areas of improvements to limit the extent of nosocomial transmission.\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.01.07.22268729","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.10.475377","pub_date":"2022-1-10","title":"Molnupiravir combined with different repurposed drugs further inhibits SARS-CoV-2 infection in human nasal epithelium in vitro","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in late 2019, has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available, and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show high clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new virus variants, and generally short duration of immunity, the development of safe and effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing and approved drugs for accelerated clinical testing and potential emergency use authorizations. However, drug-repurposing using high throughput screenings in cellular assays, often identify hits that later prove ineffective in clinical studies. Our approach was to evaluate the activity of compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Secondly, we evaluated drug combinations using sub-maximal doses of each active single compound. Here, we report the antiviral effects of 95 single compounds and 30 combinations. The data show that selected drug combinations including 10 \u03bcM of molnupiravir, a viral RNA-dependent RNA polymerase (RdRp) inhibitor, effectively inhibit SARS-CoV-2 replication. This indicates that such combinations are worthy of further evaluation as potential treatment strategies against coronavirus disease 2019 (COVID-19).","version":"1.1","doi":"10.1101/2022.01.10.475377","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475330","pub_date":"2022-1-10","title":"RelCoVax\u00ae, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice","abstract":"The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax\u00ae through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax\u00ae formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-\u03b3 release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax\u00ae demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax\u00ae through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.07.475330","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475620","pub_date":"2022-1-10","title":"Strong SARS-CoV-2 N-specific CD8+ T immunity induced by engineered extracellular vesicles associates with protection from lethal infection in mice","abstract":"SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut- based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.","version":"1.1","doi":"10.1101/2022.01.10.475620","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475248","pub_date":"2022-1-10","title":"Fusogenicity and neutralization sensitivity of the SARS-CoV-2 Delta sublineage AY.4.2","abstract":"SARS-CoV-2 lineages are continuously evolving. As of December 2021, the AY.4.2 Delta sub-lineage represented 20 % of sequenced strains in UK and has been detected in dozens of countries. It has since then been supplanted by the Omicron variant. AY.4.2 displays three additional mutations (T95I, Y145H and A222V) in the N-terminal domain (NTD) of the spike when compared to the original Delta variant (B.1.617.2) and remains poorly characterized. Here, we analyzed the fusogenicity of the AY.4.2 spike and the sensitivity of an authentic AY.4.2 isolate to neutralizing antibodies. The AY.4.2 spike exhibited similar fusogenicity and binding to ACE2 than Delta. The sensitivity of infectious AY.4.2 to a panel of monoclonal neutralizing antibodies was similar to Delta, except for the anti-RBD Imdevimab, which showed incomplete neutralization. Sensitivity of AY.4.2 to sera from individuals having received two or three doses of Pfizer or two doses of AstraZeneca vaccines was reduced by 1.7 to 2.1 fold, when compared to Delta. Our results suggest that mutations in the NTD remotely impair the efficacy of anti-RBD antibodies. The temporary spread of AY.4.2 was not associated with major changes in spike function but rather to a partially reduced neutralization sensitivity.","version":"1.1","doi":"10.1101/2022.01.07.475248","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.10.475532","pub_date":"2022-1-10","title":"Molecular basis of SARS-CoV-2 Omicron variant receptor engagement and antibody evasion and neutralization","abstract":"The SARS-CoV-2 Omicron variant exhibits striking immune evasion and is spreading globally at an unprecedented speed. Understanding the underlying structural basis of the high transmissibility and greatly enhanced immune evasion of Omicron is of high importance. Here through cryo-EM analysis, we present both the closed and open states of the Omicron spike, which appear more compact than the counterparts of the G614 strain, potentially related to the Omicron substitution induced enhanced protomer-protomer and S1-S2 interactions. The closed state showing dominant population may indicate a conformational masking mechanism of immune evasion for Omicron spike. Moreover, we capture two states for the Omicron S/ACE2 complex with S binding one or two ACE2s, revealing that the substitutions on the Omicron RBM result in new salt bridges/H-bonds and more favorable electrostatic surface properties, together strengthened interaction with ACE2, in line with the higher ACE2 affinity of the Omicron relative to the G614 strain. Furthermore, we determine cryo-EM structures of the Omicron S/S3H3 Fab, an antibody able to cross-neutralize major variants of concern including Omicron, elucidating the structural basis for S3H3-mediated broad-spectrum neutralization. Our findings shed new lights on the high transmissibility and immune evasion of the Omicron variant and may also inform design of broadly effective vaccines against emerging variants.","version":"1.1","doi":"10.1101/2022.01.10.475532","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475406","pub_date":"2022-1-08","title":"Comprehensive analysis of disease pathology in immunocompetent and immunocompromised hamster models of SARS-CoV-2 infection","abstract":"The pathogenesis of SARS-CoV-2 in the context of a specific immunological niche is not fully understood. Here, we used a golden Syrian hamster model to systematically evaluate the kinetics of host response to SARS-CoV-2 infection, following disease pathology, viral loads, antibody responses, and inflammatory cytokine expression in multiple organs. The kinetics of SARS-CoV-2 pathogenesis and genomewide lung transcriptome was also compared between immunocompetent and immunocompromised hamsters. We observed that the body weight loss was proportional to the SARS-CoV-2 infectious dose and lasted for a short time only in immunocompetent hamsters. Body weight loss was more prominent and prolonged in infected immunocompromised hamsters. While the kinetics of viral replication and peak live viral loads were not significantly different at low and high infectious doses (LD and HD), the HD-infected immunocompetent animals developed severe lung disease pathology. The immunocompetent animals cleared the live virus in all tested tissues by 12 days post-infection and generated a robust serum antibody response. In contrast, immunocompromised hamsters mounted an inadequate SARS-CoV-2 neutralizing antibody response, and the virus was detected in the pulmonary and multiple extrapulmonary organs until 16 days post-infection. These hamsters also had prolonged moderate inflammation with severe bronchiolar-alveolar hyperplasia/metaplasia. Consistent with the difference in disease presentation, distinct changes in the expression of inflammation and immune cell response pathways and network genes were seen in the lungs of infected immunocompetent and immunocompromised animals. This study highlights the interplay between the kinetics of viral replication and the dynamics of SARS-CoV-2 pathogenesis at organ-level niches and maps how COVID-19 symptoms vary in different immune contexts. Together, our data suggest that the histopathological manifestations caused by progressive SARS-CoV-2 infection may be a better predictor of COVID-19 severity than individual measures of viral load, antibody response, and cytokine storm at the systemic or local (lungs) levels in the immunocompetent and immunocompromised hosts.","version":"1.1","doi":"10.1101/2022.01.07.475406","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.06.475282","pub_date":"2022-1-07","title":"Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues","abstract":"SARS-CoV-2 primarily replicates in mucosal sites, and more information is needed about immune responses in infected tissues. We used rhesus macaques to model protective primary immune responses in tissues during mild COVID-19. Viral RNA levels were highest on days 1-2 post-infection and fell precipitously thereafter. 18F-fluorodeoxyglucose (FDG)-avid lung abnormalities and interferon (IFN)-activated myeloid cells in the bronchoalveolar lavage (BAL) were found on days \u223c3-4. Virus-specific effector CD8 and CD4 T cells were detectable in the BAL and lung tissue on days \u223c7-10, after viral RNA, lung inflammation, and IFN-activated myeloid cells had declined. Notably, SARS-CoV-2-specific T cells were not detectable in the nasal turbinates, salivary glands, and tonsils on day 10 post-infection. Thus, SARS-CoV-2 replication wanes in the lungs prior to T cell responses, and in the nasal and oral mucosa despite the apparent lack of Ag-specific T cells, suggesting that innate immunity efficiently restricts viral replication during mild COVID-19. SARS-CoV-2 infection leads to mild, focal lung inflammation, and type I IFN activated myeloid cells that mostly resolve prior to the influx of virus-specific effector T cells or antibody responses in rhesus macaques.","version":"1.1","doi":"10.1101/2022.01.06.475282","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.05.475172","pub_date":"2022-1-07","title":"High seroprevalence of SARS-CoV-2 in white-tailed deer (Odocoileus virginianus) at one of three captive cervid facilities in Texas","abstract":"Free-ranging white-tailed deer (Odocoileus virginanus) across the United States are increasingly recognized as involved in SARS-CoV-2 transmission cycles. Through a cross-sectional study of 80 deer at three captive cervid facilities in central and southern Texas, we provide evidence of 34 of 36 (94.4%) white-tailed deer at a single captive cervid facility seropositive for SARS-CoV-2 by neutralization assay (PRNT90), with endpoint titers as high as 1280. In contrast, all tested white-tailed deer and axis deer (Axis axis) at two other captive cervid facilities were seronegative, and SARS-CoV-2 RNA was not detected in respiratory swabs from deer at any of the three facilities. These data support transmission among captive deer that cannot be explained by human contact for each infected animal, as only a subset of the seropositive does had direct human contact. The facility seroprevalence was more than double of that reported from wild deer, suggesting that the confined environment may facilitate transmission. Further exploration of captive cervids and other managed animals for their role in the epizootiology of SARS-CoV-2 is critical for understanding impacts on animal health and the potential for spillback transmission to humans or other animal taxa. As SARS-CoV-2 vaccine coverge of the human population increases and variants of concern continue to emerge, identification of the epidemiologic importance of animal virus reservoirs is critical. We found that nearly all (94.4%) of the captive white-tailed deer at a cervid facility in central Texas had neutralizing antibodies for SARS-CoV-2. This seroprevalence is over double than that which has been reported from free-ranging deer from other regions of the US. Horizontal transmission among deer may be facilitated in confinement. Tracking new infections among wild and confined deer is critical for understanding the importance of animal reservoirs for both veterinary and human health.","version":"1.1","doi":"10.1101/2022.01.05.475172","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465272","pub_date":"2022-1-07","title":"Molecular Insights into the Differential Dynamics of SARS-CoV-2 Variants of Concern (VOC)","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected the lives and livelihood of millions of individuals around the world. It has mutated several times after its first inception, with an estimated two mutations occurring every month. Although we have been successful in developing vaccines against the virus, emergence of variants has enabled it to escape therapy. Few of the generated variants are also reported to be more infectious than the wild-type (WT). In this study, we analyze the attributes of all RBD/ACE2 complexes for the reported VOCs, namely, Alpha, Beta, Gamma, and Delta through computer simulations. Results indicate differences in orientation and binding energies of the VOCs from the WT. Overall, it was observed that electrostatic interactions play a major role in the binding of the complexes. Detailed residue level energetics revealed that the most prominent changes in interaction energies were seen particularly at the mutated residues which were present at RBD/ACE2 interface. We found that the Delta variant is one of the most tightly bound variants of SARS-CoV-2 with dynamics similar to WT. High binding affinity of RBD towards ACE2 is indicative of an increase in the viral transmission and infectivity. The details presented in our study would prove extremely useful for the design and development of effective therapeutic strategies for the emerging variants of the virus.","version":"1.2","doi":"10.1101/2021.10.22.465272","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.06.475303","pub_date":"2022-1-07","title":"Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape","abstract":"The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.","version":"1.1","doi":"10.1101/2022.01.06.475303","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.07.475295","pub_date":"2022-1-07","title":"Covariant Fitness Clusters Reveal Structural Evolution of SARS-CoV-2 Polymerase Across the Human Population","abstract":"Understanding the fitness landscape of viral mutations is crucial for uncovering the evolutionary mechanisms contributing to pandemic behavior. Here, we apply a Gaussian process regression (GPR) based machine learning approach that generates spatial covariance (SCV) relationships to construct stability fitness landscapes for the RNA-dependent RNA polymerase (RdRp) of SARS- CoV-2. GPR generated fitness scores capture on a residue-by-residue basis a covariant fitness cluster centered at the C487-H642-C645-C646 Zn2+ binding motif that iteratively evolves since the early phase pandemic. In the Alpha and Delta variant of concern (VOC), multi-residue SCV interactions in the NiRAN domain form a second fitness cluster contributing to spread. Strikingly, a novel third fitness cluster harboring a Delta VOC basal mutation G671S augments RdRp structural plasticity to potentially promote rapid spread through viral load. GPR principled SCV provides a generalizable tool to mechanistically understand evolution of viral genomes at atomic resolution contributing to fitness at the pathogen-host interface.","version":"1.1","doi":"10.1101/2022.01.07.475295","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.29.462344","pub_date":"2022-1-06","title":"Protection of Hamsters Challenged with SARS-CoV-2 Variants of Concern by Two Doses of MVC-COV1901 Vaccine Followed by a Single Dose of Beta Variant Version of MVC-COV1901","abstract":"The current fight against COVID-19 is compounded by the Variants of Concern (VoCs), which can diminish the effectiveness of vaccines and potentially increase viral transmission and severity of disease. MVC-COV1901 is a protein subunit vaccine based on the prefusion SARS-CoV-2 spike protein (S-2P) and is adjuvanted with CpG 1018 and aluminum hydroxide. In this study, we used the Delta variant to challenge hamsters inoculated with S-2P from the Wuhan wildtype and the Beta variant in two-dose or three-dose regimens. Two doses of wildtype S-2P followed by the third dose of Beta variant was shown to induce the highest neutralizing antibody titer against live SARS-CoV-2 of the wildtype and all current VoCs, as well as improved neutralization against Omicron variant pseudovirus compared to three doses of wildtype S-P. All regimens of vaccination were able to protect hamsters from SARS-CoV-2 Delta variant challenge and resulted in reduced lung live virus titer and pathology. Three doses of vaccination also significantly reduced lung viral RNA titer, regardless of whether the wildtype or Beta variant S-2P was used as the third dose. Based on the immunogenicity and viral challenge data, two doses of wildtype S-2P followed by the third dose of Beta variant S-2P induced potent antibody immune responses against the VoCs.","version":"1.3","doi":"10.1101/2021.09.29.462344","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.06.22268835","pub_date":"2022-01-06","title":"An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low- and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely \u201cIFAST-CRISPR\u201d, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed \u201csample-to-answer\u201d platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of \u2265 470 copies mL\n                  <jats:sup>-1</jats:sup>\n                  genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL\n                  <jats:sup>-1</jats:sup>\n                  of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.\n                </jats:p>","version":null,"doi":"10.1101/2022.01.06.22268835","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.03.458953","pub_date":"2022-1-06","title":"An Extended Motif in the SARS-CoV-2 Spike Modulates Binding and Release of Host Coatomer in Retrograde Trafficking","abstract":"\u03b2-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for \u03b1COPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. \u03b1COPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.","version":"1.2","doi":"10.1101/2021.09.03.458953","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.475015","pub_date":"2022-1-05","title":"SARS-CoV-2 Infection of Microglia Elicits Pro-inflammatory Activation and Apoptotic Cell Death","abstract":"Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in coronavirus disease 2019 (COVID-19) patients. The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we report that SARS-CoV-2 can directly infect human microglia, eliciting M1-like pro-inflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA-seq analysis also revealed that ER stress and immune responses were induced in the early and apoptotic processes in the late phase of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced pro-inflammatory cytokines such as interleukin (IL)-1\u03b2, IL-6, and tumour necrosis factor \u03b1 (TNF-\u03b1), but not the anti-inflammatory cytokine IL-10. After this pro-inflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of pro-inflammatory microglial IL-6 and TNF-\u03b1 and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in COVID-19 patients. Recent studies reported neurological manifestations and complications in COVID-19 patients, which are associated with neuroinflammation. As microglia are the dominant immune cells in brains, it needs to be elucidate the relationship between neuroinflammation and host immune response of microglia to SARS-CoV-2. Here, we suggest that SARS-CoV-2 can directly infect human microglia with cytopathic effect (CPE) using human microglial clone 3 (HMC3). The infected microglia were promoted to pro-inflammatory activation following apoptotic cell death. This pro-inflammatory activation was accompanied by the high production of pro-inflammatory cytokines, and led to neurotoxic-M1 phenotype polarization. In vivo, murine microglia were infected and produced pro-inflammatory cytokines and provoked a chronic loss using K18-hACE2 mice. Thus, our data present that SARS-CoV-2-infected microglia are potential mediators of neurological problems in COVID-19 patients. In addition, HMC3 cells are susceptible to SARS-CoV-2 and exhibit the CPE, which can be further used to investigate cellular and molecular mechanisms of neuroinflammation reported in COVID-19 patients.","version":"1.1","doi":"10.1101/2022.01.04.475015","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469906","pub_date":"2022-1-05","title":"Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines","abstract":"The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of many new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccines. To ascertain and rank the risk of VOCs and VOIs, we analyzed their ability to escape from vaccine-induced antibodies. The variants showed differential reductions in neutralization and replication titers by post-vaccination sera. Although the Omicron variant showed the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retained moderate neutralizing activity against that variant. Therefore, vaccination remains the most effective strategy to combat the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2021.11.24.469906","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.05.475095","pub_date":"2022-1-05","title":"Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2","abstract":"The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 \u00b5M and 66 \u00b5M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 \u00b5M to 46 \u00b5M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.","version":"1.1","doi":"10.1101/2022.01.05.475095","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474979","pub_date":"2022-1-05","title":"SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein","abstract":"SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.","version":"1.1","doi":"10.1101/2022.01.04.474979","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.05.474231","pub_date":"2022-1-05","title":"Genomic Perspectives on the Emerging SARS-CoV-2 Omicron Variant","abstract":"A new variant of concern for SARS-CoV-2, Omicron (B.1.1.529), was designated by the World Health Organization on November 26, 2021. This study analyzed the viral genome sequencing data of 108 samples collected from patients infected with Omicron. First, we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to. Second, the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the same time (62 vs. 45), especially in the Spike gene. Mutations in the Spike gene confer alterations in 32 amino acid residues, which was more than those observed in other SARS-CoV-2 variants. Moreover, a large number of nonsynonymous mutations occur in the codons for the amino acid residues located on the surface of the Spike protein, which could potentially affect the replication, infectivity, and antigenicity of SARS-CoV-2. Third, there are 53 mutations between the Omicron variant and its closest sequences available in public databases. Many of those mutations were rarely observed in the public database and had a low mutation rate. In addition, the linkage disequilibrium between these mutations were low, with a limited number of mutations (6) concurrently observed in the same genome, suggesting that the Omicron variant would be in a different evolutionary branch from the currently prevalent variants. To improve our ability to detect and track the source of new variants rapidly, it is imperative to further strengthen genomic surveillance and data sharing globally in a timely manner.","version":"1.1","doi":"10.1101/2022.01.05.474231","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.05.475037","pub_date":"2022-1-05","title":"An elite broadly neutralizing antibody protects SARS-CoV-2 Omicron variant challenge","abstract":"The strikingly high transmissibility and antibody evasion of SARS-CoV-2 Omicron variant have posted great challenges on the efficacy of current vaccines and antibody immunotherapy.Here, we screened 34 BNT162b2-vaccinees and cloned a public broadly neutralizing antibody (bNAb) ZCB11 from an elite vaccinee. ZCB11 neutralized all authentic SARS-CoV-2 variants of concern (VOCs), including Omicron and OmicronR346K with potent IC50 concentrations of 36.8 and 11.7 ng/mL, respectively. Functional analysis demonstrated that ZCB11 targeted viral receptor-binding domain (RBD) and competed strongly with ZB8, a known RBD-specific class II NAb. Pseudovirus-based mapping of 57 naturally occurred single mutations or deletions revealed that only S371L resulted in 11-fold neutralization resistance, but this phenotype was not observed in the Omicron variant. Furthermore,prophylactic ZCB11 administration protected lung infection against both the circulating pandemic Delta and Omicron variants in golden Syrian hamsters. These results demonstrated that vaccine-induced ZCB11 is a promising bNAb for immunotherapy against pandemic SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2022.01.05.475037","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474958","pub_date":"2022-1-05","title":"Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants","abstract":"A highly efficient and robust multiple scales in silico protocol, consisting of atomistic constant charge Molecular Dynamics (MD), constant-charge coarse-grain (CG) MD and constant-pH CG Monte Carlo (MC), has been used to study the binding affinities, the free energy of complexation of selected antigen-binding fragments of the monoclonal antibody (mAbs) CR3022 (originally derived from SARS-CoV-1 patients almost two decades ago) and 11 SARS-CoV-2 variants including the wild type. CR3022 binds strongly to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, but chooses a different site rather than the receptor-binding motif (RBM) of RBD, allowing its combined use with other mAbs against new emerging virus variants. Totally 235,000 mAbs structures were generated using the RosettaAntibodyDesign software, resulting in top 10 scored CR3022-RBD complexes with critical mutations and compared to the native one, all having the potential to block virus-host cell interaction. Of these 10 finalists, two candidates were further identified in the CG simulations to be clearly best against all virus variants, and surprisingly, all 10 candidates and the native CR3022 did exhibit a higher affinity for the Omicron variant with its highest number of mutations (15) of them all considered in this study. The multiscale protocol gives us a powerful rational tool to design efficient mAbs. The electrostatic interactions play a crucial role and appear to be controlling the affinity and complex building. Clearly, mAbs carrying a lower net charge show a higher affinity. Structural determinants could be identified in atomistic simulations and their roles are discussed in detail to further hint at a strategy towards designing the best RBD binder. Although the SARS-CoV-2 was specifically targeted in this work, our approach is generally suitable for many diseases and viral and bacterial pathogens, leukemia, cancer, multiple sclerosis, rheumatoid, arthritis, lupus, and more.","version":"1.1","doi":"10.1101/2022.01.04.474958","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.475011","pub_date":"2022-1-05","title":"Computational study of the furin cleavage domain of SARS-CoV-2: delta binds strongest of extant variants","abstract":"We demonstrate that AlphaFold and AlphaFold Multimer, implemented within the ColabFold suite, can accurately predict the structures of the furin enzyme with known six residue inhibitory peptides. Noting the similarity of the peptide inhibitors to polybasic furin cleavage domain insertion region of the SARS-CoV-2, which begins at P681, we implement this approach to study the wild type furin cleavage domain for the virus and several mutants. We introduce mutations in silico for alpha, omicron, and delta variants, for several sequences which have been rarely observed, for sequences which have not yet been observed, for other coronaviruses (NL63, OC43, HUK1a, HUK1b, MERS, and 229E), and for the H5N1 flu. We show that interfacial hydrogen bonds between the furin cleavage domain and furin are a good measure of binding strength that correlate well with endpoint binding free energy estimates, and conclude that among all candidate viral sequences studied, delta is near the very top binding strength within statistical accuracy. However, the binding strength of several rare sequences match delta within statistical accuracy. We find that the furin S1 pocket is optimized for binding arginine as opposed to lysine. This residue, typically at sequence position five, contains the most hydrogen bonds to the furin, and hydrogen bond count for just this residue shows a strong positive correlation with the overall hydrogen bond count. We demonstrate that the root mean square backbone C-alpha fluctuation of the first residue in the furin cleavage domain has a strong negative correlation with the interfacial hydrogen bond count. We show by considering the variation with the number of basic residues that the maximum mean number of interfacial hydrogen bonds expected is 15.7 at 4 basic residues.","version":"1.1","doi":"10.1101/2022.01.04.475011","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474974","pub_date":"2022-1-05","title":"Sequence assignment validation in cryo-EM models with checkMySequence","abstract":"The availability of new AI-based protein structure prediction tools radically changed the way cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register-shifts remain one of the most difficult to identify and correct. Here we introduce checkMySequence; a fast, fully automated and parameter-free method for detecting register-shifts in protein models built into cryo-EM maps. We show that the method can assist model building in cases where poorer map resolution hinders visual interpretation. We also show that checkMySequence could have helped avoid a widely discussed sequence register error in a model of SARS-CoV-2 RNA-dependent RNA polymerase that was originally detected thanks to a visual residue-by-residue inspection by members of the structural biology community. We present a new method, checkMySequence, for fast and automated detection of register errors in protein models built into cryo-EM reconstructions.","version":"1.1","doi":"10.1101/2022.01.04.474974","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.01.474713","pub_date":"2022-1-04","title":"The SARS-CoV-2 infection in Thailand: analysis of spike variants complemented by protein structure insights","abstract":"Thailand was the first country outside China to officially report COVID-19 cases. Despite the strict regulations for international arrivals, up until February 2021, Thailand had been hit by two major outbreaks. With a large number of SARS-CoV-2 sequences collected from patients, the effects of many genetic variations, especially those unique to Thai strains, are yet to be elucidated. In this study, we analysed 439,197 sequences of the SARS-CoV-2 spike protein collected from NCBI and GISAID databases. 595 sequences were from Thailand and contained 52 variants, of which 6 had not been observed outside Thailand (p.T51N, p.P57T, p.I68R, p.S205T, p.K278T, p.G832C). These variants were not predicted to be of concern. We demonstrate that the p.D614G, although already present during the first Thai outbreak, became the prevalent strain during the second outbreak, similarly to what was described in other countries. Moreover, we show that the most common variants detected in Thailand (p.A829T, p.S459F and p.S939F) do not appear to cause any major structural change to the spike trimer or the spike-ACE2 interaction. Among the variants identified in Thailand was p.N501T. This variant, which involves an asparagine critical for spike-ACE2 binding, was not predicted to increase SARS-CoV-2 binding, thus in contrast to the variant of global concern p.N501Y. In conclusion, novel variants identified in Thailand are unlikely to increase the fitness of SARS-CoV-2. The insights obtained from this study could aid SARS-CoV-2 variants prioritisations and help molecular biologists and virologists working on strain surveillance.","version":"1.1","doi":"10.1101/2022.01.01.474713","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.28.470226","pub_date":"2022-1-04","title":"Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir (PF-07321332)","abstract":"The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that nirmatrelvir has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.","version":"1.2","doi":"10.1101/2021.11.28.470226","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474908","pub_date":"2022-1-04","title":"Maternal cytokine response after SARS-CoV-2 infection during pregnancy","abstract":"Dysregulation of the immune system during pregnancy is associated with adverse pregnancy outcomes. Recent studies report cytokine changes during the acute phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We examine whether there is a lasting association between SARS-CoV-2 infection during pregnancy and peripheral blood cytokine levels. We conducted a case-control study at the Mount Sinai health system in NYC including 100 SARS-CoV-2 IgG antibody positive people matched to 100 SARS-CoV-2 IgG antibody negative people on age, race/ethnicity, parity, and insurance status. Blood samples were collected at a median gestational age of 34 weeks. Levels of 14 cytokines were measured. Individual cytokine levels and cytokine cluster Eigenvalues did not differ significantly between groups, indicating no persisting maternal cytokine changes after SARS-CoV-2 infection during pregnancy. Our findings suggest that the acute inflammatory response after SARS-CoV-2 infection may be restored to normal values during pregnancy.","version":"1.1","doi":"10.1101/2022.01.04.474908","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.03.474773","pub_date":"2022-1-04","title":"Reduced interferon antagonism but similar drug sensitivity in Omicron variant compared to Delta variant SARS-CoV-2 isolates","abstract":"The SARS-CoV-2 Omicron variant is currently causing a large number of infections in many countries. A number of antiviral agents are approved or in clinical testing for the treatment of COVID-19. Despite the high number of mutations in the Omicron variant, we here show that Omicron isolates display similar sensitivity to eight of the most important anti-SARS-CoV-2 drugs and drug candidates (including remdesivir, molnupiravir, and PF-07321332, the active compound in paxlovid), which is of timely relevance for the treatment of the increasing number of Omicron patients. Most importantly, we also found that the Omicron variant displays a reduced capability of antagonising the host cell interferon response. This provides a potential mechanistic explanation for the clinically observed reduced pathogenicity of Omicron variant viruses compared to Delta variant viruses.","version":"1.1","doi":"10.1101/2022.01.03.474773","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474803","pub_date":"2022-1-04","title":"A synthetic bispecific antibody capable of neutralizing SARS-CoV-2 Delta and Omicron","abstract":"Bispecific antibodies have emerged as a promising strategy for curtailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape. This brief report highlights RBT-0813 (also known as TB493-04), a synthetic, humanized, receptor-binding domain (RBD)-targeted bispecific antibody that retains picomolar affinity to the Spike (S) trimers of all major variants of concern and neutralizes both SARS-CoV-2 Delta and Omicron in vitro.","version":"1.1","doi":"10.1101/2022.01.04.474803","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.30.474592","pub_date":"2022-1-04","title":"The runaway evolution of SARS-CoV-2 leading to the highly evolved Delta strain","abstract":"In new epidemics after the host shift, the pathogens may experience accelerated evolution driven by novel selective pressures. When the accelerated evolution enters a positive feedback loop with the expanding epidemics, the pathogen\u2019s runaway evolution may be triggered. To test this possibility in COVID-19, we analyze the extensive databases and identify 5 major waves of strains, one replacing the previous one in 2020 \u2013 2021. The mutations differ entirely between waves and the number of mutations continues to increase, from 3-4 to 21-31. The latest wave is the Delta strain which accrues 31 new mutations to become highly prevalent. Interestingly, these new mutations in Delta strain emerge in multiple stages with each stage driven by 6 \u2013 12 coding mutations that form a fitness group. In short, the evolution of SARS-CoV-2 from the oldest to the youngest wave, and from the earlier to the later stages of the Delta wave, is a process of acceleration with more and more mutations. The global increase in the viral population size (M(t), at time t) and the mutation accumulation (R(t)) may have indeed triggered the runaway evolution in late 2020, leading to the highly evolved Alpha and then Delta strain. To suppress the pandemic, it is crucial to break the positive feedback loop between M(t) and R(t), neither of which has yet to be effectively dampened by late 2021. New waves beyond Delta, hence, should not be surprising.","version":"1.1","doi":"10.1101/2021.12.30.474592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.435705","pub_date":"2022-1-04","title":"Yeast surface display-based identification of ACE2 mutations that modulate SARS-CoV-2 spike binding across multiple mammalian species","abstract":"Understanding how SARS-CoV-2 interacts with different mammalian angiotensin-converting enzyme II (ACE2) cell entry receptors elucidates determinants of virus transmission and facilitates development of vaccines for humans and animals. Yeast display-based directed evolution identified conserved ACE2 mutations that increase spike binding across multiple species. Gln42Leu increased ACE2-spike binding for human and four of four other mammalian ACE2s; Leu79Ile had a effect for human and three of three mammalian ACE2s. These residues are highly represented, 83% for Gln42 and 56% for Leu79, among mammalian ACE2s. The above findings can be important in protecting humans and animals from existing and future SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.03.16.435705","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.03.474855","pub_date":"2022-1-04","title":"Structural and computational insights into the SARS-CoV-2 Omicron RBD-ACE2 interaction","abstract":"Since SARS-CoV-2 Omicron variant (B.1.1.529) was reported in November 2021, it has quickly spread to many countries and outcompeted the globally dominant Delta variant in several countries. The Omicron variant contains the largest number of mutations to date, with 32 mutations located at spike (S) glycoprotein, which raised great concern for its enhanced viral fitness and immune escape[. In this study, we reported the crystal structure of the receptor binding domain (RBD) of Omicron variant S glycoprotein bound to human ACE2 at a resolution of 2.6 \u00c5. Structural comparison, molecular dynamics simulation and binding free energy calculation collectively identified four key mutations (S477N, G496S, Q498R and N501Y) for the enhanced binding of ACE2 by the Omicron RBD compared to the WT RBD. Representative states of the WT and Omicron RBD-ACE2 systems were identified by Markov State Model, which provides a dynamic explanation for the enhanced binding of Omicron RBD. The effects of the mutations in the RBD for antibody recognition were analyzed, especially for the S371L/S373P/S375F substitutions significantly changing the local conformation of the residing loop to deactivate several class IV neutralizing antibodies.","version":"1.1","doi":"10.1101/2022.01.03.474855","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.03.474825","pub_date":"2022-1-04","title":"Structural basis of Omicron neutralization by affinity-matured public antibodies","abstract":"The SARS-CoV-2 Omicron Variant of Concern (B.1.1.529) has spread rapidly in many countries. With a spike that is highly diverged from that of the pandemic founder, it escapes most available monoclonal antibody therapeutics and erodes vaccine protection. A public class of IGHV3-53-using SARS-CoV-2 neutralizing antibodies typically fails to neutralize variants carrying mutations in the receptor-binding motif, including Omicron. As antibodies from this class are likely elicited in most people following SARS-CoV-2 infection or vaccination, their subsequent affinity maturation is of particular interest. Here, we isolated IGHV3-53-using antibodies from an individual seven months after infection and identified several antibodies capable of broad and potent SARS-CoV-2 neutralization, extending to Omicron without loss of potency. By introducing select somatic hypermutations into a germline-reverted form of one such antibody, CAB-A17, we demonstrate the potential for commonly elicited antibodies to develop broad cross-neutralization through affinity maturation. Further, we resolved the structure of CAB-A17 Fab in complex with Omicron spike at an overall resolution of 2.6 \u00c5 by cryo-electron microscopy and defined the structural basis for this breadth. Thus, public SARS-CoV-2 neutralizing antibodies can, without modified spike vaccines, mature to cross-neutralize exceptionally antigenically diverged SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.01.03.474825","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.04.474916","pub_date":"2022-1-04","title":"S100A4 exerts robust mucosal adjuvant activity for co-administered antigens in mice","abstract":"The lack of clinically applicable mucosal adjuvants is a major hurdle in designing effective mucosal vaccines. We hereby report that the calcium-binding protein S100A4, which regulates a wide range of biological functions, is a potent mucosal adjuvant in mice for co-administered antigens, including the SARS-CoV-2 spike protein, with comparable or even superior efficacy as cholera toxin but without causing any adverse reactions. Intranasal immunization with recombinant S100A4 elicited antigen-specific antibody and pulmonary cytotoxic T cell responses, and these responses were remarkably sustained for longer than six months. As a self-protein, S100A4 did not stimulate antibody responses against itself, a quality desired of adjuvants. S100A4 prolonged nasal residence of intranasally delivered antigens and promoted migration of antigen-presenting cells. S100A4-pulsed dendritic cells potently activated cognate T cells. Furthermore, S100A4 induced strong germinal center responses revealed by both microscopy and mass spectrometry, a novel technique for measuring germinal center activity. In conclusion, S100A4 may be a promising adjuvant in formulating mucosal vaccines, including vaccines against pathogens that infect via the respiratory tract, such as SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.04.474916","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.07.463592","pub_date":"2022-1-04","title":"Comprehensive antibody profiling of mRNA vaccination in children","abstract":"While children have been largely spared from COVID-19 disease, the emergence of viral variants of concern (VOC) with increased transmissibility, combined with fluctuating mask mandates and school re-openings have led to increased infections and disease among children. Thus, there is an urgent need to roll out COVID-19 vaccines to children of all ages. However, whether children respond equivalently to adults to mRNA vaccines and whether dosing will elicit optimal immunity remains unclear. Given the recent announcement of incomplete immunity induced by the pediatric dose of the BNT162b2 vaccine in young children, here we aimed to deeply profile and compare the vaccine-induced humoral immune response in 6-11 year old children receiving the pediatric (50\u03bcg) or adult (100\u03bcg) dose of the mRNA-1273 vaccine compared to adults and naturally infected children or children that experienced multi inflammatory syndrome in children (MIS-C) for the first time. Children elicited an IgG dominant vaccine induced immune response, surpassing adults at a matched 100\u03bcg dose, but more variable immunity at a 50\u03bcg dose. Irrespective of titer, children generated antibodies with enhanced Fc-receptor binding capacity. Moreover, like adults, children generated cross-VOC humoral immunity, marked by a decline of omicron receptor binding domain-binding, but robustly preserved omicron Spike-receptor binding, with robustly preserved Fc-receptor binding capabilities, in a dose dependent manner. These data indicate that while both 50\u03bcg and 100\u03bcg of mRNA vaccination in children elicits robust cross-VOC antibody responses, 100ug of mRNA in children results in highly preserved omicron-specific functional humoral immunity. mRNA vaccination elicits robust humoral immune responses to SARS-CoV-2 in children 6-11 years of age.","version":"1.2","doi":"10.1101/2021.10.07.463592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.30.474561","pub_date":"2022-1-03","title":"Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels","abstract":"COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post COVID condition. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevent SARS-CoV-2 entry in the brain thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Brain entry of SARS-CoV-2 through endothelial cells is prevented by melatonin through allosteric binding to human angiotensin-converting enzyme 2 (ACE2), which interferes with the cell entry receptor function of ACE2 for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.","version":"1.1","doi":"10.1101/2021.12.30.474561","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.12.439201","pub_date":"2022-1-03","title":"Impairment of SARS-CoV-2 spike-glycoprotein maturation and fusion-activity by nitazoxanide: an effect independent of spike variants emergence","abstract":"SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion-glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike-variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike biogenesis, hampering its maturation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.","version":"1.2","doi":"10.1101/2021.04.12.439201","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.02.474028","pub_date":"2022-1-03","title":"A dual-receptor mechanism between integrins and ACE2 widens SARS-CoV-2 tissue tropism","abstract":"In addition to the ACE2 receptor, SARS-CoV-2 binds to integrins to gain host cell entry and trigger pro-inflammatory integrin-mediated signalling cascades. Integrins, therefore, are likely candidates for a dual-receptor mechanism with ACE2 to explain the increased infectivity seen in SARS-CoV-2 models. As integrins are primarily expressed in vasculature and persistent vasculopathy is seen in COVID-19, examining the role of endothelial integrin involvement is crucial in uncovering the pathophysiology of SARS-CoV-2.","version":"1.1","doi":"10.1101/2022.01.02.474028","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.31.474593","pub_date":"2022-1-03","title":"Cell culture model system utilizing engineered A549 cells to express high levels of ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals","abstract":"Novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an imminent global threat since its initial outbreak in December 2019. A simple in vitro model system using cell lines highly susceptible to SARS-CoV-2 infection are critical to facilitate the study of the virus cycle and to discover effective antivirals against the virus. Human lung alveolar A549 cells are regarded as a useful and valuable model for respiratory virus infection. However, SARS-CoV-2 uses the ACE2 as receptor for viral entry and the TMPRSS2 to prime the Spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a robust human lung epithelial cell-based model by transducing ACE2 and TMPRSS2 into A549 cells and show that the ACE2 enriched A549ACE2/TMPRSS2 cells (ACE2plus) and its single-cell-derived subclone (ACE2plusC3) are highly susceptible to SARS-CoV-2 infection. These engineered ACE2plus showed higher ACE2 and TMPRSS2 mRNA expression levels than currently used Calu3 and commercial A549ACE2/TMPRSS2 cells. ACE2 and TMPRSS2 proteins were also highly and ubiquitously expressed in ACE2plusC3 cells. Additionally, antiviral drugs like Camostat mesylate, EIDD-1931, and Remdesivir strongly inhibited SARS-CoV-2 replication. Notably, multinucleated syncytia, a clinical feature commonly observed in severe COVID-19 patients was induced in ACE2plusC3 cells either by virus infection or by overexpressing the Spike proteins of different variants of SARS-CoV-2. Syncytial process was effectively blocked by the furin protease inhibitor, Decanoyl-RVKR-CMK. Taken together, we have developed a robust human A549 lung epithelial cell-based model that can be applied to probe SARS-CoV-2 replication and to facilitate the discovery of SARS-CoV-2 inhibitors.","version":"1.1","doi":"10.1101/2021.12.31.474593","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.05.466755","pub_date":"2022-1-03","title":"Scrutiny of human lung infection by SARS-CoV-2 and associated human immune responses in humanized mice","abstract":"There is an urgent need for animal models of COVID-19 to study immunopathogenesis and test therapeutic intervenes. In this study we showed that NSG mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by SARS-CoV-2, and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-seq examination identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, interferon stimulation, and pulmonary fibrosis between HL grafts from infected and control NSG-L mice. Furthermore, when infecting humanized mice with human immune system (HIS) and autologous HL grafts (HISL mice), the mice had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.","version":"1.4","doi":"10.1101/2021.11.05.466755","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.02.474743","pub_date":"2022-1-03","title":"Reduced Pathogenicity of the SARS-CoV-2 Omicron Variant in Hamsters","abstract":"The SARS-CoV-2 Omicron (B.1.1.529) variant has proven highly transmissible and has outcompeted the Delta variant in many regions of the world. Early reports have also suggested that Omicron may result in less severe clinical disease in humans. Here we show that Omicron is less pathogenic than prior SARS-CoV-2 variants in Syrian golden hamsters. Infection of hamsters with the SARS-CoV-2 WA1/2020, Alpha, Beta, or Delta strains led to 4-10% weight loss by day 4 and 10-17% weight loss by day 6, as expected. In contrast, infection of hamsters with two different Omicron challenge stocks did not result in any detectable weight loss, even at high challenge doses. Omicron infection still led to substantial viral replication in both the upper and lower respiratory tracts and pulmonary pathology, but with a trend towards higher viral loads in nasal turbinates and lower viral loads in lung parenchyma compared with WA1/2020 infection. These data suggest that the SARS-CoV-2 Omicron variant may result in more robust upper respiratory tract infection but less severe lower respiratory tract clinical disease compared with prior SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2022.01.02.474743","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.30.474613","pub_date":"2022-1-03","title":"Nonself Mutations in the Spike Protein Suggest an Increase in the Antigenicity and a Decrease in the Virulence of the Omicron Variant of SARS-CoV-2","abstract":"Despite extensive worldwide vaccination, the current COVID-19 pandemic caused by SARS-CoV-2 continues. The Omicron variant is a recently emerged variant of concern and is now taking over the Delta variant. To characterize the potential antigenicity of the Omicron variant, we examined the distributions of SARS-CoV-2 nonself mutations (in reference to the human proteome) as 5 amino acid stretches of short constituent sequences (SCSs) in the Omicron and Delta proteomes. The number of nonself SCSs did not differ much throughout the Omicron, Delta, and Reference Sequence (RefSeq) proteomes but markedly increased in the receptor binding domain (RBD) of the Omicron spike protein compared to those of the Delta and RefSeq proteins. In contrast, the number of nonself SCSs decreased in non-RBD regions in the Omicron spike protein, compensating for the increase in the RBD. Several nonself SCSs were tandemly present in the RBD of the Omicron spike protein, likely as a result of selection for higher binding affinity to the ACE2 receptor (and hence higher infectivity and transmissibility) at the expense of increased antigenicity. Taken together, the present results suggest that the Omicron variant has evolved to have higher antigenicity and less virulence in humans despite increased infectivity and transmissibility.","version":"1.1","doi":"10.1101/2021.12.30.474613","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.03.22268599","pub_date":"2022-01-03","title":"Antibody response to SARS-CoV-2 mRNA vaccine in lung cancer patients: Reactivity to vaccine antigen and variants of concern","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Purpose</jats:title>\n                  <jats:p>We investigated SARS-CoV-2 mRNA vaccine-induced binding and live-virus neutralizing antibody response in NSCLC patients to the SARS-CoV-2 wild type strain and the emerging Delta and Omicron variants.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>82 NSCLC patients and 53 healthy adult volunteers who received SARS-CoV-2 mRNA vaccines were included in the study. Blood was collected longitudinally, and SARS-CoV-2-specific binding and live-virus neutralization response to 614D (WT), B.1.617.2 (Delta), B.1.351 (Beta) and B.1.1.529 (Omicron) variants were evaluated by Meso Scale Discovery (MSD) assay and Focus Reduction Neutralization Assay (FRNT) respectively. We determined the longevity and persistence of vaccine-induced antibody response in NSCLC patients. The effect of vaccine-type, age, gender, race and cancer therapy on the antibody response was evaluated.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Binding antibody titer to the mRNA vaccines were lower in the NSCLC patients compared to the healthy volunteers (P=&lt;0.0001). More importantly, NSCLC patients had reduced live-virus neutralizing activity compared to the healthy vaccinees (P=&lt;0.0001). Spike and RBD-specific binding IgG titers peaked after a week following the second vaccine dose and declined after six months (P=&lt;0.001). While patients &gt;70 years had lower IgG titers (P=&lt;0.01), patients receiving either PD-1 monotherapy, chemotherapy or a combination of both did not have a significant impact on the antibody response. Binding antibody titers to the Delta and Beta variants were lower compared to the WT strain (P=&lt;0.0001). Importantly, we observed significantly lower FRNT\n                    <jats:sub>50</jats:sub>\n                    titers to Delta (6-fold), and Omicron (79-fold) variants (P=&lt;0.0001) in NSCLC patients.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Binding and live-virus neutralizing antibody titers to SARS-CoV-2 mRNA vaccines in NSCLC patients were lower than the healthy vaccinees, with significantly lower live-virus neutralization of B.1.617.2 (Delta), and more importantly, the B.1.1.529 (Omicron) variant compared to the wild-type strain. These data highlight the concern for cancer patients given the rapid spread of SARS-CoV-2 Omicron variant.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2022.01.03.22268599","journal":"medRxiv","score":null},{"id":"10.1101/2022.01.03.474721","pub_date":"2022-1-03","title":"Hydrodynamics of spike proteins dictate a transport-affinity competition for SARS-CoV-2 and other enveloped viruses","abstract":"Many viruses, such as SARS-CoV-2 or Influenza, possess spike-decorated envelopes. Depending on the virus type, a large variability is present in spikes number, morphology and reactivity, which remains generally unexplained. Since viruses\u2019 transmissibility depend on features beyond their genetic sequence, new tools are required to discern the effects of spikes functionality, interaction, and morphology. Here, we postulate the relevance of hydrodynamic interactions in the viral infectivity of enveloped viruses and propose micro-rheological characterization as a platform for viruses differentiation. To understand how the spikes affect virion mobility and infectivity, we investigate the diffusivity of spike-decorate structures using mesoscopic-hydrodynamic simulations. Furthermore, we explored the interplay between affinity and passive viral transport. Our results revealed that the diffusional mechanism of SARS-CoV-2 is strongly influenced by the size and distribution of its spikes. We propose and validate a universal mechanism to explain the link between optimal virion structure and maximal infectivity for many virus families.","version":"1.1","doi":"10.1101/2022.01.03.474721","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.02.473343","pub_date":"2022-1-03","title":"RG203KR mutations in SARS-CoV-2 Nucleocapsid: Assessing the impact using Virus-like particle model system","abstract":"The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on Spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of the mutations at the virion level, recombinant baculovirus expression system based VLPs were generated for the prototype Wuhan sequence along with Spike mutants like D614G, G1124V and the significant RG203KR mutation in Nucleocapsid. All the four structural proteins assembled in a particle wherein the morphology and size of the particle confirmed by TEM closely resembles the native virion. The VLP harbouring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by the immunized mice sera. Results demonstrate a non-infectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant.","version":"1.1","doi":"10.1101/2022.01.02.473343","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474084","pub_date":"2022-1-03","title":"A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron","abstract":"Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. A sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.","version":"1.2","doi":"10.1101/2021.12.24.474084","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.01.474639","pub_date":"2022-1-03","title":"In vitro evaluation of therapeutic antibodies against a SARS-CoV-2 Omicron B.1.1.529 isolate","abstract":"The emergence and rapid spread of the Omicron variant of SARS-CoV-2, which has more than 30 substitutions in the spike glycoprotein, compromises the efficacy of currently available vaccines and therapeutic antibodies. Using a clinical strain of the Omicron variant, we analyzed the neutralizing power of eight currently used monoclonal antibodies compared to the ancestral B.1 BavPat1 D614G strain. We observed that six of these antibodies have lost their ability to neutralize the Omicron variant. Of the antibodies still having neutralizing activity, Sotrovimab/Vir-7831 shows the smallest reduction in activity, with a factor change of 3.1. Cilgavimab/AZD1061 alone shows a reduction in efficacy of 15.8, resulting in a significant loss of activity for the Evusheld cocktail (42.6 fold reduction) in which the other antibody, Tixagevimab, does not retain significant activity against Omicron. Our results suggest that the clinical efficacy of the initially proposed doses should be rapidly evaluated and the possible need to modify doses or propose combination therapies should be considered.","version":"1.1","doi":"10.1101/2022.01.01.474639","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.30.474610","pub_date":"2022-1-03","title":"Robust expansion of phylogeny for fast-growing genome sequence data","abstract":"Massive sequencing of SARS-CoV-2 genomes has led to a great demand for adding new samples to a reference phylogeny instead of building the tree from scratch. To address such challenge, we proposed an algorithm \u2018TIPars\u2019 by integrating parsimony analysis with pre-computed ancestral sequences. Compared to four state-of-the-art methods on four benchmark datasets (SARS-CoV-2, Influenza virus, Newcastle disease virus and 16S rRNA genes), TIPars achieved the best performance in most tests. It took only 21 seconds to insert 100 SARS-CoV-2 genomes to a 100k-taxa reference tree using near 1.4 gigabytes of memory. Its efficient and accurate phylogenetic placements and incrementation for phylogenies with highly similar and divergent sequences suggest that it will be useful in a wide range of studies including pathogen molecular epidemiology, microbiome diversity and systematics.","version":"1.1","doi":"10.1101/2021.12.30.474610","journal":"bioRxiv","score":null},{"id":"10.1101/2022.01.03.474769","pub_date":"2022-1-03","title":"Omicron variant escapes therapeutic mAbs contrary to eight prior main VOC","abstract":"Monocolonal antibodies (mAbs) are currently used for active immunization of COVID-19 in immunocompromised patients. We herein show that in spite there are variations in susceptibility to available mAbs that are authorized for clinical use in France tested on the original B.1.1 virus and 9 variants of concern or of interest, the cocktail casirivimab/imdevimab (REGN-CoV-2) showed a major synergistic effect. However, none of the four mAbs either alone or in combination neutralized the new Omicron variant. Our data strongly warrant a reinforcement of protective measures against infection for immunocompromised patients.","version":"1.1","doi":"10.1101/2022.01.03.474769","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.28.474380","pub_date":"2021-12-30","title":"Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement","abstract":"The SARS-CoV-2 Omicron variant of concern evades antibody mediated immunity with an unprecedented magnitude due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and X-ray crystal structures of the spike and RBD bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a structural framework for understanding the marked reduction of binding of all other therapeutic mAbs leading to dampened neutralizing activity. We reveal electrostatic remodeling of the interactions within the spike and those formed between the Omicron RBD and human ACE2, likely explaining enhanced affinity for the host receptor relative to the prototypic virus.","version":"1.1","doi":"10.1101/2021.12.28.474380","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474114","pub_date":"2021-12-27","title":"Impacts of the COVID-19 Pandemic on a Human Research Islet Program","abstract":"Designated a pandemic in March 2020, the spread of severe acute respiratory syndrome virus 2 (SARS-CoV2), the virus responsible for coronavirus disease 2019 (COVID-19), led to new guidelines and restrictions being implemented for individuals, businesses, and societies in efforts to limit the impacts of COVID-19 on personal health and healthcare systems. Here we report the impacts of the COVID-19 pandemic on pancreas processing and islet isolation/distribution outcomes at the Alberta Diabetes Institute IsletCore, a facility specialising in the processing and distribution of human pancreatic islets for research. While the number of organs processed was significantly reduced, organ quality and the function of cellular outputs were minimally impacted during the pandemic when compared to an equivalent period immediately prior. Despite the maintained quality of isolated islets, recipient groups reported poorer feedback regarding the samples. Our findings suggest this is likely due to disrupted distribution which led to increased transit times to recipient labs, particularly those overseas. Thus, to improve overall outcomes in a climate of limited research islet supply, prioritization of tissue recipients based on likely tissue transit times may be needed.","version":"1.1","doi":"10.1101/2021.12.24.474114","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474138","pub_date":"2021-12-27","title":"Homologous or Heterologous Booster of Inactivated Vaccine Reduces SARS-CoV-2 Omicron Variant Escape from Neutralizing Antibodies","abstract":"The massive and rapid transmission of SARS-CoV-2 has led to the emergence of several viral variants of concern (VOCs), with the most recent one, B.1.1.529 (Omicron), which accumulated a large number of spike mutations, raising the specter that this newly identified variant may escape from the currently available vaccines and therapeutic antibodies. Using VSV-based pseudovirus, we found that Omicron variant is markedly resistant to neutralization of sera form convalescents or individuals vaccinated by two doses of inactivated whole-virion vaccines (BBIBP-CorV). However, a homologous inactivated vaccine booster or a heterologous booster with protein subunit vaccine (ZF2001) significantly increased neutralization titers to both WT and Omicron variant. Moreover, at day 14 post the third dose, neutralizing antibody titer reduction for Omicron was less than that for convalescents or individuals who had only two doses of the vaccine, indicating that a homologous or heterologous booster can reduce the Omicron escape from neutralizing. In addition, we tested a panel of 17 SARS-CoV-2 monoclonal antibodies (mAbs). Omicron resists 7 of 8 authorized/approved mAbs, as well as most of the other mAbs targeting distinct epitopes on RBD and NTD. Taken together, our results suggest the urgency to push forward the booster vaccination to combat the emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.12.24.474138","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474110","pub_date":"2021-12-27","title":"Reduced infectivity but increased immune escape of the new SARS-CoV-2 variant of concern Omicron","abstract":"A new detected SARS-CoV-2 variant Omicron (B.1.1.529) had reported from more than 80 countries. In the past few weeks, a new wave of infection driven by Omicron is in progress. Omicron Spike (S) protein pseudotyped virus was used to determine the effect of S mutations on its capacity of infectivity and immune evasion. Our results showed the lower entry efficiency and less cleavage ability of Omicron than D614G variant. Pseudotype-based neutralizing assay was performed to analyze neutralizing antibodies elicited by previously infection or the RBD-based protein subunit vaccine ZF2001 against the Omicron variant. Sera sampled at around one month after symptom onset from 12 convalescents who were previously infected by SARS-CoV-2 original strain shows a more than 20-fold decrease of neutralizing activity against Omicron variant, when compared to D614G variant. Among 12 individuals vaccinated by RBD subunit vaccine, 58.3% (7/12) sera sampled at 15-60 days after 3rd-dose vaccination did not neutralize Omicron. Geometric mean titers (GMTs, 50% inhibitory dose [ID50]) of these sera against Omicron were 9.4-fold lower than against D614G. These results suggested a higher risk of Omicron breakthrough infections and reduced efficiency of the protective immunity elicited by existing vaccines. There are important implications about the modification and optimization of the current epidemic prevention and control including vaccine strategies and therapeutic antibodies against Omicron variant.","version":"1.1","doi":"10.1101/2021.12.24.474110","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.25.445649","pub_date":"2021-12-27","title":"Structurally and functionally distinct early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response","abstract":"A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-Fc\u03b3R interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-Fc\u03b3R interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2. Divergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo.","version":"1.4","doi":"10.1101/2021.05.25.445649","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474086","pub_date":"2021-12-26","title":"The omicron (B.1.1.529) SARS-CoV-2 variant of concern does not readily infect Syrian hamsters","abstract":"The emergence of SARS-CoV-2 variants of concern (VoCs) has exacerbated the COVID-19 pandemic. End of November 2021, a new SARS-CoV-2 variant namely the omicron (B.1.1.529) emerged. Since this omicron variant is heavily mutated in the spike protein, WHO classified this variant as the 5th variant of concern (VoC). We previously demonstrated that the other SARS-CoV-2 VoCs replicate efficiently in Syrian hamsters, alike also the ancestral strains. We here wanted to explore the infectivity of the omicron variant in comparison to the ancestral D614G strain. Strikingly, in hamsters that had been infected with the omicron variant, a 3 log10 lower viral RNA load was detected in the lungs as compared to animals infected with D614G and no infectious virus was detectable in this organ. Moreover, histopathological examination of the lungs from omicron-infecetd hamsters revealed no signs of peri-bronchial inflammation or bronchopneumonia. Further experiments are needed to determine whether the omicron VoC replicates possibly more efficiently in the upper respiratory tract of hamsters than in their lungs.","version":"1.1","doi":"10.1101/2021.12.24.474086","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474081","pub_date":"2021-12-26","title":"Convalescence from prototype SARS-CoV-2 protects Syrian hamsters from disease caused by the Omicron variant","abstract":"The mutation profile of the SARS-CoV-2 Omicron variant poses a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2, 99.99% identical to Wuhan-Hu-1, to protect against disease caused by the Omicron variant. We established that infection with Omicron in na\u00efve Syrian hamsters resulted in a less severe disease than a comparable dose of prototype SARS-CoV-2 (Australia/VIC01/2020), with fewer clinical signs and less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of Omicron 50 days after an initial infection with Australia/VIC01/2020. The data provide evidence for immunity raised against prototype SARS-CoV-2 being protective against Omicron in the Syrian hamster model. Further investigation is required to conclusively determine whether Omicron is less pathogenic in Syrian hamsters and whether this is predictive of pathogenicity in humans.","version":"1.1","doi":"10.1101/2021.12.24.474081","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425128","pub_date":"2021-12-26","title":"Protein scaffold-based multimerization of soluble ACE2 efficiently blocks SARS-CoV-2 infection in vitro and in vivo","abstract":"Soluble ACE2 (sACE2) decoy receptors are promising agents to inhibit SARS-CoV-2, as their efficiency is less likely to be affected by common escape mutations in viral proteins. However, their success may be limited by their relatively poor potency. To address this challenge, we developed a large decoy library of sACE2 fusion proteins, generated with several protease inhibitors or multimerization tags. Among these decoys, multimeric sACE2 consisting of SunTag or MoonTag systems, which were originally utilized for signal amplification or gene activation systems, were extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates. These novel sACE2 fusion proteins exhibited greater than 100-fold SARS-CoV-2 neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag in combination with a more potent version of sACE2, which has multiple point mutations for greater binding (v1), achieved near complete neutralization at a sub-nanomolar range, comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutant versions of Spike, alpha, beta, gamma or delta variants, were also neutralized efficiently with SunTag or MoonTag fused sACE2(v1). Finally, therapeutic treatment of sACE2(v1)-MoonTag provided protection against SARS-CoV-2 infection in an in vivo mouse model. Overall, we suggest that the superior activity of the sACE2-SunTag or sACE2-MoonTag fusions is due to the greater occupancy of the multimeric sACE2 receptors on Spike protein as compared to monomeric sACE2. Therefore, these highly potent multimeric sACE2 decoy receptors may offer a promising treatment approach against SARS-CoV-2 infections. Multimerization of sACE2 markedly enhanced the neutralization of SARS-CoV-2 by blocking multiple viral spike proteins simultaneously.","version":"1.2","doi":"10.1101/2021.01.04.425128","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.24.474091","pub_date":"2021-12-26","title":"Two doses of mRNA vaccine elicit cross-neutralizing memory B-cells against SARS-CoV-2 Omicron variant","abstract":"SARS-CoV-2 Beta and Omicron variants have multiple mutations in the receptor-binding domain (RBD) allowing antibody evasion. Despite the resistance to circulating antibodies in those who received two doses of mRNA vaccine, the third dose prominently recalls cross-neutralizing antibodies with expanded breadth to these variants. Herein, we longitudinally profiled the cellular composition of persistent memory B-cell subsets and their antibody reactivity against these variants following the second vaccine dose. The vaccination elicited a memory B-cell subset with resting phenotype that dominated the other subsets at 4.9 months. Notably, most of the resting memory subset retained the ability to bind the Beta variant, and the memory-derived antibodies cross-neutralized the Beta and Omicron variants at frequencies of 59% and 29%, respectively. The preservation of cross-neutralizing antibody repertoires in the durable memory B-cell subset likely contributes to the prominent recall of cross-neutralizing antibodies following the third dose of the vaccine. Fully vaccinated individuals preserve cross-neutralizing memory B-cells against the SARS-CoV-2 Omicron variant.","version":"1.1","doi":"10.1101/2021.12.24.474091","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.473686","pub_date":"2021-12-25","title":"IL4I1 binds to TMPRSS13 and competes with SARS-Cov2 Spike","abstract":"The secreted enzyme interleukin four-induced gene 1 (IL4I1) is involved in the negative control of the adaptive immune response. IL4I1 expression in human cancer is frequent and correlates with poor survival and resistance to immunotherapy. Nevertheless, its mechanism of action remains partially unknown. Here, we identified transmembrane serine protease 13 (TMPRSS13) as an immune cell-expressed surface protein that binds IL4I1. TMPRSS13 is a paralog of TMPRSS2, whose protease activity participates in the cleavage of SARS-Cov2 Spike protein and facilitates virus induced-membrane fusion. We show that TMPRSS13 is expressed by human lymphocytes, monocytes and monocyte-derived macrophages, can cleave the Spike protein and allow Sars-Cov2 Spike pseudotyped virus entry into cells. We identify regions of homology between IL4I1 and Spike and demonstrate competition between the two proteins for TMPRSS13 binding. These findings may be relevant for both interfering with SARS-Cov2 infection and limiting IL4I1-dependent immunosuppressive activity in cancer. Through binding to its newly identified receptor TMPRSS13, the enzyme IL4I1 interferes with SARS-Cov2 Spike cleavage thereby blocking viral entry into host cells.","version":"1.1","doi":"10.1101/2021.12.23.473686","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.25.474052","pub_date":"2021-12-25","title":"An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants","abstract":"The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0\u00d710\u221212 M) and strong neutralizing activity (IC50 = 0.0017 nM). Together, we developed a tetravalent biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.","version":"1.1","doi":"10.1101/2021.12.25.474052","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.474055","pub_date":"2021-12-25","title":"Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening","abstract":"Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-\u03b2 but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-\u03b2. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.","version":"1.1","doi":"10.1101/2021.12.23.474055","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.473991","pub_date":"2021-12-25","title":"Analyzing the interaction of human ACE2 and RBD of spike protein of SARS-CoV-2 in perspective of Omicron variant","abstract":"The newly identified Omicron (B.1.1.529) variant of Severe Acute Respiratory Syndrome Voronavirus 2 (SARS-CoV-2) has steered concerns across the world due to the possession of large number of mutations leading to high infectivity and vaccine escape potential. The Omicron variant houses 32 mutations in S protein alone. The viral infectivity is determined mainly by the ability of spike (S) protein receptor binding domain (RBD) to bind to the human Angiotensin I Converting Enzyme 2 (hACE2) receptor. In this paper, the interaction of the RBDs of SARS-CoV-2 variants with hACE2 was analyzed by using protein-protein docking and compared with the novel Omicron variant. Our findings reveal that the Omicron RBD interacts strongly with hACE2 receptor via unique amino acid residues as compared to the Wuhan and many other variants. However, the interacting residues of RBD are found to be the same in Lamda (C.37) variant. These unique binding of Omicron RBD with hACE2 suggests an increased potential of infectivity and vaccine evasion potential of the new variant. The evolutionary drive of the SARS-CoV-2 may not be exclusively driven by RBD variants but surely provides for the platform for emergence of new variants.","version":"1.1","doi":"10.1101/2021.12.23.473991","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.474030","pub_date":"2021-12-25","title":"Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype","abstract":"The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.","version":"1.1","doi":"10.1101/2021.12.23.474030","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.23.473975","pub_date":"2021-12-25","title":"In vitro evolution predicts emerging CoV-2 mutations with high affinity for ACE2 and cross-species binding","abstract":"Emerging SARS CoV-2 variants are creating major challenges in the ongoing Covid-19 pandemic. Predicting CoV-2 mutations that increase transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines and prioritising viral monitoring and containment. Using in vitro evolution, we identify a double mutation in CoV-2 receptor binding domain (RBD) that increases affinity for ACE2 almost 20-fold. We determine the mutant:ACE2 structure to reveal the binding mechanism and show the main affinity driver, Q498H, boosts binding of other RBD variants. We find this mutation incompatible with the common N501Y mutation, but N501Y variants can acquire Q498R to access a similar bonding network and affinity gain. We show Q498H, and Q498R plus N501Y, enable variants to bind rat ACE2 with high affinity. These mutations are now emerging in CoV-2 variants, such as the Omicron variant, where they would be expected to drive increased human-to-human and cross-species transmission.","version":"1.1","doi":"10.1101/2021.12.23.473975","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.473733","pub_date":"2021-12-24","title":"Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617","abstract":"COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at \u223c3 \u00c5 resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.","version":"1.1","doi":"10.1101/2021.12.21.473733","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469842","pub_date":"2021-12-23","title":"Single-domain antibodies efficiently neutralize SARS-CoV-2 variants of concern, including Omicron variant","abstract":"Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.","version":"1.2","doi":"10.1101/2021.11.24.469842","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473914","pub_date":"2021-12-23","title":"Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice","abstract":"Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.","version":"1.1","doi":"10.1101/2021.12.22.473914","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.473702","pub_date":"2021-12-23","title":"Tutorial: Investigating SARS-CoV-2 evolution and phylogeny using MNHN-Tree-Tools","abstract":"The Covid-19 pandemic has caused at more than 3 million deaths by Mai this year [1]. It had a significant impact on the daily life and the global economy [2]. The virus has since its first recorded outbreak in China [3] mutated into new strains [4]. The Nextstrain [5] project has so far been monitoring the evolution of the virus. At the same time we were developing in our lab the MNHN-Tree-Tools [6] toolkit, primarily for the investigation of DNA repeat sequences. We have further extended MNHN-Tree-Tools [6] to guide phylogenetics. As such the toolkit has evolved into a high performance code, allowing for a fast investigation of millions of sequences. Given the context of the pandemic it became evident that we will use our versatile tool to investigate the evolution of SARS-CoV-2 sequences. Our efforts have cumulated in this tutorial that we share with the scientific community.","version":"1.1","doi":"10.1101/2021.12.21.473702","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473887","pub_date":"2021-12-23","title":"Allosteric Determinants of the SARS-CoV-2 Spike Protein Binding with Nanobodies: Examining Mechanisms of Mutational Escape and Sensitivity of the Omicron Variant","abstract":"Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with Nb6, VHH E and bi-paratopic VHH VE nanobodies. We combined atomistic dynamics and collective motions analysis with binding free energy scanning, perturbation-response scanning and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein may exploit plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH VE nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.","version":"1.1","doi":"10.1101/2021.12.22.473887","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473902","pub_date":"2021-12-23","title":"Machine learning guided design of high affinity ACE2 decoys for SARS-CoV-2 neutralization","abstract":"A potential therapeutic candidate for neutralizing SARS-CoV-2 infection is engineering high-affinity soluble ACE2 decoy proteins to compete for binding of the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant ACE2 variant, named ACE22.v.2.4, that exhibits nanomolar affinity binding to the RBD domain of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identified other ACE2 variants, namely double mutants, that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified several ACE2 double mutants that bind to RBD with tighter affinity as compared to the wild type, most notably, L79V;N90D that binds RBD with similar affinity to ACE22.v.2.4. The successful experimental validation of the double mutants demonstrated the use transfer and supervised learning approaches for engineering protein-protein interactions and identifying high affinity ACE2 peptides for targeting SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.12.22.473902","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473615","pub_date":"2021-12-23","title":"Broad and Long-lasting Immune Response against SARS-CoV-2 Omicron and Other Variants by PIKA-Adjuvanted Recombinant SARS-CoV-2 Spike (S) Protein Subunit Vaccine (YS-SC2-010)","abstract":"Recently SARS-CoV-2 Omicron (B.1.1.529) variant was identified in South Africa with numerous mutations in spike protein, and numerous community infections have been reported and raised grave concern around the world. Some studies found that the neutralization effects of several licensed vaccines against Omicron were dramatically reduced, which significantly affected antibody mediated protection, especially for individuals whose immunization were completed after extended period. In this regard, we studied the persistence and neutralization activity toward mutant strains in animal serum immunized with PIKA-adjuvanted recombinant SARS-CoV-2 spike protein subunit vaccine (YS-SC2-010). Here we are reporting that animal serum collected at 596 days after immunization with YS-SC2-010 still retains high and persistent neutralizing activity against all the Variant of Concern (VOC) variants, including Omicron variant. Although it is a blessed event to achieve 20 months long neutralization against Omicron variant after immunization with YS-SC2-010, it was also founded that the neutralization effect of immune serum on Omicron decreased by 6.29 folds as compared to D614G, more significantly when compared with other mutant strains.","version":"1.1","doi":"10.1101/2021.12.22.473615","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473949","pub_date":"2021-12-23","title":"Rapid longitudinal SARS-CoV-2 intra-host emergence of novel haplotypes regardless of immune deficiencies","abstract":"On February 2020, the municipality of Vo\u2019, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo\u2019 belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo\u2019. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events. It is of great interest and importance to understand SARS-CoV-2 ability to mutate generating new viral strains, and to assess the impact of containment strategies on viral transmission. In this study we highlight the rapid intra-host haplotype evolution regardless of symptom severity and immune deficiencies that we observed during the first wave of the pandemic in the municipality of Vo\u2019 in Italy. The confirmation that all the haplotypes found in this small community derive from a common ancestor haplotype, has allowed us to track the rapid emergence of new variants but lockdown and mass testing efficiently prevented their spread elsewhere.","version":"1.1","doi":"10.1101/2021.12.22.473949","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473892","pub_date":"2021-12-23","title":"Conformational Flexibility and Local Frustration in the Functional States of the SARS-CoV-2 Spike B.1.1.7 and B.1.351 Variants : Mutation-Induced Allosteric Modulation Mechanism of Functional Dynamics and Protein Stability","abstract":"The experimental and computational studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across variants of concern(VOCs), linking the effect of this mutation with the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies characterized the closed and open states of the B.1.1.7 (B.1.1.7) and B.1.351 (Beta) spike variants allowing for a more detailed atomistic characterization of the conformational landscapes and functional changes. In this study, we employed coarse-grained simulations of the SARS-CoV-2 spike variant trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration analysis of the conformational ensembles with collective dynamics and residue-based mutational scanning of protein stability, we determine protein stability hotspots and identify potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. Through mutational scanning of protein stability changes we quantify mutational adaptability of the S-G614, S-B.1.1.7 and S-B.1.351 variants in different functional forms. Using this analysis, we found a significant conformational and mutational plasticity of the open states for all studied variants. The results of this study suggest that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites, the inter-protomer hinges of functional motions and motions of the receptor-binding domain required for binding of the host cell receptor. The proposed mechanism of mutation-induced energetic frustration may result in the greater adaptability and the emergence of multiple conformational substates in the open form. This study also suggested functional relationships between mutation-induced modulation of protein dynamics, local frustration and allosteric regulation of the SARS-CoV-2 spike protein.","version":"1.1","doi":"10.1101/2021.12.22.473892","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473880","pub_date":"2021-12-23","title":"SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster","abstract":"The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera from individuals infected by different variants. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants.","version":"1.1","doi":"10.1101/2021.12.22.473880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.22.473934","pub_date":"2021-12-23","title":"Waning immune responses against SARS-CoV-2 among vaccinees in Hong Kong","abstract":"Nearly 4 billion doses of the BioNTech-mRNA and Sinovac-inactivated vaccines have been administrated globally, yet different vaccine-induced immunity against SARS-CoV-2 variants of concern (VOCs) remain incompletely investigated. We compare the immunogenicity and durability of these two vaccines among fully vaccinated Hong Kong people. Standard BioNTech and Sinovac vaccinations were tolerated and induced neutralizing antibody (NAb) (100% and 85.7%) and spike-specific CD4 T cell responses (96.7% and 82.1%), respectively. The geometric mean NAb IC50 and median frequencies of reactive CD4 subsets were consistently lower among Sinovac-vaccinees than BioNTech-vaccinees. Against VOCs, NAb response rate and geometric mean IC50 against B1.351 and B.1.617.2 were significantly lower for Sinovac (14.3%, 15 and 50%, 23.2) than BioNTech (79.4%, 107 and 94.1%, 131). Three months after vaccinations, NAbs to VOCs dropped near to detection limit, along with waning memory T cell responses, mainly among Sinovac-vaccinees. Our results indicate that Sinovac-vaccinees may face higher risk to pandemic VOCs breakthrough infection. This study was supported by the Hong Kong Research Grants Council Collaborative Research Fund (C7156-20GF to Z.C and C1134-20GF); the National Program on Key Research Project of China (Grant 2020YFC0860600, 2020YFA0707500 and 2020YFA0707504); Shenzhen Science and Technology Program (JSGG20200225151410198 and JCYJ20210324131610027); HKU Development Fund and LKS Faculty of Medicine Matching Fund to AIDS Institute; Hong Kong Innovation and Technology Fund, Innovation and Technology Commission and generous donation from the Friends of Hope Education Fund. Z.C.\u2019s team was also partly supported by the Theme-Based Research Scheme (T11-706/18-N).","version":"1.1","doi":"10.1101/2021.12.22.473934","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.470882","pub_date":"2021-12-23","title":"COVID-19: Salient Aspects of Coronavirus Infection, Vaccines and Vaccination Testing and their Implications","abstract":"In the present study, three basic aspects related to COVID-19 are presented. The occurrence of coronavirus infection is analyzed statistically as number of coronaviruses infected alveolar cells compared to normal alveolar cells in human lungs. The mole concept is used to estimate the number of normal alveolar cells per human lung. The number of coronavirus infections in infected alveolar cells is estimated from the published Lower Respiratory Tract (LRT) load data. The Poisson probability distribution is aptly applied to imply the incubation period of the coronavirus infection to be within day-3 to day-7, with the cumulative probability of 75%. The incubation period within day-0 to day-10 has a cumulative probability of 98%. It implies a 10-day quarantine to isolate an uninfected individual as a precautionary measure. Three vaccines to combat COVID-19, which adopt distinct paradigms while preparing them, are analyzed. These are Moderna\u2019s mRNA-1273, Oxford-AstraZeneca\u2019s ChAdOx1 nCoV-19 and Bharat BioTech\u2019s COVAXIN. The mole concept is used to estimate the antigen mass density per dose of each of these vaccines as 10 g cm-3, 0.1 g cm-3 and 1 g cm-3, respectively. The vaccines are deemed to be compatible to neutralize the infection. A statistical analysis is performed of the Moderna\u2019s mRNA-1273 vaccine efficacy of 94.1% and Oxford\u2019s ChAdOx1 nCoV-19 vaccine efficacy of 62.1% in terms of groups of volunteers testing negative to vaccine by chance. In the Moderna vaccination testing scenario, since the probability of negative response of vaccine is small, the Poisson probability distribution for 95% cumulative probability is used to describe the vaccination testing in 300 samples of 47 volunteers each. Thus, 87% of samples have average group of 3 volunteers testing negative to vaccine. About 6% of samples have all volunteers testing positive to vaccine. In the Oxford vaccination testing scenario, since the probability of negative response of vaccine is finite, the Gaussian probability distribution for 95% probability is used to describe the vaccination testing in 75 samples of 120 volunteers each. Thus, 68% of samples have average group of 45 volunteers testing negative to vaccine. No sample has all volunteers testing positive to vaccine. A vaccine, irrespective of its efficacy being high or low, is necessary for mass immunization.","version":"1.1","doi":"10.1101/2021.12.21.470882","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473447","pub_date":"2021-12-22","title":"Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by the N-terminal peptide of lactoferrin","abstract":"In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS\u2013CoV-2). The transmembrane protease serine 2 (TMPRSS2), which is responsible for the proteolytic processing of the SARS-CoV-2 spike protein as virus priming for cell entry, appears as a rational therapeutic target for the clearance of SARS-CoV-2 infection. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Here, we tested the inhibitory capacities of the human milk glycoprotein lactoferrin and its N-terminal peptide pLF1, which we identified as inhibitors of plasminogen, a serine protease homologous to TMPRSS2. In vitro proteolysis assays revealed that, unlike full-length lactoferrin, pLF1 significantly inhibited the proteolytic activity of TMPRSS2. pLF1 inhibited both the proteolytic processing of the SARS-CoV-2 spike protein and the SARS-CoV-2 infection of simian Vero cells. Because lactoferrin is a natural product and several biologically active peptides, such as the N-terminally derived lactoferricins, are produced naturally by pepsin-mediated digestion, natural or synthetic peptides from lactoferrin represent well-achievable candidates for supporting prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.12.20.473447","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.473668","pub_date":"2021-12-22","title":"Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants","abstract":"Vaccine hesitancy and continuing emergence of SARS-CoV-2 variants of concern that may escape vaccine-induced immune responses highlight the urgent need for effective COVID-19 therapeutics. Monoclonal antibodies used in the clinic have varying efficacies against distinct SARS-CoV-2 variants; thus, there is considerable interest in engineered ACE2 peptides with augmented binding affinities for SARS-CoV-2 Spike protein. These could have therapeutic benefit against multiple viral variants. Using molecular dynamics simulations, we show how three amino acid substitutions in an engineered soluble ACE2 peptide (sACE22.v2.4-IgG1) markedly increase affinity for the SARS-CoV-2 Spike (S) protein. We demonstrate high binding affinity to S protein of the early SARS-CoV-2 WA-1/2020 isolate and also to multiple variants of concern: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) SARS-CoV-2 variants. In humanized K18-hACE2 mice, prophylactic and therapeutic administration of sACE22.v2.4-IgG1 peptide prevented acute lung vascular endothelial injury and lung edema (essential features of ARDS) and significantly improved survival after infection by SARS-CoV-2 WA-1/2020 as well as P.1 variant of concern. These studies demonstrate for the first time broad efficacy in vivo of an ACE2 decoy peptide against multiple SARS-CoV-2 variants and point to its therapeutic potential.","version":"1.1","doi":"10.1101/2021.12.21.473668","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473523","pub_date":"2021-12-22","title":"Conjunctival epithelial cells resist productive SARS-CoV-2 infection","abstract":"Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-K\u03b2 activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.","version":"1.1","doi":"10.1101/2021.12.20.473523","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473584","pub_date":"2021-12-22","title":"Neutralization against Omicron SARS-CoV-2 from previous non-Omicron infection","abstract":"The explosive spread of the Omicron SARS-CoV-2 variant underscores the importance of analyzing the cross-protection from previous non-Omicron infection. We developed a high-throughput neutralization assay for Omicron SARS-CoV-2 by engineering the Omicron spike gene into an mNeonGreen USA-WA1/2020 SARS-CoV-2 (isolated in January 2020). Using this assay, we determined the neutralization titers of patient sera collected at 1- or 6-months after infection with non-Omicron SARS-CoV-2. From 1- to 6-month post-infection, the neutralization titers against USA-WA1/2020 decreased from 601 to 142 (a 4.2-fold reduction), while the neutralization titers against Omicron-spike SARS-CoV-2 remained low at 38 and 32, respectively. Thus, at 1- and 6-months after non-Omicron SARS-CoV-2 infection, the neutralization titers against Omicron were 15.8- and 4.4-fold lower than those against USA-WA1/2020, respectively. The low cross-neutralization against Omicron from previous non-Omicron infection supports vaccination of formerly infected individuals to mitigate the health impact of the ongoing Omicron surge.","version":"1.1","doi":"10.1101/2021.12.20.473584","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458225","pub_date":"2021-12-22","title":"Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape","abstract":"At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. Although a small, systematic trend towards NAb destabilization not observed for Delta or Gamma was detected for Omicron, and despite bearing significantly more RBD mutations, the epistatic landscape of the Omicron variant closely resembles that of Gamma. These results suggest that, although Omicron poses new risks not observed with Delta, structural constraints on the RBD hamper continued evolution towards more complete vaccine escape. The modest ensemble of mutations relative to the WT that are currently known to reduce vaccine efficacy is likely to comprise the majority of all possible escape mutations for future variants, predicting continued efficacy of the existing vaccines. Emergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. At the time of writing, December, 2021, Omicron is poised to replace Delta as the dominant variant worldwide. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the WT and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for the Delta variant, structural constraints on the RBD make continued evolution towards more complete vaccine escape unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.","version":"1.2","doi":"10.1101/2021.08.30.458225","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473613","pub_date":"2021-12-22","title":"The Geometry of ATG-Walks of the Omicron SARS CoV-2 Virus RNAs","abstract":"In this message, the complete RNA sequences (GISAID) of Omicron (BA.1 and BA.2) SARS CoV-2 viruses are studied using the genomic ATG-walks. These walks are compared visually and numerically with a reference RNA (Wuhan, China, 2020), and the deviation levels are estimated. Statistical characteristics of these distributions are compared, including the fractal dimension values of coding-word length distributions. Most of the 17 RNA ATG walks studied here show relatively small deviations of their characteristics and resistance to forming a new virus family.","version":"1.1","doi":"10.1101/2021.12.20.473613","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473557","pub_date":"2021-12-22","title":"mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant","abstract":"The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from na\u00efve vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.","version":"1.1","doi":"10.1101/2021.12.20.473557","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.473774","pub_date":"2021-12-22","title":"Longitudinal characterisation of phagocytic and neutralisation functions of anti-Spike antibodies in plasma of patients after SARS-CoV-2 infection","abstract":"Phagocytic responses by effector cells to antibody or complement-opsonised viruses have been recognized to play a key role in anti-viral immunity. These include antibody dependent cellular phagocytosis mediated via Fc-receptors, phagocytosis mediated by classically activated complement-fixing IgM or IgG1 antibodies and antibody independent phagocytosis mediated via direct opsonisation of viruses by complement products activated via the mannose-binding lectin pathway. Limited data suggest these phagocytic responses by effector cells may contribute to the immunological and inflammatory responses in SARS-CoV-2 infection, however, their development and clinical significance remain to be fully elucidated. In this cohort of 62 patients, acutely ill individuals were shown to mount phagocytic responses to autologous plasma-opsonised SARS-CoV-2 Spike protein-coated microbeads as early as 10 days post symptom onset. Heat inactivation of the plasma prior to use as an opsonin caused 77-95% abrogation of the phagocytic response, and pre-blocking of Fc-receptors on the effector cells showed only 18-60% inhibition. These results suggest that SARS-CoV-2 can provoke early phagocytosis, which is primarily driven by heat labile components, likely activated complements, with variable contribution from anti-Spike antibodies. During convalescence, phagocytic responses correlated significantly with anti-Spike IgG titers. Older patients and patients with severe disease had significantly higher phagocytosis and neutralisation functions when compared to younger patients or patients with asymptomatic, mild, or moderate disease. A longitudinal study of a subset of these patients over 12 months showed preservation of phagocytic and neutralisation functions in all patients, despite a drop in the endpoint antibody titers by more than 90%. Interestingly, surface plasmon resonance showed a significant increase in the affinity of the anti-Spike antibodies over time correlating with the maintenance of both the phagocytic and neutralisation functions suggesting that improvement in the antibody quality over the 12 months contributed to the retention of effector functions. Limited data suggest antibody dependent effector functions including phagocytosis may contribute to the immunological and inflammatory responses in SARS CoV-2 infection, however, their development, maintenance, and clinical significance remain unknown. In this study we show:\n\nPatients with acute SARS CoV-2 infection can mount phagocytic responses as early as 10 days post symptom onset and these responses were primarily driven by heat labile components of the autologous plasma. These results indicate that the current approach of studying phagocytosis using purified or monoclonal antibodies does not recapitulate contribution by all components in the plasma.\nIn convalescent patients, high phagocytic responses significantly correlated with increasing age, increasing disease severity, high neutralisation functions and high anti-Spike antibody titers, particularly IgG1.\nLongitudinal study of convalescent patients over a 12-month period showed maintenance of phagocytic and neutralisation functions, despite a drop in the anti-Spike endpoint antibody titers by more than 90%. However, we found significant increase in the affinity of the anti-Spike antibodies over the 12-month period and these correlated with the maintenance of functions suggesting that improvement in the antibody quality over time contributed to the retention of effector functions. Clinically, measuring antibody titers in sera but not the quality of antibodies is considered a gold standard indicator of immune protection following SARS-CoV 2 infection or vaccination. Our results challenge this notion and recommends change in the current clinical practice.\n Patients with acute SARS CoV-2 infection can mount phagocytic responses as early as 10 days post symptom onset and these responses were primarily driven by heat labile components of the autologous plasma. These results indicate that the current approach of studying phagocytosis using purified or monoclonal antibodies does not recapitulate contribution by all components in the plasma. In convalescent patients, high phagocytic responses significantly correlated with increasing age, increasing disease severity, high neutralisation functions and high anti-Spike antibody titers, particularly IgG1. Longitudinal study of convalescent patients over a 12-month period showed maintenance of phagocytic and neutralisation functions, despite a drop in the anti-Spike endpoint antibody titers by more than 90%. However, we found significant increase in the affinity of the anti-Spike antibodies over the 12-month period and these correlated with the maintenance of functions suggesting that improvement in the antibody quality over time contributed to the retention of effector functions. Clinically, measuring antibody titers in sera but not the quality of antibodies is considered a gold standard indicator of immune protection following SARS-CoV 2 infection or vaccination. Our results challenge this notion and recommends change in the current clinical practice.","version":"1.1","doi":"10.1101/2021.12.21.473774","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.18.21268018","pub_date":"2021-12-22","title":"Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The emergence of each novel SARS-CoV-2 variants of concern (VOCs) requires investigation of its potential impact on the performance of diagnostic tests in use, including Antigen-detecting rapid diagnostic tests (Ag-RDT). Although anecdotal reports have been circulating that the newly emerged Omicron variant is in principle detectable by Ag-RDTs, few data on sensitivity are available.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We have performed 1) analytical sensitivity testing with cultured virus in eight Ag-RDTs and 2) retrospective testing in duplicates with clinical samples from vaccinated individuals with Omicron (n=18) or Delta (n=17) breakthrough infection on seven Ag-RDTs.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Overall, we have found large heterogenicity between Ag-RDTs for detecting Omicron. When using cultured virus, we observed a trend towards lower sensitivity for Omicron detection compared to earlier circulating SARS-CoV-2 and the other VOCs. When comparing performance for Delta and Omicron in a comparable set of clinical samples in seven Ag-RDTs, 124/252 (49.2%) of all test performed showed a positive result for Omicron compared to 156/238 (65.6%) for Delta samples. Sensitivity for both Omicron and Delta between Ag-RDTs was highly variable. Four out of seven Ag-RDTs showed significantly lower sensitivity (p&lt;0.001) to detect Omicron when compared to Delta while three had comparable sensitivity to Delta.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Sensitivity for detecting Omicron is highly variable between Ag-RDTs, necessitating a careful consideration when using these tests to guide infection prevention measures. While analytical and retrospective testing may be a proxy and timely solution to generate performance data, it is not a replacement for clinical evaluations which are urgently needed. Biological and technical reasons for detection failure by some Ag-RDTs need to be further investigated.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This work was supported by the Swiss National Science Foundation (grant numbers 196383, 196644 and 198412), the Fondation Ancrage Bienfaisance du Groupe Pictet, the Fondation Priv\u00e9e des H\u00f4piteaux Universitaires de Gen\u00e8ve and FIND, the global alliance for diagnostics.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.12.18.21268018","journal":"medRxiv","score":null},{"id":"10.1101/2021.12.07.470392","pub_date":"2021-12-22","title":"Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies","abstract":"The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) requires immediate investigation. Here, we used high-throughput yeast display screening to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs and showed that the NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlap with ACE2-binding motif, are largely escaped by K417N, G446S, E484A, and Q493R. Group E (S309 site) and F (CR3022 site) NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, N440K, and S371L. Furthermore, Omicron pseudovirus neutralization showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, the neutralization potency of LY-CoV016/LY-CoV555, REGN10933/REGN10987, AZD1061/AZD8895, and BRII-196 were greatly reduced by Omicron, while VIR-7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron would cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.","version":"1.2","doi":"10.1101/2021.12.07.470392","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.21.473528","pub_date":"2021-12-22","title":"Immune escape of SARS-CoV-2 Omicron variant from mRNA vaccination-elicited RBD-specific memory B cells","abstract":"Memory B cells (MBCs) represent a second layer of immune protection against SARS-CoV-2. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant is of major concern. We used bio-layer interferometry to assess the affinity against the receptor-binding-domain (RBD) of Omicron spike of 313 naturally expressed monoclonal IgG that were previously tested for affinity and neutralization against VOC prior to Omicron. We report here that Omicron evades recognition from a larger fraction of these antibodies than any of the previous VOCs. Additionally, whereas 30% of these antibodies retained high affinity against Omicron-RBD, our analysis suggest that Omicron specifically evades antibodies displaying potent neutralizing activity against the D614G and Beta variant viruses. Further studies are warranted to understand the consequences of a lower memory B cell potency on the overall protection associated with current vaccines.","version":"1.1","doi":"10.1101/2021.12.21.473528","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473421","pub_date":"2021-12-22","title":"Virus-like particles (VLPs) are efficient tools for boosting mRNA-induced antibodies","abstract":"mRNA based vaccines against COVID-19 have proven most successful at keeping the SARS-CoV-2 pandemic at bay in many countries. Recently, there is an increased interest in heterologous prime-boost vaccination strategies for COVID-19 to maintain antibody response for the control of continuously emerging SARS-CoV-2 variants of concern (VoCs) and to overcome other obstacles such as supply shortage, costs and reduced safety issues or inadequate induced immune-response. In this study, we investigate the antibody responses induced by heterologous prime-boost with vaccines based on mRNA and virus-like particles (VLPs). The VLP-based mCuMVTT-RBM vaccine candidate and the approved mRNA-1273 vaccine were used for this purpose. We find that homologous prime boost regimens with either mRNA or VLP induced high levels of high avidity antibodies. Optimal antibody responses were, however, induced by heterologous regimens both for priming with mRNA and boosting with VLP and vice versa, priming with VLP and boosting with mRNA. Thus, heterologous prime boost strategies may be able to optimize efficacy and economics of novel vaccine strategies.","version":"1.1","doi":"10.1101/2021.12.20.473421","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.03.471045","pub_date":"2021-12-22","title":"Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses","abstract":"On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.","version":"1.2","doi":"10.1101/2021.12.03.471045","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472719","pub_date":"2021-12-21","title":"Striking Antibody Evasion Manifested by the Omicron Variant of SARS-CoV-2","abstract":"The Omicron (B.1.1.529) variant of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally. It is expected to become dominant in the coming weeks, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations that pose a threat to the efficacy of current COVID-19 (coronavirus disease 2019) vaccines and antibody therapies. This concern is amplified by the findings from our study. We found B.1.1.529 to be markedly resistant to neutralization by serum not only from convalescent patients, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines. Even serum from persons vaccinated and boosted with mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies to all known epitope clusters on the spike protein, we noted that the activity of 17 of the 19 antibodies tested were either abolished or impaired, including ones currently authorized or approved for use in patients. In addition, we also identified four new spike mutations (S371L, N440K, G446S, and Q493R) that confer greater antibody resistance to B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.","version":"1.3","doi":"10.1101/2021.12.14.472719","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.19.473391","pub_date":"2021-12-21","title":"SARS-CoV-2 spike conformation determines plasma neutralizing activity","abstract":"Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV-2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.","version":"1.1","doi":"10.1101/2021.12.19.473391","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473401","pub_date":"2021-12-21","title":"Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns","abstract":"Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.","version":"1.1","doi":"10.1101/2021.12.20.473401","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.19.473359","pub_date":"2021-12-21","title":"Pentosan polysulfate inhibits attachment and infection by SARS-CoV-2 in vitro: insights into structural requirements for binding","abstract":"Two years since the outbreak of the novel coronavirus SARS-CoV-2 pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side-effect. One alternative, with structural similarities to heparin is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights, inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using NMR spectroscopy and LC-MS, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective at inhibiting cell infection than low molecular weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.12.19.473359","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473542","pub_date":"2021-12-21","title":"Structural flexibility of the SARS-CoV-2 genome relevant to variation, replication, pathogenicity, and immune evasion","abstract":"The SARS-CoV-2 pandemic continues to be driven by viral variants. Most research has focused on structural proteins and on site-specific mutations. Here, we describe recombination events involving genomic terminal sequences in SARS-CoV-2 and related viruses leading to structural rearrangements in terminal and coding regions and discuss their potential contributions to viral variation, replication, pathogenicity, and immune evasion.","version":"1.1","doi":"10.1101/2021.12.20.473542","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.19.21268073","pub_date":"2021-12-21","title":"COVID-19 pandemic dynamics in South Africa and epidemiological characteristics of three variants of concern (Beta, Delta, and Omicron)","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have been key drivers of new coronavirus disease 2019 (COVID-19) pandemic waves. To better understand variant epidemiologic characteristics, here we apply a model-inference system to reconstruct SARS-CoV-2 transmission dynamics in South Africa, a country that has experienced three VOC pandemic waves (i.e. Beta, Delta, and Omicron). We estimate key epidemiologic quantities in each of the nine South African provinces during March 2020 \u2013 Feb 2022, while accounting for changing detection rates, infection seasonality, nonpharmaceutical interventions, and vaccination. Model validation shows that estimated underlying infection rates and key parameters (e.g., infection-detection rate and infection-fatality risk) are in line with independent epidemiological data and investigations. In addition, retrospective predictions capture pandemic trajectories beyond the model training period. These detailed, validated model-inference estimates thus enable quantification of both the immune erosion potential and transmissibility of three major SARS-CoV-2 VOCs, i.e., Beta, Delta, and Omicron. These findings help elucidate changing COVID-19 dynamics and inform future public health planning.</jats:p>","version":null,"doi":"10.1101/2021.12.19.21268073","journal":"medRxiv","score":null},{"id":"10.1101/2021.12.18.473303","pub_date":"2021-12-21","title":"A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors","abstract":"Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 \u00b5M range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.","version":"1.1","doi":"10.1101/2021.12.18.473303","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.18.473330","pub_date":"2021-12-21","title":"The N-Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors","abstract":"Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an \u03b1-ketoamide warhead, a P1 \u03b2-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butyl-glycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and human host cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a \u03b2-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains an P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed MPro in human 293T cells. All inhibitors including PF-07321332 with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC50 values around 1 \u03bcM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.12.18.473330","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.18.473326","pub_date":"2021-12-21","title":"The P3 O-Tert-Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors","abstract":"As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain \u03b2-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the \u03b1-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.12.18.473326","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.19.473380","pub_date":"2021-12-21","title":"SARS-CoV-2 Omicron Variant: ACE2 Binding, Cryo-EM Structure of Spike Protein-ACE2 Complex and Antibody Evasion","abstract":"The newly reported Omicron variant is poised to replace Delta as the most rapidly spread SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, explaining our finding of similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion, with greater evasion observed in sera obtained from unvaccinated convalescent patients as compared to doubly vaccinated individuals (8-vs 3-fold). The retention of strong interactions at the ACE2 interface and the increase in antibody evasion are molecular factors that likely contribute to the increased transmissibility of the Omicron variant.","version":"1.1","doi":"10.1101/2021.12.19.473380","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.20.473564","pub_date":"2021-12-21","title":"MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells","abstract":"Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS-CoV is adept at antagonizing early innate immune pathways \u2013 interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L) \u2013 generated in response to viral double-stranded (ds)RNA generated during genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes highly lethal respiratory disease. MERS-CoV encodes several innate immune antagonists, accessory proteins NS4a and NS4b unique to the merbeco lineage and the nsp15 protein endoribonuclease (EndoU), conserved among all coronaviruses. While mutation of each antagonist protein alone has little effect on innate immunity, infections with recombinant MERS-CoVs with mutations of EndoU in combination with either NS4a or NS4b, activate innate signaling pathways and are attenuated for replication. Our data indicate that EndoU and accessory proteins NS4a and NS4b together suppress innate immunity during MERS-CoV infection, to optimize viral replication. This is in contrast to SARS-CoV-2 which activates these pathways and consistent with greater mortality observed during MERS-CoV infection compared to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.12.20.473564","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472745","pub_date":"2021-12-21","title":"Sentimental Tweets Classification of Symptomatic COVID-19","abstract":"The approach I described is straightforward, related to COVID-19 SARS based tweets and the symptoms, that people tweet about. Also, social media mining for health application reports was shared in many different tasks of 2021. The motto at the back of this observe is to analyses tweets of COVID-19 based symptoms. By performing BERT model and text classification with XLNET with which uses to classify text and purpose of the texts (i.e.) tweets. So that I can get a deep understanding of the texts. When developing the system, I used two models the XLNet and DistilBERT for the text sorting task, but the outcome was XLNET out-performs the given approach to the best accuracy achieved. Now I discover a whole lot vital for as it should be categorizing tweets as encompassing self-said COVID-19 indications. Whether or not a tweets associated with COVID-19 is a non-public report or an information point out to the virus. Which gives test accuracy to an F1 score of 96%.","version":"1.1","doi":"10.1101/2021.12.15.472745","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.18.473317","pub_date":"2021-12-21","title":"Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen","abstract":"Spacing of the BNT162b2 mRNA doses beyond 3 weeks raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 na\u00efve and previously-infected donors. This regimen elicited robust RBD-specific B cell responses whose kinetics differed between cohorts, the second dose leading to increased magnitude in na\u00efve participants only. While boosting did not increase magnitude of CD4+ T cell responses further compared to the first dose, unsupervised clustering analyses of single-cell features revealed phenotypic and functional shifts over time and between cohorts. Integrated analysis showed longitudinal immune component-specific associations, with early Thelper responses post-first dose correlating with B cell responses after the second dose, and memory Thelper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.","version":"1.1","doi":"10.1101/2021.12.18.473317","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459742","pub_date":"2021-12-21","title":"Optimal strategies to protect a sub-population at risk due to an established epidemic","abstract":"Epidemics can particularly threaten certain sub-populations. For example, for SARS-CoV-2, the elderly are often preferentially protected. For diseases of plants and animals, certain sub-populations can drive mitigation because they are intrinsically more valuable for ecological, economic, socio-cultural or political reasons. Here we use optimal control theory to identify strategies to optimally protect a \u201chigh value\u201d sub-population when there is a limited budget and epidemiological uncertainty. We use protection of the Redwood National Park in California in the face of the large ongoing state-wide epidemic of sudden oak death (caused by Phytophthora ramorum) as a case study. We concentrate on whether control should be focused entirely within the National Park itself, or whether treatment of the growing epidemic in the surrounding \u201cbuffer region\u201d can instead be more profitable. We find that, depending on rates of infection and the size of the ongoing epidemic, focusing control on the high value region is often optimal. However, priority should sometimes switch from the buffer region to the high value region only as the local outbreak grows. We characterise how the timing of any switch depends on epidemiological and logistic parameters, and test robustness to systematic misspecification of these factors due to imperfect prior knowledge.","version":"1.2","doi":"10.1101/2021.09.10.459742","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.472912","pub_date":"2021-12-20","title":"Analysis of SARS-CoV-2 synonymous codon usage evolution throughout the COVID-19 pandemic","abstract":"SARS-CoV-2, the seventh coronavirus known to infect humans, can cause severe life-threatening respiratory pathologies. To better understand SARS-CoV-2 evolution, genome-wide analyses have been made, including the general characterization of its codons usage profile. Here we present a bioinformatic analysis of the evo-lution of SARS-CoV-2 codon usage over time using complete genomes collected since December 2019. Our results show that SARS-CoV-2 codon usage pattern is antagonistic to, and it is getting farther away from that of the human host. Further, a selection of deoptimized codons over time, which was accompanied by a decrease in both the codon adaptation index and the effective number of codons, was observed. All together, these findings suggest that SARS-CoV-2 could be evolving, at least from the perspective of the synonymous codon usage, to become less pathogenic.","version":"1.1","doi":"10.1101/2021.12.17.472912","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.473096","pub_date":"2021-12-20","title":"The rise and fall of SARS-CoV-2 variants and the mutational profile of Omicron","abstract":"In late December of 2019, high throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we analyze a subset (n=87,032) of all publicly available SARS-CoV-2 genomes (n=\u223c5.6 million) that were randomly selected, but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron viruses are significantly greater than those in previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike protein, while 25 other viral proteins\u2014 including those involved in SARS-CoV-2 replication\u2014are highly conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered. Omicron is the fifth SARS-CoV-2 variant to be designated a Variant of Concern (VOC) by the World Health Organization (WHO). Here we provide a retrospective analysis of SARS-CoV-2 variants and explain how the Omicron variant is distinct. Our work shows that the spike protein is a \u2018hotspot\u2019 for viral evolution in all variants, suggesting that existing vaccines and diagnostics that target this protein may become less effective against Omicron and that our therapeutic and public health strategies will have to evolve along with the virus.","version":"1.1","doi":"10.1101/2021.12.16.473096","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472982","pub_date":"2021-12-20","title":"Changes of small non-coding RNAs by severe acute respiratory syndrome coronavirus 2 infection","abstract":"The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat, with molecular mechanisms underlying its pathogenesis largely unknown. Small non-coding RNAs (sncRNAs) are known to play important roles in almost all biological processes. In the context of viral infections, sncRNAs have been shown to regulate the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK-RNA-seq, a modified next-generation sequencing (NGS), we recently found that nasopharyngeal swabs (NPS) samples from SARS-CoV-2 positive and negative subjects show a significant difference in sncRNA profiles. There are about 166 SARS-CoV-2-impacted sncRNAs. Among them, tRFs are the most significantly affected and almost all impacted tRFs are derived from the 5\u2019-end of tRNAs (tRF5). Using a modified qRT-PCR, which was recently developed to specifically quantify tRF5s by isolating the tRF signals from its corresponding parent tRNA signals, we validated that tRF5s derived from tRNA GluCTC (tRF5-GluCTC), LysCTT (tRF5-LysCTT), ValCAC (tRF5-ValCAC), CysGCA (tRF5-CysGCA) and GlnCTG (tRF5-GlnCTG) are enhanced in NPS samples of SARS-CoV2 patients and SARS-CoV2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several sncRNAs derived from the virus (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3\u2019-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs.","version":"1.1","doi":"10.1101/2021.12.16.472982","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473113","pub_date":"2021-12-20","title":"Pre-clinical evaluation of antiviral activity of nitazoxanide against Sars-CoV-2","abstract":"To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy. In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2. First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50. These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19.","version":"1.1","doi":"10.1101/2021.12.17.473113","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473223","pub_date":"2021-12-20","title":"ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection","abstract":"Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19. Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchioalveolar lavage fluid from critically ill COVID-19 patients was associated with reduced ICU and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.","version":"1.1","doi":"10.1101/2021.12.17.473223","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473170","pub_date":"2021-12-20","title":"Structure prediction of the druggable fragments in SARS-CoV-2 untranslated regions","abstract":"The outbreak of the COVID-19 pandemic has led to intensive studies of both the structure and replication mechanism of SARS-CoV-2. In spite of some secondary structure experiments being carried out, the 3D structure of the key function regions of the viral RNA has not yet been well understood. At the beginning of COVID-19 breakout, RNA-Puzzles community attempted to envisage the three-dimensional structure of 5\u2032- and 3\u2032-Un-Translated Regions (UTRs) of the SARS-CoV-2 genome. Here, we report the results of this prediction challenge, presenting the methodologies developed by six participating groups and discussing 100 RNA 3D models (60 models of 5\u2032-UTR and 40 of 3\u2032-UTR) predicted through applying both human experts and automated server approaches. We describe the original protocol for the reference-free comparative analysis of RNA 3D structures designed especially for this challenge. We elaborate on the deduced consensus structure and the reliability of the predicted structural motifs. All the computationally simulated models, as well as the development and the testing of computational tools dedicated to 3D structure analysis, are available for further study.","version":"1.1","doi":"10.1101/2021.12.17.473170","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472269","pub_date":"2021-12-20","title":"Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift","abstract":"The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab, S2X259 and S2H97, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.","version":"1.2","doi":"10.1101/2021.12.12.472269","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473180","pub_date":"2021-12-20","title":"Insights into standards of care \u2013 dexamethasone and antibodies against COVID-19 in hamster models","abstract":"In face of the ongoing SARS-CoV-2 pandemic, effective and well-understood treatment options are still scarce. While vaccines have proven instrumental in fighting SARS-CoV-2, their efficacy is challenged by vaccine hesitancy, novel variants and short-lasting immunity. Therefore, understanding and optimization of therapeutic options remains essential. We aimed at generating a deeper understanding on how currently used drugs, specifically dexamethasone and anti-SARS-CoV-2 antibodies, affect SARS-CoV-2 infection and host responses. Possible synergistic effects of both substances are investigated to evaluate combinatorial treatments. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of treatment with either dexamethasone, anti-SARS-CoV-2 spike monoclonal antibody or a combination of both. scRNA sequencing was employed to reveal transcriptional response to treatment on a single cell level. Dexamethasone treatment resulted in similar or increased viral loads compared to controls. Anti-SARS-CoV-2 antibody treatment alone or combined with dexamethasone successfully reduced pulmonary viral burden. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe COVID-19-like disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a candidate subpopulation of neutrophils specifically responsive to dexamethasone. Our analyses i) confirm the anti-inflammatory properties and indicate possible modes of action for dexamethasone, ii) validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and iii) reveal synergistic effects of a combination therapy and can thus inform more effective COVID-19 therapies.","version":"1.1","doi":"10.1101/2021.12.17.473180","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.18.473308","pub_date":"2021-12-20","title":"Neutrophil Profiles of Pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children","abstract":"Multisystem Inflammatory Syndrome in Children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory systemic illness characterized by SARS-CoV-2 antigenemia, cytokine storm and immune dysregulation; however, the role of the neutrophil has yet to be defined. In adults with severe COVID-19, neutrophil activation has been shown to be central to overactive inflammatory responses and complications. Thus, we sought to define neutrophil activation in children with MIS-C and acute COVID-19. We collected samples from 141 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 67 pediatric controls. We found that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism, which is markedly different than the neutrophil interferon-stimulated gene (ISG) response observed in pediatric patients during acute SARS-CoV-2 infection. Moreover, we identified signatures of neutrophil activation and degranulation with high levels of spontaneous neutrophil extracellular trap (NET) formation in neutrophils isolated from fresh whole blood of MIS-C patients. Mechanistically, we determined that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Overall, our findings suggest that the hyperinflammatory presentation of MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia through uncontrolled neutrophil activation and NET release in the vasculature. Circulating SARS-CoV-2 antigen:antibody immune complexes in Multisystem Inflammatory Syndrome in Children (MIS-C) drive hyperinflammatory and coagulopathic neutrophil extracellular trap (NET) formation and neutrophil activation pathways, providing insight into disease pathology and establishing a divergence from neutrophil signaling seen in acute pediatric COVID-19.","version":"1.1","doi":"10.1101/2021.12.18.473308","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.18.456880","pub_date":"2021-12-20","title":"Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-care Platform","abstract":"Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples from COVID-19 hotspots. RT\u25a1LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.","version":"1.3","doi":"10.1101/2021.08.18.456880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473105","pub_date":"2021-12-20","title":"Modeling and predicting the overlap of B- and T-cell receptor repertoires in healthy and SARS-CoV-2 infected individuals","abstract":"Adaptive immunity\u2019s success relies on the extraordinary diversity of protein receptors on B and T cell membranes. Despite this diversity, the existence of public receptors shared by many individuals gives hope for developing population wide vaccines and therapeutics. Yet many of these public receptors are shared by chance. We present a statistical approach, defined in terms of a probabilistic V(D)J recombination model enhanced by a selection factor, that describes repertoire diversity and predicts with high accuracy the spectrum of repertoire overlap in healthy individuals. The model underestimates sharing between repertoires of individuals infected with SARS-CoV-2, suggesting strong antigen-driven convergent selection. We exploit this discrepancy to identify COVID-associated receptors, which we validate against datasets of receptors with known viral specificity. We study their properties in terms of sequence features and network organization, and use them to design an accurate diagnosis tool for predicting SARS-CoV-2 status from repertoire data.","version":"1.1","doi":"10.1101/2021.12.17.473105","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473178","pub_date":"2021-12-20","title":"Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization","abstract":"Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we report the discovery of a novel antibody fragment (VH ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.","version":"1.1","doi":"10.1101/2021.12.17.473178","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473260","pub_date":"2021-12-20","title":"A Computational Dissection of Spike protein of SARS-CoV-2 Omicron Variant","abstract":"The emergence of SARS-CoV-2 omicron variant in late November, 2021 and its rapid spread to different countries, warns the health authorities to take initiative to work on containing its spread. The omicron SARS-CoV-2 variant is unusual from the other variants of concerns reported earlier as it harbors many novel mutations in its genome particularly with >30 mutations in the spike glycoprotein alone. The current study investigated the variation in binding mechanism which it carries compared to the wild type. The study also explored the interaction profile of spike-omicron with human ACE2 receptor. The structure of omicron spike glycoprotein was determined though homology modeling. The interaction analysis was performed through docking using HADDOCK followed by binding affinity calculation. Finally, the comparison of interactions were performed among spike-ACE2 complex of wild type, delta and omicron variants. The interaction analysis has revealed the involvement of highly charged and polar residues (H505, Arg498, Ser446, Arg493, and Tyr501) in the interactions. The important novel interactions in the spike-ACE2-omicron complex was observed as S494:H34, S496:D38, R498:Y41, Y501:K353, and H505:R393 and R493:D38. Moreover, the binding affinity of spike-ACE2-omicron complex (\u221217.6Kcal/mol) is much higher than wild type-ACE2 (\u221213.2Kcal/mol) and delta-ACE2 complex (\u221213.3Kcal/mol). These results indicate that the involvement of polar and charged residues in the interactions with ACE2 may have an impact on increased transmissibility of omicron variant.","version":"1.1","doi":"10.1101/2021.12.17.473260","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472155","pub_date":"2021-12-20","title":"Vandetanib Reduces Inflammatory Cytokines and Ameliorates COVID-19 in Infected Mice","abstract":"The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib) or in advanced clinical trials. We have tested 45 FDA-approved kinase inhibitors in vitro against murine hepatitis virus (MHV) as a model of SARS-CoV-2 replication and identified 12 showing inhibition in the delayed brain tumor (DBT) cell line. Vandetanib, which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), and the RET-tyrosine kinase showed the most promising results on inhibition versus toxic effect on SARS-CoV-2-infected Caco-2 and A549-hACE2 cells (IC50 0.79 \u03bcM) while also showing a reduction of > 3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, TNF-\u03b1, and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib rescued the decreased IFN-1\u03b2 caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved vandetanib is a potential therapeutic candidate for COVID-19 positioned for follow up in clinical trials either alone or in combination with other drugs to address the cytokine storm associated with this viral infection.","version":"1.1","doi":"10.1101/2021.12.16.472155","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472843","pub_date":"2021-12-20","title":"SARS-COV-2 Omicron variant predicted to exhibit higher affinity to ACE-2 receptor and lower affinity to a large range of neutralizing antibodies, using a rapid computational platform","abstract":"Rapid assessment of whether a pandemic pathogen may have increased transmissibility or be capable of evading existing vaccines and therapeutics is critical to mounting an effective public health response. Over the period of seven days, we utilized rapid computational prediction methods to evaluate potential public health implications of the emerging SARS-CoV-2 Omicron variant. Specifically, we modeled the structure of the Omicron variant, examined its interface with human angiotensin converting enzyme 2 (ACE-2) and evaluated the change in binding affinity between Omicron, ACE-2 and publicly known neutralizing antibodies. We also compared the Omicron variant to known Variants of Concern (VoC). Seven of the 15 Omicron mutations occurring in the spike protein receptor binding domain (RBD) occur at the ACE-2 cell receptor interface, and therefore may play a critical role in enhancing binding to ACE-2. Our estimates of Omicron RBD-ACE-2 binding affinities indicate that at least two of RBD mutations, Q493K/R and N501Y, contribute to enhanced ACE-2 binding, nearly doubling delta-delta-G (ddG) free energies calculated for other VoC\u2019s. Binding affinity estimates also were calculated for 54 known neutralizing SARS-CoV-2 antibodies. Analysis of the results showed that Omicron substantially degrades binding for more than half of these neutralizing SARS-CoV-2 antibodies, and for roughly twice as many of the antibodies than the currently dominant Delta variant. This early study lends support to use of rapid computational risk assessments to inform public health decision-making while awaiting detailed experimental characterization and confirmation. The recently emerged Omicron variant of SARS-CoV-2 raised significant concerns on whether this variant, compared with the currently dominant Delta variant, will increase transmissibility and degrade immunity from vaccines or protection from neutralizing antibodies. This concern is primarily driven by an exceptionally large number of mutations present on the RBD, including several mutations that have not previously been observed in widely circulating variants. Stabilized expression of this substantially modified spike/RBD followed by in-vitro assessment of affinity and neutralization to antibodies and receptors is the gold standard investigational avenue, but takes several weeks even with the heightened urgency surrounding this variant. On November 26, prior to experimental data/results being made public, we initiated an effort to apply our computational prediction platform to address the following questions.\n\nDoes Omicron affect affinity to ACE-2, with implications for transmissibility and infectivity?\nCompared to previous SARS-CoV-2 variants, does Omicron affect/alter recognition by antibodies generated via vaccination or infection/exposure, with implications for adequacy of current countermeasures (monoclonal antibodies and vaccines)?\n\n Does Omicron affect affinity to ACE-2, with implications for transmissibility and infectivity? Compared to previous SARS-CoV-2 variants, does Omicron affect/alter recognition by antibodies generated via vaccination or infection/exposure, with implications for adequacy of current countermeasures (monoclonal antibodies and vaccines)? Rapid availability of reliable predictions can help provide grounding data for decisions on preparedness and tailored response to emerging variants. We have been developing computational methods to rapidly construct structural models of modified RBD\u2019s and predict affinity to ACE-2 or neutralizing antibodies. A manuscript thoroughly describing our method, together with experimental validation demonstrating its accuracy, is in preparation and will be publicly available at a future date. The platform is designed to provide robust and reproducible computational predictions over a large number of binding complexes (multiple variant RBDs, multiple antibodies), with an interpretable confidence estimate, in a matter of days.","version":"1.1","doi":"10.1101/2021.12.16.472843","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472934","pub_date":"2021-12-20","title":"Neutralization and Stability of SARS-CoV-2 Omicron Variant","abstract":"The SARS-CoV-2 B.1.1.529/Omicron variant was first characterized in South Africa and was swiftly designated a variant of concern. Of great concern is its high number of mutations, including 30-40 mutations in the virus spike (S) protein compared to 7-10 for other variants. Some of these mutations have been shown to enhance escape from vaccine-induced immunity, while others remain uncharacterized. Additionally, reports of increasing frequencies of the Omicron variant may indicate a higher rate of transmission compared to other variants. However, the transmissibility of Omicron and its degree of resistance to vaccine-induced immunity remain unclear. Here we show that Omicron exhibits significant immune evasion compared to other variants, but antibody neutralization is largely restored by mRNA vaccine booster doses. Additionally, the Omicron spike exhibits reduced receptor binding, cell-cell fusion, S1 subunit shedding, but increased cell-to-cell transmission, and homology modeling indicates a more stable closed S structure. These findings suggest dual immune evasion strategies for Omicron, due to altered epitopes and reduced exposure of the S receptor binding domain, coupled with enhanced transmissibility due to enhanced S protein stability. These results highlight the importance of booster vaccine doses for maintaining protection against the Omicron variant, and provide mechanistic insight into the altered functionality of the Omicron spike protein.","version":"1.1","doi":"10.1101/2021.12.16.472934","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.19.473354","pub_date":"2021-12-20","title":"Variable loss of antibody potency against SARS-CoV-2 B.1.1.529 (Omicron)","abstract":"The recently-emerged SARS-CoV-2 B.1.1.529 variant (Omicron) is spreading rapidly in many countries, with a spike that is highly diverged from the pandemic founder, raising fears that it may evade neutralizing antibody responses. We cloned the Omicron spike from a diagnostic sample which allowed us to rapidly establish an Omicron pseudotyped virus neutralization assay, sharing initial neutralization results only 13 days after the variant was first reported to the WHO, 8 days after receiving the sample. Here we show that Omicron is substantially resistant to neutralization by several monoclonal antibodies that form part of clinical cocktails. Further, we find neutralizing antibody responses in pooled reference sera sampled shortly after infection or vaccination are substantially less potent against Omicron, with neutralizing antibody titers reduced by up to 45 fold compared to those for the pandemic founder. Similarly, in a cohort of convalescent sera prior to vaccination, neutralization of Omicron was low to undetectable. However, in recent samples from two cohorts from Stockholm, Sweden, antibody responses capable of cross-neutralizing Omicron were prevalent. Sera from infected-then-vaccinated healthcare workers exhibited robust cross-neutralization of Omicron, with an average potency reduction of only 5-fold relative to the pandemic founder variant, and some donors showing no loss at all. A similar pattern was observed in randomly sampled recent blood donors, with an average 7-fold loss of potency. Both cohorts showed substantial between-donor heterogeneity in their ability to neutralize Omicron. Together, these data highlight the extensive but incomplete evasion of neutralizing antibody responses by the Omicron variant, and suggest that increasing the magnitude of neutralizing antibody responses by boosting with unmodified vaccines may suffice to raise titers to levels that are protective.","version":"1.1","doi":"10.1101/2021.12.19.473354","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473179","pub_date":"2021-12-20","title":"Disrupted Peyer\u2019s patch microanatomy in COVID-19 including germinal centre atrophy independent of local virus","abstract":"Confirmed SARS-coronavirus-2 infection with gastrointestinal symptoms and changes in microbiota associated with coronavirus disease 2019 (COVID-19) severity have been previously reported, but the disease impact on the architecture and cellularity of ileal Peyer\u2019s patches (PP) remains unknown. Here we analysed post-mortem tissues from throughout the gastrointestinal (GI) tract of patients who died with COVID-19. When virus was detected by PCR in the GI tract, immunohistochemistry identified virus in epithelium and lamina propria macrophages, but not in lymphoid tissues. Immunohistochemistry and imaging mass cytometry (IMC) analysis of ileal PP revealed depletion of germinal centres (GC), disruption of B cell/T cell zonation and decreased potential B and T cell interaction and lower nuclear density in COVID-19 patients. This occurred independent of the local viral levels. The changes in PP demonstrate that the ability to mount an intestinal immune response is compromised in severe COVID-19, which could contribute to observed dysbiosis.","version":"1.1","doi":"10.1101/2021.12.17.473179","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472880","pub_date":"2021-12-20","title":"Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis","abstract":"The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.","version":"1.1","doi":"10.1101/2021.12.16.472880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472976","pub_date":"2021-12-20","title":"Single Amino Acid Change Mutation in the Hydrophobic Core of the N-terminal Domain of P22 TSP affects the Proteins Stability","abstract":"The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly shifted the attention of researchers to critically investigate most viruses to understand specific characteristics that impart their virulence. For instance, the SARS-CoV-2 has undergone several mutations, with some variants classified as \u201cvariants of concern\u201d, e.g., the Omicron and Delta variant of SARS-CoV-2 are known for their rapid transmission and antigenicity due to mutation in the Spike protein. P22 bacteriophage is a bacterial virus that has a tailspike protein (TSP) that performs similar functions as the Spike protein of SARS-COV-2. We previously carried out a site-directed mutagenesis of the P22 TSP to bear disruptive mutations in the hydrophobic core of the N-terminal Domain (NTD), then partially characterized the properties of the mutant TSPs. In this process, the valine patch (triple valine residues that formed a hydrophobic core) was replaced with charged amino acids (Asp or lysine) or hydrophobic amino acids (Leucine or isoleucine). Some of the mutant TSPs characterized showed significant differences in migration in both native and SDS-PAGE. Mutants with such disruptive mutation are known to show non-native properties, and as expected, most of these mutants obtained showed significantly different properties from the WT P22 TSP. In this work, we further characterized these mutant species by computational and in vitro assays to demonstrate the validity of our previous inference that the valine patch is a critical player in the stability of the N-terminal domain of the P22 TSP.","version":"1.1","doi":"10.1101/2021.12.16.472976","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472391","pub_date":"2021-12-20","title":"Reduced sera neutralization to Omicron SARS-CoV-2 by both inactivated and protein subunit vaccines and the convalescents","abstract":"Omicron variant continues to spread all over the world. There are lots of scientific questions remaining to be answered for such a devastating variant. There are a dozen of vaccines already in clinical use. The very urgent scientific question would be whether or not these vaccines can protect Omicron variant. Here, we tested the sera from both convalescents and vaccine recipients receiving either inactivated or protein subunits vaccines (CoronaVac from Sinovac, or BBIBP-CoV from Sinopharm, or ZF2001 from Zhifei longcom) for the binding antibody titers (ELISA) and neutralization antibodies titers (pseudovirus neutralization assay). We showed that Omicron do have severe immune escape in convalescents, with 15 of 16 were negative in neutralization. By contrast, in vaccinees who received three jabs of inactivated or protein subunit vaccine, the neutralizing activity was much better preserved. Especially in the ZF2001 group with an extended period of the second and third jab (4-6 months) remains 100% positive in Omicron neutralization, with only 3.1-folds reduction in neutralizing antibody (NAb) titer. In this case, we proposed that, the multi-boost strategy with an extended interval between the second and third jab for immune maturation would be beneficial for NAb against devastating variants such as Omicron.","version":"1.1","doi":"10.1101/2021.12.16.472391","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.473025","pub_date":"2021-12-20","title":"In vitro and computational analysis of the putative furin cleavage site (RRARS) in the divergent spike protein of the rodent coronavirus AcCoV-JC34 (sub-genus luchacovirus)","abstract":"The Coronaviridae is a highly diverse virus family, with reservoir hosts in a variety of wildlife species that encompass bats, birds and small mammals, including rodents. Within the taxonomic group alphacoronavirus, certain sub-genera (including the luchacoviruses) have phylogenetically distinct spike proteins, which remain essentially uncharacterized. Using in vitro and computational techniques, we analyzed the spike protein of the rodent coronavirus AcCoV-JC34 from the sub-genus luchacovirus, previously identified in Apodemus chevrieri (Chevrier\u2019s field mouse). We show that AcCoV-JC34\u2014unlike the other luchacoviruses\u2014has a putative furin cleavage site (FCS) within its spike S1 domain, close to the S1/S2 interface. The pattern of basic amino acids within the AcCoV-JC34 FCS (-RR-R-) is identical to that found in \u201cpre-variant\u201d SARS-CoV-2\u2014which is in itself atypical for an FCS, and suboptimal for furin cleavage. Our analysis shows that, while containing an -RR-R-motif, the AcCoV-JC34 spike \u201cFCS\u201d is not cleaved by furin (unlike for SARS-CoV-2), suggesting the possible presence of a progenitor sequence for viral emergence from a distinct wildlife host.","version":"1.1","doi":"10.1101/2021.12.16.473025","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.473030","pub_date":"2021-12-20","title":"Modeling the dynamics of within-host viral infection and evolution predicts quasispecies distributions and phase boundaries separating distinct classes of infections","abstract":"We use computational modeling to study within-host viral infection and evolution. In our model, viruses exhibit variable binding to cells, with better infection and replication countered by a stronger immune response and a high rate of mutation. By varying host conditions (permissivity to viral entry T and immune clearance intensity A) for large numbers of cells and viruses, we study the dynamics of how viral populations evolve from initial infection to steady state and obtain a phase diagram of the range of cell and viral responses. We find three distinct replicative strategies corresponding to three physiological classes of viral infections: acute, chronic, and opportunistic. We show similarities between our findings and the behavior of real viral infections such as common flu, hepatitis, and SARS-CoV-2019. The phases associated with the three strategies are separated by a phase transition of primarily first order, in addition to a crossover region. Our simulations also reveal a wide range of physical phenomena, including metastable states, periodicity, and glassy dynamics. Lastly, our results suggest that the resolution of acute viral disease in patients whose immunity cannot be boosted can only be achieved by significant inhibition of viral infection and replication. Virus, in particular RNA viruses, often produce offspring with slightly altered genetic composition. This process occurs both across host populations and within a single host over time. Here, we study the interactions of viruses with cells inside a host over time. In our model, the viruses encounter host cell defenses characterized by two parameters: permissivity to viral entry T and immune response A). The viruses then mutate upon reproduction, eventually resulting in a distribution of related viral types termed a quasi-species distribution. Across varying host conditions (T, A), three distinct viral quasi-species types emerge over time, corresponding to three classes of viral infections: acute, chronic and opportunistic. We interpret these results in terms of real viral types such as common flu, hepatitis, and also SARS-CoV-2019. Analysis of viral of viral mutant populations over a wide range of permissivity and immunity, for large numbers of cells and viruses, reveals phase transitions that separate the three classes of viruses, both in the infection-cycle dynamics and at steady state. We believe that such a multiscale approach for the study of within-host viral infections, spanning individual proteins to collections of cells, can provide insight into developing more effective therapies for viral disease.","version":"1.1","doi":"10.1101/2021.12.16.473030","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.09.471735","pub_date":"2021-12-19","title":"The T cell receptor repertoire reflects the dynamics of the immune response to vaccination","abstract":"Early, high-resolution metrics are needed to ascertain the immune response to vaccinations. The T cell receptor (TCR), a heterodimer of one \u03b1 and one \u03b2 chain, is a promising target, with the complete TCR repertoire reflecting the T cells present in an individual. To this end, we developed Tseek, an unbiased and accurate method for profiling the TCR repertoire by sequencing the TCR \u03b1 and \u03b2 chains and developing a suite of tools for repertoire analysis. An added advantage is the ability to non-invasively analyze T cells in peripheral blood mononuclear cells (PBMCs). Tseek and the analytical suite were used to explore the T cell response to both the COVID-19 mRNA vaccine (n=9) and the seasonal inactivated Influenza vaccine (n=5) at several time points. Neutralizing antibody titers were also measured in the covid vaccine samples. The COVID-19 vaccine elicited a broad T cell response involving multiple expanded clones, whereas the Influenza vaccine elicited a narrower response involving fewer clones. Many distinct T cell clones responded at each time point, over a month, providing temporal details lacking in the antibody measurements, especially before the antibodies are detectable. In individuals recovered from a SARS-CoV-2 infection, the first vaccine dose elicited a robust T cell response, while the second dose elicited a comparatively weaker response, indicating a saturation of the response. The physical symptoms experienced by the recipients immediately following the vaccinations were not indicative of the TCR/antibody responses. The TCR responses broadly presaged the antibody responses. We also found that the TCR repertoire acts as an individual fingerprint: donors of blood samples taken years apart could be identified solely based upon their TCR repertoire, hinting at other surprising uses the TCR repertoire may have. These results demonstrate the promise of TCR repertoire sequencing as an early and sensitive measure of the adaptive immune response to vaccination, which can help improve immunogen selection and optimize vaccine dosage and spacing between doses.","version":"1.2","doi":"10.1101/2021.12.09.471735","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.18.460926","pub_date":"2021-12-19","title":"A Genotype-to-Phenotype Modeling Framework to Predict Human Pathogenicity of Novel Coronaviruses","abstract":"Leveraging prior viral genome sequencing data to make predictions on whether an unknown, emergent virus harbors a \u2018phenotype-of-concern\u2019 has been a long-sought goal of genomic epidemiology. A predictive phenotype model built from nucleotide-level information alone has previously been considered un-tenable with respect to RNA viruses due to the ultra-high intra-sequence variance of their genomes, even within closely related clades. Building from our prior work developing a degenerate k-mer method to accommodate this high intra-sequence variation of RNA virus genomes for modeling frameworks, and leveraging a taxonomic \u2018group-shuffle-split\u2019 paradigm on complete coronavirus assemblies from prior to October 2018, we trained multiple regularized logistic regression classifiers at the nucleotide k-mer level capable of accurately predicting withheld SARS-CoV-2 genome sequences as human pathogens and accurately predicting withheld Swine Acute Diarrhea Syndrome coronavirus (SADS-CoV) genome sequences as non-human pathogens. LASSO feature selection identified several degenerate nucleotide predictor motifs with high model coefficients for the human pathogen class that were present across widely disparate classes of coronaviruses. However, these motifs differed in which genes they were present in, what specific codons were used to encode them, and what the translated amino acid motif was. This emphasizes the importance of a phenetic view of emerging pathogenic RNA viruses, as opposed to the canonical phylogenetic interpretations most-commonly used to track and manage viral zoonoses. Applying our model to more recent Orthocoronavirinae genomes deposited since October 2018 yields a novel contextual view of pathogen-potential across bat-related, canine-related, porcine-related, and rodent-related coronaviruses and critical adaptations which may have contributed to the emergence of the pandemic SARS-CoV-2 virus. Finally, we discuss the utility of these predictive models (and their associated predictor motifs) to novel biosurveillance protocols that substantially increase the \u2018pound-for-pound\u2019 information content of field-collected sequencing data and make a strong argument for the necessity of routine collection and sequencing of zoonotic viruses.","version":"1.2","doi":"10.1101/2021.09.18.460926","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472838","pub_date":"2021-12-18","title":"A third vaccination with a single T cell epitope protects against SARS-CoV-2 infection in the absence of neutralizing antibodies","abstract":"Understanding the mechanisms and impact of booster vaccinations can facilitate decisions on vaccination programmes. This study shows that three doses of the same synthetic peptide vaccine eliciting an exclusive CD8+ T cell response against one SARS-CoV-2 Spike epitope protected all mice against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, while only a second vaccination with this T cell vaccine was insufficient to provide protection. The third vaccine dose of the single T cell epitope peptide resulted in superior generation of effector-memory T cells in the circulation and tissue-resident memory T (TRM) cells, and these tertiary vaccine-specific CD8+ T cells were characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping showed that a substantial fraction of the tertiary effector-memory CD8+ T cells developed from remigrated TRM cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection. A third dose with a single T cell epitope-vaccine promotes a strong increase in tissue-resident memory CD8+ T cells and fully protects against SARS-CoV-2 infection, while single B cell epitope-eliciting vaccines are unable to provide protection.","version":"1.1","doi":"10.1101/2021.12.15.472838","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.19.468693","pub_date":"2021-12-18","title":"The Effect of COVID-19 on the Postdoctoral Experience: a comparison of pre-pandemic and pandemic surveys","abstract":"In the interest of advocating for the postdoctoral community in the United States, we present results from survey data collected before and during the COVID-19 pandemic on the same population of postdocs. In 2019, 5,929 postdocs in the US completed a comprehensive survey, and in 2020, a subset completed a follow-up survey several months into the pandemic. The results show that the pandemic has substantially impacted postdocs\u2019 mental health and wellness irrespective of gender, race, citizenship, or other identities. Postdocs also reported a significant impact on their career trajectories and progression, reduced confidence in achieving career goals, and negative perceptions of the job market compared to pre-COVID-19. International postdocs also reported experiencing distinct stressors due to the changes in immigration policy. Notably, having access to Postdoctoral Associations and Postdoctoral Offices positively impacted postdocs\u2019 overall well-being and helped mitigate the personal and professional stresses and career uncertainties caused by the pandemic. Graphical Abstract of survey responses to: Why or how has your research been disrupted or not disrupted due to the pandemic? Overall, postdocs responded with feelings of loss of control as the pandemic was acting upon them and taking away their ability to complete their work.","version":"1.2","doi":"10.1101/2021.11.19.468693","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.17.473121","pub_date":"2021-12-18","title":"2FAST2Q: A general-purpose sequence search and counting program for FASTQ files","abstract":"The increasingly widespread use of next generation sequencing protocols has brought the need for the development of user-friendly raw data processing tools. Here, we present 2FAST2Q, a versatile and intuitive standalone program capable of extracting and counting feature occurrences in FASTQ files. 2FAST2Q can be used in any experimental setup that requires feature extraction from raw reads, being able to quickly handle mismatch alignments, nucleotide wise Phred score filtering, custom read trimming, and sequence searching within a single program. Using published CRISPRi datasets in which Escherichia coli and Mycobacterium tuberculosis gene essentiality, as well as host-cell sensitivity towards SARS-CoV2 infectivity were tested, we demonstrate that 2FAST2Q efficiently recapitulates the output in read counts per provided feature as with traditional pipelines. Moreover, we show how different FASTQ read filtering parameters impact downstream analysis, and suggest a default usage protocol. 2FAST2Q has a familiar user interface and uses a custom sequence mismatch search algorithm, taking advantage of Python\u2019s numba module JIT runtime speeds. It is thus easier to use and faster than currently available tools, efficiently processing large CRISPRi-Seq or random-barcode sequencing datasets on any up-to-date laptop. 2FAST2Q is available as an executable file for all current operating systems without installation and as a Python3 module on the PyPI repository (available at https://veeninglab.com/2fast2q). We expect that 2FAST2Q will not only be useful for people working in microbiology but also for other fields in which amplicon sequencing data is generated.","version":"1.1","doi":"10.1101/2021.12.17.473121","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472828","pub_date":"2021-12-17","title":"An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by several therapeutic monoclonal antibodies","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic resulting in millions of deaths worldwide. Despite the development and deployment of highly effective antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. Indeed, the recent emergence of the highly-transmissible B.1.1.529 Omicron variant is especially concerning because of the number of mutations, deletions, and insertions in the spike protein. Here, using a panel of anti-receptor binding domain (RBD) monoclonal antibodies (mAbs) corresponding to those with emergency use authorization (EUA) or in advanced clinical development by Vir Biotechnology (S309, the parent mAbs of VIR-7381), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59), we report the impact on neutralization of a prevailing, infectious B.1.1.529 Omicron isolate compared to a historical WA1/2020 D614G strain. Several highly neutralizing mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987, and CT-P59) completely lost inhibitory activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (\u223c12-fold decrease, COV2-2196 and COV2-2130 combination) or minimally affected (S309). Our results suggest that several, but not all, of the antibody products in clinical use will lose efficacy against the B.1.1.529 Omicron variant and related strains.","version":"1.1","doi":"10.1101/2021.12.15.472828","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.16.472920","pub_date":"2021-12-17","title":"Amyloidogenesis of SARS-CoV-2 Spike Protein","abstract":"SARS-CoV-2 infection is associated with a surprising number of morbidities. Uncanny similarities with amyloid-disease associated blood coagulation and fibrinolytic disturbances together with neurologic and cardiac problems led us to investigate the amyloidogenicity of the SARS-CoV-2 Spike protein (S-protein). Amyloid fibril assays of peptide library mixtures and theoretical predictions identified seven amyloidogenic sequences within the S-protein. All seven peptides in isolation formed aggregates during incubation at 37\u00b0C. Three 20-amino acid long synthetic Spike peptides (sequence 191-210, 599-618, 1165-1184) fulfilled three amyloid fibril criteria: nucleation dependent polymerization kinetics by ThT, Congo red positivity and ultrastructural fibrillar morphology. Full-length folded S-protein did not form amyloid fibrils, but amyloid-like fibrils with evident branching were formed during 24 hours of S-protein co-incubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein rendering exposure of amyloidogenic segments and accumulation of the peptide 193-202, part of the most amyloidogenic synthetic Spike peptide. NE is overexpressed at inflamed sites of viral infection and at vaccine injection sites. Our data propose a molecular mechanism for amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The potential implications of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis and consequences following S-protein based vaccines should be addressed in understanding the disease, long COVID-19, and vaccine side effects.","version":"1.1","doi":"10.1101/2021.12.16.472920","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.472102","pub_date":"2021-12-17","title":"Mutations on RBD of SARS-CoV-2 Omicron variant result in stronger binding to human ACE2 receptor","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to more than 270 million infections and 5.3 million of deaths worldwide. Several major variants of SARS-CoV-2 have emerged and posed challenges in controlling the pandemic. The recently occurred Omicron variant raised serious concerns about reducing the efficacy of vaccines and neutralization antibodies due to its vast mutations. We have modelled the complex structure of the human ACE2 protein and the receptor binding domain (RBD) of Omicron Spike protein (S-protein), and conducted atomistic molecular dynamics simulations to study the binding interactions. The analysis shows that the Omicron RBD binds more strongly to the human ACE2 protein than the original strain. The mutations at the ACE2-RBD interface enhance the tight binding by increasing hydrogen bonding interaction and enlarging buried solvent accessible surface area.","version":"1.2","doi":"10.1101/2021.12.10.472102","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472864","pub_date":"2021-12-17","title":"A recurring YYDRxG pattern in broadly neutralizing antibodies to a conserved site on SARS-CoV-2, variants of concern, and related viruses","abstract":"Studying the antibody response to SARS-CoV-2 informs on how the human immune system can respond to antigenic variants as well as other SARS-related viruses. Here, we structurally and functionally characterized a potent human antibody ADI-62113 that also neutralizes SARS-CoV- 2 variants of concern and cross-reacts with many other sarbecoviruses. A YYDRxG motif encoded by IGHD3-22 in CDR H3 facilitates targeting to a highly conserved epitope on the SARS-CoV-2 receptor binding domain. A computational search for a YYDRxG pattern in publicly available sequences identified many antibodies with broad neutralization activity against SARS-CoV-2 variants and SARS-CoV. Thus, the YYDRxG motif represents a common convergent solution for the human humoral immune system to counteract sarbecoviruses. These findings also suggest an epitope targeting strategy to identify potent and broadly neutralizing antibodies that can aid in the design of pan-sarbecovirus vaccines and antibody therapeutics. Decryption of a recurrent sequence feature in anti-SARS-CoV-2 antibodies identifies how potent pan-sarbecovirus antibodies target a conserved epitope on the receptor binding domain.","version":"1.1","doi":"10.1101/2021.12.15.472864","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472450","pub_date":"2021-12-17","title":"QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis","abstract":"The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing . Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates . To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (\u0394QTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site \u2013 the FCS, loop length, and glycosylation \u2013 are required for efficient SARS-CoV-2 replication and pathogenesis.","version":"1.1","doi":"10.1101/2021.12.15.472450","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472874","pub_date":"2021-12-17","title":"Distinguishing COVID-19 infection and vaccination history by T cell reactivity","abstract":"SARS-CoV-2 infection and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of two new pools of Experimentally-defined T cell epitopes derived from the non-spike Remainder of the SARS-CoV-2 proteome (CD4RE and CD8RE). The combination of T cell responses to these new pools and Spike (S) were used to discriminate four groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status: non-infected, non-vaccinated (I\u2212V\u2212); infected and non-vaccinated (I+V\u2212); infected and then vaccinated (I+V+); and non-infected and vaccinated (I\u2212V+). The overall classification accuracy based on 30 subjects/group was 89.2% in the original cohort and 88.5% in a validation cohort of 96 subjects. The T cell classification scheme was applicable to different mRNA vaccines, and different lengths of time post-infection/post-vaccination. T cell responses from breakthrough infections (infected vaccinees, V+I+) were also effectively segregated from the responses of vaccinated subjects using the same classification tool system. When all five groups where combined, for a total of 239 different subjects, the classification scheme performance was 86.6%. We anticipate that a T cell-based immunodiagnostic scheme able to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccination and aid in establishing SARS-CoV\u22122 correlates of protection.","version":"1.1","doi":"10.1101/2021.12.15.472874","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.02.462569","pub_date":"2021-12-17","title":"De Novo Whole Genome Assembly of the Roborovski Dwarf Hamster (Phodopus roborovskii) Genome, an Animal Model for Severe/Critical COVID-19","abstract":"The Roborovski dwarf hamster Phodopus roborovskii belongs to the Phodopus genus, one of seven within Cricetinae subfamily. Like other rodents such as mice, rats or ferrets, hamsters can be important animal models for a range of diseases. Whereas the Syrian hamster from the genus Mesocricetus is now widely used as a model for mild to moderate COVID-19, Roborovski dwarf hamster show a severe to lethal course of disease upon infection with the novel human coronavirus SARS-CoV-2.","version":"1.3","doi":"10.1101/2021.10.02.462569","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472668","pub_date":"2021-12-16","title":"SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet pro-coagulant activity","abstract":"Thrombosis of the lung micro-vasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting this activity. As TMEM16F plays an important role in the stimulation of the pro-coagulant activity of platelets, and considering that platelet abnormalities are common in COVID-19 patients, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. We produced SARS-CoV-2 Spike or VSV-G protein-pseudotyped virions, or generated cells expressing Spike on their plasma membrane, and tested their effects on platelet adhesion (fluorescence), aggregation (absorbance), exposure of phosphatidylserine (flow cytometry for annexin V binding), calcium flux (flow cytometry for fluo-4 AM), and clot formation and retraction. These experiments were also conducted in the presence of the TMEM16F activity inhibitors Niclosamide and Clofazimine. Here we show that exposure to SARS-CoV-2 Spike promotes platelet activation, adhesion and spreading, both when present on the envelope of virions or upon expression on the plasma membrane of cells. Spike was effective both as a sole agonist or by enhancing the effect of known platelet activators, such as collagen and collagen-related peptide. In particular, Spike exerted a noticeable effect on the procoagulant phenotype of platelets, by enhancing calcium flux, phosphatidylserine externalisation, and thrombin generation. Eventually, this resulted in a striking increase in thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine almost abolished this Spike-induced pro-coagulant response. Together, these findings provide a pathogenic mechanism to explain thrombosis associated to COVID-19 lung disease, by which Spike present in SARS-CoV-2 virions or exposed on the surface of infected cells, leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing drugs targeting this protein, such as Niclosamide, for COVID-19 therapy.","version":"1.2","doi":"10.1101/2021.12.14.472668","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472466","pub_date":"2021-12-16","title":"Enrichment analysis on regulatory subspaces: a novel direction for the superior description of cellular responses to SARS-CoV-2","abstract":"The enrichment analysis of discriminative cell transcriptional responses to SARS-CoV-2 infection using biclustering produces a broader set of superiorly enriched GO terms and KEGG pathways against alternative state-of-the-art machine learning approaches, unraveling novel knowledge. The comprehensive understanding of the impacts of the SARS-CoV-2 virus on infected cells is still incomplete. This work identifies and analyses the main cell regulatory processes affected and induced by SARS-CoV-2, using transcriptomic data from several infectable cell lines available in public databases and in vivo samples. We propose a new class of statistical models to handle three major challenges, namely the scarcity of observations, the high dimensionality of the data, and the complexity of the interactions between genes. Additionally, we analyse the function of these genes and their interactions within cells to compare them to ones affected by IAV (H1N1), RSV and HPIV3 in the target cell lines. Gathered results show that, although clustering and predictive algorithms aid classic functional enrichment analysis, recent pattern-based biclustering algorithms significantly improve the number and quality of the detected biological processes. Additionally, a comparative analysis of these processes is performed to identify potential pathophysiological characteristics of COVID-19. These are further compared to those identified by other authors for the same virus as well as related ones such as SARS-CoV-1. This approach is particularly relevant due to a lack of other works utilizing more complex machine learning tools within this context.","version":"1.1","doi":"10.1101/2021.12.15.472466","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.22.461342","pub_date":"2021-12-16","title":"Demographic Analysis of Mutations in Indian SARS-CoV-2 Isolates","abstract":"In this study we carried out the early distribution of clades and subclades state-wise based on shared mutations in Indian SARS-CoV-2 isolates collected (27th Jan \u2013 27th May 2020). Phylogenetic analysis of these isolates indicates multiple independent sources of introduction of the virus in the country, while principal component analysis revealed some state-specific clusters. It is observed that clade 20A defining mutations C241T (ORF1ab: 5\u2019 UTR), C3037T (ORF1ab: F924F), C14408T (ORF1ab: P4715L), and A23403G (S: D614G) are predominant in Indian isolates during this period. Higher number of coronavirus cases were observed in certain states, viz., Delhi, Tamil Nadu, and Telangana. Genetic analysis of isolates from these states revealed a cluster with shared mutations, C6312A (ORF1ab: T2016K), C13730T (ORF1ab: A4489V), C23929T, and C28311T (N: P13L). Analysis of region-specific shared mutations carried out to understand the large number of deaths in Gujarat and Maharashtra identified shared mutations defining subclade, I/GJ-20A (C18877T, C22444T, G25563T (ORF3a: H57Q), C26735T, C28854T (N: S194L), C2836T) in Gujarat and two sets of co-occurring mutations C313T, C5700A (ORF1ab: A1812D) and A29827T, G29830T in Maharashtra. From the genetic analysis of mutation spectra of Indian isolates, the insights gained in its transmission, geographic distribution, containment, and impact are discussed.","version":"1.2","doi":"10.1101/2021.09.22.461342","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.17.468942","pub_date":"2021-12-16","title":"SARS-CoV-2 variants of concern remain dependent on IFITM2 for efficient replication in human lung cells","abstract":"It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several \u201cVariants of Concern\u201d (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant. Recent results showed that an early SARS-CoV-2 isolate requires endogenously expressed IFITM proteins for efficient infection. However, whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that out-competed the original strains and currently dominate the pandemic remained to be determined. Here, we demonstrate that depletion of endogenous IFITM2 expression almost entirely prevents the production of infectious Alpha, Beta, Gamma and Delta VOC SARS-CoV-2 virions in a human lung cell line. In comparison, silencing of IFITM1 had little impact, while knock-down of IFITM3 had intermediate effects on viral replication. Finally, an antibody targeting the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs including the currently dominant Delta variant are dependent on IFITM2 for efficient replication suggesting that IFITM proteins play a key role in viral transmission and pathogenicity.","version":"1.2","doi":"10.1101/2021.11.17.468942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472822","pub_date":"2021-12-16","title":"Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, will inform our understanding of this disease and its treatment. Plasma from COVID-19 patients exhibits prominent protein- and peptide-level changes Proteins from COVID-19 patient plasma exhibit prominent changes in several key posttranslational modifications","version":"1.1","doi":"10.1101/2021.12.15.472822","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.27.457964","pub_date":"2021-12-16","title":"Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding","abstract":"The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N-(NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we showed that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that was consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.","version":"1.2","doi":"10.1101/2021.08.27.457964","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.444935","pub_date":"2021-12-16","title":"Fluorescence signatures of SARS-CoV-2 spike S1 proteins and an human ACE-2: excitation-emission maps and fluorescence lifetimes","abstract":"Fast and reliable detection of infectious SARS-CoV-2 virus loads is an important issue. Fluorescence spectroscopy is a sensitive tool to do so in clean environments. This presumes a comprehensive knowledge of fluorescence data. This work aims at providing fully featured information on wavelength and time-dependent data of the fluorescence of the SARS-CoV-2 spike protein S1 subunit, its receptor binding domain (RBD) and the human angiotensinconverting enzyme 2 (hACE2), especially with respect to possible optical detection schemes. Spectrally resolved excitation-emission maps of the involved proteins and measurements of fluorescence lifetimes were recorded for excitations from 220 to 295 nm. The fluorescence decay times were extracted by using a bi-exponential kinetic approach. The binding process in the SARS-CoV-2 RBD was likewise examined for spectroscopic changes. Distinct spectral features for each protein are pointed out in relevant spectra extracted from the excitation emission maps. We also identify minor spectroscopic changes under the binding process. The decay times in the bi-exponential model are found to be (2.0\u00b1 0.1) ns and (8.0 \u00b11.0) ns. Specific material data serve as important background information for the design of optical detection and testing methods for SARS-CoV-2 loaded media.","version":"1.3","doi":"10.1101/2021.05.20.444935","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471814","pub_date":"2021-12-16","title":"Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses","abstract":"Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.","version":"1.2","doi":"10.1101/2021.12.08.471814","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.15.468283","pub_date":"2021-12-16","title":"Mutagenic distinction between the receptor-binding and fusion subunits of the SARS-CoV-2 spike glycoprotein and its upshot","abstract":"We observe that a residue R of the spike glycoprotein of SARS-CoV-2 which has mutated in one or more of the current Variants of Concern or Interest or under Monitoring rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A partial explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally. A study of the Delta and Omicron variants suggests that Delta was an energetically necessary intermediary in the evolution from Wuhan-Hu-1 to Omicron.","version":"1.2","doi":"10.1101/2021.11.15.468283","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471527","pub_date":"2021-12-16","title":"SARS-CoV-2 variants of concern remain dependent on IFITM2 for efficient replication in human lung cells","abstract":"The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2021/468942. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.2","doi":"10.1101/2021.12.06.471527","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472585","pub_date":"2021-12-15","title":"Compared with SARS-CoV2 wild type\u2019s spike protein, the SARS-CoV2 omicron\u2019s receptor binding motif has adopted a more SARS-CoV1 and/or bat/civet-like structure","abstract":"Our study focuses on free energy calculations of SARS-CoV2 spike protein receptor binding motives (RBMs) from wild type and variants-of-concern with particular emphasis on currently emerging SARS- CoV2 omicron variants of concern (VOC). Our computational free energy analysis underlines the occurrence of positive selection processes that specify omicron host adaption and bring changes on the molecular level into context with clinically relevant observations. Our free energy calculations studies regarding the interaction of omicron\u2019s RBM with human ACE2 shows weaker binding to ACE2 than alpha\u2019s, delta\u2019s, or wild type\u2019s RBM. Thus, less virus is predicted to be generated in time per infected cell. Our mutant analyses predict with focus on omicron variants a reduced spike-protein binding to ACE2-receptor protein possibly enhancing viral fitness / transmissibility and resulting in a delayed induction of danger signals as trade-off. Finally, more virus is produced but less per cell accompanied with delayed Covid-19 immunogenicity and pathogenicity. Regarding the latter, more virus is assumed to be required to initiate inflammatory immune responses.","version":"1.1","doi":"10.1101/2021.12.14.472585","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472513","pub_date":"2021-12-15","title":"Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein","abstract":"The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future. To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.","version":"1.1","doi":"10.1101/2021.12.14.472513","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472547","pub_date":"2021-12-15","title":"From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection rates approaching 40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2, WTD fawns shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD. Infectious virus was recovered from respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field. The high susceptibility of white-tailed deer (WTD) to SARS-CoV-2, their ability to transmit the virus to other deer, and the recent findings suggesting widespread SARS-CoV-2 infection in wild WTD populations in the U.S. underscore the need for a better understanding of the infection and transmission dynamics of SARS-CoV-2 in this potential reservoir species. Here we investigated the transmission dynamics of SARS-CoV-2 over time and defined the major sites of virus replication during the acute phase of infection. Additionally, we assessed the evolution of the virus as it replicated and transmitted between animals. The work provides important information on the infection dynamics of SARS-CoV-2 in WTD, an animal species that - if confirmed as a new reservoir of infection - may provide many opportunities for exposure and potential zoonotic transmission of the virus back to humans.","version":"1.1","doi":"10.1101/2021.12.14.472547","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472545","pub_date":"2021-12-15","title":"Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2","abstract":"The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug. The ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. In this present study, we screened two compound libraries of 2,864 molecules and identified a potent inhibitor (TS-984) for blocking the coronavirus S-protein and the human cell ACE2 receptor. TS-984 might have the potential to be developed into an effective anti-coronavirus drug for treating COVID-19.","version":"1.1","doi":"10.1101/2021.12.14.472545","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472632","pub_date":"2021-12-15","title":"Evidence for a mouse origin of the SARS-CoV-2 Omicron variant","abstract":"The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting the possibility of host-jumping. The molecular spectrum (i.e., the relative frequency of the twelve types of base substitutions) of mutations acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients, but was highly consistent with spectra associated with evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.","version":"1.1","doi":"10.1101/2021.12.14.472632","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472630","pub_date":"2021-12-15","title":"Considerable escape of SARS-CoV-2 variant Omicron to antibody neutralization","abstract":"The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa. It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development, and to antibodies present in 90 sera from COVID-19 vaccine recipients or convalescent individuals. Omicron was totally or partially resistant to neutralization by all mAbs tested. Sera from Pfizer or AstraZeneca vaccine recipients, sampled 5 months after complete vaccination, barely inhibited Omicron. Sera from COVID-19 convalescent patients collected 6 or 12 months post symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titers 5 to 31 fold lower against Omicron than against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and to a large extent vaccine-elicited antibodies.","version":"1.1","doi":"10.1101/2021.12.14.472630","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470470","pub_date":"2021-12-15","title":"SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 5 million people and is causing a devastating social and economic impact all over the world. The rise of new variants of concern (VOCs) represents a difficult challenge due to the loss vaccine and natural immunity, and increased transmissibility. All circulating VOCs contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes, via its receptor binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2). In an attempt to understand the effect of RBD mutations in circulating VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited, since it ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some VOCs mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect RBD conformational dynamics. To test this, we performed long atomistic (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of three circulating VOCs (alpha, beta, and delta). Our results show that in solution, wt RBD presents two distinct conformations: an \u201copen\u201d conformation where it is free to bind ACE2; and a \u201cclosed\u201d conformation, where the RBM ridge blocks the binding surface. The alpha and beta variants significantly impact the open/closed equilibrium, shifting it towards the open conformation by roughly 20%. This shift likely increases ACE2 binding affinity. Simulations of the currently predominant delta variant RBD were extreme in this regard, in that a closed conformation was never observed. Instead, the system alternated between the before mentioned open conformation and an alternative \u201creversed\u201d one, with a significantly changed orientation of the RBM ridge flanking the RBD. This alternate conformation could potentially provide a fitness advantage not only due to increased availability for ACE2 binding, but also by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the delta variant, impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.","version":"1.3","doi":"10.1101/2021.11.30.470470","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472657","pub_date":"2021-12-15","title":"Development and Characterization of Recombinant Vesicular Stomatitis Virus (rVSV)-based Bivalent Vaccine Against COVID-19 Delta Variant and Influenza Virus","abstract":"COVID-19 and influenza are both highly contagious respiratory diseases with a wide range of severe symptoms and cause great disease burdens globally. It has become very urgent and important to develop a bivalent vaccine that is able to target these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant VSV (rVSV) vaccine candidates. These rVSV-based vaccines co-express SARS-CoV-2 Delta variant spike protein (SP) or the receptor binding domain (RBD) and four copies of the highly conserved M2 ectodomain (M2e) of influenza A fused with the Ebola glycoprotein DC-targeting/activation domain. Animal studies have shown that immunization with these bivalent rVSV vaccines induced efficient but variable levels of humoral and cell-mediated immune responses against both SARS-CoV-2 and influenza M2e protein. Significantly, our vaccine candidates induced production of high levels of neutralizing antibodies that protected cells against SARS-CoV-2 Delta and other SP-pseudovirus infections in culture. Furthermore, vaccination with the bivalent VSV vaccine via either intramuscular or intranasal route efficiently protected mice from the lethal challenge of H1N1 and H3N2 influenza viruses and significantly reduced viral load in the lungs. These studies provide convincing evidence for the high efficacy of this bivalent vaccine to prevent influenza replication and initiate robust immune responses against SARS-CoV-2 Delta variants. Further investigation of its efficacy to protect against SARS-CoV-2 Delta variants will provide substantial evidence for new avenues to control two contagious respiratory infections, COVID-19 and influenza.","version":"1.1","doi":"10.1101/2021.12.14.472657","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472704","pub_date":"2021-12-15","title":"Simulation of the omicron variant of SARS-CoV-2 shows broad antibody escape, weakened ACE2 binding, and modest increase in furin binding","abstract":"The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is significant reduction of antibody binding strength corresponding to escape, the omicron spike pays a cost in terms of weaker receptor binding. The furin cleavage domain is the same or weaker binding than the alpha variant, suggesting less viral load and disease intensity than the extant delta variant.","version":"1.1","doi":"10.1101/2021.12.14.472704","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.15.472779","pub_date":"2021-12-15","title":"Genomic determinants of Furin cleavage in diverse European SARS-related bat coronaviruses","abstract":"The furin cleavage site in SARS-CoV-2 is unique within the Severe acute respiratory syndrome\u2013related coronavirus (SrC) species. We re-assessed diverse SrC from European horseshoe bats and reveal molecular determinants such as purine richness, RNA secondary structures and viral quasispecies potentially enabling furin cleavage. Furin cleavage thus likely emerged from the SrC bat reservoir via molecular mechanisms conserved across reservoir-bound RNA viruses, supporting a natural origin of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.12.15.472779","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472331","pub_date":"2021-12-15","title":"A comprehensive examination of ACE-2 receptor and prediction of spike glycoprotein and ACE-2 interaction based on in silico analysis of ACE-2 receptor","abstract":"ACE-2 receptor plays a vital role not only in the SARS-CoV-induced epidemic but also in some diseases. Studies have been carried out on the interactions of ACE-2-SARS-CoV proteins. However, comprehensive research has not been conducted on ACE2 protein by using bioinformatic tools. The present study especially two places, G104 and L108 points, which are effective in protecting the structure of the ACE-2 protein, play a critical role in the biological functioning of this protein, and play an essential role in determining the chemicalphysical properties of this protein, and play a crucial role for ACE-2 protein-SARS CoV surface glycoprotein, were determined. It was also found that the G104 and L108 regions were more prone to possible mutations or deletions than the other ACE-2 protein regions. Moreover, it was determined that all possible mutations or deletions in these regions affect the chemical-physical properties, biological functions, and structure of the ACE-2 protein. Having a negative GRAVY value, one transmembrane helix, a significant molecular weight, a long-estimated half-life as well as most having unstable are results of G104 and L108 points mutations or deletions. Finally, it was determined that LQQNGSSVLS, which belong to the ACE-2 protein, may play an active role in binding the spike protein of SARS-CoV. All possible docking score results were estimated. It is thought that this study will bring a different perspective to ACE-2 _SARS-CoV interaction and other diseases in which ACE-2 plays an important role and will also be an essential resource for studies on ACE-2 protein.","version":"1.1","doi":"10.1101/2021.12.14.472331","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472622","pub_date":"2021-12-15","title":"Computational analysis of the effect of SARS-CoV-2 variant Omicron Spike protein mutations on dynamics, ACE2 binding and propensity for immune escape","abstract":"The recently reported Omicron (B.1.1.529) SARS-CoV-2 variant has a large number of mutations in the Spike (S) protein compared to previous variants. Here we evaluate the potential effect of Omicron S mutations on S protein dynamics and ACE2 binding as contributing factors to infectivity as well as propensity for immune escape. We define a consensus set of mutations from 77 sequences assigned as Omicron in GISAID as of November 25. We create structural models of the Omicron S protein in the open and closed states, as part of a complex with ACE2 and for each of 77 complexes of S bound to different antibodies with known structures. We have previously utilized Dynamical Signatures (DS) and the Vibrational Entropy Score (VDS) to evaluate the propensity of S variants to favour the open state. Here, we introduce the Binding Influence Score (BIS) to evaluate the influence of mutations on binding affinity based on the net gain or loss of interactions within the protein-protein interface. BIS shows excellent correlation with experimental data (Pearson correlation coefficient of 0.87) on individual mutations in the ACE2 interface for the Alpha, Beta, Gamma, Delta and Omicron variants combined. On the one hand, the DS of Omicron highly favours a more rigid open state and a more flexible closed state with the largest VDS of all variants to date, suggesting a large increase in the chances to interact with ACE2. On the other hand, the BIS shows that apart from N501Y, all other mutations in the interface reduce ACE2 binding affinity. VDS and BIS show opposing effects on the overall effectiveness of Omicron mutations to promote binding to ACE2 and therefore initiate infection. To evaluate the propensity for immune escape we calculated the net change of favourable and unfavourable interactions within each S-antibody interface. The net change of interactions shows a positive score (a net increase of favourable interactions and decrease of unfavourable ones) for 41 out of 77 antibodies, a nil score for 15 and a negative score for 21 antibodies. Therefore, in only 28% of S-antibody complexes (21/77) we predict some level of immune escape due to a weakening of the interactions with Omicron S. Considering that most antibody epitopes and the mutations are within the S-ACE2 interface our results suggest that mutations within the RBD of Omicron may give rise to only partial immune escape, which comes at the expense of reduced ACE2 binding affinity. However, this reduced ACE2 affinity appears to have been offset by increasing the occupancy of the open state of the Spike protein.","version":"1.1","doi":"10.1101/2021.12.14.472622","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472614","pub_date":"2021-12-15","title":"Viral infection engenders bona fide and bystander lung memory B cell subsets through permissive selection","abstract":"Lung-resident memory B cells (MBCs) provide localized protection against reinfection in the respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. scRNA-seq analysis and confocal imaging revealed that two main transcriptionally distinct subsets of MBCs colonize the lung peribronchial niche after infection. These subsets arise from different progenitors and are both class-switched, somatically mutated and intrinsically biased in their differentiation fate towards plasma cells. Combined analysis of antigen-specificity and B cell receptor repertoire unveiled a highly permissive selection process that segregates these subsets into \u201cbona fide\u201d virus-specific MBCs and \u201cbystander\u201d MBCs with no apparent specificity for eliciting viruses. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.","version":"1.1","doi":"10.1101/2021.12.14.472614","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472526","pub_date":"2021-12-14","title":"Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India","abstract":"Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.","version":"1.1","doi":"10.1101/2021.12.13.472526","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472352","pub_date":"2021-12-14","title":"CHARM: COVID-19 Health Action Response for Marines \u2013 association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test","abstract":"SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and then bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were asked to enroll in an immune response sub-study. FluoroSpot interferon-gamma (IFN-\u03b3) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-\u03b3 responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-\u03b3 responses were significantly (p=0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic subjects; however, the difference was not statistically significant (p=0.06) for symptomatic subjects (N pool: 44.4%; M pool: 25.9%). In asymptomatic participants IFN-\u03b3 responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but symptomatic participants, IFN-\u03b3 responses were more frequent to the S pool (75.0%) than N pool (33.3%) and M pool (33.3%). The frequencies of IFN-\u03b3 responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-\u03b3 and IL2 responses to the N+M pool were significantly correlated with IFN-\u03b3 and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-\u03b3 and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.","version":"1.1","doi":"10.1101/2021.12.13.472352","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469747","pub_date":"2021-12-14","title":"Genome-wide characterization of SARS-CoV-2 cytopathogenic proteins in the search of antiviral targets","abstract":"Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify antiviral targets through genome-wide characterization of SARS-CoV-2 proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins including eight non-structural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis, and caused activation of pro-inflammatory response with production of the cytokines TNF-\u03b1, IL-6, and IFN-\u03b21, possibly through the activation of NF-\u03baB. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, \u0394G188. Compared to wild type ORF3a, the \u0394G188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19. The ongoing SARS-CoV-2 pandemic has claimed over 5 million lives with more than 250 million people infected world-wide. While vaccines are effective, the emergence of new viral variants could jeopardize vaccine protection. Antiviral drugs provide an alternative to battle against COVID-19. Our goal was to identify viral therapeutic targets that can be used in antiviral drug discovery. Utilizing a genome-wide functional analysis in a fission yeast cell-based system, we identified twelve viral candidates, including ORF3a, which cause cellular oxidative stress, inflammation and apoptosis and necrosis that contribute to COVID-19. Our findings indicate that antiviral agents targeting ORF3a could greatly impact COVID-19.","version":"1.2","doi":"10.1101/2021.11.23.469747","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472159","pub_date":"2021-12-14","title":"Mucosal and systemic responses to SARS-CoV-2 vaccination in infection na\u00efve and experienced individuals","abstract":"With much of the world infected with or vaccinated against SARS-CoV-2, understanding the immune responses to the SARS-CoV-2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS-CoV-2 mRNA vaccines in 62 individuals with and without prior SARS-CoV-2 exposure that were divided into three groups based on serostatus and/or degree of symptoms: Antibody negative, Asymptomatic, and Symptomatic. In the previously SARS-CoV-2-infected (SARS2-infected) Asymptomatic and Symptomatic groups, symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG levels peaked after 1st vaccination in the SARS2-infected groups, and were higher that the in the SARS2-naive group in the plasma and nasal samples at all time points. Neutralizing antibodies titers were also higher against the WA-1 and B.1.617.2 (Delta) variants of SARS-CoV-2 in the SARS2-infected compared to SARS2-na\u00efve vaccinees. After the first vaccination, differences in cellular immunity were not evident between groups, but the AIM+ CD4+ cell response correlated with durability of humoral immunity against the SARS-CoV-2 S protein. In those SARS2-infected, the number of vaccinations needed for protection, the durability, and need for boosters are unknown. However, the lingering differences between the SARS2-infected and SARS2-na\u00efve up to 10 months post-vaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-na\u00efve vaccinees) are needed to narrow the differences observed between these groups.","version":"1.1","doi":"10.1101/2021.12.13.472159","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.471928","pub_date":"2021-12-14","title":"Language Models for the Prediction of SARS-CoV-2 Inhibitors","abstract":"The COVID-19 pandemic highlights the need for computational tools to automate and accelerate drug design for novel protein targets. We leverage deep learning language models to generate and score drug candidates based on predicted protein binding affinity. We pre-trained a deep learning language model (BERT) on \u223c9.6 billion molecules and achieved peak performance of 603 petaflops in mixed precision. Our work reduces pre-training time from days to hours, compared to previous efforts with this architecture, while also increasing the dataset size by nearly an order of magnitude. For scoring, we fine-tuned the language model using an assembled set of thousands of protein targets with binding affinity data and searched for inhibitors of specific protein targets, SARS-CoV-2 Mpro and PLpro. We utilized a genetic algorithm approach for finding optimal candidates using the generation and scoring capabilities of the language model. Our generalizable models accelerate the identification of inhibitors for emerging therapeutic targets.","version":"1.1","doi":"10.1101/2021.12.10.471928","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472476","pub_date":"2021-12-14","title":"Potential cross-protection against SARS-CoV-2 from previous exposure to bovine coronavirus","abstract":"Humans have long shared infectious agents with cattle, and the common cold OC-43 CoV is a not-so-distant example of cross-species viral spillover. Human exposure to BCoV is certainly common, as the virus is endemic in cattle-raising regions. This article shows an in silico investigation of shared viral epitopes between BCoV and SARS-CoV-2. HLA recognition and lymphocyte reactivity were assessed using freely-available resources. Several epitopes were shared between BCoV and SARS-CoV-2, both for B and T lymphocytes. These data demonstrate that possible cross-protection is being induced by human exposure to cattle.","version":"1.1","doi":"10.1101/2021.12.13.472476","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472433","pub_date":"2021-12-14","title":"Production and secretion of functional full-length SARS-CoV-2 spike protein in Chlamydomonas reinhardtii","abstract":"The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to mammalian and insect cells, requiring complex culture media. Here we report the production of full-length SARS-CoV-2 spike protein \u2013 lacking the C-terminal membrane anchor \u2013 as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.","version":"1.1","doi":"10.1101/2021.12.13.472433","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472315","pub_date":"2021-12-14","title":"SARS-CoV-2 T cell responses are expected to remain robust against Omicron","abstract":"Omicron, the most recent SARS-CoV-2 variant of concern (VOC), harbours multiple mutations in the spike protein that were not observed in previous VOCs. Initial studies suggest Omicron to substantially reduce the neutralizing capability of antibodies induced from vaccines and previous infection. However, its effect on T cell responses remains to be determined. Here, we assess the effect of Omicron mutations on known T cell epitopes and report data suggesting T cell responses to remain broadly robust against this new variant.","version":"1.1","doi":"10.1101/2021.12.12.472315","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.14.472240","pub_date":"2021-12-14","title":"In silico evidence of superantigenic features in ORF8 protein from COVID-19","abstract":"Very early on COVID-19 pandemic outbreak, it was noted that the some of the virus-induced clinical conditions resembled features of toxaemia caused by the toxic shock syndrome toxin type 1, which is a soluble superantigen produced by Staphylococcus aureus. Among all SARS proteins, the ORF8 protein from SARS-2 virus is significantly different from other known SARS-like coronaviruses, and therefore could exhibit unique pathogenic properties. We assess if ORF8 protein bears super antigenic features using in silico tools. We show that ORF8 has properties of an extracellular soluble protein and shares a significant degree of amino acid sequence identity with toxic shock syndrome toxin. Besides, docking and binding affinity analyses between monomeric and homodimeric ORF-8 with V\u03b2 2.1 and TRBV11-2 reveal strong interaction and high binding affinity. ORF8-TRBV11-2 strong interaction can contribute to the observed clonal expansion of that chain during COVID-19-associated multisystem inflammatory syndrome. Taken together, the evidence presented here supports the hypothesis that ORF8 protein from SARS-2 bears super antigenic properties.","version":"1.1","doi":"10.1101/2021.12.14.472240","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472454","pub_date":"2021-12-14","title":"TopHap: Rapid inference of key phylogenetic structures from common haplotypes in large genome collections with limited diversity","abstract":"Building reliable phylogenies from very large collections of sequences with a limited number of phylogenetically informative sites is challenging because sequencing errors and recurrent/backward mutations interfere with the phylogenetic signal, confounding true evolutionary relationships. Massive global efforts of sequencing genomes and reconstructing the phylogeny of SARS-CoV-2 strains exemplify these difficulties since there are only hundreds of phylogenetically informative sites and millions of genomes. For such datasets, we set out to develop a method for building the phylogenetic tree of genomic haplotypes consisting of positions harboring common variants to improve the signal-to-noise ratio for more accurate phylogenetic inference of resolvable phylogenetic features. We present the TopHap approach that determines spatiotemporally common haplotypes of common variants and builds their phylogeny at a fraction of the computational time of traditional methods. To assess topological robustness, we develop a bootstrap resampling strategy that resamples genomes spatiotemporally. The application of TopHap to build a phylogeny of 68,057 genomes (68KG) produced an evolutionary tree of major SARS-CoV-2 haplotypes. This phylogeny is concordant with the mutation tree inferred using the co-occurrence pattern of mutations and recovers key phylogenetic relationships from more traditional analyses. We also evaluated alternative roots of the SARS-CoV-2 phylogeny and found that the earliest sampled genomes in 2019 likely evolved by four mutations of the most recent common ancestor of all SARS-CoV-2 genomes. An application of TopHap to more than 1 million genomes reconstructed the most comprehensive evolutionary relationships of major variants, which confirmed the 68KG phylogeny and provided evolutionary origins of major variants of concern. TopHap is available on the web at https://github.com/SayakaMiura/TopHap. s.kumar@temple.edu","version":"1.1","doi":"10.1101/2021.12.13.472454","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.11.472236","pub_date":"2021-12-14","title":"Isolation and comparative analysis of antibodies that broadly neutralize sarbecoviruses","abstract":"The devastation caused by SARS-CoV-2 has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses and thus its promise as an agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses, but also uncovered a new antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine. A monoclonal antibody that neutralizes or binds all sarbecoviruses tested and represents a reproducible antibody class.","version":"1.1","doi":"10.1101/2021.12.11.472236","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472253","pub_date":"2021-12-14","title":"Scanning the RBD-ACE2 molecular interactions in Omicron variant","abstract":"The emergence of new SARS-CoV-2 variants poses a threat to the human population where it is difficult to assess the severity of a particular variant of the virus. Spike protein and specifically its receptor binding domain (RBD) which makes direct interaction with the ACE2 receptor of the human has shown prominent amino acid substitutions in most of the Variants of Concern. Here, by using all-atom molecular dynamics simulations we compare the interaction of Wild-type RBD/ACE2 receptor complex with that of the latest Omicron variant of the virus. We observed a very interesting diversification of the charge, dynamics and energetics of the protein complex formed upon mutations. These results would help us in understanding the molecular basis of binding of the Omicron variant with that of SARS-CoV-2 Wild-type.","version":"1.1","doi":"10.1101/2021.12.12.472253","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471374","pub_date":"2021-12-14","title":"Identifying protein sites contributing to vaccine escape via statistical comparisons of short-term molecular dynamics simulations","abstract":"The identification of viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a molecular dynamics (MD) based approach that scales well to a desktop computer with a high-end modern graphics processor and enables the user to identify protein sites that are prone to vaccine escape in a viral antigen. We first implement our MD pipeline to employ site-wise calculation of Kullback-Leibler divergence in atom fluctuation over replicate sets of short-term MD production runs thus enabling a statistical comparison of the rapid motion of influenza hemagglutinin (HA) in both the presence and absence of three well-known neutralizing antibodies. Using this simple comparative method applied to motions of viral proteins, we successfully identified in silico all previously empirically confirmed sites of escape in influenza HA, predetermined via selection experiments and neutralization assays. Upon the validation of our computational approach, we then surveyed potential hot spot residues in the receptor binding domain of the SARS-CoV-2 virus in the presence of COVOX-222 and S2H97 antibodies. We identified many single sites in the antigen-antibody interface that are similarly prone to potential antibody escape and that match many of the known sites of mutations arising in the SARS-CoV-2 variants of concern. In the omicron variant, we find only minimal adaptive evolutionary shifts in the functional binding profiles of both antibodies. In summary, we provide a fast and accurate computational method to monitor hot spots of functional evolution in antibody binding footprints.","version":"1.2","doi":"10.1101/2021.12.06.471374","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465468","pub_date":"2021-12-14","title":"PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins","abstract":"Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the \u03b3-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this \u201cPermaPhosSer\u201d system \u2013 an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins \u2013 we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3\u03b2 phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli.","version":"1.2","doi":"10.1101/2021.10.22.465468","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.472134","pub_date":"2021-12-13","title":"SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern","abstract":"Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.","version":"1.1","doi":"10.1101/2021.12.10.472134","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.13.472413","pub_date":"2021-12-13","title":"Inactivation of SARS-CoV-2 and influenza A virus by spraying hypochlorous acid solution and hydrogen peroxide solution in the form of Dry Fog","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted by droplet and contact infection. SARS-CoV-2 that adheres to environmental surfaces remains infectious for several days. We herein attempted to inactivate SARS-CoV-2 and influenza A virus adhering to an environmental surface by spraying aerosolized hypochlorous acid solution and hydrogen peroxide solution in the form of Dry Fog (fog that does not wet objects even if touched). SARS-CoV-2 and influenza virus were dried on plastic plates and placed into a test chamber for inactivation by the Dry Fog spraying of disinfectants. The results obtained showed that Dry Fog spraying inactivated SARS-CoV-2 and influenza A virus in time- and exposed disinfectant amount-dependent manners. SARS-CoV-2 was more resistant to the virucidal effects of aerosolized hypochlorous acid solution and hydrogen peroxide solution than influenza A virus; therefore, higher concentrations of spray solutions were required to inactivate SARS-CoV-2 than influenza A virus. The present results provide important information for the development of a strategy that inactivates SARS-CoV-2 and influenza A virus on environmental surfaces by spatial spraying.","version":"1.1","doi":"10.1101/2021.12.13.472413","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.11.472202","pub_date":"2021-12-13","title":"Epistatic models predict mutable sites in SARS-CoV-2 proteins and epitopes","abstract":"The emergence of new variants of SARS-CoV-2 is a major concern given their potential impact on the transmissibility and pathogenicity of the virus as well as the efficacy of therapeutic interventions. Here, we predict the mutability of all positions in SARS-CoV-2 protein domains to forecast the appearance of unseen variants. Using sequence data from other coronaviruses, pre-existing to SARS-CoV-2, we build statistical models that do not only capture amino-acid conservation but more complex patterns resulting from epistasis. We show that these models are notably superior to conservation profiles in estimating the already observable SARS-CoV-2 variability. In the receptor binding domain of the spike protein, we observe that the predicted mutability correlates well with experimental measures of protein stability and that both are reliable mutability predictors (ROC AUC ~0.8). Most interestingly, we observe an increasing agreement between our model and the observed variability as more data become available over time, proving the anticipatory capacity of our model. When combined with data concerning the immune response, our approach identifies positions where current variants of concern are highly overrepresented. These results could assist studies on viral evolution, future viral outbreaks and, in particular, guide the exploration and anticipation of potentially harmful future SARS-CoV-2 variants. During the COVID pandemic, new SARS-CoV-2 variants emerge and spread, some being of major concern due to their increased infectivity or their capacity to reduce vaccine efficiency. Anticipating new mutations, which might give rise to new variants, would be of great interest. Here we construct sequence models predicting how mutable SARS-CoV-2 positions are, using a single SARS-CoV-2 sequence and databases of other coronaviruses. Predictions are tested against available mutagenesis data and the observed variability of SARS-CoV-2 proteins. Interestingly, our predictions agree increasingly with observations, as more SARS-CoV-2 sequences become available. Combining predictions with immunological data, we find a clear overrepresentation of mutations in current variants of concern. The approach may become relevant for potential outbreaks of future viral diseases.","version":"1.1","doi":"10.1101/2021.12.11.472202","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.472112","pub_date":"2021-12-13","title":"SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-\u03b2 signaling","abstract":"Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-\u03b2 signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-\u03b2 signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2021.12.10.472112","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.10.472169","pub_date":"2021-12-13","title":"Cell Surface SARS-CoV-2 Nucleocapsid Protein Modulates Innate and Adaptive Immunity","abstract":"SARS-CoV-2 nucleocapsid protein (N) induces strong antibody and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2 infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12\u03b2, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS CoV. Anti-N Abs bound to the surface of N expressing cells activate Fc receptor-expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.","version":"1.1","doi":"10.1101/2021.12.10.472169","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471857","pub_date":"2021-12-13","title":"A qualitative mathematical model of immunocompetence with applications to SARS-CoV-2 immunity","abstract":"A qualitative mathematical model of the notion of immunocompetence is developed, based on the formalism of Memory Evolutive Systems (MES), from which, immunocompetence is defined as an emergent structure of a higher order arising from the signal networks that are established between effector cells and molecules of the immune response in the presence of a given antigen. In addition, a possible mechanism of functorial nature is proposed, which may explain how immunocompetence is achieved in an organism endowed with innate and adaptive components of its immune system. Finally, a practical method to measure the immunocompetence status is established, using elements of the theory of small random graphs and taking into account the characteristics of the immune networks, established through transcriptional studies, of patients with severe COVID-19 and healthy patients, assuming that both types of patients were vaccinated with an effective biological against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.12.08.471857","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472255","pub_date":"2021-12-13","title":"Broad antiviral effects of Echinacea purpurea against SARS-CoV-2 variants of concern and potential mechanism of action","abstract":"SARS-CoV-2 variants of concern (VOC) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Some exhibit increased pathogenicity and transmissibility compared to the original wild type WUHAN (Hu-1). Broad-spectrum antivirals could complement and further enhance preventive benefits achieved through SARS-CoV-2 vaccination campaigns The anti-coronavirus activity of Echinacea purpurea (Echinaforce\u00ae extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish) and B1.525 (eta), (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild-type SARS-CoV-2 (Hu-1) were analyzed. Molecular dynamics (MD) were applied to study interaction of Echinacea\u2019s phytochemical markers with known pharmacological viral and host cell targets. EF extract broadly inhibited propagation of all investigated SARS-CoV-2 VOCs as well as entry of SARS-CoV-2 pseudoparticles at EC50\u2019s ranging from 3.62 to 12.03 \u00b5g/ml. Preventive addition of 20 \u00b5g/ml EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) as well as with the common human strain OC43. MD analyses showed constant binding affinities to Hu-1, B1.1.7, B.1.351, P.1 and B1.617.2-typic S protein variants for alkylamides, caftaric acidand feruoyl-tartaric acid in EF extract. They further indicated that the EF extract could possibly interact with TMPRSS-2, a serine protease required for virus endocytosis. EF extract demonstrated stable antiviral activity across 6 tested VOCs, which is likely due to the constant affinity of the contained phytochemical marker substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain cell protective benefits of the extract by inhibition of endocytosis. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.","version":"1.1","doi":"10.1101/2021.12.12.472255","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472313","pub_date":"2021-12-13","title":"Structural models of SARS-CoV-2 Omicron variant in complex with ACE2 receptor or antibodies suggest altered binding interfaces","abstract":"There is enormous ongoing interest in characterizing the binding properties of the SARS-CoV-2 Omicron Variant of Concern (VOC) (B.1.1.529), which continues to spread towards potential dominance worldwide. To aid these studies, based on the wealth of available structural information about several SARS-CoV-2 variants in the Protein Data Bank (PDB) and a modeling pipeline we have previously developed for tracking the ongoing global evolution of SARS-CoV-2 proteins, we provide a set of computed structural models (henceforth models) of the Omicron VOC receptor-binding domain (omRBD) bound to its corresponding receptor Angiotensin-Converting Enzyme (ACE2) and a variety of therapeutic entities, including neutralizing and therapeutic antibodies targeting previously-detected viral strains. We generated bound omRBD models using both experimentally-determined structures in the PDB as well as machine learningbased structure predictions as starting points. Examination of ACE2-bound omRBD models reveals an interdigitated multi-residue interaction network formed by omRBD-specific substituted residues (R493, S496, Y501, R498) and ACE2 residues at the interface, which was not present in the original Wuhan-Hu-1 RBD-ACE2 complex. Emergence of this interaction network suggests optimization of a key region of the binding interface, and positive cooperativity among various sites of residue substitutions in omRBD mediating ACE2 binding. Examination of neutralizing antibody complexes for Barnes Class 1 and Class 2 antibodies modeled with omRBD highlights an overall loss of interfacial interactions (with gain of new interactions in rare cases) mediated by substituted residues. Many of these substitutions have previously been found to independently dampen or even ablate antibody binding, and perhaps mediate antibody-mediated neutralization escape (e.g., K417N). We observe little compensation of corresponding interaction loss at interfaces when potential escape substitutions occur in combination. A few selected antibodies (e.g., Barnes Class 3 S309), however, feature largely unaltered or modestly affected protein-protein interfaces. While we stress that only qualitative insights can be obtained directly from our models at this time, we anticipate that they can provide starting points for more detailed and quantitative computational characterization, and, if needed, redesign of monoclonal antibodies for targeting the Omicron VOC Spike protein. In the broader context, the computational pipeline we developed provides a framework for rapidly and efficiently generating retrospective and prospective models for other novel variants of SARS-CoV-2 bound to entities of virological and therapeutic interest, in the setting of a global pandemic.","version":"1.1","doi":"10.1101/2021.12.12.472313","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471707","pub_date":"2021-12-13","title":"Delta breakthrough infections elicit potent, broad and durable neutralizing antibody responses","abstract":"The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.","version":"1.2","doi":"10.1101/2021.12.08.471707","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.03.471024","pub_date":"2021-12-13","title":"Predictions of the SARS-CoV-2 Omicron Variant (B.1.1.529) Spike Protein Receptor-Binding Domain Structure and Neutralizing Antibody Interactions","abstract":"The genome of the SARS-CoV-2 Omicron variant (B.1.1.529) was released on November 22, 2021, which has caused a flurry of media attention due the large number of mutations it contains. These raw data have spurred questions around vaccine efficacy. Given that neither the structural information nor the experimentally-derived antibody interaction of this variant are available, we have turned to predictive computational methods to model the mutated structure of the spike protein\u2019s receptor binding domain and posit potential changes to vaccine efficacy. In this study, we predict some structural changes in the receptor-binding domain that may reduce antibody interaction, but no drastic changes that would completely evade existing neutralizing antibodies (and therefore current vaccines).","version":"1.3","doi":"10.1101/2021.12.03.471024","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.12.472286","pub_date":"2021-12-13","title":"The Omicron variant is highly resistant against antibody-mediated neutralization \u2013 implications for control of the COVID-19 pandemic","abstract":"The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike-protein raised concerns that the virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by Sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent or BNT162b2-vaccinated individuals with 10- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1/BNT162b2-vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.","version":"1.1","doi":"10.1101/2021.12.12.472286","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.09.471691","pub_date":"2021-12-13","title":"Present and future distribution of bat hosts of sarbecoviruses: implications for conservation and public health","abstract":"Global changes in response to human encroachment into natural habitats and carbon emissions are driving the biodiversity extinction crisis and increasing disease emergence risk. Host distributions are one critical component to identify areas at risk of spillover, and bats act as reservoirs of diverse viruses. We developed a reproducible ecological niche modelling pipeline for bat hosts of SARS-like viruses (subgenus Sarbecovirus), given that since SARS-CoV-2 emergence several closely-related viruses have been discovered and sarbecovirus-host interactions have gained attention. We assess sampling biases and model bats\u2019 current distributions based on climate and landscape relationships and project future scenarios. The most important predictors of species distribution were temperature seasonality and cave availability. We identified concentrated host hotspots in Myanmar and projected range contractions for most species by 2100. Our projections indicate hotspots will shift east in Southeast Asia in >2 \u00b0C hotter locations in a fossil-fueled development future. Hotspot shifts have implications for conservation and public health, as loss of population connectivity can lead to local extinctions, and remaining hotspots may concentrate near human populations.","version":"1.1","doi":"10.1101/2021.12.09.471691","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471873","pub_date":"2021-12-11","title":"Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission","abstract":"Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing. However, contrary to expectation, trends observed with sub-optimal exposure timing suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.","version":"1.1","doi":"10.1101/2021.12.08.471873","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.09.21267544","pub_date":"2021-12-11","title":"Inferring variant-specific effective reproduction numbers from combined case and sequencing data","abstract":"<jats:p>Accurately estimating relative transmission rates of SARS-CoV-2 variants remains a scientific and public health priority. Recent studies have used the sample proportions of different variants from genetic sequence data to describe variant frequency dynamics and relative transmission rates, but frequencies alone cannot capture the rich epidemiological behavior of SARS-CoV-2. Here, we extend methods for infer- ring the effective reproduction number of an epidemic using confirmed case data to jointly estimate variant-specific effective reproduction numbers and frequencies of co-circulating variants using cases and sequences across states in the US from January 2021 to March 2022. Our method can be used to infer structured relationships be- tween effective reproduction numbers across time series allowing us to estimate fixed variant-specific growth advantages. We use this model to estimate the effective reproduction number of SARS-CoV-2 Variants of Concern and Variants of Interest in the United States and estimate consistent growth advantages of particular variants across different locations.</jats:p>","version":null,"doi":"10.1101/2021.12.09.21267544","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.20.436257","pub_date":"2021-12-10","title":"Interferon Resistance of Emerging SARS-CoV-2 Variants","abstract":"The emergence of SARS-CoV-2 variants with enhanced transmissibility, pathogenesis and resistance to vaccines presents urgent challenges for curbing the COVID-19 pandemic. While Spike mutations that enhance virus infectivity or neutralizing antibody evasion may drive the emergence of these novel variants, studies documenting a critical role for interferon responses in the early control of SARS-CoV-2 infection, combined with the presence of viral genes that limit these responses, suggest that interferons may also influence SARS-CoV-2 evolution. Here, we compared the potency of 17 different human interferons against multiple viral lineages sampled during the course of the global outbreak, including ancestral and four major variants of concern. Our data reveal increased interferon resistance in emerging SARS-CoV-2 variants, suggesting that evasion of innate immunity may be a significant, ongoing driving force for SARS-CoV-2 evolution. These findings have implications for the increased lethality of emerging variants and highlight the interferon subtypes that may be most successful in the treatment of early infections. In less than 2 years since its spillover into humans, SARS-CoV-2 has infected over 220 million people, causing over 4.5 million COVID-19 deaths. High infection rates provided substantial opportunities for the virus to evolve, as variants with enhanced transmissibility, pathogenesis, and resistance to vaccine-elicited neutralizing antibodies have emerged. While much focus has centered on the Spike protein which the virus uses to infect target cells, mutations were also found in other viral proteins that might inhibit innate immune responses. Specifically, viruses encounter a potent innate immune response mediated by the interferons, two of which, IFN\u03b12 and IFN\u03b2, are being repurposed for COVID-19 treatment. Here, we compared the potency of human interferons against ancestral and emerging variants of SARS-CoV-2. Our data revealed increased interferon resistance in emerging SARS-CoV-2 strains that included the alpha, beta, gamma and delta variants of concern, suggesting a significant, but underappreciated role for innate immunity in driving the next phase of the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2021.03.20.436257","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.12.448175","pub_date":"2021-12-10","title":"Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions","abstract":"The COVID-19 pandemic is an ongoing public health emergency of international concern. While a lot of efforts are being invested in vaccinating the population, there is also an emergent requirement to find potential therapeutics to effectively counter this fast mutating SARS-CoV-2 virus-induced pathogenicity. Virus-infected host cells switch their metabolism to a more glycolytic phenotype. This switch induced by the virus is needed for faster production of ATP and higher levels of anabolic intermediates, required for new virion synthesis and packaging. In this study, we used 2-Deoxy-D-glucose (2-DG) to target and inhibit the metabolic reprogramming induced by SARS-CoV-2 infection. Our results showed that virus infection induces glucose influx and glycolysis resulting in selective high accumulation of the fluorescent glucose/2-DG analogue, 2-NBDG in these cells. Subsequently, 2-DG inhibits glycolysis in infected cells thereby reducing the virus multiplication and alleviates the cells from virus induced cytopathic effect (CPE) and cell death. Herein, we demonstrate that the crucial Nglycosites (N331 and N343) of RBD in spike protein of progeny virions produced from 2-DG treated cells were found unglycosylated and defective with compromised infectivity potential. In line with earlier reported observations, our study also showed that 2-DG mediated metabolic inhibiton can attenuate SARS-COV-2 multiplication. In addition, mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced un-glycosylation of spike protein. Our findings strengthen the notion that 2-DG effectively inhibits SARS-CoV-2 multiplication. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.","version":"1.4","doi":"10.1101/2021.06.12.448175","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471153","pub_date":"2021-12-10","title":"Elucidating design principles for engineering cell-derived vesicles to inhibit SARS-CoV-2 infection","abstract":"The ability of pathogens to develop drug resistance is a global health challenge. The SARS-CoV-2 virus presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody therapies and vaccine-induced sera. Decoy nanoparticles\u2014cell-mimicking particles that bind and inhibit virions\u2014are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, we lack quantitative understanding as to how design features impact performance of these therapeutics. To address this gap, here we perform a systematic, comparative evaluation of various biologically-derived nanoscale vesicles, which may be particularly well-suited to sustained or repeated administration in the clinic due to low toxicity, and investigate their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade monoclonal antibodies and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.","version":"1.2","doi":"10.1101/2021.12.04.471153","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471499","pub_date":"2021-12-10","title":"Insights on the mutational landscape of the SARS-CoV-2 Omicron variant","abstract":"The SARS-COV2 Omicron variant has sparked global concern due to the possibility of enhanced transmissibility and escape from vaccines and therapeutics. In this study, we describe the mutational landscape of the Omicron variant using amino acid interaction (AAI) networks. AAI network analysis is particularly well suited for interrogating the impact of constellations of mutations as occur on Omicron that may function in an epistatic manner. Our analyses suggest that as compared to previous variants of concern, the Omicron variant has increased antibody escape breadth due to mutations in class 3 and 4 antibody epitopes as well as increased escape depth due to accumulated mutations in class 1 antibody epitopes. We note certain RBD mutations that might further enhance Omicron\u2019s escape, and in particular advise careful surveillance of two subclades bearing R346S/K mutations with relevance for certain therapeutic antibodies. Further, AAI network analysis suggests that the function of certain therapeutic monoclonal antibodies may be disrupted by Omicron mutations as a result of the cumulative indirect perturbations to the epitope surface properties, despite point-mutation analyses suggesting these antibodies are tolerant of the set of Omicron mutations in isolation. Finally, for several Omicron mutations that do not appear to contribute meaningfully to antibody escape, we find evidence for a plausible role in enhanced transmissibility via disruption of RBD-down conformational stability at the RBD-RBD interface.","version":"1.2","doi":"10.1101/2021.12.06.471499","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.07.471580","pub_date":"2021-12-09","title":"Predictive profiling of SARS-CoV-2 variants by deep mutational learning","abstract":"The continual evolution of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the emergence of variants that show resistance to vaccines and neutralizing antibodies (1\u20134) threaten to prolong the coronavirus disease 2019 (COVID-19) pandemic (5). Selection and emergence of SARS-CoV-2 variants are driven in part by mutations within the viral spike protein and in particular the ACE2 receptor-binding domain (RBD), a primary target site for neutralizing antibodies. Here, we develop deep mutational learning (DML), a machine learning-guided protein engineering technology, which is used to interrogate a massive sequence space of combinatorial mutations, representing billions of RBD variants, by accurately predicting their impact on ACE2 binding and antibody escape. A highly diverse landscape of possible SARS-CoV-2 variants is identified that could emerge from a multitude of evolutionary trajectories. DML may be used for predictive profiling on current and prospective variants, including highly mutated variants such as omicron (B.1.1.529), thus supporting decision making for public heath as well as guiding the development of therapeutic antibody treatments and vaccines for COVID-19.","version":"1.1","doi":"10.1101/2021.12.07.471580","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.07.471588","pub_date":"2021-12-09","title":"Orally administered epeleuton inhibits SARS-CoV-2 viral load, replication and pathology in the Syrian Hamster model","abstract":"Early treatment of patients with confirmed COVID-19 presenting mild symptoms can reduce the number that progress to more severe disease and require hospitalization. Considering the potential for the development of drug resistance to existing therapies and the emergence of new SARS-CoV-2 variants, there is a need for an expanded armamentarium of treatment options for COVID-19. Epeleuton is a novel orally administered second-generation n-3 fatty acid with potential direct antiviral and immunomodulatory actions, and a favourable clinical safety profile. In this study we show that epeleuton inhibits SARS-CoV-2 infectious viral load, replication and disease pathology in the lungs and upper airways in the Syrian hamster model of SARS-CoV-2 infection. These data support the potential utility of epeleuton in the early treatment and prevention of SARS-CoV-2 infection. Clinical trials are needed to evaluate the efficacy of epeleuton as an outpatient treatment and prevention of COVID-19.","version":"1.1","doi":"10.1101/2021.12.07.471588","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471446","pub_date":"2021-12-09","title":"Minimal cross-over between mutations associated with Omicron variant of SARS-CoV-2 and CD8+ T cell epitopes identified in COVID-19 convalescent individuals","abstract":"There is a growing concern that ongoing evolution of SARS-CoV-2 could lead to variants of concern (VOC) that are capable of avoiding some or all of the multi-faceted immune response generated by both prior infection or vaccination, with the recently described B.1.1.529 (Omicron) VOC being of particular interest. Peripheral blood mononuclear cell samples from PCR-confirmed, recovered COVID-19 convalescent patients (n=30) infected with SARS-CoV-2 in the United States collected in April and May 2020 who possessed at least one or more of six different HLA haplotypes were selected for examination of their anti-SARS-CoV-2 CD8+ T-cell responses using a multiplexed peptide-MHC tetramer staining approach. This analysis examined if the previously identified viral epitopes targeted by CD8+ T-cells in these individuals (n=52 distinct epitopes) are mutated in the newly described Omicron VOC (n=50 mutations). Within this population, only one low-prevalence epitope from the Spike protein restricted to two HLA alleles and found in 2/30 (7%) individuals contained a single amino acid change associated with the Omicron VOC. These data suggest that virtually all individuals with existing anti-SARS-CoV-2 CD8+ T-cell responses should recognize the Omicron VOC, and that SARS-CoV-2 has not evolved extensive T-cell escape mutations at this time. The newly identified Omicron variant of concern contains more mutations than any of the previous variants described to date. In addition, many of the mutations associated with the Omicron variant are found in areas that are likely bound by neutralizing antibodies, suggesting that the first line of immunological defense against COVID-19 may be compromised. However, both natural infection and vaccination develop T-cell based responses, in addition to antibodies. This study examined if the parts of the virus, or epitopes, targeted by the CD8+ T-cell response in thirty individuals who recovered from COVID-19 in 2020 were mutated in the Omicron variant. Only one of 52 epitopes identified in this population contained an amino acid that was mutated in Omicron. These data suggest that the T-cell immune response in previously infected, and most likely vaccinated individuals, should still be effective against Omicron.","version":"1.1","doi":"10.1101/2021.12.06.471446","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.07.471131","pub_date":"2021-12-09","title":"Plant-based expression of SARS-CoV-2 antigen for use in an oral vaccine","abstract":"Oral and intra-nasal vaccines represent a key means of inducing mucosal-based immunity against infection with SARS-CoV-2, yet such vaccines represent only a minority of candidates currently in development. In this brief communication, we assessed the expression of the SARS-CoV-2 Receptor Binding Domain (RBD) subunit of the surface-exposed Spike glycoprotein in the leaves of nine edible plant species (lettuce, spinach, collard greens, tomato, cucumber, radish, arugula, pepper, and Coho greens), with a goal of identifying a suitable candidate for the development of an oral vaccine against COVID-19. We report lettuce (Lactuca sativa L. cv. Hilde II Improved) to be a preferred host to support in planta expression of SARS-CoV-2 RBD, representing an important first step towards development of a plant-based oral vaccine.","version":"1.1","doi":"10.1101/2021.12.07.471131","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471664","pub_date":"2021-12-09","title":"Combinatorial mRNA vaccination enhances protection against SARS-CoV-2 delta variant","abstract":"Emergence of SARS-CoV-2 variants of concern (VOC), including the highly transmissible delta strain, has posed challenges to current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified mRNA vaccine that expresses the more conserved viral nucleoprotein (mRNA-N). We show that mRNA-N alone was able to induce a modest but significant control of SARS-CoV-2 in mice and hamsters. Critically, by combining mRNA-N with the clinically approved S-expressing mRNA vaccine (mRNA-S-2P), we found that combinatorial mRNA vaccination (mRNA-S+N) led to markedly enhanced protection against the SARS-CoV-2 delta variant compared to mRNA-S. In a hamster model, we demonstrated that while mRNA-S alone elicited significant control of the delta strain in the lungs (\u223c45-fold reduction in viral loads compared to un-vaccinated control), its effectiveness in the upper respiratory tract was weak, whereas combinatorial mRNA-S+N vaccination induced markedly more robust control of the delta variant infection in the lungs (\u223c450-fold reduction) as well as in the upper respiratory tract (\u223c20-fold reduction). Immune analyses indicated that induction of N-specific immunity as well as augmented S-specific T-cell response and neutralizing antibody activity were collectively associated the enhanced protection against SARS-CoV-2 delta strain by combinatorial mRNA vaccination. These findings suggest that the combined effects of protection in the lungs and upper respiratory tract could both reduce the risk of severe disease as well as of infection and transmission.","version":"1.1","doi":"10.1101/2021.12.08.471664","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434733","pub_date":"2021-12-09","title":"Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike","abstract":"The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct \u201cS-R/x3\u201d to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.","version":"1.2","doi":"10.1101/2021.03.10.434733","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.07.471597","pub_date":"2021-12-09","title":"Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 & 2 antigenic polymorphism","abstract":"We report in this study the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domains (RBD) and are highly neutralizing. We applied Deep Mutational Engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. These nanobodies appears as promising therapeutic candidates to fight SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.12.07.471597","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.17.440288","pub_date":"2021-12-09","title":"Emergence of a recurrent insertion in the N-terminal domain of the SARS-CoV-2 spike glycoprotein","abstract":"Tracking the evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through genomic surveillance programs is undoubtedly one of the key priorities in the current pandemic situation. Although the genome of SARS-CoV-2 acquires mutations at a slower rate compared with other RNA viruses, evolutionary pressures derived from the widespread circulation of SARS-CoV-2 in the human population have progressively favored the global emergence, though natural selection, of several variants of concern that carry multiple non-synonymous mutations in the spike glycoprotein. These are often placed in key sites within major antibody epitopes and may therefore confer resistance to neutralizing antibodies, leading to partial immune escape, or otherwise compensate infectivity deficits associated with other non-synonymous substitutions. As previously shown by other authors, several emerging variants carry recurrent deletion regions (RDRs) that display a partial overlap with antibody epitopes located in the spike N-terminal domain (NTD). Comparatively, very little attention has been directed towards spike insertion mutations prior to the emergence of the B.1.1.529 (omicron) lineage. This manuscript describes a single recurrent insertion region (RIR1) in the N-terminal domain of SARS-CoV-2 spike protein, characterized by at least 41 independent acquisitions of 1-8 additional codons between Val213 and Leu216 in different viral lineages. Even though RIR1 is unlikely to confer antibody escape, its association with two distinct formerly widespread lineages (A.2.5 and B.1.214.2), with the quickly spreading omicron and with other VOCs and VOIs warrants further investigation concerning its effects on spike structure and viral infectivity.","version":"1.4","doi":"10.1101/2021.04.17.440288","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.02.466984","pub_date":"2021-12-09","title":"Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies","abstract":"Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.","version":"1.2","doi":"10.1101/2021.11.02.466984","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.09.471885","pub_date":"2021-12-09","title":"SARS-CoV-2 Omicron Spike Glycoprotein Receptor Binding Domain Exhibits Super-Binder Ability with ACE2 but not Convalescent Monoclonal Antibody","abstract":"SARS-CoV-2, the causative virus for COVID-19 has now super-mutated into the Omicron (Om) variant. On its spike glycoprotein alone, more than 30 substitutions have been characterized with 15 within the receptor binding domain (RBD); It therefore calls to question the transmissibility and antibody escapability of Omicron. This study was setup to investigate the Omicron RBD\u2019s interaction with ACE2 (host receptor) and a SARS-CoV-2 neutralizing monoclonal antibody (mAb). In-silico mutagenesis was used to generate the Om-RBD in complex with ACE2 or mAb from the wildtype. All-atom molecular dynamics (MD) simulation trajectories were analyzed for interaction. MD trajectories showed that Omicron RBD has evolved into an efficient ACE2 binder, via pi-pi (Om-RBD-Y501/ACE2-Y41) and salt-bridge (Om-RBD-K493/ACE2-Y41) interactions. Conversely, in binding mAb, it has become less efficient (Center of mass distance of RBD from mAb complex, wildtype \u2248 30 \u00c5, Omicron \u2248 41 \u00c5). Disruption of Om-RBD/mAb complex resulted from loose interaction between Om-RBD and the light chain complementarity-determining region residues. Omicron is expected to be better transmissible and less efficiently interacting with neutralizing convalescent mAbs. Our results elucidate the mechanisms for higher transmissibility in Omicron variant.","version":"1.1","doi":"10.1101/2021.12.09.471885","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471787","pub_date":"2021-12-09","title":"A Graph Convolutional Network-based screening strategy for rapid identification of SARS-CoV-2 cell-entry inhibitors","abstract":"The cell entry of SARS-CoV-2 has emerged as an attractive drug development target. We previously reported that the entry of SARS-CoV-2 depends on the cell surface heparan sulfate proteoglycan (HSPG) and the cortex actin, which can be targeted by therapeutic agents identified by conventional drug repurposing screens. However, this drug identification strategy requires laborious library screening, which is time-consuming and often limited number of compounds can be screened. As an alternative approach, we developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in \u223c2000 potential hits. A hit confirmation assay with the uptake of a fluorescently labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to strong inhibitory activities against the entry of SARS-CoV-2 pseudotyped particles into Vero E6 cells. These results establish a GCN-based virtual screen workflow for rapid identification of new small molecule inhibitors against validated drug targets.","version":"1.1","doi":"10.1101/2021.12.08.471787","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471684","pub_date":"2021-12-09","title":"Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination","abstract":"Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.12.08.471684","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.01.127589","pub_date":"2021-12-09","title":"Reconstructed signaling and regulatory networks identify potential drugs for SARS-CoV-2 infection","abstract":"Several molecular datasets have been recently compiled to characterize the activity of SARS-CoV-2 within human cells. Here we extend computational methods to integrate several different types of sequence, functional and interaction data to reconstruct networks and pathways activated by the virus in host cells. We identify key proteins in these networks and further intersect them with genes differentially expressed at conditions that are known to impact viral activity. Several of the top ranked genes do not directly interact with virus proteins. We experimentally tested treatments for a number of the predicted targets. We show that blocking one of the predicted indirect targets significantly reduces viral loads in stem cell-derived alveolar epithelial type II cells (iAT2s). https://github.com/phoenixding/sdremsc","version":"1.3","doi":"10.1101/2020.06.01.127589","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.09.471953","pub_date":"2021-12-09","title":"The Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614 Mutant: Conformational Plasticity and Frustration-Driven Allostery as Energetic Drivers of Highly Transmissible Spike Variant","abstract":"The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern(VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model which can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the inter-protomer hinge modulate the inter-domain interactions, global mobility change and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the inter-protomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.","version":"1.1","doi":"10.1101/2021.12.09.471953","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.07.471590","pub_date":"2021-12-09","title":"A novel bacterial protease inhibitor adjuvant in RBD-based COVID-19 vaccine formulations increases neutralizing antibodies, specific germinal center B cells and confers protection against SARS-CoV-2 infection","abstract":"In this work we evaluated recombinant receptor binding domain (RBD) based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus Alum, AddaS03, AddaVax or the combination of Alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and Alum as adjuvants have a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus Alum, AddaVax or AddaS03. Antibodies induced with the formulation containing U-Omp19 not only increased their neutralization capacity against the wild-type virus but also cross neutralized alpha, lambda and gamma variants with similar potency. Also, addition of U-Omp19 to vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+Alum formulation induced RBD-specific Th1 and CD8+ T cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal SARS-CoV-2 challenge of K18-hACE2 mice.","version":"1.1","doi":"10.1101/2021.12.07.471590","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.21267454","pub_date":"2021-12-09","title":"Selection for infectivity profiles in slow and fast epidemics, and the rise of SARS-CoV-2 variants","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Evaluating the characteristics of emerging SARS-CoV-2 variants of concern is essential to inform pandemic risk assessment. A variant may grow faster if it produces a larger number of secondary infections (transmissibility advantage) or if the timing of secondary infections (generation time) is better. So far, assessments have largely focused on deriving the transmissibility advantage assuming the generation time was unchanged. Yet, knowledge of both is needed to anticipate impact. Here we develop an analytical framework to investigate the contribution of both the transmissibility advantage and generation time to the growth advantage of a variant. We find that the growth advantage depends on the epidemiological context (level of epidemic control). More specifically, variants conferring earlier transmission are more strongly favoured when the historical strains have fast epidemic growth, while variants conferring later transmission are more strongly favoured when historical strains have slow or negative growth. We develop these conceptual insights into a statistical framework to infer both the transmissibility advantage and generation time of a variant. On simulated data, our framework correctly estimates both parameters when it covers time periods characterized by different epidemiological contexts. Applied to data for the Alpha and Delta variants in England and in Europe, we find that Alpha confers a +54% [95% CI, 45-63%] transmissibility advantage compared to previous strains, and Delta +140% [98-182%] compared to Alpha, and mean generation times are similar to historical strains for both variants. This work helps interpret variant frequency and will strengthen risk assessment for future variants of concern.</jats:p>","version":null,"doi":"10.1101/2021.12.08.21267454","journal":"medRxiv","score":null},{"id":"10.1101/2021.12.08.471688","pub_date":"2021-12-09","title":"Mutations in the spike RBD of SARS-CoV-2 omicron variant may increase infectivity without dramatically altering the efficacy of current multi-dosage vaccinations","abstract":"With the continuous evolution of SARS-CoV-2, variants of concern (VOCs) and their mutations are a focus of rapid assessment. Vital mutations in the VOC are found in spike protein, particularly in the receptor binding domain (RBD), which directly interacts with ACE2 on the host cell membrane, a key determinant of the binding affinity and cell entry. With the reporting of the most recent VOC, omicron, we performed amino acid sequence alignment of the omicron spike protein with that of the wild type and other VOCs. Although it shares several conserved mutations with other variants, we found that omicron has a large number of unique mutations. We applied the Hopp-Woods scale to calculate the hydrophilicity scores of the amino acid stretches of the RBD and the entire spike protein, and found 3 new hydrophilic regions in the RBD of omicron, implying exposure to water, with the potential to bind proteins such as ACE2 increasing transmissibility and infectivity. However, careful analysis reveals that most of the exposed domains of spike protein can serve as antigenic epitopes for generating B cell and T cell-mediated immune responses. This suggests that in the collection of polyclonal antibodies to various epitopes generated after multiple doses of vaccination, some can likely still bind to the omicron spike protein and the RBD to prevent severe clinical disease. In summary, while the omicron variant might result in more infectivity, it can still bind to a reasonable repertoire of antibodies generated by multiple doses of current vaccines likely preventing severe disease. Effective vaccines may not universally prevent opportunistic infections but can prevent the sequelae of severe disease, as observed for the delta variant. This might still be the case with the omicron variant, albeit, with increased frequency of infection.","version":"1.1","doi":"10.1101/2021.12.08.471688","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.08.471777","pub_date":"2021-12-09","title":"Structural Insights of SARS-CoV-2 Spike Protein from Delta and Omicron Variants","abstract":"Given the continuing heavy toll of the COVID-19 pandemic and the emergence of the Delta (B.1.617.2) and Omicron (B.1.1.529) variants, the WHO declared both as variants of concern (VOC). There are valid concerns that the latest Omicron variant might have increased infectivity and pathogenicity. In addition, the sheer number of S protein mutations in the Omicron variant raise concerns of potential immune evasion and resistance to therapeutics such as monoclonal antibodies. However, structural insights that underpin the potential increased pathogenicity are unknown. Here we adopted an artificial intelligence (AI)-based approach to predict the structural changes induced by mutations of the Delta and Omicron variants in the spike (S) protein using Alphafold. This was followed by docking the human angiotensin-converting enzyme 2 (ACE2) with the predicted S proteins for Wuhan-Hu-1, Delta, and Omicron variants. Our in-silico structural analysis indicates that S protein for Omicron variant has a higher binding affinity to ACE-2 receptor, compared to Wuhan-Hu-1 and Delta variants. In addition, the recognition sites of the receptor binding domains for Delta and Omicron variants showed lower electronegativity compared to Wuhan-Hu-1. Importantly, further molecular insights revealed significant changes induced at fusion protein (FP) site, which may mediate enhanced viral entry. These results represent the first computational analysis of structural changes associated with Omicron variant using Alphafold, Collectively, our results highlight potential structural basis for enhanced pathogenicity of the Omicron variant, however further validation using X-ray crystallography and cryo-EM are warranted.","version":"1.1","doi":"10.1101/2021.12.08.471777","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471525","pub_date":"2021-12-08","title":"A proteomic perspective and involvement of cytokines in SARS-CoV-2 infection","abstract":"Infection with the SARS-CoV-2 virus results in manifestation of several clinical observations from asymptomatic to multi-organ failure. Biochemically, the serious effects are due to what is described as cytokine storm. The initial infection region for COVID-19 is the nasopharyngeal/oropharyngeal region which is the site where samples are taken to examine the presence of virus. We have earlier shown that several defensin genes are down regulated in cells from this region in patients who tested positive in the RTPCR test. We have now carried out detailed proteomic analysis of the nasopharyngeal/oropharyngeal swab samples collected from normal individuals and those tested positive for SARS-CoV-2 by RTPCR, involving high throughput quantitative proteomics analysis. Several proteins like annexins, cytokines and histones were found differentially regulated in the host human cells following SARS-CoV-2 infection. Genes for these proteins were also observed to be differentially regulated when their expression was analyzed. Majority of the cytokine proteins were found to be up regulated in the infected individuals. Cell to Cell signaling interaction, Immune cell trafficking and inflammatory response pathways were found associated with the differentially regulated proteins based on network pathway analysis.","version":"1.1","doi":"10.1101/2021.12.06.471525","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471464","pub_date":"2021-12-08","title":"Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target","abstract":"SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and \u03b3H2AX (another DNA damage marker) expression. We also observed an increase of PCNA and \u03b3H2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.","version":"1.1","doi":"10.1101/2021.12.06.471464","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471483","pub_date":"2021-12-08","title":"Intranasal immunization with a vaccinia virus vaccine vector expressing pre-fusion stabilized SARS-CoV-2 spike fully protected mice against lethal challenge with the heavily mutated mouse-adapted SARS2-N501YMA30 strain of SARS-CoV-2","abstract":"The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.","version":"1.1","doi":"10.1101/2021.12.06.471483","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471421","pub_date":"2021-12-08","title":"Metabolic dysregulation induces impaired lymphocyte memory formation during severe SARS-CoV-2 infection","abstract":"Cellular metabolic dysregulation is a consequence of COVID-19 infection that is a key determinant of disease severity. To understand the mechanisms underlying these cellular changes, we performed high-dimensional immune cell profiling of PBMCs from COVID-19-infected patients, in combination with single cell transcriptomic analysis of COVID-19 BALFs. Hypoxia, a hallmark of COVID-19 ARDS, was found to elicit a global metabolic reprogramming in effector lymphocytes. In response to oxygen and nutrient-deprived microenvironments, these cells shift from aerobic respiration to increase their dependence on anaerobic processes including glycolysis, mitophagy, and glutaminolysis to fulfill their bioenergetic demands. We also demonstrate metabolic dysregulation of ciliated lung epithelial cells is linked to significant increase of proinflammatory cytokine secretion and upregulation of HLA class 1 machinery. Augmented HLA class-1 antigen stimulation by epithelial cells leads to cellular exhaustion of metabolically dysregulated CD8 and NK cells, impairing their memory cell differentiation. Unsupervised clustering techniques revealed multiple distinct, differentially abundant CD8 and NK memory cell states that are marked by high glycolytic flux, mitochondrial dysfunction, and cellular exhaustion, further highlighting the connection between disrupted metabolism and impaired memory cell function in COVID-19. Our findings provide novel insight on how SARS-CoV-2 infection affects host immunometabolism and anti-viral response during COVID-19. Hypoxia and anaerobic glycolysis drive CD8, NK, NKT dysfunction Hypoxia and anaerobic glycolysis impair memory differentiation in CD8 and NK cells Hypoxia and anaerobic glycolysis cause mitochondrial dysfunction in CD8, NK, NKT cells","version":"1.1","doi":"10.1101/2021.12.06.471421","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.07.471539","pub_date":"2021-12-08","title":"No evidence of fetal defects or anti-syncytin-1 antibody induction following COVID-19 mRNA vaccination","abstract":"The impact of coronavirus disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated two of the most widely propagated claims to determine 1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities, and 2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from vaccinated murine pregnancies exhibit high circulating levels of anti-Spike and anti-RBD antibodies to SARS-CoV-2 consistent with maternal antibody status, indicating transplacental transfer. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.","version":"1.1","doi":"10.1101/2021.12.07.471539","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471215","pub_date":"2021-12-08","title":"The influence of new SARS-CoV-2 variant Omicron (B.1.1.529) on vaccine efficacy, its correlation to Delta Variants: a computational approach","abstract":"The newly discovered COVID variant B.1.1.529 in Botswana has more than 30 mutations in spike and many other in non-spike proteins, far more than any other SARS-CoV-2 variant accepted as a variant of concern by the WHO and officially named Omicron, and has sparked concern among scientists and the general public. Our findings provide insights into structural modification caused by the mutations in the Omicrons receptor-binding domain and look into the effects on interaction with the hosts neutralising antibodies CR3022, B38, CB6, P2B-2F6, and REGN, as well as ACE2R using an in silico approach. We have employed secondary structure prediction, structural superimposition, protein disorderness, molecular docking, and MD simulation to investigate host-pathogen interactions, immune evasion, and transmissibility caused by mutations in the RBD region of the spike protein of the Omicron variant and compared it to the Delta variants (AY.1, AY.2, & AY.3) and wild type. Computational analysis revealed that the Omicron variant has a higher binding affinity for the human ACE2 receptor than the wild and Delta (AY.1 and AY.2 strains), but lower than the Delta AY.3 strain. MD simulation and docking analysis suggest that the omicron and Delta AY.3 were found to have relatively unstable and compact RBD structures and hampered interactions with antibodies more than wild and Delta (AY.1 and AY.2), which may lead to relatively more pathogenicity and antibody escape. In addition, we observed lower binding affinity of Omicron for human monoclonal antibodies (CR3022, B38, CB6, and P2B2F6) when compared to wild and Delta (AY.1 & AY.2). However, the binding affinity of Omicron RBD variants for CR3022, B38, and P2B2F6 antibodies is lower as compared to Delta AY.3, which might promote immune evasion and reinfection and needs further experimental investigation.","version":"1.1","doi":"10.1101/2021.12.06.471215","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.04.455042","pub_date":"2021-12-08","title":"But Mouse, you are not alone: On some severe acute respiratory syndrome coronavirus 2 variants infecting mice","abstract":"In silico predictions combined with in vitro, in vivo and in situ observations collectively suggest that mouse adaptation of the SARS-CoV-2 virus requires an aromatic substitution in position 501 or position 498 (but not both) of the spike protein\u2019s receptor binding domain. This effect could be enhanced by mutations in positions 417, 484, and 493 (especially K417N, E484K, Q493K and Q493R), and to a lesser extent by mutations in positions 486 and 499 (such as F486L and P499T). Such enhancements due to more favourable binding interactions with residues on the complementary angiotensin-converting enzyme 2 (ACE2) interface, are however, unlikely to sustain mouse infectivity on their own based on theoretical and experimental evidence to date. Our current understanding thus points to the Alpha, Beta, Gamma, and Omicron variants of concern infecting mice, while Delta and \u2018Delta Plus\u2019 lack a similar biomolecular basis to do so. This paper identifies eleven countries (Brazil, Chile, Djibouti, Haiti, Malawi, Mozambique, Reunion, Suriname, Trinidad and Tobago, Uruguay and Venezuela) where targeted local field surveillance of mice is encouraged because they may have come in contact with humans who had the virus with adaptive mutation(s). It also provides a systematic methodology to analyze the potential for other animal reservoirs and their likely locations.","version":"1.3","doi":"10.1101/2021.08.04.455042","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471245","pub_date":"2021-12-07","title":"SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice","abstract":"Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortex\u2014 events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1Cre; LSL-hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.","version":"1.1","doi":"10.1101/2021.12.04.471245","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471394","pub_date":"2021-12-07","title":"Insertions in the SARS-CoV-2 Spike N-Terminal Domain May Aid COVID-19 Transmission","abstract":"Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is an ongoing pandemic that causes significant health/socioeconomic burden. Variants of concern (VOCs) have emerged which may affect transmissibility, disease severity and re-infection risk. Most studies focus on the receptor-binding domain (RBD) of the Spike protein. However, some studies suggest that the Spike N-terminal domain (NTD) may have a role in facilitating virus entry via sialic-acid receptor binding. Furthermore, most VOCs include novel NTD variants. Recent analyses demonstrated that NTD insertions in VOCs tend to lie close to loop regions likely to be involved in binding sialic acids. We extended the structural characterisation of these putative sugar binding pockets and explored whether variants could enhance the binding to sialic acids and therefore to the host membrane, thereby contributing to increased transmissibility. We found that recent NTD insertions in VOCs (i.e., Gamma, Delta and Omicron variants) and emerging variants of interest (VOIs) (i.e., Iota, Lambda, Theta variants) frequently lie close to known and putative sugar-binding pockets. For some variants, including the recent Omicron VOC, we find increases in predicted sialic acid binding energy, compared to the original SARS-CoV-2, which may contribute to increased transmission. We examined the similarity of NTD across a range of related Betacoronaviruses to determine whether the putative sugar-binding pockets are sufficiently similar to be exploited in drug design. Despite global sequence and structure similarity, most sialic-acid binding pockets of NTD vary across related coronaviruses. Typically, SARS-CoV-2 possesses additional loops in these pockets that increase contact with polysaccharides. Our work suggests ongoing evolutionary tuning of the sugar-binding pockets in the virus. Whilst three of the pockets are too structurally variable to be amenable to pan Betacoronavirus drug design, we detected a fourth pocket that is highly structurally conserved and could therefore be investigated in pursuit of a generic drug. Our structure-based analyses help rationalise the effects of VOCs and provide hypotheses for experiments. For example, the Omicron variant, which has increased binding to sialic acids in pocket 3, has a rather unique insertion near pocket 3. Our work suggests a strong need for experimental monitoring of VOC changes in NTD.","version":"1.1","doi":"10.1101/2021.12.06.471394","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.05.471277","pub_date":"2021-12-07","title":"A zebrafish model of COVID-19-associated cytokine storm syndrome reveals differential proinflammatory activities of Spike proteins of SARS-CoV-2 variants of concern","abstract":"The sudden and unexpected appearance of the COVID-19 pandemic turned the whole world upside down in a very short time. One of the main challenges faced has been to understand COVID-19 patient heterogeneity, as a minority develop life-threatening hyperinflammation, the so-called cytokine storm syndrome (CSS). Using the unique advantages of the zebrafish model we report here the proinflammatory role of Spike (S) proteins from different SARS-CoV-2 variants of concern after injection into the hindbrain ventricle, a cavity filled with cerebrospinal fluid to which immune cells can be easily recruited and that mimics the alveolar environment of the human lung. We found that wild type/Wuhan variant S1 (S1WT) protein promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1\u03b3 protein was more proinflammatory and S1\u03b4 was less proinflammatory than S1WT and, strikingly, S1\u03b2 promoted delayed and long-lasting inflammation. Pharmacological inhibition of the canonical inflammasome robustly alleviated S1 protein-induced inflammation and emergency myelopoiesis. In contrast, genetic inhibition of angiotensin-converting enzyme 2 strengthened the proinflammatory activity of S1, and the administration of angiopoietin (1-7) fully rescued S1-induced hyperinflammation and hemorrhages. These results shed light into the mechanisms orchestrating the COVID-19-associated CSS and the host immune response to different SARS-CoV-2 S protein variants. S proteins of SARS-CoV-2 promote hyperinflammation, neutrophilia, monocytosis and hemorrhages in zebrafish. S protein effects in zebrafish are mediated via the canonical inflammasome and the Ace2/Angiopoietin (1-7) axis. Delta S1 is less proinflammatory than wild type S1 and fails to induce emergency myelopoiesis in zebrafish. Na\u00efve and primed human white blood cells are unable to respond to S proteins.","version":"1.1","doi":"10.1101/2021.12.05.471277","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471455","pub_date":"2021-12-07","title":"Loss of Neutralizing Antibody Response to mRNA Vaccination against SARS-CoV-2 Variants: Differing Kinetics and Strong Boosting by Breakthrough Infection","abstract":"The waning efficacy of SARS-CoV-2 vaccines combined with the continued emergence of variants resistant to vaccine-induced immunity has reignited debate over the need for booster vaccines. To address this, we examined the neutralizing antibody (nAb) response against four major SARS-CoV-2 variants\u2014D614G, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2)\u2014in health care workers (HCWs) at pre-vaccination, post-first and post-second mRNA vaccine dose, and six months post-second mRNA vaccine dose. Neutralizing antibody titers against all variants, especially the Delta variant, declined dramatically from four weeks to six months post-second mRNA vaccine dose. Notably, SARS-CoV-2 infection enhanced vaccine durability, and mRNA-1273 vaccinated HCWs also exhibited ~2-fold higher nAb titers than BNT162b2 vaccinated HCWs. Together these results demonstrate possible waning of protection from infection against SARS-CoV-2 Delta variant based on decreased nAb titers, dependent on COVID-19 status and the mRNA vaccine received.","version":"1.1","doi":"10.1101/2021.12.06.471455","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471200","pub_date":"2021-12-07","title":"Omicron: A heavily mutated SARS-CoV-2 variant exhibits stronger binding to ACE2 and potently escape approved COVID-19 therapeutic antibodies","abstract":"The new SARS-CoV-2 variant of concern \u201cOmicron\u201d was recently (Nov. 24th. 2021) spotted in South Africa and already spread around the world due to its enhanced transmissibility. The variant became conspicuous as it harbors more than thirty mutations in the spike protein with 15 mutations in the RBD region alone, potentially dampening the potency of therapeutic antibodies and enhancing the ACE2 binding. More worrying, Omicron infections have been reported in individuals who have received vaccines jabs in South Africa and Hong Kong. Here, we investigated the binding strength of Omicron with ACE2 and seven monoclonal antibodies that are either approved by FDA for COVID-19 therapy or undergoing phase III clinical trials. Computational mutagenesis and binding free energies could confirm that Omicron Spike binds ACE2 stronger than prototype SARS-CoV-2. Notably, three substitutions, i.e., T478K, Q493K, and Q498R, significantly contribute to the binding energies and doubled electrostatic potential of the RBDOmic-ACE2 complex. Instead of E484K substitution that helped neutralization escape of Beta, Gamma, and Mu variants, Omicron harbors E484A substitution. Together, T478K, Q493K, Q498R, and E484A substitutions contribute to a significant drop in the electrostatic potential energies between RBDOmic-mAbs, particularly in Etesevimab, Bamlanivimab, and CT-p59. CDR diversification could help regain the neutralization strength of these antibodies; however, we could not conduct this analysis to this end. Conclusively, our findings suggest that Omicron binds ACE2 with greater affinity, enhancing its infectivity and transmissibility. Mutations in the Spike are prudently devised by the virus that enhances the receptor binding and weakens the mAbs binding to escape the immune response.","version":"1.1","doi":"10.1101/2021.12.04.471200","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434607","pub_date":"2021-12-07","title":"The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2","abstract":"VIR-7831 (sotrovimab) and VIR-7832 are dual action monoclonal antibodies (mAbs) targeting the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VIR-7831 and VIR-7832 were derived from a parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) survivor. Both mAbs contain an \u201cLS\u201d mutation in the Fc region to prolong serum half-life. In addition, VIR-7832 encodes an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection. VIR-7831 and VIR-7832 potently neutralize wild-type and variant authentic virus in vitro as well as variant pseudotyped viruses including the Omicron variant. In addition, they retain activity against monoclonal antibody resistance mutations conferring reduced susceptibility to currently authorized mAbs. The VIR-7831/VIR-7832 epitope continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro. Furthermore, both mAbs can recruit effector mechanisms in vitro that may contribute to clinical efficacy via elimination of infected host cells. In vitro studies with these mAbs demonstrated no enhancement of infection. In a Syrian Golden hamster proof-of concept wildtype SARS-CoV-2 infection model, animals treated with VIR-7831 had less weight loss, and significantly decreased total viral load and infectious virus levels in the lung compared to a control mAb. Taken together, these data indicate that VIR-7831 and VIR-7832 are key agents in the fight against COVID-19.","version":"1.9","doi":"10.1101/2021.03.09.434607","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471236","pub_date":"2021-12-07","title":"An antibody-escape calculator for mutations to the SARS-CoV-2 receptor-binding domain","abstract":"A key goal of SARS-CoV-2 surveillance is to rapidly identify viral variants with mutations that reduce neutralization by polyclonal antibodies elicited by vaccination or infection. Unfortunately, direct experimental characterization of new viral variants lags their sequence-based identification. Here we help address this challenge by aggregating deep mutational scanning data into an \u201cescape calculator\u201d that estimates the antigenic effects of arbitrary combinations of mutations to the virus\u2019s spike receptor-binding domain (RBD). The calculator can be used to intuitively visualize how mutations impact polyclonal antibody recognition, and score the expected antigenic effect of combinations of mutations. These scores correlate with neutralization assays performed on SARS-CoV-2 variants, and emphasize the ominous antigenic properties of the recently described Omicron variant. An interactive version of the calculator is at https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/escape-calc/, and we provide a Python module for batch processing.","version":"1.1","doi":"10.1101/2021.12.04.471236","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471415","pub_date":"2021-12-07","title":"Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism","abstract":"Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds the nuclear pore components Nup98-Rae1 to inhibit nuclear translocation of activated STAT1/IRF3 transcription factors. However, a direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants can still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6/Nup98 interaction domain robustly inhibited nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral-membrane protein, opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.","version":"1.1","doi":"10.1101/2021.12.06.471415","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.05.471290","pub_date":"2021-12-07","title":"Constructing a multiple-layer interactome for SARS-CoV-2 in the context of lung disease: Linking the virus with human genes and co-infecting microbes","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused millions of deaths worldwide. Many efforts have focused on unraveling the mechanism of the viral infection to develop effective strategies for treatment and prevention. Previous studies have provided some clarity on the protein-protein interaction linkages occurring during the life cycle of viral infection; however, we lack a complete understanding of the full interactome, comprising human miRNAs and protein-coding genes and co-infecting microbes. To comprehensively determine this, we developed a statistical modeling method using latent Dirichlet allocation (called MLCrosstalk, for multiple-layer crosstalk) to fuse many types of data to construct the full interactome of SARS-CoV-2. Specifically, MLCrosstalk is able to integrate samples with multiple layers of information (e.g., miRNA and microbes), enforce a consistent topic distribution on all data types, and infer individual-level linkages (i.e., differing between patients). We also implement a secondary refinement with network propagation to allow our microbe-gene linkages to address larger network structures (e.g., pathways). Using MLCrosstalk, we generated a list of genes and microbes linked to SARS-CoV-2. Interestingly, we found that two of the identified microbes, Rothia mucilaginosa and Prevotella melaninogenica, show distinct patterns representing synergistic and antagonistic relationships with the virus, respectively. We also identified several SARS-COV-2-associated pathways, including the VEGFA-VEGFR2 and immune response pathways, which may provide potential targets for drug design.","version":"1.1","doi":"10.1101/2021.12.05.471290","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.18.436013","pub_date":"2021-12-07","title":"Variants with the N501Y mutation extend SARS-CoV-2 host range to mice, with contact transmission","abstract":"Receptor recognition is a major determinant of viral host range, infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with novel hosts, and the high mutation rate of RNA viruses may explain their frequent host shifts. SARS-CoV-2 extensive circulation in humans results in the emergence of variants, including variants of concern (VOCs) with diverse mutations notably in the spike, and increased transmissibility or immune escape. Here we show that, unlike the initial and Delta variants, the three VOCs bearing the N501Y mutation can infect common laboratory mice. Contact transmission occurred from infected to naive mice through two passages. This host range expansion likely results from an increased binding of the spike to the mouse ACE2. Together with the observed contact transmission, it raises the possibility of wild rodent secondary reservoirs enabling the emergence of new variants.","version":"1.2","doi":"10.1101/2021.03.18.436013","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.06.471389","pub_date":"2021-12-07","title":"Mutational cascade of SARS-CoV-2 leading to evolution and emergence of omicron variant","abstract":"Emergence of new variant of SARS-CoV-2, namely omicron, has posed a global concern because of its high rate of transmissibility and mutations in its genome. Researchers worldwide are trying to understand the evolution and emergence of such variants to understand the mutational cascade events. We have considered all omicron genomes (n = 302 genomes) available till 2nd December 2021 in the public repository of GISAID along with representatives of variants of concern (VOC), i.e., alpha, beta, gamma, delta, and omicron; variant of interest (VOI) mu and lambda; and variant under monitoring (VUM). Whole genome-based phylogeny and mutational analysis were performed to understand the evolution of SARS CoV-2 leading to emergence of omicron variant. Whole genome-based phylogeny depicted two phylogroups (PG-I and PG-II) forming variant specific clades except for gamma and VUM GH. Mutational analysis detected 18,261 mutations in the omicron variant, majority of which were non-synonymous mutations in spike (A67, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H), followed by RNA dependent RNA polymerase (rdrp) (A1892T, I189V, P314L, K38R, T492I, V57V), ORF6 (M19M) and nucleocapsid protein (RG203KR). Delta and omicron have evolutionary diverged into distinct phylogroups and do not share a common ancestry. While, omicron shares common ancestry with VOI lambda and its evolution is mainly derived by the non-synonymous mutations.","version":"1.1","doi":"10.1101/2021.12.06.471389","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471246","pub_date":"2021-12-07","title":"The High Transmission of SARS-CoV-2 Omicron (B.1.1.529) Variant is Not Only Due to Its hACE2 binding: A Free Energy of Perturbation Study","abstract":"The mutations in the spike protein of SARS-CoV-2 Omicron variant (B.1.1.529 lineage) gave rise to questions, but the data on the mechanism of action at the molecular level is limited. In this study, we present the Free energy of perturbation (FEP) data about the RBD-hACE2 binding of this new variant. We identified two groups of mutations located close to the most contributing substitutions Q498R and Q493R, which altered significantly the RBD-hACE2 interactions. The Q498R, Y505H and G496S mutations, in addition to N501Y, highly increased the binding to hACE2. They enhanced the binding by 98, 14 and 13 folds, respectively, which transforms the S1-RBD to a picomolar binder. However, in contrast to the case in mice the Q493R/K mutations, in a combination with K417N and T478K, dramatically reduced the S1 RBD binding by over 100 folds. The N440K, G446S and T478K substitutions had lesser contribution. Thus, the total effect of these nine mutations located on the interaction surface of RBD-hACE2 turns out to be similar to that observed in the Alpha variant. In a special circumstances it could be further altered by the E484A and S477N mutations and even lower binding capacity is likely to be detected. Finally, we provide a structural basis of the observed changes in the interactions. These data may explain only partially the observed in South Africa extremely high Omicron spread and is in support to the hypothesis for multiple mechanisms of actions involved in the transmission.","version":"1.1","doi":"10.1101/2021.12.04.471246","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471206","pub_date":"2021-12-07","title":"Engineering RNA viruses with unnatural amino acid to evoke adjustable immune response in mice","abstract":"Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a model of RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) N\u03b5-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro and in vivo, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and potentially protected their neonatal offspring from lethal challenge similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.","version":"1.1","doi":"10.1101/2021.12.04.471206","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.03.471068","pub_date":"2021-12-06","title":"The Petasites hybridus CO2-extract (Ze 339) blocks SARS-CoV-2 replication in vitro","abstract":"The coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), has spread worldwide, affecting over 250 million people and resulting in over five million deaths. Antivirals that are effective are still limited. The antiviral activities of the Petasites hybdridus CO2-extract Ze 339 were previously reported. Thus, to assess the anti-SARS-CoV-2 activity of Ze 339 as well as isopetasin and neopetasin as major active compounds, a CPE- and plaque reduction assay in Vero E6 cells was used for viral output. Antiviral effects were tested using the original virus (Wuhan) and the Delta variant of SARS-CoV-2. The antiviral drug remdesivir was used as control. Pre-treatment with Ze 339 in SARS-CoV-2 infected Vero E6 cells with either virus variant significantly inhibited virus replication with IC50 values of 0.10 and 0.40 \u03bcg/mL, repectively. The IC50 values obtained for isopetasin ranged between 0.37-0.88 \u03bcM for both virus variants, that of remdesivir between 1.53-2.37 \u03bcM. In conclusion, Ze 339 as well as the petasins potently inhibited SARS-Cov-2 replication in vitro of the Wuhan and Delta variants. Since time is of essence in finding effective treatments, clinical studies will have to demonstrate if Ze339 can become a therapeutic option to treat SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2021.12.03.471068","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.03.467161","pub_date":"2021-12-06","title":"Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides","abstract":"Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV (\u201cSARS-1\u201d), SARS-CoV-2 (\u201cSARS-2\u201d) and MERS-CoV (\u201cMERS\u201d). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2\u2019 cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca2+ dependent fashion. In this study, we want to know which residues are involved in this Ca2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca2+ ions: both FP1 and FP2 bind one Ca2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca2+, the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel to combat the ongoing COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.11.03.467161","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471219","pub_date":"2021-12-06","title":"Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins","abstract":"The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.","version":"1.1","doi":"10.1101/2021.12.04.471219","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.04.471198","pub_date":"2021-12-06","title":"Modeling the trajectory of SARS-CoV-2 spike protein evolution in continuous latent space using a neural network and Gaussian process","abstract":"Viral vaccines can lose their efficacy as the genomes of targeted viruses rapidly evolve, resulting in new variants that may evade vaccine-induced immunity. This process is apparent in the emergence of new SARS-CoV-2 variants which have the potential to undermine vaccination efforts and cause further outbreaks. Predictive vaccinology points to a future of pandemic preparedness in which vaccines can be developed preemptively based in part on predictive models of viral evolution. Thus, modeling the trajectory of SARS-CoV-2 spike protein evolution could have value for mRNA vaccine development. Traditionally, in silico sequence evolution has been modeled discretely, while there has been limited investigation into continuous models. Here we present the Viral Predictor for mRNA Evolution (VPRE), an open-source software tool which learns from mutational patterns in viral proteins and models their most statistically likely evolutionary trajectories. We trained a variational autoencoder with real-time and simulated SARS-CoV-2 genome data from Australia to encode discrete spike protein sequences into continuous numerical variables. To simulate evolution along a phylogenetic path, we trained a Gaussian process model with the numerical variables to project spike protein evolution up to five months in advance. Our predictions mapped primarily to a sequence that differed by a single amino acid from the most reported spike protein in Australia within the prediction timeframe, indicating the utility of deep learning and continuous latent spaces for modeling viral protein evolution. VPRE can be readily adapted to investigate and predict the evolution of viruses other than SARS-CoV-2 in temporal, geographic, and lineage-specific pathways.","version":"1.1","doi":"10.1101/2021.12.04.471198","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.03.470766","pub_date":"2021-12-06","title":"Pandemic-scale phylogenetics","abstract":"Phylogenetics has been central to the genomic surveillance, epidemiology and contact tracing efforts during the COVD-19 pandemic. But the massive scale of genomic sequencing has rendered the pre-pandemic tools inadequate for comprehensive phylogenetic analyses. Here, we discuss the phylogenetic package that we developed to address the needs imposed by this pandemic. The package incorporates several pandemic-specific optimization and parallelization techniques and comprises four programs: UShER, matOptimize, RIPPLES and matUtils. Using high-performance computing, UShER and matOptimize maintain and refine daily a massive mutation-annotated phylogenetic tree consisting of all SARS-CoV-2 sequences available in online repositories. With UShER and RIPPLES, individual labs \u2013 even with modest compute resources \u2013 incorporate newly-sequenced SARS-CoV-2 genomes on this phylogeny and discover evidence for recombination in real-time. With matUtils, they rapidly query and visualize massive SARS-CoV-2 phylogenies. These tools have empowered scientists worldwide to study the SARS-CoV-2 evolution and transmission at an unprecedented scale, resolution and speed.","version":"1.1","doi":"10.1101/2021.12.03.470766","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.03.454981","pub_date":"2021-12-04","title":"Rapid expansion of SARS-CoV-2 variants of concern is a result of adaptive epistasis","abstract":"The SARS-CoV-2 pandemic recently entered an alarming new phase with the emergence of the variants of concern (VOC) and understanding their biology is paramount to predicting future ones. Current efforts mainly focus on mutations in the spike glycoprotein (S), but changes in other regions of the viral proteome are likely key. We analyzed more than 900,000 SARS-CoV-2 genomes with a computational systems biology approach including a haplotype network and protein structural analyses to reveal lineage-defining mutations and their critical functional attributes. Our results indicate that increased transmission is promoted by epistasis, i.e., combinations of mutations in S and other viral proteins. Mutations in the non-S proteins involve immune-antagonism and replication performance, suggesting convergent evolution. Furthermore, adaptive mutations appear in geographically disparate locations, suggesting that either independent, repeat mutation events or recombination among different strains are generating VOC. We demonstrate that recombination is a stronger hypothesis, and may be accelerating the emergence of VOC by bringing together cooperative mutations. This emphasizes the importance of a global response to stop the COVID-19 pandemic.","version":"1.4","doi":"10.1101/2021.08.03.454981","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470414","pub_date":"2021-12-04","title":"Th1, Th2 and Th17 inflammatory pathways synergistically correlate with cardiometabolic processes. A case study in COVID-19","abstract":"A predominant source of complication in SARS-CoV-2 patients arises from the cytokine storm, an elevated expression of inflammatory helper T-cell associated cytokines that can lead to tissue damage and organ failure. The high inflammatory burden of this viral infection often results in cardiovascular comorbidities. A better understanding of the interaction between the cytokine storm and cardiovascular proteins might inform medical decisions and therapeutic approaches. We hypothesized that all major helper T-cell inflammatory pathways (Th1, Th2 and Th17) synergistically contribute to cardiometabolic modifications in serum of COVID-19 patients. We proved our hypothesis by integrating Th1, Th2 and Th17 cytokines to predict expression of cardiometabolic proteins profiled by OLINK proteomics.","version":"1.2","doi":"10.1101/2021.11.29.470414","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.470924","pub_date":"2021-12-03","title":"SARS-CoV-2 wildlife surveillance in Ontario and Qu\u00e9bec, Canada","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, is capable of infecting a variety of wildlife species. Wildlife living in close contact with humans are at an increased risk of SARS-CoV-2 exposure and if infected have the potential to become a reservoir for the pathogen, making control and management more difficult. To conduct SARS-CoV-2 surveillance in urban wildlife from Ontario and Qu\u00e9bec, Canada, increasing our knowledge of the epidemiology of the virus and our chances of detecting spillover from humans into wildlife. Using a One Health approach, we leveraged activities of existing research, surveillance, and rehabilitation programs among multiple agencies to collect samples from 776 animals from 17 different wildlife species between June 2020 and May 2021. Samples from all animals were tested for the presence of SARS-CoV-2 viral RNA, and a subset of samples from 219 animals across 3 species (raccoons, Procyon lotor; striped skunks, Mephitis mephitis; and mink, Neovison vison) were also tested for the presence of neutralizing antibodies. No evidence of SARS-CoV-2 viral RNA or neutralizing antibodies was detected in any of the tested samples. Although we were unable to identify positive SARS-CoV-2 cases in wildlife, continued research and surveillance activities are critical to better understand the rapidly changing landscape of susceptible animal species. Collaboration between academic, public and animal health sectors should include experts from relevant fields to build coordinated surveillance and response capacity.","version":"1.1","doi":"10.1101/2021.12.02.470924","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.470987","pub_date":"2021-12-03","title":"Single immunization with recombinant ACAM2000 vaccinia viruses expressing the spike and the nucleocapsid proteins protect hamsters against SARS-CoV-2 caused clinical disease","abstract":"Increasing cases of SARS-CoV-2 breakthrough infections from immunization with predominantly spike protein based COVID-19 vaccines highlight the need for alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins in a novel vaccinia virus ACAM2000 platform (rACAM2000). Following a single intramuscular immunization, the rACAM2000 co-expressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and quicker recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titre and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that the rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate and further studies will investigate if the rACAM2000 vaccine candidate can induce a long lasting immunity against infection of SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2021.12.02.470987","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.03.471057","pub_date":"2021-12-03","title":"Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection","abstract":"Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.","version":"1.1","doi":"10.1101/2021.12.03.471057","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.471030","pub_date":"2021-12-03","title":"Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor","abstract":"The global COVID-19 pandemic underscores the dire need of effective antivirals. Encouraging progress has been made in developing small molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro). However, the development of papain-like protease (PLpro) inhibitors faces several obstacles. Nevertheless, PLpro represents a high-profile drug target given its multifaceted roles in viral replication. PLpro is involved in not only the cleavage of viral polyprotein but also modulation of host immune response. In this study, we conducted a drug-repurposing screening of PLpro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PLpro inhibitors with IC50 values ranging from 3.39 to 8.28 \u00b5M. The three hits showed dose-dependent binding to PLpro in the thermal shift assay. In addition, tropifexor inhibited the cellular PLpro activity in the FlipGFP assay with an IC50 of 10.6 \u00b5M. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells with an EC50 of 4.03 \u00b5M, a 7.8-fold increase compared to GRL0617 (EC50 = 31.4 \u00b5M). Overall, tropifexor represents a novel PLpro inhibitor that can be further developed as SARS-CoV-2 antivirals.","version":"1.1","doi":"10.1101/2021.12.02.471030","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470697","pub_date":"2021-12-03","title":"Macaque-human differences in SARS-CoV-2 Spike antibody response elicited by vaccination or infection","abstract":"Macaques are a commonly used model for studying immunity to human viruses, including for studies of SARS-CoV-2 infection and vaccination. However, it is unknown whether macaque antibody responses recapitulate, and thus appropriately model, the response in humans. To answer this question, we employed a phage-based deep mutational scanning approach (Phage- DMS) to compare which linear epitopes are targeted on the SARS-CoV-2 Spike protein in humans and macaques following either vaccination or infection. We also used Phage-DMS to determine antibody escape pathways within each epitope, enabling a granular comparison of antibody binding specificities at the locus level. Overall, we identified some common epitope targets in both macaques and humans, including in the fusion peptide (FP) and stem helix- heptad repeat 2 (SH-H) regions. Differences between groups included a response to epitopes in the N-terminal domain (NTD) and C-terminal domain (CTD) in vaccinated humans but not vaccinated macaques, as well as recognition of a CTD epitope and epitopes flanking the FP in convalescent macaques but not convalescent humans. There was also considerable variability in the escape pathways among individuals within each group. Sera from convalescent macaques showed the least variability in escape overall and converged on a common response with vaccinated humans in the SH-H epitope region, suggesting highly similar antibodies were elicited. Collectively, these findings suggest that the antibody response to SARS-CoV-2 in macaques shares many features with humans, but with substantial differences in the recognition of certain epitopes and considerable individual variability in antibody escape profiles, suggesting a diverse repertoire of antibodies that can respond to major epitopes in both humans and macaques. Non-human primates, including macaques, are considered the best animal model for studying infectious diseases that infect humans. Vaccine candidates for SARS-CoV-2 are first tested in macaques to assess immune responses prior to advancing to human trials, and macaques are also used to model the human immune response to SARS-CoV-2 infection. However, there may be differences in how macaque and human antibodies recognize the SARS-CoV-2 entry protein, Spike. Here we characterized the locations on Spike that are recognized by antibodies from vaccinated or infected macaques and humans. We also made mutations to the viral sequence and assessed how these affected antibody binding, enabling a comparison of antibody binding requirements between macaques and humans at a very precise level. We found that macaques and humans share some responses, but also recognize distinct regions of Spike. We also found that in general, antibodies from different individuals had unique responses to viral mutations, regardless of species. These results will yield a better understanding of how macaque data can be used to inform human immunity to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.12.01.470697","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470767","pub_date":"2021-12-03","title":"A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike","abstract":"Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that \u223c82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (\u223c0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (\u223c0.05%) and unswitched B cells (\u223c0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2021.12.01.470767","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.470946","pub_date":"2021-12-03","title":"Omicron and Delta Variant of SARS-CoV-2: A Comparative Computational Study of Spike protein","abstract":"Emerging SARS-CoV-2 variants, especially those of concern, may have an impact on the virus\u2019s transmissibility and pathogenicity, as well as diagnostic equipment performance and vaccine effectiveness. Even though the SARS-CoV-2 Delta variant (B.1.617.2) emerged during India\u2019s second wave of infections, Delta variants have grown dominant internationally and are still evolving. On November 26, 2021, WHO identified the variant B.1.1.529 as a variant of concern, naming it Omicron, based on evidence that Omicron contains numerous mutations that may influence its behaviour. However, the mode of transmission and severity of the Omicron variant remains unknown. We used computational studies to examine the Delta and Omicron variants in this work and found that the Omicron variant had a higher affinity for human ACE2 than the Delta variant due to a significant number of mutations in the SARS-CoV-2 receptor binding domain, indicating a higher potential for transmission. Based on docking studies, the Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K mutations contribute significantly to high binding affinity with human ACE2. In comparison to the Delta variant, both the entire spike protein and the RBD in Omicron include a high proportion of hydrophobic amino acids such as leucine and phenylalanine. These amino acids are located within the protein\u2019s core and are required for structural stability. Omicron has a higher percentage of alpha-helix structure than the Delta variant in both whole spike protein and RBD, indicating that it has a more stable structure. We observed a disorder-order transition in the Omicron variant between spike protein RBD regions 468-473, and it may be significant in the influence of disordered residues/regions on spike protein stability and binding to ACE2. A future study might investigate the epidemiological and biological consequences of the Omicron variant.","version":"1.1","doi":"10.1101/2021.12.02.470946","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.471028","pub_date":"2021-12-03","title":"Systems immune profiling of variant-specific vaccination against SARS-CoV-2","abstract":"Lipid-nanoparticle(LNP)-mRNA vaccines offer protection against COVID-19. However, multiple variant lineages caused widespread breakthrough infections. There is no report on variant-specific vaccines to date. Here, we generated LNP-mRNAs specifically encoding wildtype, B.1.351 and B.1.617 SARS-CoV-2 spikes, and systematically studied their immune responses in animal models. All three LNP-mRNAs induced potent antibody responses in mice. However, WT-LNP-mRNA vaccination showed reduced neutralization against B.1.351 and B.1.617; and B.1.617-specific vaccination showed differential neutralization. All three vaccine candidates elicited antigen-specific CD8 and CD4 T cell responses. Single cell transcriptomics of B.1.351-LNP-mRNA and B.1.617-LNP-mRNA vaccinated animals revealed a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induced a systemic increase in reactive CD8 T cell population, with a strong signature of transcriptional and translational machineries in lymphocytes. BCR-seq and TCR-seq unveiled repertoire diversity and clonal expansions in vaccinated animals. These data provide direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.","version":"1.1","doi":"10.1101/2021.12.02.471028","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.470852","pub_date":"2021-12-03","title":"Post-Translational Modifications Optimize the Ability of SARS-CoV-2 Spike for Effective Interaction with Host Cell Receptors","abstract":"SARS-CoV2 spike glycoprotein is prime target for vaccines and for diagnostics and therapeutic antibodies against the virus. While anchored in the viral envelope, for effective virulance, the spike needs to maintain structural flexibility to recognize the host cell surface receptors and bind to them, a property that can heavily hinge upon the dynamics of the unresolved domains, most prominently the stalk. Construction of the complete, membrane-bound spike model and the description of its dynamics remain critical steps in understanding the inner working of this key element in viral infection. Using a hybrid approach, combining homology modeling, protein-protein docking and MD simulations, guided by biochemical and glycomics data, we have developed a full-length, membrane-bound, palmitoylated and fully-glycosylated spike structure in a native membrane. Multi-microsecond MD simulations of this model, the longest known trajectory of the full-spike, reveals conformational dynamics employed by the protein to explore the crowded surface of the host cell. In agreement with cryoEM, three flexiblele hinges in stalk allow for global conformational heterogeneity of spike in the fully-glycosyslated system mediated by glycan-glycan and glycan-lipid interactions. Dynamical range of spike is considerably reduced in its non-glycosylated form, confining the area explored by the spike on the host cell surface. Furthermore, palmitoylation of the membrane domain amplify the local curvature that may prime the fusion. We show that the identified hinge regions are highly conserved in SARS coronaviruses, highlighting their functional importance in enhancing viral infection, and thereby provide novel points for discovery of alternative therapeutics against the virus. SARS-CoV2 Spike protein, which forms the basis for high pathogenicity and transmissibility of the virus, is also prime target for the development of both diagnostics and vaccines for the debilitating disease caused by the virus. We present a full model of spike methodically crafted and used to study its atomic-level dynamics by multiple-\u00b5s simulations. The results shed new light on the impact of posttranslational modifications in the pathogenicity of the virus. We show how glycan-glycan and glycan-lipid interactions broaden the protein\u2019s dynamical range, and thereby, its effective interaction with the surface receptors on the host cell. Palmitoylation of spike membrane domain, on the other hand, results in a unique deformation pattern that might prime the membrane for fusion.","version":"1.1","doi":"10.1101/2021.12.02.470852","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470353","pub_date":"2021-12-03","title":"Possible Interference in Protein \u2013 Protein interaction as a new approach in microinhibition of respiratory pathogens on nasal\u2013 oral epithelium: An early on-screen study with reference toSARS-Cov-2\u2013ACE2 binding interference","abstract":"Upper and lower respiratory pathogens \u2013 both microbes and viruses \u2013are responsible for very high morbidity, man-hour loss, residual long term clinical conditions and even mortality. In india only, high incidence of annual respiratory infections \u2013 both UT and LT \u2013 demands prophylactic intervention in addition to all therapeutic interventions available.The issue of respiratory infections is more pronounced now in the backdrop of nearly uncontrolled high incidences of SARS-Cov-2 affection resulting in death and damage of human lives to the extent of hundreds of millions spreading over entire world, with incidence variations from country to country. After the initial unanswered phase of spread of SARS-Cov-2 virus with attendant unseen mortalities, quickest invention of a series of unusual vaccines have stemmed the lethal progress to a very significant extent, although vaccinating each and every human subject \u2013 nearly 8 to 9 bn in supremely divided world \u2013economically-- is an unthinkable proposition where economic disparity dictates vaccine availability and implementation.Moreover, being of highly unstable nucleic acid composition, the original virus, by now has a thick set of variants around the globe with variable clinical outcome. Given this complex background of scanty availability and inefficient implementation, there always is a need of a preventive approach which can possibly micro-fix the pathogens, including SARS-2 on nasal epithelium so as to interfere with viral [or any pathogen] entry through specified receptor gate[s] or any other ways. The present formulation is under study -- as a candidate of interference on nasal / oral mucosa for all respiratory pathogens. This brief report describes dry on-screen studies of protein \u2013 protein interaction as well as its possible interference by an amino acid Lysine. Phospholipid bilayerresponses in presence of added loads of the same essential amino acid \u2013Lysine \u2013 showed unusual and unexplained behavior both in structural integrity as well in spatial orientation.","version":"1.1","doi":"10.1101/2021.12.01.470353","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470802","pub_date":"2021-12-03","title":"Delta variant with P681R critical mutation revealed by ultra-large atomic-scale ab initio simulation: Implications for the fundamentals of biomolecular interactions","abstract":"SARS-CoV-2 Delta variant is emerging as a globally dominant strain. Its rapid spread and high infection rate are attributed to a mutation in the spike protein of SARS-CoV-2 allowing the virus to invade human cells much faster and with increased efficiency. Particularly, an especially dangerous mutation P681R close to the furin cleavage site has been identified as responsible for increasing the infection rate. Together with the earlier reported mutation D614G in the same domain, it offers an excellent instance to investigate the nature of mutations and how they affect the interatomic interactions in the spike protein. Here, using ultra large-scale ab initio computational modeling, we study the P681R and D614G mutations in the SD2-FP domain including the effect of double mutation and compare the results with the wild type. We have recently developed a method of calculating the amino acid-amino acid bond pairs (AABP) to quantitatively characterize the details of the interatomic interactions, enabling us to explain the nature of mutation at the atomic resolution. Our most significant find is that the mutations reduce the AABP value, implying a reduced bonding cohesion between interacting residues and increasing the flexibility of these amino acids to cause the damage. The possibility of using this unique mutation quantifiers in a machine learning protocol could lead to the prediction of emerging mutations.","version":"1.1","doi":"10.1101/2021.12.01.470802","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470796","pub_date":"2021-12-03","title":"Thermal Analysis of Protein Stability and Ligand Binding in Complex Media","abstract":"Screening of ligands that can bind to biologic products of in vitro expression systems typically requires some purification of the expressed biologic target. Such purification is often laborious and time consuming and a limiting challenge. What is required, that could represent an enormous advantage, is the ability to screen expressed proteins in the crude lysate stage without purification. For that purpose, we explore here the utility of differential scanning calorimetry (DSC) measurements for detecting the presence of specific proteins and their interactions with ligands in the complex media where they were prepared, i.e. crude lysates. Model systems were designed to mimic analogous conditions comparable to those that might be encountered in actual in vitro expression systems. Results are reported for several examples where DSC measurements distinctly showed differences in the thermal denaturation behaviors of the crude lysate alone, proteins and proteins plus binding ligands added to the crude lysate. Results were obtained for Streptavidin/Biotin binding in E. coli lysate, and binding of Angiotensin Converting Enzyme 2 (ACE2) by captopril or lisinopril in the lysate supernatant derived from cultured Human Kidney cells (HEK293). ACE2 binding by the reactive binding domain (RBC) of SARS-CoV-2 was also examined. Binding of ACE2 by RBC and lisinopril were similar and consistent with the reported ACE2 inhibitory activity of lisinopril.","version":"1.1","doi":"10.1101/2021.12.01.470796","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470810","pub_date":"2021-12-03","title":"Development of Web Application for the Comparison of Segment Variability with Sequence Evolution and Immunogenic Properties for Highly Variable Proteins: An Application to Viruses","abstract":"High rate of mutation and structural flexibilities in viral proteins quickly make them resistant to the host immune system and existing antiviral strategies. For most of the pathogenic viruses, the key survival strategies lie in their ability to evolve rapidly through mutations that affects the protein structure and function. Along with the experimental research related to antiviral development, computational data mining also plays an important role in deciphering the molecular and genomic signatures of the viral adaptability. Uncovering conserved regions in viral proteins with diverse chemical and biological properties is an important area of research for developing antiviral therapeutics, though assigning those regions is not a trivial work. Advancement in protein structural information databases and repositories, made by experimental research accelerated the in-silico mining of the data to generate more integrative information. Despite of the huge effort on correlating the protein structural information with its sequence, it is still a challenge to defeat the high mutability and adaptability of the viral genomics structure. In this current study, the authors have developed a user-friendly web application interface that will allow users to study and visualize protein segment variabilities in viral proteins and may help to find antiviral strategies. The present work of web application development allows thorough mining of the surface properties and variabilities of viral proteins which in combination with immunogenicity and evolutionary properties make the visualization robust. In combination with previous research on 20-Dimensional Euclidian Geometry based sequence variability characterization algorithm, four other parameters has been considered for this platform: [1] predicted solvent accessibility information, [2] B-Cell epitopic potential, [3] T-Cell epitopic potential and [4] coevolving region of the viral protein. Uniqueness of this study lies in the fact that a protein sequence stretch is being characterized rather than single residue-based information, which helps to compare properties of protein segments with variability. In current work, as an example, beside presenting the web application platform, five proteins of SARS-CoV2 was presented with keeping focus on protein-S. Current web-application database contains 29 proteins from 7 viruses including a GitHub repository of the raw data used in this study. The web application is up and running in the following address: http://www.protsegvar.com.","version":"1.1","doi":"10.1101/2021.12.01.470810","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.02.470917","pub_date":"2021-12-03","title":"PEPPI: Whole-proteome protein-protein interaction prediction through structure and sequence similarity, functional association, and machine learning","abstract":"Proteome-wide identification of protein-protein interactions is a formidable task which has yet to be sufficiently addressed by experimental methodologies. Many computational methods have been developed to predict proteome-wide interaction networks, but few leverage both the sensitivity of structural information and the wide availability of sequence data. We present PEPPI, a pipeline which integrates structural similarity, sequence similarity, functional association data, and machine learning-based classification through a na\u00efve Bayesian classifier model to accurately predict protein-protein interactions at a proteomic scale. Through benchmarking against a set of 798 ground truth interactions and an equal number of noninteractions, we have found that PEPPI attains 4.5% higher AUROC than the best of other state-of-the-art methods. As a proteomic-scale application, PEPPI was applied to model the interactions which occur between SARS-CoV-2 and human host cells during coronavirus infection, where 403 high-confidence interactions were identified with predictions covering 73% of a gold standard dataset from PSICQUIC and demonstrating significant complementarity with the most recent high-throughput experiments. PEPPI is available both as a webserver and in a standalone version and should be a powerful and generally applicable tool for computational screening of protein-protein interactions.","version":"1.1","doi":"10.1101/2021.12.02.470917","journal":"bioRxiv","score":null},{"id":"10.1101/2021.12.01.470817","pub_date":"2021-12-03","title":"ScRNA-Seq study of neutrophils reveals vast heterogeneity and breadth of inflammatory responses in severe COVID-19 patients","abstract":"Severe cases of COVID-19 are characterized by dysregulated immune responses, but specific mechanisms contributing to the most severe outcomes remain unclear. Neutrophils are the most abundant leukocyte population in human hosts and reach markedly high numbers during severe COVID-19. However, a detailed examination of their responses has been largely overlooked in the COVID-19 literature to date. Here, we report for the first time a dedicated study of neutrophil responses using single-cell RNA sequencing (scRNA-Seq) of fresh leukocytes from 11 hospitalized adult patients with mild and severe COVID-19 disease and 5 healthy controls. We observed that neutrophils display a pronounced inflammatory profile, with dramatic disruption of predicted cell-cell interactions as the severity of the disease increases. We also identified unique mature and immature neutrophil subpopulations based on transcriptomic profiling, including an antiviral phenotype, and changes in the proportion of each population linked to the severity of the disease. Finally, pathway analysis revealed increased markers of oxidative phosphorylation and ribosomal genes, along with downregulation of many antiviral and host defense pathway genes during severe disease compared to mild infections. Collectively, our findings indicate that neutrophils are capable of mounting effective antiviral defenses but adopt a form of immune dysregulation characterized by excess cellular stress, thereby contributing to the pathogenesis of severe COVID-19.","version":"1.1","doi":"10.1101/2021.12.01.470817","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470521","pub_date":"2021-12-02","title":"SARS-CoV-2 Delta derivatives impact on neutralization of Covishield recipient sera","abstract":"The emergence of SARS-CoV-2 Delta variant and its derivatives has created grave public health problem worldwide. The high transmissibility associated with this variant has led to daily increase in the number of SARS-CoV-2 infections. Delta variant has slowly dominated the other variants of concern. Subsequently, Delta has further mutated to Delta AY.1 to Delta AY.126. Of these, Delta AY.1 has been reported from several countries including India and considered to be highly infectious and probable escape mutant. Considering the possible immune escape, we had already evaluated the efficacy of the BBV152 against Delta and Delta AY.1 variants. Here, we have evaluated the neutralizing potential of sera of COVID-19 naive vaccinees (CNV) immunized with two doses of vaccine, COVID-19 recovered cases immunized with two doses of vaccine (CRV) and breakthrough infections (BTI) post immunization with two doses of vaccine against Delta, Delta AY.1 and B.1.617.3 using 50% plaque reduction neutralization test (PRNT50). Our study observed low NAb titer in CNV group against all the variants compared to CRV and BTI groups. Delta variant has shown highest reduction of 27.3-fold in NAb titer among CNV group compared to other groups and variants. Anti-S1-RBD IgG immune response among all the groups was also substantiated with NAb response. Compromised neutralization was observed against Delta and Delta AY.1 compared B.1 in all three groups. However, it provided protection against severity of the disease and fatality.","version":"1.1","doi":"10.1101/2021.11.30.470521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470550","pub_date":"2021-12-02","title":"Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications","abstract":"An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.","version":"1.1","doi":"10.1101/2021.11.30.470550","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470640","pub_date":"2021-12-02","title":"T cell receptor repertoire signatures associated with COVID-19 severity","abstract":"T cell receptor (TCR) repertoires are critical for antiviral immunity. Determining the TCR repertoires composition, diversity, and dynamics and how they change during viral infection can inform the molecular specificity of viral infection such as SARS-CoV-2. To determine signatures associated with COVID-19 disease severity, here we performed a large-scale analysis of over 4.7 billion sequences across 2,130 TCR repertoires from COVID-19 patients and healthy donors. TCR repertoire analyses from these data identified and characterized convergent COVID-19 associated CDR3 gene usages, specificity groups, and sequence patterns. T cell clonal expansion was found to be associated with upregulation of T cell effector function, TCR signaling, NF-kB signaling, and Interferon-gamma signaling pathways. Machine learning approaches accurately predicted disease severity for patients based on TCR sequence features, with certain high-power models reaching near-perfect AUROC scores across various predictor permutations. These analyses provided an integrative, systems immunology view of T cell adaptive immune responses to COVID-19.","version":"1.1","doi":"10.1101/2021.11.30.470640","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470642","pub_date":"2021-12-02","title":"Variance in Variants: Propagating Genome Sequence Uncertainty into Phylogenetic Lineage Assignment","abstract":"Genetic sequencing is subject to many different types of errors, but most analyses treat the resultant sequences as if they are known without error. Next generation sequencing methods rely on significantly larger numbers of reads than previous sequencing methods in exchange for a loss of accuracy in each individual read. Still, the coverage of such machines is imperfect and leaves uncertainty in many of the base calls. On top of this machine-level uncertainty, there is uncertainty induced by human error, such as errors in data entry or incorrect parameter settings. In this work, we demonstrate that the uncertainty in sequencing techniques will affect downstream analysis and propose a straightforward method to propagate the uncertainty. Our method uses a probabilistic matrix representation of individual sequences which incorporates base quality scores as a measure of uncertainty that naturally lead to resampling and replication as a framework for uncertainty propagation. With the matrix representation, resampling possible base calls according to quality scores provides a bootstrap- or prior distribution-like first step towards genetic analysis. Analyses based on these re-sampled sequences will include a more complete evaluation of the error involved in such analyses. We demonstrate our resampling method on SARS-CoV-2 data. The resampling procedures adds a linear computational cost to the analyses, but the large impact on the variance in downstream estimates makes it clear that ignoring this uncertainty may lead to overly confident conclusions. We show that SARS-CoV-2 lineage designations via Pangolin are much less certain than the bootstrap support reported by Pangolin would imply and the clock rate estimates for SARS-CoV-2 are much more variable than reported.","version":"1.1","doi":"10.1101/2021.11.30.470642","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470568","pub_date":"2021-12-01","title":"Broad human and animal coronavirus neutralisation by SARS-CoV-2 S2-targeted vaccination","abstract":"Several common-cold coronaviruses (HCoVs) are endemic in humans and several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current Coronavirus disease 2019 (COVID-19) pandemic. Whilst antibody cross-reactivity with the Spike glycoproteins (S) of diverse coronaviruses has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to or mediate protection, when induced naturally or through vaccination. Using a mouse model, we show that prior HCoV-OC43 S immunity primes neutralising antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, mouse vaccination with SARS-CoV-2 S2 elicits antibodies that neutralise diverse animal and human alphacoronaviruses and betacoronaviruses in vitro, and protects against SARS-CoV-2 challenge in vivo. Lastly, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induces stronger and broader neutralising antibody response than booster Wuhan S vaccination, suggesting it may prevent repertoire focusing caused by repeated homologous vaccination. The data presented here establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern (VOCs), as well as to unpredictable, yet inevitable future coronavirus zoonoses.","version":"1.1","doi":"10.1101/2021.11.30.470568","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434968","pub_date":"2021-12-01","title":"Low ozone concentration and negative ions for rapid SARS-CoV-2 inactivation","abstract":"Ozone is a powerful anti-bacterial, anti-fungal and anti-viral agent, yet exposure to high levels of ozone can pose risks to human/animal health and, in the long term, corrode certain objects. In order to overcome these risks, we evaluated the potential of using a relatively short exposure of a low concentration of ozone to disinfect an indoor environment in the absence of individuals and animals. ICON3 by O3ZONO/M2L, a new disinfection device generating both ozone and negative ions, was selected to assess the potential of this strategy to inactivate different viral isolates of SARS-CoV-2. Tests under controlled laboratory conditions were performed in a system consisting of an ozone-proof airtight plastic box inside a biological safety cabinet, where suspensions of two strains of SARS-CoV-2 were exposed to ozone and negative ions and virucidal activity was measured by means of two complementary methodologies: viral replication capacity and viral titer determination. These studies revealed that low concentration ozone (average 3.18 ppm after the peak) inactivated up to >99% of SARS-CoV-2 within 20 minutes of exposure. Under controlled conditions, similar ozone exposure was recreated with ICON3 in different volume rooms (15, 30, 60 m3) where a linear relationship was observed between the room volume and the time of continuous ozone/ions flow required to reach and maintain the desired ozone levels used in the laboratory studies. These studies suggest that ICON3 may have the potential for use in the disinfection of SARS-CoV-2 in indoor environments in the absence of individuals and animals, under properly controlled and monitored safety conditions.","version":"1.4","doi":"10.1101/2021.03.11.434968","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.26.470157","pub_date":"2021-11-30","title":"A large-scale systematic survey of SARS-CoV-2 antibodies reveals recurring molecular features","abstract":"In the past two years, the global research in combating COVID-19 pandemic has led to isolation and characterization of numerous human antibodies to the SARS-CoV-2 spike. This enormous collection of antibodies provides an unprecedented opportunity to study the antibody response to a single antigen. From mining information derived from 88 research publications and 13 patents, we have assembled a dataset of \u223c8,000 human antibodies to the SARS-CoV-2 spike from >200 donors. Analysis of antibody targeting of different domains of the spike protein reveals a number of common (public) responses to SARS-CoV-2, exemplified via recurring IGHV/IGK(L)V pairs, CDR H3 sequences, IGHD usage, and somatic hypermutation. We further present a proof-of-concept for prediction of antigen specificity using deep learning to differentiate sequences of antibodies to SARS-CoV-2 spike and to influenza hemagglutinin. Overall, this study not only provides an informative resource for antibody and vaccine research, but fundamentally advances our molecular understanding of public antibody responses to a viral pathogen.","version":"1.1","doi":"10.1101/2021.11.26.470157","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470349","pub_date":"2021-11-30","title":"SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge","abstract":"The Covid-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus that can stimulate strong neutralizing antibody responses. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co-expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous \u223c100nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, a non-adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein, reduced virus shedding following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant) and protected against disease associated weight loss. Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data, using an established and scalable technology, suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.","version":"1.1","doi":"10.1101/2021.11.29.470349","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470346","pub_date":"2021-11-30","title":"In silico study on the effects of disulfide bonds in ORF8 of SARS-CoV-2","abstract":"The COVID-19 epidemic, caused by virus SARS-CoV-2, has been a pandemic and threatening everyone\u2019s health in the past two years. In SARS-CoV-2, the accessory protein ORF8 plays an important role in immune modulation. Here we present an in silico study on the effects of the disulfide bonds in ORF8, including the effects on the structures, the binding sites and free energy when ORF8 binds to the human leukocyte antigen (HLA-A). Using the explicit solvent Molecular Dynamics (MD) simulations, we collect the conformational ensembles on ORF8 with different disulfide bonds reduction schemes. With a new visualization technique on the local geometry, we analyze the effects of the disulfide bonds on the structure of ORF8. We find that the disulfide bonds have large influences on the loop regions of the surface. Moreover, by performing docking between HLA-A and the conformational ensembles of ORF8, we predict the preferred binding sites and find that most of them are little affected by the disulfide bonds.Further, we estimate the binding free energy between HLA-A and ORF8 with different disulfide bonds reductions. In the end, from the comparison with the available experimental results on the epitopes of ORF8, we validated our binding sites prediction. All the above observations may provide inspirations on inhibitor/drug design against ORF8 based on the binding pathway with HLA-A.","version":"1.1","doi":"10.1101/2021.11.29.470346","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.28.470250","pub_date":"2021-11-30","title":"Myeloid cell-driven nonregenerative pulmonary scarring is conserved in multiple nonhuman primate species regardless of SARS-CoV-2 infection modality","abstract":"The novel coronavirus SARS-CoV-2 has caused a worldwide pandemic resulting in widespread efforts in development of animal models that recapitulate human disease for evaluation of medical countermeasures, and to dissect COVID-19 immunopathogenesis. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM) of nonhuman primates. Species-specific cohorts of RM and AGM Rhesus macaques (Macaca mulatta, RMs) and African green monkeys (Chlorocebus aethiops, AGMs) were experimentally infected with homologous SARS-CoV-2 by either direct mucosal instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated equivalent infection initially by either exposure route although the magnitude and duration of viral loading was greater in AGMs than that of the RM. Clinical onset was nearly immediate (+1dpi) in mucosally-exposed cohorts whereas aerosol-infected animals began to show signs +7dpi. Myeloid cell responses indicative of the development of pulmonary scarring and extended lack of regenerative capacity in the pulmonary compartment was a conserved pathologic response in both species by either exposure modality. This pathological commonality may be useful in future anti-fibrosis therapeutic evaluations and expands our understanding of how SARS-CoV-2 infection leads to ARDS and functional lung damage.","version":"1.1","doi":"10.1101/2021.11.28.470250","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470362","pub_date":"2021-11-30","title":"Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants","abstract":"The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used na\u00efve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.11.29.470362","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.26.470043","pub_date":"2021-11-30","title":"A recombinant SARS-CoV-2 RBD antigen expressed in insect cells elicits immunogenicity and confirms safety in animal models","abstract":"COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus requires new immunization strategies in addition to the current vaccines approved for human administration. In the present report, the immunological and safety evaluation in mice and hamsters of a subunit vaccine based on the RBD sub-domain with two adjuvants of oil origin is described. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays in mice and hamsters, the purified RBD formulated with adjuvants based on oil-water emulsifications and squalene was able to stimulate specific neutralizing antibodies and confirm the secretion of IFN-\u03b3 after stimulating spleen cells with the purified RBD. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results demonstrate the potential of the purified RBD administered with adjuvants through an intramuscular route, to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection against infection.","version":"1.1","doi":"10.1101/2021.11.26.470043","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.29.470421","pub_date":"2021-11-30","title":"SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens","abstract":"The baseline composition of T cells directly impacts later response to a pathogen, but the complexity of precursor states remains poorly defined. Here we examined the baseline state of SARS-CoV-2 specific T cells in unexposed individuals. SARS-CoV-2 specific CD4+ T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2 specific T cells that expressed memory phenotype markers, including memory cells with gut homing receptors. T cell clones generated from tetramer-labeled cells cross-reacted with bacterial peptides and responded to stool lysates in a MHC-dependent manner. Integrated phenotypic analyses revealed additional precursor diversity that included T cells with distinct polarized states and trafficking potential to other barrier tissues. Our findings illustrate a complex pre-existing memory pool poised for immunologic challenges and implicate non-infectious stimuli from commensal colonization as a factor that shapes pre-existing immunity. Pre-existing immunity to SARS-CoV-2 contains a complex pool of precursor lymphocytes that include differentiated cells with broad tissue tropism and the potential to cross-react with commensal antigens.","version":"1.1","doi":"10.1101/2021.11.29.470421","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.28.470293","pub_date":"2021-11-30","title":"Protective immunity of the primary SARS-CoV-2 infection reduces disease severity post re-infection with Delta variants in Syrian hamsters","abstract":"Delta variant has evolved to become dominant SARS-CoV-2 lineage worldwide and there are reports of secondary infections with varying severity in vaccinated and unvaccinated naturally recovered COVID-19 patients. As the protective immunity following the infection wanes within few months, studies of re-infection after prolonged duration is needed. Hence we assessed the potential of re-infection by Delta, Delta AY.1 and B.1 in COVID-19 recovered hamsters after 3 months of infection. Re-infection with Delta and B.1 variants in hamsters showed reduced viral shedding, lung pathology and lung viral load, whereas the upper respiratory tract viral load remained similar to that of first infection. The reduction in viral load and lung pathology after re-infection with Delta AY.1 variant was not marked. Further we assessed the disease characteristics of Delta AY.1 to understand whether it has any replication advantage over Delta variant and B.1 variant, an early isolate in Syrian hamsters. Body weight changes, viral load in respiratory organs, lung pathology, cytokine response and neutralizing antibody response were assessed. Delta AY.1 variant produced milder disease in comparison to Delta variant and the neutralizing response was similar against Delta, B.1 and B.1.351 variant in contrast to Delta or B.1 infected hamsters which showed a significant reduction in neutralization titres against B.1.351. Elevation of IL-6 levels was observed post infection in hamsters after primary infection. The prior infection could not produce sterilizing immunity but the protective effect was evident following reinfection. This indicates the importance of the transmission prevention efforts even after achieving herd immunity. Secondary infections with Delta variant are being widely reported and there are reports of increased disease severity. Delta sub lineages with K417N substitution has caused concern worldwide due to the presence of the same substitution in Beta variant, a Variant of Concern known for its immune evasion. The information on the biological characteristics of this sub lineage is also scanty. The present study showed that the secondary infection with Delta variant does not show any evidence of increased disease severity in hamster model. Delta AY. 1 variant produces mild disease in Syrian hamsters in contrast to severe disease caused by Delta variant. Delta, B.1 and AY.1 variant infected hamster sera showed comparable cross neutralizing response against each other. In contrast to the lower neutralizing response shown by B.1 and Delta variant infected animals against B.1.351 variant, Delta AY.1 showed comparable response as that with other variants. SARS-CoV-2 infections do not produce sterilizing immunity but protect from developing severe disease in case of Delta variant re-infection indicating the importance of the transmission prevention efforts even after achieving herd immunity. Delta AY. 1 infection in hamsters did not show any evidence of speculated immune evasion.","version":"1.1","doi":"10.1101/2021.11.28.470293","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.28.468932","pub_date":"2021-11-30","title":"Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography","abstract":"Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in-silico drug screening studies has been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with 9 drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.","version":"1.1","doi":"10.1101/2021.11.28.468932","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.30.470527","pub_date":"2021-11-30","title":"sgDI-tector: defective interfering viral genome bioinformatics for detection of coronavirus subgenomic RNAs","abstract":"Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M>ORF3a>N>ORF6>ORF7a>ORF8>S>E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.","version":"1.1","doi":"10.1101/2021.11.30.470527","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.25.470011","pub_date":"2021-11-29","title":"Potent neutralizing anti-SARS-CoV-2 human antibodies cure infection with SARS-CoV-2 variants in hamster model","abstract":"Treatment with neutralizing monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to COVID-19 management. Unfortunately, SARS-CoV-2 variants can escape several of these recently approved mAbs, highlighting the need for additional discovery and development. In a convalescent COVID-19 patient, we identified six mAbs, classified in four epitope groups, that potently neutralized SARS-CoV-2 Wuhan, alpha, beta, gamma and delta infection in vitro. In hamsters, mAbs 3E6 and 3B8 potently cured infection with SARS-CoV-2 Wuhan, beta and delta when administered post-viral infection at 5 mg/kg. Even at 0.2 mg/kg, 3B8 still reduced viral titers. Intramuscular delivery of DNA-encoded 3B8 resulted in in vivo mAb production of median serum levels up to 90 \u03bcg/ml, and protected hamsters against delta infection. Overall, our data mark 3B8 as a promising candidate against COVID-19, and highlight advances in both the identification and gene-based delivery of potent human mAbs.","version":"1.1","doi":"10.1101/2021.11.25.470011","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469709","pub_date":"2021-11-29","title":"Genetic alteration of human MYH6 is mimicked by SARS-CoV-2 polyprotein: mapping viral variants of cardiac interest","abstract":"Acute cardiac injury has been observed in a subset of COVID-19 patients, but the molecular basis for this clinical phenotype is unknown. It has been hypothesized that molecular mimicry may play a role in triggering an autoimmune inflammatory reaction in some individuals after SARS-CoV-2 infection. Here we investigate if linear peptides contained in proteins that are primarily expressed in the heart also occur in the SARS-CoV-2 proteome. Specifically, we compared the library of 136,704 8-mer peptides from 144 human proteins (including splicing variants) to 9,926 8-mers from all 17 viral proteins in the reference SARS-CoV-2 proteome. No 8-mers were exactly identical between the reference human proteome and the reference SARS-CoV-2 proteome. However, there were 45 8-mers that differed by only one amino acid when compared to the reference SARS-CoV-2 proteome. Interestingly, analysis of protein-coding mutations from 141,456 individuals showed that one of these 8-mers from the SARS-CoV-2 Replicase polyprotein 1a/1ab (KIALKGGK) is identical to a MYH6 peptide encoded by the c.5410C>A (Q1804K) genetic variation, which has been observed at low prevalence in Africans/African Americans (0.08%), East Asians (0.3%), South Asians (0.06%) and Latino/Admixed Americans (0.003%). Furthermore, analysis of 4.85 million SARS-CoV-2 genomes from over 200 countries shows that viral evolution has already resulted in 20 additional 8-mer peptides that are identical to human heart-enriched proteins encoded by reference sequences or genetic variants. Whether such mimicry contributes to cardiac inflammation during or after COVID-19 illness warrants further experimental evaluation. We suggest that SARS-CoV-2 variants harboring peptides identical to human cardiac proteins should be investigated as \u2018viral variants of cardiac interest\u2019.","version":"1.1","doi":"10.1101/2021.11.23.469709","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.25.470044","pub_date":"2021-11-29","title":"Anticipating future SARS-CoV-2 variants of concern through ab initio quantum mechanical modeling","abstract":"Evolved SARS-CoV-2 variants are currently challenging the efficacy of first-generation vaccines, largely through the emergence of spike protein mutants. Among these variants, Delta is presently the most concerning. We employ an ab initio quantum mechanical model based on Density Functional Theory to characterize the spike protein Receptor Binding Domain (RBD) interaction with host cells and gain mechanistic insight into SARS-CoV-2 evolution. The approach is illustrated via a detailed investigation of the role of the E484K RBD mutation, a signature mutation of the Beta and Gamma variants. The simulation is employed to: predict the depleting effect of the E484K mutation on binding the RBD with select antibodies; identify residue E484 as a weak link in the original interaction with the human receptor hACE2; and describe SARS-CoV-2 Wuhan strand binding to the bat Rhinolophus macrotis ACE2 as more optimized than the human counterpart. Finally, we predict the hACE2 binding efficacy of a hypothetical E484K mutation added to the Delta variant RBD, identifying a potential future variant of concern. Results can be generalized to other mutations, and provide useful information to complement existing experimental datasets of the interaction between randomly generated libraries of hACE2 and viral spike mutants. We argue that ab initio modeling is at the point of being aptly employed to inform and predict events pertinent to viral and general evolution.","version":"1.1","doi":"10.1101/2021.11.25.470044","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.28.21264509","pub_date":"2021-11-29","title":"Estimating the transmissibility of SARS-CoV-2 during periods of high, low and zero case incidence","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Against a backdrop of widespread global transmission, a number of countries have successfully brought large outbreaks of COVID-19 under control and maintained near-elimination status. A key element of epidemic response is the tracking of disease transmissibility in near real-time. During major outbreaks, the reproduction rate can be estimated from a time-series of case, hospitalisation or death counts. In low or zero incidence settings, knowing the potential for the virus to spread is a response priority. Absence of case data means that this potential cannot be estimated directly.</jats:p>\n                <jats:p>We present a semi-mechanistic modelling framework that draws on time-series of both behavioural data and case data (when disease activity is present) to estimate the transmissibility of SARS-CoV-2 from periods of high to low \u2013 or zero \u2013 case incidence, with a coherent transition in interpretation across the changing epidemiological situations. Of note, during periods of epidemic activity, our analysis recovers the effective reproduction number, while during periods of low \u2013 or zero \u2013 case incidence, it provides an estimate of transmission risk. This enables tracking and planning of progress towards the control of large outbreaks, maintenance of virus suppression, and monitoring the risk posed by re-introduction of the virus.</jats:p>\n                <jats:p>We demonstrate the value of our methods by reporting on their use throughout 2020 in Australia, where they have become a central component of the national COVID-19 response.</jats:p>","version":null,"doi":"10.1101/2021.11.28.21264509","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.12.426404","pub_date":"2021-11-29","title":"Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis","abstract":"ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.","version":"1.4","doi":"10.1101/2021.01.12.426404","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.26.21266896","pub_date":"2021-11-28","title":"The relationship of COVID-19 related stress and media consumption with schizotypy, depression and anxiety","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Studies report a strong impact of the COVID-19 pandemic and related stressors on the mental wellbeing of general population. In this paper, we investigated whether COVID-19 related concerns and social adversity affected schizotypal traits, anxiety and depression using structural equational modelling. In mediation analyses, we furthermore explored whether these associations were mediated by healthy (sleep and physical exercise) or unhealthy behaviours (drug and alcohol consumption, excessive media use).</jats:p>\n                <jats:p>We assessed schizotypy, depression and anxiety as well as, healthy and unhealthy behaviours and a wide range of sociodemographic scores using online surveys from residents of Germany and the United Kingdom over one year during the COVID-19 pandemic. Four independent samples were collected (April/ May 2020: N=781, September/ October 2020: N=498, January/ February 2021: N=544, May 2021: N= 486). The results revealed that COVID-19 related life concerns were significantly associated with schizotypy in the autumn 2020 and spring 2021 surveys, and with anxiety and depressive symptoms in all surveys; and social adversity significantly affected the expression of schizotypal traits in all but the spring 2020 survey, and depressive and anxiety symptoms in all samples. Importantly, we found that excessive media consumption (&gt;4h per day) fully mediated the relationship of COVID-19 related life concerns and schizotypal traits in the winter 2021 survey. Furthermore, several of the surveys showed that excessive media consumption was associated with increased depressive and anxiety- related symptoms in people burdened by COVID-19 related life.</jats:p>\n                <jats:p>The ongoing uncertainties of the pandemic and the restrictions on social life have a strong impact on mental well-being and especially the expression of schizotypal traits. The negative impact is further boosted by excessive media consumption, which is especially critical for people with high schizotypal traits.</jats:p>","version":null,"doi":"10.1101/2021.11.26.21266896","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.24.469537","pub_date":"2021-11-24","title":"Neurotoxic Amyloidogenic Peptides Identified in the Proteome of SARS-COV2: Potential Implications for Neurological Symptoms in COVID-19","abstract":"COVID-19 is primarily known as a respiratory disease caused by the virus SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, cognitive and psychiatric issues, severe headaches, and even stroke are reported in as many as 30% of cases and can persist even after the infection is over (so-called \u2018long COVID\u2019). These neurological symptoms are thought to be caused by brain inflammation, triggered by the virus infecting the central nervous system of COVID-19 patients, however we still don\u2019t fully understand the mechanisms for these symptoms. The neurological effects of COVID-19 share many similarities to neurodegenerative diseases such as Alzheimer\u2019s and Parkinson\u2019s in which the presence of cytotoxic protein-based amyloid aggregates is a common etiological feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we performed a bioinformatic scan of the SARS-CoV-2 proteome, detecting peptide fragments that were predicted to be highly amyloidogenic. We selected two of these peptides and discovered that they do rapidly self-assemble into amyloid. Furthermore, these amyloid assemblies were shown to be highly toxic to a neuronal cell line. We introduce and support the idea that cytotoxic amyloid aggregates of SARS-CoV-2 proteins are causing some of the neurological symptoms commonly found in COVID-19 and contributing to long COVID, especially those symptoms which are novel to long COVID in contrast to other post-viral syndromes.","version":"1.1","doi":"10.1101/2021.11.24.469537","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469755","pub_date":"2021-11-24","title":"Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19","abstract":"Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here we show that in COVID-19 patients, circulating plasmacytoid dendritic cells (pDCs) decline early after symptom onset and this correlated with COVID-19 disease severity. This transient depletion coincides with decreased expression of antiviral type I IFN\u03b1 and the systemic inflammatory cytokines CXCL10 and IL-6. Importantly, COVID-19 disease severity correlated with decreased pDC frequency in peripheral blood. Using an in vitro stem cell-based human pDC model, we demonstrate that pDCs directly sense SARS-CoV-2 and in response produce multiple antiviral (IFN\u03b1 and IFN\u03bb1) and inflammatory (IL-6, IL-8, CXCL10) cytokines. This immune response is sufficient to protect epithelial cells from de novo SARS-CoV-2 infection. Targeted deletion of specific sensing pathways identified TLR7-MyD88 signaling as being crucial for production of the antiviral IFNs, whereas TLR2 is responsible for the inflammatory IL-6 response. Surprisingly, we found that SARS-CoV-2 engages the neuropilin-1 receptor on pDCs to mitigate the antiviral IFNs but not the IL-6 response. These results demonstrate distinct sensing pathways used by pDCs to elicit antiviral vs. immunopathological responses to SARS-CoV-2 and suggest that targeting neuropilin-1 on pDCs may be clinically relevant for mounting TLR7-mediated antiviral protection. pDCs sense SARS-CoV-2 and elicit antiviral protection of lung epithelial cells through TLR7, while recognition of TLR2 elicits an IL-6 inflammatory response associated with immunopathology.\nGraphical abstract:\nSARS-CoV-2 sensing by plasmacytoid dendritic cells.SARS-CoV-2 is internalized by pDCs via a yet unknown endocytic mechanism. The intracellular TLR7 sensor detects viral RNA and induces a signaling cascade involving MyD88-IRAK4-TRAF6 (1) to induce CXCL10 and, via IRF7 phosphorylation and translocation, inducing type I and III Interferons (2). Once secreted, type I and III IFNs initiate autocrine and paracrine signals that induce the expression of IFN stimulated genes (ISGs), thereby facilitating an antiviral response that can protect the cell against infection. However, SARS-CoV-2, has the intrinsic property to facilitate CD304 signaling, potentially by interfering with IRF7 nuclear translocation, thereby inhibiting type I IFN\u03b1 production and thus reducing the antiviral response generated by the pDC (4). Furthermore, the SARS-CoV-2 envelope (E) glycoprotein is sensed by the extracellular TLR2/6 heterodimer and this facilitates production of the inflammatory IL-6 cytokine (5). Illustration was created with BioRender.com\n\n SARS-CoV-2 is internalized by pDCs via a yet unknown endocytic mechanism. The intracellular TLR7 sensor detects viral RNA and induces a signaling cascade involving MyD88-IRAK4-TRAF6 (1) to induce CXCL10 and, via IRF7 phosphorylation and translocation, inducing type I and III Interferons (2). Once secreted, type I and III IFNs initiate autocrine and paracrine signals that induce the expression of IFN stimulated genes (ISGs), thereby facilitating an antiviral response that can protect the cell against infection. However, SARS-CoV-2, has the intrinsic property to facilitate CD304 signaling, potentially by interfering with IRF7 nuclear translocation, thereby inhibiting type I IFN\u03b1 production and thus reducing the antiviral response generated by the pDC (4). Furthermore, the SARS-CoV-2 envelope (E) glycoprotein is sensed by the extracellular TLR2/6 heterodimer and this facilitates production of the inflammatory IL-6 cytokine (5). Illustration was created with BioRender.com","version":"1.1","doi":"10.1101/2021.11.23.469755","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469775","pub_date":"2021-11-24","title":"Atazanavir is a competitive inhibitor of SARS-CoV-2 Mpro, impairing variants replication in vitro and in vivo","abstract":"Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19), however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based, and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (Mpro) was inhibited by ATV, with Morrison\u2019s inhibitory constant (Ki) 1.5-fold higher than boceprevir (GC376, a positive control). ATV was a competitive inhibition, increasing the Mpro\u2019s Michaelis-Menten (Km) more than 6-fold. Cell-based assays indicated that SARS-CoV-2 gamma is more susceptible to ATV than its predecessor strain B.1. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the Mpro/ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.","version":"1.1","doi":"10.1101/2021.11.24.469775","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469770","pub_date":"2021-11-24","title":"Characterization of the immune resistance of SARS-CoV-2 Mu variant and the immunity induced by Mu infection","abstract":"We have revealed that the SARS-CoV-2 Mu variant is highly resistant to COVID-19 convalescent sera and vaccine sera. However, it remains unclear how the immune resistance of the Mu variant is determined. Also, although the Mu variant is highly resistant to the sera obtained from COVID-19 convalescent during early pandemic (i.e., infected with prototypic virus) and vaccinated individuals (i.e., immunized based on prototypic virus), it was unaddressed how the convalescent sera from Mu-infected individuals function. In this study, we revealed that the two mutations in the spike protein of Mu variant, YY144-145TSN and E484K, are responsible for the potent immune resistance of Mu variant. Additionally, we showed that the convalescent sera obtained from the Mu-infected individuals can be broadly antiviral against the Mu variant as well as other SARS-CoV-2 variants of concern/interest. Our findings suggest that developing novel vaccines based on the Mu variant can be more effective against a broad range of SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.11.23.469770","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469714","pub_date":"2021-11-24","title":"LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans","abstract":"SARS-CoV-2 infection is mediated by the entry receptor ACE2. Although attachment factors and co-receptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory receptor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not co-expressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory receptor for SARS-CoV-2 regulating viral entry in trans.","version":"1.1","doi":"10.1101/2021.11.23.469714","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.435957","pub_date":"2021-11-24","title":"A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection","abstract":"Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies. Techniques for measuring infection with SARS-CoV-2 in the laboratory are laborious and time-consuming, and different laboratories use different approaches. There is therefore no generally agreed way to quantitate neutralising antibodies against SARS-CoV-2, which block infection with the virus and protect people from COVID-19. In this study, we describe a new way to measure SARS-CoV-2 infection, which is much simpler and faster than existing methods. It relies on the production of a specific protease enzyme by the virus, which is able to cleave and activate an engineered protein biosensor in infected cells. This biosensor emits light in the presence of viral infection, and the amount of light released is used as a readout for the amount of infectious SARS-CoV-2 present. The signal is very sensitive, so the number of infected cells required is very small, and the method can be scaled-up to test many samples at once. In particular, we demonstrate how it can be used to detect different variants of SARS-CoV-2, and quantitate neutralising antibodies against these viruses.","version":"1.3","doi":"10.1101/2021.03.22.435957","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.06.442916","pub_date":"2021-11-24","title":"Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen","abstract":"Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.","version":"1.2","doi":"10.1101/2021.05.06.442916","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444774","pub_date":"2021-11-24","title":"Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolution","abstract":"To understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. We performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. From the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.25\u00d710-6 nt-1 per infection cycle for both lineages of SARS-CoV-2. We further show that mutation accumulation is heterogeneous along the genome, with the spike gene accumulating mutations at rate five-fold higher than the genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. These results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome towards adaptation to new environments. Mutation is the ultimate source of variation. We estimated how the SARS-COV-2 virus\u2014cause of the COVID-19 pandemic\u2014mutates. Upon infecting cells, its genome can change at a rate of 0.04 per replication. We also find that this rate can change and that its spike protein can adapt, even within few replications.","version":"1.2","doi":"10.1101/2021.05.19.444774","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424617","pub_date":"2021-11-24","title":"LazySampling and LinearSampling: Fast Stochastic Sampling of RNA Secondary Structure with Applications to SARS-CoV-2","abstract":"Many RNAs fold into multiple structures at equilibrium. The classical stochastic sampling algorithm can sample secondary structures according to their probabilities in the Boltzmann ensemble, and is widely used. However, this algorithm, consisting of a bottom-up partition function phase followed by a top-down sampling phase, suffers from three limitations: (a) the formulation and implementation of the sampling phase are unnecessarily complicated; (b) the sampling phase repeatedly recalculates many redundant recursions already done during the partition function phase; (c) the partition function runtime scales cubically with the sequence length. These issues prevent stochastic sampling from being used for very long RNAs such as the full genomes of SARS-CoV-2. To address these problems, we first adopt a hypergraph framework under which the sampling algorithm can be greatly simplified. We then present three sampling algorithms under this framework, among which the LazySampling algorithm is the fastest by eliminating redundant work in the sampling phase via on-demand caching. Based on LazySampling, we further replace the cubic-time partition function by a linear-time approximate one, and derive LinearSampling, an end-to-end linear-time sampling algorithm that is orders of magnitude faster than the standard one. For instance, LinearSampling is 176\u00d7 faster (38.9s vs. 1.9h) than Vienna RNAsubopt on the full genome of Ebola virus (18,959 nt). More importantly, LinearSampling is the first RNA structure sampling algorithm to scale up to the full-genome of SARS-CoV-2 without local window constraints, taking only 69.2 seconds on its reference sequence (29,903 nt). The resulting sample correlates well with the experimentally-guided structures. On the SARS-CoV-2 genome, LinearSampling finds 23 regions of 15 nt with high accessibilities, which are potential targets for COVID-19 diagnostics and drug design. See code: https://github.com/LinearFold/LinearSampling","version":"1.3","doi":"10.1101/2020.12.29.424617","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.283358","pub_date":"2021-11-24","title":"Unsupervised clustering analysis of SARS-Cov-2 population structure reveals six major subtypes at early stage across the world","abstract":"Identifying the population structure of the newly emerged coronavirus SARS-CoV-2 has significant potential to inform public health management and diagnosis. As SARS-CoV-2 sequencing data accrued, grouping them into clusters is important for organizing the landscape of the population structure of the virus. Due to the limited prior information on the newly emerged coronavirus, we utilized four different clustering algorithms to group 16,873 SARS-CoV-2 strains, which automatically enables the identification of spatial structure for SARS-CoV-2. A total of six distinct genomic clusters were identified using mutation profiles as input features. Comparison of the clustering results reveals that the four algorithms produced highly consistent results, but the state-of-the-art unsupervised deep learning clustering algorithm performed best and produced the smallest intra-cluster pairwise genetic distances. The varied proportions of the six clusters within different continents revealed specific geographical distributions. In particular, our analysis found that Oceania was the only continent on which the strains were dispersively distributed into six clusters. In summary, this study provides a concrete framework for the use of clustering methods to study the global population structure of SARS-CoV-2. In addition, clustering methods can be used for future studies of variant population structures in specific regions of these fast-growing viruses.","version":"1.4","doi":"10.1101/2020.09.04.283358","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.22.469576","pub_date":"2021-11-24","title":"A modified porous silicon microparticle promotes mucosal delivery of SARS-CoV-2 antigen and induction of potent and durable systemic and mucosal T helper 1 skewed protective immunity","abstract":"Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM)-adjuvanted SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable SARS-CoV-2-specific systemic humoral and type 1 helper T (Th) cell-mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant infection. mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited potent systemic and lung resident memory T and B cells and SARS-CoV-2 specific IgA responses, and markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant infection. Our results suggest that mPSM can serve as potent adjuvant for SARS-CoV-2 subunit vaccine which is effective for systemic and mucosal vaccination.","version":"1.1","doi":"10.1101/2021.11.22.469576","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469823","pub_date":"2021-11-24","title":"Coronavirus Resistance Database (CoV-RDB): SARS-CoV-2 susceptibility to monoclonal antibodies, convalescent plasma, and plasma from vaccinated persons","abstract":"As novel SARS-CoV-2 variants with different patterns of spike mutations have emerged, the susceptibility of these variants to neutralization by antibodies has been rapidly assessed. However, neutralization data are generated using different approaches and are scattered across different publications making it difficult for these data to be located and synthesized. The Stanford Coronavirus Resistance Database (CoV-RDB; https://covdb.stanford.edu) is designed to house comprehensively curated published data on the neutralizing susceptibility of SARS-CoV-2 variants and spike mutations to monoclonal antibodies (mAbs), convalescent plasma (CP), and vaccinee plasma (VP). As of October 2021, CoV-RDB contains 186 publications including 64 (34%) containing 7,328 neutralizing mAb susceptibility results, 96 (52%) containing 11,390 neutralizing CP susceptibility results, and 125 (68%) containing 20,872 neutralizing VP results. The database also records which spike mutations are selected during in vitro passage of SARS-CoV-2 in the presence of mAbs and which emerge in persons receiving mAbs as treatment. The CoV-RDB interface interactively displays neutralizing susceptibility data at different levels of granularity by filtering and/or aggregating query results according to one or more experimental conditions. The CoV-RDB website provides a companion sequence analysis program that outputs information about mutations present in a submitted sequence and that also assists users in determining the appropriate mutation-detection thresholds for identifying non-consensus amino acids. The most recent data underlying the CoV-RDB can be downloaded in its entirety from a Github repository in a documented machine-readable format.","version":"1.2","doi":"10.1101/2021.11.24.469823","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.15.452507","pub_date":"2021-11-24","title":"SARS-Cov-2 Spike binding to ACE2 is stronger and longer ranged due to glycan interaction","abstract":"Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well. Glycosylation of the receptor binding domain of the Spike protein of SARS-CoV-2 as well as the ACE2 receptor leads to stronger and longer ranged binding interactions between the proteins. Particularly, at shorter distances the interactions are between residues of the proteins themselves whereas at larger distances these interactions are mediated by the glycans.","version":"1.2","doi":"10.1101/2021.07.15.452507","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469860","pub_date":"2021-11-24","title":"Nanopore ReCappable Sequencing maps SARS-CoV-2 5\u2032 capping sites and provides new insights into the structure of sgRNAs","abstract":"The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5\u2032 cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.","version":"1.1","doi":"10.1101/2021.11.24.469860","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469776","pub_date":"2021-11-24","title":"Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities","abstract":"Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses utilizing ACE2 as their receptor. Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbor structurally validated mutations that enhance spike (S) binding and remove angiotensin enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent-cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, it delayed death or effectively resolved lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.","version":"1.1","doi":"10.1101/2021.11.24.469776","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.24.469813","pub_date":"2021-11-24","title":"Dual Effects of Nanoviricides Platform Technology Based NV-CoV-2 Biomimetic Polymer Against COVID-19","abstract":"Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However its efficacy is limited in vivo due to its low stability in presence of plasma. This paper compared the stability of RDV encapsulated with our platform technology based polymer NV-387 (NV-CoV-2), in presence of plasma in vitro and in vivo. Furthermore, a non- clinical pharmacology studies of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both NL-63 infected and uninfected rats were done. In an in vitro cell culture model experiment, antiviral activity of NV-CoV-2 and NV-CoV-2-R are also compared with RDV. The results are (i) NV-CoV-2 polymer encapsulation protects RDV from plasma- mediated catabolism in vitro and in vivo, too. (ii) Body weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2, and NV-CoV-2-R) show no toxic effects on them. (iii) NL-63 infected rats body weights and their survival length were like uninfected rats after treatment with NV-CoV-2 and NV-CoV-2-R, and the efficacy as an antiviral regimen were found in the order as below: NV-CoV-2-R > NV-CoV-2 > RDV. In brief, our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect on coronaviruses. First, NV-CoV-2 itself as an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit, rendering altogether the safest and an efficient regimen against COVID-19.","version":"1.1","doi":"10.1101/2021.11.24.469813","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469663","pub_date":"2021-11-24","title":"SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Delta have extended ACE2 receptor host-ranges","abstract":"Following the emergence of SARS-CoV-2 in China in late 2019 a number of variants have emerged, with two of these \u2013 Alpha and Delta \u2013 subsequently growing to global prevalence. One characteristic of these variants are changes within the Spike protein, in particular the receptor binding domain (RBD). From a public health perspective these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host-range of the virus. Using viral pseudotyping we examined whether the variants of concern (VOCs) Alpha, Beta, Gamma and Delta have differing host ACE2 receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes which we subsequently attribute to N501Y and E484K substitutions within the Spike RBD.","version":"1.1","doi":"10.1101/2021.11.23.469663","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.23.469765","pub_date":"2021-11-24","title":"SARS-CoV-2 Delta Spike Protein Enhances the Viral Fusogenicity and Inflammatory Cytokine Production","abstract":"The Delta variant is now the most dominant and virulent SARS-CoV-2 variant of concern (VOC). In this study, we investigated several virological features of Delta spike protein (SPDelta), including protein maturation and its impact on viral entry of cell-free pseudotyped virus, cell-cell fusion ability and its induction of inflammatory cytokine production in human macrophages and dendritic cells. The results showed that SP\u0394CDelta exhibited enhanced S1/S2 cleavage in cells and pseudotyped virus-like particles (PVLPs). We further showed that SP\u0394CDelta elevated pseudovirus infection in human lung cell lines and mediated significantly enhanced syncytia formation. Furthermore, we revealed that SP\u0394CDelta-PVLPs had stronger effects on stimulating NF-\u03baB and AP-1 signaling in human monocytic THP1 cells and induced significantly higher levels of pro-inflammatory cytokine, such as TNF-\u03b1, IL-1\u03b2 and IL-6, released from human macrophages and dendritic cells. Overall, these studies provide evidence to support the important role of SP\u0394CDelta during virus infection, transmission and pathogenesis.","version":"1.1","doi":"10.1101/2021.11.23.469765","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.22.469552","pub_date":"2021-11-24","title":"Stabilization of the SARS-CoV-2 Receptor Binding Domain by Protein Core Redesign and Deep Mutational Scanning","abstract":"Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen\u2014specific antibodies at multiple distinct epitopes. This is a challenge, as stability-enhancing mutations must be focused on the protein core, whereas successful computational stabilization algorithms typically select mutations at solvent-facing positions. In this study we report the stabilization of SARS-CoV-2 Wuhan Hu-1 Spike receptor binding domain (S RBD) using a combination of deep mutational scanning and computational design, including the FuncLib algorithm. Our most successful design encodes I358F, Y365W, T430I, and I513L RBD mutations, maintains recognition by the receptor ACE2 and a panel of different anti-RBD monoclonal antibodies, is between 1-2\u00b0C more thermally stable than the original RBD using a thermal shift assay, and is less proteolytically sensitive to chymotrypsin and thermolysin than the original RBD. Our approach could be applied to the computational stabilization of a wide range of proteins without requiring detailed knowledge of active sites or binding epitopes, particularly powerful for cases when there are multiple or unknown binding sites.","version":"1.1","doi":"10.1101/2021.11.22.469552","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.22.469492","pub_date":"2021-11-23","title":"A multi-class gene classifier for SARS-CoV-2 variants based on convolutional neural network","abstract":"Surveillance of circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great importance in controlling the coronavirus disease 2019 (COVID-19) pandemic. We propose an alignment-free in silico approach for classifying SARS-CoV-2 variants based on their genomic sequences. A deep learning model was constructed utilizing a stacked 1-D convolutional neural network and multilayer perceptron (MLP). The pre-processed genomic sequencing data of the four SARS-CoV-2 variants were first fed to three stacked convolution-pooling nets to extract local linkage patterns in the sequences. Then a 2-layer MLP was used to compute the correlations between the input and output. Finally, a logistic regression model transformed the output and returned the probability values. Learning curves and stratified 10-fold cross-validation showed that the proposed classifier enables robust variant classification. External validation of the classifier showed an accuracy of 0.9962, precision of 0.9963, recall of 0.9963 and F1 score of 0.9962, outperforming other machine learning methods, including logistic regression, K-nearest neighbor, support vector machine, and random forest. By comparing our model with an MLP model without the convolution-pooling network, we demonstrate the essential role of convolution in extracting viral variant features. Thus, our results indicate that the proposed convolution-based multi-class gene classifier is efficient for the variant classification of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.11.22.469492","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.20.469409","pub_date":"2021-11-23","title":"Airway epithelial interferon response to SARS-CoV-2 is inferior to rhinovirus and heterologous rhinovirus infection suppresses SARS-CoV-2 replication","abstract":"Common alphacoronaviruses and human rhinoviruses (HRV) induce type I and III interferon (IFN) responses important to limiting viral replication in the airway epithelium. In contrast, highly pathogenic betacoronaviruses including SARS-CoV-2 may evade or antagonize RNA-induced IFN I/III responses. In airway epithelial cells (AECs) from children and older adults we compared IFN I/III responses to SARS-CoV-2 and HRV-16, and assessed whether pre-infection with HRV-16, or pretreatment with recombinant IFN-\u03b2 or IFN-\u03bb, modified SARS-CoV-2 replication. Bronchial AECs from children (ages 6-18 yrs.) and older adults (ages 60-75 yrs.) were differentiated ex vivo to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 or HRV-16, and RNA and protein was harvested from cell lysates 96 hrs. following infection and supernatant was collected 48 and 96 hrs. following infection. In additional experiments cultures were pre-infected with HRV-16, or pre-treated with recombinant IFN-\u03b21 or IFN-\u03bb2 before SARS-CoV-2 infection. Despite significant between-donor heterogeneity SARS-CoV-2 replicated 100 times more efficiently than HRV-16. IFNB1, INFL2, and CXCL10 gene expression and protein production following HRV-16 infection was significantly greater than following SARS-CoV-2. IFN gene expression and protein production were inversely correlated with SARS-CoV-2 replication. Treatment of cultures with recombinant IFN\u03b21 or IFN\u03bb2, or pre-infection of cultures with HRV-16, markedly reduced SARS-CoV-2 replication. In addition to marked between-donor heterogeneity in IFN responses and viral replication, SARS-CoV-2 elicits a less robust IFN response in primary AEC cultures than does rhinovirus, and heterologous rhinovirus infection, or treatment with recombinant IFN-\u03b21 or IFN-\u03bb2, markedly reduces SARS-CoV-2 replication.","version":"1.1","doi":"10.1101/2021.11.20.469409","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.22.469529","pub_date":"2021-11-23","title":"Simulation of aerosol dispersion during medical examinations","abstract":"The main route of transmission of the SARS-CoV2 virus has been shown to be airborne. The objective of this study is to analyze the aerosol dispersion and potential exposure to medical staff within a typical medical examination room during classical airway procedures. The multiphase simulation of the aerosol particles in the airflow is based on a Lagrangian-Eulerian approach. All simulation cases with surgical mask show partially but significantly reduced maxi-mum dispersion distances of the aerosol particles compared to the cases without surgical mask. The simulations have shown that medical examiner are exposed to large amount of aerosol particles, especially during procedures such as laryngoscopy where the examiner\u2019s head is directly in front of the the patient\u2019s face. However, exposure can be drastically reduced if the patient wears a mask which is possible for the most of the procedures studied, such as otoscopy, sonography, or anamnesis.","version":"1.1","doi":"10.1101/2021.11.22.469529","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.20.469369","pub_date":"2021-11-22","title":"Decidual immune response following COVID-19 during pregnancy varies by timing of maternal SARS-CoV-2 infection","abstract":"While COVID-19 infection during pregnancy is common, fetal transmission is rare, suggesting that intrauterine mechanisms form an effective blockade against SARS-CoV-2. Key among these is the decidual immune environment of the placenta. We hypothesized that decidual leukocytes are altered by maternal SARS-CoV-2 infection in pregnancy and that this decidual immune resonse is shaped by the timing of infection during gestation. To address this hypothesis, we collected decidua basalis tissues at delivery from women with symptomatic COVID-19 during second (2nd Tri COVID, n=8) or third trimester (3rd Tri COVID, n=8) and SARS-CoV-2-negative controls (Control, n=8). Decidual natural killer (NK) cells, macrophages and T cells were evaluated using quantitative microscopy, and pro- and anti-inflammatory cytokine mRNA expression was evaluated using quantitative reverse transcriptase PCR (qRT-PCR). When compared with the Control group, decidual tissues from 3rd Tri COVID exhibited significantly increased macrophages, NK cells and T cells, whereas 2nd Tri COVID only had significantly increased T cells. In evaluating decidual cytokine expression, we noted that IL-6, IL-8, IL-10 and TNF-\u03b1 were significantly correlated with macrophage cell abundance. However, in 2nd Tri COVID tissues, there was significant downregulation of IL-6, IL-8, IL-10, and TNF-\u03b1. Taken together, these results suggest innate and adaptive immune responses are present at the maternal-fetal interface in maternal SARS-CoV-2 infections late in pregnancy, and that infections earlier in pregnancy show evidence of a resolving immune response. Further studies are warranted to characterize the full scope of intrauterine immune responses in pregnancies affected by maternal COVID-19.","version":"1.1","doi":"10.1101/2021.11.20.469369","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.19.469335","pub_date":"2021-11-22","title":"Acute SARS-CoV-2 infection in pregnancy is associated with placental ACE-2 shedding","abstract":"Human placental tissues have variable rates of SARS-CoV-2 invasion resulting in consistently low rates of fetal transmission suggesting a unique physiologic blockade against SARS-CoV-2. Angiotensin-converting enzyme (ACE)-2, the main receptor for SARS-CoV-2, is expressed as cell surface and soluble forms regulated by a metalloprotease cleavage enzyme, ADAM17. ACE-2 is expressed in the human placenta, but the regulation of placental ACE-2 expression in relation to timing of maternal SARS-CoV-2 infection in pregnancy is not well understood. In this study, we evaluated ACE-2 expression, ADAM17 activity and serum ACE-2 abundance in a cohort of matched villous placental and maternal serum samples from Control pregnancies (SARS-CoV-2 negative, n=8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the 2nd trimester (\u201c2ndTri COVID\u201d, n=8) and 3rd trimester (\u201c3rdTri COVID\u201d, n=8). In 3rdTri COVID as compared to control and 2ndTri-COVID villous placental tissues ACE-2 mRNA expression was remarkably elevated, however, ACE-2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE-2 was also significantly increased in the maternal serum from 3rdTri COVID infections as compared to control and 2ndTri-COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE-2 protein may be the result of ACE-2 shedding. Overall, this work highlights the importance of ACE-2 for ongoing studies on SARS-CoV-2 responses at the maternal-fetal interface.","version":"1.1","doi":"10.1101/2021.11.19.469335","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.15.468720","pub_date":"2021-11-22","title":"Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cell cultures and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived cell cultures support SARS-CoV-2 replication. Furthermore, experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs primarily at 1-day post challenge (DPC), and also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was also observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled na\u00efve sheep was not highly efficient; however, viral RNA was detected in some of the respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern (VOC), to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection, and that the alpha VOC outcompeted the ancestral lineage A strain.","version":"1.2","doi":"10.1101/2021.11.15.468720","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.19.469229","pub_date":"2021-11-22","title":"Characterization of SARS-CoV-2 public CD4+ \u03b1\u03b2 T cell clonotypes through reverse epitope discovery","abstract":"The amount of scientific data and level of public sharing produced as a consequence of the COVID-19 pandemic, as well as the speed at which these data were produced, far exceeds any previous effort against a specific disease condition. This unprecedented situation allows for development and application of new research approaches. One of the major technical hurdles in immunology is the characterization of HLA-antigen-T cell receptor (TCR) specificities. Most approaches aim to identify reactive T cells starting from known antigens using functional assays. However, the need for a reverse approach identifying the antigen specificity of orphan TCRs is increasing. Utilizing large public single-cell gene expression and TCR datasets, we identified highly public CD4+ T cell responses to SARS-CoV-2, covering >75% of the analysed population. We performed an integrative meta-analysis to deeply characterize these clonotypes by TCR sequence, gene expression, HLA-restriction, and antigen-specificity, identifying strong and public CD4+ immunodominant responses with confirmed specificity. CD4+ COVID-enriched clonotypes show T follicular helper functional features, while clonotypes depleted in SARS-CoV-2 individuals preferentially had a central memory phenotype. In total we identify more than 1200 highly public CD4+ T cell clonotypes reactive to SARS-CoV-2. TCR similarity analysis showed six prominent TCR clusters, for which we predicted both HLA-restriction and cognate SARS-CoV-2 immunodominant epitopes. To validate our predictions we used an independent cohort of TCR repertoires before and after vaccination with ChAdOx1, a replication-deficient simian adenovirus-vectored vaccine, encoding the SARS-CoV-2 spike protein. We find statistically significant enrichment of the predicted spike-reactive TCRs after vaccination with ChAdOx1, while the frequency of TCRs specific to other SARS-CoV-2 proteins remains stable. Thus, the CD4-associated TCR repertoire differentiates vaccination from natural infection. In conclusion, our study presents a novel reverse epitope discovery approach that can be used to infer HLA- and antigen-specificity of orphan TCRs in any context, such as viral infections, antitumor immune responses, or autoimmune disease. Identification of highly public CD4+ T cell responses to SARS-CoV-2 Systematic prediction of exact immunogenic HLA class II epitopes for CD4+ T cell response Methodological framework for reverse epitope discovery, which can be applied to other disease contexts and may provide essential insights for future studies and clinical applications","version":"1.1","doi":"10.1101/2021.11.19.469229","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.18.469065","pub_date":"2021-11-22","title":"Immune escape facilitation by mutations of epitope residues in RdRp of SARS-CoV-2","abstract":"SARS-CoV-2 has considerably higher mutation rate. SARS-CoV-2 possesses a RNA dependent RNA polymerase (RdRp) which helps to replicate its genome. The mutation P323L in RdRp is associated with the loss of a particular epitope (321-327) from this protein which may influence the pathogenesis of the concern SARS-CoV-2 through the development of antibody escape variants. We consider the effect of mutations in some of the epitope regions including the naturally occurring mutation P323L on the structure of the epitope and their interface with paratope using all-atom molecular dynamics (MD) simulation studies. P323L mutations cause conformational changes in the epitope region by opening up the region associated with increase in the radius of gyration and intramolecular hydrogen bonds, making the region less accessible. Moreover, the fluctuations in the dihedral angles in the epitope:paratope (IgG) interface increase which destabilize the interface. Such mutations may help in escaping antibody mediated immunity of the host.","version":"1.1","doi":"10.1101/2021.11.18.469065","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.19.469276","pub_date":"2021-11-22","title":"Hypertonic saline and aprotinin based blockage of SARS-CoV-2 specific furin site cleavage by inhibition of nasal protease activity","abstract":"SARS-CoV-2 enters into the human body mainly through the nasal epithelial cells. Cell entry of SARS-CoV-2 needs to be pre-activated by S1/S2 boundary furin motif cleavage by furin and/or relevant proteases. It is important to locally block SARS-CoV-2 S1/S2 site cleavage caused by furin and other relevant protease activity in the nasal cavity. We tested hypertonic saline and aprotinin-based blockage of SARS-CoV-2 specific furin site cleavage by furin, trypsin and nasal swab samples containing nasal proteases. Our results show that saline and aprotinin block SARS-Cov-2 specific furin site cleavage and that a saline and aprotinin combination could significantly reduce SARS-Cov-2 wild-type and P681R mutant furin site cleavage by inhibition of nasal protease activity.","version":"1.1","doi":"10.1101/2021.11.19.469276","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.17.468943","pub_date":"2021-11-22","title":"SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design","abstract":"SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the host\u2019s innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 \u00c5 and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 \u00c5, show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.","version":"1.1","doi":"10.1101/2021.11.17.468943","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.19.469220","pub_date":"2021-11-22","title":"ACE2 expression and localization are regulated by CFTR: implications beyond cystic fibrosis","abstract":"As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis (CF) could be considered a comorbidity for coronavirus disease 2019 (COVID-19). Instead, CF seems to constitute an advantage in COVID-19 infection. To clarify whether host factors expressed by the CF epithelia may influence COVID-19 progression, we investigated the expression of SARS-CoV-2 receptor and coreceptors in primary airway epithelial cells. We found that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by cystic fibrosis transmembrane conductance regulator (CFTR) channels. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral infection in CF cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin (IL)-6 in healthy donor-derived primary airway epithelial cells but a very weak response in primary CF cells. Collectively, these data support the hypothesis that CF condition is unfavorable for SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.11.19.469220","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.19.469183","pub_date":"2021-11-22","title":"Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity","abstract":"The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5\u2019-C, antisense strand: 3\u2019-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.","version":"1.1","doi":"10.1101/2021.11.19.469183","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.17.468980","pub_date":"2021-11-19","title":"Nanoviricide\u2019s platform technology based NV-CoV-2 polymer increases the half-life of Remdesivir in vivo","abstract":"So far, there are seven coronaviruses identified that infect humans and only 4 of them belong to the beta family of coronavirus (HCoV-HKU1, SARS-CoV-2, MERS-CoV and SARS-CoV). SARS family are known to cause severe respiratory disease in humans. In fact, SARS-CoV-2 infection caused a pandemic COVID-19 disease with high morbidity and mortality. Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However, the efficacy of RDV in vivo is limited due to its low stability in presence of plasma. This is the report of analysis of the non-clinical pharmacology study of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both infected and uninfected rats with SARS-CoV-2. Detection and quantification of NV-CoV-2-R in plasma samples was done by MS-HPLC chromatography analyses of precipitated plasma samples from rat subjects. NV-CoV-2-R show RDV peak in MS-HPLC chromatography, whereas only NV-CoV-2 does not show any RDV-Peak, as expected. NV-CoV-2 polymer encapsulation protects RDV in vivo from plasma-mediated catabolism. Body weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2, and NV-CoV-2-R) show no toxic effects on them. Our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect on coronaviruses. First, NV-CoV-2 itself as an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit, rendering altogether the safest and an efficient regimen against COVID-19.","version":"1.1","doi":"10.1101/2021.11.17.468980","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.12.21265796","pub_date":"2021-11-19","title":"Transmission potential of vaccinated and unvaccinated persons infected with the SARS-CoV-2 Delta variant in a federal prison, July\u2014August 2021","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The extent to which vaccinated persons who become infected with SARS-CoV-2 contribute to transmission is unclear. During a SARS-CoV-2 Delta variant outbreak among incarcerated persons with high vaccination rates in a federal prison, we assessed markers of viral shedding in vaccinated and unvaccinated persons.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Consenting incarcerated persons with confirmed SARS-CoV-2 infection provided mid-turbinate nasal specimens daily for 10 consecutive days and reported symptom data via questionnaire. Real-time reverse transcription-polymerase chain reaction (RT-PCR), viral whole genome sequencing, and viral culture was performed on these nasal specimens. Duration of RT-PCR positivity and viral culture positivity was assessed using survival analysis.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>A total of 978 specimens were provided by 95 participants, of whom 78 (82%) were fully vaccinated and 17 (18%) were not fully vaccinated. No significant differences were detected in duration of RT-PCR positivity among fully vaccinated participants (median: 13 days) versus those not fully vaccinated (median: 13 days; p=0.50), or in duration of culture positivity (medians: 5 days and 5 days; p=0.29). Among fully vaccinated participants, overall duration of culture positivity was shorter among Moderna vaccine recipients versus Pfizer (p=0.048) or Janssen (p=0.003) vaccine recipients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>As this field continues to develop, clinicians and public health practitioners should consider vaccinated persons who become infected with SARS-CoV-2 to be no less infectious than unvaccinated persons. These findings are critically important, especially in congregate settings where viral transmission can lead to large outbreaks.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.11.12.21265796","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.17.21266051","pub_date":"2021-11-19","title":"The unmitigated profile of COVID-19 infectiousness","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Quantifying the temporal dynamics of infectiousness of individuals infected with SARS-CoV-2 is crucial for understanding the spread of the COVID-19 pandemic and for analyzing the effectiveness of different mitigation strategies. Many studies have tried to use data from the onset of symptoms of infector-infectee pairs to estimate the infectiousness profile of SARS-CoV-2. However, both statistical and epidemiological biases in the data could lead to an underestimation of the duration of infectiousness. We correct for these biases by curating data from the initial outbreak of the pandemic in China (when mitigation steps were still minimal), and find that the infectiousness profile is wider than previously thought. For example, our estimate for the proportion of transmissions occurring 14 days or more after infection is an order of magnitude higher - namely 19% (95% CI 10%-25%). The inferred generation interval distribution is sensitive to the definition of the period of unmitigated transmission, but estimates that rely on later periods are less reliable due to intervention effects. Nonetheless, the results are robust to other factors such as the model, the assumed growth rate and possible bias of the dataset. Knowing the unmitigated infectiousness profile of infected individuals affects estimates of the effectiveness of self-isolation and quarantine of contacts. The framework presented here can help design better quarantine policies in early stages of future epidemics using data from the initial stages of transmission.</jats:p>","version":null,"doi":"10.1101/2021.11.17.21266051","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.15.468703","pub_date":"2021-11-19","title":"Sensitive identification of known and unknown protease activities by unsupervised linear motif deconvolution","abstract":"The cleavage-site specificities for many proteases are not well-understood, restricting the utility of supervised classification methods. We present an algorithm and web interface to overcome this limitation through the unsupervised detection of overrepresented patterns in protein sequence data, providing insight into the mixture of protease activities contributing to a complex system. Here, we apply the RObust LInear Motif Deconvolution (RoLiM) algorithm to confidently detect substrate cleavage patterns for SARS-CoV-2 Mpro protease in N terminome data of an infected human cell line. Using mass spectrometry-based peptide data from a case-control comparison of 341 primary urothelial bladder cancer cases and 110 controls, we identified distinct sequence motifs indicative of increased MMP activity in urine from cancer patients. Evaluation of N terminal peptides from patient plasma post-chemotherapy detected novel Granzyme B/Corin activity. RoLiM will enhance unbiased investigation of peptide sequences to establish the composition of known and uncharacterized protease activities in biological systems.","version":"1.1","doi":"10.1101/2021.11.15.468703","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.17.468963","pub_date":"2021-11-18","title":"Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis","abstract":"Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2\u2022 and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 \u00c5 resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml\u22121 was sufficient to reconstruct a 3.10 \u00c5 resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, \u03b2-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 \u00c5 resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.","version":"1.1","doi":"10.1101/2021.11.17.468963","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.16.468893","pub_date":"2021-11-17","title":"BSG/CD147 and ACE2 receptors facilitate SARS-CoV-2 infection of human iPS cell-derived kidney podocytes","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood. In this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing. Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro. These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism. Many patients with COVID19 disease exhibit multiorgan complications, suggesting that SARS-CoV-2 infection can extend beyond the respiratory system. Acute kidney injury is a common COVID-19 complication contributing to increased morbidity and mortality. Still, SARS-Cov-2 affinity for specialized kidney cells remain less clear. By leveraging our protocol for stem cell differentiation, we show that SARS-CoV-2 can directly infect kidney glomerular podocytes by using multiple Spike-binding proteins including ACE2 and BSG/CD147. Our results also indicate that infection by SARS-CoV-2 virus can cause podocyte cell death and foot process effacement, a hallmark of podocytopathies including collapsing glomerulopathy observed in patients with severe COVID-19 disease. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.","version":"1.1","doi":"10.1101/2021.11.16.468893","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.16.468777","pub_date":"2021-11-17","title":"The Delta variant of SARS-CoV-2 maintains high sensitivity to interferons in human lung cells","abstract":"Interferons are a major part of the anti-viral innate defense system. Successful pathogens, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), need to overcome these defenses to establish an infection. Early induction of interferons (IFNs) protects against severe coronavirus disease 2019 (COVID-19). In line with this, SARS-CoV-2 is inhibited by IFNs in vitro, and IFN-based therapies against COVID-19 are investigated in clinical trials. However, SARS-CoV-2 continues to adapt to the human population resulting in the emergence of variants characterized by increased transmission fitness and/or decreased sensitivity to preventive or therapeutic measures. It has been suggested that the efficient spread of these so-called \u201cVariants of Concern\u201d (VOCs) may also involve reduced sensitivity to IFNs. Here, we examined whether the four current VOCs (Alpha, Beta, Gamma and Delta) differ in replication efficiency or IFN sensitivity from an early isolate of SARS-CoV-2. All viruses replicated in a human lung cell line and in iPSC-derived alveolar type II cells (iAT2). The Delta variant showed accelerated replication kinetics and higher infectious virus production compared to the early 2020 isolate. Replication of all SARS-CoV-2 VOCs was reduced in the presence of exogenous type I, II and III IFNs. On average, the Alpha variant was the least susceptible to IFNs and the Alpha, Beta and Gamma variants show increased resistance against type III IFN. Although the Delta variant has outcompeted all other variants in humans it remained as sensitive to IFNs as an early 2020 SARS-CoV-2 isolate. This suggests that increased replication fitness rather than IFN resistance may be a reason for its dominance. Our results may help to understand changes in innate immune susceptibility of VOCs, and inform clinical trials exploring IFN-based COVID-19 therapies.","version":"1.1","doi":"10.1101/2021.11.16.468777","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.15.468761","pub_date":"2021-11-17","title":"Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice","abstract":"Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited anti-viral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, \u223c50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine (Cxcl9, Cxcl10, Ccl2, Ccl5 and Ccl19) and cytokine (Ifn-\u03bb and Tnf-\u03b1) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease. Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.","version":"1.1","doi":"10.1101/2021.11.15.468761","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.17.468929","pub_date":"2021-11-17","title":"Predicting SARS-CoV-2 epitope-specific TCR recognition using pre-trained protein embeddings","abstract":"The COVID-19 pandemic is ongoing because of the high transmission rate and the emergence of SARS-CoV-2 variants. The P272L mutation in SARS-Cov-2 S-protein is known to be highly relevant to the viral escape associated with the second pandemic wave in Europe. Epitope-specific T-cell receptor (TCR) recognition is a key factor in determining the T-cell immunogenicity of a SARS-CoV-2 epitope. Although several data-driven methods for predicting epitope-specific TCR recognition have been proposed, they remain challenging owing to the enormous diversity of TCRs and the lack of available training data. Self-supervised transfer learning has recently been demonstrated to be powerful for extracting useful information from unlabeled protein sequences and increasing the predictive performance of the fine-tuned models in downstream tasks. Here, we present a predictive model based on Bidirectional Encoder Representations from Transformers (BERT), employing self-supervised transfer learning, to predict SARS-CoV-2 T-cell epitope-specific TCR recognition. The fine-tuned model showed notably high predictive performance for independent evaluation using the SARS-CoV-2 epitope-specific TCR CDR3\u03b2 sequence datasets. In particular, we found the proline at position 4 corresponding to the P272L mutation in the SARS-CoV-2 S-protein269-277 epitope (YLQPRTFLL) may contribute substantially to TCR recognition of the epitope through interpreting the output attention weights of our model. We anticipate that our findings will provide new directions for constructing a reliable data-driven model to predict the immunogenic T-cell epitopes using limited training data and help accelerate the development of an effective vaccine in response to SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.11.17.468929","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.16.468834","pub_date":"2021-11-17","title":"Global Mutational Sweep of SARS-CoV-2: from Chaos to Order","abstract":"Analysis of large-scale genome sequences demonstrates the mutation of SARS-CoV-2 has been undergoing significant sweeps. Driven by emerging variants, global sweeps are accelerated and purified over time. This may prolong the pandemic with repeating epidemics, presenting challenges to the control and prevention of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.11.16.468834","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.15.468633","pub_date":"2021-11-17","title":"Tunneling nanotubes provide a novel route for SARS-CoV-2 spreading between permissive cells and to non-permissive neuronal cells","abstract":"SARS-CoV-2 entry into host cells is mediated by the binding of its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor, highly expressed in several organs, but very low in the brain. The mechanism through which SARS-CoV-2 infects neurons is not understood. Tunneling nanotubes (TNTs), actin-based intercellular conduits that connect distant cells, allow the transfer of cargos, including viruses. Here, we explored the neuroinvasive potential of SARS-CoV-2 and whether TNTs are involved in its spreading between cells in vitro. We report that neuronal cells, not permissive to SARS-CoV-2 through an exocytosis/endocytosis dependent pathway, can be infected when co-cultured with permissive infected epithelial cells. SARS-CoV-2 induces TNTs formation between permissive cells and exploits this route to spread to uninfected permissive cells in co-culture. Correlative Cryo-electron tomography reveals that SARS-CoV-2 is associated with the plasma membrane of TNTs formed between permissive cells and virus-like vesicular structures are inside TNTs established both between permissive cells and between permissive and non-permissive cells. Our data highlight a potential novel mechanism of SARS-CoV-2 spreading which could serve as route to invade non-permissive cells and potentiate infection in permissive cells.","version":"1.1","doi":"10.1101/2021.11.15.468633","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.461909","pub_date":"2021-11-16","title":"Occlusion of lip movements impairs reconstruction of acoustic speech features and higher-level segmentational features in the presence of a distractor speaker","abstract":"Multisensory integration enables stimulus representation even when the sensory input in a single modality is weak. In the context of speech, when confronted with a degraded acoustic signal, congruent visual inputs promote comprehension. When this input is occluded speech comprehension consequently becomes more difficult. But it still remains inconclusive which levels of speech processing are affected under which circumstances by occlusion of the mouth area. To answer this question, we conducted an audiovisual (AV) multi-speaker experiment using naturalistic speech. In half of the trials, the target speaker wore a (surgical) face mask, while we measured the brain activity of normal hearing participants via magnetoencephalography (MEG). We additionally added a distractor speaker in half of the trials in order to create an ecologic difficult listening situation. A decoding model on the clear AV speech was trained and used to reconstruct crucial speech features in each condition. We found significant main effects of face masks on the reconstruction of acoustic features, such as the speech envelope and spectral speech features (i.e. pitch and formant frequencies), while reconstruction of higher level features of speech segmentation (phoneme and word onsets) were especially impaired through masks in difficult listening situations. As we used surgical face masks in our study, which only show mild effects on speech acoustics, we interpret our findings as the result of the occluded lip movements. This idea is in line with recent research showing that visual cortical regions track spectral modulations. Our findings extend previous behavioural results, by demonstrating the complex contextual effects of occluding relevant visual information on speech processing. Surgical face masks impair neural tracking of speech features Tracking of acoustic features is generally impaired, while higher level segmentational features show their effects especially in challenging listening situations An explanation is the prevention of a visuo-phonological transformation contributing to audiovisual multisensory integration","version":"1.2","doi":"10.1101/2021.09.28.461909","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.13.468472","pub_date":"2021-11-15","title":"A live attenuated influenza virus-vectored intranasal COVID-19 vaccine provides rapid, prolonged, and broad protection against SARS-CoV-2 infection","abstract":"Remarkable progress has been made in developing intramuscular vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they are limited with respect to eliciting local immunity in the respiratory tract, which is the primary infection site for SARS-CoV-2. To overcome the limitations of intramuscular vaccines, we constructed a nasal vaccine candidate based on an influenza vector by inserting a gene encoding the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, named CA4-dNS1-nCoV-RBD (dNS1-RBD). A preclinical study showed that in hamsters challenged 1 day and 7 days after single-dose vaccination or 6 months after booster vaccination, dNS1-RBD largely mitigated lung pathology, with no loss of body weight, caused by either the prototype-like strain or beta variant of SARS-CoV-2. Lasted data showed that the animals could be well protected against beta variant challenge 9 months after vaccination. Notably, the weight loss and lung pathological changes of hamsters could still be significantly reduced when the hamster was vaccinated 24 h after challenge. Moreover, such cellular immunity is relatively unimpaired for the most concerning SARS-CoV-2 variants. The protective immune mechanism of dNS1-RBD could be attributed to the innate immune response in the nasal epithelium, local RBD-specific T cell response in the lung, and RBD-specific IgA and IgG response. Thus, this study demonstrates that the intranasally delivered dNS1-RBD vaccine candidate may offer an important addition to fight against the ongoing COVID-19 pandemic, compensating limitations of current intramuscular vaccines, particularly at the start of an outbreak.","version":"1.1","doi":"10.1101/2021.11.13.468472","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.11.468228","pub_date":"2021-11-15","title":"Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro","abstract":"In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-\u03baB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 \u00c5 resolution crystal structure of 3CLpro C145S bound to NEMO226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro- NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.","version":"1.1","doi":"10.1101/2021.11.11.468228","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.23.393488","pub_date":"2021-11-15","title":"LinearTurboFold: Linear-Time Global Prediction of Conserved Structures for RNA Homologs with Applications to SARS-CoV-2","abstract":"The constant emergence of COVID-19 variants reduces the effectiveness of existing vaccines and test kits. Therefore, it is critical to identify conserved structures in SARS-CoV-2 genomes as potential targets for variant-proof diagnostics and therapeutics. However, the algorithms to predict these conserved structures, which simultaneously fold and align multiple RNA homologs, scale at best cubically with sequence length, and are thus infeasible for coronaviruses, which possess the longest genomes (\u223c30,000 nt) among RNA viruses. As a result, existing efforts on modeling SARS-CoV-2 structures resort to single sequence folding as well as local folding methods with short window sizes, which inevitably neglect long-range interactions that are crucial in RNA functions. Here we present LinearTurboFold, an efficient algorithm for folding RNA homologs that scales linearly with sequence length, enabling unprecedented global structural analysis on SARS-CoV-2. Surprisingly, on a group of SARS-CoV-2 and SARS-related genomes, LinearTurbo-Fold\u2019s purely in silico prediction not only is close to experimentally-guided models for local structures, but also goes far beyond them by capturing the end-to-end pairs between 5\u2019 and 3\u2019 UTRs (\u223c29,800 nt apart) that match perfectly with a purely experimental work. Furthermore, LinearTurboFold identifies novel conserved structures and conserved accessible regions as potential targets for designing efficient and mutation-insensitive small-molecule drugs, antisense oligonucleotides, siRNAs, CRISPR-Cas13 guide RNAs and RT-PCR primers. LinearTurboFold is a general technique that can also be applied to other RNA viruses and full-length genome studies, and will be a useful tool in fighting the current and future pandemics. Conserved RNA structures are critical for designing diagnostic and therapeutic tools for many diseases including COVID-19. However, existing algorithms are much too slow to model the global structures of full-length RNA viral genomes. We present LinearTurboFold, a linear-time algorithm that is orders of magnitude faster, making it the first method to simultaneously fold and align whole genomes of SARS-CoV-2 variants, the longest known RNA virus (\u223c30 kilobases). Our work enables unprecedented global structural analysis and captures long-range interactions that are out of reach for existing algorithms but crucial for RNA functions. LinearTurboFold is a general technique for full-length genome studies and can help fight the current and future pandemics.","version":"1.5","doi":"10.1101/2020.11.23.393488","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.14.468537","pub_date":"2021-11-15","title":"Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2","abstract":"Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible \u201chistone mimetic.\u201d E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.","version":"1.1","doi":"10.1101/2021.11.14.468537","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.12.468428","pub_date":"2021-11-15","title":"#COVIDisAirborne: AI-Enabled Multiscale Computational Microscopy of Delta SARS-CoV-2 in a Respiratory Aerosol","abstract":"We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus ob-scure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized. Abigail Dommer1\u2020, Lorenzo Casalino1\u2020, Fiona Kearns1\u2020, Mia Rosenfeld1, Nicholas Wauer1, Surl-Hee Ahn1, John Russo,2 Sofia Oliveira3, Clare Morris1, AnthonyBogetti4, AndaTrifan5,6, Alexander Brace5,7, TerraSztain1,8, Austin Clyde5,7, Heng Ma5, Chakra Chennubhotla4, Hyungro Lee9, Matteo Turilli9, Syma Khalid10, Teresa Tamayo-Mendoza11, Matthew Welborn11, Anders Christensen11, Daniel G. A. Smith11, Zhuoran Qiao12, Sai Krishna Sirumalla11, Michael O\u2019Connor11, Frederick Manby11, Anima Anandkumar12,13, David Hardy6, James Phillips6, Abraham Stern13, Josh Romero13, David Clark13, Mitchell Dorrell14, Tom Maiden14, Lei Huang15, John McCalpin15, Christo- pherWoods3, Alan Gray13, MattWilliams3, Bryan Barker16, HarindaRajapaksha16, Richard Pitts16, Tom Gibbs13, John Stone6, Daniel Zuckerman2*, Adrian Mulholland3*, Thomas MillerIII11,12*, ShantenuJha9*, Arvind Ramanathan5*, Lillian Chong4*, Rommie Amaro1*. 2021. #COVIDisAirborne: AI-Enabled Multiscale Computational Microscopy ofDeltaSARS-CoV-2 in a Respiratory Aerosol. In Supercomputing \u201821: International Conference for High Perfor-mance Computing, Networking, Storage, and Analysis. ACM, New York, NY, USA, 14 pages. https://doi.org/finalDOI","version":"1.1","doi":"10.1101/2021.11.12.468428","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.05.467458","pub_date":"2021-11-15","title":"Single cell RNA-seq uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19","abstract":"The systemic immune response to viral infection is shaped by master transcription factors such as NF\u03baB or PU.1. Although long non-coding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA-seq approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9 \u2013 key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling characterized PIRAT as a nuclear decoy RNA, diverting the PU.1 transcription factor from alarmin promoters to dead-end pseudogenes in na\u00efve monocytes. NF\u03baB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Our results suggest a major role of nuclear noncoding RNA circuits in systemic antiviral responses to SARS-CoV-2 in humans.","version":"1.2","doi":"10.1101/2021.11.05.467458","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.12.21266273","pub_date":"2021-11-14","title":"Maternal immune response and placental antibody transfer after COVID-19 vaccination across trimester and platforms","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The availability of three COVID-19 vaccines in the United States provides an unprecedented opportunity to examine how vaccine platforms and timing of vaccination in pregnancy impact maternal and neonatal immunity. Here, we characterized the antibody profile after Ad26.COV2.S, mRNA-1273 or BNT162b2 vaccination in 158 pregnant individuals, and evaluated transplacental antibody transfer by profiling maternal and umbilical cord blood in 175 maternal-neonatal dyads. These analyses revealed lower vaccine-induced functions and Fc-receptor binding after Ad26.COV2.S compared to mRNA vaccination, and subtle advantages in titer and function with mRNA-1273 versus BN162b2. mRNA vaccinees had higher titers and functions against SARS-CoV-2 variants of concern. First and third trimester vaccination resulted in enhanced maternal immune responses relative to second trimester. Higher cord:maternal transfer ratios following first and second trimester vaccination reflect placental compensation for waning maternal titers. These results support vaccination early in pregnancy to maximize maternal protection throughout gestation, without compromising neonatal antibody protection.</jats:p>","version":null,"doi":"10.1101/2021.11.12.21266273","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.10.468173","pub_date":"2021-11-12","title":"SARS-CoV-2 Envelope protein (E) binds and activates TLR2: A novel target for COVID-19 interventions","abstract":"In this study, we present a molecular characterization of the interaction between the SARS-CoV-2 envelope protein E with TLR2. We demonstrated that E protein interacts physically with TLR2 receptor in a specific and dose-dependent manner. Furthermore, we showed that this interaction is able to engage TLR2 pathway as demonstrated by its capacity to activate NF-\u03baB transcription factor and to stimulate the production of CXCL8 inflammatory chemokine in a TLR2-dependent manner. Furthermore, in agreement with the importance of NF-\u03baB in TLR signaling pathway, we showed that the chemical inhibition of this transcription factor led to significant inhibition of CXCL8 production, while blockade of P38 and ERK1/2 MAP kinases resulted only in a partial CXCL8 inhibition. Overall, our findings suggest considering the envelope protein E as a novel target for COVID-19 interventions: (i) either by exploring the therapeutic effect of anti-E blocking/neutralizing antibodies in symptomatic COVID-19 patients, or (ii) as a promising non-Spike SARS-CoV-2 antigen candidate to include in the development of next generation prophylactic vaccines against COVID-19 infection and disease. Although, the exact mechanisms of COVID-19 pathogenesis are unknown, recent data demonstrated that elevated levels of pro-inflammatory cytokines in serum is associated with enhanced disease pathogenesis and mortality. Thus, determining the molecular mechanisms responsible for inflammatory cytokine production in the course of SARS-CoV-2 infection could provide future therapeutic targets. In this context, to the best of our knowledge, our report is first to use a detailed molecular characterization to demonstrate that SARS-CoV-2 Envelope E protein binds to TLR2 receptor. Specifically, we showed that SARS-CoV-2 Envelope E protein binds to TLR2 in a direct, specific and dose-dependent manner. Investigating signalling events that control downstream activation of cytokine production show that E protein / TLR2 binding leads to the activation of NF-\u03baB transcription factor that control the expression of multiple pro-inflammatory cytokines including CXCL8. Overall, our findings suggest considering the envelope protein E as a novel target for COVID-19 interventions.","version":"1.1","doi":"10.1101/2021.11.10.468173","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.467990","pub_date":"2021-11-12","title":"Expression and novel alternative purification of the recombinant nucleocapsid (N) protein of SARS-CoV-2 in Escherichia coli for the serodiagnosis of COVID-19","abstract":"The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach uses SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDT\u2019s). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.","version":"1.1","doi":"10.1101/2021.11.10.467990","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.468147","pub_date":"2021-11-12","title":"Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological and pathological disease in a Syrian hamster model of COVID-19","abstract":"There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses, including the currently dominant Delta variant. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.11.10.468147","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.468174","pub_date":"2021-11-12","title":"Serum from COVID-19 patients early in the pandemic shows limited evidence of cross-neutralization against variants of concern","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in a variety of clinical symptoms ranging from no or mild to severe disease. Currently, there are multiple postulated mechanisms that may push a moderate to severe disease into a critical state. Human serum contains abundant evidence of the immune status following infection. Cytokines, chemokines, and antibodies can be assayed to determine the extent to which a patient responded to a pathogen. We examined serum and plasma from a cohort of patients infected with SARS-CoV-2 early in the pandemic and compared them to negative-control sera. Cytokine and chemokine concentrations varied depending on the severity of infection, and antibody responses were significantly increased in severe cases compared to mild to moderate infections. Neutralization data revealed that patients with high titers against an early 2020 isolate had detectable but limited neutralizing antibodies against newly circulating SARS-CoV-2 variants of concern. This study highlights the potential of re-infection for recovered COVID-19 patients.","version":"1.1","doi":"10.1101/2021.11.10.468174","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.468168","pub_date":"2021-11-12","title":"Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex","abstract":"The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.","version":"1.1","doi":"10.1101/2021.11.10.468168","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.467646","pub_date":"2021-11-12","title":"Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens","abstract":"Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence. 1725 game animals from five mammalian orders were surveyed for viruses 71 mammalian viruses were discovered, 18 with a potential risk to humans Civets harbored the highest number of potential \u2018high risk\u2019 viruses A species jump of an alphacoronavirus from bats to a civet was identified H9N2 influenza virus was detected in a civet and an Asian badger Humans viruses were also identified in game animals","version":"1.1","doi":"10.1101/2021.11.10.467646","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.415505","pub_date":"2021-11-11","title":"Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro","abstract":"Neutrophil-induced oxidative stress is a mechanism of lung injury in COVID-19, and drugs with a functional thiol group (\u201cthiol drugs\u201d), especially cysteamine, have anti-oxidant and anti-inflammatory properties that could limit this injury. Thiol drugs may also alter the redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) and thereby disrupt ACE2 binding. Using ACE2 binding assay, reporter virus pseudotyped with SARS-CoV-2 spikes (ancestral and variants) and authentic SARS-CoV-2 (Wuhan-1), we find that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus entry into cells. Pseudoviruses carrying variant spikes were less efficiently inhibited as compared to pseudotypes bearing an ancestral spike, but the most potent drugs still inhibited the Delta variant in the low millimolar range. IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. In hamsters infected with SARS-CoV-2, intraperitoneal (IP) cysteamine decreased neutrophilic inflammation and alveolar hemorrhage in the lungs but did not decrease viral infection, most likely because IP delivery could not achieve millimolar concentrations in the airways. These data show that thiol drugs inhibit SARS-CoV-2 infection in vitro and reduce SARS-CoV-2-related lung injury in vivo and provide strong rationale for trials of systemically delivered thiol drugs as COVID-19 treatments. We propose that antiviral effects of thiol drugs in vivo will require delivery directly to the airways to ensure millimolar drug concentrations and that thiol drugs with lower thiol pKa values are most likely to be effective. The effect of cysteamine to decrease SARS-CoV-2 pneumonia in vivo and of multiple thiol drugs to inhibit SARS-CoV-2 infection in vitro provides rationale for clinical trials of thiol drugs in COVID-19.","version":"1.2","doi":"10.1101/2020.12.08.415505","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.468057","pub_date":"2021-11-11","title":"SARS-CoV-2 ORF8 encoded protein contains a histone mimic, disrupts chromatin regulation, and enhances replication","abstract":"SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for a cell\u2019s ability to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.","version":"1.1","doi":"10.1101/2021.11.10.468057","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450483","pub_date":"2021-11-11","title":"Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice","abstract":"Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients. Antivirals targeting SARS-CoV-2 are sorely needed. In this study, we screened a library of drug compounds and identified clofoctol as an antiviral against SARS-CoV-2. We further demonstrated that, in vivo, this compound reduces inflammatory gene expression and lowers pulmonary pathology.","version":"1.2","doi":"10.1101/2021.06.30.450483","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438036","pub_date":"2021-11-11","title":"Fine-tuning the Spike: Role of the nature and topology of the glycan shield in the structure and dynamics of the SARS-CoV-2 S","abstract":"The dense glycan shield is an essential feature of the SARS-CoV-2 spike (S) architecture, key to immune evasion and to the activation of the prefusion conformation. Recent studies indicate that the occupancy and structures of the SARS-CoV-2 S glycans depend not only on the nature of the host cell, but also on the structural stability of the trimer; a point that raises important questions about the relative competence of different glycoforms. Moreover, the functional role of the glycan shield in the SARS-CoV-2 pathogenesis suggests that the evolution of the sites of glycosylation is potentially intertwined with the evolution of the protein sequence to affect optimal activity. Our results from multi-microsecond molecular dynamics simulations indicate that the type of glycosylation at N234, N165 and N343 greatly affects the stability of the receptor binding domain (RBD) open conformation, and thus its exposure and accessibility. Furthermore, our results suggest that the loss of glycosylation at N370, a newly acquired modification in the SARS-CoV-2 S glycan shield\u2019s topology, may have contributed to increase the SARS-CoV-2 infectivity as we find that N-glycosylation at N370 stabilizes the closed RBD conformation by binding a specific cleft on the RBD surface. We discuss how the absence of the N370 glycan in the SARS-CoV-2 S frees the RBD glycan binding cleft, which becomes available to bind cell-surface glycans, potentially increases host cell surface localization.","version":"1.2","doi":"10.1101/2021.04.01.438036","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.21266195","pub_date":"2021-11-11","title":"Disparities in COVID-19 Fatalities among Working Californians","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Information on the occupational distribution of COVID-19 mortality is limited.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To characterize COVID-19 fatalities among working Californians.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Retrospective study of laboratory-confirmed COVID-19 fatalities with dates of death from January 1 to December 31, 2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>California.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>COVID-19 accounted for 8,050 (9.9%) of 81,468 fatalities among Californians 18-64 years old. Of these decedents, 2,486 (30.9%) were matched to state employment records and classified as \u201cconfirmed working.\u201d The remainder were classified as \u201clikely working\u201d (n=4,121 [51.2%]) or \u201cnot working\u201d (n=1,443 [17.9%]) using death certificate and case registry data.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Measurements</jats:title>\n                  <jats:p>We calculated age-adjusted overall and occupation-specific COVID-19 mortality rates using 2019 American Community Survey denominators.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Confirmed and likely working COVID-19 decedents were predominantly male (76.3%), Latino (68.7%), and foreign-born (59.6%), with high school or less education (67.9%); 7.8% were Black. The overall age-adjusted COVID-19 mortality rate was 30.0 per 100,000 workers (95% confidence interval [CI], 29.3-30.8). Workers in nine occupational groups had mortality rates higher than this overall rate, including those in farming (78.0; 95% CI, 68.7-88.2); material moving (77.8; 95% CI, 70.2-85.9); construction (62.4; 95% CI, 57.7-67.4); production (60.2; 95% CI, 55.7-65.0); and transportation (57.2; 95% CI, 52.2-62.5) occupations. While occupational differences in mortality were evident across demographic groups, mortality rates were three-fold higher for male compared with female workers and three- to seven-fold higher for Latino and Black workers compared with Asian and White workers.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Limitations</jats:title>\n                  <jats:p>The requirement that fatalities be laboratory-confirmed and the use of 2019 denominator data may underestimate the occupational burden of COVID-19 mortality.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Californians in manual labor and in-person service occupations experienced disproportionate COVID-19 mortality, with the highest rates observed among male, Latino, and Black workers.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.11.10.21266195","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.09.467862","pub_date":"2021-11-10","title":"Mice infected with Mycobacterium tuberculosis are resistant to secondary infection with SARS-CoV-2","abstract":"Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to secondary CoV2 infection and its pathological consequences, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival. Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are distinct organisms which both cause lung disease. We report the surprising observation that Mtb-infected mice are resistant to secondary infection with CoV2, with no impact on Mtb burden and resistance associating with lung T and B cell expansion.","version":"1.1","doi":"10.1101/2021.11.09.467862","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.09.467693","pub_date":"2021-11-10","title":"The P681H mutation in the Spike glycoprotein confers Type I interferon resistance in the SARS-CoV-2 alpha (B.1.1.7) variant","abstract":"Variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2) threaten the global response to the COVID-19 pandemic. The alpha (B.1.1.7) variant appeared in the UK became dominant in Europe and North America in early 2021. The Spike glycoprotein of alpha has acquired a number mutations including the P681H mutation in the polybasic cleavage site that has been suggested to enhance Spike cleavage. Here, we show that the alpha Spike protein confers a level of resistance to the effects of interferon-\u03b2 (IFN\u03b2) in lung epithelial cells. This correlates with resistance to restriction mediated by interferon-induced transmembrane protein-2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is necessary for comparative resistance to IFN\u03b2 in a molecularly cloned SARS-CoV-2 encoding alpha Spike. Overall, we suggest that in addition to adaptive immune escape, mutations associated with VOCs also confer replication advantage through adaptation to resist innate immunity.","version":"1.1","doi":"10.1101/2021.11.09.467693","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.468037","pub_date":"2021-11-10","title":"Potent antibody immunity to SARS-CoV-2 variants elicited by a third dose of inactivated vaccine","abstract":"SARS-CoV-2 variants are still prevalent worldwide and continue to pose a challenge to the effectiveness of current vaccines. It remains unknown whether a third dose of inactivated vaccine elicits immune potential against SARS-CoV-2 variants. Here, we showed a significant decline in plasma neutralization against SARS-CoV-2 at seven months after a second dose of the inactivated vaccine in a large-scale cohort. However, we also found that a third vaccination with an inactivated vaccine largely increased plasma neutralization against variants including Beta, Delta, and Lambda. More importantly, the high-affinity anti-RBD memory B cells were also generated by the third vaccination, suggesting a more potent and longer protection. These findings highlighted the importance and effectiveness of a third dose of inactivated vaccine in conferring higher protection against the emerging variants in populations.","version":"1.1","doi":"10.1101/2021.11.10.468037","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.463911","pub_date":"2021-11-10","title":"Beyond Spike: Identification of nine highly prevalent SARS-CoV-2-specific CD8 T-cell epitopes in a large Norwegian cohort","abstract":"T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. As the protective role of pre-existing cross-reactivity to homologous peptides is unclear, we aimed to identify SARS-CoV-2-specific minimal epitopes recognized by CD8 T-cells among 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identified nine conserved SARS-CoV-2-specific epitopes restricted by four of the most prevalent HLA class I alleles in the Norwegian study cohort, to which responding CD8 T cells were detected in 70-100% of convalescents expressing the relevant HLA allele. Only two of these were derived from the Spike protein, included in current vaccines. We found a strong correlation between immunoprevalence and immunodominance. Thus, the CD8 T-cell response to SARS-CoV-2 is more focused than previously believed. Using a new algorithm, we predict that a vaccine including these epitopes could induce a T-cell response in 83% of Caucasians.","version":"1.2","doi":"10.1101/2021.10.13.463911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.019075","pub_date":"2021-11-10","title":"Iterative community-driven development of a SARS-CoV-2 tissue simulator","abstract":"The 2019 novel coronavirus, SARS-CoV-2, is a pathogen of critical significance to international public health. Knowledge of the interplay between molecular-scale virus-receptor interactions, single-cell viral replication, intracellular-scale viral transport, and emergent tissue-scale viral propagation is limited. Moreover, little is known about immune system-virus-tissue interactions and how these can result in low-level (asymptomatic) infections in some cases and acute respiratory distress syndrome (ARDS) in others, particularly with respect to presentation in different age groups or pre-existing inflammatory risk factors. Given the nonlinear interactions within and among each of these processes, multiscale simulation models can shed light on the emergent dynamics that lead to divergent outcomes, identify actionable \u201cchoke points\u201d for pharmacologic interventions, screen potential therapies, and identify potential biomarkers that differentiate patient outcomes. Given the complexity of the problem and the acute need for an actionable model to guide therapy discovery and optimization, we introduce and iteratively refine a prototype of a multiscale model of SARS-CoV-2 dynamics in lung tissue. The first prototype model was built and shared internationally as open source code and an online interactive model in under 12 hours, and community domain expertise is driving regular refinements. In a sustained community effort, this consortium is integrating data and expertise across virology, immunology, mathematical biology, quantitative systems physiology, cloud and high performance computing, and other domains to accelerate our response to this critical threat to international health. More broadly, this effort is creating a reusable, modular framework for studying viral replication and immune response in tissues, which can also potentially be adapted to related problems in immunology and immunotherapy.","version":"1.5","doi":"10.1101/2020.04.02.019075","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453874","pub_date":"2021-11-10","title":"Elucidation of SARS-Cov-2 Budding Mechanisms Through Molecular Dynamics Simulations of M and E Protein Complexes","abstract":"SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory\u2019s refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.","version":"1.6","doi":"10.1101/2021.07.26.453874","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.10.468025","pub_date":"2021-11-10","title":"Design of D-amino acids SARS-CoV-2 Main protease inhibitors using the cationic peptide from rattlesnake venom as a scaffold","abstract":"The C30 Endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and is hence a promising drug target. Molecules isolated from animals, insects, plants or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low \u00b5M range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.","version":"1.1","doi":"10.1101/2021.11.10.468025","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.08.467773","pub_date":"2021-11-09","title":"A self-amplifying mRNA COVID-19 vaccine drives potent and broad immune responses at low doses that protects non-human primates against SARS-CoV-2","abstract":"The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate potent cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 \u03bcg induced potent neutralizing antibody titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 \u03bcg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed at all dose levels. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. Protection was most effective with a SAM prime-boost vaccination regimen at 10 and 30 \u03bcg and with a ChAd/SAM heterologous prime-boost regimen. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime.","version":"1.1","doi":"10.1101/2021.11.08.467773","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.08.467715","pub_date":"2021-11-09","title":"SARS-CoV-2 triggered excessive inflammation and abnormal energy metabolism in gut microbiota","abstract":"Specific roles of gut microbes in COVID-19 progression are critical. However, the circumstantial mechanism remains elusive. In this study, shotgun metagenomic or metatranscriptomic sequencing were performed on fecal samples collected from 13 COVID-19 patients and controls. We analyzed the structure of gut microbiota, identified the characteristic bacteria and selected biomarkers. Further, GO, KEGG and eggNOG annotation were employed to correlate the taxon alteration and corresponding functions. The gut microbiota of COVID-19 patients was characterized by the enrichment of opportunistic pathogens and depletion of commensals. The abundance of Bacteroides spp. displayed an inverse relationship to COVID-19 severity, whereas Actinomyces oris, Escherichia coli, and Gemmiger formicilis were positively correlated with disease severity. The genes encoding oxidoreductase were significantly enriched in SARS-CoV-2 infection. KEGG annotation indicated that the expression of ABC transporter was up regulated, while the synthesis pathway of butyrate was aberrantly reduced. Furthermore, increased metabolism of lipopolysaccharide, polyketide sugar, sphingolipids and neutral amino acids was found. These results suggested the gut microbiome of COVID-19 patients was correlated with disease severity and in a state of excessive inflammatory response. Healthy gut microbiota may enhance antiviral defenses via butyrate metabolism, whereas the accumulation of opportunistic and inflammatory bacteria may exacerbate the disease progression.","version":"1.1","doi":"10.1101/2021.11.08.467715","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.464952","pub_date":"2021-11-09","title":"Major complex trait for early de novo programming \u2018CoV-MAC-TED\u2019 detected in human nasal epithelial cells infected by two SARS-CoV-2 variants is promising for designing therapeutic strategies","abstract":"Early metabolic reorganization was only recently recognized as essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels that connected to increased aerobic fermentation, which linked to alpha-tubulin-based cell restructuration and control of cell cycle progression, was identified as major complex trait for early de novo programming (\u2018CoV-MAC-TED\u2019) during SARS-CoV-2 infection. This trait was highlighted as critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named \u2018ReprogVirus\u2019, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the \u2018ReprogVirus Platform\u2019. This platform is currently under development. However, so far an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing data collection of early SARS-CoV-2 infection responses was not defined. Here, we demonstrate transcriptome level profiles of the most critical \u2018ReprogVirus\u2019 gene sets for identifying \u2018CoV-MAC-TED\u2019 in cultured human nasal epithelial cells. Our results (a) validate \u2018Cov-MAC-TED\u2019 as crucial trait for early SARS-CoV-2 reprogramming for both tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells. In vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the \u2018ReprogVirus Platform\u2019. Thus, this cell system is highly promising to advance integrative data analyses by help of Artificial Intelligence methodologies for designing anti-SARS-CoV-2 strategies.","version":"1.2","doi":"10.1101/2021.10.19.464952","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.29.466470","pub_date":"2021-11-09","title":"Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule F\u00f6rster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.","version":"1.2","doi":"10.1101/2021.10.29.466470","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.08.467648","pub_date":"2021-11-09","title":"Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease","abstract":"The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the U.S. within one year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a \u201cdesigner nanoparticle\u201d platform using phage-like particles (PLPs) derived from bacteriophage lambda for multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs, RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines.","version":"1.1","doi":"10.1101/2021.11.08.467648","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.08.21265884","pub_date":"2021-11-09","title":"Favipiravir In Adults with Moderate to Severe COVID-19: A Phase 3 Multicentre, Randomized, Double-Blinded, Placebo-Controlled Trial","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Aim</jats:title>\n                  <jats:p>To assess the efficacy and safety of favipiravir in adults with moderate to severe coronavirus disease 2019 (COVID-19).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>In this randomized, double-blind, multicenter, phase 3 trial, adults (21-80 years) with real-time reverse transcriptase polymerase chain reaction (rRT-PCR) confirmed SARS-CoV-2 infection and presenting with moderate to severe COVID-19 and requiring hospitalization were randomized 1:1 to oral favipiravir (day 1: 1800 mg BID and days 2-10: 800 mg BID) (FPV) plus standard supportive care (SoC) versus placebo plus SoC (placebo). The primary endpoint was time to resolution of hypoxia.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    In total, 353 patients were randomized to receive either FPV or placebo (175 and 178 in the FPV and placebo groups, respectively). Overall, 76% of the patients (240/315, 78% in FPV vs. 75% in placebo group) reached resolution of hypoxia on or before day 28. The median time to resolution of hypoxia was 7 days in the FPV group and 8 days in the placebo group. Treatment effect was not significant [Hazard ratio (HR) (95% CI): 0.991 (0.767, 1.280) (\n                    <jats:italic>p</jats:italic>\n                    =0.94)].\n                  </jats:p>\n                  <jats:p>\n                    Patients in the lower NEWS-2 clinical risk subgroup were more likely to achieve shorter time to resolution of hypoxia with the median time to resolution of hypoxia of 6 days in FPV and 7 days in placebo group [HR (95% CI): 1.21 (0.847, 1.731) (\n                    <jats:italic>p</jats:italic>\n                    =0.29)]; shorter time to hospital discharge with a median time to discharge of 8 and 10 days in the FPV and placebo group, respectively [HR (95% CI): 1.47 (1.081, 1.997) (\n                    <jats:italic>p=</jats:italic>\n                    0.014)]; and shorter time to improvement by 1-point improvement over baseline in WHO 10-point clinical status score with the median time to improvement by 1-point from baseline of 6 and 7 days in the FPV and placebo group, respectively [HR (95% CI): 1.16 (0.830, 1.624) (\n                    <jats:italic>p=</jats:italic>\n                    0.38)] than higher NEWS-2 clinical risk subgroup.\n                  </jats:p>\n                  <jats:p>Treatment emergent adverse event (TEAEs) were experienced by 62/334 (19%) patients [35/168 (21%) patients in FPV and 27/166 (16%) in placebo group]. Hyperuricaemia/increased blood uric acid was reported in 9 (3%)/2 (1%) patients [8 (5%)/1(1%) patients in FPV and 1 (1%)/1(1%) in placebo group], which were of mild intensity and transient. Overall, 36 serious adverse events (SAEs) were reported, 20 in FPV and 16 in placebo group.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>The trial did not find favipiravir to be effective in moderate to severe, hospitalized COVID-19 patients; favourable clinical trends were observed in patients with lower NEWS-2 risk when early administration of favipiravir could be achieved.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.11.08.21265884","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.18.464828","pub_date":"2021-11-09","title":"Transcriptome data from human nasal epithelial cells infected by H3N2 influenza virus indicate early unbalanced ROS/RNA levels, temporarily increased aerobic fermentation linked to enhanced \u03b1-tubulin and rapid energy-dependent IRF9-marked immunization","abstract":"Transcriptome studies of a selected gene set (ReprogVirus) had identified unbalanced ROS/RNS levels, which connected to increased aerobic fermentation that linked to alpha-tubulin-based cell restructuration and cell cycle control, as a major complex trait for early de novo programming (CoV-MAC-TED) upon SARS-CoV-2 infection. Recently, CoV-MAC-TED was confirmed as promising marker by using primary target human nasal epithelial cells (NECs) infected by two SARS-CoV-2 variants with different effects on disease severity. To further explore this marker/cell system as a standardized tool for identifying anti-viral targets in general, testing of further virus types is required. Results: Transcriptome level profiles of H3N2 influenza-infected NECs indicated ROS/RNS level changes and increased transcript accumulation of genes related to glycolysis, lactic fermentation and \u03b1-tubulin at 8 hours post infection. These early changes linked to energy-dependent, IRF9-marked rapid immunization. However, ReprogVirus-marker genes indicated the absence of initial cell cycle progress, which contrasted our findings during infections with two SARS-CoV-2 variants, where cell cycle progress was linked to delayed IRF9 response. Our results point to the possibility of CoV-MAC-TED-assisted, rapid individual host cell response identification upon virus infections. Conclusion: The complex trait CoV-MAC-TED can identify similar and differential early responses of SARS-CoV-2 and influenza H3N2 viruses. This indicates its appropriateness to search for anti-viral targets in view of therapeutic design strategies. For standardization, human NECs can be used. This marker/cell system is promising to identify differential early cell responses upon viral infections also depending on cell origins.","version":"1.2","doi":"10.1101/2021.10.18.464828","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467291","pub_date":"2021-11-08","title":"Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen","abstract":"The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without S, M, E genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. This achievement represents a ground-breaking discovery that will greatly accelerate the pace of developing treatments for COVID-19.","version":"1.1","doi":"10.1101/2021.11.04.467291","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.05.467537","pub_date":"2021-11-08","title":"Transcriptome analysis of SARS-CoV-2 na\u00efve and recovered individuals vaccinated with inactivated vaccine","abstract":"The urgent approval of the use of the inactivated COVID-19 vaccine is essential to reduce the threat and burden of the epidemic on global public health, however, our current understanding of the host immune response to inactivated vaccine remains limited. Herein, we performed serum IgG antibody detection and transcriptomics analysis on 20 SARS-CoV-2 na\u00efve individuals who received multiple doses of inactivated vaccine and 5 SARS-CoV-2 recovered individuals who received single dose of inactivated vaccine. Our research revealed the important role of many innate immune pathways after vaccination, identified a significant correlation with the third dose of booster vaccine and proteasome-related genes, and found that SARS-CoV-2 recovered individuals can produces a strong immune response to a single dose of inactivated vaccine. These results help us understand the reaction mechanism of the host\u2019s molecular immune system to the inactivated vaccine, and provide a basis for the choice of vaccination strategy.","version":"1.1","doi":"10.1101/2021.11.05.467537","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.05.467529","pub_date":"2021-11-08","title":"Structural basis of main proteases of coronavirus bound to drug candidate PF-07321332","abstract":"The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genus of coronaviruses is the substrate binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332 developed by Pfizer is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here we report three crystal structures of main protease of SARS-CoV-2, SARS-CoV and MERS-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of main protease harbors multiple inhibitor binding sites, where PF-07321332 occupies subsites S1, S2 and S4 and appears more restricted compared with other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of main proteases from different coronaviruses. Given the importance of main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals.","version":"1.1","doi":"10.1101/2021.11.05.467529","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.05.467523","pub_date":"2021-11-08","title":"SARS-COV-2 Delta variant displays moderate resistance to neutralizing antibodies and spike protein properties of higher soluble ACE2 sensitivity, enhanced cleavage and fusogenic activity","abstract":"The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. The spike proteins of B.1.617.1, B.1.617.2, and AY.1 variants have several substitutions in the receptor binding domain (RBD), including L452R+E484Q, L452R+T478K, and K417N+L452R+T478K, respectively, that could potentially reduce effectiveness of therapeutic antibodies and current vaccines. Here we compared B.1.617 variants, and their single and double RBD substitutions for resistance to neutralization by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. Pseudoviruses with the B.1.617.1, B.1.617.2, and AY.1 spike showed a modest 1.5 to 4.4-fold reduction in neutralization titer by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for pseudoviruses with C.37, P.1, R.1, and B.1.526 spikes, but seven- and sixteen-fold reduction for vaccine-elicited and convalescent sera, respectively, was seen for pseudoviruses with the B.1.351 spike. Four of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses due to the L452R substitution, whereas six of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.1 pseudoviruses due to either the E484Q or L452R substitution. Against AY.1 pseudoviruses, the L452R and K417N substitutions accounted for the loss of neutralization by four antibodies and one antibody, respectively, whereas one antibody lost potency that could not be fully accounted for by a single RBD substitution. The modest resistance of B.1.617 variants to vaccine-elicited sera suggest that current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants, but the therapeutic antibodies need to be carefully selected based on their resistance profiles. Finally, the spike proteins of B.1.617 variants are more efficiently cleaved due to the P681R substitution, and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.","version":"1.1","doi":"10.1101/2021.11.05.467523","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.25.220806","pub_date":"2021-11-07","title":"VIP plasma levels associate with survival in severe COVID-19 patients, correlating with protective effects in SARS-CoV-2-infected cells","abstract":"Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, VIP and PACAP, highly similar neuropeptides, decreased the SARS-CoV-2 genome replication in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.","version":"1.6","doi":"10.1101/2020.07.25.220806","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.437411","pub_date":"2021-11-07","title":"The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro)","abstract":"The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics. X-ray crystallography at variable temperature for SARS-CoV-2 Mpro reveals a complex conformational landscape, including mobile solvent at the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and an intramolecular network bridging the active site and dimer interface.","version":"1.2","doi":"10.1101/2021.05.03.437411","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440731","pub_date":"2021-11-07","title":"Evaluation of SARS-CoV-2 Entry, Inflammation and New Therapeutics in Human Lung Tissue Cells","abstract":"The development of physiological models that reproduce SARS-CoV-2 infection in primary human cells will be instrumental to identify host-pathogen interactions and potential therapeutics. Here, using cell suspensions directly from primary human lung tissues (HLT), we have developed a rapid platform for the identification of viral targets and the expression of viral entry factors, as well as for the screening of viral entry inhibitors and anti-inflammatory compounds. The direct use of HLT cells, without long-term cell culture and in vitro differentiation approaches, preserves main immune and structural cell populations, including the most susceptible cell targets for SARS-CoV-2; alveolar type II (AT-II) cells, while maintaining the expression of proteins involved in viral infection, such as ACE2, TMPRSS2, CD147 and AXL. Further, antiviral testing of 39 drug candidates reveals a highly reproducible method, suitable for different SARS-CoV-2 variants, and provides the identification of new compounds missed by conventional systems, such as VeroE6. Using this method, we also show that interferons do not modulate ACE2 expression, and that stimulation of local inflammatory responses can be modulated by different compounds with antiviral activity. Overall, we present a relevant and rapid method for the study of SARS-CoV-2. Ex vivo physiological systems for the study of SARS-CoV-2-host interactions are scarce. Here, we establish a method using primary human lung tissue (HLT) cells for the rapid analysis of cell tropism and identification of therapeutics. HLT cells preserve main cell subpopulations, including alveolar type-2 cells, and expression of SARS-CoV-2 entry factors ACE2, CD147, TMPRSS2 and AXL. HLT cells are readily susceptible to SARS-CoV-2 infection without the need of cell isolation or further cell differentiation. Antiviral testing in HLT cells allows the rapid identification of new drug candidates against SARS-CoV-2 variants, missed by conventional systems. Local inflammation is supported in HLT cells and offers the identification of relevant anti-inflammatory compounds for SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2021.04.21.440731","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423106","pub_date":"2021-11-07","title":"SARS-CoV-2 spike-glycoprotein processing at S1/S2 and S2\u2019and shedding of the ACE2 viral receptor: roles of Furin and TMPRSS2 and implications for viral infectivity and cell-to-cell fusion","abstract":"The sp\u00eeke (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic \u201cpriming\u201d at PRRAR685\u2193 into S1 and S2 (cleavage at S1/S2), and \u201cfusion-activation\u201d at a S2\u2019 site for viral entry. In vitro, Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2\u2019, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2\u2019 processing by Furin at KPSKR815\u2193 was strongly enhanced by ACE2, but not for the optimized S2\u2019 KRRKR815\u2193 mutant (\u03bcS2\u2019), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a \u201cpH-dependent\u201d endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a \u201cpH-independent\u201d viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin (BOS) and TMPRSS2 (Camostat). Consistently, these inhibitors potently reduce infectious viral titer and cytopathic effects, an outcome enhanced when both compounds were combined. Quantitative analyses of cell-to-cell fusion and sp\u00eeke processing revealed the key importance of the Furin sites for syncytia formation. Our assays showed that TMPRSS2 enhances fusion and proteolysis at S2\u2019 in the absence of cleavage at S1/S2, an effect that is linked to ACE2 shedding by TMPRSS2. Overall, our results indicate that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and in promoting viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. SARS-CoV-2 is the etiological agent of COVID-19 that resulted in >5 million deaths. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein is cleaved at two sites: S1/S2 and S2\u2019. Cleavage at S1/S2, induces a conformational change favoring the recognition of ACE2. The S2\u2019 cleavage is critical for cell-to-cell fusion and virus entry into host cells. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a non-toxic Furin inhibitor with a TMPRSS2 inhibitor could significantly reduce viral entry in lung cells, as evidenced by an average synergistic \u223c95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.","version":"1.3","doi":"10.1101/2020.12.18.423106","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444569","pub_date":"2021-11-07","title":"Spatially distributed infection increases viral load in a computational model of SARS-CoV-2 lung infection","abstract":"A key question in SARS-CoV-2 infection is why viral loads and patient outcomes vary dramatically across individuals. Because spatial-temporal dynamics of viral spread and immune response are challenging to study in vivo, we developed Spatial Immune Model of Coronavirus (SIMCoV), a scalable computational model that simulates hundreds of millions of lung cells, including respiratory epithelial cells and T cells. SIMCoV replicates viral growth dynamics observed in patients and shows how spatially dispersed infections can lead to increased viral loads. The model also shows how the timing and strength of the T cell response can affect viral persistence, oscillations, and control. By incorporating spatial interactions, SIMCoV provides a parsimonious explanation for the dramatically different viral load trajectories among patients by varying only the number of initial sites of infection, and the magnitude and timing of the T cell immune response. When the branching airway structure of the lung is explicitly represented, we find that virus spreads faster than in a 2D layer of epithelial cells, but much more slowly than in an undifferentiated 3D grid or in a well-mixed ODE model. These results illustrate how realistic spatially explicit computational models can improve understanding of within-host dynamics of SARS-CoV-2 infection. A key question in SARS-CoV-2 infection is why viral loads and patient outcomes are so different across individuals. Because it\u2019s difficult to see how the virus spreads in the lungs of infected people, we developed Spatial Immune Model of Coronavirus (SIMCoV), a computational model that simulates hundreds of millions of cells, including lung cells and immune cells. SIMCoV simulates how virus grows and then declines, and the simulations match data observed in patients. SIMCoV shows that when there are more initial infection sites, the virus grows to a higher peak. The model also shows how the timing of the immune response, particularly the T cell response, can affect how long the virus persists and whether it is ultimately cleared from the lungs. SIMCoV shows that the different viral loads in different patients can be explained by how many different places the virus is initially seeded inside their lungs. We explicitly add the branching airway structure of the lung into the model and show that virus spreads slightly faster than it would in a two-dimensional layer of lung cells, but much slower than traditional mathematical models based on differential equations. These results illustrate how realistic spatial computational models can improve understanding of how SARS-CoV-2 infection spreads in the lung.","version":"1.3","doi":"10.1101/2021.05.19.444569","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.31.466677","pub_date":"2021-11-06","title":"Multiple spillovers and onward transmission of SARS-CoV-2 in free-living and captive white-tailed deer","abstract":"Many animal species are susceptible to SARS-CoV-2 and could potentially act as reservoirs, yet transmission of the virus in non-human free-living animals has not been documented. White-tailed deer (Odocoileus virginianus), the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 may be circulating in deer, we tested 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through December of 2020 for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 deer (33.2%; 95% CI: 28, 38.9) samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, between November 23, 2020 and January 10, 2021, 80 of 97 (82.5%; 95% CI 73.7, 88.8) RPLN samples had detectable SARS-CoV-2 RNA by RT-PCR. Whole genome sequencing of the 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%), and B.1.311 (n = 19; 20%) accounting for ~75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple zooanthroponotic spillover events and deer-to-deer transmission. The discovery of sylvatic and enzootic SARS-CoV-2 transmission in deer has important implications for the ecology and long-term persistence, as well as the potential for spillover to other animals and spillback into humans. These findings highlight an urgent need for a robust and proactive \u201cOne Health\u201d approach to obtaining a better understanding of the ecology and evolution of SARS-CoV-2. SARS-CoV-2 was detected in one-third of sampled white-tailed deer in Iowa between September 2020 and January of 2021 that likely resulted from multiple human-to-deer spillover and deer-to-deer transmission events.","version":"1.2","doi":"10.1101/2021.10.31.466677","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.29.466401","pub_date":"2021-11-06","title":"SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade","abstract":"Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognised by Toll-like receptor 4 (TLR4) in innate immunity. Here, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the SARS-CoV-2 spike (S) protein. LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting its possible role as an intermediate in the TLR4 cascade. Congruently, nuclear factor-kappa B (NF-\u03baB) activation in vitro is strongly boosted by S2. In vivo, however, a boosting effect is observed for both S1 and S2, with the former potentially facilitated by proteolysis. Collectively, our study suggests the S protein may act as a delivery system for LPS in host innate immune pathways. The LPS binding pockets are highly conserved across different SARS-CoV-2 variants and therefore represent potential therapeutic targets.","version":"1.3","doi":"10.1101/2021.10.29.466401","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.03.467182","pub_date":"2021-11-05","title":"A trimeric NTD and RBD SARS-CoV-2 subunit vaccine induced protective immunity in CAG-hACE2 transgenic mice and rhesus macaques","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to significant public health, economic and social problems. Development of effective vaccines is still a priority to contain the virus and end the global pandemic. In this study, we reported that ReCOV, a recombinant trimeric NTD and RBD two-component SARS-CoV-2 subunit vaccine adjuvanted with BFA03 (an AS03-like squalene adjuvant), induced high levels of neutralizing antibodies against SARS-CoV-2 and the circulating variants in mice, rabbits and rhesus macaques. Notably, two-dose immunizations of ReCOV provided complete protection against challenge with SARS-CoV-2 in hACE2 transgenic mice and rhesus macaques, without observable antibody-dependent enhancement of infection. These results support further clinical development of ReCOV and the vaccine is currently being evaluated in a phase I clinical trial in New Zealand (NCT04818801).","version":"1.1","doi":"10.1101/2021.11.03.467182","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467308","pub_date":"2021-11-05","title":"SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus)","abstract":"Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer (Odocoileus virginianus) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. A significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.","version":"1.1","doi":"10.1101/2021.11.04.467308","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467274","pub_date":"2021-11-05","title":"Prolonged and extended impacts of SARS-CoV-2 on the olfactory neurocircuit","abstract":"The impact of SARS-CoV-2 on the olfactory pathway was studied over several time points using Syrian golden hamsters. We found an incomplete recovery of the olfactory sensory neurons, prolonged activation of glial cells in the olfactory bulb, and a decrease in the density of dendritic spines within the hippocampus. These data may be useful for elucidating the mechanism underlying long-lasting olfactory dysfunction and cognitive impairment as a post-acute COVID-19 syndrome.","version":"1.1","doi":"10.1101/2021.11.04.467274","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.21265931","pub_date":"2021-11-05","title":"Evaluation of a Machine Learning Approach Utilizing Wearable Data for Prediction of SARS-CoV-2 Infection in Healthcare Workers","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Importance</jats:title>\n                  <jats:p>Passive and non-invasive identification of SARS-CoV-2 infection remains a challenge. Widespread use of wearable devices represents an opportunity to leverage physiological metrics and fill this knowledge gap.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To determine whether a machine learning model can detect SARS-CoV-2 infection from physiological metrics collected from wearable devices.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>A multicenter observational study enrolling health care workers with remote follow-up.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>Seven hospitals from the Mount Sinai Health System in New York City</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>\n                    Eligibility criteria included health care workers who were \u226518 years, employees of one of the participating hospitals, with at least an iPhone series 6, and willing to wear an Apple Watch Series 4 or higher. We excluded participants with underlying autoimmune/inflammatory diseases, and medications known to interfere with autonomic function. We enrolled participants between April 29\n                    <jats:sup>th</jats:sup>\n                    , 2020, and March 2\n                    <jats:sup>nd</jats:sup>\n                    , 2021, and followed them for a median of 73 days (range, 3-253 days). Participants provided patient-reported outcome measures through a custom smartphone application and wore an Apple Watch, collecting heart rate variability and heart rate data, throughout the follow-up period.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Exposure</jats:title>\n                  <jats:p>Participants were exposed to SARS-CoV-2 infection over time due to ongoing community spread.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Main Outcome and Measure</jats:title>\n                  <jats:p>The primary outcome was SARS-CoV-2 infection, defined as \u00b17 days from a self-reported positive SARS-CoV-2 nasal PCR test.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We enrolled 407 participants with 49 (12%) having a positive SARS-CoV-2 test during follow-up. We examined five machine-learning approaches and found that gradient-boosting machines (GBM) had the most favorable 10-CV performance. Across all testing sets, our GBM model predicted SARS-CoV-2 infection with an average area under the receiver operating characteristic (auROC)=85% (Confidence Interval 83-88%). The model was calibrated to improve sensitivity over specificity, achieving an average sensitivity of 76% (CI \u00b1\u223c4%) and specificity of 84% (CI \u00b1\u223c0.4%). The most important predictors included parameters describing the circadian HRV mean (MESOR) and peak-timing (acrophase), and age.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions and Relevance</jats:title>\n                  <jats:p>We show that a tree-based ML algorithm applied to physiological metrics passively collected from a wearable device can identify and predict SARS-CoV2 infection. Utilizing physiological metrics from wearable devices may improve screening methods and infection tracking.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.11.04.21265931","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.05.21265853","pub_date":"2021-11-05","title":"A SARS-CoV-2 variant of concern triggers Fc effector function with increased cross-reactivity","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>SARS-CoV-2 variants of concern (VOCs) exhibit escape from neutralizing antibodies, causing concern about vaccine effectiveness. However, while non-neutralizing cytotoxic functions of antibodies are associated with decreased disease severity and vaccine protection, Fc effector function escape from VOCs is poorly defined. Furthermore, whether VOCs trigger Fc functions with altered specificity, as has been reported for neutralization, is unknown. Here, we demonstrate that the Beta VOC partially evades Fc effector activity in individuals infected with the original (D614G) variant. However, not all functions are equivalently affected, suggesting differential targeting by antibodies mediating distinct Fc functions. Furthermore, Beta infection triggered responses with significantly improved Fc cross-reactivity against global VOCs compared to either D614G infected or Ad26.COV2.S vaccinated individuals. This suggests that, as for neutralization, the infecting spike sequence impacts Fc effector function. These data have important implications for vaccine strategies that incorporate VOCs, suggesting these may induce broader Fc effector responses.</jats:p>","version":null,"doi":"10.1101/2021.11.05.21265853","journal":"medRxiv","score":null},{"id":"10.1101/2021.11.04.467342","pub_date":"2021-11-05","title":"Validation and invalidation of SARS-CoV-2 papain-like protease inhibitors","abstract":"SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. The PLpro is involved in the cleavage of viral polyproteins as well as immune modulation through removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screening. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and the cell based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of the PLpro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular inhibition of PLpro as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for specific and potent SARS-CoV-2 PLpro inhibitors.","version":"1.1","doi":"10.1101/2021.11.04.467342","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467077","pub_date":"2021-11-05","title":"The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern","abstract":"There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.","version":"1.1","doi":"10.1101/2021.11.04.467077","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467344","pub_date":"2021-11-05","title":"Brilacidin, a COVID-19 Drug Candidate, demonstrates broad-spectrum antiviral activity against human coronaviruses OC43, 229E and NL63 through targeting both the virus and the host cell","abstract":"Brilacidin, a mimetic of host defense peptides (HDPs), is currently in phase 2 clinical trial as an antibiotic drug candidate. A recent study reported that brilacidin has antiviral activity against SARS-CoV-2 by inactivating the virus. In this work, we discovered an additional mechanism of action of brilacidin by targeting heparan sulfate proteoglycans (HSPGs) on host cell surface. Brilacidin, but not acetyl brilacidin, inhibits the entry of SARS-CoV-2 pseudovirus into multiple cell lines, and heparin, a HSPG mimetic, abolishes the inhibitory activity of brilacidin on SARS-CoV-2 pseudovirus cell entry. In addition, we found that brilacidin has broad-spectrum antiviral activity against multiple human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, and HCoV-NL63. Mechanistic studies revealed that brilacidin has a dual antiviral mechanism of action including virucidal activity and binding to coronavirus attachment factor HSPGs on host cell surface. Brilacidin partially loses its antiviral activity when heparin was included in the cell cultures, supporting the host-targeting mechanism. Drug combination therapy showed that brilacidin has a strong synergistic effect with remdesivir against HCoV-OC43 in cell culture. Taken together, this study provides appealing findings for the translational potential of brilacidin as a broad-spectrum antiviral for coronaviruses including SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.11.04.467344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467378","pub_date":"2021-11-05","title":"A high-throughput, automated, cell-free expression and screening platform for antibody discovery","abstract":"Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this gap by developing an automated workflow combining cell-free DNA template generation, protein synthesis, and high-throughput binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to 119 published SARS-CoV-2 neutralizing antibodies and demonstrate rapid identification of the most potent antibody candidates.","version":"1.1","doi":"10.1101/2021.11.04.467378","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.02.466971","pub_date":"2021-11-04","title":"Protein arginylation is regulated during SARS-CoV-2 infection","abstract":"In 2019, the world witnessed the onset of an unprecedented pandemic. In September 2021, the infection by SARS-CoV-2 had already been responsible for the death of more than 4 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER-stress and activation of unfolded protein response (UPR) pathway. The degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-end rule pathway. Here we present, for the first time, data on the role of arginylation during SARS-CoV-2 infection. We studied the modulation of protein arginylation in Vero CCL-81 and Calu-3 cells infected after 2h, 6h, 12h, 24h, and 48h. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure the levels of ATE1 and arginylated proteins. This regulation is seen specifically during infections by coronaviruses. We demonstrate that during SARS-CoV-2 infection there is an increase in the expression of the ATE1 enzyme associated with regulated levels of specific arginylated proteins. On the other hand, infected macrophages showed no ATE1 regulation. An important finding revealed that modulation of the N-end rule pathway differs between different types of infected cells. We also confirmed the potential of tannic acid to reduce viral load, and furthermore, to modulate ATE1 levels during infection. In addition, the arginylation inhibitor merbromin (MER) is also capable of both reducing viral load and reducing ATE1 levels. Taken together, these data show the importance of arginylation during the progression of SARS-CoV-2 infection and open the door for future studies that may unravel the role of ATE1 and its inhibitors in pathogen infection.","version":"1.1","doi":"10.1101/2021.11.02.466971","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.03.467065","pub_date":"2021-11-04","title":"Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation","abstract":"Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal \u03b2-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.","version":"1.1","doi":"10.1101/2021.11.03.467065","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.02.463717","pub_date":"2021-11-04","title":"Comparing protein-protein interaction networks of SARS-CoV-2 and (H1N1) influenza using topological features","abstract":"SARS-CoV-2 pandemic first emerged in late 2019 in China. It has since infected more than 183 million individuals and caused about 4 million deaths globally. A protein-protein interaction network (PPIN) and its analysis can provide insight into the behavior of cells and lead to advance the procedure of drug discovery. The identification of essential proteins is crucial to understand for cellular survival. There are many centrality measures to detect influential nodes in complex networks. Since SARS-CoV-2 and (H1N1) influenza PPINs pose 553 common proteins. Analyzing influential proteins and comparing these networks together can be an effective step helping biologists in drug design. We used 21 centrality measures on SARS-CoV-2 and (H1N1) influenza PPINs to identify essential proteins. PCA-based dimensionality reduction was applied on normalized centrality values. Some measures demonstrated a high level of contribution in comparison to others in both PPINs, like Barycenter, Decay, Diffusion degree, Closeness (Freeman), Closeness (Latora), Lin, Radiality, and Residual. Using validation measures, the appropriate clustering method was chosen for centrality measures. We also investigated some graph theory-based properties like the power law, exponential distribution, and robustness. Through analysis and comparison, both networks exhibited remarkable experimental results. The network diameters were equal and in terms of heterogeneity, SARS-CoV-2 PPIN tends to be more heterogeneous. Both networks under study display a typical power-law degree distribution. Dimensionality reduction and unsupervised learning methods were so effective to reveal appropriate centrality measures.","version":"1.1","doi":"10.1101/2021.11.02.463717","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.02.467026","pub_date":"2021-11-04","title":"Marked enhancement of neutralizing antibody and IFN-\u03b3 T-cell responses by GX-19N DNA booster in mice primed with inactivated vaccine","abstract":"In response to the COVID-19 pandemic, an unprecedented level of vaccine development has occurred. As a result, various COVID-19 vaccines have been approved for use. Among these, inactivated virus particle (VP) vaccines have been widely used worldwide, but additional vaccination strategies are needed because of the short duration of immune responses elicited by these vaccines. Here, we evaluated homologous and heterologous prime\u2013boost regimens using a VP vaccine and GX-19N DNA vaccine for their ability to enhance the protective immune response against SARS-CoV-2. We demonstrated that a heterologous prime\u2013boost regimen with the VP vaccine and GX-19N DNA vaccine resulted in enhanced SRBD- & N-specific antibody responses, compared to the homologous VP vaccine prime\u2013boost vaccination. In addition, the neutralizing antibody response was significantly improved with the heterologous VP prime\u2013DNA boost regimen, and the neutralizing antibody induced with the heterologous prime\u2013boost regimen did not decrease against the SARS-CoV-2 variant of concern (VOC). The heterologous VP prime\u2013DNA boost regimen not only significantly increased S- and N-specific IFN-\u03b3 T-cell responses, but also induced an equivalent level of T-cell response against SARS-CoV-2 VOCs. Our results provide new insights into prophylactic vaccination strategies for COVID-19 vaccination.","version":"1.1","doi":"10.1101/2021.11.02.467026","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.04.467246","pub_date":"2021-11-04","title":"Coronavirus RNA synthesis takes place within membrane-bound sites","abstract":"Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Under-standing more about the replication of these viruses is imperative in order to effectively find ways to control them.","version":"1.1","doi":"10.1101/2021.11.04.467246","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.02.466951","pub_date":"2021-11-03","title":"Drug targeting Nsp1-ribosomal complex shows antiviral activity against SARS-CoV-2","abstract":"The SARS-Cov-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-Cov-2 bind in the mRNA entry channel of the 40S ribosomal subunit and block the entry of mRNAs thereby shutting down host protein synthesis. Nsp1 suppresses the host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter and find that montelukast sodium hydrate binds to Nsp1-C-ter with a binding affinity (KD) of 10.8\u00b10.2 \u03bcM in vitro and forms a stable complex with it in simulation runs with a binding energy of \u221276.71\u00b18.95 kJ/mol. The drug also rescues the inhibitory effect of Nsp1 in host protein synthesis as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, montelukast sodium hydrate demonstrates antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We therefore propose montelukast sodium hydrate may help in combatting SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.11.02.466951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.01.466865","pub_date":"2021-11-02","title":"Low selectivity index of ivermectin and macrocyclic lactones on SARS-CoV2 replication in vitro argues against their therapeutic use for COVID-19","abstract":"There are very limited antiviral therapeutic options for coronavirus infections, therefore global drug re-purposing efforts are paramount to identify available compounds that could provide clinical benefits to patients with COVID-19. Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin demonstrated anti-SARS-CoV-2 activity in vitro in human pulmonary cells in comparison to VeroE6 (with EC50 of 1-3 \u03bcM). Similarly, the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin reduced SARS-CoV-2 replication in vitro (with EC50 of 2-5 \u03bcM). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index <10 for all compounds. In conclusion, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se, due to low selectivity index. This is discussed in regards to recent clinical COVID studies on ivermectin.","version":"1.1","doi":"10.1101/2021.11.01.466865","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.01.466834","pub_date":"2021-11-02","title":"Glycan-masking spike antigen in NTD and RBD elicits broadly neutralizing antibodies against SARS-CoV-2 variants","abstract":"Glycan-masking the vaccine antigen by mutating the undesired antigenic sites with an additional N-linked glycosylation motif can refocus B-cell responses to desired/undesired epitopes, without affecting the antigen\u2019s overall-folded structure. This study examine the impact of glycan-masking mutants of the N-terminal domain (NTD) and receptor-binding domain (RBD) of SARS-CoV-2, and found that the antigenic design of the S protein increases the neutralizing antibody titers against the Wuhan-Hu-1 ancestral strain and the recently emerged SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Our results demonstrated that the use of glycan-masking Ad-S-R158N/Y160T in the NTD elicited a 2.8-fold, 6.5-fold, and 4.6-fold increase in the IC-50 NT titer against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants, respectively. Glycan-masking of Ad-S-D428N in the RBD resulted in a 3.0-fold and 2.0-fold increase in the IC50 neutralization titer against the Alpha (B.1.1.7) and Beta (B.1.351) variants, respectively. The use of glycan-masking in Ad-S-R158N/Y160T and Ad-S-D428N antigen design may help develop universal COVID-19 vaccines against current and future emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.11.01.466834","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.31.466651","pub_date":"2021-11-02","title":"Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination","abstract":"Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs). Induction of the latter is a hallmark of durable immunity after vaccination. SARS-CoV-2 mRNA vaccination induces a robust GC response in humans, but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S+ BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.","version":"1.1","doi":"10.1101/2021.10.31.466651","journal":"bioRxiv","score":null},{"id":"10.1101/2021.11.01.21265731","pub_date":"2021-11-02","title":"Report 46: Factors driving extensive spatial and temporal fluctuations in COVID-19 fatality rates in Brazilian hospitals","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The SARS-CoV-2 Gamma variant spread rapidly across Brazil, causing substantial infection and death waves. We use individual-level patient records following hospitalisation with suspected or confirmed COVID-19 to document the extensive shocks in hospital fatality rates that followed Gamma\u2019s spread across 14 state capitals, and in which more than half of hospitalised patients died over sustained time periods. We show that extensive fluctuations in COVID-19 in-hospital fatality rates also existed prior to Gamma\u2019s detection, and were largely transient after Gamma\u2019s detection, subsiding with hospital demand. Using a Bayesian fatality rate model, we find that the geographic and temporal fluctuations in Brazil\u2019s COVID-19 in-hospital fatality rates are primarily associated with geographic inequities and shortages in healthcare capacity. We project that approximately half of Brazil\u2019s COVID-19 deaths in hospitals could have been avoided without pre-pandemic geographic inequities and without pandemic healthcare pressure. Our results suggest that investments in healthcare resources, healthcare optimization, and pandemic preparedness are critical to minimize population wide mortality and morbidity caused by highly transmissible and deadly pathogens such as SARS-CoV-2, especially in low- and middle-income countries.</jats:p>\n                <jats:sec>\n                  <jats:title>Note</jats:title>\n                  <jats:p>\n                    The following manuscript has appeared as \u2018Report 46 - Factors driving extensive spatial and temporal fluctuations in COVID-19 fatality rates in Brazilian hospitals\u2019 at\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='https://spiral.imperial.ac.uk:8443/handle/10044/1/91875'>https://spiral.imperial.ac.uk:8443/handle/10044/1/91875</jats:ext-link>\n                    .\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>One sentence summary</jats:title>\n                  <jats:p>COVID-19 in-hospital fatality rates fluctuate dramatically in Brazil, and these fluctuations are primarily associated with geographic inequities and shortages in healthcare capacity.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.11.01.21265731","journal":"medRxiv","score":null},{"id":"10.1101/2020.02.05.935387","pub_date":"2021-11-01","title":"Glycopeptide antibiotic teicoplanin inhibits cell entry of SARS-CoV-2 by suppressing the proteolytic activity of cathepsin L","abstract":"Since the outbreak of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the public health worldwide has been greatly threatened. The development of an effective treatment for this infection is crucial and urgent but is hampered by the incomplete understanding of the viral infection mechanism and the lack of specific antiviral agents. We previously reported that teicoplanin, a glycopeptide antibiotic that has been commonly used in the clinic to treat bacterial infection, significantly restrained the cell entry of Ebola virus, SARS-CoV and MERS-CoV by specifically inhibiting the activity of cathepsin L (CTSL). Here, we found that the cleavage sites of CTSL on the Spike of SARS-CoV-2 were highly conserved among all the variants. The treatment with teicoplanin suppressed the proteolytic activity of CTSL on Spike and prevented the cellular infection of different pseudotyped SARS-CoV-2 viruses. Teicoplanin potently prevented the entry of authentic SARS-CoV-2 into the cellular cytoplasm with an IC50 of 2.038 \u03bcM for the Wuhan-Hu-1 reference strain and an IC50 of 2.116 \u03bcM for the SARS-CoV-2 (D614G) variant. The pre-treatment of teicoplanin also prevented SARS-CoV-2 infection in hACE2 mice. In summary, our data reveal that CTSL is required for both SARS-CoV-2 and SARS-CoV infection and demonstrate the therapeutic potential of teicoplanin for universal anti-CoVs intervention. Disease prevention and treatment are two important countermeasures to end the coronavirus disease 2019 (COVID-19). However, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, evolves all the time, resulting in the emerging of many epidemic SARS-CoV-2 mutants, which significantly impairs the effectiveness of early strain-based vaccines and antibodies. Developing universal vaccines and broad-spectrum antiviral drugs are essential to confront SARS-CoV-2 mutants including those may emerge in the future. Our study reported here showed that the cleavage sites of cellular cathepsin L (CTSL) are highly conserved among all the SARS-CoV-2 mutants and SARS-CoV. The CTSL inhibitor teicoplanin not only inhibited the cell entry of two live SARS-CoV-2 strains and various pseudotyped viruses but also prevented live virus infection in animal models. Based on our previous finding that teicoplanin also inhibited SARS-CoV and MERS-CoV infection, we believe that teicoplanin possesses the potential to become a universal anti-CoVs drug.","version":"1.2","doi":"10.1101/2020.02.05.935387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.043224","pub_date":"2021-11-01","title":"Meta-transcriptomic analysis reveals the gene expression and novel conserved sub-genomic RNAs in SARS-CoV-2 and MERS-CoV","abstract":"Fundamental to viral biology is identification and annotation of viral genes and their function. Determining the level of coronavirus gene expression is inherently difficult due to the positive stranded RNA genome and the identification of sub-genomic RNAs (sgRNAs) that are required for expression of most viral genes. In the COVID-19 epidemic so far, few genomic studies have looked at viral sgRNAs and none have systematically examined the sgRNA profiles of large numbers of SARS-CoV2 datasets in conjuction with data for other coronaviruses. We developed a bioinformatic pipeline to analyze the sgRNA profiles of coronaviruses and applied it to 588 individual samples from 20 independent studies, covering more than 10 coronavirus species. Our result showed that SARS-CoV, SARS-CoV-2 and MERS-CoV each had a core sgRNA repertoire generated via a canonical mechanism. Novel sgRNAs that encode peptides with evolutionarily conserved structures were identified in several coronaviruses and were expressed in vitro and in vivo. Two novel peptides may have direct functional relevance to disease, by alluding interferon responses and disrupting IL17E (IL25) signaling. Relevant to coronavirus infectivity and transmission, we also observed that the level of Spike sgRNAs were significantly higher in-vivo than in-vitro, while the opposite held true for the Nucleocapside protein. Our results greatly expanded the predicted number of coronaviruses proteins and identified potential viral peptide suggested to be involved in viral virulence. These methods and findings shed new light on coronavirus biology and provides a valuable resource for future genomic studies of coronaviruses.","version":"1.2","doi":"10.1101/2020.04.16.043224","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438731","pub_date":"2021-11-01","title":"Functional evaluation of the P681H mutation on the proteolytic activation the SARS-CoV-2 variant B.1.1.7 (Alpha) spike","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern (VOC) first identified in the UK in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7 or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread. SARS-CoV-2 B.1.1.7 VOC has a P681H mutation in the spike that is predicted to enhance viral infection P681H does not significantly impact furin cleavage, viral entry or cell-cell spread Other mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates","version":"1.2","doi":"10.1101/2021.04.06.438731","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.30.466586","pub_date":"2021-11-01","title":"Visceral Fat Inflammation and Fat Embolism are associated with Lung\u2019s Lipidic Hyaline Membranes in COVID-19 patients","abstract":"Visceral obesity is a critical determinant of severe coronavirus disease-2019 (COVID-19). Methods: In this study, we performed a comprehensive histomorphologic analysis of autoptic visceral adipose tissues (VAT), lungs and livers of 19 COVID-19 and 23 non-COVID-19 subjects. Although there were no between-groups differences in body-mass-index and adipocytes size, higher prevalence of CD68+ macrophages in COVID-19 subjects\u2019 VAT was detected (p=0.005) and accompanied by crown-like structures presence, signs of adipocytes stress and death. Consistently, human adipocytes were successfully infected by SARS-CoV2 in vitro and displayed lower cell viability. Being VAT inflammation associated with lipids spill-over from dead adipocytes, we studied lipids distribution employing Oil-Red-O staining (ORO). Lipids were observed within lungs and livers interstitial spaces, macrophages, endothelial cells, and vessels\u2019 lumen, features suggestive of fat embolism syndrome, more prevalent among COVID-19 individuals (p<0.001). Notably, signs of fat embolism were more prevalent among obese (p=0.03) independently of COVID-19 diagnosis, suggesting that such condition may be an obesity complication, exacerbated by SARS-CoV2 infection. Importantly, all infected subjects\u2019 lungs presented lipids-rich (ORO+) hyaline membranes, formations associated with COVID-19-related pneumonia, present only in one control with non-COVID-19 pneumonia. This study describes for the first time novel COVID-19-related features possibly underlying the unfavorable prognosis in obese SARS-CoV2-infected-subjects.","version":"1.1","doi":"10.1101/2021.10.30.466586","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.29.466519","pub_date":"2021-11-01","title":"A biosafety level 2 surrogate for studying SARS-CoV-2 survival in food processing environmental biofilms","abstract":"Meat processing plants have been at the center of the SARS-CoV-2 pandemic. There are several factors that contribute to the persistence of SARS-CoV-2 in meat processing plants and one of the factors is the formation of a multi-species biofilm with virus. Biofilm can act as a reservoir in protecting, harboring, and dispersing SARS-CoV-2 from biofilm to the meat processing facility environment. We used Murine Hepatitis Virus (MHV) as a surrogate for SARS-CoV-2 virus and meat processing facility drain samples to develop mixed-species biofilms on commonly found materials in processing facilities (Stainless-Steel (SS), PVC and tiles). The results showed that MHV was able to integrate into the environmental biofilm and survived for a period of 5 days at 7\u00b0C. There was no significate difference between the viral-environmental biofilm biovolumes developed on different materials SS, PVC, and tiles. There was a 2-fold increase in the virus-environmental biofilm biovolume when compared to environmental biofilm by itself. These results indicate a complex virus-environmental biofilm interaction which is providing enhanced protection for the survival of viral particles with the environmental biofilm community.","version":"1.1","doi":"10.1101/2021.10.29.466519","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.29.466418","pub_date":"2021-11-01","title":"SARS-CoV-2 mechanistic correlates of protection: insight from modelling response to vaccines","abstract":"The definition of correlates of protection is critical for the development of next generation SARS-CoV-2 vaccine platforms. Here, we propose a new framework for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication. A framework for modelling the immune control of viral dynamics is applied to quantify the effect of several SARS-CoV-2 vaccine platforms and to define mechanistic correlates of protection.","version":"1.1","doi":"10.1101/2021.10.29.466418","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444407","pub_date":"2021-11-01","title":"Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication","abstract":"As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5\u2019 exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3\u2032-to-5\u2032 exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14\u2019s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum. The ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.05.17.444407","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.23.461536","pub_date":"2021-11-01","title":"High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization","abstract":"Many viruses form highly pleomorphic particles; in influenza, these particles range from spheres of ~ 100 nm in diameter to filaments of several microns in length. Virion structure is of interest, not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline to image many thousands of individual influenza virions, gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. Finally, we showed that in long filaments, viral RNP complexes are located preferentially within Archetti bodies, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses.","version":"1.2","doi":"10.1101/2021.09.23.461536","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.465224","pub_date":"2021-10-29","title":"An iTSC-derived placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility in syncytiotrophoblasts","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has caused a global health crisis. The primary site of infection is in the respiratory tract but the virus has been associated with a variety of complications involving the gastrointestinal and cardiovascular systems. Since the virus affects a variety of tissue types, there has been interest in understanding SARS-CoV-2 infection in early development and the placenta. ACE2 and TMPRSS2, two genes that are critical for SARS-CoV-2 virus entry are expressed in placenta-specific cell types including extravillous trophoblasts (EVTs) and especially, syncytiotrophoblasts (STs). The potential of SARS-CoV-2 to infect these placental cells and its effect on placental development and function is still unclear. Furthermore, it is crucial to understand the possible mechanism of vertical transmission of SARS-CoV-2 through the placenta. Here, we developed an in vitro model of SARS-CoV-2 infection of placental cell types using induced trophoblast stem cells (iTSCs). This model allowed us to show that STs but not EVTs are infected. Importantly, infected STs lack the expression of key differentiation genes, lack typically observed differentiated morphology and produce significantly lower human chorionic gonadotropin (HCG) compared to non-infected controls. We also show that an anti-ACE2 antibody prevents SARS-CoV-2 infection and restores normal ST differentiation and function. We highlight the establishment of a platform to study SARS-CoV-2 infection in early placental cell types, which will facilitate investigation of antiviral therapy to protect the placenta during early pregnancy and development.","version":"1.1","doi":"10.1101/2021.10.27.465224","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.465996","pub_date":"2021-10-29","title":"Camel nanobodies broadly neutralize SARS-CoV-2 variants","abstract":"With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two VHH nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of \u22655 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. Dromedary camel (Camelus dromedarius) VHH phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.10.27.465996","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.29.466402","pub_date":"2021-10-29","title":"Inhibition of SAR S-CoV-2 infection and replication by lactoferrin, MUC1 and \u03b1-lactalbumin identified in human breastmilk","abstract":"The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and \u03b1-Lactalbumin (\u03b1-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while \u03b1-LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and \u03b1-LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and \u03b1-LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels.","version":"1.1","doi":"10.1101/2021.10.29.466402","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.466163","pub_date":"2021-10-29","title":"Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates","abstract":"The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.","version":"1.1","doi":"10.1101/2021.10.27.466163","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.466067","pub_date":"2021-10-29","title":"Novel pectin from crude polysaccharide of Syzygium aromaticum against SARS-CoV-2 activities by targeting 3CLpro","abstract":"To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 \u03bcM). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 \u00b1 1.05 \u00b5M and 0.18 \u00b1 0.01 \u00b5M, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked \u03b1-GalpA, 1, 4-linked \u03b1-GalpAOMe and 1, 2, 4-linked \u03b1-Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked \u03b2-Galp, terminal (T)-linked \u03b2-Galp, 1, 5-linked \u03b1-Araf, T-linked \u03b1-Araf, 4-linked \u03b1-GalpA and/or 4-linked \u03b2-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.","version":"1.1","doi":"10.1101/2021.10.27.466067","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.466055","pub_date":"2021-10-29","title":"Phylogenetic analysis and in silico studies link spike Q675H mutation to SARS-CoV-2 adaptive evolution","abstract":"Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants\u2019 evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region delimiting the conformational space of the highly flexible loop containing the furin cleavage site. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was found to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation is likely to confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we show an ongoing increase in the occurrence of Q675H spike mutation in the most common SARS-CoV-2 variants of concern (VOC). This finding highlights that, VOC are still evolving and start acquiring the Q675H mutation. At the same time, it suggests that our hypothesis of fitness advantage prompted by Q675H could be concrete.","version":"1.1","doi":"10.1101/2021.10.27.466055","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.28.465226","pub_date":"2021-10-29","title":"A Potential Novel COVID-19 Vaccine With RBD-HR1/HR2 Hexamer Structure","abstract":"The COVID-19 pandemic and the continued spreading of the SARS-CoV-2 variants have brought a grave public health consequence and severely devastated the global economy with recessions. Vaccination is considered as one of the most promising and efficient methods to end the COVID-19 pandemic and mitigate the disease conditions if infected. Although a few vaccines have been developed with an unprecedented speed, scientists around the world are continuing pursuing the best possible vaccines with innovations. Comparing to the expensive mRNA vaccines and attenuated/inactivated SARS-CoV-2 vaccines, recombinant protein vaccines have certain advantages, including their safety (non-virus components), potential stronger immunogenicity, broader protection, ease of scaling-up production, reduced cost, etc. In this study, we reported a novel COVID-19 vaccine generated with RBD-HR1/HR2 hexamer that was creatively fused with the RBD domain and heptad repeat 1 (HR1) or heptad repeat 2 (HR2) to form a dumbbell-shaped hexamer to target the spike S1 subunit. The novel hexamer COVID-19 vaccine induced high titers of neutralizing antibody in mouse studies (>100,000), and further experiments also showed that the vaccine also induced an alternative antibody to the HR1 region, which probably alleviated the drop of immunogenicity from the frequent mutations of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.10.28.465226","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.26.466019","pub_date":"2021-10-29","title":"Analyzing SARS CoV-2 Patient Data Using Quantum Supervised Machine Learning","abstract":"The novel coronavirus disease 2019 (COVID-19) has created a serious threat to global health. We developed a new quantum machine learning (QML) assisted diagnostic method that can provide an accurate diagnosis to aid decision processes of medical providers. One of the key elements in our method was to implement the quantum variational method to efficiently classify data, taking crucial multiple correlations among the features into account. We established and fine-tuned this quantum classifier by using a group of data drawn from publicly available COVID-19 cases. We have shown that QML is capable of processing patient information efficiently and accurately for the diagnosis of COVID-19.","version":"1.1","doi":"10.1101/2021.10.26.466019","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.466206","pub_date":"2021-10-29","title":"A multi-tissue study of immune gene expression profiling highlights the key role of the nasal epithelium in COVID-19 severity","abstract":"COVID-19 symptoms range from mild to severe illness; the cause for this differential response to infection remains unknown. Unravelling the immune mechanisms acting at different levels of the colonization process might be key to understand these differences. We carried out a multi-tissue (nasal, buccal and blood; n = 156) gene expression analysis of immune-related genes from patients affected by different COVID-19 severities, and healthy controls through the nCounter technology. We then used a differential expression approach and pathways analysis to detect tissue specific immune severity signals in COVID-19 patients. Mild and asymptomatic cases showed a powerful innate antiviral response in nasal epithelium, characterized by activation of interferon (IFN) pathway and downstream cascades, successfully controlling the infection at local level. In contrast, weak macrophage/monocyte driven innate antiviral response and lack of IFN signalling activity were shown in severe cases. Consequently, oral mucosa from severe patients showed signals of viral activity, cell arresting and viral dissemination to the lower respiratory tract, which ultimately could explain the exacerbated innate immune response and impaired adaptative immune responses observed at systemic level. Results from saliva transcriptome suggest that the buccal cavity might play a key role in SARS-CoV-2 infection and dissemination in patients with worse prognosis. We found severity-related signatures in patient tissues mainly represented by genes involved in the innate immune system and cytokine/chemokine signalling. Local immune response could be key to determine the course of the systemic response and thus COVID-19 severity. Our findings provide a framework to investigate severity host gene biomarkers and pathways that might be relevant to diagnosis, prognosis, and therapy.","version":"1.1","doi":"10.1101/2021.10.27.466206","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.28.466298","pub_date":"2021-10-29","title":"Identification of COVID-19 and COPD common key genes and pathways using a protein-protein interaction approach","abstract":"Coronavirus disease (COVID-19) is an extremely contagious and cognitive disease that could cause immense hypoxemia. The rise in critically ill patients in epidemic regions has put enormous pressure on hospitals. There is a need to define extreme COVID-19 clinical determinants to optimize clinical diagnosis and the management system is strong. Chronic obstructive pulmonary disease (COPD) is linked to a rapidly increasing risk of death rates in population pneumonia. In this research, a network of protein-protein interaction (PPI) was developed using constructed datasets of COVID-19 and COPD genes to define the interrelationship between COVID-19 and COPD, how it affects each other, and the genes that are responsible for the process. The PPI network shows the top 10 common overlapping genes, which include IL10, TLR4, TNF, IL6, CXCL8, IL4, ICAM1, IFNG, TLR2, and IL18. These are the genes that COVID-19 and high-risk COPD patients are known to be expressed. These important genes shared by COVID-19 and COPD are involved in pathways such as malaria, African trypanosomiasis, inflammatory bowel disease, Chagas disease, influenza, and tuberculosis.","version":"1.1","doi":"10.1101/2021.10.28.466298","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.466182","pub_date":"2021-10-29","title":"Efficacy of anti-microbial gel vapours against aerosolised coronavirus, bacteria, and fungi","abstract":"The urban population spends up to 90% of their time indoors. The indoor environment harbours a diverse microbial population including viruses, bacteria, and fungi. Pathogens present in the indoor environment can be transmitted to humans through aerosols. This study evaluated the efficacy of an antimicrobial gel containing a mix of essential oils against aerosols of bacteria, fungi, and coronavirus. The antimicrobial gel was allowed to vapourize inside a glass chamber for 10 or 20 minutes. Microbial aerosols of Escerichia coli, Aspergillus flavus spores or murine hepatitis virus MHV 1, a surrogate of SARS CoV-2 was passed through the gel vapours and then collected on a 6-stage Andersen sampler. The number of viable microbes present in the aerosols collected in the different stages were enumerated and compared to number of viable microbes in control microbial aerosols that were not exposed to the gel vapours. Vaporizing the antimicrobial gel for 10 and 20 minutes resulted in a 48% (p = 0.002 Vs. control) and 53% (p = 0.001 Vs. control) reduction in the number of MHV-1 in the aerosols, respectively. The antimicrobial gel vaporised for 10 minutes, reduced the number of viable E. coli by 51% (p = 0.032 Vs. control) and Aspergillus flavus spores by 72% (p=0.008 Vs. control) in the aerosols. The antimicrobial gel may be able to reduce aerosol transmission of microbes.","version":"1.1","doi":"10.1101/2021.10.27.466182","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.27.21265574","pub_date":"2021-10-28","title":"Boosting of Cross-Reactive Antibodies to Endemic Coronaviruses by SARS-CoV-2 Infection but not Vaccination with Stabilized Spike","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Pre-existing antibodies to endemic coronaviruses (CoV) that cross-react with SARS-CoV-2 have the potential to influence the antibody response to COVID-19 vaccination and infection for better or worse. In this observational study of mucosal and systemic humoral immunity in acutely infected, convalescent, and vaccinated subjects, we tested for cross reactivity against endemic CoV spike (S) protein at subdomain resolution. Elevated responses, particularly to the \u03b2-CoV OC43, were observed in all natural infection cohorts tested and were correlated with the response to SARS-CoV-2. The kinetics of this response and isotypes involved suggest that infection boosts preexisting antibody lineages raised against prior endemic CoV exposure that cross react. While further research is needed to discern whether this recalled response is desirable or detrimental, the boosted antibodies principally targeted the better conserved S2 subdomain of the viral spike and were not associated with neutralization activity. In contrast, vaccination with a stabilized spike mRNA vaccine did not robustly boost cross-reactive antibodies, suggesting differing antigenicity and immunogenicity. In sum, this study provides evidence that antibodies targeting endemic CoV are robustly boosted in response to SARS-CoV-2 infection but not to vaccination with stabilized S, and that depending on conformation or other factors, the S2 subdomain of the spike protein triggers a rapidly recalled, IgG-dominated response that lacks neutralization activity.</jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract</jats:title>\n                  <jats:fig id='figA1' position='float' fig-type='figure' orientation='portrait'>\n                    <jats:label>Graphical Abstract</jats:label>\n                    <jats:caption>\n                      <jats:p>Antibody responses to SARS-CoV-2 and endemic CoV spike proteins were measured in diverse cohorts. While antibodies to SARS-CoV-2 were induced across all isotypes, only IgA and IgG responses to endemic CoV were robustly boosted, and only among naturally-infected but not vaccinated individuals. These recalled, cross-reactive responses to endemic CoV primarily recognized the better conserved S2 domain and were non-neutralizing. While other antiviral activities of broadly cross-reactive S2-specifc antibodies are not known, the differing antigenicity of natural infection and vaccination with stabilized pre-fusion spike has potential implications for the breadth and level of protection afforded by each.</jats:p>\n                    </jats:caption>\n                    <jats:graphic xmlns:xlink='http://www.w3.org/1999/xlink' xlink:href='21265574v1_figA1' position='float' orientation='portrait'/>\n                  </jats:fig>\n                </jats:sec>","version":null,"doi":"10.1101/2021.10.27.21265574","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.26.465417","pub_date":"2021-10-28","title":"Understanding the Genetics of Viral Drug Resistance by Integrating Clinical Data and Mining of the Scientific Literature","abstract":"Drug resistance caused by mutations is a public health threat for existing and emerging viral diseases. A wealth of evidence about these mutations and their clinically-associated phenotypes is scattered across the literature, but a comprehensive perspective is usually lacking. This work aimed to produce a clinically-relevant view for the case of Hepatitis B virus (HBV) mutations by combining a chronic HBV clinical study with a compendium of genetic mutations systematically gathered from the scientific literature. We enriched clinical mutation data by systematically mining 2,472,725 scientific articles from PubMed Central in order to gather information about the HBV mutational landscape. By performing this analysis, we were able to identify mutational hotspots for each HBV genotype (A-E) and gene (C, X, P, S), as well as the location of disulfide bonds associated with these mutations. Through a modelling study, we also identified a mutational position common in both the clinical data and the literature that is located at the binding pocket for a known anti-HBV drug, namely entecavir. The results of this novel approach shows the potential of integrated analyses to assist in the development of new drugs for viral diseases that are more robust to resistance. Such analyses should be of particular interest due to the increasing importance of viral resistance in established and emerging viruses, such as for newly-developed drugs against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.10.26.465417","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438630","pub_date":"2021-10-27","title":"ADNKA overcomes SARS-CoV2-mediated NK cell inhibition through non-spike antibodies","abstract":"The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This correlated with a reduction in NK cell activation, identifying a novel mechanism by which SARS-CoV2 antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.","version":"1.2","doi":"10.1101/2021.04.06.438630","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.25.465714","pub_date":"2021-10-26","title":"Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants","abstract":"We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted\u2014most successfully, vaccination\u2014imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies) have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)\u2014namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.","version":"1.1","doi":"10.1101/2021.10.25.465714","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.26.465865","pub_date":"2021-10-26","title":"Excessive inflammatory and metabolic responses to acute SARS-CoV-2 infection are associated with a distinct gut microbiota composition","abstract":"Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and associated clinical sequelae requires well-coordinated metabolic and immune responses that limit viral spread and promote recovery of damaged systems. In order to understand potential mechanisms and interactions that influence coronavirus disease 2019 (COVID-19) outcomes, we performed a multi-omics analysis on hospitalised COVID-19 patients and compared those with the most severe outcome (i.e. death) to those with severe non-fatal disease, or mild/moderate disease, that recovered. A distinct subset of 8 cytokines and 140 metabolites in sera identified those with a fatal outcome to infection. In addition, elevated levels of multiple pathobionts and lower levels of protective or anti-inflammatory microbes were observed in the faecal microbiome of those with the poorest clinical outcomes. Weighted gene correlation network analysis (WGCNA) identified modules that associated severity-associated cytokines with tryptophan metabolism, coagulation-linked fibrinopeptides, and bile acids with multiple pathobionts. In contrast, less severe clinical outcomes associated with clusters of anti-inflammatory microbes such as Bifidobacterium or Ruminococcus, short chain fatty acids (SCFAs) and IL-17A. Our study uncovered distinct mechanistic modules that link host and microbiome processes with fatal outcomes to SARS-CoV-2 infection. These features may be useful to identify at risk individuals, but also highlight a role for the microbiome in modifying hyperinflammatory responses to SARS-CoV-2 and other infectious agents.","version":"1.1","doi":"10.1101/2021.10.26.465865","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.23.21265402","pub_date":"2021-10-26","title":"Severity of Illness Caused by Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern in Children: A Single-Center Retrospective Cohort Study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Recent surges in coronavirus 2019 disease (COVID-19) is attributed to the emergence of more transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs). However, the relative severity of SARS-CoV-2 VOCs in children is unknown.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    This retrospective single-center cohort study was performed at the Ann &amp; Robert H. Lurie Children\u2019s Hospital of Chicago, academic free-standing children\u2019s hospital. We included all children \u2264 18 years-old diagnosed with COVID-19 between October 15\n                    <jats:sup>th</jats:sup>\n                    , 2020 and August 31\n                    <jats:sup>st</jats:sup>\n                    , 2021 and whose SARS-CoV-2 isolate was sequenced using the Illumina platform. For each patient sample, we identified the SARS-CoV-2 lineage, which was assigned to one of the following groups: Non-VOC, alpha VOC, beta VOC, gamma VOC, or delta VOC. We measured frequency of 5 markers of COVID-19 severity: hospitalization; COVID-19 pharmacologic treatment; respiratory support; intensive care unit admission; and severe disease as classified by the COVID-19 World Health Organization (WHO) Clinical Progression Scale (severe disease; score \u2265 6). A series of logistic regression models were fitted to estimate odds of each severity marker with each VOC (in comparison to non-VOCs), adjusting for COVID-19 community incidence and demographic and clinical co-variates.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    During the study period, 2,025 patients tested positive for SARS-CoV-2; 1,422 (70.2%) had sufficient viral load to permit sequencing. Among the 499 (35.1%) patients whose isolate was sequenced, median (inter-quartile range) age was 7 (1,12) years; 256 (51.3%) isolates were a VOC: 96 (37.5%) alpha, 38 (14.8%) gamma, and 119 (46.5%) delta. After adjusting for age, Black race, Hispanic ethnicity, high-risk medical conditions, and COVID-19 community incidence, neither alpha nor delta was associated with severe COVID-19. Gamma was independently associated with hospitalization (OR 5.9, 95% CI 1.6-21.5,\n                    <jats:italic>p</jats:italic>\n                    =0.007), respiratory support (OR 8.3, 95% CI 1.5-56.3,\n                    <jats:italic>p</jats:italic>\n                    =0.02), and severe disease as classified by the WHO Clinical Progression Scale (OR 7.7, 95% CI 1.0-78.1,\n                    <jats:italic>p</jats:italic>\n                    =0.05).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Compared to non-VOC COVID-19 infections, the gamma VOC, but not the alpha or delta VOCs, was associated with increased severity. These data suggest that recent increased in pediatric COVID-19 hospitalizations are related to increased delta COVID-19 incidence rather than increased delta virulence in children.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.10.23.21265402","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.24.465080","pub_date":"2021-10-26","title":"Targeting the chemokine receptor CXCR4 with histamine analogue to reduce inflammation in juvenile arthritis: a proof of concept for COVID-19 therapeutic approach","abstract":"Among immune cells, activated monocytes play a detrimental role in chronic and viral-induced inflammatory pathologies. The uncontrolled activation of monocytes and the subsequent excessive production of inflammatory factors damage bone-cartilage joints in Juvenile Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis (RA) disease. Inflammatory monocytes also exert a critical role in the cytokine storm induced by SARS-CoV2 infection in severe COVID-19 patients. The moderate beneficial effect of current therapies and clinical trials highlights the need of alternative strategies targeting monocytes to treat RA and COVID-19 pathologies. Here, we show that targeting CXCR4 with small amino compound such as the histamine analogue clobenpropit (CB) inhibits spontaneous and induced-production of a set of key inflammatory cytokines by monocytes isolated from blood and synovial fluids of JIA patients. Moreover, daily intraperitoneal CB treatment of arthritic mice results in significant decrease in circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption leading to reduction of disease progression. Finally, we provide the prime evidence that the exposure of whole blood from hospitalized COVID-19 patients to CB significantly reduces levels of key cytokine-storm-associated factors including TNF-\u03b1, IL-6 and IL-1\u03b2. These overall data show that targeting CXCR4 with CB-like molecules may represent a promising therapeutic option for chronic and viral-induced inflammatory diseases.","version":"1.1","doi":"10.1101/2021.10.24.465080","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.26.21265510","pub_date":"2021-10-26","title":"Virtual reality intervention alleviates dyspnea in patients recovering from COVID pneumonia","abstract":"<jats:title>Summary</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Immersive virtual reality (iVR)-based digital therapeutics (DTx) are gaining clinical attention in the field of pain management. Based on known analogies between pain and dyspnea, we investigated the effects of visual-respiratory feedback, on persistent dyspnea in patients recovering from COVID-19 pneumonia.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We performed a controlled, randomized, single-blind, cross-over clinical study to evaluate an iVR-based intervention to alleviate dyspnea in patients recovering from COVID-19 pneumonia. Included patients reported persistent dyspnea (\u22655 on a 10-point scale) and preserved cognitive function (MoCA&gt;24). Assignment was random and concealed. Patients received synchronous (intervention) or asynchronous (control) feedback of their breathing, embodied via a gender-matched virtual body. Outcomes were assessed using questionnaires and breathing recordings. COVVR is registered with\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    (\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04844567'>NCT04844567</jats:ext-link>\n                    ).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Study enrollment was open between November 2020 and April 2021. Twenty-six patients were enrolled (27% women; age: median=55, interquartile range (IQR)=18). Data were available for 24 of 26 patients. The median (IQR) rating on a 7-point Likert-scale of breathing comfort improved from 1(2) at baseline, to 2(1) for synchronous feedback, but remained unchanged at 1(1.5) for asynchronous feedback (p&lt;0.05) between iVR conditions). Moreover, 91.2% of all patients were satisfied with the intervention (p&lt;0.0001) and 66.7% perceived it as beneficial for their breathing (p&lt;0.05). No adverse events were reported.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Based on these findings, our iVR-based DTx presents a feasible and safe respiratory rehabilitation tool that improves breathing comfort in patients recovering from COVID-19 infection presenting with persistent dyspnea. Future research should investigate the DTx\u2019s generalizability to persistent dyspnea with other etiologies and its potential for preventing chronification.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Marie Sklodowska-Curie Individual Fellowship (H2020-MSCA-IF-2019 894111/ RESPVR), Bertarelli Foundation</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.10.26.21265510","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.25.465706","pub_date":"2021-10-25","title":"Profiling RT-LAMP tolerance of sequence variation for SARS-CoV-2 RNA detection","abstract":"The ongoing SARS-CoV-2 pandemic has necessitated a dramatic increase in our ability to conduct molecular diagnostic tests, as accurate detection of the virus is critical in preventing its spread. However, SARS-CoV-2 variants continue to emerge, with each new variant potentially affecting widely-used nucleic acid amplification diagnostic tests. RT-LAMP has emerged as a quick, inexpensive diagnostic alternative to RT-qPCR, but has not been studied as thoroughly. Here we interrogate the effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification, creating 572 single point mutation \u201cvariants\u201d covering every position of the LAMP primers in 3 SARS-CoV-2 assays and analyzing their effects with over 4,500 RT-LAMP reactions. Remarkably, we observed only minimal effects on amplification speed and no effect on detection sensitivity, highlighting RT-LAMP as an extremely robust technique for viral RNA detection. Additionally, we describe the use of molecular beacons to sensitively identify variant RNA sequences. Together these data add to the growing body of knowledge on the utility of RT-LAMP and increase its potential to further our ability to conduct molecular diagnostic tests outside of the traditional clinical laboratory environment.","version":"1.1","doi":"10.1101/2021.10.25.465706","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449178","pub_date":"2021-10-25","title":"Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury","abstract":"Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from areas of interest within well preserved post-mortem lung samples from three patients. Despite substantial inter-patient heterogeneity we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies. Spatial analysis identifies IFN\u03b3 response signatures as focal to severe alveolar damage in COVID-19 pneumonitis.","version":"1.2","doi":"10.1101/2021.06.21.449178","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.24.465626","pub_date":"2021-10-24","title":"SARS-CoV-2 infects human adipose tissue and elicits an inflammatory response consistent with severe COVID-19","abstract":"The COVID-19 pandemic, caused by the viral pathogen SARS-CoV-2, has taken the lives of millions of individuals around the world. Obesity is associated with adverse COVID-19 outcomes, but the underlying mechanism is unknown. In this report, we demonstrate that human adipose tissue from multiple depots is permissive to SARS-CoV-2 infection and that infection elicits an inflammatory response, including the secretion of known inflammatory mediators of severe COVID-19. We identify two cellular targets of SARS-CoV-2 infection in adipose tissue: mature adipocytes and adipose tissue macrophages. Adipose tissue macrophage infection is largely restricted to a highly inflammatory subpopulation of macrophages, present at baseline, that is further activated in response to SARS-CoV-2 infection. Preadipocytes, while not infected, adopt a proinflammatory phenotype. We further demonstrate that SARS-CoV-2 RNA is detectable in adipocytes in COVID-19 autopsy cases and is associated with an inflammatory infiltrate. Collectively, our findings indicate that adipose tissue supports SARS-CoV-2 infection and pathogenic inflammation and may explain the link between obesity and severe COVID-19. Our work provides the first in vivo evidence of SARS-CoV-2 infection in human adipose tissue and describes the associated inflammation.","version":"1.1","doi":"10.1101/2021.10.24.465626","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465481","pub_date":"2021-10-24","title":"Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies","abstract":"In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.","version":"1.1","doi":"10.1101/2021.10.22.465481","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.23.465567","pub_date":"2021-10-24","title":"SARS-CoV-2 Infection Impacts Carbon Metabolism and Depends on Glutamine for Replication in Syrian Hamster Astrocytes","abstract":"Coronaviruses belong to a well-known family of enveloped RNA viruses and are the causative agent of the common cold. Although the seasonal coronaviruses do not pose a threat to human life, three members of this family, i.e., SARS-CoV, MERS-CoV and recently, SARS-CoV2, may cause severe acute respiratory syndrome and lead to death. Unfortunately, COVID-19 has already caused more than 4.4 million deaths worldwide. Although much is better understood about the immunopathogenesis of the lung disease, important information about systemic disease is still missing, mainly concerning neurological parameters. In this context, we sought to evaluate immunometabolic changes using in vitro and in vivo models of hamsters infected with SARS-CoV-2. Here we show that, besides infecting hamster\u2019s astrocytes, SARS-CoV-2 induces changes in protein expression and metabolic pathways involved in carbon metabolism, glycolysis, mitochondrial respiration, and synaptic transmission. Interestingly, many of the differentially expressed proteins are concurrent with proteins that correlate with neurological diseases, such as Parkinsons\u2019s disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington\u2019s disease. Metabolic analysis by high resolution real-time respirometry evidenced hyperactivation of glycolysis and mitochondrial respiration. Further metabolomics analysis confirmed the consumption of many metabolites, including glucose, pyruvate, glutamine, and alpha ketoglutarate. Interestingly, we observed that glutamine was significantly reduced in infected cultures, and the blockade of mitochondrial glutaminolysis significantly reduced viral replication and pro-inflammatory response. SARS-CoV-2 was confirmed in vivo as hippocampus, cortex, and olfactory bulb of intranasally infected hamsters were positive for viral genome several days post-infection. Altogether, our data reveals important changes in overall protein expression, mostly of those related to carbon metabolism and energy generation, causing an imbalance in important metabolic molecules and neurotransmitters. This may suggest that some of the neurological features observed during COVID-19, as memory and cognitive impairment, may rely on altered energetic profile of brain cells, as well as an unbalanced glutamine/glutamate levels, whose importance for adequate brain function is unquestionable.","version":"1.1","doi":"10.1101/2021.10.23.465567","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465294","pub_date":"2021-10-24","title":"Characterization of raloxifene as potential pharmacological agent against SARS-CoV-2 and its variants","abstract":"The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the COVID-19 pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several new vaccines for the prevention and protection of this infectious disease, which in many cases becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for new therapeutic indications, represents one of the most effective potential strategies employed to increase the success rate in the development of new drug therapies. We identified raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a potential pharmacological agent for the treatment of COVID-19 patients. Following a virtual screening campaign on the most relevant viral protein targets, in this work we report the results of the first pharmacological characterization of raloxifene in relevant cellular models of COVID-19 infection. The results obtained on all the most common viral variants originating in Europe, United Kingdom, Brazil, South Africa and India, currently in circulation, are also reported, confirming the efficacy of raloxifene and, consequently, the relevance of the proposed approach. Taken together, all the information gathered supports the clinical development of raloxifene and confirms that the drug can be proposed as a viable new option to fight the pandemic in at least some patient populations. The results obtained so far have paved the way for a first clinical study to test the safety and efficacy of raloxifene, just concluded in patients with mild to moderate COVID-19.","version":"1.1","doi":"10.1101/2021.10.22.465294","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.23.465542","pub_date":"2021-10-24","title":"Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway","abstract":"mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined \u223c3-log10 compared to control animals. In nasal swabs, sgRNA declined 1-log10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.","version":"1.1","doi":"10.1101/2021.10.23.465542","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465399","pub_date":"2021-10-23","title":"Nanoviricides Platform Technology based NV-387 polymer Protects Remdesivir from Plasma-Mediated Catabolism in vitro: Importance of its increased lifetime for in vivo action","abstract":"As of today seven coronaviruses were identified to infect humans, out of which only 4 of them belongs to beta family of coronavirus, like HCoV-HKU1, SARS-CoV-2, MERS-CoV and SARS-CoV. SARS family of viruses were known to cause severe respiratory disease in humans. SARS-CoV-2 infection causes pandemic COVID-19 disease with high morbidity and mortality. Remdesivir (RDV) is the only antiviral drug so far approved for Covid-19 therapy by FDA. However it\u2019s efficacy is limited in vivo due to it\u2019s low stability in presence of Plasma. Here we show the stability of RDV encapsulated with our platform technology based polymer NV-387 (NV-CoV-2-R), in presence of Plasma in vitro in comparison to naked RDV when incubated in plasma. The potential use of this polymer in vivo will be discussed, here.","version":"1.1","doi":"10.1101/2021.10.22.465399","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.29.458083","pub_date":"2021-10-22","title":"Allosteric regulation of 3CL protease of SARS-CoV-2 and SARS-CoV observed in the crystal structure ensemble","abstract":"The 3C-like protease (3CLpro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CLpro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CLpro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CLpro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)\u2013disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CLpro to SARS-CoV-2 3CLpro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CLpro seems to have increased activity relative to that of SARS-CoV 3CLpro.","version":"1.2","doi":"10.1101/2021.08.29.458083","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.21.465252","pub_date":"2021-10-22","title":"Nasal prevention of SARS-CoV-2 infection by intranasal influenza-based boost vaccination","abstract":"Vaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal. Since mucosal immunity is critical for nasal prevention, we investigated an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2. Substantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity. Our results demonstrated that intranasal influenza-based boost vaccination is required for inducing mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems. This study was supported by the Research Grants Council Collaborative Research Fund (C7156-20G, C1134-20G and C5110-20G), General Research Fund (17107019) and Health and Medical Research Fund (19181052 and 19181012) in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program (JSGG20200225151410198); the Health@InnoHK, Innovation and Technology Commission of Hong Kong; and National Program on Key Research Project of China (2020YFC0860600, 2020YFA0707500 and 2020YFA0707504); and donations from the Friends of Hope Education Fund. Z.C.\u2019s team was also partly supported by the Theme-Based Research Scheme (T11-706/18-N).","version":"1.1","doi":"10.1101/2021.10.21.465252","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.463185","pub_date":"2021-10-22","title":"Diversity of SARS-CoV-2 genome among various strains identified in Lucknow, Uttar Pradesh","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a significant challenge worldwide. Rapid genome sequencing of SARS-CoV-2 is going on across the globe to detect mutations and genomic modifications in SARS-CoV-2. In this study, we have sequenced twenty-three SARS-CoV-2 positive samples collected during the first pandemic from the state of Uttar Pradesh, India. We observed a range of already reported mutations (2-22), including; D614G, L452R, Q613H, Q677H, T1027I in the S gene; S194L in the N gene; Q57H, L106F, T175I in the ORF3. Few unreported mutations such as P309S in the ORF1ab gene; T379I in the N gene; and L52F, V77I in the ORF3a gene were also detected. Phylogenetic genome analysis showed similarity with other SARS-CoV-2 viruses reported from Uttar Pradesh. The observed mutations may be associated with SARS-CoV-2 virus pathogenicity or disease severity.","version":"1.3","doi":"10.1101/2021.10.05.463185","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.22.465411","pub_date":"2021-10-22","title":"Underlying selection for the diversity of Spike protein sequences of SARS-CoV-2","abstract":"The global spread of SARS-CoV-2 is fast moving and has caused a worldwide public health crisis. In the present manuscript we analyzed spike protein sequences of SARS-CoV-2 genomes to assess the impact of mutational diversity. We observed from amino acid usage patterns that spike proteins are associated with a diversity of mutational changes and most important underlying cause of variation of amino acid usage is the changes in hydrophobicity of spike proteins. The changing patterns of hydrophobicity of spike proteins over time and its influence on the receptor binding affinity provides crucial information on the SARS-CoV-2 interaction with human receptor. Our results also show that spike proteins have evolved to prefer more hydrophobic residues over time. The present study provides a comprehensive analysis of molecular sequence data to consider that mutational variants might play a crucial role in modulating the virulence and spread of the virus and has immediate implications for therapeutic strategies.","version":"1.1","doi":"10.1101/2021.10.22.465411","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.21.465254","pub_date":"2021-10-22","title":"Gramicidin S and Melittin - Potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection","abstract":"The COVID19 pandemic has resulted in multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of gramicidin S and melittin peptides obtained from Bacillus brevis and bee venom respectively. Our in vitro antiviral assay results showed significant decrease in the viral load compared to the untreated group with no/very less cytotoxicity. The EC50 values for gramicidin S and melittin are calculated as 1.571\u03bcg and 0.656\u03bcg respectively. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 hours onwards with a maximal clearance after 24 hours post infection. Based on proteome analysis it was found that more than 250 proteins were found to be differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 hours post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.10.21.465254","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.21.465386","pub_date":"2021-10-22","title":"Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variant\u2019s ability to potentially infect different animals. SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.","version":"1.1","doi":"10.1101/2021.10.21.465386","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.16.463825","pub_date":"2021-10-22","title":"\u03b41 variant of SARS-COV-2 acquires spike V1176F and yields a highly mutated subvariant in Europe","abstract":"Genomic surveillance of SARS-COV-2 has revealed that in addition to many variants of interests, this virus has yielded four variants of concern, \u03b1, \u03b2, \u03b3 and \u03b4, as designated by the World Health Organization. \u03b4 variant has recently become the predominant pandemic driver around the world and yielded four different subvariants (\u03b41, \u03b42, \u03b43 and \u03b44). Among them, \u03b41 has emerged as the key subvariant that drives the pandemic in India, Europe and the USA. A relevant question is whether \u03b41 subvariant continues to evolve and acquires additional mutations. Related to this, this subvariant has acquired spike V1176F, a signature substitution of \u03b3 variant, and yielded a new sublineage, \u03b41F. The substitution alters heptad repeat 2 of spike protein and is expected to improve interaction with heptad repeat 1 and enhance virus entry. Moreover, there are \u03b41F sublineages encoding spike N501Y, A783S, Q836E and V1264L. While N501Y is a signature substitution shared by \u03b1, \u03b2 and \u03b3 variants, V1264L is a key substitution in a \u03b41 sublineage that is a major pandemic driver in Southeast Asia. The Q836E-encoding lineage carries an average of 50 mutations per genome, making it the most mutated variant identified so far. Similar to \u03b41 subvariant, \u03b42 subvariant has also acquired spike V1176F and yielded new sublineages. Together, these results suggest that V1176F is a recurrent spike substitution that is frequently acquired by SARS-COV-2 variants to improve viral fitness. It is thus important to track the evolutionary trajectory of related variants for considering and instituting the most effective public health measures.","version":"1.2","doi":"10.1101/2021.10.16.463825","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431625","pub_date":"2021-10-22","title":"A rigorous framework for detecting SARS-CoV-2 spike protein mutational ensemble from genomic and structural features","abstract":"The recent release of SARS-CoV-2 genomic data from several countries has provided clues into the potential antigenic drift of the coronavirus population. In particular, the genomic instability observed in the spike protein necessitates immediate action and further exploration in the context of viralhost interactions. By temporally tracking 527,988 SARS-CoV-2 genomes, we identified invariant and hypervariable regions within the spike protein. We evaluated combination of mutations from SARS-CoV-2 lineages and found that maximum number of lineage-defining mutations were present in the N-terminal domain (NTD). Based on mutant 3D-structural models of known Variants of Concern (VOCs), we found that structural properties such as accessibility, secondary structural type, and intra-protein interactions at local mutation sites are greatly altered. Further, we observed significant differences between intra-protein networks of wild-type and Delta mutant, with the latter showing dense intra-protein contacts. Extensive molecular dynamics simulations of D614G mutant spike structure with hACE2 further revealed dynamic features with 47.7% of mutations mapping on flexible regions of spike protein. Thus, we propose that significant changes within spike protein structure have occurred that may impact SARS-CoV-2 pathogenesis, and repositioning of vaccine candidates is required to contain the spread of COVID-19 pathogen.","version":"1.2","doi":"10.1101/2021.02.17.431625","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.08.463334","pub_date":"2021-10-22","title":"Covid-19 genomic analysis reveals clusters of emerging sublineages within the delta variant","abstract":"The emerging SARS-CoV-2 variants may potentially have enhanced transmissibility and virulence of the virus, and impacts on performance of diagnostic tools and efficacy of vaccines. Genomic surveillance provides an opportunity to detect and characterize new mutations early enough for effective deployment of control strategies. Here, genomic data from Germany and United Kingdom were examined for genetic diversity by assessing gene mutations and inferring phylogeny, and simplifying genomic data workflow for faster visualization and interpretation. Delta variant sublineages were grouped into seven distinct clusters of spike mutations located in N-terminal domain of S1 region (T95I, D138H, *D142G, Y145H and A222V) and S2 region (T719I and *N950D). The most predominant cluster was T95I mutation, with the highest frequencies (71.1% - 83.9%) in Wales, England and Scotland, and the least frequencies (8.9% - 12.1%) in Germany. Two mutations, *D142G and *N950D here described as *reverse mutations and T719I mutation, were largely unique to Germany. In a month, frequencies of D142G had increased from 55.6% to 67.8 % in Germany. Additionally, a cluster of D142G+T719I/T mutation went up from 27.7% to 34.1%, while a T95I+ D142G+N950D/N cluster rose from 19.2% to 26.2%. Although, two distinct clusters of T95I+D138H (2.6% - 3.8%) and T95I+Y145H+A222V (2.5% - 8.5%) mutations were present in all the countries, they were most predominant in Wales and Scotland respectively. Results suggest divergent evolutionary trajectories between the clusters of T95I mutation and those of D142G mutation. These findings provide insights into underlying dynamics of evolution of the delta variant. Future studies may evaluate the epidemiological and biological implications of these sublineages.","version":"1.2","doi":"10.1101/2021.10.08.463334","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446445","pub_date":"2021-10-22","title":"Prediction of residue-specific contributions to binding and thermal stability using yeast surface display","abstract":"Accurate prediction of residue burial as well as quantitative prediction of residue-specific contributions to protein stability and activity is challenging, especially in the absence of experimental structural information. This is important for prediction and understanding of disease causing mutations, and for protein stabilization and design. Using yeast surface display of a saturation mutagenesis library of the bacterial toxin CcdB, we probe the relationship between ligand binding and expression level of displayed protein, with in vivo solubility in E.coli and in vitro thermal stability. We find that both the stability and solubility correlate well with the total amount of active protein on the yeast cell surface but not with total amount of expressed protein. We coupled FACS and deep sequencing to reconstruct the binding and expression mean fluorescent intensity of each mutant. The reconstructed mean fluorescence intensity (MFIseq) was used to differentiate between buried site, exposed non active-site and exposed active-site positions with high accuracy. The MFIseq was also used as a criterion to identify destabilized as well as stabilized mutants in the library, and to predict the melting temperatures of destabilized mutants. These predictions were experimentally validated and were more accurate than those of various computational predictors. The approach was extended to successfully identify buried and active-site residues in the receptor binding domain of the spike protein of SARS-CoV-2, suggesting it has general applicability.","version":"1.2","doi":"10.1101/2021.05.31.446445","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.20.464686","pub_date":"2021-10-21","title":"S100A8 and S100A9, biomarkers of SARS-Cov2-infected patients, suppress HIV replication in primary macrophages","abstract":"S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils and form a heterodimer complex. Recently, both proteins were identified as novel biomarkers of SARS-CoV-2 infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the \u201ccytokine storm.\u201d Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibits HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads in SARS-CoV2 co-infection.","version":"1.1","doi":"10.1101/2021.10.20.464686","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.21.465243","pub_date":"2021-10-21","title":"A novel Pan-viral prophylaxis strategy using vaccine adjuvant CAF09b protects against influenza virus infection","abstract":"The SARS-CoV-2 pandemic caused a massive health and societal crisis, although the fast development of effective vaccines reduced some of the impact. To prepare for future pandemics, a pan-viral prophylaxis could be used to control the initial virus outbreak in the period prior to vaccine approval. The liposomal vaccine adjuvant CAF\u00ae09b contains the TLR3 agonist polyinosinic:polycytidylic acid, which induces a type I interferon (IFN-I) response and an antiviral state in the affected tissues. When testing CAF09b as a potential pan-viral prophylaxis, we observed that intranasal administration of CAF09b to mice resulted in an influx of innate immune cells into the nose and lungs and upregulation of IFN-I related gene expression. When CAF09b was administered prior to challenge with mouse-adapted influenza A/Puerto Rico/8/1934 virus, it protected from severe disease, although virus was still detectable in the lungs. However, when CAF09b was administered after influenza challenge, the mice had a similar disease course to controls. In conclusion, CAF09b may be a suitable candidate as a pan-viral prophylactic treatment for epidemic viruses, but must be administered prior to virus exposure to be effective.","version":"1.1","doi":"10.1101/2021.10.21.465243","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.464931","pub_date":"2021-10-20","title":"Repurposed nystatin to inhibit SARS-CoV-2 and mutants in the GI tract","abstract":"The SARS-CoV-2 infections are considered as respiratory system diseases, mostly. In COVID-19, it might also be the infection of gastrointestinal (GI) tract too, especially at patients in severe clinical condition. SARS-CoV-2 can destroy the intestinal barrier, capable to spread into internal organs via blood and/or lymphatic circulation, and to cause serious damage there. Infected GI tract of COVID-19 patients is ideal environment for the coronavirus infection, replication and as virus reservoir might be the major source of pandemic reinfections, too. The process of virus budding is dependent on the host cell lipid rafts containing membrane-sterols, mainly cholesterol. The viral envelope may be challenged by polyene antibiotics, such as nystatin, which has strong affinity to sterols. Nystatin may block the establishment of the virus-host cell connection, too. In this study, the nystatin was investigated, as antiviral agent to SARS-CoV-2. We demonstrated by tests in Vero E6 cell based cytopathic assay, nystatin blocked the replication of SARS-CoV-2 in concentration 62.5 \u03bcg/ml (IC50) at Wuhan and British mutant strains. No efficient SARS-CoV-2 antiviral agent is known so far to alleviate pandemic, to disinfect GI tract, where vaccines might have limited effect, only. Nystatin might be the first one with emergency use authorization, either, as a safe and efficient non-systemic antiviral drug, with well-established use, since decades.","version":"1.1","doi":"10.1101/2021.10.19.464931","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.465036","pub_date":"2021-10-20","title":"Discovery of Potent Triple Inhibitors of Both SARS-CoV-2 Proteases and Human Cathepsin L","abstract":"There are currently no FDA approved inhibitors of SARS-CoV-2 viral proteases with specific treatment for post-exposure of SARS-CoV-2. Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases in SARS CoV-2 replication including SARS CoV-2 Main Protease (Mpro), SARS CoV-2 Papain Like Protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by disulfiram, a currently prescribed drug commonly used to treat chronic alcoholism that at the present time is in Phase 2 clinical trials against SARS-CoV-2. At the maximal allowed dose, disulfiram is associated with adverse effects. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, JX 06, and RI172 are the most potent inhibitors from an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L with IC50s of 330, 250 nM, and 190 nM about 4.5, 17, and 11.5-fold more potent than disulfiram, respectively. The identified protease inhibitors in this series were also tested against SARS CoV-2 in a cell-based and toxicity assay and were shown to have similar or greater antiviral effect than disulfiram. The identified triple protease inhibitors and their derivatives are promising candidates for treatment of the Covid-19 virus and related variants.","version":"1.1","doi":"10.1101/2021.10.19.465036","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.464990","pub_date":"2021-10-20","title":"An intranasally administrated SARS-CoV-2 beta variant subunit booster vaccine prevents beta variant viral replication in rhesus macaques","abstract":"Emerging of SARS-CoV-2 variants and waning of vaccine/infection-induced immunity poses threats to curbing the COVID-19 pandemic. An effective, safe, and convenient booster vaccine will be needed. We hypothesized that a variant-modified mucosal booster vaccine might induce local immunity to prevent SARS-CoV-2 infection at the port of entry. The beta-variant is hardest to cross-neutralize. Herein we assessed the protective efficacy of an intranasal booster composed of beta variant-spike protein S1 with IL-15 and TLR agonists in previously immunized macaques. The macaques were first vaccinated with Wuhan strain S1 with the same adjuvant. One year later, negligibly detectable SARS-CoV-2-specific antibody remained. Nevertheless, the booster induced vigorous humoral immunity including serum- and bronchoalveolar lavage (BAL)-IgG, secretory nasal- and BAL-IgA, and neutralizing antibody against the original strain and/or beta variant. Beta-variant S1-specifc CD4+ and CD8+ T cell responses were also elicited in PBMC and BAL. Following SARS-CoV-2 beta variant challenge, the vaccinated group demonstrated significant protection against viral replication in the upper and lower respiratory tracts, with almost full protection in the nasal cavity. The fact that one intranasal beta-variant booster administrated one year after the first vaccination provoked protective immunity against beta variant infections may inform future SARS-CoV-2 booster design and administration timing.","version":"1.1","doi":"10.1101/2021.10.19.464990","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441188","pub_date":"2021-10-20","title":"Autoantibodies against Progranulin and IL-1 receptor antagonist due to immunogenic posttranslational isoforms contribute to hyperinflammation in critically ill COVID-19","abstract":"Hyperinflammation is frequently observed in patients with severe COVID-19. Inadequate and defective IFN type I responses against SARS-CoV-2, associated with autoantibodies in a proportion of patients, lead to severe courses of disease. In addition, hyperactive responses of the humoral immune system have been described. In the current study we investigated a possible role of neutralizing autoantibodies against antiinflammatory mediators. Plasma from adult patients with severe and critical COVID-19 was screened by ELISA for antibodies against PGRN, IL-1-Ra, IL-10, IL-18BP, IL-22BP, IL-36-Ra, CD40, IFN-\u03b12, IFN-\u03b3, IFN-\u03c9 and serpinB1. Autoantibodies were characterized and the antigens were analyzed for immunogenic alterations. In a discovery cohort with severe to critical COVID-19 high titers of PGRN-autoantibodies were detected in 11 of 30 (36.7%), and of IL-1-Ra-autoantibodies in 14 of 30 (46.7%) patients. In a validation cohort of 64 patients with critical COVID-19 high-titer PGRN-Abs were detected in 25 (39%) and IL-1-Ra-Abs in 32 of 64 patients (50%). PGRN-Abs and IL-1-Ra-Abs belonged to IgM and several IgG subclasses. In separate cohorts with non-critical COVID-19, PGRN-Abs and IL-1-Ra-Abs were detected in low frequency (i.e. in < 5% of patients) and at low titers. Neither PGRN-nor IL-1-Ra-Abs were found in 40 healthy controls vaccinated against SARS-CoV-2 or 188 unvaccinated healthy controls. PGRN-Abs were not cross-reactive against SARS-CoV-2 structural proteins nor against IL-1-Ra. Plasma levels of both free PGRN and free IL-1-Ra were significantly decreased in autoantibody-positive patients compared to Ab-negative and non-COVID-19 controls. In vitro PGRN-Abs from patients functionally reduced PGRN-dependent inhibition of TNF-\u03b1 signaling, and IL-1-Ra-Abs from patients reduced IL-1-Ra- or anakinra-dependent inhibition of IL-1\u00df signaling. The pSer81 hyperphosphorylated PGRN isoform was exclusively detected in patients with high-titer PGRN-Abs; likewise, a hyperphosphorylated IL-1-Ra isoform was only found in patients with high-titer IL-1-Ra-Abs. Thr111 was identified as the hyperphophorylated amino acid of IL-1-Ra. In longitudinally collected samples hyperphosphorylated isoforms of both PGRN and IL-1-Ra emerged transiently, and preceded the appearance of autoantibodies. In hospitalized patients, the presence of IL-1-Ra-Abs or IL-1-Ra-Abs in combination with PGRN-Abs was associated with a higher morbidity and mortality. To conclude, neutralizing autoantibodies to IL-1-Ra and PGRN occur in a significant portion of patients with critical COVID-19, with a concomitant decrease in circulating free PGRN and IL-1-Ra, indicative of a misdirected, proinflammatory autoimmune response. The break of self-tolerance is likely caused by atypical hyperphosphorylated isoforms of both antigens, whose appearances precede autoantibody induction. Our data suggest that these immunogenic secondary modifications are induced by the SARS-CoV-2-infection itself or the inflammatory environment evoked by the infection and predispose for a critical course of COVID-19.","version":"1.2","doi":"10.1101/2021.04.23.441188","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.463727","pub_date":"2021-10-20","title":"Neutralization of Mu and C.1.2 SARS-CoV-2 Variants by Vaccine-elicited Antibodies in Individuals With and Without Previous History of Infection","abstract":"Recently identified SARS-CoV-2 variants Mu and C.1.2 have mutations in the receptor binding domain and N- and C-terminal domains that might confer resistance to natural and vaccine-elicited antibody. Analysis with pseudotyped lentiviruses showed that viruses with the Mu and C.1.2 spike proteins were partially resistant to neutralization by antibodies in convalescent sera and those elicited by mRNA and adenoviral vector-based vaccine-elicited antibodies. Virus with the C.1.2 variant spike, which is heavily mutated, was more neutralization-resistant than that of any of variants of concern. The resistance of the C.1.2 spike was caused by a combination of the RBD mutations N501Y, Y449H and E484K and the NTD mutations. Although Mu and C.1.2 were partially resistant to neutralizing antibody, neutralizing titers elicited by mRNA vaccination remained above what is found in convalescent sera and thus are likely to remain protective against severe disease. The neutralizing titers of sera from infection-experienced BNT162b2-vaccinated individuals, those with a history of previous SARS-CoV-2 infection, were as much as 15-fold higher than those of vaccinated individuals without previous infection and effectively neutralized all of the variants. The findings demonstrate that individuals can raise a broadly neutralizing humoral response by generating a polyclonal response to multiple spike protein epitopes that should protect against current and future variants.","version":"1.1","doi":"10.1101/2021.10.19.463727","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.20.465121","pub_date":"2021-10-20","title":"Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line","abstract":"Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.","version":"1.1","doi":"10.1101/2021.10.20.465121","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.19.464951","pub_date":"2021-10-20","title":"Preparation of ingestible antibodies to neutralize the binding of SarsCoV2 RBD (receptor binding domain) to human ACE2 Receptor","abstract":"COVID19 continues to be a serious threat to human health and mortality. There is dire need for new solutions to combat this pandemic especially for those individuals who are not vaccinated or unable to be vaccinated and continue to be exposed to the SARSCoV2. In addition, the emergence of new more transmissible variants such as delta pose additional threat from this virus. To explore another solution for prevention and treatment of COVID 19, we have produced chicken egg derived IgY antibodies against the Receptor binding domain (RBD) of SARSCoV2 spike protein which is involved in binding to human cell ACE2 receptors. The \u2013 RBD IgY effectively neutralize the binding of RBD to ACE2 and prevent pseudovirus entry in a PRNT assay. Importantly our anti-RBD IgY also neutralize the binding of Sars CoV2 delta variant RBD to ACE2. Given that chicken egg derived IgY are safe and permissible for human consumption, we plan to develop these ingestible antibodies for prevention of viral entry in the oropharyngeal and digestive tract in humans as passive immunotherapy.","version":"1.1","doi":"10.1101/2021.10.19.464951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.18.464882","pub_date":"2021-10-19","title":"Rapid and Effective Inactivation of SARS-CoV-2 by a Cationic Conjugated Oligomer with Visible Light: Studies of Antiviral Activity in Solutions and on Supports","abstract":"This paper presents results of a study of a new cationic oligomer that contains end groups and a chromophore affording inactivation of SARS-Cov-2 by visible light irradiation in solution or as a solid coating on wipes paper and glass fiber filtration substrates. A key finding of this study is that the cationic oligomer with a central thiophene ring and imidazolium charged groups give outstanding performance in both killing of E. coli bacterial cells and inactivation of the virus at very short times. Our introduction of cationic N-Methyl Imidazolium groups enhances the light-activation process for both E. coli and SARS-Cov-2 but dampens the dark killing of the bacteria and eliminates the dark inactivation of the virus. For the studies with this oligomer in solution at concentration of 1 \u03bcg/mL and E. coli we obtain 3 log killing of the bacteria with 10 min irradiation with LuzChem cool white lights (mimicking indoor illumination). With the oligomer in solution at a concentration of 10 \u03bcg/mL, we observe 4 logs inactivation (99.99 %) in 5 minutes of irradiation and total inactivation after 10 min. The oligomer is quite active against E. coli on oligomer-coated wipes papers and glass fiber filter supports. The SARS-Cov-2 is also inactivated by the oligomer coated glass fiber filter papers. This study indicates that these oligomer-coated materials may be very useful as wipes and filtration materials.","version":"1.1","doi":"10.1101/2021.10.18.464882","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.16.464641","pub_date":"2021-10-19","title":"SARS-COV-2 \u03b3 variant acquires spike P681H or P681R for improved viral fitness","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) evolves and generates different variants through a continuously branching model. Four variants of concern have been the major pandemic drivers around the world. One important question is how they may evolve and generate subvariants, some of which may be even more virulent and drive the pandemic further. While investigating how \u03b3 (or P.1) variant has been evolving, I noticed the spike substitution P681H in a group of genomes encoding a new subvariant, which has been designated P.1.7. This subvariant has become the dominant P.1 sublineage in Brazil, Italy, Spain and Peru, supporting that P681H confers evolutionary advantage to P.1.7. In Brazil and Peru, P.1.7 was still responsible for ~30% and ~40% cases, respectively, in August 2021. However, it has been competed out by \u03b41 (a \u03b4 subvariant) in both countries, Italy and Spain, suggesting that P.1.7 is not as virulent as \u03b41. In addition, 160 P.1 genomes possess a related substitution, P681R, and 120 of them encode a new subvariant, designated P.1.8. This P.1 subvariant carries two additional spike substitutions, T470N and C1235F, located at the receptor-binding pocket and cytoplasmic tail of spike protein, respectively. More P.1.8 genomes have been identified than P.1 genomes that encode P681R but not T470N and C1235F, suggesting that these two substitutions improve virulence of P.1.8 subvariant. Some P.1 genomes carry other substitutions (such as N679K, V687L and C1250F) that affect the furin cleavage site or cytoplasmic tail of spike protein. Thus, to improve viral fitness and expand its evolutionary cage, \u03b3 variant acquires mutations to finetune the furin cleavage site and cytoplasmic tail of spike protein.","version":"1.1","doi":"10.1101/2021.10.16.464641","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.16.464644","pub_date":"2021-10-19","title":"SARS-COV-2 C.1.2 variant is highly mutated but may possess reduced affinity for ACE2 receptor","abstract":"SARS-COV-2 evolution generates different variants and drives the pandemic. As the current main driver, delta variant bears little resemblance to the other three variants of concern (alpha, beta and gamma), raising the question what features the future variants of concern may possess. To address this important question, I searched through the GISAID database for potential clues. While investigating how beta variant has been evolving in South Africa, I noticed a small group of genomes mainly classified as C.1.2 variant, with one-year old boy identified in March 2021 being the index case. Over 80% patients are younger than 60. At the average, there are 46-47 mutations per genome, making this variant one of the most mutated lineages identified. A signature substitution is spike Y449H. Like beta and gamma variants, C.1.2 possesses E484K and N501Y. The genomes are heterogenous and encode different subvariants. Like alpha variant, one such subvariant encodes the spike substitution P681H at the furin cleavage site. In a related genome, this substitution is replaced by P681R, which is present in delta variant. In addition, similar to this variant of concern, three C.1.2 subvariants also encode T478K. Mechanistically, spike Y449 recognizes two key residues of the cell-entry receptor ACE2 and Y449H is known to impair the binding to ACE2 receptor, so C.1.2 variant may show reduced affinity for this receptor. If so, this variant needs other mutations to compensate for such deficiency. These results raise the question whether C.1.2 variant is as virulent as suggested by its unexpected high number of mutations.","version":"1.1","doi":"10.1101/2021.10.16.464644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.15.464595","pub_date":"2021-10-18","title":"Mitoxantrone modulates a glycosaminoglycan-spike complex to inhibit SARS-CoV-2 infection","abstract":"Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a spike-GAG complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar GAG-binding activities but with reduced affinity for DNA topoisomerase may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.","version":"1.1","doi":"10.1101/2021.10.15.464595","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.17.464700","pub_date":"2021-10-18","title":"Plant-based production of SARS-CoV-2 antigens for use in a subunit vaccine","abstract":"The COVID-19 pandemic has brought to the forefront an urgent need for the rapid development of highly efficacious vaccines, particularly in light of the ongoing emergence of multiple variants of concern. Plant-based recombinant protein platforms are emerging as cost-effective and highly scalable alternatives to conventional protein production. Viral glycoproteins, however, are historically challenging to produce in plants. Herein, we report the production of plant-expressed wild-type glycosylated SARS-CoV-2 Spike RBD (receptor-binding domain) protein that is recognized by anti-RBD antibodies and exhibits high-affinity binding to the SARS-CoV-2 receptor ACE2 (angiotensin-converting enzyme 2). Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from naturally infected convalescent, vaccinated, or convalescent and vaccinated individuals. We further demonstrate that RBD binding to the ACE2 receptor was efficiently neutralized by antibodies from sera of SARS-CoV-2 convalescent and partially and fully vaccinated individuals. Collectively, these findings demonstrate that recombinant RBD produced in planta exhibits suitable biochemical and antigenic features for use in a subunit vaccine platform.","version":"1.1","doi":"10.1101/2021.10.17.464700","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.18.464814","pub_date":"2021-10-18","title":"Bees can be trained to identify SARS-CoV-2 infected samples","abstract":"The COVID-19 pandemic has illustrated the need for the development of fast and reliable testing methods for novel, zoonotic, viral diseases in both humans and animals. Pathologies lead to detectable changes in the Volatile Organic Compound (VOC) profile of animals, which can be monitored, thus allowing the development of a rapid VOC-based test. In the current study, we successfully trained honeybees (Apis mellifera) to identify SARS-CoV-2 infected minks (Neovison vison) thanks to Pavlovian conditioning protocols. The bees can be quickly conditioned to respond specifically to infected mink\u2019s odours and could therefore be part of a wider SARS-CoV-2 diagnostic system. We tested two different training protocols to evaluate their performance in terms of learning rate, accuracy and memory retention. We designed a non-invasive rapid test in which multiple bees are tested in parallel on the same samples. This provided reliable results regarding a subject\u2019s health status. Using the data from the training experiments, we simulated a diagnostic evaluation trial to predict the potential efficacy of our diagnostic test, which yielded a diagnostic sensitivity of 92% and specificity of 86%. We suggest that a honeybee-based diagnostics can offer a reliable and rapid test that provides a readily available, low-input addition to the currently available testing methods. A honeybee-based diagnostic test might be particularly relevant for remote and developing communities that lack the resources and infrastructure required for mainstream testing methods.","version":"1.1","doi":"10.1101/2021.10.18.464814","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.16.464660","pub_date":"2021-10-18","title":"Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine","abstract":"An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.","version":"1.1","doi":"10.1101/2021.10.16.464660","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.17.464720","pub_date":"2021-10-18","title":"Mapping Molecular Gene Signatures Among Respiratory Viruses Based on Large-Scale and Genome-wide Transcriptomics Analysis","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging RNA virus causing COVID-19 disease across the globe. SARS-CoV-2 infected patients exhibit acute respiratory distress syndrome which can be compounded by endemic respiratory viruses and thus highlighting the need to understand the genetic bases of clinical outcome under multiple respiratory infections. In this study, 42 individual datasets and a multi-parametric based selected list of over 12,000 genes against five medically important respiratory viruses (SARS-CoV-2, SARS-CoV-1, influenza A, respiratory syncytial virus (RSV) and rhinovirus were collected and analysed in an attempt to understand differentially regulated gene patterns and to cast genetic markers of individual and multiple co-infections. While a certain cohort of virus-specific genes were regulated (negatively and positively), notably results revealed a greatest correlation among gene regulation by SARS-CoV-2 and RSV. Furthermore, out of analysed genes, the MAP2K5 and NFKBIL1 were specifically and highly upregulated in SARS-CoV-2 infection in vivo or in vitro. In contrast, several genes including GPBAR1 and SC5DL were specifically downregulated in SARS-CoV-2 datasets. Additionally, we catalogued a set of genes that were conserved or differentially regulated across all the respiratory viruses. These finding provide foundational and genome-wide data to gauge the markers of respiratory viral infections individually and under co-infection.","version":"1.1","doi":"10.1101/2021.10.17.464720","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.21264894","pub_date":"2021-10-17","title":"Memory B cell and humoral responses elicited by Sputnik V in na\u00efve and COVID-19-recovered vaccine recipients","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not.\n                  <jats:italic>In vitro</jats:italic>\n                  stimulated MBCs from previously infected recipients of Sputnik V secreted a significant amount of anti-RBD IgG both on days 28 and 85 from the beginning of vaccination. These antibodies demonstrated robust neutralization of the Wuhan Spike-pseudotyped lentivirus. In the na\u00efve group of vaccinees, the level of anti-RBD IgG secretion was five- to six-fold reduced compared to that of the recovered group, and maximum virus neutralization (Wuhan spike) was achieved only on day 85. Sera from all the recovered and most na\u00efve Sputnik V recipients were neutralizing against the ancestral Wuhan and mutant B.1.351 viruses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.\n                </jats:p>","version":null,"doi":"10.1101/2021.10.13.21264894","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.04.447066","pub_date":"2021-10-15","title":"Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy","abstract":"As of October 2021, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low production cost, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status.","version":"1.2","doi":"10.1101/2021.06.04.447066","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.06.442903","pub_date":"2021-10-15","title":"A phylogeny-based metric for estimating changes in transmissibility from recurrent mutations in SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally to cause the COVID-19 pandemic. Despite the constant accumulation of genetic variation in the SARS-CoV-2 population, there was little evidence for the emergence of significantly more transmissible lineages in the first half of 2020. Starting around November 2020, several more contagious and possibly more virulent \u2018Variants of Concern\u2019 (VoCs) were reported in various regions of the world. These VoCs share some mutations and deletions that haven arisen recurrently in distinct genetic backgrounds. Here, we build on our previous work modelling the association of mutations to SARS-CoV-2 transmissibility and characterise the contribution of individual recurrent mutations and deletions to estimated viral transmissibility. We then assess how patterns of estimated transmissibility in all SARS-CoV-2 clades have varied over the course of the COVID-19 pandemic by summing transmissibility estimates for all individual mutations carried by any sequenced genome analysed. Such an approach recovers the Delta variant (21A) as the most transmissible clade currently in circulation, followed by the Alpha variant (20I). By assessing transmissibility over the time of sampling, we observe a tendency for estimated transmissibility within clades to slightly decrease over time in most clades. Although subtle, this pattern is consistent with the expectation of a decay in transmissibility in mainly non-recombining lineages caused by the accumulation of weakly deleterious mutations. SARS-CoV-2 remains a highly transmissible pathogen, though such a trend could conceivably play a role in the turnover of different global viral clades observed over the pandemic so far. This work is not about the severity of disease. We do not analyse the severity of disease. We do not present any evidence that SARS-CoV-2 has decreased in severity. Lineage replacement dynamics are affected by many factors. The trend we recover for a decrease in inferred transmissibility of a clade over time is a small effect. We caution against over-interpretation. This result would not affect the management of the SARS-CoV-2 pandemic: for example, we make no claims about any impact on the efficacy of particular non-pharmaceutical interventions (NPIs). Our phylogeny-based method to infer changes in estimated transmissibility due to recurrent mutations and deletions makes a number of simplifying assumptions. These may not all be valid. The consistent trend for the slight decrease we report might be due to an as-yet-unidentified systematic bias.","version":"1.2","doi":"10.1101/2021.05.06.442903","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.14.464414","pub_date":"2021-10-15","title":"Deep learning based on stacked sparse autoencoder applied to viral genome classification of SARS-CoV-2 virus","abstract":"Since December 2019, the world has been intensely affected by the COVID-19 pandemic, caused by the SARS-CoV-2 virus, first identified in Wuhan, China. In the case of a novel virus identification, the early elucidation of taxonomic classification and origin of the virus genomic sequence is essential for strategic planning, containment, and treatments. Deep learning techniques have been successfully used in many viral classification problems associated with viral infections diagnosis, metagenomics, phylogenetic, and analysis. This work proposes to generate an efficient viral genome classifier for the SARS-CoV-2 virus using the deep neural network (DNN) based on stacked sparse autoencoder (SSAE) technique. We performed four different experiments to provide different levels of taxonomic classification of the SARS-CoV-2 virus. The confusion matrix presented the validation and test sets and the ROC curve for the validation set. In all experiments, the SSAE technique provided great performance results. In this work, we explored the utilization of image representations of the complete genome sequences as the SSAE input to provide a viral classification of the SARS-CoV-2. For that, a dataset based on k-mers image representation, with k = 6, was applied. The results indicated the applicability of using this deep learning technique in genome classification problems.","version":"1.1","doi":"10.1101/2021.10.14.464414","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091199","pub_date":"2021-10-15","title":"Recombination in Sarbecovirus lineage and mutations/insertions in spike protein linked to the emergence and adaptation of SARS-CoV-2","abstract":"The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan city, China in December 2019 and thereafter its spillover across the world has created a global pandemic and public health crisis. Researchers across the world are involved in finding the origin and evolution of SARS-CoV-2, its transmission route, molecular mechanism of interaction between SARS-CoV-2 and host cells, and the cause of pathogenicity etc. In this paper, we shed light on the origin, evolution and adaptation of SARS-CoV-2 into human systems. Our phylogenetic/evolutionary analysis supported that bat-CoV-RaTG13 is the closest relative of human SARS-CoV-2, outbreak of SARS-CoV-2 took place via inter-intra species mode of transmission, and host-specific adaptation occurred in SARS-CoV-2. Furthermore, genome recombination analysis found that Sarbecoviruses, the subgenus containing SARS-CoV and SARS-CoV-2, undergo frequent recombination. Multiple sequence alignment (MSA) of spike proteins revealed the insertion of four amino acid residues \u201cPRRA\u201d (Proline-Arginine-Arginine-Alanine) into the SARS-CoV-2 human strains. Structural modeling of spike protein of bat-CoV-RaTG13 also shows a high number of mutations at one of the receptor binding domains (RBD). Overall, this study finds that the probable origin of SARS-CoV-2 is the results of intra-species recombination events between bat coronaviruses belonging to Sarbecovirus subgenus and the insertion of amino acid residues \u201cPRRA\u201d and mutations in the RBD in spike protein are probably responsible for the adaptation of SARS-CoV-2 into human systems. Thus, our findings add strength to the existing knowledge on the origin and adaptation of SARS-CoV-2, and can be useful for understanding the molecular mechanisms of interaction between SARS-CoV-2 and host cells which is crucial for vaccine design and predicting future pandemics.","version":"1.2","doi":"10.1101/2020.05.12.091199","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.08.455272","pub_date":"2021-10-15","title":"Induction of cross-reactive antibody responses against the RBD domain of the spike protein of SARS-CoV-2 by commensal microbiota","abstract":"The commensal microflora is a source for multiple antigens that may induce cross-reactive antibodies against host proteins and pathogens. However, whether commensal bacteria can induce cross-reactive antibodies against SARS-CoV-2 remains unknown. Here we report that several commensal bacteria contribute to the generation of cross-reactive IgA antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We identified SARS-CoV-2 unexposed individuals with RBD-binding IgA antibodies at their mucosal surfaces. Conversely, neutralising monoclonal anti-RBD antibodies recognised distinct commensal bacterial species. Some of these bacteria, such as Streptococcus salivarius, induced a cross-reactive anti-RBD antibodies upon supplementation in mice. Conversely, severely ill COVID-19 patients showed reduction of Streptococcus and Veillonella in their oropharynx and feces and a reduction of anti-RBD IgA at mucosal surfaces. Altogether, distinct microbial species of the human microbiota can induce secretory IgA antibodies cross-reactive for the RBD of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.08.08.455272","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.20.427043","pub_date":"2021-10-15","title":"Design of Specific Primer Sets for the Detection of SARS-CoV-2 Variants of Concern B.1.1.7, B.1.351, P.1, B.1.617.2 using Artificial Intelligence","abstract":"As the COVID-19 pandemic continues, new SARS-CoV-2 variants with potentially dangerous features have been identified by the scientific community. Variant B.1.1.7 lineage clade GR from Global Initiative on Sharing All Influenza Data (GISAID) was first detected in the UK, and it appears to possess an increased transmissibility. At the same time, South African authorities reported variant B.1.351, that shares several mutations with B.1.1.7, and might also present high transmissibility. Earlier this year, a variant labelled P.1 with 17 non-synonymous mutations was detected in Brazil. Recently the World Health Organization has raised concern for the variants B.1.617.2 mainly detected in India but now exported worldwide. It is paramount to rapidly develop specific molecular tests to uniquely identify new variants. Using a completely automated pipeline built around deep learning and evolutionary algorithms techniques, we designed primer sets specific to variants B.1.1.7, B.1.351, P.1 and respectively. Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for each variant. In-silico tests show that the sequences in the primer sets present high accuracy and are based on 2 mutations or more. In addition, we present an analysis of key mutations for SARS-CoV-2 variants. Finally, we tested the designed primers for B.1.1.7 using RT-PCR. The presented methodology can be exploited to swiftly obtain primer sets for each new variant, that can later be a part of a multiplexed approach for the initial diagnosis of COVID-19 patients.","version":"1.3","doi":"10.1101/2021.01.20.427043","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.14.464464","pub_date":"2021-10-15","title":"Opsonization by non-neutralizing antibodies can confer protection to SARS-CoV-2 despite Spike-dependent modulation of phagocytosis","abstract":"Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization seems to affect the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phago-cytosis.","version":"1.1","doi":"10.1101/2021.10.14.464464","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.14.464416","pub_date":"2021-10-15","title":"Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir","abstract":"Remdesivir (RDV) is a direct antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, e.g., SARS-CoV-2 and hepatitis C virus (HCV) and non-segmented negative-sense RNA viruses, e.g., Nipah virus (NiV), while several segmented negative-sense RNA viruses such as influenza (Flu) virus or Crimean-Congo hemorrhagic fever virus (CCHFV) are not sensitive to the drug. The reasons for this apparent pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases (RdRp) and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. The results of this study revealed a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Delayed chain-termination is heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP is seen with all polymerases. Molecular modeling suggests a steric conflict between the 1\u2019-cyano group of RDV-MP and conserved residues of RdRp motif F. We conclude that future efforts in the development of nucleotide analogues with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1\u2019-modification.","version":"1.1","doi":"10.1101/2021.10.14.464416","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.380196","pub_date":"2021-10-15","title":"A practical PPE decontamination method using warm air and ambient humidity","abstract":"Despite a year-and-a-half of Sars-CoV-2 pandemic experience, personal protective equipment (PPE) remains in short supply. Current decontamination methods are complex, slow, expensive and particularly ill-suited for low to middle income nations where the need is greatest. We propose a low temperature, ambient humidity decontamination method (WASP-D) based on the thirty minute or less half-life of Sars-CoV-2 (and other common pathogens) at temperatures above 45\u00b0C, combined with the observation that most PPE are designed to be safely transported and stored at temperatures below 50\u00b0C. Decontamination at 12 hours, 46\u00b0C (115\u00b0F) and ambient humidity should consistently reduce SARS-CoV-2 viral load by a factor of 106, without negatively affecting PPE materials or performance.","version":"1.2","doi":"10.1101/2020.11.12.380196","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464307","pub_date":"2021-10-14","title":"A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses","abstract":"The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2-36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2-36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2-36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.","version":"1.1","doi":"10.1101/2021.10.13.464307","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464225","pub_date":"2021-10-14","title":"Increased mTOR signaling, impaired autophagic flux and cell-to-cell viral transmission are hallmarks of SARS-CoV-2 infection","abstract":"The COVID-19 disease caued by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has two characteristics that distinguish it from other viral infections. It affects more severely people with pre-existing comorbidities and viral load peaks prior to the onset of the symptoms. Investigating factors that could contribute to these characteristics, we found increased mTOR signaling and suppressed genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2. Transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients revealed that COVID-19 severity is associated with increased expression of genes related to mTOR signaling and decreased expression of genes related to autophagy, lysosome function, and vesicle fusion. SARS-CoV-2 infection in Vero E6 cells also resulted in virus retention inside the cells and trafficking of virus-bearing vesicles between neighboring cells. Our findings support a scenario where SARS-CoV-2 benefits from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling, which might relate to undetectable proliferation and evasion of the immune system.","version":"1.1","doi":"10.1101/2021.10.13.464225","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464050","pub_date":"2021-10-14","title":"A Rapid Bead-Based Assay For Screening Of SARS-CoV-2 Neutralising Antibodies","abstract":"Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Here, we describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies /metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2 - Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2\u2013GFPspark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life-time imaging of the ACE2-GFPSpark confirmed the interaction of ACE2 and S1-OFPSpark on beads. The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells.","version":"1.1","doi":"10.1101/2021.10.13.464050","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.463130","pub_date":"2021-10-14","title":"The adenosine analogue prodrug ATV006 is orally bioavailable and has potent preclinical efficacy against SARS-CoV-2 and its variants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5\u2019-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5\u2019-hydroxyl-isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.","version":"1.1","doi":"10.1101/2021.10.13.463130","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464299","pub_date":"2021-10-14","title":"Variable inhibition of unwinding rates of DNA catalyzed by the SARS-Cov-2 (COV19) helicase nsp13 by structurally distinct single DNA lesions","abstract":"The SARS 2 (Covid 19) helicase nsp13 plays a critically important role in the replication of the Corona virus by unwinding double-stranded RNA (and DNA) with a 5\u2019\u27f63\u2019 strand polarity. Here we explored the impact of single, structurally defined covalent DNA lesions on the helicase activity of nsp13 in aqueous solutions, The objectives were to derive mechanistic insights into the relationships between the structures of DNA lesions, the DNA distortions that they engender, and the inhibition of helicase activity. The lesions included two bulky stereoisomeric N2-guanine adducts derived from the reactions of benzo[a]pyrene diol epoxide with DNA. The trans-adduct assumes a minor groove conformation, while the cis-product adopts a base-displaced intercalated conformation. The non-bulky DNA lesions included the intra-strand cross-linked thymine dimers, the cis-syn-cyclobutane pyrimidine dimer, and the pyrimidine (6\u22124) pyrimidone photoproduct. All four lesions strongly inhibit the helicase activity of nsp13, The UV photolesions feature a 2 - 5-fold smaller inhibition of the nsp13 unwinding activity than the bulky DNA adducts, and the kinetics of these two pairs of DNA lesions are also different. The connections between the structural features of these four DNA lesions and their impact on nsp13 unwinding efficiencies are discussed.","version":"1.1","doi":"10.1101/2021.10.13.464299","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464254","pub_date":"2021-10-14","title":"Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry","abstract":"Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures effective against SARS-CoV-2 variants and future spillovers of other sarbecoviruses. Here we describe the isolation and characterization of a human monoclonal antibody, designated S2K146, broadly neutralizing viruses belonging to all three sarbecovirus clades known to utilize ACE2 as entry receptor and protecting therapeutically against SARS-CoV-2 beta challenge in hamsters. Structural and functional studies show that most of the S2K146 epitope residues are shared with the ACE2 binding site and that the antibody inhibits receptor attachment competitively. Viral passaging experiments underscore an unusually high barrier for emergence of escape mutants making it an ideal candidate for clinical development. These findings unveil a key site of vulnerability for the development of a next generation of vaccines eliciting broad sarbecovirus immunity.","version":"1.1","doi":"10.1101/2021.10.13.464254","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464242","pub_date":"2021-10-14","title":"ONTdeCIPHER: An amplicon-based nanopore sequencing pipeline for tracking pathogen variants","abstract":"Amplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis. Here, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology (ONT) sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogenes. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses. ONTdeCIPHER is available at https://github.com/emiracherif/ONTdeCIPHER emira.cherif@ird.fr Supplementary data are available at \u2026","version":"1.1","doi":"10.1101/2021.10.13.464242","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464180","pub_date":"2021-10-14","title":"Making Genomic Surveillance Deliver: A Lineage Classification and Nomenclature System to Inform Rabies Elimination","abstract":"The availability of pathogen sequence data and use of genomic surveillance is rapidly increasing. Genomic tools and classification systems need updating to reflect this. Here, rabies virus is used as an example to showcase the potential value of updated genomic tools to enhance surveillance to better understand epidemiological dynamics and improve disease control. Previous studies have described the evolutionary history of rabies virus; however, the resulting taxonomy lacks the definition necessary to identify incursions, lineage turnover and transmission routes at high resolution. Here we propose a lineage classification system based on the dynamic nomenclature used for SARS-CoV-2, defining a lineage by phylogenetic methods, for tracking virus spread and comparing sequences across geographic areas. We demonstrate this system through application to the globally distributed Cosmopolitan clade of rabies virus, defining 73 total lineages within the clade, beyond the 22 previously reported. We further show how integration of this tool with a new rabies virus sequence data resource (RABV-GLUE) enables rapid application, for example, highlighting lineage dynamics relevant to control and elimination programmes, such as identifying importations and their sources, and areas of persistence and transmission, including transboundary incursions. This system and the tools developed should be useful for coordinating and targeting control programmes and monitoring progress as we work towards eliminating dog-mediated rabies, as well as having potential for broad application to the surveillance of other viruses. The importance of the ability to track the diversity and spread of viruses in a universal way that can be clearly communicated has been highlighted during the SARS-CoV-2 pandemic. This, accompanied with the increase in the availability and use of pathogen sequence data, means the development of new genomic tools and classification systems can strengthen outbreak response and disease control. Here, we present an easy-to-use objective and transferable classification tool for tracking viruses at high resolution. We use rabies virus, a neglected zoonotic disease that causes around 59,000 human deaths each year, as an example use case of this tool. Applying our tool to a global clade of rabies virus, we find an over 200% increase in the definition at which we can classify the virus, allowing us to identify areas of persistence and transmission that were not previously apparent, and patterns of virus spread. Insights from the application of this tool should prove valuable in targeting vaccination campaigns and improving surveillance as countries work towards the elimination of dog-mediated rabies.","version":"1.1","doi":"10.1101/2021.10.13.464180","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.12.464152","pub_date":"2021-10-13","title":"SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy","abstract":"Blood clots are a central feature of coronavirus disease-2019 (COVID-19) and can culminate in pulmonary embolism, stroke, and sudden death. However, it is not known how abnormal blood clots form in COVID-19 or why they occur even in asymptomatic and convalescent patients. Here we report that the Spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the blood coagulation factor fibrinogen and induces structurally abnormal blood clots with heightened proinflammatory activity. SARS-CoV-2 Spike virions enhanced fibrin-mediated microglia activation and induced fibrinogen-dependent lung pathology. COVID-19 patients had fibrin autoantibodies that persisted long after acute infection. Monoclonal antibody 5B8, targeting the cryptic inflammatory fibrin epitope, inhibited thromboinflammation. Our results reveal a procoagulant role for the SARS-CoV-2 Spike and propose fibrin-targeting interventions as a treatment for thromboinflammation in COVID-19. SARS-CoV-2 spike induces structurally abnormal blood clots and thromboinflammation neutralized by a fibrin-targeting antibody.","version":"1.1","doi":"10.1101/2021.10.12.464152","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.13.464181","pub_date":"2021-10-13","title":"Low antigen abundance limits efficient T-cell recognition of highly conserved regions of SARS-CoV-2","abstract":"Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S) and ORF3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad pre-existing immunity in the community.","version":"1.1","doi":"10.1101/2021.10.13.464181","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.12.464150","pub_date":"2021-10-13","title":"AUG-3387, a Human-Derived Monoclonal Antibody Neutralizes SARS-CoV-2 Variants and Reduces Viral Load from Therapeutic Treatment of Hamsters In Vivo","abstract":"Infections from the SARS-CoV-2 virus have killed over 4.6 million people since it began spreading through human populations in late 2019. In order to develop a therapeutic or prophylactic antibody to help mitigate the effects of the pandemic, a human monoclonal antibody (mAb) that binds to the SARS-CoV-2 spike protein was isolated from a convalescent patient following recovery from COVID-19 disease. This mAb, designated AUG-3387, demonstrates a high affinity for the spike protein of the original viral strains and all variants tested to date. In vitro pseudovirus neutralization and SARS-CoV-2 neutralization activity has been demonstrated in vitro. In addition, a dry powder formulation has been prepared using a Thin-Film Freezing (TFF) process that exhibited a fine particle fraction (FPF) of 50.95 \u00b1 7.69% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 3.74 \u00b1 0.73 \u00b5m and 2.73 \u00b1 0.20, respectively. The dry powder is suitable for delivery directly to the lungs of infected patients using a dry powder inhaler device. Importantly, AUG-3387, administered as a liquid by intraperitoneal injection or the dry powder formulation delivered intratracheally into Syrian hamsters 24 hours after intranasal SARS-CoV-2 infection, demonstrated a dose-dependent reduction in the lung viral load of the virus. These data suggest that AUG-3387 formulated as a dry powder demonstrates potential to treat COVID-19.","version":"1.1","doi":"10.1101/2021.10.12.464150","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.12.464114","pub_date":"2021-10-13","title":"A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy","abstract":"Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the \u201cclass 3\u201d epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.","version":"1.1","doi":"10.1101/2021.10.12.464114","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.12.463781","pub_date":"2021-10-13","title":"The R346K Mutation in the Mu Variant of SARS-CoV-2 Alter the Interactions with Monoclonal Antibodies from Class 2: A Free Energy of Perturbation Study","abstract":"The Mu variant of SARS-CoV-2 has been recently classified as a variant of interest (VOI) by the world health organization (WHO) but limited data are available at the moment. In particular, a special attention was given to the R346K mutation located in the receptor binding domain (RBD). In the current study we performed Free energy of perturbation (FEP) calculations to elucidate it possible impact on a set of neutralizing monoclonal antibodies (mAbs) which have been shown to be strong inhibitors of the most other known COVID-19 variants. Our results show that R346K affects the class 2 antibodies but its effect is not so significant (0.66 kcal/mol); i.e. reduces the binding with RBD about 3 times. An identical value was calculated also in the presence of both class 1 and class 2 antibodies (BD-812/836). Further, a similar reduction in the binding (0.4 kcal/mol) was obtained for BD-821/771 pair of mAbs. For comparison, the addition of K417N mutation, present in the newly registered Mu variant in July 2021 in UK, affected the class 1 mAbs by 1.29 kcal/mol reducing stronger the binding by about 10 times. Thus, the resistance effect of R346K mutation in the Mu variant is possible but not so significant and is due to the additional decrease of antibody neutralization based on the reduced binding of class 2 antibodies.","version":"1.1","doi":"10.1101/2021.10.12.463781","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.09.463766","pub_date":"2021-10-12","title":"SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids","abstract":"Several studies have pointed to retinal involvement in COVID-19 disease, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate in retinal cells and its effects on the retina. Here we have used human stem cell\u2013derived retinal organoids to study retinal infection by the SARS-CoV-2 virus. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors. SARS-CoV-2 infection of retinal organoids also induces the expression of several inflammatory genes, such as interleukin 33, a gene associated with acute COVID-19 disease and retinal degeneration. Finally, we show that the use of antibodies to block the ACE2 receptor significantly reduces SARS-CoV-2 infection of retinal organoids, indicating that SARS-CoV-2 infects retinal cells in an ACE2-dependent manner. These results suggest a retinal involvement in COVID-19 and emphasize the need to monitor retinal pathologies as potential sequelae of \u201clong COVID\u201d.","version":"1.1","doi":"10.1101/2021.10.09.463766","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.11.463689","pub_date":"2021-10-12","title":"Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization","abstract":"The global pandemic of Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 has become a severe global health problem because of its rapid spread(1). Both angiotensin-converting enzyme 2 and neuropilin 1 provide initial viral binding sites for SARS-CoV-2 (2, 3). Here, we show that three cysteine residues located in a1/a2 and b1 domains of neuropilin 1 are necessary for SARS-CoV-2 spike protein internalization in human cells. Mutating cysteines C82, C104 and C147 altered neuropilin 1 stability and binding ability as well as cellular internalization and lysosomal translocation of the spike protein. This resulted in up to 4 times reduction in spike protein load in cells for the original, alpha and delta SARS-CoV-2 variants even in the presence of the endogenous angiotensin-converting enzyme 2 receptor. Transcriptome analysis of cells transfected with mutated NRP1 revealed significantly reduced expression of genes involved in viral infection and replication, including eight members of the ribosomal protein L, ten members of ribosomal protein S and five members of the proteasome \u03b2 subunit family proteins. We also observed higher expression of genes involved in suppression of inflammation and endoplasmic reticulum associated degradation. These observations suggest that these cysteines offer viable targets for therapies against COVID-19.","version":"1.1","doi":"10.1101/2021.10.11.463689","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.11.463917","pub_date":"2021-10-12","title":"Secondary structure of subgenomic RNA M of SARS-CoV-2","abstract":"SARS-CoV-2 belongs the Coronavirinae family. As other coronaviruses, SARS-CoV-2 is enveloped and possesses positive-sense, single-stranded RNA genome of \u223c 30 kb. Genome RNA is used as the template for replication and transcription. During these processes, positive-sense genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) are created. Several studies showed importance of genomic RNA secondary structure in SARS-CoV-2 replication. However, the structure of sgRNAs have remained largely unsolved so far. In this study, we performed probing of sgRNA M of SARS-CoV-2 in vitro. This is the first experimentally informed secondary structure model of sgRNA M, which presents features likely to be important in sgRNA M function. The knowledge about sgRNA M provides insights to better understand virus biology and could be used for designing new therapeutics.","version":"1.1","doi":"10.1101/2021.10.11.463917","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.11.463956","pub_date":"2021-10-12","title":"SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy","abstract":"SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses. Structural studies of soluble S variants have revealed an increased propensity towards conformations accessible to receptor human Angiotensin-Converting Enzyme 2 (hACE2). However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule F\u00f6rster Resonance Energy Transfer (smFRET) studies of S variants containing critical mutations, including D614G and E484K, in the context of virus particles. Investigated variants predominantly occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited decelerated transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.","version":"1.1","doi":"10.1101/2021.10.11.463956","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.09.463779","pub_date":"2021-10-12","title":"Intelligent Resolution: Integrating Cryo-EM with AI-driven Multi-resolution Simulations to Observe the SARS-CoV-2 Replication-Transcription Machinery in Action","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication transcription complex (RTC) is a multi-domain protein responsible for replicating and transcribing the viral mRNA inside a human cell. Attacking RTC function with pharmaceutical compounds is a pathway to treating COVID-19. Conventional tools, e.g., cryo-electron microscopy and all-atom molecular dynamics (AAMD), do not provide sufficiently high resolution or timescale to capture important dynamics of this molecular machine. Consequently, we develop an innovative workflow that bridges the gap between these resolutions, using mesoscale fluctuating finite element analysis (FFEA) continuum simulations and a hierarchy of AI-methods that continually learn and infer features for maintaining consistency between AAMD and FFEA simulations. We leverage a multi-site distributed workflow manager to orchestrate AI, FFEA, and AAMD jobs, providing optimal resource utilization across HPC centers. Our study provides unprecedented access to study the SARS-CoV-2 RTC machinery, while providing general capability for AI-enabled multi-resolution simulations at scale.","version":"1.1","doi":"10.1101/2021.10.09.463779","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.11.463936","pub_date":"2021-10-12","title":"Highly resolved spatial transcriptomics for detection of rare events in cells","abstract":"Single-cell spatial transcriptomics technologies leveraged the potential to transcriptionally landscape sophisticated reactions in cells. Current methods to delineate such complex interplay lack the flexibility in rapid target adaptation and are particularly restricted in detecting rare transcripts. We developed a multiplex single-cell RNA In-situ hybridization technique, called \u2018Molecular Cartography\u2019 (MC) that can be easily tailored to specific applications and, by providing unprecedented sensitivity, specificity and resolution, is particularly suitable in tracing rare events at a subcellular level. Using a SARS-CoV-2 infection model, MC allows the discernment of single events in host-pathogen interactions, dissects primary from secondary responses, and illustrates differences in antiviral signaling pathways affected by SARS-CoV-2, simultaneously in various cell types.","version":"1.1","doi":"10.1101/2021.10.11.463936","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.27.400788","pub_date":"2021-10-11","title":"Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA","abstract":"SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein coding-loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modelling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis. petr.pecinka@osu.cz; tlb20@cam.ac.uk","version":"1.4","doi":"10.1101/2020.11.27.400788","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.08.463699","pub_date":"2021-10-11","title":"Immunity to SARS-CoV-2 up to 15 months after infection","abstract":"Information concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset. The levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison. Following a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months. Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16- and 8-fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months. The data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-spreading Delta variant, may at least partially escape the neutralizing activity of plasma antibodies. EU-ATAC consortium, the Italian Ministry of Health, the Swedish Research Council, SciLifeLab, and KAW.","version":"1.1","doi":"10.1101/2021.10.08.463699","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425307","pub_date":"2021-10-11","title":"Decreased Interfacial Dynamics Caused by the N501Y Mutation in the SARS-CoV-2 S1 Spike:ACE2 Complex","abstract":"Corona Virus Disease of 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has caused a massive health crisis across the globe, with some genetic variants gaining enhanced infectivity and competitive fitness, and thus significantly aggravating the global health concern. In this regard, the recent SARS-CoV-2 alpha variant, B.1.1.7 lineage, reported from the United Kingdom (UK), is of great significance in that it contains several mutations that increase its infection and transmission rates as evident from clinical reports. Specifically, the N501Y mutation in the SARS-CoV-2 spike S1 receptor binding domain (S1-RBD) has been shown to possess an increased affinity for ACE2, although the basis for this is not entirely clear yet. Here, we dissect the mechanism underlying the increased affinity using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interfacial interaction of the N501Y mutant S1-RBD with ACE2 compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutant S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing drugs targeting the ACE2-S1-RBD interaction. The emergence of the new SARS-CoV-2 lineage in the UK in December 2020 has further aggravated the COVID-19 pandemic due to an increased ability of the variant to infect human hosts, likely due to mutations in the viral S1 spike protein including the N501Y S1-RBD mutation that is located at the interface of S1-RBD and ACE2, the host cell receptor for SARS-CoV-2. Given its location at the interface, N501Y S1-RBD mutation can therefore potentially alter the interfacial interaction. Multiple, all-atom, explicit solvent MD simulations of the ACE2-S1-RBD complex carried here indicated a more stable interaction between the N501Y mutant S1-RBD and ACE2 through stabilizing interfacial interactions of residues at one end of the interface that are either sequentially or physically near the mutation site. These mechanistic details will aid in better understanding the mechanism by which the alpha variant has increased infectivity as well as in designing better therapeutics including ACE2-S1 spike protein inhibitors that will, in turn, help thwarting the current and future pandemic. N501 in the wild type SARS-CoV-2 S1-RBD forms unsustained hydrogen bonds with residues in the ACE2, namely Y41 and K353 Y501 in the N501Y mutant SARS-CoV-2 S1-RBD is not capable of forming substantial hydrogen bonds with ACE2 within the time span of the current simulation Evidence from analyzing the simulation results suggests that Y501 of S1-RBD could form other types of non-covalent interactions with ACE2, such as van der Waals interactions N501Y S1-RBD mutation stabilizes the position of interfacial residues neighboring to the mutation site, as well as other non-interfacial residues that are distant from the mutation site These altered dynamics results in more stable interaction of S1-RBD with ACE2 which could be the main reason underlying the reported enhanced affinity of S1-RBD in the SARS-CoV-2 alpha variant (UK B.1.1.7 lineage) to ACE2","version":"1.2","doi":"10.1101/2021.01.07.425307","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.07.463234","pub_date":"2021-10-11","title":"High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain","abstract":"Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.","version":"1.2","doi":"10.1101/2021.10.07.463234","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449535","pub_date":"2021-10-11","title":"Integrative COVID-19 Biological Network Inference with Probabilistic Core Decomposition","abstract":"The SARS-CoV-2 coronavirus is responsible for millions of deaths around the world. To help contribute to the understanding of crucial knowledge and to further generate new hypotheses relevant to SARS-CoV-2 and human protein interactions, we make use of the information abundant Biomine probabilistic database and extend the experimentally identified SARS-CoV-2-human protein-protein interaction (PPI) network in silico. We generate an extended network by integrating information from the Biomine database, the PPI network, and other experimentally validated results. To generate novel hypotheses, we focus on the high-connectivity sub-communities that overlap most with the integrated experimentally validated results in the extended network. Therefore, we propose a new data analysis pipeline that can efficiently compute core decomposition on the extended network and identify dense subgraphs. We then evaluate the identified dense subgraph and the generated hypotheses in three contexts: literature validation for uncovered virus targeting genes and proteins, gene function enrichment analysis on subgraphs, and literature support on drug repurposing for identified tissues and diseases related to COVID-19. The majority types of the generated hypotheses are proteins with their encoding genes and we rank them by sorting their connections to the integrated experimentally validated nodes. In addition, we compile a comprehensive list of novel genes, and proteins potentially related to COVID-19, as well as novel diseases which might be comorbidities. Together with the generated hypotheses, our results provide novel knowledge relevant to COVID-19 for further validation.","version":"1.2","doi":"10.1101/2021.06.23.449535","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.363002","pub_date":"2021-10-11","title":"An insight into SARS-CoV-2 Membrane protein interaction with Spike, Envelope, and Nucleocapsid proteins","abstract":"Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, we tried to understand the interaction of membrane protein\u2019s interaction with envelope, spike, and nucleocapsid proteins using protein-protein docking. Further, simulation studies performed up to 100 ns to examine the stability of protein-protein complexes of Membrane-Envelope, Membrane-Spike, and Membrane-Nucleocapsid. Prime MM-GBSA showed high binding energy calculations than the docked complex. The interactions identified in our study will be of great importance, as it provides valuable insight into the protein-protein complex, which could be the potential drug targets for future studies.","version":"1.2","doi":"10.1101/2020.10.30.363002","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.22.449395","pub_date":"2021-10-11","title":"Super.Complex: A supervised machine learning pipeline for molecular complex detection in protein-interaction networks","abstract":"Characterization of protein complexes, i.e. sets of proteins assembling into a single larger physical entity, is important, as such assemblies play many essential roles in cells such as gene regulation. From networks of protein-protein interactions, potential protein complexes can be identified computationally through the application of community detection methods, which flag groups of entities interacting with each other in certain patterns. Most community detection algorithms tend to be unsupervised and assume that communities are dense network subgraphs, which is not always true, as protein complexes can exhibit diverse network topologies. The few existing supervised machine learning methods are serial and can potentially be improved in terms of accuracy and scalability by using better-suited machine learning models and parallel algorithms. Here, we present Super.Complex, a distributed, supervised AutoML-based pipeline for overlapping community detection in weighted networks. We also propose three new evaluation measures for the outstanding issue of comparing sets of learned and known communities satisfactorily. Super.Complex learns a community fitness function from known communities using an AutoML method and applies this fitness function to detect new communities. A heuristic local search algorithm finds maximally scoring communities, and a parallel implementation can be run on a computer cluster for scaling to large networks. On a yeast protein-interaction network, Super.Complex outperforms 6 other supervised and 4 unsupervised methods. Application of Super.Complex to a human protein-interaction network with ~8k nodes and ~60k edges yields 1,028 protein complexes, with 234 complexes linked to SARS-CoV-2, the COVID-19 virus, with 111 uncharacterized proteins present in 103 learned complexes. Super.Complex is generalizable with the ability to improve results by incorporating domain-specific features. Learned community characteristics can also be transferred from existing applications to detect communities in a new application with no known communities. Code and interactive visualizations of learned human protein complexes are freely available at: https://sites.google.com/view/supercomplex/super-complex-v3-0.","version":"1.2","doi":"10.1101/2021.06.22.449395","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433379","pub_date":"2021-10-11","title":"Citation needed? Wikipedia bibliometrics during the first wave of the COVID-19 pandemic","abstract":"With the COVID-19 pandemic\u2019s outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an \u201cinfodemic\u201d, ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedia\u2019s COVID-19-related articles before and during the pandemic\u2019s first wave (January-May 2020). We found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this \u201cscientific infrastructure\u201d facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development. Understanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its \u201ccitizen encyclopedists\u201d, it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts.","version":"1.5","doi":"10.1101/2021.03.01.433379","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.439059","pub_date":"2021-10-10","title":"Exploring zebrafish larvae as a COVID-19 model: probable SARS-COV-2 replication in the swim bladder","abstract":"Animal models are essential to understand COVID-19 pathophysiology and for pre-clinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, bloodstream, or yolk, did not result in detectable SARS-CoV-2 replication in wild-type larvae. However, when the virus was inoculated in the swim bladder, a modest increase in viral RNA was observed after 24 hours, suggesting a successful infection in some animals. This was confirmed by immunohistochemistry, with cells positive for SARS-CoV-2 nucleoprotein observed in the swim bladder. Several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. Mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.","version":"1.2","doi":"10.1101/2021.04.08.439059","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433522","pub_date":"2021-10-10","title":"High resolution profiling of MHC-II peptide presentation capacity, by Mammalian Epitope Display, reveals SARS-CoV-2 targets for CD4 T cells and mechanisms of immune-escape","abstract":"Understanding the mechanisms of immune evasion is critical for formulating an effective response to global threats like SARS-CoV2. We have fully decoded the immune synapses for multiple TCRs from acute patients, including cognate peptides and the presenting HLA alleles. Furthermore, using a newly developed mammalian epitope display platform (MEDi), we determined that several mutations present in multiple viral isolates currently expanding across the globe, resulted in reduced presentation by multiple HLA class II alleles, while some increased presentation, suggesting immune evasion based on shifting MHC-II peptide presentation landscapes. In support, we found that one of the mutations present in B1.1.7 viral strain could cause escape from CD4 T cell recognition in this way. Given the importance of understanding such mechanisms more broadly, we used MEDi to generate a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of multiple common HLA class II molecules. Unlike other strategies, our approach is sensitive and scalable, providing an unbiased and affordable high-resolution map of peptide presentation capacity for any MHC-II allele. Such information is essential to provide insight into T cell immunity across distinct HLA haplotypes across geographic and ethnic populations. This knowledge is critical for the development of effective T cell therapeutics not just against COVID-19, but any disease.","version":"1.3","doi":"10.1101/2021.03.02.433522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424619","pub_date":"2021-10-10","title":"SARS-CoV-2 spike glycoprotein S1 induces neuroinflammation in BV-2 microglia","abstract":"In addition to respiratory complications produced by SARS-CoV-2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS-CoV-2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF\u03b1, IL-6, IL-1\u03b2 and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-I\u03baB\u03b1, as well as enhancing DNA binding and transcriptional activity of NF-\u03baB. These effects of the protein were blocked in the presence of BAY11-7082 (1 \u03bcM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/mL), an action that was reduced in the presence of SKF 86002 (1 \u03bcM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 \u03bcM) and transfection with TLR4 siRNA resulted in significant reduction in TNF\u03b1 and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-\u03baB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.12.29.424619","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.21264567","pub_date":"2021-10-10","title":"Efficacy and Safety of NVX-CoV2373 in Adults in the United States and Mexico","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>BACKGROUND</jats:title>\n                  <jats:p>Vaccination using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antigen has been effective in the prevention of coronavirus disease 2019 (Covid-19). NVX-CoV2373 is an adjuvanted, recombinant S protein nanoparticle vaccine that demonstrated clinical efficacy for prevention of Covid-19 in phase 2b/3 trials in the United Kingdom and South Africa.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>This phase 3, randomized, observer-blinded, placebo-controlled trial evaluated the efficacy and safety of NVX-CoV2373 in adults \u226518 years of age in the United States and Mexico during the first quarter of 2021. Participants were randomized in a 2:1 ratio to receive two doses of NVX-CoV2373 or placebo 21 days apart. The primary end point was vaccine efficacy (VE) against reverse transcriptase-polymerase chain reaction-confirmed Covid-19 in SARS-CoV-2-na\u00efve participants \u22657 days after the second dose administration.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>Of the 29,949 participants randomized between December 27, 2020, and February 18, 2021, 29,582 (median age: 47 years, 12.6% \u226565 years) received \u22651 dose: 19,714 received vaccine and 9868 placebo. In the per-protocol population, there were 77 Covid-19 cases; 14 among vaccine and 63 among placebo recipients (VE: 90.4%, 95% confidence interval [CI] 82.9 to 94.6, P&lt;0.001). All moderate-to-severe cases occurred in placebo recipients, yielding VE of 100% (95% CI 87.0 to 100). Most sequenced viral genomes (48/61, 78.7%) were variants of concern (VOC) or interest (VOI), mainly represented by variant alpha/B.1.1.7 (31/35, 88.6% VOC identified). VE against any VOC/VOI was 92.6% (95% CI 83.6 to 96.7). Reactogenicity was mostly mild-to-moderate and transient, but more frequent in NVX-CoV2373 recipients and after the second dose. Serious adverse events were rare and evenly distributed between treatments.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>NVX-CoV2373 was well tolerated and demonstrated a high overall VE (&gt;90%) for prevention of Covid-19, with most cases due to variant strains.</jats:p>\n                  <jats:p>\n                    (Funded by the Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority and the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health; PREVENT-19\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    number,\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='https://clinicaltrials.gov/ct2/show/NCT04611802'>NCT04611802</jats:ext-link>\n                    .)\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.10.05.21264567","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.08.463665","pub_date":"2021-10-09","title":"SARS-CoV-2 infection induces inflammatory bone loss in golden Syrian hamsters","abstract":"Extrapulmonary complications of different organ systems have been increasingly recognized in patients with severe or chronic Coronavirus Disease 2019 (COVID-19). However, limited information on the skeletal complications of COVID-19 is known, even though inflammatory diseases of the respiratory tract have been known to perturb bone metabolism and cause pathological bone loss. In this study, we characterized the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on bone metabolism in an established golden Syrian hamster model for COVID-19. SARS-CoV-2 causes significant multifocal loss of bone trabeculae in the long bones and lumbar vertebrae of all infected hamsters. The bone loss progressively worsens from the acute phase to the post-recovery phase. Mechanistically, the bone loss was associated with SARS-CoV-2-induced cytokine dysregulation which upregulates osteoclastic differentiation of monocyte-macrophage lineage. The pro-inflammatory cytokines further trigger a second wave of cytokine storm in the skeletal tissues to augment their pro-osteoclastogenesis effect. Our findings in this established hamster model suggest that pathological bone loss may be a neglected complication which warrants more extensive investigations during the long-term follow-up of COVID-19 patients. The benefits of potential prophylactic and therapeutic interventions against pathological bone loss should be further evaluated. SARS-CoV-2 infection causes pathological bone loss in golden Syrian hamsters through induction of cytokine storm and inflammation-induced osteoclastogenesis.","version":"1.1","doi":"10.1101/2021.10.08.463665","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.462234","pub_date":"2021-10-09","title":"Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody","abstract":"As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of a previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryo-EM. We show that mAb J08 has low nanomolar affinity against VoCs, binds high on the receptor binding domain (RBD) ridge and is therefore unaffected by most mutations, and can bind in the RBD-up and -down conformations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy. Potent neutralizing monoclonal antibody J08 binds SARS-CoV-2 spike independent of known escape mutations.","version":"1.2","doi":"10.1101/2021.09.28.462234","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.08.463613","pub_date":"2021-10-09","title":"Gut Bacterial Dysbiosis and Instability is Associated with the Onset of Complications and Mortality in COVID-19","abstract":"There is a growing debate about the involvement of the gut microbiome in COVID-19, although it is not conclusively understood whether the microbiome has an impact on COVID-19, or vice versa, especially as analysis of amplicon data in hospitalized patients requires sophisticated cohort recruitment and integration of clinical parameters. Here, we analyzed fecal and saliva samples from SARS-CoV-2 infected and post COVID-19 patients and controls considering multiple influencing factors during hospitalization. 16S rRNA gene sequencing was performed on fecal and saliva samples from 108 COVID-19 and 22 post COVID-19 patients, 20 pneumonia controls and 26 asymptomatic controls. Patients were recruited over the first and second corona wave in Germany and detailed clinical parameters were considered. Serial samples per individual allowed intra-individual analysis. We found the gut and oral microbiota to be altered depending on number and type of COVID-19-associated complications and disease severity. The occurrence of individual complications was correlated with low-risk (e.g., Faecalibacterium prausznitzii) and high-risk bacteria (e.g., Parabacteroides). We demonstrated that a stable gut bacterial composition was associated with a favorable disease progression. Based on gut microbial profiles, we identified a model to estimate mortality in COVID-19. Gut microbiota are associated with the occurrence of complications in COVID-19 and may thereby influencing disease severity. A stable gut microbial composition may contribute to a favorable disease progression and using bacterial signatures to estimate mortality could contribute to diagnostic approaches. Importantly, we highlight challenges in the analysis of microbial data in the context of hospitalization.","version":"1.1","doi":"10.1101/2021.10.08.463613","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.08.463687","pub_date":"2021-10-09","title":"Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function","abstract":"The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 \u03bcM, 1 hour) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 \u00b5M) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.","version":"1.1","doi":"10.1101/2021.10.08.463687","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.463271","pub_date":"2021-10-09","title":"Clinical and immunological signatures of severe COVID-19 in previously healthy patients with clonal hematopoiesis","abstract":"Identifying additional risk factors for COVID-19 severity in numerous previously healthy patients without canonical clinical risk factors remains challenging. In this study, we investigate whether clonal hematopoiesis of indeterminate potential (CHIP), a common aging-related process that predisposes various inflammatory responses, may exert COVID-19 severity. We examine the clinical impact of CHIP in 143 laboratory-confirmed COVID-19 patients. Both stratified analyses and logistic regression including the interaction between canonical risk factors and CHIP show that CHIP is an independent risk factor for severe COVID-19, especially in previously healthy patients. Analyses of 60,310 single-cell immune transcriptome profiles identify distinct immunological signatures for CHIP (+) severe COVID-19 patients, particularly in classical monocytes, with a marked increase in pro-inflammatory cytokine responses and potent IFN-\u03b3 mediated hyperinflammation signature. We further demonstrate that the enhanced expression of CHIP (+) specific IFN-\u03b3 response genes is attributed to the CHIP mutation-dependent epigenetic reprogramming of poised or bivalent cis-regulatory elements. Our results highlight a unique immunopathogenic mechanism of CHIP in the progression of severe COVID-19, which could be extended to elucidate how CHIP contributes to a variety of human infectious diseases.","version":"1.2","doi":"10.1101/2021.10.05.463271","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.07.463611","pub_date":"2021-10-08","title":"A bacteria-based assay to study SARS-CoV-2 protein-protein interactions","abstract":"Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of COVID-19, an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of SARS-CoV-2-encoded proteins. Here we describe the use of a convenient bacteria-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identify sixteen distinct intraviral protein-protein interactions (PPIs), involving sixteen proteins. We find that many of the identified proteins interact with more than one partner. We further show how our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm and we use this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examine how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins. Understanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here we describe the use of a versatile bacteria-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current pandemic. We identify sixteen distinct intraviral protein-protein interactions, involving sixteen proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacteria-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically its receptor binding domain) and its cognate human cell surface receptor ACE2 and we investigate the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.","version":"1.1","doi":"10.1101/2021.10.07.463611","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.07.463402","pub_date":"2021-10-08","title":"Inhibition of SARS-CoV-2 spike protein palmitoylation reduces virus infectivity","abstract":"Spike glycoproteins of almost all enveloped viruses are known to undergo post-translational attachment of palmitic acid moieties. The precise role of such palmitoylation of the spike protein in membrane fusion and infection is not completely understood. Here, we report that palmitoylation of the first five cysteine residues of the c-terminal cysteine-rich domain of the SARS-CoV-2 spike are indispensable for infection and palmitoylation deficient spike mutants are defective in trimerization and subsequent membrane fusion. The DHHC9 palmitoyltransferase interacts with and palmitoylates the spike protein in the ER and Golgi, and knockdown of DHHC9 results in reduced fusion and infection of SARS-CoV-2. Two bis-piperazine backbone-based DHHC9 inhibitors inhibit SARS-CoV-2 spike protein palmitoylation and the resulting progeny virion particles released are defective in fusion and infection. This establishes these palmitoyltransferase inhibitors as potential new intervention strategies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.10.07.463402","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.461949","pub_date":"2021-10-08","title":"Analysis of the ARTIC version 3 and version 4 SARS-CoV-2 primers and their impact on the detection of the G142D amino acid substitution in the spike protein","abstract":"The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. ARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.","version":"1.2","doi":"10.1101/2021.09.27.461949","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.07.463533","pub_date":"2021-10-08","title":"Angiotensin converting enzyme 2 (ACE2) is expressed in murine cutaneous under single-cell transcriptome resolution","abstract":"Angiotensin converting enzyme 2 (Ace2) is widely distributed in human organs, which was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus in human beings. It was also confirmed that SARS-CoV-2 uses the same cell entry receptor, ACE2, as SARS-CoV. However, related research still not discover the expression data associated with murine skin under single cell RNA resolution. In this study, we performed single-cell RNA sequencing (scRNA-seq) on unsorted cells from mouse dorsal skin after 7 days post-wounding. 8312 sequenced cells from four skin samples met quality control metrics and were analyzed.","version":"1.1","doi":"10.1101/2021.10.07.463533","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.399154","pub_date":"2021-10-07","title":"A Novel SARS-CoV-2 Multitope Protein/Peptide Vaccine Candidate is Highly Immunogenic and Prevents Lung Infection in an AAV hACE2 Mouse Model and non-human primates","abstract":"A novel multitope protein-peptide vaccine against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and disease is described in this report. The initial development and characterization experiments are presented along with proof-of-concept studies for the vaccine candidate UB-612. UB-612 consists of eight components rationally designed for induction of potently neutralizing antibodies and broad T cell responses against SARS-CoV-2: the S1-RBD-sFc fusion protein, six synthetic peptides (one universal peptide and five SARS-CoV-2-derived peptides), a proprietary CpG TLR-9 agonist at low concentration as an excipient, and aluminum phosphate adjuvant. Through immunogenicity studies in Guinea pigs and rats, we optimized the design of protein/peptide immunogens and selected an adjuvant system, yielding a vaccine that provides excellent S1-RBD binding and high neutralizing antibody responses, robust cellular responses, and a Th1-oriented response at low doses. In challenge studies, UB- 612 vaccination reduced viral load and prevented development of disease in mouse and non-human primate challenge models. With a Phase 1 trial completed, a Phase 2 trial ongoing in Taiwan, and additional trials planned to support global authorizations, UB-612 is a highly promising and differentiated vaccine candidate for prevention of SARS-CoV-2 infection and COVID-19 disease. SARS-CoV-2 virus, the causative agent of Coronavirus Disease 2019 (COVID-19), has spread globally since its origin in 2019, causing an unprecedented public health crisis that has resulted in greater than 4.7 million deaths worldwide. Many vaccines are under development to limit disease spread and reduce the number of cases, but additional candidates that promote a robust immune response are needed. Here, we describe a multitope protein-peptide vaccine platform that is unique among COVID-19 vaccines. The advantages of our approach are induction of both high levels of neutralizing antibodies as well as a Th/CTL response in the vaccinated host, which mimics the immune response that occurs after natural infection with SARS-CoV-2. We demonstrate that our vaccine is immunogenic and effective in preventing disease in several animal models, including AAV- hACE-2 transduced mice, and both rhesus and cynomolgus macaques. Importantly, no immunopathology was observed in the lungs of immunized animals, therefore showing that antibody-dependent enhancement (ADE) does not occur. Our study provides an additional, novel vaccine candidate for advancement in clinical trials to treat and prevent SARS-CoV-2 infection and COVID-19 disease.","version":"1.2","doi":"10.1101/2020.11.30.399154","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.21264054","pub_date":"2021-10-07","title":"Poor neutralization and rapid decay of antibodies to SARS-CoV-2 variants in vaccinated dialysis patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Patients on dialysis are at risk of severe course of SARS-CoV-2 infection. Understanding the neutralizing activity and coverage of SARS-CoV-2 variants of vaccine-elicited antibodies is required to guide prophylactic and therapeutic COVID-19 interventions in this frail population. By analyzing plasma samples from 130 hemodialysis and 13 peritoneal dialysis patients after two doses of BNT162b2 or mRNA-1273 vaccines, we found that 35% of the patients had low-level or undetectable IgG antibodies to SARS-CoV-2 Spike (S). Neutralizing antibodies against the vaccine-matched SARS-CoV-2 and Delta variant were low or undetectable in 49% and 77% of patients, respectively, and were further reduced against other emerging variants. The fraction of non-responding patients was higher in SARS-CoV-2-na\u00efve hemodialysis patients immunized with BNT162b2 (66%) than those immunized with mRNA-1273 (23%). The reduced neutralizing activity correlated with low antibody avidity. Patients followed up to 7 months after vaccination showed a rapid decay of the antibody response with an average 21- and 10-fold reduction of neutralizing antibodies to vaccine-matched SARS-CoV-2 and Delta variant, which increased the fraction of non-responders to 84% and 90%, respectively. These data indicate that dialysis patients should be prioritized for additional vaccination boosts. Nevertheless, their antibody response to SARS-CoV-2 must be continuously monitored to adopt the best prophylactic and therapeutic strategy.</jats:p>","version":null,"doi":"10.1101/2021.10.05.21264054","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.03.367516","pub_date":"2021-10-07","title":"The translational landscape of SARS-CoV-2 and infected cells","abstract":"SARS-CoV-2 utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2 infected model cell lines and primary airway cells grown at the air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We find that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy in comparison to HIV-1, suggesting utilization of distinct structural elements. In the highly permissive cell models, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokines, cytokines and interferon stimulated genes, many of these mRNAs were not translated efficiently. Impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development.","version":"1.3","doi":"10.1101/2020.11.03.367516","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439482","pub_date":"2021-10-07","title":"Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.","version":"1.2","doi":"10.1101/2021.04.13.439482","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.443053","pub_date":"2021-10-07","title":"A hybrid PDE\u2013ABM model for viral dynamics with application to SARS\u2013CoV\u20132 and influenza","abstract":"We propose a hybrid partial differential equation \u2013 agent-based (PDE\u2013ABM) model to describe the spatio-temporal viral dynamics in a cell population. The virus concentration is considered as a continuous variable and virus movement is modelled by diffusion, while changes in the states of cells (i.e. healthy, infected, dead) are represented by a stochastic agent-based model. The two subsystems are intertwined: the probability of an agent getting infected in the ABM depends on the local viral concentration, and the source term of viral production in the PDE is determined by the cells that are infected. We develop a computational tool that allows us to study the hybrid system and the generated spatial patterns in detail. We systematically compare the outputs with a classical ODE system of viral dynamics, and find that the ODE model is a good approximation only if the diffusion coefficient is large. We demonstrate that the model is able to predict SARS\u2013CoV\u20132 infection dynamics, and replicate the output of in vitro experiments. Applying the model to influenza as well, we can gain insight into why the outcomes of these two infections are different.","version":"1.2","doi":"10.1101/2021.05.07.443053","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.21264581","pub_date":"2021-10-07","title":"Comparison of MIS-C Related Myocarditis, Classic Viral Myocarditis, and COVID-19 Vaccine related Myocarditis in Children","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Although rare, myocarditis in the pediatric population is a disease process that carries significant morbidity and mortality. Prior to the SARS-CoV-2 related (COVID-19) pandemic, enteroviruses were the most common cause of classic myocarditis. However, since 2020, myocarditis linked to multisystem inflammatory syndrome in children (MIS-C) is now common. In recent months, myocarditis related to COVID-19 vaccines has also been described. This study aims to compare these three different types of myocarditis with regards to clinical presentation, course, and outcomes.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>In this retrospective cohort study, we included all patients &lt;21 years of age hospitalized at our institution with classic viral myocarditis from 2015-2019, MIS-C myocarditis from 3/2020-2/2021 and COVID-19 vaccine-related myocarditis from 5/2021-6/2021. We compared demographics, initial symptomatology, treatment, laboratory data, and echocardiogram findings.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Of 201 total participants, 43 patients had classic myocarditis, 149 had MIS-C myocarditis, and 9 had COVID-19 vaccine-related myocarditis. Peak troponin was highest in the classic myocarditis group, whereas the MIS-C myocarditis group had the highest recorded brain natriuretic peptide (BNP). There were significant differences in time to recovery of normal left ventricular ejection fraction (LVEF) for the three groups: nearly all patients with MIS-C myocarditis (n=139, 93%) and all patients with COVID-19 vaccine-related myocarditis (n=9, 100%) had normal LVEF at the time of discharge, but a lower proportion of the classic myocarditis group (n=30, 70%) had a normal LVEF at discharge (p&lt;0.001). Three months post-discharge, 18 of 40 children (45%) in the classic myocarditis group still required heart failure treatment, whereas only one of the MIS-C myocarditis patients and none of the COVID-19 vaccine-associated myocarditis patients did.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Compared to those with classic myocarditis, those with MIS-C myocarditis had more significant hematologic derangements and worse inflammation at presentation, but had better clinical outcomes, including rapid recovery of cardiac function. Patients with COVID-19 vaccine-related myocarditis had similar clinical presentation to patients with classic myocarditis, but their pattern of recovery was similar to those with MIS-C, with prompt resolution of symptoms and improvement of cardiac function. Long-term follow-up should focus on cardiac and non-cardiac consequences of myocarditis associated with COVID-19 illness and vaccination.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.10.05.21264581","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.05.463205","pub_date":"2021-10-06","title":"SARS-CoV-2 causes human BBB injury and neuroinflammation indirectly in a linked organ chip platform","abstract":"COVID-19 is a multi-system disease affecting many organs outside of the lungs, and patients generally develop varying degrees of neurological symptoms. Whereas, the pathogenesis underlying these neurological manifestations remains elusive. Although in vitro models and animal models are widely used in studies of SARS-CoV-2 infection, human organ models that can reflect the pathological alterations in a multi-organ context are still lacking. In this study, we propose a new strategy to probe the effects of SARS-CoV-2 on human brains in a linked alveolus-BBB organ chip platform. The new multi-organ platform allows to recapitulate the essential features of human alveolar-capillary barrier and blood-brain barrier in a microfluidic condition by co-culturing the organ-specific cells. The results reveal direct SARS-CoV-2 exposure has no obvious effects on BBB chip alone. While, infusion of endothelial medium from infected alveolus chips can cause BBB dysfunction and neuroinflammation on the linked chip platform, including brain endothelium disruption, glial cell activation and inflammatory cytokines release. These new findings suggest that SARS-CoV-2 could induce neuropathological alterations, which might not result from direct viral infection through hematogenous route, but rather likely from systemic inflammation following lung infection. This work provides a new strategy to study the virus-host interaction and neuropathology at an organ-organ context, which is not easily obtained by other in vitro models. This will facilitate to understand the neurological pathogenesis in SARS-CoV-2 and accelerate the development of new therapeutics. A linked human alveolus-BBB chip platform is established to explore the influences of SARS-CoV-2 on human brains in an organ-organ context. SARS-CoV-2 infection could induce BBB injury and neuroinflammation. The neuropathological changes are caused by SARS-CoV-2 indirectly, which might be mediated by systemic inflammation following lung infection, but probably not by direct viral neuroinvasion.","version":"1.1","doi":"10.1101/2021.10.05.463205","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.463282","pub_date":"2021-10-06","title":"SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury","abstract":"Robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) accounts for high viral transmissibility, yet whether neutralizing IgA antibodies can control it remains unknown. Here, we evaluated receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1 and B8-dIgA2 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparably potent neutralization against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viruses in lungs, pre-exposure intranasal B8-dIgA1 or B8-dIgA2 led to 81-fold more infectious viruses and severer damage in NT than placebo. Virus-bound B8-dIgA1 and B8-dIgA2 could engage CD209 as an alternative receptor for entry into ACE2-negative cells and allowed viral cell-to-cell transmission. Cryo-EM revealed B8 as a class II neutralizing antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Therefore, RBD-specific neutralizing dIgA engages an unexpected action for enhanced SARS-CoV-2 nasal infection and injury in Syrian hamsters.","version":"1.1","doi":"10.1101/2021.10.05.463282","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.463008","pub_date":"2021-10-06","title":"How does temperature affect the dynamics of SARS-CoV-2 M proteins? Insights from Molecular Dynamics Simulations","abstract":"Enveloped viruses, in general, have several transmembrane proteins and glycoproteins, which assist the virus in entry and attachment onto the host cells. These proteins also play a significant role in determining the shape and size of the newly formed virus particles. The lipid membrane and the embedded proteins affect each other in non-trivial ways during the course of the viral life cycle. Unravelling the nature of the protein-protein and protein-lipid interactions, under various environmental and physiological conditions, could therefore prove to be crucial in development of therapeutics. Here, we study the M protein of SARS-CoV-2 to understand the effect of temperature on the properties of the protein-membrane system. The membrane embedded dimeric M proteins were studied using atomistic and coarse-grained molecular dynamics simulations at temperatures ranging between 10 and 50 \u00b0C. While temperature induced fluctuations should be monotonic, we observe a steady rise in the protein dynamics up to 40 \u00b0C, beyond which it surprisingly reverts back to the low temperature behaviour. Detailed investigation reveals disordering of the membrane lipids in the presence of the protein, which induces additional curvature around the transmembrane region. Coarse-grained simulations indicate temperature dependent aggregation of M protein dimers. Our study clearly indicates that the dynamics of membrane lipids and integral M protein of SARS-CoV-2 enables it to better associate and aggregate only at a certain temperature range (i.e., ~30 to 40 \u00b0C). This can have important implications in the protein aggregation and subsequent viral budding/fission processes.","version":"1.1","doi":"10.1101/2021.10.05.463008","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.04.462902","pub_date":"2021-10-06","title":"SARS-CoV-2 PLpro whole human proteome cleavage prediction and enrichment/depletion analysis","abstract":"A novel coronavirus (SARS-CoV-2) has caused a pandemic that has killed millions of people, worldwide vaccination and herd immunity are still far away, and few therapeutics are approved by regulatory agencies for widespread use. The coronavirus 3-chymotrypsin-like protease (3CLpro) is a commonly investigated target in COVID-19, however less work has been directed toward the equally important papain-like protease (PLpro). PLpro is less characterized due to its fewer and more diverse cleavages in coronavirus proteomes and the assumption that it mainly modulates host pathways with its deubiquitinating activity. Here, I extend my previous work on 3CLpro human cleavage prediction and enrichment/depletion analysis to PLpro.[1] Using three sets of neural networks trained on different taxonomic ranks of dataset with a maximum of 463 different putative PLpro cleavages, Matthews correlation coefficients of 0.900, 0.948, and 0.966 were achieved for Coronaviridae, Betacoronavirus, and Sarbecovirus, respectively. I predict that more than 1,000 human proteins may be cleaved by PLpro depending on diversity of the training dataset and that many of these proteins are distinct from those previously predicted to be cleaved by 3CLpro. PLpro cleavages are similarly nonrandomly distributed and result in many annotations shared with 3CLpro cleavages including ubiquitination, poly(A) tail and 5\u2019 cap RNA binding proteins, helicases, and endogenous viral proteins. Combining PLpro with 3CLpro cleavage predictions, additional novel enrichment analysis was performed on known substrates of cleaved E3 ubiquitin ligases with results indicating that many pathways including viral RNA sensing are affected indirectly by E3 ligase cleavage independent of traditional PLpro deubiquitinating activity. As with 3CLpro, PLpro whole proteome cleavage prediction revealed many novel potential therapeutic targets against coronaviruses, although experimental verification is similarly required.","version":"1.1","doi":"10.1101/2021.10.04.462902","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.06.462907","pub_date":"2021-10-06","title":"A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding","abstract":"We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently-labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle - hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the non-inhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to a known inhibitor. Finally, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.","version":"1.1","doi":"10.1101/2021.10.06.462907","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.04.463106","pub_date":"2021-10-05","title":"A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry","abstract":"Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.","version":"1.1","doi":"10.1101/2021.10.04.463106","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.463212","pub_date":"2021-10-05","title":"TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques","abstract":"The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1-population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant\u00ae), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection. Multi-omic analyses of hyperacute SARS-CoV-2 infection in rhesus macaques identified two population of infiltrating macrophages, as the primary orchestrators of inflammation in the lower airway that can be successfully treated with baricitinib","version":"1.1","doi":"10.1101/2021.10.05.463212","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.03.462919","pub_date":"2021-10-05","title":"Oral and intranasal Ad5 SARS-CoV-2 vaccines decrease disease and viral transmission in a golden hamster model","abstract":"Transmission-blocking strategies that slow the spread of SARS-CoV-2 and protect against COVID-19 are needed. We have developed a shelf-stable, orally-delivered Ad5-vectored SARS-CoV-2 vaccine candidate that expresses the spike protein. Here we demonstrated that oral and intranasal SARS-CoV-2 vaccination of this candidate protected against disease in index hamsters, and decreased aerosol transmission to unvaccinated, na\u00efve hamsters. We confirmed that mucosally-vaccinated hamsters had robust antibody responses. We then induced a post-vaccination infection by inoculating vaccinated index hamsters with SARS-CoV-2. Oral and IN-vaccinated hamsters had decreased viral RNA and infectious virus in the nose and lungs and experienced less lung pathology compared to mock-vaccinated hamsters post challenge. Naive hamsters exposed in a unidirectional air flow chamber to mucosally-vaccinated, SARS-CoV-2-infected hamsters had lower nasal swab viral RNA and exhibited less clinical symptoms of disease than control animals. Our data demonstrate that oral immunization is a viable strategy to decrease SARS-CoV-2 disease and aerosol transmission.","version":"1.1","doi":"10.1101/2021.10.03.462919","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.03.462915","pub_date":"2021-10-05","title":"Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl","abstract":"The COVID-19 pandemic persists as a global health crisis for which curative treatment has been elusive. Development of effective and safe anti-SARS-CoV-2 therapies remains an urgent need. SARS-CoV-2 entry into cells requires specific host proteases including TMPRSS2 and Cathepsin L (Ctsl), but there has been no reported success in inhibiting host proteases for treatment of SARS-CoV-2 pathogenesis in vivo. Here we have developed a lung Ctsl mRNA-targeted, CRISPR/Cas13d-based nanoparticle therapy to curb fatal SARS-CoV-2 infection in a mouse model. We show that this nanotherapy can decrease lung Ctsl expression in normal mice efficiently, specifically, and safely. Importantly, this lung-selective Ctsl-targeted nanotherapy significantly extended the survival of lethally SARS-CoV-2 infected mice by decreasing lung virus burden, reducing expression of pro-inflammatory cytokines/chemokines, and diminishing the severity of pulmonary interstitial inflammation. Additional in vitro analyses demonstrated that Cas13d-mediated Ctsl knockdown inhibited infection mediated by the spike protein of SARS-CoV-1, SARS-CoV-2, and more importantly, the authentic SARS-CoV-2 B.1.617.2 Delta variant, regardless of TMPRSS2 expression status. Our results demonstrate the efficacy and safety of a lung-selective, Ctsl-targeted nanotherapy against infection by SARS-CoV-2 and likely other emerging coronaviruses, forming a basis for investigation of this approach in clinical trials.","version":"1.1","doi":"10.1101/2021.10.03.462915","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.05.463210","pub_date":"2021-10-05","title":"Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein after infection and/or vaccination","abstract":"Control of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal. The epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions we determined potential escape mutations by comparing antibody binding of peptides containing wildtype residues versus peptides containing a mutant residue. Individuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination. The finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge, they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level. This work was supported by NIH grants AI138709 (PI Overbaugh) and AI146028 (PI Matsen). Julie Overbaugh received support as the Endowed Chair for Graduate Education (FHCRC). The research of Frederick Matsen was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.","version":"1.1","doi":"10.1101/2021.10.05.463210","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.04.463028","pub_date":"2021-10-05","title":"B.1.617.3 SARS CoV-2 spike E156G/\u0394157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity","abstract":"SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/\u0394157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (Covishield\u2122)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/\u0394157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/\u0394157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/\u0394157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/\u0394157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.","version":"1.1","doi":"10.1101/2021.10.04.463028","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.04.463014","pub_date":"2021-10-05","title":"Genetic association of TMPRSS2 rs2070788 polymorphism with COVID-19 Case Fatality Rate among Indian populations","abstract":"SARS-CoV2, the causative agent for COVID-19, an ongoing pandemic, engages the ACE2 receptor to enter the host cell through S protein priming by a serine protease, TMPRSS2. Variation in the TMPRSS2 gene may account for the difference in population disease susceptibility. The haplotype-based genetic sharing and structure of TMPRSS2 among global populations have not been studied so far. Therefore, in the present work, we used this approach with a focus on South Asia to study the haplotypes and their sharing among various populations worldwide. We have used next-generation sequencing data of 393 individuals and analysed the TMPRSS2 gene. Our analysis of genetic relatedness for this gene showed a closer affinity of South Asians with the West Eurasian populations therefore, host disease susceptibility and severity particularly in the context of TMPRSS2 will be more akin to West Eurasian instead of East Eurasian. This is in contrast to our prior study on ACE2 gene which shows South Asian haplotypes have a strong affinity towards West Eurasians. Thus ACE2 and TMPRSS2 have an antagonistic genetic relatedness among South Asians. We have also tested the SNP\u2019s frequencies of this gene among various Indian state populations with respect to the case fatality rate. Interestingly, we found a significant positive association between the rs2070788 SNP (G Allele) and the case fatality rate in India. It has been shown that the GG genotype of rs2070788 allele tends to have a higher expression of TMPRSS2 in the lung compared to the AG and AA genotypes, thus it might play a vital part in determining differential disease vulnerability. We trust that this information will be useful in underscoring the role of the TMPRSS2 variant in COVID-19 susceptibility and using it as a biomarker may help to predict populations at risk.","version":"1.1","doi":"10.1101/2021.10.04.463014","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.04.463121","pub_date":"2021-10-05","title":"Longitudinal characterization of circulating neutrophils uncovers distinct phenotypes associated with disease severity in hospitalized COVID-19 patients","abstract":"Multiple studies have identified an association between neutrophils and COVID-19 disease severity; however, the mechanistic basis of this association remains incompletely understood. Here we collected 781 longitudinal blood samples from 306 hospitalized COVID-19+ patients, 78 COVID-19\u2212 acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death. We identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature. Severe disease was associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS). We found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response. Overall, our study demonstrates neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.","version":"1.1","doi":"10.1101/2021.10.04.463121","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.02.462863","pub_date":"2021-10-04","title":"Dynamic Allostery Highlights the Evolutionary Differences between the CoV-1 and CoV-2 Main Proteases","abstract":"The SARS-CoV-2 coronavirus has become one of the most immediate and widely-studied systems since its identification and subsequent global outbreak from 2019-2020. In an effort to understand the biophysical changes as a result of mutations, the mechanics of multiple different proteins within the SARS-CoV-2 virus have been studied and compared with SARS-CoV-1. Focusing on the main protease (mPro), we first explored the long range dynamic-relationship, particularly in cross-chain dynamics, using the Dynamic Coupling Index (DCI) to investigate the dynamic coupling between the catalytic site residues and the rest of the protein, both inter and intra chain for the CoV-1 and CoV-2 mPro. We found that there is significant cross-chain coupling between these active sites and distal residues in the CoV-2 mPro but it was missing in CoV-1. The enhanced long distance interactions, particularly between the two chains, suggest subsequently enhanced cooperativity for CoV-2. A further comparative analysis of the dynamic flexibility using the Dynamic Flexibility Index (DFI) between the CoV-1 and CoV-2 mPros shows that the inhibitor binding near active sites induces change in flexibility to a distal region of the protein, opposite in behavior between the two systems; this region becomes more flexible upon inhibitor binding in CoV-1 while it becomes less flexible in the CoV-2 mPro. Upon inspection, we show that, on average, the dynamic flexibility of the sites substituted from CoV-1 to CoV-2 changes significantly less than the average calculated across all residues within the structure, indicating that the differences in behaviors between the two systems is likely the result of allosteric influence, where the new substitutions in COV-2 induce flexibility and dynamical changes elsewhere in the structure. Here we have conducted a comparative analysis between the SARS-CoV-1 and SARS-CoV-2 mPro systems to shed mechanistic insight on the biophysical changes associated with the mutations between these two enzymes. Our work shows that the CoV-2 mPro system exhibits enhanced cross-chain communication between catalytic site residues and the rest of the structure. Further, both dynamic coupling and dynamic flexibility analyses indicates that, largely, the dynamic changes as evaluated by DCI and DFI occur at sites other than the mutation sites themselves, indicating that the functional differences between these two proteins are a result of dynamic allostery","version":"1.1","doi":"10.1101/2021.10.02.462863","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.03.462911","pub_date":"2021-10-04","title":"Unique Peptide Signatures Of SARS-CoV-2 Against Human Proteome Reveal Variants\u2019 Immune Escape And Infectiveness","abstract":"SARS-CoV-2 pandemic has emerged the necessity of the identification of sequences sites in viral proteome appropriate as antigenic sites and treatment targets. In the present study, we apply a novel approach for deciphering the virus-host organism interaction, by analyzing the Unique Peptides of the virus with a minimum amino acid sequence length defined as Core Unique Peptides (CrUPs) not of the virus per se, but against the entire proteome of the host organism. The result of this approach is the identification of the CrUPs of the virus itself, which do not appear in the host organism proteome. Thereby, we analyzed the SARS-CoV-2 proteome for identification of CrUPs against the Human Proteome and they are defined as C/H-CrUPs. We found that SARS-CoV-2 include 7.503 C/H-CrUPs, with the SPIKE_SARS2 being the protein with the highest density of C/H-CrUPs. Extensive analysis indicated that the P681R mutation produces new C/H-CrUPs around the R685 cleavage site, while the L452R mutation induces the loss of antigenicity of the NF9 peptide and the strong(er) binding of the virus to ACE2 receptor protein. The simultaneous existence of these mutations in variants like Delta results in the immune escape of the virus, its massive entrance into the host cell, a notable increase in virus formation, and its massive release and thus elevated infectivity.","version":"1.1","doi":"10.1101/2021.10.03.462911","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.01.452232","pub_date":"2021-10-04","title":"Characterization and Structural Prediction of ORF10, ORF7b, ORF7a, ORF6, Membrane Glycoprotein, and Envelope Protein in SARS-CoV-2 Bangladeshi Variant through Bioinformatics Approach","abstract":"The acute respiratory disease induced by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a global epidemic in just less than a year by the first half of 2020. The subsequent efficient human-to-human transmission of this virus eventually affected millions of people worldwide. The most devastating thing is that the infection rate is continuously uprising and resulting in significant mortality especially among the older age population and those with health co-morbidities. This enveloped, positive-sense RNA virus is chiefly responsible for the infection of the upper respiratory system. The virulence of the SARS-CoV-2 is mostly regulated by its proteins like entry to the host cell through fusion mechanism, fusion of infected cells with neighboring uninfected cells to spread the virus, inhibition of host gene expression, cellular differentiation, apoptosis, mitochondrial biogenesis, etc. But very little is known about the protein structures and functionalities. Therefore, the main purpose of this study is to learn more about these proteins through bioinformatics approaches. In this study, ORF10, ORF7b, ORF7a, ORF6, membrane glycoprotein, and envelope protein have been selected from a Bangladeshi Corona-virus strain G039392 and a number of bioinformatics tools (MEGA-X-V10.1.7, PONDR\u00ae, ProtScale, ProtParam, SCRIBER, NetSurfP v2.0, IntFOLD, UCSF Chimera, and PyMol) and strategies were implemented for multiple sequence alignment and phylogeny analysis with 9 different variants, predicting hydropathicity, amino acid compositions, protein-binding propensity, protein disorders, 2D and 3D protein modeling. Selected proteins were characterized as highly flexible, structurally and electrostatically extremely stable, ordered, biologically active, hydrophobic, and closely related to the proteins of different variants. This detailed information regarding the characterization and structure of proteins of SARS-CoV-2 Bangladeshi variant was performed for the first time ever to unveil the deep mechanism behind the virulence features and also, this robust appraisal paves the future way for molecular docking, vaccine development targeting these characterized proteins.","version":"1.1","doi":"10.1101/2021.10.01.452232","journal":"bioRxiv","score":null},{"id":"10.1101/2021.10.01.462840","pub_date":"2021-10-04","title":"Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern","abstract":"Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to concentrate the antibody response to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and two variants of concern (VOC), B.1.351 and B.1.617.2. These findings demonstrate that immunogens built on structure selection can focus the response to conserved sites of vulnerability shared between wildtype virus and VOCs and induce neutralizing antibodies across variants.","version":"1.1","doi":"10.1101/2021.10.01.462840","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.21264339","pub_date":"2021-10-03","title":"Non-Markovian modelling highlights the importance of age structure on Covid-19 epidemiological dynamics","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The Covid-19 pandemic outbreak was followed by a huge amount of modelling studies in order to rapidly gain insights to implement the best public health policies. Most of these compartmental models involved ordinary differential equations (ODEs) systems. Such a formalism implicitly assumes that the time spent in each compartment does not depend on the time already spent in it, which is at odds with the clinical data. To overcome this \u201cmemoryless\u201d issue, a widely used solution is to increase and chain the number of compartments of a unique reality (\n                  <jats:italic>e</jats:italic>\n                  .\n                  <jats:italic>g</jats:italic>\n                  . have infected individual move between several compartments). This allows for greater heterogeneity and thus be closer to the observed situation, but also tends to make the whole model more difficult to apprehend and parameterize. We develop a non-Markovian alternative formalism based on partial differential equations (PDEs) instead of ODEs, which, by construction, provides a memory structure for each compartment thereby allowing us to limit the number of compartments. We apply our model to the French 2021 SARS-CoV-2 epidemic and, while accounting for vaccine-induced and natural immunity, we analyse and determine the major components that contributed to the Covid-19 hospital admissions. The results indicate that the observed vaccination rate alone is not enough to control the epidemic, and a global sensitivity analysis highlights a huge uncertainty attributable to the age-structured contact matrix. Our study shows the flexibility and robustness of PDE formalism to capture national COVID-19 dynamics and opens perspectives to study medium or long-term scenarios involving immune waning or virus evolution.\n                </jats:p>","version":null,"doi":"10.1101/2021.09.30.21264339","journal":"medRxiv","score":null},{"id":"10.1101/2021.10.01.462460","pub_date":"2021-10-01","title":"ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression","abstract":"COVID-19 caused by the SARS-CoV-2 virus remains a threat to global health. The disease severity is mediated by cell death and inflammation, which regulate both the antiviral and the pathological innate immune responses. ZBP1, an interferon-induced cytosolic nucleic acid sensor, facilitates antiviral responses via RIPK3. Although ZBP1-mediated cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway that depends on ubiquitination and RIPK3\u2019s scaffolding ability independently of cell death. In human cells, ZBP1 associates with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. RIPK1 and ZBP1 are ubiquitinated to promote TAK1- and IKK-mediated inflammatory signaling. Additionally, RIPK1 recruits the p43/41-caspase-8-p43-FLIP heterodimer to suppress RIPK3 kinase activity, which otherwise promotes inflammatory signaling in a kinase activity-dependent manner. Lastly, we show that ZBP1 contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK1-RIPK3-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspase-8. Our results suggest the ZBP1 pathway contributes to inflammation in response to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.10.01.462460","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.462687","pub_date":"2021-10-01","title":"Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern","abstract":"SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha\u2019s small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic. The global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.","version":"1.1","doi":"10.1101/2021.09.30.462687","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460613","pub_date":"2021-10-01","title":"Copper(II) Gluconate Boosts the Anti-SARS-CoV-2 Effect of Disulfiram In Vitro","abstract":"Disulfiram is a 70-year-old anti-alcoholism drug, while copper(II) gluconate (Cu(Glu)2) is a commonly used food additive or copper supplement. Here we disclose that the combination of disulfiram and copper(II) gluconate drastically enhances the anti-SARS-CoV-2 activity at the cellular level as compared to disulfiram or copper(II) gluconate alone. A 1:1 mixture of disulfiram and copper(II) gluconate shows an EC50 value of 154 nM against SARS-CoV-2 at the cellular level, much lower than the 17.45 \u03bcM reported for disulfiram alone. Notably, previous clinical trials have shown that a combination of 250 mg disulfiram (0.843 mmol) and 8 mg copper(II) gluconate (0.0176 mmol) oral capsules per day is well tolerated (NCT03034135, NCT00742911). A preliminary mechanism is proposed to rationalize the observed promotional effect.","version":"1.2","doi":"10.1101/2021.09.17.460613","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.462459","pub_date":"2021-10-01","title":"Structural mapping of antibody landscapes to human betacoronavirus spike proteins","abstract":"Preexisting immunity against seasonal coronaviruses (CoV) represents an important variable in predicting antibody responses and disease severity to Severe Acute Respiratory Syndrome CoV-2 (SARS-2) infections. We used electron microscopy based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against \u03b2-CoV spike proteins in sera from healthy donors (HDs) or SARS-2 convalescent donors (CDs). We observed that most HDs possessed antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the CDs showed reactivity across all human \u03b2-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by antibodies in preexisting and convalescent serum. Our studies provide an antigenic landscape to \u03b2-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-2 -infected individuals. We present the epitope mapping of polyclonal antibodies against beta-coronavirus spike proteins in human sera.","version":"1.1","doi":"10.1101/2021.09.30.462459","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.462514","pub_date":"2021-9-30","title":"Ad26.COV2.S Prevents SARS-CoV-2 Induced Pathways of Inflammation and Thrombosis in Hamsters","abstract":"Syrian golden hamsters exhibit features of severe disease after SARS-CoV-2 challenge and are therefore useful models of COVID-19 pathogenesis and prevention with vaccines. Recent studies have shown that SARS-CoV-2 infection stimulates type I interferon, myeloid, and inflammatory signatures similar to human disease, and that weight loss can be prevented with vaccines. However, the impact of vaccination on transcriptional programs associated with COVID-19 pathogenesis and protective adaptive immune responses is unknown. Here we show that SARS-CoV-2 challenge in hamsters stimulates antiviral, myeloid, and inflammatory programs as well as signatures of complement and thrombosis associated with human COVID-19. Notably, single dose immunization with Ad26.COV2.S, an adenovirus serotype 26 vector (Ad26)-based vaccine expressing a stabilized SARS-CoV-2 spike protein, prevents the upregulation of these pathways such that the gene expression profiles of vaccinated hamsters are comparable to uninfected animals. Finally, we show that Ad26.COV2.S vaccination induces T and B cell signatures that correlate with binding and neutralizing antibody responses. These data provide further insights into the mechanisms of Ad26.COV2.S based protection against severe COVID-19 in hamsters. In this study, we show that vaccination with Ad26.COV2.S protected SARS-CoV-2 challenged hamsters from developing severe COVID-19 disease by attenuating excessive proinflammatory responses, such as IL-6 and IL-1, macrophages and neutrophils signaling. Ad26 vaccination also prevented the upregulation of pathways associated with thrombosis such coagulation and clotting cascades associated with infection, and the transcriptomic profiles of vaccinated animals were largely comparable to control uninfected hamsters. In contrast, SARS-CoV-2 challenged unvaccinated hamsters showed significant increase of these proinflammatory and prothrombotic pathways and significant weight loss compared to vaccinated hamsters.","version":"1.1","doi":"10.1101/2021.09.30.462514","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.462449","pub_date":"2021-9-30","title":"Pyronaridine Protects Against SARS-CoV-2 in Mouse","abstract":"There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 \u00b5M) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC50 of 2.4 \u00b5M). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19. There is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg and SARS-CoV-2 has now statistically significantly reduced the viral load in mice along with IL-6, TNF-\u03b1, and IFN-\u03b2 ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically.","version":"1.1","doi":"10.1101/2021.09.30.462449","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.462156","pub_date":"2021-9-30","title":"Modulation of immunosuppressant drug treatment to improve SARS-CoV-2 vaccine efficacy in mice","abstract":"The COVID-19 pandemic dramatically demonstrated the need for improved vaccination strategies and therapeutic responses to combat infectious diseases. However, the efficacy of vaccines has not yet been demonstrated in combination with commonly used immunosuppressive drug regimens. We sought to determine how common pharmaceutical drugs used in autoimmune disorders can alter immune responses to the SARS-CoV-2 spike protein vaccination. We treated mice with five immunosuppressant drugs (cyclophosphamide, leflunomide, methotrexate, methylprednisolone, and mycophenolate mofetil), each with various mechanisms of action prior to and following immunization with SARS-CoV-2 spike protein. We assessed the functionality of antibody responses to spike protein and compared immune cell populations in mice that received no treatment with those that received continuous or temporarily suspended immune suppressive therapy. All tested immunosuppressants significantly reduced the antibody titers in serum and functional antibody response against SARS-CoV-2 spike protein in immunized mice. Temporarily halting selected immunosuppressants (methylprednisolone and methotrexate, but not cyclophosphamide) improved antibody responses significantly. Through proof-of-principle experiments utilizing a mouse model, we demonstrated that immune suppression in autoimmune disorders through pharmaceutical treatments may impair vaccine response to SARS-CoV-2, and temporary suspension of immunosuppressant treatment may be necessary to mount an effective antibody vaccine response. This work provides feasibility for future clinical assessment of the impact of immunosuppressants on vaccine efficacy in humans. Immunosuppressant regimens are widely used as therapies for a variety of diseases, including autoimmune, inflammatory, and cancer. However, immunosuppressants can impair critical immune responses to vaccination. The impact of standard immunosuppressant use on the critical, developing SARS-CoV-2 vaccination strategies has not been well-described. In this study, we use a mouse model to determine how different immunosuppressant drugs that act through different mechanisms can impair the antibody response to SARS-CoV-2 spike protein, and how modulating these drug regimens may restore antibody levels and function.","version":"1.1","doi":"10.1101/2021.09.28.462156","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.462420","pub_date":"2021-9-30","title":"SARS-CoV-2 Beta variant infection elicits potent lineage-specific and cross-reactive antibodies","abstract":"SARS-CoV-2 Beta variant of concern (VOC) resists neutralization by major classes of antibodies from non-VOC COVID-19 patients and vaccinated individuals. Here, serum of Beta variant infected patients revealed reduced cross-neutralization of non-VOC virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of novel VOC-specific clonotypes and accommodation of VOC-defining amino acids into a major non-VOC antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with non-VOC-elicited antibodies, including a public VH1-58 clonotype targeting the RBD ridge independent of VOC mutations. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift with implications for design of next-generation vaccines and therapeutics. SARS-CoV-2 Beta variant elicits lineage-specific antibodies and antibodies with neutralizing breadth against wild-type virus and VOCs.","version":"1.1","doi":"10.1101/2021.09.30.462420","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.29.21264272","pub_date":"2021-09-30","title":"Associations between SARS-CoV-2 variants and risk of COVID-19 hospitalization among confirmed cases in Washington State: a retrospective cohort study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The COVID-19 pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with seven SARS-CoV-2 variants.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Our study includes individuals with positive SARS-CoV-2 RT-PCR in the Washington Disease Reporting System with available viral genome data, from December 1, 2020 to January 14, 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>58,848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95%CI 2.40-4.26), Beta (HR 2.85, 95%CI 1.56-5.23), Delta (HR 2.28 95%CI 1.56-3.34) or Alpha (HR 1.64, 95%CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95%CI 0.56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Summary</jats:title>\n                  <jats:p>Hospitalization risk following infection with SARS-CoV-2 variant remains unclear. We find a higher hospitalization risk in cases infected with Alpha, Beta, Gamma, and Delta, but not Omicron, with vaccination lowering risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.29.21264272","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.11.443443","pub_date":"2021-9-30","title":"SARS-CoV-2 Beta Variant Substitutions Alter Spike Glycoprotein Receptor Binding Domain Structure and Stability","abstract":"The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic has visited a terrible cost on the world in the forms of disease, death, and economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 have seemingly brought within reach the end of the pandemic. However, the virus has acquired mutations; and emerging variants of concern (VOC) are more infectious and reduce the efficacy of existing vaccines. While promising efforts to combat these variants are underway, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain of three amino-acid substitutions found in several variants of concern, including alpha (B.1.1.7), beta (B.1.351), and gamma (P.1). We found that these substitutions alter the RBD structure, stability, and ability to bind to ACE2, in such a way as to possibly have opposing and compensatory effects. These findings provide new insights into how these VOC may have been selected for infectivity while maintaining the structure and stability of the receptor binding domain.","version":"1.2","doi":"10.1101/2021.05.11.443443","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.462271","pub_date":"2021-9-30","title":"Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)","abstract":"Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus. Human species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.","version":"1.1","doi":"10.1101/2021.09.28.462271","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.30.462488","pub_date":"2021-9-30","title":"Durability of immune responses to the BNT162b2 mRNA vaccine","abstract":"The development of the highly efficacious mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology. However, reports of waning vaccine efficacy, coupled with the emergence of variants of concern that are resistant to antibody neutralization, have raised concerns about the potential lack of durability of immunity to vaccination. We recently reported findings from a comprehensive analysis of innate and adaptive immune responses in 56 healthy volunteers who received two doses of the BNT162b2 vaccination. Here, we analyzed antibody responses to the homologous Wu strain as well as several variants of concern, including the emerging Mu (B.1.621) variant, and T cell responses in a subset of these volunteers at six months (day 210 post-primary vaccination) after the second dose. Our data demonstrate a substantial waning of antibody responses and T cell immunity to SARS-CoV-2 and its variants, at 6 months following the second immunization with the BNT162b2 vaccine. Notably, a significant proportion of vaccinees have neutralizing titers below the detection limit, and suggest a 3rd booster immunization might be warranted to enhance the antibody titers and T cell responses.","version":"1.1","doi":"10.1101/2021.09.30.462488","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.09.443341","pub_date":"2021-9-30","title":"Synthetic Nanobody-Functionalized Nanoparticles for Accelerated Development of Rapid, Accessible Detection of Viral Antigens","abstract":"Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays to be widely implemented at a high frequency. Here we present a generalizable assay platform, nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED), demonstrated in the detection of Ebola and COVID-19 antigens. To efficiently generate high-quality affinity reagents, synthetic nanobody co-binders and mono-binders with high affinity, specificity, and stability were selected by phage display screening of a vastly diverse, rationally randomized combinatorial library, bacterially expressed and site-specifically conjugated to gold nanoparticles (AuNPs) as multivalent in-solution sensors. Without requiring fluorescent labelling, washing, or enzymatic amplification, these AuNPs reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or simple electronic circuitry. With nanobodies against an Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (limit of detection of \u223c10 pg/mL for sGP and \u223c40 pg/mL for RBD in diluted human serum), a high specificity, and a large dynamic range (\u223c7 logs). Unlike conventional assays where slow mass transport for surface binding limits the assay time, Nano2RED features fast antigen diffusion at micrometer scale, and can be accelerated to deliver results within a few minutes. The rapid detection, low material cost (estimated < $0.01 per test), inexpensive and portable readout system (< $5 and < 100 cm3), and digital data output, make Nano2RED particularly suitable for screening of patient samples with simplified operation and accelerated data transmission. Our method is widely applicable for prototyping diagnostic assays for other antigens from new emerging viruses.","version":"1.2","doi":"10.1101/2021.05.09.443341","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.462074","pub_date":"2021-9-29","title":"A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in rhesus macaques","abstract":"Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention, post-exposure prophylaxis, or therapy. However, the titer of neutralizing antibodies required for protection against SARS-CoV-2 infection remains poorly characterized. We previously described two potently neutralizing mAbs COV2-2130 and COV2-2381 targeting non-overlapping epitopes on the receptor-binding domain of SARS-CoV-2 spike protein. Here, we engineered the Fc-region of these mAbs with mutations to extend their persistence in humans and reduce interactions with Fc gamma receptors. Passive transfer of individual or combinations of the two antibodies (designated ADM03820) given prophylactically by intravenous or intramuscular route conferred virological protection in a non-human primate (NHP) model of SARS-CoV-2 infection, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined 6,000 as a protective serum neutralizing antibody titer in NHPs against infection for passively transferred human mAbs that acted by direct viral neutralization, which corresponded to a concentration of 20 \u03bcg/mL of circulating mAb.","version":"1.1","doi":"10.1101/2021.09.27.462074","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.29.462202","pub_date":"2021-9-29","title":"A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV2 at the immune-epithelial interface","abstract":"Infection by SARS-CoV2 provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled by in vitro co-culture the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV2-specific inflammatory gene cluster distinct from that seen in influenza-A or Ebola virus-infected co-cultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV2 proteins (Spike and some non-structural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age-dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV2 induces in epithelia.","version":"1.1","doi":"10.1101/2021.09.29.462202","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.462270","pub_date":"2021-9-29","title":"Data-driven approaches for genetic characterization of SARS-CoV-2 lineages","abstract":"The genome of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has been sequenced at an unprecedented scale, leading to a tremendous amount of viral genome sequencing data. To understand the evolution of this virus in humans, and to assist in tracing infection pathways and designing preventive strategies, we present a set of computational tools that span phylogenomics, population genetics and machine learning approaches. To illustrate the utility of this toolbox, we detail an in depth analysis of the genetic diversity of SARS-CoV-2 in first year of the COVID-19 pandemic, using 329,854 high-quality consensus sequences published in the GISAID database during the pre-vaccination phase. We demonstrate that, compared to standard phylogenetic approaches, haplotype networks can be computed efficiently on much larger datasets, enabling real-time analyses. Furthermore, time series change of Tajima\u2019s D provides a powerful metric of population expansion. Unsupervised learning techniques further highlight key steps in variant detection and facilitate the study of the role of this genomic variation in the context of SARS-CoV-2 infection, with Multiscale PHATE methodology identifying fine-scale structure in the SARS-CoV-2 genetic data that underlies the emergence of key lineages. The computational framework presented here is useful for real-time genomic surveillance of SARS-CoV-2 and could be applied to any pathogen that threatens the health of worldwide populations of humans and other organisms.","version":"1.1","doi":"10.1101/2021.09.28.462270","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.29.462326","pub_date":"2021-9-29","title":"Metabolic snapshot of plasma samples reveals new pathways implicated in SARS-CoV-2 pathogenesis","abstract":"Despite of the scientific and human efforts to understand COVID-19, there are questions still unanswered. Variations in the metabolic reaction to SARS-CoV-2 infection could explain the striking differences in the susceptibility to infection and the risk of severe disease. Here, we used untargeted metabolomics to examine novel metabolic pathways related to SARS-CoV-2 susceptibility and COVID-19 clinical severity using capillary electrophoresis coupled to a time-of-flight mass spectrometer (CE-TOF-MS) in plasma samples. We included 27 patients with confirmed COVID-19 early after symptom onset who were prospectively followed and 29 healthcare workers heavily exposed to SARS-CoV-2 but with low susceptibility to infection (\u2018nonsusceptible\u2019). We found that the metabolite profile was predictive of the study group. We identified a total of 55 metabolites as biomarkers of SARS-CoV-2 susceptibility or COVID-19 clinical severity. We report the discovery of new plasma biomarkers for COVID-19 that provide mechanistic explanations for the clinical consequences of SARS-CoV-2, including mitochondrial and liver dysfunction as a consequence of hypoxemia (citrulline, citrate, and BAIBA), energy production and amino acid catabolism (L-glycine, L-alanine, L-serine, L-proline, L-aspartic acid and L-histidine), endothelial dysfunction and thrombosis (citrulline, L-ADMA, 2-AB, and Neu5Ac), and we found interconnections between these pathways. In summary, in this first report of the metabolomic profile of individuals with severe COVID-19 and SARS-CoV-2 susceptibility by CE-MS, we define several metabolic pathways implicated in SARS-CoV-2 susceptibility and COVID-19 clinical progression that could be developed as biomarkers of COVID-19.","version":"1.1","doi":"10.1101/2021.09.29.462326","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.29.462283","pub_date":"2021-9-29","title":"In silico screening of TMPRSS2 SNPs that affect its binding with SARS-CoV2 spike protein and directly involved in the interaction affinity changes","abstract":"In this paper, we used in silico analysis to shed light on the possible interaction between TMPRSS2 and SARS-CoV2 spike (S) protein by examining the role of TMPRSS2 single nucleotide polymorphisms (SNPs) in relation with susceptibility and inter-individual variability of SARS-CoV2 infection. First, we used molecular docking of human TMPRSS2 protein to predict the binding site of TMPRSS2, especially the TMPRSS2 link loops, in order to assess the effect TMPRSS2 SNPs. The latter lead to missense variants on the interaction between TMPRSS2 and SARS-CoV2 S protein. In a second step, we further refine our analysis by performing a structure-function analysis of the complexes using PyMol software, and finally by MD simulations to validate the as-obtained results. Our findings show that 17 SNPs among the 692 natural TMPRSS2 coding variants are in positions to influence the binding of TMPRSS2 with the viral S protein. All of them give more important interaction energy as assessed by docking. Among the 17 SNPs, four missense variants E389A, K392Q, T393S and Q438E lead to \u201cdirectly increasing\u201d the interaction affinity and 2 missense variants R470I and Y416C cause it \u201cdirectly decreasing\u201d. The R470I and Y416C present in African and American population, respectively. While the other 4 SNP variants (E389A; K392Q; T393S and Q438E) are present only in the European population, which could link the viral infection susceptibility to demographic, geographic and genetic factors.","version":"1.1","doi":"10.1101/2021.09.29.462283","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.462109","pub_date":"2021-9-29","title":"Use of eVLP-based vaccine candidates to broaden immunity against SARS-CoV-2 variants","abstract":"Rapid emergence of SARS-CoV-2 variants is a constant threat and a major hurdle to reach heard immunity. We produced VBI-2905a, an enveloped virus-like particle (eVLP)-based vaccine candidate expressing prefusion spike protein from the Beta variant that contains several escape mutations. VBI-2905a protected hamsters against infection with a Beta variant virus and induced high levels of neutralizing antibodies against Beta RBD. In a heterologous vaccination regimen, a single injection of VBI-2905a in animals previously immunized with VBI-2902, a vaccine candidate expressing S from ancestral SARS-CoV-2, hamsters were equally protected against Beta variant infection. As an alternate strategy to broaden immunity, we produced a trivalent vaccine expressing the prefusion spike protein from SARS-CoV-2 together with unmodifed S from SARS-CoV-1 and MERS-CoV. Relative to immunity induced against the ancestral strain, the trivalent vaccine VBI-2901a induced higher and more consistent antibody binding and neutralizing responses against a panel of variants including Beta, Delta, Kappa, and Lambda, with evidence for broadening of immunity rather than just boosting cross-reactive antibodies.","version":"1.1","doi":"10.1101/2021.09.28.462109","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.21264262","pub_date":"2021-09-29","title":"No Significant Difference in Viral Load Between Vaccinated and Unvaccinated, Asymptomatic and Symptomatic Groups When Infected with SARS-CoV-2 Delta Variant","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>We found no significant difference in cycle threshold values between vaccinated and unvaccinated, asymptomatic and symptomatic groups infected with SARS-CoV-2 Delta. Given the substantial proportion of asymptomatic vaccine breakthrough cases with high viral levels, interventions, including masking and testing, should be considered for all in settings with elevated COVID-19 transmission.</jats:p>","version":null,"doi":"10.1101/2021.09.28.21264262","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.29.462373","pub_date":"2021-9-29","title":"SARS-CoV-2 variants of concern infect the respiratory tract and induce inflammatory response in wild-type laboratory mice","abstract":"The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern poses a major threat to the public health due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage or emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared to the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-\u03b1, IL-1\u03b2) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.09.29.462373","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.29.462406","pub_date":"2021-9-29","title":"Full genome Nobecovirus sequences from Malagasy fruit bats define a unique evolutionary history for this coronavirus clade","abstract":"Bats are natural reservoirs for both Alpha- and Betacoronaviruses and the hypothesized original hosts of five of seven known zoonotic coronaviruses. To date, the vast majority of bat coronavirus research has been concentrated in Asia, though coronaviruses are globally distributed; indeed, SARS-CoV and SARS-CoV-2-related Betacoronaviruses in the subgenus Sarbecovirus have been identified circulating in Rhinolophid bats in both Africa and Europe, despite the relative dearth of surveillance in these regions. As part of a long-term study examining the dynamics of potentially zoonotic viruses in three species of endemic Madagascar fruit bat (Pteropus rufus, Eidolon dupreanum, Rousettus madagascariensis), we carried out metagenomic Next Generation Sequencing (mNGS) on urine, throat, and fecal samples obtained from wild-caught individuals. We report detection of RNA derived from Betacoronavirus subgenus Nobecovirus in fecal samples from all three species and describe full genome sequences of novel Nobecoviruses in P. rufus and R. madagascariensis. Phylogenetic analysis indicates the existence of five distinct Nobecovirus clades, one of which is defined by the highly divergent sequence reported here from P. rufus bats. Madagascar Nobecoviruses derived from P. rufus and R. madagascariensis demonstrate, respectively, Asian and African phylogeographic origins, mirroring those of their fruit bat hosts. Bootscan recombination analysis indicates significant selection has taken place in the spike, nucleocapsid, and NS7 accessory protein regions of the genome for viruses derived from both bat hosts. Madagascar offers a unique phylogeographic nexus of bats and viruses with both Asian and African phylogeographic origins, providing opportunities for unprecedented mixing of viral groups and, potentially, recombination. As fruit bats are handled and consumed widely across Madagascar for subsistence, understanding the landscape of potentially zoonotic coronavirus circulation is essential for mitigation of future zoonotic threats.","version":"1.1","doi":"10.1101/2021.09.29.462406","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.461862","pub_date":"2021-9-28","title":"ChAdOx1 nCoV-19 (AZD1222) vaccine elicits monoclonal antibodies with potent cross-neutralizing activity against SARS-CoV-2 viral variants","abstract":"Although the antibody response to COVID-19 vaccination has been studied extensively at the polyclonal level using immune sera, little has been reported on the antibody response at the monoclonal level. Here we isolate a panel of 44 anti-SARS-CoV-2 monoclonal antibodies (mAbs) from an individual who received two doses of the ChAdOx1 nCoV-19 (AZD1222) vaccine at a 12-week interval. We show that despite a relatively low serum neutralization titre, mAbs with potent neutralizing activity against the current SARS-CoV-2 variants of concern (B.1.1.7, P.1, B.1.351 and B.1.617.2) were obtained. The vaccine elicited neutralizing mAbs form 8 distinct competition groups and bind epitopes overlapping with neutralizing mAbs elicited following SARS-CoV-2 infection. AZD1222 elicited mAbs are more mutated than mAbs isolated from convalescent donors 1-2 months post infection. Spike reactive IgG+ B cells were still detectable 9-months post boost. These findings give molecular insights into AZD1222 elicited antibody response.","version":"1.1","doi":"10.1101/2021.09.27.461862","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.461930","pub_date":"2021-9-28","title":"Milk casein prevents inactivation effect of black tea galloylated theaflavins on SARS-CoV-2 in vitro","abstract":"Repeated emergence of highly contagious and potentially immune-evading variant SARS-CoV-2 is posing global health and socioeconomical threats. For suppression of the spread of the virus infection among people, a procedure to inactivate virus in saliva may be useful, because saliva of infected persons is the major origin of droplets and aerosols that mediate viral transmission to nearby persons. We previously reported that SARS-CoV-2 is rapidly and remarkably inactivated by treatment in vitro with tea including green tea, roasted green tea, oolong tea and black tea. Tea catechin-derived compounds including theaflavins (TFs) with (a) galloyl moiety(ies) showed this activity. Although black tea is popularly consumed worldwide, a lot of people consume it with sugar, milk, lemon juice, and so on. But it has not been determined whether these ingredients may influence the inactivation effect of black tea against SARS-CoV-2. Moreover, it has not been revealed whether black tea is capable of inactivating variant viruses such as delta variant. Here we examined the effect of black tea on some variants in the presence or absence of sugar, milk, and lemon juice in vitro. Black tea and galloylated TFs remarkably inactivated alpha, gamma, delta and kappa variants. Intriguingly, an addition of milk but not sugar and lemon juice totally prevented black tea from inactivating alpha and delta variant viruses. The suppressive effect was also exerted by milk casein. These results suggest the possibility that intake of black tea without milk by infected persons may result in inactivation of the virus in saliva and attenuation of spread of SARS-CoV-2 to nearby persons through droplets. Clinical studies are required to investigate this possibility.","version":"1.1","doi":"10.1101/2021.09.27.461930","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.28.461924","pub_date":"2021-9-28","title":"Nasopharyngeal Microbiota as an early severity biomarker in COVID-19 hospitalised patients: a retrospective cohort study in a Mediterranean area","abstract":"There is mounting evidence suggesting that the microbiome composition could be different in COVID-19 patients. However, the relationship between microbiota and COVID-19 severity progression is still being assessed. This study aimed to analyse the diversity and taxonomic composition of the nasopharyngeal microbiota, to determine its association with COVID-19 clinical outcome. Samples came from a retrospective cohort of adult patients with COVID-19, hospitalised in a tertiary centre. To study the nasopharyngeal microbiota, we utilized 16S rRNA sequencing. Raw sequences were processed by QIIME2. The associations between the microbiota, invasive mechanical ventilation (IMV), and all-cause mortality were analysed by multiple logistic regression (OR; 95%CI), adjusted for age, gender, and comorbidity. 177 patients were included: median age 68.0 years, 57.6% males, 59.3% had a Charlson comorbidity index \u22653, and 89.2% with pneumonia. The microbiota \u03b1 diversity indexes were lower in patients with a fatal outcome, and this association persisted after adjustment for the main confounders; whereas the \u03b2 diversity analysis showed a significant clustering, grouping the patients with a fatal outcome. After multivariate adjustment, the presence of Selenomonas spp., Filifactor spp., Actinobacillus spp., or Chroococcidiopsis spp., was associated with a reduced risk of IMV (adjusted OR 0.06[95%CI 0.01\u20130.0.47], p = 0.007). The microbiota diversity and taxonomic composition are related to COVID-19 severity. Higher diversity and the presence of certain genera in the nasopharyngeal microbiota seem to be early biomarkers of a favourable clinical evolution in hospitalised patients with moderate to severe SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2021.09.28.461924","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.462006","pub_date":"2021-9-28","title":"The BNT162b2 mRNA vaccine induces polyfunctional T cell responses with features of longevity","abstract":"Vaccination against SARS-CoV-2 infection has shown to be effective in preventing hospitalization for severe COVID-19. However, multiple reports of break-through infections and of waning antibody titers have raised concerns on the durability of the vaccine, and current discussions on vaccination strategies are centered on evaluating the opportunity of a third dose administration. Here, we monitored T cell responses to the Spike protein of SARS-CoV-2 in 71 healthy donors vaccinated with the Pfizer\u2013BioNTech mRNA vaccine (BNT162b2) for up to 6 months after vaccination. We find that vaccination induces the development of a sustained anti-viral memory T cell response which includes both the CD4+ and the CD8+ lymphocyte subsets. These lymphocytes display markers of polyfunctionality, are fit for interaction with cognate cells, show features of memory stemness, and survive in significant numbers the physiological contraction of the immune response. Collectively, this data shows that vaccination with BNT162b2 elicits an immunologically competent and potentially long-lived SARS-CoV-2-specific T cell population. Understanding the immune responses to BNT162b2 provides insights on the immunological basis of the clinical efficacy of the current vaccination campaign and may instruct future vaccination strategies.","version":"1.1","doi":"10.1101/2021.09.27.462006","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.27.461855","pub_date":"2021-9-28","title":"Mutational Signatures as Sensors of Environmental Exposures: Role of Smoking in COVID-19 Vulnerabilities","abstract":"Environmental exposures such as smoking are widely recognized risk factors in the emergence of lung diseases including lung cancer and acute respiratory distress syndrome (ARDS). However, the strength of environmental exposures is difficult to measure, making it challenging to understand their impacts. On the other hand, some COVID-19 patients develop ARDS in an unfavorable disease progression and smoking has been suggested as a potential risk factor among others. Yet initial studies on COVID-19 cases reported contradictory results on the effects of smoking on the disease \u2013 some suggest that smoking might have a protective effect against it while other studies report an increased risk. A better understanding of how the exposure to smoking and other environmental factors affect biological processes relevant to SARS-CoV-2 infection and unfavorable disease progression is needed. In this study, we utilize mutational signatures associated with environmental factors as sensors of their exposure level. Many environmental factors including smoking are mutagenic and leave characteristic patterns of mutations, called mutational signatures, in affected genomes. We postulated that analyzing mutational signatures, combined with gene expression, can shed light on the impact of the mutagenic environmental factors to the biological processes. In particular, we utilized mutational signatures from lung adenocarcinoma (LUAD) data set collected in TCGA to investigate the role of environmental factors in COVID-19 vulnerabilities. Integrating mutational signatures with gene expression in normal tissues and using a pathway level analysis, we examined how the exposure to smoking and other mutagenic environmental factors affects the infectivity of the virus and disease progression. By delineating changes associated with smoking in pathway-level gene expression and cell type proportions, our study demonstrates that mutational signatures can be utilized to study the impact of exogenous mutagenic factors on them. Consistent with previous findings, our analysis showed that smoking mutational signature (SBS4) is associated with activation of cytokine-mediated signaling pathways, leading to inflammatory responses. Smoking related changes in cell composition were also observed, including the correlation of SBS4 with the expansion of goblet cells. On the other hand, increased basal cells and decreased ciliated cells in proportion were associated with the strength of a different mutational signature (SBS5), which is present abundantly but not exclusively in smokers. In addition, we found that smoking increases the expression levels of genes that are up-regulated in severe COVID-19 cases. Jointly, these results suggest an unfavorable impact of smoking on the disease progression and also provide novel findings on how smoking impacts biological processes in lung.","version":"1.1","doi":"10.1101/2021.09.27.461855","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.24.21264045","pub_date":"2021-09-27","title":"Non-invasive Vagus Nerve Stimulation for Respiratory Symptoms of COVID-19: Results From a Randomized Controlled Trial (SAVIOR I)","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>\n                    Severe coronavirus disease 2019 (COVID-19) is characterized, in part, by an excessive inflammatory response. Evidence from animal and human studies suggests that vagus nerve stimulation can lead to reduced levels of various pro-inflammatory cytokines. We conducted a prospective randomized controlled study (SAVIOR-I) to assess the feasibility, efficacy, and safety of non-invasive vagus nerve stimulation (nVNS) for the treatment of respiratory symptoms and inflammatory markers among patients who were hospitalized for COVID-19 (\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    identifier:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04368156'>NCT04368156</jats:ext-link>\n                    ).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Participants were randomly assigned in a 1:1 allocation to receive either the standard of care (SoC) alone or nVNS therapy plus the SoC. The nVNS group received 2 consecutive 2-minute doses of nVNS 3 times daily as prophylaxis. Efficacy and safety were evaluated via the incidence of specific clinical events, inflammatory biomarker levels, and the occurrence of adverse events.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Of the 110 participants who were enrolled and randomly assigned, 97 (nVNS, n=47; SoC, n=50) had sufficient available data and comprised the evaluable population. C-reactive protein (CRP) levels decreased from baseline to a significantly greater degree in the nVNS group than in the SoC group at day 5 and overall (ie, all postbaseline data points collected through day 5, combined). Procalcitonin level also showed significantly greater decreases from baseline to day 5 in the nVNS group than in the SoC group. D-dimer levels were decreased from baseline for the nVNS group and increased from baseline for the SoC group at day 5 and overall, although the difference between the treatment groups did not reach statistical significance. No significant treatment differences were seen for clinical respiratory outcomes or any of the other biochemical markers evaluated. No serious nVNS-related adverse events occurred during the study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>nVNS therapy led to significant reductions in levels of inflammatory markers, specifically CRP and procalcitonin. Because nVNS has multiple mechanisms of action that may be relevant to COVID-19, additional research into its potential to be used earlier in the course of COVID-19 and possibly mitigate some of the symptoms associated with post-acute COVID-19 syndrome is warranted.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.24.21264045","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.25.461766","pub_date":"2021-9-26","title":"Deactivation of SARS-CoV-2 surrogate porcine epidemic diarrhea virus with electron beam irradiation under the cold chain transportation condition","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prevailed all over the world and emerged as a significant public health emergency. The rapid outbreak of SARS-CoV-2 is largely due to its high transmission capacity. Studies implied that the cold chain logistics would be a potential route for the spread of SARS-CoV-2. The low temperature condition of the cold chain is conducive to survival and transmission of virus. Thus, the virus disinfection in cold chain should not be neglected for controlling COVID-19. However, due to the low temperature feature of the cold-chain, the virus disinfecting methods suitable in cold chain are limited. Here the high-energy electron beam irradiation is proposed to disinfect the SARS-CoV-2 in cold chain logistics. We evaluated the impact of high-energy electron beam irradiation on porcine epidemic diarrhea virus (PEDV), an enveloped virus surrogate for SARS-CoV-2, and explored the possible mechanism of the action of high-energy electron beam irradiation on PEDV. The irradiation dose of 10 kGy inactivated 98.1 % PEDV on the both top and bottom surfaces of various packaging materials under cold chain frozen condition. High-energy electron beam inactivated PEDV by inducing damages on viral genome or even capsid.","version":"1.1","doi":"10.1101/2021.09.25.461766","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.26.461851","pub_date":"2021-9-26","title":"Cytotoxic T-cell-based vaccine against SARS-CoV2: a hybrid immunoinformatic approach","abstract":"This paper presents an alternative vaccination platform that provides long-term cellular immune protection mediated by cytotoxic T-cells. The immune response via cellular immunity creates superior resistance to viral mutations, which are currently the greatest threat to the global vaccination campaign. Furthermore, we also propose a safer, more facile and physiologically appropriate immunization method using either intra-nasal or oral administration. The underlying technology is an adaptation of synthetic long peptides (SLPs) previously used in cancer immunotherapy. SLPs comprising HLA class I and class II epitopes are used to stimulate antigen cross-presentation and canonical class II presentation by dendritic cells. The result is a cytotoxic T cell-mediated prompt and specific immune response against the virus-infected epithelia and a rapid and robust virus clearance. Peptides isolated from COVID-19 convalescent patients were screened for the best HLA population coverage and were tested for toxicity and allergenicity, 3D peptide folding, followed by molecular docking studies provided positive results, suggesting a favourable antigen presentation.","version":"1.1","doi":"10.1101/2021.09.26.461851","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.25.461776","pub_date":"2021-9-25","title":"LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein","abstract":"The interactions between severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to COVID-19. The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary ACE2 receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was restricted to SARS-CoV-2, suggesting LRRC15 represents a novel class of spike binding interaction. We localized the interaction to the C-terminus of the S1 domain, and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19. We present evidence from genome-wide screening that the spike protein of SARS-CoV-2 interacts with human cells expressing LRRC15. The interaction is distinct from previously known classes of spike attachment factors, and appears to have emerged recently within the coronavirus family. Although not sufficient for cell invasion, this interaction can modulate viral infection. Our data point to an unappreciated host factor for SARS-CoV-2, with potential relevance to COVID-19. - Two systematic cell-based screens for SARS-CoV-2 spike protein binding identify LRRC15 as a human host factor - Interaction with LRRC15 is reproducible in different human cell lines and independent of known glycan or ACE2 binding pathways - The C-terminal S1 domain of SARS-CoV-2 spike binds LRRC15 with sub-micromolar affinity, while related coronavirus spikes do not - LRRC15 is expressed in tissues with high ACE2 levels and may modulate infection","version":"1.1","doi":"10.1101/2021.09.25.461776","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.24.461743","pub_date":"2021-9-25","title":"Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and thrombosis","abstract":"SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed . Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice. We confirmed that protein levels of inflammatory mediators IL-1\u03b2, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.","version":"1.1","doi":"10.1101/2021.09.24.461743","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.24.461616","pub_date":"2021-9-25","title":"Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with ACE2 interface, which gives 2G1 ability to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar IC50 in vitro. In SARS-CoV-2 and Beta- and Delta-variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 could be potentially capable of dealing with emerging SARS-CoV-2 variants in future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.","version":"1.1","doi":"10.1101/2021.09.24.461616","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.24.461759","pub_date":"2021-9-25","title":"Protein Vaccine Induces a Durable, More Broadly Neutralizing Antibody Response in Macaques than Natural Infection with SARS-CoV-2 P.1","abstract":"FDA-approved and Emergency Use Authorized (EUA) vaccines using new mRNA and viral-vector technology are highly effective in preventing moderate to severe disease, however, information on their long-term efficacy and protective breadth against SARS-CoV-2 Variants of Concern (VOCs) is currently scarce. Here we describe the durability and broad-spectrum VOC immunity of a prefusion-stabilized spike (S) protein adjuvanted with liquid or lyophilized CoVaccine HT\u2122 in cynomolgus macaques. This recombinant subunit vaccine is highly immunogenic and induces robust spike-specific and broadly neutralizing antibody responses effective against circulating VOCs (B.1.351 [Beta], P.1 [Gamma], B.1.617 [Delta]) for at least 3 months after the final boost. Protective efficacy and post-exposure immunity were evaluated using a heterologous P.1 challenge nearly 3 months after the last immunization. Our results indicate that while immunization with both high and low S doses shorten and reduce viral loads in the upper and lower respiratory tract, a higher antigen dose is required to provide durable protection against disease as vaccine immunity wanes. Histologically, P.1 infection causes similar COVID-19-like lung pathology as seen with early pandemic isolates. Post-challenge IgG concentrations were restored to peak immunity levels and vaccine-matched and cross-variant neutralizing antibodies were significantly elevated in immunized macaques indicating an efficient anamnestic response. Only low levels of P.1-specific neutralizing antibodies with limited breadth were observed in control (non-vaccinated but challenged) macaques suggesting that natural infection may not prevent reinfection by other VOCs. Overall, these results demonstrate that a properly dosed and adjuvanted recombinant subunit vaccine can provide long-lasting and protective immunity against circulating VOCs. A recombinant subunit protein formulated with CoVaccine HT\u2122 adjuvant induces superior immunity than natural infection and reduces viral load while protecting cynomolgus macaques from COVID-19-like disease caused by late SARS-CoV-2 P.1 (Gamma) challenge.","version":"1.1","doi":"10.1101/2021.09.24.461759","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.24.461631","pub_date":"2021-9-25","title":"Screening key genes and signaling pathways in COVID-19 infection and its associated complications by integrated bioinformatics analysis","abstract":"Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2)/ coronavirus disease 2019 (COVID-19) infection is the leading cause of respiratory tract infection associated mortality worldwide. The aim of the current investigation was to identify the differentially expressed genes (DEGs) and enriched pathways in COVID-19 infection and its associated complications by bioinformatics analysis, and to provide potential targets for diagnosis and treatment. Valid next-generation sequencing (NGS) data of 93 COVID 19 samples and 100 non COVID 19 samples (GSE156063) were obtained from the Gene Expression Omnibus database. Gene ontology (GO) and REACTOME pathway enrichment analysis was conducted to identify the biological role of DEGs. In addition, a protein-protein interaction network, modules, miRNA-hub gene regulatory network, TF-hub gene regulatory network and receiver operating characteristic curve (ROC) analysis were used to identify the key genes. A total of 738 DEGs were identified, including 415 up regulated genes and 323 down regulated genes. Most of the DEGs were significantly enriched in immune system process, cell communication, immune system and signaling by NTRK1 (TRKA). Through PPI, modules, miRNA-hub gene regulatory network, TF-hub gene regulatory network analysis, ESR1, UBD, FYN, STAT1, ISG15, EGR1, ARRB2, UBE2D1, PRKDC and FOS were selected as hub genes, which were expressed in COVID-19 samples relative to those in non COVID-19 samples, respectively. Among them, ESR1, UBD, FYN, STAT1, ISG15, EGR1, ARRB2, UBE2D1, PRKDC and FOS were suggested to be diagonstic factors for COVID-19. The findings from this bioinformatics analysis study identified molecular mechanisms and the key hub genes that might contribute to COVID-19 infection and its associated complications.","version":"1.1","doi":"10.1101/2021.09.24.461631","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.22.21263711","pub_date":"2021-09-25","title":"Comparison of adverse events between COVID-19 and Flu vaccines","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>BACKGROUND</jats:title>\n                  <jats:p>Among the various driving factors for vaccine hesitancy, confidence in the safety associated with the vaccine constitutes as one of the key factors. This study aimed at comparing the adverse effects of COVID-19 vaccines with the Flu vaccines.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>The VAERS data from 01/01/2020 to 08/20/2021 were used. The MedDRA terms coded by VAERS were further aggregated by a clinician into clinically meaningful broader terms.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>Various common adverse events between Flu and COVID-19 vaccines have been identified. Adverse events such as headache and fever were very common across all age groups. Among the common adverse events between Flu and COVID-19 vaccine, the relative risk along with 95% CI indicated that such common adverse events were more likely to be experienced by COVID-19 vaccine users than Flu vaccine users. Our study also quantified the proportion of rare adverse events such as Guillain Barre Syndrome and Gynecological changes in the VAERS database for COVID-19 vaccines.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>Based on the available data and results, it appears that there were some common adverse events between Flu vaccines and COVID-19 vaccines. These identified common adverse events warrant further investigations based on the relative risk and 95% CI.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.22.21263711","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.23.461605","pub_date":"2021-9-24","title":"Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way","abstract":"Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (Mpro) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 Mpro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards Mpro, since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 Mpro.","version":"1.1","doi":"10.1101/2021.09.23.461605","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.23.21260526","pub_date":"2021-09-24","title":"Validation of the Panbio\u2122 COVID-19 Antigen Rapid Test (Abbott) to screen for SARS-CoV-2 infection in Sint Maarten: a diagnostic accuracy study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>Control of the pandemic has required countries to look for other forms of tests besides the gold standard real-time polymerase chain reaction (RT-PCR). Rapid antigen tests (RAT), though less sensitive than RT-PCR, offer the possibility of rapid, inexpensive and early detection of the most infectious COVID-19 cases. Only very few studies have assessed the performance of the Abbott Panbio COVID-19 RAT among asymptomatic people or in Latin America. This study set out to validate this test among people attending the public test street in Sint Maarten, Dutch Caribbean.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>People of all ages were recruited from the public COVID-19 test street regardless of COVID-19 symptoms. They received a nasopharyngeal swab for the Abbott Panbio COVID-19 RAT and the RT-PCR Qtower. Diagnostic accuracy of the RAT was compared to the RT-PCR among the overall study population and for subgroups with/without symptoms, with/without close contact and different Ct values.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Using a RT-PCR Ct cut-off value of &lt;33, 119 out of 1,411 people (8.4%) tested positive for SARS-CoV-2. Most were asymptomatic (59%). The overall sensitivity and specificity of the RAT was 84% (95% CI 76.2-90.1) and 99.9% (95% CI 99.6-100) respectively. The sensitivity reduced to 67.6% (95% CI: 49.5%, 82.6%) among people without symptoms, regardless of whether they were in close contact with a known COVID-19 case. Sensitivity reduced considerably with a Ct cut-off value of &lt;35.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The Abbott Panbio RAT is a valid and cheaper alternative to RT-PCR when used on symptomatic individuals among the general population. However, among asymptomatic people it should not be used as a stand-alone test and negative results should be confirmed with RT-PCR.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.23.21260526","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.22.461142","pub_date":"2021-9-23","title":"Identifying The \u201cCore\u201d Transcriptome of SARS-CoV-2 Infected Cells","abstract":"In 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged, causing the COVID-19 pandemic. Consequently, ongoing research has focused on better understanding the mechanisms underlying the symptoms of this disease. Although COVID-19 symptoms span a range of organ systems, the specific changes in gene regulation that lead to the variety of symptoms are still unclear. In our study, we used publicly available transcriptome data from previous studies on SARS-CoV-2 to identify commonly regulated genes across cardiomyocytes, human bronchial epithelial cells, alveolar type II cells, lung adenocarcinoma, human embryonic kidney cells, and patient samples. Additionally, using this common \u201ccore\u201d transcriptome, we could identify the genes that were specifically and uniquely regulated in bronchial epithelial cells, embryonic kidney cells, or cardiomyocytes. For example, we found that genes related to cell metabolism were uniquely upregulated in kidney cells, providing us with the first mechanistic clue about specifically how kidney cells may be affected by SARS-CoV-2. Overall, our results uncover connections between the differential gene regulation in various cell types in response to the SARS-CoV-2 infection and help identify targets of potential therapeutics.","version":"1.1","doi":"10.1101/2021.09.22.461142","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.425144","pub_date":"2021-9-23","title":"Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression","abstract":"Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences. COVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis.","version":"1.3","doi":"10.1101/2021.01.13.425144","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.22.461286","pub_date":"2021-9-23","title":"Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor","abstract":"Interferons establish an antiviral state in responding cells through the induction of hundreds of interferon-stimulated genes (ISGs). ISGs antagonize viral pathogens directly through diverse mechanisms acting at different stages of viral life cycles, and indirectly by modulating cell cycle and promoting programmed cell death. The mechanisms of action and viral specificities for most ISGs remain incompletely understood. To enable the high throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating the potentially confounding effects of endogenous IFN and potential antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG induction in isogenic cell lines with and without the capacity to respond to IFN. Engineered CRISPRa cell lines demonstrated inducible, robust, and specific gRNA-directed expression of ISGs, which are functional in restricting viral infection. Using this platform, we screened for ISGs that restrict SARS-CoV-2, the causative agent of the COVID-19 pandemic. Results included ISGs previously described to restrict SARS-CoV-2 as well as multiple novel candidate antiviral factors. We validated a subset of candidate hits by complementary targeted CRISPRa and ectopic cDNA expression infection experiments, which, among other hits, confirmed OAS1 as a SARS-CoV-2 restriction factor. OAS1 exhibited strong antiviral effects against SARS-CoV-2, and these effects required OAS1 catalytic activity. These studies demonstrate a robust, high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by the identification of multiple novel SARS-CoV-2 restriction factors.","version":"1.1","doi":"10.1101/2021.09.22.461286","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.20.461073","pub_date":"2021-9-23","title":"Personal care formulations demonstrate virucidal efficacy against multiple SARS-CoV-2 variants of concern: implications for hand hygiene","abstract":"The second and third waves of COVID-19 pandemic have largely been driven by the surge of successive SARS-CoV-2 variants of concern (VOC). These VOC have rapidly spread through multiple geographies being enabled by high transmission rates and/or high viral load compared to the original parent strain. Consequently, the altered phenotypes of these VOC have posed greater challenges to diagnostic and clinical management of COVID-19. Despite considerable progress being made on vaccine roll out, practicing proper hand hygiene has been advocated as a consistent precautionary intervention as more virulent VOC continue to emerge and spread across geographies. Two variants of concern, namely beta and delta, have recently been shown to escape antibody-mediated neutralization by virtue of acquired mutations in the receptor-binding domain of the viral spike protein which binds to the human ACE2 receptor for cellular entry. In this report we have empirically determined the efficacy of a range of personal care formulations in inactivating the beta and delta variants of SARS-CoV-2. High titres of these variants were exposed to marketed personal care formulations from Unilever under standard in-vitro suspension test-based conditions relevant to end-user habits. All the formulations demonstrated greater than 99.9% reduction in viral infective titres. The rate of inactivation by these products were comparable to that of the original strain of SARS-CoV-2 virus tested under the same conditions. Therefore, it can be concluded that well-designed personal care formulations when tested under consumer-centric conditions, and with proven efficacy against the parent strain of SARS-CoV-2 will continue to be effective against extant and emerging variants of SARS-CoV-2. This is through their broad-spectrum mode of action (disruption of lipid bilayer of the host-derived viral envelope, denaturation of envelop and nucleocapsid proteins, and disruption of genome) which is independent of the escape mutations that facilitate immune evasion or enhanced transmissibility.","version":"1.1","doi":"10.1101/2021.09.20.461073","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.22.461447","pub_date":"2021-9-23","title":"Deciphering the role of the Pancreatic Secretome in Covid-19 associated Multi-Organ Dysfunctions","abstract":"Emerging evidence indicates an intricate relationship between the SARS-CoV-2 infection and Multi-Organ Dysfunctions (MODs). Here, we have investigated the role of the Secretome of the SARS-CoV-2 infected pancreas and mechanistically linked it with the multi-organ dysfunction using the scRNA-seq analysis. We found that acinar-specific PRSS2, REG3A, REG1A, SPINK1, and ductal-specific SPP1, MMP7 genes are upregulated in alpha, beta, delta, and mesenchyme cells. Using extensive documented experimental evidence, we validated the association of upregulated pancreatic Secretome with coagulation cascade, complement activation, renin angiotensinogen system dysregulation, endothelial cell injury and thrombosis, immune system dysregulation, and fibrosis. Our finding suggests the influence of upregulated Secretome on multi-organ systems such as Nervous, Cardiovascular, Immune, Digestive, and Urogenital systems. In addition, we report that the secretory proteins IL1B, AGT, ALB, SPP1, CRP, SERPINA1, C3, TFRC, TNFSF10, and MIF are associated with diverse diseases. Thus, suggest the role of the pancreatic Secretome in SARS-CoV-2 associated MODs.","version":"1.1","doi":"10.1101/2021.09.22.461447","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435416","pub_date":"2021-9-23","title":"phastSim: efficient simulation of sequence evolution for pandemic-scale datasets","abstract":"Sequence simulators are fundamental tools in bioinformatics, as they allow us to test data processing and inference tools, as well as being part of some inference methods. The ongoing surge in available sequence data is however testing the limits of our bioinformatics software. One example is the large number of SARS-CoV-2 genomes available, which are beyond the processing power of many methods, and simulating such large datasets is also proving difficult. Here we present a new algorithm and software for efficiently simulating sequence evolution along extremely large trees (e.g. < 100, 000 tips) when the branches of the tree are short, as is typical in genomic epidemiology. Our algorithm is based on the Gillespie approach, and implements an efficient multi-layered search tree structure that provides high computational efficiency by taking advantage of the fact that only a small proportion of the genome is likely to mutate at each branch of the considered phylogeny. Our open source software is available from https://github.com/NicolaDM/phastSim and allows easy integration with other Python packages as well as a variety of evolutionary models, including indel models and new hypermutatability models that we developed to more realistically represent SARS-CoV-2 genome evolution. One of the most influential responses to the SARS-CoV-2 pandemic has been the widespread adoption of genome sequencing to keep track of viral spread and evolution. This has resulted in vast availability of genomic sequence data, that, while extremely useful and promising, is also increasingly hard to store and process efficiently. An important task in the processing of this genetic data is simulation, that is, recreating potential histories of past and future virus evolution, to benchmark data analysis methods and make statistical inference. Here, we address the problem of efficiently simulating large numbers of closely related genomes, similar to those sequenced during SARS-CoV-2 pandemic, or indeed to most scenarios in genomic epidemiology. We develop a new algorithm to perform this task, that provides not only computational efficiency, but also extreme flexibility in terms of possible evolutionary models, allowing variation in mutation rates, non-stationary evolution, and indels; all phenomena that play an important role in SARS-CoV-2 evolution, as well as many other real-life epidemiological scenarios.","version":"1.2","doi":"10.1101/2021.03.15.435416","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.21.461322","pub_date":"2021-9-22","title":"Identification of HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 and analysis of their conservation using the mutation database of SARS-CoV-2 variants","abstract":"COVID-19 vaccines are currently being administrated worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved non-structural proteins. Here, we identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.","version":"1.1","doi":"10.1101/2021.09.21.461322","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.21.461301","pub_date":"2021-9-22","title":"Design of Peptide Vaccine for COVID19: CD8+ and CD4+ T cell epitopes from SARS-CoV-2 open-reading-frame protein variants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has challenged public health at an unprecedented scale which has led to a dramatic loss of human life worldwide. To design a protective vaccine against SARS-CoV-2, it is necessary to understand which SARS-CoV-2 specific epitopes can elicit a T cell response and provide protection across a broad population. In this study, PLpro and RdRp, two immunogenic non-structural proteins from an immunodominant gene region ORF1ab, as well as ORF3a and ORF9b are identified as potential vaccine targets against SARS-CoV-2. To select top epitopes for vaccine design, we used various clinical properties, such as antigenicity, allergenicity, toxicity and IFN-y secretion. The analysis of CD8 and CD4 T cell epitopes revealed multiple potential vaccine constructs that cover a high percentage of the world population. We identified 8 immunogenic, antigenic, non-allergenic, non-toxic, stable and IFN-y inducing CD8 proteins for nsp3, 4 for nsp12, 11 for ORF3a and 3 for ORF9b that are common across four lineages of variants of concern \u2013 B.1.1.7, P.1, B.1.351 and B.1.617.2, which protect 98.12%, 87.08%, 96.07% and 63.8% of the world population, respectively. We also identified variant specific T cell epitopes that could be useful in targeting each variant strain separately. Including the prediction of mouse MHC affinity towards our top CD8 epitopes, our study revealed a total of 3 immunogenic, antigenic, non-allergenic, non-toxic, stable and IFN-y inducing CD8 epitopes overlapping with 6 antigenic, non-allergenic, non-toxic, stable and IFN-y inducing CD4 epitopes across all four variants of concern which can effectively be utilized in pre-clinical studies. The landscape of SARS-CoV-2 T cell epitopes that we identified can help lead SARS-CoV-2 vaccine development as well as epitope-based peptide vaccine research in the future.","version":"1.1","doi":"10.1101/2021.09.21.461301","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.455040","pub_date":"2021-9-22","title":"Virucidal activity of CPC-containing oral rinses against SARS-CoV-2 variants and are active in the presence of human saliva","abstract":"The role of human saliva in aerosol-based transmission of SARS-CoV-2 has highlighted the need to understand the potential of oral hygiene products to inactivate the virus. Here we examined the efficacy of mouthwashes containing cetylpyridinium chloride (CPC) or chlorhexidine (CHX) in inactivating SARS-CoV-2. After 30 seconds contact under standard aqueous conditions CPC mouthwashes achieved a \u22654.0log10 PFU/mL reduction in SARS-CoV-2 (USA-WA1/2020) titres whereas comparable products containing CHX achieved <2.0log10 PFU/mL reduction. Further testing with CPC mouthwashes demonstrated efficacy against multiple SARS-CoV-2 variants, with inactivation below the limit of detection observed against the Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1) variants. Virucidal efficacy of CPC mouthwash was also observed in the presence of human saliva with the product delivering \u22654.0log10 PFU/mL reduction in SARS-CoV-2 titres after 30 seconds providing additional evidence for the virucidal efficacy of CPC mouthwashes under simulated physiological conditions. Together these data suggest CPC-based mouthwashes are effective at inactivating SARS-CoV-2 and further supports the use of mouthwash to mitigate the risk of transmission during dentistry procedures.","version":"1.4","doi":"10.1101/2021.08.05.455040","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.11.459907","pub_date":"2021-9-22","title":"Design of T cell epitope-based vaccine candidate for SARS-CoV-2 targeting nucleocapsid and spike protein escape variants","abstract":"The current COVID-19 pandemic continues to spread and devastate in the absence of effective treatments, warranting global concern and action. Despite progress in vaccine development, the rise of novel, increasingly infectious SARS-CoV-2 variants makes it clear that our response to the virus must continue to evolve along with it. The use of immunoinformatics provides an opportunity to rapidly and efficiently expand the tools at our disposal to combat the current pandemic and prepare for future outbreaks through epitope-based vaccine design. In this study, we validated and compared the currently available epitope prediction tools, and then used the best tools to predict T cell epitopes from SARS-CoV-2 spike and nucleocapsid proteins for use in an epitope-based vaccine. We combined the mouse MHC affinity predictor and clinical predictors such as HLA affinity, immunogenicity, antigenicity, allergenicity, toxicity and stability to select the highest quality CD8 and CD4 T cell epitopes for the common SARS-CoV-2 variants of concern suitable for further preclinical studies. We also identified variant-specific epitopes to more precisely target the Alpha, Beta, Gamma, Delta, Cluster 5 and US variants. We then modeled the 3D structures of our top 4 N and S epitopes to investigate the molecular interaction between peptide-MHC and peptide-MHC-TCR complexes. Following in vitro and in vivo validation, the epitopes identified by this study may be used in an epitope-based vaccine to protect across all current variants, as well as in variant-specific booster shots to target variants of concern. Immunoinformatics tools allowed us to efficiently predict epitopes in silico most likely to prove effective in vivo, providing a more streamlined process for vaccine development in the context of a rapidly evolving pandemic.","version":"1.2","doi":"10.1101/2021.09.11.459907","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.20.21263794","pub_date":"2021-09-22","title":"Development and evaluation of a machine learning-based in-hospital COvid-19 Disease Outcome Predictor (CODOP): a multicontinental retrospective study","abstract":"<jats:title>Summary</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>More contagious SARS-CoV-2 virus variants, breakthrough infections, waning immunity, and sub-optimal rates of COVID-19 vaccination account for a new surge of infections leading to record numbers of hospitalizations and deaths in several European countries. This is a particularly concerning scenario for resource-limited countries, which have a lower vaccination rate and fewer clinical tools to fight against the next pandemic waves. There is an urgent need for clinically valuable, generalizable, and parsimonious triage tools assisting the appropriate allocation of hospital resources. We aimed to develop and extensively validate CODOP, a machine learning-based tool for accurately predicting the clinical outcome of hospitalized COVID-19 patients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>CODOP was built using modified stable iterative variable selection and linear regression with lasso regularisation. To avoid generalization problems, CODOP was trained and tested with three time-sliced and geographically distinct cohorts encompassing 40 511 blood-based analyses of COVID-19 patients from more than 110 hospitals in Spain and the USA during 2020-21. We assessed the discriminative ability of the model using the Area Under the Receiving Operative Curve (AUROC) as well as horizon and Kaplan-Meier risk stratification analyses. To reckon the fluctuating pressure levels in hospitals through the pandemic, we offer two online CODOP calculators suited for undertriage or overtriage scenarios. We challenged their generalizability and clinical utility throughout an evaluation on a cohort of patients hospitalized in five hospitals from three Latin American countries.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>CODOP uses 12 clinical parameters commonly measured at hospital admission and associated with the pathophysiology of COVID-19. CODOP reaches high discriminative ability up to nine days before clinical resolution (AUROC: 0\u00b790-0\u00b796, 95% CI 0\u00b7879-0\u00b7970), it is well calibrated, and it enables an effective dynamic risk stratification during hospitalization. The two CODOP online calculators demonstrate their potential for triage decisions when challenged with the distinctive Latin American evaluation cohorts (73-100% sensitivity and 84-100% specificity).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>The high predictive performance of CODOP in geographically disperse patient cohorts and the easiness-of-use, strongly suggest its clinical utility as a global triage tool, particularly in resource-limited countries.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>The Max Planck Society.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>We have searched PubMed for articles about the existence of in-hospital COVID-19 mortality predictive models, using the search terms \u201ccoronavirus\u201d, \u201cCOVID-19\u201d, \u201crisk\u201d, \u201cdeath\u201d, \u201cmortality\u201d, and \u201cprediction\u201d, focusing on studies published between March 1, 2020 and 31 August, 2021. The studies we identified generally used small-medium size cohorts of patients that are geographically restricted to small regions of the developed world (many times, to the same city). We haven\u2019t found studies that challenged their models in extended cohorts of patients from very distinct health system populations, particularly from resource-limited countries. Further, most of the previous models are rigid by not acknowledging the fluctuating availability of hospital resources during the pandemic (e.g., beds, oxygen supply). These and other limitations have been pointed out by expert reviews indicating that published in-hospital COVID-19 mortality predictive models are subject to high risk of bias, report an over-optimistic performance, and have limited clinical value in assisting daily triage decisions. A parsimonious, accurate and extensively validated model is yet to be developed.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>\n                      We analysed clinical data from different cohorts totalling 21 607 COVID-19 patients treated in more than 110 hospitals in Spain and the USA during three different pandemic waves extending from February 2020 to April 2021. The new CODOP in-hospital mortality prediction model is based on 11 blood biochemistry parameters (representing main biological pathways involved in the pathogenesis of SARS-CoV-2) plus Age, all of them commonly measured upon hospitalization. CODOP accurately predicted mortality risk up to nine days before clinical resolution (AUROC: 0\u00b790-0\u00b796, 95% CI 0\u00b7879-0\u00b7970), it is well calibrated, and it enables an effective dynamic risk stratification during hospitalization. We offer two online CODOP calculator subtypes (\n                      <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='https://gomezvarelalab.em.mpg.de/codop/'>https://gomezvarelalab.em.mpg.de/codop/</jats:ext-link>\n                      ) tailored to overtriage and undertriage scenarios. The online calculators were able to reach the desired prediction performance in five independent evaluation cohorts gathered in hospitals of three Latin American countries from March 7th 2020 to June 7th 2021.\n                    </jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>We present here a highly accurate, parsimonious and extensively validated COVID-19 in-hospital mortality prediction model, derived from working with the largest number and the most geographically extended representation of patients and health systems to date.</jats:p>\n                    <jats:p>The rigorous analytical methods, the generalizability of the model in distinct world regions, and its flexibility to reckon with the changing availability of hospital resources point to CODOP as a clinically useful tool potentially improving the outcome prediction and the management of COVID-19 hospitalized patients.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.20.21263794","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.14.439284","pub_date":"2021-9-22","title":"Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis","abstract":"Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present \u2018universal saturation transfer analysis\u2019 (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an \u2018end on\u2019 manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and \u2018end on\u2019 mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems. The surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of \u2018saturation transfer\u2019 protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this \u2018universal saturation transfer\u2019 analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins \u2013 SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants \u2013 by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer \u2018end on\u2019-binding to sialoside sugars was revealed contrasting with \u2018extended surface\u2019-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.","version":"1.2","doi":"10.1101/2021.04.14.439284","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.20.461023","pub_date":"2021-9-21","title":"SARS-CoV-2 preS dTM vaccine booster candidates increase functional antibody responses and cross-neutralization against SARS-CoV-2 variants of concern in non-human primates","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that partly evade neutralizing antibodies has raised concerns of reduced vaccine effectiveness and increased infection. We previously demonstrated in preclinical models and in human clinical trials that our SARS-CoV-2 recombinant spike protein vaccine adjuvanted with AS03 (CoV2 preS dTM-AS03) elicits robust neutralizing antibody responses in na\u00efve subjects. Here, the objective was to document the potency of various booster vaccine formulations in macaques previously vaccinated with mRNA or protein subunit vaccine candidates. We show that one booster dose of AS03-adjuvanted CoV2 preS dTM, D614 (parental) or B.1.351 (Beta), in monovalent or bivalent (D614 + B.1.351) formulations, significantly boosted pre-existing neutralizing antibodies and elicited high and stable cross-neutralizing antibodies covering the four known SARS-CoV-2 variants of concern (Alpha, Beta, Gamma and Delta) and, unexpectedly, SARS-CoV-1, in primed macaques. Interestingly, the non-adjuvanted CoV2 preS dTM B.1.351 vaccine formulation also significantly boosted and broadened the neutralizing antibody responses. Our findings show that these vaccine candidates used as a booster have the potential to offer cross-protection against a broad spectrum of variants. This has important implications for vaccine control of SARS-CoV-2 variants of concern and informs on the benefit of a booster with our vaccine candidates currently under evaluation in phase 2 and 3 clinical trials (NCT04762680 and NCT04904549).","version":"1.1","doi":"10.1101/2021.09.20.461023","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460814","pub_date":"2021-9-21","title":"A BioID-derived proximity interactome for SARS-CoV-2 proteins","abstract":"The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here we use BioID to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are in association with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Collectively, these studies provide a valuable resource to potentially uncover novel SARS-CoV-2 biology and inform development of antivirals.","version":"1.1","doi":"10.1101/2021.09.17.460814","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.20.461041","pub_date":"2021-9-21","title":"Mechanistic Insights into the Effects of Key Mutations on SARS-CoV-2 RBD-ACE2 Binding","abstract":"Some recent SARS-CoV-2 variants appear to have increased transmissibility than the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on target cells and infect them. In this study, we provide atomic-level insight into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities of the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.","version":"1.1","doi":"10.1101/2021.09.20.461041","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.19.460950","pub_date":"2021-9-21","title":"IL13Pred: A method for predicting immunoregulatory cytokine IL-13 inducing peptides for managing COVID-19 severity","abstract":"Interleukin 13 (IL-13) is an immunoregulatory cytokine that is primarily released by activated T-helper 2 cells. It induces the pathogenesis of many allergic diseases, such as airway hyperresponsiveness, glycoprotein hypersecretion and goblet cell hyperplasia. IL-13 also inhibits tumor immunosurveillance, which leads to carcinogenesis. In recent studies, elevated IL-13 serum levels have been shown in severe COVID-19 patients. Thus it is important to predict IL-13 inducing peptides or regions in a protein for designing safe protein therapeutics particularly immunotherapeutic. This paper describes a method developed for predicting, designing and scanning IL-13 inducing peptides. The dataset used in this study contain experimentally validated 313 IL-13 inducing peptides and 2908 non-inducing homo-sapiens peptides extracted from the immune epitope database (IEDB). We have extracted 95 key features using SVC-L1 technique from the originally generated 9165 features using Pfeature. Further, these key features were ranked based on their prediction ability, and top 10 features were used for building machine learning prediction models. In this study, we have deployed various machine learning techniques to develop models for predicting IL-13 inducing peptides. These models were trained, test and evaluated using five-fold cross-validation techniques; best model were evaluated on independent dataset. Our best model based on XGBoost achieves a maximum AUC of 0.83 and 0.80 on the training and independent dataset, respectively. Our analysis indicate that certain SARS-COV2 variants are more prone to induce IL-13 in COVID-19 patients. A standalone package as well as a web server named \u2018IL-13Pred\u2019 has been developed for predicting IL-13 inducing peptides (https://webs.iiitd.edu.in/raghava/il13pred/). Interleukin-13, an immunoregulatory cytokine plays an important role in increasing severity of COVID-19 and other diseases. IL-13Pred is a highly accurate in-silico method developed for predicting the IL-13 inducing peptides/ epitopes. IL-13 inducing peptides are reported in various SARS-CoV2 strains/variants proteins. This method can be used to detect IL-13 inducing peptides in vaccine candidates. User friendly web server and standalone software is freely available for IL-13Pred Shipra Jain is currently working as Ph.D. in Computational Biology from Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India. Anjali Dhall is currently working as Ph.D. in Computational Biology from Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India. Sumeet Patiyal is currently working as Ph.D. in Computational Biology from Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India. Gajendra P. S. Raghava is currently working as Professor and Head of Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India.","version":"1.1","doi":"10.1101/2021.09.19.460950","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.16.460616","pub_date":"2021-9-21","title":"A novel B.1.1.523 SARS-CoV-2 variant that combines many spike mutations linked to immune evasion with current variants of concern","abstract":"In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic several variants have emerged that are linked to increased transmissibility and immune evasion. These variants are recognized as variants of concern (VOC). In this study, we describe a B.1.1.523 variant that shares many spike mutations with current VOC. Receptor-binding domain mutations E484K and S494P were observed but also a deletion (position 156-158) in the N-terminal antigenic supersite that is similar to the delta-variant. These mutations are linked to immune evasion in VOC that could lead to less effective vaccines. This variant has been reported in various different countries and continents despite the dominance of B.1.1.7 (alpha) and B.1.617.2 (delta) variant. Furthermore, the B.1.1.523 pangolin lineage as a whole is recognized as a variant under monitoring since 14th of July 2021.","version":"1.1","doi":"10.1101/2021.09.16.460616","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460870","pub_date":"2021-9-21","title":"Gene networks under circadian control exhibit diurnal organization in primate organs","abstract":"Mammalian organs are individually controlled by autonomous circadian clocks. At the molecular level, this process is defined by the cyclical co-expression of both core transcription factors and off-target genes across time. While interactions between these molecular clocks are likely necessary for proper homeostasis, these features remain undefined. Here, we utilize integrative analysis of a baboon diurnal transcriptome atlas to characterize the properties of gene networks under circadian control. We found that 53.4% (8,120) of baboon genes are rhythmically expressed body-wide. In addition, >30% of gene-gene interactions exhibit periodic co-expression patterns, with core circadian genes more cyclically co-expressed than others. Moreover, two basic network modes were observed at the systems level: daytime and nighttime mode. Daytime networks were enriched for genes involved in metabolism, while nighttime networks were enriched for genes associated with growth and cellular signaling. A substantial number of diseases only form significant disease modules at either daytime or nighttime. In addition, we found that 216 of 313 genes encoding products that interact with SARS-CoV-2 are rhythmically expressed throughout the body. Importantly, more than 80% of SARS-CoV-2 related genes enriched modules are rhythmically expressed, and have significant network proximities with circadian regulators. Our data suggest that synchronization amongst circadian gene networks is necessary for proper homeostatic functions and circadian regulators have close interactions with SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.09.17.460870","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.21.461221","pub_date":"2021-9-21","title":"A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems","abstract":"Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein reversed phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, an electrophilic impurity derived from the ionizable cationic lipid component is shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.","version":"1.1","doi":"10.1101/2021.09.21.461221","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.16.460716","pub_date":"2021-9-20","title":"The supramolecular organization of SARS-CoV and SARS-CoV-2 virions revealed by coarse-grained models of intact virus envelopes","abstract":"The coronavirus disease 19 (COVID-19) pandemic is causing a global health crisis and has already caused a devastating societal and economic burden. The pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a high sequence and architecture identity with SARS-CoV, but far more people have been infected by SARS-CoV-2. Here, combining structural data from cryo-EM and structure prediction, we constructed bottom-up Martini coarse-grained models of intact SARS-CoV and SARS-CoV-2 envelopes. Microsecond molecular dynamics simulations were performed, allowing us to explore their dynamics and supramolecular organization. Both SARS-CoV and SARS-CoV-2 envelopes present a spherical morphology with structural proteins forming multiple string-like islands in the membrane and clusters between heads of spike proteins. Critical differences between the SARS-CoV and SARS-CoV-2 envelopes are the interaction pattern between spike proteins and the flexibility of spike proteins. Our models provide structural and dynamic insights in the SARS virus envelopes, and could be used for further investigation, such as drug design, and fusion and fission processes.","version":"1.1","doi":"10.1101/2021.09.16.460716","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427194","pub_date":"2021-9-20","title":"BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2","abstract":"SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.","version":"1.2","doi":"10.1101/2021.01.19.427194","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431922","pub_date":"2021-9-20","title":"ESC - a comprehensive resource for SARS-CoV-2 immune escape variants","abstract":"Ever since the breakout of COVID-19 disease, ceaseless genomic research to inspect the epidemiology and evolution of the pathogen has been undertaken globally. Large scale viral genome sequencing and analysis have uncovered the functional impact of numerous genetic variants in disease pathogenesis and transmission. Emerging evidence of mutations in spike protein domains escaping antibody neutralization is reported. We have built a database with precise collation of manually curated variants in SARS-CoV-2 from literature with potential escape mechanisms from a range of neutralizing antibodies. This comprehensive repository encompasses a total of 5258 variants accounting for 2068 unique variants tested against 230 antibodies, patient convalescent plasma and vaccine breakthrough events. This resource enables the user to gain access to an extensive annotation of SARS-CoV-2 escape variants which would contribute to exploring and understanding the underlying mechanisms of immune response against the pathogen. The resource is available at http://clingen.igib.res.in/esc/","version":"1.2","doi":"10.1101/2021.02.18.431922","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.459961","pub_date":"2021-9-20","title":"Development of a novel, pan-variant aerosol intervention for COVID-19","abstract":"To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants. Preclinical development and evaluation of aerosolized soluble recombinant human ACE2 (APN01) administered as a COVID-19 intervention is reported.","version":"1.2","doi":"10.1101/2021.09.14.459961","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.21263598","pub_date":"2021-09-20","title":"EFFICACY OF THE MEASLES-MUMPS-RUBELLA (MMR) VACCINE IN THE REDUCING THE SEVERITY OF COVID-19: AN INTERIM ANALYSIS OF A RANDOMISED CONTROLLED CLINICAL TRIAL","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>COVID-19 is still a challenge, both with regard to its treatment and to the actual efficacy of the vaccines available to date, especially with the emergence of new variants. We evaluated the efficacy of the measles-mumps-rubella (MMR) vaccine in preventing SARS-CoV-2 infection and severity of COVID-19 in health workers.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This analysis includes data from one ongoing blinded, randomized, placebo-controlled trial with participants aged 18-60 years were randomly assigned to receive the MMR vaccine or a placebo. The primary efficacy analysis included all participants with a positive nasopharyngeal RT-PCR test since their inclusion.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The MMR vaccine did not prevent the SARS-CoV-2 infection. Participants in the MMR group, compared with those in the placebo group, had a 48% risk reduction in symptomatic COVID-19 (RR = 0.52; 95% CI: 0.33\u20130.83; p=0.004) and a 76% risk reduction in COVID-19 treatment (RR = 0.24; 95% CI: 0.06 \u2013 0.88; p = 0.020) with one dose and a 51% risk reduction in COVID-19 symptoms (RR = 0.49; 95% CI: 0.31 \u2013 0.78; p = 0.001) and a 78% risk reduction in COVID-19 treatment (RR = 0.22; 95% CI: 0.06 \u2013 0.82; p = 0.015) with two doses.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>This interim analysis of an ongoing clinical trial suggests that compared with a placebo, the vaccine reduces the risk of COVID-19 symptoms and reduces the need for COVID-19 treatment.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Clinical Trials Registry</jats:title>\n                  <jats:p>\n                    Brazilian Clinical Trials Registry (ReBEC n\u00b0 RBR-2xd6dkj -\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='https://ensaiosclinicos.gov.br/rg/RBR-2xd6dkj'>https://ensaiosclinicos.gov.br/rg/RBR-2xd6dkj</jats:ext-link>\n                    ).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>HIGHLIGHTS</jats:title>\n                  <jats:list list-type='order'>\n                    <jats:list-item>\n                      <jats:p>The MMR vaccine can stimulate the innate immunity inducing a nonspecific protection against other infections, called heterologous immunity.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Repeated exposure to the antigen (innate immune response training) results in an extension of the action time of this immune response (innate immune response memory) and consequently in protection against other infections (heterologous immunity) for a longer time.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>The MMR vaccine has been used by national immunization programs in the world for many years, it is very safe and can be stored and distributed at 2-8\u00b0C, making it particularly suitable for global distribution.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Among participants who received at least one dose, compared with those in the placebo group, participants in the MMR group had a significant risk reduction in symptomatic COVID-19 and of cases requiring treatment.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>The use of MMR vaccine can be useful in several populations in the world that do not have access to the COVID-19 vaccine and in a future epidemic or pandemic as an emergency measure until specific treatments or vaccines for each case are available to the general population.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.14.21263598","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.18.460895","pub_date":"2021-9-18","title":"Sex differences in cardio-pulmonary pathology of SARS-CoV2 infected and Trypanosoma cruzi co-infected mice","abstract":"Coronavirus disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV2) is a deadly contagious infectious disease. For those who survived COVID-19, post-COVID cardiac damage poses a major threat for the progression of cardiomyopathy and heart failure. Currently, the number of COVID-related cases and deaths are increasing in Latin America, where a major COVID comorbidity is Chagas\u2019 heart disease (caused by the parasite Trypanosoma cruzi). Here, we investigated the effect of T. cruzi infection on the pathogenesis and severity of CoV2 infection and, conversely, the effect of CoV2 infection on heart pathology during coinfection. We used transgenic human angiotensin-converting enzyme 2 (huACE2) mice infected with CoV2, T. cruzi, or coinfected with both in this study. Our study shows for the first time that white adipose tissue (WAT) serves as a reservoir for CoV2 and the persistence of CoV2 in WAT alters adipose tissue morphology and adipocyte physiology. Our data demonstrate a correlation between the loss of fat cells and the pulmonary adipogenic signaling (via adiponectin isomers) and pathology in CoV2 infection. The viral load in the lungs is inversely proportional to the viral load in WAT, which differs between male and female mice. Our findings also suggest that adiponectin-PPAR signaling may differently regulate Chagas cardiomyopathy in coinfected males and females. We conclude that adipogenic signaling may play important roles in cardio-pulmonary pathogenesis during CoV2 infection and T. cruzi coinfection. The levels of adiponectin isomers differ between male and female mice during CoV2 infection and coinfection with T. cruzi, which may differently regulate inflammation, viral load, and pathology in the lungs of both the sexes. Our findings are in line with other clinical observations that reported that males are more susceptible to COVID-19 than females and suffer greater pulmonary damage.","version":"1.1","doi":"10.1101/2021.09.18.460895","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.431855","pub_date":"2021-9-17","title":"WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that cause damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the \u201ccytokine storm\u201d, strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins 6, 8, 18 and tumor necrosis factor-alpha (TNF-\u03b1) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.","version":"1.3","doi":"10.1101/2021.02.20.431855","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456066","pub_date":"2021-9-17","title":"SARS-CoV-2 Neutralization in Convalescent Plasma and Commercial Lots of Plasma-derived Immunoglobulin","abstract":"Patients suffering from primary or secondary immunodeficiency (PID or SID) face times of increased insecurity and discomfort in the light of the raging COVID-19 pandemic, not knowing if and to what extent their comorbidities impact the course of a potential SARS-CoV-2 infection. Furthermore, recently available vaccination options might not be amenable or effective for all patients of this heterogeneous population. Therefore, these patients often rely on passive immunization with plasma-derived, intravenous or subcutaneous immunoglobulin (IVIG/SCIG). Whether the ongoing COVID-19 pandemic and/or the progress in vaccination programs lead to increased and potentially protective titers in plasma-derived immunoglobulins (Ig) indicated, e.g., for humoral immunodeficiency remains a pressing question for this patient population. We investigated SARS-CoV-2 reactivity of US plasma-derived IVIG/SCIG products from the end of 2020 until 06/2021 as well as in convalescent plasma (CP) from 05/2020 to 08/2020 to determine whether potentially neutralizing antibody titers may be present. Final containers of IVIG/SCIG and CP donations were analyzed by commercial ELISA for anti-SARS-CoV-2 S1-receptor binding domain (RBD) IgG as well as microneutralization assay using a patient-derived SARS-CoV-2 (D614G) isolate. Neutralization capacities of 313 plasma single donations and 119 plasma-derived IVIG/SCIG lots were determined. Results obtained from both analytical methods were normalized against the WHO International Standard. Finally, based on dense pharmacokinetic (PK) profiles of an IVIG preparation from previously published investigations, possible steady-state plasma levels of SARS-CoV-2 neutralization capacities were approximated based on currently measured anti-SARS-CoV-2 potencies in IVIG/SCIG preparations. CP donations presented with a high variability with regards to anti-SARS-CoV-2 reactivity in ELISA as well as in neutralization testing. While approximately 50% of convalescent donations were none/low neutralizing, approximately 10% were at or above 1000 IU/mL. IVIG/SCIG lots derived from pre-pandemic plasma donations did not show neutralizing capacities of SARS-CoV-2. Lots produced between 12/2020 and 06/2021, entailing plasma donations after the emergence of SARS-CoV-2 showed a rapid and constant increase in anti-SARS-CoV-2 reactivity and neutralization capacity over time. While lot-to-lot variability was substantial, neutralization capacity increased from a mean of 20 IU/mL in 12/2020 to 505 IU/mL in 06/2021 with a maximum of 864 IU/mL for the most recent lots. Pharmacokinetic extrapolations, based on non-compartmental superposition principles using steady-state reference profiles from previously published PK investigations on IVIG in PID, yielded potential steady-state trough plasma levels of 16 IU/mL of neutralizing SARS-CoV-2 IgG based on the average final container concentration from 05/2021 of 216 IU/mL. Maximum extrapolated trough levels could reach 64 IU/mL based on the latest maximal final container potency tested in 06/2021. SARS-CoV-2 reactivity and neutralization capacity in IVIG/SCIG produced from US plasma rapidly and in part exponentially increased in the first half of 2021. The observed increase of final container potencies is likely trailing the serological status of the US donor population in terms of COVID-19 convalescence and vaccination by at least 5 months due to production lead times and should in principle continue at least until fall 2021. In summary, the data support rapidly increasing levels of anti-SARS-CoV-2 antibodies in IVIG/SCIG products implicating that a certain level of protection could be possible against COVID-19 for regularly substituted PID/SID patients. Nevertheless, more research is still needed to confirm which plasma levels are needed to provide protection against SARS-CoV-2 infection in immune-compromised patients. People with deficiencies in their immune system often have an insufficient antibody response to antigens, e.g., bacteria, viruses, or vaccines. These patients therefore often receive antibodies from healthy people to replace the missing antibodies and build a first line of defense against infections. These antibodies (also called immunoglobulins (Ig)) are prepared from plasma of healthy donors, the liquid fraction of the blood without cells. This plasma is then split up in pharmaceutical production into its protein components. One of these is immunoglobulin G (IgG), which is the protein family that neutralizes/inactivates infectious agents as well as marks these infectious agents so they can be recognized by other parts of the immune system. With the ongoing COVID-19 pandemic and the severe to fatal outcomes for certain patient groups, especially people with impaired immunity, these patients and their physicians are interested in whether their antibody replacement therapy also confers protection against SARS-CoV-2 infection. We analyzed the capability of plasma-derived Ig lots to (i) recognize SARS-CoV-2 protein by ELISA method as well as (ii) neutralize SARS-CoV-2 by neutralization studies using the actual virus under biosafety level 3 (BSL-3) conditions. Here we show increasing anti-SARS-CoV-2 activity over time of manufactured Ig lots produced between 12/2020 and 06/2021. The most recent lots had a neutralizing activity of up to 864 IU/mL. Considering that the USA represents Octapharma\u2019s main plasma source, the progress in vaccination levels together with the evolution of the COVID-19 pandemic in this country suggests that the IVIG/SCIG neutralization capacities against SARS-CoV-2 might still increase and could potentially meet a level where antibody plasma concentrations in the patient confer immune protection. Patients with humoral immunodeficiency rely on plasma-derived immunoglobulin for passive immunization against numerous pathogens. SARS-CoV-2 neutralization capacities of plasma-derived immunoglobulins have increased over time with the ongoing COVID-19 pandemic and vaccination campaigns. Plasma-derived immunoglobulin in prophylactic use for immunodeficient patients could potentially protect against SARS-CoV-2 infection in the future.","version":"1.2","doi":"10.1101/2021.08.13.456066","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460111","pub_date":"2021-9-17","title":"Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice","abstract":"The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.","version":"1.3","doi":"10.1101/2021.09.13.460111","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460782","pub_date":"2021-9-17","title":"The nuts and bolts of SARS-CoV-2 Spike Receptor Binding Domain heterologous expression","abstract":"COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful \u201ctool\u201d to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.","version":"1.1","doi":"10.1101/2021.09.17.460782","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.16.460724","pub_date":"2021-9-17","title":"ZRC3308 monoclonal antibody cocktail shows protective efficacy in Syrian hamsters against SARS-CoV-2 infection","abstract":"We have developed a monoclonal antibody (mAb) cocktail (ZRC-3308) comprising of ZRC3308-A7 and ZRC3308-B10 in the ratio 1:1 for COVID-19 treatment. The mAbs were designed to have reduced immune effector functions and increased circulation half-life. mAbs showed good binding affinities to non-competing epitopes on RBD of SARS-CoV-2 spike protein and were found neutralizing SARS-CoV-2 variants B.1, B.1.1.7, B.1.351, B.1.617.2 and B.1.617.2 AY.1 in vitro. The mAb cocktail demonstrated effective prophylactic and therapeutic activity against SARS-CoV-2 infection in Syrian hamsters. The antibody cocktail appears to be a promising candidate for the prophylactic use and for therapy in early COVID-19 cases which have not progressed to severe disease.","version":"1.1","doi":"10.1101/2021.09.16.460724","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460411","pub_date":"2021-9-17","title":"A genetic trap in yeast for inhibitors of SARS-CoV-2 main protease","abstract":"The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the E. coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor down to 10 \u03bcM by a robust growth increase. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates, and is rapidly adaptable for new variants of the protease with deviant site specificities. The COVID-19 pandemic may continue several years before vaccination campaigns can put an end it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease which is required for viral replication. To detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there, will score in the system. Moreover, by its design in yeast, the system is rapidly adaptable for tuning of detection level, eventual modification of protease cleavage site in case of future mutant variants of the SARS-CoV-2 main protease, or even for other proteases.","version":"1.2","doi":"10.1101/2021.09.14.460411","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460745","pub_date":"2021-9-17","title":"A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is strictly limited. Pseudotyped viruses that mimic SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories; however, in vivo application is inadequate. Therefore, animal models that can be instigated with animal BSL-2 will increase opportunities for in vivo evaluations. Hamsters (6-to 10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24 h after inoculation, and luminescence was measured using an in vivo imaging system. Lung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner. VSV-G (envelope [G]) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus. The SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research regarding SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.","version":"1.1","doi":"10.1101/2021.09.17.460745","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.459777","pub_date":"2021-9-17","title":"Real-time monitoring and analysis of SARS-CoV-2 nanopore sequencing with minoTour","abstract":"The ongoing SARS-CoV-2 pandemic has demonstrated the utility of real-time analysis of sequencing data, with a wide range of databases and resources for analysis now available. Here we show how the real-time nature of Oxford Nanopore Technologies sequencers can accelerate consensus generation, lineage and variant status assignment. We exploit the fact that multiplexed viral sequencing libraries quickly generate sufficient data for the majority of samples, with diminishing returns on remaining samples as the sequencing run progresses. We demonstrate methods to determine when a sequencing run has passed this point in order to reduce the time required and cost of sequencing. We extended MinoTour, our real-time analysis and monitoring platform for nanopore sequencers, to provide SARS-CoV2 analysis using ARTIC network pipelines. We additionally developed an algorithm to predict which samples will achieve sufficient coverage, automatically running the ARTIC medaka informatics pipeline once specific coverage thresholds have been reached on these samples. After testing on run data, we find significant run time savings are possible, enabling flow cells to be used more efficiently and enabling higher throughput data analysis. The resultant consensus genomes are assigned both PANGO lineage and variant status as defined by Public Health England. Samples from within individual runs are used to generate phylogenetic trees incorporating optional background samples as well as summaries of individual SNPs. As minoTour uses ARTIC pipelines, new primer schemes and pathogens can be added to allow minoTour to aid in real-time analysis of pathogens in the future. Source code and documentation is available at https://github.com/LooseLab/minotourapp. Supplementary data are available from https://github.com/LooseLab/artic_minotour_analyses.","version":"1.2","doi":"10.1101/2021.09.13.459777","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458877","pub_date":"2021-9-17","title":"Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK (Alpha), South Africa (Beta), and India (Delta)","abstract":"We report here the discovery of several highly potent small molecules that showed low nM potency against SARS-CoV (IC50: as low as 13 nM), SARS-CoV-2 (IC50: as low as 23 nM), and MERS-CoV (IC50: as low as 76 nM) in pseudovirus based assays with excellent selectivity indices (SI: as high as > 5000) demonstrating their pancoronavirus inhibition. Some compounds also show 100% inhibition of CPE (IC100) at 1.25 \u00b5M against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants of concerns (VOCs), such as the UK (B.1.1.7), South Africa (B.1.351), and Delta variant (B.1.617.2), originated in India. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data of one of the most active inhibitors, NBCoV1, show drug-like properties. In vivo PK of NBCoV1 in rats demonstrated excellent half-life (t1/2) of 11.3 h, mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.","version":"1.2","doi":"10.1101/2021.09.03.458877","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.17.460777","pub_date":"2021-9-17","title":"Altered fibrin clot structure contributes to thrombosis risk in severe COVID-19","abstract":"The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in two independent cohorts of critically ill COVID-19 patients in comparison to patients suffering from severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the kaolin clotting time was not prolonged in COVID-19 as compared to ARDS-influenza. Using confocal and electron microscopy, we show that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, we observed clot lysis in 30% of COVID-19 patients and 84% of ARDS-influenza subjects. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19. Together, our results indicate that elevated fibrinogen levels and increased FXII activation rate promote thrombosis and thrombolysis resistance via enhanced thrombus formation and stability in COVID-19.","version":"1.1","doi":"10.1101/2021.09.17.460777","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460338","pub_date":"2021-9-16","title":"Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, Fc\u03b3R activation, and corresponding COVID-19 disease severity","abstract":"Despite SARS-CoV-2 being a \u201cnovel\u201d coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (Fc\u03b3R2a & Fc\u03b3R3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2\u2019FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2\u2019FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.","version":"1.1","doi":"10.1101/2021.09.14.460338","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.16.460663","pub_date":"2021-9-16","title":"Neutralizing antibody-independent immunity to SARS-CoV-2 in hamsters and hACE-2 transgenic mice immunized with a RBD/Nucleocapsid fusion protein","abstract":"The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFN\u03b3 response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.","version":"1.1","doi":"10.1101/2021.09.16.460663","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.15.459215","pub_date":"2021-9-16","title":"Signatures of adaptive evolution during human to mink SARS CoV2 cross-species transmission inform estimates of the COVID19 pandemic timing","abstract":"One of the unique features of SARS-CoV-2 is that it mainly evolved neutrally or under purifying selection during the early pandemic. This contrasts with the preceding epidemics of the closely related SARS-CoV and MERS-CoV, both of which evolved adaptively. It is possible that the SARS-CoV-2 exhibits a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes for efficient transmission before the onset of the current pandemic. In order to test the above scenarios, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parenteral human strains. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratios per site within the spike protein was 2.93, indicating a selection process favoring adaptive amino acid changes. In addition, mutations within this protein concentrated within its receptor binding domain and receptor binding motif. Positive selection also left a trace on linked neutral variation. An excess of high frequency derived variants produced by genetic hitchhiking was found during middle (June to July 2020) and early late (August to September 2020) phases of the mink epidemic, but quickly diminished in October and November 2020. Strong positive selection found in SARS-CoV-2 from minks implies that the virus may be not unique in super-adapting to a wide range of new hosts. The mink study suggests that SARS-CoV-2 already went through adaptive evolution in humans, and likely been circulating in humans at least six months before the first case found in Wuhan, China. We also discuss circumstances under which the virus can be well-adapted to its host but fail to induce an outbreak.","version":"1.1","doi":"10.1101/2021.09.15.459215","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.21263473","pub_date":"2021-09-16","title":"Adverse reactions to BNT162b2 mRNA COVID-19 vaccine in medical staffs with a history of allergy","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) vaccination is progressing globally. Several adverse reactions have been reported with vaccination against COVID-19. It is unknown whether adverse reactions to COVID-19 vaccination are severe in individuals with allergies. We administered the COVID-19 vaccine to the medical staff at Yamagata University Hospital from March to August 2021. Subsequently, we conducted an online questionnaire-based survey to investigate the presence of allergy and adverse reactions after vaccination and examined the association between allergy and adverse reactions after immunization.</jats:p>\n                <jats:p>Responses were collected from 1586 subjects after the first vaccination and 1306 subjects after the second administration of the BNT162b2 mRNA COVID-19 vaccine. Adverse reactions included injection site pain, injection site swelling, fever, fatigue or malaise, headache, chills, nausea, muscle pain outside the injection site, and arthralgia. The frequency and severity of most adverse reactions were higher after the second vaccination compared to the first. The frequency of some adverse reactions and their severity were higher, and the duration of symptoms was longer in subjects with allergies than in subjects without allergies. Although several participants visited the emergency room for treatment after the first and second vaccinations, nobody was diagnosed with anaphylaxis.</jats:p>\n                <jats:p>Given the serious consequence of COVID-19 and the reported high efficacy of this vaccine against this disease, we conclude that vaccination of allergic individuals is generally recommended.</jats:p>","version":null,"doi":"10.1101/2021.09.13.21263473","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.15.460487","pub_date":"2021-9-15","title":"Synthetic Multiantigen MVA Vaccine COH04S1 Protects Against SARS-CoV-2 in Syrian Hamsters and Non-Human Primates","abstract":"Second-generation COVID-19 vaccines could contribute to establish protective immunity against SARS-CoV-2 and its emerging variants. We developed COH04S1, a synthetic multiantigen Modified Vaccinia Ankara-based SARS-CoV-2 vaccine that co-expresses spike and nucleocapsid antigens. Here, we report COH04S1 vaccine efficacy in animal models. We demonstrate that intramuscular or intranasal vaccination of Syrian hamsters with COH04S1 induces robust Th1-biased antigen-specific humoral immunity and cross-neutralizing antibodies (NAb) and protects against weight loss, lower respiratory tract infection, and lung injury following intranasal SARS-CoV-2 challenge. Moreover, we demonstrate that single-dose or two-dose vaccination of non-human primates with COH04S1 induces robust antigen-specific binding antibodies, NAb, and Th1-biased T cells, protects against both upper and lower respiratory tract infection following intranasal/intratracheal SARS-CoV-2 challenge, and triggers potent post-challenge anamnestic antiviral responses. These results demonstrate COH04S1-mediated vaccine protection in animal models through different vaccination routes and dose regimens, complementing ongoing investigation of this multiantigen SARS-CoV-2 vaccine in clinical trials.","version":"1.1","doi":"10.1101/2021.09.15.460487","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460408","pub_date":"2021-9-15","title":"Synthetic lethality-based prediction of anti-SARS-CoV-2 targets","abstract":"Novel strategies are needed to identify drug targets and treatments for the COVID-19 pandemic. The altered gene expression of virus-infected host cells provides an opportunity to specifically inhibit viral propagation via targeting the synthetic lethal (SL) partners of such altered host genes. Pursuing this antiviral strategy, here we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict clinically relevant candidate antiviral targets that are SL with altered host genes. The predicted SL-based targets are highly enriched for infected cell inhibiting genes reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens. Integrating our predictions with the results of these screens, we further selected a focused subset of 26 genes that we experimentally tested in a targeted siRNA screen using human Caco-2 cells. Notably, as predicted, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming non-infected cells. Our results are made publicly available, to facilitate their in vivo testing and further validation.","version":"1.1","doi":"10.1101/2021.09.14.460408","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.21263072","pub_date":"2021-09-15","title":"VALIDATION OF A SALIVA-BASED TEST FOR THE MOLECULAR DIAGNOSIS OF SARS-CoV-2 INFECTION","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>\n                    Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve screening and diagnosis of SARS-CoV-2 infection (\n                    <jats:italic>Y. Yang et al</jats:italic>\n                    .,\n                    <jats:italic>medRxiv 2020; W. Wang et al</jats:italic>\n                    .,\n                    <jats:italic>2020</jats:italic>\n                    .\n                    <jats:italic>3786; A Senok et al</jats:italic>\n                    .,\n                    <jats:italic>Infect Drug Resist 2020)</jats:italic>\n                    . Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab (N. Sethuraman et al., JAMA 2020; A.J. Jamal et al Clinical Infect Disease 2021). Saliva samples, however, offer clear practical and logistical advantages (\n                    <jats:italic>K</jats:italic>\n                    .\n                    <jats:italic>K</jats:italic>\n                    .\n                    <jats:italic>W To et al, Clinical Microb and Infect; A</jats:italic>\n                    .\n                    <jats:italic>L. Wylle et al. N Engl J Med 2020; N. Matic et al, Eur J Clin 2021</jats:italic>\n                    ) but due to lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool (D.\n                    <jats:italic>Esser et al</jats:italic>\n                    .,\n                    <jats:italic>Biomark Insights 2008; E. Kaufman et al</jats:italic>\n                    .,\n                    <jats:italic>Crit Rev Oral Biol Med2002)</jats:italic>\n                    . With this study, we aimed to validate an intra-laboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen\u2019s Kappa=0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.10.21263072","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.14.460264","pub_date":"2021-9-15","title":"Characterizing flexibility and mobility in the natural mutations of the SARS-CoV-2 spikes","abstract":"We use in silico modelling of the SARS-CoV-2 spike protein and its mutations, as deposited on the Protein Data Bank (PDB), to ascertain their dynamics, flexibility and rigidity. Identifying the precise nature of the dynamics for the spike proteins enables, in principle, the use of further in silico design methods to quickly screen for existing and novel drug molecules that might prohibit the natural protein dynamics. We employ a recent protein flexibility modeling approach, combining methods for deconstructing a protein structure into a network of rigid and flexible units with a method that explores the elastic modes of motion of this network, and a geometric modeling of flexible motion. Our results thus far indicate that the overall motion of wild-type and mutated spike protein structures remains largely the same.","version":"1.1","doi":"10.1101/2021.09.14.460264","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.15.460454","pub_date":"2021-9-15","title":"Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing","abstract":"The natural habitat of SARS-CoV-2 is the cytoplasm of a mammalian cell where it replicates its genome and expresses its proteins. While SARS-CoV-2 genes and hence its codons are presumably well optimized for mammalian protein translation, they have not been sequence optimized for nuclear expression. The cDNA of the Spike protein harbors over a hundred predicted splice sites and produces mostly aberrant mRNA transcripts when expressed in the nucleus. While different codon optimization strategies increase the proportion of full-length mRNA, they do not directly address the underlying splicing issue with commonly detected cryptic splicing events hindering the full expression potential. Similar splicing characteristics were also observed in other transgenes. By inserting multiple short introns throughout different transgenes, significant improvement in expression was achieved, including >7-fold increase for Spike transgene. Provision of a more natural genomic landscape offers a novel way to achieve multi-fold improvement in transgene expression.","version":"1.1","doi":"10.1101/2021.09.15.460454","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.15.460526","pub_date":"2021-9-15","title":"Dry heat sterilization as a method to recycle N95 respirator masks: the importance of fit","abstract":"In times of crisis, including the current COVID-19 pandemic, the supply chain of filtering facepiece respirators, such as N95 respirators, are disrupted. To combat shortages of N95 respirators, many institutions were forced to decontaminate and reuse respirators. While several reports have evaluated the impact on filtration as a measurement of preservation of respirator function after decontamination, the equally important fact of maintaining proper fit to the users\u2019 face has been understudied. In the current study, we demonstrate the complete inactivation of SARS-CoV-2 and preservation of fit test performance of N95 respirators following treatment with dry heat. We apply scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), X-ray diffraction (XRD) measurements, Raman spectroscopy, and contact angle measurements to analyze filter material changes as a consequence of different decontamination treatments. We further compared the integrity of the respirator after autoclaving versus dry heat treatment via quantitative fit testing and found that autoclaving, but not dry heat, causes the fit of the respirator onto the users face to fail, thereby rendering the decontaminated respirator unusable. Our findings highlight the importance to account for both efficacy of disinfection and mask fit when reprocessing respirators to for clinical redeployment.","version":"1.1","doi":"10.1101/2021.09.15.460526","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460185","pub_date":"2021-9-14","title":"Effect of ORF7 of SARS-CoV-2 on the chemotaxis of monocytes and neutrophils in vitro","abstract":"Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threats in worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.","version":"1.1","doi":"10.1101/2021.09.13.460185","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460191","pub_date":"2021-9-14","title":"Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery Against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques","abstract":"Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Post-pyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that post-pyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here we report that oral administration of live SARS-CoV-2 in non-human primates may offer prophylactic benefits, but that formulation and route of administration will require further optimization.","version":"1.1","doi":"10.1101/2021.09.13.460191","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460394","pub_date":"2021-9-14","title":"Anti-SARS-CoV-2 IgG and IgA antibodies in COVID-19 convalescent plasma do not facilitate antibody-dependent enhance of viral infection","abstract":"The novel coronavirus SARS-CoV2, which causes COVID-19, has resulted in the death of nearly 4 million people within the last 18 months. While preventive vaccination and monoclonal antibody therapies have been rapidly developed and deployed, early in the pandemic the use of COVID-19 convalescent plasma (CCP) was a common means of passive immunization, with the theoretical risk of antibody-dependent enhancement (ADE) of viral infection remaining undetermined. Though vaccines elicit a strong and protective immune response, and transfusion of CCP with high titers of neutralization activity are correlated with better clinical outcomes, the question of whether antibodies in CCP can enhance infection of SARS-CoV2 has not been directly addressed. In this study, we analyzed for and observed passive transfer of neutralization activity with CCP transfusion. Furthermore, to specifically understand if antibodies against the spike protein (S) enhance infection, we measured the anti-S IgG, IgA, and IgM responses and adapted retroviral-pseudotypes to measure virus neutralization with target cells expressing the ACE2 virus receptor and the Fc alpha receptor (Fc\u03b1R) or Fc gamma receptor IIA (Fc\u03b3RIIA). Whereas neutralizing activity of CCP correlated best with higher titers of anti-S IgG antibodies, the neutralizing titer was not affected when Fc receptors were present on target cells. These observations support the absence of antibody-dependent enhancement of infection (ADE) by IgG and IgA isotypes found in CCP. The results presented, therefore, support the clinical use of currently available antibody-based treatment including the continued study of CCP transfusion strategies.","version":"1.1","doi":"10.1101/2021.09.14.460394","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460356","pub_date":"2021-9-14","title":"Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display","abstract":"The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.09.14.460356","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460211","pub_date":"2021-9-14","title":"Mitoxantrone dihydrochloride, an FDA approved drug, binds with SARS-CoV-2 NSP1 C-terminal","abstract":"One of the major virulence factors of SARS-CoV-2, NSP1, is a vital drug target due to its role in host immune evasion through multiple pathways. NSP1 protein is associated with inhibiting host mRNA translation by binding to the small subunit of ribosome through its C-terminal region. Previously, we have shown the structural dynamics of NSP1 C-terminal region (NSP1-CTR) in different physiological environments. So, it would be very interesting to investigate the druggable compounds that could bind with NSP1-CTR. Here, in this article, we have performed the different spectroscopic technique-based binding assays of an anticancer drug Mitoxantrone dihydrochloride (MTX) against the NSP1-CTR. We have also performed molecular docking followed by computational simulations with two different forcefields up to one microsecond. Overall, our results have suggested good binding between NSP1-CTR and MTX and may have implications in developing therapeutic strategies targeting NSP1 protein of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.09.14.460211","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460246","pub_date":"2021-9-14","title":"Prolific Induction of IL-6 in Human Cells by SARS-CoV-2-derived Peptide is Attenuated by Recombinant Human Anti-inflammatory Cytokines made in planta","abstract":"Development of efficient therapies for COVID-19 is the focus of intense research. The cytokine release syndrome was underlined as a culprit for severe outcomes in COVID-19 patients. Interleukin-6 (IL-6) plays a crucial role in human immune responses and elevated IL-6 plasma levels have been associated with the exacerbated COVID-19 pathology. Since non-structural protein 10 (NSP10) of SARS-CoV-2 has been implicated in the induction of IL-6, we designed Peptide (P)1, containing sequences corresponding to amino acids 68-96 of NSP10, and examined its effect on cultured human cells. Treatment with P1 strongly increased IL-6 secretion by the lung cancer cell line NCI-H1792 and the breast cancer cell line MDA-MB-231 and revealed profound cytotoxic activity on Caco-2 colorectal adenocarcinoma cells. Treatment with P2, harbouring a mutation in the zinc knuckle motif of NSP10, caused no IL-6 induction and no cytotoxicity. Pre-treatment with plant-produced human anti-inflammatory cytokines IL-37b and IL-38 effectively mitigated the induction of IL-6 secretion. Our results suggest a role for the zinc knuckle motif of NSP10 in the onset of increased IL-6 plasma levels of COVID-19 patients and for IL-37b and IL-38 as therapeutics aimed at attenuating the cytokine release syndrome.","version":"1.1","doi":"10.1101/2021.09.14.460246","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.14.460275","pub_date":"2021-9-14","title":"SARS-CoV-2 Spike Protein Regulation of Angiotensin Converting Enzyme 2 and Tissue Renin-Angiotensin Systems: Influence of Biologic Sex","abstract":"Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.","version":"1.1","doi":"10.1101/2021.09.14.460275","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460163","pub_date":"2021-9-14","title":"BNT162b2-Elicited Neutralization of Delta Plus, Lambda, and Other Variants","abstract":"BNT162b2-elicited human sera are known to neutralize the currently dominant Delta SARS-CoV-2 variant. Here, we report the ability of 20 human sera, drawn 2 or 4 weeks after two doses of BNT162b2, to neutralize USA-WA1/2020 SARS-CoV-2 bearing variant spikes from Delta plus (Delta-AY.1, Delta-AY.2), Delta-\u0394144 (Delta with the Y144 deletion of the Alpha variant), Lambda, and B. 1.1.519 lineage viruses. Geometric mean plaque reduction neutralization titers against Delta-AY.1, Delta-AY.2, and Delta-\u0394144 viruses are slightly lower than against USA-WA1/2020, but all sera neutralize the variant viruses to titers of \u226580. Neutralization titers against Lambda and B. 1.1.519 variants and against USA-WA1/2020 are equivalent. The susceptibility of Delta plus, Lambda, and other variants to neutralization by the sera indicates that antigenic change has not led to virus escape from vaccine-elicited neutralizing antibodies and supports ongoing mass immunization with BNT162b2 to control the variants and to minimize the emergence of new variants.","version":"1.1","doi":"10.1101/2021.09.13.460163","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459410","pub_date":"2021-9-13","title":"Pharmacological perturbation of intracellular dynamics as a SARS-CoV-2 antiviral strategy","abstract":"SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.","version":"1.2","doi":"10.1101/2021.09.10.459410","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.166397","pub_date":"2021-9-13","title":"A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing","abstract":"Global efforts to combat the Covid-19 pandemic caused by SARS-CoV-2 still heavily rely on RT-qPCR-based diagnostic tests. However, their high cost, moderate throughput and reliance on sophisticated equipment limit widespread implementation. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. We present a rapid, robust, sensitive and versatile RT-LAMP based SARS-CoV-2 detection assay. Our forty-minute procedure bypasses a dedicated RNA isolation step, is insensitive to carry-over contamination, and uses a hydroxynaphthol blue (HNB)-based colorimetric readout, which allows robust SARS-CoV-2 detection from various sample types. Based on this assay, we have substantially increased sensitivity and scalability by a simple nucleic acid enrichment step (bead-LAMP), established a pipette-free version for home testing (HomeDip-LAMP), and developed open source enzymes that can be produced in any molecular biology setting. Our advanced, universally applicable RT-LAMP assay is a major step towards population-scale SARS-CoV-2 testing.","version":"1.3","doi":"10.1101/2020.06.23.166397","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460130","pub_date":"2021-9-13","title":"Elucidation of the interactions between SARS-CoV-2 Spike protein and wild and mutant types of IFITM proteins by in silico methods","abstract":"COVID-19 is a viral disease that has been a threat to the whole world since 2019. Although effective vaccines against the disease have been developed, there are still points to be clarified about the mechanism of SARS-CoV-2, which is the causative agent of COVID-19. In this study, we determined the binding energies and the bond types of complexes formed by open (6VYB) and closed (6VXX) forms of the Spike protein of SARS-CoV-2 and wild and mutant forms of IFITM1, IFITM2, and IFITM3 proteins using the molecular docking approach. First, all missense SNPs were found in the NCBI Single Nucleotide Polymorphism database (dbSNP) for IFITM1, IFITM2, and IFITM3 and analyzed with SIFT, PROVEAN, PolyPhen-2, SNAP2, Mutation Assessor, and PANTHER cSNP web-based tools to determine their pathogenicity. When at least four of these analysis tools showed that the SNP had a pathogenic effect on the protein product, this SNP was saved for further analysis. Delta delta G (DDG) and protein stability analysis for amino acid changes were performed in the web-based tools I-Mutant, MUpro, and SAAFEC-SEQ. The structural effect of amino acid change on the protein product was made using the HOPE web-based tool. HawkDock server was used for molecular docking and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) analysis and binding energies of all complexes were calculated. BIOVIA Discovery Studio program was utilized to visualize the complexes. Hydrogen bonds, salt bridges, and non-bonded contacts between Spike and IFITM protein chains in the complexes were detected with the PDBsum web-based tool. The best binding energy among the 6VYB-IFITM wild protein complexes belong to 6VYB-IFITM1 (-46.16 kcal/mol). Likewise, among the 6VXX-IFITM wild protein complexes, the most negative binding energy belongs to 6VXX-IFITM1 (-52.42 kcal/mol). An interesting result found in the study is the presence of hydrogen bonds between the cytoplasmic domain of the IFITM1 wild protein and the S2 domain of 6VYB. Among the Spike-IFITM mutant protein complexes, the best binding energy belongs to the 6VXX-IFITM2 N63S complex (-50.77 kcal/mol) and the worst binding energy belongs to the 6VXX-IFITM3 S50T complex (4.86 kcal/mol). The study suggests that IFITM1 protein may act as a receptor for SARS-CoV-2 Spike protein. Assays must be advanced from in silico to in vitro for the determination of the receptor-ligand interactions between IFITM proteins and SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.09.13.460130","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460054","pub_date":"2021-9-13","title":"The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression","abstract":"SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.","version":"1.1","doi":"10.1101/2021.09.13.460054","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.12.459978","pub_date":"2021-9-13","title":"Structure-activity relationships of B.1.617 and other SARS-CoV-2 spike variants","abstract":"The surge of COVID-19 infection cases is spurred by emerging SARS-CoV-2 variants such as B.1.617. Here we report 38 cryo-EM structures, corresponding to the spike protein of the Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Kappa (B.1.617.1) variants in different functional states with and without its receptor, ACE2. Mutations on the N-terminal domain not only alter the conformation of the highly antigenic supersite of the Delta variant, but also remodel the glycan shield by deleting or adding N-glycans of the Delta and Gamma variants, respectively. Substantially enhanced ACE2 binding was observed for all variants, whose mutations on the receptor binding domain modulate the electrostatics of the binding interfaces. Despite their abilities to escape host immunity, all variants can be potently neutralized by three unique antibodies.","version":"1.1","doi":"10.1101/2021.09.12.459978","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.13.460076","pub_date":"2021-9-13","title":"Elicitation of potent SARS-CoV-2 neutralizing antibody responses through immunization using a versatile adenovirus-inspired multimerization platform","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has shown that vaccine preparedness is critical to anticipate a fast response to emergent pathogens with high infectivity. To rapidly reach herd immunity, an affordable, easy to store and versatile vaccine platform is thus desirable. We previously designed a non-infectious adenovirus-inspired nanoparticle (ADDomer), and in the present work, we efficiently decorated this original vaccine platform with glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryo-Electron Microscopy structure revealed that up to 60 copies of this antigenic domain were bound on a single ADDomer particle with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated particles showed as early as the first immunization a significant anti-coronavirus humoral response, which was boosted after a second immunization. Neutralization assays with spike pseudo-typed-virus demonstrated the elicitation of strong neutralization titers. Remarkably, the existence of pre-existing immunity against adenoviral-derived particles enhanced the humoral response against SARS-CoV-2. This plug and play vaccine platform revisits the way of using adenovirus to combat emergent pathogens while potentially taking advantage of the adenovirus pre-immunity.","version":"1.1","doi":"10.1101/2021.09.13.460076","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.21262027","pub_date":"2021-09-13","title":"A simple, sensitive and quantitative FACS-based test for SARS-CoV-2 serology in humans and animals","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Serological tests are important for understanding the physiopathology and following the evolution of the Covid-19 pandemic. Assays based on flow cytometry (FACS) of tissue culture cells expressing the spike (S) protein of SARS-CoV-2 have repeatedly proven to perform slightly better than the plate-based assays ELISA and CLIA (chemiluminescent immuno-assay), and markedly better than lateral flow immuno-assays (LFIA).</jats:p>\n                <jats:p>Here, we describe an optimized and very simple FACS assay based on staining a mix of two Jurkat cell lines, expressing either high levels of the S protein (Jurkat-S) or a fluorescent protein (Jurkat-R expressing m-Cherry, or Jurkat-G, expressing GFP, which serve as an internal negative control). We show that the Jurkat-S&amp;R-flow test has a much broader dynamic range than a commercial ELISA test and performs at least as well in terms of sensitivity and specificity. Also, it is more sensitive and quantitative than the hemagglutination-based test HAT, which we described recently. The Jurkat-flow test requires only a few microliters of blood; thus, it can be used to quantify various Ig isotypes in capillary blood collected from a finger prick. It can be used also to evaluate serological responses in mice, hamsters, cats and dogs. Whilst the Jurkat-flow test is ill-suited and not intended for clinical use, it offers a very attractive solution for laboratories with access to tissue culture and flow cytometry who want to monitor serological responses in humans or in animals, and how these relate to susceptibility to infection, or re-infection, by the virus, and to protection against Covid-19.</jats:p>\n                <jats:sec>\n                  <jats:title>Note</jats:title>\n                  <jats:p>\n                    <jats:bold>This manuscript has been refereed</jats:bold>\n                    by Review Commons, and modified thanks to the comments and suggestions from two referees. Those comments, and our replies, are provided at the end of the manuscript\u2019s pdf, and can also be accessed by clicking on the box with a little green number found just above the \u201cAbstract \u201c tab in the medRXiv window.\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.06.21262027","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.08.21263276","pub_date":"2021-09-13","title":"\u201cAssociation between COVID-19 vaccination, infection, and risk of Guillain-Barre syndrome, Bell\u2019s palsy, encephalomyelitis and transverse myelitis: a population-based cohort and self-controlled case series analysis\u201d","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>OBJECTIVE</jats:title>\n                  <jats:p>We aimed to study the association between COVID-19 vaccines, SARS-CoV-2 infection, and the risk of immune-mediated neurological events.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:sec>\n                    <jats:title>Design</jats:title>\n                    <jats:p>Population-based historical rate comparison study and self-controlled case series (SCCS) analysis.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Setting</jats:title>\n                    <jats:p>Primary care records from the United Kingdom.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Participants</jats:title>\n                    <jats:p>Individuals who received the first dose of ChAdOx1 or BNT162b2 between 8 December 2020 and 6 March 2021. A cohort with a first positive RT-PCR test for SARS-CoV-2 between 1 September 2020 and 28 February 2021 was used for comparison.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Main outcome measures</jats:title>\n                    <jats:p>Outcomes included Guillain-Barre syndrome (GBS), Bell\u2019s palsy, encephalomyelitis, and transverse myelitis.</jats:p>\n                    <jats:p>Incidence rates were estimated in the 28 days post first-dose vaccine, 90 days post-COVID-19, and between 2017 to 2019 for the general population cohort for background rates. Indirectly standardised incidence ratios (SIRs) were estimated. Adjusted incidence rate ratios (IRR) were estimated from the SCCS when sufficient statistical power was reached.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Results</jats:title>\n                    <jats:p>We included 1,868,767 ChAdOx1 and 1,661,139 BNT162b2 vaccinees; 299,311 people infected with COVID-19; and 2,290,537 from the general population. SIRs for GBS were 1.91 [95% CI: 0.86 to 4.26] after ChAdOx1, 1.29 [0.49 to 3.45] after BNT162b2, and 5.20 [1.95 to 13.85] after COVID-19. In the same cohorts, SIRs for Bell\u2019s palsy were 1.34 [1.05 to 1.72], 1.15 [0.88 to 1.50], and 1.23 [0.80 to 1.89], and for encephalomyelitis 1.62 [0.61 to 4.31], 0.86 [0.22 to 3.46], and 11.05 [5.27 to 23.17], respectively. Transverse myelitis was too rare to analyse (n&lt;5 in all cohorts). SCCS analysis was only conducted for Bell\u2019s palsy due to limited statistical power. We found no association between either vaccine and Bell\u2019s palsy, with an IRR of 1.10 [0.81 to 1.46] and 1.15 [0.87 to 1.49] for BNT162b2 and ChAdOx1, respectively.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions</jats:title>\n                    <jats:p>We found no consistent association between either vaccine and any of the studied neuroimmune adverse events studied. Conversely, we found a 5-fold increase in risk of GBS and an 11-fold of encephalomyelitis following COVID-19.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.08.21263276","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.13.460032","pub_date":"2021-9-13","title":"Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector","abstract":"Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.","version":"1.1","doi":"10.1101/2021.09.13.460032","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.11.459879","pub_date":"2021-9-13","title":"Ultrafast, one-step, and microwave heating-based synthesis of DNA/RNA-AuNP conjugates","abstract":"DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here we discovered that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies showed that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of such a SNA structure. In further applications study, CRISPR/Cas9-sgRNA (135 bp), RNA from Nucleocapsid (N) gene of SARS-CoV-2 (1279 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) could be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in novel biosensing and nucleic acids delivery strategy. This novel heating-dry strategy has improved the traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.","version":"1.1","doi":"10.1101/2021.09.11.459879","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.11.459891","pub_date":"2021-9-12","title":"Phylogenetic evidence for asparagine to aspartic acid protein editing of N-glycosylated SARS-CoV-2 viral proteins by NGLY1 deglycosylation/deamidation suggests an unusual vaccination strategy","abstract":"Many viral proteins, including multiple SARS-CoV-2 proteins, are secreted via the endoplasmic reticulum, and viral particles are assembled and exported in ER-associated replication compartments. Viral coat proteins such as the SARS-CoV-2 Spike protein are N-glycosylated at NxS/T sites as they enter the ER. N-glycosylated sites in many eukaryotic proteins are deglycosylated by the NGLY1/PNG-1 deglycosylation enzyme which also deamidates the N-glycosylated asparagine to aspartic acid, thus editing the target protein sequence. Proteomic analysis of mammalian cell lines has revealed deamidation of many host N-glycosylated asparagines to aspartic acid by NGLY1/PNG-1 on peptides that are presented by mammalian HLA for immune surveillance. The key client protein for NGLY1/PNG-1 deglycosylation and N to D protein editing was revealed by genetic analysis of C. elegans proteasome regulation to be the intact endoplasmic reticulum-transiting SKN-1A transcription factor. Strikingly, an analysis of cancer cell genetic dependencies for growth revealed that the mammalian orthologue of SKN-1A, NRF1 (also called NFE2L1) is required by a highly correlated set of cell lines as NGLY1/PNG-1, supporting that NGLY1/PNG-1 and NRF1 act in the same pathway. NGLY1/PNG-1 edits N-glycosylated asparagines on the intact SKN-1 protein as it is retrieved by ERAD from the ER to in turn activate the transcription of target proteasomal genes. The normal requirement for NGLY1/PNG-1 editing of SKN-1A can be bypassed by a genomic substituion of N to D in four NxS/T N-glycosylation motifs of SKN-1A. Thus NGLY1/PNG-1-mediated N to D protein editing is more than a degradation step for the key client protein for proteasomal homeostasis in C. elegans or tumor growth in particular mammalian cell lines, SKN-1A/NRF1. In addition, such N to D substitutions in NxS/T N-glycosylation motifs occur in evolution: N to D substitutions are observed in phylogenetic comparisons of SKN-1A between nematode species that diverged hundreds of millions of years ago or of the vertebrate NRF1 between disparate vertebrates. Genomic N to D mutations bypass the many steps in N-glycosylation in the ER and deglycosylation-based editing of N to D, perhaps based on differences in the competency of divergent species for various N-glycosylation or deglycosylation steps. We surveyed the N-glycosylation sites in coronavirus proteins for such phylogenetic evidence for N to D protein editing in viral life cycles, and found evidence for preferential N to D residue substitutions in NxS/T N-glycosylation sites in comparisons of the genome sequences of hundreds of coronaviruses. This suggests that viruses use NGLY1/PNG-1 in some hosts, for example humans, to edit particular N-glycosylated residues to aspartic acid, but that in other hosts, often in bats, an N to D substitution mutation in the virus genome is selected. Single nucleotide mutations in Asp or Asn codons can produce viruses with N to D or D to N substitutions that might be selected in different animal hosts from the population of viral variants produced in any previous host. NGLY1/PNG-1 has been implicated in viral immunity in mammalian cell culture, favoring this hypothesis. Because of the phylogenetic evidence that the NGLY1/PNG-1 editing of protein sequences has functional importance for SKN-1A/NRF1 and viruses, and because most immunization protocols do not address the probable editing and functional importance of N-glycosylated aspargines to aspartic acid in normal viral infections, we suggest that immunization with viral proteins engineered to substitute D at genomically encoded NxS/T sites of N-glycosylated viral proteins that show a high frequency of N to D substitution in viral phylogeny may enhance immunological response to peptide antigens. Such genomically-edited peptides would not require ER-localization for N-glycosylation or other cell compartment localization for NGLY1/PNG-1 N to D protein editing. In addition, such N to D edited protein vaccines could be produced in bacteria since N-glycosylation and deglycosylation which do not occur in bacteria would no longer be required to immunize with a D-substituted peptide. Bacterially-expressed vaccines would be much lower cost and with fewer failure modes than attenuated viral vaccines or recombinant animal viruses produced in chicken eggs, mammalian tissue culture cells, or delivered by mRNA vectors to the patient directly. Because N to D edited peptides are clearly produced by NGLY1/PNG-1, and may be and presented by mammalian HLA, such peptides may more robustly activate T-cell killing or B-cell maturation to mediate more robust viral immunity.","version":"1.1","doi":"10.1101/2021.09.11.459891","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.11.459886","pub_date":"2021-9-12","title":"Adaptive convergent evolution of genome proofreading in SARS-CoV2: insights into the Eigen\u2019s paradox","abstract":"Evolutionary history of coronaviruses holds the key to understand mutational behavior and prepare for possible future outbreaks. By performing comparative genome analysis of nidovirales that contain the family of coronaviruses, we traced the origin of proofreading, surprisingly to the eukaryotic antiviral component ZNFX1. This common recent ancestor contributes two zinc finger (ZnF) motifs that are unique to viral exonuclease, segregating them from DNA proof-readers. Phylogenetic analyses indicate that following acquisition, genomes of coronaviruses retained and further fine-tuned proofreading exonuclease, whereas related families harbor substitution of key residues in ZnF1 motif concomitant to a reduction in their genome sizes. Structural modelling followed by simulation suggests the role of ZnF in RNA binding. Key ZnF residues strongly coevolve with replicase, and the helicase involved in duplex RNA unwinding. Hence, fidelity of replication in coronaviruses is a result of convergent evolution, that enables maintenance of genome stability akin to cellular proofreading systems.","version":"1.1","doi":"10.1101/2021.09.11.459886","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459756","pub_date":"2021-9-11","title":"Streamlined use of protein structures in variant analysis","abstract":"Variant analysis is a core task in bioinformatics that requires integrating data from many sources. This process can be helped by using 3D structures of proteins, which can provide a spatial context that can provide insight into how variants affect function. Many available tools can help with mapping variants onto structures; but each has specific restrictions, with the result that many researchers fail to benefit from valuable insights that could be gained from structural data. To address this, we have created a streamlined system for incorporating 3D structures into variant analysis. Variants can be easily specified via URLs that are easily readable and writable, and use the notation recommended by the Human Genome Variation Society (HGVS). For example, \u2018https://aquaria.app/SARS-CoV-2/S/?N501Y\u2019 specifies the N501Y variant of SARS-CoV-2 S protein. In addition to mapping variants onto structures, our system provides summary information from multiple external resources, including COSMIC, CATH-FunVar, and PredictProtein. Furthermore, our system identifies and summarizes structures containing the variant, as well as the variant-position. Our system supports essentially any mutation for any well-studied protein, and uses all available structural data \u2014 including models inferred via very remote homology \u2014 integrated into a system that is fast and simple to use. By giving researchers easy, streamlined access to a wealth of structural information during variant analysis, our system will help in revealing novel insights into the molecular mechanisms underlying protein function in health and disease. Our resource is freely available at the project home page (https://aquaria.app). After peer review, the code will be openly available via a GPL version 2 license at https://github.com/ODonoghueLab/Aquaria. PSSH2, the database of sequence-to-structure alignments, is also freely available for download at https://zenodo.org/record/4279164. sean@odonoghuelab.org None.","version":"1.1","doi":"10.1101/2021.09.10.459756","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459749","pub_date":"2021-9-10","title":"Intranasal administration of a VLP-based vaccine against COVID-19 induces neutralizing antibodies against SARS-CoV-2 and Variants of Concerns","abstract":"The highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of COVID-19 vaccines. In the current study, we tested in murine model the immunogenicity of a conventional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal vaccination. The candidate vaccine, CuMVTT-RBD, has been immunologically optimized to incorporate tetanus-toxin and is self-adjuvanted with TLR7/8 ligands. CuMVTT-RBD elicited strong RBD- and spike- specific systemic IgG and IgA antibody responses of high avidity. Local immune responses were assessed and results demonstrate strong mucosal antibody and plasma cell production in lung tissue. The induced systemic antibodies could efficiently recognize and neutralize different Variants of Concerns of mutated SARS-CoV-2 RBDs. In summary, intranasal vaccination with CuMVTT-RBD shows high immunogenicity and induces protective systemic and local specific antibody response against SARS-CoV-2 and its variants. Evaluation of an intransal administrated conventional VLP-based vaccine against COVID-19 in a murine model.","version":"1.1","doi":"10.1101/2021.09.10.459749","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.09.459504","pub_date":"2021-9-10","title":"Differential antibody dynamics to SARS-CoV-2 infection and vaccination","abstract":"Optimal immune responses furnish long-lasting (durable) antibodies protective across dynamically mutating viral variants (broad). To assess robustness of mRNA vaccine-induced immunity, we compared antibody durability and breadth after SARS-CoV-2 infection and vaccination. While vaccination delivered robust initial virus-specific antibodies with some cross-variant coverage, pre-variant SARS-CoV-2 infection-induced antibodies, while modest in magnitude, showed highly stable long-term antibody dynamics. Vaccination after infection induced maximal antibody magnitudes with enhanced longitudinal stability while infection-na\u00efve vaccinee antibodies fell with time to post-infection-alone levels. The composition of antibody neutralizing activity to variant relative to original virus also differed between groups, with infection-induced antibodies demonstrating greater relative breadth. Differential antibody durability trajectories favored COVID-19-recovered subjects with dual memory B cell features of greater early antibody somatic mutation and cross-coronavirus reactivity. By illuminating an infection-mediated antibody breadth advantage and an anti-SARS-CoV-2 antibody durability-enhancing function conferred by recalled immunity, these findings may serve as guides for ongoing vaccine strategy improvement.","version":"1.1","doi":"10.1101/2021.09.09.459504","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459744","pub_date":"2021-9-10","title":"Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants","abstract":"The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is therefore paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.","version":"1.1","doi":"10.1101/2021.09.10.459744","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459800","pub_date":"2021-9-10","title":"pH-dependent polymorphism of the structure of SARS-CoV-2 nsp7","abstract":"The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at acidic and basic conditions, SARS-CoV-2 nsp7 has a helical bundle folding at neutral pH. Remarkably, the \u03b14 helix shows gradual dislocation from the core \u03b12-\u03b13 structure as pH increases from 6.5 to 7.5. The protonation state of residue H36 contributes to the change of nsp7\u2019s intramolecular interactions, and thus, to the structural variation near-neutral pH. Spin-relaxation results revealed that all three loop regions in nsp7 possess dynamic properties associated with this structural variation.","version":"1.1","doi":"10.1101/2021.09.10.459800","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455960","pub_date":"2021-9-10","title":"SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host","abstract":"SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 \u2013 the closest bat relative \u2013 spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.","version":"1.2","doi":"10.1101/2021.08.11.455960","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.10.459786","pub_date":"2021-9-10","title":"Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity","abstract":"Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity - and not inverse similarity - between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.","version":"1.1","doi":"10.1101/2021.09.10.459786","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.446468","pub_date":"2021-9-10","title":"Pathogenic neutrophilia drives acute respiratory distress syndrome in severe COVID-19 patients","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing COVID-19 pandemic have caused \u223c40 million cases and over 648,000 deaths in the United States alone. Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African-American (AA) patients in some areas, yet targeted studies within this demographic are scant. Multi-omics single-cell analyses of immune profiles from airways and matching blood samples of Black/AA patients revealed low viral load, yet pronounced and persistent pulmonary neutrophilia with advanced features of cytokine release syndrome and acute respiratory distress syndrome (ARDS), including exacerbated production of IL-8, IL-1\u03b2, IL-6, and CCL3/4 along with elevated levels of neutrophil elastase and myeloperoxidase. Circulating S100A12+/IFITM2+ mature neutrophils are recruited via the IL-8/CXCR2 axis, which emerges as a potential therapeutic target to reduce pathogenic neutrophilia and constrain ARDS in severe COVID-19. The lung pathology due to severe COVID-19 is marked by a perpetual pathogenic neutrophilia, leading to acute respiratory distress syndrome (ARDS) even in the absence of viral burden. Circulating mature neutrophils are recruited to the airways via IL-8 (CXCL8)/CXCR2 chemotaxis. Recently migrated neutrophils further differentiate into a transcriptionally active and hyperinflammatory state, with an exacerbated expression of IL-8 (CXCL8), IL-1\u03b2 (IL1B), CCL3, CCL4, neutrophil elastase (NE), and myeloperoxidase (MPO) activity. Airway neutrophils and recruited inflammatory monocytes further increase their production of IL-8 (CXCL8), perpetuating lung neutrophilia in a feedforward loop. MdCs and T cells produce IL-1\u03b2 and TNF, driving neutrophils reprogramming and survival.","version":"1.2","doi":"10.1101/2021.06.02.446468","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437274","pub_date":"2021-9-09","title":"Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants","abstract":"Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2G\u0394HR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with the same potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2G\u0394HR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in mice. Compared with the soluble spike, the I3-01v9 SApNP showed 6-fold longer retention, 4-fold greater presentation on follicular dendritic cell dendrites, and 5-fold stronger germinal center reactions in lymph node follicles. With a well-defined mechanism, spike nanoparticle vaccines can effectively counter SARS-CoV-2 variants.","version":"1.4","doi":"10.1101/2021.03.26.437274","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.09.459577","pub_date":"2021-9-09","title":"A virus-encoded microRNA contributes to evade innate immune response during SARS-CoV-2 infection","abstract":"SARS-CoV-2 infection results in impaired interferon response in severe COVID-19 patients. However, how SARS-CoV-2 interferes with host immune response is incompletely understood. Here, we sequenced small RNAs from SARS-CoV-2-infected human cells and identified a micro-RNA (miRNA) encoded in a recently evolved region of the viral genome. We show that the virus-encoded miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer and they are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3\u2019UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 employs a virus-encoded miRNA to hijack the host miRNA machinery and evade the interferon-mediated immune response.","version":"1.1","doi":"10.1101/2021.09.09.459577","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459535","pub_date":"2021-9-09","title":"SARS-CoV-2 triggers DNA damage response in Vero E6 cells","abstract":"The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.","version":"1.1","doi":"10.1101/2021.09.08.459535","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449893","pub_date":"2021-9-09","title":"Circulating multimeric immune complexes drive immunopathology in COVID-19","abstract":"A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is traditionally considered to be protective, excessive triggering of activating Fc-gamma-receptors (Fc\u03b3Rs) could be detrimental and cause immunopathology. Here, we document that patients who develop soluble circulating IgG immune complexes (sICs) during infection are subject to enhanced immunopathology driven by Fc\u03b3R activation. Utilizing cell-based reporter systems we provide evidence that sICs are predominantly formed prior to a specific humoral response against SARS-CoV-2. sIC formation, together with increased afucosylation of SARS-CoV-2 specific IgG eventually leads to an enhanced CD16 (Fc\u03b3RIII) activation of immune cells reaching activation levels comparable active systemic lupus erythematosus (SLE) disease. Our data suggest a vicious cycle of escalating immunopathology driven by an early formation of sICs in predisposed patients. These findings reconcile the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19. The identification of sICs as drivers of an escalating immunopathology in predisposed patients opens new avenues regarding intervention strategies to alleviate critical COVID-19 progression. A vicious cycle of immunopathology in COVID-19 patients is driven by soluble multimeric immune complexes (sICs). SARS-CoV-2 infection triggers sIC formation in prone individuals. Activation of Fc\u03b3RIII/CD16 expressing immune cells by sICs precedes a humoral response to SARS-CoV2 infection. sICs and infection add to IgG afucosylation, further enhancing Fc\u03b3RIII/CD16 activation by opsonized targets. High inflammation induces further sIC mediated immune cell activation ultimately leading to an escalating immunopathology.","version":"1.4","doi":"10.1101/2021.06.25.449893","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.09.459641","pub_date":"2021-9-09","title":"Targeting Stem-loop 1 of the SARS-CoV-2 5\u2019UTR to suppress viral translation and Nsp1 evasion","abstract":"SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5\u2019 UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5\u2019 UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus\u2019 own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.","version":"1.1","doi":"10.1101/2021.09.09.459641","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.09.459664","pub_date":"2021-9-09","title":"An adjuvanted SARS-CoV-2 RBD nanoparticle elicits neutralizing antibodies and fully protective immunity in aged mice","abstract":"Development of affordable and effective vaccines that can also protect vulnerable populations such as the elderly from COVID-19-related morbidity and mortality is a public health priority. Here we took a systematic and iterative approach by testing several SARS-CoV-2 protein antigens and adjuvants to identify a combination that elicits neutralizing antibodies and protection in young and aged mice. In particular, SARS-CoV-2 receptorbinding domain (RBD) displayed as a protein nanoparticle (RBD-NP) was a highly effective antigen, and when formulated with an oil-in-water emulsion containing Carbohydrate fatty acid MonoSulphate derivative (CMS) induced the highest levels of cross-neutralizing antibodies compared to other oil-in-water emulsions or AS01B. Mechanistically, CMS induced antigen retention in the draining lymph node (dLN) and expression of cytokines, chemokines and type I interferon-stimulated genes at both injection site and dLN. Overall, CMS:RBD-NP is effective across multiple age groups and is an exemplar of a SARS-CoV-2 subunit vaccine tailored to the elderly.","version":"1.1","doi":"10.1101/2021.09.09.459664","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447308","pub_date":"2021-9-09","title":"The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 Beta (B.1.351) and other variants of concern in preclinical studies","abstract":"There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine.","version":"1.2","doi":"10.1101/2021.06.08.447308","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431591","pub_date":"2021-9-09","title":"Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2","abstract":"The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN\u03b2 or IFN\u03bb1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.","version":"1.2","doi":"10.1101/2021.02.17.431591","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.09.459634","pub_date":"2021-9-09","title":"A thermostable oral SARS-CoV-2 vaccine induces mucosal and protective immunity","abstract":"An ideal protective vaccine against SARS-CoV-2 should not only be effective in preventing disease, but also in preventing virus transmission. It should also be well accepted by the population and have a simple logistic chain. To fulfill these criteria, we developed a thermostable, orally administered vaccine that can induce a robust mucosal neutralizing immune response. We used our platform based on retrovirus-derived enveloped virus-like particles (e-VLPs) harnessed with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia, affording them resistance to degradation and the triggering of robust mucosal cellular and antibody immune responses after oral administration. We made e-VLPs expressing various forms of the SARS-CoV-2 Spike protein (S), with or without membrane protein (M) expression. We found that prime-boost administration of VSP-decorated e-VLPs expressing a pre-fusion stabilized form of S and M triggers robust mucosal responses against SARS-CoV-2 in mice and hamsters, which translate into complete protection from a viral challenge. Moreover, they dramatically boosted the IgA mucosal response of intramuscularly injected vaccines. We conclude that our thermostable orally administered e-VLP vaccine could be a valuable addition to the current arsenal against SARS-CoV-2, in a stand-alone prime-boost vaccination strategy or as a boost for existing vaccines.","version":"1.1","doi":"10.1101/2021.09.09.459634","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459486","pub_date":"2021-9-09","title":"Heat efficiently inactivates coronaviruses inside vehicles","abstract":"Heat is an established method to inactivate coronaviruses, and there is utility in using heat to reduce viral load on common touch points in vehicles exposed to a person shedding SARS-CoV-2. As SARS-CoV-2 is a Biosafety level (BSL)-3 pathogen, real world testing of heat as a sanitation method for public and private vehicles becomes a challenge, requiring a surrogate coronavirus that can be handled safely outside of a BSL-3 facility. In this study, we used Bovine Coronavirus (BCoV) as a surrogate for SARS-CoV-2 to test the efficacy of heat-based betacoronavirus inactivation. In vitro, a 30-minute exposure to 56\u00b0C completely inactivated BCoV in solution, and a 15-minute exposure reduced recovery of BCoV >1000-fold. When heated to 56\u00b0C for 15 minutes, the infectivity of BCoV spotted and dried on typical porous and non-porous automobile interior materials was reduced by 99 - 99.99%. When BCoV was spotted and dried on hard plastic (seat) material placed inside an out of service transit bus, 56\u00b0C heat for 30 minutes reduced BCoV infectivity 85 - 99.5%. Thus, 56\u00b0C is an accessible, rapid, and effective method to inactivate coronaviruses inside motor vehicles.","version":"1.1","doi":"10.1101/2021.09.08.459486","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459502","pub_date":"2021-9-09","title":"Digital Spatial Profiling of Collapsing Glomerulopathy","abstract":"Collapsing glomerulopathy is a histologically distinct variant of focal and segmental glomerulosclerosis that presents with heavy proteinuria and portends a poor prognosis. Collapsing glomerulopathy can be triggered by viral infections such as HIV and SARS-CoV-2. Transcriptional profiling of collapsing glomerulopathy lesions is difficult since only a few glomeruli may exhibit this histology within a kidney biopsy and the mechanisms driving this heterogeneity are unknown. Therefore, we used recently developed digital spatial profiling (DSP) technology which permits quantification of mRNA at the level of individual glomeruli. Using DSP, we profiled 1,852 transcripts in glomeruli from HIV and SARS-CoV-2 infected patients with biopsy confirmed collapsing glomerulopathy. The increased resolution of DSP uncovered heterogeneity in glomerular transcriptional profiles that were missed in early laser capture microdissection studies of pooled glomeruli. Focused validation using immunohistochemistry and RNA in situ hybridization showed good concordance with DSP results. Therefore, DSP represents a powerful method to dissect transcriptional programs of pathologically discernible kidney lesions.","version":"1.1","doi":"10.1101/2021.09.08.459502","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459398","pub_date":"2021-9-08","title":"No substantial pre-existing B cell immunity against SARS-CoV-2 in healthy adults","abstract":"Pre-existing immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. Various studies recently provided evidence of pre-existing T cell immunity against SARS-CoV-2 in unexposed individuals. In contrast, the presence and clinical relevance of a pre-existing B cell immunity remains to be fully elucidated. Here, we provide a detailed analysis of the B cell response to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated the memory B cell response to SARS-CoV-2 in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells on a single cell level and generated and tested 158 monoclonal antibodies. None of the isolated antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of relevant pre-existing antibody and B cell immunity against SARS-CoV-2 in unexposed adults.","version":"1.1","doi":"10.1101/2021.09.08.459398","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459464","pub_date":"2021-9-08","title":"SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2 infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named vmiR-5p (for viral miRNA), derived from the SARS-CoV-2 ORF7a transcript. Its abundance ranges from low to moderate as compared to host miRNAs. vmiR-5p functionally associates with Argonaute proteins \u2014 core components of the RNA interference pathway \u2014 leading to downregulation of host transcripts. One such host messenger RNA encodes Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which is linked to interferon signaling. We demonstrate that vmiR-5p production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence. We discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses a small viral non-coding RNA, named vmiR-5p (for viral miRNA), derived from the ORF7a transcript. vmiR-5p associates with the cellular RNA interference machinery to regulate host transcripts likely via target silencing. The production of vmiR-5p relies on cellular machinery and the formation of a strong hairpin within ORF7a sequences. This newly-described vmiR-5p may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.","version":"1.1","doi":"10.1101/2021.09.08.459464","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.07.459230","pub_date":"2021-9-08","title":"In silico, In vitro Screening of Plant Extracts for Anti-SARS-CoV-2 Activity and Evaluation of Their Acute and Sub-Acute Toxicity","abstract":"In the absence of a specific drug for COVID 19, treatment with plant extracts could be an option worthy of further investigation. To screen the phytochemicals for Anti-SARS-CoV-2 in silico and evaluate their safety and efficacy in vitro and in vivo. The phytochemicals for Anti-SARS-CoV-2 were screened in silico using molecular docking. The hits generated from in silico screening were subjected for extraction, isolation and purification. The anti-SARS-CoV-2 activity of plant extracts of Z. piperitum (ATRI-CoV-E1), W. somnifera (ATRI-CoV-E2), C. inophyllum (ATRI-CoV-E3), A. paniculata (ATRI-CoV-E4), and C. Asiatica (ATRI-CoV-E5). The in vitro safety and anti-SARS-CoV-2 activity of plant extracts were performed in VeroE6 cells using Remdesivir as positive control. The acute and sub-acute toxicity study was performed in Wistar male and female rats. The percentage of cell viability for ATRI-COV-E4, ATRI-COV-E5 and ATRI-COV-E2 treated VeroE6 cells were remarkably good on the 24th and 48th hour of treatment. The in vitro anti-SARS-CoV-2 activity of ATRI-COV-E4, ATRI-COV-E5 and ATRI-COV-E2 were significant for both E gene and N gene. The percentage of SARS-CoV-2 inhibition for ATRI-COV-E4 was better than Remdesivir. For E gene and N gene, Remdesivir showed IC50 of 0.15 \u00b5M and 0.11 \u00b5M respectively, For E gene and N gene, ATRI-CoV-E4 showed IC50 of 1.18 \u00b5g and 1.16 \u00b5g respectively. Taking the clue from in vitro findings, the plant extracts A. paniculata (ATRI-COV-E4), W. somnifera extract (ATRI-COV-E5) and C. asiatica extract (ATRI-COV-E2) were combined (ATRICOV 452) and evaluated for acute and sub-acute toxicity in Wistar male and female rats. No statistically significant difference in haematological, biochemical and histopathological parameters were noticed. The study demonstrated the Anti-SARS-CoV-2 activity in vitro and safety of plant extracts in both in vitro and in vivo experimental conditions.","version":"1.1","doi":"10.1101/2021.09.07.459230","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459428","pub_date":"2021-9-08","title":"Cigarette smoke preferentially induces full length ACE2 exposure in primary human airway cells but does not alter susceptibility to SARS-CoV-2 infection","abstract":"Cigarette smoking has multiple serious negative health consequences. However, the epidemiological relationship between cigarette smoking and SARS-CoV-2 infection is controversial; and the interaction between cigarette smoking, airway expression of the ACE2 receptor and the susceptibility of airway cells to infection is unclear. We exposed differentiated air-liquid interface cultures derived from primary human airway stem cells to cigarette smoke extract (CSE) and infected them with SARS-CoV-2. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated ACE2 that lacks the capacity to bind SARS-CoV-2 or a panel of interferon-sensitive genes. Importantly, exposure to CSE did not increase viral infectivity despite the increase in flACE2. Our data are consistent with epidemiological data suggesting current smokers are not at excess risk of SARS-CoV-2 infection. This does not detract from public health messaging emphasising the excess risk of severe COVID-19 associated with smoking-related cardiopulmonary disease.","version":"1.1","doi":"10.1101/2021.09.08.459428","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459430","pub_date":"2021-9-08","title":"Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques","abstract":"Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, particularly with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at pre-defined timepoints in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggested that age did not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.","version":"1.1","doi":"10.1101/2021.09.08.459430","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459408","pub_date":"2021-9-08","title":"An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice","abstract":"Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.","version":"1.1","doi":"10.1101/2021.09.08.459408","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459485","pub_date":"2021-9-08","title":"SARS-CoV-2 mRNA vaccination elicits robust and persistent T follicular helper cell response in humans","abstract":"SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.","version":"1.1","doi":"10.1101/2021.09.08.459485","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.08.459260","pub_date":"2021-9-08","title":"Artemisia annua hot-water extracts show potent activity in vitro against Covid-19 variants including delta","abstract":"For millennia in Southeast Asia, Artemisia annua L. was used to treat \u201cfever\u201d. This medicinal plant is effective against numerous infectious microbial and viral diseases and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria. The SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants. Using Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five fully infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1). In addition to being effective against the original wild type WA1, A. annua cultivars A3, BUR, MED and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3-8.4 \u03bcM and 1.4-25.0 \u03bcM, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0-67.7 \u03bcg DW and 59.5-160.6 \u03bcg DW, respectively. Cell toxicity was insignificant at a leaf dry weight of \u226450 \u03bcg in the extract of any cultivar. Results suggest that oral consumption of A. annua hot-water extracts (tea infusions), could provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.","version":"1.1","doi":"10.1101/2021.09.08.459260","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.459196","pub_date":"2021-9-07","title":"Both Simulation and Sequencing Data Reveal Multiple SARS-CoV-2 Variants Coinfection in COVID-19 Pandemic","abstract":"SARS-CoV-2 is a single-stranded RNA betacoronavirus with a high mutation rate. The rapidly emerged SARS-CoV-2 variants could increase the transmissibility, aggravate the severity, and even fade the vaccine protection. Although the coinfections of SARS-CoV-2 with other respiratory pathogens have been reported, whether multiple SARS-CoV-2 variants coinfection exists remains controversial. This study collected 12,986 and 4,113 SARS-CoV-2 genomes from the GISAID database on May 11, 2020 (GISAID20May11) and April 1, 2021 (GISAID21Apr1), respectively. With the single-nucleotide variants (SNV) and network clique analysis, we constructed the single-nucleotide polymorphism (SNP) coexistence networks and noted the SNP number of the maximal clique as the coinfection index. The coinfection indices of GISAID20May11 and GISAID21Apr1 datasets were 16 and 34, respectively. Simulating the transmission routes and the mutation accumulations, we discovered the linear relationship between the coinfection index and the coinfected variant number. Based on the linear relationship, we deduced that the COVID-19 cases in the GISAID20May11 and GISAID21Apr1 datasets were coinfected with 2.20 and 3.42 SARS-CoV-2 variants on average. Additionally, we performed Nanopore sequencing on 42 COVID-19 patients to explore the virus mutational characteristics. We found the heterozygous SNPs in 41 COVID-19 cases, which support the coinfection of SARS-CoV-2 variants and challenge the accuracy of phylogenetic analysis. In conclusion, our findings reported the coinfection of SARS-CoV-2 variants in COVID-19 patients, demonstrated the increased coinfected variants number in the epidemic, and provided clues for the prolonged viral shedding and severe symptoms in some cases.","version":"1.1","doi":"10.1101/2021.09.06.459196","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.459210","pub_date":"2021-9-07","title":"SARS-CoV-2 originated from SARS-CoV-1-related Bat-CoVs through Pan-CoVs rather than from SARS-CoV-2-related Bat-CoVs","abstract":"The emergence of the novel SARS-CoV-2 in 2019 sparked a dispute concerning its origin. Here, we report that the SARS-CoV-2 originated through pangolin-coronavirus (Pan-CoVs) from the SARS-CoV-related-bat-coronaviruses (SARS-CoV-1-rB-CoVs) rather than from SARS-CoV-2-related-bat-coronaviruses (SARS-CoV-2-rB-CoVs), in contrast to the previous thought. Further, our analyses strongly suggest that the Pan-CoVs evolved from the SARS-CoV-1-rB-CoVs without recombination. Further, our results suggest that the SARS-CoV-1-rB-CoVs\u2019 perhaps jumped into the pangolin, which forced the viruses to mutate and adapt to the new host, and resulted in the origin of Pan-CoVs. Surprisingly, the Pan-CoVs formed an evolutionary intermediate between SARS-CoV-2 and SARS-CoV-2-rB-CoVs at the spike gene. Our findings also suggest that the Pan-CoV/GX and Pan-CoV/Guangdong lineages recombined to form the SARS-CoV-2 spike gene. We also found evidence that the SARS-CoV-2-rB-CoVs spike gene evolved via recombination between Pan-CoV/Guangdong and SARS-CoV-1-rB-CoVs. Overall, our findings suggest that the SARS-CoV-2 emerged from SARS-CoV-1-rB-CoVs through host jumping.","version":"1.1","doi":"10.1101/2021.09.06.459210","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.459005","pub_date":"2021-9-07","title":"Ineffective neutralization of the SARS-CoV-2 Mu variant by convalescent and vaccine sera","abstract":"On August 30, 2021, the WHO classified the SARS-CoV-2 Mu variant (B.1.621 lineage) as a new variant of interest. The WHO defines \u201ccomparative assessment of virus characteristics and public health risks\u201d as primary action in response to the emergence of new SARS-CoV-2 variants. Here, we demonstrate that the Mu variant is highly resistant to sera from COVID-19 convalescents and BNT162b2-vaccinated individuals. Direct comparison of different SARS-CoV-2 spike proteins revealed that Mu spike is more resistant to serum-mediated neutralization than all other currently recognized variants of interest (VOI) and concern (VOC). This includes the Beta variant (B.1.351) that has been suggested to represent the most resistant variant to convalescent and vaccinated sera to date (e.g., Collier et al, Nature, 2021; Wang et al, Nature, 2021). Since breakthrough infection by newly emerging variants is a major concern during the current COVID-19 pandemic (Bergwerk et al., NEJM, 2021), we believe that our findings are of significant public health interest. Our results will help to better assess the risk posed by the Mu variant for vaccinated, previously infected and na\u00efve populations.","version":"1.1","doi":"10.1101/2021.09.06.459005","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.07.459123","pub_date":"2021-9-07","title":"Entrectinib - a SARS-CoV-2 inhibitor in Human Lung Tissue (HLT) cells","abstract":"Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify potential drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a promising antiviral drug. We found that part of the antiviral action of entrectinib is mediated by a non-specific mechanism, likely occurring at the viral membrane level. Such a profile could provide entrectinib with protection against the development of drug resistance by emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.09.07.459123","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.459206","pub_date":"2021-9-07","title":"The vaccinia-based Sementis Copenhagen Vector COVID-19 vaccine induces broad and durable cellular and humoral immune responses","abstract":"The ongoing COVID-19 pandemic perpetuated by SARS-CoV-2 variants, has highlighted the continued need for broadly protective vaccines that elicit robust and durable protection. Here, the vaccinia virus-based, replication-defective Sementis Copenhagen Vector (SCV) was used to develop a first-generation COVID-19 vaccine encoding the spike glycoprotein (SCV-S). Vaccination of mice rapidly induced polyfunctional CD8 T cells with cytotoxic activity and robust Th1-biased, spike-specific neutralizing antibodies, which are significantly increased following a second vaccination, and contained neutralizing activity against the alpha and beta variants of concern. Longitudinal studies indicated neutralizing antibody activity was maintained up to 9 months post-vaccination in both young and aging mice, with durable immune memory evident even in the presence of pre-existing vector immunity. This immunogenicity profile suggests a potential to expand protection generated by current vaccines in a heterologous boost format, and presents a solid basis for second-generation SCV-based COVID-19 vaccine candidates incorporating additional SARS-CoV-2 immunogens.","version":"1.1","doi":"10.1101/2021.09.06.459206","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.23.445348","pub_date":"2021-9-07","title":"Interplay between receptor binding, immune escape and protein stability determines the natural selection of SARS-CoV-2 variants","abstract":"Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain on its expression, stability, activity, and antibody escape potential-some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels with the two least stable variants showing lesser expression. Out of the 8 well-expressed mutants, only 2 (N501Y and K417T/E484K/N501Y) showed stronger affinity to ACE2, 4 (Y453F, S477N, T478I and S494P) have similar affinity, whereas the other 2 (K417N and E484K) have weaker affinity when compared to the wild-type. In terms of CC12.1 binding, when compared to the wild-type, 4 variants (K417N, Y453F, N501Y and K417T/E484K/N501Y) have weaker affinity, 2 (S477N and S494P) have similar affinity, and 2 (T478I and E484K) have stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all these factors need be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.","version":"1.2","doi":"10.1101/2021.05.23.445348","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.06.459135","pub_date":"2021-9-06","title":"Geometrical Study of Virus RNA Sequences","abstract":"In this contribution, some applications of the earlier developed fast algorithm of calculating coordinates of single nucleotides and RNA fragments are considered to create multi-scale geometrical models of RNAs and their mutations. The algorithm allows to plot single nucleotides and RNA\u2019s fragments on one figure and to track the RNA mutations of any level visually and numerically using interpolation formulas and point-to-point estimates of coordinates of ATG starting triplets and single nucleotides. The performed study of many samples of SARS CoV-2 viruses shows perturbations of ATG starting triplet coordinates in the vicinity of orf1ab gene end only.","version":"1.1","doi":"10.1101/2021.09.06.459135","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458038","pub_date":"2021-9-06","title":"Rescuing Low Frequency Variants within Intra-Host Viral Populations directly from Oxford Nanopore sequencing data","abstract":"Infectious disease monitoring on Oxford Nanopore Technologies (ONT) platforms offers rapid turnaround times and low cost, exemplified by well over a half of million ONT SARS-COV-2 datasets. Tracking low frequency intra-host variants has provided important insights with respect to elucidating within host viral population dynamics and transmission. However, given the higher error rate of ONT, accurate identification of intra-host variants with low allele frequencies remains an open challenge with no viable solutions available. In response to this need, we present Variabel, a novel approach and first method designed for rescuing low frequency intra-host variants from ONT data alone. We evaluated Variabel on both within patient and across patient paired Illumina and ONT datasets; our results show that Variabel can accurately identify low frequency variants below 0.5 allele frequency, outperforming existing state-of-the-art ONT variant callers for this task. Variabel is open-source and available for download at: www.gitlab.com/treangenlab/variabel.","version":"1.1","doi":"10.1101/2021.09.03.458038","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457644","pub_date":"2021-9-05","title":"An intranasal OMV-based vaccine induces high mucosal and systemic protecting immunity against a SARS-CoV-2 infection","abstract":"The development of more effective, accessible and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector and whole inactivated virus vaccines, is essential to curtain the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six proline stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to Outer Membrane Vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs seven days after challenge in OMV-mC-Spike vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.","version":"1.2","doi":"10.1101/2021.08.25.457644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456173","pub_date":"2021-9-05","title":"Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant","abstract":"SARS-CoV-2 Delta variant has rapidly replaced the Alpha variant around the world. The mechanism that drives this global replacement has not been defined. Here we report that Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement. In a replication competition assay, Delta SARS-CoV-2 efficiently outcompeted the Alpha variant in human lung epithelial cells and primary human airway tissues. Delta SARS-CoV-2 bearing the Alpha-spike glycoprotein replicated less efficiently than the wild-type Delta variant, suggesting the importance of Delta spike in enhancing viral replication. The Delta spike has accumulated mutation P681R located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduced the replication of Delta variant, to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhanced the cleavage of the full-length spike to S1 and S2, leading to increased infection via cell surface entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the spike cleavage from purified Alpha virions was reduced compared to the Delta spike. Collectively, our results indicate P681R as a key mutation in enhancing Delta variant replication via increased S1/S2 cleavage. Spike mutations that potentially affect furin cleavage efficiency must be closely monitored for future variant surveillance.","version":"1.3","doi":"10.1101/2021.08.12.456173","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.21262981","pub_date":"2021-09-05","title":"COVID-19 mortality in children and young people in Mexico","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To estimate COVID-19 and pre-pandemic low respiratory infection (LRI) mortality in children and young people in Mexico.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Material and methods</jats:title>\n                  <jats:p>We estimated the percentage of total mortality attributable to COVID-19 (95% confidence intervals; 95%CI) and corresponding estimates for pre-pandemic LRI mortality.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>In 2019, LRIs represented 8.6% (95%CI 8.3, 8.9) of deaths in children 0-9 years and 2.0% (95%CI 1.8, 2.3) in those aged 10-19 years. In 2020, corresponding estimates for COVID-19 were 4.4% (95%CI 4.1, 4.6) and 3.7% (95%CI 3.4, 4.1).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Relative to LRI, COVID-19 may be exerting considerable mortality burden, particularly in older children and adolescents.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.09.01.21262981","journal":"medRxiv","score":null},{"id":"10.1101/2021.09.03.458854","pub_date":"2021-9-05","title":"The genetics of eating behaviors: research in the age of COVID-19","abstract":"How much pleasure we take in eating is more than just how much we enjoy the taste of food. Food involvement \u2013 the amount of time we spend on food beyond the immediate act of eating and tasting \u2013 is key to the human food experience. We took a biological approach to test whether food-related behaviors, together capturing food involvement, have genetic components and are partly due to inherited variation. We collected data via an internet survey from a genetically informative sample of 419 adult twins (114 monozygotic twin pairs, 31 dizygotic twin pairs, and 129 singletons). Because we conducted this research during the pandemic, we also ascertained how many participants had experienced COVID-19-associated loss of taste and smell. Since these respondents had previously participated in research in person, we measured their level of engagement to evaluate the quality of their online responses. Additive genetics explained 16-44% of the variation in some measures of food involvement, most prominently various aspects of cooking, suggesting some features of the human food experience may be inborn. Other features reflected shared (early) environment, captured by respondents\u2019 twin status. About 6% of participants had a history of COVID-19 infection, many with transitory taste and smell loss, but all but one had recovered before the survey. Overall, these results suggest that people may have inborn as well as learned variations in their involvement with food. We also learned to adapt to research during a pandemic by considering COVID-19 status and measuring engagement in online studies of human eating behavior.","version":"1.1","doi":"10.1101/2021.09.03.458854","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458951","pub_date":"2021-9-04","title":"Genomic evidence for divergent co-infections of SARS-CoV-2 lineages","abstract":"Recently, patients co-infected by two SARS-CoV-2 lineages have been sporadically reported. Concerns are raised because previous studies have demonstrated co-infection may contribute to the recombination of RNA viruses and cause severe clinic symptoms. In this study, we have estimated the compositional lineage(s), tendentiousness, and frequency of co-infection events in population from a large-scale genomic analysis for SARS-CoV-2 patients. SARS-CoV-2 lineage(s) infected in each sample have been recognized from the assignment of within-host site variations into lineage-defined feature variations by introducing a hypergeometric distribution method. Of all the 29,993 samples, 53 (~0.18%) co-infection events have been identified. Apart from 52 co-infections with two SARS-CoV-2 lineages, one sample with co-infections of three SARS-CoV-2 lineages was firstly identified. As expected, the co-infection events mainly happened in the regions where have co-existed more than two dominant SARS-CoV-2 lineages. However, co-infection of two sub-lineages in Delta lineage were detected as well. Our results provide a useful reference framework for the high throughput detecting of SARS-CoV-2 co-infection events in the Next Generation Sequencing (NGS) data. Although low in average rate, the co-infection events showed an increasing tendency with the increased diversity of SARS-CoV-2. And considering the large base of SARS-CoV-2 infections globally, co-infected patients would be a nonnegligible population. Thus, more clinical research is urgently needed on these patients.","version":"1.1","doi":"10.1101/2021.09.03.458951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458946","pub_date":"2021-9-04","title":"Network analysis outlines strengths and weaknesses of emerging SARS-CoV-2 Spike variants","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered myriad efforts to dissect and understand the structure and dynamics of this complex pathogen. The Spike glycoprotein of SARS-CoV-2 has received special attention as it is the means by which the virus enters the human host cells. The N-terminal domain (NTD) is one of the targeted regions of the Spike protein for therapeutics and neutralizing antibodies against COVID-19. Though its function is not well-understood, the NTD is reported to acquire mutations and deletions that can accelerate the evolutionary adaptation of the virus driving antibody escape. Cellular processes are known to be regulated by complex interactions at the molecular level, which can be characterized by means of a graph representation facilitating the identification of key residues and critical communication pathways within the molecular complex. From extensive all-atom molecular dynamics simulations of the entire Spike for the wild-type and the dominant variant, we derive a weighted graph representation of the protein in two dominant conformations of the receptor-binding-domain; all-down and one-up. We implement graph theory techniques to characterize the relevance of specific residues at facilitating roles of communication and control, while uncovering key implications for fitness and adaptation. We find that many of the reported high-frequency mutations tend to occur away from the critical residues highlighted by our graph theory analysis, implying that these mutations tend to avoid targeting residues that are most critical for protein allosteric communication. We propose that these critical residues could be candidate targets for novel antibody therapeutics. In addition, our analysis provides quantitative insights of the critical role of the NTD and furin cleavage site and their wide-reaching influence over the protein at large. Many of our conclusions are supported by empirical evidence while others point the way towards crucial simulation-guided experiments.","version":"1.1","doi":"10.1101/2021.09.03.458946","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.445077","pub_date":"2021-9-04","title":"6\u2032,6\u2032-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6\u2032,6\u2032-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5\u2019 end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a \u223c100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication. Currently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.","version":"1.2","doi":"10.1101/2021.05.20.445077","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.02.458667","pub_date":"2021-9-03","title":"SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation and potentially multi-organ dysfunction. It remains unclear whether SARS-CoV-2 is sensed by pattern recognition receptors (PRRs) leading to immune activation. Several studies suggest that the Spike (S) protein of SARS-CoV-2 might interact with Toll-like receptor 4 (TLR4) and thereby activate immunity. Here we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor a primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived dendritic cells (DCs) express TLR4 but not ACE2, and DCs were not infected by a primary SARS-CoV-2 isolate. Notably, neither S protein nor the primary SARS-CoV-2 isolate induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs, including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2. Strikingly, infection of ACE2-positive DCs induced type I IFN and cytokine responses, which was inhibited by antibodies against ACE2. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection. The immune system needs to recognize pathogens such as SARS-CoV-2 to initiate antiviral immunity. Dendritic cells (DCs) are crucial for inducing antiviral immunity and are therefore equipped with both extracellular and intracellular pattern recognition receptors to sense pathogens. However, it is unknown if and how SARS-CoV-2 activates DCs. Recent research suggests that SARS-CoV-2 is sensed by extracellular Toll-like receptor 4 (TLR4). We have previously shown that DCs do not express ACE2, and are therefore not infected by SARS-CoV-2. Here we show that DCs do not become activated by exposure to viral Spike proteins or SARS-CoV-2 virus particles. These findings suggest that TLR4 and other extracellular TLRs do not sense SARS-CoV-2. Next, we expressed ACE2 in DCs and SARS-CoV-2 efficiently infected these ACE2-positive DCs. Notably, infection of ACE2-positive DCs induced an antiviral immune response. Thus, our study suggests that infection of DCs is required for induction of immunity, and thus that intracellular viral sensors rather than extracellular TLRs are important in sensing SARS-CoV-2. Lack of sensing by extracellular TLRs might be an escape mechanism of SARS-CoV-2 and could contribute to the aberrant immune responses observed during COVID-19.","version":"1.1","doi":"10.1101/2021.09.02.458667","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449211","pub_date":"2021-9-03","title":"Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins","abstract":"Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity and detect phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or \u03b22 glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by \u2013 among other factors \u2013 viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL to prothrombin (PT) with the strength of the antibody response against SARS-CoV-2 and that it is further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity and aPL against PT in patients with SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.06.21.449211","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.16.342097","pub_date":"2021-9-03","title":"A comparison of Remdesivir versus Au22Glutathione18 in COVID-19 golden hamsters: a better therapeutic outcome of gold compound","abstract":"We firstly disclose single compound yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from Au22Glutathione18 (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication (EC50: ~0.24 \u03bcM) and intrinsically down-regulates NF\u03baB pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The lung viral load and inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir while their lung viral load are decreased to same level. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 golden hamsters. We found that gold cluster molecule directly inhibits SARS-CoV-2 replication and intrinsically suppresses inflammatory cytokines expression in COVID-19 transgenic mouse and golden hamster model, gold cluster providing a better lung injury protection than Remdesivir in COVID-19 golden hamsters via intranasally dropping administration.","version":"1.2","doi":"10.1101/2020.10.16.342097","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458735","pub_date":"2021-9-03","title":"Optimization of single dose VSV-based COVID-19 vaccination in hamsters","abstract":"The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need the for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8+ T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4+ T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.","version":"1.1","doi":"10.1101/2021.09.03.458735","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458829","pub_date":"2021-9-03","title":"Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor","abstract":"Recently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations \u0394145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, \u0394145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.09.03.458829","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.03.458874","pub_date":"2021-9-03","title":"Wildlife in Cameroon harbor diverse coronaviruses including many isolates closely related to human coronavirus 229E","abstract":"Zoonotic spillover of animal viruses into human populations is a continuous and increasing public health risk. SARS-CoV-2 highlights the global impact emergence events can have. Considering the history and diversity of coronaviruses (CoVs), especially in bats, SARS-CoV-2 will likely not be the last to spillover from animals into human populations. We sampled and tested wildlife in the central African country Cameroon to determine which CoVs are circulating and how they relate to previously detected human and animal CoVs. We collected animal and ecological data at sampling locations and used family-level consensus PCR combined with amplicon sequencing for virus detection. Between 2003 and 2018, samples were collected from 6,580 animals of several different orders. CoV RNA was detected in 175 bats, a civet, and a shrew. The CoV RNAs detected in the bats represented 17 different genetic clusters, coinciding with alpha (n=8) and beta (n=9) CoVs. Sequences resembling human CoV-229E (HCoV-229E) were found in 40 Hipposideridae bats. Phylogenetic analyses place the human derived HCoV-229E isolates closest to those from camels in terms of the S and N genes, but closest to isolates from bats for the E, M, and RdRp genes. The CoV RNA positivity rate in bats varied significantly (p<0.001) between the wet (8.2%) and dry season (4.5%). Most sampled species accordingly had a wet season high and dry season low, while for some the opposite was found. Eight of the suspected CoV species of which we detected RNA appear to be entirely novel CoV species, which suggests that CoV diversity in African wildlife is still rather poorly understood. The detection of multiple different variants of HCoV-229E-like viruses supports the bat reservoir hypothesis for this virus, with the phylogenetic results casting some doubt on camels as an intermediate host. The findings also support the previously proposed influence of ecological factors on CoV circulation, indicating a high level of underlying complexity to the viral ecology. These results indicate the importance of investing in surveillance activities among wild animals to detect all potential threats as well as sentinel surveillance among exposed humans to determine emerging threats.","version":"1.1","doi":"10.1101/2021.09.03.458874","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458653","pub_date":"2021-9-02","title":"Nanobody-Functionalized Cellulose for Capturing and Containing SARS-CoV-2","abstract":"The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 217 million people, claiming ~ 4.5 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, safe and effective preventative measures remain urgently needed. Here, we present a generic strategy for containing SARS-CoV-2 by cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose for neutralizing virus from contaminated fluids in a continuous and cost-effective fashion. Taken together, our work leverages low-cost cellulose materials and recently developed Nbs to provide a complementary approach to addressing the pandemic. The ongoing efforts to address the COVID-19 pandemic center around the development of point-of-care diagnostics, preventative measures, and therapeutic strategies against COVID-19. In contrast to existing work, we have provided a complementary approach to target and contain SARS-CoV-2 from contaminated fluids and surfaces. Specifically, we present a generic strategy for the capture and containing of SARS-CoV-2 by cellulose-based substrates. This was archived by developing a bifunctional fusion protein consisting of both a cellulose-binding domain and a nanobody specific for the receptor-binding domain of SARS-CoV-2. As a proof-of-concept, our fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both wildtype and the D614G mutant variants, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose for neutralizing the virus from contaminated fluids in a highly continuous and cost-effective fashion.","version":"1.1","doi":"10.1101/2021.09.01.458653","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458475","pub_date":"2021-9-02","title":"Azacytidine targeting SARS-CoV-2 viral RNA as a potential treatment for COVID-19","abstract":"The COVID-19 pandemic is a global health disaster. Moreover, emerging mutated virus strains present an even greater challenge for existing vaccines and medications. One possible solution is to design drugs based on the properties of virus epigenome, which are more common among coronaviruses. Here, we reported an FDA-approved drug for myelodysplastic syndrome, azacytidine (5Aza), limited virus infection and protected mice against SARS-CoV-2. We demonstrated that this antiviral effect is related to 5Aza incorporation into viral RNA, which disrupt m5C RNA methylation modification profile. This work suggests that targeting viral epigenomes is a viable therapeutic strategy, potentially opening new pathways for treating COVID-19.","version":"1.1","doi":"10.1101/2021.09.01.458475","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.02.458740","pub_date":"2021-9-02","title":"Distinct neutralizing kinetics and magnitudes elicited by different SARS-CoV-2 variant spikes","abstract":"The rapid evolution of SARS-CoV-2 mandates a better understanding of cross-protection between variants after vaccination or infection, but studies directly evaluating such cross-protection are lacking. Here we report that immunization with different variant spikes elicits distinct neutralizing kinetics and magnitudes against other SARS-CoV-2 variants. After immunizing hamsters with wild-type or mutant SARS-CoV-2 bearing variant spikes from Alpha, Beta, Gamma, or Epsilon, the animals developed faster and greater neutralization activities against homologous SARS-CoV-2 variants than heterologous variants, including Delta. The rank of neutralizing titers against different heterologous variants varied, depending on the immunized variant spikes. The differences in neutralizing titers between homologous and heterologous variants were as large as 62-, 15-, and 9.7-fold at days 14, 28, and 45 post-immunization, respectively. Nevertheless, all immunized hamsters were protected from challenges with all SARS-CoV-2 variants, including those exhibiting the lowest neutralizing antibody titers. The results provide insights into the COVID-19 vaccine booster strategies.","version":"1.1","doi":"10.1101/2021.09.02.458740","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458647","pub_date":"2021-9-02","title":"High diversity in Delta variant across countries revealed via genome-wide analysis of SARS-CoV-2 beyond the Spike protein","abstract":"The highly contagious Delta variant of SARS-CoV-2 has emerged as the new dominant global strain, and reports of reduced effectiveness of COVID-19 vaccines against the Delta variant are highly concerning. While there has been extensive focus on understanding the amino acid mutations in the Delta variant \u2018s Spike protein, the mutational landscape of the rest of the SARS-CoV-2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS-CoV-2 proteins from nearly 2 million SARS-CoV-2 genomes from 176 countries/territories. Six highly-prevalent missense mutations in the viral life cycle-associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant (cosine similarity: meanAlpha = 0.94, S.D.Alpha = 0.05; meanDelta = 0.86, S.D.Delta = 0.1; Cohen \u2018s dAlpha-Delta = 1.17, p-value < 0.001). Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of targetable amino acid mutations in the Delta variant \u2018s proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.","version":"1.1","doi":"10.1101/2021.09.01.458647","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.02.458774","pub_date":"2021-9-02","title":"Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants","abstract":"The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.","version":"1.1","doi":"10.1101/2021.09.02.458774","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455941","pub_date":"2021-9-02","title":"Modeling the SARS-CoV-2 mutation based on geographical regions and time","abstract":"The Coronavirus Disease 2019 (COVID-19) epidemic was first detected in late-December 2019. So far, it has caused 203,815,431 confirmed cases and 4,310,623 deaths in the world. We collected sequences from 150,659 COVID-19 patients. Based on the previous phylogenomic analysis, we found three major branches of the virus RNA genomic mutation located in Asia, America, and Europe which is consistent with other studies. We selected sites with high mutation frequencies from Asia, America, and Europe. There are only 13 common mutation sites in these three regions. It infers that the viral mutations are highly dependent on their location and different locations have specific mutations. Most mutations can lead to amino acid substitutions, which occurred in 3/5\u2019UTR, S/N/M protein, and ORF1ab/3a/8/10. Thus, the mutations may affect the pathogenesis of the virus. In addition, we applied an ARIMA model to predict the short-term frequency change of these top mutation sites during the spread of the disease. We tested a variety of settings of the ARIMA model to optimize the prediction effect of three patterns. This model can provide good help for predicting short-term mutation frequency changes.","version":"1.4","doi":"10.1101/2021.08.11.455941","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458644","pub_date":"2021-9-02","title":"Co-expression analysis to identify key modules and hub genes associated with COVID19 in Platelets","abstract":"The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is a highly contagious virus that causes a severe respiratory disease known as Corona virus disease 2019 (COVID19). Indeed, COVID19 increases the risk of cardiovascular occlusive/thrombotic events and is linked to poor outcomes. The pathophysiological processes underlying COVID19-induced thrombosis are complex, and remain poorly understood. To this end, platelets play important roles in regulating our cardiovascular system, including via contributions to coagulation and inflammation. There is an ample of evidence that circulating platelets are activated in COVID19 patients, which is a primary driver of the thrombotic outcome observed in these patients. However, the comprehensive molecular basis of platelet activation in COVID19 disease remains elusive, which warrants more investigation. Hence, we employed gene co-expression network analysis combined with pathways enrichment analysis to further investigate the aforementioned issues. Our study revealed three important gene clusters/modules that were closely related to COVID19. Furthermore, enrichment analysis showed that these three modules were mostly related to platelet metabolism, protein translation, mitochondrial activity, and oxidative phosphorylation, as well as regulation of megakaryocyte differentiation, and apoptosis, suggesting a hyperactivation status of platelets in COVID19. We identified the three hub genes from each of three key modules according to their intramodular connectivity value ranking, namely: COPE, CDC37, CAPNS1, AURKAIP1, LAMTOR2, GABARAP MT-ND1, MT-ND5, and MTRNR2L12. Collectively, our results offer a new and interesting insight into platelet involvement in COVID19 disease at the molecular level, which might aid in defining new targets for treatment of COVID19\u2013induced thrombosis. Co-expression analysis of platelet RNAseq from COVID19 patients show distinct clusters of genes (modules) that are highly correlated to COVID19 disease. Identifying these modules might help in understanding the mechanism of thrombosis in COVID19 patients","version":"1.1","doi":"10.1101/2021.09.01.458644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448343","pub_date":"2021-9-01","title":"COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19","abstract":"The COVID-19 pandemic caused by the \u03b2-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax \u2013 a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD \u2013 induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.","version":"1.2","doi":"10.1101/2021.06.14.448343","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458516","pub_date":"2021-9-01","title":"Absence of severe COVID-19 in patients with clonal mast cells activation disorders: effective anti-SARS-CoV-2 immune response","abstract":"Mast cells are key actors of innate immunity and Th2 adaptive immune response which counterbalance Th1 response, critical for anti-viral immunity. Clonal Mast Cells Activation Disorders (cMCADs) such as mastocytosis and clonal mast cells activation syndrome are characterized by an abnormal mast cells accumulation and/or activation. No data have been published on the anti-viral immune response of patients with cMCADs. The aims of the study were to collected, in a comprehensive way, outcomes of cMCADs patients who experienced a biologically-proven COVID-19 and to characterize both anti-endemic coronaviruses and specific anti-SARS-CoV-2 immune responses in these patients. Clinical follow-up and outcome data were collected prospectively for one year within the French rare disease network CEREMAST encompassing patients from all over the country. Anti-SARS-CoV-2 and anti-endemic coronaviruses specific T-cells were assessed with an enzyme-linked immunospot assay (EliSpot) and anti-SARS-CoV-2 humoral response with dosage of circulating levels of specific IgG, IgA and neutralizing antibodies. Overall, 32 cMCADs patients were identified. None of them required non-invasive or mechanical ventilation; two patients were hospitalized to receive oxygen and steroid therapy. In 21 patients, a characterization of the SARS-CoV-2-specific immune response has been performed. A majority of patients showed a high proportion of circulating SARS-CoV-2-specific interferon (IFN)-\u03b3 producing T-cells and high levels of anti-Spike IgG antibodies with neutralizing activity. In addition, no defects in anti-endemic coronaviruses responses were found in patients with cMCADs compared to non-cMCADs controls. Patients with cMCADs frequently showed a spontaneous IFN-\u03b3 T-cell production in absence of any stimulation that correlated with circulating basal tryptase levels, a marker of mast cells burden. These findings underscore that patients with cMCADs might be not at risk of severe COVID-19 and the spontaneous IFN-\u03b3 production might explain this observation. Mast cells are immune cells involved in many biological processes including the anti-microbial response. However, previous studies suggest that mast cells may have a detrimental role in the response against viruses such as SARS-CoV-2, responsible for COVID-19. When a mutation occurs in mast cells, it can lead to a group of diseases called clonal mast cells activation disorders (cMCADs), characterized by deregulated activation of these cells. Hence, patients with cMCADs might be more susceptible to severe COVID-19 than general population. We therefore conducted a 1-year study in France to collect data from all cMCADs patients included in the CEREMAST rare disease French network and who experienced COVID-19. Interestingly, we did not find any severe COVID-19 (i.e. requiring non-invasive or mechanical ventilation) in spite of well-known risk factors for severe COVID-19 in a part of cMCADs patients. We then have studied the immune response against SARS-CoV-2 and other endemic coronaviruses in these patients. We did not observe any abnormalities in the immune response either at the level of T and B lymphocytes. These findings underscore that these patients might not be at risk of severe COVID-19 as one might have feared.","version":"1.1","doi":"10.1101/2021.09.01.458516","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458520","pub_date":"2021-9-01","title":"Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation","abstract":"Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses. The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.","version":"1.1","doi":"10.1101/2021.09.01.458520","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.14.435337","pub_date":"2021-9-01","title":"The virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus","abstract":"Germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit its use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the absence of exogenous photosensitizers indicating a potential porphyrin-independent alternative mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are generally safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.","version":"1.3","doi":"10.1101/2021.03.14.435337","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424232","pub_date":"2021-9-01","title":"Remdesivir-ivermectin combination displays synergistic interaction with improved in vitro antiviral activity against SARS-CoV-2","abstract":"A key element for the prevention and management of COVID-19 is the development of effective therapeutics. Drug combination strategies of repurposed drugs offer several advantages over monotherapies, including the potential to achieve greater efficacy, the potential to increase the therapeutic index of drugs and the potential to reduce the emergence of drug resistance. Here, we report on the in vitro synergistic interaction between two FDA approved drugs, remdesivir and ivermectin resulting in enhanced antiviral activity against SARS-CoV-2. These findings warrant further investigations into the clinical potential of this combination, together with studies to define the underlying mechanism.","version":"1.2","doi":"10.1101/2020.12.23.424232","journal":"bioRxiv","score":null},{"id":"10.1101/2021.09.01.458544","pub_date":"2021-9-01","title":"The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion","abstract":"Numerous enveloped viruses use specialized surface molecules called fusogens to enter host cells. During virus replication, these fusogens decorate the host cells membrane enabling them the ability to fuse with neighboring cells, forming syncytia that the viruses use to propagate while evading the immune system. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect the brain and may cause serious neurological symptoms through mechanisms which remain poorly understood. Here we show that expression of either the SARS-CoV-2 spike (S) protein or p15 protein from the baboon orthoreovirus is sufficient to induce fusion between interconnected neurons, as well as between neurons and glial cells. This phenomenon is observed across species, from nematodes to mammals, including human embryonic stem cells-derived neurons and brain organoids. We show that fusion events are progressive, can occur between distant neurites, and lead to the formation of multicellular syncytia. Finally, we reveal that in addition to intracellular molecules, fusion events allow diffusion and movement of large organelles such as mitochondria between fused neurons. Our results provide important mechanistic insights into how SARS-CoV-2 and other viruses could affect the nervous system circuitries causing neurological symptoms.","version":"1.1","doi":"10.1101/2021.09.01.458544","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.335521","pub_date":"2021-9-01","title":"Codon usage pattern reveals SARS-CoV-2 as a monomorphic pathogen of hybrid origin with role of silent mutations in rapid evolutionary success","abstract":"Viruses are dependent on the host tRNA pool, and an optimum codon usage pattern (CUP) is the driving force in its evolution. Systematic analysis of CUP of the coding sequences (CDS) of representative major pangolin lineages A and B of SARS-CoV-2 indicate a single transmission event of a codon-optimized virus from its source into humans. Here, no direct congruence could be detected in CUP of all CDS of SARS-CoV-2 with the non-human natural SARS viruses further reiterating its novelty. Several CDS show similar CUP with bat or pangolin, while others have distinct CUP pointing towards a possible hybrid nature of the virus. At the same time, phylogenetic diversity suggests the role of even silent mutations in its success by adapting to host tRNA pool. However, genomes of SARS-CoV-2 from primary infections are required to investigate the origins amongst the competing natural or lab leak theories.","version":"1.2","doi":"10.1101/2020.10.12.335521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434828","pub_date":"2021-9-01","title":"Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants","abstract":"High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called \u2018Tiled-ClickSeq\u2019, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5\u2019UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.","version":"1.2","doi":"10.1101/2021.03.10.434828","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458305","pub_date":"2021-9-01","title":"Analysis of changes occurring in Codon Positions due to mutations through the cellular automata transition rules","abstract":"Variation in the nucleotides of a codon may cause variations in the evolutionary patterns of a DNA or amino acid sequence. To address the capability of each position of a codon to have non-synonymous mutations, the concept of degree of mutation has been introduced. The degree of mutation of a particular position of codon defines the number of non-synonymous mutations occurring for the substitution of nucleotides at each position of a codon, when other two positions of that codon remain unaltered. A Cellular Automaton (CA), is used as a tool to model the mutations of any one of the four DNA bases A, C, T and G at a time where the DNA bases correspond to the states of the CA cells. Point mutation (substitution type) of a codon which characterizes changes in the amino acids, have been associated with local transition rules of a CA. Though there can be  transitions of a 4-state CA with 3-neighbourhood cells, here it has been possible to represent all possible point mutations of a codon in terms of combinations of 16 local transition functions of the CA. Further these rules are divided into 4 classes of equivalence. Also, according to the nature of mutations, the 16 local CA rules of substitutions are classified into 3 sets namely, \u2018No Mutation\u2019, \u2018Transition\u2019 and \u2018Transversion\u2019. The experiment has been carried out with three sets of single nucleotide variations(SNVs) of three different viruses but the symptoms of the diseases caused by them are to some extent similar to each other. They are SARS-CoV-1, SARS-CoV-2 and H1N1 Type A viruses. The aim is to understand the impact of nucleotide substitutions at different positions of a codon with respect to a particular disease phenotype.","version":"1.1","doi":"10.1101/2021.08.30.458305","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.31.458325","pub_date":"2021-8-31","title":"Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor","abstract":"Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.","version":"1.1","doi":"10.1101/2021.08.31.458325","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458287","pub_date":"2021-8-31","title":"Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid and Potential Utility for SARS-CoV-2 Detection","abstract":"Enzymatic beacons, or E-beacons, are 1:1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming DNA oligonucleotides equipped with a dark quencher. We prepared E-beacons biocatalytically using the promiscuous \u201chedgehog\u201d protein-cholesterol ligase, HhC. Instead of cholesterol, HhC attached nanoluciferase site-specifically to mono-sterylated hairpin DNA, prepared in high yield by solid phase synthesis. We tested three potential E-beacon dark quenchers: Iowa Black, Onyx-A, and dabcyl. Prototype E-beacon carrying each of those quenchers provided sequence-specific nucleic acid sensing through turn-on bioluminescence. For practical application, we prepared dabcyl-quenched E-beacons for potential use in detecting the COVID-19 virus, SARS-CoV-2. Targeting the E484 codon of the SARS-CoV-2 Spike protein, E-beacons (80 \u00d7 10\u221212 M) reported wild-type SARS-CoV-2 nucleic acid at \u22651 \u00d7 10\u22129 M with increased bioluminescence of 8-fold. E-beacon prepared for the E484K variant of SARS-CoV-2 functioned with similar sensitivity. These E-beacons could discriminate their complementary target from nucleic acid encoding the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with specificity, detection sensitivity with E-beacons is two to three orders of magnitude better than synthetic molecular beacons, rivaling the most sensitive nucleic acid detection agents reported to date.","version":"1.1","doi":"10.1101/2021.08.30.458287","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.31.458413","pub_date":"2021-8-31","title":"Colloidal aggregators in biochemical SARS-CoV-2 repurposing screens","abstract":"To fight the SARS-CoV-2 pandemic, much effort has been directed toward drug repurposing, testing investigational and approved drugs against several viral or human proteins in vitro. Here we investigate the impact of colloidal aggregation, a common artifact in early drug discovery, in these repurposing screens. We selected 56 drugs reported to be active in biochemical assays and tested them for aggregation by both dynamic light scattering and by enzyme counter screening with and without detergent; seventeen of these drugs formed colloids at concentrations similar to their literature reported IC50s. To investigate the occurrence of colloidal aggregators more generally in repurposing libraries, we further selected 15 drugs that had physical properties resembling known aggregators from a common repurposing library, and found that 6 of these aggregated at micromolar concentrations. An attraction of repurposing is that drugs active on one target are considered de-risked on another. This study suggests not only that many of the drugs repurposed for SARS-CoV-2 in biochemical assays are artifacts, but that, more generally, when screened at relevant concentrations, drugs can act artifactually via colloidal aggregation. Understanding the role of aggregation, and detecting its effects rapidly, will allow the community to focus on those drugs and leads that genuinely have potential for treating COVID-19. Table of Contents Graphic","version":"1.1","doi":"10.1101/2021.08.31.458413","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458303","pub_date":"2021-8-31","title":"Altered interaction between RBD and ACE2 receptor contributes towards the increased transmissibility of SARS CoV-2 delta, kappa, beta, and gamma strains with RBD double mutations","abstract":"The COVID-19 pandemics by SARS-CoV-2 causes catastrophic damage for global human health. The initial step of SARS-CoV-2 infection is the binding of the receptor-binding domain (RBD) in its spike protein to ACE2 receptor in host cell membrane. The evolving of SARS-CoV-2 constantly generates new mutations across its genome including RBD. Besides the well-known single mutation in RBD, the recent new mutation strains with RBD \u201cdouble mutation\u201d is causing new outbreaks globally, as represented by the delta strain containing RBD L452R/T478K. Although it is considered that the increased transmissibility of the double mutated strains could be attributed to the alteration of mutated RBD to ACE2 receptor, the molecular details remains to be unclear. Using the methods of molecular dynamics simulation, superimposed structural comparison, free binding energy estimation and antibody escaping, we investigated the relationship between ACE2 receptor and the RBD double mutant L452R/T478K (delta), L452R/E484Q (kappa) and E484K/N501Y (beta, gamma). The results demonstrated that each of the three RBD double mutants altered RBD structure, led to enhanced binding affinity of mutated RBD to ACE2 receptor, leading to increased transmissibility of SARS-CoV-2 to the host cells.","version":"1.1","doi":"10.1101/2021.08.30.458303","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458244","pub_date":"2021-8-31","title":"(Machine) Learning the mutation signatures of SARS-CoV-2: a primer for predictive prognosis","abstract":"Continuous emergence of new variants through appearance, accumulation and disappearance of mutations in viruses is a hallmark of many viral diseases. SARS-CoV-2 and its variants have particularly exerted tremendous pressure on global healthcare system owing to their life threatening and debilitating implications. The sheer plurality of the variants and huge scale of genome sequence data available for Covid19 have added to the challenges of traceability of mutations of concern. The latter however provides an opportunity to utilize SARS-CoV-2 genomes and the mutations therein as \u2018big data records\u2019 to comprehensively classify the variants through the (machine) learning of mutation patterns. The unprecedented sequencing effort and tracing of disease outcomes provide an excellent ground for identifying important mutations by developing machine learnt models or severity classifiers using mutation profile of SARS-CoV-2. This is expected to provide a significant impetus to the efforts towards not only identifying the mutations of concern but also exploring the potential of mutation driven predictive prognosis of SARS-CoV-2. We describe how a graduated approach of building various severity specific machine learning classifiers, using only the mutation corpus of SARS-CoV-2 genomes, can potentially lead to the identification of important mutations and guide potential prognosis of infection. We demonstrate the applicability of model derived important mutations and use of Shapley values in order to identify the significant mutations of concern as well as for developing sparse models of outcome classification. A total of 77,284 outcome traced SARS-CoV-2 genomes were employed in this study which represented a total corpus of 30346 unique nucleotide mutations and 18647 amino acid mutations. Machine learning models pertaining to graduated classifiers of target outcomes namely \u2018Asymptomatic, Mild, Symptomatic/Moderate, Severe and Fatal\u2019 were built considering the TRIPOD guidelines for predictive prognosis. Shapley values for model linked important mutations were employed to select significant mutations leading to identification of less than 20 outcome driving mutations from each classifier. We additionally describe the significance of adopting a \u2018temporal modeling approach\u2019 to benchmark the predictive prognosis linked with continuously evolving pathogens. A chronologically distinct sampling is important in evaluating the performance of models trained on \u2018past data\u2019 in accurately classifying prognosis linked with genomes of future (observed with new mutations). We conclude that while machine learning approach can play a vital role in identifying relevant mutations, caution should be exercised in using the mutation signatures for predictive prognosis in cases where new mutations have accumulated along with the previously observed mutations of concern. sharmila.mande@tcs.com Supplementary data are enclosed.","version":"1.1","doi":"10.1101/2021.08.30.458244","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458273","pub_date":"2021-8-31","title":"Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge","abstract":"Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Gu\u00e9rin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.","version":"1.1","doi":"10.1101/2021.08.30.458273","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.31.458247","pub_date":"2021-8-31","title":"Subtle immunological differences in mRNA-1273 and BNT162b2 COVID-19 vaccine induced Fc-functional profiles","abstract":"The successful development of several COVID-19 vaccines has substantially reduced morbidity and mortality in regions of the world where the vaccines have been deployed. However, in the wake of the emergence of viral variants, able to evade vaccine induced neutralizing antibodies, real world vaccine efficacy has begun to show differences across the mRNA platforms, suggesting that subtle variation in immune responses induced by the BNT162b2 and mRNA1273 vaccines may provide differential protection. Given our emerging appreciation for the importance of additional antibody functions, beyond neutralization, here we profiled the postboost binding and functional capacity of the humoral response induced by the BNT162b2 and mRNA-1273 in a cohort of hospital staff. Both vaccines induced robust humoral immune responses to WT SARS-CoV-2 and VOCs. However, differences emerged across epitopespecific responses, with higher RBD- and NTD-specific IgA, as well as functional antibodies (ADNP and ADNK) in mRNA-1273 vaccine recipients. Additionally, RBD-specific antibody depletion highlighted the different roles of non-RBD-specific antibody effector function induced across the mRNA vaccines, providing novel insights into potential differences in protective immunity generated across these vaccines in the setting of newly emerging VOCs.","version":"1.1","doi":"10.1101/2021.08.31.458247","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.28.21262748","pub_date":"2021-08-31","title":"Projected resurgence of COVID-19 in the United States in July\u2014December 2021 resulting from the increased transmissibility of the Delta variant and faltering vaccination","abstract":"<jats:title>Summary</jats:title>\n                <jats:sec>\n                  <jats:title>What is already known about this topic?</jats:title>\n                  <jats:p>The highly transmissible SARS-CoV-2 Delta variant has begun to cause increases in cases, hospitalizations, and deaths in parts of the United States. With slowed vaccination uptake, this novel variant is expected to increase the risk of pandemic resurgence in the US in July\u2014December 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>What is added by this report?</jats:title>\n                  <jats:p>Data from nine mechanistic models project substantial resurgences of COVID-19 across the US resulting from the more transmissible Delta variant. These resurgences, which have now been observed in most states, were projected to occur across most of the US, coinciding with school and business reopening. Reaching higher vaccine coverage in July\u2014December 2021 reduces the size and duration of the projected resurgence substantially. The expected impact of the outbreak is largely concentrated in a subset of states with lower vaccination coverage.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>What are the implications for public health practice?</jats:title>\n                  <jats:p>Renewed efforts to increase vaccination uptake are critical to limiting transmission and disease, particularly in states with lower current vaccination coverage. Reaching higher vaccination goals in the coming months can potentially avert 1.5 million cases and 21,000 deaths and improve the ability to safely resume social contacts, and educational and business activities. Continued or renewed non-pharmaceutical interventions, including masking, can also help limit transmission, particularly as schools and businesses reopen.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.28.21262748","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.30.458099","pub_date":"2021-8-30","title":"Strikingly different roles of SARS-CoV-2 fusion peptides uncovered by neutron scattering","abstract":"Coronavirus disease-2019 (COVID-19), a lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of the SARS-CoV-2 Spike protein, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium however re-orients the protein to the lipid leaflet in contact with the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed. We have recreated here important elements of the critical membrane fusion mechanism of the SARS-CoV-2 coronavirus by simplifying the system down to its core elements, amenable to experimental analysis by neutron scattering. Neutrons are well suited for the study of protein \u2013 membrane interactions under physiological conditions, since they allow structural and dynamics characterization at room temperature. Our results revealed strikingly different functions encoded in the viral Spike fusion domain and thereby provide a potential calcium-dependent cell entry mechanism for SARS-CoV-2. In particular, calcium drives the protein\u2019s N-terminal to harpoon through the host membrane, while removing calcium re-orients the protein so that it is able to bridge and dehydrate lipid membranes, facilitating their fusion.","version":"1.1","doi":"10.1101/2021.08.30.458099","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458208","pub_date":"2021-8-30","title":"Relationship between COVID-19 infection and neurodegeneration: Computational insight into interactions between the SARS-CoV-2 spike protein and the monoamine oxidase enzymes","abstract":"Although COVID-19 has been primarily associated with pneumonia, recent data show that its causative agent, the SARS-CoV-2 coronavirus, can infect many vital organs beyond the lungs, including the heart, kidneys and the brain. The literature agrees that COVID-19 is likely to have long-term mental health effects on infected individuals, which signifies a need to understand the role of the virus in the pathophysiology of brain disorders that is currently unknown and widely debated. Our docking and molecular dynamic simulations show that the affinity of the spike protein from the wild type (WT) and the South African B.1.351 (SA) variant towards the MAO enzymes is comparable to that for its ACE2 receptor. This allows for the WT/SA\u2026MAO complex formation, which changes MAO affinities for their neurotransmitter substrates, thus consequently impacting the rates of their metabolic conversion and misbalancing their levels. Knowing that this fine regulation is strongly linked with the etiology of various brain pathologies, these results are the first to highlight the possibility that the interference with the brain MAO catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection, thus placing a neurobiological link between these two conditions in the spotlight. Since the obtained insight suggests that a more contagious SA variant causes even larger disturbances, and with new and more problematic strains likely emerging in the near future, we firmly advise that the presented prospect of the SARS-CoV-2 induced neurological complications should not be ignored, but rather requires further clinical investigations to achieve an early diagnosis and timely therapeutic interventions. Docking and molecular dynamic simulations highlight the possibility that the interference with the brain monoamine oxidase (MAO) catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection.","version":"1.1","doi":"10.1101/2021.08.30.458208","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.29.454333","pub_date":"2021-8-30","title":"Anti- SARS-CoV-2 Receptor Binding Domain Antibody Evolution after mRNA Vaccination","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B-cell responses that continue to evolve for at least one year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested. Here, we examine memory B cell evolution 5 months after vaccination with either Moderna (mRNA-1273) or Pfizer- BioNTech (BNT162b2) mRNA vaccines in a cohort of SARS-CoV-2 na\u00efve individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge 5 months after vaccination of na\u00efve individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with breadth equivalent to those obtained by vaccinating convalescent individuals.","version":"1.2","doi":"10.1101/2021.07.29.454333","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.26.457782","pub_date":"2021-8-30","title":"Structural remodeling of SARS-CoV-2 spike protein glycans reveals the regulatory roles in receptor binding affinity","abstract":"Glycans of the SARS-CoV-2 spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. To date, the spike protein has become the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Experimental reports have described the heterogeneity of the spike protein glycosylation profile. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, there are no studies to date on the role of discrete glycoforms on the spike protein pathobiology. Building an understanding of its role in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor binding affinity revealed the regulatory effects of the RBD glycans. Remarkably, opposite effects were observed from differently remodeled glycans, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor binding affinity. Overall, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic remodeling of glycosylation offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology. The glycosylation of the SARS-CoV-2 spike protein receptor-binding domain has regulatory effects on the receptor binding affinity. Sialylation or not determines the \u201cstabilizing\u201d or \u201cdestabilizing\u201d effect of the glycans. (Protein structure model is adapted from Protein Data Bank: 6moj. The original model does not contain the glycan structure.) Glycans extensively cover the surface of SARS-CoV-2 spike (S) protein but the relationships between the glycan structures and the protein pathological behaviors remain elusive. Herein, we simplified and harmonized the glycan structures in the S protein receptor-binding domain and reported their regulatory roles in human receptor interaction. Opposite regulatory effects were observed and were determined by discrete glycan structures, which can be neutralized by the reported S309 antibody binding to the S protein. This report provides new insight into the mechanism of SARS-CoV-2 S protein infection as well as S309 neutralization.","version":"1.2","doi":"10.1101/2021.08.26.457782","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.27.457969","pub_date":"2021-8-30","title":"May a Strain Chlamydia Isolated From SARS Patient\u2019s Autopsy Issues Inhibit the Proliferation of SARS-CoV? An Early Observation in Vitro","abstract":"We found that a strain chlamydia isolated from SARS patient\u2019s autopsy issues could decrease the proliferation of SARS-CoV in vitro; The inhibitory factors distribute both in the extracellular and intracellular cultures.","version":"1.1","doi":"10.1101/2021.08.27.457969","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.30.458222","pub_date":"2021-8-30","title":"Apropos of Universal Epitope Discovery for COVID-19 Vaccines: A Framework for Targeted Phage Display-Based Delivery and Integration of New Evaluation Tools","abstract":"Targeted bacteriophage (phage) particles are potentially attractive yet inexpensive platforms for immunization. Herein, we describe targeted phage capsid display of an immunogenically relevant epitope of the SARS-CoV-2 Spike protein that is empirically conserved, likely due to the high mutational cost among all variants identified to date. This observation may herald an approach to developing vaccine candidates for broad-spectrum, towards universal, protection against multiple emergent variants of coronavirus that cause COVID-19.","version":"1.1","doi":"10.1101/2021.08.30.458222","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.21262423","pub_date":"2021-08-30","title":"Waning immunity of the BNT162b2 vaccine: A nationwide study from Israel","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Starting December 2020, Israel began a mass vaccination campaign against coronavirus administering the Pfizer BNT162b2 vaccine, which led to a sharp curtailing of the outbreak. After a period with almost no SARS-CoV-2 infections, a resurgent COVID-19 outbreak initiated mid June 2021. Possible reasons for the breakthrough were reduced vaccine effectiveness against the Delta variant, and waning immunity. The aim of this study was to quantify the extent of waning immunity using Israel\u2019s national-database.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Data on all PCR positive test results between July 11-31, 2021 of Israeli residents who became fully vaccinated before June 2021 were used in this analysis. Infection rates and severe COVID-19 outcomes were compared between individuals who were vaccinated in different time periods using a Poisson regression, stratifying by age group and adjusting for possible confounding factors.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The rates of both documented SARS-CoV-2 infections and severe COVID-19 exhibit a statistically significant increase as time from second vaccine dose elapsed. Elderly individuals (60+) who received their second dose in March 2021 were 1.6 (CI: [1.3, 2]) times more protected against infection and 1.7 (CI: [1.0, 2.7]) times more protected against severe COVID-19 compared to those who received their second dose in January 2021. Similar results were found for different age groups.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>These results indicate a strong effect of waning immunity in all age groups after six months. Quantifying the effect of waning immunity on vaccine effectiveness is critical for policy makers worldwide facing the dilemma of administering booster vaccinations.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.24.21262423","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.24.457397","pub_date":"2021-8-29","title":"Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants","abstract":"The causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3\u2019-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.","version":"1.3","doi":"10.1101/2021.08.24.457397","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437792","pub_date":"2021-8-29","title":"Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2","abstract":"The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.","version":"1.3","doi":"10.1101/2021.03.31.437792","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.22.457295","pub_date":"2021-8-29","title":"Intra-host SARS-CoV-2 evolution in the gut of mucosally-infected Chlorocebus aethiops (African green monkeys)","abstract":"In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were subsequently lost in virus recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, that were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution unique to this nonhuman primate species and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future development of primate disease models.","version":"1.2","doi":"10.1101/2021.08.22.457295","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.27.21262687","pub_date":"2021-08-29","title":"Early immune responses have long-term associations with clinical, virologic, and immunologic outcomes in patients with COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The great majority of SARS-CoV-2 infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, mild to moderate infections are an important contributor to ongoing transmission. There remains a critical need to identify host immune biomarkers predictive of clinical and virologic outcomes in SARS-CoV-2-infected patients. Leveraging longitudinal samples and data from a clinical trial of Peginterferon Lambda for treatment of SARS-CoV-2 infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients within the first 2 weeks of symptom onset. We define early immune signatures, including plasma levels of RIG-I and the CCR2 ligands (MCP1, MCP2 and MCP3), associated with control of oropharyngeal viral load, the degree of symptom severity, and immune memory (including SARS-CoV-2-specific T cell responses and spike (S) protein-binding IgG levels). We found that individuals receiving BNT162b2 (Pfizer\u2013BioNTech) vaccine had similar early immune trajectories to those observed in this natural infection cohort, including the induction of both inflammatory cytokines (e.g. MCP1) and negative immune regulators (e.g. TWEAK). Finally, we demonstrate that machine learning models using 8-10 plasma protein markers measured early within the course of infection are able to accurately predict symptom severity, T cell memory, and the antibody response post-infection.</jats:p>","version":null,"doi":"10.1101/2021.08.27.21262687","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.23.21262464","pub_date":"2021-08-29","title":"Opaganib in COVID-19 pneumonia: Results of a randomized, placebo-controlled Phase 2a trial","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>\n                    Opaganib, an oral sphingosine kinase-2 inhibitor with antiviral and anti-inflammatory properties, was shown to inhibit SARS-CoV-2 replication\n                    <jats:italic>in vitro</jats:italic>\n                    . We thus considered that opaganib could be beneficial for moderate to severe COVID-19 pneumonia. The objective of the study was to evaluate the effect of opaganib on supplemental oxygen requirements, time to hospital discharge and its safety in COVID-19 pneumonia hospitalized patients requiring supplemental oxygen.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This Phase 2a, randomized, double-blind, placebo-controlled study was conducted between July and December 2020 in eight sites in the USA. Forty-two enrolled patients received opaganib (n=23) or placebo (n=19) added to standard of care for up to 14 days and were followed up for 28 days after their last dose of opaganib/placebo.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The relative decrease in total supplemental oxygen requirement from baseline to Day 14 was 61.6% in the opaganib versus 46.7% in the placebo arms. By Day 14, 50.0% of patients in the opaganib and 22.2% in the placebo group no longer required supplemental oxygen for at least 24 hours, while 86.4% and 55.6%, respectively, were discharged from hospital. The incidence of \u2265 Grade 3 treatment-emergent adverse events was 17.4% and 33.3% in the opaganib and placebo groups, respectively. Three deaths occurred in each group.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>In this proof-of-concept study, hypoxic, hospitalized patients receiving oral opaganib required less supplementary oxygen and had earlier hospital discharge, with no safety concerns arising. These findings support further evaluation of opaganib in this population.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Summary</jats:title>\n                  <jats:p>Upon receiving opaganib, patients with COVID-19 pneumonia who were hospitalized and required supplemental oxygen showed symptomatic clinical improvement compared to placebo, with less supplemental oxygen requirement, resulting in earlier hospital discharge, and no safety concerns arising.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.23.21262464","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.25.21262636","pub_date":"2021-08-28","title":"The Unequal Burden of the Covid-19 Pandemic: Racial/Ethnic Disparities in US Cause-Specific Mortality","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>To quantify changes in all-cause and cause-specific mortality by race and ethnicity between 2019 and 2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Using 2019 and 2020 provisional death counts from the National Center for Health Statistics and population estimates from the US Census Bureau, we estimate age-standardized death rates by race/ethnicity and attribute changes in mortality to various causes of death. We also examine how patterns of change across racial/ethnic groups vary by age and sex.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Covid-19 death rates in 2020 were highest in the Hispanic community whereas Black individuals had the largest increase in all-cause mortality between 2019 and 2020. Increases in mortality from heart disease, diabetes, and external causes of death accounted for the adverse trend in all-cause mortality within the Black population. Percentage increases in all-cause mortality were similar for men and women and for ages 25-64 and 65+ for Black and White populations, but increases were greatest for working-aged men among the Hispanic population.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Examining increases in non-Covid-19 causes of death is essential for fully capturing both the direct and indirect impact of the Covid-19 pandemic on racial/ethnic mortality disparities.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.25.21262636","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.26.457874","pub_date":"2021-8-27","title":"From Alpha to Zeta: Identifying variants and subtypes of SARS-CoV-2 via clustering","abstract":"The availability of millions of SARS-CoV-2 sequences in public databases such as GISAID and EMBL-EBI (UK) allows a detailed study of the evolution, genomic diversity and dynamics of a virus like never before. Here we identify novel variants and sub-types of SARS-CoV-2 by clustering sequences in adapting methods originally designed for haplotyping intra-host viral populations. We asses our results using clustering entropy \u2014 the first time it has been used in this context. Our clustering approach reaches lower entropies compared to other methods, and we are able to boost this even further through gap filling and Monte Carlo based entropy minimization. Moreover, our method clearly identifies the well-known Alpha variant in the UK and GISAID datasets, but is also able to detect the much less represented (< 1% of the sequences) Beta (South Africa), Epsilon (California), Gamma and Zeta (Brazil) variants in the GISAID dataset. Finally, we show that each variant identified has high selective fitness, based on the growth rate of its cluster over time. This demonstrates that our clustering approach is a viable alternative for detecting even rare subtypes in very large datasets.","version":"1.1","doi":"10.1101/2021.08.26.457874","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.26.457884","pub_date":"2021-8-27","title":"A vimentin-targeting oral compound with host-directed antiviral and anti-inflammatory actions addresses multiple features of COVID-19 and related diseases","abstract":"Damage in COVID-19 results from both the SARS-CoV-2 virus and its triggered overreactive host immune responses. Therapeutic agents that focus solely on reducing viral load or hyperinflammation fail to provide satisfying outcomes in all cases. Although viral and cellular factors have been extensively profiled to identify potential anti-COVID targets, new drugs with significant efficacy remain to be developed. Here, we report the potent preclinical efficacy of ALD-R491, a vimentin-targeting small molecule compound, in treating COVID-19 through its host-directed antiviral and anti-inflammatory actions. We found that by altering the physical properties of vimentin filaments, ALD-491 affected general cellular processes as well as specific cellular functions relevant to SARS-CoV-2 infection. Specifically, ALD-R491 reduced endocytosis, endosomal trafficking, and exosomal release, thus impeding the entry and egress of the virus; increased the microcidal capacity of macrophages, thus facilitating the pathogen clearance; and enhanced the activity of regulatory T cells, therefore suppressing the overreactive immune responses. In cultured cells, ALD-R491 potently inhibited the SARS-CoV-2 spike protein and human ACE2-mediated pseudoviral infection. In aged mice with ongoing, productive SARS-CoV-2 infection, ALD-R491 reduced disease symptoms as well as lung damage. In rats, ALD-R491 also reduced bleomycin-induced lung injury and fibrosis. Our results indicate a unique mechanism and significant therapeutic potential for ALD-R491 against COVID-19. We anticipate that ALD-R491, an oral, fast-acting, and non-toxic agent targeting the cellular protein with multipart actions, will be convenient, safe, and broadly effective, regardless of viral mutations, for patients with early- or late-stage disease, post-COVID complications and other related diseases. With the Delta variant currently fueling a resurgence of new infections in the fully-vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanism-of-actions that address both the viral infection and the overactive immune system in the pathogenesis of the disease. Unlike virus-directed therapeutics that may lose efficacy due to viral mutations and immunosuppressants that require ideal timing to be effective, this agent, with its unique host-directed antiviral and anti-inflammatory actions, can work against all variants of the virus, be effective during all stages of the disease, and even resolve post-disease damage and complications. A further development of the compound will provide an important tool in the fight against COVID-19, its complications, as well as future outbreaks of new viruses.","version":"1.1","doi":"10.1101/2021.08.26.457884","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439181","pub_date":"2021-8-27","title":"Theoretical causes of the Brazilian P.1 and P.2 lineages of the SARS-CoV-2 virus through molecular dynamics","abstract":"The new \u03b2-coronavirus has been causing sad losses around the world and the emergence of new variants has caused great concern. This pandemic is of a proportion not seen since the Spanish Flu in 1918. Thus, throughout this research, the B.1.1.28 lineage of the P.1 clade (K417T, N501Y, E484K) that emerged in Brazil was studied, as well as the latest Delta variant. This is because the molecular mechanisms by which phenotypic changes in transmissibility or mortality remain unknown. Through molecular dynamics simulations with the NAMD 3 algorithm in the 50ns interval, it was possible to understand the impact on structural stabilization on the interaction of the ACE2-RBD complex, as well as simulations in 30ns for the neutralizing antibody P2B-2F6, with this antibody was derived from immune cells from patients infected with SARS-CoV-2. Although not all molecular dynamics analyzes support the hypothesis of greater stability in the face of mutations, there was a predominance of low fluctuations. Thus, 3 (three) analyzes corroborate the hypothesis of greater ACE2-RBD stability as a result of P.1, among them: Low mean RMSF values, greater formation of hydrogen bonds and low solvent exposure measured by the SASA value. An inverse behavior occurs in the interaction with neutralizing antibodies, since the mutations induce greater instability and thus hinder the recognition of antibodies in neutralizing the Spike protein, where we noticed a smaller number of hydrogen bonds as a result of P.1. Through MM-PBSA energy decomposition, we found that Van der Waals interactions predominated and were more favorable when the structure has P.1 strain mutations. Therefore, we believe that greater stabilization of the ACE2-RBD complex may be a plausible explanation for why some mutations are converging in different strains, such as E484K and N501Y. The P.1 concern variant still makes the Spike protein recognizable by antibodies, and therefore, even if the vaccines\u2019 efficacy can be diminished, there are no results in the literature that nullify them.","version":"1.2","doi":"10.1101/2021.04.09.439181","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444489","pub_date":"2021-8-27","title":"Targeted redesign of suramin analogs for novel antimicrobial lead development","abstract":"The emergence of new viral infections and drug resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets is one potential way in which to accelerate this process. Suramin was initially developed as a successful anti-parasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous towards positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure activity relationship (SAR) studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramin\u2019s target diversity, confirming that suramin preferentially binds to protein pockets which are both positively charged and enriched in aromatic or leucine residues. Further, suramin\u2019s high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity.","version":"1.2","doi":"10.1101/2021.05.17.444489","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457693","pub_date":"2021-8-26","title":"Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains","abstract":"Although mRNA vaccines prevent COVID-19, variants jeopardize their efficacy as immunity wanes. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike) or modified (mRNA-1273.351, designed for B.1.351 spike) preclinical Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Immunization with high or low dose formulations of mRNA vaccines induced neutralizing antibodies in serum against ancestral SARS-CoV-2 and several variants, although levels were lower particularly against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. Nonetheless, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, and serve as a possible model for waning immunity, showed breakthrough lung infection and pneumonia with B.1.617.2. Thus, as levels of immunity induced by mRNA vaccines decline, breakthrough infection and disease likely will occur with some SARS-CoV-2 variants, suggesting a need for additional booster regimens.","version":"1.1","doi":"10.1101/2021.08.25.457693","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.25.445523","pub_date":"2021-8-26","title":"Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2","abstract":"A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development. Removing glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine.","version":"1.2","doi":"10.1101/2021.05.25.445523","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457711","pub_date":"2021-8-26","title":"Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding","abstract":"The recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.","version":"1.1","doi":"10.1101/2021.08.25.457711","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.26.457807","pub_date":"2021-8-26","title":"Variation in synonymous nucleotide composition among genomes of sarbecoviruses and consequences for the origin of COVID-19","abstract":"The subgenus Sarbecovirus includes two human viruses, SARS-CoV and SARS-CoV-2, respectively responsible for the SARS epidemic and COVID-19 pandemic, as well as many bat viruses and two pangolin viruses. Here, the synonymous nucleotide composition (SNC) of Sarbecovirus genomes was analysed by examining third codon-positions, dinucleotides, and degenerate codons. The results show evidence for the eigth following groups: (i) SARS-CoV related coronaviruses (SCoVrC including many bat viruses from China), (ii) SARS-CoV-2 related coronaviruses (SCoV2rC; including five bat viruses from Cambodia, Thailand and Yunnan), (iii) pangolin viruses, (iv) three bat viruses showing evidence of recombination between SCoVrC and SCoV2rC genomes, (v) two highly divergent bat viruses from Yunnan, (vi) the bat virus from Japan, (vii) the bat virus from Bulgaria, and (viii) the bat virus from Kenya. All these groups can be diagnosed by specific nucleotide compositional features except the one concerned by recombination between SCoVrC and SCoV2rC. In particular, SCoV2rC genomes are characterised by the lowest percentages of cyosine and highest percentages of uracil at third codon-positions, whereas the genomes of pangolin viruses exhibit the highest percentages of adenine at third codon-positions. I suggest that latitudinal and taxonomic differences in the imbalanced nucleotide pools available in host cells during viral replication can explain the seven groups of SNC here detected among Sarbecovirus genomes. A related effect due to hibernating bats is also considered. I conclude that the two independent host switches from Rhinolophus bats to pangolins resulted in convergent mutational constraints and that SARS-CoV-2 emerged directly from a horseshoe bat virus.","version":"1.1","doi":"10.1101/2021.08.26.457807","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457692","pub_date":"2021-8-26","title":"The Lambda variant of SARS-CoV-2 has a better chance than the Delta variant to escape vaccines","abstract":"The newly emerging variants of SARS-CoV-2 from India (Delta variant) and South America (Lambda variant) have led to a higher infection rate of either vaccinated or unvaccinated people. We found that sera from Pfizer-BioNTech vaccine remain high reactivity toward the receptor binding domain (RBD) of Delta variant while it drops dramatically toward that of Lambda variant. Interestingly, the overall titer of antibodies of Pfizer-BioNTech vaccinated individuals drops 3-fold after 6 months, which could be one of major reasons for breakthrough infections, emphasizing the importance of potential third boost shot. While a therapeutic antibody, Bamlanivimab, decreases binding affinity to Delta variant by ~20 fold, it fully lost binding to Lambda variant. Structural modeling of complexes of RBD with human receptor, Angiotensin Converting Enzyme 2 (ACE2), and Bamlanivimab suggest the potential basis of the change of binding. The data suggest possible danger and a potential surge of Lambda variant in near future.","version":"1.1","doi":"10.1101/2021.08.25.457692","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457645","pub_date":"2021-8-25","title":"Structure-based screening of drug candidates targeting the SARS-CoV-2 envelope protein","abstract":"The COVID-19 (coronavirus disease 2019) pandemic is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). SARS-CoV-2 produces a small hydrophobic envelope (E) protein which shares high homology with SARS-CoV E protein. By patch-clamp recording, the E protein is demonstrated to be a cation-selective ion channel. Furthermore, the SARS-CoV-2 E protein can be blocked by a SARS-CoV E protein inhibitor hexamethylene amiloride. Using structural model and virtual screening, another E protein inhibitor AZD5153 is discovered. AZD5153 is a bromodomain protein 4 inhibitor against hematologic malignancies in clinical trial. The E protein amino acids Phe23 and Val29 are key determinants for AZD5153 sensitivity. This study provides two promising lead compounds and a functional assay of SARS-CoV-2 E protein for the future drug candidate discovery.","version":"1.1","doi":"10.1101/2021.08.25.457645","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425339","pub_date":"2021-8-25","title":"Distinct mutations and lineages of SARS-CoV-2 virus in the early phase of COVID-19 pandemic and subsequent one-year global expansion","abstract":"A novel coronavirus, SARS-CoV-2, has caused over 190 million cases and over 4 million deaths worldwide since it occurred in December 2019 in Wuhan, China. Here we conceptualized the temporospatial evolutionary and expansion dynamics of SARS-CoV-2 by taking a series of cross-sectional view of viral genomes from early outbreak in January 2020 in Wuhan to early phase of global ignition in early April, and finally to the subsequent global expansion by late December 2020. Based on the phylogenetic analysis of the early patients in Wuhan, Wuhan/WH04/2020 is supposed to be a more appropriate reference genome of SARS-CoV-2, instead of the first sequenced genome Wuhan-Hu-1. By scrutinizing the cases from the very early outbreak, we found a viral genotype from the Seafood Market in Wuhan featured with two concurrent mutations (i.e. M type) had become the overwhelmingly dominant genotype (95.3%) of the pandemic one year later. By analyzing 4,013 SARS-CoV-2 genomes from different continents by early April, we were able to interrogate the viral genomic composition dynamics of initial phase of global ignition over a timespan of 14-week. 11 major viral genotypes with unique geographic distributions were also identified. WE1 type, a descendant of M and predominantly witnessed in western Europe, consisted a half of all the cases (50.2%) at the time. The mutations of major genotypes at the same hierarchical level were mutually exclusive, which implying that various genotypes bearing the specific mutations were propagated during human-to-human transmission, not by accumulating hot-spot mutations during the replication of individual viral genomes. As the pandemic was unfolding, we also used the same approach to analyze 261,323 SARS-CoV-2 genomes from the world since the outbreak in Wuhan (i.e. including all the publicly available viral genomes) in order to recapitulate our findings over one-year timespan. By 25 December 2020, 95.3% of global cases were M type and 93.0% of M-type cases were WE1. In fact, at present all the four variants of concern (VOC) are the descendants of WE1 type. This study demonstrates the viral genotypes can be utilized as molecular barcodes in combination with epidemiologic data to monitor the spreading routes of the pandemic and evaluate the effectiveness of control measures. Moreover, the dynamics of viral mutational spectrum in the study may help the early identification of new strains in patients to reduce further spread of infection, guide the development of molecular diagnosis and vaccines against COVID-19, and help assess their accuracy and efficacy in real world at real time.","version":"1.4","doi":"10.1101/2021.01.05.425339","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428543","pub_date":"2021-8-25","title":"Genome-scale metabolic modeling reveals SARS-CoV-2-induced metabolic changes and antiviral targets","abstract":"Tremendous progress has been made to control the COVID-19 pandemic caused by the SARS-CoV-2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS-CoV-2 infection using genome-scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS-CoV-2 infection. We next applied the GEM-based metabolic transformation algorithm to predict anti-SARS-CoV-2 targets that counteract the virus-induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco-2 cells. Further generating and analyzing RNA-sequencing data of remdesivir-treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti-SARS-CoV-2 drug. Our study provides clinical data-supported candidate anti-SARS-CoV-2 targets for future evaluation, demonstrating host metabolism-targeting as a promising antiviral strategy.","version":"1.2","doi":"10.1101/2021.01.27.428543","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.457518","pub_date":"2021-8-25","title":"Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate","abstract":"SARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. The RBD203-N1 production process yielded 492.9 \u00b1 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 \u00b1 3% (total yield of purified protein: 270.5 \u00b1 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.","version":"1.1","doi":"10.1101/2021.08.24.457518","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457627","pub_date":"2021-8-25","title":"Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization","abstract":"SARS-CoV-2 is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 Spike protein (S) have arisen in circulating viral populations, culminating in the spread of several variants of concern (VOC) with varying degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we employed lentivirus-based pseudotyped viruses that express specific SARS-CoV-2 S protein substitutions and cell lines that stably express ACE2 from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the parental (prototype Wuhan-1) S at levels comparable to human ACE2. Most single S substitutions (e.g., 452R, 478K, 501Y) did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency in viral entry. Conversely, combinatorial VOC substitutions in the S protein were associated with significantly increased entry capacity of pseudotyped viruses compared to that of the parental Wuhan-1 pseudotyped virus. Similarly, infection studies using live ancestral (USA-WA1/2020), Alpha, and Beta SARS-CoV-2 viruses in hamsters revealed a higher replication potential for the Beta variant compared to the ancestral prototype virus. Moreover, neutralizing titers in sera from various animal species, including humans, were significantly reduced by single substitutions of 484K or 452R, double substitutions of 501Y-484K, 452R-484K and 452R-478K and the triple substitution of 501Y-484K-417N, suggesting that 484K and 452R are particularly important for evading neutralizing antibodies in human, cat, and rabbit sera. Cumulatively, this study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication and antibody-mediated viral neutralization. Cells stably expressing ACE2 from various animals and a lentivirus-based SARS-CoV-2 pseudotyped virus assay were established to study SARS-CoV-2 cell entry. The results demonstrated that ACE2 from a wide range of animal species facilitate S-mediated virus entry into cells, which is supported by in silico data as well as natural and experimental infection studies. Pseudotyped viruses containing mutations in the RBD of S representative of the Alpha, Gamma, and especially Beta, variants of concern demonstrated that certain mutations are associated with increased viral entry compared to the parental S. The Beta variant was also observed to have a replicative advantage in vitro and in vivo compared to the prototype virus. Pseudotyped viruses containing combinatorial substitutions of 501Y-484K-417K, 614G-501Y-484K and 614G-501Y-484K-417N increased viral entry via ACE2 across multiple species. The 501Y or 478K single substitution did not significantly affect neutralizing capacity of immune sera compared to the prototype strain, but the addition of 484K or 452R substitutions significantly reduced the neutralizing titers.","version":"1.1","doi":"10.1101/2021.08.25.457627","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457411","pub_date":"2021-8-25","title":"Woodsmoke particulates alter expression of antiviral host response genes in human nasal epithelial cells infected with SARS-CoV-2 in a sex-dependent manner","abstract":"We have previously shown that exposure to particulate air pollution, both from natural and anthropogenic sources, alters gene expression in the airways and increases susceptibility to respiratory viral infection. Additionally, we have shown that woodsmoke particulates (WSP) affect responses to influenza in a sex-dependent manner. In the present study, we used human nasal epithelial cells (hNECs) from both sexes to investigate how particulate exposure could modulate gene expression in the context of SARS-CoV-2 infection. We used diesel exhaust particulate (DEP) as well as WSP derived from eucalyptus or red oak wood. HNECs were exposed to particulates at a concentration of 22 \u03bcg/cm2 for 2 h then immediately infected with SARS-CoV-2 at a MOI (multiplicity of infection) of 0.5. Exposure to particulates had no significant effects on viral load recovered from infected cells. Without particulate exposure, hNECs from both sexes displayed a robust upregulation of antiviral host response genes, though the response was greater in males. However, WSP exposure before infection dampened expression of genes related to the antiviral host response by 72 h post infection. Specifically, red oak WSP downregulated IFIT1, IFITM3, IFNB1, MX1, CCL3, CCL5, CXCL11, CXCL10, and DDX58, among others. After sex stratification of these results, we found that exposure to WSP prior to SARS-CoV-2 infection downregulated anti-viral gene expression in hNECs from females more so than males. These data indicate that WSP, specifically from red oak, alter virus-induced gene expression in a sex-dependent manner and potentially suppress antiviral host defense responses following SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.08.23.457411","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457626","pub_date":"2021-8-25","title":"The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters","abstract":"As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel variant of concern (VOC) designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and shown to enhance infectivity in vitro and decrease antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both strains exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most body weight loss among all 3 lineages. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three strains. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the upper respiratory tract (URT) but not in the lungs. In multi-virus in-vivo competition experiments, we found that epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the URT gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) variants in hamsters. These results demonstrate enhanced virulence and high relative fitness of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) strain. In the last 12 months new variants of SARS-CoV-2 have arisen in the UK, South Africa, Brazil, India, and California. New SARS-CoV-2 variants will continue to emerge for the foreseeable future in the human population and the potential for these new variants to produce severe disease and evade vaccines needs to be understood. In this study, we used the hamster model to determine the epsilon (B.1.427/429) SARS-CoV-2 strains that emerged in California in late 2020 cause more severe disease and infected hamsters have higher viral loads in the upper respiratory tract compared to the prior B.1 (614G) strain. These findings are consistent with human clinical data and help explain the emergence and rapid spread of this strain in early 2021.","version":"1.1","doi":"10.1101/2021.08.25.457626","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.25.457699","pub_date":"2021-8-25","title":"Directing an mRNA-LNP vaccine toward lymph nodes improves humoral and cellular immunity against SARS-CoV-2","abstract":"The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic has captured the world\u2019s attention and remains an ongoing issue in order to protect against emerging variants of concern (VoCs) while generating long lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid (mRNA) encoding the spike protein in a lipid nanoparticle formulation (LNP) (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In efforts to continually improve immunity, a lymphatic drug delivery device (MuVaxx) was engineered and tested to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) to elicit adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Remarkably, a 4-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic mediated delivery using MuVaxx for SARS-CoV-2 and VoCs vaccines.","version":"1.1","doi":"10.1101/2021.08.25.457699","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457419","pub_date":"2021-8-24","title":"Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display","abstract":"Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.08.23.457419","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.457520","pub_date":"2021-8-24","title":"Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats","abstract":"The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.","version":"1.1","doi":"10.1101/2021.08.24.457520","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.457457","pub_date":"2021-8-24","title":"Defective ORF8 dimerization in delta variant of SARS CoV2 leads to abrogation of ORF8 MHC-I interaction and overcome suppression of adaptive immune response","abstract":"In India, the breakthrough infections during second wave of COVID-19 pandemic was due to SARS-COV-2 delta variant (B.1.617.2). It was reported that majority of the infections were caused by the delta variant and only 9.8% percent cases required hospitalization whereas, only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections was observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Down regulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant results in structural instability of ORF8 dimer caused by disruption of hydrogen bonding and salt bridges as revealed by structural analysis and MD simulation studies of ORF8 dimer. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 may not hindering the MHC-I expression thereby resulting in better immune response against SARS-CoV-2 delta variant, which partly explains the sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India","version":"1.1","doi":"10.1101/2021.08.24.457457","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.15.456341","pub_date":"2021-8-24","title":"Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer","abstract":"SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.","version":"1.2","doi":"10.1101/2021.08.15.456341","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441209","pub_date":"2021-8-24","title":"Insertions in SARS-CoV-2 genome caused by template switch and duplications give rise to new variants that merit monitoring","abstract":"The appearance of multiple new SARS-CoV-2 variants during the winter of 2020-2021 is a matter of grave concern. Some of these new variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the COVID-19 pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on point nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the spike protein is thought to be a determinant of SARS-CoV-2 virulence and other inserts might have contributed to coronavirus pathogenicity as well. Here, we investigate insertions in SARS-CoV-2 genomes and identify 347 unique inserts of different lengths. We present evidence that these inserts reflect actual virus variance rather than sequencing errors. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. We show that inserts in the Spike glycoprotein can affect its antigenic properties and thus merit monitoring. At least, three inserts in the N-terminal domain of the Spike (ins245IME, ins246DSWG, and ins248SSLT) that were first detected in 2021 are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity.","version":"1.2","doi":"10.1101/2021.04.23.441209","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.457448","pub_date":"2021-8-24","title":"Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption","abstract":"COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as \u201cCluster 5\u201d that contained mutations in the spike protein. However, the functional properties of these \u201cCluster 5\u201d mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.","version":"1.1","doi":"10.1101/2021.08.24.457448","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457434","pub_date":"2021-8-24","title":"Structure-based design of antisense oligonucleotides that inhibit SARS-CoV-2 replication","abstract":"Antisense oligonucleotides (ASOs) are an emerging class of drugs that target RNAs. Current ASO designs strictly follow the rule of Watson-Crick base pairing along target sequences. However, RNAs often fold into structures that interfere with ASO hybridization. Here we developed a structure-based ASO design method and applied it to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our method makes sure that ASO binding is compatible with target structures in three-dimensional (3D) space by employing structural design templates. These 3D-ASOs recognize the shapes and hydrogen bonding patterns of targets via tertiary interactions, achieving enhanced affinity and specificity. We designed 3D-ASOs that bind to the frameshift stimulation element and transcription regulatory sequence of SARS-CoV-2 and identified lead ASOs that strongly inhibit viral replication in human cells. We further optimized the lead sequences and characterized structure-activity relationship. The 3D-ASO technology helps fight coronavirus disease-2019 and is broadly applicable to ASO drug development.","version":"1.1","doi":"10.1101/2021.08.23.457434","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.457521","pub_date":"2021-8-24","title":"Gene Expression Risk Scores for COVID-19 Illness Severity","abstract":"The correlates of COVID-19 illness severity following infection with SARS-Coronavirus 2 (SARS-CoV-2) are incompletely understood. We assessed peripheral blood gene expression in 53 adults with confirmed SARS-CoV-2-infection clinically adjudicated as having mild, moderate or severe disease. Supervised principal components analysis was used to build a weighted gene expression risk score (WGERS) to discriminate between severe and non-severe COVID. Gene expression patterns in participants with mild and moderate illness were similar, but significantly different from severe illness. When comparing severe versus non-severe illness, we identified >4000 genes differentially expressed (FDR<0.05). Biological pathways increased in severe COVID-19 were associated with platelet activation and coagulation, and those significantly decreased with T cell signaling and differentiation. A WGERS based on 18 genes distinguished severe illness in our training cohort (cross-validated ROC-AUC=0.98), and need for intensive care in an independent cohort (ROC-AUC=0.85). Dichotomizing the WGERS yielded 100% sensitivity and 85% specificity for classifying severe illness in our training cohort, and 84% sensitivity and 74% specificity for defining the need for intensive care in the validation cohort. These data suggest that gene expression classifiers may provide clinical utility as predictors of COVID-19 illness severity.","version":"1.1","doi":"10.1101/2021.08.24.457521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.24.457187","pub_date":"2021-8-24","title":"Profiling Transcription Initiation in Peripheral Leukocytes Reveals Severity-Associated Cis-Regulatory Elements in Critical COVID-19","abstract":"The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the \u2019active cistrome,\u2019 offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.","version":"1.1","doi":"10.1101/2021.08.24.457187","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433919","pub_date":"2021-8-24","title":"An Essential Role of UBXN3B in B Lymphopoiesis","abstract":"Hematopoiesis is finely regulated to enable timely production of the right numbers and types of mature immune cells to maintain tissue homeostasis. Dysregulated hematopoiesis may compromise antiviral immunity and/or exacerbate immunopathogenesis. Herein, we report an essential role of UBXN3B in maintenance of hematopoietic homeostasis and restriction of immunopathogenesis during respiratory viral infection. Ubxn3b deficient (Ubxn3b\u2212/\u2212) mice are highly vulnerable to SARS-CoV-2 and influenza A infection, characterized by more severe lung immunopathology, lower virus-specific IgG, significantly fewer B cells, but more myeloid cells than Ubxn3b+/+ littermates. This aberrant immune compartmentalization is recapitulated in uninfected Ubxn3b\u2212/\u2212 mice. Mechanistically, UBXN3B controls precursor B-I (pre-BI) transition to pre-BII and subsequent proliferation in a cell-intrinsic manner, by maintaining BLNK protein stability and pre-BCR signaling. These results reveal an essential role of UBXN3B for the early stage of B cell development.","version":"1.3","doi":"10.1101/2021.03.04.433919","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457328","pub_date":"2021-8-23","title":"Presence and Stability of SARS-CoV-2 on Environmental Currency and Money Cards","abstract":"The highly contagious nature of SARS-CoV-2 has led to several studies on the transmission of the virus. A little studied potential fomite of great concern in the community is currency, which has been shown to harbor microbial pathogens in several studies. Since the onset of the COVID-19 pandemic, many businesses in the United States have limited the use of banknotes in favor of credit cards. However, SARS-CoV-2 has shown greater stability on plastic in several studies. Herein, the stability of SARS-CoV-2 at room temperature on banknotes, money cards and coins was investigated. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, showing an initial rapid reduction in viable virus and no viral detection by 24 hours. In contrast, SARS-CoV-2 was far more stable on money cards with live virus detected after 48 hours. Environmental swabbing of currency and money cards on and near the campus of Brigham Young University supported these results, with no detection of SARS-CoV-2 RNA on banknotes, and a low level on money cards. No viable virus was detected on either. These preliminary results suggest that the use of money cards over banknotes in order to slow the spread of this virus may be ill-advised. These findings should be investigated further through larger environmental studies involving more locations.","version":"1.1","doi":"10.1101/2021.08.23.457328","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457314","pub_date":"2021-8-23","title":"Switching of OAS1 splicing isoforms mitigates SARS-CoV-2 infection","abstract":"The rapidly accumulating disease susceptibility information collected from coronavirus disease (COVID-19) patient genomes must be urgently utilized to develop therapeutic interventions for SARS-CoV-2 infection. Chromosome 12q24.13, which encodes the 2\u2019-5\u2019-oligoadenylate synthetase (OAS) family of proteins that sense viral genomic RNAs and trigger an antiviral response, is identified as one of the genomic regions that contains SNPs associated with COVID-19 severity. A high-risk SNP identified at the splice acceptor site of OAS1 exon 6 is known to change the proportions of the various splicing isoforms and the activity of the enzyme. We employed in-silico motif search and RNA pull-down assay to define a factor responsible for the OAS1 splicing. Next, we rationally selected a candidate for slicing modulator to modulate this splicing. We found that inhibition of CDC-like kinase with a small chemical compound induces switching of OAS1 splice isoforms in human lung cells. In this condition, increased resistance to SARS-CoV-2 infection, enhanced RNA degradation, and transcriptional activation of interferon \u03b21, were also observed. The results indicate the possibility of using chemical splicing modifiers aided by genome-based precision medicine to boost the innate immune response against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.08.23.457314","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.456471","pub_date":"2021-8-23","title":"Single-shot rAAV5-based Vaccine Provides Long-term Protective Immunity against SARS-CoV-2 and Its Variants","abstract":"The COVID-19 pandemic and the SARS-CoV-2 with its variants have posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against the SARS-CoV-2 variants. Therefore, novel vaccines to match current mutated viral lineages with long-term protective immunity are urgently in demand. In the current study, we for the first time designed a recombinant Adeno-Associated Virus 5 (rAAV5)-based vaccine named as rAAV-COVID-19 vaccine (Covacinplus) by using RBD-plus of spike protein with both the single-stranded and the self-complementary AAV5 delivering vectors (ssAAV5 and scAAAV5), which provides excellent protection from SARS-CoV-2 infection. A single dose vaccination induced the strong immune response against SARS-CoV-2. The induced neutralizing antibodies (NAs) titers were maintained at a high peak level of over 1:1024 even after more than one year of injection and accompanied with functional T-cells responses in mice. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines exhibited high levels of serum NAs against current circulating variants including variants Alpha, Beta, Gamma and Delta. SARS-CoV-2 virus challenge test showed that ssAAV5-RBD-plus vaccine protected both young and old age mice from SARS-CoV-2 infection in the upper and the lower respiratory tracts. Moreover, whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genome of the vaccinated mice after one year vaccination, demonstrating excellent safety of the vaccine. Taken together, this study suggests that rAAV5-based vaccine is powerful against SARS-CoV-2 and its variants with long-term protective immunity and excellent safety, which has great potential for development into prophylactic vaccination in human to end this global pandemic.","version":"1.1","doi":"10.1101/2021.08.23.456471","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457229","pub_date":"2021-8-23","title":"mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern","abstract":"SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 na\u00efve and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies.","version":"1.1","doi":"10.1101/2021.08.23.457229","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.23.457408","pub_date":"2021-8-23","title":"Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection","abstract":"Pregnant women are an at-risk group for severe COVID-19, though the majority experience mild/asymptomatic disease. Although severe COVID-19 has been shown to be associated with immune activation at the maternal-fetal interface even in the absence of active viral replication, the immune response to asymptomatic/mild COVID-19 remains unknown. Here, we assessed immunological adaptations in both blood and term decidua from 9 SARS-exposed pregnant women with asymptomatic/mild disease and 15 pregnant SARS-naive women. In addition to selective loss of tissue-resident decidual macrophages, we report attenuation of antigen presentation and type I IFN signaling but upregulation of inflammatory cytokines and chemokines in blood monocyte derived decidual macrophages. On the other hand, infection was associated with remodeling of the T cell compartment with increased frequencies of activated CD69+ tissue-resident T cells and decreased abundance of Tregs. Interestingly, frequencies of cytotoxic CD4 and CD8 T cells increased only in the blood, while CD8 effector memory T cells were expanded in the decidua. In contrast to decidual macrophages, signatures of type I IFN signaling were increased in decidual T cells. Finally, T cell receptor diversity was significantly reduced with infection in both compartments, albeit to a much greater extent in the blood. The resulting aberrant immune activation in the placenta, even with asymptomatic disease may alter the exquisitely sensitive developing fetal immune system, leading to long-term adverse outcomes for offspring.","version":"1.1","doi":"10.1101/2021.08.23.457408","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.22.457114","pub_date":"2021-8-23","title":"The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines","abstract":"mRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.","version":"1.1","doi":"10.1101/2021.08.22.457114","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.20.21262389","pub_date":"2021-08-23","title":"Model-based assessment of SARS-CoV-2 Delta variant transmission dynamics within partially vaccinated K-12 school populations","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>We examined school reopening policies amidst rising transmission of the highly transmissible Delta variant, accounting for vaccination among individuals aged 12 years and older, with the goal of characterizing risk to students and teachers under various within-school non-pharmaceutical interventions (NPIs) combined with specific vaccination coverage levels.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed an individual-based transmission model to simulate transmission of the Delta variant of SARS-CoV-2 among a synthetic population, representative of Bay Area cities. We parameterized the model using community contact rates from vaccinated households ascertained from a household survey of Bay Area families with children conducted between February \u2013 April, 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interventions and outcomes</jats:title>\n                  <jats:p>We evaluated the additional infections in students and teachers/staff resulting over a 128-day semester from in-school instruction compared to remote instruction when various NPIs (mask use, cohorts, and weekly testing of students/teachers) were implemented in schools, across various community-wide vaccination coverages (50%, 60%, 70%), and student (\u226512 years) and teacher/staff vaccination coverages (50% - 95%). We quantified the added benefit of universal masking over masking among unvaccinated students and teachers, across varying levels of vaccine effectiveness (45%, 65%, 85%), and compared results between Delta and Alpha variant circulation.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The Delta variant sharply increases the risk of within-school COVID-transmission when compared to the Alpha variant. In our highest risk scenario (50% community and within-school vaccine coverage, no within-school NPIs, and predominant circulation of the Delta variant), we estimated that an elementary school could see 33-65 additional symptomatic cases of COVID-19 over a four-month semester (depending on the relative susceptibility of children &lt;10 years). In contrast, under the Bay Area reopening plan (universal mask use, community and school vaccination coverage of 70%), we estimated excess symptomatic infection attributable to school reopening among 2.0-9.7% of elementary students (8-36 excess symptomatic cases per school over the semester), 3.0% of middle school students (13 cases per school) and 0.4% of high school students (3 cases per school). Excess rates among teachers attributable to reopening were similar. Achievement of lower risk tolerances, such as &lt;5 excess infections per 1,000 students or teachers, required a cohort approach in elementary and middle school populations. In the absence of NPIs, increasing the vaccination coverage of community members from 50% to 70% or elementary teachers from 70% to 95% reduced the estimated excess rate of infection among elementary school students attributable to school transmission by 24% and 41%, respectively. We estimated that with 70% coverage of the eligible community and school population with a vaccine that is \u226465% effective, universal masking can avert more cases than masking of unvaccinated persons alone.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Amidst circulation of the Delta variant, our findings demonstrated that schools are not inherently low risk, yet can be made so with high community vaccination coverages and universal masking. Vaccination of adult community members and teachers protects unvaccinated elementary and middle school children. Elementary and middle schools that can support additional interventions, such as cohorts and testing, should consider doing so, particularly if additional studies find that younger children are equally as susceptible as adults to the Delta variant of SARS-CoV-2.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Limitations</jats:title>\n                  <jats:p>We did not consider the effect of social distancing in classrooms, or variation in testing frequency, and considerable uncertainty remains in key transmission parameters.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.20.21262389","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.21.457245","pub_date":"2021-8-22","title":"Effects of face masks and ventilation on the risk of SARS-CoV-2 respiratory transmission in public toilets: a quantitative microbial risk assessment","abstract":"Public toilets could increase the risk of COVID-19 infection via airborne transmission; however, related research is limited. We aimed to estimate SARS-CoV-2 infection risk through respiratory transmission using a quantitative microbial risk assessment framework by retrieving SARS-CoV-2 concentrations from the swab tests of 251 Thai patients. Three virus-generating scenarios were investigated: an infector breathing, breathing with a cough, and breathing with a sneeze. Infection risk (97.5th percentile) was as high as 10\u22123 with breathing and increased to 10\u22121 with a cough or sneeze, thus all higher than the risk benchmark of 5 \u00d7 10\u22125 per event. No significant gender differences for toilet users (receptors) were noted. The highest risk scenario of breathing and a sneeze was further evaluated for risk mitigation measures. Risk mitigation to lower than the benchmark succeeded only when the infector and receptor simultaneously wore an N95 respirator or surgical mask and when the receptor wore an N95 respirator and the infector wore a denim fabric mask. Ventilation up to 20 air changes per hour (ACH), beyond the 12-ACH suggested by the WHO, did not mitigate risk. Virus concentration, volume of expelled droplets, and receptor dwell time were identified as the main contributors to transmission risk. The use of public toilets poses a risk of SARS-CoV-2 respiratory transmission Highest risks generated in the order of sneezing, coughing, and breathing No gender differences in risk by counteracting dwell times and inhalation rates Ventilation did not reduce risk even at 20 ACH, beyond the WHO-recommended value N95 and surgical masks offer the most effective risk mitigation to toilet users","version":"1.1","doi":"10.1101/2021.08.21.457245","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.14.21262020","pub_date":"2021-08-22","title":"Delta variant and mRNA Covid-19 vaccines effectiveness: higher odds of vaccine infection breakthroughs","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The SARS-CoV-2 Delta variant (B.1.617.2), initially identified in India, has become predominant in several countries, including Portugal. Few studies have compared the effectiveness of mRNA vaccines against Delta versus Alpha variant of concern (VOC) and estimated variant-specific viral loads in vaccine infection breakthroughs cases. In the context of Delta dominance, this information is critical to inform decision-makers regarding the planning of restrictions and vaccination roll-out.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed a case-case study to compare mRNA vaccines\u2019 effectiveness against Delta (B.1.617.2) versus Alpha (B.1.1.7) variants. We used RT-PCR positive cases notified to the National Surveillance System between 17th of May and 4th of July 2021 (week 20 to 26) and information about demographics and vaccination status through the electronic vaccination register. Whole-genome sequencing (WGS) or spike (S) gene target failure (SGTF) data were used to classify SARS-CoV-2 variants. The odds of vaccinated individuals to become infected (odds of vaccine infection breakthrough) in Delta cases compared to Alpha SARS-CoV-2 cases was estimated by conditional logistic regression adjusted for age group, sex, and matched by the week of diagnosis. As a surrogate of viral load, mean RT-PCR Ct values were stratified and compared between vaccine status and VOC.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Of the 2 097 SARS-CoV-2 RT-PCR positive cases included in the analysis, 966 (46.1%) were classified with WGS and 1131 (53.9%) with SGTF. Individuals infected with the Delta variant were more frequently vaccinated 162 (12%) than individuals infected with the Alpha variant 38 (5%). We report a statistically significant higher odds of vaccine infection breakthrough for partial (OR=1.70; CI95% 1.18 to 2.47) and complete vaccination (OR=1.96; CI95% 1.22 to 3.14) in the Delta cases when compared to the Alpha cases, suggesting lower mRNA vaccine effectiveness against Delta cases. On our secondary analysis, we observed lower mean Ct values for the Delta VOC cases versus Alpha, regardless the vaccination status. Additionally, the Delta variant cases revealed a Ct-value mean increase of 2.24 (CI95% 0.85 to 3.64) between unvaccinated and fully vaccinated breakthrough cases contrasting with 4.49 (CI95% 2.07 to 6.91) in the Alpha VOC, suggesting a lower impact of vaccine on viral load of Delta cases.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>We found significantly higher odds of vaccine infection breakthrough in Delta cases when compared to Alpha cases, suggesting lower effectiveness of the mRNA vaccines in preventing infection with the Delta variant. Additionally, the vaccine breakthrough cases are estimated to be of higher mean Ct values, suggesting higher infectiousness with the Delta variant infection. These findings can help decision-makers weigh on the application or lifting of control measures and adjusting vaccine roll-out depending on the predominance of the Delta variant and the coverage of partial and complete mRNA vaccination.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.14.21262020","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.16.456444","pub_date":"2021-8-21","title":"A time irreversible model of nucleotide substitution for the SARS-CoV-2 evolution","abstract":"SARS-CoV-2 is the cause of the worldwide epidemic of severe acute respiratory syndrome. Evolutionary studies of the virus genome will provide a predictor of the fate of COVID-19 in the near future. Recent studies of the virus genomes have shown that C to U substitutions are overrepresented in the genome sequences of SARS-CoV-2. Traditional time-reversible substitution models cannot be applied to the evolution of SARS-CoV-2 sequences. Therefore, in this study, I propose a new time-irreversible model and a new method for estimating the nucleotide substitution rate of SARS-CoV-2. Computer simulations showed that that the new method gives good estimates. I applied the new method to estimate nucleotide substitution rates of SARS-CoV-2 sequences. The result suggests that the rate of C to U substitution of SARS-Cov-2 is ten times higher than other types of substitutions.","version":"1.4","doi":"10.1101/2021.08.16.456444","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214577","pub_date":"2021-8-21","title":"Inhibitors of L-type calcium channels show therapeutic potential for treating SARS-CoV-2 infections by preventing virus entry and spread","abstract":"COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus (CoV)-2 (SARS-CoV-2). The virus is responsible for an ongoing pandemic and concomitant public health crisis around the world. While vaccine development is proving to be highly successful, parallel drug development approaches are also critical in the response to SARS-CoV-2 and other emerging viruses. Coronaviruses require Ca2+ ions for host cell entry and we have previously shown that Ca2+ modulates the interaction of the viral fusion peptide with host cell membranes. In an attempt to accelerate drug development, we tested a panel of L-type calcium channel blocker (CCB) drugs currently developed for other conditions, to determine whether they would inhibit SARS-CoV-2 infection in cell culture. All the CCBs tested showed varying degrees of inhibition, with felodipine and nifedipine strongly limiting SARS-CoV-2 entry and infection in epithelial lung cells at concentrations where cell toxicity was minimal. Further studies with pseudo-typed particles displaying the SARS-CoV-2 spike protein suggested that inhibition occurs at the level of virus entry. Overall, our data suggest that certain CCBs have potential to treat SARS-CoV-2 infections and are worthy of further examination for possible treatment of COVID-19.","version":"1.4","doi":"10.1101/2020.07.21.214577","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.15.422900","pub_date":"2021-8-21","title":"The rapid, massive growth of COVID-19 authors in the scientific literature","abstract":"We examined the extent to which the scientific workforce in different fields was engaged in publishing COVID-19-related papers. According to Scopus (data cut, August 1, 2021), 210,183 COVID-19-related publications included 720,801 unique authors, of which 360,005 authors had published at least 5 full papers in their career and 23,520 authors were at the top 2% of their scientific subfield based on a career-long composite citation indicator. The growth of COVID-19 authors was far more rapid and massive compared with cohorts of authors historically publishing on H1N1, Zika, Ebola, HIV/AIDS and tuberculosis. All 174 scientific subfields had some specialists who had published on COVID-19. In 109 of the 174 subfields of science, at least one in ten active, influential (top-2% composite citation indicator) authors in the subfield had authored something on COVID-19. 52 hyper-prolific authors had already at least 60 (and up to 227) COVID-19 publications each. Among the 300 authors with the highest composite citation indicator for their COVID-19 publications, most common countries were USA (n=67), China (n=52), UK (n=32), and Italy (n=18). The rapid and massive involvement of the scientific workforce in COVID-19-related work is unprecedented and creates opportunities and challenges. There is evidence for hyper-prolific productivity.","version":"1.3","doi":"10.1101/2020.12.15.422900","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.19.457020","pub_date":"2021-8-20","title":"Platelet proteome analysis reveals an early hyperactive phenotype in SARS-CoV-2-infected humanized ACE2 mice","abstract":"Coronavirus disease-2019 (COVID-19) provokes a hypercoagulable state with increased incidence of thromboembolism and mortality. Platelets are major effectors of thrombosis and hemostasis. Suitable animal models are needed to better understand COVID-19-associated coagulopathy (CAC) and underlying platelet phenotypes. Here, we assessed K18-hACE2 mice undergoing a standardized SARS-CoV-2 infection protocol to study dynamic platelet responses via mass spectrometry-based proteomics. In total, we found significant changes in >1,200 proteins. Strikingly, protein alterations occurred rapidly by 2 days post-infection (dpi) and preceded outward clinical signs of severe disease. Pathway enrichment analysis of 2dpi platelet proteomes revealed that SARS-CoV-2 infection upregulated complement-coagulation networks (F2, F12, CFH, CD55/CD59), platelet activation-adhesion-degranulation proteins (PF4, SELP, PECAM1, HRG, PLG, vWF), and chemokines (CCL8, CXCL5, CXCL12). When mice started to lose weight at 4dpi, pattern recognition receptor signaling (RIG-I/MDA5, CASP8, MAPK3), and interferon pathways (IFIT1/IFIT3, STAT1) were predominant. Interestingly, SARS-CoV-2 spike protein in the lungs was observed by immunohistochemistry, but in platelets was undetected by proteomics. Similar to patients, K18-hACE2 mice during SARS-CoV-2 infection developed progressive lymphohistiocytic interstitial pneumonia with platelet aggregates in the lungs and kidneys. In conclusion, this model recapitulates activation of coagulation, complement, and interferon responses in circulating platelets, providing valuable insight into platelet pathology during COVID-19. SARS-CoV-2-infected humanized ACE2 mice recapitulate platelet reprogramming towards activation-degranulation-aggregation. Complement/coagulation pathways are dominant in platelets at 2 days post-infection (dpi), while interferon signaling is dominant at 4dpi.","version":"1.1","doi":"10.1101/2021.08.19.457020","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.20.456972","pub_date":"2021-8-20","title":"Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway","abstract":"SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.","version":"1.1","doi":"10.1101/2021.08.20.456972","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.20.457128","pub_date":"2021-8-20","title":"High-Spatiotemporal-Resolution Nanopore Sequencing of SARS-CoV-2 and Host Cell RNAs","abstract":"Recent studies have disclosed the genome, transcriptome and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for non-canonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (MOI=0.1) applied for the infection. In this study, we used direct cDNA and direct RNA nanopore sequencings for the generation of high-coverage, high-temporal-resolution transcriptomic datasets on SARS-CoV-2 and on primate host cells infected with a high virus titer (MOI=5). Sixteen sampling time points ranging from 1 to 96h with a varying time resolution and three biological replicates were used in the experiment for both the infected and the non-infected cells.","version":"1.1","doi":"10.1101/2021.08.20.457128","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.17.21262169","pub_date":"2021-08-20","title":"SARS-CoV-2 aerosol transmission in schools: the effectiveness of different interventions","abstract":"<jats:title>\n                  A\n                  <jats:sc>bstract</jats:sc>\n                </jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Indoor aerosol transmission of SARS-CoV-2 has been widely recognized, especially in schools where children remain in closed indoor spaces and largely unvaccinated. Measures such as strategic natural ventilation and high efficiency particulate air (HEPA) filtration remain poorly implemented and mask mandates are often progressively lifted as vaccination rollout is enhanced.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We adapted a previously developed aerosol transmission model to study the effect of interventions (natural ventilation, face masks, HEPA filtration, and their combinations) on the concentration of virus particles in a classroom of 160 m\n                    <jats:sup>3</jats:sup>\n                    containing one infectious individual. The cumulative dose of viruses absorbed by exposed occupants was calculated.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The most effective single intervention was natural ventilation through the full opening of six windows all day during the winter (14-fold decrease in cumulative dose), followed by the universal use of surgical face masks (8-fold decrease). In the spring/summer, natural ventilation was only effective (\u2265 2-fold decrease) when windows were fully open all day. In the winter, partly opening two windows all day or fully opening six windows at the end of each class was effective as well (\u2265 2-fold decrease). Opening windows during yard and lunch breaks only had minimal effect (\u2264 1.2-fold decrease). One HEPA filter was as effective as two windows partly open all day during the winter (2.5-fold decrease) while two filters were more effective (4-fold decrease). Combined interventions (i.e., natural ventilation, masks, and HEPA filtration) were the most effective (\u2265 30-fold decrease). Combined interventions remained highly effective in the presence of a super-spreader.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Natural ventilation, face masks, and HEPA filtration are effective interventions to reduce SARS-CoV-2 aerosol transmission. These measures should be combined and complemented by additional interventions (e.g., physical distancing, hygiene, testing, contact tracing, and vaccination) to maximize benefit.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.17.21262169","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.31.428824","pub_date":"2021-8-20","title":"Identification of a conserved neutralizing epitope present on spike proteins from all highly pathogenic coronaviruses","abstract":"Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.","version":"1.2","doi":"10.1101/2021.01.31.428824","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.18.456891","pub_date":"2021-8-19","title":"Neutralising antibodies against the SARS-CoV-2 Delta variant induced by Alhydroxyquim-II-adjuvanted trimeric spike antigens","abstract":"Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2021.08.18.456891","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.437747","pub_date":"2021-8-19","title":"XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2 Spike receptor-binding domain, targets multiple epitopes and broadly neutralizes variants","abstract":"Amino acid substitutions and deletions in Spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBM), in direct contact sites with the Angiotensin Converting Enzyme-2 (ACE-2). Therefore, in Spike/ACE2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study with the Beta strain on Vero E6 cells conducted over 1 month, no mutation was associated with addition of increasing doses XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in a phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (Covid-19) including the different variants of concern identified so far.","version":"1.2","doi":"10.1101/2021.04.02.437747","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.18.455536","pub_date":"2021-8-19","title":"Algorithm for the Quantitation of Variants of Concern for Rationally Designed Vaccines Based on the Isolation of SARS-CoV-2 Hawai\u02bbi Lineage B.1.243","abstract":"SARS-CoV-2 worldwide emergence and evolution has resulted in variants containing mutations resulting in immune evasive epitopes that decrease vaccine efficacy. We acquired clinical samples, analyzed SARS-CoV-2 genomes, used the most worldwide emerged spike mutations from Variants of Concern/Interest, and developed an algorithm for monitoring the SARS-CoV-2 vaccine platform. The algorithm partitions logarithmic-transformed prevalence data monthly and Pearson\u2019s correlation determines exponential emergence. The SARS-CoV-2 genome evaluation indicated 49 mutations. Nine of the ten most worldwide prevalent (>70%) spike protein changes have r-values >0.9. The tenth, D614G, has a prevalence >99% and r-value of 0.67. The resulting algorithm is based on the patterns these ten substitutions elucidated. The strong positive correlation of the emerged spike protein changes and algorithmic predictive value can be harnessed in designing vaccines with relevant immunogenic epitopes. SARS-CoV-2 is predicted to remain endemic and continues to evolve, so must SARS-CoV-2 monitoring and next-generation vaccine design.","version":"1.1","doi":"10.1101/2021.08.18.455536","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.442666","pub_date":"2021-8-19","title":"Longitudinal immune dynamics of mild COVID-19 define signatures of recovery and persistence","abstract":"SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).","version":"1.3","doi":"10.1101/2021.05.26.442666","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.19.456973","pub_date":"2021-8-19","title":"Modeling Coronavirus Spike Protein Dynamics: Implications for Immunogenicity and Immune Escape","abstract":"The ongoing COVID-19 pandemic is a global public health emergency requiring urgent development of efficacious vaccines. While concentrated research efforts are underway to develop antibody-based vaccines that would neutralize SARS-CoV-2, and several first-generation vaccine candidates are currently in Phase III clinical trials or have received emergency use authorization, it is forecasted that COVID-19 will become an endemic disease requiring second-generation vaccines. The SARS-CoV-2 surface Spike (S) glycoprotein represents a prime target for vaccine development because antibodies that block viral attachment and entry, i.e. neutralizing antibodies, bind almost exclusively to the receptor binding domain (RBD). Here, we develop computational models for a large subset of S proteins associated with SARS-CoV-2, implemented through coarse-grained elastic network models and normal mode analysis. We then analyze local protein domain dynamics of the S protein systems and their thermal stability to characterize structural and dynamical variability among them. These results are compared against existing experimental data, and used to elucidate the impact and mechanisms of SARS-CoV-2 S protein mutations and their associated antibody binding behavior. We construct a SARS-CoV-2 antigenic map and offer predictions about the neutralization capabilities of antibody and S mutant combinations based on protein dynamic signatures. We then compare SARS-CoV-2 S protein dynamics to SARS-CoV and MERS-CoV S proteins to investigate differing antibody binding and cellular fusion mechanisms that may explain the high transmissibility of SARS-CoV-2. The outbreaks associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 over the last two decades suggest that the threat presented by coronaviruses is ever-changing and long-term. Our results provide insights into the dynamics-driven mechanisms of immunogenicity associated with coronavirus S proteins, and present a new approach to characterize and screen potential mutant candidates for immunogen design, as well as to characterize emerging natural variants that may escape vaccine-induced antibody responses. We present novel dynamic mechanisms of coronavirus S proteins that encode antibody binding and cellular fusion properties. These mechanisms may offer an explanation for the widespread nature of SARS-CoV-2 and more limited spread of SARS-CoV and MERS-CoV. A comprehensive computational characterization of SARS-CoV-2 S protein structures and dynamics provides insights into structural and thermal stability associated with a variety of S protein mutants. These findings allow us to make recommendations about the future mutant design of SARS-CoV-2 S protein variants that are optimized to elicit neutralizing antibodies, resist structural rearrangements that aid cellular fusion, and are thermally stabilized. The integrated computational approach can be applied to optimize vaccine immunogen design and predict escape of vaccine-induced antibody responses by SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.08.19.456973","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.375972","pub_date":"2021-8-19","title":"ACE2-Targeting Monoclonal Antibody as Potent and Broad-Spectrum Coronavirus Blocker","abstract":"The evolution of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Here we report a human angiotensin-converting enzyme 2 (ACE2)-targeting monoclonal antibody, 3E8, blocked the S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2, SARS-CoV-2 mutant variants (SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617.1 and P.1), SARS-CoV and HCoV-NL63, without markedly affecting the physiological activities of ACE2 or causing severe toxicity in ACE2 \u201cknock-in\u201d mice. 3E8 also blocked live SARS-CoV-2 infection in vitro and in a prophylactic mouse model of COVID-19. Cryo-EM and \u201calanine walk\u201d studies revealed the key binding residues on ACE2 interacting with the CDR3 domain of 3E8 heavy chain. Although full evaluation of safety in non-human primates is necessary before clinical development of 3E8, we provided a potentially potent and \u201cbroad-spectrum\u201d management strategy against all coronaviruses that utilize ACE2 as entry receptors and disclosed an anti-coronavirus epitope on human ACE2.","version":"1.2","doi":"10.1101/2020.11.11.375972","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.18.456916","pub_date":"2021-8-19","title":"Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells","abstract":"The SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the virus\u2019s ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess every species susceptibility to SC2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SC2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis (cat), Equus (horse), Sus (pig), Capra (goat), Mesocricetus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SC2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SC2 and emerging variant viruses. SARS-CoV-2 (SC2) is believed to have originated in animal species and jumped into humans where it has produced the greatest viral pandemic of our time. Identification of animal species susceptible to SC2 infection would provide information on potential zoonotic reservoirs, and transmission potential at the human-animal interface. Our work provides a model system to test the ability of the virus to replicate in an otherwise non-permissive cell line by transgenic insertion of the ACE2 and TMPRSS2 genes from human and other animal species. The results from our in vitro model positively correlate with animal infection studies enhancing the predicative capability of the model. Importantly, we demonstrate that both proteins are required for successful virus replication. These findings establish a framework to test other animal species for susceptibility to infection that may be critical zoonotic reservoirs for transmission, as well as to test variant viruses that arise over time.","version":"1.1","doi":"10.1101/2021.08.18.456916","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.19.456951","pub_date":"2021-8-19","title":"Pandemic, epidemic, endemic: B cell repertoire analysis reveals unique anti-viral responses to SARS-CoV-2, Ebola and Respiratory Syncytial Virus","abstract":"Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of IGHV4-39 in both EBOV and COVID-19 infection cohorts. We also show unique characteristics absent in RSV infection or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risks off-target effects.","version":"1.1","doi":"10.1101/2021.08.19.456951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.17.456707","pub_date":"2021-8-18","title":"An Endogenously activated antiviral state restricts SARS-CoV-2 infection in differentiated primary airway epithelial cells","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and went on to cause over 3.3 million deaths in 15 months. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFN\u03bb) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) model from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFN\u03bb expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFN\u03bb secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFN\u03bb treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFN\u03bb response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFN\u03bb as a potential pharmaceutical against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.08.17.456707","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.17.456704","pub_date":"2021-8-18","title":"Protein-based RBD-C-tag COVID-19 Vaccination Candidate Elicits Protection Activity against SARS-COV-2 Variant Infection","abstract":"The identification of a vaccination candidate against COVID-19 providing protecting activity against emerging SARS-COV-2 variants remains challenging. Here, we report protection activity against a spectrum of SARS-COV-2 and variants by immunization with protein-based recombinant RBD-C-tag administered with aluminum-phosphate adjuvant intramuscularly. Immunization of C57BL/6 mice with RBD-C-tag resulted in the in vivo production of IgG antibodies recognizing the immune-critical spike protein of the SARS-COV-2 virus as well as the SARS-COV-2 variants alpha (\u201cUnited Kingdom\u201d), beta (\u201cSouth Africa\u201d), gamma (\u201cBrazil/Japan\u201d), and delta (\u201cIndia\u201d) as well as wt-spike protein. RBD-C-tag immunization led to a desired Th1 polarization of CD4 T cells producing IFN\u03b3. Importantly, RBD-C-tag immunization educated IgG production delivers antibodies that exert neutralizing activity against the highly transmissible SARS-COV-2 virus strains \u201cWashington\u201d, \u201cSouth Africa\u201d (beta), and \u201cIndia\u201d (delta) as determined by conservative infection protection experiments in vitro. Hence, the protein-based recombinant RBD-C-tag is considered a promising vaccination candidate against COVID-19 and a broad range of emerging SARS-COV-2 virus variants.","version":"1.1","doi":"10.1101/2021.08.17.456704","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.18.456769","pub_date":"2021-8-18","title":"Interaction between spike protein of SARS-CoV-2 and human virus receptor ACE2 using two-color fluorescence cross-correlation spectroscopy","abstract":"Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is initiated by the interaction between a receptor protein, angiotensin-converting enzyme type 2 (ACE2) on the cell surface, and the viral spike (S) protein. This interaction is similar to the mechanism in SARS-CoV, a close relative of SARS-CoV-2, which was identified in 2003. Drugs and antibodies that inhibit the interaction between ACE2 and S proteins could be key therapeutic methods for preventing viral infection and replication in COVID-19. Here, we demonstrate the interaction between human ACE2 and a fragment of the S protein (S1 subunit) derived from SARS-CoV-2 and SARS-CoV using two-color fluorescence cross-correlation spectroscopy (FCCS), which can detect the interaction of fluorescently labeled proteins. The S1 subunit of SARS-CoV-2 interacted in solution with soluble ACE2, which lacks a transmembrane region, more strongly than that of SARS-CoV. Furthermore, one-to-one stoichiometry of the two proteins during the interaction was indicated. Thus, we propose that this FCCS-based interaction detection system can be used to analyze the interaction strengths of various mutants of the S1 subunit that have evolved during the worldwide pandemic, and also offers the opportunity to screen and evaluate the performance of drugs and antibodies that inhibit the interaction.","version":"1.1","doi":"10.1101/2021.08.18.456769","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446571","pub_date":"2021-8-18","title":"Dynamics of SARS-CoV-2 mutations reveals regional-specificity and similar trends of N501 and High-Frequency mutation N501Y in different levels of control measures","abstract":"Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has spread globally, causing more than 161.5 million cases and 3.3 million deaths to date. Surveillance and monitoring of new mutations in the virus\u2019 genome are crucial to our understanding of the adaptation of SARS-CoV-2. Moreover, how the temporal dynamics of these mutations is influenced by control measures and non-pharmaceutical interventions (NPIs) is poorly understood. Using 1 058 020 SARS-CoV-2 from sequenced COVID-19 cases from 98 countries (totaling 714 country-month combinations), we perform a normalization by COVID-19 cases to calculate the relative frequency of SARS-CoV-2 mutations and explore their dynamics over time. We found 115 mutations estimated to be present in more than 3 % of global COVID-19 cases and determined three types of mutation dynamics: High-Frequency, Medium-Frequency, and Low-Frequency. Classification of mutations based on temporal dynamics enable us to examine viral adaptation and evaluate the effects of implemented control measures in virus evolution during the pandemic. We showed that Medium-Frequency mutations are characterized by high prevalence in specific regions and/or in constant competition with other mutations in several regions. Finally, taking N501Y mutation as representative of High-Frequency mutations, we showed that level of control measure stringency negatively correlates with the effective reproduction number of SARS-CoV-2 with High-Frequency or not-High-Frequency and both follows similar trends in different levels of stringency.","version":"1.3","doi":"10.1101/2021.06.01.446571","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441024","pub_date":"2021-8-18","title":"The Great Deceiver: miR-2392\u2019s Hidden Role in Driving SARS-CoV-2 Infection","abstract":"MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 positive patients, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters and may potentially inhibit a COVID-19 disease state in humans.","version":"1.5","doi":"10.1101/2021.04.23.441024","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456266","pub_date":"2021-8-18","title":"Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-\u03baB signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.","version":"1.2","doi":"10.1101/2021.08.13.456266","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.456012","pub_date":"2021-8-18","title":"In vitro determination of inhibitory effects by humic substances complexing Zn and Se on SARS-CoV-2 virus replication","abstract":"Humic substances are well known human nutritional supplement materials and play important performance-enhancing roles as animal feed additives, too. For decades, ingredients of humic substances have also been proven to carry potent antiviral effects against different viruses. Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern (\u201cAlpha Variant\u201d) in a VeroE6 cell line. Results show that this combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. Even picomolar concentration ranges of humic substances, vitamin C and Zn/Se ions in the given composition were enough to achieve fifty percent viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.","version":"1.2","doi":"10.1101/2021.08.11.456012","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.18.456855","pub_date":"2021-8-18","title":"Cap-independent translation and a precisely localized RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response","abstract":"Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represseses translation and induces mRNA degradation, while the host elicits anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-meidated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5\u2019UTR of SARS-CoV-2 exhibits an IRES-like activity and promotes expression of NSP1 in an eIF4E-independent and Torin-1 resistant manner. Upon expression, NSP1 enhances cap-independent translation. However, the sub-genomic 5\u2019UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin-1. The combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5\u2019UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.","version":"1.1","doi":"10.1101/2021.08.18.456855","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452194","pub_date":"2021-8-18","title":"A drug candidate for treating adverse reactions caused by pathogenic antibodies inducible by COVID-19 virus and vaccines","abstract":"In a recent study, we reported that certain anti-spike antibodies of COVID-19 and SARS-CoV viruses can have a pathogenic effect through binding to sick lung epithelium cells and misleading immune responses to attack self-cells. We termed this new pathogenic mechanism \u201cAntibody Dependent Auto-Attack\u201d (ADAA). This study explores a drug candidate for prevention and treatment of such ADAA-based diseases. The drug candidate is a formulation comprising N-acetylneuraminic acid methyl ester (NANA-Me), an analog of N-acetylneuraminic acid. NANA-Me acts through a unique mechanism of action (MOA) which is repairment of the missing sialic acid on sick lung epithelium cells. This MOA can block the antibodies\u2019 binding to sick cells, which are vulnerable to pathogenic antibodies. Our in vivo data showed that the formulation significantly reduced the sickness and deaths caused by pathogenic anti-spike antibodies. Therefore, the formulation has the potential to prevent and treat the serious conditions caused by pathogenic antibodies during a COVID-19 infection. In addition, the formulation has potential to prevent and treat the adverse reactions of COVID-19 vaccines because the vaccines can induce similar antibodies, including pathogenic antibodies. The formulation will be helpful in increasing the safety of the vaccines without reducing the vaccine\u2019s efficacy. Compared to existing antiviral drugs, the formulation has a unique MOA of targeting receptors, broad spectrum of indications, excellent safety profile, resistance to mutations, and can be easily produced.","version":"1.2","doi":"10.1101/2021.07.13.452194","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.17.456606","pub_date":"2021-8-17","title":"Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected Patients","abstract":"RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.","version":"1.1","doi":"10.1101/2021.08.17.456606","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.16.456441","pub_date":"2021-8-17","title":"T Cell Predominant Response to AAV-Spike Protects hACE2 Mice from SARS-CoV-2 Pneumonia","abstract":"Prevention of COVID-19 is widely believed to depend on neutralization of SARS-CoV-2 by vaccine-induced humoral immunity, raising concern that emerging escape variants may perpetuate the pandemic. Here we show that a single intramuscular injection of Adeno-Associated Virus-6 (AAV6) or AAV9 encoding a modified, N-terminal domain deleted (\u0394NTD) spike protein induces robust cellular immunity and provides long-term protection in k18-hACE2 transgenic mice from lethal SARS-CoV-2 challenge, associated weight loss and pneumonia independent of vaccine-induced neutralizing humoral immunity. In both mice and macaques, vaccine-induced cellular immunity results in the clearance of transduced muscle fibers coincident with macrophage and CD8+ cytotoxic T cell infiltration at the site of immunization. Additionally, mice demonstrate a strong Type-1 polarized cellular immunophenotype and equivalent ex vivo T cell reactivity to peptides of wt and alpha (B.1.1.7) variant spike. These studies demonstrate not only that AAV6 and AAV9 can function as effective vaccine platforms, but also that vaccines can provide long-term efficacy primarily through the induction of cellular immunity. The findings may provide an alternative approach to containment of the evolving COVID-19 pandemic and have broader implications for the development of variant-agnostic universal vaccines against a wider range of pathogens.","version":"1.1","doi":"10.1101/2021.08.16.456441","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.17.456689","pub_date":"2021-8-17","title":"Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant","abstract":"The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report here structure, function and antigenicity of its full-length spike (S) trimer in comparison with those of other variants, including Gamma, Kappa, and previously characterized Alpha and Beta. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2 and its pseudotyped viruses infect target cells substantially faster than all other variants tested, possibly accounting for its heightened transmissibility. Mutations of each variant rearrange the antigenic surface of the N-terminal domain of the S protein in a unique way, but only cause local changes in the receptor-binding domain, consistent with greater resistance particular to neutralizing antibodies. These results advance our molecular understanding of distinct properties of these viruses and may guide intervention strategies.","version":"1.1","doi":"10.1101/2021.08.17.456689","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.16.456587","pub_date":"2021-8-17","title":"Inhibitors of anti-apoptotic Bcl-2 family proteins exhibit potent and broad-spectrum anti-mammarenavirus activity via cell cycle arrest at G0/G1 phase","abstract":"Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their pro-apoptotic properties, but rather their ability of inducing cell arrest at G0/G1 phase. OLX and ABT-737 mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, siRNA-mediated knock down of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. Anti-apoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors mediated cell cycle arrest at the G0/G1 phase, rather than their pro-apoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate anti-mammarenavirus activity in vivo, and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals.","version":"1.1","doi":"10.1101/2021.08.16.456587","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.16.456510","pub_date":"2021-8-16","title":"Age-related susceptibility of ferrets to SARS-CoV-2 infection","abstract":"Susceptibility to SARS-CoV-2 and the outcome of COVID-19 have been linked to underlying health conditions and the age of affected individuals. Here we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18-to-39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is \u223c32 plaque forming units (PFU) per animal while in young animals it was estimated to be \u223c100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets when compared to young animals. Similar to observations in humans, this was associated with higher expressions levels of two key viral entry factors - ACE2 and TMPRSS2 - in the upper respiratory tract of aged ferrets.","version":"1.1","doi":"10.1101/2021.08.16.456510","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.15.456333","pub_date":"2021-8-16","title":"Molecular basis of a dominant SARS-CoV-2 Spike-derived epitope presented by HLA-A*02:01 recognised by a public TCR","abstract":"The data currently available on how the immune system recognizes the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus, we are lacking data on how T cells are able to recognize this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.","version":"1.1","doi":"10.1101/2021.08.15.456333","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.16.456470","pub_date":"2021-8-16","title":"Peptide Scanning of SARS-CoV and SARS-CoV-2 Spike Protein Subunit 1 Reveals Potential Additional Receptor Binding Sites","abstract":"The binding of SARS-CoV and SARS-CoV-2 to the ACE2 receptor on human cells is mediated by the spike protein subunit 1 (S1) on the virus surfaces, while the receptor binding domains (RBDs) of S1 are the major determinants for the interaction with ACE2 and dominant targets of neutralizing antibodies. However, at the virus-host interface, additional biomolecular interactions, although being relatively weak in affinity and low in specificity, could also contribute to viral attachment and play important roles in gain- or loss-of-function mutations. In this work, we performed a peptide scanning of the S1 domains of SARS-CoV and SARS-CoV-2 by synthesizing 972 16-mer native and mutated peptide fragments using a high throughput in situ array synthesis technology. By probing the array using fluorescently labelled ACE2, a number of previously unknown potential receptor binding sites of S1 have been revealed. 20 peptides were synthesized using solid phase peptide synthesis, in order to validate and quantify their binding to ACE2. Four ACE2-binding peptides were selected, to investigate whether they can be assembled through a biotinylated peptide/neutravidin system to achieve high affinity to ACE2. A number of constructs exhibited high affinity to ACE2 with Kd values of pM to low nM.","version":"1.1","doi":"10.1101/2021.08.16.456470","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456164","pub_date":"2021-8-16","title":"Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model","abstract":"COVID-19 caused by SARS-CoV-2 has been spreading worldwide. To date, several vaccine candidates moved into EUA or CA applications. Although DNA vaccine is on phase III clinical trial, it is a promised technology platform with many advantages. Here, we showed that the pGX9501 DNA vaccine encoded the spike full-length protein-induced strong humoral and cellular immune responses in mice with higher neutralizing antibodies, blocking the hACE2-RBD binding against live virus infection in vitro. Importantly, higher levels of IFN-\u03b3 expression in CD8+ and CD4+ T cell and specific cytotoxic lymphocyte (CTL) killings effect were also observed in the pGX9501-immunized group. It provided subsequent protection against virus challenges in the hACE2 transgenic mouse model. Overall, pGX9501 was a promising DNA vaccine candidate against COVID-19, inducing strong humoral immunity and cellular immunity that contributed to the vaccine\u2019s protective effects.","version":"1.1","doi":"10.1101/2021.08.13.456164","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.14.456330","pub_date":"2021-8-16","title":"Next generation infection prevention clothing: Non-woven Fabrics Coated with Cranberry Extracts Capable of Inactivating Enveloped Viruses such as SARS-CoV-2 and Multidrug-resistant Bacteria","abstract":"The Coronavirus Disease (COVID-19) pandemic is demanding rapid action of the authorities and scientific community in order to find new antimicrobial solutions that could inactivate the pathogen SARS-CoV-2 that causes this disease. Gram-positive bacteria contribute to severe pneumonia associated with COVID-19, and their resistance to antibiotics is increasing at an alarming rate. In this regard, non-woven fabrics are currently used for the fabrication of infection prevention clothing such as face masks, caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons and shoe covers as protective tools against viral and bacterial infections. However, these non-woven fabrics are made of materials that do not possess antimicrobial activity. Thus, we have developed here non-woven fabrics with antimicrobial coatings of cranberry extracts capable of inactivating enveloped viruses such as SARS-CoV-2 and the phage phi 6, and two multidrug-resistant bacteria: the methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. The non-toxicity of these advanced technology was ensured using a Caenorhabditis elegans in vivo model. These results open up a new prevention path using natural and biodegradable compounds for the fabrication of infection prevention clothing in the current COVID-19 and future pandemics.","version":"1.1","doi":"10.1101/2021.08.14.456330","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456316","pub_date":"2021-8-16","title":"Optimization of Non-Coding Regions Improves Protective Efficacy of an mRNA SARS-CoV-2 Vaccine in Nonhuman Primates","abstract":"The CVnCoV (CureVac) mRNA vaccine for SARS-CoV-2 has recently been evaluated in a phase 2b/3 efficacy trial in humans. CV2CoV is a second-generation mRNA vaccine with optimized non-coding regions and enhanced antigen expression. Here we report a head-to-head study of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in nonhuman primates. We immunized 18 cynomolgus macaques with two doses of 12 ug of lipid nanoparticle formulated CVnCoV, CV2CoV, or sham (N=6/group). CV2CoV induced substantially higher binding and neutralizing antibodies, memory B cell responses, and T cell responses as compared with CVnCoV. CV2CoV also induced more potent neutralizing antibody responses against SARS-CoV-2 variants, including B.1.351 (beta), B.1.617.2 (delta), and C.37 (lambda). While CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded robust protection with markedly lower viral loads in the upper and lower respiratory tract. Antibody responses correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of an mRNA SARS-CoV-2 vaccine in nonhuman primates.","version":"1.1","doi":"10.1101/2021.08.13.456316","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456326","pub_date":"2021-8-16","title":"Antiviral face mask functionalized with solidified hand soap: low-cost infection prevention clothing against enveloped viruses such as SARS-CoV-2","abstract":"Infection prevention clothing is becoming an essential protective tool in the current pandemic, especially because now we know that SARS-CoV-2 can easily infect humans in poorly ventilated indoor spaces. However, commercial infection prevention clothing is made of fabrics that are not capable of inactivating the virus. Therefore, viral infections of symptomatic and asymptomatic individuals wearing protective clothing such as masks can occur through aerosol transmission or by contact with the contaminated surfaces of the masks, which are suspected as an increasing source of highly infectious biological waste. Herein, we report an easy fabrication method of a novel antiviral non-woven fabric containing polymer filaments that were coated with solidified hand soap. This extra protective fabric is capable of inactivating enveloped viruses such as SARS-CoV-2 and phi 6 in one minute of contact. In this study, this antiviral fabric was used to fabricate an antiviral face mask and did not show any cytotoxic effect in human keratinocyte HaCaT cells. Furthermore, this antiviral non-woven fabric could be used for the fabrication of other infection prevention clothing such as caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons, and shoe covers. Therefore, this low-cost technology could provide a wide range of infection protective tools to combat COVID-19 and future pandemics in developed and underdeveloped countries.","version":"1.1","doi":"10.1101/2021.08.13.456326","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.454991","pub_date":"2021-8-16","title":"A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2","abstract":"A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.","version":"1.1","doi":"10.1101/2021.08.13.454991","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.15.456423","pub_date":"2021-8-16","title":"SMOG 2 and OpenSMOG: Extending the limits of structure-based models","abstract":"Applying simulations with structure-based (G\u014d-like) models has proven to be an effective strategy for investigating the factors that control biomolecular dynamics. The common element of these models is that some (or all) of the intra/inter-molecular inter-actions are explicitly defined to stabilize an experimentally-determined structure. To facilitate the development and application of this broad class of models, we previously released the SMOG 2 software package. This suite allows one to easily customize and distribute structure-based (i.e. SMOG) models for any type of polymer-ligand system. Since its original release, user feedback has driven the implementation of numerous enhancements. Here, we describe recent extensions to the software and demonstrate the capabilities of the most recent version, SMOG v2.4. Changes include new tools that aid user-defined customization of force fields, as well as an interface with the OpenMM simulation libraries (OpenSMOG v1.0). To illustrate the utility of these advances, we present several applications of SMOG2 and OpenSMOG, which include systems with millions of atoms, long polymers and explicit ions. We also highlight how one can incorporate non-structure-based (e.g. AMBER-based) energetics to define a hybrid class of models. The representative applications include large-scale rearrangements of the SARS-CoV-2 Spike protein, the HIV-1 capsid in the presence of explicit ions, and crystallographic lattices of ribosomes and proteins. In summary, SMOG 2 and OpenSMOG provide robust support for researchers who seek to apply structure-based models to large and/or intricate biomolecular systems.","version":"1.1","doi":"10.1101/2021.08.15.456423","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.14.456353","pub_date":"2021-8-16","title":"Cutting epitopes to survive: the case of lambda variant","abstract":"This manuscript concisely reports an in-silico study on the potential impact of the Spike protein mutations on immuno-escape ability of SARS-CoV-2 lambda variant. Biophysical and bioinformatics data suggest that a combination of shortening immunogenic epitope loops and generation of potential N-glycosylation sites may be a viable adaptation strategy potentially allowing this emerging viral variant escaping host immunity.","version":"1.1","doi":"10.1101/2021.08.14.456353","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.427056","pub_date":"2021-8-16","title":"Model design for non-parametric phylodynamic inference and applications to pathogen surveillance","abstract":"Inference of effective population size from genomic data can provide unique information about demographic history, and when applied to pathogen genetic data can also provide insights into epidemiological dynamics. The combination of non-parametric models for population dynamics with molecular clock models which relate genetic data to time has enabled phylodynamic inference based on large sets of time-stamped genetic sequence data. The methodology for non-parametric inference of effective population size is well-developed in the Bayesian setting, but here we develop a frequentist approach based on non-parametric latent process models of population size dynamics. We appeal to statistical principles based on out-of-sample prediction accuracy in order to optimize parameters that control shape and smoothness of the population size over time. We demonstrate the flexibility and speed of this approach in a series of simulation experiments, and apply the methodology to reconstruct the previously described waves in the seventh pandemic of cholera. We also estimate the impact of non-pharmaceutical interventions for COVID-19 in England using thousands of SARS-CoV-2 sequences. By incorporating a measure of the strength of these interventions over time within the phylodynamic model, we estimate the impact of the first national lockdown in the UK on the epidemic reproduction number.","version":"1.2","doi":"10.1101/2021.01.18.427056","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.31.429052","pub_date":"2021-8-15","title":"DRDOCK: A Drug Repurposing platform integrating automated docking, simulations and a log-odds-based drug ranking scheme","abstract":"Drug repurposing, where drugs originally approved to treat a disease are reused to treat other diseases, has received escalating attention especially in pandemic years. Structure-based drug design, integrating small molecular docking, molecular dynamic (MD) simulations and AI, has demonstrated its evidenced importance in streamlining new drug development as well as drug repurposing. To perform a sophisticated and fully automated drug screening using all the FDA drugs, intricate programming, accurate drug ranking methods and friendly user interface are very much needed. Here we introduce a new web server, DRDOCK, Drug Repurposing DOcking with Conformation-sampling and pose re-ranKing - refined by MD and statistical models, which integrates small molecular docking and molecular dynamic (MD) simulations for automatic drug screening of 2016 FDA-approved drugs over a user-submitted single-chained target protein. The drugs are ranked by a novel drug-ranking scheme using log-odds (LOD) scores, derived from feature distributions of true binders and decoys. Users can submit a selection of LOD-ranked poses for further MD-based binding affinity evaluation. We demonstrated that our platform can indeed recover one of the substrates for nsp16, a cap ribose 2\u2032-O methyltransferase, and recommends that fluralaner, tegaserod and fenoterol could be repurposed for the COVID19 treatment with the latter two being confirmed in SARS-CoV2 suppression experiments. All the sampled docking poses and trajectories can be 3D-viewed and played via our web interface. This platform shall be easy-to-use for general scientists and medicinal researchers to carry out drug repurposing within a couple of days which should add value to our timely responses to, particularly, emergent disease outbreaks. DRDOCK can be freely accessed from https://dyn.life.nthu.edu.tw/drdock/. (Due to the hardware upgrade, the service is NOT available before 7/18, 2021)","version":"1.4","doi":"10.1101/2021.01.31.429052","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456228","pub_date":"2021-8-13","title":"A diabetic milieu increases cellular susceptibility to SARS-CoV-2 infections in engineered human kidney organoids and diabetic patients","abstract":"SARS-CoV-2 infections lead to a high risk of hospitalization and mortality in diabetic patients. Why diabetic individuals are more prone to develop severe COVID-19 remains unclear. Here, we established a novel human kidney organoid model that mimics early hallmarks of diabetic nephropathy. High oscillatory glucose exposure resulted in metabolic changes, expansion of extracellular membrane components, gene expression changes determined by scRNAseq, and marked upregulation of angiotensin-converting enzyme 2 (ACE2). Upon SARS-CoV-2 infection, hyperglycemic conditions lead to markedly higher viral loads in kidney organoids compared to normoglycemia. Genetic deletion of ACE2, but not of the candidate receptor BSG/CD147, in kidney organoids demonstrated the essential role of ACE2 in SARS-CoV-2 infections and completely prevented SARS-CoV-2 infection in the diabetogenic microenvironment. These data introduce a novel organoid model for diabetic kidney disease and show that diabetic-induced ACE2 licenses the diabetic kidney to enhanced SARS-CoV-2 replication.","version":"1.1","doi":"10.1101/2021.08.13.456228","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456182","pub_date":"2021-8-13","title":"Conserved T-cell epitopes predicted by bioinformatics in SARS-COV-2 variants","abstract":"Finding conservative T cell epitopes in the proteome of numerous variants of SARS-COV-2 is required to develop T cell activating SARS-COV-2 capable of inducing T cell responses against SARS-COV-2 variants. A computational workflow was performed to find HLA restricted CD8+ and CD4+ T cell epitopes among conserved amino acid sequences across the proteome of 474727 SARS-CoV-2 strains. A batch of covserved regions in the amino acid sequences were found in the proteome of the SARS-COV-2 strains. 2852 and 847 peptides were predicted to have high binding affinity to distint HLA class I and class II molecules. Among them, 1456 and 484 peptides are antigenic. 392 and 111 of the antigenic peptides were found in the conseved amino acid sequences. Among the antigenic-conserved peptides, 6 CD8+ T cell epitopes and 7 CD4+ T cell epitopes were identifed. The T cell epitopes could be presented to T cells by high-affinity HLA molecules which are encoded by the HLA alleles with high population coverage. The T cell epitopes are conservative, antigenic and HLA presentable, and could be constructed into SARS-COV-2 vaccines for inducing protective T cell immunity against SARS-COV-2 and their variants.","version":"1.1","doi":"10.1101/2021.08.12.456182","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455384","pub_date":"2021-8-13","title":"The Inherent Flexibility of Receptor Binding Domains in SARS-CoV-2 Spike Protein","abstract":"Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large conformational changes of its receptor binding domain (RBD) to enter the host cell, as the abundant structural studies suggest. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations without pre-defined reaction coordinates. The RBDA transition from Down to one-Up is supported by transient salt-bridges between RBDA and RBDC and by the glycan at N343B. Reduced interactions between RBDA and RBDB induce the RBDB motions toward two-Up. Glycan shielding for neutralizing antibodies is the weakest in one-Open. Cryptic pockets are revealed at the RBD interfaces in intermediate structures between Down and one-Up. The inherent flexibility in S-protein is, thus, essential for the structure transition and shall be considered for antiviral drug rational design or vaccine development.","version":"1.2","doi":"10.1101/2021.08.06.455384","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456258","pub_date":"2021-8-13","title":"Covid-19 vaccine immunogenicity in people living with HIV-1","abstract":"COVID-19 vaccine efficacy has been evaluated in large clinical trials and in real-world situation. Although they have proven to be very effective in the general population, little is known about their efficacy in immunocompromised patients. HIV-infected individuals\u2019 response to vaccine may vary according to the type of vaccine and their level of immunosuppression. We evaluated immunogenicity of an mRNA anti-SARS CoV-2 vaccine in HIV-positive individuals. HIV-positive individuals (n=121) were recruited from HIV clinics in Montreal and stratified according to their CD4 counts. A control group of 20 health care workers na\u00efve to SARS CoV-2 was used. The participants\u2019 Anti-RBD IgG responses were measured by ELISA at baseline and 3 to 4 weeks after receiving the first dose of an mRNA vaccine). Eleven of 121 participants had anti-COVID-19 antibodies at baseline, and a further 4 had incomplete data for the analysis. Mean anti-RBD IgG responses were similar between between the HIV negative control group (n=20) and the combined HIV+ group (n=106) (p = 0.72). However, these responses were significantly lower in the group with <250 CD4 cells/mm3. (p<0.0001). Increasing age was independently associated with decreased immunogenicity. HIV-positive individuals with CD4 counts over 250 cells/mm3 have an anti-RBD IgG response similar to the general population. However, HIV-positive individuals with the lowest CD4 counts (<250 cells/mm3) have a weaker response. These data would support the hypothesis that a booster dose might be needed in this subgroup of HIV-positive individuals, depending on their response to the second dose.","version":"1.1","doi":"10.1101/2021.08.13.456258","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456168","pub_date":"2021-8-13","title":"SARS-CoV-2 spike opening dynamics and energetics reveal the individual roles of glycans and their collective impact","abstract":"The trimeric spike (S) glycoprotein, which protrudes from the SARS-CoV-2 viral envelope, is responsible for binding to human ACE2 receptors. The binding process is initiated when the receptor binding domain (RBD) of at least one protomer switches from a \u201cdown\u201d (closed) to an \u201cup\u201d (open) state. Here, we used molecular dynamics simulations and two-dimensional replica exchange umbrella sampling calculations to investigate the transition between the two S-protein conformations with and without glycosylation. We show that the glycosylated spike has a higher barrier to opening than the non-glycosylated one with comparable populations of the down and up states. In contrast, we observed that the up conformation is favored without glycans. Analysis of the S-protein opening pathway reveals that glycans at N165 and N122 interfere with hydrogen bonds between the RBD and the N-terminal domain in the up state. We also identify roles for glycans at N165 and N343 in stabilizing the down and up states. Finally we estimate how epitope exposure for several known antibodies changes along the opening path. We find that the epitope of the BD-368-2 antibody remains exposed irrespective of the S-protein conformation, explaining the high efficacy of this antibody.","version":"1.1","doi":"10.1101/2021.08.12.456168","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.13.456190","pub_date":"2021-8-13","title":"Screening of cell-virus, cell-cell, gene-gene cross-talks among kingdoms of life at single cell resolution","abstract":"The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) issued a significant and urgent threat to global health. The exact animal origin of SARS-CoV-2 remains obscure and understanding its host range is vital for preventing interspecies transmission. Previously, we have assessed the target cell profiles of SARS-CoV-2 in pets, livestock, poultry and wild animals. Herein, we expand this investigation to a wider range of animal species and viruses to provide a comprehensive source for large-scale screening of potential virus hosts. Single cell atlas for several mammalian species (alpaca, hamster, hedgehog, chinchilla etc.), as well as comparative atlas for lung, brain and peripheral blood mononuclear cells (PBMC) for various lineages of animals were constructed, from which we systemically analyzed the virus entry factors for 113 viruses over 20 species from mammalians, birds, reptiles, amphibians and invertebrates. Conserved cellular connectomes and regulomes were also identified, revealing the fundamental cell-cell and gene-gene cross-talks between these species. Overall, our study could help identify the potential host range and tissue tropism of SARS-CoV-2 and a diverse set of viruses and reveal the host-virus co-evolution footprints.","version":"1.1","doi":"10.1101/2021.08.13.456190","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450211","pub_date":"2021-8-12","title":"A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations","abstract":"Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, that addition of other nsps modulates expression levels, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis. The impact of the COVID-19 pandemic has made it essential to better understand the basic biology of SARS-CoV-2, and to search for compounds that can block the activity of key SARS-CoV-2 replication enzymes. However, studies with live SARS-CoV-2 require biosafety level 3 facilities, while existing replicon systems depend on long positive-sense subgenomes that are often difficult to manipulate or produce a high background signal, limiting drug-screens and a rapid analysis of emerging SARS-CoV-2 mutations during the COVID-19 pandemic. To make it easier to study emerging SARS-CoV-2 mutants and screen for inhibitors, we developed a simple mini-replicon that produces a minimal background signal, that can be used in any tissue culture lab, and that only requires four small plasmids to work.","version":"1.2","doi":"10.1101/2021.06.28.450211","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446623","pub_date":"2021-8-12","title":"Synergistic block of SARS-CoV-2 infection by combined drug inhibition of the host entry factors PIKfyve kinase and TMPRSS2 protease","abstract":"Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ~5-10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. The substantial ~5-fold or more decrease of half maximal effective concentrations (EC50 values) suggests a plausible treatment strategy based on the combined use of these inhibitors. Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include Apilimod, an inhibitor of PIKfyve kinase and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or with nafamostat mesylate.","version":"1.3","doi":"10.1101/2021.06.01.446623","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456131","pub_date":"2021-8-12","title":"Phylogenetic reconciliation reveals extensive ancestral recombination in Sarbecoviruses and the SARS-CoV-2 lineage","abstract":"An accurate understanding of the evolutionary history of rapidly-evolving viruses like SARS-CoV-2, responsible for the COVID-19 pandemic, is crucial to tracking and preventing the spread of emerging pathogens. However, viruses undergo frequent recombination, which makes it difficult to trace their evolutionary history using traditional phylogenetic methods. Here, we present a phylogenetic workflow, virDTL, for analyzing viral evolution in the presence of recombination. Our approach leverages reconciliation methods developed for inferring horizontal gene transfer in prokaryotes, and, compared to existing tools, is uniquely able to identify ancestral recombinations while accounting for several sources of inference uncertainty, including in the construction of a strain tree, estimation and rooting of gene family trees, and reconciliation itself. We apply this workflow to the Sarbecovirus subgenus and demonstrate how a principled analysis of predicted recombination gives insight into the evolution of SARS-CoV-2. In addition to providing confirming evidence for the horseshoe bat as its zoonotic origin, we identify several ancestral recombination events that merit further study.","version":"1.1","doi":"10.1101/2021.08.12.456131","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.24.449252","pub_date":"2021-8-12","title":"Lipid and nucleocapsid N-protein accumulation in COVID-19 patient lung and infected cells","abstract":"The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the corona virus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS- CoV-2 infected Vero cells, processed identically. Correlative light and electron microscopy on semi- thick cryo-sections, demonstrated induction of electron-lucent, lipid filled compartments after SARS- CoV-2 infection in both lung and cell cultures. In lung tissue, the non-structural protein 4 and the stable nucleocapsid N-protein, were detected on these novel lipid filled compartments. The induction of such lipid filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19, may explain the extensive inflammatory response and provide a new hallmark for SARS- Cov-2 infection at the final, fatal stage of infection.","version":"1.2","doi":"10.1101/2021.06.24.449252","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.455901","pub_date":"2021-8-12","title":"Robust and Functional Immunity up to 9 months after SARS-CoV-2 infection: a Southeast Asian longitudinal cohort","abstract":"Assessing the duration of humoral and cellular immunity remains key to overcome the current SARS-CoV-2 pandemic, especially in understudied populations in least developed countries. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for humoral immune response to the viral spike protein and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-spike (S) antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immunity was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased ADCC and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immunity. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection in the absence of re-infection. Functional immune memory to SARS-CoV-2, consisting of polyfunctional antibodies, memory B cells and memory T cells are maintained up to nine months in a South-East Asian cohort in the absence of re-infection.","version":"1.1","doi":"10.1101/2021.08.12.455901","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456077","pub_date":"2021-8-12","title":"Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern","abstract":"To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five na\u00efve and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from na\u00efve people. Of these, 944 from na\u00efve and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from na\u00efve people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of na\u00efve people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.","version":"1.1","doi":"10.1101/2021.08.12.456077","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.456015","pub_date":"2021-8-12","title":"Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates","abstract":"Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 \u00b5g of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.\u03b2 (heterologous), which encompasses the spike sequence of the B.1.351 (beta or \u03b2) variant. Reciprocal ID50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the \u03b2 variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost \u03b2-specific reciprocal ID50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the \u03b2 variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations. mRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.","version":"1.1","doi":"10.1101/2021.08.11.456015","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456154","pub_date":"2021-8-12","title":"Mechanisms of Coupling between Angiotensin Converting Enzyme 2 and Nicotinic Acetylcholine Receptors","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus encapsulated by a spike (S) glycoprotein envelope, binds with high affinity to angiotensin converting enzyme 2 (ACE2) during cell entry of a susceptible host. Recent studies suggest nicotinic acetylcholine receptors (nAChRs) play a role in functional ACE2 regulation and nicotine may contribute to the progression of coronavirus disease 2019 (COVID-19). Here, we present evidence for coupling between ACE2 and nAChR through bioinformatic analysis and cell culture experiments. Following molecular and structural protein comparison of over 250 ACE2 vertebrate orthologues, a region of human ACE2 at positions C542-L554 was identified to have sequence similarity to nAChR-binding neurotoxin and rabies virus glycoproteins (RBVG). Furthermore, experiments conducted in PC12 cells indicate a potential for physical interaction between ACE2 and \u03b17 nAChR proteins. Our findings support a model of nAChR involvement in in COVID-19.","version":"1.1","doi":"10.1101/2021.08.12.456154","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455956","pub_date":"2021-8-12","title":"Molecular basis of immune evasion by the delta and kappa SARS-CoV-2 variants","abstract":"Worldwide SARS-CoV-2 transmission leads to the recurrent emergence of variants, such as the recently described B.1.617.1 (kappa), B.1.617.2 (delta) and B.1.617.2+ (delta+). The B.1.617.2 (delta) variant of concern is causing a new wave of infections in many countries, mostly affecting unvaccinated individuals, and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing the impact of individual mutations on immune evasion. Mutations in the B.1.617.1 (kappa) and B.1.617.2 (delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including an unexpected remodeling of the B.1.617.2 (delta) N-terminal domain. The binding affinity of the B.1.617.1 (kappa) and B.1.617.2 (delta) receptor-binding domain for ACE2 is comparable to the ancestral virus whereas B.1.617.2+ (delta+) exhibits markedly reduced affinity. We describe a previously uncharacterized class of N-terminal domain-directed human neutralizing monoclonal antibodies cross-reacting with several variants of concern, revealing a possible target for vaccine development.","version":"1.2","doi":"10.1101/2021.08.11.455956","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.12.456045","pub_date":"2021-8-12","title":"Autophagy cargo receptors are secreted via extracellular vesicles and particles in response to endolysosomal inhibition or impaired autophagosome maturation","abstract":"The endosome-lysosome (endolysosome) system plays central roles in both autophagic degradation and secretory pathways, including the exocytic release of extracellular vesicles and particles (EVPs). Although previous work has revealed important interconnections between autophagy and EVP-mediated secretion, our molecular understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition that mediates the EVP-associated extracellular release of autophagic cargo receptors, including p62/SQSTM1. This extracellular secretion is highly regulated and critically dependent on multiple ATGs required for the progressive steps of early autophagosome formation as well as Rab27a-dependent exocytosis. Furthermore, the disruption of autophagosome maturation, either due to genetic inhibition of the autophagosome-to-autolyosome fusion machinery or blockade via the SARS-CoV2 viral protein ORF3a, is sufficient to induce robust EVP-associated secretion of autophagy cargo receptors. Finally, we demonstrate that this ATG-dependent, EVP-mediated secretion pathway buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Based on these results, we propose that secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis in response to endolysosomal dysfunction or impaired autophagosome maturation.","version":"1.1","doi":"10.1101/2021.08.12.456045","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455903","pub_date":"2021-8-11","title":"Structural Differences In 3C-like protease (Mpro) From SARS-CoV and SARS-CoV-2: Molecular Insights For Drug Repurposing Against COVID-19 Revealed by Molecular Dynamics Simulations","abstract":"A recent fatal outbreak of novel coronavirus SARS-CoV-2, identified preliminary as a causative agent for series of unusual pneumonia cases in Wuhan city, China has infected more than 20 million individuals with more than 4 million mortalities. Since, the infection crossed geographical barriers, the WHO permanently named the causing disease as COVID-2019 by declaring it a pandemic situation. SARS-CoV-2 is an enveloped single-stranded RNA virus causing a wide range of pathological conditions from common cold symptoms to pneumonia and fatal severe respiratory syndrome. Genome sequencing of SARS-CoV-2 has revealed 96% identity to the bat coronavirus and 79.6% sequence identity to the previous SARS-CoV. The main protease (known as 3C-like proteinase/ Mpro) plays a vital role during the infection with the processing of replicase polyprotein thus offering an attractive target for therapeutic interventions. SARS-CoV and SARS-CoV-2 Mpro shares 97% sequence identity, with 12 variable residues but none of them present in the catalytic and substrate binding site. With the high level of sequence and structural similarity and absence of any drug/vaccine against SARS-CoV-2, drug repurposing against Mpro is an effective strategy to combat COVID-19. Here, we report a detailed comparison of SARS-CoV-2 Mpro with SARS-CoV Mpro using molecular dynamics simulations to assess the impact of 12 divergent residues on the molecular microenvironment of Mpro. A structural comparison and analysis is made on how these variable residues affects the intra-molecular interactions between key residues in the monomer and biologically active dimer form of Mpro. The present MD simulations study concluded the change in microenvironment of active-site residues at the entrance (T25, T26, M49 and Q189), near the catalytic region (F140, H163, H164, M165 and H172) and other residues in substrate binding site (V35T, N65S, K88R and N180K) due to 12 mutation incorporated in the SARS-CoV-2 Mpro. It is also evident that SARS-CoV-2 dimer is more stable and less flexible state compared to monomer which may be due to these variable residues, mainly F140, E166 and H172 which are involved in dimerization. This also warrants a need for inhibitor design considering the more stable dimer form. The mutation accumulated in SARS-CoV-2 Mpro indirectly reconfigures the key molecular networks around the active site conferring a potential change in SARS-CoV-2, thus posing a challenge in drug repurposing SARS drugs for COVID-19. The new networks and changes in microenvironment identified by our work might guide attempts needed for repurposing and identification of new Mpro inhibitors.","version":"1.1","doi":"10.1101/2021.08.11.455903","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455959","pub_date":"2021-8-11","title":"Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization","abstract":"The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.","version":"1.1","doi":"10.1101/2021.08.11.455959","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455984","pub_date":"2021-8-11","title":"Durability of SARS-CoV-2-specific T cell responses at 12-months post-infection","abstract":"Characterizing the longevity and quality of cellular immune responses to SARS-CoV-2 is critical to understanding immunologic approaches to protection against COVID-19. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions. We identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared. SARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. Our data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.","version":"1.1","doi":"10.1101/2021.08.11.455984","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455921","pub_date":"2021-8-11","title":"SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M1 macrophage polarisation","abstract":"TLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages. Adult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFN\u03b3, S1/IFN\u03b3 or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2. TLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid\u00ae), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore\u00ae, suggesting ACE2 expression is mediated by the alternative endosomal/\u03b2-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-\u03b3 treatment of THP-1-derived macrophages induced pro-inflammatory M1 polarisation as shown by an increase in IL-1\u03b2 and IL-6 mRNA. These results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid\u00ae) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.","version":"1.1","doi":"10.1101/2021.08.11.455921","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455899","pub_date":"2021-8-11","title":"Cross-sectional genomic perspective of epidemic waves of SARS-CoV-2: a pan India study","abstract":"COVID-19 has posed unforeseen circumstances and throttled major economies worldwide. India has witnessed two waves affecting around 31 million people representing 16% of the cases globally. To date, the epidemic waves have not been comprehensively investigated to understand pandemic progress in India. In the present study, we aim for a cross-sectional analysis since its first incidence up to 26th July 2021. We have performed the pan Indian evolutionary study using 20,086 high-quality complete genomes of SARS-CoV-2. Based on the number of cases reported and mutation rates, we could divide the Indian epidemic into seven different phases. First, three phases constituting the pre-first wave had a very less average mutation rate (<11), which increased in the first wave to 17 and then doubled in the second wave (~34). In accordance with the mutation rate, variants of concern (alpha, beta, gamma and delta) and interest (eta and kappa) also started appearing in the first wave (1.5% of the genomes), which dominated the second (~96% of genomes) and post-second wave (100% of genomes) phases. Whole genome-based phylogeny could demarcate the post-first wave isolates from previous ones by the point of diversification leading to incidences of VOCs and VOIs in India. Nation-wide mutational analysis depicted more than 0.5 million events with four major mutations in ~97% of the total 20,086 genomes in the study. These included two mutations in coding (spike (D614G) and NSP 12b (P314L) of RNA dependent RNA polymerase), one silent mutation (NSP3 F106F) and one extragenic mutation (5\u2019 UTR 241). Large scale genome-wide mutational analysis is crucial in expanding knowledge on evolution of deadly variants of SARS-CoV-2 and timely management of the pandemic.","version":"1.1","doi":"10.1101/2021.08.11.455899","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.11.455942","pub_date":"2021-8-11","title":"A single shot of a hybrid hAdV5-based anti-COVID-19 vaccine induces a long-lasting immune response and broad coverage against VOC","abstract":"Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric hAdV5 vector. The vaccine (named CoroVaxG.3) is based on three pillars: i) high expression of Spike to enhance its immunodominance by using a potent promoter and a mRNA stabilizer; ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; iii) use of Spike stabilized in a prefusion conformation. Transduction with CoroVaxG.3 expressing Spike (D614G) dramatically enhanced Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3 vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha) and P.1 (gamma) Spikes, as well as an authentic WT and P.1 SARS-CoV-2 isolates. Neutralizing antibodies did not wane even after 5 months making this kind of vaccine a likely candidate to enter clinical trials","version":"1.1","doi":"10.1101/2021.08.11.455942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.10.455874","pub_date":"2021-8-11","title":"CytokineLink: a cytokine communication map to analyse immune responses in inflammatory and infectious diseases","abstract":"Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of infectious and inflammatory diseases. By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available bioinformatic resources that can model cytokine - cytokine interactions. In this effort, we built a communication map between major tissues and blood cells that reveals how cytokine-mediated intercellular networks form during homeostatic conditions. We collated the most prevalent cytokines from literature, and assigned the proteins and their corresponding receptors to source tissue and blood cell types based on enriched consensus RNA-Seq data from the Human Protein Atlas database. To assign more confidence to the interactions, we integrated literature information on cell - cytokine interactions from two systems immunology databases, immuneXpresso and ImmunoGlobe. From the collated information, we defined two metanetworks: a cell-cell communication network connected by cytokines; and a cytokine-cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Using expression data from disease states, we then applied this resource to reveal perturbations in cytokine-mediated intercellular signalling in inflammatory and infectious diseases (ulcerative colitis and COVID-19, respectively). For ulcerative colitis, with CytokineLink we demonstrated a significant rewiring of cytokine-mediated intercellular communication between non-inflamed and inflamed colonic tissues. For COVID-19, we were able to identify inactive cell types and cytokine interactions that may be important following SARS-CoV-2 infection when comparing the cytokine response with other viruses capable of initiating a cytokine storm. Such findings have potential to inform the development of novel, cytokine-targeted therapeutic strategies. CytokineLink is freely available for the scientific community through the NDEx platform and the project github repository (https://github.com/korcsmarosgroup/CytokineLink).","version":"1.1","doi":"10.1101/2021.08.10.455874","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453077","pub_date":"2021-8-11","title":"Discovery of genes that modulate flavivirus replication in an interferon-dependent manner","abstract":"Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent way, opening new perspectives to target weakness points in the life cycle of these viruses.","version":"1.2","doi":"10.1101/2021.07.20.453077","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.10.455702","pub_date":"2021-8-10","title":"Infectious SARS-CoV-2 is emitted in aerosols","abstract":"Respiratory viruses such as SARS-CoV-2 are transmitted in respiratory droplets and aerosols, which are released during talking, breathing, coughing, and sneezing. Non-contact transmission of SARS-CoV-2 has been demonstrated, suggesting transmission in aerosols. Here we demonstrate that golden Syrian hamsters emit infectious SARS-CoV-2 in aerosols, prior to and concurrent with the onset of mild clinical signs of disease. The average emission rate is 25 infectious virions/hour on days 1 and 2 post-inoculation, with average viral RNA levels 200-fold higher than infectious virus in aerosols. Female hamsters have delayed kinetics of viral shedding in aerosols compared to male hamsters, with peak viral emission for females on dpi 2 and for males on dpi 1. The majority of virus is contained within aerosols <8 \u00b5m in size. Thus, we provide direct evidence that, in hamsters, SARS-CoV-2 is an airborne virus.","version":"1.1","doi":"10.1101/2021.08.10.455702","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.10.455799","pub_date":"2021-8-10","title":"Pango lineage designation and assignment using SARS-CoV-2 spike gene nucleotide sequences","abstract":"More than 2 million SARS-CoV-2 genome sequences have been generated and shared since the start of the COVID-19 pandemic and constitute a vital information source that informs outbreak control, disease surveillance, and public health policy. The Pango dynamic nomenclature is a popular system for classifying and naming genetically-distinct lineages of SARS-CoV-2, including variants of concern, and is based on the analysis of complete or near-complete virus genomes. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. It is therefore important to understand how much information about Pango lineage status is contained in spike-only nucleotide sequences. Here we explore how Pango lineages might be reliably designated and assigned to spike-only nucleotide sequences. We survey the genetic diversity of such sequences, and investigate the information they contain about Pango lineage status. Although many lineages, including the main variants of concern, can be identified clearly using spike-only sequences, some spike-only sequences are shared among tens or hundreds of Pango lineages. To facilitate the classification of SARS-CoV-2 lineages using subgenomic sequences we introduce the notion of designating such sequences to a \u201clineage set\u201d, which represents the range of Pango lineages that are consistent with the observed mutations in a given spike sequence. These data provide a foundation for the development of software tools that can assign newly-generated spike nucleotide sequences to Pango lineage sets.","version":"1.1","doi":"10.1101/2021.08.10.455799","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.10.455627","pub_date":"2021-8-10","title":"An ultrapotent neutralizing bispecific antibody with broad spectrum against SARS-CoV-2 variants","abstract":"In spite of the successful development of effective countermeasures against Covid-19, variants have and will continue to emerge that could compromise the efficacy of currently approved neutralizing antibodies and vaccines. Consequently, novel and more efficacious agents are urgently needed. We have developed a bispecific antibody, 2022, consisting of two antibodies, 2F8 and VHH18. 2F8 was isolated from our proprietary fully synthetic human IDEAL (Intelligently Designed and Engineered Antibody Library)-VH/VL library and VHH18 is a single domain antibody isolated from IDEAL-nanobody library. 2022 was constructed by attaching VHH18 to the C-terminal of Fc of 2F8. 2022 binds two non-overlapping epitopes simultaneously on the RBD of the SARS-CoV-2 spike protein and blocks the binding of RBD to human angiotensin-converting enzyme 2 (ACE2). 2022 potently neutralizes SARS-CoV-2 and all of the variants tested in both pseudovirus and live virus assays, including variants carrying mutations known to resist neutralizing antibodies approved under EUA and that reduce the protection efficiency of current effective vaccines. The half-maximum inhibitory concentration (IC50) of 2022 is 270 pM, 30 pM, 20 pM, and 1 pM, for wild-type, alpha, beta, and delta pseudovirus, respectively. In the live virus assay, 2022 has an IC50 of 26.4 pM, 13.3 pM, and 88.6 pM, for wild-type, beta, and delta live virus, respectively. In a mouse model of SARS-CoV-2, 2022 showed strong prophylactic and therapeutic effects. A single administration of 2022 intranasal (i.n.) or intraperitoneal (i.p.) 24 hours before virus challenge completely protected all mice from bodyweight loss, as compared with up to 20% loss of bodyweight in placebo treated mice. In addition, the lung viral titers were undetectable (FRNT assay) in all mice treated with 2022 either prophylactically or therapeutically, as compared with around 1\u00d7105 pfu/g lung tissue in placebo treated mice. In summary, bispecific antibody 2022 showed potent binding and neutralizing activity across a variety of SARS-CoV-2 variants and could be an attractive weapon to combat the ongoing waves of the COVID-19 pandemic propagated mainly by variants, especially, the much more contagious delta variant.","version":"1.1","doi":"10.1101/2021.08.10.455627","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449609","pub_date":"2021-8-10","title":"Drug repurposing based on a Quantum-Inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2 including vitamin B12","abstract":"The COVID-19 pandemic has accelerated the need to identify new therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir (RDV), the only antiviral against SARS-CoV-2 currently approved for human use, using a quantum-inspired device. We modelled RDV and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. Lastly, we demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Our data reveal that BMS-986094 and different forms of vitamin B12 are effective at inhibiting replication of all these variants of SARS-CoV-2. While BMS-986094 can cause secondary effects in humans as established by phase II trials, these findings suggest that vitamin B12 deserves consideration as a SARS-CoV-2 antiviral, particularly given its extended use and lack of toxicity in humans, and its availability and affordability. Our screening method can be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.","version":"1.2","doi":"10.1101/2021.06.25.449609","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.031856","pub_date":"2021-8-10","title":"Computational search of hybrid human/ SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains","abstract":"The role of the RNAi/Dicer/Ago system to degrade RNA viruses has been elusive in mammals, which prompted authors to think that interferon (IFN) synthesis is essential in this clade relegating the RNAi defense strategy against viral infection as accessory function. We explore the theoretical possibilities that RNAi triggered by SARS-CoV-2 might degrade some host transcripts in the opposite direction although this hypothesis seems counter intuitive. SARS-CoV-2 genome was therefore computational searched for exact intra pairing within the viral RNA and also hybrid exact pairing with human transcriptome over a minimum 20 bases length. Minimal segments of 20 bases length of SARS-CoV-2 RNA were found based on the theoretical matching with existing complementary strands in the human host transcriptome. Few human genes potentially annealing with SARS-CoV-2 RNA, among them mitochondrial deubiquitinase USP30, a subunit of ubiquitin protein ligase complex FBXO21 along with two long coding RNAs were retrieved. The hypothesis that viral originated RNAi might mediate degradation of messengers of the host transcriptome was corroborated by clinical observation and phylogenetic comparative analysis indicating a strong specificity of these hybrid pairing sequences for both SARS-CoV-2 and human genomes.","version":"1.4","doi":"10.1101/2020.04.08.031856","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455494","pub_date":"2021-8-10","title":"Broad-spectrum in vitro antiviral activity of ODBG-P-RVn: an orally-available, lipid-modified monophosphate prodrug of remdesivir parent nucleoside (GS-441524)","abstract":"The intravenous administration of remdesivir for COVID-19 confines its utility to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524) against viruses that cause diseases of human public health concern, including SARS-CoV-2. ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had near-equivalent activity to remdesivir in primary-like human small airway epithelial cells. Our results warrant investigation of ODBG-P-RVn efficacy in vivo.","version":"1.2","doi":"10.1101/2021.08.06.455494","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.09.455715","pub_date":"2021-8-10","title":"Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)","abstract":"We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: (1) a Fab-fragment of an antibody directed against the nanobody, and (2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of \u223c120 kDa, called Legobody, does not perturb the nanobody-target interaction and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23 kDa membrane protein, resulting in a map at 3.2\u00c5 overall resolution with density sufficient for de novo model building, and for the 22 kDa RBD of SARS-CoV2 spike protein, resulting in a map at 3.6 \u00c5 resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.","version":"1.1","doi":"10.1101/2021.08.09.455715","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.09.455606","pub_date":"2021-8-09","title":"ACE2 is the critical in vivo receptor for SARS-CoV-2 in a novel COVID-19 mouse model with TNF- and IFN\u03b3-driven immunopathology","abstract":"Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modelling revealed how Spike mutations of maVie16 enhanced interaction with murine ACE2. MaVie16 induced profound pathology in BALB/c and C57BL/6 mice and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia and specific adaptive immunity. Inhibition of the proinflammatory cytokines IFN\u03b3 and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. The mouse-adapted SARS-CoV-2 strain maVie16 causes fatal disease in BALB/c mice and substantial inflammation, pneumonia and immunity in C57BL/6 mice TNF\u03b1/IFN\u03b3 blockade ameliorates maVie16-induced immunopathology MaVie16 infection depends on ACE2 and soluble ACE2 inhalation can prevent disease","version":"1.1","doi":"10.1101/2021.08.09.455606","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.08.455562","pub_date":"2021-8-09","title":"A broadly neutralizing biparatopic Nanobody protects mice from lethal challenge with SARS-CoV-2 variants of concern","abstract":"The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailed in vitro characterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses and in situ hybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2021.08.08.455562","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.09.455656","pub_date":"2021-8-09","title":"Reprogramming of the intestinal epithelial-immune cell interactome during SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents an unprecedented worldwide health problem. Although the primary site of infection is the lung, growing evidence points towards a crucial role of the intestinal epithelium. Yet, the exact effects of viral infection and the role of intestinal epithelial-immune cell interactions in mediating the inflammatory response are not known. In this work, we apply network biology approaches to single-cell RNA-seq data from SARS-CoV-2 infected human ileal and colonic organoids to investigate how altered intracellular pathways upon infection in intestinal enterocytes leads to modified epithelial-immune crosstalk. We point out specific epithelial-immune interactions which could help SARS-CoV-2 evade the immune response. By integrating our data with existing experimental data, we provide a set of epithelial ligands likely to drive the inflammatory response upon infection. Our integrated analysis of intra- and inter-cellular molecular networks contribute to finding potential drug targets, and suggest using existing anti-inflammatory therapies in the gut as promising drug repurposing strategies against COVID-19.","version":"1.1","doi":"10.1101/2021.08.09.455656","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.08.455468","pub_date":"2021-8-09","title":"A Tethered Ligand Assay to Probe SARS-CoV-2:ACE2 Interactions","abstract":"SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.","version":"1.1","doi":"10.1101/2021.08.08.455468","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.07.455523","pub_date":"2021-8-09","title":"Broad neutralizing nanobody against SARS-CoV-2 engineered from pre-designed synthetic library","abstract":"SARS-CoV-2 infection is initiated with Spike glycoprotein binding to the receptor of human angiotensin converting enzyme 2 via its receptor binding domain. Blocking this interaction is considered as an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody, VHH60, directly produced from a humanized synthetic nanobody library. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein with a KD of 2.56 nM, inhibits infections of both live SARS-CoV-2 and pseudotyped viruses harboring wildtype, escape mutations and prevailing variants at nanomolar level. VHH60 also suppresses SARS-CoV-2 infection and propagation 50-fold better and protects mice from death two times longer than that of control group after live virus inoculation on mice. VHH60 therefore is a powerful synthetic nanobody with a promising profile for disease control against COVID19.","version":"1.1","doi":"10.1101/2021.08.07.455523","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.11.439347","pub_date":"2021-8-09","title":"An AI-guided signature reveals the nature of the shared proximal pathways of host immune response in MIS-C and Kawasaki disease","abstract":"A significant surge in cases of multisystem inflammatory syndrome in children (MIS-C, also called Pediatric Inflammatory Multisystem Syndrome - PIMS) has been observed amidst the COVID-19 pandemic. MIS-C shares many clinical features with Kawasaki disease (KD), although clinical course and outcomes are divergent. We analyzed whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues from these patients using a computational toolbox of two gene signatures, i.e., the 166-gene viral pandemic (ViP) signature, and its 20-gene severe (s)ViP subset that were developed in the context of SARS-CoV-2 infection and a 13-transcript signature previously demonstrated to be diagnostic for KD. Our analyses revealed that KD and MIS-C are on the same continuum of the host immune response as COVID-19. While both the pediatric syndromes converge upon an IL15/IL15RA-centric cytokine storm, suggestive of shared proximal pathways of immunopathogenesis, they diverge in other laboratory parameters and cardiac phenotypes. The ViP signatures also revealed unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to monitor severity.","version":"1.2","doi":"10.1101/2021.04.11.439347","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455491","pub_date":"2021-8-08","title":"High genetic barrier to escape from human polyclonal SARS-CoV-2 neutralizing antibodies","abstract":"The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in SARS-CoV-2 convalescent and vaccinated individuals are key determinants of neutralization breadth and, consequently, the genetic barrier to viral escape. Using chimeric viruses and antibody-selected viral mutants, we show that multiple neutralizing epitopes, within and outside the viral receptor binding domain (RBD), are variably targeted by polyclonal plasma antibodies and coincide with sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic \u2018polymutant\u2019 spike proteins that resisted polyclonal antibody neutralization to a similar degree as currently circulating variants of concern (VOC). Importantly, by aggregating VOC-associated and plasma-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in SARS-CoV-2 spike are sufficient to confer near-complete resistance to the polyclonal neutralizing antibodies generated by convalescents and mRNA vaccine recipients. Strikingly however, plasma from individuals who had been infected and subsequently received mRNA vaccination, neutralized this highly resistant SARS-CoV-2 polymutant, and also neutralized diverse sarbecoviruses. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against future sarbecovirus pandemics.","version":"1.1","doi":"10.1101/2021.08.06.455491","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.21261725","pub_date":"2021-08-08","title":"Modelling the effectiveness and social costs of daily lateral flow antigen tests versus quarantine in preventing onward transmission of COVID-19 from traced contacts","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Quarantining close contacts of individuals infected with SARS-CoV-2 for 10 to 14 days is a key strategy in reducing transmission. However, quarantine requirements are often unpopular, with low adherence, especially when a large fraction of the population has been vaccinated. Daily contact testing (DCT), in which contacts are required to isolate only if they test positive, is an alternative to quarantine for mitigating the risk of transmission from traced contacts. In this study, we developed an integrated model of COVID-19 transmission dynamics and compared the strategies of quarantine and DCT with regard to reduction in transmission and social/economic costs (days of quarantine/self-isolation). Specifically, we compared 10-day quarantine to 7 days of self-testing using rapid lateral flow antigen tests, starting 3 days after exposure to a case. We modelled both incomplete adherence to quarantine and incomplete adherence to DCT. We found that DCT reduces transmission from contacts with similar effectiveness, at much lower social/economic costs, especially for highly vaccinated populations. The findings were robust across a spectrum of scenarios with varying assumptions on the speed of contact tracing, sensitivity of lateral flow antigen tests, adherence to quarantine and uptake of testing. Daily tests would also allow rapid initiation of a new round of tracing from infected contacts.</jats:p>","version":null,"doi":"10.1101/2021.08.06.21261725","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.06.455441","pub_date":"2021-8-08","title":"VPS29 exerts opposing effects on endocytic viral entry","abstract":"Emerging zoonotic viral pathogens threaten global health and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic and pandemic threat coronaviruses as well as ebolavirus. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. VPS29 deficiency caused changes endosome morphology, and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence viral susceptibility and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases.","version":"1.1","doi":"10.1101/2021.08.06.455441","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455387","pub_date":"2021-8-08","title":"Identification and Targeting Putative G-Quadruplex Sequences in Candida glabrata: A Route to Virulence and Pathogenesis Control?","abstract":"An increase in the number of Candida species that are resistant to antifungal medication and increases worldwide. Even individuals that are never exposed to antibiotics showing resistance to antifungal drugs. The increase in resistant candida species strains requires a search for novel targets for new antifungal agents. Preventing infection caused by candida species is a tremendous challenge in medicine. Although availability and use of antifungal drugs, disseminated candidiasis leads to a high mortality rate of about 40-60%, poor diagnosis, and improper disease management. Interest in G-quadruplexes as a therapeutic target has been increased in recent years, following the implication of this non-canonical G-quadruplex secondary structure in pathological diseases. However, G-quadruplex has been reported in many pathogens contributing to virulence and pathogenesis, including bacterial pathogens such as Staphylcoccus aureus and Enterococcus spp. Etc, viruses such as SARS-CoV2, HIV, HPV etc., and Fungi such as Candida species, Aspergillus fumigatus, etc. The present aim of the study is to identify and targeting G-Quadruplex forming sequences present in the Candida glabrata. PQSFinder (R Package) identified more than 5000 putative G-quadruplex forming sequences. Out of these, we have used PQS present in the SDH1 gene of Candida glabrata. It may be a key target to ameliorate the C.glabrata infection because it encodes a protein that plays a vital role in energy production in C.glabrata cells shown in the figure. The structure was built based on the already current structure that is telomeric G4 AGGG (TTAGGG)3 {PDB ID-4G0F} for identifying its stabilization by Gold carbine derivatives. Molecular docking and ADMET analysis show that Compound A has the highest binding affinity and has the best ADMET properties among the two compounds. The present study represents PQS in the SDH1 gene could be a novel antifungal target.","version":"1.2","doi":"10.1101/2021.08.06.455387","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.21259465","pub_date":"2021-08-07","title":"Distinct age-specific SARS-CoV-2 IgG decay kinetics following natural infection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Antibody responses to SARS-CoV-2 can be observed as early as 14 days post-infection, but little is known about the stability of antibody levels over time. Here we evaluate the long-term stability of anti-SARS-CoV-2 IgG antibodies following infection with SARS-CoV-2 in 402 adult donors.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We performed a multi-center study carried out at Plasma Donor Centers in the city of Heidelberg (Plasmazentrum Heidelberg, Germany) and Munich (Plasmazentrum M\u00fcnchen, Germany). We present anti-S/N and anti-N IgG antibody levels in prospective serum samples collected up to 403 days post recovery from SARS-CoV-2 infected individuals.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    The cohort includes 402 adult donors (185 female, 217 male; 17 - 68 years of age) where anti-SARS-CoV-2 IgG levels were measured in plasma samples collected between 18- and 403-days post SARS-CoV-2 infection. A linear mixed effects model demonstrated IgG decay rates that decrease over time (\u03c7\n                    <jats:sup>2</jats:sup>\n                    =176.8, p&lt;0.00001) and an interaction of time*age \u03c7 (\u03c7\n                    <jats:sup>2</jats:sup>\n                    =10.0, p&lt;0.005)), with those over 60+ years showing the highest baseline IgG levels and the fastest rate of IgG decay. Baseline viral neutralization assays demonstrated that serum IgG levels correlated with\n                    <jats:italic>in vitro</jats:italic>\n                    neutralization capacity in 91% of our cohort.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Long-term antibody levels and age-specific antibody decay rates suggest the potential need for age-specific vaccine booster guidelines to ensure long term vaccine protection against SARS-CoV-2 infection.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.08.05.21259465","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.16.452733","pub_date":"2021-8-06","title":"One mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein, spike, for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live attenuated, recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200- fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunity in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against two virus variants of concern. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in phase 1 clinical trials as a single-dose intranasal COVID-19 vaccine.","version":"1.2","doi":"10.1101/2021.07.16.452733","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.19.452771","pub_date":"2021-8-06","title":"Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants","abstract":"The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.","version":"1.3","doi":"10.1101/2021.07.19.452771","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455405","pub_date":"2021-8-06","title":"RIG-I-induced innate antiviral immunity protects mice from lethal SARS-CoV-2 infection","abstract":"The SARS-CoV-2 pandemic has underscored the need for rapidly employable prophylactic and antiviral treatments against emerging viruses. Nucleic acid agonists of the innate immune system can be administered to activate an effective antiviral program for prophylaxis in exposed populations, a measure of particular relevance for SARS-CoV-2 infection due to its efficient evasion of the host antiviral response. In this study, we utilized the K18-hACE2 mouse model of COVID-19 to examine whether prophylactic activation of the antiviral receptor RIG-I protects mice from SARS-CoV-2 infection. Systemic treatment of mice with a specific RIG-I ligand one to seven days prior to infection with a lethal dose of SARS-CoV-2 improved their survival of by up to 50 %. Improved survival was associated with lower viral load in oropharyngeal swabs and in the lungs and brain of RIG-I-treated mice. Moreover, despite antiviral protection, the surviving mice that were treated with RIG-I ligand developed adaptive SARS-CoV-2-specific immunity. These results reveal that prophylactic RIG-I activation by synthetic RNA oligonucleotides is a promising strategy to convey short-term, unspecific antiviral protection against SARS-CoV-2 infection and may be a suitable broad-spectrum approach to constraining the spread of newly emerging viruses until virus-specific therapies and vaccines become available.","version":"1.1","doi":"10.1101/2021.08.06.455405","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455382","pub_date":"2021-8-06","title":"Controlled administration of aerosolized SARS-CoV-2 to K18-hACE2 transgenic mice uncouples respiratory infection and anosmia from fatal neuroinvasion","abstract":"The development of a tractable small animal model faithfully reproducing human COVID-19 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid SARS-CoV-2 suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high CNS infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model\u2019s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction, but did not lead to fatal viral neuroinvasion. When compared to intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition and a transcriptional signature comparable to that observed in SARS-CoV-2- infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection) and therapeutic interventions.","version":"1.1","doi":"10.1101/2021.08.06.455382","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.029454","pub_date":"2021-8-06","title":"Generation of a novel SARS-CoV-2 sub-genomic RNA due to the R203K/G204R variant in nucleocapsid: homologous recombination has potential to change SARS-CoV-2 at both protein and RNA level","abstract":"Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host\u2019s anti-viral immune response, in turn affecting the frequency of variants over-time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. Deep sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Sequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for diversification and adaptation within its new host.","version":"1.4","doi":"10.1101/2020.04.10.029454","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443253","pub_date":"2021-8-06","title":"SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough","abstract":"The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.","version":"1.5","doi":"10.1101/2021.05.08.443253","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455406","pub_date":"2021-8-06","title":"Learning torus PCA based classification for multiscale RNA backbone structure correction with application to SARS-CoV-2","abstract":"Reconstructions of structure of biomolecules, for instance via X-ray crystallography or cryo-EM frequently contain clashes of atomic centers. Correction methods are usually based on simulations approximating biophysical chemistry, making them computationally expensive and often not correcting all clashes. We propose a computationally fast data-driven statistical method yielding suites free from within-suite clashes: From such a clash free training data set, devising mode hunting after torus PCA on adaptive cutting average linkage tree clustering (MINTAGE), we learn RNA suite shapes. With classification based on multiscale structure enhancement (CLEAN), for a given clash suite we determine its neighborhood on a mesoscopic scale involving several suites. As corrected suite we propose the Fr\u00e9chet mean on a torus of the largest classes in this neighborhood. We validate CLEAN MINTAGE on a benchmark data set, compare it to a state of the art correction method and apply it, as proof of concept, to two exemplary suites adjacent to helical pieces of the frameshift stimulation element of SARS-CoV-2 which are difficult to reconstruct. In contrast to a recent reconstruction proposing several different structure models, CLEAN MINTAGE unanimously proposes structure corrections within the same clash free class for all suites. https://gitlab.gwdg.de/henrik.wiechers1/clean-mintage-code","version":"1.1","doi":"10.1101/2021.08.06.455406","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.448037","pub_date":"2021-8-06","title":"\u03b1-ketoglutarate augments prolyl hydroxylase-2 mediated inactivation of phosphorylated-Akt to inhibit induced-thrombosis and inflammation","abstract":"Phosphorylation of Akt (pAkt) regulates multiple physiological and pathological processes including thrombosis and inflammation. In an approach to inhibit the pathological signalling of pAkt by prolyl-hydroxylase-2 (PHD2) we employed \u03b1-ketoglutarate (\u03b1KG), a cofactor of PHD2. Octyl-\u03b1KG supplementation to platelets promoted PHD2 activity through elevated intracellular \u03b1KG:succinate ratio and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline alongside enhanced binding of PHD2 to pAkt in \u03b1KG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-\u03b1KG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelet and leukocyte obtained from mice supplemented with dietary-\u03b1KG, unaccompanied by alteration in their counts. Dietary-\u03b1KG significantly reduced clot formation and leukocyte accumulation in various organs including lung of mice treated with thrombosis-inducing agent carrageenan. Importantly, we observed a significant rescue effect of dietary-\u03b1KG on inflamed lung of SARS-CoV-2 infected hamsters. \u03b1KG significantly reduced leukocyte accumulation, clot formation and viral load alongside downmodulation of pAkt in lung of the infected animals. Therefore, our study suggests a safe implementation of dietary-\u03b1KG in prevention of Akt-driven anomalies including thrombosis and inflammation, highlighting a better pulmonary management in COVID-19.","version":"1.2","doi":"10.1101/2021.06.11.448037","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.06.455424","pub_date":"2021-8-06","title":"Small-molecule ligands can inhibit \u22121 programmed ribosomal frameshifting in a broad spectrum of coronaviruses","abstract":"Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use \u22121 programmed ribosomal frameshifting (\u22121 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates \u22121 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs\u2014the most likely source of future zoonoses\u2014as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed \u22121 PRF significantly in several of them, while having limited to no effect on \u22121 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit \u22121 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.","version":"1.1","doi":"10.1101/2021.08.06.455424","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.14.444026","pub_date":"2021-8-05","title":"CovidExpress: an interactive portal for intuitive investigation on SARS-CoV-2 related transcriptomes","abstract":"Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans could cause coronavirus disease 2019 (COVID-19). Since its first discovery in Dec 2019, SARS-CoV-2 has become a global pandemic and caused 3.3 million direct/indirect deaths (2021 May). Amongst the scientific community\u2019s response to COVID-19, data sharing has emerged as an essential aspect of the combat against SARS-CoV-2. Despite the ever-growing studies about SARS-CoV-2 and COVID-19, to date, only a few databases were curated to enable access to gene expression data. Furthermore, these databases curated only a small set of data and do not provide easy access for investigators without computational skills to perform analyses. To fill this gap and advance open-access to the growing gene expression data on this deadly virus, we collected about 1,500 human bulk RNA-seq datasets from publicly available resources, developed a database and visualization tool, named CovidExpress (https://stjudecab.github.io/covidexpress). This open access database will allow research investigators to examine the gene expression in various tissues, cell lines, and their response to SARS-CoV-2 under different experimental conditions, accelerating the understanding of the etiology of this disease to inform the drug and vaccine development. Our integrative analysis of this big dataset highlights a set of commonly regulated genes in SARS-CoV-2 infected lung and Rhinovirus infected nasal tissues, including OASL that were under-studied in COVID-19 related reports. Our results also suggested a potential FURIN positive feedback loop that might explain the evolutional advantage of SARS-CoV-2.","version":"1.3","doi":"10.1101/2021.05.14.444026","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.455262","pub_date":"2021-8-05","title":"Identification of Potent Small Molecule Inhibitors of SARS-CoV-2 Entry","abstract":"The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, Calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals Calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.","version":"1.1","doi":"10.1101/2021.08.05.455262","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.31.454592","pub_date":"2021-8-05","title":"SARS-CoV-2 fears green: the chlorophyll catabolite Pheophorbide a is a potent antiviral","abstract":"SARS-CoV-2 pandemic is having devastating consequences worldwide. Although vaccination advances at good pace, effectiveness against emerging variants is unpredictable. The virus has displayed a remarkable resistance to treatments and no drugs have been proved fully effective against Covid-19. Thus, despite the international efforts, there is still an urgent need for new potent and safe antivirals against SARS-CoV-2. Here we exploited the enormous potential of plant metabolism using the bryophyte Marchantia polymorpha and identified a potent SARS-CoV-2 antiviral, following a bioactivity-guided fractionation and mass-spectrometry approach. We found that the chlorophyll derivative Pheophorbide a (PheoA), a porphyrin compound similar to animal Protoporphyrin IX, has an extraordinary antiviral activity against SARS-CoV-2 preventing infection of cultured monkey and human cells, without noticeable cytotoxicity. We also show that PheoA prevents coronavirus entry into the cells by directly targeting the viral particle. Besides SARS-CoV-2, PheoA also displayed a broad-spectrum antiviral activity against (+) strand RNA viral pathogens such as HCV, West Nile, and other coronaviruses, but not against (\u2212) strand RNA viruses, such as VSV. Our results indicate that PheoA displays a remarkable potency and a satisfactory therapeutic index, which together with its previous use in photoactivable cancer therapy in humans, suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.07.31.454592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.455212","pub_date":"2021-8-05","title":"Vaccination with B.1.1.7, B.1.351 and P.1 variants protects mice from challenge with wild type SARS-CoV-2","abstract":"Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against coronavirus disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild type SARS-CoV-2 and from variants B.1.1.7, B.1.351 and P.1 for their immunogenicity and protective effect in vivo against challenge with wild type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7 vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild type SARS-CoV-2 in a mouse model. The emergence of variants of SARS-CoV-2 has led to an urgency to study whether vaccines will lead to cross-protection against these variants. Here, we demonstrate that vaccination with spike proteins of various variants leads to cross-neutralizing responses, as well as protection in a mouse model against wild type SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.08.05.455212","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428353","pub_date":"2021-8-05","title":"Effect of SARS-CoV-2 spike mutations on animal ACE2 usage and in vitro neutralization sensitivity","abstract":"The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to \u223c15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.","version":"1.3","doi":"10.1101/2021.01.27.428353","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.455082","pub_date":"2021-8-05","title":"Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles","abstract":"Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M). R203M substitution within SARS-CoV-2 N, found in delta variant, improves RNA packaging into virus-like particles by 10-fold.","version":"1.1","doi":"10.1101/2021.08.05.455082","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.04.455181","pub_date":"2021-8-05","title":"Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein","abstract":"The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. Infection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD, NTD and S2. Different infected individuals generate vastly different amounts of anti-spike antibodies. By contrast, as we show here, there is a remarkable similarity in the properties of the antibodies produced. Different individuals generate the same proportions of antibodies against each domain of the spike protein. Furthermore, the relationship between the amount of anti-spike antibodies produced and their neutralization efficacy of SARS-CoV-2 is highly conserved. Therefore, the observed variation in the neutralizing activity of the antibody response in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or relative antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.","version":"1.1","doi":"10.1101/2021.08.04.455181","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.04.455157","pub_date":"2021-8-05","title":"Pandemic-Scale Phylogenomics Reveals Elevated Recombination Rates in the SARS-CoV-2 Spike Region","abstract":"Accurate and timely detection of recombinant lineages is crucial for interpreting genetic variation, reconstructing epidemic spread, identifying selection and variants of interest, and accurately performing phylogenetic analyses. During the SARS-CoV-2 pandemic, genomic data generation has exceeded the capacities of existing analysis platforms, thereby crippling real-time analysis of viral recombination. Low SARS-CoV-2 mutation rates make detecting recombination difficult. Here, we develop and apply a novel phylogenomic method to exhaustively search a nearly comprehensive SARS-CoV-2 phylogeny for recombinant lineages. We investigate a 1.6M sample tree, and identify 606 recombination events. Approximately 2.7% of sequenced SARS-CoV-2 genomes have recombinant ancestry. Recombination breakpoints occur disproportionately in the Spike protein region. Our method empowers comprehensive real time tracking of viral recombination during the SARS-CoV-2 pandemic and beyond.","version":"1.1","doi":"10.1101/2021.08.04.455157","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.455126","pub_date":"2021-8-05","title":"Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells","abstract":"A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.","version":"1.1","doi":"10.1101/2021.08.05.455126","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.05.455290","pub_date":"2021-8-05","title":"SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage","abstract":"For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2\u2019 by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.","version":"1.1","doi":"10.1101/2021.08.05.455290","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442648","pub_date":"2021-8-05","title":"Immunolocalization studies of vimentin and ACE2 on the surface of cells exposed to SARS-CoV-2 Spike proteins","abstract":"The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.","version":"1.2","doi":"10.1101/2021.05.04.442648","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.111344","pub_date":"2021-8-05","title":"Optimizing predictive models to prioritize viral discovery in zoonotic reservoirs","abstract":"Despite global investment in One Health disease surveillance, it remains difficult\u2014and often very costly\u2014to identify and monitor the wildlife reservoirs of novel zoonotic viruses. Statistical models can be used to guide sampling prioritization, but predictions from any given model may be highly uncertain; moreover, systematic model validation is rare, and the drivers of model performance are consequently under-documented. Here, we use bat hosts of betacoronaviruses as a case study for the data-driven process of comparing and validating predictive models of likely reservoir hosts. In the first quarter of 2020, we generated an ensemble of eight statistical models that predict host-virus associations and developed priority sampling recommendations for potential bat reservoirs and potential bridge hosts for SARS-CoV-2. Over more than a year, we tracked the discovery of 40 new bat hosts of betacoronaviruses, validated initial predictions, and dynamically updated our analytic pipeline. We find that ecological trait-based models perform extremely well at predicting these novel hosts, whereas network methods consistently perform roughly as well or worse than expected at random. These findings illustrate the importance of ensembling as a buffer against variation in model quality and highlight the value of including host ecology in predictive models. Our revised models show improved performance and predict over 400 bat species globally that could be undetected hosts of betacoronaviruses. Although 20 species of horseshoe bats (Rhinolophus spp.) are known to be the primary reservoir of SARS-like viruses, we find at least three-fourths of plausible betacoronavirus reservoirs in this bat genus might still be undetected. Our study is the first to demonstrate through systematic validation that machine learning models can help optimize wildlife sampling for undiscovered viruses and illustrates how such approaches are best implemented through a dynamic process of prediction, data collection, validation, and updating.","version":"1.4","doi":"10.1101/2020.05.22.111344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.24.453631","pub_date":"2021-8-04","title":"SARS-CoV-2 Delta variant pathogenesis and host response in Syrian hamsters","abstract":"B.1.617 lineage is becoming a dominant SARS-CoV-2 lineage worldwide and was the dominant lineage reported in second COVID-19 wave in India, which necessitated studying the properties of the variant. We evaluated the pathogenicity and virus shedding of B.1.617.2 (Delta) and B.1.617.3 lineage of SARS-CoV-2 and compared with that of B.1, an early virus isolate with D614G mutation in Syrian hamster model. Viral load, antibody response and lung disease were studied. No significant difference in the virus shedding pattern was observed among these variants studied. A significantly high SARS-CoV-2 sub genomic RNA could be detected in the respiratory tract of hamsters infected with Delta variant for 14 days. Delta variant induced lung disease of moderate severity in 40% of infected animals. The neutralizing capability of the B.1, Delta and B.1.617.3 variant infected animals were found significantly lower with the B.1.351 (Beta variant). The findings of the study support the attributed disease severity and the increased transmission potential of the Delta variant.","version":"1.2","doi":"10.1101/2021.07.24.453631","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.02.21261465","pub_date":"2021-08-04","title":"Severity, criticality, and fatality of the SARS-CoV-2 Beta variant","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Severity (acute-care hospitalization), criticality (ICU hospitalization), and fatality of SARS-CoV-2 Beta (B.1.351) variant was investigated through case-control studies applied to complete national cohorts of infection, disease, and death cases in Qatar. Compared to Alpha (B.1.1.7) variant, odds of progressing to severe disease were 1.24-fold (95% CI: 1.11-1.39) higher for Beta. Odds of progressing to critical disease were 1.49-fold (95% CI: 1.13-1.97) higher. Odds of COVID-19 death were 1.57-fold (95% CI: 1.03-2.43) higher. Findings highlight risks to healthcare systems, particularly to intensive care facilities and resources, with increased circulation of Beta.</jats:p>","version":null,"doi":"10.1101/2021.08.02.21261465","journal":"medRxiv","score":null},{"id":"10.1101/2021.08.04.454929","pub_date":"2021-8-04","title":"Analysis of 329,942 SARS-CoV-2 records retrieved from GISAID database","abstract":"The 31st of December 2019 was when the World Health Organization received a report about an outbreak of pneumonia of unknown etiology in the Chinese city of Wuhan. The outbreak was the result of the novel virus labeled as SARS-CoV-2, which spread to about 220 countries and caused approximately 3,311,780 deaths, infecting more than 159,319,384 people by May 12th, of 2021. The virus caused a worldwide pandemic leading to panic, quarantines, and lockdowns \u2013 although none of its predecessors from the coronavirus family have ever achieved such a scale. The key to understanding the global success of SARS-CoV-2 is hidden in its genome. We retrieved data for 329,942 SARS-CoV-2 records uploaded to the GISAID database from the beginning of the pandemic until the 8th of January 2021. To process the data, a Python variant detection script was developed, using pairwise2 from the BioPython library. Pandas, Matplotlib, and Seaborn, were applied to visualize the data. Genomic coordinates were obtained from the UCSC Genome Browser (https://genome.ucsc.edu/). Sequence alignments were performed for every gene separately. Genomes less than 26,000 nucleotides long were excluded from the research. Clustering was performed using HDBScan. Here, we addressed the genetic variability of SARS-CoV-2 using 329,942 worldwide samples. The analysis yielded 155 genome variations (SNPs and deletions) in more than 0.3% of the sequences. Nine common SNPs were present in more than 20% of the samples. Clustering results suggested that a proportion of people (2.46%) were infected with a distinct subtype of the B.1.1.7 variant. The subtype may be characterized by four to six additional mutations, with four being a more frequent option (G28881A, G28882A, and G28883\u0421 in the N gene, A23403G in S, A28095T in ORF8, G25437T in ORF3a). Two clusters were formed by mutations in the samples uploaded predominantly by Denmark and Australia, which may indicate the emergence of \u201cDanish\u201d and \u201cAustralian\u201d variants. Five clusters were linked to increased/decreased age, shifted gender ratio, or both. According to a correlation coefficient matrix, 69 mutations correlate with at least one other mutation (correlation coefficient greater than 0.7). We also addressed the completeness of the GISAID database, where between 77% and 93% of the fields were either left blank or filled incorrectly. Metadata mining analysis has led to a hypothesis about gender inequality in medical care in certain countries. Finally, we found ORF6 and E as the most conserved genes (96.15% and 94.66% of the sequences totally match the reference, respectively), making them potential targets for vaccines and treatment. Our results indicate areas of the SARS-CoV-2 genome that researchers can focus on for further structural and functional analysis.","version":"1.1","doi":"10.1101/2021.08.04.454929","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443592","pub_date":"2021-8-04","title":"Design, immunogenicity and efficacy of a Pan-SARS-CoV-2 synthetic DNA vaccine","abstract":"Here we have employed SynCon\u00ae design technology to construct a DNA vaccine expressing a pan-Spike immunogen (INO-4802) to induce broad immunity across SARS-CoV-2 variants of concern (VOC). Compared to WT and VOC-matched vaccines which showed reduced cross-neutralizing activity, INO-4802 induced potent neutralizing antibodies and T cell responses against WT as well as B.1.1.7, P.1, and B.1.351 VOCs in a murine model. In addition, a hamster challenge model demonstrated that INO-4802 conferred superior protection following intranasal B.1.351 challenge. Protection against weight loss associated with WT, B.1.1.7, P.1 and B.1.617.2 challenge was also demonstrated. Vaccinated hamsters showed enhanced humoral responses against VOC in a heterologous WT vaccine prime and INO-4802 boost setting. These results demonstrate the potential of the pan-SARS-CoV-2 vaccine, INO-4802 to induce cross-reactive immune responses against emerging VOC as either a standalone vaccine, or as a potential boost for individuals previously immunized with WT-matched vaccines.","version":"1.2","doi":"10.1101/2021.05.11.443592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.03.454782","pub_date":"2021-8-04","title":"Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity","abstract":"Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype\u2013antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.","version":"1.1","doi":"10.1101/2021.08.03.454782","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.04.455140","pub_date":"2021-8-04","title":"Contribution of single mutations to selected SARS-CoV-2 emerging variants Spike antigenicity","abstract":"Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37\u00b0C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.","version":"1.1","doi":"10.1101/2021.08.04.455140","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454526","pub_date":"2021-8-04","title":"A Master Autoantigen-ome Links Alternative Splicing, Female Predilection, and COVID-19 to Autoimmune Diseases","abstract":"Chronic and debilitating autoimmune sequelae pose a grave concern for the post-COVID-19 pandemic era. Based on our discovery that the glycosaminoglycan dermatan sulfate (DS) displays peculiar affinity to apoptotic cells and autoantigens (autoAgs) and that DS-autoAg complexes cooperatively stimulate autoreactive B1 cell responses, we compiled a database of 751 candidate autoAgs from six human cell types. At least 657 of these have been found to be affected by SARS-CoV-2 infection based on currently available multi-omic COVID data, and at least 400 are confirmed targets of autoantibodies in a wide array of autoimmune diseases and cancer. The autoantigen-ome is significantly associated with various processes in viral infections, such as translation, protein processing, and vesicle transport. Interestingly, the coding genes of autoAgs predominantly contain multiple exons with many possible alternative splicing variants, short transcripts, and short UTR lengths. These observations and the finding that numerous autoAgs involved in RNA-splicing showed altered expression in viral infections suggest that viruses exploit alternative splicing to reprogram host cell machinery to ensure viral replication and survival. While each cell type gives rise to a unique pool of autoAgs, 39 common autoAgs associated with cell stress and apoptosis were identified from all six cell types, with several being known markers of systemic autoimmune diseases. In particular, the common autoAg UBA1 that catalyzes the first step in ubiquitination is encoded by an X-chromosome escape gene. Given its essential function in apoptotic cell clearance and that X-inactivation escape tends to increase with aging, UBA1 dysfunction can therefore predispose aging women to autoimmune disorders. In summary, we propose a model of how viral infections lead to extensive molecular alterations and host cell death, autoimmune responses facilitated by autoAg-DS complexes, and ultimately autoimmune diseases. Overall, this master autoantigen-ome provides a molecular guide for investigating the myriad of autoimmune sequalae to COVID-19 and clues to the rare but reported adverse effects of the currently available COVID vaccines.","version":"1.1","doi":"10.1101/2021.07.30.454526","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.03.455003","pub_date":"2021-8-04","title":"Llamanade: an open-source computational pipeline for robust nanobody humanization","abstract":"Nanobodies (Nbs) have recently emerged as a promising class of antibody fragments for biomedical and therapeutic applications. Despite having marked physicochemical properties, Nbs are derived from camelids and may require \u201chumanization\u201d to improve translational potentials for clinical trials. Here we have systematically analyzed the sequence and structural properties of Nbs based on NGS (next-generation sequencing) databases and high-resolution structures. Our analysis reveals substantial framework diversities and underscores the key differences between Nbs and human Immunoglobulin G (IgG) antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen-binding, providing insights into Nb humanization. Based on big data analysis, we developed \u201cLlamanade\u2019\u2019, a user-friendly, open-source to facilitate rational humanization of Nbs. Using Nb sequence as input, Llamanade provides information on the sequence features, model structures, and optimizes solutions to humanize Nbs. The full analysis for a given Nb takes less than a minute on a local computer. To demonstrate the robustness of this tool, we applied it to successfully humanize a cohort of structurally diverse and highly potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a web server to support the development of a rapidly expanding repertoire of therapeutic Nbs into safe and effective trials. Camelid Nbs are characterized by small size, excellent pharmacological properties and high flexibility in bioengineering for therapeutic development. However, Nbs are \u201cxeno\u201d antibodies, which require \u201chumanization\u201d to improve their translational potential. Currently, there is a lack of systematic investigation of Nbs to rationally guide humanization. No dedicated software has been developed for this purpose. Here, we report the development of Llamanade, an open-source computational pipeline and the first dedicated software to facilitate rational humanization of Nbs. To subjectively evaluate Llamanade, we used it to humanize a cohort of structurally diverse and ultrapotent antiviral Nbs against SARS-CoV-2. Robust humanization by Llamanade significantly improved the humanness level of Nbs to closely resemble fully human IgGs. Importantly, these highly humanized antiviral Nbs remained excellent solubility and comparably high bioactivities to the non-humanized Nb precursors. We envision that Llamanade will help advance Nb research into therapeutic development.","version":"1.1","doi":"10.1101/2021.08.03.455003","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.03.454910","pub_date":"2021-8-03","title":"Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters","abstract":"COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the \u201ccold chain\u201d transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS- CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2021.08.03.454910","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.02.454546","pub_date":"2021-8-03","title":"Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern","abstract":"Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the \u201cRBD-up\u201d position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among \u03b2-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.","version":"1.1","doi":"10.1101/2021.08.02.454546","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.03.454861","pub_date":"2021-8-03","title":"SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant","abstract":"SARS-CoV-2 variants display enhanced transmissibility and/or immune evasion and can be generated in humans or animals, like minks, thus generating new reservoirs. The continuous surveillance of animal susceptibility to new variants is necessary to predict pandemic evolution. In this study we demonstrate that, compared to the B.1 SARS-CoV-2 variant, K18-hACE2 transgenic mice challenged with the B.1.351 variant displayed a faster progression of infection. Furthermore, we also report that B.1.351 can establish infection in wildtype mice, while B.1 cannot. B.1.351-challenged wildtype mice showed a milder infection than transgenic mice, confirmed by detectable viral loads in oropharyngeal swabs and tissues, lung pathology, immunohistochemistry and serology. In silico models supported these findings by demonstrating that the Spike mutations in B.1.351 resulted in increased affinity for both human and murine ACE2 receptors. Overall, this study highlights the plasticity of SARS-CoV-2 animal susceptibility landscape, which may contribute to viral persistence and expansion.","version":"1.1","doi":"10.1101/2021.08.03.454861","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.03.454858","pub_date":"2021-8-03","title":"Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization","abstract":"Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.","version":"1.1","doi":"10.1101/2021.08.03.454858","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.02.454829","pub_date":"2021-8-03","title":"Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit","abstract":"Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74\u00c5 crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development.","version":"1.1","doi":"10.1101/2021.08.02.454829","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.02.454730","pub_date":"2021-8-03","title":"The timing of natural killer cell response in coronavirus infection: a concise model perspective","abstract":"Coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2 cause respiratory diseases with remarkably heterogeneous progression. This in part reflects the viral ability to influence the cytokine secretion and thereby the innate immune system. Especially the viral interference of IFN-I signaling and the subsequent deficiency of innate immune response in the early phase have been associated with rapid virus replication and later excessive immune responses. We propose a mathematical framework to analyze IFN-I signaling and its impact on the interaction motif between virus, NK cells and macrophages. The model recapture divergent dynamics of coronavirus infections including the possibility for elevated secretion of IL-6 and IFN-\u03b3 as a consequence of exacerbated macrophage activation. Dysfunction of NK cells recruitment increase disease severity by leading to a higher viral load peak, the possibility for excessive macrophage activation, and an elevated risk of the cytokine storm. Thus the model predicts that delayed IFN-I signaling could lead to pathogenicity in the latter stage of an infection. Reversely, in case of strong NK recruitment from infected cells we predict a possible chronic disease state with moderate and potentially oscillating virus/cytokine levels.","version":"1.1","doi":"10.1101/2021.08.02.454730","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454324","pub_date":"2021-8-03","title":"Mechanism of Antiviral Activity of Nitazoxanide against the Influenza Virus: Effect of Tizoxanide on AdenosineTriphosphate in Influenza-virus Infected Madin Darby Canine Kidney Cells","abstract":"Nitazoxanide (NTZ) is a broad-spectrum antiviral undergoing clinical development for treating influenza and other viral respiratory infections such as those caused by rhinovirus/enterovirus and coronavirus including the emerging SARS-CoV-2. Nitazoxanide is a mild uncoupler of oxidative phosphorylation, which is modulating the ATP production in cells. ATP is an essential component of viral replication, and we have evaluated the effect of tizoxanide (TIZ), the active circulating metabolite of NTZ, on ATP in Madin-Darby canine kidney (MDCK) cells and in MDCK cells infected with influenza A and B viruses. TIZ decreased cellular ATP in a dose-dependent manner in MDCK cells and in MDCK cells infected with influenza A and B viruses. Maximum inhibition of ATP in influenza infected or uninfected MDCK cells reached up to 45% after 6 and 24 hours of exposure to 100\u00b5M TIZ. The decrease in cellular ATP did not affect cell viability and was reversible after eliminating TIZ from the culture. The concentrations of TIZ required to decrease cellular ATP levels were similar to those reported to inhibit replication of influenza A and B viruses in our laboratory. A decrease in ATP triggers activation of AMP-activated protein kinase, which is known to suppress the secretion of pro-inflammatory cytokines. Additional studies are warranted to evaluate the effect of TIZ on mitochondrial function.","version":"1.2","doi":"10.1101/2021.07.30.454324","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453393","pub_date":"2021-8-02","title":"Mesalamine Reduces Intestinal ACE2 Expression Without Modifying SARS-CoV-2 Infection or Disease Severity in Mice","abstract":"Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium, and can induce GI symptoms similar to the human inflammatory bowel diseases (IBD). An international surveillance epidemiology study (SECURE-IBD) reported that the standardized mortality ratio trends higher in IBD patients (1.5-1.8) and that mesalamine/sulfasalazine therapy correlates with poor outcome. The goal of our study was to experimentally address the relationship between mesalamine and SARS-CoV-2 entry, replication, and/or pathogenesis. Viral infection was performed with a chimeric vesicular stomatitis virus expressing SARS-CoV-2 spike protein and EGFP (VSV-SARS-CoV-2) and SARS-CoV-2 virus derived from an infectious cDNA clone of 2019n-CoV/USA_WA1/2020. Primary human ileal spheroids derived from healthy donors were grown as 3D spheroids or on 2D transwells. We assessed the effect of 10 mM mesalamine (Millipore Sigma) on viral RNA levels, as well as the expression of the SARS-CoV-2 receptor angiotensin II-converting enzyme 2 (ACE2), Transmembrane Serine Protease 2 (TMPRSS2), TMPRSS4, Cathepsin B (CTSB) and CTSL by qRT-PCR. 8-12 week old K18-ACE2 were treated orally with PBS or mesalamine at 200 mg/kg daily. Mice were inoculated intranasally with 1\u00d7103 FFU of SARS-CoV-2. Mice were weighed daily and viral titers were determined 7 days post infection (dpi) by qRT-PCR. For the intestinal viral entry model, VSV-SARS-CoV-2 was injected into a ligated intestinal loop of anesthetized K18-ACE2 mice and tissues were harvested 6 hours post-infection. We found no change in viral RNA levels in human intestinal epithelial cells in response to mesalamine. Expression of ACE2 was reduced following mesalamine treatment in enteroids, while CTSL expression was increased. Mice receiving mesalamine lost weight at similar rates compared to mice receiving vehicle control. Mesalamine treatment did not change viral load in the lung, heart, or intestinal tissues harvested at 7 dpi. Pretreatment with mesalamine did not modulate intestinal entry of the chimeric VSV-SARS-CoV-2 in K18-ACE2 mice. Mesalamine did not alter viral entry, replication, or pathogenesis in vitro or in mouse models. Mesalamine treatment reduced expression of the viral receptor ACE2 while concurrently increasing CTSL expression in human ileum organoids.","version":"1.2","doi":"10.1101/2021.07.23.453393","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.01.454605","pub_date":"2021-8-02","title":"The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin \u03b15\u03b21 and NF-\u03baB","abstract":"Vascular endothelial cells (EC) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, the hallmarks of the severe disease. However, the mechanisms by which EC are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin \u03b15\u03b21 signaling. Incubation of human umbilical vein EC with whole spike, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-\u03baB and enhanced the expression of leukocyte adhesion molecules VCAM1 and ICAM1, the adhesion of peripheral blood leukocytes, and the permeability of the monolayer. Inhibitors of integrin \u03b15\u03b21 activation prevented these effects. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin \u03b15\u03b21 in EC to activate Rho GTPases, eNOS pathways, and the NF-\u03baB gene expression program responsible for vascular leakage and leukocyte infiltration, respectively. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin \u03b15\u03b21 as a promising target for treating vascular inflammation in COVID-19.","version":"1.1","doi":"10.1101/2021.08.01.454605","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.02.454771","pub_date":"2021-8-02","title":"Regional Variant Analysis of Spike Glycoprotein Mutations of SARS-CoV-2 and Its Implications in COVID-19 Pandemic Control","abstract":"Mutations in the spike glycoprotein have various impacts on the receptor binding, antibody interaction, and host range of SARS-CoV-2. As the interaction of spike glycoprotein with the human ACE2 receptor is the entry point of SARS-CoV-2 in human cells, mutations in the spike protein itself contain numerous impacts on the pandemic. Here, we analysed all the mutations in the spike glycoprotein from123 strains isolated from Kerala, India. We also predicted the possible structural relevance of the unique mutations based on topological analysis of the residue interaction network of the spike glycoprotein structure.","version":"1.1","doi":"10.1101/2021.08.02.454771","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.448011","pub_date":"2021-8-02","title":"SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation","abstract":"Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighbouring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha and Beta spread and fusion in cell cultures. Alpha and Beta replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Alpha, Beta and D614G fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes differentially modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation. The Spike protein of the novel SARS-CoV-2 variants are comparative more fusogenic than the earlier strains. The mutations in the variant spike protein differential modulate syncytia formation, ACE2 binding, and antibody escape. The spike protein of Alpha, Beta and Delta, in the absence of other viral proteins, induce more syncytia than D614G The ACE2 affinity of the variant spike proteins correlates to their fusogenicity Variant associated mutations P681H, D1118H, and D215G augment cell-cell fusion, while antibody escape mutation E484K, K417N and \u0394242-244 hamper it. Variant spike-mediated syncytia formation is effectively restricted by IFITMs","version":"1.2","doi":"10.1101/2021.06.11.448011","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.01.454696","pub_date":"2021-8-02","title":"Allosteric perspective on the mutability and druggability of the SARS-CoV-2 Spike protein","abstract":"Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both diagnostics of emerging variants of concern (VOCs) and design of the variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escaping mutations via non-competitive inhibition originating from many alternative distal locations. The exhaustive allosteric signalling and probing maps provide a comprehensive picture of allostery in the Spike protein, making it possible to locate sites of potential mutations that could work as new VOCs \u201cdrivers\u201d, and to determine binding patches that may be targeted by newly developed allosteric drugs.","version":"1.1","doi":"10.1101/2021.08.01.454696","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.415596","pub_date":"2021-8-02","title":"Distant Residues Modulate Conformational Opening in SARS-CoV-2 Spike Protein","abstract":"Infection by SARS-CoV-2 involves the attachment of the receptor binding domain (RBD) of its spike proteins to the ACE2 receptors on the peripheral membrane of host cells. Binding is initiated by a down-to-up conformational change in the spike protein, the change that presents the RBD to the receptor. To date, computational and experimental studies that search for therapeutics have concentrated, for good reason, on the RBD. However, the RBD region is highly prone to mutations, and is therefore a hotspot for drug resistance. In contrast, we here focus on the correlations between the RBD and residues distant to it in the spike protein. This allows for a deeper understanding of the underlying molecular recognition events and prediction of the highest-effect key mutations in distant, allosteric sites, with implications for therapeutics. Also, these sites can appear in emerging mutants with possibly higher transmissibility and virulence, and pre-identifying them can give clues for designing pancoronavirus vaccines against future outbreaks. Our model, based on time-lagged independent component analysis (tICA) and protein graph connectivity network, is able to identify multiple residues that exhibit long-distance coupling with the RBD opening. Residues involved in the most ubiquitous D614G mutation and the A570D mutation of the highly contagious UK SARS-CoV-2 variant are predicted ab-initio from our model. Conversely, broad spectrum therapeutics like drugs and monoclonal antibodies can target these key distant-but-conserved regions of the spike protein. The novel coronavirus (SARS-CoV-2) pandemic resulted in the largest public health crisis in recent times. Significant drug design effort against SARS-CoV-2 is focused on the receptor binding domain (RBD) of the spike protein, although this region is highly prone to mutations causing therapeutic resistance. We applied deep data analysis methods on all-atom molecular dynamics simulations to identify key non-RBD residues that play a crucial role in spike-receptor binding and infection. textcol-orredBecause the non-RBD residues are typically conserved across multiple coronaviruses, they can be targeted by broad spectrum antibodies and drugs to treat infections from new strains that might appear during future epidemics.","version":"1.5","doi":"10.1101/2020.12.07.415596","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.01.454662","pub_date":"2021-8-02","title":"Langerhans cells and cDC1s play redundant roles in mRNA-LNP induced protective anti-influenza and anti-SARS-CoV-2 responses","abstract":"Nucleoside modified mRNA combined with Acuitas Therapeutics\u2019 lipid nanoparticles (LNP) have been shown to support robust humoral immune responses in many preclinical animal vaccine studies and later in humans with the SARS-CoV-2 vaccination. We recently showed that this platform is highly inflammatory due to the LNPs\u2019 ionizable lipid component. The inflammatory property is key to support the development of potent humoral immune responses. However, the mechanism by which this platform drives T follicular helper cells (Tfh) and humoral immune responses remains unknown. Here we show that lack of Langerhans cells or cDC1s neither significantly affected the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cells and humoral immune responses, nor susceptibility towards the lethal challenge of influenza and SARS-CoV-2. However, the combined deletion of these two DC subsets led to a significant decrease in the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cell and humoral immune responses. Despite these observed defects, the still high antibody titers were sufficient to confer protection towards lethal viral challenges. We further found that IL-6, but not neutrophils, was required to generate Tfh cells and antibody responses. In summary, here we bring evidence that the mRNA-LNP platform can support protective adaptive immune responses in the absence of specific DC subsets through an IL-6 dependent and neutrophil independent mechanism.","version":"1.1","doi":"10.1101/2021.08.01.454662","journal":"bioRxiv","score":null},{"id":"10.1101/2021.08.01.454686","pub_date":"2021-8-02","title":"MVsim: a toolset for quantifying and designing multivalent interactions","abstract":"Arising through multiple binding elements, multivalency can specify the avidity, duration, cooperativity, and selectivity of biomolecular interactions, but quantitative prediction and design of these properties has remained challenging. Here we present MVsim, an application suite built around a configurational network model of multivalency to facilitate the quantification, design, and mechanistic evaluation of multivalent binding phenomena through a simple graphical user interface. To demonstrate the utility and versatility of MVsim, we first show that both monospecific and multispecific multivalent ligand-receptor interactions, with their noncanonical binding kinetics, can be accurately simulated. We then quantitatively predict the ultrasensitivity and performance of multivalent-encoded protein logic gates, evaluate the inherent programmability of multispecificity for selective receptor targeting, and extract rate constants of conformational switching for the SARS-CoV-2 spike protein and model its binding to ACE2 as well as multivalent inhibitors of this interaction. MVsim is freely available at https://sarkarlab.github.io/MVsim/.","version":"1.1","doi":"10.1101/2021.08.01.454686","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454557","pub_date":"2021-8-01","title":"Evolutionary dynamics of indels in SARS-CoV-2 spike glycoprotein","abstract":"SARS-CoV-2, responsible for the current COVID-19 pandemic that claimed over 4.2 million lives, belongs to a class of enveloped viruses that undergo quick evolutionary adjustments under selection pressure. Numerous variants have emerged in SARS-CoV-2 that are currently posing a serious challenge to the global vaccination effort and COVID-19 management. The evolutionary dynamics of this virus are only beginning to be explored. In this work, we have analysed 1.79 million spike glycoprotein sequences of SARS-CoV-2 and found that the virus is fine-tuning the spike with numerous amino acid insertions and deletions (indels). Indels seem to have a selective advantage as the proportions of sequences with indels were steadily increasing over time, currently at over 89%, with similar trends across countries/variants. There were as many as 420 unique indel positions and 447 unique combinations of indels. Despite their high frequency, indels resulted in only minimal alteration, including both gain and loss, of N-glycosylation sites. As indels and point mutations are positively correlated and sequences with indels have significantly more point mutations, they have implications in the context of evolutionary dynamics of the SARS-CoV-2 spike glycoprotein.","version":"1.1","doi":"10.1101/2021.07.30.454557","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454402","pub_date":"2021-8-01","title":"Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants","abstract":"The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2021.07.30.454402","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454511","pub_date":"2021-8-01","title":"Comparable neutralization of SARS-CoV-2 Delta AY.1 and Delta in individuals sera vaccinated with BBV152","abstract":"The recent emergence of the SARS-CoV-2 Variant of Concern, B.1.617.2 (Delta) variant and its high transmissibility has led to the second wave in India. BBV152, a whole-virion inactivated SARS-CoV-2 vaccine used for mass immunization in India, showed a 65.2% protection against the Delta variant in a double-blind, randomized, multicentre, phase 3 clinical trial. Subsequently, Delta has been further mutated to Delta AY.1, AY.2, and AY.3. Of these, AY.1 variant was first detected in India in April 2021 and subsequently from twenty other countries as well. Here, we have evaluated the IgG antibody titer and neutralizing potential of sera of COVID-19 naive individual\u2019s full doses of BBV152 vaccine, COVID-19 recovered cases with full dose vaccines and breakthrough cases post-immunization BBV152 vaccines against Delta, Delta AY.1 and B.1.617.3. A reduction in neutralizing activity was observed with the COVID-19 naive individuals full vaccinated (1.3, 1.5, 1.9-fold), COVID-19 recovered cases with full BBV152 immunization (2.5, 3.5, 3.8-fold) and breakthrough cases post-immunization (1.9, 2.8, 3.5-fold) against Delta, Delta AY.1 and B.1.617.3 respectively compared to B.1 variant. A minor reduction was observed in the neutralizing antibody titer in COVID-19 recovered cases full BBV152 vaccinated and post immunized infected cases compared to COVID-19 naive vaccinated individuals. However, with the observed high titers, the sera of individuals belonging to all the aforementioned groups they would still neutralize the Delta, Delta AY.1 and B.1.617.3 variants effectively.","version":"1.1","doi":"10.1101/2021.07.30.454511","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454520","pub_date":"2021-8-01","title":"Spike protein multiorgan tropism suppressed by antibodies targeting SARS-CoV-2","abstract":"While there is clinical evidence of severe acute respiratory syndrome coronavirus 2 multiorgan tropism in severely infected coronavirus 19 patients, it\u2019s unclear if there is differential multiorgan biodistribution and organ uptake in healthy young individuals, a group that usually has asymptomatic to moderate coronavirus 19 symptoms. In addition, for antibody therapies and vaccines that target the spike protein, it\u2019s unclear if these reduce severe acute respiratory syndrome coronavirus 2 or spike protein multiorgan tropism equally. We used fluorescently labeled spike protein near infrared fluorescence to study viral behavior, using an in vivo dynamic imaging system, in young mice. We found a spike protein body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. Spike protein uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or cerebrospinal fluid. Thus, the brain vascular barriers were effective in restricting the entry of spike protein into brain parenchyma in young healthy mice. While both anti-angiotensin converting enzyme 2 and anti-spike protein antibodies suppressed spike protein biodistribution and organ uptake, anti-spike protein antibody was more effective. By extension, our data support the efficacy of these antibodies on severe acute respiratory syndrome coronavirus 2 biodistribution kinetics and multiorgan tropism that could determine coronavirus 19 organ-specific outcomes.","version":"1.1","doi":"10.1101/2021.07.30.454520","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.12.452071","pub_date":"2021-7-30","title":"TNF-\u03b1 levels in respiratory samples are associated with SARS-CoV-2 infection","abstract":"The aim of this study was to measure levels of IL-6 and TNF-\u03b1 in respiratory samples from individuals with symptoms compatible with COVID-19 and analyze their association with SARS-CoV-2 presence. SARS-CoV-2 detection was performed using the CDC (USA) real-time RT-PCR primers, probes and protocols. Cytokine concentrations were measured using commercial reagents based on enzyme linked immunosorbent assay (ELISA). TNF-\u03b1 median levels were greater in COVID19 (+) symptomatic group (5.88 (1.36 - 172.1) pg/ml) compared to COVID19 (\u2212) symptomatic individuals (2.87 (1.45 \u2013 69.9) pg/ml) (p=0.0003). No significant differences were shown in IL-6 median values between COVID-19 (+) and (\u2212) symptomatic patients (5.40 (1.7 - 467) pg/ml and 6.07 (1.57 \u2013 466.6) pg/ml respectively). In addition, increased TNF-\u03b1 levels (greater than 10 pg/ml), but not IL-6, were associated with SARS-CoV-2 presence (OR= 5.7; p=0.006; 95% CI= 1,551 to 19,11). We found a statistically significant association between the production of local TNF-\u03b1 and the presence of the virus in early stages of infection. IL-6 showed high levels in swabs from some symptomatic patients but independent from SARS-CoV-2 presence and viral load, individual\u2019s age and gender. On the contrary, TNF-\u03b1 evaluation confirmed the presence of inflammatory response but mostly related to COVID-19. More studies are required in order to characterize the cytokine profile expressed at the site of infection of SARS-CoV-2 and its implications in disease outcomes.","version":"1.2","doi":"10.1101/2021.07.12.452071","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437960","pub_date":"2021-7-30","title":"Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry","abstract":"Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases as well as coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.","version":"1.2","doi":"10.1101/2021.03.31.437960","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454437","pub_date":"2021-7-30","title":"Highly efficient SARS-CoV-2 infection of human cardiomyocytes: spike protein-mediated cell fusion and its inhibition","abstract":"Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to a bystander effect: the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) that beat spontaneously. These cardiomyocytes express the angiotensin I converting-enzyme 2 (ACE2) receptor and a subset of the proteases that mediate spike protein cleavage in the lungs, but not transmembrane protease serine 2 (TMPRSS2). Nevertheless, SARS-CoV-2 infection was productive: viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CM, smooth walled exocytic vesicles contained numerous 65-90 nm particles with typical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand the mechanisms of SARS-CoV-2 spread in hiPSC-CM we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm signal from cell to cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin abolished cell fusion. A spike mutant with the single amino acid change R682S that inactivates the furin cleavage site was fusion inactive. Thus, SARS-CoV-2 can replicate efficiently in hiPSC-CM and furin activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform provides an opportunity for target-based drug discovery in cardiac COVID-19. It is unclear whether the cardiac complications frequently observed in COVID-19 patients are due exclusively to systemic inflammation and thrombosis. Viral replication has occasionally been confirmed in cardiac tissue, but rigorous analyses are restricted to rare autopsy materials. Moreover, there are few animal models to study cardiovascular complications of coronavirus infections. To overcome these limitations, we developed an in vitro model of SARS-CoV-2 spread in induced pluripotent stem cell-derived cardiomyocytes. In these cells, infection is highly productive: viral transcription levels exceed those documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread we expressed a fluorescent version of its spike protein that allowed to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin cleavage site lost cytopathic function. The spike protein of the Middle East Respiratory Syndrome (MERS) coronavirus drove cardiomyocyte fusion with slow kinetics, whereas the spike proteins of SARS-CoV and the respiratory coronavirus 229E were inactive. These fusion activities correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 has the potential to cause cardiac damage by fusing cardiomyocytes.","version":"1.1","doi":"10.1101/2021.07.30.454437","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454436","pub_date":"2021-7-30","title":"Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity","abstract":"The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials. The pandemic caused by the SARS-CoV-2 virus has created a completely unusual situation in rapidly searching for compounds able to interfere with the biological processes exploited by the virus. This new scenario has substantially changed the timing of drug development which has also resulted in the generation of controversial results, proving that the transition from computational screening to the clinical application requires great caution and careful studies. It is therefore necessary to establish new paradigms for evaluating the efficacy of a potential active molecule. We set up a preclinical platform aimed at identifying molecules active against SARS-CoV-2 infection developing a multidisciplinary approach based on very stringent experimental criteria, comprising in-silico studies, in vitro binding tests and infection studies with pseudovirus expressing the spike protein as well as clinically isolated SARS-CoV-2 strains. We focused our attention on doxycycline which has been suggested as potential therapeutic candidate for treating COVID-19 and is currently employed in about twenty clinical trials. Doxycycline resulted effective in inhibiting the transduction of pseudovirus but it did not affect the entry and replication of SARS-CoV-2. The results obtained underline the need to define more stringent and controlled pharmacological approaches before wasting precious time and resources with clinical trials.","version":"1.1","doi":"10.1101/2021.07.30.454436","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.29.454323","pub_date":"2021-7-30","title":"SARS-CoV-2 introduction and lineage dynamics across three epidemic peaks in Southern Brazil: massive spread of P.1","abstract":"Genomic surveillance of SARS-CoV-2 is paramount for understanding viral dynamics, contributing to disease control. This study analyzed SARS-CoV-2 genomic diversity in Rio Grande do Sul (RS), Brazil, including the first case of each Regional Health Coordination and cases from three epidemic peaks. Ninety SARS-CoV-2 genomes from RS were sequenced and analyzed against SARS-CoV-2 datasets available in GISAID for phylogenetic inference and mutation analysis. SARS-CoV-2 lineages among the first cases in RS were B.1 (33.3%), B.1.1.28 (26.7%), B.1.1 (13.3%), B.1.1.33 (10.0%), and A (6.7%), evidencing SARS-CoV-2 introduction by both international origin and community-driven transmission. We found predominance of B.1.1.33 (50.0%) and B.1.1.28 (35.0%) during the first epidemic peak (July\u2013August, 2020), emergence of P.2 (55.6%) in the second peak (November\u2013December, 2020), and massive spread of P.1 and related sequences (78.4%), such as P.1-like-II, P.1.1 and P.1.2 in the third peak (February\u2013April, 2021). Eighteen novel mutation combinations were found among P.1 genomes, and 22 different spike mutations and/or deletions among P.1 and related sequences. This study shows the dispersion of SARS-CoV-2 lineages in Southern Brazil, and describes SARS-CoV-2 diversity during three epidemic peaks, highlighting the spread of P.1 and the high genetic diversity of currently circulating lineages. Genomic monitoring of SARS-CoV-2 is essential to guide health authorities\u2019 decisions to control COVID-19 in Brazil. Ninety SARS-CoV-2 genomes from Rio Grande do Sul, Brazil, were sequenced, including the first cases from 15 State Health Coordination regions and samples from three epidemic peaks. Phylogenomic inferences showed SARS-CoV-2 lineages spread, revealing its genomic diversity.","version":"1.1","doi":"10.1101/2021.07.29.454323","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454406","pub_date":"2021-7-30","title":"Identification and characterization of novel mutants of Nsp13 protein among Indian SARS-CoV-2 isolates","abstract":"SARS-CoV-2, the causative agent of COVID-19 has mutated rapidly which enabled them to adapt and evade the immune system of the host. Emerging SARS-CoV-2 variants with crucial mutations pose a global challenge in context of therapeutic drugs and vaccines being developed globally. There are currently no specific therapeutics or vaccines available to combat SARS-CoV-2 devastation. In view of this, the current study aimed to identify and characterize the mutations found in the Nsp13 of SARS-CoV-2 in Indian isolates. Non-structural protein, Nsp13 protein sequences from Indian isolates were analyzed by comparing with the first reported Severe acute respiratory syndrome Corona Virus-2 (SARS-CoV-2) protein sequence from Wuhan, China. Out of 825 Nsp13 protein sequences, a total of 38 mutations were observed among Indian isolates. Our data show that mutations in Nsp13 at various positions (H164Y, A237T, T214I, C309Y, S236I, P419S, V305E, G54S, H290Y, P53S, A308Y, and A308Y) have a significant impact on the protein\u2019s stability and flexibility. Also, the impact of Nsp13 mutations on the protein function were predicted based on PROVEAN score that includes 15 mutants as neutral and 23 mutants as deleterious effect. Furthermore, B-cell epitopes contributed by Nsp13 were identified using various predictive immunoinformatic tools. Immunological Parameters of Nsp13 such as antigenicity, allergenicity and toxicity were evaluated to predict the potential B-cell epitopes. The predicted peptide sequences were correlated with the observed mutants. Our predicted data showed that there are seven high rank linear epitopes as well as 18 discontinuous B-cell epitopes based on immunoinformatic tools. Moreover, it was observed that out of total 38 identified mutations among Indian SARS-CoV-2 Nsp13 protein, four mutant residues at position 142 (E142), 245 (H245), 247 (V247) and 419 (P419) are localised in the predicted B cell epitopic region. Altogether, the results of the present in-silico study might help to understand the impact of the identified mutations in Nsp13 protein on its stability, flexibility and function.","version":"1.1","doi":"10.1101/2021.07.30.454406","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.29.454261","pub_date":"2021-7-30","title":"COVID-ONE-humoral immune: The One-stop Database for COVID-19-specific Antibody Responses and Clinical Parameters","abstract":"Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (COVID-ONE humoral immune). COVID-ONE humoral immunity is based on a dataset that contains the IgG/IgM responses to 21 of 28 known SARS-CoV-2 proteins and 197 spike protein peptides against 2,360 COVID-19 samples collected from 783 patients. In addition, 96 clinical parameters for the 2,360 samples and information for the 783 patients are integrated into the database. Furthermore, COVID-ONE humoral immune provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the \u201cSTART\u201d button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-humoral immune is freely available at www.COVID-ONE.cn.","version":"1.1","doi":"10.1101/2021.07.29.454261","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.30.454063","pub_date":"2021-7-30","title":"A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields","abstract":"The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.","version":"1.1","doi":"10.1101/2021.07.30.454063","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.29.454385","pub_date":"2021-7-30","title":"3D printed cobalt-chromium-molybdenum porous superalloy with superior antiviral activity","abstract":"COVID-19 pandemic and associated supply-chain disruptions emphasise the requirement for antimicrobial materials for on-demand manufacturing. Besides aerosol transmission, SARS-CoV-2 is also propagated through contact with virus-contaminated surfaces. As such, the development of effective biofunctional materials that can inactivate SARS-CoV-2 are critical for pandemic preparedness. Such materials will enable the rational development of antiviral devices with prolonged serviceability reducing the environmental burden of disposable alternatives. This research reveals the novel use of Laser Powder Bed Fusion (LPBF) to 3D print porous Cobalt-Chromium-Molybdenum (Co-Cr-Mo) superalloy with potent antiviral activity (100% viral inactivation in 30 mins). The porous material was rationally conceived using a multi-objective surrogate model featuring track thickness (tt) and pore diameter (\u03d5d) as responses. The regression analysis found the most significant parameters for Co-Cr-Mo track formation to be the interaction effects of scanning rate (Vs) and laser power (Pl) in the order PlVs > Vs > Pl. Contrastively, the pore diameter was found to be primarily driven by the hatch spacing (Sh). The study is the first to demonstrate the superior antiviral properties of 3D printed Co-Cr-Mo superalloy against an enveloped virus used as biosafe viral model of SARS-CoV-2. The material significantly outperforms the viral inactivation time of other broadly used antiviral metals such as copper and silver from 5 hours to 30 minutes. As such the study goes beyond the current state-of-the-art in antiviral alloys to provide extra-protection to combat the SARS-COV-2 viral spread. The evolving nature of the COVID-19 pandemic brings new and unpredictable challenges where on-demand 3D printing of antiviral materials can achieve rapid solutions while reducing the environmental impact of disposable devices.","version":"1.1","doi":"10.1101/2021.07.29.454385","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.29.454404","pub_date":"2021-7-30","title":"Photosensitized Electrospun Nanofibrous Filters for Capturing and Killing Airborne Coronaviruses under Visible Light Irradiation","abstract":"To address the challenge of the airborne transmission of SARS-CoV-2, photosensitized electrospun nanofibrous membranes were fabricated to effectively capture and inactivate coronavirus aerosols. With an ultrafine fiber diameter (\u223c 200 nm) and a small pore size (\u223c 1.5 \u00b5m), the optimized membranes caught 99.2% of the aerosols of the murine hepatitis virus A59 (MHV-A59), a coronavirus surrogate for SARS-CoV-2. In addition, rose bengal was used as the photosensitizer for the membranes because of its excellent reactivity in generating virucidal singlet oxygen, and the membranes rapidly inactivated 98.9% of MHV-A59 in virus-laden droplets only after 15 min irradiation of simulated reading light. Singlet oxygen damaged the virus genome and impaired virus binding to host cells, which elucidated the mechanism of disinfection at a molecular level. Membrane robustness was also evaluated, and no efficiency reduction for filtering MHV-A59 aerosols was observed after the membranes being exposed to both indoor light and sunlight for days. Nevertheless, sunlight exposure photobleached the membranes, reduced singlet oxygen production, and compromised the performance of disinfecting MHV-A59 in droplets. In contrast, the membranes after simulated indoor light exposure maintained their excellent disinfection performance. In summary, photosensitized electrospun nanofibrous membranes have been developed to capture and kill airborne environmental pathogens under ambient conditions, and they hold promise for broad applications as personal protective equipment and indoor air filters. Photosensitized electrospun nanofibrous filters with excellent capture-and-kill performance against coronaviruses were designed and implemented to prevent the airborne transmission of COVID-19.","version":"1.1","doi":"10.1101/2021.07.29.454404","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.28.454232","pub_date":"2021-7-29","title":"Structural basis for recognition of two HLA-A2-restricted SARS-CoV-2 spike epitopes by public and private T cell receptors","abstract":"T cells play a vital role in combatting SARS-CoV-2 and in forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T cell receptors (TCRs) bound to their peptide\u2013MHC targets is lacking. We determined structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures revealed the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of both TCRs to recognize natural variants of YLQ and RLQ but not homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.","version":"1.1","doi":"10.1101/2021.07.28.454232","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.29.454326","pub_date":"2021-7-29","title":"SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus)","abstract":"Widespread human SARS-CoV-2 infections combined with human-wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted white-tailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have ACE2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, and can be abundant near urban centers. We evaluated 624 pre- and post-pandemic serum samples from wild deer from four U.S. states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples was tested using a SARS-CoV-2 virus neutralization test with high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2. Antibodies to SARS-CoV-2 were detected in 40% of wild white-tailed deer sampled from four U.S. states in 2021.","version":"1.1","doi":"10.1101/2021.07.29.454326","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.12.443888","pub_date":"2021-7-29","title":"mRNA vaccine-induced T cells respond identically to SARS-CoV-2 variants of concern but differ in longevity and homing properties depending on prior infection status","abstract":"While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-na\u00efve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-na\u00efve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-na\u00efve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-na\u00efve counterparts.","version":"1.2","doi":"10.1101/2021.05.12.443888","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.28.453981","pub_date":"2021-7-28","title":"Single-Cell Profiling of the Antigen-Specific Response to BNT162b2 SARS-CoV-2 RNA Vaccine","abstract":"RNA-based vaccines against SARS-CoV-2 are critical to limiting COVID-19 severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. We used single-cell technologies to identify and characterized antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 in longitudinal samples from a cohort of healthy donors. Mass cytometry and machine learning pinpointed a novel expanding, population of antigen-specific non-canonical memory CD4+ and CD8+ T cells. B cell sequencing suggested progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlated with eventual SARS-CoV-2 IgG and a donor lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms reveal an antigen-specific cellular basis of RNA vaccine-based immunity. Single-cell profiling reveals the cellular basis of the antigen-specific response to the BNT162b2 SARS-CoV-2 RNA vaccine.","version":"1.1","doi":"10.1101/2021.07.28.453981","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.28.454072","pub_date":"2021-7-28","title":"BRET-based self-cleaving biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery","abstract":"The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments and more inhibitors are being developed. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors have to be evaluated in a Biosafety Level 3 (BSL-3) or BSL-4 laboratory. Here, we design genetically encoded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After over-expression of the biosensors in HEK293 cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their anti-SARS-CoV-2 activities reported previously. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. Furthermore, our strategy can be used to design biosensors for other viral proteases that share the same activation mechanism as 3CLpro, such as HIV protease PR and HCV protease NS3. Sensitive cell-based biosensors for 3CLpro inhibitor discovery in BSL-2 laboratories. The BRET-based self-cleaving biosensors mimic the in vivo autoproteolytic activation of 3CLpro. Similar biosensors can be designed for other self-cleaving proteases, such as HIV protease PR and HCV protease NS3.","version":"1.1","doi":"10.1101/2021.07.28.454072","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.28.454098","pub_date":"2021-7-28","title":"SARS-CoV-2 in the Republic of Guinea: Fragment and Whole-Genome Sequencing, Phylogenetic Analysis","abstract":"Genetic diversity of SARS-CoV-2 isolates circulating in the Republic of Guinea in May and June 2020, as well as in March 2021, has been demonstrated using fragment (S gene) and whole genome sequencing of 14 strains. Analysis of nucleotide sequences and phylogenetic constructs make it possible to divide the studied strains into 3 groups. Comparison of the obtained data with the already available epidemiological data proves the initial importation of COVID-19 from Western European countries, and also demonstrates four independent import routes in two time periods (March 2020 and no later than March 2021).","version":"1.1","doi":"10.1101/2021.07.28.454098","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.17.452787","pub_date":"2021-7-28","title":"Unveiling Mutation Effects on the Structural Dynamics of the Main Protease from SARS-CoV-2 with Hybrid Simulation Methods","abstract":"The main protease of SARS-CoV-2 (called Mpro or 3CLpro) is essential for processing polyproteins encoded by viral RNA. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. Integrated methods combining the lowest-frequency movements obtained by Normal Mode Analysis (NMA), and the faster movements from Molecular Dynamics (MD), and data from biophysical techniques, are necessary to establish the correlation between complex structural dynamics of macromolecules and their function. In this article, we used a hybrid simulation method to sample the conformational space to characterize the structural dynamics and global motions of WT SARS-CoV-2 Mpro and 48 mutants, including several mutations that appear in P.1, B.1.1.7, B.1.351, B.1.525 and B.1.429+B.1.427 variants. Integrated Hybrid methods combining NMA and MD have been useful to study the correlation between the complex structural dynamics of macromolecules and their functioning mechanisms. Here, we applied this hybrid approach to elucidate the effects of mutation in the structural dynamics of SARS-CoV-2 Mpro, considering their flexibility, solvent accessible surface area analyses, global movements, and catalytic dyad distance. Furthermore, some mutants showed significant changes in their structural dynamics and conformation, which could lead to distinct functional properties. Single surface mutations lead to changes in Mpro structural dynamics. Mutants can be more stable than WT according to the structural dynamics properties. Mpromutants can present a distinct functionality in relation to the wild-type. Potential viral markers for more pathogenic or transmissible SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.07.17.452787","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.28.454085","pub_date":"2021-7-28","title":"SARS-CoV-2 Lambda variant exhibits higher infectivity and immune resistance","abstract":"SARS-CoV-2 Lambda, a new variant of interest, is now spreading in some South American countries; however, its virological features and evolutionary trait remain unknown. Here we reveal that the spike protein of the Lambda variant is more infectious and it is attributed to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino-acid deletion mutation in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the insertion of the RSYLTPGD246-253N mutation is closely associated with the massive infection spread of the Lambda variant in South America. Lambda S is highly infectious and T76I and L452Q are responsible for this property Lambda S is more susceptible to an infection-enhancing antibody RSYLTPGD246-253N, L452Q and F490S confer resistance to antiviral immunity","version":"1.1","doi":"10.1101/2021.07.28.454085","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.28.453844","pub_date":"2021-7-28","title":"The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics","abstract":"There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.","version":"1.1","doi":"10.1101/2021.07.28.453844","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.449251","pub_date":"2021-7-27","title":"Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase","abstract":"The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits. Here we show that water-soluble derivatives of \u03b1-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-\u03b1-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and \u03b2-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of \u03b1-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp. In summary, we conclude that solubilizing modifications to \u03b1-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2021.07.13.449251","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.09.417519","pub_date":"2021-7-27","title":"Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes","abstract":"During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying Variants Of Concern (VOC). Besides, viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host Single Nucleotide Variations (iSNVs). Analysing 1, 347 samples collected till June 2020, we recorded 18, 146 iSNV sites throughout the SARS-CoV-2 genome. Both, mutations in RdRp as well as APOBEC and ADAR mediated RNA editing seem to contribute to the differential prevalence of iSNVs in hosts. Noteworthy, 41% of all unique iSNVs were reported as SNVs by 30th September 2020 in samples submitted to GISAID, which increased to \u223c80% by 30th June 2021. Following this, analysis of another set of 1, 798 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) variations appeared as iSNVs before getting fixed in the population. We also observe hyper-editing events at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.","version":"1.3","doi":"10.1101/2020.12.09.417519","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.27.453834","pub_date":"2021-7-27","title":"Site-specific recognition of SARS-CoV-2 nsp1 protein with a tailored titanium dioxide nanoparticle","abstract":"The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In recent years nanoparticles have been shown to interact with peptides, ranging in size from single amino acids up to proteins. These nanoparticles can be tailor-made with specific functions and properties including bioavailability. To the best of our knowledge, in this study we show for the first time that a tailored titanium oxide nanoparticle interacts specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein. This can be developed potentially into a tool for selective control of viral protein functions.","version":"1.1","doi":"10.1101/2021.07.27.453834","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.27.453973","pub_date":"2021-7-27","title":"Executable Network of SARS-CoV-2-Host Interaction Predicts Drug Combination Treatments","abstract":"The COVID-19 pandemic has pushed healthcare systems globally to a breaking point. The urgent need for effective and affordable COVID-19 treatments calls for repurposing combinations of approved drugs. The challenge is to identify which combinations are likely to be most effective and at what stages of the disease. Here, we present the first disease-stage executable signalling network model of SARS-CoV-2-host interactions used to predict effective repurposed drug combinations for treating early- and late-stage severe disease. Using our executable model, we performed in silico screening of 9870 pairs of 140 potential targets and have identified 12 new drug combinations. Camostat and Apilimod were predicted to be the most promising combination in effectively supressing viral replication in the early stages of severe disease and were validated experimentally in human Caco-2 cells. Our study further demonstrates the power of executable mechanistic modelling to enable rapid pre-clinical evaluation of combination therapies tailored to disease progression. It also presents a novel resource and expandable model system that can respond to further needs in the pandemic.","version":"1.1","doi":"10.1101/2021.07.27.453973","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.27.453843","pub_date":"2021-7-27","title":"Molecular Pathophysiology of Cardiac Injury and Cardiac Microthrombi in Fatal COVID-19: Insights from Clinico-histopathologic and Single Nuclei RNA Sequencing Analyses","abstract":"Cardiac injury is associated with critical COVID-19, yet its etiology remains debated. To elucidate the pathogenic mechanisms of COVID-19-associated cardiac injury, we conducted a single-center prospective cohort study of 69 COVID-19 decedents. Of six cardiac histopathologic features, microthrombi was the most commonly detected (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak ESR and CRP during hospitalization were independently associated with higher odds of microthrombi. Using single nuclei RNA-sequence analysis, we discovered an enrichment of pro-thrombotic/anti-fibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling amongst cardiac fibroblasts in microthrombi-positive COVID-19 hearts relative to microthrombi-negative COVID-19. Non-COVID-19 non-failing hearts were used as reference controls. Our cumulative findings identify the specific transcriptomic changes in cardiac fibroblasts as salient features of COVID-19-associated cardiac microthrombi.","version":"1.1","doi":"10.1101/2021.07.27.453843","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449905","pub_date":"2021-7-26","title":"Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) Up to 15 Months Post-Infection","abstract":"The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. Non-classical monocytes are capable of causing inflammation throughout the body in response to fractalkine/CX3CL1 and RANTES/CCR5.","version":"1.3","doi":"10.1101/2021.06.25.449905","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453598","pub_date":"2021-7-26","title":"Differential Interactions Between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. It is known that the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 interacts with the human angiotensin-converting enzyme 2 (ACE2) receptor, initiating the entry of SARS-CoV-2. Since its emergence, a number of SARS-CoV-2 variants have been reported, and the variants that show high infectivity are classified as the variants of concern according to the US CDC. In this study, we performed both all-atom steered molecular dynamics (SMD) simulations and microscale thermophoresis (MST) experiments to characterize the binding interactions between ACE2 and RBD of all current variants of concern (Alpha, Beta, Gamma, and Delta) and two variants of interest (Epsilon and Kappa). We report that the RBD of the Alpha (N501Y) variant requires the highest amount of force initially to be detached from ACE2 due to the N501Y mutation in addition to the role of N90-glycan, followed by Beta/Gamma (K417N/T, E484K, and N501Y) or Delta (L452R and T478K) variant. Among all variants investigated in this work, the RBD of the Epsilon (L452R) variant is relatively easily detached from ACE2. Our results combined SMD simulations and MST experiments indicate what makes each variant more contagious in terms of RBD and ACE2 interactions. This study could help develop new drugs to inhibit SARS-CoV-2 entry effectively.","version":"1.1","doi":"10.1101/2021.07.23.453598","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453755","pub_date":"2021-7-26","title":"Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects Macaques from infection","abstract":"The SARS-CoV-2 pandemic causes an ongoing global health crisis, which requires efficient and safe vaccination programs. Here, we present synthetic SARS-CoV2 S glycoprotein-coated liposomes that resemble in size and surface structure virus-like particles. Soluble S glycoprotein trimers were stabilized by formaldehyde cross-linking and coated onto lipid vesicles (S-VLP). Immunization of cynomolgus macaques with S-VLPs induced high antibody titers and TH1 CD4+ biased T cell responses. Although antibody responses were initially dominated by RBD specificity, the third immunization boosted non-RBD antibody titers. Antibodies showed potent neutralization against the vaccine strain and the Alpha variant after two immunizations and robust neutralization of Beta and Gamma strains. Challenge of animals with SARS-CoV-2 protected all vaccinated animals by sterilizing immunity. Thus, the S-VLP approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.","version":"1.1","doi":"10.1101/2021.07.26.453755","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.22.453029","pub_date":"2021-7-26","title":"CD8+ T cell signature in acute SARS-CoV-2 infection identifies memory precursors","abstract":"Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen. Since the outbreak of the ongoing coronavirus disease 19 (COVID-19) pandemic, a key question has focused on whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells stimulated during acute infection give rise to long-lived memory T cells. Using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor (TCR) sequencing we longitudinally characterize individual SARS-CoV-2-specific CD8+ T cells of COVID-19 patients from acute infection to one year into recovery and find a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting one year after acute infection re-express CD45RA and interleukin-7 receptor \u03b1 (CD127), upregulate T cell factor-1 (TCF1), and maintain low CCR7, thus resembling CD45RA+ effector-memory T (TEMRA) cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones giving rise to long-lived cells, whereas prolonged proliferation and mammalian target of rapamycin (mTOR) signaling are associated with clone contraction and disappearance. Collectively, we identify a transcriptional signature differentiating short-from long-lived memory CD8+ T cells following an acute virus infection in humans.","version":"1.2","doi":"10.1101/2021.07.22.453029","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.06.451270","pub_date":"2021-7-26","title":"Genome Profiling of SARS-CoV-2 in Indonesia, ASEAN, and the Neighbouring East Asian Countries: Features, Challenges, and Achievements","abstract":"A year after the World Health Organisation (WHO) declared COVID-19 as a pandemic, much has been learned regarding SARS-CoV-2 epidemiology, vaccine production, and disease treatment. Whole-genome sequencing (WGS) has played a significant role in contributing to our understanding of the epidemiology and biology of this virus. In this paper, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asia and the impact of technological development, access to resources, and demography of individual countries on its uptake. Using Oxford Nanopore Technology (ONT), Nottingham-Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in Research Centre for Biotechnology, Indonesian Institute of Sciences (Lembaga Ilmu Pengetahuan Indonesia/LIPI) to carry out a small number of SARS-CoV-2 WGS in Indonesia. The ONT offers sequencing advantages that fit within the Indonesian context. Analyses of SARS-CoV-2 genomes deposited on GISAID from Southeast and East Asian countries reveal the importance of collecting clinical and demographic metadata and the importance of open access and data sharing. Lineage and phylogenetic analyses per 1 June 2021 found that: 1) B.1.466.2 variants were the most predominant in Indonesia, with mutations in the spike protein including D614G at 100%, N439K at 99.1%, and P681R at 69.7% frequency, 2) The variants of concern (VoCs) B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) were first detected in Indonesia in January 2021, 3) B.1.470 was first detected in Indonesia and spread to the neighbouring regions, and 4) The highest rate of virus transmissions between Indonesia and the rest of the world appears to be through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.","version":"1.3","doi":"10.1101/2021.07.06.451270","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453787","pub_date":"2021-7-26","title":"N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry","abstract":"N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.","version":"1.1","doi":"10.1101/2021.07.26.453787","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453327","pub_date":"2021-7-26","title":"Mutations in two SARS-CoV-2 variants of concern reflect two distinct strategies of antibody escape","abstract":"Understanding the factors that contribute to antibody escape of SARS-CoV-2 and its variants is key for the development of drugs and vaccines that provide broad protection against a variety of virus variants. Using microfluidic diffusional sizing, we determined the dissociation constant (KD) for the interaction between receptor binding domains (RBDs) of SARS-CoV-2 in its original version (WT) as well as alpha and beta variants with the host-cell receptor angiotensin converting enzyme 2 (ACE2). For RBD-alpha, the ACE2-binding affinity was increased by a factor of ten when compared with RBD-WT, while ACE2-binding of RBD-beta was largely unaffected. However, when challenged with a neutralizing antibody that binds to both RBD-WT and RBD-alpha with low nanomolar KD values, RBD-beta displayed no binding, suggesting a substantial epitope change. In SARS-CoV-2 convalescent sera, RBD-binding antibodies showed low nanomolar affinities to both wild-type and variant RBD proteins\u2014strikingly, the concentration of antibodies binding to RBD-beta was half that of RBD-WT and RBD-alpha, again indicating considerable epitope changes in the beta variant. Our data therefore suggests that one factor contributing to the higher transmissibility and antibody evasion of SARS-CoV-2 alpha and beta is a larger fraction of viruses that can form a complex with ACE2. However, the two variants employ different mechanisms to achieve this goal. While SARS-CoV-2 alpha RBD binds with greater affinity to ACE2 and is thus more difficult to displace from the receptor by neutralizing antibodies, RBD-beta is less accessible to antibodies due to epitope changes which increases the chances of ACE2-binding and infection.","version":"1.1","doi":"10.1101/2021.07.23.453327","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.25.453717","pub_date":"2021-7-26","title":"ETAS\u00ae50 Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1\u03b2 Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages","abstract":"Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. ETAS\u00ae50, a standardized extract of Asparagus officinalis stem, is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide- and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of ETAS\u00ae50 on pro-inflammatory responses in S1-stimulated murine peritoneal exudate macrophages. Co-treatment of the cells with ETAS\u00ae50 significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. ETAS\u00ae50 also markedly suppressed the S1-induced transcription of IL-6 and IL-1\u03b2. However, among the TLR4 signaling proteins, ETAS\u00ae50 did not affect the degradation of inhibitor \u03baB\u03b1, nuclear translocation of nuclear factor-\u03baB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, ETAS\u00ae50 significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1\u03b2 by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that ETAS\u00ae50 attenuates S1-induced IL-6 and IL-1\u03b2 production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, ETAS\u00ae50 may be beneficial in regulating excessive inflammation in patients with COVID-19.","version":"1.1","doi":"10.1101/2021.07.25.453717","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453805","pub_date":"2021-7-26","title":"CIGB-300 synthetic peptide, an antagonist of CK2 kinase activity, as a treatment for Covid-19. A computational biology approach","abstract":"Drug repositioning became the first choice for treating Covid-19 patients due to the urgent need to deal with the pandemic. Similarities in the hijacking mechanisms used by SARS-CoV-2 and several type of cancer, suggest the repurposing of cancer drugs to treat Covid-19. CK2 kinase antagonists have been proposed for the treatment of cancer. A recent study in cells infected with SARS-CoV-2 virus found a significant CK2 kinase activity, and the use of a CK2 inhibitor showed antiviral responses. CIGB-300, originally designed as an anticancer peptide, is an antagonist of CK2 kinase activity that binds to CK2 phospho-acceptor sites. Recent preliminary results show an antiviral activity of CIGB-300 versus a surrogate model of coronavirus. Here we present a computational biology study that provides evidences at the molecular level of how CIGB-300 might interfere with SARS-CoV-2 life cycle inside infected human cells. First, from SARS-CoV studies, we infer the potential incidence of CIGB-300 in SARS-CoV-2 interference on immune response. Next, from the analysis of multiple Omics data, we propose the action of CIGB-300 since early stage of viral infections perturbing the virus hijacking of RNA splicing machinery. It was also predicted the interference of CIGB-300 in virus-host interactions responsible for the high infectivity and the particular immune response to SARS-CoV-2 infection. Further, we provide evidences of CIGB-300 attenuation of phenotypes related to muscle, bleeding, coagulation and respiratory disorders.","version":"1.1","doi":"10.1101/2021.07.26.453805","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.25.453673","pub_date":"2021-7-26","title":"Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies","abstract":"Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 \u03bcg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.","version":"1.1","doi":"10.1101/2021.07.25.453673","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453840","pub_date":"2021-7-26","title":"Protection of human ACE2 transgenic Syrian hamsters from SARS CoV-2 variants by human polyclonal IgG from hyper-immunized transchromosomic bovines","abstract":"Pandemic SARS CoV-2 has been undergoing rapid evolution during spread throughout the world resulting in the emergence of many Spike protein variants, some of which appear to either evade antibody neutralization, transmit more efficiently, or potentially exhibit increased virulence. This raises significant concerns regarding the long-term efficacy of protection elicited after primary infection and/or from vaccines derived from single virus Spike (S) genotypes, as well as the efficacy of anti-S monoclonal antibody based therapeutics. Here, we used fully human polyclonal human IgG (SAB-185), derived from hyperimmunization of transchromosomic bovines with DNA plasmids encoding the SARS-CoV-2 Wa-1 strain S protein or purified ectodomain of S protein, to examine the neutralizing capacity of SAB-185 in vitro and the protective efficacy of passive SAB-185 antibody (Ab) transfer in vivo. The Ab preparation was tested for neutralization against five variant SARS-CoV-2 strains: Munich (Spike D614G), UK (B.1.1.7), Brazil (P.1) and SA (B.1.3.5) variants, and a variant isolated from a chronically infected immunocompromised patient (Spike \u0394144-146). For the in vivo studies, we used a new human ACE2 (hACE2) transgenic Syrian hamster model that exhibits lethality after SARS-Cov-2 challenge and the Munich, UK, SA and \u0394144-146 variants. SAB-185 neutralized each of the SARS-CoV-2 strains equivalently on Vero E6 cells, however, a control convalescent human serum sample was less effective at neutralizing the SA variant. In the hamster model, prophylactic SAB-185 treatment protected the hamsters from fatal disease and minimized clinical signs of infection. These results suggest that SAB-185 may be an effective treatment for patients infected with SARS CoV-2 variants.","version":"1.1","doi":"10.1101/2021.07.26.453840","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.26.453518","pub_date":"2021-7-26","title":"Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS-CoV-2","abstract":"Airborne transmission, a term combining both large droplet and aerosol transmission, is thought to be the main transmission route of SARS-CoV-2. Here we investigated the relative efficiency of aerosol transmission of two variants of SARS-CoV-2, B.1.1.7 (alpha) and lineage A, in the Syrian hamster. A novel transmission caging setup was designed and validated, which allowed the assessment of transmission efficiency at various distances. At 2 meters distance, only particles <5 \u00b5m traversed between cages. In this setup, aerosol transmission was confirmed in 8 out of 8 (N = 4 for each variant) sentinels after 24 hours of exposure as demonstrated by respiratory shedding and seroconversion. Successful transmission occurred even when exposure time was limited to one hour, highlighting the efficiency of this transmission route. Interestingly, the B.1.1.7 variant outcompeted the lineage A variant in an airborne transmission chain after mixed infection of donors. Combined, this data indicates that the infectious dose of B.1.1.7 required for successful transmission may be lower than that of lineage A virus. The experimental proof for true aerosol transmission and the increase in the aerosol transmission potential of B.1.1.7 underscore the continuous need for assessment of novel variants and the development or preemptive transmission mitigation strategies.","version":"1.1","doi":"10.1101/2021.07.26.453518","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.22.21261000","pub_date":"2021-07-25","title":"Impact of SARS-CoV-2 variant on the severity of maternal infection and perinatal outcomes: Data from the UK Obstetric Surveillance System national cohort","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>In the UK, the Alpha variant of SARS-CoV-2 became dominant in late 2020, rapidly succeeded by the Delta variant in May 2021. The aim of this study was to compare the impact of these variants on severity of maternal infection and perinatal outcomes within the time-periods in which they predominated.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This national, prospective cohort study collated data on hospitalised pregnant women with symptoms of confirmed SARS-CoV-2 infection and compared the severity of infection and perinatal outcomes across the Wildtype (01/03/20-30/11/20), Alpha (01/12/20-15/05/21) and Delta dominant periods (16/05/21-11/07/21), using multivariable logistic regression.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Of 3371 pregnant women, the proportion that experienced moderate to severe infection significantly increased between Wildtype and Alpha periods (24.4% vs. 35.8%; aOR1.75 95%CI 1.48-2.06), and between Alpha and Delta periods (35.8% vs. 45.0%; aOR1.53, 95%CI 1.07-2.17). Compared to the Wildtype period, symptomatic women admitted in the Alpha period were more likely to require respiratory support (27.2% vs. 20.3%, aOR1.39, 95%CI 1.13-1.78), have pneumonia (27.5% vs. 19.1%, aOR1.65, 95%CI 1.38-1.98) and be admitted to intensive care (11.3% vs. 7.7%, aOR1.61, 95%CI 1.24-2.10). Women admitted during the Delta period had further increased risk of pneumonia (36.8% vs. 27.5%, aOR1.64 95%CI 1.14-2.35). No fully vaccinated pregnant women were admitted between 01/02/2021 when vaccination data collection commenced and 11/07/2021. The proportion of women receiving pharmacological therapies for SARS-CoV-2 management was low, even in those critically ill.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>\n                    SARS-CoV-2 infection during Alpha and Delta dominant periods was associated with more severe infection and worse pregnancy outcomes compared to the Wildtype infection, which itself increased risk compared to women without SARS-CoV-2 infection.\n                    <jats:sup>1</jats:sup>\n                    Clinicians need to be aware and implement COVID-specific therapies in keeping with national guidance. Urgent action to tackle vaccine misinformation and policy change to prioritise uptake in pregnancy is essential.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>National Institute for Health Research HS&amp;DR Programme (11/46/12).</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.07.22.21261000","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.24.21261007","pub_date":"2021-07-25","title":"Indomethacin Use for Mild &amp; Moderate hospitalised Covid-19 patients: An open label randomized clinical trial","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been presented as a broad-spectrum antiviral agent. This randomised clinical trial in a hospital setting evaluated the efficacy and safety of this drug in RT-PCR-positive coronavirus disease 2019 (COVID-19) patients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Materials &amp; Methods</jats:title>\n                  <jats:p>\n                    A total of 210 RT-PCR-positive COVID-19 patients, who provided consent were allotted, to control or case arm, based on block randomisation. The control arm received standard of care comprising paracetamol, ivermectin, and other adjuvant therapies. The patients in the case arm received indomethacin instead of paracetamol, with other medications retained. The primary endpoint was the development of hypoxia/desaturation with SpO\n                    <jats:sub>2</jats:sub>\n                    \u2264 93, while time to become afebrile and time for cough and myalgia resolution were the secondary endpoints.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The results of 210 patients were available, with 103 and 107 patients in the indomethacin and paracetamol arms, respectively. We monitored patient profiles along with everyday clinical parameters. Blood chemistry at the time of admission and discharge was assessed.</jats:p>\n                  <jats:p>\n                    As no one in either of the arms required high-flow oxygen, desaturation with SpO\n                    <jats:sub>2</jats:sub>\n                    level of 93 and below was an important goal. In the indomethacin group, none of the 103 patients developed desaturation. On the other hand, 20 of the 107 patients in the paracetamol arm developed desaturation. Patients who received indomethacin also experienced more rapid symptomatic relief than those in the paracetamol arm, with most symptoms disappearing in half the time. 56 patients out of 107 in the paracetamol arm had fever on the seventh day, while no patient in the indomethacin group had fever. Neither arm reported any adverse event. The fourteenth-day follow-up revealed that the paracetamol arm patients had faced several discomforts, including myalgia, joint pain, and tiredness; indomethacin arm patients mostly complained only of tiredness.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Indomethacin is a safe and effective drug for treating patients with mild and moderate covid-19.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.07.24.21261007","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.22.21259587","pub_date":"2021-07-24","title":"High-throughput Mutational Surveillance of the SARS-CoV-2 Spike Gene","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>SARS-CoV-2 has evolved rapidly towards higher infectivity and partial immune escape over the course of the pandemic. This evolution is driven by the enormous virus population, that has infected close to 200 million people by now. Therefore, cost effective and scalable methods are needed to monitor viral evolution globally. Mutation-specific PCR approaches have become inadequate to distinguish the variety of circulating SARS-CoV-2 variants and are unable to detect novel ones. Conversely, whole genome sequencing protocols remain too labor- and cost-intensive to monitor SARS-CoV-2 at the required density. By adapting SARSeq we present a simple, fast, and scalable S-gene tiling pipeline for focused sequencing of the S-gene encoding for the spike protein. This method reports on all sequence positions with known importance for infectivity and immunity, yet scales to &gt;20K samples per run. S-gene tiling is used for nationwide surveillance of SARS-CoV-2 at a density of 10% to 50% of all cases of infection in Austria. SARSeq S-tiling uncovered several infection clusters with variants of concern such as the biggest known cluster of Beta/B.1.351 outside Africa and successfully informed public health measures in a timely manner, allowing their successful implementation. Our close monitoring of mutations further highlighted evolutionary constraints and freedom of the spike protein ectodomain and sheds light on foreseeable evolutionary trajectories.</jats:p>","version":null,"doi":"10.1101/2021.07.22.21259587","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.20.21260813","pub_date":"2021-07-24","title":"Association of lipid-lowering drugs with COVID-19 outcomes: A Mendelian Randomization study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Lipid metabolism plays an important role in viral infections. Large cohort study suggested a protective potential of lipid-lowering drugs in COVID-19 outcomes, but the nature of observational study precludes it to draw a causal inference.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>To assess the causal effect of lipid-lowering drugs (HMGCR inhibitors, PCSK9 inhibitors and NPC1L1 inhibitors) on COVID-19 outcomes using 2-sample Mendelian Randomization (MR) study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We used two kinds of genetic instruments to proxy the exposure of lipid-lowering drugs, including expression quantitative trait loci (eQTLs) of drugs target genes, and genetic variants within or nearby drugs target genes associated with low-density lipoprotein (LDL) cholesterol from genome-wide association study (GWAS). GWASs of COVID-19 outcomes (susceptibility, hospitalization and very severe disease) were obtained from the COVID-19 Host Genetics Initiative. Summary-data-based MR (SMR) and inverse-variance weighted MR (IVW-MR) were used to calculate the effect estimates.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>SMR analysis found that a higher expression of HMGCR was associated with a higher risk of COVID-19 hospitalization (OR=1.38, 95%CI=1.06-1.81; P=0.019). Similarly, IVW-MR analysis observed a positive association between HMGCR-mediated LDL cholesterol and COVID-19 hospitalization (OR=1.32, 95%CI=1.00-1.74; P=0.049). No consistent evidence from both SMR and IVW-MR analyses was found for the association of HMGCR inhibitors with COVID-19 susceptibility or very severe disease, or for the association of PCSK9 inhibitors and NPC1L1 inhibitor with COVID-19 outcomes.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>In this 2-sample MR study, we found potential causal evidence that HMGCR inhibitors could reduce the risk of COVID-19 hospitalization. Further research is needed to explore the therapeutic role of statins for COVID-19.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.07.20.21260813","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.22.453309","pub_date":"2021-7-23","title":"A Highly Potent SARS-CoV-2 Blocking Lectin Protein","abstract":"COVID-19 pandemic effected more than 180 million people around the globe causing more than four million deaths as of July 2021. Sars-CoV-2, the new coronavirus, has been identified as the primary cause of the infection. The number of vaccinated people is increasing however prophylactic drugs are highly demanded to ensure a secure social contact. There have been a number of drug molecules repurposed to fight against Sars-CoV-2, however the proofs for the effectiveness of these drug candidates is limited. Here we demonstrated griffithsin (GRFT), a lectin protein, to block the entry of the Sars-CoV2 into the Vero6 cell lines and IFNAR-/-mouse models by attaching to spike protein of the Sars-CoV-2. Given the current mutation frequency of the Sars-CoV-2 we believe that GRFT protein-based drugs will have a high impact in preventing the transmission both on Wuhan strain as well as any other emerging variants including delta variant causing high speed spread of COVID-19.","version":"1.1","doi":"10.1101/2021.07.22.453309","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453352","pub_date":"2021-7-23","title":"Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2","abstract":"Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potential beneficial as well as detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be re-activated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Understanding the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be deconvoluted by surface-based assays like enzyme-linked immunosorbent assays (ELISAs) which are routinely used to assess cross-reactivity. Here, we have used microfluidic antibody-affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory re-activation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.07.23.453352","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453488","pub_date":"2021-7-23","title":"TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction","abstract":"Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike\u2019s polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.","version":"1.1","doi":"10.1101/2021.07.23.453488","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453521","pub_date":"2021-7-23","title":"Probiotic consortia improve anti-viral immunity to SARS-CoV-2 in Ferrets","abstract":"Probiotics have been suggested as one solution to counter detrimental health effects by SARS-CoV-2, however, data so far is scarce. We tested the effect of two probiotic consortia, OL-1 and OL-2, against SARS-CoV-2 in ferrets and assessed their effect on cytokine production and transcriptome in a human monocyte-derived macrophage (Mf) and dendritic cell (DC) model. The results showed that the consortia significantly reduced the viral load, modulated immune response, and regulated viral receptor expression in ferrets compared to placebo. In human Mf and DC model, OL-1 and OL-2 induced cytokine production and genes related to SARS-CoV-2 anti-viral immunity. The study results indicate that probiotic stimulation of the ferret immune system leads to improved anti-viral immunity against SARS-COV-2 and that critical genes and cytokines for anti-SARS-CoV-2 immunity are stimulated in human immune cells in vitro. The effect of the consortia against SARS-CoV-2 warrants further investigations in human clinical trials.","version":"1.1","doi":"10.1101/2021.07.23.453521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453505","pub_date":"2021-7-23","title":"SARS-CoV-2 Exploits Sexually Dimorphic and Adaptive IFN and TNFa Signaling to Gain Entry into Alveolar Epithelium","abstract":"Infection of the alveolar epithelium constitutes a bottleneck in the progression of COVID-19 to SARS presumably due to the paucity of viral entry receptors in alveolar epithelial type 1 and 2 cells. We have found that the male alveolar epithelial cells express twice as many ACE2 and TMPRSS2 entry receptors as the female ones. Intriguingly, IFN and TNF-\u03b1 signaling are preferentially active in male alveolar cells and induce binding of the cognate transcription factors to the promoters and lung-active enhancers of ACE2 and TMPRSS2. Cotreatment with IFN-I and III dramatically increases expression of the receptors and viral entry in alveolar epithelial cells. TNF\u03b1 and IFN-II, typically overproduced during the cytokine storm, similarly collaborate to induce these events. Whereas JAK inhibitors suppress viral entry induced by IFN-I/III, simultaneous inhibition of IKK/NF-\u03baB is necessary to block viral entry induced by TNF\u03b1 and IFN-II. In addition to explaining the increased incidence of SARS in males, these findings indicate that SARS-Cov-2 hijacks epithelial immune signaling to promote infection of the alveolar epithelium and suggest that JAK inhibitors, singly and in combination with NF-KB inhibitors, may exhibit efficacy in preventing or treating COVID-19 SARS.","version":"1.1","doi":"10.1101/2021.07.23.453505","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446065","pub_date":"2021-7-23","title":"No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing","abstract":"A recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50\u00d7) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolved 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events are, at most, extremely rare in vivo, and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.","version":"1.2","doi":"10.1101/2021.05.28.446065","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453545","pub_date":"2021-7-23","title":"Improving RT-LAMP Detection of SARS-CoV-2 RNA through Primer Set Selection and Combination","abstract":"Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a viable molecular diagnostic method to expand the breadth and reach of nucleic acid testing, particularly for SARS-CoV-2 detection and surveillance. While rapidly growing in prominence, RT-LAMP remains a relatively new method compared to the standard RT-qPCR, and contribution to our body of knowledge on designing LAMP primer sets and assays can have significant impact on its utility and adoption. Here we evaluate 18 LAMP primer sets for SARS-CoV-2, comparing speed and sensitivity with different LAMP formulations and conditions across more than 5,000 RT-LAMP reactions and identifying several primer sets with similar high sensitivity for different SARS-CoV-2 gene targets. Significantly we observe a consistent sensitivity enhancement by combining primer sets for different targets, confirming and building on earlier work to create a simple, general approach to building better and more sensitive RT-LAMP assays.","version":"1.1","doi":"10.1101/2021.07.23.453545","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.424956","pub_date":"2021-7-23","title":"In-Vitro Fluorescence Microscopy Studies Show Retention of Spike-Protein (SARS-Cov-2) on Cell Membrane in the Presence of Amodiaquin Dihydrochloride Dihydrate Drug","abstract":"The ability of S-glycoprotein (S-protein) in SARS-Cov-2 to bind to the host cell receptor protein (angiotensin-converting enzyme 2 (ACE2)) leading to its entry in the cellular system determines its contagious index and global spread. Three available drugs (Riboflavin, Amodiaquin dihydrochloride dihydrate (ADD), and Remidesivir) were investigated to understand the kinetics of S-protein and its entry inside a cellular environment. Optical microscopy and fluorescence-based assays on 293T cells (transfected with ACE2 plasmid) were used as the preamble for assessing the behavior of S-protein in the presence of these drugs for the first 12 hours post-S-protein - ACE2 binding. Preliminary results suggest relatively long retention of S-protein on the cell membrane in the presence of ADD drug. Evident from the %-overlap and colocalization of S-protein with endosome studies, a significant fraction of S-protein entering the cell escape endosomal degradation process, suggesting S-protein takes non-endocytic mediated entry in the presence of ADD. In contrast, in the presence of Riboflavin, S-protein carries out a normal endocytic pathway, comparable to the control (no drug) group. Therefore, the present study indicates ADD potentially affects S-protein\u2019s entry mechanism (endocytic pathway) in addition to its reported target action mechanism. Hence, ADD substantially interferes with S-protein cellular entrance mechanism. This is further strengthened by 24 hrs study. However, detailed studies at the molecular scale are necessary to clarify our understanding of exact intermediate molecular processes. The present study (based on limited data) reveals ADD could be a potential candidate to manage Covid-19 functions through the yet unknown molecular mechanism.","version":"1.2","doi":"10.1101/2021.01.05.424956","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453472","pub_date":"2021-7-23","title":"The in vitro and in vivo potency of CT-P59 against Delta and its associated variants of SARS-CoV-2","abstract":"The Delta variant originally from India is rapidly spreading across the world and causes to resurge infections of SARS-CoV-2. We previously reported that CT-P59 presented its in vivo potency against Beta and Gamma variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on the Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal study. CT-P59 showed reduced antiviral activity but enabled neutralization against Delta, Epsilon, and Kappa variants in cells. In line with in vitro results, the mouse challenge experiment with the Delta variant substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against the Delta variant infection, hinting that CT-P59 has therapeutic potency for patients infected with Delta and its associated variants. CT-P59 exerts the antiviral effect on authentic Delta, Epsilon and Kappa variants in cell-based experiments. CT-P59 showed neutralizing potency against variants including Delta, Epsilon, Kappa, L452R, T478K and P681H pseudovirus variants. The administration of clinically relevant dose of CT-P59 showed in vivo protection against Delta variants in animal challenge experiment.","version":"1.1","doi":"10.1101/2021.07.23.453472","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453470","pub_date":"2021-7-23","title":"PDZ-containing proteins targeted by the ACE2 receptor","abstract":"Angiotensin converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residues segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants values ranging from 3 to 81 \u03bcM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role on ACE2 internalization and recycling that could benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in neurological symptoms of COVID-19.","version":"1.1","doi":"10.1101/2021.07.23.453470","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.23.453524","pub_date":"2021-7-23","title":"US Dog Importations during the COVID-19 Pandemic: Do we have an erupting problem?","abstract":"Dog importation data from 2018-2020 were evaluated to ascertain whether the dog importation patterns in the United States changed during the COVID-19 pandemic, specifically with regard to denial of entry. Dog denial of entry reports from January 1, 2018, to December 31, 2020, stored within the Centers for Disease Control and Prevention (CDC) Quarantine Activity Reporting System, were reviewed. Basic descriptive statistics were used to analyze the data. Reason for denial, country of origin, and month of importation were all examined to determine which countries of origin resulted in the largest number of denials, and whether there was a seasonal change in importations during the COVID-19 pandemic (2020), compared to previous years (2018 and 2019). During 2020, CDC denied entry to 458 dogs. This represents a 52% increase in dogs denied entry compared to the averages in 2018 and 2019. Dogs were primarily denied entry for falsified rabies vaccination certificates (56%). Three countries exported 74% of all dogs denied entry into the United States, suggesting that targeted interventions may be needed for certain countries. Increased attempts to import inadequately vaccinated dogs from countries with canine rabies in 2020 may have been due to the increased demand for domestic pets during the COVID-19 pandemic. Educational messaging should highlight the risk of rabies and the importance of making informed pet purchases from foreign entities to protect pet owners, their families, and the public.","version":"1.1","doi":"10.1101/2021.07.23.453524","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.453304","pub_date":"2021-7-22","title":"Impact of human airway epithelial cellular composition on SARS-CoV-2 infection biology","abstract":"Infection biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), are incompletely understood. Here, we assessed the impact of airway epithelial cellular composition on infection in air-liquid interface (ALI) cultures of differentiated primary human tracheal (PTEC) and bronchial epithelial cells (PBEC). We first compared SARS-CoV-2 infection kinetics, related antiviral and inflammatory responses, and viral entry factors in PTEC and PBEC. Next, the contribution of differentiation time was investigated by differentiating ALI-PTEC/PBEC for 3-5 weeks and comparing dynamics of viral replication/spread, cellular composition and epithelial responses. We observed a gradual increase in viral load with prolonged culture duration. Ciliated and goblet cells were predominantly infected in both PTEC and PBEC. Immunofluorescence analysis and RT-qPCR showed that compared to other cell types mainly ciliated and goblet cell numbers were affected by increased culture duration. An increased proportion of these two target cell types was associated with increased viral load. Furthermore, modulation of cellular composition using IL-13 and the Notch signaling inhibitor DAPT, underlined the importance of both ciliated and goblet cells for infection. DAPT treatment resulted in a lower viral load and a relative increase in ciliated cells at the expense of goblet cells, compared to IL-13 treated cultures in which both cell types were present and viral load was higher. In conclusion, our results identify cellular composition as a contributing factor to airway epithelial susceptibility to SARS-CoV-2. In this study, we determined an effect of culture duration and airway cellular composition of ALI-PBEC and ALI-PTEC cultures on SARS-CoV-2 infection. We found that SARS-CoV-2 infection was increased with prolonged cell culture time and the total percentage and proportion of ciliated and goblet cells played an important role in infection level, suggesting that airway epithelial differentiation/maturation levels may in part determine susceptibility of SARS-CoV-2 infection. The development of effective therapies either targeting virus replication or pathogenesis against SARS-CoV-2 requires robust cell culture-based infection models to test small molecules and biologicals. Therefore, it is important to identify factors that are essential for reliably modeling SARS-CoV-2-airway epithelial cell interactions. This study sheds light on virus-airway epithelial cell interactions and adds to the complexity of SARS-CoV-2 cell tropism in the airways. In addition, the effect of IL-13 on viral infection hints at a causal connection between SARS-CoV-2 infection and (allergic) asthma.","version":"1.1","doi":"10.1101/2021.07.21.453304","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.22.453345","pub_date":"2021-7-22","title":"ADAR mediated A-to-I RNA editing affects SARS-CoV-2 characteristics and fuels its evolution","abstract":"Upon SARS-CoV-2 infection, viral intermediates activate the Type I interferon (IFN) response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to A-to-I RNA editing of SARS-CoV-2. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, and on average, approximately 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans. Collectively, our data suggest that SARS-CoV-2 hijacks components of the host antiviral machinery to edit its genome and fuel its evolution.","version":"1.1","doi":"10.1101/2021.07.22.453345","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.12.452099","pub_date":"2021-7-22","title":"Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication","abstract":"Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with the viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can bind comparatively stabilized to the viral RdRp active site, as it primarily forms base stacking with the template Uracil nucleotide (at +1), which is under freely fluctuations and supports a low free energy barrier of the RDV-TP insertion (\u223c 1.5 kcal/mol). In comparison, the barrier (\u223c 2.6 kcal/mol), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be particularly stabilized by motif B-N691, S682, and motif F-K500 with the sugar, base, and the template backbone, respectively. Although the RDV-TP insertion can be hindered by motif-F R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif-A D623, respectively.","version":"1.3","doi":"10.1101/2021.07.12.452099","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.453232","pub_date":"2021-7-22","title":"Structure insights, thermodynamic profiles, dsDNA melting activity, and liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid N-terminal domain binding to DNA","abstract":"The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD), either with or without the SR-rich motif (SR), upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on specificity for N-NTD/N-NTD-SR interaction with TRS, including an unfavorable energetic contribution to binding along with hydrogen bonds between the triple-thymidine (TTT) motif in the dsTRS and \u03b2-sheet II due to the defined position and orientation of the DNA duplex, a well-defined pattern (\u0394H > 0 and \u0394S > 0 for ssTRS, and \u0394H < 0 and \u0394S < 0 for dsTRS) for the thermodynamic profile of binding, and a preference for TRS in the formation of liquid condensates when compared to a non-specific sequence. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.","version":"1.1","doi":"10.1101/2021.07.21.453232","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.452479","pub_date":"2021-7-22","title":"The infinite alleles model revisited: a Gibbs sampling approach","abstract":"The SARS-CoV-2 outbreak started in late 2019 in the Hubei province in China and the first viral sequence was made available to the scientific community on early January 2020. From there, viral genomes from all over the world have followed at an outstanding rate, reaching already more than 105 on early May 2020, and more than 106 by early March 2021. Phylodynamics methods have been designed in recent years to process such datasets and infer population dynamics and sampling intensities in the past. However, the unprecedented scale of the SARS-CoV-2 dataset now calls for new methodological developments, relying e.g. on simplifying assumptions of the mutation process. In this article, I build on the infinite alleles model stemming from the field of population genetics to develop a new Bayesian statistical method allowing the joint reconstruction of the outbreak\u2019s effective population sizes and sampling intensities through time. This relies on prior conjugacy properties that prove useful both to develop a Gibbs sampler and to gain intuition on the way different parameters of the model are linked and inferred. I finally illustrate the use of this method on SARS-CoV-2 genomes sequenced during the first wave of the outbreak in four distinct European countries, thus offering a new perspective on the evolution of the sampling intensity through time in these countries from genetic data only.","version":"1.1","doi":"10.1101/2021.07.21.452479","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.453274","pub_date":"2021-7-21","title":"Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics","abstract":"SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.","version":"1.1","doi":"10.1101/2021.07.21.453274","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453160","pub_date":"2021-7-21","title":"Immunogenic SARS-CoV2 Epitopes Defined by Mass Spectrometry","abstract":"SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, T cell responses are important in mediating recovery and immune-protection. The identification of immunogenic epitopes that can elicit a meaningful T cell response can be elusive. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes; however, our previous studies find that \u2018immunodominant\u2019 SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing SARS-CoV-2. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined by directly analyzing peptides eluted from the peptide-MHC complex and then validating immunogenicity empirically by determining if such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes of SARS-CoV-2 derived not only from structural but also non-structural genes in regions highly conserved among SARS-CoV-2 strains including recently recognized variants. We report here, for the first time, several novel SARS-CoV-2 epitopes from membrane glycol-protein (MGP) and non-structure protein-13 (NSP13) defined by mass-spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity to induce T cell response. Current state of the art uses putative epitope peptides based on in silico prediction algorithms to evaluate the T cell response among COVID-19 patients. However, none of these peptides have been tested for immunogenicity, i.e. the ability to elicit a T cell response capable of recognizing endogenously presented peptide. In this study, we used MHC immune-precipitation, acid elution and tandem mass spectrometry to define the SARS-CoV-2 immunopeptidome for membrane glycol-protein and the non-structural protein. Furthermore, taking advantage of a highly robust endogenous T cell (ETC) workflow, we verify the immunogenicity of these MS-defined peptides by in vitro generation of MGP and NSP13 peptide-specific T cells and confirm T cell recognition of MGP or NSP13 endogenously expressing cell lines.","version":"1.1","doi":"10.1101/2021.07.20.453160","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.451321","pub_date":"2021-7-21","title":"Unlike Chloroquine, mefloquine inhibits SARS-CoV-2 infection in physiologically relevant cells and does not induce viral variants","abstract":"Repositioning of clinical approved drugs could represent the fastest way to identify therapeutic options during public health emergencies, the majority of drugs explored for repurposing as antivirals for 2019 coronavirus disease (COVID-19) have failed to demonstrate clinical benefit. Without specific antivirals, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Antimalarial drugs, such as chloroquine (CQ)/hydroxychloroquine (HCQ) and mefloquine have emerged as potential anti-SARS-CoV-2 antivirals. CQ/HCQ entered the Solidarity and RECOVERY clinical trials against COVID-19 and showed lack of efficacy. Importantly, mefloquine is not a 4-aminoquinoline like CQ and HCQ and has been previously repurposed for other respiratory diseases. Unlike the 4-aminoquinolines that accumulate in the high pH of intracellular lysosomes of the lung, the high respiratory tract penetration of mefloquine is driven by its high lipophilicity. While CQ and HCQ exhibit activity in Vero E6 cells, their activity is obviated in TMPRSS2-expressing cells, such as Calu-3 cells, which more accurately recapitulate in vivo entry mechanisms for SARS-CoV-2. Accordingly, here we report the anti-SARS-CoV-2 activity of mefloquine in Calu-3 type II pneumocytes and primary human monocytes. Mefloquine inhibited SARS-CoV-2 replication in Calu-3 cells with low cytotoxicity and EC50 and EC90 values of 1.2 and 5.3 \u00b5M, respectively. In addition, mefloquine reduced up to 68% the SARS-CoV-2 RNA levels in infected monocytes, reducing viral-induced inflammation. Mefloquine blocked early steps of the SARS-CoV-2 replicative cycle and was less prone than CQ to induce drug-associated viral mutations and synergized with RNA polymerase inhibitor. The pharmacological parameters of mefloquine are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points that this drug may accumulate in the lungs. These data indicate that mefloquine could represent an orally available clinically approved drug option against COVID-19 and should not be neglected on the basis of the failure of CQ and HCQ.","version":"1.1","doi":"10.1101/2021.07.21.451321","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.256339","pub_date":"2021-7-21","title":"Highly potent anti-SARS-CoV-2 multivalent DARPin therapeutic candidates","abstract":"Globally accessible therapeutics against SARS-CoV-2 are urgently needed. Here, we report the generation of the first anti-SARS-CoV-2 DARPin molecules with therapeutic potential as well as rapid large-scale production capabilities. Highly potent multivalent DARPin molecules with low picomolar virus neutralization efficacies were generated by molecular linkage of three different monovalent DARPin molecules. These multivalent DARPin molecules target various domains of the SARS-CoV-2 spike protein, thereby limiting possible viral escape. Cryo-EM analysis of individual monovalent DARPin molecules provided structural explanations for the mode of action. Analysis of the protective efficacy of one multivalent DARPin molecule in a hamster SARS-CoV-2 infection model demonstrated a significant reduction of pathogenesis. Taken together, the multivalent DARPin molecules reported here, one of which has entered clinical studies, constitute promising therapeutics against the COVID-19 pandemic.","version":"1.3","doi":"10.1101/2020.08.25.256339","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444723","pub_date":"2021-7-21","title":"Presence of a SARS-COV-2 protein enhances Amyloid Formation of Serum Amyloid A","abstract":"A marker for the severeness and disease progress of COVID-19 is overexpression of serum amyloid A (SAA) to levels that in other diseases are associated with a risk for SAA amyloidosis. In order to understand whether SAA amyloidosis could also be a long-term risk of SARS-COV-2 infections we have used long all-atom molecular dynamic simulations to study the effect of a SARS-COV-2 protein segment on SAA amyloid formation. Sampling over 40 \u00b5s we find that presence of the nine-residue segment SK9, located at the C-terminus of the Envelope protein, increases the propensity for SAA fibril formation by three mechanisms: it reduces the stability of the lipid-transporting hexamer shifting the equilibrium toward monomers, it increases the frequency of aggregation-prone configurations in the resulting chains, and it raises the stability of SAA fibrils. Our results therefore suggest that SAA amyloidosis and related pathologies may be a long-term risk of SARS-COV-2 infections.","version":"1.2","doi":"10.1101/2021.05.18.444723","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453146","pub_date":"2021-7-21","title":"SARS-CoV-2 Restructures the Host Chromatin Architecture","abstract":"SARS-CoV-2 has made >190-million infections worldwide, thus it is pivotal to understand the viral impacts on host cells. Many viruses can significantly alter host chromatin, but such roles of SARS-CoV-2 are largely unknown. Here, we characterized the three-dimensional (3D) genome architecture and epigenome landscapes in human cells after SARS-CoV-2 infection, revealing remarkable restructuring of host chromatin architecture. High-resolution Hi-C 3.0 uncovered widespread A compartmental weakening and A-B mixing, together with a global reduction of intra-TAD chromatin contacts. The cohesin complex, a central organizer of the 3D genome, was significantly depleted from intra-TAD regions, supporting that SARS-CoV-2 disrupts cohesin loop extrusion. Calibrated ChIP-Seq verified chromatin restructuring by SARS-CoV-2 that is particularly manifested by a pervasive reduction of euchromatin modifications. Built on the rewired 3D genome/epigenome maps, a modified activity-by-contact model highlights the transcriptional weakening of antiviral interferon response genes or virus sensors (e.g., DDX58) incurred by SARS-CoV-2. In contrast, pro-inflammatory genes (e.g. IL-6) high in severe infections were uniquely regulated by augmented H3K4me3 at their promoters. These findings illustrate how SARS-CoV-2 rewires host chromatin architecture to confer immunological gene deregulation, laying a foundation to characterize the long-term epigenomic impacts of this virus.","version":"1.1","doi":"10.1101/2021.07.20.453146","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453127","pub_date":"2021-7-21","title":"An Orally Available Cathepsin L Inhibitor Protects Lungs Against SARS-CoV-2-Induced Diffuse Alveolar Damage in African Green Monkeys","abstract":"The COVID-19 pandemic resulted from global infection by the SARS-CoV-2 coronavirus and rapidly emerged as an urgent health issue requiring effective treatments. To initiate infection, the Spike protein of SARS-CoV-2 requires proteolytic processing mediated by host proteases. Among the host proteases proposed to carry out this activation is the cysteine protease cathepsin L. Inhibiting cathepsin L has been proposed as a therapeutic strategy for treating COVID-19. SLV213 (K777) is an orally administered small molecule protease inhibitor that exhibits in vitro activity against a range of viruses, including SARS-CoV-2. To confirm efficacy in vivo, K777 was evaluated in an African green monkey (AGM) model of COVID-19. A pilot experiment was designed to test K777 in a prophylactic setting, animals were pre-treated with 100mg/kg K777 (N=4) or vehicle (N=2) before inoculation with SARS-CoV-2. Initial data demonstrated that K777 treatment reduced pulmonary pathology compared to vehicle-treated animals. A second study was designed to test activity in a therapeutic setting, with K777 treatment (33 mg/kg or 100 mg/kg) initiated 8 hours after exposure to the virus. In both experiments, animals received K777 daily via oral gavage for 7 days. Vehicle-treated animals exhibited higher lung weights, pleuritis, and diffuse alveolar damage. In contrast, lung pathology was reduced in K777-treated monkeys, and histopathological analyses confirmed the lack of diffuse alveolar damage. Antiviral effects were further demonstrated by quantitative reductions in viral load of samples collected from upper and lower airways. These preclinical data support the potential for early SLV213 treatment in COVID-19 patients to prevent severe lung pathology and disease progression.","version":"1.1","doi":"10.1101/2021.07.20.453127","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453118","pub_date":"2021-7-21","title":"Integrin activation is an essential component of SARS-CoV-2 infection","abstract":"Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the \u03b1I MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: \u2018inside-out\u2019 signaling primes the ligand binding function of integrins via a talin dependent mechanism and \u2018outside-in\u2019 signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by G\u03b113 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and G\u03b113 binding to the cytoplasmic tail of an integrin\u2019s \u03b2 subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.07.20.453118","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453162","pub_date":"2021-7-21","title":"PRE-CLINICAL IMMUNE RESPONSE AND SAFETY EVALUATION OF THE PROTEIN SUBUNIT VACCINE NANOCOVAX FOR COVID-19","abstract":"The Coronavirus disease-2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has become a dire global health concern. The development of vaccines with high immunogenicity and safety is crucial for control of the global COVID-19 pandemic and prevention of further illness and fatalities. Here, we report development of SARS-CoV-2 vaccine candidate, Nanocovax, based on recombinant protein production of the extracellular (soluble) portion of the S protein of SARS-CoV-2. The results showed that Nanocovax induced high levels of S protein-specific IgG, as well neutralizing antibody in three animal models including Balb/C mice, Syrian hamsters, and non-human primate (Macaca leonina). In addition, the viral challenge study using the hamster model showed that Nanocovax protected the upper respiratory tract from SARS-CoV-2 infection. No adverse effects were induced by Nanocovax in swiss mice (Musmusculus var. Albino), Rats (Rattus norvegicus), and New Zealand rabbits. These pre-clinical results indicated that Nanocovax is safe and effective.","version":"1.1","doi":"10.1101/2021.07.20.453162","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453054","pub_date":"2021-7-21","title":"Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism","abstract":"SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50 of 0.086 \u03bcg mL-1 (DL4) and 0.385 \u03bcg mL-1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50 of 0.414 \u03bcg mL-1 for Alpha, 1.060 \u03bcg mL-1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.07.20.453054","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453042","pub_date":"2021-7-21","title":"An untargeted metabolomic approach to identify antiviral defense mechanisms in memory leukocytes secreting in vitro IgG anti-SARS-Cov-2","abstract":"Available knowledge shows that individuals infected by SARS-CoV-2 undergo an altered metabolic state in multiple organs. Metabolic activities are directly involved in modulating the immune responses against infectious diseases, yet our understanding remains limited on how host metabolism relates with inflammatory responses. To better elucidate the underlying biochemistry of leukocytes response, we focused our analysis on the possible relationships between SARS-CoV-2 post-infection stages and distinct metabolic pathways. Indeed, in cultures of peripheral blood mononuclear cells (PBMC, n=48) obtained 60-90 days after infection and showing in vitro IgG antibody memory for spike-S1 antigen (n=19), we observed a significant altered metabolism of tryptophan and urea cycle pathways. This work for the first time identifies metabolic routes in cell metabolism possibly related to later stages of immune defense against SARS-Cov-2 infection, namely when circulating antibodies may be absent, but an antibody memory is present. The results suggest a reprogramming of leukocyte metabolism after viral pathogenesis through activation of specific amino acid pathways possibly related to protective immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.07.20.453042","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.452858","pub_date":"2021-7-21","title":"Cornering an Ever-Evolving Coronavirus: TATX-03, a fully human synergistic multi-antibody cocktail targeting the SARS-CoV-2 Spike Protein with in vivo efficacy","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an ongoing global human health crisis and will likely become endemic, requiring novel sustainable therapeutic strategies. We report on the discovery of a fully human multi-antibody cocktail (TATX-03) targeting diversified non-overlapping epitopes on the SARS-CoV-2 spike protein that suppressed replication-competent viral titers to undetectable levels in the lungs of SARS-CoV-2 challenged hamsters upon both prophylactic and therapeutic administration. While monotherapy with two of the individual cocktail components also showed clear in vivo protection, neither recapitulated the efficacy of TATX-03. This synergistic effect was further supported by examining in vivo efficacy of these individual antibodies and corresponding combination therapy at a lower dose. Furthermore, in vitro screenings using VSV-particles pseudo-typed with spike proteins representing the SARS-CoV-2 variants of concern Alpha, Beta, and Delta showed that TATX-03 maintained its neutralization potency. These results merit further development of TATX-03 as a potential therapy for SARS-CoV-2 infection with resistance to mutagenic escape.","version":"1.1","doi":"10.1101/2021.07.20.452858","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453079","pub_date":"2021-7-21","title":"CoVizu: Rapid analysis and visualization of the global diversity of SARS-CoV-2 genomes","abstract":"Phylogenetics has played a pivotal role in the genomic epidemiology of SARS-CoV-2, such as tracking the emergence and global spread of variants, and scientific communication. However, the rapid accumulation of genomic data from around the world \u2014 with over two million genomes currently available in the GISAID database \u2014 is testing the limits of standard phylogenetic methods. Here, we describe a new approach to rapidly analyze and visualize large numbers of SARS-CoV-2 genomes. Using Python, genomes are filtered for problematic sites, incomplete coverage, and excessive divergence from a strict molecular clock. All differences from the reference genome, including indels, are extracted using minimap2, and compactly stored as a set of features for each genome. For each Pango lineage (https://cov-lineages.org), we collapse genomes with identical features into \u2018variants\u2019, generate 100 bootstrap samples of the feature set union to generate weights, and compute the symmetric differences between the weighted feature sets for every pair of variants. The resulting distance matrices are used to generate neigihbor-joining trees in RapidNJ and converted into a majority-rule consensus tree for the lineage. Branches with support values below 50% or mean lengths below 0.5 differences are collapsed, and tip labels on affected branches are mapped to internal nodes as directly-sampled ancestral variants. Currently, we process about million genomes in approximately nine hours on 34 cores. The resulting trees are visualized using the JavaScript framework D3.js as \u2018beadplots\u2019, in which variants are represented by horizontal line segments, annotated with beads representing samples by collection date. Variants are linked by vertical edges to represent branches in the consensus tree. These visualizations are published at https://filogeneti.ca/CoVizu. All source code was released under an MIT license at https://github.com/PoonLab/covizu.","version":"1.1","doi":"10.1101/2021.07.20.453079","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.453244","pub_date":"2021-7-21","title":"Lung Epithelial Regulation of BCL2 Related Protein A1 (BCL2A1) by Coronaviruses (SARS-CoV) and Type I Interferon Signaling","abstract":"Highly pathogenic respiratory viruses such as 1918 influenza (HIN1) and coronavirus (SARS-CoV-2) induce significant lung injury with diffuse alveolar damage, capillary leak, and extensive cell death resulting in acute respiratory distress syndrome (ARDS). Direct effects of the virus, as well as host immune response such as proinflammatory cytokine production, contribute to programmed cell death or apoptosis. Alveolar lung epithelial type II (AT2) cells play a major role in the clearance of respiratory viruses, secretion of surfactant proteins and antimicrobial substances into the bronchoalveolar fluid as well as repair of lung injury. Gene expression in AT2 cells is regulated in a tissue and cell-specific manner and in a temporal fashion. The availability of tissue and cell-specific RNA datasets in Human Protein Atlas led to the identification of localized expression patterns of BCL-2 family members such as BCL2 related protein A1 (BCL2A1) in AT2 cells and immune cells of the lung. BCL2A1 expression was regulated by multiple stimuli including Toll-like receptor (TLR) ligands, interferons (IFNs), inflammatory cytokines, and inhibited by the steroid dexamethasone. In this study, regulation of BCL2A1 gene expression in human lung epithelial cells by several respiratory viruses and type I interferon signaling was investigated. SARS-CoV-2 infection significantly induced BCL2A1 expression in human lung epithelial cells within 24 hours that required the expression of Angiotensin-converting enzyme 2 (ACE2). BCL2A1 mRNA induction by SARS-CoV-2 was correlated with the induced expression of IFN-\u03b2 and IFN-regulated transcription factor mRNA. BCL2A1 was induced by IFN-\u03b2 treatment or by infection with influenza virus lacking the non-structural protein1(NS1) in NHBE cells. Furthermore, bioinformatics revealed that a subset of BCL-2 family members involved in the control of apoptosis and transcription such as BCL2A1, BCL2L14, BCL3, and BCL6 were regulated in the lung epithelial cells by coronaviruses and in the lung tissue samples of COVID-19 patients. Transcriptomic data also suggested that these genes were differentially regulated by the steroid drug dexamethasone.","version":"1.1","doi":"10.1101/2021.07.21.453244","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.453140","pub_date":"2021-7-21","title":"Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants","abstract":"Extensive glycosylation of the spike protein of SARS-CoV-2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the COVID-19 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific N-terminal domain site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.","version":"1.1","doi":"10.1101/2021.07.21.453140","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.452903","pub_date":"2021-7-21","title":"Viral receptor profiles of masked palm civet revealed by single-cell transcriptomics","abstract":"Civets are small mammals belonging to the family Viverridae. The masked palm civets (Paguma larvata) served as an intermediate host in the bat-to-human transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. Because of their unique role in the SARS outbreak, civets were suspected as a potential intermediate host of SARS-CoV-2, the etiological pathogen of the COVID-19 pandemic. Besides their susceptibility to coronaviruses, civets can also be infected by other viruses, such as canine distemper viruses, parvoviruses, influenza viruses, etc. Regarding the ecological and economical role of civets, it is vital to evaluate the potential threats from different pathogens to these animals. Receptor binding is a necessary step for virus entry into host cells. Understanding the distribution of receptors of various viruses provides hints to their potential tissue tropisms. Herein, we characterized the cell atlas of five important organs (the frontal lobe, lung, liver, spleen and kidney) of masked palm civets (Paguma larvata) and described the expression profiles of receptor associated genes of 132 viruses from 25 families, including 16 viruses from 10 families reported before that can attack civets and 116 viruses with little infection record.","version":"1.1","doi":"10.1101/2021.07.20.452903","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.21.453241","pub_date":"2021-7-21","title":"A single oral immunization with a replication-competent adenovirus-vectored vaccine protects mice from influenza respiratory infection","abstract":"The development of effective and flexible vaccine platforms is a major public health challenge as recently highlighted by the COVID-19 pandemic. Adenoviruses (AdVs) are easy to produce and have a good safety and efficacy profile when administered orally as demonstrated by the long-term use of oral AdV 4 and 7 vaccines in the US military. These viruses therefore appear to be the ideal backbone for the development of oral replicative vector vaccines. However, research on these vaccines is limited by the ineffective replication of human AdVs in laboratory animals. The use of mouse AdV type 1 (MAV-1) in its natural host allows infection to be studied under replicative conditions. Here, we orally vaccinated mice with MAV-1 vectors expressing the full length or the \u201cheadless\u201d hemagglutinin (HA) of influenza to assess the protection conferred against an intranasal challenge of influenza. We showed that while the headless HA vector did not generate a significant humoral or cellular immune response to influenza, a single oral immunisation with the full-length HA vaccine generated influenza-specific and neutralizing antibodies and completely protected the mice against clinical signs and viral replication. Given the constant threat of pandemics and the need for annual vaccination against influenza and possibly emerging agents such as SARS-CoV-2, new types of vaccines that are easier to produce and administer and therefore more widely accepted are a critical public health need. Here, using a relevant animal model, we have shown that replicative oral AdV vaccine vectors can help make vaccination against major respiratory diseases more available, better accepted and therefore more effective. These results could be of major importance in the coming years in the fight against emerging diseases such as COVID-19.","version":"1.1","doi":"10.1101/2021.07.21.453241","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.17.452778","pub_date":"2021-7-20","title":"HLA-dependent variation in SARS-CoV-2 CD8+ T cell cross-reactivity with human coronaviruses","abstract":"Pre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure to SARS-CoV-2 has been reported in several studies. While emerging evidence hints toward prior exposure to common-cold human coronaviruses (HCoV), the extent of- and conditions for-cross-protective immunity between SARS-CoV-2 and HCoVs remain open. Here, by leveraging a comprehensive pool of publicly available functionally evaluated SARS-CoV-2 peptides, we report 126 immunogenic SARS-CoV-2 peptides with high sequence similarity to 285 MHC-presented target peptides from at least one of four HCoV, thus providing a map describing the landscape of SARS-CoV-2 shared and private immunogenic peptides with functionally validated T cell responses. Using this map, we show that while SARS-CoV-2 immunogenic peptides in general exhibit higher level of dissimilarity to both self-proteome and -microbiomes, there exist several SARS-CoV-2 immunogenic peptides with high similarity to various human protein coding genes, some of which have been reported to have elevated expression in severe COVID-19 patients. We then combine our map with a SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls and show that whereas the public repertoire for the majority of convalescent patients are dominated by TCRs cognate to private SARS-CoV-2 peptides, for a subset of patients, more than 50% of their public repertoires that show reactivity to SARS-CoV-2, consist of TCRs cognate to shared SARS-CoV-2-HCoV peptides. Further analyses suggest that the skewed distribution of TCRs cognate to shared and private peptides in COVID-19 patients is likely to be HLA-dependent. Finally, by utilising the global prevalence of HLA alleles, we provide 10 peptides with known cognate TCRs that are conserved across SARS-CoV-2 and multiple human coronaviruses and are predicted to be recognised by a high proportion of the global population. Overall, our work indicates the potential for HCoV-SARS-CoV-2 reactive CD8+ T cells, which is likely dependent on differences in HLA-coding genes among individuals. These findings may have important implications for COVID-19 heterogeneity and vaccine-induced immune responses as well as robustness of immunity to SARS-CoV-2 and its variants.","version":"1.1","doi":"10.1101/2021.07.17.452778","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423721","pub_date":"2021-7-20","title":"The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147-receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease","abstract":"Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known whether heart vascular pericytes (PCs) are infected by SARS-CoV-2, and if the S protein alone provokes PC dysfunction. Here, we aimed to investigate the effects of the S protein on primary human cardiac PC signalling and function. Results show, for the first time, that cardiac PCs are not permissive to SARS-CoV-2 infection in vitro, whilst a recombinant S protein alone elicits functional alterations in PCs. This was documented as: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors responsible for EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation and rescued PC function in the presence of the S protein. In conclusion, our findings suggest that circulating S protein prompts vascular PC dysfunction, potentially contributing to establishing microvascular injury in organs distant from the site of infection. This mechanism may have clinical and therapeutic implications. Severe COVID-19 manifests as a microvascular syndrome, but whether SARS-CoV-2 infects and damages heart vascular pericytes (PCs) remains unknown. We provide evidence that cardiac PCs are not infected by SARS-CoV-2. Importantly, we show that the recombinant S protein alone elicits cellular signalling through the CD147 receptor in cardiac PCs, thereby inducing cell dysfunction and microvascular disruption in vitro. This study suggests that soluble S protein can potentially propagate damage to organs distant from sites of infection, promoting microvascular injury. Blocking the CD147 receptor in patients may help protect the vasculature not only from infection, but also from the collateral damage caused by the S protein.","version":"1.2","doi":"10.1101/2020.12.21.423721","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.20.453027","pub_date":"2021-7-20","title":"The SARS-CoV-2 reproduction number R0 in cats","abstract":"Domestic cats are susceptible to SARS-CoV-2 virus infection and given that they are in close contact with people, assessing the potential risk cats represent for the transmission and maintenance of SARS-CoV-2 is important. Assessing this risk implies quantifying transmission from humans-to-cats, from cats-to-cats and from cats-to-humans. Here we quantified the risk of cat-to-cat transmission by reviewing published literature describing transmission either experimentally or under natural conditions in infected households. Data from these studies were collated to quantify the SARS-CoV-2 reproduction number R0 among cats. The estimated R0 was significantly higher than 1, hence cats could play a role in the transmission and maintenance of SARS-CoV-2. Questions that remain to be addressed are the risk of transmission from humans-to-cats and cats-to-humans. Further data on household transmission and data on virus levels in both the environment around infected cats and their exhaled air could be a step towards assessing these risks.","version":"1.1","doi":"10.1101/2021.07.20.453027","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448820","pub_date":"2021-7-19","title":"SARS-CoV-2 spike P681R mutation, a hallmark of the Delta variant, enhances viral fusogenicity and pathogenicity","abstract":"During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and currently, four variants of concerns (VOCs) are considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2/Delta variant, closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617.2/Delta variant is highly fusogenic, and notably, more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates the spike protein cleavage and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity than the parental virus. Our data suggest that the P681R mutation is a hallmark that characterizes the virological phenotype of the B.1.617.2/Delta variant and is closely associated with enhanced pathogenicity.","version":"1.2","doi":"10.1101/2021.06.17.448820","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452571","pub_date":"2021-7-19","title":"Molecular evolution and structural analyses of the spike glycoprotein from Brazilian SARS-CoV-2 genomes: the impact of the fixation of selected mutations","abstract":"The COVID-19 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached by July 2021 almost 200 million cases and more than 4 million deaths worldwide since its beginning in late 2019, leading to enhanced concern in the scientific community and the general population. One of the most important pieces of this host-pathogen interaction is the spike protein, which binds to the human Angiotensin-converting enzyme 2 (hACE2) cell receptor, mediates the membrane fusion and is the major target of neutralizing antibodies against SARS-CoV-2. The multiple amino acid substitutions observed in this region, specially in the Receptor Binding Domain (RBD), mainly after almost one year of its emergence (late 2020), have enhanced the hACE2 binding affinity and led to several modifications in the mechanisms of SARS-CoV-2 pathogenesis, improving the viral fitness and/or promoting immune evasion, with potential impact in the vaccine development. In this way, the present work aimed to evaluate the effect of positively selected mutations fixed in the Brazilian SARS-CoV-2 lineages and to check for mutational evidence of coevolution. Additionally, we evaluated the impact of selected mutations identified in some of the VOC and VOI lineages (C.37, B.1.1.7, P.1, and P.2) of Brazilian samples on the structural stability of the spike protein, as well as their possible association with more aggressive infection profiles by estimating the binding affinity in the RBD-hACE2 complex. We identified 48 sites under selective pressure in Brazilian spike sequences, 17 of them with the strongest evidence by the HyPhy tests, including VOC related mutation sites 138, 142, 222, 262, 484, 681, and 845, among others. The coevolutionary analysis identified a number of 28 coevolving sites that were found not to be conditionally independent, such as the couple E484K - N501Y from P.1 and B.1.351 lineages. Finally, the molecular dynamics and free energy estimates showed the structural stabilizing effect and the higher impact of E484K for the improvement of the binding affinity between the spike RBD and the hACE2 in P.1 and P.2 lineages, as well as the stabilizing and destabilizing effects for the positively selected sites.","version":"1.1","doi":"10.1101/2021.07.16.452571","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452721","pub_date":"2021-7-19","title":"Single-dose respiratory mucosal delivery of next-generation viral-vectored COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2","abstract":"The emerging SARS-CoV-2 variants of concern (VOC) increasingly threaten the effectiveness of current first-generation COVID-19 vaccines that are administered intramuscularly and are designed to only target the spike protein. There is thus a pressing need to develop next-generation vaccine strategies to provide more broad and long-lasting protection. By using adenoviral vectors (Ad) of human and chimpanzee origin, we developed Ad-vectored trivalent COVID-19 vaccines expressing Spike-1, Nucleocapsid and RdRp antigens and evaluated them following single-dose intramuscular or intranasal immunization in murine models. We show that respiratory mucosal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the three-arm immunity, consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells, and mucosal trained innate immunity. We further show that single-dose intranasal immunization provides robust protection against not only the ancestral strain of SARS-CoV-2, but also two emerging VOC, B.1.1.7 and B.1.351. Our findings indicate that single-dose respiratory mucosal delivery of an Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy against current and future VOC. This strategy has great potential to be used not only to boost first-generation vaccine-induced immunity but also to expand the breadth of protective T cell immunity at the respiratory mucosa.","version":"1.1","doi":"10.1101/2021.07.16.452721","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452709","pub_date":"2021-7-19","title":"SARS-CoV-2 Spike Pseudoviruses: A Useful tool to study virus entry and address emerging neutralization escape phenotypes","abstract":"SARS-CoV-2 genetic variants are emerging around the globe. Unfortunately, several SARS-CoV-2 variants, especially, variants of concern (VOC) are less susceptible to neutralization by the convalescent and post-vaccination sera, raising concerns of increased disease transmissibility and severity. Recent data suggests the SARS-CoV-2 neutralizing anti-body levels as a good correlate of vaccine mediated protection. However, currently used BSL3 based virus micro-neutralization (MN) assays are more laborious, time consuming and expensive, underscoring the need for BSL2 based, cost effective neutralization assays against SARS-CoV-2 variants. In light of this unmet need, we have developed a BSL2 pseudovirus based neutralization assay (PBNA) in cells expressing Angiotensin Converting Enzyme-2 (ACE2) receptor for SARS-CoV-2. The assay is reproducible (R2=0.96), demonstrates a good dynamic range and high sensitivity. Our data suggests that the biological Anti-SARS-CoV-2 research reagents such as NIBSC 20/130 show lower neutralization against B.1.351 RSA and B1.1.7 UK VOC, whereas a commercially available monoclonal antibody MM43 retains activity against both these variants. SARS-CoV-2 Spike Pseudovirus based neutralization assays for VOC would be useful tools to measure the neutralization ability of candidate vaccines in both preclinical models and clinical trials and further help develop effective prophylactic countermeasures against emerging neutralization escape phenotypes.","version":"1.1","doi":"10.1101/2021.07.16.452709","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.17.452554","pub_date":"2021-7-19","title":"Macrophages govern antiviral responses in human lung tissues protected from SARS-CoV-2 infection","abstract":"The majority of SARS-CoV-2 infections among healthy individuals result in asymptomatic to mild disease. However, the immunological mechanisms defining effective lung tissue protection from SARS-CoV-2 infection remain elusive. Unlike mice solely engrafted with human fetal lung xenograft (fLX), mice co-engrafted with fLX and a myeloid-enhanced human immune system (HNFL mice) are protected against SARS-CoV-2 infection, severe inflammation, and histopathology. Effective control of viral infection in HNFL mice associated with significant macrophage infiltration, and the induction of a potent macrophage-mediated interferon response. The pronounced upregulation of the USP18-ISG15 axis (a negative regulator of IFN responses), by macrophages was unique to HNFL mice and represented a prominent correlate of reduced inflammation and histopathology. Altogether, our work shed light on unique cellular and molecular correlates of lung tissue protection during SARS-CoV-2 infection, and underscores macrophage IFN responses as prime targets for developing immunotherapies against coronavirus respiratory diseases. Mice engrafted with human fetal lung xenografts (fLX-mice) are highly susceptible to SARS-CoV-2. Co-engraftment with a human myeloid-enriched immune system protected fLX-mice against infection. Tissue protection was defined by a potent and well-balanced antiviral response mediated by infiltrating macrophages. Protective IFN response was dominated by the upregulation of the USP18-ISG15 axis.","version":"1.1","doi":"10.1101/2021.07.17.452554","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452756","pub_date":"2021-7-19","title":"Comparison of heat-inactivated and infectious SARS-CoV-2 across indoor surface materials shows comparable RT-qPCR viral signal intensity and persistence","abstract":"Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with COVID-19 and inform appropriate infection mitigation responses. Research groups have reported detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2 positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over seven days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle (Cq)) can be correlated to surface viral load using only one linear regression model per material category. The same experiment was performed with infectious viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods. Environmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made towards containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) [1], a large-scale environmental monitoring effort in elementary school and child care settings, has processed > 13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT - qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.","version":"1.1","doi":"10.1101/2021.07.16.452756","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452748","pub_date":"2021-7-19","title":"Key substitutions in the spike protein of SARS-CoV-2 variants can predict resistance to monoclonal antibodies, but other substitutions can modify the effects","abstract":"Mutations in the spike protein of SARS-CoV-2 variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), A.23.1 and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside of the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies. Therapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant, but that other substitutions can affect the degree of resistance in unpredictable ways. These finding highlight complex interactions among substitutions in the spike protein affecting virus neutralization and potentially virus entry into cells.","version":"1.1","doi":"10.1101/2021.07.16.452748","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.19.452910","pub_date":"2021-7-19","title":"Integrative multi-omics landscape of non-structural protein 3 of severe acute respiratory syndrome coronaviruses","abstract":"The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently a global pandemic. Extensive investigations have been performed to study the clinical and cellular effects of SARS-CoV-2 infection. Mass spectrometry-based proteomics studies have revealed the cellular changes due to the infection and identified a plethora of interactors for all SARS-CoV-2 components, except for the longest non-structural protein 3 (NSP3). Here, we expressed the full-length NSP3 proteins of SARS-CoV and SARS-CoV-2 to investigate their unique and shared functions using multi-omics methods. We conducted interactome, phosphoproteome, ubiquitylome, transcriptome, and proteome analyses of NSP3-expressing cells. We found that NSP3 plays essential roles in cellular functions such as RNA metabolism and immune response such as NF-\u03baB signal transduction. Interestingly, we showed that SARS-CoV-2 NSP3 has both endoplasmic reticulum and mitochondrial localizations. In addition, SARS-CoV-2 NSP3 is more closely related to mitochondrial ribosomal proteins, whereas SARS-CoV NSP3 is related to the cytosolic ribosomal proteins. In summary, our multi-omics studies of NSP3 enhance our understanding of the functions of NSP3 and offer valuable insights for the development of anti-SARS strategies.","version":"1.1","doi":"10.1101/2021.07.19.452910","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.14.452401","pub_date":"2021-7-19","title":"Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of \u223c107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of \u223c106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.","version":"1.2","doi":"10.1101/2021.07.14.452401","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.18.452826","pub_date":"2021-7-19","title":"Susceptibilities of human ACE2 genetic variants in coronavirus infection","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 1603 million cases worldwide and 3.4 million deaths (as of May 2021), with varying incidences and death rates among regions/ethnicities. Human genetic variation can affect disease progression and outcome, but little is known about genetic risk factors for SARS-CoV-2 infection. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 all utilize the human protein angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2 gene. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike protein and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity.","version":"1.1","doi":"10.1101/2021.07.18.452826","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.17.452576","pub_date":"2021-7-19","title":"Mutation-induced Changes in the Receptor-binding Interface of the SARS-CoV-2 Delta Variant B.1.617.2 and Implications for Immune Evasion","abstract":"While the vaccination efforts against SARS-CoV-2 infections are ongoing worldwide, new genetic variants of the virus are emerging and spreading. Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variant\u2019s receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding \u03b2-loop-\u03b2 motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.","version":"1.1","doi":"10.1101/2021.07.17.452576","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.17.452804","pub_date":"2021-7-19","title":"ACE2 binding is an ancestral and evolvable trait of sarbecoviruses","abstract":"Two different sarbecoviruses have caused major human outbreaks in the last two decades. Both these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 via the spike receptor-binding domain (RBD). However, binding to ACE2 orthologs from humans, bats, and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses. Here, we use high-throughput assays to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologs. We find that ACE2 binding is an ancestral trait of sarbecovirus RBDs that has subsequently been lost in some clades. Furthermore, we demonstrate for the first time that bat sarbecoviruses from outside Asia can bind ACE2. In addition, ACE2 binding is highly evolvable: for many sarbecovirus RBDs there are single amino-acid mutations that enable binding to new ACE2 orthologs. However, the effects of individual mutations can differ markedly between viruses, as illustrated by the N501Y mutation which enhances human ACE2 binding affinity within several SARS-CoV-2 variants of concern but severely dampens it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening consideration of the range of sarbecoviruses with spillover potential.","version":"1.1","doi":"10.1101/2021.07.17.452804","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.19.452918","pub_date":"2021-7-19","title":"Rosin Soap Exhibits Virucidal Activity","abstract":"Chemical methods of virus inactivation are used routinely to prevent viral transmission in both a personal hygiene capacity but also in at-risk environments like hospitals. Several \u2018virucidal\u2019 products exist, including hand soaps, gels and surface disinfectants. Resin acids, which can be derived from Tall oil produced from trees, have been shown to exhibit anti-bacterial activity. However, whether these products or their derivatives have virucidal activity is unknown. Here, we assessed the capacity of Rosin soap to inactivate a panel of pathogenic mammalian viruses in vitro. We show that Rosin soap can inactivate the human enveloped viruses: influenza A virus (IAV), respiratory syncytial virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For IAV, rosin soap could provide a 100,000-fold reduction in infectivity. However, Rosin soap failed to affect the non-enveloped encephalomyocarditis virus (EMCV). The inhibitory effect of Rosin soap against IAV infectivity was dependent on its concentration but not dependent on incubation time nor temperature. Together, we demonstrate a novel chemical inactivation method against enveloped viruses, which could be of use in preventing virus infections in certain settings. Viruses remain a significant cause of human disease and death, most notably illustrated through the current Covid-19 pandemic. Control of virus infection continues to pose a significant global health challenge to the human population. Viruses can spread through multiple routes, including via environmental and surface contamination where viruses can remain infectious for days. Methods to inactivate viruses on such surfaces may help mitigate infection. Here we present evidence identifying a novel \u2018virucidal\u2019 product in Rosin soap, which is produced from Tall oil from coniferous trees. Rosin soap was able to rapidly and potently inactivate influenza virus and other enveloped viruses.","version":"1.1","doi":"10.1101/2021.07.19.452918","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.14.21260544","pub_date":"2021-07-18","title":"Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g. subgenomic RNA [sgRNA]) and replicative intermediates (e.g. negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE C\n                  <jats:sub>T</jats:sub>\n                  values ranging from 12.3-37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charit\u00e9 primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a C\n                  <jats:sub>T</jats:sub>\n                  above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 C\n                  <jats:sub>T</jats:sub>\n                  compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cut-off of &gt;31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.\n                </jats:p>","version":null,"doi":"10.1101/2021.07.14.21260544","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.17.452802","pub_date":"2021-7-18","title":"Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network","abstract":"Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with CRISPR/Cas12a based visual colorimetric assays. Among them, 9 ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for naked-eye read-out of assay results (no transducing component required for visualization). Subsequently, we developed a convolutional neural network algorithm standardize and to automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube towards an instrument-less colorimetric POCT format that requires only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the single-copy detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this novel RAVI-CRISPR system for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen-side testing.","version":"1.1","doi":"10.1101/2021.07.17.452802","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.128876","pub_date":"2021-7-17","title":"Room Temperature Isothermal Colorimetric Padlock Probe Rolling Circle Amplification for Viral DNA and RNA Detection","abstract":"Seasonal flu and pandemics, which account for millions of infections and hundreds of thousands of deaths, require rapid and reliable detection mechanisms to implement preventive and therapeutic measures. Current detection methods of viral infections have limitations in speed, accuracy, accessibility, and usability. This project presents a novel, widely applicable viral diagnostic test that uses a modified version of rolling circle amplification (RCA) to be sensitive, specific, direct RNA targeted, colorimetric and operable at room temperature. We are specifically detecting the following high-impact viruses: SARS-CoV-2, Influenza A (H1N1pdm09), and Influenza B (Victoria Lineage), although our test can be adapted to any viral infection. Results using synthetic viral DNA and RNA sequences show that our diagnostic test takes approximately one hour, detects femtomolar concentrations of RNA strands, and differentiates between virus strains. We believe implementing our diagnostic test will provide faster responses to future viral-related outbreaks for quicker societal recovery.","version":"1.3","doi":"10.1101/2020.06.12.128876","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452441","pub_date":"2021-7-16","title":"A single de novo substitution in SARS-CoV-2 spike informs enhanced adherence to human ACE2","abstract":"SARS-CoV-2 initiates colonization of host cells by binding to cell membrane ACE2 receptor. This binding is mediated by the viral spike receptor binding domain (RBD). The COVID-19 pandemic has brought devastating consequences at a clinical, social and economical levels. Therefore, anticipation of potential novel SARS-causing species or SARS-CoV-2 variants with enhanced binding to ACE2 is key in the prevention of future threats to come. We have characterized a de novo single substitution, Q498Y, in SARS-CoV-2 RBD that confers stronger adherence to ACE2. While the SARS-CoV-2 \u03b2 variant, which includes three simultaneous amino acid replacements, induces a 4-fold stronger affinity, a single Q498Y substitution results in 2.5-fold tighter binding, compared to the Wuhan-Hu-1 SARS-CoV-2 2019 strain. Additionally, we crystallized RBDQ498Y complexed with ACE2 and provide here the structural basis for this enhanced affinity. These studies inform a rationale for prevention of potential SARS-causing viruses to come.","version":"1.1","doi":"10.1101/2021.07.16.452441","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.16.452629","pub_date":"2021-7-16","title":"Collaboration Between Host and Viral Factors Shape SARS-CoV-2 Evolution","abstract":"SARS-CoV-2 continues to evolve, resulting in several \u2018variants of concern\u2019 with novel properties. The factors driving SARS-CoV-2 fitness and evolution in the human respiratory tract remain poorly defined. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change. Through viral whole-genome sequencing, we explored the evolution of two clinical isolates of SARS-CoV-2 during passage in unmodified Vero-derived cell lines and in parallel, in well-differentiated primary nasal epithelial cell (WD-PNEC) cultures. We identify a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near-fixation with 2 passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with Vero-derived cells facilitating more profound genetic changes. However, most mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike while the strain with mutations in NSP12 (T293I), Spike (P812R) and a truncation of ORF7a remained viable in WD-PNECs. Our work highlights the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and fitness in human respiratory epithelium.","version":"1.1","doi":"10.1101/2021.07.16.452629","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.15.452504","pub_date":"2021-7-16","title":"Neutralization of recombinant RBD-subunit vaccine ZF2001-elicited antisera to SARS-CoV-2 variants including Delta","abstract":"SARS-CoV-2 variants brought new waves of infection worldwide. In particular, Delta variant (B.1.617.2 lineage) has become predominant in many countries. These variants raised the concern for their potential immune escape to the currently approved vaccines. ZF2001 is a subunit vaccine received emergency use authorization (EUA) in both China and Uzbekistan, with more than 100-million doses administrated with a three-dose regimen. The tandem-repeat dimer of SARS-CoV-2 spike protein receptor binding domain (RBD) was used as the antigen. In this work, we evaluated the neutralization of ZF2001-elicited antisera to SARS-CoV-2 variants including all four variants of concern (Alpha, Beta, Gamma and Delta) and other three variants of interest (Epsilon, Eta and Kappa) by pseudovirus-based assay. We found antisera preserved majority of the neutralizing activity against these variants. E484K/Q substitution is the key mutation to reduce the RBD-elicited sera neutralization. Moreover, ZF2001-elicited sera with a prolonged intervals between the second and third dose enhanced the neutralizing titers and resilience to SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.07.15.452504","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.07.449660","pub_date":"2021-7-15","title":"Defining the Immune Responses for SARS-CoV-2-Human Macrophage Interactions","abstract":"Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end organ malfunctions. All follow an abortive viral infection. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding consequent end-organ tissue damage.","version":"1.3","doi":"10.1101/2021.07.07.449660","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.15.452488","pub_date":"2021-7-15","title":"Monitoring the spread of SARS-CoV-2 variants in Moscow and the Moscow region using targeted high-throughput sequencing","abstract":"Since the outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus, the international community has been concerned about the emergence of mutations that alter the biological properties of the pathogen, for example, increasing its infectivity or virulence. In particular, since the end of 2020, several variants of concern have been identified around the world, including variants \u201calpha\u201d (B.1.1.7, \u201cBritish\u201d), \u201cbeta\u201d (B.1.351, \u201cSouth African\u201d), \u201cgamma\u201d (P.1, \u201cBrazilian\u201d) and \u201cdelta\u201d (B.1.617.2, \u201cIndian\u201d). However, the existing mechanism for searching for important mutations and identifying strains may not be effective enough, since only a relatively small fraction of all identified pathogen samples can be examined for genetic changes by whole genome sequencing due to its high cost. In this study, we used the method of targeted high-throughput sequencing of the most significant regions of the gene encoding the S-glycoprotein of the SARS-CoV-2 virus, for which a primer panel was developed. Using this technique, we examined 579 random samples obtained from patients in Moscow and the Moscow region with coronavirus infection from February to June 2021. The study demonstrated the dynamics of the representation in the Moscow region of a number of SARS-CoV-2 strains and its most significant individual mutations in the period from February to June 2021. It was found that the strain B.1.617.2 began to spread rapidly in Moscow and the Moscow region in May, and in June it became dominant, partially displacing other varieties of the virus. The results obtained make it possible to accurately determine the belonging of the samples to the abovementioned and some other strains. The approach can be used to standardize the procedure for searching for new and existing epidemiologically significant mutations in certain regions of the SARS-CoV-2 genome, which allows studying a large number of samples in a short time and to get a more detailed picture of the epidemiological situation in the region.","version":"1.1","doi":"10.1101/2021.07.15.452488","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.308098","pub_date":"2021-7-15","title":"Rapid, dose-dependent and efficient inactivation of surface dried SARS-CoV-2 by 254 nm UV-C irradiation","abstract":"The SARS-CoV-2 pandemic urges for cheap, reliable, and rapid technologies for disinfection and decontamination. One frequently proposed method is UV-C irradiation. However, UV-C doses necessary to achieve inactivation of high-titer SARS-CoV-2 are poorly defined. Using a box and two handheld systems designed to decontaminate objects and surfaces we evaluated the efficacy of 254 nm UV-C treatment to inactivate surface dried SARS-CoV-2. Drying for two hours did not have a major impact on the infectivity of SARS-CoV-2, indicating that exhaled virus in droplets or aerosols stays infectious on surfaces at least for a certain amount of time. Short exposure of high titer surface dried virus (3-5*10^6 IU/ml) with UV-C light (16 mJ/cm2) resulted in a total inactivation of SARS-CoV-2. Dose-dependency experiments revealed that 3.5 mJ/cm2 were still effective to achieve a > 6-log reduction in viral titers whereas 1.75 mJ/cm2 lowered infectivity only by one order of magnitude. Our results demonstrate that SARS-CoV-2 is rapidly inactivated by relatively low doses of UV-C irradiation. Furthermore, the data reveal that the relationship between UV-C dose and log-viral titer reduction of surface residing SARS-CoV-2 is non-linear. In the context of UV-C-based technologies used to disinfect surfaces, our findings emphasize the necessity to assure sufficient and complete exposure of all relevant areas by integrated UV-C doses of at least 3.5 mJ/cm2 at 254 nm. Altogether, UV-C treatment is an effective non-chemical possibility to decontaminate surfaces from high-titer infectious SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.09.22.308098","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.14.452381","pub_date":"2021-7-15","title":"Single cell profiling of T and B cell repertoires following SARS-CoV-2 mRNA vaccine","abstract":"mRNA based vaccines for SARS-CoV-2 have shown exceptional clinical efficacy providing robust protection against severe disease. However, our understanding of transcriptional and repertoire changes following full vaccination remains incomplete. We used single-cell RNA sequencing and functional assays to compare humoral and cellular responses to two doses of mRNA vaccine with responses observed in convalescent individuals with asymptomatic disease. Our analyses revealed enrichment of spike-specific B cells, activated CD4 T cells, and robust antigen-specific polyfunctional CD4 T cell responses in all vaccinees. On the other hand, CD8 T cell responses were both weak and variable. Interestingly, clonally expanded CD8 T cells were observed in every vaccinee, as observed following natural infection. TCR gene usage, however, was variable, reflecting the diversity of repertoires and MHC polymorphism in the human population. Natural infection induced expansion of larger CD8 T cell clones occupied distinct clusters, likely due to the recognition of a broader set of viral epitopes presented by the virus not seen in the mRNA vaccine. Our study highlights a coordinated adaptive immune response where early CD4 T cell responses facilitate the development of the B cell response and substantial expansion of effector CD8 T cells, together capable of contributing to future recall responses.","version":"1.1","doi":"10.1101/2021.07.14.452381","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.15.452072","pub_date":"2021-7-15","title":"Chromatin alterations in the aging lung change progenitor cell activity","abstract":"The lung contains multiple progenitor cell types that respond to damage, but how their responses are choreographed and why they decline with age is poorly understood. We report that histone H3 lysine 9 di-methylation (K9me2), mediated by histone methyltransferase G9a, regulates the dynamics of lung epithelial progenitor cells, and this regulation deteriorates with age. In aged mouse lungs, K9me2 loss coincided with lower frequency and activity of alveolar type 2 (AT2) cell progenitors. In contrast, K9me2 loss resulted in increased frequency and activity of multipotent progenitor cells with bronchiolar and alveolar potential (BASCs) and bronchiolar progenitors. K9me2 depletion in young mice through deletion or inhibition of G9a decreased AT2 progenitor activity and impaired alveolar injury regeneration. Conversely, K9me2 depletion increased chromatin accessibility of bronchiolar cell genes, increased BASC frequency and accelerated bronchiolar repair. K9me2 depletion also resulted in increased bronchiolar cell expression of the SARS-CoV2 receptor Ace2 in aged lungs. These data point to K9me2 and G9a as a critical regulator of the balance of lung progenitor cell regenerative responses and prevention of susceptibility to age-related lung diseases. These findings indicate that epigenetic regulation coordinates progenitor cell populations to expedite regeneration in the most efficient manner and disruption of this regulation presents significant challenges to lung health.","version":"1.1","doi":"10.1101/2021.07.15.452072","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452256","pub_date":"2021-7-14","title":"Tissue Specific Age Dependence of the Cell Receptors Involved in the SARS-CoV-2 Infection","abstract":"The coronavirus disease 2019 (COVID-19) pandemic has affected tens of millions of individuals and caused hundreds of thousands of deaths worldwide. Due to its rapid surge, there is a shortage of information on viral behavior and host response after SARS-CoV-2 infection. Here we present a comprehensive, multiscale network analysis of the transcriptional response to the virus. We particularly focus on key-regulators, cell-receptors, and host-processes that are hijacked by the virus for its advantage. ACE2-controlled processes involve a key-regulator CD300e (a TYROBP receptor) and the activation of IL-2 pro-inflammatory cytokine signaling. We further investigate the age-dependency of such receptors and identify the adipose and the brain as potentially contributing tissues for the disease\u2019s severity in old patients. In contrast, several other tissues in the young population are more susceptible to SARS-CoV-2 infection. In summary, this present study provides novel insights into the gene regulatory organization during the SARS-CoV-2 infection and the tissue-specific age dependence of the cell receptors involved in COVID-19.","version":"1.1","doi":"10.1101/2021.07.13.452256","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452288","pub_date":"2021-7-14","title":"hnRNPA1 regulates early translation to replication switch in SARS-CoV-2 life cycle","abstract":"Our study suggests that methylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for its optimal replication in the target cells. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1, an RNA-binding protein) was shown to mediate deposition of N6-methyladenosine (m6A) in internal SARS-CoV-2 RNA. The levels of hNRNPA1 expression and extent of methylation varied, depending on the course of SARS-CoV-2 life cycle. The recruitment of eIF4E (translational initiation factor) facilitated viral RNA translation at 1 hour post infection (1 hpi). However, at 2 hpi, methylation of internal SARS-CoV-2 RNA recruited hNRNPA1 which facilitated viral RNA transcription but resulted in translational repression, a phenomenon contributing in understanding the early translation to replication switch in the viral life cycle. Besides, the abrogation of methylation also produced a defective 5\u2019 cap of viral RNA which failed to interact with eIF4E, thereby resulting in a decreased synthesis of viral proteins. To conclude, methylation of the internal and 5\u2019 cap of SARS-CoV-2 RNA was shown to regulate transcription and translation of SARS-CoV-2 in a time dependent manner. RNA modifications are found in all life forms and have been linked to development, health and diseases. Our study reveals that internal SARS-CoV-2 RNA methylation (m6A) is essential for interaction with hNRNPA1 to effectively synthesize viral genome. Besides, m6A-marked RNA and hRNPA1 interaction was also shown to regulate early translation to replication switch in SARS-CoV-2 life cycle. Blocking SARS-CoV-2 RNA methylation resulted in reduced virus yield, suggesting epitranscriptomic machinery (methylation) facilitates SARS-CoV-2 replication and might represent potential target for new antiviral drugs against COVID-19.","version":"1.1","doi":"10.1101/2021.07.13.452288","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452160","pub_date":"2021-7-14","title":"Adaptation, spread and transmission of SARS-CoV-2 in farmed minks and related humans in the Netherlands","abstract":"In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and related humans on farms. High number of farm infections (68/126) in minks and farm related personnel (>50% of farms) were detected, with limited spread to the general human population. Three of five initial introductions of SARS-CoV-2 lead to subsequent spread between mink farms until November 2020. The largest cluster acquired a mutation in the receptor binding domain of the Spike protein (position 486), evolved faster and spread more widely and longer. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combing genetic information with epidemiological information at the animal-human interface.","version":"1.1","doi":"10.1101/2021.07.13.452160","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452251","pub_date":"2021-7-14","title":"A modular protein subunit vaccine candidate produced in yeast confers protection against SARS-CoV-2 in non-human primates","abstract":"Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>104) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log10 reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.","version":"1.1","doi":"10.1101/2021.07.13.452251","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.14.452313","pub_date":"2021-7-14","title":"Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant","abstract":"Since its outbreak in 2019 SARS-CoV-2 has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure [1, 2]. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been widely replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates [3, 4]. In order to find an explanation, why SARS-CoV-2 and its emerging mutated versions showed enhanced transfection efficiency as compared to SARS-CoV 2002, an improved binding affinity of the spike protein to human ACE has been proposed by crystal structure analysis and was identified in cell culture models [5-7]. Kinetic analysis of the interaction of various spike protein constructs with the human ACE2 was considered to be best described by a Langmuir based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with ACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of \u223c190 with an overall KD of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wt but is also found in B.1.1.7 where its transition rate is five-fold increased.","version":"1.1","doi":"10.1101/2021.07.14.452313","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452259","pub_date":"2021-7-14","title":"Xeno-nucleic Acid (XNA) 2\u2019-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding","abstract":"The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor binding domain (RBD) on the viral S protein with angiotensin converting enzyme 2 (ACE2) on the surface of human host cells. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2\u2019-fluoroarabinonucleic acid (FANA). The best selected ~ 79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~ 10-20 nM and a binding half-life for the RBD of 53 \u00b1 18 minutes. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.","version":"1.1","doi":"10.1101/2021.07.13.452259","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.14.452343","pub_date":"2021-7-14","title":"Repurposing screen highlights broad-spectrum coronavirus antivirals and their host targets","abstract":"Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiologic processes in humans, thus providing unique opportunities for discovery of host targeting antivirals. We interrogated the ReFRAME repurposing library with 12,993 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus, mapping to 59 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor and dopamine receptor and cyclin-dependent kinase inhibitors. Counter-screening with SARS-CoV-2 and validation in primary cells identified Phortress, an aryl hydrocarbon receptor (AHR) ligand, Bardoxolone and Omaveloxolone, two nuclear factor, erythroid 2 like 2 (NFE2L2) activators as inhibitors of both alpha- and betacoronaviruses. The landscape of coronavirus targeting molecules provides important information for the development of broad-spectrum antivirals reinforcing pandemic preparedness.","version":"1.1","doi":"10.1101/2021.07.14.452343","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.12.452076","pub_date":"2021-7-13","title":"Analysis of amino acid change dynamics reveals SARS-CoV-2 variant emergence","abstract":"Since its emergence in late 2019, the diffusion of SARS-CoV-2 is associated with the evolution of its viral genome. The co-occurrence of specific amino acid changes, collectively named \u2018virus variant\u2019, requires scrutiny (as variants may hugely impact the agent\u2019s transmission, pathogenesis, or antigenicity); variant evolution is studied using phylogenetics. Yet, never has this problem been tackled by digging into data with ad hoc analysis techniques. Here we show that the emergence of variants can in fact be traced through data-driven methods, further capitalizing on the value of large collections of SARS-CoV-2 sequences. For all countries with sufficient data, we compute weekly counts of amino acid changes, unveil time-varying clusters of changes with similar \u2013 rapidly growing \u2013 dynamics, and then follow their evolution. Our method succeeds in timely associating clusters to variants of interest/concern, provided their change composition is well characterized. This allows us to detect variants\u2019 emergence, rise, peak, and eventual decline under competitive pressure of another variant. Our early warning system, exclusively relying on deposited sequences, shows the power of big data in this context, and concurs to calling for the wide spreading of public SARS-CoV-2 genome sequencing for improved surveillance and control of the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.07.12.452076","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.15.448551","pub_date":"2021-7-13","title":"Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro","abstract":"Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with Zinc demonstrate better clinical outcome. The molecular target and mechanistic details of anti-coronaviral activity of Zinc remain obscure. We show that ionic Zinc not only inhibits SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of Mpro-Zn2+ complex at 1.9 \u00c5 and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.","version":"1.2","doi":"10.1101/2021.06.15.448551","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.12.452027","pub_date":"2021-7-13","title":"Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine","abstract":"The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.","version":"1.1","doi":"10.1101/2021.07.12.452027","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.03.438321","pub_date":"2021-7-13","title":"A daily-updated database and tools for comprehensive SARS-CoV-2 mutation-annotated trees","abstract":"The vast scale of SARS-CoV-2 sequencing data has made it increasingly challenging to comprehensively analyze all available data using existing tools and file formats. To address this, we present a database of SARS-CoV-2 phylogenetic trees inferred with unrestricted public sequences, which we update daily to incorporate new sequences. Our database uses the recently-proposed mutation-annotated tree (MAT) format to efficiently encode the tree with branches labeled with parsimony-inferred mutations as well as Nextstrain clade and Pango lineage labels at clade roots. As of June 9, 2021, our SARS-CoV-2 MAT consists of 834,521 sequences and provides a comprehensive view of the virus\u2019 evolutionary history using public data. We also present matUtils \u2013 a command-line utility for rapidly querying, interpreting and manipulating the MATs. Our daily-updated SARS-CoV-2 MAT database and matUtils software are available at http://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/ and https://github.com/yatisht/usher, respectively.","version":"1.2","doi":"10.1101/2021.04.03.438321","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449831","pub_date":"2021-7-13","title":"Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses","abstract":"The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.","version":"1.2","doi":"10.1101/2021.06.25.449831","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452154","pub_date":"2021-7-13","title":"Interaction between SARS-CoV-2 spike glycoprotein and human skin models: a molecular dynamics study","abstract":"The possibility of contamination of human skin by infectious virions plays an important role in indirect transmission of respiratory viruses but little is known about the fundamental physico-chemical aspects of the virus-skin interactions. In the case of coronaviruses, the interaction with surfaces (including the skin surface) is mediated by their large glycoprotein spikes that protrude from (and cover) the viral envelope. Here, we perform all atomic simulations between the SARS-CoV-2 spike glycoprotein and human skin models. We consider an \u201coily\u201d skin covered by sebum and a \u201cclean\u201d skin exposing the stratum corneum. The simulations show that the spike tries to maximize the contacts with stratum corneum lipids, particularly ceramides, with substantial hydrogen bonding. In the case of \u201coily\u201d skin, the spike is able to retain its structure, orientation and hydration over sebum with little interaction with sebum components. Comparison of these results with our previous simulations of the interaction of SARS-CoV-2 spike with hydrophilic and hydrophobic solid surfaces, suggests that the\u201dsoft\u201d or \u201chard\u201d nature of the surface plays an essential role in the interaction of the spike protein with materials.","version":"1.1","doi":"10.1101/2021.07.13.452154","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.13.452175","pub_date":"2021-7-13","title":"Development of the Inactivated QazCovid-in Vaccine: Protective Efficacy of the Vaccine in Syrian Hamsters","abstract":"In March 2020, the first cases of human coronavirus infection COVID-19 were registered in Kazakhstan. We isolated the SARS-CoV-2 virus from the clinical material from the patients. Subsequently, a whole virion inactivated candidate vaccine, QazCovid-in, was developed based on this virus. To obtain the vaccine, a virus grown in Vero cell culture was used, which was inactivated with formaldehyde, purified, concentrated, sterilized by filtration, and then sorbed on aluminum hydroxide gel particles. The formula virus and adjuvant in buffer saline solution was used as a vaccine. The safety and protective effectiveness of the developed vaccine was studied on Syrian hamsters. The results of the studies showed the absolute safety of the candidate vaccine on the Syrian hamsters. When studying the protective effectiveness, the developed vaccine with an immunizing dose of 5 mcg/dose of a specific antigen protected animals from wild virus at a dose of 104.5 TCID50/ml. The candidate vaccine formed virus-neutralizing antibodies in vaccinated hamsters in titers from 3.3 \u00b1 1.45 log2 to 7.25 \u00b1 0.78 log2, which were retained for 6 months (observation period) in the indicated titers. The candidate vaccine suppressed the replication of the wild virus in the body of vaccinated hamsters, protected against the development of acute pneumonia and ensured 100% survival of the animals. At the same time, no replicative virus was isolated from the lungs of vaccinated animals. At the same time, a virulent virus was isolated from the lungs of unvaccinated animals in relatively high titers, reaching 4.5 \u00b1 0.7 lg TCID50/ml. After challenge infection, 100% of unvaccinated hamsters became ill with clinical signs (stress state, passivity, tousled coat, decreased body temperature and body weight, and the development of acute pneumonia), of which 25 \u00b1 5% were fatal. The findings paved the way for testing the candidate vaccine in humans in clinical trials.","version":"1.1","doi":"10.1101/2021.07.13.452175","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.11.451964","pub_date":"2021-7-12","title":"Pathogenic and transcriptomic differences of emerging SARS-CoV-2 variants in the Syrian golden hamster model","abstract":"Following the discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid spread throughout the world, new viral variants of concern (VOC) have emerged. There is a critical need to understand the impact of the emerging variants on host response and disease dynamics to facilitate the development of vaccines and therapeutics. Syrian golden hamsters are the leading small animal model that recapitulates key aspects of severe coronavirus disease 2019 (COVID-19). In this study, we show that intranasal inoculation of SARS-CoV-2 into hamsters with the ancestral virus (nCoV-WA1-2020) or VOC first identified in the United Kingdom (B.1.1.7) and South Africa (B.1.351) led to similar gross and histopathologic pulmonary lesions. Although differences in viral genomic copy numbers were noted in the lungs and oral swabs of challenged animals, infectious titers in the lungs were comparable. Antibody neutralization capacities varied, dependent on the original challenge virus and cross-variant protective capacity. Transcriptional profiling indicated significant induction of antiviral pathways in response to all three challenges with a more robust inflammatory signature in response to B.1.1.7. Furthermore, no additional mutations in the spike protein were detected at peak disease. In conclusion, the emerging VOC showed distinct humoral responses and transcriptional profiles in the hamster model compared to the ancestral virus.","version":"1.1","doi":"10.1101/2021.07.11.451964","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.11.451951","pub_date":"2021-7-12","title":"Niclosamide reverses SARS-CoV-2 control of lipophagy","abstract":"The global effort to combat COVID-19 rapidly produced a shortlist of approved drugs with anti-viral activities for clinical repurposing. However, the jump to clinical testing was lethal in some cases as a full understanding of the mechanism of antiviral activity as opposed to pleiotropic activity/toxicity for these drugs was lacking. Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of infected Vero E6 cells, especially plasmalogens that correlated with increased levels of virus replication. Niclosamide (NIC), a poorly soluble anti-helminth drug identified for repurposed treatment of COVID-19, reduced the total lipid profile that would otherwise amplify during virus infection. NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which are required for virus production. Future screens of approved drugs may identify more druggable compounds than NIC that can safely but effectively counter SARS-CoV-2 subversion of lipid metabolism thereby reducing virus replication. However, these data support the consideration of niclosamide as a potential COVID-19 therapeutic given its modulation of lipophagy leading to the reduction of virus egress and the subsequent regulation of key lipid mediators of pathological inflammation.","version":"1.1","doi":"10.1101/2021.07.11.451951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.10.451922","pub_date":"2021-7-12","title":"High rate of mutational events in SARS-CoV-2 genomes across Brazilian geographical regions, February 2020 to June 2021","abstract":"Brazil has been considered as one of the emerging epicenters of the coronavirus pandemic in 2021, experiencing over 3,000 daily deaths caused by the virus at the peak of the second wave. In total, the country has more than 19.2 million confirmed cases of Covid-19, including over 533,509 fatalities. A set of emerging variants arose in the country, some of them posing new challenges for COVID-19 control. The goal of this study was to describe mutational events across samples from Brazilian SARS-CoV-2 sequences openly publicly obtainable on the Global Initiative on Sharing Avian Influenza Data-EpiCoV (GISAID-EpiCoV) platform and generate an index of new mutant by each genome. A total of 16,953 SARS-CoV-2 genomes were obtained and are not proportionally representative of the five Brazilian geographical regions. A comparative sequence analysis was conducted to identify common mutations located at 42 positions of the genome (38 were in coding regions whereas two in 5\u2019 and two in 3\u2019 UTR). Moreover, 11 were synonymous and 27 missense variants, and more than 44.4% were located in the spike gene. Across the total of single nucleotide variations (SNVs) identified, 32 were found in genomes obtained from all five Brazilian regions. While a high genomic diversity is reported in Europe given the large number of sequenced genomes, Africa is emerging as a hotspot for new variants. In South America, Brazil and Chile, rates are similar to those found in South Africa and India, giving enough space to generate new viral mutations. Genomic surveillance is the central key to identifying the emerging variants of SARS-CoV-2 in Brazil and has shown that the country is one of the \u201chotspots\u201d in the generation of new variants.","version":"1.1","doi":"10.1101/2021.07.10.451922","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.12.452002","pub_date":"2021-7-12","title":"SARS-CoV-2 Spike Protein Mutations and Escape from Antibodies: a Computational Model of Epitope Loss in Variants of Concern","abstract":"The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 Spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.","version":"1.1","doi":"10.1101/2021.07.12.452002","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.12.452021","pub_date":"2021-7-12","title":"Drug-free nasal spray as a barrier against SARS-CoV-2 infection: safety and efficacy in human nasal airway epithelia","abstract":"For SARS-CoV-2 and other respiratory viruses, the nasal epithelium is a key portal for infection. Therefore, the nose is an important target of prophylactic and therapeutic interventions against these viruses. We developed a nasal spray (AM-301, a medical device marketed as Bentrio) to protect against infection by SARS-CoV-2 and potentially other viruses. To test the safety and efficacy of AM-301 against SARS-CoV-2 infection. AM-301 was tested on an in vitro 3D model of primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in prophylaxis and infection mitigation assays. AM-301 did not have any detrimental effect on the nasal epithelium. Prophylactic treatment with AM-301 reduced viral titer significantly vs. controls over 4 days, reaching a maximum reduction of 99%. When treatment with AM-301 was started 24 or 30 h after infection, epithelia that received the formulation had a 12- or 14-fold lower titer than controls. AM-301 was found to be safe in vitro, and it significantly decelerated viral titer growth in experimental models of prophylaxis and mitigation. Its physical (non-pharmaceutical) mechanism of action, safety and efficacy pave the way for further investigation of its possible use against a broad spectrum of viruses, allergens and pollutants.","version":"1.1","doi":"10.1101/2021.07.12.452021","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.11.451855","pub_date":"2021-7-11","title":"The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes","abstract":"Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation\u2014an open trimer\u2014 contains easily accessible RBDs. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines. An alternative conformation of SARS-CoV-2 spike ectodomain modulated by temperature, binding, and sequence variants.","version":"1.1","doi":"10.1101/2021.07.11.451855","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.10.451880","pub_date":"2021-7-10","title":"A mouse-adapted SARS-CoV-2 strain replicating in standard laboratory mice","abstract":"SARS-CoV-2 has infected almost 200 million humans and caused over 4 million deaths worldwide. Evaluating countermeasures and improving our understanding of COVID-19 pathophysiology require access to animal models that replicate the hallmarks of human disease. Mouse infection with SARS-CoV-2 is limited by poor affinity between the virus spike protein and its cellular receptor ACE2. We have developed by serial passages the MACo3 virus strain which efficiently replicates in the lungs of standard mouse strains and induces age-dependent lung lesions. Compared to other mouse-adapted strains and severe mouse models, infection with MACo3 results in mild to moderate disease and will be useful to investigate the role of host genetics and other factors modulating COVID-19 severity.","version":"1.1","doi":"10.1101/2021.07.10.451880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.24.449840","pub_date":"2021-7-10","title":"Phylogenetic network analysis revealed the recombinant origin of the SARS-CoV-2 VOC202012/01 (B.1.1.7) variant first discovered in U.K.","abstract":"The emergence of new variants of the SARS-CoV-2 virus poses serious problems to the control of the current COVID-19 pandemic. Understanding how the variants originate is critical for effective control of the spread of the virus and the global pandemic. The study of the virus evolution so far has been dominated by phylogenetic tree analysis, which however is inappropriate for a few important reasons. Here we used phylogenetic network approach to study the origin of the VOC202012/01 (Alpha) or so-called UK variant (PANGO Lineage B.1.1.7). The multiple network analyses using different methods consistently revealed that the VOC202012/01 variant was a result of recombination, in contrast to the common assumption that the variant evolved from step-wise mutations in a linear order. The study provides an example for the power and application of phylogenetic network analysis in studying virus evolution, which can be applied to study the evolutionary processes leading to the emergence of other variants of the SARS-CoV-2 virus as well as many other viruses. The emergence of new variants of the SARS-CoV-2 virus, including the Alpha variant first found in U.K., poses serious challenges to the control of the current COVID-19 pandemic. Understanding how new variant originated is paramount to end the pandemic as effectively and quickly as possible. The dominant phylogenetic tree approach to study virus evolution has been inadequate and even misleading. Here we used a phylogenetic network approach to study the origin of the VOC202012/01 (Alpha) variant which was first reported in U.K. last year but has soon spread into many other countries, leading to dramatic increase in infection and death. Multiple analyses consistently revealed that the variant originated through recombination of pre-existing virus strains, highlighting an important but largely ignored mechanism in the evolution of the SARS-CoV-2 virus so far.","version":"1.2","doi":"10.1101/2021.06.24.449840","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.09.451732","pub_date":"2021-7-09","title":"Reduced neutralization of SARS-CoV-2 B.1.617 variant by inactivated and RBD-subunit vaccine","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.07.09.451732","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.01.450756","pub_date":"2021-7-09","title":"Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E","abstract":"Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus, however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1\u2019, S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from \u22128.8 to \u22127.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from \u22127.1 to \u22127.8. In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 \u00b5M. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.","version":"1.2","doi":"10.1101/2021.07.01.450756","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435440","pub_date":"2021-7-09","title":"Rationally designed immunogens enable immune focusing to the SARS-CoV-2 receptor binding motif","abstract":"Eliciting antibodies to surface-exposed viral glycoproteins can lead to protective responses that ultimately control and prevent future infections. Targeting functionally conserved epitopes may help reduce the likelihood of viral escape and aid in preventing the spread of related viruses with pandemic potential. One such functionally conserved viral epitope is the site to which a receptor must bind to facilitate viral entry. Here, we leveraged rational immunogen design strategies to focus humoral responses to the receptor binding motif (RBM) on the SARS-CoV-2 spike. Using glycan engineering and epitope scaffolding, we find an improved targeting of the serum response to the RBM in context of SARS-CoV-2 spike imprinting. Furthermore, we observed a robust SARS-CoV-2-neutralizing serum response with increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses and represents an adaptable design approach for targeting conserved epitopes on other viral glycoproteins. SARS-CoV-2 immune focusing with engineered immunogens","version":"1.2","doi":"10.1101/2021.03.15.435440","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.09.451812","pub_date":"2021-7-09","title":"Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2","abstract":"The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.","version":"1.1","doi":"10.1101/2021.07.09.451812","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450133","pub_date":"2021-7-09","title":"Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences","abstract":"Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. By detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain. \n\nSingle molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics\nThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replication\nGenomic RNA is stable and persistent during the initial stages of infection\nB.1.1.7 variant replicates more slowly than the Victoria strain\n Single molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics There is substantial heterogeneity between cells in rates of SARS-CoV-2 replication Genomic RNA is stable and persistent during the initial stages of infection B.1.1.7 variant replicates more slowly than the Victoria strain","version":"1.2","doi":"10.1101/2021.06.29.450133","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448421","pub_date":"2021-7-09","title":"MMMVI: Detecting SARS-CoV-2 Variants of Concern in Metagenomic Wastewater Samples","abstract":"SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Variants of Concern (VOCs) and Variants of Interest (VOIs) are lineages that represent a greater risk to public health, and can be differentiated from the wildtype virus based on unique profiles of signature mutations. Metagenomic sequence analysis of wastewater represents an emerging form of surveillance that can capture early signal for these variants in a community prior to detection through public health testing or genomic surveillance activities. However, because multiple viral genomes are likely to be present in a metagenomic sample, additional analytical scrutiny of the sequencing reads beyond variant calling is needed to increase confidence in diagnostic determinations. Where multiple signature mutations are present on a given read, they can be used as enhanced biomarkers to confirm the presence of a VOC/VOI in the sample. We have developed MMMVI, a tool to aggregate and report on the likely presence of a VOC/VOI in a set of metagenomic reads based on the detection of reads bearing multiple signature mutations. MMMVI is implemented in Python, and is available under the MIT licence from https://github.com/dorbarker/voc-identify/ dillon.barker@canada.ca","version":"1.2","doi":"10.1101/2021.06.14.448421","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429458","pub_date":"2021-7-09","title":"Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses","abstract":"Exposure to a pathogen elicits an adaptive immune response aimed to control and eradicate. Interrogating the abundance and specificity of the naive B cell repertoire contributes to understanding how to potentially elicit protective responses. Here, we isolated naive B cells from 8 seronegative human donors targeting the SARS-CoV-2 receptor-binding domain (RBD). Single B cell analysis showed diverse gene usage with no restricted complementarity determining region lengths. We show that recombinant antibodies engage SARS-CoV-2 RBD, circulating variants, and pre-emergent coronaviruses. Representative antibodies signal in a B cell activation assay and can be affinity matured through directed evolution. Structural analysis of a naive antibody in complex with spike shows a conserved mode of recognition shared with infection-induced antibodies. Lastly, both naive and affinity-matured antibodies can neutralize SARS-CoV-2. Understanding the naive repertoire may inform potential responses recognizing variants or emerging coronaviruses enabling the development of pan-coronavirus vaccines aimed at engaging germline responses. Isolation of antibody germline precursors targeting the receptor binding domain of coronaviruses.","version":"1.2","doi":"10.1101/2021.02.02.429458","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438035","pub_date":"2021-7-09","title":"The neutralization potency of anti-SARS-CoV-2 therapeutic human monoclonal antibodies is retained against novel viral variants","abstract":"A wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported to date, most of which target the spike glycoprotein and in particular its receptor binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit. The therapeutic implementation of these antibodies has been recently challenged by emerging SARS-CoV-2 variants that harbor extensively mutated spike versions. Consequently, the re-assessment of mAbs, previously reported to neutralize the original early-version of the virus, is of high priority. Four previously selected mAbs targeting non-overlapping epitopes, were evaluated for their binding potency to RBD versions harboring individual mutations at spike positions 417, 439, 453, 477, 484 and 501. Mutations at these positions represent the prevailing worldwide distributed modifications of the RBD, previously reported to mediate escape from antibody neutralization. Additionally, the in vitro neutralization potencies of the four RBD-specific mAbs, as well as two NTD-specific mAbs, were evaluated against two frequent SARS-CoV-2 variants of concern (VOCs): (i) the B.1.1.7 variant, emerged in the UK and (ii) the B.1.351 variant, emerged in South Africa. Variant B.1.351 was previously suggested to escape many therapeutic mAbs, including those authorized for clinical use. The possible impact of RBD mutations on recognition by mAbs is addressed by comparative structural modelling. Finally, we demonstrate the therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each of the virus strains. Our results clearly indicate that despite the accumulation of spike mutations, some neutralizing mAbs preserve their potency against SARS-CoV-2. In particular, the highly potent MD65 and BL6 mAbs are shown to retain their ability to bind the prevalent novel viral mutations and to effectively protect against B.1.1.7 and B.1.351 variants of high clinical concern.","version":"1.3","doi":"10.1101/2021.04.01.438035","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439051","pub_date":"2021-7-09","title":"Host cell membrane capture by the SARS CoV-2 spike protein fusion intermediate","abstract":"Cell entry by SARS-CoV-2 is accomplished by the S2 subunit of the spike S protein on the virion surface by capture of the host cell membrane and fusion with the viral envelope. Capture and fusion require the prefusion S2 to transit to its potent, fusogenic form, the fusion intermediate (FI). However, the FI structure is unknown, detailed computational models of the FI are unavailable, and the mechanisms and timing of membrane capture and fusion are not established. Here, we constructed a full-length model of the CoV-2 FI by extrapolating from known CoV-2 pre- and postfusion structures. In atomistic and coarse-grained molecular dynamics simulations the FI was remarkably flexible and executed large bending and extensional fluctuations due to three hinges in the C-terminal base. Simulations suggested a host cell membrane capture time of \u223c 2 ms. Isolated fusion peptide simulations identified an N-terminal helix that directed and maintained binding to the membrane but grossly underestimated the binding time, showing that the fusion peptide environment is radically altered when attached to its host fusion protein. The large configurational fluctuations of the FI generated a substantial exploration volume that aided capture of the target membrane, and may set the waiting time for fluctuation-triggered refolding of the FI that draws the viral envelope and host cell membrane together for fusion. These results describe the FI as a machinery designed for efficient membrane capture and suggest novel potential drug targets.","version":"1.3","doi":"10.1101/2021.04.09.439051","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.09.451770","pub_date":"2021-7-09","title":"A photoactivable natural product with broad antiviral activity against enveloped viruses including highly pathogenic coronaviruses","abstract":"The SARS-CoV-2 outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against HCoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and MERS-CoV, and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane as demonstrated by cryo-electron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses, and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2 contaminated surfaces.","version":"1.1","doi":"10.1101/2021.07.09.451770","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451696","pub_date":"2021-7-09","title":"Therapeutic efficacy of CT-P59 against P.1 variant of SARS-CoV-2","abstract":"P.1. or gamma variant also known as the Brazil variant, is one of the variants of concern (VOC) which appears to have high transmissibility and mortality. To explore the potency of the CT-P59 monoclonal antibody against P.1 variant, we tried to conduct binding affinity, in vitro neutralization, and in vivo animal tests. In in vitro assays revealed that CT-P59 is able to neutralize P.1 variant in spite of reduction in its binding affinity against a RBD (receptor binding domain) mutant protein including K417T/E484K/N501Y and neutralizing activity against P.1 pseudoviruses and live viruses. In contrast, in vivo hACE2 (human angiotensin-converting enzyme 2)-expressing TG (transgenic) mouse challenge experiment demonstrated that a clinically relevant or lower dosages of CT-P59 is capable of lowering viral loads in the respiratory tract and alleviates symptoms such as body weight losses and survival rates. Therefore, a clinical dosage of CT-P59 could compensate for reduced in vitro antiviral activity in P.1-infected mice, implying that CT-P59 has therapeutic potency for COVID-19 patients infected with P.1 variant. CT-P59 could bind to and neutralize P.1 variant, but CT-P59 showed reduced susceptibility in in vitro tests. The clinical dosage of CT-P59 demonstrated in vivo therapeutic potency against P.1 variants in hACE2-expressing mice challenge study. CT-P59 ameliorates their body weight loss and prevents the lethality in P.1 variant-infected mice.","version":"1.1","doi":"10.1101/2021.07.08.451696","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451607","pub_date":"2021-7-08","title":"Ultrastructural analysis of nasopharyngeal epithelial cells from patients with SARS-CoV-2 infection","abstract":"The nasal epithelium is an initial site for SARS-CoV-2 infection, responsible for the ongoing COVID-19 pandemic. However, the pathogenicity and morphological impact of SARS-CoV-2 on the nasopharynx cells from symptomatic patients with different viral loads remain poorly understood. Here, we investigated the ultrastructure of nasal cells obtained from individuals at distinct disease days and with high and low SARS-CoV-2 loads. Squamous and ciliated cells were the main cells observed in SARS-CoV-2 negative samples. We identified virus-like particles (VLPs) and replication organelles (RO)-like structures in the squamous cells from high viral load samples after 3- and 4-days of symptoms. Ultrastructural changes were found in those cells, such as the loss of microvilli and primary cilium, the increase of multivesicular bodies and autophagosomes, and signs of cell death. No ciliated cells were found in those samples. Squamous cells from low viral load sample after 5 days of symptoms showed few microvilli and no primary cilium. VLPs and RO-like structures were found in the ciliated cells only. No ultrastructural alterations were seen in the cells from low viral load individuals after 10- and 14-days of symptoms. Our results shed light on the ultrastructural effects of SARS-CoV-2 infection on the human nasopharyngeal cells.","version":"1.1","doi":"10.1101/2021.07.08.451607","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451555","pub_date":"2021-7-08","title":"In Silico Defined SARS-CoV2 Epitopes May Not Predict Immunogenicity to COVID19","abstract":"SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally, if not more important than humoral responses in mediating recovery and immune-protection. One of the major challenges in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities and consensus data. Our studies find that, in contrast to current dogma, \u2018immunodominant\u2019 SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes.","version":"1.1","doi":"10.1101/2021.07.08.451555","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451654","pub_date":"2021-7-08","title":"Evaluation of intranasal nafamostat or camostat for SARS-CoV-2 chemoprophylaxis in Syrian golden hamsters","abstract":"Successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention implementable alongside vaccination programmes. Camostat and nafamostat are serine protease inhibitors that inhibit SARS-CoV-2 viral entry in vitro but have not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and while camostat is orally available, both drugs have extremely short plasma half-lives. This study sought to determine whether intranasal dosing at 5 mg/kg twice daily was able to prevent airborne transmission of SARS-CoV-2 from infected to uninfected Syrian golden hamsters. SARS-CoV-2 viral RNA was above the limits of quantification in both saline- and camostat-treated hamsters 5 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, intranasal nafamostat-treated hamsters remained RNA negative for the full 7 days of cohabitation. Changes in body weight over the course of the experiment were supportive of a lack of clinical symptomology in nafamostat-treated but not saline- or camostat-treated animals. These data are strongly supportive of the utility of intranasally delivered nafamostat for prevention of SARS-CoV-2 infection and further studies are underway to confirm absence of pulmonary infection and pathological changes.","version":"1.1","doi":"10.1101/2021.07.08.451654","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451640","pub_date":"2021-7-08","title":"Insights into the mutation T1117I in the spike and the lineage B.1.1.389 of SARS-CoV-2 circulating in Costa Rica","abstract":"Emerging mutations and genotypes of the SARS-CoV-2 virus, responsible for the COVID-19 pandemic, have been reported globally. In Costa Rica during the year 2020, a predominant genotype carrying the mutation T1117I in the spike (S:T1117I) was previously identified. To investigate the possible effects of this mutation on the function of the spike, i.e. the biology of the virus, different bioinformatic pipelines based on phylogeny, natural selection and co-evolutionary models, molecular docking and epitopes prediction were implemented. Results of the phylogeny of sequences carrying the S:T1117I worldwide showed a polyphyletic group, with the emergency of local lineages. In Costa Rica, the mutation is found in the lineage B.1.1.389 and it is suggested to be a product of positive/adaptive selection. Different changes in the function of the spike protein and more stable interaction with a ligand (nelfinavir drug) were found. Only one epitope out 742 in the spike was affected by the mutation, with some different properties, but suggesting scarce changes in the immune response and no influence on the vaccine effectiveness. Jointly, these results suggest a partial benefit of the mutation for the spread of the virus with this genotype during the year 2020 in Costa Rica, although possibly not strong enough with the introduction of new lineages during early 2021 which became predominant later. In addition, the bioinformatics pipeline offers an integrative and exhaustive in silico strategy to eventually study other mutations of interest for the SARS-CoV-2 virus and other pathogens. In Costa Rica during the year 2020, a predominant SARS-CoV-2 genotype carrying the mutation T1117I in the spike (S:T1117I) was identified. The S:T1117I was assessed for possible effects of this mutation on the function of the spike with a in silico approach. Phylogeny revealed that sequences carrying the S:T1117I worldwide define a polyphyletic group, with the emergency of local lineages, including the lineage B.1.1.389 in Costa Rica. A positive/adaptive selection was identified for S:T1117I, with different changes in the function of the spike protein, more stable interaction with ligands and scarce changes in the immune response. The bioinformatics pipeline can be eventually used to study other mutations of the SARS-CoV-2 virus and other pathogens.","version":"1.1","doi":"10.1101/2021.07.08.451640","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451426","pub_date":"2021-7-08","title":"Antibody and T cell memory immune response after two doses of the BNT162b2 mRNA vaccine in older adults with and without prior SARS-CoV-2 infection","abstract":"We quantified S1-specific IgG, neutralizing antibody titers, specific IFN\u03b3 secreting T cells and functionality of specific CD4+ and CD8+ T cells in 130 young adults (median age 44.0 years) and 106 older residents living in a long-term care facility (86.5 years) after 2 doses of BNT162b2. Three months after the first injection, humoral and cellular memory responses were dramatically impaired in the 54 COVID-19-naive older compared to the 121 COVID-19-naive younger adults. Notably, older participants\u2019 neutralizing antibodies, detected in 76.5% (versus 100% in young adults, P < 0.0001), were ten times lower than the younger\u2019s antibody titers (P < 0.0001). Antibody and T cell responses were greater among the 52 COVID-19-recovered than among the 54 COVID-19-naive older adults (P < 0.0001). Our study shows that 2 doses of BNT162b2 does not guarantee long-term protection against SARS-CoV-2 in the older. An additional dose should be considered to boost their specific memory response.","version":"1.1","doi":"10.1101/2021.07.08.451426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.08.451649","pub_date":"2021-7-08","title":"Structure of a germline-like human antibody defines a neutralizing epitope on the SARS-CoV-2 spike NTD","abstract":"Structural characterization of infection- and vaccination-elicited antibodies in complex with antigen provides insight into the evolutionary arms race between the host and the pathogen and informs rational vaccine immunogen design. We isolated a germline-like monoclonal antibody (mAb) from plasmablasts activated upon mRNA vaccination against SARS-CoV-2 and determined its structure in complex with the spike glycoprotein by cryo-EM. We show that the mAb engages a previously uncharacterized neutralizing epitope on the spike N-terminal domain (NTD). The high-resolution structure reveals details of the intermolecular interactions and shows that the mAb inserts its HCDR3 loop into a hydrophobic NTD cavity previously shown to bind a heme metabolite, biliverdin. We demonstrate direct competition with biliverdin and that - because of the conserved nature of the epitope \u2013 the mAb maintains binding to viral variants B.1.1.7 and B.1.351. Our study illustrates the feasibility of targeting the NTD to achieve broad neutralization against SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.07.08.451649","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.07.451538","pub_date":"2021-7-08","title":"Atomistic Simulations and Deep Mutational Scanning of Protein Stability and Binding Interactions in the SARS-CoV-2 Spike Protein Complexes with Nanobodies: Molecular Determinants of Mutational Escape Mechanisms","abstract":"Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this work, we combined atomistic simulations and conformational dynamics analysis with the ensemble-based mutational profiling of binding interactions for a diverse panel of SARS-CoV-2 spike complexes with nanobodies. Using this computational toolkit we identified dynamic signatures and binding affinity fingerprints for the SARS-CoV-2 spike protein complexes with nanobodies Nb6 and Nb20, VHH E, a pair combination VHH E+U, a biparatopic nanobody VHH VE, and a combination of CC12.3 antibody and VHH V/W nanobodies. Through ensemble-based deep mutational profiling of stability and binding affinities, we identify critical hotspots and characterize molecular mechanisms of SARS-CoV-2 spike protein binding with single ultra-potent nanobodies, nanobody cocktails and biparatopic nanobodies. By quantifying dynamic and energetic determinants of the SARS-CoV-2 S binding with nanobodies, we also examine the effects of circulating variants and escaping mutations. We found that mutational escape mechanisms may be controlled through structurally and energetically adaptable binding hotspots located in the host receptor-accessible binding epitope that are dynamically coupled to the stability centers in the distant epitope targeted by VHH U/V/W nanobodies. The results of this study suggested a mechanism in which through cooperative dynamic changes, nanobody combinations and biparatopic nanobody can modulate the global protein response and induce the increased resilience to common escape mutants.","version":"1.1","doi":"10.1101/2021.07.07.451538","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.07.451411","pub_date":"2021-7-07","title":"CAT, AGTR2, L-SIGN and DC-SIGN are potential receptors for the entry of SARS-CoV-2 into human cells","abstract":"Since December 2019, the COVID-19 caused by SARS-CoV-2 has been widely spread all over the world. It is reported that SARS-CoV-2 infection affects a series of human tissues, including lung, gastrointestinal tract, kidney, etc. ACE2 has been identified as the primary receptor of the SARS-CoV-2 Spike (S) protein. The relatively low expression level of this known receptor in the lungs, which is the predominantly infected organ in COVID-19, indicates that there may be some other co-receptors or alternative receptors of SARS-CoV-2 to work in coordination with ACE2. Here, we identified twenty-one candidate receptors of SARS-CoV-2, including ACE2-interactor proteins and SARS-CoV receptors. Then we investigated the protein expression levels of these twenty-one candidate receptors in different human tissues and found that five of which CAT, MME, L-SIGN, DC-SIGN, and AGTR2 were specifically expressed in SARS-CoV-2 affected tissues. Next, we performed molecular simulations of the above five candidate receptors with SARS-CoV-2 S protein, and found that the binding affinities of CAT, AGTR2, L-SIGN and DC-SIGN to S protein were even higher than ACE2. Interestingly, we also observed that CAT and AGTR2 bound to S protein in different regions with ACE2 conformationally, suggesting that these two proteins are likely capable of the co-receptors of ACE2. Conclusively, we considered that CAT, AGTR2, L-SIGN and DC-SIGN were the potential receptors of SARS-CoV-2. Moreover, AGTR2 and DC-SIGN tend to be highly expressed in the lungs of smokers, which is consistent with clinical phenomena of COVID-19, and further confirmed our conclusion. Besides, we also predicted the binding hot spots for these putative protein-protein interactions, which would help develop drugs against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.07.07.451411","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.07.451375","pub_date":"2021-7-07","title":"Multivalent designed proteins protect against SARS-CoV-2 variants of concern","abstract":"Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2021.07.07.451375","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.06.451227","pub_date":"2021-7-07","title":"T-CoV: a comprehensive portal of HLA-peptide interactions affected by SARS-CoV-2 mutations","abstract":"Rapidly appearing SARS-CoV-2 mutations can affect T cell epitopes, which can help the virus to evade either CD8 or CD4 T-cell responses. We developed T-cell COVID-19 Atlas (T-CoV, https://t-cov.hse.ru) \u2013 the comprehensive web portal, which allows one to analyze how SARS-CoV-2 mutations alter the presentation of viral peptides by HLA molecules. The data are presented for common virus variants and the most frequent HLA class I and class II alleles. Binding affinities of HLA molecules and viral peptides were assessed with accurate in silico methods. The obtained results highlight the importance of taking HLA alleles diversity into account: mutation-mediated alterations in HLA-peptide interactions were highly dependent on HLA alleles. For example, we found that the essential number of peptides tightly bound to HLA-B*07:02 in the reference Wuhan variant ceased to be tight binders for the Indian (Delta) and the UK (Alpha) variants. In summary, we believe that T-CoV will help researchers and clinicians to predict the susceptibility of individuals with different HLA genotypes to infection with variants of SARS-CoV-2 and/or forecast its severity.","version":"1.1","doi":"10.1101/2021.07.06.451227","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.06.451340","pub_date":"2021-7-07","title":"Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection","abstract":"There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors ACE2 and TMPRSS2. Multiciliated cells are the primary initial target of SARS-CoV-2 infection. Upon infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses and treatment with remdesivir or camostat methylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia. Subsets of human iPSC-airway epithelial cells express SARS-Co-V entry factors ACE2 and TMPRSS2. iPSC-airway cells are permissive to SARS-CoV-2 infection via multiciliated cells. SARS-CoV-2 infection of iPSC-airway leads to a robust interferon and inflammatory response. iPSC-airway is a physiologically relevant model to study SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.07.06.451340","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.21259973","pub_date":"2021-07-07","title":"Critical Role of the Subways in the Initial Spread of SARS-CoV-2 in New York City","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>We studied the possible role of the subways in the spread of SARS-CoV-2 in New York City during late February and March 2020. Data on cases and hospitalizations, along with phylogenetic analyses of viral isolates, demonstrate rapid community transmission throughout all five boroughs within days. The near collapse of subway ridership during the second week of March was followed within 1-2 weeks by the flattening of COVID-19 incidence curve. We observed persistently high entry into stations located along the subway line serving a principal hotspot of infection in Queens. We used smartphone tracking data to estimate the volume of subway visits originating from each zip code tabulation area (ZCTA). Across ZCTAs, the estimated volume of subway visits on March 16 was strongly predictive of subsequent COVID-19 incidence during April 1-8. In a spatial analysis, we distinguished between the conventional notion of geographic contiguity and a novel notion of contiguity along subway lines. We found that the March 16 subway-visit volume in subway-contiguous ZCTAs had an increasing effect on COVID-19 incidence during April 1-8 as we enlarged the radius of influence up to 5 connected subway stops. By contrast, the March 31 cumulative incidence of COVID-19 in geographically-contiguous ZCTAs had an increasing effect on subsequent COVID-19 incidence as we expanded the radius up to 3 connected ZCTAs. The combined evidence points to the initial citywide dissemination of SARS-CoV-2 via a subway-based network, followed by percolation of new infections within local hotspots.</jats:p>","version":null,"doi":"10.1101/2021.07.03.21259973","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.06.451301","pub_date":"2021-7-07","title":"A Newcastle disease virus-vector expressing a prefusion-stabilized spike protein of SARS-CoV-2 induces protective immune responses against prototype virus and variants of concern in mice and hamsters","abstract":"Rapid development of coronavirus disease 2019 (COVID-19) vaccines and expedited authorization for use and approval has been proven beneficial to mitigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread and given hope in this desperate situation. It is believed that sufficient supplies and equitable allocations of vaccines are necessary to limit the global impact of the COVID-19 pandemic and the emergence of additional variants of concern. We have developed a COVID-19 vaccine based on Newcastle disease virus (NDV) that can be manufactured at high yields in embryonated eggs. Here we provide evidence that the NDV vector expressing an optimized spike antigen (NDV-HXP-S), upgraded from our previous construct, is a versatile vaccine that can be used live or inactivated to induce strong antibody responses and to also cross-neutralize variants of concern. The immunity conferred by NDV-HXP-S effectively counteracts SARS-CoV-2 infection in mice and hamsters. It is noteworthy that vaccine lots produced by existing egg-based influenza virus vaccine manufacturers in Vietnam, Thailand and Brazil exhibited excellent immunogenicity and efficacy in hamsters, demonstrating that NDV-HXP-S vaccines can be quickly produced at large-scale to meet global demands.","version":"1.1","doi":"10.1101/2021.07.06.451301","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.06.451357","pub_date":"2021-7-07","title":"SARS-CoV-2 genome sequencing with Oxford Nanopore Technology and Rapid PCR Barcoding in Bolivia","abstract":"SARS-CoV-2 genomic surveillance has Illumina technology as the golden standard. However, Oxford Nanopore Technology (ONT) provides significant improvements in accessibility, turnaround time and portability. Characteristics that gives developing countries the opportunity to perform genome surveillance. The most used protocol to sequence SARS-CoV-2 with ONT is an amplicon-sequencing protocol provided by the ARTIC Network which requires DNA ligation. Ligation reagents can be difficult to obtain in countries like Bolivia. Thus, here we provide an alternative for library preparation using the rapid PCR barcoding kit (ONT). We mapped more than 3.9 million sequence reads that allowed us to sequence twelve SARS-CoV-2 genomes from three different Bolivian cities. The average sequencing depth was 324X and the average genome length was 29527 bp. Thus, we could cover in average a 98,7% of the reference genome. The twelve genomes were successfully assigned to four different nextstrain clades (20A, 20B, 20E and 20G) and we could observe two main lineages of SARS-CoV-2 circulating in Bolivia. Therefore, this alternative library preparation for SARS-CoV-2 genome sequencing is effective to identify SARS-CoV-2 variants with high accuracy and without the need of DNA ligation. Hence, providing another tool to perform SARS-CoV-2 genome surveillance in developing countries.","version":"1.1","doi":"10.1101/2021.07.06.451357","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.05.451203","pub_date":"2021-7-07","title":"Mouse Antibodies with Activity Against the SARS-CoV-2 D614G and B.1.351 Variants","abstract":"With the rapid spread of SARS-CoV-2 variants, including those that are resistant to antibodies authorized for emergency use, it is apparent that new antibodies may be needed to effectively protect patients against more severe disease. Differences between the murine and human antibody repertoires may allow for the isolation of murine monoclonal antibodies that recognize a different or broader range of SARS-CoV-2 variants than the human antibodies that have been characterized so far. We describe mouse antibodies B13 and O24 that demonstrate neutralizing potency against SARS-CoV-2 Wuhan (D614G) and B.1.351 variants. Such murine antibodies may have advantages in protecting against severe symptoms when individuals are exposed to new SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.07.05.451203","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.06.451119","pub_date":"2021-7-07","title":"Nonclinical Safety and Immunogenicity of an rVSV-\u0394G-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs","abstract":"rVSV-\u0394G-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. Nonclinical safety, immunogenicity and efficacy studies were conducted in 4 animal species, using multiple dose levels (up to 108 PFU/animal) and various dosing regimens. There were no treatment related mortalities in any study, or any noticeable clinical signs. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings gave cause for safety concern in any of the studies. There was no viral shedding in urine, nor viral RNA detected in whole blood or serum samples 7 days post vaccination. The rVSV-\u0394G-SARS-CoV-2-S vaccine immune response gave rise to neutralizing antibodies, cellular immune response, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph node. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive IFNAR KO mice. Vaccine virus replication and distribution in K18 hACE2 transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The rVSV-\u0394G-SARS-CoV-2-S vaccine was well tolerated locally and systemically and elicited an effective immunogenic response up to the highest dose tested, supporting further clinical development.","version":"1.1","doi":"10.1101/2021.07.06.451119","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.01.21259583","pub_date":"2021-07-07","title":"Transmission networks of SARS-CoV-2 in coastal Kenya during the first two waves: a retrospective genomic study","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The transmission networks of SARS-CoV-2 in sub-Saharan Africa remain poorly understood.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We undertook phylogenetic analysis of 747 SARS-CoV-2 positive samples collected across six counties in coastal Kenya during the first two waves (March 2020 - February 2021). Viral imports and exports from the region were inferred using ancestral state reconstruction (ASR) approach.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The genomes were classified into 35 Pango lineages, six of which accounted for 79% of the sequenced infections: B.1 (49%), B.1.535 (11%), B.1.530 (6%), B.1.549 (4%), B.1.333 (4%) and B.1.1 (4%). Four identified lineages were Kenya specific. In a contemporaneous global subsample, 990 lineages were documented, 261 for Africa and 97 for Eastern Africa. ASR analysis identified &gt;300 virus location transition events during the period, these comprising: 69 viral imports into Coastal Kenya; 93 viral exports from coastal Kenya; and 191 inter-county import/export events. Most international viral imports (58%) and exports (92%) occurred through Mombasa City, a key touristic and commercial Coastal Kenya center; and many occurred prior to June 2020, when stringent local COVID-19 restriction measures were enforced. After this period, local virus transmission dominated, and distinct local phylogenies were seen.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Our analysis supports moving control strategies from a focus on international travel to local transmission.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This work was funded by Wellcome (grant#: 220985) and the National Institute for Health Research (NIHR), project references: 17/63/and 16/136/33 using UK aid from the UK Government to support global health research, The UK Foreign, Commonwealth and Development Office.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.07.01.21259583","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.05.451199","pub_date":"2021-7-07","title":"An Autoantigen Profile from Jurkat T-Lymphoblasts Provides a Molecular Guide for Investigating Autoimmune Sequelae of COVID-19","abstract":"In order to understand autoimmune phenomena contributing to the pathophysiology of COVID-19 and post-COVID syndrome, we have been profiling autoantigens (autoAgs) from various cell types. Although cells share numerous autoAgs, each cell type gives rise to unique COVID-altered autoAg candidates, which may explain the wide range of symptoms experienced by patients with autoimmune sequelae of SARS-CoV-2 infection. Based on the unifying property of affinity between autoantigens (autoAgs) and the glycosaminoglycan dermatan sulfate (DS), this paper reports 140 candidate autoAgs identified from proteome extracts of human Jurkat T-cells, of which at least 105 (75%) are known targets of autoantibodies. Comparison with currently available multi-omic COVID-19 data shows that 125 (89%) of DS-affinity proteins are altered at protein and/or RNA levels in SARS-CoV-2-infected cells or patients, with at least 94 being known autoAgs in a wide spectrum of autoimmune diseases and cancer. Protein alterations by ubiquitination and phosphorylation in the viral infection are major contributors of autoAgs. The autoAg protein network is significantly associated with cellular response to stress, apoptosis, RNA metabolism, mRNA processing and translation, protein folding and processing, chromosome organization, cell cycle, and muscle contraction. The autoAgs include clusters of histones, CCT/TriC chaperonin, DNA replication licensing factors, proteasome and ribosome proteins, heat shock proteins, serine/arginine-rich splicing factors, 14-3-3 proteins, and cytoskeletal proteins. AutoAgs such as LCP1 and NACA that are altered in the T cells of COVID patients may provide insight into T-cell responses in the viral infection and merit further study. The autoantigen-ome from this study contributes to a comprehensive molecular map for investigating acute, subacute, and chronic autoimmune disorders caused by SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.07.05.451199","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.07.434295","pub_date":"2021-7-07","title":"AI-driven prediction of SARS-CoV-2 variant binding trends from atomistic simulations","abstract":"We present a novel technique to predict binding affinity trends between two molecules from atomistic molecular dynamics simulations. The technique uses a neural network algorithm applied to a series of images encoding the distance between two molecules in time. We demonstrate that our algorithm is capable of separating with high accuracy non-hydrophobic mutations with low binding affinity from those with high binding affinity. Moreover, we show high accuracy in prediction using a small subset of the simulation, therefore requiring a much shorter simulation time. We apply our algorithm to the binding between several variants of the SARS-CoV-2 spike protein and the human receptor ACE2.","version":"1.2","doi":"10.1101/2021.03.07.434295","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.242206","pub_date":"2021-7-07","title":"Heat-treated virus inactivation rate depends strongly on treatment procedure: illustration with SARS-CoV-2","abstract":"Decontamination helps limit environmental transmission of infectious agents. It is required for the safe re-use of contaminated medical, laboratory and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), virus inactivation rate under heat treatment at 70\u00b0C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval: [0.09, 1.77]) in closed vials in a heat block to 37.00 min ([12.65, 869.82]) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature temperature-dependent coronavirus stability and found that specimen containers, and whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines. Heat is a powerful weapon against most infectious agents. It is widely used for decontamination of medical, laboratory and personal protective equipment, and for biological samples. There are many methods of heat treatment, and methodological details can affect speed and efficacy of decontamination. We applied four different heat-treatment procedures to liquid specimens containing SARS-CoV-2. Our results show that the container used to store specimens during decontamination can substantially affect inactivation rate: for a given initial level of contamination, decontamination time can vary from a few minutes in closed vials to several hours in uncovered plates. Reviewing the literature, we found that container choices and heat treatment methods are only rarely reported explicitly in methods sections. Our study shows that careful consideration of heat-treatment procedure \u2014 in particular the choice of specimen container, and whether it is covered \u2014 can make results more consistent across studies, improve decontamination practice, and provide insight into the mechanisms of virus inactivation.","version":"1.3","doi":"10.1101/2020.08.10.242206","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.07.451505","pub_date":"2021-7-07","title":"Immunogenicity of low dose prime-boost vaccination of mRNA vaccine CV07050101 in non-human primates","abstract":"Many different vaccine candidates against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of COVID-19, are currently approved and under development. Vaccine platforms vary from mRNA vaccines to viral-vectored vaccines, and several candidates have been shown to produce humoral and cellular responses in small animal models, non-human primates and human volunteers. In this study, six non-human primates received a prime-boost intramuscular vaccination with 4 \u00b5g of mRNA vaccine candidate CV07050101, which encodes a pre-fusion stabilized spike (S) protein of SARS-CoV-2. Boost vaccination was performed 28 days post prime vaccination. As a control, six animals were similarly injected with PBS. Humoral and cellular immune responses were investigated at time of vaccination, and two weeks afterwards. No antibodies could be detected two and four weeks after prime vaccination. Two weeks after boost vaccination, binding but no neutralizing antibodies were detected in 4 out of 6 non-human primates. SARS-CoV-2 S protein specific T cell responses were detected in these 4 animals. In conclusion, prime-boost vaccination with 4 \u00b5g of vaccine candidate CV07050101 resulted in limited immune responses in 4 out of 6 non-human primates.","version":"1.1","doi":"10.1101/2021.07.07.451505","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.06.451329","pub_date":"2021-7-07","title":"Investigating the conformational dynamics of SARS-CoV-2 NSP6 protein with emphasis on non-transmembrane 91-112 & 231-290 regions","abstract":"The NSP6 protein of SARS-CoV-2 is a transmembrane protein, with some regions lying outside the membrane. Besides, a brief role of NSP6 in autophagosome formation, this is not studied significantly. Also, there is no structural information available till date. Based on the prediction by TMHMM server for transmembrane prediction, it is found that the N-terminal residues (1-11), middle region residues (91-112) and C-terminal residues (231-290) lies outside the membrane. Molecular Dynamics (MD) simulations showed that NSP6 consisting of helical structures, whereas membrane outside lying region (91-112) showed partial helicity, which further used as model and obtain disordered type conformation after 1.5 microsecond. Whereas, the residues 231-290 has both helical and beta sheet conformations in its structure model. A 200ns simulations resulted in the loss of beta sheet structures, while helical regions remained intact. Further, we have characterized the residue 91-112 by using reductionist approaches. The NSP6 (91-112) was found disordered like in isolation, which gain helical conformation in different biological mimic environmental conditions. These studies can be helpful to study NSP6 (91-112) interactions with host proteins, where different protein conformation might play significant role. The present study adds up more information about NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis. The schematic representation of NSP6 membrane topology and conformational dynamics of residue 91-112. The N-terminal and C-terminal are shown in cytoplasmic side based on the experimental evidence on coronaviruses reported by Oostra et al., 2008. The membrane anchoring domain are shown based on the TMHMM server prediction.","version":"1.1","doi":"10.1101/2021.07.06.451329","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.450938","pub_date":"2021-7-05","title":"A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening","abstract":"Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV\u20112) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 \u03bcM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, \u03b2), Brazilian/Japanese variant P.1 (Gamma, \u03b3), and Californian (Epsilon, \u03b5), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.","version":"1.1","doi":"10.1101/2021.07.03.450938","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.05.451089","pub_date":"2021-7-05","title":"Mutation signatures inform the natural host of SARS-CoV-2","abstract":"The before-outbreak evolutionary history of SARS-CoV-2 is enigmatic because it shares only \u223c96% genomic similarity with RaTG13, the closest relative so far found in wild animals (horseshoe bats). Since mutations on single-stranded viral RNA are heavily shaped by host factors, the viral mutation signatures can in turn inform the host. By comparing publically available viral genomes we here inferred the mutations SARS-CoV-2 accumulated before the outbreak and after the split from RaTG13. We found the mutation spectrum of SARS-CoV-2, which measures the relative rates of 12 mutation types, is 99.9% identical to that of RaTG13. It is also similar to that of two other bat coronaviruses but distinct from that evolved in non-bat hosts. The viral mutation spectrum informed the activities of a variety of mutation-associated host factors, which were found almost identical between SARS-CoV-2 and RaTG13, a pattern difficult to create in laboratory. All the findings are robust after replacing RaTG13 with RshSTT182, another coronavirus found in horseshoe bats with \u223c93% similarity to SARS-CoV-2. Our analyses suggest SARS-CoV-2 shared almost the same host environment with RaTG13 and RshSTT182 before the outbreak.","version":"1.1","doi":"10.1101/2021.07.05.451089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.451026","pub_date":"2021-7-05","title":"Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection","abstract":"The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19\u2122). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. The Advax-SM adjuvanted vaccine induced high titers of binding antibody against spike protein that were able to neutralise the original wildtype virus on which the vaccine was based as well as the variant B.1.1.7 lineage virus. The Covax-19 vaccine also induced potent spike-specific CD4+ and CD8+ memory T-cells with a dominant Th1 phenotype, and this was shown to be associated with cytotoxic T lymphocyte killing of spike labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus 2 weeks after the second immunisation. Notably, ferrets that received two 25 or 50\u03bcg doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting the possibility that Covax-19 vaccine may in addition to protection against lung infection also have the potential to block virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.","version":"1.1","doi":"10.1101/2021.07.03.451026","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437955","pub_date":"2021-7-05","title":"To knot or not to knot: Multiple conformations of the SARS-CoV-2 frameshifting RNA element","abstract":"The SARS-CoV-2 frameshifting RNA element (FSE) is an excellent target for therapeutic intervention against Covid-19. This small gene element employs a shifting mechanism to pause and backtrack the ribosome during translation between Open Reading Frames 1a and 1b, which code for viral polyproteins. Any interference with this process has profound effect on viral replication and propagation. Pinpointing the structures adapted by the FSE and associated structural transformations involved in frameshifting has been a challenge. Using our graph-theory-based modeling tools for representing RNA secondary structures, \u201cRAG\u201d (RNA-As-Graphs), and chemical structure probing experiments, we show that the 3-stem H-type pseudoknot (3_6 dual graph), long assumed to be the dominant structure has a viable alternative, an HL-type 3-stem pseudoknot (3_3) for longer constructs. In addition, an unknotted 3-way junction RNA (3_5) emerges as a minor conformation. These three conformations share Stems 1 and 3, while the different Stem 2 may be involved in a conformational switch and possibly associations with the ribo-some during translation. For full-length genomes, a stem-loop motif (2_2) may compete with these forms. These structural and mechanistic insights advance our understanding of the SARS-CoV-2 frameshifting process and concomitant virus life cycle, and point to three avenues of therapeutic intervention.","version":"1.2","doi":"10.1101/2021.03.31.437955","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.04.451040","pub_date":"2021-7-05","title":"Predicted Impact of the Viral Mutational Landscape on the Cytotoxic Response against SARS-CoV-2","abstract":"The massive assessment of immune evasion due to viral mutations that potentially increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Potential epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 HLA class I families. Allele families showed extreme differences in number of recognized epitopes, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Among them, the B27 supertype showed the lowest number of epitopes. Epitope escape mutations identified in ~118,000 NCBI isolates mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% evaded epitopes for the same supertype in the same isolate, except for B27, which reached up to 33%. In contrast to most supertypes, eight particular allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles. Collectively, these data indicate that independent escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. This study highlights the automated integration of genomic, geographical and immunoinformatic information for surveillance of SARS-CoV-2 variants potentially affecting the population as a whole, as well as minority subpopulations. The cytotoxic T response, a type of immune response dependent upon an individual\\'s genetics that does not require antibodies, is critical for neutralizing SARS-CoV-2 infection. The potential bypass of the cytotoxic T response by mutations acquired by the virus after one year of the pandemic is therefore of maximal concern. We have approached the complexity of human variability and more than 100.000 viral genomes in this respect using a computational strategy. We have detected numerous mutations in these genomes that mask some viral regions involved in the cytotoxic response. However, the accumulation of these changes in independent isolates is still too low to threaten the global human population. In contrast, our protocol has identified mutations that may be relevant for specific populations and minorities with cytotoxic genetic backgrounds susceptible to SARS-CoV-2 infection. Some viral variants co-existed in the same country with these human communities which warrants deeper surveillance in these cases to prevent local outbreaks. Our study support the integration of massive data of different natures in the surveillance of viral pandemics.","version":"1.1","doi":"10.1101/2021.07.04.451040","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.05.21260003","pub_date":"2021-07-05","title":"Soft drinks can be misused to give false \u201cfalse positive\u201d SARS-CoV-2 lateral flow device results","abstract":"<jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The COVID-19 pandemic created the need for very large scale, rapid testing to prevent and contain transmission of the SARS-CoV-2 virus. Lateral flow device (LFD) immunoassays meet this need by indicating the presence of SARS-CoV-2 antigen from nose/throat swab washings in 30 minutes without laboratory processing, and can be manufactured quickly at low cost. Since March 2021, UK schools have asked pupils without symptoms to test twice weekly. Pupils have posted on social media about using soft drinks to create positive results. The aim of this study was to systematically test a variety soft drinks to determine whether they can cause false \u201cfalse positive\u201d LFD results.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This study used 14 soft drinks and 4 artificial sweeteners to determine the outcome of misusing them as analyte for the Innova SARS-CoV-2 antigen rapid qualitative LFD. The pH value, sugar content and ingredients of each sample are described. The LFD results were double read and a subset was repeated using the same devices and fake analytes but differently sourced.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>One sample (1/14; 7%), spring water, produced a negative result. Ten drinks (10/14; 71%) produced a positive or weakly positive result. Three samples (3/14; 21%) produced void results, mostly the fruit concentrate drinks. There was no apparent correlation between the pH value (pH 5.0 in 13/14, 93%; pH 6.5 in 1/14; 7%) or the sugar content (range 0-10.7 grams per 100mls) of the drinks and their LFD result. The 4 artificial sweeteners all produced negative results. A subset of the results was fully replicated with differently sourced materials.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Several soft drinks can be misused to give false positive SARS-CoV-2 LFD results. Daily LFD testing should be performed first thing in the morning, prior to the consumption of any food or drinks, and supervised where feasible.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This work was self-funded by author LO and the LFD were gifted for use in this study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Lateral flow devices (LFD) for SARS-CoV-2 antigen testing have been used extensively in the UK and internationally in COVID-19 pandemic responses, providing rapid testing at low cost</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Recent reports from young people on social media suggested soft drinks might be misused as LFD analyte and produce a seemingly positive result</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Various common soft drinks used as fake analyte can produce false positive SARS-CoV-2 LFD results</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Artificial sweeteners alone in fake analyte solution did not produce false positive results</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>Soft drinks misused as analyte can produce false \u201cfalse positive\u201d SARS-CoV-2 LFD results</jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Daily testing is best done first thing in the morning, prior to any food or drink, and under supervision where possible</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.07.05.21260003","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.04.450986","pub_date":"2021-7-05","title":"Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses","abstract":"The worldwide spread of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) caused an urgent need for an in-depth understanding of interactions between the virus and its host. Here, we dissected the dynamics of virus replication and the host cell transcriptional response to SARS-CoV-2 infection at a systems level by combining time-resolved RNA sequencing with mathematical modeling. We observed an immediate transcriptional activation of inflammatory pathways linked to the anti-viral response followed by increased expression of genes involved in ribosome and mitochondria function, thus hinting at rapid alterations in protein production and cellular energy supply. At later stages, metabolic processes, in particular those depending on cytochrome P450 enzymes, were downregulated. To gain a deeper understanding of the underlying transcriptional dynamics, we developed an ODE model of SARS-CoV-2 infection and replication. Iterative model reduction and refinement revealed that a negative feedback from virus proteins on the expression of anti-viral response genes was essential to explain our experimental dataset. Our study provides insights into SARS-CoV-2 virus-host interaction dynamics and facilitates the identification of druggable host pathways supporting virus replication.","version":"1.1","doi":"10.1101/2021.07.04.450986","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.450992","pub_date":"2021-7-05","title":"Increased Histone-DNA Complexes and Endothelial-Dependent Thrombin Generation in Severe COVID-19","abstract":"Coagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. We studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Blood samples were assayed for levels of histone-DNA complexes. Confocal microscopy was used to evaluate fibrin structure in clots formed from recalcified plasma samples using fluorescently-labeled fibrinogen. Endogenous thrombin potential was measured by calibrated automated thrombin assays in the presence of tissue factor and phospholipid (PCPS) or cultured human endothelial cells. Circulating nucleosomes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). COVID-19 patient plasma thrombin generation was also altered. Despite having an increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. Control plasma samples did not generate measurable thrombin (lag time >60 min); in contrast, plasma samples from COVID-19+ patients generated thrombin (mean lag time \u223c20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. Elevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation in COVID-19 may contribute to coagulopathy. Histone-DNA complexes are significantly elevated in the plasma of patients with severe SARS-CoV-2 infection. Measures of thrombin generation by calibrated automated thrombography and fibrin clots formed in situ are altered in severe COVID-19. Plasma from COVID-19 patients promotes thrombin generation on cultured endothelial cells in the absence of added tissue factor or phospholipids. The additive effects of histones on thrombin generation and endothelial cell function may play a major role in the thrombotic complications observed in severe SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.07.03.450992","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.04.451027","pub_date":"2021-7-05","title":"Increased frequency of recurrent in-frame deletions in new expanding lineages of SARS CoV-2 reflects immune selective pressure","abstract":"Most of the attention in the surveillance of evolution of SARS-CoV-2 has been centered on single nucleotide substitutions in the spike glycoprotein. We show that in-frame deletions (IFDs) also play a significant role in the evolution of viral genome. The percentage of genomes and lineages with IFDs is growing rapidly and they co-occur independently in multiple lineages, including emerging variants of concerns. IFDs distribution is correlated with spike mutations associated with immune escape and concentrated in proteins involved in interactions with the host immune system. Structural analysis suggests that IFDs remodel viral proteins\u2019 surfaces at common epitopes and interaction interfaces, affecting the virus\u2019 interactions with the immune system. We hypothesize that the increased frequency of IFDs is an adaptive response to elevated global population immunity. Monitoring of SARS-CoV-2 genome evolution uncovers increased frequency and non-random distribution of in-frame deletions in recently emerged lineages.","version":"1.1","doi":"10.1101/2021.07.04.451027","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.05.451222","pub_date":"2021-7-05","title":"Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques","abstract":"To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. Broadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the na\u00efve macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.","version":"1.1","doi":"10.1101/2021.07.05.451222","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.18.440366","pub_date":"2021-7-05","title":"An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amenable to Dexamethasone Therapy","abstract":"SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative, but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNA-Seq and plasma proteomics atlas (biernaskielab.ca/COVID_neutrophil). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFNactive state, downregulated interferon responsive genes, and activated IL1R2+ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFNactive neutrophils, a greater degree of steroid-induced immature neutrophil expansion, and increased mortality benefit compared to females in the dexamethasone era. Indeed, the highest proportion of IFNactive neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined, and the identified pathways and plasma proteins can now be targeted to develop improved therapeutics.","version":"1.2","doi":"10.1101/2021.04.18.440366","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.450989","pub_date":"2021-7-04","title":"Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 Spike Glycoprotein","abstract":"Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both SARS-CoV-2 infection severity and long-term immunity, either after recovery or vaccination. A hallmark of COVID-19 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides by 10 common allotypes of ER aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for MHC class I molecules, including known SARS-CoV-2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses to COVID-19.","version":"1.1","doi":"10.1101/2021.07.03.450989","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.03.451001","pub_date":"2021-7-04","title":"Network-based identification and pharmacological targeting of host cell master regulators induced by SARS-CoV-2 infection","abstract":"Precise characterization and targeting of host cell transcriptional machinery hijacked by SARS-CoV-2 remains challenging. To identify therapeutically targetable mechanisms that are critical for SARS-CoV-2 infection, here we elucidated the Master Regulator (MR) proteins representing mechanistic determinants of the gene expression signature induced by SARS-CoV-2. The analysis revealed coordinated inactivation of MR-proteins linked to regulatory programs potentiating efficiency of viral replication (detrimental host MR-signature) and activation of MR-proteins governing innate immune response programs (beneficial MR-signature). To identify MR-inverting compounds capable of rescuing activity of inactivated host MR-proteins, with-out adversely affecting the beneficial MR-signature, we developed the ViroTreat algorithm. Overall, >80% of drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 infection, without affecting cell viability. ViroTreat is fully generalizable and can be extended to identify drugs targeting the host cell-based MR signatures induced by virtually any pathogen.","version":"1.1","doi":"10.1101/2021.07.03.451001","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.21259420","pub_date":"2021-07-03","title":"Effectiveness of mRNA and ChAdOx1 COVID-19 vaccines against symptomatic SARS-CoV-2 infection and severe outcomes with variants of concern in Ontario","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>SARS-CoV-2 variants of concern (VOC) are more transmissible and have the potential for increased disease severity and decreased vaccine effectiveness. We estimated the effectiveness of BNT162b2 (Pfizer-BioNTech Comirnaty), mRNA-1273 (Moderna Spikevax), and ChAdOx1 (AstraZeneca Vaxzevria) vaccines against symptomatic SARS-CoV-2 infection and COVID-19 hospitalization or death caused by the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) VOCs in Ontario, Canada using a test-negative design study. Effectiveness against symptomatic infection \u22657 days after two doses was 89\u201392% against Alpha, 87% against Beta, 88% against Gamma, 82\u201389% against Beta/Gamma, and 87\u201395% against Delta across vaccine products. The corresponding estimates \u226514 days after one dose were lower. Effectiveness estimates against hospitalization or death were similar to, or higher than, against symptomatic infection. Effectiveness against symptomatic infection is generally lower for older adults (\u226560 years) compared to younger adults (&lt;60 years) for most of the VOC-vaccine combinations.</jats:p>","version":null,"doi":"10.1101/2021.06.28.21259420","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.02.450959","pub_date":"2021-7-03","title":"SARS-CoV-2 Lambda Variant Remains Susceptible to Neutralization by mRNA Vaccine-elicited Antibodies and Convalescent Serum","abstract":"The SARS-CoV-2 lambda variant (lineage C.37) was designated by the World Health Organization as a variant of interest and is currently increasing in prevalence in South American and other countries. The lambda spike protein contains novel mutations within the receptor binding domain (L452Q and F490S) that may contribute to its increased transmissibility and could result in susceptibility to re-infection or a reduction in protection provided by current vaccines. In this study, the infectivity and susceptibility of viruses with the lambda variant spike protein to neutralization by convalescent sera and vaccine-elicited antibodies was tested. Virus with the lambda spike had higher infectivity and was neutralized by convalescent sera and vaccine-elicited antibodies with a relatively minor 2.3-3.3-fold decrease in titer on average. The virus was neutralized by the Regeneron therapeutic monoclonal antibody cocktail with no loss of titer. The results suggest that vaccines in current use will remain protective against the lambda variant and that monoclonal antibody therapy will remain effective.","version":"1.1","doi":"10.1101/2021.07.02.450959","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.02.450857","pub_date":"2021-7-03","title":"Dasabuvir inhibits human norovirus infection in human intestinal enteroids","abstract":"Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, with a human intestinal enteroid (HIE) culture system where HuNoVs are able to replicate reproducibly, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity. Dasabuvir, which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human A rotavirus (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation.","version":"1.1","doi":"10.1101/2021.07.02.450857","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.02.450915","pub_date":"2021-7-02","title":"Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples","abstract":"In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.","version":"1.1","doi":"10.1101/2021.07.02.450915","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.02.450663","pub_date":"2021-7-02","title":"Natural infection of SARS-CoV-2 delta variant in Asiatic lions (Panthera leo persica) in India","abstract":"In May 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in nine Asiatic lions (Panthera leo persica) in Arignar Anna Zoological Park, Chennai, Tamil Nadu, India. Sequence and phylogenetic analysis showed that the SARS-CoV-2 viruses belong to a variant of concern (VOC, delta variant, B.1.617.2 lineage) and that these viruses clustered with B.1.617.2 lineage viruses of the same geographical region detected in the same month.","version":"1.1","doi":"10.1101/2021.07.02.450663","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.02.450920","pub_date":"2021-7-02","title":"Betacoronavirus-specific alternate splicing","abstract":"Viruses can subvert a number of cellular processes in order to block innate antiviral responses, and many viruses interact with cellular splicing machinery. SARS-CoV-2 infection was shown to suppress global mRNA splicing, and at least 10 SARS-CoV-2 proteins bind specifically to one or more human RNAs. Here, we investigate 17 published experimental and clinical datasets related to SARS-CoV-2 infection as well as datasets from the betacoronaviruses SARS-CoV and MERS as well as Streptococcus pneumonia, HCV, Zika virus, Dengue virus, influenza H3N2, and RSV. We show that genes showing differential alternative splicing in SARS-CoV-2 have a similar functional profile to those of SARS-CoV and MERS and affect a diverse set of genes and biological functions, including many closely related to virus biology. Additionally, the differentially spliced transcripts of cells infected by coronaviruses were more likely to undergo intron-retention, contain a pseudouridine modification and a smaller number of exons than differentially spliced transcripts in the control groups. Viral load in clinical COVID-19 samples was correlated with isoform distribution of differentially spliced genes. A significantly higher number of ribosomal genes are affected by DAS and DGE in betacoronavirus samples, and the betacoronavirus differentially spliced genes are depleted for binding sites of RNA-binding proteins. Our results demonstrate characteristic patterns of differential splicing in cells infected by SARS-CoV-2, SARS-CoV, and MERS, potentially modifying a broad range of cellular functions and affecting a diverse set of genes and biological functions.","version":"1.1","doi":"10.1101/2021.07.02.450920","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.01.450676","pub_date":"2021-7-02","title":"Neutralization of Delta variant with sera of Covishield vaccinees and COVID-19 recovered vaccinated individuals","abstract":"The recent emergence of B.1.617 lineage has created grave public health problem in India. The lineage further mutated to generate sub-lineages B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3. Apparently, the Delta variant has slowly dominated the other variants including B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3. With this, World Health Organization has described this sub-lineage as variant of concern. The high transmissibility associated with Delta variant has led to second wave of pandemic in India which affected millions of people. Besides this, variant of concerns has been reported to show lower neutralization to several approved vaccines. This has led to breakthrough infections after completion of vaccination regimen. There is limited information available on the duration of protective immune response post-infection, vaccination or breakthrough infection with SARS-CoV-2. In this study, we have evaluated immune response in sera of the Covishield vaccinated individuals belonging to category: I. one dose vaccinated, II. two doses vaccinated, III. COVID-19 recovered plus one dose vaccinated, IV. COVID-19 recovered plus two doses vaccinated and V. breakthrough COVID-19 cases. The findings of the study demonstrated that the breakthrough cases and the COVID-19 recovered individuals with one or two dose of vaccine had relatively higher protection against Delta variant in comparison to the participants who were administered either one or two doses of Covishield\u2122. Prior vaccination results in less severe disease against subsequent infection provide evidence that both humoral and cellular immune response play an important role in protection.","version":"1.1","doi":"10.1101/2021.07.01.450676","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.21259544","pub_date":"2021-07-02","title":"Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>mRNA COVID-19 vaccines are playing a key role in controlling the COVID-19 pandemic. The relationship between post-vaccination symptoms and strength of antibody responses is unclear.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Single center, prospective, observational cohort study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>Participants worked at Walter Reed National Military Medical Center and were seen monthly at the Naval Medical Research Center Clinical Trials Center.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>Generally healthy adults that were not severely immunocompromised, had no history of COVID-19, and were seronegative for SARS-CoV-2 spike protein prior to vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Measures</jats:title>\n                  <jats:p>Severity of vaccine-associated symptoms was obtained through participant completed questionnaires. Testing for IgG antibodies against SARS-CoV-2 spike protein and receptor binding domain was conducted using microsphere-based multiplex immunoassays.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>206 participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers one month after vaccination. We also observed that 1) post-vaccination symptoms were inversely correlated with age and weight and more common in women, 2) systemic symptoms were more frequent after the second vaccination, 3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and 4) older age was associated with lower titers.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Limitations</jats:title>\n                  <jats:p>Study only observes antibody responses and consists of healthy participants.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Lack of post-vaccination symptoms following receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies one month after vaccination. This study also suggests that it may be possible to design future mRNA vaccines that confer robust antibody responses with lower frequencies of vaccine-associated symptoms.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This study was executed by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed by the Uniformed Services University of the Health Sciences (USUHS) through a cooperative agreement by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). This project has been funded by the Defense Health Program, U.S. DoD, under award HU00012120067. Project funding for JHP was in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The funding bodies have had no role in the study design or the decision to submit the manuscript for publication.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.25.21259544","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.27.21259271","pub_date":"2021-07-02","title":"SARS-CoV-2 antibody prevalence in Sierra Leone, March 2021: a cross-sectional, nationally representative, age-stratified serosurvey","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>As of 26 March 2021, the Africa CDC had reported 4,159,055 cases of COVID-19 and 111,357 deaths among the 55 African Union Member States; however, no country has published a nationally representative serosurvey as of May 2021. Such data are vital for understanding the pandemic\u2019s progression on the continent, evaluating containment measures, and policy planning.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a cross-sectional, nationally representative, age-stratified serosurvey in Sierra Leone in March 2021 by randomly selecting 120 Enumeration Areas throughout the country and 10 randomly selected households in each of these. One to two persons per selected household were interviewed to collect information on socio-demographics, symptoms suggestive of COVID-19, exposure history to laboratory-confirmed COVID-19 cases, and history of COVID-19 illness. Capillary blood was collected by fingerstick, and blood samples were tested using the Hangzhou Biotest Biotech RightSign COVID-19 IgG/IgM Rapid Test Cassette. Total seroprevalence was was estimated after applying sampling weights.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>The overall weighted seroprevalence was 2.6% (95% CI 1.9-3.4). This is 43 times higher than the reported number of cases. Rural seropositivity was 1.8% (95% CI 1.0-2.5), and urban seropositivity was 4.2% (95% CI 2.6-5.7).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Although overall seroprevalence was low compared to countries in Europe and the Americas (suggesting relatively successful containment in Sierra Leone), our findings indicate enormous underreporting of active cases. This has ramifications for the country\u2019s third wave (which started in June 2021), where the average number of daily reported cases was 87 by the end of the month\u2014this could potentially be on the order of 3,700 actual infections, calling for stronger containment measures in a country with only 0.2% of people fully vaccinated. It may also reflect significant underreporting of incidence and mortality across the continent.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This study was supported by NIAID K08 AI139361, the Sierra Leone Ministry of Health and Sanitation, and the Africa CDC.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.27.21259271","journal":"medRxiv","score":null},{"id":"10.1101/2021.07.01.450707","pub_date":"2021-7-01","title":"Ad26.COV2.S elicited neutralizing activity against Delta and other SARS-CoV-2 variants of concern","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and recently emerging variants with substitutions in the Spike protein have led to growing concerns over increased transmissibility and decreased vaccine coverage due to immune evasion. Here, sera from recipients of a single dose of our Ad26.COV2.S COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants of concern. All tested variants demonstrated susceptibility to Ad26.COV2.S-induced serum neutralization albeit mainly reduced as compared to the B.1 strain. Most pronounced reduction was observed for the B.1.351 (Beta; 3.6-fold) and P.1 (Gamma; 3.4-fold) variants that contain similar mutations in the receptor-binding domain (RBD) while only a 1.6-fold reduction was observed for the widely spreading B.1.617.2 (Delta) variant.","version":"1.1","doi":"10.1101/2021.07.01.450707","journal":"bioRxiv","score":null},{"id":"10.1101/2021.07.01.450701","pub_date":"2021-7-01","title":"Thiol-based mucolytics exhibit antiviral activity against SARS-CoV-2 through allosteric disulfide disruption in the spike glycoprotein","abstract":"Small molecule therapeutics targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have lagged far behind the development of vaccines in the fight to control the COVID-19 pandemic. Here, we show that thiol-based mucolytic agents, P2119 and P2165, potently inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity of repurposed mucolytic agents to the reduction of key disulfides, specifically, by disruption of the Cys379\u2013Cys432 and Cys391\u2013Cys525 pairs distal to the receptor binding motif (RBM) in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol warhead pointed directly towards Cys432. These collective findings establish the vulnerability of human coronaviruses to repurposed thiol-based mucolytics and lay the groundwork for developing these compounds as a potential treatment, preventative and/or adjuvant against infection.","version":"1.1","doi":"10.1101/2021.07.01.450701","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.21259673","pub_date":"2021-07-01","title":"Infectivity and immune escape of the new SARS-CoV-2 variant of interest Lambda","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The newly described SARS-CoV-2 lineage C.37 was recently classified as a variant of interest by the WHO (Lambda variant) based on its high circulation rates in South American countries and the presence of critical mutations in the spike protein. The impact of such mutations in infectivity and immune escape from neutralizing antibodies are entirely unknown.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We performed a pseudotyped virus neutralization assay and determined the impact of the Lambda variant on infectivity and immune escape using plasma samples from healthcare workers (HCW) from two centers in Santiago, Chile who received the two-doses scheme of the inactivated virus vaccine CoronaVac.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We observed an increased infectivity mediated by the Lambda spike protein that was even higher than that of the D614G (lineage B) or the Alpha and Gamma variants. Compared to the Wild type (lineage A), neutralization was decreased by 3.05-fold for the Lambda variant while it was 2.33-fold for the Gamma variant and 2.03-fold for the Alpha variant.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Our results indicate that mutations present in the spike protein of the Lambda variant of interest confer increased infectivity and immune escape from neutralizing antibodies elicited by CoronaVac. These data reinforce the idea that massive vaccination campaigns in countries with high SARS-CoV-2 circulation must be accompanied by strict genomic surveillance allowing the identification of new isolates carrying spike mutations and immunology studies aimed to determine the impact of these mutations in immune escape and vaccines breakthrough.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.28.21259673","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.11.435000","pub_date":"2021-6-30","title":"ChAdOx1 nCoV-19 (AZD1222) protects Syrian hamsters against SARS-CoV-2 B.1.351 and B.1.1.7","abstract":"We investigated ChAdOx1 nCoV-19 (AZD1222) vaccine efficacy against SARS-CoV-2 variants of concern (VOCs) B.1.1.7 and B.1.351 in Syrian hamsters. We previously showed protection against SARS-CoV-2 disease and pneumonia in hamsters vaccinated with a single dose of ChAdOx1 nCoV-19. Here, we observed a 9.5-fold reduction of virus neutralizing antibody titer in vaccinated hamster sera against B.1.351 compared to B.1.1.7. Vaccinated hamsters challenged with B.1.1.7 or B.1.351 did not lose weight compared to control animals. In contrast to control animals, the lungs of vaccinated animals did not show any gross lesions. Minimal to no viral subgenomic RNA (sgRNA) and no infectious virus was detected in lungs of vaccinated animals. Histopathological evaluation showed extensive pulmonary pathology caused by B.1.1.7 or B.1.351 replication in the control animals, but none in the vaccinated animals. These data demonstrate the effectiveness of the ChAdOx1 nCoV-19 vaccine against clinical disease caused by B.1.1.7 or B.1.351 VOCs.","version":"1.3","doi":"10.1101/2021.03.11.435000","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450453","pub_date":"2021-6-30","title":"Highly-Neutralizing COVID-19-Convalescent-Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters","abstract":"Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19. Convalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.","version":"1.1","doi":"10.1101/2021.06.29.450453","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450298","pub_date":"2021-6-30","title":"Pathology and immunity after SARS-CoV-2 infection in male ferrets is affected by age and inoculation route","abstract":"Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nose was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.","version":"1.1","doi":"10.1101/2021.06.30.450298","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450547","pub_date":"2021-6-30","title":"Computational saturation mutagenesis of SARS-CoV-1 spike glycoprotein: stability, binding affinity, and comparison with SARS-CoV-2","abstract":"Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we performed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003-2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003-2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the S glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2. Severe acute respiratory syndrome coronavirus (SARS-CoV-1) is an RNA virus that undergoes frequent mutations, which may result in more virulent SARS-CoV-1 variants. To prevent another pandemic in the future, scientists must understand the mechanisms of viral mutations and predict if any variants could become a dominant. The infection of SARS-CoV-1 in cells is largely depending on the interactions of the viral Spike (S) and human angiotensin-converting enzyme 2 (hACE2). We applied a computational method to predict S missense mutations that will make SARS-CoV-1 more virulent. We are interested in the variants that can change SARS-CoV-1 spike protein stability and/or change the virus-receptor interactions. We mutated each residue of SARS-CoV-1 spike to all possible amino acids; we calculated the differences between the folding energy and binding energy of each variant and the wildtype and identified the target S mutations with significant effects on protein stability and protein-protein interaction. We found some viral mutations could destabilize S and weaken S-hACE2 binding of SARS-CoV-1 isolate. Our results show that the computational saturation mutagenesis is a reliable approach in the analysis and prediction of missense mutations.","version":"1.1","doi":"10.1101/2021.06.30.450547","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450452","pub_date":"2021-6-30","title":"Host cellular RNA helicases regulate SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has largest RNA genome of approximately 30kb among RNA viruses. The DDX DEAD-box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. As well, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to utilize own viral replication and overcomes their anti-viral effect of DDX21 and MOV10 through as interaction with host cellular RNA helicase. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection. SARS-CoV-2 has large RNA genome of approximately 30kb. To regulate and maintain such large viral RNA genome, host RNA helicases may involve in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5 and DDX6 are required for the SARS-CoV-2 infection. Interestingly, the SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and, by suppressing host innate immune system.","version":"1.1","doi":"10.1101/2021.06.29.450452","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450426","pub_date":"2021-6-30","title":"Varenicline Prevents SARS-CoV-2 Infection In Vitro and in Rhesus Macaques","abstract":"SARS-CoV-2 infections have resulted in a global pandemic, but an antiviral therapy for this novel strain of coronavirus does not currently exist. The objective of our study was to investigate the antiviral potential of the nicotinic acetylcholine receptor (nACHR) agonist varenicline tartrate against SARS-CoV-2. We assessed antiviral activity using in vitro human cell assays and we assessed in vivo efficacy in a rhesus macaque model. In vitro studies found that varenicline tartrate, over a range of concentrations, reduced the infectivity of SARS-CoV-2 wildtype, alpha, and beta variants in Calu-3 cells and Caco-2 cells, with maintenance of cell viability. In vivo studies found that varenicline tartrate, administered as a nasal spray to rhesus macaques, reduced SARS-CoV-2 wildtype viral load and inhibited viral replication in the nasal mucosa and upper airway. Although the study reported here was exploratory, we have confirmed that the nAChR agonist varenicline has the potential to interact with and inhibit SARS-CoV-2 infection and replication.","version":"1.1","doi":"10.1101/2021.06.29.450426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450531","pub_date":"2021-6-30","title":"Early detection of SARS-CoV-2 in circulating immune cells in a mouse model","abstract":"SARS-CoV-2 infects the respiratory tract, lung and then other organs. However, its pathogenesis remains largely unknown. We used RareScope\u2122 Fluorescence Light Sheet Microscopy (FLSM) and fluorescent in situ hybridization of RNA (RNA-FISH) to detect SARS-CoV-2 RNA and dissemination kinetics in mouse blood circulation. By RNA-FISH, we found that SARS-CoV-2 RNA-positive leukocytes, including CD11c cells, appeared as early as one day after infection and continued through day 10 post infection. Our data suggest that SARS-CoV-2-permissive leukocytes contribute to systemic viral dissemination, and RNA-FISH combined with FLSM can be utilized as a sensitive tool for SARS-CoV-2 detection in blood specimens.","version":"1.1","doi":"10.1101/2021.06.30.450531","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450617","pub_date":"2021-6-30","title":"Detection of potential new SARS-CoV-2 Gamma-related lineage in Tocantins shows the spread and ongoing evolution of P.1 in Brazil","abstract":"After more than a year of the pandemic situation of COVID-19, the United Kingdom (UK), South Africa, and Brazil became the epicenter of new lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Variants of Concern (VOCs) were identified through a continuous genomic surveillance global effort, the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and P.1 (Gamma) harboring a constellation set of mutations. This research aims to: (i) report the predominance of the Gamma (P.1) lineage presenting the epidemiological situation of the SARS-CoV-2 genomic surveillance at the state of Tocantins, and (ii) describe the emergence of possible new mutations and viral variants with the potential new lineage (P1-related) represented by 8 genomes from the Tocantins harboring the mutation L106F in ORF3a. At the moment, 6,687 SARS-CoV-2 genomes from GISAID carry this mutation. The whole-genome sequencing has an important role in understanding the evolution and genomic diversity of SARS-CoV-2, thus, the continuous monitoring will help in the control measures and restrictions imposed by the secretary of health of the state to prevent the spread of variants.","version":"1.1","doi":"10.1101/2021.06.30.450617","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.446813","pub_date":"2021-6-30","title":"Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking","abstract":"SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody\u2013ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen\u2013ligand interactions.","version":"1.2","doi":"10.1101/2021.06.02.446813","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.16.448653","pub_date":"2021-6-30","title":"Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection by targeting cooperatively host TMPRSS2 in vitro and in vivo","abstract":"SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNa) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNa and that both Serpin E1 and camostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.","version":"1.2","doi":"10.1101/2021.06.16.448653","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.30.450490","pub_date":"2021-6-30","title":"The SARS-CoV-2 host cell membrane fusion protein TMPRSS2 is a tumor suppressor and its downregulation correlates with increased antitumor immunity and immunotherapy response in lung adenocarcinoma","abstract":"TMPRSS2 is a host cell membrane fusion protein for SARS-CoV-2 invading human host cells. It also has an association with cancer, particularly prostate cancer. However, its association with lung cancer remains insufficiently explored. Thus, an in-depth investigation into the association between TMPRSS2 and lung cancer is significant, considering that lung cancer is the leading cause of cancer death and that the lungs are the primary organ SARS-CoV-2 attacks. Using five lung adenocarcinoma (LUAD) genomics datasets, we explored associations between TMPRSS2 expression and immune signatures, cancer-associated pathways, tumor progression phenotypes, and clinical prognosis in LUAD by the bioinformatics approach. Furthermore, we validated the findings from the bioinformatics analysis by performing in vitro experiments with the human LUAD cell line A549 and in vivo experiments with mouse tumor models. We also validated our findings in LUAD patients from Jiangsu Cancer Hospital, China. TMPRSS2 expression levels were negatively correlated with the enrichment levels of CD8+ T and NK cells and immune cytolytic activity in LUAD, which represent antitumor immune signatures. Meanwhile, TMPRSS2 expression levels were negatively correlated with the enrichment levels of CD4+ regulatory T cells and myeloid-derived suppressor cells and PD-L1 expression levels in LUAD, which represent antitumor immunosuppressive signatures. However, TMPRSS2 expression levels showed a significant positive correlation with the ratios of immune-stimulatory/immune-inhibitory signatures (CD8+ T cells/PD-L1) in LUAD. It indicated that TMPRSS2 levels had a stronger negative correlation with immune-inhibitory signatures than with immune-stimulatory signatures. TMPRSS2 downregulation correlated with elevated activities of many oncogenic pathways in LUAD, including cell cycle, mismatch repair, p53, and extracellular matrix (ECM) signaling. TMPRSS2 downregulation correlated with increased proliferation, stemness, genomic instability, tumor advancement, and worse survival in LUAD. In vitro and in vivo experiments validated the association of TMPRSS2 deficiency with increased tumor cell proliferation and invasion and antitumor immunity in LUAD. Moreover, in vivo experiments demonstrated that TMPRSS2-knockdown tumors were more sensitive to BMS-1, an inhibitor of PD-1/PD-L1. TMPRSS2 is a tumor suppressor, while its downregulation is a positive biomarker of immunotherapy in LUAD. Our data provide a connection between lung cancer and pneumonia caused by SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.06.30.450490","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450372","pub_date":"2021-6-30","title":"Epitope order Matters in multi-epitope-based peptide (MEBP) vaccine design: An in silico study","abstract":"With different countries facing multiple waves, with some SARS-CoV-2 variants more deadly and virulent, the COVID-19 pandemic is becoming more dangerous by the day and the world is facing an even more dreadful extended pandemic with exponential positive cases and increasing death rates. There is an urgent need for more efficient and faster methods of vaccine development against SARS-CoV-2. Compared to experimental protocols, the opportunities to innovate are very high in immunoinformatics/in silico approaches especially with the recent adoption of structural bioinformatics in peptide vaccine design. In recent times, multi-epitope-based peptide vaccine candidates (MEBPVCs) have shown extraordinarily high humoral and cellular responses to immunization. Most of the publications claim that respective reported MEBPVC(s) assembled using a set of in silico predicted epitopes, to be the computationally validated potent vaccine candidate(s) ready for experimental validation. However, in this article, for a given set of predicted epitopes, it is shown that the published MEBPVC is one among the many possible variants and there is high likelihood of finding more potent MEBPVCs than the published candidate. To test the same, a methodology is developed where novel MEBP variants are derived by changing the epitope order of the published MEBPVC. Further, to overcome the limitations of current qualitative methods of assessment of MEBPVC, to enable quantitative comparison, ranking, and the discovery of more potent MEBPVCs, novel predictors, Percent Epitope Accessibility (PEA), Receptor specific MEBP vaccine potency(RMVP), MEBP vaccine potency(MVP) are introduced. The MEBP variants indeed showed varied MVP scores indicating varied immunogenicity. When the MEBP variants were ranked in descending order of their MVP scores, the published MEBPVC had the least MVP score. Further, the MEBP variants with IDs, SPVC_387 and SPVC_206, had the highest MVP scores indicating these variants to be more potent MEBPVCs than the published MEBPVC and hence should be prioritized for experimental testing and validation. Through this method, more vaccine candidates will be available for experimental validation and testing. This study also opens the opportunity to develop new software tools for designing more potent MEBPVCs in less time. The computationally validated top-ranked MEBPVCs must be experimentally tested, validated, and verified. The differences and deviations between experimental results and computational predictions provide an opportunity for improving and developing more efficient algorithms and reliable scoring schemes and software.","version":"1.1","doi":"10.1101/2021.06.29.450372","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450335","pub_date":"2021-6-30","title":"Classifying COVID-19 variants based on genetic sequences using deep learning models","abstract":"The COrona VIrus Disease (COVID-19) pandemic led to the occurrence of several variants with time. This has led to an increased importance of understanding sequence data related to COVID-19. In this chapter, we propose an alignment-free k-mer based LSTM (Long Short-Term Memory) deep learning model that can classify 20 different variants of COVID-19. We handle the class imbalance problem by sampling a fixed number of sequences for each class label. We handle the vanishing gradient problem in LSTMs arising from long sequences by dividing the sequence into fixed lengths and obtaining results on individual runs. Our results show that one-vs-all classifiers have test accuracies as high as 92.5% with tuned hyperparameters compared to the multi-class classifier model. Our experiments show higher overall accuracies for B.1.1.214, B.1.177.21, B.1.1.7, B.1.526, and P.1 on the one-vs-all classifiers, suggesting the presence of distinct mutations in these variants. Our results show that embedding vector size and batch sizes have insignificant improvement in accuracies, but changing from 2-mers to 3-mers mostly improves accuracies. We also studied individual runs which show that most accuracies improved after the 20th run, indicating that these sequence positions may have more contributions to distinguishing among different COVID-19 variants.","version":"1.1","doi":"10.1101/2021.06.29.450335","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450330","pub_date":"2021-6-29","title":"Genetic diversity and evolution of SARS-CoV-2 in Belgium during the first wave outbreak","abstract":"SARS-CoV-2, the causative agent of COVID-19 was first detected in Belgium on 3rd February 2020, albeit the first epidemiological wave started in March and ended in June 2020. One year after the first epidemiological wave hit the country data analyses reveled the temporal and variant distribution of SARS-CoV-2 and its implication with Belgian epidemiological measures. In this study, 766 complete SARS-CoV-2 genomes of samples originating from the first epidemiological were sequenced to characterize the temporal and geographic distribution of the COVID-19 pandemic in Belgium through phylogenetic and variant analysis. Our analysis reveals the presence of the major circulating SARS-CoV-2 clades (G, GH and GR) and lineages circulating in Belgium at that time. Moreover, it contextualizes the density of SARS-CoV-2 cases over time with non-intervention measures taken to prevent the spread of SARS-CoV-2 in Belgium, specific international case imports and the functional implications of the most representative non-synonymous mutations present in Belgium between February to June 2020.","version":"1.1","doi":"10.1101/2021.06.29.450330","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431844","pub_date":"2021-6-29","title":"Predicting the zoonotic capacity of mammals to transmit SARS-CoV-2","abstract":"Back and forth transmission of SARS-CoV-2 between humans and animals may lead to wild reservoirs of virus that can endanger efforts toward long-term control of COVID-19 in people, and protecting vulnerable animal populations that are particularly susceptible to lethal disease. Predicting high risk host species is key to targeting field surveillance and lab experiments that validate host zoonotic potential. A major bottleneck to predicting animal hosts is the small number of species with available molecular information about the structure of ACE2, a key cellular receptor required for viral cell entry. We overcome this bottleneck by combining species\u2019 ecological and biological traits with 3D modeling of virus and host cell protein interactions using machine learning methods. This approach enables predictions about the zoonotic capacity of SARS-CoV-2 for over 5,000 mammals \u2014 an order of magnitude more species than previously possible. The high accuracy predictions achieved by this approach are strongly corroborated by in vivo empirical studies. We identify numerous common mammal species whose predicted zoonotic capacity and close proximity to humans may further enhance the risk of spillover and spillback transmission of SARS-CoV-2. Our results reveal high priority areas of geographic overlap between global COVID-19 hotspots and potential new mammal hosts of SARS-CoV-2. With molecular sequence data available for only a small fraction of potential host species, predictive modeling integrating data across multiple biological scales offers a conceptual advance that may expand our predictive capacity for zoonotic viruses with similarly unknown and potentially broad host ranges.","version":"1.3","doi":"10.1101/2021.02.18.431844","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.377333","pub_date":"2021-6-29","title":"Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2","abstract":"The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain, named MASCp36, that causes severe acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was 58 PFU in 9-month-old, male BALB/c mice. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three mutations in RBD significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of human ACE2 (hACE2) or mACE2 in complex with the RBD of MASCp36 at 3.1 to 3.7 angstrom resolution elucidates molecular basis for the receptor-binding switch driven by specific amino acid substitutions. Interestingly, N501Y and Q493H enhanced the binding affinity to human ACE2 (hACE2); while triple mutations N501Y/Q493H/K417N decreased affinity to hACE2, thus led to the reduced infectivity of MASCp36 to human cells. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in human and animals. A mouse adapted SARS-CoV-2 strain that harbored specific amino acid substitutions in the RBD of S protein showed 100% mortality in aged, male BALB/c mice.","version":"1.2","doi":"10.1101/2020.11.10.377333","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450293","pub_date":"2021-6-29","title":"Rapid determination of the wide dynamic range of SARS-CoV-2 Spike T cell responses in whole blood of vaccinated and naturally infected","abstract":"Antibodies and T cells cooperate to control virus infections. The definition of the correlates of protection necessary to manage the COVID-19 pandemic, require both immune parameters but the complexity of traditional tests limits virus-specific T cell measurements. We test the sensitivity and performance of a simple and rapid SARS-CoV-2 Spike-specific T cell test based on stimulation of whole blood with peptides covering the SARS-CoV-2 Spike protein followed by cytokine (IFN-\u03b3, IL-2) measurement in different cohorts including BNT162b2 vaccinated (n=112; 201 samples), convalescent asymptomatic (n=62; 62 samples) and symptomatic (n=68; 115 samples) COVID-19 patients and SARS-CoV-1 convalescent individuals (n=12; 12 samples). The sensitivity of the rapid cytokine whole blood test equates traditional methods of T cell analysis (ELISPOT, Activation Induced Markers). Utilizing this test we observed that Spike-specific T cells in vaccinated preferentially target the S2 region of Spike and that their mean magnitude is similar between them and SARS-CoV-2 convalescents at 3 months after vaccine or virus priming respectively. However, a wide heterogeneity of Spike-specific T cell magnitude characterizes the individual responses irrespective of the time of analysis. No correlation between neutralizing antibody levels and Spike-specific T cell magnitude were found. Rapid measurement of cytokine production in whole blood after peptide activation revealed a wide dynamic range of Spike-specific T cell response after vaccination that cannot be predicted from neutralizing antibody quantities. Both Spike-specific humoral and cellular immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies.","version":"1.1","doi":"10.1101/2021.06.29.450293","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.449051","pub_date":"2021-6-29","title":"Recovery of deleted deep sequencing data sheds more light on the early Wuhan SARS-CoV-2 epidemic","abstract":"The origin and early spread of SARS-CoV-2 remains shrouded in mystery. Here I identify a data set containing SARS-CoV-2 sequences from early in the Wuhan epidemic that has been deleted from the NIH\u2019s Sequence Read Archive. I recover the deleted files from the Google Cloud, and reconstruct partial sequences of 13 early epidemic viruses. Phylogenetic analysis of these sequences in the context of carefully annotated existing data further supports the idea that the Huanan Seafood Market sequences are not fully representative of the viruses in Wuhan early in the epidemic. Instead, the progenitor of currently known SARS-CoV-2 sequences likely contained three mutations relative to the market viruses that made it more similar to SARS-CoV-2\u2019s bat coronavirus relatives.","version":"1.2","doi":"10.1101/2021.06.18.449051","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450244","pub_date":"2021-6-29","title":"Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2","abstract":"SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. SARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. - Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types","version":"1.1","doi":"10.1101/2021.06.28.450244","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450356","pub_date":"2021-6-29","title":"Thrombocytopenia and splenic platelet directed immune responses after intravenous ChAdOx1 nCov-19 administration","abstract":"Vaccines against SARS-CoV-2 are based on a range of novel vaccine platforms, with adenovirus-based approaches (like ChAdOx1 nCov-19) being one of them. Recently a rare and novel complication of SARS-CoV-2 targeted adenovirus vaccines has emerged: thrombosis with thrombocytopenia syndrome (TTS). TTS is characterized by low platelet counts, clot formation at unusual anatomic sites and platelet-activating PF4-polyanion antibodies reminiscent of heparin-induced thrombocytopenia. Here, we employ in vitro and in vivo models to characterize the possible mechanisms of this platelet-targeted autoimmunity. We show that intravenous but not intramuscular injection of ChAdOx1 nCov-19 triggers platelet-adenovirus aggregate formation and platelet activation. After intravenous injection, these aggregates are phagocytosed by macrophages in the spleen and platelet remnants are found in the marginal zone and follicles. This is followed by a pronounced B-cell response with the emergence of circulating antibodies binding to platelets. Our work contributes to the understanding of TTS and highlights accidental intravenous injection as potential mechanism for post-vaccination TTS. Hence, safe intramuscular injection, with aspiration prior to injection, could be a potential preventive measure when administering adenovirus-based vaccines.","version":"1.1","doi":"10.1101/2021.06.29.450356","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450274","pub_date":"2021-6-29","title":"Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage","abstract":"One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo. The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1\u2018 position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.","version":"1.1","doi":"10.1101/2021.06.28.450274","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.29.450397","pub_date":"2021-6-29","title":"Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain","abstract":"Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the 3.7 \u00c5 resolution cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD \u03b2-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition. Cryo-EM structure of neutralizing antibody 5-7 in complex with SARS CoV-2 spike 5-7 recognizes NTD outside of the previously identified antigenic supersite 5-7 binds to a site known to accommodate numerous hydrophobic ligands Structural basis of 5-7 neutralization tolerance to some variants of concern","version":"1.1","doi":"10.1101/2021.06.29.450397","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.415216","pub_date":"2021-6-29","title":"Engineered receptor binding domain immunogens elicit pan-sarbecovirus neutralizing antibodies outside the receptor binding motif","abstract":"Effective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. In mice, we find that a cocktail of these homotrimeric sarbecovirus RBDs elicits antibodies to conserved viral epitopes outside of the ACE2 receptor binding motif (RBM). Importantly, these responses neutralize all sarbecovirus components even in context of prior SARS-CoV-2 imprinting. We further show that a substantial fraction of the neutralizing antibodies elicited after vaccination in humans also engages non-RBM epitopes on the RBD. Collectively, our results suggest a strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine. Immunity to SARS-CoV-2 in the human population will be widespread due to natural infection and vaccination. However, another novel coronavirus will likely emerge in the future and may cause a subsequent pandemic. Humoral responses induced by SARS-CoV-2 infection and vaccination provide limited protection against even closely related coronaviruses. We show immunization with a cocktail of trimeric coronavirus receptor binding domains induces a neutralizing antibody response that is broadened to related coronaviruses with pandemic potential. Importantly, this broadening occurs in context of an initial imprinted SARS-CoV-2 spike immunization showing that preexisting immunity can be expanded to recognize other related coronaviruses. Our immunogens focused the serum antibody response to conserved epitopes on the receptor binding domain outside of the ACE2 receptor binding motif; this contrasts with current SARS-CoV-2 therapeutic antibodies, which predominantly target the receptor binding motif.","version":"1.2","doi":"10.1101/2020.12.07.415216","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450214","pub_date":"2021-6-28","title":"A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS- CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, \u03b2) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent- expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS- CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.06.28.450214","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450153","pub_date":"2021-6-28","title":"Myeloid cell interferon responses correlate with clearance of SARS-CoV-2","abstract":"The emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. While studies have reported immune profiling using single cell RNA sequencing in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. We performed longitudinal single-cell RNA sequencing of bronchoalveolar lavage (BAL) cell suspensions from adult rhesus macaques infected with SARS-CoV-2 (n=6) to delineate the early dynamics of immune cells changes. The bronchoalveolar compartment exhibited dynamic changes in transcriptional landscape 3 days post-SARS-CoV-2-infection (3dpi) (peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline). We observed the accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I IFN response was highly induced in the plasmacytoid dendritic cells. The presence of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin converting enzyme 2 (ACE2) expression was also observed. These macrophages were significantly recruited to the lungs of macaques at 3dpi and harbored SARS-CoV-2, while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery. The recruitment of a myeloid cell-mediated Type I IFN response is associated with the rapid clearance of SARS-CoV-2 infection in macaques.","version":"1.1","doi":"10.1101/2021.06.28.450153","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449918","pub_date":"2021-6-28","title":"Progression and Resolution of SARS-CoV-2 Infection in Golden Syrian Hamsters","abstract":"To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.","version":"1.2","doi":"10.1101/2021.06.25.449918","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450163","pub_date":"2021-6-28","title":"N4-hydroxycytidine and inhibitors of dihydroorotate dehydrogenase synergistically suppress SARS-CoV-2 replication","abstract":"Effective therapeutics to inhibit the replication of SARS-CoV-2 in infected individuals are still under development. The nucleoside analogue N4-hydroxycytidine (NHC), also known as EIDD-1931, interferes with SARS-CoV-2 replication in cell culture. It is the active metabolite of the prodrug Molnupiravir (MK-4482), which is currently being evaluated for the treatment of COVID-19 in advanced clinical studies. Meanwhile, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, counteract virus replication and are also being clinically evaluated for COVID-19 therapy. Here we show that the combination of NHC and DHODH inhibitors such as teriflunomide, IMU-838/vidofludimus, and BAY2402234, strongly synergizes to inhibit SARS-CoV-2 replication. While single drug treatment only mildly impaired virus replication, combination treatments reduced virus yields by at least two orders of magnitude. We determined this by RT-PCR, TCID50, immunoblot and immunofluorescence assays in Vero E6 and Calu-3 cells infected with wildtype and the Alpha and Beta variants of SARS-CoV-2. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC in nascent viral RNA, thus precluding the correct synthesis of the viral genome in subsequent rounds of replication, thereby inhibiting the production of replication competent virus particles. This concept was further supported by the rescue of replicating virus after addition of pyrimidine nucleosides to the media. Based on our results, we suggest combining these drug candidates, which are currently both tested in clinical studies, to counteract the replication of SARS-CoV-2, the progression of COVID-19, and the transmission of the disease within the population. The strong synergy displayed by DHODH inhibitors and the active compound of Molnupiravir might enable lower concentrations of each drug to antagonize virus replication, with less toxicity. Both Molnupiravir and DHODH inhibitors are currently being tested in advanced clinical trials or are FDA-approved for different purposes, raising the perspective of rapidly testing their combinatory efficacy in clinical studies. Molnupiravir is currently a promising candidate for treating early stages of COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it appears only moderately useful in treating severe COVID-19. Since the combination inhibits virus replication far more strongly, and since DHODH inhibitors may also suppress excessive immune responses, the combined clinical application bears the potential of alleviating the disease burden even at later stages.","version":"1.1","doi":"10.1101/2021.06.28.450163","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.20.436163","pub_date":"2021-6-28","title":"The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism\u2019s response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.03.20.436163","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.449914","pub_date":"2021-6-28","title":"Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated as D614G). Results showed minimal effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), B.1.617.2 (Delta), and P.1 (Gamma) showed decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273\u2013elicited serum neutralization.","version":"1.1","doi":"10.1101/2021.06.28.449914","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449990","pub_date":"2021-6-28","title":"Effect of prophylactic use of intra-nasal oil formulations in the hamster model of Covid-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection initiates with viral entry in upper respiratory tract leading to coronavirus disease 2019 (Covid-19). Severe Covid-19 is characterized by pulmonary pathologies associated with respiratory failure. Thus, therapeutics aimed at inhibiting entry of the virus or its internalization in the upper respiratory tract, are of interest. Herein, we report the prophylactic application of two intra-nasal formulations provided by the National Medicinal Plant Board (NMPB), Anu oil and Til tailya in SARS-CoV2 infection hamster model. Prophylactic nasal instillation of these oil formulations exhibited reduced viral load in lungs, and resulted in reduced body weight loss and pneumonitis. In line with reduced viral load, histopathlogical analysis revealed a reduction in lung pathology in Anu oil group as compared to the control infected group. However, Til tailya group did not show a significant reduction in lung pathology. Furthermore, molecular analysis using mRNA expression profiling indicated reduced expression of pro-inflammatory cytokines genes, including Th1 and Th17 cytokines for both the intra-nasal formulations as a result of decreased viral load. Together, the prophylactic intra-nasal application of Annu oil seems to be useful in limiting both the viral load and disease severity disease in SARS-CoV2 infection in hamster model.","version":"1.1","doi":"10.1101/2021.06.25.449990","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436337","pub_date":"2021-6-28","title":"Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy","abstract":"Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.03.22.436337","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.28.450190","pub_date":"2021-6-28","title":"Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models","abstract":"Emerging variants of concern (VOCs) drive the SARS-CoV-2 pandemic. We assessed VOC B.1.1.7, now prevalent in several countries, and VOC B.1.351, representing the greatest threat to populations with immunity to the early SARS-CoV-2 progenitors. B.1.1.7 showed a clear fitness advantage over the progenitor variant (wt-S614G) in ferrets and two mouse models, where the substitutions in the spike glycoprotein were major drivers for fitness advantage. In the \u201csuperspreader\u201d hamster model, B.1.1.7 and wt-S614G had comparable fitness, whereas B.1.351 was outcompeted. The VOCs had similar replication kinetics as compared to wt-S614G in human airway epithelial cultures. Our study highlights the importance of using multiple models for complete fitness characterization of VOCs and demonstrates adaptation of B.1.1.7 towards increased upper respiratory tract replication and enhanced transmission in vivo. B.1.1.7 VOC outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo.","version":"1.1","doi":"10.1101/2021.06.28.450190","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437907","pub_date":"2021-6-28","title":"A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells","abstract":"We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal \u201clate pathway\u201d, the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.","version":"1.3","doi":"10.1101/2021.03.31.437907","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.09.086249","pub_date":"2021-6-28","title":"The role of high cholesterol in age-related COVID19 lethality","abstract":"Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China in 2019. The disease is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.","version":"1.5","doi":"10.1101/2020.05.09.086249","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449320","pub_date":"2021-6-28","title":"The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus entry inhibitor","abstract":"The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective antiviral countermeasures are urgently needed as vaccine development and deployment against an emerging coronavirus takes time, even in the best-case scenario. The green tea catechin, epigallocatechin gallate (EGCG), has broad spectrum antiviral activity. We demonstrate here that EGCG prevents murine and human coronavirus infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from highly pathogenic coronaviruses, as well as a bat coronavirus poised to emerge into humans. We show that EGCG treatment reduces coronavirus attachment to target cell surfaces. Our results demonstrate the potential for the development of pan-coronavirus attachment inhibitors to protect against current and future emerging coronaviruses.","version":"1.2","doi":"10.1101/2021.06.21.449320","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429646","pub_date":"2021-6-28","title":"Extensive recombination-driven coronavirus diversification expands the pool of potential pandemic pathogens","abstract":"The ongoing SARS-CoV-2 pandemic is the third zoonotic coronavirus identified in the last twenty years. Enzootic and epizootic coronaviruses of diverse lineages also pose a significant threat to livestock, as most recently observed for virulent strains of porcine epidemic diarrhea virus (PEDV) and swine acute diarrhea-associated coronavirus (SADS-CoV). Unique to RNA viruses, coronaviruses encode a proofreading exonuclease (ExoN) that lowers point mutation rates to increase the viability of large RNA virus genomes, which comes with the cost of limiting virus adaptation via point mutation. This limitation can be overcome by high rates of recombination that facilitate rapid increases in genetic diversification. To compare dynamics of recombination between related sequences, we developed an open-source computational workflow (IDPlot) to measure nucleotide identity, locate recombination breakpoints, and infer phylogenetic relationships. We analyzed recombination dynamics among three groups of coronaviruses with noteworthy impacts on human health and agriculture: SARSr-CoV, Betacoronavirus-1, and SADSr-CoV. We found that all three groups undergo recombination with highly diverged viruses from sparsely sampled or undescribed lineages, which can disrupt the inference of phylogenetic relationships. In most cases, no parental origin of recombinant regions could be found in genetic databases, suggesting that much coronavirus diversity remains unknown. These patterns of recombination expand the genetic pool that may contribute to future zoonotic events. Our results also illustrate the limitations of current sampling approaches for anticipating zoonotic threats to human and animal health.","version":"1.2","doi":"10.1101/2021.02.03.429646","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.19.431972","pub_date":"2021-6-25","title":"Antiviral activity of influenza A virus defective interfering particles against SARS-CoV-2 replication in vitro through stimulation of innate immunity","abstract":"SARS-CoV-2 causing COVID-19 emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 only show limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.02.19.431972","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449882","pub_date":"2021-6-25","title":"SARS-CoV-2 Variants are Selecting for Spike Protein Mutations that Increase Protein Stability","abstract":"The emergence of SARS-CoV-2 in 2019 has caused severe disruption and a huge number of human deaths across the globe. As the pandemic spreads, a natural result is the emergence of variants with a variety of amino acid mutations. Variants of SARS-CoV-2 with mutations in their spike protein may result in an increased infectivity, increased lethality, or immune escape, and whilst many of these properties can be explained through changes to binding affinity or changes to post-translational modification, many mutations have no known biophysical impact on the structure of protein. The Gibbs free energy of a protein represents a measure of protein stability, with an increased stability resulting in a protein that is more thermodynamically stable, and more robust to changes in external environment. Here we show that mutations in the spike proteins of SARS-CoV-2 are selecting for amino acid changes that result in a more stable protein than expected by chance. We calculate all possible mutations in the SARS-CoV-2 spike protein, and show that many variants are more stable than expected when compared to the background, indicating that protein stability is an important consideration for the understanding of SARS-CoV-2 evolution. Variants exhibit a range of stabilities, and we further suggest that some stabilising mutations may be acting as a \u201ccounterbalance\u201d to destabilising mutations that have other properties, such as increasing binding site affinity for the human ACE2 receptor. We suggest that protein folding calculations offer a useful tool for early identification of advantageous mutations.","version":"1.1","doi":"10.1101/2021.06.25.449882","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449871","pub_date":"2021-6-25","title":"Engineered chimeric T cell receptor fusion construct (TRuC)-expressing T cells prevent translational shutdown in SARS-CoV-2-infected cells","abstract":"SARS-CoV-2, the causative agent of Covid-19, is known to evade the immune system by several mechanisms. This includes the shutdown of the host cellular protein synthesis, which abrogates the induction of antiviral interferon responses. The virus initiates the infection of susceptible cells by binding with its spike protein (S) to the host angiotensin-converting enzyme 2 (ACE2). Here we applied the T cell receptor fusion construct (TRuC) technology to engineer T cells against such infected cells. In our TRuCs an S-binding domain is fused to the CD3\u03b5 component of the T cell receptor (TCR) complex, enabling recognition of S-containing cells in an HLA independent manner. This domain either consists of the S-binding part of ACE2 or a single-chain variable fragment of an anti-S antibody. We show that the TRuC T cells are activated by and kill cells that express S of SARS-CoV-2 and its alpha (B.1.1.7) and beta (B.1.351) variants at the cell surface. Treatment of SARS-CoV-2 infected cells with our engineered T cells did not lead to massive cytotoxicity towards the infected cells, but resulted in a complete rescue of the translational shutdown despite ongoing viral replication. Our data show that engineered TRuC T cell products might be used against SARS-CoV-2 by exposing infected cells to the host innate immune system.","version":"1.1","doi":"10.1101/2021.06.25.449871","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449750","pub_date":"2021-6-25","title":"A random priming amplification method for whole genome sequencing of SARS-CoV-2 and H1N1 influenza A virus","abstract":"Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of COVID that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.","version":"1.1","doi":"10.1101/2021.06.25.449750","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.25.449913","pub_date":"2021-6-25","title":"Use of hiPSC-derived cardiomyocytes to rule out proarrhythmic effects of drugs: the case of hydroxychloroquine in COVID-19","abstract":"In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 patients due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro. While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring may be challenging. We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after subchronic exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients. We demonstrate that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation in vitro and triggered arrhythmias that halted the beating at high concentration. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD values. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to quickly assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.","version":"1.1","doi":"10.1101/2021.06.25.449913","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.22.21259316","pub_date":"2021-06-25","title":"Investigating phenotypes of pulmonary COVID-19 recovery \u2013 a longitudinal observational prospective multicenter trial","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>COVID-19 is associated with long-term pulmonary symptoms and may result in chronic pulmonary impairment. The optimal procedures to prevent, identify, monitor, and treat these pulmonary sequelae are elusive.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research question</jats:title>\n                  <jats:p>To characterize the kinetics of pulmonary recovery, risk factors and constellations of clinical features linked to persisting radiological lung findings after COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Study design and methods</jats:title>\n                  <jats:p>A longitudinal, prospective, multicenter, observational cohort study including COVID-19 patients (n = 108). Longitudinal pulmonary imaging and functional readouts, symptom prevalence, clinical and laboratory parameters were collected during acute COVID-19 and at 60-, 100- and 180-days follow-up visits. Recovery kinetics and risk factors were investigated by logistic regression. Classification of clinical features and study participants was accomplished by k-means clustering, the k-nearest neighbors (kNN), and naive Bayes algorithms.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>At the six-month follow-up, 51.9% of participants reported persistent symptoms with physical performance impairment (27.8%) and dyspnea (24.1%) being the most frequent. Structural lung abnormalities were still present in 45.4% of the collective, ranging from 12% in the outpatients to 78% in the subjects treated at the ICU during acute infection. The strongest risk factors of persisting lung findings were elevated interleukin-6 (IL6) and C-reactive protein (CRP) during recovery and hospitalization during acute COVID-19. Clustering analysis revealed association of the lung lesions with increased anti-S1/S2 antibody, IL6, CRP, and D-dimer levels at the early follow-up suggesting non-resolving inflammation as a mechanism of the perturbed recovery.</jats:p>\n                  <jats:p>Finally, we demonstrate the robustness of risk class assignment and prediction of individual risk of delayed lung recovery employing clustering and machine learning algorithms.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Severity of acute infection, and systemic inflammation is strongly linked to persistent post-COVID-19 lung abnormality. Automated screening of multi-parameter health record data may assist the identification of patients at risk of delayed pulmonary recovery and optimize COVID-19 follow-up management.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Clinical Trial Registration</jats:title>\n                  <jats:p>\n                    ClinicalTrials.gov:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04416100'>NCT04416100</jats:ext-link>\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.22.21259316","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.15.448419","pub_date":"2021-6-24","title":"Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19","abstract":"Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.","version":"1.2","doi":"10.1101/2021.06.15.448419","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.24.449680","pub_date":"2021-6-24","title":"SARS-CoV-2-Triggered Mast Cell Rapid Degranulation Induces Alveolar Epithelial Inflammation and Lung Injury","abstract":"SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. Here we showed that SARS-CoV-2-triggeed MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation alterred various signaling pathways in alveolar epithelial cells, particularly, led to the production of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments. SARS-CoV-2 triggers an immediate mast cell (MC) degranulation, which initiates the alveolar epithelial inflammation and disrupts the tight junction. MC stabilizers that block degranulation reduce virus-induced lung inflammation and injury. The binding of RBD of Spike protein of SARS-CoV-2-to ACE2 receptor protein triggers an immediate MC degranulation MC degranulation induces transcriptomic changes include an upregulated inflammatory signaling and a downregulated cell-junction signaling MC degranulation leads to alveolar epithelial inflammation and disruption of tight junctions MC stabilizer that inhibits degranulation reduces SARS-CoV-2-induced lung inflammation and injury in vivo","version":"1.1","doi":"10.1101/2021.06.24.449680","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.24.449811","pub_date":"2021-6-24","title":"Pyrimidine biosynthesis inhibitors synergize with nucleoside analogs to block SARS-CoV-2 infection","abstract":"The ongoing COVID-19 pandemic has highlighted the dearth of approved drugs to treat viral infections, with only \u223c90 FDA approved drugs against human viral pathogens. To identify drugs that can block SARS-CoV-2 replication, extensive drug screening to repurpose approved drugs is underway. Here, we screened \u223c18,000 drugs for antiviral activity using live virus infection in human respiratory cells. Dose-response studies validate 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Amongst these drug candidates are 16 nucleoside analogs, the largest category of clinically used antivirals. This included the antiviral Remdesivir approved for use in COVID-19, and the nucleoside Molnupirivir, which is undergoing clinical trials. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral, and we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibits SARS-CoV-2 infection in vitro and in vivo suggesting a clinical path forward.","version":"1.1","doi":"10.1101/2021.06.24.449811","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.22.441041","pub_date":"2021-6-24","title":"Prefusion conformation of SARS-CoV-2 receptor-binding domain favours interactions with human receptor ACE2","abstract":"A new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2), poses a serious threat across continents, leading the World Health Organization to declare a Public Health Emergency of International Concern. In order to block the entry of the virus into human host cells, major therapeutic and vaccine design efforts are now targeting the interactions between the SARS-CoV-2 spike (S) glycoprotein and the human cellular membrane receptor angiotensin-converting enzyme, hACE2. By analyzing cryo-EM structures of SARS-CoV-2 and SARS-CoV-1, we report here that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) that binds with hACE2 has expanded in size, undergoing a large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits higher intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bonds in the CoV-2 complex. Further interface analysis has shown that interfacial water promotes and stabilizes the formation of CoV-2/hACE2 complex. This interaction has caused a significant structural rigidification, favoring proteolytic processing of S protein for the fusion of the viral and cellular membrane. Moreover, conformational dynamics simulations of RBD motions in SARS-CoV-2 and SARS-CoV-1 point to the role in modification in the RBD dynamics and their likely impact on infectivity.","version":"1.2","doi":"10.1101/2021.04.22.441041","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449282","pub_date":"2021-6-24","title":"Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-CoV-2 activation","abstract":"Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-coronavirus-2 (SARS-CoV-2). Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) Protein, enabling virus-host membrane fusion and infection of the lungs. We present here an efficient recombinant production strategy for enzymatically active TMPRSS2 ectodomain enabling enzymatic characterization, and the 1.95 \u00c5 X-ray crystal structure. To stabilize the enzyme for co-crystallization, we pre-treated TMPRSS2 with the synthetic protease inhibitor nafamosat to form a stable but slowly reversible (15 hour half-life) phenylguanidino acyl-enzyme complex. Our study provides a structural basis for the potent but non-specific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that will guide future generations of inhibitors to improve selectivity. TMPRSS2 cleaved recombinant SARS-CoV-2 S protein ectodomain at the canonical S1/S2 cleavage site and at least two additional minor sites previously uncharacterized. We established enzymatic activity and inhibition assays that enabled ranking of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.7 nM to 120 \u03bcM and determination of inhibitor mechanisms of action. These results provide a body of data and reagents to support future drug development efforts to selectively inhibit TMPRSS2 and other type 2 transmembrane serine proteases involved in viral glycoprotein processing, in order to combat current and future viral threats. Viruses hijack the biochemical activity of host proteins for viral invasion and replication. Transmembrane protease, serine-2 (TMPRSS2) is a surface-expressed protease implicated in the activation of influenza A, influenza B, and coronaviruses, including SARS-CoV-2, to drive efficient infection of the lungs. TMPRSS2 is an attractive target for antiviral therapies, as inhibiting its proteolytic activity blocks efficient viral entry. However, a structural and biochemical understanding of the protease has remained elusive and no selective inhibitors are available. We engineered on-demand activatable TMPRSS2 ectodomain and determined the 1.95 \u00c5 X-ray crystal structure of the stabilized acyl-enzyme after treatment with nafamostat, a protease inhibitor under investigation as a COVID-19 therapeutic. The structure reveals unique features of the TMPRSS2 substrate recognition pocket and domain architecture, and explains the potent, but nonselective inhibition by nafamostat. TMPRSS2 efficiently cleaved the SARS-CoV-2 S protein at the canonical S1/S2 site as well as two minor sites previously uncharacterized. We further established a robust enzymatic assay system and characterized inhibition by two additional clinical protease inhibitors under study for COVID-19, camostat and bromhexine. Our results provide a body of data and reagents to enable ongoing drug development efforts to selectively inhibit TMPRSS2 and other TTSPs involved in viral glycoprotein processing, in order to combat current and future viral threats.","version":"1.1","doi":"10.1101/2021.06.23.449282","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.444275","pub_date":"2021-6-24","title":"Allicin inhibits SARS-CoV-2 replication and abrogates the antiviral host response in the Calu-3 proteome","abstract":"The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health burden. Volatile garlic organosulfur compounds, such as the thiol-reactive allicin (diallyl thiosulfinate) exert strong antimicrobial activity against various respiratory pathogens. Here, we investigated the antiviral activity of allicin against SARS-CoV-2 in infected Vero E6 and Calu-3 lung cells. Calu-3 cells showed greater allicin tolerance due >4-fold increased GSH levels compared to Vero E6. However, biocompatible allicin doses efficiently inhibited viral replication in both cell lines. Proteome analyses of SARS-CoV-2 infected Calu-3 cells revealed a strong induction of the antiviral interferon-stimulated gene (ISG) signature (e.g. cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, 2\u20195\u2019OAS and ISG15), pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTC1, ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin abrogated the ISG host response and reverted the host cellular pathways to levels of uninfected Calu-3 cells, confirming the antiviral and immunomodulatory activity of allicin in the host proteome. Thus, biocompatible doses of allicin could be promising for protection of lung cells against SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.05.15.444275","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449627","pub_date":"2021-6-24","title":"E484K and N501Y SARS-CoV 2 Spike Mutants Increase ACE2 Recognition but Reduce Affinity for Neutralizing Antibody","abstract":"SARS-CoV2 mutants emerge as variants of concern (VOC) due to altered selection pressure and rapid replication kinetics. Among them, lineages B.1.1.7, B.1.351, and P.1 contain a key mutation N501Y. B.1.135 and P.1 lineages have another mutation, E484K. Here, we decode the effect of these two mutations on the host receptor, ACE2, and neutralizing antibody (B38) recognition. The gain in binding affinity for the N501Y RBD mutant to the ACE2 is attributed to improved \u03c0-\u03c0 stacking and \u03c0-cation interactions. The enhanced receptor affinity of the E484K mutant is caused due to the formation of a specific hydrogen bond and salt-bridge interaction with Glu75 of ACE2. Notably, both the mutations reduce the binding affinity for B38 due to the loss of several hydrogen-bonding interactions. The insights obtained from the study are crucial to interpret the increased transmissibility and reduction in the neutralization efficacy of rapidly emerging SARS-CoV2 VOCs.","version":"1.1","doi":"10.1101/2021.06.23.449627","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449594","pub_date":"2021-6-23","title":"SARS-CoV-2 neutralising antibodies in Dogs and Cats in the United Kingdom","abstract":"Companion animals are susceptible to SARS-CoV-2 infection and sporadic cases of pet infections have occurred in the United Kingdom. Here we present the first large-scale serological survey of SARS-CoV-2 neutralising antibodies in dogs and cats in the UK. Results are reported for 688 sera (454 canine, 234 feline) collected by a large veterinary diagnostic laboratory for routine haematology during three time periods; pre-COVID-19 (January 2020), during the first wave of UK human infections (April-May 2020) and during the second wave of UK human infections (September 2020-February 2021). Both pre-COVID-19 sera and those from the first wave tested negative. However, in sera collected during the second wave, 1.4% (n=4) of dogs and 2.2% (n=2) cats tested positive for neutralising antibodies. The low numbers of animals testing positive suggests pet animals are unlikely to be a major reservoir for human infection in the UK. However, continued surveillance of in-contact susceptible animals should be performed as part of ongoing population health surveillance initiatives.","version":"1.1","doi":"10.1101/2021.06.23.449594","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.22.449540","pub_date":"2021-6-23","title":"Escherichia coli recombinant expression of SARS-CoV-2 protein fragments","abstract":"We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach used the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All fusion proteins were expressed in E. coli at about 0.1 g/L, and could be purified with a single affinity binding step using inexpensive cellulose powder. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibody directed to the appropriate SARS-CoV-2 antigenic region. The largest intact CBM9 fusion protein incorporates spike protein amino acids 540-588, which is a conserved region immediately C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.","version":"1.1","doi":"10.1101/2021.06.22.449540","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449639","pub_date":"2021-6-23","title":"Comparative Structural Analyses of Selected Spike Protein-RBD Mutations in SARS-CoV-2 Lineages","abstract":"The severity of the covid 19 has been observed throughout the world as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had spread globally claiming more than 2 million lives and left a devastating impact on peoples\u2019 life. Recently several virulent mutant strains of this virus, such as the B.1.1.7, B.1.351, and P1 lineages have emerged. These strains are predominantly observed in UK, South Africa and Brazil. Another extremely pathogenic B.1.617 lineage and its sub-lineages, first detected in India, are now affecting some countries at notably stronger spread-rates. This paper computationally examines the time-based structures of B.1.1.7, B.1.351, P1 lineages with selected spike protein mutations. Additionally, the mutations in the more recently found B.1.617 lineage and some of its sub-lineages are explored, and the implications for multiple point mutations of the spike protein\u2019s receptor-binding domain (RBD) are described.","version":"1.1","doi":"10.1101/2021.06.23.449639","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449558","pub_date":"2021-6-23","title":"Unsupervised classification of SARS-CoV-2 genomic sequences uncovers hidden genetic diversity and suggests an efficient strategy for genomic surveillance","abstract":"Accurate and timely monitoring of emerging genomic diversity is crucial for limiting the spread of potentially more transmissible/pathogenic strains of SARS-CoV-2. At the time of writing, over 1.8M distinct viral genome sequences have been made publicly available, and a sophisticated nomenclature system based on phylogenetic evidence and expert manual curation has allowed the relatively rapid classification of emerging lineages of potential concern. Here, we propose a complementary approach that integrates fine-grained spatiotemporal estimates of allele frequency with unsupervised clustering of viral haplotypes, and demonstrate that multiple highly frequent genetic variants, arising within large and/or rapidly expanding SARS-CoV-2 lineages, have highly biased geographic distributions and are not adequately captured by current SARS-CoV-2 nomenclature standards. Our results advocate a partial revision of current methods used to track SARS-CoV-2 genomic diversity and highlight the importance of the application of strategies based on the systematic analysis and integration of regional data. Here we provide a complementary, completely automated and reproducible framework for the mapping of genetic diversity in time and across different geographic regions, and for the prioritization of virus variants of potential concern. We believe that the approach outlined in this study will contribute to relevant advances to current genomic surveillance methods.","version":"1.1","doi":"10.1101/2021.06.23.449558","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426626","pub_date":"2021-6-23","title":"Immunogenicity and protective efficacy of a highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative","abstract":"The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of \u223c80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 \u00b0C and were stable to long term storage of over 4 weeks at 37 \u00b0C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were \u223c three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.","version":"1.3","doi":"10.1101/2021.01.13.426626","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.23.449568","pub_date":"2021-6-23","title":"Increased lung cell entry of B.1.617.2 and evasion of antibodies induced by infection and BNT162b2 vaccination","abstract":"The delta variant of SARS-CoV-2, B.1.617.2, emerged in India and has subsequently spread to over 80 countries. B.1.617.2 rapidly replaced B.1.1.7 as the dominant virus in the United Kingdom, resulting in a steep increase in new infections, and a similar development is expected for other countries. Effective countermeasures require information on susceptibility of B.1.617.2 to control by antibodies elicited by vaccines and used for COVID-19 therapy. We show, using pseudotyping, that B.1.617.2 evades control by antibodies induced upon infection and BNT162b2 vaccination, although with lower efficiency as compared to B.1.351. Further, we found that B.1.617.2 is resistant against Bamlanivimab, a monoclonal antibody with emergency use authorization for COVID-19 therapy. Finally, we show increased Calu-3-lung cell entry and enhanced cell-to-cell fusion of B.1.617.2, which may contribute to augmented transmissibility and pathogenicity of this variant. These results identify B.1.617.2 as an immune evasion variant with increased capacity to enter and fuse lung cells.","version":"1.1","doi":"10.1101/2021.06.23.449568","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449352","pub_date":"2021-6-22","title":"Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation","abstract":"The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein\u2019s function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we therefore recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration, if higher titer vectors are required.","version":"1.1","doi":"10.1101/2021.06.21.449352","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.22.449355","pub_date":"2021-6-22","title":"Detection of antibodies neutralizing historical and emerging SARS-CoV-2 strains using a thermodynamically coupled de novo biosensor system","abstract":"With global vaccination efforts against SARS-CoV-2 underway, there is a need for rapid quantification methods for neutralizing antibodies elicited by vaccination and characterization of their strain dependence. Here, we describe a designed protein biosensor that enables sensitive and rapid detection of neutralizing antibodies against wild type and variant SARS-CoV-2 in serum samples. More generally, our thermodynamic coupling approach can better distinguish sample to sample differences in analyte binding affinity and abundance than traditional competition based assays.","version":"1.1","doi":"10.1101/2021.06.22.449355","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.22.449398","pub_date":"2021-6-22","title":"The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity","abstract":"The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNA and, through the recruitment of TRIM25, KHNYN and other cellular RNA degradation machinery, target them for degradation or prevent their translation. ZAP\u2019s activity requires the N-terminal RNA binding domain that selectively binds CpG-containing RNA. However, much less is known about the functional contribution of the remaining domains. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain of the long ZAP isoform (ZAP-L) is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the inactive catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks CpG-dependent antiviral activity despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L\u2019s CpG-directed antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in the ZAP-L-mediated antiviral activity. Cell-intrinsic antiviral factors, such as the zinc-finger antiviral protein (ZAP), provide a first line of defence against viral pathogens. ZAP acts by selectively binding CpG dinucleotide-rich RNAs, which are more common in some viruses than their vertebrate hosts, leading to their degradation. Here, we show that the ability to target these foreign elements is not only dependent on ZAP\u2019s N-terminal RNA-binding domain, but additional determinants in the central and C-terminal regions also regulate this process. The PARP domain and its associated CaaX box, are crucial for ZAP\u2019s CpG-specific activity and required for optimal binding to cofactors TRIM25 and KHNYN. Furthermore, a CaaX box, known to mediate post-translational modification by a hydrophobic S-farnesyl group, caused re-localization of ZAP from the cytoplasm and increased its association with intracellular membranes. This change in ZAP\u2019s distribution was essential for inhibition of both a ZAP-sensitized HIV-1 and SARS-CoV-2. Our work unveils how the determinants outside the CpG RNA-binding domain assist ZAP\u2019s antiviral activity and highlights the role of S-farnesylation and membrane association in this process.","version":"1.1","doi":"10.1101/2021.06.22.449398","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449284","pub_date":"2021-6-21","title":"SARS-CoV-2 activates ER stress and Unfolded protein response","abstract":"Coronavirus disease-2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus infection is a major global public health concern affecting millions of people worldwide. The scientific community has joint efforts to provide effective and rapid solutions to this disease. Knowing the molecular, transmission and clinical features of this disease is of paramount importance to develop effective therapeutic and diagnostic tools. Here, we provide evidence that SARS-CoV-2 hijacks the glycosylation biosynthetic, ER-stress and UPR machineries for viral replication using a time-resolved (0-48 hours post infection, hpi) total, membrane as well as glycoproteome mapping and orthogonal validation. We found that SARS-CoV-2 induces ER stress and UPR is observed in Vero and Calu-3 cell lines with activation of the PERK-eIF2\u03b1-ATF4-CHOP signaling pathway. ER-associated protein upregulation was detected in lung biopsies of COVID-19 patients and associated with survival. At later time points, cell death mechanisms are triggered. The data show that ER stress and UPR pathways are required for SARS-CoV-2 infection, therefore representing a potential target to develop/implement anti-CoVID-19 drugs.","version":"1.1","doi":"10.1101/2021.06.21.449284","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444467","pub_date":"2021-6-21","title":"Moxidectin and ivermectin inhibit SARS-CoV-2 replication in Vero E6 cells but not in human primary airway epithelium cells","abstract":"Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 \u00b5M. These in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. In April 2021, the World Health Organization stated, however, the following: \u201cthe current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive\u201d. It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to head comparison of the antiviral activity of ivermectin and a structurally related, but metabolically more stable, moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that the compounds predominantly act on a step after virus cell entry. Surprisingly, however, in human airway-derived cell models, moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at a concentration of 10 \u00b5M. These disappointing results calls for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on Vero cells. Altogether, these findings suggest that, even by using a high-dose regimen of ivermectin or switching to another drug in the same class are unlikely to be useful for treatment against SARS-CoV-2 in humans.","version":"1.2","doi":"10.1101/2021.05.17.444467","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.20.436243","pub_date":"2021-6-21","title":"A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo","abstract":"Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we isolated a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the B.1.351 variant, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the B.1.351 variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes a novel strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.03.20.436243","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.20.449191","pub_date":"2021-6-21","title":"The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor","abstract":"Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community \u2013 the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of structural heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native mass spectrometry (MS) as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.","version":"1.1","doi":"10.1101/2021.06.20.449191","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449182","pub_date":"2021-6-21","title":"Immunological features that determine the strength of antibody responses to BNT162b2 mRNA vaccine against SARS-CoV-2","abstract":"We analyzed peripheral blood mononuclear cells (PBMCs) of each 20 individuals with a high anti-SARS-CoV-2 antibody titer and a low antibody titer out of 1,774 healthcare workers who received BNT162b2 mRNA vaccine. A higher antibody titer was associated with the frequencies of na\u00efve and transitional B cells before vaccination. In addition, fold changes in the frequency of activated CD8+ T cells upon vaccination were correlated with the antibody titers.","version":"1.1","doi":"10.1101/2021.06.21.449182","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.21.449205","pub_date":"2021-6-21","title":"One viral sequence for each host? \u2013 The neglected within-host diversity as the main stage of SARS-CoV-2 evolution","abstract":"The standard practice of presenting one viral sequence for each infected individual implicitly assumes low within-host genetic diversity. It places the emphasis on the viral evolution between, rather than within, hosts. To determine this diversity, we collect SARS-CoV-2 samples from the same patient multiple times. Our own data in conjunction with previous reports show that two viral samples collected from the same individual are often very different due to the substantial within-host diversity. Each sample captures only a small part of the total diversity that is transiently and locally released from infected cells. Hence, the global SARS-CoV-2 population is a meta-population consisting of the viruses in all the infected hosts, each of which harboring a genetically diverse sub-population. Advantageous mutations must be present first as the within-host diversity before they are revealed as between-host polymorphism. The early detection of such diversity in multiple hosts could be an alarm for potentially dangerous mutations. In conclusion, the main forces of viral evolution, i.e., mutation, drift, recombination and selection, all operate within hosts and should be studied accordingly. Several significant implications are discussed.","version":"1.1","doi":"10.1101/2021.06.21.449205","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447321","pub_date":"2021-6-21","title":"E156G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.617.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape","abstract":"Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type.","version":"1.3","doi":"10.1101/2021.06.07.447321","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.20.448993","pub_date":"2021-6-20","title":"Selectively expressing SARS-CoV-2 Spike protein S1 subunit in cardiomyocytes induces cardiac hypertrophy in mice","abstract":"Cardiac injury is common in hospitalized COVID-19 patients and portends poorer prognosis and higher mortality. To better understand how SARS-CoV-2 (CoV-2) damages the heart, it is critical to elucidate the biology of CoV-2 encoded proteins, each of which may play multiple pathological roles. For example, CoV-2 Spike glycoprotein (CoV-2-S) not only engages ACE2 to mediate virus infection, but also directly impairs endothelial function and can trigger innate immune responses in cultured murine macrophages. Here we tested the hypothesis that CoV-2-S damages the heart by activating cardiomyocyte (CM) innate immune responses. HCoV-NL63 is another human coronavirus with a Spike protein (NL63-S) that also engages ACE2 for virus entry but is known to only cause moderate respiratory symptoms. We found that CoV-2-S and not NL63-S interacted with Toll-like receptor 4 (TLR4), a crucial pattern recognition receptor that responsible for detecting pathogen and initiating innate immune responses. Our data show that the S1 subunit of CoV-2-S (CoV-2-S1) interacts with the extracellular leucine rich repeats-containing domain of TLR4 and activates NF-kB. To investigate the possible pathological role of CoV-2-S1 in the heart, we generated a construct that expresses membrane-localized CoV-2-S1 (S1-TM). AAV9-mediated, selective expression of the S1-TM in CMs caused heart dysfunction, induced hypertrophic remodeling, and elicited cardiac inflammation. Since CoV-2-S does not interact with murine ACE2, our study presents a novel ACE2-independent pathological role of CoV-2-S, and suggests that the circulating CoV-2-S1 is a TLR4-recognizable alarmin that may harm the CMs by triggering their innate immune responses.","version":"1.1","doi":"10.1101/2021.06.20.448993","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448816","pub_date":"2021-6-19","title":"Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with Pol \u03b1 - Primase","abstract":"The molecular mechanisms that drive the infection by the SARS-CoV-2 coronavirus \u2013 the causative agent of the COVID-19 (Coronavirus disease-2019) pandemic \u2013 are under intense current scrutiny, to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent cell-based analyses of SARS-CoV-2 protein - protein interactions have mapped the human proteins targeted by the virus. The DNA polymerase \u03b1 - primase complex or primosome \u2013 responsible for initiating DNA synthesis in genomic duplication \u2013 was identified as a target of nsp1 (non structural protein 1), a major virulence factor in the SARS-CoV-2 infection. Here, we report the biochemical characterisation of the interaction between nsp1 and the primosome and the cryoEM structure of the primosome - nsp1 complex. Our data provide a structural basis for the reported interaction between the primosome and nsp1. They suggest that Pol \u03b1 - primase plays a part in the immune response to the viral infection, and that its targeting by SARS-CoV-2 aims to interfere with such function.","version":"1.1","doi":"10.1101/2021.06.17.448816","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.19.449100","pub_date":"2021-6-19","title":"SARS-CoV-2 mRNA Vaccine Induces Robust Specific and Cross-reactive IgG and Unequal Strain-specific Neutralizing Antibodies in Na\u00efve and Previously Infected Recipients","abstract":"With the advance of SARS-CoV-2 vaccines, the outlook for overcoming the global COVID-19 pandemic has improved. However, understanding of immunity and protection offered by the SARS-CoV-2 vaccines against circulating variants of concern (VOC) is rapidly evolving. We investigated the mRNA vaccine-induced antibody responses against the referent WIV04 (Wuhan) strain, circulating variants, and human endemic coronaviruses in 168 na\u00efve and previously infected people at three-time points. Samples were collected prior to vaccination, after the first and after the second doses of one of the two available mRNA-based vaccines. After full vaccination, both na\u00efve and previously infected participants developed comparable robust SARS-CoV-2 specific spike IgG levels, modest IgM and IgA binding antibodies, and varying degrees of HCoV cross-reactive antibodies. However, the strength and frequency of neutralizing antibodies produced in na\u00efve people were significantly lower than in the previously infected group. We also found that 1/3rd of previously infected people had undetectable neutralizing antibodies after the first vaccine dose; 40% of this group developed neutralizing antibodies after the second dose. In all subjects neutralizing antibodies produced against the B.1.351 and P.1 variants were weaker than those produced against the reference and B.1.1.7 strains. Our findings provide support for future booster vaccinations modified to be active against the circulating variants.","version":"1.1","doi":"10.1101/2021.06.19.449100","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.19.449092","pub_date":"2021-6-19","title":"Mapping the host protein interactome of non-coding regions in SARS-CoV-2 genome","abstract":"A deep understanding of SARS-CoV-2-host interactions is crucial to the development of effective therapeutics. The role of non-coding regions of viral RNA (ncrRNAs) has not been scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry (MAMS) to systematically map the interactome of SARS-CoV-2 ncrRNA in different human cell lines. Integration of the results defined the core and cell-type-specific ncrRNA-host protein interactomes. The majority of ncrRNA-binding proteins were involved in RNA biogenesis, protein translation, viral infection, and stress response. The 5\u2032 UTR interactome is enriched with proteins in the snRNP family and is a target for the regulation of viral replication and transcription. The 3\u2032 UTR interactome is enriched with proteins involved in the cytoplasmic RNP granule (stress granule) and translation regulation. We show that the ORF10 is likely to be a part of 3\u2032 UTR. Intriguingly, the interactions between negative-sense ncrRNAs and host proteins, such as translation initiation factors and antiviral factors, suggest a pathological role of negative-sense ncrRNAs. Moreover, the cell-type-specific interactions between ncrRNAs and mitochondria may explain the differences of cell lines in viral susceptibility. Our study unveils a comprehensive landscape of the functional SARS-CoV-2 ncrRNA-host protein interactome, providing a new perspective on virus-host interactions and the design of future therapeutics.","version":"1.1","doi":"10.1101/2021.06.19.449092","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.449083","pub_date":"2021-6-19","title":"Sequencing using a two-steps strategy reveals high genetic diversity in the S gene of SARS-CoV-2 after a high transmission period in Tunis, Tunisia","abstract":"Recent efforts have reported numerous variants that influence SARS-CoV-2 viral characteristics including pathogenicity, transmission rate and ability of detection by molecular tests. Whole genome sequencing based on NGS technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low and middle income countries. To decrease sequencing cost, we developed a couple of primers allowing to generate partial sequences in the viral S gene allowing rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred and one nasopharyngeal specimens collected during the decreasing phase of a high transmission COVID-19 wave in T unisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allowed the detection of first introduction in the country of already known VOCs and VOIs as well as others variants that have interesting genomic mutations and need to be kept under surveillance. The method of choice for SARS-CoV-2 variants detection is whole genome sequencing using NGS technologies. Resources for this technology remain limited in many low and middle income countries where it is not possible to perform whole genome sequencing for representative number of SARS-CoV-2 positive cases. In the present work, we developed a novel strategy based on a first partial sanger screening in the S gene including key mutations of the already known VOCs and VOIs for rapid identification of these VOCs and VOIs and helps to better select specimens that need to be sequenced by NGS technologies. The second step consisting in whole genome sequencing allowed to have a holistic view of all variants within the selected viral strains and confirmed the initial classification of the strains based on partial S gene sequencing.","version":"1.1","doi":"10.1101/2021.06.18.449083","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.449054","pub_date":"2021-6-19","title":"The percentage of Monocytes CD39+ is higher in Pregnant COVID-19 than in Non-Pregnant COVID-19 patients","abstract":"Current medical guidelines consider COVID-19 pregnant women a high-risk group. Physiological gestation down regulates the immunological response to maintain \u201cmaternal-fetal tolerance\u201d; hence, a SARS-CoV-2 infection constitutes a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients a cross-sectional study was conducted. Leukocyte immunophenotype, mononuclear leukocyte response to polyclonal stimulus and cytokine/chemokine serum concentration were analyzed in pregnant fifteen COVID-19+ and control groups (fifteen non-pregnant COVID-19+, and thirteen pregnant COVID-19-women). Pregnant COVID-19+ patients exhibit lower percentages of monocytes HLA-DR+ compared with control groups. Nevertheless, pregnant COVID-19+ women show a higher percentage of monocytes CD39+ than controls. Furthermore, a higher concentration of TNF-\u03b1, IL-6, MIP1b and IL-4 was observed within the pregnant COVID-19+ group. Our result shows that pregnant women express immunological characteristics that potentially mediate the immune response in COVID-19.","version":"1.1","doi":"10.1101/2021.06.18.449054","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.446355","pub_date":"2021-6-19","title":"Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding","abstract":"The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.","version":"1.1","doi":"10.1101/2021.06.18.446355","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.19.449095","pub_date":"2021-6-19","title":"Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs","abstract":"MicroRNA are short and non-coding RNA, 18-25 nucleotides in length. They are produced at the early stage of viral infection. The roles played by cellular miRNAs and miRNA-mediated gene-silencing in the COVID-19 epidemic period is critical in order to develop novel therapeutics. We analysed SARS-CoV-2 Surface Glycoprotein (S) nucleotide sequence originating from India as well as Iran, Australia, Germany, Italy, Russia, China, Japan and Turkey and identified mutation in potential seed location of several human miRNA. Seventy single nucleotide polymorphisms (SNP) were detected in the S gene out of which, 36, 32 and 2 were cases of transitions, transversions and deletions respectively. Eleven human miRNA targets were identified on the reference S gene sequence with a score >80 in the miRDB database. Mutation A845S erased a common binding site of 7 human miRNA (miR-195-5p, miR-16-5p, miR-15b-5p, miR-15a-5p, miR-497-5p, miR-424-5p and miR-6838-5p). A synonymous mutation altered the wild type Ochre stop codon within the S gene sequence (Italy) to Opal thereby changing the seed sequence of miR-511-3p. Similar (synonymous) mutations were detected at amino acid position 659 and 1116 of the S gene where amino acids serine and threonine were retained, abolishing potential seed location for miR-219a-1-3p and miR-20b-3p respectively. The significance of this finding in reference to the strategy to use synthetic miRNA combinations as a novel therapeutic tool is discussed.","version":"1.1","doi":"10.1101/2021.06.19.449095","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.449086","pub_date":"2021-6-19","title":"Differences in IgG antibody responses following BNT162b2 and mRNA-1273 Vaccines","abstract":"Studies examining antibody responses by vaccine brand are lacking and may be informative for optimizing vaccine selection, dosage, and regimens. The purpose of this study is to assess IgG antibody responses following immunization with BNT162b2 (30 \u03bcg S protein) and mRNA-1273 (100 \u03bcg S protein) vaccines. A cohort of clinicians at a non-for-profit organization is being assessed clinically and serologically following immunization with BNT162b2 or mRNA-1273. IgG responses were measured at the Remington Laboratory by an IgG against the SARS-CoV-2 spike protein-receptor binding domain. Mixed-effect linear (MEL) regression modeling was used to examine whether the SARS-CoV-2 IgG level differed by vaccine brand, dosage, or days since vaccination. Among 532 SARS-CoV-2 seronegative participants, 530 (99.6%) seroconverted with either vaccine. After adjustments for age and gender MEL regression modeling revealed that the average IgG increased after the second dose compared to the first dose (p<0.001). Overall, titers peaked at week six for both vaccines. Titers were significantly higher for mRNA-1273 vaccine on days 14-20 (p < 0.05), 42-48 (p < 0.01), 70-76 (p < 0.05), 77-83 (p < 0.05), and higher for BNT162b2 vaccine on days 28-34 (p < 0.001). In two participants taking immunosuppressive drugs SARS-CoV-2 IgG remained negative. mRNA-1273 elicited both earlier and higher IgG antibody responses than BNT162b2, possibly due to the higher S-protein delivery. Prospective clinical and serological follow-up of defined cohorts such as this may prove useful in determining antibody protection and whether differences in antibody kinetics between the vaccines have manufacturing relevance and clinical significance.","version":"1.1","doi":"10.1101/2021.06.18.449086","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448890","pub_date":"2021-6-18","title":"No evidence of SARS-CoV-2 infection in Neotropical Primates sampled during COVID-19 pandemic in Minas Gerais and Rio Grande do Sul, Brazil","abstract":"In 2019, a new coronavirus disease (COVID-19) was detected in China. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was capable to infect domestic and captive mammals like cats, tigers and minks. Due to genetic similarities, concern about the infection of Non-Human Primates (NHPs) and the establishment of a sylvatic cycle has grown in the Americas. In this study, neotropical primates (NP) were sampled in different areas from Brazil to investigate whether they were infected by SARS-CoV-2. A total of 89 samples from 51 NP of four species were examined. No positive samples were detected via RT-qPCR, regardless of the NHP species, tissue or habitat tested. This work provides the first report on the lack of evidence of circulation of SARS-CoV-2 in NP. The expand of wild animals sampling is necessary to understand their role in the epidemiology of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.06.17.448890","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.448958","pub_date":"2021-6-18","title":"Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2","abstract":"The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the \u201cimmune-escape\u201d Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.","version":"1.1","doi":"10.1101/2021.06.18.448958","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.448982","pub_date":"2021-6-18","title":"Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids","abstract":"The events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.","version":"1.1","doi":"10.1101/2021.06.18.448982","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429495","pub_date":"2021-6-18","title":"Molecular dynamics simulation study reveals effects of key mutations on spike protein structure in SARS-CoV-2","abstract":"SARS-CoV-2 has been spreading rapidly since 2019 and has produced large-scale mutations in the genomes. The mutation in genes may lead to changes in protein structure, which would have a great impact on the epidemiological characteristics. In this study, we selected the key mutations of SARS-CoV-2 from a real-time monitoring tool, including D614G, A222V, N501Y, T716I, S982A, D1118H of spike (S) protein, and performed molecular dynamics (MD) simulations on single-site mutant D614G, double-site mutant D614G&A222V and penta-site mutant N501Y&D614G&T716I&S982A&D1118H to investigate their effects on protein structure and stability using molecular dynamics (MD) simulations. The results suggested that D614G improved the stability of S protein, while D614G&A222V and N501Y&D614G&T716I&S982A&D1118H showed an increased solvent accessible surface area and they might enhance the ability of protein to react with the outside environment. Our findings could complement the mechanistic link between genotype--phenotype--epidemiological characteristics in the study of SARS-CoV-2. We also found no significant difference between the antigenicity of S protein and the mutants through Ellipro, which may reference for vaccine development and application.","version":"1.3","doi":"10.1101/2021.02.03.429495","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.448939","pub_date":"2021-6-18","title":"Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses","abstract":"Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behaviour of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behaviour arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e. Fabs). Surprisingly, these sub-stoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.","version":"1.1","doi":"10.1101/2021.06.18.448939","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448891","pub_date":"2021-6-18","title":"A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global health. One attractive antiviral target is the membrane fusion mechanism employed by the virus to gain access to the host cell. Here we report a robust protein-based fluorescent polarization assay, that mimicking the formation of the six-helix bundle (6-HB) process during the membrane fusion, for the evaluation and screening of SARS-CoV-2 fusion Inhibitors. The IC50 of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal C) were measured to be 6 nM, 2.5 nM, and 8.9 \u00b5M respectively. In addition, we found Sal A has a slightly lower IC50 (3.9 \u00b5M) than Sal C. Interesting, simple caffeic acid can also disrupt the formation of 6-HB with sub-mM concentration. A pilot high throughput screening (HTS) a small marine natural product library validates the assay with a Z\u2019 factor close to 0.8. We envision the current assay provides a convenient way to screen SARS-CoV-2 fusion inhibitor and assess their binding affinity.","version":"1.1","doi":"10.1101/2021.06.17.448891","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.21258689","pub_date":"2021-06-18","title":"Generation and transmission of inter-lineage recombinants in the SARS-CoV-2 pandemic","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern, but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of two months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7\u2019s set of mutations.</jats:p>","version":null,"doi":"10.1101/2021.06.18.21258689","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.18.448921","pub_date":"2021-6-18","title":"Identification of SGLT2 inhibitor Ertugliflozin as a treatment for COVID-19 using computational and experimental paradigm","abstract":"Drug repurposing can expedite the process of drug development by identifying known drugs which are effective against SARS-CoV-2. The RBD domain of SARS-CoV-2 Spike protein is a promising drug target due to its pivotal role in viral-host attachment. These specific structural domains can be targeted with small molecules or drug to disrupt the viral attachment to the host proteins. In this study, FDA approved Drugbank database were screened using a virtual screening approach and computational chemistry methods. Five drugs were short listed for further profiling based on docking score and binding energies. Further these selected drugs were tested for their in vitro biological activity. There was significant correlation between the prediction from computational studies and the actual RBD-ACE2 binding inhibition by the drugs. Then, we performed a series of studies that mimic some of the biological events seen in COVID-19 patients such as secretion of IL1\u03b2, presentation of a more thrombogenic endothelium by production of thrombomodulin and accumulation of inflammatory cells such as monocytes in the lungs. Of all the drugs, most promising drug was Ertugliflozin which is used for type-2 diabetes. This drug possesses several desired properties and may be a good candidate for immediate repurposing for treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.06.18.448921","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.18.448935","pub_date":"2021-6-18","title":"Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients","abstract":"Infection with SARS-CoV-2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID-19 patients. In addition, immune dysfunction occurs in patients characterized by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets, and platelet-derived microparticles (PMP), during the clinical course of COVID-19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID-19 diagnosis. Elevated frequencies of PS+PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition, and coagulation, currently used for COVID-19 clinical outcome prognosis. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death-ligand 1 (PD-L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes toward lymphocytes and cause immune dysfunction in COVID-19.","version":"1.1","doi":"10.1101/2021.06.18.448935","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.21258919","pub_date":"2021-06-18","title":"Age-Based Disparities in Hospitalizations and Mortality for Coronavirus Disease 2019 (COVID-19)","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Purpose</jats:title>\n                  <jats:p>Evidence suggests that older adults, racial/ethnic minorities, and those with comorbidities all face elevated risk for morbidity and mortality from COVID-19; but there are limited reports describing the potential for interactions between these factors.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We sought to evaluate age-based heterogeneity in observed disparities in hospitalization, ICU admission, and mortality related to COVID-19 using CDC public use surveillance data on 3,662,325 COVID-19 cases reported from January 1 to August 30, 2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Racial/ethnic and comorbidity disparities in hospitalization were most pronounced during ages 20-29 and ages 10-19, with similar elevation seen for disparities in ICU risk.</jats:p>\n                  <jats:p>Racial/ethnic disparities in mortality were most pronounced during ages 20-29 while risk from comorbidity peaks among ages 10-39.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>As COVID-19 continues to affect younger populations, special attention to the implications for the most vulnerable subgroups are clearly warranted.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Implications and Contribution</jats:title>\n                  <jats:p>Adolescents and young adults appear to have experienced the greatest inequities in COVID-19 outcomes by race/ethnicity and comorbidity. Careful monitoring of trends in this population is warranted as they re-enter school, work, and social settings while being the last group to receive priority for vaccination.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.14.21258919","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.11.293951","pub_date":"2021-6-17","title":"Non-permissive SARS-CoV-2 infection in human neurospheres","abstract":"Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.","version":"1.4","doi":"10.1101/2020.09.11.293951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446676","pub_date":"2021-6-17","title":"SARS-CoV-2 Infects Syncytiotrophoblast and Activates Inflammatory Responses in the Placenta","abstract":"SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.","version":"1.2","doi":"10.1101/2021.06.01.446676","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.16.448772","pub_date":"2021-6-17","title":"Designing a Novel Multi-Epitope Vaccine against SARS-CoV-2; Implication for Viral Binds and Fusion Inhibition through Inducing Neutralizing Antibodies","abstract":"Recently the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pervasive threat to public health so it is an emergency to vaccine development. The SARS-CoV-2 spike (S) glycoprotein plays a vital role in binds and fusion to the angiotensin-converting enzyme 2 (ACE2). The multi-epitope peptide vaccines are capable of inducing the specific humoral or cellular immune responses. In this regard, the RBD and spike cleavage site is the most probable target for vaccine development to inducing binds and fusion inhibitors neutralizing antibodies. In the present study, several immunoinformatics tools are used for analyzing the spike (S) glycoprotein sequence including the prediction of the potential linear B-cell epitopes, B-cell multi-epitope design, secondary and tertiary structures, physicochemical properties, solubility, antigenicity, and allergenicity for the promising vaccine candidate against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.06.16.448772","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.16.448754","pub_date":"2021-6-17","title":"A stem-loop RNA RIG-I agonist confers prophylactic and therapeutic protection against acute and chronic SARS-CoV-2 infection in mice","abstract":"As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral replication in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I) dependent manner. SLR14 demonstrated remarkable protective capacity against lethal SARS-CoV-2 infection when used prophylactically and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity by inducing IFN-I responses in the absence of the adaptive immune system. In the context of infection with variants of concern (VOC), SLR14 conferred broad protection and uncovered an IFN-I resistance gradient across emerging VOC. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and for treatment of chronically infected immunosuppressed patients.","version":"1.1","doi":"10.1101/2021.06.16.448754","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448459","pub_date":"2021-6-17","title":"Memory B cells control SARS-CoV-2 variants upon mRNA vaccination of naive and COVID-19 recovered individuals","abstract":"How a previous SARS-CoV-2 infection may amplify and model the memory B cell (MBC) response elicited by mRNA vaccines was addressed by a comparative longitudinal study of two cohorts, naive individuals and disease-recovered patients, up to 2 months after vaccination. The quality of the memory response was assessed by analysis of the VDJ repertoire, affinity and neutralization against variants of concerns (VOC), using unbiased cultures of 2452 MBCs. Upon boost, the MBC pool of recovered patients selectively expanded, further matured and harbored potent neutralizers against VOC. Maturation of the MBC response in naive individuals was much less pronounced. Nevertheless, and as opposed to their weaker neutralizing serum response, half of their RBD-specific MBCs displayed high affinity towards multiple VOC and one-third retained neutralizing potency against B.1.351. Thus, repeated vaccine challenges could reduce these differences by recall of affinity-matured MBCs and allow naive vaccinees to cope efficiently with VOC.","version":"1.1","doi":"10.1101/2021.06.17.448459","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448882","pub_date":"2021-6-17","title":"Post-Infection Entry Mechanism of Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) Chimeras is Mediated by Viroporins","abstract":"The limitations of virus-specific antiviral drugs became apparent during the current COVID-19 pandemic. The search for broad range antiviral proteins of a new kind to answer current and future pandemics has become an even more pressing matter. Here, the author further describes the expected anti-SARS-CoV-2 mechanisms of a novel broad range antiviral chimeric protein constructed between ricin A chain and pokeweed antiviral proteins. The latest in protein-ligand docking software were used to determine binding affinity of RTA-PAPs to SARS-CoV-2 frameshift stimulation element and elucidate the preferential post-infection entry mechanisms of RTA-PAPs into virus infected cells over non-infected ones, by doing a comparative analysis between in vitro and in silico results on numerous viruses. The results obtained strongly suggest that the post-infection preferential entry of RTA-PAPs into infected cells is mediated by the presence of viroporins integrated into the host cell membrane. The discovery of this mechanism revealed RTA-PAPs, and proteins like them, to be a new class of broad range antivirals that target with high specificity viroporin producing viruses, and with gain of functions in antiviral activities, post-infection.","version":"1.1","doi":"10.1101/2021.06.17.448882","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.17.448814","pub_date":"2021-6-17","title":"Western diet increases COVID-19 disease severity in the Syrian hamster","abstract":"Pre-existing comorbidities such as obesity or metabolic diseases can adversely affect the clinical outcome of COVID-19. Chronic metabolic disorders are globally on the rise and often a consequence of an unhealthy diet, referred to as a Western Diet. For the first time in the Syrian hamster model, we demonstrate the detrimental impact of a continuous high-fat high-sugar diet on COVID-19 outcome. We observed increased weight loss and lung pathology, such as exudate, vasculitis, hemorrhage, fibrin, and edema, delayed viral clearance and functional lung recovery, and prolonged viral shedding. This was accompanied by an increased trend of systemic IL-10 and IL-6, as well as a dysregulated serum lipid response dominated by polyunsaturated fatty acid-containing phosphatidylethanolamine, recapitulating cytokine and lipid responses associated with severe human COVID-19. Our data support the hamster model for testing restrictive or targeted diets and immunomodulatory therapies to mediate the adverse effects of metabolic disease on COVID-19.","version":"1.1","doi":"10.1101/2021.06.17.448814","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423920","pub_date":"2021-6-16","title":"Evolutionary tracking of SARS-CoV-2 genetic variants highlights an intricate balance of stabilizing and destabilizing mutations","abstract":"The currently ongoing COVID-19 pandemic caused by SARS-CoV-2 has accounted for millions of infections and deaths across the globe. Genome sequences of SARS-CoV-2 are being published daily in public databases and the availability of this genome datasets has allowed unprecedented access into the mutational patterns of SARS-CoV-2 evolution. We made use of the same genomic information for conducting phylogenetic analysis and identifying lineage-specific mutations. The catalogued lineage defining mutations were analysed for their stabilizing or destabilizing impact on viral proteins. We recorded persistence of D614G, S477N, A222V V1176F variants and a global expansion of the PANGOLIN variant B.1. In addition, a retention of Q57H (B.1.X), R203K/G204R (B.1.1.X), T85I (B.1.2-B.1.3), G15S+T428I (C.X) and I120F (D.X) variations was observed. Overall, we recorded a striking balance between stabilizing and destabilizing mutations, therefore well-maintained protein structures. With selection pressures in the form of newly developed vaccines and therapeutics to mount soon in coming months, the task of mapping of viral mutations and recording of their impact on key viral proteins would be crucial to pre-emptively catch any escape mechanism that SARS-CoV-2 may evolve for. As large numbers of the SARS CoV-2 genome sequences are shared in publicly accessible repositories, it enables scientists a detailed evolutionary analysis since its initial isolation in Wuhan, China. We investigated the evolutionarily associated mutational diversity overlaid on the major phylogenetic lineages circulating globally, using 513 representative genomes. We detailed phylogenetic persistence of key variants facilitating global expansion of the PANGOLIN variant B.1, including the recent, fast expanding, B.1.1.7 lineage. The stabilizing or destabilizing impact of the catalogued lineage defining mutations on viral proteins indicates their possible involvement in balancing the protein function and structure. A clear understanding of this mutational profile is of high clinical significance to catch any vaccine escape mechanism, as the same proteins make crucial components of vaccines recently approved and in development. In this direction, our study provides an imperative framework and baseline data upon which further analysis could be built as newer variants of SARS-CoV-2 continue to appear.","version":"1.2","doi":"10.1101/2020.12.22.423920","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.15.448611","pub_date":"2021-6-16","title":"SARS-CoV-2 Viral Replication in a High Throughput Human Primary Epithelial Airway Organ Model","abstract":"COVID-19 emerged as a worldwide pandemic early in 2020, and at this writing has caused over 170 million cases and 3.7 million deaths worldwide, and almost 600,000 deaths in the United States. The rapid development of several safe and highly efficacious vaccines stands as one of the most extraordinary achievements in modern medicine, but the identification and administration of efficacious therapeutics to treat patients suffering from COVID-19 has been far less successful. A major factor limiting progress in the development of effective treatments has been a lack of suitable preclinical models for the disease, currently reliant upon various animal models and in vitro culture of immortalized cell lines. Here we report the first successful demonstration of SARS-CoV-2 infection and viral replication in a human primary cell-based organ-on-chip, leveraging a recently developed tissue culture platform known as PREDICT96. This successful demonstration of SARS-CoV-2 infection in human primary airway epithelial cells derived from a living donor represents a powerful new pathway for disease modeling and an avenue for screening therapeutic candidates in a high throughput platform.","version":"1.1","doi":"10.1101/2021.06.15.448611","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447477","pub_date":"2021-6-16","title":"In depth analysis of Cyprus-specific mutations of SARS-CoV-2 strains using computational approaches","abstract":"This study aims to characterize SARS-CoV-2 mutations which are primarily prevalent in the Cypriot population. Moreover, using computational approaches, we assess whether these mutations are associated with changes in viral virulence. We utilize genetic data from 144 sequences of SARS-CoV-2 strains from the Cypriot population obtained between March 2020 and January 2021, as well as all data available from GISAID. We combine this with countries\u2019 regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of Cyprus-specific mutations are obtained by mutation tracking analysis. This entails calculating specific mutation frequencies within the Cypriot population and comparing these with their prevalence world-wide throughout the course of the pandemic. We further make use of linear regression models to extrapolate additional information that may be missed through standard statistical analysis. We report a single mutation found in the ORF1ab gene (S6059F) that appears to be significantly enriched within the Cypriot population. We further analyse this mutation using regression models to investigate possible associations with increased deaths and cases per million. Moreover, protein structure prediction tools show that the mutation infers a conformational change to the protein that significantly alters its structure when compared to the reference protein. Investigating Cyprus-specific mutations for SARS-CoV-2 can not only lead to important findings from which to battle the pandemic on a national level, but also provide insights into viral virulence worldwide.","version":"1.2","doi":"10.1101/2021.06.08.447477","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427579","pub_date":"2021-6-16","title":"Ongoing recombination in SARS-CoV-2 revealed through genealogical reconstruction","abstract":"The evolutionary process of genetic recombination has the potential to rapidly change the properties of a viral pathogen, and its presence is a crucial factor to consider in the development of treatments and vaccines. It can also significantly affect the results of phylogenetic analyses and the inference of evolutionary rates. The detection of recombination from samples of sequencing data is a very challenging problem, and is further complicated for SARS-CoV-2 by its relatively slow accumulation of genetic diversity. The extent to which recombination is ongoing for SARS-CoV-2 is not yet resolved. To address this, we use a parsimony-based method to reconstruct possible genealogical histories for samples of SARS-CoV-2 sequences, which enables us to pinpoint specific recombination events that could have generated the data. We propose a statistical framework for disentangling the effects of recurrent mutation from recombination in the history of a sample, and hence provide a way of estimating the probability that ongoing recombination is present. We apply this to samples of sequencing data collected in England and South Africa, and find evidence of ongoing recombination.","version":"1.3","doi":"10.1101/2021.01.21.427579","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.16.448640","pub_date":"2021-6-16","title":"SARS-CoV-2 envelope-protein corruption of homeostatic signaling mechanisms in mammalian cells","abstract":"During a SARS-CoV2 infection, host cells produce large amounts of the viral envelope protein (Ep-CoV2). Ep-CoV2 is partially inserted into the membrane of nascent viral particles and into cellular membranes. To mimic the pathophysiological impact of the cellular protein fraction, Ep-CoV2 was overexpressed in mammalian cells and effects on key signaling parameters were monitored. By tagging with green fluorescent protein (GFP), we found that Ep-CoV2 protein is mostly present in the endoplasmic reticulum with additional trace amounts in the plasma membrane. We observed that wild-type Ep-CoV2 and, to a lesser extent, its mutants (N15A, V25F) corrupted some of the most important homeostatic mechanisms in cells. The same was observed with isolated transmembrane domains of the protein. The Ep-CoV2-evoked elevation of intracellular Ca2+ and pH as well as the induced membrane depolarization produced by the presence of the protein interfere with major signal transduction cascades in host cells. These functions of Ep-CoV2, which likely contribute to the pathogenesis of the viral protein, result from the ion-channel activity of the viral protein. Two independent assays, a functional reconstitution of Ep-CoV2 protein in artificial membranes and a rescue of K+-deficient yeast mutants, confirm that Ep-CoV2 generates a cation-conducting channel with a low unitary conductance and a complex ion selectivity. The data presented here suggest that specific channel function inhibitors of Ep-CoV2 can provide cell protection and virostatic effects.","version":"1.1","doi":"10.1101/2021.06.16.448640","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.16.448525","pub_date":"2021-6-16","title":"A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters","abstract":"The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 \u03bcg) or low (0.2 \u03bcg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.","version":"1.1","doi":"10.1101/2021.06.16.448525","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447287","pub_date":"2021-6-16","title":"Implicating Gene and Cell Networks Responsible for Differential COVID-19 Host Responses via an Interactive Single Cell Web Portal","abstract":"Numerous studies have provided single-cell transcriptome profiles of host responses to SARS-CoV-2 infection. Critically lacking however is a datamine that allows users to compare and explore cell profiles to gain insights and develop new hypotheses. To accomplish this, we harmonized datasets from COVID-19 and other control condition blood, bronchoalveolar lavage, and tissue samples, and derived a compendium of gene signature modules per cell type, subtype, clinical condition, and compartment. We demonstrate approaches to probe these via a new interactive web portal (http://toppcell.cchmc.org/ COVID-19). As examples, we develop three hypotheses: (1) a multicellular signaling cascade among alternatively differentiated monocyte-derived macrophages whose tasks include T cell recruitment and activation; (2) novel platelet subtypes with drastically modulated expression of genes responsible for adhesion, coagulation and thrombosis; and (3) a multilineage cell activator network able to drive extrafollicular B maturation via an ensemble of genes strongly associated with risk for developing post-viral autoimmunity.","version":"1.2","doi":"10.1101/2021.06.07.447287","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448461","pub_date":"2021-6-15","title":"Protective efficacy of rhesus adenovirus COVID-19 vaccines against mouse-adapted SARS-CoV-2","abstract":"The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibits a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative Ad vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to i) evaluate the protective efficacy of RhAd52 vaccines and ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.","version":"1.1","doi":"10.1101/2021.06.14.448461","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448426","pub_date":"2021-6-15","title":"Proinflammatory responses in SARS-CoV-2 infected and soluble spike glycoprotein S1 subunit activated human macrophages","abstract":"Critically ill COVID-19 patients infected with SARS-CoV-2 display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We used SARS-CoV-2 infected and glycosylated soluble SARS-CoV-2 Spike S1 subunit (S1) treated THP-1 human-derived macrophage-like cell line to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-\u03b1 and CXCL10 mRNAs, and induced secretion of TNF-\u03b1 from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication, virus infection resulted in upregulation of both TNF-\u03b1 and CXCL10 genes. Our study shows that S1 is a key viral component inducing inflammatory response in macrophages, independently of virus replication. Thus, virus-infected or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.","version":"1.1","doi":"10.1101/2021.06.14.448426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448464","pub_date":"2021-6-15","title":"Increased sensitivity of SARS-CoV-2 to type III interferon in human intestinal epithelial cells","abstract":"The coronavirus SARS-CoV-2 caused the COVID-19 global pandemic leading to 3.5 million deaths worldwide as of June 2021. The human intestine was found to be a major viral target which could have a strong impact on virus spread and pathogenesis since it is one of the largest organs. While type I interferons (IFNs) are key cytokines acting against systemic virus spread, in the human intestine type III IFNs play a major role by restricting virus infection and dissemination without disturbing homeostasis. Recent studies showed that both type I and III IFNs can inhibit SARS-CoV-2 infection, but it is not clear if one IFN controls SARS-CoV-2 infection of the human intestine better or with a faster kinetics. In this study, we could show that both type I and III IFNs possess antiviral activity against SARS-CoV-2 in human intestinal epithelial cells (hIECs), however type III IFN is more potent. Shorter type III IFN pretreatment times and lower concentrations were required to efficiently reduce virus load when compared to type I IFNs. Moreover, type III IFNs significantly inhibited SARS-CoV-2 even 4 hours post-infection and induced a long-lasting antiviral effect in hIECs. Importantly, the sensitivity of SARS-CoV-2 to type III IFNs was virus-specific since type III IFN did not control VSV infection as efficiently. Together these results suggest that type III IFNs have a higher potential for IFN-based treatment of SARS-CoV-2 intestinal infection as compared to type I IFNs.","version":"1.1","doi":"10.1101/2021.06.14.448464","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.15.448497","pub_date":"2021-6-15","title":"The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress","abstract":"Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.","version":"1.1","doi":"10.1101/2021.06.15.448497","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.15.448391","pub_date":"2021-6-15","title":"Low-dose fluvoxamine modulates endocytic trafficking of SARS-CoV-2 spike protein: a potential mechanism for anti-COVID-19 protection by antidepressants","abstract":"Commonly prescribed antidepressants may be associated with protection against severe COVID-19, with one drug (fluvoxamine) currently undergoing a Phase 3 clinical trial. The mechanism of their action, however, remains unknown. Here, I investigated the effect of fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking may provide a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.","version":"1.1","doi":"10.1101/2021.06.15.448391","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448452","pub_date":"2021-6-15","title":"Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures","abstract":"Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein\u2019s two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein\u2019s two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.","version":"1.1","doi":"10.1101/2021.06.14.448452","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.435741","pub_date":"2021-6-15","title":"Structural basis of anti-SARS-CoV-2 activity of HCQ: specific binding to N protein to disrupt its interaction with nucleic acids and LLPS","abstract":"Great efforts have led to successfully developing the spike-based vaccines but challenges still exist to completely terminate the SARS-CoV-2 pandemic. SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in almost all key steps of the viral life cycle, thus representing a top drug target. Almost all key functions of N protein including liquid-liquid phase separation (LLPS) depend on its capacity in interacting with nucleic acids. Therefore, only the variants with their N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Very recently, hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of SARS-CoV-2 N protein with Kd of 112.1 and 57.1 \u03bcM respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS essential for the viral life cycle. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to them all. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex decodes a promising strategy for further design of better anti-SARS-CoV-2 drugs from HCQ. Therefore, HCQ is a promising candidate to help terminate the pandemic.","version":"1.3","doi":"10.1101/2021.03.16.435741","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.15.448568","pub_date":"2021-6-15","title":"Deep Mutational Scanning of Dynamic Interaction Networks in the SARS-CoV-2 Spike Protein Complexes: Allosteric Hotspots Control Functional Mimicry and Resilience to Mutational Escape","abstract":"We develop a computational approach for deep mutational scanning of residue interaction networks in the SARS-CoV-2 spike protein complexes to characterize mechanisms of functional mimicry and resilience to mutational escape by miniprotein inhibitors. Using a dynamic mutational profiling and sensitivity analysis of protein stability, binding interactions and global network parameters describing allosteric signaling, we identify regulatory hotspots in the SARS-CoV-2 S complexes with the ACE2 host receptor and ultra-potent miniproteins. The results revealed that global circulating variants are associated with allosteric control points that are dynamically coupled to structural stability hotspots. In this mechanism, variant-induced perturbations of flexible allosteric sites can result in global network changes and elicit specific protein responses. The binding affinity fingerprints and allosteric signatures of the SARS-CoV-2 complexes with miniproteins are determined by a dynamic cross-talk between regulatory control points and conformationally adaptable allosteric hotspots that collectively control structure-functional mimicry, signal transmission and resilience to mutational escape.","version":"1.1","doi":"10.1101/2021.06.15.448568","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.15.448495","pub_date":"2021-6-15","title":"Tracking SARS-CoV-2 Spike Protein Mutations in the United States (2020/01 \u2013 2021/03) Using a Statistical Learning Strategy","abstract":"The emergence and establishment of SARS-CoV-2 variants of interest (VOI) and variants of concern (VOC) highlight the importance of genomic surveillance. We propose a statistical learning strategy (SLS) for identifying and spatiotemporally tracking potentially relevant Spike protein mutations. We analyzed 167,893 Spike protein sequences from US COVID-19 cases (excluding 21,391 sequences from VOI/VOC strains) deposited at GISAID from January 19, 2020 to March 15, 2021. Alignment against the reference Spike protein sequence led to the identification of viral residue variants (VRVs), i.e., residues harboring a substitution compared to the reference strain. Next, generalized additive models were applied to model VRV temporal dynamics, to identify VRVs with significant and substantial dynamics (false discovery rate q-value <0.01; maximum VRV proportion > 10% on at least one day). Unsupervised learning was then applied to hierarchically organize VRVs by spatiotemporal patterns and identify VRV-haplotypes. Finally, homology modelling was performed to gain insight into potential impact of VRVs on Spike protein structure. We identified 90 VRVs, 71 of which have not previously been observed in a VOI/VOC, and 35 of which have emerged recently and are durably present. Our analysis identifies 17 VRVs \u223c91 days earlier than their first corresponding VOI/VOC publication. Unsupervised learning revealed eight VRV-haplotypes of 4 VRVs or more, suggesting two emerging strains (B1.1.222 and B.1.234). Structural modeling supported potential functional impact of the D1118H and L452R mutations. The SLS approach equally monitors all Spike residues over time, independently of existing phylogenic classifications, and is complementary to existing genomic surveillance methods.","version":"1.1","doi":"10.1101/2021.06.15.448495","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448436","pub_date":"2021-6-15","title":"Molecular dynamics analysis of fast-spreading severe acute respiratory syndrome coronavirus 2 variants and their effects in the interaction with human angiotensin-converting enzyme 2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving with mutations in the Spike protein, especially in the receptor-binding domain (RBD). The failure of public health measures to contain the spread of the disease in many countries has given rise to novel viral variants with increased transmissibility. However, key questions about how quickly the variants can spread and whether they can cause a more severe disease remain unclear. Herein, we performed a structural investigation using molecular dynamics simulations and determined dissociation constant (KD) values using surface plasmon resonance (SPR) assays of three fastspreading SARS-CoV-2 variants, Alpha, Beta and Gamma ones, as well as genetic factors in the host cells that may be related to the viral infection. Our results suggest that the SARS-CoV-2 variants facilitate their entry into the host cell by moderately increased binding affinities to the human ACE2 receptor, different torsions in hACE2 mediated by RBD variants, and an increased Spike exposure time to proteolytic enzymes. We also found that other host cell aspects, such as gene and isoform expression of key genes for the infection (ACE2, FURIN and TMPRSS2), may have few contributions to the SARS-CoV-2 variants infectivity. In summary, we concluded that a combination of viral and host cell factors allows SARS-CoV-2 variants to increase their abilities to spread faster than wild-type.","version":"1.1","doi":"10.1101/2021.06.14.448436","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.14.448413","pub_date":"2021-6-15","title":"Stepwise evolution and exceptional conservation of ORF1a/b overlap in coronaviruses","abstract":"The programmed frameshift element (PFE) rerouting translation from ORF1a to ORF1b is essential for propagation of coronaviruses. A combination of genomic features that make up PFE\u2014the overlap between the two reading frames, a slippery sequence, as well as an ensemble of complex secondary structure elements\u2014puts severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these elements. The vast amount of SARS-CoV-2 sequencing data generated within the past year provides an opportunity to assess evolutionary dynamics of PFE in great detail. Here we performed a comparative analysis of all available coronaviral genomic data available to date. We show that the overlap between ORF1a and b evolved as a set of discrete 7, 16, 22, 25, and 31 nucleotide stretches with a well defined phylogenetic specificity. We further examined sequencing data from over 350,000 complete genomes and 55,000 raw read datasets to demonstrate exceptional conservation of the PFE region.","version":"1.1","doi":"10.1101/2021.06.14.448413","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.21258766","pub_date":"2021-06-15","title":"Out-of-Pocket Spending Within 90 Days of Discharge from COVID-19 Hospitalization","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>INTRODUCTION</jats:title>\n                  <jats:p>Millions of U.S. patients have been hospitalized for COVID-19. After discharge, these patients often have extensive health care needs, but out-of-pocket burden for this care is poorly described. We assessed out-of-pocket spending within 90 days of discharge from COVID-19 hospitalization among privately insured and Medicare Advantage patients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>\n                    In May 2021, we conducted a cross-sectional analysis of the IQVIA PharMetrics\n                    <jats:sup>\u00ae</jats:sup>\n                    Plus for Academics Database, a national de-identified claims database. Among privately insured and Medicare Advantage patients hospitalized for COVID-19 between March-June 2020, we calculated mean out-of-pocket spending for care within 90 days of discharge. For context, we repeated analyses for patients hospitalized for pneumonia.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>Among 1,465 COVID-19 patients included, 516 (35.2%) and 949 (64.8%) were covered by private insurance and Medicare Advantage plans. Among these patients, mean (SD) post-discharge out-of-pocket spending was $534 (1,045) and $680 (1,360); spending exceeded $2,000 for 7.0% and 10.3%. Compared with pneumonia patients, mean post-discharge out-of-pocket spending among COVID-19 patients was higher among the privately insured ($534 vs $445) and lower among Medicare Advantage patients ($680 vs $918).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>For the privately insured, post-discharge out-of-pocket spending was higher among patients hospitalized for COVID-19 than among patients hospitalized for pneumonia. The opposite was true for Medicare Advantage patients, potentially because insurer cost-sharing waivers for COVID-19 treatment covered the costs of some post-discharge care, such as COVID-19 readmissions. Nonetheless, given the high volume of U.S. COVID-19 hospitalizations to date, our findings suggest a large number of Americans have experienced substantial financial burden for post-discharge care.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.11.21258766","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.15.448583","pub_date":"2021-6-15","title":"Structural Modeling of the TMPRSS Subfamily of Host Cell Proteases Reveals Potential Binding Sites","abstract":"The transmembrane protease serine subfamily (TMPRSS) has at least eight members with known protein sequence: TMPRSS2, TMPRRS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, TMPRSS11, TMPRSS12 and TMPRSS13. A majority of these TMPRSS proteins have key roles in human hemostasis as well as promoting certain pathologies, including several types of cancer. In addition, TMPRSS proteins have been shown to facilitate the entrance of respiratory viruses into human cells, most notably TMPRSS2 and TMPRSS4 activate the spike protein of the SARS-CoV-2 virus. Despite the wide range of functions that these proteins have in the human body, none of them have been successfully crystallized. The lack of structural data has significantly hindered any efforts to identify potential drug candidates with high selectivity to these proteins. In this study, we present homology models for all members of the TMPRSS family including any known isoform (the homology model of TMPRSS2 is not included in this study as it has been previously published). The atomic coordinates for all homology models have been refined and equilibrated through molecular dynamic simulations. The structural data revealed potential binding sites for all TMPRSS as well as key amino acids that can be targeted for drug selectivity.","version":"1.1","doi":"10.1101/2021.06.15.448583","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165803","pub_date":"2021-6-14","title":"CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2","abstract":"As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.","version":"1.3","doi":"10.1101/2020.06.22.165803","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.297945","pub_date":"2021-6-14","title":"Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets with therapeutic potential","abstract":"SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.","version":"1.3","doi":"10.1101/2020.09.16.297945","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.12.448080","pub_date":"2021-6-14","title":"High efficacy of therapeutic equine hyperimmune antibodies against SARS-CoV-2 variants of concern","abstract":"SARS-CoV-2 variants of concern (VoC) show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies. We tested therapeutic equine polyclonal antibodies (pAbs) against four VoC (alpha, beta, epsilon and gamma). We show that equine pAbs efficiently neutralize VoC, suggesting they are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.","version":"1.1","doi":"10.1101/2021.06.12.448080","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.13.448258","pub_date":"2021-6-14","title":"NMPylation and de-NMPylation of SARS-CoV-2 Nsp9 by the NiRAN domain","abstract":"Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all RNA viruses and has been a focus of mechanistic studies and drug discovery. The second active site resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. Both catalytic reactions are essential for viral replication, but the mechanism and targets of NiRAN are poorly characterized. One recent study showed that NiRAN transfers NMP to the first residue of RNA-binding protein Nsp9. Another study reported a structure of SARS-CoV-2 replicase with an extended Nsp9 in the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 Nsp12 efficiently and reversibly NMPylates the native but not the extended Nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas a substitution of a catalytic RdRp Asp residue does not. NMPylation is inhibited by nucleotide analogs, pyrophosphate, and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We hypothesize that Nsp9 remodels both active sites of Nsp12 to support initiation of RNA synthesis by RdRp and subsequent capping of the product RNA by the NiRAN domain.","version":"1.1","doi":"10.1101/2021.06.13.448258","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.12.448149","pub_date":"2021-6-14","title":"Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals","abstract":"Individuals infected with the SARS-CoV-2 virus present with a wide variety of phenotypes ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe COVID-19 phenotype. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the severe COVID-19 phenotype. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform an locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify 4 introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. These variants likely drive the locus\u2019 impact on severity by putatively modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.","version":"1.1","doi":"10.1101/2021.06.12.448149","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.12.448196","pub_date":"2021-6-13","title":"Exposure route, sex, and age influence disease outcome in a golden Syrian hamster model of SARS-CoV-2 infection","abstract":"The emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant pandemic of coronavirus disease 2019 (COVID-19) has led to over one hundred million confirmed infections, greater than three million deaths, and severe economic and social disruption. Animal models of SARS-CoV-2 are critical tools for the pre-clinical evaluation of antivirals, vaccines, and candidate therapeutics currently under urgent development to curb COVID-19-associated morbidity and mortality. The golden (Syrian) hamster model of SARS-CoV-2 infection recapitulates key characteristics of severe COVID-19, including high-titer viral replication in the upper and lower respiratory tract and the development of pathogenic lesions in the lungs. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in golden hamsters. We report that delivery of SARS-CoV-2 primarily to the nasal passages (low-volume intranasal), the upper and lower respiratory tract (high-volume intranasal), or the digestive tract (intragastric) results in comparable viral titers in the lung tissue and similar levels of viral shedding during acute infection. However, low-volume intranasal exposure results in milder weight loss during acute infection while intragastric exposure leads to a diminished capacity to regain body weight following the period of acute illness. Further, we examined both sex and age differences in response to SARS-CoV-2 infection. Male hamsters, and to a greater extent older male hamsters, display an impaired capacity to recover from illness and a delay in viral clearance compared to females. Lastly, route of exposure, sex, and age were found to influence the nature of the host inflammatory cytokine response, but they had a minimal effect on both the quality and durability of the humoral immune response as well as the susceptibility of hamsters to SARS-CoV-2 re-infection. Together, these data indicate that the route of exposure, sex, and age have a meaningful impact SARS-CoV-2 pathogenesis in hamsters and that these variables should be considered when designing pre-clinical challenge studies.","version":"1.1","doi":"10.1101/2021.06.12.448196","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.448155","pub_date":"2021-6-13","title":"Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by Connectivity map-based analysis and in vitro studies","abstract":"Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds. An accelerated repurposing potential of antiviral herbs can provide usable drugs and identify druggable targets. In this study, we dissect the anti-coronavirus activity of Cissampelos pareira L (Cipa). using an integrative approach. We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map (https://clue.io/). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa.","version":"1.1","doi":"10.1101/2021.06.11.448155","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.448134","pub_date":"2021-6-13","title":"UK B.1.1.7 variant exhibits increased respiratory replication and shedding in nonhuman primates","abstract":"The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, African green monkeys were infected intranasally with either a contemporary D614G or the UK B.1.1.7 variant. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tract tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases. UK B.1.1.7 infection of African green monkeys exhibits increased respiratory replication and shedding but no disease enhancement","version":"1.1","doi":"10.1101/2021.06.11.448134","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.29.446267","pub_date":"2021-6-11","title":"Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes","abstract":"The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV-2 proteins, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our results show that these peptides and proteins form aggregates via a nucleation-dependent mechanism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome. The aggregation of proteins is linked with human disease in a variety of ways. In the case of viral infections, one could expect that the aberrant aggregation of viral proteins may damage the host cells, and also that viral particles may trigger the misfolding and aggregation of host proteins, resulting in damage to the host organism. Here we investigate the aggregation propensity of SARS-CoV-2 proteins and show that many of them can form aggregates that are potentially cytotoxic. In perspective, these results suggest that a better understanding of the effects of viruses on the human protein homeostasis system could help future therapeutic efforts.","version":"1.2","doi":"10.1101/2021.05.29.446267","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.447942","pub_date":"2021-6-11","title":"Anti-SARS-CoV-2 hyperimmune immunoglobulin provides potent and robust neutralization capacity and antibody-dependent cellular cytotoxicity and phagocytosis induction through N and S proteins","abstract":"Although progressive COVID-19 vaccinations provide a significant reduction of infection rate in the short-to mid-term, effective COVID-19 treatments will continue to be an urgent need. We have functionally characterized the anti-SARS-CoV-2 hyperimmune immunoglobulin (hIG) prepared from human COVID-19 convalescent plasma. SARS-CoV-2 virus neutralization was evaluated by four different methodologies (plaque reduction, virus induced cytotoxicity, TCID50 reduction and immunofluorimetry-based methodology) performed at four different laboratories and using four geographically different SARS-CoV-2 isolates (one each from USA and Italy; two from Spain). Two of the isolates contained the D614G mutation. Neutralization capacity against the original Wuhan SARS-CoV-2 straom and variants (D614G mutant, B.1.1.7, P.1 and B.1.351 variants) was evaluated using a pseudovirus platform expressing the corresponding spike (S) protein. The capacity to induce antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) was also evaluated. All the SARS-CoV-2 isolates tested were effectively neutralized by hIG solutions. This was confirmed by all four methodologies showing potent neutralization capacity. Wild-type SARS-CoV-2 and variants were effectively neutralized as demonstrated using the pseudovirus platform. The hIG solutions had the capacity to induce ADCC and ADCP against SARS-CoV-2 N and S proteins but not the E protein. Under our experimental conditions, very low concentrations (25-100 \u00b5g IgG/mL) were required to induce both effects. Besides the S protein, we observed a clear and potent effect triggered by antibodies in the hIG solutions against the SARS-CoV-2 N protein. These results show that, beyond neutralization, other IgG Fc-dependent pathways may play a role in the protection from and/or resolution of SARS-CoV-2 infection when using hIG COVID-19 products. This could be especially relevant for the treatment of more neutralization resistant SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2021.06.11.447942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.10.447982","pub_date":"2021-6-11","title":"High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells","abstract":"T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation. Complementary pathways of antibody production mediate neutralizing responses to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.06.10.447982","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.10.447999","pub_date":"2021-6-11","title":"A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants","abstract":"Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. The US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.","version":"1.1","doi":"10.1101/2021.06.10.447999","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447722","pub_date":"2021-6-11","title":"Neutralizing antibodies elicited by the Ad26.COV2.S COVID-19 vaccine show reduced activity against 501Y.V2 (B.1.351), despite protection against severe disease by this variant","abstract":"The emergence of SARS-CoV-2 variants, such as 501Y.V2, with immune evasion mutations in the spike has resulted in reduced efficacy of several COVID-19 vaccines. However, the efficacy of the Ad26.COV2.S vaccine, when tested in South Africa after the emergence of 501Y.V2, was not adversely impacted. We therefore assessed the binding and neutralization capacity of n=120 South African sera (from Day 29, post-vaccination) from the Janssen phase 3 study, Ensemble. Spike binding assays using both the Wuhan-1 D614G and 501Y.V2 Spikes showed high levels of cross-reactivity. In contrast, in a subset of 27 sera, we observed significantly reduced neutralization of 501Y.V2 compared to Wuhan-1 D614G, with 22/27 (82%) of sera showing no detectable neutralization of 501Y.V2 at Day 29. These data suggest that even low levels of neutralizing antibodies may contribute to protection from moderate/severe disease. In addition, Fc effector function and T cells may play an important role in protection by this vaccine against 501Y.V2.","version":"1.2","doi":"10.1101/2021.06.09.447722","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.11.448032","pub_date":"2021-6-11","title":"Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques","abstract":"The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The DNA-only vaccine regimens were compared to a regimen that included co- immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques. Anti-Spike neutralizing antibodies provide strong protection against SARS-CoV-2 infection in animal models, and correlate with protection in humans, supporting the notion that induction of strong humoral immunity is key to protection. We show induction of robust antibody and T cell responses by different Spike DNA-based vaccine regimens able to effectively mediate protection and to control SARS-CoV-2 infection in the rhesus macaque model. This study provides the opportunity to compare vaccines able to induce different humoral and cellular immune responses in an effort to develop durable immunity against the SARS-CoV-2. A vaccine regimen comprising simultaneous co-immunization of DNA and Protein at the same anatomical site showed best neutralizing abilities and was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. Thus, an expansion of the DNA vaccine regimen to include co-immunization with Spike protein may be of advantage also for SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.06.11.448032","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447491","pub_date":"2021-6-11","title":"ROS/RNS balancing, aerobic fermentation regulation and cell cycle control \u2013 a complex early trait (\u2018CoV-MAC-TED\u2019) for combating SARS-CoV-2-induced cell reprogramming","abstract":"In a perspective entitled \u2018From plant survival under severe stress to anti-viral human defense\u2019 we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named \u2018ReprogVirus\u2019 was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the \u2018ReprogVirus platform\u2019 was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to \u2018RegroVirus\u2019 complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called \u2018CoV-MAC-TED\u2019. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target \u2018CoV-MAC-TED\u2019 in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that \u2018de-stressing\u2019 disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.","version":"1.2","doi":"10.1101/2021.06.08.447491","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.14.382697","pub_date":"2021-6-11","title":"Gene Expression Meta-Analysis Reveals Interferon-induced Genes","abstract":"Severe Acute Respiratory Syndrome (SARS) corona virus (CoV) infections are a serious public health threat because of their pandemic-causing potential. This work analyzes mRNA expression data from SARS infections through meta-analysis of gene signatures, possibly identifying therapeutic targets associated with major SARS infections. This work defines 37 gene signatures representing SARS-CoV, Middle East Respiratory Syndrome (MERS)-CoV, and SARS-CoV2 infections in human lung cultures and/or mouse lung cultures or samples and compares them through Gene Set Enrichment Analysis (GSEA). To do this, positive and negative infectious clone SARS (icSARS) gene panels are defined from GSEA-identified leading-edge genes between two icSARS-CoV derived signatures, both from human cultures. GSEA then is used to assess enrichment and identify leading-edge icSARS panel genes between icSARS gene panels and 27 other SARS-CoV gene signatures. The meta-analysis is expanded to include five MERS-CoV and three SARS-CoV2 gene signatures. Genes associated with SARS infection are predicted by examining the intersecting membership of GSEA-identified leading-edges across gene signatures. Significant enrichment (GSEA p<0.001) is observed between two icSARS-CoV derived signatures, and those leading-edge genes defined the positive (233 genes) and negative (114 genes) icSARS panels. Non-random significant enrichment (null distribution p<0.001) is observed between icSARS panels and all verification icSARSvsmock signatures derived from human cultures, from which 51 over- and 22 under-expressed genes are shared across leading-edges with 10 over-expressed genes already associated with icSARS infection. For the icSARSvsmock mouse signature, significant, non-random significant enrichment held for only the positive icSARS panel, from which nine genes are shared with icSARS infection in human cultures. Considering other SARS strains, significant, non-random enrichment (p<0.05) is observed across signatures derived from other SARS strains for the positive icSARS panel. Five positive icSARS panel genes, CXCL10, OAS3, OASL, IFIT3, and XAF1, are found across mice and human signatures regardless of SARS strains. The GSEA-based meta-analysis approach used here identifies genes with and without reported associations with SARS-CoV infections, highlighting this approach\u2019s predictability and usefulness in identifying genes that have potential as therapeutic targets to preclude or overcome SARS infections.","version":"1.2","doi":"10.1101/2020.11.14.382697","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427813","pub_date":"2021-6-11","title":"Severe COVID-19 associated variants linked to chemokine receptor gene control in monocytes and macrophages","abstract":"Genome-wide association studies have identified 3p21.31 as the main risk locus for severe disease in COVID-19 patients, although underlying biological mechanisms remain elusive. We performed a comprehensive epigenomic dissection of the 3p21.31 locus, identifying a CTCF-dependent tissue-specific 3D regulatory chromatin hub that controls the activity of several tissue-homing chemokine receptor (CCR) genes in monocytes and macrophages. Risk SNPs colocalized with regulatory elements and were linked to increased expression of CCR1, CCR2 and CCR5 in monocytes and macrophages. As excessive organ infiltration of inflammatory monocytes and macrophages is a hallmark of severe COVID-19, our findings provide a rationale for the genetic association of 3p21.31 variants with elevated risk of hospitalization upon SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2021.01.22.427813","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.10.447951","pub_date":"2021-6-10","title":"Safety and Potency of COVIran Barekat Inactivated Vaccine Candidate for SARS-CoV-2: A Preclinical Study","abstract":"There is an urgent demand to manufacture an effective and safe vaccine to prevent SARS-CoV2 infection, which resulted in a global pandemic. In this study, we developed an inactivated whole-virus SARS-CoV-2 candidate vaccine named COVIran Barekat. Immunization at two different doses (3 \u00b5g or 5 \u00b5g per dose) elicited a high level of SARS-CoV-2 specific neutralizing antibodies in mice, rabbits, and non-human primates. The results show the safety profile in studied animals (include guinea pig, rabbit, mice, and monkeys). Rhesus macaques were immunized with the two-dose of 5 \u00b5g and 3 \u00b5g of the COVIran Barekat vaccine and showed highly efficient protection against 104 TCID50 of SARS-CoV-2 intratracheal challenge compared with the control group. These results highlight the COVIran Barekat vaccine as a potential candidate to induce a strong and potent immune response which may be a promising and feasible vaccine to protect against SARS-CoV2 infection.","version":"1.1","doi":"10.1101/2021.06.10.447951","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.10.447950","pub_date":"2021-6-10","title":"MPI8 is Potent Against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L","abstract":"A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10- 3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.","version":"1.1","doi":"10.1101/2021.06.10.447950","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.10.447768","pub_date":"2021-6-10","title":"Identification of ACE2 modifiers by CRISPR screening","abstract":"SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.","version":"1.1","doi":"10.1101/2021.06.10.447768","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447484","pub_date":"2021-6-09","title":"DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is known to be more common in the elderly, who show also more severe symptoms and a higher risk of hospitalization and death. Here we show that the expression of the Angiotensin Converting Enzyme 2 (ACE2), the SARS-CoV2 cell receptor, increases during aging in mouse and human lungs, and following telomere shortening or dysfunction in mammalian cells and in mouse models. This increase is regulated at the transcription level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Indeed, ATM inhibition or the selective inhibition of telomeric DDR, through the use of antisense oligonucleotides, prevents Ace2 upregulation following telomere damage, in cultured cells and in mice. We propose that during aging telomeric shortening, by triggering DDR activation, causes the upregulation of ACE2, the SARS-CoV2 cell receptor, thus making the elderly likely more susceptible to the infection.","version":"1.1","doi":"10.1101/2021.06.09.447484","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447527","pub_date":"2021-6-09","title":"Second-Generation Antibodies Neutralize Emerging SARS-CoV-2 Variants of Concern","abstract":"Recently emerged SARS-CoV-2 variants show resistance to some antibodies that were authorized for emergency use. We employed hybridoma technology combined with authentic virus assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize new variants of SARS-CoV-2. AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 and B.1.351. Finally, the combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. The neutralizing properties were fully reproduced in chimeric mouse-human versions, which may represent a promising tool for COVID-19 therapy.","version":"1.1","doi":"10.1101/2021.06.09.447527","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445201","pub_date":"2021-6-09","title":"Evidence for Deleterious Antigenic Imprinting in SARS-CoV-2 Immune Response","abstract":"A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous potential antigen, a sequence derived from the neuraminidase protein of H3N2 Influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIM from a previous infection could underlie some cases of COVID-19 disease severity. Infections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples\u2019 immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.","version":"1.2","doi":"10.1101/2021.05.21.445201","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447365","pub_date":"2021-6-09","title":"Country-wide genomic surveillance of SARS-CoV-2 strains","abstract":"Genomic surveillance has enabled the identification of several SARS-CoV-2 variants, allowing the formulation of appropriate public health policies. However, surveillance could be made more effective. We have determined that the time taken from strain collection to genome submission for over 1.7 million SARS-CoV-2 strains available at GISAID. We find that strain-wise, time lag in this process ranges from one day to over a year. Country-wise, the UK has taken a median of 16 days (for 417,287 genomes), India took 57 days (for 15,614 genomes), whereas Qatar spent 289 days (for 2298 genomes). We strongly emphasize that along with increasing the number of genomes of COVID-19 positive cases sequenced, their accelerated submission to GISAID should also be strongly encouraged and facilitated. This will enable researchers across the globe to track the spreading of variants in a timely manner; analyse their biology, epidemiology, and re-emerging infections; and define effective public health policies.","version":"1.1","doi":"10.1101/2021.06.08.447365","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447613","pub_date":"2021-6-09","title":"Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics","abstract":"As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an MPro-eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of MPro inhibitors. In comparison to an antiviral assay in which MPro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular MPro inhibition information for assessment and optimization of MPro inhibitors. We used this assay to analyze 30 literature reported MPro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of MPro, GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit MPro. Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 \u03bcM. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit MPro. Kinetic recharacterization of these two compounds rules out their possibility as MPro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC50 and EC50 values respectively below 1 \u03bcM. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.06.08.447613","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445786","pub_date":"2021-6-09","title":"Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis","abstract":"The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology, viral replication or cytokine expression were observed between lean and obese groups. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection is inadequate for performing nutrition studies, and caution should be used when interpreting resulting data.","version":"1.2","doi":"10.1101/2021.05.26.445786","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447662","pub_date":"2021-6-09","title":"Intranasal administration of a monoclonal neutralizing antibody protects mice against SARS-CoV-2 infection","abstract":"Despite recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both, prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2 and retains activity against the variants of concern B.1.1.7 and B.1.351. Importantly, not only systemic but also intranasal application of DZIF-10c abolished presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies. Monoclonal neutralizing antibodies are important in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection. However, their intravenous application might lead to suboptimal bioavailability in the lung. We here precisely characterize a new monoclonal neutralizing antibody (DZIF-10c) that binds to the receptor binding domain of the spike protein of SARS-CoV-2. DZIF-10c neutralizes SARS-CoV-2 with exceptionally high potency and maintains activity against circulating variants of concern. The antibody has a favorable pharmacokinetic profile and protects mice from SARS-CoV-2 infection. Importantly, we show that intranasal administration of DZIF-10c generates protective efficacy. These results not only identify DZIF-10c as a novel highly potent neutralizing antibody, but further pave the way for a topical application of anti-SARS-CoV-2 antibodies.","version":"1.1","doi":"10.1101/2021.06.09.447662","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447754","pub_date":"2021-6-09","title":"An outbreak of SARS-CoV-2 with high mortality in mink (Neovison vison) on multiple Utah farms","abstract":"The breadth of animal hosts that are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may serve as reservoirs for continued viral transmission are not known entirely. In August 2020, an outbreak of SARS-CoV-2 occurred in multiple mink farms in Utah and was associated with high mink mortality and rapid viral transmission between animals. The outbreak\u2019s epidemiology, pathology, molecular characterization, and tissue distribution of virus within infected mink is provided. Infection of mink was likely by reverse zoonosis. Once established, infection spread rapidly between independently housed animals and farms, and caused severe respiratory disease and death. Clinical signs were most notably sudden death, anorexia, and increased respiratory effort. Gross pathology examination revealed severe pulmonary congestion and edema. Microscopically there was pulmonary edema with moderate vasculitis, perivasculitis, and fibrinous interstitial pneumonia. Reverse transcriptase polymerase chain reaction (RT-PCR) of tissues collected at necropsy demonstrated the presence of SARS-CoV-2 viral RNA in multiple organs including nasal turbinates, lung, tracheobronchial lymph node, epithelial surfaces, and others. Whole genome sequencing from multiple mink was consistent with published SARS-CoV-2 genomes with few polymorphisms. The Utah mink SARS-CoV-2 strain fell into Clade GH, which is unique among mink and other animal strains sequenced to date and did not share other spike RBD mutations Y453F and F486L found in mink. Localization of viral RNA by in situ hybridization revealed a more localized infection, particularly of the upper respiratory tract. Mink in the outbreak reported herein had high levels of virus in the upper respiratory tract associated with mink-to-mink transmission in a confined housing environment and were particularly susceptible to disease and death due to SARS-CoV-2 infection. The recent emergence and worldwide spread of the novel coronavirus has resulted in worldwide disease and economic hardship. The virus, known as SARS-CoV-2 is believed to have originated in bats and has spread worldwide through human-to-human virus transmission. It remains unclear which animal species, other than humans, may also be susceptible to viral infection and could naturally transmit the virus to susceptible hosts. In this study, we describe an outbreak of disease and death due to SARS-CoV-2 infection in farmed mink in Utah, United States. The investigation reveals that mink can spread the virus rapidly between animals and that the disease in mink is due to the viral infection and damage to tissues of the upper and lower respiratory system. The determination that mink are susceptible to SARS-CoV-2 indicates the need for strict biosecurity measures on mink farms to remediate mink-to-mink and human-to-mink transmission for the protection of mink, as well as prevent potential transmission from mink to humans.","version":"1.1","doi":"10.1101/2021.06.09.447754","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447631","pub_date":"2021-6-09","title":"Immunogenicity and In vivo protection of a variant nanoparticle vaccine that confers broad protection against emerging SARS-CoV-2 variants","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to spread globally. As SARS-CoV-2 has transmitted from person to person, variant viruses have emerged with elevated transmission rates and higher risk of infection for vaccinees. We present data showing that a recombinant prefusion-stabilized Spike (rS) protein based on the B.1.351 sequence (rS-B.1.351) was highly immunogenic in mice and produced neutralizing antibodies against SARS-CoV-2/WA1, B.1.1.7, and B.1.351. Mice vaccinated with our prototype vaccine NVX-CoV2373 (rS-WU1) or rS-B.1.351 alone, in combination, or as a heterologous prime boost, were protected when challenged with live SARS-CoV-2/B.1.1.7 or SARS-CoV-2/B.1.351. Virus titer was reduced to undetectable levels in the lungs post-challenge in all vaccinated mice, and Th1-skewed cellular responses were observed. A strong anamnestic response was demonstrated in baboons boosted with rS-B.1.351 approximately one year after immunization with NVX-CoV2373 (rS-WU1). An rS-B.1.351 vaccine alone or in combination with prototype rS-WU1 induced protective antibody- and cell-mediated responses that were protective against challenge with SARS-CoV-2 variant viruses.","version":"1.1","doi":"10.1101/2021.06.08.447631","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447687","pub_date":"2021-6-09","title":"Monitoring group activity of hamsters and mice as a novel tool to evaluate COVID-19 progression, convalescence and rVSV-\u0394G-spike vaccination efficacy","abstract":"COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models were the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of the human COVID-19. Group activity of animals in their home cage continuously monitored by the HCMS100 was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters re-challenged with SARS-CoV-2, exhibited minor reduction in group activity compared to naive hamsters. To evaluate rVSV-\u0394G-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor group activity of hamsters in their home cage. Single-dose rVSV-\u0394G-spike vaccination of immunized group showed a faster recovery compared to the non-immunized infected hamsters, substantiating the efficacy of rVSV-\u0394G-spike vaccine. HCMS100 offers non-intrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19.","version":"1.1","doi":"10.1101/2021.06.09.447687","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.09.447656","pub_date":"2021-6-09","title":"Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine","abstract":"The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-\u03b3 and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs. MigVax-101 is a multi-epitope oral vaccine for SARS-CoV-2. MigVax-101 elicits neutralizing IgG and IgA production and cellular responses in mice MigVax-101 serves as an effective booster in rats to a parenteral anti-S1 vaccine.","version":"1.1","doi":"10.1101/2021.06.09.447656","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447341","pub_date":"2021-6-09","title":"The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour","abstract":"The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. The results also show significant differences in behaviour between clinical variants of the spike: e.g. the D614G mutation shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.","version":"1.1","doi":"10.1101/2021.06.07.447341","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433618","pub_date":"2021-6-09","title":"A Pharmacophore Model for SARS-CoV-2 3CLpro Small Molecule Inhibitors and in Vitro Experimental Validation of Computationally Screened Inhibitors","abstract":"Among the biomedical efforts in response to the current coronavirus (COVID-19) pandemic, pharmacological strategies to reduce viral load in patients with severe forms of the disease are being studied intensively. One of the main drug target proteins proposed so far is the SARS-CoV-2 viral protease 3CLpro (also called Mpro), an essential component for viral replication. Ongoing ligand- and receptor-based computational screening efforts would be facilitated by an improved understanding of the electrostatic, hydrophobic and steric features that characterize small molecule inhibitors binding stably to 3CLpro, as well as by an extended collection of known binders. Here, we present combined virtual screening, molecular dynamics simulation, machine learning and in vitro experimental validation analyses which have led to the identification of small molecule inhibitors of 3CLpro with micromolar activity, and to a pharmacophore model that describes functional chemical groups associated with the molecular recognition of ligands by the 3CLpro binding pocket. Experimentally validated inhibitors using a ligand activity assay include natural compounds with available prior knowledge on safety and bioavailability properties, such as the natural compound rottlerin (IC50 = 37 \u00b5M), and synthetic compounds previously not characterized (e.g. compound CID 46897844, IC50 = 31 \u00b5M). In combination with the developed pharmacophore model, these and other confirmed 3CLpro inhibitors may provide a basis for further similarity-based screening in independent compound databases and structural design optimization efforts, to identify 3CLpro ligands with improved potency and selectivity. Overall, this study suggests that the integration of virtual screening, molecular dynamics simulations and machine learning can facilitate 3CLpro-targeted small molecule screening investigations. Different receptor-, ligand- and machine learning-based screening strategies provided complementary information, helping to increase the number and diversity of identified active compounds. Finally, the resulting pharmacophore model and experimentally validated small molecule inhibitors for 3CLpro provide resources to support follow-up computational screening efforts for this drug target.","version":"1.2","doi":"10.1101/2021.03.02.433618","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.445535","pub_date":"2021-6-09","title":"SARS-CoV-2 B.1.617 Indian variants: are electrostatic potential changes responsible for a higher transmission rate?","abstract":"Lineage B.1.617+, also known as G/452R.V3, is a recently described SARS-CoV-2 variant under investigation (VUI) firstly identified in October 2020 in India. As of May 2021, three sublineages labelled as B.1.617.1, B.1.617.2 and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R and P681R. Here we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the Receptor Binding Domain (RBD) of the spike protein. Enhanced RBD-EP is particularly noticeable in the B.1.617.2 sublineage, which shows multiple replacements of neutral or negatively-charged amino acids with positively-charged amino acids. We here hypothesize that this EP change can favor the interaction between the B.1.617+RBD and the negatively-charged ACE2 thus conferring a potential increase in the virus transmission.","version":"1.1","doi":"10.1101/2021.06.08.445535","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447437","pub_date":"2021-6-08","title":"FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID-19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.","version":"1.1","doi":"10.1101/2021.06.07.447437","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447224","pub_date":"2021-6-08","title":"Predicted Coronavirus Nsp5 Protease Cleavage Sites in the Human Proteome: A Resource for SARS-CoV-2 Research","abstract":"The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.06.08.447224","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447530","pub_date":"2021-6-08","title":"Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro","abstract":"Single domain shark VNAR antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the SARS-CoV-2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant ACE2 receptor in vitro. In addition, 3 antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies due to their low complexity, small size, unique epitope recognition and formatting flexibility should be a useful adjunct to existing antibody approaches to treat COVID-19.","version":"1.1","doi":"10.1101/2021.06.08.447530","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.446560","pub_date":"2021-6-08","title":"Mapping Potential Antigenic Drift Sites (PADS) on SARS-CoV-2 Spike in Continuous Epitope-Paratope Space","abstract":"SARS-CoV-2 mutations with antigenic effects pose a risk to immunity developed through vaccination and natural infection. While vaccine updates for current variants of concern (VOCs) are underway, it is likewise important to prepare for further antigenic mutations as the virus navigates the heterogeneous global landscape of host immunity. Toward this end, a wealth of data and tools exist that can augment existing genetic surveillance of VOC evolution. In this study, we integrate published datasets describing genetic, structural, and functional constraints on mutation along with computational analyses of antibody-spike co-crystal structures to identify a set of potential antigenic drift sites (PADS) within the receptor binding domain (RBD) and N-terminal domain (NTD) of SARS-CoV-2 spike protein. Further, we project the PADS set into a continuous epitope-paratope space to facilitate interpretation of the degree to which newly observed mutations might be antigenically synergistic with existing VOC mutations, and this representation suggests that functionally convergent and synergistic antigenic mutations are accruing across VOC NTDs. The PADS set and synergy visualization serve as a reference as new mutations are detected on VOCs, enable proactive investigation of potentially synergistic mutations, and offer guidance to antibody and vaccine design efforts.","version":"1.1","doi":"10.1101/2021.06.07.446560","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447588","pub_date":"2021-6-08","title":"Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection","abstract":"Coronaviruses constitute a global threat to human population since three highly pathogenic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have crossed species to cause severe human respiratory disease. Considering the worldwide emergency status due to the current COVID-19 pandemic, effective pan-coronavirus antiviral drugs are required to tackle the ongoing as well as future (re)emerging virus outbreaks. Protein kinase CK2 has been deemed a promising therapeutic target in COVID-19 supported by its in vitro pharmacologic inhibition and molecular studies on SARS-CoV-2 infected cells. CIGB-325 is a first-in-class synthetic peptide impairing the CK2-mediated signaling whose safety and clinical benefit have been evidenced in Covid-19 and cancer patients after intravenous administration. Here, we explored the putative antiviral effect of CIGB-325 over MDBK cells infected by bovine coronavirus (BCoV) Mebus. Importantly, CIGB-325 inhibited both the cytopathic effect and the number of plaques forming units with a half-inhibitory concentrations IC50 = 3.5 \u03bcM and 17.7 \u03bcM, respectively. Accordingly, viral protein accumulation at the cytoplasm was clearly reduced by treating BCoV-infected cells with CIGB-325 over time, as determined by immunocytochemistry. Of note, data from pull-down assay followed by western blot and/or mass spectrometry identification revealed physical interaction of CIGB-325 with nucleocapsid (N) protein and a bona fide cellular CK2 substrates. Functional enrichment and network analysis from the CIGB-325 interacting proteins indicated cytoskeleton reorganization and protein folding as the most represented biological processes disturbed by this anti-CK2 peptide. Altogether, our findings not only unveil the direct antiviral activity of CIGB-325 on coronavirus infection but also provide molecular clues underlying such effect. Also, our data reinforce the scientific rationality behind the pharmacologic inhibition of CK2 to treat coronavirus infections.","version":"1.1","doi":"10.1101/2021.06.08.447588","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422772","pub_date":"2021-6-08","title":"Transfer Learning for Predicting Virus-Host Protein Interactions for Novel Virus Sequences","abstract":"Viruses such as SARS-CoV-2 infect the human body by forming interactions between virus proteins and human proteins. However, experimental methods to find protein interactions are inadequate: large scale experiments are noisy, and small scale experiments are slow and expensive. Inspired by the recent successes of deep neural networks, we hypothesize that deep learning methods are well-positioned to aid and augment biological experiments, hoping to help identify more accurate virus-host protein interaction maps. Moreover, computational methods can quickly adapt to predict how virus mutations change protein interactions with the host proteins. We propose DeepVHPPI, a novel deep learning framework combining a self-attention-based transformer architecture and a transfer learning training strategy to predict interactions between human proteins and virus proteins that have novel sequence patterns. We show that our approach outperforms the state-of-the-art methods significantly in predicting Virus\u2013Human protein interactions for SARS-CoV-2, H1N1, and Ebola. In addition, we demonstrate how our framework can be used to predict and interpret the interactions of mutated SARS-CoV-2 Spike protein sequences. We make all of our data and code available on GitHub https://github.com/QData/DeepVHPPI. Jack Lanchantin, Tom Weingarten, Arshdeep Sekhon, Clint Miller, and Yanjun Qi. 2021. Transfer Learning for Predicting Virus-Host Protein Interactions for Novel Virus Sequences. In Proceedings of ACM Conference (ACM-BCB). ACM, New York, NY, USA, 10 pages. https://doi.org/??","version":"1.2","doi":"10.1101/2020.12.14.422772","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.08.447516","pub_date":"2021-6-08","title":"An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15","abstract":"Using available cryo-EM and x-ray crystal structures of the nonstructural proteins that are responsible for SARS-CoV-2 viral RNA replication and transcription, we have constructed an atomistic model of how the proteins assemble into a functioning superstructure. Our principal finding is that the complex is hexameric, centered around nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, where nsp14 is seen to undergo a large conformational change between its two domains. This conformational change facilitates binding of six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Two of the six polymerase subunits are each proposed to carry dimers of nsp13, while two others are proposed to carry monomers. The polymerase subunits that coordinate nsp13 dimers also bind the nucleocapsid, which positions the 5\u2019-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analyzing the path of the viral RNA indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping (the nsp12 NiRAN and the nsp14 and nsp16 methyltransferases). The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping and the role of the endonuclease. It provides a platform to guide biochemical and structural research to address the stoichiometric and spatial configuration of the replication-transcription complex. The replication of the coronavirus genome and the synthesis of subgenomic mRNA is a complex process involving multiple viral proteins. Despite a fairly complete structural picture of the individual proteins that are believed to coalesce into a larger replication-transcription complex, there is no clear model of how these proteins interact. Here we present the first detailed atomistic model of a complete replication-transcription complex for SARS-CoV-2, made up of the non-structural proteins nsp7-nsp16, as well as the nucleocapsid. Forming a large, hexameric superstructure centered around nsp15, the model provides new perspective on the function of its individual components, including the exonuclease, the endonuclease, the NiRAN site, the helicase, the multiple zinc fingers, and the nucleocapsid. It offers a cohesive view of replication, proofreading, template switching and mRNA capping, which should serve as a guide for future experimental exploration.","version":"1.1","doi":"10.1101/2021.06.08.447516","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447286","pub_date":"2021-6-07","title":"Dual roles of a novel oncolytic viral vector-based SARS-CoV-2 vaccine: preventing COVID-19 and treating tumor progression","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cancer patients are usually immunocompromised and thus are particularly susceptible to SARS-CoV-2 infection resulting in COVID-19. Although many vaccines against COVID-19 are being preclinically or clinically tested or approved, none have yet been specifically developed for cancer patients or reported as having potential dual functions to prevent COVID-19 and treat cancer. Here, we confirmed that COVID-19 patients with cancer have low levels of antibodies against the spike (S) protein, a viral surface protein mediating the entry of SARS-CoV-2 into host cells, compared with COVID-19 patients without cancer. We developed an oncolytic herpes simplex virus-1 vector-based vaccine named oncolytic virus (OV)-spike. OV-spike induced abundant anti-S protein neutralization antibodies in both tumor-free and tumor-bearing mice, which inhibit infection of VSV-SARS-CoV-2 and wild-type (WT) live SARS-CoV-2 as well as the B.1.1.7 variant in vitro. In the tumor-bearing mice, OV-spike also inhibited tumor growth, leading to better survival in multiple preclinical tumor models than the untreated control. Furthermore, OV-spike induced anti-tumor immune response and SARS-CoV-2-specific T cell response without causing serious adverse events. Thus, OV-spike is a promising vaccine candidate for both preventing COVID-19 and enhancing the anti-tumor response. A herpes oncolytic viral vector-based vaccine is a promising vaccine with dual roles in preventing COVID-19 and treating tumor progression","version":"1.1","doi":"10.1101/2021.06.07.447286","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.06.446781","pub_date":"2021-6-07","title":"FXR inhibition reduces ACE2 expression, SARS-CoV-2 infection and may improve COVID-19 outcome","abstract":"Prevention of SARS-CoV-2 entry in cells through the modulation of viral host receptors, such as ACE2, could represent a new therapeutic approach complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive. Here, we identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19-affected tissues, including the gastrointestinal and respiratory systems. We demonstrate that FXR antagonists, including the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulate ACE2 levels, and reduce susceptibility to SARS-CoV-2 infection in lung, cholangiocyte and gut organoids. We then show that therapeutic levels of UDCA downregulate ACE2 in human organs perfused ex situ and reduce SARS-CoV-2 infection ex vivo. Finally, we perform a retrospective study using registry data and identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death. In conclusion, we identify a novel function of FXR in controlling ACE2 expression and provide evidence that this approach could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials.","version":"1.1","doi":"10.1101/2021.06.06.446781","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.06.447293","pub_date":"2021-6-07","title":"Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome","abstract":"SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didn\u2019t identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2\u2019s integration into host genome is lacking. SARS-CoV-2 does not integrate into host genome through whole genome sequencing.","version":"1.1","doi":"10.1101/2021.06.06.447293","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447351","pub_date":"2021-6-07","title":"Intronic regulation of SARS-CoV-2 receptor (ACE2) expression mediated by immune signaling and oxidative stress pathways","abstract":"The angiotensin-converting enzyme 2 (ACE2) protein has been highly studied as a key catalytic regulator of the renin-angiotensin system (RAS), involved in fluid homeostasis and blood pressure modulation. In addition to its important physiological role as a broadly-expressed membrane-bound protein, ACE2 serves as a cell-surface receptor for some viruses - most notably, coronaviruses such as SARS-CoV and SARS-CoV-2. Differing levels of ACE2 expression may impact viral susceptibility and subsequent changes to expression may be a pathogenic mechanism of disease risk and manifestation. Therefore, an improved understanding of how ACE2 expression is regulated at the genomic and transcriptional level may help us understand not only how the effects of pre-existing conditions (e.g., chronic obstructive pulmonary disease) may manifest with increased COVID-19 incidence, but also the mechanisms that regulate ACE2 levels following viral infection. Here, we initially perform bioinformatic analyses of several datasets to generate hypotheses about ACE2 gene-regulatory mechanisms in the context of immune signaling and chronic oxidative stress. We then identify putative non-coding regulatory elements within ACE2 intronic regions as potential determinants of ACE2 expression activity. We perform functional validation of our computational predictions in vitro via targeted CRISPR-Cas9 deletions of the identified ACE2 cis-regulatory elements in the context of both immunological stimulation and oxidative stress conditions. We demonstrate that intronic ACE2 regulatory elements are responsive to both immune signaling and oxidative-stress pathways, and this contributes to our understanding of how expression of this gene may be modulated at both baseline and during immune challenge. Our work supports the further pursuit of these putative mechanisms in our understanding, prevention, and treatment of infection and disease caused by ACE2-utilizing viruses such as SARS-CoV, SARS-CoV-2, and future emerging SARS-related viruses. The recent emergence of the virus SARS-CoV-2 which has caused the COVID-19 pandemic has prompted scientists to intensively study how the virus enters human host cells. This work has revealed a key protein, ACE2, that acts as a receptor permitting the virus to infect cells. Much research has focused on how the virus physically interacts with ACE2, yet little is known on how ACE2 is turned on or off in human cells at the level of the DNA molecule. Understanding this level of regulation may offer additional ways to prevent or lower viral entry into human hosts. Here, we have examined the control of the ACE2 gene, the DNA sequence that instructs ACE2 protein receptor formation, and we have done so in the context of immune stimulation. We have indeed identified a number of DNA on/off switches for ACE2 that appear responsive to immuno-logical and oxidative stress. These switches may fine-tune how ACE2 is turned on or off before, during, and/or after infection by SARS-CoV-2 or other related coronaviruses. Our studies help pave the way for additional functional studies on these switches, and their potential therapeutic targeting in the future.","version":"1.1","doi":"10.1101/2021.06.07.447351","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.05.447221","pub_date":"2021-6-07","title":"B.1.1.7 and B.1.351 variants are highly virulent in K18-ACE2 transgenic mice and show different pathogenic patterns from early SARS-CoV-2 strains","abstract":"SARS-CoV-2 continues to circulate globally resulting in emergence of several variants of concern (VOC), including B.1.1.7 and B.1.351 that show increased transmissibility and enhanced resistance to antibody neutralization. In a K18-hACE2 transgenic mouse model, we demonstrate that Both B.1.1.7 and B.1.351 are 100 times more lethal than the original SARS-CoV-2 bearing 614D. Mice infected with B.1.1.7 and B.1.351 exhibited more severe lesions in internal organs than those infected with early SARS-CoV-2 strains bearing 614D or 614G. Infection of B.1.1.7 and B.1.351 also results in distinct tissue-specific cytokine signatures, significant D-dimer depositions in vital organs and less pulmonary hypoxia signaling before death as compared to the mice infected with early SARS-CoV-2 strains. However, K18-hACE2 mice with the pre-existing immunity from prior infection or immunization were resistant to the lethal reinfection of B.1.1.7 or B.1.351, despite having reduced neutralization titers against these VOC. Our study reveals distinguishing pathogenic patterns of B.1.1.7 and B.1.351 variants from those early SARS-CoV-2 strains in K18-hACE2 mice, which will help to inform potential medical interventions for combatting COVID-19.","version":"1.1","doi":"10.1101/2021.06.05.447221","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447338","pub_date":"2021-6-07","title":"In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR","abstract":"A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases \u2013 as seen with the B.1.1.7 variant \u2013 can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from \u22120.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charit\u00e9 RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.","version":"1.1","doi":"10.1101/2021.06.07.447338","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.07.447335","pub_date":"2021-6-07","title":"The local anaesthetic procaine prodrugs ProcCluster\u00ae and Procaine-hydrochloride impair SARS-CoV-2 replication in vitro","abstract":"The SARS-CoV-2 pandemic has had the world in suspense for more than a year. Even if more and more vaccines are approved there is still an urgent need for efficient antiviral treatment strategies. Here, we present data on the inhibitory effect of the local anaesthetic procaine, especially the prodrugs ProcCluster\u00ae and Procaine-hydrochloride on SARS-CoV-2 infection in vitro. Remarkably, similar effects could be shown on the replication of influenza A viruses in cell culture systems. Since the active ingredient procaine is well-tolerated and already used in the clinics for anaesthetic purposes, the further investigation of this substance could enable its reuse in antiviral therapy, including SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.06.07.447335","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.05.447177","pub_date":"2021-6-07","title":"Neutralization against B.1.351 and B.1.617.2 with sera of COVID-19 recovered cases and vaccinees of BBV152","abstract":"Recently, multiple SARS-CoV-2 variants have been detected across the globe. The recent emergence of B.1.617 lineage has created serious public health problem in India. The high transmissibility was observed with this lineage which has led to daily increase in the number of SARS-CoV-2 infections. Apparently, the sub-lineage B.1.617.2 has slowly dominated the other variants including B1617.1, B.617.3 and B.1.1.7. With this, World Health Organization has described B.1.617.2 as variant of concern. Besides this, variant of concern B.1.351 has been also reported from India, known to showreducedefficacyfor many approved vaccines. With the increasing threat of the SARS-CoV-2 variants, it is imperative to assess the efficacy of the currently available vaccines against these variants. Here, we have evaluated the neutralization potential of sera collected from COVID-19 recovered cases (n=20) and vaccinees with two doses of BBV152 (n=17) against B.1.351 and B.1.617.2 compared to the prototype B.1 (D614G) variant.The finding of the study demonstrated a reduction in neutralization titers with sera of COVID-19 recovered cases(3.3-fold and 4.6-fold) and BBV152 vaccinees (3. 0 and 2.7 fold) against B.1.351 and B.1.617.2 respectively.Although, there is reduction in neutralization titer, the whole-virion inactivated SARS-CoV-2 vaccine (BBV152) demonstrates protective response against VOC B.1351 and B.1.617.2.","version":"1.1","doi":"10.1101/2021.06.05.447177","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445443","pub_date":"2021-6-07","title":"Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core","abstract":"The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind, fuse, and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes the preparation of the fusion machinery by dissociation of the S1 domains. We report the development of bottom-up coarse-grained (CG) models validated with cryo-electron tomography (cryo-ET) data, and the use of CG molecular dynamics simulations to investigate the dynamical mechanisms involved in viral binding and exposure of the S2 trimeric core. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces. The multivalent interaction cyclically and processively induces S1 dissociation, thereby exposing the S2 core containing the fusion machinery. Our simulations thus reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.","version":"1.2","doi":"10.1101/2021.05.24.445443","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445517","pub_date":"2021-6-07","title":"Evolutionary Inference Predicts Novel ACE2 Protein Interactions Relevant to COVID-19 Pathologies","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the cell receptor that the coronavirus SARS-CoV-2 binds to and uses to enter and infect human cells. COVID-19, the pandemic disease caused by the coronavirus, involves diverse pathologies beyond those of a respiratory disease, including micro-thrombosis (micro-clotting), cytokine storms, and inflammatory responses affecting many organ systems. Longer-term chronic illness can persist for many months, often well after the pathogen is no longer detected. A better understanding of the proteins that ACE2 interacts with can reveal information relevant to these disease manifestations and possible avenues for treatment. We have undertaken an approach to predict candidate ACE2 interacting proteins which uses evolutionary inference to identify a set of mammalian proteins that \u201ccoevolve\u201d with ACE2. The approach, called evolutionary rate correlation (ERC), detects proteins that show highly correlated evolutionary rates during mammalian evolution. Such proteins are candidates for biological interactions with the ACE2 receptor. The approach has uncovered a number of key ACE2 protein interactions of potential relevance to COVID-19 pathologies. Some proteins have previously been reported to be associated with severe COVID-19, but are not currently known to interact directly with ACE2, while additional predicted novel ACE2 interactors are of potential relevance to the disease. Using reciprocal rankings of protein ERCs, we have identified strongly interconnected ACE2 associated protein networks relevant to COVID-19 pathologies. ACE2 has clear connections to coagulation pathway proteins, such as Coagulation Factor V and fibrinogen components FGG, FGB, and FGA, the latter possibly mediated through ACE2 connections to Clusterin (which clears misfolded extracellular proteins) and GPR141 (whose functions are relatively unknown). ACE2 also connects to proteins involved in cytokine signaling and immune response (e.g. IFNAR2, XCR1, and TLR8), and to Androgen Receptor (AR). The ERC prescreening approach has also elucidated possible functions for previously uncharacterized proteins and possible new functions for well-characterized ones. Suggestions are made for the validation of ERC predicted ACE2 protein interactions. We propose that ACE2 has novel protein interactions that are disrupted during SARS-CoV-2 infection, contributing to the spectrum of COVID-19 pathologies.","version":"1.3","doi":"10.1101/2021.05.24.445517","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.06.446935","pub_date":"2021-6-07","title":"Hot water extract of Arthrospira maxima (AHWE) has broad-spectrum antiviral activity against RNA virus including coronavirus SARS-CoV2, and the antivirus spray application","abstract":"The emergence and re-emergence of RNA virus outbreaks highlight the urgent need for the development of broad-spectrum antiviral agents. Arthrospira maxima has be used as a food source for a long time, and the protein or polysaccharide fractions were evidenced to have antiviral activity, therefore we examined the antiviral efficacy of hot water extract from Arthrospira maxima (AHWE), on Enterovirus 71 (EV71), Influenza virus, Herpes simplex virus (HSV), Respiratory syncytial virus (RSV), Ebola virus, and Coronavirus for antiviral spray application. In this study, we demonstrated that the AHWE shown 90 to 100% inhibition rate on the plaque formation of EV71, HSV-1, HSV-2, influenza virus, RSV, 229E and SARS-COV2 at virus attachment stage, and the long-lasting protection study also found while the AHWE was pre-exposed to the open air for more than 4 hours in plaque reduction assay. In addition, AHWE also had inhibitory effect on Ebola virus replication at 500 ug/ml. Finally, AHWE also shown no toxicity and skin sensitivity that imply it could be safe for future clinical use if approved by FDA. In conclusion, this study suggests that AHWE could be developed as a potential broad-spectrum antivirus spray product and therapeutic agent.","version":"1.1","doi":"10.1101/2021.06.06.446935","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.06.446826","pub_date":"2021-6-07","title":"Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant","abstract":"Emergence of SARS-CoV-2 variants, including the globally successful B.1.1.7 lineage, suggests viral adaptations to host selective pressures resulting in more efficient transmission. Although much effort has focused on Spike adaptation for viral entry and adaptive immune escape, B.1.1.7 mutations outside Spike likely contribute to enhance transmission. Here we used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells. We found that B.1.1.7 isolates have dramatically increased subgenomic RNA and protein levels of Orf9b and Orf6, both known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation, and Orf9b binding and activity was regulated via phosphorylation. We conclude that B.1.1.7 has evolved beyond the Spike coding region to more effectively antagonise host innate immune responses through upregulation of specific subgenomic RNA synthesis and increased protein expression of key innate immune antagonists. We propose that more effective innate immune antagonism increases the likelihood of successful B.1.1.7 transmission, and may increase in vivo replication and duration of infection.","version":"1.1","doi":"10.1101/2021.06.06.446826","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.269159","pub_date":"2021-6-07","title":"Rethinking Remdesivir: Synthesis, Antiviral Activity and Pharmacokinetics of Oral Lipid Prodrugs","abstract":"Remdesivir (RDV, GS-5734) is currently the only FDA-approved antiviral drug for the treatment of SARS CoV-2 infection. The drug is approved for use in adults or children 12-years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2 infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed and several including molnupiravir and PF-07321332 are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of Remdesivir nucleoside (RVn, GS-441524) that are processed to RVn-monophosphate, the precursor of the active RVn-triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells and Huh7.5 cells. In Syrian hamsters oral treatment with ODBG-P-RVn was well tolerated and achieved therapeutic levels in plasma above the EC90 for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.","version":"1.2","doi":"10.1101/2020.08.26.269159","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.06.447235","pub_date":"2021-6-07","title":"Predicted pH-dependent stability of SARS-CoV-2 spike protein trimer from interfacial acidic groups","abstract":"Transition between receptor binding domain (RBD) up and down forms of the SARS-CoV-2 spike protein trimer is coupled to receptor binding and is one route by which variants can alter viral properties. It is becoming apparent that key roles in the transition are played by pH and a more compact closed form, termed locked. Calculations of pH-dependence are made for a large set of spike trimers, including locked form trimer structures that have recently become available. Several acidic sidechains become sufficiently buried in the locked form to give a predicted pH-dependence in the mild acidic range, with stabilisation of the locked form as pH reduces from 7.5 to 5, consistent with emerging characterisation by cryo-electron microscopy. The calculated pH effects in pre-fusion spike trimers are modulated mainly by aspartic acid residues, rather than the more familiar histidine role at mild acidic pH. These acidic sidechains are generally surface located and weakly interacting when not in a locked conformation. In this model, their replacement (perhaps with asparagine) would remove the pH-dependent destabilisation of locked spike trimer conformations, and increase their recovery at neutral pH. This would provide an alternative or supplement to the insertion of disulphide linkages for stabilising spike protein trimers, with potential relevance for vaccine design.","version":"1.1","doi":"10.1101/2021.06.06.447235","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.11.439398","pub_date":"2021-6-07","title":"Expression of the ACE2 virus entry protein in the nervus terminalis reveals the potential for an alternative route to brain infection in COVID-19","abstract":"Previous studies suggested that the SARS-CoV-2 virus may gain access to the brain by using a route along the olfactory nerve. However, there is a general consensus that the obligatory virus entry receptor, angiotensin converting enzyme 2 (ACE2), is not expressed in olfactory receptor neurons, and the timing of arrival of the virus in brain targets is inconsistent with a neuronal transfer along olfactory projections. We determined whether nervus terminalis neurons and their peripheral and central projections should be considered as a potential alternative route from the nose to the brain. Nervus terminalis neurons in postnatal mice were double-labeled with antibodies against ACE2 and two nervus terminalis markers, gonadotropin-releasing hormone (GnRH) and choline acetyltransferase (CHAT). We show that a small fraction of CHAT-labeled nervus terminalis neurons, and the large majority of GnRH-labeled nervus terminalis neurons with cell bodies in the region between the olfactory epithelium and the olfactory bulb express ACE2 and cathepsins B and L. Nervus terminalis neurons therefore may provide a direct route for the virus from the nasal epithelium, possibly via innervation of Bowman\u2019s glands, to brain targets, including the telencephalon and diencephalon. This possibility needs to be examined in suitable animal models and in human tissues.","version":"1.2","doi":"10.1101/2021.04.11.439398","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.445948","pub_date":"2021-6-07","title":"Integrating digital and field surveillance to complement efforts to manage epidemic diseases of livestock: African swine fever as a case study","abstract":"The SARS-CoV-2 pandemic has unveiled the importance of stakeholders and ordinary citizens in managing infectious disease emergencies. Efficient management of infectious diseases requires a top-down approach which must be complemented with a bottom-up response to be effective. Here we investigate a novel approach to surveillance for transboundary animal diseases using African Swine fever as a model. We were able to collect data at a population level on information-seeking behavior and at a local level through a targeted questionnaire-based survey to relevant stakeholders such as farmers and veterinary authorities. Our study shows how information-seeking behavior and resulting public attention during an epidemic, can be addressed through novel data streams from digital platforms such as Wikipedia. We also bring evidence on how field surveys aimed at local workers (e.g. farmers) and public authorities remain a crucial tool to assess more in-depth preparedness and awareness among front-line actors. We conclude that they should be used in combination to maximize the outcome of surveillance and prevention activities for selected transboundary animal diseases.","version":"1.2","doi":"10.1101/2021.05.27.445948","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.04.447156","pub_date":"2021-6-06","title":"Combustible and electronic cigarette exposures increase ACE2 activity and SARS-CoV-2 Spike binding","abstract":"The outbreak of coronavirus disease 2019 (COVID-19) has extensively impacted global health. The causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a transmembrane metallo-carboxypeptidase that is expressed in both membrane-anchored (mACE2) and soluble (sACE2) forms in the lung. Tobacco use has been speculated as a vulnerability factor for contracting SARS-CoV-2 infection and subsequent disease severity, whilst electronic cigarettes (e-cigarettes) have been shown to induce harmful proteomic and immune changes in the lungs of vapers. We therefore tested the hypothesis that combustible tobacco (e.g. cigarettes) and non-combustible e-cigarettes could affect ACE2 activity and subsequent SARS-CoV-2 infection. We observed that sACE2 activity was significantly higher in bronchoalveolar lavage fluid from both smokers and vapers compared to age-matched non-smokers. Exposure to cigarette smoke increased ACE2 levels, mACE2 activity, and sACE2 in primary bronchial epithelial cultures. Finally, treatment with either cigarette smoke condensate or JUUL e-liquid increased infections with a spike-coated SARS-CoV-2 pseudovirus. Overall, these observations suggest that tobacco product use elevates ACE2 activity and increases the potential for SARS-CoV-2 infection through enhanced spike protein binding.","version":"1.1","doi":"10.1101/2021.06.04.447156","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.04.447160","pub_date":"2021-6-06","title":"Drug Repurposing for the SARS-CoV-2 Papain-Like Protease","abstract":"As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen\u2019s two large polypeptide translates ORF1a and ORF1ab in human host cells to form 15 functionally important, mature nonstructural proteins. One of the two enzymes, papain-like protease or PLpro, also possesses deubiquitination and deISGylation activities that suppresses host innate immune responses toward SARS-CoV-2 infection. Therefore, PLpro is a potential COVID-19 drug target. To repurpose drugs for PLpro, we experimentally screened 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLpro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit potent inhibition of PLpro at 200 \u03bcM. More comprehensive characterizations revealed 7 inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 60 \u03bcM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 \u03bcM. Among four inhibitors with an IC50 value below 10 \u03bcM, SJB2-043 is the most unique in that it doesn\u2019t fully inhibit PLpro but has an outstanding IC50 value of 0.56 \u03bcM. SJB2-043 likely binds to an allosteric site of PLpro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLpro holds promises but in-depth analysis of repurposed drugs is necessary to avoid omitting allosteric inhibitors.","version":"1.1","doi":"10.1101/2021.06.04.447160","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.03.21258241","pub_date":"2021-06-05","title":"Multisystemic cellular tropism of SARS-CoV-2 in autopsies of COVID-19 patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n=8), using detailed histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA\n                    <jats:italic>in situ</jats:italic>\n                    hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. Finally, we confirmed these findings in an independent external autopsy cohort (n=9).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>\n                    SARS-CoV-2 RNA was mainly localized in epithelial cells, endothelial and mesenchymal cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. The findings were validated using\n                    <jats:italic>in situ</jats:italic>\n                    hybridization on external COVID-19 autopsy samples. Finally, apart from the lung, correlation of virus detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>SARS-CoV-2 could be observed in virtually all organs, colocalizing with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells, and viral replication was found across all organ systems. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>SARS-CoV-2 has been shown to infect the respiratory tract and affect several other major organs. However, on a cellular level, the localization of SARS-CoV-2 and its targets ACE2 and TMPRSS2 have not been described comprehensively.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>\n                      We have analyzed tissue SARS-CoV-2 RNA using RT-PCR and visualized its localization together with ACE2 and TMPRSS2 using\n                      <jats:italic>in situ</jats:italic>\n                      hybridization (ISH) in 25 different autopsy tissues. SARS-CoV-2 sense and antisense RNA were detected in 16 tissues/organs, mainly in epithelial cells and, to a lesser extent, in endothelial or stromal cells. Detection of viral protein using immunohistochemistry or viral particles using transmission electron microscopy did not yield specific results. Interestingly, apart from the respiratory tract and specifically the lungs, we have not found a specific pathology that would be associated with extrapulmonary viral spread.\n                    </jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>We provide a recommendation on using these methods in autopsy diagnostics for SARS-CoV-2. Our data extend the current hypothesis of severe COVID-19 being multisystemic diseases. Our data also provide clear evidence of infection and replication of SARS-CoV-2 in the endothelial cell across all organs, extending the hypothesis on the (micro)vascular involvement in COVID-19.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.03.21258241","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.03.447021","pub_date":"2021-6-04","title":"Screening of Botanical Drugs against SARS-CoV-2 Entry","abstract":"An escalating pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is impacting global health. Specific treatment options for diseases caused by SARS-CoV-2 are largely lacking. Herein, we used a pseudotype virus (pv) bearing the SARS-CoV-2 S glycoprotein to screen a botanical drug library to identify an agent against SARS-CoV-2 entry. All the four hits, including angeloylgomisin O, schisandrin B, procyanidin, and oleanonic acid, were identified for effective inhibition of SARS-CoV-2 S pv entry in the micromolar range. A mechanistic study revealed that these four agents inhibit SARS-CoV-2 S pv entry by blocking S-mediated membrane fusion. Furthermore, angeloylgomisin O, schisandrin B, and oleanonic acid inhibited authentic SARS-CoV-2 with a high selective index (SI). We also showed that all the four hits could also inhibit the entry of pv of Middle East respiratory syndrome coronavirus (MERS-CoV) and newly emerged SARS-CoV-2 variants (D614G, K417N/E484K/N501Y/D614G). In drug combination studies performed in cellular antiviral assays, angeloylgomisin O and schisandrin B displayed synergistic effects in combination with remdesivir. These results indicated that angeloylgomisin O, schisandrin B, procyanidin, and oleanonic acid can inhibit SARS-CoV-2 and that they are potential therapeutic agents for COVID-19.","version":"1.1","doi":"10.1101/2021.06.03.447021","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429940","pub_date":"2021-6-04","title":"Protein N-glycosylation is essential for SARS-CoV-2 infection","abstract":"SARS-CoV-2 extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These sugars are predicted to help mediate spike binding to the host receptor by stabilizing its \u2018open\u2019 conformation and evading host immunity. Here, we investigated both the essentiality of the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Inhibition of host N-glycosylation using RNAi or FDA-approved drugs reduced virus infectivity, including that of several variants. Under these conditions, cells produced less virions and some completely lost their infectivity. Furthermore, partial deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, spike N-glycosylation is a targetable pathway with clinical potential for treatment or prevention of COVID-19.","version":"1.2","doi":"10.1101/2021.02.05.429940","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.03.446942","pub_date":"2021-6-04","title":"Visualization of SARS-CoV-2 infection dynamic","abstract":"Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.","version":"1.1","doi":"10.1101/2021.06.03.446942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.21258097","pub_date":"2021-06-04","title":"Rapid and Sensitive Detection of SARS-CoV-2 Infection Using Quantitative Peptide Enrichment LC-MS Analysis","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples.</jats:p>\n                <jats:p>The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout (r=0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct \u226430). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.</jats:p>","version":null,"doi":"10.1101/2021.06.02.21258097","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.04.447130","pub_date":"2021-6-04","title":"A hybrid in silico approach reveals novel inhibitors of multiple SARS-CoV-2 variants","abstract":"The National Center for Advancing Translational Sciences (NCATS) has been actively generating SARS-CoV-2 high-throughput screening data and disseminates it through the OpenData Portal (https://opendata.ncats.nih.gov/covid19/). Here, we provide a hybrid approach that utilizes NCATS screening data from the SARS-CoV-2 cytophatic effect reduction assay to build predictive models, using both machine learning and pharmacophore-based modeling. Optimized models were used to perform two iterative rounds of virtual screening to predict small molecules active against SARS-CoV-2. Experimental testing with live virus provided 100 (~16% of predicted hits) active compounds (Efficacy > 30%, IC50 \u2264 15 \u03bcM). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further analysis identified allosteric binders to host receptor angiotensin-converting enzyme 2, which were able to inhibit the entry of pseudoparticles bearing spike protein of wild type SARS-CoV-2 as well as South African B.1.351 and UK B.1.1.7 variants.","version":"1.1","doi":"10.1101/2021.06.04.447130","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.03.21258228","pub_date":"2021-06-04","title":"Persistent SARS-CoV-2 infection and intra-host evolution in association with advanced HIV infection","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>While most people effectively clear SARS-CoV-2, there are several reports of prolonged infection in immunosuppressed individuals. Here we present a case of prolonged infection of greater than 6 months with shedding of high titter SARS-CoV-2 in an individual with advanced HIV and antiretroviral treatment failure. Through whole genome sequencing at multiple time-points, we demonstrate the early emergence of the E484K substitution associated with escape from neutralizing antibodies, followed by other escape mutations and the N501Y substitution found in most variants of concern. This provides support to the hypothesis of intra-host evolution as one mechanism for the emergence of SARS-CoV-2 variants with immune evasion properties.</jats:p>","version":null,"doi":"10.1101/2021.06.03.21258228","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.04.447114","pub_date":"2021-6-04","title":"Sub-Picomolar Detection of SARS-CoV-2 RBD via Computationally-Optimized Peptide Beacons","abstract":"The novel coronavirus SARS-CoV-2 continues to pose a significant global health threat. Along with vaccines and targeted therapeutics, there is a critical need for rapid diagnostic solutions. In this work, we employ deep learning-based protein design to engineer molecular beacons that function as conformational switches for high sensitivity detection of the SARS-CoV-2 spike protein receptor binding domain (S-RBD). The beacons contain two peptides, together forming a heterodimer, and a binding ligand between them to detect the presence of S-RBD. In the absence of S-RBD (OFF), the peptide beacons adopt a closed conformation that opens when bound to the S-RBD and produces a fluorescence signal (ON), utilizing a fluorophore-quencher pair at the two ends of the heterodimer stems. Two candidate beacons, C17LC21 and C21LC21, can detect the S-RBD with limits of detection (LoD) in the sub-picomolar range. We envision that these beacons can be easily integrated with on-chip optical sensors to construct a point-of-care diagnostic platform for SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.06.04.447114","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.312165","pub_date":"2021-6-04","title":"JIB-04 has broad-spectrum antiviral activity and inhibits SARS-CoV-2 replication and coronavirus pathogenesis","abstract":"Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens.","version":"1.2","doi":"10.1101/2020.09.24.312165","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.04.447090","pub_date":"2021-6-04","title":"Genome-scale CRISPR Screens Identify Host Factors that Promote Human Coronavirus Infection","abstract":"The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths as of May 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as potentially antigenically distinct SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development. Genome-wide CRISPR screens identified host factors that promote human coronavirus infection, revealing novel antiviral drug targets.","version":"1.1","doi":"10.1101/2021.06.04.447090","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.03.447023","pub_date":"2021-6-04","title":"Immunological profiling of COVID-19 patients with pulmonary sequelae","abstract":"Cellular immunity may be involved in organ damage and rehabilitation in patients with coronavirus disease 2019 (COVID-19). We aimed to delineate immunological features of COVID-19 patients with pulmonary sequelae (PS) one year after discharge. 50 COVID-19 survivors were recruited and classified according to radiological characteristics: 24 patients with PS and 26 patients without PS. Phenotypic and functional characteristics of immune cells were evaluated by multiparametric flow cytometry. Patients with PS had an increased proportion of natural killer (NK) cells and lower percentage of B cells compared to patients without PS. Phenotypic and functional features of T cells in patients with PS were predominated by the accumulation of CD4+ T cells secreting IL-17A, short-lived effector-like CD8+ T cells (CD27-CD62L-) and senescent T cells with excessive secretion of granzyme-B/perforin/IFN-\u03b3. NK cells were characterized by the excessive secretion of granzyme-B and perforin and the downregulation of NKP30 and NKP46; highly activated NKT and \u03b3\u03b4 T cells exhibited NKP30 and TIM-3 upregulation and NKB1 downregulation in patients with PS. However, immunosuppressive cells were comparable between the two groups. The interrelation of immune cells in COVID-19 was intrinsically identified, whereby T cells secreting IL-2, IL-4 and IL-17A were enriched among CD28+ and CD57-cells and cells secreting perforin/granzyme-B/IFN-\u03b3/TNF-\u03b1 expressed markers of terminal differentiation. CD57+NK cells, CD4+perforin+ T cells and CD8+CD27+CD62L+ T cells were identified as the independent predictors for residual lesions. Overall, our findings unveil the profound imbalance of immune landscape that may correlate with organ damage and rehabilitation in COVID-19. A considerable proportion of COVID-19 survivors have residual lung lesions, such as ground glass opacity and fiber streak shadow. To determine the relationship between host immunity and residual lung lesions, we performed an extensive analysis of immune responses in convalescent patients with COVID-19 one year after discharge. We found significant differences in immunological characteristics between patients with pulmonary sequelae and patients without pulmonary sequelae one year after discharge. Our study highlights the profound imbalance of immune landscape in the COVID-19 patients with pulmonary sequelae, characterized by the robust activation of cytotoxic T cells, NK cells and \u03b3\u03b4 T cells as well as the deficiencies of immunosuppressive cells. Importantly, CD57+NK cells, CD4+perforin+ T cells and CD8+CD27+CD62L+ T cells were identified as the independent predictors for residual lesions.","version":"1.1","doi":"10.1101/2021.06.03.447023","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.21258176","pub_date":"2021-06-04","title":"Necessity of COVID-19 Vaccination in Persons who have Already had COVID-19","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The purpose of this study was to evaluate the necessity of COVID-19 vaccination in persons with prior COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Employees of Cleveland Clinic working in Ohio on Dec 16, 2020, the day COVID-19 vaccination was started, were included. Anyone who tested positive for COVID-19 at least once before the study start date was considered previously infected. One was considered vaccinated 14 days after receipt of the second dose of a COVID-19 mRNA vaccine. The cumulative incidence of COVID-19, symptomatic COVID-19, and hospitalizations for COVID-19, were examined over the next 10.5 months.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Among the 52238 employees, 4718 (9%) had prior COVID-19 at the start of the study, and 35113 (67%) had received at least two doses of the vaccine by the end of the study. Of the 4284 COVID-19 infections during the study, 3476 (81.1%) occurred in persons who were unvaccinated, and 4263 (99.5%) occurred among those without prior COVID-19. In Cox proportional hazards regression, both prior COVID-19 and vaccination were independently associated with significantly lower risk of COVID-19. Vaccination was associated with lower risk of COVID-19 among those without prior COVID-19 (HR 0.24, 95% CI 0.22\u20130.26) but not among those with prior COVID-19 (HR 0.86, 95% CI 0.33\u20132.29).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Both previous infection and vaccination provide substantial protection against COVID-19. Vaccination reduces risk of COVID-19 among those without prior COVID-19 but not among those with prior COVID-19, at least not within one year following infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Summary</jats:title>\n                  <jats:p>Cumulative incidence of COVID-19 over 10.5 months, including the Delta phase, was examined among 52238 employees at Cleveland Clinic. Vaccination was associated with significantly lower risk of COVID-19 among those without prior COVID-19 but not among those with prior COVID-19.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.06.01.21258176","journal":"medRxiv","score":null},{"id":"10.1101/2020.12.10.419044","pub_date":"2021-6-03","title":"A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilizing immunity against SARS-CoV-2 infection in mice","abstract":"Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Gu\u00e9rin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilized, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralizing antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralized B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.","version":"1.2","doi":"10.1101/2020.12.10.419044","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.437224","pub_date":"2021-6-03","title":"Twelve-month specific IgG response to SARS-CoV-2 receptor-binding domain among COVID-19 convalescent plasma donors in Wuhan","abstract":"To investigate the duration of humoral immune response in convalescent coronavirus disease 2019 (COVID-19) patients, we conduct a 12-month longitudinal study through collecting a total of 1,782 plasma samples from 869 convalescent plasma donors in Wuhan, China and test specific antibody responses. The results show that positive rate of IgG antibody against receptor-binding domain of spike protein (RBD-IgG) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the COVID-19 convalescent plasma donors exceeded 70% for 12 months post diagnosis. The level of RBD-IgG decreases with time, with the titer stabilizing at 64.3% of the initial level by the 9th month. Moreover, male plasma donors produce more RBD-IgG than female, and age of the patients positively correlates with the RBD-IgG titer. A strong positive correlation between RBD-IgG and neutralizing antibody titers is also identified. These results facilitate our understanding of SARS-CoV-2-induced immune memory to promote vaccine and therapy development.","version":"1.2","doi":"10.1101/2021.04.05.437224","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.19.435806","pub_date":"2021-6-03","title":"Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir","abstract":"Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3CL main protease inhibitor in vitro (IC50 value of 29 \u00b5M) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.","version":"1.4","doi":"10.1101/2021.03.19.435806","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.03.446959","pub_date":"2021-6-03","title":"The mutational landscape of SARS-CoV-2 variants diversifies T cell targets in an HLA supertype-dependent manner","abstract":"The rapid, global dispersion of SARS-CoV-2 since its initial identification in December 2019 has led to the emergence of a diverse range of variants. The initial concerns regarding the virus were quickly compounded with concerns relating to the impact of its mutated forms on viral infectivity, pathogenicity and immunogenicity. To address the latter, we seek to understand how the mutational landscape of SARS-CoV-2 has shaped HLA-restricted T cell immunity at the population level during the first year of the pandemic, before mass vaccination. We analyzed a total of 330,246 high quality SARS-CoV-2 genome assemblies sampled across 143 countries and all major continents. Strikingly, we found that specific mutational patterns in SARS-CoV-2 diversify T cell epitopes in an HLA supertype-dependent manner. In fact, we observed that proline residues are preferentially removed from the proteome of prevalent mutants, leading to a predicted global loss of SARS-CoV-2 T cell epitopes in individuals expressing HLA-B alleles of the B7 supertype family. In addition, we show that this predicted global loss of epitopes is largely driven by a dominant C-to-U mutation type at the RNA level. These results indicate that B7 supertype-associated epitopes, including the most immunodominant ones, were more likely to escape CD8+ T cell immunosurveillance during the first year of the pandemic. Together, our study lays the foundation to help understand how SARS-CoV-2 mutants shape the repertoire of T cell targets and T cell immunity across human populations. The proposed theoretical framework has implications in viral evolution, disease severity, vaccine resistance and herd immunity.","version":"1.1","doi":"10.1101/2021.06.03.446959","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.21258025","pub_date":"2021-06-03","title":"SARS-CoV-2 specific memory B-cells from individuals with diverse disease severities recognize SARS-CoV-2 variants of concern","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  In this investigation we examined the magnitude, breadth, and durability of SARS-CoV-2 specific antibodies in two distinct B-cell compartments: long-lived plasma cell-derived antibodies in the plasma, and peripheral memory B-cells along with their associated antibody profiles elicited after\n                  <jats:italic>in vitro</jats:italic>\n                  stimulation. We found that magnitude varied amongst individuals, but was the highest in hospitalized subjects. Variants of concern (VoC) -RBD-reactive antibodies were found in the plasma of 72% of samples in this investigation, and VoC-RBD-reactive memory B-cells were found in all but 1 subject at a single time-point. This finding, that VoC-RBD-reactive MBCs are present in the peripheral blood of all subjects including those that experienced asymptomatic or mild disease, provides a reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to limit disease severity and transmission of variants of concern as they continue to arise and circulate.\n                </jats:p>","version":null,"doi":"10.1101/2021.05.28.21258025","journal":"medRxiv","score":null},{"id":"10.1101/2021.06.03.446968","pub_date":"2021-6-03","title":"Enteric coronavirus infection and treatment modeled with an immunocompetent human intestine-on-a-chip","abstract":"Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PMBCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.","version":"1.1","doi":"10.1101/2021.06.03.446968","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.379917","pub_date":"2021-6-03","title":"Identifying and prioritizing potential human-infecting viruses from their genome sequences","abstract":"Determining which animal viruses may be capable of infecting humans is currently intractable at the time of their discovery, precluding prioritization of high-risk viruses for early investigation and outbreak preparedness. Given the increasing use of genomics in virus discovery and the otherwise sparse knowledge of the biology of newly-discovered viruses, we developed machine learning models that identify candidate zoonoses solely using signatures of host range encoded in viral genomes. Within a dataset of 861 viral species with known zoonotic status, our approach outperformed models based on the phylogenetic relatedness of viruses to known human-infecting viruses (AUC = 0.773), distinguishing high-risk viruses within families that contain a minority of human-infecting species and identifying putatively undetected or so far unrealized zoonoses. Analyses of the underpinnings of model predictions suggested the existence of generalisable features of viral genomes that are independent of virus taxonomic relationships and that may preadapt viruses to infect humans. Our model reduced a second set of 645 animal-associated viruses that were excluded from training to 272 high and 41 very high-risk candidate zoonoses and showed significantly elevated predicted zoonotic risk in viruses from non-human primates, but not other mammalian or avian host groups. A second application showed that our models could have identified SARS-CoV-2 as a relatively high-risk coronavirus strain and that this prediction required no prior knowledge of zoonotic SARS-related coronaviruses. Genome-based zoonotic risk assessment provides a rapid, low-cost approach to enable evidence-driven virus surveillance and increases the feasibility of downstream biological and ecological characterisation of viruses.","version":"1.2","doi":"10.1101/2020.11.12.379917","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.446831","pub_date":"2021-6-03","title":"CD4+ T cell lymphopenia and dysfunction in severe COVID-19 disease is autocrine TNF-\u03b1/TNFRI-dependent","abstract":"Lymphopenia is common in severe COVID-19 disease, yet the mechanisms are poorly understood. In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared to recovered, mild disease (p<0.0001). In severe disease, immunodominant CD4+ T cell responses to Spike-1(S1) produced increased in vitro TNF-\u03b1, but impaired proliferation and increased susceptibility to activation-induced cell death (AICD). CD4+TNF-\u03b1+ T cell responses inversely correlated with absolute CD4+ counts from severe COVID-19 patients (n=76; R=-0.744, P<0.0001). TNF-\u03b1 blockade including infliximab or anti-TNFRI antibodies strikingly rescued S1-specific CD4+ proliferation and abrogated S1-AICD in severe COVID-19 patients (P<0.001). Single-cell RNAseq demonstrated downregulation of Type-1 cytokines and NF\u03baB signaling in S1-stimulated CD4+ cells with infliximab treatment. Lung CD4+ T cells in severe COVID-19 were reduced and produced higher TNF-\u03b1 versus PBMC. Together, our findings show COVID-19-associated CD4+ lymphopenia and dysfunction is autocrine TNF-\u03b1/TNFRI-dependent and therapies targeting TNF-\u03b1 may be beneficial in severe COVID-19. Autocrine TNF-\u03b1/TNFRI regulates CD4+ T cell lymphopenia and dysfunction in severe COVID-19 disease.","version":"1.1","doi":"10.1101/2021.06.02.446831","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.446845","pub_date":"2021-6-03","title":"3D-Scaffold: Deep Learning Framework to Generate 3D Coordinates of Drug-like Molecules with Desired Scaffolds","abstract":"The prerequisite of therapeutic drug design is to identify novel molecules with desired biophysical and biochemical properties. Deep generative models have demonstrated their ability to find such molecules by exploring a huge chemical space efficiently. An effective way to obtain molecules with desired target properties is the preservation of critical scaffolds in the generation process. To this end, we propose a domain aware generative framework called 3D-Scaffold that takes 3D coordinates of the desired scaffold as an input and generates 3D coordinates of novel therapeutic candidates as an output while always preserving the desired scaffolds in generated structures. We show that our framework generates predominantly valid, unique, novel, and experimentally synthesizable molecules that have drug-like properties similar to the molecules in the training set. Using domain specific datasets, we generate covalent and non-covalent antiviral inhibitors. To measure the success of our framework in generating therapeutic candidates, generated structures were subjected to high throughput virtual screening via docking simulations, which shows favorable interaction against SARS-CoV-2 main protease and non-structural protein endoribonuclease (NSP15) targets. Most importantly, our model performs well with relatively small volumes of training data and generalizes to new scaffolds, making it applicable to other domains.","version":"1.2","doi":"10.1101/2021.06.02.446845","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446640","pub_date":"2021-6-02","title":"SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator","abstract":"SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2-positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.","version":"1.1","doi":"10.1101/2021.06.01.446640","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.443175","pub_date":"2021-6-02","title":"Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year","abstract":"Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of na\u00efve individuals. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.05.07.443175","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.21256092","pub_date":"2021-06-02","title":"Cross-reactive antibodies after SARS-CoV-2 infection and vaccination","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Current SARS-CoV-2 vaccines are losing efficacy against emerging variants and may not protect against future novel coronavirus outbreaks, emphasizing the need for more broadly protective vaccines. To inform the development of a pan-coronavirus vaccine, we investigated the presence and specificity of cross-reactive antibodies against the spike (S) proteins of human coronaviruses (hCoV) after SARS-CoV-2 infection and vaccination. We found an 11 to 123-fold increase in antibodies binding to SARS-CoV and MERS-CoV as well as a 2 to 4-fold difference in antibodies binding to seasonal hCoVs in COVID-19 convalescent sera compared to pre-pandemic healthy donors, with the S2 subdomain of the S protein being the main target for cross-reactivity. In addition, we detected cross-reactive antibodies to all hCoV S proteins after SARS-CoV-2 S protein immunization in macaques, with higher responses for hCoV more closely related to SARS-CoV-2. These findings support the feasibility of and provide guidance for development of a pan-coronavirus vaccine.</jats:p>","version":null,"doi":"10.1101/2021.05.26.21256092","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.28.446009","pub_date":"2021-6-02","title":"Combination of a Sindbis-SARS-CoV-2 spike vaccine and \u03b1OX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity","abstract":"The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world\u2019s population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (\u03b1OX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus \u03b1OX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus \u03b1OX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.","version":"1.2","doi":"10.1101/2021.05.28.446009","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.446698","pub_date":"2021-6-02","title":"Efficacy of ancestral receptor-binding domain, S1 and trimeric spike protein vaccines against SARS-CoV-2 variants B.1.1.7, B.1.351, and B.1.617.1","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current SARS-CoV-2 vaccines are based on spike (S) protein, S1 subunit, or receptor-binding domain (RBD) of prototype strain. Emergence of several novel SARS-CoV-2 variants has raised concern about potential immune escape. In this study, we performed an immunogenicity comparison of ancestral RBD, S1, and S ectodomain trimer (S-trimer) antigens and tested the efficacy of these prototype vaccines against the circulating variants, especially B.1.617 that has been linked to India\u2019s current COVID-19 surge. We found that RBD and S-trimer proteins could induce significantly higher neutralizing antibody titers than S1 protein. For the three vaccines, the neutralizing titers decreased over time, but still remained high for at least five months after immunization. Importantly, the three prototype vaccines were still effective in neutralizing the variants of concern, although B.1.351 and B.1.617.1 lineages showed varying degrees of reduction in neutralization by the immune sera. The vaccines-induced sera were shown to block receptor binding and inhibit S protein-mediated membrane fusion. In addition, the immune sera did not promote antibody-dependent enhancement (ADE) in vitro. Our work provides valuable information for development of SARS-CoV-2 subunit vaccines and also supports the continued use of ancestral RBD or S-based vaccines to fight the COVID-19 epidemic.","version":"1.1","doi":"10.1101/2021.06.02.446698","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.02.446343","pub_date":"2021-6-02","title":"Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration into human lung cells","abstract":"To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin are high-affinity competitive inhibitors of ACE2 binding to the Wuhan S1 and the European [E614G] S1 proteins. These drugs also inhibit ACE2 binding to the Wuhan RBD, as well as to RBD proteins containing the S. Africa [E484K], Mink [Y453F] and UK [N501Y] mutations. As hypothesized, we also found that ouabain and digitoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:Spike binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.","version":"1.1","doi":"10.1101/2021.06.02.446343","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446639","pub_date":"2021-6-02","title":"Effects of the COVID-19 pandemic on publication landscape in chimeric antigen receptor-modified immune cell research","abstract":"Chimeric antigen receptors (CARs) are artificial receptors introduced mainly into T cells. CAR-induced immune cell (CARi) products have achieved impressive success rates in treating some difficult-to-treat hematological malignancies. Here, we describe effects of the global COVID-19 pandemic on CARi publication landscape. Due to the pandemic, the total number of publications decreased in 2020 compared to 2019 in all fields of cancer immunotherapy except CARi. Nearly exponential increases in the number of CARi publications slowed-down in 2020 for the first time in the past 11 years. There were more CARi than coronavirus publications until 2020 when coronavirus publications increased over 5,000% compared to 2019 (575 publications in 2019 vs. 30,390 in 2020). Unlike cancer immunotherapy where the majority of the publications consist of conference abstracts and review articles, majority of the coronavirus publications are original research articles. There are more coronavirus publications in Pubmed than Embase. The opposite is true for CARi publications. Our analysis of the data from the FDA Adverse Event Reporting System (FAERS) show significantly higher death rate in patients treated with Kymirah than Yescarta (28.14% vs. 16.02%). Kymirah and Yescarta are the two main CAR T cell products for treatment of DLBCL and/or B-ALL. However, despite being highly significant, this result is not easily interpretable due to multiple confounding variables in the FAERS data. Our analysis additionally suggest that the significant effects of co-stimulatory domains (4-1BB vs. CD28) consistently reported in preclinical studies do not translate into clinical results. Our manual curation of the CARi publications in PubMed shows that only 5.2% of the publications report results from CARi clinical trials, although we found 663 clinical trials listed on ClinicalTrials.gov database. In conclusion, publication landscape in CARi as well as other fields of cancer immunotherapy has changed due to the global COVID-19 pandemic. This trend will likely continue in the near future. CARi research is now in need of increased measures by publishers to reduce repetitive and/or duplicate publications and more stringent criteria for data entry into public databases including PubMed, Embase, ClinicalTrials.gov, and FAERS to advance this important field of medical research.","version":"1.1","doi":"10.1101/2021.06.01.446639","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446579","pub_date":"2021-6-01","title":"SARS-CoV-2 Spreads through Cell-to-Cell Transmission","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While ACE2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the variants of concern (VOC) B.1.1.7 and B.1.351 have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccine sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.","version":"1.1","doi":"10.1101/2021.06.01.446579","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446555","pub_date":"2021-6-01","title":"Endomembrane systems are reorganized by ORF3a and Membrane (M) of SARS-CoV-2","abstract":"The endomembrane reticulum (ER) is largely reorganized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 ORF3a and membrane (M) protein expression affects ER-derived structures including cubic membrane and double membrane vesicles in coronavirus-infected cells; however, the molecular mechanisms underlying ER remodeling remain unclear. We introduced a \u201cplug and playable\u201d proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged SARS-CoV-2 ORF3a and M proteins. Through mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using Spot-TurboID workflow, 117 and 191 proteins were robustly determined as ORF3a and M interactomes, respectively, and many, including RNF5 (E3 ubiquitin ligase), overlap with the mitochondrial-associated membrane (MAM) proteome. RNF5 expression was correlated to ORF3a ubiquitination. MAM formation and secreted proteome profiles were largely affected by ORF3a expression. Thus, SARS-CoV-2 may utilize MAM as a viral assembly site, suggesting novel anti-viral treatment strategies for blocking viral replication in host cells. \nSARS-CoV-2 proteins ORF3a and M alter endoplasmic reticulum proteome profile\nORF3a affects mitochondrial-associated membrane formation\nSARS-CoV-2 may utilize mitochondrial-associated membrane as viral assembly site\nORF3a and M interactome proteins may serve as targets for COVID-19 treatment\n SARS-CoV-2 proteins ORF3a and M alter endoplasmic reticulum proteome profile ORF3a affects mitochondrial-associated membrane formation SARS-CoV-2 may utilize mitochondrial-associated membrane as viral assembly site ORF3a and M interactome proteins may serve as targets for COVID-19 treatment ER remodelling by SARS-CoV-2 ORF3a and M protein","version":"1.1","doi":"10.1101/2021.06.01.446555","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446591","pub_date":"2021-6-01","title":"Structural basis for SARS-CoV-2 Nucleocapsid protein recognition by single-domain antibodies","abstract":"The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly-conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.06.01.446591","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446136","pub_date":"2021-6-01","title":"First evidence of SARS-CoV-2 genome detection in zebra mussel (Dreissena polymorpha)","abstract":"The uses of bivalve molluscs in environmental biomonitoring have recently gained momentum due to their ability to indicate and concentrate human pathogenic microorganisms. In the context of the health crisis caused by the COVID-19 epidemic, the objective of this study was to determine if the SARS-CoV-2 ribonucleic acid genome can be detected in zebra mussels (Dreissena polymorpha) exposed to raw and treated urban wastewaters from two separate plants to support its interest as bioindicator of the SARS-CoV-2 genome contamination in water. The zebra mussels were exposed to treated wastewater through caging at the outlet of two plants located in France, as well as to raw wastewater at laboratory scale in controlled conditions. Within their digestive tissues, our results showed that SARS-CoV-2 genome was detected in zebra mussels, whether in raw and treated wastewaters. Moreover, the detection of the SARS-CoV-2 genome in such bivalve molluscans appeared even with low concentrations in raw wastewaters. This is the first detection of the SARS-CoV-2 genome in the tissues of a sentinel species exposed to raw and treated urban wastewaters. Despite the need for development for quantitative approaches, these results support the importance of such invertebrate organisms, especially zebra mussel, for the active surveillance of pathogenic microorganisms and their indicators in environmental waters.","version":"1.1","doi":"10.1101/2021.05.28.446136","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446420","pub_date":"2021-6-01","title":"Virucidal activity of a proprietary blend of plant-based oils (Viruxal) against SARS-CoV-2 and influenza viruses \u2013 an in vitro study","abstract":"The emergence of a novel coronavirus known as SARS-CoV-2 resulting in a global pandemic COVID-19 has led to a dramatic loss of life worldwide and presented an unprecedented challenge to public health. Viruxal is a medical device in a form of a nasal and oral spay containing a a proprietary blend of plant-based oils which acts against enveloped viruses. The aim of this study was to evaluate the virus deactivation activity of Viruxal against SARS-CoV-2 and Influenza A(H1N1) viruses. An assay to detect virucidal activity was performed with four concentrations of Viruxal on two virus suspensions. Assessments were made based on log reduction values measured from the assay. Viruxal exhibited virucidal activity by reducing virus titer more than 90% for the enveloped viruses SARS-CoV-2 and influenza A(H1N1) after 30 minutes contact. Viruxal was validated for its potential usefulness as a medical device for treatment and prevention of enveloped respiratory viruses.","version":"1.1","doi":"10.1101/2021.05.31.446420","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443384","pub_date":"2021-6-01","title":"Structural basis for cell-type specific evolution of viral fitness by SARS-CoV-2","abstract":"As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS\u0394 variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.","version":"1.2","doi":"10.1101/2021.05.11.443384","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446516","pub_date":"2021-6-01","title":"SARS-CoV-2 cell-to-cell spread occurs rapidly and is insensitive to antibody neutralization","abstract":"Viruses increase the efficiency of close-range transmission between cells by manipulating cellular physiology and behavior, and SARS-CoV-2 uses cell fusion as one mechanism for cell-to-cell spread. Here we visualized infection using time-lapse microscopy of a human lung cell line and used live virus neutralization to determine the sensitivity of SARS-CoV-2 cell-to-cell spread to neutralizing antibodies. SARS-CoV-2 infection rapidly led to cell fusion, forming multinucleated cells with clustered nuclei which started to be detected at 6h post-infection. To compare sensitivity of cell-to-cell spread to neutralization, we infected either with cell-free virus or with single infected cells expressing on their surface the SARS-CoV-2 spike protein. We tested two variants of SARS-CoV-2: B.1.117 containing only the D614G substitution, and the escape variant B.1.351. We used the much smaller area of single infected cells relative to infection foci to exclude any input infected cells which did not lead to transmission. The monoclonal antibody and convalescent plasma we tested neutralized cell-free SARS-CoV-2, with the exception of B.1.351 virus, which was poorly neutralized with plasma from non-B.1.351 infections. In contrast, cell-to-cell spread of SARS-CoV-2 showed no sensitivity to monoclonal antibody or convalescent plasma neutralization. These observations suggest that, once cells are infected, SARS-CoV-2 may be more difficult to neutralize in cell types and anatomical compartments permissive for cell-to-cell spread.","version":"1.1","doi":"10.1101/2021.06.01.446516","journal":"bioRxiv","score":null},{"id":"10.1101/2021.06.01.446491","pub_date":"2021-6-01","title":"SARS coronavirus vaccines protect against different coronaviruses","abstract":"Although SARS-CoV-2 vaccines have shown efficacy against SARS-CoV-2, it is unclear if they can also protect against other coronaviruses that may infect humans in the future. Here, we show that SARS-CoV-2 vaccination in humans elicits cross-reactive antibodies against other coronaviruses. Our studies in mice demonstrate that SARS-CoV-2 vaccination protects against a common cold coronavirus, and that SARS-CoV-1 vaccination protects against SARS-CoV-2. Similarly, infection with a common cold coronavirus also conferred enhanced protection from subsequent infections with other coronaviruses. Mechanistically, both T cells and antibodies mediated cross-protection. This is the first direct demonstration that coronavirus-specific immunity can confer heterologous protection in vivo, providing a rationale for universal coronavirus vaccines. SARS-CoV-2 vaccination elicits cross-reactive antibody against other coronaviruses in humans. COVID-19 patients generate cross-reactive antibody against other coronaviruses. A SARS-CoV-1 vaccine protects against SARS-CoV-2. Prior coronavirus infections improve immune protection following heterologous coronavirus challenges.","version":"1.1","doi":"10.1101/2021.06.01.446491","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.29.445137","pub_date":"2021-5-31","title":"Functional characterization of SARS-CoV-2 vaccine elicited antibodies in immunologically na\u00efve and pre-immune humans","abstract":"As the COVID-19 pandemic continues, the authorization of vaccines for emergency use has been crucial in slowing down the rate of infection and transmission of the SARS-CoV-2 virus that causes COVID-19. In order to investigate the longitudinal serological responses to SARS-CoV-2 natural infection and vaccination, a large-scale, multi-year serosurveillance program entitled SPARTA (SARS SeroPrevalence and Respiratory Tract Assessment) was initiated at 4 locations in the U.S. The serological assay presented here measuring IgG binding to the SARS-CoV-2 receptor binding domain (RBD) detected antibodies elicited by SARS-CoV-2 infection or vaccination with a 95.5% sensitivity and a 95.9% specificity. We used this assay to screen more than 3100 participants and selected 20 previously infected pre-immune and 32 immunologically na\u00efve participants to analyze their antibody binding to RBD and viral neutralization (VN) responses following vaccination with two doses of either the Pfizer-BioNTech BNT162b2 or the Moderna mRNA-1273 vaccine. Vaccination not only elicited a more robust immune reaction than natural infection, but the level of neutralizing and anti-RBD antibody binding after vaccination is also significantly higher in pre-immune participants compared to immunologically na\u00efve participants (p<0.0033). Furthermore, the administration of the second vaccination did not further increase the neutralizing or binding antibody levels in pre-immune participants (p=0.69). However, ~46% of the immunologically na\u00efve participants required both vaccinations to seroconvert.","version":"1.1","doi":"10.1101/2021.05.29.445137","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.444482","pub_date":"2021-5-31","title":"Investigating the possible origin and transmission routes of SARS-CoV-2 genomes and variants of concern in Bangladesh","abstract":"The COVID-19 pandemic induced by the SARS-CoV-2 virus and its variants has ravaged most countries around the world including Bangladesh. We have analyzed publicly available genomic data to understand the current COVID-19 outbreak scenario as well as the evolutionary origin and transmission routes of SARS-CoV-2 isolates in Bangladesh. All the early isolates as well as recent B.1.1.7 and B.1.351 variants had already spread across the major divisional cities of Bangladesh. A sex biasness towards male COVID-19 patient samples sequencing has observed over female in all age-group, that could be the trend in infection rate. Phylogenetic analysis indicated a total of 13 estimated countries, including Italy, India, United Kingdom, Saudi Arabia, United Arab Emirates, Germany, Australia, New Zealand, South Africa, Democratic Republic of the Congo, United States, Russia, and Denmark, could be the possible origin introduced SARS-CoV-2 isolates in Bangladesh due to regional and intercontinental travel. Recent, B.1.1.7 variant could be imported from a total of 7 estimated countries including UK, India, Nigeria, Spain, Ireland, Australia, and Indonesia, while South Africa and the United States are the most likely sources of B.1351 variant in Bangladesh. Based on these findings, public health strategies could be designed and implemented to reduce the local transmission of the virus.","version":"1.3","doi":"10.1101/2021.05.24.444482","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.30.446357","pub_date":"2021-5-31","title":"Complete protection by a single dose skin patch delivered SARS-CoV-2 spike vaccine","abstract":"SARS-CoV-2 has infected over 160 million people and resulted in more than 3.3 million deaths, and we still face many challenges in the rollout of vaccines. Here, we use the high-density microarray patch to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show the vaccine, dry-coated on the patch is thermostable, and delivery of spike via HD-MAP induced greater cellular and antibody immune responses, with serum able to potently neutralize clinically relevant isolates including those from the B.1.1.7 and B.1.351 lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge, demonstrating that HD-MAP delivery of a SARS-CoV-2 vaccine is superior to traditional needle-and-syringe vaccination and has the potential to greatly impact the ongoing COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.05.30.446357","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.445667","pub_date":"2021-5-31","title":"Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting","abstract":"Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.","version":"1.1","doi":"10.1101/2021.05.31.445667","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.445871","pub_date":"2021-5-31","title":"Correlation of vaccine-elicited antibody levels and neutralizing activities against SARS-CoV-2 and its variants","abstract":"Both Pfizer-BNT162b2 and Moderna-mRNA-1273 vaccines can elicit an effective immune response against SARS-CoV-2 infection. However, the elicited serum antibody levels vary substantially and longitudinally decrease after vaccination. We examined the correlation of vaccination-induced IgG levels and neutralization titers against newly emerged variants remains and demonstrate a significant reduction of neutralization activities against the variants (B.1.1.7, B.1.525, and B.1.351) in Pfizer or Moderna vaccined sera. There was a significant and positive correlation between serum IgG levels and ID50 titers for not only SARS-CoV-2 WT but also the variants. These findings indicate that a high level of anti-spike IgG may offer better protection against infection from SARS-CoV-2 and its variants. Therefore, it is necessary to longitudinally monitor specific serum IgG level for evaluating the protective efficacy of the vaccines against SARS-CoV-2 and its new variants.","version":"1.1","doi":"10.1101/2021.05.31.445871","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.29.446272","pub_date":"2021-5-31","title":"Kite-shaped molecules block SARS-CoV-2 cell entry at a post-attachment step","abstract":"Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 1-5\u00b5M range, were identified. Further studies demonstrated that these \u2018kite-shaped\u2019 molecules were surprisingly specific for SARS-CoV and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.","version":"1.1","doi":"10.1101/2021.05.29.446272","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446374","pub_date":"2021-5-31","title":"Species-specific molecular barriers to SARS-CoV-2 replication in bat cells","abstract":"Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the susceptibility of primary cells from Rhinolophus ferrumequinum and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis and Nyctalus noctula, to SARS-CoV-2 infection. None of these cells were sensitive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication. This ability to control viral replication correlated with a potent interferon response. Our data highlight the existence of species-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. Bats host ancestors of several viruses that cause serious disease in humans, as illustrated by the on-going SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions have been hampered by a limited number of bat cell lines. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The varying susceptibilities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by over-expression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bats and viruses.","version":"1.1","doi":"10.1101/2021.05.31.446374","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.30.446322","pub_date":"2021-5-31","title":"SARS-CoV-2-specific memory B cells can persist in the elderly despite loss of neutralising antibodies","abstract":"Memory B cells (MBC) can provide a recall response able to supplement waning antibodies with an affinity-matured response better able to neutralise variant viruses. We studied a cohort of vulnerable elderly care home residents and younger staff, a high proportion of whom had lost neutralising antibodies (nAb), to investigate their reserve immunity from SARS-CoV-2-specific MBC. Class-switched spike and RBD-tetramer-binding MBC with a classical phenotype persisted five months post-mild/asymptomatic SARS-CoV-2 infection, irrespective of age. Spike/RBD-specific MBC remained detectable in the majority who had lost nAb, although at lower frequencies and with a reduced IgG/IgA isotype ratio. Functional spike/S1/RBD-specific recall was also detectable by ELISpot in some who had lost nAb, but was significantly impaired in the elderly, particularly to RBD. Our findings demonstrate persistence of SARS-CoV-2-specific MBC beyond loss of nAb, but highlight the need for careful monitoring of functional defects in RBD-specific B cell immunity in the elderly. Circulating class-switched spike and RBD-specific memory B cells can outlast detectable neutralising antibodies but are functionally constrained in the elderly.","version":"1.1","doi":"10.1101/2021.05.30.446322","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446421","pub_date":"2021-5-31","title":"Accelerated Antibody Discovery Targeting the SARS-CoV-2 Spike Protein for COVID-19 Therapeutic Potential","abstract":"Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in an aggressive timeline. In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the SARS-CoV-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb\u2122 mouse model for increased diversity. The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.","version":"1.1","doi":"10.1101/2021.05.31.446421","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446386","pub_date":"2021-5-31","title":"Spike mutation T403R allows bat coronavirus RaTG13 to use human ACE2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.","version":"1.1","doi":"10.1101/2021.05.31.446386","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429674","pub_date":"2021-5-31","title":"Pharmacokinetics of Orally Administered GS-441524 in Dogs","abstract":"Despite being FDA-approved for COVID-19, the clinical efficacy of remdesivir (Veklury\u00ae) remains contentious. We previously pointed out pharmacokinetic, pharmacodynamic and toxicology reasons for why its parent nucleoside GS-441524, is better suited for COVID-19 treatment. Here, we assess the oral bioavailability of GS-441524 in beagle dogs and show that plasma concentrations \u223c24-fold higher than the EC50 against SARS-CoV-2 are easily and safely sustained. These data support translation of GS-441524 as an oral agent for COVID-19.","version":"1.3","doi":"10.1101/2021.02.04.429674","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446403","pub_date":"2021-5-31","title":"Irradiation of UVC LED at 277 nm inactivates coronaviruses by photodegradation of spike protein","abstract":"To interrupt SARS-CoV-2 transmission chains, Ultraviolet-C (UVC) irradiation has emerged as a potential disinfection tool to aid in blocking the spread of coronaviruses. While conventional 254-nm UVC mercury lamps have been used for disinfection purposes, other UVC wavelengths have emerged as attractive alternatives but a direct comparison of these tools is lacking with the inherent mechanistic properties unclear. Our results using human coronaviruses, hCoV-229E and hCoV-OC43, have indicated that 277-nm UVC LED is most effective in viral inactivation, followed by 222-nm far UVC and 254-nm UVC mercury lamp. While UVC mercury lamp is more effective in degrading viral genomic content compared to 277-nm UVC LED, the latter results in a pronounced photo-degradation of spike proteins which potentially contributed to the higher efficacy of coronavirus inactivation. Hence, inactivation of coronaviruses by 277-nm UVC LED irradiation constitutes a more promising method for disinfection.","version":"1.1","doi":"10.1101/2021.05.31.446403","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.31.446476","pub_date":"2021-5-31","title":"COVID-19 Mortality is Associated with Impaired Innate Immunity in Pre-existing Health Conditions","abstract":"COVID-19 poses a life-threatening endangerment to individuals with chronic diseases. However, not all comorbidities affect COVID-19 prognosis equally. Some increase the risk of COVID-19 related death by more than six folds while others show little to no impact. To prevent severe outcomes, it is critical that we comprehend pre-existing molecular abnormalities in common health conditions that predispose patients to poor prognoses. In this study, we aim to discover some of these molecular risk factors by associating gene expression dysregulations in common health conditions with COVID-19 mortality rates in different cohorts. We focused on fourteen pre-existing health conditions, for which age-and-sex-adjusted hazard ratios of COVID-19 mortality have been documented. For each health condition, we analyzed existing transcriptomics data to identify differentially expressed genes (DEGs) between affected individuals and unaffected individuals. We then tested if fold changes of any DEG in these pre-existing conditions were correlated with hazard ratios of COVID-19 mortality to discover molecular risk factors. We performed gene set enrichment analysis to identify functional groups overrepresented in these risk factor genes and examined their relationships with the COVID-19 disease pathway. We found that upregulated expression of 70 genes and downregulated expression of 181 genes in pre-existing health conditions were correlated with increased risk of COVID-19 related death. These genes were significantly enriched with endoplasmic reticulum (ER) function, proinflammatory reaction, and interferon production that participate in viral transcription and immune responses to viral infections. Impaired innate immunity in pre-existing health conditions are associated with increased hazard of COVID-19 mortality. The discovered molecular risk factors are potential prognostic biomarkers and targets for therapeutic interventions.","version":"1.1","doi":"10.1101/2021.05.31.446476","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.29.443900","pub_date":"2021-5-30","title":"Broadening a SARS-CoV-1 neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution","abstract":"The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.","version":"1.1","doi":"10.1101/2021.05.29.443900","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446250","pub_date":"2021-5-30","title":"Computational genomic analysis of the lung tissue microenvironment in COVID-19 patients","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus has affected over 170 million people, and caused over 3.5 million deaths throughout the world as of May 2021. Although over 150 million people around the world have recovered from this disease, the long term effects of the disease are still under study. A year after the start of the pandemic, data from COVID-19 recovered patients shows multiple organs affected with a broad spectrum of manifestations. Long term effects of SARS-CoV-2 infection includes fatigue, chest pain, cellular damage, and robust innate immune response with inflammatory cytokine production. More clinical studies and clinical trials are needed to not only document, but also to understand and determine the factors that predispose certain people to the long term side effects of his infection. In this manuscript, our goal was to explore the multidimensional landscape of infected lung tissue microenvironment to better understand complex interactions between SARS-CoV-2 viral infection, immune response and the lungs microbiome of COVID-19 patients. Each sample was analyzed with several machine learning tools allowing simultaneous detection and quantification of viral RNA amount at genome and gene level; human gene expression and fractions of major types of immune cells, as well as metagenomic analysis of bacterial and viral abundance. To contrast and compare specific viral response to SARS-COV-2 we have analyzed deep sequencing data from additional cohort of patients infected with NL63 strain of corona virus. Our correlation analysis of three types of measurements in patients i.e. fraction of viral RNA (at genome and gene level), Human RNA (transcripts and gene level) and bacterial RNA (metagenomic analysis), showed significant correlation between viral load as well as level of specific viral gene expression with the fractions of immune cells present in lung lavage as well as with abundance of major fractions of lung microbiome in COVID-19 patients. Our exploratory study has provided novel insights into complex regulatory signaling interactions and correlative patterns between the viral infection, inhibition of innate and adaptive immune response as well as microbiome landscape of the lung tissue. These initial findings could provide better understanding of the diverse dynamics of immune response and the side effects of the SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.05.28.446250","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.21257936","pub_date":"2021-05-30","title":"Inference of SARS-CoV-2 generation times using UK household data","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The distribution of the generation time (the interval between individuals becoming infected and passing on the virus) characterises changes in the transmission risk during SARS-CoV-2 infections. Inferring the generation time distribution is essential to plan and assess public health measures. We previously developed a mechanistic approach for estimating the generation time, which provided an improved fit to SARS-CoV-2 data from January-March 2020 compared to existing models. However, few estimates of the generation time exist based on data from later in the pandemic. Here, using data from a household study conducted from March-November 2020 in the UK, we provide updated estimates of the generation time. We consider both a commonly used approach in which the transmission risk is assumed to be independent of when symptoms develop, and our mechanistic model in which transmission and symptoms are linked explicitly. Assuming independent transmission and symptoms, we estimated a mean generation time (4.2 days, 95% CrI 3.3-5.3 days) similar to previous estimates from other countries, but with a higher standard deviation (4.9 days, 3.0-8.3 days). Using our mechanistic approach, we estimated a longer mean generation time (6.0 days, 5.2-7.0 days) and a similar standard deviation (4.9 days, 4.0-6.3 days). Both models suggest a shorter mean generation time in September-November 2020 compared to earlier months. Since the SARS-CoV-2 generation time appears to be changing, continued data collection and analysis is necessary to inform future public health policy decisions.</jats:p>","version":null,"doi":"10.1101/2021.05.27.21257936","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.29.446300","pub_date":"2021-5-30","title":"Antibody Display of cell surface receptor Tetraspanin12 and SARS-CoV-2 spike protein","abstract":"In previous work, Hsieh and Higgins presented a novel structure of antibodies identified from malaria-exposed individuals, in which the extracellular immunoglobulin (Ig)-like domain of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) is presented on the third complementarity determining regions (CDR3) of the Ig heavy chain. Here we develop an Antibody Display technology based on this LAIR1-containing antibody, by grafting proteins of interest (POI) onto the heavy chain CDR3 while retaining the biological properties of the POI. As a proof of principle, we displayed the second extracellular domain of Tetraspanin12 (Tspan12EC2) and the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the heavy chain CDR3. Our data revealed that Antibody Display Tspan12EC2 bound to Norrie Disease Protein (Norrin) and Antibody Display SARS-CoV-2 RBD bound to angiotensin-converting enzyme 2 (ACE2) and neutralizing nanobodies. Collectively, Antibody Display technology offers the general strategy of designing novel antibodies by grafting POI onto the CDR3.","version":"1.1","doi":"10.1101/2021.05.29.446300","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.446089","pub_date":"2021-5-29","title":"SARS-CoV-2 spike glycoprotein-reactive T cells can be readily expanded from COVID-19 vaccinated donors","abstract":"The COVID-19 vaccine was designed to provide protection against infection by the severe respiratory coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19). However, the vaccine\u2019s efficacy can be compromised in patients with immunodeficiencies or the vaccine-induced immunoprotection suppressed by other comorbidity treatments, such as chemotherapy or immunotherapy. To enhance the protective role of the COVID-19 vaccine, we have investigated a combination of the COVID-19 vaccination with ex vivo enrichment and large-scale expansion of SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T cells. SARS-CoV-2-unexposed donors were vaccinated with two doses of the BNT162b2 SARS-CoV-2 vaccine. The peripheral blood mononuclear cells of the vaccinated donors were cell culture-enriched with T cells reactive to peptides derived from SARS-CoV-2 spike glycoprotein. The enriched cell cultures were large-scale expanded using the rapid expansion protocol (REP) and the peptide-reactive T cells evaluated. We show that vaccination with the SARS-CoV-2 spike glycoprotein-based mRNA COVID-19 vaccine induced humoral response against SARS-CoV-2 spike glycoprotein in all tested healthy SARS-CoV-2-unexposed donors. This humoral response was found to correlate with the ability of the donors\u2019 PBMCs to become enriched with SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T cells. Using an 11-day rapid expansion protocol, the enriched cell cultures were expanded nearly a thousand fold, and the proportions of the SARS-CoV-2 spike glycoprotein-reactive T cells increased. These findings show for the first time that the combination of the COVID-19 vaccination and ex vivo T cell large-scale expansion of SARS-CoV-2-reactive T cells could be a powerful tool for developing T cell-based adoptive cellular immunotherapy of COVID-19.","version":"1.1","doi":"10.1101/2021.05.27.446089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.21257860","pub_date":"2021-05-29","title":"Cellular and humoral immunogenicity of a SARS-CoV-2 mRNA vaccine in patients on hemodialysis","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Patients with chronic renal insufficiency on intermittent hemodialysis face an increased risk of COVID-19 induced mortality and impaired vaccine responses. To date, only few studies addressed SARS-CoV-2 vaccine elicited immunity in this immunocompromised population.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We assessed immunogenicity of the mRNA vaccine BNT162b2 in at risk dialysis patients and characterized systemic cellular and humoral immune responses in serum and saliva using interferon \u03b3 release assay and multiplex-based cytokine and immunoglobulin measurements. We further compared binding capacity and neutralization efficacy of vaccination-induced immunoglobulins against emerging SARS-CoV-2 variants of concern B.1.1.7, B.1.351, B.1.429 and Cluster 5 by ACE2-RBD competition assay.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Patients on intermittent hemodialysis exhibit detectable but variable cellular and humoral immune responses against SARS-CoV-2 and variants of concern after a two-dose regimen of BNT162b2. Although vaccination-induced immunoglobulins were detectable in saliva and plasma, both anti-SARS-CoV-2 IgG and neutralization efficacy was reduced compared to controls. Similarly, T-cell mediated interferon \u03b3 release after stimulation with SARS-CoV-2 spike peptides was significantly diminished.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Quantifiable humoral and cellular immune responses after BNT162b2 vaccination in individuals on intermittent dialysis are encouraging, but urge for longitudinal follow-up to assess longevity of immunity. Diminished virus neutralization and interferon \u03b3 responses in face of emerging variants of concern may favor this at risk population for re-vaccination using modified vaccines at the earliest opportunity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Initiative and Networking Fund of the Helmholtz Association of German Research Centers, EU Horizon 2020 research and innovation program, State Ministry of Baden-W\u00fcrttemberg for Economic Affairs, Labor and Tourism.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in the context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>Patients on dialysis tend to have a reduced immune response to both infection and vaccination. We searched PubMed and MedRxiv for studies including search terms such as \u201cCOVID-19\u201d, \u201cvaccine\u201d, and \u201cdialysis\u201d but no peer-reviewed studies to date assessed both SARS-CoV-2 specific B- and T-cell responses, mucosal immunoglobulins, and considered the impact of SARS-CoV-2 variants of concern in this at risk population.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of the study</jats:title>\n                    <jats:p>We provide a comprehensive functional characterization of both T- and B-cell responses following a two-dose regimen of BNT162b2 in at risk patients on maintenance hemodialysis. More importantly, to the best of our knowledge, we assess for the first time binding and neutralization capacity of vaccination-induced circulation and mucosal antibodies towards emerging SARS-CoV-2 variants of concern in an immunocompromised population.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>Patients on maintenance hemodialysis develop a substantial cellular and humoral immune response following the BNT162b2 vaccine. These findings should encourage patients on intermittent hemodialysis to receive the vaccine. However, we suggest continuing additional protection measures against variants of concern in this at risk population until longevity of the vaccine response is fully evaluated.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.05.26.21257860","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.28.446200","pub_date":"2021-5-29","title":"A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories","abstract":"Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options of in vivo models are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice. In vitro studies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.","version":"1.1","doi":"10.1101/2021.05.28.446200","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.446014","pub_date":"2021-5-28","title":"The legacy of maternal SARS-CoV-2 infection on the immunology of the neonate","abstract":"Despite extensive and ongoing studies of SARS-CoV-2 and evidence that pregnant women are at increased risk of severe COVID-19, the effect of maternal infection on the developing infant remains unclear. To determine the potential impact of exposure to SARS-CoV-2 in utero on the neonate, we have assessed the immunological status of infants born to mothers with confirmed SARS-CoV-2 infection during gestation. No evidence of vertical transmission of SARS-CoV-2 was observed, but transfer of maternal SARS-CoV-2 specific IgG to infants was apparent, although to a lesser extent in cases of active or recent maternal infection. Infants born to mothers with recent/ongoing infection had elevated circulating pro-inflammatory cytokines and enhanced percentages of innate immune cells compared to that seen in infants born to uninfected mothers. In tandem, higher frequencies of FOXP3+ regulatory T cells and circulating IL-10 demonstrated a further nuance to the neonatal effector response. Interestingly, cytokine functionality was enhanced in infants born to mothers exposed to SARS-CoV-2 at any time during pregnancy. This indicates that maternal SARS-CoV-2 infection influences in utero priming of the fetal immune system.","version":"1.1","doi":"10.1101/2021.05.27.446014","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.445991","pub_date":"2021-5-28","title":"Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 and Spike Protein Subunit 1","abstract":"A hallmark of COVID-19 is a hyperinflammatory state that is associated with severity. Various anti-inflammatory therapeutics have shown mixed efficacy in treating COVID-19, and the mechanisms by which hyperinflammation occurs are not well understood. Previous research indicated that monocytes, a key innate immune cell, undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response in monocytes. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism that was associated with production of pro-inflammatory cytokines including interleukin-6 and tumor necrosis factor-\u03b1. This response was dependent on hypoxia-inducible factor-1\u03b1, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production in monocytes, and abrogated glycolytic and mitochondrial metabolism. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments in monocytes. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.","version":"1.1","doi":"10.1101/2021.05.27.445991","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446020","pub_date":"2021-5-28","title":"Evaluating the risk of SARS-CoV-2 transmission to bats using a decision analytical framework","abstract":"Preventing wildlife disease outbreaks is a priority issue for natural resource agencies, and management decisions can be urgent, especially in epidemic circumstances. With the emergence of SARS-CoV-2, wildlife agencies were concerned whether the activities they authorize might increase the risk of viral transmission from humans to North American bats but had a limited amount of time in which to make decisions. We provide a description of how decision analysis provides a powerful framework to analyze and re-analyze complex natural resource management problems as knowledge evolves. Coupled with expert judgment and avenues for the rapid release of information, risk assessment can provide timely scientific information for evolving decisions. In April 2020, the first rapid risk assessment was conducted to evaluate the risk of transmission of SARS-CoV-2 from humans to North American bats. Based on the best available information, and relying heavily on formal expert judgment, the risk assessment found a small possibility of transmission during summer work activities. Following that assessment, additional knowledge and data emerged, such as bat viral challenge studies, that further elucidated the risks of human-to-bat transmission and culminated in a second risk assessment in the fall of 2020. We update the first SARS-CoV-2 risk assessment with new estimates of little brown bat (Myotis lucifugus) susceptibility and new management alternatives, using findings from the prior two risk assessments and other empirical studies. We highlight the strengths of decision analysis and expert judgment not only to frame decisions and produce useful science in a timely manner, but also to serve as a framework to reassess risk as understanding improves. For SARS-CoV-2 risk, new knowledge led to an 88% decrease in the median number of bats estimated to be infected per 1000 encountered when compared to earlier results. The use of facemasks during, or a negative COVID-19 test prior to, bat encounters further reduced those risks. Using a combination of decision analysis, expert judgment, rapid risk assessment, and efficient modes of information distribution, we provide timely science support to decision makers for summer bat work in North America.","version":"1.1","doi":"10.1101/2021.05.28.446020","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446149","pub_date":"2021-5-28","title":"Decomposition of the SARS-CoV-2-ACE2 interface reveals a common trend among emerging viral variants","abstract":"New viral variants of the SARS-CoV-2 virus show enhanced infectivity compared to wild type, resulting in an altered pandemic situation in affected areas. These variants are the B.1.1.7 (United Kingdom), B.1.1.7 with the additional E484K mutation, the B.1.351 variant (South Africa) and the P.1 variant (Brazil). Understanding the binding modalities between these viral variants and the host cell receptor ACE2 allows depicting changes, but also common motifs of virus-host cell interaction. The trimeric spike protein expressed at the viral surface contains the receptor-binding domain (RBD) that forms the molecular interface with ACE2. All the above-mentioned variants carry between one and three amino acid exchanges within the interface-forming region of the RBD, thereby altering the binding interface with ACE2. Using molecular dynamics simulations and decomposition of the interaction energies between the RBD and ACE2, we identified phenylalanine 486, glutamine 498, threonine 500 and tyrosine 505 as important interface-forming residues across viral variants. We also suggest a reduced binding energy between RBD and ACE2 in viral variants with higher infectivity, attributed to residue-specific differences in electrostatic interaction energy. Importantly, individual amino acid exchanges not only influence the affected position, but also alter the conformation of surrounding residues and affect their interaction potential as well. We demonstrate how computational methods can help to identify changed as well as common motifs across viral variants. These identified motifs might play a crucial role, in the strategical development of therapeutic interventions against the fast mutating SARS-CoV-2 virus. The COVID-19 pandemic caused by the SARS-CoV-2 virus has significantly changed our lives. To date, there is a lack of neutralizing drugs that specifically target SARS-CoV-2. Hope lies in newly developed vaccines that effectively prevent severe cases of acute respiratory syndrome. However, emerging viral variants escape vaccine-induced immune-protection. Therefore, identification of appropriate molecular targets across viral variants is important for the development of second- and third-generation vaccines and inhibitory antibodies. In this study, we identify residues across viral variants that are important for viral binding to the host cell. As such residues cannot be replaced without diminishing infectivity of the virus, these residues represent primary targets for intervention, for example by neutralizing antibodies.","version":"1.1","doi":"10.1101/2021.05.28.446149","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446159","pub_date":"2021-5-28","title":"SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia","abstract":"The beta-coronavirus SARS-CoV-2 is at the origin of a persistent worldwide pandemic. SARS-CoV-2 infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract eventually causing acute severe respiratory syndrome with a high degree of mortality in the elderly. Here we use reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2. We show that in bronchial epithelia derived from adult donors, infections initiate in multi-ciliated cells. Then, infection rapidly spread within 24-48h throughout the whole epithelia. Within 3-4 days, large apical syncytia form between multi-ciliated cells and basal cells, which dissipate into the apical lumen. We show that these syncytia are a significant source of the released infectious dose. In stark contrast to these findings, bronchial epithelia reconstituted from children donors are intrinsically more resistant to virus infection and show active restriction of virus spread. This restriction is paired with accelerated release of IFN compared to adult donors. Taken together our findings reveal apical syncytia formation as an underappreciated source of infectious virus for either local dissemination or release into the environment. Furthermore, we provide direct evidence that children bronchial epithelia are more resistant to infection with SARS-CoV-2 providing experimental support for epidemiological observations that SARS-CoV-2 cases\u2019 fatality is linked to age. Bronchial epithelia are the primary target for SARS-CoV-2 infections. Our work uses reconstituted bronchial epithelia from adults and children. We show that infection of adult epithelia with SARS-CoV-2 is rapid and results in the synchronized release of large clusters of infected cells and syncytia into the apical lumen contributing to the released infectious virus dose. Infection of children derived bronchial epithelia revealed an intrinsic resistance to infection and virus spread, probably as a result of a faster onset of interferon secretion. Thus, our data provide direct evidence for the epidemiological observation that children are less susceptible to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.28.446159","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446163","pub_date":"2021-5-28","title":"The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin","abstract":"The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence 681PRRAR/S686. This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.","version":"1.1","doi":"10.1101/2021.05.28.446163","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446137","pub_date":"2021-5-28","title":"Mutational hotspot in the SARS-CoV-2 Spike protein N-terminal domain conferring immune escape potential","abstract":"Global efforts are being taken to monitor the evolution of SARS-CoV-2, aiming at early identification of mutations with the potential of increasing viral infectivity or virulence. We report a striking increase in the frequency of recruitment of diverse substitutions at a critical residue (W152), positioned in the N-terminal domain (NTD) of the Spike protein, observed repeatedly across independent phylogenetic and geographical contexts. We investigate the impact these mutations might have on the evasion of neutralizing antibodies. Finally, we uncover that NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path on which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape.","version":"1.1","doi":"10.1101/2021.05.28.446137","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446155","pub_date":"2021-5-28","title":"Digital PCR to quantify ChAdOx1 nCoV-19 copies in blood and tissues","abstract":"Vaccination with the adenoviral-vector based Astra Zeneca ChAdOx1 nCov-19 vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR is an excellent tool to quantify transgene copies in vivo. Here we present a highly sensitive digital PCR for in-situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human serum 24, 72 and 168 hours post vaccination, and in a variety of murine tissues in an experimental vaccination model 30 minutes post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.","version":"1.1","doi":"10.1101/2021.05.28.446155","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.28.446204","pub_date":"2021-5-28","title":"The N-terminal and central domains of CoV-2 nsp1 play key functional roles in suppression of cellular gene expression and preservation of viral gene expression","abstract":"Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus.","version":"1.1","doi":"10.1101/2021.05.28.446204","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.445500","pub_date":"2021-5-28","title":"Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research","abstract":"Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Unlike many other candidates being investigated, tizoxanide (the active metabolite of nitazoxanide) plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbeccos Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Therefore, a robustly validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6ng/mL to 1000ng/mL. Accuracy and precision ranged between 102.2% and 113.5%, 100.1% and 105.4%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.27.445500","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445356","pub_date":"2021-5-28","title":"Reprogrammed tracrRNAs enable repurposing RNAs as crRNAs and detecting RNAs","abstract":"In type II CRISPR systems, the guide RNA (gRNA) consists of a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA) which interacts directly with Cas9 and is essential to its guided DNA targeting function. Though tracrRNAs are diverse in sequences and structures across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for particular Cas9 has not been studied adequately. Here, we revealed the high programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9. By reprogramming the crRNA-tracrRNA hybridized sequence, reprogrammed tracrRNAs can repurpose various RNAs as crRNAs to trigger CRISPR function. We showed that the engineered crRNA-tracrRNA pairs enable design of orthogonal cellular computing devices and hijacking of endogenous RNAs as crRNAs. We next designed novel RNA sensors that can monitor the transcriptional activity of specific genes on the host genome and detect SARS-CoV-2 RNA in vitro. The engineering potential of crRNA-tracrRNA interaction has therefore redefined the capabilities of CRISPR/Cas9 system.","version":"1.2","doi":"10.1101/2021.05.24.445356","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.23.445305","pub_date":"2021-5-27","title":"Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19","abstract":"Diagnostics has played a significant role in effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays. Thus far, limited knowledge exists about the antigenic properties of the N protein. In this paper, we demonstrate the significant impact of dimerization of SARS-CoV-2 nucleocapsid protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics of COVID-19. The expressed purified protein from E.coli consists of two forms, dimeric and monomeric forms, which have been further characterized by biophysical and immunological means. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form of the protein. These findings have also been confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. It is evident that use of the dimeric form will increase the sensitivity of the current nucleocapsid dependent ELISA for rapid COVID-19 diagnostic. Further, the results indicate that monitoring and maintaining of the monomerdimer composition is critical for accurate and robust diagnostics.","version":"1.2","doi":"10.1101/2021.05.23.445305","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.445985","pub_date":"2021-5-27","title":"SARS-CoV-2 inactivation by human defensin HNP1 and retrocyclin RC-101","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 respiratory disease pandemic. While induction of adaptive antiviral immunity via vaccination holds promise for combatting the pandemic, the emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that are active against the virus. One component of the innate immune system with broad anti-pathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The defensins\u2019 ability to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of thermodynamically pliable proteins. Accordingly, we found that human neutrophil a-defensin HNP1 and retrocyclin RC-101 destabilize SARS-CoV-2 Spike protein and interfere with Spike-mediated membrane fusion and SARS-CoV-2 infection in cell culture. We show that HNP1 binds to Spike with submicromolar affinity. Although binding of HNP1 to serum albumin is more than 20-fold weaker, serum reduces the anti-SARS-CoV-2 activity of HNP1. At high concentrations of HNP1, its ability to inactivate the virus was preserved even in the presence of serum. These results suggest that specific a- and 8-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.","version":"1.1","doi":"10.1101/2021.05.27.445985","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427830","pub_date":"2021-5-27","title":"Exploring the natural origins of SARS-CoV-2 in the light of recombination","abstract":"The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (Sarbecoviruses) have been identified, is fuelling speculation on the natural origins of SARS-CoV-2. We have comprehensively analysed phylogenetic relations between SARS-CoV-2, and the related bat and pangolin Sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Clustering of the inferred recombination events is non-random with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift in the ancestry of bat Sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm horseshoe bats, Rhinolophus, are the likely SARS-CoV-2 progenitor reservoir species. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and co-circulation of these viruses\u2019 ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years or so. We confirm that a direct proximal ancestor to SARS-CoV-2 is yet to be sampled, since the closest relative shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for more wildlife sampling to (i) pinpoint the exact origins of SARS-CoV-2\u2019s animal progenitor, and (ii) survey the extent of the diversity in the related Sarbecoviruses\u2019 phylogeny that present high risk for future spillover. The origin of SARS-CoV-2 can be traced to horseshoe bats, genus Rhinolophus, with ranges in both China and Southeast Asia. The closest known relatives of SARS-CoV-2 exhibit frequent transmission among their Rhinolophus host species. Sarbecoviruses have undergone extensive recombination throughout their evolutionary history. Accounting for the mosaic patterns of these recombinants is important when inferring relatedness to SARS-CoV-2. Breakpoint patterns are consistent with recombination hotspots in the coronavirus genome, particularly upstream of the pike open reading frame with a coldspot in S1.","version":"1.3","doi":"10.1101/2021.01.22.427830","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.445918","pub_date":"2021-5-27","title":"An altered metabolism in leukocytes showing in vitro igG memory from SARS-CoV-2-infected patients","abstract":"Coronavirus disease 2019 (COVID 19) is a systemic infection that exerts a significant impact on cell metabolism. In this study we performed metabolomic profiling coupled with multivariate statistics analysis obtained from 43 in vitro cultures of peripheral blood mononuclear cells (PBMC), 19 of which displaying IgG memory for spike-S1 antigen 60-90 days after infection. By using mass spectrometry analysis, a significant up regulation of S-adenosyl-Homocysteine, Sarcosine and Arginine was found in leukocytes showing IgG memory. These metabolites are known to be involved in physiological recovering from viral infections and immune activities, and our findings might represent a novel and easy measure that could be of help in understanding SARS-Cov-2 effects on leukocytes.","version":"1.1","doi":"10.1101/2021.05.27.445918","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445185","pub_date":"2021-5-27","title":"Single-Molecule Dynamics of SARS-CoV-2 5\u2019 Cap Recognition by Human eIF4F","abstract":"Coronaviruses initiate translation through recognition of the viral RNA 5\u2019 m7GpppAm cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5\u2019-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E\u2013cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5\u2019 UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E\u2013RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E\u2013cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F\u2013cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.","version":"1.1","doi":"10.1101/2021.05.26.445185","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445838","pub_date":"2021-5-27","title":"Reduced sensitivity of infectious SARS-CoV-2 variant B.1.617.2 to monoclonal antibodies and sera from convalescent and vaccinated individuals","abstract":"The SARS-CoV-2 B.1.617 lineage emerged in October 2020 in India. It has since then become dominant in some indian regions and further spread to many countries. The lineage includes three main subtypes (B1.617.1, B.1617.2 and B.1.617.3), which harbour diverse Spike mutations in the N-terminal domain (NTD) and the receptor binding domain (RBD) which may increase their immune evasion potential. B.1.617.2 is believed to spread faster than the other versions. Here, we isolated infectious B.1.617.2 from a traveller returning from India. We examined its sensitivity to monoclonal antibodies (mAbs) and to antibodies present in sera from COVID-19 convalescent individuals or vaccine recipients, in comparison to other viral lineages. B.1.617.2 was resistant to neutralization by some anti-NTD and anti-RBD mAbs, including Bamlanivimab, which were impaired in binding to the B.1.617.2 Spike. Sera from convalescent patients collected up to 12 months post symptoms and from Pfizer Comirnaty vaccine recipients were 3 to 6 fold less potent against B.1.617.2, relative to B.1.1.7. Sera from individuals having received one dose of AstraZeneca Vaxzevria barely inhibited B.1.617.2. Thus, B.1.617.2 spread is associated with an escape to antibodies targeting non-RBD and RBD Spike epitopes.","version":"1.1","doi":"10.1101/2021.05.26.445838","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.27.445958","pub_date":"2021-5-27","title":"A new method to study genome mutations using the information entropy","abstract":"We report a non-clinical, mathematical method of studying genetic sequences based on the information theory. Our method involves calculating the information entropy spectrum of genomes by splitting them into \u201cwindows\u201d containing a fixed number of nucleotides. The information entropy value of each window is computed using the m-block information entropy formula. We show that the information entropy spectrum of genomes contains sufficient information to allow detection of genetic mutations, as well as possibly predicting future ones. Our study indicates that the best m-block size is 2 and the optimal window size should contain more than 9, and less than 33 nucleotides. In order to implement the proposed technique, we created specialized software, which is freely available. Here we report the successful test of this method on the reference RNA sequence of the SARS-CoV-2 virus collected in Wuhan, Dec. 2019 (MN908947) and one of its randomly selected variants from Taiwan, Feb. 2020 (MT370518), displaying 7 mutations.","version":"1.1","doi":"10.1101/2021.05.27.445958","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.215251","pub_date":"2021-5-27","title":"Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples","abstract":"Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. We developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative molecular diagnosis with the simplicity of point-of-care testing. This handheld device employs a single reaction-pot for amplification and a mini-camera for detection. Competitive features are the rapid analysis (<30min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (<5copies/reaction), smartphone-operation and fast prototyping (3D-printing). The device\u2019s clinical utility is demonstrated in cancer-mutations and COVID-19 testing. Excellent performance includes: detection of 0.01% of BRAF-V600E-to-wild-type molecules; 97% sensitivity to SARS-CoV-2 RNA detection (89 samples); 83% (Ct<34), 98% (Ct<30) and 100% (Ct<25) to 163 nasopharyngeal-swabs; 100% specificity in all cases. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.","version":"1.2","doi":"10.1101/2020.07.22.215251","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.25.445557","pub_date":"2021-5-26","title":"An Issue of Concern: Unique Truncated ORF8 Protein Variants of SARS-CoV-2","abstract":"Open reading frame 8 (ORF8) protein is one of the most evolving accessory proteins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits presentation of viral antigens by the major histocompatibility complex class I (MHC-I) and interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 to evade immunity and replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein defines the B.1.1.7 lineage of SARS-CoV-2, which is engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) due to the Q27STOP mutations were identified among 49055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents which includes Africa, Asia, Europe and South America. Based on various quantitative features such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, a collection of nine possible T-ORF8 unique variants were defined. The question of whether T-ORF8 variants work similarly to ORF8 has yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.","version":"1.2","doi":"10.1101/2021.05.25.445557","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255810","pub_date":"2021-5-26","title":"Infection and transmission of SARS-CoV-2 depends on heparan sulfate proteoglycans","abstract":"The current pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and outbreaks of new variants highlight the need for preventive treatments. Here we identified heparan sulfate proteoglycans as attachment receptors for SARS-CoV-2. Notably, neutralizing antibodies against SARS-CoV-2 isolated from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS-CoV-2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS-CoV-2, both DC subsets efficiently captured SARS-CoV-2 via heparan sulfate proteoglycans, and transmitted the virus to ACE2-positive cells. Moreover, human primary nasal cells were infected by SARS-CoV-2 and infection was blocked by pre-treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.08.18.255810","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445878","pub_date":"2021-5-26","title":"Effective prophylaxis of COVID-19 in rhesus macaques using a combination of two parentally-administered SARS-CoV-2 neutralizing antibodies","abstract":"SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.","version":"1.1","doi":"10.1101/2021.05.26.445878","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445787","pub_date":"2021-5-26","title":"SARS-CoV-2 Nsp14 activates NF-\u03baB signaling and induces IL-8 upregulation","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-\u03baB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-\u03baB signaling. Nsp14 caused the nuclear translocation of NF-\u03baB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5\u2019-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-\u03baB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-\u03baB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-\u03baB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-\u03baB.","version":"1.1","doi":"10.1101/2021.05.26.445787","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445880","pub_date":"2021-5-26","title":"Evidence of neutralizing antibodies against SARS-CoV-2 in domestic cats living with owners with a history of COVID-19 in Lima \u2013 Peru","abstract":"SARS-CoV-2 can infect a variety of wild and domestic animals worldwide. Of these, domestic cats are highly susceptible species and potential viral reservoirs. As such, it is important to investigate disease exposure in areas with active community transmission and high disease prevalence. In this report we demonstrate the presence of serum neutralizing antibodies against the receptor binding-domain (RBD) of the SARS-CoV-2 in cats whose owners had been infected with SARS-CoV-2 in Lima, Peru, using a commercial competitive ELISA SARS-CoV-2 Surrogate Virus Neutralization Test. Out of 41 samples, 17.1% (7/41) and 31.7% (13/41) were positive, using the cut-off inhibition value of 30% and 20%, respectively. Not all cats living in a single house had detectable neutralizing antibodies showing that heterogenous exposure and immune among cohabiting animals. This is the first report of SARS-COV-2 exposure of domestic cats in Lima, Peru. Further studies are required to ascertain the prevalence of SARS-COV-2 exposure among domestic cats of Lima, Peru.","version":"1.1","doi":"10.1101/2021.05.26.445880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445769","pub_date":"2021-5-26","title":"Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques","abstract":"SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure. SARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.","version":"1.1","doi":"10.1101/2021.05.26.445769","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445843","pub_date":"2021-5-26","title":"Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo","abstract":"Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of endotoxin, which correlated directly with their abilities to induce cytokine production. The lipopolysaccharide (LPS) inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.","version":"1.1","doi":"10.1101/2021.05.26.445843","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445422","pub_date":"2021-5-26","title":"A modular molecular framework for quickly estimating the binding affinity of the spike protein of SARS-CoV-2 variants for ACE2, in presence of mutations at the spike receptor binding domain","abstract":"The rapid spread of new SARS-CoV-2 variants needs the development of rapid tools for predicting the affinity of the mutated proteins responsible for the infection, i.e., the SARS-CoV-2 spike protein, for the human ACE2 receptor, aiming to understand if a variant can be more efficient in invading host cells. Here we show how our computational pipeline, previously used for studying SARS-CoV-2 spike receptor binding domain (RBD)/ACE2 interactions and pre-/post-fusion conformational changes, can be used for predicting binding affinities of the human ACE2 receptor for the spike protein RBD of the characterized infectious variants of concern/interest B.1.1.7-UK (carrying the mutations N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417N/T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 variant (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Furthermore, we searched for ACE2 structurally related proteins that might be involved in interactions with the SARS-CoV-2 spike protein, in those tissues showing low ACE2 expression, revealing two new proteins, THOP1 and NLN, deserving to be investigated for their possible inclusion in the group of host-cell entry factors responsible for host-cell SARS-CoV-2 invasion and immunity response.","version":"1.1","doi":"10.1101/2021.05.26.445422","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434834","pub_date":"2021-5-26","title":"REGEN-COV protects against viral escape in preclinical and human studies","abstract":"Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of minor virus variants in SARS-COV-2 isolates found in COVID-19 patients or identified from preclinical in vitro and in vivo studies. This study demonstrates that a combination of non-competing antibodies, REGEN-COV, not only provides full coverage against current variants of concern/interest but also protects against emergence of new such variants and their potential seeding into the population in a clinical setting.","version":"1.4","doi":"10.1101/2021.03.10.434834","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.26.445809","pub_date":"2021-5-26","title":"Impaired function and delayed regeneration of dendritic cells in COVID-19","abstract":"Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DC) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute disease to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage- HLADR+ cells lacking DC markers. Increased frequency of the recently discovered CD163+ CD14+ DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of PD-L1 in conventional DC (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate na\u00efve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.","version":"1.1","doi":"10.1101/2021.05.26.445809","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445532","pub_date":"2021-5-25","title":"Infection of brain pericytes underlying neuropathology of COVID-19 patients","abstract":"A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69,5 years; and n = 7 control, median age = 68 years), and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in peri-vascular cells. Viral particles were identified in the vascular wall and paralleled by peri-vascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from an additional 16 individuals (n = 8 COVID-19, median age = 67 years; and n = 8 control, median age = 69,5 years) exhibited significantly lower levels of the pericyte marker PDGFR\u03b2 in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to peri-vascular inflammation and a locally compromised blood-brain barrier.","version":"1.1","doi":"10.1101/2021.05.24.445532","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445534","pub_date":"2021-5-25","title":"SARS-CoV-2 convergent evolution as a guide to explore adaptive advantage","abstract":"Much can be learned from 1.2 million sequences of SARS-CoV-2 generated during the last 15 months. Out of the overwhelming number of mutations sampled so far, only few rose to prominence in the viral population. Many of these emerged recently and independently in multiple lineages. Such a textbook example of convergent evolution at the molecular level is not only curiosity but a guide to uncover the basis for adaptive advantage behind these events. Focusing on the extent of the convergent evolution in the spike (S) protein, our report confirms that the most concerning SARS-CoV-2 lineages carry the heaviest burden of convergent S-protein mutations, suggesting their fundamental adaptive advantage. The great majority (21/25) of S-protein sites under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. We further show that among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favored. While the probed mutation space in the S protein covered all amino-acids reachable by single nucleotide changes, substitutions requiring two nucleotide changes or epistatic mutations of multiple-residues have only recently started to emerge. Unfortunately, despite their convergent emergence and physical association, most of these adaptive mutations and their combinations remain understudied. We aim to promote research of current variants which are currently understudied but may become important in the future.","version":"1.1","doi":"10.1101/2021.05.24.445534","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439891","pub_date":"2021-5-25","title":"An mRNA SARS-CoV-2 vaccine employing Charge-Altering Releasable Transporters with a TLR-9 agonist induces neutralizing antibodies and T cell memory","abstract":"The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.","version":"1.2","doi":"10.1101/2021.04.14.439891","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.13.295493","pub_date":"2021-5-25","title":"Genes with 5\u2032 terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein","abstract":"Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes are preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5\u2032 terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5\u2019 UTRs from TOP transcripts can drive preferential expression in the presence of NSP1. Finally, we found that LARP1, a key effector protein in the mTOR pathway may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.","version":"1.3","doi":"10.1101/2020.09.13.295493","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444362","pub_date":"2021-5-25","title":"SARS-like coronaviruses in horseshoe bats (Rhinolophus spp.) in Russia, 2020","abstract":"We found and genetically described two novel SARS-like coronaviruses in feces and oral swabs of the great (R. ferrumequinum) and the lesser (R. hipposideros) horseshoe bats in southern region of Russia. The viruses, named Khosta-1 and Khosta-2, together with related viruses from Bulgaria and Kenya, form a separate phylogenetic lineage. We found an evidence of recombination events in evolutionary history of Khosta-1, which involved the acquisition of structural proteins S, E, and M as well as nonstructural genes ORF3, ORF6, ORF7a, and ORF7b from a virus that is closely related to Kenyan isolate BtKY72. Examination of bats by RT-PCR revealed that 62,5% of great horseshoe bats in one of the caves were positive for Khosta-1 virus while its overall prevalence was 14%. The prevalence of Khosta-2 was 1,75%. Our results show that SARS-like coronaviruses circulate in horseshoe bats in the region and provide a new data on their genetic diversity.","version":"1.2","doi":"10.1101/2021.05.17.444362","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142570","pub_date":"2021-5-24","title":"Comparative analysis of non structural protein 1 of SARS-CoV2 with SARS-CoV1 and MERS-CoV: An in silico study","abstract":"The recently emerged SARS-CoV2 caused a major pandemic of coronavirus disease (COVID-19). Non structural protein 1 (nsp1) is found in all beta coronavirus that causes several severe respiratory diseases. This protein is considered as a virulence factor and has an important role in pathogenesis. This study aims to elucidate the structural conformations of non structural protein 1 (nsp1), prediction of epitope sites and identification of important residues for targeted therapy against COVID-19. In this study, molecular modelling coupled with molecular dynamics simulations were performed to analyse the conformational change of nsp1 of SARS-CoV1, SARS-CoV2 and MERS-CoV at molecular level. Principal component analysis escorted by free energy landscape revealed that SARS-CoV2 nsp1 protein shows greater flexibility, compared to SARS-CoV1 and MERS-CoV nsp1. From the sequence alignment, it was observed that 28 mutations are present in SERS-CoV2 nsp1 protein compared to SERS-CoV1 nsp1. Several B-cell and T-cell epitopes were identified by immunoinformatics approach. SARS-CoV2 nsp1 protein binds with the interface region of the palm and finger domain of POLA1 by using hydrogen bond and salt bridge interactions. These findings can be used to develop therapeutics specific against COVID-19.","version":"1.2","doi":"10.1101/2020.06.09.142570","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438020","pub_date":"2021-5-24","title":"Screening of HLA-A restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A transgenic mice","abstract":"While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8+ T cell epitopes from E, M, N, S and RdRp proteins were validated. Among them, 110 epitopes have not been reported previously; 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; 4 epitopes from S protein displayed one amino acid distinct from the current variants of SARS-CoV-2. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8+ T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. Unlike previous researches, this study established a modified cell co-culture system of DC-peptide-PBL using healthy donor\u2019s PBMCs to validate the CD8+ T cell epitope on-silicon predicted; provided a library of CD8+ T cell epitopes restricted by a series of high-frequency HLA-A allotypes which covering broad Asian populations; identified the HLA-A cross-restrictions of these CD8+ T cell epitopes using competitive binding experiments with HMy2.CIR cell lines expressing indicated HLA-A molecules; and initially confirmed the in vivo feasibility of 9 or 10-mer peptide cocktail vaccines of SARS-CoV2. These data will facilitate the development of vaccines inducing antiviral CD8+ T cell responses.","version":"1.2","doi":"10.1101/2021.04.01.438020","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444622","pub_date":"2021-5-24","title":"Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the world\u2019s population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.","version":"1.2","doi":"10.1101/2021.05.18.444622","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445386","pub_date":"2021-5-24","title":"Nucleocapsid mutation R203K/G204R increases the infectivity, fitness and virulence of SARS-CoV-2","abstract":"In addition to the mutations on the spike protein (S), co-occurring mutations on nucleocapsid (N) protein are also emerging in SARS-CoV-2 world widely. Mutations R203K/G204R on N, carried by high transmissibility SARS-CoV-2 lineages including B.1.1.7 and P.1, has a rapid spread in the pandemic during the past year. In this study, we performed comprehensive population genomic analyses and virology experiment concerning on the evolution, causation and virology consequence of R203K/G204R mutations. The global incidence frequency (IF) of 203K/204R has rose up from nearly zero to 76% to date with a shrinking from August to November in 2020 but bounced later. Our results show that the emergence of B.1.1.7 is associated with the second growth of R203K/G204R mutants. We identified positive selection evidences that support the adaptiveness of 203K/204R variants. The R203K/G204R mutant virus was created and compared with the native virus. The virus competition experiments show that 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly in relation to the ribonucleocapsid (RNP) assemble during the virus replication. Moreover, the 203K/204R virus increased the infectivity in a human lung cell line and induced an enhanced damage to blood vessel of infected hamsters\u2019 lungs. In consistence, we observed a positive association between the increased severity of COVID-19 and the IF of 203K/204R from in silicon analysis of global clinical and epidemic data. In combination with the informatics and virology experiment, our work suggested the contribution of 203K/204R to the increased transmission and virulence of the SARS-CoV-2. In addition to mutations on the S protein, the mutations on the N protein are also important to virus spread during the pandemic.","version":"1.1","doi":"10.1101/2021.05.24.445386","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.23.445341","pub_date":"2021-5-24","title":"Genome-wide identification and prediction of SARS-CoV-2 mutations show an abundance of variants: Integrated study of bioinformatics and deep neural learning","abstract":"Genomic data analysis is a fundamental system for monitoring pathogen evolution and the outbreak of infectious diseases. Based on bioinformatics and deep learning, this study was designed to identify the genomic variability of SARS-CoV-2 worldwide and predict the impending mutation rate. Analysis of 259044 SARS-CoV-2 isolates identify 3334545 mutations (14.01 mutations per isolate), suggesting a high mutation rate. Strains from India showed the highest no. of mutations (48) followed by Scotland, USA, Netherlands, Norway, and France having up to 36 mutations. Besides the most prominently occurring mutations (D416G, F106F, P314L, and UTR:C241T), we identify L93L, A222V, A199A, V30L, and A220V mutations which are in the top 10 most frequent mutations. Multi-nucleotide mutations GGG>AAC, CC>TT, TG>CA, and AT>TA have come up in our analysis which are in the top 20 mutational cohort. Future mutation rate analysis predicts a 17%, 7%, and 3% increment of C>T, A>G, and A>T, respectively in the future. Conversely, 7%, 7%, and 6% decrement is estimated for T>C, G>A, and G>T mutations, respectively. T>G\\A, C>G\\A, and A>T\\C are not anticipated in the future. Since SARS-CoV-2 is evolving continuously, our findings will facilitate the tracking of mutations and help to map the progression of the COVID-19 intensity worldwide.","version":"1.1","doi":"10.1101/2021.05.23.445341","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445424","pub_date":"2021-5-24","title":"Isolation of SARS-CoV-2 B.1.1.28.2 P2 variant and pathogenicity comparison with D614G variant in hamster model","abstract":"Considering the potential threat from emerging SARS-CoV-2 variants and the rising COVID-19 cases, SARS-CoV-2 genomic surveillance is ongoing in India. We report herewith the isolation of the P.2 variant (B.1.1.28.2) from international travelers and further its pathogenicity evaluation and comparison with D614G variant (B.1) in hamster model. Virus isolation was performed in Vero CCL81 cells and genomic characterization by next generation sequencing. The pathogenicity of the isolate was assessed in Syrian hamster model and compared with B.1 variant. B.1.1.28.2 variant was isolated from nasal/throat swabs of international travelers returned to India from United Kingdom and Brazil. The B.1.1.28.2 variant induced body weight loss, viral replication in the respiratory tract, lung lesions and caused severe lung pathology in infected Syrian hamster model in comparison, with B.1 variant infected hamsters. The sera from B.1.1.28.2 infected hamsters efficiently neutralized the D614G variant virus whereas 6-fold reduction in the neutralization was seen in case of D614G variant infected hamsters\u2019 sera with the B.1.1.28.2 variant. B.1.1.28.2 lineage variant could be successfully isolated and characterization could be performed. Pathogenicity of the isolate was demonstrated in Syrian hamster model and in comparison, with B.1 variant was found more pathogenic. The findings of increased disease severity and neutralization reduction is of great concern and point towards the need for screening the vaccines for efficacy.","version":"1.1","doi":"10.1101/2021.05.24.445424","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444646","pub_date":"2021-5-24","title":"Effects of common mutations in the SARS-CoV-2 Spike RBD domain and its ligand the human ACE2 receptor on binding affinity and kinetics","abstract":"The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD domain are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the more transmissible B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD domain that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations, and combinations found in new SARS-CoV-2 variants first identified in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P1). Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations primarily enhance transmission, the K417N/T mutations facilitate immune escape, and the E484K mutation facilitates both transmission and immune escape.","version":"1.2","doi":"10.1101/2021.05.18.444646","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445374","pub_date":"2021-5-24","title":"Severe Acute Respiratory Syndrome Coronavirus-2 genome sequence variations relate to morbidity and mortality in Coronavirus Disease-19","abstract":"Outcome of infection with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) may depend on the host, virus or the host-virus interaction-related factors. Complete SARS-CoV-2 genome was sequenced using Illumina and Nanopore platforms from naso-/oro-pharyngeal ribonucleic acid (RNA) specimens from COVID-19 patients of varying severity and outcomes, including patients with mild upper respiratory symptoms (n=35), severe disease ad-mitted to intensive care with respiratory and gastrointestinal symptoms (n=21), fatal COVID-19 outcome (n=17) and asymptomatic (n=42). Of a number of genome variants observed, p.16L>L (Nsp1), p.39C>C (Nsp3), p.57Q>H (ORF3a), p.71Y>Y (Membrane glycoprotein), p.194S>L (Nucleocapsid protein) were observed in similar frequencies in different patient subgroups. However, seventeen other variants were observed only in symptomatic patients with severe and fatal COVID-19. Out of the latter, one was in the 5\u2019UTR (g.241C>T), eight were synonymous (p.14V>V and p.92L>L in Nsp1 protein, p.226D>D, p.253V>V, and p.305N>N in Nsp3, p.34G>G and p.79C>C in Nsp10 protein, p.789Y>Y in Spike protein), and eight were non-synonymous (p.106P>S, p.157V>F and p.159A>V in Nsp2, p.1197S>R and p.1198T>K in Nsp3, p.97A>V in RdRp, p.614D>G in Spike protein, p.13P>L in nucleocapsid). These were completely absent in the asymptomatic group. SARS-CoV-2 genome variations have a significant impact on COVID-19 presentation, severity and outcome.","version":"1.1","doi":"10.1101/2021.05.24.445374","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.23.445114","pub_date":"2021-5-24","title":"ABO blood group is involved in the quality of the specific immune response","abstract":"Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12\u2013305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the Spike (S), Nucleocapsid (N), and Membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that A-group individuals presented significantly higher frequencies of specific CD4+ T-cell responses against Pep-M than O-group individuals. The A-group subjects also needed longer to clear the virus and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors determine the sustainability of the body\u2019s defences, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity. This work describes that cellular responses against SARS-CoV-2 M-protein can be detected after 10 months but were lost against S- and N-proteins. Moreover, the individual factors; ABO-group and age influence the sustainability of the specific humoral and cellular immunity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.23.445114","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.24.445335","pub_date":"2021-5-24","title":"SARS-CoV-2 spike protein unlikely to bind to integrins via the Arg-Gly-Asp (RGD) motif of the Receptor Binding Domain: evidence from structural analysis and microscale accelerated molecular dynamics","abstract":"The Receptor Binding Domain (RBD) of SARS-CoV-2 virus harbors a sequence of Arg-Gly-Asp tripeptide named RGD motif, which has also been identified in extracellular matrix proteins that bind integrins as well as other disintegrins and viruses. Accordingly, integrins have been proposed as host receptors for SARS-CoV-2. The hypothesis was supported by sequence and structural analysis. However, given that the microenvironment of the RGD motif imposes structural hindrance to the protein-protein association, the validity of this hypothesis is still uncertain. Here, we used normal mode analysis, accelerated molecular dynamics microscale simulation, and protein-protein docking to investigate the putative role of RGD motif of SARS-CoV-2 RBD for interacting with integrins. We found, by molecular dynamics, that neither RGD motif nore its microenvironment show any significant conformational shift in the RBD structure. Highly populated clusters were used to run a protein-protein docking against three RGD-binding integrin types, showing no capability of the RBD domain to interact with the RGD binding site. Moreover, the free energy landscape revealed that the RGD conformation within RBD could not acquire an optimal geometry to allow the interaction with integrins. Our results highlighted different structural features of the RGD motif that may prevent its involvement in the interaction with integrins. We, therefore, suggest, in the case where integrins are confirmed to be the direct host receptors for SARS-CoV-2, a possible involvement of other residues to stabilize the interaction.","version":"1.1","doi":"10.1101/2021.05.24.445335","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445189","pub_date":"2021-5-24","title":"Evaluation of mRNA-1273 against SARS-CoV-2 B.1.351 Infection in Nonhuman Primates","abstract":"Vaccine efficacy against the B.1.351 variant following mRNA-1273 vaccination in humans has not been determined. Nonhuman primates (NHP) are a useful model for demonstrating whether mRNA-1273 mediates protection against B.1.351. Nonhuman primates received 30 or 100 \u00b5g of mRNA-1273 as a prime-boost vaccine at 0 and 4 weeks, a single immunization of 30 \u00b5g at week 0, or no vaccine. Antibody and T cell responses were assessed in blood, bronchioalveolar lavages (BAL), and nasal washes. Viral replication in BAL and nasal swabs were determined by qRT-PCR for sgRNA, and histopathology and viral antigen quantification were performed on lung tissue post-challenge. Eight weeks post-boost, 100 \u00b5g x2 of mRNA-1273 induced reciprocal ID50 neutralizing geometric mean titers against live SARS-CoV-2 D614G and B.1.351 of 3300 and 240, respectively, and 430 and 84 for the 30 \u00b5g x2 group. There were no detectable neutralizing antibodies against B.1351 after the single immunization of 30 \u00b5g. On day 2 following B.1.351 challenge, sgRNA in BAL was undetectable in 6 of 8 NHP that received 100 \u00b5g x2 of mRNA-1273, and there was a \u223c2-log reduction in sgRNA in NHP that received two doses of 30 \u00b5g compared to controls. In nasal swabs, there was a 1-log10 reduction observed in the 100 \u00b5g x2 group. There was limited inflammation or viral antigen in lungs of vaccinated NHP post-challenge. Immunization with two doses of mRNA-1273 achieves effective immunity that rapidly controls lower and upper airway viral replication against the B.1.351 variant in NHP.","version":"1.1","doi":"10.1101/2021.05.21.445189","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444882","pub_date":"2021-5-24","title":"The Structure of ChAdOx1/AZD-1222 Reveals Interactions with CAR and PF4 with Implications for Vaccine-induced Immune Thrombotic Thrombocytopenia","abstract":"Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combatting the 2019 SARS-CoV-2 pandemic. Following emergency use authorisation, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination. To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation. We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT. We present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2\u00c5 resolution. ChAdOx1 is in global use in the AstraZeneca vaccine, ChAdOx1 nCoV-19/AZD-1222, to combat the SARS-CoV-2 coronavirus pandemic. Recently observed, rare, adverse events make detailed mechanistic understanding of this vector key to informing proper treatment of affected patients and the development of safer viral vectors. Here, we determine a primary mechanism ChAdOx1 uses to attach to cells is coxsackie and adenovirus receptor (CAR), a protein which is identical in humans and chimpanzees. We demonstrate the vector does not form a stable CD46 interaction, a common species B adenovirus receptor, via its primary attachment protein. Further, we reveal the surface of the ChAdOx1 viral capsid has a strong electronegative potential. Molecular simulations suggest this charge, together with shape complementarity, are a mechanism by which an oppositely charged protein, platelet factor 4 (PF4) may bind the vector surface. PF4 is a key protein involved in the formation of blood clots, and the target of auto-antibodies in heparin-induced immune thrombotic thrombocytopenia (HITT), an adverse reaction to heparin therapy which presents similarly to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare complication of ChAdOx1 nCoV-19 vaccination. We propose a mechanism in which the ChAdOx1-PF4 complex may stimulate the production of antibodies against PF4, leading to delayed blood clot formation, as observed in VITT.","version":"1.2","doi":"10.1101/2021.05.19.444882","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.23.445371","pub_date":"2021-5-24","title":"Unsupervised explainable AI for the collective analysis of a massive number of genome sequences: various examples from the small genome of pandemic SARS-CoV-2 to the human genome","abstract":"In genetics and related fields, huge amounts of data, such as genome sequences, are accumulating, and the use of artificial intelligence (AI) suitable for big data analysis has become increasingly important. Unsupervised AI that can reveal novel knowledge from big data without prior knowledge or particular models is highly desirable for analyses of genome sequences, particularly for obtaining unexpected insights. We have developed a batch-learning self-organizing map (BLSOM) for oligonucleotide compositions that can reveal various novel genome characteristics. Here, we explain the data mining by the BLSOM: unsupervised and explainable AI. As a specific target, we first selected SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) because a large number of the viral genome sequences have been accumulated via worldwide efforts. We analyzed more than 0.6 million sequences collected primarily in the first year of the pandemic. BLSOMs for short oligonucleotides (e.g., 4~6-mers) allowed separation into known clades, but longer oligonucleotides further increased the separation ability and revealed subgrouping within known clades. In the case of 15-mers, there is mostly one copy in the genome; thus, 15-mers appeared after the epidemic start could be connected to mutations. Because BLSOM is an explainable AI, BLSOM for 15-mers revealed the mutations that contributed to separation into known clades and their subgroups. After introducing the detailed methodological strategies, we explained BLSOMs for various topics. The tetranucleotide BLSOM for over 5 million 5-kb fragment sequences derived from almost all microorganisms currently available and its use in metagenome studies. We also explained BLSOMs for various eukaryotes, such as fishes, frogs and Drosophila species, and found a high separation ability among closely related species. When analyzing the human genome, we found evident enrichments in transcription factor-binding sequences (TFBSs) in centromeric and pericentromeric heterochromatin regions. The tDNAs (tRNA genes) were separated by the corresponding amino acid.","version":"1.1","doi":"10.1101/2021.05.23.445371","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445119","pub_date":"2021-5-21","title":"Probenecid Inhibits SARS-CoV-2 Replication In Vivo and In Vitro","abstract":"Effective vaccines are slowing the COVID-19 pandemic, but SARS-CoV-2 will likely remain an issue in the future making it important to have therapeutics to treat patients. There are few options for treating patients with COVID-19. We show probenecid potently blocks SARS-CoV-2 replication in mammalian cells and virus replication in a hamster model. Furthermore, we demonstrate that plasma concentrations up to 50-fold higher than the protein binding adjusted IC90 value are achievable for 24h following a single oral dose. These data support the potential clinical utility of probenecid to control SARS-CoV-2 infection in humans.","version":"1.1","doi":"10.1101/2021.05.21.445119","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.409318","pub_date":"2021-5-21","title":"A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to most known recently emerging RBD mutations","abstract":"The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 \u00c5 resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with Fc\u03b3R silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. Human antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.","version":"1.2","doi":"10.1101/2020.12.03.409318","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445118","pub_date":"2021-5-21","title":"Missense variants in human ACE2 modify binding to SARS-CoV-2 Spike","abstract":"SARS-CoV-2 infection begins with the interaction of the SARS-CoV-2 Spike (Spike) and human angiotensin-converting enzyme 2 (ACE2). To explore whether population variants in ACE2 might influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities for Spike receptor binding domain through surface plasmon resonance (SPR). We discovered variants that enhance and reduce binding, including two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (\u0394\u0394G = \u00b1 0.59 0.08 kcal mol\u22121) is often seen in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (\u0394\u0394G = 0.26 0.09 kcal mol\u22121) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. Carriers of these alleles may be more susceptible to infection or severe disease and these variants may influence the global epidemiology of Covid-19. We also identified three rare ACE2 variants that strongly inhibited (p.Glu37Lys, \u0394\u0394G = \u22121.33 \u00b1 0.15 kcal mol\u22121 and p.Gly352Val, predicted \u0394\u0394G = \u22121.17 kcal mol\u22121) or abolished (p.Asp355Asn) Spike binding. These variants may confer resistance to infection. Finally, we calibrated the mCSM-PPI2 \u0394\u0394G prediction algorithm against our SPR data, give new predictions for all possible ACE2 missense variants at the Spike interface and estimate the overall burden of ACE2 variants on Covid-19 phenotypes.","version":"1.1","doi":"10.1101/2021.05.21.445118","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445152","pub_date":"2021-5-21","title":"The Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase","abstract":"The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but non-structural protein 13 (nsp13) stands out as a highly conserved and yet under studied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound presenting a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 \u03bcs of Gaussian accelerated molecular dynamics simulation (at least 6 \u03bcs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key aspects of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inch-worm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV and V and suggest a powerstroke\u2013like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.","version":"1.1","doi":"10.1101/2021.05.21.445152","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445156","pub_date":"2021-5-21","title":"ESCA pipeline: Easy-to-use SARS-CoV-2 genome Assembler","abstract":"Early sequencing and quick analysis of SARS-CoV-2 genome are contributing to understand the dynamics of COVID19 epidemics and to countermeasures design at global level. Amplicon-based NGS methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession, harboring multiple deletions and amino acid changing mutations. To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here we propose an easy-to-use SARS-CoV-2 genome Assembler: the ESCA pipeline. Results showed that ESCA can perform high quality genome assembly from IonTor-rent and Illumina raw data, and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility to compare assembled genomes of multi sample runs through an easy table format. Script and manuals are available on GitHub: https://github.com/cesaregruber/ESCA","version":"1.1","doi":"10.1101/2021.05.21.445156","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.444848","pub_date":"2021-5-21","title":"Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity","abstract":"Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (\u223c80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.","version":"1.2","doi":"10.1101/2021.05.20.444848","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444823","pub_date":"2021-5-21","title":"Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses","abstract":"Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.","version":"1.2","doi":"10.1101/2021.05.19.444823","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.445042","pub_date":"2021-5-21","title":"Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif","abstract":"The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 \u00b0C, respectively. This process is significantly slowed down at 4 \u00b0C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.","version":"1.1","doi":"10.1101/2021.05.20.445042","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445091","pub_date":"2021-5-21","title":"Identification of a novel lineage bat SARS-related coronaviruses that use bat ACE2 receptor","abstract":"Severe respiratory disease coronavirus-2 (SARS-CoV-2) causes the most devastating disease, COVID-19, of the recent century. One of the unsolved scientific questions around SARS-CoV-2 is the animal origin of this virus. Bats and pangolins are recognized as the most probable reservoir hosts that harbor the highly similar SARS-CoV-2 related viruses (SARSr-CoV-2). Here, we report the identification of a novel lineage of SARSr-CoVs, including RaTG15 and seven other viruses, from bats at the same location where we found RaTG13 in 2015. Although RaTG15 and the related viruses share 97.2% amino acid sequence identities to SARS-CoV-2 in the conserved ORF1b region, but only show less than 77.6% to all known SARSr-CoVs in genome level, thus forms a distinct lineage in the Sarbecovirus phylogenetic tree. We then found that RaTG15 receptor binding domain (RBD) can bind to and use Rhinolophus affinis bat ACE2 (RaACE2) but not human ACE2 as entry receptor, although which contains a short deletion and has different key residues responsible for ACE2 binding. In addition, we show that none of the known viruses in bat SARSr-CoV-2 lineage or the novel lineage discovered so far use human ACE2 efficiently compared to SARSr-CoV-2 from pangolin or some of the SARSr-CoV-1 lineage viruses. Collectively, we suggest more systematic and longitudinal work in bats to prevent future spillover events caused by SARSr-CoVs or to better understand the origin of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.21.445091","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.445060","pub_date":"2021-5-21","title":"Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant","abstract":"The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum, amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.","version":"1.1","doi":"10.1101/2021.05.20.445060","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.444918","pub_date":"2021-5-21","title":"Structure and dynamics of RNA guanine quadruplexes in SARS-CoV-2 genome. Original strategies against emerging viruses","abstract":"Guanine quadruplexes (G4) structures in viral genome have a key role in modulating viruses\u2019 biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modelling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.","version":"1.1","doi":"10.1101/2021.05.20.444918","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.21.445090","pub_date":"2021-5-21","title":"Water-triggered, irreversible conformational change of SARS-CoV-2 main protease on passing from the solid state to aqueous solution","abstract":"The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1 ms long molecular dynamics simulation shows that it readily undergoes a symmetry breaking event on passing from the solid state to the aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analysing this long time simulation, here we uncover a previously unrecognised role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling methods performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.","version":"1.1","doi":"10.1101/2021.05.21.445090","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444889","pub_date":"2021-5-20","title":"An AAV-ie based Vaccine effectively protects against SARS-CoV-2 and Circulating Variants","abstract":"Prophylactic vaccines against SARS-CoV-2 have been extensively developed globally to overcome the COVID-19 pandemic. However, recently emerging SARS-CoV-2 variants B.1.1.7 and B.1.351 limit the vaccine protection effects and successfully escape antibody cocktail treatment. Herein, based on our previously engineered adeno-associated viral (AAV) vector, AAV-ie, and systematic immunogen screening, we developed an AAV-ie-S1 vaccine with thermostability, high efficiency, safety, and single-dose vaccination advantage. Importantly, the AAV-ie-S1 immune sera efficiently neutralize B.1.1.7 and B.1.351, indicating a potential to circumvent the spreading of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.19.444889","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.445008","pub_date":"2021-5-20","title":"Acute SARS-CoV-2 infection is associated with an expansion of bacteria pathogens in the nose including Pseudomonas Aeruginosa","abstract":"Much of the research conducted on SARS-CoV-2 and COVID-19 has focused on the systemic host response, especially that generated by severely ill patients. Very few studies have investigated the impact of acute SARS-CoV-2 within the nasopharynx, the site of initial infection and viral replication. In this study we profiled changes in the nasal microbial communities as well as in host transcriptional profile during acute SARS-CoV-2 infection using 16S amplicon sequencing and RNA sequencing. These analyses were coupled to viral genome sequencing. Our microbiome analysis revealed that the nasal microbiome of COVID patients was unique and was marked by an expansion of bacterial pathogens. Some of these microbes (i.e. Acinetobacter) were shared with COVID negative health care providers from the same medical center but absent in COVID negative outpatients seeking care at the same institutions suggesting acquisition of nosocomial respiratory pathogens. Specifically, we report a distinct increase in the prevalence and abundance of the pathogen Pseudomonas aeruginosa in COVID patients that correlated with viral RNA load. These data suggest that the inflammatory environment caused by SARS-CoV-2 infection and potentially exposure to the hospital environment leads to an expansion of bacterial pathogens in the nasal cavity that could contribute to increased incidence of secondary bacterial infections. Additionally, we observed a robust host transcriptional response in the nasal epithelia of COVID patients, indicative of an antiviral innate immune repones and neuronal damage. Finally, analysis of viral genomes did not reveal an association between viral loads and viral sequences.","version":"1.1","doi":"10.1101/2021.05.20.445008","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444875","pub_date":"2021-5-20","title":"4\u2019-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication","abstract":"The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4\u2019-fluorouridine (4\u2019-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i+3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4\u2019-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. 4\u2019-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo.","version":"1.1","doi":"10.1101/2021.05.19.444875","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.423942","pub_date":"2021-5-20","title":"SARS-CoV-2 infecting the inner ear results in potential hearing damage at the early stage or prognosis of COVID-19 in rodents","abstract":"In order to find out the association between the sensorineural hearing loss and COVID-19, we detected the expression ACE2 and TMPRSS2 in the mouse the hamster. Using the public data from NCBI and GISAID, we assessed the expression of ACE2 and TMPRSS2 at the transcriptomic, DNA, and protein levels of ACE2 in the brain, inner ear, and muscle from the golden Syrian hamster (Mesocricetus auratus) and mouse (Mus musculus). We identified ACE2 and TMPRSS2 expressed at different level in the inner ear and brain at DNA and transcriptomic levels of both mouse and the hamster. The protein expression shows a similar pattern of the brain and inner ear, while the expression of ACE2 from the inner ear was relatively higher than it from the muscle. SARS-CoV-2 could infect the hearing system potentially and SSNHL could be a characteristic to detect asymptomatic patients of COVID-19.","version":"1.2","doi":"10.1101/2020.12.23.423942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.444757","pub_date":"2021-5-20","title":"Differential interferon-\u03b1 subtype immune signatures suppress SARS-CoV-2 infection","abstract":"Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha (\u03b1) subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN\u03b1 subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN\u03b1 subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN\u03b15 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN\u03b1 subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN\u03b15. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.20.444757","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.02.442358","pub_date":"2021-5-20","title":"Vitamin C Binds to SARS Coronavirus-2 Main Protease Essential for Viral Replication","abstract":"There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS-Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the protein\u2019s active site at 2.5 \u00c5ngstrom resolution. The crystal structure of the Vitamin C 3CLpro complex may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but on related proteases of other infectious viruses.","version":"1.2","doi":"10.1101/2021.05.02.442358","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.20.444932","pub_date":"2021-5-20","title":"Topic: Hyper immune Bovine Colostrum as a Low-Cost, Large-Scale Source of Antibodies against COVID-19","abstract":"Many different strategies have been used to fight against COVID-19 pandemic as a therapeutics or prophylaxis approaches. However, none of them so far have used, passive immune transfer using products from immunized farm animals. Hyper immune bovine colostrums (HBC) have been used against many different respiratory and gastrointestinal tracts infections during past decades. Six mixed Holstein X Semental dairy cattle\u2019s in their 6-7 months of gestation period years were chosen for hyper immunization with COVID-19 vaccine. An isolated and very well protected site was selected and equipped according to animal husbandry code of practice, used for animal experimentation. Specific IgG level against SARS-CoV-2 virus was measured before and after vaccination in the sera, and in the colostrum following parturition. Very high specific IgG level was detected one week following second vaccination in the sera and in first colostrums after parturition. Safety of the product was approved following phase 1 of clinical trials in 40 healthy volunteers. Phase 2 of the clinical trials is underway. Early results show effectiveness of the product in reducing sore throat and cough in early stages of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.05.20.444932","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.21256584","pub_date":"2021-05-20","title":"Neutrophils cause critical illness in COVID-19 and reveal CDK6 inhibitors as potential treatment","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Despite recent development of vaccines and monoclonal antibodies to prevent SARS-CoV-2 infection, treatment of critically ill COVID-19 patients remains an important goal. In principle, genome-wide association studies (GWAS) could shortcut the clinical evidence needed to repurpose drugs - the use of an existing drug for a new indication. However, it has been shown that the genes found in GWA studies usually do not encode an established drug target and the causal role for disease, a key requirement for drug efficacy, is unclear. We report here an alternative method for finding and testing causal target candidates for drug repurposing.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Rather than focusing on the genetics of the disease, we looked for disease-causing traits by searching for significant differences in 33 blood cell types, 30 blood biochemistries, and body mass index between an infectious disease phenotype and healthy controls. We then matched critically ill COVID-19 cases with controls that exhibited mild or no symptoms after SARS-CoV-2 infection in order to identify disease-causing traits by applying causal inference methods.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We found high neutrophil cell count as a causal trait for the immune overreaction in critical illness due to COVID-19. Based on these findings, we identified the enzyme CDK6 as a potential drug target to prevent the immune overreaction in critical illness due to COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    The genetics of disease-causing traits turns out to be a rich reservoir for the identification of known drug targets. This is due to the usually larger datasets of traits, as they also cover healthy ones. A clinical trial testing CDK6 inhibitor palbociclib in critically ill COVID-19 patients is currently ongoing (\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    Identifier:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT05371275'>NCT05371275</jats:ext-link>\n                    ).\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.05.18.21256584","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.20.444952","pub_date":"2021-5-20","title":"A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses","abstract":"Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids (IOs) are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.","version":"1.1","doi":"10.1101/2021.05.20.444952","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.15.440089","pub_date":"2021-5-19","title":"Distinct SARS-CoV-2 Antibody Responses Elicited by Natural Infection and mRNA Vaccination","abstract":"We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8,476 finger stick blood specimens were collected before and after an aggressive mRNA vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing 10 SARS-CoV-2 antigens, 4 SARS, 3 MERS, 12 Common CoV, and 8 Influenza antigens. Twenty-six percent of 3,347 specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive. The Ab response was predominantly against nucleocapsid (NP), full length spike and the spike S2 domain. Anti-receptor binding domain (RBD) reactivity was low and there was no cross-reactivity against SARS S1 or SARS RBD. An aggressive mRNA vaccination campaign at the UCI Medical Center started on December 16, 2020 and 6,724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% in December to 79% in January, 93% in February and 99% in March. mRNA vaccination induced much higher Ab levels especially against the RBD domain and significant cross-reactivity against SARS RBD and S1 was also observed. Nucleocapsid protein Abs can be used to distinguish individuals in a population of vaccinees to classify those who have been previously infected and those who have not, because nucleocapsid is not in the vaccine. Previously infected individuals developed higher Ab titers to the vaccine than those who have not been previously exposed. These results indicate that mRNA vaccination rapidly induces a much stronger and broader Ab response than SARS-CoV-2 infection.","version":"1.4","doi":"10.1101/2021.04.15.440089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.19.444825","pub_date":"2021-5-19","title":"Adaptive immune determinants of viral clearance and protection in mouse models of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 160 million infections and more than 3 million deaths worldwide. While effective vaccines are currently being deployed, the adaptive immune determinants which promote viral clearance and confer protection remain poorly defined. Using mouse models of SARS-CoV-2, we demonstrate that both humoral and cellular adaptive immunity contributes to viral clearance in the setting of primary infection. Furthermore, we find that either convalescent mice, or mice that receive mRNA vaccination are protected from both homologous infection and infection with a variant of concern, B.1.351. Additionally, we find this protection to be largely mediated by antibody response and not cellular immunity. These results highlight the in vivo protective capacity of antibodies generated to both vaccine and natural infection. Defining the roles of humoral and cellular adaptive immunity in viral clearance and protection from SARS-CoV-2 and a variant of concern.","version":"1.1","doi":"10.1101/2021.05.19.444825","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444742","pub_date":"2021-5-19","title":"Landscape-Based Mutational Sensitivity Cartography and Network Community Analysis of the SARS-CoV-2 Spike Protein Structures: Quantifying Functional Effects of the Circulating Variants","abstract":"Structural and biochemical studies SARS-CoV-2 spike mutants with the enhanced infectivity have attracted significant attention and offered several mechanisms to explain the experimental data. In this study, we used an integrative computational approach to examine molecular mechanisms underlying functional effects of the D614G mutation by exploring atomistic modeling of the SARS-CoV-2 spike proteins as allosteric regulatory machines. We combined atomistic simulations, deep mutational scanning and sensitivity mapping together with the network-based community analysis to examine structures of the native and mutant SARS-CoV-2 spike proteins in different functional states. Conformational dynamics and analysis of collective motions in the SARS-CoV-2 spike proteins demonstrated that the D614 position anchors a key regulatory cluster that dictates functional transitions between open and closed states. Using mutational scanning and sensitivity analysis of the spike residues, we identified the evolution of stability hotspots in the SARS-CoV-2 spike structures of the mutant trimers. The results offer support to the reduced shedding mechanism of as a driver of the increased infectivity triggered by the D614G mutation. By employing the landscape-based network community analysis of the SARS-CoV-2 spike proteins, our results revealed that the D614G mutation can promote the increased number of stable communities in the open form by enhancing the stability of the inter-domain interactions. This study provides atomistic view of the interactions and stability hotspots in the SARS-CoV-2 spike proteins, offering a useful insight into the molecular mechanisms of the D614G mutation that can exert its functional effects through allosterically induced changes on stability of the residue interaction networks.","version":"1.1","doi":"10.1101/2021.05.18.444742","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444690","pub_date":"2021-5-19","title":"Garcinia kola and garcinoic acid suppress SARS-CoV-2 spike glycoprotein S1-induced hyper-inflammation in human PBMCs through inhibition of NF-\u03baB activation","abstract":"Symptoms and complications associated with severe SARS-CoV-2 infection such as acute respiratory distress syndrome (ARDS) and organ damage have been linked to SARS-CoV-2 spike glycoprotein S1-induced increased production of pro-inflammatory cytokines by immune cells. In this study, the effects of an extract of Garcinia kola seeds and garcinoic acid were investigated in SARS-CoV-2 spike glycoprotein S1-stimulated human PBMCs. Results of ELISA experiments revealed that Garcinia kola extract (6.25, 12.5 and 25 \u03bcg/mL) and garcinoic acid (1.25, 2.5 and 5 \u03bcM) significantly reduced SARS-CoV-2 spike glycoprotein S1-induced increased secretion of TNF\u03b1, IL-6, IL-1\u03b2 and IL-8 in PBMCs. In-cell western assays showed that pre-treatment with Garcinia kola extract and garcinoic acid reduced elevated expressions of both phospho-p65 and phospho-\u03baB\u03b1 proteins, as well as NF-\u03baB DNA binding capacity and NF-\u03baB-driven luciferase expression following stimulation of PBMCs with spike glycoprotein S1. Furthermore, pre-treatment of PBMCs with Garcinia kola extract prior to stimulation with SARS-CoV-2 spike glycoprotein S1 resulted in reduced damage to adjacent A549 lung epithelial cells. Gas Chromatography-Mass Spectrometry (GCMS) and HPLC-PDA confirmed the presence of garcinoic acid in the Garcinia kola extract used in this study. These results suggest that the seed of Garcinia kola and garcinoic acid are natural products which may possess pharmacological/therapeutic benefits in reducing cytokine storm during the late stage of severe SARS-CoV-2 and other coronavirus infections.","version":"1.1","doi":"10.1101/2021.05.18.444690","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.02.424917","pub_date":"2021-5-18","title":"SARS-CoV-2 infects lung epithelial cells and induces senescence and an inflammatory response in patients with severe COVID-19","abstract":"SARS-CoV-2 infection of the respiratory system can progress to a life threatening multi-systemic disease, mediated via an excess of cytokines (\u201ccytokine storm\u201d), but the molecular mechanisms are poorly understood. To investigate whether SARS-CoV-2 may induce cellular senescence in lung epithelial cells, leading to secretion of inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). Autopsy lung tissue samples from eleven COVID-19 patients and sixty age-matched non-infected controls were analysed by immunohistochemistry for SARS-CoV-2 and markers of cellular senescence (SenTraGor, p16INK4A) and key SASP cytokines (interleukin-1\u03b2, interleukin-6). We also investigated whether SARS-CoV-2 infection of an epithelial cell line induces senescence and cytokine secretion. SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in alveolar type-2 (AT2) cells, which also expressed the angiotensin-converting-enzyme 2 (ACE2), a critical entry receptor for this virus. In COVID-19 samples, AT2 cells displayed increased markers of senescence [p16INK4A, SenTraGor staining positivity in 12\u00b11.2% of cells compared to 1.7\u00b10.13% in non-infected controls (p<0.001)], with markedly increased expression of interleukin-1\u03b2 and interleukin-6 (p<0.001). Infection of epithelial cells (Vero E6) with SARS-CoV-2 in-vitro induced senescence and DNA damage (increased SenTraGor and \u03b3-H2AX), and reduced proliferation (Ki67) compared to uninfected control cells (p<0.01). We demonstrate that in severe COVID-19 patients, AT2 cells are infected with SARS-CoV-2 and show senescence and expression of proinflammatory cytokines. We also show that SARS-CoV-2 infection of epithelial cells may induce senescence and inflammation, indicating that cellular senescence may be an important molecular mechanism of severe COVID-19.","version":"1.2","doi":"10.1101/2021.01.02.424917","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444397","pub_date":"2021-5-18","title":"An intranasal ASO therapeutic targeting SARS-CoV-2","abstract":"The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identified an LNA ASO binding to the 5\u2019 leader sequence of SARS-CoV-2 ORF1a/b that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the K18-hACE2 humanized COVID-19 mouse model potently (98-99%) suppressed viral replication in the lungs of infected mice, revealing strong prophylactic and treatment effects. We found that the LNA ASO also represses viral infection in golden Syrian hamsters, and is highly efficacious in countering all SARS-CoV-2 \u201cvariants of concern\u201d tested in vitro and in vivo, including B.1.427, B.1.1.7, and B.1.351 variants. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce transmission of variants partially resistant to vaccines and monoclonal antibodies, and could be deployed intranasally for prophylaxis or via lung delivery by nebulizer to decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences, and they may have particular impact in areas where vaccine distribution is a challenge, and could be stockpiled for future coronavirus pandemics.","version":"1.1","doi":"10.1101/2021.05.17.444397","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.19.440435","pub_date":"2021-5-18","title":"SARS-CoV-2 20I/501Y.V1 variant in the hamster model","abstract":"Late 2020, SARS-CoV-2 20I/501Y.V1 variant from lineage B.1.1.7 emerged in United Kingdom and gradually replaced the D614G strains initially involved in the global spread of the pandemic. In this study, we used a Syrian hamster model to compare a clinical strain of 20I/501Y.V1 variant with an ancestral D614G strain. The 20I/501Y.V1 variant succeeded to infect animals and to induce a pathology that mimics COVID-19. However, both strains induced replicated to almost the same level and induced a comparable disease and immune response. A slight fitness advantage was noted for the D614G strain during competition and transmission experiments. These data do not corroborate the current epidemiological situation observed in humans nor recent reports that showed a more rapid replication of the 20I/501Y.V1 variant in human reconstituted bronchial epithelium.","version":"1.3","doi":"10.1101/2021.04.19.440435","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444675","pub_date":"2021-5-18","title":"Implications Derived from S-Protein Variants of SARS-CoV-2 from Six Continents","abstract":"Spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical determinants of the infectivity and antigenicity of the virus. Several mutations in the spike protein of SARS-CoV-2 have already been detected, and their effect in immune system evasion and enhanced transmission as a cause of increased morbidity and mortality are being investigated. From pathogenic and epidemiological perspectives, spike proteins are of prime interest to researchers. This study focused on the unique variants of S proteins from six continents Asia, Africa, Europe, Oceania, South America, and North America. In comparison to the other five continents, Africa (29.065%) had the highest percentage of unique S proteins. Notably, only North America had 87% (14046) of the total (16143) specific S proteins available in the NCBI database(across all continents). Based on the amino acid frequency distributions in the S protein variants from all the continents, the phylogenetic relationship implies that unique S proteins from North America were significantly different from those of the other five continents. Overtime, the unique variants originating from North America are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. Hence it is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of COVID-19 pandemic. It is also further suggested that the efficacy of existing vaccines and future vaccine development must be reviewed with careful scrutiny, and if needed, further re-engineered based on requirements dictated by new emerging S protein variants.","version":"1.1","doi":"10.1101/2021.05.18.444675","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.18.444605","pub_date":"2021-5-18","title":"In Silico Molecular-Based Rationale for SARS-CoV-2 Spike Circulating Mutations Able to Escape Bamlanivimab and Etesevimab Monoclonal Antibodies","abstract":"The purpose of this work was to provide an in silico molecular rationale of the role eventually played by currently circulating S-RBDCoV-2 mutations in evading the immune surveillance effects elicited by the two Eli Lilly LY-CoV555/bamlanivimab and LY-CoV016/etesevimab monoclonal antibodies. The main findings from this study and shows that, compared to the wild-type SARS-CoV-2 spike protein, mutations E484A/G/K/Q/R/V, Q493K/L/R, S494A/P/R, L452R and F490S are predicted to be markedly resistant to neutralization by LY-CoV555, while mutations K417E/N/T, D420A/G/N, N460I/K/S/T, T415P, and Y489C/S are predicted to confer LY-CoV016 escaping advantage to the viral protein. A challenge of our global in silico results against the relevant experimental data resulted in an overall 90% agreement. This achievement not only constitutes a further, robust validation of our computer-based approach but also yields a molecular-based rationale for all relative experimental findings, and leads us to conclude that the current circulating SARS-CoV-2 and all possible emergent variants carrying these mutations in the spike protein can present new challenges for mAb-based therapies and ultimately threaten the fully-protective efficacy of currently available vaccines.","version":"1.1","doi":"10.1101/2021.05.18.444605","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444592","pub_date":"2021-5-18","title":"Higher concentrations of bacterial enveloped virus Phi6 can protect the virus from environmental decay","abstract":"Phage Phi6 is an enveloped virus considered as a possible non-pathogenic surrogate for SARS-CoV-2 and other viral pathogens in transmission studies. Higher input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phage are dried in plastic tubes, and when they are stored in saline solution at 4\u00b0C. When bacteriophage Phi6 are placed in LB (Luria-Bertani) growth medium prior to placement on the plastic surface, viral recovery is not influenced by the starting concentration. The protection is reflected in longer half-lives of the phage at higher concentrations compared to lower. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon survival of infectious virus following inoculation of various surfaces, high initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at the higher concentrations, but also because the half-lives themselves are extended at the higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus, and to consider the role of the carrier and matrix if the results are to be clinically relevant. During the COVID-19 pandemic, a lot of attention has been paid to the environmental decay of SARS-CoV-2 due to proposed transmission of the virus via fomites. However, published experiments have commenced with very high virus titer inoculums, an experimental design not representative of real-life conditions. The study described here evaluated the impact of initial virus titer on environmental decay of an enveloped virus, using a non-pathogenic surrogate for SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.","version":"1.1","doi":"10.1101/2021.05.17.444592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.16.444324","pub_date":"2021-5-17","title":"Verification of SARS-CoV-2-Encoded small RNAs and contribution to Infection-Associated lung inflammation","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 pandemic. SARS-CoV-2 is a positive-stranded RNA virus belongs to Coronaviridae family. The viral genome of SARS-CoV-2 contains around 29.8 kilobase with a 5\u2032-cap structure and 3\u2032-poly-A tail, and shows 79.2% nucleotide identity with human SARS-CoV-1, which caused the 2002-2004 SARS outbreak. As the successor to SARS-CoV-1, SARS-CoV-2 now has circulated across the globe. There is a growing understanding of SARS-CoV-2 in virology, epidemiology, and clinical management strategies. In this study, we verified the existence of two 18-22 nt small viral RNAs (svRNAs) derived from the same precursor in human specimens infected with SARS-CoV-2, including nasopharyngeal swabs and formalin-fixed paraffin-embedded (FFPE) explanted lungs from lung transplantation of COVID-19 patients. We then simulated and confirmed the formation of these two SARS-CoV-2-Encoded small RNAs in human lung epithelial cells. And the potential pro-inflammatory effects of the splicing and maturation process of these two svRNAs in human lung epithelial cells were also explored. By screening cytokine storm genes and the characteristic expression profiling of COVID-19 in the explanted lung tissues and the svRNAs precursor transfected human lung epithelial cells, we found that the maturation of these two small viral RNAs contributed significantly to the infection associated lung inflammation, mainly via the activation of the CXCL8, CXCL11 and type I interferon signaling pathway. Taken together, we discovered two SARS-CoV-2-Encoded small RNAs and investigated the pro-inflammatory effects during their maturation in human lung epithelial cells, which might provide new insight into the pathogenesis and possible treatment options for COVID-19.","version":"1.1","doi":"10.1101/2021.05.16.444324","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.16.444369","pub_date":"2021-5-17","title":"Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate","abstract":"Beginning in late 2020, the emergence and spread of multiple variant SARS-CoV-2 strains harboring mutations which may enable immune escape necessitates the rapid evaluation of second generation COVID-19 vaccines, with the goal of inducing optimized immune responses that are broadly protective. Here we demonstrate in a mouse immunogenicity study that two doses of a modified B.1.351 spike (S)-Trimer vaccine (B.1.351 S-Trimer) candidate can induce strong humoral immune responses that can broadly neutralize both the original SARS-CoV-2 strain (Wuhan-Hu-1) and Variants of Concern (VOCs), including the UK variant (B.1.1.7), South African variant (B.1.351) and Brazil variant (P.1). Furthermore, while immunization with two doses (prime-boost) of Prototype S-Trimer vaccine (based on the original SARS-CoV-2 strain) induced lower levels of cross-reactive neutralization against the B.1.351 variant, a third dose (booster) administered with either Prototype S-Trimer or B.1.351 S-Trimer was able to increase neutralizing antibody titers against B.1.351 to levels comparable to neutralizing antibody titers against the original strain elicited by two doses of Prototype S-Trimer.","version":"1.1","doi":"10.1101/2021.05.16.444369","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.09.426021","pub_date":"2021-5-17","title":"Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate","abstract":"A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1\u03b1-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signaling pathway might offer therapeutic opportunities for the treatment of COVID-19.","version":"1.3","doi":"10.1101/2021.01.09.426021","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.17.444533","pub_date":"2021-5-17","title":"High level production and characterization of truncated human angiotensin converting enzyme 2 in Nicotiana benthamiana plant as a potential therapeutic target in COVID-19","abstract":"The COVID-19 pandemic, which is caused by SARS-CoV-2 has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs safe, a cost-effective SARS-CoV-2 coronavirus vaccine, therapeutic and antiviral drugs to combat the COVID-19. Angiotensin-converting enzyme 2 (ACE2), as a key receptor for SARS-CoV-2 infections, has been proposed as a potential therapeutic target in COVID-19 patients. In this study, we report high level production (about \u223c0.75 g /kg leaf biomass) of glycosylated and non-glycosylated forms of recombinant human truncated ACE2 in Nicotiana benthamiana plant. The plant produced recombinant human truncated ACE2s successfully bind to the SARC-CoV-2 spike protein, but deglycosylated ACE2 binds more strongly than the glycosylated counterpart. Importantly, both deglycosylated and glycosylated forms of AEC2 stable at elevated temperatures for prolonged periods and demonstrated strong anti-SARS-CoV-2 activity in vitro. The IC50 values of glycosylated and deglycosylated AEC2 were 0.4 and 24 \u03bcg/ml, respectively, for the pre-entry infection, when incubated with 100TCID50 of SARS-CoV-2. Thus, plant produced truncated ACE2s are promising cost-effective and safe candidate as a potential therapeutic targets in the treatment of COVID-19 patients.","version":"1.1","doi":"10.1101/2021.05.17.444533","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.444301","pub_date":"2021-5-17","title":"SARS-CoV-2 convergent evolution cannot be reliably inferred from phylogenetic analyses","abstract":"A homoplasy is a trait shared between individuals that did not arise in a common ancestor, but rather is the result of convergent evolution. SARS-CoV-2 homoplasic mutations are important to characterise, because the evidence for a mutation conferring a fitness advantage is strengthened if this mutation has evolved independently and repeatedly in separate viral lineages. Yet detecting homoplasy is difficult due to insufficient variation between sequences to construct reliable phylogenetic trees. Here, we develop a method to more robustly identify confident homoplasies. We derive a maximum likelihood (ML) tree, with taxa bearing seemingly recurrent mutations dispersed across the tree, and then, for each potentially homoplasic mutation, we derive an alternative tree where the same taxa are constrained to one clade such that the mutation is no longer homoplasic. We then compare how well the two trees fit the sequence data. Applying this method to SARS-CoV-2 yields only a few instances where the constrained trees have significantly less statistical support than unconstrained tree, suggesting phylogenetics can provide limited support for homoplasy in SARS-CoV-2 and that caution is needed when inferring evidence of convergent evolution from phylogenetic methods in the absence of evidence from other sources.","version":"1.1","doi":"10.1101/2021.05.15.444301","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.15.431212","pub_date":"2021-5-17","title":"A glycan gate controls opening of the SARS-CoV-2 spike protein","abstract":"SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded \u201cdown\u201d to an exposed \u201cup\u201d state in order to bind the human ACE2 receptor and infect cells. While snapshots of the \u201cup\u201d and \u201cdown\u201d states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 \u03bcs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.","version":"1.2","doi":"10.1101/2021.02.15.431212","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426227","pub_date":"2021-5-17","title":"Microsecond simulations and CD spectroscopy reveals the intrinsically disordered nature of SARS-CoV-2 Spike-C-terminal cytoplasmic tail (residues 1242-1273) in isolation","abstract":"All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions. Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region. This curiosity has led us to investigate the C terminal cytosolic region of the spike glycoprotein of SARS-CoV-2 in isolation. The cytosolic regions is supposed to be from 1235-1273 residues or 1242-1273 residues depending on what prediction tool we use. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown its conformational dynamics. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm which is also adding the analysis as its intrinsically disordered nature. We believe that our findings will surely help us understand the structure-function relationship of the spike protein\u2019s cytosolic region.","version":"1.2","doi":"10.1101/2021.01.11.426227","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432861","pub_date":"2021-5-17","title":"Antiviral Resistance against Viral Mutation: Praxis and Policy for SARS-CoV-2","abstract":"New tools developed by Moderna, BioNTech/Pfizer, and Oxford/Astrazeneca, among others, provide universal solutions to previously problematic aspects of drug or vaccine delivery, uptake and toxicity, portending new tools across the medical sciences. A novel method is presented based on estimating protein backbone free energy via geometry to predict effective antiviral targets, antigens and vaccine cargos that are resistant to viral mutation. This method, partly described in earlier work of the author, is reviewed and reformulated here in light of the recent proliferation of structural data on the SARS-CoV-2 spike glycoprotein and its latest mutations in the variants of concern and several further variants of interest including all international lineages. Particular attention to structures computed with Cryo Electron Microscopy allows the novel approach of probing the pH dependence of free energy in order to infer function. Key findings include: collections of recurring mutagenic residues occur across strains, presumably through cooperative convergent evolution; the preponderance of mutagenic residues do not participate in backbone hydrogen bonds; metastability of the spike glycoprotein limits the change of free energy from before to after mutation and thereby constrains selective pressure; and there are mRNA or virus-vector cargos which target low free energy peptides proximal to conserved high free energy peptides providing specific recipes for vaccines with greater specificity than the current full-spike approach. These results serve to limit peptides in the spike glycoprotein with high mutagenic potential and thereby provide a priori constraints on viral and attendant vaccine evolution. Scientific and regulatory challenges to nucleic acid therapeutic and vaccine development and deployment are finally discussed.","version":"1.3","doi":"10.1101/2021.02.25.432861","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.13.444021","pub_date":"2021-5-17","title":"Global mapping of RNA homodimers in living cells","abstract":"RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding of RNA homodimerization has been hampered by the lack of systematic in vivo detection methods. Here we show that PARIS, COMRADES, and other RNA proximity ligation methods can detect RNA homodimers transcriptome-wide as \u201coverlapping\u201d chimeric reads that contain more than one copy of the same sequence. Analysing published proximity ligation datasets, we show that RNA:RNA homodimers mediated by direct base-pairing interactions are rare across the transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA and a subset of tRNAs. Analysis of data from virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N protein in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.","version":"1.1","doi":"10.1101/2021.05.13.444021","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.137380","pub_date":"2021-5-17","title":"ScRNA-seq discover cell cluster change under OAB\u2014\u2014ACE2 expression reveal possible alternation of 2019-nCoV infectious pathway","abstract":"Previous study indicated that bladder cells which express ACE2 were a potential infection route of 2019-nCov. This study observed some differences of bladder cell cluster and their ACE2 expression between OAB mice and healthy mice, indicating the change of infectious possibility and pathway under overactive bladder (OAB) circumstance. Pubic dataset acquisition was used to get ACE2 expression in normal human bladder and mice bladder (GSE129845). We built up over OAB model and studied the impact on cell typing and ACE2 expression. By way of using single-cell RNA sequencing (scRNA-seq) technique, bladder cell clustering and ACE2 expression in various cell types were measured respectively. In pubic database (healthy human and mice bladder), ACE2 expression in humans and mice is concentrated in bladder epithelial cells. The disappearance of umbrella cells, a component of bladder epithelial, was found in our OAB model. In the two mouse bladder samples, ACE2 expression of epithelial cells is 34.1%, also the highest of all cell types. The disappearance of umbrella cell may alternate the infection pathway of 2019-nCov and relate to the onset and progression of OAB.","version":"1.2","doi":"10.1101/2020.06.05.137380","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.16.444344","pub_date":"2021-5-17","title":"Key informant perceptions on wildlife hunting in India during the COVID-19 lockdown","abstract":"Lockdowns intended to control the COVID-19 pandemic resulted in major socioeconomic upheavals across the world. While there were numerous reports of these lockdowns benefiting wildlife by reducing human movement and habitat disturbance, increased hunting during these lockdowns emerged as a conservation concern, particular in tropical Asia and Africa. We used online interviews with key informants including wildlife researchers, enforcement staff and NGO employees (N=99), and media reports (N=98), to examine the impacts of India\u2019s COVID-19 lockdown (March-May 2020) on wildlife hunting across the country. We asked whether and how hunting patterns changed during the lockdown, and explored socioeconomic and institutional factors underlying these changes. Over half the interviewees spread over 43 administrative districts perceived hunting (mammals, in particular) to have increased during the lockdown relative to a pre-lockdown reference period. Interviewees identified household consumption (53% of respondents) and sport and recreation (34%) as main motivations for hunting during the lockdown, and logistical challenges for enforcement (36%), disruption of food supply (32%), and need for recreational opportunities (32%) as key factors associated with hunting during this period. These insights were corroborated by statements by experts extracted from media articles. Collectively, our findings suggest that the COVID-19 lockdown potentially increased hunting across much of India, and emphasize the role of livelihood and food security in mitigating threats to wildlife during such periods of acute socioeconomic perturbation.","version":"1.1","doi":"10.1101/2021.05.16.444344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.16.444004","pub_date":"2021-5-16","title":"Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition","abstract":"The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2021.05.16.444004","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.13.444010","pub_date":"2021-5-16","title":"Durability of mRNA-1273-induced antibodies against SARS-CoV-2 variants","abstract":"SARS-CoV-2 mutations may diminish vaccine-induced protective immune responses, and the durability of such responses has not been previously reported. Here, we present a comprehensive assessment of the impact of variants B.1.1.7, B.1.351, P.1, B.1.429, and B.1.526 on binding, neutralizing, and ACE2-blocking antibodies elicited by the vaccine mRNA-1273 over seven months. Cross-reactive neutralizing responses were rare after a single dose of mRNA-1273. At the peak of response to the second dose, all subjects had robust responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of mRNA-1273. Across all assays, B.1.351 had the greatest impact on antibody recognition, and B.1.1.7 the least. These data complement ongoing studies of clinical protection to inform the potential need for additional boost vaccinations. Most mRNA-1273 vaccinated individuals maintained binding and functional antibodies against SARS-CoV-2 variants for 6 months.","version":"1.1","doi":"10.1101/2021.05.13.444010","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.444222","pub_date":"2021-5-16","title":"Stabilization of the SARS-CoV-2 Spike receptor-binding domain using deep mutational scanning and structure-based design","abstract":"The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40\u00b0C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.","version":"1.1","doi":"10.1101/2021.05.15.444222","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.14.444076","pub_date":"2021-5-16","title":"The Spike Proteins of SARS-CoV-2 B.1.617 and B.1.618 Variants Identified in India Provide Partial Resistance to Vaccine-elicited and Therapeutic Monoclonal Antibodies","abstract":"Highly transmissible SARS-CoV-2 variants recently identified in India designated B.1.617 and B.1.618 have mutations within the spike protein that may contribute to their increased transmissibility and that could potentially result in re-infection or resistance to vaccine-elicited antibody. B.1.617 encodes a spike protein with mutations L452R, E484Q, D614G and P681R while the B.1.618 spike has mutations \u0394145-146, E484K and D614G. We generated lentiviruses pseudotyped by the variant proteins and determined their resistance to neutralization by convalescent sera, vaccine-elicited antibodies and therapeutic monoclonal antibodies. Viruses with B.1.617 and B.1.618 spike were neutralized with a 2-5-fold decrease in titer by convalescent sera and vaccine-elicited antibodies. The E484Q and E484K versions were neutralized with a 2-4-fold decrease in titer. Virus with the B.1.617 spike protein was neutralized with a 4.7-fold decrease in titer by the Regeneron monoclonal antibody cocktail as a result of the L452R mutation. The modest neutralization resistance of the variant spike proteins to vaccine elicited antibody suggests that current vaccines will remain protective against the B.1.617 and B.1.618 variants.","version":"1.1","doi":"10.1101/2021.05.14.444076","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.444318","pub_date":"2021-5-16","title":"The Unconventional Self-Cleavage of Selenoprotein K","abstract":"Through known association with other proteins, human selenoprotein K (selenok) is currently implicated in the palmitoylation of proteins, degradation of misfolded proteins, innate immune response, and the life cycle of SARS-CoV-2 virus. However, neither the catalytic function of selenok\u2019s selenocysteine (Sec), which, curiously, resides in an intrinsically disordered protein segment nor selenok\u2019s specific role in these pathways are known to date. This report casts these questions in a new light as it describes that selenok is able -both in vitro and in vivo- to cleave some of its own peptide bonds. The cleavages not only release selenok segments that contain its reactive Sec, but as the specific cleavage sites were identified, they proved to cluster tightly near sites through which selenok interacts with protein partners. Furthermore, it is shown that selenok\u2019s cleavage activity is neither restricted to itself nor promiscuous but selectively extends to at least one of its protein partners. Together, selenok\u2019s cleavage ability and its features have all hallmarks of a regulatory mechanism that could play a central role in selenok\u2019s associations with other proteins and its cellular functions overall.","version":"1.1","doi":"10.1101/2021.05.15.444318","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.443978","pub_date":"2021-5-15","title":"Fc-independent neutralization of SARS-CoV-2 by recombinant human monoclonal antibodies","abstract":"The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in vivo neutralization of SARS-CoV-2 is not yet clear. Delineating the role this process plays in antibody-mediated protection will have a great impact on the design of such therapeutics. Here, the Fc of two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, targeting distinct domains of the spike, was engineered to abrogate their Fc-dependent functions. The protective activity of these antibodies was tested against lethal SARS-CoV-2 infections in K18-hACE2 transgenic mice, both before or two days post-exposure in comparison to their original, Fc-active antibodies. Antibody treatment with both Fc-variants similarly rescued the mice from death, reduced viral load and prevented signs of morbidity. In addition, surviving animals developed a significant endogenous immune response towards the virus. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in antibody-mediated protection, which should aid in future design of effective antibody-based therapies.","version":"1.1","doi":"10.1101/2021.05.15.443978","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.15.444262","pub_date":"2021-5-15","title":"Safety, immunogenicity and protection provided by unadjuvanted and adjuvanted formulations of recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in non-human primates","abstract":"Although antivirals are important tools to control the SARS-CoV-2 infection, effective vaccines are essential to control the current pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here we report the immunogenicity and protection induced in macaques by intramuscular injections of VLP bearing SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytosine phosphoguanine (CpG) 1018. Although a single dose of unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after prime) and adjuvants significantly improved both responses with a higher immunogenicity and protection provided by AS03 adjuvanted CoVLP. Fifteen microgram CoVLP adjuvanted with AS03 induced a balanced IL-2 driven response along with IL-4 expression in CD4 T cells and mobilization of CD4 follicular helper cells (Tfh). Animals were challenged by multiple routes (i.e. intratracheal, intranasal and ocular) with a total viral dose of 106 plaque forming units of SARS-CoV-2. Lower viral replication in nasal swabs and broncho-alveolar lavage (BAL) as well as fewer SARS-CoV-2 infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of pro-inflammatory cytokines and chemotactic factors in BAL were observed in the animals immunized with CoVLP adjuvanted with AS03. No clinical, pathologic or virologic evidences of vaccine associated enhanced disease (VAED) were observed in vaccinated animals. CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.","version":"1.1","doi":"10.1101/2021.05.15.444262","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.14.444111","pub_date":"2021-5-14","title":"CD147 antibody specifically and effectively inhibits infection and cytokine storm of SARS-CoV-2 variants","abstract":"SARS-CoV-2 and its variants are raging worldwide. Unfortunately, the global vaccination is not efficient enough to attain a vaccine-based herd-immunity and yet no special and effective drug is developed to contain the spread of the disease. Previously we have identified CD147 as a novel receptor for SARS-CoV-2 infection. Here, we demonstrated that CD147 antibody effectively inhibits infection and cytokine storm caused by SARS-CoV-2 variants. In CD147KO VeroE6 cells, infections of SARS-CoV-2, its variants (B.1.1.7, B.1.351) and pseudovirus mutants (B.1.1.7, B.1.351, B.1.525, B.1.526 (S477N), B.1.526 (E484K), P.1, P.2, B.1.617.1, B.1.617.2) were decreased. Meanwhile, CD147 antibody effectively blocked the entry of variants and pseudomutants in VeroE6 cells, and inhibited the expression of cytokines. A model of SARS-CoV-2-infected hCD147 transgenic mice was constructed, which recapitulated the features of exudative diffuse alveolar damage and dynamic immune responses of COVID-19. CD147 antibody could effectively clear the virus and alveolar exudation, resolving the pneumonia. We found the elevated level of cyclophilin A (CyPA) in plasma of severe/critical cases, and identified CyPA as the most important proinflammatory intermediate causing cytokine storm. Mechanistically, spike protein of SARS-CoV-2 bound to CD147 and initiated the JAK-STAT pathway, which induced expression of CyPA. CyPA reciprocally bound to CD147, triggered MAPK pathway and consequently mediated the expression of cytokine and chemokine. In conclusion, CD147 is a critical target for SARS-CoV-2 variants and CD147 antibody is a promising drug to control the new wave of COVID-19 epidemic.","version":"1.1","doi":"10.1101/2021.05.14.444111","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.14.444205","pub_date":"2021-5-14","title":"Common Mechanism of SARS-CoV and SARS-CoV-2 Pathogenesis across Species","abstract":"Sarbecovirus (CoV) infections, including Severe Acute Respiratory CoV (SARS-CoV) and SARS-CoV-2, are considerable human threats. Human GWAS studies have recently identified loci associated with variation in SARS-CoV-2 susceptibility. However, genetically tractable models that reproduce human CoV disease outcomes are needed to mechanistically evaluate genetic determinants of CoV susceptibility. We used the Collaborative Cross (CC) and human GWAS datasets to elucidate host susceptibility loci that regulate CoV infections and to identify host quantitative trait loci that modulate severe CoV and pan-CoV disease outcomes including a major disease regulating loci including CCR9. CCR9 ablation resulted in enhanced titer, weight loss, respiratory dysfunction, mortality, and inflammation, providing mechanistic support in mitigating protection from severe SARS-CoV-2 pathogenesis across species. This study represents a comprehensive analysis of susceptibility loci for an entire genus of human pathogens conducted, identifies a large collection of susceptibility loci and candidate genes that regulate multiple aspects type-specific and cross-CoV pathogenesis, and also validates the paradigm of using the CC platform to identify common cross-species susceptibility loci and genes for newly emerging and pre-epidemic viruses.","version":"1.1","doi":"10.1101/2021.05.14.444205","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443572","pub_date":"2021-5-14","title":"Siglec-1 on dendritic cells mediates SARS-CoV-2 trans-infection of target cells while on macrophages triggers proinflammatory responses","abstract":"COVID-19 pandemic is not yet under control by vaccination, and effective antivirals are critical for preparedness. Here we report that macrophages and dendritic cells, key antigen presenting myeloid cells (APCs), are largely resistant to SARS-CoV-2 infection. APCs effectively captured viruses within cellular compartments that lead to antigen degradation. Macrophages sense SARS-CoV-2 and released higher levels of cytokines, including those related to cytokine storm in severe COVID-19. The sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) present on APCs, which interacts with sialylated gangliosides on membranes of retroviruses or filoviruses, also binds SARS-CoV-2 via GM1. Blockage of Siglec-1 receptors by monoclonal antibodies reduces SARS-CoV-2 uptake and transfer to susceptible target cells. APCs expressing Siglec-1 and carrying SARS-CoV-2 are found in pulmonary tissues of non-human primates. Single cell analysis reveals the in vivo induction of cytokines in those macrophages. Targeting Siglec-1 could offer cross-protection against SARS-CoV-2 and other enveloped viruses that exploit APCs for viral dissemination, including those yet to come in future outbreaks.","version":"1.1","doi":"10.1101/2021.05.11.443572","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.14.444190","pub_date":"2021-5-14","title":"Glucosidase inhibitors suppress SARS-CoV-2 in tissue culture and may potentiate","abstract":"Iminosugar glucosidase inhibitors prevent the folding of a range of viral N-linked glycoproteins, ranging from hepatitis B to Ebola. We recently showed they inhibit folding and function of the ACE2 protein, which is the receptor for SARS-CoV-2, and they have also inhibited the SARS Spike polypeptides. Here we report that the imino sugar glucosidase inhibitors, N-butyl deoxynojirimycin (NBDNJ), which is approved for management of lysosomal storage disease (sold as Zavesca), and ureido-N-hexyl deoxynojirimycine (BSBI-19029), suppress the replication of SARS-ncCoV-2/USA/WA1/2020 strain, in tissue culture. Moreover, combinations of either of these iminosugars with Remdesivir were particularly potent in suppressing SARS-CoV-2. Briefly, NBDNJ, 19029 and Remdesivir suppressed SARS-CoV-2 production in A549ACE2 human lung cells with IC90s of ~130 \u03bcM, ~4.0 \u03bcM, and 0.006 \u03bcM respectively. The combination of as little as 0.037 \u03bcM of NBDNJ or 0.04 \u03bcM 19029, respectively and 0.002 \u03bcM Remdesivir yielded IC90s. Medical strategies to manage SARS-CoV-2 infection of people are urgently needed, and although Remdesivir and Favipiravir have shown efficacy, it is limited. NBDNJ was recently reported by others to have tissue culture activity against SARS-CoV-2, so our report confirms this, and extends the findings to a more potent iminosugar, 19029 and combination with Remdesivir. Since both NBDNJ and Remdesivir are both approved and available for human use, the possibility that NBDNJ has mono therapeutic value against SARS-CoV-2 as well as can enhance Remdesivir, may have clinical implications, which are discussed, here.","version":"1.1","doi":"10.1101/2021.05.14.444190","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.14.443968","pub_date":"2021-5-14","title":"Isolation and characterization of SARS-CoV-2 VOC, 20H/501Y.V2, from UAE travelers","abstract":"Multiple SARS-CoV-2 variants have been emerged and created serious public health in the affected countries. The variant of Concern associated with high transmissibility, disease severity and escape mutations is threat to vaccination program across the globe. Travel has been important factor in spread of SARS-CoV-2 variants worldwide. India has also witnessed the dreadful effect of these SARS-CoV-2 variants. Here, we report the Isolation and characterization of SARS-CoV-2 VOC, 20H/501Y.V2 (B.1.351), from UAE travelers to India. The virus isolate would be useful to determine the efficacy of the currently available vaccines in India.","version":"1.1","doi":"10.1101/2021.05.14.443968","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.22.441007","pub_date":"2021-5-14","title":"Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding","abstract":"Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics. Amino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme","version":"1.2","doi":"10.1101/2021.04.22.441007","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.12.443826","pub_date":"2021-5-14","title":"in silico Assessment of Antibody Drug Resistance to Bamlanivimab of SARS-CoV-2 Variant B.1.617","abstract":"The highly infectious SARS-CoV-2 variant B.1.617 with double mutations E484Q and L452R in the receptor binding domain (RBD) of SARS-CoV-2\u2019s spike protein is worrisome. Demonstrated in crystal structures, the residues 452 and 484 in RBD are not in direct contact with interfacial residues in the angiotensin converting enzyme 2 (ACE2). This suggests that albeit there are some possibly nonlocal effects, the E484Q and L452R mutations might not significantly affect RBD\u2019s binding with ACE2, which is an important step for viral entry into host cells. Thus, without the known molecular mechanism, these two successful mutations (from the point of view of SARS-CoV-2) can be hypothesized to evade human antibodies. Using in silico all-atom molecular dynamics (MD) simulation as well as deep learning (DL) approaches, here we show that these two mutations significantly reduce the binding affinity between RBD and the antibody LY-CoV555 (also named as Bamlanivimab) that was proven to be efficacious for neutralizing the wide-type SARS-CoV-2. With the revealed molecular mechanism on how L452R and E484K evade LY-CoV555, we expect that more specific therapeutic antibodies can be accordingly designed and/or a precision mixing of antibodies can be achieved in a cocktail treatment for patients infected with the variant B.1.617.","version":"1.1","doi":"10.1101/2021.05.12.443826","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422739","pub_date":"2021-5-14","title":"Decomposing the sources of SARS-CoV-2 fitness variation in the United States","abstract":"The fitness of a pathogen is a composite phenotype determined by many different factors influencing growth rates both within and between hosts. Determining what factors shape fitness at the host population-level is especially challenging because both intrinsic factors like pathogen genetics and extrinsic factors such as host behaviour influence between-host transmission potential. These challenges have been highlighted by controversy surrounding the population-level fitness effects of mutations in the SARS-CoV-2 genome and their relative importance when compared against non-genetic factors shaping transmission dynamics. Building upon phylodynamic birth-death models, we develop a new framework to learn how hundreds of genetic and non-genetic factors have shaped the fitness of SARS-CoV-2. We estimate the fitness effects of all amino acid variants and several structural variants that have circulated in the United States between February 2020 and March 2021 from viral phylogenies. We also estimate how much fitness variation among pathogen lineages is attributable to genetic versus non-genetic factors such as spatial heterogeneity in transmission rates. Before September 2020, most fitness variation between lineages can be explained by background spatial heterogeneity in transmission rates across geographic regions. Starting in late 2020, genetic variation in fitness increased dramatically with the emergence of several new lineages including B.1.1.7, B.1.427, B.1.429 and B.1.526. Our analysis also indicates that genetic variants in less well-explored genomic regions outside of Spike may be contributing significantly to overall fitness variation in the viral population.","version":"1.2","doi":"10.1101/2020.12.14.422739","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.441906","pub_date":"2021-5-14","title":"Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany, 2020","abstract":"SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with many new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August to October 2020, indicating the major cause of the second wave was the introduction of new variants.","version":"1.2","doi":"10.1101/2021.04.29.441906","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.13.443721","pub_date":"2021-5-13","title":"Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients","abstract":"One of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds. Analysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients. We report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection. SARS-CoV-2 infection results in the robust activation of the body\u2019s innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.","version":"1.1","doi":"10.1101/2021.05.13.443721","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.12.443948","pub_date":"2021-5-13","title":"Plasmacytoid dendritic cells produce type I interferon and reduce viral replication in airway epithelial cells after SARS-CoV-2 infection","abstract":"Infection with SARS-CoV-2 has caused a pandemic of unprecedented dimensions. SARS-CoV-2 infects airway and lung cells causing viral pneumonia. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with inborn errors of type I IFN response or auto-antibodies against IFN-\u03b1. Plasmacytoid dendritic cells (pDCs) are a unique immune cell population specialized in recognizing and controlling viral infections through the production of high concentrations of type I IFN. In this study, we isolated pDCs from healthy donors and showed that pDCs are able to recognize SARS-CoV-2 and rapidly produce large amounts of type I IFN. Sensing of SARS-CoV-2 by pDCs was independent of viral replication since pDCs were also able to recognize UV-inactivated SARS-CoV-2 and produce type I IFN. Transcriptional profiling of SARS-CoV-2 and UV-SARS-CoV-2 stimulated pDCs also showed a rapid type I and III IFN response as well as induction of several chemokines, and the induction of apoptosis in pDCs. Moreover, we modeled SARS-CoV-2 infection in the lung using primary human airway epithelial cells (pHAEs) and showed that co-culture of pDCs with SARS-CoV-2 infected pHAEs induces an antiviral response and upregulation of antigen presentation in pHAE cells. Importantly, the presence of pDCs in the co-culture results in control of SARS-CoV-2 replication in pHAEs. Our study identifies pDCs as one of the key cells that can recognize SARS-CoV-2 infection, produce type I and III IFN and control viral replication in infected cells. Type I interferons (IFNs) are a major part of the innate immune defense against viral infections. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with defects in the type I IFN response. Interestingly, many cells are not able to produce type I IFN after being infected with SARS-CoV-2 and cannot control viral infection. In this study we show that plasmacytoid dendritic cells are able to recognize SARS-CoV-2 and produce type I IFN, and that pDCs are able to help control viral infection in SARS-CoV-2 infected airway epithelial cells.","version":"1.1","doi":"10.1101/2021.05.12.443948","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.12.21257080","pub_date":"2021-05-13","title":"A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The progression of the SARS-CoV-2 pandemic in Africa has so far been heterogeneous and the full impact is not yet well understood. Here, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations, predominantly from Europe, which diminished following the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1 and C.1.1. Although distorted by low sampling numbers and blind-spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a breeding ground for new variants.</jats:p>","version":null,"doi":"10.1101/2021.05.12.21257080","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.03.438330","pub_date":"2021-5-13","title":"Driving potent neutralization of a SARS-CoV-2 Variant of Concern with a heterotypic boost","abstract":"The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.","version":"1.2","doi":"10.1101/2021.04.03.438330","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438675","pub_date":"2021-5-12","title":"Unique protein features of SARS-CoV-2 relative to other Sarbecoviruses","abstract":"Defining the unique properties of SARS-CoV-2 protein sequences, has potential to explain the range of Coronavirus Disease 2019 (COVID-19) severity. To achieve this we compared proteins encoded by all Sarbecoviruses using profile Hidden Markov Model similarities to identify protein features unique to SARS-CoV-2. Consistent with previous reports, a small set of bat and pangolin-derived Sarbecoviruses show the greatest similarity to SARS-CoV-2 but unlikely to be the direct source of SARS-CoV-2. Three proteins (nsp3, spike and orf9) showed differing regions between the bat Sarbecoviruses and SARS-CoV-2 and indicate virus protein features that might have evolved to support human infection and/or transmission. Spike analysis identified all regions of the protein that have tolerated change and revealed that the current SARS-CoV-2 variants of concern (VOCs) have sampled only a fraction (~31%) of the possible spike domain changes which have occurred historically in Sarbecovirus evolution. This result emphasises the evolvability of these coronaviruses and potential for further change in virus replication and transmission properties over the coming years.","version":"1.2","doi":"10.1101/2021.04.06.438675","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443286","pub_date":"2021-5-12","title":"Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B","abstract":"Safe and effective vaccines are needed to end the COVID-19 pandemic caused by SARS-CoV-2. Here we report the preclinical development of a lipid nanoparticle (LNP) formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern (VOCs), including the B.1.1.7, B.1.351 and P.1 lineages. No adverse effects were induced by PTX-COVID19-B in both mice and hamsters. These preclinical results indicate that PTX-COVID19-B is safe and effective. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 1 clinical trial ongoing (ClinicalTrials.gov number: NCT04765436). PTX-COVID19-B is a SARS-CoV-2 mRNA vaccine that is highly immunogenic, safe, and effective in preventing SARS-CoV-2 infection in mice and hamsters and is currently being evaluated in human clinical trials.","version":"1.1","doi":"10.1101/2021.05.11.443286","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.12.443228","pub_date":"2021-5-12","title":"Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used solely for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLN. A low dose of I-R-F induces not only high titer long-lasting neutralizing antibodies but also comprehensive T cell responses than RBD, and even provides comprehensive protection in one dose without adjuvant. This study shows that the I-R-F vaccine provides rapid and complete protection throughout upper and lower respiratory tracts against high dose SARS-CoV-2 challenge in rhesus macaques. Due to its potency and safety, this engineered vaccine may become one of the next-generation vaccine candidates in the global race to defeat COVID-19.","version":"1.1","doi":"10.1101/2021.05.12.443228","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443693","pub_date":"2021-5-12","title":"Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.","version":"1.1","doi":"10.1101/2021.05.11.443693","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443609","pub_date":"2021-5-12","title":"Early cross-coronavirus reactive signatures of protective humoral immunity against COVID-19","abstract":"The introduction of vaccines has inspired new hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against COVID-19, thus we profiled the earliest humoral signatures in a large cohort of severe and asymptomatic COVID-19 individuals. While a SARS-CoV-2-specific immune response evolved rapidly in survivors of COVID-19, non-survivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibody evolution. Given the conservation of S2 across \u03b2-coronaviruses, we found the early development of SARS-CoV-2-specific immunity occurred in tandem with pre-existing common \u03b2-coronavirus OC43 humoral immunity in survivors, which was selectively also expanded in individuals that develop paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.","version":"1.1","doi":"10.1101/2021.05.11.443609","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.12.443357","pub_date":"2021-5-12","title":"First description of two immune escape indian B.1.1.420 and B.1.617.1 SARS-CoV2 variants in France","abstract":"Following the outbreak of the SARS-CoV2 virus worldwide in 2019, the rapid widespread overtime of variants suggests today an undergoing positive selection of variants which could potentially provide advantageous genetic property of the virus. Numerous variants have already been described across different countries including N501Y, E484K or L452R mutations on gene coding to spike protein. Most recently, 2 new Indian variants with N440K and E484Q and L452R mutations associated with impaired antibody response and immune reactions were identified in India. The potential consequences of emerging variants are increased transmissibility, increased pathogenicity and the ability to escape natural or vaccine-induced immunity. We described for the first time in France both variants: the N440K immune escape variant within a new strain detected in France in a couple of patients who did not have any history of travel abroad and the new E484Q and L452R Indian variant from a patient travelling from Indian to Marseille to embark on a ship as a crew member. Such study of the circulating viral strains and their variants within the increasing number of infected people worldwide will provide further insights into the viral dissemination. Hence, real time close monitoring variant could help the scientific community to prevent fast-spreading and raise alarms towards potentially harmful variants.","version":"1.1","doi":"10.1101/2021.05.12.443357","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443686","pub_date":"2021-5-12","title":"Impacts on the structure-function relationship of SARS-CoV-2 spike by B.1.1.7 mutations","abstract":"The UK variant of the severe acute respiratory syndrome coronavirus (SARS-CoV-2), known as B.1.1.7, harbors several point mutations and deletions on the spike (s) protein, which potentially alter its structural epitopes to evade host immunity while enhancing host receptor binding. Here we report the cryo-EM structures of the S protein of B.1.1.7 in its apo form and in the receptor ACE2-bound form. One or two of the three receptor binding domains (RBDs) were in the open conformation but no fully closed form was observed. In the ACE-bound form, all three RBDs were engaged in receptor binding. The B.1.1.7-specific A570D mutation introduced a salt bridge switch that could modulate the opening and closing of the RBD. Furthermore, the N501Y mutation in the RBD introduced a favorable \u03c0-\u03c0 interaction manifested in enhanced ACE2 binding affinity. The N501Y mutation abolished the neutralization activity of one of the three potent neutralizing antibodies (nAbs). Cryo-EM showed that the cocktail of other two nAbs simultaneously bound to all three RBDs. Furthermore, the nAb cocktail synergistically neutralized different SARS-CoV-2 pseudovirus strains, including the B.1.1.7.","version":"1.1","doi":"10.1101/2021.05.11.443686","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443708","pub_date":"2021-5-12","title":"Energy Landscape of the SARS-CoV-2 Reveals Extensive Conformational Heterogeneity","abstract":"Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As for any protein, some of the spike\u2019s conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the spike protein conformations, and identify molecular rearrangements along the most-likely conformational path in the vicinity of the open (so called 1RBD-up) state. The resulting global and local atomic refinements reveal larger movements than those expected by comparing the reported 1RBD-up and 1RBD-down cryo-EM models. Here we report greater degrees of \u201copenness\u201d in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these conformations along the minimum free-energy pathway. A local analysis of seven key binding pockets reveals that six out them, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, and protein-glycan interaction sites, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This is reminiscent of a conformational pre-equilibrium. We found only one binding pocket, namely antibody AB-C135 to remain closed along the entire minimum free energy path, suggesting an induced fit mechanism for this enzyme.","version":"1.1","doi":"10.1101/2021.05.11.443708","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443659","pub_date":"2021-5-11","title":"SARS-CoV-2 Variant Identification Using a Genome Tiling Array and Genotyping Probes","abstract":"With over three million deaths worldwide attributed to the respiratory disease COVID-19 caused by the novel coronavirus SARS-CoV-2, it is essential that continued efforts be made to track the evolution and spread of the virus globally. We previously presented a rapid and cost-effective method to sequence the entire SARS-CoV-2 genome with 95% coverage and 99.9% accuracy. This method is advantageous for identifying and tracking variants in the SARS-CoV-2 genome when compared to traditional short read sequencing methods which can be time consuming and costly. Herein we present the addition of genotyping probes to our DNA chip which target known SARS-CoV-2 variants. The incorporation of the genotyping probe sets along with the advent of a moving average filter have improved our sequencing coverage and accuracy of the SARS-CoV-2 genome.","version":"1.1","doi":"10.1101/2021.05.11.443659","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443555","pub_date":"2021-5-11","title":"Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis","abstract":"Molnupiravir is an orally available antiviral drug candidate that is in phase III trials for the treatment of COVID-19 patients. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in patients. Here we establish the molecular mechanisms that underlie molnupiravir-induced RNA mutagenesis by the RNA-dependent RNA polymerase (RdRp) of the coronavirus SARS-CoV-2. Biochemical assays show that the RdRp readily uses the active form of molnupiravir, \u03b2-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. Incorporation of NHC monophosphate into nascent RNA does not impair further RdRp progression. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes containing mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.","version":"1.1","doi":"10.1101/2021.05.11.443555","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.21249238","pub_date":"2021-05-11","title":"multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL\n                  <jats:sup>\u22121</jats:sup>\n                  RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.\n                </jats:p>","version":null,"doi":"10.1101/2021.05.07.21249238","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.11.443477","pub_date":"2021-5-11","title":"Viral shedding and transmission after natural infection and vaccination in an animal model of SARS-CoV-2 propagation","abstract":"At present, global immunity to SARS-CoV-2 resides within a heterogeneous combination of susceptible, naturally infected and vaccinated individuals. The extent to which viral shedding and transmission occurs on re-exposure to SARS-CoV-2 after prior natural exposure or vaccination is an emerging area of understanding. We used Sialodacryoadenitis Virus (SDAV) in rats to model the extent to which immune protection afforded by prior natural infection via high risk (inoculation; direct contact) or low risk (fomite) exposure, or by vaccination, influenced viral shedding and transmission on re-exposure. On initial infection, we confirmed that amount, duration and consistency of viral shedding were correlated with exposure risk. Animals were reinfected after 3.7-5.5 months using the same exposure paradigm. Amount and duration of viral shedding were correlated with re-exposure type and serologic status. 59% of seropositive animals shed virus. Previously exposed seropositive reinfected animals were able to transmit virus to 25% of naive recipient rats after 24-hour exposure by direct contact. Rats vaccinated intranasally with a related virus (Parkers Rat Coronavirus) were able to transmit SDAV to only 4.7% of naive animals after a 7-day direct contact exposure, despite comparable viral shedding. Observed cycle threshold values associated with transmission in both groups ranged from 29-36 cycles, however observed shedding was not a prerequisite for transmission. Results indicate that low-level shedding in both naturally infected and vaccinated seropositive animals can propagate infection in susceptible individuals. Extrapolated to COVID-19, our results suggest that continued propagation of SARS-CoV-2 by seropositive previously infected or vaccinated individuals is possible.","version":"1.1","doi":"10.1101/2021.05.11.443477","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443532","pub_date":"2021-5-11","title":"Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2","abstract":"Human angiotensin I-converting enzyme 2 (hACE2) is a type-I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV and SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation plays no role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell to cell fusion mediated by SARS-CoV S protein but not SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which could be the result of low cell surface expression of the deglycosylated ACE2 receptor. Elucidating the role of glycosylation in the virus-receptor interaction is important for the development of approaches that disrupt infection. In this study, we show that deglycosylation of both ACE2 and S had a minimal effect on the Spike-ACE2 interaction. In addition, we found that removal of N-glycans of ACE2 impaired its ability to support an efficient transduction of SARS-CoV and SARS-CoV-2 S pseudotyped viruses. Our data suggest that the role of deglycosylation of ACE2 on reducing infection is likely due to a reduced expression of the viral receptor on the cell surface. These findings offer insight into the glycan structure and function of ACE2, and potentially suggest that future antiviral therapies against coronaviruses and other coronavirus-related illnesses involving inhibition of ACE2 recruitment to the cell membrane could be developed.","version":"1.1","doi":"10.1101/2021.05.10.443532","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443519","pub_date":"2021-5-11","title":"Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies","abstract":"The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.","version":"1.1","doi":"10.1101/2021.05.10.443519","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443524","pub_date":"2021-5-11","title":"CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes","abstract":"The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.","version":"1.1","doi":"10.1101/2021.05.10.443524","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434872","pub_date":"2021-5-11","title":"Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice","abstract":"The emergence of SARS-CoV in 2003 and SARS-CoV-2 in 2019 highlights the need to develop universal vaccination strategies against the broader Sarbecovirus subgenus. Using chimeric spike designs, we demonstrate protection against challenge from SARS-CoV, SARS-CoV-2, SARS-CoV-2 B.1.351, bat CoV (Bt-CoV) RsSHC014, and a heterologous Bt-CoV WIV-1 in vulnerable aged mice. Chimeric spike mRNAs induced high levels of broadly protective neutralizing antibodies against high-risk Sarbecoviruses. In contrast, SARS-CoV-2 mRNA vaccination not only showed a marked reduction in neutralizing titers against heterologous Sarbecoviruses, but SARS-CoV and WIV-1 challenge in mice resulted in breakthrough infection. Chimeric spike mRNA vaccines efficiently neutralized D614G, UK B.1.1.7., mink cluster five, and the South African B.1.351 variant of concern. Thus, multiplexed-chimeric spikes can prevent SARS-like zoonotic coronavirus infections with pandemic potential. Chimerized RBD, NTD, and S2 spike mRNA-LNPs protect mice against epidemic, zoonotic, and pandemic SARS-like viruses","version":"1.2","doi":"10.1101/2021.03.11.434872","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.11.443549","pub_date":"2021-5-11","title":"Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells even at a distance from the light source","abstract":"Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E, and that UVA treatment results in an increase in intracellular levels of MAVS. In this study, we set out to determine the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (such as via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 minutes and in unexposed controls, at 30-40% and at 100% confluency. MAVS levels were also compared in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. Also, MAVS was assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. The results show that UVA increases the expression of MAVS protein. Cells in a confluent monolayer exposed to UVA were able to confer an elevation in MAVS in cells adjacent to the exposed section, and even cells in the most distant sections not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to distant confluent cells not exposed to light.","version":"1.1","doi":"10.1101/2021.05.11.443549","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.443114","pub_date":"2021-5-10","title":"Following the Trail of One Million Genomes: Footprints of SARS-CoV-2 Adaptation to Humans","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accumulated genomic mutations at an approximately linear rate since it first infected human populations in late 2019. Controversies remain regarding the identity, proportion, and effects of adaptive mutations as SARS-CoV-2 evolves from a bat-to a human-adapted virus. The potential for vaccine-escape mutations poses additional challenges in pandemic control. Despite being of great interest to therapeutic and vaccine development, human-adaptive mutations in SARS-CoV-2 are masked by a genome-wide linkage disequilibrium under which neutral and even deleterious mutations can reach fixation by chance or through hitchhiking. Furthermore, genome-wide linkage equilibrium imposes clonal interference by which multiple adaptive mutations compete against one another. Informed by insights from microbial experimental evolution, we analyzed close to one million SARS-CoV-2 genomes sequenced during the first year of the COVID-19 pandemic and identified putative human-adaptive mutations according to the rates of synonymous and missense mutations, temporal linkage, and mutation recurrence. Furthermore, we developed a forward-evolution simulator with the realistic SARS-CoV-2 genome structure and base substitution probabilities able to predict viral genome diversity under neutral, background selection, and adaptive evolutionary models. We conclude that adaptive mutations have emerged early, rapidly, and constantly to dominate SARS-CoV-2 populations despite clonal interference and purifying selection. Our analysis underscores a need for genomic surveillance of mutation trajectories at the local level for early detection of adaptive and immune-escape variants. Putative human-adaptive mutations are over-represented in viral proteins interfering host immunity and binding host-cell receptors and thus may serve as priority targets for designing therapeutics and vaccines against human-adapted forms of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.07.443114","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.09.443299","pub_date":"2021-5-10","title":"Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant","abstract":"SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.","version":"1.1","doi":"10.1101/2021.05.09.443299","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443480","pub_date":"2021-5-10","title":"Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation","abstract":"Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. Phosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.05.10.443480","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.09.443331","pub_date":"2021-5-10","title":"SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity","abstract":"The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of other coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2 in mice. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, along with high magnitude neutralizing titers against major VoC. These results provide design strategies for pan-coronavirus vaccine development. Iterative structure-based design of four Spike-domain Ferritin nanoparticle classes of immunogens SpFN-ALFQ and RFN-ALFQ immunization elicits potent neutralizing activity against SARS-CoV-2, variants of concern, and SARS-CoV-1 Passively transferred IgG from immunized C57BL/6 mice protects K18-hACE2 mice from lethal SARS-CoV-2 challenge","version":"1.1","doi":"10.1101/2021.05.09.443331","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443438","pub_date":"2021-5-10","title":"SARS-COV-2 Recombinant Receptor-Binding-Domain (RBD) Induces Neutralising Antibodies Against Variant Strains of SARS-CoV-2 and SARS-CoV-1","abstract":"SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them are targeting the spike protein (or virion) in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested the utility of SARS-CoV-2 spike RBD as a vaccine antigen and explore the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results indicate RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralisation of variants B1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.","version":"1.1","doi":"10.1101/2021.05.10.443438","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.09.442808","pub_date":"2021-5-10","title":"A human antibody that broadly neutralizes betacoronaviruses protects against SARS-CoV-2 by blocking the fusion machinery","abstract":"The repeated spillovers of \u03b2-coronaviruses in humans along with the rapid emergence of SARS-CoV-2 escape variants highlight the need to develop broad coronavirus therapeutics and vaccines. Five monoclonal antibodies (mAbs) were isolated from COVID-19 convalescent individuals and found to cross-react with multiple \u03b2-coronavirus spike (S) glycoproteins by targeting the stem helix. One of these mAbs, S2P6, cross-reacts with more than twenty human and animal \u03b2-coronavirus S glycoproteins and broadly neutralizes SARS-CoV-2 and pseudotyped viruses from the sarbecovirus, merbecovirus and embecovirus subgenera. Structural and functional studies delineate the molecular basis of S2P6 cross-reactivity and broad neutralization and indicate that this mAb blocks viral entry by inhibiting membrane fusion. S2P6 protects hamsters challenged with SARS-CoV-2 (including the B.1.351 variant of concern) through direct viral neutralization and Fc-mediated effector functions. Serological and B cell repertoire analyses indicate that antibodies targeting the stem helix are found in some convalescent donors and vaccinees but are predominantly of narrow specificity. Germline reversion of the identified cross-reactive mAbs revealed that their unmutated ancestors are specific for the endemic OC43 or HKU1 viruses and acquired enhanced affinity and breadth through somatic mutations. These data demonstrate that conserved epitopes in the coronavirus fusion machinery can be targeted by protective antibodies and provide a framework for structure-guided design of pan-\u03b2-coronavirus vaccines eliciting broad protection.","version":"1.1","doi":"10.1101/2021.05.09.442808","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.09.443238","pub_date":"2021-5-10","title":"Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response","abstract":"The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all vaccine candidates. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms, are compatible with established changes in SARS-CoV-2 vaccines, and are compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation vaccines may enhance the neutralizing antibody response and lead to pan-SARS-CoV-2 protection.","version":"1.1","doi":"10.1101/2021.05.09.443238","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443474","pub_date":"2021-5-10","title":"Binding of SARS-CoV-2 fusion peptide to host membranes","abstract":"During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. Initial contact with the host cell and membrane fusion are both mediated by the viral spike (S) protein. Proteolytic cleavage of S at the S2\u2032 site exposes its 40 amino acid long fusion peptide (FP). Binding of the FP to the host membrane anchors the S2 domain of S in both the viral and the host membrane. The reorganization of S2 then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 \u03bcs-long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than ten-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.","version":"1.1","doi":"10.1101/2021.05.10.443474","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443422","pub_date":"2021-5-10","title":"Rapid inactivation of SARS-CoV-2 variants by continuous and intermittent irradiation with a deep-ultraviolet light-emitting diode (DUV-LED) device","abstract":"More than 1 year has passed since social activities have been restricted due to the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More recently, novel SARS-CoV-2 variants have been spreading around the world, and there is growing concern of higher transmissibility of the variants and weaker protective efficacy of vaccine against the variants. Immediate measures are needed to reduce human exposure to the virus. In this study, the antiviral efficacy of deep-ultraviolet light-emitting diode (DUV-LED) irradiation (280 \u00b1 5 nm, 3.75 mW/cm2) against three SARS-CoV-2 variants was evaluated. For the B.1.1.7, B.1.351, and P.1 strains, the infectious titer reduction rates of 96.3%, 94.6%, and 91.9%, respectively, were already recognized with the irradiation of virus stocks for 1 s, and the rates increased to 99.9%, 99.9%, and 99.8%, respectively, with irradiation for 5 s. We also tested the effect of pulsed DUV-LED irradiation (7.5 mW/cm2, duty rate: 50%, frequency: 1 KHz) under the same output conditions as continuous irradiation, and found that the antiviral efficacy of pulsed and continuous irradiation was the same. These findings suggest that SARS-CoV-2 may be instantly inactivated by DUV-LED irradiation if the DUV-LED device is further developed and optimized to increase its output.","version":"1.1","doi":"10.1101/2021.05.10.443422","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443377","pub_date":"2021-5-10","title":"SARS-CoV-2 receptor ACE2 identifies immuno-hot tumors in breast cancer","abstract":"Angiotensin-converting enzyme 2 (ACE2) is known as a host cell receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is identified to be dysregulated in multiple tumors. Although the characterization of abnormal ACE2 expression in malignancies has been preliminarily explored, in-depth analysis of ACE2 in breast cancer (BRCA) has not been elucidated. A systematic pan-cancer analysis was conducted to assess the expression pattern and immunological role of ACE2 based on RNA-sequencing (RNA-seq) data downloaded from The Cancer Genome Atlas (TCGA). Next, correlations between ACE2 expression immunological characteristics in the BRCA tumor microenvironment (TME) were evaluated. Also, the role of ACE2 in predicting the clinical features and the response to therapeutic options in BRCA was estimated. These findings were subsequently validated in another public transcriptomic cohort as well as a recruited cohort. ACE2 was lowly expressed in most cancers compared with adjacent tissues. ACE2 was positively correlated with immunomodulators, tumor-infiltrating immune cells (TIICs), cancer immunity cycles, immune checkpoints, and tumor mutation burden (TMB). Besides, high ACE2 levels indicated the triple-negative breast cancer (TNBC) subtype of BRCA, lower response to endocrine therapy and higher response to chemotherapy, anti-ERBB therapy, antiangiogenic therapy and immunotherapy. To sum up, ACE2 correlates with an inflamed TME and identifies immuno-hot tumors, which may be used as an auxiliary biomarker for the identification of immunological characteristics in BRCA.","version":"1.1","doi":"10.1101/2021.05.10.443377","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443404","pub_date":"2021-5-10","title":"In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum","abstract":"Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (\u2265 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.","version":"1.1","doi":"10.1101/2021.05.10.443404","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443494","pub_date":"2021-5-10","title":"Nanopore dwell time analysis permits sequencing and conformational assignment of pseudouridine in SARS-CoV-2","abstract":"Nanopore devices can directly sequence RNA, and the method has the potential to determine locations of epitranscriptomic modifications that have grown in significance because of their roles in cell regulation and stress response. Pseudouridine (\u03a8), the most common modification in RNA, was sequenced with a nanopore system using a protein sensor with a helicase brake in synthetic RNAs with 100% modification at 18 known human pseudouridinylation sites. The new signals were compared to native uridine (U) control strands to characterize base calling and associated errors as well as ion current and dwell time changes. The data point to strong sequence context effects in which \u03a8 can easily be detected in some contexts while in others \u03a8 yields signals similar to U that would be false negatives in an unknown sample. We identified that the passage of \u03a8 through the helicase brake slowed the translocation kinetics compared to U and showed a smaller sequence bias that could permit detection of this modification in RNA. The unique signals from \u03a8 relative to U are proposed to reflect the syn-anti conformational flexibility of \u03a8 not found in U, and the difference in \u03c0 stacking between these bases. This observation permitted analysis of SARS-CoV-2 nanopore sequencing data to identify five conserved \u03a8 sites on the 3\u2019 end of the viral sub-genomic RNAs, and other less conserved \u03a8 sites. Using the helicase as a sensor protein in nanopore sequencing experiments enables detection of this modification in a greater number of relevant sequence contexts. The data are discussed concerning their analytical and biological significance.","version":"1.1","doi":"10.1101/2021.05.10.443494","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.09.443289","pub_date":"2021-5-10","title":"Hyperinflammation evokes different antiviral TMPRSS2 and ADAM17 expression responses in human gut xenograft versus host mouse gut although overall genomic responses are similar","abstract":"The global spread of the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the pandemic outbreak of coronavirus disease 2019 (COVID-19), an inflammatory disease that is primarily affecting the respiratory system. However, gastrointestinal symptoms in COVID-19 patients suggests that the gut may present another viral target organ. Disease development and severity is dependent on viral interaction with two cell surface human proteins, ACE2 and TMPRSS2, and on antiviral response which may lead to systemic hyperinflammatory syndrome and multiorgan dysfunction. Understanding the host response to SARS-CoV-2 infection and the pathology of the disease will be greatly enhanced by the development of appropriate animal models. Laboratory mice have been the mainstay of therapeutic and vaccine development, however, the virus does not grow in wild type mice and only induced mild disease in transgenic animals expressing human ACE2. As there are known differences between immune response in laboratory mice and humans we evaluated the response of human gut developed as xenografts and host mouse gut following systemic LPS injections as a hyperinflammation model system. The orthologous gene expression levels in the mouse and human gut were highly correlated (Spearman\u2019s rank correlation coefficient: 0.28\u20130.76) and gene set enrichment analysis of significantly upregulated human and mouse genes revealed that a number of inflammatory and immune response pathways are commonly regulated in the two species. However, species differences were also observed, most importantly, in the inflamed human gut but not in the mouse gut, there was clear upregulation of mRNAs coding for TMPRSS2, ADAM17 and for RIG-I-like receptors, which are involved in the recognition of viruses and in antiviral innate immune response. Moreover, using species-specific immunofluorescence microscopy, we demonstrated the expression and localization of human ACE2 and TMPRSS2 proteins, which are essential elements of the molecular machinery that enables SARS-CoV-2 to infect and replicate in human gut cells. Our findings demonstrate that the intestinal immune response to inflammation in humans and mice are generally very similar. However, certain human-specific diseases, such as COVID-19, can only be successfully studied in an experimental model of human tissue, such as the gut xenograft.","version":"1.1","doi":"10.1101/2021.05.09.443289","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.10.443496","pub_date":"2021-5-10","title":"Receptor binding may directly activate the fusion machinery in coronavirus spike glycoproteins","abstract":"SARS-CoV-2, the causative agent of the COVID-19 pandemic, is an enveloped RNA virus. Trimeric spike glycoproteins extend outward from the virion; these class I viral membrane fusion proteins mediate entry of the virus into a host cell and are the dominant antigen for immune response. Cryo-EM studies have generated a large number of structures for the spike either alone, or bound to the cognate receptor ACE2 or antibodies, with the three receptor binding domains (RBDs) seen closed, open, or in various combinations. Binding to ACE2 requires an open RBD, and is believed to trigger the series of dramatic conformational changes in the spike that lead to the shedding of the S1 subunit and transition of the spring-loaded S2 subunit to the experimentally observed post-fusion structure. The steps following ACE2 binding are poorly understood despite extensive characterization of the spike through X-ray, cryo-EM, and computation. Here, we use all-atom simulations, guided by analysis of 81 existing experimental structures, to develop a model for the structural and energetic coupling that connects receptor binding to activation of the membrane fusion machinery.","version":"1.1","doi":"10.1101/2021.05.10.443496","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443244","pub_date":"2021-5-09","title":"The K18-hACE2 Transgenic Mouse Model Recapitulates Non-Severe and Severe COVID-19 in Response to Infectious Dose of SARS-CoV-2 Virus","abstract":"A comprehensive analysis and characterization of a SARS-CoV-2 infection model that mimics non-severe and severe COVID-19 in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2\u00d7103 and 2\u00d7104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lungs, liver, and kidney, while lower doses (2\u00d7101 and 2\u00d7102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this humanized hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in biopsy samples from COVID-19 patients. Finally, the mice that recovered after infection with a low dose of virus also survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human biopsy samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. The pandemic of COVID-19 has reached 112,589,814 cases and caused 2,493,795 deaths worldwide as of February 23, 2021, has raised an urgent need for development of novel drugs and therapeutics to prevent the spread and pathogenesis of SARS-CoV-2. To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 virus to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of anti-viral drugs and therapeutics.","version":"1.1","doi":"10.1101/2021.05.08.443244","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443267","pub_date":"2021-5-09","title":"An intranasal vaccine durably protects against SARS-CoV-2 variants in mice","abstract":"SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy and the end of the COVID-19 pandemic. We recently reported the protective activity of a single-dose intranasally-administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose-response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351 and B.1.1.28 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains.","version":"1.1","doi":"10.1101/2021.05.08.443267","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443275","pub_date":"2021-5-09","title":"In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin \u03b15\u03b21, and human ACE2 to facilitate viral entry into host cells in vitro. We previously found that inhibition of integrin \u03b15\u03b21 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro. In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo. We discovered that treatment with single- or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 hours after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence and improved lung histology in a majority of mice 72 hours post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin \u03b15 and \u03b1v (an \u03b15-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C\u2013X\u2013C motif chemokine ligand 10 (Cxcl10), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin \u03b15\u03b21 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.","version":"1.1","doi":"10.1101/2021.05.08.443275","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.21256675","pub_date":"2021-05-09","title":"Relative role of border restrictions, case finding and contact tracing in controlling SARS-CoV-2 in the presence of undetected transmission","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Several countries have controlled the spread of COVID-19 through varying combinations of border restrictions, case finding, contact tracing and careful calibration on the resumption of domestic activities. However, evaluating the effectiveness of these measures based on observed cases alone is challenging as it does not reflect the transmission dynamics of missed infections.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Combining data on notified local COVID-19 cases with known and unknown sources of infections (i.e. linked and unlinked cases) in Singapore in 2020 with a transmission model, we reconstructed the incidence of missed infections and estimated the relative effectiveness of different types of outbreak control. We also examined implications for estimation of key real-time metrics \u2014 the reproduction number and ratio of unlinked to linked cases, using observed data only as compared to accounting for missed infections.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Prior to the partial lockdown in Singapore, initiated in April 2020, we estimated 89% (95%CI 75\u201399%) of the infections caused by notified cases were contact traced, but only 12.5% (95%CI 2\u201369%) of the infections caused by missed infectors were identified. We estimated that the reproduction number was 1.23 (95%CI 0.98\u20131.54) after accounting for missed infections but was 0.90 (95%CI 0.79-1.1) based on notified cases alone. At the height of the outbreak, the ratio of missed to notified infections was 34.1 (95%CI 26.0\u201346.6) but the ratio of unlinked to linked infections was 0.81 (95%CI 0.59\u20131.36). Our results suggest that when case finding and contact tracing identifies at least 50% and 20% of the infections caused by missed and notified cases respectively, the reproduction number could be reduced by more than 14%, rising to 20% when contact tracing is 80% effective.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Depending on the relative effectiveness of border restrictions, case finding and contact tracing, unobserved outbreak dynamics can vary greatly. Commonly used metrics to evaluate outbreak control \u2014 typically based on notified data \u2014 could therefore misrepresent the true underlying outbreak.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Ministry of Health, Singapore.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>We searched PubMed, BioRxiv and MedRxiv for articles published in English up to Mar 20, 2021 using the terms: (2019-nCoV OR \u201cnovel coronavirus\u201d OR COVID-19 OR SARS-CoV-2) AND (border OR travel OR restrict* OR import*) AND (\u201ccase finding\u201d OR surveillance OR test*) AND (contact trac*) AND (model*). The majority of modelling studies evaluated the effectiveness of various combinations of interventions in the absence of outbreak data. For studies that reconstructed the initial spread of COVID-19 with outbreak data, they further simulated counterfactual scenarios in the presence or absence of these interventions to quantify the impact to the outbreak trajectory. None of the studies disentangled the effects of case finding, contact tracing, introduction of imported cases and the reproduction number, in order to reproduce an observed SARS-CoV-2 outbreak trajectory.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>Notified COVID-19 cases with unknown and known sources of infection are identified through case finding and contact tracing respectively. Their respective daily incidence and the growth rate over time may differ. By capitalising on these differences in the outbreak data and the use of a mathematical model, we could identify the key drivers behind the growth and decline of both notified and missed COVID-19 infections in different time periods \u2014 e.g. domestic transmission vs external introductions, relative role of case finding and contact tracing in domestic transmission. Estimating the incidence of missed cases also allows us to evaluate the usefulness of common surveillance metrics that rely on observed cases.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>Comprehensive outbreak investigation data integrated with mathematical modelling helps to quantify the strengths and weaknesses of each outbreak control intervention during different stages of the pandemic. This would allow countries to better allocate limited resources to strengthen outbreak control. Furthermore, the data and modelling approach allows us to estimate the extent of missed infections in the absence of population wide seroprevalence surveys. This allows us to compare the growth dynamics of notified and missed infections as reliance on the observed data alone may create the illusion of a controlled outbreak.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.05.05.21256675","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.05.21256681","pub_date":"2021-05-09","title":"A Prenylated dsRNA Sensor Protects Against Severe COVID-19 and is Absent in Horseshoe Bats","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Cell autonomous antiviral defenses can inhibit the replication of viruses and reduce transmission and disease severity. To better understand the antiviral response to SARS-CoV-2, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that while some people can express a prenylated OAS1 variant, that is membrane-associated and blocks SARS-CoV-2 infection, other people express a cytosolic, nonprenylated OAS1 variant which does not detect SARS-CoV-2 (determined by the splice-acceptor SNP Rs10774671). Alleles encoding nonprenylated OAS1 predominate except in people of African descent. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response. Remarkably, approximately 55 million years ago, retrotransposition ablated the OAS1 prenylation signal in horseshoe bats (the presumed source of SARS-CoV-2). Thus, SARS-CoV-2 never had to adapt to evade this defense. As prenylated OAS1 is widespread in animals, the billions of people that lack a prenylated OAS1 could make humans particularly vulnerable to the spillover of coronaviruses from horseshoe bats.</jats:p>","version":null,"doi":"10.1101/2021.05.05.21256681","journal":"medRxiv","score":null},{"id":"10.1101/2021.05.07.443177","pub_date":"2021-5-09","title":"Mathematical Analysis and Topology of SARS-CoV-2, Bonding with Cells and Unbonding","abstract":"We consider the structure of the novel coronavirus (SARS-Cov-2) in terms of the number of spikes that are critical in bonding with the cells in the host. Bonding formation is considered for selection criteria with and without any treatments. Functional mappings from the discrete space of spikes and cells and their analysis are performed. We found that careful mathematical constructions help in understanding the treatment impacts, and the role of vaccines within a host. Smale\u2019s famous 2-D horseshoe examples inspired us to create 3-D visualizations and understand the topological diffusion of spikes from one human organ to another organ. The pharma industry will benefit from such an analysis for designing efficient treatment and vaccine strategies.","version":"1.1","doi":"10.1101/2021.05.07.443177","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443212","pub_date":"2021-5-09","title":"The role of host cell glycans on virus infectivity: The SARS-CoV-2 case","abstract":"Long and complex chains of sugars, called glycans, often coat both the cell and protein surface. Glycans both modulate specific interactions and protect cells. On the cell surface, these sugars form a cushion known as the glycocalyx. Here, we show that Heparan Sulfate (HS) chains \u2013 part of the glycocalyx \u2013 and other glycans \u2013 expressed on the surface of both host and virus proteins \u2013 have a critical role in modulating both attractive and repulsive potentials during viral infection. We analyse the SARS-CoV-2 virus, modelling its spike proteins binding to HS chains and two key entry receptors, ACE2 and TMPRSS2. We include the volume exclusion effect imposed on the HS chains impose during virus insertion into glycocalyx and the steric repulsion caused by changes in the conformation of the ACE2 glycans involved in binding to the spike. We then combine all these interactions, showing that the interplay of all these components is critical to the behaviour of the virus. We show that the virus tropism depends on the combinatorial expression of both HS chains and receptors. Finally, we demonstrate that when both HS chains and entry receptors express at high density, steric effects dominate the interaction, preventing infection.","version":"1.1","doi":"10.1101/2021.05.08.443212","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.442971","pub_date":"2021-5-09","title":"Expansion of tissue-resident CD8+ T cells and CD4+ Th17 cells in the nasal mucosa following mRNA COVID-19 vaccination","abstract":"Vaccines against SARS-CoV-2 have shown high efficacy in clinical trials, yet a full immunologic characterization of these vaccines, particularly within the upper respiratory tract, remains lacking. We enumerated and phenotyped T cells in nasal mucosa and blood before and after vaccination with the Pfizer-BioNTech COVID-19 vaccine (n =21). Tissue-resident memory (Trm) CD8+ T cells expressing CD69+CD103+ expanded \u223c12 days following the first and second doses, by 0.31 and 0.43 log10 cells per swab respectively (p=0.058 and p=0.009 in adjusted linear mixed models). CD69+CD103+CD8+ T cells in the blood decreased post-vaccination. Similar increases in nasal CD8+CD69+CD103-T cells were observed, particularly following the second dose. CD4+ Th17 cells were also increased in abundance following both doses. Following stimulation with SARS-CoV-2 spike peptides, CD8+ T cells increased expression of CD107a and CD154. These data suggest that nasal T cells may be induced and contribute to the protective immunity afforded by this vaccine.","version":"1.1","doi":"10.1101/2021.05.07.442971","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.08.443047","pub_date":"2021-5-09","title":"AutoVEM2: a flexible automated tool to analyze candidate key mutations and epidemic trends for virus","abstract":"In our previous work, we developed an automated tool, AutoVEM, for real-time monitoring the candidate key mutations and epidemic trends of SARS-CoV-2. In this research, we further developed AutoVEM into AutoVEM2. AutoVEM2 is composed of three modules, including call module, analysis module, and plot module, which can be used modularly or as a whole for any virus, as long as the corresponding reference genome is provided. Therefore, it\u2019s much more flexible than AutoVEM. Here, we analyzed three existing viruses by AutoVEM2, including SARS-CoV-2, HBV and HPV-16, to show the functions, effectiveness and flexibility of AutoVEM2. We found that the N501Y locus was almost completely linked to the other 16 loci in SARS-CoV-2 genomes from the UK and Europe. Among the 17 loci, 5 loci were on the S protein and all of the five mutations cause amino acid changes, which may influence the epidemic traits of SARS-CoV-2. And some candidate key mutations of HBV and HPV-16, including T350G of HPV-16 and C659T of HBV, were detected. In brief, we developed a flexible automated tool to analyze candidate key mutations and epidemic trends for any virus, which would become a standard process for virus analysis based on genome sequences in the future. An automatic tool to quickly analyze candidate key mutations and epidemic trends for any virus was developed. Our integrated analysis method and tool could become a standard process for virus mutation and epidemic trend analysis based on genome sequences in the future. N501Y with the other 16 highly linked mutation sites of SARS-CoV-2 in the UK and Europe were further confirmed, and some valuable mutation sites of HBV and HPV-16 were detected.","version":"1.1","doi":"10.1101/2021.05.08.443047","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442742","pub_date":"2021-5-07","title":"Sequencing SARS-CoV-2 in a malaria research laboratory in Mali, West Africa: the road to sequencing the first SARS-CoV-2 genome in Mali","abstract":"Next generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, the genomic surveillance has never been so important until the COVID 19 pandemic. This has been evidenced with the detection of new variants of SARS-CoV-2 in the United Kingdom, South Africa and Brazil recently using genomic surveillance. Until recently, Malian scientists did not have access to any local NGS platform and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform following by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200x. We have demonstrated our capability of generating a high quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.","version":"1.2","doi":"10.1101/2021.05.05.442742","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.443055","pub_date":"2021-5-07","title":"Infection, recovery and re-infection of farmed mink with SARS-CoV-2","abstract":"Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene. The infected mink recovered and after free-testing of the mink, the animals remained seropositive. During follow-up studies, after a period of more than 2 months without virus detection, over 75% of tested animals scored positive again for SARS-CoV-2 RNA. Whole genome sequencing showed that the virus circulating during this re-infection was most closely related to the virus identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies after re-infection than at free-testing. Thus, following recovery from an initial infection, seropositive mink rapidly became susceptible to re-infection by SARS-CoV-2. Following widespread infection with SARS-CoV-2 of mink on a farm, all tested animals had seroconverted and the farm was then tested free of infection; however, less than 3 months later, a further round of infection affected more than 75% of tested animals.","version":"1.1","doi":"10.1101/2021.05.07.443055","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.443089","pub_date":"2021-5-07","title":"poreCov - an easy to use, fast, and robust workflow for SARS-CoV-2 genome reconstruction via nanopore sequencing","abstract":"In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.","version":"1.1","doi":"10.1101/2021.05.07.443089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.01.442286","pub_date":"2021-5-07","title":"Phylodynamic insights on the early spread of the COVID-19 pandemic and the efficacy of intervention measures","abstract":"We performed phylodynamic analyses of all available SARS-CoV-2 genomes from the early phase of the COVID-19 pandemic\u2014combined with a novel dataset on contemporary global air-travel volume\u2014to assess the efficacy of public-health measures on viral geographic spread. Globally, viral dispersal rates are significantly correlated with air-travel volume, and widespread international air-travel bans imposed against China by early February coincide with a significant reduction in geographic viral spread. In North America, the efficacy of this travel ban was temporary, possibly due to the lack of both containment measures against other infected regions and domestic mitigation measures. By contrast, in China, domestic mitigation measures were correlated with a long-term reduction in viral spread, despite repeated international introductions. Our study supports a role for both targeted international containment and domestic mitigation measures as critical components of a more comprehensive public-health strategy to mitigate future outbreaks caused by the emergence of novel SARS-CoV-2 variants. Phylodynamic analyses reveal that variation in rates of early geographic spread of COVID-19 are correlated with intervention measures.","version":"1.1","doi":"10.1101/2021.05.01.442286","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.443115","pub_date":"2021-5-07","title":"Subtle differences in the pathogenicity of SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 in rhesus macaques","abstract":"The emergence of several SARS-CoV-2 variants has caused global concerns about increased transmissibility, increased pathogenicity, and decreased efficacy of medical countermeasures. Animal models can be used to assess phenotypical changes in the absence of confounding factors that affect observed pathogenicity and transmissibility data in the human population. Here, we studied the pathogenicity of variants of concern (VOC) B.1.1.7 and B.1.351 in rhesus macaques and compared it to a recent clade B.1 SARS-CoV-2 isolate containing the D614G substitution in the spike protein. The B.1.1.7 VOC behaved similarly to the D614G with respect to clinical disease, virus shedding and virus replication in the respiratory tract. Inoculation with the B.1.351 isolate resulted in lower clinical scores in rhesus macaques that correlated with lower virus titers in the lungs, less severe histologic lung lesions and less viral antigen detected in the lungs. We observed differences in the local innate immune response to infection. In bronchoalveolar lavages, cytokines and chemokines were upregulated on day 4 in animals inoculated with D614G and B.1.1.7 but not in those inoculated with B.1.351. In nasal samples, we did not detect upregulation of cytokines and chemokines in D614G or B.1.351-inoculated animals. However, cytokines and chemokines were upregulated in the noses of B.1.1.7-inoculated animals. Taken together, our comparative pathogenicity study suggests that ongoing circulation under diverse evolutionary pressure favors transmissibility and immune evasion rather than an increase in intrinsic pathogenicity.","version":"1.1","doi":"10.1101/2021.05.07.443115","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.07.441534","pub_date":"2021-5-07","title":"iProMix: A decomposition model for studying the function of ACE2 based on bulk proteogenomic data for coronavirus pathogenesis","abstract":"Both SARS-CoV and SARS-CoV-2 use ACE2 receptors to enter epithelial cells in lung and many other tissues to cause human diseases. Genes and pathways that regulate ACE2 may facilitate/inhibit viral entry and replication, and genes and pathways that are controlled by ACE2 may be perturbed during infection, both affecting disease severity and outcomes. It is critical to understand how genes and pathways are associated with ACE2 in epithelial cells by leveraging proteomic data, but an accurate large-scale proteomic profiling at cellular resolution is not feasible at current stage. Therefore, we propose iProMix, a novel framework that decomposes bulk tissue proteomic data to identify epithelial cell component specific associations between ACE2 and other proteins. Unlike existing decomposition based association analyses, iProMix allows both predictors and outcomes to be impacted by cell type composition of the tissue and accounts for the impacts of decomposition variations and errors on hypothesis tests. It also builds in the functions to improve cell type estimation if estimates from existing literature are unsatisfactory. Simulations demonstrated that iProMix has well-controlled false discovery rate and large power in non-asymptotic settings with both correctly and mis-specified cell-type composition. We applied iProMix to the 110 adjacent normal tissue samples of patients with lung adenocarcinoma from Clinical Proteomic Tumor Analysis Consortium, and identified that interferon \u03b1 and \u03b3 pathways were most significantly associated with ACE2 protein abundances in epithelial cells. Interestingly, the associations were sex-specific that the positive associations were only observed in men, while in women the associations were negative.","version":"1.1","doi":"10.1101/2021.05.07.441534","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442875","pub_date":"2021-5-06","title":"The Effect of Minnelide against SARS-CoV-2 in a Murine Model","abstract":"Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of coronavirus disease 2019, COVID-19, and the current COVID-19 pandemic. Even as more vaccine candidates are released, more treatment options are critically needed. Here, we investigated the use of Minnelide, a water soluble pro-drug with anti-inflammatory properties, for the treatment of COVID-19. To do this, k18-hACE2 mice were infected with SARS-CoV-2 or given PBS control intranasally. The next day mice were either treated daily with low dose (0.0025mg/day) or high dose Minnelide (0.005mg/day), or given vehicle control intraperitoneal. Mice were weighed daily, and sacrificed at day 6 and 10 post-infection to analyze viral burden, cytokine response, and pathology. We observed a reduction in viral load in the lungs of Minnelide-treated mice infected with SARS-CoV-2 at day 10 post-infection compared to day 6 post-infection. All SARS-CoV-2 infected non-treated mice were moribund six days post-infection while treatment with Minnelide extended survival for both low (60% survival) and high (100% survival) dose treated mice ten days post-infection. Interestingly, cytokine analysis demonstrated a significant reduction in IL-6 (lung and heart) and D-dimer (serum) in high dose treated SARS-CoV-2 infected mice compared to mice infected with SARS-CoV-2 alone at day 6 post-infection. Additionally, histology analysis revealed that Minnelide treatment significantly improved lung pathology ten days post-infection with SARS-CoV-2 with all the mice exhibiting normal lung tissue with thin alveolar septa and no inflammatory cells. Overall, our study exhibits potential for the use of Minnelide to improve survival in COVID-19 patients.","version":"1.1","doi":"10.1101/2021.05.05.442875","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.14.431177","pub_date":"2021-5-06","title":"Endothelial cells elicit a pro-inflammatory response to SARS-CoV-2 without productive viral infection","abstract":"Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious. Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier to show that primary human endothelial cells express very low levels the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2. Accordingly, endothelial cells can only be infected when SARS-CoV-2 is present at very high concentrations. However, this is not a productive infection (i.e. no infectious virus is produced) and viral entry induces an inflammatory response. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. They do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection is only likely to occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells are still likely to play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.02.14.431177","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.06.442935","pub_date":"2021-5-06","title":"A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells","abstract":"The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.","version":"1.1","doi":"10.1101/2021.05.06.442935","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.06.442911","pub_date":"2021-5-06","title":"Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies","abstract":"The respiratory virus responsible for Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-2), has impacted nearly every aspect of life worldwide, claiming the lives of over 2.5 million people globally, at the time of this publication. Neutralizing nanobodies (VHH) represent a promising therapeutic intervention strategy to address the current SARS-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH antibodies were identified and evaluated for their capacity to tightly bind to the SARS-2 receptor-binding domain (RBD), to prevent binding of SARS-2 spike (S) to the cellular receptor Angiotensin-converting enzyme 2 (ACE2), and to neutralize viral infection. Preliminary preclinical evaluation of multiple nanobody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-2. The identified and characterized nanobodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic. To fully address the on-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-2), it will be important to have both vaccines and therapeutic strategies to prevent and mitigate the effects of SARS-2. In this study, we describe the identification and characterization of potently neutralizing humanized single domain heavy chain (VHH) antibodies that have binding affinity for both the original Wuhan strain and widely circulating B.1.1.7/UK strain. VHH antibodies have the same therapeutic potential as conventional antibodies in half the size and with greater stability and solubility. Using a synthetic humanized high-diversity VHH phage library we identified several candidates with strong affinity for the SARS-2 spike that block the interaction of SARS-2 spike with the cellular receptor ACE2, and effectively neutralize infection with SARS-2 in vitro. By sequencing viral escape mutants generated in the presence of each VHH we mapped the binding sites of the VHH antibodies and assessed their affinity against newly emerging SARS-2 variants. Finally, we demonstrate that two of these VHH antibodies show prophylactic and therapeutic efficacy in vivo against challenge with SARS-2. This study establishes that screening highly diverse VHH phage libraries against viral threats can yield highly effective therapeutic agents in real time.","version":"1.1","doi":"10.1101/2021.05.06.442911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.424003","pub_date":"2021-5-06","title":"Paired SARS-CoV-2 Spike Protein Mutations Observed During Ongoing SARS-CoV-2 Viral Transfer from Humans to Minks and Back to Humans","abstract":"A mutation analysis of SARS-CoV-2 genomes collected around the world sorted by sequence, date, geographic location, and species has revealed a large number of variants from the initial reference sequence in Wuhan. This analysis also reveals that humans infected with SARS-CoV-2 have infected mink populations in the Netherlands, Denmark, United States, and Canada. In these animals, a small set of mutations in the spike protein receptor binding domain (RBD), often occurring in specific combinations, has transferred back into humans. The viral genomic mutations in minks observed in the Netherlands and Denmark show the potential for new mutations on the SARS-CoV-2 spike protein RBD to be introduced into humans by zoonotic transfer. Our data suggests that close attention to viral transfer from humans to farm animals and pets will be required to prevent build-up of a viral reservoir for potential future zoonotic transfer.","version":"1.3","doi":"10.1101/2020.12.22.424003","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.06.441046","pub_date":"2021-5-06","title":"Structure-based design of a highly stable, covalently-linked SARS-CoV-2 spike trimer with improved structural properties and immunogenicity","abstract":"The continued threat of SARS-CoV-2 to global health necessitates development of improved research tools and vaccines. We present an improved SARS-CoV-2 spike ectodomain, \u201cVFLIP\u201d, bearing five proline substitutions, a flexible cleavage site linker, and an inter-protomer disulfide bond. VFLIP displays significantly improved stability, high-yield production and retains its trimeric state without exogenous trimerization motifs. High-resolution cryo-EM and glycan profiling reveal that the VFLIP quaternary structure and glycosylation mimic the native spike on the viral surface. Further, VFLIP has enhanced affinity and binding kinetics relative to other stabilized spike proteins for antibodies in the Coronavirus Immunotherapeutic Consortium (CoVIC), and mice immunized with VFLIP exhibit potent neutralizing antibody responses against wild-type and B.1.351 live SARS-CoV-2. Taken together, VFLIP represents an improved tool for diagnostics, structural biology, antibody discovery, and vaccine design.","version":"1.1","doi":"10.1101/2021.05.06.441046","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440861","pub_date":"2021-5-06","title":"InterARTIC: an interactive web application for whole-genome nanopore sequencing analysis of SARS-CoV-2 and other viruses","abstract":"InterARTIC is an interactive web application for the analysis of viral whole-genome sequencing (WGS) data generated on Oxford Nanopore Technologies (ONT) devices. A graphical interface enables users with no bioinformatics expertise to analyse WGS experiments and reconstruct consensus genome sequences from individual isolates of viruses, such as SARS-CoV-2. InterARTIC is intended to facilitate widespread adoption and standardisation of ONT sequencing for viral surveillance and molecular epidemiology. We demonstrate the use of InterARTIC for the analysis of ONT viral WGS data from SARS-CoV-2 and Ebola virus, using a laptop computer or the internal computer on an ONT GridION sequencing device. We showcase the intuitive graphical interface, workflow customisation capabilities and job-scheduling system that facilitate execution of small- and large-scale WGS projects on any common virus. InterARTIC is a free, open-source web application implemented in Python. The application can be downloaded as a set of pre-compiled binaries that are compatible with all common Ubuntu distributions, or built from source. For further details please visit: https://github.com/Psy-Fer/interARTIC/.","version":"1.3","doi":"10.1101/2021.04.21.440861","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442701","pub_date":"2021-5-05","title":"SARS-CoV-2 cell-to-cell infection is resistant to neutralizing antibodies","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. Based on available studies, this is the first attempt to quantitatively measure SARS-CoV-2 cell-to-cell infection, for which the mechanisms are largely unknown. The findings suggest that this route of SARS-CoV-2 transmission could be of great importance for treatment and prevention of COVID-19. Immune surveillance of viral or bacterial infections is largely mediated by neutralizing antibodies. Antibodies against the SARS-CoV-2 spike protein are produced after vaccination or infection, but their titers only partly reflect the degree of protection against infection. To identify protective antibodies, a neutralization test with replicating viruses or pseudoviruses (PVs) is required. This study developed lentiviral-based PV neutralization assays that, unlike similar systems reported earlier, enable quantitative measurement of SARS-CoV-2 neutralization in cell cocultures. Using both PVs and replication-competent virus, it was demonstrated that SARS-CoV-2 cell-to-cell infection is considerably more resistant to serum neutralization than infection with purified viral particles. The tests are easy to set up in many labs, and are believed to be more informative for monitoring SARS-CoV-2 collective immunity or entry inhibitor screening.","version":"1.1","doi":"10.1101/2021.05.04.442701","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.441029","pub_date":"2021-5-05","title":"Sex-biased response to and brain cell infection by SARS-CoV-2 in a highly susceptible human ACE2 transgenic model","abstract":"The COVID-19 pandemic is caused by SARS-CoV-2 infection. Human angiotensin-converting enzyme II (hACE2) has been identified as the receptor enabling SARS-CoV-2 host entry. To establish a mouse model for COVID-19, we generated transgenic mouse lines using the (HS4)2-pCAG-hACE2-HA-(HS4)2 transgene cassette, which expresses HA-tagged hACE2 under control of the CAG promoter and is flanked by HS4 insulators. Expression levels of the hACE2 transgene are respectively higher in lung, brain and kidney of our CAG-hACE2 transgenic mice and relatively lower in duodenum, heart and liver. The CAG-hACE2 mice are highly susceptibility to SARS-CoV-2 infection, with 100 PFU of SARS-CoV-2 being sufficient to induce 87.5% mortality at 9 days post-infection and resulting in a sole (female) survivor. Mortality was 100% at the higher titer of 1000 PFU. At lower viral titers, we also found that female mice exposed to SARS-CoV-2 infection suffered much less weight loss than male mice, implying sex-biased responses to SARS-CoV-2 infection. We subjected neuronal cultures to SARS-CoV-2 pseudovirus infection to ascertain the susceptibilities of neurons and astrocytes. Moreover, we observed that expression of SARS-CoV-2 Spike protein alters the synaptic responses of cultured neurons. Our transgenic mice may serve as a model for severe COVID-19 and sex-biased responses to SARS-CoV-2 infection, aiding in the development of vaccines and therapeutic treatments for this disease.","version":"1.2","doi":"10.1101/2021.05.04.441029","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442779","pub_date":"2021-5-05","title":"ProLung\u2122-budesonide Inhibits SARS-CoV-2 Replication and Reduces Lung Inflammation","abstract":"Inhaled budesonide benefits patients with COVID-19. ProLung\u2122-budesonide enables the sustained, low dose administration of budesonide within a delivery vehicle similar to lung surfactant. ProLung\u2122-budesonide may offer anti-inflammatory and protective effects to the lung in COVID-19, yet it\u2019s effect on SARS-CoV-2 replication is unknown. To determine the efficacy of ProLung\u2122-budesonide against SARS-CoV-2 infection in vitro, evaluate its ability to decrease inflammation, and airway hyperresponsiveness in an animal model of lung inflammation. SARS-CoV-2-infected Vero 76 cells were treated with ProLung\u2122-budesonide ([0.03\u2013 100 \u03bcg/ml]) for 3 days, and virus yield in the supernatant was measured. Ovalbumin-sensitized C57BL/6 mice received aerosolized (a) ProLung\u2122-budesonide weekly, (b) only budesonide, either daily or weekly, or (c) weekly empty ProLung\u2122-carrier (without budesonide). All treatment groups were compared to sensitized untreated, or normal mice using histopathologic examination, electron microscopy (EM), airway hyperresponsiveness (AHR) to Methacholine (Mch) challenge, and eosinophil peroxidase activity (EPO) measurements in bronchioalveolar lavage (BAL). ProLung\u2122-budesonide showed significant inhibition on viral replication of SARS-CoV-2-infected cells with the selectivity index (SI) value > 24. Weekly ProLung\u2122-budesonide and daily budesonide therapy significantly decreased lung inflammation and EPO in BAL. ProLung\u2122-budesonide localized in type II pneumocytes, and was the only group to significantly decrease AHR, and EPO in BAL with Mch challenge ProLung\u2122-budesonide significantly inhibited viral replication in SARS-CoV-2 infected cells. It localized into type II pneumocytes, decreased lung inflammation, AHR and EPO activity with Mch challenge. This novel drug formulation may offer a potential inhalational treatment for COVID-19.","version":"1.1","doi":"10.1101/2021.05.05.442779","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442782","pub_date":"2021-5-05","title":"Combination Respiratory Vaccine Containing Recombinant SARS-CoV-2 Spike and Quadrivalent Seasonal Influenza Hemagglutinin Nanoparticles with Matrix-M Adjuvant","abstract":"The 2019 outbreak of a severe respiratory disease caused by an emerging coronavirus, SARS-CoV-2, has spread globally with high morbidity and mortality. Co-circulating seasonal influenza has greatly diminished recently, but expected to return with novel strains emerging, thus requiring annual strain adjustments. We have developed a recombinant hemagglutinin (HA) quadrivalent nanoparticle influenza vaccine (qNIV) produced using an established recombinant insect cell expression system to produce nanoparticles. Influenza qNIV adjuvanted with Matrix-M was well-tolerated and induced robust antibody and cellular responses, notably against both homologous and drifted A/H3N2 viruses in Phase 1, 2, and 3 trials. We also developed a full-length SARS-CoV-2 spike protein vaccine which is stable in the prefusion conformation (NVX-CoV2373) using the same platform technology. In phase 3 clinical trials, NVX-CoV2373 is highly immunogenic and protective against the prototype strain and B.1.1.7 variant. Here we describe the immunogenicity and efficacy of a combination quadrivalent seasonal flu and COVID-19 vaccine (qNIV/CoV2373) in ferret and hamster models. The combination qNIV/CoV2373 vaccine produces high titer influenza hemagglutination inhibiting (HAI) and neutralizing antibodies against influenza A and B strains. The combination vaccine also elicited antibodies that block SARS-CoV-2 spike protein binding to the human angiotensin converting enzyme-2 (hACE2) receptor. Significantly, hamsters immunized with qNIV/CoV2373 vaccine and challenged with SARS-CoV-2 were protected against weight loss and were free of replicating SARS-CoV-2 in the upper and lower respiratory tract with no evidence of viral pneumonia. This study supports evaluation of qNIV/CoV2373 combination vaccine as a preventive measure for seasonal influenza and CoVID-19. Combination qNIV/CoV2373 vaccine induced protective hemagglutination inhibition (HAI) responses to seasonal influenza A and B unchanged when formulated with recombinant spike. Combination qNIV/CoV2373 vaccine maintained clinical and virologic protection against experimental challenge with SARS-CoV-2. Combination qNIV/CoV2373 vaccine showed no clinical or histological sign of enhanced disease following experimental challenge with SARS-CoV-2. Combination qNIV/CoV2373 vaccine induced antibodies against SARS-CoV-2 neutralizing epitopes common between US-WA and B.1.352 variant.","version":"1.1","doi":"10.1101/2021.05.05.442782","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442536","pub_date":"2021-5-05","title":"Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein","abstract":"The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.","version":"1.1","doi":"10.1101/2021.05.05.442536","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442780","pub_date":"2021-5-05","title":"Prior aerosol infection with lineage A SARS-CoV-2 variant protects hamsters from disease, but not reinfection with B.1.351 SARS-CoV-2 variant","abstract":"The circulation of SARS-CoV-2 has resulted in the emergence of variants of concern (VOCs). It is currently unclear whether previous infection with SARS-CoV-2 provides protection against reinfection with VOCs. Here, we show that low dose aerosol exposure to hCoV-19/human/USA/WA-CDC-WA1/2020 (WA1, lineage A), resulted in a productive mild infection. In contrast, low dose of SARS-CoV-2 via fomites did not result in productive infection in the majority of exposed hamsters and these animals remained non-seroconverted. After recovery, hamsters were re-exposed to hCoV-19/South African/KRISP-K005325/2020 (VOC B.1.351) via an intranasal challenge. Seroconverted rechallenged animals did not lose weight and shed virus for 3 days. They had little infectious virus and no pathology in the lungs. In contrast, shedding, weight loss and extensive pulmonary pathology caused by B.1.351 replication was observed in the non-seroconverted animals. The rechallenged seroconverted animals did not transmit virus to na\u00efve sentinels via direct contact transmission, in contrast to the non-seroconverted animals. Reinfection with B.1.351 triggered an anamnestic response that boosted not only neutralizing titers against lineage A, but also titers against B.1.351. Our results confirm that aerosol exposure is a more efficient infection route than fomite exposure. Furthermore, initial infection with SARS-CoV-2 lineage A does not prevent heterologous reinfection with B.1.351 but prevents disease and onward transmission. These data suggest that previous SARS-CoV-2 exposure induces partial protective immunity. The reinfection generated a broadly neutralizing humoral response capable of effectively neutralizing B.1.351 while maintaining its ability to neutralize the virus to which the initial response was directed against.","version":"1.1","doi":"10.1101/2021.05.05.442780","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442663","pub_date":"2021-5-05","title":"SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination","abstract":"The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19 pandemic. The number of COVID-19 cases and deaths in India has risen steeply in recent weeks and a novel SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the ACE2 receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyzed whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 entered two out of eight cell lines tested with slightly increased efficiency and was blocked by entry inhibitors. In contrast, B.1.617 was resistant against Bamlanivimab, an antibody used for COVID-19 treatment. Finally, B.1.617 evaded antibodies induced by infection or vaccination, although with moderate efficiency. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.","version":"1.1","doi":"10.1101/2021.05.04.442663","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423965","pub_date":"2021-5-05","title":"Development of a novel hybrid alphavirus-SARS-CoV-2 particle for rapid in vitro screening and quantification of neutralization antibodies, viral variants, and antiviral drugs","abstract":"Timely development of vaccines and antiviral drugs is critical to control the COVID-19 pandemic . Current methods for quantifying vaccine-induced neutralizing antibodies involve the use of pseudoviruses, such as the SARS-CoV-2 spike protein (S) pseudotyped lentivirus. However, these pseudoviruses contain structural proteins foreign to SARS-CoV-2, and require days to infect and express reporter genes. Here we describe the development of a new hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) particle for rapid and accurate quantification of neutralization antibodies and viral variants. Ha-CoV-2 is a non-replicating SARS-CoV-2 virus-like particle, composed of SARS-CoV-2 structural proteins (S, M, N, and E) and a RNA genome derived from a fast expressing alphavirus vector . We demonstrated that Ha-CoV-2 can rapidly and robustly express reporter genes in target cells within 3-6 hours. We further validated Ha-CoV-2 for rapid quantification of neutralization antibodies, viral variants, and antiviral drugs. In addition, as a proof-of-concept, we assembled and compared the relative infectivity of a panel of 10 Ha-CoV-2 variant isolates (D614G, P.1, B.1.1.207, B.1.351, B.1.1.7, B.1.429, B.1.258, B.1.494, B.1.2, B.1.1298), and demonstrated that these variants in general are 2-10 fold more infectious. Furthermore, we quantified the anti-serum from an infected and vaccinated individual; the one dose vaccination with Moderna mRNA-1273 has greatly increased the anti-serum titer for approximately 6 fold. The post-vaccination serum has also demonstrated various neutralizing activities against all 9 variants tested. These results demonstrated that Ha-CoV-2 can be used as a robust platform for rapid quantification of neutralizing antibodies against SARS-CoV-2 and its variants.","version":"1.2","doi":"10.1101/2020.12.22.423965","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442784","pub_date":"2021-5-05","title":"SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy","abstract":"SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.","version":"1.1","doi":"10.1101/2021.05.05.442784","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432474","pub_date":"2021-5-05","title":"SARS-CoV-2 causes brain inflammation and induces Lewy body formation in macaques","abstract":"SARS-CoV-2 may cause acute respiratory disease, but the infection can also initiate neurological symptoms. Here we show that SARS-CoV-2 infection causes brain inflammation in the macaque model. An increased metabolic activity in the pituitary gland of two macaques was observed by longitudinal positron emission tomography-computed tomography (PET-CT). Post-mortem analysis demonstrated infiltration of T-cells and activated microglia in the brain, and viral RNA was detected in brain tissues from one animal. We observed Lewy bodies in brains of all rhesus macaques. These data emphasize the virus\u2019 capability to induce neuropathology in this nonhuman primate model for SARS-CoV-2 infection. As in humans, Lewy body formation is an indication for the development of Parkinson\u2019s disease, this data represents a warning for potential long-term neurological effects after SARS-CoV-2 infection. SARS-CoV-2 causes brain inflammation and Lewy bodies, a hallmark for Parkinson, after an asymptomatic infection in macaques.","version":"1.2","doi":"10.1101/2021.02.23.432474","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.02.442052","pub_date":"2021-5-05","title":"Preliminary Immunogenicity of a Pan-COVID-19 T Cell Vaccine in HLA-A*02:01 Mice","abstract":"New strains of SARS-CoV-2 have emerged, including B.1.351 and P.1, that demonstrate increased transmissibility and the potential of rendering current SARS-CoV-2 vaccines less effective. A concern is that existing SARS-CoV-2 spike subunit vaccines produce neutralizing antibodies to three dimensional spike epitopes that are subject to change during viral drift. Here we provide an initial report on the hypothesis that adaptive T cell based immunity may provide a path for a pan-COVID-19 vaccine that is resilient to viral drift. T cell based adaptive immunity can be based on short peptide sequences selected from the viral proteome that are less subject to drift, and can utilize multiple such epitopes to provide redundancy in the event of drift. We find that SARS-CoV-2 peptides contained in a mRNA-LNP T cell vaccine for SARS-CoV-2 are immunogenic in mice transgenic for the human HLA-A*02:01 gene. We plan to test the efficacy of this vaccine with SARS-CoV-2 B.1.351 challenge trials with HLA-A*02:01 mice.","version":"1.2","doi":"10.1101/2021.05.02.442052","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442760","pub_date":"2021-5-05","title":"SARS CoV-2 variant B.1.617.1 is highly pathogenic in hamsters than B.1 variant","abstract":"The recent emergence of new SARS-CoV-2 lineage B.1.617 in India has been associated with a surge in the number of daily infections. This variant has combination of specific mutations L452R, E484Q and P681R reported to possibly enhance the transmissibility with likelihood of escaping the immunity. We investigated the viral load and pathogenic potential of B.1.617.1 in Syrian golden hamsters. Two groups of Syrian golden hamsters (9 each) were inoculated intranasally with SARS CoV-2 isolates, B.1 (D614G) and B.1.617.1 respectively. The animals were monitored daily for the clinical signs and body weight. The necropsy of three hamsters each was performed on 3, 5- and 7-days post-infection (DPI). Throat swab (TS), nasal wash (NW) and organ samples (lungs, nasal turbinate, trachea) were collected and screened using SARS-CoV-2 specific Real-time RT-PCR. The hamsters infected with B.1.617.1 demonstrated increased body weight loss compared to B.1 variant. The highest viral load was observed in nasal turbinate and lung specimens of animals infected with B.1.167.1 on 3 DPI. Neutralizing antibody (NAb) and IgG response in hamsters of both the groups were observed from 5 and 7 DPI respectively. However, higher neutralizing antibody titers were observed against B.1.167.1. Gross pathology showed pronounced lung lesions and hemorrhage with B.1.671 compared to B.1. B.1617.1 and B.1 variant varied greatly in their infectiousness, pathogenesis in hamster model. This study demonstrates higher pathogenicity in hamsters evident with reduced body weight, higher viral load in lungs and pronounced lung lesions as compared to B.1 variant. B.1.617.1 is the new SARS-CoV-2 lineage that emerged in India. Maximal body weight loss and higher viral load in hamsters infected with B.1.617.1. It caused pronounced lung lesions in hamsters compared to B.1 variant which demonstrates the pathogenic potential of B.1.617.1.","version":"1.1","doi":"10.1101/2021.05.05.442760","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442686","pub_date":"2021-5-05","title":"Emerging genetic diversity of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) alters its B-cell epitopes","abstract":"The RNA dependent RNA polymerase (RdRp) plays crucial role in virus life cycle by replicating the viral RNA genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and during this process acquired mutations. This study was carried out to identify mutations in RdRp as the SARS-CoV-2 spread in India. We compared the 668 RdRp sequences reported from India with the first reported RdRp sequence from Wuhan, China. Our data revealed that RdRp have acquired sixty mutations among Indian isolates. Our protein modelling study also revealed that several mutants including D833Y, A699S, Y149C and C464F can potentially alter stability and flexibility of RdRp. We also predicted the potential B cell epitopes contributed by RdRp and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among sixty RdRp mutants identified in this study, 40% of them localises in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp to understand the impact of these mutations on SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.05.04.442686","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.05.442725","pub_date":"2021-5-05","title":"1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution","abstract":"Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the C\u03b2 of the apo form of the full-lengthSARS-CoV-2 nsp1 including folded domain together with the flaking N- and C-terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.","version":"1.1","doi":"10.1101/2021.05.05.442725","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.17.344002","pub_date":"2021-5-05","title":"Adult Stem Cell-derived Complete Lung Organoid Models Emulate Lung Disease in COVID-19","abstract":"SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type-II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Infected ALO-monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2\u2192AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. Human lung organoids with mixed proximodistal epithelia are created Proximal airway cells are critical for viral infectivity Distal alveolar cells are important for emulating host response Both are required for the overzealous response in severe COVID-19 An integrated stem cell-based disease modeling and computational approach demonstrate how both proximal airway epithelium is critical for SARS-CoV-2 infectivity, but distal differentiation of alveolar pneumocytes is critical for simulating the overzealous host response in fatal COVID-19.","version":"1.4","doi":"10.1101/2020.10.17.344002","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442699","pub_date":"2021-5-05","title":"Durable antibody responses in staff at two long-term care facilities, during and post SARS-CoV-2 outbreaks","abstract":"SARS-CoV-2 has had a disproportionate impact on non-hospital healthcare settings such as long-term care facilities (LTCFs). The communal nature of these facilities, paired with the high-risk profile of residents, has resulted in thousands of infections and deaths and a high case fatality rate. To detect pre-symptomatic infections and identify infected workers, we performed weekly surveillance testing of staff at two LTCFs which revealed a large outbreak at one of the sites. We collected serum from staff members throughout the study and evaluated it for binding and neutralization to measure seroprevalence, seroconversion, and type and functionality of antibodies. At the site with very few incident infections, we detected that over 40% of the staff had preexisting SARS-CoV-2 neutralizing antibodies, suggesting prior exposure. At the outbreak site, we saw rapid seroconversion following infection. Neutralizing antibody levels were stable for many weeks following infection, suggesting a durable, long-lived response. Receptor-binding domain antibodies and neutralizing antibodies were strongly correlated. The site with high seroprevalence among staff had two unique introductions of SARS-CoV-2 into the facility through seronegative infected staff during the period of study but these did not result in workplace spread or outbreaks. Together our results reveal that high seroprevalence rate among staff can contribute to herd immunity within a workplace and protect against subsequent infection and spread within a facility.","version":"1.1","doi":"10.1101/2021.05.04.442699","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.442357","pub_date":"2021-5-04","title":"Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge","abstract":"The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse-adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.","version":"1.1","doi":"10.1101/2021.05.03.442357","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.28.437369","pub_date":"2021-5-04","title":"Preliminary report on SARS-CoV-2 Spike mutation T478K","abstract":"Several SARS-CoV-2 variants have emerged, posing a renewed threat to COVID-19 containment and to vaccine and drug efficacy. In this study, we analyzed more than 1,000,000 SARS-CoV-2 genomic sequences deposited up to April 27, 2021 on the GISAID public repository, and identified a novel T478K mutation located on the SARS-CoV-2 Spike protein. The mutation is structurally located in the region of interaction with human receptor ACE2 and was detected in 11,435 distinct cases. We show that T478K has appeared and risen in frequency since January 2021, predominantly in Mexico and USA, but we could also detect it in several European countries.","version":"1.3","doi":"10.1101/2021.03.28.437369","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.442538","pub_date":"2021-5-04","title":"CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection","abstract":"SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.05.03.442538","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424739","pub_date":"2021-5-04","title":"Meta-analysis of virus-induced host gene expression reveals unique signatures of immune dysregulation induced by SARS-CoV-2","abstract":"The clinical outcome of COVID-19 has an extreme age, genetic and comorbidity bias that is thought to be driven by an impaired immune response to SARS-CoV-2, the causative agent of the disease. The unprecedented impact of COVID-19 on global health has resulted in multiple studies generating extensive gene expression datasets in a relatively short period of time. In order to better understand the immune dysregulation induced by SARS-CoV-2, we carried out a meta-analysis of these transcriptomics data available in the published literature. Datasets included both those available from SARS-CoV-2 infected cell lines in vitro and those from patient samples. We focused our analysis on the identification of viral perturbed host functions as captured by co-expressed gene module analysis. Transcriptomics data from lung biopsies and nasopharyngeal samples, as opposed to those available from other clinical samples and infected cell lines, provided key signatures on the role of the host\u2019s immune response on COVID-19 pathogenesis. For example, severity of infection and patients\u2019 age are linked to the absence of stimulation of the RIG-I-like receptor signaling pathway, a known critical immediate line of defense against RNA viral infections that triggers type-I interferon responses. In addition, co-expression analysis of age-stratified transcriptional data provided evidence that signatures of key immune response pathways are perturbed in older COVID-19 patients. In particular, dysregulation of antigen-presenting components, down-regulation of cell cycle mechanisms and signatures of hyper-enriched monocytes were strongly correlated with the age of older individuals infected with SARS-CoV-2. Collectively, our meta-analysis highlights the ability of transcriptomics and gene-module analysis of aggregated datasets to aid our improved understanding of the host-specific disease mechanisms underpinning COVID-19.","version":"1.2","doi":"10.1101/2020.12.29.424739","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.442520","pub_date":"2021-5-04","title":"A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein . Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >106 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids . Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.","version":"1.1","doi":"10.1101/2021.05.03.442520","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.05.369413","pub_date":"2021-5-04","title":"The post-acute phase of SARS-CoV-2 infection in two macaques species is associated with signs of ongoing virus replication and pathology in pulmonary and extrapulmonary tissues","abstract":"The post-acute phase of SARS-CoV-2 infection was investigated in rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, as suggested by the absence of viral RNA in nasal and tracheal swabs, computed tomography (CT) and positron emission tomography (PET)-CT were able to reveal pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, like conjunctiva, cervical and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans. More than a year after the start of the pandemic, the long-term consequences of SARS-CoV-2 infection start to surface. The variety of clinical manifestations associated with post-acute COVID-19 suggests the involvement of multiple biological mechanisms. In this study, we show that rhesus and cynomolgus macaques shed virus from their respiratory tract, generate virus-specific humoral immune responses, and show signs of SARS-CoV-2-induced lung pathology. PET-CT revealed that both species showed ongoing mild to moderate pulmonary disease, even after the virus was no longer detectable in nasal and tracheal swabs. Five to six weeks after infection, necropsy confirmed minimal to mild histopathological manifestations in various tissues, like the lungs, heart, lymph nodes, and conjunctiva. We detected Viral RNA in the heart, respiratory tract, and tracheobronchial lymph nodes, and subgenomic messenger RNA in the lungs and surrounding lymph nodes, indicative of ongoing virus replication. We show widespread tissue dissemination of SARS-CoV-2 in infected macaques and the presence of replicating virus in lungs and surrounding lymph nodes after alleged convalescence of infection. This finding is intriguing in the light of long-COVID disease symptoms seen in humans as it has been hypothesized that persistent infection may contribute to this phenomenon.","version":"1.2","doi":"10.1101/2020.11.05.369413","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.440524","pub_date":"2021-5-04","title":"Semi-supervised identification of SARS-CoV-2 molecular targets","abstract":"SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. In this work, we analyzed a corpus of 66,000 SARS-CoV-2 genome sequences. We developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on use of a single reference genome and by overcoming atypical genome traits. Using this method, we identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction compared to proteome references including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools such as Prokka (base) and VAPiD, we yielded an 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 molecular target sequences\u2014 some conserved across time and geography while others represent emerging variants. We observed 3,362 non-redundant sequences per protein on average within this corpus and describe key D614G and N501Y variants spatiotemporally. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized Receptor Binding Domain variants. Here, we comprehensively present the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable high-accuracy method to analyze newly sequenced infections.","version":"1.1","doi":"10.1101/2021.05.03.440524","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.438781","pub_date":"2021-5-04","title":"Highly functional Cellular Immunity in SARS-CoV-2 Non-Seroconvertors is associated with immune protection","abstract":"The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total na\u00efve and low effector CD8+ T cells in NSC. Moreover, polyfunctional Nucleocapsid (NP)-specific CD8+ T-cell responses, as well as reduced levels of T-cell activation monitored by PD-1 and activation-induced markers, were distinctive immunological traits in NSC. Longitudinal data support the stability of the NSC phenotype over three months. Our results implicate highly functional SARS-CoV-2 Spike and NP T-cell responses with low immune activation in protection from disease severity in the absence of seroconversion. To understand SARS-CoV-2 specific T-cell immunity in the absence of seroconversion, we characterized immunological features of Non-Seroconvertors recovered from infection. Highly functional specific T-cell responses and low immune activation were determinants of immune protection from severe disease.","version":"1.1","doi":"10.1101/2021.05.04.438781","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.04.442548","pub_date":"2021-5-04","title":"ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling","abstract":"ORAI1 and STIM1 are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using ORAI1 and STIM1 knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, ORAI1 knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca2+-dependent transcription factors of the activator protein 1 (AP-1) family and MEF2C. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.05.04.442548","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.441323","pub_date":"2021-5-04","title":"Protective heterologous T cell immunity in COVID-19 induced by MMR and Tdap vaccine antigens","abstract":"T cells are critical for control of viral infection and effective vaccination. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diphtheria-pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. Using co-cultures of antigen presenting cells (APC) loaded with antigens and autologous T cells, we found a high correlation between responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap vaccine proteins in both SARS-CoV-2 infected individuals and individuals immunized with mRNA-based SARS-CoV-2 vaccines. The overlapping T cell population contained effector memory T cells (TEMRA) previously implicated in anti-viral immunity and their activation required APC-derived IL-15. TCR- and scRNA-sequencing detected cross-reactive clones with TEMRA features among the cells recognizing SARS-CoV-2, MMR and Tdap epitopes. A propensity-weighted analysis of 73,582 COVID-19 patients revealed that severe disease outcomes (hospitalization and transfer to intensive care unit or death) were reduced in MMR or Tdap vaccinated individuals by 38-32% and 23-20% respectively. In summary, SARS-CoV-2 re-activates memory T cells generated by Tdap and MMR vaccines, which may reduce disease severity.","version":"1.1","doi":"10.1101/2021.05.03.441323","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.442371","pub_date":"2021-5-04","title":"Impaired T-cell and antibody immunity after COVID-19 infection in chronically immunosuppressed transplant recipients","abstract":"Assessment of T-cell immunity to the COVID-19 coronavirus requires reliable assays and is of great interest, given the uncertain longevity of the antibody response. Some recent reports have used immunodominant spike (S) antigenic peptides and anti-CD28 co-stimulation in varying combinations to assess T-cell immunity to SARS-CoV-2. These assays may cause T-cell hyperstimulation and could overestimate antiviral immunity in chronically immunosuppressed transplant recipients, who are predisposed to infections and vaccination failures. Here, we evaluate CD154-expressing T-cells induced by unselected S antigenic peptides in 204 subjects-103 COVID-19 patients and 101 healthy unexposed subjects. Subjects included 72 transplanted and 130 non-transplanted subjects. S-reactive CD154+T-cells co-express and can thus substitute for IFN\u03b3 (n=3). Assay reproducibility in a variety of conditions was acceptable with coefficient of variation of 2-10.6%. S-reactive CD154+T-cell frequencies were a) higher in 42 healthy unexposed transplant recipients who were sampled pre-pandemic, compared with 59 healthy non-transplanted subjects (p=0.02), b) lower in Tr COVID-19 patients compared with healthy transplant patients (p<0.0001), c) lower in Tr patients with severe COVID-19 (p<0.0001), or COVID-19 requiring hospitalization (p<0.05), compared with healthy Tr recipients. S-reactive T-cells were not significantly different between the various COVID-19 disease categories in NT recipients. Among transplant recipients with COVID-19, cytomegalovirus co-infection occurred in 34%; further, CMV-specific T-cells (p<0.001) and incidence of anti-receptor-binding-domain IgG (p=0.011) were lower compared with non-transplanted COVID-19 patients. Healthy unexposed transplant recipients exhibit pre-existing T-cell immunity to SARS-CoV-2. COVID-19 infection leads to impaired T-cell and antibody responses to SARS-CoV-2 and increased risk of CMV co-infection in transplant recipients.","version":"1.1","doi":"10.1101/2021.05.03.442371","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.442038","pub_date":"2021-5-03","title":"ACE2 glycans preferentially interact with the RBD of SARS-CoV-2 over SARS-CoV","abstract":"We report a distinct difference in the interactions of the glycans of the host-cell receptor, ACE2, with SARS-CoV-2 and SARS-CoV S-protein receptor-binding domains (RBDs). Our analysis demonstrates that the ACE2 glycan at N90 may offer protection against infections of both coronaviruses, while the ACE2 glycan at N322 enhances interactions with the SARS-CoV-2 RBD. The interactions of the ACE2 glycan at N322 with SARS-CoV RBD are blocked by the presence of the RBD glycan at N357 of the SARS-CoV RBD. The absence of this glycosylation site on SARS-CoV-2 RBD may enhance its binding with ACE2.","version":"1.2","doi":"10.1101/2021.04.29.442038","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.441080","pub_date":"2021-5-03","title":"Human organoid systems reveal in vitro correlates of fitness for SARS-CoV-2 B.1.1.7","abstract":"A new phase of the COVID-19 pandemic has started as several SARS-CoV-2 variants are rapidly emerging globally, raising concerns for increased transmissibility. As animal models and traditional in vitro systems may fail to model key aspects of the SARS-CoV-2 replication cycle, representative in vitro systems to assess variants phenotypically are urgently needed. We found that the British variant (clade B.1.1.7), compared to an ancestral SARS-CoV-2 clade B virus, produced higher levels of infectious virus late in infection and had a higher replicative fitness in human airway, alveolar and intestinal organoid models. Our findings unveil human organoids as powerful tools to phenotype viral variants and suggest extended shedding as a correlate of fitness for SARS-CoV-2. British SARS-CoV-2 variant (clade B.1.1.7) infects organoids for extended time and has a higher fitness in vitro.","version":"1.1","doi":"10.1101/2021.05.03.441080","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.442194","pub_date":"2021-5-03","title":"SARS-CoV-2 spike protein induces brain pericyte immunoreactivity in absence of productive viral infection","abstract":"COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via still elusive mechanisms. SARS-CoV-2 infects host cells by binding to angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor that recognizes the viral spike (S) protein. Brain pericytes were recently shown to express ACE2 at the neurovascular interface, outlining their possible implication in microvasculature injury in COVID-19. Yet, pericyte responses to SARS-CoV-2 is still to be fully elucidated. Using cell-based assays, we report that ACE2 expression in human brain vascular pericytes is highly dynamic and is increased upon S protein stimulation. Pericytes exposed to S protein underwent profound phenotypic changes translated by increased expression of contractile and myofibrogenic proteins, namely \u03b1-smooth muscle actin (\u03b1-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). These changes were associated to an altered intracellular calcium (Ca2+) dynamic. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-\u03baB) signalling pathway, which was potentiated by hypoxia, a condition associated to vascular comorbidities, which exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely interleukin-8 (IL-8), IL-18, macrophage migration inhibitory factor (MIF), and stromal cell-derived factor-1 (SDF-1). Finally, we found that S protein could reach the mouse brain via the intranasal route and that reactive ACE2-expressing pericytes are recruited to the damaged tissue undergoing fibrotic scarring in a mouse model of cerebral multifocal micro-occlusions, a main reported vascular-mediated neurological condition associated to COVID-19. Our data demonstrate that the released S protein is sufficient to mediate pericyte immunoreactivity, which may contribute to microvasculature injury in absence of a productive viral infection. Our study provides a better understanding for the possible mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.","version":"1.1","doi":"10.1101/2021.04.30.442194","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.442455","pub_date":"2021-5-03","title":"SARS -CoV-2 T-cell immunity to variants of concern following vaccination","abstract":"Recently, two mRNA vaccines to severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) have become available, but there is also an emergence of SARS-CoV-2 variants with increased transmissibility and virulence. A major concern is whether the available vaccines will be equally effective against these variants. The vaccines are designed to induce an immune response against the SARS-CoV-2 spike protein, which is required for viral entry to host cells. Immunity to SARS-CoV-2 is often evaluated by antibody production, while less is known about the T-cell response. Here we developed, characterized, and implemented two standardized, functional assays to measure T-cell immunity to SARS-CoV-2 in uninfected, convalescent, and vaccinated individuals. We found that vaccinated individuals had robust T-cell responses to the wild type spike and nucleocapsid proteins, even more so than convalescent patients. We also found detectable but diminished T-cell responses to spike variants (B.1.1.7, B.1.351, and B.1.1.248) among vaccinated but otherwise healthy donors. Since decreases in antibody neutralization have also been observed with some variants, investigation into the T-cell response to these variants as an alternative means of viral control is imperative. Standardized measurements of T-cell responses to SARS-CoV-2 are feasible and can be easily adjusted to determine changes in response to variants.","version":"1.1","doi":"10.1101/2021.05.03.442455","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431484","pub_date":"2021-5-03","title":"A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine","abstract":"Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.","version":"1.2","doi":"10.1101/2021.02.18.431484","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.01.442304","pub_date":"2021-5-03","title":"SARS-CoV-2 Genome Sequencing Methods Differ In Their Ability To Detect Variants From Low Viral Load Samples","abstract":"SARS-CoV-2 genomic surveillance has been vital in understanding the spread of COVID-19, the emergence of viral escape mutants and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low frequency variants. Our analyses highlight that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina respiratory viral oligo panel, and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high quality sequence data from low viral load samples for public health and genomic surveillance purposes.","version":"1.1","doi":"10.1101/2021.05.01.442304","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.442229","pub_date":"2021-5-03","title":"The immunological factors predisposing to severe COVID-19 are already present in healthy elderly and men","abstract":"Male sex and old age are risk factors for COVID-19 severity, but the underlying causes are unknown. A possible explanation for this might be the differences in immunological profiles in males and the elderly before the infection. Given the seasonal profile of COVID-19, the seasonal response against SARS-CoV-2 could also be different in these groups. The abundance of circulating proteins and immune populations associated with severe COVID-19 was analyzed in 2 healthy cohorts. PBMCs of female, male, young, and old subjects in different seasons of the year were stimulated with heat-inactivated SARS-CoV-2. Several T cell subsets, which are known to be depleted in severe COVID-19 patients, were intrinsically less abundant in men and older individuals. Plasma proteins increasing with disease severity, including HGF, IL-8, and MCP-1, were more abundant in the elderly and males. The elderly produced significantly more IL-1RA and had a dysregulated IFN\u03b3 response with lower production in the summer compared with young individuals. The immune characteristics of severe COVID-19, described by a differential abundance of immune cells and circulating inflammatory proteins, are intrinsically present in healthy men and the elderly. This might explain the susceptibility of men and the elderly to SARS-CoV-2 infection. Immunological profile of severe COVID-19, characterized by altered immune cell populations and inflammatory plasma proteins is intrinsically present in healthy men and the elderly. Different age and sex groups show distinct seasonal responses to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.04.30.442229","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.335562","pub_date":"2021-5-03","title":"Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation","abstract":"The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. We have applied proteomics to identify cellular factors that interact with the cytoplasmic tail of S. We confirmed interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding-site promotes exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.","version":"1.2","doi":"10.1101/2020.10.12.335562","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.22.440932","pub_date":"2021-5-03","title":"Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra, India","abstract":"As the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expands, genomic epidemiology and whole genome sequencing are being constantly used to investigate its transmissions and evolution. In the backdrop of the global emergence of \u201cvariants of concern\u201d (VOCs) during December 2020 and an upsurge in a state in the western part of India since January 2021, whole genome sequencing and analysis of spike protein mutations using sequence and structural approaches was undertaken to identify possible new variants and gauge the fitness of current circulating strains. Phylogenetic analysis revealed that the predominant clade in circulation was a distinct newly identified lineage B.1.617 possessing common signature mutations D111D, G142D, L452R, E484Q, D614G and P681R, in the spike protein including within the receptor binding domain (RBD). Of these, the mutations at residue positions 452, 484 and 681 have been reported in other globally circulating lineages. The structural analysis of RBD mutations L452R and E484Q along with P681R in the furin cleavage site, revealed that these may possibly result in increased ACE2 binding and rate of S1-S2 cleavage resulting in better transmissibility. The same two RBD mutations indicated decreased binding to select monoclonal antibodies (mAbs) and may affect their neutralization potential. Experimental validation against a wider panel of mAbs, sera from vaccinees and those that recovered from natural infection needs to be studied. The emergence of such local variants through the accumulation of convergent mutations during the COVID-19 second wave needs to be further investigated for their public health impact in the rest of the country and its possibility of becoming a VOC.","version":"1.2","doi":"10.1101/2021.04.22.440932","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.02.442326","pub_date":"2021-5-03","title":"Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals","abstract":"Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.","version":"1.1","doi":"10.1101/2021.05.02.442326","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.01.442279","pub_date":"2021-5-03","title":"Signaling through Fc\u03b3RIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19","abstract":"Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the Fc\u03b3RIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. The Fc\u03b3RIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19","version":"1.1","doi":"10.1101/2021.05.01.442279","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.02.442384","pub_date":"2021-5-03","title":"In silico and in vitro Demonstration of Homoharrintonine\u2019s Antagonism of RBD-ACE2 Binding and its Anti-inflammatory and anti-thrombogenic Properties in a 3D human vascular lung model","abstract":"Since 2019 the world has seen severe onslaught of SARS-CoV-2 viral pandemic. There is an urgent need for drugs that can be used to either prevent or treat the potentially fatal disease COVD-19. To this end, we screened FDA approved antiviral drugs which could be repurposed for COVID-19 through molecular docking approach in the various active sites of receptor binding domain (RBD). The RBD domain of SARS-CoV-2 spike protein is a promising drug target due to its pivotal role in viral-host attachment. Specifically, we focussed on identifying antiviral drugs which could a) block the entry of virus into host cells, b) demonstrate anti-inflammatory and/or anti-thrombogenic properties. Drugs which poses both properties could be useful for prevention and treatment of the disease. While we prioritized a few antiviral drugs based on molecular docking, corroboration with in vitro studies including a new 3D human vascular lung model strongly supported the potential of Homoharringtonine, a drug approved for chronic myeloid leukaemia to be repurposed for COVID-19. This natural product drug not only antagonized the biding of SARS-CoV-2 spike protein RBD binding to human angiotensin receptor 2 (ACE-2) protein but also demonstrated for the first time anti-thrombogenic and anti-leukocyte adhesive properties in a human cell model system. Overall, this work provides an important lead for development of rapid treatment of COVID-19 and also establishes a screening paradigm using molecular modelling and 3D human vascular lung model of disease to identify drugs with multiple desirable properties for prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.05.02.442384","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.03.442392","pub_date":"2021-5-03","title":"The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties","abstract":"One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.","version":"1.1","doi":"10.1101/2021.05.03.442392","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.01.442293","pub_date":"2021-5-03","title":"ACE2 expression in rat brain: implications for COVID-19 associated neurological manifestations","abstract":"We examined cell type-specific expression and distribution of rat brain angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, in rodent brain. ACE2 is ubiquitously present in brain vasculature, with the highest density of ACE2 expressing capillaries found in the olfactory bulb, the hypothalamic paraventricular, supraoptic and mammillary nuclei, the midbrain substantia nigra and ventral tegmental area, and the hindbrain pontine nucleus, pre-B\u00f6tzinger complex, and nucleus of tractus solitarius. ACE2 was expressed in astrocytes and astrocytic foot processes, pericytes and endothelial cells, key components of the blood-brain-barrier. We found discrete neuronal groups immunopositive for ACE2 in brainstem respiratory rhythm generating centers including the pontine nucleus, the parafascicular/retrotrapezoid nucleus, the parabrachial nucleus, the B\u00f6tzinger and pre-B\u00f6tzinger complex and the nucleus of tractus solitarius; in arousal-related pontine reticular nucleus and in gigantocellular reticular nuclei; in brainstem aminergic nuclei, including substantia nigra, ventral tegmental area, dorsal raphe, and locus coeruleus; in the epithalamic habenula, hypothalamic paraventricular and suprammamillary nuclei; and in the hippocampus. Identification of ACE2-expressing neurons in rat brain within well-established functional circuits facilitates prediction of possible neurological manifestations of brain ACE2 dysregulation during and after COVID-19 infection. ACE2 is present in astrocytes, pericytes, and endothelia of the blood brain barrier. Neuronal ACE2 expression is shown in discrete nuclei through the brain. Brainstem breathing, arousal-related, hypothalamic and limbic nuclei express ACE2. ACE2 is expressed in circuits potentially involved in COVID-19 pathophysiology.","version":"1.1","doi":"10.1101/2021.05.01.442293","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.02.441948","pub_date":"2021-5-03","title":"Inhibiting LSD1 suppresses coronavirus-induced inflammation but spares innate antiviral activity","abstract":"Tissue-resident macrophages exert critical but conflicting effects on the progression of coronavirus infections by secreting both anti-viral type I Interferons and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for Covid19, indiscriminately suppress both responses, possibly impairing viral clearance, and provide limited clinical benefit. Here we set up a mouse in vitro co-culture system that reproduces the macrophage response to SARS-CoV2 seen in patients and allows quantitation of inflammatory and antiviral activities. We show that the NFKB-dependent inflammatory response can be selectively inhibited by ablating the lysine-demethylase LSD1, which additionally unleashed interferon-independent ISG activation and blocked viral egress through the lysosomal pathway. These results provide a rationale for repurposing LSD1 inhibitors, a class of drugs extensively studied in oncology, for Covid-19 treatment. Targeting a chromatin-modifying enzyme in coronavirus infections curbs tissue-damage without affecting antiviral response","version":"1.1","doi":"10.1101/2021.05.02.441948","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.442222","pub_date":"2021-5-03","title":"Possible role of accessory proteins in the viral replication for the 20I/501Y.V1 (B.1.1.7) SARS-CoV-2 variant","abstract":"The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 variant. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher infectivity.","version":"1.1","doi":"10.1101/2021.04.30.442222","journal":"bioRxiv","score":null},{"id":"10.1101/2021.05.02.442313","pub_date":"2021-5-02","title":"Immunoinformatics approach for a novel multi-epitope vaccine construct against spike protein of human coronaviruses","abstract":"Spike (S) proteins are an attractive target as it mediates the binding of the SARS-CoV-2 to the host through ACE-2 receptors. We hypothesize that the screening of S protein sequences of all the HCoVs would result in the identification of potential multi-epitope vaccine candidates capable of conferring immunity against various HCoVs. In the present study, several machine learning-based in-silico tools were employed to design a broad-spectrum multi-epitope vaccine candidate against S protein of human coronaviruses. To the best of our knowledge, it is one of the first study, where multiple B-cell epitopes and T-cell epitopes (CTL and HTL) were predicted from the S protein sequences of all seven known HCoVs and linked together with an adjuvant to construct a potential broad-spectrum vaccine candidate. Secondary and tertiary structures were predicted, validated and the refined 3D-model was docked with an immune receptor. The vaccine candidate was evaluated for antigenicity, allergenicity, solubility, and its ability to achieve high-level expression in bacterial hosts. Finally, the immune simulation was carried out to evaluate the immune response after three vaccine doses. The designed vaccine is antigenic (with or without the adjuvant), non-allergenic, binds well with TLR-3 receptor and might elicit a diverse and strong immune response.","version":"1.1","doi":"10.1101/2021.05.02.442313","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.442063","pub_date":"2021-4-30","title":"Comparing Expression of OAS-RNaseL Pathway-Related Genes in SARS-CoV-2 and Similar Viruses","abstract":"The COVID-19 pandemic, caused by the virus SARS-CoV-2, has been a major public health emergency and has caused millions of deaths worldwide to date. Due to the novel nature of the virus, efforts across the world are underway to better understand the molecular pathogenesis of SARS-CoV-2 and how it interacts with host immune responses. One important branch of the innate immune response, the interferon system, triggers the expression of many effector mechanisms known to be powerful antagonists against many pathogenic viruses. One such interferon stimulated mechanism is the OAS-RNaseL pathway, which is known to trigger the degradation of viral RNA in infected host cells. Our study seeks to utilize publicly available transcriptomic data to analyze the host cell OAS-RNaseL pathway to SARS-CoV-2 infection. We hoped to gain an understanding of the importance of the pathway in controlling SARS-CoV-2 infection and whether or not the pathway could be exploited therapeutically. Our findings demonstrated that upregulation of OAS-RNaseL pathway genes in response to SARS-CoV-2 infection varies based on cell type and appeared to correlate with ACE2 receptor expression. Pathway responses to other viruses like SARS-CoV and MERS-CoV were found to parallel those to SARS-CoV-2, suggesting common response patterns by the pathway to these viruses. Overall, these results demonstrate that the OAS-RNaseL pathway could contribute to control of SARS-CoV-2 infection. Further studies on various mechanistic actions by the pathway would need to be conducted to fully understand its role in host defense and therapy.","version":"1.1","doi":"10.1101/2021.04.29.442063","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091397","pub_date":"2021-4-30","title":"Origin of Novel Coronavirus (COVID-19): A Computational Biology Study using Artificial Intelligence","abstract":"Origin of the COVID-19 virus (SARS-CoV-2) has been intensely debated in the scientific community since the first infected cases were detected in December 2019. The disease has caused a global pandemic, leading to deaths of thousands of people across the world and thus finding origin of this novel coronavirus is important in responding and controlling the pandemic. Recent research results suggest that bats or pangolins might be the hosts for SARS-CoV-2 based on comparative studies using its genomic sequences. This paper investigates the SARS-CoV-2 origin by using artificial intelligence (AI) and raw genomic sequences of the virus. More than 300 genome sequences of COVID-19 infected cases collected from different countries are explored and analysed using unsupervised clustering methods. The results obtained from various AI-enabled experiments using clustering algorithms demonstrate that all examined SARS-CoV-2 genomes belong to a cluster that also contains bat and pangolin coronavirus genomes. This provides evidence strongly supporting scientific hypotheses that bats and pangolins are probable hosts for SARS-CoV-2. At the whole genome analysis level, our findings also indicate that bats are more likely the hosts for the COVID-19 virus than pangolins.","version":"1.4","doi":"10.1101/2020.05.12.091397","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.441093","pub_date":"2021-4-30","title":"Allosteric Cross-Talk Among SARS-CoV-2 Spike\u2019s Receptor-Binding Domain Mutations Triggers an Effective Hijacking of Human Cell Receptor","abstract":"The rapid and relentless emergence of novel highly transmissible SARS-CoV-2 variants, possibly decreasing vaccine efficacy, currently represents a formidable medical and societal challenge. These variants frequently hold mutations on the Spike protein\u2019s Receptor-Binding Domain (RBD), which, binding to the Angiotensin-Converting Enzyme 2 (ACE2) receptor, mediates viral entry into the host cells. Here, all-atom Molecular Dynamics simulations and Dynamical Network Theory of the wild-type and mutant RBD/ACE2 adducts disclose that while the N501Y mutation (UK variant) enhances the Spike\u2019s binding affinity towards ACE2, the N501Y, E484K and K417N mutations (South African variant) aptly adapt to increase SARS-CoV-2 propagation via a two-pronged strategy: (i) effectively grasping ACE2 through an allosteric signaling between pivotal RBD structural elements; and (ii) impairing the binding of antibodies elicited by infected/vaccinated patients. This information, unlocking the molecular terms and evolutionary strategies underlying the increased virulence of emerging SARS-CoV-2 variants, set the basis for developing the next-generation anti-COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2021.04.30.441093","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.442060","pub_date":"2021-4-30","title":"SARS-CoV-2 bearing a mutation at the S1/S2 cleavage site exhibits attenuated virulence and confers protective immunity","abstract":"Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by host furin protease. Proteolytic cleavage activates the spike protein and influences both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogensis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited a low growth property. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, indicating the attenuated variant nature of S gene mutants. We reproduced the attenuated growth of S gene mutants in primary differenciated human airway epithelial cells. This transient infection was enough to induce protective neutralizing antibodies crossreacting with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of SARS-CoV-2 and highlights the potential benefits of S gene mutants as potential immunogens.","version":"1.1","doi":"10.1101/2021.04.29.442060","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.442139","pub_date":"2021-4-30","title":"Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein","abstract":"The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human \u03b2-1,2 N-acetlyglucosamintransferases I and II (MGAT1\u2206TM and MGAT2\u2206TM) and the \u03b2-1,4-galactosyltransferase (GalT\u2206TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.","version":"1.1","doi":"10.1101/2021.04.30.442139","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.440988","pub_date":"2021-4-30","title":"Limited within-host diversity and tight transmission bottlenecks limit SARS-CoV-2 evolution in acutely infected individuals","abstract":"The recent emergence of divergent SARS-CoV-2 lineages has raised concerns about the role of selection within individual hosts in propagating novel variants. Of particular concern are variants associated with immune escape and/or enhanced transmissibility. Though growing evidence suggests that novel variants can arise during prolonged infections, most infections are acute. Understanding the extent to which variants emerge and transmit among acutely infected hosts is therefore critical for predicting the pace at which variants resistant to vaccines or conferring increased transmissibility might emerge in the majority of SARS-CoV-2 infections. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely infected individuals. We find that within-host diversity during acute infection is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household. Accordingly, we also find that within-host variants are rarely detected along phylogenetically linked infections in the broader community. Together, these findings suggest that efficient selection and transmission of novel SARS-CoV-2 variants is unlikely during typical, acute infection. Patterns of SARS-CoV-2 within hosts suggest efficient selection and transmission of novel variants is unlikely during typical, acute infection.","version":"1.1","doi":"10.1101/2021.04.30.440988","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.441968","pub_date":"2021-4-30","title":"Influenza viral particles harboring the SARS-CoV-2 spike RBD as a combination respiratory disease vaccine","abstract":"Vaccines targeting SARS-CoV-2 have gained emergency FDA approval, however the breadth against emerging variants and the longevity of protection remains unknown. Post-immunization boosting may be required, perhaps on an annual basis if the virus becomes an endemic pathogen. Seasonal influenza virus vaccines are already developed every year, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed a recombinant influenza A virus (IAV) genetic platform that \u201creprograms\u201d the virus to package an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicits neutralizing antibodies and provides protection from lethal challenge with both pathogens. This technology may allow for leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses.","version":"1.1","doi":"10.1101/2021.04.30.441968","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.441559","pub_date":"2021-4-30","title":"Neutralization of B.1.1.28 P2 variant with sera of natural SARS-CoV-2 infection and recipients of BBV152 vaccine","abstract":"The emergence of new SARS-CoV-2 variants has been a serious threat to the public health system and vaccination program. The variant of concerns have been the under investigation for their neutralizing potential against the currently available COVID-19 vaccines. Here, we have determined the neutralization efficacy of B.1.1.28.2 variant with the convalescent sera of individuals with natural infection and BBV152 vaccination. The two-dose vaccine regimen significantly boosted the IgG titer and neutralizing efficacy against both B.1.1.28.2 and D614G variants compared to that seen with natural infection. The study demonstrated 1.92 and 1.09 fold reductions in the neutralizing titer against B.1.1.28.2 variant in comparison with prototype D614G variant with sera of vaccine recipients and natural infection respectively.","version":"1.1","doi":"10.1101/2021.04.30.441559","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.30.441434","pub_date":"2021-4-30","title":"N440K variant of SARS-CoV-2 has Higher Infectious Fitness","abstract":"Several variants of SARS-CoV-2 have been emerging across the globe, continuing to threaten the efforts to end COVID-19 pandemic. Recent data indicate the prevalence of variants with N440K Spike substitution in several parts of India, which is under the second wave of the pandemic. Here, we first analyze the prevalence of N440K variants within the sequences submitted from India and identify a rising trend of its spread across various clusters. We then compare the replicative fitness and infectivity of a prototype of this variant with two other previously prevalent strains. The N440K variant produced ten times higher infectious viral titers than a prevalent A2a strain, and over 1000 folds higher titers than a much less prevalent A3i strain prototype in Caco2 cells. Similar results were detected in Calu-3 cells as well, confirming the increased potency of the N440K variant. Interestingly, A3i strain showed the highest viral RNA levels, but the lowest infectious titers in the culture supernatants, indicating the absence of correlation between the RNA content and the infectivity of the sample. N440K mutation has been reported in several viral sequences across India and based on our results, we predict that the higher infectious titers achieved by N440K variant could possibly lead to its higher rate of transmission. Availability of more sequencing data in the immediate future would help understand the potential spread of this variant in more detail.","version":"1.1","doi":"10.1101/2021.04.30.441434","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427206","pub_date":"2021-4-30","title":"Azithromycin Plus Zinc Sulfate Rapidly and Synergistically Suppresses I\u03baB\u03b1-Mediated In Vitro Human Airway Cell ACE2 Expression for SARS-CoV-2 Entry","abstract":"Large-scale efforts have been persistently undertaken for medical prophylaxis and treatment of COVID-19 disasters worldwide. A variety of novel viral spike protein-targeted vaccine preparations have recently been clinically distributed based on accelerated approval. We revisited the early but inconclusive clinical interest in the combination of azithromycin and zinc sulfate repurposing with safety advantages. In vitro proof of concept was provided for rapid and synergistic suppression of ACE2 expression following treatments in human airway cells, Calu-3 and H322M. The two representative ACE2-expressing human airway cells indicate the upper and lower respiratory tracts. Prophylactic and early therapeutic roles of azithromycin combined with zinc are proposed for virus cellular entry prevention potential bridging to effective antibody production.","version":"1.2","doi":"10.1101/2021.01.19.427206","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.442061","pub_date":"2021-4-30","title":"Decitabine Reactivation of FoxM1-Dependent Endothelial Regeneration and Vascular Repair for Potential Treatment of Elderly ARDS and COVID-19 Patients","abstract":"Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS) and COVID-19. We repot that aging impairs the intrinsic FoxM1-dependent endothelial regeneration and vascular repair program and causes persistent lung injury and high mortality following sepsis. Therapeutic gene transduction of FOXM1 in vascular endothelium or treatment with FDA-approved drug Decitabine was sufficient to reactivate FoxM1-dependent lung endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. In COVID-19 lung autopsy samples, FOXM1 expression was not induced in vascular endothelial cells of elderly patients in contrast to mid-age patients. Thus, Decitabine reactivation of FoxM1-dependent vascular repair represents a potential effective therapy for elderly COVID-19 and non-COVID-19 ARDS patients.","version":"1.1","doi":"10.1101/2021.04.29.442061","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441880","pub_date":"2021-4-29","title":"Protracted yet coordinated differentiation of long-lived SARS-CoV-2-specific CD8+ T cells during COVID-19 convalescence","abstract":"CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc322-331, a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38- parameter CyTOF phenotyping on tetramer-identified Nuc322-331-specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS- CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322-331-specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a \u223c6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.","version":"1.1","doi":"10.1101/2021.04.28.441880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441832","pub_date":"2021-4-29","title":"An enveloped virus-like particle vaccine expressing a stabilized prefusion form of the SARS-CoV-2 spike protein elicits potent immunity after a single dose","abstract":"Development of efficacious single dose vaccines would substantially aid efforts to stop the uncontrolled spread of the COVID-19 pandemic. We evaluated enveloped virus-like particles (eVLPs) expressing various forms of the SARS-CoV-2 spike protein and several adjuvants in an effort to identify a COVID-19 vaccine candidate efficacious after a single dose. The eVLPs expressing a modified prefusion form of SARS-CoV-2 spike protein were selected as they induced the highest antibody binding titers and neutralizing activity after a single injection in mice. Formulation of SARS-CoV-2 S eVLPs with aluminum phosphate resulted in balanced induction of IgG2 and IgG1 isotypes and antibody binding and neutralization titers were undiminished for more than 3 months after a single immunization. A single dose of this candidate, VBI-2902a (prefusion S eVLPs formulated with aluminum phosphate), protected Syrian golden hamsters from challenge with SARS-CoV-2 and supports the on-going clinical evaluation of VBI-2902a as a potential single dose vaccine against COVID-19. VBI-2902a is a VLP-based vaccine candidate against SARS-COV-2 VBI-2902a contains VLPs pseudotyped with a modified prefusion SARS-COV-2 S in Alum. VBI-2902a induces robust neutralization antibody response against SARS-COV-2 S VBI-2902a protects hamsters from SARS-CoV-2 induced lung inflammation A single dose of VBI-2902a provides protective benefit in hamsters","version":"1.1","doi":"10.1101/2021.04.28.441832","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.442018","pub_date":"2021-4-29","title":"Molecular interactions of the M and E integral membrane proteins of SARS-CoV-2","abstract":"Specific lipid-protein interactions are key for cellular processes, and even more so for the replication of pathogens. The COVID-19 pandemic has drastically changed our lives and cause the death of nearly three million people worldwide, as of this writing. SARS-CoV-2 is the virus that causes the disease and has been at the center of scientific research over the past year. Most of the research on the virus is focused on key players during its initial attack and entry into the cellular host; namely the S protein, its glycan shield, and its interactions with the ACE2 receptors of human cells. As cases continue to raise around the globe, and new mutants are identified, there is an urgent need to understand the mechanisms of this virus during different stages of its life cycle. Here, we consider two integral membrane proteins of SARS-CoV-2 known to be important for viral assembly and infectivity. We have used microsecond-long all-atom molecular dynamics to examine the lipid-protein and protein-protein interactions of the membrane (M) and envelope (E) structural proteins of SARS-CoV-2 in a complex membrane model. We contrast the two proposed protein complexes for each of these proteins, and quantify their effect on their local lipid environment. This ongoing work also aims to provide molecular-level understanding of the mechanisms of action of this virus to possibly aid in the design of novel treatments.","version":"1.1","doi":"10.1101/2021.04.29.442018","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.442010","pub_date":"2021-4-29","title":"The natural stilbenoid (\u2013)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants","abstract":"Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host ACE2 receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (\u2013)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (\u2013)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 \u03bcM in contrast to an IC50 of 28.3 \u03bcM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index = 257.3). When assessed against the USA-WA1/2020 variant, (\u2013)-hopeaphenol also inhibited entry of a VSV\u0394G-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect assays (IC50 = 10.2 \u03bcM) without cytotoxicity. Notably, (\u2013)- hopeaphenol also inhibited two emerging variants of concern originating from the United Kingdom (B.1.1.7) and South Africa (B.1.351) in both cytopathic effect and spike-containing pseudovirus assays with similar (B.1.1.7) or improved (B.1.351) efficacies over the USA- WA1/2020 variant. These results identify (\u2013)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants including those with increased infectivity and/or reduced susceptibility to existing vaccines. SARS-CoV-2 antivirals are needed to supplement existing vaccine efforts and target emerging viral variants with increased infectivity or reduced susceptibility to existing vaccines. Here we show that (\u2013)-hopeaphenol, a naturally-occurring stilbenoid compound, in addition to its analogues vatalbinoside A and vaticanol B, inhibit SARS-CoV-2 by blocking the interaction of the viral spike protein with the cellular ACE2 entry receptor. Importantly, in addition to inhibiting the early USA-WA1/2020 SARS-CoV-2 variant, hopeaphenol also inhibits emerging variants of concern including B.1.1.7 (\u201cUnited Kingdom variant\u201d) and B.1.351 (\u201cSouth Africa variant\u201d), with improved efficacy against B.1.351. (\u2013)-Hopeaphenol therefore represents a new antiviral lead against infection from multiple SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.04.29.442010","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.441258","pub_date":"2021-4-29","title":"Allelic variation in Class I HLA determines pre-existing memory responses to SARS-CoV-2 that shape the CD8+ T cell repertoire upon viral exposure","abstract":"Effective presentation of antigens by HLA class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multi-omic technology to generate the first unified ex vivo characterization of the CD8+ T cell response to SARS-CoV-2 across 4 major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCR \u03b1/\u03b2 sequence diversity, and the utilization of pre-existing SARS-CoV-2 reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations influence pre-existing immunity to SARS-CoV-2 and shape the immune repertoire upon subsequent viral exposure. We perform a unified, multi-omic characterization of the CD8+ T cell response to SARS-CoV-2, revealing pre-existing immunity conditioned by HLA genotype.","version":"1.1","doi":"10.1101/2021.04.29.441258","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.29.441933","pub_date":"2021-4-29","title":"Possible link between higher transmissibility of B.1.617 and B.1.1.7 variants of SARS-CoV-2 and increased structural stability of its spike protein and hACE2 affinity","abstract":"The Severe Acute syndrome corona Virus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has caused alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests, which also might lead to inefficacy of the available drugs. It has also increased the chances of reinfection and immune escape. We have performed Molecular Dynamic simulations of three different Spike-ACE2 complexes, namely Wildtype (WT), B.1.1.7 variant (N501Y Spike mutant) and B.1.617 variant (L452R, E484Q Spike mutant) and compared their dynamics, binding energy and molecular interactions. Our result shows that mutation has caused the increase in the binding energy between the Spike and hACE2. In the case of B.1.617 variant, the mutations at L452R and E484Q increased the stability and intra-chain interactions in the Spike protein, which may change the interaction ability of human antibodies to this Spike variant. Further, we found that the B.1.1.7 variant had increased hydrogen interaction with LYS353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.04.29.441933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.056283","pub_date":"2021-4-29","title":"SwabExpress: An end-to-end protocol for extraction-free COVID-19 testing","abstract":"The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral activation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. After optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.","version":"1.2","doi":"10.1101/2020.04.22.056283","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.057810","pub_date":"2021-4-28","title":"Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens","abstract":"The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry based (MS) proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.","version":"1.3","doi":"10.1101/2020.04.23.057810","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441655","pub_date":"2021-4-28","title":"A broadly neutralizing antibody protects against SARS-CoV, pre-emergent bat CoVs, and SARS-CoV-2 variants in mice","abstract":"SARS-CoV in 2003, SARS-CoV-2 in 2019, and SARS-CoV-2 variants of concern (VOC) can cause deadly infections, underlining the importance of developing broadly effective countermeasures against Group 2B Sarbecoviruses, which could be key in the rapid prevention and mitigation of future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV, bat CoVs WIV-1 and RsSHC014, and SARS-CoV-2 variants D614G, B.1.1.7, B.1.429, B1.351 by a receptor-binding domain (RBD)-specific antibody DH1047. Prophylactic and therapeutic treatment with DH1047 demonstrated protection against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B1.351infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among Sarbecoviruses. We conclude that DH1047 is a broadly neutralizing and protective antibody that can prevent infection and mitigate outbreaks caused by SARS-like strains and SARS-CoV-2 variants. Our results argue that the RBD conserved epitope bound by DH1047 is a rational target for pan Group 2B coronavirus vaccines.","version":"1.1","doi":"10.1101/2021.04.27.441655","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.440939","pub_date":"2021-4-28","title":"Massively Multiplexed Affinity Characterization of Therapeutic Antibodies Against SARS-CoV-2 Variants","abstract":"Antibody therapies represent a valuable tool to reduce COVID-19 deaths and hospitalizations. Multiple antibody candidates have been granted emergency use authorization by the FDA and many more are in clinical trials. Most antibody therapies for COVID-19 are engineered to bind to the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein and disrupt its interaction with ACE2. Notably, several SARS-CoV-2 strains have accrued mutations throughout the RBD that improve ACE2 binding affinity, enhance viral transmission, and escape some existing antibody therapies. Here, we measure the binding affinity of 33 therapeutic antibodies against a large panel of SARS-CoV-2 variants and related strains of clinical significance to determine epitopic residues, determine which mutations result in loss of binding, and predict how future RBD variants may impact antibody efficacy. By measuring protein binding in vitro, we identify which clinical antibodies retain binding to various mutant SARS-CoV-2 strains.","version":"1.1","doi":"10.1101/2021.04.27.440939","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.29.925867","pub_date":"2021-4-28","title":"Insights into Cross-species Evolution of Novel Human Coronavirus SARS-CoV-2 and Defining Immune Determinants for Vaccine Development","abstract":"Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in the city of Wuhan, China during December 2019, has now spread to various countries across the globe triggering a heightened containment effort. This human pathogen is a member of betacoronavirus genus carrying 30 kilobase of single positive-sense RNA genome. Understanding the evolution, zoonotic transmission, and source of this novel virus would help accelerating containment and prevention efforts. The present study reported detailed analysis of SARS-CoV-2 genome evolution and potential candidate peptides for vaccine development. This new coronavirus genotype might have been evolved from a bat-coronavirus by accumulating non-synonymous mutations, indels, and recombination events. Structural proteins Spike (S), and Membrane (M) had extensive mutational changes, whereas Envelope (E) and Nucleocapsid (N) proteins were very conserved suggesting differential selection pressures exerted on SARS-CoV-2 during evolution. Interestingly, SARS-CoV-2 Spike protein contains a 39 nucleotide sequence insertion relative to SARS-like bat-SL-CoVZC45/2017. Furthermore, we identified eight high binding affinity (HBA) CD4 T-cell epitopes in the S, E, M and N proteins, which can be commonly recognized by HLA-DR alleles of Asia and Asia-Pacific Region population. These immunodominant epitopes can be incorporated in universal subunit SARS-CoV-2 vaccine. Diverse HLA types and variations in the epitope binding affinity may contribute to the wide range of immunopathological outcomes of circulating virus in humans. Our findings emphasize the requirement for continuous surveillance of SARS-CoV-2 strains in live animal markets to better understand the viral adaptation to human host and to develop practical solutions to prevent the emergence of novel pathogenic SARS-CoV-2 strains.","version":"1.3","doi":"10.1101/2020.01.29.925867","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.440619","pub_date":"2021-4-28","title":"Topical TMPRSS2 inhibition prevents SARS-CoV-2 infection in differentiated primary human airway cells","abstract":"There are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection. The likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection. We first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection. These experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.","version":"1.2","doi":"10.1101/2021.04.23.440619","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425331","pub_date":"2021-4-28","title":"Interferon alpha-based combinations suppress SARS-CoV-2 infection in vitro and in vivo","abstract":"There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggers intrinsic and extrinsic cellular antiviral responses, as well as reduces replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Although IFNa alone was insufficient to completely abolish SARS-CoV-2 replication, combinations of IFNa with remdesivir or other antiviral agents (EIDD-2801, camostat, cycloheximide, or convalescent serum) showed strong synergy and effectively inhibited SARS-CoV-2 infection in human lung epithelial Calu-3 cells. Furthermore, we showed that the IFNa-remdesivir combination suppressed virus replication in human lung organoids, and that its single prophylactic dose attenuated SARS-CoV-2 infection in lungs of Syrian hamsters. Transcriptome and metabolomic analyses showed that the combination of IFNa-remdesivir suppressed virus-mediated changes in infected cells, although it affected the homeostasis of uninfected cells. We also demonstrated synergistic antiviral activity of IFNa2a-based combinations against other virus infections in vitro. Altogether, our results indicate that IFNa2a-based combination therapies can achieve higher efficacy while requiring lower dosage compared to monotherapies, making them attractive targets for further pre-clinical and clinical development.","version":"1.5","doi":"10.1101/2021.01.05.425331","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441695","pub_date":"2021-4-28","title":"SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels","abstract":"Diarrhea occurs in 2-50% of cases of COVID-19 (\u223c8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for \u223c24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by \u223c8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of \u223c10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).","version":"1.1","doi":"10.1101/2021.04.27.441695","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441840","pub_date":"2021-4-28","title":"Regulation of Lysosome-Associated Membrane Protein 3 (LAMP3) in Lung Epithelial Cells by Coronaviruses (SARS-CoV-1/2) and Type I Interferon Signaling","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is a major risk factor for mortality and morbidity in critical care hospitals around the world. Lung epithelial type II cells play a major role in several physiological processes, including recognition and clearance of respiratory viruses as well as repair of lung injury in response to environmental toxicants. Gene expression profiling of lung epithelial type II-specific genes led to the identification of lysosomal-associated membrane protein 3 (LAMP3). Intracellular locations of LAMP3 include plasma membrane, endosomes, and lysosomes. These intracellular organelles are involved in vesicular transport and facilitate viral entry and release of the viral RNA into the host cell cytoplasm. In this study, regulation of LAMP3 expression in human lung epithelial cells by several respiratory viruses and type I interferon signaling was investigated. Coronaviruses including SARS-CoV-1 and SARS-CoV-2 significantly induced LAMP3 expression in lung epithelial cells within 24 hours after infection that required the presence of ACE2 viral entry receptor. Time-course experiments revealed that the induced expression of LAMP3 by SARS-CoV-2 was correlated with the induced expression of interferon-beta1 (IFNB1) and signal transducers and activator of transcription 1 (STAT1) mRNA levels. LAMP3 was also induced by direct IFN-beta treatment or by infection with influenza virus lacking the non-structural protein1(NS1) in NHBE bronchial epithelial cells. LAMP3 expression was induced in human lung epithelial cells by several respiratory viruses, including respiratory syncytial virus (RSV) and the human parainfluenza virus 3 (HPIV3). Location in lysosomes and endosomes as well as induction by respiratory viruses and type I Interferon suggests that LAMP3 may have an important role in inter-organellar regulation of innate immunity and a potential target for therapeutic modulation in health and disease. Furthermore, bioinformatics revealed that a subset of lung type II cell genes were differentially regulated in the lungs of COVID-19 patients.","version":"1.1","doi":"10.1101/2021.04.28.441840","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441763","pub_date":"2021-4-28","title":"A spike-ferritin nanoparticle vaccine induces robust innate immune activity and drives polyfunctional SARS-CoV-2-specific T cells","abstract":"Potent cellular responses to viral infections are pivotal for long -lived protection. Evidence is growing that these responses are critical in SARS -CoV-2 immunity. Assessment of a SARS -CoV-2 spike ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel\u00ae (AH) or Army Liposome Formulation containing QS-21 (ALFQ) demonstrated unique vaccine evoked immune signatures. SpFN+ALFQ enhanced recruitment of highly activated classical and non -classical antigen presenting cells (APCs) to the vaccine-draining lymph nodes of mice. The multifaceted APC response of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike -specific T cells with a bias towards TH1 responses and more robust SARS-CoV-2 spike-specific recall response. In addition, SpFN+ALFQ induced Kb spike(539-546)-specific memory CD8+ T cells with effective cytolytic function and distribution to the lungs. This epitope is also present in SARS-CoV, thus suggesting that generation of cross-reactive T cells may provide protection against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant, generates effective SARS-CoV-2 specific innate and adaptive immune T cell responses that are key components to inducing long-lived immunity. SpFN vaccine generates multifactorial cellular immune responses.","version":"1.1","doi":"10.1101/2021.04.28.441763","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441474","pub_date":"2021-4-28","title":"Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drive rapid and potent immunogenicity capable of single-dose protection","abstract":"Antibodies from SARS-CoV-2 vaccines may target epitopes which reduce durability or increase the potential for escape from vaccine-induced immunity. Using a novel synthetic vaccinology pipeline, we developed rationally immune focused SARS-CoV-2 Spike-based vaccines. N-linked glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and comprehensive in vitro screening, we incorporated glycans into the Spike Receptor-Binding Domain (RBD) and assessed antigenic profiles. We developed glycan coated RBD immunogens and engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicited potent neutralizing antibodies in guinea pigs, hamsters and multiple mouse models, including human ACE2 and human B cell repertoire transgenics. RBD nanoparticles encoding wild-type and the P.1 SARS-CoV-2 variant induced high levels of cross-neutralizing antibodies. Single, low dose immunization protected against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of novel, potent coronavirus vaccines.","version":"1.1","doi":"10.1101/2021.04.28.441474","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.436907","pub_date":"2021-4-28","title":"A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents","abstract":"There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35\u00b0C than 37\u00b0C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (\u03b21 and \u03bb1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.","version":"1.3","doi":"10.1101/2021.03.25.436907","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.428004","pub_date":"2021-4-28","title":"Allosteric activation of SARS-CoV-2 RdRp by remdesivir triphosphate and other phosphorylated nucleotides","abstract":"The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), Nsp12, has a unique NiRAN domain that transfers nucleoside monophosphates to the Nsp9 protein. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation) Nsp12 exists in inactive state in which NiRAN/RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or NTPs partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN/RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically-linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and design of its inhibitors. Codon-optimization of Nsp12 triggers misfolding and activity loss Slow translation, accessory Nsp7 and Nsp8 subunits, and NTPs rescue Nsp12 Non-substrate nucleotides activate RNA chain synthesis, likely via NiRAN domain Crosstalk between two Nsp12 active sites that bind the same ligands","version":"1.2","doi":"10.1101/2021.01.24.428004","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441707","pub_date":"2021-4-28","title":"Therapeutic effect of CT-P59 against SARS-CoV-2 South African variant","abstract":"The global circulation of newly emerging variants of SARS-CoV-2 is a new threat to public health due to their increased transmissibility and immune evasion. Moreover, currently available vaccines and therapeutic antibodies were shown to be less effective against new variants, in particular, the South African (SA) variant, termed 501Y.V2 or B.1.351. To assess the efficacy of the CT-P59 monoclonal antibody against the SA variant, we sought to perform as in vitro binding and neutralization assays, and in vivo animal studies. CT-P59 neutralized B.1.1.7 variant to a similar extent as to wild type virus. CT-P59 showed reduced binding affinity against a RBD (receptor binding domain) triple mutant containing mutations defining B.1.351 (K417N/E484K/N501Y) also showed reduced potency against the SA variant in live virus and pseudovirus neutralization assay systems. However, in vivo ferret challenge studies demonstrated that a therapeutic dosage of CT-P59 was able to decrease B.1.351 viral load in the upper and lower respiratory tracts, comparable to that observed for the wild type virus. Overall, although CT-P59 showed reduced in vitro neutralizing activity against the SA variant, sufficient antiviral effect in B.1.351-infected animals was confirmed with a clinical dosage of CT-P59, suggesting that CT-P59 has therapeutic potential for COVID-19 patients infected with SA variant. CT-P59 significantly inhibit B.1.1.7 variant to a similar extent as to wild type virus CT-P59 showed reduced potency against the B.1.351 variant in in vitro studies Therapeutic dosage of CT-P59 showed in vivo neutralizing potency against B.1.351 in ferret challenge study.","version":"1.1","doi":"10.1101/2021.04.27.441707","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.28.441797","pub_date":"2021-4-28","title":"Prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine","abstract":"The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (\u0394NS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of \u0394NS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a \u0394NS1 virus results in an immediate antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that \u0394NS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on \u0394NS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.","version":"1.1","doi":"10.1101/2021.04.28.441797","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441606","pub_date":"2021-4-27","title":"Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. - Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors","version":"1.1","doi":"10.1101/2021.04.27.441606","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441510","pub_date":"2021-4-27","title":"Aerosol Exposure of Cynomolgus Macaques to SARS-CoV-2 Results in More Severe Pathology than Existing Models","abstract":"The emergence of SARS-CoV-2 pandemic has highlighted the need for animal models that faithfully recapitulate the salient features of COVID-19 disease in humans; these models are necessary for the rapid down-selection, testing, and evaluation of medical countermeasures. Here we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure, combined intratracheal/intranasal and small particle aerosol, in two nonhuman primate species: rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at the time of necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models were suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.","version":"1.1","doi":"10.1101/2021.04.27.441510","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.26.441517","pub_date":"2021-4-27","title":"Relative Mutant N501Y SARS-CoV-2 Spike Protein RBD Inhibition of Anti-Spike Protein IgG and ACE-2 Binding to Spike Protein Species","abstract":"In the SARS-CoV-2 coronavirus pandemic of 2019 (COVID-19), it has become evident that the ACE-2 receptor-binding domain (RBD) of the viral spike protein (SP) is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. The most definitive confirmation of this contention is that the two mRNA COVID-19 vaccines in general use, which elicit antibodies specific for the RBD, exhibit approximately 95% protective efficacy against COVID-19. A potential challenge to vaccine efficacy is the emergence of SARS-CoV-2 variants possessing multiple mutations affecting amino acid residues in the RBD. Of concern are variants that arose in the United Kingdom, Brazil and South Africa. One of the variants, designated B.1.351, has shown a higher transmissibility due to greater affinity for the ACE-2 receptor and decreased neutralization by convalescent plasma, therapeutic monoclonal antibodies, and post-vaccination plasma. Common to several of the variants is the N501Y mutation in the RBD, which may be responsible for at least part of the observed variant properties. To test this hypothesis, we measured the ability of the Y501 RBD to inhibit binding of the wild type RBD and full SP (S1 + S2) to the ACE-2 protein and a human monoclonal IgG antibody elicited to the wild type RBD, relative to the wild type RBD in two enzyme-linked immunosorbent assays (ELISAs). We found no significant difference in the IC50 of the two RBD species\u2019 inhibition of ACE-2 binding, but unexpectedly found that the IC50 of the wild type RBD inhibition of antibody binding was nearly twice that of the Y501 RBD, reflecting a lower affinity. These results suggest that the individual N501Y mutation does not contribute to altered viral properties by itself, but may contribute to a collective conformational shift produced by multiple mutations.","version":"1.1","doi":"10.1101/2021.04.26.441517","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.26.441518","pub_date":"2021-4-27","title":"Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.","version":"1.1","doi":"10.1101/2021.04.26.441518","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441512","pub_date":"2021-4-27","title":"Iota-carrageenan prevents the replication of SARS-CoV-2 on an in vitro respiratory epithelium model","abstract":"There are, except for remdesivir, no approved antivirals for the treatment or prevention of SARS-CoV-2 infections. Iota-carrageenan formulated into a nasal spray has already been proven safe and effective in viral respiratory infections. We explored this antiviral activity in Calu-3, a human respiratory model cell line. A formula of iota-carrageenan and sodium chloride, as a nasal spray, already approved for human use, effectively inhibited SARS-CoV-2 infection in vitro, providing a more substantial reference for further clinical studies or developments.","version":"1.1","doi":"10.1101/2021.04.27.441512","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441589","pub_date":"2021-4-27","title":"Long-lasting humoral immunity in Covid-19 infected patients at a University Hospital Clinic in \u00d6sterg\u00f6tland County Council during 2020-2021","abstract":"Longitudinal serum samples and nasopharyngeal/nasal swab samples were collected from forty-eight individuals (median age 66yrs) with Covid-19 PCR-positive test results at Link\u00f6ping University Hospital. Samples were collected from initial visit and for 6 months follow up. Presence of serum IgG and IgA against SARS-CoV-2 antigens (S1-spike, nucleocapsid and NSP3) were analyzed. Nasal swabs were tested for presence of IgA against the outer envelope S1 spike protein. Ninety-two percent of participants were seropositive against SARS-CoV-2 recombinant proteins at day 28 from study entry and all (100%) were seropositive from samples collected at 2 months or later. The most common antibody responses (both serum IgG, mainly IgG1 and IgA) were detected against the S1-spike protein and the nucleoprotein. In samples collected from nasal tissues considerably lower frequencies of IgA-positive reactivities were detected. Sixteen to 18 percent of study participants showed detectable IgA levels in nasal samples, except at day 60 when 36% of tested individuals showed presence of IgA against the S1-spike protein. The study suggests that the absolute majority of studied naturally infected Covid-19 patient in the Linkoping, Ostergotland health region develop over 6 months lasting detectable levels of serum IgG and IgA responses towards the SARS-CoV-2 S1-spike protein as well as against the nucleoprotein, but not against the non-structural protein 3.","version":"1.1","doi":"10.1101/2021.04.27.441589","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.27.441521","pub_date":"2021-4-27","title":"Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs","abstract":"Designed Ankyrin Repeat Proteins (DARPins) are a class of antibody mimetics with a high and mostly unexplored potential in drug development. They are clinically validated and thus represent a true alternative to classical immunoglobulin formats. In contrast to immunoglobulins, they are built from solenoid protein domains comprising an N-terminal capping repeat, one or more internal repeats and a C-terminal capping repeat. By using in silico analysis and a rationally guided Ala-Scan, we identified position 17 of the N-terminal capping repeat to play a key role for the overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by about 8\u00b0C to 10\u00b0C when the original Asp17 was replaced by Leu, Val, Ile, Met, Ala or Thr, as shown by high-temperature unfolding experiments at equilibrium. We then transferred the Asp17Leu mutation to various backgrounds, including different N- and C-terminal capping repeats and clinically validated DARPin domains, such as the VEGF-binding ankyrin repeat domain of abicipar pegol. In all cases, the proteins remained monomeric and showed improvements in the thermostability of about 8\u00b0C to 16\u00b0C. Thus, the replacement of Asp17 seems to be generically applicable to this drug class. Molecular dynamics simulations show that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, such a beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development. This mutation is likely responsible, at least in part, for the very high melting temperature (>90\u00b0C) of this promising anti-Covid-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.","version":"1.1","doi":"10.1101/2021.04.27.441521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.25.441372","pub_date":"2021-4-26","title":"Control-theoretic immune tradeoffs explain SARS-CoV-2 virulence and transmission variation","abstract":"Dramatic variation in SARS-CoV-2 virulence and transmission between hosts has driven the COVID-19 pandemic. The complexity and dynamics of the immune response present a challenge to understanding variation in SARS-CoV-2 infections. To address this challenge, we apply control theory, a framework used to study complex feedback systems, to establish rigorous mathematical bounds on immune responses. Two mechanisms of SARS-CoV-2 biology are sufficient to create extreme variation between hosts: (1) a sparsely expressed host receptor and (2) potent, but not unique, suppression of interferon. The resulting model unifies disparate and unexplained features of the SARS-CoV-2 pandemic, predicts features of future viruses that threaten to cause pandemics, and identifies potential interventions.","version":"1.1","doi":"10.1101/2021.04.25.441372","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.26.440920","pub_date":"2021-4-26","title":"A SARS CoV-2 nucleocapsid vaccine protects against distal viral dissemination","abstract":"The SARS CoV-2 pandemic has killed millions of people. This viral infection can also result in substantial morbidity, including respiratory insufficiency and neurological manifestations, such as loss of smell and psychiatric diseases. Most SARS CoV-2 vaccines are based on the spike antigen, and although they have shown extraordinary efficacy at preventing severe lung disease and death, they do not always confer sterilizing immune protection. We performed studies in K18-hACE2 mice to evaluate whether the efficacy of SARS CoV-2 vaccines could be augmented by incorporating nucleocapsid as a vaccine antigen. We vaccinated mice with adenovirus-based vaccines encoding spike antigen alone, nucleocapsid antigen alone, or combined spike and nucleocapsid antigens. Mice were then challenged intranasally with SARS CoV-2, and acute viral loads were quantified at a proximal site of infection (lung) and a distal site of infection (brain). Interestingly, the spike-based vaccine conferred acute protection in the lung, but not in the brain. The spike-based vaccine conferred acute protection in the brain only if combined with the nucleocapsid-based vaccine. These findings suggest that nucleocapsid-specific immunity is important for the distal control of SARS CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.","version":"1.1","doi":"10.1101/2021.04.26.440920","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.24.441228","pub_date":"2021-4-26","title":"A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV-2 challenge in rhesus macaques","abstract":"Persistent transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has given rise to a COVID-19 pandemic. Several vaccines, evoking protective spike antibody responses, conceived in 2020, are being deployed in mass public health vaccination programs. Recent data suggests, however, that as sequence variation in the spike genome accumulates, some vaccines may lose efficacy. Using a macaque model of SARS-CoV-2 infection, we tested the efficacy of a peptide-based vaccine targeting MHC Class I epitopes on the SARS-CoV-2 nucleocapsid protein. We administered biodegradable microspheres with synthetic peptides and adjuvants to rhesus macaques. Unvaccinated control and vaccinated macaques were challenged with 1 x 108 TCID50 units of SARS-CoV-2, followed by assessment of clinical symptoms, viral load, chest radiographs, sampling of peripheral blood and bronchoalveolar lavage (BAL) fluid for downstream analysis. Vaccinated animals were free of pneumonia-like infiltrates characteristic of SARS-CoV-2 infection and presented with lower viral loads relative to controls. Gene expression in cells collected from BAL samples of vaccinated macaques revealed a unique signature associated with enhanced development of adaptive immune responses relative to control macaques. We demonstrate that a room temperature stable peptide vaccine based on known immunogenic HLA Class I bound CTL epitopes from the nucleocapsid protein can provide protection against SARS-CoV-2 infection in non-human primates.","version":"1.1","doi":"10.1101/2021.04.24.441228","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.26.441457","pub_date":"2021-4-26","title":"Niclosamide shows strong antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency against the UK B.1.1.7 and SA B.1.351 variant","abstract":"SARS-CoV-2 variants are emerging with potential increased transmissibility highlighting the great unmet medical need for new therapies. Niclosamide is a potent anti-SARS-CoV-2 agent that has advanced in clinical development. We validate the potent antiviral efficacy of niclosamide in a SARS-CoV-2 human airway model. Furthermore, niclosamide is effective against the D614G, B.1.1.7 and B.1.351 variants. Our data further support the potent anti-SARS-CoV-2 properties of niclosamide and highlights its great potential as a therapeutic agent for COVID-19.","version":"1.1","doi":"10.1101/2021.04.26.441457","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.26.441501","pub_date":"2021-4-26","title":"A potently neutralizing anti-SARS-CoV-2 antibody inhibits variants of concern by binding a highly conserved epitope","abstract":"With the emergence of SARS-CoV-2 variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here we developed a panel of neutralizing anti-SARS-CoV-2 mAbs that bind the receptor binding domain of the spike protein at distinct epitopes and block virus attachment to cells and its receptor, human angiotensin converting enzyme-2 (hACE2). While several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by historical SARS-CoV-2 strains, others induced escape variants in vivo and lost activity against emerging strains. We identified one mAb, SARS2-38, that potently neutralizes all SARS-CoV-2 variants of concern tested and protects mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engages a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of inhibitory antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.04.26.441501","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.03.438113","pub_date":"2021-4-26","title":"Bioinformatics analysis of SARS-CoV-2 RBD mutant variants and insights into antibody and ACE2 receptor binding","abstract":"Prevailing COVID-19 vaccines are based on the spike protein of earlier SARS-CoV-2 strain that emerged in Wuhan, China. The continuously evolving nature of SARS-CoV-2 resulting emergence of new variants raises the risk of immune absconds. During the last few months, several RBD (receptor-binding domain) variants have been reported to affect the vaccine efficacy considerably. Soon after reporting of a new double mutant variant (L452R & E484Q) in India, the country facing a deadlier second wave of infections which prompts researchers to suspects this variant to be accountable. To address the relevant concerns about this new variant affecting vaccine efficacy, we performed molecular simulation dynamics based structural analysis of spike protein of double mutant (L452R & E484Q) along with K417G variants and earlier reported RBD variants and found structural changes in RBD region after comparing with the wild type. Comparison of the binding affinity of the double mutant and earlier reported RBD variant for ACE2 (angiotensin 2 altered enzymes) receptor and CR3022 antibody with the wild-type strain revealed the lowest binding affinity of the double mutant for CR3022 among all other variants. These findings suggest that the newly emerged double mutant could significantly reduce the impact of the current vaccine which threatens the protective efficacy of current vaccine therapy.","version":"1.2","doi":"10.1101/2021.04.03.438113","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.26.441280","pub_date":"2021-4-26","title":"SARS-CoV-2 Entry Protein TMPRSS2 and Its Homologue, TMPRSS4 Adopts Structural Fold Similar to Blood Coagulation and Complement Pathway Related Proteins","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes TMPRSS2 receptor to enter target human cells and subsequently causes coronavirus disease 19 (COVID-19). TMPRSS2 belongs to the type II serine proteases of subfamily TMPRSS, which is characterized by the presence of the serine-protease domain. TMPRSS4 is another TMPRSS member, which has a domain architecture similar to TMPRSS2. TMPRSS2 and TMPRSS4 have been shown to be involved in SARS-CoV-2 infection. However, their normal physiological roles have not been explored in detail. In this study, we analyzed the amino acid sequences and predicted 3D structures of TMPRSS2 and TMPRSS4 to understand their functional aspects at the protein domain level. Our results suggest that these proteins are likely to have common functions based on their conserved domain organization. Furthermore, we show that the predicted 3D structure of their serine protease domain has significant similarity to that of plasminogen which dissolves blood clot, and of other blood coagulation related proteins. Additionally, molecular docking analyses of inhibitors of four blood coagulation and anticoagulation factors show the same high specificity to TMPRSS2 and TMPRSS4 3D structures. Hence, our observations are consistent with the blood coagulopathy observed in COVID-19 patients and their predicted functions based on the sequence and structural analyses offer avenues to understand better and explore therapeutic approaches for this disease.","version":"1.1","doi":"10.1101/2021.04.26.441280","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441186","pub_date":"2021-4-26","title":"Binding mechanism of neutralizing Nanobodies targeting SARS-CoV-2 Spike Glycoprotein","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors. Several nanobodies neutralize SARS-CoV-2 infection by binding to the receptor-binding domain (RBD) of S protein, but the underlying mechanism is not well understood. Here, we identified an extended network of pairwise interactions between RBD and nanobodies H11-H4, H11-D4, and Ty1 by performing all-atom molecular dynamics (MD) simulations. Simulations of the nanobody-RBD-ACE2 complex revealed that H11-H4 more strongly binds to RBD without overlapping with ACE2 and triggers dissociation of ACE2 due to electrostatic repulsion. In comparison, Ty1 binding results in dissociation of ACE2 from RBD due to an overlap with the ACE2 binding site, whereas H11-D4 binding does not trigger ACE2 dissociation. Mutations in SARS-CoV-2 501Y.V1 and 501.V2 variants resulted in a negligible effect on RBD-ACE2 binding. However, the 501.V2 variant weakened H11-H4 and H11-D4 binding while strengthening Ty1 binding to RBD. Our simulations indicate that all three nanobodies can neutralize 501Y.V1 while Ty1 is more effective against the 501.V2 variant.","version":"1.1","doi":"10.1101/2021.04.23.441186","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.425605","pub_date":"2021-4-26","title":"Role of spatial patterning of N-protein interactions in SARS-CoV-2 genome packaging","abstract":"Viruses must efficiently and specifically package their genomes while excluding cellular nucleic acids and viral sub-genomic fragments. Some viruses use specific packaging signals, which are conserved sequence/structure motifs present only in the full-length genome. Recent work has shown that viral proteins important for packaging can undergo liquid-liquid phase separation (LLPS), where one or two viral nucleic acid binding proteins condense with the genome. The compositional simplicity of viral components lends itself well to theoretical modeling compared to more complex cellular organelles. Viral LLPS can be limited to one or two viral proteins and a single genome that is enriched in LLPS-promoting features. In our previous study, we observed that LLPS-promoting sequences of SARS-CoV-2 are located at the 5\u02b9 and 3\u02b9 ends of the genome, whereas the middle of the genome is predicted to consist mostly of solubilizing elements. Is this arrangement sufficient to drive single genome packaging, genome compaction, and genome cyclization? We addressed these questions using a coarse-grained polymer model, LASSI, to study the LLPS of nucleocapsid protein with RNA sequences that either promote LLPS or solubilization. With respect to genome cyclization, we find the most optimal arrangement restricts LLPS-promoting elements to the 5\u02b9 and 3\u02b9 ends of the genome, consistent with the native spatial patterning. Genome compaction is enhanced by clustered LLPS-promoting binding sites, while single genome packaging is most efficient when binding sites are distributed throughout the genome. These results suggest that many and variably positioned LLPS-promoting signals can support packaging in the absence of a singular packaging signal which argues against necessity of such a feature. We hypothesize that this model should be generalizable to multiple viruses as well as cellular organelles like paraspeckles, which enrich specific, long RNA sequences in a defined arrangement. The COVID-19 pandemic has motivated research of the basic mechanisms of coronavirus replication. A major challenge faced by viruses such as SARS-CoV-2 is the selective packaging of a large genome in a relatively small capsid while excluding host and sub-genomic nucleic acids. Genomic RNA of SARS-CoV-2 can condense with the Nucleocapsid (N-protein), a structural protein component critical for packaging of many viruses. Notably, certain regions of the genomic RNA drive condensation of N-protein while other regions solubilize it. Here, we explore how the spatial patterning of these opposing elements promotes single genome compaction, packaging, and cyclization. This model informs future in silico experiments addressing spatial patterning of genomic features that are experimentally intractable because of the length of the genome.","version":"1.2","doi":"10.1101/2021.01.06.425605","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441195","pub_date":"2021-4-26","title":"Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies","abstract":"Many anti-SARS-CoV-2 neutralizing antibodies target the ACE2-binding site on viral spike receptor-binding domains (RBDs). The most potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly-emergent zoonotic sarbecoviruses and variants, but usually show only weak neutralization potencies. We characterized two class 4 anti-RBD antibodies derived from COVID-19 donors that exhibited broad recognition and potent neutralization of zoonotic coronavirus and SARS-CoV-2 variants. C118-RBD and C022-RBD structures revealed CDRH3 mainchain H-bond interactions that extended an RBD \u03b2-sheet, thus reducing sensitivity to RBD sidechain changes, and epitopes that extended from the cryptic epitope to occlude ACE2 binding. A C118-spike trimer structure revealed rotated RBDs to allow cryptic epitope access and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.","version":"1.1","doi":"10.1101/2021.04.23.441195","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.25.441271","pub_date":"2021-4-26","title":"Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier","abstract":"Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.","version":"1.1","doi":"10.1101/2021.04.25.441271","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.25.441361","pub_date":"2021-4-26","title":"Conserved in 186 countries the RBD fraction of SARS CoV-2 S-protein with in-silicoT500S mutation strongly blocks ACE2 rejecting the viral spike; A Molecular-docking analysis","abstract":"SARS-CoV-2 developed global-pandemic with millions of infections/deaths. Blocker/inhibitor of ACE2 and viral-spikes Receptor-Binding-Domain RBD-blockers are helpful. Here, conserved RBD (CUTs) from 186-countries were compared with WUHAN-Hu-1 wild-type by CLUSTAL-X2 and Structural-alignment using Pymol. The RBD of ACE2-bound nCOV2 crystal-structure (2.68\u00c5) 6VW1 was analyzed by Haddock-PatchDock. Extensive structural study/trial to introduce point/double/triple mutations in the following locations (Y489S/Y453S/T500S/T500Y)/(Y489S,Y453S/Y489S,T500S/Y489S,T500Y/Y453S,T500S/Y453S, T500Y)/ (Y489S,Y453S,T500S/Y489S,Y453S,T500Y) of CUT4 (most-effective) were tested with Swiss-Model-Expacy. Blind-docking of mutated-CUTs to ACE2 (6VW1) by Haddock-Hawkdock was performed and optimally complete-rejection of nCOV2 to ACE2 was noticed. Further, competitive-docking/binding-analyses were done by PRODIGY. Present results suggest that compared to the wild-spike, CUT4 showed extra LYS31-PHE490/GLN42-GLN498 bonding and lack of TYR41-THR500 interaction (in wild H-bond:2.639\u00c5) with ACE2 RBD. Mutated-CUT4 strongly binds with the ACE2-RBD, promoting TYR41-T500S (H-bond: 2.0\u00c5 and 1.8\u00c5)/T500Y (H-bond:2.6\u00c5) interaction and complete inhibition of ACE2 RBD-nCOV2. Mutant combinations T500S,Y489S,T500S and Y489S,Y453S,T500Y mostly blocked ACE2. Conclusively, CUT4-mutant rejects whole glycosylated-nCoV2 pre-dock/post-dock/competitive-docking conditions.","version":"1.1","doi":"10.1101/2021.04.25.441361","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.25.441316","pub_date":"2021-4-26","title":"Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery","abstract":"RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses. Interactome analyses of host proteins and the SARS-CoV-2, EBOV, and ZIKV RNA genomes unveil viral biology and drug targets.","version":"1.1","doi":"10.1101/2021.04.25.441316","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441187","pub_date":"2021-4-26","title":"Predicted structural mimicry of spike receptor-binding motifs from highly pathogenic human coronaviruses","abstract":"Viruses often encode proteins that mimic host proteins in order to facilitate infection. Little work has been done to understand the potential mimicry of the SARS-CoV-2, SARS-CoV, and MERS-CoV spike proteins, particularly the receptor-binding motifs, which could be important in determining tropism of the virus. Here, we use structural bioinformatics software to characterize potential mimicry of the three coronavirus spike protein receptor-binding motifs. We utilize sequence-independent alignment tools to compare structurally known or predicted three-dimensional protein models with the receptor-binding motifs and verify potential mimicry with protein docking simulations. Both human and non-human proteins were found to be similar to all three receptor-binding motifs. Similarity to human proteins may reveal which pathways the spike protein is co-opting, while analogous non-human proteins may indicate shared host interaction partners and overlapping antibody cross-reactivity. These findings can help guide experimental efforts to further understand potential interactions between human and coronavirus proteins. Potential coronavirus spike protein mimicry revealed by structural comparison Human and non-human protein potential interactions with virus identified Predicted structural mimicry corroborated by protein-protein docking Epitope-based alignments may help guide vaccine efforts","version":"1.1","doi":"10.1101/2021.04.23.441187","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439863","pub_date":"2021-4-23","title":"Clinicopathologic features of a feline SARS-CoV-2 infection model parallel acute COVID-19 in humans","abstract":"The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. This study validates a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with severe COVID-19 in humans. Intra-tracheal inoculation of concentrated SARS-CoV-2 caused infected cats to develop clinical disease consistent with that observed in the early exudative phase of COVID-19. A novel clinical scoring system for feline respiratory disease was developed and utilized, documenting a significant degree of lethargy, fever, dyspnea, and dry cough in infected cats. In addition, histopathologic pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were observed due to SARS-CoV-2 infection, imitating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation exists between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were quantified in nasal turbinates, distal trachea, lung, and various other organs. Natural ACE2 expression, paired with clinicopathologic correlates between this feline model and human COVID-19, encourage use of this model for future translational studies. Identifying an ideal animal model to study COVID-19 has been difficult, and current models come with challenges that restrict their potential in translational studies. Few lab animals naturally express the receptors necessary for viral infection (ACE2), and many fail to manifest clinical signs or pathology similar to that seen in humans. Other models (non-human primates, mink) are ideal for disease and transmission studies, but are restricted by cost, husbandry challenges, and scarce availability. Alternatively, cats naturally express ACE2 receptors, are naturally infected with SARS-CoV-2 and can transmit virus from cat-to-cat. Prior to this study, cats infected by oral/nasal routes have not displayed significant clinical disease or lung pathology. However, we demonstrate that direct inoculation of concentrated SARS-CoV-2 virus in the trachea of cats induces analogous clinical and pathologic features to hospitalized patients with acute COVID-19. Our results show that infected cats exhibit significant clinical signs during experimental infection (coughing, increased respiratory effort, lethargy, and fever) and exhibit extensive lung lesions that mimic severe COVID-19 pathology such as diffuse alveolar damage and hyaline membrane formation \u2013 highlighting the immeasurable potential for this feline model to address translational approaches for COVID-19 and to better understand the role of cats in transmission and disease.","version":"1.2","doi":"10.1101/2021.04.14.439863","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.22.440848","pub_date":"2021-4-23","title":"Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection","abstract":"SARS-CoV-2 can cause a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of the host factors mediating viral infection or restriction is critical to elucidate SARS-CoV-2 host-pathogen interactions and the progression of COVID-19. To this end, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. These screens uncovered proviral and antiviral host factors across highly interconnected host pathways, including components implicated in clathrin transport, inflammatory signaling, cell cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high-molecular weight glycoproteins, as a prominent viral restriction network. We demonstrate that multiple membrane-anchored mucins are critical inhibitors of SARS-CoV-2 entry and are upregulated in response to viral infection. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and suggests interactions between SARS-CoV-2 and airway mucins of COVID-19 patients as a host defense mechanism.","version":"1.1","doi":"10.1101/2021.04.22.440848","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.441125","pub_date":"2021-4-23","title":"Computational investigation reveals that the mutant strains of SARS-CoV2 are highly infectious than wildtype","abstract":"Remarkable infectivity of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) is due to the rapid emergence of various strains, thus enable the virus to rule the world. Over the course of SARS-CoV2 pandemic, the scientific communities worldwide are responding to newly emerging genetic variants. However, the mechanism behind the persistent infection of these variants is still not known due to the paucity of study of these variants at molecular level. In this scenario, computational methods have immense utility in understanding the molecular and functional properties of different variants. Therefore, in this study various mutants (MTs) of SpikeS1 receptor binding domain (RBD) of highly infectious SARS-CoV2 strains were carried and elucidated the protein structure and dynamics using molecular dynamics (MD) approach. MD simulation study showed that all MTs exhibited stable structures with altered functional properties. Furthermore, the binding strength of different MTs along with WT (wildtype) was revealed through protein-protein docking and observed that MTs showed high binding affinities than WT. Hence, this study shed light on the molecular basis of infection caused by different variants of SARS-CoV2, which might play an important role in to cease the transmission and pathogenesis of virus and also implicate in rational designing of a specific drug.","version":"1.1","doi":"10.1101/2021.04.23.441125","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.23.21255523","pub_date":"2021-04-23","title":"COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  The COVID-19 pandemic has created an urgent need for rapid, effective, and low-cost SARS-CoV-2 diagnostic testing. Here, we describe COV-ID, an approach that combines RT-LAMP with deep sequencing to detect SARS-CoV-2 in unprocessed human saliva with high sensitivity (5\u201310 virions). Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human\n                  <jats:italic>STATHERIN</jats:italic>\n                  , and an artificial SARS spike-in. The approach was validated on clinical saliva samples, where it showed 100% agreement with RT-qPCR. COV-ID can also be performed directly on saliva adsorbed on filter paper, simplifying collection logistics and sample handling.\n                </jats:p>","version":null,"doi":"10.1101/2021.04.23.21255523","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.22.441045","pub_date":"2021-4-23","title":"Inactivation of SARS-CoV-2 by \u03b2-propiolactone Causes Aggregation of Viral Particles and Loss of Antigenic Potential","abstract":"Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. \u03b2-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly causing undesirable outcomes including a potential immune escape by infectious virions, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.04.22.441045","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.435056","pub_date":"2021-4-23","title":"Persistence of SARS-CoV-2 virus and viral RNA on hydrophobic and hydrophilic surfaces and investigating contamination concentration","abstract":"The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used RT-PCR analysis to detect SARS-CoV-2 RNA on surfaces but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered, and whether virus viability is concentration dependant. Viable virus persisted for the longest time on surgical mask material and stainless steel with a 99.9% reduction in viability by 124 and 113 hours respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 hours. Viability on cotton was reduced second fastest, with 99.9% reduction in 72 hours. RNA on all the surfaces exhibited a one log reduction in genome copy recovery over 21 days. The findings show that SARS-CoV-2 is most stable on non-porous hydrophobic surfaces. RNA is highly stable when dried on surfaces with only one log reduction in recovery over three weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within two days. Therefore, when RNA is detected on surfaces it does not directly indicate presence of viable virus even at high CT values. This study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface. Unless sampled immediately after contamination it is difficult to align RNA copy numbers to quantity of viable virus on a surface.","version":"1.2","doi":"10.1101/2021.03.11.435056","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440647","pub_date":"2021-4-23","title":"Immune Correlates of Protection by mRNA-1273 Immunization against SARS-CoV-2 Infection in Nonhuman Primates","abstract":"Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 \u2013 100 \u03bcg of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to na\u00efve hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP. mRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.","version":"1.2","doi":"10.1101/2021.04.20.440647","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440658","pub_date":"2021-4-23","title":"Gut microbiota diversity and C-Reactive Protein are predictors of disease severity in COVID-19 patients","abstract":"Risk factors for COVID-19 disease severity are still poorly understood. Considering the pivotal role of gut microbiota on host immune and inflammatory functions, we investigated the association between changes in gut microbiota composition and the clinical severity of COVID-19. We conducted a multicentre cross-sectional study prospectively enrolling 115 COVID-19 patients categorized according to: 1) WHO Clinical Progression Scale - mild 19 (16.5%), moderate 37 (32.2%) or severe 59 (51.3%); and 2) location of recovery from COVID-19 - ambulatory 14 (household isolation; 12.2%), hospitalized in ward 40 (34.8%) or intensive care unit 61 (53.0%). Gut microbiota analysis was performed through 16S rRNA gene sequencing and data obtained was further related with clinical parameters of COVID-19 patients. Risk factors for COVID-19 severity were identified by univariate and multivariable logistic regression models. In comparison with mild COVID-19 patients, the gut microbiota of moderate and severe patients has: a) lower Firmicutes/Bacteroidetes ratio, b) higher abundance of Proteobacteria; and c) lower abundance of beneficial butyrate-producing bacteria such as Roseburia and Lachnospira genera. Multivariable regression analysis showed that Shannon index diversity (odds ratio [OR] 2.85 [95% CI 1.09-7.41]; p=0.032) and C-Reactive Protein (OR 3.45 [95% CI 1.33-8.91]; p=0.011) were risk factors for COVID-19 severe disease (a score of 6 or higher in WHO clinical progression scale). In conclusion, our results demonstrated that hospitalised moderate and severe COVID-19 patients have microbial signatures of gut dysbiosis and for the first time, the gut microbiota diversity is pointed out as a prognostic biomarker for COVID-19 disease severity.","version":"1.1","doi":"10.1101/2021.04.20.440658","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.04.438417","pub_date":"2021-4-22","title":"Substitutions and codon usage in SARS-CoV-2 in mammals indicate natural selection and host adaptation","abstract":"The outbreak of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, rapidly spread to create a global pandemic and has continued to spread across hosts from humans to animals, transmitting particularly effectively in mink. How SARS-CoV-2 evolves in animals and humans and the differences in the separate evolutionary processes remain unclear. We analyzed the composition and codon usage bias of SARS-CoV-2 in infected humans and animals. Compared with other animals, SARS-CoV-2 in mink had the most substitutions. The substitutions of cytidine in SARS-CoV-2 in mink account for nearly 50% of the substitutions, while those in other animals represent only 30% of the substitutions. The incidence of adenine transversion in SARS-CoV-2 in other animals is threefold higher than that in mink-CoV (the SARS-CoV-2 virus in mink). A synonymous codon usage analysis showed that SARS-CoV-2 is optimized to adapt in the animals in which it is currently reported, and all the animals showed decreased adaptability relative to that of humans, except for mink. A binding affinity analysis indicated that the spike protein of the SARS-CoV-2 variant in mink showed a greater preference for binding with the mink receptor ACE2 than with the human receptor, especially as the mutation Y453F and F486L in mink-CoV lead to improvement of binding affinity for mink receptor. Our study focuses on the divergence of SARS-CoV-2 genome composition and codon usage in humans and animals, indicating possible natural selection and current host adaptation.","version":"1.2","doi":"10.1101/2021.04.04.438417","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.14.431043","pub_date":"2021-4-22","title":"Detection and characterization of the SARS-CoV-2 lineage B.1.526 in New York","abstract":"Wide-scale SARS-CoV-2 genome sequencing is critical to tracking viral evolution during the ongoing pandemic. Variants first detected in the United Kingdom, South Africa, and Brazil have spread to multiple countries. We developed the software tool, Variant Database (VDB), for quickly examining the changing landscape of spike mutations. Using VDB, we detected an emerging lineage of SARS-CoV-2 in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage (now designated as B.1.526) are L5F, T95I, D253G, E484K or S477N, D614G, and A701V. This lineage was first sequenced in late November 2020 when it represented <1% of sequenced coronavirus genomes that were collected in New York City (NYC). By February 2021, genomes from this lineage accounted for ~32% of 3288 sequenced genomes from NYC specimens. Phylodynamic inference confirmed the rapid growth of the B.1.526 lineage in NYC, notably the sub-clade defined by the spike mutation E484K, which has outpaced the growth of other variants in NYC. Pseudovirus neutralization experiments demonstrated that B.1.526 spike mutations adversely affect the neutralization titer of convalescent and vaccinee plasma, indicating the public health importance of this lineage.","version":"1.3","doi":"10.1101/2021.02.14.431043","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440862","pub_date":"2021-4-22","title":"Rapid induction of antigen-specific CD4+ T cells guides coordinated humoral and cellular immune responses to SARS-CoV-2 mRNA vaccination","abstract":"The SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses in healthy individuals following mRNA vaccination. Vaccination induced rapid near-maximal antigen-specific CD4+ T cell responses in all subjects after the first vaccine dose. CD8+ T cell responses developed gradually after the first and second dose and were variable. Vaccine-induced T cells had central memory characteristics and included both Tfh and Th1 subsets, similar to natural infection. Th1 and Tfh responses following the first dose predicted post-boost CD8+ T cell and neutralizing antibody levels, respectively. Integrated analysis of 26 antigen-specific T cell and humoral responses revealed coordinated features of the immune response to vaccination. Lastly, whereas booster vaccination improved CD4+ and CD8+ T cell responses in SARS-CoV-2 na\u00efve subjects, the second vaccine dose had little effect on T cell responses in SARS-CoV-2 recovered individuals. Thus, longitudinal analysis revealed robust T cell responses to mRNA vaccination and highlighted early induction of antigen-specific CD4+ T cells.","version":"1.1","doi":"10.1101/2021.04.21.440862","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440697","pub_date":"2021-4-22","title":"Endomembrane targeting of human OAS1 p46 augments antiviral activity","abstract":"Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.","version":"1.1","doi":"10.1101/2021.04.21.440697","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440680","pub_date":"2021-4-21","title":"Human Taste Cells Express ACE2: a Portal for SARS-CoV-2 Infection","abstract":"Loss and changes in taste and smell are well-reported symptoms of SARS-CoV-2 infection. The virus targets cells for entry by high affinity binding of its spike protein to cell-surface angiotensin-converting enzyme-2 (ACE2). It was not known whether ACE2 is expressed on taste receptor cells (TRCs) nor if TRCs are infected directly. Using an in-situ hybridization (ISH) probe and an antibody specific to ACE2, it seems evident that ACE2 is present on a subpopulation of specialized TRCs, namely, PLC\u03b22 positive, Type II cells in taste buds in taste papillae. Fungiform papillae (FP) of a SARS-CoV-2+ patient exhibiting symptoms of COVID-19, including taste changes, were biopsied. Based on ISH, replicating SARS-CoV-2 was present in Type II cells of this patient. Therefore, taste Type II cells provide a portal for viral entry that predicts vulnerabilities to SARS-CoV-2 in the oral cavity. The continuity and cell turnover of the FP taste stem cell layer of the patient were disrupted during infection and had not fully recovered 6 weeks post symptom onset. Another patient suffering post-COVID-19 taste disturbances also had disrupted stem cells. These results indicate that a COVID-19 patient who experienced taste changes had replicating virus in their taste buds and that SARS-CoV-2 infection results in deficient stem cell turnover needed for differentiation into TRCs.","version":"1.1","doi":"10.1101/2021.04.21.440680","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440783","pub_date":"2021-4-21","title":"Deep time course proteomics of SARS-CoV- and SARS-CoV-2-infected human lung epithelial cells (Calu-3) reveals strong induction of interferon-stimulated gene (ISG) expression by SARS-CoV-2 in contrast to SARS-CoV","abstract":"SARS-CoV and SARS-CoV-2 infections are characterized by remarkable differences, including contagiosity and case fatality rate. The underlying mechanisms are not well understood, illustrating major knowledge gaps of coronavirus biology. In this study, protein expression of SARS-CoV- and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was analysed using data-independent acquisition mass spectrometry (DIA-MS). This resulted in the so far most comprehensive map of infection-related proteome-wide expression changes in human cells covering the quantification of 7478 proteins across 4 time points. Most notably, the activation of interferon type-I response was observed, which surprisingly is absent in other recent proteome studies, but is known to occur in SARS-CoV-2-infected patients. The data reveal that SARS-CoV-2 triggers interferon-stimulated gene (ISG) expression much stronger than SARS-CoV, which reflects the already described differences in interferon sensitivity. Potentially, this may be caused by the enhanced expression of viral M protein of SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I interferon expression. This study expands the knowledge on the host response to SARS-CoV-2 infections on a global scale using an infection model, which seems to be well suited to analyse innate immunity.","version":"1.1","doi":"10.1101/2021.04.21.440783","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440716","pub_date":"2021-4-21","title":"Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease","abstract":"The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. It is required for infection, it cleaves the viral polyprotein at multiple sites, and it is conserved among coronaviruses and distinct from human proteases. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes substrates and how the peptide sequence can dictate catalytic efficiency by influencing the position of the scissile bond. One peptide, constituting the junction between viral non-structural proteins 8 and 9 (nsp8/9), has P1\u2019 and P2\u2019 residues that are unique among SARS-CoV-2 cleavage sites but conserved among nsp8/9 junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1\u2019 or P2\u2019 can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.","version":"1.1","doi":"10.1101/2021.04.20.440716","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440654","pub_date":"2021-4-21","title":"Rapid and Efficient Inactivation of SARS-CoV-2 from Surfaces using UVC Light Emitting Diode Device","abstract":"Efforts are underway to develop countermeasures to prevent the environmental spread of COVID-19 pandemic caused by SARS-CoV-2. Physical decontamination methods like Ultraviolet radiation has shown to be promising. Here, we describe a novel device emitting ultraviolet C radiation (UVC), called NuvaWave, to rapidly and efficiently inactivate SARS-CoV-2. SARS-CoV-2 was dried on a chambered glass slides and introduced in a NuvaWave robotic testing unit. The robot simulated waving NuvaWave over the virus at a pre-determined UVC radiation dose of 1, 2, 4 and 8 seconds. Post-UVC exposure, virus was recovered and titered by plaque assay in Vero E6 cells. We observed that relative control (no UVC exposure), exposure of the virus to UVC for one or two seconds resulted in a >2.9 and 3.8 log10 reduction in viral titers, respectively. Exposure of the virus to UVC for four or eight seconds resulted in a reduction of greater than 4.7-log10 reduction in viral titers. The NuvaWave device inactivates SARS-CoV-2 on surfaces to below the limit of detection within one to four seconds of UVC irradiation. This device can be deployed to rapidly disinfect surfaces from SARS-CoV-2, and to assist in mitigating its spread in a variety of settings.","version":"1.1","doi":"10.1101/2021.04.20.440654","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440722","pub_date":"2021-4-21","title":"Mild and severe SARS-CoV-2 infection induces respiratory and intestinal microbiome changes in the K18-hACE2 transgenic mouse model","abstract":"Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and declining economies around the world. K18-hACE2 mice develop disease resembling severe SARS-CoV-2 infection in a virus dose-dependent manner. The relationship between SARS-CoV-2 and the intestinal or respiratory microbiome is not fully understood. In this context, we characterized the cecal and lung microbiome of SARS-CoV-2 challenged K18-hACE2 transgenic mice in the presence or absence of treatment with the Mpro inhibitor GC376. Cecum microbiome showed decreased Shannon and Inv Simpson diversity index correlating with SARS-CoV-2 infection dosage and a difference of Bray-Curtis dissimilarity distances among control and infected mice. Bacterial phyla such as Firmicutes, particularly Lachnospiraceae and Oscillospiraceae, were significantly less abundant while Verrucomicrobiota, particularly the family Akkermansiaceae, were increasingly more prevalent during peak infection in mice challenged with a high virus dose. In contrast to the cecal microbiome, the lung microbiome showed similar microbial diversity among the control, low and high challenge virus groups, independent of antiviral treatment. Bacterial phyla in the lungs such as Bacteroidota decreased while Firmicutes and Proteobacteria were significantly enriched in mice challenged with a high dose of SARS-CoV-2. In summary, we identified changes in the cecal and lung microbiome of K18-hACE2 mice with severe clinical signs of SARS-CoV-2 infection. The COVID-19 pandemic has resulted in millions of deaths. The host\u2019s respiratory and intestinal microbiome can affect directly or indirectly the immune system during viral infections. We characterized the cecal and lung microbiome in a relevant mouse model challenged with a low and high dose of SARS-CoV-2 in the presence or absence of an antiviral Mpro inhibitor, GC376. Decreased microbial diversity and taxonomic abundances of the phyla Firmicutes, particularly Lachnospiraceae, correlating with infection dosage was observed in the cecum. In addition, microbes within the family Akkermansiaceae were increasingly more prevalent during peak infection, which is observed in other viral infections. The lung microbiome showed similar microbial diversity to the control, independent of antiviral treatment. Decreased Bacteroidota and increased Firmicutes and Proteobacteria were observed in the lungs in a virus dose-dependent manner. These studies add to a better understanding of the complexities associated with the intestinal microbiome during respiratory infections.","version":"1.1","doi":"10.1101/2021.04.20.440722","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440676","pub_date":"2021-4-21","title":"Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19","abstract":"Coronavirus disease 2019 (COVID-19) is especially severe in aged populations. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC). Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions. (Words: 254)","version":"1.1","doi":"10.1101/2021.04.20.440676","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.21.440801","pub_date":"2021-4-21","title":"Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 isolates from Pennsylvania","abstract":"Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote \u201cescape\u201d from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.","version":"1.1","doi":"10.1101/2021.04.21.440801","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440588","pub_date":"2021-4-21","title":"MARCH8 targets cytoplasmic lysine residues of various viral envelope glycoproteins","abstract":"The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoproteins but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we investigate the antiviral spectrum of MARCH8 using HIV-1 pseudotyped with a variety of viral envelope glycoproteins. Pseudotyping experiments revealed that viral envelopes derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelopes by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor.","version":"1.1","doi":"10.1101/2021.04.20.440588","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.439992","pub_date":"2021-4-20","title":"Drug repurposing to face Covid-19: Celastrol, a potential leading drug capable of inhibiting SARS-CoV-2 replication and induced inflammation","abstract":"The global emergence of Covid-19 has caused huge human casualties. Clinical manifestations of the disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and non-homeostatic inflammatory response. In face of the urgent demand for effective drugs to treat Covid-19, we have searched for candidate compounds using a drug repurposing approach based on in silico analysis followed by biological validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F \u2013 a plant used in traditional Chinese medicine \u2013 as one of the best compounds out of 39 repurposed drug candidates. Celastrol reverted gene expression signature from SARS-CoV-2-infected cells; bound with high-affinity energy to viral molecular targets such as main protease (Mpro) and receptor-biding domain (RBD); inhibited SARS-CoV-2 replication in monkey (Vero and Vero-ACE2) and human (Caco-2 and Calu-3) cell lines; and decreased interleukin-6 (IL-6) secretion in SARS-CoV-2-infected human cell lines. Interestingly, celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity. Therefore, celastrol is a promising lead drug candidate to treat Covid-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells, two critical events in the pathophysiology of this disease.","version":"1.1","doi":"10.1101/2021.04.20.439992","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.19.440481","pub_date":"2021-4-20","title":"Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants","abstract":"Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.04.19.440481","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440678","pub_date":"2021-4-20","title":"Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein","abstract":"The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.","version":"1.1","doi":"10.1101/2021.04.20.440678","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.19.440446","pub_date":"2021-4-20","title":"Inactivation of SARS-CoV-2 in chlorinated swimming pool water","abstract":"SARS-CoV-2 transmission remains a global problem which exerts a significant direct cost to public health. Additionally, other aspects of physical and mental health can be affected by limited access to social and exercise venues as a result of lockdowns in the community or personal reluctance due to safety concerns. Swimming pools have reopened in the UK as of April 12th, but the effect of swimming pool water on inactivation of SARS-CoV-2 has not yet been directly demonstrated. Here we demonstrate that water which adheres to UK swimming pool guidelines is sufficient to reduce SARS-CoV-2 infectious titre by at least 3 orders of magnitude.","version":"1.1","doi":"10.1101/2021.04.19.440446","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.28.428642","pub_date":"2021-4-20","title":"Coronavirus-associated molecular mimicry through homology to a SARS-CoV-2 peptide could be leading to susceptibility in patients with HLA-A*02:01 and HLA-A*24:02 serotypes","abstract":"This study aims to predict autoimmunity-related pathological mechanisms that possess risk for individuals with specific human leukocyte antigen (HLA) serotypes and shared by certain coronaviruses including SARS-CoV-2, based on homology to a SARS-CoV-2 peptide. With the given aim, 1-) coronavirus-associated sequences, which are homologous to the 15mer SARS-CoV-2 peptide CFLGYFCTCYFGLFC, are obtained. 2-) Human peptides that have at least 7 residue matches with those coronavirus sequences, and the SARS-CoV-2 15mer, are found. 3-) Epitope pairs, which are sourced by those aligned coronavirus and human sequences are identified. 4-) Epitope pairs that are predicted to bind strongly not only to the same HLA allele with each other but also to the same HLA allele as those of the respective alignment of the SARS-CoV-2 peptide are selected. Following are the identified proteins or peptides (with HLA-A*02:01 or HLA-A*24:02 epitopes), as described in 1-to-4: Immunoglobulin heavy chain junction regions, CRB1 isoform I precursor, slit homolog 2 protein, hCG1995581, hCG2028737, phospholipid phosphatase-related protein type 2. Among those, CRB1 isoform I precursor sequence with the predicted HLA-A*24:02 epitope aligns with the highest number of different sequences. Results imply autoimmunity risk in COVID-19 patients with HLA-A*02:01 and HLA-A*24:02 serotypes, through molecular mimicry, as a shared pathogenicity risk that can be prevalent upon getting infected with certain coronaviruses. These can pave way to improved risk groups\u2019 assessment and autoimmunity treatment options, for COVID-19 and its associated diseases. Also, the approach in this study can be used to predict prospective pathologies of the transmissible variants in susceptible humans.","version":"1.3","doi":"10.1101/2021.01.28.428642","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440593","pub_date":"2021-4-20","title":"Antibody Conversion rates to SARS-CoV-2 in Saliva from Children Attending Summer Schools in Barcelona, Spain","abstract":"Surveillance tools to estimate infection rates in young populations are essential to guide recommendations for school reopening and management during viral epidemics. Ideally, field-deployable non-invasive, sensitive techniques are required to detect low viral load exposures among asymptomatic children. We determined SARS-CoV-2 antibody conversion by high-throughput Luminex assays in saliva samples collected weekly in 1,509 children and 396 adults in 22 Summer schools and 2 pre-schools in 27 venues in Barcelona, Spain, from June 29th to July 31st 2020, between the first and second COVID-19 pandemic waves. Saliva antibody conversion defined as \u22654-fold increase in IgM, IgA and/or IgG levels to SARS-CoV-2 antigens between two visits over a 5-week period was 3.22% (49/1518), or 2.36% if accounting for potentially cross-reactive antibodies, six times higher than the cumulative infection rate (0.53%) by weekly saliva RT-PCR screening. IgG conversion was higher in adults (2.94%, 11/374) than children (1.31%, 15/1144) (p=0.035), IgG and IgA levels moderately increased with age, and antibodies were higher in females. Most antibody converters increased both IgG and IgA antibodies but some augmented either IgG or IgA, with a faster decay over time for IgA than IgG. Nucleocapsid rather than spike was the main antigen target. Anti-spike antibodies were significantly higher in individuals not reporting symptoms than symptomatic individuals, suggesting a protective role against COVID-19. To conclude, saliva antibody profiling including three isotypes and multiplexing antigens is a useful and more user-friendly tool for screening pediatric populations to determine SARS-CoV-2 exposure and guide public health policies during pandemics.","version":"1.1","doi":"10.1101/2021.04.20.440593","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.16.440141","pub_date":"2021-4-20","title":"Potential transmission chains of variant B.1.1.7 and co-mutations of SARS-CoV-2","abstract":"The presence of SARS-CoV-2 mutants, including the emerging variant B.1.1.7, has raised great concerns in terms of pathogenesis, transmission, and immune escape. Characterizing SARS-CoV-2 mutations, evolution, and effects on infectivity and pathogenicity is crucial to the design of antibody therapies and surveillance strategies. Here we analyzed 454,443 SARS-CoV-2 spike genes/proteins and 14,427 whole-genome sequences. We demonstrated that the early variant B.1.1.7 may not have evolved spontaneously in the United Kingdom or within human populations. Our extensive analyses suggested that Canidae, Mustelidae or Felidae, especially the Canidae family (for example, dog) could be a possible host of the direct progenitor of variant B.1.1.7. An alternative hypothesis is that the variant was simply yet to be sampled. Notably, the SARS-CoV-2 whole genome represents a large number of potential co-mutations with very strong statistical significances (p value<E\u2013 44). In addition, we used an experimental SARS-CoV-2 reporter replicon system to introduce the dominant co-mutations NSP12_c14408t, 5\u2019UTR_c241t, and NSP3_c3037t into the viral genome, and to monitor the effect of the mutations on viral replication. Our experimental results demonstrated that the co-mutations significantly attenuated the viral replication. The study provides valuable clues for discovering the transmission chains of variant B.1.1.7 and understanding the evolutionary process of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.04.16.440141","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.20.440651","pub_date":"2021-4-20","title":"Comparison of Mucosal and Intramuscular Immunization against SARS-CoV-2 with Replication-Defective and Replicating Single-cycle Adenovirus Vaccines","abstract":"SARS-CoV-2 enters the body at mucosal surfaces, such as the nose and lungs. These events involve a small number of virions at these mucosal barriers and are therefore a strategic point to stop a COVID-19 infection before it starts. Despite this, most vaccines against COVID-19 are being injected into the muscle where they will not generate the highest levels of mucosal protection. The vaccines that are approved for use in humans are all replication-defective (RD) mRNA, DNA, or adenovirus (Ad) vaccines that do not amplify antigen transgenes. We developed single cycle adenovirus (SC-Ad) vectors that replicate antigen genes up to 10,000-fold in human cells, but that are disabled from producing infectious Ad particles. We show here that SC-Ad expressing the full-length SARS-CoV-2 spike protein produces 100-fold more spike protein than a matched RD-Ad-Spike vector. When Ad-permissive hamsters were immunized with these vaccines by intranasal (IN) or intramuscular (IM) routes, SC-Ad produced significantly stronger antibody responses as compared to RD-Ad against the spike protein that rose over 14 weeks after one immunization. Single IN or IM immunizations generated significant antibody responses in serum and in bronchoalveolar lavages (BALs). IN priming, but not IM priming, generated HLA-restricted CD8 T cell responses in BALs. SC-Ad-Spike generated antibodies that retain binding to spike receptor binding domains (RBDs) with mutations from new viral variants. These data suggest empowering the genomes of gene-based vaccines with the ability to amplify antigen genes can increase potency. This may be particularly advantageous when applying mucosal vaccines to combat mucosal pathogens like SARS-CoV-2. Arming adenovirus vaccines with the ability to replicate vaccine antigen genes may increase potency for systemic, or more importantly, mucosal immunization against mucosal pathogens.","version":"1.1","doi":"10.1101/2021.04.20.440651","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.21255452","pub_date":"2021-04-20","title":"A Real World Evaluation of the safety and immunogenicity of the Covishield vaccine, ChAdOx1 nCoV- 19 Corona Virus Vaccine (Recombinant) in Health Care Workers (HCW) in National Capital Region (NCR) of India: A preliminary report","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The SARS-CoV-2 pandemic has severely impacted health systems, economic and social progress globally in 2020. The rollout of vaccines in several parts of the world is being hailed as a solution to the crisis. With newer and more virulent serotypes on the horizon and limited vaccine available, evaluation of safety and immunogenicity is critical for rationalization of vaccine use in public health.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To evaluate real world safety, and, immunogenicity of the Covishield vaccine, ChAdOx1 nCoV-19 Corona Virus Vaccine (Recombinant) in Health Care Workers (HCW) during the national vaccine roll out in the NCR, New Delhi. The safety is evaluated through Adverse Events and Serious Adverse Events reported though enhanced pharmacovigilance protocols, and, the immunogenicity by quantitative determination of anti-S1 and anti-S2 specific IgG antibodies to SARS-CoV-2 in serum samples collected before the receipt of the vaccine and 14 days after dose 1, using the fully automated LIAISON\u00ae SARS-CoV-2 S1/S2 IgG test using the chemiluminescence immunoassay (CLIA)</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>In the two weeks after immunization with the Covishield vaccine {ChAdOx1 nCoV-19 Corona Virus Vaccine (Recombinant)}, none of the 1638 evaluated participants reported any serious adverse events (ie require hospitalization or emergency room visit). Solicited adverse events reported via daily diary cards included pain (62.7%) and soreness (24.1%) at injection site as most common, whereas fever (48.4%), headache (43.4%), myalgia (38.4%), fatigue (33.4%), joint pain (27.0%) and nausea (16.0%) were most common solicited systemic adverse events on day 1. Majority of local and systemic adverse events were seen in first 2 days post vaccination and thereafter they resolved. Lesser reactogenicity was observed in subjects with age &gt;50 years. No major difference was observed in adverse events when subjects were stratified based on history of COVID 19 disease or baseline seropositivity. In our study serostatus improved from 48.2% positive at baseline to 79.0% positive 2 weeks following first dose of vaccination. After first dose of vaccination overall higher percentage (98.2%) of seropositivity rates were observed in those with past history of COVID 19 disease</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>The Covishield vaccine {ChAdOx1 nCoV-19 Corona Virus Vaccine (Recombinant)}, was safe and reported mild self limiting adverse events over 2-4 days and had an good early (within 2 weeks) seroresponse. This holds the promise of far reaching impact on vaccine availability for a larger population and thereby providing a widespread coverage.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.04.14.21255452","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.03.131987","pub_date":"2021-4-20","title":"Analyzing hCov Genome Sequences: Predicting Virulence and Mutation","abstract":"Covid-19 pandemic, caused by the SARS-CoV-2 genome sequence of coronavirus, has affected millions of people all over the world and taken thousands of lives. It is of utmost importance that the character of this deadly virus be studied and its nature be analyzed. We present here an analysis pipeline comprising a classification exercise to identify the virulence of the genome sequences and extraction of important features from its genetic material that are used subsequently to predict mutation at those interesting sites using deep learning techniques. We have classified the SARS-CoV-2 genome sequences with high accuracy and predicted the mutations in the sites of Interest. In a nutshell, we have prepared an analysis pipeline for hCov genome sequences leveraging the power of machine intelligence and uncovered what remained apparently shrouded by raw data.","version":"1.2","doi":"10.1101/2020.06.03.131987","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.293258","pub_date":"2021-4-20","title":"Functional binding dynamics relevant to the evolution of zoonotic spillovers in endemic and emergent Betacoronavirus strains","abstract":"Comparative functional analysis of the dynamic interactions between various Betacoronavirus mutant strains and broadly utilized target proteins such as ACE2 and CD26, is crucial for a more complete understanding of zoonotic spillovers of viruses that cause diseases such as COVID-19. Here, we employ machine learning to replicated sets of nanosecond scale GPU accelerated molecular dynamics simulations to statistically compare and classify atom motions of these target proteins in both the presence and absence of different endemic and emergent strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. Machine learning was used to identify functional binding dynamics that are evolutionarily conserved from bat CoV-HKU4 to human endemic/emergent strains. Conserved dynamics regions of ACE2 involve both the N-terminal helices, as well as a region of more transient dynamics encompassing K353, Q325 and a novel motif AAQPFLL 386-92 that appears to coordinate their dynamic interactions with the viral RBD at N501. We also demonstrate that the functional evolution of Betacoronavirus zoonotic spillovers involving ACE2 interaction dynamics are likely pre-adapted from two precise and stable binding sites involving the viral bat progenitor strain\u2019s interaction with CD26 at SAMLI 291-5 and SS 333-334. Our analyses further indicate that the human endemic strains hCoV-HKU1 and hCoV-OC43 have evolved more stable N-terminal helix interactions through enhancement of an interfacing loop region on the viral RBD, whereas the highly transmissible SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1) have evolved more stable viral binding via more focused interactions between the viral N501 and ACE2 K353 alone.","version":"1.4","doi":"10.1101/2020.09.11.293258","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.21255512","pub_date":"2021-04-20","title":"Assessing the Mortality Impact of the COVID-19 Pandemic in Florida State Prisons","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The increased risk of COVID-19 infection among incarcerated individuals due to environmental hazards is well known and recent studies have highlighted the higher rates of infection and mortality prisoners in the United States face due to COVID-19. However, the impact of COVID-19 on all-cause mortality rates in incarcerated populations has not been studied.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Using data reported by the Florida Department of Corrections on prison populations and mortality events we conducted a retrospective cohort study of all individuals incarcerated in Florida state prisons between 2015 and 2020. We calculated excess deaths by estimating age-specific expected deaths from mortality trends in 2015 through 2019 and taking the difference between observed and expected deaths during the pandemic period. We calculated life table measures using standard demographic techniques and assessed significant yearly changes using bootstrapping.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>The Florida Department of Corrections reported 510 total deaths from March 1, 2020 to December 31, 2020 among the state prison population. This was 42% higher (rate ratio 1.42, 95% CI 1.15 to 1.89) than the expected number of deaths in light of mortality rates for previous years. Reported COVID-19 deaths in a month were positively correlated with estimated excess deaths (80.4%, p &lt;.01). Using age-specific mortality estimates, we found that life expectancy at age 20 declined by 4 years (95% CI 2.06-6.57) between 2019 and 2020 for the Florida prison population.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>The Florida prison population saw a significant increase in all-cause mortality during the COVID-19 pandemic period, leading to a decrease in life expectancy of more than four years. Life years lost by the Florida prison population were likely far greater than those lost by the general United States population, as reported by other studies. This difference in years lost highlights the need for increased interventions to protect vulnerable incarcerated populations during pandemics.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Vital Projects Fund, Arnold Ventures, US Centers for Disease Control, Eunice Kennedy Shriver National Institute of Child Health and Human Development</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.04.14.21255512","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.19.440531","pub_date":"2021-4-19","title":"A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.","version":"1.1","doi":"10.1101/2021.04.19.440531","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.18.440296","pub_date":"2021-4-19","title":"Molecular relationships between SARS-CoV-2 Spike protein and LIFR, a pneumonia protective IL-6 family cytokine receptor","abstract":"The fine-tuned control of immune responses is attained by pairs of activating and inhibitory signaling receptors which modulate the quality and magnitude of immune responses. Some viruses exploit these pathways to enter host cells as well as interfere with immune responses. Here, we report that the SARS-CoV-1/2 Spike proteins (S) contain a potential inhibitory tyrosine- based immunoreceptor motif (ITIM) with the D614G variant occurring within this motif. Through an in silico screen of ITIM-containing human proteins, we find that the S-located ITIM is closely related to a previously reported ITIM in the cytoplasmic tail of the human Leukemia Inhibitory Factor Receptor (LIFR), a pneumonia protective IL-6 family cytokine receptor. To infer potential functional interactions between SARS-CoV-2 infection and LIFR expression, we performed single-cell transcriptome analysis of public datasets of lung tissues from healthy individuals and COVID-19 patients. We show that transcripts of LIFR and its ligand LIF are highly expressed in SARS-CoV-2 susceptible lung cells from mild and severe COVID-19 patients but not in healthy individuals. In addition, the human endogenous retroviral envelope gene (ERVW-1) encoding a fusogenic protein of the same functional class as the S protein, is induced in SARS-CoV-2 susceptible cell subpopulations in COVID-19 patients with no detectable expression in healthy individuals. We also report that pulmonary epithelial cells express transcripts of several immunoreceptors including the ITIM-containing antibody receptor FCGR2B which is detectable in healthy and severe COVID-19 cases but not in mild cases. These results suggest that molecular dysregulation of ITIM-mediated inhibitory signaling by the SARS-CoV-2 S protein may play a role in COVID-19 immunopathology.","version":"1.1","doi":"10.1101/2021.04.18.440296","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.18.440302","pub_date":"2021-4-19","title":"SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, causing health and economic problems. Currently, as dangerous mutations emerge there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus membrane binds to the angiotensin converting enzyme 2 (ACE2) receptor on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in vitro and in vivo SARS-CoV-2 infection, better than ACE2-Ig. Mechanistically we show that anti-spike antibodies generation, ACE2 enzymatic activity and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus concentration infection. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients. SARS-CoV-2 infection caused serious socio-economic and health problems around the globe. As dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. SARS-CoV-2 infection starts via binding of SARS-CoV-2 spike protein receptor binding domain (RBD) to its receptor, ACE2, on host cells. To intercept this binding, we generated Ig-fusion proteins. ACE2-Ig was generated to possibly block RBD by binding to it and RBD-Ig to block ACE2. We indeed showed that the fusion proteins bind to their respective target. We found that it is more efficient to inhibit SARS-CoV-2 infection by blocking ACE2 receptor with RBD-Ig. We also showed that RBD-Ig does not interfere with ACE2 activity or surface expression. Importantly, as our treatment does not target the virus directly, it may be efficient against any emerging variant. We propose here that RBD-Ig physically blocks virus infection by binding to ACE2 and thus it may be used for the treatment of SARS-CoV-2-infected patients.","version":"1.1","doi":"10.1101/2021.04.18.440302","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.19.440452","pub_date":"2021-4-19","title":"Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export","abstract":"A key feature of the mammalian innate immune response to viral infection is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response and limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. In addition, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both activation of the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. These findings argue that inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response.","version":"1.1","doi":"10.1101/2021.04.19.440452","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439793","pub_date":"2021-4-19","title":"Neuropilin-1 Mediates SARS-CoV-2 Infection in Bone Marrow-derived Macrophages","abstract":"SARS-CoV-2 infection in human can cause medical complications across various tissues and organs. Despite of the advances to understanding the pathogenesis of SARS-CoV-2, its tissue tropism and interactions with host cells have not been fully understood. Existing clinical data have suggested possible SARS-CoV-2 infection in human skeleton system. In the present study, we found that authentic SARS-CoV-2 could efficiently infect human and mouse bone marrow-derived macrophages (BMMs) and alter the expression of macrophage chemotaxis and osteoclast-related genes. Importantly, in a mouse SARS-CoV-2 infection model that was enabled by the intranasal adenoviral (AdV) delivery of human angiotensin converting enzyme 2 (hACE2), SARS-CoV-2 was found to be present in femoral BMMs as determined by in situ immunofluorescence analysis. Using single-cell RNA sequencing (scRNA-Seq), we characterized SARS-CoV-2 infection in BMMs. Importantly, SARS-CoV-2 entry on BMMs appeared to be dependent on the expression of neuropilin-1 (NRP1) rather than the widely recognized receptor ACE2. It was also noted that unlike brain macrophages which displayed aging-dependent NRP1 expression, BMMs from neonatal and aged mice had constant NRP1 expression, making BMMs constantly vulnerable target cells for SARS-CoV-2. Furthermore, it was found that the abolished SARS-CoV-2 entry in BMM-derived osteoclasts was associated with the loss of NRP1 expression during BMM-to-osteoclast differentiation. Collectively, our study has suggested that NRP1 can mediate SARS-CoV-2 infection in BMMs, which precautions the potential impact of SARS-CoV-2 infection on human skeleton system.","version":"1.2","doi":"10.1101/2021.04.14.439793","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.18.440358","pub_date":"2021-4-19","title":"Network controllability enrichment analysis reveals that SARS-CoV-2 infection tends to target indispensable nodes of a directed human protein-protein interaction network","abstract":"The COVID-19 disease has been a global threat caused by the new coronavirus species, SARS-CoV-2, since early 2020 with an urgent need for therapeutic interventions. In order to provide insight into human proteins targeted by SARS-CoV-2, here we study a directed human protein-protein interaction network (dhPPIN) based on our previous work on network controllability of virus targets. We previously showed that human proteins targeted by viruses tend to be those whose removal in a dhPPIN requires more control of the network dynamics, which were classified as indispensable nodes. In this study we introduce a more comprehensive rank-based enrichment analysis of our previous dhPPIN for SARS-CoV-2 infection and show that SARS-CoV-2 also tends to target indispensable nodes in the dhPPIN using multiple proteomics datasets, supporting validity and generality of controllability analysis of viral infection in humans. Also, we find differential controllability among SARS-CoV-2, SARS-CoV-1, and MERS-CoV from a comparative proteomics study. Moreover, we show functional significance of indispensable nodes by analyzing heterogeneous datasets from a genome-wide CRISPR screening study, a time-course phosphoproteomics study, and a genome-wide association study. Specifically, we identify SARS-CoV-2 ORF3A as most frequently interacting with indispensable proteins in the dhPPIN, which are enriched in TGF-beta signaling and tend to be sources nodes and interact with each other. Finally, we built an integrated network model of ORF3A-interacting indispensable proteins with multiple functional supports to provide hypotheses for experimental validation as well as therapeutic opportunities. Therefore, a sub-network of indispensable proteins targeted by SARS-CoV-2 could serve as a prioritized network of drug targets and a basis for further functional and mechanistic studies from a network controllability perspective.","version":"1.1","doi":"10.1101/2021.04.18.440358","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.17.440246","pub_date":"2021-4-19","title":"Live Virus Neutralisation of the 501Y.V1 and 501Y.V2 SARS-CoV-2 Variants following INO-4800 Vaccination of Ferrets","abstract":"The ongoing COVID-19 pandemic has resulted in significant global morbidity and mortality on a scale similar to the influenza pandemic of 1918. Over the course of the last few months, a number of SARS-CoV-2 variants have been identified against which vaccine-induced immune responses may be less effective. These \u201cvariants-of-concern\u201d have garnered significant attention in the media, with discussion around their impact on the future of the pandemic and the ability of leading COVID-19 vaccines to protect against them effectively. To address concerns about emerging SARS-CoV-2 variants affecting vaccine-induced immunity, we investigated the neutralisation of representative \u2018G614\u2019, \u2018501Y.V1\u2019 and \u2018501Y.V2\u2019 virus isolates using sera from ferrets that had received prime-boost doses of the DNA vaccine, INO-4800. Neutralisation titres against G614 and 501Y.V1 were comparable, but titres against the 501Y.V2 variant were approximately 4-fold lower, similar to results reported with other nucleic acid vaccines and supported by in silico biomolecular modelling. The results confirm that the vaccine-induced neutralising antibodies generated by INO-4800 remain effective against current variants-of-concern, albeit with lower neutralisation titres against 501Y.V2 similar to other leading nucleic acid-based vaccines.","version":"1.1","doi":"10.1101/2021.04.17.440246","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.17.440278","pub_date":"2021-4-19","title":"Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection","abstract":"SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients. SARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.04.17.440278","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.18.440345","pub_date":"2021-4-19","title":"Inferring the stabilization effects of SARS-CoV-2 variants on the binding with ACE2 receptor","abstract":"With the progression of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic, several variants of the virus are emerging with mutations distributed all over the viral sequence. While most of them are expected to have little to no effects at the phenotype level, some of these variants presenting specific mutations on the Spike protein are rapidly spreading, making urgent the need of characterizing their effects on phenotype features like contagiousness and antigenicity. With this aim, we performed extensive molecular dynamics simulations on a selected set of possible Spike variants in order to assess the stabilizing effect of particular amino acid substitutions, with a special focus on the mutations that are both characteristic of the top three most worrying variants at the moment, i.e the English, South African and Amazonian ones, and that occur at the molecular interface between SARS-CoV-2 Spike protein and its human ACE2 receptor. We characterize these variants\u2019 effect in terms of (i) residues mobility, (ii) compactness, studying the network of interactions at the interface, and (iii) variation of shape complementarity via expanding the molecular surfaces in the Zernike basis. Overall, our analyses highlighted greater stability of the three variant complexes with respect to both the wild type and two negative control systems, especially for the English and Amazonian variants. In addition, in the three variants, we investigate the effects a not-yet observed mutation in position 501 could provoke on complex stability. We found that a phenylalanine mutation behaves similarly to the English variant and may cooperate in further increasing the stability of the South African one, hinting at the need for careful surveillance for the emergence of such kind of mutations in the population. Ultimately, we show that the observables we propose describe key features for the stability of the ACE2-spike complex and can help to monitor further possible spike variants.","version":"1.1","doi":"10.1101/2021.04.18.440345","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.19.440086","pub_date":"2021-4-19","title":"Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities","abstract":"Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, current methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a scalable viral peptide discovery approach covering 229 RNA viruses that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an \u03a6xFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its \u03a6xFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction blocks SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.","version":"1.1","doi":"10.1101/2021.04.19.440086","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.19.440492","pub_date":"2021-4-19","title":"The SARS CoV-1 3a protein disrupts Golgi complex morphology and cargo trafficking","abstract":"Coronaviruses assemble by budding into the endoplasmic reticulum-Golgi intermediate compartment, but the pathway of egress from infected cells is not well understood. Efficient egress of infectious bronchitis virus (a gamma coronavirus, CoV) requires neutralization of Golgi pH by the envelope (E) protein. This results in reduced rates of cargo traffic and disrupts Golgi morphology, but it protects the spike protein from aberrant proteolysis. The severe acute respiratory syndrome (SARS) CoV-1 E protein does not disrupt the Golgi, however. We show here that in transfected cells, the ORF3a protein of SARS CoV-1 disrupts Golgi morphology, cargo trafficking and luminal pH. Unlike the infectious bronchitis virus E protein, these functions of the SARS CoV-1 3a protein appear to require its viroporin activity. Thus, neutralization of acidic compartments may be a universal feature of CoV infection, although different viral proteins and mechanisms may be used to achieve this outcome.","version":"1.1","doi":"10.1101/2021.04.19.440492","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.10.21255248","pub_date":"2021-04-17","title":"Retrospective cohort study of Ivermectin as a SARS-CoV-2 pre-exposure prophylaxis method in Healthcare Workers","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>This observational and retrospective cohort study, carried out in two medical centers, Centro Medico Bournigal (CMBO) in Puerto Plata and Centro Medico Punta Cana (CMPC) in Punta Cana, Dominican Republic, sought to determine whether Ivermectin, at a weekly oral (PO) dose of 0.2 mg/kg, is an effective pre-exposure prophylaxis (PrEP) method preventing the spread of Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2), in the healthcare workers. The study began on June 29, 2020 and ended on July 26, 2020, where 713 active healthcare personnel were included for the analysis, from which 326 healthcare personnel adhered to a weekly prophylactic program with Ivermectin for Coronavirus Disease-19 (COVID-19), designed by Grupo Rescue task force, that runs both medical centers, and 387 healthcare personnel who did not adhere to the program and were assigned as control group. A SPSS Propensity Score Matching procedure was applied in a 1:1 ratio to homogeneously evaluate the groups that were made up of 271 participants each.</jats:p>\n                <jats:p>In 28 days of follow-up, a significant protection of Ivermectin as a PrEP method preventing the infection from SARS-CoV-2 was observed 1.8% compared to those who did not take it 6.6%, (p-value = 0.006) with a risk reduction of a positive Chain Reaction of Real-Time Polymerase Transcription (RT-PCR) COVID-19 test by 74% (HR 0.26, 95% CI [0.10,0.71]). These results support the use of weekly Ivermectin as a compassionate preventive method in the healthcare personnel.</jats:p>\n                <jats:p>\n                  This study protocol is available at\n                  <jats:bold>Clinicaltrials</jats:bold>\n                  .\n                  <jats:bold>gov</jats:bold>\n                  Identifier:\n                  <jats:bold>NCT04832945</jats:bold>\n                </jats:p>","version":null,"doi":"10.1101/2021.04.10.21255248","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.16.440124","pub_date":"2021-4-16","title":"SARS-CoV-2 proteins bind heme and hemoglobin","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins\u2019 possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemoglobin\u2019s porphyrin coordination region. Different hemin binding capacities of mock and SARS-CoV-2-infected culture extracts were noticed using gel electrophoresis and TMB staining. Hemin-binding proteins were isolated from SARS-CoV-2-infected cells by affinity chromatography and identified by shotgun proteomics, indicating that structural (nucleoprotein, spike, and membrane protein) and non-structural (Nsp3 and Nsp7) viral proteins interact with hemin. In vitro analyses of virus adsorption to host cells and viral replication studies in Vero cells demonstrated inhibitory activities - at different levels - by hemin, protoporphyrin IX (PpIX) Hb. Strikingly, free Hb at 1\u03bcM suppressed viral replication (99 %), and its interaction with SARS-CoV-2 was localized to the RBD region of the Spike protein. The findings showed clear evidence of new avenues to disrupt viral replication and understand virus physiopathology that warrants further investigation.","version":"1.1","doi":"10.1101/2021.04.16.440124","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.16.440083","pub_date":"2021-4-16","title":"Antibody Cocktail Exhibits Broad Neutralization against SARS-CoV-2 and SARS-CoV-2 variants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them, two RBD-binding antibodies (F61 and H121) showed high affinity neutralization against SARS-CoV-2, whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis, F61 identified a linear epitope located in residues G446 -S494, which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites, while H121 recognized a conformational epitope located on the side face of RBD, outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly, F61 and H121 exhibited efficient neutralizing activity against variants B.1.1.7 and B.1.351, those showed immune escape. Efficient neutralization of F61 and H121 against multiple mutations within RBD revealed a broad neutralizing activity against SARS-CoV-2 variants, which mitigated the risk of viral escape. Our findings defined the basis of therapeutic cocktails of F61 and H121 with broad neutralization and delivered a guideline for the current and future vaccine design, therapeutic antibody development, and antigen diagnosis of SARS-CoV-2 and its novel variants.","version":"1.1","doi":"10.1101/2021.04.16.440083","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.16.440101","pub_date":"2021-4-16","title":"A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model","abstract":"TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly generate human monoclonal antibodies. After immunizing these mice against the spike protein of SARS-CoV-2, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralized SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of neutralizing antibodies binds to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2 induced weight loss. Thus, we report two clusters of potent non-competing SARS-CoV-2 neutralizing antibodies providing potential candidates for therapy and prophylaxis of COVID-19. The study further supports the use of transgenic animals with human immunoglobulin gene repertoires in pandemic preparedness initiatives.","version":"1.1","doi":"10.1101/2021.04.16.440101","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.16.440215","pub_date":"2021-4-16","title":"SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response","abstract":"Increased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-\u03b2-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.","version":"1.1","doi":"10.1101/2021.04.16.440215","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.16.440104","pub_date":"2021-4-16","title":"Homo-harringtonine (HHT) \u2013 A highly effective drug against coronaviruses and the potential for large-scale clinical applications","abstract":"In the search for treatment schemes of COVID-19, we start by examining the general weakness of coronaviruses and then identify approved drugs attacking that weakness. The approach, if successful, should identify drugs with a specific mechanism that is at least as effective as the best drugs proposed and are ready for clinical trials. All coronaviruses translate their non-structural proteins (\u223c16) in concatenation, resulting in a very large super-protein. Homo-harringtonine (HHT), which has been approved for the treatment of leukemia, blocks protein elongation very effectively. Hence, HHT can repress the replication of many coronaviruses at the nano-molar concentration. In two mouse models, HHT clears SARS-CoV-2 in 3 days, especially by nasal dripping of 40 ug per day. We also use dogs to confirm the safety of HHT delivered by nebulization. The nebulization scheme could be ready for large-scale applications at the onset of the next epidemics. For the current COVID-19, a clinical trial has been approved by the Ditan hospital of Beijing but could not be implemented for want of patients. The protocol is available to qualified medical facilities.","version":"1.1","doi":"10.1101/2021.04.16.440104","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.15.439839","pub_date":"2021-4-15","title":"SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8","abstract":"The inflammatory and IFN pathways of innate immunity play a key role in both resistance and pathogenesis of Coronavirus Disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-Associated Molecular Patterns (SAMPs) remain to be completely defined. Here we identify single-stranded RNA (ssRNA) fragments from SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and functions, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identify TLR7/8 as crucial cellular sensors of ssRNAs encoded by SARS-CoV-2 involved in host resistance and disease pathogenesis of COVID-19.","version":"1.1","doi":"10.1101/2021.04.15.439839","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.28.437363","pub_date":"2021-4-15","title":"SARS-CoV-2 variant B.1.1.7 caused HLA-A2+ CD8+ T cell epitope mutations for impaired cellular immune response","abstract":"The rapid spreading of the newly emerged SARS-CoV-2 variant, B.1.1.7, highlighted the requirements to better understand adaptive immune responses to this virus. Since CD8+ T cell responses play an important role in disease resolution and modulation in COVID-19 patients, it is essential to address whether these newly emerged mutations would result in altered immune responses. Here we evaluated the immune properties of the HLA-A2 restricted CD8+ T cell epitopes containing mutations from B.1.1.7, and furthermore performed a comprehensive analysis of the SARS-CoV-2 specific CD8+ T cell responses from COVID-19 convalescent patients and SARS-CoV-2 vaccinees recognizing the ancestral Wuhan strain compared to B.1.1.7. First, most of the predicted CD8+ T cell epitopes showed proper binding with HLA-A2, while epitopes from B.1.1.7 had lower binding capability than those from the ancestral strain. In addition, these peptides could effectively induced the activation and cytotoxicity of CD8+ T cells. Our results further showed that at least two site mutations in B.1.1.7 resulted in a decrease in CD8+ T cell activation and a possible immune evasion, namely A1708D mutation in ORF1ab1707-1716 and I2230T mutation in ORF1ab2230-2238. Our current analysis provides information that contributes to the understanding of SARS-CoV-2-specific CD8+ T cell responses elicited by infection of mutated strains or vaccination.","version":"1.2","doi":"10.1101/2021.03.28.437363","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.15.439956","pub_date":"2021-4-15","title":"Time-series trend of pandemic SARS-CoV-2 variants visualized using batch-learning self-organizing map for oligonucleotide compositions","abstract":"To confront the global threat of coronavirus disease 2019, a massive number of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences have been decoded, with the results promptly released through the GISAID database. Based on variant types, eight clades have already been defined in GISAID, but the diversity can be far greater. Owing to the explosive increase in available sequences, it is important to develop new technologies that can easily grasp the whole picture of the big-sequence data and support efficient knowledge discovery. An ability to efficiently clarify the detailed time-series changes in genome-wide mutation patterns will enable us to promptly identify and characterize dangerous variants that rapidly increase their population frequency. Here, we collectively analyzed over 150,000 SARS-CoV-2 genomes to understand their overall features and time-dependent changes using a batch-learning self-organizing map (BLSOM) for oligonucleotide composition, which is an unsupervised machine learning method. BLSOM can separate clades defined by GISAID with high precision, and each clade is subdivided into clusters, which shows a differential increase/decrease pattern based on geographic region and time. This allowed us to identify prevalent strains in each region and to show the commonality and diversity of the prevalent strains. Comprehensive characterization of the oligonucleotide composition of SARS-CoV-2 and elucidation of time-series trends of the population frequency of variants can clarify the viral adaptation processes after invasion into the human population and the time-dependent trend of prevalent epidemic strains across various regions, such as continents.","version":"1.1","doi":"10.1101/2021.04.15.439956","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439928","pub_date":"2021-4-15","title":"Evaluation of the SARS-CoV-2 inactivation efficacy associated with buffers from three kits used on high-throughput RNA extraction platforms","abstract":"Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QiaCube HT and the ThermoFisher Kingfisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type; SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer from the MagMax Pathogen RNA/DNA kit (ThermoFisher), used on the Kingfisher Flex, which included guanidinium isothiocycnate (GITC), a detergent, and isopropanol demonstrated a minimum inactivation efficacy of 1 x 105 TCID50/ml. An alternative lysis buffer from the MagMax Viral/Pathogen Nucleic Acid kit (Thermofisher) also used on the Kingfisher Flex and the lysis buffer from QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QiaCube HT (both of which contained GITC and a detergent) reduced titres by 1 x 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 minutes, 68 \u00b0C) did not completely inactivate the virus, demonstrating a reduction of 1 x 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.","version":"1.1","doi":"10.1101/2021.04.14.439928","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.15.440035","pub_date":"2021-4-15","title":"Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii","abstract":"Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.","version":"1.1","doi":"10.1101/2021.04.15.440035","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.15.440004","pub_date":"2021-4-15","title":"Computationally prioritized drugs inhibit SARS-CoV-2 infection and syncytia formation","abstract":"New affordable therapeutic protocols for COVID-19 are urgently needed despite the increasing number of effective vaccines and monoclonal antibodies. To this end, there is increasing attention towards computational methods for drug repositioning and de novo drug design. Here, we systematically integrated multiple data-driven computational approaches to perform virtual screening and prioritize candidate drugs for the treatment of COVID-19. From the set of prioritized drugs, we selected a subset of representative candidates to test in human cells. Two compounds, 7-hydroxystaurosporine and bafetinib, showed synergistic antiviral effects in our in vitro experiments, and strongly inhibited viral-induced syncytia formation. Moreover, since existing drug repositioning methods provide limited usable information for de novo drug design, we extracted and prioritized the chemical substructures of the identified drugs, providing a chemical vocabulary that may help to design new effective drugs.","version":"1.1","doi":"10.1101/2021.04.15.440004","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428478","pub_date":"2021-4-14","title":"Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice","abstract":"Improving the standard of clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAb) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of COVID-19. However, it is not known if combination RDV/mAb will improve outcomes over single agent therapies or whether antibody therapies will remain efficacious against variants. In kinetic studies in a mouse-adapted model of ancestral SARS-CoV-2 pathogenesis, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 hours after infection. The same antibody combination was also effective in prevention and therapy against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared to single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and support the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.","version":"1.2","doi":"10.1101/2021.01.27.428478","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439641","pub_date":"2021-4-14","title":"Spike Protein Targeting \u201cNano-Glue\u201d that Captures and Promotes SARS-CoV-2 Elimination","abstract":"The global emergency caused by the SARS-CoV-2 pandemics can only be solved with adequate preventive and therapeutic strategies, both currently missing. The electropositive Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein with abundant \u03b2-sheet structure serves as target for COVID-19 therapeutic drug design. Here, we discovered that ultrathin 2D CuInP2S6 (CIPS) nanosheets as a new agent against SARS-CoV-2 infection, which also able to promote viral host elimination. CIPS exhibits extremely high and selective binding capacity with the RBD of SARS-CoV-2 spike protein, with consequent inhibition of virus entry and infection in ACE2-bearing cells and human airway epithelial organoids. CIPS displays nano-viscous properties in selectively binding with spike protein (KD < 1 pM) with negligible toxicity in vitro and in vivo. Further, the CIPS-bound SARS-CoV-2 was quickly phagocytosed and eliminated by macrophages, suggesting CIPS could be successfully used to capture and facilitate the virus host elimination with possibility of triggering anti-viral immunization. Thus, we propose CIPS as a promising nanodrug for future safe and effective anti-SARS-CoV-2 therapy, as well as for use as disinfection agent and surface coating material to constrain the SARS-CoV-2 spreading.","version":"1.1","doi":"10.1101/2021.04.13.439641","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439709","pub_date":"2021-4-14","title":"Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants","abstract":"Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains that continue to fuel the COVID-19 pandemic despite intensive vaccination efforts throughout the world. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Mutations in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement can account for the increased transmissibility and risk of mortality as the variant may begin to infect efficiently infect additional cell types expressing low levels of ACE2. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, rendering complete resistance to some potent neutralizing antibodies. These findings provide structural details on how the wide spread of SARS-CoV-2 enables rapid evolution to enhance viral fitness and immune evasion. They may guide intervention strategies to control the pandemic.","version":"1.1","doi":"10.1101/2021.04.13.439709","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439719","pub_date":"2021-4-14","title":"INO-4800 DNA Vaccine Induces Neutralizing Antibodies and T cell Activity Against Global SARS-CoV-2 Variants","abstract":"Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with broadened host specificity, pathogenicity, and immune evasion to vaccine induced immunity. Here we compared humoral and cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFN\u03b3 T cell responses were fully maintained against all variants tested.","version":"1.1","doi":"10.1101/2021.04.14.439719","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439840","pub_date":"2021-4-14","title":"Pattern Detection in Multiple Genome Sequences with Applications: The Case of All SARS-CoV-2 Complete Variants","abstract":"Pattern detection and string matching are fundamental problems in computer science and the accelerated expansion of bioinformatics and computational biology have made them a core topic for both disciplines. The SARS-CoV-2 pandemic has made such problems more demanding with hundreds or thousands of new genome variants discovered every week, because of constant mutations, and the need for fast and accurate analyses. Medicines and, mostly, vaccines must be altered to adapt and efficiently address mutations. The need of computational tools for genomic analysis, such as sequence alignment, is very important, although, in most cases the resources and computational power needed is vast. The presented data structures and algorithms, specifically built for text mining and pattern detection, can help to address efficiently several bioinformatics problems. With a single execution of advanced algorithms, with limited space and time complexity, it is possible to acquire knowledge on all repeated patterns that exist in multiple genome sequences and this information can be used for further meta analyses. The potentials of the presented solutions are demonstrated with the analysis of more than 55,000 SARS-CoV-2 genome sequences (collected on March 10, 2021) and the detection of all repeated patterns with length up to 60 nucleotides in these sequences, something practically impossible with other algorithms due to its complexity. These results can be used to help provide answers to questions such as all variants common patterns, sequence alignment, palindromes and tandem repeats detection, genome comparisons, etc.","version":"1.1","doi":"10.1101/2021.04.14.439840","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439743","pub_date":"2021-4-14","title":"Dynamic Profiling of Binding and Allosteric Propensities of the SARS-CoV-2 Spike Protein with Different Classes of Antibodies: Mutational and Perturbation-Based Scanning Reveal Allosteric Duality of Functionally Adaptable Hotspots","abstract":"Structural and biochemical studies of the SARS-CoV-2 spike complexes with highly potent antibodies have revealed multiple conformation-dependent epitopes and a broad range of recognition modes linked to different neutralization responses In this study, we combined atomistic simulations with mutational and perturbation-based scanning approaches to perform in silico profiling of binding and allosteric propensities of the SARS-CoV-2 spike protein residues in complexes with B38, P2B-2F6, EY6A and S304 antibodies representing three different classes. Conformational dynamics analysis revealed that binding-induced modulation of soft modes can elicit the unique protein response to different classes of antibodies. Mutational scanning heatmaps and sensitivity analysis revealed the binding energy hotspots for different classes of antibodies that are consistent with the experimental deep mutagenesis, showing that differences in the binding affinity caused by global circulating variants in spike positions K417, E484 and N501 are relatively moderate and may not fully account for the observed antibody resistance effects. Through functional dynamics analysis and perturbation-response scanning of the SARS-CoV-2 spike protein residues in the unbound form and antibody-bound forms, we examine how antibody binding can modulate allosteric propensities of spike protein residues and determine allosteric hotspots that control signal transmission and global conformational changes. These results show that residues K417, E484, and N501 targeted by circulating mutations correspond to a group of versatile allosteric centers in which small perturbations can modulate collective motions, alter the global allosteric response and elicit binding resistance. We suggest that SARS-CoV-2 S protein may exploit plasticity of specific allosteric hotspots to generate escape mutants that alter response to antibody binding without compromising activity of the spike protein.","version":"1.1","doi":"10.1101/2021.04.13.439743","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.14.439844","pub_date":"2021-4-14","title":"The SARS-CoV-2 mRNA-1273 vaccine elicits more RBD-focused neutralization, but with broader antibody binding within the RBD","abstract":"The emergence of SARS-CoV-2 variants with mutations in key antibody epitopes has raised concerns that antigenic evolution will erode immunity. The susceptibility of immunity to viral evolution is shaped in part by the breadth of epitopes targeted. Here we compare the specificity of antibodies elicited by the mRNA-1273 vaccine versus natural infection. The neutralizing activity of vaccine-elicited antibodies is even more focused on the spike receptor-binding domain (RBD) than for infection-elicited antibodies. However, within the RBD, binding of vaccine-elicited antibodies is more broadly distributed across epitopes than for infection-elicited antibodies. This greater binding breadth means single RBD mutations have less impact on neutralization by vaccine sera than convalescent sera. Therefore, antibody immunity acquired by different means may have differing susceptibility to erosion by viral evolution. Deep mutational scanning shows the mRNA-1273 RBD-binding antibody response is less affected by single viral mutations than the infection response.","version":"1.1","doi":"10.1101/2021.04.14.439844","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.435706","pub_date":"2021-4-14","title":"Fast and accurate genome-wide predictions and structural modeling of protein-protein interactions using Galaxy","abstract":"Protein-protein interactions play a crucial role in almost all cellular processes. Identifying interacting proteins reveals insight into living organisms and yields novel drug targets for disease treatment. Here, we present a publicly available, automated pipeline to predict genome-wide protein-protein interactions and produce high-quality multimeric structural models. Application of our method to the Human and Yeast genomes yield protein-protein interaction networks similar in quality to common experimental methods. We identified and modeled Human proteins likely to interact with the papain-like protease of SARS-CoV2\u2019s non-structural protein 3 (Nsp3). We also produced models of SARS-CoV2\u2019s spike protein (S) interacting with myelin-oligodendrocyte glycoprotein receptor (MOG) and dipeptidyl peptidase-4 (DPP4). The presented method is capable of confidently identifying interactions while providing high-quality multimeric structural models for experimental validation. The interactome modeling pipeline is available at usegalaxy.org and usegalaxy.eu.","version":"1.2","doi":"10.1101/2021.03.17.435706","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439586","pub_date":"2021-4-13","title":"Elevated anti-SARS-CoV-2 antibodies and IL-6, IL-8, MIP-1\u03b2, early predictors of severe COVID-19","abstract":"Viral and host immune kinetics during acute COVID-19 and after remission of acute symptoms need better characterization. SARS-CoV-2 RNA, anti-SARS-CoV-2 IgA, IgM, and IgG antibodies, and pro-inflammatory cytokines were measured in sequential samples among hospitalized COVID-19 patients during acute infection and 6 months following diagnosis. 24 laboratory-confirmed COVID-19 patients with mild/moderate and severe COVID-19 were included. Most were males 83%, median age of 61 years. 21% were admitted to the ICU and 8 of them (33.3%) met criteria for severe COVID-19 disease. A delay in SARS-CoV-2 levels decline during the first 6 days of follow-up and viral load persistence until month 3 were related with severe COVID-19, but not viral load levels at the diagnosis. Higher levels of anti-SARS-CoV-2 IgA, IgM, IgG and the cytokines IL-6, IL-8 and MIP-1\u03b2 at the diagnosis time were related with severe COVID-19 outcome. Higher levels of MIP-1\u03b2, IL-1\u03b2, MIP-1\u03b1 and IFN-\u03b3 were observed at month 1/3 during mild/moderate disease compared to severe COVID-19. IgG persisted at low levels after 6 months of diagnosis. Higher concentrations of IgA, IgM, and IgG, and IL-6, IL-8 and MIP-1\u03b2 are identified as early predictors of COVID-19 severity, but not SARS-CoV-2 RNA levels at diagnosis.","version":"1.1","doi":"10.1101/2021.04.13.439586","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.21255131","pub_date":"2021-04-13","title":"Early and ongoing importations of SARS-CoV-2 in Canada","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Tracking the emergence and spread of SARS-CoV-2 is critical to inform public health interventions. Phylodynamic analyses have quantified SARS-CoV-2 migration on global and local scales\n                  <jats:sup>1\u20135</jats:sup>\n                  , yet they have not been applied to determine transmission dynamics in Canada. We quantified SARS-CoV-2 migration into, within, and out of Canada in the context of COVID-19 travel restrictions. To minimize sampling bias, global sequences were subsampled with probabilities corrected for their countries\u2019 monthly contribution to global new diagnoses. A time-scaled maximum likelihood tree was used to estimate most likely ancestral geographic locations (country or Canadian province), enabling identification of sublineages, defined as introduction events into Canada resulting in domestic transmission. Of 402 Canadian sublineages identified, the majority likely originated from the USA (54%), followed by Russia (7%), India (6%), Italy (6%), and the UK (5%). International introductions were mostly into Ontario (39%) and Quebec (38%). Among Pango lineages\n                  <jats:sup>6</jats:sup>\n                  , B.1 was imported at least 191 separate times from 11 different countries. Introduction rates peaked in late March then diminished but were not eliminated following national interventions including restrictions on non-essential travel. We further identified 1,380 singleton importations, international importations that did not result in further sampled transmission, whereby representation of lineages and location were comparable to sublineages. Although proportion of international transmission decreased over time, this coincided with exponential growth of within-province transmission \u2013 in fact, total number of sampled transmission events from international or interprovincial sources increased from winter 2020 into spring 2020 in many provinces. Ontario, Quebec, and British Columbia acted as sources of transmission more than recipients, within the caveat of higher sequence representation. We present strong evidence that international introductions and interprovincial transmission of SARS-CoV-2 contributed to the Canadian COVID-19 burden throughout 2020, despite initial reductions mediated by travel restrictions in 2020. More stringent border controls and quarantine measures may have curtailed introductions of SARS-CoV-2 into Canada and may still be warranted.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement</jats:title>\n                  <jats:p>By analyzing SARS-CoV-2 genomes from Canada in the context of the global pandemic, we illuminate the extent to which the COVID-19 burden in Canada was perpetuated by ongoing international importations and interprovincial transmission throughout 2020. Although travel restrictions enacted in March 2020 reduced the importation rate and proportion of transmission from abroad across all Canadian provinces, SARS-CoV-2 introductions from the USA, India, Russia, and other nations were detectable through the summer and fall of 2020.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.04.09.21255131","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.11.439379","pub_date":"2021-4-13","title":"One Health Investigation of SARS-CoV-2 Infection and Seropositivity among Pets in Households with Confirmed Human COVID-19 Cases \u2014 Utah and Wisconsin, 2020","abstract":"Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pet cohabitants as a sub-study of an ongoing COVID-19 household transmission investigation. Mammalian pets from households with \u22651 person with laboratory-confirmed COVID-19 were eligible for inclusion from April\u2013May 2020. Demographic/exposure information, oropharyngeal, nasal, rectal, and fur swabs, feces, and blood were collected from enrolled pets and tested by rRT-PCR and virus neutralization assays. We enrolled 37 dogs and 19 cats from 34 of 41 eligible households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog\u2019s fur swabs (2%) tested positive by rRT-PCR at the first animal sampling. Among 47 pets with serological results from 30 households, eight (17%) pets (4 dogs, 4 cats) from 6 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40\u2013100%) compared to households with no seropositive pet (median 37%; range: 13\u2013100%) (p=0.01). Thirty-three pets with serologic results had frequent daily contact (\u22651 hour) with the human index patient before the person\u2019s COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the human index patient after diagnosis and none (0%) were seropositive; of the 19 (58%) pets with continued contact, 4 (21%) were seropositive. Seropositive pets likely acquired infection from humans, which may occur more frequently than previously recognized. People with COVID-19 should restrict contact with animals. Centers for Disease Control and Prevention, U.S. Department of Agriculture","version":"1.1","doi":"10.1101/2021.04.11.439379","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439681","pub_date":"2021-4-13","title":"Epitope classification and RBD binding properties of neutralizing antibodies against SARS-CoV-2 variants of concern","abstract":"Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.","version":"1.1","doi":"10.1101/2021.04.13.439681","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439668","pub_date":"2021-4-13","title":"From partial to whole genome imputation of SARS-CoV-2 for epidemiological surveillance","abstract":"the current SARS-CoV-2 pandemic has emphasized the utility of viral whole genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is increasingly producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and therefore useless, sequences. However, viral sequences evolve in the context of a complex phylogeny and therefore different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data. We developed impuSARS, an application that includes Minimac, the most widely used strategy for genomic data imputation and, taking advantage of the enormous amount of SARS-CoV-2 whole genome sequences available, a reference panel containing 239,301 sequences was built. The impuSARS application was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing) showing great fidelity when reconstructing the original sequences. The impuSARS application is also able to impute whole genomes from commercial kits covering less than 20% of the genome or only from the Spike protein with a precision of 0.96. It also recovers the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (< 20%) imputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. impuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole genome sequencing.","version":"1.1","doi":"10.1101/2021.04.13.439668","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.305698","pub_date":"2021-4-13","title":"AI-guided discovery of the invariant host response to viral pandemics","abstract":"We sought to define the host immune response, a.k.a, the \u201ccytokine storm\u201d that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a \u2018seed\u2019 gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2\u2022ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs. The host immune response in COVID-19. The SARS-CoV-2 pandemic has inspired many groups to find innovative methodologies that can help us understand the host immune response to the virus; unchecked proportions of such immune response have been implicated in fatality. We searched GEO and ArrayExpress that provided many publicly available gene expression data that objectively measure the host immune response in diverse conditions. However, challenges remain in identifying a set of host response events that are common to every condition. There are no studies that provide a reproducible assessment of prognosticators of disease severity, the host response, and therapeutic goals. Consequently, therapeutic trials for COVID-19 have seen many more \u2018misses\u2019 than \u2018hits\u2019. This work used multiple (> 45,000) gene expression datasets from GEO and ArrayExpress and analyzed them using an unbiased computational approach that relies upon fundamentals of gene expression patterns and mathematical precision when assessing them. This work identifies a signature that is surprisingly conserved in all viral pandemics, including Covid-19, inspiring the nomenclature ViP-signature. A subset of 20-genes classified disease severity in respiratory pandemics. The ViP signatures pinpointed the nature and source of the \u2018cytokine storm\u2019 mounted by the host. They also helped formulate precise therapeutic goals and rationalized the repurposing of FDA-approved drugs. The ViP signatures provide a quantitative and qualitative framework for assessing the immune response in viral pandemics when creating pre-clinical models; they serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.","version":"1.3","doi":"10.1101/2020.09.21.305698","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.13.439274","pub_date":"2021-4-13","title":"Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease","abstract":"The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.","version":"1.1","doi":"10.1101/2021.04.13.439274","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.12.439425","pub_date":"2021-4-12","title":"The Seroprevalence of SARS-CoV-2 in Europe: A Systematic Review","abstract":"A year following the onset of the COVID-19 pandemic, new infections and deaths continue to increase in Europe. Serological studies, through providing evidence of past infection, can aid understanding of the population dynamics of SARS-CoV-2 infection. This systematic review of SARS-CoV-2 seroprevalence studies in Europe was undertaken to inform public health strategies including vaccination, that aim to accelerate population immunity. We searched the databases Web of Science, MEDLINE, EMBASE, SCOPUS, Cochrane Database of Systematic Reviews and grey literature sources for studies reporting seroprevalence of SARS-CoV-2 antibodies in Europe published between 01/12/2019 - 30/09/20. We provide a narrative synthesis of included studies. Studies were categorized into subgroups including healthcare workers (HCWs), community, outbreaks, pregnancy and children/school. Due to heterogeneity in other subgroups, we only performed a random effects meta-analysis of the seroprevalence amongst HCWs stratified by their country. 109 studies were included spanning 17 European countries, that estimated the seroprevalence of SAR-CoV2 from samples obtained between November 2019 \u2013 August 2020. A total of 53/109 studies included HCWs with a reported seroprevalence among HCWs ranging from 0.7% to 45.3%, which did not differ significantly by country. In community studies significant heterogeneity was reported in the seroprevalence among different age groups and the majority of studies reported there was no significant difference by gender. This review demonstrates a wide heterogeneity in reported seroprevalence of SARS-CoV-2 antibodies between populations. Continued evaluation of seroprevalence is required to understand the impact of public health measures and inform interventions including vaccination programmes.","version":"1.1","doi":"10.1101/2021.04.12.439425","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439149","pub_date":"2021-4-12","title":"Intranasal gene therapy to prevent infection by SARS-CoV-2 variants","abstract":"SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis. SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis.","version":"1.2","doi":"10.1101/2021.04.09.439149","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.12.439549","pub_date":"2021-4-12","title":"Interactions between SARS-CoV-2 N-protein and \u03b1-synuclein accelerate amyloid formation","abstract":"First cases that point at a correlation between SARS-CoV-2 infections and the development of Parkinson\u2019s disease have been reported. Currently it is unclear if there also is a direct causal link between these diseases. To obtain first insights into a possible molecular relation between viral infections and the aggregation of \u03b1-synuclein protein into amyloid fibrils characteristic for Parkinson\u2019s disease, we investigated the effect of the presence of SARS-CoV-2 proteins on \u03b1-synuclein aggregation. We show, in test tube experiments, that SARS-CoV-2 S-protein has no effect on \u03b1-synuclein aggregation while SARS-CoV-2 N-protein considerably speeds up the aggregation process. We observe the formation of multi-protein complexes, and eventually amyloid fibrils. Microinjection of N-protein in SHSY-5Y cells disturbed the \u03b1-synuclein proteostasis and increased cell death. Our results point toward direct interactions between the N-protein of SARS-CoV-2 and \u03b1-synuclein as molecular basis for the observed coincidence between SARS-CoV-2 infections and Parkinsonism.","version":"1.1","doi":"10.1101/2021.04.12.439549","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.10.439300","pub_date":"2021-4-12","title":"Combinatorial approach with mass spectrometry and lectin microarray dissected glycoproteomic features of virion-derived spike protein of SARS-CoV-2","abstract":"The COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2, has a global impact on public health. Since glycosylation of the viral envelope glycoproteins is known to be deeply associated with their immunogenicity, intensive studies on the glycans of its major glycoprotein, S protein, have been conducted. Nevertheless, the detailed site-specific glycan compositions of virion-associated S protein have not yet been clarified. Here, we conducted intensive glycoproteomic analyses of SARS-CoV-2 S protein using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Using our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, we identified 1,759 site-specific glycan compositions. The most frequent was HexNAc:Hex:Fuc:NeuAc:NeuGc = 6:6:1:0:0, suggesting a tri-antennary N-glycan terminating with LacNAc and having bisecting GlcNAc and a core fucose, which was found in 20 of 22 glycosylated sites. The subsequent lectin microarray analysis emphasized intensive outer arm fucosylation of glycans, which efficiently complemented the glycoproteomic features. The present results illustrate the high-resolution glycoproteomic features of SARS-CoV-2 S protein and significantly contribute to vaccine design, as well as the understanding of viral protein synthesis.","version":"1.1","doi":"10.1101/2021.04.10.439300","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.11.439322","pub_date":"2021-4-12","title":"Prediction and evolution of the molecular fitness of SARS-CoV-2 variants: Introducing SpikePro","abstract":"The understanding of the molecular mechanisms driving the fitness of the SARS-CoV-2 virus and its mutational evolution is still a critical issue. We built a simplified computational model, called SpikePro, to predict the SARS-CoV-2 fitness from the amino acid sequence and structure of the spike protein. It contains three contributions: the viral transmissibility predicted from the stability of the spike protein, the infectivity computed in terms of the affinity of the spike protein for the ACE2 receptor, and the ability of the virus to escape from the human immune response based on the binding affinity of the spike protein for a set of neutralizing antibodies. Our model reproduces well the available experimental, epidemiological and clinical data on the impact of variants on the biophysical characteristics of the virus. For example, it is able to identify circulating viral strains that, by increasing their fitness, recently became dominant at the population level. SpikePro is a useful instrument for the genomic surveillance of the SARS-CoV-2 virus, since it predicts in a fast and accurate way the emergence of new viral strains and their dangerousness. It is freely available in the GitHub repository github.com/3BioCompBio/SpikeProSARS-CoV-2.","version":"1.1","doi":"10.1101/2021.04.11.439322","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.12.439473","pub_date":"2021-4-12","title":"Revealing the threat of emerging SARS-CoV-2 mutations to antibody therapies","abstract":"The ongoing massive vaccination and the development of effective intervention offer the long-awaited hope to end the global rage of the COVID-19 pandemic. However, the rapidly growing SARS-CoV-2 variants might compromise existing vaccines and monoclonal antibody (mAb) therapies. Although there are valuable experimental studies about the potential threats from emerging variants, the results are limited to a handful of mutations and Eli Lilly and Regeneron mAbs. The potential threats from frequently occurring mutations on the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) to many mAbs in clinical trials are largely unknown. We fill the gap by developing a topology-based deep learning strategy that is validated with tens of thousands of experimental data points. We analyze 261,348 genome isolates from patients to identify 514 non-degenerate RBD mutations and investigate their impacts on 16 mAbs in clinical trials. Our findings, which are highly consistent with existing experimental results about variants from the UK, South Africa, Brazil, US-California, and Mexico shed light on potential threats of 95 high-frequency mutations to mAbs not only from Eli Lilly and Regeneron but also from Celltrion and Rockefeller University that are in clinical trials. We unveil, for the first time, that high-frequency mutations R346K/S, N439K, G446V, L455F, V483F/A, E484Q/V/A/G/D, F486L, F490L/V/S, Q493L, and S494P/L might compromise some of mAbs in clinical trials. Our study gives rise to a general perspective about how mutations will affect current vaccines.","version":"1.1","doi":"10.1101/2021.04.12.439473","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.11.439360","pub_date":"2021-4-12","title":"Development of highly potent neutralising nanobodies against multiple SARS-CoV-2 variants including the variant of concern B.1.351","abstract":"The pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. During the years of 2020-2021, millions of humans have died due to SARS-CoV-2 infection and severe economic damage to the global economy has occurred. Unprecedented rapid investments in vaccine development have been made to counter the spread of SARS-CoV-2 among humans. While vaccines are a key pillar of modern medicine, SARS-CoV-2 has mutated as it spread among humans. Vaccines previously developed and approved by regulators are becoming less effective against new variants. One variant of SARS-CoV-2 known as B.1.351 that was first reported to be present in South Africa significantly reduces the efficacy of vaccines developed to date. Therapeutic options that work against the B.1.351 variant are therefore urgently needed to counteract reduced vaccine efficacy. We present here the discovery of recombinant alpaca antibodies that neutralise live virus of B.1.351 and other SARS-CoV-2 variants potently. The antibodies described here may be a useful tool for clinicians who are treating patients infected with B.1.351 and other SARS-CoV-2 for which there is currently no known highly effective treatment.","version":"1.1","doi":"10.1101/2021.04.11.439360","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439169","pub_date":"2021-4-12","title":"Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300","abstract":"SARS-CoV-2 encodes main protease (Mpro), an attractive target for therapeutic interventions. We show Mpro is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of Mpro revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S Mpro mutant revealed that Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.","version":"1.2","doi":"10.1101/2021.04.09.439169","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431825","pub_date":"2021-4-12","title":"Identification of COVID-19 prognostic markers and therapeutic targets through meta-analysis and validation of Omics data from nasopharyngeal samples","abstract":"While our battle with the COVID-19 pandemic continues, a multitude of Omics data has been generated from patient samples in various studies, which remains to be translated. We conducted a meta-analysis of published transcriptome and proteome profiles of nasal swab and bronchioalveolar lavage fluid (BALF) samples of COVID-19 patients, to shortlist high confidence upregulated host factors. Subsequently, mRNA overexpression of selected genes was validated in nasal swab/BALF samples from a cohort of COVID-19 positive/negative, symptomatic/asymptomatic individuals. Analysis of these data revealed S100 family genes (S100A6, S100A8, S100A9, and S100P) as prognostic markers of COVID-19 disease. Furthermore, Thioredoxin gene (TXN) was identified as a significant upregulated host factor in our overlap analysis. An FDA-approved drug Auranofin, which inhibits Thioredoxin reduction, was found to mitigate SARS-CoV-2 replication in vitro and in vivo in the hamster challenge model. Overall, this study translates COVID-19 host response Big Data into potential clinical interventions.","version":"1.2","doi":"10.1101/2021.02.18.431825","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.11.439401","pub_date":"2021-4-12","title":"RESIC: A tool for comprehensive adenosine to inosine RNA Editing Site Identification and Classification","abstract":"Adenosine to inosine (A-to-I) RNA editing, the most prevalent type of RNA editing in metazoans, is carried out by adenosine deaminases (ADARs) in double-stranded RNA regions. Several computational approaches have been recently developed to identify A-to-I RNA editing sites from sequencing data, each addressing a particular issue. Here we present RESIC, an efficient pipeline that combines several approaches for the detection and classification of RNA editing sites. The pipeline can be used for all organisms and can use any number of RNA-sequencing datasets as input. RESIC provides 1. The detection of editing sites in both repetitive and non-repetitive genomic regions; 2. The identification of hyper-edited regions; 3. Optional exclusion of polymorphism sites to increase reliability, based on DNA, and ADAR-mutant RNA sequencing datasets, or SNP databases. We demonstrate the utility of RESIC by applying it to human, successfully overlapping and extending the list of known putative editing sites. We further tested changes in the patterns of A-to-I RNA editing, and RNA abundance of ADAR enzymes, following SARS-CoV-2 infection in human cell lines. Our results suggest that upon SARS-CoV-2 infection, compared to mock, the number of hyper editing sites is increased, and in agreement, the activity of ADAR1, which catalyzes hyper-editing, is enhanced. These results imply the involvement of A-to-I RNA editing in conceiving the unpredicted phenotype of COVID-19 disease. RESIC code is open-source and is easily extendable.","version":"1.1","doi":"10.1101/2021.04.11.439401","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437622","pub_date":"2021-4-12","title":"Rapid characterization of spike variants via mammalian cell surface display","abstract":"The SARS-CoV-2 spike (S) protein is a critical component of subunit vaccines and a target for neutralizing antibodies. Spike is also undergoing immunogenic selection with clinical variants that increase infectivity and partially escape convalescent plasma. Here, we describe spike display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed \u223c200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by thirteen neutralizing antibodies (nAbs). An alanine scan of all five N-terminal domain (NTD) loops highlights a public class of epitopes in the N1, N3, and N5 loops that are recognized by most of the NTD-binding nAbs. Some clinical NTD substitutions abrogate binding to these epitopes but are circulating at low frequencies around the globe. NTD mutations in variants of concern B.1.1.7 (United Kingdom), B.1.351 (South Africa), B.1.1.248 (Brazil), and B.1.427/B.1.429 (California) impact spike expression and escape most NTD-targeting nAbs. However, two classes of NTD nAbs still bind B.1.1.7 spikes and neutralize in pseudoviral assays. B.1.351 and B.1.1.248 include compensatory mutations that either increase spike expression or increase ACE2 binding affinity. Finally, B.1.351 and B.1.1.248 completely escape a potent ACE2 peptide mimic. We anticipate that spike display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.","version":"1.2","doi":"10.1101/2021.03.30.437622","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.12.439478","pub_date":"2021-4-12","title":"Epitope profiling of coronavirus-binding antibodies using computational structural modelling","abstract":"Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. It is well-established in the literature that sequence-based clonal clustering can identify antibodies with similar epitope complementarity. However, there is growing evidence that antibodies from markedly different lineages but with similar structures can engage the same epitope with near-identical binding modes. Here, we describe a novel computational method for epitope profiling based on structural modelling and clustering, and show how it can identify sequence-dissimilar antibodies that engage the same epitope. We start by searching for evidence of structural conservation across the latest solved SARS-CoV-2\u2014binding antibody crystal structures. Despite the relatively small number of solved structures, we find numerous examples of sequence-diverse but structurally-similar coronavirus-binding antibodies engaging the same epitope. We therefore developed a high-throughput structural modeling and clustering method to identify functionally-similar antibodies across the set of thousands of coronavirus-binding antibody sequences in the Coronavirus Antibody Database (CoV-AbDab). In the resulting multiple-occupancy structural clusters, 92% bind to consistent domains based on CoV-AbDab metadata. Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than would be suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.","version":"1.1","doi":"10.1101/2021.04.12.439478","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428847","pub_date":"2021-4-12","title":"Information retrieval in an infodemic: the case of COVID-19 publications","abstract":"The COVID-19 pandemic has led to an exponential surge and an enormous amount of published literature, both accurate and inaccurate, a term usually coined as an infodemic. In the context of searching for COVID-19 related scientific literature, we present an information retrieval methodology for effectively finding relevant publications for different information needs. Our multi-stage information retrieval architecture combines probabilistic weighting models and re-ranking algorithms based on neural masked language models. The methodology was evaluated in the context of the TREC-COVID challenge, achieving competitive results with the top ranking teams participating in the competition. Particularly, the ranking combination of bag-of-words and language models significantly outperformed a BM25-based baseline model (16 percentage points for the NDCG@20 metric), correctly retrieving more than 16 out of the top 20 documents retrieved. The proposed pipeline could thus support the effective search and discovery of relevant information in the case of an infodemic.","version":"1.2","doi":"10.1101/2021.01.29.428847","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.10.439161","pub_date":"2021-4-11","title":"Quantatitive Analysis of Conserved Sites on the SARS-CoV-2 Receptor-Binding Domain to Promote Development of Universal SARS-Like Coronavirus Vaccines","abstract":"Although vaccines have been successfully developed and approved against SARS-CoV-2, it is still valuable to perform studies on conserved antigenic sites for preventing possible pandemic-risk of other SARS-like coronavirus in the future and prevalent SARS-CoV-2 variants. By antibodies obtained from convalescent COVID-19 individuals, receptor binding domain (RBD) were identified as immunodominant neutralizing domain that efficiently elicits neutralizing antibody response with on-going affinity mature. Moreover, we succeeded to define a quantitative antigenic map of neutralizing sites within SARS-CoV-2 RBD, and found that sites S2, S3 and S4 (new-found site) are conserved sites and determined as subimmunodominant sites, putatively due to their less accessibility than SARS-CoV-2 unique sites. P10-6G3, P07-4D10 and P05-6H7, respectively targeting S2, S3 and S4, are relatively rare antibodies that also potently neutralizes SARS-CoV, and the last mAbs performing neutralization without blocking S protein binding to receptor. Further, we have tried to design some RBDs to improve the immunogenicity of conserved sites. Our studies, focusing on conserved antigenic sites of SARS-CoV-2 and SARS-CoV, provide insights for promoting development of universal SARS-like coronavirus vaccines therefore enhancing our pandemic preparedness.","version":"1.1","doi":"10.1101/2021.04.10.439161","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.10.439279","pub_date":"2021-4-11","title":"Ultrastructural insight into SARS-CoV-2 attachment, entry and budding in human airway epithelium","abstract":"Ultrastructural studies of SARS-CoV-2 infected cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Many studies are limited by the lack of access to appropriate cellular models. As the airway epithelium is the primary site of infection it is essential to study SARS-CoV-2 infection of these cells. Here, we examined human airway epithelium, grown as highly differentiated air-liquid interface cultures and infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant (Variant of Concern 202012/01) by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Two key SARS-CoV-2 entry molecules, ACE2 and TMPRSS2, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistent with these observations, extracellular virions were frequently seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion at the apical plasma membrane demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein. Profiles strongly suggesting viral budding from the membrane was observed in these compartments and this may explain how virions gain their S glycoprotein containing envelope.","version":"1.1","doi":"10.1101/2021.04.10.439279","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.10.439288","pub_date":"2021-4-11","title":"ADAM17 inhibition prevents neutrophilia and lung injury in a mouse model of Covid-19","abstract":"Severe coronavirus disease 2019 (Covid-19) is characterized by lung injury, cytokine storm and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe Covid-19 in infected individuals. Angiotensin-converting enzyme-2 ACE-2) is the receptor for SARS-CoV-2, the virus causing Covid-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE-2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates Covid-19-related lung inflammation. We employed a pre-clinical mouse model using intra-tracheal instillation of a combination of polyinosinic:polycytidylic acid (poly-I:C) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with Covid-19. Histological analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill Covid-19 patients. Administration of the ADAM17 inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of pro-inflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17 inhibition. Thus, we propose inhibition of ADAM17 as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression towards severe Covid-19.","version":"1.1","doi":"10.1101/2021.04.10.439288","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.10.439275","pub_date":"2021-4-11","title":"CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity","abstract":"Safe, economical and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and halt the pandemic. We have constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains two immunodominant peptides screened from receptor-binding domain (RBD) and is fully chemically synthesized. And the vaccine has optimized nanoemulsion formulation, outstanding stability and safety. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of RBD-specific and protective neutralizing antibodies (NAbs), which were also effective to RBD mutations. CoVac501 was found to elicit the increase of memory T cells, antigen-specific CD8+ T cell responses and Th1-biased CD4+ T cell immune responses in NHPs. More importantly, the sera from the immunized NHPs can prevent infection of live SARS-CoV-2 in vitro. A novel SARS-CoV-2 vaccine we developed, CoVac501, which is a fully chemically synthesized and self-adjuvanting peptides conjugated with TLR7 agonists, can induce high-efficient humoral and cellular immune responses against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.04.10.439275","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.438911","pub_date":"2021-4-10","title":"Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2","abstract":"Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.","version":"1.1","doi":"10.1101/2021.04.08.438911","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439166","pub_date":"2021-4-10","title":"Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques","abstract":"Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 \u00b5g RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only \u223c2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. The emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.","version":"1.1","doi":"10.1101/2021.04.09.439166","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439154","pub_date":"2021-4-10","title":"Atypical N-glycosylation of SARS-CoV-2 impairs the efficient binding of Spike-RBM to the human-host receptor hACE2","abstract":"SARS-CoV-2 internalization by human host cells relies on the molecular binding of its spike glycoprotein (SGP) to the angiotensin-converting-enzyme-2 (hACE2) receptor. It remains unknown whether atypical N-glycosylation of SGP modulates SARS-CoV-2 tropism for infections. Here, we address this question through an extensive bioinformatics analysis of publicly available structural and genetic data. We identified two atypical sequons (sequences of N-glycosylation: NGV 481-483 and NGV 501-503), strategically located on the receptor-binding motif (RBM) of SGP and facing the hACE2 receptor. Interestingly, the cryo-electron microscopy structure of trimeric SGP in complex with potent-neutralizing antibodies from convalescent patients revealed covalently-linked N-glycans in NGV 481-483 atypical sequons. Furthermore, NGV 501-503 atypical sequon involves the asparagine-501 residue, whose highly-transmissible mutation N501Y is present in circulating variants of major concerns and affects the SGP-hACE2 binding-interface through the well-known hotspot-353. These findings suggest that atypical SGP post-translational modifications modulate the SGP-hACE2 binding-affinity affecting consequently SARS-CoV-2 transmission and pathogenesis.","version":"1.1","doi":"10.1101/2021.04.09.439154","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439203","pub_date":"2021-4-10","title":"A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike","abstract":"The S protein of the SARS-CoV-2 is a Type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the nuclear magnetic resonance (NMR) structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane \u03b1-helix (TMH) trimer that assembles spontaneously in membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeat. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.","version":"1.1","doi":"10.1101/2021.04.09.439203","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.21255089","pub_date":"2021-04-10","title":"Convalescent Plasma Use in the United States was inversely correlated with COVID-19 Mortality: Did Plasma Hesitancy cost lives?","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The US Food and Drug Administration authorized Convalescent Plasma (CCP) therapy for hospitalized COVID-19 patients via the Expanded Access Program (EAP) and the Emergency Use Authorization (EUA), leading to use in about 500,000 patients during the first year of the pandemic for the US.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We tracked the number of CCP units dispensed to hospitals by blood banking organizations and correlated that usage with hospital admission and mortality data.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>CCP usage per admission peaked in Fall 2020, with more than 40% of inpatients estimated to have received CCP between late September and early November 2020. However, after randomized controlled trials failed to show a reduction in mortality, CCP usage per admission declined steadily to a nadir of less than 10% in March 2021. We found a strong inverse correlation (r = \u22120.52, P = 0.002) between CCP usage per hospital admission and deaths occurring two weeks after admission, and this finding was robust to examination of deaths taking place one, two or three weeks after admission. Changes in the number of hospital admissions, SARS-CoV-2 variants, and age of patients could not explain these findings. The retreat from CCP usage might have resulted in as many as 29,000 excess deaths from mid-November 2020 to February 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>A strong inverse correlation between CCP use and mortality per admission in the USA provides population level evidence consistent with the notion that CCP reduces mortality in COVID-19 and suggests that the recent decline in usage could have resulted in excess deaths.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.04.07.21255089","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.08.439088","pub_date":"2021-4-10","title":"The homology analysis of ACE2 gene and its distinct expression in laboratory and wild animals","abstract":"Angiotensin-converting enzyme-2 (ACE2) has been recognized as an entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cells while bats has been suspected as natural host of SARS-CoV-2. However, the detail of intermediate host or the route of transmission of SARS-CoV-2 is still unclear. In this study, we analyze the conservation of ACE2 gene in 11 laboratory and wild animals that live in close proximity either with Bats or human and further investigated its RNA and protein expression pattern in wild bats, mice and tree shrew. We verified that the wild-bats and mice were belonged to Hipposideros pomona and Rattus norvegicus, respectively. ACE2 gene is highly conserved among all 11 animals species at the DNA level. Phylogenetic analysis based on the ACE2 nucleotide sequences revealed that wild bat and Tree shrew were forming a cluster close to human. We further report that ACE2 RNA expression pattern is highly species-specific in different tissues of different animals. Most notably, we found that the expression pattern of ACE2 RNA and protein are very different in each animal species. In summary, our results suggested that ACE2 gene is highly conserved among all 11 animals species. However, different relative expression pattern of ACE2 RNA and protein in each animal species is interesting. Further research is needed to clarify the possible connection between different relative expression pattern of ACE2 RNA and protein in different laboratory and wild animal species and the susceptibility to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.04.08.439088","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.438924","pub_date":"2021-4-09","title":"Genome-wide CRISPR activation screen identifies novel receptors for SARS-CoV-2 entry","abstract":"The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been endangering worldwide public health and economy. SARS-CoV-2 infects a variety of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative pathways for virus entry. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. In addition to known host proteins, i.e. ACE2, TMPRSS2 and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike\u2019s N-terminal domain (NTD). Their essential and physiological roles have all been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in a fashion independent of ACE2. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of the therapeutic countermeasures against COVID-19.","version":"1.2","doi":"10.1101/2021.04.08.438924","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438849","pub_date":"2021-4-09","title":"Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality","abstract":"The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.","version":"1.2","doi":"10.1101/2021.04.07.438849","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.09.439147","pub_date":"2021-4-09","title":"Nasal delivery of single-domain antibodies improves symptoms of SARS-CoV-2 infection in an animal model","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung via a nebulizer could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients. Vaccines for COVID-19 are now available but therapeutic drugs are still needed to treat life-threatening cases and those who cannot be vaccinated. We discovered a new heavy-chain single-domain antibody that can effectively neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. Unlike other drug candidates, which prevent the virus from attaching to the receptor on the host cell, this new antibody acts by blocking the virus membrane from fusing with the host cell membrane. We studied the behavior of the new antibody in vitro using VeroE6/TMPRSS2 cells and human lung organoids. When delivered through the nose to infected Syrian hamsters, we found that this antibody could prevent the typical symptoms caused by SARS-CoV-2. Our results are significant because delivering simple drugs directly to infected lungs using a nebulizer could increase the potency of the drugs while reducing the risk of immune reaction that could occur if the drugs escape or are delivered through the blood stream.","version":"1.1","doi":"10.1101/2021.04.09.439147","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433466","pub_date":"2021-4-09","title":"Increased Resistance of SARS-CoV-2 Variant P.1 to Antibody Neutralization","abstract":"The relative resistance of SARS-CoV-2 variants B.1.1.7 and B.1.351 to antibody neutralization has been described recently. We now report that another emergent variant from Brazil, P.1, is not only refractory to multiple neutralizing monoclonal antibodies, but also more resistant to neutralization by convalescent plasma (3.4 fold) and vaccinee sera (3.8-4.8 fold). The cryo-electron microscopy structure of a soluble prefusion-stabilized spike reveals the P.1 trimer to adopt exclusively a conformation in which one of the receptor-binding domains is in the \u201cup\u201d position, with the functional impact of mutations appearing to arise from local changes instead of global conformational alterations. The P.1 variant threatens current antibody therapies but less so the protective efficacy of our vaccines.","version":"1.2","doi":"10.1101/2021.03.01.433466","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.21254882","pub_date":"2021-04-09","title":"Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2 mRNA vaccinated individuals","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>The SARS-CoV-2 pandemic has been raging for over a year, creating global detrimental impact. The BNT162b2 mRNA vaccine has demonstrated high protection levels, yet apprehension exists that several variants of concerns (VOCs) can surmount the immune defenses generated by the vaccines. Neutralization assays have revealed some reduction in neutralization of VOCs B.1.1.7 and B.1.351, but the relevance of these assays in real life remains unclear. We performed a case-control study that examined the distribution of SARS-CoV-2 variants observed in infections of vaccinated individuals (\u201cbreakthrough cases\u201d) and matched infections of unvaccinated individuals. We hypothesized that if there is lower vaccine effectiveness against one of the VOCs, its proportion among the breakthrough cases should be higher than among unvaccinated cases. Our results show that vaccinees that tested positive at least a week after the second dose were indeed disproportionally infected with B.1.351, as compared with unvaccinated individuals (odds ratio of 8:1). Those who tested positive between two weeks after the first dose and one week after the second dose, were disproportionally infected by B.1.1.7 (odds ratio of 26:10), suggesting reduced vaccine effectiveness against both VOCs at particular time windows following vaccination. Nevertheless, the B.1.351 incidence in Israel to-date remains low and vaccine effectiveness remains high among those fully vaccinated. These results overall suggest that vaccine breakthrough infection may be more frequent with both VOCs, yet a combination of mass-vaccination with two doses coupled with non-pharmaceutical interventions control and contain their spread.</jats:p>","version":null,"doi":"10.1101/2021.04.06.21254882","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.31.437918","pub_date":"2021-4-09","title":"Structural dynamics of the \u03b2-coronavirus Mpro protease ligand binding sites","abstract":"\u03b2-coronaviruses alone have been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a back-up against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensible role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all non-redundant ligand-binding sites available for SARS-CoV2, SARS-CoV and MERS-CoV Mpro. Extensive adaptive sampling has been used to explore conformational dynamics employing convolutional variational auto encoder-based deep learning, and investigates structural conservation of the ligand binding sites using Markov state models across \u03b2-coronavirus homologs. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across \u03b2-coronavirus homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.","version":"1.2","doi":"10.1101/2021.03.31.437918","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438812","pub_date":"2021-4-08","title":"Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp14/nsp10 Exoribonuclease","abstract":"SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.","version":"1.1","doi":"10.1101/2021.04.07.438812","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438808","pub_date":"2021-4-08","title":"Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp13 Helicase","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterised pharmaceuticals for nsp13 inhibitors using a FRET-based high-throughput screening (HTS) approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.","version":"1.1","doi":"10.1101/2021.04.07.438808","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438807","pub_date":"2021-4-08","title":"Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase","abstract":"The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.","version":"1.1","doi":"10.1101/2021.04.07.438807","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438540","pub_date":"2021-4-08","title":"TMPRSS2 and RNA-dependent RNA polymerase are effective targets of therapeutic intervention for treatment of COVID-19 caused by SARS-CoV-2 variants (B.1.1.7 and B.1.351)","abstract":"SARS-CoV-2 is a causative agent of COVID-19 pandemic and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development and this effort was partially successful. Since the beginning of COVID-19 pandemic, the emergence of SARS-CoV-2 variants has been reported in many parts of the world and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. The viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development, thus the inhibitory effects of TMPRSS2 and RdRp inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the UK and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 and RdRp are equally effective against the two variants of concern.","version":"1.1","doi":"10.1101/2021.04.06.438540","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.305490","pub_date":"2021-4-08","title":"Critical Interactions Between the SARS-CoV-2 Spike Glycoprotein and the Human ACE2 Receptor","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects human cells upon binding of its spike (S) glycoproteins to ACE2 receptors and causes the coronavirus disease 2019 (COVID-19). Therapeutic approaches to prevent SARS-CoV-2 infection are mostly focused on blocking S-ACE2 binding, but critical residues that stabilize this interaction are not well understood. By performing all-atom molecular dynamics (MD) simulations, we identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonding between the receptor-binding domain (RBD) of the S protein and ACE2. Mutagenesis of these residues on the RBD was not sufficient to destabilize binding but reduced the average work to unbind the S protein from ACE2. In particular, the hydrophobic end of RBD serves as the main anchor site and unbinds last from ACE2 under force. We propose that blocking the hydrophobic surface of RBD via neutralizing antibodies could prove an effective strategy to inhibit S-ACE2 interactions.","version":"1.3","doi":"10.1101/2020.09.21.305490","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438843","pub_date":"2021-4-08","title":"SARS-CoV-2 infection in the Syrian hamster model causes inflammation as well as type I interferon dysregulation in both respiratory and non-respiratory tissues including the heart and kidney","abstract":"COVID-19 (coronavirus disease 2019) caused SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection is a disease affecting several organ systems. A model that captures all clinical symptoms of COVID-19 as well as long-haulers disease is needed. We investigated the host responses associated with infection in several major organ systems including the respiratory tract, the heart, and the kidneys after SARS-CoV-2 infection in Syrian hamsters. We found significant increases in inflammatory cytokines (IL-6, IL-1beta, and TNF) and type II interferons whereas type I interferons were inhibited. Examination of extrapulmonary tissue indicated inflammation in the kidney, liver, and heart which also lacked type I interferon upregulation. Histologically, the heart had evidence of mycarditis and microthrombi while the kidney had tubular inflammation. These results give insight into the multiorgan disease experienced by people with COVID-19 and possibly the prolonged disease in people with post-acute sequelae of SARS-CoV-2 (PASC).","version":"1.1","doi":"10.1101/2021.04.07.438843","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438811","pub_date":"2021-4-08","title":"Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp15 Endoribonuclease","abstract":"SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered the economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.","version":"1.1","doi":"10.1101/2021.04.07.438811","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438871","pub_date":"2021-4-08","title":"Machine Learning Identifies Ponatinib as a Potent Inhibitor of SARS-CoV2-induced Cytokine Storm","abstract":"Although 15-20% of COVID-19 patients experience hyper-inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N-terminal domain (NTD) of the spike protein from the SARS-CoV-2 and emerging variants B1.1.7 and B.1.351 substantially induces multiple inflammatory molecules in human monocytes and PBMCs. Further, we identified several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD-induced cytokine release. Additionally, we found that the FDA-approved, multi-kinase inhibitor Ponatinib is a potent inhibitor of the NTD-mediated cytokine storm. Taken together, we propose that agents targeting multiple kinases required for the SARS-CoV-2-mediated cytokine storm, such as Ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID-19.","version":"1.1","doi":"10.1101/2021.04.07.438871","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.439006","pub_date":"2021-4-08","title":"Intranasal HD-Ad Vaccine Protects the Upper and Lower Respiratory Tracts of hACE2 Mice against SARS-CoV-2","abstract":"The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.","version":"1.1","doi":"10.1101/2021.04.08.439006","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438810","pub_date":"2021-4-08","title":"Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase","abstract":"The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order to identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play a key role in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2\u2019-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.","version":"1.1","doi":"10.1101/2021.04.07.438810","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.438884","pub_date":"2021-4-08","title":"Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity","abstract":"A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.","version":"1.1","doi":"10.1101/2021.04.08.438884","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438866","pub_date":"2021-4-08","title":"Seasonal stability of SARS-CoV-2 in biological fluids","abstract":"Transmission of SARS-CoV-2 occurs by close contact with infected persons through droplets, the inhalation of infectious aerosols and the exposure to contaminated surface. Previously, we determined the virus stability on different types of surfaces under indoor and seasonal climatic conditions. SARS-CoV-2 survived the longest on surfaces under winter conditions, followed by spring/fall and summer conditions, suggesting the seasonal pattern of stability on surfaces. However, under natural conditions, the virus is secreted in various biological fluids from infected humans. In this respect, it remains unclear how long the virus survives in various types of biological fluids. This study explored the SARS-CoV-2 stability in human biological fluids under different environmental conditions and estimated the half-life. The virus was stable for up to 21 days in nasal mucus, sputum, saliva, tear, urine, blood, and semen; it remained infectious significantly longer under winter and spring/fall conditions than under summer conditions. In contrast, the virus was only stable up to 24 hours in feces and breast milk. These findings demonstrate the potential risk of infectious biological fluids in SARS-CoV-2 transmission and have implications for its seasonality.","version":"1.1","doi":"10.1101/2021.04.07.438866","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438806","pub_date":"2021-4-08","title":"Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease","abstract":"The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.","version":"1.1","doi":"10.1101/2021.04.07.438806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.240325","pub_date":"2021-4-08","title":"Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective","abstract":"The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. We revise Remdesivir\u2019s mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP","version":"1.2","doi":"10.1101/2020.08.06.240325","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438709","pub_date":"2021-4-08","title":"Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape","abstract":"An ideal anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse SARS-related coronaviruses, and be highly protective through viral neutralization and effector functions. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S309, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.","version":"1.1","doi":"10.1101/2021.04.06.438709","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.437687","pub_date":"2021-4-08","title":"Human pulmonary artery endothelial cells upregulate ACE2 expression in response to iron-regulatory elements: potential implications for SARS-CoV-2 infection of vascular endothelial cells","abstract":"Emerging studies from the ongoing covid-19 pandemic have implicated vascular dysfunction and endotheliitis in many patients presenting with severe disease. However, there is limited evidence for the expression of ACE2 (the principal co-receptor for Sars-Cov-2 cellular attachment) in vascular endothelial cells which has prompted the suggestion that the virus does not infect these cell types. However, the studies presented here demonstrate enhanced expression of ACE2 at the level of both mRNA and protein, in human pulmonary artery endothelial cells (PAECs) challenged with either IL-6 or hepcidin. Notably elevated levels both these iron-regulatory elements have been described in Covid-19 patients with severe disease and are further associated with morbidity and mortality. Additionally, levels of both IL-6 and hepcidin are often elevated in the elderly and in chronic disease settings, these populations being at greater risk of adverse outcomes from Sars-Cov-2 infection. A role for IL-6 and hepcidin as modulators of ACE2 expression seems plausible, additional, studies are required to determine if viral infection can be demonstrated in PAECs challenged with either of these iron-regulatory elements.","version":"1.1","doi":"10.1101/2021.04.08.437687","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438804","pub_date":"2021-4-08","title":"Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease","abstract":"The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.","version":"1.1","doi":"10.1101/2021.04.07.438804","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.438833","pub_date":"2021-4-08","title":"A Comparison of Persistence of SARS-CoV-2 Variants on Stainless Steel","abstract":"The survival of newer variants of SARS-CoV-2 on a representative surface has been compared to the established UK circulating isolate to determine whether enhanced environmental stability could play a part in their increased transmissibility. Stainless-steel coupons were inoculated with liquid cultures of the three variants, with coupons recovered over seven days and processed for recoverable viable virus using plaque assay. After drying, there was no significant difference in inactivation rates between variants. Indicating there is no increased environmental persistence from the new variants.","version":"1.1","doi":"10.1101/2021.04.08.438833","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438820","pub_date":"2021-4-08","title":"Comparable environmental stability and disinfection profiles of the currently circulating SARS-CoV-2 variants of concern B.1.1.7 and B.1.351","abstract":"The emergence of novel SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern with increased transmission dynamics has raised questions regarding stability and disinfection of these viruses. In this study, we analyzed surface stability and disinfection of the currently circulating SARS-CoV-2 variants B.1.1.7 and B.1.351 compared to the wildtype. Treatment with heat, soap and ethanol revealed similar inactivation profiles indicative of a comparable susceptibility towards disinfection. Furthermore, we observed comparable surface stability on steel, silver, copper and face masks. Overall, our data support the application of currently recommended hygiene concepts to minimize the risk of B.1.1.7 and B.1.351 transmission.","version":"1.1","doi":"10.1101/2021.04.07.438820","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.438904","pub_date":"2021-4-08","title":"The impact of viral mutations on recognition by SARS-CoV-2 specific T-cells","abstract":"We identify amino acid variants within dominant SARS-CoV-2 T-cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T-cells assessed by IFN-\u03b3 and cytotoxic killing assays. These data demonstrate the potential for T-cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T-cell as well as humoral immunity.","version":"1.1","doi":"10.1101/2021.04.08.438904","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.08.439071","pub_date":"2021-4-08","title":"A repurposed drug screen identifies compounds that inhibit the binding of the COVID-19 spike protein to ACE2","abstract":"Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding by <90%, measured the EC50 of binding inhibition, and computationally modeled the docking of the best inhibitors to both Spike and ACE2. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction as well as identifying several potential inhibitors that could serve as templates for future drug discovery efforts.","version":"1.1","doi":"10.1101/2021.04.08.439071","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.438818","pub_date":"2021-4-08","title":"Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody","abstract":"The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2\u00d7259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2\u00d7259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2\u00d7259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2\u00d7259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.","version":"1.1","doi":"10.1101/2021.04.07.438818","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.07.21255081","pub_date":"2021-04-07","title":"Effectiveness of CoronaVac among healthcare workers in the setting of high SARS-CoV-2 Gamma variant transmission in Manaus, Brazil: A test-negative case-control study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, Gamma, emerged in the city of Manaus in late 2020 during a large resurgence of coronavirus disease (COVID-19), and has spread throughout Brazil. The effectiveness of vaccines in settings with widespread Gamma variant transmission has not been reported.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We performed a matched test-negative case-control study to estimate the effectiveness of an inactivated vaccine, CoronaVac, in healthcare workers (HCWs) in Manaus, where the Gamma variant accounted for 86% of genotyped SARS-CoV-2 samples at the peak of its epidemic. We performed an early analysis of effectiveness following administration of at least one vaccine dose and an analysis of effectiveness of the two-dose schedule. The primary outcome was symptomatic SARS-CoV-2 infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>For the early at-least-one-dose and two-dose analyses the study population was, respectively, 53,176 and 53,153 HCWs residing in Manaus and aged 18 years or older, with complete information on age, residence, and vaccination status. Among 53,153 HCWs eligible for the two-dose analysis, 47,170 (89%) received at least one dose of CoronaVac and 2,656 individuals (5%) underwent RT-PCR testing from 19 January, 2021 to 13 April, 2021. Of 3,195 RT-PCR tests, 885 (28%) were positive. 393 and 418 case- control pairs were selected for the early and two-dose analyses, respectively, matched on calendar time, age, and neighbourhood. Among those who had received both vaccine doses before the RT-PCR sample collection date, the average time from second dose to sample collection date was 14 days (IQR 7-24). In the early analysis, vaccination with at least one dose was associated with a 0.50-fold reduction (adjusted vaccine effectiveness (VE), 49.6%, 95% CI 11.3 to 71.4) in the odds of symptomatic SARS-CoV-2 infection during the period 14 days or more after receiving the first dose. However, we estimated low effectiveness (adjusted VE 36.8%, 95% CI -54.9 to 74.2) of the two-dose schedule against symptomatic SARS-CoV-2 infection during the period 14 days or more after receiving the second dose. A finding that vaccinated individuals were much more likely to be infected than unvaccinated individuals in the period 0-13 days after first dose (aOR 2.11, 95% CI 1.36-3.27) suggests that unmeasured confounding led to downward bias in the vaccine effectiveness estimate.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Evidence from this test-negative study of the effectiveness of CoronaVac was mixed, and likely affected by bias in this setting. Administration of at least one vaccine dose showed effectiveness against symptomatic SARS-CoV-2 infection in the setting of epidemic Gamma variant transmission. However, the low estimated effectiveness of the two-dose schedule underscores the need to maintain non-pharmaceutical interventions while vaccination campaigns with CoronaVac are being implemented.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Funda\u00e7\u00e3o Oswaldo Cruz (Fiocruz); Municipal Health Secretary of Manaus</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in Context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>We searched PubMed for articles published from inception of the pandemic until April 3, 2021, with no language restrictions, using the search terms \u201cP.1\u201d AND \u201cvaccine\u201d AND \u201cSARS-CoV-2\u201d. Additionally, we searched for \u201cCoronaVac\u201d AND \u201cSARS-CoV-2\u201d. Early studies have found plasma from convalescent COVID-19 patients and sera from vaccinated individuals have reduced neutralisation of the SARS-CoV-2 variant, Gamma or P.1, compared with strains isolated earlier in the pandemic. Pfizer BNT162b2 mRNA, Oxford-AstraZeneca ChAdOx1, and CoronaVac are the only vaccines for which such data has been published to date.</jats:p>\n                    <jats:p>No studies reported effectiveness of any vaccine on reducing the risk of infection or disease among individuals exposed to P.1 or in settings of high P.1 transmission.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>This study finds that vaccination with CoronaVac was 49.4% (95% CI 13.2 to 71.9) effective at preventing COVID-19 in a setting with likely high prevalence of the Gamma Variant of Concern. However, an analysis of effectiveness by dose was underpowered and failed to find significant effectiveness of the two-dose schedule of CoronaVac (estimated VE 37.1%, 95% CI -53.3 to 74.2).</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>These findings are suggestive for the effectiveness of CoronaVac in healthcare workers in the setting of widespread P.1 transmission but must be strengthened by observational studies in other settings and populations. Based on this evidence, there is a need to implement sustained non-pharmaceutical interventions even as vaccination campaigns continue.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.04.07.21255081","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.25.437113","pub_date":"2021-4-07","title":"Evolutionary differences in the ACE2 reveals the molecular origins of COVID-19 susceptibility","abstract":"We explore the energetic frustration patterns associated with the binding between the SARS-CoV-2 spike protein and the ACE2 receptor protein in a broad selection of animals. Using energy landscape theory and the concept of energy frustration\u2014theoretical tools originally developed to study protein folding\u2014we are able to identify interactions among residues of the spike protein and ACE2 that result in COVID-19 resistance. This allows us to identify whether or not a particular animal is susceptible to COVID-19 from the protein sequence of ACE2 alone. Our analysis predicts a number of experimental observations regarding COVID-19 susceptibility, demonstrating that this feature can be explained, at least partially, on the basis of theoretical means.","version":"1.2","doi":"10.1101/2021.03.25.437113","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.21254767","pub_date":"2021-04-07","title":"Neutrophil-mediated Oxidative Stress and Albumin Structural Damage Predict COVID-19-associated Mortality","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 25 patients who were followed up for a median of 12.5 days (1-35 days), among them 14 had died. Analyzing blood samples from patients and healthy individuals (n=10), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance (EPR) spectra of spin labelled fatty acids (SLFA) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10-11). Non-survivors\u2019 HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Stratified at the means, Kaplan\u2013Meier survival analysis indicated that lower values of S/W ratio and accumulated H\n                  <jats:sub>2</jats:sub>\n                  O\n                  <jats:sub>2</jats:sub>\n                  in plasma significantly predicted in-hospital mortality (S/W&lt;0.16, 80% (9/12) vs. S/W&gt;0.16, 20% (2/10),\n                  <jats:italic>p</jats:italic>\n                  =0.008; plasma [H\n                  <jats:sub>2</jats:sub>\n                  O\n                  <jats:sub>2</jats:sub>\n                  ]&gt;7.1 \u03bcM, 83.3% (5/6) vs. 16.7% (1/6),\n                  <jats:italic>p</jats:italic>\n                  =0.049). When we combined these two parameters as the ratio ((S/W)/[H\n                  <jats:sub>2</jats:sub>\n                  O\n                  <jats:sub>2</jats:sub>\n                  ]) to derive a risk score, the resultant risk score lower than the mean (&lt; 0.0253) predicted mortality with 100% accuracy (100% (6/6) vs. 0% (0/6), logrank \u03c7\n                  <jats:sup>2</jats:sup>\n                  = 12.01, p = 5\u00d710\n                  <jats:sup>\u22124</jats:sup>\n                  ). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements.\n                </jats:p>","version":null,"doi":"10.1101/2021.04.01.21254767","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.05.437453","pub_date":"2021-4-06","title":"Ageing impairs the airway epithelium defence response to SARS-CoV-2","abstract":"Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the \u201ccytokine storm\u201d seen in severe COVID-19. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.","version":"1.1","doi":"10.1101/2021.04.05.437453","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.10.942748","pub_date":"2021-4-06","title":"Recombination and lineage-specific mutations linked to the emergence of SARS-CoV-2","abstract":"The emergence of SARS-CoV-2 underscores the need to better understand the evolutionary processes that drive the emergence and adaptation of zoonotic viruses in humans. In the betacoronavirus genus, which also includes SARS-CoV and MERS-CoV, recombination frequently encompasses the Receptor Binding Domain (RBD) of the Spike protein, which, in turn, is responsible for viral binding to host cell receptors. Here, we find evidence of a recombination event in the RBD involving ancestral linages to both SARS-CoV and SARS-CoV-2. Although we cannot specify the recombinant nor the parental strains, likely due to the ancestry of the event and potential undersampling, our statistical analyses in the space of phylogenetic trees support such an ancestral recombination. Consequently, SARS-CoV and SARS-CoV-2 share an RBD sequence that includes two insertions (positions 432-436 and 460-472), as well as the variants 427N and 436Y. Both 427N and 436Y belong to a helix that interacts directly with the human ACE2 (hACE2) receptor. Reconstruction of ancestral states, combined with protein-binding affinity analyses using the physics-based trRosetta algorithm, reveal that the recombination event involving ancestral strains of SARS-CoV and SARS-CoV-2 led to an increased affinity for hACE2 binding, and that alleles 427N and 436Y significantly enhanced affinity as well. Structural modeling indicates that ancestors of SARS-CoV-2 may have acquired the ability to infect humans decades ago. The binding affinity with the human receptor was subsequently boosted in SARS-CoV and SARS-CoV-2 through further mutations in RBD. In sum, we report an ancestral recombination event affecting the RBD of both SARS-CoV and SARS-CoV-2 that was associated with an increased binding affinity to hACE2. This paper addresses critical questions about the origin of the SARS-CoV-2 virus: what are the evolutionary mechanisms that led to the emergence of the virus, and how can we leverage such knowledge to assess the potential of SARS-like viruses to become pandemic strains? In this work, we demonstrate common mechanisms involved in the emergence of human-infecting SARS-like viruses: first, by acquiring a common haplotype in the RBD through recombination, and further, through increased specificity to the human ACE2 receptor through lineage specific mutations. We also show that the ancestors of SARS-CoV-2 already had the potential to infect humans at least a decade ago, suggesting that SARS-like viruses currently circulating in wild animal species constitute a source of potential pandemic re-emergence.","version":"1.3","doi":"10.1101/2020.02.10.942748","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438579","pub_date":"2021-4-06","title":"Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity","abstract":"SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA \u2248 EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5\u2019 position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein. The novel coronavirus SARS-CoV-2, the causative agent of the world-wide pandemic of COVID-19, has become one of the greatest threats to human health. While rapid progress has been made in the development of vaccines, drug discovery has lagged, partly due to the lack of atomic-resolution structures of the free and drug-bound forms of the viral proteins. The SARS-CoV-2 envelope (E) protein, with its multiple activities that contribute to viral replication, is widely regarded as a potential target for COVID-19 treatment. As structural information is essential for drug discovery, we established an efficient sample preparation system for biochemical and structural studies of intact full-length SARS-CoV-2 E protein and characterized its structure and dynamics. We also characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of the amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein.","version":"1.1","doi":"10.1101/2021.04.06.438579","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438547","pub_date":"2021-4-06","title":"Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner","abstract":"After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro. Crossneutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood and very little is known about the potential protective effect of non-neutralizing antibodies in vivo. Using a panel of mouse monoclonal antibodies, we investigate both of these aspects.","version":"1.1","doi":"10.1101/2021.04.05.438547","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438479","pub_date":"2021-4-06","title":"SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques","abstract":"Early life SARS-CoV-2 vaccination has the potential to provide lifelong protection and achieve herd immunity. To evaluate SARS-CoV-2 infant vaccination, we immunized two groups of 8 infant rhesus macaques (RMs) at weeks 0 and 4 with stabilized prefusion SARS-CoV-2 S-2P spike (S) protein, either encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or mixed with 3M-052-SE, a TLR7/8 agonist in a squalene emulsion (Protein+3M-052-SE). Neither vaccine induced adverse effects. High magnitude S-binding IgG and neutralizing infectious dose 50 (ID50) >103 were elicited by both vaccines. S-specific T cell responses were dominated by IL-17, IFN-\u03b3, or TNF-\u03b1. Antibody and cellular responses were stable through week 22. The S-2P mRNA-LNP and Protein-3M-052-SE vaccines are promising pediatric SARS-CoV-2 vaccine candidates to achieve durable protective immunity. SARS-CoV-2 vaccines are well-tolerated and highly immunogenic in infant rhesus macaques","version":"1.1","doi":"10.1101/2021.04.05.438479","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438328","pub_date":"2021-4-06","title":"On the many advantages of using the VariantExperiment class to store, exchange and analyze SARS-CoV-2 genomic data and associated metadata","abstract":"On Friday, 19 March 2021, WHO organized a virtual global workshop highlighting the need for a globally coordinated plan to increase SARS-CoV-2 genetic sequencing capacities to detect SARS-CoV-2 mutations and variants, and to monitor virus genomic evolution worldwide. One week later, in another virtual meeting, it focused on sero epidemiology for SARS-CoV-2 variants of concern and variants of interest. Efficient monitoring of the virus relies on the storage, handling and sharing of the genomic data and the associated metadata. In this manuscript, we demonstrate how the Bioconductor VariantExperiment class addresses these needs, offering a robust and efficient solution to the requirements laid out by the WHO.","version":"1.1","doi":"10.1101/2021.04.05.438328","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438465","pub_date":"2021-4-06","title":"The Up state of the SARS-COV-2 Spike homotrimer favors an increased virulence for new variants","abstract":"The COVID-19 pandemic has spread widely worldwide. However, as soon as the vaccines were released \u2013 the only scientifically verified and efficient therapeutic option thus far \u2013 a few mutations combined into variants of SARS-CoV-2 that are more transmissible and virulent emerged raising doubts about their efficiency. Therefore, this work aims to explain possible molecular mechanisms responsible for the increased transmissibility and the increased rate of hospitalizations related to the new variants. A combination of theoretical methods was employed. Constant-pH Monte Carlo simulations were carried out to quantify the stability of several spike trimeric structures at different conformational states and the free energy of interactions between the receptor binding domain (RBD) and Angiotensin Converting Enzyme 2 (ACE2) for the most worrying variants. Electrostatic epitopes were mapped using the PROCEEDpKa method. These analyses showed that the increased virulence is more likely to be due to the improved stability to the S trimer in the opened state (the one in which the virus can interact with the cellular receptor ACE2) than due to alterations in the complexation RBD-ACE2, once the increased observed in the free energy values is small. Conversely, the South African variant (B.1.351), when compared with the wild type SARS-CoV-2, is much more stable in the opened state (either with one or two RBDs in the up position) than in the closed state (with the three RBDs in the down position). Such results contribute to the understanding of the natural history of disease and also to indicate possible strategies to both develop new therapeutic molecules and to adjust the vaccine doses for a higher production of B cells antibodies.","version":"1.1","doi":"10.1101/2021.04.05.438465","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438552","pub_date":"2021-4-06","title":"The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signalling","abstract":"SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.","version":"1.1","doi":"10.1101/2021.04.06.438552","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438584","pub_date":"2021-4-06","title":"Mutations in the B.1.1.7 SARS-CoV-2 spike protein reduce receptor-binding affinity and induce a flexible link to the fusion peptide","abstract":"The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. A number of single amino acid exchanges and deletions within the trimeric viral spike protein characterize this new SARS-CoV-2 variant. Crucial for viral entry into the host cell is the interaction of the spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) as well as integration of the viral fusion peptide into the host membrane. Respective amino acid exchanges within the SARS-CoV-2 variant B.1.1.7 affect inter-monomeric contact sites within the spike protein (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the viral spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability, the fusion peptide, and ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. In addition to reduced flexibility in the N-terminal domain of the spike protein, we found increased flexibility in direct spatial proximity of the fusion peptide. This increase in flexibility is due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7 found in pre- and post-cleavage state at the S2\u2019 site. Our results also imply a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface, which, in combination, might explain the higher infectivity of B.1.1.7. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7, but also other highly contagious SARS-CoV-2 variants, as many of them likewise exhibit a combination of the D614G and N501Y mutation.","version":"1.1","doi":"10.1101/2021.04.06.438584","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438352","pub_date":"2021-4-06","title":"A new SARS-CoV-2 lineage that shares mutations with known Variants of Concern is rejected by automated sequence repository quality control","abstract":"We report a SARS-CoV-2 lineage that shares N501Y, P681H, and other mutations with known variants of concern, such as B.1.1.7. This lineage, which we refer to as B.1.x (COG-UK sometimes references similar samples as B.1.324.1), is present in at least 20 states across the USA and in at least six countries. However, a large deletion causes the sequence to be automatically rejected from repositories, suggesting that the frequency of this new lineage is underestimated using public data. Recent dynamics based on 339 samples obtained in Santa Cruz County, CA, USA suggest that B.1.x may be increasing in frequency at a rate similar to that of B.1.1.7 in Southern California. At present the functional differences between this variant B.1.x and other circulating SARS-CoV-2 variants are unknown, and further studies on secondary attack rates, viral loads, immune evasion and/or disease severity are needed to determine if it poses a public health concern. Nonetheless, given what is known from well-studied circulating variants of concern, it seems unlikely that the lineage could pose larger concerns for human health than many already globally distributed lineages. Our work highlights a need for rapid turnaround time from sequence generation to submission and improved sequence quality control that removes submission bias. We identify promising paths toward this goal.","version":"1.1","doi":"10.1101/2021.04.05.438352","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.437914","pub_date":"2021-4-06","title":"Uneven growth of SARS-CoV-2 clones evidenced by more than 500,000 whole-genome sequences","abstract":"We have computed the frequencies of the alleles of the \u201cUK variant\u201d (B.1.1.7) and \u201cSouth Africa variant\u201d (B.1.351) of SARS-CoV-2 from the large GISAID repository. We find that the frequencies of the mutations in UK variant overall rose towards the end of 2020, as widely reported in the literature and in the general press. However, we also find that these frequencies vary in different patterns rather than in concert. For South Africa variant we find a more complex scenario with frequencies of some mutations rising and some remaining close to zero. Our results point to that what is generally reported as one variant is in fact a collection of variants with different genetic characteristics.","version":"1.1","doi":"10.1101/2021.04.06.437914","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438634","pub_date":"2021-4-06","title":"Plasma microbiome in COVID-19 subjects: an indicator of gut barrier defects and dysbiosis","abstract":"The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) symptoms. We asked whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n=30) and healthy control (n=16) were collected during hospitalization. Plasma microbiome was analyzed using 16S rRNA sequencing, metatranscriptomic analysis, and gut permeability markers including FABP-2, PGN and LPS in both patient cohorts. Almost 65% (9 out 14) COVID-19 patients showed abnormal presence of gut microbes in their bloodstream. Plasma samples contained predominately Proteobacteria, Firmicutes, and Actinobacteria. The abundance of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than the gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 subjects. The levels of plasma gut permeability markers FABP2 (1282\u00b1199.6 vs 838.1\u00b191.33; p=0.0757), PGN (34.64\u00b13.178 vs 17.53\u00b12.12; p<0.0001), and LPS (405.5\u00b148.37 vs 249.6\u00b117.06; p=0.0049) were higher in COVID-19 patients compared to healthy subjects. These findings support that the intestine may represent a source for bacteremia and may contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.","version":"1.1","doi":"10.1101/2021.04.06.438634","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438500","pub_date":"2021-4-06","title":"An Autoantigen-ome from HS-Sultan B-Lymphoblasts Offers a Molecular Map for Investigating Autoimmune Sequelae of COVID-19","abstract":"To understand how COVID-19 may induce autoimmune diseases, we have been compiling an atlas of COVID-autoantigens (autoAgs). Using dermatan sulfate (DS) affinity enrichment of autoantigenic proteins extracted from HS-Sultan lymphoblasts, we identified 362 DS-affinity proteins, of which at least 201 (56%) are confirmed autoAgs. Comparison with available multi-omic COVID data shows that 315 (87%) of the 362 proteins are affected in SARS-CoV-2 infection via altered expression, interaction with viral components, or modification by phosphorylation or ubiquitination, at least 186 (59%) of which are known autoAgs. These proteins are associated with gene expression, mRNA processing, mRNA splicing, translation, protein folding, vesicles, and chromosome organization. Numerous nuclear autoAgs were identified, including both classical ANAs and ENAs of systemic autoimmune diseases and unique autoAgs involved in the DNA replication fork, mitotic cell cycle, or telomerase maintenance. We also identified many uncommon autoAgs involved in nucleic acid and peptide biosynthesis and nucleocytoplasmic transport, such as aminoacyl-tRNA synthetases. In addition, this study found autoAgs that potentially interact with multiple SARS-CoV-2 Nsp and Orf components, including CCT/TriC chaperonin, insulin degrading enzyme, platelet-activating factor acetylhydrolase, and the ezrin-moesin-radixin family. Furthermore, B-cell-specific IgM-associated ER complex (including MBZ1, BiP, heat shock proteins, and protein disulfide-isomerases) is enriched by DS-affinity and up-regulated in B-cells of COVID-19 patients, and a similar IgH-associated ER complex was also identified in autoreactive pre-B1 cells in our previous study, which suggests a role of autoreactive B1 cells in COVID-19 that merits further investigation. In summary, this study demonstrates that virally infected cells are characterized by alterations of proteins with propensity to become autoAgs, thereby providing a possible explanation for infection-induced autoimmunity. The COVID autoantigen-ome provides a valuable molecular resource and map for investigation of COVID-related autoimmune sequelae and considerations for vaccine design.","version":"1.1","doi":"10.1101/2021.04.05.438500","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.06.438614","pub_date":"2021-4-06","title":"Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure","abstract":"The trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.","version":"1.1","doi":"10.1101/2021.04.06.438614","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.05.438524","pub_date":"2021-4-06","title":"Antibody response to SARS-CoV-2 mRNA vaccines in pregnant women and their neonates","abstract":"Pregnant women were excluded from initial clinical trials for COVID-19 vaccines, thus the immunologic response to vaccination in pregnancy and the transplacental transfer of maternal antibodies are just beginning to be studied.","version":"1.1","doi":"10.1101/2021.04.05.438524","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.04.438420","pub_date":"2021-4-05","title":"Rational Selection of PCR Primer/Probe Design Sites for SARS-CoV-2","abstract":"Various reports of decreased analytical sensitivities of real-time PCR-based detection of Coronavirus Disease 2019 (COVID-19) have been associated with occurrence of mutations in the target area of primer/probe binding. Knowledge about propensities of different genes to undergo mutation can inform researchers to select optimal genes to target for the qPCR design. We analyzed supplementary data from over 45 thousand SARS-CoV-2 genomes provided by Mercatelli et al to calculate the unique and prevalent mutations in different genes of SARS-CoV-2. We found that non-structural proteins in the ORF1ab region were more conserved compared to structural genes. Further factors which need to be relied upon for proper selection of genes for qPCR design are discussed.","version":"1.1","doi":"10.1101/2021.04.04.438420","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438288","pub_date":"2021-4-05","title":"An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity","abstract":"During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic.","version":"1.1","doi":"10.1101/2021.04.02.438288","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.296715","pub_date":"2021-4-05","title":"Single-component, self-assembling, protein nanoparticles presenting the receptor binding domain and stabilized spike as SARS-CoV-2 vaccine candidates","abstract":"Vaccination against SARS-CoV-2 provides an effective tool to combat the COIVD-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2G\u0394HR2), and displayed S2G\u0394HR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2G\u0394HR2 elicited two-fold-higher NAb titers than S2P, while S2G\u0394HR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2G\u0394HR2-presenting I3-01v9 SApNP also induced critically needed T-cell immunity, thereby providing a promising vaccine candidate. The SARS-CoV-2 receptor binding domain and S2G\u0394HR2 spike elicited potent immune responses when displayed on protein nanoparticles as vaccine candidates.","version":"1.2","doi":"10.1101/2020.09.14.296715","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438292","pub_date":"2021-4-04","title":"Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model","abstract":"In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105 TCID50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFN\u03b2 and TNF\u03b1, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population. Men experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection. Following SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.","version":"1.1","doi":"10.1101/2021.04.02.438292","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.03.438258","pub_date":"2021-4-04","title":"Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies","abstract":"Investigating the mechanisms of SARS-CoV-2 cellular infection is key to better understand COVID-19 immunity and pathogenesis. Infection, which involves both cell attachment and membrane fusion, relies on the ACE2 receptor that is paradoxically found at low levels in the respiratory tract, suggesting that additional mechanisms facilitating infection may exist. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding Ig-like lectin 1 (SIGLEC1) function as auxiliary receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the N-terminal domain (NTD) or to the conserved proteoglycan site at the base of the Receptor Binding Domain (RBD), while poorly neutralizing infection of ACE2 over-expressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the Receptor Binding Motif (RBM), while potently neutralizing infection of ACE2 over-expressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.","version":"1.1","doi":"10.1101/2021.04.03.438258","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438262","pub_date":"2021-4-04","title":"Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques","abstract":"Severe COVID-19 has been associated with T cell lymphopenia , but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.","version":"1.1","doi":"10.1101/2021.04.02.438262","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438274","pub_date":"2021-4-04","title":"Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN","abstract":"High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3\u2032-to-5\u2032 exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-\u00c5 resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3\u2032 end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.","version":"1.1","doi":"10.1101/2021.04.02.438274","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.21254660","pub_date":"2021-04-02","title":"Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 \u2018variants of concern\u2019 (VOC) across diverse geographic locales suggests herd immunity may fail to eliminate the virus. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC\n                  <jats:sub>90</jats:sub>\n                  ) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.\n                </jats:p>","version":null,"doi":"10.1101/2021.03.31.21254660","journal":"medRxiv","score":null},{"id":"10.1101/2021.04.02.438204","pub_date":"2021-4-02","title":"Discovery and in-vitro evaluation of potent SARS-CoV-2 entry inhibitors","abstract":"SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked \u223c8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their in vitro efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC50 in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.","version":"1.1","doi":"10.1101/2021.04.02.438204","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438155","pub_date":"2021-4-02","title":"An immunoinformatics approach to study the epitopes contributed by Nsp13 of SARS-CoV-2","abstract":"The on-going coronavirus disease-19 (COVID-19) pandemic caused by SARS-CoV-2 has infected hundreds of millions of people and killed more than two million people worldwide. Currently, there are no effective drugs available for treating SARS-CoV-2 infections; however, vaccines are now being administered worldwide to control this virus. In this study, we have studied SARS-CoV-2 helicase, Nsp13, which is critical for viral replication. We compared the Nsp13 sequences reported from India with the first reported sequence from Wuhan province, China to identify and characterize the mutations occurring in this protein. To correlate the functional impact of these mutations, we characterised the most prominent B cell and T cell epitopes contributed by Nsp13. Our data revealed twenty-one epitopes, which exhibited high antigenicity, stability and interactions with MHC class-I and class-II molecules. Subsequently, the physiochemical properties of these epitopes were also analysed. Furthermore, several of these Nsp13 epitopes harbour mutations, which were further characterised by secondary structure and per-residue disorderness, stability and dynamicity predictions. Altogether, we report the candidate epitopes of Nsp13 that may help the scientific community to understand the evolution of SARS-CoV-2 variants and their probable implications.","version":"1.1","doi":"10.1101/2021.04.02.438155","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.282780","pub_date":"2021-4-02","title":"SARS-CoV-2 infection paralyzes cytotoxic and metabolic functions of the immune cells","abstract":"The SARS-CoV-2 virus is the causative agent of the global COVID-19 infectious disease outbreak, which can lead to acute respiratory distress syndrome (ARDS). However, it is still unclear how the virus interferes with immune cell and metabolic functions in the human body. In this study, we investigated the immune response in acute or convalescent COVID19 patients. We characterized the peripheral blood mononuclear cells (PBMCs) using flow cytometry and found that CD8+ T cells were significantly subsided in moderate COVID-19 and convalescent patients. Furthermore, characterization of CD8+ T cells suggested that patients with a mild and moderate course of the COVID-19 disease and convalescent patients have significantly diminished expression of both perforin and granzyme A in CD8+ T cells. Using 1H-NMR spectroscopy, we characterized the metabolic status of their autologous PBMCs. We found that fructose, lactate and taurine levels were elevated in infected (mild and moderate) patients compared with control and convalescent patients. Glucose, glutamate, formate and acetate levels were attenuated in COVID-19 (mild and moderate) patients. In summary, our report suggests that SARS-CoV-2 infection leads to disrupted CD8+ T cytotoxic functions and changes the overall metabolic functions of immune cells.","version":"1.2","doi":"10.1101/2020.09.04.282780","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438063","pub_date":"2021-4-02","title":"COVIDOUTCOME \u2013 Estimating COVID Severity Based on Mutation Signatures in the SARS-CoV-2 Genome","abstract":"Numerous studies demonstrate frequent mutations in the genome of SARS-CoV-2. Our goal was to statistically link mutations to severe disease outcome. We used an automated machine learning approach where 1,594 viral genomes with available clinical follow-up data were used as the training set (797 \u201csevere\u201d and 797 \u201cmild\u201d). The best algorithm, based on random forest classification combined with the LASSO feature selection algorithm was employed to the training set to link mutation signatures and outcome. The performance of the final model was estimated by repeated, stratified, 10-fold cross validation (CV), then adjusted for multiple testing with Bootstrap Bias Corrected CV. We identified 26 protein and UTR mutations significantly linked to severe outcome. The best classification algorithm uses a mutation signature of 22 mutations as well as the patient\u2019s age as the input and shows high classification efficiency with an AUC of 0.94 (CI: [0.912, 0.962]) and a prediction accuracy of 87% (CI: [0.830, 0.903]). Finally, we established an online platform (https://covidoutcome.com/) which is capable to use a viral sequence and the patient\u2019s age as the input and provides a percentage estimation of disease severity. We demonstrate a statistical association between mutation signatures of SARS-CoV-2 and severe outcome of COVID-19. The established analysis platform enables a real-time analysis of new viral genomes. A statistical link between SARS-Cov-2 mutation status and severe COVID outcome was established using automated machine learning techniques based on random forest and logistic regression combined with feature selection algorithms. A mutation signature based on 3,779 protein coding and 36 UTR mutations capable to identify severe outcome cases was established. The trained model showed high classification performance (AUC=0.94 (CI: [0.912, 0.962]), accuracy=0.87 (CI: [0.830, 0.903])). A registration-free web-server for automated classification of new samples was set up and is accessible at http://www.covidoutcome.com. The established pipeline provides a quick assessment of future patients warranting a prospective clinical validation.","version":"1.1","doi":"10.1101/2021.04.01.438063","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438218","pub_date":"2021-4-02","title":"One dose of COVID-19 nanoparticle vaccine REVC-128 provides protection against SARS-CoV-2 challenge at two weeks post immunization","abstract":"A COVID-19 vaccine with capability to induce early protection is needed to efficiently eliminate viral spread. Here, we demonstrate the development of a nanoparticle vaccine candidate, REVC-128, in which multiple trimeric spike ectodomain subunits with glycine (G) at position 614 were multimerized onto a nanoparticle. In-vitro characterization of this vaccine confirms its structural and antigenic integrity. In-vivo immunogenicity evaluation in mice indicates that a single dose of this vaccine induces potent serum neutralizing antibody titer at two weeks post immunization, which is significantly higher than titer induced by trimeric spike protein without nanoparticle presentation. The comparison of serum binding to spike subunits between animals immunized by spike with and without nanoparticle presentation indicates that nanoparticle prefers the display of spike RBD (Receptor-Binding Domain) over S2 subunit, likely resulting in a more neutralizing but less cross-reactive antibody response. Moreover, a Syrian golden hamster in-vivo model for SARS-CoV-2 virus challenge was implemented at two weeks post a single dose of REVC-128 immunization. The results show that vaccination protects hamsters against SARS-CoV-2 virus challenge with evidence of steady body weight, suppressed viral loads and alleviation of tissue damage (lung and nares) for protected animals, compared with ~10% weight loss, higher viral loads and tissue damage in unprotected animals. Furthermore, the data show that vaccine REVC-128 is thermostable at up to 37\u00b0C for at least 4 weeks. These findings, along with a long history of safety for protein vaccines, suggest that the REVC-128 is a safe, stable and efficacious single-shot vaccine candidate to induce the earliest protection against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.04.02.438218","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.27.437323","pub_date":"2021-4-02","title":"High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor","abstract":"Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 \u00b5M [95% CI 2.2, 4.0]. Further, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple \u00b5s-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design. The ongoing novel coronavirus pandemic (COVID-19) has prompted a global race towards finding effective therapeutics that can target the various viral proteins. Despite many virtual screening campaigns in development, the discovery of validated inhibitors for SARS-CoV-2 protein targets has been limited. We discover a novel inhibitor against the SARS-CoV-2 main protease. Our integrated platform applies downstream biochemical assays, X-ray crystallography, and atomistic simulations to obtain a comprehensive characterization of its inhibitory mechanism. Inhibiting Mpro can lead to significant biomedical advances in targeting SARS-CoV-2 treatment, as it plays a crucial role in viral replication.","version":"1.2","doi":"10.1101/2021.03.27.437323","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438182","pub_date":"2021-4-02","title":"A realistic touch-transfer method reveals low risk of transmission for SARS-CoV-2 by contaminated coins and bank notes","abstract":"The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as a potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus (BCoV), as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to skin. Although we observed prolonged virus stability, our results, including a novel touch transfer method, indicate that the transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.","version":"1.1","doi":"10.1101/2021.04.02.438182","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433597","pub_date":"2021-4-02","title":"Maturation signatures of conventional dendritic cell subtypes in COVID-19 reflect direct viral sensing","abstract":"Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19 and this negatively affects T cell activation. The presence of functional effector T cells in mild patients and dysfunctional T cells in severely ill patients suggests that adequate T cell responses are needed to limit disease severity. Therefore, understanding how cDCs cope with SARS-CoV-2 infections can help elucidate the mechanism of generation of protective immune responses. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures with the up-regulation of interferon-stimulated genes and IL-6 signaling pathways. The main difference observed between DC2s and DC3s is the up-regulation of anti-apoptotic genes in DC3s, which explains their accumulation during infection. Furthermore, comparing cDCs between severe and mild patients, we find in the former a profound down-regulation of genes encoding molecules involved in antigen presentation, such as major histocompatibility complex class II (MHCII) molecules, \u03b22 microglobulin, TAP and costimulatory proteins, while an opposite trend is observed for proinflammatory molecules, such as complement and coagulation factors. Therefore, as the severity of the disease increases, cDC2s enhance their inflammatory properties and lose their main function, which is the antigen presentation capacity. In vitro, direct exposure of cDC2s to the virus recapitulates the type of activation observed in vivo. Our findings provide evidence that SARS-CoV-2 can interact directly with cDC2s and, by inducing the down-regulation of crucial molecules required for T cell activation, implements an efficient immune escape mechanism that correlates with disease severity.","version":"1.2","doi":"10.1101/2021.03.03.433597","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.438186","pub_date":"2021-4-02","title":"SARS-CoV-2 B.1.1.7 infection of Syrian hamster does not cause more severe disease and is protected by naturally acquired immunity","abstract":"Epidemiological studies have revealed the emergence of multiple SARS-CoV-2 variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality (1). To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and re-infection studies in na\u00efve and convalescent Syrian hamsters (>10 months old). Hamsters infected by either a B.1.1.7 variant or a B.1 (G614) variant exhibited comparable viral loads and pathology. Convalescent hamsters that were previously infected by the original D614 variant were protected from disease following B.1.1.7 challenge with no observable clinical signs or lung pathology. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in hamsters and that natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant.","version":"1.1","doi":"10.1101/2021.04.02.438186","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430519","pub_date":"2021-4-02","title":"Transformations, Lineage Comparisons, and Analysis of Down to Up Protomer States of Variants of the SARS-CoV-2 Prefusion Spike Protein Including the UK Variant B.1.1.7","abstract":"Monitoring and strategic response to variants in SARS-CoV-2 represents a considerable challenge in the current pandemic, as well as potentially future viral outbreaks of similar magnitude. In particular mutations and deletions involving the virion\u2019s prefusion Spike protein have significant potential impact on vaccines and therapeutics that utilize this key structural viral protein in their mitigation strategies. In this study, we have demonstrated how dominant energetic landscape mappings (\u201cglue points\u201d) coupled with sequence alignment information can potentially identify or flag key residue mutations and deletions associated with variants. Surprisingly, we also found excellent homology of stabilizing residue glue points across the lineage of \u03b2 coronavirus Spike proteins, and we have termed this as \u201csequence homologous glue points\u201d. In general, these flagged residue mutations and/or deletions are then computationally studied in detail using all-atom biocomputational molecular dynamics over approximately one microsecond in order to ascertain structural and energetic changes in the Spike protein associated variants. Specifically, we examined both a theoretically-based triple mutant and the so-called UK or B.1.1.7 variant. For the theoretical triple mutant, we demonstrated through Alanine mutations, which help \u201cunglue\u201d key residue-residue interactions, that these three key stabilizing residues could cause the transition of Down to Up protomer states, where the Up protomer state allows binding of the prefusion Spike protein to hACE2 host cell receptors, whereas the Down state is believed inaccessible. Thus, we are able to demonstrate the importance of glue point residue identification in the overall stability of the prefusion Spike protein. For the B.1.1.7 variant, we demonstrated the critical importance of D614G and N5017 on the structure and binding, respectively, of the Spike protein. Notably, we had previously identified D614 as a key glue point in the inter-protomer stabilization of the Spike protein prior to the emergence of its mutation. The mutant D614G is a structure breaking Glycine mutation demonstrating a relatively more distal Down state RBD and a more stable conformation in general. In addition, we demonstrate that the mutation N501Y may significantly increase the Spike protein binding to hACE2 cell receptors through its interaction with Y41 of hACE2 forming a potentially strong hydrophobic residue binding pair. We note that these two key mutations, D614G and N501Y, are also found in the so-called South African (SA; B.1.351) variant of SARS-CoV-2. Future studies along these lines are, therefore, aimed at mapping glue points to residue mutations and deletions of associated prefusion Spike protein variants in order to help identify and analyze possible \u201cvariants of interest\u201d and optimize efforts aimed at the mitigation of this current and future virions.","version":"1.2","doi":"10.1101/2021.02.09.430519","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.02.437736","pub_date":"2021-4-02","title":"A high-throughput fluorescence polarization assay to discover inhibitors of arenavirus and coronavirus exoribonucleases","abstract":"Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses. Arenaviridae and Coronaviridae viral families share an exoribonuclease activity of common evolutionary origin Arenaviridae and Coronaviridae exoribonuclease is an attractive target for drug development We present a high-throughput assay in 384 well-plates for the screening of inhibitors using fluorescence polarization We validated the assay by screening of a focused library of 23 metal chelators against SARS-CoV-2, Lymphocytic Choriomeningitis virus and Machupo virus exoribonucleases We determined the IC50 by fluorescence polarization of one inhibitor common to the three viruses.","version":"1.1","doi":"10.1101/2021.04.02.437736","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.437942","pub_date":"2021-4-01","title":"Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants","abstract":"The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.","version":"1.1","doi":"10.1101/2021.04.01.437942","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438120","pub_date":"2021-4-01","title":"Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein","abstract":"The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.","version":"1.1","doi":"10.1101/2021.04.01.438120","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437647","pub_date":"2021-4-01","title":"A recombinant receptor-binding domain in trimeric form generates completely protective immunity against SARS-CoV-2 infection in nonhuman primates","abstract":"Safe and effective vaccination is critical to combatting the COVID-19 pandemic. Here, we developed a trimeric SARS-CoV-2 receptor-binding domain (RBD) subunit vaccine candidate that simulates the natural structure of the spike (S) trimer glycoprotein. Immunization with RBD-trimer induced robust humoral and cellular immune responses and a high level of neutralizing antibodies that were maintained for at least 4 months. Moreover, the antibodies that were produced in response to the vaccine effectively neutralized the SARS-CoV-2 501Y.V2 variant. Of note, when the titers of the antibodies dropped to a sufficiently low level, only one boost quickly activated the anamnestic immune response, resulting in complete protection against the SARS-CoV-2 challenge in rhesus macaques without typical histopathological changes or viral replication in the lungs and other respiratory tissues. Our results indicated that immunization with SARS-CoV-2 RBD-trimer could raise long-term and broad immunity protection in nonhuman primates, thereby offering an optimal vaccination strategy against COVID-19.","version":"1.2","doi":"10.1101/2021.03.30.437647","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437931","pub_date":"2021-4-01","title":"Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity","abstract":"Since late 2019, SARS-CoV-2 has caused a global pandemic that has infected 128 million people worldwide. Although several vaccine candidates have received emergency use authorization (EUA), there are still a limited number of vaccine doses available. To increase the number of vaccinated individuals, there are ongoing discussions about administering partial vaccine doses, but there is still a paucity of data on how vaccine fractionation affects vaccine-elicited immunity. We performed studies in mice to understand how the priming dose of a SARS CoV-2 vaccine affects long-term immunity to SARS CoV-2. We first primed C57BL/6 mice with an adenovirus-based vaccine encoding SARS CoV-2 spike protein (Ad5-SARS-2 spike), similar to that used in the CanSino and Sputnik V vaccines. This prime was administered either at a low dose (LD) of 106 PFU or at a standard dose (SD) of 109 PFU, followed by a SD boost in all mice four weeks later. As expected, the LD prime induced lower immune responses relative to the SD prime. However, the LD prime elicited immune responses that were qualitatively superior, and upon boosting, mice that were initially primed with a LD exhibited significantly more potent immune responses. Overall, these data demonstrate that limiting the priming dose of a SARS CoV-2 vaccine may confer unexpected benefits. These findings may be useful for improving vaccine availability and for rational vaccine design.","version":"1.1","doi":"10.1101/2021.03.31.437931","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438087","pub_date":"2021-4-01","title":"Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites","abstract":"New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. All 22 N-glycan sites of SARS-CoV-2 Spike remain highly conserved among the variants B.1.1.7, 501Y.V2 and P.1, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.","version":"1.1","doi":"10.1101/2021.04.01.438087","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438089","pub_date":"2021-4-01","title":"Placental expression of ACE2 and TMPRSS2 in maternal SARS-CoV-2 infection: are placental defenses mediated by fetal sex?","abstract":"Sex differences in vulnerability to and severity of SARS-CoV-2 infection have been described in non-pregnant populations. ACE2 and TMPRSS2, host molecules required for viral entry, are regulated by sex steroids and expressed in the placenta. We sought to investigate whether placental ACE2 and TMPRSS2 expression vary by fetal sex and in the presence of maternal SARS-CoV-2 infection. Placental ACE2 and TMPRSS2 were quantified in 68 pregnant individuals (38 SARS-CoV-2 positive, 30 SARS-CoV-2 negative) delivering at Mass General Brigham from April to June 2020. Maternal SARS-CoV-2 status was determined by nasopharyngeal RT-PCR. Placental SARS-CoV-2 viral load was quantified. RTqPCR was performed to quantify expression of ACE2 and TMPRSS2 relative to the reference gene YWHAZ. Western blots were performed on placental homogenates to quantify protein levels. The impact of fetal sex and SARS-CoV-2 exposure on ACE2 and TMPRSS2 expression was analyzed by 2-way ANOVA. SARS-CoV-2 virus was undetectable in all placentas. Maternal SARS-CoV-2 infection impacted TMPRSS2 placental gene and protein expression in a sexually dimorphic fashion (2-way ANOVA interaction p-value: 0.002). We observed no impact of fetal sex or maternal SARS-CoV-2 status on placental ACE2 gene or protein expression. Placental TMPRSS2 expression was significantly correlated with ACE2 expression in males (Spearman\u2019s \u03c1=0.54, p=0.02) but not females (\u03c1=0.23, p=0.34) exposed to maternal SARS-CoV-2. Sex differences in placental TMPRSS2 but not ACE2 were observed in the setting of maternal SARS-CoV-2 infection. These findings may have implications for offspring vulnerability to placental infection and vertical transmission.These findings may have implications for offspring vulnerability to placental infection and vertical transmission.","version":"1.1","doi":"10.1101/2021.04.01.438089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429536","pub_date":"2021-4-01","title":"Induction of exaggerated cytokine production in human peripheral blood mononuclear cells by a recombinant SARS-CoV-2 spike glycoprotein S1 and its inhibition by dexamethasone","abstract":"An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNF\u03b1, IL-6, IL-1\u03b2 and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-\u03baB p65 and I\u03baB\u03b1, and I\u03baB\u03b1 degradation. DNA binding of NF-\u03baB p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 \u00b5M) resulted in inhibition of spike glycoprotein S1-induced NF-\u03baB activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNF\u03b1, IL-6, IL-1\u03b2 and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1\u03b2 production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-\u03baB, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 is possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm.","version":"1.2","doi":"10.1101/2021.02.03.429536","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.438122","pub_date":"2021-4-01","title":"SARS-CoV-2 binding to ACE2 triggers pericyte-mediated angiotensin-evoked cerebral capillary constriction","abstract":"The SARS-CoV-2 receptor, ACE2, is found on pericytes, contractile cells enwrapping capillaries that regulate brain, heart and kidney blood flow. ACE2 converts vasoconstricting angiotensin II into vasodilating angiotensin-(1-7). In brain slices from hamster, which has an ACE2 sequence similar to human ACE2, angiotensin II alone evoked only a small capillary constriction, but evoked a large pericyte-mediated capillary constriction generated by AT1 receptors in the presence of the SARS-CoV-2 receptor binding domain (RBD). The effect of the RBD was mimicked by blocking ACE2. A mutated non-binding RBD did not potentiate constriction. A similar RBD-potentiated capillary constriction occurred in human cortical slices. This constriction reflects an RBD-induced decrease in the conversion of angiotensin II to angiotensin-(1-7). The clinically-used drug losartan inhibited the RBD-potentiated constriction. Thus AT1 receptor blockers could be protective in SARS-CoV-2 infection by reducing pericyte-mediated blood flow reductions in the brain, and perhaps the heart and kidney.","version":"1.1","doi":"10.1101/2021.04.01.438122","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437925","pub_date":"2021-4-01","title":"SARS-CoV-2 immune evasion by variant B.1.427/B.1.429","abstract":"SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.","version":"1.1","doi":"10.1101/2021.03.31.437925","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.421784","pub_date":"2021-4-01","title":"Conformational Ensembles of Non-Coding Elements in the SARS-CoV-2 Genome from Molecular Dynamics Simulations","abstract":"The 5\u2032 untranslated region (UTR) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is a conserved, functional and structured genomic region consisting of several RNA stem-loop elements. While the secondary structure of such elements has been determined experimentally, their three-dimensional structures are not known yet. Here, we predict structure and dynamics of five RNA stem loops in the 5\u2032-UTR of SARS-CoV-2 by extensive atomistic molecular dynamics simulations, more than 0.5ms of aggregate simulation time, in combination with enhanced sampling techniques. We compare simulations with available experimental data, describe the resulting conformational ensembles, and identify the presence of specific structural rearrangements in apical and internal loops that may be functionally relevant. Our atomic-detailed structural predictions reveal a rich dynamics in these RNA molecules, could help the experimental characterisation of these systems, and provide putative three-dimensional models for structure-based drug design studies.","version":"1.3","doi":"10.1101/2020.12.11.421784","journal":"bioRxiv","score":null},{"id":"10.1101/2021.04.01.436305","pub_date":"2021-4-01","title":"An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces sterile immunity and prevents transmission of SARS-CoV-2 between cats","abstract":"Early in the global SARS-CoV-2 pandemic concerns were raised regarding infection of other animal hosts and whether these could play a significant role in the viral epidemiology. Infection of animals could be detrimental by causing clinical disease but also of concern if they become a viral reservoir allowing further mutations, plus having the potential to infect other animals or humans. The first reported animals to be infected both under experimental conditions and from anecdotal field evidence were cats described in China early in 2020. Given the concerns this finding raised and the close contacts between humans and cats, we aimed to determine whether a vaccine candidate could be developed that was suitable for use in multiple susceptible animal species and whether this vaccine could reduce infection of cats in addition to preventing spread to other cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus (VEEV), known to be safe and efficacious for use in a variety of animals, expressing a stabilised Spike antigen, could induce neutralising antibody titers in guinea pigs and cats. After two intramuscular vaccinations, virus neutralising antibodies were detected in the respiratory tract of the guinea pigs and a cell mediated immune response was induced. The design of the SARS-CoV-2 antigen was shown to be critical in developing a strong neutralising antibody response. Vaccination of cats was able to induce a serum neutralising antibody response which lasted for the course of the experiment. Interestingly, in contrast to control animals, infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after challenge. Moreover, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit virus. The results show that the RP vaccine induces sterile immunity preventing SARS-CoV-2 infection and transmission. This data suggests that this RP vaccine could be a multi-species vaccine useful for preventing spread to and between other animals should that approach be required.","version":"1.1","doi":"10.1101/2021.04.01.436305","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.27.437352","pub_date":"2021-4-01","title":"Modeling Substrate Coordination to Zn-Bound Angiotensin Converting Enzyme 2","abstract":"The spike protein in the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with the receptor Angiotensin Converting Enzyme 2 (ACE2) on the host cell to facilitate the viral uptake. Angiotensin II (Ang II) peptide, which has a naturally high affinity for ACE2, may be useful in inhibiting this interaction. In this study, we computationally designed several Ang II mutants to find a strong binding sequence to ACE2 receptor and examined the role of ligand substitution in the docking of native as well as mutant Ang II to the ACE2 receptor. The peptide in the ACE2-peptide complex was coordinated to zinc in the ACE2 cleft. Exploratory molecular dynamics (MD) simulations were used to measure the time-based stability of the native and mutant peptides and their receptor complexes. The MD-generated root-mean-square deviation (RMSD) values are mostly similar between the native and seven mutant peptides considered in this work, although the values for free peptides demonstrated higher variation, and often were higher in amplitude than peptides associated with the ACE2 complex. An observed lack of a strong secondary structure in the short peptides is attributed to the latter\u2019s greater flexibility and movement. The strongest binding energies within the ACE2-peptide complexes were observed in the native Ang II and only one of its mutant variants, suggesting ACE2 cleft is designed to provide optimal binding to the native sequence. An examination of the S1 binding site on ACE2 suggests that complex formation alone with these peptides may not be sufficient to allosterically inhibit the binding of SARS-CoV-2 spike proteins. However, it opens up the potential for utilizing AngII-ACE2 binding in the future design of molecular and supramolecular structures to prevent spike protein interaction with the receptor through creation of steric hindrance.","version":"1.2","doi":"10.1101/2021.03.27.437352","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437823","pub_date":"2021-3-31","title":"Linking Diabetes mellitus to SARS-CoV-2 infection through differential targeting of the microRNAs in the Pancreas tissue","abstract":"Coronavirus Disease 2019 (COVID-19) severity and Diabetes mellitus affect each other bidirectionally. The plus-sense single-stranded RNA (+ssRNA) genome of the SARS-CoV-2 virus can be targeted and suppressed by the host cell\u2019s microRNAs (miRNAs). Using the differential gene expression analysis between the mock-infected and the SARS-CoV-2-infected pancreatic tissue, we report five Diabetes-associated genes that are upregulated due to SARS-CoV-2 infection in the hESC pancreas tissues. Ten miRNAs regulating these five genes can potentially target the SARS-CoV-2 genome. We hypothesize that the SARS-CoV-2 genome copies in the infected human pancreas cell compete with the host cell\u2019s native genes in being regulated by the native miRNAs. It leads to the reduced miRNA-regulation and, thus, the upregulation of the Diabetes-associated native genes. Thus, the resultant new-onset or elevated Diabetic symptoms may worsen the condition of COVID-19 patients.","version":"1.1","doi":"10.1101/2021.03.31.437823","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437666","pub_date":"2021-3-31","title":"Emerging SARS-CoV-2 mutation hotspots associated with clinical outcomes","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding the influence of mutations in the SARS-CoV-2 gene on clinical outcomes and related factors is critical for treatment and prevention. Here, we analyzed 209,551 high-coverage complete virus sequences and 321 RNA-seq samples to mine the mutations associated with clinical outcome in the SARS-CoV-2 genome. Several important hotspot variants were found to be associated with severe clinical outcomes. Q57H variant in ORF3a protein were found to be associated with higher mortality rate, and was high proportion in severe cases (39.36%) and 501Y.V2 strains (100%) but poorly proportional to asymptomatic cases (10.04%). T265I could change nsp2 structure and mitochondrial permeability, and evidently higher in severe cases (20.12%) and 501Y.V2 strains (100%) but lower in asymptomatic cases (1.43%). Additionally, R203K and G204R could decrease the flexibility and immunogenic property of N protein with high frequency among severe cases, VUI 202012/01 and 484K.V2 strains. Interestingly, the SARS-CoV-2 genome was more susceptible to mutation because of the high frequency of nt14408 mutation (which located in RNA polymerase) and the high expression levels of ADAR and APOBEC in severe clinical outcomes. In conclusion, several important mutation hotspots in the SARS-CoV-2 genome associated with clinical outcomes was found in our study, and that might correlate with different SARS-CoV-2 mortality rates.","version":"1.1","doi":"10.1101/2021.03.31.437666","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.31.437815","pub_date":"2021-3-31","title":"Differential mutational profile of SARS-CoV-2 proteins across deceased and asymptomatic patients","abstract":"The SARS-CoV-2 infection spread at an alarming rate with many places showed multiple peaks in incidence. Present study involves a total of 332 SARS-CoV-2 sequences from 114 Asymptomatic and 218 Deceased patients from twenty-one different countries. The mining of mutations was done using the GISAID CoVSurver (www.gisaid.org/epiflu-applications/covsurver-mutations-app) with the reference sequence \u2018hCoV-19/Wuhan/WIV04/2019\u2019 present in NCBI with Accession number NC-045512.2. The impact of the mutations on SARS-CoV-2 proteins mutation was predicted using PredictSNP1(loschmidt.chemi.muni.cz/predictsnp1) which is a meta-server integrating six predictor tools: SIFT, PhD-SNP, PolyPhen-1, PolyPhen-2, MAPP and SNAP. The iStable integrated server (predictor.nchu.edu.tw/iStable) was used to predict shifts in the protein stability due to mutations. A total of 372 variants were observed in the 332 SARS-CoV-2 sequences with several variants incident in multiple patients accounting for a total of 1596 incidences. Asymptomatic and Deceased specific mutants constituted 32% and 62% of the repertoire respectively indicating their exclusivity. However, the most prevalent mutations were those present in both. Though some parts of the genome are more variable than others but there was clear difference between incidence and prevalence. NSP3 with 68 variants had total occurrence of only 105 whereas Spike protein had 346 occurrences with just 66 variants. For Deleterious variants, NSP3 had the highest incidence of 25 followed by NSP2 (16), ORF3a (14) and N (14). Spike protein had just 7 Deleterious variants out of 66. Deceased patients have more Deleterious than Neutral variants as compared to the symptomatic ones. Further, it appears that the Deleterious variants which decrease protein stability are more significant in pathogenicity of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.31.437815","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437771","pub_date":"2021-3-31","title":"Arrayed multicycle drug screens identify broadly acting chemical inhibitors for repurposing against SARS-CoV-2","abstract":"Coronaviruses (CoVs) circulate in humans and animals, and expand their host range by zoonotic and anthroponotic transmissions. Endemic human CoVs, such as 229E and OC43 cause limited respiratory disease, and elicit short term anti-viral immunity favoring recurrent infections. Yet, severe acute respir-atory syndrome (SARS)-CoV-2 spreads across the globe with unprecedented impact on societies and economics. The world lacks broadly effective and affordable anti-viral agents to fight the pandemic and reduce the death toll. Here, we developed an image-based multicycle replication assay for focus for-mation of \u03b1-coronavirus hCoV-229E-eGFP infected cells for screening with a chemical library of 5440 compounds arrayed in 384 well format. The library contained about 39% clinically used compounds, 26% in phase I, II or III clinical trials, and 34% in preclinical development. Hits were counter-selected against toxicity, and challenged with hCoV-OC43 and SARS-CoV-2 in tissue culture and human bronchial and nasal epithelial explant cultures from healthy donors. Fifty three compounds inhibited hCoV-229E-GFP, 39 of which at 50% effective concentrations (EC50) < 2\u03bcM, and were at least 2-fold separated from toxicity. Thirty nine of the 53 compounds inhibited the replication of hCoV-OC43, while SARS-CoV-2 was inhibited by 11 compounds in at least two of four tested cell lines. Six of the 11 compounds are FDA-approved, one of which is used in mouth wash formulations, and five are systemic and orally available. Here, we demonstrate that methylene blue (MB) and mycophenolic acid (MPA), two broadly available low cost compounds, strongly inhibited shedding of infectious SARS-CoV-2 at the apical side of the cultures, in either pre- or post-exposure regimens, with somewhat weaker effects on viral RNA release indicated by RT-qPCR measurements. Our study illustrates the power of full cycle screens in repurposing clinical compounds against SARS-CoV-2. Importantly, both MB and MPA reportedly act as immunosuppressants, making them interesting candidates to counteract the cytokine storms affecting COVID-19 patients.","version":"1.1","doi":"10.1101/2021.03.30.437771","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437783","pub_date":"2021-3-31","title":"Exploring the role of glycans in the interaction of SARS-CoV-2 RBD and human receptor ACE2","abstract":"COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.","version":"1.1","doi":"10.1101/2021.03.30.437783","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437757","pub_date":"2021-3-31","title":"Supramolecular cylinders target bulge structures in the 5\u2019 UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication","abstract":"The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with Molecular Dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5\u2019 UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in the stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel antiviral agents.","version":"1.1","doi":"10.1101/2021.03.30.437757","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.21254568","pub_date":"2021-03-31","title":"Nonpharmaceutical Interventions Remain Essential to Reducing COVID-19 Burden Even in a Well-Vaccinated Society: A Modeling Study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Vaccination and non-pharmaceutical interventions (NPIs) reduce transmission of SARS-CoV-2 infection, but their effectiveness depends on coverage and adherence levels. We used scenario modeling to evaluate their effects on cases and deaths averted and herd immunity. NPIs and vaccines worked synergistically in different parts of the pandemic to reduce disease burden.</jats:p>","version":null,"doi":"10.1101/2021.03.29.21254568","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.30.437796","pub_date":"2021-3-31","title":"An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication","abstract":"All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-\u03b2 transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics. In the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate, and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within non-structural protein 3. It has received significant attention as a potential drug target as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the various roles and functions of Mac1 during infection remain largely unknown. Here, utilizing recombinant Mac1 mutant viruses, we have determined that different biochemical functions of Mac1 have distinct roles in the replication of MHV, a model CoV. These results indicate that Mac1 is more important for CoV replication than previously appreciated, and could help guide the development of inhibitory compounds that target unique regions of this protein domain.","version":"1.1","doi":"10.1101/2021.03.30.437796","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425340","pub_date":"2021-3-30","title":"Natural variants in SARS-CoV-2 S protein pinpoint structural and functional hotspots: implications for prophylaxis and therapeutic strategies","abstract":"In December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3,540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2021.01.04.425340","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437173","pub_date":"2021-3-30","title":"Severity of SARS-CoV-2 infection as a function of the interferon landscape across the respiratory tract of COVID-19 patients","abstract":"The COVID-19 outbreak driven by SARS-CoV-2 has caused more than 2.5 million deaths globally, with the most severe cases characterized by over-exuberant production of immune-mediators, the nature of which is not fully understood. Interferons of the type I (IFN-I) or type III (IFN-III) families are potent antivirals, but their role in COVID-19 remains debated. Our analysis of gene and protein expression along the respiratory tract shows that IFNs, especially IFN-III, are over-represented in the lower airways of patients with severe COVID-19, while high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity; also, IFN expression varies with abundance of the cell types that produce them. Our data point to a dynamic process of inter- and intra-family production of IFNs in COVID-19, and suggest that IFNs play opposing roles at distinct anatomical sites.","version":"1.1","doi":"10.1101/2021.03.30.437173","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.30.437704","pub_date":"2021-3-30","title":"Sequence analysis of SARS-CoV-2 in nasopharyngeal samples from patients with COVID-19 illustrates population variation and diverse phenotypes, placing the in vitro growth properties of B.1.1.7 and B.1.351 lineage viruses in context","abstract":"New variants of SARS-CoV-2 are continuing to emerge and dominate the regional and global sequence landscapes. Several variants have been labelled as Variants of Concern (VOCs) because of perceptions or evidence that these may have a transmission advantage, increased risk of morbidly and/or mortality or immune evasion in the context of prior infection or vaccination. Placing the VOCs in context and also the underlying variability of SARS-CoV-2 is essential in understanding virus evolution and selection pressures. Sequences of SARS-CoV-2 in nasopharyngeal swabs from hospitalised patients in the UK were determined and virus isolated. The data indicated the virus existed as a population with a consensus level and non-synonymous changes at a minor variant. For example, viruses containing the nsp12 P323L variation from the Wuhan reference sequence, contained minor variants at the position including P and F and other amino acids. These populations were generally preserved when isolates were amplified in cell culture. In order to place VOCs B.1.1.7 (the UK \u2018Kent\u2019 variant) and B.1.351 (the \u2018South African\u2019 variant) in context their growth was compared to a spread of other clinical isolates. The data indicated that the growth in cell culture of the B.1.1.7 VOC was no different from other variants, suggesting that its apparent transmission advantage was not down to replicating more quickly. Growth of B.1.351 was towards the higher end of the variants. Overall, the study suggested that studying the biology of SARS-CoV-2 is complicated by population dynamics and that these need to be considered with new variants. SARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet causing a global pandemic. In common with other coronaviruses, SARS-CoV-2 genetic material (genomes) can become quite diverse as a consequence of replicating inside cells. This has given rise to multiple variants from the original virus that infected humans. These variants may have different properties and in the context of a widespread vaccination program may render vaccines less ineffective. Our research confirms the degree of genetic diversity of SARS-CoV-2 in patients. By isolating viruses from these patients, we show that there is a 100-fold range in growth of even normal variants. Interestingly, by comparing this to the pattern seen with two Variants of Concern (UK and South African variants), we show that at least in cells the ability of the B.1.1.7 variant to grow is not substantially different to many of the previous variants.","version":"1.1","doi":"10.1101/2021.03.30.437704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.422865","pub_date":"2021-3-29","title":"The Ensembl COVID-19 resource: Ongoing integration of public SARS-CoV-2 data","abstract":"The COVID-19 pandemic has seen unprecedented use of SARS-CoV-2 genome sequencing for epidemiological tracking and identification of emerging variants. Understanding the potential impact of these variants on the infectivity of the virus and the efficacy of emerging therapeutics and vaccines has become a cornerstone of the fight against the disease. To support the maximal use of genomic information for SARS-CoV-2 research, we launched the Ensembl COVID-19 browser, incorporating a new Ensembl gene set, multiple variant sets (including novel variation calls), and annotation from several relevant resources integrated into the reference SARS-CoV-2 assembly. This work included key adaptations of existing Ensembl genome annotation methods to model ribosomal slippage, stringent filters to elucidate the highest confidence variants and utilisation of our comparative genomics pipelines on viruses for the first time. Since May 2020, the content has been regularly updated and tools such as the Ensembl Variant Effect Predictor have been integrated. The Ensembl COVID-19 browser is freely available at https://covid-19.ensembl.org.","version":"1.2","doi":"10.1101/2020.12.18.422865","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.437540","pub_date":"2021-3-29","title":"SARS-CoV2, a threat to marine mammals? A study from Italian seawaters","abstract":"Zoonotically transmitted coronaviruses were responsible for three disease outbreaks since 2002, with the \u201cSevere Acute Respiratory Syndrome Coronavirus-2\u201d (SARS-CoV-2) causing the dramatic \u201cCoronavirus Disease-2019\u201d (CoViD-19) pandemic, which affected public health, economy, and society on a global scale. The impacts of the SARS-CoV-2 pandemic permeate into our environment and wildlife as well; in particular, concern has been raised about the viral occurrence and persistence in aquatic and marine ecosystems. The discharge of untreated wastewaters carrying infectious SARS-CoV-2 into natural water systems that are home of sea mammals may have dramatic consequences on vulnerable species. The efficient transmission of coronaviruses raise questions regarding the contributions of virus-receptors interactions. The main receptor of SARS-CoV-2 is Angiotensin Converting Enzyme-2 (ACE-2), serving as a functional receptor for the viral spike (S) protein. This study was aimed, through the comparative analysis of the ACE-2 receptor with the human one, at assessing the susceptibility to SARS-CoV-2 of the different species of marine mammals living in Italian waters. We also determined, by means of immunohistochemistry, ACE-2 receptor localization in the lung tissue from different cetacean species, in order to provide a preliminary characterization of ACE-.2 expression in the marine mammals\u2019 respiratory tract. Furthermore, in order to evaluate if and how wastewater management in Italy may lead to susceptible marine mammal populations being exposed to the virus, geo-mapping data of wastewater plants, associated to the identification of specific stretches of coast more exposed to extreme weather events, overlapped to marine mammal population data, were carried out. Results showed the SARS-CoV-2 exposure for marine mammals inhabiting Italian coastal waters. Thus, we highlight the potential hazard of reverse zoonotic transmission of SARS-CoV-2 infection, along with its impact on marine mammals regularly inhabiting the Mediterranean Sea, whilst also stressing the need of appropriate action to prevent further damage to specific vulnerable populations. Growing concern exists that SARS-CoV-2, as already ascertained for its SARS-CoV and MERS-CoV \u201cpredecessors\u201d, originated from an animal \u201creservoir\u201d, performing thereafter its spillover into mankind, that was possibly anticipated by viral \u201cpassage\u201d into a secondary animal host. Within the dramatic SARS-CoV-2 pandemic context, hitherto characterized by over 110 million cases and almost 2,500,000 deaths on a global scale, several domestic and wild animal species have been reported as susceptible to natural and/or experimental SARS-CoV-2 infection. In this respect, while some marine mammal species are deemed as potentially susceptible to SARS-CoV-2 infection on the basis of the sequence homology of their ACE-2 viral receptor with the human one, this study addresses such a critical issue also in stranded sea mammal specimens.","version":"1.1","doi":"10.1101/2021.03.29.437540","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.28.437426","pub_date":"2021-3-29","title":"COVID-19 dominant D614G mutation in the SARS-CoV-2 spike protein desensitizes its temperature-dependent denaturation","abstract":"The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, making it the dominant form in the COVID-19 pandemic. Here we demonstrated by cryo-electron microscopy that the D614G mutation does not significantly perturb the structure of the spike protein, but multiple receptor binding domains are in an upward conformation poised for host receptor binding. The impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics, and to significantly increase the thermal stability under physiological pH. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation, and suggest an approach for better design of spike-protein based conjugates for vaccine development.","version":"1.1","doi":"10.1101/2021.03.28.437426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.437515","pub_date":"2021-3-29","title":"Enrichment of SARS-CoV-2 entry factors and interacting intracellular genes in peripheral immune cells","abstract":"SARS-CoV-2 uses ACE2 and TMPRSS2 to gain entry into the cell. However, recent studies have shown that SARS-CoV-2 may use additional host factors that are required for the viral lifecycle. Here we used publicly available datasets, CoV associated genes and machine learning algorithms to explore the SARS-CoV-2 interaction landscape in different tissues. We find that in general a small fraction of cells expresses ACE2 in the different tissues including nasal, bronchi and lungs. We show that a small fraction of immune cells (including T-cells, macrophages, dendritic cells) found in tissues also express ACE2. We show that healthy circulating immune cells do not express ACE2 and TMPRSS2. However, a small fraction of circulating immune cells (including dendritic cells, monocytes, T-cells) in the PBMC of COVID-19 patients express ACE2 and TMPRSS2. Additionally, we found that a large spectrum of cells (in circulation and periphery) in both healthy and COVID-19 positive patients were significantly enriched for SARS-CoV-2 factors. Thus, we propose that further research is needed to explore if SARS-CoV-2 can directly infect peripheral immune cells to better understand the virus\u2019 mechanism of action.","version":"1.1","doi":"10.1101/2021.03.29.437515","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.437516","pub_date":"2021-3-29","title":"Sexually dimorphic placental responses to maternal SARS-CoV-2 infection","abstract":"There is a persistent male bias in the prevalence and severity of COVID-19 disease. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of disease in adults, and play a key role in the placental anti-viral response. Moreover, the interferon response has been shown to alter Fc-receptor expression, and therefore may impact placental antibody transfer. Here we examined the intersection of viral-induced placental interferon responses, maternal-fetal antibody transfer, and fetal sex. Placental interferon stimulated genes (ISGs), Fc-receptor expression, and SARS-CoV-2 antibody transfer were interrogated in 68 pregnancies. Sexually dimorphic placental expression of ISGs, interleukin-10, and Fc receptors was observed following maternal SARS-CoV-2 infection, with upregulation in males. Reduced maternal SARS-CoV-2-specific antibody titers and impaired placental antibody transfer were noted in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.29.437516","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.295824","pub_date":"2021-3-29","title":"SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. Phase separation is driven, in part, by hydrophobic and electrostatic interactions. While the N protein forms spherical assemblies with unstructured RNA, it forms asymmetric condensates with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.","version":"1.3","doi":"10.1101/2020.09.14.295824","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.436639","pub_date":"2021-3-29","title":"A lipid nanoparticle RBD-hFc mRNA vaccine protects hACE2 transgenic mice against lethal SARS-CoV-2 infection","abstract":"The current global COVID-19 pandemic led to an unprecedented effort to develop effective vaccines against SARS-CoV-2. mRNA vaccines were developed very rapidly during the last year, and became the leading immunization platform against the virus, with highly promising phase-3 results and remarkable efficacy data. Since most animal models are not susceptible to SARS CoV-2 infection, pre-clinical studies are often limited to infection-prone animals such as hamsters and non-human primates. In these animal models, SARS-CoV-2 infection results in viral replication and a mild disease disease. Therefore, the protective efficacy of the vaccine in these animals is commonly evaluated by its ability to elicit immunologic responses, diminish viral replication and prevent weight loss. Our lab recently reported the design of a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). These experiments demonstrated the development of a robust and specific immunologic response in RBD-hFc mRNA-vaccinated BALB/c mice. In the current study, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18-hACE2 mouse model. We report that administration of RBD-hFc mRNA vaccine to K18-hACE2 mice led to a robust humoral response comprised of both binding and neutralizing antibodies. In accordance with the recorded immunologic immune response, 70% of vaccinated mice were protected against a lethal dose (3000 plaque forming units) of SARS-CoV-2, while all control animals succumbed to infection. To the best of our knowledge, this is the first non-replicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.03.29.436639","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.437530","pub_date":"2021-3-29","title":"Age-dependent appearance of SARS-CoV-2 entry cells in mouse chemosensory systems reflects COVID-19 anosmia and ageusia symptoms","abstract":"COVID-19 pandemic has given rise to a collective scientific effort to study its viral causing agent SARS-CoV-2. Research is focusing in particular on its infection mechanisms and on the associated-disease symptoms. Interestingly, this environmental pathogen directly affects the human chemosensory systems leading to anosmia and ageusia. Evidence for the presence of the cellular entry sites of the virus, the ACE2/TMPRSS2 proteins, has been reported in non-chemosensory cells in the rodent\u2019s nose and mouth, missing a direct correlation between the symptoms reported in patients and the observed direct viral infection in human sensory cells. Here, mapping the gene and protein expression of ACE2/TMPRSS2 in the mouse olfactory and gustatory cells, we precisely identified the virus target cells to be of basal and sensory origin and we revealed their age-dependent appearance. Our results not only clarify human viral-induced sensory symptoms but also propose new investigative perspectives based on ACE2-humanized mouse models.","version":"1.1","doi":"10.1101/2021.03.29.437530","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.437479","pub_date":"2021-3-29","title":"Description and comparison of PIMS-TS innate cell signature and immunophenotype with a cohort of healthy children, severe viral and bacterial infections and Kawasaki Disease","abstract":"A new clinical syndrome associated to SARS-CoV-2 has been described in children. It has been named as Pediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS). This new disease is a main cause of hospital and pediatric intensive care unit (PICU). In this work we describe the innate cell signature and immunophenotype of children admitted to PICU because of PIMS-TS. Also, we compare it with healthy controls and children admitted to PICU because bacterial infection, viral infection and Kawasaki disease. We made a prospective-retrospective observational study in a tertiary pediatric hospital. Children admitted to PICU because of PIMS-TS from March 2020 to September 2020 were consecutively included. They were compare with previous cohorts from our center. A total of 247 children were included: 183 healthy controls, 25 viral infections, 20 bacterial infections, 6 Kawasaki disease and 13 PIMS-TS. PIMT-TS showed the lowest percentage of lymphocytes and monocytes with higher relative numbers of CD4+ (p =0,000). At the same time, we describe a differential expression of CD64, CD11a and CD11b. Monocytes and neutrophils in PIMS-TS showed higher levels of CD64 expression compared to all groups (p = 0,000). Also, proteins involved in leukocyte tissue migration, like CD11a and CD11b were highly expressed compare to other severe viral or bacterial infections (p = 0,000). In PIMS-TS this increased CD11a expression could be a sign of the activation and trafficking of these leukocytes. These findings are congruent with an inflammatory process and the trend of these cells to leave the bloodstream. In conclusion, we compare for the first time the innate cellular response of children with PIMS-TS with other severe forms of viral or bacterial infection and Kawasaki disease. Our findings define a differential cell innate signature. These data should be further studied and may facilitate the diagnosis and management of these patients.","version":"1.1","doi":"10.1101/2021.03.29.437479","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.29.437576","pub_date":"2021-3-29","title":"Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection","abstract":"Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are typically detected using DNA sequencing, which requires advanced sample preparation and instrumentation, and thus cannot be deployed for on-site testing or in low-resource settings. In this work we have developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines LAMP-based target amplification with fluorescent probes to detect SNPs with high specificity in a one-pot reaction format. A competitive \u201csink\u201d strand preferentially binds to off-target products and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible SARS-CoV-2 variants. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity.","version":"1.1","doi":"10.1101/2021.03.29.437576","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.28.437376","pub_date":"2021-3-28","title":"A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection","abstract":"The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.","version":"1.1","doi":"10.1101/2021.03.28.437376","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437180","pub_date":"2021-3-27","title":"The RNA sensor MDA5 detects SARS-CoV-2 infection","abstract":"Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.","version":"1.1","doi":"10.1101/2021.03.26.437180","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.27.437300","pub_date":"2021-3-27","title":"Rapid Evolution of SARS-CoV-2 Challenges Human Defenses","abstract":"Evolutionary ecology theory provides an avenue to anticipate the future behavior of SARS-CoV-2. Here we quantify the accelerating evolution of SARS-CoV-2 by tracking the SARS-CoV-2 mutation globally, with a focus on the Receptor Binding Domain (RBD) of the spike protein believed to determine infectivity. We estimate that 384 million people were infected by March 1st, 2021, producing up to 1021 copies of the virus, with one new RBD variant appearing for every 600,000 human infections, resulting in approximately three new effective RBD variants produced daily. Doubling the number of RBD variants every 71.67 days followed by selection of the most infective variants challenges our defenses and calls for a shift to anticipatory, rather than reactive tactics. Accelerating evolution of SARS-CoV-2 demands formulating universal vaccines and treatments based on big-data simulations of possible new variants.","version":"1.1","doi":"10.1101/2021.03.27.437300","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436465","pub_date":"2021-3-27","title":"TMPRSS2 inhibitor discovery facilitated through an in silico and biochemical screening platform","abstract":"The COVID-19 pandemic has highlighted the need for new antiviral targets, as many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a highly promising antiviral target, as it plays a direct role in priming the spike protein before viral entry occurs. Further, unlike other targets such as ACE2, TMPRSS2 has no known biological role. Here we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we demonstrate that serine protease inhibitors camostat, nafamostat, and gabexate inhibit through a covalent mechanism. We further identify new non-covalent compounds as TMPRSS2 protease inhibitors, demonstrating the utility of a combined virtual and experimental screening campaign in rapid drug discovery efforts.","version":"1.2","doi":"10.1101/2021.03.22.436465","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.27.437309","pub_date":"2021-3-27","title":"The nucleotide addition cycle of the SARS-CoV-2 polymerase","abstract":"Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.","version":"1.1","doi":"10.1101/2021.03.27.437309","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437123","pub_date":"2021-3-26","title":"Occurrence of COVID-19 symptoms during SARS-CoV-2 infection defines waning of humoral immunity","abstract":"Approximately half of the SARS-CoV-2 infections occur without apparent symptoms, raising questions regarding long-term humoral immunity in asymptomatic individuals. Plasma levels of immunoglobulin G (IgG) and M (IgM) against the viral spike or nucleoprotein were determined for 25,091 individuals enrolled in a surveillance program in Wuhan, China. We compared 405 asymptomatic individuals with 459 symptomatic COVID-19 patients. The well-defined duration of the SARS-CoV-2 endemic in Wuhan allowed a side-by-side comparison of antibody responses following symptomatic and asymptomatic infections without subsequent antigen re-exposure. IgM responses rapidly declined in both groups. However, both the prevalence and durability of IgG responses and neutralizing capacities correlated positively with symptoms. Regardless of sex, age, and body weight, asymptomatic individuals lost their SARS-CoV-2-specific IgG antibodies more often and rapidly than symptomatic patients. These findings have important implications for immunity and favour immunization programs including individuals after asymptomatic infections. Prevalence and durability of SARS-CoV-2-specific IgG responses and neutralizing capacities correlate with COVID-19 symptoms.","version":"1.1","doi":"10.1101/2021.03.26.437123","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436522","pub_date":"2021-3-26","title":"The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection","abstract":"We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone\u2019s translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.","version":"1.3","doi":"10.1101/2021.03.22.436522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.373449","pub_date":"2021-3-26","title":"Genetic Modification to Design a Stable Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain as a COVID-19 Vaccine Candidate","abstract":"Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a \u201cwild type\u201d RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.","version":"1.2","doi":"10.1101/2020.11.09.373449","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437014","pub_date":"2021-3-26","title":"Aberrant glycosylation of anti-SARS-CoV-2 IgG is a pro-thrombotic stimulus for platelets","abstract":"A subset of patients with COVID-19 become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in severely ill COVID-19 patients are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesised that platelets might be susceptible to activation by anti-SARS-CoV-2 antibodies and contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike IgG enhanced platelet-mediated thrombosis on von Willebrand Factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcyRIIA and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by therapeutic small molecules R406 (fostamatinib) and ibrutinib that inhibit tyrosine kinases syk and btk respectively or by the P2Y12 antagonist cangrelor.","version":"1.1","doi":"10.1101/2021.03.26.437014","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.241414","pub_date":"2021-3-26","title":"Rapid SARS-CoV-2 Adaptation to Available Cellular Proteases","abstract":"Since the pandemic spread of SARS-CoV-2, the virus has exhibited remarkable genome stability, but recent emergence of novel variants show virus evolution potential. Here we show that SARS-CoV-2 rapidly adapts to Vero E6 cells that leads to loss of furin cleavage motif in spike protein. The adaptation is achieved by asymptotic expansion of minor virus subpopulations to dominant genotype, but wildtype sequence is maintained at low percentage in the virus swarm, and mediate reverse adaptation once the virus is passaged on human lung cells. The Vero E6-adapted virus show defected cell entry in human lung cells and the mutated spike variants cannot be processed by furin or TMPRSS2. However, the mutated S1/S2 site is cleaved by cathepsins with higher efficiency. Our data show that SARS-CoV-2 can rapidly adapt spike protein to available proteases and advocate for deep sequence surveillance to identify virus adaptation potential and novel variant emergence. Recently emerging SARS-CoV-2 variants B1.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor spike mutation and have been linked to increased virus pathogenesis. The emergence of these novel variants highlight coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt upon selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genome with original genotype are maintained in virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.","version":"1.2","doi":"10.1101/2020.08.10.241414","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.436314","pub_date":"2021-3-26","title":"Immunoinformatic approach to design a vaccine against SARS-COV-2 membrane glycoprotein","abstract":"SARS-COV-2 is a pandemic virus causing COVID-19 disease which affects lungs and upper respiratory tract leading to progressive increase in the death rate worldwide. Currently, there are more than 123 million cases and over 2.71 million confirmed death caused by this virus. In this study, by utilizing an immunoinformatic approach, multiepitope-based vaccine is designed from the membrane protein which plays a vital role in the virion assembly of the novel-CoV. A total of 19 MHC class- I binders with HLA-A and HLA-B alleles have been selected with NetMHC pan EL 4.0 method from IEDB MHC-I prediction server. Four epitopes candidates from M-protein were selected based on the antigenicity, stability, immunogenicity, Ramachandran plot and scores with 100 % was taken for docking analysis with alleles HLA-A (PDB ID: 1B0R) and HLA-B (PDB ID: 3C9N) using ClusPro server. Among the four epitopes, the epitope FVLAAVYRI has the least binding energy and forms electrostatic, hydrogen and hydrophobic interactions with HLA-A (\u2212932.8 Kcal/mol) and HLA-B (\u2212860.7 Kcal/mol) which induce the T-cell response. Each HLA-A and HLA-B complex in the system environment achieves stable backbone configuration between 45-100 ns of MD simulation. This study reports a potent antigenic and immunogenic profile of FVLAAVYRI epitope from M-protein and further in vitro and in vivo validation is needed for its adaptive use as vaccine against COVID-19.","version":"1.1","doi":"10.1101/2021.03.26.436314","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416297","pub_date":"2021-3-26","title":"Complete Protection of Nasal and Lung Airways Against SARS-CoV-2 Challenge by Antibody Plus Th1 Dominant N- and S-Specific T-Cell Responses to Subcutaneous Prime and Thermally-Stable Oral Boost Bivalent hAd5 Vaccination in an NHP Study","abstract":"We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike (S-Fusion) protein and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) with the potential to increase MHC class I/II responses. The adenovirus serotype 5 platform used, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity, can be delivered in an oral formulation that overcomes cold-chain limitations. The hAd5 S-Fusion + N-ETSD vaccine was evaluated in rhesus macaques showing that a subcutaneous prime followed by oral boosts elicited both humoral and Th1 dominant T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge. hAd5 spike + nucleocapsid SC prime/oral boost vaccine elicits humoral and T-cell responses and protects rhesus macaques from SARS-CoV-2 challenge.","version":"1.3","doi":"10.1101/2020.12.08.416297","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437153","pub_date":"2021-3-26","title":"Comparison of SARS-CoV-2 VOC 202012/01 (UK variant) and D614G variant transmission by different routes in Syrian hamsters","abstract":"Many SARS-CoV-2 variants of concern has been reported recently which were linked to increased transmission. In our earlier study on virus shedding using VOC 202012/01(UK variant) and D614G variant in hamster model, we observed significantly higher viral RNA shedding through nasal wash in case of UK variant. Hence, we compared the transmission of both the UK and D614G variant by various routes in Syrian hamsters to understand whether the high viral RNA shedding could enhance the transmission efficiency of the variant. The current study demonstrated comparable transmission efficiency of both UK and D614G variants of SARS-CoV-2 in Syrian hamsters.","version":"1.1","doi":"10.1101/2021.03.26.437153","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436476","pub_date":"2021-3-26","title":"The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice","abstract":"In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted (hAd5[E1-, E2b-, E3-]) that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell-surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity \u2013 including mucosal immunity \u2013 against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.","version":"1.2","doi":"10.1101/2021.03.22.436476","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.149229","pub_date":"2021-3-26","title":"Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza","abstract":"Infection of certain influenza viruses is triggered when its hemagglutinin (HA) is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, while the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R\u2193 sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal \u03b1-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.","version":"1.3","doi":"10.1101/2020.06.12.149229","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.436894","pub_date":"2021-3-26","title":"Thermal Fogging with Disinfectant Didecyl Dimethyl Ammonium Bromide Effectively Kills A Coronavirus, An Influenza Virus and Two Indicator Bacteria in Subzero Cold-Chain Environment","abstract":"The origin of several local COVID-19 outbreaks in China in 2020 were confirmed to be frozen food or packages contaminated with SARS-CoV-2, revealing the lack of effective disinfection measures in the frozen food chain. To evaluate the disinfection efficacy at \u221220\u00b0C of a recently marketed thermal fogging disinfection product, with disinfectant-antifreeze combination being didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG). Carriers with porcine epidemic diarrhea virus (PEDV) YN1, a coronavirus, and a swine influenza virus H1N1 (SIV-H1N1), and three indicator bacteria, E. coli, S. aureus, and B. subtilis endospores, were respectively placed in a \u221220\u00b0C freezer warehouse with or without DDAB-PPG fogging and activities of the microorganisms were tested. At \u221220\u00b0C, DDAB-PPG fogging, which fully settles in 3.5\u22124.5 hours, fully inactivated PEDV of 10\u22123.5 TCID50/0.1ml and SIV-H1N1 of 26 hemagglutination titer within 15-30 min, and inactivated S. aureus and E. coli vegetative cells (106/ml) within 15 or 60 min, respectively, but had little effect on B. subtilis spores. The bactericidal effect lasted at least 3 hours for bacteria on carrier plates and for 6 hours for airborne bacteria. A practical subzero temperature disinfection technology was confirmed its efficacy in killing enveloped viruses and vegetative bacteria. It would help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity. First report of efficacy of disinfectant didecyl dimethyl ammonium bromide (DDAB) at subzero temperatures The customized thermal fogging machine makes fine droplets of DDAB-propylene glycol (PPG) fog which can suspend for ~4 hours at \u221220\u00b0C DDAB-PPG thermal fogging at \u221220\u00b0C effectively inactivated a coronavirus and an influenza virus DDAB-PPG thermal fogging at \u221220\u00b0C for effectively killed S. aureus and E. coli DDAB-PPG thermal fogging at \u221220\u00b0C inactivated airborne microorganism for up to 6 h","version":"1.1","doi":"10.1101/2021.03.25.436894","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.26.437218","pub_date":"2021-3-26","title":"ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes","abstract":"The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, with investigation at the protein level limited by uncertain specificity of commercial ACE2 antibodies. Here, we report our development of a sensitive and specific flow cytometry-based assay for cellular ACE2 protein abundance. Application of this approach to multiple cell lines revealed an unexpected degree of cellular heterogeneity, with detectable ACE2 protein in only a subset of cells in each isogenic population. This heterogeneity was mediated at the mRNA level by transcripts predominantly initiated from the ACE2 proximal promoter. ACE2 expression was heritable but not fixed over multiple generations of daughter cells, with gradual drift toward the original heterogeneous background. RNA-seq profiling identified distinct transcriptomes of ACE2-expressing relative cells to non-expressing cells, with enrichment in functionally related genes and transcription factor target sets. Our findings provide a validated approach for the specific detection of ACE2 protein at the surface of single cells, support an epigenetic mechanism ACE2 gene regulation, and identify specific pathways associated with ACE2 expression in HuH7 cells.","version":"1.1","doi":"10.1101/2021.03.26.437218","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436523","pub_date":"2021-3-25","title":"Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates","abstract":"The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 \u00b5g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFN\u2019s potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. A SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.","version":"1.1","doi":"10.1101/2021.03.24.436523","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.437060","pub_date":"2021-3-25","title":"Sulforaphane exhibits in vitro and in vivo antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are no orally available medications for prophylaxis for those exposed to SARS-CoV-2 and limited therapeutic options for those who develop COVID-19. We evaluated the antiviral activity of sulforaphane (SFN), a naturally occurring, orally available, well-tolerated, nutritional supplement present in high concentrations in cruciferous vegetables with limited side effects. SFN inhibited in vitro replication of four strains of SARS-CoV-2 as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN is a promising treatment for prevention of coronavirus infection or treatment of early disease.","version":"1.1","doi":"10.1101/2021.03.25.437060","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.437083","pub_date":"2021-3-25","title":"Target Capture Sequencing of SARS-CoV-2 Genomes Using the ONETest Coronaviruses Plus","abstract":"Genomic sequencing is important to track and monitor genetic changes in SARS-CoV-2. We introduce a target capture next-generation sequencing methodology, the ONETest Coronaviruses Plus, to sequence SARS-CoV-2 genomes and select genes of other respiratory viruses simultaneously. We applied the ONETest on 70 respiratory samples (collected in Florida, USA between May and July, 2020), in which SARS-CoV-2 had been detected by a qualitative PCR assay. For 48 (69%) of the samples, we also applied the ARTIC protocol for Illumina sequencing. All the libraries were sequenced as 2\u00d7150 nucleotide reads on an Illumina instrument. The ONETest data were analyzed using an in-house pipeline and the ARTIC data using a published pipeline to produce consensus SARS-CoV-2 genome sequences, to which lineages were assigned using pangolin. Of the 70 ONETest libraries, 45 (64%) had a complete or near-complete SARS-CoV-2 genome sequence (> 29,000 bases and with > 90% of its bases covered by at least 10 reads). Of the 48 ARTIC libraries, 25 (52%) had a complete or near-complete SARS-CoV-2 genome sequence. In 24 out of 34 (71%) samples in which both the ONETest and ARTIC sequences were complete or near-complete and in which lineage could be assigned to both the ONETest and ARTIC sequences, the SARS-CoV-2 lineage identified was the same. The ONETest can be used to sequence the SARS-CoV-2 genomes in archived samples and thereby enable detection of circulating and emerging SARS-CoV-2 variants. Target capture approaches, such as the ONETest, are less prone to loss of sequence coverage probably due to amplicon dropouts encountered in amplicon approaches, such as ARTIC. With its added value of characterizing other major respiratory pathogens, although not assessed in this study, the ONETest can help to better understand the epidemiology of infectious respiratory disease in the post COVID-19 era.","version":"1.1","doi":"10.1101/2021.03.25.437083","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.260190","pub_date":"2021-3-25","title":"Virucidal and antiviral activity of astodrimer sodium against SARS-CoV-2 in vitro","abstract":"An effective response to the ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will involve a range of complementary preventive modalities. The current studies were conducted to evaluate the in vitro SARS-CoV-2 antiviral and virucidal activity of astodrimer sodium, a dendrimer with broad spectrum antimicrobial activity, including against enveloped viruses in in vitro and in vivo models, that is marketed for antiviral and antibacterial applications. We report that astodrimer sodium inhibits replication of SARS-CoV-2 in Vero E6 and Calu-3 cells, with 50% effective concentrations (EC50) for i) reducing virus-induced cytopathic effect of 0.002 to 0.012 mg/mL in Vero E6 cells, and ii) infectious virus release by plaque assay of 0.019 to 0.031 mg/mL in Vero E6 cells and 0.031 to 0.045 mg/mL in Calu-3 cells. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also virucidal, reducing SARS-CoV-2 infectivity by >99.9% (>3 log10) within 1 minute of exposure, and up to >99.999% (>5 log10) shown at astodrimer sodium concentrations of 10 to 30 mg/mL in Vero E6 and Calu-3 cell lines. Astodrimer sodium also inhibited infection in a primary human airway epithelial cell line. The data were similar for all investigations and were consistent with the potent antiviral and virucidal activity of astodrimer sodium being due to inhibition of virus-host cell interactions, as previously demonstrated for other viruses. Further studies will confirm if astodrimer sodium binds to SARS-CoV-2 spike protein and physically blocks initial attachment of the virus to the host cell. Given the in vitro effectiveness and significantly high SI, astodrimer sodium warrants further investigation for potential as a nasally administered or inhaled antiviral agent for SARS-CoV-2 prevention and treatment applications.","version":"1.2","doi":"10.1101/2020.08.20.260190","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.437035","pub_date":"2021-3-25","title":"Computational assessment of the spike protein antigenicity reveals diversity in B cell epitopes but stability in T cell epitopes across SARS-CoV-2 variants","abstract":"Since its emergence into the human population at the end of 2019, SARS-CoV-2 has caused significant morbidity and mortality worldwide. Efforts to develop a protective vaccine against COVID-19 have yielded several vaccine platforms currently in distribution targeting the original SARS-CoV-2 spike protein sequence from the first cases of infection. In recent months, variants of SARS-CoV-2 have raised concerns that viral mutation may undermine vaccination efforts through viral escape of host immune memory acquired from infection or vaccination. We therefore used a computational approach to predict changes in spike protein antigenicity with respect to host B cell and CD8+ T cell immunity across six SARS-CoV-2 variants (D614G, B.1.1.7, B.1.351, P.1, B.1.429, and mink-related). Our epitope analysis using DiscoTope suggests possible changes in B cell epitopes in the S1 region of the spike protein across variants, in particular the B.1.1.7 and B.1.351 lineages, which may influence immunodominance. Additionally, we show that high-affinity MHC-I-binding peptides and glycosylation sites on the spike protein appear consistent between variants with the exception of an extra glycosylation site in the P.1 variant. Together, these analyses suggests T cell vaccine strategies have the most longevity before reformulation.","version":"1.1","doi":"10.1101/2021.03.25.437035","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.436930","pub_date":"2021-3-25","title":"Genomic surveillance and phylodynamic analyses reveal emergence of novel mutation and co-mutation patterns within SARS-CoV2 variants prevalent in India","abstract":"Emergence of distinct viral clades has been observed in SARS-CoV2 variants across the world and India. Identification of the genomic diversity and the phylodynamic profiles of the prevalent strains of the country are critical to understand the evolution and spread of the variants. We performed whole-genome sequencing of 54 SARS-CoV2 strains collected from COVID-19 patients in Kolkata, West Bengal during August to October 2020. Phylogeographic and phylodynamic analyses were performed using these 54 and other sequences from India and abroad available in GISAID database. Spatio-temporal evolutionary dynamics of the pathogen across various regions and states of India over three different time periods in the year 2020 were analyzed. We estimated the clade dynamics of the Indian strains and compared the clade specific mutations and the co-mutation patterns across states and union territories of India over the time course. We observed that GR, GH and G (GISAID) or 20B and 20A (Nextstrain) clades were the prevalent clades in India during middle and later half of the year 2020. However, frequent mutations and co-mutations observed within the major clades across time periods do not show much overlap, indicating emergence of newer mutations in the viral population prevailing in the country. Further, we explored the possible association of specific mutations and co-mutations with the infection outcomes manifested within the Indian patients.","version":"1.1","doi":"10.1101/2021.03.25.436930","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.436935","pub_date":"2021-3-25","title":"COVID-19: A need for new rather than repurposed antiviral drugs","abstract":"SARS-CoV-2 infection, the causative agent of COVID-19, has resulted in over 2,500,000 deaths to date. Although vaccines are becoming available, treatment options remain limited. Repurposing of compounds could reduce the time, cost, and risks associated with the development of new drugs and has been the focus of many clinical studies. Here, we summarise available evidence on 29 FDA-approved compounds, from in vitro results to clinical trials, focussing on remdesivir, galidesivir and favipiravir, and test 29 antiviral compounds\u2019 activity in vitro. A comprehensive search strategy was used to retrieve trials and publications related to antiviral compounds with potential efficacy to treat coronaviruses. These data were used to prioritise testing of a panel of antiviral drugs in vitro against patient isolates of SARS-CoV-2. An in vitro screen was carried out to determine the activity of 29 FDA-approved compounds. 625 clinical trials investigated 16 repurposed antiviral candidate compounds for the treatment of COVID-19. In vitro studies identified ten drug candidates with demonstrable anti-SARS-CoV-2 activity, including favipiravir, remdesivir, and galidesivir. To validate these findings, a drug screen was conducted using two cell lines and wildtype isolates of SARS-CoV-2 isolated from patients in the UK. While eight drugs with anti-SARS-CoV-2 activity were identified in vitro, activity in clinical trials has, as yet failed to demonstrate a strong effect on mortality. So far, no repurposed antiviral has shown a strong effect on mortality in clinical studies. The urgent need for novel antivirals in this pandemic is clear, despite the costs and time associated with their development. Repurposing of existing compounds for the treatment of COVID-19 has been the focus of many in vitro studies and clinical trials, saving time, costs and risks associated with the research and development of new compounds. We reviewed the literature for 29 FDA-approved compounds with previously reported (or suspected) anti-SARS-CoV-2 activity and found 625 clinical trials that have been undertaken on 16 different drugs. We determined if repurposed antivirals are suitable for clinical trials based on previously published data, and conducted an additional in vitro screen using locally circulating strains in the UK (PHE2 and GLA1). We report the difference in IC50 from published data using Wuhan1/Wash1 strains with PHE2 and GLA1, including IC50 values below 100\u03bcM for galidesivir in wild-type virus. Given the limited success of repurposed compounds in the treatment of COVID-19, we comment on the urgent need for new antivirals specifically targeting SARS-CoV-2. Our data show that most prospective compounds for repurposing show no anti-SARS-CoV-2 activity, and antiviral activity in vitro does not always translate to clinical benefit. So far, no repurposed compound has shown a strong effect on mortality in clinical studies. Drugs, including monoclonal antibody therapies, that have been developed to target SARS-CoV-2 virus itself have shown most promise.","version":"1.1","doi":"10.1101/2021.03.25.436935","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.25.437046","pub_date":"2021-3-25","title":"Freely accessible ready to use global infrastructure for SARS-CoV-2 monitoring","abstract":"The COVID-19 pandemic is the first global health crisis to occur in the age of big genomic data.Although data generation capacity is well established and sufficiently standardized, analytical capacity is not. To establish analytical capacity it is necessary to pull together global computational resources and deliver the best open source tools and analysis workflows within a ready to use, universally accessible resource. Such a resource should not be controlled by a single research group, institution, or country. Instead it should be maintained by a community of users and developers who ensure that the system remains operational and populated with current tools. A community is also essential for facilitating the types of discourse needed to establish best analytical practices. Bringing together public computational research infrastructure from the USA, Europe, and Australia, we developed a distributed data analysis platform that accomplishes these goals. It is immediately accessible to anyone in the world and is designed for the analysis of rapidly growing collections of deep sequencing datasets. We demonstrate its utility by detecting allelic variants in high-quality existing SARS-CoV-2 sequencing datasets and by continuous reanalysis of COG-UK data. All workflows, data, and documentation is available at https://covid19.galaxyproject.org.","version":"1.1","doi":"10.1101/2021.03.25.437046","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436885","pub_date":"2021-3-25","title":"Computational prediction of the effect of amino acid changes on the binding affinity between SARS-CoV-2 spike protein and the human ACE2 receptor","abstract":"The association of the receptor binding domain (RBD) of SARS-CoV-2 viral spike with human angiotensin converting enzyme (hACE2) represents the first required step for viral entry. Amino acid changes in the RBD have been implicated with increased infectivity and potential for immune evasion. Reliably predicting the effect of amino acid changes in the ability of the RBD to interact more strongly with the hACE2 receptor can help assess the public health implications and the potential for spillover and adaptation into other animals. Here, we introduce a two-step framework that first relies on 48 independent 4-ns molecular dynamics (MD) trajectories of RBD-hACE2 variants to collect binding energy terms decomposed into Coulombic, covalent, van der Waals, lipophilic, generalized Born electrostatic solvation, hydrogen-bonding, \u03c0-\u03c0 packing and self-contact correction terms. The second step implements a neural network to classify and quantitatively predict binding affinity using the decomposed energy terms as descriptors. The computational base achieves an accuracy of 82.2% in terms of correctly classifying single amino-acid substitution variants of the RBD as worsening or improving binding affinity for hACE2 and a correlation coefficient r of 0.69 between predicted and experimentally calculated binding affinities. Both metrics are calculated using a 5-fold cross validation test. Our method thus sets up a framework for effectively screening binding affinity change with unknown single and multiple amino-acid changes. This can be a very valuable tool to predict host adaptation and zoonotic spillover of current and future SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.03.24.436885","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436901","pub_date":"2021-3-25","title":"CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus","abstract":"Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T-cell based vaccines against respiratory viral pathogens such as influenza A virus (IAV). Chemokine receptor 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs, to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendrtitic cells (DCs) and monocyte-derived DCs internalized and processed vaccine antigen in lungs. We also found that Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF-3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127HI/KLRG-1LO, OX40+veCD62L+ve and mucosally imprinted CD69+veCD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs, induced by CCR2 deficiency was linked to dampened expression of T-bet, but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens including IAV and SARS-CoV-2. While antibody-based immunity to influenza A virus (IAV) is type and sub-type specific, lung and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T-cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of anti-viral lung-resident memory T cells, following intranasal vaccination. These findings suggested that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses such as IAV and SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.24.436901","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436830","pub_date":"2021-3-24","title":"New detection of SARS-CoV-2 in two cats height months after COVID-19 outbreak appearance in France","abstract":"Although there are several reports in the literature of SARS-CoV-2 infection in cats, few SARS-CoV-2 sequences from infected cats have been published. In this report, SARS-CoV-2 infection was evaluated in two cats by clinical observation, molecular biology (qPCR and NGS), and serology (Microsphere immunoassay and seroneutralization). Following the observation of symptomatic SARS-CoV-2-infection in two cats, infection status was confirmed by RT-qPCR and, in one cat, serological analysis for antibodies against N-protein and S-protein, as well as neutralizing antibodies. Comparative analysis of five SARS-CoV-2 sequence-fragments obtained from one of the cats showed that this infection was not with one of the three recently emerged variants of SARS-CoV-2. This study provides additional information on the clinical, molecular, and serological aspects of SARS-CoV-2 infection in cats.","version":"1.1","doi":"10.1101/2021.03.24.436830","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436850","pub_date":"2021-3-24","title":"Factors Associated with Emerging and Re-emerging of SARS-CoV-2 Variants","abstract":"Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background of D614G in Spike protein and P323L in RNA polymerase. The correlation analyses showed that all mutations in specific variants did not evolve simultaneously. Instead, some mutations evolved most likely to compensate for the viral fitness.","version":"1.1","doi":"10.1101/2021.03.24.436850","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436375","pub_date":"2021-3-24","title":"An mRNA vaccine against SARS-CoV-2: Lyophilized, liposome-based vaccine candidate EG-COVID induces high levels of virus neutralizing antibodies","abstract":"In addition to the traditional method of vaccine development, the mRNA coronavirus vaccine, which is attractive as a challenging vaccination, recently opened a new era in vaccinology. Here we describe the EG-COVID which is a novel liposome-based mRNA candidate vaccine that encodes the spike (S) protein of SARS-CoV-2 with 2P-3Q substitution in European variant. We developed the mRNA vaccine platform that can be lyophilized using liposome-based technology. Intramuscular injection of the EG-COVID elicited robust humoral and cellular immune response to SARS-CoV-2. Furthermore, sera obtained from mice successfully inhibited SARS-CoV-2 viral infection into Vero cells. We developed EG-COVID and found it to be effective based on in vitro data, and we plan to initiate a clinical trial soon. Since EG-COVID is a lyophilized mRNA vaccine that is convenient for transportation and storage, accessibility to vaccines will be significantly improved.","version":"1.2","doi":"10.1101/2021.03.22.436375","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436864","pub_date":"2021-3-24","title":"A public vaccine-induced human antibody protects against SARS-CoV-2 and emerging variants","abstract":"The emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility is a public health threat. Some of these variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies, which principally target the receptor binding domain (RBD) on the virus spike glycoprotein. Here, we describe 2C08, a SARS-CoV-2 mRNA vaccine-induced germinal center B cell-derived human monoclonal antibody that binds to the receptor binding motif within the RBD. 2C08 broadly neutralizes SARS-CoV-2 variants with remarkable potency and reduces lung inflammation, viral load, and morbidity in hamsters challenged with either an ancestral SARS-CoV-2 strain or a recent variant of concern. Clonal analysis identified 2C08-like public clonotypes among B cell clones responding to SARS-CoV-2 infection or vaccination in at least 20 out of 78 individuals. Thus, 2C08-like antibodies can be readily induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern. Protection against SARS-CoV-2 variants by a potently neutralizing vaccine-induced human monoclonal antibody.","version":"1.1","doi":"10.1101/2021.03.24.436864","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436686","pub_date":"2021-3-24","title":"Untangling the cell immune response dynamic for severe and critical cases of SARS-CoV-2 infection","abstract":"COVID-19 is a global pandemic leading high death tolls worldwide day by day. Clinical evidence suggests that COVID-19 patients can be classified as non-severe, severe and critical cases. In particular, studies have highlighted the relationship between the lymphopenia and the severity of the illness, where CD8+ T cells have the lowest levels in critical cases. In this work, we aim to elucidate the key parameters that define the course of the disease deviating from severe to critical case. To this end, several mathematical models are proposed to represent the dynamic of the immune response in patients with SARS-CoV-2 infection. The best model had a good fit to reported experimental data, and in accordance with values found in the literature. Our results suggest that a rapid proliferation of CD8+ T cells is decisive in the severity of the disease.","version":"1.1","doi":"10.1101/2021.03.23.436686","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436822","pub_date":"2021-3-24","title":"Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication","abstract":"Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5\u2019-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.","version":"1.1","doi":"10.1101/2021.03.24.436822","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.426895","pub_date":"2021-3-24","title":"E484K as an innovative phylogenetic event for viral evolution: Genomic analysis of the E484K spike mutation in SARS-CoV-2 lineages from Brazil","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people since its beginning in 2019. The propagation of new lineages and the discovery of key mechanisms adopted by the virus to overlap the immune system are central topics for the entire public health policies, research and disease management. Since the second semester of 2020, the mutation E484K has been progressively found in the Brazilian territory, composing different lineages over time. It brought multiple concerns related to the risk of reinfection and the effectiveness of new preventive and treatment strategies due to the possibility of escaping from neutralizing antibodies. To better characterize the current scenario we performed genomic and phylogenetic analyses of the E484K mutated genomes sequenced from Brazilian samples in 2020. From October, 2020, more than 40% of the sequenced genomes present the E484K mutation, which was identified in three different lineages (P1, P2 and B.1.1.33) in four Brazilian regions. We also evaluated the presence of E484K associated mutations and identified selective pressures acting on the spike protein, leading us to some insights about adaptive and purifying selection driving the virus evolution.","version":"1.2","doi":"10.1101/2021.01.27.426895","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.435771","pub_date":"2021-3-24","title":"3D visualization of SARS-CoV-2 infection and receptor distribution in Syrian hamster lung lobes display distinct spatial arrangements","abstract":"SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRRS2) to facilitate viral-host membrane fusion. ACE2 and TMPRRS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq level, however, biologically relevant protein receptor organization in whole tissues is still poorly understood. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRRS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals were stained using antibodies against ACE2 and TMPRRS2, combined with fluorescent spike protein and SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize expression patterns. The data demonstrates that infection is restricted to sites with both ACE2 and TMPRRS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the terminal bronchioles and alveoli. Conversely, infection completely overlaps at these sites where ACE2 and TMPRSS2 co-localize.\n\n\n","version":"1.1","doi":"10.1101/2021.03.24.435771","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436684","pub_date":"2021-3-24","title":"Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects","abstract":"SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.","version":"1.1","doi":"10.1101/2021.03.23.436684","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.24.436620","pub_date":"2021-3-24","title":"B.1.526 SARS-CoV-2 variants identified in New York City are neutralized by vaccine-elicited and therapeutic monoclonal antibodies","abstract":"DNA sequence analysis recently identified the novel SARS-CoV-2 variant B.1.526 that is spreading at an alarming rate in the New York City area. Two versions of the variant were identified, both with the prevalent D614G mutation in the spike protein together with four novel point mutations and with an E484K or S477N mutation in the receptor binding domain, raising concerns of possible resistance to vaccine-elicited and therapeutic antibodies. We report that convalescent sera and vaccine-elicited antibodies retain full neutralizing titer against the S477N B.1.526 variant and neutralize the E484K version with a modest 3.5-fold decrease in titer as compared to D614G. The E484K version was neutralized with a 12-fold decrease in titer by the REGN10933 monoclonal antibody but the combination cocktail with REGN10987 was fully active. The findings suggest that current vaccines and therapeutic monoclonal antibodies will remain protective against the B.1.526 variants. The findings further support the value of wide-spread vaccination.","version":"1.1","doi":"10.1101/2021.03.24.436620","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436648","pub_date":"2021-3-24","title":"Phospholipidosis is a shared mechanism underlying the in vitro antiviral activity of many repurposed drugs against SARS-CoV-2","abstract":"Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs\u2014including those from phenotypic screens and others that we ourselves had found\u2014for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost. Drug-induced phospholipidosis is a single mechanism that may explain the in vitro efficacy of a wide-variety of therapeutics repurposed for COVID-19.","version":"1.1","doi":"10.1101/2021.03.23.436648","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425198","pub_date":"2021-3-24","title":"The impact of COVID-19 vaccination campaigns accounting for antibody-dependent enhancement","abstract":"COVID-19 vaccines are approved, vaccination campaigns are launched, and worldwide return to normality seems within close reach. Nevertheless, concerns about the safety of COVID-19 vaccines arose, due to their fast emergency approval. In fact, the problem of antibody-dependent enhancement was raised in the context of COVID-19 vaccines. We introduce a complex extension of the model underlying the pandemic preparedness tool CovidSim 1.1 (http://covidsim.eu/) to optimize vaccination strategies with regard to the onset of campaigns, vaccination coverage, vaccination schedules, vaccination rates, and efficiency of vaccines. Vaccines are not assumed to immunize perfectly. Some individuals fail to immunize, some reach only partial immunity, and \u2013 importantly \u2013 some develop antibody-dependent enhancement, which increases the likelihood of developing symptomatic and severe episodes (associated with higher case fatality) upon infection. Only a fraction of the population will be vaccinated, reflecting vaccination hesitancy or contraindications. The model is intended to facilitate decision making by exploring ranges of parameters rather than to be fitted by empirical data. We parameterized the model to reflect the situation in Germany and predict increasing incidence (and prevalence) in early 2021 followed by a decline by summer. Assuming contact reductions (curfews, social distancing, etc.) to be lifted in summer, disease incidence will peak again. Fast vaccine deployment contributes to reduce disease incidence in the first quarter of 2021, and delay the epidemic outbreak after the summer season. Higher vaccination coverage results in a delayed and reduced epidemic peak. A coverage of 75% - 80% is necessary to prevent an epidemic peak without further drastic contact reductions. With the vaccine becoming available, compliance with contact reductions is likely to fade. To prevent further economic damage from COVID-19, high levels of immunization need to be reached before next year\u2019s flu season, and vaccination strategies and disease management need to be flexibly adjusted. The predictive model can serve as a refined decision support tool for COVID-19 management.","version":"1.3","doi":"10.1101/2021.01.04.425198","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436613","pub_date":"2021-3-23","title":"Application of an integrated computational antibody engineering platform to design SARS-CoV-2 neutralizers","abstract":"As the COVID-19 pandemic continues to spread, hundreds of new initiatives including studies on existing medicines are running to fight the disease. To deliver a potentially immediate and lasting treatment to current and emerging SARS-CoV-2 variants, new collaborations and ways of sharing are required to create as many paths forward as possible. Here we leverage our expertise in computational antibody engineering to rationally design/optimize three previously reported SARS-CoV neutralizing antibodies and share our proposal towards anti-SARS-CoV-2 biologics therapeutics. SARS-CoV neutralizing antibodies, m396, 80R, and CR-3022 were chosen as templates due to their diversified epitopes and confirmed neutralization potency against SARS. Structures of variable fragment (Fv) in complex with receptor binding domain (RBD) from SARS-CoV or SARS-CoV2 were subjected to our established in silico antibody engineering platform to improve their binding affinity to SARS-CoV2 and developability profiles. The selected top mutations were ensembled into a focused library for each antibody for further screening. In addition, we convert the selected binders with different epitopes into the trispecific format, aiming to increase potency and to prevent mutational escape. Lastly, to avoid antibody induced virus activation or enhancement, we applied NNAS and DQ mutations to the Fc region to eliminate effector functions and extend half-life.","version":"1.1","doi":"10.1101/2021.03.23.436613","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436611","pub_date":"2021-3-23","title":"Characterization and functional interrogation of SARS-CoV-2 RNA interactome","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 pandemic, which has caused a devastating global health crisis. The emergence of highly transmissible novel viral strains that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology and to develop additional therapeutic strategies. Using a comprehensive identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-2 viral genome during infection. By systematically knocking down their expression in a human lung epithelial cell line, we found that the majority of the RBPs identified in our study are proviral factors that regulate SARS-CoV-2 genome replication. We showed that some of these proteins represented drug targets of interest for inhibiting SARS-CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-CoV-2 RNA interactome during infection and highlights candidates for host-centered antiviral therapies.","version":"1.1","doi":"10.1101/2021.03.23.436611","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436642","pub_date":"2021-3-23","title":"Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2","abstract":"The identification of CD4+ T cell epitopes is essential for the design of effective vaccines capable of inducing neutralizing antibodies and long-term immunity. Here we demonstrate in COVID-19 patients a robust CD4+ T cell response to naturally processed SARS-CoV-2 Spike and Nucleoprotein, including effector, helper and memory T cells. By characterizing 2,943 Spike-reactive T cell clones, we found that 34% of the clones and 93% of the patients recognized a conserved immunodominant region encompassing residues S346-365 in the RBD and comprising three nested HLA-DR and HLA-DP restricted epitopes. By using pre- and post-COVID-19 samples and Spike proteins from alpha and beta coronaviruses, we provide in vivo evidence of cross-reactive T cell responses targeting multiple sites in the SARS-CoV-2 Spike protein. The possibility of leveraging immunodominant and cross-reactive T helper epitopes is instrumental for vaccination strategies that can be rapidly adapted to counteract emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.03.23.436642","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436637","pub_date":"2021-3-23","title":"The SARS-CoV-2 replication-transcription complex is a priority target for broad-spectrum pan-coronavirus drugs","abstract":"In the absence of effective treatment, COVID-19 is likely to remain a global disease burden.\nCompounding this threat is the near certainty that novel coronaviruses with pandemic potential will emerge in years to come. Pan-coronavirus drugs \u2013 agents active against both SARS-CoV-2 and other coronaviruses \u2013 would address both threats. A strategy to develop such broad-spectrum inhibitors is to pharmacologically target binding sites on SARS-CoV-2 proteins that are highly conserved in other known coronaviruses, the assumption being that any selective pressure to keep a site conserved across past viruses will apply to future ones. Here, we systematically mapped druggable binding pockets on the experimental structure of fifteen SARS-CoV-2 proteins and analyzed their variation across twenty-seven \u03b1- and \u03b2-coronaviruses and across thousands of SARS-CoV-2 samples from COVID-19 patients. We find that the two most conserved druggable sites are a pocket overlapping the RNA binding site of the helicase nsp13, and the catalytic site of the RNA-dependent RNA polymerase nsp12, both components of the viral replication-transcription complex. We present the data on a public web portal (https://www.thesgc.org/SARSCoV2_pocketome/) where users can interactively navigate individual protein structures and view the genetic variability of drug binding pockets in 3D.","version":"1.1","doi":"10.1101/2021.03.23.436637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.15.991844","pub_date":"2021-3-23","title":"V367F mutation in SARS-CoV-2 spike RBD emerging during the early transmission phase enhances viral infectivity through increased human ACE2 receptor binding affinity","abstract":"The current global pandemic of COVID-19 is caused by a novel coronavirus SARS-CoV-2. The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring mutations in the RBD during the early transmission phase have altered the receptor binding affinity and infectivity, firstly we analyzed in silico the binding dynamics between mutated SARS-CoV-2 RBDs and the human ACE2 receptor. Among 32,123 genomes of SARS-CoV-2 isolates (January through March, 2020), 302 non-synonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations. The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding ELISA, surface plasmon resonance, and pseudotyped virus assays. Genome phylogenetic analysis of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F + D614G) which emerged later and formed a distinct sub-cluster. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of SARS-CoV-2 under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines. A novel coronavirus SARS-CoV-2 has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether the mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and caused them more infectious, should be paid more attentions to. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase that have increased human ACE2 receptor binding affinity and infectivity. The RBD mutation analysis provides insights into SARS-CoV-2 evolution. The continuous surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is important and necessary, particularly when the direct correlation between the virus variations and vaccine effectiveness is underdetermined during the sustained COVID-19 pandemic.","version":"1.6","doi":"10.1101/2020.03.15.991844","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434584","pub_date":"2021-3-23","title":"New SARS-CoV-2 lineages could evade CD8+ T-cells response","abstract":"Many SARS-CoV-2 variants of concern have emerged since the Covid-19 outburst, notably the lineages detected in the UK, South Africa, and Brazil. Their increased transmissibility and higher viral load put them in the spotlight. Much has been investigated on the ability of those new variants to evade antibody recognition. However, not enough attention has been given to pre-existing and induced SARS-CoV-2-specific CD8+ T cell responses during the natural course of infection by new lineages. In this work, we investigated the SARS-CoV-2-specific CD8+ T cell epitopes from the main variants of concern and the potential of associated mutations to trigger or hinder CD8+ T-cells response. We also estimated the population\u2019s coverage of these different lineages, considering peptide binding predictions to class I HLA alleles from 29 countries to investigate differences in the fraction of individuals expected to respond to a given epitope set from new and previous lineages. We observed a lower populational coverage for 20B/S.484K (P.2 lineage) in contrast to an increased coverage found for 20H/501Y.V2 (B.1.351 Lineage) and 20J/501Y.V3 (P.1 lineage) compared to a reference lineage. Moreover, mutations such as Spike N501Y and Nucleocapsid T205I were predicted to have an overall higher affinity through HLA-I than the reference sequence. In summary, the data in this work provided evidence for the existence of potentially immunogenic and conserved epitopes across new SARS-CoV-2 variants, but also highlights the reduced populational\u2019s coverage for the Brazilian lineage P.2, suggesting its potential to evade from CD8+ T-cell responses. Our results also may guide efforts to characterize and validate relevant peptides to trigger CD8+ T-cell responses, and design new universal T-cell-inducing vaccine candidates that minimize detrimental effects of viral diversification and at the same time induce responses to a broad human population.","version":"1.2","doi":"10.1101/2021.03.09.434584","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436379","pub_date":"2021-3-23","title":"Antidepressant and antipsychotic drugs reduce viral infection by SARS-CoV-2 and fluoxetine show antiviral activity against the novel variants in vitro","abstract":"Repurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants. Several antidepressant drugs and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7 and B.1.351. Several clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the (B.1) lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudo viruses with N501Y, K417N, and E484K spike mutations, and the VoC-1 (B.1.1.7) and VoC-2 (B.1.351) variants of SARS-CoV-2. Our study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern.","version":"1.1","doi":"10.1101/2021.03.22.436379","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436564","pub_date":"2021-3-23","title":"Protein-primed RNA synthesis in SARS-CoVs and structural basis for inhibition by AT-527","abstract":"How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and \u22122 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5\u2019-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 \u00c5 resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and \u22122, representing a promising drug for COVID-19 treatment.","version":"1.1","doi":"10.1101/2021.03.23.436564","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436573","pub_date":"2021-3-23","title":"Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients","abstract":"T cells play pivotal roles in protective immunity against SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate the production of antigen-specific antibodies; however, T cell receptor (TCR) clonotypes used by SARS-CoV-2-specific Tfh cells have not been well characterized. Here, we first identified and crystallized public TCR of Tfh clonotypes that are shared and expanded in unhospitalized COVID-19-recovered patients. These clonotypes preferentially recognized SARS-CoV-2 spike (S) protein epitopes which are conserved among emerging SARS-CoV-2 variants. These clonotypes did not react with S proteins derived from common cold human coronaviruses, but cross-reacted with symbiotic bacteria, which might confer the publicity. Among SARS-CoV-2 S epitopes, S864-882, presented by frequent HLA-DR alleles, could activate multiple public Tfh clonotypes in COVID-19-recovered patients. Furthermore, S864-882-loaded HLA tetramer preferentially bound to CD4+ T cells expressing CXCR5. In this study, we identified and crystallized public TCR for SARS-CoV-2 that may contribute to the prevention of COVID-19 aggravation.","version":"1.1","doi":"10.1101/2021.03.23.436573","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436481","pub_date":"2021-3-23","title":"Neutralization of European, South African, and United States SARS-CoV-2 mutants by a human antibody and antibody domains","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission with several emerging variants remain uncontrolled in many countries, indicating the pandemic remains severe. Recent studies showed reduction of neutralization against these emerging SARS-CoV-2 variants by vaccine-elicited antibodies. Among those emerging SARS-CoV-2 variants, a panel of amino acid mutations was characterized including those in the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. In the present study, we evaluated our previously identified antibody and antibody domains for binding to these RBD variants with the emerging mutations, and neutralization of pseudo typed viruses carrying spike proteins with such mutations. Our results showed that one previously identified antibody domain, ab6, can bind 32 out of 35 RBD mutants tested in an ELISA assay. All three antibodies and antibody domains can neutralize pseudo typed B.1.1.7 (UK variant), but only the antibody domain ab6 can neutralize the pseudo typed virus with the triple mutation (K417N, E484K, N501Y). This domain and its improvements have potential for therapy of infections caused by SARS-CoV-2 mutants.","version":"1.1","doi":"10.1101/2021.03.22.436481","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436644","pub_date":"2021-3-23","title":"Dimeric form of SARS-CoV-2 polymerase","abstract":"The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 \u00c5 resolution. In this structure, the two RdRps contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs for transcription.","version":"1.1","doi":"10.1101/2021.03.23.436644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.23.436593","pub_date":"2021-3-23","title":"Dry Swab Method of sample collection for SARS-CoV2 testing can be used for culturing virus","abstract":"Earlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated. Swabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25\u00b0C\u00b11\u00b0C) or at 4\u00b0C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells. The RT-qPCR data suggests that SD incubated both at RT and 4\u00b0C harbors viral particles that are viable and culturable at par with SVTM and STE. The dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.","version":"1.1","doi":"10.1101/2021.03.23.436593","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.28.428665","pub_date":"2021-3-23","title":"Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce robust immune responses in mice","abstract":"Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two dose heterologous vaccination regimens than single dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.","version":"1.2","doi":"10.1101/2021.01.28.428665","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435423","pub_date":"2021-3-22","title":"Network medicine links SARS-CoV-2/COVID-19 infection to brain microvascular injury and neuroinflammation in dementia-like cognitive impairment","abstract":"Dementia-like cognitive impairment is an increasingly reported complication of SARS-CoV-2 infection. However, the underlying mechanisms responsible for this complication remain unclear. A better understanding of causative processes by which COVID-19 may lead to cognitive impairment is essential for developing preventive interventions. In this study, we conducted a network-based, multimodal genomics comparison of COVID-19 and neurologic complications. We constructed the SARS-CoV-2 virus-host interactome from protein-protein interaction assay and CRISPR-Cas9 based genetic assay results, and compared network-based relationships therein with those of known neurological manifestations using network proximity measures. We also investigated the transcriptomic profiles (including single-cell/nuclei RNA-sequencing) of Alzheimer\u2019s disease (AD) marker genes from patients infected with COVID-19, as well as the prevalence of SARS-CoV-2 entry factors in the brains of AD patients not infected with SARS-CoV-2. We found significant network-based relationships between COVID-19 and neuroinflammation and brain microvascular injury pathways and processes which are implicated in AD. We also detected aberrant expression of AD biomarkers in the cerebrospinal fluid and blood of patients with COVID-19. While transcriptomic analyses showed relatively low expression of SARS-CoV-2 entry factors in human brain, neuroinflammatory changes were pronounced. In addition, single-nucleus transcriptomic analyses showed that expression of SARS-CoV-2 host factors (BSG and FURIN) and antiviral defense genes (LY6E, IFITM2, IFITM3, and IFNAR1) was significantly elevated in brain endothelial cells of AD patients and healthy controls relative to neurons and other cell types, suggesting a possible role for brain microvascular injury in COVID-19-mediated cognitive impairment. Notably, individuals with the AD risk allele APOE E4/E4 displayed reduced levels of antiviral defense genes compared to APOE E3/E3 individuals. Our results suggest significant mechanistic overlap between AD and COVID-19, strongly centered on neuroinflammation and microvascular injury. These results help improve our understanding of COVID-19-associated neurological manifestations and provide guidance for future development of preventive or treatment interventions.","version":"1.1","doi":"10.1101/2021.03.15.435423","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431721","pub_date":"2021-3-22","title":"Immune characterization and profiles of SARS-CoV-2 infected patients reveals potential host therapeutic targets and SARS-CoV-2 oncogenesis mechanism","abstract":"The spread of SARS-CoV-2 and the increasing mortality rates of COVID-19 create an urgent need for treatments, which are currently lacking. Although vaccines have been approved by the FDA for emergency use in the U.S., patients will continue to require pharmacologic intervention to reduce morbidity and mortality as vaccine availability remains limited. The rise of new variants makes the development of therapeutic strategies even more crucial to combat the current pandemic and future outbreaks. Evidence from several studies suggests the host immune response to SARS-CoV-2 infection plays a critical role in disease pathogenesis. Consequently, host immune factors are becoming more recognized as potential biomarkers and therapeutic targets for COVID-19. To develop therapeutic strategies to combat current and future coronavirus outbreaks, understanding how the coronavirus hijacks the host immune system during and after the infection is crucial. In this study, we investigated immunological patterns or characteristics of the host immune response to SARS-CoV-2 infection that may contribute to the disease severity of COVID-19 patients. We analyzed large bulk RNASeq and single cell RNAseq data from COVID-19 patient samples to immunoprofile differentially expressed gene sets and analyzed pathways to identify human host protein targets. We observed an immunological profile of severe COVID-19 patients characterized by upregulated cytokines, interferon-induced proteins, and pronounced T cell lymphopenia, supporting findings by previous studies. We identified a number of host immune targets including PERK, PKR, TNF, NF-kB, and other key genes that modulate the significant pathways and genes identified in COVID-19 patients. Finally, we identified genes modulated by COVID-19 infection that are implicated in oncogenesis, including E2F transcription factors and RB1, suggesting a mechanism by which SARS-CoV-2 infection may contribute to oncogenesis. Further clinical investigation of these targets may lead to bonafide therapeutic strategies to treat the current COVID-19 pandemic and protect against future outbreaks and viral escape variants.","version":"1.2","doi":"10.1101/2021.02.17.431721","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436441","pub_date":"2021-3-22","title":"Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in na\u00efve and COVID-19 recovered individuals","abstract":"The rapid development and deployment of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in na\u00efve individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in na\u00efve individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has been questioned. Here we characterized SARS-CoV-2 spike-specific humoral and cellular immunity in na\u00efve and previously infected individuals during full BNT162b2 vaccination. Our results demonstrate that the second dose increases both the humoral and cellular immunity in na\u00efve individuals. On the contrary, the second BNT162b2 vaccine dose results in a reduction of cellular immunity in COVID-19 recovered individuals, which suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.22.436441","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.384040","pub_date":"2021-3-22","title":"Rapidly self-sterilizing PPE capable of 99.9% SARS-CoV-2 deactivation in 30 seconds","abstract":"The coronavirus disease 2019 (COVID-19) has created an acute worldwide demand for sustained broadband pathogen suppression in households, hospitals, and public spaces. The US recently passed a new sad milestone of 500,000 deaths due to COVID-19, the highest rate anywhere in the world. In response, we have created a rapid-acting, self-sterilizing PPE configurations capable of killing SARS-CoV-2 and other microbes in seconds. The highly active material destroys pathogens faster than any conventional copper configuration. The material maintains its antimicrobial efficacy over sustained use and is shelf stable. We have performed rigorous testing in accordance with guidelines from U.S. governing authorities and believe that the material could offer broad spectrum, non-selective defense against most microbes via integration into masks and other protective equipment. A novel configuration of copper offering continued fast-acting protection against viruses and bacteria, including SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.11.16.384040","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.21.436312","pub_date":"2021-3-22","title":"Predicting Hosts Based on Early SARS-CoV-2 Samples and Analyzing Later World-wide Pandemic in 2020","abstract":"The SARS-CoV-2 pandemic has raised the concern for identifying hosts of the virus since the early-stage outbreak. To address this problem, we proposed a deep learning method, DeepHoF, based on extracting the viral genomic features automatically, to predict host likelihood scores on five host types, including plant, germ, invertebrate, non-human vertebrate and human, for novel viruses. DeepHoF made up for the lack of an accurate tool applicable to any novel virus and overcame the limitation of the sequence similarity-based methods, reaching a satisfactory AUC of 0.987 on the five-classification. Additionally, to fill the gap in the efficient inference of host species for SARS-CoV-2 using existed tools, we conducted a deep analysis on the host likelihood profile calculated by DeepHoF. Using the isolates sequenced in the earliest stage of COVID-19, we inferred minks, bats, dogs and cats were potential hosts of SARS-CoV-2, while minks might be one of the most noteworthy hosts. Several genes of SARS-CoV-2 demonstrated their significance in determining the host range. Furthermore, the large-scale genome analysis, based on DeepHoF\u2019s computation for the later world-wide pandemic in 2020, disclosed the uniformity of host range among SARS-CoV-2 samples and the strong association of SARS-CoV-2 between humans and minks.","version":"1.1","doi":"10.1101/2021.03.21.436312","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436468","pub_date":"2021-3-22","title":"Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2","abstract":"The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARS-CoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.","version":"1.1","doi":"10.1101/2021.03.22.436468","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.147074","pub_date":"2021-3-22","title":"Evolution and spread of SARS-CoV-2 likely to be affected by climate","abstract":"COVID-19 pandemic has been extensively studied by many researchers. However, it is still unclear why it was restricted to higher latitudes during the initial days and later cascaded in the tropics. Here, we analyzed 176 SARS-CoV-2 genomes across different latitudes and climate (Koppen\u2019s climate) that provided insights about within species virus evolution and its relation to abiotic factors. Two genetically variant groups, named as G1 and G2 were identified, well defined by four mutations. The G1 group (ancestor), is mainly restricted to warm and moist, temperate climate (Koppen\u2019s C climate) while its descendent G2 group surpasses the climatic restrictions of G1, initially cascading into neighboring cold climate (D) of higher latitudes and later into hot climate of the tropics (A). It appears that the gradation of temperate climate (Cfa-Cfb) to cold climate (Dfa-Dfb) drives the evolution of G1 into G2 variant group which later adapted to tropical climate (A) as well. It seems this virus followed inverse latitudinal gradient in the beginning due to its preference towards temperate (C) and cold climate (D). Our work elucidates virus evolutionary studies combined with climatic studies can provide crucial information about the pathogenesis and natural spreading pathways in such outbreaks which is hard to achieve through individual studies. Mutational insights gained may help design an efficacious vaccine. The authors elucidate adaptation of SARS-CoV-2 to different climates by studying phylogenetics and the distribution of strains on Koppen\u2019s climate map. Phylogenetic network divides SARS-CoV-2 strains into two variant groups, G1 and G2. G1 strains is restricted to Koppen\u2019s \u201ctemperate\u201d climate (mainly Cfa-Cfb). G2 strains has evolved from G1 to sustain in other climates mainly \u201chumid-continental\u201d (Dfa-Dfb) and \u201ctropical-savannah\u201d (Aw) climate.","version":"1.3","doi":"10.1101/2020.06.18.147074","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.435960","pub_date":"2021-3-22","title":"CVnCoV protects human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2","abstract":"The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines as the primary control option. Recently, viral mutants termed \u201cvariants of concern\u201d (VOC) have emerged with the potential to escape host immunity. VOC B.1.351 was first discovered in South Africa in late 2020, and causes global concern due to poor neutralization with propensity to evade preexisting immunity from ancestral strains. We tested the efficacy of a spike encoding mRNA vaccine (CVnCoV) against the ancestral strain BavPat1 and the novel VOC B.1.351 in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice developed elevated SARS-CoV-2 RBD-specific antibody as well as neutralization titers against the ancestral strain BavPat1. Neutralization titers against VOC B.1.351 were readily detectable but significantly reduced compared to BavPat1. VOC B.1.351-infected control animals experienced a delayed course of disease, yet nearly all SARS-CoV-2 challenged na\u00efve mice succumbed with virus dissemination and high viral loads. CVnCoV vaccine completely protected the animals from disease and mortality caused by either viral strain. Moreover, SARS-CoV-2 was not detected in oral swabs, lung, or brain in these groups. Only partial protection was observed in mice receiving the formalin-inactivated virus preparation. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV shows complete disease protection against the novel VOC B.1.351 in our studies.","version":"1.1","doi":"10.1101/2021.03.22.435960","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436427","pub_date":"2021-3-22","title":"Replicative fitness SARS-CoV-2 20I/501Y.V1 variant in a human reconstituted bronchial epithelium","abstract":"Since its emergence in 2019, circulating populations of the new coronavirus continuously acquired genetic diversity. At the end of 2020, a variant named 20I/501Y.V1 (lineage B.1.1.7) emerged and replaced other circulating strains in several regions. This phenomenon has been poorly associated to biological evidence that this variant and original strain exhibit different phenotypic characteristics. Here, we analyse the replication ability of this new variant in different cellular models using for comparison an ancestral D614G European strain (lineage B1). Results from comparative replication kinetics experiments in vitro and in a human reconstituted bronchial epithelium showed no difference. However, when both viruses were put in competition in a human reconstituted bronchial epithelium, the 20I/501Y.V1 variant outcompeted the ancestral strain. Altogether, these findings demonstrate that this new variant replicates more efficiently and could contribute to better understand the progressive replacement of circulating strains by the SARS-CoV-2 20I/501Y.V1 variant. The emergence of several SARS-CoV-2 variants raised numerous questions concerning the future course of the pandemic. We are currently observing a replacement of the circulating viruses by the variant from the United Kingdom known as 20I/501Y.V1 from B.1.1.7 lineage but there is little biological evidence that this new variant exhibit a different phenotype. In the present study, we used different cellular models to assess the replication ability of the 20I/501Y.V1 variant. Our results showed that this variant replicate more efficiently in a human reconstituted bronchial epithelium, which may explain why it spreads so rapidly in human populations.","version":"1.1","doi":"10.1101/2021.03.22.436427","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.06.434059","pub_date":"2021-3-22","title":"Structural Analysis of Spike Protein Mutations in the SARS-CoV-2 P.3 Variant","abstract":"A SARS-CoV-2 lineage designated as P.3 with multiple signature mutations in the Spike protein region was recently reported with cases from the Central Visayas Region of the Philippines. Whole genome sequencing revealed that the 33 samples under this lineage all contain the E484K, N501Y, and P681H Spike mutations previously found in variants of concern (VOC) such as the B.1.351, the P.1 and B.1.1.7 variants first reported in South Africa, Brazil, and the United Kingdom, respectively. The possible implications of the mutations found in the Spike protein of P.3 were analyzed for their potential effects on structure, stability, and molecular surface character. The analysis suggests that these mutations could significantly impact the possible interactions of the Spike protein with the ACE2 receptor and neutralizing antibodies, and warrants further clinical investigation. Some of the mutations affecting the N and C terminal domains may have effects on Spike monomer and trimer stability. This report provides insights on relevant targets for the design of future diagnostics, therapeutics and vaccines against the evolving SARS-CoV-2 variants in the Philippines.","version":"1.2","doi":"10.1101/2021.03.06.434059","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436454","pub_date":"2021-3-22","title":"Structural modeling of the SARS-CoV-2 Spike/human ACE2 complex interface can identify high-affinity variants associated with increased transmissibility","abstract":"The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are conformationally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S447N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S447N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.","version":"1.1","doi":"10.1101/2021.03.22.436454","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427676","pub_date":"2021-3-22","title":"Personalized Virus Load Curves of SARS-CoV-2 Infection","abstract":"This manuscript has been withdrawn due to submission errors. Therefore, the authors do not wish this work to be cited as a reference. Please see and cite for reference the manuscript posted on medRxiv https://doi.org/10.1101/2021.01.21.21250268 If you have any questions, please contact the corresponding author.","version":"1.2","doi":"10.1101/2021.01.21.427676","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.22.436387","pub_date":"2021-3-22","title":"A method for intuitively extracting macromolecular dynamics from structural disorder","abstract":"Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.","version":"1.1","doi":"10.1101/2021.03.22.436387","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.20.436259","pub_date":"2021-3-21","title":"Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.","version":"1.1","doi":"10.1101/2021.03.20.436259","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.21.436311","pub_date":"2021-3-21","title":"Structural and energetic profiling of SARS-CoV-2 antibody recognition and the impact of circulating variants","abstract":"The SARS-CoV-2 pandemic highlights the need for a detailed molecular understanding of protective antibody responses. This is underscored by the emergence and spread of SARS-CoV-2 variants, including B.1.1.7, P1, and B.1.351, some of which appear to be less effectively targeted by current monoclonal antibodies and vaccines. Here we report a high resolution and comprehensive map of antibody recognition of the SARS-CoV-2 spike receptor binding domain (RBD), which is the target of most neutralizing antibodies, using computational structural analysis. With a dataset of nonredundant experimentally determined antibody-RBD structures, we classified antibodies by RBD residue binding determinants using unsupervised clustering. We also identified the energetic and conservation features of epitope residues and assessed the capacity of viral variant mutations to disrupt antibody recognition, revealing sets of antibodies predicted to effectively target recently described viral variants. This detailed structure-based reference of antibody RBD recognition signatures can inform therapeutic and vaccine design strategies.","version":"1.1","doi":"10.1101/2021.03.21.436311","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.19.436231","pub_date":"2021-3-21","title":"Critical interactions for SARS-CoV-2 spike protein binding to ACE2 identified by machine learning","abstract":"Both SARS-CoV and SARS-CoV-2 bind to the human ACE2 receptor. Based on high-resolution structures, the two viruses bind in practically identical conformations, although several residues of the receptor-binding domain (RBD) differ between them. Here we have used molecular dynamics (MD) simulations, machine learning (ML), and free energy perturbation (FEP) calculations to elucidate the differences in RBD binding by the two viruses. Although only subtle differences were observed from the initial MD simulations of the two RBD-ACE2 complexes, ML identified the individual residues with the most distinctive ACE2 interactions, many of which have been highlighted in previous experimental studies. FEP calculations quantified the corresponding differences in binding free energies to ACE2, and examination of MD trajectories provided structural explanations for these differences. Lastly, the energetics of emerging SARS-CoV-2 mutations were studied, showing that the affinity of the RBD for ACE2 is increased by N501Y and E484K mutations but is slightly decreased by K417N.","version":"1.1","doi":"10.1101/2021.03.19.436231","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.21253731","pub_date":"2021-03-20","title":"The Spike-specific IgA in milk commonly-elicited after SARS-Cov-2 infection is concurrent with a robust secretory antibody response, exhibits neutralization potency strongly correlated with IgA binding, and is highly durable over time","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Approximately 10% of infants will experience COVID-19 illness requiring advanced care (1). A potential mechanism to protect this population could be provided by passive immunity through the milk of a previously infected mother. We and others have reported on the presence of SARS-CoV-2-specific antibodies in human milk (2-5). We now report the prevalence of SARS-CoV-2 IgA in the milk of 75 COVID-19-recovered participants, and find that 88% of samples are positive for Spike-specific IgA. In a subset of these samples, 95% exhibited robust IgA activity as determined by endpoint binding titer, with 50% considered high-titer. These IgA positive specimens were also positive for Spike-specific antibodies bearing the secretory component. Levels of IgA antibodies and antibodies bearing secretory component were shown to be strongly positively correlated. The secretory IgA response was dominant among the milk samples tested compared to the IgG response, which was present in 75% of samples and found to be of high-titer in only 13% of cases. Our IgA durability analysis using 28 paired samples, obtained 4-6 weeks and 4-10 months after infection, found that all samples exhibited persistently significant Spike-specific IgA, with 43% of donors exhibiting increasing IgA titers over time. Finally, COVID-19 and pre-pandemic control milk samples were tested for the presence of neutralizing antibodies; 6 of 8 COVID-19 samples exhibited neutralization of Spike-pseudotyped VSV (IC\n                  <jats:sub>50</jats:sub>\n                  range, 2.39 \u2013 89.4ug/mL) compared to 1 of 8 controls. IgA binding and neutralization capacities were found to be strongly positively correlated. These data are highly relevant to public health, not only in terms of the protective capacity of these antibodies for breastfed infants, but also for the potential use of such antibodies as a COVID-19 therapeutic, given that secretory IgA is highly stable not only in milk and the infant mouth and gut, but in all mucosa including the gastrointestinal tract, upper airway, and lungs (6).\n                </jats:p>","version":null,"doi":"10.1101/2021.03.16.21253731","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.17.435823","pub_date":"2021-3-19","title":"A novel SARS-CoV-2 related virus with complex recombination isolated from bats in Yunnan province, China","abstract":"A novel beta-coronavirus, SARS-CoV-2, emerged in late 2019 and rapidly spread throughout the world, causing the COVID-19 pandemic. However, the origin and direct viral ancestors of SARS-CoV-2 remain elusive. Here, we discovered a new SARS-CoV-2-related virus in Yunnan province, in 2018, provisionally named PrC31, which shares 90.7% and 92.0% nucleotide identities with SARS-CoV-2 and the bat SARSr-CoV ZC45, respectively. Sequence alignment revealed that several genomic regions shared strong identity with SARS-CoV-2, phylogenetic analysis supported that PrC31 shares a common ancestor with SARS-CoV-2. The receptor binding domain of PrC31 showed only 64.2% amino acid identity with SARS-CoV-2. Recombination analysis revealed that PrC31 underwent multiple complex recombination events within the SARS-CoV and SARS-CoV-2 sub-lineages, indicating the evolution of PrC31 from yet-to-be-identified intermediate recombination strains. Combination with previous studies revealed that the beta-CoVs may possess more complicated recombination mechanism. The discovery of PrC31 supports that bats are the natural hosts of SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.03.17.435823","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.19.436183","pub_date":"2021-3-19","title":"Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19","abstract":"Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 (\u22642-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.03.19.436183","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.18.436001","pub_date":"2021-3-19","title":"Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection","abstract":"Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-\u03b1). In COVID-19 there is a deficit in DC numbers and IFN-\u03b1 production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and non-hospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers as CD86 and CD4 were only restored in previously non-hospitalized patients while integrin \u03b27 and indoleamine 2,3- dyoxigenase (IDO) no restoration was observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19.","version":"1.1","doi":"10.1101/2021.03.18.436001","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.19.435959","pub_date":"2021-3-19","title":"Common dandelion (Taraxacum officinale) efficiently blocks the interaction between ACE2 cell surface receptor and SARS-CoV-2 spike protein D614, mutants D614G, N501Y, K417N and E484K in vitro","abstract":"On 11th March 2020, coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, was declared as a global pandemic by the World Health Organization (WHO). To date, there are rapidly spreading new \u201cvariants of concern\u201d of SARS-CoV-2, the United Kingdom (B.1.1.7), the South African (B.1.351) or Brasilian (P.1) variant. All of them contain multiple mutations in the ACE2 receptor recognition site of the spike protein, compared to the original Wuhan sequence, which is of great concern, because of their potential for immune escape. Here we report on the efficacy of common dandelion (Taraxacum officinale) to block protein-protein interaction of spike S1 to the human ACE2 cell surface receptor. This could be shown for the original spike D614, but also for its mutant forms (D614G, N501Y, and mix of K417N, E484K, N501Y) in human HEK293-hACE2 kidney and A549-hACE2-TMPRSS2 lung cells. High molecular weight compounds in the water-based extract account for this effect. Infection of the lung cells using SARS-CoV-2 spike pseudotyped lentivirus particles was efficiently prevented by the extract and so was virus-triggered pro-inflammatory interleukin 6 secretion. Modern herbal monographs consider the usage of this medicinal plant as safe. Thus, the in vitro results reported here should encourage further research on the clinical relevance and applicability of the extract as prevention strategy for SARS-CoV-2 infection. SARS-CoV-2 is steadily mutating during continuous transmission among humans. This might eventually lead the virus into evading existing therapeutic and prophylactic approaches aimed at the viral spike. We found effective inhibition of protein-protein interaction between the human virus cell entry receptor ACE2 and SARS-CoV-2 spike, including five relevant mutations, by water-based common dandelion (Taraxacum officinale) extracts. This was shown in vitro using human kidney (HEK293) and lung (A549) cells, overexpressing the ACE2 and ACE2/TMPRSS2 protein, respectively. Infection of the lung cells using SARS-CoV-2 pseudotyped lentivirus was efficiently prevented by the extract. The results deserve more in-depth analysis of dandelions\u2019 effectiveness in SARS-CoV-2 prevention and now require confirmatory clinical evidence.","version":"1.1","doi":"10.1101/2021.03.19.435959","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434722","pub_date":"2021-3-19","title":"Structure and dynamics of the SARS-CoV-2 envelope protein monomer","abstract":"Coronaviruses, especially SARS-CoV-2, present an ongoing threat for human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic \u03b1-helical transmembrane domain (TMD) and amphiphilic \u03b1-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43 and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane. This may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate budding of the viral particles. The presented results may be helpful for better understanding of the function of coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.","version":"1.2","doi":"10.1101/2021.03.10.434722","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.19.435740","pub_date":"2021-3-19","title":"The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2, in vitro","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the mucosa of the tongue and gingiva, the oral cavity seems like it is an entry point for SARS-CoV-2. Daily oral care using mouthwash seems to play an important role in preventing SARS-CoV-2 infection. However, the relationship between daily oral care and the mechanisms of virus entry into host cells is unclear. In this study, we evaluated the inhibitory effects of ingredients that are generally contained in toothpaste and mouthwash on the interaction between the spike protein and ACE2 and on the serine protease activity of TMPRSS2 using an enzyme-linked immunosorbent assay and in vitro enzyme assay, respectively. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to the inhibitor-binding site of ACE2. In addition, tranexamic acid and 6-aminohexanoic acid, which act as serine protease inhibitors, exerted inhibitory effects on TMPRSS2 protease activity. Further experimental and clinical studies are needed to further elucidate these mechanisms. Our findings support the possibility that toothpaste and mouthwash contain ingredients that inhibit SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.03.19.435740","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424260","pub_date":"2021-3-19","title":"TCR meta-clonotypes for biomarker discovery with tcrdist3: identification of public, HLA-restricted SARS-CoV-2 associated TCR features","abstract":"As the mechanistic basis of adaptive cellular antigen recognition, T cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages antigen-enriched repertoires to form meta-clonotypes \u2013 groups of biochemically similar TCRs \u2013 that can be used to robustly identify and quantify functionally similar TCRs in bulk repertoires. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the 17 SARS-CoV-2 antigen-enriched repertoires with the strongest evidence of HLA-restriction. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (FDR < 0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.","version":"1.2","doi":"10.1101/2020.12.24.424260","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.12.435194","pub_date":"2021-3-19","title":"Antibody evasion by the Brazilian P.1 strain of SARS-CoV-2","abstract":"Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.","version":"1.2","doi":"10.1101/2021.03.12.435194","journal":"bioRxiv","score":null},{"id":"10.1101/292979","pub_date":"2021-3-19","title":"Manipulation of the unfolded protein response: a pharmacological strategy against coronavirus infection","abstract":"Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1\u03b1 and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release \u223c1,000-fold. Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection. SARS-CoV-2 is the novel coronavirus responsible for the COVID-19 pandemic which has resulted in over 100 million cases since the end of 2019. Most people infected with the virus will experience mild to moderate respiratory illness and recover without any special treatment. However, older people, and those with underlying medical problems like chronic respiratory disease are more likely to develop a serious illness. So far, more than 2 million people have died of COVID-19. Unfortunately, there is no specific medication for this viral disease. In order to produce viral proteins and to replicate their genetic information, all coronaviruses use a cellular structure known as the endoplasmic reticulum or ER. However, the massive production and modification of viral proteins stresses the ER and this activates a compensatory cellular response that tries to reduce ER protein levels. This is termed the unfolded protein response or UPR. We believe that coronaviruses take advantage of the activation of the UPR to enhance their replication. The UPR is also activated in some types of cancer and neurodegenerative disorders and UPR inhibitor drugs have been developed to tackle these diseases. In this work, we have tested some of these compounds in human lung cells infected with SARS-CoV-2 and found that virus production was reduced 1000-fold in human lung cells.","version":"1.4","doi":"10.1101/292979","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424644","pub_date":"2021-3-19","title":"Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation","abstract":"Coagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. We performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced Factor XIII (FXII) activation using a chromogenic substrate assay. FXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasma FXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro. Collectively, our study supports that the NETs/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both, NETs and FXIIa, could provide a strategy to mitigate COVID-19-induced thrombo-inflammation. This study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).","version":"1.2","doi":"10.1101/2020.12.29.424644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.19.436166","pub_date":"2021-3-19","title":"Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients","abstract":"Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.","version":"1.1","doi":"10.1101/2021.03.19.436166","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.435913","pub_date":"2021-3-18","title":"Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase","abstract":"SARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5\u2019 capping of viral mRNAs. The formation of the 5\u2019 cap-1 structure is known to require a guanylyltransferase (GTase) as well as 5\u2019 triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5\u2019 end of viral RNA via a 5\u2019 to 5\u2019 triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2. SARS-CoV-2 is a respiratory RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Coronaviruses encode an RNA polymerase which, in combination with other viral proteins, is responsible for synthesising capped viral mRNA. mRNA cap synthesis requires a guanylyltransferase enzyme; here we show that the SARS-CoV-2 guanylyltransferase is located in the viral RNA polymerase, and we identify the protein domain responsible for guanylyltransferase activity. Furthermore we demonstrate that remdesivir triphosphate, the active metabolite of remdesivir, inhibits both the guanylyltransferase and RNA polymerase functions of the SARS-CoV-2 RNA polymerase. These findings improve understanding of the coronavirus mRNA cap synthesis mechanism, in addition to highlighting a new target for the development of therapeutics to treat SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.03.17.435913","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434000","pub_date":"2021-3-18","title":"Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2","abstract":"The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid\u2013liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid\u2013liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid\u2013liquid phase separation, suggesting a therapeutic route for Covid-19. Atomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.","version":"1.2","doi":"10.1101/2021.03.05.434000","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.425793","pub_date":"2021-3-18","title":"Extracellular vimentin as a target against SARS-CoV-2 host cell invasion","abstract":"Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. In the case of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) has been identified as a necessary receptor, but not all ACE2-expressing cells are equally infected, suggesting that other extracellular factors are involved in host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens\u2019 cellular uptake. Here, we present evidence that extracellular vimentin might act as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry. We demonstrate direct binding between vimentin and SARS-CoV-2 pseudovirus coated with the SARS-CoV-2 spike protein and show that antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. Our results suggest new therapeutic strategies for preventing and slowing SARS-CoV-2 infection, focusing on targeting cell host surface vimentin.","version":"1.2","doi":"10.1101/2021.01.08.425793","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.433449","pub_date":"2021-3-18","title":"Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in rodents","abstract":"We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and \u22122, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and \u22122 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.","version":"1.2","doi":"10.1101/2021.03.08.433449","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.433877","pub_date":"2021-3-18","title":"A map of binary SARS-CoV-2 protein interactions implicates host immune regulation and ubiquitination","abstract":"Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly.","version":"1.2","doi":"10.1101/2021.03.15.433877","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.18.435945","pub_date":"2021-3-18","title":"Myocarditis in naturally infected pets with the British variant of COVID-19","abstract":"Domestic pets can contract SARS-CoV-2 infection but, based on the limited information available to date, it is unknown whether the new British B.1.1.7 variant can more easily infect certain animal species or increase the possibility of human-to-animal transmission. In this study, we report the first cases of infection of domestic cats and dogs by the British B.1.1.7 variant of SARS-CoV-2 diagnosed at a specialist veterinary hospital in the South-East of England. Furthermore, we discovered that many owners and handlers of these pets had developed Covid-19 respiratory symptoms 3-6 weeks before their pets became ill and had also tested PCR positive for Covid-19. Interestingly, all these B.1.1.7 infected pets developed atypical clinical manifestations, including severe cardiac abnormalities secondary to myocarditis and a profound impairment of the general health status of the patient but without any primary respiratory signs. Together, our findings demonstrate for the first time the ability for companion animals to be infected by the B.1.1.7 variant of SARS-CoV-2 and raise questions regarding its pathogenicity in these animals. Moreover, given the enhanced infectivity and transmissibility of B.1.1.7 variant for humans, these findings also highlights more than ever the risk that companion animals may potentially play a significant role in SARS-CoV-2 outbreak dynamics than previously appreciated.","version":"1.1","doi":"10.1101/2021.03.18.435945","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.435863","pub_date":"2021-3-18","title":"Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies","abstract":"Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.","version":"1.1","doi":"10.1101/2021.03.17.435863","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.18.435972","pub_date":"2021-3-18","title":"A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses","abstract":"The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 na\u00efve individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.18.435972","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.435802","pub_date":"2021-3-17","title":"Characterisation of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV2 variants","abstract":"The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7 and B.1.351 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralising antibody approaches. Furthermore, we report a pre-clinical characterisation package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralisation capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralised four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections.","version":"1.1","doi":"10.1101/2021.03.17.435802","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.21253625","pub_date":"2021-03-17","title":"A proteome-wide genetic investigation identifies several SARS-CoV-2-exploited host targets of clinical relevance","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The virus SARS-CoV-2 can exploit biological vulnerabilities in susceptible hosts that predispose to development of severe COVID-19. Previous reports have identified several host proteins related to the interferon response (e.g. OAS1), interleukin-6 signalling (IL-6R), and the coagulation cascade (linked via ABO) that were associated with risk of COVID-19. In the present study, we performed proteome-wide genetic colocalisation tests leveraging publicly available protein and COVID-19 datasets, to identify additional proteins that may contribute to COVID-19 risk. Our analytic approach identified several known targets (e.g. ABO, OAS1), but also nominated new proteins such as soluble FAS (colocalisation probability &gt; 0.9, p = 1 \u00d7 10\n                  <jats:sup>\u22124</jats:sup>\n                  ), implicating FAS-mediated apoptosis as a potential target for COVID-19 risk. We also undertook polygenic (pan) and cis-Mendelian randomisation analyses that showed consistent associations of genetically predicted ABO protein with several COVID-19 phenotypes. The ABO signal was associated with plasma concentrations of several proteins, with the strongest association observed with CD209 in several proteomic datasets. We demonstrated experimentally that CD209 directly interacts with the spike protein of SARS-CoV-2, suggesting a mechanism that could explain the ABO association with COVID-19. Our work provides a prioritised list of host targets potentially exploited by SARS-CoV-2 and is a precursor for further research on CD209 and FAS as therapeutically tractable targets for COVID-19.\n                </jats:p>","version":null,"doi":"10.1101/2021.03.15.21253625","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.16.435700","pub_date":"2021-3-17","title":"SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-\u03baB pathway","abstract":"Pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induces inflammatory cytokines and chemokines including IL-6, IL-1\u00df, TNFa, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and neucleocapsid (N) proteins. When stimulated with extracellular S protein, human lung epithelial cells A549 also produce inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly are non-inflammatory, but elicit an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-\u03baB pathway in a MyD88-dependent manner. Further, such an activation of the NF-\u03baB pathway is abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein induces IL-6, TNF-a, and IL-1 \u00df in wild-type, but not Tlr2-deficient mice. Together these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.","version":"1.1","doi":"10.1101/2021.03.16.435700","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.434488","pub_date":"2021-3-17","title":"SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2","abstract":"Since SARS-CoV-2 emerged in 2019, genomic sequencing has identified mutations in the viral RNA including in the receptor-binding domain of the Spike protein. Structural characterisation of the Spike carrying point mutations aids in our understanding of how these mutations impact binding of the protein to its human receptor, ACE2, and to therapeutic antibodies. The Spike G485R mutation has been observed in multiple isolates of the virus and mutation of the adjacent residue E484 to lysine is known to contribute to antigenic escape. Here, we have crystallised the SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2. The crystal structure shows that while the G485 residue does not have a direct interaction with ACE2, its mutation to arginine affects the structure of the loop made by residues 480-488 in the receptor-binding motif, disrupting the interactions of neighbouring residues with ACE2 and with potential implications for antigenic escape from vaccines, antibodies and other biologics directed against SARS-CoV-2 Spike.","version":"1.1","doi":"10.1101/2021.03.16.434488","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.435637","pub_date":"2021-3-17","title":"Epigallocatechin Gallate from Green Tea Effectively Blocks Infection of SARS-CoV-2 and New Variants by Inhibiting Spike Binding to ACE2 Receptor","abstract":"As the COVID-19 pandemic rages on, the new SARS-CoV-2 variants have emerged in the different regions of the world. These newly emerged variants have mutations in their spike (S) protein that may confer resistance to vaccine-elicited immunity and existing neutralizing antibody therapeutics. Therefore, there is still an urgent need of safe, effective, and affordable agents for prevention/treatment of SARS-CoV-2 and its variant infection. Here, we demonstrated that green tea beverage (GTB) or its major ingredient, epigallocatechin gallate (EGCG), were highly effective in inhibiting infection of live SARS-CoV-2 and human coronavirus (HCoV OC43). In addition, infection of the pseudoviruses with spikes of the new variants (UK-B.1.1.7, SA-B.1.351, and CA-B.1.429) was efficiently blocked by GTB or EGCG. Among the 4 active green tea catechins at noncytotoxic doses, EGCG was the most potent in the action against the viruses. The highest inhibitory activity was observed when the viruses or the cells were pre-incubated with EGCG prior to the infection. Mechanistic studies revealed that EGCG blocked infection at the entry step through interfering with the engagement of the receptor binding domain (RBD) of the viral spikes to angiotensin-converting enzyme 2 (ACE2) receptor of the host cells. These data support further clinical evaluation and development of EGCG as a novel, safe, and cost-effective natural product for prevention/treatment of SARS-CoV-2 transmission and infection.","version":"1.1","doi":"10.1101/2021.03.17.435637","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.435581","pub_date":"2021-3-17","title":"Longitudinal characterization of humoral and cellular immunity in hospitalized COVID-19 patients reveal immune persistence up to 9 months after infection","abstract":"Insights into early, specific humoral and cellular responses to infection with SARS-CoV-2, as well as the persistence and magnitude of resulting immune memory is important amidst the ongoing pandemic. The combination of humoral and cellular immunity will most likely contribute to protection from reinfection or severe disease. Here, we conducted a longitudinal study on hospitalized moderate and severe COVID-19 patients from the acute phase of disease into convalescence at five- and nine-months post symptom onset. Utilizing flow cytometry, serological assays as well as B cell and T cell FluoroSpot assays, we assessed the magnitude and specificity of humoral and cellular immune memory during and after human SARS-CoV-2 infection. During acute COVID-19, we observed an increase in germinal center activity, a substantial expansion of antibodysecreting cells, and the generation of SARS-CoV-2-neutralizing antibodies. Despite gradually decreasing antibody levels, we show persistent, neutralizing antibody titers as well as robust specific memory B cell responses and polyfunctional T cell responses at five- and nine-months after symptom onset in both moderate and severe COVID-19 patients. Long-term SARS-CoV-2 specific responses were marked by preferential targeting of spike over nucleocapsid protein. Our findings describe the initiation and, importantly, persistence of cellular and humoral SARS-CoV-2 specific immunological memory in hospitalized COVID-19 patients long after recovery, likely contributing towards protection against reinfection.","version":"1.1","doi":"10.1101/2021.03.17.435581","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.409714","pub_date":"2021-3-17","title":"Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intra-host viral population structure of infected individuals. Most mutations will follow a neutral molecular evolution and won\u2019t make significant contributions to variations within and between infected hosts. Herein, we profiled the intra-sample genetic diversity of SARS-CoV-2 variants using high-throughput sequencing datasets from 15,289 infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing and/or sequencing protocols. Despite high mutational background, we identified recurrent intra-variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral Spike (S) protein. Strikingly, we observed a high-frequency C->A missense mutations resulting in the S protein lacking the last 20 amino acids (S\u039420). We found that this truncated S protein undergoes increased processing and increased syncytia formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intra-host disease cytopathic effects. The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not have significant contributions to viral molecular evolution, some are naturally selected and cause a genetic drift through positive selection. Many recent SARS-CoV-2 variants have been recently described and show phenotypic selection towards more infectious viruses. Our study describes another type of variant that does not contribute to inter-host heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the Spike protein removes an important ER-retention signal, which consequently results in a Spike variant that easily travels through the Golgi toward the plasma membrane in a pre-activated conformation, leading to increased syncytia formation.","version":"1.2","doi":"10.1101/2020.12.03.409714","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424413","pub_date":"2021-3-17","title":"Discovery of TMPRSS2 inhibitors from virtual screening","abstract":"The SARS-CoV-2 pandemic has prompted researchers to pivot their efforts to finding antiviral compounds and vaccines. In this study, we focused on the human host cell transmembrane protease serine 2 (TMPRSS2), which plays an important role in the viral life cycle by cleaving the spike protein to initiate membrane fusion. TMPRSS2 is an attractive target and has received attention for the development of drugs against SARS and MERS. Starting with comparative structural modeling and binding model analysis, we developed an efficient pharmacophore-based approach and applied a large-scale in silico database screening for small molecule inhibitors against TMPRSS2. The hits were evaluated in the TMPRSS2 biochemical assay and the SARS-CoV-2 pseudotyped particle (PP) entry assay. A number of novel inhibitors were identified, providing starting points for further development of drug candidates for the treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.12.28.424413","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.17.21253775","pub_date":"2021-03-17","title":"Estimating the elevated transmissibility of the B.1.1.7 strain over previously circulating strains in England using GISAID sequence frequencies","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The B.1.1.7 strain, also referred to as Alpha variant, is a variant strain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Alpha variant is considered to possess higher transmissibility compared to the strains previously circulating in England. This paper proposes a new method to estimate the selective advantage of a mutant strain over another strain using the time course of strain frequencies and the distribution of the serial interval of infections. This method allows the instantaneous reproduction numbers of infections to vary over calendar time. The proposed method also assumes that the selective advantage of a mutant strain over previously circulating strains is constant. Applying the method to SARS-CoV-2 sequence data from England, the instantaneous reproduction number of the B.1.1.7 strain was estimated to be 26.6\u201345.9% higher than previously circulating strains in England. This result indicates that control measures should be strengthened by 26.6\u201345.9% when the B.1.1.7 strain is newly introduced to a country where viruses with similar transmissibility to the preexisting strain in England are predominant.</jats:p>","version":null,"doi":"10.1101/2021.03.17.21253775","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.15.435391","pub_date":"2021-3-16","title":"Immunoinformatic Approach for the identification of T Cell and B Cell Epitopes in the Surface Glycoprotein and Designing a Potent Multiepitope Vaccine Construct Against SARS-CoV-2 including the new UK variant","abstract":"The emergence of a novel coronavirus in China in late 2019 has turned into a SARS-CoV-2 pandemic affecting several millions of people worldwide in a short span of time with high fatality. The crisis is further aggravated by the emergence and evolution of new variant SARS-CoV-2 strains in UK during December, 2020 followed by their transmission to other countries. A major concern is that prophylaxis and therapeutics are not available yet to control and prevent the virus which is spreading at an alarming rate, though several vaccine trials are in the final stage. As vaccines are developed through various strategies, their immunogenic potential may drastically vary and thus pose several challenges in offering both arms of immunity such as humoral and cell-mediated immune responses against the virus. In this study, we adopted an immunoinformatics-aided identification of B cell and T cell epitopes in the Spike protein, which is a surface glycoprotein of SARS-CoV-2, for developing a new Multiepitope vaccine construct (MEVC). MEVC has 575 amino acids and comprises adjuvants and various cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes that possess the highest affinity for the respective HLA alleles, assembled and joined by linkers. The computational data suggest that the MEVC is non-toxic, non-allergenic and thermostable with the capability to elicit both humoral and cell-mediated immune responses. The population coverage of various countries affected by COVID-19 with respect to the selected B and T cell epitopes in MEVC was also investigated. Subsequently, the biological activity of MEVC was assessed by bioinformatic tools using the interaction between the vaccine candidate and the innate immune system receptors TLR3 and TLR4. The epitopes of the construct were analyzed with that of the strains belonging to various clades including the new variant UK strain having multiple unique mutations in S protein. Due to the advantageous features, the MEVC can be tested in vitro for more practical validation and the study offers immense scope for developing a potential vaccine candidate against SARS-CoV-2 in view of the public health emergency associated with COVID-19 disease caused by SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.15.435391","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435472","pub_date":"2021-3-16","title":"Replication kinetic, cell tropism and associated immune responses in SARS-CoV-2 and H5N1 virus infected human iPSC derived neural models","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite ACE2, TMPRSS2 and NPR1 expression in all cultures. However, this sparse infection did result in the production of type-III-interferons and IL-8. In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS.","version":"1.1","doi":"10.1101/2021.03.15.435472","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.435601","pub_date":"2021-3-16","title":"Cluster Analysis of SARS-CoV-2 Gene using Deep Learning Autoencoder: Gene Profiling for Mutations and Transitions","abstract":"We report on a method for analyzing the variant of coronavirus genes using autoencoder. Since coronaviruses have mutated rapidly and generated a large number of genotypes, an appropriate method for understanding the entire population is required. The method using autoencoder meets this requirement and is suitable for understanding how and when the variants emarge and disappear. For the over 30,000 SARS-CoV-2 ORF1ab gene sequences sampled globally from December 2019 to February 2021, we were able to represent a summary of their characteristics in a 3D plot and show the expansion, decline, and transformation of the virus types over time and by region. Based on ORF1ab genes, the SARS-CoV-2 viruses were classified into five major types (A, B, C, D, and E in the order of appearance): the virus type that originated in China at the end of 2019 (type A) practically disappeared in June 2020; two virus types (types B and C) have emerged in the United States and Europe since February 2020, and type B has become a global phenomenon. Type C is only prevalent in the U.S. and is suspected to be associated with high mortality, but this type also disappeared at the end of June. Type D is only found in Australia. Currently, the epidemic is dominated by types B and E.","version":"1.1","doi":"10.1101/2021.03.16.435601","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435551","pub_date":"2021-3-16","title":"Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay","abstract":"The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 \u00b5M. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 \u00b5M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 \u00b5M). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.","version":"1.1","doi":"10.1101/2021.03.15.435551","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.435654","pub_date":"2021-3-16","title":"DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing","abstract":"The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortium\u2019s amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.","version":"1.1","doi":"10.1101/2021.03.16.435654","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435496","pub_date":"2021-3-16","title":"Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain","abstract":"Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed \u201cdual-display\u201d), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. The ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.","version":"1.1","doi":"10.1101/2021.03.15.435496","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435497","pub_date":"2021-3-16","title":"Killed whole genome-reduced bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model","abstract":"As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible Gram-negative autotransporter to express vaccine antigens on surface of genome-reduced bacteria to enhance interaction of vaccine antigen with immune system. As a proof of principle, we utilized genome-reduced E. coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as a killed whole cell vaccine. The FP sequence is highly conserved across coronaviruses; the 6 FP core amino acid residues along with the 4 adjacent residues upstream and the 3 residues downstream the core are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated IFN-\u03b3 responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly-protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens. We report a new vaccine platform to express vaccine antigens on surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrated the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface of E.coli to produce killed whole cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates IFN-\u03b3 responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly-protective coronavirus vaccine since a Betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against Alphacoronavirus PEDV. When using a vaccine appropriate bacteria vector, this inexpensive new vaccine platform offers the potential for use in developing countries.","version":"1.1","doi":"10.1101/2021.03.15.435497","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.14.435295","pub_date":"2021-3-16","title":"3D genomic capture of regulatory immuno-genetic profiles in COVID-19 patients for prognosis of severe COVID disease outcome","abstract":"Human infection with the SARS-CoV-2 virus leads to coronavirus disease (COVID-19). A striking characteristic of COVID-19 infection in humans is the highly variable host response and the diverse clinical outcomes, ranging from clinically asymptomatic to severe immune reactions leading to hospitalization and death. Here we used a 3D genomic approach to analyse blood samples at the time of COVID diagnosis, from a global cohort of 80 COVID-19 patients, with different degrees of clinical disease outcomes. Using 3D whole genome EpiSwitch\u00ae arrays to generate over 1 million data points per patient, we identified a distinct and measurable set of differences in genomic organization at immune-related loci that demonstrated prognostic power at baseline to stratify patients with mild forms of illness and those with severe forms that required hospitalization and intensive care unit (ICU) support. Further analysis revealed both well established and new COVID-related dysregulated pathways and loci, including innate and adaptive immunity; ACE2; olfactory, G\u03b2\u03c8, Ca2+ and nitric oxide (NO) signalling; prostaglandin E2 (PGE2), the acute inflammatory cytokine CCL3, and the T-cell derived chemotactic cytokine CCL5. We identified potential therapeutic agents for mitigation of severe disease outcome, with several already being tested independently, including mTOR inhibitors (rapamycin and tacrolimus) and general immunosuppressants (dexamethasone and hydrocortisone). Machine learning algorithms based on established EpiSwitch\u00ae methodology further identified a subset of 3D genomic changes that could be used as prognostic molecular biomarker leads for the development of a COVID-19 disease severity test.","version":"1.1","doi":"10.1101/2021.03.14.435295","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435528","pub_date":"2021-3-16","title":"Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines","abstract":"Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.","version":"1.1","doi":"10.1101/2021.03.15.435528","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.16.435618","pub_date":"2021-3-16","title":"Multicellular Spatial Model of RNA Virus Replication and Interferon Responses Reveals Factors Controlling Plaque Growth Dynamics","abstract":"Respiratory viruses present major health challenges, as evidenced by the 2009 influenza pandemic and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestation at the cell and tissue levels are vital to understanding the mechanisms of immunopathology and developing improved, strain independent treatments. Here, we present a novel spatialized multicellular spatial computational model of two principal components of tissue infection and response: RNA virus replication and type-I interferon mediated antiviral response to infection within lung epithelial cells. The model is parameterized using data from influenza virus infected cell cultures and, consistent with experimental observations, exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. Modulating the phosphorylation of STAT or altering the ratio of the diffusion constants of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of arrest on diffusion constants highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro experiments to measure these diffusion constants. Sensitivity analyses were performed under conditions creating both continuous plaque growth and arrested plaque growth. Findings suggest that plaque growth and cytokine assay measurements should be collected during arrested plaque growth, as the model parameters are significantly more sensitive and more likely to be identifiable. The model\u2019s metrics replicate experimental immunostaining imaging and titer based sampling assays. The model is easy to extend to include SARS-CoV-2-specific mechanisms as they are discovered or to include as a component linking epithelial cell signaling to systemic immune models. COVID-19 is possibly the defining healthcare crisis of the current generation, with tens of millions of global cases and more than a million reported deaths. Respiratory lung infections form lesions in the lungs, whose number and size correlate with severity of illness. In some severe cases, the disease triggers a severe inflammatory condition known as cytokine storm. Given the complexity of the immune system, computational modeling is needed to link molecular signaling at the site of inflection to the signaling impact on the overall immune system, ultimately revealing how severe inflammatory conditions may emerge. Here, we created a computational model of the early stages of infection that simulates lung cells infected with RNA viruses, such those responsible for COVID-19 and influenza, to help explore how the disease forms viral plaques, an in vitro analog to lesion growth in the lung. Our model recapitulates in vitro observations that pretreatment of biological signaling molecules called with type-I interferons, which are currently being evaluated for treatment of COVID-19. Analyzing the model, we, can stop viral plaque growth. We found that enhancing certain aspects of the innate immune system, such as the JAK/STAT pathway, may be able to stop viral plaque growth, suggesting molecules involved in this pathway as possible drug candidates. Quantifying the parameters needed to model interferon signaling and viral replication, experiments should be performed under conditions that inhibit viral growth, such as pretreating cells with interferon. We present a computational framework that is essential to constructing larger models of respiratory infection induced immune responses, can be used to evaluate drugs and other medical interventions quickly, cheaply, and without the need for animal testing during the initial phase, and that defines experiments needed to improve our fundamental understanding of the mechanisms regulating the immune response.","version":"1.1","doi":"10.1101/2021.03.16.435618","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.156455","pub_date":"2021-3-15","title":"Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV","abstract":"The global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. We therefore conducted a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactome of both viruses, as well as their influence on transcriptome, proteome, ubiquitinome and phosphoproteome in a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon SARS-CoV-2 and SARS-CoV infections at different layers and identified unique and common molecular mechanisms of these closely related coronaviruses. The TGF-\u03b2 pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org) highlights many hotspots that can be targeted by existing drugs and it can guide rational design of virus- and host-directed therapies, which we exemplify by identifying kinase and MMPs inhibitors with potent antiviral effects against SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.06.17.156455","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429604","pub_date":"2021-3-15","title":"Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH","abstract":"The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescence in situ hybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.","version":"1.2","doi":"10.1101/2021.02.04.429604","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435326","pub_date":"2021-3-15","title":"Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase","abstract":"The global COVID-19 pandemic is caused by the SARS-CoV-2 virus and has infected over 100 million and caused over 2 million fatalities worldwide at the point of writing. There is currently a lack of effective drugs to treat people infected with SARS-CoV-2. The SARS-CoV-2 Non-structural protein 13 (NSP13) is a superfamily1B helicase that has been identified as a possible target for anti-viral drugs due to its high sequence conservation and essential role in viral replication. In this study we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and the non-hydrolysable ATP analogue (AMP-PNP). Comparisons of these structures reveal details of global and local conformational changes that are induced by nucleotide binding and hydrolysis and provide insights into the helicase mechanism and possible modes of inhibition. Structural analysis reveals two pockets on NSP13 that are classified as \u201cdruggable\u201d and include one of the most conserved sites in the entire SARS-CoV-2 proteome. To identify possible starting points for anti-viral drug development we have performed a crystallographic fragment screen against SARS-CoV-2 NSP13 helicase. The fragment screen reveals 65 fragment hits across 52 datasets, with hot spots in pockets predicted to be of functional importance, including the druggable nucleotide and nucleic acid binding sites, opening the way to structure guided development of novel antiviral agents.","version":"1.1","doi":"10.1101/2021.03.15.435326","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429765","pub_date":"2021-3-15","title":"Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates","abstract":"Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 146,917 SARS-CoV-2 genome assemblies and 2,393 NGS datasets from GISAID, NCBI Virus and NCBI SRA archives focusing on non-synonymous mutations in the spike protein. Only around 13.8% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.98% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 2,592 distinct variants were found in total. The majority of the observed variants were recurrent, but only nine and 23 recurrent variants were found in at least 0.5% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 15.1% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the increasing importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies.","version":"1.2","doi":"10.1101/2021.02.04.429765","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.14.435322","pub_date":"2021-3-15","title":"Effects of Mutations in the Receptor-Binding Domain of SARS-CoV-2 Spike on its Binding Affinity to ACE2 and Neutralizing Antibodies Revealed by Computational Analysis","abstract":"SARS-CoV-2 causing coronavirus disease 2019 (COVID-19) is responsible for one of the most deleterious pandemics of our time. The interaction between the ACE2 receptors at the surface of human cells and the viral Spike (S) protein triggers the infection making the receptor-binding domain (RBD) of the SARS-CoV-2 S-protein a focal target for the neutralizing antibodies (Abs). Despite the recent progress in the development and deployment of vaccines, the emergence of novel variants of SARS-CoV-2 insensitive to Abs produced in response to the vaccine administration and/or monoclonal ones represents upcoming jeopardy. Here, we assessed the possible effects of single and multiple mutations in the RBD of SARS-CoV-2 S-protein on its binding energy to various antibodies and the human ACE2 receptor. The performed computational analysis indicates that while single amino acid replacements in RBD may only cause partial impairment of the Abs binding, moreover, limited to specific epitopes, some variants of SARS-CoV-2 (with as few as 8 mutations), which are already present in the population, may potentially result in a much broader antigenic escape. We also identified a number of point mutations, which, in contrast to the majority of replacements, reduce RBD affinity to various antibodies without affecting its binding to ACE2. Overall, the results provide guidelines for further experimental studies aiming at the identification of the high-risk RBD mutations allowing for an antigenic escape.","version":"1.1","doi":"10.1101/2021.03.14.435322","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.15.435309","pub_date":"2021-3-15","title":"One-shot identification of SARS-CoV-2 S RBD escape mutants using yeast screening","abstract":"The potential emergence of SARS-CoV-2 Spike (S) escape mutants is a threat to reduce the efficacy of existing vaccines and neutralizing antibody (nAb) therapies. An understanding of the antibody/S escape mutations landscape is urgently needed to preemptively address this threat. Here we describe a rapid method to identify escape mutants for nAbs targeting the S receptor binding site. We identified escape mutants for five nAbs, including three from the public germline class VH3-53 elicited by natural COVID-19 infection. Escape mutations predominantly mapped to the periphery of the ACE2 recognition site on the RBD with K417, D420, Y421, F486, and Q493 as notable hotspots. We provide libraries, methods, and software as an openly available community resource to accelerate new therapeutic strategies against SARS-CoV-2. We present a facile method to identify antibody escape mutants on SARS-CoV-2 S RBD.","version":"1.1","doi":"10.1101/2021.03.15.435309","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423779","pub_date":"2021-3-15","title":"Cetylpyridinium chloride-containing mouthwashes reduce the infectivity of SARS-CoV-2 variants in vitro","abstract":"Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity present in these oral rinses and their exact mechanism of action remain unknown. Here we show that Cetylpyridinium chloride (CPC), a quaternary ammonium compound present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting the viral fusion step with target cells after disrupting the integrity of the viral envelope. We also found that CPC-containing mouth rinses decreased more than a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. This activity was effective for different SARS-CoV-2 variants, including the B.1.1.7 variant, predominant in UK, also in the presence of sterilized saliva. CPC-containing mouth rinses could therefore represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.","version":"1.2","doi":"10.1101/2020.12.21.423779","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.12.435191","pub_date":"2021-3-15","title":"A novel soluble ACE2 protein totally protects from lethal disease caused by SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2), which is membrane bound, as its initial cell contact receptor preceding viral entry. Here we report a human soluble ACE2 variant fused with a 5kD albumin binding domain (ABD) and bridged via a dimerization motif hinge-like 4-cysteine dodecapeptide, which we term ACE2 1-618-DDC-ABD. This protein is enzymatically active, has increased duration of action in vivo conferred by the ABD-tag, and displays 20-30-fold higher binding affinity to the SARS-CoV-2 receptor binding domain than its des-DDC monomeric form (ACE2 1-618-ABD) due to DDC-linked dimerization. ACE2 1-618-DDC-ABD was administered for 3 consecutive days to transgenic k18-hACE2 mice, a model that develops lethal SARS-CoV-2 infection, to evaluate the preclinical preventative/ therapeutic value for COVID-19. Mice treated with ACE2 1-618-DDC-ABD developed a mild to moderate disease for the first few days assessed by a clinical score and modest weight loss. The untreated control animals, by contrast, became severely ill and had to be sacrificed by day 6/7 and lung histology revealed extensive pulmonary alveolar hemorrhage and mononuclear infiltrates. At 6 days, mortality was totally prevented in the treated group, lung histopathology was improved and viral titers markedly reduced. This demonstrates for the first time in vivo the preventative/ therapeutic potential of a novel soluble ACE2 protein in a preclinical animal model.","version":"1.2","doi":"10.1101/2021.03.12.435191","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.238386","pub_date":"2021-3-15","title":"Recombinant SARS-CoV-2 genomes are currently circulating at low levels","abstract":"Viral recombination can generate novel genotypes with unique phenotypic characteristics, including transmissibility and virulence. Although the capacity for recombination among betacoronaviruses is well documented, there is limited evidence of recombination between SARS-CoV-2 strains. By identifying the mutations that primarily determine SARS-CoV-2 clade structure, we developed a lightweight approach for detecting recombinant genomes. Among the over 537,000 genomes queried, we detect 1175 putative recombinants that contain multiple mutational markers from distinct clades. Additional phylogenetic analysis and the observed co-circulation of predicted parent clades in the geographic regions of exposure further support the feasibility of recombination in these detected cases. An analysis of these detected cases did not reveal any evidence for recombination hotspots in the SARS-CoV-2 genome. Although most recombinant genotypes were detected a limited number of times, at least two recombinants are now widely transmitted. Recombinant genomes were also found to contain substitutions of concern for elevated transmissibility and lower vaccine efficacy, including D614G, N501Y, E484K, and L452R. Adjusting for an unequal probability of detecting recombinants derived from different parent clades, and for geographic variation in clade abundance, we estimate that at most 5% of circulating viruses in the USA and UK are recombinant. While the phenotypic characterization of detected recombinants was beyond the scope of our analysis, the identification of transmitted recombinants involving substitutions of concern underscores the need to sustain efforts to monitor the emergence of new genotypes generated through recombination.","version":"1.2","doi":"10.1101/2020.08.05.238386","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434497","pub_date":"2021-3-15","title":"SARS-CoV-2 variants resist antibody neutralization and broaden host ACE2 usage","abstract":"New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS-CoV-2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS-CoV-2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N-E484K-N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection.","version":"1.2","doi":"10.1101/2021.03.09.434497","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.14.435299","pub_date":"2021-3-15","title":"S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity","abstract":"SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S-acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.","version":"1.1","doi":"10.1101/2021.03.14.435299","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.14.435180","pub_date":"2021-3-15","title":"Differential gene expression by RNA-Seq in Sigma-2 Receptor/TMEM97 knockout cells reveals its role in complement activation and SARS-CoV-2 viral uptake","abstract":"Our lab has recently shown that the Sigma-2 Receptor/Transmembrane Protein 97 (sigma-2R/TMEM97) interacts with the low-density lipoprotein receptor (LDLR) and facilitates the enhanced uptake of various ligands including lipoproteins and intrinsically disordered proteins. TMEM97 has been recently been shown to interact with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins, highlighting its potential involvement with viral entry into the cell. We hypothesized that sigma-2R/TMEM97 may play a role in facilitating viral uptake, and with the regulation of inflammatory and thrombotic pathways that are involved with viral infection. In this study, we identified the top differentially expressed genes upon the knockout of sigma-2R/TMEM97, and analyzed the genes involved with the inflammatory and thrombotic cascades, effects that are observed in patients infected with SARS-CoV-2. We found that the ablation of sigma-2R/TMEM97 resulted in an increase in Complement Component 4 Binding Protein (C4BP) proteins, at both the translational and transcriptional levels. We also showed that sigma-2R/TMEM97 interacts with the cellular receptor for SARS-CoV-2, the human angiotensin-converting enzyme 2 (ACE2) receptor, forming a protein complex, and that disruption of this complex results in the inhibition of viral uptake. The results of this study suggest that sigma-2R/TMEM97 may be a novel therapeutic target to inhibit SARS-CoV-2 viral uptake, as well as to decrease inflammatory and thrombotic effects through the modulation of the complement cascade.","version":"1.1","doi":"10.1101/2021.03.14.435180","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.435037","pub_date":"2021-3-15","title":"Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity","abstract":"New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes. Cryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion. Adaptation to mink resulted in increased ACE2 binding and spike destabilization. B.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding. E484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation. S protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning.","version":"1.2","doi":"10.1101/2021.03.11.435037","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.13.435222","pub_date":"2021-3-15","title":"BNT162b2 mRNA COVID-19 vaccine induces antibodies of broader cross-reactivity than natural infection but recognition of mutant viruses is up to 10-fold reduced","abstract":"Several new variants of SARS-CoV-2 have emerged since fall 2020 which have multiple mutations in the receptor binding domain (RBD) of the spike protein. We aimed to assess how mutations in RBD affected recognition of immune sera by antibodies induced by natural infection versus immunization with BNT162b2, a mRNA-based vaccine against COVID-19. We produced SARS-CoV-2 RBD mutants with single mutations in the receptor binding domain (RBD) region (E484K, K417N, N501Y) or with all 3 mutations combined, as occurring in the newly emerged variants B.1.351 (South Africa) and P.1 (Brazil). Using standard and avidity ELISAs, we determined the binding capacities to mutant RBDs of antibodies induced by infection versus vaccination. These binding assays showed that vaccination induced antibodies recognize both wildtype and mutant RBDs with higher avidities than those raised by infection. Nevertheless, recognition of mutants RBDK417N and RBDN501Y was 2.5-3-fold reduced while RBDE484K and the triple mutant were 10-fold less well recognized, demonstrating that the mutation at position 484 was key for the observed loss in cross-reactivity. Our binding data demonstrate improved recognition of mutant viruses by BNT162b2-induced antibodies compared to those induced by natural infection. Recognition may, however, be 10-fold reduced for the variants B.1.351/P.1, suggesting that the development of a new vaccine is warranted. The E484K mutation is an key hurdle for immune recognition, convalescent plasma and monoclonal antibody therapy as well as serological assays based on the wildtype sequence may therefore seriously impaired. BNT162b2 mRNA COVID-19 vaccine-induced antibodies recognize mutant viruses with up to 10-fold lower efficiency","version":"1.1","doi":"10.1101/2021.03.13.435222","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.13.435221","pub_date":"2021-3-14","title":"Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry","abstract":"Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.","version":"1.1","doi":"10.1101/2021.03.13.435221","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.13.435256","pub_date":"2021-3-14","title":"Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex","abstract":"Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3\u2019-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3\u2019-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals. The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.","version":"1.1","doi":"10.1101/2021.03.13.435256","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.12.435186","pub_date":"2021-3-14","title":"SARS-CoV-2 Nsp8 N-terminal domain dimerizes and harbors autonomously folded elements","abstract":"The SARS-CoV-2 Nsp8 protein is a critical component of the RNA replicase, as its N-terminal domain (NTD) anchors Nsp12, the RNA, and Nsp13. Whereas its C-terminal domain (CTD) structure is well resolved, there is an open debate regarding the conformation adopted by the NTD as it is predicted as disordered but found in a variety of complex-dependent conformations or missing from many other structures. Using NMR spectroscopy, we show that the SARS CoV-2 Nsp8 NTD features both well folded secondary structure and disordered segments. Our results suggest that while part of this domain corresponding to two long \u03b1-helices forms autonomously, the folding of other segments would require interaction with other replicase components. When isolated, the \u03b1-helix population progressively declines towards the C-termini, and dynamics measurements indicate that the Nsp8 NTD behaves as a dimer under our conditions.","version":"1.1","doi":"10.1101/2021.03.12.435186","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.12.434969","pub_date":"2021-3-13","title":"A potential SARS-CoV-2 variant of interest (VOI) harboring mutation E484K in the Spike protein was identified within lineage B.1.1.33 circulating in Brazil","abstract":"The SARS-CoV-2 epidemic in Brazil was dominated by two lineages designated as B.1.1.28 and B.1.1.33. Two SARS-CoV-2 variants harboring mutations at the receptor-binding domain of the Spike (S) protein, designated as lineages P.1 and P.2, evolved within lineage B.1.1.28 and are rapidly spreading in Brazil. Lineage P.1 is considered a Variant of Concern (VOC) because of the presence of multiple mutations in the S protein (including K417T, E484K, N501Y), while lineage P.2 only harbors mutation S:E484K and is considered a Variant of Interest (VOI). Here we report the identification of a new SARS-CoV-2 VOI within lineage B.1.1.33 that also harbors mutation S:E484K and was detected in Brazil between November 2020 and February 2021. This VOI displayed four non-synonymous lineage-defining mutations (NSP3:A1711V, NSP6:F36L, S:E484K, and NS7b:E33A) and was designated as lineage N.9. The VOI N.9 probably emerged in August 2020 and has spread across different Brazilian states from the Southeast, South, North and Northeast regions.","version":"1.1","doi":"10.1101/2021.03.12.434969","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.21253235","pub_date":"2021-03-12","title":"Wastewater Surveillance of SARS-CoV-2 across 40 U.S. states","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Wastewater-based disease surveillance is a promising approach for monitoring community outbreaks. Here we describe a nationwide campaign to monitor SARS-CoV-2 in the wastewater of 159 counties in 40 U.S. states, covering 13% of the U.S. population from February 18 to June 2, 2020. Out of 1,751 total samples analyzed, 846 samples were positive for SARS-CoV-2 RNA, with overall viral concentrations declining from April to May. Wastewater viral titers were consistent with, and appeared to precede, clinical COVID-19 surveillance indicators, including daily new cases. Wastewater surveillance had a high detection rate (&gt;80%) of SARS-CoV-2 when the daily incidence exceeded 13 per 100,000 people. Detection rates were positively associated with wastewater treatment plant catchment size. To our knowledge, this work represents the largest-scale wastewater-based SARS-CoV-2 monitoring campaign to date, encompassing a wide diversity of wastewater treatment facilities and geographic locations. Our findings demonstrate that a national wastewater-based approach to disease surveillance may be feasible and effective.</jats:p>","version":null,"doi":"10.1101/2021.03.10.21253235","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.11.434589","pub_date":"2021-3-12","title":"An Innovative antibody-based Plug-and-Play strategy for SARS-CoV-2","abstract":"The novel and highly pathogenic coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human host lung cells via its spike protein binding to angiotensin-converting enzyme 2 (ACE2) in a process critical dependent on host protease-mediated fusion event. Thus, effective targeted therapies blocking the first step of viral fusion and cellular entry remains a critical unmet medical need to overcome disease pathology. Here we engineered and describe an antibody-based novel and targeted plug-and-play strategy, which directly competes with the proteolytic activation function of SAR-CoV-2 spike protein. The described strategy involves the engineering of furin substrate residues in IgG1 Fc-extended flexible region of spike targeting antibody. Our results with spike receptor-binding domain (RBD) targeting CR3022 antibody support blockade of the viral function using proof of concept ACE2 overexpressing cells. Our study reveals analytical, safe, and selective mechanistic insights for SARS-CoV-2 therapeutic design and is broadly applicable to the future coronaviridae family members (including mutant variants) exploiting the host protease system for cellular entry.","version":"1.1","doi":"10.1101/2021.03.11.434589","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.12.435174","pub_date":"2021-3-12","title":"A metal ion orients mRNA to ensure accurate 2\u2019-O ribosyl methylation of the first nucleotide of the SARS-CoV-2 genome","abstract":"The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2\u2019-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2\u2019-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in the host cell. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2\u2019-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.","version":"1.1","doi":"10.1101/2021.03.12.435174","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.12.435176","pub_date":"2021-3-12","title":"TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins","abstract":"TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its over-expression being associated with familial frontotemporal lobar degeneration, and under-expression linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a non-standard way to show that the lumenal portion of TMEM106B is a member of the LEA-2 domain superfamily. The non-standard tools (HMMER, HHpred and trRosetta) extended this to predict two yeast LEA-2 proteins in the lumenal domains of the degradative vacuole, equivalent to the lysosome: one in Vac7, a regulator of PI(3,5)P2 production, and three in Tag1 which signals to terminate autophagy. Further analysis of previously unreported LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid binding groove. This implies that TMEM106B, Vac7 and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles.","version":"1.1","doi":"10.1101/2021.03.12.435176","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434891","pub_date":"2021-3-11","title":"Phodopus roborovskii SH101 as a systemic infection model of SARS-CoV-2","abstract":"Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide threat with its unusually high transmission rates and rapid evolution into diverse strains. Unlike typical respiratory viruses, SARS-CoV-2 frequently causes systemic infection by breaking the boundaries of the respiratory systems. The development of animal models recapitulating the clinical manifestations of COVID-19 is of utmost importance not only for the development of vaccines and antivirals but also for understanding the pathogenesis. However, there has not been developed an animal model for systemic infection of SARS-CoV-2 representing most aspects of the clinical manifestations of COVID-19 with systemic symptoms. Here we report that a hamster strain of Phodopus roborovskii SH101, a laboratory inbred hamster strain of P. roborovskii, displayed most symptoms of systemic infection upon SARS-CoV-2 infection as in the case of the human counterpart, unlike current COVID-19 animal models. P. roborovskii SH101 post-infection of SARS-CoV-2 represented most clinical symptoms of COVID-19 such as snuffling, dyspnea, cough, labored breathing, hunched posture, progressive weight loss, and ruffled fur, in addition to high fever following shaking chills. Histological examinations also revealed a serious right-predominated pneumonia as well as slight organ damages in the brain and liver, manifesting systemic COVID-19 cases. Considering the merit of a small animal as well as its clinical manifestations of SARS-CoV-2 infection in human, this hamster model seems to provide an ideal tool to investigate COVID-19. Although the current animal models supported SARS-CoV-2 replication and displayed varying degrees of illness after SARS-CoV-2 infection, the infections of SARS-CoV-2 were mainly limited to the respiratory systems of these animals, including hACE2 transgenic mice, hamsters, ferrets, fruit bats, guinea pigs, African green monkey, Rhesus macaques, and Cynomolgus macaques. While these animal models can be a modest model for the respiratory infection, there is a clear limit for use them in the study of COVID-19 that also displays multiple systemic symptoms. Therefore, the development of an animal model recapitulating COVID-19-specific symptoms such as the right-predominated pneumonia would be the utmost need to overcome the imminent threat posed by COVID-19. We identified a very interesting hamster strain, Phodopus roborovskii SH101, which mimics almost all aspects of the clinical manifestations of COVID-19 upon SARS-CoV-2 infection. Unlike the current animal models, SARS-CoV-2-infected P. roborovskii SH101 not only displayed the symptoms of respiratory infection but also clinical manifestations specific to human COVID-19 such as high fever following shaking chills, serious right-predominated pneumonia, and minor organ damages in the brain and liver.","version":"1.1","doi":"10.1101/2021.03.10.434891","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434764","pub_date":"2021-3-11","title":"Novel inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives","abstract":"The global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic. The main protease of SARS-CoV-2 (Mpro), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro catalytic dyad has been revealed by crystallographic studies of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.","version":"1.1","doi":"10.1101/2021.03.11.434764","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430547","pub_date":"2021-3-11","title":"ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions","abstract":"SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development. SARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.","version":"1.3","doi":"10.1101/2021.02.09.430547","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434937","pub_date":"2021-3-11","title":"SARS-CoV-2 comprehensive receptor profiling: mechanistic insight to drive new therapeutic strategies","abstract":"Here we describe a hypothesis free approach to screen for interactions of SARS-CoV-2 spike (S) protein with human cell surface receptors. We used a library screening approach to detect binding interactions across one of the largest known panels of membrane-bound and soluble receptors, comprising 5845 targets, expressed recombinantly in human cells. We were able confirm and replicate SARS-CoV-2 binding to ACE2 and other putative coreceptors such as CD209 and CLEC4M. More significantly, we identified interactions with a number of novel SARS-CoV-2 S binding proteins. Three of these novel receptors, NID1, CNTN1 and APOA4 were specific to SARS-CoV-2, and not SARS-COV, with APOA4 binding the S-protein with equal affinity as ACE2. With this knowledge we may further understand the disease pathogenesis of COVID-19 patients and how infection by SARS-CoV-2 may lead to differences in pathology in specific organs or indeed the virulence observed in different ethnicities. Importantly we illustrate a methodology which can be used for rapid, unbiassed identification of cell surface receptors, to support drug screening and drug repurposing approaches for this and future pandemics.","version":"1.1","doi":"10.1101/2021.03.11.434937","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.356667","pub_date":"2021-3-11","title":"Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice","abstract":"The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A, oral polio vaccine, and smallpox proved to be reliable approaches for immunization for prolonged periods. During the pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate, having the advantages of being manufactured rapidly and tested easily in comparison with recombinant vaccines. In this study, an inactivated virus vaccine that includes a gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required has been optimized. The vaccine candidate (OZG-38.61.3) was then applied in mice by employing the intradermal route, which decreased the requirement of a higher concentration of inactivated virus for proper immunization, unlike most of the classical inactivated vaccine treatments. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral copy per dose) of OZG-38.61.3 was initially determined in Balb/c mice. This was followed by testing the immunogenicity and protective efficacy of OZG-38.61.3. Human ACE2-encoding transgenic mice were immunized and then infected with a dose of infective SARS-CoV-2 virus for the challenge test. Findings of this study show that vaccinated mice have lower SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.","version":"1.3","doi":"10.1101/2020.10.28.356667","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434841","pub_date":"2021-3-11","title":"Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva","abstract":"Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within \u223c1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.","version":"1.1","doi":"10.1101/2021.03.11.434841","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.432949","pub_date":"2021-3-11","title":"Bromodomain and extraterminal protein inhibitor, apabetalone (RVX-208), reduces ACE2 expression and attenuates SARS-CoV-2 infection in vitro","abstract":"Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.","version":"1.1","doi":"10.1101/2021.03.10.432949","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429108","pub_date":"2021-3-11","title":"Nelfinavir markedly improves lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect","abstract":"In response to the ongoing COVID-19 pandemic, repurposing of drugs for the treatment of SARS-CoV-2 infections is being explored. The HIV protease inhibitor Nelfinavir, widely prescribed in combination with other HIV inhibitors, has been shown to inhibit in vitro SARS-CoV-2 replication. We here report on the effect of Nelfinavir in the Syrian hamster SARS-CoV-2 infection model. Although treatment of infected hamsters with either 15 or 50 mg/kg BID Nelfinavir [for four consecutive days, initiated on the day of infection] does not reduce viral RNA loads nor infectious virus titres in the lungs compared to the vehicle control, the drug reduced virus-induced lung pathology to nearly the baseline scores of healthy animals. A substantial interstitial infiltration of neutrophils is observed in the lungs of treated (both infected and uninfected) animals. The protective effect of Nelfinavir on SARS-CoV-2-induced lung pathology (at doses that are well tolerated and that result in exposures nearing those observed in HIV-infected patients) may lay the foundation for clinical studies with this widely used drug.","version":"1.3","doi":"10.1101/2021.02.01.429108","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.18.304139","pub_date":"2021-3-11","title":"The stem loop 2 motif is a site of vulnerability for SARS-CoV-2","abstract":"RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stemloop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3\u2019 UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 infection in cell culture. Our results thus permit us to suggest that the s2m element is a site of vulnerability readily targeted by ASOs, which show promise as anti-viral agents.","version":"1.2","doi":"10.1101/2020.09.18.304139","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.06.434193","pub_date":"2021-3-11","title":"SARS-CoV-2 501Y.V2 (B.1.351) elicits cross-reactive neutralizing antibodies","abstract":"Neutralization escape by SARS-CoV-2 variants, as has been observed in the 501Y.V2 (B.1.351) variant, has impacted the efficacy of first generation COVID-19 vaccines. Here, the antibody response to the 501Y.V2 variant was examined in a cohort of patients hospitalized with COVID-19 in early 2021 - when over 90% of infections in South Africa were attributed to 501Y.V2. Robust binding and neutralizing antibody titers to the 501Y.V2 variant were detected and these binding antibodies showed high levels of cross-reactivity for the original variant, from the first wave. In contrast to an earlier study where sera from individuals infected with the original variant showed dramatically reduced potency against 501Y.V2, sera from 501Y.V2-infected patients maintained good cross-reactivity against viruses from the first wave. Furthermore, sera from 501Y.V2-infected patients also neutralized the 501Y.V3 (P.1) variant first described in Brazil, and now circulating globally. Collectively these data suggest that the antibody response in patients infected with 501Y.V2 has a broad specificity and that vaccines designed with the 501Y.V2 sequence may elicit more cross-reactive responses.","version":"1.2","doi":"10.1101/2021.03.06.434193","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434928","pub_date":"2021-3-11","title":"A tandem-repeat dimeric RBD protein-based COVID-19 vaccine ZF2001 protects mice and nonhuman primates","abstract":"A safe, efficacious and deployable vaccine is urgently needed to control COVID-19 pandemic. We report here the preclinical development of a COVID-19 vaccine candidate, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and NHPs, and also elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 \u03bcg or 50 \u03bcg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea and bronchi, with milder lung lesions. No evidence of disease enhancement is observed in both models. ZF2001 is being evaluated in the ongoing international multi-center Phase 3 trials (NCT04646590) and has been approved for emergency use in Uzbekistan.","version":"1.1","doi":"10.1101/2021.03.11.434928","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.11.434758","pub_date":"2021-3-11","title":"Emerging variants of concern in SARS-CoV-2 membrane protein: a highly conserved target with potential pathological and therapeutic implications","abstract":"Mutations in the SARS-CoV-2 Membrane (M) gene are relatively uncommon. The M gene encodes the most abundant viral structural protein, and is implicated in multiple viral functions, including initial attachment to the host cell via heparin sulfate proteoglycan, viral protein assembly in conjunction with the N and E genes, and enhanced glucose transport. We have identified a recent spike in the frequency of reported SARS-CoV-2 genomes carrying M gene mutations. This is associated with emergence of a new sub-B.1 clade defined by the previously unreported M:I82T mutation within TM3, the third of three membrane spanning helices implicated in glucose transport. The frequency of this mutation increased in the USA from 0.014% in October 2020 to 1.62% in February 2021, a 116-fold change. While constituting 0.7% of the isolates overall, M:I82T sub-B.1 lineage accounted for 14.4% of B.1 lineage isolates in February 2021, similar to the rapid initial increase previously seen with the B.1.1.7 and B.1.429 lineages, which quickly became the dominant lineages in Europe and California over a period of several months. A similar increase in incidence was also noted in another related mutation, V70L, also within the TM2 transmembrane helix. The rapid emergence of this sub-B.1 clade with recurrent I82T mutation suggests that this M gene mutation is more biologically fit, perhaps related to glucose uptake during viral replication, and should be included in ongoing genomic surveillance efforts and warrants further evaluation for potentially increased pathogenic and therapeutic implications.","version":"1.1","doi":"10.1101/2021.03.11.434758","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432189","pub_date":"2021-3-11","title":"Acquisition of the L452R mutation in the ACE2-binding interface of Spike protein triggers recent massive expansion of SARS-Cov-2 variants","abstract":"The recent rise in mutational variants of SARS-CoV-2, especially with changes in the Spike protein, is of significant concern due to the potential ability for these mutations to increase viral infectivity, virulence and/or ability to escape protective antibodies. Here, we investigated genetic variations in a 414-583 amino acid region of the Spike protein, partially encompassing the ACE2 receptor-binding domain (RBD), across a subset of 570 nasopharyngeal samples isolated between April 2020 and February 2021, from Washington, California, Arizona, Colorado, Minnesota and Illinois. We found that samples isolated since November have an increased number of amino acid mutations in the region, with L452R being the dominant mutation. This mutation is associated with a recently discovered CAL.20C viral variant from clade 20C, lineage B.1.429, that since November-December 2020 is associated with multiple outbreaks and is undergoing massive expansion across California. In some samples, however, we found a distinct L452R-carrying variant of the virus that, upon detailed analysis of the GISAID database genomes, is also circulating primarily in California, but emerged even more recently. The newly identified variant derives from the clade 20A (lineage B.1.232) and is named CAL.20A. We also found that the SARS-CoV-2 strain that caused the only recorded case of infection in an ape - gorillas in the San Diego Zoo, reported in January 2021 - is CAL.20A. In contrast to CAL.20C that carries two additional to L452R mutations in the Spike protein, L452R is the only mutation found in CAL.20A. According to the phylogenetic analysis, however, emergence of CAL.20C was also specifically triggered by acquisition of the L452R mutation. Further analysis of GISAID-deposited genomes revealed that several independent L452R-carrying lineages have recently emerged across the globe, with over 90% of the isolates reported between December 2020 \u2013 February 2021. Taken together, these results indicate that the L452R mutation alone is of significant adaptive value to SARS-CoV-2 and, apparently, the positive selection for this mutation became particularly strong only recently, possibly reflecting viral adaptation to the containment measures or increasing population immunity. While the functional impact of L452R has not yet been extensively evaluated, leucine-452 is positioned in the receptor-binding motif of RBD, in the interface of direct contact with the ACE2 receptor. Its replacement with arginine is predicted to result in both a much stronger binding to the receptor and escape from neutralizing antibodies. If true, this in turn might lead to significantly increased infectivity of the L452R variants, warranting their close surveillance and in-depth functional studies.","version":"1.2","doi":"10.1101/2021.02.22.432189","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434696","pub_date":"2021-3-10","title":"Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice","abstract":"Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient (\u0394ORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of \u0394ORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the \u0394ORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while \u0394ORF7a, \u0394ORF7b and \u0394ORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.09.434696","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.31.428851","pub_date":"2021-3-10","title":"Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells","abstract":"COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2- DOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in- line with reported proteinuria and liver damage in patients with COVID-19. We identified 35 drugs that reduced viral replication in Vero and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.","version":"1.4","doi":"10.1101/2021.01.31.428851","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434030","pub_date":"2021-3-10","title":"Development of Equine Immunoglobulin Fragment F(ab\u2019)2 with High Neutralizing Capability against SARS-CoV-2","abstract":"The ongoing pandemic, COVID-19, caused by SARS-CoV-2 has taken the world, and especially the scientific community by storm. While vaccines are being introduced into the market, there is also a pressing need to find potential drugs and therapeutic modules. Remdesivir is one of the antivirals currently being used with a limited window of action. As more drugs are being vetted, passive immunotherapy in the form of neutralizing antibodies can provide immediate action to combat the increasing numbers of COVID-positive cases. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 generate high titers of antibody with a strong virus neutralizing potential. ELISA performed with pooled antisera displayed highest immunoglobulin titer on 42 days post-immunization, at 1:51,200 dilutions. F(ab\u2019)2 immunoglobulin fragments generated from the pools also showed very high, antigen-specific affinity at 1:102,400 dilutions. Finally, in vitro virus neutralization assays confirmed that different pools of F(ab\u2019)2 fragments could successfully neutralize SARS-CoV-2 with titers well above 25,000, indicating the potential of this strategy in treating severe COVID-19 cases with high titers. The F(ab\u2019)2 was able to cross neutralize another SARS-CoV-2 strain, demonstrating its efficacy against the emerging viral variants and the importance of this approach in our efforts of eradication of COVID-19. In conclusion, this study demonstrates that virus-neutralizing antibodies raised in equines can potentially be used as a treatment regimen in the form of effective passive immunotherapy to combat COVID-19.","version":"1.1","doi":"10.1101/2021.03.09.434030","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.432967","pub_date":"2021-3-10","title":"Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response","abstract":"The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.","version":"1.1","doi":"10.1101/2021.03.10.432967","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434363","pub_date":"2021-3-10","title":"Genome-Wide Covariation in SARS-CoV-2","abstract":"The SARS-CoV-2 virus causing the global pandemic is a coronavirus with a genome of about 30Kbase length [Song et al., 2019]. The design of vaccines and choice of therapies depends on the structure and mutational stability of encoded proteins in the open reading frames(ORFs) of this genome. In this study, we computed, using Expectation Reflection, the genome-wide covariation of the SARS-CoV-2 genome based on an alignment of \u2248 130000 SARS-CoV-2 complete genome sequences obtained from GISAID[Shu & McCauley, 2017]. We used this covariation to compute the Direct Information between pairs of positions across the whole genome, investigating potentially important relationships within the genome, both within each encoded protein and between encoded proteins. We then computed the covariation within each clade of the virus. The covariation detected recapitulates all clade determinants and each clade exhibits distinct covarying pairs.","version":"1.2","doi":"10.1101/2021.03.08.434363","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434840","pub_date":"2021-3-10","title":"Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike","abstract":"Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.03.10.434840","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.419242","pub_date":"2021-3-10","title":"The combined treatment of Molnupiravir and Favipiravir results in a marked potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model","abstract":"Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV2. In recent days preliminary efficacy data have been reported in COVID-19 patients. We here studied the combined antiviral effect of the drugs in the SARS-CoV2 hamster infection model. We first demonstrate that Molnupiravir can reduce infectious virus titers in lungs of infected animals in a dose-dependent manner by up to 3.5 log10 which is associated with a marked improvement of virus-induced lung pathology. When animals are treated with a combination of suboptimal doses of Molnupiravir and Favipiravir (that each alone result in respectively a 1.3 log10 and 1.1 log10 reduction of infectious virus titers in the lungs), a marked combined potency is observed. Infectious virus titers in the lungs of animals treated with the combo are on average reduced by 4.5 log10 and infectious virus are no longer detected in the lungs of 60% of treated infected animals. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs. In the combo-treated hamsters an increased frequency of C-to-T and G-to-A mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir and Favipiravir in the treatment of COVID-19.","version":"1.5","doi":"10.1101/2020.12.10.419242","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.10.434447","pub_date":"2021-3-10","title":"Naturally-acquired immunity in Syrian Golden Hamsters provides protection from re-exposure to emerging heterosubtypic SARS-CoV-2 variants B.1.1.7 and B.1.351","abstract":"The ability of acquired immune responses against SARS-CoV-2 to protect after subsequent exposure to emerging variants of concern (VOC) such as B1.1.7 and B1.351 is currently of high significance. Here, we use a hamster model of COVID-19 to show that prior infection with a strain representative of the original circulating lineage B of SARS-CoV-2 induces protection from clinical signs upon subsequent challenge with either B1.1.7 or B1.351 viruses, which recently emerged in the UK and South Africa, respectively. The results indicate that these emergent VOC may be unlikely to cause disease in individuals that are already immune due to prior infection, and this has positive implications for overall levels of infection and COVID-19 disease.","version":"1.1","doi":"10.1101/2021.03.10.434447","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.07.434276","pub_date":"2021-3-10","title":"Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate","abstract":"The COVID-19 pandemic has claimed the lives of millions of people worldwide and threatens to become an endemic problem, therefore the need for as many types of vaccines as possible is of high importance. Because of the millions of doses required, it is desirable that vaccines are not only safe and effective, but also easy to administer, store, and inexpensive to produce. Newcastle Disease Virus (NDV) is responsible for a respiratory disease in chickens. It has no pathogenic homologue in humans. NDV is recognized as an oncolytic virus, and its use in humans for oncological treatment is being evaluated. In the present work, we have developed two types of NDV-vectored candidate vaccines, which carry the surface-exposed RBD and S1 antigens of SARS-CoV-2, respectively. These vaccine candidates were produced in specific-pathogen-free embryonating chicken eggs, and purified from allantoic fluid before lyophilization. These vaccines were administered intranasally to three different animal models: mice, rats and hamsters, and evaluated for safety, toxicity, immunogenicity, stability and efficacy. Efficacy was evaluated in a challenge assay against active SARS-CoV-2 virus in the Golden Syrian hamster model. The NDV-vectored vaccine based on the S1 antigen was shown to be safe and highly immunogenic, with the ability to neutralize SARS-CoV-2 in-vitro, even with an extreme dilution of 1/640. Our results reveal that this vaccine candidate protects the lungs of the animals, preventing cellular damage in this tissue. In addition, this vaccine reduces the viral load in the lungs, suggesting that it may significantly reduce the likelihood of transmission. Being lyophilized, this vaccine candidate is very stable and can be stored for several months at 4-8\u2070C. In conclusion, our NDV-based vaccine candidate has shown a very favorable performance in the pre-clinical study, serving as evidence for a future evaluation in a Phase-I human clinical trial. This candidate represents a promising tool in the fight against COVID-19.","version":"1.2","doi":"10.1101/2021.03.07.434276","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434592","pub_date":"2021-3-10","title":"Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes","abstract":"There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.","version":"1.1","doi":"10.1101/2021.03.09.434592","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434433","pub_date":"2021-3-09","title":"Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection revealed in COVID-19 Autopsies and Spatial Omics Tissue Maps","abstract":"The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.","version":"1.1","doi":"10.1101/2021.03.08.434433","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434371","pub_date":"2021-3-09","title":"Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host-factors having implication in the disease pathogenesis and severity","abstract":"Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology that resembles human COVID-19 patients. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples shows a similar type of pathophysiological manifestation of SARS-CoV-2 infection as reported earlier in COVID-19 patients and hamsters infected with other isolates. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14dpi. Here, we carried out quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters at day 4 and day 14 post infection. This resulted in the identification of 1,585 differentially expressed proteins of which 68 proteins were significantly altered among both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in the identification of candidate biomarkers and understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis.","version":"1.1","doi":"10.1101/2021.03.09.434371","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.21252839","pub_date":"2021-03-09","title":"Transmission of SARS-CoV-2 by children to contacts in schools and households: a prospective cohort and environmental sampling study in London","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Assessing transmission of SARS-CoV-2 by children in schools is of critical importance to inform public health action. We assessed frequency of acquisition of SARS-CoV-2 by contacts of children with COVID-19 in schools and households, as well as the amount of virus shed into the air and onto fomites in both settings.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Cases of COVID-19 in children in London schools were identified via notification. Weekly sampling for 3-4 weeks and PCR testing for SARS-CoV-2 of immediate classroom contacts (the \u201cbubble\u201d), non-bubble school contacts, and household contacts was undertaken supported by genome sequencing, along with surface and air sampling in the school and home environment.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Within schools, secondary transmission was not detected in 28 individual bubble contacts, representing 10 distinct bubble classes. Across 8 non-bubble classes, 3/62 pupils tested positive\u2013 all three were asymptomatic and tested positive in one setting on the same day, unrelated to the original index case. In contrast, the secondary attack rate in na\u00efve household contacts was 14.3% (5/35) rising to 19.1% (9/47) when considering all household contacts. Environmental contamination with SARS-CoV-2 was rare in schools, regardless of school type; fomite SARS-CoV-2 RNA was identified in 4/189 (2.1%) samples in bubble classrooms, 2/127 (1.6%) samples in non-bubble classrooms, and 5/130 (3.8%) samples in washrooms. This contrasted with fomites in households, where SARS-CoV-2 RNA was identified in 60/248 (24.2%) bedroom samples, 66/241 (27.4%) communal room samples, and 21/188 (11.2%) bathroom samples. Air sampling identified SARS-CoV-2 RNA in just 1/68 (1.5%) of school air samples, compared with 21/85 (24.7%) of air samples taken in homes.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Summary</jats:title>\n                  <jats:p>The low levels of environmental contamination in schools are consistent with low transmission frequency and adequate levels of cleaning and ventilation in schools during the period of study. Secondary transmission in schools was rare. The high frequency of secondary transmission in households associated with evident viral shedding throughout the home suggests a need to improve advice to households with infection in children in order to prevent onward community spread by sibling and adult contacts. The data highlight that transmission from children is very likely to occur when precautions are reduced.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.03.08.21252839","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.09.434219","pub_date":"2021-3-09","title":"The FDA-approved drug cobicistat synergizes with remdesivir to inhibit SARS-CoV-2 replication","abstract":"Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) and testing of a number of drug regimens has led to conflicting results. Here we show that cobicistat, which is an-FDA approved drug-booster that blocks the activity of the drug metabolizing proteins Cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Cell-to-cell membrane fusion assays indicated that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with the putative CYP3A target and nucleoside analog remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. The cobicistat/remdesivir combination was able to potently abate viral replication to levels comparable to mock-infected cells leading to an almost complete rescue of infected cell viability. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19.","version":"1.1","doi":"10.1101/2021.03.09.434219","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434135","pub_date":"2021-3-09","title":"SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection","abstract":"SARS-CoV-2 spillback from humans into domestic and wild animals has been well-documented. We compared variants of cell culture-expanded SARS-CoV-2 inoculum and virus recovered from four species following experimental exposure. Five nonsynonymous changes in nsp12, S, N and M genes were near fixation in the inoculum, but reverted to wild-type sequences in RNA recovered from dogs, cats and hamsters within 1-3 days post-exposure. Fourteen emergent variants were detected in viruses recovered from animals, including substitutions at spike positions H69, N501, and D614, which also vary in human lineages of concern. The rapidity of in vitro and in vivo SARS-CoV-2 selection reveals residues with functional significance during host-switching, illustrating the potential for spillback reservoir hosts to accelerate evolution, and demonstrating plasticity of viral adaptation in animal models. SARS-CoV-2 variants rapidly arise in non-human hosts, revealing viral evolution and potential risk for human reinfection.","version":"1.2","doi":"10.1101/2021.03.05.434135","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.433764","pub_date":"2021-3-09","title":"Site-specific steric control of SARS-CoV-2 spike glycosylation","abstract":"A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.","version":"1.1","doi":"10.1101/2021.03.08.433764","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434641","pub_date":"2021-3-09","title":"A recombinant \u2018ACE2 Triple Decoy\u2019 that traps and neutralizes SARS-CoV-2 shows enhanced affinity for highly transmissible SARS-CoV-2 variants","abstract":"The highly-transmissible SARS-CoV-2 variants now replacing the first wave strain pose an increased threat to human health by their ability, in some instances, to escape existing humoral protection conferred by previous infection, neutralizing antibodies, and possibly vaccination. Thus, other therapeutic options are necessary. One such therapeutic option that leverages SARS-CoV-2 initiation of infection by binding of its spike receptor binding domain (S RBD) to surface-expressed host cell angiotensin-converting enzyme 2 (ACE2) is an ACE2 \u2018decoy\u2019 that would trap the virus by competitive binding and thus inhibit propagation of infection. Here, we used Molecular Dynamic (MD) simulations to predict ACE2 mutations that might increase its affinity for S RBD and screened these candidates for binding affinity in vitro. A double mutant ACE2(T27Y/H34A)-IgG1FC fusion protein was found to have very high affinity for S RBD and to show greater neutralization of SARS-CoV-2 in a live virus assay as compared to wild type ACE2. We further modified the double mutant ACE2 decoy by addition of an H374N mutation to inhibit ACE2 enzymatic activity while maintaining high S RBD affinity. We then confirmed the potential efficacy of our ACE2(T27Y/H34A/H374N)-IgG1FC Triple Decoy against S RBD expressing variant-associated E484K, K417N, N501Y, and L452R mutations and found that our ACE2 Triple Decoy not only maintains its high affinity for S RBD expressing these mutations, but shows enhanced affinity for S RBD expressing the N501Y or L452R mutations and the highest affinity for S RBD expressing both the E484K and N501Y mutations. The ACE2 Triple Decoy also demonstrates the ability to compete with wild type ACE2 in the cPass\u2122 surrogate virus neutralization in the presence of S RBD with these mutations. Additional MD simulation of ACE2 WT and decoy interactions with S RBD WT or B.1.351 variant sequence S RBD provides insight into the enhanced affinity of the ACE2 decoy for S RBD and reveals its potential as a tool to predict affinity and inform therapeutic design. The ACE2 Triple Decoy is now undergoing continued assessment, including expression by a human adenovirus serotype 5 (hAd5) construct to facilitate delivery in vivo. An ACE2(N27Y/H34A/H374N)-IgG1FC fusion protein decoy sustains high affinity to all SARS-CoV-2 spike receptor binding domain (RBD) protein variants tested, shows enhanced affinity for the N501Y and L452R variants, and the highest affinity for combined N501Y and E484K variants.","version":"1.1","doi":"10.1101/2021.03.09.434641","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434499","pub_date":"2021-3-09","title":"The N501Y spike substitution enhances SARS-CoV-2 transmission","abstract":"Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.","version":"1.1","doi":"10.1101/2021.03.08.434499","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.09.434359","pub_date":"2021-3-09","title":"In vitro rapid inactivation of SARS-CoV-2 by visible light photocatalysis using boron-doped bismuth oxybromide","abstract":"Inactivation of SARS-CoV-2 in wastewater and on surfaces is critical to prevent the fecal-oral and fomite transmission, respectively. We hypothesized that visible light active photocatalysts could dramatically enhance the rate or extent of virus inactivation and enable the use of visible light rather than shorter wavelength ultraviolet light. A novel visible light active photocatalyst, boron-doped bismuth oxybromide (B-BiOBr), was synthesized and tested for its SARS-CoV-2 inactivation towards Vero E6 cell lines in dark and under irradiation at 426 nm by a light emitting diode (LED) in water. SARS-CoV-2 inactivation in the presence of B-BiOBr (0.8 g/L) under LED irradiation reached 5.32-log in 5 min, which was 400 to 10,000 times higher than those achieved with conventional photocatalysts of tungsten or titanium oxide nanomaterials, respectively. Even without LED irradiation, B-BiOBr inactivated 3.32-log of SARS-CoV-2 in the dark due to the ability of bismuth ions interfering with the SARS-CoV-2 helicase function. LED irradiation at 426 nm alone, without the photocatalyst, contributed to 10% of the observed inactivation and was attributed to production of reactive oxygen species due to blue-light photoexcitation of molecules in the culture media, which opens further modes of action to engineer disinfection strategies. The visible light active B-BiOBr photocatalyst, with its rapid SARS-CoV-2 inactivation in the presence and absence of light, holds tremendous opportunities to build a healthy environment by preventing the fecal-oral and fomite transmission of emerging pathogens.","version":"1.1","doi":"10.1101/2021.03.09.434359","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.433897","pub_date":"2021-3-09","title":"COVIDrugNet: a network-based web tool to investigate the drugs currently in clinical trial to contrast COVID-19","abstract":"The COVID-19 pandemic poses a huge problem of public health that requires the implementation of all available means to contrast it, and drugs are one of them. In this context, we observed an unmet need of depicting the continuously evolving scenario of the ongoing drug clinical trials through an easy-to-use, freely accessible online tool. Starting from this consideration, we developed COVIDrugNet (http://compmedchem.unibo.it/covidrugnet), a web application that allows users to capture a holistic view and keep up to date on how the clinical drug research is responding to the SARS-CoV-2 infection. Here, we describe the web app and show through some examples how one can explore the whole landscape of medicines in clinical trial for the treatment of COVID-19 and try to probe the consistency of the current approaches with the available biological and pharmacological evidence. We conclude that careful analyses of the COVID-19 drug-target system based on COVIDrugNet can help to understand the biological implications of the proposed drug options, and eventually improve the search for more effective therapies.","version":"1.1","doi":"10.1101/2021.03.05.433897","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434440","pub_date":"2021-3-08","title":"Comparative host interactomes of the SARS-CoV-2 nonstructural protein 3 and human coronavirus homologs","abstract":"Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) dataset for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.","version":"1.1","doi":"10.1101/2021.03.08.434440","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.371419","pub_date":"2021-3-08","title":"Exosome-Mediated mRNA Delivery For SARS-CoV-2 Vaccination","abstract":"Expression-dependent, Spike-only vaccines have been developed, deployed, and shown to be effective in the fight against SARS-CoV-2. However, additional approaches to vaccine development may be needed to meet existing and future challenges posed by emerging Spike variant strains, as well as a likely need for different antigen-delivery systems that are safe and effective for regular, periodic re-administration. We report here the development of mRNA-loaded exosomes, demonstrate that they can mediate the functional expression of heterologous proteins in vitro and in vivo, and have fewer adverse effects than comparable doses of lipid nanoparticles. Furthermore, we applied this approach to the development of an exosome-based, multiplexed mRNA vaccine that drives expression of immunogenic SARS-CoV-2 Nucleocapsid and Spike proteins. This vaccine elicited long-lasting cellular and humoral responses to Nucleocapsid and to Spike, demonstrating that exosome-based mRNA formulations represent a previously unexplored platform in the fight against COVID-19 and other infectious diseases.","version":"1.2","doi":"10.1101/2020.11.06.371419","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434390","pub_date":"2021-3-08","title":"Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses","abstract":"Although a variety of SARS-CoV-2 related coronaviruses have been identified, the evolutionary origins of this virus remain elusive. We describe a meta-transcriptomic study of 411 samples collected from 23 bat species in a small (~1100 hectare) region in Yunnan province, China, from May 2019 to November 2020. We identified coronavirus contigs in 40 of 100 sequencing libraries, including seven representing SARS-CoV-2-like contigs. From these data we obtained 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related genomes. Of these viruses, RpYN06 exhibited 94.5% sequence identity to SARS-CoV-2 across the whole genome and was the closest relative of SARS-CoV-2 in the ORF1ab, ORF7a, ORF8, N, and ORF10 genes. The other three SARS-CoV-2 related coronaviruses were nearly identical in sequence and clustered closely with a virus previously identified in pangolins from Guangxi, China, although with a genetically distinct spike gene sequence. We also identified 17 alphacoronavirus genomes, including those closely related to swine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species in Southeast Asia and southern China, with the largest contiguous hotspots extending from South Lao and Vietnam to southern China. Our study highlights both the remarkable diversity of bat viruses at the local scale and that relatives of SARS-CoV-2 and SARS-CoV circulate in wildlife species in a broad geographic region of Southeast Asia and southern China. These data will help guide surveillance efforts to determine the origins of SARS-CoV-2 and other pathogenic coronaviruses.","version":"1.1","doi":"10.1101/2021.03.08.434390","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434404","pub_date":"2021-3-08","title":"Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract","abstract":"The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.","version":"1.1","doi":"10.1101/2021.03.08.434404","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.07.434287","pub_date":"2021-3-08","title":"A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic","abstract":"The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.","version":"1.1","doi":"10.1101/2021.03.07.434287","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434228","pub_date":"2021-3-08","title":"Sialic acid-Dependent Binding and Viral Entry of SARS-CoV-2","abstract":"Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 \u03bcM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral coreceptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.03.08.434228","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432807","pub_date":"2021-3-08","title":"Modeling the SARS-CoV-2 nsp1\u20135\u2019-UTR complex via extended ensemble simulations","abstract":"Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2\u2019s own RNA evades nsp1\u2019s host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results showed that the stem loop structure of the SARS-CoV-2 RNA 5\u2019-untranslated region (SL1) is recognized by both nsp1\u2019s globular region and intrinsically disordered region. The recognition presumably enables selective translation of viral RNAs. Cluster analysis of the binding mode and detailed analysis of the binding poses revealed several residues involved in the SL1 recognition mechanism. The simulation results imply that the nsp1 C-terminal helices are lifted from the 40S ribosome upon the binding of SL1 to nsp1, unblocking translation of the viral RNA.","version":"1.2","doi":"10.1101/2021.02.24.432807","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.06.434226","pub_date":"2021-3-08","title":"Experimental susceptibility of North American raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) to SARS-CoV-2","abstract":"Skunks and raccoons were intranasally inoculated or indirectly exposed to SARS-CoV-2. Both species are susceptible to infection; however, the lack of, and low quantity of infectious virus shed by raccoons and skunks, respectively, and lack of cage mate transmission in both species, suggest that neither species are competent SARS-CoV-2 reservoirs. Experimental SARS-CoV-2 inoculation of North American raccoons and striped skunks showed susceptibility to infection, but transient, low-level shedding suggests that neither species is likely to be a competent natural reservoir.","version":"1.1","doi":"10.1101/2021.03.06.434226","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.07.434227","pub_date":"2021-3-08","title":"Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies","abstract":"Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.","version":"1.1","doi":"10.1101/2021.03.07.434227","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434168","pub_date":"2021-3-08","title":"Deletion disrupts a conserved antibody epitope in a SARS-CoV-2 variant of concern","abstract":"Multiple SARS-CoV-2 variants with altered antigenicity have emerged and spread internationally. In one lineage of global concern, we identify a transmitted variant with a deletion in its receptor binding domain (RBD) that disrupts an epitope which is conserved across sarbecoviruses. Overcoming antigenic variation by selectively focusing immune pressure on this conserved site may, ultimately, drive viral resistance.","version":"1.1","doi":"10.1101/2021.03.05.434168","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422555","pub_date":"2021-3-08","title":"Recurrent emergence and transmission of a SARS-CoV-2 spike deletion H69/V70","abstract":"SARS-CoV-2 amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide out of frame deletion in the S gene, which results in loss of two amino acids: H69 and V70. We report that in human infections \u0394H69/V70 often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F, and in the latter two cases has followed the RBD mutation. One of the \u0394H69/V70+ N501Y lineages, now known as B.1.1.7, has undergone rapid expansion and includes eight S gene mutations: RBD (N501Y and A570D), S1 (\u0394H69/V70 and \u0394144) and S2 (P681H, T716I, S982A and D1118H). In vitro, we show that \u0394H69/V70 does not reduce serum neutralisation across multiple convalescent sera. However, \u0394H69/V70 increases infectivity and is associated with increased incorporation of cleaved spike into virions. \u0394H69/V70 is able to compensate for small infectivity defects induced by RBD mutations N501Y, N439K and Y453F. In addition, replacement of H69 and V70 residues in the B.1.1.7 spike reduces its infectivity and spike mediated cell-cell fusion. Based on our data \u0394H69/V70 likely acts as a permissive mutation that allows acquisition of otherwise deleterious immune escape mutations. Enhanced surveillance for the \u0394H69/V70 deletion with and without RBD mutations should be considered as a global priority not only as a marker for the B.1.1.7 variant, but potentially also for other emerging variants of concern. Vaccines designed to target the deleted spike protein could mitigate against its emergence as increased selective forces from immunity and vaccines increase globally. \u0394H69/V70 is present in at least 28 SARS-CoV-2 lineages \u0394H69/V70 does not confer escape from convalescent sera \u0394H69/V70 increases spike infectivity and compensates for RBD mutations \u0394H69/V70 is associated with greater spike cleavage B.1.1.7 requires \u0394H69/V70 for optimal spike cleavage and infectivity","version":"1.6","doi":"10.1101/2020.12.14.422555","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.025981","pub_date":"2021-3-08","title":"SARS-COV-2 RBD Oral Vaccine Boosted by Mucosal Immune Adjuvant LTB26 via DCs and B Cells Activation in Mice","abstract":"Although several SARS-COV-2 vaccines have been approved, no one oral live vaccine is available. Here, an oral SARS-COV-2 RBD live vaccine containing LTB26 adjuvant has been developed. BALB/c mice are oral vaccinated with attenuated Salmonella typhimurium SL7207 containing pcDNA3.1-LTB26RBD or pcDNA3.1-RBD plasmids. The result shows that the high level of RBD specific antibody is produced in pcDNA3.1- LTB26RBD treatment. The mechanism indicates that LTB26 enhances RBD antibody production by significantly upregulating the activity of MHC II+ DCs and CD19+CD45+ B cells. LTB26 mutant is derived from heat-labile enterotoxin B subunit (LTB) wild type of Escherichia coli with enhanced immune adjuvanticity. Based on the pre-experiment result that SL7207 interferes the function of LTB26, the purified LTB26 was mixed with purified human rotavirus VP8 antigen to explore the mechanism of adjuvant. The results suggests that LTB26 enhances mucosal immune responses via increased of BCR and MHC II+ expression. Furthermore, LTB26 promotes both Th1 and Th2 cell mediated immunity. Therefore, LTB26 maybe a potent adjuvant for mucosal vaccine development in view of the safety of LTB26 than LT toxin.","version":"1.2","doi":"10.1101/2020.04.06.025981","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.341743","pub_date":"2021-3-08","title":"Predicting COVID-19 Severity with a Specific Nucleocapsid Antibody plus Disease Risk Factor Score","abstract":"Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, ELISA and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide-range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the ICU, requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within six days post-symptom onset and sometimes within one day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient\u2019s comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 Likelihood Ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention. The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.","version":"1.3","doi":"10.1101/2020.10.15.341743","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.08.434384","pub_date":"2021-3-08","title":"Comparative studies of the seven human coronavirus envelope proteins using topology prediction and molecular modelling to understand their pathogenicity","abstract":"Human (h) coronaviruses (CoVs) 229E, NL63, OC43, and HKU1 are less virulent and cause mild, self-limiting respiratory tract infections, while SARS-CoV, MERS-CoV, and SARS-CoV-2, are more virulent and have caused severe outbreaks. The CoV envelope (E) protein, an important contributor to the pathogenesis of severe hCoVs infections, may provide insight into this disparate severity of the disease. Topology prediction programs and 3D modelling software was used to predict and visualize structural aspects of the hCoV E protein related to its functions. All seven hCoV E proteins largely adopted different topologies, with some distinction between the more virulent and less virulent ones. The 3D models refined this distinction, showing the PDZ-binding motif (PBM) of SARS-CoV, MERS-CoV, and SARS-CoV-2 to be more flexible than the PBM of hCoVs 229E, NL63, OC43, and HKU1. We speculate that the increased flexibility of the PBM may provide the more virulent hCoVs with a greater degree of freedom, which can allow them to bind to different host proteins and can contribute to a more severe form of the disease. This is the first paper to predict the topologies and model 3D structures of all seven hCoVs E proteins, providing novel insights for possible drug and/or vaccine development.","version":"1.1","doi":"10.1101/2021.03.08.434384","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.07.433083","pub_date":"2021-3-08","title":"Knowledge, Attitude and Practices towards COVID-19 Guidelines among Students in Bangladesh","abstract":"This paper explores the level of knowledge, attitude and practices of COVID-19 guidelines among the students in Bangladesh. In achieving this objective, this paper uses primary data collected from 1822 students and three different Likert scales and a one-way ANOVA test are used to assess knowledge, attitudes, and practice (KAP) scores and mean differences with respect to different variables. The research reveals that the majority of students have a higher level of knowledge and a positive attitude towards the COVID-19 guidelines. In contrary, only 0.22 percent students show strong compliance towards COVID-19 guidelines while majority students (60.54 percent) have rather a poor adherence which is an alarming finding. Reopening the educational institutions in Bangladesh is, therefore, not advisable yet and reinforcing the preventive measures through campaigns and online discussion to persuade people to follow the preventive guidelines is highly recommended to contain this global disease. Majority papers revealed the KAP of general people or medical related people. Majority people have moderate level of knowledge towards COVID-19. No research is found on KAP of the students in Bangladesh. The KAP of COVID-19 guidelines among the students in Bangladesh. Majority students have the higher level of knowledge towards COVID-19 guideline among students. Only 0.22 percent students show strong compliance towards COVID-19 guidelines which recommends not reopening educational institutions now.","version":"1.1","doi":"10.1101/2021.03.07.433083","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.06.433708","pub_date":"2021-3-06","title":"SARS-CoV-2 surveillance in Norway rats (Rattus norvegicus) from Antwerp sewer system, Belgium","abstract":"SARS-CoV-2 human-to-animal transmission can lead to the establishment of novel reservoirs and the evolution of new variants with the potential to start new outbreaks in humans. We tested Norway rats inhabiting the sewer system of Antwerp, Belgium, for the presence of SARS-CoV-2 following a local COVID-19 epidemic peak. In addition, we discuss the use and interpretation of SARS-CoV-2 serological tests on non-human samples. Between November and December 2020, Norway rat oral swabs, feces and tissues from the sewer system of Antwerp were collected to be tested by RT-qPCR for the presence of SARS-CoV-2. Serum samples were screened for the presence of anti-SARS-CoV-2 IgG antibodies using a Luminex microsphere immunoassay (MIA). Samples considered positive were then checked for neutralizing antibodies using a conventional viral neutralization test (cVNT). The serum of 35 rats was tested by MIA showing 3 potentially positive sera that were later shown to be negative by cVNT. All tissue samples of 39 rats analyzed tested negative for SARS-CoV-2 RNA. This is the first study that evaluates SARS-CoV-2 infection in urban rats. We can conclude that the sample of 39 rats had never been infected with SARS-CoV-2. We show that diagnostic serology tests can give misleading results when applied on non-human samples. SARS-CoV-2 monitoring activities should continue due to the emergence of new variants prone to infect Muridae rodents.","version":"1.1","doi":"10.1101/2021.03.06.433708","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434150","pub_date":"2021-3-06","title":"Long-read sequencing of SARS-CoV-2 reveals novel transcripts and a diverse complex transcriptome landscape","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2 (COVID-19), is a positive single-stranded RNA virus with a 30 kb genome that is responsible for the current pandemic. To date, the genomes of global COVID-19 variants have been primarily characterized via short-read sequencing methods. Here, we devised a long-read RNA (IsoSeq) sequencing approach to characterize the COVID-19 transcript landscape and expression of its \u223c27 coding regions. Our analysis identified novel COVID-19 transcripts including a) a short \u223c65-70 nt 5\u2019-UTR fused to various downstream ORFs encoding accessory proteins such as the envelope, ORF 8, and ORF 9 (nucleocapsid) proteins, that are relatively highly expressed, b) novel SNVs that are differentially expressed, whereby a subset are suggestive of partial RNA editing events, and c) SNVs at functional sites, whereby at least one is associated with a differentially expressed spike protein isoform. These previously uncharacterized COVID-19 isoforms, expressed genes, and gene variants were corroborated using ddPCR. Understanding this transcriptional complexity may help provide insight into the biology and pathogenicity of SARS-CoV-2 compared to other coronaviruses.","version":"1.1","doi":"10.1101/2021.03.05.434150","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.06.434214","pub_date":"2021-3-06","title":"Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics","abstract":"Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.","version":"1.1","doi":"10.1101/2021.03.06.434214","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.433713","pub_date":"2021-3-05","title":"Zoonotic spillover of SARS-CoV-2: mink-adapted virus in humans","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 started in fall 2019. A range of different mammalian species, including farmed mink, have been confirmed as susceptible to infection with this virus. We report here the spillover of mink-adapted SARS-CoV-2 from farmed mink to humans after extensive adaptation that lasted at least 3 months. We found the presence of four mutations in the S gene (that gave rise to variant: G75V, M177T, Y453F and C1247F) and others in an isolate obtained from SARS-CoV-2 positive patient.","version":"1.1","doi":"10.1101/2021.03.05.433713","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427567","pub_date":"2021-3-05","title":"Bispecific antibody neutralizes circulating SARS-CoV-2 variants, prevents escape and protects mice from disease","abstract":"Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19). We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C135. CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.","version":"1.2","doi":"10.1101/2021.01.22.427567","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434089","pub_date":"2021-3-05","title":"Neutralization heterogeneity of United Kingdom and South-African SARS-CoV-2 variants in BNT162b2-vaccinated or convalescent COVID-19 healthcare workers","abstract":"There are concerns about neutralizing antibodies (NAb) potency against the newly emerged VOC202012/01 (UK) and 501Y.V2 (SA) SARS-CoV-2 variants in mRNA-vaccinated subjects and in recovered COVID-19 patients. We used a viral neutralization test with a strict 100% neutralizing criterion on UK and SA clinical isolates in comparison with a globally distributed D614G SARS-CoV-2 strain. In two doses BNT162b2-vaccinated healthcare workers (HCW), despite heterogeneity in neutralizing capacity against the three SARS-CoV-2 strains, most of the sera harbored at least a NAb titer \u2265 1:10 suggesting a certain humoral protection activity either on UK or SA variants. However, six months after mild forms of COVID-19, an important proportion of HCW displayed no neutralizing activity against SA strain. This result supports strong recommendations for vaccination of previously infected subjects.","version":"1.1","doi":"10.1101/2021.03.05.434089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433604","pub_date":"2021-3-05","title":"Blockade of SARS-CoV-2 infection in vitro by highly potent PI3K-\u03b1/mTOR/BRD4 inhibitor","abstract":"Pathogenic viruses like SARS-CoV-2 and HIV hijack the host molecular machinery to establish infection and survival in infected cells. This has led the scientific community to explore the molecular mechanisms by which SARS-CoV-2 infects host cells, establishes productive infection, and causes life-threatening pathophysiology. Very few targeted therapeutics for COVID-19 currently exist, such as remdesivir. Recently, a proteomic approach explored the interactions of 26 of 29 SARS-CoV-2 proteins with cellular targets in human cells and identified 67 interactions as potential targets for drug development. Two of the critical targets, the bromodomain and extra-terminal domain proteins (BETs): BRD2/BRD4 and mTOR, are inhibited by the dual inhibitory small molecule SF2523 at nanomolar potency. SF2523 is the only known mTOR PI3K-\u03b1/(BRD2/BRD4) inhibitor with potential to block two orthogonal pathways necessary for SARS-CoV-2 pathogenesis in human cells. Our results demonstrate that SF2523 effectively blocks SARS-CoV-2 replication in lung bronchial epithelial cells in vitro, showing an IC50 value of 1.5 \u00b5M, comparable to IC50 value of remdesivir (1.1 \u00b5M). Further, we demonstrated that the combination of doses of SF2523 and remdesivir is highly synergistic: it allows for the reduction of doses of SF2523 and remdesivir by 25-fold and 4-fold, respectively, to achieve the same potency observed for a single inhibitor. Because SF2523 inhibits two SARS-CoV-2 driven pathogenesis mechanisms involving BRD2/BRD4 and mTOR signaling, our data suggest that SF2523 alone or in combination with remdesivir could be a novel and efficient therapeutic strategy to block SARS-CoV-2 infection and hence be beneficial in preventing severe COVID-19 disease evolution. Evidence of in silico designed chemotype (SF2523) targeting PI3K-\u03b1/mTOR/BRD4 inhibits SARS-CoV-2 infection and is highly synergistic with remdesivir.","version":"1.2","doi":"10.1101/2021.03.02.433604","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434152","pub_date":"2021-3-05","title":"Scalable, Micro-Neutralization Assay for Qualitative Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples","abstract":"As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was expanding, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5,000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.","version":"1.1","doi":"10.1101/2021.03.05.434152","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428122","pub_date":"2021-3-05","title":"Profiling transcription factor sub-networks in type I interferon signaling and in response to SARS-CoV-2 infection","abstract":"Type I interferons (IFN \u03b1/\u03b2) play a central role in innate immunity to respiratory viruses, including coronaviruses. Genetic defects in type I interferon signaling were reported in a significant proportion of critically ill COVID-19 patients. Extensive studies on interferon-induced intracellular signal transduction pathways led to the elucidation of the Jak-Stat pathway. Furthermore, advances in gene expression profiling by microarrays have revealed that type I interferon rapidly induced multiple transcription factor mRNA levels. In this study, transcription factor profiling in the transcriptome was used to gain novel insights into the role of inducible transcription factors in response to type I interferon signaling in immune cells and in lung epithelial cells after SARS-CoV-2 infection. Modeling the interferon-inducible transcription factor mRNA data in terms of distinct sub-networks based on biological functions such as antiviral response, immune modulation, and cell growth revealed enrichment of specific transcription factors in mouse and human immune cells. The evolutionarily conserved core type I interferon gene expression consists of the inducible transcriptional factor mRNA of the antiviral response sub-network and enriched in granulocytes. Analysis of the type I interferon-inducible transcription factor sub-networks as distinct protein-protein interaction pathways revealed insights into the role of critical hubs in signaling. Interrogation of multiple microarray datasets revealed that SARS-CoV-2 induced high levels of IFN-beta and interferon-inducible transcription factor mRNA in human lung epithelial cells. Transcription factor mRNA of the three major sub-networks regulating antiviral, immune modulation, and cell growth were differentially regulated in human lung epithelial cell lines after SARS-CoV-2 infection and in the tissue samples of COVID-19 patients. A subset of type I interferon-inducible transcription factors and inflammatory mediators were specifically enriched in the lungs and neutrophils of COVID-19 patients. The emerging complex picture of type I IFN transcriptional regulation consists of a rapid transcriptional switch mediated by the Jak-Stat cascade and a graded output of the inducible transcription factor activation that enables temporal regulation of gene expression.","version":"1.2","doi":"10.1101/2021.01.25.428122","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.434119","pub_date":"2021-3-05","title":"SARS-CoV-2-host chimeric RNA-sequencing reads do not necessarily signify virus integration into the host DNA","abstract":"The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, partly based on the detection of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA was highly enriched for host exonic, than intronic or intergenic sequences and often involved the same, highly expressed host genes. These findings suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.","version":"1.1","doi":"10.1101/2021.03.05.434119","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433753","pub_date":"2021-3-05","title":"Identification and quantification of SARS-CoV-2 leader subgenomic mRNA gene junctions in nasopharyngeal samples shows phasic transcription in animal models of COVID-19 and dysregulation at later time points that can also be identified in humans","abstract":"SARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5\u2019 sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing. High resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19. LeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.","version":"1.2","doi":"10.1101/2021.03.03.433753","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.13.431090","pub_date":"2021-3-05","title":"A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation","abstract":"While SARS-CoV-2 likely has animal origins, the viral genetic changes necessary to adapt this animal-derived ancestral virus to humans are largely unknown, mostly due to low levels of sequence polymorphism and the notorious difficulties in experimental manipulations of coronavirus genomes. We scanned more than 182,000 SARS-CoV-2 genomes for selective sweep signatures and found that a distinct footprint of positive selection is located around a non-synonymous change (A1114G; T372A) within the Receptor-Binding Domain of the Spike protein, which likely played a critical role in overcoming species barriers and accomplishing interspecies transmission from animals to humans. Structural analysis indicated that the substitution of threonine with an alanine in SARS-CoV-2 concomitantly removes a predicted glycosylation site at N370, resulting in more favorable binding predictions to human ACE2, the cellular receptor. Using a novel bacteria-free cloning system for manipulating RNA virus genomes, we experimentally validated that this SARS-CoV-2-unique substitution significantly increases replication in human cells relative to its putative ancestral variant. Notably, this mutation\u2019s impact on virus replication in human cells was much greater than that of the Spike D614G mutant, which has been widely reported to have been selected for during human-to-human transmission.","version":"1.2","doi":"10.1101/2021.02.13.431090","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.21252767","pub_date":"2021-03-05","title":"Geographic disparities in COVID-19 case rates are not reflected in seropositivity rates using a neighborhood survey in Chicago","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>To date, COVID-19 case rates are disproportionately higher in Black and Latinx communities across the U.S., leading to more hospitalizations and deaths in those communities. These differences in case rates are evident in comparisons of Chicago neighborhoods with differing race/ethnicities of their residents. Disparities could be due to neighborhoods with more adverse health outcomes associated with poverty and other social determinants of health experiencing higher prevalence of SARS-CoV-2 infection or due to greater morbidity and mortality resulting from equivalent SARS-CoV-2 infection prevalence. We surveyed five pairs of adjacent ZIP codes in Chicago with disparate COVID-19 case rates for highly specific and quantitative serological evidence of any prior infection by SARS-CoV-2 to compare with their disparate COVID-19 case rates. Dried blood spot samples were self-collected at home by internet-recruited participants in summer 2020, shortly after Chicago\u2019s first wave of the COVID-19 pandemic. Pairs of neighboring ZIP codes with very different COVID-19 case rates had similar seropositivity rates for anti-SARS-CoV-2 receptor binding domain IgG antibodies. Overall, these findings of comparable exposure to SARS-CoV-2 across neighborhoods with very disparate COVID-19 case rates are consistent with social determinants of health, and the comorbidities related to them, driving differences in COVID-19 rates across neighborhoods.</jats:p>","version":null,"doi":"10.1101/2021.03.02.21252767","journal":"medRxiv","score":null},{"id":"10.1101/2021.03.05.434038","pub_date":"2021-3-05","title":"Scent dog identification of SARS-CoV-2 infections, similar across different body fluids","abstract":"The main strategy to contain the current SARS-CoV-2 pandemic remains to implement a comprehensive testing, tracing and quarantining strategy until vaccination of the population is adequate. Ten dogs were trained to detect SARS-CoV-2 infections in beta-propiolactone inactivated saliva samples. The subsequent cognitive transfer performance for the recognition of non-inactivated samples were tested on saliva, urine, and sweat in a randomised, double-blind controlled study. Dogs were tested on a total of 5242 randomised sample presentations. Dogs detected non-inactivated saliva samples with a diagnostic sensitivity of 84% and specificity of 95%. In a subsequent experiment to compare the scent recognition between the three non-inactivated body fluids, diagnostic sensitivity and specificity were 95% and 98% for urine, 91% and 94% for sweat, 82%, and 96% for saliva respectively. The scent cognitive transfer performance between inactivated and non-inactivated samples as well as between different sample materials indicates that global, specific SARS-CoV-2-associated volatile compounds are released across different body secretions, independently from the patient\u2019s symptoms. The project was funded as a special research project of the German Armed Forces. The funding source DZIF-Fasttrack 1.921 provided us with means for biosampling.","version":"1.1","doi":"10.1101/2021.03.05.434038","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433970","pub_date":"2021-3-05","title":"Molecular strategies for antibody binding and escape of SARS-CoV-2 and its mutations","abstract":"The COVID19 pandemic, caused by SARS-CoV-2, has infected more than 100 million people worldwide. Due to the rapid spreading of SARS-CoV-2 and its impact, it is paramount to find effective treatments against it. Human neutralizing antibodies are an effective method to fight viral infection. However, the recent discovery of new strains that substantially change the S-protein sequence has raised concern about vaccines and antibodies\u2019 effectiveness. Here, we investigated the binding mechanisms between the S-protein and several antibodies. Multiple mutations were included to understand the strategies for antibody escape in new variants. We found that the combination of mutations K417N and E484K produced higher binding energy to ACE2 than the wild type, suggesting higher efficiency to enter host cells. The mutations\u2019 effect depends on the antibody class. While Class I enhances the binding avidity in the presence of N501Y mutation, class II antibodies showed a sharp decline in the binding affinity. Our simulations suggest that Class I antibodies will remain effective against the new strains. In contrast, Class II antibodies will have less affinity to the S-protein, potentially affecting these antibodies\u2019 efficiency.","version":"1.1","doi":"10.1101/2021.03.04.433970","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.433666","pub_date":"2021-3-05","title":"Analysis of SARS-CoV-2 Mutations Over Time Reveals Increasing Prevalence of Variants in the Spike Protein and RNA-Dependent RNA Polymerase","abstract":"Amid the ongoing COVID-19 pandemic, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein and the proteins comprising the RNA-Dependent RNA Polymerase (RdRP) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences for the spike proteins and RNA-dependent RNA polymerase proteins were downloaded from the GISAID database, aligned, and the variants identified. Polymorphisms in the protein sequence were investigated at the protein structural level and examined longitudinally in order to identify sequence and strain variants that are emerging over time. Our analysis revealed a group of variants in the spike protein and the polymerase complex that appeared in August, and account for around five percent of the genomes analyzed up to the last week of October. A structural analysis also facilitated investigation of several unique variants in the receptor binding domain and the N-terminal domain of the spike protein, with high-frequency mutations occurring more commonly in these regions. The identification of new variants emphasizes the need for further study on the effects of these mutations and the implications of their increased prevalence, particularly as these mutations may impact vaccine or therapeutic efficacy.","version":"1.1","doi":"10.1101/2021.03.05.433666","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.05.433800","pub_date":"2021-3-05","title":"Previous SARS-CoV-2 infection increases B.1.1.7 cross-neutralization by vaccinated individuals","abstract":"To assess the potential impact of predominant circulating SARS-CoV-2 variants on neutralizing activity of infected and/or vaccinated individuals, we analyzed neutralization of pseudoviruses expressing the spike of the original Wuhan strain, the D614G and B.1.1.7 variants. Our data show that parameters of natural infection (time from infection and infecting variant) determined cross-neutralization. Importantly, upon vaccination, previously infected individuals developed equivalent B.1.1.7 and Wuhan neutralizing responses. In contrast, uninfected vaccinees showed reduced neutralization against B.1.1.7. This study was funded by Grifols, the Departament de Salut of the Generalitat de Catalunya, the Spanish Health Institute Carlos III, CERCA Programme/Generalitat de Catalunya, and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos.","version":"1.1","doi":"10.1101/2021.03.05.433800","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.26.399436","pub_date":"2021-3-05","title":"Host-directed FDA-approved drugs with antiviral activity against SARS-CoV-2 identified by hierarchical in silico/in vitro screening methods","abstract":"The unprecedent situation generated by the COVID-19 global emergency prompted us to actively work to fight against this pandemic by searching for repurposable agents among FDA approved drugs to shed light into immediate opportunities for the treatment of COVID-19 patients. In the attempt to proceed toward a proper rationalization of the search for new antivirals among approved drugs, we carried out a hierarchical in silico/in vitro protocol which successfully combines virtual and biological screening to speed up the identification of host-directed therapies against COVID-19 in an effective way. To this end a multi-target virtual screening approach focused on host-based targets related to viral entry followed by the experimental evaluation of the antiviral activity of selected compounds has been carried out. As a result, five different potentially repurposable drugs interfering with viral entry, cepharantine, clofazimine, metergoline, imatinib and efloxate, have been identified.","version":"1.2","doi":"10.1101/2020.11.26.399436","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.422591","pub_date":"2021-3-05","title":"Collaborative Cross Founder Expression Analysis (CCFEA)","abstract":"To understand the role of host genetic variation in infection by SARS-CoV and influenza A virus we developed the Collaborative Cross Founder Expression Analysis (CCFEA), a shiny visualization tool using public RNAseq data from the collaborative cross (CC) founder strains (A/J, C57BL/6J, 129s1/SvImJ, NOD/ShILtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) that underwent infection by either virus, linked with genetic analyses to define loci linked to infection, immunity, and disease phenotypes. Individual gene expression data is displayed across founders, viral infections and days post infection.","version":"1.1","doi":"10.1101/2021.03.04.422591","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433768","pub_date":"2021-3-04","title":"Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants","abstract":"Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and \u201cnanomice\u201d we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.","version":"1.1","doi":"10.1101/2021.03.04.433768","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433849","pub_date":"2021-3-04","title":"Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation","abstract":"There is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3\u2019 antisense sequence for target recognition and a 5\u2019-phosphorylated 2\u2019-5\u2019 poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or \u0394H69/\u0394V70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.","version":"1.1","doi":"10.1101/2021.03.04.433849","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.350207","pub_date":"2021-3-04","title":"SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells","abstract":"Since entering the human population, SARS-CoV-2 (the causative agent of COVID-19) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from non-human Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally-occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E, and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin-cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific virus replication, and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants-of-concern.","version":"1.3","doi":"10.1101/2020.10.22.350207","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433658","pub_date":"2021-3-04","title":"Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal","abstract":"Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including CSE, FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.","version":"1.1","doi":"10.1101/2021.03.04.433658","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433887","pub_date":"2021-3-04","title":"SARS-CoV-2 variant with higher affinity to ACE2 shows reduced sera neutralization susceptibility","abstract":"Several new variants of SARS-CoV-2 have emerged since fall 2020 which have multiple mutations in the receptor binding domain (RBD) of the spike protein. We aimed to assess how mutations in the SARS-CoV-2 RBD affect receptor affinity to angiotensin-converting enzyme 2 (ACE2) and neutralization by anti-RBD serum antibodies. We produced a SARS-CoV-2 RBD mutant (RBDmut) with key mutations (E484K, K417N, N501Y) from the newly emerged Brazilian variant. Using Biolayer Interferometry, we analyzed the binding of this mutant to ACE2, and the susceptibility to neutralization by sera from vaccinated mice and COVID-19 convalescent patients. Kinetic profiles showed increased RBDmut - ACE2 affinity compared to RBDwt, and binding of vaccine-elicited or convalescent sera was significantly reduced. Likewise, both sera types showed significantly reduced ability to block RBDmut - ACE2 binding indicating that antibodies induced by RBDwt have reduced capability to neutralize mutant virus. Our physiochemical data show enhanced infectivity and reduced neutralization by polyclonal antibodies of the Brazilian variant of SARS-CoV-2. SARS-CoV-2 variant with Brazilian RBD mutations shows increased ACE2 affinity and reduced susceptibility to blockage by vaccine-elicited and convalescent sera.","version":"1.1","doi":"10.1101/2021.03.04.433887","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433931","pub_date":"2021-3-04","title":"Published Anti-SARS-CoV-2 In Vitro Hits Share Common Mechanisms of Action that Synergize with Antivirals","abstract":"The global efforts in the past few months have led to the discovery of around 200 drug repurposing candidates for COVID-19. Although most of them only exhibited moderate anti- SARS-CoV-2 activity, gaining more insights into their mechanisms of action could facilitate a better understanding of infection and the development of therapeutics. Leveraging large-scale drug-induced gene expression profiles, we found 36% of the active compounds regulate genes related to cholesterol homeostasis and microtubule cytoskeleton organization. The expression change upon drug treatment was further experimentally confirmed in human lung primary small airway. Following bioinformatics analysis on COVID-19 patient data revealed that these genes are associated with COVID-19 patient severity. The expression level of these genes also has predicted power on anti-SARS-CoV-2 efficacy in vitro, which led to the discovery of monensin as an inhibitor of SARS-CoV-2 replication in Vero-E6 cells. The final survey of recent drug- combination data indicated that drugs co-targeting cholesterol homeostasis and microtubule cytoskeleton organization processes more likely present a synergistic effect with antivirals. Therefore, potential therapeutics should be centered around combinations of targeting these processes and viral proteins.","version":"1.1","doi":"10.1101/2021.03.04.433931","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433852","pub_date":"2021-3-04","title":"Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain","abstract":"In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.","version":"1.1","doi":"10.1101/2021.03.04.433852","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433846","pub_date":"2021-3-04","title":"SARS-CoV-2 viability in time on experimental surfaces","abstract":"We evaluated the SARS-CoV-2 viability preservation on different model surfaces over time. It was found that the SARS-CoV-2 RNA was detected on all studied surfaces for 360 minutes, while the viability of the virus was completely lost after 120 minutes. Type of experimental surface significantly affects viability preservation.","version":"1.1","doi":"10.1101/2021.03.04.433846","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433675","pub_date":"2021-3-04","title":"Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection","abstract":"The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.03.03.433675","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433156","pub_date":"2021-3-04","title":"Altered Subgenomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections","abstract":"SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3\u2019 of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. The recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.","version":"1.2","doi":"10.1101/2021.03.02.433156","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433882","pub_date":"2021-3-04","title":"A novel conformational state for SARS-CoV-2 main protease","abstract":"The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of coronavirus, making it a promising target for drugs against Covid-19 pandemic. Here we present a crystal structure of Mpro disclosing new structural features of key regions of the enzyme. We show that the oxyanion loop, involved in substrate recognition and enzymatic activity, can adopt a new conformation, which is stable and significantly different from the known ones. In this new state the S1 subsite of the substrate binding region is completely reshaped and a new cavity near the S2\u2019 subsite is created. This new structural information expands the knowledge of the conformational space available to Mpro, paving the way for the design of novel classes of inhibitors specifically designed to target this unprecedented binding site conformation, thus enlarging the chemical space for urgent antiviral drugs against Covid-19 pandemic.","version":"1.1","doi":"10.1101/2021.03.04.433882","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433725","pub_date":"2021-3-04","title":"Disinfection of SARS-CoV-2 contaminated surfaces of personal items with UVC-LED disinfection boxes","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted from person-to-person by close contact, small aerosol respiratory droplets and potentially via contact with contaminated surfaces. Here, we investigated the effectiveness of commercial UVC-LED disinfection boxes in inactivating SARS-CoV-2 contaminated surfaces of personal items. We contaminated glass, metal and plastic samples representing the surfaces of personal items such as smartphones, coins or credit cards with SARS-CoV-2 formulated in an organic matrix mimicking human respiratory secretions. For disinfection, the samples were placed at different distances from UVC emitting LEDs inside commercial UVC-LED disinfection boxes and irradiated for different time periods (up to 10 minutes). High viral loads of SARS-CoV-2 were effectively inactivated on all surfaces after 3 minutes of irradiation. Even 10 seconds of UVC-exposure strongly reduced viral loads. Thus, UVC-LED boxes proved to be an effective method for disinfecting SARS-CoV-2 contaminated surfaces that are typically found on personal items.","version":"1.1","doi":"10.1101/2021.03.03.433725","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.352344","pub_date":"2021-3-04","title":"A computational approach to evaluate the combined effect of SARS-CoV-2 RBD mutations and ACE2 receptor genetic variants on infectivity: The COVID-19 host-pathogen nexus","abstract":"SARS-CoV-2 infectivity is largely determined by the virus Spike protein binding to the ACE2 receptor. Meanwhile, marked infection rate differences were reported between populations and individuals. To understand the disease dynamic, we developed a computational approach to study the implications of both SARS-CoV-2 RBD mutations and ACE2 polymorphism on the stability of the virus-receptor complex. We used the 6LZG PDB RBD/ACE2 3D model, the mCSM platform, the LigPlot+ and PyMol software to analyze the data on SARS-CoV-2 mutations and ACE variants retrieved from GISAID and Ensembl/GnomAD repository. We observed that out of 351 RBD point mutations, 83% destabilizes the complex according to free energy (\u0394\u0394G) differences. We also spotted variations in the patterns of polar and hydrophobic interactions between the mutations occurring in 15 out of 18 contact residues. Similarly, comparison of the effect on the complex stability of different ACE2 variants showed that the pattern of molecular interactions and the complex stability varies also according to ACE2 polymorphism. We infer that it is important to consider both ACE2 variants and circulating SARS-CoV-2 RBD mutations to assess the stability of the virus-receptor association and evaluate infectivity. This approach might offers a good molecular ground to mitigate the virus spreading.","version":"1.4","doi":"10.1101/2020.10.23.352344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433558","pub_date":"2021-3-04","title":"Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters","abstract":"Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.","version":"1.1","doi":"10.1101/2021.03.03.433558","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.04.433966","pub_date":"2021-3-04","title":"Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain","abstract":"The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.","version":"1.1","doi":"10.1101/2021.03.04.433966","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433824","pub_date":"2021-3-04","title":"A novel, anatomy-similar in vitro model of 3D airway epithelial for anti-coronavirus drug discovery","abstract":"SARS-CoV-2 and its induced COVID-19 remains as a global health calamity. Severe symptoms and high mortality, caused by cytokine storm and acute respiratory distress syndrome in the lower respiratory airway, are always associated with elderly individuals and those with comorbidities; whereas mild or moderate COVID-19 patients have limited upper respiratory flu-like symptoms. There is an urgent need to investigate SARS-CoV-2 and other coronaviruses replication and immune responses in human respiratory systems. The human reconstituted airway epithelial air-liquid interface (ALI) models are the most physiologically relevant model for the investigation of coronavirus infection and virus-triggered innate immune signatures. We established ALI models representing both the upper and the lower respiratory airway to characterize the coronavirus infection kinetics, tissue pathophysiology, and innate immune signatures from upper and lower respiratory tract perspective. Our data suggested these in vitro ALI models maintain high physiological relevance with human airway tissues. The coronavirus induced immune response observed in these upper and lower respiratory airway models are similar to what has been reported in COVID-19 patients. The antiviral efficacy results of a few promising anti-coronavirus drugs in these models were consistent with previous reports and could be valuable for the human dose prediction. Taken together, our study demonstrates the importance of 3D airway epithelial ALI model for the understanding of coronavirus pathogenesis and the discovery and development of anti-coronavirus drugs.","version":"1.1","doi":"10.1101/2021.03.03.433824","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433767","pub_date":"2021-3-04","title":"SPOT: a web-tool enabling Swift Profiling Of Transcriptomes","abstract":"The increasing number of single cell and bulk RNAseq data sets describing complex gene expression profiles in different organisms, organs or cell types calls for an intuitive tool allowing rapid comparative analysis. Here we present Swift Profiling Of Transcriptomes (SPOT) as a web tool that allows not only differential expression analysis but also fast ranking of genes fitting transcription profiles of interest. Based on a heuristic approach the spot algorithm ranks the genes according to their proximity to the user-defined gene expression profile of interest. The best hits are visualized as a table, bar chart or dot plot and can be exported as an Excel file. While the tool is generally applicable, we tested it on RNAseq data from malaria parasites that undergo multiple stage transformations during their complex life cycle as well as on data from multiple human organs during development and cell lines infected by the SARS-CoV-2 virus. SPOT should enable non-bioinformaticians to easily analyse their own and any available dataset.","version":"1.1","doi":"10.1101/2021.03.03.433767","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433579","pub_date":"2021-3-04","title":"T-cell Repertoire Characteristics of Asymptomatic and Re-detectable Positive COVID-19 Patients","abstract":"The prevention of COVID-19 pandemic is highly complicated by the prevalence of asymptomatic and recurrent infection. Many previous immunological studies have focused on symptomatic and convalescent patients, while the immune responses in asymptomatic patients and re-detectable positive cases remain unclear. Here we comprehensively analyzed the peripheral T-cell receptor (TCR) repertoire of 54 COVID-19 patients in different phases, including asymptomatic, symptomatic, convalescent and re-detectable positive cases. We found progressed immune responses from asymptomatic to symptomatic phase. Furthermore, the TCR profiles of re-detectable positive cases were highly similar to those of asymptomatic patients, which could predict the risk of recurrent infection. Therefore, TCR repertoire surveillance has the potential to strengthen the clinical management and the immunotherapy development for COVID-19. The Science and Technology Innovation Project of Foshan Municipality (2020001000431) and the National Key Research and Development Project (2020YFA0708001).","version":"1.1","doi":"10.1101/2021.03.03.433579","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433699","pub_date":"2021-3-03","title":"Comparative analysis of codon usage patterns in SARS-CoV-2, its mutants and other respiratory viruses","abstract":"The Coronavirus disease 2019 (COVID-19) outbreak caused by Severe Acute Respiratory Syndrome Coronavirus 2 virus (SARS-CoV-2) poses a worldwide human health crisis, causing respiratory illness with a high mortality rate. To investigate the factors governing codon usage bias in all the respiratory viruses, including SARS-CoV-2 isolates from different geographical locations (~62K), including two recently emerging strains from the United Kingdom (UK), i.e., VUI202012/01 and South Africa (SA), i.e., 501.Y.V2 codon usage bias (CUBs) analysis was performed. The analysis includes RSCU analysis, GC content calculation, ENC analysis, dinucleotide frequency and neutrality plot analysis. We were motivated to conduct the study to fulfil two primary aims: first, to identify the difference in codon usage bias amongst all SARS-CoV-2 genomes and, secondly, to compare their CUBs properties with other respiratory viruses. A biased nucleotide composition was found as most of the highly preferred codons were A/U-ending in all the respiratory viruses studied here. Compared with the human host, the RSCU analysis led to the identification of 11 over-represented codons and 9 under-represented codons in SARS-CoV-2 genomes. Correlation analysis of ENC and GC3s revealed that mutational pressure is the leading force determining the CUBs. The present study results yield a better understanding of codon usage preferences for SARS-CoV-2 genomes and discover the possible evolutionary determinants responsible for the biases found among the respiratory viruses, thus unveils a unique feature of the SARS-CoV-2 evolution and adaptation. To the best of our knowledge, this is the first attempt at comparative CUBs analysis on the worldwide genomes of SARS-CoV-2, including novel emerged strains and other respiratory viruses.","version":"1.1","doi":"10.1101/2021.03.03.433699","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.432690","pub_date":"2021-3-03","title":"Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell epitope mapping","abstract":"The current COVID-19 pandemic represents a global challenge. A better understanding of the immune response against SARS-CoV-2 is key to unveil the differences in disease severity and to develop future vaccines targeting novel SARS-CoV-2 variants. Feature barcode technology combined with CITE-seq antibodies and DNA-barcoded peptide-MHC I Dextramer reagents enabled us to identify relevant SARS-CoV-2-derived epitopes and compare epitope-specific CD8+ T cell populations between mild and severe COVID-19. We identified a strong CD8+ T cell response against an S protein-derived epitope. CD8+ effector cells in severe COVID-19 displayed hyperactivation, T cell exhaustion and were missing characteristics of long-lived memory T cells. We identify A*0101 WTAGAAAYY as an immunogenic CD8+ T cell epitope with the ability to drive clonal expansion. We provide an in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection which will be relevant for the development of molecular and targeted therapies and potential adjustments of vaccination strategies.","version":"1.1","doi":"10.1101/2021.03.03.432690","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433614","pub_date":"2021-3-03","title":"Recombinant protein subunit SARS-CoV-2 vaccines formulated with CoVaccine HT adjuvant induce broad, Th1 biased, humoral and cellular immune responses in mice","abstract":"The speed at which several COVID-19 vaccines went from conception to receiving FDA and EMA approval for emergency use is an achievement unrivaled in the history of vaccine development. Mass vaccination efforts using the highly effective vaccines are currently underway to generate sufficient herd immunity and reduce transmission of the SARS-CoV-2 virus. Despite the most advanced vaccine technology, global recipient coverage, especially in resource-poor areas remains a challenge as genetic drift in na\u00efve population pockets threatens overall vaccine efficacy. In this study, we described the production of insect-cell expressed SARS-CoV-2 spike protein ectodomain and examined its immunogenicity in mice. We demonstrated that, when formulated with CoVaccine HT\u2122adjuvant, an oil-in-water nanoemulsion compatible with lyophilization, our vaccine candidates elicit a broad-spectrum IgG response, high neutralizing antibody titers, and a robust, antigen-specific IFN-\u03b3 secreting response from immune splenocytes in outbred mice. Our findings lay the foundation for the development of a dry-thermostabilized vaccine that is deployable without refrigeration.","version":"1.1","doi":"10.1101/2021.03.02.433614","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.432977","pub_date":"2021-3-03","title":"Neutralizing IFNL3 Autoantibodies in Severe COVID-19 Identified Using Molecular Indexing of Proteins by Self-Assembly","abstract":"Unbiased antibody profiling can identify the targets of an immune reaction. A number of likely pathogenic autoreactive antibodies have been associated with life-threatening SARS-CoV-2 infection; yet, many additional autoantibodies likely remain unknown. Here we present Molecular Indexing of Proteins by Self Assembly (MIPSA), a technique that produces ORFeome-scale libraries of proteins covalently coupled to uniquely identifying DNA barcodes for analysis by sequencing. We used MIPSA to profile circulating autoantibodies from 55 patients with severe COVID-19 against 11,076 DNA-barcoded proteins of the human ORFeome library. MIPSA identified previously known autoreactivities, and also detected undescribed neutralizing interferon lambda 3 (IFN-\u03bb3) autoantibodies. At-risk individuals with anti-IFN-\u03bb3 antibodies may benefit from interferon supplementation therapies, such as those currently undergoing clinical evaluation. Molecular Indexing of Proteins by Self Assembly (MIPSA) identifies neutralizing IFNL3 autoantibodies in patients with severe COVID-19.","version":"1.1","doi":"10.1101/2021.03.02.432977","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.03.433704","pub_date":"2021-3-03","title":"Low-dose lung radiotherapy for COVID-19 lung disease: a preclinical efficacy study in a bleomycin model of pneumonitis","abstract":"Low-dose whole lung radiotherapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling and mechanisms of action. Female C57BL/6 mice were treated with intranasal bleomycin sulphate (7.5 or 11.25 units/kg, day 0), then exposed to whole lung radiation therapy (0.5, 1.0, 1.5 Gy or sham, day 3). Bodyweight was measured daily and lung tissue harvested for histology and flow cytometry on day 10. Computed tomography (CT) lung imaging was performed pre-radiation (day 3) and pre-endpoint (day 10). Bleomycin caused pneumonitis of variable severity which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight and a proportion of these mice exhibited less severe histopathological lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. Additionally, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids. Overall,bleomycin-treated mice exhibited significantly higher percentages of non-aerated lung in left than right lungs and LDLR (1.0 Gy) prevented further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not modulate bodyweight or flow cytometric readouts of bleomycin-induced pneumonitis. Our data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose and provide preliminary evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids.","version":"1.1","doi":"10.1101/2021.03.03.433704","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433498","pub_date":"2021-3-02","title":"Atorvastatin effectively inhibits late replicative cycle steps of SARS-CoV-2 in vitro","abstract":"SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a pandemic of historic proportions and continues to spread worldwide. Currently, there is no effective therapy against this virus. This article evaluated the in vitro antiviral effect of Atorvastatin against SARS-CoV-2 and also identified the interaction affinity between Atorvastatin and three SARS-CoV-2 proteins, using in silico structure-based molecular docking approach. The antiviral activity of Atorvastatin against SARS-CoV-2 was evaluated by three different treatment strategies using a clinical isolate of SARS-CoV-2. The interaction of Atorvastatin with Spike, RNA-dependent RNA polymerase (RdRp) and 3C-like protease (3CLpro) was evaluated by molecular docking. Atorvastatin showed anti-SARS-CoV-2 activity of 79%, 54.8%, 22.6% and 25% at 31.2, 15.6, 7.9, and 3.9 \u00b5M, respectively, by pre-post-treatment strategy. In addition, atorvastatin demonstrated an antiviral effect of 26.9% at 31.2 \u00b5M by pre-infection treatment. This compound also inhibited SARS-CoV-2 in 66.9%, 75%, 27.9% and 29.2% at concentrations of 31.2, 15.6, 7.9, and 3.9 \u00b5M, respectively, by post-infection treatment. The interaction of atorvastatin with SARS-CoV-2 Spike, RdRp and 3CL protease yielded a binding affinity of \u22128.5 Kcal/mol, \u22126.2 Kcal/mol, and \u22127.5 Kcal/mol, respectively. Our study demonstrated the in vitro anti-SARS-CoV-2 activity of Atorvastatin, mainly against the late steps of the viral replicative cycle. A favorable binding affinity with viral proteins by bioinformatics methods was also shown. Due to its low cost, availability, well-established safety and tolerability, and the extensive clinical experience of atorvastatin, it could prove valuable in reducing morbidity and mortality from COVID-19. The COVID-19 pandemic constitutes the largest global public health crisis in a century, with enormous health and socioeconomic challenges. Therefore, it is necessary to search for specific antivirals against its causative agent (SARS-CoV-2). In this sense, the use of existing drugs may represent a useful treatment option in terms of safety, cost-effectiveness, and timeliness. Atorvastatin is widely used to prevent cardiovascular events. This compound modulates the synthesis of cholesterol, a molecule necessary in different stages of the viral replicative cycle. Our study demonstrated the antiviral potential of atorvastatin against SARS-CoV-2, using an in vitro model. Furthermore, the ability of Atorvastatin to directly interfere with three viral targets (Spike, RdRp and 3CL protease) was demonstrated by bioinformatic methods. This compound is a well-studied, low-cost, and generally well-tolerated drug, so it could be a promising antiviral for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.03.01.433498","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.430255","pub_date":"2021-3-02","title":"A chicken IgY can efficiently inhibit the entry and replication of SARS-CoV-2 by targeting the ACE2 binding domain in vitro","abstract":"COVID-19 pneumonia has now spread widely in the world. Currently, no specific antiviral drugs are available. The vaccine is the most effective way to control the epidemic. Passive immune antibodies are also an effective method to prevent and cure COVID-19 pneumonia. We used the SARS-CoV-2 S receptor-binding domain (RBD) as an antigen to immunize layers in order to extract, separate, and purify SARS-CoV-2-IgY from egg yolk. SARS-CoV-2-IgY (S-IgY)can block the entry of SARS-CoV-2 into the Cells and reduce the viral load in cells. The Half effective concentration (EC50) of W3-IgY (S-IgY in the third week after immunization) is 1.35 \u00b1 0.15nM. The EC50 of W9-IgY (S-IgY in the ninth week after immunization) is 2.76 \u00b1 1.54 nM. When the dose of S-IgY is 55 nM, the fluorescence representing intracellular viral protein is obviously weakened in Immunofluorescence microscopy. Results of Sars-CoV-2 /Vero E6 cell experiment confirmed that S-IgY has a strong antiviral effect on SARS-CoV-2, and its (EC50) is 27.78 \u00b11.54 nMvs 3,259 \u00b1 159.62 nM of Redesivir (differ > 106 times P<0.001). S-IgY can inhibit the entry and replication of SARS-CoV-2, which is related to its targeting the ACE2 binding domain. S-IgY is safe, efficient, stable, and easy to obtain. This antibody can be an effective tool for preventing and treating COVID-19 pneumonia. The figure briefly illustrates that the preparation and extraction of S-IgY and its anti-S-CoV-2 mechanism is to inhibit the entry and replication of SARS-CoV-2 by targeting the ACE2.","version":"1.3","doi":"10.1101/2021.02.16.430255","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.336644","pub_date":"2021-3-02","title":"Ongoing Global and Regional Adaptive Evolution of SARS-CoV-2","abstract":"Understanding the trends in SARS-CoV-2 evolution is paramount to control the COVID- 19 pandemic. We analyzed more than 300,000 high quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the nuclear localization signal (NLS) associated region of the nucleocapsid protein are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region- specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS associated variants across multiple partitions rose to global prominence in March-July, during a period of stasis in terms of inter- regional diversity. Finally, beginning July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation. Understanding the ongoing evolution of SARS-CoV-2 is essential to control and ultimately end the pandemic. We analyzed more than 300,000 SARS-CoV-2 genomes available as of January 2021 and demonstrate adaptive evolution of the virus that affects, primarily, multiple sites in the spike and nucleocapsid protein. Selection appears to act on combinations of mutations in these and other SARS-CoV-2 genes. Evolution of the virus is accompanied by ongoing adaptive diversification within and between geographic regions. This diversification could substantially prolong the pandemic and the vaccination campaign, in which variant-specific vaccines are likely to be required.","version":"1.3","doi":"10.1101/2020.10.12.336644","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433130","pub_date":"2021-3-02","title":"Safety and Immunogenicity Evaluation of Inactivated whole-virus-SARS-COV-2 As Emerging Vaccine Development In Egypt","abstract":"The current worldwide pandemic COVID-19 is causing severe human health problems, with high numbers of mortality rates and huge economic burdens that require an urgent demand for safe, and effective and vaccine development. Our study was the first trail to development and evaluation of safety and immune response to inactivated whole SARS-COV-2 virus vaccine adjuvanted with aluminium hydroxide. We used characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069 and MW250352 at GenBank that isolated from Egyptian patients SARS-CoV-2-positive. Development of the vaccine was carried out in a BSL - 3 facilities and the immunogenicity was determined in mice at two doses (55\u00b5g and 100\u00b5g per dose). All vaccinated mice were received a booster dose 14 days post first immunization. Our results demonstrated distinct cytopathic effect on the vero cell monolayers induced through SARS-COV-2 propagation and the viral particles were identified as Coronaviridae by transmission electron microscopy. SARS-CoV-2 was identified by RT-PCR performed on the cell culture. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless the dose concentration, with excellent safety profiles.However, no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by wild virus challenge the vaccinated mice and detection of viral replication in lung tissues. Vaccinated mice recorded complete protection from challenge infection three weeks post second dose. SARS-COV-2 replication was not observed in the lungs of mice following SARS-CoV-2 challenge, regardless of the level of serum neutralizing antibodies. This finding will support the future trials for evaluation an applicable SARS-CoV-2 vaccine candidate.","version":"1.1","doi":"10.1101/2021.03.01.433130","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433503","pub_date":"2021-3-02","title":"Cytoplasmic domain and enzymatic activity of ACE2 is not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells","abstract":"The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor- human angiotensin converting enzyme 2 (ACE2). Here, we used lentivirus based pseudotypes bearing spike protein to demonstrate that entry of SARS-CoV-2 into host cells is dependent on clathrin-mediated endocytosis, and phosphoinositides play essential role during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 is not required for efficient virus entry. These results provide new insights into SARS-CoV-2 cellular entry and present potential targets for drug development.","version":"1.1","doi":"10.1101/2021.03.01.433503","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433434","pub_date":"2021-3-02","title":"High-content screening of coronavirus genes for innate immune suppression reveals enhanced potency of SARS-CoV-2 proteins","abstract":"Suppression of the host intracellular innate immune system is an essential aspect of viral replication. Here, we developed a suite of medium-throughput high-content cell-based assays to reveal the effect of individual coronavirus proteins on antiviral innate immune pathways. Using these assays, we screened the 196 protein products of seven coronaviruses (SARS-CoV-2, SARS-CoV-1, 229E, NL63, OC43, HKU1 and MERS). This includes a previously unidentified gene in SARS-CoV-2 encoded within the Spike gene. We observe immune-suppressing activity in both known host-suppressing genes (e.g., NSP1, Orf6, NSP3, and NSP5) as well as other coronavirus genes, including the newly identified SARS-CoV-2 protein. Moreover, the genes encoded by SARS-CoV-2 are generally more potent immune suppressors than their homologues from the other coronaviruses. This suite of pathway-based and mechanism-agnostic assays could serve as the basis for rapid in vitro prediction of the pathogenicity of novel viruses based on provision of sequence information alone.","version":"1.1","doi":"10.1101/2021.03.02.433434","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433608","pub_date":"2021-3-02","title":"Perturbation of ACE2 structural ensembles by SARS-CoV-2 spike protein binding","abstract":"The human ACE2 enzyme serves as a critical first recognition point of coronaviruses, including SARS-CoV-2. In particular, the extracellular domain of ACE2 interacts directly with the S1 tailspike protein of the SARS-CoV-2 virion through a broad protein-protein interface. Although this interaction has been characterized by X-ray crystallography and Cryo-EM, these structures do not reveal significant differences in ACE2 structure upon S1 protein binding. In this work, using several all-atom molecular dynamics simulations, we show persistent differences in ACE2 structure upon binding. These differences are determined with the Linear Discriminant Analysis (LDA) machine learning method and validated using independent training and testing datasets, including long trajectories generated by D. E. Shaw Research on the Anton 2 supercomputer. In addition, long trajectories for 78 potent ACE2-binding compounds, also generated by D. E. Shaw Research, were projected onto the LDA classification vector in order to determine whether the ligand-bound ACE2 structures were compatible with S1 protein binding. This allows us to predict which compounds are \u201capo-like\u201d vs \u201ccomplex-like\u201d, as well as to pinpoint long-range ligand-induced allosteric changes of ACE2 structure.","version":"1.1","doi":"10.1101/2021.03.02.433608","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.02.433390","pub_date":"2021-3-02","title":"Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates","abstract":"Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3\u00d7106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.","version":"1.1","doi":"10.1101/2021.03.02.433390","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.08.430146","pub_date":"2021-3-02","title":"SARS-CoV-2 RBD-Tetanus toxoid conjugate vaccine induces a strong neutralizing immunity in preclinical studies","abstract":"Controlling the global COVID-19 pandemic depends, among other measures, on developing preventive vaccines at an unprecedented pace. Vaccines approved for use and those in development intend to use neutralizing antibodies to block viral sites binding to the host\u2019s cellular receptors. Virus infection is mediated by the spike glycoprotein trimer on the virion surface via its receptor binding domain (RBD). Antibody response to this domain is an important outcome of the immunization and correlates well with viral neutralization. Here we show that macromolecular constructs with recombinant RBD conjugated to tetanus toxoid induce a potent immune response in laboratory animals. Some advantages of the immunization with the viral antigen coupled to tetanus toxoid have become evident such as predominant IgG immune response due to affinity maturation and long-term specific B-memory cells. This paper demonstrates that subunit conjugate vaccines can be an alternative for COVID-19, paving the way for other viral conjugate vaccines based on the use of small viral proteins involved in the infection process.","version":"1.2","doi":"10.1101/2021.02.08.430146","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428529","pub_date":"2021-3-01","title":"Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail","abstract":"The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-2130, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an \u201caromatic cage\u201d at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2-2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.","version":"1.2","doi":"10.1101/2021.01.27.428529","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.27.433180","pub_date":"2021-3-01","title":"Negligible impact of SARS-CoV-2 variants on CD4+ and CD8+ T cell reactivity in COVID-19 exposed donors and vaccinees","abstract":"The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.02.27.433180","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.27.433186","pub_date":"2021-3-01","title":"Effect of SARS-CoV-2 proteins on vascular permeability","abstract":"SARS-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5) and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein\u2013protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by COVID-19. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.","version":"1.1","doi":"10.1101/2021.02.27.433186","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.427166","pub_date":"2021-3-01","title":"SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma","abstract":"SARS-CoV-2 501Y.V2 (B.1.351), a novel lineage of coronavirus causing COVID-19, contains substitutions in two immunodominant domains of the spike protein. Here, we show that pseudovirus expressing 501Y.V2 spike protein completely escapes three classes of therapeutically relevant antibodies. This pseudovirus also exhibits substantial to complete escape from neutralization, but not binding, by convalescent plasma. These data highlight the prospect of reinfection with antigenically distinct variants and foreshadows reduced efficacy of spike-based vaccines.","version":"1.2","doi":"10.1101/2021.01.18.427166","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433110","pub_date":"2021-3-01","title":"Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice","abstract":"Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.03.01.433110","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169334","pub_date":"2021-3-01","title":"Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 By Disrupting The Spike-ACE2 interaction","abstract":"Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV-2 virus, providing a tractable new target for therapeutic intervention. Clinically-used heparins demonstrate inhibitory activity, but world supplies are limited, necessitating alternative solutions. Synthetic HS mimetic pixatimod is a drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds to and destabilizes the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), and directly inhibits its binding to human ACE2, consistent with molecular modelling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of monkey Vero E6 and human bronchial epithelial cells at concentrations within its safe therapeutic dose range. Furthermore, in a K18-hACE2 mouse model pixatimod demonstrates that pixatimod markedly attenuates SARS-CoV-2 viral titer and COVID-19-like symptoms. This demonstration of potent anti-SARS-CoV-2 activity establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics. Together with other known activities of pixatimod our data provides a strong rationale for its clinical investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.","version":"1.3","doi":"10.1101/2020.06.24.169334","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.27.433054","pub_date":"2021-3-01","title":"Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2","abstract":"Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistically friendly are critically needed for global equity, especially for middle to low income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported with the use of the receptor binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, so called \u201cHexaPro\u201d, has been shown for its physical property to possess two RBD in the \u201cup\u201d conformation, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminium hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. In addition, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.","version":"1.1","doi":"10.1101/2021.02.27.433054","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433340","pub_date":"2021-3-01","title":"The impact of mutations on the structural and functional properties of SARS-CoV-2 proteins: A comprehensive bioinformatics analysis","abstract":"An in-depth analysis of first wave SARS-CoV-2 genome is required to identify various mutations that significantly affect viral fitness. In the present study, we have performed comprehensive in-silico mutational analysis of 3C-like protease (3CLpro), RNA dependent RNA polymerase (RdRp), and spike (S) proteins with the aim of gaining important insights into first wave virus mutations and their functional and structural impact on SARS-CoV-2 proteins. Our integrated analysis gathered 3465 SARS-CoV-2 sequences and identified 92 mutations in S, 37 in RdRp, and 11 in 3CLpro regions. The impact of those mutations was also investigated using various in silico approaches. Among these 32 mutations in S, 15 in RdRp, and 3 in 3CLpro proteins are found to be deleterious in nature and could alter the structural and functional behavior of the encoded proteins. D614G mutation in spike and P323L in RdRp are the globally dominant variants with a high frequency. Most of them have also been found in the binding moiety of the viral proteins which determine their critical involvement in the host-pathogen interactions and drug targets. The findings of the current study may facilitate better understanding of COVID-19 diagnostics, vaccines, and therapeutics.","version":"1.1","doi":"10.1101/2021.03.01.433340","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433344","pub_date":"2021-3-01","title":"In vitro screening of herbal medicinal products for their supportive curing potential in the context of SARS-CoV-2","abstract":"Herbal medicinal products have a long-standing history of use in the therapy of common respiratory infections. In the COVID-19 pandemic, they may have the potential for symptom relief in non-severe or moderate disease cases. Here we describe the results derived by in vitro screening of five herbal medicinal products with regard to their potential to i) interfere with the binding of the human Angiotensin-converting enzyme 2 (ACE2) receptor with the SARS-CoV-2 Spike S1 protein, ii) modulate the release of the human defensin HBD1 and cathelicidin LL-37 from human A549 lung cells upon Spike S1 protein stimulation and iii) modulate the release of IFN-\u03b3 from activated human peripheral blood mononuclear cells (PBMC). The investigated extracts were: Sinupret extract (SINx), Bronchipret thyme-ivy (BRO TE), Bronchipret thyme-primrose (BRO TP), Imupret (IMU), and Tonsipret (TOP). The inhibitory effect of the herbal medicinal products on the binding interaction of Spike S1 protein and the human ACE2 receptor was measured by ELISA. The effects on intracellular IFN-\u03b3 expression in stimulated human PBMCs were measured by flow cytometry. Regulation on HBD1 and LL-37 expression and secretion was assessed in 25d long-term cultured human lung A549 epithelial cells by RT-PCR and ELISA. IMU and BRO TE concentration-dependently inhibited the interaction between spike protein and the ACE2 Receptor. However, this effect was only observed in the cell-free assay at a concentration range which was later on determined as cytotoxic to human PBMC. SINx, TOP and BRO TP significantly upregulated the intracellular expression of antiviral IFN\u03b3 from stimulated PBMC. Co-treatment of A549 cells with IMU or BRO TP together with SARS-CoV-2 spike protein significantly upregulated mRNA expression (IMU) and release (IMU and BRO TP) of HBD1 and LL-37 (BRO TP). The in vitro screening results provide first evidence for an immune activating potential of some of the tested herbal medicinal extracts in the context of SARS-CoV-2. Whether these could be helpful in prevention of SARS-CoV-2 invasion or supportive in recovery from SARS-CoV-2 infection needs deeper understanding of the observations.","version":"1.1","doi":"10.1101/2021.03.01.433344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433431","pub_date":"2021-3-01","title":"Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell","abstract":"Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.","version":"1.1","doi":"10.1101/2021.03.01.433431","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433428","pub_date":"2021-3-01","title":"A Comparison of Performance for Different SARS-Cov-2 Sequencing Protocols","abstract":"SARS-Cov-2 genome sequencing has been identified as a fundamental tool for fighting the COVID-19 pandemic. It is used, for example, for identifying new variants of the virus and for elaborating phylogenetic trees that help to trace the spread of the virus. In the present study we provide a comprehensive comparison between the quality of the assemblies obtained from different sequencing protocols. We demonstrate how some protocols actively promoted by different high-level administrations are inefficient and how less-used alternative protocols show a significant increased performance. This increase of performance could lead to cheaper sequencing protocols and therefore to a more convenient escalation of the sequencing efforts around the world.","version":"1.1","doi":"10.1101/2021.03.01.433428","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.28.433287","pub_date":"2021-3-01","title":"The E3 Ubiquitin Ligase RNF5 Facilitates SARS-CoV-2 Membrane Protein-Mediated Virion Release","abstract":"As enveloped virus, SARS-CoV-2 membrane protein (M) mediates viral release from cellular membranes, but the molecular mechanisms of SARS-CoV-2 virions release remain poorly understood. Here, we performed RNAi screening and identified the E3 ligase RNF5 which mediates ubiquitination of SARS-CoV-2 M at residue K15 to enhance the interaction of viral envelope (E) with M. M-E complex ensures the uniform size of viral particles for viral maturation and mediates viral release. Moreover, overexpression of M induces complete autophagy which is dependent on RNF5-mediated ubiquitin modification. M inhibits the activity of lysosome protease, and uses autolysosomes for virion release. Consequently, all these results demonstrate that RNF5 mediates ubiquitin modification of SARS-CoV-2 M to stabilize the M-E complex and induce autophagy for virion release.","version":"1.1","doi":"10.1101/2021.02.28.433287","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433404","pub_date":"2021-3-01","title":"CD47 as a potential biomarker for the early diagnosis of severe COVID-19","abstract":"The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. Antiviral interventions are only effective prior to the onset of hyperinflammation. Hence, biomarkers are needed for the early identification and treatment of high-risk patients. Here, we show in a range of model systems and data from post mortem samples that SARS-CoV-2 infection results in increased levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells. Systematic literature searches also indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions which may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, CD47 is a candidate biomarker for severe COVID-19. Further research will have to show whether CD47 is a reliable diagnostic marker for the early identification of COVID-19 patients requiring antiviral therapy.","version":"1.1","doi":"10.1101/2021.03.01.433404","journal":"bioRxiv","score":null},{"id":"10.1101/2021.03.01.433314","pub_date":"2021-3-01","title":"Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains","abstract":"We examined the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 that arose in the United Kingdom and spread globally. Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa B.1.351 variant, than of the infecting variant. The drop in cross-reactivity was more pronounced following B.1.1.7 than parental strain infection, indicating asymmetric heterotypic immunity induced by SARS-CoV-2 variants.","version":"1.1","doi":"10.1101/2021.03.01.433314","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.25.424008","pub_date":"2021-3-01","title":"Differential Dynamic Behavior of Prefusion Spike Proteins of SARS Coronaviruses 1 and 2","abstract":"The coronavirus spike protein, which binds to the same human receptor in both SARS-CoV-1 and 2, has been implied to be a potential source of their differential transmissibility. However, the mechanistic details of spike protein binding to its human receptor remain elusive at the molecular level. Here, we have used an extensive set of unbiased and biased microsecond-level all-atom molecular dynamics (MD) simulations of SARS-CoV-1 and 2 spike proteins to determine the differential dynamic behavior of prefusion spike protein structure in the two viruses. Our results indicate that the active form of the SARS-CoV-2 spike protein is more stable than that of SARS-CoV-1 and the energy barrier associated with the activation is higher in SARS-CoV-2. Our results also suggest that not only the receptor binding domain (RBD) but also other domains such as the N-terminal domain (NTD) could play a role in the differential binding behavior of SARS-CoV-1 and 2 spike proteins.","version":"1.2","doi":"10.1101/2020.12.25.424008","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432969","pub_date":"2021-3-01","title":"Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants","abstract":"The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 \u03bcg/mL; IC80 < 0.0006 to 0.0251 \u03bcg/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs. Ultrapotent antibodies from convalescent donors neutralize and mitigate resistance of SARS-CoV-2 variants of concern.","version":"1.2","doi":"10.1101/2021.02.25.432969","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432576","pub_date":"2021-3-01","title":"Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape","abstract":"Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.","version":"1.2","doi":"10.1101/2021.02.24.432576","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.28.433291","pub_date":"2021-3-01","title":"Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.","version":"1.1","doi":"10.1101/2021.02.28.433291","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.21251531","pub_date":"2021-02-28","title":"Histopathological assessments reveal retinal vascular changes, inflammation and gliosis in patients with lethal COVID-19","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Purpose</jats:title>\n                  <jats:p>To assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Comparative analysis of human eyes.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Subjects</jats:title>\n                  <jats:p>Eleven donor eyes from COVID-19 positive donors and similar age-matched donor eyes from patients with a negative test for SARS-CoV-2 were assessed.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Globes were evaluated ex-vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for epon-embedding and immunocytochemistry</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Main Outcome Measures</jats:title>\n                  <jats:p>Retinal thickness and histopathology, detection of SARS-CoV-2 Spike protein, changes in vascular density, gliosis, and degree of inflammation.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the control. OCT based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes, however the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    <jats:italic>In situ</jats:italic>\n                    analysis of the retinal tissue suggested that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.25.21251531","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.26.433111","pub_date":"2021-2-27","title":"VCFCons: a versatile VCF-based consensus sequence generator for small genomes","abstract":"We had developed VCFCons to address urgent need for a robust consensus sequence generator for SARS-CoV-2 viral surveillance, which presented several unique requirements, including: (a) low coverage areas should be noted with \u2018N\u2019s, (b) low frequency or suspicious variant calls need to be filtered. We have found that, while some existing tools such as bcftools can generate the desired consensus sequence, it required multiple filtering steps and additional scripting. VCFCons can generate consensus sequences based on variant calls in a VCF format with versatile filtering criteria based on coverage and estimated variant frequency. We applied VCFCons to the Labcorp SARS-CoV-2 sequencing data and showed that it generated correct consensus sequences that were successfully submitted to GISAID and NCBI. We hope the community will find value in this tool and aim to continue developing VCFCons to handle more complex viral data in the future.","version":"1.1","doi":"10.1101/2021.02.26.433111","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.26.433062","pub_date":"2021-2-26","title":"Comparative infectivity and pathogenesis of emerging SARS-CoV-2 variants in Syrian hamsters","abstract":"Within one year after its emergence, more than 108 million people contracted SARS-CoV-2 and almost 2.4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1), variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics and new spikes in excess mortality. We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 lineages in Syrian golden hamsters to assess their relative infectivity and pathogenicity in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Overall, we established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC.","version":"1.1","doi":"10.1101/2021.02.26.433062","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.356014","pub_date":"2021-2-26","title":"COVID-19 Disease Map, a computational knowledge repository of SARS-CoV-2 virus-host interaction mechanisms","abstract":"We describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. We describe the contents of the Map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases and highlight new testable hypotheses.","version":"1.3","doi":"10.1101/2020.10.26.356014","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.21251664","pub_date":"2021-02-26","title":"OBESITY MAY HAMPER SARS-CoV-2 VACCINE IMMUNOGENICITY","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The first goal of the study was to analyse the antibody titre 7 days after the second dose of BNT162b2 vaccine in a group of 248 healthcare workers (HCW). The second goal was to analyse how the antibody titre changes in correlation with age, gender and BMI.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Participants were assigned to receive the priming dose at baseline and booster dose at day 21. Blood and nasopharyngeal swabs were collected at baseline and 7 days after second dose of vaccine.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>248 HWCs were analysed, 158 women (63.7%) and 90 men (36.3%). After the second dose of BNT162b2 vaccine, 99.5% of participants developed a humoral immune response.</jats:p>\n                  <jats:p>The geometric mean concentration of antibodies among the vaccinated subjects after booster dose (285.9 AU/mL 95% CI: 249.5-327.7); was higher than that of human convalescent sera (39.4 AU/mL, 95% CI: 33.1-46.9), with p&lt;0.0001. The antibody titre was found to be higher in young and female participants. A strong correlation of BMI classes with antibody titres was noticed: humoral response was more efficient in the group with under- and normal-weight vs the group with pre- and obesity participants (p&lt;0.0001 at T1).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>These findings imply that females, lean and young people have an increased capacity to mount humoral immune responses compared to males, overweight and the older population. Although further studies are needed, this data may have important implications for the development of vaccination strategies for COVID-19, particularly in obese people.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>None</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.24.21251664","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.23.21252299","pub_date":"2021-02-26","title":"The Public Health Impact of Delaying a Second Dose of the BNT162b2 or mRNA-1273 COVID-19 Vaccine","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>To estimate population health outcomes under delayedsecond dose versus standard schedule SARS-CoV-2 mRNA vaccination.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Agent-based modeling on a simulated population of 100,000 based on a real-world US county. The simulation runs were replicated 10 times. To test the robustness of these findings, simulations were performed under different estimates for single-dose efficacy and vaccine administration rates, and under the possibility that a vaccine prevents only symptoms but not asymptomatic spread.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>population level simulation.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>100,000 agents are included in the simulation, with a representative distribution of demographics and occupations. Networks of contacts are established to simulate potentially infectious interactions though occupation, household, and random interactions</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interventions</jats:title>\n                  <jats:p>we simulate standard Covid-19 vaccination, versus delayed-second-dose vaccination prioritizing first dose. Sensitivity analyses include first-dose vaccine efficacy of 70%, 80% and 90% after day 12 post-vaccination; vaccination rate of 0.1%, 0.3%, and 1% of population per day; assuming the vaccine prevents only symptoms but not asymptomatic spread; and an alternative vaccination strategy that implements delayed-second-dose only for those under 65 years of age.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Main outcome measures</jats:title>\n                  <jats:p>cumulative Covid-19 mortality over 180 days, cumulative infections and hospitalizations.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Over all simulation replications, the median cumulative mortality per 100,000 for standard versus delayed second dose was 226 vs 179; 233 vs 207; and 235 vs 236; for 90%, 80% and 70% first-dose efficacy, respectively. The delayed-second-dose strategy was optimal for vaccine efficacies at or above 80%, and vaccination rates at or below 0.3% population per day, both under sterilizing and non-sterilizing vaccine assumptions, resulting in absolute cumulative mortality reductions between 26 and 47 per 100,000. The delayed-second-dose for those under 65 performed consistently well under all vaccination rates tested.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>A delayed-second-dose vaccination strategy, at least for those under 65, could result in reduced cumulative mortality under certain conditions.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.23.21252299","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.25.432762","pub_date":"2021-2-25","title":"The type 2 asthma mediator IL-13 inhibits SARS-CoV-2 infection of bronchial epithelium","abstract":"Asthma is associated with chronic changes in the airway epithelium, a key target of SARS-CoV-2. Many epithelial changes are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. We sought to discover how IL-13 and other cytokines affect expression of genes encoding SARS-CoV-2-associated host proteins in human bronchial epithelial cells (HBECs) and determine whether IL-13 stimulation alters susceptibility to SARS-CoV-2 infection. We used bulk and single cell RNA-seq to identify cytokine-induced changes in SARS-CoV-2-associated gene expression in HBECs. We related these to gene expression changes in airway epithelium from individuals with mild-moderate asthma and chronic obstructive pulmonary disease (COPD). We analyzed effects of IL-13 on SARS-CoV-2 infection of HBECs. Transcripts encoding 332 of 342 (97%) SARS-CoV-2-associated proteins were detected in HBECs (\u22651 RPM in 50% samples). 41 (12%) of these mRNAs were regulated by IL-13 (>1.5-fold change, FDR < 0.05). Many IL-13-regulated SARS-CoV-2-associated genes were also altered in type 2 high asthma and COPD. IL-13 pretreatment reduced viral RNA recovered from SARS-CoV-2 infected cells and decreased dsRNA, a marker of viral replication, to below the limit of detection in our assay. Mucus also inhibited viral infection. IL-13 markedly reduces susceptibility of HBECs to SARS-CoV-2 infection through mechanisms that likely differ from those activated by type I interferons. Our findings may help explain reports of relatively low prevalence of asthma in patients diagnosed with COVID-19 and could lead to new strategies for reducing SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.02.25.432762","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432853","pub_date":"2021-2-25","title":"Pyroptosis of syncytia formed by fusion of SARS-CoV-2 Spike and ACE2 expressing cells","abstract":"SARS-Cov-2 infected cells fused with the ACE2-positive neighboring cells forming syncytia. However, the effect of syncytia in disease development is largely unknown. We established an in vitro cell-cell fusion system and used it to mimic the fusion of SARS-CoV-2 infected cells with ACE2-expressing cells to form syncytia. We found that Caspase-9 was activated after syncytia formation, and Caspase-3/7 was activated downstream of Caspase-9, but it triggered GSDME-dependent pyroptosis rather than apoptosis. What is more, single cell RNA-sequencing data showed that both ACE2 and GSDME were expression in alveolar type 2 cells in human lung. We propose that pyroptosis is the fate of syncytia formed by SARS-CoV-2 infected host cells and ACE2-positive cells, which indicated that lytic death of syncytia may contribute to the excessive inflammatory responses in severe COVID-19 patients.","version":"1.1","doi":"10.1101/2021.02.25.432853","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.430130","pub_date":"2021-2-25","title":"A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2","abstract":"The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients\u2019 demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.","version":"1.1","doi":"10.1101/2021.02.25.430130","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432136","pub_date":"2021-2-25","title":"Comparison of the pathogenicity and virus shedding of SARS CoV-2 VOC 202012/01 and D614G variant in hamster model","abstract":"The emergence of SARS-CoV-2 variants has posed a serious challenge to public health system and vaccination programs across the globe. We have studied the pathogenicity and virus shedding pattern of the SARS-CoV-2 VOC 202012/01 and compared with D614G variant in Syrian hamsters. VOC 202012/01 could produce disease in hamsters characterized by body weight loss and respiratory tract tropism but mild lung pathology. Further, we also documented that neutralizing antibodies developed against VOC 202012/01 could equally neutralize D614G variant. Higher load of VOC 202012/01 in the nasal wash specimens was observed during the first week of infection outcompeting the D614G variant. The findings suggest increased fitness of VOC 202012/01 to the upper respiratory tract which could lead to higher transmission. Further investigations are needed to understand the transmissibility of new variants. SARS-CoV-2 VOC 202012/01 infected hamsters demonstrated high viral RNA shedding through the nasal secretions and significant body weight loss with mild lung pathology compared to the D614G variant.","version":"1.1","doi":"10.1101/2021.02.25.432136","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432265","pub_date":"2021-2-25","title":"Visualization of SARS-CoV-2 Infection Scenes by \u2018Zero-Shot\u2019 Enhancements of Electron Microscopy Images","abstract":"Electron microscopy (EM) recordings of infected tissues serve to diagnose a disease, and they can contribute to our understanding of infection processes. Consequently, a large number of EM images of the interaction of SARS-CoV-2 viruses with cells have been made available by numerous labs. However, due to EM recording techniques at high resolution, images of infection scenes are very noisy and they appear two dimensional (\u2018flat\u2019). Current research consequently aims (A) at methods that can remove noise, and (B) at techniques that allow for recovering a 3D impression of the virus or its parts. Here we discuss a novel method which can recover a spatial impression of a whole infection scene at high resolution. In contrast to previous approaches which aim at the reconstruction of single spike proteins or a single virus, the here used method can be applied to a single noisy EM image of an infection scene. As one example image, we show a high resolution image of SARS-CoV-2 viruses in Vero cell cultures (Fig. 1). The method we use is based on probabilistic machine learning algorithms which can operate in a \u2018zero-shot\u2019 setting, i.e., in a setting when just one noisy image (and no large and clean image corpus) is available. The probabilistic method we apply can estimate non-noisy images by inferring first order statistics (pixel means) across image patches using a previously learned probabilistic image representation. Estimating higher order statistics and appropriately chosen probabilistic models then allow for the generation of images that enhance details and give a spatial impression of a full nanoscopic scene.","version":"1.1","doi":"10.1101/2021.02.25.432265","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.155267","pub_date":"2021-2-25","title":"Transcriptogram analysis reveals relationship between viral titer and gene sets responses during Corona-virus infection","abstract":"To understand the difference between benign and severe outcomes after Coronavirus infection, we urgently need ways to clarify and quantify the time course of tissue and immune responses. Here we re-analyze 72-hour time-series microarrays generated in 2013 by Sims and collaborators for SARS-CoV-1 in vitro infection of a human lung epithelial cell line. Transcriptograms, a Bioinformatics tool to analyze genome-wide gene expression data, allow us to define an appropriate context-dependent threshold for mechanistic relevance of gene differential expression. Without knowing in advance which genes are relevant, classical analyses detect every gene with statistically-significant differential expression, leaving us with too many genes and hypotheses to be useful. Using a Transcriptogram-based top-down approach, we identified three major, differentially-expressed gene sets comprising 219 mainly immune-response-related genes. We identified timescales for alterations in mitochondrial activity, signaling and transcription regulation of the innate and adaptive immune systems and their relationship to viral titer. At the individual-gene level, EGR3 was significantly upregulated in infected cells. Similar activation in T-cells and fibroblasts in infected lung could explain the T-cell anergy and eventual fibrosis seen in SARS-CoV-1 infection. The methods can be applied to RNA data sets for SARS-CoV-2 to investigate the origin of differential responses in different tissue types, or due to immune or preexisting conditions or to compare cell culture, organoid culture, animal models, and human-derived samples.","version":"1.3","doi":"10.1101/2020.06.16.155267","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.25.432838","pub_date":"2021-2-25","title":"Misinformation, Perceptions Towards COVID-19 and Willingness to be Vaccinated: A Population-Based Survey in Yemen","abstract":"Since the beginning of the COVID-19 outbreak, many pharmaceutical companies were racing to develop a safe and effective COVID-19 vaccine. Simultaneously, rumors and misinformation about COVID-19 were and still widely spreading. Therefore, this study aimed to investigate the prevalence of COVID-19 misinformation among the Yemeni population and its association with vaccine acceptance and perceptions. A cross-sectional online survey was conducted in four major cities in Yemen. The constructed questionnaire consisted of four main sections (sociodemographic data, misinformation, perceptions (perceived susceptibility, severity and worry), and vaccination acceptance evaluation). Subject recruitment and data collection were conducted online utilizing social websites and using the snowball sampling technique. Descriptive and inferential analyses were performed using SPSS version 27. The total number of respondents was 484. Over 60% of them were male and had a university education, more than half had less than 100$ monthly income and were Khat chewers, while only 18% were smokers. Misinformation prevalence ranged from 8.9% to 38.9%, depending on the statement being asked. Men, university education, higher income, employment, and living in urban areas were associated with a lower misinformation level (p <0.05). Statistically significant association (p <0.05) between university education, living in urban areas, and being employed with perceived susceptibility were observed. The acceptance rate was 61.2% for free vaccines, but it decreased to 43% if they had to purchase it. Females, respondents with lower monthly income, and those who believed that pharmaceutical companies made the virus for financial gains were more likely to reject the vaccination (p <0.05). The study revealed that the acceptance rate to take a vaccine was suboptimal and significantly affected by gender, misinformation, cost, and income. Furthermore, being female, Nonuniversity educated, low-income, and living in rural areas were associated with higher susceptibility to misinformation about COVID-19. These findings show a clear link between misinformation susceptibility and willingness to vaccinate. Focused awareness campaigns to decrease misinformation and emphasize the vaccination\u2019s safety and efficacy might be fundamental before initiating any mass vaccination in Yemen.","version":"1.1","doi":"10.1101/2021.02.25.432838","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432203","pub_date":"2021-2-24","title":"Discovery of a AhR flavonoid agonist that counter-regulates ACE2 expression in rodent models of inflammation and attenuates ACE2-SARS-CoV2 interaction in vitro","abstract":"The severe acute respiratory syndrome (SARS)-CoV-2, a newly emerged coronavirus first identified in 2019, is the pathogenetic agent od Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE) 2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of ACE2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin, a natural flavonoid bind and activates the Aryl hydrocarbon Receptor (AhR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent DSS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of ACE2 mRNA expression. Colon levels of ACE2 mRNA were directly correlated with TNF\u03b1 mRNA levels. In contrast to ACE2 the angiotensin 1-7 receptor MAS was downregulated in the inflamed tissues. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV2 Spike protein to ACE2 and reduces the SARS-CoV2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.","version":"1.1","doi":"10.1101/2021.02.24.432203","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432759","pub_date":"2021-2-24","title":"Implications of central carbon metabolism in SARS-CoV-2 replication and disease severity","abstract":"Viruses hijack host metabolic pathways for their replicative advantage. Several observational trans-omics analyses associated carbon and amino acid metabolism in coronavirus disease 2019 (COVID-19) severity in patients but lacked mechanistic insights. In this study, using patient- derived multi-omics data and in vitro infection assays, we aimed to understand i) role of key metabolic pathways in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) reproduction and ii) its association with disease severity. Our data suggests that monocytes are key to the altered immune response during COVID-19. COVID-19 infection was associated with increased plasma glutamate levels, while glucose and mannose levels were determinants of the disease severity. Monocytes showed altered expression pattern of carbohydrate and amino acid transporters, GLUT1 and xCT respectively in severe COVID-19. Furthermore, lung epithelial cells (Calu-3) showed a strong acute metabolic adaptation following infection in vitro by modulating central carbon metabolism. We found that glycolysis and glutaminolysis are essential for virus replication and blocking these metabolic pathways caused significant reduction in virus production. Taken together, our study highlights that the virus utilizes and re-wires pathways governing central carbon metabolism leading to metabolic toxicity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.","version":"1.1","doi":"10.1101/2021.02.24.432759","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.425825","pub_date":"2021-2-24","title":"Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants","abstract":"Artemisia annua L. has been used for millennia in Southeast Asia to treat \u201cfever\u201d. Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. Using Vero E6 and Calu-3 cells, we measured anti viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated. Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 \u03bcM, 0.01-0.14 \u03bcg, and 23.4-57.4 \u03bcg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet the hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 \u03bcM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 \u03bcM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections. \nAmodiaquine\nArtemisinin\nArtesunate\nArtemether\nDeoxyartemisinin\nDihydroartemisinin\n\n Amodiaquine Artemisinin Artesunate Artemether Deoxyartemisinin Dihydroartemisinin \nArtemisia annua is effective in stopping replication of SARS-CoV-2 including 2 new variants.\nThe anti-viral effect does not correlate to artemisinin, nor to the total flavonoid content.\nThe anti-viral mechanism does not appear to involve blockade virus entry into cell.\nThe plant offers two additional benefits: a decreased inflammatory response and blunting of fibrosis.\nA. annua may provide a safe, low-cost alternative for treating patients infected with SARS-CoV-2.\n\n Artemisia annua is effective in stopping replication of SARS-CoV-2 including 2 new variants. The anti-viral effect does not correlate to artemisinin, nor to the total flavonoid content. The anti-viral mechanism does not appear to involve blockade virus entry into cell. The plant offers two additional benefits: a decreased inflammatory response and blunting of fibrosis. A. annua may provide a safe, low-cost alternative for treating patients infected with SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.01.08.425825","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432656","pub_date":"2021-2-24","title":"SARS-CoV-2 ORF6 disturbs nucleocytoplasmic trafficking to advance the viral replication","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 pandemic. ORF6 is known to antagonize the interferon signaling by inhibiting the nuclear translocation of STAT1. Here we show that ORF6 acts as a virulence factor through two distinct strategies. First, ORF6 directly interacts with STAT1 in an IFN-independent manner to inhibit its nuclear translocation. Second, ORF6 directly binds to importin \u03b11, which is a nuclear transport factor encoded by KPNA2, leading to a significant suppression of importin \u03b11-mediated nuclear transport. Furthermore, we found that KPNA2 knockout enhances the viral replication, suggesting that importin \u03b11 suppresses the viral propagation. Additionally, the analyses of gene expression data revealed that importin \u03b11 levels decreased significantly in the lungs of older individuals. Taken together, SARS-CoV-2 ORF6 disrupts the nucleocytoplasmic trafficking to accelerate the viral replication, resulting in the disease progression, especially in older individuals.","version":"1.1","doi":"10.1101/2021.02.24.432656","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432490","pub_date":"2021-2-24","title":"Targeted Drug Repurposing Against the SARS-CoV-2 E channel Identifies Blockers With in vitro Antiviral Activity","abstract":"It is difficult to overstate the impact that COVID-19 had on humankind. The pandemic\u2019s etiological agent, SARS-CoV-2, is a member of the Coronaviridae, and as such, is an enveloped virus with ion channels in its membrane. Therefore, in an attempt to provide an option to curb the viral spread, we searched for blockers of its E protein viro-porin. Using three bacteria-based assays, we identified eight compounds that exhibited activity after screening a library of ca. 3000 approved-for-human-use drugs. Reassuringly, analysis of viral replication in tissue culture indicated that most of the compounds could reduce infectivity to varying extents. In conclusion, targeting a particular channel in the virus for drug repurposing may increase our arsenal of treatment options to combat COVID-19 virulence. The goal of our study was to expand the treatment arsenal against COVID-19. To that end, we have decided to focus on drug therapy, and as a target - the E protein, an ion channel in the virus. Ion channels as a family are excellent drug targets, but viral channels have been underexploited for pharmaceutical point intervention. To hasten future regulatory requirements and focus the chemical search space, we screened a library of ca. 3000 approved-for-human-use drugs using three independent bacteria-based assays. Our results yielded eight compounds, which were subsequently tested for antiviral activity in tissue culture. Gratifyingly, most compounds were able to reduce viral replication, and as such, both validate our approach and potentially augment our anti-COVID tool kit.","version":"1.1","doi":"10.1101/2021.02.24.432490","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432734","pub_date":"2021-2-24","title":"Targeting of the NLRP3 Inflammasome for early COVID-19","abstract":"Following entry and replication of Severe Acute Respiratory Syndrome-coronavirus-2 (SARS-CoV-2) into ACE2 expressing cells, the infected cells undergo lysis releasing more virus but also cell contents. In the lung, constitutive cytokines such as IL-1\u03b1 are released together with other cell contents. A cascade of inflammatory cytokines ensues, including chemokines and IL-1\u03b2, triggering both local as well as systemic inflammation. This cascade of inflammatory cytokines in patients with COVID-19 is termed \u201cCytokine Release Syndrome\u201d (CRS), and is associated with poor outcomes and death. Many studies reveal that blocking IL-1 activities in COVID-19 patients reduces disease severity and deaths. Here we report highly significant circulating levels of IL-1\u03b2, IL-1 Receptor antagonist, IL-6, TNF\u03b1, IL-10 and soluble urokinase plasminogen activator receptor in COVID-19 patients with mild or no symptoms. We also report that in circulating myeloid cells from the same patients, there is increased expression of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) early in the infection. We observed increased NLRP3 gene expression in myeloid cells correlated with IL-1\u03b2 gene expression and also with elevated circulating IL-1\u03b2 levels. We conclude that early in SARS-CoV-2 infection, NLRP3 activation takes place and initiates the CRS. Thus, NLRP3 is a target to reduce the organ damage of inflammatory cytokines of the CRS.","version":"1.1","doi":"10.1101/2021.02.24.432734","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432721","pub_date":"2021-2-24","title":"A missense variant effect prediction and annotation resource for SARS-CoV-2","abstract":"The COVID19 pandemic is a global crisis severely impacting many people across the world. An important part of the response is monitoring viral variants and determining the impact they have on viral properties, such as infectivity, disease severity and interactions with drugs and vaccines. In this work we generate and make available computational variant effect predictions for all possible single amino-acid substitutions to SARS-CoV-2 in order to complement and facilitate experiments and expert analysis. The resulting dataset contains predictions from evolutionary conservation and protein and complex structural models, combined with viral phosphosites, experimental results and variant frequencies. We demonstrate predictions\u2019 effectiveness by comparing them with expectations from variant frequency and prior experiments. We then identify higher frequency variants with significant predicted effects as well as finding variants measured to impact antibody binding that are least likely to impact other viral functions. A web portal is available at sars.mutfunc.com, where the dataset can be searched and downloaded.","version":"1.1","doi":"10.1101/2021.02.24.432721","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432634","pub_date":"2021-2-24","title":"A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets","abstract":"Over the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) \u2013 SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 \u2013 has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.","version":"1.1","doi":"10.1101/2021.02.24.432634","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432649","pub_date":"2021-2-24","title":"Could Italy host the new coronavirus before China?","abstract":"The pandemic of the COVID-19 disease caused by the SARS-CoV-2 virus has been believed to originate in China and spread later to other parts of the world. It is well acknowledged that the first diseased individuals appeared in China as early as in December 2019, and possibly even earlier in November. It has also been well established that the virus stroke Italy later in January or in February 2020, distinctly after the outbreak in China. Paper by Apolone et al. published in a local Italian medical journal in November 2020 however exposed this chronology to doubt. By fitting early part of the epidemic curve with the exponential model and extrapolating it backwards in time, we could estimate the day-zero of the epidemic and calculated its confidence intervals in Italy and China. We also calculated how probable it is that Italy encountered the virus prior 1 January 2020. We determined an early portion of the epidemic curve representing unhindered exponential growth which fit the exponential model with high determination >0.97 in both countries. We suggest that the day-zero in China and Italy was 8 December (95% CI: 3 Dec., 20 Dec.) and 22 January (95% CI: 16 Jan., 29 Jan.), respectively. We could with high confidence reject that Italy encountered the virus earlier than China (p <0.01). Based on our analysis we oppose the findings published by Apolone at al. and view the proposed pre-pandemic presence of the virus in Italy as very unlikely.","version":"1.2","doi":"10.1101/2021.02.24.432649","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432418","pub_date":"2021-2-24","title":"The nonstructural protein 5 of coronaviruses antagonizes GSDMD-mediated pyroptosis by cleaving and inactivating its pore-forming p30 fragment","abstract":"Coronaviruses (CoV) are a family of RNA viruses that typically cause respiratory, enteric and hepatic diseases in animals and humans. Here, we used porcine epidemic diarrhea virus (PEDV) as a model of coronaviruses (CoVs) to illustrate the reciprocal regulation between CoVs infection and pyroptosis. For the first time, we clarified the molecular mechanism of porcine Gasdermin D (pGSDMD)-mediated pyroptosis and demonstrated that amino acids T239 and F240 within pGSDMD-p30 are critical for pyroptosis. Furthermore, 3C-like protease Nsp5 from SARS-CoV-2, MERS-CoV, PDCoV and PEDV can cleave human/porcine GSDMD at the Q193-G194 junction upstream of the caspase-1 cleavage site to produce two fragments which fail to trigger pyroptosis or inhibit viral replication. Thus, we provide clear evidence that coronoviruses may utilize viral Nsp5-GSDMD pathway to help their host cells escaping from pyroptosis, protecting the replication of the virus during the initial period, which suggest an important strategy for coronoviruses infection and sustain.","version":"1.1","doi":"10.1101/2021.02.23.432418","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.24.432694","pub_date":"2021-2-24","title":"Rapid adaptation and remote delivery of undergraduate research training during the COVID 19 Pandemic","abstract":"COVID-19 continues to alter daily life around the globe. Education is particularly affected by shifts to distance learning. This change has poignant effects on all aspects of academic life, including the consequence of increased mental stress reported specifically for students. COVID-19 cancellations of many summer fellowships and internships for undergraduates across the country increased students\u2019 uncertainty about their educational opportunities and careers. When the pandemic necessitated elimination of on-campus programming at Mayo Clinic, a new program was developed for remote delivery. Summer Foundations in Research (SFIR) was drafted around 4 aims: 1) support the academic trajectory gap in research science created by COVID-19; 2) build sustainable scientific relationships with mentors, peers, and the community; 3) create opportunities for participants to share and address concerns with their own experiences in the pandemic; and 4) provide support for individual wellbeing. SFIR included research training, but also training in communication through generative Dialogue and resilience through Amit Sood\u2019s SMART program. 170 participants were followed for outcomes in these spaces. Knowledge of and interest in careers involving biomedical research rose significantly following SFIR. Participants\u2019 mean confidence levels in 12 Key areas of research rose between 0.08 to 1.32 points on a 7-point scale. The strongest gains in mean confidence levels were seen in designing a study and collaborating with others. SFIR participants demonstrated gains in perceived happiness, and measured resilience and a reduction in stress. Participants\u2019 qualitative responses indicated exceptionally positive mentor relationships and specific benefit of both the SMART program and Dialogue.","version":"1.1","doi":"10.1101/2021.02.24.432694","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432407","pub_date":"2021-2-23","title":"At the Intersection Between SARS-CoV-2, Macrophages and the Adaptive Immune Response: A Key Role for Antibody-Dependent Pathogenesis But Not Enhancement of Infection in COVID-19","abstract":"Since entering the world stage in December of 2019, SARS-CoV-2 has impacted every corner of the globe with over 1.48 million deaths and caused untold economic damage. Infections in humans range from asymptomatic to severe disease associated with dysregulation of the immune system leading to the development of acute respiratory distress syndrome (ARDs). The distinct shift in peripheral monocyte activation and infiltration of these cells into the respiratory tract in ARDs patients suggests severe COVID-19 may largely result from damage to the respiratory epithelia by improperly activated macrophages. Here, we present evidence that dysregulation of the immune response in COVID-19 begins with activation of macrophages by non-neutralizing antibodies and induction of ACE2 expression, rendering these cells susceptible to killing by SARS-CoV-2. Death of macrophages occurs independently of viral replication and leads to the release of inflammatory mediators and modulation of the susceptibility of downstream epithelial cells to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.22.432407","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.21251504","pub_date":"2021-02-23","title":"Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppress macrophage anti-inflammation and efficient tissue repair programs and provide mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.</jats:p>","version":null,"doi":"10.1101/2021.02.18.21251504","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.22.432402","pub_date":"2021-2-23","title":"Human basigin (CD147) does not directly interact with SARS-CoV-2 spike glycoprotein","abstract":"Basigin, or CD147, has been reported as a co-receptor used by SARS-CoV-2 to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes where it is bound by one of the parasite\u2019s invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (either expressed in E. coli or mammalian cells). Given the immense interest in SARS-CoV-2 therapeutic targets, we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein. Reducing the mortality and morbidity associated with COVID-19 remains a global health priority. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a co-receptor for SARS-COV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a co-receptor and plausibility as a therapeutic target.","version":"1.1","doi":"10.1101/2021.02.22.432402","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432373","pub_date":"2021-2-23","title":"Structural Basis for SARS-CoV-2 Envelope Protein in Recognition of Human Cell Junction Protein PALS1","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus has created a global health and economic emergency. SARS-CoV-2 viruses hijack human proteins to promote their spread and virulence including the interactions involving the viral envelope (E) protein and human proteins. To understand the structural basis for SARS-CoV-2 viral-host recognition, we used cryo-electron microscopy to determine a structure for the human cell junction protein PALS1 and SARS-CoV-2 E protein complex. The structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains in PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions resulting in vascular leakage, lung damage, viral spread, and virulence. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce the E-mediated damage to vascular structures.","version":"1.1","doi":"10.1101/2021.02.22.432373","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432450","pub_date":"2021-2-23","title":"The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation","abstract":"Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2. SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.","version":"1.1","doi":"10.1101/2021.02.23.432450","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432177","pub_date":"2021-2-23","title":"Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection","abstract":"Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials. Complement has been implicated in COVID-19. However, whether this is distinctive of COVID-19 remains unanswered. Ma et al report increased complement activation in COVID-19 compared to influenza and non-COVID respiratory failure, and demonstrate alternative pathway activation as a key marker of multiorgan failure and death.","version":"1.1","doi":"10.1101/2021.02.22.432177","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432535","pub_date":"2021-2-23","title":"Metagenomic diagnosis and pathogenic network profile of SARS-CoV-2 in patients co-morbidly affected by type 2 diabetes","abstract":"The mortality of COVID-19 disease is very high among males or elderly or individuals having comorbidities with obesity, cardiovascular diseases, lung infections, hypertension, and/or diabetes. Our study characterizes SARS-CoV-2 infected patients\u2019 metagenomic features with or without type 2 diabetes to identify the microbial interactions associated with its fatal consequences. This study compared the baseline nasopharyngeal microbiome of SARS-CoV-2 infected diabetic and non-diabetic patients with controls adjusted with age and gender. The mNGS were performed using Ion GeneStudio S5 Series and the data were analyzed by the Vegan-package in R. All three groups possessed significant bacterial diversity and dissimilarity indexes (p<0.05). Spearman\u2019s correlation coefficient network analysis illustrated 183 significant positive correlations and 13 negative correlations of pathogenic bacteria (r=0.6-1.0, p<0.05), and 109 positive correlations among normal-flora and probiotic bacteria (r>0.6, p<0.05). The SARS-CoV-2 diabetic group exhibited a significant increase of pathogens (p<0.05) and opportunistic pathogens (p<0.05) with a simultaneous decrease of normal-flora (p<0.05). The molecular docking analysis of Salivaricin, KLD4 (alpha), and enterocin produced by several enriched probiotic strains presented strong binding affinity with Shiga toxin, outer membrane proteins (ompA, omp33) or hemolysin. The dysbiosis of the bacterial community might be linked with severe consequences of COVID-19 infected diabetic patients, although few probiotic strains inhibited numerous pathogens in the same pathological niches. This study suggested that the promotion of normal-flora and probiotics through dietary changes and reduction of excessive pro-inflammatory states by preventing pathogenic environment might lead to a better outcome for those co-morbid patients.","version":"1.1","doi":"10.1101/2021.02.23.432535","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432207","pub_date":"2021-2-23","title":"Targeting CoV-2 Spike RBD and ACE-2 Interaction with Flavonoids of Anatolian Propolis by in silico and in vitro Studies in terms of possible COVID-19 therapeutics","abstract":"Propolis is a multi-functional bee product with a rich in polyphenols. In this study, the inhibition effect of Anatolian propolis against SARS coronavirus-2 (SARS CoV-2) was investigated as in vitro and in silico. Raw and commercial of propolis samples were used in the study and it was found that both of were rich in caffeic acid, p-coumaric acid, ferulic acid, t-cinnamic acid, hesperetin, chrysin, pinocembrin and caffeic acid phenethyl ester (CAPE) by HPLC-UV analysis. The ethanolic propolis extracts (EPE) were used in the screening ELISA test against the spike S1 protein (SARS Cov-2): ACE-2 inhibition KIT for in vitro study. Binding energy constants of these polyphenols to the CoV-2 Spike S1 RBD and ACE-2proteinwere calculated separately as molecular docking study using AutoDock 4.2 molecular docking software. In addition, pharmacokinetics and drug-likeness properties of these eight polyphenols were calculated according to the SwissADME tool. Binding energy constant of pinocembrin was the highest for both of the receptors, followed by chrysin, CAPE and hesperetin. In silico ADME behavior of the eight polyphenols were found potential ability to work effectively as novel drugs. The findings of both studies showed that propolis has a high inhibitory potential against Covid-19 virus. However, further studies are needed.","version":"1.2","doi":"10.1101/2021.02.22.432207","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432379","pub_date":"2021-2-23","title":"Using mixed-effects modeling to estimate decay kinetics of response to SARS-CoV-2 infection","abstract":"The duration of natural immunity in response to SARS-CoV-2 is a matter of some debate in the literature at present. For example, in a recent publication characterizing SARS-CoV-2 immunity over time, the authors fit pooled longitudinal data, using fitted slopes to infer the duration of SARS-CoV-2 immunity. In fact, such approaches can lead to misleading conclusions as a result of statistical model-fitting artifacts. To exemplify this phenomenon, we reanalyzed one of the markers (pseudovirus neutralizing titer) in the publication, using mixed-effects modeling, a methodology better suited to longitudinal datasets like these. Our findings showed that the half-life was both longer and more variable than reported by the authors. The example selected by us here illustrates the utility of mixed-effects modeling in provide more accurate estimates of the duration and heterogeneity of half-lives of molecular and cellular biomarkers of SARS-CoV-2 immunity.","version":"1.1","doi":"10.1101/2021.02.22.432379","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432479","pub_date":"2021-2-23","title":"Oral Hsp90 inhibitor, SNX-5422, attenuates SARS-CoV-2 replication and dampens inflammation in airway cells","abstract":"Currently available SARS-CoV-2 therapeutics are targeted towards moderately to severely ill patients and require intravenous infusions, with limited options for exposed or infected patients with no or mild symptoms. While vaccines have demonstrated protective efficacy, vaccine hesitancy and logistical distribution challenges will delay their ability to end the pandemic. Hence, there is a need for rapidly translatable, easy-to-administer-therapeutics, that can prevent SARS-CoV-2 disease progression, when administered in the early stages of infection. We demonstrate that an orally bioavailable Hsp90 inhibitor, SNX-5422, currently in clinical trials as an anti-cancer therapeutic, inhibits SARS-CoV-2 replication in vitro at a high selectivity index. SNX-5422 treatment of human primary airway epithelial cells dampened expression of inflammatory pathways associated with poor SARS-CoV-2 disease outcomes. Additionally, SNX-5422 interrupted expression of host factors that are crucial for SARS-CoV-2 replication machinery. Development of SNX-5422 as SARS-CoV-2-early-therapy will dampen disease severity, resulting in better clinical outcomes and reduced hospitalizations.","version":"1.1","doi":"10.1101/2021.02.23.432479","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432569","pub_date":"2021-2-23","title":"Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses","abstract":"Globally there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals\u2019 weight loss post-infection and decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology and prevents viral pneumonia. Combined with the marked stability and low production cost, this novel therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.","version":"1.1","doi":"10.1101/2021.02.23.432569","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432486","pub_date":"2021-2-23","title":"A monocyte/dendritic cell molecular signature of SARS-CoV2-related multisystem inflammatory syndrome in children (MIS-C) with severe myocarditis","abstract":"SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-\u03baB activity, TNF-\u03b1 signaling, associated with decreased gene expression of NF-\u03baB inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1\u03b1 and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.","version":"1.1","doi":"10.1101/2021.02.23.432486","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432424","pub_date":"2021-2-23","title":"Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells","abstract":"As the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.","version":"1.1","doi":"10.1101/2021.02.23.432424","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.378828","pub_date":"2021-2-23","title":"Local topology, bifurcations and mutation hot-spots in proteins with SARS-CoV-2 spike protein as an example","abstract":"Novel topological methods are introduced to protein research. The aim is to identify hot-spot sites where a bifurcation can change the local topology of the protein backbone. Since the shape of a protein is intimately related to its biological function, a mutation that takes place at such a bifurcation hot-spot has an enhanced capacity to change the protein\u2019s biological function. The methodology applies to any protein but it is developed with the SARS-CoV-2 spike protein as a timely example. First, topological criteria are introduced to identify and classify potential mutation hot-spot sites along the protein backbone. Then, the expected outcome of a substitution mutation is estimated for a general class of hot-spots, by a comparative analysis of the backbone segments that surround the hot-spot sites. This analysis employs the statistics of commensurable amino acid fragments in the Protein Data Bank, in combination with general stereochemical considerations. It is observed that the notorious D614G substitution of the spike protein is a good example of such a mutation hot-spot. Several topologically similar examples are then analyzed in detail, some of them are even better candidates for a mutation hot-spot than D614G. The local topology of the recently observed N501Y mutation is also inspected, and it is found that this site is prone to a different kind of local topology changing bifurcation.","version":"1.3","doi":"10.1101/2020.11.11.378828","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.23.432460","pub_date":"2021-2-23","title":"Comprehensive evaluation of ACE2 expression in female ovary by single-cell RNA-seq analysis","abstract":"Pneumonia induced by severe acute respiratory coronavirus 2 (SARS-CoV-2) via ACE2 receptor may affect many organ systems like lung, heart and kidney. An autopsy report revealed positive SARS-Cov-2 detection results in ovary, however, the developmental-stage-specific and cell-type-specific risk in fetal primordial germ cells (PGCs) and adult women ovary remained unclear. In this study, we used single-cell RNA-sequencing (scRNA-seq) datasets spanning several developmental stages of ovary including PGCs and cumulus-oocyte complex (COC) to investigate the potential risk of SARS-CoV-2 infection. We found that PGCs and COC exhibited high ACE2 expression. More importantly, the ratio of ACE2-positive cells was sharply up-regulated in primary stage and ACE2 was expressed in all oocytes and cumulus cells in preovulatory stage, suggesting the possible risk of SARS-CoV-2 infection in follicular development. CatB/L, not TMPRSS2, was identified to prime for SARS-CoV-2 entry in follicle. Our findings provided insights into the potential risk of SARS-CoV-2 infection during folliculogenesis in adulthood and the possible risk in fetal PGCs.","version":"1.1","doi":"10.1101/2021.02.23.432460","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432271","pub_date":"2021-2-23","title":"BiSEK: a platform for a reliable differential expression analysis","abstract":"Differential Expression Analysis (DEA) of RNA-sequencing data is frequently performed for detecting key genes, affected across different conditions. Although DEA-workflows are well established, preceding reliability-testing of the input material, which is crucial for consistent and strong results, is challenging and less straightforward. Here we present Biological Sequence Expression Kit (BiSEK), a graphical user interface-based platform for DEA, dedicated to a reliable inquiry. BiSEK is based on a novel algorithm to track discrepancies between the data and the statistical model design. Moreover, BiSEK enables differential-expression analysis of groups of genes, to identify affected pathways, without relying on the significance of genes comprising them. Using BiSEK, we were able to improve previously conducted analysis, aimed to detect genes affected by FUBP1 depletion in chronic myeloid leukemia cells of mice bone-marrow. We found affected genes that are related to the regulation of apoptosis, supporting in-vivo experimental findings. We further tested the host response following SARS-CoV-2 infection. We identified a substantial interferon-I reaction and low expression levels of TLR3, an inducer of interferon-III (IFN-III) production, upon infection with SARS-CoV-2 compared to other respiratory viruses. This finding may explain the low IFN-III response upon SARS-CoV-2 infection. BiSEK is open-sourced, available as a web-interface.","version":"1.1","doi":"10.1101/2021.02.22.432271","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.21.432184","pub_date":"2021-2-22","title":"High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19","abstract":"Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting. Mutant sequences of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) arising during any individual case of coronavirus disease 2019 (COVID-19) could theoretically enable the virus to evade immune responses or antiviral therapies that target the predominant infecting virus sequence. However, commonly used sequencing technologies are not optimally designed to detect variant virus sequences within each sample. To address this issue, we developed novel technology for sequencing large numbers of individual SARS-CoV-2 genomic RNA molecules across the region encoding the virus surface proteins. This technology revealed extensive genetic diversity in cultured viruses from a clinical isolate of SARS-CoV-2, but lower diversity in samples from 7 individuals with COVID-19. Importantly, concurrent analysis of paired serum samples in selected individuals revealed relatively low levels of antibody binding to the SARS-CoV-2 spike protein at the time of initial sequencing. With increased serum binding to spike protein, we detected multiple SARS-CoV-2 variants bearing independent mutations in a single epitope, as well as a transient increase in virus burden. These findings suggest that SARS-CoV-2 replication creates sufficient virus genetic diversity to allow immune-mediated selection of variants within the time frame of acute COVID-19. Large-scale studies of SARS-CoV-2 variation and specific immune responses will help define the contributions of intra-individual SARS-CoV-2 evolution to COVID-19 clinical outcomes and antiviral drug susceptibility.","version":"1.1","doi":"10.1101/2021.02.21.432184","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432359","pub_date":"2021-2-22","title":"SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity","abstract":"SARS-CoV2 being highly infectious has been particularly effective in causing widespread infection globally and more variants of SARS-CoV2 are constantly being reported with increased genomic surveillance. In particular, the focus is on mutations of Spike protein, which binds human ACE2 protein enabling SARS-CoV2 entry and infection. Here we present a rapid experimental method leveraging the speed and flexibility of Mircoscale Thermopheresis (MST) to characterize the interaction between Spike Receptor Binding Domain (RBD) and human ACE2 protein. The B.1.351 variant harboring three mutations, (E484K, N501Y, and K417N) binds the ACE2 at nearly five-fold greater affinity than the original SARS-COV-2 RBD. We also find that the B.1.1.7 variant, binds two-fold more tightly to ACE2 than the SARS-COV-2 RBD.","version":"1.1","doi":"10.1101/2021.02.22.432359","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430478","pub_date":"2021-2-22","title":"High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples","abstract":"In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking movement and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using online visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable \u2018how-to\u2019 reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and next-generation nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.","version":"1.2","doi":"10.1101/2021.02.09.430478","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432218","pub_date":"2021-2-22","title":"Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells","abstract":"Genetic differences are a primary reason for differences in the susceptibility and severity of coronavirus disease 2019 (COVID-19). Because induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Notably, undifferentiated human iPS cells themselves cannot be infected bySARS-CoV-2. Using adenovirus vectors, here we found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected with SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, the budding of viral particles, the production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We also evaluated COVID-19 therapeutic drugs in ACE2-iPS cells and confirmed the strong antiviral effects of Remdesivir, EIDD-2801, and interferon-beta. In addition, we performed SARS-CoV-2 infection experiments on ACE2-iPS/ES cells from 8 individuals. Male iPS/ES cells were more capable of producing the virus as compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro, but they are also a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences.","version":"1.1","doi":"10.1101/2021.02.22.432218","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.432046","pub_date":"2021-2-22","title":"Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant","abstract":"The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies to neutralize these variants. We compared antibody binding and live virus neutralization of sera from naturally infected and spike mRNA vaccinated individuals against a circulating SARS-CoV-2 B.1 variant and the emerging B.1.351 variant. In acutely-infected (5-19 days post-symptom onset), convalescent COVID-19 individuals (through 8 months post-symptom onset) and mRNA-1273 vaccinated individuals (day 14 post-second dose), we observed an average 4.3-fold reduction in antibody titers to the B.1.351-derived receptor binding domain of the spike protein and an average 3.5-fold reduction in neutralizing antibody titers to the SARS-CoV-2 B.1.351 variant as compared to the B.1 variant (spike D614G). However, most acute and convalescent sera from infected and all vaccinated individuals neutralize the SARS-CoV-2 B.1.351 variant, suggesting that protective immunity is retained against COVID-19.","version":"1.1","doi":"10.1101/2021.02.20.432046","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.432110","pub_date":"2021-2-22","title":"A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2","abstract":"Since its initial discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID19, has spread worldwide and despite significant research efforts, treatment options remain limited. Replication of SARS-CoV-2 in lung is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses triggered, in part, by heightened production of interleukin-6 (IL-6) that recruits lymphocytes to the site of infection that amplify tissue injury. Antagonists of the glucocorticoid and androgen receptors have shown promise in experimental models of COVID19 and in clinical studies, because cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2), are transcriptionally regulated by these receptors. We therefore postulated that the glucocorticoid (GR) and androgen receptor (AR) antagonist, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID19. Animals were infected intranasally with 2.5 \u00d7 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/ 2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were then examined at days 3, 5 and 7 post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the initiation and progression of SARS-CoV-2 infection. Results of these studies indicated that oral administration of PT150 decreased replication of SARS-CoV-2 in lung, as well as expression of ACE2 and TMPRSS2 protein. Hypercellularity and inflammatory cell infiltration driven by macrophage responses were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular modeling suggested that PT150 binds to the co-activator interface of the ligand binding domain of both AR and GR and thereby acts as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR across multiple species permissive to SARS-CoV-2 infection revealed a high degree of sequence identity maintained across species, including human, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID19 across variants of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.20.432110","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.21.432165","pub_date":"2021-2-22","title":"Comparative Perturbation-Based Modeling of the SARS-CoV-2 Spike Protein Binding with Host Receptor and Neutralizing Antibodies : Structurally Adaptable Allosteric Communication Hotspots Define Spike Sites Targeted by Global Circulating Mutations","abstract":"In this study, we used an integrative computational approach focused on comparative perturbation-based modeling to examine molecular mechanisms and determine functional signatures underlying role of functional residues in the SARS-CoV-2 spike protein that are targeted by novel mutational variants and antibody-escaping mutations. Atomistic simulations and functional dynamics analysis are combined with alanine scanning and mutational sensitivity profiling for the SARS-CoV-2 spike protein complexes with the ACE2 host receptor are REGN-COV2 antibody cocktail (REG10987+REG10933). Using alanine scanning and mutational sensitivity analysis, we have shown that K417, E484 and N501 residues correspond to key interacting centers with a significant degree of structural and energetic plasticity that allow mutants in these positions to afford the improved binding affinity with ACE2. Through perturbation-based network modeling and community analysis of the SARS-CoV-2 spike protein complexes with ACE2 we demonstrate that E406, N439, K417 and N501 residues serve as effector centers of allosteric interactions and anchor major inter-molecular communities that mediate long-range communication in the complexes. The results provide support to a model according to which mutational variants and antibody-escaping mutations constrained by the requirements for host receptor binding and preservation of stability may preferentially select structurally plastic and energetically adaptable allosteric centers to differentially modulate collective motions and allosteric interactions in the complexes with the ACE2 enzyme and REGN-COV2 antibody combination. This study suggests that SARS-CoV-2 spike protein may function as a versatile and functionally adaptable allosteric machine that exploits plasticity of allosteric regulatory centers to fine-tune response to antibody binding without compromising activity of the spike protein.","version":"1.1","doi":"10.1101/2021.02.21.432165","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.21251905","pub_date":"2021-02-22","title":"Methodological Approach for Wastewater Based Epidemiological Studies for SARS-CoV-2","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Post COVID-19 outbreak, wastewater-based epidemiology (WBE) studies as surveillance system is becoming an emerging interest due to its functional advantage as tool for early warning signal and to catalyze effective disease management strategies based on the community diagnosis. A comprehensive attempt was made in this study to define a methodological approach for conducting WBE studies in the framework of identifying/selection of surveillance sites, standardizing sampling policy, designing sampling protocols to improve sensitivity, adopting safety protocol, and interpreting the data. The methodology was applied to a community and studied its epidemiological status with reference to occurrence, persistence, and variation of SARS-CoV-2 genome load in wastewater system to understand the prevalence of infection. Hourly and daily grab samples were analyzed and compared with the composite samples over a surveillance window of 7 days. Based on the SARS-CoV-2 RNA copies/L, faeces shedding, and volume of sewage generated the infected individuals and the population who are in active phase in the studied community was estimated.</jats:p>","version":null,"doi":"10.1101/2021.02.17.21251905","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.17.431683","pub_date":"2021-2-22","title":"Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016","abstract":"Monoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for COVID-19. However, ongoing evolution of SARS-CoV-2 can render monoclonal antibodies ineffective. Here we completely map all mutations to the SARS-CoV-2 spike receptor binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages (E484K escapes LY-CoV555, K417N/T escape LY-CoV016). Additionally, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts should diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to antigenic evolution of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.17.431683","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432259","pub_date":"2021-2-22","title":"Laser-facilitated epicutaneous immunization with SARS-CoV-2 spike protein induces ACE2 blocking antibodies in mice","abstract":"The skin represents an attractive target tissue for vaccination against respiratory viruses such as SARS-CoV-2. Laser-facilitated epicutaneous immunization (EPI) has been established as a novel technology to overcome the skin barrier, which combines efficient delivery via micropores with an inherent adjuvant effect due to the release of danger-associated molecular patterns. Here we delivered the S1 subunit of the Spike protein of SARS-CoV-2 to the skin of BALB/c mice via laser-generated micropores with or without CpG-ODN1826 or the B subunit of heat-labile enterotoxin of E.coli (LT-B). EPI induced serum IgG titers of 1:3200 that could be boosted 5 to 10-fold by co-administration of LT-B and CpG, respectively. Sera were able to inhibit binding of the spike protein to its receptor ACE2. Our data indicate that delivery of recombinant spike protein via the skin may represent an alternative route for vaccines against Covid-19.","version":"1.1","doi":"10.1101/2021.02.22.432259","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432096","pub_date":"2021-2-22","title":"Reanalysis of deep-sequencing data from Austria points towards a small SARS-COV-2 transmission bottleneck on the order of one to three virions","abstract":"An early analysis of SARS-CoV-2 deep-sequencing data that combined epidemiological and genetic data to characterize the transmission dynamics of the virus in and beyond Austria concluded that the size of the virus\u2019s transmission bottleneck was large \u2013 on the order of 1000 virions. We performed new computational analyses using these deep-sequenced samples from Austria. Our analyses included characterization of transmission bottleneck sizes across a range of variant calling thresholds and examination of patterns of shared low-frequency variants between transmission pairs in cases where de novo genetic variation was present in the recipient. From these analyses, among others, we found that SARS-CoV-2 transmission bottlenecks are instead likely to be very tight, on the order of 1-3 virions. These findings have important consequences for understanding how SARS-CoV-2 evolves between hosts and the processes shaping genetic variation observed at the population level.","version":"1.1","doi":"10.1101/2021.02.22.432096","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.432081","pub_date":"2021-2-22","title":"Nanoceutical Fabric Prevents COVID-19 Spread through Expelled Respiratory Droplets: A Combined Computational, Spectroscopic and Anti-microbial Study","abstract":"Centers for Disease Control and Prevention (CDC) warns the use of one-way valves or vents in free masks for potential threat of spreading COVID-19 through expelled respiratory droplets. Here, we have developed a nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanomaterial for potential use as membrane filter in the one way valve for the ease of breathing without the threat of COVID-19 spreading. A detailed computational study revealed that zinc oxide nanoflowers (ZnO NF) with almost two-dimensional petals trap SARS-CoV-2 spike proteins, responsible to attach to ACE-2 receptors in human lung epithelial cells. The study also confirm significant denaturation of the spike proteins on the ZnO surface, revealing removal of virus upon efficient trapping. Following the computational study, we have synthesized ZnO NF on cotton matrix using hydrothermal assisted strategy. Electron microscopic, steady-state and picosecond resolved spectroscopic studies confirm attachment of ZnO NF to the cotton (i.e., cellulose) matrix at atomic level to develop the nanoceutical fabric. A detailed antimicrobial assay using Pseudomonas aeruginosa bacteria (model SARS-CoV-2 mimic) reveals excellent anti-microbial efficiency of the developed nanoceutical fabric. To our understanding the novel nanoceutical fabric used in one-way valve of a face mask would be the choice to assure breathing comfort along with source control of COVID-19 infection. The developed nanosensitized cloth can also be used as antibacterial/anti CoV-2 washable dress material in general. A novel nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanoflower can potentially be used as membrane filter in the one way valve of face mask to assure breathing comfort along with source control of COVID-19 infection. The nanoceutical fabric denatures the SARS-CoV-2 spike protein and makes the microorganism ineffective.","version":"1.1","doi":"10.1101/2021.02.20.432081","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.22.432357","pub_date":"2021-2-22","title":"Experimental evidence for enhanced receptor binding by rapidly spreading SARS-CoV-2 variants","abstract":"Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 9-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation does not significantly influence the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 3-fold weaker than N501Y. The recently emerging double mutant E484K/N501Y binds as tight as N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.","version":"1.1","doi":"10.1101/2021.02.22.432357","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.21.432168","pub_date":"2021-2-22","title":"Structural Basis for Accommodation of Emerging B.1.351 and B.1.1.7 Variants by Two Potent SARS-CoV-2 Neutralizing Antibodies","abstract":"Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented a response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for both ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies like 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.","version":"1.1","doi":"10.1101/2021.02.21.432168","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.21.432171","pub_date":"2021-2-22","title":"An Autoantigen Profile of Human A549 Lung Cells Reveals Viral and Host Etiologic Molecular Attributes of Autoimmunity in COVID-19","abstract":"We aim to establish a comprehensive COVID-19 autoantigen atlas in order to understand autoimmune diseases caused by SARS-CoV-2 infection. Based on the unique affinity between dermatan sulfate and autoantigens, we identified 348 proteins from human lung A549 cells, of which 198 are known targets of autoantibodies. Comparison with current COVID data identified 291 proteins that are altered at protein or transcript level in SARS-CoV-2 infection, with 191 being known autoantigens. These known and putative autoantigens are significantly associated with viral replication and trafficking processes, including gene expression, ribonucleoprotein biogenesis, mRNA metabolism, translation, vesicle and vesicle-mediated transport, and apoptosis. They are also associated with cytoskeleton, platelet degranulation, IL-12 signaling, and smooth muscle contraction. Host proteins that interact with and that are perturbed by viral proteins are a major source of autoantigens. Orf3 induces the largest number of protein alterations, Orf9 affects the mitochondrial ribosome, and they and E, M, N, and Nsp proteins affect protein localization to membrane, immune responses, and apoptosis. Phosphorylation and ubiquitination alterations by viral infection define major molecular changes in autoantigen origination. This study provides a large list of autoantigens as well as new targets for future investigation, e.g., UBA1, UCHL1, USP7, CDK11A, PRKDC, PLD3, PSAT1, RAB1A, SLC2A1, platelet activating factor acetylhydrolase, and mitochondrial ribosomal proteins. This study illustrates how viral infection can modify host cellular proteins extensively, yield diverse autoantigens, and trigger a myriad of autoimmune sequelae.","version":"1.1","doi":"10.1101/2021.02.21.432171","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.432085","pub_date":"2021-2-22","title":"KLF2 is a therapeutic target for COVID-19 induced endothelial dysfunction","abstract":"Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its mechanism being incompletely understood. Emerging evidence has demonstrated that the endothelium represents the Achilles\\' heel in COVID-19 patients and that endothelial dysfunction precipitates COVID-19 and accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. Primary human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were treated with serum from control subjects or COVID-19 patients. Downstream monocyte adhesion and associated gene/protein expression was evaluated in endothelial cells exposed to COVID-19 patient serum in the presence of KLF2 activator (Atorvastatin) or KLF2 overexpression by an adenoviral vector. Here, we demonstrate that the expression of KLF2 was significantly reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1\u03b2 and TNF-\u03b1, two cytokines observed in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis and reduced angiogenesis). Treatment of HPMECs with atorvastatin or KLF2 adenovirus ameliorate COVID-19 serum-induced increase in endothelial inflammation and monocyte adhesion by increasing KLF2 expression. Altogether, the present study demonstrates that genetic and pharmacological activation of KLF2 represses COVID-19 associated endothelial dysfunction, heralding a potentially new direction to treat endothelialitis accompanying COVID-19.","version":"1.2","doi":"10.1101/2021.02.20.432085","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.432092","pub_date":"2021-2-21","title":"Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility","abstract":"Heart dysfunction, represented by conditions such as myocarditis and arrhythmia, has been reported in COVID-19 patients. Therapeutic strategies focused on the cardiovascular system, however, remain scarce. The Sigma-1 receptor (S1R) has been recently proposed as a therapeutic target because its inhibition reduces SARS-CoV-2 replication. To investigate the role of S1R in SARS-CoV-2 infection in the heart, we used human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) as an experimental model. Here we show that the S1R antagonist NE-100 decreases SARS-CoV-2 infection and viral replication in hiPSC-CMs. Also, NE-100 reduces cytokine release and cell death associated with infection. Because S1R is involved in cardiac physiology, we investigated the effects of NE-100 in cardiomyocyte morphology and function. We show that NE-100 compromises cytoskeleton integrity and reduces beating frequency, causing contractile impairment. These results show that targeting S1R to challenge SARS-CoV-2 infection may be a useful therapeutic strategy but its detrimental effects in vivo on cardiac function should not be ignored.","version":"1.1","doi":"10.1101/2021.02.20.432092","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.21.432120","pub_date":"2021-2-21","title":"Finding Druggable Sites in Proteins using TACTICS","abstract":"Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (Trajectory-based Analysis of Conformations To Identify Cryptic Sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.","version":"1.1","doi":"10.1101/2021.02.21.432120","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.20.431155","pub_date":"2021-2-20","title":"Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19","abstract":"Infection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, KRT13+ \u201chillock\u201d-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., CTSL, TMPRSS2) or response (e.g., MX1, IFITM3, EIF2AK2) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.","version":"1.1","doi":"10.1101/2021.02.20.431155","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.21251926","pub_date":"2021-02-19","title":"SARS-CoV-2 shedding dynamics across the respiratory tract, sex and disease severity for adult and pediatric COVID-19","abstract":"<jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>SARS-CoV-2 shedding dynamics influence the risk of transmission and clinical manifestations of COVID-19. Yet, the relationships between SARS-CoV-2 shedding dynamics in the upper (URT) and lower respiratory tract (LRT) and age, sex and COVID-19 severity remain unclear.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Using systematic review, we developed a dataset of case characteristics (age, sex and COVID-19 severity) and quantitative respiratory viral loads (rVLs). We then conducted stratified analyses to assess SARS-CoV-2 shedding across disease course, COVID-19 severity, the respiratory tract, sex and age groups (aged 0 to 17 years, 18 to 59 years, and 60 years or older).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    The systematic dataset included 1,266 adults and 136 children with COVID-19. In the URT, adults with severe COVID-19 had higher rVLs at 1 day from symptom onset (DFSO) than adults (\n                    <jats:italic>P</jats:italic>\n                    = 0.005) or children (\n                    <jats:italic>P</jats:italic>\n                    = 0.017) with nonsevere illness. Between 1-10 DFSO, severe adults had comparable rates of SARS-CoV-2 clearance from the URT as nonsevere adults (\n                    <jats:italic>P</jats:italic>\n                    = 0.479) and nonsevere children (\n                    <jats:italic>P</jats:italic>\n                    = 0.863). In the LRT, severe adults showed higher rVLs post-symptom onset than nonsevere adults (\n                    <jats:italic>P</jats:italic>\n                    = 0.006). In the analyzed period (4-10 DFSO), severely affected adults had no significant trend in SARS-CoV-2 clearance from LRT (\n                    <jats:italic>P</jats:italic>\n                    = 0.105), whereas nonsevere adults showed a clear trend (\n                    <jats:italic>P</jats:italic>\n                    &lt; 0.001). After stratifying for disease severity, sex and age (including child vs. adult) were not predictive of the duration of respiratory shedding. The estimated accuracy for using URT shedding as a prognostic indicator for COVID-19 severity was up to 65%, whereas it was up to 81% for LRT shedding.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>High, persistent LRT shedding of SARS-CoV-2 characterized severe COVID-19 in adults. After symptom onset, severe cases tended to have slightly higher URT shedding than their nonsevere counterparts. Disease severity, rather than age or sex, predicted SARS-CoV-2 kinetics. LRT specimens more accurately prognosticate COVID-19 severity than do URT specimens.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant, NSERC Senior Industrial Research Chair and the Toronto COVID-19 Action Fund.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.17.21251926","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.28.272880","pub_date":"2021-2-19","title":"A comparative analysis of SARS-CoV-2 antivirals in human airway models characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231\u2019s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231\u2019s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.08.28.272880","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.19.431973","pub_date":"2021-2-19","title":"Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance, and characterized six FRET substrates with a 2-Abz/Tyr(3-NO2) FRET pair. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and a new substrate 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at the position P5 improved the catalytic efficacy. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.","version":"1.1","doi":"10.1101/2021.02.19.431973","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178343","pub_date":"2021-2-19","title":"Insights into the secondary structural ensembles of the full SARS-CoV-2 RNA genome in infected cells","abstract":"SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on native RNA structures in SARS-CoV-2 and most putative regulatory sequences are functionally uncharacterized. Here we report secondary structure ensembles of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and the algorithm \u2018detection of RNA folding ensembles using expectation\u2013maximization\u2019 clustering (DREEM). Our results reveal previously undescribed alternative RNA conformations across the genome, including structures of the frameshift stimulating element (FSE), a major drug target, that are drastically different from prevailing in vitro population average models. Importantly, we find that this structural ensemble promotes frameshifting rates (~40%) similar to in vivo ribosome profiling studies and much higher than the canonical minimal FSE (~20%). Overall, our result highlight the value of studying RNA folding in its native, dynamic and cellular context. The genomic structures detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.","version":"1.2","doi":"10.1101/2020.06.29.178343","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.19.431311","pub_date":"2021-2-19","title":"SARS-CoV-2 variant evolution in the United States: High accumulation of viral mutations over time likely through serial Founder Events and mutational bursts","abstract":"Since the first case of COVID-19 in December 2019 in Wuhan, China, SARS-CoV-2 has spread worldwide and within a year has caused 2.29 million deaths globally. With dramatically increasing infection numbers, and the arrival of new variants with increased infectivity, tracking the evolution of its genome is crucial for effectively controlling the pandemic and informing vaccine platform development. Our study explores evolution of SARS-CoV-2 in a representative cohort of sequences covering the entire genome in the United States, through all of 2020 and early 2021. Strikingly, we detected many accumulating Single Nucleotide Variations (SNVs) encoding amino acid changes in the SARS-CoV-2 genome, with a pattern indicative of RNA editing enzymes as major mutators of SARS-CoV-2 genomes. We report three major variants through October of 2020. These revealed 14 key mutations that were found in various combinations among 14 distinct predominant signatures. These signatures likely represent evolutionary lineages of SARS-CoV-2 in the U.S. and reveal clues to its evolution such as a mutational burst in the summer of 2020 likely leading to a homegrown new variant, and a trend towards higher mutational load among viral isolates, but with occasional mutation loss. The last quartile of 2020 revealed a concerning accumulation of mostly novel low frequency replacement mutations in the Spike protein, and a hypermutable glutamine residue near the putative furin cleavage site. Finally, the end of the year data revealed the presence of known variants of concern including B.1.1.7, which has acquired additional Spike mutations. Overall, our results suggest that predominant viral sequences are dynamically evolving over time, with periods of mutational bursts and unabated mutation accumulation. This high level of existing variation, even at low frequencies and especially in the Spike-encoding region may be become problematic when superspreader events, akin to serial Founder Events in evolution, drive these rare mutations to prominence. The pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the death of more than 2.29 million people and continues to be a severe threat internationally. Although simple measures such as social distancing, periodic lockdowns and hygiene protocols were immediately put into force, the infection rates were only temporarily minimized. When infection rates exploded again new variants of the virus began to emerge. Our study focuses on a representative set of sequences from the United States throughout 2020 and early 2021. We show that the driving force behind the variants of public health concern, is widespread infection and superspreader events. In particular, we show accumulation of mutations over time with little loss from genetic drift, including in the Spike region, which could be problematic for vaccines and therapies. This lurking accumulated genetic variation may be a superspreader event from becoming more common and lead to variants that can escape the immune protection provided by the existing vaccines.","version":"1.1","doi":"10.1101/2021.02.19.431311","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431897","pub_date":"2021-2-19","title":"Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies","abstract":"Monoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations.","version":"1.1","doi":"10.1101/2021.02.18.431897","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431684","pub_date":"2021-2-19","title":"Delivery of recombinant SARS-CoV-2 envelope protein into human cells","abstract":"SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of \u201cTrojan Horse\u201d anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.","version":"1.1","doi":"10.1101/2021.02.18.431684","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.19.424337","pub_date":"2021-2-19","title":"Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors","abstract":"The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 \u00b1 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 \u00b1 0.2 \u03bcM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.","version":"1.1","doi":"10.1101/2021.02.19.424337","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.423113","pub_date":"2021-2-19","title":"Long SARS-CoV-2 nucleocapsid sequences in blood monocytes collected soon after hospital admission","abstract":"Many viruses infect circulating mononuclear cells thereby facilitating infection of diverse organs. Blood monocytes (PBMC) are being intensively studied as immunologic and pathologic responders to the new SARS-CoV-2 virus (CoV19) but direct evidence showing CoV19 in monocytes is lacking. Circulating myeloid cells that take up residence in various organs can harbor viral genomes for many years in lymphatic tissues and brain, and act as a source for re-infection and/or post-viral organ pathology. Because nucleocapsid (NC) proteins protect the viral genome we tested PBMC from acutely ill patients for the diagnostic 72bp NC RNA plus adjacent longer (301bp) transcripts. In 2/11 patient PBMC, but no uninfected controls, long NCs were positive as early as 2-6 days after hospital admission as validated by sequencing. Pathogenic viral fragments, or the infectious virus, are probably disseminated by rare myeloid migratory cells that incorporate CoV19 by several pathways. Predictably, these cells carried CoV19 to heart and brain educing the late post-viral pathologies now evident.","version":"1.2","doi":"10.1101/2020.12.16.423113","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431919","pub_date":"2021-2-19","title":"Adequate knowledge of COVID-19 impacts good practices amongst health profession students in the Philippines","abstract":"The spread of the coronavirus disease 2019 (COVID-19) in the Philippines started with its first suspected case on January 22, 2020. The government reacted by imposing several measures including community quarantine, class suspensions, drug therapy and vaccine development, and travel restrictions. This online survey was done amongst Filipino health professions undergraduate students to uncover the relationship between their knowledge, attitude, and practice during this pandemic. Cross-sectional data were obtained from an online survey done on students of medicine, dentistry, optometry, rehabilitative sciences, and pharmacy. At a response rate of 100% (n=1257), the results show that healthcare profession students in the Philippines have good knowledge (87.6%) and practices (63.6%) regarding COVID-19, yet attitude (63.6%) was just passable. This study also shows that a strong correlation exists between knowledge and practice concerning the current pandemic, r(2) = 0.08, P = 0.004. Adequate knowledge of COVID-19 impacts good practices of avoiding crowded places and misuse of steam inhalation amongst health profession students in the Philippines. Knowledge and practice pertaining to the current pandemic have been found to be good, but attitude remains low.","version":"1.1","doi":"10.1101/2021.02.18.431919","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.22.262931","pub_date":"2021-2-19","title":"Theoretical basis for stabilizing messenger RNA through secondary structure design","abstract":"RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. These computational tests were carried out on both model mRNAs and COVID-19 mRNA vaccine candidates. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term \u2018superfolder\u2019 mRNAs. These designs exhibit wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity, and their folding is robust to temperature, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1, and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable. Messenger RNA (mRNA) medicines that encode and promote translation of a target protein have shown promising use as vaccines in the current SARS-CoV-2 pandemic as well as infectious diseases due to their speed of design and manufacturing. However, these molecules are intrinsically prone to hydrolysis, leading to poor stability in aqueous buffer and major challenges in distribution. Here, we present a principled biophysical model for predicting RNA degradation, and demonstrate that the stability of any mRNA can be increased at least two-fold over conventional design techniques. Furthermore, the predicted stabilization is robust to post-design modifications. This conceptual framework and accompanying algorithm can be immediately deployed to guide re-design of mRNA vaccines and therapeutics to increase in vitro stability.","version":"1.2","doi":"10.1101/2020.08.22.262931","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431554","pub_date":"2021-2-19","title":"Association of CXCR6 with COVID-19 severity: Delineating the host genetic factors in transcriptomic regulation","abstract":"The coronavirus disease 2019 (COVID-19) is an infectious disease that mainly affects the host respiratory system with \u223c80% asymptomatic or mild cases and \u223c5% severe cases. Recent genome-wide association studies (GWAS) have identified several genetic loci associated with the severe COVID-19 symptoms. Delineating the genetic variants and genes is important for better understanding its biological mechanisms. We implemented integrative approaches, including transcriptome-wide association studies (TWAS), colocalization analysis and functional element prediction analysis, to interpret the genetic risks using two independent GWAS datasets in lung and immune cells. To understand the context-specific molecular alteration, we further performed deep learning-based single cell transcriptomic analyses on a bronchoalveolar lavage fluid (BALF) dataset from moderate and severe COVID-19 patients. We discovered and replicated the genetically regulated expression of CXCR6 and CCR9 genes. These two genes have a protective effect on the lung and a risk effect on whole blood, respectively. The colocalization analysis of GWAS and cis-expression quantitative trait loci highlighted the regulatory effect on CXCR6 expression in lung and immune cells. In the lung resident memory CD8+ T (TRM) cells, we found a 3.32-fold decrease of cell proportion and lower expression of CXCR6 in the severe than moderate patients using the BALF transcriptomic dataset. Pro-inflammatory transcriptional programs were highlighted in TRM cells trajectory from moderate to severe patients. CXCR6 from the 3p21.31 locus is associated with severe COVID-19. CXCR6 tends to have a lower expression in lung TRM cells of severe patients, which aligns with the protective effect of CXCR6 from TWAS analysis. We illustrate one potential mechanism of host genetic factor impacting the severity of COVID-19 through regulating the expression of CXCR6 and TRM cell proportion and stability. Our results shed light on potential therapeutic targets for severe COVID-19.","version":"1.2","doi":"10.1101/2021.02.17.431554","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431722","pub_date":"2021-2-18","title":"Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2","abstract":"The increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the serological response to SARS-CoV-2 RBD, the SARS-CoV-2 NTD response is less cross-reactive with SARS-CoV. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers an alternative strategy for serology testing, it may not be suitable as an immunogen for vaccine development.","version":"1.1","doi":"10.1101/2021.02.17.431722","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431750","pub_date":"2021-2-18","title":"A glimpse into the diverse cellular immunity against SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific cellular immune response may prove to be essential for long-term immune protection against the novel coronavirus disease 2019 (COVID-19). To assess COVID-19-specific immunity in the population, we synthesized selected peptide pools of SARS-CoV-2 structural and functional proteins, including Spike (S), Membrane (M), Envelope (E), Nucleocapsid (N) and Protease (P) as target antigens. Survey of the T cell precursur frequencies in healthy individuals specific to these viral antigens demonstrated a diverse cellular immunity, including high, medium, low and no responders. This was further confirmed by in vitro induction of anti-SARS-CoV-2 T cell immune responses using dendritic cell (DC)/T cell coculture, which supported the corresponding T cell precursor frequencies in each of the individuals tested. In general, the combination of all five viral antigen pools induced the strongest cellular immune response, yet individual donors responded differently to different viral antigens. Importantly, in vitro restimulation of the T cells with the DC-peptides induced increased anti-viral immune responses in all individuals even in the no responders, suggesting that repeated antigen stimulation could elicit a broad protection in immune na\u00efve population. Our analysis recapitulates the critical role of cellular immunity in fighting COVID-19 and the importance of analyzing anti-SARS-CoV-2 T cell response in addition to antibody response in the population. Facing the rapid evolving SARS-CoV-2 variants in the world, current emphasis on antibody-producing vaccines needs a quick revisit. The virus-specific cellular immunity may prove to be essential for long-term protection against COVID-19. This study designed a series of antigenic peptides encompassing the conserved and/or essential domains of Spike (S), Membrane (M), envelope (E), Nucleocapsid (N) and Protease (P) as targets to assess Covid-19-specific immunity in the population. The results demonstrated a diverse cellular immunity, including high, medium, low and no responders. This was verified by in vitro generation of anti-SARS-CoV-2 T-cells from these subjects. The study suggested that individuals responded differently to the different viral antigens, and importantly, repeated stimulation could produce virus specific T cells in all individuals, including the no responders. This study illustrates the needs for assessing anti-viral cellular immunity in addition to antibody response in the general population.","version":"1.1","doi":"10.1101/2021.02.17.431750","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.14.431174","pub_date":"2021-2-18","title":"Direct activation of endothelial cells by SARS-CoV-2 nucleocapsid protein is blocked by Simvastatin","abstract":"Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multi-organ failure in patients with COVID-19. However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the SARS-CoV-2 viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved with endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through TLR2/NF-\u03baB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkablely, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, MERS-CoV, HUB1-CoV and influenza virus H1N1 did not affect endothelial activation. These findings are well consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.","version":"1.2","doi":"10.1101/2021.02.14.431174","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431755","pub_date":"2021-2-18","title":"SARS-CoV-2 nucleocapsid protein dually regulates innate immune responses","abstract":"The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing global pandemic of COVID-19, may trigger immunosuppression in the early stage and a cytokine storm in the late stage of infection, however, the underlying mechanisms are not well understood. Here we demonstrated that the SARS-CoV-2 nucleocapsid (N) protein dually regulated innate immune responses, i.e., the low-dose N protein suppressed type I interferon (IFN-I) signaling and inflammatory cytokines, whereas high-dose N protein promoted IFN-I signaling and inflammatory cytokines. Mechanistically, the SARS-CoV-2 N protein interacted with the tripartite motif protein 25 (TRIM25), thereby dually regulating the phosphorylation and nuclear translocation of IRF3, STAT1 and STAT2. Additionally, low-dose N protein combined with TRIM25 could suppress retinoic acid-inducible gene I (RIG-I) ubiquitination and activation. Our findings revealed a regulatory mechanism of innate immune responses by the SARS-CoV-2 N protein, which would contribute to understanding the pathogenesis of SARS-CoV-2 and other SARS-like coronaviruses, and development of more effective strategies for controlling COVID-19.","version":"1.1","doi":"10.1101/2021.02.17.431755","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.31.424729","pub_date":"2021-2-18","title":"The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates","abstract":"SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-\u03b3 (Fc\u03b3R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc\u03b3R-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.","version":"1.2","doi":"10.1101/2020.12.31.424729","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431835","pub_date":"2021-2-18","title":"Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein","abstract":"Rotavirus, a segmented double-stranded RNA virus, is a major cause of acute gastroenteritis in young children. The introduction of live oral rotavirus vaccines has reduced the incidence of rotavirus disease in many countries. To explore the possibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding sequences for NSP3 and FLAG-tagged portions of the SARS-CoV-2 spike (S) protein. A 2A translational element was used to drive separate expression of NSP3 and the S product. rSA11 viruses were recovered that encoded the S-protein S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended receptor-binding domain (ExRBD), and S2 core (CR) domain (rSA11/NSP3-fS1, -fNTD, -fRBD, -fExRBD, and -fCR, respectively). Generation of rSA11/fS1 required a foreign-sequence insertion of 2.2-kbp, the largest such insertion yet made into the rotavirus genome. Based on isopycnic centrifugation, rSA11 containing S sequences were denser than wildtype virus, confirming the capacity of the rotavirus to accommodate larger genomes. Immunoblotting showed that rSA11/-fNTD, -fRBD, -fExRBD, and -fCR viruses expressed S products of expected size, with fExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, rSA11/NSP3-fS1 failed to express its expected 80-kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of portions of the SARS-CoV-2 S protein (e.g., NTD, RBD, ExRBD, and CR) with sizes smaller than the S1 fragment. Among the vaccines administered to children in the US and many other countries are those targeting rotavirus, a segmented double-stranded RNA virus that is a major cause of severe gastroenteritis. In this study, we have examined the feasibility of modifying the rotavirus genome by reverse genetics, such that the virus could serve as an expression vector of the SARS-CoV-2 spike protein. Results were obtained showing that recombinant rotaviruses can be generated that express domains of the SARS CoV-2 spike protein, including the receptor-binding domain (RBD), a common target of neutralizing antibodies produced in individuals infected by the virus. Our findings raise the possibility of creating a combined rotavirus-COVID-19 vaccine that could be used in place of current rotavirus vaccines.","version":"1.1","doi":"10.1101/2021.02.18.431835","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.18.431811","pub_date":"2021-2-18","title":"Alzheimer\u2019s-like remodeling of neuronal ryanodine receptor in COVID-19","abstract":"COVID-19, caused by SARS-CoV-2 involves multiple organs including cardiovascular, pulmonary and central nervous system. Understanding how SARS-CoV-2 infection afflicts diverse organ systems remains challenging. Particularly vexing has been the problem posed by persistent organ dysfunction known as \u201clong COVID,\u201d which includes cognitive impairment. Here we provide evidence linking SARS-CoV-2 infection to activation of TGF-\u00df signaling and oxidative overload. One consequence is oxidation of the ryanodine receptor/calcium (Ca2+) release channels (RyR) on the endo/sarcoplasmic (ER/SR) reticuli in heart, lung and brains of patients who succumbed to COVID-19. This depletes the channels of the stabilizing subunit calstabin2 causing them to leak Ca2+ which can promote heart failure, pulmonary insufficiency  and cognitive and behavioral defects. Ex-vivo treatment of heart, lung, and brain tissues from COVID-19 patients using a Rycal drug (ARM210) prevented calstabin2 loss and fixed the channel leak. Of particular interest is that neuropathological pathways activated downstream of leaky RyR2 channels in Alzheimer\u2019s Disease (AD) patients were activated in COVID-19 patients. Thus, leaky RyR2 Ca2+ channels may play a role in COVID-19 pathophysiology and could be a therapeutic target for amelioration of some comorbidities associated with SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.02.18.431811","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.14.21251704","pub_date":"2021-02-18","title":"Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity","abstract":"<jats:title>SUMMARY</jats:title>\n                <jats:p>Vaccination elicits immune responses capable of potently neutralizing SARS-CoV-2. However, ongoing surveillance has revealed the emergence of variants harboring mutations in spike, the main target of neutralizing antibodies. To understand the impact of these variants, we evaluated the neutralization potency of 99 individuals that received one or two doses of either BNT162b2 or mRNA-1273 vaccines against pseudoviruses representing 10 globally circulating strains of SARS-CoV-2. Five of the 10 pseudoviruses, harboring receptor-binding domain mutations, including K417N/T, E484K, and N501Y, were highly resistant to neutralization. Cross-neutralization of B.1.351 variants was comparable to SARS-CoV and bat-derived WIV1-CoV, suggesting that a relatively small number of mutations can mediate potent escape from vaccine responses. While the clinical impact of neutralization resistance remains uncertain, these results highlight the potential for variants to escape from neutralizing humoral immunity and emphasize the need to develop broadly protective interventions against the evolving pandemic.</jats:p>","version":null,"doi":"10.1101/2021.02.14.21251704","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.15.21251623","pub_date":"2021-02-18","title":"FDA-authorized COVID-19 vaccines are effective per real-world evidence synthesized across a multi-state health system","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Large Phase 3 clinical trials of the two FDA-authorized COVID-19 vaccines, mRNA-1273 (Moderna) and BNT162b2 (Pfizer/BioNTech), have demonstrated efficacies of 94.1% (n = 30,420, 95% CI: 89.3-96.8) and 95% (n = 43,448, 95% CI: 90.3-97.6) in preventing symptomatic COVID-19, respectively. Given the ongoing vaccine rollout to healthcare personnel and residents of long-term care facilities, here we provide a preliminary assessment of real-world vaccination efficacy in 62,138 individuals from the Mayo Clinic and associated health system (Arizona, Florida, Minnesota, Wisconsin) between December 1\n                  <jats:sup>st</jats:sup>\n                  2020 and February 8\n                  <jats:sup>th</jats:sup>\n                  2021. Our retrospective analysis contrasts 31,069 individuals receiving at least one dose of either vaccine with 31,069 unvaccinated individuals who are propensity-matched based on demographics, location (zip code), and number of prior SARS-CoV-2 PCR tests. 8,041 individuals received two doses of a COVID-19 vaccine and were at risk for infection at least 36 days after their first dose. Administration of two COVID-19 vaccine doses was 88.7% effective in preventing SARS-CoV-2 infection (95% CI: 68.4-97.1%) with onset at least 36 days after the first dose. Furthermore, vaccinated patients who were subsequently diagnosed with COVID-19 had significantly lower 14-day hospital admission rates than propensity-matched unvaccinated COVID-19 patients (3.7% vs. 9.2%; Relative Risk: 0.4; p-value: 0.007). Building upon the previous randomized trials of these vaccines, this study demonstrates their real-world effectiveness in reducing the rates of SARS-CoV-2 infection and COVID-19 severity among individuals at highest risk for infection.\n                </jats:p>","version":null,"doi":"10.1101/2021.02.15.21251623","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.14.431117","pub_date":"2021-2-18","title":"Mutation N501Y in RBD of Spike Protein Strengthens the Interaction between COVID-19 and its Receptor ACE2","abstract":"SARS-CoV-2 is spreading around the world for the past year. Enormous efforts have been taken to understand its mechanism of transmission. It is well established now that the receptor-binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) as its first step of entry. Being a single-stranded RNA virus, SARS-CoV-2 is evolving rapidly. Recently, several variants such as B.1.1.7, B.1.351, and P.1, with a key mutation N501Y on the RBD, appear to be more infectious to humans. To understand its mechanism, we combined cell surface binding assay, kinetics study, single-molecule technique, and computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction characterized from all these methodologies, while the other two mutations from B.1.351 contributed to a less effect. Fluorescence-activated cell scan (FACS) assays found that RBD N501Y mutations are of higher binding affinity to ACE2 than the wild type. Surface plasmon resonance further indicated that N501Y mutation had a faster association rate and slower dissociation rate. Consistent with the kinetics study, atomic force microscopy-based single-molecule force microscopy quantify their strength on living cells, showing a higher binding probability and unbinding force for the mutation. Finally, Steered Molecular Dynamics (SMD) simulations on the dissociation of RBD-ACE2 complexes revealed that the N501Y introduced additional \u03c0-\u03c0 and \u03c0-cation interaction for the higher force/interaction. Taken together, we suggested that the reinforced interaction from N501Y mutation in RBD should play an essential role in the higher transmission of COVID-19 variants.","version":"1.2","doi":"10.1101/2021.02.14.431117","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.05.979658","pub_date":"2021-2-18","title":"Bridging the gap: Using reservoir ecology and human serosurveys to estimate Lassa virus spillover in West Africa","abstract":"Forecasting the risk of pathogen spillover from reservoir populations of wild or domestic animals is essential for the effective deployment of interventions such as wildlife vaccination or culling. Due to the sporadic nature of spillover events and limited availability of data, developing and validating robust, spatially explicit, predictions is challenging. Recent efforts have begun to make progress in this direction by capitalizing on machine learning methodologies. An important weakness of existing approaches, however, is that they generally rely on combining human and reservoir infection data during the training process and thus conflate risk attributable to the prevalence of the pathogen in the reservoir population with the risk attributed to the realized rate of spillover into the human population. Because effective planning of interventions requires that these components of risk be disentangled, we developed a multi-layer machine learning framework that separates these processes. Our approach begins by training models to predict the geographic range of the primary reservoir and the subset of this range in which the pathogen occurs. The spillover risk predicted by the product of these reservoir specific models is then fit to data on realized patterns of historical spillover into the human population. The result is a geographically specific spillover risk forecast that can be easily decomposed and used to guide effective intervention. Applying our method to Lassa virus, a zoonotic pathogen that regularly spills over into the human population across West Africa, results in a model that explains a modest but statistically significant portion of geographic variation in historical patterns of spillover. When combined with a mechanistic mathematical model of infection dynamics, our spillover risk model predicts that 897,700 humans are infected by Lassa virus each year across West Africa, with Nigeria accounting for more than half of these human infections. The 2019 emergence of SARS-CoV-2 is a grim reminder of the threat animal-borne pathogens pose to human health. Even prior to SARS-CoV-2, the spillover of pathogens from animal reservoirs was a persistent problem, with pathogens such as Ebola, Nipah, and Lassa regularly but unpredictably causing outbreaks. Machine-learning models that anticipate when and where pathogen transmission from animals to humans is likely to occur would help guide surveillance efforts and preemptive countermeasures like information campaigns or vaccination programs. We develop a novel machine learning framework that uses datasets describing the distribution of a virus within its host and the range of its animal host, along with data on spatial patterns of human immunity, to infer rates of animal-to-human transmission across a region. By training the model on data from the animal host alone, our framework allows rigorous validation of spillover predictions using human data. We apply our framework to Lassa fever, a viral disease of West Africa that is spread to humans by rodents, and use the predictions to update estimates of Lassa virus infections in humans. Our results suggest that Nigeria is most at risk for the emergence of Lassa virus, and should be prioritized for outbreak-surveillance.","version":"1.3","doi":"10.1101/2020.03.05.979658","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431704","pub_date":"2021-2-17","title":"Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual","abstract":"Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background \u201cnoise\u201d. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.","version":"1.1","doi":"10.1101/2021.02.17.431704","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.430500","pub_date":"2021-2-17","title":"Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants","abstract":"The protective efficacy of neutralizing antibodies (nAbs) elicited during natural infection with SARS-CoV-2 and by vaccination based on its spike protein has been compromised with emergence of the recent SARS-CoV-2 variants. Residues E484 and K417 in the receptor-binding site (RBS) are both mutated in lineages first described in South Africa (B.1.351) and Brazil (B.1.1.28.1). The nAbs isolated from SARS-CoV-2 patients are preferentially encoded by certain heavy-chain germline genes and the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2) can each bind the RBS in two different binding modes. However, their binding and neutralization are abrogated by either the E484K or K417N mutation, whereas nAbs to the cross-reactive CR3022 and S309 sites are largely unaffected. This structural and functional analysis illustrates why mutations at E484 and K417 adversely affect major classes of nAbs to SARS-CoV-2 with consequences for next-generation COVID-19 vaccines.","version":"1.1","doi":"10.1101/2021.02.16.430500","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431437","pub_date":"2021-2-17","title":"Phase transitions may explain why SARS-CoV-2 spreads so fast and why new variants are spreading faster","abstract":"The novel coronavirus SARS CoV-2 responsible for the COVID-19 pandemic and SARS CoV-1 responsible for the SARS epidemic of 2002-2003 share an ancestor yet evolved to have much different transmissibility and global impact. A previously developed thermodynamic model of protein conformations predicted that SARS CoV-2 is very close to a thermodynamic critical point, which makes it highly infectious but also easily displaced by a spike-based vaccine because there is a tradeoff between transmissibility and robustness. The model identified a small cluster of four key mutations of SARS CoV-2 that promotes much stronger viral attachment and viral spreading. Here we apply the model to two new strains (B.1.1.7 and B.1.351) and predict, using no free parameters, how the new mutations can further enhance infectiousness.","version":"1.1","doi":"10.1101/2021.02.16.431437","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431617","pub_date":"2021-2-17","title":"Multiple Sites on SARS-CoV-2 Spike Protein are Susceptible to Proteolysis by Cathepsins B, K, L, S, and V","abstract":"SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic. Proteases are central to the infection process of SARS-CoV-2. Cleavage of the spike protein on the virus\u2019s capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS-CoV-2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS-CoV-2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID-19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.","version":"1.1","doi":"10.1101/2021.02.17.431617","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431492","pub_date":"2021-2-17","title":"SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys","abstract":"Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.","version":"1.1","doi":"10.1101/2021.02.17.431492","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431630","pub_date":"2021-2-17","title":"A measles-vectored COVID-19 vaccine induces long-term immunity and protection from SARS-CoV-2 challenge in mice","abstract":"In light of the expanding SARS-CoV-2 pandemic, developing efficient vaccines that can provide sufficient coverage for the world population is a global health priority. The measles virus (MV)-vectored vaccine is an attractive candidate given the measles vaccine\u2019s extensive safety history, well-established manufacturing process, and induction of strong, long-lasting immunity. We developed an MV-based SARS-CoV-2 vaccine using either the full-length spike (S) or S2 subunit as the antigen. While the S2 antigen failed to induce neutralizing antibodies, the prefusion-stabilized, full-length S (MV-ATU2-SF-2P-dER) construct proved to be an attractive vaccine candidate, eliciting strong Th1-dominant T-cell and neutralizing antibody responses against the S antigen while minimizing reactivity to the vector itself. Neutralizing antibody titers remained high three months after homologous prime-boost immunization, and infectious virus was undetectable in all animals after challenge with a mouse-adapted SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2021.02.17.431630","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.17.431566","pub_date":"2021-2-17","title":"E484K mutation in SARS-CoV-2 RBD enhances binding affinity with hACE2 but reduces interactions with neutralizing antibodies and nanobodies: Binding free energy calculation studies","abstract":"The pandemic of the COVID-19 disease caused by SARS-CoV-2 has led to more than 100 million infections and over 2 million deaths worldwide. The progress in the developments of effective vaccines and neutralizing antibody therapeutics brings hopes to eliminate the threat of COVID-19. However, SARS-CoV-2 continues to mutate, and several new variants have been emerged. Among the various naturally-occurring mutations, the E484K mutation shared by both the 501Y.V2 and 501Y.V3 variants attracted serious concerns, which may potentially enhance the receptor binding affinity and reduce the immune response. In the present study, the molecular mechanism behind the impacts of E484K mutation on the binding affinity of the receptor-binding domain (RBD) with the receptor human angiotensin-converting enzyme 2 (hACE2) was investigated by using the molecular dynamics (MD) simulations combined with the molecular mechanics-generalized Born surface area (MMGBSA) method. Our results indicate that the E484K mutation results in more favorable electrostatic interactions compensating the burial of the charged and polar groups upon the binding of RBD with hACE2, which significantly improves the RBD-hACE2 binding affinity. Besides that, the E484K mutation also causes the conformational rearrangements of the loop region containing the mutant residue, which leads to more tight binding interface of RBD with hACE2 and formation of some new hydrogen bonds. The more tight binding interface and the new hydrogen bonds formation also contribute to the improved binding affinity of RBD to the receptor hACE2. In addition, six neutralizing antibodies and nanobodies complexed with RBD were selected to explore the effects of E484K mutation on the recognition of these antibodies to RBD. The simulation results show that the E484K mutation significantly reduces the binding affinities to RBD for most of the studied neutralizing antibodies, and the decrease in the binding affinities is mainly owing to the unfavorable electrostatic interactions caused by the mutation. Our studies revealed that the E484K mutation may improve the binding affinity between RBD and the receptor hACE2, implying more transmissibility of the E484K-containing variants, and weaken the binding affinities between RBD and the studied neutralizing antibodies, indicating reduced effectiveness of these antibodies. Our results provide valuable information for the effective vaccine development and antibody drugs design.","version":"1.1","doi":"10.1101/2021.02.17.431566","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431395","pub_date":"2021-2-17","title":"HLA binding of self-peptides is biased towards proteins with specific molecular functions","abstract":"Human leukocyte antigen (HLA) is highly polymorphic and plays a key role in guiding adaptive immune responses by presenting foreign and self peptides to T cells. Each HLA variant selects a minor fraction of peptides that match a certain motif required for optimal interaction with the peptide-binding groove. These restriction rules define the landscape of peptides presented to T cells. Given these limitations, one might suggest that the choice of peptides presented by HLA is non-random and there is preferential presentation of an array of peptides that is optimal for distinguishing self and foreign proteins. In this study we explore these preferences with a comparative analysis of self peptides enriched and depleted in HLA ligands. We show that HLAs exhibit preferences towards presenting peptides from certain proteins while disfavoring others with specific functions, and highlight differences between various HLA genes and alleles in those preferences. We link those differences to HLA anchor residue propensities and amino acid composition of preferentially presented proteins. The set of proteins that peptides presented by a given HLA are most likely to be derived from can be used to distinguish between class I and class II HLAs and HLA alleles. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Finally, we show that the reported self peptidome preferences of distinct HLA variants can be compensated by combinations of HLA-A/HLA-B and HLA-A/HLA-C alleles in frequent haplotypes.","version":"1.1","doi":"10.1101/2021.02.16.431395","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431310","pub_date":"2021-2-17","title":"Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization","abstract":"Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2 and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non or weakly neutralizing (\u201coff-target\u201d) antibodies. Using our recently developed electron microscopy epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.","version":"1.1","doi":"10.1101/2021.02.16.431310","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.15.431237","pub_date":"2021-2-16","title":"SARS-CoV2 envelop proteins reshape the serological responses of COVID-19 patients","abstract":"The SARS-CoV-2 pandemic has elicited a unique international mobilization of the scientific community to better understand this coronavirus and its associated disease and to develop efficient tools to combat infection. Similar to other coronavirae, SARS-CoV-2 hijacks the host cell complex secretory machinery to produce properly folded viral proteins that will compose the nascent virions; including Spike, Envelope and Membrane proteins, the most exposed membrane viral proteins to the host immune system. Antibody response is part of the anti-viral immune arsenal that infected patients develop to fight viral particles in the body. Herein, we investigate the immunogenic potential of Spike (S), Envelope (E) and Membrane (M) proteins using a human cell-based system to mimic membrane insertion and N-glycosylation. We show that both S and M proteins elicit the production of specific IgG, IgM and IgA in SARS-CoV-2 infected patients. Elevated Ig responses were observed in COVID+ patients with moderate and severe forms of the disease. Finally, when SARS-CoV-2 Spike D614 and G614 variants were compared, reduced Ig binding was observed with the Spike G614 variant. Altogether, this study underlines the needs for including topological features in envelop proteins to better characterize the serological status of COVID+ patients, points towards an unexpected immune response against the M protein and shows that our assay could represent a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness.","version":"1.2","doi":"10.1101/2021.02.15.431237","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431318","pub_date":"2021-2-16","title":"The Host Interactome of Spike Expands the Tropism of SARS-CoV-2","abstract":"The SARS-CoV-2 virus causes severe acute respiratory syndrome (COVID-19) and has rapidly created a global pandemic. Patients that survive may face a slow recovery with long lasting side effects that can afflict different organs. SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor Angiotensin Converting Enzyme 2 (ACE2) which binds to spike protein trimers on the surface of SARS-CoV-2 virions. However, SARS-CoV-2 can spread to other tissues even though they are negative for ACE2. To gain insight into the molecular constituents that might influence SARS-CoV-2 tropism, we determined which additional host factors engage with the viral spike protein in disease-relevant human bronchial epithelial cells (16HBEo\u2212). We found that spike recruited the extracellular proteins laminin and thrombospondin and was retained in the endoplasmatic reticulum (ER) by the proteins DJB11 and FBX2 which support re-folding or degradation of nascent proteins in the ER. Because emerging mutations of the spike protein potentially impact the virus tropism, we compared the interactome of D614 spike with that of the rapidly spreading G614 mutated spike. More D614 than G614 spike associated with the proteins UGGT1, calnexin, HSP7A and GRP78/BiP which ensure glycosylation and folding of proteins in the ER. In contrast to G614 spike, D614 spike was endoproteolytically cleaved, and the N-terminal S1 domain was degraded in the ER even though C-terminal \u2018S2 only\u2019 proteoforms remained present. D614 spike also bound more laminin than G614 spike, which suggested that extracellular laminins may function as co-factor for an alternative, \u2018S2 only\u2019 dependent virus entry. Because the host interactome determines whether an infection is productive, we developed a novel proteome-based cell type set enrichment analysis (pCtSEA). With pCtSEA we determined that the host interactome of the spike protein may extend the tropism of SARS-CoV-2 beyond mucous epithelia to several different cell types, including macrophages and epithelial cells in the nephron. An \u2018S2 only\u2019 dependent, alternative infection of additional cell types with SARS-CoV-2 may impact vaccination strategies and may provide a molecular explanation for a severe or prolonged progression of disease in select COVID-19 patients.","version":"1.1","doi":"10.1101/2021.02.16.431318","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.15.431215","pub_date":"2021-2-16","title":"Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV","abstract":"There is an urgent need to understand the nature of immune responses generated against SARS-CoV-2, to better inform risk-mitigation strategies for people living with HIV (PLWH). Although not all PLWH are considered immunosuppressed, residual cellular immune deficiency and ongoing inflammation could influence COVID-19 disease severity, the evolution and durability of protective memory responses. Here, we performed an integrated analysis, characterizing the nature, breadth and magnitude of SARS-CoV-2-specific immune responses in PLWH, controlled on ART, and HIV negative subjects. Both groups were in the convalescent phase of predominately mild COVID-19 disease. The majority of PLWH mounted SARS-CoV-2 Spike- and Nucleoprotein-specific antibodies with neutralizing activity and SARS-CoV-2-specific T cell responses, as measured by ELISpot, at levels comparable to HIV negative subjects. T cell responses against Spike, Membrane and Nucleocapsid were the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. Notably, the overall magnitude of SARS-CoV-2-specific T cell responses related to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses were observed. Both humoral and cellular responses to SARS-CoV-2 were detected at 5-7 months post-infection, providing evidence of medium-term durability of responses irrespective of HIV serostatus. Incomplete immune reconstitution on ART and a low CD4:CD8 ratio could, however, hamper the development of immunity to SARS-CoV-2 and serve as a useful tool for risk stratification of PLWH. These findings have implications for the individual management and potential effectiveness of vaccination against SARS-CoV-2 in PLWH. Adaptive immune responses to SARS-CoV-2 in the setting of HIV infection","version":"1.1","doi":"10.1101/2021.02.15.431215","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431021","pub_date":"2021-2-16","title":"N-Terminal finger stabilizes the reversible feline drug GC376 in SARS-CoV-2 Mpro","abstract":"The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug\u2019s properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug\u2019s P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.","version":"1.1","doi":"10.1101/2021.02.16.431021","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.15.431198","pub_date":"2021-2-16","title":"Single-Domain SARS-CoV-2 S1 and RBD Antibodies Isolated from Immunized Llama Effectively Bind Targets of the Wuhan, UK, and South African Strains in vitro","abstract":"The spreading of SARS-CoV-2 variants has become a major challenge of the current fight against the pandemic. Of particular concerns are the strains that have arisen from the United Kingdom (UK) and South Africa. The UK variant spreads rapidly and is projected to overtake the original strain in the US as early as in March 2021, while the South African variant appears to evade some effects of the current vaccines. Potential false-negative diagnosis using currently available antigen kits that may not recognize these variants could cause another wave of community infection. Therefore, it is imperative that antibodies used in the detection kits are validated for binding against these variants. Here we report that the nanoantibodies (nAbs in our terminology, also referred to as VHH fragments, single domain antibodies, nanobodies\u2122) that we have developed for rapid antigen detection test bind the receptor binding domain (RBD) of the S1 protein from the original COVID-SARS-2 virus as well as those from the UK and South African variants. This finding validates our antibodies used in our assay for the detection of these major variant strains.","version":"1.1","doi":"10.1101/2021.02.15.431198","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.079830","pub_date":"2021-2-16","title":"Development of a vaccine against the newly emerging COVID-19 virus based on the receptor binding domain displayed on virus-like particles","abstract":"The ongoing coronavirus COVID-19 pandemic is caused by a new coronavirus (SARS-CoV-2) with its origin in the city of Wuhan in China. From there it has been rapidly spreading to many cities inside and outside China. Nowadays more than 33 millions with deaths surpassing 1 million have been recorded worldwide thus representing a major health issue. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrated that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus (CuMVTT) resulted in a vaccine candidate that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized COVID-19 virus in vitro.","version":"1.3","doi":"10.1101/2020.05.06.079830","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425478","pub_date":"2021-2-16","title":"Chitinase 3-like-1 is a Therapeutic Target That Mediates the Effects of Aging in COVID-19","abstract":"COVID-19 is caused by the SARS-CoV-2 (SC2) virus and is more prevalent and severe in the elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor ACE2 and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging and that anti-CHI3L1, kasugamycin and inhibitors of phosphorylation, abrogate these ACE2- and SPP-inductive events. Human studies also demonstrated that the levels of circulating CHI3L1 are increased in the elderly and patients with CM where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP; that this induction is a major mechanism contributing to the effects of aging during SC2 infection and that CHI3L1 coopts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.","version":"1.2","doi":"10.1101/2021.01.05.425478","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431364","pub_date":"2021-2-16","title":"Identification of common key genes and pathways between Covid-19 and lung cancer by using protein-protein interaction network analysis","abstract":"COVID-19 is indeed an infection that is caused by a recently found coronavirus group, a type of virus proven to cause human respiratory diseases. The high mortality rate was observed in patients who had pre-existing health conditions like cancer. However, the molecular mechanism of SARS-CoV-2 infection in lung cancer patients was not discovered yet at the pathway level. This study was about determining the common key genes of COVID-19 and lung cancer through network analysis. The hub genes associated with COVID-19 and lung cancer were identified through Protein-Protein interaction analysis. The hub genes are ALB, CXCL8, FGF2, IL6, INS, MMP2, MMP9, PTGS2, STAT3 and VEGFA. Through gene enrichment, it is identified both COVID-19 and lung cancer have a common pathway in EGFR tyrosine kinase inhibitor resistance, IL-17 signalling pathway, AGE-RAGE signalling pathway in diabetic complications, HIF-1 signalling pathway and pathways in cancer.","version":"1.1","doi":"10.1101/2021.02.16.431364","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.15.431291","pub_date":"2021-2-16","title":"Favourable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases","abstract":"Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained and the impact of underlying immune dysfunction or suppression unknown. Here, we examined the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM) and juvenile systemic lupus erythematosus (JSLE), against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group. Despite immune dysfunction and immunosuppressive treatment, JIA, JDM and JSLE patients mounted comparable or stronger responses than healthier peers, dominated by IgG antibodies to HCoV-OC43 spike, and harboured IgG antibodies that cross-reacted with SARS-CoV-2 spike. In contrast, responses to HCoV-OC43 and SARS-CoV-2 nucleoproteins exhibited delayed age-dependent class-switching and were not elevated in JIA, JDM and JSLE patients, arguing against increased exposure. Consequently, autoimmune rheumatic diseases and their treatment were associated with a favourable ratio of spike to nucleoprotein antibodies.","version":"1.1","doi":"10.1101/2021.02.15.431291","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431305","pub_date":"2021-2-16","title":"501Y.V2 and 501Y.V3 variants of SARS-CoV-2 lose binding to Bamlanivimab in vitro","abstract":"We generated several versions of the receptor binding domain (RBD) of the Spike protein with mutations existing within newly emerging variants from South Africa and Brazil. We found that the mutant RBD with K417N, E484K, and N501Y exchanges has higher binding affinity to the human receptor compared to the wildtype RBD. This mutated version of RBD also completely abolishes the binding to a therapeutic antibody, Bamlanivimab, in vitro.","version":"1.1","doi":"10.1101/2021.02.16.431305","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.16.431420","pub_date":"2021-2-16","title":"Transfer learning via multi-scale convolutional neural layers for human-virus protein-protein interaction prediction","abstract":"To predict interactions between human and viral proteins, we combine evolutionary sequence profile features with a Siamese convolutional neural network (CNN) architecture and a multi-layer perceptron (MLP). Our architecture outperforms various feature encodings-based machine learning and state-of-the-art prediction methods. As our main contribution, we introduce two types of transfer learning methods (i.e., \u2018frozen\u2019 type and \u2018fine-tuning\u2019 type) that reliably predict interactions in a target human-virus domain based on training in a source human-virus domain, by retraining CNN layers. Our transfer learning strategies can effectively apply prior knowledge transfer from large source dataset/task to small target dataset/task to improve prediction performance. Finally, we utilize the \u2018frozen\u2019 type of transfer learning to predict human-SARS-CoV-2 PPIs, indicating that our predictions are topologically and functionally similar to experimentally known interactions. Source code and datasets are available at https://github.com/XiaodiYangCAU/TransPPI/.","version":"1.1","doi":"10.1101/2021.02.16.431420","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430446","pub_date":"2021-2-16","title":"GABA administration limits viral replication and pneumonitis in a mouse model of COVID-19","abstract":"Despite the availability of vaccines for COVID-19, serious illness and death induced by coronavirus infection will remain a global health burden because of vaccination hesitancy, possible virus mutations, and the appearance of novel coronaviruses. Accordingly, there is a need for new approaches to limit severe illness stemming from coronavirus infections. Cells of the immune system and lung epithelia express receptors for GABA (GABA-Rs), a widely used neurotransmitter within the CNS. GABA-R agonists have anti-inflammatory effects and can limit acute lung injury. We previously showed that GABA treatment effectively reduced disease severity and death rates in mice following infection with a coronavirus (MHV-1) which provides a potentially lethal model of COVID-19. Here, we report that GABA treatment also reduced viral load in the lungs, suggesting that GABA-Rs may provide a new druggable target to limit pulmonary coronavirus replication. Histopathological analysis revealed that GABA treatment reduced lung inflammatory infiltrates and damages. Since GABA is safe for human consumption, inexpensive, and available worldwide, it is a promising candidate to help treat COVID-19.","version":"1.2","doi":"10.1101/2021.02.09.430446","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.25.313148","pub_date":"2021-2-16","title":"Rapid behavioural response of urban birds to COVID-19 lockdown","abstract":"Biodiversity is threatened by the growth of urban areas. However, it is still poorly understood how animals can cope with and adapt to these rapid and dramatic transformations of natural environments. The COVID-19 pandemic provides us with a unique opportunity to unveil the mechanisms involved in this process. Lockdown measures imposed in most countries are causing an unprecedented reduction of human activities giving us an experimental setting to assess the effects of our lifestyle on biodiversity. We studied the birds\u2019 response to the population lockdown by using more than 126,000 bird records collected by a citizen science project in north eastern Spain. We compared the occurrence and detectability of birds during the spring 2020 lockdown with baseline data from previous years in the same urban areas and dates. We found that birds did not increase their probability of occurrence in urban areas during the lockdown, refuting the hypothesis that nature has recovered its space in human emptied urban areas. However, we found an increase in bird detectability, especially during early morning, suggesting a rapid change in the birds\u2019 daily routines in response to quieter and less crowded cities. In conclusion, urban birds showed high behavioural plasticity to rapidly adjust to novel environmental conditions, as those imposed by the COVID-19.","version":"1.3","doi":"10.1101/2020.09.25.313148","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426080","pub_date":"2021-2-15","title":"Immunological and cardio-vascular pathologies associated with SARS-CoV-2 infection in golden syrian hamster","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection in golden Syrian hamster (GSH) causes lung pathology and resembles human coronavirus disease (Covid-19). However, extra-pulmonary pathologies of SARS-CoV-2 infection that result in long Covid remains undefined in GSH. Here, using in silico modelling we show that hamster angiotensin-converting enzyme-2 (ACE-2) and neuropilin-1 (NRP-1) interaction with SARS-CoV-2 is similar to human. Intranasal SARS-CoV-2 infection in GSH resulted in early onset of lung pathologies marked by aggressive inflammatory response. Remarkably, late phase of SARS-CoV2 infection in GSH showed cardiovascular complications (CVC) characterized by ventricular hypertrophy, ventricular wall thickening, interstitial coronary fibrosis and altered lipidomics with elevated cholesterol, low-density lipoprotein and long chain fatty acid triglycerides. Moreover, serum metabolomics profile of infected GSH correlated with Covid19 patients. Together, we propose GSH as a suitable animal model to study immediate and long Covid19 pathologies that could be extended to therapeutics against Covid19 related CVC.","version":"1.2","doi":"10.1101/2021.01.11.426080","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.14.431122","pub_date":"2021-2-15","title":"In vitro efficacy of Artemisia extracts against SARS-CoV-2","abstract":"Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts of Artemisia annua and Artemisia afra have been widely used in Africa in efforts to prevent and fight COVID-19 infections. We sought to study the ability of different A. annua and A. afra extracts and the Covid-Organics drink produced in Madagascar to inhibit SARS-CoV-2 and feline coronavirus (FCoV) replication in vitro. Several extracts as well as Covid-Organics inhibit SARS-CoV-2 and FCoV replication at concentrations that did not affect cell viability. It remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral replication following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment in patients.","version":"1.1","doi":"10.1101/2021.02.14.431122","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.25.221135","pub_date":"2021-2-15","title":"SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation","abstract":"The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2\u2019 site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. Cell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2\u2019 cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.","version":"1.4","doi":"10.1101/2020.07.25.221135","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.14.431129","pub_date":"2021-2-15","title":"ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model","abstract":"\n\n There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 \u03bcM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.","version":"1.1","doi":"10.1101/2021.02.14.431129","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.398909","pub_date":"2021-2-15","title":"Broad-spectrum antiviral activity of 3D8, a nucleic acid-hydrolyzing single chain variable fragment (scFv), targeting SARS-CoV-2 and multiple coronaviruses in vitro","abstract":"The current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of Coronavirus-induced disease 19 (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single chain variable fragment (scFv), a recombinant antibody exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. This study is aimed to investigate an antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses. 3D8, a recombinant scFv antibody was evaluated for antiviral activity against SARS-CoV-2, HCoV-OC43 and PEDV in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. Nucleic acid hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles respectively in 3D8 scFv-treated cells. This data demonstrates the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.","version":"1.2","doi":"10.1101/2020.11.25.398909","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.23.258574","pub_date":"2021-2-15","title":"Bromodomain and Extraterminal Inhibition Blocks Inflammation-Induced Cardiac Dysfunction and SARS-CoV-2 Infection (Pre-Clinical)","abstract":"Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory \u2018cytokine-storm\u2019, a cocktail of interferon gamma, interleukin 1\u03b2 and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.","version":"1.3","doi":"10.1101/2020.08.23.258574","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.431032","pub_date":"2021-2-15","title":"Pulsed broad-spectrum UV light effectively inactivates SARS-CoV-2 on multiple surfaces","abstract":"The ongoing SARS-CoV-2 pandemic has resulted in an increased need for technologies capable of efficiently disinfecting public spaces as well as personal protective equipment. UV light disinfection is a well-established method for inactivating respiratory viruses. Here, we have determined that broad-spectrum, pulsed UV light is effective at inactivating SARS-CoV-2 on multiple surfaces. For hard, non-porous surfaces we observed that SARS-CoV-2 was inactivated to undetectable levels on plastic and glass with a UV dose of 34.9 mJ/cm2 and stainless steel with a dose of 52.5 mJ/cm2. We also observed that broad-spectrum, pulsed UV light is effective at reducing SARS-CoV-2 on N95 respirator material to undetectable levels with a dose of 103 mJ/cm2. We included UV dosimeter cards that provide a colorimetric readout of UV dose and demonstrated their utility as a means to confirm desired levels of exposure were reached. Together, the results present here demonstrate that broad-spectrum, pulsed UV light is an effective technology for the inactivation of SARS-CoV-2 on multiple surfaces.","version":"1.1","doi":"10.1101/2021.02.12.431032","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.11.430757","pub_date":"2021-2-15","title":"SARS CoV-2 nucleoprotein enhances the infectivity of lentiviral spike particles","abstract":"The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that this augmentation by N renders LV spike particles less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Biochemical analysis revealed that the spike protein is better enriched in virions when the particles are produced in the presence of SARS CoV-2 nucleoprotein. Importantly, this improvement in infectivity is achieved without a concomitant increase in sensitivity towards RBD binding-based neutralization. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.","version":"1.1","doi":"10.1101/2021.02.11.430757","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.13.431008","pub_date":"2021-2-15","title":"Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures","abstract":"Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 \u00b5M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a \u201cBL2 groove\u201d that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.","version":"1.1","doi":"10.1101/2021.02.13.431008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.16.342782","pub_date":"2021-2-15","title":"Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19","abstract":"Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in mouse and human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Further, Camostat \u2013 a TMPRSS2 inhibitor, blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction. Thus providing evidence for the first time a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or Camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors, androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.","version":"1.2","doi":"10.1101/2020.10.16.342782","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.430907","pub_date":"2021-2-15","title":"A single-cell atlas of lymphocyte adaptive immune repertoires and transcriptomes reveals age-related differences in convalescent COVID-19 patients","abstract":"COVID-19 disease outcome is highly dependent on adaptive immunity from T and B lymphocytes, which play a critical role in the control, clearance and long-term protection against SARS-CoV-2. To date, there is limited knowledge on the composition of the T and B cell immune receptor repertoires [T cell receptors (TCRs) and B cell receptors (BCRs)] and transcriptomes in convalescent COVID-19 patients of different age groups. Here, we utilize single-cell sequencing (scSeq) of lymphocyte immune repertoires and transcriptomes to quantitatively profile the adaptive immune response in COVID-19 patients of varying age. We discovered highly expanded T and B cells in multiple patients, with the most expanded clonotypes coming from the effector CD8+ T cell population. Highly expanded CD8+ and CD4+ T cell clones show elevated markers of cytotoxicity (CD8: PRF1, GZMH, GNLY; CD4: GZMA), whereas clonally expanded B cells show markers of transition into the plasma cell state and activation across patients. By comparing young and old convalescent COVID-19 patients (mean ages = 31 and 66.8 years, respectively), we found that clonally expanded B cells in young patients were predominantly of the IgA isotype and their BCRs had incurred higher levels of somatic hypermutation than elderly patients. In conclusion, our scSeq analysis defines the adaptive immune repertoire and transcriptome in convalescent COVID-19 patients and shows important age-related differences implicated in immunity against SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.02.12.430907","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.431026","pub_date":"2021-2-15","title":"Jumper Enables Discontinuous Transcript Assembly in Coronaviruses","abstract":"Genes in SARS-CoV-2 and, more generally, in viruses in the order of Nidovirales are expressed by a process of discontinuous transcription mediated by the viral RNA-dependent RNA polymerase. This process is distinct from alternative splicing in eukaryotes, rendering current transcript assembly methods unsuitable to Nidovirales sequencing samples. Here, we introduce the Discontinuous Transcript Assembly problem of finding transcripts  and their abundances c given an alignment  under a maximum likelihood model that accounts for varying transcript lengths. Underpinning our approach is the concept of a segment graph, a directed acyclic graph that, distinct from the splice graph used to characterize alternative splicing, has a unique Hamiltonian path. We provide a compact characterization of solutions as subsets of non-overlapping edges in this graph, enabling the formulation of an efficient mixed integer linear program. We show using simulations that our method, Jumper, drastically outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1 and SARS-CoV-2 samples, we find that Jumper not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are well supported by direct evidence from long-read data, presence in multiple, independent samples or a conserved core sequence. Jumper enables detailed analyses of Nidovirales transcriptomes. Software is available at https://github.com/elkebir-group/Jumper","version":"1.1","doi":"10.1101/2021.02.12.431026","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.431018","pub_date":"2021-2-13","title":"HaVoC, a bioinformatic pipeline for reference-based consensus assembly and lineage assignment for SARS-CoV-2 sequences","abstract":"SARS-CoV-2 related research has increased in importance worldwide since December 2019. Several new variants of SARS-CoV-2 have emerged globally, of which the most notable and concerning currently are the UK variant B.1.1.7, the South African variant B1.351 and the Brazilian variant P.1. Detecting and monitoring novel variants is essential in SARS-CoV-2 surveillance. While there are several tools for assembling virus genomes and performing lineage analyses to investigate SARS-CoV-2, each is limited to performing singular or a few functions separately. Due to the lack of publicly available pipelines, which could perform fast reference-based assemblies on raw SARS-CoV-2 sequences in addition to identifying lineages to detect variants of concern, we have developed an open source bioinformatic pipeline called HaVoC (Helsinki university Analyzer for Variants Of Concern). HaVoC can reference assemble raw sequence reads and assign the corresponding lineages to SARS-CoV-2 sequences. HaVoC is a pipeline utilizing several bioinformatic tools to perform multiple necessary analyses for investigating genetic variance among SARS-CoV-2 samples. The pipeline is particularly useful for those who need a more accessible and fast tool to detect and monitor the spread of SARS-CoV-2 variants of concern during local outbreaks. HaVoC is currently being used in Finland for monitoring the spread of SARS-CoV-2 variants. HaVoC user manual and source code are available at https://www.helsinki.fi/en/projects/havoc and https://bitbucket.org/auto_cov_pipeline/havoc, respectively.","version":"1.1","doi":"10.1101/2021.02.12.431018","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.430950","pub_date":"2021-2-13","title":"Experimental Control of a Reaction Occurring during the Interaction between Chicken Anemia Virus (CAV) and Its Corresponding Antibodies","abstract":"In our ongoing investigations, we have studied a specific interaction between electromagnetic fields and matter \u2013 the so-called Electromagnetic echo effect (EMEE). It enables rapid and contactless investigations of gases, liquids and solids to be performed, since the signal generated as a result of the effect is quite sensitive to all kinds of changes occurring within the studied samples. The effect can be considered universal for all matter and provides analysis in real time. We use this phenomenon to demonstrate the practical possibility to control reactions, occurring between Chicken anemia virus (CAV) and the corresponding antibodies. This methodology can be used for simple but reliable control of similar, otherwise hard to detect, antigen-antibody reactions, in order to confirm the presence of a certain viral species. The approach offers a high level of safety, since it enables measurements to be taken remotely, thus limiting exposure to contagion. We further discuss the possibility to register the presence of SARS-nCoV-2 in an attempt to address current global pandemic.","version":"1.1","doi":"10.1101/2021.02.12.430950","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.11.430866","pub_date":"2021-2-12","title":"A combination of cross-neutralizing antibodies synergizes to prevent SARS-CoV-2 and SARS-CoV pseudovirus infection","abstract":"Coronaviruses have caused several epidemics and pandemics including the ongoing coronavirus disease 2019 (COVID-19). Some prophylactic vaccines and therapeutic antibodies have already showed striking effectiveness against COVID-19. Nevertheless, concerns remain about antigenic drift in SARS-CoV-2 as well as threats from other sarbecoviruses. Cross-neutralizing antibodies to SARS-related viruses provide opportunities to address such concerns. Here, we report on crystal structures of a cross-neutralizing antibody CV38-142 in complex with the receptor binding domains from SARS-CoV-2 and SARS-CoV. Our structural findings provide mechanistic insights into how this antibody can accommodate antigenic variation in these viruses. CV38-142 synergizes with other cross-neutralizing antibodies, in particular COVA1-16, to enhance neutralization of SARS-CoV-2 and SARS-CoV. Overall, this study provides valuable information for vaccine and therapeutic design to address current and future antigenic drift in SARS-CoV-2 and to protect against zoonotic coronaviruses.","version":"1.1","doi":"10.1101/2021.02.11.430866","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.430998","pub_date":"2021-2-12","title":"SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization","abstract":"Transmission of SARS-CoV-2 from humans to farmed mink was observed in Europe and the US. In the infected animals viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink were mostly compatible with efficient entry into human cells and its inhibition by soluble ACE2. In contrast, mutation Y453F reduced neutralization by an antibody with emergency use authorization for COVID-19 therapy and by sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.","version":"1.1","doi":"10.1101/2021.02.12.430998","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.430940","pub_date":"2021-2-12","title":"Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2","abstract":"Isolation and characterization of antibodies in COVID-19 patients has largely focused on memory B cells, however it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in controlling and resolving SARS-CoV-2 infection. To date there is little known about the specificity of plasma cells in COVID-19 patients. This is largely because plasma cells lack surface antibody expression, which complicates their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and high-throughput mammalian display screening to interrogate the specificity of plasma cells from 16 convalescent COVID-19 patients. Single-cell sequencing allows us to profile antibody repertoire features in these patients and identify highly expanded clonal lineages. Mammalian display screening is employed to reveal that 37 antibodies (out of 132 candidates) derived from expanded plasma cell clonal lineages are specific for SARS-CoV-2 antigens, including antibodies that target the receptor binding domain (RBD) with high affinity and exhibit potent neutralization of SARS-CoV-2. Single-cell antibody repertoire sequencing and high-throughput screening identifies highly expanded plasma cells from convalescent COVID-19 patients that produce SARS-CoV-2-specific antibodies capable of potent neutralization.","version":"1.1","doi":"10.1101/2021.02.12.430940","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429199","pub_date":"2021-2-12","title":"In vitro evolution of Remdesivir resistance reveals genome plasticity of SARS-CoV-2","abstract":"Remdesivir (RDV) is used widely for COVID-19 patients despite varying results in recent clinical trials. Here, we show how serially passaging SARS-CoV-2 in vitro in the presence of RDV selected for drug-resistant viral populations. We determined that the E802D mutation in the RNA-dependent RNA polymerase was sufficient to confer decreased RDV sensitivity without affecting viral fitness. Analysis of more than 200,000 sequences of globally circulating SARS-CoV-2 variants show no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we also observed changes in the Spike (i.e., H69 E484, N501, H655) corresponding to mutations identified in emerging SARS-CoV-2 variants indicating that they can arise in vitro in the absence of immune selection. This study illustrates SARS-CoV-2 genome plasticity and offers new perspectives on surveillance of viral variants. SARS-CoV-2 drug resistance & genome plasticity","version":"1.2","doi":"10.1101/2021.02.01.429199","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428137","pub_date":"2021-2-12","title":"Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7","abstract":"The COVID-19 pandemic has ravaged the globe, and its causative agent, SARS-CoV-2, continues to rage. Prospects of ending this pandemic rest on the development of effective interventions. Single and combination monoclonal antibody (mAb) therapeutics have received emergency use authorization, with more in the pipeline. Furthermore, multiple vaccine constructs have shown promise, including two with ~95% protective efficacy against COVID-19. However, these interventions were directed toward the initial SARS-CoV-2 that emerged in 2019. The recent emergence of new SARS-CoV-2 variants B.1.1.7 in the UK and B.1.351 in South Africa is of concern because of their purported ease of transmission and extensive mutations in the spike protein. We now report that B.1.1.7 is refractory to neutralization by most mAbs to the N-terminal domain (NTD) of spike and relatively resistant to a few mAbs to the receptor-binding domain (RBD). It is not more resistant to convalescent plasma or vaccinee sera. Findings on B.1.351 are more worrisome in that this variant is not only refractory to neutralization by most NTD mAbs but also by multiple individual mAbs to the receptor-binding motif on RBD, largely due to an E484K mutation. Moreover, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4 fold) and vaccinee sera (10.3-12.4 fold). B.1.351 and emergent variants with similar spike mutations present new challenges for mAb therapy and threaten the protective efficacy of current vaccines.","version":"1.3","doi":"10.1101/2021.01.25.428137","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.430933","pub_date":"2021-2-12","title":"Rapidly Increasing SARS-CoV-2 Neutralization by Intravenous Immunoglobulins Produced from Plasma Collected During the 2020 Pandemic","abstract":"Immunoglobulin (IG) lots (N=176) released since March 2020 were tested for SARS-CoV-2 neutralizing antibodies, with first positive results for September 2020 lots, mean = 1.8 IU/ml, 46% of lots positive. From there, values steadily increased, in correlation with the cumulative COVID-19 incidence, to reach a mean of 36.7 IU/ml and 93% of lots positive by January 2021. Extrapolating the correlation, IGs could reach an anti-SARS-CoV-2 potency of ~400 IU/ml by July 2021. At that stage, prophylactic IG treatment for primary/secondary immunodeficiency could contain similar doses of anti-SARS-CoV-2 as convalescent plasma which is used for treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.02.12.430933","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.430472","pub_date":"2021-2-12","title":"Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies","abstract":"SARS-CoV-2 B.1.1.7 and B.1.351 variants emerged respectively in United Kingdom and South Africa and spread in many countries. Here, we isolated infectious B.1.1.7 and B.1.351 strains and examined their sensitivity to anti-SARS-CoV-2 antibodies present in sera and nasal swabs, in comparison with a D614G reference virus. We established a novel rapid neutralization assay, based on reporter cells that become GFP+ after overnight infection. B.1.1.7 was neutralized by 79/83 sera from convalescent patients collected up to 9 months post symptoms, almost similar to D614G. There was a mean 6-fold reduction in titers and even loss of activity against B.1.351 in 40% of convalescent sera after 9 months. Early sera from 19 vaccinated individuals were almost as potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Nasal swabs from vaccine recipients were not neutralizing, except in individuals who were diagnosed COVID-19+ before vaccination. Thus, faster-spreading variants acquired a partial resistance to humoral immunity generated by natural infection or vaccination, mostly visible in individuals with low antibody levels.","version":"1.1","doi":"10.1101/2021.02.12.430472","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.23.308239","pub_date":"2021-2-12","title":"The COVID-19 PHARMACOME: A method for the rational selection of drug repurposing candidates from multimodal knowledge harmonization","abstract":"The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community\u2019s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2 / COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.","version":"1.4","doi":"10.1101/2020.09.23.308239","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.12.293498","pub_date":"2021-2-12","title":"Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19","abstract":"COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.","version":"1.3","doi":"10.1101/2020.09.12.293498","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.12.429482","pub_date":"2021-2-12","title":"Physical phenotype of blood cells is altered in COVID-19","abstract":"Clinical syndrome coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by rapid spreading and high mortality worldwide. While the pathology is not yet fully understood, hyper-inflammatory response and coagulation disorders leading to congestions of microvessels are considered to be key drivers of the still increasing death toll. Until now, physical changes of blood cells have not been considered to play a role in COVID-19 related vascular occlusion and organ damage. Here we report an evaluation of multiple physical parameters including the mechanical features of five frequent blood cell types, namely erythrocytes, lymphocytes, monocytes, neutrophils, and eosinophils. More than 4 million blood cells of 17 COVID-19 patients at different levels of severity, 24 volunteers free from infectious or inflammatory diseases, and 14 recovered COVID-19 patients were analyzed. We found significant changes in erythrocyte deformability, lymphocyte stiffness, monocyte size, and neutrophil size and deformability. While some of these changes recovered to normal values after hospitalization, others persisted for months after hospital discharge, evidencing the long-term imprint of COVID-19 on the body.","version":"1.1","doi":"10.1101/2021.02.12.429482","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.10.21251247","pub_date":"2021-02-12","title":"Safety and efficacy of the ChAdOx1 nCoV-19 (AZD1222) Covid-19 vaccine against the B.1.351 variant in South Africa","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Assessing safety and efficacy of Covid-19 vaccines in different populations is essential, as is investigation of efficacy against emerging SARS-CoV-2 variants of concern including the B.1.351 (501Y.V2) variant first identified in South Africa.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We conducted a randomized multicentre, double blinded controlled trial on safety and efficacy of ChAdOx1-nCoV19 in HIV-uninfected people in South Africa. Participants age 18 to &lt;65 years randomized (1:1) to two doses of vaccine containing 5\u00d710\n                    <jats:sup>10</jats:sup>\n                    viral particles or placebo (0.9%NaCl) 21-35 days apart. Post 2\n                    <jats:sup>nd</jats:sup>\n                    -dose serum samples (n=25) were tested by pseudotyped (PSVNA) and live virus (LVNA) neutralization assays against the D614G and B.1.351 variants. Primary endpoints were safety and vaccine efficacy (VE) &gt;14 days following second dose against laboratory confirmed symptomatic Covid-19.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    2026 HIV-uninfected adults were enrolled between June 24\n                    <jats:sup>th</jats:sup>\n                    and Nov 9\n                    <jats:sup>th</jats:sup>\n                    , 2020; 1010 and 1011 received at least one dose of placebo or vaccine, respectively. Median age was 31 years. The B.1.351 variant showed increased resistance to vaccinee sera using the PSVNA and LVNA. In the primary endpoint analysis, 23/717 (3.2%) placebo and 19/750 (2.5%) vaccine recipients developed mild-moderate Covid-19; VE 21.9% (95%Confidence Interval: \u221249.9; 59.8). Of the primary endpoint cases, 39/42 (92.9%) were the B.1.351 variant; against which VE was 10.4% (95%CI: \u221276.8; 54.8) analyzed as a secondary objective. The incidence of serious adverse events was balanced between the vaccine and placebo groups.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>A two-dose regimen of ChAdOx1-nCoV19 did not show protection against mild-moderate Covid-19 due to B.1.351 variant, however, VE against severe Covid-19 is undetermined.</jats:p>\n                  <jats:p>\n                    (Funded by The Bill &amp; Melinda Gates Foundation and South African Medical Research Council; ClinicalTrails.gov number,\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04444674'>NCT04444674</jats:ext-link>\n                    ).\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.10.21251247","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.11.429193","pub_date":"2021-2-12","title":"Surface-aerosol stability and pathogenicity of diverse MERS-CoV strains from 2012 - 2018","abstract":"Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a coronavirus that infects both humans and dromedary camels and is responsible for an ongoing outbreak of severe respiratory illness in humans in the Middle East. While some mutations found in camel-derived MERS-CoV strains have been characterized, the majority of natural variation found across MERS-CoV isolates remains unstudied. Here we report on the environmental stability, replication kinetics and pathogenicity of several diverse isolates of MERS-CoV as well as SARS-CoV-2 to serve as a basis of comparison with other stability studies. While most of the MERS-CoV isolates exhibited similar stability and pathogenicity in our experiments, the camel derived isolate, C/KSA/13, exhibited reduced surface stability while another camel isolate, C/BF/15, had reduced pathogenicity in a small animal model. These results suggest that while betacoronaviruses may have similar environmental stability profiles, individual variation can influence this phenotype, underscoring the importance of continual, global viral surveillance.","version":"1.1","doi":"10.1101/2021.02.11.429193","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.11.430787","pub_date":"2021-2-11","title":"SARS-CoV-2 variants B.1.351 and B.1.1.248: Escape from therapeutic antibodies and antibodies induced by infection and vaccination","abstract":"The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of UK, South Africa and Brazil variant into human cells is susceptible to blockade by entry inhibitors. In contrast, entry of the South Africa and Brazil variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment and was less efficiently inhibited by serum/plasma from convalescent or BNT162b2 vaccinated individuals. These results suggest that SARS-CoV-2 may escape antibody responses, which has important implications for efforts to contain the pandemic.","version":"1.1","doi":"10.1101/2021.02.11.430787","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.10.430677","pub_date":"2021-2-11","title":"Differential roles of RIG-I-like receptors in SARS-CoV-2 infection","abstract":"The retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are the major viral RNA sensors that are essential for activation of antiviral immune responses. However, their roles in severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) infection are largely unknown. Herein we investigate their functions in human epithelial cells, the primary and initial target of SARS-CoV-2, and the first line of host defense. A deficiency in MDA5 (MDA5\u2212/\u2212), RIG-I or mitochondrial antiviral signaling protein (MAVS) greatly enhanced viral replication. Expression of the type I/III interferons (IFN) was upregulated following infection in wild-type cells, while this upregulation was severely abolished in MDA5\u2212/\u2212 and MAVS\u2212/\u2212, but not in RIG-I\u2212/\u2212 cells. Of note, ACE2 expression was ~2.5 fold higher in RIG-I\u2212/\u2212 than WT cells. These data demonstrate a dominant role of MDA5 in activating the type I/III IFN response to SARS-CoV-2, and an IFN-independent anti-SARS-CoV-2 role of RIG-I.","version":"1.1","doi":"10.1101/2021.02.10.430677","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.10.430696","pub_date":"2021-2-11","title":"Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates","abstract":"The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.","version":"1.1","doi":"10.1101/2021.02.10.430696","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.10.430668","pub_date":"2021-2-11","title":"Data-driven analysis of COVID-19 reveals specific severity patterns distinct from the temporal immune response","abstract":"Key immune signatures of SARS-CoV-2 infection may associate with either adverse immune reactions (severity) or simply an ongoing anti-viral response (temporality); how immune signatures contribute to severe manifestations and/or temporal progression of disease and whether longer disease duration correlates with severity remain unknown. Patient blood was comprehensively immunophenotyped via mass cytometry and multiplex cytokine arrays, leading to the identification of 327 basic subsets that were further stratified into more than 5000 immunotypes and correlated with 28 plasma cytokines. Low-density neutrophil abundance was closely correlated with hepatocyte growth factor levels, which in turn correlated with disease severity. Deep analysis also revealed additional players, namely conventional type 2 dendritic cells, natural killer T cells, plasmablasts and CD16+ monocytes, that can influence COVID-19 severity independent of temporal progression. Herein, we provide interactive network analysis and data visualization tools to facilitate data mining and hypothesis generation for elucidating COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2021.02.10.430668","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.11.430789","pub_date":"2021-2-11","title":"Accelerating COVID-19 research with graph mining and transformer-based learning","abstract":"In 2020, the White House released the, \u201cCall to Action to the Tech Community on New Machine Readable COVID-19 Dataset,\u201d wherein artificial intelligence experts are asked to collect data and develop text mining techniques that can help the science community answer high-priority scientific questions related to COVID-19. The Allen Institute for AI and collaborators announced the availability of a rapidly growing open dataset of publications, the COVID-19 Open Research Dataset (CORD-19). As the pace of research accelerates, biomedical scientists struggle to stay current. To expedite their investigations, scientists leverage hypothesis generation systems, which can automatically inspect published papers to discover novel implicit connections. We present an automated general purpose hypothesis generation systems AGATHA-C and AGATHA-GP for COVID-19 research. The systems are based on graph-mining and the transformer model. The systems are massively validated using retrospective information rediscovery and proactive analysis involving human-in-the-loop expert analysis. Both systems achieve high-quality predictions across domains (in some domains up to 0.97% ROC AUC) in fast computational time and are released to the broad scientific community to accelerate biomedical research. In addition, by performing the domain expert curated study, we show that the systems are able to discover on-going research findings such as the relationship between COVID-19 and oxytocin hormone. All code, details, and pre-trained models are available at https://github.com/IlyaTyagin/AGATHA-C-GP \u2022 Applied computing \u2192 Bioinformatics; Document management and text processing; \u2022 Computing methodologies \u2192 Learning latent representations; Neural networks; Information extraction; Semantic networks.","version":"1.1","doi":"10.1101/2021.02.11.430789","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429917","pub_date":"2021-2-10","title":"Catching SARS-CoV-2 by sequence hybridization: a comparative analysis","abstract":"Controlling and monitoring the still ongoing SARS-CoV-2 pandemic regarding geographical distributions, evolution and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and worldwide sequence data sharing. Efficient sequencing strategies enabling the retrieval of the maximum number of high quality, full-length genomes are hence indispensable. Here, we describe for the first time a combined approach of digital droplet PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were not only able to determine the most sensitive and specific capture panel, but to discriminate their mode of action and number of read pairs needed to recover a high quality full length genome. Thereby, we are providing essential information for all sequencing laboratories worldwide striving for maximizing the sequencing output and simultaneously minimizing time, costs and sequencing resources.","version":"1.2","doi":"10.1101/2021.02.05.429917","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.355784","pub_date":"2021-2-10","title":"Characterization and structural prediction of the putative ORF10 protein in SARS-CoV-2","abstract":"Upstream of the 3\u2019-untranslated region in the SARS-CoV-2 genome is ORF10 which has been proposed to encode for the ORF10 protein. Current research is still unclear on whether this protein is synthesized, but further investigations are still warranted. Herein, this study uses multiple bioinformatic tools to biochemically and functionally characterize the ORF10 protein, along with predicting its tertiary structure. Results indicate a highly ordered, hydrophobic, and thermally stable protein that contains at least one transmembrane region. This protein also possesses high residue protein-binding propensity, primarily in the N-terminal half. An assessment of forty-one missense mutations reveal slight changes in residue flexibility, mainly in the C-terminal half. However, these same mutations do not inflict significant changes on protein stability and other biochemical features. The predicted model suggests the ORF10 protein contains a \u03b2-\u03b1-\u03b2 motif with a \u03b2-molecular recognition feature occurring in the first \u03b2-strand. Functionally, the ORF10 protein could be a membrane protein. A single pocket was identified in this protein but found to possess low druggability. The ORF10 itself consists of two distinct lineages: the SARS-CoV lineage and the SARS-CoV-2 lineage. Evidence of strong positive selection (dN/dS = 4.01) and purifying selection (dN/dS = 0.713) were found within the SARS-CoV-2 lineage and SARS-CoV lineage, respectively. Collectively, these results continue to assess the biological relevance of ORF10 and its putatively encoded protein, thereby aiding in diagnostic and possibly vaccine development.","version":"1.4","doi":"10.1101/2020.10.26.355784","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429627","pub_date":"2021-2-10","title":"Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells","abstract":"Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S \u2013 insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.02.03.429627","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.10.430499","pub_date":"2021-2-10","title":"Bifurcated monocyte states are predictive of mortality in severe COVID-19","abstract":"Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by CCL4 (MIP-1\u03b2) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1\u03b2 and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.","version":"1.1","doi":"10.1101/2021.02.10.430499","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430395","pub_date":"2021-2-10","title":"Mutation bias implicates RNA editing in a wide range of mammalian RNA viruses","abstract":"The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, potentially driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in the more genetically diverse datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 32 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x\u20137.5x). C->U transitions showed a favoured 5\u2019 U upstream context consistent with previous analyses of APOBEC-mediated RNA targeting. Amongst several genomic compositional and structural parameters, the presence of genome scale RNA secondary structure (GORS) was associated with C->U/U->C transition asymmetries (p < 0.001), potentially reflecting the documented structure dependence of APOBEC-mediated RNA editing. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, consistent with extensive homoplasy documented in SARS-CoV-2. Excess C->U substitutions accounted for 15-20% of standing sequence variability of HCV and other RNA viruses; RNA editing may therefore represent a potent driver of RNA virus sequence diversification and longer term evolution. The rapid evolution of RNA viruses is thought to arise from high mutation frequencies during replication and the rapid accumulation of genetic changes over time in response to its changing environments. This study describes an additional potent factor that contributes to the evolution of RNA infecting mammals, the deliberate mutation of the viral genome by host antiviral pathways active within the cell when it becomes infected. This so called \u201cgenome editing\u201d by one or more APOBEC enzymes leads to characteristic C->U mutations that damage the virus\u2019s ability to replicate. While this pathway is well characterised as an antiviral defence against HIV and other retroviruses, this study provides evidence for its activity against a wide range of human and veterinary viruses, including HCV and foot and mouth disease virus. APOBEC-driven mutations accounted for 15-20% of standing sequence variability of RNA virus groups, representing a potent driver of RNA virus sequence diversification.","version":"1.2","doi":"10.1101/2021.02.09.430395","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430269","pub_date":"2021-2-10","title":"Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19","abstract":"The biological determinants of the wide spectrum of COVID-19 clinical manifestations are not fully understood. Here, over 1400 plasma proteins and 2600 single-cell immune features comprising cell phenotype, basal signaling activity, and signaling responses to inflammatory ligands were assessed in peripheral blood from patients with mild, moderate, and severe COVID-19, at the time of diagnosis. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identified and independently validated a multivariate model classifying COVID-19 severity (multi-class AUCtraining = 0.799, p-value = 4.2e-6; multi-class AUCvalidation = 0.773, p-value = 7.7e-6). Features of this high-dimensional model recapitulated recent COVID-19 related observations of immune perturbations, and revealed novel biological signatures of severity, including the mobilization of elements of the renin-angiotensin system and primary hemostasis, as well as dysregulation of JAK/STAT, MAPK/mTOR, and NF-\u03baB immune signaling networks. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for the prevention of COVID-19 progression. Feyaerts et al. demonstrate that an integrated analysis of plasma and single-cell proteomics differentiates COVID-19 severity and reveals severity-specific biological signatures associated with the dysregulation of the JAK/STAT, MAPK/mTOR, and NF-\u03baB immune signaling networks and the mobilization of the renin-angiotensin and hemostasis systems.","version":"1.1","doi":"10.1101/2021.02.09.430269","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.08.429275","pub_date":"2021-2-09","title":"A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration","abstract":"The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing antibodies are a potential therapeutic strategy for treating COVID-19. A number of neutralizing nanobodies (Nbs) were reported for their in vitro activities. However, in vivo protection of these nanobodies was not reported in animal models. In the current report, we characterized several RBD-specific Nbs isolated from a screen of an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibited picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and 15 circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs were engineered. Nb15-NbH-Nb15, a novel trimer constituted of three Nbs, was constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibited sub-ng/ml neutralization potency against the wild-type and currently circulating variants of SARS-CoV-2 with a long half-life in vivo. In addition, we showed that intranasal administration of Nb15-NbH-Nb15 provided 100% protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration. Nb15-NbH-Nb15, with a novel heterotrimeric bispecific configuration, exhibited potent and broad neutralization potency against SARS-CoV-2 in vitro and provided in vivo protection against SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery. We described a novel heterotrimeric configuration of Nb-NbH-Nb (Nb15-NbH-Nb15) that exhibited improved viral inhibition and stability. Nb15-NbH-Nb15 provides ultrahigh neutralization potency against SARS-CoV-2 wild type and 18 mutant variants, including the current circulating variants of D614G and N501Y predominantly in the UK and South Africa. It is the first to demonstrate the Nbs efficacy in preventing and treating SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery.","version":"1.1","doi":"10.1101/2021.02.08.429275","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.351775","pub_date":"2021-2-09","title":"mRNA based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus neutralizing antibodies and mediates protection in rodents","abstract":"mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 (COVID-19) and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle encapsulated mRNA vaccine that encodes full length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus neutralizing antibodies and robust T cell responses. CVnCoV vaccination protected hamsters from challenge with wild type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.10.23.351775","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.367359","pub_date":"2021-2-09","title":"SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice","abstract":"There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel\u00ae were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-\u03b3, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel\u00ae containing formulation and possibly in combination with other immunostimulants.","version":"1.2","doi":"10.1101/2020.11.04.367359","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430349","pub_date":"2021-2-09","title":"A Human 3D neural assembloid model for SARS-CoV-2 infection","abstract":"Clinical evidence suggests the central nervous system (CNS) is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although mechanisms remain unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Prior to infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral \u2018replication hubs\u2019, with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new \u2018assembloid\u2019 model that supports SARS-CoV-2 entry and replication in neural tissue, and PCCOs serve as an experimental model for neural infection.","version":"1.1","doi":"10.1101/2021.02.09.430349","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430458","pub_date":"2021-2-09","title":"Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model","abstract":"Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-\u03b1 starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-\u03b1 starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatments are beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease. The timing of type I interferon treatment is a critical determinant of its efficacy against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.02.09.430458","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430451","pub_date":"2021-2-09","title":"A novel antibody against the furin cleavage site of SARS-CoV-2 spike protein: effects on proteolytic cleavage and ACE2 binding","abstract":"SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.","version":"1.1","doi":"10.1101/2021.02.09.430451","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430517","pub_date":"2021-2-09","title":"Dual RNA-Seq analysis of SARS-CoV-2 correlates specific human transcriptional response pathways directly to viral expression","abstract":"The SARS-CoV-2 pandemic has challenged humankind\u2019s ability to quickly determine the cascade of health effects caused by a novel infection. Even with the unprecedented speed at which vaccines were developed and introduced into society, identifying therapeutic interventions and drug targets for patients infected with the virus remains important as new strains of the virus may evolve, or future coronaviruses may emerge, that are resistant to current vaccines. The application of transcriptomic RNA sequencing of infected samples may shed new light on the pathways involved in viral mechanisms and host responses. We describe the application of \u201cdual RNA-seq\u201d analysis to consider both the host and pathogen transcriptomes simultaneously, to investigate for the first time the co-regulation of human and SARS-CoV-2 genes. Together with differential expression analysis, we describe the tissue specificity of SARS-CoV-2 expression, an inferred lipopolysaccharide response, and co-regulation of CXCL\u2019s, SPRR\u2019s, S100\u2019s with SARS-CoV-2 expression. Lipopolysaccharide response pathways in particular offer promise for future therapeutic research and the prospect of subgrouping patients based on chemokine expression that may help explain the vastly different reactions patients have to infection. Taken together these findings illuminate previously unappreciated SARS-CoV-2 expression signatures, identify new therapeutic considerations, and contribute a pipeline for studying multi-transcriptome systems.","version":"1.1","doi":"10.1101/2021.02.09.430517","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.08.430369","pub_date":"2021-2-09","title":"Exosomes from COVID-19 patients carry tenascin-C and fibrinogen-\u03b2 in triggering inflammatory signals in distant organ cells","abstract":"SARS-CoV-2 infection causes cytokine storm and overshoot immunity in humans; however, it remains to be determined whether genetic material of SARS-CoV-2 and/or virus induced soluble mediators from lung epithelial cells as natural host are carried out by macrophages or other vehicles at distant organs causing tissue damage. We speculated that exosomes as extracellular vesicles are secreted from SARS-CoV-2 infected cells may transport messages to other cells of distant organs leading to pathogenic consequences. For this, we took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and SARS-CoV-2 infected patients. Our results revealed that tenascin-C (TNC) and fibrinogen-\u03b2 (FGB) are highly abundant in exosomes from SARS-CoV-2 infected patient\u2019s plasma as compared to that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via NF-\u03baB pathway, we examined the status of TNF-\u03b1, IL-6 and CCL5 expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase when compared with that from healthy subjects. Together, our results demonstrated that soluble mediators, like TNC and FGB, are transported through plasma exosomes in SARS-CoV-2 infected patients and trigger pro-inflammatory cytokine expression in cells of distant organs in COVID-19 patients. Exosomes play an important role in intercellular communication by inducing physiological changes in recipient cells by transferring bioactive proteins. Little is known about exosomes from SARS-CoV-2 infected cells and their role in pathogenesis. Here, we have carefully examined and analyzed this aspect of SARS-CoV-2 infection. Our results uncovered the potential mechanisms by which SARS-CoV-2 communicates with other cells of distant organs and promotes pathogenesis. We expect to detect whether other factors are modulated in the presence of COVID-19 exosomes. Our exosomes related proteomic experiments prioritize after initial verification to further examine their role in SARS-CoV-2 associated other pathogenic mechanisms to target for therapeutic modalities.","version":"1.1","doi":"10.1101/2021.02.08.430369","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.09.430314","pub_date":"2021-2-09","title":"Disruption of nuclear architecture as a cause of COVID-19 induced anosmia","abstract":"Olfaction relies on a coordinated partnership between odorant flow and neuronal communication. Disruption in our ability to detect odors, or anosmia, has emerged as a hallmark symptom of infection with SARS-CoV-2, yet the mechanism behind this abrupt sensory deficit remains elusive. Here, using molecular evaluation of human olfactory epithelium (OE) from subjects succumbing to COVID-19 and a hamster model of SARS-CoV-2 infection, we discovered widespread downregulation of olfactory receptors (ORs) as well as key components of their signaling pathway. OR downregulation likely represents a non-cell autonomous effect, since SARS-CoV-2 detection in OSNs is extremely rare both in human and hamster OEs. A likely explanation for the reduction of OR transcription is the striking reorganization of nuclear architecture observed in the OSN lineage, which disrupts multi-chromosomal compartments regulating OR expression in humans and hamsters. Our experiments uncover a novel molecular mechanism by which a virus with a very selective tropism can elicit persistent transcriptional changes in cells that evade it, contributing to the severity of COVID-19.","version":"1.1","doi":"10.1101/2021.02.09.430314","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.08.430344","pub_date":"2021-2-09","title":"Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids","abstract":"Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within molecular condensates.","version":"1.1","doi":"10.1101/2021.02.08.430344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426269","pub_date":"2021-2-09","title":"Cryo-EM Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and Two Potent Neutralizing Antibodies","abstract":"The recently reported \u201cUK variant\u201d of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-\u00c5 resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments. The N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.","version":"1.2","doi":"10.1101/2021.01.11.426269","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.07.21251287","pub_date":"2021-02-09","title":"Modelling the impact of reopening schools in the UK in early 2021 in the presence of the alpha variant and with roll-out of vaccination against SARS-CoV-2","abstract":"<jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Following the resurgence of the COVID-19 epidemic in the UK in late 2020 and the emergence of the alpha (also known as B117) variant of the SARS-CoV-2 virus, a third national lockdown was imposed from January 4, 2021. Following the decline of COVID-19 cases over the remainder of January 2021, the question of when and how to reopen schools became an increasingly pressing one in early 2021. This study models the impact of a partial national lockdown with social distancing measures enacted in communities and workplaces under different strategies of reopening schools from March 8, 2021 and compares it to the impact of continual full national lockdown remaining until April 19, 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We used our previously published agent-based model, Covasim, to model the emergence of the alpha variant over September 1, 2020 to January 31, 2021 in presence of Test, Trace and Isolate (TTI) strategies. We extended the model to incorporate the impacts of the roll-out of a two-dose vaccine against COVID-19, with 200,000 daily vaccine doses prioritised by age starting with people 75 years or older, assuming vaccination offers a 95% reduction in disease acquisition risk and a 30% reduction in transmission risk. We used the model, calibrated until January 25, 2021, to simulate the impact of a full national lockdown (FNL) with schools closed until April 19, 2021 versus four different partial national lockdown (PNL) scenarios with different elements of schooling open: 1) staggered PNL with primary schools and exam-entry years (years 11 and 13) returning on March 8, 2021 and the rest of the schools years on March 15, 2020; 2) full-return PNL with both primary and secondary schools returning on March 8, 2021; 3) primary-only PNL with primary schools and exam critical years (years 11 and 13) going back only on March 8, 2021 with the rest of the secondary schools back on April 19, 2021 and 4) part-rota PNL with both primary and secondary schools returning on March 8, 2021 with primary schools remaining open continuously but secondary schools on a two-weekly rota-system with years alternating between a fortnight of face-to-face and remote learning until April 19, 2021. Across all scenarios, we projected the number of new daily cases, cumulative deaths and effective reproduction number R until April 30, 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Our calibration across different scenarios is consistent with alpha variant being around 60% more transmissible than the wild type. We find that strict social distancing measures, i.e. national lockdowns, were essential in containing the spread of the virus and controlling hospitalisations and deaths during January and February 2021. We estimated that a national lockdown over January and February 2021 would reduce the number of cases by early March to levels similar to those seen in October 2020, with R also falling and remaining below 1 over this period. We estimated that infections would start to increase when schools reopened, but found that if other parts of society remain closed, this resurgence would not be sufficient to bring R above 1. Reopening primary schools and exam critical years only or having primary schools open continuously with secondary schools on rotas was estimated to lead to lower increases in cases and R than if all schools opened. Without an increase in vaccination above the levels seen in January and February, we estimate that R could have increased above 1 following the reopening of society, simulated here from April 19, 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Our findings suggest that stringent measures were integral in mitigating the increase in cases and bringing R below 1 over January and February 2021. We found that it was plausible that a PNL with schools partially open from March 8, 2021 and the rest of the society remaining closed until April 19, 2021 would keep R below 1, with some increase evident in infections compared to continual FNL until April 19, 2021. Reopening society in mid-April, without an increase in vaccination levels, could push R above 1 and induce a surge in infections, but the effect of vaccination may be able to control this in future depending on the transmission blocking properties of the vaccines.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.07.21251287","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.09.430424","pub_date":"2021-2-09","title":"Tankyrase-mediated ADP-ribosylation is a novel regulator of TNF-induced death","abstract":"Tumor necrosis factor (TNF) is an inflammatory cytokine that, upon binding to its receptor TNFR1, can drive cytokine production, cell survival, or cell death and is a major component of an organism\u2019s anti-pathogen repetoire. TNF stimulation leads to the formation of two distinct signalling complexes, a well-defined membrane bound complex (complex 1), and a less well characterised cytosolic death inducing complex (complex 2). Using mass spectrometry, we identified the ADP-ribosyltransferase, tankyrase-1 (TNKS1/TNKS/ARTD5/PARP5a) as a novel native complex 2 component. Following a TNF-induced death stimulus TNKS1 is recruited to complex 2, resulting in complex 2 poly(ADP-ribosyl)ation (PARylation). Tankyrase inhibitors sensitise cells to TNF-induced death, which is correlated with increased complex 2 assembly. Tankyrase-mediated PARylation promotes recruitment of the E3 ligase RNF146 and RNF146 deficiency or proteasome inhibition results in increased levels of complex 2, suggesting that RNF146 causes proteasomal degradation of complex 2. Several viruses express ADP-ribose binding macrodomain proteins, and expression of the SARS-CoV-2 or VEEV macrodomain markedly sensitises cells to TNF-induced death. This suggests that ADP-ribosylation serves as yet another mechanism to detect pathogenic interference of TNF signalling and retaliate with an inflammatory cell death.","version":"1.1","doi":"10.1101/2021.02.09.430424","journal":"bioRxiv","score":null},{"id":"10.1101/696724","pub_date":"2021-2-09","title":"Mapping Transcriptomic Vector Fields of Single Cells","abstract":"Single-cell RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these data, however, requires dynamical models capable of predicting cell fate and unveiling the governing regulatory mechanisms. Here, we introduce dynamo, an analytical framework that reconciles intrinsic splicing and labeling kinetics to estimate absolute RNA velocities, reconstructs velocity vector fields that predict future cell fates, and finally employs differential geometry analyses to elucidate the underlying regulatory networks. We applied dynamo to a wide range of disparate biological processes including prediction of future states of differentiating hematopoietic stem cell lineages, deconvolution of glucocorticoid responses from orthogonal cell-cycle progression, characterization of regulatory networks driving zebrafish pigmentation, and identification of possible routes of resistance to SARS-CoV-2 infection. Our work thus represents an important step in going from qualitative, metaphorical conceptualizations of differentiation, as exemplified by Waddington\u2019s epigenetic landscape, to quantitative and predictive theories.","version":"1.2","doi":"10.1101/696724","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.07.429299","pub_date":"2021-2-08","title":"A human antibody with blocking activity to RBD proteins of multiple SARS-CoV-2 variants including B.1.351 showed potent prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus macaques","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), interacts with the host cell receptor angiotensin-converting enzyme 2 (hACE2) via its spike 1 protein for infection. After the virus sequence was published, we identified two potent antibodies against SARS-CoV-2 RBD from antibody libraries using a phage-to-yeast (PtY) display platform in only 10 days. Our lead antibody JMB2002, now in a phase I clinical trial, showed broad-spectrum in vitro blocking activity against hACE2 binding to the RBD of multiple SARS-CoV-2 variants including B.1.351 that was reportedly much more resistant to neutralization by convalescent plasma, vaccine sera and some clinical stage neutralizing antibodies. Furthermore, JMB2002 has demonstrated complete prophylactic and potent therapeutic efficacy in a rhesus macaque disease model. Prophylactic and therapeutic countermeasure intervention of SARS-CoV-2 using JMB2002 would likely slow down the transmission of currently emerged SARS-CoV-2 variants and result in more efficient control of the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2021.02.07.429299","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052217","pub_date":"2021-2-08","title":"SARS-CoV-2 Encodes a PPxY Late Domain Motif Known to Enhance Budding and Spread in Enveloped RNA Viruses","abstract":"The current COVID-19 (Coronavirus Disease-2019) pandemic is affecting the health and/or socioeconomic welfare of almost everyone in the world. Finding vaccines and therapeutics is therefore urgent, but elucidation of the molecular mechanisms that allow some viruses to cross host species boundaries, becoming a threat to human health, must also be given close attention. Here, analysis of all proteins of SARS-CoV-2 revealed a unique PPxY Late (L) domain motif, 25PPAY28, in a spike (S) protein inside a predicted hot disordered loop subject to phosphorylation and binding. PPxY motifs in enveloped RNA viruses are known to recruit Nedd4 E3 ubiquitin ligases and ultimately the ESCRT complex to enhance virus budding and release, resulting in higher viral loads, hence facilitating new infections. Interestingly, proteins of SARS-CoV-1 do not feature PPxY motifs, which could explain why SARS-CoV-2 is more contagious than SARS-CoV-1. Should an experimental assessment of this hypothesis show that the PPxY motif plays the same role in SARS-CoV-2 as it does in other enveloped RNA viruses, this motif will become a promising target for the development of novel host-oriented antiviral therapeutics for preventing S proteins from recruiting Nedd4 E3 ubiquitin ligase partners.","version":"1.3","doi":"10.1101/2020.04.20.052217","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.08.430254","pub_date":"2021-2-08","title":"Neural epidermal growth factor-like 1 protein variant increases survival and modulates the inflammatory and immune responses in human ACE-2 transgenic mice infected with SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19) is a viral illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is a worsening global pandemic. COVID-19 has caused at least 1.7 million deaths worldwide and over 300,000 in the United States. Recently, two promising vaccines are being administered in several countries. However, there remains an urgent need for a therapeutic treatment for COVID-19 patients with severe respiratory damage that can lead to intensive care, prolonged hospitalization, or mortality. Moreover, an increasing population of patients manifest lingering disabling symptoms (called Long Haulers). Here, we tested the efficacy of a recombinant neural epidermal growth factor like 1 protein variant (NELL1-NV1) in a COVID-19 mouse model, transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor (tg-mice hACE2) infected with SARS-CoV-2. The administration of NELL1-NV1 to SARS-CoV-2-infected tg-mice hACE2 significantly improved clinical health score and increased survival. Analyses of bronchoalveolar (BAL) fluid demonstrated decreased levels of several cytokines and chemokines (IFN-\u03b3, IL-10, IL-12 p70, CXCL-10/IP-10, MIG and Rantes), in NV1-treated treated mice compared to controls. Cytokines including IL-1\u03b1, IL-9, IL-6, LIX/CXCL5, KC/CXCL1, MIP-2/CXCL2, MIP-1\u03b1/CCL3, and G-CSF, critical to immune responses such as neutrophil recruitment, viral clearance and vascularization, were increased compared to controls. Our data suggest the potential of NELL1-NV1-based therapy to mitigate the cytokine storm, modulate the abnormal immune response and repair respiratory tissue damage in COVID-19 patients.","version":"1.1","doi":"10.1101/2021.02.08.430254","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.28.428521","pub_date":"2021-2-08","title":"Modeling mutational effects on biochemical phenotypes using convolutional neural networks: application to SARS-CoV-2","abstract":"Biochemical phenotypes are major indexes for protein structure and function characterization. They are determined, at least in part, by the intrinsic physicochemical properties of amino acids and may be reflected in the protein three-dimensional structure. Modeling mutational effects on biochemical phenotypes is a critical step for understanding protein function and disease mechanism as well as enabling drug discovery. Deep Mutational Scanning (DMS) experiments have been performed on SARS-CoV-2\u2019s spike receptor binding domain and the human ACE2 zinc-binding peptidase domain \u2013 both central players in viral infection and evolution and antibody evasion - quantifying how mutations impact binding affinity and protein expression. Here, we modeled biochemical phenotypes from massively parallel assays, using convolutional neural networks trained on protein sequence mutations in the virus and human host. We found that neural networks are significantly predictive of binding affinity, protein expression, and antibody escape, learning complex interactions and higher-order features that are difficult to capture with conventional methods from structural biology. Integrating the intrinsic physicochemical properties of amino acids, including hydrophobicity, solvent-accessible surface area, and long-range non-bonded energy per atom, significantly improved prediction (empirical p<0.01) though there was such a strong dependence on the sequence data alone to yield reasonably good prediction. We observed concordance of the DMS data and our neural network predictions with an independent study on intermolecular interactions from molecular dynamics (multiple 500 ns or 1 \u03bcs all-atom) simulations of the spike protein-ACE2 interface, with critical implications for the use of deep learning to dissect molecular mechanisms. The mutation- or genetically-determined component of a biochemical phenotype estimated from the neural networks has improved causal inference properties relative to the original phenotype and can facilitate crucial insights into disease pathophysiology and therapeutic design.","version":"1.3","doi":"10.1101/2021.01.28.428521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.21251134","pub_date":"2021-02-08","title":"Inhaled budesonide in the treatment of early COVID-19 illness: a randomised controlled trial","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Multiple early hospital cohorts of coronavirus disease 2019 (COVID-19) showed that patients with chronic respiratory disease were significantly under-represented. We hypothesised that the widespread use of inhaled glucocorticoids was responsible for this finding and tested if inhaled glucorticoids would be an effective treatment for early COVID-19 illness.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a randomised, open label trial of inhaled budesonide, compared to usual care, in adults within 7 days of the onset of mild Covid-19 symptoms. The primary end point was COVID-19-related urgent care visit, emergency department assessment or hospitalisation. The trial was stopped early after independent statistical review concluded that study outcome would not change with further participant enrolment.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>146 patients underwent randomisation. For the per protocol population (n=139), the primary outcome occurred in 10 participants and 1 participant in the usual care and budesonide arms respectively (difference in proportion 0.131, p=0.004). The number needed to treat with inhaled budesonide to reduce COVID-19 deterioration was 8. Clinical recovery was 1 day shorter in the budesonide arm compared to the usual care arm (median of 7 days versus 8 days respectively, logrank test p=0.007). Proportion of days with a fever and proportion of participants with at least 1 day of fever was lower in the budesonide arm. Fewer participants randomised to budesonide had persistent symptoms at day 14 and day 28 compared to participants receiving usual care.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Early administration of inhaled budesonide reduced the likelihood of needing urgent medical care and reduced time to recovery following early COVID-19 infection.</jats:p>\n                  <jats:p>\n                    (Funded by Oxford NIHR Biomedical Research Centre and AstraZeneca;\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    number,\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04416399'>NCT04416399</jats:ext-link>\n                    )\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>The majority of interventions studied for the COVID-19 pandemic are focused on hospitalised patients. Widely available and broadly relevant interventions for mild COVID-19 are urgently needed.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>In this open label randomised controlled trial, inhaled budesonide, when given to adults with early COVID-19 illness, reduces the likelihood of requiring urgent care, emergency department consultation or hospitalisation. There was also a quicker resolution of fever, a known poor prognostic marker in COVID-19 and a faster self-reported and questionnaire reported symptom resolution. There were fewer participants with persistent COVID-19 symptoms at 14 and 28 days after budesonide therapy compared to usual care.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>The STOIC trial potentially provides the first easily accessible effective intervention in early COVID-19. By assessing health care resource utilisation, the study provides an exciting option to help with the worldwide pressure on health care systems due to the COVID-19 pandemic. Data from this study also suggests a potentially effective treatment to prevent the long term morbidity from persistent COVID-19 symptoms.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.02.04.21251134","journal":"medRxiv","score":null},{"id":"10.1101/2021.02.04.429815","pub_date":"2021-2-08","title":"Activin A correlates with the worst outcomes in COVID-19 patients, and can be induced by cytokines via the IKK/NF-kappa B pathway","abstract":"A fraction of COVID-19 patients develop the most severe form, characterized by Acute Respiratory Disease Syndrome (ARDS). The molecular mechanisms causing COVID-19-induced ARDS have yet to be defined, though many studies have documented an increase in cytokines known as a \u201ccytokine storm.\u201d Here, we demonstrate that cytokines that activate the NF-kappaB pathway can induce Activin A and its downstream marker, FLRG. In hospitalized COVID-19 patients elevated Activin A/FLRG at baseline were predictive of the most severe longitudinal outcomes of COVID-19, including the need for mechanical ventilation, lack of clinical improvement and all-cause mortality. Patients with Activin A/FLRG above the sample median were 2.6/2.9 times more likely to die, relative to patients with levels below the sample median, respectively. The study indicates high levels of Activin A and FLRG put patients at risk of ARDS, and blockade of Activin A may be beneficial in treating COVID-19 patients experiencing ARDS.","version":"1.1","doi":"10.1101/2021.02.04.429815","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.06.430072","pub_date":"2021-2-07","title":"Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.","version":"1.1","doi":"10.1101/2021.02.06.430072","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.06.430094","pub_date":"2021-2-07","title":"The local topological free energy of the SARS-CoV-2 Spike protein","abstract":"The novel coronavirus SARS-CoV-2 infects human cells using a mechanism that involves binding and structural rearrangement of its spike protein. Understanding protein rearrangement and identifying specific residues where mutations affect protein rearrangement has attracted a lot of attention for drug development. We use a mathematical method introduced in [9] to associate a local topological/geometrical free energy along the SARS-CoV-2 spike protein backbone. Our results show that the total local topological free energy of the SARS-CoV-2 spike protein monotonically decreases from pre-to post-fusion and that its distribution along the protein domains is related to their activity in protein rearrangement. By using density functional theory (DFT) calculations with inclusion of solvent effects, we show that high local topological free energy conformations are unstable compared to those of low topological free energy. By comparing to experimental data, we find that the high local topological free energy conformations in the spike protein are associated with mutations which have the largest experimentally observed effect to protein rearrangement.","version":"1.1","doi":"10.1101/2021.02.06.430094","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429959","pub_date":"2021-2-07","title":"Targeting CTP Synthetase 1 to Restore Interferon Induction and Impede Nucleotide Synthesis in SARS-CoV-2 Infection","abstract":"The newly emerged SARS-CoV-2 caused a global pandemic with astonishing mortality and morbidity. The mechanisms underpinning its highly infectious nature remain poorly understood. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. Screening a SARS-CoV-2 expression library identified ORF7b and ORF8 that suppressed IFN induction via inducing the deamidation of interferon regulatory factor 3 (IRF3). Deamidated IRF3 fails to bind the promoters of classic IRF3-responsible genes, thus muting IFN induction. Conversely, a shRNA-mediated screen focused on cellular glutamine amidotransferases corroborated that CTPS1 deamidates IRF3 to inhibit IFN induction. Functionally, ORF7b and ORF8 activate CTPS1 to promote de novo CTP synthesis while shutting down IFN induction. De novo synthesis of small-molecule inhibitors of CTPS1 enabled CTP depletion and IFN induction in SARS-CoV-2 infection, thus impeding SARS-CoV-2 replication. Our work uncovers a strategy that a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means for developing antiviral therapy that would be resistant to SARS-CoV-2 mutation.","version":"1.1","doi":"10.1101/2021.02.05.429959","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.430022","pub_date":"2021-2-07","title":"Impact of cobas PCR Media Freezing on SARS-CoV-2 Viral RNA Integrity and Whole Genome Sequencing Analyses","abstract":"SARS-CoV-2 whole genome sequencing is an important molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swab samples and shipment to reference laboratories is usually required since RNA sequencing is rarely available in routine clinical microbiology laboratories where initial diagnosis and support to outbreak investigations occur. The cobas PCR Media transport medium developed by Roche facilitates high throughput analyses on cobas multianalyzer PCR platforms. There is no data on the stability of SARS-CoV-2 RNA after freezing and thawing of clinical samples in this transport medium, but potential denaturing of the molecular template could impair test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral whole genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Genomic coverage and sequencing depth did not significantly differ between fresh and frozen samples (n=10). For samples with lower viral inoculum and PCR cycle threshold above 30, sequencing quality scores and detection of single nucleotide polymorphisms did not differ either. Freezing of cobas PCR Media does not negatively affect the quality of SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories. Those results support secondary use of diagnostic nasopharyngeal swab material for viral sequencing without requirement for additional clinical samples.","version":"1.1","doi":"10.1101/2021.02.05.430022","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.06.430088","pub_date":"2021-2-07","title":"Insights on SARS-CoV-2\u2019s Mutations for Evading Human Antibodies: Sacrifice and Survival","abstract":"Recent mutations on the receptor binding domain (RBD) of the SARS-CoV-2\u2019s spike protein have been manifested as the major cause of the wide and rapid spread of the virus. Especially, the variant B.1.351 in South Africa with the hallmark of triple mutations (N501Y, K417N and E484K) is worrisome. Quickly after the outbreak of this new variant, several studies showed that both N501Y and E484K can enhance the binding between RBD and the human ACE2 receptor. However, the mutation K417N seems to be unfavorable because it removes one interfacial salt-bridge. So far, it is still not well understood why the K417N mutation is selected in the viral evolution. Here, we show that despite the loss in the binding affinity (1.48 kcal/mol) between RBD and ACE2 the K417N mutation abolishes a buried interfacial salt-bridge between RBD and the neutralizing antibody CB6 and thus substantially reduces their binding energy by 9.59 kcal/mol, facilitating the variants to efficiently elude CB6 (as well as many other antibodies). Thus, when proliferating from person to person the virus might have adapted to the human immune system through evasive mutations. Taking into account limited and relevant experimental works in the field, we show that our theoretical predictions are consistent with existing experimental findings. By harnessing the revealed molecular mechanism for variants, it becomes feasible to redesign therapeutic antibodies accordingly to make them more efficacious. \n\n","version":"1.1","doi":"10.1101/2021.02.06.430088","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.430003","pub_date":"2021-2-07","title":"Neutralization of viruses with European, South African, and United States SARS-CoV-2 variant spike proteins by convalescent sera and BNT162b2 mRNA vaccine-elicited antibodies","abstract":"The increasing prevalence of SARS-CoV-2 variants with mutations in the spike protein has raised concerns that recovered individuals may not be protected from reinfection and that current vaccines will become less effective. The B.1.1.7 isolate identified in the United Kingdom and B.1.351 isolate identified in the Republic of South Africa encode spike proteins with multiple mutations in the S1 and S2 subunits. In addition, variants have been identified in Columbus, Ohio (COH.20G/677H), Europe (20A.EU2) and in domesticated minks. Analysis by antibody neutralization of pseudotyped viruses showed that convalescent sera from patients infected prior to the emergence of the variant viruses neutralized viruses with the B.1.1.7, B.1.351, COH.20G/677H Columbus Ohio, 20A.EU2 Europe and mink cluster 5 spike proteins with only a minor decrease in titer compared to that of the earlier D614G spike protein. Serum specimens from individuals vaccinated with the BNT162b2 mRNA vaccine neutralized D614G virus with titers that were on average 7-fold greater than convalescent sera. Vaccine elicited antibodies neutralized virus with the B.1.1.7 spike protein with titers similar to D614G virus and neutralized virus with the B.1.351 spike with, on average, a 3-fold reduction in titer (1:500), a titer that was still higher than the average titer with which convalescent sera neutralized D614G (1:139). The reduction in titer was attributable to the E484K mutation in the RBD. The B.1.1.7 and B.1.351 viruses were not more infectious than D614G on ACE2.293T cells in vitro but N501Y, an ACE2 contacting residue present in the B.1.1.7, B.1.351 and COH.20G/677H spike proteins caused higher affinity binding to ACE2, likely contributing to their increased transmissibility. These findings suggest that antibodies elicited by primary infection and by the BNT162b2 mRNA vaccine are likely to maintain protective efficacy against B.1.1.7 and most other variants but that the partial resistance of virus with the B.1.351 spike protein could render some individuals less well protected, supporting a rationale for the development of modified vaccines containing E484K.","version":"1.1","doi":"10.1101/2021.02.05.430003","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.06.430041","pub_date":"2021-2-06","title":"Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection","abstract":"In coronaviridae such as SARS-CoV-2, subgenomic RNAs (sgRNA) are replicative intermediates, therefore, their abundance and structures could infer viral replication activity and severity of host infection. Here, we systematically characterized the sgRNA expression and their structural variation in 81 clinical specimens collected from symptomatic and asymptomatic individuals with a goal of assessing viral genomic signatures of disease severity. We demonstrated the highly coordinated and consistent expression of sgRNAs from individuals with robust infections that results in symptoms, and found their expression is significantly repressed in the asymptomatic infections, indicating that the ratio of sgRNAs to genomic RNA (sgRNA/gRNA) is highly correlated with the severity of the disease. Using long read sequencing technologies to characterize full-length sgRNA structures, we also observed widespread deletions in viral RNAs, and identified unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Furthermore, based on the sgRNA structures, the frequently occurred structural variants in SARS-CoV-2 genomes serves as a mechanism to further induce SARS-CoV-2 proteome complexity. Taken together, our results show that differential sgRNA expression and structural mutational burden both appear to be correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.","version":"1.1","doi":"10.1101/2021.02.06.430041","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429860","pub_date":"2021-2-06","title":"Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat RBD of SARS-CoV-2 spike protein protects mice against COVID-19","abstract":"A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus vectored vaccines expressing spike (S) protein have advanced into phase 3 trials, with three approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD) or dimeric tandem-repeat RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular (Th1-based) immune responses. AdC7-RBD-tr2 showed higher antibody responses compared with both AdC7-S and AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.","version":"1.1","doi":"10.1101/2021.02.05.429860","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429891","pub_date":"2021-2-06","title":"CD8+ T cell immunogenicity induced by endogenous EVs engineered by antigens fused to a truncated Nefmut EV-anchoring protein","abstract":"Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicits a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective to a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. Furthermore, the EV-anchoring function was preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. By analyzing the immune responses induced after intramuscular injection of DNA vectors expressing fusion products based on the four viral antigens, we found that the Nefmut C-terminal deletion did not impact on the levels of antigen \u2013specific CD8+ T lymphocytes as evaluated by IFN-\u03b3 EliSpot analysis and intracellular cytokine staining. In addition, immune responses at distal sites remained unaffected, as indicated by the similar percentages of SARS-CoV-2 S1- and S2-specific CD8+ T cells detected in spleens and lung airways of mice injected with DNA vectors expressing the viral antigens fused with either Nefmut or NefmutPL. We concluded that the C-terminal Nefmut truncation does not affect stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, NefmutPL represents a safer alternative to full-length Nefmut for the design of CD8+ T cell vaccines for humans.","version":"1.1","doi":"10.1101/2021.02.05.429891","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429901","pub_date":"2021-2-06","title":"Active microrheology using pulsed optical tweezers to probe viscoelasticity of Lamin A towards diagnosis of laminopathies","abstract":"Lamins are nucleoskeletal proteins of mammalian cells that stabilize the structure and maintain the rigidity of the nucleus. These type V intermediate filament proteins which are predominantly of A and B types provide necessary tensile strength to the nucleus. Single amino acid missense mutations occurring all over the lamin A protein form a cluster of human diseases termed as laminopathies, a few of which principally affect the muscle and cardiac tissues responsible for load bearing functionalities of the body. One such mutation is lamin A350P which causes dilated cardiomyopathy in patients. It is likely that a change from alanine to proline in the \u03b1-helical 2B rod domain of the protein might severely disrupt the propensity of the filaments to polymerise into functional higher order structures required to form a fully functional lamina with its characteristic elasticity. In this study, we validate for the very first time, the application of active microrheology employing oscillating optical tweezers to investigate any alterations in the visco-elastic parameters of the mutant protein meshwork in vitro, which might translate into possible changes in nuclear plasticity. We confirm our findings from this robust yet fast method by imaging both the wild type and mutant lamins using a super resolution microscope, and observe changes in the mesh size which explain our measured changes in the viscoelastic parameters of the lamins. This method could naturally be extended to conduct microrheological measurements on any intermediate filament protein or any protein endowed with elastic behavior, with minor schematic modifications, thus bearing significant implications in laminopathies and other diseases which are associated with changes in structural rigidity of any cellular organelle. Lamin A mutations produce an array of diseases termed as laminopathies which are primarily characterized by alteration of elastic behavior of the nucleus which in turn leads to defects in mechanotransduction. This is the first report in the lamin arena which shows a fast, accurate and direct quantification of elastic moduli of lamin A using optical tweezers-based microrheology. This has very significant implications and can be registered to be a robust and universal method that could also be suitably used for probing changes in elastic properties of any proteins or surfactants in a disease scenario such as SARS-Cov2 (Covid-19), which is pandemic at this time.","version":"1.1","doi":"10.1101/2021.02.05.429901","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429566","pub_date":"2021-2-05","title":"Serological Profile Of Specific Antibodies Against Dominant Antigens Of SARS-CoV-2 In Chilean COVID-19 Patients.","abstract":"Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has been a pandemic since March 2020. Currently, the virus has infected more than 50 million people worldwide and more than half a million in Chile. For many coronaviruses, Spike (S) and Nucleocapsid (N) proteins are described as major antigenic molecules, inducing seroconversion and production of neutralizing antibodies. In this work, we evaluated the presence in serum of IgM, IgA and IgG antibodies against N and S proteins of SARS-CoV-2 using western blot, and developed an ELISA test for the qualitative characterization of COVID-19 patients. Patients with an active infection or who have recovered from COVID-19 showed specific immunoblotting patterns for the recombinants S protein and its domains S1 and S2, as well as for the N protein of SARS-CoV-2. Anti-N antibodies were more frequently detected than anti-S or anti-S1-RBD antibodies. People who were never exposed to SARS-CoV-2 did not show reactivity. Finally, indirect ELISA assays using N and S1-RBD proteins, alone or in combination, were established with variable sensitivity and specificity depending on the antigen bound to the solid phase. Overall, Spike showed higher specificity than the nucleocapsid, and comparable sensitivity for both antigens. Both approaches confirmed the seroconversion after infection and allowed us to implement the analysis of antibodies in blood for research purposes in a local facility.","version":"1.1","doi":"10.1101/2021.02.05.429566","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.16.425365","pub_date":"2021-2-05","title":"SARS-CoV-2 RECoVERY: a multi-platform open-source bioinformatic pipeline for the automatic construction and analysis of SARS-CoV-2 genomes from NGS sequencing data","abstract":"Since its first appearance in December 2019, the novel Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), spread worldwide causing an increasing number of cases and deaths (35,537,491 and 1,042,798, respectively at the time of writing, https://covid19.who.int). Similarly, the number of complete viral genome sequences produced by Next Generation Sequencing (NGS), increased exponentially. NGS enables a rapid accumulation of a large number of sequences. However, bioinformatics analyses are critical and require combined approaches for data analysis, which can be challenging for non-bioinformaticians. A user-friendly and sequencing platform-independent bioinformatics pipeline, named SARS-CoV-2 RECoVERY (REconstruction of CoronaVirus gEnomes & Rapid analYsis) has been developed to build SARS-CoV-2 complete genomes from raw sequencing reads and to investigate variants. The genomes built by SARS-CoV-2 RECoVERY were compared with those obtained using other software available and revealed comparable or better performances of SARS\u2013CoV2 RECoVERY. Depending on the number of reads, the complete genome reconstruction and variants analysis can be achieved in less than one hour. The pipeline was implemented in the multi-usage open-source Galaxy platform allowing an easy access to the software and providing computational and storage resources to the community. SARS-CoV-2 RECoVERY is a piece of software destined to the scientific community working on SARS-CoV-2 phylogeny and molecular characterisation, providing a performant tool for the complete reconstruction and variants\u2019 analysis of the viral genome. Additionally, the simple software interface and the ability to use it through a Galaxy instance without the need to implement computing and storage infrastructures, make SARS-CoV-2 RECoVERY a resource also for virologists with little or no bioinformatics skills. The pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) is implemented in the Galaxy instance ARIES (https://aries.iss.it).","version":"1.2","doi":"10.1101/2021.01.16.425365","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.428650","pub_date":"2021-2-05","title":"SARS-CoV-2 ORF7b: is a bat virus protein homologue a major cause of COVID-19 symptoms?","abstract":"ORF7b is an accessory protein of SARS-CoV-2, the virus behind the COVID-19 pandemic. Using cell-free synthesized ORF7b, we experimentally show that ORF7b assembles into stable multimers. The ORF7b sequence shows a transmembrane segment, which multimerizes through a leucine zipper. We hypothesize that ORF7b has the potential to interfere with important cellular processes that involve leucine-zipper formation, and present two particularly striking examples. First, leucine zippers are central in heart rhythm regulation through multimerization of phospholamban in cardiomyocytes. Second, epithelial cell-cell adhesion relies on E-cadherins, which dimerize using a transmembrane leucine zipper. Most common symptoms of SARS-CoV-2 infection, including heart arrythmias, odor loss, impaired oxygen uptake and intestinal problems, up to multiorgan failure, can be rationalized by a possible interference of ORF7b with the functions of these proteins. We ask whether this is pure coincidence, or whether our observations point to disruption by ORF7b of vital processes in COVID-19.","version":"1.1","doi":"10.1101/2021.02.05.428650","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.24.113423","pub_date":"2021-2-05","title":"A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point-of-care","abstract":"COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS-CoV-2, COVID-19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 detection based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS-CoV-2 genomic-RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies/reaction. The results can be read with the naked eye within 45 minutes, without cross-reactivity to closely related coronaviruses. Moreover, the direct detection of SARS-CoV-2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready-to-use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS-CoV-2 nucleic acids at point-of-care.","version":"1.2","doi":"10.1101/2020.05.24.113423","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428808","pub_date":"2021-2-05","title":"The evolutionary making of SARS-CoV-2","abstract":"A mechanistic understanding of how SARS-CoV-2 (sarbecovirus, betacoronavirus) infects human cells is emerging, but the evolutionary trajectory that gave rise to this pathogen is poorly understood. Here we scan SARS-CoV-2 protein sequences in-silico for innovations along the evolutionary lineage starting with the last common ancestor of coronaviruses. SARS-CoV-2 substantially differs from viruses outside sarbecovirus both in its set of encoded proteins and in their domain architectures, indicating divergent functional demands. Within sarbecoviruses, sub-domain level profiling using predicted linear epitopes reveals how the primary interface between host cell and virus, the spike, was gradually reshaped. The only epitope that is private to SARS-CoV-2 overlaps with the furin cleavage site, a \u201cswitch\u201d that modulates spike\u2019s conformational landscape in response to host-cell interaction. This cleavage site has fundamental relevance for both immune evasion and cell infection, and the apparently ongoing evolutionary fine-tuning of its use by SARS-CoV-2 should be monitored.","version":"1.2","doi":"10.1101/2021.01.29.428808","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429937","pub_date":"2021-2-05","title":"Post-infection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and was proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 hr post infection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.05.429937","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271684","pub_date":"2021-2-05","title":"Tropism of SARS-CoV-2 in commonly used laboratory cell lines and their proteomic landscape during infection","abstract":"The present pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is driving intense research activities to understand the basic biology of the virus and determine effective therapeutic strategies. The commonly used laboratory cell lines of human origin are the first line of experimental models to study the pathogenicity and performing antiviral assays. Thus, to find suitable cell models to study SARS-CoV-2, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different cell lines of human origin and compared their growth characteristics to other globally isolated strains. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high to moderate level of susceptibility to the majority of virus isolates. In Caco2 cells the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein expression profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3 and Huh7 cells. Overall, cell-type specific variability was noted for cytopathogenicity, susceptibility and cellular response to SARS-CoV-2. This study provides important clues regarding SARS-CoV-2 pathogenesis and can represent as a guide for future studies to design therapeutics.","version":"1.3","doi":"10.1101/2020.08.28.271684","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429982","pub_date":"2021-2-05","title":"Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation","abstract":"The SARS-CoV-2 coronavirus responsible for the global pandemic contains a unique furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation. Here, we show that O-glycosylation near the furin cleavage site is mediated by specific members of the GALNT enzyme family and is dependent on the novel proline at position 681 (P681). We further demonstrate that O-glycosylation of S decreases furin cleavage. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the potential role of P681 mutations in the recently identified, highly transmissible B.1.1.7 variant.","version":"1.1","doi":"10.1101/2021.02.05.429982","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.05.429759","pub_date":"2021-2-05","title":"Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 in nonhuman primates following NVX-CoV2373 subunit vaccine with Matrix-M\u2122 vaccination","abstract":"Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M\u2122. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants. NVX-CoV2373 subunit vaccine elicits receptor blocking, virus neutralizing antibodies, and Fc-effector functional antibodies. The vaccine protects against respiratory tract infection and virus shedding in non-human primates (NHPs). Both neutralizing and Fc-effector functions contribute to protection, potentially through different mechanisms in the upper and lower respiratory tract. Both macaque and human vaccine-induced antibodies exhibit altered Fc-receptor binding to emerging mutants.","version":"1.1","doi":"10.1101/2021.02.05.429759","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.21251108","pub_date":"2021-02-05","title":"To what extent do we need to rely on non-pharmaceutical interventions while COVID-19 vaccines roll out in 2021?","abstract":"<jats:title>\n                  <jats:bold>Abstract</jats:bold>\n                </jats:title>\n                <jats:p>\n                  COVID-19 vaccination is being conducted in over 190 countries/regions to control SARS-CoV-2 transmission and return to a pre-pandemic lifestyle. However, understanding when non-pharmaceutical interventions (NPIs) can be lifted as immunity builds up remain a key question for policy makers. To address it, we built a data-driven model of SARS-CoV-2 transmission for China. We estimated that to prevent the escalation of local outbreaks to widespread epidemics, stringent NPIs need to remain in place at least one year after the start of vaccination. Should NPIs alone be capable to keep the reproduction number (R\n                  <jats:sub>t</jats:sub>\n                  ) around 1.3, the synergetic effect of NPIs and vaccination could reduce up to 99% of COVID-19 burden and bring R\n                  <jats:sub>t</jats:sub>\n                  below the epidemic threshold in about 9 months. Maintaining strict NPIs throughout 2021 is of paramount importance to reduce COVID-19 burden while vaccines are distributed to the population, especially in large populations with little natural immunity.\n                </jats:p>","version":null,"doi":"10.1101/2021.02.03.21251108","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.22.111294","pub_date":"2021-2-05","title":"Preprinting the COVID-19 pandemic","abstract":"The world continues to face a life-threatening viral pandemic. The virus underlying the COVID-19 disease, SARS-CoV-2, has caused over 98 million confirmed cases and 2.2 million deaths since January 2020. Although the most recent respiratory viral pandemic swept the globe only a decade ago, the way science operates and responds to current events has experienced a paradigm shift in the interim. The scientific community has responded rapidly to the COVID-19 pandemic, releasing over 125,000 COVID-19 related scientific articles within 10 months of the first confirmed case, of which more than 30,000 were hosted by preprint servers. We focused our analysis on bioRxiv and medRxiv, two growing preprint servers for biomedical research, investigating the attributes of COVID-19 preprints, their access and usage rates, as well as characteristics of their propagation on online platforms. Our data provides evidence for increased scientific and public engagement with preprints related to COVID-19 (COVID-19 preprints are accessed more, cited more, and shared more on various online platforms than non-COVID-19 preprints), as well as changes in the use of preprints by journalists and policymakers. We also find evidence for changes in preprinting and publishing behaviour: COVID-19 preprints are shorter and reviewed faster. Our results highlight the unprecedented role of preprints and preprint servers in the dissemination of COVID-19 science, and the impact of the pandemic on the scientific communication landscape.","version":"1.3","doi":"10.1101/2020.05.22.111294","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429628","pub_date":"2021-2-05","title":"Development of a highly sensitive bioanalytical assay for the quantification of favipiravir","abstract":"Favipiravir (FAV; T-705) has been approved for use as an anti-influenza therapeutic and has reports against a wide range of viruses (e.g., Ebola virus, rabies and norovirus). Most recently FAV has been reported to demonstrate activity against SARS-CoV-2. Repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Pre-clinical and clinical investigations of FAV require robust, reproducible and sensitive bioanalytical assay. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 0.78-200 ng/mL Accuracy and precision ranged between 89% and 110%, 101% and 106%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of FAV against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.03.429628","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429738","pub_date":"2021-2-04","title":"Type-I interferon signatures in SARS-CoV-2 infected Huh7 cells","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-\u03b2 production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon stimulated genes and their role in inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.","version":"1.1","doi":"10.1101/2021.02.04.429738","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429670","pub_date":"2021-2-04","title":"The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia","abstract":"The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the devastating ongoing coronavirus disease-2019 (COVID-19) pandemic which poses a great threat to global public health. The spike (S) polypeptide of SARS-CoV-2 consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary. The inclusion of the 4 amino acids (PRRA) at the S1/S2 boundary forms a furin cleavage site (FCS), 682RRAR\u2193S686, distinguishing SARS-CoV-2 from its closest relative, the SARS-CoV. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, the virus acquired various deletions surrounding the FCS. In the present study, we studied the viral transcriptome in SARS-CoV-2 infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability at the S1/S2 boundary using RNA-seq. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the S1/S2 boundary of the S gene: one is a deletion of 12 amino acids, 678TNSPRRAR\u2193SVAS689, which contains the FCS, another is a deletion of 5 amino acids, 675QTQTN679, which is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions revealed that the selective pressure for the FCS maintains the S gene stability in HAE-ALI but with exceptions, in which the FCS deletions are remained at a high rate. Thus, our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is donor-dependent.","version":"1.1","doi":"10.1101/2021.02.03.429670","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.427991","pub_date":"2021-2-04","title":"Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant","abstract":"Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.","version":"1.2","doi":"10.1101/2021.01.24.427991","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.31.363176","pub_date":"2021-2-04","title":"A model for pH coupling of the SARS-CoV-2 spike protein open/closed equilibrium","abstract":"SARS-CoV-2, causative agent of the COVID-19 pandemic, is thought to release its RNA genome at either the cell surface or within endosomes, the balance being dependent on spike protein stability, and the complement of receptors, co-receptors and proteases. To investigate possible mediators of pH-dependence, pKa calculations have been made on a set of structures for spike protein ectodomain and fragments from SARS-CoV-2 and other coronaviruses. Dominating a heat map of the aggregated predictions, 3 histidine residues in S2 are consistently predicted as destabilising in pre-fusion (all 3) and post-fusion (2 of 3) structures. Other predicted features include the more moderate energetics of surface salt-bridge interactions, and sidechain-mainchain interactions. Two aspartic acid residues in partially buried salt-bridges (D290 \u2013 R273 and R355 \u2013 D398) have pKas that are calculated to be elevated and destabilising in more open forms of the spike trimer. These aspartic acids are most stabilised in a tightly closed conformation that has been observed when linoleic acid is bound, and which also affects the interactions of D614. The D614G mutation is known to modulate the balance of closed to open trimer. It is suggested that D398 in particular contributes to a pH-dependence of the open/closed equilibrium, potentially coupled to the effects of linoleic acid binding and D614G mutation, and possibly also A570D mutation. These observations are discussed in the context of SARS-CoV-2 infection, mutagenesis studies, and other human coronaviruses.","version":"1.2","doi":"10.1101/2020.10.31.363176","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429819","pub_date":"2021-2-04","title":"Codon arrangement modulates MHC-I peptides presentation: implications for a SARS-CoV-2 peptide-based vaccine","abstract":"Among various vaccination strategies, peptide-based vaccines appear as excellent candidates because they are cheap to produce, are highly stable and harbor low toxicity. However, predicting which MHC-I Associated Peptide (MAP) will ultimately reach cell surface remains challenging, due to high false discovery rates. Previously, we demonstrated that synonymous codon arrangement (usage and placement) is predictive of, and modulates MAP presentation. Here, we apply CAMAP (Codon Arrangement MAP Predictor), the artificial neural network we used to unveil the role of codon arrangement in MAP presentation, to predict SARS-CoV MAPs. We report that experimentally identified SARS-CoV-1 and SARS-CoV-2 MAPs are associated with significantly higher CAMAP scores. Based on CAMAP scores and binding affinity, we identified 48 non-overlapping MAP candidates for a peptide-based vaccine, ensuring coverage for a high proportion of HLA haplotypes in the US population (>78%) and SARS-CoV-2 strains (detected in >98% of SARS-CoV-2 strains present in the GISAID database). Finally, we built an interactive web portal (https://www.epitopes.world) where researchers can freely explore CAMAP predictions for SARS-CoV-1/2 viruses. Collectively, we present an analysis framework that can be generalizable to empower the rapid identification of virus-specific MAPs, including in the context of an emergent virus, to help accelerate target identification for peptide-based vaccine designs that could be critical in safely attaining group immunity in the context of a global pandemic.","version":"1.1","doi":"10.1101/2021.02.04.429819","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429761","pub_date":"2021-2-04","title":"Bronchoalveolar lavage affects thorax computed tomography of healthy and SARS-CoV-2 infected rhesus macaques (Macaca mulatta)","abstract":"Medical imaging as method to assess the longitudinal process of a SARS-CoV-2 infection in non-human primates is commonly used in research settings. Bronchoalveolar lavage (BAL) is also regularly used to determine the local virus production and immune effects of SARS-CoV-2 in the lower respiratory tract. However, the potential interference of those two diagnostic modalities with each other is unknown in non-human primates. The current study investigated the effect and duration of BAL on computed tomography (CT) in both healthy and experimentally SARS-CoV-2-infected female rhesus macaques (Macaca mulatta). In addition, the effect of subsequent BALs was reviewed. Thorax CTs and BALs were obtained from four healthy animals and 11 experimentally SARS-CoV-2-infected animals. From all animals, CTs were obtained just before BAL, and 24 hours post-BAL. Additionally, from the healthy animals, CTs immediately after and four hours post-BAL were obtained. Thorax CTs were evaluated for alterations in lung density, measured in Hounsfield units, and a visual semi-quantitative scoring system. An increase in the lung density was observed on the immediately post-BAL CT but resolved within 24 hours in the healthy animals. In the infected animals, a significant difference in both the lung density and CT score was still found 24 hours after BAL. Furthermore, the differences between timepoints in CT score were increased for the second BAL. These results indicate that the effect of BAL on infected lungs is not completed within the first 24 hours. Therefore, it is of importance to acknowledge the interference between BAL and CT in rhesus macaques.","version":"1.1","doi":"10.1101/2021.02.04.429761","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429769","pub_date":"2021-2-04","title":"Modulation of SARS-CoV-2 Spike-induced Unfolded Protein Response (UPR) in HEK293T cells by selected small chemical molecules","abstract":"Coronaviruses (CoV) exploits the endoplasmic reticulum (ER) of the host cells for replication and in doing so, increases ER stress. evokes Unfolded Protein Response (UPR) and possibly autophagy, which could all attribute to the pathophysiology of the viral infections. To date, little is known about the roles of ER stress, UPR, and autophagy in SARS-CoV-2 infection. Here we over-expressed the viral Spike (S) protein in cultured HEK293T cells, as it has been shown that such protein is largely responsible for UPR activation in other CoV-infected cells. We noticed, in the transfected cells, heightened ER stress, activation of the PERK-eIF2\u03b1 arm of the UPR, induction of autophagy and cell death. When we treated the transfected cells with Tauroursodeoxycholic acid (TUDCA), 4-phenyl butyric acid (PBA), Salubrinal, Trazadone hydrochloride, and Dibenzoylmethane (DBM), we saw reduced the BiP/GRP78 levels, but only PBA and TUDCA could significantly diminish the levels of peIF2\u03b1 and autophagy expression.","version":"1.1","doi":"10.1101/2021.02.04.429769","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429711","pub_date":"2021-2-04","title":"Follow-up of a hospital cohort during the first 3,530 suspected cases of COVID-19 in Sao Jose do Rio Preto, Sao Paulo, Brazil","abstract":"In a global context, COVID-19 is the most significant health threat in the present days, evidenced by the fact that, in just over four months, SARS-CoV-2 has spread to 171 countries, reaching a Pandemic status. Most patients with COVID-19 have a mild course of the disease. However, approximately 20% develop severe illness with a high mortality rate which is associated with age, comorbidities, and immunosuppression. Epidemiological studies are used to reveal the extent of viral spread in homes, communities, and hospitals. Thus, preventive and control measures can be established by the authorities. In this study, patients with suspect COVID-19 symptoms who search for hospital care at the city of Sao Jos\u00e9 do Rio Preto (Sao Paulo, Brazil) were monitored, in order to identify the first case of this new disease in the region. In the first two months (March and April), more than 3000 individuals looked for the public and private health system with suspected respiratory symptoms, but only 164 (8.4%) were COVID-19 confirmed. From those, males (56.1%) and patients of the age distribution of 16-59 (91.2%), with diarrhea (22.2%), runny nose (25%), altered taste (15.9%), and anosmia (11.6%) presented statistical significance, although none comorbidities were related with COVID-19 occurrence. The odds ratio analysis supports this finding. Days of onset of symptoms are positively associated with whit viral load, and the same happens with the occurrence of symptoms (dyspnea and low saturation).","version":"1.1","doi":"10.1101/2021.02.04.429711","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.04.429751","pub_date":"2021-2-04","title":"Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2","abstract":"Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.","version":"1.1","doi":"10.1101/2021.02.04.429751","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429486","pub_date":"2021-2-03","title":"The mutation profile of SARS-CoV-2 is primarily shaped by the host antiviral defense","abstract":"Understanding SARS-CoV-2 evolution is a fundamental effort in coping with the COVID-19 pandemic. The virus genomes have been broadly evolving due to the high number of infected hosts world-wide. Mutagenesis and selection are the two inter-dependent mechanisms of virus diversification. However, which mechanisms contribute to the mutation profiles of SARS-CoV-2 remain under-explored. Here, we delineate the contribution of mutagenesis and selection to the genome diversity of SARS-CoV-2 isolates. We generated a comprehensive phylogenetic tree with representative genomes. Instead of counting mutations relative to the reference genome, we identified each mutation event at the nodes of the phylogenetic tree. With this approach, we obtained the mutation events that are independent of each other and generated the mutation profile of SARS-CoV-2 genomes. The results suggest that the heterogeneous mutation patterns are mainly reflections of host (i) antiviral mechanisms that are achieved through APOBEC, ADAR, and ZAP proteins and (ii) probable adaptation against reactive oxygen species. SARS-CoV-2 genomes are evolving worldwide. Revealing the evolutionary characteristics of SARS-CoV-2 is essential to understand host-virus interactions. Here, we aim to understand whether mutagenesis or selection is the primary driver of SARS-CoV-2 evolution. This study provides an unbiased computational method for profiling and analyzing independently occurring SARS-CoV-2 mutations. The results point out three host antiviral mechanisms shaping the mutational profile of SARS-CoV-2 through APOBEC, ADAR, and ZAP proteins. Besides, reactive oxygen species might have an impact on the SARS-CoV-2 mutagenesis.","version":"1.1","doi":"10.1101/2021.02.02.429486","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429351","pub_date":"2021-2-03","title":"Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection","abstract":"The immune system of most SARS-CoV-2 infected individuals limits viral spread to the upper airways without pulmonary involvement. This prevents the development of pneumonic COVID-19. However, the protective immunological responses causative of successful viral containment in the upper airways remain unclear. Here, we combine longitudinal single-cell RNA sequencing, proteomic profiling, multidimensional flow cytometry, RNA-Seq of FACS-sorted leukocyte subsets and multiplex plasma interferon profiling to uncover temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients. We compare host responses in a high-risk patient population infected with SARS-CoV-2 but without pulmonary involvement to patients with COVID-19 pneumonia. Our data reveal a distinct immunological signature of successful viral containment, characterized by an early prominent interferon stimulated gene (ISG) upregulation across immune cell subsets. In addition, reduced cytotoxic potential of Natural Killer (NK) and T cells, as well as a monocyte phenotype with immune-modulatory potential are hallmarks of protective immunity. Temporal resolution across disease trajectories highlights ISG upregulation as particularly prominent early in the disease and confirms increased expression also in comparison to healthy controls. We validate this distinct temporal ISG signature by in-depth RNA-seq of FACS-sorted leukocyte subsets in a large prospective ambulatory SARS-CoV-2 infected cohort confirming early and robust ISG upregulation particularly in monocytes and T cells. In conclusion, our data demonstrate a protective ISG phenotype in patients with successful containment of SARS-CoV-2 infection without progression to COVID-19. This early protective interferon response might be exploited as a therapeutic approach and for disease course prediction.","version":"1.1","doi":"10.1101/2021.02.03.429351","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429311","pub_date":"2021-2-03","title":"Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain","abstract":"Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL (\u223c0.043 nM) and 3.18 ng/mL (\u223c0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. To date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.","version":"1.1","doi":"10.1101/2021.02.02.429311","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429601","pub_date":"2021-2-03","title":"Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.","version":"1.1","doi":"10.1101/2021.02.03.429601","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429555","pub_date":"2021-2-03","title":"In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human ACE2 complex, viral entry and cell fusion","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a novel computational model of the SARS-CoV-2 S/ACE2 complex as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context, and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.","version":"1.1","doi":"10.1101/2021.02.03.429555","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429510","pub_date":"2021-2-03","title":"Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2","abstract":"Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness as well as DFT properties. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, the proposed kinase inhibitors and a few of the predicted nucleotidyl transferase inhibitors significantly inhibited the aforementioned enzymatic activity. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.03.429510","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429625","pub_date":"2021-2-03","title":"A high-throughput radioactivity-based assay for screening SARS-CoV-2 nsp10-nsp16 complex","abstract":"Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current SARS-CoV-2 pandemic, necessitate the development of therapeutics that could be easily and effectively administered world-wide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA-capping through its 2\u2019-O-methylation activity. Like other methyltransferases, SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for methyltransferase activity of nsp10-nsp16 complex in a 384-well format, and kinetic characterization, and optimization of the assay for HTS (Z\u2032-factor: 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.","version":"1.1","doi":"10.1101/2021.02.03.429625","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429540","pub_date":"2021-2-03","title":"Variation in predicted COVID-19 risk among lemurs and lorises","abstract":"The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 1.5 million fatalities since it first emerged in late 2019. As we write, infection rates are currently at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary viral target is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predicts that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results and finds additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and Endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.03.429540","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429211","pub_date":"2021-2-03","title":"Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters","abstract":"Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.02.03.429211","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429355","pub_date":"2021-2-03","title":"Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike","abstract":"The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ~45% showed neutralizing activity of which ~20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.","version":"1.1","doi":"10.1101/2021.02.03.429355","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429480","pub_date":"2021-2-03","title":"Assessment of immunogenicity and protective efficacy of ZyCoV-D DNA vaccine candidates in Rhesus macaques against SARS-CoV-2 infection","abstract":"Vaccines remain the key protective measure to achieve herd immunity to control the disease burden and stop COVID-19 pandemic. We have developed and assessed the immunogenicity and protective efficacy of two formulations (1mg and 2mg) of ZyCoV-D (a plasmid DNA based vaccine candidates) administered through Needle Free Injection System (NFIS) and syringe-needle (intradermal) in rhesus macaques with three dose vaccine regimens. The vaccine candidate 2mg dose administered using Needle Free Injection System (NFIS) elicited a significant immune response with development of SARS-CoV-2 S1 spike region specific IgG and neutralizing antibody (NAb) titers during the immunization phase and significant enhancement in the levels after the virus challenge. In 2 mg NFIS group the IgG and NAb titers were maintained and showed gradual rise during the immunization period (15 weeks) and till 2 weeks after the virus challenge. It also conferred better protection to macaques evident by the viral clearance from nasal swab, throat swab and bronchoalveolar lavage fluid specimens in comparison with macaques from other immunized groups. In contrast, the animals from placebo group developed high levels of viremia and lung disease following the virus challenge. Besides this, the vaccine candidate also induced increase lymphocyte proliferation and cytokines response (IL-6, IL-5).The administration of the vaccine candidate with NFIS generated a better immunogenicity response in comparison to syringe-needle (intradermal route). The study demonstrated immunogenicity and protective efficacy of the vaccine candidate, ZyCoV-D in rhesus macaques.","version":"1.1","doi":"10.1101/2021.02.02.429480","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.357418","pub_date":"2021-2-03","title":"High-throughput fluorescent assay for inhibitor screening of proteases from RNA viruses","abstract":"Spanish flu and other influenza outbreaks, the recent Zika epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases that are caused by RNA viruses. Perhaps less well known yet dangerous RNA viruses cause deadly diseases such as polio, Ebola, measles, rubella, yellow fever, dengue fever and many others. To combat a particular viral disease by diminishing its spread and number of fatal cases, effective vaccines and antivirals are indispensable. Therefore, quick access to the means of discovery of new treatments for any epidemic outbreak is of great interest and in vitro biochemical assays are the basis of drug discovery. The recent outbreak of the coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands an affordable and reliable assay for testing antivirals. Here, we developed a quick and inexpensive high-throughput fluorescent assay to test inhibitors of viral proteases. Accordingly, we employed this assay to sample inhibitors for papain-like protease from SARS-CoV-2. In addition, we validated this assay for screening inhibitors of flaviviral protease from the tick-borne encephalitis virus to emphasize a broad range of applications of our approach. This fluorescent high-throughput assay is based on fluorescent energy transfer (FRET) between two distinct fluorescent proteins (eGFP and mCherry) connected via a substrate polypeptide. When the substrate is cleaved, FRET is abolished and the change in fluorescence corresponds to reaction progress. Our data show that this assay can be used for testing the inhibitors in the 96 or 384 well plates format with robust and reproducible outcomes.","version":"1.2","doi":"10.1101/2020.10.27.357418","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.03.429522","pub_date":"2021-2-03","title":"Knowledge and Awareness of coronavirus disease 2019 (COVID-19) among Chinese dental students\u2014\u2014a comparison study","abstract":"This study aimed to measure the knowledge and awareness of COVID-19 among Chinese dental students during the global outbreak recently. A descriptive cross-sectional study was performed among dental students and nonmedical college students in China. All the participants were required to anonymously answer a reliable online questionnaire, which covered 3 different fields of COVID-19. Average scores of dental students (D group), including junior (JD group) and senior dental students (SD group), and nonmedical college students (N group) were compared respectively. Chi-square test and independent sample T test were taken for statistical analysis with SPSS.12. Totally 497 questionnaires were collected, including 224 from dental students and 273 from non-medical students. The overall average score was 57\u00b119.2. The average scores of dental students were 64.5\u00b118. The D group had significantly higher scores on the total score, section scores, and 20 questions respectively than with the N group. No significant differences were found on 5 questions. Compared with the N group, the SD group won on all three sections while JD group failed to win on the diagnose section. Although the dental student showed good awareness regarding the clinical aspects of COVID-19 than non-medical students, there are still some weakness in the part of treatment and prevention, which need to be strengthened for better prepare during work. Besides, the low accuracy rate of lower grade dental students is also worth noting.","version":"1.1","doi":"10.1101/2021.02.03.429522","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429431","pub_date":"2021-2-03","title":"Potential global impact of the N501Y mutation on MHC-II presentation and immune escape","abstract":"The B.1.1.7 SARS-CoV-2 variant, characterized by the N501Y mutation, is rapidly emerging, raising concerns about its effectiveness on natural as well as vaccine-induced adaptive viral immunity at the population level. Since CD4 T cell responses are of critical importance to the antibody response, we examined the global effects of N501Y mutation on MHC-II presentation compared to the N501 wildtype and found poorer presentation across the majority of MHC-II alleles. This suggests that the N501Y mutation may not only diminish binding of antibodies to the RBD but also interfere with their production by weakening the cooperation between T and B cells, facilitating immune escape.","version":"1.1","doi":"10.1101/2021.02.02.429431","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429469","pub_date":"2021-2-03","title":"Identification of the SHREK family of proteins as broad-spectrum host antiviral factors","abstract":"Mucins and mucin-like molecules are highly glycosylated, high-molecular-weight cell surface proteins that possess a semi-rigid and highly extended extracellular domain. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein, has recently been found to restrict HIV-1 infectivity through virion incorporation that sterically hinders virus particle attachment to target cells. Here, we report the identification of a family of antiviral cellular proteins, named the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators (PSGL-1, CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123), that share similar structural characteristics with PSGL-1. We demonstrate that SHREK proteins block HIV-1 infectivity by inhibiting virus particle attachment to target cells. In addition, we demonstrate that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. Furthermore, we demonstrate that a subset of SHREKs also blocks the infectivity of a hybrid alphavirus-SARS-CoV-2 virus-like particle. These results suggest that SHREK proteins may be a part of host innate immunity against enveloped viruses.","version":"1.1","doi":"10.1101/2021.02.02.429469","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.28.428568","pub_date":"2021-2-02","title":"Evolution of ACE2 and SARS-CoV-2 Interplay Across 247 Vertebrates","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause the most serious pandemics of Coronavirus Disease 2019 (COVID-19), which threatens human health and public safety. SARS-CoV-2 spike (S) protein uses angiotensin-converting enzyme 2 (ACE2) as recognized receptor for its entry into host cell that contributes to the infection of SARS-CoV-2 to hosts. Using computational modeling approach, this study resolved the evolutionary pattern of bonding affinity of ACE2 in 247 jawed vertebrates to the spike (S) protein of SARS-CoV-2. First, high-or-low binding affinity phenotype divergence of ACE2 to the S protein of SARS-CoV-2 has appeared in two ancient species of jawed vertebrates, Scyliorhinus torazame (low-affinity, Chondrichthyes) and Latimeria chalumnae (high-affinity, Coelacanthimorpha). Second, multiple independent affinity divergence events recur in fishes, amphibians-reptiles, birds, and mammals. Third, high affinity phenotypes go up in mammals, possibly implying the rapid expansion of mammals might accelerate the evolution of coronaviruses. Fourth, we found natural mutations at eight amino acid sites of ACE2 can determine most of phenotype divergences of bonding affinity in 247 vertebrates and resolved their related structural basis. Moreover, we also identified high-affinity or low-affinity-associated concomitant mutation group.The group linked to extremely high affinity may provide novel potentials for the development of human recombinant soluble ACE2 (hrsACE2) in treating patients with COVID-19 or for constructing genetically modified SARS-CoV-2 infection models promoting vaccines studies. These findings would offer potential benefits for the treatment and prevention of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.28.428568","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.13.420406","pub_date":"2021-2-02","title":"Identification of bis-benzylisoquinoline alkaloids as SARS-CoV-2 entry inhibitors from a library of natural products in vitro","abstract":"Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major public health issue. To screen for antiviral drugs for COVID-19 treatment, we constructed a SARS-CoV-2 spike (S) pseudovirus system using an HIV-1-based lentiviral vector with a luciferase reporter gene to screen 188 small potential antiviral compounds. Using this system, we identified nine compounds, specifically, bis-benzylisoquinoline alkaloids, that potently inhibited SARS-CoV-2 pseudovirus entry, with EC50 values of 0.1\u201310 \u03bcM. Mechanistic studies showed that these compounds, reported as calcium channel blockers (CCBs), inhibited Ca2+-mediated membrane fusion and consequently suppressed coronavirus entry. These candidate drugs showed broad-spectrum efficacy against the entry of several coronavirus pseudotypes (SARS-CoV, MERS-CoV, SARS-CoV-2 [S-D614, S-G614, N501Y.V1 and N501Y.V2]) in different cell lines (293T, Calu-3, and A549). Antiviral tests using native SARS-CoV-2 in Vero E6 cells confirmed that four of the drugs (SC9/cepharanthine, SC161/hernandezine, SC171, and SC185/neferine) reduced cytopathic effect and supernatant viral RNA load. Among them, cepharanthine showed the strongest anti-SARS-CoV-2 activity. Collectively, this study offers new lead compounds for coronavirus antiviral drug discovery.","version":"1.2","doi":"10.1101/2020.12.13.420406","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.285940","pub_date":"2021-2-02","title":"Resveratrol And Pterostilbene Potently Inhibit SARS-CoV-2 Replication In Vitro","abstract":"The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory and anti-oxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. Resveratrol and pterostilbene showed antiviral activity in African green monkey kidney cells and in human primary bronchial epithelial cells cultured in an air-liquid interface system. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly 5 rounds of replication, demonstrating the long-lasting effect of these compounds. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to treat SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether or not these drugs are advantageous for COVID-19 treatment.","version":"1.2","doi":"10.1101/2020.09.24.285940","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.428884","pub_date":"2021-2-02","title":"The basis of a more contagious 501Y.V1 variant of SARS-COV-2","abstract":"Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing a world-wide pandemic. A variant of SARS-COV-2 (20I/501Y.V1) recently discovered in the United Kingdom has a single mutation from N501 to Y501 within the receptor binding domain (Y501-RBD), of the Spike protein of the virus. This variant is much more contagious than the original version (N501-RBD). We found that this mutated version of RBD binds to human Angiotensin Converting Enzyme 2 (ACE2) a ~10 times more tightly than the native version (N501-RBD). Modeling analysis showed that the N501Y mutation would allow a potential aromatic ring-ring interaction and an additional hydrogen bond between the RBD and ACE2. However, sera from individuals immunized with the Pfizer-BioNTech vaccine still efficiently block the binding of Y501-RBD to ACE2 though with a slight compromised manner by comparison with their ability to inhibit binding to ACE2 of N501-RBD. This may raise the concern whether therapeutic anti-RBD antibodies used to treat COVID-19 patients are still efficacious. Nevertheless, a therapeutic antibody, Bamlanivimab, still binds to the Y501-RBD as efficiently as its binds to N501-RBD.","version":"1.1","doi":"10.1101/2021.02.02.428884","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.428995","pub_date":"2021-2-02","title":"Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice","abstract":"Since the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.","version":"1.1","doi":"10.1101/2021.02.02.428995","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.02.429327","pub_date":"2021-2-02","title":"Computational insights into differential interaction of mamalian ACE2 with the SARS-CoV-2 spike receptor binding domain","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causing agent of the COVID-19 pandemic, has spread globally. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals and predicted the structures of ACE2-RBD complexes. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The comparison results suggest that the ACE2 of bovine, cat and panda form strong binding with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse and civet, the ACE2 proteins interact weakly with RBD.","version":"1.1","doi":"10.1101/2021.02.02.429327","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429283","pub_date":"2021-2-02","title":"A Thermostable, Flexible RNA Vaccine Delivery Platform for Pandemic Response","abstract":"Current RNA vaccines against SARS-CoV-2 are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at \u221220\u00b0C or \u221270\u00b0C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) RNA vaccine delivery system for use in pandemic preparedness and pandemic response. Liquid NLC is stable at refrigerated temperatures for \u2265 1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for \u2265 8 months or refrigerated temperature for \u2265 21 months. This thermostable RNA vaccine platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings. An RNA vaccine delivery system stable at room temperature for 8+ months and refrigerated for 21+ months.","version":"1.1","doi":"10.1101/2021.02.01.429283","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429110","pub_date":"2021-2-01","title":"Sequential delivery of LAIV and SARS-CoV-2 in the ferret model can reduce SARS-CoV-2 shedding and does not result in enhanced lung pathology","abstract":"Co-circulation of SARS-CoV-2 and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in co-infected patients. The lack of a readily available COVID-19 vaccine has reinforced the importance of influenza vaccine programmes during the COVID-19 pandemic. Live Attenuated Influenza Vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration might influence the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV (QLAIV) was administered to ferrets 3 days pre- or post-SARS-CoV-2 infection. LAIV administration did not exacerbate SARS-CoV-2 disease course or lung pathology with either regimen. Additionally, LAIV administered prior to SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract (URT). We conclude that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.","version":"1.1","doi":"10.1101/2021.02.01.429110","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.31.429010","pub_date":"2021-2-01","title":"Environmentally-induced mdig is a major contributor to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan metabolism","abstract":"The novel \u03b2-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 101 million people and resulted in 2.2 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provide evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is a master regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. These data, accordingly, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.","version":"1.1","doi":"10.1101/2021.01.31.429010","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.31.429001","pub_date":"2021-2-01","title":"Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro","abstract":"Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 \u03bcM and 99% at 15 \u03bcM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 \u03bcM), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.","version":"1.1","doi":"10.1101/2021.01.31.429001","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.30.428979","pub_date":"2021-2-01","title":"Production of SARS-CoV-2 virus-like particles in insect cells","abstract":"Coronavirus disease (COVID-19) causes a serious threat to human health. To production of SARS-COV-2 virus-like particles (VLPs) in insect cells for vaccine development and scientific research. The E, M and S genes were cloned into multiple cloning sites of the new triple expression plasmid with one p10 promoter, two pPH promoters and three multiple cloning sites. The plasmid was transformed into DH10 BacTM Escherichia coli competent cells to obtain recombinant bacmid. Then the recombinant bacmid was transfected in ExpiSf9\u2122 insect cells to generate recombinant baculovirus. After ExpiSf9\u2122 infected with the recombinant baculovirus, the E, M, and S protein co-expressed in insect cells. Finally, SARS-CoV-2 VLPs were self-assembled in insect cells after infection. The morphology and the size of SARS-CoV-2 VLPs are similar to the native virions.","version":"1.1","doi":"10.1101/2021.01.30.428979","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.428871","pub_date":"2021-2-01","title":"Nanotraps for the containment and clearance of SARS-CoV-2","abstract":"SARS-CoV-2 enters host cells through its viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells. Here we show functionalized nanoparticles, termed \u201cNanotraps\u201d, completely inhibited SARS-CoV-2 infection by blocking the interaction between the spike protein of SARS-CoV-2 and the ACE2 of host cells. The liposomal-based Nanotrap surfaces were functionalized with either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies and phagocytosis-specific phosphatidylserines. The Nanotraps effectively captured SARS-CoV-2 and completely blocked SARS-CoV-2 infection to ACE2-expressing human cell lines and primary lung cells; the phosphatidylserine triggered subsequent phagocytosis of the virus-bound, biodegradable Nanotraps by macrophages, leading to the clearance of pseudotyped and authentic virus in vitro. Furthermore, the Nanotraps demonstrated excellent biosafety profile in vitro and in vivo. Finally, the Nanotraps inhibited pseudotyped SARS-CoV-2 infection in live human lungs in an ex vivo lung perfusion system. In summary, Nanotraps represent a new nanomedicine for the inhibition of SARS-CoV-2 infection. Nanotraps block interaction between SARS-CoV-2 spike protein and host ACE2 receptors Nanotraps trigger macrophages to engulf and clear virus without becoming infected Nanotraps showed excellent biosafety profiles in vitro and in vivo Nanotraps blocked infection to living human lungs in ex vivo lung perfusion system To address the global challenge of creating treatments for SARS-CoV-2 infection, we devised a nanomedicine termed \u201cNanotraps\u201d that can completely capture and eliminate the SARS-CoV-2 virus. The Nanotraps integrate protein engineering, immunology, and nanotechnology and are effective, biocompatible, safe, stable, feasible for mass production. The Nanotraps have the potential to be formulated into a nasal spray or inhaler for easy administration and direct delivery to the respiratory system, or as an oral or ocular liquid, or subcutaneous, intramuscular or intravenous injection to target different sites of SARS-CoV-2 exposure, thus offering flexibility in administration and treatment. More broadly, the highly versatile Nanotrap platform could be further developed into new vaccines and therapeutics against a broad range of diseases in infection, autoimmunity and cancer, by incorporating with different small molecule drugs, RNA, DNA, peptides, recombinant proteins, and antibodies.","version":"1.1","doi":"10.1101/2021.02.01.428871","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429176","pub_date":"2021-2-01","title":"Host PDZ-containing proteins targeted by SARS-Cov-2","abstract":"Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 \u03bcM to 82 \u03bcM. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.","version":"1.1","doi":"10.1101/2021.02.01.429176","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.28.21250421","pub_date":"2021-02-01","title":"Viral sequencing reveals US healthcare personnel rarely become infected with SARS-CoV-2 through patient contact","abstract":"<jats:title>Summary</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Healthcare personnel (HCP) are at increased risk of infection with the severe acute respiratory coronavirus 2019 virus (SARS-CoV-2). Between 12 March 2020 and 10 January 2021, &gt;1,170 HCP tested positive for SARS-CoV-2 at a major academic medical institution in the Upper Midwest of the United States. We aimed to understand the sources of infections in HCP and to evaluate the efficacy of infection control procedures used at this institution to protect HCP from healthcare-associated transmission.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>In this retrospective case series, we used viral genomics to investigate the likely source of SARS-CoV-2 infection in 96 HCP where epidemiological data alone could not be used to rule out healthcare-associated transmission. We obtained limited epidemiological data through informal interviews and review of the electronic health record. We combined viral sequence data and available epidemiological information to infer the most likely source of HCP infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>We investigated 32 SARS-CoV-2 infection clusters involving 96 HCP, 140 possible patient contacts, and 1 household contact (total n = 237). Of these, 182 sequences met quality standards and were used for downstream analysis. We found the majority of HCP infections could not be linked to a patient or co-worker and therefore likely occurred in the outside community (58/96; 60.4%). We found a smaller percentage could be traced to a coworker (10/96; 10.4%) or were part of a patient-employee cluster (12/96; 12.5%). Strikingly, the smallest proportion of HCP infections could be clearly traced to a patient source (4/96; 4.2%).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Infection control procedures, consistently followed, offer significant protection to HCP caring for COVID-19 patients in a representative American academic medical institution. Rapid SARS-CoV-2 genome sequencing in healthcare settings can be used retrospectively to reconstruct the likely source of HCP infection when epidemiological data are not available or are inconclusive. Understanding the source of SARS-CoV-2 infection can then be used prospectively to adjust and improve infection control practices and guidelines.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This project was funded in part through a COVID-19 Response grant from the Wisconsin Partnership Program at the University of Wisconsin School of Medicine and Public Health to T.C.F. and D.H.O. Author N.S. is supported by the National Institute of Allergy and Infectious Diseases Institute (NIAID) Grant 1DP2AI144244-01.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>\n                      On 16 January 2021 we searched for \u201cSARS-CoV-2\u201d AND \u201chealthcare workers\u201d AND \u201cviral sequencing\u201d in Google Scholar. This search returned 57 results, and included a number of preprint articles. We found two studies that used viral sequencing to investigate healthcare-associated outbreaks in the Netherlands\n                      <jats:sup>1</jats:sup>\n                      and the United Kingdom\n                      <jats:sup>2</jats:sup>\n                      . To our knowledge, no study has used viral sequencing to specifically investigate the source of SARS-CoV-2 infections in healthcare workers in the United States. Although we and others have written about the potential utility of sequencing as an infection control asset\n                      <jats:sup>3\u20136</jats:sup>\n                      , few have demonstrated the practical application of such efforts.\n                    </jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>Our study suggests infection control measures in place at the institution evaluated in this case series are largely protecting healthcare personnel (HCP) from healthcare-associated SARS-CoV-2 infections. Even so, the majority of healthcare-associated infections we did identify appeared to be linked to HCP-to-HCP spread so additional messaging and guidelines to reduce HCP-to-HCP spread in and out of the workplace may be warranted. In addition, we demonstrated how rapid viral sequencing can be combined with, even limited, epidemiological information to reconstruct healthcare-associated SARS-CoV-2 outbreaks.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>Healthcare-associated SARS-CoV-2 infections negatively affect HCP, patients, and communities. Infections among HCP add further strain to the healthcare system and put patients and other HCP at risk. We found the majority of HCP infections appeared to be acquired through community exposure so measures to reduce community spread are critical. This further emphasizes the importance of mask-wearing, physical distancing, robust testing programs, and the rapid distribution of vaccines.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2021.01.28.21250421","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.31.426979","pub_date":"2021-2-01","title":"In vitro inactivation of SARS-CoV-2 with 0.5% povidone iodine nasal spray (Nasodine) at clinically relevant concentrations and timeframes using tissue culture and PCR based assays","abstract":"There has been considerable speculation regarding the potential of PVP-I nasal disinfection as an adjunct to other countermeasures during the ongoing SARS-CoV-2 pandemic. Nasodine is a commercial formulation of 0.5% PVP-I that has been evaluated for safety and efficacy in human trials as a treatment for the common cold, including a Phase III trial (ANZCTR: ACTRN12619000764134). This study presents the first report of the in vitro efficacy of this formulation against SARS-CoV-2. We conducted in vitro experiments to determine if the PVP-I formulation inactivated SARS-CoV-2 using two independent assays and virus isolates, and incorporating both PCR-based detection and cell culture methods to assess residual virus after exposure to the formulation. Based on cell culture results, the PVP-I formulation was found to rapidly inactivate SARS-CoV-2 isolates in vitro in short timeframes (15 seconds to 15 minutes) consistent with the minimum and maximum potential residence time in the nose. The Nasodine formula was found to be more effective than 0.5% PVP-I in saline. Importantly, it was found that the formulation inactivated culturable virus but had no effect on PCR-detectable viral RNA. The PVP-I formulation eliminated the viability of SARS-CoV-2 virus with short exposure times consistent with nasal use. PCR alone may not be adequate for viral quantification in nasal PVP-I studies; future studies should incorporate cell culture to assess viral viability. Nasal disinfection with PVP-I may be a useful intervention for newly-diagnosed COVID-19 patients to reduce transmission risk and disease progression to the lower respiratory tract.","version":"1.1","doi":"10.1101/2021.01.31.426979","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429219","pub_date":"2021-2-01","title":"Enhanced immunogenicity of a synthetic DNA vaccine expressing consensus SARS-CoV-2 Spike protein using needle-free immunization","abstract":"The ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 \u03bcg of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 \u03bcg or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-\u03b3, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.","version":"1.1","doi":"10.1101/2021.02.01.429219","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.31.429007","pub_date":"2021-2-01","title":"Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A","abstract":"The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ~30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and initiation of infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions the RNA is protected by viral capsid proteins, membrane proteins and nucleocapsid proteins. To overcome this protection we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We constructed six proteins expressed in E. coli cells: 1) MBP-ACE2NTD, 2) ACE2NTD-GFP, 3) RNase I (6xHis), 4) RNase III (6xHis), 5) RNase I-ACE2NTD (6xHis), and 6) human RNase A-ACE2NTD150 (6xHis). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6xHis) and hRNase A-ACE2NTD (6xHis) fusions are active in cleaving COVID-19 RNA in vitro. The recombinant RNase I (6xHis) and RNase III (6xHis) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human cell receptor and nuclease that may be used to degrade viral nucleic acids in our environment.","version":"1.1","doi":"10.1101/2021.01.31.429007","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429069","pub_date":"2021-2-01","title":"Neutralization of SARS-CoV-2 VOC 501Y.V2 by human antisera elicited by both inactivated BBIBP-CorV and recombinant dimeric RBD ZF2001 vaccines","abstract":"Recently, the emerged and rapidly spreading SARS-CoV-2 variant of concern (VOC) 501Y.V2 with 10 amino acids in spike protein were found to escape host immunity induced by infection or vaccination. Global concerns have been raised for its potential to affect vaccine efficacy. Here, we evaluated the neutralization activities of two vaccines developed in China against 501Y.V2. One is licensed inactivated vaccine BBIBP-CorV and the other one is recombinant dimeric receptor-binding domain (RBD) vaccine ZF2001. Encouragingly, both vaccines largely preserved neutralizing titres, with slightly reduction, against 501Y.V2 authentic virus compare to their titres against both original SARS-CoV-2 and the currently circulating D614G virus. These data indicated that 501Y.V2 variant will not escape the immunity induced by vaccines targeting whole virus or RBD.","version":"1.1","doi":"10.1101/2021.02.01.429069","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.31.429023","pub_date":"2021-2-01","title":"Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2\u2032-O-methyl transfer by SARS-CoV-2 nsp16","abstract":"Capping viral messenger RNAs is essential for efficient translation and prevents their detection by host innate immune responses. For SARS-CoV-2, RNA capping includes 2\u2032-O-methylation of the first ribonucleotide by methyltransferase nsp16 in complex with activator nsp10. The reaction requires substrates, a short RNA and SAM, and is catalyzed by divalent cations, with preference for Mn2+. Crystal structures of nsp16-nsp10 with capped RNAs revealed a critical role of metal ions in stabilizing interactions between ribonucleotides and nsp16, resulting in precise alignment of the substrates for methyl transfer. An aspartate residue that is highly conserved among coronaviruses alters the backbone conformation of the capped RNA in the binding groove. This aspartate is absent in mammalian methyltransferases and is a promising site for designing coronavirus-specific inhibitors.","version":"1.1","doi":"10.1101/2021.01.31.429023","journal":"bioRxiv","score":null},{"id":"10.1101/2021.02.01.429135","pub_date":"2021-2-01","title":"SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics","abstract":"Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3\u2019 end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5\u2019 end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range.","version":"1.1","doi":"10.1101/2021.02.01.429135","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.30.428921","pub_date":"2021-1-30","title":"SARS-CoV-2 binding and neutralizing antibody levels after vaccination with Ad26.COV2.S predict durable protection in rhesus macaques","abstract":"The first COVID-19 vaccines have recently gained authorization for emergency use. At this moment, limited knowledge on duration of immunity and efficacy of these vaccines is available. Data on other coronaviruses after natural infection suggest that immunity to SARS-CoV-2 might be short lived, and preliminary evidence indicates waning antibody titers following SARS-CoV-2 infection. Here we model the relationship between immunogenicity and protective efficacy of a series of Ad26 vectors encoding stabilized variants of the SARS-CoV-2 Spike (S) protein in rhesus macaques and validate the analyses by challenging macaques 6 months after immunization with the Ad26.COV2.S vaccine candidate that has been selected for clinical development. We find that Ad26.COV2.S confers durable protection against replication of SARS-CoV-2 in the lungs that is predicted by the levels of S-binding and neutralizing antibodies. These results suggest that Ad26.COV2.S could confer durable protection in humans and that immunological correlates of protection may enable the prediction of durability of protection.","version":"1.1","doi":"10.1101/2021.01.30.428921","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428442","pub_date":"2021-1-30","title":"A single dose of replication-competent VSV-vectored vaccine expressing SARS-CoV-2 S1 protects against virus replication in a hamster model of severe COVID-19","abstract":"The development of effective countermeasures against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic, is a priority. We designed and produced ConVac, a replication-competent vesicular stomatitis virus (VSV) vaccine vector that expresses the S1 subunit of SARS-CoV-2 spike protein. We used golden Syrian hamsters as animal model of severe COVID-19 to test the efficacy of the ConVac vaccine. A single vaccine dose elicited high levels of SARS-CoV-2 specific binding and neutralizing antibodies; following intranasal challenge with SARS-CoV-2, animals were protected from weight loss and viral replication in the lungs. No enhanced pathology was observed in vaccinated animals upon challenge, but some inflammation was still detected. The data indicate rapid control of SARS-CoV-2 replication by the S1-based VSV-vectored SARS-CoV-2 ConVac vaccine.","version":"1.1","doi":"10.1101/2021.01.29.428442","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.30.428920","pub_date":"2021-1-30","title":"Impact of Prior Infection on Protection and Transmission of SARS-CoV-2 in Golden Hamsters","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 100 million confirmed human infections, and 2 million more deaths globally since its emergence in the end of 2019. Several studies have shown that prior infection provided protective immunity against SARS-CoV-2 in non-human primate models. However, the effect of prior infection on blocking SARS-CoV-2 transmission is not clear. Here, we evaluated the impact of prior infection on protection and transmission of the SARS-CoV-2 virus in golden hamsters. Our results showed that prior infection provided protective immunity against SARS-CoV-2 re-challenge, but it was not sterizing immunity. The transmission experiment results showed that SARS-CoV-2 was efficiently transmitted from naive hamsters to prior infected hamsters by direct contact and airborne route, but not by indirect contact. Further, the virus was efficiently transmitted from prior infected hamsters to naive hamsters by direct contact, but not by airborne route and indirect contact. Surprisingly, the virus can be transmitted between prior infected hamsters by direct contact during a short period of early infection. Taken together, our study demonstrated that prior infected hamsters with good immunity can still be naturally re-infected, and the virus can be transmitted between prior infected hamsters and the naive through different transmission routes, implying the potential possibility of human re-infection and the risk of virus transmission between prior infected population and the healthy. Our study will help to calculate the herd immunity threshold more accurately, make more reasonable public health decisions, formulate an optimized population vaccination program, as well as aid the implementation of appropriate public health and social measures to control COVID-19.","version":"1.1","doi":"10.1101/2021.01.30.428920","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426911","pub_date":"2021-1-30","title":"mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants","abstract":"To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines1,2. These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known3\u20136. Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers3,5,6. Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection7,8. However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.","version":"1.2","doi":"10.1101/2021.01.15.426911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.357350","pub_date":"2021-1-30","title":"Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular Membrane","abstract":"Infection of human cells by the SARS-CoV2 relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics (MD) simulations taking advantage of the Highly Mobile Membrane Mimetic (HMMM) model, to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level, and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration where the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane binding modes (binding modes 1-3) where binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the \u201cfusion active core residues\u201d, in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection with potential implications in designing novel inhibitors. A key step in cellular infection by the SARS-CoV2 virus is its attachment to and penetration into the plasma membrane of human cells. These processes hinge upon the membrane interaction of the viral fusion peptide, a segment exposed by the spike protein upon its conformational changes after encountering the host cell. In this study, using molecular dynamics simulations, we describe how the fusion peptide from the SARS-CoV2 virus binds human cellular membranes and characterize, at an atomic level, lipid-protein interactions important for the stability of its membrane-bound state.","version":"1.2","doi":"10.1101/2020.10.27.357350","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.19.423600","pub_date":"2021-1-30","title":"Genetic Conservation of SARS-CoV-2 RNA Replication Complex in Globally Circulating Isolates and Recently Emerged Variants from Humans and Minks Suggests Minimal Pre-Existing Resistance to Remdesivir","abstract":"Remdesivir (RDV) exhibits potent antiviral activity against SARS-CoV-2 and is currently the only drug approved for the treatment of COVID-19. However, little is currently known about the potential for pre-existing resistance to RDV and the possibility of SARS-CoV-2 genetic diversification that might impact RDV efficacy as the virus continue to spread globally. In this study, >90,000 SARS-CoV-2 sequences from globally circulating clinical isolates, including sequences from recently emerged United Kingdom and South Africa variants, and >300 from mink isolates were analyzed for genetic diversity in the RNA replication complex (nsp7, nsp8, nsp10, nsp12, nsp13, and nsp14) with a focus on the RNA-dependent RNA polymerase (nsp12), the molecular target of RDV. Overall, low genetic variation was observed with only 12 amino acid substitutions present in the entire RNA replication complex in \u22650.5% of analyzed sequences with the highest overall frequency (82.2%) observed for nsp12 P323L that consistently increased over time. Low sequence variation in the RNA replication complex was also observed among the mink isolates. Importantly, the coronavirus Nsp12 mutations previously selected in vitro in the presence of RDV were identified in only 2 isolates (0.002%) within all the analyzed sequences. In addition, among the sequence variants observed in \u22650.5% clinical isolates, including P323L, none were located near the established polymerase active site or sites critical for the RDV mechanism of inhibition. In summary, the low diversity and high genetic stability of the RNA replication complex observed over time and in the recently emerged SARS-CoV-2 variants suggests a minimal global risk of pre-existing SARS-CoV-2 resistance to RDV.","version":"1.2","doi":"10.1101/2020.12.19.423600","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428890","pub_date":"2021-1-30","title":"Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii","abstract":"Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated, lacking the amino terminus. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.","version":"1.1","doi":"10.1101/2021.01.29.428890","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.28.428743","pub_date":"2021-1-29","title":"Development of spike receptor-binding domain nanoparticle as a vaccine candidate against SARS-CoV-2 infection in ferrets","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of COVID-19 pandemic, enters host cells via the interaction of its Receptor-Binding Domain (RBD) of Spike protein with host Angiotensin-Converting Enzyme 2 (ACE2). Therefore, RBD is a promising vaccine target to induce protective immunity against SARS-CoV-2 infection. In this study, we report the development of RBD protein-based vaccine candidate against SARS-CoV-2 using self-assembling H. pylori-bullfrog ferritin nanoparticles as an antigen delivery. RBD-ferritin protein purified from mammalian cells efficiently assembled into 24-mer nanoparticles. 16-20 months-old ferrets were vaccinated with RBD-ferritin nanoparticles (RBD-nanoparticles) by intramuscular or intranasal inoculation. All vaccinated ferrets with RBD-nanoparticles produced potent neutralizing antibodies against SARS-CoV-2. Strikingly, vaccinated ferrets demonstrated efficient protection from SARS-CoV-2 challenge, showing no fever, body weight loss and clinical symptoms. Furthermore, vaccinated ferrets showed rapid clearance of infectious viruses in nasal washes and lungs as well as viral RNA in respiratory organs. This study demonstrates the Spike RBD-nanoparticle as an effective protein vaccine candidate against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.28.428743","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428516","pub_date":"2021-1-29","title":"SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by ancestral Spike vaccines","abstract":"The SARS-CoV-2 Spike glycoprotein mediates virus entry and is a major target for neutralizing antibodies. All current vaccines are based on the ancestral Spike with the goal of generating a protective neutralizing antibody response. Several novel SARS-CoV-2 variants with multiple Spike mutations have emerged, and their rapid spread and potential for immune escape have raised concerns. One of these variants, first identified in the United Kingdom, B.1.1.7 (also called VUI202012/01), contains eight Spike mutations with potential to impact antibody therapy, vaccine efficacy and risk of reinfection. Here we employed a lentivirus-based pseudovirus assay to show that variant B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (~2-fold), by serum samples from convalescent individuals and recipients of two different vaccines based on ancestral Spike: mRNA-1273 (Moderna), and protein nanoparticle NVX-CoV2373 (Novavax). Some monoclonal antibodies to the receptor binding domain (RBD) of Spike were less effective against the variant while others were largely unaffected. These findings indicate that B.1.1.7 is not a neutralization escape variant that would be a major concern for current vaccines, or for an increased risk of reinfection.","version":"1.2","doi":"10.1101/2021.01.27.428516","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.425392","pub_date":"2021-1-29","title":"SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor","abstract":"SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the \u201cEuropean\u201d (20E-EU1), \u201cBritish\u201d (501.V1),\u201dSouth African\u201d (501.V2), and \u2018\u2018Brazilian\u201d variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9\u00c5 Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.","version":"1.3","doi":"10.1101/2021.01.06.425392","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.251249","pub_date":"2021-1-29","title":"A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2R\u03b3c KO. NOD) for COVID-19","abstract":"We report the first Human Immune System (HIS)-humanized mouse model (\u201cDRAGA\u201d: HLA-A2.HLA-DR4.Rag1KO.IL-2R\u03b3cKO.NOD) for COVID-19 research. This mouse is reconstituted with human cord blood-derived, HLA-matched hematopoietic stem cells. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and TMPRSS2 serine protease co-localized on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing deteriorated clinical condition, replicating virus in the lungs, and human-like lung immunopathology including T-cell infiltrates, microthrombi and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103+) CD8+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice also developed antibodies against the SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.","version":"1.3","doi":"10.1101/2020.08.19.251249","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428834","pub_date":"2021-1-29","title":"The SARS-CoV-2 Y453F mink variant displays a striking increase in ACE-2 affinity but does not challenge antibody neutralization","abstract":"SARS-CoV-2 transmission from humans to animals has been reported for many domesticated species, including cats, dogs and minks. Identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. The genetic variant, known as \u201ccluster-five\u201d, arose among farmed minks in Denmark and resulted in a complete shutdown of the world\u2019s largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the Y453F cluster-five receptor-binding domain (RBD) and show that it does not decrease established humoral immunity or affect the neutralizing response in a vaccine model based on wild-type RBD or spike. However, it binds the human ACE-2 receptor with a four-fold higher affinity suggesting an enhanced transmission capacity and a possible challenge for viral control.","version":"1.1","doi":"10.1101/2021.01.29.428834","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428535","pub_date":"2021-1-29","title":"Recombination and low-diversity confound homoplasy-based methods to detect the effect of SARS-CoV-2 mutations on viral transmissibility","abstract":"The SARS-CoV-2 variant carrying the Spike protein mutation G614 was first detected in late January 2020 and within a few months became the dominant form globally. In the months that followed, many studies, both in vitro and in animal models, showed that variants carrying this mutation were more infectious and more readily transmitted than the ancestral Wuhan form. Here we investigate why a recently published study by van Dorp et al. failed to detect such higher transmissibility of the G614 variant using homoplasy-based methods. We show that both low diversity and recombination confound the methods utilized by van Dorp et al. and significantly decrease their sensitivity. Furthermore, though they claim no evidence of recombination in their dataset, we and several other studies identify a subset of the sequences as recombinants, possibly enough to affect their statistic adversely.","version":"1.1","doi":"10.1101/2021.01.29.428535","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.29.428773","pub_date":"2021-1-29","title":"Characterization of Critical Determinants of ACE2-RBD Interaction","abstract":"Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. Utilizing our recently developed NanoBiT technology-based biosensor, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, before and after the addition of competitive inhibitors, as well as neutralizing antibody activity. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.","version":"1.1","doi":"10.1101/2021.01.29.428773","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.21250604","pub_date":"2021-01-29","title":"The World Mortality Dataset: Tracking excess mortality across countries during the COVID-19 pandemic","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Comparing the impact of the COVID-19 pandemic between countries or across time is difficult because the reported numbers of cases and deaths can be strongly affected by testing capacity and reporting policy. Excess mortality, defined as the increase in all-cause mortality relative to the expected mortality, is widely considered as a more objective indicator of the COVID-19 death toll. However, there has been no global, frequently-updated repository of the all-cause mortality data across countries. To fill this gap, we have collected weekly, monthly, or quarterly all-cause mortality data from 94 countries and territories, openly available as the regularly-updated World Mortality Dataset. We used this dataset to compute the excess mortality in each country during the COVID-19 pandemic. We found that in several worst-affected countries (Peru, Ecuador, Bolivia, Mexico) the excess mortality was above 50% of the expected annual mortality. At the same time, in several other countries (Australia, New Zealand) mortality during the pandemic was below the usual level, presumably due to social distancing measures decreasing the non-COVID infectious mortality. Furthermore, we found that while many countries have been reporting the COVID-19 deaths very accurately, some countries have been substantially underreporting their COVID-19 deaths (e.g. Nicaragua, Russia, Uzbekistan), sometimes by two orders of magnitude (Tajikistan). Our results highlight the importance of open and rapid all-cause mortality reporting for pandemic monitoring.</jats:p>","version":null,"doi":"10.1101/2021.01.27.21250604","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.28.122291","pub_date":"2021-1-28","title":"Lung cancer models reveal SARS-CoV-2-induced EMT contributes to COVID-19 pathophysiology","abstract":"COVID-19 is an infectious disease caused by SARS-CoV-2, which enters host cells via the cell surface proteins ACE2 and TMPRSS2. Using a variety of normal and malignant models and tissues from the aerodigestive and respiratory tracts, we investigated the expression and regulation of ACE2 and TMPRSS2. We find that ACE2 expression is restricted to a select population of highly epithelial cells. Notably, infection with SARS-CoV-2 in cancer cell lines, bronchial organoids, and patient nasal epithelium, induces metabolic and transcriptional changes consistent with epithelial to mesenchymal transition (EMT), including upregulation of ZEB1 and AXL, resulting in an increased EMT score. Additionally, a transcriptional loss of genes associated with tight junction function occurs with SARS-CoV-2 infection. The SARS-CoV-2 receptor, ACE2, is repressed by EMT via TGFbeta, ZEB1 overexpression and onset of EGFR TKI inhibitor resistance. This suggests a novel model of SARS-CoV-2 pathogenesis in which infected cells shift toward an increasingly mesenchymal state, associated with a loss of tight junction components with acute respiratory distress syndrome-protective effects. AXL-inhibition and ZEB1-reduction, as with bemcentinib, offers a potential strategy to reverse this effect. These observations highlight the utility of aerodigestive and, especially, lung cancer model systems in exploring the pathogenesis of SARS-CoV-2 and other respiratory viruses, and offer important insights into the potential mechanisms underlying the morbidity and mortality of COVID-19 in healthy patients and cancer patients alike.","version":"1.2","doi":"10.1101/2020.05.28.122291","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428191","pub_date":"2021-1-28","title":"Identification and analysis of novel variants in SARS-COV-2 genomes isolated from the Kingdom of Bahrain","abstract":"The challenges imposed by the ongoing outbreak of severe acute respiratory syndrome coronavirus-2 affects every aspect of our modern world, ranging from our health to our socio-economic needs. Our existence highly depends on the vaccine\u2019s availability, which demands in-depth research of the available strains and their mutations. In this work, we have analyzed all the available SERS-CoV2 genomes isolated from the Kingdom of Bahrain in terms of their variance and origin analysis. We have predicted various known and unique mutations in the SERS-CoV2 isolated from Bahrain. The complexity of the phylogenetic tree and dot plot representation of the strains mentioned above with other isolates of Asia indicates the versatility and multiple origins of Bahrain\u2019s SERS-CoV2 isolates. We have also identified two high impact spike mutations from these strains which increase the virulence of SARS-CoV2. Our research could have a high impact on vaccine development and distinguishes the source of SERS-CoV2 in the Kingdom of Bahrain.","version":"1.2","doi":"10.1101/2021.01.25.428191","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428534","pub_date":"2021-1-28","title":"Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein","abstract":"Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.27.428534","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428042","pub_date":"2021-1-28","title":"Ubiquitin ligase RIPLET mediates polyubiquitination of RIG-I and LGP2 and regulates the innate immune responses to SARS-CoV-2 infection","abstract":"RIG-I, a cytoplasmic viral RNA sensor, is crucial for innate antiviral immune responses; however, there are controversies about RIG-I\u2019s regulatory mechanism by several ubiquitin ligases and LGP2. Our genetic study revealed that the RIPLET ubiquitin ligase was a general activating factor for RIG-I signaling, whereas another ubiquitin ligase, TRIM25, activated RIG-I in a cell-type-specific manner. These RIPLET and TRIM25 functions were modulated by accessory factors, such as ZCCH3C and NLRP12. Interestingly, we found an additional role of RIPLET in innate immune responses. RIPLET induced delayed polyubiquitination of LGP, resulting in the attenuation of excessive cytokine expression at the late phase. Moreover, RIPLET was involved in the innate immune responses against SARS-CoV-2 infection, a cause of the recent COVID-19 pandemic. Our data indicate that RIPLET fine-tunes innate immune responses via polyubiquitination of RIG-I and LGP2 against virus infection, including SARS-CoV-2.","version":"1.2","doi":"10.1101/2021.01.25.428042","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.26.424450","pub_date":"2021-1-28","title":"The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins","abstract":"The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention. SARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a \u03bcM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.","version":"1.2","doi":"10.1101/2020.12.26.424450","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428541","pub_date":"2021-1-28","title":"D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Viral Transmission via Enhanced Furin-mediated Spike Cleavage","abstract":"Since the D614G substitution in the spike (S) of SARS-CoV-2 emerged, the variant strain underwent rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage of viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614. Both the virus titer and syncytial phenotype were significantly increased in S-G614 than in S-D614 isolates. We further showed increased cleavage of S at the furin substrate site, a key event that promotes syncytium, in S-G614 isolates. These functions of the D614G substitution were validated in cells expressing S protein. The effect on syncytium was abolished by furin inhibitor treatment and mutation of the furin-cleavage site, suggesting its dependence on cleavage by furin. Our study provides a mechanistic explanation for the increased transmissibility of S-G614 containing SARS-CoV-2 through enhanced furin-mediated S cleavage, which increases membrane fusion and virus infectivity.","version":"1.1","doi":"10.1101/2021.01.27.428541","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428380","pub_date":"2021-1-27","title":"Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques","abstract":"We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. In this study, we evaluated the immunogenicity and protective efficacy of reduced doses of Ad26.COV2.S. 30 rhesus macaques were immunized once with 1\u00d71011, 5\u00d71010, 1.125\u00d71010, or 2\u00d7109 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes. Vaccine doses as low as 2\u00d7109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125\u00d71010 vp were required for protection in nasal swabs. Activated memory B cells as well as binding and neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show evidence of virologic, immunologic, histopathologic, or clinical enhancement of disease compared with sham controls. These data demonstrate that a single immunization with a relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques. Moreover, our findings show that a higher vaccine dose may be required for protection in the upper respiratory tract compared with the lower respiratory tract.","version":"1.1","doi":"10.1101/2021.01.27.428380","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.426655","pub_date":"2021-1-27","title":"An Updated Investigation Prior To COVID-19 Vaccination Program In Indonesia: Full-Length Genome Mutation Analysis Of SARS-CoV-2","abstract":"Indonesia kick-started the big project of COVID-19 vaccination program in January 2021 by employed vaccine to the president of Indonesia. The outbreak and rapid transmission of COVID-19 have endangered the global health and economy. This study aimed to investigate the full-length genome mutation analysis of 166 Indonesian SARS-CoV-2 isolates as 12 January 2021. All data of isolates was extracted from the Global Initiative on Sharing All Influenza Data (GISAID) EpiCoV database. CoVsurver was employed to investigate the full-length genome mutation analysis of all isolates. Furthermore, this study also focused on the unlocking of mutation in Indonesian SARS-CoV-2 isolates S protein. WIV04 isolate that was originated from Wuhan, China was used as a virus reference according to CoVsurver default. All data was visualized using GraphPad Prism software, PyMOL, and BioRender. This study result showed that a full-length genome mutation analysis of 166 Indonesian SARS-CoV-2 isolates was successfully discovered. Every single mutation in S protein was described and then visualised by employing BioRender. Furthermore, it also found that D614G mutation appeared in 103 Indonesian SARS-CoV-2 isolates. To sum up, this study helps to observe the spread of the COVID-19 transmission. However, it would like to propose that the epidemiological surveillance and genomics studies might be improved on COVID-19 pandemic in Indonesia.","version":"1.1","doi":"10.1101/2021.01.26.426655","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428428","pub_date":"2021-1-27","title":"Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model","abstract":"The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice and produced milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls, most notably in the brain in mice challenged with a low virus dose. Although GC-376 was not sufficient to improve neither clinical symptoms nor survival, it did show a positive effect against SARS-CoV-2 in vivo. This study supports the notion that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2, and GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.01.27.428428","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428356","pub_date":"2021-1-27","title":"Sequence signatures of two IGHV3-53/3-66 public clonotypes to SARS-CoV-2 receptor binding domain","abstract":"Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short CDR H3. Germline-encoded sequence motifs in CDRs H1 and H2 play a major role, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, have not been elucidated. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that appear to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. Overall, our results advance fundamental understanding of the antibody response to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.26.428356","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428466","pub_date":"2021-1-27","title":"Synthetic nanobody\u2013SARS-CoV-2 receptor-binding domain structures identify distinct epitopes","abstract":"The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16\u2013RBD and Sb45\u2013RBD; a ternary complex of Sb45\u2013RBD\u2013Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design. X-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in recognition of distinct epitopic sites.","version":"1.1","doi":"10.1101/2021.01.27.428466","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428331","pub_date":"2021-1-27","title":"Computational Analysis of Protein Stability and Allosteric Interaction Networks in Distinct Conformational Forms of the SARS-CoV-2 Spike D614G Mutant: Reconciling Functional Mechanisms through Allosteric Model of Spike Regulation","abstract":"Structural and biochemical studies SARS-CoV-2 spike mutants with the enhanced infectivity have attracted significant attention and offered several mechanisms to explain the experimental data. The development of a unified view and a working model which is consistent with the diverse experimental data is an important focal point of the current work. In this study, we used an integrative computational approach to examine molecular mechanisms underlying functional effects of the D614G mutation by exploring atomistic modeling of the SARS-CoV-2 spike proteins as allosteric regulatory machines. We combined coarse-grained simulations, protein stability and dynamic fluctuation communication analysis along with network-based community analysis to simulate structures of the native and mutant SARS-CoV-2 spike proteins in different functional states. The results demonstrated that the D614 position anchors a key regulatory cluster that dictates functional transitions between open and closed states. Using molecular simulations and mutational sensitivity analysis of the SARS-CoV-2 spike proteins we showed that the D614G mutation can improve stability of the spike protein in both closed and open forms, but shifting thermodynamic preferences towards the open mutant form. The results offer support to the reduced shedding mechanism of S1 domain as a driver of the increased infectivity triggered by the D614G mutation. Through distance fluctuations communication analysis, we probed stability and allosteric communication propensities of protein residues in the native and mutant SARS-CoV-2 spike proteins, providing evidence that the D614G mutation can enhance long-range signaling of the allosteric spike engine. By employing network community analysis of the SARS-CoV-2 spike proteins, our results revealed that the D614G mutation can promote the increased number of stable communities and allosteric hub centers in the open form by reorganizing and enhancing the stability of the S1-S2 inter-domain interactions and restricting mobility of the S1 regions. This study provides atomistic-based view of the allosteric interactions and communications in the SARS-CoV-2 spike proteins, suggesting that the D614G mutation can exert its primary effect through allosterically induced changes on stability and communications in the residue interaction networks.","version":"1.1","doi":"10.1101/2021.01.26.428331","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428394","pub_date":"2021-1-27","title":"Lysosomal-Immune Axis Is Associated with COVID 19 Disease Severity: Insights from Patient Single Cell Data","abstract":"SARS-COV-2 has become a leading cause of illness, hospitalizations, and deaths worldwide yet heterogeneity in disease morbidity remains a conundrum. In this study, we analyzed publicly available single-cell RNA-seq data from 75076 cells sequenced from clinically staged COVID-19 patients using a network approach and identified lysosomal-immune axis as a factor significantly associated with disease severity. Our results suggest modulation of lysosomal-immune pathways may present a novel drug-targeting strategy to attenuate SARS-Cov-2 infections.","version":"1.1","doi":"10.1101/2021.01.27.428394","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.427998","pub_date":"2021-1-27","title":"Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K, and N501Y variants by BNT162b2 vaccine-elicited sera","abstract":"We engineered three SARS-CoV-2 viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion+N501Y+D614G from UK; and E484K+N501Y+D614G from SA. Neutralization geometric mean titers (GMTs) of twenty BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.","version":"1.1","doi":"10.1101/2021.01.27.427998","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428384","pub_date":"2021-1-27","title":"SARS-CoV-2 emerging complexity","abstract":"The novel SARS_CoV-2 virus, prone to variation when interacting with spatially extended ecosystems and within hosts can be considered a complex dynamic system. Therefore, it behaves creating several space-time manifestations of its dynamics. However, these physical manifestations in nature have not yet been fully disclosed or understood. Here we show 4-3 and 2-D space-time patterns of rate of infected individuals on a global scale, giving quantitative measures of transitions between different dynamical behaviour. By slicing the spatio-temporal patterns, we found manifestations of the virus behaviour such as cluster formation and bifurcations. Furthermore, by analysing the morphogenesis processes by entropy, we have been able to detect the virus phase transitions, typical of adaptive biological systems. Our results for the first time describe the virus patterning behaviour processes all over the world, giving for them quantitative measures. We know that the outcomes of this work are still partial and more advanced analyses of the virus behaviour in nature are necessary. However, we think that the set of methods implemented can provide significant advantages to better analyse the viral behaviour in the approach of system biology, thus expanding knowledge and improving pandemic problem solving.","version":"1.1","doi":"10.1101/2021.01.27.428384","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.27.428372","pub_date":"2021-1-27","title":"Zinc supplement augments the suppressive effects of repurposed drugs of NF-kappa B inhibitor on ACE2 expression in human lung cell lines in vitro","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a vast number of infections and fatalities worldwide. As the development and safety validation of effective vaccines are ongoing, drug repurposing is most efficient approach to search FDA approved agents against coronavirus disease 2019 (COVID-19). In the present study, we found that endogenous ACE2 expressions could be detected in H322M and Calu-3 cell lines, as well as their ACE2 mRNA and protein expressions were suppressed by pyrrolidine dithiocarbamate (PDTC), a NF-kappa B inhibitor, in dose- and time-dependent manners. Moreover, N-acetyl-cysteine (NAC) pretreatment reversed PDTC-induced ACE2 suppression, as well as the combined treatment of hydrogen peroxide and knockdown of p50 subunit of NF-kappa B by siRNA reduced ACE2 expression in H322M cells. In addition, anthelmintic drug triclabendazole and antiprotozoal drug emetine, repurposed drugs of NF-kappa B inhibitor, also inhibited ACE2 mRNA and protein expressions in H322M cells. Moreover, zinc supplement augmented the suppressive effects of triclabendazole and emetine on ACE2 suppression in H322M and Calu-3 cells. Taken together, these results indicate that ACE2 expression is modulated by reactive oxygen species (ROS) and NF-kappa B signal in human lung cell lines, and zinc combination with triclabendazole or emetine has the clinical potential for the prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2021.01.27.428372","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428293","pub_date":"2021-1-27","title":"Niclosamide Inhalation Powder Made by Thin-Film Freezing: Pharmacokinetic and Toxicology Studies in Rats and Hamsters","abstract":"In this work, we have developed and tested in vivo a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters. The niclosamide dry powder, suitable for inhalation, is being developed as a therapeutic agent against COVID-19 infection. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with a specific pharmacological effect against SARS-CoV-2 infection. In the past, clinical trials for other indications were terminated prior to completion due to low and highly variable oral bioavailability. In order to quickly address the current pandemic, targeting niclosamide directly to the lungs is rational to address the COVID-19 main clinical complications. Thin-film freezing technology was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 \u03bcm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose pharmacokinetic study in rats as evidenced by histopathology analysis, but also was able to achieve lung concentrations above the required IC50 and IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation formulation by thin-film freezing for further scale-up and clinical testing against the COVID-19 infection. This approach overcomes niclosamide\u2019s limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.","version":"1.1","doi":"10.1101/2021.01.26.428293","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428208","pub_date":"2021-1-27","title":"Do examinations prepare students for higher education? A lesson from the Covid-19 lockdown","abstract":"The COVID-19 pandemic caused severe disruption to education in the UK in 2020, with most of the school teaching moving online and national school examinations being cancelled. This was particularly disruptive for those taking end of school examinations in preparation for higher education. Biological science courses require students to absorb a lot of new vocabulary and concepts, with examinations traditionally focusing on content recall rather than reasoning. Students who had entered university in September 2019 were compared with those arriving in September 2020 with respect to their knowledge of bioscience vocabulary and understanding of key concepts. Results showed no significant difference between those who had gone through the examination process in 2019 relative to those who had not, in 2020. This suggests the cramming of information for examinations has no detectable effect on the knowledge and understanding of biology that students take with them to university.","version":"1.1","doi":"10.1101/2021.01.26.428208","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428149","pub_date":"2021-1-27","title":"TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor","abstract":"Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. We report here that TREM2 is a thyroid hormone regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone. Both endogenous thyroid hormone and sobetirome, a synthetic thyroid hormone agonist drug, suppress pro-inflammatory cytokine production from myeloid cells including macrophages that have been treated with the SARS-CoV-2 spike protein which produces a strong, pro-inflammatory phenotype. Thyroid hormone agonism was also found to induce phagocytic behavior in microglia, a phenotype consistent with activation of the TREM2 pathway. The thyroid hormone antagonist NH-3 blocks the anti-inflammatory effects of thyroid hormone agonists and suppresses microglia phagocytosis. Finally, in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model, treatment with Sob-AM2, a CNS-penetrating sobetirome prodrug, results in increased Trem2 expression in disease lesion resident myeloid cells which correlates with therapeutic benefit in the EAE clinical score and reduced damage to myelin. Our findings represent the first report of endocrine regulation of TREM2 and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small molecule therapeutic agents.","version":"1.1","doi":"10.1101/2021.01.25.428149","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.427990","pub_date":"2021-1-26","title":"Molecular Dynamics Reveals the Effects of Temperature on Critical SARS-CoV-2 Proteins","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a newly identified RNA virus that causes the serious infection Coronavirus Disease 2019 (COVID-19). The incidence of COVID-19 is still increasing worldwide despite the summer heat and cool winter. However, little is known about seasonal stability of SARS-CoV-2. Herein, we employ Molecular Dynamics (MD) simulations to explore the effect of temperature on four critical SARS-CoV-2 proteins. Our work demonstrates that the spike Receptor Binding Domain (RBD), Main protease (Mpro), and nonstructural protein 3 (macro X) possesses extreme thermos-stability when subjected to temperature variations rendering them attractive drug targets. Furthermore, our findings suggest that these four proteins are well adapted to habitable temperatures on earth and are largely insensitive to cold and warm climates. Furthermore, we report that the critical residues in SARS-CoV-2 RBD were less responsive to temperature variations as compared to the critical residues in SARS-CoV. As such, extreme summer and winter climates, and the transition between the two seasons, are expected to have a negligible effect on the stability of SARS-CoV-2 which will marginally suppress transmission rates until effective therapeutics are available world-wide.","version":"1.2","doi":"10.1101/2021.01.24.427990","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428207","pub_date":"2021-1-26","title":"Kinetics and correlates of the neutralizing antibody response to SARS-CoV-2","abstract":"A detailed understanding of antibody-based SARS-CoV-2 immunity has critical implications for overcoming the COVID-19 pandemic and for informing on vaccination strategies. In this study, we evaluated the dynamics of the SARS-CoV-2 antibody response in a cohort of 963 recovered individuals over a period of 10 months. Investigating a total of 2,146 samples, we detected an initial SARS-CoV-2 antibody response in 94.4% of individuals, with 82% and 79% exhibiting serum and IgG neutralization, respectively. Approximately 3% of recovered patients demonstrated exceptional SARS-CoV-2 neutralizing activity, defining them as \u2018elite neutralizers\u2019. These individuals also possessed effective cross-neutralizing IgG antibodies to SARS-CoV-1 without any known prior exposure to this virus. By applying multivariate statistical modeling, we found that sero-reactivity, age, time since disease onset, and fever are key factors predicting SARS-CoV-2 neutralizing activity in mild courses of COVID-19. Investigating longevity of the antibody response, we detected loss of anti-spike reactivity in 13% of individuals 10 months after infection. Moreover, neutralizing activity had an initial half-life of 6.7 weeks in serum versus 30.8 weeks in purified IgG samples indicating the presence of a more stable and long-term memory IgG B cell repertoire in the majority of individuals recovered from COVID-19. Our results demonstrate a broad spectrum of the initial SARS-CoV-2 neutralizing antibody response depending on clinical characteristics, with antibodies being maintained in the majority of individuals for the first 10 months after mild course of COVID-19.","version":"1.1","doi":"10.1101/2021.01.26.428207","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428212","pub_date":"2021-1-26","title":"A novel SARS-CoV-2 related coronavirus in bats from Cambodia","abstract":"Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identified nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a small region corresponding to the spike N terminal domain. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously understood, and suggests that Southeast Asia represents a key area to consider in the ongoing search for the origins of SARS-CoV-2, and in future surveillance for coronaviruses.","version":"1.1","doi":"10.1101/2021.01.26.428212","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428251","pub_date":"2021-1-26","title":"A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques","abstract":"The deployment of a vaccine that limits transmission and disease likely will be required to end the Coronavirus Disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally-administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S) in the upper and lower respiratory tract of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged one month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induced neutralizing antibodies and T cell responses and limited or prevented infection in the upper and lower respiratory tract after SARS-CoV-2 challenge. As this single intranasal dose vaccine confers protection against SARS-CoV-2 in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans.","version":"1.1","doi":"10.1101/2021.01.26.428251","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.156554","pub_date":"2021-1-26","title":"Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma. 3a has been implicated in viral release, inhibition of autophagy, inflammasome activation, and cell death and its deletion reduces viral titer and morbidity in mice, raising the possibility that 3a could be an effective vaccine or therapeutic target. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 \u00c5 resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.","version":"1.3","doi":"10.1101/2020.06.17.156554","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428240","pub_date":"2021-1-26","title":"Immunogenic Potential of DNA Vaccine candidate, ZyCoV-D against SARS-CoV-2 in Animal Models","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500\u03bcg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-\u03b3 levels.","version":"1.1","doi":"10.1101/2021.01.26.428240","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.386904","pub_date":"2021-1-26","title":"Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2","abstract":"Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of Imatinib in multiple models. Our results indicated that Imatinib and another TKI, the Masitinib, exhibit an antiviral activity in VeroE6 cells. However, Imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, Imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of Imatinib and similar TKIs as antivirals in the treatment of Covid-19.","version":"1.2","doi":"10.1101/2020.11.17.386904","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428190","pub_date":"2021-1-26","title":"Computational Investigation of Increased Virulence and Pathogenesis of SARS-CoV-2 Lineage B.1.1.7","abstract":"New variants of SARS-CoV-2 are being reported worldwide. More specifically, the variants reported in South Africa (501Y.V2) and United Kingdom (B.1.1.7) were found to be more contagious than the wild type. There are also speculations that the variants might evade the host immune responses induced by currently available vaccines and develop resistance to drugs under consideration. The first step of viral infection in COVID-19, occurs through the interaction of receptor binding domain (RBD) of the spike protein with peptidase domain of the human ACE-2 (hACE-2) receptor. So, possibly the mutations in the RBD domain of spike protein in the new variants could modulate the protein-protein interaction with hACE-2 receptor leading to the increased virulence. In this study, we aim to get molecular level understanding into the mechanism behind the increased infection rate due to such mutations in these variants. We have computationally studied the interaction of the spike protein in both wild-type and B.1.1.7 variant with hACE-2 receptor using combined molecular dynamics and binding free energy calculations using molecular mechanics-Generalized Born surface area (MM-GBSA) approach. The binding free energies computed using configurations from minimization run and low temperature simulation show that mutant variant of spike protein has increased binding affinity for hACE-2 receptor (i.e. \u0394\u0394G(N501Y,A570D) is in the range \u221220.4 to \u221221.4 kcal/mol)The residue-wise decomposition analysis and intermolecular hydrogen bond analysis evidenced that the N501Y mutation has increased interaction between RBD of spike protein with ACE-2 receptor. We have also carried out calculations using density functional theory and the results evidenced the increased interaction between three pairs of residues (TYR449 (spike)-ASP38 (ACE-2), TYR453-HIE34 and TYR501-LYS353) in the variant that could be attributed to its increased virulence. The free energies of wild-type and mutant variants of the spike protein computed from MM-GBSA approach suggests that latter variant is stable by about \u221210.4 kcal/mol when compared to wild type suggesting that it will be retained in the evolution due to increased stability. We demonstrate that with the use of the state-of-the art of computational approaches, we can in advance predict the more virulent nature of variants of SARS-CoV-2 and alert the world health-care system.","version":"1.1","doi":"10.1101/2021.01.25.428190","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.426407","pub_date":"2021-1-26","title":"Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio","abstract":"Following the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa) that manifest multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20G variants. One variant, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His (Q677H), affecting a consensus QTQTN domain near the S1/S2 furin cleavage site, nucleocapsid (N) p.Asp377Tyr (D377Y) and membrane glycoprotein (M) p.Ala85Ser (A85S) mutations, with additional S mutations in subsets. The other variant present in two samples, contains S N501Y, which is a marker of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. The Ohio variant is from a different clade and shares multiple mutations with the clade 20G viruses circulating in the area prior to December 2020. These two SARS-CoV-2 viruses, which we show are also present and evolving currently in several other parts of North America, add to the diversity of S gene shifts occurring worldwide. These and other shifts in this period of the pandemic support multiple independent acquisition of functionally significant and potentially complementing mutations affecting the S QTQTN site (Q675H or Q677H) and certain receptor binding domain mutations (e.g., E484K and N501Y).","version":"1.3","doi":"10.1101/2021.01.12.426407","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.343673","pub_date":"2021-1-26","title":"Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy","abstract":"The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 \u201cAchilles heel,\u201d the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e.g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential \u201cAchilles heels\u201d for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.","version":"1.3","doi":"10.1101/2020.10.22.343673","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428296","pub_date":"2021-1-26","title":"Variability in codon usage in Coronaviruses is mainly driven by mutational bias and selective constraints on CpG dinucleotide","abstract":"The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus within the Orthocoronavirinae causing an emergent infectious disease in humans, the ongoing coronavirus disease 2019 pandemic (COVID-19). Due to the high zoonotic potential of these viruses, it is critical to unravel their evolutionary history of host species shift, adaptation and emergence. Only such knowledge can guide virus discovery, surveillance and research efforts to identify viruses posing a pandemic risk in humans. We present a comprehensive analysis of the composition and codon usage bias of the 82 Orthocoronavirinae members, infecting 47 different avian and mammalian hosts. Our results clearly establish that synonymous codon usage varies widely among viruses and is only weakly dependent on the type of host they infect. Instead, we identify mutational bias towards AT-enrichment and selection against CpG dinucleotides as the main factors responsible of the codon usage bias variation. Further insight on the mutational equilibrium within Orthocoronavirinae revealed that most coronavirus genomes are close to their neutral equilibrium, the exception is the three recently-infecting human coronaviruses, which lie further away from the mutational equilibrium than their endemic human coronavirus counterparts. Finally, our results suggest that while replicating in humans SARS-CoV-2 is slowly becoming AT-richer, likely until attaining a new mutational equilibrium.","version":"1.1","doi":"10.1101/2021.01.26.428296","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.26.428302","pub_date":"2021-1-26","title":"40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations","abstract":"A one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants\u2019 prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.","version":"1.1","doi":"10.1101/2021.01.26.428302","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428049","pub_date":"2021-1-26","title":"Haploflow: Strain-resolved de novo assembly of viral genomes","abstract":"In viral infections often multiple related viral strains are present, due to coinfection or within-host evolution. We describe Haploflow, a de Bruijn graph-based assembler for de novo genome assembly of viral strains from mixed sequence samples using a novel flow algorithm. We assessed Haploflow across multiple benchmark data sets of increasing complexity, showing that Haploflow is faster and more accurate than viral haplotype assemblers and generic metagenome assemblers not aiming to reconstruct strains. Haplotype reconstructed high-quality strain-resolved assemblies from clinical HCMV samples and SARS-CoV-2 genomes from wastewater metagenomes identical to genomes from clinical isolates.","version":"1.1","doi":"10.1101/2021.01.25.428049","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428025","pub_date":"2021-1-26","title":"Mechanism of Viral Glycoprotein Targeting by Membrane-associated-RING-CH Proteins","abstract":"An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins. Viral envelope glycoproteins are an important structural component on the surface of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.","version":"1.2","doi":"10.1101/2021.01.25.428025","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.427896","pub_date":"2021-1-25","title":"A multiscale model suggests that a moderately weak inhibition of SARS-CoV-2 replication by type I IFN could accelerate the clearance of the virus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that emerged in China at the end of 2019 and caused a large global outbreak. The interaction between SARS-CoV-2 and the immune response is complex because it is regulated by various processes taking part at the intracellular, tissue, and host levels. To gain a better understanding of the pathogenesis and progression of COVID-19, we formulate a multiscale model that integrate the main mechanisms which regulate the immune response to SARS-CoV-2 across multiple scales. The model describes the effect of type I interferon on the replication of SARS-CoV-2 inside cells. At the tissue level, we simulate the interactions between infected cells and immune cells using a hybrid agent-based representation. At the same time, we model the dynamics of virus spread and adaptive immune response in the host organism. After model validation, we demonstrate that a moderately weak inhibition of virus replication by type I IFN could elicit a strong adaptive immune response which accelerates the clearance of the virus. Furthermore, numerical simulations suggest that the deficiency of lymphocytes and not dendritic cells could lead to unfavourable outcomes in the elderly population.","version":"1.1","doi":"10.1101/2021.01.25.427896","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428097","pub_date":"2021-1-25","title":"Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent individuals up to 8 months post-symptom onset","abstract":"Functional and lasting immune responses to the novel coronavirus (SARS-CoV-2) are currently under intense investigation as antibody titers in plasma have been shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we sought to determine the presence of SARS-CoV-2-specific memory B cells in COVID-19 convalescent patients. In this study, we report on the evolution of the overall humoral immune responses on 101 blood samples obtained from 32 COVID-19 convalescent patients between 16 and 233 days post-symptom onset. Our observations indicate that anti-Spike and anti-RBD IgM in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity in convalescent plasma declines rapidly compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which increase over time, and the number of IgG+ memory B cells which remain stable thereafter for up to 8 months after symptoms onset. With the recent approval of highly effective vaccines for COVID-19, data on the persistence of immune responses are of central importance. Even though overall circulating SARS-CoV-2 Spike-specific antibodies contract over time during convalescence, we demonstrate that RBD-specific B cells increase and persist up to 8 months post symptom onset. We also observe modest increases in RBD-specific IgG+ memory B cells and importantly, detectable IgG and sustained Fc-effector activity in plasma over the 8-month period. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for the prevention of secondary infections, vaccine efficacy and herd immunity against COVID-19.","version":"1.1","doi":"10.1101/2021.01.25.428097","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.427846","pub_date":"2021-1-25","title":"SARS-CoV-2 receptor binding mutations and antibody mediated immunity","abstract":"SARS-CoV-2 mutations can impact infectivity, viral load, and overall morbidity/mortality during infection. In this analysis, we look at the mutational landscape of the SARS-CoV-2 receptor binding domain, a structure that is antigenic and allows for viral binding to the host. We analyze 104193 GISAID sequences acquired on October 15th, 2020 with a majority of sequences (96%) containing point mutations. We report high frequency mutations with improved binding affinity to ACE2 including S477N, N439K, V367F, and N501Y and address the potential impact of RBD mutations on antibody binding. The high frequency S477N mutation is present in 6.7% of all SARS-CoV-2 sequences, co-occurs with D614G, and is currently present in 14 countries. To address RBD-antibody interactions we take a subset of human derived antibodies and define their interacting residues using PDBsum. Our analysis shows that adaptive immunity against SARS-CoV-2 enlists broad coverage of the RBD suggesting that antibody mediated immunity should be sufficient to resolve infection in the presence of RBD point mutations that conserve structure.","version":"1.1","doi":"10.1101/2021.01.25.427846","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.427729","pub_date":"2021-1-25","title":"Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine","abstract":"Multiple successful vaccines against SARS-CoV-2 are urgently needed to address the ongoing Covid-19 pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein co-administered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising of polybutadiene-b-polyethylene glycol and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells, which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of memory CD4+ and CD8+ T cells that produce Th1 cytokines. This study is an important step towards the development of an efficacious vaccine in humans.","version":"1.1","doi":"10.1101/2021.01.24.427729","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.428007","pub_date":"2021-1-25","title":"Effect of mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell-cell fusion function","abstract":"The SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S cell-cell fusion. Additionally, we examine S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this highly sought-after therapeutic target.","version":"1.1","doi":"10.1101/2021.01.24.428007","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.427948","pub_date":"2021-1-25","title":"mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative infection of a global pandemic that has led to more than 2 million deaths worldwide. The Moderna mRNA-1273 vaccine has demonstrated ~94% efficacy in a Phase 3 study and has been approved under Emergency Use Authorization. The emergence of SARS-CoV-2 variants with mutations in the spike protein, most recently circulating isolates from the United Kingdom (B.1.1.7) and Republic of South Africa (B.1.351), has led to lower neutralization from convalescent serum by pseudovirus neutralization (PsVN) assays and resistance to certain monoclonal antibodies. Here, using two orthogonal VSV and lentivirus PsVN assays expressing spike variants of 20E (EU1), 20A.EU2, D614G-N439, mink cluster 5, B.1.1.7, and B.1.351 variants, we assessed the neutralizing capacity of sera from human subjects or non-human primates (NHPs) that received mRNA-1273. No significant impact on neutralization against the B.1.1.7 variant was detected in either case, however reduced neutralization was measured against the mutations present in B.1.351. Geometric mean titer (GMT) of human sera from clinical trial participants in VSV PsVN assay using D614G spike was 1/1852. VSV pseudoviruses with spike containing K417N-E484K-N501Y-D614G and full B.1.351 mutations resulted in 2.7 and 6.4-fold GMT reduction, respectively, when compared to the D614G VSV pseudovirus. Importantly, the VSV PsVN GMT of these human sera to the full B.1.351 spike variant was still 1/290, with all evaluated sera able to fully neutralize. Similarly, sera from NHPs immunized with 30 or 100\u03bcg of mRNA-1273 had VSV PsVN GMTs of ~ 1/323 or 1/404, respectively, against the full B.1.351 spike variant with a ~ 5 to 10-fold reduction compared to D614G. Individual mutations that are characteristic of the B.1.1.7 and B.1.351 variants had a similar impact on neutralization when tested in VSV or in lentivirus PsVN assays. Despite the observed decreases, the GMT of VSV PsVN titers in human vaccinee sera against the B.1.351 variant remained at ~1/300. Taken together these data demonstrate reduced but still significant neutralization against the full B.1.351 variant following mRNA-1273 vaccination.","version":"1.1","doi":"10.1101/2021.01.25.427948","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428125","pub_date":"2021-1-25","title":"The CXCR6/CXCL16 axis links inflamm-aging to disease severity in COVID-19 patients","abstract":"Advancing age and chronic health conditions, significant risk factors for severe COVID-19, are associated with a pro-inflammatory state, termed inflamm-aging. CXCR6+ T cells are known to traffic to the lung and have been reported to increase with age. The ligand of CXCR6, CXCL16, is constitutively expressed in the lung and upregulated during inflammatory responses and the CXCR6/CXCL16 axis is associated with severe lung disease and pneumonia. Genome-wide association studies have also recently identified 3p21.31, encompassing the CXCR6 gene, as a susceptibility locus for severe COVID-19. We assessed numbers T cells expressing the chemokine receptor CXCR6 and plasma levels of CXCL16, in control and COVID-19 patients. Results demonstrated that circulating CD8+CXCR6+ T cells were significantly elevated with advancing age, yet virtually absent in patients with severe COVID-19. Peripheral levels of CXCL16 were significantly upregulated in severe COVID-19 patients compared to either mild COVID-19 patients or SARS-CoV-2 negative controls. This study supports a significant role of the CXCR6/CXCL16 axis in the immunopathogenesis of severe COVID-19.","version":"1.1","doi":"10.1101/2021.01.25.428125","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.25.428136","pub_date":"2021-1-25","title":"mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge","abstract":"The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.25.428136","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427863","pub_date":"2021-1-24","title":"CD8+ T cell epitope variations suggest a potential antigen presentation deficiency for spike protein of SARS-CoV-2","abstract":"COVID-19 is caused by a newly identified coronavirus, SARS-CoV-2, and has become a pandemic around the world. The illustration of the immune responses against SARS-CoV-2 is urgently needed for understanding the pathogenesis of the disease and its vaccine development. CD8+ T cells are critical for virus clearance and induce long lasting protection in the host. Here we identified specific HLA-A2 restricted T cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides (n-Sp1, 2, 6, 7, 11, 13, 14) were confirmed to bind with HLA-A2 and potentially be presented by antigen presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. In addition, these epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytotoxicity to target cells. Meanwhile, all these epitopes exhibited high frequency of variations. Among them, n-Sp1 epitope variation 5L>F significantly decreased the proportion of specific T cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A2 and decreased the proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SAR-CoV-2.","version":"1.1","doi":"10.1101/2021.01.22.427863","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.23.427885","pub_date":"2021-1-24","title":"Variation analysis of SARS-CoV-2 complete sequences from Iran","abstract":"The SARS-CoV-2 is a new emerging coronavirus initially reported in China at the late December 2019 and rapidly spread to the whole of the world. To date, 1261903 total case and 55830 deaths are reported from Iran as 8 January. In this study, we investigated all the complete sequences of SARS-CoV-2 that publicly reported from Iran. Twenty-four sequences between March to September 2020 were analyzed to identify genome variations and phylogenetic relationships. Furthermore, we assessed the amino acid changes related to the spike glycoprotein as an important viral factor associated with the entry to the host cells and as a vaccine target. Most of the variations are occurred in the ORF1ab, S, N, intergenic and ORF7 regions. The analysis of spike protein mutations demonstrated that D614G mutation could be detected from the May and beyond. Phylogenetic analysis showed that most of the circulated viruses in Iran are belong to the B.4 lineage. Although, we found a limited number of variants associated to the B.1 lineage carrying D614G mutation. Furthermore, we detected a variant characterize as the B.1.36 lineage with sixteen mutations in the spike protein region. This study showed the frequency of the viral populations in Iran as September, therefore, there is an emergent need to genomic surveillance to track viral lineage shift in the country beyond the September. These data would help to predict future situation and apply better strategy to control of the pandemic.","version":"1.1","doi":"10.1101/2021.01.23.427885","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.427089","pub_date":"2021-1-24","title":"Meta-analysis reveals consistent immune response patterns in COVID-19 infected patients at single-cell resolution","abstract":"A number of single-cell RNA studies looking at the human immune response to the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recently published. However, the number of samples used in each individual study typically is small, moreover the technologies and protocols used in different studies vary, thus somewhat restricting the range of conclusions that can be made with high confidence. To better capture the cellular gene expression changes upon SARS-CoV-2 infection at different levels and stages of disease severity and to minimise the effect of technical artefacts, we performed meta-analysis of data from 9 previously published studies, together comprising 143 human samples, and a set of 16 healthy control samples (10X). In particular, we used generally accepted immune cell markers to discern specific cell subtypes and to look at the changes of the cell proportion over different disease stages and their consistency across the studies. While half of the observations reported in the individual studies can be confirmed across multiple studies, half of the results seem to be less conclusive. In particular, we show that the differentially expressed genes consistently point to upregulation of type I Interferon signal pathway and downregulation of the mitochondrial genes, alongside several other reproducibly consistent changes. We also confirm the presence of expanded B-cell clones in COVID-19 patients, however, no consistent trend in T-cell clonal expansion was observed.","version":"1.1","doi":"10.1101/2021.01.24.427089","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.427939","pub_date":"2021-1-24","title":"In silico investigation of the new UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 variants with a focus at the ACE2-Spike RBD interface","abstract":"SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor and several in silico methods to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both UK and South African strains) should be favorable for the interaction with ACE2 while the K417N and E484K substitutions (South African) would seem unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the predicted less favorable (regarding binding) K417N and E484K Spike replacements. Our finding suggests that, if indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions outside the direct Spike-ACE2 interface. Transmission of the UK and possibly South African SARS-CoV-2 strains appears substantially increased compared to other variants This could be due, in part, to increased affinity between the variant Spike proteins and ACE2 We investigated in silico the 3D structure of the Spike-ACE2 complex with a focus on Spike K417N, E484K and N501Y The N501Y substitution is predicted to increase the affinity toward ACE2 (UK strain) with subsequent enhanced transmissibility and possibly pathogenicity Additional substitutions at positions 417 and 484 (South African strain) may pertub the interaction with ACE2 raising questions about transmissibility and pathogenicity","version":"1.1","doi":"10.1101/2021.01.24.427939","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.24.427965","pub_date":"2021-1-24","title":"An Autoantigen Atlas from Human Lung HFL1 Cells Offers Clues to Neurological and Diverse Autoimmune Manifestations of COVID-19","abstract":"COVID-19 is accompanied by a myriad of both transient and long-lasting autoimmune responses. Dermatan sulfate (DS), a glycosaminoglycan crucial for wound healing, has unique affinity for autoantigens (autoAgs) from apoptotic cells. DS-autoAg complexes are capable of stimulating autoreactive B cells and autoantibody production. Using DS affinity, we identified an autoantigenome of 408 proteins from human fetal lung fibroblast HFL11 cells, at least 231 of which are known autoAgs. Comparing with available COVID data, 352 proteins of the autoantigenome have thus far been found to be altered at protein or RNA levels in SARS-Cov-2 infection, 210 of which are known autoAgs. The COVID-altered proteins are significantly associated with RNA metabolism, translation, vesicles and vesicle transport, cell death, supramolecular fibrils, cytoskeleton, extracellular matrix, and interleukin signaling. They offer clues to neurological problems, fibrosis, smooth muscle dysfunction, and thrombosis. In particular, 150 altered proteins are related to the nervous system, including axon, myelin sheath, neuron projection, neuronal cell body, and olfactory bulb. An association with the melanosome is also identified. The findings from our study illustrate a strong connection between viral infection and autoimmunity. The vast number of COVID-altered proteins with propensity to become autoAgs offers an explanation for the diverse autoimmune complications in COVID patients. The variety of autoAgs related to mRNA metabolism, translation, and vesicles raises concerns about potential adverse effects of mRNA vaccines. The COVID autoantigen atlas we are establishing provides a detailed molecular map for further investigation of autoimmune sequelae of the pandemic. An autoantigenome by dermatan sulfate affinity from human lung HFL1 cells may explain neurological and autoimmune manifestations of COVID-19","version":"1.1","doi":"10.1101/2021.01.24.427965","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.171769","pub_date":"2021-1-22","title":"Genomic Mutations and Changes in Protein Secondary Structure and Solvent Accessibility of SARS-CoV-2 (COVID-19 Virus)","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic virus that has caused the global COVID-19 pandemic. Tracing the evolution and transmission of the virus is crucial to respond to and control the pandemic through appropriate intervention strategies. This paper reports and analyses genomic mutations in the coding regions of SARS-CoV-2 and their probable protein secondary structure and solvent accessibility changes, which are predicted using deep learning models. Prediction results suggest that mutation D614G in the virus spike protein, which has attracted much attention from researchers, is unlikely to make changes in protein secondary structure and relative solvent accessibility. Based on 6,324 viral genome sequences, we create a spreadsheet dataset of point mutations that can facilitate the investigation of SARS-CoV-2 in many perspectives, especially in tracing the evolution and worldwide spread of the virus. Our analysis results also show that coding genes E, M, ORF6, ORF7a, ORF7b and ORF10 are most stable, potentially suitable to be targeted for vaccine and drug development.","version":"1.2","doi":"10.1101/2020.07.10.171769","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427775","pub_date":"2021-1-22","title":"Insights from Genomes and Genetic Epidemiology of SARS-CoV-2 isolates from the state of Andhra Pradesh","abstract":"Coronavirus disease (COVID-19) emerged from a city in China and has now spread as a global pandemic affecting millions of individuals. The causative agent, SARS-CoV-2 is being extensively studied in terms of its genetic epidemiology using genomic approaches. Andhra Pradesh is one of the major states of India with the third-largest number of COVID-19 cases with limited understanding of its genetic epidemiology. In this study, we have sequenced 293 SARS-CoV-2 genome isolates from Andhra Pradesh with a mean coverage of 13,324X. We identified 564 high-quality SARS-CoV-2 variants, out of which 15 are novel. A total of 18 variants mapped to RT-PCR primer/probe sites, and 4 variants are known to be associated with an increase in infectivity. Phylogenetic analysis of the genomes revealed the circulating SARS-CoV-2 in Andhra Pradesh majorly clustered under the clade A2a (94%), while 6% fall under the I/A3i clade, a clade previously defined to be present in large numbers in India. To the best of our knowledge, this is the most comprehensive genetic epidemiological analysis performed for the state of Andhra Pradesh.","version":"1.1","doi":"10.1101/2021.01.22.427775","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427315","pub_date":"2021-1-22","title":"DINC-COVID: A webserver for ensemble docking with flexible SARS-CoV-2 proteins","abstract":"Recent efforts to computationally identify inhibitors for SARS-CoV-2 proteins have largely ignored the issue of receptor flexibility. We have implemented a computational tool for ensemble docking with the SARS-CoV-2 proteins, including the main protease (Mpro), papain-like protease (PLpro) and RNA-dependent RNA polymerase (RdRp). Ensembles of other SARS-CoV-2 proteins are being prepared and made available through a user-friendly docking interface. Plausible binding modes between conformations of a selected ensemble and an uploaded ligand are generated by DINC, our parallelized meta-docking tool. Binding modes are scored with three scoring functions, and account for the flexibility of both the ligand and receptor. Additional details on our methods are provided in the supplementary material. dinc-covid.kavrakilab.org Details on methods for ensemble generation and docking are provided as supplementary data online. geancarlo.zanatta@ufc.br, kavraki@rice.edu","version":"1.1","doi":"10.1101/2021.01.21.427315","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424149","pub_date":"2021-1-22","title":"A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process","abstract":"SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral main protease is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. Although several mechanisms of self-cleavage had been proposed for SARS-CoV, the lack of structural information for each step is a setback to the understanding of this process. Herein, we used X-ray crystallography to characterize an immature form of the main protease, which revealed major conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis-cleavage of N-terminal residues within the dimer, leading to the mature active site. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the main protease bound to its endogenous N and C-terminal residues during the formation of the tetramer. This quaternary form is also present in solution, suggesting a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during maturation, which can guide the development of new inhibitors targeting its intermediary states.","version":"1.2","doi":"10.1101/2020.12.23.424149","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.123976","pub_date":"2021-1-22","title":"Temporal evolution and adaptation of SARS-COV-2 codon usage","abstract":"The outbreak of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has caused an unprecedented pandemic. Since the first sequenced whole-genome of SARS-CoV-2 on January 2020, the identification of its genetic variants has become crucial in tracking and evaluating their spread across the globe. In this study, we compared 134,905 SARS-CoV-2 genomes isolated from all affected countries since the outbreak of this novel coronavirus with the first sequenced genome in Wuhan, China to quantify the evolutionary divergence of SARS-CoV-2. Thus, we compared the codon usage patterns of SARS-CoV-2 genes encoding the membrane protein (M), envelope (E), spike surface glycoprotein (S), nucleoprotein (N), RNA-dependent RNA polymerase (RdRp). The polyproteins ORF1a and ORF1b were examined separately. We found that SARS-CoV-2 tends to diverge over time by accumulating mutations on its genome and, specifically, on the sequences encoding proteins N and S. Interestingly, different patterns of codon usage were observed among these genes. Genes S and N tend to use a narrower set of synonymous codons that are better optimized to the human host. Conversely, genes E and M consistently use the broader set of synonymous codons, which does not vary in respect to the reference genome. CAI and SiD time evolutions show a tendency to decrease that emerge for most genes. Forsdyke plots are used to study the nature of mutations and they show a rapid evolutionary divergence of each gene, due to the low values of x-intercepets.","version":"1.2","doi":"10.1101/2020.05.29.123976","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427749","pub_date":"2021-1-22","title":"Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies","abstract":"SARS-CoV-2 Spike-specific antibodies contribute the majority of the neutralizing activity in most convalescent human sera. Two SARS-CoV-2 variants, N501Y.V1 (also known as B.1.1.7 lineage or VOC-202012/01) and N501Y.V2 (B.1.351 lineage), reported from the United Kingdom and South Africa, contain several mutations in the receptor binding domain of Spike and are of particular concern. To address the infectivity and neutralization escape phenotypes potentially caused by these mutations, we used SARS-CoV-2 pseudovirus system to compare the viral infectivity, as well as the neutralization activities of convalescent sera and monoclonal antibodies (mAbs) against SARS-CoV-2 variants. Our results showed that N501Y Variant 1 and Variant 2 increase viral infectivity compared to the reference strain (wild-type, WT) in vitro. At 8 months after symptom onset, 17 serum samples of 20 participants (85%) retaining titers of ID50 >40 against WT pseudovirus, whereas the NAb titers of 8 samples (40%) and 18 samples (90%) decreased below the threshold against N501Y.V1 and N501Y.V2, respectively. In addition, both N501Y Variant 1 and Variant 2 reduced neutralization sensitivity to most (6/8) mAbs tested, while N501Y.V2 even abrogated neutralizing activity of two mAbs. Taken together the results suggest that N501Y.V1 and N501Y.V2 reduce neutralization sensitivity to some convalescent sera and mAbs.","version":"1.1","doi":"10.1101/2021.01.22.427749","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427802","pub_date":"2021-1-22","title":"Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation","abstract":"Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein that facilitates serine protease-mediated entry into human airway cells. We report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents MBCS mutations. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.","version":"1.1","doi":"10.1101/2021.01.22.427802","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.22.427737","pub_date":"2021-1-22","title":"Human embryonic stem cell-derived cardiomyocytes express SARS-CoV-2 host entry proteins: screen to identify inhibitors of infection","abstract":"Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.01.22.427737","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.171975","pub_date":"2021-1-22","title":"Increased Expression of Chondroitin Sulfotransferases following AngII may Contribute to Pathophysiology Underlying Covid-19 Respiratory Failure: Impact may be Exacerbated by Decline in Arylsulfatase B Activity","abstract":"The precise mechanisms by which Covid-19 infection leads to hypoxia and respiratory failure have not yet been elucidated. Interactions between sulfated glycosaminoglycans (GAGs) and the SARS-CoV-2 spike glycoprotein have been identified as participating in viral adherence and infectivity. The spike glycoprotein binds to respiratory epithelium through the angiotensin converting enzyme 2 (ACE2) receptor, which endogenously interacts with Angiotensin (Ang) II to yield Angiotensin 1-7. In this report, we show that stimulation of human vascular smooth muscle cells by Ang II leads to increased mRNA expression of two chondroitin sulfotransferases (CHST11 and CHST15), which are required for synthesis of chondroitin 4-sulfate (C4S) and chondroitin 4,6-disulfate (CSE), respectively. Also, increased total sulfated GAGs, increased sulfotransferase activity, and increased expression of the proteoglycans biglycan, syndecan, perlecan, and versican followed treatment by Ang II. Candesartan, an Angiotensin II receptor blocker (Arb), largely, but incompletely, inhibited these increases, and the differences from baseline remained significant. These results suggest that another effect of Ang II also contributes to the increased expression of chondroitin sulfotransferases, total sulfated GAGs, and proteoglycans. We hypothesize that activation of ACE2 may contribute to these increases and suggest that the SARS-CoV-2 spike glycoprotein interaction with ACE2 may also increase chondroitin sulfotransferases, sulfated GAGs, and proteoglycans and thereby contribute to viral adherence to bronchioalveolar cells and to respiratory compromise in SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.06.25.171975","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.190546","pub_date":"2021-1-22","title":"The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus","abstract":"SARS-CoV-1 and SARS-CoV-2 are not phylogenetically closely related; however, both use the ACE2 receptor in humans for cell entry. This is not a universal sarbecovirus trait; for example, many known sarbecoviruses related to SARS-CoV-1 have two deletions in the receptor binding domain of the spike protein that render them incapable of using human ACE2. Here, we report three sequences of a novel sarbecovirus from Rwanda and Uganda which are phylogenetically intermediate to SARS-CoV-1 and SARS-CoV-2 and demonstrate via in vitro studies that they are also unable to utilize human ACE2. Furthermore, we show that the observed pattern of ACE2 usage among sarbecoviruses is best explained by recombination not of SARS-CoV-2, but of SARS-CoV-1 and its relatives. We show that the lineage that includes SARS-CoV-2 is most likely the ancestral ACE2-using lineage, and that recombination with at least one virus from this group conferred ACE2 usage to the lineage including SARS-CoV-1 at some time in the past. We argue that alternative scenarios such as convergent evolution are much less parsimonious; we show that biogeography and patterns of host tropism support the plausibility of a recombination scenario; and we propose a competitive release hypothesis to explain how this recombination event could have occurred and why it is evolutionarily advantageous. The findings provide important insights into the natural history of ACE2 usage for both SARS-CoV-1 and SARS-CoV-2, and a greater understanding of the evolutionary mechanisms that shape zoonotic potential of coronaviruses. This study also underscores the need for increased surveillance for sarbecoviruses in southwestern China, where most ACE2-using viruses have been found to date, as well as other regions such as Africa, where these viruses have only recently been discovered.","version":"1.2","doi":"10.1101/2020.07.07.190546","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.21250266","pub_date":"2021-01-22","title":"Excess mortality associated with the COVID-19 pandemic among Californians 18\u201365 years of age, by occupational sector and occupation: March through October 2020","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Though SARS-CoV-2 outbreaks have been documented in occupational settings and though there is speculation that essential workers face heightened risks for COVID-19, occupational differences in excess mortality have, to date, not been examined. Such information could point to opportunities for intervention, such as workplace modifications and prioritization of vaccine distribution.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods and findings</jats:title>\n                  <jats:p>Using death records from the California Department of Public Health, we estimated excess mortality among Californians 18\u201365 years of age by occupational sector and occupation, with additional stratification of the sector analysis by race/ethnicity. During the COVID-19 pandemic, working age adults experienced a 22% increase in mortality compared to historical periods. Relative excess mortality was highest in food/agriculture workers (39% increase), transportation/logistics workers (28% increase), facilities (27%) and manufacturing workers (23% increase). Latino Californians experienced a 36% increase in mortality, with a 59% increase among Latino food/agriculture workers. Black Californians experienced a 28% increase in mortality, with a 36% increase for Black retail workers. Asian Californians experienced an 18% increase, with a 40% increase among Asian healthcare workers. Excess mortality among White working-age Californians increased by 6%, with a 16% increase among White food/agriculture workers.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Certain occupational sectors have been associated with high excess mortality during the pandemic, particularly among racial and ethnic groups also disproportionately affected by COVID-19. In-person essential work is a likely venue of transmission of coronavirus infection and must be addressed through strict enforcement of health orders in workplace settings and protection of in-person workers. Vaccine distribution prioritizing in-person essential workers will be important for reducing excess COVID mortality.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.01.21.21250266","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.21.427574","pub_date":"2021-1-21","title":"Evolving Insights from SARS-CoV-2 Genome from 200K COVID-19 Patients","abstract":"We present an updated version of our automated computational pipeline, Infection Pathogen Detector IPD 2.0 with a SARS-CoV-2 module, to perform genomic analysis to understand the pathogenesis and virulence of the virus. Analysing the currently available 208911 SARS-CoV2 genome sequences (as accessed on 28 Dec 2020), we generate an extensive database of sample- wise variants and clade annotation, which forms the core of the SARS-CoV-2 analysis module of the analysis pipeline. A comparative account of lineage-specific mutations in the newer SARS-CoV-2 strains emerging in the UK, South Africa and Brazil along with data reported from India identify overlapping and lineages specific acquired mutations suggesting a repetitive convergent and adaptive evolution. Thus, the persistence of pandemic may lead to the emergence of newer regional strains with improved fitness. IPD 2.0 also adopts the recent dynamic clade nomenclature and shows improvement in accuracy of clade assignment, processing time and portability, to its predecessor and thus could be a vital tool to help facilitate genomic surveillance in a population to identify variants involved in breakthrough infections.","version":"1.1","doi":"10.1101/2021.01.21.427574","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427657","pub_date":"2021-1-21","title":"High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity","abstract":"SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 \u03bcM range in vitro. Furthermore, Exebryl-1, a \u03b2-amyloid anti-aggregation molecule for Alzheimer\u2019s therapy, was shown to have antiviral activity between 10 to 66 \u03bcM, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral. Drugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.","version":"1.1","doi":"10.1101/2021.01.21.427657","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.20.427541","pub_date":"2021-1-21","title":"In vitro infection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response","abstract":"Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the in vitro study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 16 donors, most of whom had risk factors for severe illness from COVID-19. Cryopreserved tissues preserved 90% of cell viability and contained heterogeneous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectible with HCoV-OC43 and SARS-CoV-2 coronavirus strains, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL-6, CXCL8 and IFN\u03b2 in response to SARS-CoV-2 infection. Treatment of tissues with dexamethasone and the experimental drug, N-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted antiviral activity, suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the in vitro study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis. The current biological systems for the study of COVID-19 are in vitro systems that differ from the human lung in many respects, and animal hosts to which the virus is not adapted. We developed another alternative for studying pathogenesis and drug susceptibility of SARS-CoV-2 in a cryopreserved bank of human lung tissues. We consider the importance of this work to relate to the practical use of this culture system as a repeatable and scalable approach that allows for the study of an important infection in relevant tissues. The tissue bank highlights the heterogeneous response to SARS-CoV-2 infection and treatment, which allows researchers to investigate why treatments work in some donors but not others.","version":"1.1","doi":"10.1101/2021.01.20.427541","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427629","pub_date":"2021-1-21","title":"Survey of peridomestic mammal susceptibility to SARS-CoV-2 infection","abstract":"Wild animals have been implicated as the origin of SARS-CoV-2, but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. Here we show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our work expands upon the existing knowledge base of susceptible species and provides evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.21.427629","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.21.427563","pub_date":"2021-1-21","title":"Publication practices during the COVID-19 pandemic: Biomedical preprints and peer-reviewed literature","abstract":"The coronavirus pandemic introduced many changes to our society, and deeply affected the established in biomedical sciences publication practices. In this article, we present a comprehensive study of the changes in scholarly publication landscape for biomedical sciences during the COVID-19 pandemic, with special emphasis on preprints posted on bioRxiv and medRxiv servers. We observe the emergence of a new category of preprint authors working in the fields of immunology, microbiology, infectious diseases, and epidemiology, who extensively used preprint platforms during the pandemic for sharing their immediate findings. The majority of these findings were works-in-progress unfitting for a prompt acceptance by refereed journals. The COVID-19 preprints that became peer-reviewed journal articles were often submitted to journals concurrently with the posting on a preprint server, and the entire publication cycle, from preprint to the online journal article, took on average 63 days. This included an expedited peer-review process of 43 days and journal\u2019s production stage of 15 days, however there was a wide variation in publication delays between journals. Only one third of COVID-19 preprints posted during the first nine months of the pandemic appeared as peer-reviewed journal articles. These journal articles display high Altmetric Attention Scores further emphasizing a significance of COVID-19 research during 2020. This article will be relevant to editors, publishers, open science enthusiasts, and anyone interested in changes that the 2020 crisis transpired to publication practices and a culture of preprints in life sciences.","version":"1.1","doi":"10.1101/2021.01.21.427563","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427324","pub_date":"2021-1-20","title":"Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein","abstract":"Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.","version":"1.1","doi":"10.1101/2021.01.19.427324","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.20.427368","pub_date":"2021-1-20","title":"GRP78 binds SARS-CoV-2 Spike protein and ACE2 and GRP78 depleting antibody blocks viral entry and infection in vitro","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors may also play major roles in viral infection. Here we report that the stress-inducible molecular chaperone GRP78 can form a complex with the SARS-CoV-2 Spike protein and ACE2 intracellularly and on the cell surface, and that the substrate binding domain of GRP78 is critical for this function. Knock-down of GRP78 by siRNA dramatically reduced cell surface ACE2 expression. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159), selected for its ability to cause GRP78 endocytosis and its safe clinical profile in preclinical models, reduces cell surface ACE2 expression, SARS-CoV-2 Spike-driven viral entry, and significantly inhibits SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78.","version":"1.1","doi":"10.1101/2021.01.20.427368","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427330","pub_date":"2021-1-20","title":"Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts","abstract":"Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified a viral load threshold that minimizes false positives. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.","version":"1.1","doi":"10.1101/2021.01.19.427330","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427355","pub_date":"2021-1-20","title":"Increased elastase sensitivity and decreased intramolecular interactions in the more transmissible SARS-CoV-2 variants\u2019 spike protein: Analysis of the new UK and SA SARS-CoV-2 variants","abstract":"Two SARS-CoV-2 variants showing increased transmissibility relative to the Wuhan virus have recently been identified. Although neither variant causes more severe illness or increased risk of death, the faster spread of the virus is a major threat. Using computational tools, we found that the new SARS-CoV-2 variants may acquire an increased transmissibility by increasing the propensity of its spike protein to expose the receptor binding domain. This information leads to the identification of potential treatments to avert the imminent threat of these more transmittable SARS-CoV-2 variants. The more infective SARS-CoV-2 variants may expose its Achilles Heel \u2013 an opportunity to reduce their spreading.","version":"1.1","doi":"10.1101/2021.01.19.427355","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.21249786","pub_date":"2021-01-20","title":"Emergence of a novel SARS-CoV-2 strain in Southern California, USA","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Since October 2020, novel strains of SARS-CoV-2 including B.1.1.7, have been identified to be of global significance from an infection and surveillance perspective. While this strain (B.1.1.7) may play an important role in increased COVID rates in the UK, there are still no reported strains to account for the spike of cases in Los Angeles (LA) and California as a whole, which currently has some of the highest absolute and per-capita COVID transmission rates in the country. From the early days of the pandemic when LA only had a single viral genome uploaded onto GISAID we have been at the forefront of generating and analyzing the SARS-CoV-2 sequencing data from the LA region. We report a novel strain emerging in Southern California. Most current cases in the catchment population in LA fall into two distinct subclades: 1) 20G (24% of total) is the predominant subclade currently in the United States 2) a relatively novel strain in clade 20C, CAL.20C strain (\u223c36% of total) is defined by five concurrent mutations. After an analysis of all of the publicly available data and a comparison to our recent sequences, we see a dramatic growth in the relative percentage of the CAL.20C strain beginning in November of 2020. The predominance of this strain coincides with the increased positivity rate seen in this region. Unlike 20G, this novel strain CAL.20C is defined by multiple mutations in the S protein, a characteristic it shares with both the UK and South African strains, both of which are of significant clinical and scientific interest</jats:p>","version":null,"doi":"10.1101/2021.01.18.21249786","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.19.426622","pub_date":"2021-1-20","title":"Evaluation of the effects of SARS-CoV-2 genetic mutations on diagnostic RT-PCR assays","abstract":"Several mutant strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging. Mismatch(es) in primer/probe binding regions would decrease the detection sensitivity of the PCR test, thereby affecting the results of clinical testing. In this study, we conducted an in silico survey on SARS-CoV-2 sequence variability within the binding regions of primer/probe published by the Japan National Institute of Infectious Diseases (NIID) and Centers for Disease Control and Prevention (CDC). In silico analysis revealed the presence of mutations in the primer/probe binding regions. We performed RT-PCR assays using synthetic RNAs containing the mutations and showed that some mutations significantly decreased the detection sensitivity of the RT-PCR assays. Our results highlight the importance of genomic monitoring of SARS-CoV-2 and evaluating the effects of mismatches on PCR testing sensitivity.","version":"1.1","doi":"10.1101/2021.01.19.426622","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427373","pub_date":"2021-1-20","title":"Inactivated rabies virus vectored SARS-CoV-2 vaccine prevents disease in a Syrian hamster model","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent coronavirus that has caused a worldwide pandemic. Although human disease is often asymptomatic, some develop severe illnesses such as pneumonia, respiratory failure, and death. There is an urgent need for a vaccine to prevent its rapid spread as asymptomatic infections accounting for up to 40% of transmission events. Here we further evaluated an inactivated rabies vectored SARS-CoV-2 S1 vaccine CORAVAX in a Syrian hamster model. CORAVAX adjuvanted with MPLA-AddaVax, a TRL4 agonist, induced high levels of neutralizing antibodies and generated a strong Th1-biased immune response. Vaccinated hamsters were protected from weight loss and viral replication in the lungs and nasal turbinates three days after challenge with SARS-CoV-2. CORAVAX also prevented lung disease, as indicated by the significant reduction in lung pathology. This study highlights CORAVAX as a safe, immunogenic, and efficacious vaccine that warrants further assessment in human trials.","version":"1.1","doi":"10.1101/2021.01.19.427373","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427320","pub_date":"2021-1-20","title":"Coevolutionary Analysis and Perturbation-Based Network Modeling of the SARS-CoV-2 Spike Protein Complexes with Antibodies: Binding-Induced Control of Dynamics, Allosteric Interactions and Signaling","abstract":"The structural and biochemical studies of the SARS-CoV-2 spike glycoproteins and complexes with highly potent antibodies have revealed multiple conformation-dependent epitopes highlighting the link between conformational plasticity of spike proteins and capacity for eliciting specific binding and broad neutralization responses. In this study, we used coevolutionary analysis, molecular simulations, and perturbation-based hierarchical network modeling of the SARS-CoV-2 S complexes with H014, S309, S2M11 and S2E12 antibodies targeting distinct epitopes to explore molecular mechanisms underlying binding-induced modulation of dynamics, stability and allosteric signaling in the spike protein trimers. The results of this study revealed key regulatory centers that can govern allosteric interactions and communications in the SARS-CoV-2 spike proteins. Through coevolutionary analysis of the SARS-CoV-2 spike proteins, we identified highly coevolving hotspots and functional clusters forming coevolutionary networks. The results revealed significant coevolutionary couplings between functional regions separated by the medium-range distances which may help to facilitate a functional cross-talk between distant allosteric regions in the SARS-CoV-2 spike complexes with antibodies. We also discovered a potential mechanism by which antibody-specific targeting of coevolutionary centers can allow for efficient modulation of allosteric interactions and signal propagation between remote functional regions. Using a hierarchical network modeling and perturbation-response scanning analysis, we demonstrated that binding of antibodies could leverage direct contacts with coevolutionary hotspots to allosterically restore and enhance couplings between spatially separated functional regions, thereby protecting the spike apparatus from membrane fusion. The results of this study also suggested that antibody binding can induce a switch from a moderately cooperative population-shift mechanism, governing structural changes of the ligand-free SARS-CoV-2 spike protein, to antibody-induced highly cooperative mechanism that can better withstand mutations in the functional regions without significant deleterious consequences for protein function. This study provides a novel insight into allosteric regulatory mechanisms of SARS-CoV-2 S proteins, showing that antibodies can modulate allosteric interactions and signaling of spike proteins, providing a plausible strategy for therapeutic intervention by targeting specific hotspots of allosteric interactions in the SARS-CoV-2 proteins.","version":"1.1","doi":"10.1101/2021.01.19.427320","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.20.427105","pub_date":"2021-1-20","title":"Use Of Canine Olfactory Detection For COVID-19 Testing Study On U.A.E. Trained Detection Dog Sensitivity","abstract":"This study aimed to evaluate the sensitivity of 21 dogs belonging to different United Arab Emirates (UAE) Ministry of Interior (MOI), trained for COVID-19 olfactory detection. The study involved 17 explosives detection dogs, two cadaver detection dogs and two dogs with no previous detection training. Training lasted two weeks before starting the validation protocol. Sequential five and seven-cone line-ups were used with axillary sweat samples from symptomatic COVID-19 individuals (SARS-CoV-2 PCR positive) and from asymptomatic COVID-19 negative individuals (SARS-CoV-2 PCR negative). A total of 1368 trials were performed during validation, including 151 positive and 110 negative samples. Each line-up had one positive sample and at least one negative sample. The dog had to mark the positive sample, randomly positioned behind one of the cones. The dog, handler and data recorder were blinded to the positive sample location. The calculated overall sensitivities were between 71% and 79% for three dogs, between83% and 87% for three other dogs, and equal to or higher than 90% for the remaining 15 dogs (more than two thirds of the 21 dogs). After calculating the overall sensitivity for each dog using all line-ups, \u201cmatched\u201d sensitivities were calculated only including line-ups containing COVID-19 positive and negative samples strictly comparable on confounding factors such as diabetes, anosmia, asthma, fever, body pain, diarrhoea, sex, hospital, method of sweat collection and sampling duration. Most of the time, the sensitivities increased after matching. Pandemic conditions in the U.A.E., associated with the desire to use dogs as an efficient mass-pretesting tool has already led to the operational deployment of the study dogs. Future studies will focus on comparatives fields-test results including the impact of the main COVID-19 comorbidities and other respiratory tract infections.","version":"1.1","doi":"10.1101/2021.01.20.427105","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427310","pub_date":"2021-1-20","title":"A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering","abstract":"A \u201cuniversal\u201d vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both TH1 and TH2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.","version":"1.1","doi":"10.1101/2021.01.19.427310","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.21249884","pub_date":"2021-01-20","title":"Estimating the effects of non-pharmaceutical interventions on the number of new infections with COVID-19 during the first epidemic wave","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The novel coronavirus (SARS-CoV-2) has rapidly developed into a global epidemic. To control its spread, countries have implemented non-pharmaceutical interventions (NPIs), such as school closures, gathering bans, or even stay-at-home orders. Here we study the effectiveness of seven NPIs in reducing the number of new infections, which was inferred from the reported cases of COVID-19 using a semi-mechanistic Bayesian hierarchical model. Based on data from the first epidemic wave of\n                  <jats:italic>n</jats:italic>\n                  = 20 countries (i.e., the United States, Canada, Australia, the EU-15 countries, Norway, and Switzerland), we estimate the relative reduction in the number of new infections attributed to each NPI. Among the NPIs considered, event bans were most effective, followed by venue and school closures, whereas stay-at-home orders and work bans were least effective. With this retrospective cross-country analysis, we provide estimates regarding the effectiveness of different NPIs during the first epidemic wave.\n                </jats:p>","version":null,"doi":"10.1101/2021.01.15.21249884","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.19.427282","pub_date":"2021-1-19","title":"SARS-CoV-2 infection of circulating immune cells is not responsible for virus dissemination in severe COVID-19 patients","abstract":"In late 2019 a novel coronavirus (SARS-CoV-2) emerged, and has since caused a global pandemic. Understanding the pathogenesis of COVID-19 disease is necessary to inform development of therapeutics, and management of infected patients. Using scRNAseq of blood drawn from SARS-CoV-2 patients, we asked whether SARS-CoV-2 may exploit immune cells as a \u2018Trojan Horse\u2019 to disseminate and access multiple organ systems. Our data suggests that circulating cells are not actively infected with SARS-CoV-2, and do not appear to be a source of viral dissemination.","version":"1.1","doi":"10.1101/2021.01.19.427282","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426463","pub_date":"2021-1-19","title":"Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine","abstract":"Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2021.01.15.426463","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.311845","pub_date":"2021-1-19","title":"An evolutionary portrait of the progenitor SARS-CoV-2 and its dominant offshoots in COVID-19 pandemic","abstract":"We report the likely most recent common ancestor of SARS-CoV-2 \u2013 the coronavirus that causes COVID-19. This progenitor SARS-CoV-2 genome was recovered through a novel application and advancement of computational methods initially developed to reconstruct the mutational history of tumor cells in a patient. The progenitor differs from the earliest coronaviruses sampled in China by three variants, implying that none of the earliest patients represent the index case or gave rise to all the human infections. However, multiple coronavirus infections in China and the USA harbored the progenitor genetic fingerprint in January 2020 and later, suggesting that the progenitor was spreading worldwide as soon as weeks after the first reported cases of COVID-19. Mutations of the progenitor and its offshoots have produced many dominant coronavirus strains, which have spread episodically over time. Fingerprinting based on common mutations reveals that the same coronavirus lineage has dominated North America for most of the pandemic. There have been multiple replacements of predominant coronavirus strains in Europe and Asia and the continued presence of multiple high-frequency strains in Asia and North America. We provide a continually updating dashboard of global evolution and spatiotemporal trends of SARS-CoV-2 spread (http://sars2evo.datamonkey.org/).","version":"1.3","doi":"10.1101/2020.09.24.311845","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.427182","pub_date":"2021-1-19","title":"Experimental re-infected cats do not transmit SARS-CoV-2","abstract":"SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to na\u00efve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Histologic lesions that characterized primary SARS-CoV-2 infected cats at 4 DPC were absent in the re-challenged cats. Na\u00efve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed na\u00efve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against reinfection.","version":"1.1","doi":"10.1101/2021.01.18.427182","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426526","pub_date":"2021-1-19","title":"In vivo structure and dynamics of the RNA genome of SARS-Cov-2","abstract":"The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3\u2019-UTR and 5\u2019-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide direct evidence of interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE, forming a \u201chigh-order pseudoknot\u201d embedding FSE, which might help ribosome stalling at frameshift sites. More importantly, we found that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome cyclization is weakened and genome domains remain stable. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, describes dynamics of RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.","version":"1.2","doi":"10.1101/2021.01.15.426526","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.16.426970","pub_date":"2021-1-19","title":"A trans-complementation system for SARS-CoV-2","abstract":"The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.","version":"1.1","doi":"10.1101/2021.01.16.426970","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426849","pub_date":"2021-1-19","title":"The impact of Spike mutations on SARS-CoV-2 neutralization","abstract":"Multiple SARS-CoV-2 vaccines have shown protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, Spike. Antibodies from SARS-CoV-2 infection neutralize the virus by focused targeting of Spike and there is limited serum cross-neutralization of the closely-related SARS-CoV. As new SARS-CoV-2 variants are rapidly emerging, exemplified by the B.1.1.7, 501Y.V2 and P.1 lineages, it is critical to understand if antibody responses induced by infection with the original SARS-CoV-2 virus or the current vaccines will remain effective against virus variants. In this study we evaluate neutralization of a series of mutated Spike pseudotypes including a B.1.1.7 Spike pseudotype. The analyses of a panel of Spike-specific monoclonal antibodies revealed that the neutralizing activity of some antibodies was dramatically reduced by Spike mutations. In contrast, polyclonal antibodies in the serum of patients infected in early 2020 remained active against most mutated Spike pseudotypes. The majority of serum samples were equally able to neutralize the B.1.1.7 Spike pseudotype, however potency was reduced in a small number of samples (3 of 36) by 5\u201310-fold. This work highlights that changes in the SARS-CoV-2 Spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their impact on vaccine efficacy.","version":"1.1","doi":"10.1101/2021.01.15.426849","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.426984","pub_date":"2021-1-19","title":"Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera","abstract":"Recently, a new SARS-CoV-2 lineage called B.1.1.7 has emerged in the United Kingdom that was reported to spread more efficiently than other strains. This variant has an unusually large number of mutations with 10 amino acid changes in the spike protein, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we investigated SARS-CoV-2-S pseudoviruses bearing either the Wuhan reference strain or the B.1.1.7 lineage spike protein with sera of 16 participants in a previously reported trial with the mRNA-based COVID-19 vaccine BNT162b2. The immune sera had equivalent neutralizing titers to both variants. These data, together with the combined immunity involving humoral and cellular effectors induced by this vaccine, make it unlikely that the B.1.1.7 lineage will escape BNT162b2-mediated protection.","version":"1.1","doi":"10.1101/2021.01.18.426984","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.17.427000","pub_date":"2021-1-19","title":"Comprehensive mapping of SARS-CoV-2 interactions in vivo reveals functional virus-host interactions","abstract":"SARS-CoV-2 has emerged as a major threat to global public health, resulting in global societal and economic disruptions. Here, we investigate the intramolecular and intermolecular RNA interactions of wildtype (WT) and a mutant (\u0394382) SARS-CoV-2 virus in cells using high throughput structure probing on Illumina and Nanopore platforms. We identified twelve potentially functional structural elements within the SARS-CoV-2 genome, observed that identical sequences can fold into divergent structures on different subgenomic RNAs, and that WT and \u0394382 virus genomes can fold differently. Proximity ligation sequencing experiments identified hundreds of intramolecular and intermolecular pair-wise interactions within the virus genome and between virus and host RNAs. SARS-CoV-2 binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2\u2019-O-methylated. 2\u2019-O-methylation sites in the virus genome are enriched in the untranslated regions and are associated with increased pair-wise interactions. SARS-CoV-2 infection results in a global decrease of 2\u2019-O-methylation sites on host mRNAs, suggesting that binding to snoRNAs could be a pro-viral mechanism to sequester methylation machinery from host RNAs towards the virus genome. Collectively, these studies deepen our understanding of the molecular basis of SARS-CoV-2 pathogenicity, cellular factors important during infection and provide a platform for targeted therapy.","version":"1.1","doi":"10.1101/2021.01.17.427000","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.034942","pub_date":"2021-1-19","title":"Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and emergence of RBD mutant with lower ACE2 binding affinity","abstract":"Monitoring the mutation dynamics of SARS-CoV-2 is critical for the development of effective approaches to contain the pathogen. By analyzing 106 SARS-CoV-2 and 39 SARS genome sequences, we provided direct genetic evidence that SARS-CoV-2 has a much lower mutation rate than SARS. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. The discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of SARS-CoV-2. Despite that we found the spike glycoprotein of SARS-CoV-2 is particularly more conserved, we identified a receptor binding domain mutation that leads to weaker ACE2 binding capability based on in silico simulation, which concerns a SARS-CoV-2 sample collected on 27th January 2020 from India. This represents the first report of a significant SARS-CoV-2 mutant, and requires attention from researchers working on vaccine development around the world. Based on the currently available genome sequence data, we provided direct genetic evidence that the SARS-COV-2 genome has a much lower mutation rate and genetic diversity than SARS during the 2002-2003 outbreak. The spike (S) protein encoding gene of SARS-COV-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. We confirmed a previously reported rearrangement in the S protein arrangement of SARS-COV-2, and propose that this rearrangement should have occurred between human SARS-CoV and a bat SARS-CoV, at a time point much earlier before SARS-COV-2 transmission to human. We provided first evidence that a mutated SARS-COV-2 with reduced human ACE2 receptor binding affinity have emerged in India based on a sample collected on 27th January 2020.","version":"1.2","doi":"10.1101/2020.04.09.034942","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423733","pub_date":"2021-1-19","title":"Evidence of a dysregulated Vitamin D pathway in SARS-CoV-2 infected patient\u2019s lung cells","abstract":"Although a defective vitamin D pathway has been widely suspected to be associated in SARS-CoV-2 pathobiology, the status of the vitamin D pathway and vitamin D-modulated genes in lung cells of patients infected with SARS-CoV-2 remains unknown. To understand the significance of the vitamin D pathway in SARS-CoV-2 pathobiology, computational approaches were applied to transcriptomic datasets from bronchoalveolar lavage fluid (BALF) cells of such patients or healthy individuals. Levels of vitamin D receptor, retinoid X receptor, and CYP27A1 in BALF cells of patients infected with SARS-CoV-2 were found to be reduced. Additionally, 107 differentially expressed, predominantly downregulated genes modulated by vitamin D were identified in transcriptomic datasets from patient\u2019s cells. Further analysis of differentially expressed genes provided eight novel genes with a conserved motif with vitamin D-responsive elements, implying the role of both direct and indirect mechanisms of gene expression by the dysregulated vitamin D pathway in SARS-CoV-2-infected cells. Network analysis of differentially expressed vitamin D-modulated genes identified pathways in the immune system, NF-KB/cytokine signaling, and cell cycle regulation as top predicted pathways that might be affected in the cells of such patients. In brief, the results provided computational evidence to implicate a dysregulated vitamin D pathway in the pathobiology of SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2020.12.21.423733","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.10.426114","pub_date":"2021-1-19","title":"The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-CoV-2 in cell culture models","abstract":"The SARS-CoV-2 pandemic has caused a significant number of fatalities and worldwide disruption. To identify drugs to repurpose to treat SARS-CoV-2 infections, we established a screen to measure dimerization of ACE2, the primary receptor for the virus. This screen identified fenofibric acid, the active metabolite of fenofibrate. Fenofibric acid also destabilized the receptor binding domain (RBD) of the viral spike protein and inhibited RBD binding to ACE2 in ELISA and whole cell binding assays. Fenofibrate and fenofibric acid were tested by two independent laboratories measuring infection of cultured Vero cells using two different SARS-CoV-2 isolates. In both settings at drug concentrations which are clinically achievable, fenofibrate and fenofibric acid reduced viral infection by up to 70%. Together with its extensive history of clinical use and its relatively good safety profile, these studies identify fenofibrate as a potential therapeutic agent requiring urgent clinical evaluation to treat SARS-CoV-2 infection. The approved drug fenofibrate inhibits infection by SARS-COV-2","version":"1.2","doi":"10.1101/2021.01.10.426114","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426287","pub_date":"2021-1-19","title":"Emergence and Evolution of a Prevalent New SARS-CoV-2 Variant in the United States","abstract":"Genomic surveillance can lead to early identification of novel viral variants and inform pandemic response. Using this approach, we identified a new variant of the SARS-CoV-2 virus that emerged in the United States (U.S.). The earliest sequenced genomes of this variant, referred to as 20C-US, can be traced to Texas in late May of 2020. This variant circulated in the U.S. uncharacterized for months and rose to recent prevalence during the third pandemic wave. It initially acquired five novel, relatively unique non-synonymous mutations. 20C-US is continuing to acquire multiple new mutations, including three independently occurring spike protein mutations. Monitoring the ongoing evolution of 20C-US, as well as other novel emerging variants, will be essential for understanding SARS-CoV-2 host adaptation and predicting pandemic outcomes.","version":"1.2","doi":"10.1101/2021.01.11.426287","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423850","pub_date":"2021-1-19","title":"SARS-CoV-2 Genomic Surveillance in Costa Rica: Evidence of a Divergent Population and an Increased Detection of a Spike T1117I Mutation","abstract":"Genome sequencing is a key strategy in the surveillance of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Latin America is the hardest hit region of the world, accumulating almost 20% of COVID-19 cases worldwide. Costa Rica was first exemplary for the region in its pandemic control, declaring a swift state of emergency on March 16th that led to a low quantity of cases, until measures were lifted in early May. From the first detected case in March 6th to December 31st almost 170 000 cases have been reported in Costa Rica, 99.5% of them from May onwards. We analyzed the genomic variability during the SARS-CoV-2 pandemic in Costa Rica using 185 sequences, 52 from the first months of the pandemic, and 133 from the current wave. Three GISAID clades (G, GH, and GR) and three PANGOLIN lineages (B.1, B.1.1, and B.1.291) are predominant, with phylogenetic relationships that are in line with the results of other Latin American countries, suggesting introduction and multiple re-introductions from other regions of the world. The whole-genome variant calling analysis identified a total of 283 distinct nucleotide variants. These correspond mostly to non-synonymous mutations (51.6%, 146) but 45.6% (129) corresponded to synonymous mutations. The 283 variants showed an expected power-law distribution: 190 single nucleotide mutations were identified in single sequences, only 16 single nucleotide mutations were found in >5% sequences, and only two mutations in >50% genomes. These mutations were distributed through the whole genome. However, 63.6% were present in ORF1ab, 11.7% in Spike gene and 10.6% in the Nucleocapsid gene. Additionally, the prevalence of worldwide-found variant D614G in the Spike (98.9% in Costa Rica), ORF8 L84S (1.1%) is similar to what is found elsewhere. Interestingly, the frequency of mutation T1117I in the Spike has increased during the current pandemic wave beginning in May 2020 in Costa Rica, reaching 29.2% detection in the full genome analyses in November 2020. This variant has been observed in less than 1% of the GISAID reported sequences worldwide in all the 2020. Structural modeling of the Spike protein with the T1117I mutation suggest a potential effect on the viral oligomerization needed for cell infection, but no differences with other genomes on transmissibility, severity nor vaccine effectiveness are predicted. Nevertheless, in-vitro experiments are required to support these in-silico findings. In conclusion, genome analyses of the SARS-CoV-2 sequences over the course of COVID-19 pandemic in Costa Rica suggest introduction of lineages from other countries as travel bans and measures were lifted, similar to results found in other studies, as well as an increase in the Spike-T1117I variant that needs to be monitored and studied in further analyses as part of the surveillance program during the pandemic.","version":"1.3","doi":"10.1101/2020.12.21.423850","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.19.389916","pub_date":"2021-1-19","title":"Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape","abstract":"Zoonotic pandemics, like that caused by SARS-CoV-2, can follow the spillover of animal viruses into highly susceptible human populations. Their descendants have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses, due to a proofreading polymerase. In the spike glycoprotein, we find recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution.","version":"1.2","doi":"10.1101/2020.11.19.389916","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.426885","pub_date":"2021-1-19","title":"Rapid protection from COVID-19 in nonhuman primates vaccinated intramuscularly but not intranasally with a single dose of a recombinant vaccine","abstract":"The ongoing pandemic of Coronavirus disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single dose, fast-acting vesicular stomatitis virus-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (IM) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, intranasal (IN) vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to control animals. While IM and IN vaccination both induced neutralizing antibody titers, only IM vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of IM-vaccinated animals only. Overall, the data demonstrates that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study. VSV vaccine protects NHPs from COVID-19 in 10 days","version":"1.1","doi":"10.1101/2021.01.19.426885","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.425627","pub_date":"2021-1-19","title":"Neuropilin-1 Assists SARS-CoV-2 Infection by Stimulating the Separation of Spike Protein Domains S1 and S2","abstract":"The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for SARS-CoV-2 entry. As the Spike protein of SARS-CoV-2 is cleaved into the S1 and the S2 domain by furin protease, Nrp1 binds to the newly created C-terminal RRAR amino acid sequence of the S1 domain. In this study, we model the association of a Nrp1 (a2-b1-b2) protein with the Spike protein computationally and analyze the topological constraints in the SARS-CoV-2 Spike protein for binding with Nrp1 and ACE2. Importantly, we study the exit mechanism of S2 from the S1 domain with the assistance of ACE2 as well as Nrp1 by molecular dynamics pulling simulations. In the presence of Nrp1, by binding the S1 more strongly to the host membrane, there is a high probability of S2 being pulled out, rather than S1 being stretched. Thus, Nrp1 binding could stimulate the exit of S2 from the S1 domain, which will likely increase virus infectivity as the liberated S2 domain mediates the fusion of virus and host membranes. Understanding of such a Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future combination therapy.","version":"1.2","doi":"10.1101/2021.01.06.425627","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.330068","pub_date":"2021-1-19","title":"Conformational Dynamics of NSP11 Peptide of SARS-CoV-2 Under Membrane Mimetics and Different Solvent Conditions","abstract":"The intrinsically disordered proteins/regions (IDPs/IDPRs) are known to be responsible for multiple cellular processes and are associated with many chronic diseases. In viruses, the existence of a disordered proteome is also proven and is related to its conformational dynamics inside the host. The SARS-CoV-2 has a large proteome, in which, structure and functions of many proteins are not known yet, along with nsp11. In this study, we have performed extensive experimentation on nsp11. Our results based on the CD spectroscopy gives characteristic disordered spectrum for IDPs. Further, we investigated the conformational behavior of nsp11 in the presence of membrane mimetic environment, \u03b1-helix inducer, and natural osmolyte. In the presence of negatively charged and neutral liposomes, nsp11 remains disordered. However, with SDS micelle, it adopted an \u03b1-helical conformation, suggesting the helical propensity of nsp11. Finally, we again confirmed the IDP behavior of nsp11 using MD simulations. In future, this conformational dynamic study could help to clarify its functional importance in SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.10.07.330068","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427256","pub_date":"2021-1-19","title":"An all-solid-state heterojunction oxide transistor for the rapid detection of biomolecules and SARS-CoV-2 spike S1 protein","abstract":"Solid-state transistor sensors that can detect biomolecules in real time are highly attractive for emerging bioanalytical applications. However, combining cost-effective manufacturing with high sensitivity, specificity and fast sensing response, remains challenging. Here we develop low-temperature solution-processed In2O3/ZnO heterojunction transistors featuring a geometrically engineered tri-channel architecture for rapid real-time detection of different biomolecules. The sensor combines a high electron mobility channel, attributed to the quasi-two-dimensional electron gas (q2DEG) at the buried In2O3/ZnO heterointerface, in close proximity to a sensing surface featuring tethered analyte receptors. The unusual tri-channel design enables strong coupling between the buried q2DEG and the minute electronic perturbations occurring during receptor-analyte interactions allowing for robust, real-time detection of biomolecules down to attomolar (aM) concentrations. By functionalizing the tri-channel surface with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) antibody receptors, we demonstrate real-time detection of the SARS-CoV-2 spike S1 protein down to attomolar concentrations in under two minutes.","version":"1.1","doi":"10.1101/2021.01.19.427256","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.427189","pub_date":"2021-1-19","title":"Sterically-Confined Rearrangements of SARS-CoV-2 Spike Protein Control Cell Invasion","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and transmission involves a series of processes that may be targeted by vaccines and therapeutics. During transmission, host cell invasion is controlled by a large-scale conformational change of the Spike protein. This conformational rearrangement leads to membrane fusion, which creates transmembrane pores through which the viral genome is passed to the host. During Spike-protein-mediated fusion, the fusion peptides must be released from the core of the protein and associate with the host membrane. Interestingly, the Spike protein possesses many post-translational modifications, in the form of branched glycans that flank the surface of the assembly. Despite the large number of glycosylation sites, until now, the specific role of glycans during cell invasion has been unclear. Here, we propose that glycosylation is needed to provide sufficient time for the fusion peptides to reach the host membrane, otherwise the viral particle would fail to enter the host. To understand this process, an all-atom model with simplified energetics was used to perform thousands of simulations in which the protein transitions between the prefusion and postfusion conformations. These simulations indicate that the steric composition of the glycans induces a pause during the Spike protein conformational change. We additionally show that this glycan-induced delay provides a critical opportunity for the fusion peptides to capture the host cell. This previously-unrecognized role of glycans reveals how the glycosylation state can regulate infectivity of this pervasive pathogen.","version":"1.1","doi":"10.1101/2021.01.18.427189","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.422952","pub_date":"2021-1-19","title":"Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mouse and non-human primates","abstract":"The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.","version":"1.3","doi":"10.1101/2021.01.05.422952","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426908","pub_date":"2021-1-19","title":"Interferon-regulated genetic programs and JAK/STAT pathway activate the intronic promoter of the short ACE2 isoform in renal proximal tubules","abstract":"Recently, a short, interferon-inducible isoform of Angiotensin-Converting Enzyme 2 (ACE2), dACE2 was identified. ACE2 is a SARS-Cov-2 receptor and changes in its renal expression have been linked to several human nephropathies. These changes were never analyzed in context of dACE2, as its expression was not investigated in the kidney. We used Human Primary Proximal Tubule (HPPT) cells to show genome-wide gene expression patterns after cytokine stimulation, with emphasis on the ACE2/dACE2 locus. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq and RNA-seq. qRT-PCR differentiating between ACE2 and dACE2 revealed 300- and 600-fold upregulation of dACE2 by IFN\u03b1 and IFN\u03b2, respectively, while full length ACE2 expression was almost unchanged. JAK inhibitor ruxolitinib ablated STAT1 and dACE2 expression after interferon treatment. Finally, with RNA-seq, we identified a set of genes, largely immune-related, induced by cytokine treatment. These gene expression profiles provide new insights into cytokine response of proximal tubule cells.","version":"1.1","doi":"10.1101/2021.01.15.426908","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.19.427250","pub_date":"2021-1-19","title":"COVID-19 Knowledge, Attitudes, and Practices of United Arab Emirates Medical and Health Sciences Students: A Cross Sectional Study","abstract":"COVID-19 pandemic is the largest unprecedented viral pandemic of the 21st century. We aimed to study the COVID-19 knowledge, attitudes, and practices (KAP) among medical and health sciences students in the United Arab Emirates (UAE). We performed a cross-sectional study between 2nd June and 19th August 2020. The survey was developed using online Survey Monkey. The link was distributed via UAE University to all students and via WhatsApp\u00a9 groups. The self-administered questionnaire was conducted in English and comprised of two parts: socio-demographic characteristics and KAP towards COVID-19. A total of 712 responses to the questionnaire were collected. 90% (n=695) were under-graduate, while 10% (n=81) were post-graduate students. Majority (87%, n=647) stated that they obtained COVID-19 information from multiple reliable sources. They were highly knowledgeable about COVID-19 pandemic but 76% (n=539) did not recognize its routes of transmission. 63% (n=431) were worried of getting COVID-19, while 92% (n=633)) were worried that a family member could get infected with the virus. 97% (n=655) took precautions when accepting home deliveries, 94% (n=637) had been washing their hands more frequently, and 95% (n=643) had been wearing face masks. In conclusion, participants showed high levels of knowledge and awareness about COVID-19. They were worried about getting infected themselves or their family members, and had good practices against COVID-19.","version":"1.1","doi":"10.1101/2021.01.19.427250","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426691","pub_date":"2021-1-18","title":"SARS-CoV-2 infection reduces Kr\u00fcppel-Like Factor 2 in human lung autopsy","abstract":"Acute respiratory distress syndrome (ARDS) occurred in ~12% of hospitalized COVID-19 patients in a recent New York City cohort. Pulmonary endothelial dysfunction, characterized by increased expression of inflammatory genes and increased monolayer permeability, is a major component of ARDS. Vascular leak results in parenchymal accumulation of leukocytes, protein, and extravascular water, leading to pulmonary edema, ischemia, and activation of coagulation associated with COVID-19. Endothelial inflammation further contributes to uncontrolled cytokine storm in ARDS. We have recently demonstrated that Kr\u00fcppel-like factor 2 (KLF2), a transcription factor which promotes endothelial quiescence and monolayer integrity, is significantly reduced in experimental models of ARDS. Lung inflammation and high-tidal volume ventilation result in reduced KLF2, leading to pulmonary endothelial dysfunction and acute lung injury. Mechanistically, we found that KLF2 is a potent transcriptional activator of Rap guanine nucleotide exchange factor 3 (RAPGEF3) which orchestrates and maintains vascular integrity. Moreover, KLF2 regulates multiple genome-wide association study (GWAS)-implicated ARDS genes. Whether lung KLF2 is regulated by SARS-CoV-2 infection is unknown. Here we report that endothelial KLF2 is significantly reduced in human lung autopsies from COVID-19 patients, which supports that ARDS due to SARS-CoV-2 is a vascular phenotype possibly attributed to KLF2 down-regulation. We provide additional data demonstrating that KLF2 is down-regulated in SARS-CoV infection in mice.","version":"1.1","doi":"10.1101/2021.01.15.426691","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.17.427024","pub_date":"2021-1-18","title":"Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.","version":"1.1","doi":"10.1101/2021.01.17.427024","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.17.425424","pub_date":"2021-1-18","title":"Large scale genomic and evolutionary study reveals SARS-CoV-2 virus isolates from Bangladesh strongly correlate with European origin and not with China","abstract":"The global public health is in serious crisis due to emergence of SARS-CoV-2 virus. Studies are ongoing to reveal the genomic variants of the virus circulating in various parts of the world. However, data generated from low- and middle-income countries are scarce due to resource limitation. This study was focused to perform whole genome sequencing of 151 SARS-CoV-2 isolates from COVID-19 positive Bangladeshi patients. The goal of this study was to identify the genomic variants among the SARS-CoV-2 virus isolates in Bangladesh, to determine the molecular epidemiology and to develop a relationship between host clinical trait with the virus genomic variants. Suspected patients were tested for COVID-19 using one step commercial qPCR kit for SARS-CoV-2 Virus. Viral RNA was extracted from positive patients, converted to cDNA which was amplified using Ion AmpliSeq\u2122 SARS-CoV-2 Research Panel. Massive parallel sequencing was carried out using Ion AmpliSeq\u2122 Library Kit Plus. Assembly of raw data is done by aligning the reads to a pre-defined reference genome (NC_045512.2) while retaining the unique variations of the input raw data by creating a consensus genome. A random forest-based association analysis was carried out to correlate the viral genomic variants with the clinical traits present in the host. Among the 151 viral isolates, we observed the 413 unique variants. Among these 8 variants occurred in more than 80 % of cases which include 241C to T, 1163A to T, 3037C to T,14408C to T, 23403A to G, 28881G to A, 28882 G to A, and finally the 28883G to C. Phylogenetic analysis revealed a predominance of variants belonging to GR clade, which have a strong geographical presence in Europe, indicating possible introduction of the SARS-CoV-2 virus into Bangladesh through a European channel. However, other possibilities like a route of entry from China cannot be ruled out as viral isolate belonging to L clade with a close relationship to Wuhan reference genome was also detected. We observed a total of 37 genomic variants to be strongly associated with clinical symptoms such as fever, sore throat, overall symptomatic status, etc. (Fisher\u2019s Exact Test p-value<0.05). The most mention-worthy among those were the 3916CtoT (associated with causing sore throat, p-value 0.0005), the 14408C to T (associated with protection from developing cough, p-value= 0.027), and the 28881G to A, 28882G to A, and 28883G to C variant (associated with causing chest pain, p-value 0.025). To our knowledge, this study is the first large scale phylogenomic studies of SARS-CoV-2 virus circulating in Bangladesh. The observed epidemiological and genomic features may inform future research platform for disease management, vaccine development and epidemiological study.","version":"1.1","doi":"10.1101/2021.01.17.425424","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.16.426965","pub_date":"2021-1-18","title":"Fluorescent Glycan Fingerprinting of SARS2 Spike Proteins","abstract":"Glycosylation is the most common post-translational modification and has myriad biological functions. However, glycan analysis and research has always been a challenge. Here, we would like to present new techniques of glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS-2 spike (S) glycoproteins. SARS-2, a novel coronavirus and the causative agent of COVID-19 pandemic, has devastated the world since the end of 2019. To obtain the N-glycan fingerprint of a S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, and then separated on acrylamide gel through electrophoresis, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders that are enzymatically generated are run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. Due to lack of enzyme for broad O-glycans releasing, O-glycans on the RBD protein are labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated on gel. Glycan fingerprinting could serve as a quick way for global assessment of the glycosylation of a glycoprotein.","version":"1.1","doi":"10.1101/2021.01.16.426965","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.18.427092","pub_date":"2021-1-18","title":"A national analysis of trends in COVID-19 infection and clinical management in Veterans Health Administration medical facilities","abstract":"We explored longitudinal trends in sociodemographic characteristics, reported symptoms, laboratory findings, pharmacological and non-pharmacological treatment, comorbidities, and 30-day in-hospital mortality among hospitalized patients with coronavirus disease 2019 (COVID-19). This retrospective cohort study included 43,267 patients diagnosed with COVID-19 in the Veterans Health Administration between 03/01/20 and 08/31/20 and followed until 09/30/20. We focused our analysis on patients that were subsequently hospitalized, and categorized them into groups based on the month of hospitalization. We summarized our findings through descriptive statistics. We used a nonparametric rank-sum test for trend to examine any differences in the distribution of our study variables across the six months. During our study period, 8,240 patients were hospitalized, and 1,081 (13.1%) died within 30 days of admission. Hospitalizations increased over time, but the proportion of patients that died consistently declined from March (N=221/890, 24.8%) to August (N=111/1,396, 8.0%). Patients hospitalized in March compared to August were younger on average, mostly black, and symptomatic. They also had a higher frequency of baseline comorbidities, including hypertension and diabetes, and were more likely to present with abnormal laboratory findings including low lymphocyte counts and elevated creatinine. Lastly, receipt of mechanical ventilation and Hydroxychloroquine declined from March to August, while treatment with Dexamethasone and Remdesivir increased. We found evidence of declining COVID-19 severity and fatality over time within a national health care system.","version":"1.1","doi":"10.1101/2021.01.18.427092","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.21249713","pub_date":"2021-01-16","title":"Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV-2","abstract":"<jats:title>Summary</jats:title>\n                <jats:p>To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India), conducted a sero-survey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS CoV2 anti-nucleocapsid (anti-NC) antibodies; 95% with surrogate neutralization activity. Three-fourth recalled no symptoms. Repeat serology tests at 3 (n=346) and 6 (n=35) months confirmed stability of antibody response and neutralization potential. Local sero-positivity was higher in densely populated cities and was inversely correlated with a 30 day change in regional test positivity rates (TPR). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of sero-positivity were high-exposure work (Odds Ratio, 95% CI, p value; 2\u00b723, 1\u00b792\u20132\u00b759, 6\u00b75E-26), use of public transport (1\u00b779, 1\u00b743\u20132\u00b724, 2\u00b78E-06), not smoking (1\u00b752, 1\u00b716\u20131\u00b799, 0\u00b702), non-vegetarian diet (1\u00b767, 1\u00b741\u20131\u00b799, 3\u00b70E-08), and B blood group (1\u00b736,1\u00b715-1\u00b761, 0\u00b7001).</jats:p>\n                <jats:sec>\n                  <jats:title>Impact Statement</jats:title>\n                  <jats:p>Widespread asymptomatic and undetected SARS-CoV2 infection affected more than a 100 million Indians by September 2020. Declining new cases thereafter may be due to persisting humoral immunity amongst sub-communities with high exposure.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Council of Scientific and Industrial Research, India (CSIR)</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.01.12.21249713","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.14.426742","pub_date":"2021-1-15","title":"CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating \u2018off-the-shelf\u2019 S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.","version":"1.1","doi":"10.1101/2021.01.14.426742","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.21249839","pub_date":"2021-01-15","title":"Innate lymphoid cells and disease tolerance in SARS-CoV-2 infection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on abundance of specific lymphoid subsets explain these correlations. This study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan \u2014 T cell subsets decrease less than 2-fold \u2014 and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis, and the percentage of amphiregulin-producing ILCs was higher in females than in males. These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance accounts for increased COVID-19 severity with age and in males.</jats:p>","version":null,"doi":"10.1101/2021.01.14.21249839","journal":"medRxiv","score":null},{"id":"10.1101/2020.12.18.413344","pub_date":"2021-1-15","title":"Progressing adaptation of SARS-CoV-2 to humans","abstract":"The second and subsequent waves of coronavirus disease 2019 (COVID-19) have caused problems worldwide . Here, using an objective analytical method , we present the changes that occurred in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19, over time. The virus has mutated in three major directions, resulting in three groups to date. Analysis of the basic structure of the group of viruses was completed by April and shared across all continents. However, the virus continued to mutate independently in each country after the borders were closed. In particular, the virus mutated before the occurrence of the second and subsequent peaks. It seems that the mutations conferred higher infectivity to the virus, because of which the virus overcame previously effective protection and caused second waves of the disease. Currently, each country may possess such a unique, stronger variant. Some of them slowly entered other countries and caused epidemics. These viruses could also serve as sources of further mutations by exchanging parts of the genome, which could create variants with superior infectivity.","version":"1.2","doi":"10.1101/2020.12.18.413344","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425674","pub_date":"2021-1-15","title":"CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge","abstract":"The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 \u00b5g or 5 \u00b5g of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 \u03bcg or 5 \u03bcg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.","version":"1.2","doi":"10.1101/2021.01.07.425674","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.426521","pub_date":"2021-1-15","title":"Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities invitro","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 \u00b5M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 \u00d7 10\u22126). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 \u00b5M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 \u00b5g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.","version":"1.1","doi":"10.1101/2021.01.14.426521","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.424792","pub_date":"2021-1-15","title":"Hydroxyzine inhibits SARS-CoV-2 Spike protein binding to ACE2 in a qualitative in vitro assay","abstract":"COVID-19 currently represents a major public health problem. Multiple efforts are being performed to control this disease. Vaccinations are already in progress. However, no effective treatments have been found so far. The disease is caused by the SARS-CoV-2 coronavirus that through the Spike protein interacts with its cell surface receptor ACE2 to enter into the host cells. Therefore, compounds able to block this interaction may help to stop disease progression. In this study, we have analyzed the effect of compounds reported to interact and modify the activity of ACE2 on the binding of the Spike protein. Among the compounds tested, we found that hydroxyzine could inhibit the binding of the receptor-binding domain of Spike protein to ACE2 in a qualitative in vitro assay. This finding supports the reported clinical data showing the benefits of hydroxyzine on COVID-19 patients, raising the need for further investigation into its effectiveness in the treatment of COVID-19 given its well-characterized medical properties and affordable cost.","version":"1.2","doi":"10.1101/2021.01.04.424792","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.162560","pub_date":"2021-1-15","title":"Comparative Genomics and Integrated Network Approach Unveiled Undirected Phylogeny Patterns, Co-mutational Hotspots, Functional Crosstalk and Regulatory Interactions in SARS-CoV-2","abstract":"SARS-CoV-2 pandemic resulted in 92 million cases in a span of one year. The study focuses on understanding population specific variations attributing its high rate of infections in specific geographical regions particularly in USA. Rigorous phylogenomic network analysis of complete SARS-CoV-2 genomes (245) inferred five central clades named a (ancestral), b, c, d and e (subtype e1 & e2). The clade d & e2 were found exclusively comprising of USA. Clades were distinguished by 10 co-mutational combinations in Nsp3, ORF8, Nsp13, S, Nsp12, Nsp2 and Nsp6. Our analysis revealed that only 67.46% of SNP mutations were at amino acid level. T1103P mutation in Nsp3 was predicted to increase protein stability in 238 strains except 6 strains which were marked as ancestral type; whereas co-mutation (P409L & Y446C) in Nsp13 were found in 64 genomes from USA highlighting its 100% co-occurrence. Docking highlighted mutation (D614G) caused reduction in binding of Spike proteins with ACE2, but it also showed better interaction with TMPRSS2 receptor contributing to high transmissibility among USA strains. We also found host proteins, MYO5A, MYO5B, MYO5C had maximum interaction with viral proteins (N, S, M). Thus, blocking the internalization pathway by inhibiting MYO5 proteins which could be an effective target for COVID-19 treatment. The functional annotations of the HPI network were found to be closely associated with hypoxia and thrombotic conditions confirming the vulnerability and severity of infection. We also screened CpG islands in Nsp1 & N conferring ability of SARS-CoV-2 to enter and trigger ZAP activity inside host cell. In the current study we presented a global view of mutational pattern observed in SARS-CoV-2 virus transmission. This provided a who-infect-whom geographical model since the early pandemic. This is hitherto the most comprehensive comparative genomics analysis of full-length genomes for co-mutations at different geographical regions specially in USA strains. Compositional structural biology results suggested that mutations have balance of contrary forces effect on pathogenicity suggesting only few mutations to effective at translation level but not all. Novel HPI analysis and CpG predictions elucidates the proof of concept of hypoxia and thrombotic conditions in several patients. Thus, the current study focuses the understanding of population specific variations attributing high rate of SARS-CoV-2 infections in specific geographical regions which may eventually be vital for the most severely affected countries and regions for sharp development of custom-made vindication strategies.","version":"1.2","doi":"10.1101/2020.06.20.162560","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424267","pub_date":"2021-1-15","title":"SARS-CoV-2 mutations among minks show reduced lethality and infectivity to humans","abstract":"SARS-CoV-2 infection in minks has become a serious problem, as the virus may mutate and reinfect humans; some countries have decided to cull minks. Here, the virus sequencing data in minks were analysed and compared to those of human-virus. Although the mink-virus maintained the characteristics of human-virus, some variants rapidly mutated, adapting to minks. Some mink-derived variants infected humans, which accounted for 40% of the total SARS-CoV-2 cases in the Netherlands. These variants appear to be less lethal and infective compared to those in humans. Variants that have mutated further among minks were not found in humans. Such mink-viruses might be suitable for vaccination for humans, such as in the case of the smallpox virus, which is less infective and toxic to humans.","version":"1.2","doi":"10.1101/2020.12.23.424267","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.21249694","pub_date":"2021-01-15","title":"Optimal SARS-CoV-2 vaccine allocation using real-time seroprevalence estimates in Rhode Island and Massachusetts","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  As three SARS-CoV-2 vaccines come to market in Europe and North America in the winter of 2020-2021, distribution networks will be in a race against a major epidemiological wave of SARS-CoV-2 that began in autumn 2020. Rapid and optimized vaccine allocation is critical during this time. With 95% efficacy reported for two of the vaccines, near-term public health needs require that distribution is prioritized to the elderly, health-care workers, teachers, essential workers, and individuals with co-morbidities putting them at risk of severe clinical progression. Here, we evaluate various age-based vaccine distributions using a validated mathematical model based on current epidemic trends in Rhode Island and Massachusetts. We allow for varying waning efficacy of vaccine-induced immunity, as this has not yet been measured. We account for the fact that known COVID-positive cases may not be included in the first round of vaccination. And, we account for current age-specific immune patterns in both states. We find that allocating a substantial proportion (\n                  <jats:italic>&gt;</jats:italic>\n                  75%) of vaccine supply to individuals over the age of 70 is optimal in terms of reducing total cumulative deaths through mid-2021. As we do not explicitly model other high mortality groups, this result on vaccine allocation applies to all groups at high risk of mortality if infected. Our analysis confirms that for an easily transmissible respiratory virus, allocating a large majority of vaccinations to groups with the highest mortality risk is optimal. Our analysis assumes that health systems during winter 2020-2021 have equal staffing and capacity to previous phases of the SARS-CoV-2 epidemic; we do not consider the effects of understaffed hospitals or unvaccinated medical staff. Vaccinating only seronegative individuals avoids redundancy in vaccine use on individuals that may already be immune, and will result in 1% to 2% reductions in cumulative hospitalizations and deaths by mid-2021. Assuming high vaccination coverage (\n                  <jats:italic>&gt;</jats:italic>\n                  28%) and no major relaxations in distancing, masking, gathering size, or hygiene guidelines between now and spring 2021, our model predicts that a combination of vaccination and population immunity will lead to low or near-zero transmission levels by the second quarter of 2021.\n                </jats:p>","version":null,"doi":"10.1101/2021.01.12.21249694","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.15.21249881","pub_date":"2021-01-15","title":"Modeling the impact of racial and ethnic disparities on COVID-19 epidemic dynamics","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The impact of variable infection risk by race and ethnicity on the dynamics of SARS-CoV-2 spread is largely unknown. Here, we fit structured compartmental models to seroprevalence data from New York State and analyze how herd immunity thresholds (HITs), final sizes, and epidemic risk changes across groups. A simple model where interactions occur proportionally to contact rates reduced the HIT, but more realistic models of preferential mixing within groups increased the threshold toward the value observed in homogeneous populations. Across all models, the burden of infection fell disproportionately on minority populations: in a model fit to Long Island serosurvey and census data, 81% of Hispanics or Latinos were infected when the HIT was reached compared to 34% of non-Hispanic whites. Our findings, which are meant to be illustrative and not best estimates, demonstrate how racial and ethnic disparities can impact epidemic trajectories and result in unequal distributions of SARS-CoV-2 infection.</jats:p>","version":null,"doi":"10.1101/2021.01.15.21249881","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.14.426613","pub_date":"2021-1-15","title":"Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart","abstract":"Membrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I viruses contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion, and is termed as \u2018fusion peptide\u2019. However, Severe Acute Respiratory Syndrome Coronavirus (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion, but fails to open pore.","version":"1.1","doi":"10.1101/2021.01.14.426613","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426426","pub_date":"2021-1-15","title":"Optimisation and validation of a sensitive bioanalytical method for niclosamide","abstract":"The SARS-CoV-2 pandemic has spread at an unprecedented rate, and repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Niclosamide has been proposed as a candidate for repurposing for SARS-CoV-2 based upon the observation that it is amongst the most potent antiviral molecules evaluated in vitro. To investigate the pharmacokinetics of niclosamide, reliable, reproducible and sensitive bioanalytical assays are required. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 31.25-2000 ng/mL (high dynamic range) and 0.78-100 ng/mL (low dynamic range). Accuracy and precision ranged between 97.2% and 112.5%, 100.4% and 110.0%, respectively. The presented assay should have utility in preclinical evaluation of the exposure-response relationship and may be adapted for later evaluation of niclosamide in clinical trials.","version":"1.1","doi":"10.1101/2021.01.13.426426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.424628","pub_date":"2021-1-15","title":"Accurate bulk quantitation of droplet digital PCR","abstract":"Droplet digital PCR provides superior accuracy in nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource or clinical settings. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR.","version":"1.1","doi":"10.1101/2021.01.13.424628","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.15.426787","pub_date":"2021-1-15","title":"Correlation of Computerized Tomography (CT) Severity Score for COVID-19 pneumonia with Clinical Outcomes","abstract":"Various CT severity scores have already been described in literature since the start of this pandemic. One pertinent issue with all of the previously described severity scores is their relative challenging calculation and variance in inter-observer agreement. The severity score proposed in our study is relatively simpler, easier to calculate and apart from a trained radiologist, can easily be calculated even by physicians with good inter-observer agreement. Therefore, a rapid CT severity score calculation can give a clue to physician about possible clinical outcome without being dependent on radiologist who may not be readily available especially in third world countries. The objective of this study is to develop a simple CT severity score (CT-SS) with good inter-observer agreement and access its correlation with clinical outcome. This retrospective study was conducted by the Department of Radiology and Internal Medicine, at the Aga Khan University Hospital Karachi, from April 2020 to August 2020. Non-probability consecutive sampling was used to include all patients who were positive for COVID-19 on PCR, and underwent CT chest examination at AKUH. Severity of disease was calculated in each lobe on the basis of following proposed CT severity scoring system (CT-SS). For each lobe the percentage of involvement by disease was scored \u2013 0% involvement was scored 0, <50% involvement was scored 1 and >50% involvement was scored 2. Maximum score for one lobe was 2 and hence total maximum overall score for all lobes was 10. Continuous data was represented using mean and standard deviation, and compared using independent sample t-tests. Categorical data was represented using frequencies and percentages, and compared using Chi-squared tests. Inter-observer reliability between radiologist and COVID intensivist for the 10 point CT-SS rated on 0-10 was assessed using the Kappa statistic. A p-value < 0.05 was considered significant for all analyses. A total of 73 patients were included, the majority male (58.9%) with mean age 55.8 \u00b1 13.93 years. The CT-SS rated on 0-10 showed substantial inter-observer reliability between radiologist and intensivist with a Kappa statistic of 0.78. Patients with CT-SS 8-10 had a significantly higher ICU admission & intubation rate (53.8% vs. 23.5%) and mortality rate (35.9% vs. 11.8%; p = 0.017), as compared to those with CT-SS 0-7. We conclude that the described CT severity score (CT-SS) is a quick, effective and easily reproducible tool for prediction of adverse clinical outcome in patients with COVID 19 pneumonia. The tool shows good inter-observer agreement when calculated by radiologist and physician independently.","version":"1.1","doi":"10.1101/2021.01.15.426787","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.426726","pub_date":"2021-1-14","title":"Impacts of 203/204: RG>KR mutation in the N protein of SARS-CoV-2","abstract":"We present a structure-based model of phosphorylation-dependent binding and sequestration of SARS-CoV-2 nucleocapsid protein and the impact of two consecutive amino acid changes R203K and G204R. Additionally, we studied how mutant strains affect HLA-specific antigen presentation and correlated these findings with HLA allelic population frequencies. We discovered RG>KR mutated SARS-CoV-2 expands the ability for differential expression of the N protein epitope on Major Histocompatibility Complexes (MHC) of varying Human Leukocyte Antigen (HLA) origin. The N protein LKR region K203, R204 of wild type (SARS-CoVs) and (SARS-CoV-2) observed HLA-A*30:01 and HLA-A*30:21, but mutant SARS-CoV-2 observed HLA-A*31:01 and HLA-A*68:01. Expression of HLA-A genotypes associated with the mutant strain occurred more frequently in all populations studied. The novel coronavirus known as SARS-CoV-2 causes a disease renowned as 2019-nCoV (or COVID-19). HLA allele frequencies worldwide could positively correlate with the severity of coronavirus cases and a high number of deaths.","version":"1.1","doi":"10.1101/2021.01.14.426726","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426628","pub_date":"2021-1-14","title":"Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2","abstract":"The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) \u2013 the SARS-CoV-2 receptor \u2013 shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus. Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.","version":"1.1","doi":"10.1101/2021.01.13.426628","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426436","pub_date":"2021-1-14","title":"Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation","abstract":"There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.01.13.426436","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.426475","pub_date":"2021-1-14","title":"N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2","abstract":"SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.","version":"1.1","doi":"10.1101/2021.01.14.426475","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426548","pub_date":"2021-1-14","title":"Surveillance of genetic diversity and evolution in locally transmitted SARS-CoV-2 in Pakistan during the first wave of the COVID-19 pandemic","abstract":"Surveillance of genetic diversity in the SARS-CoV-2 is extremely important to detect the emergence of more infectious and deadly strains of the virus. In this study, we monitored mutational events in the SARS-CoV-2 genome through whole genome sequencing. The samples (n=48) were collected from the hot spot regions of the metropolitan city Karachi, Pakistan during the four months (May 2020 to August 2020) of first wave of the COVID-19 pandemic. The data analysis highlighted 122 mutations, including 120 single nucleotide variations (SNV), and 2 deletions. Among the 122 mutations, there were 71 singletons, and 51 recurrent mutations. A total of 16 mutations, including 5 nonsynonymous mutations, were detected in spike protein. Notably, the spike protein missense mutation D614G was observed in 31 genomes. The phylogenetic analysis revealed majority of the genomes (36) classified as B lineage, where 2 genomes were from B.6 lineage, 5 genomes from B.1 ancestral lineage and remaining from B.1 sub-lineages. It was noteworthy that three clusters of B.1 sub-lineages were observed, including B.1.36 lineage (10 genomes), B.1.160 lineage (11 genomes), and B.1.255 lineage (5 genomes), which represent independent events of SARS-CoV-2 transmission within the city. The sub-lineage B.1.36 had higher representation from the Asian countries and the UK, B.1.160 correspond to the European countries with highest representation from the UK, Denmark, and lesser representation from India, Saudi Arabia, France and Switzerland, and the third sub-lineage (B.1.255) correspond to the USA. Collectively, our study provides meaningful insight into the evolution of SARS-CoV-2 lineages in spatio-temporal local transmission during the first wave of the pandemic.","version":"1.1","doi":"10.1101/2021.01.13.426548","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.426705","pub_date":"2021-1-14","title":"Mutation rates and selection on synonymous mutations in SARS-CoV-2","abstract":"The COVID-19 pandemic has seen an unprecedented response from the sequencing community. Leveraging the sequence data from more than 140,000 SARS-CoV-2 genomes, we study mutation rates and selective pressures affecting the virus. Understanding the processes and effects of mutation and selection has profound implications for the study of viral evolution, for vaccine design, and for the tracking of viral spread. We highlight and address some common genome sequence analysis pitfalls that can lead to inaccurate inference of mutation rates and selection, such as ignoring skews in the genetic code, not accounting for recurrent mutations, and assuming evolutionary equilibrium. We find that two particular mutation rates, G\u2192U and C\u2192U, are similarly elevated and considerably higher than all other mutation rates, causing the majority of mutations in the SARS-CoV-2 genome, and are possibly the result of APOBEC and ROS activity. These mutations also tend to occur many times at the same genome positions along the global SARS-CoV-2 phylogeny (i.e., they are very homoplasic). We observe an effect of genomic context on mutation rates, but the effect of the context is overall limited. While previous studies have suggested selection acting to decrease U content at synonymous sites, we bring forward evidence suggesting the opposite.","version":"1.1","doi":"10.1101/2021.01.14.426705","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.426652","pub_date":"2021-1-14","title":"Natural SARS-CoV-2 infection in kept ferrets, Spain","abstract":"We found SARS-CoV-2 RNA in 6 of 71 ferrets (8.4%) and isolated the virus from one rectal swab. Natural SARS-CoV-2 infection does occur in kept ferrets, at least under circumstances of high viral circulation in the human population. However, small ferret collections are probably unable to maintain prolonged virus circulation.","version":"1.1","doi":"10.1101/2021.01.14.426652","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.426365","pub_date":"2021-1-14","title":"The lethal triad: SARS-CoV-2 Spike, ACE2 and TMPRSS2. Mutations in host and pathogen may affect the course of pandemic","abstract":"Variants of SARS-CoV-2 have been identified rapidly after the beginning of pandemic. One of them, involving the spike protein and called D614G, represents a substantial percentage of currently isolated strains. While research on this variant was ongoing worldwide, on December 20th 2020 the European Centre for Disease Prevention and Control reported a Threat Assessment Brief describing the emergence of a new variant of SARS-CoV-2, named B.1.1.7, harboring multiple mutations mostly affecting the Spike protein. This viral variant has been recently associated with a rapid increase in COVID-19 cases in South East England, with alarming implications for future virus transmission rates. Specifically, of the nine amino acid replacements that characterize the Spike in the emerging variant, four are found in the region between the Fusion Peptide and the RBD domain (namely the already known D614G, together with A570D, P681H, T716I), and one, N501Y, is found in the Spike Receptor Binding Domain \u2013 Receptor Binding Motif (RBD-RBM). In this study, by using in silico biology, we provide evidence that these amino acid replacements have dramatic effects on the interactions between SARS-CoV-2 Spike and the host ACE2 receptor or TMPRSS2, the protease that induces the fusogenic activity of Spike. Mostly, we show that these effects are strongly dependent on ACE2 and TMPRSS2 polymorphism, suggesting that dynamics of pandemics are strongly influenced not only by virus variation but also by host genetic background.","version":"1.1","doi":"10.1101/2021.01.12.426365","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.14.426695","pub_date":"2021-1-14","title":"SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses","abstract":"The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.","version":"1.1","doi":"10.1101/2021.01.14.426695","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.046565","pub_date":"2021-1-13","title":"Functional and Genetic Analysis of Viral Receptor ACE2 Orthologs Reveals a Broad Potential Host Range of SARS-CoV-2","abstract":"The pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat. Epidemiological studies suggest that bats are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. We found that ACE2 is expressed in a wide range of species, with especially high conservation in mammals. By analyzing amino acid residues of ACE2 critical for virus entry, based on structure of SARS-CoV spike protein interaction with human, bat, palm civet, pig and ferret ACE2, we identified approximately eighty ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 representative ACE2 orthologs among eighty orthologs for functional analysis and it showed that 44 of these mammalian ACE2 orthologs, including those of domestic animals, pets, livestock, and animals commonly found in zoos and aquaria, could bind SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. In summary, our study demonstrates that ACE2 from a remarkably broad range of species can facilitate SARS-CoV-2 entry. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.","version":"1.4","doi":"10.1101/2020.04.22.046565","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426537","pub_date":"2021-1-13","title":"Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity","abstract":"We selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65\u00b0C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.","version":"1.1","doi":"10.1101/2021.01.13.426537","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.425914","pub_date":"2021-1-13","title":"An insight into neurotoxic and toxicity of spike fragments SARS-CoV-2 by exposure environment: A threat to aquatic health?","abstract":"The Spike protein (S protein) is a critical component in the infection of the new coronavirus (SARS-CoV-2). The objective of this work was to evaluate whether peptides from S protein could cause negative impact in the aquatic animals. The aquatic toxicity of SARS-CoV-2 spike protein peptides derivatives has been evaluated in tadpoles (n = 50 tadpoles / 5 replicates of 10 animals) from species Physalaemus cuvieri (Leptodactylidae). After synthesis, purification, and characterization of peptides (PSDP2001, PSDP2002, PSDP2003) an aquatic contamination has been simulatedwith these peptides during 24 hours of exposure in two concentrations (100 and 500 ng/mL). The control group (\u201cC\u201d) was composed of tadpoles kept in polyethylene containers containing de-chlorinated water. Oxidative stress, antioxidant biomarkers and neurotoxicity activity were assessed. In both concentrations, PSPD2002 and PSPD2003 increased catalase and superoxide dismutase antioxidants enzymes activities, as well as oxidative stress (nitrite levels, hydrogen peroxide and reactive oxygen species). All three peptides also increased acetylcholinesterase activity in the highest concentration. These peptides showed molecular interactions in silico with acetylcholinesterase and antioxidant enzymes. Aquatic particle contamination of SARS-CoV-2 has neurotoxics effects in P. cuvieri tadpoles. These findings indicate that the COVID-19 can constitute environmental impact or biological damage potential. SARS-CoV-2 spike protein peptides (PSDP) were synthesized, purified, and characterized by solid phase peptide synthesis. PSDP peptides promoted REDOX imbalance and acute neurotoxicity in tadpoles (Physalaemus cuvieri) In silico studies have shown interactionsbetween peptides and acetylcholinesterase and antioxidant enzymes Aquatic particle contamination of SARS-CoV-2 can constitute additional environmental damage","version":"1.1","doi":"10.1101/2021.01.11.425914","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426571","pub_date":"2021-1-13","title":"Using image-based haplotype alignments to map global adaptation of SARS-CoV-2","abstract":"Quantifying evolutionary change among viral genomes is an important clinical device to track critical adaptations geographically and temporally. We built image-based haplotype-guided evolutionary inference (ImHapE) to quantify adaptations in expanding populations of non-recombining SARS-CoV-2 genomes. By combining classic population genetic summaries with image-based deep learning methods, we show that different rates of positive selection are driving evolutionary fitness and dispersal of SARS-CoV-2 globally. A 1.35-fold increase in evolutionary fitness is observed within the UK, associated with expansion of both the B.1.177 and B.1.1.7 SARS-CoV-2 lineages.","version":"1.1","doi":"10.1101/2021.01.13.426571","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426295","pub_date":"2021-1-13","title":"In vitro screening of anti-viral and virucidal effects against SARS-CoV-2 by Hypericum perforatum and Echinacea","abstract":"Special Infectious Agent Unit in King Fahd Medical Research Center at King Abdulaziz University, Jeddah, Saudi Arabia, has pursed the anti-viral project field to optimize the group of medicinal plants for human-infectious diseases. We have begun virtually in this field since COVID-19 pandemic, besides our divergence in the infectious agents\u2019. In this study and based on the previous review, Hypericum perforatum (St. John\u2019s Wort) and Echinacea (gaia HERBS\u00ae) were tested in vitro using Vero E6 cells for their anti-viral effects against the newly identified Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) through its infectious cycle from 0 to 48 hours post infection. The hypericin (0.9 mg) of H. perforatum and the different parts (roots, seeds, aerial) of two types of Echinacea species (Echinacea purpurea and Echinacea angustifolia) were examined their efficacy in certain concentration and under light-dependent anti-viral activities to measure the inhibition of the SARS-CoV-2 mRNA expression of RNA-dependent RNA polymerase (RdRP) gene and the viral load with quantitative real-time polymerase chain reaction (qRT-PCR), and to assess the neutralization of the SARS-CoV-2 spike receptor binding on cell culture assay. Interestingly, the mixture (H.E.) of 100 mg/mL of H. perforatum and Echinacea was tested too on SARS-CoV-2 and showed crucial anti-viral activity competing H. perforatum then Echinacea effects as anti-viral treatment. Therefore, the results of gaia HERBS\u00ae products, H. perforatum and Echinacea species, applied in this study showed significant anti-viral and virucidal effects in the following order of potency: H. perforatum, H.E., and Echinacea on SARS-CoV-2 infectious cycle; and will definitely required a set up of clinical trial with specific therapeutic protocol based on the outcome of this study. After an outbreak of Rift Valley Fever in the Southern region of Saudi Arabia, particularly in May 2003, Special Infectious Agents Unit (SIAU) was established and founded by Prof. Esam Ibraheem Azhar. This unit contains a full range of facilities including Biosafety Level 3, allows him and his research groups to ambulate and culture risk group 3 viruses in Saudi Arabia & Gulf States for the first time. Since that time, SIAU and our international collaboration have been extended to implement a standard protocols in the infectious agents diagnostics procedure through different mode of collaboration including exchange of expertise, joint research program and more recently a technology transfer agreements with number of international institute sharing same interests. Furthermore, we have been engaged in number of researches related to Hajj & Umrah plus number of national services with the Ministry of Health (MOH) through which, we utilize our Mobile biosafety level 3 Lab to enhance the diagnostics of MERS CoV in the Holly sites during Hajj since 2014. In our SIAU and with a powerful team, we have excellent researches made valuable contributions through in vivo and in vitro animal and human studies, and several human viral pathogens which are a threat to global health security due to millions of pilgrims visiting Saudi Arabia every year from 182 countries: with particular areas of interests in: Alkhurma Viral Hemorrhagic Fever, Dengue Hemorrhagic Fever Viruses, Rift Valley Fever Virus, MERS-CoV and more recently the new global infectious diseases threat, Sever Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2).","version":"1.1","doi":"10.1101/2021.01.11.426295","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426597","pub_date":"2021-1-13","title":"Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein","abstract":"Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs\u2019 flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.","version":"1.1","doi":"10.1101/2021.01.13.426597","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426553","pub_date":"2021-1-13","title":"Distinct lung-homing receptor expression and activation profiles on NK cell and T cell subsets in COVID-19 and influenza","abstract":"Respiratory viral infections with SARS-CoV-2 or influenza viruses commonly induce a strong infiltration of immune cells into the lung, with potential detrimental effects on the integrity of the lung tissue. Despite comprising the largest fractions of circulating lymphocytes in the lung, little is known about how blood natural killer (NK) cells and T cell subsets are equipped for lung-homing in COVID-19 and influenza. Using 28-colour flow cytometry and re-analysis of published RNA-seq datasets, we provide a detailed comparative analysis of NK cells and T cells in peripheral blood from moderately sick COVID-19 and influenza patients, focusing on the expression of chemokine receptors known to be involved in leukocyte recruitment to the lung. The results reveal a predominant role for CXCR3, CXCR6, and CCR5 in COVID-19 and influenza patients, mirrored by scRNA-seq signatures in peripheral blood and bronchoalveolar lavage from publicly available datasets. NK cells and T cells expressing lung-homing receptors displayed stronger phenotypic signs of activation as compared to cells lacking lung-homing receptors, and activation was overall stronger in influenza as compared to COVID-19. Together, our results indicate migration of functionally competent CXCR3+, CXCR6+, and/or CCR5+ NK cells and T cells to the lungs in moderate COVID-19 and influenza patients, identifying potential common targets for future therapeutic interventions in respiratory viral infections. The composition of in particular CXCR3+ and/or CXCR6+ NK cells and T cells is altered in peripheral blood upon infection with SARS-CoV-2 or influenza virus in patients with moderate disease. Lung-homing receptor-expression is biased towards phenotypically activated NK cells and T cells, suggesting a functional role for these cells co-expressing in particular CXCR3 and/or CXCR6 upon homing towards the lung.","version":"1.1","doi":"10.1101/2021.01.13.426553","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.13.426558","pub_date":"2021-1-13","title":"Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant","abstract":"Rapidly spreading SARS-CoV-2 variants present not only an increased threat to human health due to the confirmed greater transmissibility of several of these new strains but, due to conformational changes induced by the mutations, may render first-wave SARS-CoV-2 convalescent sera, vaccine-induced antibodies, or recombinant neutralizing antibodies (nAbs) ineffective. To be able to assess the risk of viral escape from neutralization by first-wave antibodies, we leveraged our capability for Molecular Dynamic (MD) simulation of the spike receptor binding domain (S RBD) and its binding to human angiotensin-converting enzyme 2 (hACE2) to predict alterations in molecular interactions resulting from the presence of the E484K, K417N, and N501Y variants found in the South African 501Y.V2 strain \u2013 alone and in combination. We report here the combination of E484K, K417N and N501Y results in the highest degree of conformational alterations of S RBD when bound to hACE2, compared to either E484K or N501Y alone. Both E484K and N501Y increase affinity of S RBD for hACE2 and E484K in particular switches the charge on the flexible loop region of RBD which leads to the formation of novel favorable contacts. Enhanced affinity of S RBD for hACE2 very likely underpins the greater transmissibility conferred by the presence of either E484K or N501Y; while the induction of conformational changes may provide an explanation for evidence that the 501Y.V2 variant, distinguished from the B.1.1.7 UK variant by the presence of E484K, is able to escape neutralization by existing first-wave anti-SARS-CoV-2 antibodies and re-infect COVID-19 convalescent individuals.","version":"1.1","doi":"10.1101/2021.01.13.426558","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.385401","pub_date":"2021-1-13","title":"An ancient viral epidemic involving host coronavirus interacting genes more than 20,000 years ago in East Asia","abstract":"The current SARS-CoV-2 pandemic has emphasized the vulnerability of human populations to novel viral pressures, despite the vast array of epidemiological and biomedical tools now available. Notably, modern human genomes contain evolutionary information tracing back tens of thousands of years, which may help identify the viruses that have impacted our ancestors \u2013 pointing to which viruses have future pandemic potential. Here, we apply evolutionary analyses to human genomic datasets to recover selection events involving tens of human genes that interact with coronaviruses, including SARS-CoV-2, that likely started more than 20,000 years ago. These adaptive events were limited to the population ancestral to East Asian populations. Multiple lines of functional evidence support an ancient viral selective pressure, and East Asia is the geographical origin of several modern coronavirus epidemics. An arms race with an ancient coronavirus, or with a different virus that happened to use similar interactions as coronaviruses with human hosts, may thus have taken place in ancestral East Asian populations. By learning more about our ancient viral foes, our study highlights the promise of evolutionary information to better predict the pandemics of the future. Importantly, adaptation to ancient viral epidemics in specific human populations does not necessarily imply any difference in genetic susceptibility between different human populations, and the current evidence points toward an overwhelming impact of socioeconomic factors in the case of COVID-19.","version":"1.2","doi":"10.1101/2020.11.16.385401","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.426388","pub_date":"2021-1-12","title":"Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease","abstract":"The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the \u03b1-ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other \u03b1-ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.","version":"1.1","doi":"10.1101/2021.01.12.426388","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.425991","pub_date":"2021-1-12","title":"Novel RT-ddPCR Assays for determining the transcriptional profile of SARS-CoV-2","abstract":"The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics. We developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2 Our panel targets nongenic and genic regions in genomic and subgenomic RNAs All assays detect 1-10 copies and are linear over 3-4 orders of magnitude All assays correlated with the clinical Abbott SARS-CoV-2 Viral Load Assay Clinical samples showed higher copy numbers for targets at the 3\u2019 end of the genome","version":"1.1","doi":"10.1101/2021.01.12.425991","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.426042","pub_date":"2021-1-12","title":"Snake venom phospholipases A2 possess a strong virucidal activity against SARS-CoV-2 in vitro and block the cell fusion mediated by spike glycoprotein interaction with the ACE2 receptor","abstract":"A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the absence of specific therapeutic and prophylactic agents, the virus has infected almost hundred million people, of whom nearly two million have died from the viral disease COVID-19. The ongoing COVID-19 pandemic is a global threat requiring new therapeutic strategies. Among them, antiviral studies based on natural molecules are a promising approach. The superfamily of phospholipases A2 (PLA2s) consists of a large number of members that catalyze the hydrolysis of phospholipids at a specific position. Here we show that secreted PLA2s from the venom of various snakes protect to varying degrees the Vero E6 cells widely used for the replication of viruses with evident cytopathic action, from SARS-CoV-2 infection PLA2s showed low cytotoxicity to Vero E6 cells and the high antiviral activity against SARS-CoV-2 with IC50 values ranged from 0.06 to 7.71 \u03bcg/ml. Dimeric PLA2 HDP-2 from the viper Vipera nikolskii, as well as its catalytic and inhibitory subunits, had potent virucidal (neutralizing) activity against SARS-CoV-2. Inactivation of the enzymatic activity of the catalytic subunit of dimeric PLA2 led to a significant decrease in antiviral activity. In addition, dimeric PLA2 inhibited cell-cell fusion mediated by SARS-CoV-2 spike glycoprotein. These results suggest that snake PLA2s, in particular dimeric ones, are promising candidates for the development of antiviral drugs that target lipid bilayers of the viral envelope and may be good tools to study the interaction of viruses with host cell membranes.","version":"1.1","doi":"10.1101/2021.01.12.426042","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.12.426381","pub_date":"2021-1-12","title":"Male sex and age biases viral burden, viral shedding, and type 1 and 2 interferon responses during SARS-CoV-2 infection in ferrets","abstract":"SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and the elderly. Here we investigated the impact of male sex and age by infecting adult male, aged male, and adult female ferrets with SARS-CoV-2. Aged male ferrets had a decrease in temperature which was accompanied by prolonged viral replication with increased pathology in the upper respiratory tract after infection. Transcriptome analysis of the nasal turbinates and lungs indicated that female ferrets had significant increases in interferon response genes (OASL, MX1, ISG15, etc.) on day 2 post infection which was delayed in aged males. In addition, genes associated with taste and smell such as RTP1, CHGA, and CHGA1 at later time points were upregulated in males but not in females. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.","version":"1.1","doi":"10.1101/2021.01.12.426381","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.09.426058","pub_date":"2021-1-11","title":"Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces shedding of SARS-CoV-2 D614G in rhesus macaques","abstract":"Intramuscular vaccination with ChAdOx1 nCoV-19/AZD1222 protected rhesus macaques against pneumonia but did not reduce shedding of SARS-CoV-2. Here we investigate whether intranasally administered ChAdOx1 nCoV-19 reduces shedding, using a SARS-CoV-2 virus with the D614G mutation in the spike protein. Viral load in swabs obtained from intranasally vaccinated hamsters was significantly decreased compared to controls and no viral RNA or infectious virus was found in lung tissue, both in a direct challenge and a transmission model. Intranasal vaccination of rhesus macaques resulted in reduced shedding and a reduction in viral load in bronchoalveolar lavage and lower respiratory tract tissue. In conclusion, intranasal vaccination reduced shedding in two different SARS-CoV-2 animal models, justifying further investigation as a potential vaccination route for COVID-19 vaccines.","version":"1.1","doi":"10.1101/2021.01.09.426058","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.09.426032","pub_date":"2021-1-11","title":"Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA SARS 2 S in preclinical vaccination","abstract":"The severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on vaccinia virus MVA against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust synthesis of S antigen, make it a suitable candidate vaccine for industrial scale production. Vaccinated mice produced S antigen-specific CD8+ T cells and serum antibodies binding to S glycoprotein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19. The highly attenuated vaccinia virus MVA is licensed as smallpox vaccine, and as vector it is a component of the approved Adenovirus-MVA-based prime-boost vaccine against Ebola virus disease. Here we provide results from testing the COVID-19 candidate vaccine MVA-SARS-2-S, a poxvirus-based vector vaccine that proceeded to clinical evaluation. When administered by intramuscular inoculation, MVA-SARS-2-S expresses and safely delivers the full-length SARS-CoV-2 spike (S) protein, inducing balanced SARS-CoV-2-specific cellular and humoral immunity, and protective efficacy in vaccinated mice. Substantial clinical experience has already been gained with MVA vectors using homologous and heterologous prime-boost applications, including the immunization of children and immunocompromised individuals. Thus, MVA-SARS-2-S represents an important resource for developing further optimized COVID-19 vaccines.","version":"1.1","doi":"10.1101/2021.01.09.426032","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.425965","pub_date":"2021-1-11","title":"Molecular Dynamics Analysis of a Flexible Loop at the Binding Interface of the SARS-CoV-2 Spike Protein Receptor-Binding Domain","abstract":"Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.","version":"1.1","doi":"10.1101/2021.01.08.425965","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.425974","pub_date":"2021-1-11","title":"Immunogenicity and Protective Efficacy of an Intranasal Live-attenuated Vaccine Against SARS-CoV-2 in Preclinical Animal Models","abstract":"The global deployment of an effective and safe vaccine is currently a public health priority to curtail the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based intranasal vectored-vaccine in mice and hamsters for its immunogenicity, safety and protective efficacy in challenge studies with SARS-CoV-2. The recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 administrated via intranasal route in mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T cell-mediated immunity. Hamsters vaccinated with two doses of vaccine showed complete protection from clinical disease including lung infection, inflammation, and pathological lesions after SARS-CoV-2 challenge. Importantly, a single or double dose of intranasal rNDV-S vaccine completely blocked SARS-CoV-2 shedding in nasal turbinate and lungs within 4 days of vaccine administration in hamsters. Taken together, intranasal administration of rNDV-S has the potential to control infection at the site of inoculation, which should prevent both the clinical disease and transmission to halt the spread of the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2021.01.08.425974","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425716","pub_date":"2021-1-11","title":"Comprehensive comparison of transcriptomes in SARS-CoV-2 infection: alternative entry routes and innate immune responses","abstract":"The pathogenesis of COVID-19 emerges as complex, with multiple factors leading to injury of different organs. Several studies on underlying cellular processes have produced contradictory claims, e.g. on SARS-CoV-2 cell entry or innate immune responses. However, clarity in these matters is imperative for therapy development. We therefore performed a meta-study with a diverse set of transcriptomes under infections with SARS-CoV-2, SARS-CoV and MERS-CoV, including data from different cells and COVID-19 patients. Using these data, we investigated viral entry routes and innate immune responses. First, our analyses support the existence of cell entry mechanisms for SARS and SARS-CoV-2 other than the ACE2 route with evidence of inefficient infection of cells without expression of ACE2; expression of TMPRSS2/TPMRSS4 is unnecessary for efficient SARS-CoV-2 infection with evidence of efficient infection of A549 cells transduced with a vector expressing human ACE2. Second, we find that innate immune responses in terms of interferons and interferon simulated genes are strong in relevant cells, for example Calu3 cells, but vary markedly with cell type, virus dose, and virus type.","version":"1.2","doi":"10.1101/2021.01.07.425716","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.162156","pub_date":"2021-1-11","title":"CoronaHiT: High throughput sequencing of SARS-CoV-2 genomes","abstract":"The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 96 samples at a time. There is a need, however, for a flexible, platform agnostic, method that can provide multiple throughput options depending on changing requirements as the pandemic peaks and troughs. Here we present CoronaHiT, a method capable of multiplexing up to 96 small genomes on a single MinION flowcell or >384 genomes on Illumina NextSeq, using transposase mediated addition of adapters and PCR based addition of barcodes to ARTIC PCR products. We demonstrate the method by sequencing 95 and 59 SARS-CoV-2 genomes for routine and rapid outbreak response runs, respectively, on Nanopore and Illumina platforms and compare to the standard ARTIC LoCost nanopore method. Of the 154 samples sequenced using the three approaches, genomes with \u2265 90% coverage (GISAID criteria) were generated for 64.3% of samples for ARTIC LoCost, 71.4% for CoronaHiT-ONT, and 76.6% for CoronaHiT-Illumina and have almost identical clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 96 SARS-CoV-2 genomes per MinION flowcell and that Illumina sequencing can be performed on the same libraries, which will allow significantly higher throughput. CoronaHiT provides increased coverage for higher Ct samples, thereby increasing the number of high quality genomes that pass the GISAID QC threshold. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.","version":"1.2","doi":"10.1101/2020.06.24.162156","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.354811","pub_date":"2021-1-11","title":"Post-exposure protection of SARS-CoV-2 lethal infected K18-hACE2 transgenic mice by neutralizing human monoclonal antibody","abstract":"Coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterized and further evaluated the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection while administration of the MD65 antibody as late as 3 days after exposure, rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data unprecedentedly demonstrate, the therapeutic value of human monoclonal antibodies as a life-saving treatment of severe COVID-19 infection.","version":"1.2","doi":"10.1101/2020.10.26.354811","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.410472","pub_date":"2021-1-11","title":"Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide","abstract":"Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the \u201cfusion peptide\u201d (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry. SARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed \u201cfusion peptide\u201d (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca2+-dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.","version":"1.2","doi":"10.1101/2020.12.03.410472","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.372037","pub_date":"2021-1-11","title":"Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization","abstract":"Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics, viral escape mutants could compromise their efficacy. To define the immune-selected mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) to generate 50 different escape mutants. The variants were mapped onto the RBD structure and evaluated for cross-resistance to mAbs and convalescent human sera. Each mAb had a unique resistance profile, although many shared residues within an epitope. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera, suggesting some humans induce a narrow repertoire of neutralizing antibodies. Comparing the antibody-mediated mutational landscape in S with sequence variation in circulating SARS-CoV-2, we define substitutions that may attenuate neutralizing immune responses in some humans.","version":"1.2","doi":"10.1101/2020.11.06.372037","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.423670","pub_date":"2021-1-11","title":"From infection to immunity: understanding the response to SARS-CoV2 through in-silico modeling","abstract":"Immune system conditions of the patient is a key factor in COVID-19 infection survival. A growing number of studies have focused on immunological determinants to develop better biomarkers for therapies. The dynamics of the insurgence of immunity is at the core of the both SARS-CoV-2 vaccine development and therapies. This paper addresses a fundamental question in the management of the infection: can we describe the insurgence (and the span) of immunity in COVID-19? The in-silico model developed here answers this question at individual (personalized) and population levels. We simulate the immune response to SARS-CoV-2 and analyze the impact of infecting viral load, affinity to the ACE2 receptor and age in the artificially infected population on the course of the disease. We use a stochastic agent-based immune simulation platform to construct a virtual cohort of infected individuals with age-dependent varying degree of immune competence. We use a parameter setting to reproduce known inter-patient variability and general epidemiological statistics. We reproduce in-silico a number of clinical observations and we identify critical factors in the statistical evolution of the infection. In particular we evidence the importance of the humoral response over the cytotoxic response and find that the antibody titers measured after day 25 from the infection is a prognostic factor for determining the clinical outcome of the infection. Our modeling framework uses COVID-19 infection to demonstrate the actionable effectiveness of simulating the immune response at individual and population levels. The model developed is able to explain and interpret observed patterns of infection and makes verifiable temporal predictions. Within the limitations imposed by the simulated environment, this work proposes in a quantitative way that the great variability observed in the patient outcomes in real life can be the mere result of subtle variability in the infecting viral load and immune competence in the population. In this work we i) show the power of model predictions, ii) identify the clinical end points that could be more suitable for computational modeling of COVID-19 immune response, iii) define the resolution and amount of data required to empower this class of models for translational medicine purposes and, iv) we exemplify how computational modeling of immune response provides an important view to discuss hypothesis and design new experiments, in particular paving the way to further investigations about the duration of vaccine-elicited immunity especially in the view of the blundering effect of immunesenescence.","version":"1.2","doi":"10.1101/2020.12.20.423670","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426209","pub_date":"2021-1-11","title":"COVID-19 Severity Is Associated with Differential Antibody Fc-mediated Innate Immune Functions","abstract":"Beyond neutralization, antibodies elicit several innate immune functions including complement deposition (ADCD), phagocytosis (ADCP), and cytotoxicity (ADCC). These functions can be both beneficial (by clearing pathogens) and/or detrimental (by inducing inflammation). We tested the possibility that qualitative differences in SARS-CoV-2 specific antibody-mediated innate immune functions contribute to Coronavirus disease 2019 (COVID-19) severity. We found that antibodies from hospitalized COVID-19 patients elicited higher ADCD but lower ADCP compared to antibodies from non-hospitalized COVID-19 patients. Consistently, higher ADCD was associated with higher systemic inflammation during COVID-19. Our study points to qualitative, differential features of anti-SARS-CoV-2 antibodies as potential contributors to COVID-19 severity.","version":"1.1","doi":"10.1101/2021.01.11.426209","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.425999","pub_date":"2021-1-11","title":"The landscape of human brain immune response in patients with severe COVID-19","abstract":"In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between brain tropism, neuroinflammation and host immune response has not been well characterized. We analyzed 68,557 single-nucleus transcriptomes from three brain regions (dorsolateral prefrontal cortex, medulla oblongata and choroid plexus) and identified an increased proportion of stromal cells and monocytes in the choroid plexus of COVID-19 patients. Differential gene expression, pseudo-temporal trajectory and gene regulatory network analyses revealed microglial transcriptome perturbations, mediating a range of biological processes, including cellular activation, mobility and phagocytosis. Quantification of viral spike S1 protein and SARS-CoV-2 transcripts did not support the notion of brain tropism. Overall, our findings suggest extensive neuroinflammation in patients with acute COVID-19. Single-nucleus transcriptome analysis suggests extensive neuroinflammation in human brain tissue of patients with acute coronavirus disease 2019.","version":"1.1","doi":"10.1101/2021.01.08.425999","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.11.426218","pub_date":"2021-1-11","title":"Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class","abstract":"Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilized VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486RBD. Despite these similarities, class members bound both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N343 on a neighboring RBD \u2013 a quaternary interaction accommodated by an increase in RBD separation of up to 12 \u00c5. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions. Determine structures of VH1-2-derived antibodies 2-43, 2-15, and H4 in complex with SARS-CoV-2 spike Define a multi-donor VH1-2-antibody class with modular components for RBD and quaternary recognition Reveal structural basis of RBD-up and RBD-down recognition within the class Show somatic hypermutations and avidity to be critical for potency Delineate changes in spike conformation induced by CDRH3-mediated quaternary recognition","version":"1.1","doi":"10.1101/2021.01.11.426218","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.423118","pub_date":"2021-1-11","title":"Modelling conformational state dynamics and its role on infection for SARS-CoV-2 Spike protein variants","abstract":"The SARS-CoV-2 Spike protein needs to be in an open-state conformation to interact with ACE2 as part of the viral entry mechanism. We utilise coarse-grained normal-mode analyses to model the dynamics of Spike and calculate transition probabilities between states for 17081 Spike variants. Our results correctly model an increase in open-state occupancy for the more infectious D614G via an increase in flexibility of the closed-state and decrease of flexibility of the open-state. We predict the same effect for several mutations on Glycine residues (404, 416, 504, 252) as well as residues K417, D467 and N501, including the N501Y mutation, explaining the higher infectivity of the B.1.1.7 and 501.V2 strains. This is, to our knowledge, the first use of normal-mode analysis to model conformational state transitions and the effect of mutations thereon. The specific mutations of Spike identified here may guide future studies to increase our understanding of SARS-CoV-2 infection mechanisms and guide public health in their surveillance efforts.","version":"1.3","doi":"10.1101/2020.12.16.423118","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.10.426120","pub_date":"2021-1-11","title":"Potent SARS-CoV-2 Neutralizing Antibodies Directed Against Spike N-Terminal Domain Target a Single Supersite","abstract":"Numerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycans N17, N74, N122, and N149. This site \u2013 formed primarily by a mobile \u03b2-hairpin and several flexible loops \u2013 was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single supersite.","version":"1.1","doi":"10.1101/2021.01.10.426120","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.10.426143","pub_date":"2021-1-11","title":"Impact of South African 501.V2 Variant on SARS-CoV-2 Spike Infectivity and Neutralization: A Structure-based Computational Assessment","abstract":"The SARS-CoV-2 variants emerging from South Africa (501.V2) and the UK (B.1.1.7) necessitate rapid assessment of the effects of the corresponding amino acid substitutions in the spike (S) receptor-binding domain (RBD) of the variants on the interactions with the human ACE2 receptor and monoclonal antibodies (mAbs) reported earlier to neutralize the spike. Molecular modeling and simulations reveal that N501Y, shared by both variants, increases ACE2 binding affinity, and may impact the collective dynamics of the ACE2-RBD complex, occupying a central hinge site that modulates the overall dynamics of the complex. In contrast, the substitutions K417N and E484K in the South African variant 501.V2 would reduce the ACE2-binding affinity by abolishing two interfacial salt bridges that facilitate RBD binding to ACE2, K417(S)-D30(ACE2) and E484 (S)-K31(ACE2). These two mutations may thus be more than compensating the attractive effect induced by N501Y, overall resulting in an ACE2-binding affinity comparable to that of the wildtype RBD. Further analysis of the impact of these mutations on the interactions with mAbs targeting the spike indicate that the substitutions K417N and E484K may also abolish the salt bridges between the spike and selected mAbs, such as REGN10933, BD23, H11_H4, and C105, thus reducing the binding affinity and effectiveness of these mAbs. bahar@pitt.edu Supplementary data are available at Bioinformatics online.","version":"1.1","doi":"10.1101/2021.01.10.426143","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.425915","pub_date":"2021-1-08","title":"Ad26.COV2.S-elicited immunity protects against G614 spike variant SARS-CoV-2 infection in Syrian hamsters and does not enhance respiratory disease in challenged animals with breakthrough infection after sub-optimal vaccine dosing","abstract":"Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1\u00d7109 vp and 1\u00d71010 vp elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers which was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these pre-clinical data confirm efficacy of a 1-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.","version":"1.1","doi":"10.1101/2021.01.08.425915","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425801","pub_date":"2021-1-08","title":"Fibrinolysis influences SARS-CoV-2 infection in ciliated cells","abstract":"Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host proteases, may cleave the furin site of SARS-CoV-2 S protein and \u03b3 subunits of epithelial sodium channels (\u03b3 ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. To address this issue, we analyzed single-cell RNA sequence (scRNA-seq) datasets, and found the PLAU (encoding urokinase plasminogen activator), SCNN1G (\u03b3ENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in alveolar epithelial, basal, club, and ciliated epithelial cells. The relative expression level of PLAU, TMPRSS2, and ACE2 were significantly upregulated in severe COVID-19 patients and SARS-CoV-2 infected cell lines using Seurat and DESeq2 R packages. Moreover, the increments in PLAU, FURIN, TMPRSS2, and ACE2 were predominately observed in different epithelial cells and leukocytes. Accordingly, SARS-CoV-2 may share and strengthen the ENaC fibrinolytic proteases network in ACE2 positive airway and alveolar epithelial cells, which may expedite virus infusion into the susceptible cells and bring about ENaC associated edematous respiratory condition.","version":"1.1","doi":"10.1101/2021.01.07.425801","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197343","pub_date":"2021-1-08","title":"SARS-CoV-2 induces human plasmacytoid pre-dendritic cell diversification via UNC93B and IRAK4","abstract":"Several studies have analyzed antiviral immune pathways in late-stage severe COVID-19. However, the initial steps of SARS-CoV-2 antiviral immunity are poorly understood. Here, we have isolated primary SARS-CoV-2 viral strains, and studied their interaction with human plasmacytoid pre-dendritic cells (pDC), a key player in antiviral immunity. We show that pDC are not productively infected by SARS-CoV-2. However, they efficiently diversified into activated P1-, P2-, and P3-pDC effector subsets in response to viral stimulation. They expressed CD80, CD86, CCR7, and OX40 ligand at levels similar to influenza virus-induced activation. They rapidly produced high levels of interferon-\u03b1, interferon-\u03bb1, IL-6, IP-10, and IL-8. All major aspects of SARS-CoV-2-induced pDC activation were inhibited by hydroxychloroquine. Mechanistically, SARS-CoV-2-induced pDC activation critically depended on IRAK4 and UNC93B1, as established using pDC from genetically deficient patients. Overall, our data indicate that human pDC are efficiently activated by SARS-CoV-2 particles and may thus contribute to type I IFN-dependent immunity against SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.07.10.197343","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.10.334292","pub_date":"2021-1-08","title":"The landscape of antibody binding in SARS-CoV-2 infection","abstract":"The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which one epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the six other known human CoVs. We also confirm reactivity against four of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to nine SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.","version":"1.2","doi":"10.1101/2020.10.10.334292","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.21249345","pub_date":"2021-01-08","title":"Clinical Validation of a Novel T-cell Receptor Sequencing Assay for Identification of Recent or Prior SARS-CoV-2 Infection","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>While diagnostic, therapeutic, and vaccine development in the COVID-19 pandemic has proceeded at unprecedented speed and scale, critical gaps remain in our understanding of the immune response to SARS-CoV-2. Current diagnostic strategies, including serology, have numerous limitations in addressing these gaps. Here we describe clinical performance of T- Detect\u2122 COVID, the first reported assay to determine recent or prior SARS-CoV-2 infection based on T-cell receptor (TCR) sequencing and immune repertoire profiling from whole blood samples.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    Methods for high-throughput immunosequencing of the TCR\u03b2 gene from blood specimens have been described\n                    <jats:sup>1</jats:sup>\n                    . We developed a statistical classifier showing high specificity for identifying prior SARS-CoV-2 infection\n                    <jats:sup>2</jats:sup>\n                    , utilizing &gt;4,000 SARS-CoV-2-associated TCR sequences from 784 cases and 2,447 controls across 5 independent cohorts. The T-Detect COVID Assay comprises immunosequencing and classifier application to yield a qualitative positive or negative result. Several retrospective and prospective cohorts were enrolled to assess assay performance including primary and secondary Positive Percent Agreement (PPA; N=205, N=77); primary and secondary Negative Percent Agreement (NPA; N=87, N=79); PPA compared to serology (N=55); and pathogen cross-reactivity (N=38).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>T-Detect COVID demonstrated high PPA in subjects with prior PCR-confirmed SARS-CoV-2 infection (97.1% 15+ days from diagnosis; 94.5% 15+ days from symptom onset), high NPA (\u223c100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than two commercial EUA serology tests, and no evidence of pathogen cross-reactivity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>T-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance to identify recent or prior SARS-CoV-2 infection from standard blood samples. This assay can provide critical insights on the SARS-CoV-2 immune response, with potential implications for clinical management, risk stratification, surveillance, assessing protective immunity, and understanding long-term sequelae.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.01.06.21249345","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.07.425806","pub_date":"2021-1-08","title":"Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a na\u00efve human antibody library","abstract":"Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that na\u00efve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human na\u00efve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of na\u00efve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.","version":"1.1","doi":"10.1101/2021.01.07.425806","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.08.21249426","pub_date":"2021-01-08","title":"Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostic strategies, that are easily adapatable to the changing virus. Here, we have designed and developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. In our assay, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter-protein e.g. LacZ or Nano-lantern that is easily detected using color/luminescence. By optimizing RNA-amplification and biosensor-design, we have generated a highly-sensitive diagnostic assay; with sensitivity down to attomolar SARS-CoV-2 RNA. As low as 100 copies of viral RNA are detected with development of bright color that is easily visualized by the human eye, or a simple cell phone camera as well as quantified using a spectrophotometer. This makes our assay deployable all the way from a well equiped laboratory to a low-resource setting anywhere in the world. Finally, this\n                  <jats:bold>PHA</jats:bold>\n                  sed\n                  <jats:bold>N</jats:bold>\n                  ASBA-\n                  <jats:bold>T</jats:bold>\n                  ranslation\n                  <jats:bold>O</jats:bold>\n                  ptical\n                  <jats:bold>M</jats:bold>\n                  ethod (PHANTOM) using our engineered RNA biosensors efficiently detects the presence of viral RNA in human patient samples, correlating well with Ct values from RT-qPCR tests. This work presents a powerful and universally accessible strategy for detecting Covid-19 and its prominent variants. This strategy is easily adaptable to further viral evolution and brings RNA-based bioengineering to centerstage.\n                </jats:p>","version":null,"doi":"10.1101/2021.01.08.21249426","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.08.084327","pub_date":"2021-1-08","title":"SARS-CoV-2 Spike Glycoprotein and ACE2 interaction reveals modulation of viral entry in wild and domestic animals","abstract":"SARS-CoV-2 is a viral pathogen causing life-threatening disease in human. Interaction between spike protein of SARS-CoV-2 and ACE2 receptor on the cells is a potential factor in the infectivity of a host. The interaction of SARS-CoV-2 spike receptor-binding domain with its receptor - ACE2, in different hosts was evaluated to understand and predict viral entry. The protein and nucleotide sequences of ACE2 were initially compared across different species to identify key differences among them. The ACE2 receptor of various species was homology modeled (6LZG, 6M0J, and 6VW1 as a reference), and its binding ability to the spike ACE2 binding domain of SARS-CoV-2 was assessed. Initially, the spike binding parameters of ACE2 of known infected and uninfected species were compared with each Order (of animals) as a group. Finally, a logistic regression model vis-a-vis the spike binding parameters of ACE2 (considering data against 6LZG and 6M0J) was constructed to predict the probability of viral entry in different hosts. Phylogeny and alignment comparison did not lead to any meaningful conclusion on viral entry in different hosts. Out of several spike binding parameters of ACE2, a significant difference between the known infected and uninfected species was observed for six parameters. However, these parameters did not specifically categorize the Orders (of animals) into infected or uninfected. The logistic regression model constructed revealed that in the mammalian class, most of the species of Carnivores, Artiodactyls, Perissodactyls, Pholidota, and Primates had high probability of viral entry. However, among the primates, African Elephant had low probability of viral entry. Among rodents, hamsters were highly probable for viral entry with rats and mice having a medium to low probability. Rabbits have a high probability of viral entry. In Birds, ducks have a very low probability, while chickens seemed to have medium probability and turkey showed the highest probability of viral entry. Most of the species considered in this study showed high probability of viral entry. This study would prompt us to closely follow certain species of animals for determining pathogenic insult by SARS-CoV-2 and for determining their ability to act as a carrier and/or disseminator.","version":"1.4","doi":"10.1101/2020.05.08.084327","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.21249190","pub_date":"2021-01-08","title":"Host genome analysis of structural variations by Optical Genome Mapping provides clinically valuable insights into genes implicated in critical immune, viral infection, and viral replication pathways in patients with severe COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The varied clinical manifestations and outcomes in patients with SARS-CoV-2 infections implicate a role of host-genetics in the predisposition to disease severity. This is supported by evidence that is now emerging, where initial reports identify common risk factors and rare genetic variants associated with high risk for severe/ life-threatening COVID-19. Impressive global efforts have focused on either identifying common genetic factors utilizing short-read sequencing data in Genome-Wide Association Studies (GWAS) or whole-exome and genome studies to interrogate the human genome at the level of detecting single nucleotide variants (SNVs) and short indels. However, these studies lack the sensitivity to accurately detect several classes of variants, especially large structural variants (SVs) including copy number variants (CNVs), which account for a substantial proportion of variation among individuals. Thus, we investigated the host genomes of individuals with severe/life-threatening COVID-19 at the level of large SVs (500bp-Mb level) to identify events that might provide insight into the inter-individual clinical variability in clinical course and outcomes of COVID-19 patients.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Optical genome mapping using Bionano\u2019s Saphyr\u00ae system was performed on thirty-seven severely ill COVID-19 patients admitted to intensive care units (ICU). To extract candidate SVs, three distinct analyses were undertaken. First, an unbiased whole-genome analysis of SVs was performed to identify rare/unique genic SVs in these patients that did not appear in population datasets to determine candidate loci as decisive predisposing factors associated with severe COVID-19. Second, common SVs with a population frequency filter was interrogated for possible association with severe COVID-19 based on literature surveys. Third, genome-wide SV enrichment in severely ill patients versus the general population was investigated by calculating odds ratios to identify top-ranked genes/loci. Candidate SVs were confirmed using qPCR and an independent bioinformatics tool (FaNDOM).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Our patient-centric investigation identified 11 SVs involving 38 genes implicated in three key host-viral interaction pathways: (1) innate immunity and inflammatory response, (2) airway resistance to pathogens, and (3) viral replication, spread, and RNA editing. These included seven rare/unique SVs (not present in the control dataset), identified in 24.3% (9/37) of patients, impacting up to 31 genes, of which\n                    <jats:italic>STK26</jats:italic>\n                    and\n                    <jats:italic>DPP4</jats:italic>\n                    are the most promising candidates. A duplication partially overlapping\n                    <jats:italic>STK26</jats:italic>\n                    was corroborated with data showing upregulation of this gene in severely ill patients. Further, using a population frequency filter of less than 20% in the Bionano control dataset, four SVs involving seven genes were identified in 56.7% (21/37) of patients.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>\n                    This study is the first to systematically assess and highlight SVs\u2019 potential role in the pathogenesis of COVID-19 severity. The genes implicated here identify novel SVs, especially\n                    <jats:italic>STK26</jats:italic>\n                    , and extend previous reports involving innate immunity and type I interferon response in the pathogenesis of COVID-19. Our study also shows that optical genome mapping can be a powerful tool to identify large SVs impacting disease outcomes with split survival and add valuable genomic information to the existing sequencing-based technology databases to understand the inter-individual variability associated with SARS-CoV-2 infections and COVID-19 mortality.\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.01.05.21249190","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.06.20248960","pub_date":"2021-01-08","title":"Impact of B.1.1.7 variant mutations on antibody recognition of linear SARS-CoV-2 epitopes","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>In 579 COVID patients\u2019 samples collected between March and July of 2020, we examined the effects of non-synonymous mutations harbored by the circulating B.1.1.7 strain on linear antibody epitope signal for spike glycoprotein and nucleoprotein. At the antigen level, the mutations only substantially reduced signal in 0.5% of the population. Although some epitope mutations reduce measured signal in up to 6% of the population, these are not the dominant epitopes for their antigens. Given dominant epitope patterns observed, our data suggest that the mutations would not result in immune evasion of linear epitopes for a large majority of these COVID patients.</jats:p>","version":null,"doi":"10.1101/2021.01.06.20248960","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.06.425652","pub_date":"2021-1-08","title":"Combined Ventilation Of Two Subjects With A Single Mechanical Ventilator Using A New Medical Device: An In Vitro Study","abstract":"The SARS-CoV2 pandemic has created a sudden lack of ventilators. DuplicAR\u00ae is a novel device that allows simultaneous and independent ventilation of two subjects with a single ventilator. The aims of this study are: a) to determine the efficacy of DuplicAR\u00ae to independently regulate the peak and positive-end expiratory pressures in each subject, both under pressure-controlled ventilation and volume-controlled ventilation, and b) to determine the ventilation mode in which DuplicAR\u00ae presents the best performance and safety. Two test lungs are connected to a single ventilator using DuplicAR\u00ae. Three experimental stages are established: 1) two identical subjects, 2) two subjects with the same weight but different lung compliance, and 3) two subjects with different weight and lung compliance. In each stage, the test lungs are ventilated in two ventilation modes. The positive-end expiratory pressure requirements are increased successively in one of the subjects. The goal is to achieve a tidal volume of 7 ml/kg for each subject in all different stages through manipulation of the ventilator and the DuplicAR\u00ae controllers. DuplicAR\u00ae allows adequate ventilation of two subjects with different weight and/or lung compliance and/or PEEP requirements. This is achieved by adjusting the total tidal volume for both subjects (in volume-controlled ventilation) or the highest peak pressure needed (in pressure-controlled ventilation) along with the basal positive-end expiratory pressure on the ventilator, and simultaneously manipulating the DuplicAR\u00ae controllers to decrease the tidal volume or the peak pressure in the subject that needs less and/or to increase the positive-end expiratory pressure in the subject that needs more. While ventilatory goals can be achieved in any of the ventilation modes, DuplicAR\u00ae performs better in pressure-controlled ventilation, as changes experienced in the variables of one subject do not modify the other one. DuplicAR\u00ae is an effective tool to manage the peak inspiratory pressure and the positive-end expiratory pressure independently in two subjects connected to a single ventilator. The driving pressure can be adjusted to meet the requirements of subjects with different weight and lung compliance. Pressure-controlled ventilation has advantages over volume-controlled ventilation and is therefore the recommended ventilation mode.","version":"1.1","doi":"10.1101/2021.01.06.425652","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425705","pub_date":"2021-1-07","title":"Sequencing of SARS CoV2 in local transmission cases through oxford nanopore MinION platform from Karachi Pakistan","abstract":"The first case of severe acute respiratory syndrome 2 (SARS CoV2) was imported to Pakistan in February 2020 since then 10,258 deaths have been witnessed. The virus has been mutating and local transmission cases from different countries vary due to host dependent viral adaptation. Many distinct clusters of variant SARS CoV2 have been defined globally. In this study, the epidemiology of SARS CoV2 was studied and locally transmitted SARS CoV2 isolates from Karachi were sequenced to compared and identify any possible variants.The real time PCR was performed on nasopharyngeal specimen to confirm SARSCoV2 with Orf 1ab and E gene as targets. The viral sequencing was performed through oxford nanopore technology MinION platform. Isolates from first and second wave of COVID-19 outbreak in Karachi were compared. The overall positivity rate for PCR was 26.24% with highest number of positive cases in June. Approximately, 37.45% PCR positive subjects aged between 19-40 years. All the isolates belonged to GH clade and shared missense mutation D614G in spike protein linked to increased transmission rate worldwide. Another spike protein mutation A222V coexisted with D614G in the virus from second wave of COVID-19. Based on the present findings it is suggested that the locally transmitted virus from Karachi vary from those reported from other parts of Pakistan. Slight variability was also observed between viruses from first and second wave. Variability in any potential vaccine target may result in failed trials therefore information on any local viral variants is always useful for effective vaccine design and/or selection. Despite precautionary measures the COVID-19 pandemic is causing deaths all over the world. The continuous mutations in viral genome is making it difficult to design vaccines. Variability in genome is host dependent and data sharing has revealed that variant for different geographical locations may harbor different mutations. Keeping this in mind the current study was focused on the epidemiology of SARS CoV2 in symptomatic and asymptomatic COVID \u201319 suspected cases with impact of age and gender. The locally transmitted SARS CoV2 isolates from Karachi were sequenced to compared and identify any possible variants. The sequenced viral genome varied from the already submitted sequences from Pakistan thereby confirming that slightly different viruses were causing infections during different time periods in Karachi. All belonged to GH clade with D614G, P323L and Q57H mutations. The virus from second wave had A222V mutation making it more different. This information can be useful in selecting or designing a vaccine.","version":"1.1","doi":"10.1101/2021.01.07.425705","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425729","pub_date":"2021-1-07","title":"Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in hamsters","abstract":"Reinfections with SARS-CoV-2 have already been documented in humans, although its real incidence is currently unknown. Besides having great impact on public health, this phenomenon raises the question if immunity generated by a single infection is sufficient to provide sterilizing/protective immunity to a subsequent SARS-CoV-2 re-exposure. The Golden Syrian hamster is a manageable animal model to explore immunological mechanisms able to counteract COVID-19, as it recapitulates pathological aspects of mild to moderately affected patients. Here, we report that SARS-CoV-2-inoculated hamsters resolve infection in the upper and lower respiratory tracts within seven days upon inoculation with the Cat01 (G614) SARS-CoV-2 isolate. Three weeks after primary challenge, and despite high titers of neutralizing antibodies, half of the animals were susceptible to reinfection by both identical (Cat01, G614) and variant (WA/1, D614) SARS-CoV-2 isolates. However, upon re-inoculation, only nasal tissues were transiently infected with much lower viral replication than those observed after the first inoculation. These data indicate that a primary SARS-CoV-2 infection is not sufficient to elicit a sterilizing immunity in hamster models but protects against lung disease.","version":"1.1","doi":"10.1101/2021.01.07.425729","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425740","pub_date":"2021-1-07","title":"Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera","abstract":"Rapidly spreading variants of SARS-CoV-2 that have arisen in the United Kingdom and South Africa share the spike N501Y substitution, which is of particular concern because it is located in the viral receptor binding site for cell entry and increases binding to the receptor (angiotensin converting enzyme 2). We generated isogenic N501 and Y501 SARS-CoV-2. Sera of 20 participants in a previously reported trial of the mRNA-based COVID-19 vaccine BNT162b2 had equivalent neutralizing titers to the N501 and Y501 viruses.","version":"1.1","doi":"10.1101/2021.01.07.425740","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425621","pub_date":"2021-1-07","title":"Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells","abstract":"New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD \u223c 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4\u00b1 0.1 \u03bcM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2021.01.07.425621","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.425497","pub_date":"2021-1-07","title":"Genetic Characteristics and Phylogeny of 969-bp S Gene Sequence of SARS-CoV-2 from Hawaii Reveals the Worldwide Emerging P681H Mutation","abstract":"COVID-19 pandemic has ravaged the world, caused over 1.8 million deaths in the first year, and severely affected the global economy. Hawaii is not spared from the transmission of SARS-CoV-2 in the local population, including high infection rates in racial and ethnic minorities. Early in the pandemic, we described in this journal various technologies used for the detection of SARS-CoV-2. Herein we characterize a 969-bp SARS-CoV-2 segment of the S gene downstream of the receptor-binding domain. At the John A. Burns School of Medicine Biocontainment Facility, RNA was extracted from an oropharyngeal swab and a nasal swab from two patients from Hawaii who were infected with the SARS-CoV-2 in August 2020. Following PCR, the two viral strains were sequenced using Sanger sequencing, and phylogenetic trees were generated using MEGAX. Phylogenetic tree results indicate that the virus has been introduced to Hawaii from multiple sources. Further, we decoded 13 single nucleotide polymorphisms across 13 unique SARS-CoV-2 genomes within this region of the S gene, with one non-synonymous mutation (P681H) found in the two Hawaii strains. The P681H mutation has unique and emerging characteristics with a significant exponential increase in worldwide frequency when compared to the plateauing of the now universal D614G mutation. The P681H mutation is also characteristic of the new SARS-CoV-2 variants from the United Kingdom and Nigeria. Additionally, several mutations resulting in cysteine residues were detected, potentially resulting in disruption of the disulfide bridges in and around the receptor-binding domain. Targeted sequence characterization is warranted to determine the origin of multiple introductions of SARS-CoV-2 circulating in Hawaii.","version":"1.1","doi":"10.1101/2021.01.06.425497","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425745","pub_date":"2021-1-07","title":"New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3\u2019-5\u2019 exoribonucleolytic activity on SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has triggered a global pandemic with devastating consequences for health-care and social-economic systems. Thus, the understanding of fundamental aspects of SARS-CoV-2 is of extreme importance. In this work, we have focused our attention on the viral ribonuclease (RNase) nsp14, since this protein was considered one of the most interferon antagonists from SARS-CoV-2, and affects viral replication. This RNase is a multifunctional protein that harbors two distinct activities, an N-terminal 3\u2019-to-5\u2019 exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both with critical roles in coronaviruses life cycle. Namely, SARS-CoV-2 nsp14 ExoN knockout mutants are non-viable, indicating nsp14 as a prominent target for the development of antiviral drugs. Nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein, which has a pleiotropic function during viral replication. In this study, we have performed the first biochemical characterization of the complex nsp14-nsp10 from SARS-CoV-2. Here we confirm the 3\u2019-5\u2019 exoribonuclease and MTase activities of nsp14 in this new Coronavirus, and the critical role of nsp10 in upregulating the nsp14 ExoN activity in vitro. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity. The nsp14 MTase activity also seems to be independent of the presence of nsp10 cofactor, contrarily to nsp14 ExoN. Until now, there is no available structure for the SARS-CoV-2 nsp14-nsp10 complex. As such, we have modelled the SARS-CoV-2 nsp14-nsp10 complex based on the 3D structure of the complex from SARS-CoV (PDB ID 5C8S). We also have managed to map key nsp10 residues involved in its interaction with nsp14, all of which are also shown to be essential for stimulation of the nsp14 ExoN activity. This reinforces the idea that a stable interaction between nsp10 and nsp14 is strictly required for the nsp14-mediated ExoN activity of SARS-CoV-2, as observed for SARS-CoV. We have studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function contrasting to the functionality of these conserved catalytic residues in SARS-CoV, and in the Middle East respiratory syndrome coronav\u00edrus (MERS). The differences here revealed can have important implications regarding the specific pathogenesis of SARS-CoV-2. The nsp10-nsp14 interface is a recognized attractive target for antivirals against SARS-CoV-2 and other coronaviruses. This work has unravelled a basis for discovering inhibitors targeting the specific amino acids here reported, in order to disrupt the assembly of this complex and interfere with coronaviruses replication.","version":"1.1","doi":"10.1101/2021.01.07.425745","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.26.424429","pub_date":"2021-1-07","title":"Analysis and Forecasting of Global RT-PCR Primers for SARS-CoV-2","abstract":"Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.","version":"1.2","doi":"10.1101/2020.12.26.424429","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425724","pub_date":"2021-1-07","title":"Immunoinformatic based analytics on T-cell epitope from spike protein of SARS-CoV-2 concerning Indian population","abstract":"The whole world is drastically affected by the current pandemic due to severe virus, SARS-CoV-2 and scientists are rigorously looking for the efficient vaccine against it that become an emergent issue. Reverse vaccinology approach may provide us with significant therapeutic leads in this direction and further determination of T-cell / B-cell response to antigen. In the present study, we conducted population coverage analysis referring to the diverse Indian population. By using tools from Immune epitope database (IEDB), HLA- distribution analysis was performed to find the most promiscuous T-cell epitope out of In silico determined epitope of Spike protein from SARS-CoV-2. Selection of these epitopes have been conducted based on their binding affinity with the maximum number of HLA alleles belong to the highest population coverage rate values for the chosen geographical area in India. 404 cleavage sites within the 1288 amino acids sequence of spike glycoprotein were determined by NetChop proteasomal cleavage prediction suggesting that this protein has adequate sites in the protein sequence for cleaving into appropriate epitopes. For population coverage analysis, 221 selected epitopes are considered that shows the projected population coverage as 83.08% with 19.29 average hit (average number of epitope hits/HLA combinations recognized by the population) and 5.91 pc90 (minimum number of epitope hits/HLA combinations recognized by 90% of the population). 54 epitopes are found with the highest coverage among the Indian population and highly conserved within the given spike RBD domain sequence. Docking analysis of each epitope with their respective allele suggests that the epitope NSFTRGVYY represents highest binding affinity with docking score -7.6 kcal/mol with its allele HLA-C*07:01 among all the epitopes. Since the Covid-19 cases are still in progress and seem to remain like this until we find an effective vaccine, moreover in countries like India, vast diversity in the population may present a hindrance to particular vaccine efficiency. Outcomes from this study could be critical to design vaccine against SARS-CoV-2 for a different set of the population within the country.","version":"1.1","doi":"10.1101/2021.01.07.425724","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.425698","pub_date":"2021-1-07","title":"The peripheral T cell population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus 2","abstract":"The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that began in December 2019. Lymphopenia is a common feature in severe cases of COVID-19; however, the role of T cell responses during infection is unclear. Here, we inoculated six cynomolgus monkeys, divided into two groups according to the CD3+ T cell population in peripheral blood, with two clinical isolates of SARS-CoV-2: one of East Asian lineage and one of European lineage. After initial infection with the isolate of East Asian lineage, all three monkeys in the CD3+ low group showed clinical symptoms, including loss of appetite, lethargy, and transient severe anemia with/without short-term fever, within 14 days post-infection (p.i.). By contrast, all three monkeys in the CD3+ high group showed mild clinical symptoms such as mild fever and loss of appetite within 4 days p.i. and then recovered. After a second inoculation with the isolate of European lineage, three of four animals in both groups showed mild clinical symptoms but recovered quickly. Hematological, immunological, and serological tests suggested that the CD3+ high and low groups mounted different immune responses during the initial and second infection stages. In both groups, anti-viral and innate immune responses were activated during the early phase of infection and re-infection. However, in the CD3+ low group, inflammatory responses, such as increased production of monocytes and neutrophils, were stronger than those in the CD3+ high group, leading to more severe immunopathology and failure to eliminate the virus. Taken together, the data suggest that the peripheral T lymphocyte population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with SARS-CoV-2. SARS-CoV-2 infection causes an illness with clinical manifestations that vary from asymptomatic or mild to severe; examples include severe pneumonia and acute respiratory distress syndrome. Lymphopenia, which is common in severe COVID-19 cases, is characterized by markedly reduced numbers of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells. Here, we showed that cynomolgus monkeys selected according to the T cell populations in peripheral blood have different outcomes after experimental infection with SARS-CoV-2. These findings will increase our understanding of disease pathogenesis and may facilitate the development of animal models for vaccine evaluation.","version":"1.1","doi":"10.1101/2021.01.07.425698","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.07.21249390","pub_date":"2021-01-07","title":"Interleukin-6 Receptor Antagonists in Critically Ill Patients with Covid-19 \u2013 Preliminary report","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The efficacy of interleukin-6 receptor antagonists in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We evaluated tocilizumab and sarilumab in an ongoing international, multifactorial, adaptive platform trial. Adult patients with Covid-19, within 24 hours of commencing organ support in an intensive care unit, were randomized to receive either tocilizumab (8mg/kg) or sarilumab (400mg) or standard care (control). The primary outcome was an ordinal scale combining in-hospital mortality (assigned \u22121) and days free of organ support to day 21. The trial uses a Bayesian statistical model with pre-defined triggers to declare superiority, efficacy, equivalence or futility.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Tocilizumab and sarilumab both met the pre-defined triggers for efficacy. At the time of full analysis 353 patients had been assigned to tocilizumab, 48 to sarilumab and 402 to control. Median organ support-free days were 10 (interquartile range [IQR] \u22121, 16), 11 (IQR 0, 16) and 0 (IQR \u22121, 15) for tocilizumab, sarilumab and control, respectively. Relative to control, median adjusted odds ratios were 1.64 (95% credible intervals [CrI] 1.25, 2.14) for tocilizumab and 1.76 (95%CrI 1.17, 2.91) for sarilumab, yielding &gt;99.9% and 99.5% posterior probabilities of superiority compared with control. Hospital mortality was 28.0% (98/350) for tocilizumab, 22.2% (10/45) for sarilumab and 35.8% (142/397) for control. All secondary outcomes and analyses supported efficacy of these IL-6 receptor antagonists.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    In critically ill patients with Covid-19 receiving organ support in intensive care, treatment with the IL-6 receptor antagonists, tocilizumab and sarilumab, improved outcome, including survival. (\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='http://ClinicalTrials.gov'>ClinicalTrials.gov</jats:ext-link>\n                    number:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT02735707'>NCT02735707</jats:ext-link>\n                    )\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2021.01.07.21249390","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.06.425622","pub_date":"2021-1-06","title":"Self-organized stem cell-derived human lung buds with proximo-distal patterning and novel targets of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic and the lack of therapeutics hinders pandemic control. Although lung disease is the primary clinical outcome in COVID-19 patients, how SARS-CoV-2 induces tissue pathology in the lung remains elusive. Here we describe a high-throughput platform to generate tens of thousands of self-organizing, nearly identical, and genetically matched human lung buds derived from human pluripotent stem cells (hPSCs) cultured on micropatterned substrates. Strikingly, in vitro-derived human lung buds resemble fetal human lung tissue and display in vivo-like proximo-distal coordination of alveolar and airway tissue differentiation whose 3D epithelial self-organization is directed by the levels of KGF. Single-cell transcriptomics unveiled the cellular identities of airway and alveolar tissue and the differentiation of WNThi cycling alveolar stem cells, a human-specific lung cell type. These synthetic human lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-dependent susceptibilities to infection, intercellular transmission and cytopathology in airway and alveolar tissue in individual lung buds. Interestingly, we detected an increased susceptibility to infection in alveolar cells and identified cycling alveolar stem cells as targets of SARS-CoV-2. We used this platform to test neutralizing antibodies isolated from convalescent plasma that efficiently blocked SARS-CoV-2 infection and intercellular transmission. Our platform offers unlimited, rapid and scalable access to disease-relevant lung tissue that recapitulate key hallmarks of human lung development and can be used to track SARS-CoV-2 infection and identify candidate therapeutics for COVID-19.","version":"1.1","doi":"10.1101/2021.01.06.425622","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.156679","pub_date":"2021-1-06","title":"A COVID Moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.","version":"1.2","doi":"10.1101/2020.06.17.156679","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425516","pub_date":"2021-1-06","title":"RNA-protein interaction analysis of SARS-CoV-2 5\u2019- and 3\u2019-untranslated regions identifies an antiviral role of lysosome-associated membrane protein-2","abstract":"Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is a positive-strand RNA virus. Viral genome is capped at the 5\u2019-end, followed by an untranslated region (UTR). There is poly-A tail at 3\u2019-end, preceded by an UTR. Self-interaction between the RNA regulatory elements present within 5\u2019- and 3\u2019-UTRs as well as their interaction with host/virus-encoded proteins mediate the function of 5\u2019- and 3\u2019-UTRs. Using RNA-protein interaction detection (RaPID) assay coupled to liquid chromatography with tandem mass-spectrometry, we identified host interaction partners of SARS-CoV-2 5\u2019- and 3\u2019-UTRs and generated an RNA-protein interaction network. By combining these data with the previously known protein-protein interaction data proposed to be involved in virus replication, we generated the RNA-protein-protein interaction (RPPI) network, likely to be essential for controlling SARS-CoV-2 replication. Notably, bioinformatics analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism. Lysosome-associated membrane protein-2a (Lamp2a) was one of the host proteins that interact with the 5\u2019-UTR. Further studies showed that Lamp2 level is upregulated in SARS-CoV-2 infected cells and overexpression of Lamp2a and Lamp2b variants reduced viral RNA level in infected cells and vice versa. In summary, our study provides an useful resource of SARS-CoV-2 5\u2019- and 3\u2019-UTR binding proteins and reveal the antiviral function of host Lamp2 protein. Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5\u2019- and 3\u2019-UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication. Here, we identified the host proteins that associate with the UTRs of SARS-CoV-2, combined those data with the previously known protein-protein interaction data (expected to be involved in virus replication) and generated the RNA-protein-protein interaction (RPPI) network. Analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism, which are important for virus replication. Analysis of one of the interaction partners of the 5\u2019-UTR (Lamp2a) demonstrated its antiviral role in SARS-CoV-2 infected cells. Collectively, our study provides a resource of SARS-CoV-2 UTR-binding proteins and identifies an antiviral role of host Lamp2a protein.","version":"1.1","doi":"10.1101/2021.01.05.425516","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425508","pub_date":"2021-1-06","title":"Human cell-dependent, directional, time-dependent changes in the mono- and oligonucleotide compositions of SARS-CoV-2 genomes","abstract":"When a virus that has grown in a nonhuman host starts an epidemic in the human population, human cells may not provide growth conditions ideal for the virus. Therefore, the invasion of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is usually prevalent in the bat population, into the human population is thought to have necessitated changes in the viral genome for efficient growth in the new environment. In the present study, to understand host-dependent changes in coronavirus genomes, we focused on the mono- and oligonucleotide compositions of SARS-CoV-2 genomes and investigated how these compositions changed time-dependently in the human cellular environment. We also compared the oligonucleotide compositions of SARS-CoV-2 and other coronaviruses prevalent in humans or bats to investigate the causes of changes in the host environment. Time-series analyses of changes in the nucleotide compositions of SARS-CoV-2 genomes revealed a group of mono- and oligonucleotides whose compositions changed in a common direction for all clades, even though viruses belonging to different clades should evolve independently. Interestingly, the compositions of these oligonucleotides changed towards those of coronaviruses that have been prevalent in humans for a long period and away from those of bat coronaviruses. Clade-independent, time-dependent changes are thought to have biological significance and should relate to viral adaptation to a new host environment, providing important clues for understanding viral host adaptation mechanisms.","version":"1.1","doi":"10.1101/2021.01.05.425508","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.365833","pub_date":"2021-1-06","title":"Zinc-embedded fabrics inactivate SARS-CoV-2 and influenza A virus","abstract":"Infections with respiratory viruses can spread via liquid droplets and aerosols, and cause diseases such as influenza and COVID-19. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of respiratory droplets containing these viruses. However, influenza A viruses and coronaviruses are stable for hours on various materials, which makes frequent and correct disposal of these PPE important. Metal ions embedded into PPE may inactivate respiratory viruses, but confounding factors such as absorption of viruses make measuring and optimizing the inactivation characteristics difficult. Here we used polyamide 6.6 (PA66) fibers that had zinc ions embedded during the polymerisation process and systematically investigated if these fibers can absorb and inactivate pandemic SARS-CoV-2 and influenza A virus H1N1. We find that these viruses are readily absorbed by PA66 fabrics and inactivated by zinc ions embedded into this fabric. The inactivation rate (pfu\u00b7gram\u22121\u00b7min\u22121) exceeds the number of active virus particles expelled by a cough and supports a wide range of viral loads. Moreover, we found that the zinc content and the virus inactivating property of the fabric remain stable over 50 standardized washes. Overall, these results provide new insight into the development of \u201cpathogen-free\u201d PPE and better protection against RNA virus spread.","version":"1.3","doi":"10.1101/2020.11.02.365833","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425420","pub_date":"2021-1-06","title":"COVID-19 virtual patient cohort reveals immune mechanisms driving disease outcomes","abstract":"To understand the diversity of immune responses to SARS-CoV-2 and distinguish features that predispose individuals to severe COVID-19, we developed a mechanistic, within-host mathematical model and virtual patient cohort. Our results indicate that virtual patients with low production rates of infected cell derived IFN subsequently experienced highly inflammatory disease phenotypes, compared to those with early and robust IFN responses. In these in silico patients, the maximum concentration of IL-6 was also a major predictor of CD8+ T cell depletion. Our analyses predicted that individuals with severe COVID-19 also have accelerated monocyte-to-macrophage differentiation that was mediated by increased IL-6 and reduced type I IFN signalling. Together, these findings identify biomarkers driving the development of severe COVID-19 and support early interventions aimed at reducing inflammation. Understanding of how the immune system responds to SARS-CoV-2 infections is critical for improving diagnostic and treatment approaches. Identifying which immune mechanisms lead to divergent outcomes can be clinically difficult, and experimental models and longitudinal data are only beginning to emerge. In response, we developed a mechanistic, mathematical and computational model of the immunopathology of COVID-19 calibrated to and validated against a broad set of experimental and clinical immunological data. To study the drivers of severe COVID-19, we used our model to expand a cohort of virtual patients, each with realistic disease dynamics. Our results identify key processes that regulate the immune response to SARS-CoV-2 infection in virtual patients and suggest viable therapeutic targets, underlining the importance of a rational approach to studying novel pathogens using intra-host models.","version":"1.1","doi":"10.1101/2021.01.05.425420","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.21249236","pub_date":"2021-01-06","title":"Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality","abstract":"<jats:p>While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load \u2013 as measured by saliva but not nasopharyngeal \u2014 is a dynamic unifying correlate of disease presentation, severity, and mortality over time.</jats:p>","version":null,"doi":"10.1101/2021.01.04.21249236","journal":"medRxiv","score":null},{"id":"10.1101/2021.01.06.425542","pub_date":"2021-1-06","title":"Incidence and Characteristics of Co-infection and Secondary Infection in Patients with COVID-19","abstract":"The etiology and epidemiology of co-infection and secondary infection in COVID-19 patients remain unknown. The study aims to investigate the occurrence and characteristics of co-infection and secondary infection in COVID-19 patients, mainly focusing on Streptococcus pneumoniae co-infections. This study was a prospective, observational cohort study of the inpatients diagnosed with COVID-19 in two designated hospitals in south China enrolled between Jan 11 and Feb 22, 2020. The urine specimen was collected on admission and applied for pneumococcal urinary antigen tests (PUATs). Demographic, clinical and microbiological data of patients were recorded simultaneously. A total of 146 patients with a confirm diagnosis of COVID-19 at the median age of 50.0 years (IQR 36.0-61.0) were enrolled, in which, 16 (11.0%) were classified as severe cases and 130 (89.0%) as non-severe cases. Of the enrolled patients, only 3 (2.1%) were considered to present the co-infection, in which 1 was co-infected with S.pneumoniae, 1 with B. Ovatus infection and the other one with Influenza A virus infection. Secondary infection occurred in 16 patients, with S. maltophilia as the most commonly isolated pathogen (43.8%), followed by P. aeruginosa (25.0%), E. aerogenes (25.0%), C. parapsilosis (25.0%) and A. fumigates (18.8%). Patients with confirmed COVID-19 were rarely co-infected with Streptococcus pneumoniae or other pathogens, indicating that the application of antibiotics against CAP on admission may not be necessary in the treatment of COVID-19 cases.","version":"1.1","doi":"10.1101/2021.01.06.425542","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.06.425543","pub_date":"2021-1-06","title":"Heterogeneity versus the COVID-19 Pandemic","abstract":"In this paper, heterogeneity is formally defined, and its properties are explored. We define and distinguish observable versus non-observable heterogeneity. It is proposed that heterogeneity among the vulnerable is a significant factor in the contagion impact of COVID-19, as demonstrated with incidence rates on a Diamond Princess Cruise ship in February 2020. Given the nature of the disease, its heterogeneity and human social norms, pre-voyage and post-voyage quick testing procedures may become the new standard for cruise ship passengers and crew. The technological advances in testing available today would facilitate more humanistic treatment as compared to more archaic quarantine and isolation practices for all onboard ship. With quick testing, identification of those infected and thus not allowed to embark on a cruise or quarantining those disembarking and other mitigation strategies, the popular cruise adventure could be available safely again. Whatever the procedures implemented, the methodological purpose of this study should add valuable insight in the modeling of disease and specifically, the COVID-19 virus.","version":"1.1","doi":"10.1101/2021.01.06.425543","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424533","pub_date":"2021-1-05","title":"Rapid inactivation in vitro of SARS-CoV-2 in saliva by black tea and green tea","abstract":"Saliva plays major roles in human-to-human transmission of the SARS-CoV-2. Recently we reported that black, green and oolong tea significantly inactivated SARS-CoV-2 within 1 min. Theaflavin-3,3\u2019-di-gallate (TFDG), theasinensin A (TSA) and (-) epigallocatechin gallate (EGCG) were involved in the anti-viral activities. Here we examined how long period is required for the compounds to inactivate the virus. We also assessed whether tea inactivates SARS-CoV-2 diluted in human saliva. Treatment of SARS-CoV-2 with 500 \u03bcM TFDG or TSA for 10 sec reduced the virus titer to undetectable levels (less than 1/1,000). Black and green tea decreased virus titer to less than 1/100 within 10 sec even in saliva. These findings suggest a possibility that intake of, or gargling with, tea may inactivate SARS-CoV-2 in saliva in infected individuals, which may eventually attenuate spread of COVID-19 within a population, although clinical studies are required to test this hypothesis by determining the intensity and duration of the anti-viral effect of tea in saliva in humans.","version":"1.2","doi":"10.1101/2020.12.28.424533","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425336","pub_date":"2021-1-05","title":"SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses","abstract":"Coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses is inevitable as the COVID-19 pandemic continues. This study aimed to evaluate cell lines commonly used in virus diagnosis and isolation for their susceptibility to SARS-CoV-2. While multiple kidney cell lines from monkeys were susceptible and permissive to SARS-CoV-2, many cell types derived from human, dog, mink, cat, mouse, or chicken were not. Analysis of MDCK cells, which are most commonly used for surveillance and study of influenza viruses, demonstrated that they were insusceptible to SARS-CoV-2 and that the cellular barrier to productive infection was due to low expression level of the angiotensin converting enzyme 2 (ACE2) receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by over-expression of canine ACE2 in trans. Moreover, SARS-CoV-2 cell tropism did not appear to be affected by a D614G mutation in the spike protein.","version":"1.1","doi":"10.1101/2021.01.04.425336","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.390039","pub_date":"2021-1-05","title":"Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor","abstract":"In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2 the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation/insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum, antiviral agents.","version":"1.2","doi":"10.1101/2020.11.24.390039","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.289074","pub_date":"2021-1-05","title":"Structural Genetics of circulating variants affecting the SARS-CoV-2 Spike / human ACE2 complex","abstract":"SARS-CoV-2 entry in human cells is mediated by the interaction between the viral Spike protein and the human ACE2 receptor. This mechanism evolved from the ancestor bat coronavirus and is currently one of the main targets for antiviral strategies. However, there currently exist several Spike protein variants in the SARS-CoV-2 population as the result of mutations, and it is unclear if these variants may exert a specific effect on the affinity with ACE2 which, in turn, is also characterized by multiple alleles in the human population. In the current study, the GBPM analysis, originally developed for highlighting host-guest interaction features, has been applied to define the key amino acids responsible for the Spike/ACE2 molecular recognition, using four different crystallographic structures. Then, we intersected these structural results with the current mutational status, based on more than 295,000 sequenced cases, in the SARS-CoV-2 population. We identified several Spike mutations interacting with ACE2 and mutated in at least 20 distinct patients: S477N, N439K, N501Y, Y453F, E484K, K417N, S477I and G476S. Among these, mutation N501Y in particular is one of the events characterizing SARS-CoV-2 lineage B.1.1.7, which has recently risen in frequency in Europe. We also identified five ACE2 rare variants that may affect interaction with Spike and susceptibility to infection: S19P, E37K, M82I, E329G and G352V. We developed a method to identify key amino acids responsible for the initial interaction between SARS-CoV-2 (the COVID-19 virus) and human cells, through the analysis of Spike/ACE2 complexes. We further identified which of these amino acids show variants in the viral and human populations. Our results will facilitate scientists and clinicians alike in identifying the possible role of present and future Spike and ACE2 sequence variants in cell entry and general susceptibility to infection.","version":"1.3","doi":"10.1101/2020.09.09.289074","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425316","pub_date":"2021-1-05","title":"Molecular Mechanism of the N501Y Mutation for Enhanced Binding between SARS-CoV-2\u2019s Spike Protein and Human ACE2 Receptor","abstract":"Coronavirus disease 2019 (COVID-19) has been an ongoing global pandemic for over a year. Recently, an emergent SARS-CoV-2 variant (B.1.1.7) with an unusually large number of mutations had become highly contagious and wide-spreading in United Kingdom. From genome analysis, the N501Y mutation within the receptor binding domain (RBD) of the SARS-CoV-2\u2019s spike protein might have enhanced the viral protein\u2019s binding with the human angiotensin converting enzyme 2 (hACE2). The latter is the prelude for the virus\u2019 entry into host cells. So far, the molecular mechanism of this enhanced binding is still elusive, which prevents us from assessing its effects on existing therapeutic antibodies. Using all atom molecular dynamics simulations, we demonstrated that Y501 in mutated RBD can be well coordinated by Y41 and K353 in hACE2 through hydrophobic interactions, increasing the overall binding affinity between RBD and hACE2 by about 0.81 kcal/mol. We further explored how the N501Y mutation might affect the binding between a neutralizing antibody (CB6) and RBD. We expect that our work can help researchers design proper measures responding to this urgent virus mutation, such as adding a modified/new neutralizing antibody specifically targeting at this variant in the therapeutic antibody cocktail.","version":"1.1","doi":"10.1101/2021.01.04.425316","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424332","pub_date":"2021-1-05","title":"Genetic epidemiology of variants associated with immune escape from global SARS-CoV-2 genomes","abstract":"Many antibody and immune escape variants in SARS-CoV-2 are now documented in literature. The availability of SARS-CoV-2 genome sequences enabled us to investigate the occurrence and genetic epidemiology of the variants globally. Our analysis suggests that a number of genetic variants associated with immune escape have emerged in global populations.","version":"1.2","doi":"10.1101/2020.12.24.424332","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161687","pub_date":"2021-1-05","title":"Short sequence motif dynamics in the SARS-CoV-2 genome suggest a role for cytosine deamination in CpG reduction","abstract":"RNA viruses use CpG reduction to evade the host cell defense, but the driving mechanisms are still largely unknown. In an attempt to address this we used a rapidly growing genomic dataset of SARS-CoV-2 with relevant metadata information. Remarkably, by simply ordering SARS-CoV-2 genomes by their date of collection, we find a progressive increase of C-to-U substitutions resulting in 5\\'-UCG-3\\' motif reduction that in turn have reduced the CpG frequency over just a few months of observation. This is consistent with APOBEC-mediated RNA editing resulting in CpG reduction, thus allowing the virus to escape ZAP-mediated RNA degradation. Our results thus link the dynamics of target sequences in the viral genome for two known host molecular defense mechanisms, mediated by the APOBEC and ZAP proteins.","version":"1.4","doi":"10.1101/2020.06.19.161687","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425441","pub_date":"2021-1-05","title":"Crystallographic molecular replacement using an in silico-generated search model of SARS-CoV-2 ORF8","abstract":"The majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre-existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for molecular replacement. The ORF8 protein of SARS-CoV-2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all \u03b2-sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.","version":"1.1","doi":"10.1101/2021.01.05.425441","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.387092","pub_date":"2021-1-05","title":"Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice","abstract":"Protection against SARS-CoV-2 and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor-binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles; 4-8 distinct RBDs). Mice immunized with RBD-nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic-RBD-nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs compared to sera from immunizations with homotypic SARS-CoV-2\u2013RBD-nanoparticles or COVID-19 convalescent human plasmas. Moreover, sera from mosaic-RBD\u2013immunized mice neutralized heterologous pseudotyped coronaviruses equivalently or better after priming than sera from homotypic SARS-CoV-2\u2013RBD-nanoparticle immunizations, demonstrating no immunogenicity loss against particular RBDs resulting from co-display. A single immunization with mosaic-RBD-nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses. Nanoparticle strategy for pan-sarbecovirus vaccine Immunizing with nanoparticles displaying diverse coronavirus RBDs elicits cross-reactive and neutralizing antibody responses.","version":"1.3","doi":"10.1101/2020.11.17.387092","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.05.425384","pub_date":"2021-1-05","title":"Covid19Risk.ai: An open source repository and online calculator of prediction models for early diagnosis and prognosis of Covid-19","abstract":"The current pandemic has led to a proliferation of predictive models being developed to address various aspects of COVID-19 patient care. We aimed to develop an online platform that would serve as an open source repository for a curated subset of such models, and provide a simple interface for included models to allow for online calculation. This platform would support doctors during decision-making regarding diagnoses, prognoses, and follow-up of COVID-19 patients, expediting the models\u2019 transition from research to clinical practice. In this proof-of-principle study, we performed a literature search in PubMed and WHO database to find suitable models for implementation on our platform. All selected models were publicly available (peer reviewed publications or open source repository) and had been validated (TRIPOD type 3 or 2b). We created a method for obtaining the regression coefficients if only the nomogram was available in the original publication. All predictive models were transcribed on a practical graphical user interface using PHP 8.0.0, and published online together with supporting documentation and links to the associated articles. The open source website https://covid19risk.ai/ currently incorporates nine models from six different research groups, evaluated on datasets from different countries. The website will continue to be populated with other models related to COVID-19 prediction as these become available. This dynamic platform allows COVID-19 researchers to contact us to have their model curated and included on our website, thereby increasing the reach and real-world impact of their work. We have successfully demonstrated in this proof-of-principle study that our website provides an inclusive platform for predictive models related to COVID-19. It enables doctors to supplement their judgment with patient-specific predictions from externally-validated models in a user-friendly format. Additionally, this platform supports researchers in showcasing their work, which will increase the visibility and use of their models.","version":"1.1","doi":"10.1101/2021.01.05.425384","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.368258","pub_date":"2021-1-04","title":"Immunogenicity and protective efficacy of one- and two-dose regimens of the Ad26.COV2.S COVID-19 vaccine candidate in adult and aged rhesus macaques","abstract":"Safe and effective coronavirus disease (COVID)-19 vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged non-human primates (NHP). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared to a single dose. In one-dose regimens neutralizing antibody responses were stable for at least 14 weeks, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and Th1 skewed cellular responses in aged NHP that were comparable to adult animals. Importantly, aged Ad26.COV2.S-vaccinated animals challenged 3 months post -dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. These are the first NHP data showing COVID-19 vaccine protection against the SARS-CoV-2 spike G614 variant and support ongoing clinical Ad26.COV2.S development. COVID-19 vaccines are urgently needed and while single-dose vaccines are preferred, two-dose regimens may improve efficacy. We show improved Ad26.COV2.S immunogenicity in non-human primates after a second vaccine dose, while both regimens protected aged animals against SARS-CoV-2 disease.","version":"1.2","doi":"10.1101/2020.11.17.368258","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.367391","pub_date":"2021-1-04","title":"Evolution of Antibody Immunity to SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.","version":"1.2","doi":"10.1101/2020.11.03.367391","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.01.425028","pub_date":"2021-1-04","title":"Structure-function investigation of a new VUI-202012/01 SARS-CoV-2 variant","abstract":"The SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus) has accumulated multiple mutations during its global circulation. Recently, a new strain of SARS-CoV-2 (VUI 202012/01) had been identified leading to sudden spike in COVID-19 cases in South-East England. The strain has accumulated 23 mutations which have been linked to its immune evasion and higher transmission capabilities. Here, we have highlighted structural-function impact of crucial mutations occurring in spike (S), ORF8 and nucleocapsid (N) protein of SARS-CoV-2. Some of these mutations might confer higher fitness to SARS-CoV-2. Since initial outbreak of COVID-19 in Wuhan city of central China, its causative agent; SARS-CoV-2 virus has claimed more than 1.7 million lives out of 77 million populations and still counting. As a result of global research efforts involving public-private-partnerships, more than 0.2 million complete genome sequences have been made available through Global Initiative on Sharing All Influenza Data (GISAID). Similar to previously characterized coronaviruses (CoVs), the positive-sense single-stranded RNA SARS-CoV-2 genome codes for ORF1ab non-structural proteins (nsp(s)) followed by ten or more structural/nsps [1, 2]. The structural proteins include crucial spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins. The S protein mediates initial contacts with human hosts while the E and M proteins function in viral assembly and budding. In recent reports on evolution of SARS-CoV-2, three lineage defining non-synonymous mutations; namely D614G in S protein (Clade G), G251V in ORF3a (Clade V) and L84S in ORF 8 (Clade S) were observed [2\u20134]. The latest pioneering works by Plante et al and Hou et al have shown that compared to ancestral strain, the ubiquitous D614G variant (clade G) of SARS-CoV-2 exhibits efficient replication in upper respiratory tract epithelial cells and transmission, thereby conferring higher fitness [5, 6]. As per latest WHO reports on COVID-19, a new strain referred as SARS-CoV-2 VUI 202012/01 (Variant Under Investigation, year 2020, month 12, variant 01) had been identified as a part of virological and epidemiological analysis, due to sudden rise in COVID-19 detected cases in South-East England [7]. Preliminary reports from UK suggested higher transmissibility (increase by 40-70%) of this strain, escalating Ro (basic reproduction number) of virus to 1.5-1.7 [7, 8]. This apparent fast spreading variant inculcates 23 mutations; 13 non-synonymous, 6 synonymous and 4 amino acid deletions [7]. In the current scenario, where immunization programs have already commenced in nations highly affected by COVID-19, advent of this new strain variant has raised concerns worldwide on its possible role in disease severity and antibody responses. The mutations also could also have significant impact on diagnostic assays owing to S gene target failures.","version":"1.1","doi":"10.1101/2021.01.01.425028","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.03.425115","pub_date":"2021-1-04","title":"The ancient cardioprotective mechanisms of ACE2 bestow SARS-CoV-2 with a wide host range","abstract":"SARS-CoV-2 infects a broader range of mammalian species than previously anticipated, suggesting there may be additional unknown hosts wherein the virus can evolve and potentially circumvent effective vaccines. We find that SARS-CoV-2 gains a wide host range by binding ACE2 sites essential for ACE2 carboxypeptidase activity. Six mutations found only in rodent species immune to SARS-CoV-2 are sufficient to abolish viral binding to human and dog ACE2. This is achieved through context-dependent mutational effects (intramolecular epistasis) conserved despite ACE2 sequence divergence between species. Across mammals, this epistasis generates sequence-function diversity, but through structures all bound by SARS-CoV-2. Mutational trajectories to the mouse conformation not bound by SARS-CoV-2 are blocked, by single mutations functionally deleterious in isolation, but compensatory in combination, explaining why human polymorphisms at these sites are virtually non-existent. Closed to humans, this path was opened to rodents via permissive cardiovascular phenotypes and ancient increases to ACE2 activity, serendipitously granting SARS-CoV-2 immunity. This reveals how ancient evolutionary trajectories are linked with unprecedented phenotypes such as COVID-19 and suggests extreme caution should be taken to monitor and prevent emerging animal reservoirs of SARS-CoV-2. A conserved mechanism essential for ACE2 catalytic activity is exploited by SARS-CoV-2 binding, allowing the virus to infect a wide range of species.","version":"1.1","doi":"10.1101/2021.01.03.425115","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425289","pub_date":"2021-1-04","title":"TMPRSS2 structure-phylogeny repositions Avoralstat for SARS-CoV-2 prophylaxis in mice","abstract":"Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.","version":"1.1","doi":"10.1101/2021.01.04.425289","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.277426","pub_date":"2021-1-04","title":"Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates","abstract":"COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2+ mice.","version":"1.2","doi":"10.1101/2020.09.04.277426","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.03.425141","pub_date":"2021-1-04","title":"Glycan reactive anti-HIV-1 antibodies bind the SARS-CoV-2 spike protein but do not block viral entry","abstract":"The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via Fc\u03b3RII receptor engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate the ability for cross-reactive antibodies to interfere in immunoassays.","version":"1.1","doi":"10.1101/2021.01.03.425141","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.055756","pub_date":"2021-1-04","title":"Identification of Plitidepsin as Potent Inhibitor of SARS-CoV-2-Induced Cytopathic Effect after a Drug Repurposing Screen","abstract":"There is an urgent need to identify therapeutics for the treatment of Coronavirus diseases 2019 (COVID-19). Although different antivirals are given for the clinical management of SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract SARS-CoV-2-induced cytopathic effect and viral replication in vitro. Among the potential 72 antivirals tested herein that were previously proposed to inhibit SARS-CoV-2 infection, only 18% had an IC50 below 25 \u03bcM or 102 IU/mL. These included plitidepsin, novel cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-alpha, interferon-gamma, fenofibrate, camostat along the well-known remdesivir and chloroquine derivatives. Plitidepsin was the only clinically approved drug displaying nanomolar efficacy. Four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. Since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. Although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. Thus, these combinations could decrease the potential emergence of resistant viruses. Antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. Combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials.","version":"1.3","doi":"10.1101/2020.04.23.055756","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.10.087288","pub_date":"2021-1-04","title":"Heparan sulfate proteoglycans as attachment factor for SARS-CoV-2","abstract":"Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length-and sequence-dependent manner. Hexa- and octa-saccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with much higher affinity to heparin (KD = 55 nM) compared to the RBD (KD = 1 \u03bcM) alone. We also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. Our data supports a model in which HS functions as the point of initial attachment for SARS-CoV-2 infection. Tissue staining studies using biologically relevant tissues indicate that heparan sulfate proteoglycan (HSPG) is a critical attachment factor for the virus. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells.","version":"1.2","doi":"10.1101/2020.05.10.087288","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.20.347187","pub_date":"2021-1-04","title":"Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity","abstract":"Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.10.20.347187","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.04.425297","pub_date":"2021-1-04","title":"SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2+","abstract":"Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2\u2019 cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue difference, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.","version":"1.1","doi":"10.1101/2021.01.04.425297","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.384917","pub_date":"2021-1-04","title":"Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck","abstract":"The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems. Through ongoing human adaptation, spill-back events from other animal intermediates, or with the distribution of vaccines and therapeutics, the landscape of SARS-CoV-2 genetic variation is certain to change. The evolutionary mechanisms by which SARS-CoV-2 will continue to adapt to mammalian hosts depend on genetic variation generated within and between hosts. Here, using domestic cats as a model, we show that within-host SARS-CoV-2 genetic variation is predominantly influenced by genetic drift and purifying selection. Transmission of SARS-CoV-2 between hosts is defined by a narrow transmission bottleneck, involving 2-5 viruses. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which arises rapidly and is transmitted in cats. Spike H655Y has been previously shown to confer escape from human monoclonal antibodies and is currently found in over 1000 human sequences. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge, underscoring the importance of continued genomic surveillance in humans and non-human mammalian hosts.","version":"1.2","doi":"10.1101/2020.11.16.384917","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424530","pub_date":"2021-1-04","title":"Host\u2019s Specific SARS-CoV-2 Mutations: Insertion of the Phenylalanine in the NSP6 Linked to the United Kingdom and Premature Termination of the ORF-8 Associated with the European and the United States of America Derived Samples","abstract":"The coronavirus belongs to the order Nidovirales, which is known for the longest RNA genome virus. The polymerase enzyme of SARS-CoV-2 has proofreading functions, but still, the RNA viruses have a higher mutation rate than DNA viruses. The mutations in the viral genome provide a replication advantage in any population/geographical location and that may have profound consequences in the outcome and pathogenesis, diagnosis and patient management of the viral infection. In the present study, we have analysed full-length SARS-CoV-2 genome sequences, derived from symptomatic/asymptomatic COVID-19 patients from all six continents to investigate the common mutations globally. Our results revealed that SARS-CoV-2 is mutating independently, we identified total 313 mutations and some (21 mutations) of them are prevailing over time irrespective of geographical location. Another important finding, we are reporting here is, the mutation rate of the virus varies in different geographical locations suggesting the virus is adapting different strategies in the infected populations, having different genetic backgrounds across the globe. We have identified 11085TTT insertion (insertion of the Phenylalanine in NSP6 at position 38) mutation, which is mainly linked to the UK derived SARS-CoV-2 samples, we have also discovered non-sense mutation in ORF-8 after 17 amino acid is linked to the European and the USA derived SARS-CoV-2 samples.","version":"1.2","doi":"10.1101/2020.12.29.424530","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.377739","pub_date":"2021-1-04","title":"Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination","abstract":"The Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.","version":"1.2","doi":"10.1101/2020.11.11.377739","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.04.325522","pub_date":"2021-1-04","title":"Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2\u2019 site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.10.04.325522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.31.425021","pub_date":"2021-1-04","title":"Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies","abstract":"The evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent serum antibodies are impacted by all mutations to the spike\u2019s receptor-binding domain (RBD), the main target of serum neutralizing activity. Binding by polyclonal serum antibodies is affected by mutations in three main epitopes in the RBD, but there is substantial variation in the impact of mutations both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBD\u2019s receptor binding motif. The most important site is E484, where neutralization by some sera is reduced >10-fold by several mutations, including one in emerging viral lineages in South Africa and Brazil. Going forward, these serum escape maps can inform surveillance of SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2020.12.31.425021","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.02.425099","pub_date":"2021-1-04","title":"Spike protein disulfide disruption as a potential treatment for SARS-CoV-2","abstract":"The coronaviral pandemic is exerting a tremendously detrimental impact on global health, quality of life and the world economy, emphasizing the need for effective medications for current and future coronaviral outbreaks as a complementary approach to vaccines. The Spike protein, responsible for cell receptor binding and viral internalization, possesses multiple disulfide bonds raising the possibility that disulfide-reducing agents might disrupt Spike function, prevent viral entry and serve as effective drugs against SARS-CoV-2. Here we show the first experimental evidence that reagents capable of reducing disulfide bonds can inhibit viral infection in cell-based assays. Molecular dynamics simulations of the Spike receptor-binding domain (RBD) predict increased domain flexibility when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the surface loop, comprised of residues 456-490, which interacts with the Spike cell receptor ACE2. Consistent with this finding, the addition of exogenous disulfide bond reducing agents affects the RBD secondary structure, lowers its melting temperature from 52 to 36-39\u00b0C and decreases its binding affinity to ACE2 by two orders of magnitude at 37\u00b0C. Finally, the reducing agents dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) inhibit viral replication at high \u00b5M \u2013 low mM levels with a negligible effect on cell viability at these concentrations. The antiviral effect of monothiol-based reductants N-Acetyl-L-cysteine (NAC) and reduced glutathione (GSH) was not observed due to decreases in cell viability. Our research demonstrates the clear potential for medications that disrupt Spike disulfides as broad-spectrum anticoronaviral agents and as a first-line defense against current and future outbreaks.","version":"1.1","doi":"10.1101/2021.01.02.425099","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424482","pub_date":"2021-1-04","title":"Structural basis for broad coronavirus neutralization","abstract":"Three highly pathogenic \u03b2-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARS-CoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight \u03b2-coronavirus spike glycoproteins, including all five human-infecting \u03b2-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryo-electron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with \u03b2-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.","version":"1.2","doi":"10.1101/2020.12.29.424482","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.29.925354","pub_date":"2021-1-04","title":"Interpretable detection of novel human viruses from genome sequencing data","abstract":"Viruses evolve extremely quickly, so reliable methods for viral host prediction are necessary to safeguard biosecurity and biosafety alike. Novel human-infecting viruses are difficult to detect with standard bioinformatics workflows. Here, we predict whether a virus can infect humans directly from next-generation sequencing reads. We show that deep neural architectures significantly outperform both shallow machine learning and standard, homology-based algorithms, cutting the error rates in half and generalizing to taxonomic units distant from those presented during training. Further, we develop a suite of interpretability tools and show that it can be applied also to other models beyond the host prediction task. We propose a new approach for convolutional filter visualization to disentangle the information content of each nucleotide from its contribution to the final classification decision. Nucleotide-resolution maps of the learned associations between pathogen genomes and the infectious phenotype can be used to detect regions of interest in novel agents, for example the SARS-CoV-2 coronavirus, unknown before it caused a COVID-19 pandemic in 2020. All methods presented here are implemented as easy-to-install packages enabling analysis of NGS datasets without requiring any deep learning skills, but also allowing advanced users to easily train and explain new models for genomics.","version":"1.7","doi":"10.1101/2020.01.29.925354","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.31.424987","pub_date":"2021-1-03","title":"Paired heavy and light chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses","abstract":"Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2. A molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralization Cryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimer Cryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spike Sequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chains IGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires","version":"1.1","doi":"10.1101/2020.12.31.424987","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.31.424961","pub_date":"2021-1-02","title":"Comprehensive analysis of the host-virus interactome of SARS-CoV-2","abstract":"Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.","version":"1.1","doi":"10.1101/2020.12.31.424961","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.02.425072","pub_date":"2021-1-02","title":"Sequencing of Sars-CoV-2 genome using different Nanopore chemistries","abstract":"Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and Rapid Barcoding Kit (RBK) vs Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in timine and adenine homopolymeric regions than R9, at expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than RBK, and provided longer reads than RBK. It also resulted in a larger percentage of aligned reads (99% vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK used on a R9 flow cell could maximize the yield and accuracy of the consensus sequence when used in epidemiological surveillance of SARS-CoV-2. Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome Ligation sequencing is preferred rather than the rapid kit using transposases","version":"1.1","doi":"10.1101/2021.01.02.425072","journal":"bioRxiv","score":null},{"id":"10.1101/2021.01.02.424974","pub_date":"2021-1-02","title":"Rapid inactivation of SARS-CoV-2 on copper touch surfaces determined using a cell culture infectivity assay","abstract":"COVID-19, caused by SARS-CoV-2, was first reported in China in 2019 and has transmitted rapidly around the world, currently responsible for 83 million reported cases and over 1.8 million deaths. The mode of transmission is believed principally to be airborne exposure to respiratory droplets from symptomatic and asymptomatic patients but there is also a risk of the droplets contaminating fomites such as touch surfaces including door handles, stair rails etc, leading to hand pick up and transfer to eyes, nose and mouth. We have previously shown that human coronavirus 229E survives for more than 5 days on inanimate surfaces and another laboratory reproduced this for SARS-CoV-2 this year. However, we showed rapid inactivation of Hu-CoV-229E within 10 minutes on different copper surfaces while the other laboratory indicated this took 4 hours for SARS-CoV-2. So why the difference? We have repeated our work with SARS-CoV-2 and can confirm that this coronavirus can be inactivated on copper surfaces in as little as 1 minute. We discuss why the 4 hour result may be technically flawed.","version":"1.1","doi":"10.1101/2021.01.02.424974","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424906","pub_date":"2021-1-02","title":"Unbuttoning the impact of N501Y mutant RBD on viral entry mechanism: A computational insight","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic has become a serious global threat. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for this pandemic has imposed a severe burden on the medical settings. The spike (S) protein of SARS-CoV-2 is an important structural protein playing a key role in the viral entry. This protein is responsible for the receptor recognition and cell membrane fusion process. The recent reports of the appearance and spread of new SARS-CoV-2 strain has raised alarms. It was reported that this new variant containing the prominent active site mutation in the RBD (N501Y) was rapidly spreading within the population. The reported N501Y mutation within the spike\u2019s essential part, known as the \u2018receptor-binding domain\u2019 has raised several questions. Here in this study we have tried to explore the effect of N501Y mutation within the spike protein using several in silico approaches","version":"1.1","doi":"10.1101/2020.12.30.424906","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424708","pub_date":"2021-1-01","title":"The high infectivity of SARS-CoV-2 B.1.1.7 is associated with increased interaction force between Spike-ACE2 caused by the viral N501Y mutation","abstract":"The Spike glycoprotein receptor-binding domain (RBD) of SARS-CoV-2 mediates the viral particle\u2019s binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells. Therefore, Spike-ACE2 interaction is a crucial determining factor for viral infectivity. A new phylogenetic group of SARS-CoV-2 (lineage B.1.1.7) has been recently identified in the COVID-19 Genomics UK Consortium dataset, which features an amino acid substitution in the Spike RBD (N501Y mutation). Infections with the SARS-CoV-2 lineage B.1.1.7 have been overgrowing in recent weeks in the United Kingdom, indicating an even greater spread capacity than that seen with previous strains of the novel coronavirus. We hypothesized that this rapid spreading/infectivity of the B.1.1.7 lineage might be due to changes in the interaction force between the mutant Spike RBD and ACE2. This study employed in silico methods involving mutagenesis (N501Y mutation) and interface analysis focusing on the Spike RDB-ACE2 interaction. The results showed that the SARS-CoV-2 N501Y mutant (lineage B.1.1.7) establishes a more significant number of interactions relating to the mutant residue Y501 (Spike RDB) with residues Y41 and K353 (ACE2). This finding shows that the increased infectivity of SARS-CoV-2 lineage B.1.1.7 is associated with the interaction force between the Spike RBD Y501 mutant residue with the ACE2 receptor, which in this strain is increased.","version":"1.1","doi":"10.1101/2020.12.29.424708","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424878","pub_date":"2021-1-01","title":"MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease","abstract":"Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals. Vaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.","version":"1.1","doi":"10.1101/2020.12.30.424878","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424283","pub_date":"2020-12-31","title":"The N501Y and K417N mutations in the spike protein of SARS-CoV-2 alter the interactions with both hACE2 and human derived antibody: A Free energy of perturbation study","abstract":"The N501Y and K417N mutations in spike protein of SARS-CoV-2 and their combination arise questions but the data about their mechanism of action at molecular level is limited. Here, we present Free energy perturbation (FEP) calculations for the interactions of the spike S1 receptor binding domain (RBD) with both the ACE2 receptor and an antibody derived from COVID-19 patients. Our results shown that the S1 RBD-ACE2 interactions were significantly increased whereas those with the STE90-C11 antibody dramatically decreased; about over 100 times. The K417N mutation had much more pronounced effect and in a combination with N501Y fully abolished the antibody effect. This may explain the observed in UK and South Africa more spread of the virus but also raise an important question about the possible human immune response and the success of already available vaccines.","version":"1.2","doi":"10.1101/2020.12.23.424283","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424745","pub_date":"2020-12-31","title":"Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates","abstract":"Development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we found that mRNA-LNP immunization compared to protein immunization elicited either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope (prM-E) or HIV-1 Env gp160 induced durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 \u03bcg were immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.","version":"1.1","doi":"10.1101/2020.12.30.424745","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424862","pub_date":"2020-12-30","title":"Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.","version":"1.1","doi":"10.1101/2020.12.30.424862","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424779","pub_date":"2020-12-30","title":"Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS\u2013CoV-2 in Nicotiana benthamiana as vaccine candidates against COVID-19","abstract":"The COVID-19 pandemic, which is caused by SARS-CoV-2 has rapidly spread to more than 216 countries and has put global public health at high risk. The world urgently needs a cost-effective and safe SARS-CoV-2 coronavirus vaccine, antiviral and therapeutic drugs to control the COVID-19 pandemic. In this study, we engineered the Nucleocapsid (N) and Spike protein (S) variants (Receptor binding domain, RBD and S1 domain) of SARS-CoV-2 genes and produced in Nicotiana benthamiana plant. The purification yields were at least 20 mg of pure protein/kg of plant biomass for each target protein. The S protein variants of SARS-CoV-2 showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. The purified plant produced N and S variants were recognized by N and S protein specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant produced N and S protein variants. In addition, IgG responses of plant produced N and S antigens elicited significantly high titers of antibody in mice. This is the first report demonstrating production of functional active S1 domain and Nucleocapsid protein of SARC-CoV-2 in plants. In addition, in this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies compared to RBD or N proteins. Thus, obtained data support that plant produced N and S antigens, developed in this study, are promising vaccine candidates against COVID-19.","version":"1.1","doi":"10.1101/2020.12.29.424779","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424534","pub_date":"2020-12-30","title":"A novel fermented Yi traditional medicine efficiently suppresses the replication of SARS-CoV-2 in vitro","abstract":"Currently, the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a worldwide epidemic, causing more than 80 million infections and more than 1.7 million deaths. The pandemic has led to the closure of enterprises and schools in many countries, resulting in serious disruption of the global economy and social activities. Remdesivir is currently approved by the FDA for the treatment of COVID-19, but the WHO declared that Remdesivir is almost ineffective against COVID-19. The research and development of vaccines has made great progress, but it will take at least several months for safe and effective vaccines to be widely used clinically. Clinical studies revealed that some Traditional Chinese Medicines, such as Lianhua Qingwen Capsule and Huoxiang Zhengqi Water, exhibited excellent therapeutic effect on COVID-19. However, until now, there is still no cure for COVID-19. Therefore, there is an urgent need to find medicines that can effectively fight against the SARS-CoV-2. In this study, JIE BEN No. 10 (JB10), a fermentation broth produced by Yi traditional medicine fermentation technology, was explored for its anti-coronavirus activity. The in vitro data showed that JB10 could significantly suppresses the replication of the SARS-CoV-2 with an EC50 of 769.1 times dilution and a selection index of 42.68. Further studies indicated that JB10 had significant anti-inflammatory and antioxidant activities. The analysis of active components suggested that JB10 contained a large amount of superoxide dismutase (SOD), flavones, polyphenols, crude polysaccharide, etc. which may explain the anti-coronavirus activity, anti-inflammatory and antioxidant effects. Our study provides a new potentially therapeutic strategy for COVID-19.","version":"1.1","doi":"10.1101/2020.12.29.424534","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424801","pub_date":"2020-12-30","title":"Pharmacophore-based peptide biologics neutralize SARS-CoV-2 S1 and deter S1-ACE2 interaction in vitro","abstract":"Effective therapeutics and stable vaccine are the urgent need of the day to combat COVID-19 pandemic. SARS-CoV-2 spike protein has a pivotal role in cell-entry and host immune response, thus regarded as potential drug- and vaccine-target. As the virus utilizes the S1 domain of spike to initiate cell-attachment and S2 domain for membrane fusion, several attempts have been made to design viral-receptor and viral-fusion blockers. Here, by deploying interactive structure-based design and pharmacophore-based approaches, we designed short and stable peptide-biologics i.e. CoV-spike-neutralizing peptides (CSNPs) including CSNP1, CSNP2, CSNP3, CSNP4. We could demonstrate in cell culture experiments that CSNP2 binds to S1 at submicromolar concentration and abrogates the S1-hACE2 interaction. CSNP3, a modified and downsized form of CSNP2, could neither interfere with the S1-hACE2 interaction nor bind to S1. CSNP4 exhibited dose-dependent binding to both S1 and hACE2 and abolished the S1-hACE2 interaction in vitro. CSNP4 possibly enhance the mAb-based S1 neutralization by limiting the spontaneous movement of spike receptor-binding domain (RBD), whereas CSNP2 allowed RBD-mAb binding without any steric hindrance. Taken together, we suggest that CSNP2 and CSNP4 are potent and stable candidate peptides that can neutralize the SARS-CoV-2 spike and possibly pose the virus to host immune surveillance.","version":"1.1","doi":"10.1101/2020.12.30.424801","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424829","pub_date":"2020-12-30","title":"Process Development and Scale-up Optimization of the SARS-CoV-2 Receptor Binding Domain-Based Vaccine Candidate, RBD219-N1C1","abstract":"A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel\u00ae has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMP) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1 and 5 L scale in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.","version":"1.1","doi":"10.1101/2020.12.30.424829","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.30.424641","pub_date":"2020-12-30","title":"Characterization of cell-cell communication in COVID-19 patients","abstract":"COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use it to examine five public single-cell RNA-seq datasets with a total of 85 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients.","version":"1.1","doi":"10.1101/2020.12.30.424641","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424728","pub_date":"2020-12-30","title":"In vitro Targeting of Transcription Factors to Control the Cytokine Release Syndrome in COVID-19","abstract":"Treatment of the cytokine release syndrome (CRS) has become an important part of rescuing hospitalized COVID-19 patients. Here, we systematically explored the transcriptional regulators of inflammatory cytokines involved in the COVID-19 CRS to identify candidate transcription factors (TFs) for therapeutic targeting using approved drugs. We integrated a resource of TF-cytokine gene interactions with single-cell RNA-seq expression data from bronchoalveolar lavage fluid cells of COVID-19 patients. We found 581 significantly correlated interactions, between 95 TFs and 16 cytokines upregulated in the COVID-19 patients, that may contribute to pathogenesis of the disease. Among these, we identified 19 TFs that are targets of FDA approved drugs. We investigated the potential therapeutic effect of 10 drugs and 25 drug combinations on inflammatory cytokine production in peripheral blood mononuclear cells, which revealed two drugs that inhibited cytokine production and numerous combinations that show synergistic efficacy in downregulating cytokine production. Further studies of these candidate repurposable drugs could lead to a therapeutic regimen to treat the CRS in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.12.29.424728","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424590","pub_date":"2020-12-29","title":"Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma","abstract":"As the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread around the globe, effective vaccination protocols are under deployment. Alternatively, the use of convalescent plasma (CP) therapy relies on the transfer of the immunoglobulin repertoire of a donor that has recovered from the disease as a means of passive vaccination. While the lack of an effective antiviral treatment inadvertently increases the interest in CP products, initial clinical evaluation on COVID-19 patients revealed that critical factors determining the outcome of CP therapy need to be defined clearly if clinical efficacy is to be expected. Measurement of neutralizing activity against SARS-CoV-2 using wildtype virus presents a reliable functional assay but the availability of suitable BSL3 facilities for virus culture restricts its applicability. Instead, the use of pseudovirus particles containing elements from the SARS-CoV-2 virus is widely applied to determine the activity of CP or other neutralizing agents such as monoclonal antibodies. In this study, we present our approach to optimize GFP-encoding lentiviral particles pseudotyped with the SARS-CoV-2 Spike and Membrane proteins for use in neutralization assays. Our results show the feasibility of pseudovirus production using a C-terminal truncated Spike protein which is greatly enhanced by the incorporation of the D614G mutation. Moreover, we report that the use of Sodium Butyrate during lentiviral vector production dramatically increases pseudovirus titers. Analysis of CP neutralizing activity against particles pseudotyped with wildtype or D614G mutant Spike protein in the presence or absence the M protein revealed differential activity in CP samples that did not necessarily correlate with the amount of anti-SARS-CoV-2 antibodies. Our results indicate that the extent of neutralizing activity in CP samples depends on the quality rather than the quantity of the humoral immune responses and varies greatly between donors. Functional screening of neutralizing activity using pseudovirus-based neutralization assays must be accepted as a critical tool for choosing CP donors if clinical efficacy is to be maximized.","version":"1.1","doi":"10.1101/2020.12.28.424590","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424698","pub_date":"2020-12-29","title":"An Ultrasensitive Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and in vitro","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus-host interaction in order to quickly evaluate infectious ability of mutant virus and develop or validate virus-inhibiting drugs. Here we have developed an ultrasensitive bioluminescent biosensor to evaluate virus-cell interaction by quantifying the interaction between SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants, and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid and HTP biosensor tool will significantly expedite detection of viral mutants and anti-COVID-19 drug discovery processes.","version":"1.1","doi":"10.1101/2020.12.29.424698","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424715","pub_date":"2020-12-29","title":"Design of Specific Primer Set for Detection of B.1.1.7 SARS-CoV-2 Variant using Deep Learning","abstract":"The SARS-CoV-2 variant B.1.1.7 lineage, also known as clade GR from Global Initiative on Sharing All Influenza Data (GISAID), Nextstrain clade 20B, or Variant Under Investigation in December 2020 (VUI \u2013 202012/01), appears to have an increased transmissability in comparison to other variants. Thus, to contain and study this variant of the SARS-CoV-2 virus, it is necessary to develop a specific molecular test to uniquely identify it. Using a completely automated pipeline involving deep learning techniques, we designed a primer set which is specific to SARS-CoV-2 variant B.1.1.7 with >99% accuracy, starting from 8,923 sequences from GISAID. The resulting primer set is in the region of the synonymous mutation C16176T in the ORF1ab gene, using the canonical sequence of the variant B.1.1.7 as a reference. Further in-silico testing shows that the primer set\u2019s sequences do not appear in different viruses, using 20,571 virus samples from the National Center for Biotechnology Information (NCBI), nor in other coronaviruses, using 487 samples from National Genomics Data Center (NGDC). In conclusion, the presented primer set can be exploited as part of a multiplexed approach in the initial diagnosis of Covid-19 patients, or used as a second step of diagnosis in cases already positive to Covid-19, to identify individuals carrying the B.1.1.7 variant.","version":"1.1","doi":"10.1101/2020.12.29.424715","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424622","pub_date":"2020-12-29","title":"Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient\u2019s immunological status, and found dramatic changes in the IGH within the patients\u2019 immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones during 2-3 weeks of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34 and IGHV4-39 in COVID-19 patients were more abundant than that of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.","version":"1.1","doi":"10.1101/2020.12.28.424622","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424733","pub_date":"2020-12-29","title":"SARS-CoV-2 highly conserved s2m element dimerizes via a kissing complex and interacts with host miRNA-1307-3p","abstract":"The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the 3\u2019 UTR is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the s2m element of SARS-CoV-2 dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular miRNA-1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.","version":"1.1","doi":"10.1101/2020.12.29.424733","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.267724","pub_date":"2020-12-29","title":"Epitopes targeted by T cells in convalescent COVID-19 patients","abstract":"Knowledge of the epitopes of SARS-CoV-2 that are targeted by T cells in convalescent patients is important for understanding T cell immunity against COVID-19. This information can aid the design, development and assessment of COVID-19 vaccines, and inform novel diagnostic technologies. Here we provide a unified description and meta-analysis of emerging data of SARS-CoV-2 T cell epitopes compiled from 15 independent studies of cohorts of convalescent COVID-19 patients. Our analysis demonstrates the broad diversity of T cell epitopes that have been collectively recorded for SARS-CoV-2, while also identifying a selected set of immunoprevalent epitopes that induced consistent T cell responses in multiple cohorts and in a large fraction of tested patients. The landscape of SARS-CoV-2 T cell epitopes that we describe can help guide future immunological studies, including those related to vaccines and diagnostics. A web-based platform has been developed to help complement these efforts.","version":"1.2","doi":"10.1101/2020.08.26.267724","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424630","pub_date":"2020-12-29","title":"Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers","abstract":"The RNA pseudoknot that stimulates \u22121 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frame-shifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5\u2032 end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5\u2032 end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.","version":"1.1","doi":"10.1101/2020.12.28.424630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424646","pub_date":"2020-12-29","title":"Environmental Dependence of the Structure of the C-terminal Domain of the SARS-CoV-2 Envelope Protein","abstract":"The SARS-CoV-2 envelope protein (E) is involved in a broad spectrum of functions in the cycle of the virus, including assembly, budding, envelope formation, and pathogenesis. To enable these activities, E is likely to be capable of changing its conformation depending on environmental cues. To investigate this issue, here we characterised the structural properties of the C-terminal domain of E (E-CTD), which has been reported to interact with host cell membranes. We first studied the conformation of the E-CTD in solution, finding characteristic features of a disordered protein. By contrast, in the presence of large unilamellar vesicles and micelles, which mimic cell membranes, the E-CTD was observed to become structured. The E-CTD was also found to display conformational changes with osmolytes. Furthermore, prolonged incubation of the E-CTD under physiological conditions resulted in amyloid-like fibril formation. Taken together, these findings indicate that the E-CTD can change its conformation depending on its environment, ranging from a disordered state, to a membrane-bound folded state, and an amyloid state. Our results thus provide insight into the structural basis of the role of E in the viral infection process. The E-CTD of SARS-CoV-2 is intrinsically disordered in solution The E-CTD folds into an ordered structure in presence of membrane mimetics The E-CTD displays conformational changes in the presence of osmolytes Prolonged incubation of the E-CTD leads to its self-assembly into amyloid-like fibrils The E-CTD shows a disordered secondary structure in an aqueous solution and converts into an ordered structure in the presence of membrane mimetics (neutral and negative lipids) and natural osmolytes (TMAO). Incubation at physiological condition shows typical amyloid-like fibrils. The yellow-colored structure represents a predicted structure of the E-CTD by PEP-FOLD.","version":"1.1","doi":"10.1101/2020.12.29.424646","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424711","pub_date":"2020-12-29","title":"Deep mining of early antibody response in COVID-19 patients yields potent neutralisers and reveals high level of convergence","abstract":"Passive immunisation using monoclonal antibodies will play a vital role in the fight against COVID-19. Until now, the majority of anti-SARS-CoV-2 antibody discovery efforts have relied on screening B cells of patients in the convalescent phase. Here, we describe deep-mining of the antibody repertoires of hospitalised COVID-19 patients using a combination of phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralising antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded potent neutralising antibodies with distinct mechanisms of action, including the identification of a novel non-ACE2 receptor blocking antibody that is not expected to be affected by any of the major viral variants reported. The study highlighted the presence of potent neutralising antibodies with near germline sequences within both the IgG and IgM pools at early stages of infection. Furthermore, we highlight a highly convergent antibody response with the same sequences occurring both within this study group and also within the responses described in previously published anti-SARS-CoV-2 studies.","version":"1.1","doi":"10.1101/2020.12.29.424711","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424682","pub_date":"2020-12-29","title":"ACE2 peptide fragment interacts with several sites on the SARS-CoV-2 spike protein S1","abstract":"The influence of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2(ACE2) was studied to model the interaction of the virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 \u03b11 domain, which is the binding site for the S1 protein. The on-rate and off-rate of S1-ACE2 complex formation were measured in the presence of various peptide concentrations using Bio-Layer Interferometry (BLI). The formation of the S1-ACE2 complex was inhibited when the S1 protein was preincubated with the peptide, however, no significant inhibitory effect was observed in the absence of preincubation. Dissociation kinetics revealed that the peptide remained bound to the S1-ACE2 complex and stabilized this complex. Computational mapping of the S1 protein surface for peptide binding revealed two additional sites, located at some distance from the receptor binding domain (RBD) of S1. These additional binding sites affect the interaction between the peptide, the S1 protein, and ACE2.","version":"1.1","doi":"10.1101/2020.12.29.424682","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.29.424712","pub_date":"2020-12-29","title":"Rapid expression of COVID-19 proteins by transient expression in tobacco","abstract":"In 2020 we suffered from a major global pandemic caused by the SARS-CoV-2 coronavirus. Efforts to contain the virus include the development of rapid tests and vaccines, which require a ready supply of viral proteins. Here we report the production of two SARS-CoV-2 proteins by transient transformation of tobacco, leading to high expression within three days, and subsequent purification of the intact proteins. Such efforts may help to develop testing resources to alleviate the major impacts of this global pandemic.","version":"1.1","doi":"10.1101/2020.12.29.424712","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.27.424507","pub_date":"2020-12-28","title":"Losartan promotes cell survival following SARS-CoV-2 infection in vitro","abstract":"Coronavirus disease 2019 (COVID-19) can be associated with mortality and high morbidity worldwide. There is an extensive effort to control infection and disease caused by SARS-CoV-2. This study addressed the hypothesis that angiotensin II type I receptor blocker, Losartan, may restrict pathogenesis caused by SARS-CoV-2 by decreasing viral-induced cytopathological changes by blocking angiotensin II type 1 receptor (AT1R), thus reducing the affinity of the virus for ACE2, and inhibiting papain-like protease of the virus. Losartan inhibitory effect on deubiquitination and deISGylation properties of papain-like protease was investigated using a fluorescence method and gel shift analysis determining its inhibitory effects. The inhibitory effect of Losartan on SARS-CoV-2 cell replication was investigated both when losartan was added to the cell culture 1 hour before (pre-infection group) and 1 hour after (post-infection group) SARS-CoV-2 infection of Vero E6 cells. Losartan treatment of Vero E6 cells prior to and after SARS-CoV-2 infection reduced SARS-CoV-2 replication by 80% and 70% respectively. Losartan was not a strong deubiquitinase and deISGylase inhibitor of PLpro. Losartan added pre- and post-infection to the Vero E6 cell culture significantly prevents cell destruction and replication by SARS-CoV2. Losartan has low side-effects, is readily available, and can be produced at high levels globally, all features of a promising drug in treatment of COVID-19 if validated by clinical trials.","version":"1.1","doi":"10.1101/2020.12.27.424507","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424451","pub_date":"2020-12-28","title":"SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma","abstract":"To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed. Three mutations allowed SARS-CoV-2 to evade the polyclonal antibody response of a highly neutralizing COVID-19 convalescent plasma.","version":"1.1","doi":"10.1101/2020.12.28.424451","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255935","pub_date":"2020-12-28","title":"IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition","abstract":"Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.","version":"1.2","doi":"10.1101/2020.08.18.255935","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424554","pub_date":"2020-12-28","title":"Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection","abstract":"SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease.","version":"1.1","doi":"10.1101/2020.12.28.424554","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424565","pub_date":"2020-12-28","title":"SARS-CoV-2 disease severity and transmission efficiency is increased for airborne but not fomite exposure in Syrian hamsters","abstract":"Transmission of SARS-CoV-2 is driven by contact, fomite, and airborne transmission. The relative contribution of different transmission routes remains subject to debate. Here, we show Syrian hamsters are susceptible to SARS-CoV-2 infection through intranasal, aerosol and fomite exposure. Different routes of exposure presented with distinct disease manifestations. Intranasal and aerosol inoculation caused more severe respiratory pathology, higher virus loads and increased weight loss. Fomite exposure led to milder disease manifestation characterized by an anti-inflammatory immune state and delayed shedding pattern. Whereas the overall magnitude of respiratory virus shedding was not linked to disease severity, the onset of shedding was. Early shedding was linked to an increase in disease severity. Airborne transmission was more efficient than fomite transmission and dependent on the direction of the airflow. Carefully characterized of SARS-CoV-2 transmission models will be crucial to assess potential changes in transmission and pathogenic potential in the light of the ongoing SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2020.12.28.424565","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424029","pub_date":"2020-12-28","title":"SPINT2 controls SARS-CoV-2 viral infection and is associated to disease severity","abstract":"COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2. Here, we explored the existence of a co-regulation between SPINT2/TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.","version":"1.1","doi":"10.1101/2020.12.28.424029","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.055467","pub_date":"2020-12-28","title":"In silico design of Multi-epitope-based peptide vaccine against SARS-CoV-2 using its spike protein","abstract":"SARS-CoV-2 has been efficient in ensuring that many countries are brought to a standstill. With repercussions ranging from rampant mortality, fear, paranoia, and economic recession, the virus has brought together countries to look at possible therapeutic countermeasures. With prophylactic interventions possibly months away from being particularly effective, a slew of measures and possibilities concerning the design of vaccines are being worked upon. We attempted a structure-based approach utilizing a combination of epitope prediction servers and Molecular dynamic (MD) simulations to develop a multi-epitope-based subunit vaccine that involves the two subunits of the spike glycoprotein of SARS-CoV-2 (S1 and S2) coupled with a substantially effective chimeric adjuvant to create stable vaccine constructs. The designed constructs were evaluated based on their docking with Toll-Like Receptor (TLR) 4. Our findings provide an epitope-based peptide fragment that can be a potential candidate for the development of a vaccine against SARS-CoV-2. Recent experimental studies based on determining immunodominant regions across the spike glycoprotein of SARS-CoV-2 indicate the presence of the predicted epitopes included in this study.","version":"1.4","doi":"10.1101/2020.04.23.055467","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.28.424582","pub_date":"2020-12-28","title":"Emerging SARS-CoV-2 diversity revealed by rapid whole genome sequence typing","abstract":"Discrete classification of SARS-CoV-2 viral genotypes can identify emerging strains and detect geographic spread, viral diversity, and transmission events. We developed a tool (GNUVID) that integrates whole genome multilocus sequence typing and a supervised machine learning random forest-based classifier. We used GNUVID to assign sequence type (ST) profiles to each of 69,686 SARS-CoV-2 complete, high-quality genomes available from GISAID as of October 20th 2020. STs were then clustered into clonal complexes (CCs), and then used to train a machine learning classifier. We used this tool to detect potential introduction and exportation events, and to estimate effective viral diversity across locations and over time in 16 US states. GNUVID is a scalable tool for viral genotype classification (available at https://github.com/ahmedmagds/GNUVID) that can be used to quickly process tens of thousands of genomes. Our genotyping ST/CC analysis uncovered dynamic local changes in ST/CC prevalence and diversity with multiple replacement events in different states. We detected an average of 20.6 putative introductions and 7.5 exportations for each state. Effective viral diversity dropped in all states as shelter-in-place travel-restrictions went into effect and increased as restrictions were lifted. Interestingly, our analysis showed correlation between effective diversity and the date that state-wide mask mandates were imposed. Our classification tool uncovered multiple introduction and exportation events, as well as waves of expansion and replacement of SARS-CoV-2 genotypes in different states. Combined with future genomic sampling the GNUVID system could be used to track circulating viral diversity and identify emerging clones and hotspots.","version":"1.1","doi":"10.1101/2020.12.28.424582","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.189944","pub_date":"2020-12-28","title":"Mutational signatures and heterogeneous host response revealed via large-scale characterization of SARS-CoV-2 genomic diversity","abstract":"To dissect the mechanisms underlying the inflation of variants in the SARS-CoV-2 genome, we present one of the largest up-to-date analyses of intra-host genomic diversity, which reveals that most samples present heterogeneous genomic architectures, due to the interplay between host-related mutational processes and transmission dynamics. The deconvolution of the set of intra-host minor variants unveils the existence of non overlapping mutational signatures related to specific nucleotide substitutions, which prove that distinct hosts respond differently to SARS-CoV-2 infections, and which are likely ruled by APOBEC, Reactive Oxygen Species (ROS) and ADAR. Thanks to a corrected-for-signatures dN/dS analysis we demonstrate that the mutational processes underlying such signatures are affected by purifying selection, with important exceptions. In fact, several mutations linked to low-rate mutational processes appear to transit to clonality in the population, eventually leading to the definition of new clonal genotypes and to a statistically significant increase of overall genomic diversity. Importantly, the analysis of the phylogenetic model shows the presence of multiple homoplasies, due to mutational hotspots, phantom mutations or positive selection, and supports the hypothesis of transmission of minor variants during infections. Overall, the results of this study pave the way for the integrated characterization of intra-host genomic diversity and clinical outcome of SARS-CoV-2 hosts.","version":"1.2","doi":"10.1101/2020.07.06.189944","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.328112","pub_date":"2020-12-28","title":"Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response","abstract":"The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.","version":"1.2","doi":"10.1101/2020.10.06.328112","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.26.424449","pub_date":"2020-12-28","title":"MHC-II constrains the natural neutralizing antibody response to the SARS-CoV-2 spike RBM in humans","abstract":"SARS-CoV-2 antibodies develop within two weeks of infection, but wane relatively rapidly post-infection, raising concerns about whether antibody responses will provide protection upon re-exposure. Here we revisit T-B cooperation as a prerequisite for effective and durable neutralizing antibody responses centered on a mutationally constrained RBM B cell epitope. T-B cooperation requires co-processing of B and T cell epitopes by the same B cell and is subject to MHC-II restriction. We evaluated MHC-II constraints relevant to the neutralizing antibody response to a mutationally-constrained B cell epitope in the receptor binding motif (RBM) of the spike protein. Examining common MHC-II alleles, we found that peptides surrounding this key B cell epitope are predicted to bind poorly, suggesting a lack MHC-II support in T-B cooperation, impacting generation of high-potency neutralizing antibodies in the general population. Additionally, we found that multiple microbial peptides had potential for RBM cross-reactivity, supporting previous exposures as a possible source of T cell memory.","version":"1.1","doi":"10.1101/2020.12.26.424449","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.307546","pub_date":"2020-12-28","title":"Making the invisible enemy visible","abstract":"During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, 548 structures covering 16 of the SARS-CoV-2 viral proteins have been released in a span of only 6 months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination - and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others.","version":"1.2","doi":"10.1101/2020.10.07.307546","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.417758","pub_date":"2020-12-28","title":"Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols Towards Downstream Isolation and Analysis","abstract":"Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction haven\u2019t been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.","version":"1.3","doi":"10.1101/2020.12.10.417758","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423869","pub_date":"2020-12-28","title":"In Vitro Safety \u201cClinical Trial\u201d of the Cardiac Liability of Hydroxychloroquine and Azithromycin as COVID19 Polytherapy","abstract":"Despite global efforts, there are no effective FDA-approved medicines for the treatment of SARS-CoV-2 infection. Potential therapeutics focus on repurposed drugs, some with cardiac liabilities. Here we report on a preclinical drug screening platform, a cardiac microphysiological system (MPS), to assess cardiotoxicity associated with hydroxychloroquine (HCQ) and azithromycin (AZM) polytherapy in a mock clinical trial. The MPS contained human heart muscle derived from patient-specific induced pluripotent stem cells. The effect of drug response was measured using outputs that correlate with clinical measurements such as QT interval (action potential duration) and drug-biomarker pairing. Chronic exposure to HCQ alone elicited early afterdepolarizations (EADs) and increased QT interval from day 6 onwards. AZM alone elicited an increase in QT interval from day 7 onwards and arrhythmias were observed at days 8 and 10. Monotherapy results closely mimicked clinical trial outcomes. Upon chronic exposure to HCQ and AZM polytherapy, we observed an increase in QT interval on days 4-8.. Interestingly, a decrease in arrhythmias and instabilities was observed in polytherapy relative to monotherapy, in concordance with published clinical trials. Furthermore, biomarkers, most of them measurable in patients\u2019 serum, were identified for negative effects of single drug or polytherapy on tissue contractile function, morphology, and antioxidant protection. The cardiac MPS can predict clinical arrhythmias associated with QT prolongation and rhythm instabilities. This high content system can help clinicians design their trials, rapidly project cardiac outcomes, and define new monitoring biomarkers to accelerate access of patients to safe COVID-19 therapeutics.","version":"1.2","doi":"10.1101/2020.12.21.423869","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.26.424423","pub_date":"2020-12-27","title":"The neutralization effect of Montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies","abstract":"Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of SARS-CoV-2. In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist Montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of Montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the Fluorescent Resonance Energy Transfer (FRET)-based main protease enzyme inhibition assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T / hACE2, and virus neutralization assay using xCELLigence MP real time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of the Montelukast both on virus entry into the host cell (Spike/ACE2) and on the main protease enzyme inhibition. The virus neutralization assay results showed that while no cytotoxicity of the Montelukast was observed at 12 \u03bcM concentration, the cell index time 50 (CIT50) value was delayed for 12 hours. Moreover, it was also shown that Favipiravir, a well-known antiviral used in COVID-19 therapy, should be used by 16-fold higher concentrations than Montelukast in order to have the same effect of Montelukast. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and if its effect is proven in clinical phase studies, it should be used against COVID-19.","version":"1.1","doi":"10.1101/2020.12.26.424423","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424326","pub_date":"2020-12-26","title":"Influence of HLA class II polymorphism on predicted cellular immunity against SARS-CoV-2 at the population and individual level","abstract":"Development of effective adaptive immune responses after coronavirus disease 2019 (COVID-19) and after vaccination against SARS-CoV-2 is predicated on recognition of viral peptides, presented in the context of HLA class II molecules, by CD4+ T-cells. We capitalised on extensive high resolution HLA data deposited in the National Marrow Donor Program registry to obtain detailed information on human HLA haplotype frequencies of twenty five human populations and used a bioinformatics approach to investigate the role of HLA polymorphism on SARS-CoV-2 immunogenicity at the population and at the individual level. Within populations, we identify wide inter-individual variability in predicted CD4+ T-cell reactivity against structural, non-structural and accessory SARS-CoV-2 proteins, according to expressed HLA genotype. However, we find similar potential for anti-SARS-CoV-2 cellular immunity at the population level, across all ethnic groups examined, suggesting that HLA polymorphism is unlikely to account for observed disparities in clinical outcomes after COVID-19 among different race and ethnic groups. We predict robust immune reactivity against SARS-CoV-2 Spike protein, the basis for the majority of current vaccination efforts, both at the population and individual level, despite significant variation in Spike-derived peptide presentation by individual HLA genotypes. Finally, we provide comprehensive maps of SARS-CoV-2 proteome immunogenicity accounting for population coverage in major ethnic groups. Our findings provide important insight on the potential role of HLA polymorphism on development of protective immunity after SARS-CoV-2 infection and after vaccination and a firm basis for further experimental studies in this field.","version":"1.1","doi":"10.1101/2020.12.24.424326","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.20248822","pub_date":"2020-12-26","title":"Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England","abstract":"<jats:p>A novel SARS-CoV-2 variant, VOC 202012/01 (lineage B.1.1.7), emerged in southeast England in November 2020 and is rapidly spreading towards fixation. Using a variety of statistical and dynamic modelling approaches, we estimate that this variant has a 43\u201390% (range of 95% credible intervals 38\u2013130%) higher reproduction number than preexisting variants. A fitted two-strain dynamic transmission model shows that VOC 202012/01 will lead to large resurgences of COVID-19 cases. Without stringent control measures, including limited closure of educational institutions and a greatly accelerated vaccine roll-out, COVID-19 hospitalisations and deaths across England in 2021 will exceed those in 2020. Concerningly, VOC 202012/01 has spread globally and exhibits a similar transmission increase (59\u201374%) in Denmark, Switzerland, and the United States.</jats:p>","version":null,"doi":"10.1101/2020.12.24.20248822","journal":"medRxiv","score":null},{"id":"10.1101/2020.12.26.424422","pub_date":"2020-12-26","title":"Extensive High-Order Complexes within SARS-CoV-2 Proteome Revealed by Compartmentalization-Aided Interaction Screening","abstract":"Bearing the largest single-stranded RNA genome in nature, SARS-CoV-2 utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. Here, we developed an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we mapped the interaction network among RTC-related viral proteins. We identified a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC led to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient, and robust interaction screening strategy, and indicate the existence of a complex interaction network among RTC-related factors, thus opening up new opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.","version":"1.1","doi":"10.1101/2020.12.26.424422","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.26.424442","pub_date":"2020-12-26","title":"Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2","abstract":"Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.","version":"1.1","doi":"10.1101/2020.12.26.424442","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.25.424300","pub_date":"2020-12-26","title":"Disruption of nasal bacteria enhances protective immune responses to influenza A virus and SARS-CoV-2 infection in mice","abstract":"Gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here we demonstrate that while intranasal administration of influenza virus hemagglutinin vaccine alone was insufficient to induce the vaccine-specific antibody responses, disruption of nasal bacteria by lysozyme or addition of culturable oral bacteria from a healthy human volunteer rescued inability of the nasal bacteria to generate antibody responses to intranasally administered the split-virus vaccine. Myd88-depdnent signaling in the hematopoietic compartment was required for adjuvant activity of intranasally administered oral bacteria. In addition, we found that the oral bacteria-combined intranasal vaccine induced protective antibody response to influenza virus and SARS-CoV-2 infection. Our findings here have identified a previously unappreciated role for nasal bacteria in the induction of the virus-specific adaptive immune responses.","version":"1.1","doi":"10.1101/2020.12.25.424300","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424245","pub_date":"2020-12-26","title":"Single point mutations can potentially enhance infectivity of SARS-CoV-2 revealed by in silico affinity maturation and SPR assay","abstract":"The RBD (receptor binding domain) of the SARS-CoV-2 virus S (spike) protein mediates the viral cell attachment and serves as a promising target for therapeutics development. Mutations on the S-RBD may alter its affinity to cell receptor and affect the potency of vaccines and antibodies. Here we used an in-silico approach to predict how mutations on RBD affect its binding affinity to hACE2 (human angiotensin-converting enzyme2). The effect of all single point mutations on the interface was predicted. SPR assay result shows that 6 out of 9 selected mutations can strengthen binding affinity. Our prediction has reasonable agreement with the previous deep mutational scan results and recently reported mutants. Our work demonstrated in silico method as a powerful tool to forecast more powerful virus mutants, which will significantly benefit for the development of broadly neutralizing vaccine and antibody.","version":"1.1","doi":"10.1101/2020.12.24.424245","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424172","pub_date":"2020-12-26","title":"Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of Covid-19 death","abstract":"Most Covid-19 victims are old and die from unrelated causes. Here we present twelve complete autopsies, including two rapid autopsies of young patients where the cause of death was Covid-19 ARDS. The main virus induced pathology was in the lung parenchyma and not in the airways. Most coagulation events occurred in the intra-alveolar and not in the intra-vascular space and the few thrombi were mainly composed of aggregated thrombocytes. The dominant inflammatory response was the massive accumulation of CD163+ macrophages and the disappearance of T killer, NK and B-cells. The virus was replicating in the pneumocytes and macrophages but not in bronchial epithelium, endothel, pericytes or stromal cells. The lung consolidations were produced by a massive regenerative response, stromal and epithelial proliferation and neovascularization. We suggest that thrombocyte aggregation inhibition, angiogenesis inhibition and general proliferation inhibition may have a roll in the treatment of advanced Covid-19 ARDS.","version":"1.1","doi":"10.1101/2020.12.23.424172","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424254","pub_date":"2020-12-24","title":"Cell-type apoptosis in lung during SARS-CoV-2 infection","abstract":"The SARS-CoV-2 pandemic has inspired renewed interest in understanding the fundamental pathology of acute respiratory distress syndrome (ARDS) following infection because fatal COVID-19 cases are commonly linked to respiratory failure due to ARDS. The pathologic alteration known as diffuse alveolar damage in endothelial and epithelial cells is a critical feature of acute lung injury in ARDS. However, the pathogenesis of ARDS following SRAS-CoV-2 infection remains largely unknown. In the present study, we examined apoptosis in post-mortem lung sections from COVID-19 patients and lung tissues from a non-human primate model of SARS-CoV-2 infection, in a cell-type manner, including type 1 and 2 alveolar cells and vascular endothelial cells (ECs), macrophages, and T cells. Multiple-target immunofluorescence (IF) assays and western blotting suggest both intrinsic and extrinsic apoptotic pathways are activated during SARS-CoV-2 infection. Furthermore, we observed that SARS-CoV-2 fails to induce apoptosis in human bronchial epithelial cells (i.e., BEAS2B cells) and primary human umbilical vein endothelial cells (HUVECs), which are refractory to SARS-CoV-2 infection. However, infection of co-cultured Vero cells and HUVECs or Vero cells and BEAS2B cells with SARS-CoV-2 induced apoptosis in both Vero cells and HUVECs/BEAS2B cells, but did not alter the permissiveness of HUVECs or BEAS2B cells to the virus. Post-exposure treatment of the co-culture of Vero cells and HUVECs with an EPAC1-specific activator ameliorated apoptosis in HUVECs. These findings may help to delineate a novel insight into the pathogenesis of ARDS following SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.23.424254","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.422670","pub_date":"2020-12-24","title":"Detection and molecular characterisation of SARS-CoV-2 in farmed mink (Neovision vision) in Poland","abstract":"SARS-CoV-2 is the aetiological agent of COVID-19 disease and has been spreading worldwide since December 2019. The virus has been shown to infect different animal species under experimental conditions. Also, minks have been found to be susceptible to SARS-CoV-2 infection in fur farms in Europe and the USA. Here we investigated 91 individual minks from a farm located in Northern Poland. Using RT-PCR, antigen detection and NGS, we confirmed 15 animals positive for SARS-CoV-2. The result was verified by sequencing of full viral genomes, confirming SARS-CoV-2 infection in Polish mink. Country-scale monitoring conducted by veterinary inspection so far has not detected the presence of SARS-CoV-2 on other mink farms. Taking into consideration that Poland has a high level of positive diagnostic tests among its population, there is a high risk that more Polish mink farms become a source for SARS-CoV-2. Findings reported here and from other fur producing countries urge the assessment of SARS-CoV-2 prevalence in animals bred in Polish fur farms.","version":"1.1","doi":"10.1101/2020.12.24.422670","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424171","pub_date":"2020-12-24","title":"A SARS-CoV-2 spike binding DNA aptamer that inhibits pseudovirus infection in vitro by an RBD independent mechanism","abstract":"The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Notwithstanding, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.","version":"1.1","doi":"10.1101/2020.12.23.424171","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424111","pub_date":"2020-12-24","title":"Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L","abstract":"The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.","version":"1.1","doi":"10.1101/2020.12.23.424111","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424189","pub_date":"2020-12-24","title":"Berberine and obatoclax inhibit SARS-CoV-2 replication in primary human nasal epithelial cells in vitro","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and has infected an estimated 10% of the global population in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of a specific set of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.","version":"1.1","doi":"10.1101/2020.12.23.424189","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424229","pub_date":"2020-12-24","title":"Patterns of within-host genetic diversity in SARS-CoV-2","abstract":"Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1,181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within and between host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.","version":"1.1","doi":"10.1101/2020.12.23.424229","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424203","pub_date":"2020-12-24","title":"Experimental SARS-CoV-2 infection of bank voles - general susceptibility but lack of direct transmission","abstract":"After experimental inoculation, SARS-CoV-2 infection was proven for bank voles by seroconversion within eight days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Therefore, bank voles are unlikely to establish effective SARS-CoV-2 transmission cycles in nature. Bank voles show low-level viral replication and seroconversion upon infection with SARS-CoV-2, but lack transmission to contact animals.","version":"1.1","doi":"10.1101/2020.12.24.424203","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424271","pub_date":"2020-12-24","title":"Real-time monitoring epidemic trends and key mutations in SARS-CoV-2 evolution by an automated tool","abstract":"With the global epidemic of SARS-CoV-2, it is important to monitor the variation, haplotype subgroup epidemic trends and key mutations of SARS-CoV-2 over time effectively, which is of great significance to the development of new vaccines, the update of therapeutic drugs, and the improvement of detection reagents. The AutoVEM tool developed in the present study could complete all mutations detections, haplotypes classification, haplotype subgroup epidemic trends and key mutations analysis for 131,576 SARS-CoV-2 genome sequences in 18 hours on a 1 core CPU and 2G internal storage computer. Through haplotype subgroup epidemic trends analysis of 131,576 genome sequences, the great significance of the previous 4 specific sites (C241T, C3037T, C14408T and A23403G) was further revealed, and 6 new mutation sites of highly linked (T445C, C6286T, C22227T, G25563T, C26801G and G29645T) were discovered for the first time that might be related to the infectivity, pathogenicity or host adaptability of SARS-CoV-2. In brief, we proposed an integrative method and developed an efficient automated tool to monitor haplotype subgroup epidemic trends and screen out the key mutations in the evolution of SARS-CoV-2 over time for the first time, and all data could be updated quickly to track the prevalence of previous key mutations and new key mutations because of high efficiency of the tool. In addition, the idea of combinatorial analysis in the present study can also provide a reference for the mutation monitoring of other viruses.","version":"1.1","doi":"10.1101/2020.12.24.424271","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424322","pub_date":"2020-12-24","title":"Inferring Toll-Like Receptor induced epitope subunit vaccine candidate against SARS-CoV-2: A Reverse Vaccinology approach","abstract":"Toll-Like Receptors (TLRs) are a group of Pattern Recognition Receptors (PRRs) which bind to the exogenous pathogen associated molecular patterns (PAMPs) like other PRRs; hence the main function is to sense the harmness and mediate the innate immune response to pathogens. TLRs play an important role in innate immune responses to infection. The host has evolved to use other TLR and PAMP agonists as agents to stimulate a protective inflammatory immune response against infection. Because only a small number of doses are given, TLR agonists appear to have greater potential and fewer safety concerns than other uses as vaccine adjuvants. In the present days, development of peptides targeting immune response can be approved for survival in biological monitoring systems before vaccine exposures. Peptide vaccines are easy to synthesize, more stable and relatively safe. In addition, production of peptides becomes simple, easily reproducible, fast and cost effective. Getting vaccinated against Covid-19, which has become a pandemic in the human population, is the most practical way to control the outbreak. The new coronavirus does not contain a drug or vaccine to prevent it from spreading to humans. To getting a proper vaccine candidate against the novel coronavirus, the present study used the reverse vaccinology approach by using a complete set of SARS-CoV-2 proteins; such as: Spike, Envelope, Nucleocapsid, Membrane, NSPs, and ORFs to extract the antigenic elements that produce B-cell, T-cell and IFN positive epitopes. These epitopes with precise binding to the Toll-Like receptors (1-10) have developed epitope based vaccine candidates. We have prioritized a set of epitopes based on their antigenicity, allergenicity, sequence conservation and projected population coverage world-wide. The selected epitopes were employed for in-silico docking interactions with Toll-Like receptors and molecular dynamic simulation confirmed the stability of the vaccine candidates resulting epitope of spike proteins with both the TLR 7 and 8 shows the best binding affinity. We believe that this ideal epitope vaccine candidate could enhance the immune response of the host and reduce the reinfection risk.","version":"1.1","doi":"10.1101/2020.12.24.424322","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.24.424209","pub_date":"2020-12-24","title":"ace2 expression is higher in intestines and liver while being tightly regulated in development and disease in zebrafish","abstract":"Human Angiotensin I Converting Enzyme 2 (ACE2) that acts as a receptor for SARS-CoV-2 entry is highly expressed in human type II pneumocytes and enterocytes and similarly in other mammals and zebrafish (Danio rerio). The zebrafish genome has a highly conserved, one-to-one ortholog of ACE2, i.e., ace2, whose expression profile however has not yet been studied during development or in pathologies relevant to COVID-19. Herein, we identified significant development-, tissue- and gender-specific modulations in ace2 expression based on meta-analysis of zebrafish Affymetrix transcriptomics datasets (ndatasets=107, GPL1319 in GEO database). Co-expression network analysis of ace2 revealed distinct positively correlated (carboxypeptidase activity and fibrin clot formation), and negatively correlated (cilia biogenesis/transport and chromatin modifications) STRING network modules. Using additional transcriptomics datasets, we showed zebrafish embryos before 3 days post fertilization (dpf) exhibited low levels of ace2 that increased significantly until 4 dpf implicating a role for ace2 in organogenesis. Re-analysis of RNA-seq datasets from zebrafish adult tissues demonstrated ace2 was expressed highly in intestines, variably in liver, and at lower levels in other organs. In addition, zebrafish females and males showed significant dimorphism in their age-dependent expression of ace2, and between ovary and testis where the latter had higher levels. Moreover, we demonstrated ace2 expression was significantly modulated under different physiological and pathological conditions associated with development, diet, infection, and inflammation. Our findings implicate a novel translational role for zebrafish ace2 in differentiation and pathologies predominantly found in intestines and liver, in which the effects of SARS-CoV-2 could be detrimental.","version":"1.1","doi":"10.1101/2020.12.24.424209","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424199","pub_date":"2020-12-24","title":"Exploring epitope and functional diversity of anti-SARS-CoV2 antibodies using AI-based methods","abstract":"Since the beginning of the COVID19 pandemics, an unprecedented research effort has been conducted to analyze the antibody responses in patients, and many trials based on passive immunotherapy \u2014 notably monoclonal antibodies \u2014 are ongoing. Twenty-one antibodies have entered clinical trials, 6 having reached phase 2/3, phase 3 or having received emergency authorization. These represent only the tip of the iceberg, since many more antibodies have been discovered and represent opportunities either for diagnosis purposes or as drug candidates. The main problem facing laboratories willing to develop such antibodies is the huge task of analyzing them and choosing the best candidate for exhaustive experimental validation. In this work we show how artificial intelligence-based methods can help in analyzing large sets of antibodies in order to determine in a few hours the best candidates in few hours. The MAbCluster method, which only requires knowledge of the amino acid sequences of the antibodies, allows to group the antibodies having the same epitope, considering only their amino acid sequences and their 3D structures (actual or predicted), and to infer some of their functional properties. We then use MAbTope to predict the epitopes for all antibodies for which they are not already known. This allows an exhaustive comparison of the available epitopes, but also gives a synthetic view of the possible combinations. Finally, we show how these results can be used to predict which antibodies might be affected by the different mutations arising in the circulating strains of the virus, such as the N501Y mutation that has started to spread in Great-Britain.","version":"1.1","doi":"10.1101/2020.12.23.424199","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423909","pub_date":"2020-12-23","title":"Platycodin D prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection in vitro by hindering membrane fusion","abstract":"An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to the global public health. Herbal medicines and their derived natural products have drawn much attention to treat COVID-19, but there has been no natural product showing inhibitory activity against SARS-CoV-2 infection with detailed mechanism. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection-routes via lysosome- and transmembrane protease, serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host-entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by a pharmacological inhibition or gene-silencing of NPC1, which is mutated in Niemann-Pick type C (NPC) patients displaying disrupted membrane cholesterol. Finally, readily available local foods or herbal medicines containing PG root show the similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that a brief disruption of membrane cholesterol can be a novel therapeutic approach against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.22.423909","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423940","pub_date":"2020-12-23","title":"No evidence for human monocyte-derived macrophage infection and antibody-mediated enhancement of SARS-CoV-2 infection","abstract":"Vaccines are essential to control the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and to protect the vulnerable population. However, one safety concern of vaccination is the possible development of antibody-dependent enhancement (ADE) of SARS-CoV-2 infection. The potential infection of Fc receptor bearing cells such as macrophages, would support continued virus replication and inflammatory responses, and thereby potentially worsen the clinical outcome of COVID-19. Here we demonstrate that SARS-CoV-2 and SARS-CoV-1 neither infect human monocyte-derived macrophages nor induce inflammatory cytokines in these cells, in sharp contrast to Middle East respiratory syndrome (MERS) coronavirus and the common cold human coronavirus 229E. Furthermore, serum from convalescent COVID-19 patients neither induced enhancement of SARS-CoV-2 infection nor innate immune response in human macrophages. These results support the view that ADE may not be involved in the immunopathological processes associated with COVID-19, however, more studies are necessary to understand the potential contribution of antibodies-virus complexes with other cells expressing FcR receptors.","version":"1.1","doi":"10.1101/2020.12.22.423940","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424169","pub_date":"2020-12-23","title":"SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation","abstract":"SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through activation of cytoplasmic RNA-ensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing, or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19. SARS-CoV-2 activates RNA sensors and consequent inflammatory responses in lung epithelial cells Epithelial RNA sensing responses drive pro-inflammatory macrophage activation Exogenous inflammatory stimuli exacerbate responses to SARS-CoV-2 in both eplithelial cells and macrophages Immunomodulators inhibit RNA sensing responses and consequent macrophage inflammation","version":"1.1","doi":"10.1101/2020.12.23.424169","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.424069","pub_date":"2020-12-23","title":"Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a \u03b2-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.","version":"1.1","doi":"10.1101/2020.12.22.424069","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.422708","pub_date":"2020-12-23","title":"SARS-CoV-2 Envelope (E) Protein Interacts with PDZ-Domain-2 of Host Tight Junction Protein ZO1","abstract":"Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe respiratory dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway barrier damage and mitigate virus spread.","version":"1.1","doi":"10.1101/2020.12.22.422708","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423906","pub_date":"2020-12-23","title":"Host Cell Proteases Drive Early or Late SARS-CoV-2 Penetration","abstract":"SARS-CoV-2 is a newly emerged coronavirus (CoV) that spread through human populations worldwide in early 2020. CoVs rely on host cell proteases for activation and infection. The trypsin-like protease TMPRSS2 at the cell surface, cathepsin L in endolysosomes, and furin in the Golgi have all been implicated in the SARS-CoV-2 proteolytic processing. Whether SARS-CoV-2 depends on endocytosis internalization and vacuolar acidification for infectious entry remains unclear. Here, we examined the dynamics of SARS-CoV-2 activation during the cell entry process in tissue culture. Using four cell lines representative of lung, colon, and kidney epithelial tissues, we found that TMPRSS2 determines the SARS-CoV-2 entry pathways. In TMPRSS2-positive cells, infection was sensitive to aprotinin, a TMPRSS2 inhibitor, but not to SB412515, a drug that impairs cathepsin L. Infectious penetration was marginally dependent on endosomal acidification, and the virus passed the protease-sensitive step within 10 min. In a marked contrast, in TMPRSS2-negative cells cathepsin L and low pH were required for SARS-CoV-2 entry. The cathepsin L-activated penetration occurred within 40-60 min after internalization and required intact endolysosomal functions. Importantly, pre-activation of the virus allowed it to bypass the need for endosomal acidification for viral fusion and productive entry. Overall, our results indicate that SARS-CoV-2 shares with other CoVs a strategy of differential use of host cell proteases for activation and infectious penetration. This study also highlights the importance of TMPRSS2 in dictating the entry pathway used by SARS-CoV-2. Preventing SARS-CoV-2 spread requires approaches affecting early virus-host cell interactions before the virus enters and infects target cells. Host cell proteases are critical for coronavirus activation and infectious entry. Here, we reconcile apparent contradictory observations from recent reports on endosomal acidification and the role of furin, TMPRSS2, and cathepsin L in the productive entry and fusion process of SARS-CoV-2. Investigating authentic virus in various cell types, we demonstrated that SARS-CoV-2 developed the ability to use different entry pathways, depending on the proteases expressed by the target cell. Our results have strong implications for future research on the apparent broad tropism of the virus in vivo. This study also provides a handle to develop novel antiviral strategies aiming to block virus entry, as illustrated with the several drugs that we identified to prevent SARS-CoV-2 infection, some with low IC50.","version":"1.1","doi":"10.1101/2020.12.22.423906","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424138","pub_date":"2020-12-23","title":"mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection","abstract":"The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines to meet a worldwide need. mRNA-based vaccines are the most promising technology for rapid and safe SARS-CoV-2 vaccine development and production. We have designed CVnCoV, a lipid-nanoparticle (LNP) encapsulated, sequence optimised mRNA-based SARS-CoV-2 vaccine that encodes for full length, pre-fusion stabilised Spike protein. Unlike other mRNA-based approaches, CVnCoV exclusively consists of non-chemically modified nucleotides and can be applied at comparatively low doses. Here we demonstrate that CVnCoV induces robust humoral and cellular responses in non-human primates (NHPs). Animals vaccinated with 8 \u03bcg of CVnCoV were protected from challenge infection with SARS-CoV-2. Comprehensive analyses of pathological changes in challenged animals via lung histopathology and Computed Tomography (CT) scans gave no indication of enhanced disease upon CVnCoV vaccination. These results demonstrate safety, immunogenicity, and protective efficacy of CVnCoV in NHPs that extend our previously published preclinical data and provide strong support for further clinical testing in ongoing phase 2b/3 efficacy studies.","version":"1.1","doi":"10.1101/2020.12.23.424138","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423894","pub_date":"2020-12-23","title":"Ferritin nanoparticle based SARS-CoV-2 RBD vaccine induces persistent antibody response and long-term memory in mice","abstract":"Since the outbreak of COVID-19, over 200 vaccine candidates have been documented and some of them have advanced to clinical trials with encouraging results. However, the antibody persistence over 3 months post immunization and the long-term memory have been rarely reported. Here, we report that a ferritin nanoparticle based SARS-CoV-2 RBD vaccine induced in mice an efficient antibody response which lasts for at least 7 months post immunization. Significantly higher number of memory B cells were maintained and a significantly higher level of recall response was induced upon antigen challenge. Thus, we believe our current study provide the first information about the long-term antibody persistence and memory response of a COVID-19 vaccine. This information would be also timely useful for the development and evaluation of other vaccines.","version":"1.1","doi":"10.1101/2020.12.22.423894","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.23.424177","pub_date":"2020-12-23","title":"Identification of COVID-19-relevant transcriptional regulatory networks and associated kinases as potential therapeutic targets","abstract":"Identification of transcriptional regulatory mechanisms and signaling networks involved in the response of host to infection by SARS-CoV-2 is a powerful approach that provides a systems biology view of gene expression programs involved in COVID-19 and may enable identification of novel therapeutic targets and strategies to mitigate the impact of this disease. In this study, we combined a series of recently developed computational tools to identify transcriptional regulatory networks involved in the response of epithelial cells to infection by SARS-CoV-2, and particularly regulatory mechanisms that are specific to this virus. In addition, using network-guided analyses, we identified signaling pathways that are associated with these networks and kinases that may regulate them. The results identified classical antiviral response pathways including Interferon response factors (IRFs), interferons (IFNs), and JAK-STAT signaling as key elements upregulated by SARS-CoV-2 in comparison to mock-treated cells. In addition, comparing SARS-Cov-2 infection of airway epithelial cells to other respiratory viruses identified pathways associated with regulation of inflammation (MAPK14) and immunity (BTK, MBX) that may contribute to exacerbate organ damage linked with complications of COVID-19. The regulatory networks identified herein reflect a combination of experimentally validated hits and novel pathways supporting the computational pipeline to quickly narrow down promising avenue of investigations when facing an emerging and novel disease such as COVID-19.","version":"1.1","doi":"10.1101/2020.12.23.424177","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.424071","pub_date":"2020-12-23","title":"In Vivo Pharmacokinetic Study of Remdesivir Dry Powder for Inhalation in Hamsters","abstract":"Remdesivir dry powder for inhalation was previously developed using thin film freezing (TFF). A single-dose 24-hour pharmacokinetic study in hamsters, a small animal model for SARS-CoV-2, demonstrated that pulmonary delivery of TFF remdesivir can achieve plasma remdesivir and GS-441524 levels higher than the reported EC50s of both remdesivir and GS-441524 (in human epithelial cells) over 20 hours. The half-life of GS-4412524 following dry powder insufflation was about 7 hours, suggesting the dosing regimen would be twice daily administration. Although the remdesivir-Captisol\u00ae (80/20 w/w) formulation showed faster and greater absorption of remdesivir and GS-4412524 in the lung, remdesivir-leucine (80/20 w/w) exhibited a greater Cmax with shorter Tmax and lower AUC of GS-441524, indicating lower total drug exposure is required to achieve a high effective concentration against SAR-CoV-2. In conclusion, remdesivir dry powder for inhalation would be a promising alternative dosage form for the treatment of COVID-19 disease.","version":"1.1","doi":"10.1101/2020.12.22.424071","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.424026","pub_date":"2020-12-23","title":"WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization","abstract":"In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology and the response to therapeutic drugs. On March 15, 2020, a stay-at-home order was put into effect in the state of Massachusetts, USA, to flatten the curve of the spread of the novel SARS-CoV2 virus that causes COVID-19. For biomedical researchers in our state, this meant putting a hold on experiments for nine weeks until May 18, 2020. To keep the lab engaged and productive, and to enhance communication and collaboration, we embarked on an in-lab project that we all found important but that we never had the time for: the detailed annotation and drawing of C. elegans metabolic pathways. As a result, we present WormPaths, which is composed of two parts: 1) the careful manual annotation of metabolic genes into pathways, categories and levels, and 2) 66 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on our WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the unfortunate event of additional lockdowns, we envision further developing these maps to be more interactive, with an analogy of road maps that are available on mobile devices.","version":"1.1","doi":"10.1101/2020.12.22.424026","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.044404","pub_date":"2020-12-23","title":"VERSO: a comprehensive framework for the inference of robust phylogenies and the quantification of intra-host genomic diversity of viral samples","abstract":"We introduce VERSO, a two-step framework for the characterization of viral evolution from sequencing data of viral genomes, which improves over phylogenomic approaches for consensus sequences. VERSO exploits an efficient algorithmic strategy to return robust phylogenies from clonal variant profiles, also in conditions of sampling limitations. It then leverages variant frequency patterns to characterize the intra-host genomic diversity of samples, revealing undetected infection chains and pinpointing variants likely involved in homoplasies. On simulations, VERSO outperforms state-of-the-art tools for phylogenetic inference. Notably, the application to 6726 Amplicon and RNA-seq samples refines the estimation of SARS-CoV-2 evolution, while co-occurrence patterns of minor variants unveil undetected infection paths, which are validated with contact tracing data. Finally, the analysis of SARS-CoV-2 mutational landscape uncovers a temporal increase of overall genomic diversity, and highlights variants transiting from minor to clonal state and homoplastic variants, some of which falling on the spike gene. Available at: https://github.com/BIMIB-DISCo/VERSO.","version":"1.4","doi":"10.1101/2020.04.22.044404","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423898","pub_date":"2020-12-22","title":"SARS-CoV-2 RNA quantification using droplet digital RT-PCR","abstract":"Quantitative viral load assays have transformed our understanding of \u2013 and ability to manage \u2212 viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time reverse transcriptase-polymerase chain reaction (RT-PCR), yield semi-quantitative results only. Reverse transcriptase droplet digital PCR (RT-ddPCR), a technology that partitions each reaction into 20,000 nanolitre-sized droplets prior to amplification, offers an attractive platform for SARS-CoV-2 RNA quantification. We evaluated eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests for use in RT-ddPCR, and identified three (Charit\u00e9-Berlin E-Sarbeco and Pasteur Institute IP2 and IP4) as the most efficient, precise and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. Analytical efficiency of the E-Sarbeco primer/probe set, for example, was ~83%, while assay precision, as measured by the coefficient of variation, was ~2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. We further calibrated RT-ddPCR-derived SARS-CoV-2 E gene copy numbers against cycle threshold (Ct) values from a commercial real-time RT-PCR diagnostic platform. The resulting log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from Ct values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.","version":"1.1","doi":"10.1101/2020.12.21.423898","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.423682","pub_date":"2020-12-22","title":"Analysis of 46,046 SARS-CoV-2 whole-genomes leveraging principal component analysis (PCA)","abstract":"Since the beginning of the global SARS-CoV-2 pandemic, there have been a number of efforts to understand the mutations and clusters of genetic lines of the SARS-CoV-2 virus. Until now, phylogenetic analysis methods have been used for this purpose. Here we show that Principal Component Analysis (PCA), which is widely used in population genetics, can not only help us to understand existing findings about the mutation processes of the virus, but can also provide even deeper insights into these processes while being less sensitive to sequencing gaps. Here we describe a comprehensive analysis of a 46,046 SARS-CoV-2 genome sequence dataset downloaded from the GISAID database in June of this year. PCA provides deep insights into the analysis of large data sets of SARS-CoV-2 genomes, revealing virus lineages that have thus far been unnoticed.","version":"1.1","doi":"10.1101/2020.12.20.423682","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423893","pub_date":"2020-12-22","title":"Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection","abstract":"SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3\u2019UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/.","version":"1.1","doi":"10.1101/2020.12.22.423893","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199414","pub_date":"2020-12-22","title":"Global Geographic and Temporal Analysis of SARS-CoV-2 Haplotypes Normalized by COVID-19 Cases during the Pandemic","abstract":"Since the identification of SARS-CoV-2, a large number of genomes have been sequenced with unprecedented speed around the world. This marks a unique opportunity to analyze virus spreading and evolution in a worldwide context. Currently, there is not a useful haplotype description to help to track important and globally scattered mutations. Also, differences in the number of sequenced genomes between countries and/or months make it difficult to identify the emergence of haplotypes in regions where few genomes are sequenced but a large number of cases are reported. We propose an approach based on the normalization by COVID-19 cases of relative frequencies of mutations using all the available data to identify major haplotypes. Furthermore, we can use a similar normalization approach to tracking the temporal and geographic distribution of haplotypes in the world. Using 171 461 genomes, we identify five major haplotypes (OTUs) based on nine high-frequency mutations. OTU_3 characterized by mutations R203K and G204R is currently the most frequent haplotype circulating in four of the six continents analyzed. On the other hand, during almost all months analyzed, OTU_5 characterized by the mutation T85I in nsp2 is the most frequent in North America. Recently (since September), OTU_2 has been established as the most frequent in Europe. OTU_1, the ancestor haplotype is near to extinction showed by its low number of isolations since May. Also, we analyzed whether age, gender, or patient status is more related to a specific OTU. We did not find OTU\u2019s preference for any age group, gender, or patient status. Finally, we discuss structural and functional hypotheses in the most frequently identified mutations, none of those mutations show a clear effect on the transmissibility or pathogenicity.","version":"1.4","doi":"10.1101/2020.07.12.199414","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.244996","pub_date":"2020-12-22","title":"Lactoferrin as potential supplementary nutraceutical agent in COVID-19 patients: in vitro and in vivo preliminary evidences","abstract":"Lactoferrin, a multifunctional cationic glycoprotein, secreted by exocrine glands and neutrophils, possesses an antiviral activity extendable to SARS-CoV-2. We performed in vitro assays proving lactoferrin antiviral activity through direct attachment to both virus and cell surface components. This activity varied according to concentration (100/500\u03bcg/ml), multiplicity of infection (0.1/0.01) and cell type (Vero E6/Caco-2 cells). Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between the lactoferrin and the Spike S glycoprotein, thus hindering the viral entry into the cells. Hence, we conducted a clinical trial to investigate effect and tolerability of a liposomal lactoferrin formulation as a supplementary nutraceutical agent in mild-to-moderate and asymptomatic COVID-19 patients. A total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided in 3 groups according to the administered regimen. Thirty-two patients, 14 hospitalised and 18 in home-based insolation received oral and intranasal liposomal bovine lactoferrin (bLf), 32 hospitalised patients were treated with standard of care treatment (hydroxychloroquine, azitromicin and lopinavir/darunavir), and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, not-treated, healthy subjects were added as a control group for ancillary analysis. bLf-supplemented COVID-19 patients obtained an earlier and significant (p < 0,0001.) median rRT-PCR SARS-COV-2 RNA negative conversion than standard of care-treated and non-treated COVID-19 patients (14.25 vs 27.13 vs 32.61 days, respectively). In addition, bLf-supplemented COVID-19 patients showed significant fast clinical symptoms recovery than standard of care-treated and non-treated COVID-19 patients. Moreover, in bLf-supplemented patients, a significant decrease of either serum ferritin or IL-6 levels or host iron overload, all parameters characterizing inflammatory processes, were observed. Serum D-dimers was also found significantly decreased following bLf supplement. No adverse events were reported. These in vitro and in vivo observations led us to speculate a potential and safe supplementary role of Blf in the management of mild-to-moderate and asymptomatic COVID-19 patients.","version":"1.4","doi":"10.1101/2020.08.11.244996","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423917","pub_date":"2020-12-22","title":"T cell activation, highly armed cytotoxic cells and a sharp shift in monocytes CD300 receptors expression is characteristic of patients with severe COVID-19","abstract":"COVID-19 manifests with a wide diversity of clinical phenotypes characterized by dysfunctional and exaggerated host immune responses. Many results have been described on the status of the immune system of patients infected with SARS-CoV-2, but there are still aspects that have not been fully characterized. In this study, we have analyzed a cohort of patients with mild, moderate and severe disease. We performed flow cytometric studies and correlated the data with the clinical features and clinical laboratory values of patients. Both conventional and unsupervised data analyses concluded that patients with severe disease are characterized, among others, by a higher state of activation in all T cell subsets, higher expression of perforin and granzyme B in cytotoxic cells, expansion of adaptive NK cells and the accumulation of activated and immature dysfunctional monocytes which are identified by a low expression of HLA-DR and an intriguing abrupt change in the expression pattern of CD300 receptors. More importantly, correlation analysis showed a strong association between the alterations in the immune cells and the clinical signs of severity. These results indicate that patients with severe COVID-19 have a broad perturbation of their immune system, and they will help to understand the immunopathogenesis of severe COVID-19 as well as could be of special value for physicians to decide which specific therapeutic options are most effective for their patients.","version":"1.1","doi":"10.1101/2020.12.22.423917","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423860","pub_date":"2020-12-22","title":"Analyzing the vast coronavirus literature with CoronaCentral","abstract":"The global SARS-CoV-2 pandemic has caused a surge in research exploring all aspects of the virus and its effects on human health. The overwhelming rate of publications means that human researchers are unable to keep abreast of the research. To ameliorate this, we present the CoronaCentral resource which uses machine learning to process the research literature on SARS-CoV-2 along with articles on SARS-CoV and MERS-CoV. We break the literature down into useful categories and enable analysis of the contents, pace, and emphasis of research during the crisis. These categories cover therapeutics, forecasting as well as growing areas such as \u201cLong Covid\u201d and studies of inequality and misinformation. Using this data, we compare topics that appear in original research articles compared to commentaries and other article types. Finally, using Altmetric data, we identify the topics that have gained the most media attention. This resource, available at https://coronacentral.ai, is updated multiple times per day and provides an easy-to-navigate system to find papers in different categories, focussing on different aspects of the virus along with currently trending articles.","version":"1.1","doi":"10.1101/2020.12.21.423860","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.22.423939","pub_date":"2020-12-22","title":"Type I and III IFNs produced by the nasal epithelia and dimmed inflammation are key features of alpacas resolving MERS-CoV infection","abstract":"While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, provoking the induction of interferon stimulated genes (ISGs) along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-\u03baB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, is central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10. Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of a respiratory disease causing high mortality in humans. In camelids, the main MERS-CoV reservoir host, viral infection leads to subclinical disease. Our study describes transcriptional regulations of innate immunological pathways underlying asymptomatic clinical manifestations of alpacas in response to MERS-CoV. Concomitant to the peak of infection, these animals elicited a strong transient interferon response and induction of the anti-inflammatory cytokine IL10 in the nasal mucosa. This was associated to a dimmed regulation of pro-inflammatory cytokines and induction of interferon stimulated genes along the whole respiratory mucosa, leading to the rapid clearance of the virus. Thus, innate immune responses occurring in the nasal mucosa appear to be the key in controlling MERS-CoV disease by avoiding a cytokine storm. Understanding on how asymptomatic host reservoirs counteract MERS-CoV infection will aid in the development of antiviral drugs and vaccines.","version":"1.1","doi":"10.1101/2020.12.22.423939","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.410357","pub_date":"2020-12-21","title":"pH and Receptor Induced Conformational Changes-Implications Towards S1 Dissociation of SARS-CoV2 Spike Glycoprotein","abstract":"Viruses, being obligate intracellular parasites, must first attach themselves and gain entry into host cells. Viral fusion machinery is the central player in the viral attachment process in almost every viral disease. Viruses have incorporated an array of efficient fusion proteins on their surfaces to bind efficiently to host cell receptors. They make use of the host proteolytic enzymes to rearrange their surface protein(s) into the form which facilitates their binding to host-cell membrane proteins and subsequently, fusion. This stage of viral entry is very critical and has many therapeutic implications. The current global pandemic of COVID-19 has sparked severe health crisis and economic shutdowns. SARS-CoV2, the etiological agent of the disease has led to millions of deaths and brought the scientific community together in an attempt to understand the mechanisms of SARS-CoV2 pathogenesis and mortality. Like other viral fusion machinery, CoV2 spike (S) glycoprotein- \u2018The Demogorgon\u2019 poses the same questions about viral-host cell fusion. The intermediate stages of S protein-mediated viral fusion are unclear owing to the lack of structural insights and concrete biochemical evidence. The mechanism of conformational transition is still unclear. S protein binding and fusion with host cell receptors, Eg., angiotensin-converting enzyme-2 (ACE2) is accompanied by cleavage of S1/S2 subunits. To track the key events of viral-host cell fusion, we have identified (in silico) that low pH-induced conformational change and ACE-2 binding events promote S1 dissociation. Deciphering key mechanistic insights of SARS-CoV2 fusion will further our understanding of other class-I fusion proteins","version":"1.1","doi":"10.1101/2020.12.21.410357","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423746","pub_date":"2020-12-21","title":"Immunological and pathological outcomes of SARS-CoV-2 challenge after formalin-inactivated vaccine immunisation of ferrets and rhesus macaques","abstract":"There is an urgent requirement for safe and effective vaccines to prevent novel coronavirus disease (COVID-19) caused by SARS-CoV-2. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferret and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine in ferrets or unvaccinated macaques. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen at seven days post infection in ferrets. This increased lung pathology was observed early in the infection (day 7) but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.","version":"1.1","doi":"10.1101/2020.12.21.423746","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.423630","pub_date":"2020-12-21","title":"Mutation Landscape of SARS COV2 in Africa","abstract":"COVID-19 disease has had a relatively less severe impact in Africa. To understand the role of SARS CoV2 mutations on COVID-19 disease in Africa, we analysed 282 complete nucleotide sequences from African isolates deposited in the NCBI Virus Database. Sequences were aligned against the prototype Wuhan sequence (GenBank accession: NC_045512.2) in BWA v. 0.7.17. SAM and BAM files were created, sorted and indexed in SAMtools v. 1.10 and marked for duplicates using Picard v. 2.23.4. Variants were called with mpileup in BCFtools v. 1.11. Phylograms were created using Mr. Bayes v 3.2.6. A total of 2,349 single nucleotide polymorphism (SNP) profiles across 294 sites were identified. Clades associated with severe disease in the United States, France, Italy, and Brazil had low frequencies in Africa (L84S=2.5%, L3606F=1.4%, L3606F/V378I/=0.35, G251V=2%). Sub Saharan Africa (SSA) accounted for only 3% of P323L and 4% of Q57H mutations in Africa. Comparatively low infections in SSA were attributed to the low frequency of the D614G clade in earlier samples (25% vs 67% global). Higher disease burden occurred in countries with higher D614G frequencies (Egypt=98%, Morocco=90%, Tunisia=52%, South Africa) with D614G as the first confirmed case. V367F, D364Y, V483A and G476S mutations associated with efficient ACE2 receptor binding and severe disease were not observed in Africa. 95% of all RdRp mutations were deaminations leading to CpG depletion and possible attenuation of virulence. More genomic and experimental studies are needed to increase our understanding of the temporal evolution of the virus in Africa, clarify our findings, and reveal hot spots that may undermine successful therapeutic and vaccine interventions.","version":"1.1","doi":"10.1101/2020.12.20.423630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.07.286906","pub_date":"2020-12-21","title":"The combination of Bromelain and Acetylcysteine (BromAc) synergistically inactivates SARS-CoV-2","abstract":"SARS-CoV-2 infection is the cause of a worldwide pandemic, currently with limited therapeutic options. Whilst vaccines are at the forefront of the therapeutic initiative, drug repurposing remains a promising approach for SARS-CoV-2 treatment. BromAc (Bromelain & Acetylcysteine) has synergistic action against glycoproteins by the synchronous breakage of glycosidic linkages and disulfide bonds. The spike protein of SARS-CoV-2, formed of glycoprotein and disulfide bridges for stabilization, represents an attractive target as it is essential for binding to the ACE2 receptor in host cells present in nasal mucosa. We sought to determine the effect of BromAc on the Spike and Envelope proteins and its potential to reduce infectivity in host cells. Recombinant Spike and Envelope proteins were treated by single agent and combination BromAc at 50 and 100 \u00b5g/20mg/mL and analyzed by electrophoresis. Ultraviolet analysis of disulfide bond reduction was performed for both Spike and Envelope proteins after treatment with Acetylcysteine. In vitro whole virus culture inactivation of pre-treated wild type and an S1/S2 Spike mutant SARS-CoV-2 with BromAc from 25 to 250 \u00b5g/20mg/mL was measured by cytopathic effect, cell lysis assay, and replication capacity by RT-PCR. Recombinant Spike and Envelope SARS-CoV-2 proteins were fragmented by BromAc at both 50 and 100 \u00b5g/20mg/mL whilst single agents had minimal effect. Spike and Envelope protein disulfide bonds were reduced by Acetylcysteine. In vitro whole virus culture of both wild type and Spike mutant SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. BromAc disintegrates SARS-CoV-2 Spike and Envelope proteins. In vitro tests on whole virus support this finding with inactivation of its replication capacity most strongly at 100 and 250 \u00b5g/20mg/mL BromAc, even in Spike mutant virus. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent. There is currently no suitable therapeutic treatment for early SARS-CoV-2 aimed to prevent disease progression. BromAc is under clinical development by the authors for mucinous cancers due to its ability to alter complex glycoproteins structure. The potential of BromAc on SARS-CoV-2 Spike and Envelope glycoproteins stabilized by disulfide bonds was examined and found to disintegrate recombinant Spike and Envelope proteins whilst reducing disulfide stabilizer bridges. BromAc also showed an inhibitory effect on wild-type and Spike mutant SARS-CoV-2 by inactivation of its replication capacity in vitro. Hence, BromAc may be an effective therapeutic agent for early SARS-CoV-2 infection, despite mutations, and even have potential as a prophylactic in people at high risk of infection.","version":"1.2","doi":"10.1101/2020.09.07.286906","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.408575","pub_date":"2020-12-21","title":"In Vitro Analysis of the Anti-viral Potential of nasal spray constituents against SARS-CoV-2","abstract":"Viral pandemics have taken a significant toll on humanity and the world now is contending with the SARS-CoV-2 epidemic. Readily available economical preventive measures should be immediately explored. Xylitol has been reported to reduce the severity of viral infections as well as the severity of pneumonia, and increase the survivability of animal subjects. Since pneumonia and acute respiratory distress syndrome are potentially fatal complications of COVID-19, the present study tested the in vitro effectiveness of xylitol against SARS-CoV-2. Virus titers and LRV of SARS-CoV-2, were incubated with a single concentration of nasal spray. Toxicity was observed in the top dilution (1/10). Virus was seen below that dilution so it did not affect calculations of virus titer or LRV. After a 25-minute contact time, the nasal spray (11% Pure Xylitol, 0.85%NaCL (Saline), and 0.20% grapefruit seed extract) reduced virus from 4.2 to 1.7 log10 CCID50 per 0.1 mL, a statistically significant reduction (P<0.001) of 2.5 log10 CCID50. STEM Images obtained at the BIoCryo Laboratory revealed virus contained on the cell wall but none intra-cellular, possibly due to D-xylose (xylitol) production of glycoaminoglycans decoy targets. Xylitol and grapefruit seed extract are not exotic nor expensive rare high technology answers to viral epidemics. The potential in saving lives and the economies of the world by using X-GSE combination therapy should inspire large clinical trials, especially in those nations whereas the healthcare system would be dangerously compromised by the adoption of less effective and significantly more financially demanding therapies. Because there are no risk factors in using the X/GSE combination therapy, and the nasal spray is over the counter available without a prescription, and the spray allows for comfortable long term mask-wearing, adoption of this preventive anti-viral therapy should be encouraged.","version":"1.3","doi":"10.1101/2020.12.02.408575","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.31.362848","pub_date":"2020-12-21","title":"Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations","abstract":"Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.","version":"1.2","doi":"10.1101/2020.10.31.362848","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.423708","pub_date":"2020-12-21","title":"Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma","abstract":"Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq) to the spike ectodomain (S-ECD) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 \u00c5). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.20.423708","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.422693","pub_date":"2020-12-21","title":"\u201cA recombinant protein SARS-CoV-2 candidate vaccine elicits high-titer neutralizing antibodies in macaques\u201d","abstract":"Vaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. We assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain (NTD) or an extended C-terminal domain containing the receptor-binding domain (RBD) and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. A robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the NTD alone lacked this activity. Crucially, sera from animals immunized with the RBD but not the NTD had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. These data support the utility of spike subunit-based antigens as a vaccine for use in humans.","version":"1.1","doi":"10.1101/2020.12.20.422693","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.422820","pub_date":"2020-12-21","title":"In vitro measurements of protein\u2013protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum","abstract":"The humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2\u2013spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the binding energies of the binary interactions between the ACE2 receptor and the spike protein, and the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential areas of application ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development.","version":"1.1","doi":"10.1101/2020.12.20.422820","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.158121","pub_date":"2020-12-21","title":"The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA","abstract":"The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.","version":"1.2","doi":"10.1101/2020.06.17.158121","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423787","pub_date":"2020-12-21","title":"Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody","abstract":"The global battle against the Covid-19 pandemic relies strongly on the human defence of antibody, which is assumed to bind the antigen\u2019s Receptor Binding Domain with its Hypervariable Region. Due to the similarity to other viruses such as SARS, however, our understanding of the antibody-virus interaction has been largely limited to the genomic sequencing, which poses serious challenges to the containment, vaccine exploration and rapid serum testing. Based on the physical/chemical nature of the interaction, infrared spectroscopy was employed to reveal the binding disparity, when unusual temperature dependence was discovered from the 1550cm-1 absorption band, attributed to the hydrogen bonds by carboxyl/amino groups, binding the SARS-CoV-2 spike protein and closely resembled SARS-CoV-2 or SARS-CoV-1 antibodies. The infrared absorption intensity, associated with the number of hydrogen bonds, was found to increase sharply between 27\u00b0C and 31\u00b0C, with the relative absorbance matches at 37\u00b0C the hydrogen bonding numbers of the two antibody types (19 vs 12). Meanwhile the ratio of bonds at 27\u00b0C, calculated by thermodynamic exponentials rather than by the layman\u2019s guess, produces at least 5% inaccuracy. As a result, the specificity of the SARS-CoV-2 antibody will be more conclusive beyond 31\u00b0C, instead of at the usual room temperature of 20\u00b0C - 25\u00b0C, when the vaccine research and antibody diagnosis would likely be undermined. Beyond genomic sequencing, the temperature dependence, as well as the bond number match at 37\u00b0C between relative absorbance and the hydrogen bonding numbers of the two antibody types, are not only of clinical significance in particular, but also of a sample for the physical/chemical understanding of the vaccine-antibody interactions in general.","version":"1.1","doi":"10.1101/2020.12.21.423787","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.18.344622","pub_date":"2020-12-21","title":"An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants","abstract":"The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain (RBD) followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor. Variant N501Y in the RBD, which has emerged in a rapidly spreading lineage (B.1.1.7) in England, enhances affinity for wild type ACE2 20-fold but remains tightly bound to engineered sACE22.v2.4. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.","version":"1.2","doi":"10.1101/2020.10.18.344622","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.265645","pub_date":"2020-12-21","title":"SARS-CoV-2 3CLpro whole human proteome cleavage prediction and enrichment/depletion analysis","abstract":"A novel coronavirus (SARS-CoV-2) has devastated the globe as a pandemic that has killed more than 1,600,000 people. Widespread vaccination is still uncertain, so many scientific efforts have been directed toward discovering antiviral treatments. Many drugs are being investigated to inhibit the coronavirus main protease, 3CLpro, from cleaving its viral polyprotein, but few publications have addressed this protease\u2019s interactions with the host proteome or their probable contribution to virulence. Too few host protein cleavages have been experimentally verified to fully understand 3CLpro\u2019s global effects on relevant cellular pathways and tissues. Here, I set out to determine this protease\u2019s targets and corresponding potential drug targets. Using a neural network trained on cleavages from 388 coronavirus proteomes with a Matthews correlation coefficient of 0.983, I predict that a large proportion of the human proteome is vulnerable to 3CLpro, with 4,460 out of approximately 20,000 human proteins containing at least one putative cleavage site. These cleavages are nonrandomly distributed and are enriched in the epithelium along the respiratory tract, brain, testis, plasma, and immune tissues and depleted in olfactory and gustatory receptors despite the prevalence of anosmia and ageusia in COVID-19 patients. Affected cellular pathways include cytoskeleton/motor/cell adhesion proteins, nuclear condensation and other epigenetics, host transcription and RNAi, ribosomal stoichiometry and nascent-chain detection and degradation, coagulation, pattern recognition receptors, growth factors, lipoproteins, redox, ubiquitination, and apoptosis. This whole proteome cleavage prediction demonstrates the importance of 3CLpro in expected and nontrivial pathways affecting virulence, lead me to propose more than a dozen potential therapeutic targets against coronaviruses, and should therefore be applied to all viral proteases and subsequently experimentally verified.","version":"1.2","doi":"10.1101/2020.08.24.265645","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.423607","pub_date":"2020-12-21","title":"In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2","abstract":"Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.","version":"1.1","doi":"10.1101/2020.12.20.423607","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.20.347641","pub_date":"2020-12-21","title":"Viral surface geometry shapes influenza and coronavirus spike evolution through antibody pressure","abstract":"The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the glycoprotein (spike). However, not all antigenic sites are targeted equally by antibodies, leading to complex immunodominance patterns. We used 3D computational models to estimate antibody pressure on the seasonal influenza H1N1 and SARS spikes. Analyzing publically available sequences, we show that antibody pressure, through the geometrical organization of spikes on the viral surface, shaped their mutability. Studying the mutability patterns of SARS-CoV-2 and the 2009 H1N1 pandemic spikes, we find that they are not predominantly shaped by antibody pressure. However, for SARS-CoV-2, we find that over time, it acquired mutations at antibody-accessible positions, which could indicate possible escape as define by our model. We offer a geometry-based approach to predict and rank the probability of surface resides of SARS-CoV-2 spike to acquire antibody escaping mutations.","version":"1.2","doi":"10.1101/2020.10.20.347641","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.21.423761","pub_date":"2020-12-21","title":"Predicting COVID-19 cases with unknown homogeneous or heterogeneous resistance to infectivity","abstract":"This article constructs a restricted infection rate inverse binomial-based approach to predict COVID-19 cases after a family gathering. The traditional inverse binomial (IB) model is unqualified to match the reality of COVID-19, because the data contradicts the model\u2019s requirement that variance should be greater than expected value. A refined version of the IB model is a necessity to predict COVID-19 cases after family gatherings. Our refined version of an IB model is more appropriate and versatile, as it accommodates all potential data scenarios: equal, lesser, or greater variance than expected value. Application of the approach is based on a restricted infectivity rate and methodology on Fan et al.\u2019s COVID-19 data, which exhibits two clusters of infectivity. Cluster 1 has a smaller number of primary cases and exhibits larger variance than the expected cases with a negative correlation of 28%, implying that the number of secondary cases is lesser when the number of primary cases increases and vice versa. The traditional inverse binomial (IB) model is appropriate for Cluster 1. The probability of contracting COVID-19 is estimated to be 0.13 among the primary, but is 0.75 among the secondary in Cluster 1, with a wider gap. Conversely, Cluster 2, exhibits smaller variance than the expected cases with a correlation of 79%, implying the number of primary and secondary cases increase or decrease together. Cluster 2 disqualifies the traditional IB model and demands its refined version. Probability of contracting COVID-19 is estimated to be 0.74 among the primary, but is 0.72 among the secondary in Cluster 2, with a narrower gap. The model\u2019s ability to estimate the community\u2019s health system memory for future policies to be developed is an asset of this approach. The current hazard level to be infected with COVID-19 among the primary and secondary groups are estimable and interpretable. Current statistical models are not able to accurately predict disease infection spread in the COVID-19 pandemic. We have applied a widely-used inverse binomial method to predict rates of infection after small gatherings, going from primary (original) cases to secondary (later) cases after family gatherings or social events, using the data from the Wuhan and Gansu provinces in China, where the virus first spread. The advantages of the proposed approach include that the model\u2019s ability to estimate the community\u2019s health system memory for future policies to be developed, as such policies might reduce COVID\u2019s spread if not its control. In our approach, as demonstrated, the current hazard level of becoming infected with COVID-19 and the odds of contracting COVID-19 among the primary in comparison to the secondary groups are estimable and interpretable. We hope the proposed approach will be used in future epidemics.","version":"1.1","doi":"10.1101/2020.12.21.423761","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423104","pub_date":"2020-12-20","title":"Glycyrrhizin effectively neutralizes SARS-CoV-2 in vitro by inhibiting the viral main protease","abstract":"The newly emerged coronavirus, which was designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 disease. High effective and well-tolerated medication for hospitalized and non-hospitalized patients is urgently needed. Traditional herbal medicine substances were discussed as promising candidates for the complementary treatment of viral diseases and recently suggested for the treatment of COVID-19. In the present study, we investigated aqueous licorice root extract for its neutralizing activity against SARS-CoV-2 in vitro, identified the active compound glycyrrhizin and uncovered the respective mechanism of viral neutralization. We demonstrated that glycyrrhizin, the primary active ingredient of the licorice root, potently neutralizes SARS-CoV-2 by inhibiting the viral main protease. Our experiments highlight glycyrrhizin as a potential antiviral compound that should be further investigated for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.12.18.423104","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.19.423584","pub_date":"2020-12-20","title":"Identification of NPC1 as a novel SARS-CoV-2 intracellular target","abstract":"Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol trafficking, which is crucial in the Ebola virus (EBOV) cycle. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cell by binding of the viral spike (S) protein to the ACE2 receptor. This requires S-protein processing either by the surface transmembrane serine protease TMPRSS2 for plasma membrane fusion or cathepsin L for endosomal entry. Additional host factors are required for viral fusion at endosomes. Here, we report a novel interaction of the SARS-CoV-2 nucleoprotein (N) with the cholesterol transporter NPC1. Moreover, small molecules interfering with NPC1 that inhibit EBOV entry, also inhibited human coronavirus. Our findings suggest an important role for NPC1 in SARS-CoV-2 infection, a common strategy shared with EBOV, and a potential therapeutic target to fight against COVID-19.","version":"1.1","doi":"10.1101/2020.12.19.423584","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423507","pub_date":"2020-12-20","title":"SARS-CoV-2 escapes CD8 T cell surveillance via mutations in MHC-I restricted epitopes","abstract":"CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control, though direct evidence has been lacking so far. Here, we identified non-synonymous mutations in MHC-I restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV- 2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding, which was associated with a loss of recognition and functional responses by CD8+ T cells isolated from HLA-matched COVID-19 patients. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through escape mutations in MHCI-restricted viral epitopes. This provides evolutionary evidence for CD8+ T cell immunity controlling SARS-CoV-2 with consequences for COVID-19 vaccine design.","version":"1.1","doi":"10.1101/2020.12.18.423507","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423552","pub_date":"2020-12-20","title":"Sterilizing immunity against SARS-CoV-2 in hamsters conferred by a novel recombinant subunit vaccine","abstract":"A safe and effective SARS-CoV-2 vaccine is essential to avert the on-going COVID-19 pandemic. Here, we developed a subunit vaccine, which is comprised of CHO-expressed spike ectodomain protein (StriFK) and nitrogen bisphosphonates-modified zinc-aluminum hybrid adjuvant (FH002C). This vaccine candidate rapidly elicited the robust humoral response, Th1/Th2 balanced helper CD4 T cell and CD8 T cell immune response in animal models. In mice, hamsters, and non-human primates, 2-shot and 3-shot immunization of StriFK-FH002C generated 28- to 38-fold and 47- to 269-fold higher neutralizing antibody titers than the human COVID-19 convalescent plasmas, respectively. More importantly, the StriFK-FH002C immunization conferred sterilizing immunity to prevent SARS-CoV-2 infection and transmission, which also protected animals from virus-induced weight loss, COVID-19-like symptoms, and pneumonia in hamsters. Vaccine-induced neutralizing and cell-based receptor-blocking antibody titers correlated well with protective efficacy in hamsters, suggesting vaccine-elicited protection is immune-associated. The StriFK-FH002C provided a promising SARS-CoV-2 vaccine candidate for further clinical evaluation.","version":"1.1","doi":"10.1101/2020.12.18.423552","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.19.423592","pub_date":"2020-12-20","title":"The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2","abstract":"The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein (S). The presence of a polybasic cleavage site in SARS-CoV-2 S at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the S precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons, and more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2 and the degree of this restriction is governed by route of viral entry. Removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH- and cathepsin-dependent in late endosomes where, like SARS-CoV-1 S, it is more sensitive to IFITM2 restriction. Furthermore, we find that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 S may reduce viral replication through the activity of type I IFNs. The furin cleavage site in the S protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2, and that the sensitivity is exacerbated by deletion of the furin cleavage site which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus may represent a potential therapeutic target for COVID-19 treatment development.","version":"1.1","doi":"10.1101/2020.12.19.423592","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.19.423537","pub_date":"2020-12-20","title":"An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity","abstract":"The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 \u03bcM and EC50 of 1.27 \u03bcM, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the \u03b1-methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.","version":"1.1","doi":"10.1101/2020.12.19.423537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.20.414748","pub_date":"2020-12-20","title":"Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions","abstract":"The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.","version":"1.1","doi":"10.1101/2020.12.20.414748","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423358","pub_date":"2020-12-18","title":"An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies","abstract":"SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.","version":"1.1","doi":"10.1101/2020.12.18.423358","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.15.383323","pub_date":"2020-12-18","title":"Immunological memory to SARS-CoV-2 assessed for up to eight months after infection","abstract":"Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at \u2265 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.","version":"1.2","doi":"10.1101/2020.11.15.383323","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.062315","pub_date":"2020-12-18","title":"Disparate temperature-dependent virus \u2013 host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium","abstract":"Since its emergence in December 2019, SARS-CoV-2 has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human respiratory tract have been shown to affect the replication kinetics of several viruses, as well as host immune response dynamics, we investigated the impact of temperatures during SARS-CoV-2 and SARS-CoV infection in the human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated more efficiently at temperatures encountered in the upper respiratory tract, and displayed higher sensitivity to type I and type III IFNs. Time-resolved transcriptome analysis highlighted a temperature-dependent and virus-specific induction of the IFN-mediated antiviral response. These data reflect clinical features of SARS-CoV-2 and SARS-CoV, as well as their associated transmission efficiencies, and provide crucial insight on pivotal virus - host interaction dynamics.","version":"1.2","doi":"10.1101/2020.04.27.062315","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423415","pub_date":"2020-12-18","title":"A recombinant fragment of Human surfactant protein D binds Spike protein and inhibits infectivity and replication of SARS-CoV-2 in clinical samples","abstract":"COVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known. This study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. rfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp gene of the virus using qPCR. In-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5\u03bcM rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 \u03bcM). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5\u03bcM rfhSP-D. These results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and a significant reduction in SARS-CoV-2 infection and replication in-vitro.","version":"1.1","doi":"10.1101/2020.12.18.423415","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423427","pub_date":"2020-12-18","title":"SARS-CoV-2 spike protein interacts with and activates TLR4","abstract":"The onset of sepsis is an important feature of COVID19 and a main cause of death. It is unknown how SARS-CoV-2 infection results in viral sepsis in human. We recently found that SARS-CoV-2 provoked an anti-bacterial like response and activation of TLR4 pathway at the very early stage of infection in animal models. This abnormal immune response led to emergency granulopoiesis and sepsis. However, the original trigger of TLR4 signaling by SARS-CoV-2 is unknown. We here identified that the trimeric spike protein of SARS-CoV-2 could bind to TLR4 directly and robustly activate downstream signaling in monocytes and neutrophils. Moreover, specific TLR4 or NFKB inhibitor, or knockout of MyD88 could significantly block IL-1B induction by spike protein. We thus reveal that spike protein of SARS-CoV-2 functions as a potent stimulus causing TLR4 activation and sepsis related abnormal responses.","version":"1.1","doi":"10.1101/2020.12.18.423427","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422634","pub_date":"2020-12-18","title":"A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics","abstract":"The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unfortunately, unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ~30,000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ~200 virtual screenings of compound libraries on selected protein structures, we redefine the protein\u2019s druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.","version":"1.2","doi":"10.1101/2020.12.14.422634","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.201905","pub_date":"2020-12-18","title":"Dynamic tracking of variant frequencies depicts the evolution of mutation sites amongst SARS-CoV-2 genomes from India","abstract":"With the exponential spread of the COVID-19 pandemic across the world within the twelve months, SARS-CoV-2 strains are continuously trying to adapt themselves in the host environment by random mutations. While doing so, some variants with evolutionary advantages such as better human to human transmissibility potential might get naturally selected. This short communication demonstrates how the mutation frequency patterns are evolving in 2,457 SAR-CoV-2 strains isolated from COVID-19 patients across diverse Indian states. We have identified 19 such variants showing contrasting mutational probabilities in the span of seven months. Out of these, 14 variants are showing increasing mutational probabilities suggesting their propagation with time due to their unexplored evolutionary advantages. Whereas mutational probabilities of five variants have significantly decreased in June onwards as compared to March/April, suggesting their termination with time. Further in-depth investigation of these identified variants will provide valuable knowledge about the evolution, infection strategies, transmission rates, and epidemiology of SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.07.14.201905","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.074039","pub_date":"2020-12-18","title":"The heterogeneous landscape and early evolution of pathogen-associated CpG dinucleotides in SARS-CoV-2","abstract":"COVID-19 can lead to acute respiratory syndrome, which can be due to dysregulated immune signaling. We analyze the distribution of CpG dinucleotides, a pathogen-associated molecular pattern, in the SARS-CoV-2 genome. We find that the CpG content, which we characterize by a force parameter that accounts for statistical constraints acting on the genome at the nucleotidic and amino-acid levels, is, on average, low compared to other pathogenic betacoronaviruses. However, the CpG force widely fluctuates along the genome, with a particularly low value, comparable to the circulating seasonal HKU1, in the spike coding region and a greater value, comparable to SARS and MERS, in the highly expressed nucleocapside coding region (N ORF), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3\u2019UTRs of all subgenomic RNA. This dual nature of CpG content could confer to SARS-CoV-2 the ability to avoid triggering pattern recognition receptors upon entry, while eliciting a stronger response during replication. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic, finding a signature of CpG loss in regions with a greater CpG force. Sequence motifs preceding the CpG-loss-associated loci in the N ORF match recently identified binding patterns of the Zinc finger Anti-viral Protein. Using a model of the viral gene evolution under human host pressure, we find that synonymous mutations seem driven in the SARS-CoV-2 genome, and particularly in the N ORF, by the viral codon bias, the transition-transversion bias and the pressure to lower CpG content.","version":"1.4","doi":"10.1101/2020.05.06.074039","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.041962","pub_date":"2020-12-18","title":"De novo 3D models of SARS-CoV-2 RNA elements and small-molecule-binding RNAs to aid drug discovery","abstract":"The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta\u2019s FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5\u2019 UTR; the reverse complement of the 5\u2019 UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3\u2019 UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m, and 3\u2019 UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets (\u2018FARFAR2-SARS-CoV-2\u2019, https://github.com/DasLab/FARFAR2-SARS-CoV-2; and \u2018FARFAR2-Apo-Riboswitch\u2019, at https://github.com/DasLab/FARFAR2-Apo-Riboswitch\u2019) include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.","version":"1.2","doi":"10.1101/2020.04.14.041962","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423418","pub_date":"2020-12-18","title":"Human Surfactant Protein D Binds S1 and Receptor Binding Domain of Spike protein and acts as an entry inhibitor of SARS-CoV-2 Pseudotyped viral particles in vitro","abstract":"Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows immune surveillance role against pulmonary pathogens. Higher levels of serum SP-D have been reported in patients with severe acute respiratory syndrome coronavirus-1 (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo models. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing Angiotensin Converting Enzyme 2. The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following rfhSP-D treatment (10 \u03bcg/ml). The results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merits pre-clinical studies in murine models.","version":"1.1","doi":"10.1101/2020.12.18.423418","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.16.341883","pub_date":"2020-12-18","title":"Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses","abstract":"Environmental conditions affect virus inactivation rate and transmission potential. Understanding those effects is critical for anticipating and mitigating epidemic spread. Ambient temperature and humidity strongly affect the inactivation rate of enveloped viruses, but a mechanistic, quantitative theory of those effects has been elusive. We measure the stability of the enveloped respiratory virus SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities; median estimated virus half-life is over 24 hours at 10 \u00b0C and 40 % RH, but approximately 1.5 hours at 27 \u00b0C and 65 % RH. Our mechanistic model uses simple chemistry to explain the increase in virus inactivation rate with increased temperature and the U-shaped dependence of inactivation rate on relative humidity. The model accurately predicts quantitative measurements from existing studies of five different human coronaviruses (including SARS-CoV-2), suggesting that shared mechanisms may determine environmental stability for many enveloped viruses. Our results indicate scenarios of particular transmission risk, point to pandemic mitigation strategies, and open new frontiers in the mechanistic study of virus transmission.","version":"1.3","doi":"10.1101/2020.10.16.341883","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423420","pub_date":"2020-12-18","title":"A new concept on anti-SARS-CoV-2 vaccines: strong CD8+ T-cell immune response in both spleen and lung induced in mice by endogenously engineered extracellular vesicles","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is spreading rapidly in the absence of validated tools to control the growing epidemic besides social distancing and masks. Many efforts are ongoing for the development of vaccines against SARS-CoV-2 since there is an imminent need to develop effective interventions for controlling and preventing SARS-CoV-2 spread. Essentially all vaccines in most advanced phases are based on the induction of antibody response against either whole or part of spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). We exploited this technology with the aim to identify a clinical candidate defined as DNA vectors expressing SARS-CoV-2 antigens inducing a robust CD8+ T-cell response. This is a new vaccination approach employing a DNA expression vector encoding a biologically inactive HIV-1 Nef protein (Nefmut) showing an unusually high efficiency of incorporation into EVs even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells are internalized by antigen-presenting cells leading to cross-presentation of the associated antigens thereby priming of antigen-specific CD8+ T-cells. To apply this technology to a design of anti-SARS-CoV-2 vaccine, we recovered DNA vectors expressing the products of fusion between Nefmut and four viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. All fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity was generated. Most important, high levels of virus-specific CD8+ T cells were found in bronchoalveolar lavages of immunized mice. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lung. EVs engineered with SARS-CoV-2 antigens proved immunogenic also in the human system through cross-priming assays carried out with ex vivo human cells. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed as anti-SARS-CoV-2 vaccine candidates.","version":"1.1","doi":"10.1101/2020.12.18.423420","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423467","pub_date":"2020-12-18","title":"assayM: a web application to monitor mutations in COVID-19 diagnostic assays","abstract":"Reverse Transcriptase \u2013 Polymerase Chain Reaction (RT-PCR) is the gold standard as diagnostic assays for the detection of COVID-19 and the specificity and sensitivity of these assays depend on the complementarity of the RT-PCR primers to the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the virus mutates over time during replication cycles, there is an urgent need to continuously monitor the virus genome for appearances of mutations and mismatches in the PCR primers used in these assays. Here we present assayM, a web application to explore and monitor mutations introduced in the primer and probe sequences published by the World Health Organisation (WHO) or in any custom-designed assay primers for SARS-CoV-2 detection assays in globally available SARS-CoV-2 genome datasets. assayM is available on https://grafnet.kaust.edu.sa/assayM as a web application and also as an open-source R shiny application, downloadable from https://github.com/raeece/assayM arnab.pain@kaust.edu.sa","version":"1.1","doi":"10.1101/2020.12.18.423467","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423363","pub_date":"2020-12-18","title":"Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19","abstract":"Our understanding of protective vs. pathologic immune responses to SARS-CoV-2, the virus that causes Coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses reveal widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, with the most profound disturbances including a prominent neutrophil hyperactivation signature and monocytes with anti-inflammatory features. We further demonstrate that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention. Single-cell profiling demonstrates multifarious dysregulation of innate immune phenotype associated with COVID-19 severity.","version":"1.1","doi":"10.1101/2020.12.18.423363","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.18.423439","pub_date":"2020-12-18","title":"SNPnexus COVID: Facilitating the analysis of COVID-19 host genetics","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demanded an unprecedented scientific response, with researchers collaborating on a global scale to better understand how host genetics can influence susceptibility to coronavirus infection and the severity of COVID-19 symptoms. The number of projects directed towards sequencing patients\u2019 genomes has increased rapidly during this time with the rate of data generation outpacing the resources available for analysis and biological interpretation of these datasets. SNPnexus COVID is a cutting-edge web-based analytical platform that allows researchers to analyse and interpret the functional implications of genetic variants in COVID-19 patient genomes and to prioritise those that demonstrate clinical utility for the prevention, management and/or treatment of COVID-19. Our resource links to diverse multifactorial datasets and information resources that would require substantial time and computational power to otherwise mine independently. This streamlines biological data interpretation and allows researchers to better understand the multidimensional characteristics of their data. Importantly, SNPnexus COVID is powered by the SNPnexus software and follows its intuitive infrastructure, which precludes the need for programmatic experience in its users. SNPnexus COVID is freely available at https://www.snp-nexus.org/v4/covid/","version":"1.1","doi":"10.1101/2020.12.18.423439","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.17.423313","pub_date":"2020-12-18","title":"A human coronavirus evolves antigenically to escape antibody immunity","abstract":"There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these \u201cfuture\u201d viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8\u201317 years later. The decreased neutralization of \u201cfuture\u201d viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2020.12.17.423313","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.17.423376","pub_date":"2020-12-18","title":"Temporal kinetics of RNAemia and associated systemic cytokines in hospitalized COVID-19 patients","abstract":"COVID-19 is associated to a wide range of extra-respiratory complications, of which the pathogenesis is currently not fully understood. In this study we report the temporal kinetics of viral RNA and inflammatory cytokines and chemokines in serum during the course of COVID-19. We show that a RNAemia occurs more frequently and lasts longer in patients that develop critical disease compared to patients that develop moderate or severe disease. Furthermore we show that concentrations of IL-10 and MCP-1\u2014but not IL-6\u2014are associated with viral load in serum. However, higher levels of IL-6 were associated with the development of critical disease. The direct association of inflammatory cytokines with viral load or disease severity highlights the complexity of systemic inflammatory response and the role of systemic viral spread.","version":"1.1","doi":"10.1101/2020.12.17.423376","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.423178","pub_date":"2020-12-17","title":"Genomic and phylogenetic analyses of SARS-CoV-2 strains isolated in the city of Gwangju, South Korea","abstract":"Since the first identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in late December 2019, the coronavirus disease 2019 (COVID-19) has spread fast around the world. RNA viruses, including SARS-CoV-2, have higher gene mutations than DNA viruses during virus replication. Variations in SARS-CoV-2 genome could contribute to efficiency of viral spread and severity of COVID-19. In this study, we analyzed the locations of genomic mutations to investigate the genetic diversity among isolates of SARS-CoV-2 in Gwangju. We detected non-synonymous and frameshift mutations in various parts of SARS-CoV-2 genome. The phylogenetic analysis for whole genome showed that SARS-CoV-2 genomes in Gwangju isolates are clustered within clade V and G. Our findings not only provide a glimpse into changes of prevalent virus clades in Gwangju, South Korea, but also support genomic surveillance of SARS-CoV-2 to aid in the development of efficient therapeutic antibodies and vaccines against COVID-19.","version":"1.1","doi":"10.1101/2020.12.16.423178","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.17.423130","pub_date":"2020-12-17","title":"Suppression of miR-155 attenuates lung cytokine storm induced by SARS-CoV-2 infection in human ACE2-transgenic mice","abstract":"Coronavirus disease 2019 (COVID-19) is a recent global pandemic. It is a deadly human viral disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with a high rate of infection, morbidity and mortality. Therefore, there is a great urgency to develop new therapies to control, treat and prevent this disease. Endogenous microRNAs (miRNAs, miRs) of the viral host are key molecules in preventing viral entry and replication, and building an antiviral cellular defense. Here, we have analyzed the role of miR-155, one of the most powerful drivers of host antiviral responses including immune and inflammatory responses, in the pathogenicity of SARS-CoV-2 infection. Subsequently, we have analyzed the potency of anti-miR-155 therapy in a COVID-19 mouse model (mice transgenic for human angiotensin I-converting enzyme 2 receptor (tg-mice hACE2)). We report for the first time that miR-155 expression is elevated in COVID-19 patients. Further, our data indicate that the viral load as well as miR-155 levels are higher in male relative to female patients. Moreover, we find that the delivery of anti-miR-155 to SARS-CoV-2-infected tg-mice hACE2 effectively suppresses miR-155 expression, and leads to improved survival and clinical scores. Importantly, anti-miR-155-treated tg-mice hACE2 infected with SARS-CoV-2 not only exhibit reduced levels of pro-inflammatory cytokines, but also have increased anti-viral and anti-inflammatory cytokine responses in the lungs. Thus, our study suggests anti-miR-155 as a novel therapy for mitigating the lung cytokine storm induced by SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.17.423130","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.423122","pub_date":"2020-12-17","title":"Immune-Based Prediction of COVID-19 Severity and Chronicity Decoded Using Machine Learning","abstract":"Individuals with systemic symptoms long after COVID-19 has cleared represent approximately ~10% of all COVID-19 infected individuals. Here we present a bioinformatics approach to predict and model the phases of COVID so that effective treatment strategies can be devised and monitored. We investigated 144 individuals including normal individuals and patients spanning the COVID-19 disease continuum. We collected plasma and isolated PBMCs from 29 normal individuals, 26 individuals with mild-moderate COVID-19, 25 individuals with severe COVID-19, and 64 individuals with Chronic COVID-19 symptoms. Immune subset profiling and a 14-plex cytokine panel were run on all patients. Data was analyzed using machine learning methods to predict and distinguish the groups from each other.Using a multi-class deep neural network classifier to better fit our prediction model, we recapitulated a 100% precision, 100% recall and F1 score of 1 on the test set. Moreover, a first score specific for the chronic COVID-19 patients was defined as S1 = (IFN-\u03b3 + IL-2)/ CCL4-MIP-1\u03b2. Second, a score specific for the severe COVID-19 patients was defined as S2 = (10*IL-10 + IL-6) - (IL-2 + IL-8). Severe cases are characterized by excessive inflammation and dysregulated T cell activation, recruitment, and counteracting activities. While chronic patients are characterized by a profile able to induce the activation of effector T cells with pro-inflammatory properties and the capacity of generating an effective immune response to eliminate the virus but without the proper recruitment signals to attract activated T cells. Immunologic Modeling of Severity and Chronicity of COVID-19","version":"1.1","doi":"10.1101/2020.12.16.423122","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.423166","pub_date":"2020-12-17","title":"Molecular diversity analysis of the spike glycoprotein (S) gene from Hong Kong - China","abstract":"In this work, 37 haplotypes of spike glycoprotein of SARS-CoV-2 from Hong Kong, China, were used. All sequences were publicly available on the Platform of the National Center for Biotechnology Information (NCBI) and were analyzed for their Molecular Variance (AMOVA), haplotypic diversity, mismatch, demographic and spatial expansion, molecular diversity and time of evolutionary divergence. The results suggested that there was a low diversity among haplotypes, with very low numbers of transitions, transversions, indels-type mutations and with total absence of population expansion perceived in the neutrality tests. The estimators used in this study supported the uniformity among all the results found and confirm the evolutionary conservation of the gene, as well as its protein product, a fact that stimulates the use of therapies based on neutralizing antibodies, such as vaccines based on protein S.","version":"1.1","doi":"10.1101/2020.12.16.423166","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.423071","pub_date":"2020-12-16","title":"One Year of SARS-CoV-2: How Much Has the Virus Changed?","abstract":"SARS-CoV-2 coronavirus has caused a world-wide crisis with profound effects on both healthcare and the economy. In order to combat the COVID-19 pandemic, research groups have shared viral genome sequence data through the GISAID initiative. We collected and computationally profiled \u223c223,000 full SARS-CoV-2 proteome sequences from GISAID over one year for emergent nonsynonymous mutations. Our analysis shows that SARS-CoV-2 proteins are mutating at substantially different rates, with most viral proteins exhibiting little mutational variability. As anticipated, our calculations capture previously reported mutations occurred in the first period of the pandemic, such as D614G (Spike), P323L (NSP12), and R203K/G204R (Nucleocapsid), but also identify recent mutations like A222V and L18F (Spike) and A220V (Nucleocapsid). Our comprehensive temporal and geographical analyses show two periods with different mutations in the SARS-CoV-2 proteome: December 2019 to June 2020 and July to November 2020. Some mutation rates differ also by geography; the main mutations in the second period occurred in Europe. Furthermore, our structure-based molecular analysis provides an exhaustive assessment of mutations in the context of 3D protein structure. Emerging sequence-to-structure data is beginning to reveal the site-specific mutational tolerance of SARS-CoV2 proteins as the virus continues to spread around the globe.","version":"1.1","doi":"10.1101/2020.12.16.423071","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.15.422939","pub_date":"2020-12-16","title":"Choice of assemblers has a critical impact on de novo assembly of SARS-CoV-2 genome and characterizing variants","abstract":"Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic following its initial emergence in China. Using next-generation sequencing technologies, a large number of SARS-CoV-2 genomes are being sequenced at an unprecedented rate and being deposited in public repositories. For the de novo assembly of the SARS-CoV-2 genomes, a myriad of assemblers is being used, although their impact on the assembly quality has not been characterized for this virus. In this study, we aim to understand the variabilities on assembly qualities due to the choice of the assemblers. We performed 6,648 de novo assemblies of 416 SARS-CoV-2 samples using 8 different assemblers with different k-mers. We used Illumina paired-end sequencing reads and compared the genome assembly quality to that of different assemblers. We showed the choice of assemblers plays a significant role in reconstructing the SARS-CoV-2 genome. Two metagenomic assemblers e.g. MEGAHIT and metaSPAdes performed better compared to others in most of the assembly quality metrics including, recovery of a larger fraction of the genome, constructing larger contigs and higher N50, NA50 values etc. We showed that at least 09% (259/2,873) of the variants present in the assemblies between MEGAHIT and metaSPAdes are unique to the assembly methods. Our analyses indicate the critical role of assembly methods for assembling SARS-CoV-2 genome using short reads and their impact on variant characterization. This study could help guide future studies to determine which assembler is best suited for the de novo assembly of virus genomes.","version":"1.1","doi":"10.1101/2020.12.15.422939","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.251207","pub_date":"2020-12-16","title":"The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro","abstract":"While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3\u03b1/\u03b2 and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.","version":"1.4","doi":"10.1101/2020.08.14.251207","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.422677","pub_date":"2020-12-16","title":"Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen","abstract":"Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 \u03bcM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 \u00c5, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity. Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray.","version":"1.1","doi":"10.1101/2020.12.16.422677","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.16.422529","pub_date":"2020-12-16","title":"Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway","abstract":"Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.","version":"1.1","doi":"10.1101/2020.12.16.422529","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.15.412809","pub_date":"2020-12-16","title":"Cyclic gallium-68 labeled peptides for specific detection of human angiotensin-converting enzyme 2","abstract":"In this study, we developed ACE2-specific, peptide-derived 68Ga-labeled radiotracers, motivated by the hypotheses that (1) ACE2 is an important determinant of SARS-CoV-2 susceptibility, and (2) that modulation of ACE2 in COVID-19 drives severe organ injury. A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears two cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with 68Ga to generate peptide radiotracers ([68Ga]NOTA-PEP). The aminocaproate-derived radiotracer [68Ga]NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. Cyclic DX-600 derived peptides had markedly lower IC50\u2019s than their linear counterparts. The three cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated IC50\u2019s similar to, or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga, and the biodistribution of [68Ga]NOTA-PEP4 was determined in a hACE2 transgenic murine cohort. Pharmacologic concentrations of co-administered NOTA-PEP (\u201cblocking\u201d) showed significant reduction of [68Ga]NOTA-PEP4 signals in the in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. NOTA-conjugated, cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for 68Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer [68Ga]NOTA-PEP4 showed specific binding in the heart, liver, lungs and intestine - organs known to be affected in SARS-CoV-2 infection. These results suggest that [68Ga]NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2 infected murine models and COVID-19 patients. For Table of Contents use only","version":"1.1","doi":"10.1101/2020.12.15.412809","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422793","pub_date":"2020-12-15","title":"Genomic diversity analysis of SARS-CoV-2 genomes in Rwanda","abstract":"COVID-19 (Coronavirus disease 2019) is an emerging pneumonia-like respiratory disease of humans and is recently spreading across the globe. To analyze the genome sequence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) isolated from Rwanda with other viral strains from African countries. We downloaded 75 genomes sequences of clinical SARS-CoV-2 from the GISAID (global initiative on sharing all influenza data) database and we comprehensively analyzed these SARS-CoV-2 genomes sequences alongside with Wuhan SARS-CoV-2 sequences as the reference strains. We analyzed 75 genomes sequences of SARS-CoV-2 isolated in different African countries including 10 samples of SARS-CoV-2 isolated in Rwanda between July and August 2020. The phylogenetic analysis of the genome sequence of SARS-CoV-2 revealed a strong identity with reference strains between 90-95%. We identified a missense mutation in four proteins including orf1ab polyprotein, NSP2, 2\u2019-O-ribose methyltransferase and orf1a polyprotein. The most common changes in the base are C > T. We also found that all clinically SARS-CoV-2 isolated from Rwanda had genomes belonging to clade G and lineage B.1. Tracking the genetic evolution of SARS-CoV-2 over time is important to understand viral evolution pathogenesis. These findings may help to implement public health measures in curbing COVID-19 in Rwanda.","version":"1.1","doi":"10.1101/2020.12.14.422793","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.386714","pub_date":"2020-12-15","title":"Two mutations in the SARS-CoV-2 spike protein and RNA polymerase complex are associated with COVID-19 mortality risk","abstract":"SARS-CoV-2 mortality has been extensively studied in relation to host susceptibility. How sequence variations in the SARS-CoV-2 genome affect pathogenicity is poorly understood. Whole-genome sequencing (WGS) of the virus with death in SARS-CoV-2 patients is one potential method of early identification of highly pathogenic strains to target for containment. We analyzed 7,548 single stranded RNA-genomes of SARS-CoV-2 patients in the GISAID database (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) and associated variants with reported patient\u2019s health status from COVID-19, i.e. deceased versus non-deceased. We probed each locus of the single stranded RNA of the SARS-CoV-2 virus for direct association with host/patient mortality using a logistic regression. In total, evaluating 29,891 loci of the viral genome for association with patient/host mortality, two loci, at 12,053bp and 25,088bp, achieved genome-wide significance (p-values of 4.09e-09 and 4.41e-23, respectively). Mutations at 25,088bp occur in the S2 subunit of the SARS-CoV-2 spike protein, which plays a key role in viral entry of target host cells. Additionally, mutations at 12,053bp are within the ORF1ab gene, in a region encoding for the protein nsp7, which is necessary to form the RNA polymerase complex responsible for viral replication and transcription. Both mutations altered amino acid coding sequences, potentially imposing structural changes that could enhance viral infectivity and symptom severity, and may be important to consider as targets for therapeutic development. Identification of these highly significant associations, unlikely to occur by chance, may assist with COVID-19 early containment of strains that are potentially highly pathogenic.","version":"1.2","doi":"10.1101/2020.11.17.386714","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.381194","pub_date":"2020-12-15","title":"In Vitro Activity of Itraconazole Against SARS-CoV-2","abstract":"As long as there is no vaccine available, having access to inhibitors of SARS-CoV-2 will be of utmost importance. Antivirals against coronaviruses do not exist, hence global drug re-purposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have potential activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with SARS-CoV-2. Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 \u03bcM; MTT assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 \u03bcM). Remdesivir inhibited viral replication with an EC50 = 0.4 \u03bcM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3 log10 drop and >4 log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15). Itraconazole exerted in vitro low micromolar activity against SARS-CoV-2 (EC50 = 2.3 \u03bcM) Remdesivir demonstrated potent antiviral activity, confirming validity of the assay Itraconazole has since shown no efficacy in a clinical study in hospitalized COVID-19 patients","version":"1.2","doi":"10.1101/2020.11.13.381194","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.15.422866","pub_date":"2020-12-15","title":"No detectable signal for ongoing genetic recombination in SARS-CoV-2","abstract":"The COVID-19 pandemic has led to an unprecedented global sequencing effort of its viral agent SARS-CoV-2. The first whole genome assembly of SARS-CoV-2 was published on January 5 2020. Since then, over 150,000 high-quality SARS-CoV-2 genomes have been made available. This large genomic resource has allowed tracing of the emergence and spread of mutations and phylogenetic reconstruction of SARS-CoV-2 lineages in near real time. Though, whether SARS-CoV-2 undergoes genetic recombination has been largely overlooked to date. Recombination-mediated rearrangement of variants that arose independently can be of major evolutionary importance. Moreover, the absence of recombination is a key assumption behind the application of phylogenetic inference methods. Here, we analyse the extant genomic diversity of SARS-CoV-2 and show that, to date, there is no detectable hallmark of recombination. We assess our detection power using simulations and validate our method on the related MERS-CoV for which we report evidence for widespread genetic recombination.","version":"1.1","doi":"10.1101/2020.12.15.422866","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422791","pub_date":"2020-12-15","title":"Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies","abstract":"Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC50) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.","version":"1.1","doi":"10.1101/2020.12.14.422791","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422601","pub_date":"2020-12-15","title":"Rapid SARS-CoV-2 Detection and Classification Using Phase Imaging with Computational Specificity","abstract":"Efforts to mitigate the COVID-19 crisis revealed that fast, accurate, and scalable testing is crucial for curbing the current impact and that of future pandemics. We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification. An ultrasensitive interferometric method was used to image four virus types with nanoscale optical pathlength sensitivity. Pairing these data with fluorescence images for ground truth, we trained semantic segmentation models based on U-Net, a particular type of convolutional neural network. The trained network was applied to classify the viruses from the interferometric images only, containing simultaneously SARS-CoV-2, H1N1 (influenza-A), HAdV (adenovirus), and ZIKV (Zika). Remarkably, due to the nanoscale sensitivity in the input data, the neural network was able to identify SARS-CoV-2 vs. the other viruses with 96% accuracy. The inference time for each image is 60 ms, on a common graphic processing unit. This approach of directly imaging unlabeled viral particles may provide an extremely fast test, of less than a minute per patient. As the imaging instrument operates on regular glass slides, we envision this method as potentially testing on patient breath condensates. The necessary high throughput can be achieved by translating concepts from digital pathology, where a microscope can scan hundreds of slides automatically. This work proposes a rapid (<1 min.), label-free testing method for SARS-CoV-2 detection, using quantitative phase imaging and deep learning.","version":"1.1","doi":"10.1101/2020.12.14.422601","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422718","pub_date":"2020-12-15","title":"Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro","abstract":"Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-\u00c5-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.","version":"1.1","doi":"10.1101/2020.12.14.422718","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.416180","pub_date":"2020-12-15","title":"Profound Treg perturbations correlate with COVID-19 severity","abstract":"The hallmark of severe COVID-19 disease has been an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We explored the hypothesis that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in both Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, which correlated with poor outcomes. Accordingly, these Tregs over-expressed a range of suppressive effectors, but also pro-inflammatory molecules like IL32. Most strikingly, they acquired similarity to tumor-infiltrating Tregs, known to suppress local anti-tumor responses. These traits were most marked in acute patients with severe disease, but persisted somewhat in convalescent patients. These results suggest that Tregs may play nefarious roles in COVID-19, via suppressing anti-viral T cell responses during the severe phase of the disease, and/or via a direct pro-inflammatory role.","version":"1.2","doi":"10.1101/2020.12.11.416180","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.420489","pub_date":"2020-12-14","title":"A traditional medicine, Respiratory Detox Shot (RDS), inhibits the infection of SARS-CoV, SARS-CoV-2, and the Influenza A virus in vitro","abstract":"The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has resulted in the infection of over 60 million people and has caused over 1.4 million deaths as of December 2020 in more than 220 countries and territories. Currently, there is no effective treatment for COVID-19 to reduce mortality. We investigated the potential anti-coronavirus activities from an oral liquid of traditional medicine, Respiratory Detox Shot (RDS), which contains mostly herbal ingredients traditionally used to manage lung diseases. Here we report that RDS inhibited the infection of target cells by SARS-CoV and SARS-CoV-2 pseudoviruses, and by infectious wild-type SARS-CoV-2. We further demonstrated that RDS inhibits viral early infection steps. In addition, we found that RDS can also block the infection of target cells by Influenza A virus. These results suggest that RDS may broadly inhibit the infection of respiratory viruses.","version":"1.2","doi":"10.1101/2020.12.10.420489","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.420133","pub_date":"2020-12-14","title":"Targeting Scavenger Receptor Type B-1 (SR-B1) and Cholesterol Inhibits Entry of SARS-CoV-2 Pseudovirus in Cell Culture","abstract":"The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China in late 2019 and has now caused a global pandemic. The disease caused by SARS-CoV-2 is known as COVID-19. To date, few treatments for COVID-19 have proven effective, and the current standard of care is primarily supportive. As a result, novel therapeutic strategies are in high demand. Viral entry into target cells is frequently sensitive to cell membrane lipid composition and membrane organization. Evidence suggests that cell entry of SARS-CoV-2 is most efficient when the target cell plasma membrane is replete with cholesterol; and recent data implicate cholesterol flux through the high-affinity receptor for cholesterol-rich high-density lipoprotein (HDL), called scavenger receptor type B-1 (SR-B1), as critical for SARS-CoV-2 entry. Here, we demonstrate that a cholesterol-poor synthetic biologic high-density lipoprotein (HDL NP) targets SR-B1 and inhibits cell entry of a SARS-CoV-2 spike protein pseudovirus. Human cells expressing SR-B1 are susceptible to SARS-CoV-2 infection, and viral entry can be inhibited by 50-80% using HDL NPs in an SR-B1-dependent manner. These results indicate that HDL NP targeting of SR-B1 is a powerful potential therapy to combat COVID-19 and other viral diseases.","version":"1.1","doi":"10.1101/2020.12.14.420133","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422737","pub_date":"2020-12-14","title":"SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation","abstract":"Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen \u223c6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses. \nCell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19\nDrug screen reveals critical role for membrane lipid composition in fusion\nSpike\u2019s unusual membrane-proximal cysteines and aromatics are essential for fusion\nCholesterol tunes relative infectivity of SARS-CoV-2 viral particles\n Cell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19 Drug screen reveals critical role for membrane lipid composition in fusion Spike\u2019s unusual membrane-proximal cysteines and aromatics are essential for fusion Cholesterol tunes relative infectivity of SARS-CoV-2 viral particles","version":"1.1","doi":"10.1101/2020.12.14.422737","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422710","pub_date":"2020-12-14","title":"Surface proteins of SARS-CoV-2 drive airway epithelial cells to induce interferon-dependent inflammation","abstract":"SARS-CoV-2, the virus that has caused the COVID-19 pandemic, robustly activates the host immune system in critically ill patients. Understanding how the virus engages the immune system will facilitate the development of needed therapeutic strategies. Here we demonstrate both in vitro and in vivo that the SARS-CoV-2 surface proteins Spike (S) and Envelope (E) activate the key immune signaling interferon (IFN) pathway in both immune and epithelial cells independent of viral infection and replication. These proteins induce reactive oxidative species generation and increases in human and murine specific IFN-responsive cytokines and chemokines, similar to their upregulation in critically ill COVID-19 patients. Induction of IFN signaling is dependent on canonical but discrepant inflammatory signaling mediators as the activation induced by S is dependent on IRF3, TBK1, and MYD88 while that of E is largely MYD88 independent. Furthermore, these viral surface proteins, specifically E, induced peribronchial inflammation and pulmonary vasculitis in a mouse model. Finally we show that the organized inflammatory infiltrates are dependent on type I IFN signaling, specifically in lung epithelial cells. These findings underscore the role of SARS-CoV-2 surface proteins, particularly the understudied E protein, in driving cell specific inflammation and their potential for therapeutic intervention. SARS-CoV-2 robustly activates widespread inflammation, but we do not understand mechanistically how the virus engages the immune system. This knowledge will facilitate the development of critically needed therapeutic strategies to promote beneficial immune responses will dampening harmful inflammation. Here we demonstrate that SARS-CoV-2 surface proteins spike and envelope alone activated innate cell function and the interferon signaling pathway. This activation occurred in both immune and epithelial cells, and mechanistic studies demonstrated dependence on known key inflammatory signaling mediators, IRF3, TBK1, and MYD88. In animal studies, we showed that these viral surface proteins induce epithelial cell IFN-dependent lung pathology, reminiscent to acute COVID-19 pulmonary infection. These findings underscore the need for further investigation into the role of SARS-CoV-2 surface proteins, particularly the understudied E protein, in driving cell specific inflammation.","version":"1.1","doi":"10.1101/2020.12.14.422710","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.13.422511","pub_date":"2020-12-14","title":"Hepatitis C Virus Drugs Simeprevir and Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV-2 Replication in Cell Culture","abstract":"Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS- CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold. Several HCV protease inhibitors are predicted to inhibit SARS-CoV-2 Mpro and PLpro. Seven HCV drugs inhibit Mpro enzyme activity, four HCV drugs inhibit PLpro. Seven HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human cells. HCV drugs simeprevir and grazoprevir synergize with remdesivir to inhibit SARS- CoV-2. Bafna, White and colleagues report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases and SARS-CoV-2 replication in cell culture. Two drugs, simeprevir and grazoprevir, synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir antiviral activity as much as 10-fold.","version":"1.1","doi":"10.1101/2020.12.13.422511","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.14.422714","pub_date":"2020-12-14","title":"Neuroinvasion and encephalitis following intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system. Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed at day 3 and declined at days 5 and 6 after infection. In contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals at days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.","version":"1.1","doi":"10.1101/2020.12.14.422714","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.12.422477","pub_date":"2020-12-14","title":"Computational Analysis of Dynamic Allostery and Control in the three SARS-CoV-2 non-structural proteins","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the COVID-19 pandemic, has no vaccine or antiviral drugs available to the public, at the time of writing. The virus\u2019 non-structural proteins are promising drug targets because of their vital role in the viral cycle. A significant body of work has been focused on finding inhibitors which covalently and competitively bind the active site of the non-structural proteins, but little has been done to address regions other than the active site, i.e. for non-competitive inhibition. Here we extend previous work on the SARS-CoV-2 Mpro (nsp5) to three other SARS-CoV-2 proteins: host shutoff factor (nsp1), papain-like protease (nsp3, also known as PLpro) and RNA-dependent RNA-polymerase (nsp12, also known as RdRp) in complex with nsp7 and nsp8 cofactors. Using open-source software (DDPT) to construct Elastic Network Models (ENM) of the chosen proteins we analyse their fluctuation dynamics and thermodynamics, as well as using this protein family to study convergence and robustness of the ENM. Exhaustive 2-point mutational scans of the ENM and their effect on fluctuation free energies suggest several new candidate regions, distant from the active site, for control of the proteins\u2019 function, which may assist the drug development based on the current small molecule binding screens. The results also provide new insights, including non-additive effects of double-mutation or inhibition, into the active biophysical research field of protein fluctuation allostery and its underpinning dynamical structure.","version":"1.1","doi":"10.1101/2020.12.12.422477","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.299891","pub_date":"2020-12-14","title":"High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters","abstract":"The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor via receptor binding domain (RBD) to enter into the cell and inhibiting this interaction is a main approach to inhibit SARS-CoV-2 infection. We engineered ACE2 to enhance the affinity with directed evolution in 293T cells. Three cycles of random mutation and cell sorting achieved 100-fold higher affinity to RBD than wild-type ACE2. The extracellular domain of modified ACE2 fused to the human IgG1-Fc region had stable structure and neutralized SARS-CoV-2 without the emergence of mutational escape. Therapeutic administration protected hamsters from SARS-CoV-2 infection, decreasing lung virus titers and pathology. Engineering ACE2 decoy receptors with human cell-based directed evolution is a promising approach to develop a SARS-CoV-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus. Engineered ACE2 decoy receptor has a therapeutic potential against COVID-19 without viral escape mutation.","version":"1.2","doi":"10.1101/2020.09.16.299891","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.13.422589","pub_date":"2020-12-14","title":"Profiling of oral microbiota and cytokines in COVID-19 patients","abstract":"SARS-CoV-2 presence has been recently demonstrated in the sputum or saliva, suggesting how the shedding of viral RNA outlasts the end of symptoms. Recent data from transcriptome analysis show that oral cavity mucosa harbors high levels of ACE2 and TMPRSS2, highlighting its role as a double-edged sword for SARS-CoV-2 body entrance or interpersonal transmission. In the present study, for the first time, we demonstrate the oral microbiota structure and inflammatory profile of COVID-19 patients. Hospitalized COVID-19 patients and matched healthy controls underwent naso/oral-pharyngeal and oral swabs. Microbiota structure was analyzed by 16S rRNA V2 automated targeted sequencing, while oral and sera concentrations of 27 cytokines were assessed using magnetic bead-based multiplex immunoassays. A significant diminution in species richness was observed in COVID-19 patients, along with a marked difference in beta-diversity. Species such as Prevotella salivae and Veillonella infantium were distinctive for COVID-19 patients, while Neisseria perflava and Granulicatella elegans were predominant in controls. Interestingly, these two groups of oral species oppositely clustered within the bacterial network, defining two distinct Species Interacting Group (SIGs). Pro-inflammatory cytokines were distinctive for COVID-19 in both oral and serum samples, and we found a specific bacterial consortium able to counteract them, following a novel index called C4 firstly proposed here. We even introduced a new parameter, named CytoCOV, able to predict COVID-19 susceptibility for an unknown subject at 71% of power with an AUC equal to 0.995. This pilot study evidenced a distinctive oral microbiota composition in COVID-19 subjects, with a definite structural network in relation to secreted cytokines. Our results would pave the way for a theranostic approach in fighting COVID-19, trying to enlighten the intimate relationship among microbiota and SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.13.422589","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.12.20248070","pub_date":"2020-12-14","title":"Social Media Study of Public Opinions on Potential COVID-19 Vaccines: Informing Dissent, Disparities, and Dissemination","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The current development of vaccines for SARS-CoV-2 is unprecedented. Little is known, however, about the nuanced public opinions on the coming vaccines. We adopt a human-guided machine learning framework (using more than 40,000 rigorously selected tweets from more than 20,000 distinct Twitter users) to capture public opinions on the potential vaccines for SARS-CoV-2, classifying them into three groups: pro-vaccine, vaccine-hesitant, and anti-vaccine. We aggregate opinions at the state and country levels, and find that the major changes in the percentages of different opinion groups roughly correspond to the major pandemic-related events. Interestingly, the percentage of the pro-vaccine group is lower in the Southeast part of the United States. Using multinomial logistic regression, we compare demographics, social capital, income, religious status, political affiliations, geo-locations, sentiment of personal pandemic experience and non-pandemic experience, and county-level pandemic severity perception of these three groups to investigate the scope and causes of public opinions on vaccines. We find that socioeconomically disadvantaged groups are more likely to hold polarized opinions on potential COVID-19 vaccines. The anti-vaccine opinion is the strongest among the people who have the worst personal pandemic experience. Next, by conducting counterfactual analyses, we find that the U.S. public is most concerned about the safety, effectiveness, and political issues regarding potential vaccines for COVID-19, and improving personal pandemic experience increases the vaccine acceptance level. We believe this is the first large-scale social media-based study to analyze public opinions on potential COVID-19 vaccines that can inform more effective vaccine distribution policies and strategies.</jats:p>","version":null,"doi":"10.1101/2020.12.12.20248070","journal":"medRxiv","score":null},{"id":"10.1101/2020.12.13.422548","pub_date":"2020-12-13","title":"Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SRAS-CoV-2. Vero E6 cells were treated with copper gluconate 18 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were maintained in fresh medium containing copper gluconate for an additional 48-hour period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT assay. The viability of Vero E6 cells treated with copper gluconate up to 200 \u03bcM was found to be similar to that of untreated cells, but it dropped below 40% with 400 \u03bcM of this agent. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3%,5.2% in cells treated with 0, 2, 10, 25, 50 and 100 \u03bcM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 \u03bcM of copper gluconate respectively (p < 0.05). Copper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells. Furthers studies are needed to determine whether copper homeostasis could play a role in SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.13.422548","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.13.422469","pub_date":"2020-12-13","title":"A novel cell culture system modeling the SARS-CoV-2 life cycle","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19, and no effective antiviral agents and vaccines are available. SARS-CoV-2 is classified as a biosafety level-3 (BLS-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2-GFP/\u0394N trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.","version":"1.1","doi":"10.1101/2020.12.13.422469","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.12.422532","pub_date":"2020-12-13","title":"Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition","abstract":"SARS-CoV-2 research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a non-infectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlights the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.","version":"1.1","doi":"10.1101/2020.12.12.422532","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.13.422567","pub_date":"2020-12-13","title":"The Potential for SARS-CoV-2 to Evade Both Natural and Vaccine-induced Immunity","abstract":"SARS-CoV-2 attaches to the surface of susceptible cells through extensive interactions between the receptor binding domain (RBD) of its spike protein and angiotensin converting enzyme type 2 (ACE2) anchored in cell membranes. To investigate whether naturally occurring mutations in the spike protein are able to prevent antibody binding, yet while maintaining the ability to bind ACE2 and viral infectivity, mutations in the spike protein identified in cases of human infection were mapped to the crystallographically-determined interfaces between the spike protein and ACE2 (PDB entry 6M0J), antibody CC12.1 (PDB entry 6XC2), and antibody P2B-2F6 (PDB entry 7BWJ). Both antibody binding interfaces partially overlap with the ACE2 binding interface. Among 16 mutations that map to the RBD:CC12.1 interface, 11 are likely to disrupt CC12.1 binding but not ACE2 binding. Among 12 mutations that map to the RBD:P2B-2F6 interface, 8 are likely to disrupt P2B-2F6 binding but not ACE2 binding. As expected, none of the mutations observed to date appear likely to disrupt the RBD:ACE2 interface. We conclude that SARS-CoV-2 with mutated forms of the spike protein may retain the ability to bind ACE2 while evading recognition by antibodies that arise in response to the original wild-type form of the spike protein. It seems likely that immune evasion will be possible regardless of whether the spike protein was encountered in the form of infectious virus, or as the immunogen in a vaccine. Therefore, it also seems likely that reinfection with a variant strain of SARS-CoV-2 may occur among people who recover from Covid-19, and that vaccines with the ability to generate antibodies against multiple variant forms of the spike protein will be necessary to protect against variant forms of SARS-CoV-2 that are already circulating in the human population.","version":"1.1","doi":"10.1101/2020.12.13.422567","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.12.422516","pub_date":"2020-12-13","title":"SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome","abstract":"Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious. Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.","version":"1.1","doi":"10.1101/2020.12.12.422516","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.13.422550","pub_date":"2020-12-13","title":"Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies","abstract":"SARS-CoV-2 unprecedentedly threatens the public health at worldwide level. There is an urgent need to develop an effective vaccine within a highly accelerated time. Here, we present the most comprehensive S-protein-based linear B-cell epitope candidate list by combining epitopes predicted by eight widely-used immune-informatics methods with the epitopes curated from literature published between Feb 6, 2020 and July 10, 2020. We find four top prioritized linear B-cell epitopes in the hotspot regions of S protein can specifically bind with serum antibodies from horse, mouse, and monkey inoculated with different SARS-CoV-2 vaccine candidates or a patient recovering from COVID-19. The four linear B-cell epitopes can induce neutralizing antibodies against both pseudo and live SARS-CoV-2 virus in immunized wild-type BALB/c mice. This study suggests that the four linear B-cell epitopes are potentially important candidates for serological assay or vaccine development.","version":"1.1","doi":"10.1101/2020.12.13.422550","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.422139","pub_date":"2020-12-12","title":"The MERS-CoV receptor gene is among COVID-19 risk factors inherited from Neandertals","abstract":"In the current SARS-CoV-2 pandemic, two genetic regions derived from Neandertals have been shown to increase and decrease, respectively, the risk of falling severely ill upon infection. Here, we show that 2-8% of people in Eurasia carry a variant promoter region of the DPP4 gene inherited from Neandertals. This gene encodes an enzyme that serves as a receptor for the coronavirus MERS-CoV and is currently not believed to be a receptor for SARS-CoV-2. However, the Neandertal DPP4 variant doubles the risk to become critically ill in COVID-19.","version":"1.1","doi":"10.1101/2020.12.11.422139","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.422055","pub_date":"2020-12-12","title":"Millisecond-scale molecular dynamics simulation of spike RBD structure reveals evolutionary adaption of SARS-CoV-2 to stably bind ACE2","abstract":"The Receptor Binding Domain (RBD) of the SARS-CoV-2 surface spike (S) protein interacts with host angiotensin converting enzyme 2 (ACE2) to gain entry to host cells and initiate infection. Detailed, accurate understanding of key interactions between S RBD and ACE2 provides critical information that may be leveraged in the development of strategies for the prevention and treatment of COVID-19. Utilizing the published sequences and cryo-EM structures of both the viral S RBD and ACE2, we performed in silico molecular dynamics (MD) simulations of free S RBD and of its interaction with ACE2 over the exceptionally long durations of 2.9 and 2 milliseconds, respectively, to elucidate the nature and relative affinity of S RBD surface residues for the ACE2 binding region. Our findings reveal that free S RBD has assumed an optimized ACE2 binding-ready conformation, incurring little entropic penalty for binding, an evolutionary adaptation that contributes to its high affinity for the receptor. We further identified high probability molecular binding interactions that inform both vaccine design and therapeutic development, which may include recombinant ACE2-based spike decoys and/or allosteric S RBD-ACE2 binding inhibitors to prevent or arrest infection and thus disease.","version":"1.1","doi":"10.1101/2020.12.11.422055","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.421057","pub_date":"2020-12-11","title":"Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals","abstract":"The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.","version":"1.1","doi":"10.1101/2020.12.11.421057","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.421008","pub_date":"2020-12-11","title":"BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2","abstract":"A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD \u2018down,\u2019 one-RBD \u2018up\u2019 state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFN\u03b3+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).","version":"1.1","doi":"10.1101/2020.12.11.421008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.05.413377","pub_date":"2020-12-11","title":"Whole Genome Sequencing for Revealing the Point Mutations of SARS-CoV-2 Genome in Bangladeshi Isolates and their Structural Effects on Viral Proteins","abstract":"Coronavirus disease-19 (COVID-19) is the recent global pandemic caused by the virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The virus has already killed more than one million people worldwide and billions are at risk of getting infected. As of now, there is neither any drug nor any vaccine in sight with conclusive scientific evidence that it can cure or provide protection against the illness. Since novel coronavirus is a new virus, mining its genome sequence is of crucial importance for drug/vaccine(s) development. Whole genome sequencing is a helpful tool in identifying genetic changes that occur in a virus when it spreads through the population. In this study, we performed complete genome sequencing of SARS-CoV-2 to unveil the genomic variation and indel, if present. We discovered thirteen (13) mutations in Orf1ab, S and N gene where seven (7) of them turned out to be novel mutations from our sequenced isolate. Besides, we found one (1) insertion and seven (7) deletions from the indel analysis among the 323 Bangladeshi isolates. However, the indel did not show any effect on proteins. Our energy minimization analysis showed both stabilizing and destabilizing impact on viral proteins depending on the mutation. Interestingly, all the variants were located in the binding site of the proteins. Furthermore, drug binding analysis revealed marked difference in interacting residues in mutants when compared to the wild type. Our analysis also suggested that eleven (11) mutations could exert damaging effects on their corresponding protein structures. The analysis of SARS-CoV-2 genetic variation and their impacts presented in this study might be helpful in gaining a better understanding of the pathogenesis of this deadly virus.","version":"1.2","doi":"10.1101/2020.12.05.413377","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.418855","pub_date":"2020-12-11","title":"Identification of eight SARS-CoV-2 ORF7a deletion variants in 2,726 clinical specimens","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF7a, the ortholog of SARS-CoV ORF7a, is a type I transmembrane protein and plays an important role in virus-host interactions. Deletion variants in ORF7a may influence virulence, but only a few such isolates have been reported. Here, we report 8 unique ORF7a deletion variants of 6 to 96 nucleotides in length identified from 2,726 clinical specimens collected in March of 2020.","version":"1.1","doi":"10.1101/2020.12.10.418855","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.06.413443","pub_date":"2020-12-11","title":"Efficient inhibition of SARS-CoV-2 strains by a novel ACE2-IgG4-Fc fusion protein with a stabilized hinge region","abstract":"The novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) enters its host cells after binding to the angiotensin-converting enzyme 2 (ACE2) via its spike glycoprotein. This interaction is critical for virus entry and virus-host membrane fusion. Soluble ACE2 ectodomains bind and neutralize the virus but the short in vivo half-lives of soluble ACE2 limits its therapeutic use. Fusion of the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain can prolong the in vivo half-life but bears the risk of unwanted Fc-receptor activation and antibody-dependent disease enhancement. Here, we describe optimized ACE2-Fc fusion constructs that avoid Fc-receptor binding by using IgG4-Fc as a fusion partner. The engineered ACE2-IgG4-Fc fusion proteins described herein exhibit promising pharmaceutical properties and a broad antiviral activity at single-digit nanomolar concentration. In addition, they allow to maintain the beneficial enzymatic activity of ACE2 and thus are very promising candidate antivirals broadly acting against coronaviruses.","version":"1.2","doi":"10.1101/2020.12.06.413443","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.11.416818","pub_date":"2020-12-11","title":"Duplex formation between the template and the nascent strand in the transcription-regulating sequences determines the site of template switching in SARS \u2013 CoV-2","abstract":"Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis where the polymerase switches template from a 3\u2019 proximal genome body sequence to a 5\u2019 untranslated leader sequence. This leads to a fusion between the common 5\u2019 leader sequence and a 3\u2019 proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesising the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs.","version":"1.1","doi":"10.1101/2020.12.11.416818","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.23.394577","pub_date":"2020-12-11","title":"Publishing of COVID-19 Preprints in Peer-reviewed Journals, Preprinting Trends, Public Discussion and Quality Issues","abstract":"COVID-19-related (vs. non-related) articles appear to be more expeditiously processed and published in peer-reviewed journals. We aimed to evaluate: (i) whether COVID-19-related preprints were favored for publication, (ii) preprinting trends and public discussion of the preprints, and (iii) the relationship between the publication topic (COVID-19-related or not) and quality issues. Manuscripts deposited at bioRxiv and medRxiv between January 1 and September 27 were assessed for the probability of publishing in peer-reviewed journals, and those published were evaluated for submission-to-acceptance time. The extent of public discussion was assessed based on Altmetric and Disqus data. The Retraction Watch Database and PubMed were used to explore the retraction of COVID-19 and non-COVID-19 articles and preprints. With adjustment for the preprinting server and number of deposited versions, COVID-19-related preprints were more likely to be published within 120 days since the deposition of the first version (OR=2.04, 95%CI 1.87-2.23) as well as over the entire observed period (OR=1.42, 95%CI 1.33-1.52). Submission-to-acceptance was by 38.67 days (95%CI 34.96-42.39) shorter for COVID-19 articles. Public discussion of preprints was modest and COVID-19 articles were overrepresented in the pool of retracted articles in 2020. Current data suggest a preference for publication of COVID-19-related preprints over the observed period.","version":"1.3","doi":"10.1101/2020.11.23.394577","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.118992","pub_date":"2020-12-10","title":"Within-host genomics of SARS-CoV-2","abstract":"Extensive global sampling and whole genome sequencing of the pandemic virus SARS-CoV-2 have enabled researchers to characterise its spread, and to identify mutations that may increase transmission or enable the virus to escape therapies or vaccines. Two important components of viral spread are how frequently variants arise within individuals, and how likely they are to be transmitted. Here, we characterise the within-host diversity of SARS-CoV-2, and the extent to which genetic diversity is transmitted, by quantifying variant frequencies in 1390 clinical samples from the UK, many from individuals in known epidemiological clusters. We show that SARS-CoV-2 infections are characterised by low levels of within-host diversity across the entire viral genome, with evidence of strong evolutionary constraint in Spike, a key target of vaccines and antibody-based therapies. Although within-host variants can be observed in multiple individuals in the same phylogenetic or epidemiological cluster, highly infectious individuals with high viral load carry only a limited repertoire of viral diversity. Most viral variants are either lost, or occasionally fixed, at the point of transmission, consistent with a narrow transmission bottleneck. These results suggest potential vaccine-escape mutations are likely to be rare in infectious individuals. Nonetheless, we identified Spike variants present in multiple individuals that may affect receptor binding or neutralisation by antibodies. Since the fitness advantage of escape mutations in highly-vaccinated populations is likely to be substantial, resulting in rapid spread if and when they do emerge, these findings underline the need for continued vigilance and monitoring.","version":"1.4","doi":"10.1101/2020.05.28.118992","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.292581","pub_date":"2020-12-10","title":"Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings","abstract":"The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3. In this study, the stability of the virus in cell culture media at 4\u00b0C and on touch panel surfaces used in laboratory environment was analyzed. In addition, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation. In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.","version":"1.2","doi":"10.1101/2020.09.11.292581","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.09.418806","pub_date":"2020-12-10","title":"Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma","abstract":"Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile. In the current ongoing COVID-19 pandemic, neutralizing antibodies have been shown to be a critical feature of recovered patients. HIV-1 bnAbs recognize extensively diverse cross-reactive epitopes and tolerate diversity within their core epitope. Given the unique nature of HIV-1 bnAbs and their ability to recognize and/or accommodate viral glycans, we reasoned that the glycan shield of SARS-CoV-2 spike protein can be targeted by HIV-1 specific bnAbs. Herein, we showed that HIV-1 specific antibodies cross-react and neutralize SARS-CoV-2. Understanding cross-reactive neutralization epitopes of antibodies generated in divergent viral infections will provide key evidence for engineering so called super-antibodies (antibodies that can potently neutralize diverse pathogens with similar antigenic features). Such cross-reactive antibodies can provide a blueprint upon which synthetic variants can be generated in the face of future pandemics.","version":"1.1","doi":"10.1101/2020.12.09.418806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.09.417121","pub_date":"2020-12-10","title":"An interactive viral genome evolution network analysis system enabling rapid large-scale molecular tracing of SARS-CoV-2","abstract":"Comprehensive analyses of viral genomes can provide a global picture on SARS-CoV-2 transmission and help to predict the oncoming trends of pandemic. This molecular tracing is mainly conducted through extensive phylogenetic network analyses. However, the rapid accumulation of SARS-CoV-2 genomes presents an unprecedented data size and complexity that has exceeded the capacity of existing methods in constructing evolution network through virus genotyping. Here we report a Viral genome Evolution Network Analysis System (VENAS), which uses Hamming distances adjusted by the minor allele frequency to construct viral genome evolution network. The resulting network was topologically clustered and divided using community detection algorithm, and potential evolution paths were further inferred with a network disassortativity trimming algorithm. We also employed parallel computing technology to achieve rapid processing and interactive visualization of >10,000 viral genomes, enabling accurate detection and subtyping of the viral mutations through different stages of Covid-19 pandemic. In particular, several core viral mutations can be independently identified and linked to early transmission events in Covid-19 pandemic. As a general platform for comprehensive viral genome analysis, VENAS serves as a useful computational tool in the current and future pandemics.","version":"1.1","doi":"10.1101/2020.12.09.417121","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.308338","pub_date":"2020-12-10","title":"NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response","abstract":"As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay (\u201cNeutrobodyPlex\u201d) for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide.","version":"1.4","doi":"10.1101/2020.09.22.308338","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.420109","pub_date":"2020-12-10","title":"SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential","abstract":"Coronaviruses have caused multiple epidemics in the past two decades, in addition to the current COVID-19 pandemic that is severely damaging global health and the economy. Coronaviruses employ between twenty and thirty proteins to carry out their viral replication cycle including infection, immune evasion, and replication. Among these, nonstructural protein 16 (Nsp16), a 2\u2019-O-methyltransferase, plays an essential role in immune evasion. Nsp16 achieves this by mimicking its human homolog, CMTr1, which methylates mRNA to enhance translation efficiency and distinguish self from other. Unlike human CMTr1, Nsp16 requires a binding partner, Nsp10, to activate its enzymatic activity. The requirement of this binding partner presents two questions that we investigate in this manuscript. First, how does Nsp10 activate Nsp16? While experimentally-derived structures of the active Nsp16/Nsp10 complex exist, structures of inactive, monomeric Nsp16 have yet to be solved. Therefore, it is unclear how Nsp10 activates Nsp16. Using over one millisecond of molecular dynamics simulations of both Nsp16 and its complex with Nsp10, we investigate how the presence of Nsp10 shifts Nsp16\u2019s conformational ensemble in order to activate it. Second, guided by this activation mechanism and Markov state models (MSMs), we investigate if Nsp16 adopts inactive structures with cryptic pockets that, if targeted with a small molecule, could inhibit Nsp16 by stabilizing its inactive state. After identifying such a pocket in SARS-CoV-2 Nsp16, we show that this cryptic pocket also opens in SARS-CoV-1 and MERS, but not in human CMTr1. Therefore, it may be possible to develop pan-coronavirus antivirals that target this cryptic pocket. Coronaviruses are a major threat to human health. These viruses employ molecular machines, called proteins, to infect host cells and replicate. Characterizing the structure and dynamics of these proteins could provide a basis for designing small molecule antivirals. In this work, we use computer simulations to understand the moving parts of an essential SARS-CoV-2 protein, understand how a binding partner turns it on and off, and identify a novel pocket that antivirals could target to shut this protein off. The pocket is also present in other coronaviruses but not in the related human protein, so it could be a valuable target for pan-coronavirus antivirals.","version":"1.1","doi":"10.1101/2020.12.10.420109","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.413609","pub_date":"2020-12-10","title":"SARS-CoV-2 inhibition in human airway epithelial cells using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray","abstract":"There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of \u22123 to \u22124 at a concentration of 10 \u2013 100 \u03bcg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 \u03bcg/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQ\u2019s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.","version":"1.1","doi":"10.1101/2020.12.10.413609","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.10.419440","pub_date":"2020-12-10","title":"Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion","abstract":"Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.","version":"1.1","doi":"10.1101/2020.12.10.419440","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416750","pub_date":"2020-12-09","title":"Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases","abstract":"T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4+ and CD8+ T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.","version":"1.1","doi":"10.1101/2020.12.08.416750","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.09.416586","pub_date":"2020-12-09","title":"Identification of a Novel Susceptibility Marker for SARS-CoV-2 Infection in Human Subjects and Risk Mitigation with a Clinically Approved JAK Inhibitor in Human/Mouse Cells","abstract":"Coronavirus disease (COVID-19), caused by SARS-CoV-2, has affected over 65 million individuals and killed over 1.5 million persons (December 8, 2020; www.who.int). While fatality rates are higher among the elderly and those with underlying comorbidities, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases. We report that the autoimmune PTPN2 risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry mediated by SARS-CoV-2 spike protein. Elevated ACE2 expression and viral entry were mediated by increased JAK-STAT signalling, and were reversed by the JAK inhibitor, tofacitinib. Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry, and identify a clinically approved therapeutic agent to mitigate this risk.","version":"1.1","doi":"10.1101/2020.12.09.416586","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416677","pub_date":"2020-12-09","title":"Intranasal administration of SARS-CoV-2 neutralizing human antibody prevents infection in mice","abstract":"Prevention of SARS-CoV-2 infection at the point of nasal entry is a novel strategy that has the potential to help contain the ongoing pandemic. Using our proprietary technologies, we have engineered a human antibody that recognizes SARS-CoV-2 S1 spike protein with an enhanced affinity for mucin to improve the antibody\u2019s retention in respiratory mucosa. The modified antibody, when administered into mouse nostrils, was shown to block infection in mice that were exposed to high titer SARS-CoV-2 pseudovirus 10 hours after the initial antibody treatment. Our data show that the protection against SARS-CoV-2 infection is effective in both nasal and lung areas 7 days after viral exposure. The modified antibody is stable in a nasal spray formulation and maintains its SARS-CoV-2 neutralizing activity. Nasal spray of the modified antibody can be developed as an affordable and effective prophylactic product to protect people from infection by exposure to SARS-CoV-2 virus in the air. A Fc-modified human antibody prevents SARS-CoV-2 viral infection via nasal administration","version":"1.1","doi":"10.1101/2020.12.08.416677","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416636","pub_date":"2020-12-09","title":"Persistent Cellular Immunity to SARS-CoV-2 Infection","abstract":"SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4+ and CD8+ T cells, expression of activation/exhaustion markers, and cell division. We show that SARS-CoV-2 infection elicits broadly reactive and highly functional memory T cell responses that persist 6 months after infection. In addition, recovered individuals show enduring immune alterations in CD4+ and CD8+ T cells compartments.","version":"1.1","doi":"10.1101/2020.12.08.416636","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.417022","pub_date":"2020-12-09","title":"The D614G Mutation Enhances the Lysosomal Trafficking of SARS-CoV-2 Spike","abstract":"The spike D614G mutation increases SARS-CoV-2 infectivity, viral load, and transmission but the molecular mechanism underlying these effects remains unclear. We report here that spike is trafficked to lysosomes and that the D614G mutation enhances the lysosomal sorting of spike and the lysosomal accumulation of spike-positive punctae in SARS-CoV-2-infected cells. Spike trafficking to lysosomes is an endocytosis-independent, V-ATPase-dependent process, and spike-containing lysosomes drive lysosome clustering but display poor lysotracker labeling and reduced uptake of endocytosed materials. These results are consistent with a lysosomal pathway of coronavirus biogenesis and raise the possibility that a common mechanism may underly the D614G mutation\u2019s effects on spike protein trafficking in infected cells and the accelerated entry of SARS-CoV-2 into uninfected cells.","version":"1.1","doi":"10.1101/2020.12.08.417022","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.293464","pub_date":"2020-12-09","title":"Exploratory neuroimmune profiling identifies CNS-specific alterations in COVID-19 patients with neurological involvement","abstract":"One third of COVID-19 patients develop significant neurological symptoms, yet SARS-CoV-2 is rarely detected in central nervous system (CNS) tissue, suggesting a potential role for parainfectious processes, including neuroimmune responses. We therefore examined immune parameters in cerebrospinal fluid (CSF) and blood samples from a cohort of patients with COVID-19 and significant neurological complications. We found divergent immunological responses in the CNS compartment, including increased levels of IL-12 and IL-12-associated innate and adaptive immune cell activation. Moreover, we found increased proportions of B cells in the CSF relative to the periphery and evidence of clonal expansion of CSF B cells, suggesting a divergent intrathecal humoral response to SARS-CoV-2. Indeed, all COVID-19 cases examined had anti-SARS-CoV-2 IgG antibodies in the CSF whose target epitopes diverged from serum antibodies. We directly examined whether CSF resident antibodies target self-antigens and found a significant burden of CNS autoimmunity, with the CSF from most patients recognizing neural self-antigens. Finally, we produced a panel of monoclonal antibodies from patients\u2019 CSF and show that these target both anti-viral and anti-neural antigens\u2014including one mAb specific for the spike protein that also recognizes neural tissue. This exploratory immune survey reveals evidence of a compartmentalized and self-reactive immune response in the CNS meriting a more systematic evaluation of neurologically impaired COVID-19 patients. A subset of COVID-19 patients with neurologic impairment show cerebrospinal fluid-specific immune alterations that point to both neuroinvasion and anti-neural autoimmunity as potential causes of impairment.","version":"1.2","doi":"10.1101/2020.09.11.293464","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.415703","pub_date":"2020-12-09","title":"Pre-existing T cell-mediated cross-reactivity to SARS-CoV-2 cannot solely be explained by prior exposure to endemic human coronaviruses","abstract":"Several studies have reported the presence of pre-existing humoral or cell-mediated cross-reactivity to SARS-CoV-2 peptides in healthy individuals unexposed to SARS-CoV-2. In particular, the current literature suggests that this pre-existing cross-reactivity could, in part, derive from prior exposure to \u2018common cold\u2019 endemic human coronaviruses (HCoVs). In this study, we characterised the sequence homology of SARS-CoV-2-derived T-cell epitopes reported in the literature across the entire diversity of the Coronaviridae family. Slightly over half (54.8%) of the tested epitopes did not have noticeable homology to any of the human endemic coronaviruses (HKU1, OC43, NL63 and 229E), suggesting prior exposure to these viruses cannot explain the full cross-reactive profiles observed in healthy unexposed individuals. Further, we find that the proportion of cross-reactive SARS-CoV-2 epitopes with noticeable sequence homology is extremely well predicted by the phylogenetic distance to SARS-CoV-2 (R2 = 96.6%). None of the coronaviruses sequenced to date showed a statistically significant excess of T-cell epitope homology relative to the proportion of expected random matches given the sequence similarity of their core genome to SARS-CoV-2. Taken together, our results suggest that the repertoire of cross-reactive epitopes reported in healthy adults cannot be primarily explained by prior exposure to any coronavirus known to date, or any related yet-uncharacterised coronavirus.","version":"1.1","doi":"10.1101/2020.12.08.415703","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.09.417741","pub_date":"2020-12-09","title":"Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies","abstract":"Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.","version":"1.1","doi":"10.1101/2020.12.09.417741","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.412155","pub_date":"2020-12-09","title":"Chromatin remodeling in peripheral blood cells reflects COVID-19 symptom severity","abstract":"SARS-CoV-2 infection triggers highly variable host responses and causes varying degrees of illness in humans. We sought to harness the peripheral blood mononuclear cell (PBMC) response over the course of illness to provide insight into COVID-19 physiology. We analyzed PBMCs from subjects with variable symptom severity at different stages of clinical illness before and after IgG seroconversion to SARS-CoV-2. Prior to seroconversion, PBMC transcriptomes did not distinguish symptom severity. In contrast, changes in chromatin accessibility were associated with symptom severity. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif occupancy for individual PBMC cell types. The most extensive remodeling occurred in CD14+ monocytes where sub-populations with distinct chromatin accessibility profiles were associated with disease severity. Our findings indicate that pre-seroconversion chromatin remodeling in certain innate immune populations is associated with divergence in symptom severity, and the identified transcription factors, regulatory elements, and downstream pathways provide potential prognostic markers for COVID-19 subjects. Chromatin accessibility in immune cells from COVID-19 subjects is remodeled prior to seroconversion to reflect disease severity.","version":"1.3","doi":"10.1101/2020.12.04.412155","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416875","pub_date":"2020-12-09","title":"Single dose immunization with a COVID-19 DNA vaccine encoding a chimeric homodimeric protein targeting receptor binding domain (RBD) to antigen-presenting cells induces rapid, strong and long-lasting neutralizing IgG, Th1 dominated CD4+ T cells and strong CD8+ T cell responses in mice","abstract":"The pandemic caused by the SARS-CoV-2 virus in 2020 has led to a global public health emergency, and non-pharmaceutical interventions required to limit the viral spread are severely affecting health and economies across the world. A vaccine providing rapid and persistent protection across populations is urgently needed to prevent disease and transmission. We here describe the development of novel COVID-19 DNA plasmid vaccines encoding homodimers consisting of a targeting unit that binds chemokine receptors on antigen-presenting cells (human MIP-1\u03b1 /LD78\u03b2), a dimerization unit (derived from the hinge and CH3 exons of human IgG3), and an antigenic unit (Spike or the receptor-binding domain (RBD) from SARS-CoV-2). The candidate encoding the longest RBD variant (VB2060) demonstrated high secretion of a functional protein and induced rapid and dose-dependent RBD IgG antibody responses that persisted up to at least 3 months after a single dose of the vaccine in mice. Neutralizing antibody (nAb) titers against the live virus were detected from day 7 after one dose. All tested dose regimens reached titers that were higher or comparable to those seen in sera from human convalescent COVID-19 patients from day 28. T cell responses were detected already at day 7, and were subsequently characterized to be multifunctional CD8+ and Th1 dominated CD4+ T cells. Responses remained at sustained high levels until at least 3 months after a single vaccination, being further strongly boosted by a second vaccination at day 89. These findings, together with the simplicity and scalability of plasmid DNA manufacturing, safety data on the vaccine platform in clinical trials, low cost of goods, data indicating potential long term storage at +2\u00b0 to 8\u00b0C and simple administration, suggests the VB2060 candidate is a promising second generation candidate to prevent COVID-19.","version":"1.1","doi":"10.1101/2020.12.08.416875","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416164","pub_date":"2020-12-09","title":"Conformational diversity of CDR region during affinity maturation determines the affinity and stability of Sars-Cov-1 VHH-72 nanobody","abstract":"The affinity maturation of Sars-Cov-1 VHH-72 nanobody from its germline predecessor has been studied at the molecular level. The effect of somatic mutations accumulated during affinity maturation process on flexibility, stability and affinity of the germline and affinity matured nanobody was studied. Affinity maturation results in loss of local flexibility in CDR of H3 and this resulted in a gain of affinity towards the antigen. Further affinity maturation was found to destabilize the nanobody. Mechanistically the loss of flexibility of the CDR H3 is due to the redistribution of hydrogen bond network due to somatic mutation A50T, also this contributes significantly to the destability of the nanobody. Unlike antibody, in nanobody the framework region is highly conserved and structural diversity in CDR is the determining factor in diverse antigen binding and also a factor contributing to the stability. This study provide insights into the interrelationship between flexibility, stability and affinity during affinity maturation in a nanobody.","version":"1.1","doi":"10.1101/2020.12.08.416164","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.415422","pub_date":"2020-12-08","title":"Circular RNA profiling reveals abundant and diverse circRNAs of SARS-CoV-2, SARS-CoV and MERS-CoV origin","abstract":"Circular RNAs (circRNAs) encoded by DNA genomes have been identified across host and pathogen species as parts of the transcriptome. Accumulating evidences indicate that circRNAs play critical roles in autoimmune diseases and viral pathogenesis. Here we report that RNA viruses of the Betacoronavirus genus of Coronaviridae, SARS-CoV-2, SARS-CoV and MERS-CoV, encode a novel type of circRNAs. Through de novo circRNA analyses of publicly available coronavirus-infection related deep RNA-Sequencing data, we identified 351, 224 and 2,764 circRNAs derived from SARS-CoV-2, SARS-CoV and MERS-CoV, respectively, and characterized two major back-splice events shared by these viruses. Coronavirus-derived circRNAs are more abundant and longer compared to host genome-derived circRNAs. Using a systematic strategy to amplify and identify back-splice junction sequences, we experimentally identified over 100 viral circRNAs from SARS-CoV-2 infected Vero E6 cells. This collection of circRNAs provided the first line of evidence for the abundance and diversity of coronavirus-derived circRNAs and suggested possible mechanisms driving circRNA biogenesis from RNA genomes. Our findings highlight circRNAs as an important component of the coronavirus transcriptome. We report for the first time that abundant and diverse circRNAs are generated by SARS-CoV-2, SARS-CoV and MERS-CoV and represent a novel type of circRNAs that differ from circRNAs encoded by DNA genomes.","version":"1.1","doi":"10.1101/2020.12.07.415422","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.415836","pub_date":"2020-12-08","title":"Anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component Andrographolide in human lung epithelial cells and cytotoxicity evaluation in major organ cell representatives","abstract":"The coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem affecting more than fifty million cases with over one million deaths globally. The effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine anti-SARS-CoV\u20132 activity of Andrographis paniculata extract and its major component andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Post-infection treatment of A. paniculata and andrographolide in SARS-CoV\u20132 infected Calu-3 cells significantly inhibited the production of infectious virions with the IC50 of 0.036 \u03bcg/mL and 0.034 \u03bcM, respectively, as determined by plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3) and brain (SH-SY5Y), showed the CC50 of >100 \u03bcg/mL for A. paniculata extract and 13.2-81.5 \u03bcM for andrographolide, respectively, corresponding to the selectivity index over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV\u20132 infection.","version":"1.1","doi":"10.1101/2020.12.08.415836","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.415018","pub_date":"2020-12-08","title":"Extended in vitro inactivation of SARS-CoV-2 by titanium dioxide surface coating","abstract":"SARS-CoV-2 transmission occurs via airborne droplets and surface contamination. We show tiles coated with TiO2 120 days previously can inactivate SARS-CoV-2 under ambient indoor lighting with 87% reduction in titres at 1h and complete loss by 5h exposure. TiO2 coatings could be an important tool in containing SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.12.08.415018","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.08.416339","pub_date":"2020-12-08","title":"Natural SARS-CoV-2 infections, including virus isolation, among serially tested cats and dogs in households with confirmed human COVID-19 cases in Texas, USA","abstract":"The natural infections and epidemiological roles of household pets in SARS-CoV-2 transmission are not understood. We conducted a longitudinal study of dogs and cats living with at least one SARS-CoV-2 infected human in Texas and found 47.1% of 17 cats and 15.3% of 59 dogs from 25.6% of 39 households were positive for SARS-CoV-2 via RT-PCR and genome sequencing or neutralizing antibodies. Virus was isolated from one cat. The majority (82.4%) of infected pets were asymptomatic. Re-sampling of one infected cat showed persistence of viral RNA at least 32 d-post human diagnosis (25 d-post initial test). Across 15 antibody-positive animals, titers increased (33.3%), decreased (33.3%) or were stable (33.3%) over time. A One Health approach is informative for prevention and control of SARS-CoV-2 transmission.","version":"1.1","doi":"10.1101/2020.12.08.416339","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.20245225","pub_date":"2020-12-08","title":"Relative Sensitivity of ID NOW and RT-PCR for Detection of SARS-CoV-2 in an Ambulatory Population: Clinical Evaluation, Systematic Review and Meta-analysis","abstract":"<jats:p>Importance: The SARS-CoV-2 (COVID-19) virus has infected over 65 million people worldwide and caused over one and half million deaths.  Definitive diagnosis and treatment require laboratory confirmation, which is generally based upon Reverse-Transcription Polymerase Chain Reaction (RT-PCR) tests carried out in a high-complexity laboratory.  The Abbott ID NOW platform provides fast results but has been criticized for low sensitivity.\n\nObjective:  To determine the sensitivity of SARS-CoV-2 detection by ID NOW in an ambulatory population presenting for initial diagnostic testing.\n\nDesign:  Consecutive patients presenting at the Febrile Upper Respiratory Infection clinics and other clinics at The Everett Clinic between April 8th and 22nd, 2020 provided two nasal swab samples, which were tested for SARS-CoV-2 using the ID NOW platform and an RT-PCR platform.  Patients were excluded if they were unable to demonstrate an understanding of the study, unwilling to commit to having all samples collected, had a history of nosebleed in the past 24 hours, nasal surgery in the past two weeks, chemotherapy treatment with documented low platelet and low white blood cell counts, or acute facial trauma.  The sensitivity for each platform was compared with a composite reference standard based upon all tests.  In addition, a systematic review (subset of review CRD420202044410 registered on PROSPERO) was conducted using papers identified by searching PubMed, bioRxiv and medRxiv for investigations in which at least 20 subjects were simultaneously tested using ID NOW and RT-PCR, and which appeared between January 1, 2020 and August 16, 2020. Bias was assessed using the QUADAS2 instrument.  Meta-analysis was conducted in which the results of this evaluation were combined with those of results from the systematic review to estimate the performance of ID NOW by comparison with RT-PCR.\n\nResults:  The study enrolled 785 symptomatic patients, 21 of whom tested positive for SARS-CoV-2 by both the ID NOW and RT-PCR assays, and 2 of whom tested positive only with the RT-PCR assay.  In addition, the study enrolled 189 asymptomatic patients, none of whom tested positive by either ID NOW or RT-PCR.  An invalid ID NOW assay result was reported for 9 subjects (2 asymptomatic, 7 symptomatic), all of whom tested negative by RT-PCR.  Thus, the positive percent agreement between the ID NOW assay and the RT-PCR assay was 91.3%, and the negative percent agreement was 100%.  The meta-analysis, which included the results of this evaluation together with results from our systematic review, allows us to estimate the sensitivity of the ID NOW assay to be 84% (95% CI 55-96%), and identifies patients with viral loads most likely to be associated with transmissible infections.\n\nDiscussion:  This clinical study and meta-analysis show that although the ID NOW platform is not as sensitive as RT-PCR for identification of SARS-CoV-2, it nevertheless provides a high positive and negative predictive value in many populations.  Conclusions are limited by the relatively small sizes of many of the studies included in the systematic review and meta-analysis, together with the heterogeneity of populations enrolled and nucleic acid amplification tests utilized for comparison.\u2003</jats:p>","version":null,"doi":"10.1101/2020.12.07.20245225","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.27.117184","pub_date":"2020-12-07","title":"Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19","abstract":"The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10-15 years from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 FDA-approved compounds and clinical candidates, we identified 17 dose-responsive compounds with in vitro antiviral efficacy in human liver Huh7 cells and confirmed antiviral efficacy in human colon carcinoma Caco-2, human prostate adenocarcinoma LNCaP, and in a physiologic relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein classically found in secretory fluids, including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19. Since its emergence in China in December 2019, SARS-CoV-2 has caused a global pandemic. Repurposing of FDA-approved drugs is a promising strategy for identifying rapidly deployable treatments for COVID-19. Herein, we developed a pipeline for quantitative high-throughput image-based screening of SARS-CoV-2 infection in human cells that led to the identification of several FDA-approved drugs and clinical candidates with in vitro antiviral activity.","version":"1.4","doi":"10.1101/2020.05.27.117184","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.413252","pub_date":"2020-12-07","title":"Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease","abstract":"How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells. In Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA. In both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr. ACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.","version":"1.1","doi":"10.1101/2020.12.07.413252","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.411744","pub_date":"2020-12-07","title":"Online biophysical predictions for SARS-CoV-2 proteins","abstract":"The SARS-CoV-2 virus, the causative agent of COVID-19, consists of an assembly of proteins that determine its infectious and immunological behavior, as well as its response to therapeutics. Major structural biology efforts on these proteins have already provided essential insights into the mode of action of the virus, as well as avenues for structure-based drug design. However, not all of the SARS-CoV-2 proteins, or regions thereof, have a well-defined three-dimensional structure, and as such might exhibit ambiguous, dynamic behaviour that is not evident from static structure representations, nor from molecular dynamics simulations using these structures. We here present a website (http://sars2.bio2byte.be/) that provides protein sequence-based predictions of the backbone and side-chain dynamics and conformational propensities of these proteins, as well as derived early folding, disorder, \u03b2-sheet aggregation and protein-protein interaction propensities. These predictions attempt to capture the \u2018emergent\u2019 properties of the proteins, so the inherent biophysical propensities encoded in the sequence, rather than context-dependent behaviour such as the final folded state. In addition, we provide an indication of the biophysical variation that is observed in homologous proteins, which give an indication of the limits of the functionally relevant biophysical behaviour of these proteins. With this website, we therefore hope to provide researchers with further clues on the behaviour of SARS-CoV-2 proteins.","version":"1.2","doi":"10.1101/2020.12.04.411744","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.414706","pub_date":"2020-12-07","title":"Spike Protein of SARS-CoV-2 Activates Macrophages and Contributes to Induction of Acute Lung Inflammations in Mice","abstract":"Coronavirus disease 2019 (COVID-19) patients exhibit multiple organ malfunctions with a primary manifestation of acute and diffuse lung injuries. The Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to mediate viral entry into host cells; however, whether it can be cellularly pathogenic and contribute to pulmonary hyper-inflammations in COVID-19 is not well known. In this study, we developed a Spike protein-pseudotyped (Spp) lentivirus with the proper tropism of SARS-CoV-2 Spike protein on the surface and tracked down the fate of Spp in wild type C57BL/6J mice receiving intravenous injection of the virus. A lentivirus with vesicular stomatitis virus glycoprotein (VSV-G) was used as the control. Two hours post-infection (hpi), Spp showed more than 27-75 times more viral burden in the lungs than other organs; it also exhibited about 3-5 times more viral burden than VSV-G lentivirus in the lungs, liver, kidney and spleen. Acute pneumonia was evident in animals 24 hpi. Spp lentivirus was mainly found in LDLR+ macrophages and pneumocytes in the lungs, but not in MARC1+ macrophages. IL6, IL10, CD80 and PPAR-\u03b3 were quickly upregulated in response to infection of Spp lentivirus in the lungs in vivo as well as in macrophage-like RAW264.7 cells in vitro. We further confirmed that forced expression of the Spike protein in RAW264.7 cells could significantly increase the mRNA levels of the same panel of inflammatory factors. Our results demonstrate that the Spike protein of SARS-CoV-2 alone can induce cellular pathology, e.g. activating macrophages and contributing to induction of acute inflammatory responses.","version":"1.1","doi":"10.1101/2020.12.07.414706","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.325910","pub_date":"2020-12-07","title":"Production of ORF8 protein from SARS-CoV-2 using an inducible virus-mediated expression system in suspension-cultured tobacco BY-2 cells","abstract":"COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread worldwide in 2020, is an urgent problem to be overcome. The ORF8 of SARS-CoV-2 has been suggested to be associated with the symptoms of COVID-19, according to reports of clinical studies. However, little is known about the function of ORF8. As one of the ways to advance the functional analysis of ORF8, mass production of ORF8 with the correct three-dimensional structure is necessary. In this study, we attempted to produce ORF8 protein by chemically inducible protein production system using tobacco BY-2 cells. An ORF8-producing line was generated by the Agrobacterium method. As a result, the production of ORF8 of 8.8 \u00b1 1.4 mg/L of culture medium was confirmed. SDS-PAGE and nuclear magnetic resonance (NMR) analysis confirmed that the ORF8 produced by this system is a dimeric form with a single structure, unlike that produced in Escherichia coli. Furthermore, it was suggested that the ORF8 produced by this system was glycosylated. Through this study, we succeeded in producing ORF8 folded into a single structure in a chemically inducible protein production system using tobacco BY-2 cells. It is expected that the functional analysis of ORF8 will be advanced using the ORF8 produced by this system and that it will greatly contribute to the development of antibodies and therapeutic agents targeting ORF8.","version":"1.2","doi":"10.1101/2020.10.07.325910","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.06.412759","pub_date":"2020-12-07","title":"The new generation hDHODH inhibitor MEDS433 hinders the in vitro replication of SARS-CoV-2","abstract":"Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. Here, we report on the anti-SARS-CoV-2 activity of MEDS433, a novel inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidines biosynthesis. MEDS433 inhibits in vitro virus replication in the low nanomolar range, and through a mechanism that stems from its ability to block hDHODH activity. MEDS433 thus represents an attractive candidate to develop novel anti-SARS-CoV-2 agents.","version":"1.1","doi":"10.1101/2020.12.06.412759","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.406637","pub_date":"2020-12-07","title":"Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic","abstract":"Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.","version":"1.3","doi":"10.1101/2020.12.01.406637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.414631","pub_date":"2020-12-07","title":"Identification of potential coagulation pathway abnormalities in SARS-Cov-2 infection; insights from bioinformatics analysis","abstract":"Abnormal coagulation parameters have been explored in a significant number of severe COVID-19 patients, linked to poor prognosis and increased risk of organ failure. Here, to uncover the potential abnormalities in coagulation pathways, we analyzed the RNA-seq data (GEO147507) obtained from the treatment of three pulmonary epithelial cell lines with SARS-CoV-2. The significant differentially expressed genes (DEGs) were subjected to Enrichr database for KEGG pathway enrichment analysis and gene ontology (GO) functional annotation. The STRING database was used to generate PPI networks for identified DEGs. We found three upregulated procoagulant genes (SERPINE1, SERPINA5, and SERPINB2) belong to the serine protease inhibitor (serpin) superfamily that inhibit tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) in the fibrinolysis process. In conclusion, we suggest the fibrinolysis process, especially the blockage of t-PA and u-PA inhibitors, a potential target for more study in treating coagulopathy in severe COVID-19 cases.","version":"1.1","doi":"10.1101/2020.12.07.414631","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.410340","pub_date":"2020-12-07","title":"First-described recently discovered non-toxic vegetal-derived furocoumarin preclinical efficacy against SARS-CoV-2: a promising antiviral herbal drug","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiology of coronavirus disease 2019 (COVID19) pandemic. ICEP4 purified compound (ICEP4) is a recently discovered furocoumarin-related purified compound derived from the roots and seeds of Angelica archangelica (herbal drug). ICEP4-related herbal preparations have been extensively used as active herbal ingredients in traditional medicine treatments in several European countries. Extraction method of patent pending ICEP4 (patent application no. GB2017123.7) has previously shown strong manufacturing robustness, long-lasting stability, and repeated chemical consistency. Here we show that ICEP4 presents a significant in vitro cytoprotective effect in highly virulent-SARS-CoV-2 challenged Vero E6 cellular cultures, using doses of 34.5 and 69 \u03bcM. No dose-related ICEP4 toxicity was observed in Vero E6 cells, M0 macrophages, B, CD4+ T and CD8+ T lymphocytes, Natural Killer (NK) or Natural Killer T (NKT) cells. No dose-related ICEP4 inflammatory response was observed in M0 macrophages quantified by IL6 and TNF\u03b1 release in cell supernatant. No decrease in survival rate was observed after either 24 hr acute or 21-day chronic exposure in in vivo toxicity studies performed in C. elegans. Therefore, ICEP4 toxicological profile has demonstrated marked differences compared to others vegetal furocoumarins. Successful ICEP4 doses against SARS-CoV-2-challenged cells are within the maximum threshold of toxicity concern (TTC) of furocoumarins as herbal preparation, stated by European Medicines Agency (EMA). The characteristic chemical compounding of ICEP4, along with its safe TTC, allow us to assume that the first-observation of a natural antiviral compound has occurred. The potential druggability of a new synthetic ICEP4-related compound remains to be elucidated. However, well-established historical use of ICEP4-related compounds as herbal preparations may point towards an already-safe, widely extended remedy, which may be ready-to-go for large-scale clinical trials under the EMA emergency regulatory pathway. To the best of the authors\u2019 knowledge, ICEP4-related herbal drug can be postulated as a promising therapeutic treatment for COVID19.","version":"1.2","doi":"10.1101/2020.12.04.410340","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.360545","pub_date":"2020-12-07","title":"Antiviral activity of plant juices and green tea against SARS-CoV-2 and influenza virus in vitro","abstract":"Many plant juices, extracts and teas have been shown to possess antiviral activity. We here analyzed the virucidal activity of black chokeberry (Aronia melanocarpa), pomegranate (Punica granatum), and elderberry (Sambucus nigra) juice, as well as green tea (Camellia sinensis) against different respiratory viruses. We found that all tested plant derived products effectively inactivated influenza virus, whereas only chokeberry juice diminished SARS-CoV-2 and vaccinia virus infectivity. None of the products inactivated non-enveloped human adenovirus type 5. Thus, black chokeberry juice exerts virucidal activity against different enveloped viral pathogens under in vitro conditions. Whether application of virucidal juices or green tea as oral rinses may lower viral loads in the oral cavity in vivo remains to be evaluated.","version":"1.2","doi":"10.1101/2020.10.30.360545","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.07.414292","pub_date":"2020-12-07","title":"A framework for predicting potential host ranges of pathogenic viruses based on receptor ortholog analysis","abstract":"Viral zoonoses are a serious threat to public health and global security, as reflected by the current scenario of the growing number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases. However, as pathogenic viruses are highly diverse, identification of their host ranges remains a major challenge. Here, we present a combined computational and experimental framework, called REceptor ortholog-based POtential virus hoST prediction (REPOST), for the prediction of potential virus hosts. REPOST first selects orthologs from a diverse species by identity and phylogenetic analyses. Secondly, these orthologs is classified preliminarily as permissive or non-permissive type by infection experiments. Then, key residues are identified by comparing permissive and non-permissive orthologs. Finally, potential virus hosts are predicted by a key residue\u2013specific weighted module. We performed REPOST on SARS-CoV-2 by studying angiotensin-converting enzyme 2 orthologs from 287 vertebrate animals. REPOST efficiently narrowed the range of potential virus host species (with 95.74% accuracy).","version":"1.1","doi":"10.1101/2020.12.07.414292","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.20242073","pub_date":"2020-12-07","title":"Sulodexide in the treatment of patients with early stages of COVID-19: a randomised controlled trial","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Targeting endothelial cells has been suggested for the treatment of patients with COVID-19 and sulodexide has pleiotropic properties within the vascular endothelium that can prove beneficial to the same. We aimed to evaluate the effect of sulodexide when used in the early clinical stages of COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a single-centre, outpatient setting, randomised controlled trial with a parallel-group design in Mexico. Including patients within three days of clinical symptom onset, who were at a high risk of severe clinical progression due to chronic comorbidities. Participants were randomly allocated to receive an oral dose of sulodexide (500 LRU twice a day) or the placebo for 21 days. Primary outcomes were need and length of hospitalisation, need and length of oxygen support.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Between June 5 and August 30, 2020, 243 patients were included in the \u201cper-protocol\u201d analysis. One hundred twenty-four of them received sulodexide, while 119 received placeboes. At 21 days follow-up, 22 of 124 patients required hospitalisation in the sulodexide group compared to 35 of 119 in the placebo group [relative risk (RR), 0\u00b76; 95% confidence interval (CI), 0\u00b737-0\u00b796; p=0\u00b703]. Fewer patients required oxygen support in the sulodexide group [37 of 124 vs. 50 of 119; RR, 0\u00b771; 95% CI, 0\u00b75 to 1; p=0\u00b705], and for fewer days (9\u00b17\u00b72 in the sulodexide group vs. 11\u00b75\u00b19\u00b76 in the placebo group; p=0\u00b702). There was no between-group difference concerning the length of hospital stay.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Early intervention in COVID-19 patients with sulodexide reduced hospital admissions and oxygen support requirements, although with no significant effect on mortality. This has beneficial implications in the patient well-being, making sulodexide a favourable medication until an effective vaccine or an antiviral becomes available.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Researcher independently initiated, partially funded by Alfasigma, Mexico.</jats:p>\n                  <jats:p>\n                    Listed in the ISRCTN registry under ID\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='isrctn' xlink:href='59048638'>ISRCTN59048638</jats:ext-link>\n                    .\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.12.04.20242073","journal":"medRxiv","score":null},{"id":"10.1101/2020.12.04.410589","pub_date":"2020-12-06","title":"Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection","abstract":"SARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential. We developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD). To diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values \u22120.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months. This SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness. In contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.","version":"1.1","doi":"10.1101/2020.12.04.410589","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.412585","pub_date":"2020-12-06","title":"Decontamination of Common Healthcare Facility Surfaces Contaminated with SARS-CoV-2 using Peracetic Acid Dry Fogging","abstract":"The SARS-Cov-2 pandemic has highlighted the urgent need for safe and effective surface decontamination methods, particularly in healthcare settings. The effectiveness of peracetic acid (PAA) dry fogging in decontaminating common healthcare setting surfaces was evaluated after experimentally contaminating nine surfaces (stainless steel, latex painted wood, unsealed hardwood, melamine countertop, vinyl flooring, clear plastic, faux leather, computer keyboard button and smartphone touch screen) with more than 106 TCID50 of SARS-CoV-2. When fumigated with PAA dry fog for an hour, no infectious SARS-CoV-2 virus was recovered from experimentally inoculated coupons of representing nine different surface types. In contrast, high titer recovery of infectious virus was demonstrated for corresponding untreated drying controls of the same materials. Standard surface decontaminating processes, including sprays and wipes, are laborious and often cannot completely decontaminate sensitive electronic equipment. The ease of use, low cost and overall effectiveness of a PAA dry fogging suggest it should be considered for decontaminating settings, particularly intensive care units where severely ill SARS-CoV-2 patients are cared for.","version":"1.1","doi":"10.1101/2020.12.04.412585","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.412098","pub_date":"2020-12-06","title":"Significant inactivation of SARS-CoV-2 by a green tea catechin, a catechin-derivative and galloylated theaflavins in vitro","abstract":"Potential effects of teas and their constituents on SARS-CoV-2 infection were studied in vitro. Infectivity of SARS-CoV-2 was significantly reduced by a treatment with green tea, roasted green tea or oolong tea. Most remarkably, exposure to black tea for 1 min decreased virus titer to an undetectable level (less than 1/1,000 of untreated control). An addition of (-) epigallocatechin gallate (EGCG) significantly inactivated SARS-CoV-2, while theasinensin A (TSA) and galloylated theaflavins including theaflavin 3, 3\u2019-di-gallate (TFDG) had more remarkable anti-viral activities. Virus treated with TSA at 500 \u03bcM or TFDG at 100 \u03bcM showed less than 1/10,000 infectivity compared with untreated virus. TSA and TFDG significantly inhibited interaction between recombinant ACE2 and RGD of S protein. These results strongly suggest that EGCG, and more remarkably TSA and galloylated theaflavins, inactivate the novel coronavirus.","version":"1.1","doi":"10.1101/2020.12.04.412098","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.05.409821","pub_date":"2020-12-06","title":"Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures","abstract":"The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5\u2019-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5\u2019-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5\u2019-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5\u2019-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.","version":"1.1","doi":"10.1101/2020.12.05.409821","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.411736","pub_date":"2020-12-06","title":"Genetic variability in COVID-19-related genes in the Brazilian population","abstract":"SARS-CoV-2 employs the angiotensin-converting enzyme 2 (ACE2) receptor and the transmembrane serine protease (TMPRSS2) to infect human lung cells. Previous studies have suggested that different host genetic backgrounds in ACE2 and TMPRSS2 could contribute to differences in the rate of infection or severity of COVID-19. Recent studies also showed that variants in 15 genes related to type I interferon immunity to influenza virus could predispose to life-threatening COVID-19 pneumonia. Additional genes (SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, IL6, CTSL, ABO, and FURIN) and HLA alleles have also been implicated in response to infection with SARS-CoV-2. Currently, Brazil has recorded the third-highest number of COVID-19 patients worldwide. We aim to investigate the genetic variation present in COVID-19-related genes in the Brazilian population. We analysed 27 candidate genes and HLA alleles in 954 admixed Brazilian exomes. We used the information available in two public databases (http://www.bipmed.org and http://abraom.ib.usp.br/), and additional exomes from individuals born in southeast Brazil, the region with the highest number of COVID-19 patients in the country. Variant allele frequencies were compared with the 1000 Genomes Project phase 3 (1KGP) and the gnomAD databases. We found 395 non-synonymous variants; of these, 325 were also found in the 1000 Genome Project phase 3 (1KGP) and/or gnomAD. Six of these variants were previously reported as putatively influencing the rate of infection or clinical prognosis for COVID-19. The remaining 70 variants were identified exclusively in the Brazilian sample, with a mean allele frequency of 0.0025. In silico prediction of the impact in protein function revealed that three of these rare variants were pathogenic. Furthermore, we identified HLA alleles that were previously associated with COVID-19 response at loci DQB1 and DRB1. Our results showed genetic variability common to other populations, but also rare and ultra-rare variants exclusively found in the Brazilian population. These findings could potentially lead to differences in the rate of infection or response to infection by SARS-CoV-2 and should be further investigated in patients with the disease.","version":"1.1","doi":"10.1101/2020.12.04.411736","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.412494","pub_date":"2020-12-06","title":"The aging whole blood transcriptome reveals a potential role of FASLG in COVID-19","abstract":"The risk for severe illness from COVID-19 increases with age as older patients are at the highest risk. Although it is still unclear whether the virus is blood-transmitted, the viral RNA is detected in serum. Identifying how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interacts with specific blood components during aging is expected to guide proper therapies. Considering that all human coronavirus require host cellular molecules to promote infection, we investigated the aging whole blood transcriptome from the Genotype-Tissue Expression (GTEx) database to explore differentially expressed genes (DEGs) translated into proteins potentially interacting with viral proteins. From a total of 22 DEGs in aged blood, five genes (FASLG, CTSW, CTSE, VCAM1, and BAG3) changed expression during aging. These age-related genes are involved in immune response, inflammation, cell component and cell adhesion, and platelet activation/aggregation. Both males and females older than 50 overexpress FASLG compared with younger adults (20-30 years old), possibly inducing a hyper-inflammatory cascade that activates specific immune cells. Furthermore, the expression of cathepsins (CTSW and CTSE) and the anti-apoptotic co-chaperone molecule BAG3 was significantly increased throughout aging in both gender. By exploring publicly available Single-Cell RNA-Sequencing (scRNA-Seq) data on peripheral blood of SARS-CoV-2-infected patients, we found FASLG and CTSW expressed mainly in natural killer (NK) cells and CD8+ (cytotoxic) T lymphocytes whereas BAG3 was expressed in CD4+ T cells, naive T cells, and CD14+ monocytes. The increased expression of FASLG in blood during aging may explain why older patients are more prone to severe acute viral infection complications. These results indicate FASLG as a prognostic candidate and potential therapeutic target for more aggressive clinical manifestation of COVID-19.","version":"1.1","doi":"10.1101/2020.12.04.412494","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.391763","pub_date":"2020-12-05","title":"Hacking The Diversity Of SARS-CoV-2 And SARS-Like Coronaviruses In Human, Bat And Pangolin Populations","abstract":"In 2019, a novel coronavirus, SARS-CoV-2/nCoV-19, emerged in Wuhan, China, and has been responsible for the current COVID-19 pandemic. The evolutionary origins of the virus remain elusive and understanding its complex mutational signatures could guide vaccine design and development. As part of the international \u201cCoronaHack\u201d in April 2020 (https://www.coronahack.co.uk/), we employed a collection of contemporary methodologies to compare the genomic sequences of coronaviruses isolated from human (SARS-CoV-2;n=163), bat (bat-CoV;n=215) and pangolin (pangolin-CoV;n=7) available in public repositories. Following de novo gene annotation prediction, analyses of gene-gene similarity network, codon usage bias and variant discovery were undertaken. Strong host-associated divergences were noted in ORF3a, ORF6, ORF7a, ORF8 and S, and in codon usage bias profiles. Lastly, we have characterised several high impact variants (inframe insertion/deletion or stop gain) in bat-CoV and pangolin-CoV populations, some of which are found in the same amino acid position and may be highlighting loci of potential functional relevance.","version":"1.2","doi":"10.1101/2020.11.24.391763","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.181123","pub_date":"2020-12-05","title":"Do the Moroccan SARS-CoV-2 genetic diversity hamper the use of the developed universal vaccines in Morocco?","abstract":"The SARS-CoV-2 identified as coronavirus species associated with severe acute respiratory syndrome. At the time of writing, the genetic diversity of Moroccan strains of SARS-CoV-2 is poorly documented. The present study aims to analyze and identify the genetic variants of fortyeight Moroccan strains of SARS-CoV-2 collected from mid-March to the end of May and the prediction of their possible sources. Our results revealed 108 mutations in Moroccan SARS-CoV-2, 50% were non-synonymous were present in seven genes (S, M, N, E, ORF1ab, ORF3a, and ORF8) with variable frequencies. Remarkably, eight non-synonymous mutations were predicted to have a deleterious effect for (ORF1ab, ORF3a, and the N protein. The analysis of the haplotype network of Moroccan strains suggests different sources of SARS-CoV-2 infection in Morocco. Likewise, the phylogenetic analysis revealed that these Moroccan strains were closely related to those belonging to the five continents, indicating no specific strain dominating in Morocco. These findings have the potential to lead to new comprehensive investigations combining genomic data, epidemiological information, and clinical characteristics of SARS-CoV-2 patients in Morocco and could indicate that the developed vaccines are likely to be effective against Moroccan strains.","version":"1.2","doi":"10.1101/2020.06.30.181123","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.410092","pub_date":"2020-12-05","title":"Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 In Vitro","abstract":"Ribavirin is a guanosine analog and has a broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection. In silico analysis suggested that Ribarivin has a broad-spectrum impact on Vero E6 cells. According to the detailed molecular techniques, Ribavirin was shown to decrease TMPRSS2 expression both at mRNA and protein level 48 hours after treatment. The suppressive effect of Ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that Ribavirin also showed an inhibitory effect on TMPRSS2 enzyme. As a conclusion, Ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effect of TMPRSS2 and ACE2 expression.","version":"1.1","doi":"10.1101/2020.12.04.410092","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.407510","pub_date":"2020-12-04","title":"Comparative analysis of antigen-specific anti-SARS-CoV-2 antibody isotypes in COVID-19 patients","abstract":"Serological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.","version":"1.1","doi":"10.1101/2020.12.04.407510","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.407031","pub_date":"2020-12-04","title":"Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.12.03.407031","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.408153","pub_date":"2020-12-04","title":"LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin D\u2019s beneficial effects are mediated by up-regulating LL-37, a well-known antimicrobial peptide with antiviral effects. Recombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37\u2019s ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2. We could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/SStrep KD = 410 nM, LL-37/SHis KD = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/SStrep KD = 370 nM, hACE2/SHis KD = 370 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 290 nM. Further, inhibition of the binding of SStrep (IC50 = 740 nM), S1e (IC50 = 170 nM), and RBD (IC50 = 130 nM) to hACE2 by LL-37 was demonstrated. We have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.","version":"1.2","doi":"10.1101/2020.12.02.408153","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.172833","pub_date":"2020-12-04","title":"Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples","abstract":"The ongoing spread of pandemic coronavirus disease (COVID-19) is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). In the lack of specific drugs or vaccines for SARS-CoV-2, demands rapid diagnosis and management are crucial for controlling the outbreak in the community. Here we report the development of the first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 minutes. We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and changes in gold-colloid color from pink to blue in visible range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus (HPV) infected women. The results were validated using nasopharangeal RNA samples from suspected COVID-19 subjects (n=136). Using RT-PCR as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. Overall, the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.","version":"1.4","doi":"10.1101/2020.06.30.172833","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.409144","pub_date":"2020-12-04","title":"SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE2","abstract":"Coronavirus disease 2019 (COVID-19) includes the cardiovascular complications in addition to respiratory disease. SARS-CoV-2 infection impairs endothelial function and induces vascular inflammation, leading to endotheliitis. SARS-CoV-2 infection relies on the binding of Spike glycoprotein (S protein) to angiotensin converting enzyme 2 (ACE2) in the host cells. We show here that S protein alone can damage vascular endothelial cells (ECs) in vitro and in vivo, manifested by impaired mitochondrial function, decreased ACE2 expression and eNOS activity, and increased glycolysis. The underlying mechanism involves S protein downregulation of AMPK and upregulation of MDM2, causing ACE2 destabilization. Thus, the S protein-exerted vascular endothelial damage via ACE2 downregulation overrides the decreased virus infectivity.","version":"1.1","doi":"10.1101/2020.12.04.409144","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.04.411389","pub_date":"2020-12-04","title":"Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of the induction of IFN-mediated innate immune defences","abstract":"The pandemic spread of SARS-CoV-2, the etiological agent of COVID-19, represents a significant and ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41\u00b0C. Fever is an evolutionarily conserved host response to microbial infection and inflammation that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 tropism and replication. Utilizing a 3D air-liquid interface (ALI) model that closely mimics the natural tissue physiology and cellular tropism of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. We show that temperature elevation induces wide-spread transcriptome changes that impact upon the regulation of multiple pathways, including epigenetic regulation and lncRNA expression, without disruption of general cellular transcription or the induction of interferon (IFN)-mediated antiviral immune defences. Respiratory tissue incubated at temperatures >37\u00b0C remained permissive to SARS-CoV-2 infection but severely restricted the initiation of viral transcription, leading to significantly reduced levels of intraepithelial viral RNA accumulation and apical shedding of infectious virus. To our knowledge, we present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication. Our data identify an important role for temperature elevation in the epithelial restriction of SARS-CoV-2 that occurs independently of the induction of canonical IFN-mediated antiviral immune defences and interferon-stimulated gene (ISG) expression.","version":"1.1","doi":"10.1101/2020.12.04.411389","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.331645","pub_date":"2020-12-04","title":"SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity","abstract":"The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search for treatments and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. We investigated the breadth and potency of antibody-, and T-cell immune responses, in 203 recovered SARS-CoV-2 infected patients who presented with asymptomatic to severe infections. We report very broad serological profiles with cross-reactivity to other human coronaviruses. Further, >99% had SARS-CoV-2 epitope specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 95% of individuals. A significant positive correlation between spike-ACE2 blocking antibody titers and neutralization potency was observed. SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 90% of HLA-A2+ individuals. The viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of disease severity. SARS-CoV-2 can cause severe and deadly infections. However, the immunological understanding of this viral infection is limited. Currently, several vaccines are being developed to help limit transmission and prevent the current pandemic. However, basic understanding of the adaptive immune response developed during SARS-CoV-2 infections is needed to inform further vaccine development and to understand the protective properties of the developed immune response. We investigated, the adaptive immune response developed during SARS-CoV-2 infections in recovered patients experiencing a full spectrum of disease severity, from asymptomatic infections to severe cases requiring hospitalization. We used a novel multiplex serological platform, cell-based neutralization assays and dextramer flow cytometry assays to characterize a broad and robust humoral and cellular immune response towards SARS-CoV-2. We found that the vast majority of recovered individuals have clear detectable and functional SARS-CoV-2 spike specific adaptive immune responses, despite diverse disease severities. The detection of both a humoral and cellular functional spike specific immune response in the vast majority of the individuals, irrespective of asymptomatic manifestations, supports vaccine designs currently underway, and encourages further exploration of whether primary infections provide protection to reinfection.","version":"1.2","doi":"10.1101/2020.10.08.331645","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.402487","pub_date":"2020-12-04","title":"Clade GR and Clade GH Isolates in Asia Show Highest Amount of SNPs","abstract":"Clades are monophyletic groups composed of a common ancestor and all its lineal descendants. As the propensity of virulence of a disease depends upon the type of clade the virus belongs to and it causes different fatality rates of disease in different countries, so the clade-wise analysis of SARS-CoV-2 isolates collected from different countries can illuminate the actual evolutionary relationships between them. In this study, 1566 SARS-CoV-2 genome sequences across ten Asian countries are collected, clustered, and characterized based on the clade they belong to. The isolates are compared to the Wuhan reference sequence (Accession no:MN996528.1) to identify the mutations that occurred at different protein regions. Structural changes in amino acids due to mutations lead to functional instability of the proteins. Detailed clade-wise functional assessments are carried out to quantify the stability and vulnerability of the mutations occurring in SARS-CoV-2 genomes which can shade light on personalized prevention and treatment of the disease and encourage towards the invention of clade-specific vaccines.","version":"1.2","doi":"10.1101/2020.11.30.402487","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.409458","pub_date":"2020-12-03","title":"Molecular basis of the logical evolution of the novel coronavirus SARS-CoV-2: A comparative analysis","abstract":"A novel disease, COVID-19, is sweeping the world since end of 2019. While in many countries, the first wave is over, but the pandemic is going through its next phase with a significantly higher infectability. COVID-19 is caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that seems to be more infectious than any other previous human coronaviruses. To understand any unique traits of the virus that facilitate its entry into the host, we compared the published structures of the viral spike protein of SARS-CoV-2 with other known coronaviruses to determine the possible evolutionary pathway leading to the higher infectivity. The current report presents unique information regarding the amino acid residues that were a) conserved to maintain the binding with ACE2 (Angiotensin-converting enzyme 2), and b) substituted to confer an enhanced binding affinity and conformational flexibility to the SARS-CoV-2 spike protein. The present study provides novel insights into the evolutionary nature and molecular basis of higher infectability and perhaps the virulence of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.12.03.409458","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.20242149","pub_date":"2020-12-03","title":"Variants in SARS-CoV-2 Associated with Mild or Severe Outcome","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>The coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency causing a disparate burden of death and disability around the world. The molecular characteristics of the virus that predict better or worse outcome are largely still being discovered.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We downloaded 155,958 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from GISAID and evaluated whether variants improved prediction of reported severity beyond age and region. We also evaluated specific variants to determine the magnitude of association with severity and the frequency of these variants among the genomes.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Logistic regression models that included viral genomic variants outperformed other models (AUC=0.91 as compared with 0.68 for age and gender alone; p&lt;0.001). Among individual variants, we found 17 single nucleotide variants in SARS-CoV-2 have more than two-fold greater odds of being associated with higher severity and 67 variants associated with \u2264 0.5 times the odds of severity. The median frequency of associated variants was 0.15% (interquartile range 0.09%-0.45%). Altogether 85% of genomes had at least one variant associated with patient outcome.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Numerous SARS-CoV-2 variants have two-fold or greater association with odds of mild or severe outcome and collectively, these variants are common. In addition to comprehensive mitigation efforts, public health measures should be prioritized to control the more severe manifestations of COVID-19 and the transmission chains linked to these severe cases.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.12.01.20242149","journal":"medRxiv","score":null},{"id":"10.1101/2020.12.03.409763","pub_date":"2020-12-03","title":"SARS-CoV-2 D614 and G614 spike variants impair neuronal synapses and exhibit differential fusion ability","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) exhibits two major variants based on mutations of its spike protein, i.e., the D614 prototype and G614 variant. Although neurological symptoms have been frequently reported in patients, it is still unclear whether SARS-CoV-2 impairs neuronal activity or function. Here, we show that expression of both D614 and G614 spike proteins is sufficient to induce phenotypes of impaired neuronal morphology, including defective dendritic spines and shortened dendritic length. Using spike protein-specific monoclonal antibodies, we found that D614 and G614 spike proteins show differential S1/S2 cleavage and cell fusion efficiency. Our findings provide an explanation for higher transmission of the G614 variant and the neurological manifestations observed in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.12.03.409763","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.408823","pub_date":"2020-12-03","title":"A single intranasal or intramuscular immunization with chimpanzee adenovirus vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters","abstract":"The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable or neutralizing the virus, antibody levels in serum were higher in hamsters immunized by an intranasal compared to intramuscular route. Accordingly, ChAd-SARS-CoV-2-S immunized hamsters were protected against a challenge with a high dose of SARS-CoV-2. After challenge, ChAd-SARS-CoV-2-S-immunized hamsters had less weight loss and showed reductions in viral RNA and infectious virus titer in both nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.","version":"1.1","doi":"10.1101/2020.12.02.408823","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.243220","pub_date":"2020-12-03","title":"Rapid endothelial infection, endothelialitis and vascular damage characterise SARS-CoV-2 infection in a human lung-on-chip model","abstract":"Severe cases of COVID-19 present with hypercoagulopathies and systemic endothelialitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of immune cell mediated cytokine storms is unknown. This is in part because in vitro models are typically epithelial cell monocultures or fail to recapitulate vascular physiology. We use a vascularised lung-on-chip model where, consistent with monoculture reports, low numbers of SARS-CoV-2 virions are released apically from alveolar epithelial cells. However, rapid infection of the underlying endothelial layer leads to the generation of clusters of endothelial cells with low or no CD31 expression, a progressive loss of endothelial barrier integrity, and a pro-coagulatory microenvironment. These morphological changes do not occur if these cells are exposed to the virus apically. Viral RNA persists in individual cells, which generates a response that is skewed towards NF-KB mediated inflammation, is typified by IL-6 secretion even in the absence of immune cells, and is transient in epithelial cells but persistent in endothelial cells. Perfusion with Tocilizumab, an inhibitor of trans IL-6 signalling slows the loss of barrier integrity but does not prevent the formation of endothelial cell clusters with reduced CD31 expression. SARS-CoV-2 mediated endothelial cell damage occurs despite a lack of rapid viral replication, in a cell-type specific manner and independently of immune-cell mediated cytokine storms, whose effect would only exacerbate the damage.","version":"1.2","doi":"10.1101/2020.08.10.243220","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.410233","pub_date":"2020-12-03","title":"Horizontal gene transfer and recombination analysis of SARS-CoV-2 genes helps discover its close relatives and shed light on its origin","abstract":"The SARS-CoV-2 pandemic is among the most dangerous infectious diseases that have emerged in recent history. Human CoV strains discovered during previous SARS outbreaks have been hypothesized to pass from bats to humans using intermediate hosts, e.g. civets for SARS-CoV and camels for MERS-CoV. The discovery of an intermediate host of SARS-CoV-2 and the identification of specific mechanism of its emergence in humans are topics of primary evolutionary importance. In this study we investigate the evolutionary patterns of 11 main genes of SARS-CoV-2. Previous studies suggested that the genome of SARS-CoV-2 is highly similar to the horseshoe bat coronavirus RaTG13 for most of the genes and to some Malayan pangolin coronavirus (CoV) strains for the receptor binding (RB) domain of the spike protein. We provide a detailed list of statistically significant horizontal gene transfer and recombination events (both intergenic and intragenic) inferred for each of 11 main genes of the SARS-Cov-2 genome. Our analysis reveals that two continuous regions of genes S and N of SARS-CoV-2 may result from intragenic recombination between RaTG13 and Guangdong (GD) Pangolin CoVs. Statistically significant gene transfer-recombination events between RaTG13 and GD Pangolin CoV have been identified in region [1215-1425] of gene S and region [534-727] of gene N. Moreover, some significant recombination events between the ancestors of SARS-CoV-2, RaTG13, GD Pangolin CoV and bat CoV ZC45-ZXC21 coronaviruses have been identified in genes ORF1ab, S, ORF3a, ORF7a, ORF8 and N. Furthermore, topology-based clustering of gene trees inferred for 25 CoV organisms revealed a three-way evolution of coronavirus genes, with gene phylogenies of ORF1ab, S and N forming the first cluster, gene phylogenies of ORF3a, E, M, ORF6, ORF7a, ORF7b and ORF8 forming the second cluster, and phylogeny of gene ORF10 forming the third cluster. The results of our horizontal gene transfer and recombination analysis suggest that SARS-Cov-2 could not only be a chimera resulting from recombination of the bat RaTG13 and Guangdong pangolin coronaviruses but also a close relative of the bat CoV ZC45 and ZXC21 strains. They also indicate that a GD pangolin may be an intermediate host of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.12.03.410233","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.405738","pub_date":"2020-12-03","title":"Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples","abstract":"SARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. We used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.","version":"1.2","doi":"10.1101/2020.12.01.405738","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.03.409441","pub_date":"2020-12-03","title":"Identification of low micromolar SARS-CoV-2 Mpro inhibitors from hits identified by in silico screens","abstract":"Mpro, also known as 3CLpro, is the main protease of the SARS-CoV-2 coronavirus and, as such, is essential for the viral life cycle. Two studies have each screened and ranked in silico more than one billion chemical compounds in an effort to identify putative inhibitors of Mpro. More than five hundred of the seven thousand top-ranking hits were synthesized by an external supplier and examined with respect to their activity in two biochemical assays: a protease activity assay and a thermal shift assay. Two clusters of chemical compounds with Mpro inhibitory activity were identified. An additional five hundred molecules, analogues of the compounds in the two clusters described above, were also synthesized and characterized in vitro. The study of the analogues revealed that the compounds of the first cluster acted by denaturing Mpro and might denature other proteins as well. In contrast, the compounds of the second cluster targeted Mpro with much greater specificity and enhanced its melting temperature, consistent with the formation of stable Mpro-inhibitor complexes. The most active compounds of the second cluster exhibited IC50 values between 4 and 7 \u03bcM and their chemical structure suggests that they could serve as leads for the development of potent Mpro inhibitors.","version":"1.1","doi":"10.1101/2020.12.03.409441","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.409037","pub_date":"2020-12-03","title":"SARS-CoV-2 in Brazil: analysis of molecular variance and genetic diversity in viral haplotypes found in the states of Rio de Janeiro, S\u00e3o Paulo, Paran\u00e1 and Tocantins","abstract":"In this work, 18 sequences of the SARS-CoV-2 virus were used, from four Brazilian states (Rio de Janeiro, S\u00e3o Paulo, Paran\u00e1 and Tocantins) with 09, 04, 04, 8 and 01 haplotypes, respectively, with lengths ranging from 234 to 29,903 bp. All sequences were publicly available on the National Biotechnology Information Center (NCBI) platform and were previously aligned with the MEGA X software, where all gaps and ambiguous sites were extracted for the construction of the phylogenetic tree. Of the 301 sites analyzed, 68% varied, 131 of which were parsimonium-informative sites. Phylogenetic analyses revealed the presence of two distinct subgroups, corroborated by the high FST (80%). The high degree of polymorphism found among these samples helped to establish a clear pattern of non-genetic structuring, based on the time of divergence between the groups. All molecular variance estimators confirmed that there was no consensus in the conservation of the studied sequences, also indicating a high variation for the protein products of the virus. In a highly miscegenational and diverse population such as the Brazilian population, this observation draws our attention to the need for an urgent increase in public health actions, awareness strategies, hygiene and distancing practices and not the other way around.","version":"1.1","doi":"10.1101/2020.12.02.409037","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.202275","pub_date":"2020-12-03","title":"Transcriptional response modules characterise IL-1\u03b2 and IL-6 activity in COVID-19","abstract":"Dysregulated IL-1\u03b2 and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1\u03b2 and IL-6 in COVID-19. We show that the expression of IL-1\u03b2 or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1\u03b2 and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood, but is not associated with severity of COVID-19 disease, length of stay or mortality. We propose that IL-1\u03b2 and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.","version":"1.2","doi":"10.1101/2020.07.22.202275","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.407486","pub_date":"2020-12-02","title":"Nasopharyngeal microbial communities of patients infected with SARS-COV-2 that developed COVID-19","abstract":"SARS-CoV-2 is an RNA virus causing COVID-19. The clinical characteristics and epidemiology of COVID-19 have been extensively investigated, however studies focused on the patient\u2019s microbiota are still lacking. In this study, we investigated the nasopharyngeal microbiome composition of patients who developed different severity levels of COVID-19. We performed Rdna-SSU (16S) sequencing from nasopharyngeal swab samples obtained from SARS-CoV-2 positive (56) and negative (18) patients in the province of Alicante (Spain) in their first visit to the hospital. Positive SARS-CoV-2 patients were observed and later categorized in mild (symptomatic without hospitalization), moderate (hospitalization) and severe (admission to ICU). We compared the microbiome diversity and OTU composition among severity groups using Similarity Percentage (SIMPER) analysis and Maaslin2. We also built bacterial co-abundance networks for each group using Fastpar. Statistical analysis indicated differences in the nasopharyngeal microbiome of COVID19 patients. 62 OTUs were found exclusively in SARS-CoV-2 positive patients, mostly classified as members of the phylum Bacteroidetes (18) and Firmicutes (25). OTUs classified as Prevotella were found to be significantly more abundant in patients that developed more severe COVID-19. Furthemore, co-abundance analysis indicated a loss of network complexity among samples from patients that later developed more severe symptoms. Our preliminary study shows that the nasopharyngeal microbiome of COVID-19 patients showed differences in the composition of specific OTUs and complexity of co-abundance networks. These microbes with differential abundances among groups could serve as biomarkers for COVID-19 severity. Nevertheless, further studies with larger sample sizes should be conducted to validate these results. This work has studied the microbiota of the nasopharyngeal tract in COVID19 patients using advanced techniques of molecular microbiology. Diverse microorganisms, most of which are harmless or even beneficial to the host, colonize the nasopharyngeal tract. These microorganisms are the microbiota, and they are present in every people. However, changes in this microbiota could be related to different diseases as cancer, gastrointestinal pathologies or even COVID19. This study has been performed to investigate the microbiota from patients with COVID19, in order to determinate its implication in the pathology severity. The results obtained showed that it is possible that several specific microorganisms are present only in patients with severe COVID19. These data, could be used as a prognostic biomarker to early detect whose patients will develop a severe COVID19 and improve their clinical management.","version":"1.1","doi":"10.1101/2020.12.01.407486","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.408047","pub_date":"2020-12-02","title":"Effective in-vitro inactivation of SARS-CoV-2 by commercially available mouthwashes","abstract":"Infectious SARS-CoV-2 can be recovered from the oral cavities and saliva of COVID-19 patients with potential implications for disease transmission. Reducing viral load in patient saliva using antiviral mouthwashes may therefore have a role as a control measure in limiting virus spread, particularly in dental settings. Here, the efficacy of SARS-CoV-2 inactivation by seven commercially available mouthwashes with a range of active ingredients were evaluated in vitro. We demonstrate \u22654.1 to \u22655.5 log10 reduction in SARS-CoV-2 titre following a one minute treatment with commercially available mouthwashes containing 0.01-0.02% stabilised hypochlorous acid or 0.58% povidone iodine, and non-specialist mouthwashes with both alcohol-based and alcohol-free formulations designed for home use. In contrast, products containing 1.5% hydrogen peroxide or 0.2% chlorhexidine gluconate were ineffective against SARS-CoV-2 in these tests. This study contributes to the growing body of evidence surrounding virucidal efficacy of mouthwashes/oral rinses against SARS-CoV-2, and has important applications in reducing risk associated with aerosol generating procedures in dentistry and potentially for infection control more widely.","version":"1.1","doi":"10.1101/2020.12.02.408047","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.406934","pub_date":"2020-12-02","title":"MTX-COVAB, a human-derived antibody with potent neutralizing activity against SARS-CoV-2 infection in vitro and in a hamster model of COVID-19","abstract":"Fast track microfluidic screening of the antibody repertoires of 12 convalescent COVID-19 donors comprising 2.8mio antibodies yielded MTX-COVAB, a human-derived monoclonal antibody with low picomolar neutralization IC50 of SARS-CoV-2. COVAB neutralization potency is on par with the Regeneron cocktail as demonstrated in a comparative neutralization assay. MTX-COVAB shows strong efficacy in vivo and binds to all currently identified clinically relevant variants of SARS-CoV-2. MTX-COVAB completes GMP manufacturing by the end of this year and will be tested in the clinic in March 2021.","version":"1.1","doi":"10.1101/2020.12.01.406934","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.351353","pub_date":"2020-12-02","title":"Insight into the origin of 5\u2019UTR and source of CpG reduction in SARS-CoV-2 genome","abstract":"SARS-CoV-2, the causative agent of COVID-19, has an RNA genome, which is, overall, closely related to the bat coronavirus sequence RaTG13. However, the ACE2-binding domain of this virus is more similar to a coronavirus isolated from a Guangdong pangolin. In addition to this unique feature, the genome of SARS-CoV-2 (and its closely related coronaviruses) has a low CpG content. This has been postulated to be the signature of an evolutionary pressure exerted by the host antiviral protein ZAP. Here, we analyzed the sequences of a wide range of viruses using both alignment-based and alignment free approaches to investigate the origin of SARS-CoV-2 genome. Our analyses revealed a high level of similarity between the 5\u2019UTR of SARS-CoV-2 and that of the Guangdong pangolin coronavirus. This suggests bat and pangolin coronaviruses might have recombined at least twice (in the 5\u2019UTR and ACE2 binding regions) to seed the formation of SARS-CoV-2. An alternative hypothesis is that the lineage preceding SARS-CoV-2 is a yet to be sampled bat coronavirus whose ACE2 binding domain and 5\u2019UTR are distinct from other known bat coronaviruses. Additionally, we performed a detailed analysis of viral genome compositions as well as expression and RNA binding data of ZAP to show that the low CpG abundance in SARS-CoV-2 is not related to an evolutionary pressure from ZAP.","version":"1.2","doi":"10.1101/2020.10.23.351353","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.408229","pub_date":"2020-12-02","title":"Novel Mutations in NSP1 and PLPro of SARS-CoV-2 NIB-1 Genome Mount for Effective Therapeutics","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the etiologic agent of Coronavirus Disease-2019 (COVID-19), is rapidly accumulating new mutations. Analysis of these mutations is necessary for gaining knowledge regarding different aspects of therapeutic development. Recently, we have reported a Sanger method based genome sequence of a viral isolate named SARS-CoV-2 NIB-1, circulating in Bangladesh. The genome has four novel mutations in V121D, V843F, A889V and G1691C positions. V121D substitution has the potential to destabilize the Non-Structural Protein (NSP-1) which inactivates the type-1 Interferon-induced antiviral system hence this mutant could be the basis of attenuated vaccines against SARS-CoV-V843F, A889V and G1691C are all located in NSP3. G1691C can decrease the flexibility of the protein while V843F and A889V changed the binding pattern of SARS-CoV-2 Papain-Like protease (PLPro) inhibitor GRL0617. V843F PLPro showed reduced affinity for Interferon Stimulating Gene-15 (ISG-15) protein whereas V843F+A889V double mutants exhibited the same binding affinity as wild type PLPro. Here, V843F is a conserved position of PLPro that damaged the structure but A889V, a less conserved residue, most probably neutralized that damage. Mutants of NSP1 could provide attenuated vaccines against coronavirus. Also, these mutations of PLPro could be targeted to develop anti-SARS therapeutics.","version":"1.1","doi":"10.1101/2020.12.02.408229","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.391664","pub_date":"2020-12-02","title":"Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells","abstract":"A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.","version":"1.1","doi":"10.1101/2020.12.02.391664","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.02.408112","pub_date":"2020-12-02","title":"Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro","abstract":"Antivirals targeting SARS-CoV-2 could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PI) against SARS-CoV-2 and their interactions with remdesivir, the only antiviral approved for treatment of COVID-19. HCV PI showed differential potency in VeroE6 cell-based antiviral assays based on detection of the SARS-CoV-2 Spike protein. Linear PI boceprevir, telaprevir and narlaprevir had 50% effective concentrations (EC50) of ~40 \u03bcM. Among macrocyclic PI simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir and deldeprevir, simeprevir had the highest (EC50 15 \u03bcM) and glecaprevir the lowest (EC50 >178 \u03bcM) potency. Acyclic PI asunaprevir and faldaprevir had EC50 of 72 and 23 \u03bcM, respectively. ACH-806, an HCV NS3 protease co-factor NS4A inhibitor, had EC50 of 46 \u03bcM. For selected PI, potency was similar in human hepatoma Huh7.5 cells. Selectivity indexes, based on antiviral and cell viability assays, were highest for linear PI. In combination with remdesivir, linear PI boceprevir and narlaprevir showed antagonism, while macrocyclic PI simeprevir, paritaprevir and grazoprevir showed synergism with drug reduction indexes of up to 27 for simeprevir. Treatment of infected cultures with equipotent concentrations (1-fold EC50) of HCV PI revealed minor differences in barrier to SARS-CoV-2 escape. Complete viral suppression was achieved treating with \u22653-fold EC50 boceprevir or combination of 1-fold EC50 simeprevir with 0.4-fold EC50 remdesivir, not leading to significant viral suppression in single treatments. Considering potency, human plasma concentrations and synergism with remdesivir, simeprevir seemed the most promising compound for optimization of future antiviral treatments of COVID-19.","version":"1.1","doi":"10.1101/2020.12.02.408112","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.160630","pub_date":"2020-12-02","title":"Assessing uncertainty in the rooting of the SARS-CoV-2 phylogeny","abstract":"The rooting of the SARS-CoV-2 phylogeny is important for understanding the origin and early spread of the virus. Previously published phylogenies have used different rootings that do not always provide consistent results. We investigate several different strategies for rooting the SARS-CoV-2 tree and provide measures of statistical uncertainty for all methods. We show that methods based on the molecular clock tend to place the root in the B clade, while methods based on outgroup rooting tend to place the root in the A clade. The results from the two approaches are statistically incompatible, possibly as a consequence of deviations from a molecular clock or excess back-mutations. We also show that none of the methods provide strong statistical support for the placement of the root in any particular edge of the tree. Our results suggest that inferences on the origin and early spread of SARS-CoV-2 based on rooted trees should be interpreted with caution.","version":"1.3","doi":"10.1101/2020.06.19.160630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.407361","pub_date":"2020-12-02","title":"Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms","abstract":"SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola virus and demonstrated activity against SARS-CoV-2 in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in vitro in HeLa cells and of mouse adapted Ebola virus in mouse in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with Kd values of 339 nM and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 hence justifying in vivo evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.","version":"1.1","doi":"10.1101/2020.12.01.407361","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.407015","pub_date":"2020-12-02","title":"Shared B cell memory to coronaviruses and other pathogens varies in human age groups and tissues","abstract":"Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells encoding humoral immune memory. We evaluated convergent antigen-specific antibody genes of similar sequences shared between individuals in pediatric and adult blood, and deceased organ donor tissues. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults\u2019 convergent clones often express mutated IgM or IgD in blood and are class-switched in lymphoid tissues; in contrast, children have abundant class-switched convergent clones in blood. Consistent with serological reports, pre-pandemic children had class-switched convergent clones to SARS-CoV-2, enriched in cross-reactive clones for seasonal coronaviruses, while adults showed few such clones in blood or lymphoid tissues. These results extend age-related and anatomical mapping of human humoral pathogen-specific immunity. Children have elevated frequencies of pathogen-specific class-switched memory B cells, including SARS-CoV-2-binding clones.","version":"1.1","doi":"10.1101/2020.12.01.407015","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.407429","pub_date":"2020-12-02","title":"COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types","abstract":"Common genetic polymorphisms associated with severity of COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Here, we assessed the effects of 679 COVID-19-risk variants on gene expression in a wide-range of immune cell types. Severe COVID-19-risk variants were significantly associated with the expression of 11 protein-coding genes, and overlapped with either target gene promoter or cis-regulatory regions that interact with target promoters in the cell types where their effects are most prominent. For example, we identified that the association between variants in the 3p21.31 risk locus and the expression of CCR2 in classical monocytes is likely mediated through an active cis-regulatory region that interacted with CCR2 promoter specifically in monocytes. The expression of several other genes showed prominent genotype-dependent effects in non-classical monocytes, NK cells, B cells, or specific T cell subtypes, highlighting the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.","version":"1.1","doi":"10.1101/2020.12.01.407429","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.405340","pub_date":"2020-12-01","title":"Role of Long-range Allosteric Communication in Determining the Stability and Disassembly of SARS-COV-2 in Complex with ACE2","abstract":"Severe acute respiratory syndrome (SARS) and novel coronavirus disease (COVID-19) are caused by two closely related beta-coronaviruses, SARS-CoV and SARS-CoV-2, respectively. The envelopes surrounding these viruses are decorated with spike proteins, whose receptor binding domains (RBDs) initiate invasion by binding to the human angiotensin-converting enzyme 2 (ACE2). Subtle changes at the interface with ACE2 seem to be responsible for the enhanced affinity for the receptor of the SARS-CoV-2 RBD compared to SARS-CoV RBD. Here, we use Elastic Network Models (ENMs) to study the response of the viral RBDs and ACE2 upon dissassembly of the complexes. We identify a dominant detachment mode, in which the RBD rotates away from the surface of ACE2, while the receptor undergoes a conformational transition which stretches the active-site cleft. Using the Structural Perturbation Method, we determine the network of residues, referred to as the Allostery Wiring Diagram (AWD), which drives the large-scale motion activated by the detachment of the complex. The AWD for SARS-CoV and SARS-CoV-2 are remarkably similar, showing a network that spans the interface of the complex and reaches the active site of ACE2, thus establishing an allosteric connection between RBD binding and receptor catalytic function. Informed in part by the AWD, we used Molecular Dynamics simulations to probe the effect of interfacial mutations in which SARS-CoV-2 residues are replaced by their SARS-CoV counterparts. We focused on a conserved glycine (G502 in SARS-CoV-2, G488 in SARS-CoV) because it belongs to a region that initiates the dissociation of the complex along the dominant detachment mode, and is prominent in the AWD. Molecular Dynamics simulations of SARS-CoV-2 wild-type and G502P mutant show that the affinity for the human receptor of the mutant is drastically diminished. Our results suggest that in addition to residues that are in direct contact with the interface those involved in long range allosteric communication are also a determinant of the stability of the RBD-ACE2 complex.","version":"1.1","doi":"10.1101/2020.11.30.405340","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.405472","pub_date":"2020-12-01","title":"Prospective mapping of viral mutations that escape antibodies used to treat COVID-19","abstract":"Antibodies are becoming a frontline therapy for SARS-CoV-2, but the risk of viral evolutionary escape remains unclear. Here we map how all mutations to SARS-CoV-2\u2019s receptor-binding domain (RBD) affect binding by the antibodies in Regeneron\u2019s REGN-COV2 cocktail and Eli Lilly\u2019s LY-CoV016. These complete maps uncover a single amino-acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2, as well as in lab viral escape selections. Finally, the maps reveal that mutations escaping each individual antibody are already present in circulating SARS-CoV-2 strains. Overall, these complete escape maps enable immediate interpretation of the consequences of mutations observed during viral surveillance.","version":"1.1","doi":"10.1101/2020.11.30.405472","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.404483","pub_date":"2020-12-01","title":"Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models","abstract":"The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.","version":"1.1","doi":"10.1101/2020.12.01.404483","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.406116","pub_date":"2020-12-01","title":"A pomegranate peel extract as inhibitor of SARS-CoV-2 Spike binding to human ACE2 (in vitro): a promising source of novel antiviral drugs","abstract":"Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes and triterpenes, with potential antiviral activities. As the dramatic outbreak of the pandemic COVID-19, caused by the SARS-CoV-2 virus, thousands of scientists are working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal to discover effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. In this study, by using different in vitro approaches, we uncovered the role of a pomegranate peel extract in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human Angiotensin-Converting Enzyme 2 (ACE2) receptor, and in inhibiting the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results open up new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.12.01.406116","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.405662","pub_date":"2020-12-01","title":"Differential effects of antiseptic mouth rinses on SARS-CoV-2 infectivity in vitro","abstract":"SARS-CoV-2 is detectable in saliva from asymptomatic individuals, suggesting a potential benefit from the use of mouth rinses to suppress viral load and reduce virus spread. Published studies on reduction of SARS-CoV-2-induced cytotoxic effects by antiseptics do not exclude antiseptic-associated cytotoxicity. Here, we determined the effect of commercially available mouth rinses and antiseptic povidone-iodine on the infectivity of SARS-CoV-2 virus and of a non-pathogenic, recombinant, SARS-CoV-2 infection vector (pseudotyped SARS-CoV-2 virus). We first determined the effect of mouth rinses on cell viability to ensure that antiviral activity was not a consequence of mouth rinse-induced cytotoxicity. Colgate Peroxyl (hydrogen peroxide) exhibited the most cytotoxicity, followed by povidone-iodine, chlorhexidine gluconate (CHG), and Listerine (essential oils and alcohol). Potent anti-viral activities of povidone iodine and Colgate peroxyl mouth rinses was the consequence of rinse-mediated cellular damage. The potency of CHG was greater when the product was not washed off after virus attachment, suggesting that the prolonged effect of mouth rinses on cells impacts anti-viral activity. To minimalize mouth rinse-associated cytotoxicity, mouth rinse was largely removed from treated-viruses by centrifugation prior to infection of cells. A 5% (v/v) dilution of Colgate Peroxyl or povidone-iodine completely blocked viral infectivity. A similar 5% (v/v) dilution of Listerine or CHG had a moderate suppressive effect on the virus, but a 50% (v/v) dilution of Listerine or CHG blocked viral infectivity completely. Prolonged incubation of virus with mouth rinses was not required for viral inactivation. Our results indicate that mouth rinses can significantly reduce virus infectivity, suggesting a potential benefit for reducing SARS-CoV-2 spread. SARS-CoV-2 is detectable in saliva from asymptomatic individuals, suggesting the potential necessity for the use of mouth rinses to suppress viral load to reduce virus spread. Published studies on anti-SARS-CoV-2 activities of antiseptics determined by virus-induced cytotoxic effects cannot exclude antiseptic-associated cytotoxicity. We found that all mouth rinses tested inactivated SARS-CoV-2 viruses. Listerine and CHG were less cytotoxic than Colgate Peroxyl or povidone-iodine and were active against the virus. When mouth rinses were present in the cell culture during the infection, the potent anti-viral effect of mouth rinses were in part due to the mouth rinse-associated cytotoxicity. Our results suggest that assessing anti-viral candidates including mouth rinses with minimal potential disruption of cells may help identify active agents that can reduce SARS-CoV-2 spread.","version":"1.1","doi":"10.1101/2020.12.01.405662","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.406025","pub_date":"2020-12-01","title":"The N-glycosylation sites and Glycan-binding ability of S-protein in SARS-CoV-2 Coronavirus","abstract":"The emerging acute respiratory disease, COVID-19, caused by SARS-CoV-2 Coronavirus (SARS2 CoV) has spread fastly all over the word. As a member of RNA viruses, the glycosylation of envelope glycoprotein plays the crucial role in protein folding, evasing host immune system, invading host cell membrane, even affecting host preference. Therefore, detail glyco-related researches have been adopted in the Spike protein (S-protein) of SARS2 CoV from the bioinformatic perspective. Phylogenic analysis of S-protein sequences revealed the evolutionary relationship of N-glycosylation sites in different CoVs. Structural comparation of S-proteins indicated their similarity and distributions of N-glycosylation sites. Further potential sialic acid or galactose affinity domains have been described in the S-protein by docking analysis. Molecular dynamic simulation for the glycosylated complexus of S-protein-ACE2 implied that the complicate viral binding of receptor-binding domain may be influenced by peripheric N-glycans from own and adjacent monoers. These works will contribute to investigate the N-glycosylation in S-protein and explain the highly contagious of COVID-19.","version":"1.1","doi":"10.1101/2020.12.01.406025","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.407007","pub_date":"2020-12-01","title":"Multimodal Single-Cell Omics Analysis of COVID-19 Sex Differences in Human Immune Systems","abstract":"Sex differences in the risk of SARS-CoV-2 infection have been controversial and the underlying mechanisms of COVID-19 sexual dimorphism remain understudied. Here we inspected sex differences in SARS-CoV-2 positivity, hospitalization, admission to the intensive care unit (ICU), sera immune profiling, and two single-cell RNA-sequencing (snRNA-seq) profiles from nasal tissues and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with varying degrees of disease severity. Our propensity score-matching observations revealed that male individuals have a 29% increased likelihood of SARS-CoV-2 positivity, with a hazard ration (HR) 1.32 (95% confidence interval [CI] 1.18-1.48) for hospitalization and HR 1.51 (95% CI 1.24-1.84) for admission to ICU. Sera from male patients at hospital admission had decreased lymphocyte count and elevated inflammatory markers (C-reactive protein, procalcitonin, and neutrophils). We found that SARS-CoV-2 entry factors, including ACE2, TMPRSS2, FURIN and NRP1, have elevated expression in nasal squamous cells from males with moderate and severe COVID-19. Cell-cell network proximity analysis suggests possible epithelium-immune cell interactions and immune vulnerability underlying a higher mortality in males with COVID-19. Monocyte-elevated expression of Toll like receptor 7 (TLR7) and Bruton tyrosine kinase (BTK) is associated with severe outcomes in males with COVID-19. These findings provide basis for understanding immune responses underlying sex differences, and designing sex-specific targeted treatments and patient care for COVID-19.","version":"1.1","doi":"10.1101/2020.12.01.407007","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.406306","pub_date":"2020-12-01","title":"Neutrophil and monocyte dysfunctional effector response towards bacterial challenge in critically-ill COVID-19 patients","abstract":"COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections","version":"1.1","doi":"10.1101/2020.12.01.406306","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.096016","pub_date":"2020-12-01","title":"Transcriptional profiling reveals potential involvement of microvillous TRPM5-expressing cells in viral infection of the olfactory epithelium","abstract":"Understanding viral infection of the olfactory epithelium is essential because the olfactory nerve is an important route of entry for viruses to the central nervous system. Specialized chemosensory epithelial cells that express the transient receptor potential cation channel subfamily M member 5 (TRPM5) are found throughout the airways and intestinal epithelium and are involved in responses to viral infection. Herein we performed deep transcriptional profiling of olfactory epithelial cells sorted by flow cytometry based on the expression of mCherry as a marker for olfactory sensory neurons and for eGFP in OMP-H2B::mCherry/TRPM5-eGFP transgenic mice (Mus musculus). We find profuse expression of transcripts involved in inflammation, immunity and viral infection in TRPM5-expressing microvillous cells. Our study provides new insights into a potential role for TRPM5-expressing microvillous cells in viral infection of the olfactory epithelium. We find that, as found for solitary chemosensory cells (SCCs) and brush cells in the airway epithelium, and for tuft cells in the intestine, the transcriptome of TRPM5-expressing microvillous cells indicates that they are likely involved in the inflammatory response elicited by viral infection of the olfactory epithelium.","version":"1.3","doi":"10.1101/2020.05.14.096016","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.046144","pub_date":"2020-12-01","title":"A scientometric overview of CORD-19","abstract":"As the COVID-19 pandemic unfolds, researchers from all disciplines are coming together and contributing their expertise. CORD-19, a dataset of COVID-19 and coronavirus publications, has been made available along-side calls to help mine the information it contains and to create tools to search it more effectively. We analyse the delineation of the publications included in CORD-19 from a scientometric perspective. Based on a comparison to the Web of Science database, we find that CORD-19 provides an almost complete coverage of research on COVID-19 and coronaviruses. CORD-19 contains not only research that deals directly with COVID-19 and coronaviruses, but also research on viruses in general. Publications from CORD-19 focus mostly on a few well-defined research areas, in particular: coronaviruses (primarily SARS-CoV, MERS-CoV and SARS-CoV-2); public health and viral epidemics; molecular biology of viruses; influenza and other families of viruses; immunology and antivirals; clinical medicine. CORD-19 publications that appeared in 2020, especially editorials and letters, are disproportionately popular on social media. While we fully endorse the CORD-19 initiative, it is important to be aware that CORD-19 extends beyond research on COVID-19 and coronaviruses.","version":"1.3","doi":"10.1101/2020.04.20.046144","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.406611","pub_date":"2020-12-01","title":"Designed proteins assemble antibodies into modular nanocages","abstract":"Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by \u03b1-SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.","version":"1.1","doi":"10.1101/2020.12.01.406611","journal":"bioRxiv","score":null},{"id":"10.1101/2020.12.01.407148","pub_date":"2020-12-01","title":"Mucosal Associated Invariant T (MAIT) Cell Responses Differ by Sex in COVID-19","abstract":"Sexual dimorphisms in immune responses contribute to coronavirus disease 2019 (COVID-19) outcomes, yet the mechanisms governing this disparity remain incompletely understood. We carried out sex-balanced sampling of peripheral blood mononuclear cells from confirmed COVID-19 inpatients and outpatients, uninfected close contacts, and healthy controls for 36-color flow cytometry and single cell RNA-sequencing. Our results revealed a pronounced reduction of circulating mucosal associated invariant T (MAIT) cells in infected females. Integration of published COVID-19 airway tissue datasets implicate that this reduction represented a major wave of MAIT cell extravasation during early infection in females. Moreover, female MAIT cells possessed an immunologically active gene signature, whereas male counterparts were pro-apoptotic. Collectively, our findings uncover a female-specific protective MAIT profile, potentially shedding light on reduced COVID-19 susceptibility in females.","version":"1.1","doi":"10.1101/2020.12.01.407148","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.29.402339","pub_date":"2020-11-30","title":"Recombinant Fc-fusion vaccine of RBD induced protection against SARS-CoV-2 in non-human primate and mice","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to infect people globally. The increased COVID-19 cases and no licensed vaccines highlight the need to develop safe and effective vaccines against SARS-CoV-2 infection. Multiple vaccines candidates are under pre-clinical or clinical trails with different strengths and weaknesses. Here we developed a pilot scale production of a recombinant subunit vaccine (RBD-Fc Vacc) with the Receptor Binding Domain of SARS-CoV-2 S protein fused with the Fc domain of human IgG1. RBD-Fc Vacc induced SARS-CoV-2 specific neutralizing antibodies in non-human primates and human ACE2 transgenic mice. The antibodies induced in macaca fascicularis neutralized three divergent SARS-CoV2 strains, suggesting a broader neutralizing ability. Three times immunizations protected Macaca fascicularis (20ug or 40ug per dose) and mice (10ug or 20ug per dose) from SARS-CoV-2 infection respectively. These data support clinical development of SARS-CoV-2 vaccines for humans. RBD-Fc Vacc is currently being assessed in randomized controlled phase 1/II human clinical trails. This study confirms protective efficacy of a SARS-CoV-2 RBD-Fc subunit vaccine.","version":"1.1","doi":"10.1101/2020.11.29.402339","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.404624","pub_date":"2020-11-30","title":"A Neutralizing Antibody-Conjugated Photothermal Nanoparticle Captures and Inactivates SARS-CoV-2","abstract":"The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research including several clinical trials, currently there are no completely safe or effective therapeutics to cure the disease. Here we report a strategy incorporating neutralizing antibodies conjugated on the surface of a photothermal nanoparticle to actively capture and inactivate SARS-CoV-2. The photothermal nanoparticle is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with neutralizing antibodies. Such nanoparticles displayed efficient capture of SARS-CoV-2 pseudoviruses, excellent photothermal effect, and complete inhibition of viral entry into ACE2-expressing host cells via simultaneous blocking and inactivating of the virus. This photothermal nanoparticle is a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus providing a broad range of protection against multiple strains of SARS-CoV-2.\n\n\n","version":"1.1","doi":"10.1101/2020.11.30.404624","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.404905","pub_date":"2020-11-30","title":"Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain","abstract":"Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.","version":"1.1","doi":"10.1101/2020.11.30.404905","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.29.401984","pub_date":"2020-11-30","title":"Emetine as an antiviral agent suppresses SARS-CoV-2 replication by inhibitinginteraction of viral mRNAwith eIF4E: An in vitro study","abstract":"Emetine is a FDA-approved drug for the treatment of amebiasis. In the recent times we had also demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. Following emergence of the COVID-19, we further evaluated thein vitro antiviral activity of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The therapeutic index of emetine was determined to be 10910.4, at a cytotoxic concentration 50 (CC50) of 1603.8 nM and effective concentration 50 (EC50) of 0.147 nM.Besides, we also demonstrated the protective efficacy of emetine against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment was shown to decrease viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding.In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 RNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation ofprotein translation). Further, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathwayfor its effective replication in the target cells. To conclude, emetine targets SARS-CoV-2 protein synthesis which is mediated via inhibiting the interaction of SARS-CoV-2 RNA with eIF4E. This is a novel mechanistic insight on the antiviral efficacy of emetine. In vitro antiviral efficacy against SARS-CoV-2 and its ability to protect chicken embryos against IBV suggests that emetine could be repurposed to treat COVID-19.","version":"1.1","doi":"10.1101/2020.11.29.401984","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.404384","pub_date":"2020-11-30","title":"pH Effect on the Dynamics of SARS-CoV-2 Main Protease (Mpro)","abstract":"The SARS-CoV-2 main protease (Mpro) is a crucial enzyme responsible for the maturation of novel coronavirus, thus it serves as an excellent target for drug discovery. SARS-CoV-2 is found to have similarity with SARS-CoV, which showed conformational changes upon varying pH. There is no study till date on how pH change affect the conformtional flexibilty of SARS-CoV-2 Mpro, therefore, we attempt to find the effect of pH variation through constant pH molecular dynamics simulation studies. Protein is found to be most stable at neutral pH and as pH turns basic protein structure becomes most destabilized. Acidic pH also tends to change the structural properties of Mpro. Our study provides evidence that the flexibility of Mpro is pH dependent like SARS-CoV Mpro.","version":"1.1","doi":"10.1101/2020.11.30.404384","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.26.20239368","pub_date":"2020-11-30","title":"Estimation and worldwide monitoring of the effective reproductive number of SARS-CoV-2","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The effective reproductive number\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>e</jats:italic>\n                  </jats:sub>\n                  is a key indicator of the growth of an epidemic. Since the start of the SARS-CoV-2 pandemic, many methods and online dashboards have sprung up to monitor this number through time. However, these methods are not always thoroughly tested, correctly placed in time, or are overly confident during high incidence periods. Here, we present a method for timely estimation of\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>e</jats:italic>\n                  </jats:sub>\n                  , applied to COVID-19 epidemic data from 170 countries. We thoroughly evaluate the method on simulated data, and present an intuitive web interface for interactive data exploration. We show that, in early 2020, in the majority of countries the estimated\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>e</jats:italic>\n                  </jats:sub>\n                  dropped below 1 only after the introduction of major non-pharmaceutical interventions. For Europe the implementation of non-pharmaceutical interventions was broadly associated with reductions in the estimated\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>e</jats:italic>\n                  </jats:sub>\n                  . Globally though, relaxing non-pharmaceutical interventions had more varied effects on subsequent\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>e</jats:italic>\n                  </jats:sub>\n                  estimates. Our framework is useful to inform governments and the general public on the status of epidemics in their country, and is used as the official source of\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>e</jats:italic>\n                  </jats:sub>\n                  estimates for SARS-CoV-2 in Switzerland. It further allows detailed comparison between countries and in relation to covariates such as implemented public health policies, mobility, behaviour, or weather data.\n                </jats:p>","version":null,"doi":"10.1101/2020.11.26.20239368","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.27.20240051","pub_date":"2020-11-30","title":"The impact of vaccination on COVID-19 outbreaks in the United States","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Global vaccine development efforts have been accelerated in response to the devastating COVID-19 pandemic. We evaluated the impact of a 2-dose COVID-19 vaccination campaign on reducing incidence, hospitalizations, and deaths in the United States (US).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed an agent-based model of SARS-CoV-2 transmission and parameterized it with US demographics and age-specific COVID-19 outcomes. Healthcare workers and high-risk individuals were prioritized for vaccination, while children under 18 years of age were not vaccinated. We considered a vaccine efficacy of 95% against disease following 2 doses administered 21 days apart achieving 40% vaccine coverage of the overall population within 284 days. We varied vaccine efficacy against infection, and specified 10% pre-existing population immunity for the base-case scenario. The model was calibrated to an effective reproduction number of 1.2, accounting for current non-pharmaceutical interventions in the US.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Vaccination reduced the overall attack rate to 4.6% (95% CrI: 4.3% - 5.0%) from 9.0% (95% CrI: 8.4% - 9.4%) without vaccination, over 300 days. The highest relative reduction (54-62%) was observed among individuals aged 65 and older. Vaccination markedly reduced adverse outcomes, with non-ICU hospitalizations, ICU hospitalizations, and deaths decreasing by 63.5% (95% CrI: 60.3% - 66.7%), 65.6% (95% CrI: 62.2% - 68.6%), and 69.3% (95% CrI: 65.5% - 73.1%), respectively, across the same period.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Our results indicate that vaccination can have a substantial impact on mitigating COVID-19 outbreaks, even with limited protection against infection. However, continued compliance with non-pharmaceutical interventions is essential to achieve this impact.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Key points</jats:title>\n                  <jats:p>Vaccination with a 95% efficacy against disease could substantially mitigate future attack rates, hospitalizations, and deaths, even if only adults are vaccinated. Non-pharmaceutical interventions remain an important part of outbreak response as vaccines are distributed over time.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.11.27.20240051","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.30.403824","pub_date":"2020-11-30","title":"First computational design of Covid-19 coronavirus vaccine using lambda superstrings","abstract":"In this work we have developed, by employing lambda superstrings, a map of candidate vaccines against SARS-CoV-2 with lengths between 9 and 200, based on estimations of the immunogenicity of the epitopes and the binding affinity of epitopes to MHC class I molecules using tools from the IEDB Analysis Resource, as well as the overall predictions obtained using the VaxiJen tool. We have synthesized one of the peptides, specifically the one of length 22, and we have carried out an immunogenicity assay and a cytokine assay, which has given positive results in both cases.","version":"1.1","doi":"10.1101/2020.11.30.403824","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.30.403873","pub_date":"2020-11-30","title":"Multi-parameter formulation development for an HIV-vaccine protein with direct validation of epitope binding integrity and stoichiometry","abstract":"Vaccines are on the front line-of-defense against infectious diseases, ranging from threats we are familiar with, including polio, tuberculosis, or HIV, to novel and emerging threats such as SARS-CoV-2. Successful development of new protein-based vaccines requires sophisticated and efficient development of storage and formulation conditions. Here we demonstrate the combined power of 2bind\u2019s sophisticated buffer matrix FORMOscreen\u00ae and NanoTemper Technologies\u2019 novel Prometheus Panta high-throughput Dynamic Light Scattering/Nano Differential Scanning Fluorimetry instrument. We show that this combination can comprehensively improve critical biophysical parameters for the HIV-1 vaccine BG505-SOSIP and find the optimal formulation condition with unmatched efficiency.","version":"1.1","doi":"10.1101/2020.11.30.403873","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.398008","pub_date":"2020-11-29","title":"Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.","version":"1.2","doi":"10.1101/2020.11.25.398008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.29.402404","pub_date":"2020-11-29","title":"Genome Scale-Differential Flux Analysis reveals deregulation of lung cell metabolism on SARS Cov2 infection","abstract":"The COVID-19 pandemic is posing an unprecedented threat to the whole world. In this regard, it is absolutely imperative to understand the mechanism of metabolic reprogramming of host human cells by SARS Cov2. A better understanding of the metabolic alterations would aid in design of better therapeutics to deal with COVID-19 pandemic. We developed an integrated genome-scale metabolic model of normal human bronchial epithelial cells (NHBE) infected with SARS Cov2 using gene-expression and macromolecular make-up of the virus. The reconstructed model predicts growth rates of the virus in high agreement with the experimental measured values. Furthermore, we report a method for conducting genome-scale differential flux analysis (GS-DFA) in context-specific metabolic models. We apply the method to the context-specific model and identify severely affected metabolic modules predominantly comprising of lipid metabolism. We conduct an integrated analysis of the flux-altered reactions, host-virus protein-protein interaction network and phospho-proteomics data to understand the mechanism of flux alteration in host cells. We show that several enzymes driving the altered reactions inferred by our method to be directly interacting with viral proteins and also undergoing differential phosphorylation under diseased state. In case of SARS Cov2 infection, lipid metabolism particularly fatty acid oxidation and beta-oxidation cycle along with arachidonic acid metabolism are predicted to be most affected which confirms with clinical metabolomics studies. GS-DFA can be applied to existing repertoire of high-throughput proteomic or transcriptomic data in diseased condition to understand metabolic deregulation at the level of flux.","version":"1.1","doi":"10.1101/2020.11.29.402404","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.29.402677","pub_date":"2020-11-29","title":"Deconvoluting the T cell response to SARS-CoV-2: specificity versus chance- and cognate cross-reactivity","abstract":"SARS-CoV-2 infection takes a mild or clinically inapparent course in the majority of humans who contract this virus. After such individuals have cleared the virus, only the detection of SARS-CoV-2-specific immunological memory can reveal the exposure, and hopefully the establishment of immune protection. With most viral infections, the presence of specific serum antibodies has provided a reliable biomarker for the exposure to the virus of interest. SARS-CoV-2 infection, however, does not reliably induce a durable antibody response, especially in sub-clinically infected individuals. Consequently, it is plausible for a recently infected individual to yield a false negative result within only a few months after exposure. Immunodiagnostic attention has therefore shifted to studies of specific T cell memory to SARS-CoV-2. Most reports published so far agree that a T cell response is engaged during SARS-CoV-2 infection, but they also state that in 20-81% of non-SARS-CoV-2-exposed individuals, T cells respond to SARS-CoV-2 antigens (mega peptide pools), allegedly due to T cell cross-reactivity with coronaviruses causing Common Cold (CCC), or other antigens. Here we show that by introducing irrelevant mega peptide pools as negative controls to account for chance cross-reactivity, and by establishing the antigen dose-response characteristic of the T cells, one can clearly discern between cognate T cell memory induced by SARS-CoV-2 infection vs. cross-reactive T cell responses in individuals who had not been infected with SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.29.402677","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.12.294504","pub_date":"2020-11-29","title":"S494 O-glycosylation site on the SARS-COV-2 RBD Affects the Virus Affinity to ACE2 and its Infectivity; A Molecular Dynamics Study","abstract":"SARS-COV-2 is a strain of Coronavirus family which caused the extensive pandemic of COVID-19, which is still going on. Several studies showed that the glycosylation of virus spike (S) protein and the Angiotensin-Converting Enzyme 2 (ACE2) receptor on the host cell is critical for the virus infectivity. Molecular Dynamics (MD) simulations were used to explore the role of a novel mutated O-glycosylation site (D494S) on the Receptor Binding Domain (RBD) of S protein. This site was suggested as a key mediator of virus-host interaction. We showed that the decoration of S494 with elongated O-glycans results in stabilized interactions on the direct RBD-ACE2 interface with more favorable binding free energies for longer oligosaccharides. Hence, this crucial factor must be taken into account for any further inhibitory approaches towards RBD-ACE2 interaction.","version":"1.2","doi":"10.1101/2020.09.12.294504","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.28.400671","pub_date":"2020-11-29","title":"Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (MERS)","abstract":"Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional spill overs into humans. The severity of infection in humans is influenced by numerous factors and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified \u2013 which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on clinical samples from patients with MERS. The data illustrated that whole genome or near whole genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants including deletion mutants. Whereas, the metagenomic analysis provided information of the background microbiome.","version":"1.1","doi":"10.1101/2020.11.28.400671","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.27.401893","pub_date":"2020-11-28","title":"Effect of RBD mutation (Y453F) in spike glycoprotein of SARS-CoV-2 on neutralizing antibody affinity","abstract":"Natural selection \u201cadaptation\u201d in the coronavirus can occur during coronavirus amplification in vivo in farmed minks. Natural selection in such viruses is observed by introduction of mutations in SARS- CoV-2 that are not observed during the growth process in humans. Infection with a mutant (Y453F) of SARS-CoV-2 from farmed minks is known to widely spread among humans. We investigated the virological characteristics of this SARS-CoV-2 mutant (Y453F) using three-dimensional protein structural analysis. Our experimental study suggests that virus variants with the Y453F mutation partially escaped detection by four neutralizing monoclonal antibodies. The spread of SARS-CoV-2 variants mediated by millions of infected farmed minks is uncontrolled; consequently, raising a concern that infection of SARS-CoV-2 mutants that cause serious symptoms in humans may spread globally.","version":"1.1","doi":"10.1101/2020.11.27.401893","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.394288","pub_date":"2020-11-27","title":"Inactivation of SARS-CoV-2 on surfaces and in solution with Virusend (TX-10), a novel disinfectant","abstract":"Until an effective vaccine against SARS-CoV-2 is available on a widespread scale, the control of the COVID-19 pandemic is reliant upon effective pandemic control measures. The ability of SARS-CoV-2 to remain viable on surfaces and in aerosols, means indirect contact transmission can occur and so there is an opportunity to reduce transmission using effective disinfectants in public and communal spaces. Virusend (TX-10), a novel disinfectant, has been developed as a highly effective disinfectant against a range of microbial agents. Here we investigate the ability of Virusend (TX-10) to inactivation SARS-CoV-2. Using surface and solution inactivation assays, we show that Virusend (TX-10) is able to reduce SARS-CoV-2 viral titre by 4log10 PFU/mL within 1 minute of contact. Ensuring disinfectants are highly effective against SARS-CoV-2 is important in eliminating environmental sources of the virus to control the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.11.25.394288","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.399139","pub_date":"2020-11-27","title":"Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection","abstract":"The efficacy of virus-specific T cells in clearing pathogens involves a fine balance between their antiviral and inflammatory features. SARS-CoV-2-specific T cells in individuals who clear SARS-CoV-2 infection without symptoms or disease could reveal non-pathological yet protective characteristics. We therefore compared the quantity and function of SARS-CoV-2-specific T cells in a cohort of asymptomatic individuals (n=85) with that of symptomatic COVID-19 patients (n=76), at different time points after antibody seroconversion. We quantified T cells reactive to structural proteins (M, NP and Spike) using ELISpot assays, and measured the magnitude of cytokine secretion (IL-2, IFN-\u03b3, IL-4, IL-6, IL-1\u03b2, TNF-\u03b1 and IL-10) in whole blood following T cell activation with SARS-CoV-2 peptide pools as a functional readout. Frequencies of T cells specific for the different SARS-CoV-2 proteins in the early phases of recovery were similar between asymptomatic and symptomatic individuals. However, we detected an increased IFN-\u03b3 and IL-2 production in asymptomatic compared to symptomatic individuals after activation of SARS-CoV-2-specific T cells in blood. This was associated with a proportional secretion of IL-10 and pro-inflammatory cytokines (IL-6, TNF-\u03b1 and IL-1\u03b2) only in asymptomatic infection, while a disproportionate secretion of inflammatory cytokines was triggered by SARS-CoV-2-specific T cell activation in symptomatic individuals. Thus, asymptomatic SARS-CoV-2 infected individuals are not characterized by a weak antiviral immunity; on the contrary, they mount a robust and highly functional virus-specific cellular immune response. Their ability to induce a proportionate production of IL-10 might help to reduce inflammatory events during viral clearance.","version":"1.1","doi":"10.1101/2020.11.25.399139","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.26.400390","pub_date":"2020-11-27","title":"A Novel Cell Therapy for COVID-19 and Potential Future Pandemics: Virus Induced Lymphocytes (VIL)","abstract":"The a priori T cell repertoire and immune response against SARS-CoV-2 viral antigens may explain the varying clinical course and prognosis of patients having a mild COVID-19 infection as opposed to those developing more fulminant multisystem organ failure and associated mortality. Using a novel SARS-Cov-2-specific artificial antigen presenting cell (aAPC), coupled with a rapid expansion protocol (REP) as practiced in tumor infiltrating lymphocytes (TIL) therapy, we generate an immune catalytic quantity of Virus Induced Lymphocytes (VIL). Using T cell receptor (TCR)-specific aAPCs carrying co-stimulatory molecules and major histocompatibility complex (MHC) class-I immunodominant SARS-CoV-2 peptide-pentamer complexes, we expand virus-specific VIL derived from peripheral blood mononuclear cells (PBMC) of convalescent COVID-19 patients up to 1,000-fold. This is achieved in a clinically relevant 7-day vein-to-vein time-course as a potential adoptive cell therapy (ACT) for COVID-19. We also evaluate this approach for other viral pathogens using Cytomegalovirus (CMV)-specific VIL from donors as a control. Rapidly expanded VIL are enriched in virus antigen-specificity and show an activated, polyfunctional cytokine profile and T effector memory phenotype which may contribute to a robust immune response. Virus-specific T cells can also be delivered allogeneically via MHC-typing and patient human leukocyte antigen (HLA)-matching to provide pragmatic treatment in a large-scale therapeutic setting. These data suggest that VIL may represent a novel therapeutic option that warrants further clinical investigation in the armamentarium against COVID-19 and other possible future pandemics.","version":"1.1","doi":"10.1101/2020.11.26.400390","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.25.965582","pub_date":"2020-11-27","title":"Screening of FDA-approved drugs using a MERS-CoV clinical isolate from South Korea identifies potential therapeutic options for COVID-19","abstract":"Therapeutic options for coronavirus remain limited. To address this unmet medical need, we screened 5,406 compounds, including United States Food and Drug Administration (FDA)- approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6. Time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI>100), atovaquone, an anti-malarial (TI>34), and ciclosonide, an inhalable corticosteroid (TI>6). Furthermore, utilizing the severe acute respiratory syndrome CoV-2 (SARS-CoV-2), combinations of remedesivir with selected dugs were evaluated, which identified ciclosonide and nelfinavir to be additive and synergistic drugs in vitro, respectively. Together, we screened FDA-approved drugs using patient-derived MERS-CoV, triaged hits to discriminate between early and late viral life cycle inhibitors, confirmed selected drugs using SARS-CoV-2, and demonstrated the added value of selected medications in combination with remedesivir. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.","version":"1.3","doi":"10.1101/2020.02.25.965582","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.398859","pub_date":"2020-11-26","title":"Pharmacologic profiling reveals lapatinib as a novel antiviral against SARS-CoV-2 in vitro","abstract":"The emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but an effective antiviral therapy has yet to be discovered. To improve treatment options, we conducted a high-throughput drug repurposing screen to uncover compounds that block the viral activity of SARS-CoV-2. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed at 10 dose points (ranging from 20 \u03bcM to 1 nM). Our screening revealed several FDA-approved agents that act as novel antivirals that block both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 replication by over 50,000-fold without any toxicity and at doses readily achievable in human tissues. Further, both lapatinib and doramapimod could be combined with remdesivir to dramatically improve antiviral activity in cells. These findings reveal novel treatment options for people infected with SARS-CoV-2 that can be readily implemented during the pandemic.","version":"1.1","doi":"10.1101/2020.11.25.398859","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.399055","pub_date":"2020-11-26","title":"Triple Combination Nitazoxanide, Ribavirin, and Hydroxychloroquine results in the multiplicative reduction of in vitro SARS-CoV-2 viral replication","abstract":"An immediate unmet medical need exists to test and develop existing approved drugs against SARS-COV-2. Despite many efforts, very little progress has been made regarding finding low-cost oral medicines that can be made widely available worldwide to address the global pandemic. We sought to examine if a triple combination of nitazoxanide (using its active metabolite tizoxanide), ribavirin, and hydroxychloroquine would lead to a multiplicative effects on viral replication of SARS-COV-2 resulting in a significant reduction of virus yield using VERO E6 cells as a model of viral replication. Virus yield measured in PFU/ml was ~ 2 logs lower with triple combination versus either drug alone, resulting in the prolongation of time to peak cytopathic effects (CPE). The time to produce 50% CPE increased from 2.8 days for viral controls versus 5.3 days for triple combination therapy. Finally, for each 1-log reduction in virus yield 24 hours post-infection, there was an additional 0.7-day delay in onset of CPE. A triple combination of tizoxanide, ribavirin, and hydroxychloroquine produced a reduction in SARS-COV-2 viral replication in Vero E6 cells, warranting exploration in additional cell lines as well as human clinical trials.","version":"1.1","doi":"10.1101/2020.11.25.399055","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.25.398578","pub_date":"2020-11-25","title":"SARS-CoV-2 utilizes a multipronged strategy to suppress host protein synthesis","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. Here, we use RNA-sequencing and ribosome profiling along SARS-CoV-2 infection and comprehensively define the mechanisms that are utilized by SARS-CoV-2 to shutoff cellular protein synthesis. We show SARS-CoV-2 infection leads to a global reduction in translation but that viral transcripts are not preferentially translated. Instead, we reveal that infection leads to accelerated degradation of cytosolic cellular mRNAs which facilitates viral takeover of the mRNA pool in infected cells. Moreover, we show that the translation of transcripts whose expression is induced in response to infection, including innate immune genes, is impaired, implying infection prevents newly transcribed cellular mRNAs from accessing the ribosomes. Overall, our results uncover the multipronged strategy employed by SARS-CoV-2 to commandeer the translation machinery and to suppress host defenses.","version":"1.1","doi":"10.1101/2020.11.25.398578","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.389627","pub_date":"2020-11-25","title":"The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus in the subgenus Sarbecovirus causes a respiratory disease with varying symptoms referred to as coronavirus disease 2019 (COVID-19) and is responsible for a pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, and infection and fatality numbers continuing to increase globally, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against positive- and negative-sense RNA viruses. This compound class inhibits eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. The synthetic rocaglate CR-31-B (-) has previously been shown to inhibit the replication of human coronaviruses, such as HCoV-229E and MERS-CoV, as well as Zika-, Lassa-, Crimean Congo hemorrhagic fever virus in primary cells. Here, we assessed the antiviral activity of CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In African green monkey Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In line with this, viral protein accumulation and replication/transcription complex formation were found to be strongly reduced by this compound. In an ex vivo infection system using human airway epithelial cells, CR-31-B (-) was found to cause a massive reduction of SARS-CoV-2 titers by about 4 logs to nearly non-detectable levels. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.","version":"1.2","doi":"10.1101/2020.11.24.389627","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.396671","pub_date":"2020-11-25","title":"Evolutionary analysis of SARS-CoV-2 spike protein for its different clades","abstract":"The spike protein of SARS-CoV-2 has become the main target for antiviral and vaccine development. Despite its relevance, there is scarce information about its evolutionary traces. The aim of this study was to investigate the diversification patterns of the spike for each clade of SARS-CoV-2 through different approaches. Two thousand and one hundred sequences representing the seven clades of the SARS-CoV-2 were included. Patterns of genetic diversifications and nucleotide evolutionary rate were estimated for the spike genomic region. The haplotype networks showed a star shape, where multiple haplotypes with few nucleotide differences diverge from a common ancestor. Four hundred seventy nine different haplotypes were defined in the seven analyzed clades. The main haplotype, named Hap-1, was the most frequent for clades G (54%), GH (54%), and GR (56%) and a different haplotype (named Hap-252) was the most important for clades L (63.3%), O (39.7%), S (51.7%), and V (70%). The evolutionary rate for the spike protein was estimated as 1.08 x 10\u22123 nucleotide substitutions/site/year. Moreover, the nucleotide evolutionary rate after nine months of pandemic was similar for each clade. In conclusion, the present evolutionary analysis is relevant since the spike protein of SARS-CoV-2 is the target for most therapeutic candidates; besides, changes in this protein could have consequences on viral transmission, response to antivirals and efficacy of vaccines. Moreover, the evolutionary characterization of clades improves knowledge of SARS-CoV-2 and deserves to be assessed in more detail since re-infection by different phylogenetic clades has been reported.","version":"1.1","doi":"10.1101/2020.11.24.396671","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.360578","pub_date":"2020-11-25","title":"Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables easy, fast and accurate outbreak analysis in hospital and community settings","abstract":"Current transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole-genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report the use of reverse complement polymerase chain reaction (RC-PCR), a novel technology for WGS of SARS-CoV-2 enabling library preparation in a single PCR saving time, resources and enables high throughput screening. Additionally, we show SARS-CoV-2 diversity and possible transmission within the Radboud university medical center (Radboudumc) during September 2020 using RC-PCR WGS. A total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were selected for whole-genome sequencing. Ct values of the samples ranged from 16 to 42. They were collected from 83 healthcare workers and three patients at the Radboudumc, in addition to 64 people living in the area around the hospital and tested by the local health services. For validation purposes, nineteen of the included samples were previously sequenced using Oxford Nanopore Technologies and compared to RC-PCR WGS results. The applicability of RC-PCR WGS in outbreak analysis for public health service and hospitals was tested on six suspected clusters containing samples of healthcare workers and patients with an epidemiological link. RC-PCR resulted in sequencing data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Comparison to Oxford Nanopore technologies gives a near-perfect agreement on 95% of the samples (18 out of 19). Three out of six clusters with a suspected epidemiological link were fully confirmed, in the others, four healthcare workers were not associated. In the public health service samples, a previously unknown chain of transmission was confirmed. SAR-CoV-2 whole-genome sequencing using RC-PCR is a reliable technique and applicable for use in outbreak analysis and surveillance. Its ease of use, high-trough screening capacity and wide applicability makes it a valuable addition or replacement during this ongoing SARS-CoV-2 pandemic. None At present whole genome sequencing techniques for SARS-CoV-2 have a large turnover time and are not widely available. Only a few laboratories are currently able to perform large scale SARS-CoV-2 sequencing. This restricts the use of sequencing to aid hospital and community infection prevention. Here we present clinical and technical data on a novel Whole Genome Sequencing technology, implementing reverse-complement PCR. It is able to obtain high genome coverage of SARS-CoV-2 and confirm and exclude epidemiological links in 173 healthcare workers and patients. The RC-PCR technology simplifies the workflow thereby reducing hands on time. It combines targeted PCR and sequence library construction in a single PCR, which normally takes several steps. Additionally, this technology can be used in concordance with the widely available range of Illumina sequencers. RC-PCR whole genome sequencing technology enables rapid and targeted surveillance and response to an ongoing outbreak that has great impact on public health and society. Increased use of sequencing technologies in local laboratories can help prevent increase of SARS-CoV-2 spreading by better understanding modes of transmission.","version":"1.2","doi":"10.1101/2020.10.29.360578","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.20.392126","pub_date":"2020-11-24","title":"Timing the SARS-CoV-2 Index Case in Hubei Province","abstract":"Understanding when SARS-CoV-2 emerged is critical to evaluating our current approach to monitoring novel zoonotic pathogens and understanding the failure of early containment and mitigation efforts for COVID-19. We employed a coalescent framework to combine retrospective molecular clock inference with forward epidemiological simulations to determine how long SARS-CoV-2 could have circulated prior to the time of the most recent common ancestor. Our results define the period between mid-October and mid-November 2019 as the plausible interval when the first case of SARS-CoV-2 emerged in Hubei province. By characterizing the likely dynamics of the virus before it was discovered, we show that over two-thirds of SARS-CoV-2-like zoonotic events would be self-limited, dying out without igniting a pandemic. Our findings highlight the shortcomings of zoonosis surveillance approaches for detecting highly contagious pathogens with moderate mortality rates.","version":"1.1","doi":"10.1101/2020.11.20.392126","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.393629","pub_date":"2020-11-24","title":"Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2","abstract":"Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10-fold of effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the RBD, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19. SARS-CoV-2 specific antibody, HB27, blocks viral receptor binding and membrane fusion HB27 confers prophylactic and therapeutic protection against SARS-CoV-2 in mice models Rhesus macaques showed no adverse side effects when administered with HB27 Cryo-EM studies suggest that HB27 sterically occludes SARS-CoV-2 from its receptor","version":"1.1","doi":"10.1101/2020.11.24.393629","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.23.20236828","pub_date":"2020-11-24","title":"HIV status alters disease severity and immune cell responses in\n                  <i>\u03b2</i>\n                  variant SARS-CoV-2 infection wave","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  There are conflicting reports on the effects of HIV on COVID-19. Here we analyzed disease severity and immune cell changes during and after SARS-CoV-2 infection in 236 participants from South Africa, of which 39% were people living with HIV (PLWH), during the first and second (\n                  <jats:italic>\u03b2</jats:italic>\n                  dominated) infection waves. The second wave had more PLWH requiring supplemental oxygen relative to HIV negative participants. Higher disease severity was associated with low CD4 T cell counts and higher neutrophil to lymphocyte ratios (NLR). Yet, CD4 counts recovered and NLR stabilized after SARS-CoV-2 clearance in wave 2 infected PLWH, arguing for an interaction between SARS-CoV-2 and HIV infection leading to low CD4 and high NLR. The first infection wave, where severity in HIV negative and PLWH was similar, still showed some HIV modulation of SARS-CoV-2 immune responses. Therefore, HIV infection can synergize with the SARS-CoV-2 variant to change COVID-19 outcomes.\n                </jats:p>","version":null,"doi":"10.1101/2020.11.23.20236828","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.24.395079","pub_date":"2020-11-24","title":"A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro","abstract":"We recently discovered a superantigen-like motif, similar to Staphylococcal enterotoxin B (SEB), near the S1/S2 cleavage site of SARS-CoV-2 Spike protein, which might explain the multisystem-inflammatory syndrome (MIS-C) observed in children and cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif, and in particular its PRRA insert, to inhibit infection by blocking the access of host cell proteases, TMPRSS2 or furin, to the cleavage site. The high affinity of 6D3 for the furin-cleavage site originates from a poly-acidic segment at its heavy chain CDR2, a feature shared with SARS-CoV-2-neutralizing mAb 4A8. The affinity of 6D3 and 4A8 for this site points to their potential utility as therapeutics for treating COVID-19, MIS-C, or common cold caused by human coronaviruses (HCoVs) that possess a furin-like cleavage site.","version":"1.1","doi":"10.1101/2020.11.24.395079","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.21.392605","pub_date":"2020-11-24","title":"Phycobilins as potent food bioactive broad-spectrum inhibitor compounds against Mpro and PLpro of SARS-CoV-2 and other coronaviruses: A preliminary Study","abstract":"In the twenty first century, we have witnessed three corona virus outbreaks; SARS in 2003, MERS in 2012 and ongoing pandemic COVID-19. To prevent outbreaks by novel mutant strains, we need broad-spectrum antiviral agents that are effective against wide array of coronaviruses. In this study, we scientifically investigated potent food bioactive broad-spectrum antiviral compounds by targeting Mpro and PLpro proteases of CoVs using in silico and in vitro approaches. The results revealed that phycocyanobilin (PCB) showed potential inhibitor activity against both proteases. PCB had best binding affinity to Mpro and PLpro with IC50 values of 71 \u03bcm and 62 \u03bcm, respectively. In addition, in silico studies of Mpro and PLpro enzymes of other human and animal CoVs indicated broad spectrum inhibitor activity of the PCB. Like PCB, other phycobilins such as phycourobilin (PUB), Phycoerythrobilin (PEB) and Phycoviolobilin (PVB) showed similar binding affinity to SARS-CoV-2 Mpro and PLpro","version":"1.1","doi":"10.1101/2020.11.21.392605","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.396028","pub_date":"2020-11-24","title":"Protective face mask filter capable of inactivating SARS-CoV-2, and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis","abstract":"Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating neither SARS-CoV-2 nor multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2 mediated pneumonia disease complex and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a novel protective non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating SARS-CoV-2 in one minute of contact, and the life-threatening methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many health care workers and researchers working in this urgent and challenging field.","version":"1.1","doi":"10.1101/2020.11.24.396028","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.24.393405","pub_date":"2020-11-24","title":"Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking","abstract":"The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.","version":"1.1","doi":"10.1101/2020.11.24.393405","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.133355","pub_date":"2020-11-24","title":"Design of a Companion Bioinformatic Tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants","abstract":"Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. This work provides a framework able to track down SARS-CoV-2 genomic variability.","version":"1.2","doi":"10.1101/2020.06.22.133355","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.22.389056","pub_date":"2020-11-24","title":"Stable neutralizing antibody levels six months after mild and severe COVID-19 episode","abstract":"Understanding mid-term kinetics of immunity to SARS-CoV-2 is the cornerstone for public health control of the pandemic and vaccine development. However, current evidence is rather based on limited measurements, thus losing sight of the temporal pattern of these changes. In this longitudinal analysis, conducted on a prospective cohort of COVID-19 patients followed up to 242 days, we found that individuals with mild or asymptomatic infection experienced an insignificant decay in neutralizing activity that persisted six months after symptom onset or diagnosis. Hospitalized individuals showed higher neutralizing titers, which decreased following a two-phase pattern, with an initial rapid decline that significantly slowed after day 80. Despite this initial decay, neutralizing activity at six months remained higher among hospitalized individuals. The slow decline in neutralizing activity at mid-term contrasted with the steep slope of antibody titers change, reinforcing the hypothesis that the quality of immune response evolves over the post-convalescent stage.","version":"1.2","doi":"10.1101/2020.11.22.389056","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.23.20236968","pub_date":"2020-11-24","title":"Deciphering early-warning signals of SARS-CoV-2 elimination and resurgence from limited data at multiple scales","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Inferring the transmission potential of an infectious disease during low-incidence periods following epidemic waves is crucial for preparedness. In such periods, scarce data may hinder existing inference methods, blurring early-warning signals essential for discriminating between the likelihoods of resurgence versus elimination. Advanced insight into whether elevating caseloads (requiring swift community-wide interventions) or local elimination (allowing controls to be relaxed or refocussed on case-importation) might occur, can separate decisive from ineffective policy. By generalising and fusing recent approaches, we propose a novel early-warning framework that maximises the information extracted from low-incidence data to robustly infer the chances of sustained local-transmission or elimination in real time, at any scale of investigation (assuming sufficiently good surveillance). Applying this framework, we decipher hidden disease-transmission signals in prolonged low-incidence COVID-19 data from New Zealand, Hong Kong and Victoria, Australia. We uncover how timely interventions associate with averting resurgent waves, support official elimination declarations and evidence the effectiveness of the rapid, adaptive COVID-19 responses employed in these regions.</jats:p>","version":null,"doi":"10.1101/2020.11.23.20236968","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.23.20233155","pub_date":"2020-11-24","title":"Disaggregating Asian Race Reveals COVID-19 Disparities among Asian Americans at New York City\u2019s Public Hospital System","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>There is growing recognition of the burden of COVID-19 among Asian Americans, but data on outcomes among Asian ethnic subgroups remain extremely limited. We conducted a retrospective analysis of 85,328 patients tested for COVID-19 at New York City\u2019s public hospital system between March 1 and May 31, 2020, to describe characteristics and COVID-19 outcomes of Asian ethnic subgroups compared to Asians overall and other racial/ethnic groups. South Asians had the highest rates of positivity and hospitalization among Asians, second only to Hispanics for positivity and Blacks for hospitalization. Chinese patients had the highest mortality rate of all groups and were nearly 1.5 times more likely to die than Whites. The high burden of COVID-19 among South Asian and Chinese Americans underscores the urgent needs for improved data collection and reporting as well as public health program and policy efforts to mitigate the disparate impact of COVID-19 among these communities.</jats:p>","version":null,"doi":"10.1101/2020.11.23.20233155","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.23.395301","pub_date":"2020-11-24","title":"Type I and III IFNs produced by the nasal epithelia and dimmed inflammation are key features of alpacas resolving MERS-CoV infection","abstract":"While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, provoking the induction of interferon stimulated genes (ISGs) along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-\u03baB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, is central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10. Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of a respiratory disease causing high mortality in humans. In camelids, the main MERS-CoV reservoir host, viral infection leads to subclinical disease. Our study describes transcriptional regulations of innate immunological pathways underlying asymptomatic clinical manifestations of alpacas in response to MERS-CoV. Concomitant to the peak of infection, these animals elicited a strong transient interferon response and induction of the anti-inflammatory cytokine IL10 in the nasal mucosa. This was associated to a dimmed regulation of pro-inflammatory cytokines and induction of interferon stimulated genes along the whole respiratory mucosa, leading to the rapid clearance of the virus. Thus, innate immune responses occurring in the nasal mucosa appear to be the key in controlling MERS-CoV disease by avoiding a cytokine storm. Understanding on how asymptomatic host reservoirs counteract MERS-CoV infection will aid in the development of antiviral drugs and vaccines.","version":"1.1","doi":"10.1101/2020.11.23.395301","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.21.392407","pub_date":"2020-11-23","title":"N439K variant in spike protein may alter the infection efficiency and antigenicity of SARS-CoV-2 based on molecular dynamics simulation","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing an outbreak of coronavirus disease 2019 (COVID-19), has been undergoing various mutations. The analysis of the structural and energetic effects of mutations on protein-protein interactions between the receptor binding domain (RBD) of SARS-CoV-2 and angiotensin converting enzyme 2 (ACE2) or neutralizing monoclonal antibodies will be beneficial for epidemic surveillance, diagnosis, and optimization of neutralizing agents. According to the molecular dynamics simulation, a key mutation N439K in the SARS-CoV-2 RBD region created a new salt bridge which resulted in greater electrostatic complementarity. Furthermore, the N439K-mutated RBD bound hACE2 with a higher affinity than wild-type, which may lead to more infectious. In addition, the N439K-mutated RBD was markedly resistant to the SARS-CoV-2 neutralizing antibody REGN10987, which may lead to the failure of neutralization. These findings would offer guidance on the development of neutralizing antibodies and the prevention of COVID-19.","version":"1.1","doi":"10.1101/2020.11.21.392407","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.23.394114","pub_date":"2020-11-23","title":"A Nasal Spray Solution of Grapefruit Seed Extract plus Xylitol Displays Virucidal Activity Against SARS-Cov-2 In Vitro","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the ongoing pandemic coronavirus disease 2019 (COVID-19) has triggered worldwide concerted efforts in an attempt to identify effective therapies. In the present study, we have identified two candidate agents with potential activity against SARS-CoV-2 which can be administered intranasally, namely, xylitol and grape seed fruit extract (GSE). A commercially available nasal spray (Xlear) combining xylitol and GSE has been available for years, but the antiviral effects of this solution have not been documented. This in vitro study examined the virucidal effect of Xlear against SARS-CoV-2. To this end, two independent sets of experiments were carried out to test the hypothesis that Xlear is an effective (Experiment I) and replicable (Experiment II) means to deactivate SARS-CoV-2. When tested against SARS-CoV-2, the test compound GSE 0.2% was the only compound effective at reducing >3 log10 CCID50 infectious virus from, 3.67 log10 CCID50/0.1 mL to an undetectable amount of infectious virus. The present results validated by two independent sets of experiments, performed by different labs, on different viral strains, provide early evidence to encourage further pilot and clinical studies aimed at investigating the use of Xlear as a potential treatment for COVID-19","version":"1.1","doi":"10.1101/2020.11.23.394114","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.21.392753","pub_date":"2020-11-23","title":"Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19","abstract":"Two proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.","version":"1.1","doi":"10.1101/2020.11.21.392753","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.22.393587","pub_date":"2020-11-23","title":"A Synthetic Defective Interfering SARS-CoV-2","abstract":"Viruses thrive by exploiting the cells they infect but must also produce viral proteins to replicate and infect other cells. As a consequence, they are also susceptible to exploitation by defective versions of themselves that do not produce such proteins. A defective viral genome with deletions in protein-coding genes could still replicate in cells coinfected with full-length viruses, and even replicate faster due to its shorter size, interfering with the replication of the virus. We have created a synthetic defective interfering version of SARS-CoV-2, the virus causing the recent Covid-19 pandemic, assembling parts of the viral genome that do not code for any functional protein but enable it to be replicated and packaged. This synthetic defective genome replicates three times faster than SARS-CoV-2 in coinfected cells, and interferes with it, reducing the viral load of a cell by half in 24 hours. The synthetic genome is transmitted as efficiently as the full-length genome, confirming the location of the putative packaging signal of SARS-CoV-2. A version of such a synthetic construct could be used as a self-promoting antiviral therapy: by enabling replication of the synthetic genome, the virus promotes its own demise.\n\nGraphic summary\n\n","version":"1.1","doi":"10.1101/2020.11.22.393587","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.22.393009","pub_date":"2020-11-23","title":"In-Silico analysis reveals lower transcription efficiency of C241T variant of SARS-CoV-2 with host replication factors MADP1 and HNRNP-1","abstract":"Novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has claimed more than 1.5 million lives worldwide and counting. As per the GISAID database, the genomics of SARS-CoV2 is extensively studied with more than 500 genome submissions per day. Out of several hotspot mutations within the SARS-CoV-2 genome, researchers have focused a lot on missense variants but the least work is done on the UTRs. One of the most frequent 5\u2019 UTR variants in the SARS-CoV-2 genome is the C241T with a global frequency of more than 0.9. In the present study, the effect of the C241T mutation has been studied with respect to change in RNA structure and its interaction with the host replication factors MADP1 Zinc finger CCHC-type and RNA-binding motif 1 (hnRNP1). The results obtained from molecular docking and molecular dynamics simulation indicated weaker interaction of C241T mutant stem loops with host transcription factor MADP1 indicating reduced replication efficiency. The results are also correlated with increased recovery rates and decreased death rates of global SARS-CoV-2 cases.","version":"1.1","doi":"10.1101/2020.11.22.393009","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.378422","pub_date":"2020-11-23","title":"Inhibition of SARS-CoV-2 main protease by allosteric drug-binding","abstract":"The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.11.12.378422","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.21.392670","pub_date":"2020-11-23","title":"A repurposed, blood gene signature is associated with poor outcomes in SARS-CoV-2","abstract":"Poor outcomes after SARS-CoV-2 infection are difficult to predict. Survivors may develop pulmonary fibrosis. We previously identified a 52-gene signature in peripheral blood, predictive of mortality in Idiopathic Pulmonary Fibrosis. In this study, we analyzed this signature in SARS-CoV-2 infected individuals and identified genomic risk profiles with significant differences in outcomes. Analysis of single cell expression data shows that monocytes, red blood cells, neutrophils and dendritic cells are the cellular source of the high risk gene signature.","version":"1.1","doi":"10.1101/2020.11.21.392670","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.23.394312","pub_date":"2020-11-23","title":"The multidisciplinary nature of COVID-19 research","abstract":"We analyzed the scientific output after COVID-19 and contrasted it with studies published in the aftermath of seven epidemics/pandemics: Severe Acute Respiratory Syndrome (SARS), Influenza A virus H5N1 and Influenza A virus H1N1 human infections, Middle East Respiratory Syndrome (MERS), Ebola virus disease, Zika virus disease, and Dengue. We examined bibliometric measures for COVID-19 and the rest of studied epidemics/pandemics. Data were extracted from Web of Science, using its journal classification scheme as a proxy to quantify the multidisciplinary coverage of scientific output. We proposed a novel Thematic Dispersion Index (TDI) for the analysis of pandemic early stages. The literature on the seven epidemics/pandemics before COVID-19 has shown explosive growth of the scientific production and continuous impact during the first three years following each emergence or re-emergence of the specific infectious disease. A subsequent decline was observed with the progressive control of each health emergency. We observed an unprecedented growth in COVID-19 scientific production. TDI measured for COVID-19 (29,4) in just six months, was higher than TDI of the rest (7,5 to 21) during the first three years after epidemic initiation. COVID-19 literature showed the broadest subject coverage, which is clearly a consecuence of its social, economic, and political impact. The proposed indicator (TDI), allowed the study of multidisciplinarity, differentiating the thematic complexity of COVID-19 from the previous seven epidemics/pandemics. The multidisciplinary nature and thematic complexity of COVID-19 research were successfully analyzed through a scientometric perspective.","version":"1.1","doi":"10.1101/2020.11.23.394312","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.21.392639","pub_date":"2020-11-22","title":"Anti-COVID-19 efficacy of ivermectin in the golden hamster","abstract":"The devastating coronavirus disease 2019 (COVID-19) pandemic, due to SARS-CoV-2, has caused more than 47 million confirmed cases and more than 1.2 million human deaths around the globe, and most of the severe cases of COVID-19 in humans are associated with neurological symptoms such as anosmia and ageusia, and uncontrolled inflammatory immune response. Among therapeutic options, the use of the anti-parasitic drug ivermectin (IVM), has been proposed, given its possible anti-SARS-CoV-2 activity. Ivermectin is a positive allosteric modulator of the \u03b1-7 nicotinic acetylcholine receptor, which has been suggested to represent a target for the control of Covid-19 infection, with a potential immunomodulatory activity. We assessed the effects of IVM in SARS-CoV-2-intranasally-inoculated golden Syrian hamsters. Even though ivermectin had no effect on viral load, SARS-Cov-2-associated pathology was greatly attenuated. IVM had a sex-dependent and compartmentalized immunomodulatory effect, preventing clinical deterioration and reducing olfactory deficit in infected animals. Importantly, ivermectin dramatically reduced the Il-6/Il-10 ratio in lung tissue, which likely accounts for the more favorable clinical presentation in treated animals. Our data support IVM as a promising anti-COVID-19 drug candidate.","version":"1.1","doi":"10.1101/2020.11.21.392639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.20.392381","pub_date":"2020-11-22","title":"Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate","abstract":"SARS-CoV-2 has initiated a global pandemic and vaccines are being rapidly developed. Using the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the ability of prior infection or vaccine-induced immunity to protect against infection in ferrets. Ferrets were semi-permissive to infection with the USA-WA1/2020 isolate. When transmission was assessed via the detection of vRNA at multiple timepoints, direct contact transmission was efficient to 3/3 and 3/4 contact animals in two respective studies, while respiratory transmission was poor to only 1/4 contact animals. To assess the durability of immunity, ferrets were re-challenged 28 or 56 days post-primary infection. Following viral challenge, no infectious virus was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared. To determine if intramuscular vaccination protected ferrets against infection, ferrets were vaccinated using a prime-boost strategy with the S-protein receptor-binding domain formulated with an oil-in-water adjuvant. Upon viral challenge, none of the mock or vaccinated animals were protected against infection, and there were no significant differences in vRNA or infectious virus titers in the nasal wash. Combined these studies demonstrate that in ferrets direct contact is the predominant mode of transmission of the SARS-CoV-2 USA-WA1/2020 isolate and immunity to SARS-CoV-2 is maintained for at least 56 days. Our studies also indicate protection of the upper respiratory tract against SARS-CoV-2 will require vaccine strategies that mimic natural infection or induce site-specific immunity. The SARS-CoV-2 USA-WA1/2020 strain is a CDC reference strain used by multiple research laboratories. Here, we show the predominant mode of transmission of this isolate in ferrets is by direct contact. We further demonstrate ferrets are protected against re-infection for at least 56 days even when levels of neutralizing antibodies are low or undetectable. Last, we show that when ferrets were vaccinated by the intramuscular route to induce antibodies against SARS-CoV-2, ferrets remain susceptible to infection of the upper respiratory tract. Collectively, these studies suggest protection of the upper respiratory tract will require vaccine approaches that mimic natural infection.","version":"1.1","doi":"10.1101/2020.11.20.392381","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.19.389726","pub_date":"2020-11-20","title":"Anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) potency of Mefloquine as an entry inhibitor in vitro","abstract":"Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 \u03bcM, IC90 = 2.31 \u03bcM, and IC99 = 4.39 \u03bcM in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.","version":"1.1","doi":"10.1101/2020.11.19.389726","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079061","pub_date":"2020-11-20","title":"Unsupervised cluster analysis of SARS-CoV-2 genomes reflects its geographic progression and identifies distinct genetic subgroups of SARS-CoV-2 virus","abstract":"Over 10,000 viral genome sequences of the SARS-CoV-2 virus have been made readily available during the ongoing coronavirus pandemic since the initial genome sequence of the virus was released on the open access Virological website (http://virological.org/) early on January 11. We utilize the published data on the single stranded RNAs of 11, 132 SARS-CoV-2 patients in the GISAID (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) database, which contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Among many important research questions which are currently being investigated, one aspect pertains to the genetic characterization/classification of the virus. We analyze data on the nucleotide sequencing of the virus and geographic information of a subset of 7, 640 SARS-CoV-2 patients without missing entries that are available in the GISAID database. Instead of modelling the mutation rate, applying phylogenetic tree approaches, etc., we here utilize a model-free clustering approach that compares the viruses at a genome-wide level. We apply principal component analysis to a similarity matrix that compares all pairs of these SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index (Jaccard, 1901; Tan et al., 2005; Prokopenko et al., 2016; Schlauch et al., 2017). Our analysis results of the SARS-CoV-2 genome data illustrates the geographic and chronological progression of the virus, starting from the first cases that were observed in China to the current wave of cases in Europe and North America. This is in line with a phylogenetic analysis which we use to contrast our results. We also observe that, based on their sequence data, the SARS-CoV-2 viruses cluster in distinct genetic subgroups. It is the subject of ongoing research to examine whether the genetic subgroup could be related to diseases outcome and its potential implications for vaccine development.","version":"1.3","doi":"10.1101/2020.05.05.079061","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388991","pub_date":"2020-11-20","title":"Production of anti-SARS-CoV-2 hyperimmune globulin from convalescent plasma","abstract":"In late 2019, the SARS-CoV-2 virus emerged in China and quickly spread into a world-wide pandemic. Prior to the development of specific drug therapies or a vaccine, more immediately available treatments were sought including convalescent plasma. A potential improvement from convalescent plasma could be the preparation of anti-SARS-CoV-2 hyperimmune globulin (hIVIG). Convalescent plasma was collected from an existing network of plasma donation centers. A caprylate/chromatography purification process was used to manufacture hIVIG. Initial batches of hIVIG were manufactured in a versatile, small-scale facility designed and built to rapidly address emerging infectious diseases. Processing convalescent plasma into hIVIG resulted in a highly purified IgG product with more concentrated neutralizing antibody activity. hIVIG will allow for the administration of greater antibody activity per unit of volume with decreased potential for several adverse events associated with plasma administration. IgG concentration and IgG antibody specific to SARS-CoV-2 were increased over 10-fold from convalescent plasma to the final product. Normalized ELISA activity (per mg/mL IgG) was maintained throughout the process. Protein content in these final product batches was 100% IgG, consisting of 98% monomer and dimer forms. Potentially hazardous proteins (IgM, IgA, and anti-A, anti-B and anti-D antibodies) were reduced to minimal levels. Multiple batches of anti-SARS-CoV-2 hyperimmune globulin (hIVIG) that met regulatory requirements were manufactured from human convalescent plasma. The first clinical study in which the hIVIG will be evaluated will be Inpatient Treatment with Anti-Coronavirus Immunoglobulin (ITAC) [NCT04546581].","version":"1.2","doi":"10.1101/2020.11.18.388991","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.19.390187","pub_date":"2020-11-20","title":"AI-Driven Multiscale Simulations Illuminate Mechanisms of SARS-CoV-2 Spike Dynamics","abstract":"We develop a generalizable AI-driven workflow that leverages heterogeneous HPC resources to explore the time-dependent dynamics of molecular systems. We use this workflow to investigate the mechanisms of infectivity of the SARS-CoV-2 spike protein, the main viral infection machinery. Our workflow enables more efficient investigation of spike dynamics in a variety of complex environments, including within a complete SARS-CoV-2 viral envelope simulation, which contains 305 million atoms and shows strong scaling on ORNL Summit using NAMD. We present several novel scientific discoveries, including the elucidation of the spike\u2019s full glycan shield, the role of spike glycans in modulating the infectivity of the virus, and the characterization of the flexible interactions between the spike and the human ACE2 receptor. We also demonstrate how AI can accelerate conformational sampling across different systems and pave the way for the future application of such methods to additional studies in SARS-CoV-2 and other molecular systems. Lorenzo Casalino1\u2020, Abigail Dommer1\u2020, Zied Gaieb1\u2020, Emilia P. Barros1, Terra Sztain1, Surl-Hee Ahn1, Anda Trifan2,3, Alexander Brace2, Anthony Bogetti4, Heng Ma2, Hyungro Lee5, Matteo Turilli5, Syma Khalid6, Lillian Chong4, Carlos Simmerling7, David J. Hardy3, Julio D. C. Maia3, James C. Phillips3, Thorsten Kurth8, Abraham Stern8, Lei Huang9, John McCalpin9, Mahidhar Tatineni10, Tom Gibbs8, John E. Stone3, Shantenu Jha5, Arvind Ramanathan2\u2217, Rommie E. Amaro1\u2217. 2020. AI-Driven Multiscale Simulations Illuminate Mechanisms of SARS-CoV-2 Spike Dynamics. In Supercomputing \u201920: International Conference for High Performance Computing, Networking, Storage, and Analysis. ACM, New York, NY, USA, 14 pages. https://doi.org/finalDOI","version":"1.1","doi":"10.1101/2020.11.19.390187","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.11.037473","pub_date":"2020-11-20","title":"Multiscale 3-dimensional pathology findings of COVID-19 diseased lung using high-resolution cleared tissue microscopy","abstract":"The study of pulmonary samples from individuals who have died as a direct result of COVID-19 infection is vital to our understanding of the pathogenesis of this disease. Histopathologic studies of lung tissue from autopsy of patients with COVID-19 specific mortality are only just emerging. All existing reports have relied on traditional 2-dimensional slide-based histological methods for specimen preparation. However, emerging methods for high-resolution, massively multiscale imaging of tissue microstructure using fluorescence labeling and tissue clearing methods enable the acquisition of tissue histology in 3-dimensions, that could open new insights into the nature of SARS-Cov-2 infection and COVID-19 disease processes. In this article, we present the first 3-dimensional images of lung autopsy tissues taken from a COVID-19 patient, including 3D \u201cvirtual histology\u201d of cubic-millimeter volumes of the diseased lung, providing unique insights into disease processes contributing to mortality that could inform frontline treatment decisions.","version":"1.3","doi":"10.1101/2020.04.11.037473","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.365551","pub_date":"2020-11-20","title":"COVID-19 genetic risk and Neanderthals: A case study highlighting the importance of scrutinizing diversity","abstract":"Recent genome wide association studies (GWAS) have identified genetic risk factors for developing severe COVID-19 symptoms. The first published study reported a 1bp insertion rs11385942 on chromosome 3 (1) and subsequent studies single nucleotide variants (SNVs) such as rs35044562, rs67959919 (2) and rs13078854 (3), all highly correlated with each other. Zeberg and P\u00e4\u00e4bo (4) subsequently traced them back to Neanderthal origin. They found that a 49.4 kb genomic region including the risk allele of rs35044562 is inherited from Neanderthals of Vindija in Croatia. Here we add a differently focused evaluation of this major genetic risk factor to these recent analyses. We show that (i) COVID-19-related genetic factors of three previously assessed Neanderthals deviate from those of modern humans and that (ii) they differ among world-wide human populations, which compromises risk prediction in non-Europeans. Currently, caution is thus advised in the genetic risk assessment of non-Europeans during this world-wide COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.11.02.365551","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.20.391011","pub_date":"2020-11-20","title":"Modeling the frequency and number of persons to test to detect and control COVID-19 outbreaks in congregate settings","abstract":"Congregate settings are at risk for coronavirus disease 2019 (COVID-19) outbreaks. Diagnostic testing can be used as a tool in these settings to identify outbreaks and to control transmission. We used transmission modeling to estimate the minimum number of persons to test and the optimal frequency to detect small outbreaks of COVID-19 in a congregate facility. We also estimated the frequency of testing needed to interrupt transmission within a facility. The number of people to test and frequency of testing needed depended on turnaround time, facility size, and test characteristics. Parameters are calculated for a variety of scenarios. In a facility of 100 people, 26 randomly selected individuals would need to be tested at least every 6 days to identify a true underlying prevalence of at least 5%, with test sensitivity of 85%, and greater than 95% outbreak detection sensitivity. Disease transmission could be interrupted with universal, facility-wide testing with rapid turnaround every three days. Testing a subset of individuals in congregate settings can improve early detection of small outbreaks of COVID-19. Frequent universal diagnostic testing can be used to interrupt transmission within a facility, but its efficacy is reliant on rapid turnaround of results for isolation of infected individuals.","version":"1.1","doi":"10.1101/2020.11.20.391011","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.20.391318","pub_date":"2020-11-20","title":"Arena3Dweb: Interactive 3D visualization of multilayered networks","abstract":"Efficient integration and visualization of heterogeneous biomedical information in a single view is a key challenge. In this study, we present Arena3Dweb, the first, fully interactive and dependency-free, web application which allows the visualization of multilayered graphs in 3D space. With Arena3Dweb, users can integrate multiple networks in a single view along with their intra- and inter-layer connections. For clearer and more informative views, users can choose between a plethora of layout algorithms and apply them on a set of selected layers either individually or in combination. Users can align networks and highlight node topological features, whereas each layer as well as the whole scene can be translated, rotated and scaled in 3D space. User-selected edge colors can be used to highlight important paths, while node positioning, coloring and resizing can be adjusted on-the-fly. In its current version, Arena3Dweb supports weighted and unweighted undirected graphs and is written in R, Shiny and JavaScript. We demonstrate the functionality of Arena3Dweb using two different use-case scenarios; one regarding drug repurposing for SARS-CoV-2 and one related to GPCR signaling pathways implicated in melanoma. Arena3Dweb is available at http://bib.fleming.gr:3838/Arena3D.","version":"1.1","doi":"10.1101/2020.11.20.391318","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.389312","pub_date":"2020-11-19","title":"Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance","abstract":"Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.","version":"1.1","doi":"10.1101/2020.11.18.389312","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388850","pub_date":"2020-11-19","title":"SARS-CoV-2 infection suppresses ACE2 function and antiviral immune response in the upper respiratory tract of infected patients","abstract":"There is an urgent need to elucidate the molecular mechanisms underlying the transmissibility and pathogenesis of SARS-CoV-2. ACE2 is a host ectopeptidase with well-described anti-inflammatory and tissue protective functions and the receptor for the virus. Understanding SARS-CoV-2-ACE2 interaction and the expression of antiviral host genes in early infection phase is crucial for fighting the pandemic. We tested the significance of soluble ACE2 enzymatic activity longitudinally in positive nasopharyngeal swabs at two time points after symptom consultation, along with gene expression profiles of ACE2, its proteases, ADAM17 and TMPRRS2, and interferon-stimulated genes (ISGs), DDX58, CXCL10 and IL-6. Soluble ACE2 activity decreased during infection course, in parallel to ACE2 gene expression. On the contrary, SARS-CoV-2 infection induced expression of the ISG genes in positive SARS-CoV-2 samples at baseline compared to negative control subjects, although this increase wanes with time. These changes positively correlated with viral load. Our results demonstrate the existence of mechanisms by which SARS-CoV-2 suppress ACE2 expression and function casting doubt on the IFN-induced upregulation of the receptor. Moreover, we show that initial intracellular viral sensing and subsequent ISG induction is also rapidly downregulated. Overall, our results offer new insights into ACE2 dynamics and inflammatory response in the human upper respiratory tract that may contribute to understand the early antiviral host response to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.11.18.388850","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388819","pub_date":"2020-11-18","title":"COVID-19-associated olfactory dysfunction reveals SARS-CoV-2 neuroinvasion and persistence in the olfactory system","abstract":"While recent investigations have revealed viral, inflammatory and vascular factors involved in SARS-CoV-2 lung pathogenesis, the pathophysiology of neurological disorders in COVID-19 remains poorly understood. Yet, olfactory and taste dysfunction are rather common in COVID-19, especially in pauci-symptomatic patients which constitutes the most frequent clinical manifestation of the infection. We conducted a virologic, molecular, and cellular study of the olfactory system from COVID-19 patients presenting acute loss of smell, and report evidence that the olfactory epithelium represents a highly significant infection site where multiple cell types, including olfactory sensory neurons, support cells and immune cells, are infected. Viral replication in the olfactory epithelium is associated with local inflammation. Furthermore, we show that SARS-CoV-2 induces acute anosmia and ageusia in golden Syrian hamsters, both lasting as long as the virus remains in the olfactory epithelium and the olfactory bulb. Finally, olfactory mucosa sampling in COVID-19 patients presenting with persistent loss of smell reveals the presence of virus transcripts and of SARS-CoV-2-infected cells, together with protracted inflammation. Viral persistence in the olfactory epithelium therefore provides a potential mechanism for prolonged or relapsing symptoms of COVID-19, such as loss of smell, which should be considered for optimal medical management and future therapeutic strategies.","version":"1.1","doi":"10.1101/2020.11.18.388819","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.259531","pub_date":"2020-11-18","title":"Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures","abstract":"SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.","version":"1.3","doi":"10.1101/2020.08.20.259531","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.387555","pub_date":"2020-11-18","title":"Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters","abstract":"An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS \u0394capB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS \u0394capB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing \u03b2-coronaviruses.","version":"1.1","doi":"10.1101/2020.11.17.387555","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.387902","pub_date":"2020-11-18","title":"Sequential ER stress and inflammatory responses are induced by SARS-CoV-2 ORF3 through ERphagy","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have resulted in a number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, diseases and therapy remains limited, underlining the urgency of fundamental studies and drug development. Studies have shown that induction of autophagy and hijacking of autophagic machinery are essential for infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3 as an inducer of autophagy and revealed that ORF3 localizes to the ER and induces FAM134B-related ERphagy through the HMGB1-Beclin1 pathway. As a consequence, ORF3 induces ER stress and inflammatory responses through ERphagy and sensitizes cells to ER stress-induced cell death, suggesting that SARS-CoV-2 ORF3 hijacks ERphagy and then harms ER homeostasis to induce inflammatory responses through excessive ER stress. These findings reveal a sequential induction of ERphagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and provide therapeutic potential for ERphagy and ER stress-related drugs for COVID-19 treatment and prevention. SARS-CoV-2 infection and replication require autophagosome-like double-membrane vacuoles. Inhibition of autophagy suppresses viral replication, indicating the essential role of autophagy in SARS-CoV-2 infection. However, how SARS-CoV-2 hijacks autophagy and the function of autophagy in the disease progression remain unknown. Here, we reveal that SARS-CoV-2 ORF3 induces ERphagy and consequently induces ER stress to trigger acute inflammatory responses and enhance sensitivity to ER stress-induced apoptosis. Our studies uncover ERphagy-induced inflammatory responses during SARS-CoV-2 infection and provide a promising therapeutic approach for treating SARS-CoV-2 infection and inflammatory responses in COVID-19 by manipulating autophagy and ER stress.","version":"1.1","doi":"10.1101/2020.11.17.387902","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.377713","pub_date":"2020-11-18","title":"In silico analyses on the comparative sensing of SARS-CoV-2 mRNA by intracellular TLRs of human","abstract":"The worldwide outbreak of COVID-19 pandemic caused by SARS-CoV-2 leads to loss of mankind and global economic stability. The continuous spreading of the disease and its pathogenesis takes millions of lives of peoples and the unavailability of appropriate therapeutic strategy makes it much more severe. Toll-like receptors (TLRs) are the crucial mediators and regulators of host immunity. The role of several TLRs in immunomodulation of host by SARS-CoV-2 is recently demonstrated. However, the functionality of human intracellular TLRs including TLR3,7,8 and 9 is still being untested for sensing of viral RNA. This study is hoped to rationalize the comparative binding and sensing of SARS-CoV-2 mRNA towards the intracellular TLRs, considering the solvent-based force-fields operational in the cytosolic aqueous microenvironment that predominantly drive these reactions. Our in-silico study on the binding of all mRNAs with the intracellular TLRs shown that the mRNA of NSP10, S2, and E proteins of SARS-CoV-2 are potent enough to bind with TLR3, TLR9, and TLR7 and trigger downstream cascade reactions, and may be used as an option for validation of therapeutic option and immunomodulation against COVID-19.","version":"1.2","doi":"10.1101/2020.11.11.377713","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.377432","pub_date":"2020-11-18","title":"An assessment of efficacy of Iodine complex (Renessans) against SARS-CoV-2 in non-human primates (Rhesus macaque)","abstract":"Renessans is an iodine complex which has proven in vitro antiviral activity including Anti-SARS-CoV-2 activity. The present study was designed to determine its efficacy against SARS-CoV-2 in monkeys (Rhesus macaque). A total of 14 monkeys were divided into four groups: A) Prophylactic group (n=03), (B) Treatment group (n=03), (C) infection control group (n=04) and (D) negative control group (n=04) and were housed in BSL-3 Animal facility while group D was housed at another animal house. Group A was administered with Renessans @ 2.85 mg/7 kg from 5 days prior to the infection to 08 days post infections (DPI). Group B was administered with Renessans from 03-08 DPI @ 2.85 mg/7 kg. Group C was administered with WIF only. The infection @ 2 \u00d7 106 TCID of SARS-CoV-2 was given to all group monkeys through intranasal and oral route under anesthesia. Nasal swab samples (at different times) and fecal matter on daily basis were collected for the detection of SARS-CoV-2 through real-time quantitative PCR. Three monkeys (one from each of group A, B and C) were euthanized at 07 DPI to determine the gross pathological lesions and SARS-CoV-2 detection from internal tissues. Nasal swabs from all the monkeys from group A, B and C were positive for SARS-CoV-2 at 02 and 07 DPI (Day 05 of treatment). At 14 DPI, all (100%) nasal swabs from group A were negative for SARS-CoV-2 while 50% and 100% were positive from group B and C, respectively. At 21 DPI, monkeys from group B were negative and all in group C were still positive for SARS-CoV-2. Similarly, fecal matter of monkeys in group A and B was returned negative in significantly lesser time as compared to monkeys from infection control group. Based on these research findings it is concluded that the Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2. Therefore, a clinical trial of the drug in COVID-19 patients may reveal its anti-COVID-19 potential.","version":"1.1","doi":"10.1101/2020.11.17.377432","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388140","pub_date":"2020-11-18","title":"Detecting SARS-CoV-2 variants with SNP genotyping","abstract":"Tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020- and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 76% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with our marker panel at a cost of < \u00a31.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.","version":"1.1","doi":"10.1101/2020.11.18.388140","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.20231597","pub_date":"2020-11-18","title":"Declining Trend in the Initial SARS-CoV-2 Viral Load During the Pandemic: Preliminary Observations from Detroit, Michigan","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>We report a downward trend in the initial SARS-CoV-2 viral load in nasopharyngeal swab samples of hospitalized patients with COVID-19 in Detroit, Michigan, coinciding with a decrease in the number of deaths during April-June 2020. A gradual decrease in the initial viral load reflected the downward progression of the pandemic.</jats:p>","version":null,"doi":"10.1101/2020.11.16.20231597","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.18.388934","pub_date":"2020-11-18","title":"An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines","abstract":"The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2020.11.18.388934","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388413","pub_date":"2020-11-18","title":"Long-chain polyphosphates impair SARS-CoV-2 infection and replication: a route for therapy in man","abstract":"Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme (ACE)2 and RNA-dependent RNA polymerase (RdRp) amino acids. We thus tested PolyPs for functional interactions in vitro in SARS-CoV-2\u2013infected Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/RdRP complex, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.","version":"1.1","doi":"10.1101/2020.11.18.388413","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388868","pub_date":"2020-11-18","title":"Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays","abstract":"The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights:\n\nImproved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters\nTwo versions of RBD show different sensitivity in serology assays\nYields of greater than 50 mg/l obtained under optimal conditions\nMagnetic bead purification technology improves throughput of protein production\n Improved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters Two versions of RBD show different sensitivity in serology assays Yields of greater than 50 mg/l obtained under optimal conditions Magnetic bead purification technology improves throughput of protein production","version":"1.1","doi":"10.1101/2020.11.18.388868","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.378992","pub_date":"2020-11-18","title":"The expression of PDCD1 and CD274 in T cells and macrophages correlated positively with COVID-19 severity","abstract":"The immune responses underlying the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To help understand the pathology of coronavirus disease 2019 (COVID-19) pandemics, public data were analyzed and the expression of PDCD1 (encoding PD-1) and CD274 (encoding PD-L1) in T cells and macrophages were identified to correlate positively with COVID-19 severity.","version":"1.1","doi":"10.1101/2020.11.17.378992","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388983","pub_date":"2020-11-18","title":"HLA class I genotypes customize vaccination strategies in immune simulation to combat COVID-19","abstract":"Memory CD8+ T cells are associated with a better outcome in Coronavirus Disease 2019 (COVID-19) and recognized as promising vaccine targets against viral infections. This study determined the efficacy of population-dominant and infection-relevant human leukocyte antigens (HLA) class I proteins to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides through calculating binding affinities and simulating CD8+ T cell responses. As a result, HLA class I proteins distinguished or shared various viral peptides derived from viruses. HLA class I supertypes clustered viral peptides through recognizing anchor and preferred residues. SARS-CoV-2 peptides overlapped significantly with SARS but minimally with common human coronaviruses. Immune simulation of CD8+ T cell activation using predicted SARS-CoV-2 peptide antigens depended on high-affinity peptide binding, anchor residue interaction, and synergistic presentation of HLA class I proteins in individuals. Results demonstrated that multi-epitope vaccination, employing a strong binding affinity, viral adjuvants, and heterozygous HLA class I genes, induced potent immune responses. Therefore, optimal CD8+ T cell responses can be achieved and customized contingent on HLA class I genotypes in human populations, supporting a precise vaccination strategy to combat COVID-19.","version":"1.1","doi":"10.1101/2020.11.18.388983","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388280","pub_date":"2020-11-18","title":"Single-cell Transcriptome of Bronchoalveolar Lavage Fluid Reveals Dynamic Change of Macrophages During SARS-CoV-2 Infection in Ferrets","abstract":"Although the profile of immune cells changes during the natural course of SARS-CoV-2 inflection in human patients, few studies have used a longitudinal approach to reveal their dynamic features. Here, we performed single-cell RNA sequencing of bronchoalveolar lavage fluid cells longitudinally obtained from SARS-CoV-2-infected ferrets. Landscape analysis of the lung immune microenvironment showed dynamic changes in cell proportions and characteristics in uninfected control, at 2 days post-infection (dpi) (early stage of SARS-CoV-2 infection with peak viral titer), and 5 dpi (resolution phase). NK cells and CD8+ T cells exhibited activated subclusters with interferon-stimulated features, which were peaked at 2 dpi. Intriguingly, macrophages were classified into 10 distinct subpopulations, and their relative proportions changed over the time. We observed prominent transcriptome changes among monocyte-derived infiltrating macrophages and differentiated M1/M2 macrophages, especially at 2 dpi. Moreover, trajectory analysis revealed gene expression changes from monocyte-derived infiltrating macrophages toward M1 or M2 macrophages and identified the distinct macrophage subpopulation that had rapidly undergone SARS-CoV-2-mediated activation of inflammatory responses. Finally, we found that different spectrums of M1 or M2 macrophages showed distinct patterns of gene modules downregulated by immune-modulatory drugs. Overall, these results elucidate fundamental aspects of the immune response dynamics provoked by SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.11.18.388280","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.387571","pub_date":"2020-11-18","title":"In-vitro virucidal activity of hypothiocyanite and hypothiocyanite/lactoferrin mix against SARS-CoV-2","abstract":"SARS-CoV-2 replicates efficiently in the upper airway during prodromal stage with resulting viral shedding into the environment from patients with active disease as well as from asymptomatic individuals. So far, virus spread has been exclusively contained by non-pharmacological interventions (social distancing, face masks, hand washing and several measures limiting business activities or movement of individuals). There is a need to find pharmacological interventions to mitigate the viral spread, supporting yet limiting the existing health protection measures while an effective and safe vaccine will hopefully become available. Hypothiocyanite and lactoferrin as part of the innate human immune system were shown to have a large spectrum of cidal activity against bacteria, fungi and viruses. To test their virucidal activity against SARS-CoV-2 we conducted an in-vitro study. Here we show a dose-dependent virucidal activity of hypothiocyanite at micromolar concentrations, slightly improved by the presence of lactoferrin. The two substances are devoid of any cytotoxicity and may be administered combined by aerosol to exploit their antiviral activity at the port of entry (mouth, nasal cavity, conjunctiva) or exit (mouth, through emission of respiratory droplets) of SARS-CoV-2 in the human body. Furthermore, aerosol with hypothiocyanite and lactoferrin combined could also have a therapeutic effect in the lower respiratory tract, at the level of gas exchange units of the lung, preventing the devastating infection of alveolar type II cells where ACE2 is highly expressed. An in-vivo validation of in-vitro results is urgently required.","version":"1.1","doi":"10.1101/2020.11.17.387571","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.381079","pub_date":"2020-11-18","title":"Brief Report: The Virucidal Efficacy of Oral Rinse Components Against SARS-CoV-2 In Vitro","abstract":"The ability of widely-available mouthwashes to inactivate SARS-CoV-2 in vitro was tested using a protocol capable of detecting a 5-log10 reduction in infectivity, under conditions mimicking the naso/oropharynx. During a 30 second exposure, two rinses containing cetylpyridinium-chloride and a third with ethanol/ethyl lauroyl arginate eliminated live virus to EN14476 standards (>4-log10 reduction), while others with ethanol/essential oils and povidone-iodine (PVP-I) eliminated virus by 2-3-log10. Chlorhexidine or ethanol alone displayed little or no ability to inactivate virus. Studies are warranted to determine whether these formulations can inactivate virus in the human oropharynx in vivo, and whether this might impact transmission risk.","version":"1.2","doi":"10.1101/2020.11.13.381079","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.089409","pub_date":"2020-11-18","title":"An integrated in silico immuno-genetic analytical platform provides insights into COVID-19 serological and vaccine targets","abstract":"The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a major global health and socio-economic burden. It has instigated the mobilisation of resources into the development of control tools, such as diagnostics and vaccines. The poor performance of some diagnostic serological tools has emphasised the need for up to date immune-informatic analyses to inform the selection of viable targets for further study. This requires the integration and analysis of genetic and immunological data for SARS-CoV-2 and its homology with other human coronavirus species to understand cross-reactivity. We have developed an online \u201cimmuno-analytics\u201d resource to facilitate SARS-CoV-2 research, combining an extensive B/T-cell epitope mapping and prediction meta-analysis, and human CoV sequence homology mapping and protein database annotation, with an updated variant database and geospatial tracking for >7,800 non-synonymous mutation positions derived from >150,000 whole genome sequences. To demonstrate its utility, we present an integrated analysis of SARS-CoV-2 spike and nucleocapsid proteins, both being vaccine and serological diagnostic targets, including an analysis of changes in relevant mutation frequencies over time. Our analysis reveals that the nucleocapsid protein in its native form appears to be a sub-optimal target for use in serological diagnostic platforms. The most frequent mutations were the spike protein D614G and nsp12 L314P, which were common (>86%) across all the geographical regions. Some mutations in the spike protein (e.g. A222V and L18F) have increased in frequency in Europe during the latter half of 2020, detected using our automated algorithms. The tool also suggests that orf3a proteins may be a suitable alternative target for diagnostic serologic assays in a post-vaccine surveillance setting. The immuno-analytics tool can be accessed online (http://genomics.lshtm.ac.uk/immuno) and will serve as a useful resource for biological discovery and surveillance in the fight against SARS-CoV-2. Further, the tool may be adapted to inform on biological targets in future outbreaks, including potential emerging human coronaviruses that spill over from animal hosts.","version":"1.2","doi":"10.1101/2020.05.11.089409","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.18.388645","pub_date":"2020-11-18","title":"Formulation of a composite nasal spray enabling enhanced surface coverage and prophylaxis of SARS-COV-2","abstract":"Airborne pathogens pose high risks in terms of both contraction and transmission within the respiratory pathways, in particular the nasal region. Although knowledge of airborne transmission has long been known, there is little in the way of adequate intervention that can protect the individual, or even prevent further spread. This study focuses on a nasal applicant with the capacity to combat such issues, by focussing on the SARS-CoV-2 virus. Formulation of a spray containing polysaccharides known for their mucoadhesive properties was undertaken and characterised for their mechanical, spray patterns and antiviral properties. The ability to engineer key behaviours such as yielding have been shown, through systematic understanding of a composite mixture containing two polymers: gellan and \u03bbcarrageenan. Furthermore, spray systems demonstrated highly potent antiviral capacities, resulting in complete inhibition of the virus when studied for both prophylaxis and prevention of spread. Finally, a mechanism has been proposed to explain such findings. Therefore, demonstrating the first fully preventative device, targeted to protect the lining of the upper respiratory pathways.","version":"1.1","doi":"10.1101/2020.11.18.388645","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.386003","pub_date":"2020-11-17","title":"Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.16.386003","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.386532","pub_date":"2020-11-17","title":"The Development of a Novel Nanobody Therapeutic for SARS-CoV-2","abstract":"Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD with a Kd of 15.7picomolar (\u223c3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND50 of 0.16microgram/milliliter (\u223c6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic. Potent and low-cost Nanosota-1 drugs block SARS-CoV-2 infections both in vitro and in vivo and act both preventively and therapeutically.","version":"1.1","doi":"10.1101/2020.11.17.386532","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.385252","pub_date":"2020-11-17","title":"Maturation and persistence of the anti-SARS-CoV-2 memory B cell response","abstract":"Memory B cells play a fundamental role in host defenses against viruses, but to date, their role have been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 Spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including preexisting cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late remarkably stable memory B-cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.","version":"1.1","doi":"10.1101/2020.11.17.385252","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.385849","pub_date":"2020-11-17","title":"The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2","abstract":"Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with SARS-CoV-2 among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although RaACE2 binding to the receptor binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but limited for mouse, ACE2s enter cells. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.","version":"1.1","doi":"10.1101/2020.11.16.385849","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.385500","pub_date":"2020-11-17","title":"An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19","abstract":"The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.","version":"1.1","doi":"10.1101/2020.11.17.385500","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.15.383661","pub_date":"2020-11-17","title":"Multiscale PHATE Exploration of SARS-CoV-2 Data Reveals Multimodal Signatures of Disease","abstract":"The biomedical community is producing increasingly high dimensional datasets, integrated from hundreds of patient samples, which current computational techniques struggle to explore. To uncover biological meaning from these complex datasets, we present an approach called Multiscale PHATE, which learns abstracted biological features from data that can be directly predictive of disease. Built on a continuous coarse graining process called diffusion condensation, Multiscale PHATE creates a tree of data granularities that can be cut at coarse levels for high level summarizations of data, as well as at fine levels for detailed representations on subsets. We apply Multiscale PHATE to study the immune response to COVID-19 in 54 million cells from 168 hospitalized patients. Through our analysis of patient samples, we identify CD16hi CD66blo neutrophil and IFN\u03b3+GranzymeB+ Th17 cell responses enriched in patients who die. Further, we show that population groupings Multiscale PHATE discovers can be directly fed into a classifier to predict disease outcome. We also use Multiscale PHATE-derived features to construct two different manifolds of patients, one from abstracted flow cytometry features and another directly on patient clinical features, both associating immune subsets and clinical markers with outcome.","version":"1.1","doi":"10.1101/2020.11.15.383661","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.380899","pub_date":"2020-11-17","title":"Impact of COVID-19: Decrease in the Number of Fledging Barn Swallow Chicks in Tokyo","abstract":"Barn swallows that have crossed the sea from Southeast Asia usually appear in the Kyushu Region of Japan around March after passing through Okinawa Prefecture. When the climate becomes warmer, these birds then move further north, nesting and raising their chicks in various parts of Japan. It is worth noting that barn swallows typically nest on man-made objects, for example, the roofs of houses and barns. It is believed that this is because barn swallows protect their eggs and chicks from foreign enemies such as sparrows and crows so they build their nests in populated areas. The barn swallow\u2019s behavior of using the presence of people to keep foreign enemies away shows that barn swallows are quite wise. However, it has been reported that from the spring to summer of 2020, barn swallows, nesting and raising their chicks, which were seen every year, were not found in various parts of Japan. Therefore, we investigated the relationship between people\u2019s self-restraint from going out and the fledging of barn swallow chicks in Tokyo metropolitan during the corona virus disease 2019 (COVID-19) era. The results of the survey showed a link between people\u2019s refraining from going out and the fledging of barn swallow chicks. Next spring of 2021, the termination of COVID-19 is an important environment for swallow chick fledging.","version":"1.1","doi":"10.1101/2020.11.16.380899","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.17.387068","pub_date":"2020-11-17","title":"Assessment of protein-protein interfaces in cryo-EM derived assemblies","abstract":"Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for the map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we have assessed protein-protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed PI-score, a density-independent machine learning-based metric, trained using protein-protein interfaces\u2019 features in high-resolution crystal structures. Using PI-score, we were able to identify errors at interfaces in the PDB-deposited cryo-EM structures (including SARS-CoV-2 complexes) and in the models submitted for cryo-EM targets in CASP13 and the EM model challenge. Some of the identified errors, especially at medium-to-low resolution structures, were not captured by density-based assessment scores. Our method can therefore provide a powerful complementary assessment tool for the increasing number of complexes solved by cryo-EM.","version":"1.1","doi":"10.1101/2020.11.17.387068","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.384222","pub_date":"2020-11-17","title":"Comparative analysis of antibody- and lipid-based multiplexing methods for single-cell RNA-seq","abstract":"Multiplexing of samples in single-cell RNA-seq studies allows significant reduction of experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or - lipids allow barcoding sample-specific cells, a process called \u2018hashing\u2019. Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines. Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. Antibody hashing was further evaluated on clinical samples using PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects.","version":"1.1","doi":"10.1101/2020.11.16.384222","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.15.383463","pub_date":"2020-11-16","title":"SARS-CoV-2-specific T cell memory is long-lasting in the majority of convalsecent COVID-19 individuals","abstract":"An unaddressed key question in the current coronavirus disease 2019 (COVID-19) pandemic is the duration of immunity for which specific T cell responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an indispensable element. Being situated in Wuhan where the pandemic initiated enables us to conduct the longest analyses of memory T cell responses against SARS-CoV-2 in COVID-19 convalescent individuals (CIs). Magnitude and breadth of SARS-CoV-2 memory CD4 and CD8 T cell responses were heterogeneous between patients but robust responses could be detected up to 9 months post disease onset in most CIs. Loss of memory CD4 and CD8 T cell responses were observed in only 16.13% and 25.81% of CIs, respectively. Thus, the overall magnitude and breadth of memory CD4 and CD8 T cell responses were quite stable and not inversely correlated with the time from disease onset. Interestingly, the only significant decrease in the response was found for memory CD4 T cells in the first 6-month post COVID-19 disease onset. Longitudinal analyses revealed that the kinetics of SARS-CoV-2 memory CD4 and CD8 T cell responses were quite heterogenous between patients. Loss of memory CD4 T cell responses was observed more frequently in asymptomatic cases than after symptomatic COVID-19. Interestingly, the few CIs in which SARS-CoV-2-specific IgG responses disappeared showed more durable memory CD4 T cell responses than CIs who remained IgG-positive for month. Collectively, we provide the first comprehensive characterization of the long-term memory T cell response in CIs, suggesting that SARS-CoV-2-specific T cell immunity is long-lasting in the majority of individuals.","version":"1.1","doi":"10.1101/2020.11.15.383463","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.378257","pub_date":"2020-11-16","title":"Intranasal Administration of ACIS KEPTIDE\u2122 Prevents SARS-CoV2-Induced Acute Toxicity in K18-hACE2 Humanized Mouse Model of COVID-19: A Mechanistic Insight for the Prophylactic Role of KEPTIDE\u2122 in COVID-19","abstract":"Previously, we have demonstrated that ACIS KEPTIDE\u2122, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE\u2122 attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*106) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 \u03bcg/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities. In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo, significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice. ACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 & 8). Intranasal inoculation of SARS-CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6) and kidney tubular cells (Fig.8). ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8). ACIS KEPTIDE\u2122 completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8). ACIS KEPTIDE\u2122 improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult. Intranasal administration of 0.05% Beta-propiolactone (\u03b2PL)-inactivated SARS-CoV2 (1 *106) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation (Supplemental videos) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality. The peptide backbone of ACIS KEPTIDE\u2122 provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8). Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8) of SARS-CoV2-treated K18-hACE2 mice.","version":"1.1","doi":"10.1101/2020.11.13.378257","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.15.383927","pub_date":"2020-11-16","title":"Transcriptome Profiling of different types of human respiratory tract cells infected by SARS-CoV-2 Highlight an unique Role for Inflammatory and Interferon Response","abstract":"The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) at the end of 2019 has caused a large global outbreak and now become a major public health issue. Lack of data underlying how the human host interacts with SARS-CoV-2 virus. In the current study, We performed Venn-analysis, Gene ontology (GO), KEGG pathway analysis and Protein-protein interaction analysis of whole transcriptome studies with the aim of clarifying the genes and pathways potentially altered during human respiratory tract cells infected with SARS-CoV-2. We selected four studies through a systematic search of the Gene Expression Omnibus (GEO) database or published article about SARS-CoV-2 infection in different types of respiratory tract cells. We found 36 overlapping upregulated genes among different types of cells after viral infection. Further functional enrichment analysis revealed these DEGs are most likely involved in biological processes related to inflammatory response and response to cytokine, cell component related to extracellular space and I-kappaB/NF-kappaB complex, molecular function related to protein binding and cytokine activity. KEGG pathways analysis highlighted altered conical and casual pathways related to TNF, NF-kappa B, Cytokine-cytokine receptor interaction and IL17 signaling pathways during SARS-CoV-2 infection with CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, IL32, CX3CL1, CCL20, IRF1, NFKB2 and NFKB1A up-regulated which may explain the inflammatory cytokine storms associated with severe cases of COVID-19.","version":"1.1","doi":"10.1101/2020.11.15.383927","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.385278","pub_date":"2020-11-16","title":"High resolution profiling of pathways of escape for SARS-CoV-2 spike-binding antibodies","abstract":"Defining long-term protective immunity to SARS-CoV-2 is one of the most pressing questions of our time and will require a detailed understanding of potential ways this virus can evolve to escape immune protection. Immune protection will most likely be mediated by antibodies that bind to the viral entry protein, Spike (S). Here we used Phage-DMS, an approach that comprehensively interrogates the effect of all possible mutations on binding to a protein of interest, to define the profile of antibody escape to the SARS-CoV-2 S protein using COVID-19 convalescent plasma. Antibody binding was common in two regions: the fusion peptide and linker region upstream of the heptad repeat region 2. However, escape mutations were variable within these immunodominant regions. There was also individual variation in less commonly targeted epitopes. This study provides a granular view of potential antibody escape pathways and suggests there will be individual variation in antibody-mediated virus evolution.","version":"1.1","doi":"10.1101/2020.11.16.385278","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.380394","pub_date":"2020-11-16","title":"Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerase (PARP), blocks replication of the SARS-CoV-2 human coronavirus in vitro","abstract":"By late 2020, the coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 has caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new PARP inhibitor, stenoparib, which was recently advanced to Stage II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro. Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the HCoV-NL63 human seasonal respiratory coronavirus. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and post-entry processes as determined by time-of-addition (TOA) experiments. Moreover, a 10 \u03bcM dosage of stenoparib \u2013 below the approximated 25.5 \u03bcM half-maximally effective concentration (EC50), combined with 0.5 \u03bcM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a standalone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19. New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in Stage II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans has already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and post-entry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection.","version":"1.2","doi":"10.1101/2020.11.12.380394","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.384594","pub_date":"2020-11-16","title":"Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine candidate","abstract":"Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, which may have implications for the resulting immune response and future vaccine design.","version":"1.1","doi":"10.1101/2020.11.16.384594","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.14.383026","pub_date":"2020-11-16","title":"Effects of inactivation method on SARS-CoV-2 virion protein and structure","abstract":"The risk posed by Severe Acute Respiratory Syndrome Coronavirus \u22122 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting destructive effects of inactivation. We observed that 15 minutes of incubation at 65\u00b0C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 \u03bcJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody recognition. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the resulting viral proteins and virions were negatively impacted by inactivation method and time. We outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.","version":"1.1","doi":"10.1101/2020.11.14.383026","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.14.382416","pub_date":"2020-11-16","title":"Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics","abstract":"Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.","version":"1.1","doi":"10.1101/2020.11.14.382416","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.15.382044","pub_date":"2020-11-16","title":"Trimeric SARS-CoV-2 Spike proteins produced from CHO-cells in bioreactors are high quality antigens","abstract":"The Spike protein of SARS-CoV-2 is essential for virus entry into human cells. In fact, most neutralizing antibodies against SARS-CoV-2 are directed against the Spike, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for Spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus Spikes are large, heavily glycosylated, homotrimeric complexes, with inherent instability. Their poor manufacturability now threatens availability of these proteins for vaccines and diagnostic tests. Here, we outline a scalable, GMP-compliant, chemically defined process for production of a cell secreted, stabilized form of the trimeric Spike protein. The process is chemically defined and based on clonal, suspension-CHO cell populations and on protein purification via a two-step, scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2 specific IgG1 was investigated and found to exceed that of Spike fragments (S1 and RBD). The process described here will enable production of sufficient high-quality trimeric Spike protein to meet the global demand for SARS-CoV-2 vaccines and diagnostic tests.","version":"1.1","doi":"10.1101/2020.11.15.382044","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.14.382770","pub_date":"2020-11-16","title":"Discovery of rhodomyrtone as a broad-spectrum antiviral inhibitor with anti-SARS-CoV-2 activity","abstract":"The outbreak of new viruses, such as serve acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as the emerging of drug-resistance viruses highlight the urgent need for the development of broad-spectrum antiviral drugs. Herein, we report the discovery of a plant-derived small molecule, 6,8-dihydroxy-9-isobutyl-2,2,4,4-tetramethyl-7-(3-methylbutanoyl)-4,9-dihydro-1H-xanthene-1,3(2H)-dione (rhodomyrtone, RDT), which exhibited potent broad-spectrum antiviral activities against several RNA and DNA viruses, including SARS-CoV-2, respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Kaposi\u2019s sarcoma-associated herpesvirus (KSHV). RDT can significantly suppress viral gene expression and show the low possibility to elicit drug-resistant variants. Mechanistic study implied that RDT inhibited viral infection by disturbing the cellular factors that essential for viral gene expression. Our results suggested that RDT might be a promising lead compound for the development of broad-spectrum antiviral drugs.","version":"1.1","doi":"10.1101/2020.11.14.382770","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.16.385468","pub_date":"2020-11-16","title":"Examining the Persistence of Human Coronaviruses on Fresh Produce","abstract":"Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, to date, there is no data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes and cucumbers. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i.","version":"1.1","doi":"10.1101/2020.11.16.385468","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.20230326","pub_date":"2020-11-15","title":"Rapid feedback on hospital onset SARS-CoV-2 infections combining epidemiological and sequencing data","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Rapid identification and investigation of healthcare-associated infections (HCAIs) is important for suppression of SARS-CoV-2, but the infection source for hospital onset COVID-19 infections (HOCIs) cannot always be readily identified based only on epidemiological data. Viral sequencing data provides additional information regarding potential transmission clusters, but the low mutation rate of SARS-CoV-2 can make interpretation using standard phylogenetic methods difficult.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed a novel statistical method and sequence reporting tool (SRT) that combines epidemiological and sequence data in order to provide a rapid assessment of the probability of HCAI among HOCI cases (defined as first positive test &gt;48 hours following admission) and to identify infections that could plausibly constitute outbreak events. The method is designed for prospective use, but was validated using retrospective datasets from hospitals in Glasgow and Sheffield collected February-May 2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We analysed data from 326 HOCIs. Among HOCIs with time-from-admission \u22658 days the SRT algorithm identified close sequence matches from the same ward for 160/244 (65.6%) and in the remainder 68/84 (81.0%) had at least one similar sequence elsewhere in the hospital, resulting in high estimated probabilities of within-ward and within-hospital transmission. For HOCIs with time-from-admission 3-7 days, the SRT probability of healthcare acquisition was &gt;0.5 in 33/82 (40.2%).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The methodology developed can provide rapid feedback on HOCIs that could be useful for infection prevention and control teams, and warrants further prospective evaluation. The integration of epidemiological and sequence data is important given the low mutation rate of SARS-CoV-2 and its variable incubation period.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.11.12.20230326","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.13.381228","pub_date":"2020-11-14","title":"Analysis of the Dynamics and Distribution of SARS-CoV-2 Mutations and its Possible Structural and Functional Implications","abstract":"After eight months of the pandemic declaration, COVID-19 has not been globally controlled. Several efforts to control SARS-CoV-2 dissemination are still running including vaccines and drug treatments. The effectiveness of these procedures depends, in part, that the regions to which these treatments are directed do not vary considerably. Although, it is known that the mutation rate of SARS-CoV-2 is relatively low it is necessary to monitor the adaptation and evolution of the virus in the different stages of the pandemic. Thus, identification, analysis of the dynamics, and possible functional and structural implication of mutations are relevant. Here, we first estimate the number of COVID-19 cases with a virus with a specific mutation and then calculate its global relative frequency (NRFp). Using this approach in a dataset of 100 924 genomes from GISAID, we identified 41 mutations to be present in viruses in an estimated number of 750 000 global COVID-19 cases (0.03 NRFp). We classified these mutations into three groups: high-frequent, low-frequent non-synonymous, and low-frequent synonymous. Analysis of the dynamics of these mutations by month and continent showed that high-frequent mutations appeared early in the pandemic, all are present in all continents and some of them are almost fixed in the global population. On the other hand, low-frequent mutations (non-synonymous and synonymous) appear late in the pandemic and seems to be at least partially continent-specific. This could be due to that high-frequent mutation appeared early when lockdown policies had not yet been applied and low-frequent mutations appeared after lockdown policies. Thus, preventing global dissemination of them. Finally, we present a brief structural and functional review of the analyzed ORFs and the possible implications of the 25 identified non-synonymous mutations.","version":"1.1","doi":"10.1101/2020.11.13.381228","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.370387","pub_date":"2020-11-14","title":"A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities","abstract":"One goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols. Raw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX). To allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.","version":"1.1","doi":"10.1101/2020.11.13.370387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.346197","pub_date":"2020-11-14","title":"Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype","abstract":"In malaria-na\u00efve children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of monocytes collected from uninfected, asymptomatic Malian individuals before the malaria season revealed an inverse relationship between age and Pf-iRBC-inducible inflammatory cytokine (IL-1\u03b2, IL-6 and TNF) production, whereas Malian infants and malaria-na\u00efve U.S. adults produced similarly high levels of inflammatory cytokines. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. These findings also suggest that past malaria exposure could mitigate monocyte-associated immunopathology induced by other pathogens such as SARS-CoV-2. The malaria parasite is mosquito-transmitted and causes fever and other inflammatory symptoms while circulating in the bloodstream. However, in regions of high malaria transmission the parasite is less likely to cause fever as children age and enter adulthood, even though adults commonly have malaria parasites in their blood. Monocytes are cells of the innate immune system that secrete molecules that cause fever and inflammation when encountering microorganisms like malaria. Although inflammation is critical to initiating normal immune responses, too much inflammation can harm infected individuals. In Mali, we conducted a study of a malaria-exposed population from infants to adults and found that participants\u2019 monocytes produced less inflammation as age increases, whereas monocytes of Malian infants and U.S. adults, who had never been exposed to malaria, both produced high levels of inflammatory molecules. Accordingly, monocytes exposed to malaria in the laboratory became less inflammatory when re-exposed to malaria again later, and these monocytes \u2018turned down\u2019 their inflammatory genes. This study helps us understand how people become immune to inflammatory symptoms of malaria and may also help explain why people in malaria-endemic areas appear to be less susceptible to the harmful effects of inflammation caused by other pathogens such as SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.10.21.346197","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.20228890","pub_date":"2020-11-13","title":"Anti-SARS-CoV-2 IgG responses are powerful predicting signatures for the outcome of COVID-19 patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The COVID-19 global pandemic is far from ending. There is an urgent need to identify applicable biomarkers for early predicting the outcome of COVID-19. Growing evidences have revealed that SARS-CoV-2 specific antibodies evolved with disease progression and severity in COIVD-19 patients. We assumed that antibodies may serve as biomarkers for predicting disease outcome. By taking advantage of a newly developed SARS-CoV-2 proteome microarray, we surveyed IgG responses against 20 proteins of SARS-CoV-2 in 1,034 hospitalized COVID-19 patients on admission and followed till 66 days. The microarray results were further correlated with clinical information, laboratory test results and patient outcomes. Cox proportional hazards model was used to explore the association between SARS-CoV-2 specific antibodies and COVID-19 mortality. We found that nonsurvivors induced higher levels of IgG responses against most of non-structural proteins than survivors on admission. In particular, the magnitude of IgG antibodies against 8 non-structural proteins (NSP1, NSP4, NSP7, NSP8, NSP9, NSP10, RdRp, and NSP14) and 2 accessory proteins (ORF3b and ORF9b) possessed significant predictive power for patient death, even after further adjustments for demographics, comorbidities, and common laboratory biomarkers for disease severity (all with\n                  <jats:italic>p</jats:italic>\n                  trend &lt; 0.05). Additionally, IgG responses to all of these 10 non-structural/accessory proteins were also associated with the severity of disease, and differential kinetics and serum positive rate of these IgG responses were confirmed in COVID-19 patients of varying severities within 20 days after symptoms onset. The AUCs for these IgG responses, determined by computational cross-validations, were between 0.62 and 0.71. Our findings have important implications for improving clinical management, and especially for developing medical interventions and vaccines.\n                </jats:p>","version":null,"doi":"10.1101/2020.11.10.20228890","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.12.380931","pub_date":"2020-11-13","title":"The Preclinical Inhibitor GS441524 in Combination with GC376 Efficaciously Inhibited the Proliferation of SARS-CoV-2 in the Mouse Respiratory Tract","abstract":"The unprecedented coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious threat to global public health. Development of effective therapies against SARS-CoV-2 is urgently needed. Here, we evaluated the antiviral activity of a remdesivir parent nucleotide analog, GS441524, which targets the coronavirus RNA-dependent RNA polymerase enzyme, and a feline coronavirus prodrug, GC376, which targets its main protease, using a mouse-adapted SARS-CoV-2 infected mouse model. Our results showed that GS441524 effectively blocked the proliferation of SARS-CoV-2 in the mouse upper and lower respiratory tracts via combined intranasal (i.n.) and intramuscular (i.m.) treatment. However, the ability of high-dose GC376 (i.m. or i.n. and i.m.) was weaker than GS441524. Notably, low-dose combined application of GS441524 with GC376 could effectively protect mice against SARS-CoV-2 infection via i.n. or i.n. and i.m. treatment. Moreover, we found that the pharmacokinetic properties of GS441524 is better than GC376, and combined application of GC376 and GS441524 had a synergistic effect. Our findings support the further evaluation of the combined application of GC376 and GS441524 in future clinical studies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which has seriously threatened global public health and economic development. Currently, effective therapies to treat COVID-19 are urgently needed. In this study, we assessed the efficacy of the preclinical inhibitors GC376 and GS441524 using a mouse-adapted SARS-CoV-2 infected mouse model for the first time. Our results showed that low-dose combined application of GC376 and GS441524 could effectively protect mice from HRB26M infection in the upper and lower respiratory tracts. Hence, the combined application should be developed and considered for future clinic practice.","version":"1.1","doi":"10.1101/2020.11.12.380931","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.381319","pub_date":"2020-11-13","title":"Plasma irradiation efficiently inactivates the coronaviruses mouse hepatitis virus and SARS-CoV-2","abstract":"Many inactivation methods have been shown to inactivate SARS-CoV-2 for safe and efficient diagnostic methods. COVID-19 is caused by airborne infection of SARS-CoV-2, and therefore, methods of inactivating the virus efficiently and safely are crucial for reducing the risk of airborne infection. In this regard, the effect of plasma discharge on the infectivity of the coronaviruses mouse hepatitis virus (MHV) and SARS-CoV-2 was tested. Plasma discharge efficiently reduced the infectivity of both coronaviruses. Treatment of SARS-CoV-2 in culture medium with a plasma discharge resulted in 95.17% viral inactivation after plasma irradiation after 1 hour (hr), 99.54% inactivation after 2 hrs and 99.93% inactivation after 3 hrs. Similar results were obtained for MHV. The results indicated that plasma discharge effectively and safely inactivated the airborne coronaviruses and may be useful in minimizing the risk of airborne infection of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.13.381319","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.381533","pub_date":"2020-11-13","title":"Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2","abstract":"The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.13.381533","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.201509","pub_date":"2020-11-13","title":"SARS-CoV-2 infects cells following viral entry via clathrin-mediated endocytosis","abstract":"With more than 51 million cases and 1.3 million deaths, and with the resulting social upheaval, the COVID-19 pandemic presents one of the greatest challenges ever to human society. It is thus vital to fully understand the biology of SARS-CoV-2, the causative agent of COVID-19. SARS-CoV-2 uses its spike glycoprotein to interact with the cell surface as a first step in the infection process. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, we now demonstrate that following engagement with the plasma membrane, SARS-CoV-2 undergoes rapid clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system, and importantly clathrin-heavy chain knockdown, which blocks clathrin-mediated endocytosis, reduces viral infectivity. This discovery reveals important new information about the basic biology of SARS-CoV-2 infectivity.","version":"1.3","doi":"10.1101/2020.07.13.201509","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.381343","pub_date":"2020-11-13","title":"ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2","abstract":"The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domain, previously C6orf106) is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we have utilised RNA-seq to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection. We find that inhibition of ILRUN expression increases cellular expression of several members of the renin-angiotensin aldosterone system (RAAS), including the SARS-CoV-2 entry receptor angiotensin converting enzyme 2 (ACE2). Furthermore, inhibition of ILRUN results in increased SARS-CoV-2 replication. These data identify ILRUN as a novel inhibitor of SARS-CoV-2 replication and represents, to our knowledge, the first report of ILRUN as a regulator of the RAAS. There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions and the development of innovative and exciting therapeutic strategies and new knowledge and tools to better protect against the impacts of disease. The human protein-coding gene ILRUN is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we present the first evidence that ILRUN modulation has implications for SARS-CoV-2 infections. Virus infectivity assays confirmed that gene silencing of ILRUN had a proviral effect and increased SARS-CoV-2 replication, whilst over-expression of ILRUN inhibited SARS-CoV-2 production. Additionally, we observed that ILRUN also regulates the expression of key elements of the RAAS. These data have important implications for the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.","version":"1.1","doi":"10.1101/2020.11.13.381343","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.074930","pub_date":"2020-11-13","title":"Design of an Epitope-Based Peptide Vaccine against the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2): A Vaccine-informatics Approach","abstract":"The recurrent and recent global outbreak of SARS-CoV-2 has turned into a global concern which has infected more than 19-million people all over the globe, and this number is increasing in hours. Unfortunate no vaccine or specific treatment is available, which make it more deadly. A vaccine-informatics approach has shown significant breakthrough in peptide-based epitope mapping and opens the new horizon in vaccine development. In this study, we have identified a total of 15 antigenic peptides (including T and B cells) in the surface glycoprotein of SARS-CoV-2 which showed non-toxic nature, non-allergenic, highly antigenic and non-mutated in other SARS-CoV-2 virus strains. The population coverage analysis has found that CD4+ T-cell peptides showed higher cumulative population coverage over to CD8+ peptides in the 16 different geographical regions of the world. We identified twelve peptides (LTDEMIAQY, WTAGAAAYY, WMESEFRVY, IRASANLAA, FGAISSVLN, VKQLSSNFG, FAMQMAYRF, FGAGAALQI, YGFQPTNGVGYQ, LPDPSKPSKR, QTQTNSPRRARS and VITPGTNTSN) that are 80% - 90% identical with experimentally determined epitopes of SARS-CoV, and this will likely be beneficial for a quick progression of the vaccine design. Moreover, docking analysis suggested that identified peptides are tightly bound in the groove of HLA molecules which can induce the T-cell response. Overall this study allows us to determine potent peptide antigen targets in surface glycoprotein on intuitive grounds which open up a new horizon in COVID-19 research. However, this study needs experimental validation by in vitro and in vivo.","version":"1.2","doi":"10.1101/2020.05.03.074930","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.361048","pub_date":"2020-11-13","title":"Synergism of TNF-\u03b1 and IFN-\u03b3 triggers inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syndromes","abstract":"The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF-\u03b1 and IFN-\u03b3 specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the JAK/STAT1/IRF1 axis activated by TNF-\u03b1 and IFN-\u03b3 co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF-\u03b1 and IFN-\u03b3-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF-\u03b1 and IFN-\u03b3 in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF-\u03b1 and IFN-\u03b3 synergism play key pathological roles.","version":"1.3","doi":"10.1101/2020.10.29.361048","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.13.20231076","pub_date":"2020-11-13","title":"SARS-CoV-2 serological tests can generate false positive results for samples from patients with chronic inflammatory diseases","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless the tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays with samples from patients with chronic inflammatory diseases collected before April 2019, thus defined as negative.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and RF +/- systemic lupus erythematosus (SLE, n=10), were tested with 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed multiplex bead-based assay.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Six LFA and the in-house IgG assay gave the correct negative results for all samples. However, the majority of assays (n=13), gave false positive signal with samples from patients with RA and SLE. This was most notable in RF positive RA samples. MS samples did not give any false positive in any of the assays.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>The majority of the verified serological assays were sensitive to interfering antibodies in samples from patients with chronic inflammatory diseases and therefore may have poor specificity in this context. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.11.13.20231076","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.13.381335","pub_date":"2020-11-13","title":"A whole virion vaccine for COVID-19 produced via a novel inactivation method: results from animal challenge model studies","abstract":"The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX\u2122) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this study provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have utility in the preparation of one such vaccine candidate. We have developed a vaccine for COVID-19 which is prepared by a novel method for inactivation of a whole virion particle and tested it in a hamster animal model for its ability to protect against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.11.13.381335","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.380816","pub_date":"2020-11-13","title":"An evolutionary analysis of the SARS-CoV-2 genomes from the countries in the same meridian","abstract":"In the current study we analyzed the genomes of SARS-CoV-2 strains isolated from Italy, Sweden, Congo (countries in the same meridian) and Brazil, as outgroup country. Evolutionary analysis revealed codon 9628 under episodic selective pressure for all four countries, suggesting it as a key site for the virus evolution. Belonging to the P0DTD3 (Y14_SARS2) uncharacterized protein 14, further investigation has been conducted showing the codon mutation as responsible for the helical modification in the secondary structure. According to the predictions done, the codon is placed into the more ordered region of the gene (41-59) and close the area acting as transmembrane (54-67), suggesting its involvement into the attachment phase of the virus. The predicted structures of P0DTD3 mutated and not confirmed the importance of the codon to define the protein structure and the ontological analysis of the protein emphasized that the mutation enhances the binding probability.","version":"1.1","doi":"10.1101/2020.11.12.380816","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.374777","pub_date":"2020-11-13","title":"Epidemiological transcriptomic data supports BCG protection in viral diseases including COVID-19","abstract":"Epidemiological and clinical evidence suggests that Bacille Calmette-Gu\u00e9rin (BCG) vaccine induced trained immunity protects against non-specific infections. Multiple clinical trials are currently underway to assess effectiveness of the vaccine in the coronavirus disease 2019 (COVID-19). However, the durability and mechanism of BCG trained immunity remain unclear. Here, an integrative analysis of available epidemiological transcriptomic data related to BCG vaccination and respiratory tract viral infections, and transcriptomic alterations reported in COVID-19 is presented toward addressing this gap. Results suggest that the vaccine induces very long-lasting transcriptomic changes that, unsurprisingly, mimic viral infections by upregulated antiviral defense response, and, counterintuitively. oppose viral infections by downregulated myeloid cell activation. These durability and mechanistic insights have immediate implications in fight against the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.11.10.374777","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.378018","pub_date":"2020-11-12","title":"The repurposed drugs suramin and quinacrine inhibit cooperatively in vitro SARS-CoV-2 3CLpro","abstract":"Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) up to now WHO reported more than 50 million confirmed cases and more than one million losses, globally. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide resulting in a pandemic of unprecedented magnitude. To date, no clinically safe drug or vaccine is available and the development of molecules to combat SARS-CoV-2 infections is imminent. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is the repurposing of clinically developed drugs, e.g., anti-parasitic drugs. The results described in this study demonstrate the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules present a competitive and non-competitive mode of inhibition, respectively, with IC50 and KD values in low \u03bcM range. Using docking and molecular dynamics simulations we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin in combination with quinacrine showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. The identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease. Drug repositioning offers hope to the SARS-CoV-2 control.","version":"1.1","doi":"10.1101/2020.11.11.378018","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.380709","pub_date":"2020-11-12","title":"ORF3a mediated-incomplete autophagy facilitates SARS-CoV-2 replication","abstract":"SARS-CoV-2 is the causative agent for the COVID-19 pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin-1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 28 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate Beclin-1-Vps34-Atg14 complex but selectively inhibit Beclin-1-Vps34-UVRAG complex. Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raises the possibility of targeting the autophagic pathway for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.11.12.380709","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.344424","pub_date":"2020-11-12","title":"Molecular Mimicry Map (3M) of SARS-CoV-2: Prediction of potentially immunopathogenic SARS-CoV-2 epitopes via a novel immunoinformatic approach","abstract":"Currently, more than 33 million peoples have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and more than a million people died from coronavirus disease 2019 (COVID-19), a disease caused by the virus. There have been multiple reports of autoimmune and inflammatory diseases following SARS-CoV-2 infections. There are several suggested mechanisms involved in the development of autoimmune diseases, including cross-reactivity (molecular mimicry). A typical workflow for discovering cross-reactive epitopes (mimotopes) starts with a sequence similarity search between protein sequences of human and a pathogen. However, sequence similarity information alone is not enough to predict cross-reactivity between proteins since proteins can share highly similar conformational epitopes whose amino acid residues are situated far apart in the linear protein sequences. Therefore, we used a hidden Markov model-based tool to identify distant viral homologs of human proteins. Also, we utilized experimentally determined and modeled protein structures of SARS-CoV-2 and human proteins to find homologous protein structures between them. Next, we predicted binding affinity (IC50) of potentially cross-reactive T-cell epitopes to 34 MHC allelic variants that have been associated with autoimmune diseases using multiple prediction algorithms. Overall, from 8,138 SARS-CoV-2 genomes, we identified 3,238 potentially cross-reactive B-cell epitopes covering six human proteins and 1,224 potentially cross-reactive T-cell epitopes covering 285 human proteins. To visualize the predicted cross-reactive T-cell and B-cell epitopes, we developed a web-based application \u201cMolecular Mimicry Map (3M) of SARS-CoV-2\u201d (available at https://ahs2202.github.io/3M/). The web application enables researchers to explore potential cross-reactive SARS-CoV-2 epitopes alongside custom peptide vaccines, allowing researchers to identify potentially suboptimal peptide vaccine candidates or less ideal part of a whole virus vaccine to design a safer vaccine for people with genetic and environmental predispositions to autoimmune diseases. Together, the computational resources and the interactive web application provide a foundation for the investigation of molecular mimicry in the pathogenesis of autoimmune disease following COVID-19.","version":"1.1","doi":"10.1101/2020.11.12.344424","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.379099","pub_date":"2020-11-12","title":"Goblet Cell Hyperplasia Increases SARS-CoV-2 Infection in COPD","abstract":"SARS-CoV-2 has become a major problem across the globe, with approximately 50 million cases and more than 1 million deaths and currently no approved treatment or vaccine. Chronic obstructive pulmonary disease (COPD) is one of the underlying conditions in adults of any age that place them at risk for developing severe illness associated with COVID-19. We established an airway epithelium model to study SARS-CoV-2 infection in healthy and COPD lung cells. We found that both the entry receptor ACE2 and the co-factor transmembrane protease TMPRSS2 are expressed at higher levels on nonciliated goblet cell, a novel target for SARS-CoV-2 infection. We observed that SARS-CoV-2 infected goblet cells and induced syncytium formation and cell sloughing. We also found that SARS-CoV-2 replication was increased in the COPD airway epithelium likely due to COPD associated goblet cell hyperplasia. Our results reveal goblet cells play a critical role in SARS-CoV-2 infection in the lung.","version":"1.1","doi":"10.1101/2020.11.11.379099","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.379958","pub_date":"2020-11-12","title":"Combined in silico docking and in vitro antiviral testing for drug repurposing identified lurasidone and elbasvir as SARS-CoV-2 and HCoV-OC43 inhibitors","abstract":"The current emergency of the novel coronavirus SARS-CoV-2 urged the need for broad-spectrum antiviral drugs as the first line of treatment. Coronaviruses are a large family of viruses that already challenged humanity in at least two other previous outbreaks and are likely to be a constant threat for the future. In this work we developed a pipeline based on in silico docking of known drugs on SARS-CoV RNA-dependent RNA polymerase combined with in vitro antiviral assays on both SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Results showed that certain drugs displayed activity for both viruses at a similar inhibitory concentration, while others were specific. In particular, the antipsychotic drug lurasidone and the antiviral drug elbasvir showed promising activity in the low micromolar range against both viruses with good selective index.","version":"1.1","doi":"10.1101/2020.11.12.379958","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.377598","pub_date":"2020-11-12","title":"Nanodroplet-Benzalkonium Chloride Formulation Demonstrates In Vitro and Ex-Vivo Broad-Spectrum Antiviral Activity Against SARS-CoV-2 and other Enveloped Viruses","abstract":"The Covid-19 pandemic has highlighted the importance of aerosolized droplets inhaled into the nose in the transmission of respiratory viral disease. Inactivating pathogenic viruses at the nasal portal of entry may reduce viral loads, thereby reducing transmission and contagion. We have developed an oil-in-water nanoemulsion (nanodroplet) formulation containing the potent antiseptic 0.13% Benzalkonium Chloride (NE-BZK) which demonstrates safe and broad anti-viral activity. While The Centers for Disease Control and Prevention (CDC) have reported that BZK may have less reliable activity than ethyl alcohol against certain viruses, including coronaviruses, we have demonstrated that NE-BZK exhibits broad-spectrum, long-lasting antiviral activity with >99.99% in vitro killing of enveloped viruses including SARS-CoV-2, human coronavirus, RSV and influenza B. Furthermore, in vitro studies demonstrated that NE-BZK continues to kill >99.99% of human coronavirus even when diluted 20-fold, while 0.13% aqueous BZK solution (AQ-BZK) did not. Ex vivo studies of NE-BZK on human cadaver skin demonstrated persistent >99.99% killing of human coronavirus for at least 8 hours after application. AQ-BZK failed to demonstrate durable antimicrobial activity on skin over time. The repeated application of NE-BZK, twice daily for 2 weeks on to rabbit nostrils indicated safety with no irritation. These findings demonstrate that formulating BZK on the surface of proprietary nanodroplets offers a safe and effective antiviral as a significant addition to strategies to combat the spread of respiratory viral infectious diseases.","version":"1.1","doi":"10.1101/2020.11.12.377598","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.12.379537","pub_date":"2020-11-12","title":"A retrospective cluster analysis of COVID-19 cases by county","abstract":"The COVID-19 pandemic in the U.S. has exhibited distinct waves, the first beginning in March 2020, the second beginning in early June, and additional waves currently emerging. Paradoxically, almost no county has exhibited this multi-wave pattern. We aim to answer three research questions: (1) How many distinct clusters of counties exhibit similar COVID-19 patterns in the time-series of daily confirmed cases?; (2) What is the geographic distribution of the counties within each cluster? and (3) Are county-level demographic, socioeconomic and political variables associated with the COVID-19 case patterns? We analyzed data from counties in the U.S. from March 1 to October 24, 2020. Time series clustering identified clusters in the daily confirmed cases of COVID-19. An explanatory model was used to identify demographic, socioeconomic and political variables associated the cluster patterns. Four patterns were identified from the timing of the outbreaks including counties experiencing a spring, an early summer, a late summer, and a fall outbreak. Several county-level demographic, socioeconomic, and political variables showed significant associations with the identified clusters. The timing of the outbreak is related both to the geographic location within the U.S. and several variables including age, poverty distribution, and political association. These results show that the reported pattern of cases in the U.S. is observed through aggregation of the COVID-19 cases, suggesting that local trends may be more informative. The timing of the outbreak varies by county, and is associated with important demographic, socioeconomic and geographic factors.","version":"1.1","doi":"10.1101/2020.11.12.379537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.375394","pub_date":"2020-11-11","title":"Network-based Virus-Host Interaction Prediction with Application to SARS-CoV-2","abstract":"COVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has quickly become a global health crisis since the first report of infection in December of 2019. However, the infection spectrum of SARS-CoV-2 and its comprehensive protein-level interactions with hosts remain unclear. There is a massive amount of under-utilized data and knowledge about RNA viruses highly relevant to SARS-CoV-2 and their hosts\u2019 proteins. More in-depth and more comprehensive analyses of that knowledge and data can shed new insight into the molecular mechanisms underlying the COVID-19 pandemic and reveal potential risks. In this work, we constructed a multi-layer virus-host interaction network to incorporate these data and knowledge. A machine learning-based method, termed Infection Mechanism and Spectrum Prediction (IMSP), was developed to predict virus-host interactions at both protein and organism levels. Our approach revealed five potential infection targets of SARS-CoV-2, which deserved public health attention, and eight highly possible interactions between SARS-CoV-2 proteins and human proteins. Given a new virus, IMSP can utilize existing knowledge and data about other highly relevant viruses to predict multi-scale interactions between the new virus and potential hosts.","version":"1.1","doi":"10.1101/2020.11.09.375394","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.254375","pub_date":"2020-11-11","title":"Potent neutralizing equine antibodies raised against recombinant SARS-CoV-2 spike protein for COVID-19 passive immunization therapy","abstract":"We used the trimeric spike (S) glycoprotein (residues 1-1208) in the prefusion conformation to immunize horses for production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by anti-spike ELISA were above 1:1,000,000, and neutralizing antibody titer was 1:14,604 (average PRNT90), which is 140-fold higher than the average neutralizing titer of plasma from three convalescent COVID-19 patients analyzed for comparison. Using the same technology routinely used for industrial production of other horse hyperimmune products, plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding a F(ab\u2019)2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Repeating the hyperimmunization in a second group of horses confirmed the very high neutralizing titers in serum and in a GMP clinical F(ab\u2019)2 lot. Virus-neutralizing activity in samples from mice that received the F(ab\u2019)2 preparation was detected even three days after injection, indicating an appropriate half-life for therapeutic intervention. These results supported the design of a clinical trial (identifier NCT04573855) to evaluate safety and efficacy of this horse F(ab\u2019)2 preparation.","version":"1.3","doi":"10.1101/2020.08.17.254375","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.377606","pub_date":"2020-11-11","title":"Recombinant ACE2 Expression is Required for SARS-CoV-2 to Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses","abstract":"SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS) characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on dysregulated EC responses. Whether SARS-CoV-2 directly or indirectly affects functions of the endothelium remains to be resolved and critical to understanding SARS-CoV-2 pathogenesis and therapeutic targets. We demonstrate that primary human ECs lack ACE2 receptors at protein and RNA levels, and that SARS-CoV-2 is incapable of directly infecting ECs derived from pulmonary, cardiac, brain, umbilical vein or kidney tissues. In contrast, pulmonary ECs transduced with recombinant ACE2 receptors are infected by SARS-CoV-2 and result in high viral titers (\u223c1\u00d7107/ml), multinucleate syncytia and EC lysis. SARS-CoV-2 infection of ACE2-expressing ECs elicits procoagulative and inflammatory responses observed in COVID-19 patients. The inability of SARS-CoV-2 to directly infect and lyse ECs without ACE2 expression explains the lack of vascular hemorrhage in COVID-19 patients and indicates that the endothelium is not a primary target of SARS-CoV-2 infection. These findings are consistent with SARS-CoV-2 indirectly activating EC programs that regulate thrombosis and endotheliitis in COVID-19 patients, and focus strategies on therapeutically targeting epithelial and inflammatory responses that activate the endothelium or initiate limited ACE2 independent EC infection. SARS-CoV-2 infects pulmonary epithelial cells through ACE2 receptors and causes ARDS. COVID-19 causes progressive respiratory failure resulting from diffuse alveolar damage and systemic coagulopathy, thrombosis and capillary inflammation that tie alveolar responses to EC dysfunction. This has prompted theories that SARS-CoV-2 directly infects ECs through ACE2 receptors, yet SARS-CoV-2 antigen has not been co-localized with ECs and prior studies indicate that ACE2 co-localizes with alveolar epithelial cells and vascular smooth muscle cells, not ECs. Here we demonstrate that primary human ECs derived from lung, kidney, heart, brain and umbilical veins require expression of recombinant ACE2 receptors in order to be infected by SARS-CoV-2. However, SARS-CoV-2 lytically infects ACE2-ECs and elicits procoagulative and inflammatory responses observed in COVID-19 patients. These findings suggest a novel mechanism of COVID-19 pathogenesis resulting from indirect EC activation, or infection of a small subset of ECs by an ACE2 independent mechanism, that transform rationales and targets for therapeutic intervention.","version":"1.1","doi":"10.1101/2020.11.10.377606","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.377408","pub_date":"2020-11-11","title":"Ozone exposure upregulates the expression of host susceptibility protein TMPRSS2 to SARS-CoV-2","abstract":"SARS-CoV-2, a novel coronavirus, and the etiologic agent for the current global health emergency, causes acute infection of the respiratory tract leading to severe disease and significant mortality. Ever since the start of SARS-CoV-2, also known as COVID-19 pandemic, countless uncertainties have been revolving around the pathogenesis and epidemiology of the SARS-CoV-2 infection. While air pollution has been shown to be strongly correlated to increased SARS-CoV-2 morbidity and mortality, whether environmental pollutants such as ground level ozone affects the susceptibility of individuals to SARS-CoV-2 is not yet established. To investigate the impact of ozone inhalation on the expression levels of signatures associated with host susceptibility to SARS-CoV-2. We analyzed lung tissues collected from mice that were sub-chronically exposed to air or 0.8ppm ozone for three weeks (4h/night, 5 nights/week), and analyzed the expression of signatures associated with host susceptibility to SARS-CoV-2. SARS-CoV-2 entry into the host cells requires proteolytic priming by the host-derived protease, transmembrane protease serine 2 (TMPRSS2). The TMPRSS2 protein and Tmprss2 transcripts were significantly elevated in the extrapulmonary airways, parenchyma, and alveolar macrophages from ozone-exposed mice. A significant proportion of additional known SARS-CoV-2 host susceptibility genes were upregulated in alveolar macrophages and parenchyma from ozone-exposed mice. Our data indicate that the unhealthy levels of ozone in the environment may predispose individuals to severe SARS-CoV-2 infection. Given the severity of this pandemic, and the challenges associated with direct testing of host-environment interactions in clinical settings, we believe that this mice-ozone-exposure based study informs the scientific community of the potentially detrimental effects of the ambient ozone levels determining the host susceptibility to SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.10.377408","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.266437","pub_date":"2020-11-11","title":"Stapled peptides based on Human Angiotensin-Converting Enzyme 2 (ACE2) potently inhibit SARS-CoV-2 infection in vitro","abstract":"SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is a \u223c30 aa long helix. Here we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50-94% helicity). On the contrary, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus based single-cycle assay in HT1080/ACE2 and human lung cells A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (IC50: 1.9 \u2013 4.1 \u00b5M) and A549/ACE2 cells (IC50: 2.2 \u2013 2.8 \u00b5M). The linear peptides NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient preventing the complete formation of cytopathic effects (CPEs) at an IC100 17.2 \u00b5M. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 \u00b5M. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T1/2) of >289 min.","version":"1.2","doi":"10.1101/2020.08.25.266437","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.368092","pub_date":"2020-11-11","title":"High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector","abstract":"The spike (S) protein is one of the three proteins forming the coronaviruses\u2019 viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests \u2013 the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 \u2013 human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and similar applications.","version":"1.2","doi":"10.1101/2020.11.04.368092","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.377366","pub_date":"2020-11-11","title":"Unification of the M/ORF3-related proteins points to a diversified role for ion conductance in pathogenesis of coronaviruses and other nidoviruses","abstract":"The new coronavirus, SARS-CoV-2, responsible for the COVID-19 pandemic has emphasized the need for a better understanding of the evolution of virus-host conflicts. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) and involved in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. By sequence analysis and structural modeling, we show that these viral families utilize specific conserved polar residues to constitute an ion-conducting pore in the membrane. We reconstruct the evolutionary history of these families, objectively establish the common origin of the M proteins of CoVs and Toroviruses. We show that the divergent ORF3a/ORF3b/ORF5 families represent a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a model for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly, membrane fusion and budding. We show that the M and ORF3 are under differential evolutionary pressures; in contrast to the slow evolution of M as core structural component, the CoV-ORF3 clade is under selection for diversification, which indicates it is likely at the interface with host molecules and/or immune attack. Coronaviruses (CoVs) have become a major threat to human welfare as the causative agents of several severe infectious diseases, namely Severe Acute Respiratory Syndrome (SARS), Middle Eastern Respiratory Syndrome (MERS), and the recently emerging human coronavirus disease 2019 (COVID-19). The rapid spread, severity of these diseases, as well as the potential re-emergence of other CoV-associated diseases have imposed a strong need for a thorough understanding of function and evolution of these CoVs. By utilizing robust domain-centric computational strategies, we have established homologous relationships between many divergent families of CoV proteins, including SARS-CoV/SARS-CoV-2 ORF3a, MERS-CoV ORF5, proteins from both beta-CoVs (ORF3c) and alpha-CoVs (ORF3b), the typical CoV Matrix proteins, and many distant homologs from other nidoviruses. We present evidence that they are active ion channel proteins, and the Cov-specific ORF3 clade proteins are under selection for rapid diversification, suggesting they might have been involved in interfering host molecules and/or immune attack.","version":"1.1","doi":"10.1101/2020.11.10.377366","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.11.378778","pub_date":"2020-11-11","title":"Rapid generation of potent antibodies by autonomous hypermutation in yeast","abstract":"The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source na\u00efve nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw \u223c580-fold and \u223c925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.","version":"1.1","doi":"10.1101/2020.11.11.378778","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.376905","pub_date":"2020-11-10","title":"Protective efficacy of a SARS-CoV-2 DNA Vaccine in wild-type and immunosuppressed Syrian hamsters","abstract":"A worldwide effort to counter the COVID-19 pandemic has resulted in hundreds of candidate vaccines moving through various stages of research and development, including several vaccines in phase 1, 2 and 3 clinical trials. A relatively small number of these vaccines have been evaluated in SARS-CoV-2 disease models, and fewer in a severe disease model. Here, a SARS-CoV-2 DNA targeting the spike protein and delivered by jet injection, nCoV-S(JET), elicited neutralizing antibodies in hamsters and was protective in both wild-type and transiently immunosuppressed hamster models. This study highlights the DNA vaccine, nCoV-S(JET), we developed has a great potential to move to next stage of preclinical studies, and it also demonstrates that the transiently-immunosuppressed Syrian hamsters, which recapitulate severe and prolonged COVID-19 disease, can be used for preclinical evaluation of the protective efficacy of spike-based COVID-19 vaccine.","version":"1.1","doi":"10.1101/2020.11.10.376905","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.376673","pub_date":"2020-11-10","title":"SARS-CoV-2 infection causes transient olfactory dysfunction in mice","abstract":"Olfactory dysfunction caused by SARS-CoV-2 infection represents as one of the most predictive and common symptoms in COVID-19 patients. However, the causal link between SARS-CoV-2 infection and olfactory disorders remains lacking. Herein we demonstrate intranasal inoculation of SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), resulting in transient olfactory dysfunction in humanized ACE2 mice. The sustentacular cells and Bowman\u2019s gland cells in OE were identified as the major targets of SARS-CoV-2 before the invasion into olfactory sensory neurons. Remarkably, SARS-CoV-2 infection triggers cell death and immune cell infiltration, and impairs the uniformity of OE structure. Combined transcriptomic and proteomic analyses reveal the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptors in OE from the infected animals. Overall, our mouse model recapitulates the olfactory dysfunction in COVID-19 patients, and provides critical clues to understand the physiological basis for extrapulmonary manifestations of COVID-19.","version":"1.1","doi":"10.1101/2020.11.10.376673","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.07.370726","pub_date":"2020-11-10","title":"In Vitro Efficacy of \u201cEssential Iodine Drops\u201d Against Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2)","abstract":"Aerosolization of respiratory droplets is considered the main route of coronavirus disease 2019 (COVID-19). Therefore, reducing the viral load of Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) shed via respiratory droplets is potentially an ideal strategy to prevent the spread of the pandemic. The in vitro virucidal activity of intranasal Povidone-Iodine (PVP-I) has been demonstrated recently to reduce SARS-CoV-2 viral titres. This study evaluated the virucidal activity of the aqueous solution of Iodine-V (a clathrate complex formed by elemental iodine and fulvic acid) as in Essential Iodine Drops (EID) with 200 \u03bcg elemental iodine/ml content against SARS-CoV-2 to ascertain whether it is a better alternative to PVP-I. SARS-CoV-2 (USAWA1/2020 strain) virus stock was prepared by infecting Vero 76 cells (ATCC CRL-1587) until cytopathic effect (CPE). The virucidal activity of EID against SARS-CoV-2 was tested in three dilutions (1:1; 2:1 and 3:1) in triplicates by incubating at room temperature (22 \u00b1 2\u00b0C) for either 60 or 90 seconds. The surviving viruses from each sample were quantified by a standard end-point dilution assay. EID (200 \u03bcg iodine/ml) after exposure for 60 and 90 seconds was compared to controls. In both cases, the viral titre was reduced by 99% (LRV 2.0). The 1:1 dilution of EID with virus reduced SARS-CoV-2 virus from 31,623 cell culture infectious dose 50% (CCCID50) to 316 CCID50 within 90 seconds. Substantial reductions in LRV by Iodine-V in EID confirmed the activity of EID against SARS-CoV-2 in vitro, demonstrating that Iodine-V in EID is effective at inactivating the virus in vitro and therefore suggesting its potential application intranasally to reduce SARS-CoV-2 transmission from known or suspected COVID-19 patients.","version":"1.1","doi":"10.1101/2020.11.07.370726","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.375022","pub_date":"2020-11-10","title":"A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples","abstract":"The COVID-19 pandemic is a once-in-a-lifetime event, exceeding mortality rates of the flu pandemics from the 1950\u2019s and 1960\u2019s. Whole-genome sequencing (WGS) of SARS-CoV-2 plays a critical role in understanding the disease. Performance variation exists across SARS-CoV-2 viral WGS technologies, but there is currently no benchmarking study comparing different WGS sequencing protocols. We compared seven different SARS-CoV-2 WGS library protocols using RNA from patient nasopharyngeal swab samples under two storage conditions. We constructed multiple WGS libraries encompassing three different viral inputs: 1,000,000, 250,000 and 1,000 copies. Libraries were sequenced using two distinct platforms with varying sequencing depths and read lengths. We found large differences in mappability and genome coverage, and variations in sensitivity, reproducibility and precision of single-nucleotide variant calling across different protocols. We ranked the performance of protocols based on six different metrics. Our results indicated that the most appropriate protocol depended on viral input amount and sequencing depth. Our findings offer guidance in choosing appropriate WGS protocols to characterize SARS-CoV-2 and its evolution.","version":"1.1","doi":"10.1101/2020.11.10.375022","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.372201","pub_date":"2020-11-10","title":"The hydroalcoholic extract of Uncaria tomentosa (Cat\u2019s claw) inhibits the replication of novel coronavirus (SARS-CoV-2) in vitro","abstract":"The coronavirus disease 2019 (COVID-19) has become a serious problem for public health since it was identified in the province of Wuhan (China) and spread around the world producing high mortality rates and economic losses. Nowadays, WHO recognizes traditional, complementary, and alternative medicine for treating COVID-19 symptoms. Therefore, we investigated the antiviral potential of the hydroalcoholic extract of Uncaria tomentosa stem bark from Peru against SARS-CoV-2 in vitro. The antiviral activity of U. tomentosa against SARS-CoV-2 in vitro was assessed in Vero E6 cells using cytopathic effect (CPE) and plaque reduction assay. After 48h of treatment, U. tomentosa showed an inhibition of 92.7 % of SARS-CoV-2 at 25.0 \u00b5g/mL (p<0.0001) by plaque reduction assay on Vero E6 cells. In addition, U. tomentosa, induced a reduction of 98.6 % (p=0.02) and 92.7 % (p=0.03) in the CPE caused by SARS-CoV-2 on Vero E6 cells at 25 \u00b5g/mL and 12.5 \u00b5g/mL, respectively. The EC50 calculated for U. tomentosa extract by plaque reduction assay was 6.6 \u00b5g/mL (4.89 \u2013 8.85 \u00b5g/mL) for a selectivity index of 4.1. The EC50 calculated for U. tomentosa extract by TCID50 assay was 2.57 \u00b5g/mL (1.05 \u2013 3.75 \u00b5g/mL) for a selectivity index of 10.54. These results showed that U. tomentosa known as Cat\u2019s claw has antiviral effect against SARS-CoV-2 observed as a reduction in the viral titer and CPE after 48h of treatment on Vero E6 cells. Therefore, we hypothesized that U. tomentosa stem bark, could be promissory to the development of new therapeutic strategies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.09.372201","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.376822","pub_date":"2020-11-10","title":"Highly potent bispecific sybodies neutralize SARS-CoV-2","abstract":"The COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. Cryo-EM analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. In the symmetric complex each of the three RBDs were bound by both sybodies, and adopted the up conformation. The asymmetric conformation, with three Sb#15 and two Sb#68 bound, contained one down RBD, one up-out RBD and one up RBD. Bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. Our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent SARS-CoV-2 inhibitors for potential therapeutic applications.","version":"1.1","doi":"10.1101/2020.11.10.376822","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.10.374587","pub_date":"2020-11-10","title":"Susceptibility of well-differentiated airway epithelial cell cultures from domestic and wildlife animals to SARS-CoV-2","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.10.374587","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.08.372581","pub_date":"2020-11-09","title":"Resistance of endothelial cells to SARS-CoV-2 infection in vitro","abstract":"The secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium activating thrombotic pathways, disrupting barrier function and allowing access of the virus to other organs of the body. However, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed. To determine infectibility of primary endothelial cell lines with live SARS-CoV-2 and pseudoviruses expressing SARS-CoV-2 spike protein. Expression of ACE2 and BSG pathways genes was determined in three types of endothelial cells; blood outgrowth, lung microvascular and aortic endothelial cells. For comparison nasal epithelial cells, Vero E6 cells (primate kidney fibroblast cell line) and HEK 293T cells (human embryonic kidney cells) transfected with either ACE2 or BSG were used as controls. Endothelial and Vero E6 cells were treated with live SARS-CoV-2 virus for 1 hour and imaged at 24 and 72 hours post infection. Pseudoviruses containing SARS-CoV-2, Ebola and Vesicular Stomatis Virus glycoproteins were generated and added to endothelial cells and HEK 239Ts for 2 hours and infection measured using luminescence at 48 hours post infection. Compared to nasal epithelial cells, endothelial cells expressed low or undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG, PPIA and PPIB. Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudovirus (but showed susceptibility to Ebola and Vesicular Stomatitis Virus). Overexpression of ACE2 but not BSG in HEK 239T cells conferred SARS-CoV-2 pseudovirus entry. Endothelial cells primed with IL-1\u00df remained resistant to SARS-CoV-2. Endothelial cells are resistant to infection with SARS-CoV-2 virus, in line with relatively low levels of ACE2 and TMPRSS2, suggesting that the vascular dysfunction and thrombosis seen in severe COVID-19 is a result of factors released by adjacent infected cells (e.g. epithelial cells) and/or circulating, systemic inflammatory mediators.","version":"1.1","doi":"10.1101/2020.11.08.372581","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.368431","pub_date":"2020-11-09","title":"Identification, Isolation, Propagation and Inactivation of SARS-CoV2 Isolated from Egypt","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 causes the novel pandemic Pneumonia disease. It is a positive single strand ssRNA virus that infect human. COVID-19 appeared in Egypt in Feb 2020. The samples were taken from patients with COVID-19 symptoms at military hospital in Egypt and transported to the main chemical laboratories under all the biosafety measures according to WHO guidelines. All samples were tested with RT-PCR. Positive samples were cultured using VeroE6 cell lines. The propagated virus was isolated and inactivated. The isolated virus was sequenced using next generation sequencing and submitted into gene bank. This study provides an isolation, propagation and inactivation methodology which is valuable for production of inactivated vaccines against SARS-CoV2 in Egypt.","version":"1.3","doi":"10.1101/2020.11.04.368431","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.372375","pub_date":"2020-11-09","title":"Nafamostat Mesylate in lipid carrier for nasal SARS-CoV2 titer reduction in a hamster model","abstract":"Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) has been responsible for the largest pandemic in recent decades. After seemingly being in control due to consequent lock-downs and social distancing, the majority of countries faces currently a second wave of exponentially increasing infections, hospital referrals and deaths due to SARS-CoV-2-mediated disease (COVID-19). To date, no effective vaccination has been found, and wearing masks and social distancing are the only effective approaches to reduce further spreading. However, unwillingness in the societies to distance again and consequently wear masks might be reasons for the second SARS-CoV-2 infection wave. User-friendly chemicals interfering at the host site with viral entry might be an approach to contain the pandemic. In addition, such an approach would work synergistic with vaccinations that miss new virus mutants. Nafamostat (NM) has been shown in vitro to interfere with cellular virus entry by inhibition of the host transmembrane protease serine 2 (TMPRSS2), an enzyme required for SARS-CoV-2 spike protein cleavage, a prerequisite for cell entry. We hypothesized that nasal application of NM in a liposomal layer (as additional mechanical barrier) could lower the nasal viral load and subsequently reduce the severity of COVID-19. We found, indeed, that nasal viral load one day post single NM application, was lowered in a hamster SARS-CoV-2 infection model. However, severity of subsequent local tissue destruction and weight loss due to pneumonitis was not favorably altered. In conclusion, a single NM application reduced nasal viral load, but did not favorably improve the outcome of COVID-19, likely due to the short half-time of NM. Improvement of NM stability or repetitive application (which was not permitted in this animal model according to Dutch law) might circumvent these challenges.","version":"1.1","doi":"10.1101/2020.11.09.372375","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.358614","pub_date":"2020-11-09","title":"Mice with Induced Pulmonary Comorbidities Display Severe Lung Inflammation and Mortality following Exposure to SARS-CoV-2","abstract":"Severe manifestations of COVID-19 are mostly restricted to people with comorbidities. Here we report that induced mild pulmonary morbidities render SARS-CoV-2-refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Notably, the deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel mice-based animal model for the study of comorbidity-dependent severe COVID-19.","version":"1.3","doi":"10.1101/2020.10.28.358614","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.335513","pub_date":"2020-11-09","title":"Alternate primers for whole-genome SARS-CoV-2 sequencing","abstract":"As the world is struggling to control the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there is an urgency to develop effective control measures. Essential information is encoded in the virus genome sequence with accurate and complete SARS-CoV-2 sequences essential for tracking the movement and evolution of the virus and for guiding efforts to develop vaccines and antiviral drugs. While there is unprecedented SARS-CoV-2 sequencing efforts globally, approximately 19 to 43% of the genomes generated monthly are gapped, reducing their information content. The current study documents the genome gap frequencies and their positions in the currently available data and provides an alternative primer set and a sequencing scheme to help improve the quality and coverage of the genomes.","version":"1.3","doi":"10.1101/2020.10.12.335513","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.374082","pub_date":"2020-11-09","title":"SARS-CoV-2 epitope mapping on microarrays highlights strong immune-response to N protein region","abstract":"A workflow for SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 Spike (S), Nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-171) providing 92% sensitivity and 100% specificity of IgG detection in Covid-19 samples thus being a promising candidate for rapid implementation in serological tests.","version":"1.1","doi":"10.1101/2020.11.09.374082","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.374603","pub_date":"2020-11-09","title":"The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways","abstract":"The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33\u00b0C instead of 37\u00b0C. Even stronger preference for the upper airway temperature of 33\u00b0C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37\u00b0C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37\u00b0C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology. The rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33\u00b0C) or lower (37\u00b0C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.","version":"1.1","doi":"10.1101/2020.11.09.374603","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.08.373738","pub_date":"2020-11-09","title":"Virucidal efficacy of different formulations for hand and surface disinfection targeting SARS CoV-2","abstract":"In the ongoing SARS CoV-2 pandemic effective measures are needed, and guidance based on the methodological framework of the European committee for standardization (CEN) can help to choose effective disinfectants on an immediate basis. This study demonstrates that two commercially available formulations for surface disinfection and one formulation for hand disinfection claiming \u201cvirucidal activity against enveloped viruses\u201d are effectively inactivating SARS-CoV-2. This study emphasizes that chemical disinfectants claiming \u201cvirucidal activity against enveloped viruses\u201d are an effective choice to target enveloped SARS-CoV-2 as a preventive measure.","version":"1.1","doi":"10.1101/2020.11.08.373738","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.05.370767","pub_date":"2020-11-09","title":"Nodosome inhibition as a novel broad-spectrum antiviral strategy against arboviruses and SARS-CoV-2","abstract":"In the present report, we describe two small molecules with broad-spectrum antiviral activity. These drugs block formation of the nodosome. The studies were prompted by the observation that infection of human fetal brain cells with Zika virus (ZIKV) induces expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a host factor that was found to promote ZIKV replication and spread. A drug that targets NOD2 was shown to have potent broad-spectrum antiviral activity against other flaviviruses, alphaviruses and SARS-CoV-2, the causative agent of COVID-19. Another drug that inhibits the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) which functions downstream of NOD2, also decreased replication of these pathogenic RNA viruses. The broad-spectrum action of nodosome targeting drugs is mediated, at least in part, by enhancement of the interferon response. Together, these results suggest that further preclinical investigation of nodosome inhibitors as potential broad-spectrum antivirals is warranted.","version":"1.2","doi":"10.1101/2020.11.05.370767","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.375139","pub_date":"2020-11-09","title":"Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells","abstract":"The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here we demonstrate a quantitative reporter for Mpro function in living cells, in which protease inhibition by genetic or chemical methods results in strong eGFP fluorescence. This robust gain-of-function system readily distinguishes between inhibitor potencies and can be scaled-up to high-throughput platforms for drug testing.","version":"1.1","doi":"10.1101/2020.11.09.375139","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.374272","pub_date":"2020-11-09","title":"Single cell transcriptomic re-analysis of immune cells in bronchoalveolar lavage fluids reveals the correlation of B cell characteristics and disease severity of patients with SARS-CoV-2 infection","abstract":"The COVID-19 pandemic (SARS-CoV-2) is a global infectious disease with rapid spread. Some patients have severe symptoms and clinical signs caused by an excessive inflammatory response, which increases the risk of mortality. In this study, we reanalyzed scRNA-seq data of cells from bronchoalveolar lavage fluids of patients with COVID-19 with mild and severe symptoms, focusing on antibody-producing cells. In patients with severe disease, B cells seemed to be more activated and expressed more immunoglobulin genes compared with cells from patients with mild disease, and macrophages expressed higher levels of the TNF superfamily member B-cell activating factor but not of APRIL (a proliferation-inducing ligand). In addition, macrophages from patients with severe disease had increased pro-inflammatory features and pathways associated with Fc receptor-mediated signaling, compared with patients with mild disease. CCR2-positive plasma cells accumulated in patients with severe disease, probably because of increased CCL2 expression on macrophages from patients with severe disease. Together, these results support that different characteristics of B cells might affect the severity of COVID-19 infection.","version":"1.1","doi":"10.1101/2020.11.09.374272","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.374769","pub_date":"2020-11-09","title":"Neutrophil extracellular traps induce the epithelial-mesenchymal transition: implications in post-COVID-19 fibrosis","abstract":"The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Neutrophils activated with PMA (PMA-Neu), a stimulus known to induce NETs formation, induce both EMT and cell death in the lung epithelial cell line, A549. Notably, NETs isolated from PMA-Neu induce EMT without cell damage. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs. Thus, we tested in an in vitro alveolar model the hypothesis that virus-induced NET may drive EMT. Co-culturing A549 at air-liquid interface with alveolar macrophages, neutrophils and SARS-CoV2, we demonstrated a significant induction of the EMT in A549 together with high concentration of NETs, IL8 and IL1\u03b2, best-known inducers of NETosis. Lung tissues of COVID-19 deceased patients showed that epithelial cells are characterized by increased mesenchymal markers. These results show for the first time that NETosis plays a major role in triggering lung fibrosis in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.11.09.374769","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.374173","pub_date":"2020-11-09","title":"Towards Targeting the Disordered SARS-CoV-2 Nsp2 C-terminal Region: Partial Structure and Dampened Mobility Revealed by NMR Spectroscopy","abstract":"Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many virus use their own IDPs to \u201chack\u201d these processes to disactive host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational perferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2 interacts with human proteins that regulate translation initiation and endosome vesicle sorting, and the C-terminal region of this protein is predicted to be disordered. Molecules that block these interactions could be valuable leads for drug development. To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13C,15N-labeled C-terminal region of Nsp2. The 13C\u03b2 and backbone 13CO, 1HN, 13C\u03b1 and 15N nuclei were assigned by analysis of a series of 2D 1H-15N HSQC and 13C-15N CON as well as 3D HNCO, HNCA, CBCAcoNH and HncocaNH spectra. Overall, the chemical shift data confirm that this region is chiefly disordered, but contains two five-residue segments that adopt a small population of \u03b2-strand structure. Whereas the region is flexible on ms/ms timescales as gauged by T1\u03c1 measurements, the {1H}-15N NOEs reveal a flexibility on ns/ps timescales that is midway between a fully flexible and a completely rigid chain.","version":"1.1","doi":"10.1101/2020.11.09.374173","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.09.372169","pub_date":"2020-11-09","title":"Identification of a unique TCR repertoire, consistent with a superantigen selection process in Children with Multi-system Inflammatory Syndrome","abstract":"Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares many clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. The superantigen specificity for binding different V\u03b2-chains results in V\u03b2-skewing, whereby T cells with specific V\u03b2-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Beta Variable gene (TRBV)11-2. Furthermore, TRBV11-2 skewing was remarkably correlated with MIS-C severity and serum cytokine levels. Further analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanding TRBV11-2 clones revealed extensive junctional diversity, indicating a superantigen-mediated selection process for TRBV expansion. In silico modelling indicates that polyacidic residues in TCR V\u03b211-2 engage in strong interactions with the superantigen-like motif of SARS-CoV-2 spike glycoprotein. Overall, our data indicate that the immune response in MIS-C is consistent with superantigenic activation. Multisystem Inflammatory Disease in Children (MIS-C) patients exhibit T cell receptor (TCR) repertoire skewing, with expansion of T cell Receptor Beta Variable gene (TRBV)11-2 TRBV11-2 skewing correlates with MIS-C severity and cytokine storm J gene/CDR3 diversity in MIS-C patients is compatible with a superantigen selection process In silico modelling indicates TCR V\u03b211-2 engages in CDR3-independent interactions with the polybasic insert P681RRAR in the SAg-like motif of SARS-CoV-2 spike","version":"1.1","doi":"10.1101/2020.11.09.372169","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.372227","pub_date":"2020-11-08","title":"Variability of Accessory Proteins Rules the SARS-CoV-2 Pathogenicity","abstract":"The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) which is pandemic with an estimated fatality rate less than 1% is ongoing. SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 with putative functions to manipulate host immune mechanisms such as interferons, immune signaling receptor NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) inflammasome, inflammatory cytokines such as interleukin 1\u03b2 (IL-1\u03b2) are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins of all complete proteomes (available as of October 26, 2020, in the National Center for Biotechnology Information depository) of SARS-CoV-2, were observed across six continents. Across all continents, the decreasing order of percentage of unique variations in the accessory proteins was found to be ORF3a>ORF8>ORF7a>ORF6>ORF10>ORF7b. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. This finding suggests that the wide variations of accessory proteins seem to govern the pathogenicity of SARS-CoV-2, and consequently, certain propositions and recommendations can be made in the public interest.","version":"1.1","doi":"10.1101/2020.11.06.372227","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.368191","pub_date":"2020-11-08","title":"A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2","abstract":"The COVID-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with SARS-CoV-2 and allow rapid and high throughput testing of drugs and vaccines. Exposure of larvae to SARS-CoV-2 Spike (S) receptor binding domain (RBD) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ACE inhibitor, reverted this effect. Intranasal administration of SARS-CoV-2 S RBD in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. Zebrafish intranasally treated with SARS-CoV-2 S RBD became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. Single cell RNA-Seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. Combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of SARS-CoV-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model.","version":"1.1","doi":"10.1101/2020.11.06.368191","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.07.367649","pub_date":"2020-11-08","title":"In vitro: Natural Compounds (Thymol, Carvacrol, Hesperidine, And Thymoquinone) Against Sars-Cov2 Strain Isolated From Egyptian Patients","abstract":"The current pandemic of the coronavirus disease-2019 (COVID-19) has badly affected our life during the year 2020. SARS-CoV-2 is the primary causative agent of the newly emerged pandemic. Natural flavonoids, Terpenoid and Thymoquinone are tested against different viral and host-cell protein targets. These natural compounds have a good history in treating Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Molecular docking combined with cytotoxicity and plaque reduction assay is used to test the natural compounds against different viral (Spike, RdRp, and Mpro) and host-cell (TMPRSS II, keap 1, and ACE2) targets. The results demonstrate the binding possibility of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) to the viral main protease (Mpro). Some of these natural compounds were approved to start clinical trail from Egypt Center for Research and Regenerative Medicine ECRRM IRB (Certificate No.IRB00012517)","version":"1.1","doi":"10.1101/2020.11.07.367649","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.07.365726","pub_date":"2020-11-08","title":"Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection","abstract":"The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.","version":"1.1","doi":"10.1101/2020.11.07.365726","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.264564","pub_date":"2020-11-06","title":"Flagellin from Pseudomonas aeruginosa modulates SARS-CoV-2 infectivity in CF airway epithelial cells by increasing TMPRSS2 expression","abstract":"The major challenge of the COVID-19 health crisis is to identify the factors of susceptibility to SARS-Cov2 in order to adapt the recommendations to the populations and to reduce the risk of getting COVID-19 to the most vulnerable people especially those having chronic respiratory diseases including cystic fibrosis (CF). Airway epithelial cells (AEC) are playing a critical role in the immune response and in COVID-19 severity. SARS-CoV-2 infects the airways through ACE2 receptor and the host protease TMPRSS2 was shown to play a major role in SARS-CoV-2 infectivity. Here, we show that the main component of P. aeruginosa flagella, ie. flagellin is able to increase TMPRSS2 expression in AEC, and even more in those deficient for CFTR. Importantly, this increased TMPRSS2 expression is associated with an increase in the level of SARS-CoV-2 infection. Considering the urgency of the health situation, this result is of major significance for patients with CF which are frequently infected and colonized by P. aeruginosa during the course of the disease.","version":"1.2","doi":"10.1101/2020.08.24.264564","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.371617","pub_date":"2020-11-06","title":"Persistence of SARS-CoV-2 specific B- and T-cell responses in convalescent COVID-19 patients 6-8 months after the infection","abstract":"The longevity of the immune response against SARS-CoV-2 is currently debated. We thus profiled the serum anti-SARS-CoV-2 antibody levels and virus specific memory B- and T-cell responses over time in convalescent COVID-19 patients. A cohort of COVID-19 patients from the Lombardy region in Italy who experienced mild to critical disease and Swedish volunteers with mild symptoms, were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, specific memory B- and T-cell responses were tested in selected patient samples. Anti-SARS-CoV-2 antibodies were present in 85% samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM or IgA antibodies declined after 1 month while levels of specific IgG antibodies remained stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses were developed in vast majority of the patients tested, regardless of disease severity, and remained detectable up to 6-8 months after infection. Although the serum levels of anti-SARS-CoV-2 IgG antibodies started to decline, virus-specific T and/or memory B cell responses increased with time and maintained during the study period (6-8 months after infection). European Union\u2019s Horizon 2020 research and innovation programme (ATAC), the Italian Ministry of Health, CIMED, the Swedish Research Council and the China Scholarship Council.","version":"1.1","doi":"10.1101/2020.11.06.371617","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.370676","pub_date":"2020-11-06","title":"Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago","abstract":"Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.06.370676","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.369439","pub_date":"2020-11-06","title":"Allosteric Hotspots in the Main Protease of SARS-CoV-2","abstract":"Inhibiting the main protease of SARS-CoV-2 is of great interest in tackling the COVID-19 pandemic caused by the virus. Most efforts have been centred on inhibiting the binding site of the enzyme. However, considering allosteric sites, distant from the active or orthosteric site, broadens the search space for drug candidates and confers the advantages of allosteric drug targeting. Here, we report the allosteric communication pathways in the main protease dimer by using two novel fully atomistic graph theoretical methods: Bond-to-bond propensity analysis, which has been previously successful in identifying allosteric sites without a priori knowledge in benchmark data sets, and, Markov transient analysis, which has previously aided in finding novel drug targets in catalytic protein families. We further score the highest ranking sites against random sites in similar distances through statistical bootstrapping and identify four statistically significant putative allosteric sites as good candidates for alternative drug targeting.","version":"1.1","doi":"10.1101/2020.11.06.369439","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.181867","pub_date":"2020-11-06","title":"periscope: sub-genomic RNA identification in SARS-CoV-2 Genomic Sequencing Data","abstract":"We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed \u201csub-genomic RNAs\u201d. sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5\u2019 UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5\u2019 end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/\u2212 cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.","version":"1.2","doi":"10.1101/2020.07.01.181867","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.370999","pub_date":"2020-11-06","title":"Differing impacts of global and regional responses on SARS-CoV-2 transmission cluster dynamics","abstract":"Although the global response to COVID-19 has not been entirely unified, the opportunity arises to assess the impact of regional public health interventions and to classify strategies according to their outcome. Analysis of genetic sequence data gathered over the course of the pandemic allows us to link the dynamics associated with networks of connected individuals with specific interventions. In this study, clusters of transmission were inferred from a phylogenetic tree representing the relationships of patient sequences sampled from December 30, 2019 to April 17, 2020. Metadata comprising sampling time and location were used to define the global behavior of transmission over this earlier sampling period, but also the involvement of individual regions in transmission cluster dynamics. Results demonstrate a positive impact of international travel restrictions and nationwide lockdowns on global cluster dynamics. However, residual, localized clusters displayed a wide range of estimated initial secondary infection rates, for which uniform public health interventions are unlikely to have sustainable effects. Our findings highlight the presence of so-called \u201csuper-spreaders\u201d, with the propensity to infect a larger-than-average number of people, in countries, such as the USA, for which additional mitigation efforts targeting events surrounding this type of spread are urgently needed to curb further dissemination of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.06.370999","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.299537","pub_date":"2020-11-06","title":"Tissue Level Profiling of SARS-CoV-2 antivirals in mice to predict their effects: comparing Remdesivir\u2019s active metabolite GS-441 524 vs. the clinically failed Hydroxychloroquine","abstract":"Remdesivir and hydroxychloroquine are or were among the most promising therapeutic options to tackle the current SARS-CoV-2 pandemic. Besides the use of the prodrug remdesivir itself, the direct administration of GS-441 524, the resulting main metabolite of remdesivir, could be advantageous and even more effective. All substances were not originally developed for the treatment of COVID-19 and especially for GS-441 524 little is known about its pharmacokinetic and physical-chemical properties. To justify the application of new or repurposed drugs in humans, pre-clinical in vivo animal models are mandatory to investigate relevant PK and PD properties and their relationship to each other. In this study, an adapted mouse model was chosen to demonstrate its suitability to provide sufficient information on the model substances GS-441 524 and HCQ regarding plasma concentration and distribution into relevant tissues a prerequisite for treatment effectiveness. GS-441 524 and HCQ were administered intravenously as a single injection to male mice. Blood and organ samples were taken at several time points and drug concentrations were quantified in plasma and tissue homogenates by two liquid chromatography/tandem mass spectrometry methods. In vitro experiments were conducted to investigate the degradation of remdesivir in human plasma and blood. All pharmacokinetic analyses were performed with R Studio using non-compartmental analysis. High tissue to plasma ratios for GS-441 524 and HCQ were found, indicating a significant distribution into the examined tissue, except for the central nervous system and fat. For GS-441 524, measured tissue concentrations exceeded the reported in vitro EC50 values by more than 10-fold and in consideration of its high efficacy against feline infectious peritonitis, GS-441 524 could indeed be effective against SARS-CoV-2 in vivo. For HCQ, relatively high in vitro EC50 values are reported, which were not reached in all tissues. Facing its slow tissue distribution, HCQ might not lead to sufficient tissue saturation for a reliable antiviral effect. The mouse model was able to characterise the PK and tissue distribution of both model substances and is a suitable tool to investigate early drug candidates against SARS-CoV-2. Furthermore, we could demonstrate a high tissue distribution of GS-441 524 even if not administered as the prodrug remdesivir.","version":"1.3","doi":"10.1101/2020.09.16.299537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.20225805","pub_date":"2020-11-06","title":"Development of an automated chemiluminescence assay system for quantitative measurement of multiple anti-SARS-CoV-2 antibodies","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>Serological tests for COVID-19 have been instrumental in studying the epidemiology of the disease. However, the performance of the currently available tests is plagued by the problem of variability. We have developed a high-throughput serological test capable of simultaneously detecting total immunoglobulins (Ig) and immunoglobulin G (IgG) against two of the most immunologically relevant SARS-CoV-2 antigens, nucleocapsid protein (NP) and spike protein (SP) and report its performance in detecting COVID-19 in clinical samples.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We designed and prepared reagents for measuring NP-IgG, NP-Total Ig, SP-IgG, and SP-Total Ig (using N-terminally truncated NP (\u0394N-NP) or receptor-binding domain (RBD) antigen) on the advanced chemiluminescence enzyme immunoassay system TOSOH AIA-CL. After determining the basal thresholds based on 17 sera obtained from confirmed COVID-19 patients and 600 negative sera. Subsequently, the clinical validity of the assay was evaluated using independent 202 positive samples and 1,000 negative samples from healthy donors.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>All of the four test parameters showed 100% specificity individually (1,000/1,000; 95%CI, 99.63-100). The sensitivity of the assay increased proportionally to the elapsed time from symptoms onset, and all the tests achieved 100% sensitivity (153/153; 95%CI, 97.63-100) after 13 days from symptoms onset. NP-Total Ig was the earliest to attain maximal sensitivity among the other antibodies tested.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Our newly developed serological testing exhibited 100% sensitivity and specificity after 13 days from symptoms onset. Hence, it could be used as a reliable method for accurate detection of COVID-19 patients and to evaluate seroprevalence and possibly for surrogate assessment of herd immunity.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.11.04.20225805","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.06.370916","pub_date":"2020-11-06","title":"Acrylamide Fragment Inhibitors that Induce Unprecedented Conformational Distortions in Enterovirus 71 3C and SARS-CoV-2 Main Protease","abstract":"RNA viruses are critically dependent upon virally encoded proteases that cleave the viral polyproteins into functional mature proteins. Many of these proteases are structurally conserved with an essential catalytic cysteine and this offers the opportunity to irreversibly inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that selectively target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we effectively re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold as well as similar catalytic-triad. We demonstrate that the hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. In addition, we provide the first demonstration that targeting the active site cysteine of Mpro can also have profound allosteric effects, distorting secondary structures required for formation of the active dimeric unit of Mpro. These new data provide novel mechanistic insights into the design of EV71 3Cpro and SARS-CoV-2 Mpro inhibitors and identify acrylamide-tagged pharmacophores for elaboration into more selective agents of therapeutic potential.","version":"1.1","doi":"10.1101/2020.11.06.370916","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.06.371971","pub_date":"2020-11-06","title":"Genomic and phenotypic analysis of COVID-19-associated pulmonary aspergillosis isolates of Aspergillus fumigatus","abstract":"The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) first described from Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as fungal opportunistic pathogens from the genus Aspergillus. To gain insight into COVID-19 associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit major differences from the genome of the Af293 reference strain. By examining virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, and the minimum inhibitory concentration of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss of function mutations in genes known to increase virulence when deleted (e.g., in the FLEA gene, which encodes a lectin recognized by macrophages). Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains tested. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA.","version":"1.1","doi":"10.1101/2020.11.06.371971","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.04.325415","pub_date":"2020-11-06","title":"Regulation of the ACE2 locus in human airways cells","abstract":"The angiotensin-converting enzyme 2 (ACE2) receptor is the gateway for SARS-CoV-2 to airway epithelium and the strong inflammatory response after viral infection is a hallmark in COVID-19 patients. Deciphering the regulation of the ACE2 gene is paramount for understanding the cell tropism of SARS-CoV-2 infection. Here we identify candidate regulatory elements in the ACE2 locus in human primary airway cells and lung tissue. Activating histone and promoter marks and Pol II loading characterize the intronic dACE2 and define novel candidate enhancers distal to the genuine ACE2 promoter and within additional introns. dACE2, and to a lesser extent ACE2, RNA levels increased in primary bronchial cells treated with interferons and this induction was mitigated by Janus kinase (JAK) inhibitors that are used therapeutically in COVID-19 patients. Our analyses provide insight into regulatory elements governing the ACE2 locus and highlight that JAK inhibitors are suitable tools to suppress interferon-activated genetic programs in bronchial cells.","version":"1.2","doi":"10.1101/2020.10.04.325415","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.336487","pub_date":"2020-11-06","title":"Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality","abstract":"Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. Using a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.","version":"1.2","doi":"10.1101/2020.10.12.336487","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.364315","pub_date":"2020-11-05","title":"SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis","abstract":"Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.","version":"1.1","doi":"10.1101/2020.11.04.364315","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.361576","pub_date":"2020-11-05","title":"Human Identical Sequences of SARS-CoV-2 Promote Clinical Progression of COVID-19 by Upregulating Hyaluronan via NamiRNA-Enhancer Network","abstract":"The COVID-19 pandemic is a widespread and deadly public health crisis. The pathogen SARS-CoV-2 replicates in the lower respiratory tract and causes fatal pneumonia. Although tremendous efforts have been put into investigating the pathogeny of SARS-CoV-2, the underlying mechanism of how SARS-CoV-2 interacts with its host is largely unexplored. Here, by comparing the genomic sequences of SARS-CoV-2 and human, we identified five fully conserved elements in SARS-CoV-2 genome, which were termed as \u201chuman identical sequences (HIS)\u201d. HIS are also recognized in both SARS-CoV and MERS-CoV genome. Meanwhile, HIS-SARS-CoV-2 are highly conserved in the primate. Mechanically, HIS-SARS-CoV-2, behaving as virus-derived miRNAs, directly target to the human genomic loci and further interact with host enhancers to activate the expression of adjacent and distant genes, including cytokines gene and angiotensin converting enzyme II (ACE2), a well-known cell entry receptor of SARS-CoV-2, and hyaluronan synthase 2 (HAS2), which further increases hyaluronan formation. Noteworthily, hyaluronan level in plasma of COVID-19 patients is tightly correlated with severity and high risk for acute respiratory distress syndrome (ARDS) and may act as a predictor for the progression of COVID-19. HIS antagomirs, which downregulate hyaluronan level effectively, and 4-Methylumbelliferone (MU), an inhibitor of hyaluronan synthesis, are potential drugs to relieve the ARDS related ground-glass pattern in lung for COVID-19 treatment. Our results revealed that unprecedented HIS elements of SARS-CoV-2 contribute to the cytokine storm and ARDS in COVID-19 patients. Thus, blocking HIS-involved activating processes or hyaluronan synthesis directly by 4-MU may be effective strategies to alleviate COVID-19 progression.","version":"1.1","doi":"10.1101/2020.11.04.361576","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.361154","pub_date":"2020-11-05","title":"Intranasal fusion inhibitory lipopeptide prevents direct contact SARS-CoV-2 transmission in ferrets","abstract":"Containment of the COVID-19 pandemic requires reducing viral transmission. SARS-CoV-2 infection is initiated by membrane fusion between the viral and host cell membranes, mediated by the viral spike protein. We have designed a dimeric lipopeptide fusion inhibitor that blocks this critical first step of infection for emerging coronaviruses and document that it completely prevents SARS-CoV-2 infection in ferrets. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour co-housing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and non-toxic and thus readily translate into a safe and effective intranasal prophylactic approach to reduce transmission of SARS-CoV-2. A dimeric form of a SARS-CoV-2-derived lipopeptide is a potent inhibitor of fusion and infection in vitro and transmission in vivo.","version":"1.1","doi":"10.1101/2020.11.04.361154","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.368449","pub_date":"2020-11-05","title":"Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes","abstract":"DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primers or probes regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-COV-2, SARS-COV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 \u00b0C to the fully matched reference temperature. We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.","version":"1.1","doi":"10.1101/2020.11.04.368449","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.367375","pub_date":"2020-11-05","title":"Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals","abstract":"The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the RBD and the non-RBD domain of the spike antigen using a novel TCR-binding algorithm. A selected pool of 11 predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools containing 157 and 158 peptides both in unexposed donors and in convalescent patients suggesting that strong T-cell epitopes are likely to be missed when larger peptide pools are used in assays. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. Whether the presence of pre-existing T-cell immunity provides protection against COVID-19 or contributes to severe disease phenotype remains to be determined in a larger cohort. However, our findings raise the expectation that a significant majority of the global population is likely to have SARS-CoV-2 reactive T-cells because of prior exposure to flu and CMV viruses, in addition to common cold-causing coronaviruses.","version":"1.1","doi":"10.1101/2020.11.03.367375","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.05.369264","pub_date":"2020-11-05","title":"The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN","abstract":"The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.","version":"1.1","doi":"10.1101/2020.11.05.369264","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.369165","pub_date":"2020-11-05","title":"Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing","abstract":"The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here we report a simple and efficient workflow for whole genome sequencing utilizing one-step RT-PCR amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm IFC and instruments to amplify 48 samples with 39 pairs of primers in a single step. Application of this method on RNA samples from both viral isolate and clinical specimens demonstrate robustness and efficiency of this method in obtaining the full genome sequence of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.04.369165","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.05.370239","pub_date":"2020-11-05","title":"SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging","abstract":"Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.","version":"1.1","doi":"10.1101/2020.11.05.370239","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.355842","pub_date":"2020-11-05","title":"The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity","abstract":"SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.","version":"1.1","doi":"10.1101/2020.11.04.355842","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.369041","pub_date":"2020-11-05","title":"A Workflow of Integrated Resources to Catalyze Network Pharmacology Driven COVID-19 Research","abstract":"In the event of an outbreak due to an emerging pathogen, time is of the essence to contain or to mitigate the spread of the disease. Drug repositioning is one of the strategies that has the potential to deliver therapeutics relatively quickly. The SARS-CoV-2 pandemic has shown that integrating critical data resources to drive drug-repositioning studies, involving host-host, hostpathogen and drug-target interactions, remains a time-consuming effort that translates to a delay in the development and delivery of a life-saving therapy. Here, we describe a workflow we designed for a semi-automated integration of rapidly emerging datasets that can be generally adopted in a broad network pharmacology research setting. The workflow was used to construct a COVID-19 focused multimodal network that integrates 487 host-pathogen, 74,805 host-host protein and 1,265 drug-target interactions. The resultant Neo4j graph database named \u201cNeo4COVID19\u201d is accessible via a web interface and via API calls based on the Bolt protocol. We believe that our Neo4COVID19 database will be a valuable asset to the research community and will catalyze the discovery of therapeutics to fight COVID-19. https://neo4covid19.ncats.io","version":"1.1","doi":"10.1101/2020.11.04.369041","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.25.354548","pub_date":"2020-11-04","title":"Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain","abstract":"COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing tens of millions of infections and over a million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species\u2019 ACE2 and human ACE2. As cattle are a known reservoir for coronaviruses with previous human zoonotic transfer, and has a relatively divergent ACE2 sequence, we compared the binding kinetics of bovine and human ACE2 to SARS-CoV-2 RBD. This revealed a nanomolar binding affinity for bovine ACE2 but an approximate ten-fold reduction of binding compared to human ACE2. Since cows have been experimentally infected by SARS-CoV-2, this lower affinity sets a threshold for sequences with lower homology to human ACE2 to be able to serve as a productive viral receptor for SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.10.25.354548","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.20222778","pub_date":"2020-11-04","title":"SARS-CoV-2 responsive T cell numbers and anti-Spike IgG levels are both associated with protection from COVID-19: A prospective cohort study in keyworkers","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Immune correlates of protection from COVID-19 are incompletely understood. 2,826 keyworkers had T-SPOT\n                  <jats:sup>\u00ae</jats:sup>\n                  <jats:italic>Discovery</jats:italic>\n                  SARS-CoV-2 tests (measuring interferon-\u03b3 secreting, SARS-CoV-2 responsive T cells, Oxford Immunotec Ltd), and anti-Spike S1 domain IgG antibody levels (EuroImmun AG) performed on recruitment into a cohort study. 285/2,826 (10.1%) of participants had positive SARS-CoV-2 RT-PCR tests, predominantly associated with symptomatic illness, during 200 days followup. T cell responses to Spike, Nucleoprotein and Matrix proteins (SNM responses) were detected in some participants at recruitment, as were anti-Spike S1 IgG antibodies; higher levels of both were associated with protection from subsequent SARS-CoV-2 test positivity. In volunteers with moderate antibody responses, who represented 39% (252/654) of those with detectable anti-Spike IgG, protection was partial, and higher with higher circulating T cell SNM responses. SARS-CoV-2 responsive T cell numbers predict protection in individuals with low anti-Spike IgG responses; serology alone underestimates the proportion of the population protected after infection.\n                </jats:p>","version":null,"doi":"10.1101/2020.11.02.20222778","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.04.20225573","pub_date":"2020-11-04","title":"Household Transmission of SARS-CoV-2: Insights from a Population-based Serological Survey","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Knowing the transmissibility of asymptomatic infections and risk of infection from household- and community-exposures is critical to SARS-CoV-2 control. Limited previous evidence is based primarily on virologic testing, which disproportionately misses mild and asymptomatic infections. Serologic measures are more likely to capture all previously infected individuals.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>Estimate the risk of SARS-CoV-2 infection from household and community exposures, and identify key risk factors for transmission and infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Cross-sectional household serosurvey and transmission model.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>Geneva, Switzerland</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>4,524 household members \u22655 years from 2,267 households enrolled April-June 2020.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Measurements</jats:title>\n                  <jats:p>Past SARS-CoV-2 infection confirmed through IgG ELISA. Chain-binomial models based on the number of infections within households used to estimate the cumulative extra-household infection risk and infection risk from exposure to an infected household member by demographics and infector\u2019s symptoms.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>The chance of being infected by a SARS-CoV-2 infected household member was 17.3% (95%CrI,13.7-21.7%) compared to a cumulative extra-household infection risk of 5.1% (95%CrI,4.5-5.8%). Infection risk from an infected household member increased with age, with 5-9 year olds having 0.4 times (95%CrI, 0.07-1.4) the odds of infection, and \u226565 years olds having 2.7 (95%CrI,0.88-7.4) times the odds of infection of 20-49 year olds. Working-age adults had the highest extra-household infection risk. Seropositive asymptomatic household members had 69.6% lower odds (95%CrI,33.7-88.1%) of infecting another household member compared to those reporting symptoms, accounting for 14.7% (95%CrI,6.3-23.2%) of all household infections.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Limitations</jats:title>\n                  <jats:p>Self-reported symptoms, small number of seropositive kids and imperfect serologic tests.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>The risk of infection from exposure to a single infected household member was more than three-times that of extra-household exposures over the first pandemic wave. Young children had a lower risk of infection from household members. Asymptomatic infections are far less likely to transmit than symptomatic ones but do cause infections.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding Source</jats:title>\n                  <jats:p>Swiss Federal Office of Public Health, Swiss School of Public Health (Corona Immunitas research program), Fondation de Bienfaisance du Groupe Pictet, Fondation Ancrage, Fondation Priv\u00e9e des H\u00f4pitaux Universitaires de Gen\u00e8ve, and Center for Emerging Viral Diseases.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.11.04.20225573","journal":"medRxiv","score":null},{"id":"10.1101/2020.11.02.365536","pub_date":"2020-11-04","title":"Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients","abstract":"COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.","version":"1.2","doi":"10.1101/2020.11.02.365536","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.190454","pub_date":"2020-11-04","title":"Unsupervised machine learning reveals key immune cell subsets in COVID-19, rhinovirus infection, and cancer therapy","abstract":"For an emerging disease like COVID-19, systems immunology tools may quickly identify and quantitatively characterize cells associated with disease progression or clinical response. With repeated sampling, immune monitoring creates a real-time portrait of the cells reacting to a novel virus before disease specific knowledge and tools are established. However, single cell analysis tools can struggle to reveal rare cells that are under 0.1% of the population. Here, the machine learning workflow Tracking Responders Expanding (T-REX) was created to identify changes in both very rare and common cells in diverse human immune monitoring settings. T-REX identified cells that were highly similar in phenotype and localized to hotspots of significant change during rhinovirus and SARS-CoV-2 infections. Specialized reagents used to detect the rhinovirus-specific CD4+ cells, MHCII tetramers, were not used during unsupervised analysis and instead \u2018left out\u2019 to serve as a test of whether T-REX identified biologically significant cells. In the rhinovirus challenge study, T-REX identified virus-specific CD4+ T cells based on these cells being a distinct phenotype that expanded by \u226595% following infection. T-REX successfully identified hotspots containing virus-specific T cells using pairs of samples comparing Day 7 of infection to samples taken either prior to infection (Day 0) or after clearing the infection (Day 28). Mapping pairwise comparisons in samples according to both the direction and degree of change provided a framework to compare systems level immune changes during infectious disease or therapy response. This revealed that the magnitude and direction of systemic immune change in some COVID-19 patients was comparable to that of blast crisis acute myeloid leukemia patients undergoing induction chemotherapy and characterized the identity of the immune cells that changed the most. Other COVID-19 patients instead matched an immune trajectory like that of individuals with rhinovirus infection or melanoma patients receiving checkpoint inhibitor therapy. T-REX analysis of paired blood samples provides an approach to rapidly identify and characterize mechanistically significant cells and to place emerging diseases into a systems immunology context.","version":"1.2","doi":"10.1101/2020.07.31.190454","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.04.367896","pub_date":"2020-11-04","title":"Risk perceptions and preventive practices of COVID-19 among healthcare professionals in public hospitals in Ethiopia","abstract":"Healthcare professionals are at higher risk of contracting the novel coronavirus due to their work exposure in the healthcare settings. Practicing appropriate preventive measures to control COVID-19 infection is one of the most important interventions that healthcare workers are expected to use. The aim of this study was to assess the level of risk perception and practices of preventive measures of COVID-19 among health workers in Addis Ababa, Ethiopia. A hospital-based cross-sectional study was conducted from 9th to 26th June 2020 among healthcare professionals working at six public hospitals in Addis Ababa. Data were collected using a self-administered structured questionnaire. Frequency, percentage, and mean were used to summarize the data. A binary logistic regression analyses were performed to identify factors associated with risk perception about COVID-19. A total of 1,134 participants were surveyed. Wearing facemask (93%), hand washing for at least 20 seconds (93%), covering mouth and nose while coughing or sneezing (91%), and avoiding touching eyes, nose, and mouth (91%) were the commonly self-reported preventive practices. About 88% perceived that they were worried about the risk of becoming infected with coronavirus, and majority (91%) worried about the risk of infection to their family. The mean score of overall fear and worry of COVID-19 was 2.37 on a scale of 1 to 3. Respondents who ever provided clinical care to COVID-19 patients were more likely to report fear and worry (adjusted OR=1.34, 95% CI:1.02-1.91), however those who ever participated in Ebola or SARS outbreaks were less likely to report fear and worry due to COVID-19 crisis (adjusted OR=0.66, 95% CI:0.48-0.90). This study has revealed widespread practices of preventive measures and the highest perceived risk of COVID-19 among healthcare workers. Therefore, an effective risk communication intervention should be implemented to ensure the maintenance of appropriate practices during the current COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.11.04.367896","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.285007","pub_date":"2020-11-04","title":"Brain volumetric changes in the general population following the COVID-19 outbreak and lockdown","abstract":"The COVID-19 outbreak introduced unprecedented health-risks, as well as pressure on the financial, social, and psychological well-being due to the response to the outbreak. Here, we examined the manifestations of the COVID-19 outbreak on the brain structure in the healthy population, following the initial phase of the pandemic in Israel. We pre-registered our hypothesis that the intense experience of the outbreak potentially induced stress-related brain modifications. Volumetric changes in n = 50 participants who were scanned before and after the COVID-19 outbreak and lockdown, were compared with n = 50 control participants who were scanned twice prior to the pandemic. The pandemic provided a rare opportunity to examine brain plasticity in a natural experiment. We found volumetric increases in bilateral amygdalae, putamen, and the anterior temporal cortices. Changes in the amygdalae diminished as time elapsed from lockdown relief, suggesting that the intense experience associated with the pandemic outbreak induced transient volumetric changes in brain regions commonly associated with stress and anxiety.","version":"1.3","doi":"10.1101/2020.09.08.285007","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.366666","pub_date":"2020-11-03","title":"Looking for pathways related to COVID-19 phenotypes: Confirmation of pathogenic mechanisms by SARS-CoV-2 - Host interactome","abstract":"In the last months, many studies have clearly described several mechanisms of SARS-CoV-2 infection at cell and tissue level. Host conditions and comorbidities were identified as risk factors for severe and fatal disease courses, but the mechanisms of interaction between host and SARS-CoV-2 determining the grade of COVID- 19 severity, are still unknown. We provide a network analysis on protein\u2013protein interactions (PPI) between viral and host proteins to better identify host biological responses, induced by both whole proteome of SARS-CoV-2 and specific viral proteins. A host-virus interactome was inferred on published PPI, using an explorative algorithm (Random Walk with Restart) triggered by all the 28 proteins of SARS-CoV-2, or each single viral protein one-by-one. The functional analysis for all proteins, linked to many aspects of COVID-19 pathogenesis, allows to identify the subcellular districts, where SARS-CoV-2 proteins seem to be distributed, while in each interactome built around one single viral protein, a different response was described, underlining as ORF8 and ORF3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. Finally, an explorative network-based approach was applied to Bradykinin Storm, highlighting a possible direct action of ORF3a and NS7b to enhancing this condition. This network-based model for SARS-CoV-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. We identified possible host responses induced by specific proteins of SARS-CoV-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe COVID-19 patients.","version":"1.1","doi":"10.1101/2020.11.03.366666","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.365049","pub_date":"2020-11-03","title":"Computational prediction of SARS-CoV-2 encoded miRNAs and their putative host targets","abstract":"Over the past two decades, there has been a continued research on the role of small non-coding RNAs including microRNAs (miRNAs) in various diseases. Studies have shown that viruses modulate the host cellular machinery and hijack its metabolic and immune signalling pathways by miRNA mediated gene silencing. Given the immensity of coronavirus disease 19 (COVID-19) pandemic and the strong association of viral encoded miRNAs with their pathogenesis, it is important to study Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) miRNAs. To address this unexplored area, we identified 8 putative novel miRNAs from SARS-CoV-2 genome and explored their possible human gene targets. A significant proportion of these targets populated key immune and metabolic pathways such as MAPK signalling pathway, maturity-onset diabetes, Insulin signalling pathway, endocytosis, RNA transport, TGF-\u03b2 signalling pathway, to name a few. The data from this work is backed up by recently reported high-throughput transcriptomics datasets obtains from SARS-CoV-2 infected samples. Analysis of these datasets reveal that a significant proportion of the target human genes were down-regulated upon SARS-CoV-2 infection. The current study brings to light probable host metabolic and immune pathways susceptible to viral miRNA mediated silencing in a SARS-CoV-2 infection, and discusses its effects on the host pathophysiology.","version":"1.1","doi":"10.1101/2020.11.02.365049","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.366138","pub_date":"2020-11-03","title":"Rapid development of SARS-CoV-2 receptor binding domain-conjugated nanoparticle vaccine candidate","abstract":"The ongoing of coronavirus disease 2019 (COVID-19) pandemic caused by novel SARS-CoV-2 coronavirus, resulting in economic losses and seriously threating the human health in worldwide, highlighting the urgent need of a stabilized, easily produced and effective preventive vaccine. The SARS-COV-2 spike protein receptor binding region (RBD) plays an important role in the process of viral binding receptor angiotensin-converting enzyme 2 (ACE2) and membrane fusion, making it an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticles vaccine candidates, RBD-Ferritin (24-mer), RBD-mi3 (60-mer) and RBD-I53-50 (120-mer), with the application of covalent bond linking by SpyTag-SpyCatcher system. It was demonstrated that the neutralizing capability of sera from mice immunized with three RBD-conjugated nanoparticles adjuvanted with AddaVax or Sigma Systerm Adjuvant (SAS) after each immunization was ~8-to 120-fold greater than monomeric RBD group in SARS-CoV-2 pseudovirus and authentic virus neutralization assay. Most importantly, sera from RBD-conjugated NPs groups more efficiently blocked the binding of RBD to ACE2 or neutralizing antibody in vitro, a further proof of promising immunization effect. Besides, high physical stability and flexibility in assembly consolidated the benefit for rapid scale-up production of vaccine. These results supported that our designed SARS-CoV-2 RBD-conjugated nanoparticle was competitive vaccine candidate and the carrier nanoparticles could be adopted as universal platform for future vaccine development.","version":"1.1","doi":"10.1101/2020.11.03.366138","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.366757","pub_date":"2020-11-03","title":"Structure of nonstructural protein 1 from SARS-CoV-2","abstract":"The periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burden worldwide. The most recent occurrence originated in the city of Wuhan, China where a novel coronavirus (SARS-CoV-2) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1\u2019s importance in the CoV lifecycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral lifecycle. Here we report the high-resolution crystal structure of the amino, globular portion of SARS-CoV-2 nsp1 (residues 10 \u2013 127) at 1.77\u00c5 resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 \u2013 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral lifecycle. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. One protein known to play a critical role in the coronavirus lifecycle is nonstructural protein1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and for the design of live-attenuated vaccines. Here we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77\u00c5 resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.","version":"1.1","doi":"10.1101/2020.11.03.366757","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.365270","pub_date":"2020-11-03","title":"The ACE2-binding interface of SARS-CoV-2 Spike inherently deflects immune recognition","abstract":"The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.11.03.365270","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.364729","pub_date":"2020-11-03","title":"Identification of Cross-Reactive CD8+ T Cell Receptors with High Functional Avidity to a SARS-CoV-2 Immunodominant Epitope and Its Natural Mutant Variants","abstract":"Despite the growing knowledge of T cell responses and their epitopes in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Using a peptide library predicted with HLA class I-restriction, specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients. Among the 15 SARS-CoV-2 epitopes identified from the S and N proteins, N361-369 (KTFPPTEPK) was the most dominant epitope. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity, and they exhibited complementary cross-reactivity to reported N361-369 mutant variants. In dendritic cells (DCs) and the lung organoid model, we found that the N361-369 epitope could be processed and endogenously presented to elicit the activation and cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, illuminating natural ways of viral clearance with high relevancy in the vaccine development.","version":"1.1","doi":"10.1101/2020.11.02.364729","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.366609","pub_date":"2020-11-03","title":"Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design","abstract":"Having claimed over 1 million lives worldwide to date, the ongoing COVID-19 pandemic has created one of the biggest challenges to develop an effective drug to treat infected patients. Among all the proteins expressed by the virus, RNA helicase is a fundamental protein for viral replication, and it is highly conserved among the coronaviridae family. To date, there is no high-resolution structure of helicase bound with ATP and RNA. We present here structural insights and molecular dynamics (MD) simulation results of the SARS-CoV-2 RNA helicase both in its apo form and in complex with its natural substrates. Our structural information of the catalytically competent helicase complex provides valuable insights for the mechanism and function of this enzyme at the atomic level, a key to develop specific inhibitors for this potential COVID-19 drug target.","version":"1.1","doi":"10.1101/2020.11.03.366609","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.363598","pub_date":"2020-11-03","title":"Potential Opportunity of Antisense Therapy of COVID-19 on an in Vitro Model","abstract":"Data on potential effectiveness and prospects of treatment of new coronavirus infection of COVID-19 caused by virus SARS-CoV-2 with the help of antisense oligonucleotides acting against RNA of virus on an in vitro model are given. The ability of antisense oligonucleotides to suppress viral replication in diseases caused by coronaviruses using the example of SARS and MERS is shown. The identity of the initial regulatory section of RNA of various coronaviruses was found within 50 - 100 nucleotides from the 5\u2019-end, which allows using antisense suppression of this RNA fragment. A new RNA fragment of the virus present in all samples of coronovirus SARS-CoV-2 has been identified, the suppression of which with the help of an antisense oligonucleotide can be effective in the treatment of COVID-19. The study of the synthesized antisense oligonucleotide 5\u2019 - AGCCGAGTGACAGCC ACACAG, complementary to the selected virus RNA sequence, was carried out. The low toxicity of the preparations of this group in the cell culture study and the ability to reduce viral load at high doses according to real time-PCR data are shown. The cytopathogenic dose exceeds 2 mg / ml. At a dosage of 1 mg / ml, viral replication is reduced by 5-13 times. Conclusions are made about the prospects of this direction and the feasibility of using the inhalation way of drug administration into the body.","version":"1.1","doi":"10.1101/2020.11.02.363598","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.20221804","pub_date":"2020-11-03","title":"A catalog of associations between rare coding variants and COVID-19 outcomes","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19), a respiratory illness that can result in hospitalization or death. We investigated associations between rare genetic variants and seven COVID-19 outcomes in 543,213 individuals, including 8,248 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome-wide or when specifically focusing on (i) 14 interferon pathway genes in which rare deleterious variants have been reported in severe COVID-19 patients; (ii) 167 genes located in COVID-19 GWAS risk loci; or (iii) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, with results publicly browsable at\n                  <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='uri' xlink:href='https://rgc-covid19.regeneron.com'>https://rgc-covid19.regeneron.com</jats:ext-link>\n                  .\n                </jats:p>","version":null,"doi":"10.1101/2020.10.28.20221804","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.31.363044","pub_date":"2020-11-03","title":"RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays","abstract":"Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a \u201cmix-and-measure\u201d homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.","version":"1.1","doi":"10.1101/2020.10.31.363044","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.03.366641","pub_date":"2020-11-03","title":"Early immune response in mice immunized with a semi-split inactivated vaccine against SARS-CoV-2 containing S protein-free particles and subunit S protein","abstract":"The development of a vaccine against COVID-19 is a hot topic for many research laboratories all over the world. Our aim was to design a semi-split inactivated vaccine offering a wide range of multi-epitope determinants important for the immune system including not only the spike (S) protein but also the envelope, membrane and nucleocapsid proteins. We designed a semi-split vaccine prototype consisting of S protein-depleted viral particles and free S protein. Next, we investigated its immunogenic potential in BALB/c mice. The animals were immunized intradermally or intramuscularly with the dose adjusted with buffer or addition of aluminum hydroxide, respectively. The antibody response was evaluated by plasma analysis at 7 days after the first or second dose. The immune cell response was studied by flow cytometry analysis of splenocytes. The data showed a very early onset of both S protein-specific antibodies and virus-neutralizing antibodies at 90% inhibition regardless of the route of vaccine administration. However, significantly higher levels of neutralizing antibodies were detected in the intradermally (geometric mean titer - GMT of 7.8 \u00b1 1.4) than in the intramuscularly immunized mice (GMT of 6.2 \u00b1 1.5). In accordance with this, stimulation of cellular immunity by the semi-split vaccine was suggested by elevated levels of B and T lymphocyte subpopulations in the murine spleens. These responses were more predominant in the intradermally immunized mice compared with the intramuscular route of administration. The upward trend in the levels of plasmablasts, memory B cells, Th1 and Th2 lymphocytes, including follicular helper T cells, was confirmed even in mice receiving the vaccine intradermally at a dose of 0.5 \u03bcg. We demonstrated that the semi-split vaccine is capable of eliciting both humoral and cellular immunity early after vaccination. Our prototype thus represents a promising step toward the development of an efficient anti-COVID-19 vaccine for human use.","version":"1.1","doi":"10.1101/2020.11.03.366641","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.363739","pub_date":"2020-11-02","title":"Temporal patterns in the evolutionary genetic distance of SARS-CoV-2 during the COVID-19 pandemic","abstract":"During the pandemic of coronavirus disease 2019 (COVID-19), the genetic mutations occurred in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cumulatively or sporadically. In this study, we employed a computational approach to identify and trace the emerging patterns of the SARS-CoV-2 mutations, and quantify accumulative genetic distance across different periods and proteins. Full-length human SARS-CoV-2 strains in United Kingdom were collected. We investigated the temporal variation in the evolutionary genetic distance defined by the Hamming distance since the start of COVID-19 pandemic. Our results showed that the SARS-CoV-2 was in the process of continuous evolution, mainly involved in spike protein (S protein), the RNA-dependent RNA polymerase (RdRp) region of open reading frame 1 (ORF1) and nucleocapsid protein (N protein). By contrast, mutations in other proteins were sporadic and genetic distance to the initial sequenced strain did not show an increasing trend.","version":"1.1","doi":"10.1101/2020.11.01.363739","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.201277","pub_date":"2020-11-02","title":"Single-nucleotide conservation state annotation of the SARS-CoV-2 genome","abstract":"Given the global impact and severity of COVID-19, there is a pressing need for a better understanding of the SARS-CoV-2 genome and mutations. Multi-strain sequence alignments of coronaviruses (CoV) provide important information for interpreting the genome and its variation. We apply a comparative genomics method, ConsHMM, to the multi-strain alignments of CoV to annotate every base of the SARS-CoV-2 genome with conservation states based on sequence alignment patterns among CoV. The learned conservation states show distinct enrichment patterns for genes, protein domains, and other regions of interest. Certain states are strongly enriched or depleted of SARS-CoV-2 mutations, which can be used to predict potentially consequential mutations. We expect the conservation states to be a resource for interpreting the SARS-CoV-2 genome and mutations.","version":"1.3","doi":"10.1101/2020.07.13.201277","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.359042","pub_date":"2020-11-02","title":"Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models","abstract":"SARS-CoV-2 is the coronavirus that causes the respiratory disease COVID-19, which is now the third-leading cause of death in the United States. The FDA has recently approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though recent data from the WHO shows little to no benefit with use of this anti-viral agent. Here we report the discovery of ethacridine, a safe antiseptic use in humans, as a potent drug for use against SARS-CoV-2 (EC50 ~ 0.08 \u03bcM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Indeed, ethacridine is effective in various cell types, including primary human nasal epithelial cells. Taken together, these data identify a promising, potent, and new use of the old drug possessing a distinct mode of action for inhibiting SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.10.28.359042","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.364497","pub_date":"2020-11-02","title":"The SARS-CoV-2 RNA interactome","abstract":"SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its ability to repurpose host RNA-binding proteins (RBPs) to form its own RNA interactome. Here, we developed and applied a robust ribonucleoprotein capture protocol to uncover the SARS-CoV-2 RNA interactome. We report 109 host factors that directly bind to SARS-CoV-2 RNAs including general antiviral factors such as ZC3HAV1, TRIM25, and PARP12. Applying RNP capture on another coronavirus HCoV-OC43 revealed evolutionarily conserved interactions between viral RNAs and host proteins. Network and transcriptome analyses delineated antiviral RBPs stimulated by JAK-STAT signaling and proviral RBPs responsible for hijacking multiple steps of the mRNA life cycle. By knockdown experiments, we further found that these viral-RNA-interacting RBPs act against or in favor of SARS-CoV-2. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.","version":"1.1","doi":"10.1101/2020.11.02.364497","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.364661","pub_date":"2020-11-02","title":"Liquid chalk is an antiseptic against SARS-CoV-2 and influenza A respiratory viruses","abstract":"The COVID-19 pandemic has impacted and enforced significant restrictions within our societies, including the attendance of the public and professional athletes in gyms. Liquid chalk is a commonly used accessories in gyms and is comprised of magnesium carbonate and alcohol that quickly evaporates on the hands to leave a layer of dry chalk. We investigated whether liquid chalk is an antiseptic against highly pathogenic human viruses including, SARS-CoV-2, influenza virus and noroviruses. Chalk was applied before or after virus inoculum and recovery of infectious virus was determined to mimic the use in the gym. We observed that addition of chalk before or after virus contact lead to a significant reduction on recovery of infectious SARS-CoV-2 and influenza but had little impact on norovirus. These observations suggest that the use and application of liquid chalk can be an effective and suitable antiseptic for major sporting events, such as the Olympic Games.","version":"1.1","doi":"10.1101/2020.11.02.364661","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.361766","pub_date":"2020-11-02","title":"Rapid inactivation of SARS-CoV-2 with ozone water","abstract":"Although ozone water is one of the promising candidates for hand hygiene to prevent fomite infection, the detailed effects of ozone water on SARS-CoV-2 have not been clarified. We evaluated the inactivating effect of ozone water against SARS-CoV-2 by its concentration and exposure time. The reduction rates of virus titer after 5 sec treatment with ozone concentrations of 1, 4, 7, and 10 mg/L were 81.4%, 93.2%, 96.6%, and 96.6%, respectively. No further decrease in virus titer was observed by the extended exposure time over 5 sec. High-concentration ozone water was considered to be effective in promptly inactivating SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2020.11.01.361766","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.363499","pub_date":"2020-11-02","title":"Characterization of structural and energetic differences between conformations of the SARS-CoV-2 spike protein","abstract":"The novel coronavirus disease 2019 (COVID-19) pandemic has disrupted modern societies and their economies. The resurgence in COVID-19 cases as part of the second wave is observed across Europe and the Americas. The scientific response has enabled a complete structural characterization of the Severe Acute Respiratory Syndrome \u2013 novel Coronavirus 2 (SARS-CoV-2). Among the most relevant proteins required by the novel coronavirus to facilitate the cell entry mechanism is the spike protein trimer. This protein possesses a receptor-binding domain (RBD) that binds the cellular angiotensin-converting enzyme 2 (ACE2) and then triggers the fusion of viral and host cell membranes. In this regard, a comprehensive characterization of the structural stability of the spike protein is a crucial step to find new therapeutics to interrupt the process of recognition. On the other hand, it has been suggested the participation of more than one RBD as a possible mechanism to enhance cell entry. Here we discuss the protein structural stability based on the computational determination of the dynamic contact map and the energetic difference of the spike protein conformations via the mapping of the hydration free energy by the Poisson-Boltzmann method. We expect our result to foster the discussion of the number of RBD involved during recognition and the repurposing of new drugs to disable the recognition by discovering new hotspots for drug targets apart from the flexible loop in the RBD that binds the ACE2.","version":"1.1","doi":"10.1101/2020.11.01.363499","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.364364","pub_date":"2020-11-02","title":"Photocatalyst under visible light irradiation inactivates SARS-CoV-2 on an abiotic surface","abstract":"There is a worldwide attempt to develop prevention strategies against SARS-CoV-2 transmission. Here we examined the effectiveness of visible light-responsive photocatalyst RENECAT\u2122 on the inactivation of SARS-CoV-2 under different temperatures and exposure durations. The viral activation on the photocatalyst-coated glass slides decreased from 5.93\u00b10.38 logTCID50/ml to 3.05\u00b10.25 logTCID50/ml after exposure to visible light irradiation for 6h at 20\u00b0C. On the other hand, lighting without the photocatalyst, or the photocatalyst-coat without lighting retained viral stability. Immunoblotting and electron microscopic analyses showed the reduced amounts of spike protein on the viral surface after the photocatalyst treatment. Our data suggest a possible implication of the photocatalyst on the decontamination of the SARS-CoV-2 in indoor environments, thereby preventing indirect viral spread.","version":"1.1","doi":"10.1101/2020.11.01.364364","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.312553","pub_date":"2020-11-02","title":"SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes","abstract":"Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase\u2013ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase\u2013ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.09.24.312553","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.362954","pub_date":"2020-11-02","title":"Genetic diversity analysis of the D614G mutation in SARS-CoV-2","abstract":"In this work, we evaluated the levels of genetic diversity in 18 genomes of SARS-CoV-2 carrying the D614G mutation, coming from Malaysia and Venezuela and publicly available at the National Center of Biotechnology and Information (NCBI). These haplotypes were previously used for phylogenetic analysis, following the LaBECom protocols. All gaps and unconserved sites were extracted for the construction of a phylogenetic tree. As specific methodologies for paired FST estimators, Molecular Variance (AMOVA), Genetic Distance, mismatch, demographic and spatial expansion analyses, molecular diversity and evolutionary divergence time analyses, 20,000 random permutations were always used. The results revealed the presence of only 57 sites of polymorphic and parsimonium-informative among the 29,827bp analyzed and the analyses based on FST values confirmed the presence of two distinct genetic entities with fixation index of 22% and with a higher component of population variation (78.14%). Tau variations revealed a significant time of divergence, supported by mismatch analysis of the observed distribution (\u03c4 = 42%). It is safe to say that the small number of existing polymorphisms should not reflect major changes in the protein products of viral populations in both countries and this consideration provides the safety that, although there are differences in the haplotypes studied, these differences are minimal for both regions analyzed geographically and, therefore, it seems safe to extrapolate the levels of polymorphism and molecular diversity found in the samples for other mutant genomes of SARS-CoV-2 in other countries. This reduces speculation about the possibility of large differences between mutant strains of SARS-CoV-2 (D614G) and wild strains, at least at the level of their protein products, although the mutant form has higher transmission speed and infection. The analyses suggest that possible variations in protein products, of the wild virus in relation to its mutant form, should be minimal, bringing peace of mind as to the increased risk of death from the new form of the virus, as well as possible problems of gradual adjustments in some molecular targets for vaccines.","version":"1.1","doi":"10.1101/2020.10.30.362954","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.362319","pub_date":"2020-11-02","title":"Robust SARS-CoV-2-specific T-cell immunity is maintained at 6 months following primary infection","abstract":"The immune response to SARS-CoV-2 is critical in both controlling primary infection and preventing re-infection. However, there is concern that immune responses following natural infection may not be sustained and that this may predispose to recurrent infection. We analysed the magnitude and phenotype of the SARS-CoV-2 cellular immune response in 100 donors at six months following primary infection and related this to the profile of antibody level against spike, nucleoprotein and RBD over the previous six months. T-cell immune responses to SARS-CoV-2 were present by ELISPOT and/or ICS analysis in all donors and are characterised by predominant CD4+ T cell responses with strong IL-2 cytokine expression. Median T-cell responses were 50% higher in donors who had experienced an initial symptomatic infection indicating that the severity of primary infection establishes a \u2018setpoint\u2019 for cellular immunity that lasts for at least 6 months. The T-cell responses to both spike and nucleoprotein/membrane proteins were strongly correlated with the peak antibody level against each protein. The rate of decline in antibody level varied between individuals and higher levels of nucleoprotein-specific T cells were associated with preservation of NP-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T-cell responses are retained at six months following infection although the magnitude of this response is related to the clinical features of primary infection.","version":"1.1","doi":"10.1101/2020.11.01.362319","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.364273","pub_date":"2020-11-02","title":"D614G substitution enhances the stability of trimeric SARS-CoV-2 spike protein","abstract":"SARS-CoV-2 spike protein with D614G substitution has become the dominant variant in the ongoing COVID-19 pandemic. Several studies to characterize the new virus expressing G614 variant show that it exhibits increased infectivity compared to the ancestral virus having D614 spike protein. Here, using in-silico mutagenesis and energy calculations, we analyzed inter-residue interaction energies and thermodynamic stability of the dominant (G614) and the ancestral (D614) variants of spike protein trimer in \u2018closed\u2019 and \u2018partially open\u2019 conformations. We find that the local interactions mediated by aspartate at the 614th position are energetically frustrated and create unfavourable environment. Whereas, glycine at the same position confers energetically favourable environment and strengthens intra-as well as inter-protomer association. Such changes in the local interaction energies enhance the thermodynamic stability of the spike protein trimer as free energy difference (\u0394\u0394G) upon glycine substitution is \u22122.6 kcal/mol for closed conformation and \u22122.0 kcal/mol for open conformation. Our results on the structural and energetic basis of enhanced stability hint that G614 may confer increased availability of functional form of spike protein trimer and consequent in higher infectivity than the D614 variant.","version":"1.1","doi":"10.1101/2020.11.02.364273","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.363812","pub_date":"2020-11-02","title":"Structural basis for repurpose and design of nucleoside drugs for treating COVID-19","abstract":"SARS-CoV-2 has caused a global pandemic of COVID-19 that urgently needs an effective treatment. Nucleoside analog drugs including favipiravir have been repurposed for COVID-19 despite of unclear mechanism of their inhibition of the viral RNA polymerase (RdRp). Here we report the cryo-EM structures of the viral RdRp in complex with favipiravir and two other nucleoside inhibitor drugs ribavirin and penciclovir. Ribavirin and the ribosylated form of favipiravir share a similar ribose scaffold that is distinct from penciclovir. However, the structures reveal that all three inhibitors are covalently linked to the primer strand in a monophosphate form despite the different chemical scaffolds between favipiravir and penciclovir. Surprisingly, the base moieties of these inhibitors can form mismatched pairs with the template strand. Moreover, in view of the clinical disadvantages of remdesivir mainly associated with its prodrug form, we designed several orally-available remdesivir parent nucleoside derivatives, including VV16 that showed 5-fold more potent than remdesivir in inhibition of viral replication. Together, these results demonstrate an unexpected promiscuity of the viral RNA polymerase and provide a basis for repurpose and design of nucleotide analog drugs for COVID-19. Cryo-EM structures of the RNA polymerase of SARS-CoV-2 reveals the basis for repurposing of old nucleotide drugs to treat COVID-19.","version":"1.1","doi":"10.1101/2020.11.01.363812","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.18.303420","pub_date":"2020-11-02","title":"The COVID-19 immune landscape is dynamically and reversibly correlated with disease severity","abstract":"Despite a rapidly growing body of literature on COVID-19, our understanding of the immune correlates of disease severity, course and outcome remains poor. Using mass cytometry, we assessed the immune landscape in longitudinal whole blood specimens from 59 patients presenting with acute COVID-19, and classified based on maximal disease severity. Hospitalized patients negative for SARS-CoV-2 were used as controls. We found that the immune landscape in COVID-19 forms three dominant clusters, which correlate with disease severity. Longitudinal analysis identified a pattern of productive innate and adaptive immune responses in individuals who have a moderate disease course, whereas those with severe disease have features suggestive of a protracted and dysregulated immune response. Further, we identified coordinate immune alterations accompanying clinical improvement and decline that were also seen in patients who received IL-6 pathway blockade. The hospitalized COVID-19 negative cohort allowed us to identify immune alterations that were shared between severe COVID-19 and other critically ill patients. Collectively, our findings indicate that selection of immune interventions should be based in part on disease presentation and early disease trajectory due to the profound differences in the immune response in those with mild to moderate disease and those with the most severe disease.","version":"1.3","doi":"10.1101/2020.09.18.303420","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.363242","pub_date":"2020-11-02","title":"Ivermectin reduces coronavirus infection in vivo: a mouse experimental model","abstract":"SARS-CoV2 is a single strand RNA virus member of the type 2 coronavirus family, responsible for causing COVID-19 disease in humans. The objective of this study was to test the ivermectin drug in a murine model of coronavirus infection using a type 2 family RNA coronavirus similar to SARS-CoV2, the mouse hepatitis virus (MHV). BALB/cJ female mice were infected with 6,000 PFU of MHV-A59 (Group Infected; n=20) and immediately treated with one single dose of 500 \u03bcg/kg of ivermectin (Group Infected + IVM; n=20), or were not infected and treated with PBS (Control group; n=16). Five days after infection/treatment, mice were euthanized to obtain different tissues to check general health status and infection levels. Overall results demonstrated that viral infection induces the typical MHV disease in infected animals, with livers showing severe hepatocellular necrosis surrounded by a severe lymphoplasmacytic inflammatory infiltration associated with a high hepatic viral load (52,158 AU), while ivermectin administration showed a better health status with lower viral load (23,192 AU; p<0.05) and few livers with histopathological damage (p<0.05), not showing statistical differences with control mice (P=NS). Furthermore, serum transaminase levels (aspartate aminotransferase and alanine aminotransferase) were significantly lower in treated mice compared to infected animals. In conclusion, ivermectin seems to be effective to diminish MHV viral load and disease in mice, being a useful model for further understanding new therapies against coronavirus diseases.","version":"1.1","doi":"10.1101/2020.11.02.363242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.365015","pub_date":"2020-11-02","title":"The effect of heat on SARS-CoV-2 viability and RNA integrity as determined by plaque assay, virus culture and real-time RT-PCR","abstract":"The effect of heat on SARS-CoV-2/England/2/2020 viability was assessed by plaque assay and virus culture. Heating to 56\u00b0C and 60\u00b0C for 15, 30 and 60 minutes led to a reduction in titre of between 2.1 and 4.9 log10 pfu/ml but complete inactivation was not observed. At 80\u00b0C plaques were observed after 15 and 30 minutes of heating, however after 60 minutes viable virus was only detected following virus culture. Heating to 80\u00b0C for 90 minutes and 95\u00b0C for 1 and 5 minutes resulted in no viable virus being detected. At 56\u00b0C and 60\u00b0C significant variability between replicates was observed and the titre often increased with heat-treatment time. Nucleic acids were extracted and tested by RT-PCR. Sensitivity of the RT-PCR was not compromised by heating to 56\u00b0C and 60\u00b0C. Heating to 80\u00b0C for 30 minutes or more and 95\u00b0C for 1 or 5 minutes however, resulted in an increase of at least three Ct values. This increase remained constant when different dilutions of virus underwent heat treatment. This indicates that high temperature heat inactivation of clinical samples prior to nucleic acid extraction could significantly affect the ability to detect virus in clinical samples from patients with lower viral loads by RT-PCR.","version":"1.1","doi":"10.1101/2020.11.02.365015","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.02.350439","pub_date":"2020-11-02","title":"Predicting the animal hosts of coronaviruses from compositional biases of spike protein and whole genome sequences through machine learning","abstract":"The COVID-19 pandemic has demonstrated the serious potential for novel zoonotic coronaviruses to emerge and cause major outbreaks. The immediate animal origin of the causative virus, SARS-CoV-2, remains unknown, a notoriously challenging task for emerging disease investigations. Coevolution with hosts leads to specific evolutionary signatures within viral genomes that can inform likely animal origins. We obtained a set of 650 spike protein and 511 whole genome nucleotide sequences from 225 and 187 viruses belonging to the family Coronaviridae, respectively. We then trained random forest models independently on genome composition biases of spike protein and whole genome sequences, including dinucleotide and codon usage biases in order to predict animal host (of nine possible categories, including human). In hold-one-out cross-validation, predictive accuracy on unseen coronaviruses consistently reached \u223c73%, indicating evolutionary signal in spike proteins to be just as informative as whole genome sequences. However, different composition biases were informative in each case. Applying optimised random forest models to classify human sequences of MERS-CoV and SARS-CoV revealed evolutionary signatures consistent with their recognised intermediate hosts (camelids, carnivores), while human sequences of SARS-CoV-2 were predicted as having bat hosts (suborder Yinpterochiroptera), supporting bats as the suspected origins of the current pandemic. In addition to phylogeny, variation in genome composition can act as an informative approach to predict emerging virus traits as soon as sequences are available. More widely, this work demonstrates the potential in combining genetic resources with machine learning algorithms to address long-standing challenges in emerging infectious diseases.","version":"1.1","doi":"10.1101/2020.11.02.350439","journal":"bioRxiv","score":null},{"id":"10.1101/2020.11.01.363788","pub_date":"2020-11-01","title":"Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV","abstract":"The outbreak of SARS in 2002-2003 caused by SARS-CoV, and the pandemic of COVID-19 in 2020 caused by 2019-nCoV (SARS-CoV-2), have threatened human health globally and raised the urgency to develop effective antivirals against the viruses. In this study, we expressed and purified the RNA-dependent RNA polymerase (RdRp) nsp12 of SARS-CoV and developed a primer extension assay for the evaluation of nsp12 activity. We found that nsp12 could efficiently extend single-stranded RNA, while having low activity towards double-stranded RNA. Nsp12 required a catalytic metal (Mg2+ or Mn2+) for polymerase activity and the activity was also K+-dependent, while Na+ promoted pyrophosphorylation, the reverse process of polymerization. To identify antivirals against nsp12, a competitive assay was developed containing 4 natural rNTPs and a nucleotide analog, and the inhibitory effects of 24 FDA-approved nucleotide analogs were evaluated in their corresponding active triphosphate forms. Ten of the analogs, including 2 HIV NRTIs, could inhibit the RNA extension of nsp12 by more than 40%. The 10 hits were verified which showed dose-dependent inhibition. In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12. Furthermore, the 2 HIV NRTIs were evaluated on 2019-nCoV nsp12 which showed inhibition as well. Then we expanded the evaluation to all 8 FDA-approved HIV NRTIs and discovered 5 of them, tenofovir, stavudine, abacavir, zidovudine and zalcitabine, could inhibit the RNA extension by nsp12 of SARS-CoV and 2019-nCoV. In conclusion, 5 FDA-approved HIV NRTIs inhibited the RNA extension by nsp12 and were promising candidates for the treatment of SARS and COVID-19.","version":"1.1","doi":"10.1101/2020.11.01.363788","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.20220905","pub_date":"2020-11-01","title":"Longitudinal monitoring of SARS-CoV-2 RNA on high-touch surfaces in a community setting","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Environmental surveillance of surface contamination is an unexplored tool for understanding transmission of SARS-CoV-2 in community settings. We conducted longitudinal swab sampling of high-touch non-porous surfaces in a Massachusetts town during a COVID-19 outbreak from April to June 2020. Twenty-nine of 348 (8.3 %) surface samples were positive for SARS-CoV-2, including crosswalk buttons, trash can handles, and door handles of essential business entrances (grocery store, liquor store, bank, and gas station). The estimated risk of infection from touching a contaminated surface was low (less than 5 in 10,000), suggesting fomites play a minimal role in SARS-CoV-2 community transmission. The weekly percentage of positive samples (out of\n                  <jats:italic>n</jats:italic>\n                  =33 unique surfaces per week) best predicted variation in city-level COVID-19 cases using a 7-day lead time. Environmental surveillance of SARS-CoV-2 RNA on high-touch surfaces could be a useful tool to provide early warning of COVID-19 case trends.\n                </jats:p>","version":null,"doi":"10.1101/2020.10.27.20220905","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.31.363473","pub_date":"2020-11-01","title":"TMPRSS2 and ADAM17 interactions with ACE2 complexed with SARS-CoV-2 and B0AT1 putatively in intestine, cardiomyocytes, and kidney","abstract":"COVID-19 outcomes reflect organ-specific interplay of SARS-CoV-2 and its receptor, ACE2, with TMPRSS2 and ADAM17. Confirmed active tropism of SARS-CoV-2 in epithelial cells of intestine and kidney proximal tubule, and in aging cardiomyocytes, capriciously manifests extra-pulmonary organ-related clinical symptoms in about half of COVID-19 patients, occurring by poorly understood mechanisms. We approached this knowledge gap by recognizing a clue that these three particular cell types share a common denominator kindred of uniquely expressing the SLC6A19 neutral amino acid transporter B0AT1 protein (alternatively called NBB, B, B0) serving glutamine and tryptophan uptake. B0AT1 is a cellular trafficking chaperone partner of ACE2, shown by cryo-EM to form a thermodynamically-favored stabilized 2ACE2:2B0AT1 dimer-of-heterodimers. The gut is the body\u2019s site of greatest magnitude expression depot of both ACE2 and B0AT1. This starkly contrasts with pulmonary pneumocyte expression of monomeric ACE2 with conspicuously undetectable B0AT1. We hypothesized that B0AT1 steers the organ-related interplay amongst ACE2, TMPRSS2, ADAM17, and SARS-CoV-2 RBD. The present study employed molecular docking modeling that indicated active site catalytic pocket residues of TMPRSS2 and ADAM17 each formed bonds \u2264 2 A with monomer ACE2 specific residues within a span R652-D713 involved in cleaving sACE2 soluble ectodomain release. These bonds are consistent with competitive binding interactions of experimental anti-SARS-CoV-2 drug small molecules including Camostat and Nafamostat. Without B0AT1, ACE2 residues K657 and N699 dominated docking bonding with TMPRSS2 or ADAM17 active sites, with ACE2 R710 and R709 contributing electrostatic attractions, but notably ACE2 S708 never closer than 16-44 A. However, in the dimer-of-heterodimers arrangement all ACE2 neck region residues were limited to TMPRSS2 or ADAM17 approaches 35 A, with the interference directly attributed to the presence of a neighboring B0AT1 subunit complexed to the partnering ACE2 subunit of 2ACE2:2B0AT1; ADAM17 failed to dock by bumping its active site pocket oriented dysfunctionally outwardly facing 1800 away. Results were the same whether the dimer-of-heterodimers was in either the \u201cclosed\u201d or \u201copen\u201d conformation, or whether or not SARS-CoV-2 RBD was complexed to ACE2. The results implicate B0AT1-and in particular the 2ACE2:2B0AT1 complex-as a maJor player in the landscape of COVID-19 pathophysiology engaging TMPRSS2 and ADAM17, consistent with experimental evidence in the literature and in clinical reports. These findings provide a gateway to understanding the roles of B0AT1 relating to COVID-19 manifestations putatively assigned to intestinal and renal epithelial cells and cardiomyocytes, with underpinnings useful for considerations in public hygiene policy and drug development.","version":"1.1","doi":"10.1101/2020.10.31.363473","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.20220897","pub_date":"2020-11-01","title":"The effect of eviction moratoria on the transmission of SARS-CoV-2","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Massive unemployment during the COVID-19 pandemic could result in an eviction crisis in US cities. Here we model the effect of evictions on SARS-CoV-2 epidemics, simulating viral transmission within and among households in a theoretical metropolitan area. We recreate a range of urban epidemic trajectories and project the course of the epidemic under two counterfactual scenarios, one in which a strict moratorium on evictions is in place and enforced, and another in which evictions are allowed to resume at baseline or increased rates. We find, across scenarios, that evictions lead to significant increases in infections. Applying our model to Philadelphia using locally-specific parameters shows that the increase is especially profound in models that consider realistically heterogenous cities in which both evictions and contacts occur more frequently in poorer neighborhoods. Our results provide a basis to assess municipal eviction moratoria and show that policies to stem evictions are a warranted and important component of COVID-19 control.</jats:p>","version":null,"doi":"10.1101/2020.10.27.20220897","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.31.363309","pub_date":"2020-11-01","title":"Molecular dynamics and in silico mutagenesis on the reversible inhibitor-bound SARS-CoV-2 Main Protease complexes reveal the role of a lateral pocket in enhancing the ligand affinity","abstract":"The 2019 novel coronavirus pandemic caused by SARS-CoV-2 remains a serious health threat to humans and a number of countries are already in the middle of the second wave of infection. There is an urgent need to develop therapeutics against this deadly virus. Recent scientific evidences have suggested that the main protease (Mpro) enzyme in SARS-CoV-2 can be an ideal drug target due to its crucial role in the viral replication and transcription processes. Therefore, there are ongoing research efforts to identify drug candidates against SARS-CoV-2 Mpro that resulted in hundreds of X-ray crystal structures of ligand bound Mpro complexes in the protein data bank (PDB) that describe structural details of different chemotypes of fragments binding within different sites in Mpro. In this work, we perform rigorous molecular dynamics (MD) simulation of 62 reversible ligand-Mpro complexes in the PDB to gain mechanistic insights about their interactions at atomic level. Using a total of ~2.25 \u03bcs long MD trajectories, we identified and characterized different pockets and their conformational dynamics in the apo Mpro structure. Later, using the published PDB structures, we analyzed the dynamic interactions and binding affinity of small ligands within those pockets. Our results identified the key residues that stabilize the ligands in the catalytic sites and other pockets in Mpro. Our analyses unraveled the role of a lateral pocket in the catalytic site in Mpro that is critical for enhancing the ligand binding to the enzyme. We also highlighted the important contribution from HIS163 in this lateral pocket towards ligand binding and affinity against Mpro through computational mutation analyses. Further, we revealed the effects of explicit water molecules and Mpro dimerization in the ligand association with the target. Thus, comprehensive molecular level insights gained from this work can be useful to identify or design potent small molecule inhibitors against SARS-CoV-2 Mpro.","version":"1.1","doi":"10.1101/2020.10.31.363309","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.362335","pub_date":"2020-11-01","title":"Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture","abstract":"As the COVID-19 pandemic continues to fold out, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. Despite the weaker enzymatic inhibition of calpain inhibitors II and XII against Mpro compared to GC-376, calpain inhibitors II and XII had more potent cellular antiviral activity. This observation promoted us to hypothesize that the cellular antiviral activity of calpain inhibitors II and XII might also involve the inhibition of cathepsin L in addition to Mpro. To test this hypothesis, we tested calpain inhibitors II and XII in the SARS-CoV-2 pseudovirus neutralization assay in Vero E6 cells and found that both compounds significantly decreased pseudoviral particle entry into cells, indicating their role in inhibiting cathepsin L. The involvement of cathepsin L was further confirmed in the drug time-of-addition experiment. In addition, we found that these four compounds not only inhibit SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift binding assay and enzymatic FRET assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 are not only promising antiviral drug candidates against existing human coronaviruses, but also might work against future emerging CoVs.","version":"1.1","doi":"10.1101/2020.10.30.362335","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.362749","pub_date":"2020-11-01","title":"Mechanism of ligand recognition by human ACE2 receptor","abstract":"Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of lungs, heart, kidney and pancreas. It is considered an important drug target for treating SARS-CoV-2, as well as pulmonary diseases, heart failure, hypertension, renal diseases and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we address this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond LiGaMD simulations have successfully captured both binding and unbinding of the MLN-4760 inhibitor in the ACE2 receptor. In the ligand unbound state, the ACE2 receptor samples distinct Open, Partially Open and Closed conformations. Ligand binding biases the receptor conformational ensemble towards the Closed state. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the ACE2 receptor. Our simulation findings are expected to facilitate rational drug design of ACE2 against coronaviruses and other related human diseases.","version":"1.1","doi":"10.1101/2020.10.30.362749","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.352450","pub_date":"2020-10-30","title":"Brilacidin, a COVID-19 Drug Candidate, Exhibits Potent In Vitro Antiviral Activity Against SARS-CoV-2","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than one million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin\u2014a de novo designed synthetic small molecule that captures the biological properties of HDPs\u2014on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is primarily a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin has demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 and thus supports brilacidin as a promising COVID-19 drug candidate. Brilacidin potently inhibits SARS-CoV-2 in an ACE2 positive human lung cell line. Brilacidin achieved a high Selectivity Index of 426 (CC50=241\u03bcM/IC50=0.565\u03bcM). Brilacidin\u2019s main mechanism appears to disrupt viral integrity and impact viral entry. Brilacidin and remdesivir exhibit excellent synergistic activity against SARS-CoV-2. SARS-CoV-2, the emergent novel coronavirus, has led to the current global COVID-19 pandemic, characterized by extreme contagiousness and high mortality rates. There is an urgent need for effective therapeutic strategies to safely and effectively treat SARS-CoV-2 infection. We demonstrate that brilacidin, a synthetic small molecule with peptide-like properties, is capable of exerting potent in vitro antiviral activity against SARS-CoV-2, both as a standalone treatment and in combination with remdesivir, which is currently the only FDA-approved drug for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.10.29.352450","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.355677","pub_date":"2020-10-30","title":"The genomic epidemiology of SARS-CoV-2 in Palestine","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the COVID-19 pandemic, continues to cause significant public health burden and disruption globally. Genomic epidemiology approaches point to most countries in the world having experienced many independent introductions of SARS-CoV-2 during the early stages of the pandemic. However, this situation may change with local lockdown policies and restrictions on travel leading to the emergence of more geographically structured viral populations and lineages transmitting locally. Here, we report the first SARS-CoV-2 genomes from Palestine sampled from early March, when the first cases were observed, through to August of 2020. SARS-CoV-2 genomes from Palestine fall across the diversity of the global phylogeny, consistent with at least nine independent introductions into the region. We identify one locally predominant lineage in circulation represented by 50 Palestinian SARS-CoV-2, grouping with isolated viral samples from patients in Israel and the UK. We estimate the age of introduction of this lineage to 05/02/2020 (16/01/2020 - 19/02/2020), suggesting SARS-CoV-2 was already in circulation in Palestine predating its first detection in Bethlehem in early March. Our work highlights the value of ongoing genomic surveillance and monitoring to reconstruct the epidemiology of COVID-19 at both local and global scales.","version":"1.3","doi":"10.1101/2020.10.26.355677","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.347534","pub_date":"2020-10-30","title":"A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells","abstract":"K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 \u03bcM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of \u2264 100 \u03bcM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing. The virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.","version":"1.2","doi":"10.1101/2020.10.23.347534","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.250217","pub_date":"2020-10-30","title":"Hydroxychloroquine: mechanism of action inhibiting SARS-CoV2 entry","abstract":"Hydroxychloroquine (HCQ) has been proposed in the treatment of SARS-coronavirus 2 (SARS-CoV-2) infection, albeit with much controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ\u2019s mechanism of actions in vitro is needed to resolve these conflicts. Recently, anesthetics were shown to disrupt ordered monosialotetrahexosylganglioside1 (GM1) lipid rafts. These same lipid rafts recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point, away from phosphatidylinositol 4,5 bisphosphate (PIP2) domains. Here we employed super resolution imaging of cultured mammalian cells to show HCQ directly perturbs GM1 lipid rafts and inhibits the ability of ACE2 receptor to associate with the endocytic pathway. HCQ also disrupts PIP2 domains and their ability to cluster and sequester ACE2. Similarly, the antibiotic erythromycin inhibits viral entry and both HCQ and erythromycin decrease the antimicrobial host defense peptide amyloid beta in cultured cells. We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to anesthetics. The disruption likely decreases viral clustering at both endocytic and putative PIP2 entry points. Question: What is the molecular basis for antiviral activity of hydroxychloroquine? Findings: Hydroxychloroquine disrupt lipid rafts similar to general anesthetics. Meaning: Since lipids cluster ACE2 and facilitate viral entry, hydroxychloroquine appears to inhibit viral entry by disrupting the lipid clustering of the SARS-CoV2 receptor.","version":"1.2","doi":"10.1101/2020.08.13.250217","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.20220996","pub_date":"2020-10-30","title":"Durable SARS-CoV-2 B cell immunity after mild or severe disease","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Multiple studies have shown loss of SARS-CoV-2 specific antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from COVID-19. However, memory B cells (MBC) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multi-dimensional flow cytometric analysis of S protein receptor binding domain (S-RBD)-specific MBC in cohorts of ambulatory COVID-19 patients with mild disease, and hospitalized patients with moderate to severe disease, at a median of 54 (39-104) days after onset of symptoms. We detected S-RBD-specific class-switched MBC in 13 out of 14 participants, including 4 of the 5 participants with lowest plasma levels of anti-S-RBD IgG and neutralizing antibodies. Resting MBC (rMBC) made up the largest proportion of S-RBD-specific class-switched MBC in both cohorts. FCRL5, a marker of functional memory when expressed on rMBC, was dramatically upregulated on S-RBD-specific rMBC. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD-specific, class-switched MBC that phenotypically resemble germinal center-derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell-mediated immunity against SARS-CoV-2 after recovery from mild or severe COVID-19 disease.</jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract</jats:title>\n                  <jats:fig id='ufig1' position='float' fig-type='figure' orientation='portrait'>\n                    <jats:graphic xmlns:xlink='http://www.w3.org/1999/xlink' xlink:href='20220996v1_ufig1' position='float' orientation='portrait'/>\n                  </jats:fig>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.28.20220996","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.13.249904","pub_date":"2020-10-30","title":"Pandemic danger to the deep: the risk of marine mammals contracting SARS-CoV-2 from wastewater","abstract":"We are in unprecedented times with the ongoing COVID-19 pandemic. The pandemic has impacted public health, the economy and our society on a global scale. In addition, the impacts of COVID-19 permeate into our environment and wildlife as well. Here, we discuss the essential role of wastewater treatment and management during these times. A consequence of poor wastewater management is the discharge of untreated wastewater carrying infectious SARS-CoV-2 into natural water systems that are home to marine mammals. Here, we predict the susceptibility of marine mammal species using a modelling approach. We identified that many species of whale, dolphin and seal, as well as otters, are predicted to be highly susceptible to infection by the SARS-CoV-2 virus. In addition, geo-mapping highlights how current wastewater management in Alaska may lead to susceptible marine mammal populations being exposed to the virus. Localities such as Cold Bay, Naknek, Dillingham and Palmer may require additional treatment of their wastewater to prevent virus spillover through sewage. Since over half of these susceptibility species are already at risk worldwide, the release of the virus via untreated wastewater could have devastating consequences for their already declining populations. For these reasons, we discuss approaches that can be taken by the public, policymakers and wastewater treatment facilities to reduce the risk of virus spillover in our natural water systems. Thus, we indicate the potential for reverse zoonotic transmission of COVID-19 and its impact on marine wildlife; impacts that can be mitigated with appropriate action to prevent further damage to these vulnerable populations.","version":"1.2","doi":"10.1101/2020.08.13.249904","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.340612","pub_date":"2020-10-30","title":"Imperfect innate immune antagonism renders SARS-CoV-2 vulnerable towards IFN-\u03b3 and -\u03bb","abstract":"The innate immune system constitutes a powerful barrier against viral infections. However, it may fail because successful emerging pathogens, like SARS-CoV-2, evolved strategies to counteract it. Here, we systematically assessed the impact of 29 SARS-CoV-2 proteins on viral sensing, type I, II and III interferon (IFN) signaling, autophagy and inflammasome formation. Mechanistic analyses show that autophagy and type I IFN responses are effectively counteracted at different levels. For example, Nsp14 induces loss of the IFN receptor, whereas ORF3a disturbs autophagy at the Golgi/endosome interface. Comparative analyses revealed that antagonism of type I IFN and autophagy is largely conserved, except that SARS-CoV-1 Nsp15 is more potent in counteracting type I IFN than its SARS-CoV-2 ortholog. Altogether, however, SARS-CoV-2 counteracts type I IFN responses and autophagy much more efficiently than type II and III IFN signaling. Consequently, the virus is relatively resistant against exogenous IFN-\u03b1/\u03b2 and autophagy modulation but remains highly vulnerable towards IFN-\u03b3 and -\u03bb treatment. In combination, IFN-\u03b3 and -\u03bb act synergistically, and drastically reduce SARS-CoV-2 replication at exceedingly low doses. Our results identify ineffective type I and II antagonism as weakness of SARS-CoV-2 that may allow to devise safe and effective anti-viral therapies based on targeted innate immune activation.","version":"1.2","doi":"10.1101/2020.10.15.340612","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.361873","pub_date":"2020-10-30","title":"Amino acid transporter B0AT1 influence on ADAM17 interactions with SARS-CoV-2 receptor ACE2 putatively expressed in intestine, kidney, and cardiomyocytes","abstract":"SARS-CoV-2 exhibits significant experimental and clinical gastrointestinal, renal, and cardiac muscle tropisms responsible for local tissue-specific and systemic pathophysiology capriciously occurring in about half of COVID-19 patients. The underlying COVID-19 mechanisms engaged by these extra-pulmonary organ systems are largely unknown. We approached this knowledge gap by recognizing that neutral amino acid transporter B0AT1 (alternately called NBB, B, B0 in the literature) is a common denominator expressed nearly exclusively by three particular cell types: intestinal epithelia, renal proximal tubule epithelium, and cardiomyocytes. B0AT1 provides uptake of glutamine and tryptophan. The gut is the main depot expressing over 90% of the body\u2019s entire pool of SARS-CoV-2 receptor angiotensin converting enzyme-2 (ACE2) and B0AT1. Recent cryo-EM studies established that ACE2 forms a thermodynamically favored dimer-of-heterodimers complex with B0AT1 assembled in the form of a dimer of two ACE2:B0AT1 heterodimers anchored in plasma membranes. Prior epithelial cell studies demonstrated ACE2 chaperone trafficking of B0AT1. This contrasts with monomeric expression of ACE2 in lung pneumocytes, in which B0AT1 is undetectable. The cell types in question also express a disintegrin and metalloproteinase-17 (ADAM17) known to cleave and shed the ectodomain of monomeric ACE2 from the cell surface, thereby relinquishing protection against unchecked renin-angiotensin-system (RAS) events of COVID-19. The present study employed molecular docking modeling to examine the interplaying assemblage of ACE2, ADAM17 and B0AT1. We report that in the monomer form of ACE2, neck region residues R652-N718 provide unimpeded access to ADAM17 active site pocket, but notably R708 and S709 remained >10-15 \u00c5 distant. In contrast, interference of ADAM17 docking to ACE2 in a dimer-of-heterodimers arrangement was directly correlated with the presence of a neighboring B0AT1 subunit complexed to the partnering ACE2 subunit of the 2ACE2:2B0AT1] dimer of heterodimers, representing the expression pattern putatively exclusive to intestinal, renal and cardiomyocyte cell types. The monomer and dimer-of-heterodimers docking models were not influenced by the presence of SARS-CoV-2 receptor binding domain (RBD) complexed to ACE2. The results collectively provide the underpinnings for understanding the role of B0AT1 involvement in COVID-19 and the role of ADAM17 steering ACE2 events in intestinal and renal epithelial cells and cardiomyocytes, with implications useful for consideration in pandemic public hygiene policy and drug development.","version":"1.1","doi":"10.1101/2020.10.30.361873","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.30.360115","pub_date":"2020-10-30","title":"PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2","abstract":"The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the world\u2019s first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-CryptTM platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-\u03b3 levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)\u2013biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.","version":"1.1","doi":"10.1101/2020.10.30.360115","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.361287","pub_date":"2020-10-30","title":"A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies","abstract":"Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.","version":"1.1","doi":"10.1101/2020.10.29.361287","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.360800","pub_date":"2020-10-29","title":"Epitope profiling reveals binding signatures of SARS-CoV-2 immune response and cross-reactivity with endemic HCoVs","abstract":"A major goal of current SARS-CoV-2 vaccine efforts is to elicit antibody responses that confer protection. Mapping the epitope targets of the SARS-CoV-2 antibody response is critical for innovative vaccine design, diagnostics, and development of therapeutics. Here, we developed a phage display library to map antibody binding sites at high resolution within the complete viral proteomes of all human-infecting coronaviruses in patients with mild or moderate/severe COVID-19. The dominant immune responses to SARS-CoV-2 were targeted to regions spanning the Spike protein, Nucleocapsid, and ORF1ab. Some epitopes were identified in the majority of samples while others were rare, and we found variation in the number of epitopes targeted by different individuals. We also identified a set of cross-reactive sequences that were bound by antibodies in SARS-CoV-2 unexposed individuals. Finally, we uncovered a subset of enriched epitopes from commonly circulating human coronaviruses with significant homology to highly reactive SARS-CoV-2 sequences.","version":"1.1","doi":"10.1101/2020.10.29.360800","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.361261","pub_date":"2020-10-29","title":"Modeling the Opening SARS-CoV-2 Spike: an Investigation of its Dynamic Electro-Geometric Properties","abstract":"The recent COVID-19 pandemic has brought about a surge of crowd-sourced initiatives aimed at simulating the proteins of the SARS-CoV-2 virus. A bottleneck currently exists in translating these simulations into tangible predictions that can be leveraged for pharmacological studies. Here we report on extensive electrostatic calculations done on an exascale simulation of the opening of the SARS-CoV-2 spike protein, performed by the Folding@home initiative. We compute the electric potential as the solution of the non-linear Poisson-Boltzmann equation using a parallel sharp numerical solver. The inherent multiple length scales present in the geometry and solution are reproduced using highly adaptive Octree grids. We analyze our results focusing on the electro-geometric properties of the receptor-binding domain and its vicinity. This work paves the way for a new class of hybrid computational and data-enabled approaches, where molecular dynamics simulations are combined with continuum modeling to produce high-fidelity computational measurements serving as a basis for protein bio-mechanism investigations.","version":"1.1","doi":"10.1101/2020.10.29.361261","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.359935","pub_date":"2020-10-29","title":"Global Absence and Targeting of Protective Immune States in Severe COVID-19","abstract":"While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense. In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.","version":"1.1","doi":"10.1101/2020.10.28.359935","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.359836","pub_date":"2020-10-29","title":"Protective Effects of STI-2020 Antibody Delivered Post-Infection by the Intranasal or Intravenous Route in a Syrian Golden Hamster COVID-19 Model","abstract":"We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.","version":"1.1","doi":"10.1101/2020.10.28.359836","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.360479","pub_date":"2020-10-29","title":"Large-scale single-cell analysis reveals critical immune characteristics of COVID-19 patients","abstract":"Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 205 COVID-19 patients and controls to create a comprehensive immune landscape. Lymphopenia and active T and B cell responses were found to coexist and associated with age, sex and their interactions with COVID-19. Diverse epithelial and immune cell types were observed to be virus-positive and showed dramatic transcriptomic changes. Elevation of ANXA1 and S100A9 in virus-positive squamous epithelial cells may enable the initiation of neutrophil and macrophage responses via the ANXA1-FPR1 and S100A8/9-TLR4 axes. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and designing effective therapeutic strategies for COVID-19. Large-scale scRNA-seq analysis depicts the immune landscape of COVID-19 Lymphopenia and active T and B cell responses coexist and are shaped by age and sex SARS-CoV-2 infects diverse epithelial and immune cells, inducing distinct responses Cytokine storms with systemic S100A8/A9 are associated with COVID-19 severity","version":"1.1","doi":"10.1101/2020.10.29.360479","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.29.360586","pub_date":"2020-10-29","title":"Distinct cellular immune profiles in the airways and blood of critically ill patients with COVID-19","abstract":"Our understanding of the coronavirus disease-19 (COVID-19) immune response is almost exclusively derived from studies that examined blood. To gain insight in the pulmonary immune response we analysed BALF samples and paired blood samples from 17 severe COVID-19 patients. Macrophages and T cells were the most abundant cells in BALF. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells and expressed higher levels of the exhaustion marker PD-1 than in peripheral blood. Prolonged ICU stay associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. In conclusion, the bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood. The bronchoalveolar immune response in severe COVID-19 strongly differs from the peripheral blood immune profile. Fatal COVID-19 associated with T cell activation blood, but not in BALF.","version":"1.1","doi":"10.1101/2020.10.29.360586","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.089375","pub_date":"2020-10-28","title":"The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low \u03bcM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.","version":"1.3","doi":"10.1101/2020.05.11.089375","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.358259","pub_date":"2020-10-28","title":"SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway","abstract":"SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.","version":"1.1","doi":"10.1101/2020.10.27.358259","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.358945","pub_date":"2020-10-28","title":"SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic coronavirus to spill over to humans in less than 20 years, after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome (MERS)-CoV in 2012. SARS-CoV-2 is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to severe lung injury and death in the most severe cases. The COVID-19 pandemic is currently a major health issue worldwide. Immune dysregulation characterized by altered innate cytokine responses is thought to contribute to the pathology of COVID-19 patients, which is a testimony of the fundamental role of the innate immune response against SARS-CoV-2. Here, we further characterized the host cell antiviral response against SARS-CoV-2 by using primary human airway epithelia and immortalized model cell lines. We mainly focused on the type I and III interferon (IFN) responses, which lead to the establishment of an antiviral state through the expression of IFN-stimulated genes (ISGs). Our results demonstrate that both primary airway epithelial cells and model cell lines elicit a robust immune response characterized by a strong induction of type I and III IFN through the detection of viral pathogen molecular patterns (PAMPs) by melanoma differentiation associated gene (MDA)-5. However, despite the high levels of type I and III IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication, whereas IFN pre-treatment strongly inhibited viral replication and de novo production of infectious virions. Taken together, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.","version":"1.1","doi":"10.1101/2020.10.28.358945","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.25.217158","pub_date":"2020-10-28","title":"High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2","abstract":"Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal \u03b11,3-galactose (\u03b1Gal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and \u03b11,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and \u03b1-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1\u03bcg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.","version":"1.3","doi":"10.1101/2020.07.25.217158","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.355305","pub_date":"2020-10-28","title":"5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro","abstract":"The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is a candidate as an oral antiviral drug for COVID-19.","version":"1.1","doi":"10.1101/2020.10.28.355305","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.20.958272","pub_date":"2020-10-28","title":"A Multiscale and Comparative Model for Receptor Binding of 2019 Novel Coronavirus and the Implication of its Life Cycle in Host Cells","abstract":"The respiratory syndrome caused by a new type of coronavirus has been emerging from China and caused more than one million death globally since December 2019. This new virus, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the same receptor called Angiotensin-converting enzyme 2 (ACE2) to attack humans as the coronavirus that caused the severe acute respiratory syndrome (SARS) seventeen years ago. Both viruses recognize ACE2 through the spike proteins (S-protein) on their surfaces. It was found that the S-protein from the SARS coronavirus (SARS-CoV) bind stronger to ACE2 than SARS-CoV-2. However, function of a bio-system is often under kinetic, rather than thermodynamic, control. To address this issue, we constructed a structural model for complex formed between ACE2 and the S-protein from SARS-CoV-2, so that the rate of their association can be estimated and compared with the binding of S-protein from SARS-CoV by a multiscale simulation method. Our simulation results suggest that the association of new virus to the receptor is slower than SARS, which is consistent with the experimental data obtained very recently. We further integrated this difference of association rate between virus and receptor into a mathematical model which describes the life cycle of virus in host cells and its interplay with the innate immune system. Interestingly, we found that the slower association between virus and receptor can result in longer incubation period, while still maintaining a relatively higher level of viral concentration in human body. Our computational study therefore provides, from the molecular level, one possible explanation that this new pandemic by far spread much faster than SARS.","version":"1.2","doi":"10.1101/2020.02.20.958272","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.358481","pub_date":"2020-10-28","title":"Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir","abstract":"Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1\u20134. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25\u20137. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3\u2019-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3\u2019-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3\u2019-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.","version":"1.1","doi":"10.1101/2020.10.28.358481","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.354563","pub_date":"2020-10-28","title":"Aglycone polyether ionophores as broad-spectrum agents inhibit multiple enveloped viruses including SARS-CoV-2 in vitro and successfully cure JEV infected mice","abstract":"Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo. Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.","version":"1.1","doi":"10.1101/2020.10.27.354563","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.357137","pub_date":"2020-10-28","title":"Extracellular vesicle-based vaccine platform displaying native viral envelope proteins elicits a robust anti-SARS-CoV-2 response in mice","abstract":"Extracellular vesicles (EVs) emerge as essential mediators of intercellular communication. DNA vaccines encoding antigens presented on EVs efficiently induce T-cell responses and EV-based vaccines containing the Spike (S) proteins of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) are highly immunogenic in mice. Thus, EVs may serve as vaccine platforms against emerging diseases, going beyond traditional strategies, with the antigen displayed identically to the original protein embedded in the viral membrane and presented as such to the immune system. Compared to their viral and pseudotyped counterparts, EV-based vaccines overcome many safety issues including pre-existing immunity against these vectors. Here, we applied our technology in natural EV\u2019s engineering, to express the S proteins of SARS-CoV-2 embedded in the EVs, which mimic the virus with its fully native spikes. Immunizations with a two component CoVEVax vaccine, comprising DNA vector (DNAS-EV) primes, allowing in situ production of Spike harbouring EVs, and a boost using S-EVs produced in mammalian cells, trigger potent neutralizing and cellular responses in mice, in the absence of any adjuvants. CoVEVax would be the prototype of vaccines, where the sole exchange of the envelope proteins on EVs leads to the generation of new vaccine candidates against emerging viruses.","version":"1.1","doi":"10.1101/2020.10.28.357137","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.28.359257","pub_date":"2020-10-28","title":"In vitro assessment of the virucidal activity of four mouthwashes containing Cetylpyridinium Chloride, ethanol, zinc and a mix of enzyme and proteins against a human coronavirus","abstract":"saliva is established to contain high counts SARS-CoV-2 virus and contact with saliva droplets, contaminated surfaces or airborne particles are sources of viral transmission. The generation of infective aerosols during clinical procedures is of particular concern. Therefore, a fuller understanding of the potential of mouthwash to reduce viral counts and modulate the risk of transmission in medical professional and public context is an important research topic. we determined the virucidal activity of four anti-bacterial mouthwashes against a surrogate for SARS-CoV-2, Human CoV-SARS 229E, using a standard ASTM suspension test, with dilution and contact times applicable to recommended mouthwash use. the mouthwash formulated with 0.07% Cetylpyridinium Chloride exhibited virucidal effects providing a \u22653.0 log reduction HCoV-229E viral count. Mouthwashes containing 15.7% ethanol, 0.2% zinc sulphate heptahydrate and a mix of enzymes and proteins did not demonstrate substantive virucidal activity in this test. mouthwash containing 0.07% Cetylpyridinium Chloride warrants further laboratory and clinical assessment to determine their potential benefit in reducing the risk of SARS-CoV-2. SARS-CoV-2 can be transmitted through contact with infective saliva. Studies are needed to understand if mouthwash can lower SARS-CoV-2 transmission risk. 0.07% Cetylpyridinium Chloride (CPC) mouthwash exhibited virucidal effects against HCoV-SARS 229E. Further studies on potential of 0.07% CPC mouthwash against SARS-CoV-2 are warranted.","version":"1.1","doi":"10.1101/2020.10.28.359257","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.353300","pub_date":"2020-10-27","title":"Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models","abstract":"The outbreak of coronavirus disease 2019 (COVID-19) rapidly spreads across worldwide and becomes a global pandemic. Remdesivir is the only COVID-19 treatment approved by U.S. Food and Drug Administration (FDA); however, its effectiveness is still under questioning as raised by the results of a large WHO Solidarity Trial. Herein, we report that the parent nucleotide of remdesivir, GS-441524, potently inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero E6 and other cells. It exhibits good plasma distribution and longer half-life (t1/2=4.8h) in rat PK study. GS-441524 is highly efficacious against SARS-CoV-2 in AAV-hACE2 transduced mice and murine hepatitis virus (MHV) in mice, reducing the viral titers in CoV-attacked organs, without noticeable toxicity. Given that GS-441524 was the predominant metabolite of remdesivir in the plasma, the anti-COVID-19 effect of remdesivir may partly come from the effect of GS-441524. Our results also supported that GS-441524 as a promising and inexpensive drug candidate in the treatment of COVID-19 and future emerging CoVs diseases.","version":"1.1","doi":"10.1101/2020.10.26.353300","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.20219089","pub_date":"2020-10-27","title":"Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing data-sets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS-CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting\n                  <jats:italic>ACE2</jats:italic>\n                  expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection and implicates saliva in viral transmission.\n                </jats:p>","version":null,"doi":"10.1101/2020.10.26.20219089","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.27.357558","pub_date":"2020-10-27","title":"SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility","abstract":"During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.","version":"1.1","doi":"10.1101/2020.10.27.357558","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.357731","pub_date":"2020-10-27","title":"SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway","abstract":"Cytokine storm resulting from a heightened inflammatory response is a prominent feature of severe COVID-19 disease. This inflammatory response results from assembly/activation of a cell-intrinsic defense platform known as the inflammasome. We report that the SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. ORF3a triggers IL-1\u03b2 expression via NF\u03baB, thus priming the inflammasome while also activating it via ASC-dependent and -independent modes. ORF3a-mediated inflammasome activation requires efflux of potassium ions and oligomerization between NEK7 and NLRP3. With the selective NLRP3 inhibitor MCC950 able to block ORF3a-mediated inflammasome activation and key ORF3a residues needed for virus release and inflammasome activation conserved in SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention. Development of anti-SARS-CoV-2 therapies is aimed predominantly at blocking infection or halting virus replication. Yet, the inflammatory response is a significant contributor towards disease, especially in those severely affected. In a pared-down system, we investigate the influence of ORF3a, an essential SARS-CoV-2 protein, on the inflammatory machinery and find that it activates NLRP3, the most prominent inflammasome by causing potassium loss across the cell membrane. We also define key amino acid residues on ORF3a needed to activate the inflammatory response, and likely to facilitate virus release, and find that they are conserved in virus isolates across continents. These findings reveal ORF3a and NLRP3 to be attractive targets for therapy.","version":"1.1","doi":"10.1101/2020.10.27.357731","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.27.357426","pub_date":"2020-10-27","title":"A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice","abstract":"The spread of SARS-CoV-2 has caused a global pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection, and may enable the relaxation of social distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection, therefore it is desirable that any new vaccine candidates should elicit a robust immune response in older adults. Here, we test the immunogenicity of the adenoviral vectored vaccine ChAdOx1 nCoV-19 (AZD-1222) in aged mice. We find that a single dose of this vaccine induces cellular and humoral immunity in aged mice, but at a reduced magnitude than in younger adult mice. Furthermore, we report that a second dose enhances the immune response to this vaccine in aged mice, indicating that a primeboost strategy may be a rational approach to enhance immunogenicity in older persons.","version":"1.1","doi":"10.1101/2020.10.27.357426","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.356048","pub_date":"2020-10-27","title":"ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease","abstract":"Activation of the RIG-I-like receptors, RIG-I and MDA5, establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15 whose mechanistic roles in innate immunity still remain enigmatic. Here we report that ISGylation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISG15 conjugation to the caspase activation and recruitment domains of MDA5 promotes the formation of higher-order assemblies of MDA5 and thereby triggers activation of innate immunity against a range of viruses including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease (PLpro) of SARS-CoV-2, a recently emerged coronavirus that causes the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a novel immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.","version":"1.1","doi":"10.1101/2020.10.26.356048","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.25.20219048","pub_date":"2020-10-27","title":"Ct threshold values, a proxy for viral load in community SARS-CoV-2 cases, demonstrate wide variation across populations and over time","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>Information on SARS-CoV-2 in representative community surveillance is limited, particularly cycle threshold (Ct) values (a proxy for viral load). Of 3,312,159 nose and throat swabs taken 26-April-2020 to 13-March-2021 in the UK\u2019s national COVID-19 Infection Survey, 27,902(0.83%) were RT-PCR-positive, 10,317(37%), 11,012(40%) and 6,550(23%) for 3, 2 or 1 of the N, S and ORF1ab genes respectively, with median Ct=29.2 (\u223c215 copies/ml; IQR Ct=21.9-32.8, 14-56,400 copies/ml). Independent predictors of lower Cts (i.e. higher viral load) included self-reported symptoms and more genes detected, with at most small effects of sex, ethnicity and age. Single-gene positives almost invariably had Ct&gt;30, but Cts varied widely in triple-gene positives, including without symptoms. Population-level Cts changed over time, with declining Ct preceding increasing SARS-CoV-2 positivity. Of 6,189 participants with IgG S-antibody tests post-first RT-PCR-positive, 4,808(78%) were ever antibody-positive; Cts were significantly higher in those remaining antibody-negative. Community SARS-CoV-2 Ct values could be a useful epidemiological early-warning indicator.</jats:p>\n                <jats:sec>\n                  <jats:title>IMPACT STATEMENT</jats:title>\n                  <jats:p>Ct values from SARS-CoV-2 RT-PCR tests vary widely and over calendar time. They have the potential to be used more broadly in public testing programmes as an \u201cearly-warning\u201d system for shifts in infectious load and hence transmission.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.25.20219048","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.26.356279","pub_date":"2020-10-27","title":"Genetic determinants of COVID-19 drug efficacy revealed by genome-wide CRISPR screens","abstract":"Immunomodulatory agents dexamethasone and colchicine, antiviral drugs remdesivir, favipiravir and ribavirin, as well as antimalarial drugs chloroquine phosphate and hydroxychloroquine are currently used in the combat against COVID-19. However, whether some of these drugs have clinical efficacy for COVID-19 is under debate. Moreover, these drugs are applied in COVID-19 patients with little knowledge of genetic biomarkers, which will hurt patient outcome. To answer these questions, we designed a screen approach that could employ genome-wide sgRNA libraries to systematically uncover genes crucial for these drugs\u2019 action. Here we present our findings, including genes crucial for the import, export, metabolic activation and inactivation of remdesivir, as well as genes that regulate colchicine and dexamethasone\u2019s immunosuppressive effects. Our findings provide preliminary information for developing urgently needed genetic biomarkers for these drugs. Such biomarkers will help better interpret COVID-19 clinical trial data and point to how to stratify COVID-19 patients for proper treatment with these drugs.","version":"1.1","doi":"10.1101/2020.10.26.356279","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.352294","pub_date":"2020-10-26","title":"SARS-CoV-2 specific memory T lymphocytes from COVID-19 convalescent donors: identification, biobanking and large-scale production for Adoptive Cell Therapy","abstract":"SARS-CoV-2 is causing a second outbreak so the hope for its complete eradication is far from happening. In the absence of effective vaccines, it is mandatory to find effective treatments with low adverse effects able to treat hospitalized patients with COVID-19 disease. In this work, we determined the existence of SARS-CoV-2 specific T cells within the CD45RA\u2212 T memory cells from the blood of convalescent donors. Memory T cells can respond quickly to the infection and provide long-term immune protection to reduce the severity of the COVID-19 symptoms. Also, CD45RA\u2212 memory T cells confer protection from other pathogens the donors encountered in their life. This is vital to clear other secondary infections usually developed in hospitalized COVID-19 patients. SARS-CoV-2 specific memory T cells were found within all the CD45RA\u2212 subsets CD3+, CD4+, CD8+, and in the central memory and effector memory subpopulations. The procedure to obtain the cells is feasible, easy to implement for small scale manufacture, quick and cost-effective involving minimal manipulation, and without GMP condition requirements. This biobank of specific SARS-CoV-2 memory T cells would be immediately available \u2018off-the-shelf\u2019 to treat moderate/severe cases of COVID-19 increasing the therapeutic options available for these patients.","version":"1.1","doi":"10.1101/2020.10.23.352294","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.354969","pub_date":"2020-10-26","title":"Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a positive-sense single stranded RNA virus with high human transmissibility. This study generated Whole Genome data to determine the origin and pattern of transmission of SARS-CoV-2 from the first six cases tested in The Gambia. Total RNA from SARS-CoV-2 was extracted from inactivated nasopharyngeal-oropharyngeal swabs of six cases and converted to cDNA following the ARTIC COVID-19 sequencing protocol. Libraries were constructed with the NEBNext ultra II DNA library prep kit for Illumina and Oxford Nanopore Ligation sequencing kit and sequenced on Illumina MiSeq and Nanopore GridION, respectively. Sequencing reads were mapped to the Wuhan reference genome and compared to eleven other SARS-CoV-2 strains of Asian, European and American origins. A phylogenetic tree was constructed with the consensus genomes for local and non-African strains. Three of the Gambian strains had a European origin (UK and Spain), two strains were of Asian origin (Japan). In The Gambia, Nanopore and Illumina sequencers were successfully used to identify the sources of SARS-CoV-2 infection in COVID-19 cases.","version":"1.1","doi":"10.1101/2020.10.26.354969","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.355107","pub_date":"2020-10-26","title":"The Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing antibody, SC31, isolated from an early convalescent COVID-19 patient, are essential for the optimal therapeutic efficacy of the antibody","abstract":"SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFN\u03b3-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite Fc\u03b3R binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development. Anti-SARS-CoV-2 IgG1 antibody SC31 controls infection in vivo by blocking SP:ACE2 binding and triggering a Fc-mediated anti-viral response.","version":"1.1","doi":"10.1101/2020.10.26.355107","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.354787","pub_date":"2020-10-26","title":"Tracking cytosine depletion in SARS-CoV-2","abstract":"Danchin et al. have pointed out that cytosine drives the evolution of SARS-CoV-2. A depletion of cytosine might lead to the attenuation of SARS-CoV-2. We built a website to track the composition change of mono-, di-, and tri-nucleotide of SARS-CoV-2 over time. The website downloads new strains available from GISAID and updates its results daily. Our analysis suggests that the composition of cytosine in coronaviruses is related to their reported mortality. Using 137,315 SARS-CoV-2 strains collected in ten months, we observed cytosine depletion at a rate of about one cytosine loss per month from the whole genome. The website is available at http://www.bio8.cs.hku.hk/sarscov2/. rbluo@cs.hku.hk Supplementary data are available at Bioinformatics online.","version":"1.1","doi":"10.1101/2020.10.26.354787","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.351916","pub_date":"2020-10-26","title":"Direct visualization of native infectious SARS-CoV-2 and its inactivation forms using high resolution Atomic Force Microscopy","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID19, a new emerging pandemic affecting humans. Here, single viruses were analyze by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess infectious virus morphology in its native conformation, or upon inactivation treatments, at the nanoscale level and in 3D. High resolution AFM reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory, as revealed by combining AFM and plaque assays, to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-atomic force microscopy is a remarkable toolbox for rapid and direct virus identification and characterization.","version":"1.2","doi":"10.1101/2020.10.23.351916","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.25.354571","pub_date":"2020-10-26","title":"Comparison of the in vitro-efficacy of different mouthwash solutions targeting SARS-CoV-2 based on the European Standard EN 14476","abstract":"The SARS-Cov-2 pandemic is triggering a global health emergency alert, and recent research is indicating the relevance of aerosols in the spread of SARS-CoV-2. Thus, in this study antiseptic mouthwashes based on the actives chlorhexidine (CHX) and octenidine (OCT) were investigated regarding their efficacy against SARS-CoV-2 using EN 14476. Based on the requirement of EN 14476 (i.e. reduction of viral titer by \u2265 4 log 10), the OCT-based formulation was effective within only 15 sec against SARS-CoV-2, and thus constitutes an interesting candidate for future clinical studies to prove its effectiveness in a potential prevention of SARS-CoV-2 transmission by aerosols.","version":"1.1","doi":"10.1101/2020.10.25.354571","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.324145","pub_date":"2020-10-26","title":"SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs","abstract":"T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.","version":"1.2","doi":"10.1101/2020.10.02.324145","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.355099","pub_date":"2020-10-26","title":"Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome","abstract":"Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.","version":"1.1","doi":"10.1101/2020.10.26.355099","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.250464","pub_date":"2020-10-26","title":"At least seven distinct rotavirus genotype constellations in bats with evidence of reassortment and zoonotic transmissions","abstract":"Bats host many viruses pathogenic to humans, and increasing evidence suggests that Rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America and Africa were PCR-positive for RVA and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, including evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite varying levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses. The increased research on bat coronaviruses after SARS-CoV and MERS-CoVallowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents shed light on the vast genetic diversity of rotaviruses and also hinted at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.","version":"1.2","doi":"10.1101/2020.08.13.250464","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.26.355206","pub_date":"2020-10-26","title":"Positive outcomes of COVID-19 research-related gender policy changes","abstract":"The COVID-19 pandemic has exposed and exacerbated gender biases in science, technology, engineering, mathematics, and medicine. Accumulating evidence suggests that female scientists\u2019 productivity dropped during the initial lockdown period. With more time being spent on caregiving responsibilities, women may be struggling to collaborate on grant applications and launch new experiments. Scientists with disabilities or who belong to Indigenous nations or communities of color may have less time to devote to research due to health, family, or community needs. Collateral damage in this situation, the appropriate integration of sex, gender, and other identity characteristics in research content may also suffer. Sex and gender are better attended to when female scientists form part of the research team. Research funding agencies have a role to play in mitigating these effects by putting in place gender equity policies that support all applicants and ensure research quality. Accordingly, a national health research funder implemented gender policy changes that included extending deadlines and factoring sex and gender into COVID-19 grant requirements. Following these changes, the funder received more applications from female scientists, awarded a greater proportion of grants to female compared to male scientists, and received and funded more grant applications that considered sex and gender in the content of COVID-19 research. Whether or not these strategies will be sufficient in the long-term to prevent widening of the gender gap in science, technology, engineering, mathematics and medicine requires continued monitoring and oversight. Further work is urgently required to mitigate inequities associated with identity characteristics beyond gender.","version":"1.1","doi":"10.1101/2020.10.26.355206","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.353219","pub_date":"2020-10-25","title":"The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy","abstract":"The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected more than 29 million individuals worldwide with nearly ~1 million fatalities. Understanding how SARS-CoV-2 initiates viral pathogenesis is of the utmost importance for development of antiviral drugs. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks underline the urgent need for better understanding the mechanism of viral subversion of autophagy. Using murine hepatitis virus-A59 (MHV-A59) as a model betacoronavirus, time-course infections revealed a significant loss in the protein level of ULK1, a canonical autophagy regulating serine-threonine kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target this protein, we conducted in vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PLpro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PLpro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from the C-terminal substrate recognition region. Consistent with this, over-expression of SARS-CoV-2 PLpro is sufficient to impair starvation-induced canonical autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PLpro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagic pathway (Word count=250) The recent COVID-19 global pandemic alongside the 2003 SARS and 2012 MERS outbreaks underscore an urgent need to better understand betacoronaviruses as pathogens that pose global challenge to human health. Studying the underlying biology of how betacoronaviruses subvert innate cellular defense pathways such as autophagy will help to guide future efforts to develop anti-viral therapy. (Word count= 55)","version":"1.1","doi":"10.1101/2020.10.23.353219","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.353177","pub_date":"2020-10-25","title":"Dysregulation of Pulmonary Responses in Severe COVID-19","abstract":"Patients with coronavirus disease 2019 (COVID-19) predominantly have a respiratory tract infection with various symptoms and high mortality is associated with respiratory failure second to severe disease. The risk factors leading to severe disease remain unclear. Here, we reanalyzed a published single-cell RNA-Seq (scRNA-Seq) dataset and found that bronchoalveolar lavage fluid (BALF) of patients with severe disease compared to those with mild disease contained decreased TH17 type cells, decreased IFNA1 expressing cells with lower expression of toll-like receptor 7 (TLR7) and TLR8, increased IgA expressing B cells, and increased hyperactive epithelial cells (and/or macrophages) expressing matrix metalloproteinases (MMPs), Hyaluronan synthase 2 (HAS2), and Plasminogen activator inhibitor-1 (PAI-1), which may together contribute to the pulmonary pathology in severe COVID-19. We propose IFN-I (and TLR7/TLR8) and PAI-1 as potential biomarkers to predict the susceptibility to severe COVID19.","version":"1.1","doi":"10.1101/2020.10.23.353177","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.25.353227","pub_date":"2020-10-25","title":"1Cross Sectional Study of Middle East Respiratory Syndrome (Mers-Cov Infection) in Camels at Selected Sites of Amibara District, Afar Region, Ethiopia","abstract":"A Cross sectional study of Middle East Respiratory Syndrome Corona virus (MERS-CoV) in Camel was conducted between February 2018 to April 2019 in three selected sites of Amibara district of Afar region, Northeast Ethiopia. The study was aimed to observe the current sero-prevalence status of MERS-CoV, assess the presence of active cases through detection RNA Viral particle and investigate possible risk factors of MERS-CoV in camels. A total of 589 sera were collected and tested with indirect Enzyme linked ImmunoSorbent Assay (iELISA). The overall seroprevalance of MERS-CoV was 87.3% (n=514/589, 95% CI: 84.5-89.9). Association of different risk factors with seroprevalance revealed that origin (X2=13.39,P=0.001), sex (X2=4.5 P=0.034), age ((X2=185.7, P=0.001) season (X2=41.7, P=0.000) and reproduction status (X2=96.1, P=0.001) displayed a statistical significant difference among the groups (P<0.05) while herd size did not show a Significant difference among groups (p>0.05). In multivariable logistic regression analysis, age (OR=7.39, 95% CI:3.43-15.91), season (OR=4.83, 95% CI:-2.14-10.90), and in adult female camel reproduction status (OR=7.39,95% C I:3.43-15.91) showed statistically significant difference among the groups for MERS CoV antibody detection while risk factors of origin, animal sex and herd size difference were statistically insignificant. A total of 857 nasal swab samples were collected for the detection of MERS-CoV RNA particle. However, all swab samples tested by Real-time reverse transcription polymerase chain reaction (RT-PCR) technique were Negative for the virus. In conclusion, the present study revealed a high seroprevalance of MERS CoV in adult camels. However, in spite of high seroprevalance the lack of any RNA viral particle in the study suggests the need for further in depth longitudinal study to detect the circulating virus focusing on juveniles and young camels whereby seroprevalance of antibody is low when compared with adult camel in order to get the active virus before the camel develop antibody. Moreover, the zoonotic significance and potential transmission routes of MERS CoV to pastoral communities should also be investigated and design strategy for the preparedness in control of the diseases in Ethiopia.","version":"1.1","doi":"10.1101/2020.10.25.353227","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.348854","pub_date":"2020-10-24","title":"Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut","abstract":"Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. Despite the evidence about SARS-CoV-2 infecting the human gut, little is known about the importance of the enteric phase of SARS-CoV-2 for the viral lifecycle and for the development of COVID-19-associated pathologies. Similarly, it remains unknown whether the innate immune response triggered in this organ to combat viral infection is similar or distinct compared to the one triggered in other organs. We exploited human ileum- and colon-derived organoids as a non-transformed culture model supporting SARS-CoV-2 infection. We characterized the replication kinetics of SARS-CoV-2 in intestinal epithelial cells and correlated the expression of the viral receptor ACE2 with infection. We performed conventional and targeted single-cell transcriptomics and multiplex single-molecule RNA fluorescence in situ hybridization and used IFN-reporter bioassays to characterize the response of primary human intestinal epithelial cells to SARS-CoV-2 infection. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2. We found the lack of positive correlation between susceptibility to infection and the expression of ACE2 and revealed that SARS-CoV-2 downregulates ACE2 expression upon infection. Infected cells activated strong proinflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon in infected cells. Our findings reveal that SARS-CoV-2 curtails the immune response in primary human intestinal epithelial cells to promote its replication and spread and this highlights the gut as a proinflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis. What is already known about this subject?\n\nCOVID-19 patients have gastrointestinal symptoms which likely correlates with SARS-CoV-2 infection of the intestinal epithelium\nSARS-CoV-2 replicates in human intestinal epithelial cells.\nIntestinal organoids are a good model to study SARS-CoV-2 infection of the gastrointestinal tract\nThere is a limited interferon response in human lung epithelial cells upon SARS-CoV-2 infection.\n\n COVID-19 patients have gastrointestinal symptoms which likely correlates with SARS-CoV-2 infection of the intestinal epithelium SARS-CoV-2 replicates in human intestinal epithelial cells. Intestinal organoids are a good model to study SARS-CoV-2 infection of the gastrointestinal tract There is a limited interferon response in human lung epithelial cells upon SARS-CoV-2 infection. What are the new findings?\n\nA specific subpopulation of enterocytes are the prime targets of SARS-CoV-2 infection of the human gut.\nThere is a lack of correlation between ACE2 expression and susceptibility to SARS-CoV-2 infection. SARS-CoV-2 downregulates ACE2 expression upon infection.\nHuman intestinal epithelium cells produce interferon upon SARS-CoV-2 infection.\nInterferon acts in a paracrine manner to induce interferon stimulated genes that control viral infection only in bystander cells.\nSARS-CoV-2 actively blocks interferon signaling in infected cells.\n\n A specific subpopulation of enterocytes are the prime targets of SARS-CoV-2 infection of the human gut. There is a lack of correlation between ACE2 expression and susceptibility to SARS-CoV-2 infection. SARS-CoV-2 downregulates ACE2 expression upon infection. Human intestinal epithelium cells produce interferon upon SARS-CoV-2 infection. Interferon acts in a paracrine manner to induce interferon stimulated genes that control viral infection only in bystander cells. SARS-CoV-2 actively blocks interferon signaling in infected cells. How might it impact on clinical practice in the foreseeable future?\n\nThe absence of correlation between ACE2 levels and susceptibility suggest that medications influencing ACE2 levels (e.g. high blood pressure drugs) will not make patients more susceptible to SARS-CoV-2 infection.\nThe restricted cell tropism and the distinct immune response mounted by the GI tract, suggests that specific cellular restriction/replication factors and organ specific intrinsic innate immune pathways can represent unique therapeutic targets to treat COVD-19 patients by considering which organ is most infected/impacted by SARS-CoV-2.\nThe strong pro-inflammatory signal mounted by the intestinal epithelium can fuel the systemic inflammation observed in COVID-19 patients and is likely participating in the lung specific pathology.\n\n The absence of correlation between ACE2 levels and susceptibility suggest that medications influencing ACE2 levels (e.g. high blood pressure drugs) will not make patients more susceptible to SARS-CoV-2 infection. The restricted cell tropism and the distinct immune response mounted by the GI tract, suggests that specific cellular restriction/replication factors and organ specific intrinsic innate immune pathways can represent unique therapeutic targets to treat COVD-19 patients by considering which organ is most infected/impacted by SARS-CoV-2. The strong pro-inflammatory signal mounted by the intestinal epithelium can fuel the systemic inflammation observed in COVID-19 patients and is likely participating in the lung specific pathology.","version":"1.1","doi":"10.1101/2020.10.21.348854","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.352831","pub_date":"2020-10-24","title":"Ivermectin repurposing for COVID-19 therapy: Safety and pharmacokinetic assessment of a novel nasal spray formulation in a pig model","abstract":"High ivermectin (IVM) concentrations suppress in vitro SARS-CoV-2 replication. Nasal IVM spray (N-IVM-spray) administration may contribute to attaining high drug concentrations in nasopharyngeal (NP) tissue, a primary site of virus entrance/replication. The safety and pharmacokinetic performance of a new N-IVM spray formulation in a piglet model were assessed. Crossbred piglets (10\u201312 kg) were treated with either one or two (12 h apart) doses of N-IVM-spray (2 mg, 1 puff/nostril) or orally (0.2 mg/kg). The overall safety of N-IVM-spray was assessed (clinical, haematological, serum biochemical determinations), and histopathology evaluation of the application site tissues performed. The IVM concentration profiles measured in plasma and respiratory tract tissues (nasopharynx and lungs) after the nasal spray treatment (one and two applications) were compared with those achieved after the oral administration. Animals tolerated well the novel N\u2013IVM-spray formulation. No local/systemic adverse events were observed. After nasal administration, the highest IVM concentrations were measured in NP and lung tissues. Significant increases in IVM concentration profiles in both NP-tissue and lungs were observed after the 2-dose nasal administrations. The nasal/oral IVM concentration ratios in NP and lung tissues (at 6 h post-dose) markedely increased by repeating the spray application. The fast attainment of high and persistent IVM concentrations in NP tissue is the main advantage of the nasal over the oral route. These original results are encouraging to support the undertaking of further clinical trials to evaluate the safety/efficacy of the nasal IVM spray application in the treatment and/or prevention of COVID-19.","version":"1.1","doi":"10.1101/2020.10.23.352831","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.353169","pub_date":"2020-10-24","title":"Identification bioactive compounds from marine microorganism and exploration of structure\u2013activity relationships (SARs)","abstract":"Marine natural products (MNPs) have become new strong leads for antimicrobial drug discovery and an effective alternative to control drug resistant infections. Herein we report the bioassay guided fractionation of marine extracts from sponges Lendenfeldia, Ircinia and Dysidea that led us to identify novel compounds with antimicrobial properties. Tertiary amines or quaternary amine salts: anilines 1, benzylamines 2, tertiary amines 3 and 4, and quaternary amine salt 5, along with three known compounds (6-8) were isolated from a crude extract and MeOH eluent marine extracts. The absolute configurations of the new compounds were assigned based on tandem mass spectrometry (MS) analysis. Several of the compounds exhibited potent in-vitro antibacterial activity, especially against Methicillin-resistant Staphylococcus aureus (MRSA) (MICs from 15.6 to 62.5 micro g/mL). Herein, we also, report structure activity relationships of a diverse range of commercial structurally similar compounds. The structure activity relationships (SARs) results clearly demonstrate that modification of the amines through linear chain length, and inclusion of aromatic rings, modifies the observed antimicrobial activity towards different biological activity. Several commercially available compounds, which are structurally related to the molecules we discovered showed broad spectrum antimicrobial activity against different test pathogens with an MIC50 range of 50 to 0.01 microM. The results of cross-referencing antimicrobial activity and cytotoxicity establish that these compounds are promising potential lead molecules, with a favourable therapeutic index for antimicrobial drug development. Additionally, the SAR studies show that simplified analogues of the isolated compounds with increased bioactivity","version":"1.1","doi":"10.1101/2020.10.23.353169","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.350348","pub_date":"2020-10-23","title":"Transferrin receptor is another receptor for SARS-CoV-2 entry","abstract":"Angiotensin-converting enzyme 2 (ACE2) has been suggested as a receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to cause coronavirus disease 2019 (COVID-19). However, no ACE2 inhibitors have shown definite beneficiaries for COVID-19 patients, applying the presence of another receptor for SARS-CoV-2 entry. Here we show that ACE2 knockout dose not completely block virus entry, while TfR directly interacts with virus Spike protein to mediate virus entry and SARS-CoV-2 can infect mice with over-expressed humanized transferrin receptor (TfR) and without humanized ACE2. TfR-virus co-localization is found both on the membranes and in the cytoplasma, suggesting SARS-CoV-2 transporting by TfR, the iron-transporting receptor shuttling between cell membranes and cytoplasma. Interfering TfR-Spike interaction blocks virus entry to exert significant anti-viral effects. Anti-TfR antibody (EC50 ~16.6 nM) shows promising anti-viral effects in mouse model. Collectively, this report indicates that TfR is another receptor for SARS-CoV-2 entry and a promising anti-COVID-19 target.","version":"1.1","doi":"10.1101/2020.10.23.350348","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.351569","pub_date":"2020-10-23","title":"Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects","abstract":"SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.","version":"1.1","doi":"10.1101/2020.10.22.351569","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.184374","pub_date":"2020-10-23","title":"Single source of pangolin CoVs with a near identical Spike RBD to SARS-CoV-2","abstract":"Multiple publications have independently described pangolin CoV genomes from the same batch of smuggled pangolins confiscated in Guangdong province in March, 2019. We analyzed the three metagenomic datasets that sampled this batch of pangolins and found that the two complete pangolin CoV genomes, GD_1 by Xiao et al. Nature and MP789 by Liu et al. PLoS Pathogens, were both built primarily using the 2019 dataset first described by Liu et al. Viruses. Other publications, such as Zhang et al. Current Biology and Lam et al. Nature, have also relied on this same dataset by Liu et al. Viruses for their assembly of the Guangdong pangolin CoV sequences and comparisons to SARS-CoV-2. To our knowledge, all of the published pangolin CoV genome sequences that share a highly similar Spike receptor binding domain with SARS-CoV-2 originate from this singular batch of smuggled pangolins. This raises the question of whether pangolins are truly reservoirs or hosts of SARS-CoV-2-related coronaviruses in the wild, or whether the pangolins may have contracted the CoV from another host species during trafficking. Our observations highlight the importance of requiring authors to publish their complete genome assembly pipeline and all contributing raw sequence data, particularly those supporting epidemiological investigations, in order to empower peer review and independent analysis of the sequence data. This is necessary to ensure both the accuracy of the data and the conclusions presented by each publication.","version":"1.2","doi":"10.1101/2020.07.07.184374","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.208041","pub_date":"2020-10-23","title":"SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 (ACE2) is expressed in human pancreatic \u03b2-cells and in the human pancreas microvasculature","abstract":"Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2), is a necessary step for SARS-CoV-2 infection permissiveness. In the light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyse ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing \u03b2-cells. ACE2 is also is highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human \u03b2-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increases ACE2 expression in the \u03b2-cell line EndoC-\u03b2H1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct \u03b2-cell virus tropism.","version":"1.2","doi":"10.1101/2020.07.23.208041","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.344085","pub_date":"2020-10-23","title":"Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Modified Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine","abstract":"The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses.","version":"1.1","doi":"10.1101/2020.10.23.344085","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.352666","pub_date":"2020-10-23","title":"Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19","abstract":"The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated sixteen of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.10.23.352666","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.23.342113","pub_date":"2020-10-23","title":"SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs","abstract":"The COVID-19 pandemic has caused over one million deaths thus far. There is an urgent need for the development of specific viral therapeutics and a vaccine. SARS-CoV-2 nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for both antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we initially catalogued the protein-protein interactions of 21 SARS-CoV-2 proteins in HEK293 cells, finding that the stress granule resident proteins G3BP1 and G3BP2 copurify with N with high specificity. We demonstrate that N protein expression in human cells sequesters G3BP1 and G3BP2 through its physical interaction with these proteins, attenuating stress granule (SG) formation. The ectopic expression of G3BP1 in N-expressing cells was sufficient to reverse this phenotype. Since N is an RNA-binding protein, we performed iCLIP-sequencing experiments in cells, with or without exposure to oxidative stress, to identify the host RNAs targeted by N. Our results indicate that SARS-CoV-2 N protein binds directly to thousands of mRNAs under both conditions. Like the G3BPs stress granule proteins, N was found to predominantly bind its target mRNAs in their 3\u2019UTRs. RNA sequencing experiments indicated that expression of N results in wide-spread gene expression changes in both unstressed and oxidatively stressed cells. We suggest that N regulates host gene expression by both attenuating stress granules and binding directly to target mRNAs.","version":"1.1","doi":"10.1101/2020.10.23.342113","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.20217208","pub_date":"2020-10-23","title":"Early use of nitazoxanide in mild Covid-19 disease: randomized, placebo-controlled trial","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The antiparasitic drug nitazoxanide is widely available and exerts broad-spectrum antiviral activity\n                  <jats:italic>in vitro</jats:italic>\n                  . However, there is no evidence of its impact on SARS-CoV-2 infection.\n                </jats:p>\n                <jats:p>In a multicenter, randomized, double-blind, placebo-controlled trial, adult patients who presented up to 3 days after onset of Covid-19 symptoms (dry cough, fever, and/or fatigue) were enrolled. After confirmation of SARS-CoV2 infection by RT-PCR on nasopharyngeal swab, patients were randomized 1:1 to receive either nitazoxanide (500 mg) or placebo, TID, for 5 days. The primary outcome was complete resolution of symptoms. Secondary outcomes were viral load, general laboratory tests, serum biomarkers of inflammation, and hospitalization rate. Adverse events were also assessed.</jats:p>\n                <jats:p>\n                  From June 8 to August 20, 2020, 1,575 patients were screened. Of these, 392 (198 placebo, 194 nitazoxanide) were analyzed. Median time from symptom onset to first dose of study drug was 5 (4-5) days. At the 5-day study visit, symptom resolution did not differ between the nitazoxanide and placebo arms. However, at the 1-week follow-up, 78% in the nitazoxanide arm and 57% in the placebo arm reported complete resolution of symptoms (\n                  <jats:italic>p</jats:italic>\n                  =0.048). Swabs collected were negative for SARS-CoV-2 in 29.9% of patients in the nitazoxanide arm\n                  <jats:italic>versus</jats:italic>\n                  18.2% in the placebo arm (p=0.009). Viral load was also reduced after nitazoxanide compared to placebo (p=0.006). No serious adverse events were observed.\n                </jats:p>\n                <jats:p>In patients with mild Covid-19, symptom resolution did not differ between the nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly.</jats:p>\n                <jats:sec>\n                  <jats:title>Take home message</jats:title>\n                  <jats:p>This was the first study to evaluate the effect of early nitazoxanide therapy in mild Covid-19. Nitazoxanide did not accelerate symptom resolution after 5 days of therapy; however, reduced viral load significantly with no serious adverse events.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.21.20217208","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.17.343749","pub_date":"2020-10-23","title":"On Classification and Taxonomy of Coronaviruses (Riboviria, Nidovirales, Coronaviridae) with special focus on severe acute respiratory syndrome-related coronavirus 2 (SARS-Cov-2)","abstract":"Coronaviruses are highly pathogenic and therefore important human and veterinary pathogens viruses worldwide (1). Members of family Coronaviridae have previously been analysed phylogenetically, resulting in proposals of virus interrelationships (2\u20135). However, available Coronavirus phylogenies remain unrooted, based on limited sampling, and normally depend on a single method (2\u201311). The main subjects of this study are the taxonomy and systematics of coronaviruses and our goal is to build the first natural classification of Coronaviridae using several methods of cladistic analyses (12), Maximum Likelihood method, as well as rigorous taxonomic sampling, making the most accurate representation of Coronaviridae\u2019s relationships to date. Nomenclature recommendations to help effectively incorporate principles of binary nomenclature into Coronaviridae taxonomy are provided. We have stressed that no member of Sarbecovirus clade is an ancestor of SARS-Cov-2, and humans are the only known host. Multiple comprehensive phylogenetic analyses of all coronavirus species enabled testing of critical proposals on virus interrelationships.","version":"1.2","doi":"10.1101/2020.10.17.343749","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.20216192","pub_date":"2020-10-23","title":"Relaxed peripheral tolerance drives broad\n                  <i>de novo</i>\n                  autoreactivity in severe COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  An emerging feature of COVID-19 is the identification of autoreactivity in patients with severe disease that may contribute to disease pathology, however the origin and resolution of these responses remain unclear. Previously, we identified strong extrafollicular B cell activation as a shared immune response feature between both severe COVID-19 and patients with advanced rheumatic disease. In autoimmune settings, this pathway is associated with relaxed peripheral tolerance in the antibody secreting cell compartment and the generation of\n                  <jats:italic>de novo</jats:italic>\n                  autoreactive responses. Investigating these responses in COVID-19, we performed single-cell repertoire analysis on 7 patients with severe disease. In these patients, we identify the expansion of a low-mutation IgG1 fraction of the antibody secreting cell compartment that are not memory derived, display low levels of selective pressure, and are enriched for autoreactivity-prone\n                  <jats:italic>IGHV4-34</jats:italic>\n                  expression. Within this compartment, we identify B cell lineages that display specificity to both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against glomerular basement membrane, and describe progressive, broad, clinically relevant autoreactivity within these patients correlated with disease severity. Importantly, we identify anti-carbamylated protein responses as a common hallmark and candidate biomarker of broken peripheral tolerance in severe COVID-19. Finally, we identify the contraction of this pathway upon recovery, and re-establishment of tolerance standards coupled with a concomitant loss of acute-derived ASCs irrespective of antigen specificity. In total, this study reveals the origins, breadth, and resolution of acute-phase autoreactivity in severe COVID-19, with significant implications in both early interventions and potential treatment of patients with post-COVID sequelae.\n                </jats:p>","version":null,"doi":"10.1101/2020.10.21.20216192","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.22.349951","pub_date":"2020-10-22","title":"Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19","abstract":"The COVID-19 pandemic caused by the emergent SARS-CoV-2 coronavirus threatens global public health and there is an urgent need to develop safe and effective vaccines. Here we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV2. We show that our vaccine candidate, GRAd- COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies which neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, Th1- dominated cellular response in the periphery and in the lung. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV2 neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of GRAd-COV2 vaccine in a currently ongoing Phase I clinical trial (NCT04528641).","version":"1.1","doi":"10.1101/2020.10.22.349951","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.351056","pub_date":"2020-10-22","title":"Small-Molecule In Vitro Inhibitors of the Coronavirus Spike \u2013 ACE2 Protein-Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2","abstract":"Inhibitors of the protein-protein interaction (PPI) between the SARS-CoV-2 spike protein and ACE2, which acts as a ligand-receptor pair that initiates the viral attachment and cellular entry of this coronavirus causing the ongoing COVID-19 pandemic, are of considerable interest as potential antiviral agents. While blockade of such PPIs with small molecules is more challenging than with antibodies, small-molecule inhibitors (SMIs) might offer alternatives that are less strain- and mutation-sensitive, suitable for oral or inhaled administration, and more controllable / less immunogenic. Here, we report the identification of SMIs of this PPI by screening our compound-library that is focused on the chemical space of organic dyes. Among promising candidates identified, several dyes (Congo red, direct violet 1, Evans blue) and novel drug-like compounds (DRI-C23041, DRI-C91005) inhibited the interaction of hACE2 with the spike proteins of SARS-CoV-2 as well as SARS-CoV with low micromolar activity in our cell-free ELISA-type assays (IC50s of 0.2-3.0 \u03bcM); whereas, control compounds, such as sunset yellow FCF, chloroquine, and suramin, showed no activity. Protein thermal shift assays indicated that the SMIs identified here bind SARS-CoV-2-S and not ACE2. Selected promising compounds inhibited the entry of a SARS-CoV-2-S expressing pseudovirus into ACE2-expressing cells in concentration-dependent manner with low micromolar IC50s (6-30 \u03bcM). This provides proof-of-principle evidence for the feasibility of small-molecule inhibition of PPIs critical for coronavirus attachment/entry and serves as a first guide in the search for SMI-based alternative antiviral therapies for the prevention and treatment of diseases caused by coronaviruses in general and COVID-19 in particular.","version":"1.1","doi":"10.1101/2020.10.22.351056","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.349415","pub_date":"2020-10-22","title":"Broad transcriptional dysregulation of brain and choroid plexus cell types with COVID-19","abstract":"Though SARS-CoV-2 primarily targets the respiratory system, it is increasingly appreciated that patients may suffer neurological symptoms of varied severity. However, an unbiased understanding of the molecular processes across brain cell types that could contribute to these symptoms in COVID-19 patients is still missing. Here, we profile 47,678 droplet-based single-nucleus transcriptomes from the frontal cortex and choroid plexus across 10 non-viral, 4 COVID-19, and 1 influenza patient. We complement transcriptomic data with immunohistochemical staining for the presence of SARS-CoV-2. We find that all major cortex parenchymal and choroid plexus cell types are affected transcriptionally with COVID-19. This arises, in part, from SARS-CoV-2 infection of the cortical brain vasculature, meninges, and choroid plexus, stimulating increased inflammatory signaling into the brain. In parallel, peripheral immune cells infiltrate the brain, microglia activate programs mediating the phagocytosis of live neurons, and astrocytes dysregulate genes involved in neurotransmitter homeostasis. Among neurons, layer 2/3 excitatory neurons\u2014evolutionarily expanded in humans\u2014show a specific downregulation of genes encoding major SNARE and synaptic vesicle components, predicting compromised synaptic transmission. These perturbations are not observed in terminal influenza. Many COVID-19 gene expression changes are shared with those in chronic brain disorders and reside in genetic variants associated with cognitive function, schizophrenia, and depression. Our findings and public dataset provide a molecular framework and new opportunities to understand COVID-19 related neurological disease.","version":"1.1","doi":"10.1101/2020.10.22.349415","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.349522","pub_date":"2020-10-22","title":"Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites","abstract":"SARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer\u2019s cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain\u2019s potential permeation pathway has six constrictions narrowing to about 1 \u00c5 in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity\u2019s electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.","version":"1.1","doi":"10.1101/2020.10.22.349522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.328864","pub_date":"2020-10-22","title":"Databiology Lab CORONAHACK: Collection of Public COVID-19 Data","abstract":"COVID-19 has had an unprecedented global impact in health and economy affecting millions of persons world-wide. To support and enable a collaborative response from the global research communities, we created a data collection for different public sources for anonymized patient clinical data, imaging datasets, molecular data as nucleotide and protein sequences for the SARS-CoV-2 virus, reports of count of cases and deaths per city/country, and other economic indicators in Databiology Lab (https://www.lab.databiology.net/) where researchers could access these data assets and use the hundreds of available open source bioinformatic applications to analyze them. These data assets are regularly updated and was used in a successful virtual 3-day hackathon organized by Databiology Ltd and Mindstream-AI where hundreds of attendees to work collaboratively to analyze these data collections.","version":"1.1","doi":"10.1101/2020.10.22.328864","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.22.349944","pub_date":"2020-10-22","title":"OmicLoupe: Facilitating biological discovery by interactive exploration of multiple omic datasets and statistical comparisons","abstract":"Visual exploration of gene product behavior across multiple omic datasets can pinpoint technical limitations in data and reveal biological trends. The OmicLoupe software was developed to facilitate such exploration and provides more than 15 interactive cross-dataset visualizations for omic data. It expands visualizations to multiple datasets for quality control, statistical comparisons and overlap and correlation analyses, while allowing for rapid inspection and downloading of selected features. The usage of OmicLoupe is demonstrated in three diverse studies, including an analysis of SARS-CoV-2 infection across omic layers, based on previously published proteomics and transcriptomics studies. OmicLoupe is available at quantitativeproteomics.org/omicloupe","version":"1.1","doi":"10.1101/2020.10.22.349944","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.10.334664","pub_date":"2020-10-21","title":"The human brain vasculature shows a distinct expression pattern of SARS-CoV-2 entry factors","abstract":"A large number of hospitalized COVID-19 patients show neurological symptoms such as ischemic- and hemorrhagic stroke as well as encephalitis, and SARS-CoV-2 can directly infect endothelial cells leading to endotheliitis across multiple vascular beds. These findings suggest an involvement of the brain- and peripheral vasculature in COVID-19, but the underlying molecular mechanisms remain obscure. To understand the potential mechanisms underlying SARS-CoV-2 tropism for brain vasculature, we constructed a molecular atlas of the expression patterns of SARS-CoV-2 viral entry-associated genes (receptors and proteases) and SARS-CoV-2 interaction partners in human (and mouse) adult and fetal brain as well as in multiple non-CNS tissues in single-cell RNA-sequencing data across various datasets. We observed a distinct expression pattern of the cathepsins B (CTSB) and -L (CTSL) - which are able to substitute for the ACE2 co-receptor TMPRSS2 - in the human vasculature with CTSB being mainly expressed in the brain vasculature and CTSL predominantly in the peripheral vasculature, and these observations were confirmed at the protein level in the Human Protein Atlas and using immunofluorescence stainings. This expression pattern of SARS-CoV-2 viral-entry associated proteases and SARS-CoV-2 interaction partners was also present in endothelial cells and microglia in the fetal brain, suggesting a developmentally established SARS-CoV-2 entry machinery in the human vasculature. At both the adult and fetal stages, we detected a distinct pattern of SARS-CoV-2 entry associated genes\u2019 transcripts in brain vascular endothelial cells and microglia, providing a potential explanation for an inflammatory response in the brain endothelium upon SARS-CoV-2 infection. Moreover, CTSB was co-expressed in adult and fetal brain endothelial cells with genes and pathways involved in innate immunity and inflammation, angiogenesis, blood-brain-barrier permeability, vascular metabolism, and coagulation, providing a potential explanation for the role of brain endothelial cells in clinically observed (neuro)vascular symptoms in COVID-19 patients. Our study serves as a publicly available single-cell atlas of SARS-CoV-2 related entry factors and interaction partners in human and mouse brain endothelial- and perivascular cells, which can be employed for future studies in clinical samples of COVID-19 patients.","version":"1.2","doi":"10.1101/2020.10.10.334664","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.347799","pub_date":"2020-10-21","title":"Preclinical study of DNA vaccines targeting SARS-CoV-2","abstract":"To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFN\u03b3 expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.10.21.347799","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.349225","pub_date":"2020-10-21","title":"Restriction of SARS-CoV-2 Replication by Targeting Programmed \u22121 Ribosomal Frameshifting In Vitro","abstract":"Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed \u22121 ribosomal frameshifting (\u22121 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in \u22121 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a \u22121 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on \u22121 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting \u22121 PRF as an effective antiviral strategy for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.10.21.349225","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.078758","pub_date":"2020-10-21","title":"A phylodynamic workflow to rapidly gain insights into the dispersal history and dynamics of SARS-CoV-2 lineages","abstract":"Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of the causative virus (SARS-CoV-2) have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analysed with gold-standard phylogeographic approaches. We here describe and apply an analytical pipeline that is a compromise between fast and rigorous analytical steps. As a proof of concept, we focus on the Belgium epidemic, with one of the highest spatial density of available SARS-CoV-2 genomes. At the global scale, our analyses confirm the importance of external introduction events in establishing multiple transmission chains in the country. At the country scale, our spatially-explicit phylogeographic analyses highlight that the national lockdown had a relatively low impact on both the lineage dispersal velocity and the long-distance dispersal events within Belgium. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.","version":"1.5","doi":"10.1101/2020.05.05.078758","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.21.347690","pub_date":"2020-10-21","title":"Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP","abstract":"The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 \u00c5. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.","version":"1.1","doi":"10.1101/2020.10.21.347690","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.200691","pub_date":"2020-10-21","title":"Predicted Cellular Immunity Population Coverage Gaps for SARS-CoV-2 Subunit Vaccines and their Augmentation by Compact Peptide Sets","abstract":"Subunit vaccines induce immunity to a pathogen by presenting a component of the pathogen and thus inherently limit the representation of pathogen peptides for cellular immunity based memory. We find that SARS-CoV-2 subunit peptides may not be robustly displayed by the Major Histocompatibility Complex (MHC) molecules in certain individuals. We introduce an augmentation strategy for subunit vaccines that adds a small number of SARS-CoV-2 peptides to a vaccine to improve the population coverage of pathogen peptide display. Our population coverage estimates integrate clinical data on peptide immunogenicity in convalescent COVID-19 patients and machine learning predictions. We evaluate the population coverage of 9 different subunits of SARS-CoV-2, including 5 functional domains and 4 full proteins, and augment each of them to fill a predicted coverage gap.","version":"1.2","doi":"10.1101/2020.08.04.200691","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.022384","pub_date":"2020-10-21","title":"One-step RNA extraction for RT-qPCR detection of 2019-nCoV","abstract":"The global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.","version":"1.3","doi":"10.1101/2020.04.02.022384","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.20.20215863","pub_date":"2020-10-21","title":"Cognitive deficits in people who have recovered from COVID-19 relative to controls: An N=84,285 online study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Case studies have revealed neurological problems in severely affected COVID-19 patients. However, there is little information regarding the nature and broader prevalence of cognitive problems post-infection or across the full spread of severity. We analysed cognitive test data from 84,285 Great British Intelligence Test participants who completed a questionnaire regarding suspected and biologically confirmed COVID-19 infection. People who had recovered, including those no longer reporting symptoms, exhibited significant cognitive deficits when controlling for age, gender, education level, income, racial-ethnic group and pre-existing medical disorders. They were of substantial effect size for people who had been hospitalised, but also for mild but biologically confirmed cases who reported no breathing difficulty. Finer grained analyses of performance support the hypothesis that COVID-19 has a multi-system impact on human cognition.</jats:p>\n                <jats:sec>\n                  <jats:title>Significance statement</jats:title>\n                  <jats:p>There is evidence that COVID-19 may cause long term health changes past acute symptoms, termed \u2018long COVID\u2019. Our analyses of detailed cognitive assessment and questionnaire data from tens thousands of datasets, collected in collaboration with BBC2 Horizon, align with the view that there are chronic cognitive consequences of having COVID-19. Individuals who recovered from suspected or confirmed COVID-19 perform worse on cognitive tests in multiple domains than would be expected given their detailed age and demographic profiles. This deficit scales with symptom severity and is evident amongst those without hospital treatment. These results should act as a clarion call for more detailed research investigating the basis of cognitive deficits in people who have survived SARS-COV-2 infection.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.20.20215863","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.20.346783","pub_date":"2020-10-20","title":"High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples","abstract":"The SARS-CoV-2 virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than a million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike specific antibodies in the plasma of convalescent donors. We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations, but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.","version":"1.1","doi":"10.1101/2020.10.20.346783","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.20.347021","pub_date":"2020-10-20","title":"Impact of clade specific mutations on structural fidelity of SARS-CoV-2 proteins","abstract":"The SARS-CoV-2 is a positive stranded RNA virus with a genome size of ~29.9 kilobase pairs which spans 29 open reading frames. Studies have revealed that the genome encodes about 16 non-structural proteins (nsp), four structural proteins, and six or seven accessory proteins. Based on prevalent knowledge on SARS-CoV and other coronaviruses, functions have been assigned for majority of the proteins. While, researchers across the globe are engrossed in identifying a potential pharmacological intervention to control the viral outbreak, none of the work has come up with new antiviral drugs or vaccines yet. One possible approach that has shown some positive results is by treating infected patients with the plasma collected from convalescent COVID-19 patients. Several vaccines around the world have entered their final trial phase in humans and we expect that these will in time be available for application to worldwide population to combat the disease. In this work we analyse the effect of prevalent mutations in the major pathogenesis related proteins of SARS-COV2 and attempt to pinpoint the effects of those mutations on the structural stability of the proteins. Our observations and analysis direct us to identify that all the major mutations have a negative impact in context of stability of the viral proteins under study and the mutant proteins suffer both structural and functional alterations as a result of the mutations. Our binary scoring scheme identifies L84S mutation in ORF8 as the most disruptive of the mutations under study. We believe that, the virus is under the influence of an evolutionary phenomenon similar to Muller\u2019s ratchet where the continuous accumulation of these mutations is making the virus less virulent which may also explain the reduction in fatality rates worldwide.","version":"1.1","doi":"10.1101/2020.10.20.347021","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.236893","pub_date":"2020-10-20","title":"Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis","abstract":"Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, we performed viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. Despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.","version":"1.2","doi":"10.1101/2020.08.04.236893","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.275719","pub_date":"2020-10-20","title":"SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but are Not Enriched in Islet Endocrine Cells","abstract":"Reports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet \u03b2 cells. This would require binding and entry of SARS-CoV-2 into host \u03b2 cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. ACE2 and TMPRSS2 transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in \u03b1 or \u03b2 cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet \u03b2 cells through these cell entry proteins.","version":"1.2","doi":"10.1101/2020.08.31.275719","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.20.346262","pub_date":"2020-10-20","title":"Zebrafish studies on the vaccine candidate to COVID-19, the Spike protein: Production of antibody and adverse reaction","abstract":"Establishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2020.10.20.346262","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337980","pub_date":"2020-10-20","title":"Structural impact on SARS-CoV-2 spike protein by D614G substitution","abstract":"Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.","version":"1.2","doi":"10.1101/2020.10.13.337980","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.076992","pub_date":"2020-10-20","title":"MALVIRUS: an integrated web application for viral variant calling","abstract":"Being able to efficiently call variants from the increasing amount of sequencing data daily produced from multiple viral strains is of the utmost importance, as demonstrated during the COVID-19 pandemic, in order to track the spread of the viral strains across the globe. We present MALVIRUS, an easy-to-install and easy-to-use web application that assists users in two tasks:\n\ncomputing a variant catalog consisting in a set of population SNP loci from the population sequences and\nefficiently calling variants of the catalog from a read sample.\n computing a variant catalog consisting in a set of population SNP loci from the population sequences and efficiently calling variants of the catalog from a read sample. Tests on Illumina and Nanopore samples prove the efficiency and the effectiveness of MALVIRUS in genotyping SARS-CoV-2 strain samples with respect to GISAID data.","version":"1.2","doi":"10.1101/2020.05.05.076992","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.20.346916","pub_date":"2020-10-20","title":"Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein","abstract":"The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses.","version":"1.1","doi":"10.1101/2020.10.20.346916","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.345363","pub_date":"2020-10-19","title":"SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance","abstract":"SARS-CoV-2 emerged in late 2019 and caused a pandemic, whereas the closely related SARS-CoV was contained rapidly in 2003. Here, a newly developed experimental set-up was used to study transmission of SARS-CoV and SARS-CoV-2 through the air between ferrets over more than a meter distance. Both viruses caused a robust productive respiratory tract infection resulting in transmission of SARS-CoV-2 to two of four indirect recipient ferrets and SARS-CoV to all four. A control pandemic A/H1N1 influenza virus also transmitted efficiently. Serological assays confirmed all virus transmission events. Although the experiments did not discriminate between transmission via small aerosols, large droplets and fomites, these results demonstrate that SARS-CoV and SARS-CoV-2 can remain infectious while travelling through the air. Efficient virus transmission between ferrets is in agreement with frequent SARS-CoV-2 outbreaks in mink farms. Although the evidence for airborne virus transmission between humans under natural conditions is absent or weak for SARS-CoV and SARS-CoV-2, ferrets may represent a sensitive model to study interventions aimed at preventing virus transmission.","version":"1.1","doi":"10.1101/2020.10.19.345363","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.344911","pub_date":"2020-10-19","title":"SARS-CoV-2 genome-wide mapping of CD8 T cell recognition reveals strong immunodominance and substantial CD8 T cell activation in COVID-19 patients","abstract":"To understand the CD8+ T cell immunity related to viral protection and disease severity in COVID-19, we evaluated the complete SARS-CoV-2 genome (3141 MHC-I binding peptides) to identify immunogenic T cell epitopes, and determine the level of CD8+ T cell involvement using DNA-barcoded peptide-major histocompatibility complex (pMHC) multimers. COVID-19 patients showed strong T cell responses, with up to 25% of all CD8+ lymphocytes specific to SARS-CoV-2-derived immunodominant epitopes, derived from ORF1 (open reading frame 1), ORF3, and Nucleocapsid (N) protein. A strong signature of T cell activation was observed in COVID-19 patients, while no T cell activation was seen in the \u2018non-exposed\u2019 and \u2018high exposure risk\u2019 healthy donors. Interestingly, patients with severe disease displayed the largest T cell populations with a strong activation profile. These results will have important implications for understanding the T cell immunity to SARS-CoV-2 infection, and how T cell immunity might influence disease development.","version":"1.1","doi":"10.1101/2020.10.19.344911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.17.343863","pub_date":"2020-10-19","title":"Development and pre-clinical characterization of two therapeutic equine formulations towards SARS-CoV-2 proteins for the potential treatment of COVID-19","abstract":"In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.","version":"1.1","doi":"10.1101/2020.10.17.343863","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.267526","pub_date":"2020-10-19","title":"Breadth and function of antibody response to acute SARS-CoV-2 infection in humans","abstract":"Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-RBD, three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.08.28.267526","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.345470","pub_date":"2020-10-19","title":"Structural basis of SARS-CoV-2 polymerase inhibition by Favipiravir","abstract":"The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into an unprecedented global pandemic. Nucleoside analogues, such as Remdesivir and Favipiravir, can serve as the first-line broad-spectrum antiviral drugs against the newly emerging viral diseases. Recent clinical trials of these two drugs for SARS-CoV-2 treatment revealed antiviral efficacies as well as side effects with different extents. As a pyrazine derivative, Favipiravir could be incorporated into the viral RNA products by mimicking both adenine and guanine nucleotides, which may further lead to mutations in progeny RNA copies due to the non-conserved base-pairing capacity. Here, we determined the cryo-EM structure of Favipiravir bound to the replicating polymerase complex of SARS-CoV-2 in the pre-catalytic state. This structure provides a missing snapshot for visualizing the catalysis dynamics of coronavirus polymerase, and reveals an unexpected base-pairing pattern between Favipiravir and pyrimidine residues which may explain its capacity for mimicking both adenine and guanine nucleotides. These findings shed lights on the mechanism of coronavirus polymerase catalysis and provide a rational basis for developing antiviral drugs to combat the SARS-CoV-2 pandemic.","version":"1.1","doi":"10.1101/2020.10.19.345470","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.343954","pub_date":"2020-10-19","title":"Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium","abstract":"The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2020.10.19.343954","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.345702","pub_date":"2020-10-19","title":"Diversity and genomic determinants of the microbiomes associated with COVID-19 and non-COVID respiratory diseases","abstract":"The novel coronavirus disease 2019 (COVID-19) is a rapidly emerging and highly transmissible disease caused by the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2). Understanding the microbiomes associated with the upper respiratory tract infection (URTI), chronic obstructive pulmonary disease (COPD) and COVID-19 diseases has clinical interest. We hypothesized that the diversity of microbiome compositions and their genomic features are associated with different pathological conditions of these human respiratory tract diseases (COVID-19 and non-COVID; URTI and COPD). To test this hypothesis, we analyzed 21 whole metagenome sequences (WMS) including eleven COVID-19 (BD = 6 and China = 5), six COPD (UK = 6) and four URTI (USA = 4) samples to unravel the diversity of microbiomes, their genomic features and relevant metabolic functions. The WMS data mapped to 534 bacterial, 60 archaeal and 61 viral genomes with distinct variation in the microbiome composition across the samples (COVID-19>COPD>URTI). Notably, 94.57%, 80.0% and 24.59% bacterial, archaeal and viral genera shared between the COVID-19 and non-COVID samples, respectively, however, the COVID-19 related samples had sole association with 16 viral genera other than SARS-CoV-2. Strain-level virome profiling revealed 660 and 729 strains in COVID-19 and non-COVID sequence data, respectively and of them 34.50% strains shared between the conditions. Functional annotation of metagenomics sequences of thevCOVID-19 and non-COVID groups identified the association of several biochemical pathways related to basic metabolism (amino acid and energy), ABC transporters, membrane transport, replication and repair, clustering-based subsystems, virulence, disease and defense, adhesion, regulation of virulence, programmed cell death, and primary immunodeficiency. We also detected 30 functional gene groups/classes associated with resistance to antibiotics and toxic compounds (RATC) in both COVID-19 and non-COVID microbiomes. Furthermore, a predominant higher abundance of cobalt-zinc-cadmium resistance (CZCR) and multidrug resistance to efflux pumps (MREP) genes were detected in COVID-19 metagenome. The profiles of microbiome diversity and associated microbial genomic features found in both COVID-19 and non-COVID (COPD and URTI) samples might be helpful for developing the microbiome-based diagnostics and therapeutics for COVID-19 and non-COVID respiratory diseases. However, future studies might be carried out to explore the microbiome dynamics and the cross-talk between host and microbiomes employing larger volume of samples from different ethnic groups and geoclimatic conditions.","version":"1.1","doi":"10.1101/2020.10.19.345702","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.345066","pub_date":"2020-10-19","title":"Mutational analysis and assessment of its impact on proteins of SARS-CoV-2 genomes from India","abstract":"The ongoing global pandemic of SARS-CoV-2 implies a corresponding accumulation of mutations. Herein the mutational status of 611 genomes from India along with their impact on proteins was ascertained. After excluding gaps and ambiguous sequences, a total of 493 variable sites (152 parsimony informative and 341 singleton) were observed. The most prevalent reference nucleotide was C (209) and substituted one was T (293). NSP3 had the highest incidence of 101 sites followed by S protein (74 sites), NSP12b (43 sites) and ORF3a (31 sites). The average number of mutations per sample for males and females was 2.56 and 2.88 respectively suggesting a higher contribution of mutations from females. Non-uniform geographical distribution of mutations implied by Odisha (30 samples, 109 mutations) and Tamil Nadu (31 samples, 40 mutations) suggests that sequences in some regions are mutating faster than others. There were 281 mutations (198 \u2018Neutral\u2019 and 83 \u2018Disease\u2019) affecting amino acid sequence. NSP13 has a maximum of 14 \u2018Disease\u2019 variants followed by S protein and ORF3a with 13 each. Further, constitution of \u2018Disease\u2019 mutations in genomes from asymptomatic people was mere 11% but those from deceased patients was over three folds higher at 38% indicating contribution of these mutations to the pathophysiology of the SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.10.19.345066","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.345140","pub_date":"2020-10-19","title":"COVID-19: Variant screening, an important step towards precision epidemiology","abstract":"Precision epidemiology using genomic technologies allows for a more targeted approach to COVID-19 control and treatment at individual and population level, and is the urgent need of the day. It enables identification of patients who may be at higher risk than others to COVID-19-related mortality, due to their genetic architecture, or who might respond better to a COVID-19 treatment. The COVID-19 virus, similar to SARS-CoV, uses the ACE2 receptor for cell entry and employs the cellular serine protease TMPRSS2 for viral S protein priming. This study aspires to present a multi-omics view of how variations in the ACE2 and TMPRSS2 genes affect COVID-19 infection and disease progression in affected individuals. It reports, for both genes, several variant and gene expression analysis findings, through (i) comparison analysis over single nucleotide polymorphisms (SNPs), that may account for the difference of COVID-19 manifestations among global sub-populations; (ii) calculating prevalence of structural variations (copy number variations (CNVs) / insertions), amongst populations; and (iii) studying expression patterns stratified by gender and age, over all human tissues. This work is a good first step to be followed by additional studies and functional assays towards informed treatment decisions and improved control of the infection rate.","version":"1.1","doi":"10.1101/2020.10.19.345140","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.205567","pub_date":"2020-10-19","title":"COVID-19 Detection on Chest X-Ray and CT Scan Images Using Multi-image Augmented Deep Learning Model","abstract":"COVID-19 is posed as very infectious and deadly pneumonia type disease until recent time. Despite having lengthy testing time, RT-PCR is a proven testing methodology to detect coronavirus infection. Sometimes, it might give more false positive and false negative results than the desired rates. Therefore, to assist the traditional RT-PCR methodology for accurate clinical diagnosis, COVID-19 screening can be adopted with X-Ray and CT scan images of lung of an individual. This image based diagnosis will bring radical change in detecting coronavirus infection in human body with ease and having zero or near to zero false positives and false negatives rates. This paper reports a convolutional neural network (CNN) based multi-image augmentation technique for detecting COVID-19 in chest X-Ray and chest CT scan images of coronavirus suspected individuals. Multi-image augmentation makes use of discontinuity information obtained in the filtered images for increasing the number of effective examples for training the CNN model. With this approach, the proposed model exhibits higher classification accuracy around 95.38% and 98.97% for CT scan and X-Ray images respectively. CT scan images with multi-image augmentation achieves sensitivity of 94.78% and specificity of 95.98%, whereas X-Ray images with multi-image augmentation achieves sensitivity of 99.07% and specificity of 98.88%. Evaluation has been done on publicly available databases containing both chest X-Ray and CT scan images and the experimental results are also compared with ResNet-50 and VGG-16 models.","version":"1.2","doi":"10.1101/2020.07.15.205567","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.19.344713","pub_date":"2020-10-19","title":"A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development","abstract":"The ongoing Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic has acutely highlighted the need to identify new treatment strategies for viral infections. Here we present a pivotal molecular mechanism of viral protein translation that relies on the mitochondrial translation machinery. We found that rare codons such as Leu-TTA are highly enriched in many viruses, including SARS-CoV-2, and these codons are essential for the regulation of viral protein expression. SARS-CoV-2 controls the translation of its spike gene by hijacking host mitochondria through 5\u2019 leader and 3\u2019UTR sequences that contain mitochondrial localization signals and activate the EGR1 pathway. Mitochondrial-targeted drugs such as lonidamine and polydatin significantly repress rare codon-driven gene expression and viral replication. This study identifies an unreported viral protein translation mechanism and opens up a novel avenue for developing antiviral drugs. Mitochondria are a potential target for antiviral therapy","version":"1.1","doi":"10.1101/2020.10.19.344713","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.16.343459","pub_date":"2020-10-18","title":"Molecular mechanism of SARS-CoV-2 inactivation by temperature","abstract":"Recent studies have shown that SARS-CoV-2 virus can be inactivated by effect of heat, even though, little is known about the molecular changes induced by the temperature. Here, we unravel the basics of such inactivation mechanism over the SARS-CoV-2 spike glycoprotein by executing atomistic molecular dynamics simulations. Both the closed down and open up states, which determine the accessibility to the receptor binding domain, were considered. Results suggest that the spike undergoes drastic changes in the topology of the hydrogen bond network while salt bridges are mainly preserved. Reorganization in the hydrogen bonds structure produces conformational variations in the receptor binding subunit and explain the thermal inactivation of the virus. Conversely, the macrostructure of the spike is preserved at high temperature because of the retained salt bridges. The proposed mechanism has important implications for engineering new approaches to inactivate the SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2020.10.16.343459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.17.339051","pub_date":"2020-10-18","title":"3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract","abstract":"The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (> 150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and emphasize a distinct focal infection pattern in nasal turbinates. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.10.17.339051","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.18.343715","pub_date":"2020-10-18","title":"Structure, Dynamics, Receptor Binding, and Antibody Binding of Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane","abstract":"The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2), and is considered the major target for drug and vaccine development. We previously built fully-glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of receptor binding domain (RBD) and different templates for the stalk region. In this work, multiple \u03bcs-long all-atom molecular dynamics simulations were performed to provide deeper insight into the structure and dynamics of S protein, and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of RBD, and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody-glycan clash analysis instead of a commonly-used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibody. Overall, our observations offer structural and dynamic insight into SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.","version":"1.1","doi":"10.1101/2020.10.18.343715","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.039198","pub_date":"2020-10-16","title":"Identification of potential vaccine candidates against SARS-CoV-2, A step forward to fight COVID-19: A Reverse Vaccinology Approach","abstract":"The recent Coronavirus Disease 2019 (COVID-19) causes an immense health crisis to global public health. The COVID-19 is the etiologic agent of a recently arose disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Presently, there is no vaccine available against this emerged viral disease. Therefore, it is indeed a need of the hour to develop an effectual and safe vaccine against this decidedly pandemic disease. In the current study, we collected SARS-CoV-2 genome which is prominent in India against human host, further more using reverse vaccinology here we claim effective vaccine candidates that can be mile stone in battle against COVID19. This novel study divulged one promising antigenic peptide GVYFASTEK from surface glycoprotein (protein accession no. - QIA98583.1) of SARS-CoV-2, which was predicated to be interacted with MHC alleles and showed up to 90% conservancy and high value of antigenicity. Subsequently, the molecular docking and simulation studies were verified molecular interaction of this prime antigenic peptide with the residues of HLA-A*11\u201301 allele for MHC Class I. After vigorous analysis, this peptide was predicted to be suitable epitope which is capable to induce the strong cell-mediated immune response against the SARS-CoV-2. Consequences from the current study could facilitate selecting SARS-CoV-2 epitopes for vaccine production pipelines in the immediate future. This novel research will certainly pave the way for a fast, reliable and virtuous platform to provide timely countermeasure of this dangerous pandemic disease, COVID-19.","version":"1.2","doi":"10.1101/2020.04.13.039198","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.341479","pub_date":"2020-10-16","title":"Abnormal antibodies to self-carbohydrates in SARS-CoV-2 infected patients","abstract":"SARS-CoV-2 is a deadly virus that is causing the global pandemic coronavirus disease 2019 (COVID-19). Our immune system plays a critical role in preventing, clearing, and treating the virus, but aberrant immune responses can contribute to deleterious symptoms and mortality. Many aspects of immune responses to SARS-CoV-2 are being investigated, but little is known about immune responses to carbohydrates. Since the surface of the virus is heavily glycosylated, pre-existing antibodies to glycans could potentially recognize the virus and influence disease progression. Furthermore, antibody responses to carbohydrates could be induced, affecting disease severity and clinical outcome. In this study, we used a carbohydrate antigen microarray with over 800 individual components to profile serum anti-glycan antibodies in COVID-19 patients and healthy control subjects. In COVID-19 patients, we observed abnormally high IgG and IgM antibodies to numerous self-glycans, including gangliosides, N-linked glycans, LacNAc-containing glycans, blood group H, and sialyl Lewis X. Some of these anti-glycan antibodies are known to play roles in autoimmune diseases and neurological disorders, which may help explain some of the unusual and prolonged symptoms observed in COVID-19 patients. The detection of antibodies to self-glycans has important implications for using convalescent serum to treat patients, developing safe and effective SARS-CoV-2 vaccines, and understanding the risks of infection. In addition, this study provides new insight into the immune responses to SARS-CoV-2 and illustrates the importance of including host and viral carbohydrate antigens when studying immune responses to viruses.","version":"1.1","doi":"10.1101/2020.10.15.341479","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.341636","pub_date":"2020-10-16","title":"Multivalency transforms SARS-CoV-2 antibodies into broad and ultrapotent neutralizers","abstract":"The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus Disease 2019 (COVID-19), has caused a global pandemic. Antibodies are powerful biotherapeutics to fight viral infections; however, discovery of the most potent and broadly acting clones can be lengthy. Here, we used the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 \u00d7 10\u221214 M were achieved as a result of up to 10,000-fold potency enhancements. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and Ig-like in vivo bioavailability. This MULTi-specific, multi-Affinity antiBODY (Multabody; or MB) platform contributes a new class of medical countermeasures against COVID-19 and an efficient approach to rapidly deploy potent and broadly-acting therapeutics against infectious diseases of global health importance. multimerization platform transforms antibodies emerging from discovery screens into potent neutralizers that can overcome SARS-CoV-2 sequence diversity.","version":"1.1","doi":"10.1101/2020.10.15.341636","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153411","pub_date":"2020-10-16","title":"The in vitro antiviral activity of the anti-hepatitis C virus (HCV) drugs daclatasvir and sofosbuvir against SARS-CoV-2","abstract":"Current approaches of drugs repurposing against 2019 coronavirus disease (COVID-19) have not proven overwhelmingly successful and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Daclatasvir (DCV) and sofosbuvir (SFV) are clinically approved against hepatitis C virus (HCV), with satisfactory safety profile. DCV and SFV target the HCV enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. We thus tested whether SARS-COV-2 would be susceptible these anti-HCV drugs. DCV consistently inhibited the production of infectious SARS-CoV-2 in Vero cells, in the hepatoma cell line (HuH-7) and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 \u03bcM, respectively. Although less potent than DCV, SFV and its nucleoside metabolite inhibited replication in Calu-3 cells. Moreover, SFV/DCV combination (1:0.15 ratio) inhibited SARS-CoV-2 with EC50 of 0.7:0.1 \u03bcM in Calu-3 cells. SFV and DCV prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Both drugs inhibited independent events during RNA synthesis and this was particularly the case for DCV, which also targeted secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial DCV in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Doses higher than those approved may ultimately be required, but these data provide a basis to further explore these agents as COVID-19 antiviral candidates.","version":"1.2","doi":"10.1101/2020.06.15.153411","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.16.342410","pub_date":"2020-10-16","title":"Drug synergy of combinatory treatment with remdesivir and the repurposed drugs fluoxetine and itraconazole effectively impairs SARS-CoV-2 infection in vitro","abstract":"The SARS-COV-2 pandemic and the global spread of coronavirus disease 2019 (COVID-19) urgently calls for efficient and safe antiviral treatment strategies. A straightforward approach to speed up drug development at lower costs is drug repurposing. Here we investigated the therapeutic potential of targeting the host- SARS-CoV-2 interface via repurposing of clinically licensed drugs and evaluated their use in combinatory treatments with virus- and host-directed drugs. We tested the antiviral potential of repurposing the antifungal itraconazole and the antidepressant fluoxetine on the production of infectious SARS-CoV-2 particles in the polarized Calu-3 cell culture model and evaluated the added benefit of a combinatory use of these host-directed drugs with remdesivir, an inhibitor of viral RNA polymerase. Drug treatments were well-tolerated and potent impaired viral replication was observed with all drug treatments. Importantly, both itraconazole-remdesivir and fluoxetine-remdesivir combinations inhibited the production of infectious SARS-CoV-2 particles > 90% and displayed synergistic effects in commonly used reference models for drug interaction. Itraconazole-Remdesivir and Fluoxetine-Remdesivir combinations are promising therapeutic options to control SARS-CoV-2 infection and severe progression of COVID-19.","version":"1.1","doi":"10.1101/2020.10.16.342410","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.325050","pub_date":"2020-10-16","title":"The Strand-biased Transcription of SARS-CoV-2 and Unbalanced Inhibition by Remdesivir","abstract":"SARS-CoV-2, a positive single-stranded RNA virus, caused the COVID-19 pandemic. During the viral replication and transcription, the RNA dependent RNA polymerase (RdRp) \u201cjumps\u201d along the genome template, resulting in discontinuous negative-stranded transcripts. In other coronaviruses, the negative strand RNA was found functionally relevant to the activation of host innate immune responses. Although the sense-mRNA architectures of SARS-CoV-2 were reported, its negative strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand. During negative strand synthesis, apart from canonical sub-genomic ORFs, numerous non-canonical fusion transcripts are formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor Remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the anti-viral vaccine development and drug design. Strand-biased transcription of SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.10.15.325050","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.158154","pub_date":"2020-10-16","title":"Experimental and natural evidence of SARS-CoV-2 infection-induced activation of type I interferon responses","abstract":"Type I interferons (IFNs) are our first line of defence against a virus. Protein over-expression studies have suggested the ability of SARS-CoV-2 proteins to block IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wildtype SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are lacking. Here we demonstrate that SARS-CoV-2 infection induces a mild type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Our data demonstrate that SARS-CoV-2 is not adept in blocking type I IFN responses and provide support for ongoing IFN clinical trials.","version":"1.2","doi":"10.1101/2020.06.18.158154","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.341958","pub_date":"2020-10-16","title":"Early induction of SARS-CoV-2 specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients","abstract":"Virus-specific humoral and cellular immunity act synergistically to protect the host from viral infection. We interrogated the dynamic changes of virological and immunological parameters in 12 patients with symptomatic acute SARS-CoV-2 infection from disease onset to convalescence or death. We quantified SARS-CoV-2 viral RNA in the respiratory tract in parallel with antibodies and circulating T cells specific for various structural (NP, M, ORF3a and spike) and non-structural proteins (ORF7/8, NSP7 and NSP13). We observed that while rapid induction and quantity of humoral responses were associated with increased disease severity, an early induction of SARS-CoV-2 specific T cells was present in patients with mild disease and accelerated viral clearance. These findings provide further support for a protective role of SARS-CoV-2 specific T cells over antibodies during SARS-CoV-2 infection with important implications in vaccine design and immune-monitoring.","version":"1.1","doi":"10.1101/2020.10.15.341958","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.213777","pub_date":"2020-10-16","title":"Cellular events of acute, resolving or progressive COVID-19 in SARS-CoV-2 infected non-human primates","abstract":"We investigated the immune events following SARS-CoV-2 infection, from the acute inflammatory state up to four weeks post infection, in non-human primates (NHP) with heterogeneous pulmonary pathology. The acute phase was characterized by a robust and rapid migration of monocytes expressing CD16 from the blood and concomitant increase in CD16+ macrophages in the lungs. We identified two subsets of interstitial macrophages (HLA-DR+ CD206\u2013), a transitional CD11c+ CD16+ cell population that was directly associated with IL-6 levels in plasma, and one long lasting CD11b+ CD16+ cell population. Strikingly, levels of monocytes were a correlate of viral replication in bronchial brushes and we discovered TARC (CCL17) as a new potential mediator of myeloid recruitment to the lungs. Worse disease outcomes were associated with high levels of cell infiltration in lungs including CD11b+ CD16hi macrophages and CD11b+ neutrophils. Accumulation of macrophages was long-lasting and detectable even in animals with mild or no signs of disease. Interestingly, animals with anti-inflammatory responses including high IL-10:IL-6 and kynurenine to tryptophan ratios had less signs of disease. Our results unravel cellular mechanisms of COVID-19 and suggest that NHP may be appropriate models to test immune therapies.","version":"1.2","doi":"10.1101/2020.07.21.213777","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.16.339937","pub_date":"2020-10-16","title":"Peptide vaccine candidate mimics the heterogeneity of natural SARS-CoV-2 immunity in convalescent humans and induces broad T cell responses in mice models","abstract":"We developed a global peptide vaccine against SARS-CoV-2 that addresses the dual challenges of heterogeneity in the immune responses of different individuals and potential heterogeneity of the infecting virus. PolyPEPI-SCoV-2 is a polypeptide vaccine containing nine 30-mer peptides derived from all four major structural proteins of the SARS-CoV-2. Vaccine peptides were selected based on their frequency as HLA class I and class II personal epitopes (PEPIs) restricted to multiple autologous HLA alleles of individuals in an in silico cohort of 433 subjects of different ethnicities. PolyPEPI-SCoV-2 vaccine administered with Montanide ISA 51VG adjuvant generated robust, Th1-biased CD8+ and CD4+ T cell responses against all four structural proteins of the virus, as well as binding antibodies upon subcutaneous injection into BALB/c and CD34+ transgenic mice. In addition, PolyPEPI-SCoV-2-specific, polyfunctional CD8+ and CD4+ T cells were detected ex vivo in each of the 17 asymptomatic/mild COVID-19 convalescents\u2019 blood investigated, 1\u20135 months after symptom onset. The PolyPEPI-SCoV-2-specific T cell repertoire used for recovery from COVID-19 was extremely diverse: donors had an average of seven different peptide-specific T cells, against the SARS-CoV-2 proteins; 87% of donors had multiple targets against at least three SARS-CoV-2 proteins and 53% against all four. In addition, PEPIs determined based on the complete HLA class I genotype of the convalescent donors were validated, with 84% accuracy, to predict PEPI-specific CD8+ T cell responses measured for the individuals. Extrapolation of the above findings to a US bone marrow donor cohort of 16,000 HLA-genotyped individuals with 16 different ethnicities (n=1,000 each ethnic group) suggest that PolyPEPI-SCoV-2 vaccination in a general population will likely elicit broad, multi-antigenic CD8+ and CD4+ T cell responses in 98% of individuals, independent of ethnicity, including Black, Asian, and Minority Ethnic (BAME) cohorts.","version":"1.1","doi":"10.1101/2020.10.16.339937","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.20212498","pub_date":"2020-10-16","title":"The effect of influenza vaccination on trained immunity: impact on COVID-19","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  Every year, influenza causes 290.000 to 650.000 deaths worldwide and vaccination is encouraged to prevent infection in high-risk individuals. Interestingly, cross-protective effects of vaccination against heterologous infections have been reported, and long-term boosting of innate immunity (also termed\n                  <jats:italic>trained immunity</jats:italic>\n                  ) has been proposed as the underlying mechanism. Several epidemiological studies also suggested cross-protection between influenza vaccination and COVID-19 during the current pandemic. However, the mechanism behind such an effect is unknown. Using an established\n                  <jats:italic>in-vitro</jats:italic>\n                  model of trained immunity, we demonstrate that the quadrivalent inactivated influenza vaccine used in the Netherlands in the 2019-2020 influenza season can induce a trained immunity response, including an improvement of cytokine responses after stimulation of human immune cells with SARS-CoV-2. In addition, we found that SARS-CoV-2 infection was less common among Dutch hospital employees who had received influenza vaccination during the 2019/2020 winter season (RR = 0,61 (95% CI, 0.4585 - 0.8195,\n                  <jats:italic>P</jats:italic>\n                  = 0.001). In conclusion, a quadrivalent inactivated influenza vaccine can induce trained immunity responses against SARS-CoV-2, which may result in relative protection against COVID-19. These data, coupled with similar recent independent reports, argue for a beneficial effect of influenza vaccination against influenza as well as COVID-19, and suggests its effective deployment in the 2020-2021 influenza season to protect against both infections.\n                </jats:p>","version":null,"doi":"10.1101/2020.10.14.20212498","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.16.342428","pub_date":"2020-10-16","title":"The Theory and Practice of the viral dose in neutralization assay: insights on SARS-CoV-2 \u201cdoublethink\u201d effect","abstract":"Due to the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for reliable high-throughput serological assays in order to evaluate the immunological responses against SARS-COV-2 virus and to enable population screening, as well as vaccines and drug\u2019s efficacy testing. Several serological assays for SARS-CoV-2 are now becoming available in the market. However, it has also become extremely important to have well-established assays with desirable high sensitivity and specificity. To date, the micro-neutralization (MN) assay, is currently considered the gold-standard being capable of evaluating and detecting, functional neutralizing antibodies (nAbs). Several protocols exist for microneutralization assays which vary in several steps of the protocol: cell seeding conditions, number of cells seeded, virus amount used in the infection step, virus-serum-cells incubation period etc. These potential differences account for a high degree of variability and inconsistency of the results and using a harmonized protocol for the micro-neutralization assay could potentially solve this. Given this situation, the main aim of our study was to carry out SARS-CoV-2 wild type virus MN assay in order to investigate which optimal tissue culture infective dose 50 (TCID50) infective dose in use is the most adequate choice for implementation in terms of reproducibility, standardization possibilities and comparability of results. Therefore, we assessed the MN by using two different viral infective doses: a standard dose of 100 TCID50/well and a lower dose of 25 TCID50/well. The results obtained, yielded by MN on using the lower infective dose (25 TCID50), were in line with those obtained with the standard infective dose; in some cases, however, we detected a titre that was one or two dilution steps higher, which maintained all negative samples negative. This suggesting that the lower dose can potentially have a positive impact on the detection and estimation of neutralizing antibodies present in a given sample, showing higher sensitivity but similar specificity and therefore, it would require a more accurate assessment and cross-laboratories standardisation especially when MN is employed as serological assay of choice for pre-clinical and clinical studies.","version":"1.1","doi":"10.1101/2020.10.16.342428","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.336099","pub_date":"2020-10-16","title":"Molecular characterization of SARS-CoV-2 from Bangladesh: Implications in genetic diversity, possible origin of the virus, and functional significance of the mutations","abstract":"In a try to understand the pathogenesis, evolution and epidemiology of the SARS-CoV-2 virus, scientists from all over the world are tracking its genomic changes in real-time. Genomic studies can be helpful in understanding the disease dynamics. We have downloaded 324 complete and near complete SARS-CoV-2 genomes submitted in GISAID database from Bangladesh which were isolated between 30 March to 7 September, 2020. We then compared these genomes with Wuhan reference sequence and found 4160 mutation events including 2253 missense single nucleotide variations, 38 deletions and 10 insertions. The C>T nucleotide change was most prevalent (41% of all muations) possibly due to selective mutation pressure to reduce CpG sites to evade CpG targeted host immune response. The most frequent mutation that occurred in 98% isolates was 3037C>T which is a synonymous change that almost always accompanied 3 other mutations that include 241C>T, 14408C>T (P323L in RdRp) and 23403A>G (D614G in spike protein). The P323L was reported to increase mutation rate and D614G is associated with increased viral replication and currently most prevalent variant circulating all over the world. We identified multiple missense mutations in B-cell and T-cell predicted epitope regions and/or PCR target regions (including R203K and G204R that occurred in 86% of the isolates) that may impact immunogenicity and/or RT-PCR based diagnosis. Our analysis revealed 5 large deletion events in ORF7a and ORF8 gene products that may be associated with less severity of the disease and increased viral clearance. Our phylogeny analysis identified most of the isolates belonged to the Nextstrain clade 20B (86%) and GISAID clade GR (88%). Most of our isolates shared common ancestors either directly with European countries or jointly with middle eastern countries as well as Australia and India. Interestingly, the 19B clade (GISAID S clade) was unique to Chittagong which was originally prevalent in China. This reveals possible multiple introduction of the virus in Bangladesh via different routes. Hence more genome sequencing and analysis with related clinical data is needed to interpret functional significance and better predict the disease dynamics that may be helpful for policy makers to control the COVID-19 pandemic in Bangladesh.","version":"1.2","doi":"10.1101/2020.10.12.336099","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.340455","pub_date":"2020-10-16","title":"Progressive and accurate assembly of multi-domain protein structures from cryo-EM density maps","abstract":"Progress in cryo-electron microscopy (cryo-EM) has provided the potential for large-size protein structure determination. However, the solution rate for multi-domain proteins remains low due to the difficulty in modeling inter-domain orientations. We developed DEMO-EM, an automatic method to assemble multi-domain structures from cryo-EM maps through a progressive structural refinement procedure combining rigid-body domain fitting and flexible assembly simulations with deep neural network inter-domain distance profiles. The method was tested on a large-scale benchmark set of proteins containing up to twelve continuous and discontinuous domains with medium-to-low-resolution density maps, where DEMO-EM produced models with correct inter-domain orientations (TM-score >0.5) for 98% of cases and significantly outperformed the state-of-the-art methods. DEMO-EM was applied to SARS-Cov-2 coronavirus genome and generated models with average TM-score/RMSD of 0.97/1.4\u00c5 to the deposited structures. These results demonstrated an efficient pipeline that enables automated and reliable large-scale multi-domain protein structure modeling with atomic-level accuracy from cryo-EM maps.","version":"1.1","doi":"10.1101/2020.10.15.340455","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.340794","pub_date":"2020-10-15","title":"Integrated characterization of SARS-CoV-2 genome, microbiome, antibiotic resistance and host response from single throat swabs","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a newly-developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.","version":"1.1","doi":"10.1101/2020.10.15.340794","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.339952","pub_date":"2020-10-15","title":"Multiplexed proteomics and imaging of resolving and lethal SARS-CoV-2 infection in the lung","abstract":"Normal tissue physiology and repair depends on communication with the immune system. Understanding this communication at the molecular level in intact tissue requires new methods. The consequences of SARS-CoV-2 infection, which can result in acute respiratory distress, thrombosis and death, has been studied primarily in accessible liquid specimens such as blood, sputum and bronchoalveolar lavage, all of which are peripheral to the primary site of infection in the lung. Here, we describe the combined use of multiplexed deep proteomics with multiplexed imaging to profile infection and its sequelae directly in fixed lung tissue specimens obtained from necropsy of infected animals and autopsy of human decedents. We characterize multiple steps in disease response from cytokine accumulation and protein phosphorylation to activation of receptors, changes in signaling pathways, and crosslinking of fibrin to form clots. Our data reveal significant differences between naturally resolving SARS-CoV-2 infection in rhesus macaques and lethal COVID-19 in humans. The approach we describe is broadly applicable to other tissues and diseases. Proteomics of infected tissue reveals differences in inflammatory and thrombotic responses between resolving and lethal COVID-19.","version":"1.1","doi":"10.1101/2020.10.14.339952","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.339986","pub_date":"2020-10-15","title":"A Common Methodological Phylogenomics Framework for intra-patient heteroplasmies to infer SARS-CoV-2 sublineages and tumor clones","abstract":"We present a common methodological framework to infer the phylogenomics from genomic data, be it reads of SARS-CoV-2 of multiple COVID-19 patients or bulk DNAseq of the tumor of a cancer patient. The commonality is in the phylogenetic retrodiction based on the genomic reads in both scenarios. While there is evidence of heteroplasmy, i.e., multiple lineages of SARS-CoV-2 in the same COVID-19 patient; to date, there is no evidence of sublineages recombining within the same patient. The heterogeneity in a patient\u2019s tumor is analogous to intra-patient heteroplasmy and the absence of recombination in the cells of tumor is a widely accepted assumption. Just as the different frequencies of the genomic variants in a tumor presupposes the existence of multiple tumor clones and provides a handle to computationally infer them, we postulate that so do the different variant frequencies in the viral reads, offering the means to infer the multiple co-infecting sublineages. We describe the Concerti computational framework for inferring phylogenies in each of the two scenarios. To demonstrate the accuracy of the method, we reproduce some known results in both scenarios. We also make some additional discoveries. We uncovered new potential parallel mutation in the evolution of the SARS-CoV-2 virus. In the context of cancer, we uncovered new clones harboring resistant mutations to therapy from clinically plausible phylogenetic tree in a patient.","version":"1.1","doi":"10.1101/2020.10.14.339986","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.341537","pub_date":"2020-10-15","title":"Design of SARS-CoV-2 RBD mRNA Vaccine Using Novel Ionizable Lipids","abstract":"The novel coronavirus SARS-CoV-2 has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple and rapid platform for immunization, and therefore have been employed in recent studies towards the development of a SARS-CoV-2 vaccine. In this study, we present the design of a lipid nanoparticles (LNP)-encapsulated receptor binding domain (RBD) mRNA vaccine. Several ionizable lipids have been evaluated in vivo in a luciferase mRNA reporter assay, and two leading LNPs formulation have been chosen for the subsequent RBD mRNA vaccine experiment. Intramuscular administration of LNP RBD mRNA elicited robust humoral response, high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. These novel lipids open new avenues for mRNA vaccines in general and for a COVID19 vaccine in particular.","version":"1.1","doi":"10.1101/2020.10.15.341537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.340034","pub_date":"2020-10-15","title":"A high throughput RNA displacement assay for screening SARS-CoV-2 nsp10-nsp16 complex towards developing therapeutics for COVID-19","abstract":"SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2\u2019-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in 384-well format with a Z\u2032-Factor of 0.6, suitable for high throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, product of the reaction, SAH, and a common methyltransferase inhibitor, sinefungin using Isothermal Titration Calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high throughput method for screening nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2020.10.14.340034","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.339838","pub_date":"2020-10-15","title":"Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration","abstract":"The Spike protein of SARS-CoV-2, its receptor binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.","version":"1.1","doi":"10.1101/2020.10.15.339838","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.338053","pub_date":"2020-10-15","title":"Shark conservation risks associated with the use of shark liver oil in SARS-CoV-2 vaccine development","abstract":"The COVID-19 pandemic may create new demand for wildlife-generated products for human health, including a shark-derived ingredient used in some vaccines. Adjuvants are a vaccine component that increases efficacy, and some adjuvants contain squalene, a natural compound derived from shark liver oil which is found most abundantly in deep-sea sharks. In recent decades, there has been growing conservation concern associated with the sustainability of many shark fisheries. The need for a potentially massive number of adjuvant-containing SARS-CoV-2 vaccines may increase global demand for shark-derived squalene, with possible consequences for shark conservation, especially of vulnerable and understudied deep-sea species. A shift to non-animal-derived sources of squalene, which are similar in cost and identical in effectiveness, or an emphasis on increasing traceability and sustainability of shark-derived squalene from existing well-managed fisheries, could better support conservation and public health goals.","version":"1.1","doi":"10.1101/2020.10.14.338053","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.340604","pub_date":"2020-10-15","title":"Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without","abstract":"Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split \u03b2-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.","version":"1.1","doi":"10.1101/2020.10.15.340604","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.339465","pub_date":"2020-10-15","title":"Evidence of antigenic imprinting in sequential Sarbecovirus immunization","abstract":"Antigenic imprinting, which describes the bias of antibody response due to previous immune history, can influence vaccine effectiveness and has been reported in different viruses. Give that COVID-19 vaccine development is currently a major focus of the world, there is a lack of understanding of how background immunity influence antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting in Sarbecovirus, which is the subgenus that SARS-CoV-2 belongs to. Specifically, we sequentially immunized mice with two antigenically distinct Sarbecovirus strains, namely SARS-CoV and SARS-CoV-2. We found that the neutralizing antibodies triggered by the sequentially immunization are dominantly against the one that is used for priming. Given that the impact of the background immunity on COVID-19 is still unclear, our results will provide important insights into the pathogenesis of this disease as well as COVID-19 vaccination strategy.","version":"1.1","doi":"10.1101/2020.10.14.339465","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.15.20209817","pub_date":"2020-10-15","title":"Repurposed antiviral drugs for COVID-19 \u2013interim WHO SOLIDARITY trial results","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>BACKGROUND</jats:title>\n                  <jats:p>WHO expert groups recommended mortality trials in hospitalized COVID-19 of four re-purposed antiviral drugs.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>Study drugs were Remdesivir, Hydroxychloroquine, Lopinavir (fixed-dose combination with Ritonavir) and Interferon-\u03b21a (mainly subcutaneous; initially with Lopinavir, later not). COVID-19 inpatients were randomized equally between whichever study drugs were locally available and open control (up to 5 options: 4 active and local standard-of-care). The intent-to-treat primary analyses are of in-hospital mortality in the 4 pairwise comparisons of each study drug vs its controls (concurrently allocated the same management without that drug, despite availability). Kaplan-Meier 28-day risks are unstratified; log-rank death rate ratios (RRs) are stratified for age and ventilation at entry.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>In 405 hospitals in 30 countries 11,266 adults were randomized, with 2750 allocated Remdesivir, 954 Hydroxychloroquine, 1411 Lopinavir, 651 Interferon plus Lopinavir, 1412 only Interferon, and 4088 no study drug. Compliance was 94-96% midway through treatment, with 2-6% crossover. 1253 deaths were reported (at median day 8, IQR 4-14). Kaplan-Meier 28-day mortality was 12% (39% if already ventilated at randomization, 10% otherwise). Death rate ratios (with 95% CIs and numbers dead/randomized, each drug vs its control) were: Remdesivir RR=0.95 (0.81-1.11, p=0.50; 301/2743 active vs 303/2708 control), Hydroxychloroquine RR=1.19 (0.89-1.59, p=0.23; 104/947 vs 84/906), Lopinavir RR=1.00 (0.79-1.25, p=0.97; 148/1399 vs 146/1372) and Interferon RR=1.16 (0.96-1.39, p=0.11; 243/2050 vs 216/2050). No study drug definitely reduced mortality (in unventilated patients or any other subgroup of entry characteristics), initiation of ventilation or hospitalisation duration.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>\n                    These Remdesivir, Hydroxychloroquine, Lopinavir and Interferon regimens appeared to have little or no effect on hospitalized COVID-19, as indicated by overall mortality, initiation of ventilation and duration of hospital stay. The mortality findings contain most of the randomized evidence on Remdesivir and Interferon, and are consistent with meta-analyses of mortality in all major trials. (Funding: WHO. Registration:\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='isrctn' xlink:href='83971151'>ISRCTN83971151</jats:ext-link>\n                    ,\n                    <jats:ext-link xmlns:xlink='http://www.w3.org/1999/xlink' ext-link-type='clintrialgov' xlink:href='NCT04315948'>NCT04315948</jats:ext-link>\n                    )\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.15.20209817","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.13.337584","pub_date":"2020-10-14","title":"ACIS, A Novel KepTide\u2122, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19","abstract":"Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide\u2122 (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein \u201cspike\u201d (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide\u2122. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide\u2122 efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide\u2122 therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally.","version":"1.1","doi":"10.1101/2020.10.13.337584","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.336818","pub_date":"2020-10-14","title":"Structural stability of SARS-CoV-2 degrades with temperature","abstract":"SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter. The economic and public health impact of the COVID-19 pandemic are very significant. However scientific information needed to underpin policy decisions are limited partly due to novelty of the SARS-CoV-2 pathogen. There is therefore an urgent need for mechanistic studies of both COVID-19 disease and the SARS-CoV-2 virus. We show that individual virus particles suffer structural destabilization at relatively mild but elevated temperatures. Our nanoscale results are consistent with recent observations at larger scales. Our work strengthens the case for COVID-19 resurgence in winter.","version":"1.1","doi":"10.1101/2020.10.12.336818","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.335893","pub_date":"2020-10-14","title":"Co-infection of influenza A virus enhances SARS-CoV-2 infectivity","abstract":"The upcoming flu season in the northern hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental co-infection of IAV with either pseudotyped or SARS-CoV-2 live virus, we found that IAV pre-infection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice co-infected with IAV in vivo. Moreover, such enhancement of SARS-CoV-2 infectivity was not seen with several other viruses probably due to a unique IAV segment as an inducer to elevate ACE2 expression. This study illustrates that IAV has a special nature to aggravate SARS-CoV-2 infection, and prevention of IAV is of great significance during the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.10.14.335893","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.338095","pub_date":"2020-10-14","title":"Rapid Development of Neutralizing and Diagnostic SARS-COV-2 Mouse Monoclonal Antibodies","abstract":"The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nanomolar-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.","version":"1.1","doi":"10.1101/2020.10.13.338095","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.338038","pub_date":"2020-10-14","title":"The emergence of inter-clade hybrid SARS-CoV-2 lineages revealed by 2D nucleotide variation mapping","abstract":"I performed whole-genome sequencing on SARS-CoV-2 collected from COVID-19 samples at Mayo Clinic Rochester in mid-April, 2020, generated 85 consensus genome sequences and compared them to other genome sequences collected worldwide. I proposed a novel illustrating method using a 2D map to display populations of co-occurring nucleotide variants for intra- and inter-viral clades. This method is highly advantageous for the new era of \u201cbig-data\u201d when high-throughput sequencing is becoming readily available. Using this method, I revealed the emergence of inter-clade hybrid SARS-CoV-2 lineages that are potentially caused by homologous genetic recombination.","version":"1.1","doi":"10.1101/2020.10.13.338038","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.339150","pub_date":"2020-10-14","title":"Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters","abstract":"SARS-CoV-2 infection results in viral burden in the upper and lower respiratory tract, enabling transmission and often leading to substantial lung pathology. Delivering the antiviral treatment directly to the lungs has the potential to improve lung bioavailability and dosing efficiency. As the SARS-CoV-2 Receptor Binding Domain (RBD) of the Spike (S) is increasingly deemed to be a clinically validated target, RBD-specific B cells were isolated from patients following SARS-CoV-2 infection to derive a panel of fully human monoclonal antibodies (hmAbs) that potently neutralize SARS-CoV-2. The most potent hmAb, 1212C2 was derived from an IgM memory B cell, has high affinity for SARS-CoV-2 RBD which enables its direct inhibition of RBD binding to ACE2. The 1212C2 hmAb exhibits in vivo prophylactic and therapeutic activity against SARS-CoV-2 in hamsters when delivered intraperitoneally, achieving a meaningful reduction in upper and lower respiratory viral burden and lung pathology. Furthermore, liquid nebulized inhale treatment of SARS-CoV-2 infected hamsters with as low as 0.6 mg/kg of inhaled dose, corresponding to approximately 0.03 mg/kg of lung deposited dose, mediated a reduction in respiratory viral burden that is below the detection limit, and mitigated lung pathology. The therapeutic efficacy achieved at an exceedingly low-dose of inhaled 1212C2 supports the rationale for local lung delivery and achieving dose-sparing benefits as compared to the conventional parenteral route of administration. Taken together, these results warrant an accelerated clinical development of 1212C2 hmAb formulated and delivered via inhalation for the prevention and treatment of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.10.14.339150","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.339515","pub_date":"2020-10-14","title":"The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation","abstract":"SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5\u2019UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5\u2019UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine \u2018Achille heel\u2019 of the virus.","version":"1.1","doi":"10.1101/2020.10.14.339515","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.338541","pub_date":"2020-10-14","title":"Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives","abstract":"The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19 patients. The initial step to identifying potential candidates usually involves in vitro screening. Here we describe a simple rapid bioassay for drug screening using Vero E6 cells and inhibition of cytopathic effects (CPE) measured using crystal violet staining. The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. A key refinement involves a simple growth assay to identify drug concentrations that cause cellular stress or \u201ccytomorbidity\u201d, as distinct from cytotoxicity or loss of viability. For instance, hydroxychloroquine shows anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities.","version":"1.1","doi":"10.1101/2020.10.13.338541","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.340091","pub_date":"2020-10-14","title":"Use of convalescent serum reduces severity of COVID-19 in nonhuman primates","abstract":"Passive transfer of convalescent plasma or serum is a time-honored strategy for treating infectious diseases. Human convalescent plasma containing antibodies against SARS-CoV-2 is currently being used to treat COVID-19 patients. However, most patients have been treated outside of randomized clinical trials making it difficult to determine the efficacy of this approach. Here, we assessed the efficacy of convalescent sera in a newly developed African green monkey model of COVID-19. Groups of SARS-CoV-2-infected animals were treated with pooled convalescent sera containing either high or low to moderate anti-SARS-CoV-2 neutralizing antibody titers. Differences in viral load and disease pathology were minimal between monkeys that received the lower titer convalescent sera and untreated controls. However, and importantly, lower levels of SARS-CoV-2 in respiratory compartments, reduced gross and histopathological lesion severity in the lungs, and reductions in several parameters associated with coagulation and inflammatory processes were observed in monkeys that received convalescent sera versus untreated controls. Our data support human studies suggesting that convalescent plasma therapy is an effective strategy if donors with high level of antibodies against SARS-CoV-2 are employed and if recipients are at an early stage of disease.","version":"1.1","doi":"10.1101/2020.10.14.340091","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.337535","pub_date":"2020-10-14","title":"Immunogenicity of novel mRNA COVID-19 vaccine MRT5500 in mice and non-human primates","abstract":"An effective vaccine to address the global pandemic of coronavirus disease 2019 (COVID-19) is an urgent public health priority. Novel synthetic mRNA and vector-based vaccine technologies offer an expeditious development path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on Spike (S) glycoprotein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus causing COVID-19. Several mRNA constructs expressing various structural conformations of S-protein, including wild type (WT), a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), were tested in a preclinical animal model for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques. The selected 2P/GSAS vaccine formulation, now designated MRT5500, elicited potent nAbs as measured in two types of neutralization assays. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate species, a result that helps to address a hypothetical concern regarding potential vaccine-associated enhanced respiratory diseases associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for clinical development against COVID-19.","version":"1.1","doi":"10.1101/2020.10.14.337535","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.338558","pub_date":"2020-10-14","title":"Methyltransferase-like 3 modulates severe acute respiratory syndrome coronavirus-2 RNA N6-methyladenosine modification and replication","abstract":"The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection not only increased the expression of methyltransferase-like 3 (METTL3) but also altered its distribution. Modification of METTL3 expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp interacted with METTL3, and METTL3 was distributed in both the nucleus and cytoplasm in the presence of RdRp. RdRp appeared to modulate the sumoylation and ubiquitination of METTL3 via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication.","version":"1.1","doi":"10.1101/2020.10.14.338558","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.051201","pub_date":"2020-10-14","title":"Sex, androgens and regulation of pulmonary AR, TMPRSS2 and ACE2","abstract":"The sex discordance in COVID-19 outcomes has been widely recognized, with males generally faring worse than females and a potential link to sex steroids. A plausible mechanism is androgen-induced expression of TMPRSS2 and/or ACE2 in pulmonary tissues that may increase susceptibility or severity in males. This hypothesis is the subject of several clinical trials of anti-androgen therapies around the world. Here, we investigated the sex-associated TMPRSS2 and ACE2 expression in human and mouse lungs and interrogated the possibility of pharmacologic modification of their expression with anti-androgens. We found no evidence for increased TMPRSS2 expression in the lungs of males compared to females in humans or mice. Furthermore, in male mice, treatment with the androgen receptor antagonist enzalutamide did not decrease pulmonary TMPRSS2. On the other hand, ACE2 and AR expression was sexually dimorphic and higher in males than females. ACE2 was moderately suppressible with enzalutamide therapy. Our work suggests that sex differences in COVID-19 outcomes attributable to viral entry are independent of TMPRSS2. Modest changes in ACE2 could account for some of the sex discordance.","version":"1.2","doi":"10.1101/2020.04.21.051201","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337907","pub_date":"2020-10-14","title":"Obesity alters Ace2 and Tmprss2 expression in lung, trachea, and esophagus in a sex-dependent manner: Implications for COVID-19","abstract":"Obesity is a major risk factor for SARS-CoV-2 infection and COVID-19 severity. The underlying basis of this association is likely complex in nature. The host-cell receptor angiotensin converting enzyme 2 (ACE2) and the type II transmembrane serine protease (TMPRSS2) are important for viral cell entry. It is unclear whether obesity alters expression of Ace2 and Tmprss2 in the lower respiratory tract. Here, we show that: 1) Ace2 expression is elevated in the lung and trachea of diet-induced obese male mice and reduced in the esophagus of obese female mice relative to lean controls; 2) Tmprss2 expression is increased in the trachea of obese male mice but reduced in the lung and elevated in the trachea of obese female mice relative to lean controls; 3) in chow-fed lean mice, females have higher expression of Ace2 in the lung and esophagus as well as higher Tmprss2 expression in the lung but lower expression in the trachea compared to males; and 4) in diet-induced obese mice, males have higher expression of Ace2 in the trachea and higher expression of Tmprss2 in the lung compared to females, whereas females have higher expression of Tmprss2 in the trachea relative to males. Our data indicate diet- and sex-dependent modulation of Ace2 and Tmprss2 expression in the lower respiratory tract and esophagus. Given the high prevalence of obesity worldwide and a sex-biased mortality rate, we discuss the implications and relevance of our results for COVID-19.","version":"1.1","doi":"10.1101/2020.10.13.337907","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.100214","pub_date":"2020-10-14","title":"Normalization of single-cell RNA-seq counts by log(x+1)* or log(1+x)*","abstract":"Single-cell RNA-seq technologies have been successfully employed over the past decade to generate many high resolution cell atlases. These have proved invaluable in recent efforts aimed at understanding the cell type specificity of host genes involved in SARS-CoV-2 infections. While single-cell atlases are based on well-sampled highly-expressed genes, many of the genes of interest for understanding SARS-CoV-2 can be expressed at very low levels. Common assumptions underlying standard single-cell analyses don\u2019t hold when examining low-expressed genes, with the result that standard workflows can produce misleading results. Lowly expressed genes in single-cell RNA-seq can be easliy misanalyzed. log(1+x) count normalization introduces errors for lowly expressed genes The average log(1+x) expression differs considerably from log(x) when x is small An alternative approach is to use the fraction of cells with non-zero expression","version":"1.3","doi":"10.1101/2020.05.19.100214","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337691","pub_date":"2020-10-14","title":"Reducing SAR in 7T brain fMRI by circumventing fat suppression while removing the lipid signal through a parallel acquisition approach","abstract":"Ultra-high-field functional magnetic resonance imaging (fMRI) offers the way to new insights while increasing the spatial and temporal resolution. However, a crucial concern in 7T human MRI is the increase in power deposition, supervised through the specific absorption rate (SAR). The SAR limitation can restrict the brain coverage or the minimal repetition time of fMRI experiments. fMRI is based on the well-known gradient-echo echo-planar imaging (GRE-EPI) sequence, which offers ultrafast acquisition. Commonly, the GRE-EPI sequence comprises two pulses: fat suppression and excitation. This work provides the means for a significant reduction in the SAR by circumventing the fat-suppression pulse. Without this fat-suppression, however, lipid signal can result in artifacts due to the chemical shift between the lipid and water signals. Our approach exploits a reconstruction similar to the simultaneous-multi-slice (SMS) method to separate the lipid and water images, thus avoiding undesired lipid artifacts in brain images. The lipid-water separation is based on the known spatial shift of the lipid signal, which can be detected by the multi-channel coils sensitivity profiles. Our study shows robust human imaging, offering greater flexibility to reduce the SAR, shorten the repetition time or increase the volume coverage with substantial benefit for brain functional studies.","version":"1.1","doi":"10.1101/2020.10.13.337691","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.14.339689","pub_date":"2020-10-14","title":"COVID19: Exploring uncommon epitopes for a stable immune response through MHC1 binding","abstract":"The COVID19 pandemic has resulted in 1,092,342 deaths as of 14th October 2020, indicating the urgent need for a vaccine. This study highlights novel protein sequences generated by shot gun sequencing protocols that could serve as potential antigens in the development of novel subunit vaccines and through a stringent inclusion criterion, we characterized these protein sequences and predicted their 3D structures. We found distinctly antigenic sequences from the SARS-CoV-2 that have led to identification of 4 proteins that demonstrate an advantageous binding with Human leukocyte antigen-1 molecules. Results show how previously unexplored proteins may serve as better candidates for subunit vaccine development due to their high stability and immunogenicity, reinforce by their HLA-1 binding propensities and low global binding energies. This study thus takes a unique approach towards furthering the development of vaccines by employing multiple consensus strategies involved in immuno-informatics technique.","version":"1.1","doi":"10.1101/2020.10.14.339689","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.321596","pub_date":"2020-10-13","title":"A small interfering RNA (siRNA) database for SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19) rapidly transformed into a global pandemic, for which a demand for developing antivirals capable of targeting the SARS-CoV-2 RNA genome and blocking the activity of its genes has emerged. In this work, we propose a database of SARS-CoV-2 targets for siRNA approaches, aiming to speed the design process by providing a broad set of possible targets and siRNA sequences. Beyond target sequences, it also displays more than 170 features, including thermodynamic information, base context, target genes and alignment information of sequences against the human genome, and diverse SARS-CoV-2 strains, to assess whether siRNAs targets bind or not off-target sequences. This dataset is available as a set of four tables in a single spreadsheet file, each table corresponding to sequences of 18, 19, 20, and 21 nucleotides length, respectively, aiming to meet the diversity of technology and expertise among labs around the world concerning siRNAs design of varied sizes, more specifically between 18 and 21nt length. We hope that this database helps to speed the development of new target antivirals for SARS-CoV-2, contributing to more rapid and effective responses to the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.09.30.321596","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337287","pub_date":"2020-10-13","title":"Sero-prevalence of anti-SARS-CoV-2 Antibodies in Addis Ababa, Ethiopia","abstract":"Anti-SARS-CoV-2 antibody tests are being increasingly used for sero-epidemiological purposes to provide better understanding of the extent of the infection in the community, and monitoring the progression of the COVID-19 epidemic. We conducted sero-prevalence study to estimate prior infection with with SARS-CoV-2 in Addis Ababa. A cross-sectional study was done from April 23 to 28, 2020 among 301 randomly selected residents of Addis Ababa; with no known history of contact with confirmed COVID-19 person. Interviews on socio demographic and behavioural risk factor followed by serological tests were performed for SARS-CoV-2 IgM, and IgG antibodies, using COVID-19 IgG/IgM Rapid Test Cassette. The test has sensitivity of 87\u00b79% and specificity of 100% for lgM; and a sensitivity of 97\u00b72% and specificity of 100% for IgG. RT-PCR test was also done on combined nasopharyngeal and oropharengeal swabs as an important public health consideration. The unadjusted antibody-based crude SARS-CoV-2 prevalence was 7\u00b76% and the adjusted true SARS-CoV-2 prevalence was estimated at 8\u00b78% (95% CI 5\u00b75%-11\u00b76%) for the study population. Higher sero-prevalence were observed for males (9.0%), age below 50 years (8.2%), students and unemployed (15.6%), those with primary education (12.1%), smokers (7.8%), alcohol consumers (8.6%), chatt-chewers (13.6%) and shish smokers (18.8%). Seroprevalence was not significantly associated neither with socio-demographic not behavioral characteristics. According to the findings, possibly more individuals had been infected in Addis Ababa than what was being detected and reported by RT-PCR test suggestive of community transmission. The use of serological test for epidemiological estimation of the extent of SARS-CoV-2 epidemic gives a more precise estimate of magnitude which would be used for further monitoring and surveillance of the magnitude of the SARS CoV-2 infection.","version":"1.1","doi":"10.1101/2020.10.13.337287","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199588","pub_date":"2020-10-13","title":"Cold sensitivity of the SARS-CoV-2 spike ectodomain","abstract":"The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible \u201cup\u201d or receptor-inaccessible \u201cdown\u201d conformations. Here, we report that the commonly used stabilized S ectodomain construct \u201c2P\u201d is sensitive to cold temperature, and that this cold sensitivity is resolved in a \u201cdown\u201d state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.","version":"1.3","doi":"10.1101/2020.07.12.199588","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.264077","pub_date":"2020-10-13","title":"Human ACE2 peptide mimics block SARS-CoV-2 Pulmonary Cells Infection","abstract":"In the light of the recent accumulated knowledge on SARS-CoV-2 and its mode of human cells invasion, the binding of viral spike glycoprotein to human Angiotensin Converting Enzyme 2 (hACE2) receptor plays a central role in cell entry. We designed a series of peptides mimicking the N-terminal helix of hACE2 protein which contains most of the contacting residues at the binding site and have a high helical folding propensity in aqueous solution. Our best peptide mimics bind to the virus spike protein with high affinity and are able to block SARS-CoV-2 human pulmonary cell infection with an inhibitory concentration (IC50) in the nanomolar range. These first in class blocking peptide mimics represent powerful tools that might be used in prophylactic and therapeutic approaches to fight the coronavirus disease 2019 (COVID-19).","version":"1.2","doi":"10.1101/2020.08.24.264077","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.336800","pub_date":"2020-10-13","title":"Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies","abstract":"Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus virus diseases 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2 infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). In order to understand the mechanism of these nAbs on neutralizing SARS-CoV-2 virus infections, we have performed cryo-EM analysis and here report cryo-EM structures of the ten most potent nAbs in their native full-length IgG or Fab forms bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBD in the \u201cup\u201d conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and their correlation with more potent neutralization and the shedding of S1 subunit.","version":"1.1","doi":"10.1101/2020.10.13.336800","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337774","pub_date":"2020-10-13","title":"Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells","abstract":"An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4-18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 Spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.10.13.337774","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.308676","pub_date":"2020-10-13","title":"A 3D Structural Interactome to Explore the Impact of Evolutionary Divergence, Population Variation, and Small-molecule Drugs on SARS-CoV-2-Human Protein-Protein Interactions","abstract":"The recent COVID-19 pandemic has sparked a global public health crisis. Vital to the development of informed treatments for this disease is a comprehensive understanding of the molecular interactions involved in disease pathology. One lens through which we can better understand this pathology is through the network of protein-protein interactions between its viral agent, SARS-CoV-2, and its human host. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV can be explained by rapid evolution along the interface between the Spike protein and its human receptor (ACE2) leading to increased binding affinity. Sequence divergences that modulate other protein-protein interactions may further explain differences in transmission and virulence in this novel coronavirus. To facilitate these comparisons, we combined homology-based structural modeling with the ECLAIR pipeline for interface prediction at residue resolution, and molecular docking with PyRosetta. This enabled us to compile a novel 3D structural interactome meta-analysis for the published interactome network between SARS-CoV-2 and human. This resource includes docked structures for all interactions with protein structures, enrichment analysis of variation along interfaces, predicted \u0394\u0394G between SARS-CoV and SARS-CoV-2 variants for each interaction, predicted impact of natural human population variation on binding affinity, and a further prioritized set of drug repurposing candidates predicted to overlap with protein interfaces\u2020. All predictions are available online\u2020 for easy access and are continually updated when new interactions are published. \u2020 Some sections of this pre-print have been redacted to comply with current bioRxiv policy restricting the dissemination of purely in silico results predicting potential therapies for SARS-CoV-2 that have not undergone thorough peer-review. The results section titled \u201cPrioritization of Candidate Inhibitors of SARS-CoV-2-Human Interactions Through Binding Site Comparison,\u201d Figure 4, Supplemental Table 9, and all links to our web resource have been removed. Blank headers left in place to preserve structure and item numbering. Our full manuscript will be published in an appropriate journal following peer-review.","version":"1.1","doi":"10.1101/2020.10.13.308676","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.331306","pub_date":"2020-10-13","title":"Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities","abstract":"The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world\u2019s capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were all engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and its Receptor Binding Domain (RBD) in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.","version":"1.1","doi":"10.1101/2020.10.13.331306","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337212","pub_date":"2020-10-13","title":"SARS-CoV-2 S protein ACE2 interaction reveals novel allosteric targets","abstract":"The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells through surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, we have mapped the S:ACE2 interface and uncovered long-range allosteric propagation of ACE2 binding to sites critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 \u00c5 away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~ 130 \u00c5 away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a mechanistic basis for S:ACE2 complex formation, critical for proteolytic processing and viral-host membrane fusion and highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as allosterically exposed cryptic hotspots for potential therapeutic development. SARS-CoV-2 spike protein binding to receptor ACE2 allosterically enhances furin proteolysis at distal S1/S2 cleavage sites","version":"1.1","doi":"10.1101/2020.10.13.337212","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.05.284224","pub_date":"2020-10-13","title":"Vapur: A Search Engine to Find Related Protein - Compound Pairs in COVID-19 Literature","abstract":"Coronavirus Disease of 2019 (COVID-19) created dire consequences globally and triggered an intense scientific effort from different domains. The resulting publications created a huge text collection in which finding the studies related to a biomolecule of interest is challenging for general purpose search engines because the publications are rich in domain specific terminology. Here, we present Vapur: an online COVID-19 search engine specifically designed to find related protein - chemical pairs. Vapur is empowered with a relation-oriented inverted index that is able to retrieve and group studies for a query biomolecule with respect to its related entities. The inverted index of Vapur is automatically created with a BioNLP pipeline and integrated with an online user interface. The online interface is designed for the smooth traversal of the current literature by domain researchers and is publicly available at https://tabilab.cmpe.boun.edu.tr/vapur/.","version":"1.2","doi":"10.1101/2020.09.05.284224","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.13.337303","pub_date":"2020-10-13","title":"Elucidating the viral and host factors enabling the cross-species transmission of primate lentiviruses from simians to humans","abstract":"The HIV-1 epidemic originated from a cross-species transmission of a primate lentivirus from chimpanzees to humans near the turn of the 18th century. Simian immunodeficiency viruses have been jumping between old world monkeys in West/Central Africa for thousands of years. So why did HIV-1 only emerge in the past century? This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 26 primate lentiviruses. Pairwise competitions of these primate lentiviruses revealed that SIVcpz had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for 37 million infections worldwide. In contrast the \u201cHIV-2 lineage\u201d (SIVsmm, SIVmac, SIVagm, and HIV-2) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the \u201cHIV-2 lineage\u201d strains, a restriction that was inversely correlated with replicative fitness. SIVcpz from the chimpanzee subspecies Pan troglodytes troglodytes (Ptt) was slightly more fit in human cells than the strains from Pt schweinfurthii (Pts). However, unlike all other primate lentiviruses (including the HIV-2 lineage), SIVcpz was nonpathogenic in human tonsillar tissue and did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in chimpanzees. Despite the close phylogenetic relationship between SIVcpz_Ptt and HIV-1, this epidemic was either caused by cross species transmission of a rare, undiscovered SIVcpz strain of higher virulence or higher virulence differentially evolved among HIV-1 subtypes during the human epidemic. Invasion of wild animal habitats by humans can have devastating consequences for the human population as evident by the HIV-1 and SARS-CoV-2 epidemics. With SARS-CoV-2, a recent zoonotic jump, likely from bats, will help to identify a coronavirus progenitor. In contrast, simian immunodeficiency virus (SIV) jumped into humans over 100 years ago from a possibly extinct sub-species of chimpanzees and/or extinct lineage of SIV. We examined replicative fitness and pathogenesis of 26 different primate lentiviruses in human and chimpanzee primary lymphoid cells from blood and within tonsils. SIV from a specific chimpanzee species and lowland gorillas were the most capable of infecting and replicating in human and chimp lymphoid cells but they did not result in the pathogenesis related to disease in humans. In contrast, SIV from other old world monkeys were pathogenic but could not replicate efficiently in human cells. We propose the main HIV-1 is derived from a distinct jump of a very rare SIV strain in chimps leading to AIDS pandemic.","version":"1.1","doi":"10.1101/2020.10.13.337303","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.335083","pub_date":"2020-10-12","title":"SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells","abstract":"Currently, the world is suffering from the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. So far, 30 million people have been infected with SARS-CoV-2, and nearly 1 million people have died because of COVID-19 worldwide, causing serious health, economical, and sociological problems. However, the mechanism of the effect of SARS-CoV-2 on human host cells has not been defined. The present study reports that the SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in lung vascular cells. The treatment of human pulmonary artery smooth muscle cells or human pulmonary artery endothelial cells with recombinant SARS-CoV-2 spike protein S1 subunit (Val16 \u2013 Gln690) at 10 ng/ml (0.13 nM) caused an activation of MEK phosphorylation. The activation kinetics was transient with a peak at 10 min. The recombinant protein that contains only the ACE2 receptor-binding domain of SARS-CoV-2 spike protein S1 subunit (Arg319 \u2013 Phe541), on the other hand, did not cause this activation. Consistent with the activation of cell growth signaling in lung vascular cells by SARS-CoV-2 spike protein, pulmonary vascular walls were found to be thickened in COVID-19 patients. Thus, SARS-CoV-2 spike protein-mediated cell growth signaling may participate in adverse cardiovascular/pulmonary outcomes, and this mechanism may provide new therapeutic targets to combat COVID-19.","version":"1.1","doi":"10.1101/2020.10.12.335083","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.10.334458","pub_date":"2020-10-12","title":"SARS-CoV-2 infects carotid arteries: implications for vascular disease and organ injury in COVID-19","abstract":"Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.10.10.334458","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.335919","pub_date":"2020-10-12","title":"Within-patient genetic diversity of SARS-CoV-2","abstract":"SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, is evolving into different genetic variants by accumulating mutations as it spreads globally. In addition to this diversity of consensus genomes across patients, RNA viruses can also display genetic diversity within individual hosts, and co-existing viral variants may affect disease progression and the success of medical interventions. To systematically examine the intra-patient genetic diversity of SARS-CoV-2, we processed a large cohort of 3939 publicly-available deeply sequenced genomes with specialised bioinformatics software, along with 749 recently sequenced samples from Switzerland. We found that the distribution of diversity across patients and across genomic loci is very unbalanced with a minority of hosts and positions accounting for much of the diversity. For example, the D614G variant in the Spike gene, which is present in the consensus sequences of 67.4% of patients, is also highly diverse within hosts, with 29.7% of the public cohort being affected by this coexistence and exhibiting different variants. We also investigated the impact of several technical and epidemiological parameters on genetic heterogeneity and found that age, which is known to be correlated with poor disease outcomes, is a significant predictor of viral genetic diversity. Since it arose in late 2019, the new coronavirus (SARS-CoV-2) behind the COVID-19 pandemic has mutated and evolved during its global spread. Individual patients may host different versions, or variants, of the virus, hallmarked by different mutations. We examine the diversity of genetic variants coexisting within patients across a cohort of 3939 publicly accessible samples and 749 recently sequenced samples from Switzerland. We find that a small number of patients carry most of the diversity, and that patients with more diversity tend to be older. We also find that most of the diversity is concentrated in certain regions and positions of the virus genome. In particular, we find that a variant reported to increase infectivity is among the most diverse positions. Our study provides a large-scale survey of within-patient diversity of the SARS-CoV-2 genome.","version":"1.1","doi":"10.1101/2020.10.12.335919","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.336016","pub_date":"2020-10-12","title":"Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein","abstract":"The human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.10.12.336016","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052019","pub_date":"2020-10-12","title":"Synonymous mutations and the molecular evolution of SARS-Cov-2 origins","abstract":"Human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is most closely related, by average genetic distance, to two coronaviruses isolated from bats, RaTG13 and RmYN02. However, there is a segment of high amino acid similarity between human SARS-CoV-2 and a pangolin isolated strain, GD410721, in the receptor binding domain (RBD) of the spike protein, a pattern that can be caused by either recombination or by convergent amino acid evolution driven by natural selection. We perform a detailed analysis of the synonymous divergence, which is less likely to be affected by selection than amino acid divergence, between human SARS-CoV-2 and related strains. We show that the synonymous divergence between the bat derived viruses and SARS-CoV-2 is larger than between GD410721 and SARS-CoV-2 in the RBD, providing strong additional support for the recombination hypothesis. However, the synonymous divergence between pangolin strain and SARS-CoV-2 is also relatively high, which is not consistent with a recent recombination between them, instead it suggests a recombination into RaTG13. We also find a 14-fold increase in the dN/dS ratio from the lineage leading to SARS-CoV-2 to the strains of the current pandemic, suggesting that the vast majority of non-synonymous mutations currently segregating within the human strains have a negative impact on viral fitness. Finally, we estimate that the time to the most recent common ancestor of SARS-CoV-2 and RaTG13 or RmYN02 based on synonymous divergence, is 51.71 years (95% C.I., 28.11-75.31) and 37.02 years (95% C.I., 18.19-55.85), respectively.","version":"1.3","doi":"10.1101/2020.04.20.052019","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.11.335299","pub_date":"2020-10-12","title":"D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction","abstract":"The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design. SARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability. The D614G mutation alters S protein conformational dynamics. D614G enhances protease cleavage susceptibility at the S protein furin cleavage site. Cryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics.","version":"1.1","doi":"10.1101/2020.10.11.335299","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.12.336339","pub_date":"2020-10-12","title":"Genomic Similarity of Nucleotides in SARS CoronaVirus using K-Means Unsupervised Learning Algorithm","abstract":"The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us a great opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb) among all known RNA viruses, with G + C contents varying from 32% to 43%. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C were known to exist but now a new genus D also exists,namely the Deltacoronavirus. In such a situation, it becomes highly important for efficient classification of all virus data so that it helps us in suitable planning,containment and treatment. The objective of this paper is to classify SARS corona-virus nucleotide sequences based on parameters such as sequence length,percentage similarity between the sequence information,open and closed gaps in the sequence due to multiple mutations and many others.By doing this,we will be able to predict accurately the similarity of SARS CoV-2 virus with respect to other corona-viruses like the Wuhan corona-virus,the bat corona-virus and the pneumonia virus and would help us better understand about the taxonomy of the corona-virus family. In addition to the guidelines provided in the abstract above,the following points summarizes the article below:\n\nThe article discusses an application of Machine Learning in the field of virology.\nIt aims to classify the SARS CoV2 virus as per the already known sequences of the bat-coronavirus, the Wuhan Sea Food Market pneumonia virus and the Wuhan coronavirus.\nTo solve and predict the similarity of the SARS CoV2 coronavirus w.r.t other viruses discussed above,K-Means Unsupervised Learning Algorithm has been chosen.\nThe data-set used is MN997409.1-4NY0T82X016-Alignment-HitTable.csv found on www.kaggle.com.(Complete link shared in the references section).[17]\nThe results have been validated by using a simple data-correlation technique namely Spearman\u2019s Rank Correlation Coeffecient.\nI have also discussed my future work using Deep Neural Nets that can help predict new virus sequences and effectively find similarity if any with already discovered viruses.\n\n The article discusses an application of Machine Learning in the field of virology. It aims to classify the SARS CoV2 virus as per the already known sequences of the bat-coronavirus, the Wuhan Sea Food Market pneumonia virus and the Wuhan coronavirus. To solve and predict the similarity of the SARS CoV2 coronavirus w.r.t other viruses discussed above,K-Means Unsupervised Learning Algorithm has been chosen. The data-set used is MN997409.1-4NY0T82X016-Alignment-HitTable.csv found on www.kaggle.com.(Complete link shared in the references section).[17] The results have been validated by using a simple data-correlation technique namely Spearman\u2019s Rank Correlation Coeffecient. I have also discussed my future work using Deep Neural Nets that can help predict new virus sequences and effectively find similarity if any with already discovered viruses.","version":"1.1","doi":"10.1101/2020.10.12.336339","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.11.335406","pub_date":"2020-10-12","title":"Unsupervised explainable AI for simultaneous molecular evolutionary study of forty thousand SARS-CoV-2 genomes","abstract":"Unsupervised AI (artificial intelligence) can obtain novel knowledge from big data without particular models or prior knowledge and is highly desirable for unveiling hidden features in big data. SARS-CoV-2 poses a serious threat to public health and one important issue in characterizing this fast-evolving virus is to elucidate various aspects of their genome sequence changes. We previously established unsupervised AI, a BLSOM (batch-learning SOM), which can analyze five million genomic sequences simultaneously. The present study applied the BLSOM to the oligonucleotide compositions of forty thousand SARS-CoV-2 genomes. While only the oligonucleotide composition was given, the obtained clusters of genomes corresponded primarily to known main clades and internal divisions in the main clades. Since the BLSOM is explainable AI, it reveals which features of the oligonucleotide composition are responsible for clade clustering. The BLSOM has powerful image display capabilities and enables efficient knowledge discovery about viral evolutionary processes.","version":"1.1","doi":"10.1101/2020.10.11.335406","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.10.331348","pub_date":"2020-10-11","title":"Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice","abstract":"The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.","version":"1.1","doi":"10.1101/2020.10.10.331348","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.20210245","pub_date":"2020-10-11","title":"Evaluating the Sensitivity of SARS-CoV-2 Infection Rates on College Campuses to Wastewater Surveillance","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>As college campuses reopen, we are in the midst of a large-scale experiment on the efficacy of various strategies to contain the SARS-CoV-2 virus. Traditional individual surveillance testing via nasal swabs and/or saliva is among the measures that colleges are pursuing to reduce the spread of the virus on campus. Additionally, some colleges are testing wastewater on their campuses for signs of infection, which can provide an early warning signal for campuses to locate COVID-positive individuals. However, a representation of wastewater surveillance has not yet been incorporated into epidemiological models for college campuses, nor has the efficacy of wastewater screening been evaluated relative to traditional individual surveillance testing, within the structure of these models. Here, we implement a new model component for wastewater surveillance within an established epidemiological model for college campuses. We use a hypothetical residential university to evaluate the efficacy of wastewater surveillance to maintain low infection rates. We find that wastewater sampling with a 1-day lag to initiate individual screening tests, plus completing the subsequent tests within a 4-day period can keep overall infections within 5% of the infection rates seen with traditional individual surveillance testing. Our results also indicate that wastewater surveillance can be an effective way to dramatically reduce the number of false positive cases by identifying subpopulations for surveillance testing where infectious individuals are more likely to be found. Through a Monte Carlo risk analysis, we find that surveillance testing that relies solely on wastewater sampling can be fragile against scenarios with high viral reproductive numbers and high rates of infection of campus community members by outside sources. These results point to the practical importance of additional surveillance measures to limit the spread of the virus on campus and the necessity of a proactive response to the initial signs of outbreak.</jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary</jats:title>\n                  <jats:p>College campuses have employed a variety of measures to keep their communities safe amid the SARS-CoV-2 pandemic. Many colleges are implementing surveillance testing programs wherein students are randomly selected to be tested for SARS-CoV-2. These strategies aim to manage the number of infections among the student population by isolating infected individuals. Some colleges are monitoring wastewater on their campuses for signs of the virus, which has been found to be capable of detecting viral RNA. If a wastewater sample shows signs of viral RNA, then screening tests are administered to the individuals who live or work in the buildings that contribute to the sewer in question. We present a model for such wastewater surveillance within a larger model for the spread of SARS-CoV-2 on a college campus. We show that wastewater surveillance can reduce the number of false positive cases and the associated disruptions to student life, while maintaining similar overall numbers of infections. However, we find that surveillance testing strategies that rely solely on wastewater sampling may be less effective if the local transmission rate of the virus is high, or if the rate of infection of members of the campus community by outside sources is high.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.09.20210245","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.08.20209510","pub_date":"2020-10-11","title":"Global projections of lives saved from COVID-19 with universal mask use","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>BACKGROUND</jats:title>\n                  <jats:p>Social distancing mandates (SDM) have reduced health impacts from COVID-19 but also resulted in economic downturns that have led many nations to relax SDM. Until deployment of an efficacious and equitable vaccine, intervention options to reduce COVID-19 mortality and minimize restrictive SDM are sought by society.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>A susceptible-exposed-infectious-recovered (SEIR) deterministic transmission model was parameterized with data on reported deaths, cases, and select covariates to predict infections and deaths from COVID-19 through March 01, 2021. We explore three scenarios: a \u201cnon-adaptive\u201d scenario where neither mask use or SDM adapt to changing conditions, a \u201creference\u201d where current national levels of mask use are maintained and SDM reintroduced when deaths rise, and an increase in mask use to 95% coverage levels (\u201cuniversal mask\u201d). We reviewed published studies to set priors on the magnitude of reduction in transmission through increasing mask use.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>Mask use was estimated at 59.0% of people globally on October 19, 2020. Universal mask use could avert 733,310 deaths (95% UI 385,981 to 1,107,759) between October 27, 2020 and March 01, 2021, the difference between the predicted 2.95 million deaths (95% UI 2.70 to 3.35) in the reference scenario and 2.22 million deaths (95% UI 2.00 to 2.45) in the universal mask scenario over this time period.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>The cumulative toll of the COVID-19 pandemic could be substantially reduced by the universal adoption of masks before the availability of a vaccine. This low-cost, low-barrier policy, whether customary or mandated, has enormous health benefits with presumed marginal economic costs.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.08.20209510","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.09.334052","pub_date":"2020-10-10","title":"Kidney injury molecule-1 is a potential receptor for SARS-CoV-2","abstract":"COVID-19 patients present high incidence of kidney abnormalities, which are associated with poor prognosis and high mortality. Identification of SARS-CoV-2 in kidney of COVID-19 patients suggests renal tropism and direct infection. Presently, it is generally recognized that SARS-CoV-2 initiates invasion through binding of receptor-binding domain (RBD) of spike protein to host cell-membrane receptor ACE2, however, whether there is additional target of SARS-CoV-2 in kidney remains unclear. Kidney injury molecule-1 (KIM1) is a transmembrane protein that drastically up-regulated after renal injury. Here, binding between SARS-CoV2-RBD and the extracellular Ig V domain of KIM1 was identified by molecular simulations and co-immunoprecipitation, which was comparable in affinity to that of ACE2 to SARS-CoV-2. Moreover, KIM1 facilitated cell entry of SARS-CoV2-RBD, which was potently blockaded by a rationally designed KIM1-derived polypeptide. Together, the findings suggest KIM1 may mediate and exacerbate SARS-CoV-2 infection in a \u2018vicious cycle\u2019, and KIM1 could be further explored as a therapeutic target.","version":"1.1","doi":"10.1101/2020.10.09.334052","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.334136","pub_date":"2020-10-10","title":"Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates","abstract":"The unprecedented and rapid spread of SARS-CoV-2 has motivated the need for a rapidly producible and scalable vaccine. Here, we developed a synthetic soluble SARS-CoV-2 spike (S) DNA-based vaccine candidate, GX-19. In mice, immunization with GX-19 elicited not only S-specific systemic and pulmonary antibody responses but also Th1-biased T cell responses in a dose-dependent manner. GX-19 vaccinated nonhuman primate seroconverted rapidly and exhibited detectable neutralizing antibody response as well as multifunctional CD4+ and CD8+ T cell responses. Notably, when the immunized nonhuman primates were challenged at 10 weeks after the last vaccination with GX-19, they did not develop fever and reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides durable protective immune response and also support further development of GX-19 as a vaccine candidate for SARS-CoV-2 in human clinical trials.","version":"1.1","doi":"10.1101/2020.10.09.334136","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.333948","pub_date":"2020-10-10","title":"The effect of salt on the dynamics of CoV-2 RBD at ACE2","abstract":"In this article, we investigate the effect of electrolytes on the stability of the complex between the coronavirus type 2 spike protein receptor domain (CoV-2 RBD) and ACE2, which plays an important role in the activation cascade at the viral entry of CoV-2 into human cells. At the cellular surface, electrolytes play an important role, especially in the interaction of proteins near the membrane surface. Additionally, the binding interface of the CoV-2 RBD - ACE2 complex is highly hydrophilic. We simulated the CoV-2 RBD - ACE2 complex at varying salt concentrations over the concentration range from 0.03 M to 0.3 M of calcium and sodium chloride over an individual simulation length of 750 ns in 9 independent simulations (6.75 \u00b5s total). We observe that the CoV-2 RBD - ACE2 complex is stabilized independent of the salt concentration. We identify a strong negative electrostatic potential at the N-terminal part of CoV-2 RBD and we find that CoV-2 RBD binds even stronger at higher salt concentrations. We observe that the dynamics of the N-terminal part of CoV-2 RBD stabilize the protein complex leading to strong collective motions and a stable interface between CoV-2 RBD and ACE2. We state that the sequence of CoV-2 RBD might be optimized for a strong binding to ACE2 at varying salt concentrations at the cellular surface, which acts as a key component in the activation of CoV-2 for its viral entry. A novel coronavirus, coronavirus type 2 (CoV-2), was identified as primary cause for a worldwide pandemic of the severe acute respiratory syndrome (SARS CoV-2). The CoV-2 spike protein is a major target for the development of a vaccine and potential strategies to inhibit the viral entry into human cells. At the cellular surface, CoV-2 activation involves the direct interaction between ACE2 and CoV-2 RBD. At the cellular surface, electrolytes play an important role, especially in the interaction of proteins near the membrane surface. We thus investigate the effect of ion conditions on the interaction of the CoV-2 RBD - ACE2 complex and find stabilizing effects. We speculate that CoV-2 RBD is optimized for strong binding to ACE2 at varying salt concentrations.","version":"1.1","doi":"10.1101/2020.10.09.333948","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.332452","pub_date":"2020-10-09","title":"Possible transmission flow of SARS-CoV-2 based on ACE2 features","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.","version":"1.1","doi":"10.1101/2020.10.08.332452","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.332734","pub_date":"2020-10-09","title":"Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic has raised an urgent need to develop effective therapeutics against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As a potential antiviral drug target, the nucleocapsid (N) protein of SARS-CoV-2 functions as a viral RNA chaperone and plays vital and multifunctional roles during the life cycle of coronavirus. In this study, we discovered that the N protein of SARS-CoV-2 undergoes liquid-liquid phase separation (LLPS) both in vitro and in vivo, which is further modulated by viral RNA. In addition, we found that, the core component of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, nsp12, preferentially partitions into the N protein condensates. Moreover, we revealed that, two small molecules, i.e., CVL218 and PJ34, can be used to intervene the N protein driven phase separation and loosen the compact structures of the condensates of the N-RNA-nsp12 complex of SARS-CoV-2. The discovery of the LLPS-mediated interplay between N protein and nsp12 and the corresponding modulating compounds illuminates a feasible way to improve the accessibility of antiviral drugs (e.g., remdesivir) to their targets (e.g., nsp12/RdRp), and thus may provide useful hints for further development of effective therapeutic strategies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.10.09.332734","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.333278","pub_date":"2020-10-09","title":"Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2","abstract":"Characterization of the humoral response to SARS-CoV-2, the etiological agent of Covid-19, is essential to help control the infection. In this regard, we and others recently reported that the neutralization activity of plasma from COVID-19 patients decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is still not well understood. In this study, we selected plasma from a cohort of Covid-19 convalescent patients and selectively depleted immunoglobulin A, M or G before testing the remaining neutralizing capacity of the depleted plasma. We found that depletion of immunoglobulin M was associated with the most substantial loss of virus neutralization, followed by immunoglobulin G. This observation may help design efficient antibody-based COVID-19 therapies and may also explain the increased susceptibility to SARS-CoV-2 of autoimmune patients receiving therapies that impair the production of IgM.","version":"1.1","doi":"10.1101/2020.10.09.333278","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.318972","pub_date":"2020-10-09","title":"LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection","abstract":"SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2020.09.30.318972","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.332544","pub_date":"2020-10-09","title":"Neutralizing antibodies from early cases of SARS-CoV-2 infection offer cross-protection against the SARS-CoV-2 D614G variant","abstract":"The emergence of a SARS-CoV-2 variant with a point mutation in the spike (S) protein, D614G, has taken precedence over the original Wuhan isolate by May 2020. With an increased infection and transmission rate, it is imperative to determine whether antibodies induced against the D614 isolate may cross-neutralize against the G614 variant. In this report, profiling of the anti-SARS-CoV-2 humoral immunity reveals similar neutralization profiles against both S protein variants, albeit waning neutralizing antibody capacity at the later phase of infection. These findings provide further insights towards the validity of current immune-based interventions. Random mutations in the viral genome is a naturally occurring event that may lead to enhanced viral fitness and immunological resistance, while heavily impacting the validity of licensed therapeutics. A single point mutation from aspartic acid (D) to glycine (G) at position 614 of the SARS-CoV-2 spike (S) protein, termed D614G, has garnered global attention due to the observed increase in transmissibility and infection rate. Given that a majority of the developing antibody-mediated therapies and serological assays are based on the S antigen of the original Wuhan reference sequence, it is crucial to determine if humoral immunity acquired from the original SARS-CoV-2 isolate is able to induce cross-detection and cross-protection against the novel prevailing D614G variant.","version":"1.1","doi":"10.1101/2020.10.08.332544","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.332692","pub_date":"2020-10-09","title":"Variant analysis of SARS-CoV-2 genomes in the Middle East","abstract":"Coronavirus (COVID-19) was introduced into society in late 2019 and has now reached over 26 million cases and 850,000 deaths. The Middle East has a death toll of \u223c50,000 and over 20,000 of these are in Iran, which has over 350,000 confirmed cases. We expect that Iranian cases caused outbreaks in the neighbouring countries and that variant mapping and phylogenetic analysis can be used to prove this. We also aim to analyse the variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to characterise the common genome variants and provide useful data in the global effort to prevent further spread of COVID-19. The approach uses bioinformatics approaches including multiple sequence alignment, variant calling and annotation and phylogenetic analysis to identify the genomic variants found in the region. The approach uses 122 samples from the 13 countries of the Middle East sourced from the Global Initiative on Sharing All Influenza Data (GISAID). We identified 2200 distinct genome variants including 129 downstream gene variants, 298 frame shift variants, 789 missense variants, 1 start lost, 13 start gained, 1 stop lost, 249 synonymous variants and 720 upstream gene variants. The most common, high impact variants were 10818delTinsG, 2772delCinsC, 14159delCinsC and 2789delAinsA. Variant alignment and phylogenetic tree generation indicates that samples from Iran likely introduced COVID-19 to the rest of the Middle East. The phylogenetic and variant analysis provides unique insight into mutation types in genomes. Initial introduction of COVID-19 was most likely due to Iranian transmission. Some countries show evidence of novel mutations and unique strains. Increased time in small populations is likely to contribute to more unique genomes. This study provides more in depth analysis of the variants affecting in the region than any other study. None","version":"1.1","doi":"10.1101/2020.10.09.332692","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.09.332858","pub_date":"2020-10-09","title":"SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets","abstract":"Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved.","version":"1.1","doi":"10.1101/2020.10.09.332858","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.094714","pub_date":"2020-10-09","title":"ACE2 interaction networks in COVID-19: a physiological framework for prediction of outcome in patients with cardiovascular risk factors","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (coronavirus disease 2019; COVID-19) is associated with adverse outcomes in patients with cardiovascular disease (CVD). The aim of the study was to characterize the interaction between SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) functional networks with a focus on CVD.; Using the network medicine approach and publicly available datasets, we investigated ACE2 tissue expression and described ACE2 interaction networks which could be affected by SARS-CoV-2 infection in the heart, lungs and nervous system. We compared them with changes in ACE-2 networks following SARS-CoV-2 infection by analyzing public data of stem cell-derived cardiomyocytes (hiPSC-CMs). This analysis was performed using the NERI algorithm, which integrates protein-protein interaction with co-expression networks. We also performed miRNA-target predictions to identify which ones regulate ACE2-related networks and could play a role in the COVID19 outcome. Finally, we performed enrichment analysis for identifying the main COVID-19 risk groups. We found similar ACE2 expression confidence levels in respiratory and cardiovascular systems, supporting that heart tissue is a potential target of SARS-CoV-2. Analysis of ACE2 interaction networks in infected hiPSC-CMs identified multiple hub genes with corrupted signalling which can be responsible for cardiovascular symptoms. The most affected genes were EGFR, FN1, TP53, HSP90AA1, and APP, while the most affected interactions were associated with MAST2 and CALM1. Enrichment analysis revealed multiple diseases associated with the interaction networks of ACE2, especially cancerous diseases, obesity, hypertensive disease, Alzheimer\u2019s disease, non-insulin-dependent diabetes mellitus, and congestive heart failure. Among affected ACE2-network components connected with SARS-Cov-2 interactome, we identified AGT, CAT, DPP4, CCL2, TFRC and CAV1, associated with cardiovascular risk factors. We described for the first time miRNAs which were common regulators of ACE2 networks and virus-related proteins in all analyzed datasets. The top miRNAs were miR-27a-3p, miR-26b-5p, miR-10b-5p, miR-302c-5p, hsa-miR-587, hsa-miR-1305, hsa-miR-200b-3p, hsa-miR-124-3p, and hsa-miR-16-5p.; Our study provides a complete mechanistic framework for investigating the ACE2 network which was validated by expression data. This framework predicted risk groups, including the established ones, thus providing reliable novel information regarding the complexity of signalling pathways affected by SARS-CoV-2. It also identified miR which could be used in personalized diagnosis in COVID-19.","version":"1.3","doi":"10.1101/2020.05.13.094714","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.330456","pub_date":"2020-10-09","title":"idCOV: a pipeline for quick clade identification of SARS-CoV-2 isolates","abstract":"idCOV is a phylogenetic pipeline for quickly identifying the clades of SARS-CoV-2 virus isolates from raw sequencing data based on a selected clade-defining marker list. Using a public dataset, we show that idCOV can make equivalent calls as annotated by Nextstrain.org on all three common clade systems using user uploaded FastQ files directly. Web and equivalent command-line interfaces are available. It can be deployed on any Linux environment, including personal computer, HPC and the cloud. The source code is available at https://github.com/xz-stjude/idcov. A documentation for installation can be found at https://github.com/xz-stjude/idcov/blob/master/README.md.","version":"1.1","doi":"10.1101/2020.10.08.330456","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.05.327197","pub_date":"2020-10-09","title":"A genetic variant protective against severe COVID-19 is inherited from Neandertals","abstract":"It was recently shown that the major genetic risk factor associated with becoming severely ill with COVID-19 when infected by SARS-CoV-2 is inherited from Neandertals. Thanks to new genetic association studies additional risk factors are now being discovered. Using data from a recent genome-wide associations from the Genetics of Mortality in Critical Care (GenOMICC) consortium, we show that a haplotype at a region associated with requiring intensive care is inherited from Neandertals. It encodes proteins that activate enzymes that are important during infections with RNA viruses. As compared to the previously described Neandertal risk haplotype, this Neandertal haplotype is protective against severe COVID-19, is of more moderate effect, and is found at substantial frequencies in all regions of the world outside Africa.","version":"1.3","doi":"10.1101/2020.10.05.327197","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255646","pub_date":"2020-10-09","title":"First report of antiviral activity of carbon nanofibers: Enhancement of the viral inhibition capacity of calcium alginate films","abstract":"The World Health Organization has called for new effective and affordable alternative antiviral materials for the prevention and treatment of viral infections. In this regard, calcium alginate has previously shown to possesses antiviral activity against the enveloped double-stranded DNA herpes simplex virus type 1. However, non-enveloped viruses are more resistant to inactivation than enveloped ones. Thus, the viral inhibition capacity of calcium alginate and the effect of adding a minuscule amount of carbon nanomaterials (0.1% w/w) have been explored here against a non-enveloped double-stranded DNA virus model for the first time. The results of this study showed that neat calcium alginate films are able to inactivate this type of non-enveloped virus and that including that extremely low percentage of carbon nanofibers significantly enhanced its viral inhibition from ~55.6% to 96.33%. This is the first published study to demonstrate CNFs\u2019 antiviral activity. However, adding this small percentage of graphene oxide did not improve the antiviral activity of calcium alginate, although both composite biomaterials possess antiviral and other outstanding properties very promising for biomedical applications.","version":"1.2","doi":"10.1101/2020.08.18.255646","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.326462","pub_date":"2020-10-08","title":"Genome-scale identification of SARS-CoV-2 and pan-coronavirus host factor networks","abstract":"The COVID-19 pandemic has claimed the lives of more than one million people worldwide. The causative agent, SARS-CoV-2, is a member of the Coronaviridae family, which are viruses that cause respiratory infections of varying severity. The cellular host factors and pathways co-opted by SARS-CoV-2 and other coronaviruses in the execution of their life cycles remain ill-defined. To develop an extensive compendium of host factors required for infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E), we performed parallel genome-scale CRISPR knockout screens. These screens uncovered multiple host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis, as well as an unexpected requirement for several poorly characterized proteins. We identified an absolute requirement for the VTT-domain containing protein TMEM41B for infection by SARS-CoV-2 and all other coronaviruses. This human Coronaviridae host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus spillover events. Genome-wide CRISPR screens for SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E coronavirus host factors. Parallel genome-wide CRISPR screening uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles. Coronaviruses co-opt multiple biological pathways, including glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis and anchoring, among others. TMEM41B - a poorly understood factor with roles in autophagy and lipid mobilization - is a critical pan-coronavirus host factor.","version":"1.1","doi":"10.1101/2020.10.07.326462","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.330688","pub_date":"2020-10-08","title":"CD8+ T cell responses in convalescent COVID-19 individuals target epitopes from the entire SARS-CoV-2 proteome and show kinetics of early differentiation","abstract":"Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.","version":"1.1","doi":"10.1101/2020.10.08.330688","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.331751","pub_date":"2020-10-08","title":"Preferential recognition and antagonism of SARS-CoV-2 spike glycoprotein binding to 3-O-sulfated heparan sulfate","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 is in immediate need of an effective antidote. Although the Spike glycoprotein (SgP) of SARS-CoV-2 has been shown to bind to heparins, the structural features of this interaction, the role of a plausible heparan sulfate proteoglycan (HSPG) receptor, and the antagonism of this pathway through small molecules remain unaddressed. Using an in vitro cellular assay, we demonstrate HSPGs modified by the 3-O-sulfotransferase isoform-3, but not isoform-5, preferentially increased SgP-mediated cell-to-cell fusion in comparison to control, unmodified, wild-type HSPGs. Computational studies support preferential recognition of the receptor-binding domain of SgP by 3-O-sulfated HS sequences. Competition with either fondaparinux, a 3-O-sulfated HS-binding oligopeptide, or a synthetic, non-sugar small molecule, blocked SgP-mediated cell-to-cell fusion. Finally, the synthetic, sulfated molecule inhibited fusion of GFP-tagged pseudo SARS-CoV-2 with human 293T cells with sub-micromolar potency. Overall, overexpression of 3-O-sulfated HSPGs contribute to fusion of SARS-CoV-2, which could be effectively antagonized by a synthetic, small molecule.","version":"1.1","doi":"10.1101/2020.10.08.331751","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.331157","pub_date":"2020-10-08","title":"Angiotensin-converting enzyme 2 (ACE2) is upregulated in Alzheimer\u2019s disease brain","abstract":"Alzheimer\u2019s disease is a chronic neurodegenerative disorder and represents the main cause of dementia. Currently, the world is suffering from the pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. In COVID-19, neurological manifestations have been reported to occur. The present study demonstrates that the protein expression level of ACE2 is upregulated in the brain of Alzheimer\u2019s disease patients. The increased ACE2 expression is not age-dependent, suggesting the direct relationship between Alzheimer\u2019s disease and the ACE2 expression. Oxidative stress has been implicated in the pathogenesis of Alzheimer\u2019s disease, and Alzheimer\u2019s disease brains examined in this study also exhibited higher carbonylated proteins as well as increased thiol oxidation state of peroxiredoxin 6 (Prx6). The positive correlation was found between the increased ACE2 protein expression and oxidative stress in Alzheimer\u2019s disease brain. Thus, the present study reveals the relationships between Alzheimer\u2019s disease and ACE2, the receptor for SARS-CoV-2. These results warrant monitoring Alzheimer\u2019s disease patients with COVID-19 carefully for the possible higher viral load in the brain and long-term adverse neurological consequences.","version":"1.1","doi":"10.1101/2020.10.08.331157","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.08.331421","pub_date":"2020-10-08","title":"Psychiatric Genomics Research During the COVID-19 Pandemic: A Survey of Psychiatric Genomics Consortium Researchers","abstract":"Between April 20, 2020 and June 19, 2020 we conducted a survey of the membership of the Psychiatric Genomics Consortium (PGC) to explore the impact of COVID-19 on their research and academic careers. A total of 123 individuals responded representing academic ranks from trainee to full professor, tenured and fixed-term appointments, and all genders. The survey included both quantitative and free text responses. Results revealed considerable concern about the impact of COVID-19 on research with the greatest concern reported by individuals in non-permanent positions and female researchers. Concerns about the availability of funding and the impact of the pandemic on career progression were commonly reported by early career researchers. We provide recommendations for institutions, organizations such as the PGC, as well as individual senior investigators to ensure that the futures of early career investigators, especially those underrepresented in academic medicine such as women and underrepresented minorities, are not disproportionately disadvantaged by the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.10.08.331421","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.077826","pub_date":"2020-10-07","title":"Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2\u2013Priming Protease TMPRSS2","abstract":"Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. The main cellular location where the SARS-CoV-2 S protein priming occurs remains debatable, therefore hampering the development of targeted treatments. Herein, we identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease\u2013inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Our data support the key role of extracellular serine proteases in SARS-CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells. Delivery of extracellular serine protease inhibitors (serpins) such as A1AT has the capacity to reduce SARS-CoV-2 dissemination by binding and inhibiting extracellular proteases on the host cells, thus, inhibiting the first step in SARS-CoV-2 cell cycle (i.e. cell entry).","version":"1.2","doi":"10.1101/2020.05.04.077826","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.19.211110","pub_date":"2020-10-07","title":"The short- and long-range RNA-RNA Interactome of SARS-CoV-2","abstract":"The Coronaviridae is a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome and subgenomes adopt alternative topologies inside cells, and engage in different interactions with host RNAs. Notably, we discovered a long-range RNA-RNA interaction - the FSE-arch - that encircles the programmed ribosomal frameshifting element. The FSE-arch is conserved in the related MERS-CoV and is under purifying selection. Our findings illuminate RNA structure based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies.","version":"1.2","doi":"10.1101/2020.07.19.211110","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.329771","pub_date":"2020-10-07","title":"SARS-CoV-2 has observably higher propensity to accept uracil as nucleotide substitution: Prevalence of amino acid substitutions and their predicted functional implications in circulating SARS-CoV-2 in India up to July, 2020","abstract":"SARS-CoV-2 has emerged as pandemic all over the world since late 2019. In this study, we investigated the diversity of the virus in the context of SARS-CoV-2 spread in India. Full-length SARS-CoV-2 genome sequences of the circulating viruses from all over India were collected from GISAID, an open data repository, until 25thJuly, 2020. We have focused on the non-synonymous changes across the genome that resulted in amino acid substitutions. Analysis of the genomic signatures of the non-synonymous mutations demonstrated a strong association between the time of sample collection and the accumulation of genetic diversity. Most of these isolates from India belonged to the A2a clade (63.4%) which has overcome the selective pressure and is spreading rapidly across several continents. Interestingly a new clade I/A3i has emerged as the second-highest prevalent type among the Indian isolates, comprising 25.5% of the Indian sequences. Emergence of new mutations in the S protein was observed. Major SARS-CoV-2 clades in India have defining mutations in the RdRp. Maximum accumulation of mutations was observed in ORF1a. Other than the clade-defining mutations, few representative non-synonymous mutations were checked against the available crystal structures of the SARS-CoV-2 proteins in the DynaMut server to assess their thermodynamic stability. We have observed that SARS-CoV-2 genomes contain more uracil than any other nucleotide. Furthermore, substitution of nucleotides to uracil was highest among the non-synonymous mutations observed. The A+U content in SARS-CoV-2 genome is much higher compared to other RNA viruses, suggesting that the virus RdRp has a propensity towards uracil incorporation in the genome. This implies that thymidine analogues may have a better chance to competitively inhibit SARS-CoV-2 RNA replication than other nucleotide analogues.","version":"1.1","doi":"10.1101/2020.10.07.329771","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.328328","pub_date":"2020-10-07","title":"MAJORA: Continuous integration supporting decentralised sequencing for SARS-CoV-2 genomic surveillance","abstract":"Genomic epidemiology has become an increasingly common tool for epidemic response. Recent technological advances have made it possible to sequence genomes rapidly enough to inform outbreak response, and cheaply enough to justify dense sampling of even large epidemics. With increased availability of sequencing it is possible for agile networks of sequencing facilities to collaborate on the sequencing and analysis of epidemic genomic data. In response to the ongoing SARS-CoV-2 pandemic in the United Kingdom, the COVID-19 Genomics UK (COG-UK) consortium was formed with the aim of rapidly sequencing SARS-CoV-2 genomes as part of a national-scale genomic surveillance strategy. The network consists of universities, academic institutes, regional sequencing centres and the four UK Public Health Agencies. We describe the development and deployment of Majora, an encompassing digital infrastructure to address the challenge of collecting and integrating both genomic sequencing data and sample-associated metadata produced across the COG-UK network. The system was designed and implemented pragmatically to stand up capacity rapidly in a pandemic caused by a novel virus. This approach has underpinned the success of COG-UK, which has rapidly become the leading contributor of SARS-CoV-2 genomes to international databases and has generated over 60,000 sequences to date.","version":"1.1","doi":"10.1101/2020.10.06.328328","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.175430","pub_date":"2020-10-07","title":"SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome","abstract":"SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of \u2018cryptic\u2019 epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 \u2018cryptic\u2019 pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.","version":"1.3","doi":"10.1101/2020.06.27.175430","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.330324","pub_date":"2020-10-07","title":"Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2","abstract":"Coronaviruses, like SARS-CoV-2, encode a nucleotidyl transferase in the N-terminal NiRAN domain of the non-structural protein (nsp) 12 protein within the RNA dependent RNA polymerase (RdRP) . Though the substrate targets of the viral nucleotidyl transferase are unknown, NiRAN active sites are highly conserved and essential for viral replication . We show, for the first time, the detection and sequence location of GMP-modified amino acids in nidovirus RdRP-associated proteins using heavy isotope-assisted MS and MS/MS peptide sequencing. We identified lys-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of nucleotidylation in vitro that uses a phosphoramide bond to covalently attach with GMP. In SARS-CoV-2 replicase proteins, we demonstrate a unique O-linked GMP attachment on nsp7 ser-1, whose formation required the presence of nsp12. It is clear that additional nucleotidylation sites remain undiscovered, which includes the possibility that nsp12 itself may form a transient GMP adduct in the NiRAN active site that has eluted detection in these initial studies due to instability of the covalent attachment. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.","version":"1.1","doi":"10.1101/2020.10.07.330324","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.152587","pub_date":"2020-10-07","title":"Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera","abstract":"Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents numerous questions and challenges that demand immediate attention. Among these is the urgent need for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. For this, sensitive, specific and quantitative serological assays are required. Here we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay which complements the portfolio of SARS-CoV-2 serological assays. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.","version":"1.3","doi":"10.1101/2020.06.15.152587","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255570","pub_date":"2020-10-07","title":"Spike protein mutational landscape in India: Could Muller\u2019s ratchet be a future game-changer for COVID-19?","abstract":"The dire need of effective preventive measures and treatment approaches against SARS-CoV-2 virus, causing COVID-19 pandemic, calls for an in-depth understanding of its evolutionary dynamics with attention to specific geographic locations, since lockdown and social distancing to prevent the virus spread could lead to distinct localized dynamics of virus evolution within and between countries owing to different environmental and host-specific selection pressures. To decipher any correlation between SARS-CoV-2 evolution and its epidemiology in India, we studied the mutational diversity of spike glycoprotein, the key player for the attachment, fusion and entry of virus to the host cell. For this, we analyzed the sequences of 630 Indian isolates as available in GISAID database till June 07, 2020, and detected the spike protein variants to emerge from two major ancestors \u2013 Wuhan-Hu-1/2019 and its D614G variant. Average stability of the docked spike protein \u2013 host receptor (S-R) complexes for these variants correlated strongly (R2=0.96) with the fatality rates across Indian states. However, while more than half of the variants were found unique to India, 67% of all variants showed lower stability of S-R complex than the respective ancestral variants, indicating a possible fitness loss in recently emerged variants, despite a continuous increase in mutation rate. These results conform to the sharply declining fatality rate countrywide (>7-fold during April 11 \u2013 June 28, 2020). Altogether, while we propose the potential of S-R complex stability to track disease severity, we urge an immediate need to explore if SARS-CoV-2 is approaching mutational meltdown in India. Epidemiological features are intricately linked to evolutionary diversity of rapidly evolving pathogens, and SARS-CoV-2 is no exception. Our work suggests the potential of average stability of complexes formed by the circulating spike mutational variants and the human host receptor to track the severity of SARS-CoV-2 infection in a given region. In India, the stability of these complexes for recent variants tend to decrease relative to their ancestral ones, following countrywide declining fatality rate, in contrast to an increasing mutation rate. We hypothesize such a scenario as nascent footprints of Muller\u2019s ratchet, proposing large-scale population genomics study for its validation, since this understanding could lead to therapeutic approaches for facilitating mutational meltdown of SARS-CoV-2, as experienced earlier for influenza A virus.","version":"1.2","doi":"10.1101/2020.08.18.255570","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.328302","pub_date":"2020-10-07","title":"Extremely potent human monoclonal antibodies from convalescent Covid-19 patients","abstract":"Human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2. Extremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.","version":"1.1","doi":"10.1101/2020.10.07.328302","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.07.329748","pub_date":"2020-10-07","title":"Umbilical cord blood derived microglia-like cells to model COVID-19 exposure","abstract":"Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.","version":"1.1","doi":"10.1101/2020.10.07.329748","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.310490","pub_date":"2020-10-07","title":"Broad-Spectrum, Patient-Adaptable Inhaled Niclosamide-Lysozyme Particles are Efficacious Against Coronaviruses in Lethal Murine Infection Models","abstract":"Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, the oral formulation produces systemic drug levels that are too low to inhibit SARS-CoV-2. As an alternative, direct delivery of NIC to the respiratory tract as an aerosol could target the primary site of for SARS-CoV-2 acquisition and spread. We have developed a niclosamide powder suitable for delivery via dry powder inhaler, nebulizer, and nasal spray through the incorporation of human lysozyme (hLYS) as a carrier molecule. This novel formulation exhibits potent in vitro and in vivo activity against MERS-CoV and SARS-CoV-2 and may protect against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to CoV infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure wide-spread utilization","version":"1.2","doi":"10.1101/2020.09.24.310490","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.320762","pub_date":"2020-10-07","title":"Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes","abstract":"Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.","version":"1.2","doi":"10.1101/2020.09.30.320762","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.04.325266","pub_date":"2020-10-07","title":"Reference ontology and database annotation of the COVID-19 Open Research Dataset (CORD-19)","abstract":"The COVID-19 Open Research Dataset (CORD-19) was released in March 2020 to allow the machine learning and wider research community to develop techniques to answer scientific questions on COVID-19. The dataset consists of a large collection of scientific literature, including over 100,000 full text papers. Annotating training data to normalise variability in biological entities can improve the performance of downstream analysis and interpretation. To facilitate and enhance the use of the CORD-19 data in these applications, in late March 2020 we performed a comprehensive annotation process using named entity recognition tool, TERMite, along with a number of large reference ontologies and vocabularies including domains of genes, proteins, drugs and virus strains. The additional annotation has identified and tagged over 45 million entities within the corpus made up of 62,746 unique biomedical entities. The latest updated version of the annotated data, as well as older versions, is made openly available under GPL-2.0 License for the community to use at: https://github.com/SciBiteLabs/CORD19","version":"1.2","doi":"10.1101/2020.10.04.325266","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.323634","pub_date":"2020-10-06","title":"Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors","abstract":"The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.","version":"1.1","doi":"10.1101/2020.10.06.323634","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.327635","pub_date":"2020-10-06","title":"GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition","abstract":"Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.10.06.327635","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.328369","pub_date":"2020-10-06","title":"SARS-CoV-2 infection damages airway motile cilia and impairs mucociliary clearance","abstract":"Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.","version":"1.1","doi":"10.1101/2020.10.06.328369","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.252437","pub_date":"2020-10-06","title":"Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment immunogen","abstract":"Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100\u00b0C when lyophilized, upto 70\u00b0C in solution and stable for over four weeks at 37\u00b0C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax\u2122, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.","version":"1.2","doi":"10.1101/2020.08.15.252437","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.327445","pub_date":"2020-10-06","title":"Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. \u00b7 ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species \u00b7 Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways \u00b7 Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function \u00b7 Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection","version":"1.1","doi":"10.1101/2020.10.06.327445","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.328336","pub_date":"2020-10-06","title":"Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19","abstract":"The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 \u00c5 cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site, therefore inhibiting the viral RNA replication. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19. Discovery and mechanism of suramin as potent SARS-CoV-2 RNA polymerase inhibitor and its repurposing for treating COVID-19.","version":"1.1","doi":"10.1101/2020.10.06.328336","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.327742","pub_date":"2020-10-06","title":"Cytoplasmic short linear motifs in ACE2 and integrin \u03b23 link SARS-CoV-2 host cell receptors to endocytosis and autophagy","abstract":"The spike protein of the SARS-CoV-2 interacts with angiotensin converting enzyme 2 (ACE2) and enters the host cell by receptor-mediated endocytosis. Concomitantly, evidence is pointing to the involvement of additional host cell receptors, such as integrins. The cytoplasmic tails of ACE2 and integrin \u03b23 contain a plethora of predicted binding motifs. Here, we confirm the functionality of some of these motifs through affinity measurements. The class I PDZ binding motif in the ACE2 cytoplasmic tail binds the first PDZ domain of the scaffold protein NHERF3. The clathrin-adaptor subunit AP2 \u03bc2 interacts with an endocytic motif in the ACE2 with low affinity and the interaction is abolished by phosphorylation of Tyr781. Furthermore, the C-terminal region of integrin b3 contains a LC3-interacting region, and its interaction with ATG8 domains is enhanced by phosphorylation. Together, our data provides possible molecular links between host cell receptors and endocytosis and autophagy. Affinity measurements confirmed binding of short linear motifs in the cytoplasmic tails of ACE2 and integrin \u03b23, thereby linking the receptors to endocytosis and autophagy.","version":"1.1","doi":"10.1101/2020.10.06.327742","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.06.328138","pub_date":"2020-10-06","title":"The strength of a NES motif in the nucleocapsid protein of human coronaviruses is related to genus, but not to pathogenic capacity","abstract":"Seven members of the Coronaviridae family infect humans, but only three (SARS-CoV, SARS-CoV-2 and MERS-CoV) cause severe disease with a high case fatality rate. Using in silico analyses (machine learning techniques and comparative genomics), several features associated to coronavirus pathogenicity have been recently proposed, including a potential increase in the strength of a predicted novel nuclear export signal (NES) motif in the nucleocapsid protein. Here, we have used a well-established nuclear export assay to experimentally establish whether the recently proposed nucleocapsid NESs are capable of mediating nuclear export, and to evaluate if their activity correlates with coronavirus pathogenicity. The six NES motifs tested were functional in our assay, but displayed wide differences in export activity. Importantly, these differences in NES strength were not related to strain pathogenicity. Rather, we found that the NESs of the strains belonging to the genus Alphacoronavirus were markedly stronger than the NESs of the strains belonging to the genus Betacoronavirus. We conclude that, while some of the genomic features recently identified in silico could be crucial contributors to coronavirus pathogenicity, this does not appear to be the case of nucleocapsid NES activity, as it is more closely related to coronavirus genus than to pathogenic capacity.","version":"1.1","doi":"10.1101/2020.10.06.328138","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.047480","pub_date":"2020-10-06","title":"Coronavirus infection and PARP expression dysregulate the NAD Metabolome: an actionable component of innate immunity","abstract":"Poly-ADP-ribose polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using nicotinamide adenine dinucleotide (NAD) as the source of ADPR. While the well-known poly-ADP-ribosylating (PARylating) PARPs primarily function in the DNA damage response, many non-canonical mono-ADP-ribosylating (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust upregulation of several PARPs following infection with Murine Hepatitis Virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly upregulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while downregulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM and NR dramatically decrease the replication of an MHV virus that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD, and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.","version":"1.6","doi":"10.1101/2020.04.17.047480","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.05.327528","pub_date":"2020-10-06","title":"Sub-second heat inactivation of coronavirus","abstract":"Heat treatment denatures viral proteins that comprise the virion, making virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature of as low as 75 \u00b0C and treatment duration of 15 min can effectively inactivate CoV. The applicability of a CoV heat inactivation method greatly depends on the length of time of a heat treatment and the temperature needed to inactivate the virus. With the goal of finding conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple system that can measure sub-second heat inactivation of CoV. The system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can be flowed at various speeds. Flowing virus solution at different speeds, along with a real-time temperature monitoring system, allows the virus to be accurately exposed to a desired temperature for various durations of time. Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 85.2 \u00b0C for 0.48 s exposure is sufficient to obtain > 5 Log10 reduction in viral titer (starting titer: 5 \u00d7 107 PFU/mL), and that when exposed to 83.4 \u00b0C for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log10 reduction). Three coronaviruses (CoVs) have now caused global outbreaks within the past 20 years, with the COVID19 pandemic caused by SARS-CoV-2 still ongoing. Methods that can rapidly inactivate viruses, especially CoVs, can play critical roles in ensuring public safety and safeguarding personal health. Heat treatment of viruses to inactive them can be an efficient and inexpensive method, with the potential to be incorporated into various human-occupied spaces. In this work, a simple system that can heat-treat viruses for extremely short period was developed and utilized to show that sub-second exposure of CoV to heat is sufficient to inactivate CoV. This opens up the possibility of developing instruments and methods of disinfecting CoV in diverse settings, including rapid liquid disinfection and airborne virus disinfection. The developed method can also be broadly utilized to assess heat sensitivity of viruses other viral pathogens of interest and develop sub-second rapid heat inactivation approaches.","version":"1.1","doi":"10.1101/2020.10.05.327528","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.05.326637","pub_date":"2020-10-05","title":"In vitro efficacy of Artemisinin-based treatments against SARS-CoV-2","abstract":"Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Proof-of-concept for prophylactic efficacy of the extracts was obtained using a plaque-reduction assay in VeroE6 cells. Subsequent concentration-response studies using a high-throughput antiviral assay, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that pretreatment and treatment with extracts, artemisinin, and artesunate inhibited SARS-CoV-2 infection of VeroE6 cells. In treatment assays, artesunate (50% effective concentration (EC50): 7 \u03bcg/mL) was more potent than the tested plant extracts (128-260 \u03bcg/mL) or artemisinin (151 \u03bcg/mL) and artemether (>179 \u03bcg/mL), while generally EC50 in pretreatment assays were slightly higher. The selectivity index (SI), calculated based on treatment and cell viability assays, was highest for artemisinin (54), and roughly equal for the extracts (5-10), artesunate (6) and artemether (<7). Similar results were obtained in human hepatoma Huh7.5 cells. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.","version":"1.1","doi":"10.1101/2020.10.05.326637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.20205708","pub_date":"2020-10-05","title":"Antigen-based testing but not real-time PCR correlates with SARS-CoV-2 virus culture","abstract":"<jats:title>SUMMARY</jats:title>\n                <jats:p>\n                  Individuals can test positive for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR) after no longer being infectious.\n                  <jats:sup>1-8</jats:sup>\n                  Positive SARS-CoV-2 antigen-based testing exhibits a temporal pattern that corresponds with active, replicating virus and could therefore be a more accurate predictor of an individual\u2019s potential to transmit SARS-CoV-2.\n                  <jats:sup>2,3,9</jats:sup>\n                  Using the BD Veritor System for Rapid Detection of SARS-CoV-2 later flow antigen detection test, we demonstrate a higher concordance of antigen-positive test results with the presence of cultured, infectious virus when compared to RT-PCR. When compared to infectious virus isolation, the sensitivity of antigen-based testing is similar to RT-PCR. The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. Coupled with a rapid time-to-result, low cost, and scalability, antigen-based testing should facilitate effective implementation of testing and public health interventions that will better contain COVID-19.\n                </jats:p>","version":null,"doi":"10.1101/2020.10.02.20205708","journal":"medRxiv","score":null},{"id":"10.1101/2020.10.04.325662","pub_date":"2020-10-05","title":"Motif Analysis in k-mer Networks: An Approach towards Understanding SARS-CoV-2 Geographical Shifts","abstract":"With an increasing number of SARS-CoV-2 sequences available day by day, new genomic information is getting revealed to us. As SARS-CoV-2 sequences highlight wide changes across the samples, we aim to explore whether these changes reveal the geographical origin of the corresponding samples. The k-mer distributions, denoting normalized frequency counts of all possible combinations of nucleotide of size upto k, are often helpful to explore sequence level patterns. Given the SARS-CoV-2 sequences are highly imbalanced by its geographical origin (relatively with a higher number samples collected from the USA), we observe that with proper under-sampling k-mer distributions in the SARS-CoV-2 sequences predict its geographical origin with more than 90% accuracy. The experiments are performed on the samples collected from six countries with maximum number of sequences available till July 07, 2020. This comprises SARS-CoV-2 sequences from Australia, USA, China, India, Greece and France. Moreover, we demonstrate that the changes of genomic sequences characterize the continents as a whole. We also highlight that the network motifs present in the sequence similarity networks have a significant difference across the said countries. This, as a whole, is capable of predicting the geographical shift of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.10.04.325662","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.04.325316","pub_date":"2020-10-04","title":"Efficient culture of SARS-CoV-2 in human hepatoma cells enhances viability of the virus in human lung cancer cell lines permitting the screening of antiviral compounds","abstract":"Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log10 increase in infectivity titers (TCID50) in Huh7.5 cells, with titers of ~8 Log10TCID50/mL, and >2-Log10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log10TCID50/mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log10TCID50/mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity. The cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.","version":"1.1","doi":"10.1101/2020.10.04.325316","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.04.325423","pub_date":"2020-10-04","title":"GABA administration prevents severe illness and death following coronavirus infection in mice","abstract":"There is an urgent need for new treatments to prevent and ameliorate severe illness and death induced by SARS-CoV-2 infection in COVID-19 patients. The coronavirus mouse hepatitis virus (MHV)-1 causes pneumonitis in mice which shares many pathological characteristics with human SARS-CoV infection. Previous studies have shown that the amino acid gamma-aminobutyric acid (GABA) has anti-inflammatory effects. We tested whether oral treatment with GABA could modulate the MHV-1 induced pneumonitis in susceptible A/J mice. As expected, MHV-1-inoculated control mice became severely ill (as measured by weight loss, clinical score, and the ratio of lung weight to body weight) and >60% of them succumbed to the infection. In contrast, mice that received GABA immediately after MHV-1 inoculation became only mildly ill and all of them recovered. When GABA treatment was initiated after the appearance of illness (3 days post-MHV-1 infection), we again observed that GABA treatment significantly reduced the severity of illness and greatly increased the frequency of recovery. Therefore, the engagement of GABA receptors (GABA-Rs) prevented the MHV-1 infection-induced severe pneumonitis and death in mice. Given that GABA-R agonists, like GABA and homotaurine, are safe for human consumption, stable, inexpensive, and available worldwide, they are promising candidates to help prevent severe illness stemming from SARS-CoV-2 infection and other coronavirus strains.","version":"1.1","doi":"10.1101/2020.10.04.325423","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.01.20204073","pub_date":"2020-10-04","title":"COVID-19 Classification of X-ray Images Using Deep Neural Networks","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>In the midst of the coronavirus disease 2019 (COVID-19) outbreak, chest X-ray (CXR) imaging is playing an important role in diagnosis and monitoring of patients with COVID-19. Machine learning solutions have been shown to be useful for X-ray analysis and classification in a range of medical contexts. In this study, we propose a machine learning model for detection of patients tested positive for COVID-19 from CXRs that were collected from inpatients hospitalized in four different hospitals. We additionally present a tool for retrieving similar patients according to the model\u2019s results on their CXRs.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>In this retrospective study, 1384 frontal CXRs, of COVID-19 confirmed patients imaged between March-August 2020, and 1024 matching CXRs of non-COVID patients imaged before the pandemic, were collected and used to build a deep learning classifier for detecting patients positive for COVID-19. The classifier consists of an ensemble of pre-trained deep neural networks (DNNS), specifically, ReNet34, ReNet50, ReNet152, vgg16, and is enhanced by data augmentation and lung segmentation. We further implemented a nearest-neighbors algorithm that uses DNN-based image embeddings to retrieve the images most similar to a given image.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Our model achieved accuracy of 90.3%, (95%CI: 86.3%-93.7%) specificity of 90% (95%CI: 84.3%-94%), and sensitivity of 90.5% (95%CI: 85%-94%) on a test dataset comprising 15% (350/2326) of the original images. The AUC of the ROC curve is 0.96 (95%CI: 0.93-0.97).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>We provide deep learning models, trained and evaluated on CXRs that can assist medical efforts and reduce medical staff workload in handling COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Key Points</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>A machine learning model was able to detect chest X-ray (CXR) images of patients tested positive for COVID-19 with accuracy and detection rate above 90%.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>A tool was created for finding existing CXR images with imaging characteristics most similar to a given CXR, according to the model\u2019s image embeddings.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.01.20204073","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.16.151555","pub_date":"2020-10-03","title":"Identifying persistent structures in multiscale \u2018omics data","abstract":"In any \u2018omics study, the scale of analysis can dramatically affect the outcome. For instance, when clustering single-cell transcriptomes, is the analysis tuned to discover broad or specific cell types? Likewise, protein communities revealed from protein networks can vary widely in sizes depending on the method. Here we use the concept of \u201cpersistent homology\u201d, drawn from mathematical topology, to identify robust structures in data at all scales simultaneously. Application to mouse single-cell transcriptomes significantly expands the catalog of identified cell types, while analysis of SARS-COV-2 protein interactions suggests hijacking of WNT. The method, HiDeF, is available via Python and Cytoscape.","version":"1.3","doi":"10.1101/2020.06.16.151555","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.324228","pub_date":"2020-10-02","title":"SARS-CoV-2 proteins and anti-COVID-19 drugs induce lytic reactivation of an oncogenic virus","abstract":"An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of Coronavirus Disease-2019 (COVID-19), a respiratory disease, has infected over 34,000,000 people since the end of 2019, killed over 1,000,000, and caused worldwide social and economic disruption. Due to the mechanisms of SARS-CoV-2 infection to host cells and its pathogenesis remain largely unclear, there are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Besides severe respiratory and systematic symptoms, several comorbidities may also increase risk of fatal disease outcome. Therefore, it is required to investigate the impacts of COVID-19 on pre-existing diseases of patients, such as cancer and other infectious diseases. In the current study, we have reported that SARS-CoV-2 encoded proteins and some anti-COVID-19 drugs currently used are able to induce lytic reactivation of Kaposi\u2019s sarcoma-associated herpesvirus (KSHV), one of major human oncogenic viruses through manipulation of intracellular signaling pathways. Our data indicate that those KSHV+ patients especially in endemic areas exposure to COVID-19 or undergoing the treatment may have increased risks to develop virus-associated cancers, even after they have fully recovered from COVID-19.","version":"1.1","doi":"10.1101/2020.10.02.324228","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.01.323220","pub_date":"2020-10-02","title":"Potent mouse monoclonal antibodies that block SARS-CoV-2 infection","abstract":"Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies in molecular pathology research is not fully addressed. Here, we produced mouse anti-SARS-CoV-2 spike monoclonal antibodies that exhibit not only robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also neutralizing activity against SARS-CoV-2 infection in vitro. Our monoclonal antibodies are of mouse origin, making them compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, and large-scale production and easy distribution are guaranteed by conventional mouse hybridoma technology.","version":"1.1","doi":"10.1101/2020.10.01.323220","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.323915","pub_date":"2020-10-02","title":"A Multiscale Coarse-grained Model of the SARS-CoV-2 Virion","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Computer simulations of complete viral particles can provide theoretical insights into large-scale viral processes including assembly, budding, egress, entry, and fusion. Detailed atomistic simulations, however, are constrained to shorter timescales and require billion-atom simulations for these processes. Here, we report the current status and on-going development of a largely \u201cbottom-up\u201d coarse-grained (CG) model of the SARS-CoV-2 virion. Structural data from a combination of cryo-electron microscopy (cryo-EM), x-ray crystallography, and computational predictions were used to build molecular models of structural SARS-CoV-2 proteins, which were then assembled into a complete virion model. We describe how CG molecular interactions can be derived from all-atom simulations, how viral behavior difficult to capture in atomistic simulations can be incorporated into the CG models, and how the CG models can be iteratively improved as new data becomes publicly available. Our initial CG model and the detailed methods presented are intended to serve as a resource for researchers working on COVID-19 who are interested in performing multiscale simulations of the SARS-CoV-2 virion. This study reports the construction of a molecular model for the SARS-CoV-2 virion and details our multiscale approach towards model refinement. The resulting model and methods can be applied to and enable the simulation of SARS-CoV-2 virions.","version":"1.1","doi":"10.1101/2020.10.02.323915","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.318261","pub_date":"2020-10-02","title":"A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core","abstract":"SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.","version":"1.2","doi":"10.1101/2020.09.30.318261","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.20205641","pub_date":"2020-10-02","title":"Seroprevalence of SARS-COV-2 Antibodies in Scottish Healthcare Workers","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>Healthcare workers are believed to be at increased risk of SARS-CoV-2 infection. The extent of that increased risk compared to the general population and the groups most at risk have not been extensively studied.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>A prospective observational study of health and social care workers in NHS Tayside (Scotland, UK) from May to September 2020. The Siemens SARS-CoV-2 total antibody assay was used to establish seroprevalence in this cohort. Patients provided clinical information including demographics and workplace information. Controls, matched for age and sex to the general Tayside population, were studied for comparison.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>A total of 2062 health and social care workers were recruited for this study. The participants were predominantly female (81.7%) and 95.2% were white. 299 healthcare workers had a positive antibody test (14.5%). 11 out of 231 control sera tested positive (4.8%). Healthcare workers therefore had an increased likelihood of a positive test (odds ratio 3.4 95% CI 1.85-6.16, p&lt;0.0001). Dentists, healthcare assistants and porters were the job roles most likely to test positive. Those working in front-line roles with COVID-19 patients were more likely to test positive (17.4% vs. 13.4%, p=0.02). 97.1% of patients who had previously tested positive for SARS-CoV-2 by RT-PCR had positive antibodies, compared to 11.8% of individuals with a symptomatic illness who had tested negative. Anosmia was the symptom most associated with the presence of detectable antibodies.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>In this study, healthcare workers were three times more likely to test positive for SARS-CoV-2 than the general population. The seroprevalence data in different populations identified in this study will be useful to protect healthcare staff during future waves of the pandemic.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.10.02.20205641","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.05.135921","pub_date":"2020-10-02","title":"SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface","abstract":"COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human na\u00efve universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.","version":"1.2","doi":"10.1101/2020.06.05.135921","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.323519","pub_date":"2020-10-02","title":"Transmission of SARS-COV-2 from China to Europe and West Africa: a detailed phylogenetic analysis","abstract":"SARS-CoV-2, the virus causing the Covid-19 pandemic emerged in December 2019 in China and raised fears that it could overwhelm healthcare systems worldwide. In June 2020, all African countries registered human infections with SARS-CoV-2. The virus is mutating steadily and this is monitored by a well curated database of viral nucleotide sequences from samples taken from infected individual thus enabling phylogenetic analysis and phenotypic associations. We downloaded from the GISAID database, SARS-CoV-2 sequences established from four West African countries Ghana, Gambia, Senegal and Nigeria and then performed phylogenetic analysis employing the nextstrain pipeline. Based on mutations found within the sequences we calculated and visualized statistics characterizing clades according to the GISAID nomenclature. We found country-specific patterns of viral clades: the later Europe-associated G-clades predominantly in Senegal and Gambia, and combinations of the earlier (L, S, V) and later clades in Ghana and Nigeria. Contrary to our expectations, the later Europe-associated G-clades emerged before the earlier clades. Detailed analysis of distinct samples showed that some of the earlier clades might have circulated latently and some reflect migration routes via Mali and Tunisia. The distinct patterns of viral clades in the West African countries point at its emergence from Europe and China via Asia and Europe. The observation that the later clades emerged before the earlier clades could be simply due to founder effects or due to latent circulation of the earlier clades. Only a marginal correlation of the G-clades associated with the D614G mutation could be identified with the relatively low case fatality (0.6-3.2). Ghana and Nigeria have a combination of earlier (L, V, S) and later Europe-associated G-clades of SARS-CoV-2, therefore pointing to multiple introductions while in Senegal and Gambia Europe-associated G-clades predominate pointing to introductions mainly from Europe. Surprisingly, the later G-clades emerged before the earlier clades (L, V, S) Detailed phylogenetic analysis points at latent circulation of earlier clades before the first registered cases. Phylogenetic analysis of some cases points at migration routes to Europe via Tunisia, Egypt and Mali. A marginal correlation of r=0.28 between the percentage of the D614G mutation defining the G-clades and case-fatality can be detected.","version":"1.1","doi":"10.1101/2020.10.02.323519","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.068999","pub_date":"2020-10-02","title":"Estimating cumulative incidence of SARS-CoV-2 with imperfect serological tests: exploiting cutoff-free approaches","abstract":"Large-scale serological testing in the population is essential to determine the true extent of the current SARS-CoV-2 pandemic. Serological tests measure antibody responses against pathogens and use predefined cutoff levels that dichotomize the quantitative test measures into sero-positives and negatives and use this as a proxy for past infection. With the imperfect assays that are currently available to test for past SARS-CoV-2 infection, the fraction of seropositive individuals in serosurveys is a biased estimator of the cumulative incidence and is usually corrected to account for the sensitivity and specificity. Here we use an inference method \u2014 referred to as mixture-model approach \u2014 for the estimation of the cumulative incidence that does not require to define cutoffs by integrating the quantitative test measures directly into the statistical inference procedure. We confirm that the mixture model outperforms the methods based on cutoffs, leading to less bias and error in estimates of the cumulative incidence. We illustrate how the mixture model can be used to optimize the design of serosurveys with imperfect serological tests. We also provide guidance on the number of control and case sera that are required to quantify the test\u2019s ambiguity sufficiently to enable the reliable estimation of the cumulative incidence. Lastly, we show how this approach can be used to estimate the cumulative incidence of classes of infections with an unknown distribution of quantitative test measures. This is a very promising application of the mixture-model approach that could identify the elusive fraction of asymptomatic SARS-CoV-2 infections. An R-package implementing the inference methods used in this paper is provided. Our study advocates using serological tests without cutoffs, especially if they are used to determine parameters characterizing populations rather than individuals. This approach circumvents some of the shortcomings of cutoff-based methods at exactly the low cumulative incidence levels and test accuracies that we are currently facing in SARS-CoV-2 serosurveys. As other pathogens, SARS-CoV-2 elicits antibody responses in infected people that can be detected in their blood serum as early as a week after the infection until long after recovery. The presence of SARS-CoV-2 specific antibodies can therefore be used as a marker of past infection, and the prevalence of seropositive people, i.e. people with specific antibodies, is a key measure to determine the extent of the SARS-CoV-2 pandemic. The serological tests, however, are usually not perfect, yielding false positive and false negative results. Here we exploit an approach that refrains from classifying people as seropositive or negative, but rather compares the antibody level of an individual to that of confirmed cases and controls. This approach leads to more reliable estimates of cumulative incidence, especially for the low prevalence and low test accuracies that we face during the current SARS-CoV-2 pandemic. We also show how this approach can be extended to infer the presence of specific types of cases that have not been used for validating the test, such as people that underwent a mild or asymptomatic infection.","version":"1.3","doi":"10.1101/2020.04.29.068999","journal":"bioRxiv","score":null},{"id":"10.1101/2020.10.02.324046","pub_date":"2020-10-02","title":"Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19","abstract":"The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which may cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine, VSV\u0394G-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients. Boost immunization with the identical replicon further enhanced neutralizing activity. These results demonstrate that rhabdovirus minispike replicons represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen. SARS-CoV-2 S RBD antigen is preferred over entire S to preclude potential disease enhancing antibodies construction of a chimeric rhabdovirus minispike protein presenting RBD in native conformation construction of single round VSV and rabies virus replicon vaccines presentation of minispike antigen on cells and on noninfectious VLPs strong induction of SARS-CoV-2 neutralizing antibodies by the VSV replicon/VLP system in vaccinated mice","version":"1.1","doi":"10.1101/2020.10.02.324046","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.317818","pub_date":"2020-10-01","title":"SARS-CoV-2 Spike protein promotes hyper-inflammatory response that can be ameliorated by Spike-antagonistic peptide and FDA-approved ER stress and MAP kinase inhibitors in vitro","abstract":"SARS-CoV-2 infection causes an inflammatory cytokine storm and acute lung injury. Currently there are no effective antiviral and/or anti-inflammatory therapies. Here we demonstrate that 2019 SARS-CoV-2 spike protein subunit 1 (CoV2-S1) induces high levels of NF-\u03baB activations, production of pro-inflammatory cytokines and mild epithelial damage, in human bronchial epithelial cells. CoV2-S1-induced NF-\u03baB activation requires S1 interaction with human ACE2 receptor and early activation of endoplasmic reticulum (ER) stress, and associated unfolded protein response (UPR), and MAP kinase signalling pathways. We developed an antagonistic peptide that inhibits S1-ACE2 interaction and CoV2-S1-induced productions of pro-inflammatory cytokines. The existing FDA-approved ER stress inhibitor, 4-phenylburic acid (4-PBA), and MAP kinase inhibitors, trametinib and ulixertinib, ameliorated CoV2-S1-induced inflammation and epithelial damage. These novel data highlight the potentials of peptide-based antivirals for novel ACE2-utilising CoVs, while repurposing existing drugs may be used as treatments to dampen elevated inflammation and lung injury mediated by SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.30.317818","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.080606","pub_date":"2020-10-01","title":"Multiple early introductions of SARS-CoV-2 into a global travel hub in the Middle East","abstract":"International travel played a significant role in the early global spread of SARS-CoV-2. Understanding transmission patterns from different regions of the world will further inform global dynamics of the pandemic. Using data from Dubai in the United Arab Emirates (UAE), a major international travel hub in the Middle East, we establish SARS-CoV-2 full genome sequences from the index and early COVID-19 patients in the UAE. The genome sequences are analysed in the context of virus introductions, chain of transmissions, and possible links to earlier strains from other regions of the world. Phylogenetic analysis showed multiple spatiotemporal introductions of SARS-CoV-2 into the UAE from Asia, Europe, and the Middle East during the early phase of the pandemic. We also provide evidence for early community-based transmission and catalogue new mutations in SARS-CoV-2 strains in the UAE. Our findings contribute to the understanding of the global transmission network of SARS-CoV-2.","version":"1.4","doi":"10.1101/2020.05.06.080606","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.320903","pub_date":"2020-10-01","title":"SARS-CoV-2 viral budding and entry can be modeled using virus-like particles","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. While a select agent designation has not been made for SARS-CoV-2, closely related SARS-CoV-1 and MERS coronaviruses are classified as Risk Group 3 select agents, which restricts use of the live viruses to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form viruslike particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions.","version":"1.1","doi":"10.1101/2020.09.30.320903","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.071811","pub_date":"2020-10-01","title":"Mutational landscape and in silico structure models of SARS-CoV-2 Spike Receptor Binding Domain reveal key molecular determinants for virus-host interaction","abstract":"Protein-protein interactions between virus and host are crucial for infection. SARS-CoV-2, the causative agent of COVID-19 pandemic is an RNA virus prone to mutations. Formation of a stable binding interface between the Spike (S) protein Receptor Binding Domain (RBD) of SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) of host actuates viral entry. Yet, how this binding interface evolves as virus acquires mutations during pandemic remains elusive. Here, using a high fidelity bioinformatics pipeline, we analysed 31,403 SARS-CoV-2 genomes across the globe, and identified 444 non-synonymous mutations that cause 49 distinct amino acid substitutions in the RBD. Molecular phylogenetic analysis suggested independent emergence of these RBD mutants during pandemic. In silico structure modelling of interfaces induced by mutations on residues which directly engage ACE2 or lie in the near vicinity revealed molecular rearrangements and binding energies unique to each RBD mutant. Comparative structure analysis using binding interface from mouse that prevents SARS-CoV-2 entry uncovered minimal molecular determinants in RBD necessary for the formation of stable interface. We identified that interfacial interaction involving amino acid residues N487 and G496 on either ends of the binding scaffold are indispensable to anchor RBD and are well conserved in all SARS-like corona viruses. All other interactions appear to be required to locally remodel binding interface with varying affinities and thus may decide extent of viral transmission and disease outcome. Together, our findings propose the modalities and variations in RBD-ACE2 interface formation exploited by SARS-CoV-2 for endurance. COVID-19, so far the worst hit pandemic to mankind, started in January 2020 and is still prevailing globally. Our study identified key molecular arrangements in RBD-ACE2 interface that help virus to tolerate mutations and prevail. In addition, RBD mutations identified in this study can serve as a molecular directory for experimental biologists to perform functional validation experiments. The minimal molecular requirements for the formation of RBD-ACE2 interface predicted using in silico structure models may help precisely design neutralizing antibodies, vaccines and therapeutics. Our study also proposes the significance of understanding evolution of protein interfaces during pandemic.","version":"1.3","doi":"10.1101/2020.05.02.071811","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.100545","pub_date":"2020-10-01","title":"Longitudinal high-throughput TCR repertoire profiling reveals the dynamics of T cell memory formation after mild COVID-19 infection","abstract":"COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T cell response nor the diversity of resulting immune memory are well understood. In this study we use longitudinal high-throughput T cell receptor (TCR) sequencing to track changes in the T cell repertoire following two mild cases of COVID-19. In both donors we identified CD4+ and CD8+ T cell clones with transient clonal expansion after infection. The antigen specificity of CD8+ TCR sequences to SARS-CoV-2 epitopes was confirmed by both MHC tetramer binding and presence in large database of SARS-CoV-2 epitope-specific TCRs. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T cell clones were detected in the memory fraction at the pre-infection timepoint, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.05.18.100545","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.320242","pub_date":"2020-9-30","title":"Host range projection of SARS-CoV-2: South Asia perspective","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causing agent of Coronavirus Disease-2019 (COVID-19), is likely to be originated from bat and transmitted through intermediate hosts. However, the immediate source species of SARS-CoV-2 has not yet been confirmed. Here, we used diversity analysis of the angiotensin I converting enzyme 2 (ACE2) that serves as cellular receptor for SARS-CoV-2 and transmembrane protease serine 2 (TMPRSS2), which has been proved to be utilized by SARS-CoV-2 for spike protein priming. We also simulated the structure of receptor binding domain of SARS-CoV-2 spike protein (SARS-CoV-2 S RBD) with the ACE2s to investigate their binding affinity to determine the potential intermediate animal hosts that could spread the SARS-CoV-2 virus to humans in South Asia. We identified cow, buffalo, goat and sheep, which are predominant species in the household farming system in South Asia that can potentially be infected by SARS-CoV-2. All the bird species studied along with rat and mouse were considered less potential to interact with SARS-CoV-2. The interaction interfaces of SARS-CoV-2 S RBD and ACE2 protein complex suggests pangolin as a potential intermediate host in SARS-CoV-2. Our results provide a valuable resource for the identification of potential hosts for SARS-CoV-2 in South Asia and henceforth reduce the opportunity for a future outbreak of COVID-19.","version":"1.1","doi":"10.1101/2020.09.30.320242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.29.319566","pub_date":"2020-9-30","title":"Functional Landscape of SARS-CoV-2 Cellular Restriction","abstract":"A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.","version":"1.1","doi":"10.1101/2020.09.29.319566","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.287201","pub_date":"2020-9-30","title":"Analysis of SARS-CoV-2 genomes from across Africa reveals potentially clinically relevant mutations","abstract":"SARS-CoV-2 is a betacoronavirus, the etiologic agent of the novel Coronavirus disease 2019 (COVID-19). The World Health Organization officially declared COVID-19 as a pandemic in March 2020 after the outbreak in Wuhan, China, in late 2019. Across the continents and specifically in Africa, all index cases were travel-related. Understanding how the virus\u2019s transportation across continents and different climatic conditions affect the genetic composition and the consequent effects on transmissibility, infectivity, and virulence of the virus is critical. Thus, it is crucial to compare COVID-19 genome sequences from the African continent with sequences from selected COVID-19 hotspots/countries in Asia, Europe, North and South America and Oceania. To identify possible distinguishing mutations in the African SARS-CoV-2 genomes compared to those from these selected countries, we conducted in silico analyses and comparisons. Complete African SARS-CoV-2 genomes deposited in GISAID and NCBI databases as of June 2020 were downloaded and aligned with genomes from Wuhan, China and other SARS-CoV-2 hotspots. Using phylogenetic analysis and amino acid sequence alignments of the spike and replicase (NSP12) proteins, we searched for possible vaccine coverage targets or potential therapeutic agents. Identity plots for the alignments were created with BioEdit software and the phylogenetic analyses with the MEGA X software. Our results showed mutations in the spike and replicate proteins of the SARS-Cov-2 virus. Phylogenetic tree analyses demonstrated variability across the various regions/countries in Africa as there were different clades in the viral proteins. However, a substantial proportion of these mutations (90%) were similar to those described in all the other settings, including the Wuhan strain. There were, however, novel mutations in the genomes of the circulating strains of the virus in African. To the best of our knowledge, this is the first study reporting these findings from Africa. However, these findings\u2019 implications on symptomatic or asymptomatic manifestations, progression to severe disease and case fatality for those affected, and the cross efficacy of vaccines developed from other settings when applied in Africa are unknown.","version":"1.2","doi":"10.1101/2020.09.08.287201","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.29.319731","pub_date":"2020-9-30","title":"Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino-oligomers","abstract":"SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense global public health importance. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense agents composed of a phosphordiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5\u2019-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method and viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays. PPMO designed to base-pair with sequence in the 5\u2019-terminal region or the leader transcription regulatory sequence-region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, in a non-toxic and dose-responsive manner. The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further pre-clinical development.","version":"1.1","doi":"10.1101/2020.09.29.319731","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.29.318931","pub_date":"2020-9-30","title":"Computational identification of human biological processes and protein sequence motifs putatively targeted by SARS-CoV-2 proteins using protein-protein interaction networks","abstract":"While the COVID-19 pandemic is causing important loss of life, knowledge of the effects of the causative SARS-CoV-2 virus on human cells is currently limited. Investigating protein-protein interactions (PPIs) between viral and host proteins can provide a better understanding of the mechanisms exploited by the virus and enable the identification of potential drug targets. We therefore performed an in-depth computational analysis of the interactome of SARS-CoV-2 and human proteins in infected HEK293 cells published by Gordon et al. to reveal processes that are potentially affected by the virus and putative protein binding sites. Specifically, we performed a set of network-based functional and sequence motif enrichment analyses on SARS-CoV-2-interacting human proteins and on a PPI network generated by supplementing viral-host PPIs with known interactions. Using a novel implementation of our GoNet algorithm, we identified 329 Gene Ontology terms for which the SARS-CoV-2-interacting human proteins are significantly clustered in the network. Furthermore, we present a novel protein sequence motif discovery approach, LESMoN-Pro, that identified 9 amino acid motifs for which the associated proteins are clustered in the network. Together, these results provide insights into the processes and sequence motifs that are putatively implicated in SARS-CoV-2 infection and could lead to potential therapeutic targets.","version":"1.1","doi":"10.1101/2020.09.29.318931","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.25.313270","pub_date":"2020-9-30","title":"SARS-CoV-2 and Malayan pangolin coronavirus infect human endoderm, ectoderm and induced lung progenitor cells","abstract":"Since the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in several somatic cells, little is known about the infection of SASRS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that SARS-CoV-2 pseudovirus and GX_P2V could infect lung progenitor and even anterior foregut endoderm cells causing these cells death, which differentiated from human embryonic stem cells (hESCs). The infection and replication of SARS-CoV-2 and GX_P2V were inhibited when treated with whey protein of breastmilk and Remdesivir, confirming that these two viruses could infect lung progenitor and even anterior foregut endoderm. Moreover, we found that SARS-CoV-2 pseudovirus could infect endoderm and ectoderm. We found that whey protein blocked SARS-CoV-2 infecting these cells. In line with the SARS-CoV-2 results, GX_P2V could also infected endoderm and ectoderm, and also was inhibited by Remdesivir treatment. Although expressing coronavirus related receptor such as ACE2 and TMPRSS2, mesoderm cells are not permissive for SARS-CoV-2 and GX_P2V infection, which needed further to study the mechanisms. Interestingly, we also found that hESCs, which also express ACE2 and TMPRSS2 markers, are permissive for GX_P2V but not SARS-CoV-2 pseudovirus infection and replication, indicating the widespread cell types for GX_P2V infection. Heparin treatment blocked efficiently viral infection. These results provided insight that these stem cells maybe provided a stable repository of coronavirus function or genome. The potential consequence of SARS-CoV-2 and animal coronavirus such as GX_P2V infection in hESCs, germ layer and induced progenitors should be closely monitored.","version":"1.2","doi":"10.1101/2020.09.25.313270","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.30.318311","pub_date":"2020-9-30","title":"The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells","abstract":"SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2\u2019 cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.","version":"1.1","doi":"10.1101/2020.09.30.318311","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.29.319061","pub_date":"2020-9-30","title":"BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response","abstract":"Effective vaccine against SARS-CoV-2 is the utmost importance in the current world. More than 1 million deaths are accounted for relevant pandemic disease COVID-19. Recent data showed that D614G genotype of the virus is highly infectious and responsible for almost all infection for 2nd wave. Despite of multiple vaccine development initiatives, there are currently no report that has addressed this critical variant D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine considering the D614G variant and characterization of the vaccine in preclinical trial. The surface plasmon resonance (SPR) data with spike protein as probe and competitive neutralization with RBD and S2 domain revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice on day 7 and 14 neutralized SARS-CoV-2 pseudovirus in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, immunization protected mice lungs from pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The IgG2a to IgG1 and (IgG2a+IgG2b) to (IgG1+IgG3) ratios were found 1\u00b10.2 and 1.24\u00b10.1, respectively. These values are comparatively higher than relevant values for other published SARS-CoV-2 vaccine in development, and suggesting higher viral clearance capacity for our vaccine. The data suggested great promise for immediate translation of the technology to the clinic.","version":"1.1","doi":"10.1101/2020.09.29.319061","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.250647","pub_date":"2020-9-30","title":"Sampling SARS-CoV-2 proteomes for predicted CD8 T-cell epitopes as a tool for understanding immunogenic breadth and rational vaccine design","abstract":"Predictive models for vaccine design have become a powerful and necessary resource for the expeditiousness design of vaccines to combat the ongoing SARS-CoV-2 global pandemic. Here we use the power of these predicted models to assess the sequence diversity of circulating SARS-CoV-2 proteomes in the context of an individual\u2019s CD8 T-cell immune repertoire to identify potential. defined regions of immunogenicity. Using this approach of expedited and rational CD8 T-cell vaccine design, it may be possible to develop a therapeutic vaccine candidate with the potential for both global and local coverage.","version":"1.3","doi":"10.1101/2020.08.15.250647","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.317685","pub_date":"2020-9-29","title":"SARS-CoV-2 D614G Variant Exhibits Enhanced Replication ex vivo and Earlier Transmission in vivo","abstract":"The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.","version":"1.1","doi":"10.1101/2020.09.28.317685","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.29.317289","pub_date":"2020-9-29","title":"Susceptibility of midge and mosquito vectors to SARS-CoV-2 by natural route of infection","abstract":"SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2infected blood meal. Species tested included Culicoides sonorensis biting midges, as well as Culex tarsalis and Culex quinquefasciatus mosquitoes, all known biological vectors for numerous RNA viruses. Arthropods were allowed to feed on SARS-CoV-2 spiked blood and at various time points post infection analyzed for the presence of viral RNA and infectious virus. Additionally, cell lines derived from C. sonorensis (W8a), Ae. aegypti (C6/36), Cx. quinquefasciatus (HSU), and Cx. tarsalis (CxTrR2) were tested for SARS-CoV-2 susceptibility. Our results indicate that none of the biting insects, nor the insect cell lines support SARS-CoV-2 replication. We conclude, that biting insect do not pose a risk for transmission of SARS-CoV-2 to humans or animals following a SARS-CoV-2 infected blood meal.","version":"1.1","doi":"10.1101/2020.09.29.317289","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.27.316174","pub_date":"2020-9-29","title":"Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach","abstract":"SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a na\u00efve phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.","version":"1.2","doi":"10.1101/2020.09.27.316174","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.29.317131","pub_date":"2020-9-29","title":"De Novo Discovery of High Affinity Peptide Binders for the SARS-CoV-2 Spike Protein","abstract":"The \u03b2-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein, the most exposed surface structure of the virus, are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants Kd = 80 to 970 nM) for RBD and selectivity over human serum proteins. Picomolar RBD concentrations in biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides might associate with the SARS-CoV-2-spike-RBD at a site unrelated to ACE2 binding, making them potential orthogonal reagents for sandwich immunoassays. We envision our discovery as a robust starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus directed delivery of therapeutics.","version":"1.1","doi":"10.1101/2020.09.29.317131","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.311480","pub_date":"2020-9-29","title":"Prime-boost vaccination of mice and Rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates","abstract":"COVID-19 vaccines are being developed urgently worldwide, among which single-shot adenovirus vectored vaccines represent a major approach. Here, we constructed two novel adenovirus vectored COVID-19 vaccine candidates on simian adenovirus serotype 23 (Sad23L) and human adenovirus serotype 49 vectors (Ad49L) carrying the full-length gene of SARS-CoV-2 spike protein (S), designated Sad23L-nCoV-S and Ad49L-nCoV-S vaccines, respectively. The immunogenicity elicited by these two vaccine strains was individually evaluated in mice. Specific humoral and cellular immune responses were proportionally observed in a dose-dependent manner, and stronger response was obtained by boosting. Furthermore, five rhesus macaques were intramuscularly injected with a dose of 5\u00d7109 PFU Sad23L-nCoV-S vaccine for prime vaccination, followed by boosting with 5\u00d7109 PFU of Ad49L-nCoV-S vaccine at 4-week interval. Three macaques were injected with Sad23L-GFP and Ad49L-GFP vectorial viruses as negative controls. Both mice and macaques tolerated well the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost vaccination regimen induced high titers of 103.16 S-binding antibody (S-BAb), 102.75 cell receptor binding domain (RBD)-BAb and 102.38 neutralizing antibody (NAb) to pseudovirus a week after boosting injection, followed by sustained high levels over 10 weeks of observation. Robust IFN-\u03b3 secreting T-cell response (712.6 SFCs/106 cells), IL-2 secreting T-cell response (334 SFCs/106 cells) and intracellular IFN-\u03b3 expressing CD4+/CD8+ T cell response (0.39%/0.55%) to S peptides were detected in the vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.","version":"1.1","doi":"10.1101/2020.09.28.311480","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.20200915","pub_date":"2020-09-29","title":"SARS-CoV-2 seroprevalence survey estimates are affected by anti-nucleocapsid antibody decline","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>We analyzed 21,676 residual specimens from Ontario, Canada collected between March-August, 2020 to investigate the effect of antibody decline on SARS-CoV-2 seroprevalence estimates. Testing specimens orthogonally using the Abbott (anti-nucleocapsid) and then the Ortho (anti-spike) assays, seroprevalence estimates ranged from 0.4%-1.4%, despite ongoing disease activity. The geometric mean concentration (GMC) of antibody-positive specimens decreased over time (p=0.015), and the GMC of antibody-negative specimens increased over time (p=0.0018). The association between the two tests decreased each month (p&lt;0.001), suggesting anti-N antibody decline. Lowering the Abbott index cut-off from 1.4 to 0.7 resulted in a 16% increase in positive specimens.</jats:p>","version":null,"doi":"10.1101/2020.09.28.20200915","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.29.318196","pub_date":"2020-9-29","title":"Design, expression, purification and characterization of a YFP-tagged 2019-nCoV spike receptor-binding domain construct","abstract":"2019-nCoV is the causative agent of the serious, still ongoing, worldwide COVID-19 pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFN\u03b12) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA IMAC purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.","version":"1.1","doi":"10.1101/2020.09.29.318196","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.20203166","pub_date":"2020-09-29","title":"Comparison of infection control strategies to reduce COVID-19 outbreaks in homeless shelters in the United States: a simulation study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>COVID-19 outbreaks have occurred in homeless shelters across the US, highlighting an urgent need to identify the most effective infection control strategy to prevent future outbreaks.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed a microsimulation model of SARS-CoV-2 transmission in a homeless shelter and calibrated it to data from cross-sectional polymerase-chain-reaction (PCR) surveys conducted during COVID-19 outbreaks in five shelters in three US cities from March 28 to April 10, 2020. We estimated the probability of averting a COVID-19 outbreak when an exposed individual is introduced into a representative homeless shelter of 250 residents and 50 staff over 30 days under different infection control strategies, including daily symptom-based screening, twice-weekly PCR testing and universal mask wearing.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    The proportion of PCR-positive residents and staff at the shelters with observed outbreaks ranged from 2.6% to 51.6%, which translated to basic reproduction number (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>0</jats:sub>\n                    ) estimates of 2.9\u20136.2. The probability of averting an outbreak diminished with higher transmissibility (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>0</jats:sub>\n                    ) within the simulated shelter and increasing incidence in the local community. With moderate community incidence (~30 confirmed cases/1,000,000 people/day), the estimated probabilities of averting an outbreak in a low-risk (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>0</jats:sub>\n                    =1.5), moderate-risk (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>0</jats:sub>\n                    =2.9), and high-risk (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>0</jats:sub>\n                    =6.2) shelter were, respectively: 0.35, 0.13 and 0.04 for daily symptom-based screening; 0.53, 0.20, and 0.09 for twice-weekly PCR testing; 0.62, 0.27 and 0.08 for universal masking; and 0.74, 0.42 and 0.19 for these strategies combined.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>In high-risk homeless shelter environments and locations with high community incidence of COVID-19, even intensive infection control strategies (incorporating daily symptom-screening, frequent PCR testing and universal mask wearing) are unlikely to prevent outbreaks, suggesting a need for non-congregate housing arrangements for people experiencing homelessness. In lower-risk environments, combined interventions should be employed to reduce outbreak risk.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.28.20203166","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.28.20202937","pub_date":"2020-09-29","title":"The potential contribution of face coverings to the control of SARS-CoV-2 transmission in schools and broader society in the UK: a modelling study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Recent findings suggest that an adequate test-trace-isolate (TTI) strategy is needed to prevent a secondary COVID-19 wave with the reopening of society in the UK. Here we assess the potential importance of mandatory masks in the parts of community and in secondary schools. We show that, assuming current TTI levels, adoption of masks in secondary schools in addition to community settings can reduce the size of a second wave, but will not prevent it; more testing of symptomatic people, tracing and isolating of their contacts is also needed. To avoid a second wave, with masks mandatory in secondary schools and in certain community settings, under current tracing levels, 68% or 46% of those with symptomatic infection would need to be tested if masks\u2019 effective coverage were 15% or 30% respectively, compared to 76% and 57% if masks are mandated in community settings but not secondary schools.</jats:p>","version":null,"doi":"10.1101/2020.09.28.20202937","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.29.20204115","pub_date":"2020-09-29","title":"A Sew-Free Origami Mask for Improvised Respiratory Protection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Recently, respiratory aerosols with diameters smaller than 100\n                  <jats:italic>\u00b5</jats:italic>\n                  m have been confirmed as important vectors for the spread of SARS-CoV-2. While cloth masks afford some protection for larger ballistic droplets, they are typically inefficient at filtering these aerosols and require specialized fabrication devices to produce. We describe a fabrication technique that makes use of a folding procedure (origami) to transform a filtration material into a mask. These origami masks can be fabricated by non-experts at minimal cost and effort, provide adequate filtration efficiencies, and are easily scaled to different facial sizes. Using a mannequin fit test simulator, we demonstrate that these masks can provide optimal filtration efficiency and ease of breathing with minimal leakage. Because this mask provides greater comfort compared to commercial alternatives, it is likely to promote greater mask wearing tolerance and acceptance.\n                </jats:p>","version":null,"doi":"10.1101/2020.09.29.20204115","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.25.20200329","pub_date":"2020-09-28","title":"SARS-CoV-2 Uses CD4 to Infect T Helper Lymphocytes","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality\n                  <jats:sup>1,2</jats:sup>\n                  . The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here we show that SARS-CoV-2 infects human CD4\n                  <jats:sup>+</jats:sup>\n                  T helper cells, but not CD8\n                  <jats:sup>+</jats:sup>\n                  T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.\n                </jats:p>","version":null,"doi":"10.1101/2020.09.25.20200329","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.23.310565","pub_date":"2020-9-28","title":"COVID-19 CG: Tracking SARS-CoV-2 mutations by locations and dates of interest","abstract":"COVID-19 CG is an open resource for tracking SARS-CoV-2 single-nucleotide variations (SNVs) and lineages while filtering by location, date, gene, and mutation of interest. COVID-19 CG provides significant time, labor, and cost-saving utility to diverse projects on SARS-CoV-2 transmission, evolution, emergence, immune interactions, diagnostics, therapeutics, vaccines, and intervention tracking. Here, we describe case studies in which users can interrogate (1) SNVs in the SARS-CoV-2 Spike receptor binding domain (RBD) across different geographic regions to inform the design and testing of therapeutics, (2) SNVs that may impact the sensitivity of commonly used diagnostic primers, and (3) the recent emergence of a dominant lineage harboring an S477N RBD mutation in Australia. To accelerate COVID-19 research and public health efforts, COVID-19 CG will be continually upgraded with new features for users to quickly and reliably pinpoint mutations as the virus evolves throughout the pandemic and in response to therapeutic and public health interventions.","version":"1.2","doi":"10.1101/2020.09.23.310565","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.316281","pub_date":"2020-9-28","title":"Identification of TMEM106B as proviral host factor for SARS-CoV-2","abstract":"The ongoing COVID-19 pandemic is responsible for worldwide economic damage and nearly one million deaths. Potent drugs for the treatment of severe SARS-CoV-2 infections are not yet available. To identify host factors that support coronavirus infection, we performed genome-wide functional genetic screens with SARS-CoV-2 and the common cold virus HCoV-229E in non-transgenic human cells. These screens identified PI3K type 3 as a potential drug target against multiple coronaviruses. We discovered that the lysosomal protein TMEM106B is an important host factor for SARS-CoV-2 infection. Furthermore, we show that TMEM106B is required for replication in multiple human cell lines derived from liver and lung and is expressed in relevant cell types in the human airways. Our results identify new coronavirus host factors that may potentially serve as drug targets against SARS-CoV-2 or to quickly combat future zoonotic coronavirus outbreaks.","version":"1.1","doi":"10.1101/2020.09.28.316281","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.27.315796","pub_date":"2020-9-28","title":"A Tethered Ligand Assay to Probe the SARS-CoV-2 ACE2 Interaction under Constant Force","abstract":"The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within \u22641 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2020.09.27.315796","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109280","pub_date":"2020-9-28","title":"Dynamically evolving novel overlapping gene as a factor in the SARS-CoV-2 pandemic","abstract":"Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics, but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.","version":"1.3","doi":"10.1101/2020.05.21.109280","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.25.313601","pub_date":"2020-9-28","title":"A potent SARS-CoV-2 neutralizing human monoclonal antibody that reduces viral burden and disease severity in Syrian hamsters","abstract":"The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.","version":"1.2","doi":"10.1101/2020.09.25.313601","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.27.316018","pub_date":"2020-9-28","title":"Genome-Wide Asymptomatic B-Cell, CD4+ and CD8+ T-Cell Epitopes, that are Highly Conserved Between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Immune Targets for Pre-Emptive Pan-Coronavirus Vaccines","abstract":"Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal \u201cflu-like\u201d global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+ and CD8+ T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines . In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+ and CD8+ T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the \u201cCommon Cold\u201d; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+ and CD8+ T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in \u201chumanized\u201d Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.","version":"1.1","doi":"10.1101/2020.09.27.316018","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165787","pub_date":"2020-9-28","title":"Detailed phylogenetic analysis of SARS-CoV-2 reveals latent capacity to bind human ACE2 receptor","abstract":"SARS-CoV-2 is a unique event, having emerged suddenly as a highly infectious viral pathogen for human populations. Previous phylogenetic analyses show its closest known evolutionary relative to be a virus detected in bats (RaTG13), with a common assumption that SARS-CoV-2 evolved from a zoonotic ancestor via recent genetic changes (likely in the Spike protein receptor binding domain \u2013 or RBD) that enabled it to infect humans. We used detailed phylogenetic analysis, ancestral sequence reconstruction, and in situ molecular dynamics simulations to examine the Spike-RBD\u2019s functional evolution, finding that the common ancestral virus with RaTG13, dating to at least 2013, possessed high binding affinity to the human ACE2 receptor. This suggests that SARS-CoV-2 likely possessed a latent capacity to bind to human cellular targets (though this may not have been sufficient for successful infection) and emphasizes the importance to expand the cataloging and monitoring of viruses circulating in both human and non-human populations.","version":"1.3","doi":"10.1101/2020.06.22.165787","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.129775","pub_date":"2020-9-28","title":"Optimizing the molecular diagnosis of Covid-19 by combining RT-PCR and a pseudo-convolutional machine learning approach to characterize virus DNA sequences","abstract":"The proliferation of the SARS-Cov-2 virus to the whole world caused more than 250,000 deaths worldwide and over 4 million confirmed cases. The severity of Covid-19, the exponential rate at which the virus proliferates, and the rapid exhaustion of the public health resources are critical factors. The RT-PCR with virus DNA identification is still the benchmark Covid-19 diagnosis method. In this work we propose a new technique for representing DNA sequences: they are divided into smaller sequences with overlap in a pseudo-convolutional approach, and represented by co-occurrence matrices. This technique analyzes the DNA sequences obtained by the RT-PCR method, eliminating sequence alignment. Through the proposed method, it is possible to identify virus sequences from a large database: 347,363 virus DNA sequences from 24 virus families and SARS-Cov-2. Experiments with all 24 virus families and SARS-Cov-2 (multi-class scenario) resulted 0.822222 \u00b1 0.05613 for sensitivity and 0.99974 \u00b1 0.00001 for specificity using Random Forests with 100 trees and 30% overlap. When we compared SARS-Cov-2 with similar-symptoms virus families, we got 0.97059 \u00b1 0.03387 for sensitivity, and 0.99187 \u00b1 0.00046 for specificity with MLP classifier and 30% overlap. In the real test scenario, in which SARS-Cov-2 is compared to Coronaviridae and healthy human DNA sequences, we got 0.98824 \u00b1 001198 for sensitivity and 0.99860 \u00b1 0.00020 for specificity with MLP and 50% overlap. Therefore, the molecular diagnosis of Covid-19 can be optimized by combining RT-PCR and our pseudo-convolutional method to identify SARS-Cov-2 DNA sequences faster with higher specificity and sensitivity.","version":"1.2","doi":"10.1101/2020.06.02.129775","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.207308","pub_date":"2020-9-28","title":"SARS-CoV-2 structural coverage map reveals state changes that disrupt host immunity","abstract":"In response to the COVID-19 pandemic, many life scientists are focused on SARS-CoV-2. To help them use available structural data, we systematically modeled all viral proteins using all related 3D structures, generating 872 models that provide detail not available elsewhere. To organise these models, we created a structural coverage map: a novel, one-stop visualization summarizing what is \u2014 and is not \u2014 known about the 3D structure of the viral proteome. The map highlights structural evidence for viral protein interactions, mimicry, and hijacking; it also helps researchers find 3D models of interest, which can then be mapped with UniProt, PredictProtein, or CATH features. The resulting Aquaria-COVID resource (https://aquaria.ws/covid) helps scientists understand molecular mechanisms underlying coronavirus infection. Based on insights gained using our resource, we propose mechanisms by which the virus may enter immune cells, sense the cell type, then switch focus from viral reproduction to disrupting host immune responses. Currently, much of the COVID-19 viral proteome has unknown molecular structure. To improve this, we generated \u223c1,000 structural models, designed to capture multiple states for each viral protein. To organise these models, we created a structure coverage map: a novel, one-stop visualization summarizing what is \u2014 and is not \u2014 known about viral protein structure. We used these data to create an online resource, designed to help COVID-19 researchers gain insight into the key molecular processes that drive infection. Based on insights gained using our resource, we speculate that the virus may sense the type of cells it infects and, within certain cells, it may switch from reproduction to disruption of the immune system.","version":"1.5","doi":"10.1101/2020.07.16.207308","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.073171","pub_date":"2020-9-28","title":"Functional profiling of COVID-19 respiratory tract microbiomes","abstract":"In response to the ongoing global pandemic, progress has been made in understanding the molecular-level host interactions of the new coronavirus SARS-CoV-2 responsible for COVID-19. However, when the virus enters the body it interacts not only with the host but also with the micro-organisms already inhabiting the host. Understanding the virus-host-microbiome interactions can yield additional insights into the biological processes perturbed by viral invasion. With this aim we carry out a functional analysis of previously published RNA sequencing data of bronchoalveolar lavage fluid from eight COVID-19 patients, twenty-five community-acquired pneumonia patients, and twenty healthy controls. The resulting microbiome functional profiles and their top differentiating features clearly separate the cohorts. By examining the functional features in connection with their associated metabolic pathways, differentially abundant pathways are indicated, compared to both the community-acquired pneumonia and healthy cohorts. From this analysis, distinguishing signatures in COVID-19 respiratory tract microbiomes are identified, including decreased lipid and glycan metabolism pathways, and increased carbohydrate metabolism pathways. Here we present a framework for comparative functional analysis of microbiomes, the results from which can lead to new hypotheses on the host-microbiome interactions in healthy versus afflicted cohorts. The findings from this analysis call for further research on microbial functions and host-microbiome interactions during SARS-CoV-2 infection.","version":"1.5","doi":"10.1101/2020.05.01.073171","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.26.314971","pub_date":"2020-9-28","title":"Ultrafast Sample Placement on Existing Trees (UShER) Empowers Real-Time Phylogenetics for the SARS-CoV-2 Pandemic","abstract":"As the SARS-CoV-2 virus spreads through human populations, the unprecedented accumulation of viral genome sequences is ushering a new era of \u201cgenomic contact tracing\u201d \u2013 that is, using viral genome sequences to trace local transmission dynamics. However, because the viral phylogeny is already so large \u2013 and will undoubtedly grow many fold \u2013 placing new sequences onto the tree has emerged as a barrier to real-time genomic contact tracing. Here, we resolve this challenge by building an efficient, tree-based data structure encoding the inferred evolutionary history of the virus. We demonstrate that our approach improves the speed of phylogenetic placement of new samples and data visualization by orders of magnitude, making it possible to complete the placements under real-time constraints. Our method also provides the key ingredient for maintaining a fully-updated reference phylogeny. We make these tools available to the research community through the UCSC SARS-CoV-2 Genome Browser to enable rapid cross-referencing of information in new virus sequences with an ever-expanding array of molecular and structural biology data. The methods described here will empower research and genomic contact tracing for laboratories worldwide. USHER is available to users through the UCSC Genome Browser at https://genome.ucsc.edu/cgi-bin/hgPhyloPlace. The source code and detailed instructions on how to compile and run UShER are available from https://github.com/yatisht/usher.","version":"1.1","doi":"10.1101/2020.09.26.314971","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.317206","pub_date":"2020-9-28","title":"Exploring dynamics and network analysis of spike glycoprotein of SARS-COV-2","abstract":"The ongoing pandemic caused by coronavirus SARS-COV-2 continues to rage with devastating consequences on human health and global economy. The spike glycoprotein on the surface of coronavirus mediates its entry into host cells and is the target of all current antibody design efforts to neutralize the virus. The glycan shield of the spike helps the virus to evade the human immune response by providing a thick sugar-coated barrier against any antibody. To study the dynamic motion of glycans in the spike protein, we performed microsecond-long MD simulation in two different states that correspond to the receptor binding domain in open or closed conformations. Analysis of this microsecond-long simulation revealed a scissoring motion on the N-terminal domain of neighboring monomers in the spike trimer. Role of multiple glycans in shielding of spike protein in different regions were uncovered by a network analysis, where the high betweenness centrality of glycans at the apex revealed their importance and function in the glycan shield. Microdomains of glycans were identified featuring a high degree of intra-communication in these microdomains. An antibody overlap analysis revealed the glycan microdomains as well as individual glycans that inhibit access to the antibody epitopes on the spike protein. Overall, the results of this study provide detailed understanding of the spike glycan shield, which may be utilized for therapeutic efforts against this crisis.","version":"1.1","doi":"10.1101/2020.09.28.317206","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.292078","pub_date":"2020-9-28","title":"Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition","abstract":"Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails\u2013including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.","version":"1.2","doi":"10.1101/2020.09.10.292078","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.316448","pub_date":"2020-9-28","title":"Impact of COVID-19 on Hospital Admission of Acute Stroke patients in Bangladesh","abstract":"With the proposed pathophysiologic mechanism of neurologic injury by SARS COV-2 the frequency of stroke and henceforth the related hospital admissions were expected to rise. In this paper we investigate this presumption by comparing the frequency of admissions of stroke cases in Bangladesh before and during the pandemic. We conducted a retrospective analysis of stroke admissions in a 100-bed stroke unit at the National Institute of Neurosciences and Hospital (NINS&H) which is considerably a large stroke unit. We considered all the admitted cases from the 1st January to the 30th June, 2020. We used Poisson regressions to determine whether statistically significant changes in admission counts can be found before and after 25 March since when there is a surge in COVID-19 infections. A total of 1394 stroke patients got admitted during the study period. Half of the patients were older than 60 years, whereas only 2.6% were 30 years old or younger with a male-female ratio of 1.06:1. From January to March, 2020 the mean rate of admission was 302.3 cases per month which dropped to 162.3 cases per month from April to June with an overall reduction of 46.3% in acute stroke admission per month. In those two periods, reductions in average admission per month for ischemic stroke (IST), intracerebral hemorrhage (ICH), subarachnoid hemorrhage (SAH) and venous stroke (VS) were 45.5%, 37.2%, 71.4% and 39.0%, respectively. Based on weekly data, results of Poisson regressions confirm that the average number of admissions per week dropped significantly during the last three months of the sample period. Further, in the first three months, a total of 22 cases of hyperacute stroke management were done whereas in the last three months there was an 86.4% reduction in the number of hyperacute stroke patients getting reperfusion treatment. Only 38 patients (2.7%) were later found to be RT- PCR for SARS Cov-2 positive based on nasal swab testing. Our study revealed more than fifty percent reduction in acute stroke admission during the COVID-19 pandemic. It is still elusive whether the reduction is related to the fear of getting infected by COVID-19 from hospitalization or the overall restriction on public movement and stay-home measures.","version":"1.1","doi":"10.1101/2020.09.28.316448","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.316307","pub_date":"2020-9-28","title":"Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins","abstract":"Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.","version":"1.1","doi":"10.1101/2020.09.28.316307","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.27.312538","pub_date":"2020-9-28","title":"No evidence that plasmablasts transdifferentiate into developing neutrophils in severe COVID-19 disease","abstract":"A recent study by Wilk et al. of the transcriptome of peripheral blood mononuclear cells (PBMCs) in seven patients hospitalized with COVID-19 described a population of \u201cdeveloping neutrophils\u201d that were \u201cphenotypically related by dimensionality reduction\u201d to plasmablasts, and that these two cell populations represent a \u201clinear continuum of cellular phenotype\u201d. The authors suggest that, in the setting of acute respiratory distress syndrome (ARDS) secondary to severe COVID-19, a \u201cdifferentiation bridge from plasmablasts to developing neutrophils\u201d connected these distantly related cell types. This conclusion is controversial as it appears to violate several basic principles in cell biology relating to cell lineage identity and fidelity. Correctly classifying cells and their developmental history is an important issue in cell biology and we suggest that this conclusion is not supported by the data as we show here that: (1) regressing out covariates such as unique molecular identifiers (UMIs) can lead to overfitting; and (2) that UMAP embeddings may reflect the expression of similar genes but not necessarily direct cell lineage relationships.","version":"1.1","doi":"10.1101/2020.09.27.312538","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.28.316604","pub_date":"2020-9-28","title":"Host transcriptomic profiling of COVID-19 patients with mild, moderate, and severe clinical outcomes","abstract":"Characterizing key molecular and cellular pathways involved in COVID-19 is essential for disease prognosis and management. We perform shotgun transcriptome sequencing of human RNA obtained from nasopharyngeal swabs of patients with COVID-19, and identify a molecular signature associated with disease severity. Specifically, we identify globally dysregulated immune related pathways, such as cytokine-cytokine receptor signaling, complement and coagulation cascades, JAK-STAT, and TGF-\u03b2 signaling pathways in all, though to a higher extent in patients with severe symptoms. The excessive release of cytokines and chemokines such as CCL2, CCL22, CXCL9 and CXCL12 and certain interferons and interleukins related genes like IFIH1, IFI44, IFIT1 and IL10 were significantly higher in patients with severe clinical presentation compared to mild and moderate presentations. Moreover, early induction of the TGF-\u03b2 signaling pathway might be the primary cause of pulmonary fibrosis in patients with severe disease. Differential gene expression analysis identified a small set of regulatory genes that might act as strong predictors of patient outcome. Our data suggest that rapid transcriptome analysis of nasopharyngeal swabs can be a powerful approach to quantify host molecular response and may provide valuable insights into COVID-19 pathophysiology.","version":"1.1","doi":"10.1101/2020.09.28.316604","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.25.314070","pub_date":"2020-9-27","title":"Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection","abstract":"Following emergence in late 2019, SARS-CoV-2 rapidly became pandemic and is presently responsible for millions of infections and hundreds of thousands of deaths worldwide. There is currently no approved vaccine to halt the spread of SARS-CoV-2 and only very few treatment options are available to manage COVID-19 patients. For development of preclinical countermeasures, reliable and well-characterized small animal disease models will be of paramount importance. Here we show that intranasal inoculation of SARS-CoV-2 into Syrian hamsters consistently caused moderate broncho-interstitial pneumonia, with high viral lung loads and extensive virus shedding, but animals only displayed transient mild disease. We determined the infectious dose 50 to be only five infectious particles, making the Syrian hamster a highly susceptible model for SARS-CoV-2 infection. Neither hamster age nor sex had any impact on the severity of disease or course of infection. Finally, prolonged viral persistence in interleukin 2 receptor gamma chain knockout hamsters revealed susceptibility of SARS-CoV-2 to adaptive immune control. In conclusion, the Syrian hamster is highly susceptible to SARS-CoV-2 making it a very suitable infection model for COVID-19 countermeasure development. The Syrian hamster is highly susceptible to SARS-CoV-2 making it an ideal infection model for COVID-19 countermeasure development.","version":"1.1","doi":"10.1101/2020.09.25.314070","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.26.314385","pub_date":"2020-9-27","title":"Wide variabilities identified among spike proteins of SARS Cov2 globally-dominant variant identified","abstract":"SARS Cov2 is a newly emerged virus causing pandemic with fatality and co-morbidity. The greatest limitations emerged is the lack of effective treatment and vaccination due to frequent mutations and reassortment of the virus, leading to evolvement of different strains. We identified a wide variability in the whole genome sequences as well as spike protein variants (responsible for binding with ACE2 receptor) of SARS Cov2 identified globally. Structural variations of spike proteins identified from representative countries from all the continents, seven of them have revealed genetically similar, may be regarded as the dominant type. Novel non-synonymous mutations as S247R, R408I, G612D, A930V and deletion detected at amino acid position 144. RMSD values ranging from 4.45 to 2.25 for the dominant variant spike1 with other spike proteins. This study is informative for future vaccine research and drug development with the dominant type.","version":"1.1","doi":"10.1101/2020.09.26.314385","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.25.20201889","pub_date":"2020-09-27","title":"Prioritising COVID-19 vaccination in changing social and epidemiological landscapes","abstract":"<jats:title>Summary</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>During the COVID-19 pandemic, authorities must decide which groups to prioritise for vaccination. These decision will occur in a constantly shifting social-epidemiological landscape where the success of large-scale non-pharmaceutical interventions (NPIs) like physical distancing requires broad population acceptance.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We developed a coupled social-epidemiological model of SARS-CoV-2 transmission. Schools and workplaces are closed and re-opened based on reported cases. We used evolutionary game theory and mobility data to model individual adherence to NPIs. We explored the impact of vaccinating 60+ year-olds first; &lt;20 year-olds first; uniformly by age; and a novel contact-based strategy. The last three strategies interrupt transmission while the first targets a vulnerable group. Vaccination rates ranged from 0.5% to 4.5% of the population per week, beginning in January or July 2021.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>Case notifications, NPI adherence, and lockdown periods undergo successive waves during the simulated pandemic. Vaccination reduces median deaths by 32% \u2013 77% (22% \u2013 63%) for January (July) availability, depending on the scenario. Vaccinating 60+ year-olds first prevents more deaths (up to 8% more) than transmission-interrupting strategies for January vaccine availability across most parameter regimes. In contrast, transmission-interrupting strategies prevent up to 33% more deaths than vaccinating 60+ year-olds first for July availability, due to higher levels of natural immunity by that time. Sensitivity analysis supports the findings.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>Further research is urgently needed to determine which populations can benefit from using SARS-CoV-2 vaccines to interrupt transmission.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>Ontario Ministry of Colleges and Universities.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Research in context</jats:title>\n                  <jats:sec>\n                    <jats:title>Evidence before this study</jats:title>\n                    <jats:p>Whether to vaccinate individuals who cause the most transmission or those who are at highest risk of death is relevant to prioritizing COVID-19 vaccination. We searched PubMed and medRxiv for the terms COVID19, vaccin*, model, and priorit* up to September 24, 2020, with no date or language restrictions. We identified 4 papers on mathematical models of COVID-19 vaccine prioritization that explored the conditions under which different age groups should be vaccinated first. We did not find any coupled social-epidemiological models that capture feedback between social dynamics and epidemic trajectories.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Added value of this study</jats:title>\n                    <jats:p>The dynamic interaction between SARS-CoV-2 epidemics and the population response through scalable non-pharmaceutical interventions will continue to play a large role in the course of the pandemic, both before and after vaccines become available. Hence, social-epidemiological models may be useful. Our social-epidemiological model identifies the conditions under which COVID-19 deaths can be reduced most effectively by prioritizing older individuals first, versus other strategies designed to interrupt transmission. We explore how the best vaccination strategy varies depending on a wide range of socio-epidemiological and vaccine program parameters. We identify clear and interpretable conditions under which using COVID-19 vaccines to interrupt transmission can reduce mortality most effectively.</jats:p>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>Implications of all the available evidence</jats:title>\n                    <jats:p>Seroprevalence surveys before the onset of vaccination could measure population-level SARS-CoV-2 immunity. In populations where seropositivity is high due to previous waves, vaccinating to interrupt transmission may reduce deaths more effectively than targeting older individuals. More research is urgently required to evaluate how to prioritise vaccination in populations that have experienced one or more waves of COVID-19.</jats:p>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.25.20201889","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.25.310078","pub_date":"2020-9-25","title":"Sequence Analysis for SNP Detection and Phylogenetic Reconstruction of SARS-CoV-2 Isolated from Nigerian COVID-19 Cases","abstract":"Coronaviruses are a group of viruses that belong to the Family Coronaviridae, Genus Betacoronavirus. In December 2019, a new coronavirus disease (COVID-19) characterized by severe respiratory symptoms was discovered. The causative pathogen was a novel coronavirus known as 2019-nCoV and later as SARS-CoV-2. Within two months of its discovery, COVID-19 became a pandemic causing widespread morbidity and mortality. Whole genome sequence data of SARS-CoV-2 isolated from Nigerian COVID-19 cases were retrieved by downloading from GISAID database. A total of 18 sequences that satisfied quality assurance (length \u2265 29700 nts and number of unknown bases denoted as \u2018N\u2019 \u2264 5%) were used for the study. Multiple sequence alignment (MSA) was done in MAFFT (Version 7.471) while SNP calling was implemented in DnaSP (Version 6.12.03) respectively and then visualized in Jalview (Version 2.11.1.0). Phylogenetic analysis was with MEGA X software. Nigerian SARS-CoV-2 had 99.9% genomic similarity with four large conserved genomic regions. A total of 66 SNPs were identified out of which 31 were informative. Nucleotide diversity assessment gave Pi = 0.00048 and average SNP frequency of 2.22 SNPs per 1000 nts. Non-coding genomic regions particularly 5\u2019UTR and 3\u2019UTR had a SNP density of 3.77 and 35.4 respectively. The region with the highest SNP density was ORF10 with a frequency of 8.55 SNPs/1000 nts). Majority (72.2%) of viruses in Nigeria are of L lineage with preponderance of D614G mutation which accounted for 11 (61.1%) out of the 18 viral sequences. Nigeria SARS-CoV-2 revealed 3 major clades namely Oyo, Ekiti and Osun on a maximum likelihood phylogenetic tree. Nigerian SARS-CoV-2 reveals high mutation rate together with preponderance of L lineage and D614G mutants. Implication of these mutations for SARS-CoV-2 virulence and the need for more aggressive testing and treatment of COVID-19 in Nigeria is discussed. Additionally, attempt to produce testing kits for COVID-19 in Nigeria should consider the conserved regions identified in this study. Strict adherence to COVID-19 preventive measure is recommended in view of Nigerian SARS-CoV-2 phylogenetic clustering pattern, which suggests intensive community transmission possibly rooted in communal culture characteristic of many ethnicities in Nigeria.","version":"1.1","doi":"10.1101/2020.09.25.310078","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.312769","pub_date":"2020-9-25","title":"Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication","abstract":"Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE2, one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE2 receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE2 signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE2 signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients. Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NSAIDs function by inhibiting the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes are critical for the generation of prostaglandins, lipid molecules with diverse roles in maintaining homeostasis as well as regulating the inflammatory response. While COX-1/COX-2 signaling pathways have been shown to affect the replication of many viruses, their effect on SARS-CoV-2 infection remains unknown. We found that SARS-CoV-2 infection induced COX-2 expression in both human cell culture systems and mouse models. However, inhibition of COX-2 activity with NSAIDs did not affect SARS-CoV-2 entry or replication. Our findings suggest that COX-2 signaling may instead regulate the lung inflammation observed in COVID-19 patients, which is an important area for future studies.","version":"1.1","doi":"10.1101/2020.09.24.312769","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.20200394","pub_date":"2020-09-25","title":"Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of effective antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones providing an opportunity to integrate with contract tracing apps, medical providers, and electronic medical records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within one hour, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach can provide an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.</jats:p>","version":null,"doi":"10.1101/2020.09.24.20200394","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.25.309914","pub_date":"2020-9-25","title":"Prophylactic intranasal administration of a TLR2 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model","abstract":"Respiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. Rapid spread of virus through populations poses an enormous challenge for outbreak control. Like all respiratory viruses, the most recent novel human coronavirus SARS-CoV-2, initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19.","version":"1.1","doi":"10.1101/2020.09.25.309914","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.312595","pub_date":"2020-9-25","title":"A high-affinity RBD-targeting nanobody improves fusion partner\u2019s potency against SARS-CoV-2","abstract":"A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and \u2018greasy\u2019 site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.","version":"1.1","doi":"10.1101/2020.09.24.312595","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.230888","pub_date":"2020-9-25","title":"Computer Simulations of the interaction between SARS-CoV-2 spike glycoprotein and different surfaces","abstract":"A prominent feature of coronaviruses is the presence of a large glycoprotein spike protruding from a lipidic membrane. This glycoprotein spike determines the interaction of coronaviruses with the environment and the host. In this paper, we perform all atomic Molecular Dynamics simulations of the interaction between the SARS-CoV-2 trimeric glycoprotein spike and surfaces of materials. We considered a material with high hydrogen bonding capacity (cellulose) and a material capable of strong hydrophobic interactions (graphite). Initially, the spike adsorbs to both surfaces through essentially the same residues belonging to the receptor binding subunit of its three monomers. Adsorption onto cellulose stabilizes in this configuration, with the help of a large number of hydrogen bonds developed between cellulose and the three receptor binding domains (RBD) of the glycoprotein spike. In the case of adsorption onto graphite, the initial adsorption configuration is not stable and the surface induces a substantial deformation of the glycoprotein spike with a large number of adsorbed residues not pertaining to the binding subunits of the spike monomers.","version":"1.2","doi":"10.1101/2020.07.31.230888","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.20201061","pub_date":"2020-09-25","title":"Quantifying the impact of quarantine duration on COVID-19 transmission","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The numbers of confirmed cases of SARS-CoV-2 infection are increasing in many places. Consequently, the number of individuals placed into quarantine is increasing too. The large number of individuals in quarantine has high societal and economical costs, as well as infringing on the freedom of the individual. This has led to a vigorous debate about the duration of quarantine, particularly in light of the potentially low specificity of quarantine (i.e. low probability of quarantined individuals indeed being infected). We present a mathematical model that leverages empirically determined distributions of incubation period, infectivity, and generation time to quantify how the duration of quarantine affects transmission. With this model we address the impact of shortening the quarantine for returning travellers and traced contacts of confirmed cases, both in terms of prevented transmission and the ratio of prevented transmission to days spent in quarantine. We also consider the impact of i) test-and-release strategies; ii) additional hygiene measures imposed upon release after a negative test; iii) the development of symptoms during quarantine; iv) the relationship between quarantine duration and adherence; and v) the specificity of quarantine. When considering the benefit versus cost utility of quarantine, we find that the diminishing impact of longer quarantine on transmission prevention may support a quarantine duration below 10 days, particularly for returning travellers. A greater gain of utility can be achieved through a test-and-release strategy, and this can be even further strengthened by imposed hygiene measures post-release. We also find that unless a test-and-release strategy is considered, the specificity of quarantine does not affect the optimal duration of quarantine. Therefore, the argument that we should shorten quarantine because of lack of specificity is misguided.</jats:p>","version":null,"doi":"10.1101/2020.09.24.20201061","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.24.298851","pub_date":"2020-9-25","title":"ADPriboDB v2.0: An Updated Database of ADP-ribosylated Proteins","abstract":"ADP-ribosylation is a protein modification responsible for biological processes such as DNA repair, RNA regulation, cell cycle, and biomolecular condensate formation. Dysregulation of ADP-ribosylation is implicated in cancer, neurodegeneration, and viral infection. We developed ADPriboDB (adpribodb.leunglab.org) to facilitate studies in uncovering insights into the mechanisms and biological significance of ADP-ribosylation. ADPriboDB 2.0 serves as a one-stop repository comprising 48,346 entries and 9,097 ADP-ribosylated proteins, of which 6,708 were newly identified since the original database release. In this updated version, we provide information regarding the sites of ADP-ribosylation in 32,946 entries. The wealth of information allows us to interrogate existing databases or newly available data. For example, we found that ADP-ribosylated substrates are significantly associated with the recently identified human protein interaction networks associated with SARS-CoV-2, which encodes a conserved protein domain called macrodomain that binds and removes ADP-ribosylation. In addition, we create a new interactive tool to visualize the local context of ADP-ribosylation, such as structural and functional features as well as other post-translational modifications (e.g., phosphorylation, methylation and ubiquitination). This information provides opportunities to explore the biology of ADP-ribosylation and generate new hypotheses for experimental testing.","version":"1.1","doi":"10.1101/2020.09.24.298851","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.164384","pub_date":"2020-9-24","title":"Functional characterization of SARS-CoV-2 infection suggests a complex inflammatory response and metabolic alterations","abstract":"Covid-19, caused by the SARS-CoV-2 virus, has reached the category of a worldwide pandemic. Even though intensive efforts, no effective treatments or a vaccine are available. Molecular characterization of the transcriptional response in Covid-19 patients could be helpful to identify therapeutic targets. In this study, RNAseq data from peripheral blood mononuclear cell samples from Covid-19 patients and healthy controls was analyzed from a functional point of view using probabilistic graphical models. Two networks were built: one based on genes differentially expressed between healthy and infected individuals and another one based on the 2,000 most variable genes in terms of expression in order to make a functional characterization. In the network based on differentially expressed genes, two inflammatory response nodes with different tendencies were identified, one related to cytokines and chemokines, and another one related to bacterial infections. In addition, differences in metabolism, which were studied in depth using Flux Balance Analysis, were identified. SARS-CoV2-infection caused alterations in glutamate, methionine and cysteine, and tetrahydrobiopterin metabolism. In the network based on 2,000 most variable genes, also two inflammatory nodes with different tendencies between healthy individuals and patients were identified. Similar to the other network, one was related to cytokines and chemokines. However, the other one, lower in Covid-19 patients, was related to allergic processes and self-regulation of the immune response. Also, we identified a decrease in T cell node activity and an increase in cell division node activity. In the current absence of treatments for these patients, functional characterization of the transcriptional response to SARS-CoV-2 infection could be helpful to define targetable processes. Therefore, these results may be relevant to propose new treatments.","version":"1.2","doi":"10.1101/2020.06.22.164384","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.311977","pub_date":"2020-9-24","title":"Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2","abstract":"Understanding the pathogenesis of the SARS-CoV-2 infection is key to develop preventive and therapeutic strategies against COVID-19, in the case of severe illness but also when the disease is mild. The use of appropriate experimental animal models remains central in the in-vivo exploration of the physiopathology of infection and antiviral strategies. This study describes SARS-CoV-2 intra-nasal infection in ferrets and hamsters with low doses of low-passage SARS-CoV-2 clinical French isolate UCN19, describing infection levels, excretion, immune responses and pathological patterns in both animal species. Individual infection with 103 pfu SARS-CoV-2 induced a more severe disease in hamsters than in ferrets. Viral RNA was detected in the lungs of hamsters but not of ferrets and in the brain (olfactive and/or spinal bulbs) of both species. Overall, the clinical disease remained mild, with serological responses detected from 7 days and 10 days post inoculation in hamsters and ferrets respectively. Virus became undetectable and pathology resolved within 14 days. The kinetics and levels of infection can be used in ferrets and hamsters as experimental models for understanding the pathogenicity of SARS-CoV-2, and testing the protective effect of drugs.","version":"1.1","doi":"10.1101/2020.09.24.311977","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.312355","pub_date":"2020-9-24","title":"A vaccine built from potential immunogenic pieces derived from the SARS-CoV-2 spike glycoprotein","abstract":"Coronavirus Disease 2019 (COVID-19) represents a new global threat demanding a multidisciplinary effort to fight its etiological agent\u2014severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this regard, immunoinformatics may aid to predict prominent immunogenic regions from critical SARS-CoV-2 structural proteins, such as the spike (S) glycoprotein, for their use in prophylactic or therapeutic interventions against this rapidly emerging coronavirus. Accordingly, in this study, an integrated immunoinformatics approach was applied to identify cytotoxic T cell (CTC), T helper cell (THC), and Linear B cell (BC) epitopes from the S glycoprotein in an attempt to design a high-quality multi-epitope vaccine. The best CTC, THC, and BC epitopes showed high viral antigenicity, lack of allergenic or toxic residues, and suitable HLA-viral peptide interactions. Remarkably, SARS-CoV-2 receptor-binding domain (RBD) and its receptor-binding motif (RBM) harbour several potential epitopes. The structure prediction, refinement, and validation data indicate that the multi-epitope vaccine has an appropriate conformation and stability. Three conformational epitopes and an efficient binding between Toll-like receptor 4 (TLR4) and the vaccine model were observed. Importantly, the population coverage analysis showed that the multi-epitope vaccine could be used globally. Notably, computer-based simulations suggest that the vaccine model has a robust potential to evoke and maximize both immune effector responses and immunological memory to SARS-CoV-2. Further research is needed to accomplish with the mandatory international guidelines for human vaccine formulations.","version":"1.1","doi":"10.1101/2020.09.24.312355","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.143438","pub_date":"2020-9-24","title":"A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection","abstract":"SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE2. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development. Llama-derived single-domain antibodies (nanobodies, \u223c15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 \u03bcg mL\u22121). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL\u22121) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.","version":"1.2","doi":"10.1101/2020.06.09.143438","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.312298","pub_date":"2020-9-24","title":"Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses","abstract":"The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.","version":"1.1","doi":"10.1101/2020.09.24.312298","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.24.311027","pub_date":"2020-9-24","title":"S-Trimer, a COVID-19 subunit vaccine candidate, induces protective immunity in nonhuman primates","abstract":"SARS-CoV-2 is the underlying cause for the COVID-19 pandemic. Like most enveloped RNA viruses, SARS-CoV-2 uses a homotrimeric surface antigen to gain entry into host cells. Here we describe S-Trimer, a native-like trimeric subunit vaccine candidate for COVID-19 based on Trimer-Tag technology. Immunization of S-Trimer with either AS03 (oil-in-water emulsion) or CpG 1018 (TLR9 agonist) plus alum adjuvants induced high-levels of neutralizing antibodies and Th1-biased cellular immune responses in animal models. Moreover, rhesus macaques immunized with adjuvanted S-Trimer were protected from SARS-CoV-2 challenge compared to vehicle controls, based on clinical observations and reduction of viral loads in lungs. Trimer-Tag may be an important new platform technology for scalable production and rapid development of safe and effective subunit vaccines against current and future emerging RNA viruses.","version":"1.1","doi":"10.1101/2020.09.24.311027","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.23.309849","pub_date":"2020-9-23","title":"Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.23.309849","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.23.309948","pub_date":"2020-9-23","title":"Respiratory disease in cats associated with human-to-cat transmission of SARS-CoV-2 in the UK","abstract":"Two cats from different COVID-19-infected households in the UK were found to be infected with SARS-CoV-2 from humans, demonstrated by immunofluorescence, in situ hybridisation, reverse transcriptase quantitative PCR and viral genome sequencing. Lung tissue collected post-mortem from cat 1 displayed pathological and histological findings consistent with viral pneumonia and tested positive for SARS-CoV-2 antigens and RNA. SARS-CoV-2 RNA was detected in an oropharyngeal swab collected from cat 2 that presented with rhinitis and conjunctivitis. High throughput sequencing of the virus from cat 2 revealed that the feline viral genome contained five single nucleotide polymorphisms (SNPs) compared to the nearest UK human SARS-CoV-2 sequence. An analysis of cat 2\u2019s viral genome together with nine other feline-derived SARS-CoV-2 sequences from around the world revealed no shared catspecific mutations. These findings indicate that human-to-cat transmission of SARS-CoV-2 occurred during the COVID-19 pandemic in the UK, with the infected cats developing mild or severe respiratory disease. Given the versatility of the new coronavirus, it will be important to monitor for human-to-cat, cat-to-cat and cat-to-human transmission.","version":"1.1","doi":"10.1101/2020.09.23.309948","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.23.309294","pub_date":"2020-9-23","title":"A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2","abstract":"Mutations and transient conformational movements of receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable, present immune escape routes to SARS-CoV-2. To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, combination of RBD-targeting NAbs and NTD-binding NAb, FC05, dramatically enhanced the neutralization potency in cell-based assays and animal model. Results of competitive SPR assays and cryo-EM structures of Fabs bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in NTD and RBD of S, when immunized in rabbits elicited potent protective immune responses against SARS-CoV-2. These results provide a proof-of-concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD. Immunogens identified in the NTD and RBD of the SARS-CoV-2 spike protein using a cocktail of non-competing NAbs when injected in rabbits elicited a potent protective immune response against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.23.309294","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.308783","pub_date":"2020-9-23","title":"In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein","abstract":"Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.","version":"1.1","doi":"10.1101/2020.09.22.308783","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.308965","pub_date":"2020-9-23","title":"Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection","abstract":"Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.22.308965","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.20198911","pub_date":"2020-09-23","title":"Environmental impact of Personal Protective Equipment supplied to health and social care services in England in the first six months of the COVID-19 pandemic","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>Use of Personal Protective Equipment (PPE) has been central to controlling spread of SARS-CoV2. This study aims to quantify the environmental impact of this, and to model strategies for its reduction.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Life cycle assessment was used to determine environmental impacts of PPE supplied to health and social care in England during the first six months of the COVID-19 pandemic. The base scenario assumed all products were single-use, air freighted, and disposed via clinical waste. Scenario modelling was used to determine the effect of 1) switching mode of, or eliminating, international travel during supply, 2) reducing glove use 3) using reusable alternatives, 4) maximal recycling.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    The carbon footprint of PPE supplied during the study period totalled 158,838 tonnes CO\n                    <jats:sub>2</jats:sub>\n                    e, with greatest contributions from gloves, aprons, face shields, and Type IIR surgical masks. The estimated damage to human health was 314 DALYs (disability adjusted life years), impact on ecosystems was 0.67 species.year (loss of local species per year), and impact on resource depletion costing US $ 20.4 million.\n                  </jats:p>\n                  <jats:p>Scenario modelling indicated one-third of the carbon footprint could be avoided through switching to shipping, and by 41% through manufacturing PPE in the UK. The carbon footprint was reduced by 83% compared with the base scenario through a combination of UK manufacturing, reducing glove use, using reusable gowns and reuse of face shields, and maximal recycling, estimated to save 259 DALYS, 0.54 species.year, and US $ 15 million due to resource depletion.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The environmental impact of PPE could be reduced through shipping supplies or domestic manufacture, rationalising glove use, using reusables where possible, and optimising waste management.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>SUMMARY BOX</jats:title>\n                  <jats:sec>\n                    <jats:title>What is already known on this topic</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>The current COVID-19 pandemic has seen a massive global increase in the use and manufacture of PPE which has contributed, with other measures, to the reduction in transmission of the virus in many countries.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                  <jats:sec>\n                    <jats:title>What this study adds</jats:title>\n                    <jats:list list-type='bullet'>\n                      <jats:list-item>\n                        <jats:p>\n                          The carbon footprint of PPE supplied to health and social care in England in the first six months of the COVD-19 pandemic was 158,848 tonnes CO\n                          <jats:sub>2</jats:sub>\n                          e, equivalent to around 65,500 return flights from London to New York.\n                        </jats:p>\n                      </jats:list-item>\n                      <jats:list-item>\n                        <jats:p>The environmental impact of PPE could be reduced through shipping supplies or domestic manufacture, rationalising glove use, using reusables where possible, and optimising waste processing.</jats:p>\n                      </jats:list-item>\n                    </jats:list>\n                  </jats:sec>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.21.20198911","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.22.20196048","pub_date":"2020-09-23","title":"College Openings in the United States Increased Mobility and COVID-19 Incidence","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>\n                  School and college reopening-closure policies are considered one of the most promising non-pharmaceutical interventions for mitigating infectious diseases. Nonetheless, the effectiveness of these policies is still debated, largely due to the lack of empirical evidence on behavior during implementation. We examined U.S. college reopenings\u2019 association with changes in human mobility within campuses and in COVID-19 incidence in the counties of the campuses over a twenty-week period around college reopenings in the Fall of 2020. We used an integrative framework, with a difference-in-differences design comparing areas with a college campus, before and after reopening, to areas without a campus and a Bayesian approach to estimate the daily reproductive number (\n                  <jats:italic>R</jats:italic>\n                  <jats:sub>\n                    <jats:italic>t</jats:italic>\n                  </jats:sub>\n                  ). We found that college reopenings were associated with increased campus mobility, and increased COVID-19 incidence by 3.4 cases per 100,000 (95% confidence interval [CI]: 1.5 \u2013 5.4), or a 25% increase relative to the pre-period mean. This reflected our estimate of increased transmission locally after reopening. A greater increase in county COVID-19 incidence resulted from campuses that drew students from counties with high COVID-19 incidence in the weeks before reopening (\n                  <jats:italic>\u03c7</jats:italic>\n                  <jats:sup>2</jats:sup>\n                  (2) = 10.19,\n                  <jats:italic>p</jats:italic>\n                  = 0.006). Even by Fall of 2021, large shares of populations remained unvaccinated, increasing the relevance of understanding non-pharmaceutical decisions over an extended period of a pandemic. Our study sheds light on movement and social mixing patterns during the closure-reopening of colleges during a public health threat, and offers strategic instruments for benefit-cost analyses of school reopening/closure policies.\n                </jats:p>","version":null,"doi":"10.1101/2020.09.22.20196048","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.22.308023","pub_date":"2020-9-22","title":"Humoral response to SARS-CoV-2 by healthy and sick dogs during COVID-19 pandemic in Spain","abstract":"COVID-19 is a zoonotic disease originated by SARS-CoV-2. Infection of animals with SARS-CoV-2 are being reported during last months, and also an increase of severe lung pathologies in domestic dogs has been detected by veterinarians in Spain. Therefore it is necessary to describe the pathological processes in those animals that show symptoms similar to those described in humans affected by COVID-19. The potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. Forty animals with pulmonary pathologies were studied by chest X-ray, ultrasound study, and computed tomography. Nasopharyngeal and rectal swab were analyzed to detect canine pathogens, including SARS-CoV-2. Twenty healthy dogs living in SARS-CoV-2 positive households were included. Immunoglobulin detection by different immunoassays was performed. Our findings show that sick dogs presented severe alveolar or interstitial pattern, with pulmonary opacity, parenchymal abnormalities, and bilateral lesions. Forty dogs were negative for SARS-CoV-2 but Mycoplasma spp. was detected in 26 of 33 dogs. Five healthy and one pathological dog presented IgG against SARS-CoV-2. Here we report that despite detecting dogs with IgG \u03b1-SARS-CoV-2, we never obtained a positive RT-qPCR, not even in dogs with severe pulmonary disease; suggesting that even in the case of a canine infection transmission would be unlikely. Moreover, dogs living in COVID-19 positive households could have been more exposed to be infected during outbreaks.","version":"1.1","doi":"10.1101/2020.09.22.308023","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.307751","pub_date":"2020-9-22","title":"High prevalence of SARS-CoV-2 antibodies in pets from COVID-19+ households","abstract":"In a survey of household cats and dogs of laboratory-confirmed COVID-19 patients, we found a high seroprevalence of SARS-CoV-2 antibodies, ranging from 21% to 53%, depending on the positivity criteria chosen. Seropositivity was significantly greater among pets from COVID-19+ households compared to those with owners of unknown status. Our results highlight the potential role of pets in the spread of the epidemic.","version":"1.1","doi":"10.1101/2020.09.22.307751","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.308668","pub_date":"2020-9-22","title":"Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography","abstract":"The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication, possessing an unusual catalytic dyad composed of His41 and Cys145. A long-standing question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are. Here, we present the room-temperature neutron structure of 3CL Mpro from SARS-CoV-2, which allows direct determination of hydrogen atom positions and, hence, protonation states. The catalytic site natively adopts a zwitterionic reactive state where His41 is doubly protonated and positively charged, and Cys145 is in the negatively charged thiolate state. The neutron structure also identified the protonation states of other amino acid residues, mapping electrical charges and intricate hydrogen bonding networks in the SARS-CoV-2 3CL Mpro active site cavity and dimer interface. This structure highlights the ability of neutron protein crystallography for experimentally determining protonation states at near-physiological temperature \u2013 the critical information for structure-assisted and computational drug design.","version":"1.1","doi":"10.1101/2020.09.22.308668","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.190348","pub_date":"2020-9-22","title":"Role of SARS-CoV-2 in altering the RNA binding protein and miRNA directed post-transcriptional regulatory networks in humans","abstract":"The outbreak of a novel coronavirus SARS-CoV-2 responsible for COVID-19 pandemic has caused worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2 mediated alteration on post-transcriptional gene regulation across human tissues remains elusive. In this study, we analyze publicly available genomic datasets to systematically dissect the crosstalk and dysregulation of human post-transcriptional regulatory networks governed by RNA binding proteins (RBPs) and micro-RNAs (miRs), due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2 encoded proteins directly interact with 51 human RBPs of which majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed a functional analysis of differentially expressed genes in mock-treated versus SARS-CoV-2 infected lung cells that revealed enrichment for immune response, cytokine-mediated signaling, and metabolism associated genes. This study also characterized the alternative splicing events in SARS-CoV-2 infected cells compared to control demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to viral infection. Motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed the enrichment for RBPs such as SRSFs, PCBPs, ELAVs, and HNRNPs suggesting the sponging of RBPs by SARS-CoV-2 genome. A similar analysis to study the interactions of miRs with SARS-CoV-2 revealed functionally important miRs that were highly expressed in immune cells, suggesting that these interactions may contribute to the progression of the viral infection and modulate host immune response across other human tissues. Given the need to understand the interactions of SARS-CoV-2 with key post-transcriptional regulators in the human genome, this study provides a systematic computational analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs, across tissues types during SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.07.06.190348","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.300038","pub_date":"2020-9-22","title":"Positive selection within the genomes of SARS-CoV-2 and other Coronaviruses independent of impact on protein function","abstract":"The emergence of a novel coronavirus (SARS-CoV-2) associated with severe acute respiratory disease (COVID-19) has prompted efforts to understand the genetic basis for its unique characteristics and its jump from non-primate hosts to humans. Tests for positive selection can identify apparently nonrandom patterns of mutation accumulation within genomes, highlighting regions where molecular function may have changed during the origin of a species. Several recent studies of the SARS-CoV-2 genome have identified signals of conservation and positive selection within the gene encoding Spike protein based on the ratio of synonymous to nonsynonymous substitution. Such tests cannot, however, detect changes in the function of RNA molecules. Here we apply a test for branch-specific oversubstitution of mutations within narrow windows of the genome without reference to the genetic code. We recapitulate the finding that the gene encoding Spike protein has been a target of both purifying and positive selection. In addition, we find other likely targets of positive selection within the genome of SARS-CoV-2, specifically within the genes encoding Nsp4 and Nsp16. Homology-directed modeling indicates no change in either Nsp4 or Nsp16 protein structure relative to the most recent common ancestor. Thermodynamic modeling of RNA stability and structure, however, indicates that RNA secondary structure within both genes in the SARS-CoV-2 genome differs from those of RaTG13, the reconstructed common ancestor, and Pan-CoV-GD (Guangdong). These SARS-CoV-2-specific mutations may affect molecular processes mediated by the positive or negative RNA molecules, including transcription, translation, RNA stability, and evasion of the host innate immune system. Our results highlight the importance of considering mutations in viral genomes not only from the perspective of their impact on protein structure, but also how they may impact other molecular processes critical to the viral life cycle.","version":"1.2","doi":"10.1101/2020.09.16.300038","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.307439","pub_date":"2020-9-22","title":"Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution","abstract":"In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the \u201cdown\u201d conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.","version":"1.1","doi":"10.1101/2020.09.21.307439","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.307975","pub_date":"2020-9-22","title":"Mass cytometry and artificial intelligence define CD169 as a specific marker of SARS-CoV2-induced acute respiratory distress syndrome","abstract":"Acute respiratory distress syndrome (ARDS) is the main complication of COVID-19, requiring admission to Intensive Care Unit (ICU). Despite recent immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS specifically differs from other causes of ARDS remains unknown, To address this question, we built 3 cohorts of patients categorized in COVID-19negARDSpos, COVID-19posARDSpos, and COVID-19posARDSneg, and compared their immune landscape analyzed by high-dimensional mass cytometry on peripheral blood followed by artificial intelligence analysis. A cell signature associating S100A9/calprotectin-producing CD169pos monocytes, plasmablasts, and Th1 cells was specifically found in COVID-19posARDSpos, unlike COVID-19negARDSpos patients. Moreover, this signature was shared by COVID-19posARDSneg patients, suggesting severe COVID-19 patients, whatever they experienced or not ARDS, displayed similar immune dysfunctions. We also showed an increase in CD14posHLA-DRlow and CD14lowCD16pos monocytes correlated to the occurrence of adverse events during ICU stay. Our study demonstrates that COVID-19-associated ARDS display a specific immune profile, and might benefit from personalized therapy in addition to standard ARDS management. COVID-19-associated ARDS is biologically distinct from other causes of ARDS.","version":"1.1","doi":"10.1101/2020.09.22.307975","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222109","pub_date":"2020-9-22","title":"Development of Remdesivir as a Dry Powder for Inhalation by Thin Film Freezing","abstract":"Remdesivir exhibits in vitro activity against SARS-CoV-2 and was granted approval for Emergency Use. To maximize delivery to the lungs, we formulated remdesivir as a dry powder for inhalation using thin film freezing (TFF). TFF produces brittle matrix nanostructured aggregates that are sheared into respirable low-density microparticles upon aerosolization from a passive dry powder inhaler. In vitro aerodynamic testing demonstrated that drug loading and excipient type affected the aerosol performance of remdesivir. Remdesivir combined with optimal excipients exhibited desirable aerosol performance (up to 93.0% FPF; 0.82\u03bcm MMAD). Remdesivir was amorphous after the TFF process, which benefitted drug dissolution in simulated lung fluid. TFF remdesivir formulations are stable after one-month storage at 25 \u00b0C/60%RH. In vivo pharmacokinetic evaluation showed that TFF-remdesivir-leucine was poorly absorbed into systemic circulation while TFF-remdesivir-Captisol\u00ae demonstrated increased systemic uptake compared to leucine. Remdesivir was hydrolyzed to the nucleoside analog GS-441524 in lung, and levels of GS-441524 were greater in lung with the leucine formulation compared to Captisol\u00ae. In conclusion, TFF technology produces high potency remdesivir dry powder formulations for inhalation suitable to treat patients with COVID-19 on an outpatient basis and earlier in the disease course where effective antiviral therapy can reduce related morbidity and mortality.","version":"1.2","doi":"10.1101/2020.07.26.222109","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.22.307637","pub_date":"2020-9-22","title":"Why the COVID-19 pandemic is a traumatic stressor","abstract":"The COVID-19 pandemic does not fit into prevailing Post-traumatic Stress Disorder (PTSD) models, or diagnostic criteria, yet emerging research shows traumatic stress symptoms as a result of this ongoing global stressor. Current pathogenic event models focus on past, and largely direct, trauma exposure to certain kinds of life-threatening events. Nevertheless, among a sample of online participants (N = 1,040) in five western countries, we found participants had PTSD-like symptoms for events that had not happened and when participants had been directly (e.g., contact with virus) or indirectly exposed to COVID-19 (e.g., via media). Moreover, 13.2% of our sample were likely PTSD-positive, despite types of COVID-19 \u201cexposure\u201d (e.g., lockdown) not fitting DSM-5 criteria. The emotional impact of \u201cworst\u201d experienced/anticipated events best predicted PTSD-like symptoms. Our findings add to existing literature supporting a pathogenic event memory model of traumatic stress.","version":"1.1","doi":"10.1101/2020.09.22.307637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.306720","pub_date":"2020-9-21","title":"Investigation of COVID-19 comorbidities reveals genes and pathways coincident with the SARS-CoV-2 viral disease","abstract":"The emergence of the SARS-CoV-2 virus and subsequent COVID-19 pandemic initiated intense research into the mechanisms of action for this virus. It was quickly noted that COVID-19 presents more seriously in conjunction with other human disease conditions such as hypertension, diabetes, and lung diseases. We conducted a bioinformatics analysis of COVID-19 comorbidity-associated gene sets, identifying genes and pathways shared among the comorbidities, and evaluated current knowledge about these genes and pathways as related to current information about SARS-CoV-2 infection. We performed our analysis using GeneWeaver (GW), Reactome, and several biomedical ontologies to represent and compare common COVID-19 comorbidities. Phenotypic analysis of shared genes revealed significant enrichment for immune system phenotypes and for cardiovascular-related phenotypes, which might point to alleles and phenotypes in mouse models that could be evaluated for clues to COVID-19 severity. Through pathway analysis, we identified enriched pathways shared by comorbidity datasets and datasets associated with SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.09.21.306720","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.213926","pub_date":"2020-9-21","title":"The discovery of a recombinant SARS2-like CoV strain provides insights into SARS and COVID-19 pandemics","abstract":"In December 2019, the world awoke to a new zoonotic strain of coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the present study, we identified key recombination regions and mutation sites cross the SARS-CoV-2, SARS-CoV and SARS-like CoV clusters of betacoronavirus subgroup B. Based on the analysis of these recombination events, we proposed that the Spike protein of SARS-CoV-2 may have more than one specific receptor for its function. In addition, we reported\u2014for the first time\u2014a recombination event of ORF8 at the whole-gene level in a bat and ultimately determined that ORF8 enhances the viral replication. In conjunction with our previous discoveries, we found that receptor binding abilities, junction furin cleavage sites (FCSs), strong first ribosome binding sites (RBSs) and enhanced ORF8s are main factors contributing to transmission, virulence and host adaptability of CoVs. Junction FCSs and enhanced ORF8s increase the efficiencies in viral entry into cells and replication, respectively while strong first RBSs enhance the translational initiation. The strong recombination ability of CoVs integrated these factors to generate multiple recombinant strains, two of which evolved into SARS-CoV and SARS-CoV-2 by nature selection, resulting in the SARS and COVID-19 pandemics.","version":"1.2","doi":"10.1101/2020.07.22.213926","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.23.107334","pub_date":"2020-9-21","title":"Lung epithelial stem cells express SARS-CoV-2 entry factors: implications for COVID-19","abstract":"SARS-CoV-2 can infiltrate the lower respiratory tract, resulting in severe respiratory failure and a high death rate. Normally, the airway and alveolar epithelium can be rapidly reconstituted by multipotent stem cells after episodes of infection. Here, we analyzed published RNA-seq datasets and demonstrated that cells of four different lung epithelial stem cell types express SARS-CoV-2 entry factors, including Ace2. Thus, stem cells can be potentially infected by SARS-CoV-2, which may lead to defects in regeneration capacity partially accounting for the severity of SARS-CoV-2 infection and its consequences.","version":"1.2","doi":"10.1101/2020.05.23.107334","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.20.300574","pub_date":"2020-9-21","title":"Common genetic variation in humans impacts in vitro susceptibility to SARS-CoV-2 infection","abstract":"The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant inter-individual variability. In addition to showing more disease in males, the elderly, and individuals with underlying co-morbidities, SARS-CoV-2 can seemingly render healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants also impact vulnerability to infection. Here we apply human induced pluripotent stem cell (hiPSC)-based models and CRISPR-engineering to explore the host genetics of SARS-CoV-2. We demonstrate that a single nucleotide polymorphism (rs4702), common in the population at large, and located in the 3\u2019UTR of the protease FURIN, impacts alveolar and neuron infection by SARS-CoV-2 in vitro. Thus, we provide a proof-of-principle finding that common genetic variation can impact viral infection, and thus contribute to clinical heterogeneity in SARS-CoV-2. Ongoing genetic studies will help to better identify high-risk individuals, predict clinical complications, and facilitate the discovery of drugs that might treat disease.","version":"1.2","doi":"10.1101/2020.09.20.300574","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.305441","pub_date":"2020-9-21","title":"A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody","abstract":"Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.","version":"1.1","doi":"10.1101/2020.09.21.305441","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.296814","pub_date":"2020-9-21","title":"Phylogenomics reveals multiple introductions and early spread of SARS-CoV-2 into Peru","abstract":"Peru has become one of the countries with the highest mortality rate from the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To investigate early transmission event and genomic diversity of SARS-CoV-2 isolates circulating in Peru, we analyzed a total of 3472 SARS-CoV-2 genomes, from which 149 ones were from Peru. Phylogenomic analysis revealed multiple and independent introductions of the virus mainly from Europe and Asia. In addition, we found evidence for community-driven transmission of SARS-CoV-2 as suggested by clusters of related viruses found in patients living in different Peru regions.","version":"1.2","doi":"10.1101/2020.09.14.296814","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.306837","pub_date":"2020-9-21","title":"Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection","abstract":"The COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans, from asymptomatic or mild disease to severe disease that can require mechanical ventilation. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from indiviuals that go on to become infected with SARS-CoV-2. Here, we utilized data from a screen of genetically diverse mice from the Collaborative Cross (CC) infected with SARS-CoV to determine whether circulating baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Further, early control of virus in the lung correlates with an increased abundance of activated CD4 and CD8 T cells and regulatory T cells prior to infections across strains. A basal propensity of T cells to express IFNg and IL17 over TNFa also correlated with early viral control. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. While future studies of human samples prior to infection with SARS-CoV-2 are required, our studies in mice with SARS-CoV serve as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease. We used a screen of genetically diverse mice from the Collaborative Cross infected with mouse-adapted SARS-CoV in combination with comprehensive pre-infection immunophenotyping to identify baseline circulating immune correlates of severe virologic and clinical outcomes upon SARS-CoV infection.","version":"1.1","doi":"10.1101/2020.09.21.306837","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.207563","pub_date":"2020-9-21","title":"Neutralization Assay with SARS-CoV-1 and SARS-CoV-2 Spike Pseudotyped Murine Leukemia Virions","abstract":"Antibody neutralization is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal MLV genome encoding firefly luciferase. These pseudovirions provide a practical means of assessing immune responses under laboratory conditions consistent with biocontainment level 2.","version":"1.2","doi":"10.1101/2020.07.17.207563","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.299776","pub_date":"2020-9-21","title":"An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor","abstract":"Designing covalent inhibitors is a task of increasing importance in drug discovery. Efficiently designing irreversible inhibitors, though, remains challenging. Here, we present covalentizer, a computational pipeline for creating irreversible inhibitors based on complex structures of targets with known reversible binders. For each ligand, we create a custom-made focused library of covalent analogs. We use covalent docking, to dock these tailored covalent libraries and to find those that can bind covalently to a nearby cysteine while keeping some of the main interactions of the original molecule. We found ~11,000 cysteines in close proximity to a ligand across 8,386 protein-ligand complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In prospective evaluation against a panel of kinases, five out of nine predicted covalent inhibitors showed IC50 between 155 nM - 4.2 \u03bcM. Application of the protocol to an existing SARS-CoV-1 Mpro reversible inhibitor led to a new acrylamide inhibitor series with low micromolar IC50 against SARS-CoV-2 Mpro. The docking prediction was validated by 11 co-crystal structures. This is a promising lead series for COVID-19 antivirals. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.","version":"1.1","doi":"10.1101/2020.09.21.299776","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.20.297242","pub_date":"2020-9-21","title":"Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry","abstract":"Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. The single mutation that allowed for mouse ACE2 to serve as a viral receptor provides a potential avenue for the development of SARS-CoV-2 mouse model.","version":"1.1","doi":"10.1101/2020.09.20.297242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.21.300913","pub_date":"2020-9-21","title":"Rapid detection of inter-clade recombination in SARS-CoV-2 with Bolotie","abstract":"The ability to detect recombination in pathogen genomes is crucial to the accuracy of phylogenetic analysis and consequently to forecasting the spread of infectious diseases and to developing therapeutics and public health policies. However, previous methods for detecting recombination and reassortment events cannot handle the computational requirements of analyzing tens of thousands of genomes, a scenario that has now emerged in the effort to track the spread of the SARS-CoV-2 virus. Furthermore, the low divergence of near-identical genomes sequenced in short periods of time presents a statistical challenge not addressed by available methods. In this work we present Bolotie, an efficient method designed to detect recombination and reassortment events between clades of viral genomes. We applied our method to a large collection of SARS-CoV-2 genomes and discovered hundreds of isolates that are likely of a recombinant origin. In cases where raw sequencing data was available, we were able to rule out the possibility that these samples represented co-infections by analyzing the underlying sequence reads. Our findings further show that several recombinants appear to have persisted in the population.","version":"1.2","doi":"10.1101/2020.09.21.300913","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.083048","pub_date":"2020-9-21","title":"A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR","abstract":"The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.","version":"1.3","doi":"10.1101/2020.05.07.083048","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.20194787","pub_date":"2020-09-21","title":"COVID-19 herd immunity in the Brazilian Amazon","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The herd immunity threshold is the proportion of a population that must be immune to an infectious disease, either by natural infection or vaccination such that, in the absence of additional preventative measures, new cases decline and the effective reproduction number falls below unity\n                  <jats:sup>1</jats:sup>\n                  . This fundamental epidemiological parameter is still unknown for the recently-emerged COVID-19, and mathematical models have predicted very divergent results\n                  <jats:sup>2,3</jats:sup>\n                  . Population studies using antibody testing to infer total cumulative infections can provide empirical evidence of the level of population immunity in severely affected areas. Here we show that the transmission of SARS-CoV-2 in Manaus, located in the Brazilian Amazon, increased quickly during March and April and declined more slowly from May to September. In June, one month following the epidemic peak, 44% of the population was seropositive for SARS-CoV-2, equating to a cumulative incidence of 52%, after correcting for the false-negative rate of the antibody test. The seroprevalence fell in July and August due to antibody waning. After correcting for this, we estimate a final epidemic size of 66%. Although non-pharmaceutical interventions, plus a change in population behavior, may have helped to limit SARS-CoV-2 transmission in Manaus, the unusually high infection rate suggests that herd immunity played a significant role in determining the size of the epidemic.\n                </jats:p>","version":null,"doi":"10.1101/2020.09.16.20194787","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.21.306357","pub_date":"2020-9-21","title":"Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19","abstract":"Less than a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 22 million people worldwide with a death toll approaching 1 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 \u00c5 and 2.6 \u00c5 respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.","version":"1.1","doi":"10.1101/2020.09.21.306357","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.18.304493","pub_date":"2020-9-19","title":"An immunodominance hierarchy exists in CD8+ T cell responses to HLA-A*02:01-restricted epitopes identified from the non-structural polyprotein 1a of SARS-CoV-2","abstract":"COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-\u03b3-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. For the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.","version":"1.1","doi":"10.1101/2020.09.18.304493","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.18.302901","pub_date":"2020-9-18","title":"SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism","abstract":"The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5\u2019 untranslated region (5\u2019 UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5\u2019 UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5\u2019 UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5\u2019 UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5\u2019 UTR may be targeted for anti-COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2020.09.18.302901","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.18.301952","pub_date":"2020-9-18","title":"A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro","abstract":"Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.","version":"1.1","doi":"10.1101/2020.09.18.301952","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.20190694","pub_date":"2020-09-18","title":"KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein\n                  <jats:italic>in vitro</jats:italic>\n                  . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.\n                </jats:p>","version":null,"doi":"10.1101/2020.09.16.20190694","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.14.20186494","pub_date":"2020-09-18","title":"Efficacy of commercial mouth-rinses on SARS-CoV-2 viral load in saliva: Randomized Control Trial in Singapore","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The presence of high SARS-coronavirus 2 (SARS-CoV-2) titres in saliva may result in transmission of the virus and increase the risk of COVID-19 infection. This is particularly important as significant amounts of aerosols are generated during dental procedures, posing risk to dental care personnel and patients. Thus, reducing the titres of SARS-CoV-2 in the saliva of infected patients could be one of the key approaches to reduce the risk of COVID-19 transmission during dental procedures. In this randomised control trial, the efficacy of three commercial mouth-rinse viz. povidone-iodine (PI), chlorhexidine gluconate (CHX) and cetylpyridinium chloride (CPC), in reducing the salivary SARS-CoV-2 viral load in COVID-19 positive patients were compared with water. A total of 36 COVID-19 positive patients were recruited, of which 16 patients were randomly assigned to four groups\u2014 PI group (n=4), CHX group (n=6), CPC group (n=4) and water as control group (n=2). Saliva samples were collected from all patients at baseline and at 5 min, 3 h and 6 h post-application of mouth-rinses/water. The samples were subjected to SARS-CoV-2 RT-PCR analysis. The fold change of Ct values were significantly increased in CPC group at 5 minutes and 6 h time points (p&lt;0.05), while it showed significant increase at 6 h time point for PI group (p&lt;0.01). Considering Ct values as an indirect method of arbitrarily quantifying the viral load, it can be postulated that CPC mouth-rinse can decrease the salivary SARS-CoV-2 levels within 5 minutes of use, compared to water rinsing. The effect of decreasing salivary load with CPC and PI mouth-rinsing was observed to be sustained at 6 h time point. Within the limitation of the current study, it can be concluded that use of CPC and PI formulated commercial mouth-rinses, with its sustained effect on reducing salivary SARS-CoV-2 level, may be useful as a pre-procedural rinse to help reduce the transmission of COVID-19.</jats:p>","version":null,"doi":"10.1101/2020.09.14.20186494","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.18.302398","pub_date":"2020-9-18","title":"Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells","abstract":"The SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with currently 29 million confirmed cases and close to a million deaths. At this time, there are no FDA-approved vaccines or therapeutics for COVID-19, but Emergency Use Authorization has been granted for remdesivir, a broad-spectrum antiviral nucleoside analog. However, remdesivir is only moderately efficacious against SARS-CoV-2 in the clinic, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. We identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5 A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir\u2019s apparent potency 25-fold. We therefore suggest that the FDA-approved Hepatitis C therapeutics Epclusa (velpatasvir/sofosbuvir) and Zepatier (elbasvir/grazoprevir) should be fast-tracked for clinical evaluation in combination with remdesivir to improve treatment of acute SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2020.09.18.302398","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.202549","pub_date":"2020-9-18","title":"Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro","abstract":"The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-CoV and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, facilitates the attachment of viral particles to the cell surface to promote cell entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry, and reveals drugs capable of targeting this important step in the viral life cycle.","version":"1.2","doi":"10.1101/2020.07.14.202549","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.272328","pub_date":"2020-9-18","title":"Potential impact on coagulopathy of gene variants of coagulation related proteins that interact with SARS-CoV-2","abstract":"Thrombosis has been one of the complications of the Coronavirus disease of 2019 (COVID-19), often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms by which some of these variants may contribute to disease are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches. Increased blood clotting, especially in the lungs, is a common complication of COVID-19. Infectious diseases cause inflammation which in turn can contribute to increased blood clotting. However, the extent of clot formation that is seen in the lungs of COVID-19 patients suggests that there may be a more direct link. We identified three human proteins that are involved indirectly in the blood clotting cascade and have been shown to interact with proteins of SARS virus, which is closely related to the novel coronavirus. We examined computationally the interaction of these human proteins with the viral proteins. We looked for genetic variants of these proteins and examined how these variants are distributed across populations. We investigated whether variants of these genes could impact severity of COVID-19. Further investigation around these variants may provide clues for the pathogenesis of COVID-19 particularly in minority groups.","version":"1.2","doi":"10.1101/2020.09.08.272328","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.229781","pub_date":"2020-9-18","title":"Effect of \u03b2-chitosan on the binding interaction between SARS-CoV-2 S-RBD and ACE2","abstract":"SARS-CoV-2 invades human respiratory epithelial cells via an interaction between its spike RBD protein (SARS-CoV-2 S-RBD) and the host cell receptor angiotensin converting enzyme II (ACE2). Blocking this interaction provides a potent approach to preventing and controlling SARS-CoV-2 infection. In this work, the ability of \u03b2-chitosan to block the binding interaction between SARS-CoV-2 S-RBD and ACE2 was investigated. The inhibitory effect of \u03b2-chitosan on inflammation induced by the SARS-CoV-2 S-RBD was also studied. Native-PAGE analysis indicated that \u03b2-chitosan could bind with ACE2 and the SARS-CoV-2 S-RBD and a conjugate of \u03b2-chitosan and ACE2 could no longer bind with the SARS-CoV-2 S-RBD. HPLC analysis suggested that a conjugate of \u03b2-chitosan and the SARS-CoV-2 S-RBD displayed high binding affinity without dissociation under high pressure (40 MPa) compared with that of \u03b2-chitosan and ACE2. Furthermore, immunofluorescent staining of Vero E6 cells and lungs from hACE2 mice showed that the presence of \u03b2-chitosan prevented SARS-CoV-2 S-RBD from binding to ACE2. Meanwhile, \u03b2-chitosan could dramatically suppress the inflammation caused by the presence of the SARS-CoV-2 S-RBD both in vitro and vivo. Moreover, the decreased expression of ACE2 caused by \u03b2-chitosan treatment was restored by addition of TAPI-1, an inhibitor of the transmembrane protease ADAM17. Our findings demonstrated that \u03b2-chitosan displays an antibody-like function capable of neutralizing the SARS-CoV-2 S-RBD and effectively preventing the binding of the SARS-CoV-2 S-RBD to ACE2. Moreover, ADAM17 activation induced by \u03b2-chitosan treatment can enhance the cleavage of the extracellular domain of ACE2, releasing the active ectodomain into the extracellular environment, which can prevent the binding, internalization, and degradation of ACE2 bound to the SARS-CoV-2 S-RBD and thus diminish inflammation. Our study provides an alternative avenue for preventing SARS-CoV-2 infection using \u03b2-chitosan.","version":"1.3","doi":"10.1101/2020.07.31.229781","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214056","pub_date":"2020-9-18","title":"Implications of the COVID-19 lockdown on dengue transmission in Malaysia","abstract":"The impact of movement restrictions during the COVID-19 lockdown on the existing endemic infectious disease dengue fever has generated considerable research interest. We compared the Malaysia weekly epidemiological records of dengue incidences during the period of lockdown to the trend of previous years (2015 to 2019) and a simulation at the corresponding period that expected no movement restrictions. We found that the dengue incidence declined significantly with a greater magnitude at phase 1 of lockdown, with a negative gradient of 3.2-fold steeper than the trend observed in previous years and 6.5-fold steeper than the simulation, indicating that the control of population movement did reduce dengue transmission. However, starting from phase 2 of lockdown, the dengue incidences demonstrated an elevation and earlier rebound by at least 4 weeks and grew with an exponential pattern compared to the simulation and previous years. Together with our data on Aedes mosquitoes from a district of Penang, Malaysia, we revealed that Aedes albopictus is the predominant species for both indoor and outdoor environments. The abundance of the mosquito was increasing steadily during the period of lockdown, and demonstrated strong correlation with the locally reported dengue incidences; therefore, we proposed the possible diffusive effect of vector that led to a higher acceleration of incidence rate. These findings would help authorities review the direction and efforts of the vector control strategy.","version":"1.2","doi":"10.1101/2020.07.21.214056","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.302877","pub_date":"2020-9-18","title":"Matrix metalloproteinases and tissue inhibitors of metalloproteinases in murine coronavirus-induced neuroinflammation","abstract":"Mouse hepatitis virus (MHV) belongs to the same beta-coronavirus family as SARS-CoV-2, MERS-CoV, and SARS-CoV. Studies have shown the requirement of host cellular proteases for priming the surface spike protein during viral entry and transmission in coronaviruses. The metzincin family of metal-dependent endopeptidases called matrix metalloproteinases (MMPs) is involved in virus encephalitis, enhanced blood-brain barrier permeability, or cell-to-cell fusion upon viral infection. Here we show the role of MMPs as mediators of virus-induced host neuroinflammatory response in the MHV model. Infection of mice with wild-type MHV-A59 or its isogenic recombinant strains, RSA59 or RSMHV2 significantly upregulated MMP-3, MMP-8, and MMP-14 transcript levels. Functional network assessment with Ingenuity Pathway Analysis revealed a direct involvement of these MMPs in disrupting junctional assembly between endothelial cells via interaction with junctional adhesion molecules and thereby facilitating transmigration of peripheral lymphocytes. Our findings also suggest mRNA upregulation of Park7, which is involved in NADPH oxidase-dependent ROS production, following RSA59 infection. RSA59 infection resulted in elevated mRNA levels of RelA, a subunit of NF-\u03baB. Infection with MHV-A59 is known to generate ROS, and oxidative stress can activate NF-\u03baB. Thus, our findings indicate the existence of a possible nexus between ROS, NF-\u03baB, and MMPs in RSA59-induced neuroinflammation. We also assessed the expression of endogenously produced regulators of MMP activities. Elevated mRNA and protein levels of tissue inhibitors of metalloproteinases 1 (TIMP-1) in MHV-A59 infection are suggestive of a TIMP-1 mediated host antiviral response. The newly emergent coronavirus has brought the world to a near standstill. In the past, studies have focused on the function of host proteases in virus attachment and entry. Our research indicates the involvement of a group of metal-dependent host proteases in inflammation associated with coronavirus infection. Inflammation is the first response of the host to virus infection. While it helps in restricting the spread and clearance of viral particles, uncontrolled inflammation results in several inflammatory consequences. Therefore, it becomes vital to limit unchecked host immune response. The inhibition of specific metalloproteases represents a potential new therapeutic approach in coronavirus infection and disease outcome.","version":"1.1","doi":"10.1101/2020.09.17.302877","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.20183392","pub_date":"2020-09-18","title":"Disparities in COVID-19 Related Mortality in U.S. Prisons and the General Population","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>We provide an analysis of COVID-19 mortality data to assess the potential magnitude of COVID-19 among prison residents. Data were pooled from Covid Prison Project and multiple publicly available national and state level sources. Data analyses consisted of standard epidemiologic and demographic estimates. A single case study was included to generate a more in-depth and multi-faceted understanding of COVID-19 mortality in prisons. The increase in crude COVID-19 mortality rates for the prison population has outpaced the rates for the general population. People in prison experienced a significantly higher mortality burden compared to the general population (standardized mortality ratio (SMR) = 2.75; 95% confidence interval = 2.54, 2.96). For a handful of states (n = 5), these disparities were more extreme, with SMRs ranging from 5.55 to 10.56. Four states reported COVID-19 related death counts that are more than 50% of expected deaths from all-causes in a calendar year. The case study suggested there was also variation in mortality among units within prison systems, with geriatric facilities potentially at highest risk. Understanding the dynamic trends in COVID-19 mortality in prisons as they move in and out of \u201chotspot\u201d status is critical.</jats:p>","version":null,"doi":"10.1101/2020.09.17.20183392","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.16.300871","pub_date":"2020-9-17","title":"Distinct SARS-CoV-2 Antibody Reactivity Patterns in Coronavirus Convalescent Plasma Revealed by a Coronavirus Antigen Microarray","abstract":"A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and Random Forest (RF) prediction model, to classify individual specimens as either Reactive or Non-Reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS\u00ae Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the 3 assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 95%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed 3 main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified according to these reactivity patterns may be better associated with CCP treatment efficacy than antibody levels alone. The use of a SARS-CoV-2 antigen array may be useful to qualify CCP for administration as a treatment for acute COVID-19 and to interrogate vaccine immunogenicity and performance in preclinical and clinical studies to understand and recapitulate antibody responses associated with protection from infection and disease.","version":"1.2","doi":"10.1101/2020.09.16.300871","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.300996","pub_date":"2020-9-17","title":"Monocytes and macrophages, targets of SARS-CoV-2: the clue for Covid-19 immunoparalysis","abstract":"To date, the Covid-19 pandemic affected more than 18 million individuals and caused more than 690, 000 deaths. Its clinical expression is pleiomorphic and severity is related to age and comorbidities such as diabetes and hypertension. The pathophysiology of the disease relies on aberrant activation of immune system and lymphopenia that has been recognized as a prognosis marker. We wondered if the myeloid compartment was affected in Covid-19 and if monocytes and macrophages could be infected by SARS-CoV-2. We show here that SARS-CoV-2 efficiently infects monocytes and macrophages without any cytopathic effect. Infection was associated with the secretion of immunoregulatory cytokines (IL-6, IL-10, TGF-\u03b2) and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In addition, we found that in vitro macrophage polarization did not account for the permissivity to SARS-CoV-2, since M1-and M2-type macrophages were similarly infected. Finally, in a cohort of 76 Covid-19 patients ranging from mild to severe clinical expression, all circulating monocyte subsets were decreased, likely related to massive emigration into tissues. Monocytes from Covid-19 patients exhibited decreased expression of HLA-DR and increased expression of CD163, irrespective of the clinical status. Hence, SARS-CoV-2 drives circulating monocytes and macrophages inducing immunoparalysis of the host for the benefit of Covid-19 disease progression.","version":"1.1","doi":"10.1101/2020.09.17.300996","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.301614","pub_date":"2020-9-17","title":"Mutational signatures in countries affected by SARS-CoV-2: Implications in host-pathogen interactome","abstract":"We are in the midst of the third severe coronavirus outbreak caused by SARS-CoV-2 with unprecedented health and socio-economic consequences due to the COVID-19. Globally, the major thrust of scientific efforts has shifted to the design of potent vaccine and anti-viral candidates. Earlier genome analyses have shown global dominance of some mutations purportedly indicative of similar infectivity and transmissibility of SARS-CoV-2 worldwide. Using high-quality large dataset of 25k whole-genome sequences, we show emergence of new cluster of mutations as result of geographic evolution of SARS-CoV-2 in local population (\u226510%) of different nations. Using statistical analysis, we observe that these mutations have either significantly co-occurred in globally dominant strains or have shown mutual exclusivity in other cases. These mutations potentially modulate structural stability of proteins, some of which forms part of SARS-CoV-2-human interactome. The high confidence druggable host proteins are also up-regulated during SARS-CoV-2 infection. Mutations occurring in potential hot-spot regions within likely T-cell and B-cell epitopes or in proteins as part of host-viral interactome, could hamper vaccine or drug efficacy in local population. Overall, our study provides comprehensive view of emerging geo-clonal mutations which would aid researchers to understand and develop effective countermeasures in the current crisis. Our comparative analysis of globally dominant mutations and region-specific mutations in 25k SARS-CoV-2 genomes elucidates its geo-clonal evolution. We observe locally dominant mutations (co-occurring or mutually exclusive) in nations with contrasting COVID-19 mortalities per million of population) besides globally dominant ones namely, P314L (ORF1b) and D164G (S) type. We also see exclusive dominant mutations such as in Brazil (I33T in ORF6 and I292T in N protein), England (G251V in ORF3a), India (T2016K and L3606F in ORF1a) and in Spain (L84S in ORF8). The emergence of these local mutations in ORFs within SARS-CoV-2 genome could have interventional implications and also points towards their potential in modulating infectivity of SARS-CoV-2 in regional population.","version":"1.1","doi":"10.1101/2020.09.17.301614","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.302380","pub_date":"2020-9-17","title":"Topography, spike dynamics and nanomechanics of individual native SARS-CoV-2 virions","abstract":"SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, displays a corona-shaped layer of spikes which play fundamental role in the infection process. Recent structural data suggest that the spikes possess orientational freedom and the ribonucleoproteins segregate into basketlike structures. How these structural features regulate the dynamic and mechanical behavior of the native virion, however, remain unknown. By imaging and mechanically manipulating individual, native SARS-CoV-2 virions with atomic force microscopy, here we show that their surface displays a dynamic brush owing to the flexibility and rapid motion of the spikes. The virions are highly compliant and able to recover from drastic mechanical perturbations. Their global structure is remarkably temperature resistant, but the virion surface becomes progressively denuded of spikes upon thermal exposure. Thus, both the infectivity and thermal sensitivity of SARS-CoV-2 rely on the dynamics and the mechanics of the virus. The native coronavirus 2 displays a dynamic surface layer of spikes, a large mechanical compliance and unique self-healing capacity.","version":"1.1","doi":"10.1101/2020.09.17.302380","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.297846","pub_date":"2020-9-17","title":"Emergence and expansion of highly infectious spike:D614G mutant SARS-CoV-2 in central India","abstract":"COVID 19 has emerged as global pandemic with largest damage to the economy and human psyche. The genomic signature deciphered during the ongoing pandemic period is valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating strain in our country will enable to generate selective details in virulent and non virulent markers to prophylaxis and therapeutic interventions. The first case of SARS CoV-2 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of COVID-19 cases within 3 months in this region. We analyzed around 5000 COVID -19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh. A total of 136 cases were found positive over a span of three months period this includes virus introduction to region and further spread. Whole genome sequences employing Oxford nanopore technology were deciphered for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh State of India. The region witnessed index cases with multiple travel history responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were observed. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades A2a, A4 and B. The cluster wise segregation was observed suggesting multiple introduction links and evolution of virus in the region. This is the first comprehensive details of whole genome sequence analysis from central India region, which will add genome wide knowledge towards diagnostic and therapeutic interventions.","version":"1.2","doi":"10.1101/2020.09.15.297846","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.301861","pub_date":"2020-9-17","title":"Discovery of human ACE2 variants with altered recognition by the SARS-CoV-2 spike protein","abstract":"Understanding how human ACE2 genetic variants differ in their recognition by SARS-CoV-2 can have a major impact in leveraging ACE2 as an axis for treating and preventing COVID-19. In this work, we experimentally interrogate thousands of ACE2 mutants to identify over one hundred human single-nucleotide variants (SNVs) that are likely to have altered recognition by the virus, and make the complementary discovery that ACE2 residues distant from the spike interface can have a strong influence upon the ACE2-spike interaction. These findings illuminate new links between ACE2 sequence and spike recognition, and will find wide-ranging utility in SARS-CoV-2 fundamental research, epidemiological analyses, and clinical trial design.","version":"1.1","doi":"10.1101/2020.09.17.301861","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.300319","pub_date":"2020-9-17","title":"A soluble ACE2 microbody protein fused to a single immunoglobulin Fc domain is a potent inhibitor of SARS-CoV-2 infection in cell culture","abstract":"Soluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. We report on an improved soluble ACE2, termed a \u201cmicrobody\u201d in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibited entry of lentiviral SARS-CoV-2 spike protein pseudotyped virus and live SARS-CoV-2 with a potency 10-fold higher than unmodified soluble ACE2 and was active after initial virus binding to the cell. The ACE2 microbody inhibited the entry of ACE2-specific \u03b2 coronaviruses and viruses with the high infectivity variant D614G spike. The ACE2 microbody may be a valuable therapeutic for COVID-19 that is active against SARS-CoV-2 variants and future coronaviruses that may arise.","version":"1.1","doi":"10.1101/2020.09.16.300319","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.301093","pub_date":"2020-9-17","title":"Comparison of Rhesus and Cynomolgus macaques as an authentic model for COVID-19","abstract":"A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques, resembling the mild clinical cases of COVID-19 in humans. Immune responses against SARS-CoV-2 were also similar in both species and equivalent to those reported in milder infections and convalescent human patients. Importantly, we have devised a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the optimal study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of novel and repurposed interventions against SARS-CoV-2. Accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.","version":"1.1","doi":"10.1101/2020.09.17.301093","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.300970","pub_date":"2020-9-17","title":"Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model","abstract":"The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model. A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design.","version":"1.1","doi":"10.1101/2020.09.16.300970","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.300335","pub_date":"2020-9-17","title":"Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells","abstract":"The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 \u00b0C and 52 \u00b0C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.17.300335","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.17.299933","pub_date":"2020-9-17","title":"Identification of novel antiviral drug combinations in vitro and tracking their development","abstract":"Combination therapies have become a standard for the treatment for HIV and HCV infections. They are advantageous over monotherapies due to better efficacy and reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify several new synergistic combinations against emerging and re-emerging viral infections in vitro. We observed synergistic activity of nelfinavir with investigational drug EIDD-2801 and convalescent serum against SARS-CoV-2 infection in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of vemurafenib combination with emetine, homoharringtonine, gemcitabine, or obatoclax against echovirus 1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar and niclosamide were synergistic against HCV infection in hepatocyte derived Huh-7.5 cells, whereas combinations of monensin with lamivudine and tenofovir were synergistic against HIV-1 infection in human cervical TZM-bl cells. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status. Overall, the development of combinational therapies could have a global impact improving the preparedness and protection of the general population from emerging and re-emerging viral threats.","version":"1.1","doi":"10.1101/2020.09.17.299933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.297366","pub_date":"2020-9-16","title":"Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19. Bromelain inhibits / cleaves the expression of ACE-2 and TMPRSS2 Bromelain cleaves / degrades SARS-CoV-2 spike protein Bromelain inhibits S-Ectodomain binding and SARS-CoV-2 infection","version":"1.1","doi":"10.1101/2020.09.16.297366","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.300277","pub_date":"2020-9-16","title":"Baricitinib treatment resolves lower airway inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques","abstract":"Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV-2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.09.16.300277","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.299800","pub_date":"2020-9-16","title":"Lopinavir-ritonavir is not an effective inhibitor of the main protease activity of SARS-CoV-2 in vitro","abstract":"COVID-19 has caused over 900,000 deaths worldwide as of September 2020, and effective medicines are urgently needed. Lopinavir was identified as an inhibitor of the HIV protease, and a lopinavir-ritonavir combination therapy was reported to be beneficial for the treatment of SARS and MERS. However, recent clinical tests could not prove that lopinavir-ritonavir therapy was an effective treatment for COVID-19. In this report, we examined the effect of lopinavir and ritonavir to the activity of the purified main protease (Mpro) protein of SARS- CoV-2, the causative virus of COVID-19. Unexpectedly, lopinavir and ritonavir did not inhibit Mpro activity. These results will aid the drug candidate selection for ongoing and future COVID-19 clinical trials.","version":"1.1","doi":"10.1101/2020.09.16.299800","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.300459","pub_date":"2020-9-16","title":"The flexibility of ACE2 in the context of SARS-CoV-2 infection","abstract":"The COVID-19 pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine developments efforts are underway, many questions remain outstanding on the mechanism of SARS-CoV-2 viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and entry in the cell. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular Spike glycoprotein and at the receptor binding domain-receptor interface, suggesting a role in infection. Here, we perform all-atom molecular dynamics simulations of the glycosylated, full-length membrane-bound ACE2 receptor, in both an apo and spike receptor binding domain (RBD) bound state, in order to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix, while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer, and suggests a mechanical contribution of the host receptor towards the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can be exploited for the rational design of effective SARS-CoV-2 therapeutics. As the host receptor of SARS-CoV-2, ACE2 has been the subject of extensive structural and antibody design efforts in aims to curtail COVID-19 spread. Here, we perform molecular dynamics simulations of the homodimer ACE2 full-length structure to study the dynamics of this protein in the context of the cellular membrane. The simulations evidence exceptional plasticity in the protein structure due to flexible hinge motions in the head-transmembrane domain linker region and helix mobility in the membrane, resulting in a varied ensemble of conformations distinct from the experimental structures. Our findings suggest a dynamical contribution of ACE2 to the spike glycoprotein shedding required for infection, and contribute to the question of stoichiometry of the Spike-ACE2 complex.","version":"1.1","doi":"10.1101/2020.09.16.300459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.300483","pub_date":"2020-9-16","title":"Evaluation of nafamostat mesylate safety and inhibition of SARS-CoV-2 replication using a 3-dimensional human airway epithelia model","abstract":"In the current COVID-19 pandemic context, Ensysce and its subsidiary Covistat have been working to repurpose nafamostat mesylate as an effective oral and inhalation treatment against SARS-CoV-2 infection. Prior reports used cell lines to demonstrate the antiviral potential of nafamostat against coronaviral infections and determined its mechanism of action through inhibition of transmembrane protease serine 2 (TMPRSS2). We selected a biologically relevant pre-clinical experimental model of SARS-CoV-2 lung infection using a 3D human reconstituted airway epithelial model of nasal origin to characterize the effects of nafamostat on tissue-level cellular ultrastructure and viral infection kinetics. Our results confirm the not only the relevance of this model for the preclinical evaluation of safety and efficacy of antiviral candidates, but also the highly potent nature of nafamostat SARS-CoV-2 antiviral activity. The studies described herein provided evidence demonstrating the therapeutic potential of nafamostat against COVID-19, as well as its safety upon exposure to lung airway cellular. Using a human airway model, study demonstrates the powerful inhibitory effect of nafamostat on SARS-CoV-2 genome copy detection when applied apically.","version":"1.1","doi":"10.1101/2020.09.16.300483","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.298992","pub_date":"2020-9-16","title":"Efficient production of Moloney murine leukemia virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that started in China at the end of 2019 has rapidly spread to become pandemic. Several investigational vaccines that have already been tested in animals and humans were able to induce neutralizing antibodies against the SARS-CoV-2 spike (S) protein, however protection and long-term efficacy in humans remain to be demonstrated. We have investigated if a virus-like particle (VLP) derived from Moloney murine leukemia virus (MLV) could be engineered to become a candidate SARS-CoV-2 vaccine amenable to mass production. First, we showed that a codon optimized version of the S protein could migrate efficiently to the cell membrane. However, efficient production of infectious viral particles was only achieved with stable expression of a shorter version of S in its C-terminal domain (\u0394S) in 293 cells that express MLV Gag-Pol (293GP). The incorporation of \u0394S was 15-times more efficient into VLPs as compared to the full-length version, and that was not due to steric interference between the S cytoplasmic tail and the MLV capsid. Indeed, a similar result was also observed with extracellular vesicles released from parental 293 and 293GP cells. The amount of \u0394S incorporated into VLPs released from producer cells was robust, with an estimated 1.25 \u03bcg/ml S2 equivalent (S is comprised of S1 and S2). Thus, a scalable platform that has the potential for production of pan-coronavirus VLP vaccines has been established. The resulting nanoparticles could potentially be used alone or as a boost for other immunization strategies for COVID-19. Several candidate COVID-19 vaccines have already been tested in humans, but their protective effect and long-term efficacy are uncertain. Therefore, it is necessary to continue developing new vaccine strategies that could be more potent and/or that would be easier to manufacture in large-scale. Virus-like particle (VLP) vaccines are considered highly immunogenic and have been successfully developed for human papilloma virus as well as hepatitis and influenza viruses. In this study, we report the generation of a robust Moloney murine leukemia virus platform that produces VLPs containing the spike of SARS-CoV-2. This vaccine platform that is compatible with lyophilization could simplify storage and distribution logistics immensely.","version":"1.1","doi":"10.1101/2020.09.16.298992","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.299164","pub_date":"2020-9-16","title":"Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors","abstract":"There is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (Mpro) is one of the most extensively studied drug targets. Mpro is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites and it is highly conserved among coronaviruses. In addition, Mpro has a unique substrate preference for glutamine in the P1 position. Taken together, it appears that Mpro inhibitors can achieve both broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as Mpro inhibitors, with several of which also showed antiviral activity in cell culture. In this study, we investigated the mechanism of action of six previously reported Mpro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of Mpro by these six compounds is non-specific and the inhibition is abolished or greatly reduced with the addition of reducing reagent DTT. In the absence of DTT, these six compounds not only inhibit Mpro, but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease, the 2Apro and 3Cpro from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC50 values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARS-CoV-2 Mpro inhibitors, and urge the scientific community to be stringent with hit validation.\n\n\n\n","version":"1.1","doi":"10.1101/2020.09.15.299164","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.16.299362","pub_date":"2020-9-16","title":"Structure Model Analysis Of Phosphorylation Dependent Binding And Sequestration Of SARS-COV-2 Encoded Nucleocapsid Protein By Protein 14-3-3","abstract":"Phosphorylation of serines 197 and 206 of SARS-COV-2 Nucleocapsid protein (NCp) enhanced the stability and binding efficiency and sequestration of NCp to Protein 14-3-3 by increasing the Stability Energy (\u0394Gstability energy) and Binding Energy (\u0394\u0394Gbinding energy) from ~545 Kcal/mol to ~616 Kcal/mol, and from 108 Kcal/mol to ~228 Kcal/mol respectively. The calculated Binding Energy Difference (\u0394\u0394Gbinding energy difference) between dephospho-NCp-14-3-3 complex and phospho-NCp-13-3-3 complex was ~72 Kcal/mol. Phosphorylations of serines 186, 197, 202 and 206, and threonines 198 and 205 NCp also caused an increase in the Stability Energy (\u0394Gstability energy) and Binding Energy (\u0394\u0394Gbinding energy) from ~545 Kcal/mol to ~617, 616, 583, 580, 574, 564 and 566 Kcal/mol and from ~108 Kcal/mol to ~228, 216, 184, 188, 184, 174 and 112 Kcal/mol respectively. Phosphorylation of NCp on serines 197 and 206 caused a decrease in Stability Energy and Binding Energy from ~698 Kcal/mol to 688 Kcal/mol, and from ~91 Kcal/mol to ~82 Kcal/mol for the dimerization of NCp. These results support the existence of a phosphorylation dependent cellular mechanism to bind and sequester NCp.","version":"1.1","doi":"10.1101/2020.09.16.299362","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.298067","pub_date":"2020-9-15","title":"Antibody potency, effector function and combinations in protection from SARS-CoV-2 infection in vivo","abstract":"SARS-CoV-2, the causative agent of COVID-19, is responsible for over 24 million infections and 800,000 deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a mouse adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). In vitro antibody neutralization potency did not uniformly correlate with in vivo activity, and some hu-mAbs were more potent in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors is essential for optimal protection against SARS-CoV-2 MA. The data indicate that hu-mAb protective activity is dependent on intact effector function and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.","version":"1.1","doi":"10.1101/2020.09.15.298067","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.298547","pub_date":"2020-9-15","title":"Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy","abstract":"Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.","version":"1.1","doi":"10.1101/2020.09.15.298547","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.295956","pub_date":"2020-9-15","title":"Ceftazidime Is a Potential Drug to Inhibit SARS-CoV-2 Infection In Vitro by Blocking Spike Protein-ACE2 Interaction","abstract":"Coronavirus Disease 2019 (COVID-19) spreads globally as a sever pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cell entry of SARS-CoV-2 mainly depends on binding of the viral spike (S) proteins to angiotensin converting enzyme 2 (ACE2) on host cells. Therefore, repurposing of known drugs to inhibit S protein-ACE2 interaction could be a quick way to develop effective therapy for COVID-19. Using a high-throughput screening system to investigate the interaction between spike receptor binding domain (S-RBD) and ACE2 extracellular domain, we screened 3581 FDA-approved drugs and natural small molecules and identified ceftazidime as a potent compound to inhibit S-RBD\u2013ACE2 interaction by binding to S-RBD. In addition to significantly inhibit S-RBD binding to HPAEpiC cells, ceftazidime efficiently prevented SARS-CoV-2 pseudovirus to infect ACE2-expressing 293T cells. The inhibitory concentration (IC50) was 113.2 \u03bcM, which is far below the blood concentration (over 300 \u03bcM) of ceftazidime in patients when clinically treated with recommended dose. Notably, ceftazidime is a drug clinically used for the treatment of pneumonia with minimal side effects compared with other antiviral drugs. Thus, ceftazidime has both anti-bacterial and anti-SARS-CoV-2 effects, which should be the first-line antibiotics used for the clinical treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.09.14.295956","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.298604","pub_date":"2020-9-15","title":"SARS-CoV-2 ribosomal frameshifting pseudoknot: Improved secondary structure prediction and detection of inter-viral structural similarity","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the COVID-19 pandemic; a pandemic of a scale that has not been seen in the modern era. Despite over 29 million reported cases and over 900, 000 deaths worldwide as of September 2020, herd immunity and widespread vaccination efforts by many experts are expected to be insufficient in addressing this crisis for the foreseeable future. Thus, there is an urgent need for treatments that can lessen the effects of SARS-CoV-2 in patients who become seriously affected. Many viruses including HIV, the common cold, SARS-CoV and SARS-CoV-2 use a unique mechanism known as \u22121 programmed ribosomal frameshifting (\u22121 PRF) to successfully replicate and infect cells in the human host. SARS-CoV (the coronavirus responsible for SARS) and SARS-CoV-2 possess a unique RNA structure, a three-stemmed pseudoknot, that stimulates \u22121 PRF. Recent experiments identified that small molecules can be introduced as antiviral agents to bind with the pseudoknot and disrupt its stimulation of \u22121 PRF. If successfully developed, small molecule therapy that targets \u22121 PRF in SARS-CoV-2 is an excellent strategy to improve patients\u2019 prognoses. Crucial to developing these successful therapies is modeling the structure of the SARS-CoV-2 \u22121 PRF pseudoknot. Following a structural alignment approach, we identify similarities in the \u22121 PRF pseudoknots of the novel coronavirus SARS-CoV-2, the original SARS-CoV, as well as a third coronavirus: MERS-CoV, the coronavirus responsible for Middle East Respiratory Syndrome (MERS). In addition, we provide a better understanding of the SARS-CoV-2 \u22121 PRF pseudoknot by comprehensively investigating the structural landscape using a hierarchical folding approach. Since understanding the impact of mutations is vital to long-term success of treatments that are based on predicted RNA functional structures, we provide insight on SARS-CoV-2 \u22121 PRF pseudoknot sequence mutations and their effect on the resulting structure and its function.","version":"1.1","doi":"10.1101/2020.09.15.298604","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.296228","pub_date":"2020-9-15","title":"The Anti-histamine Azelastine, Identified by Computational Drug Repurposing, Inhibits SARS-CoV-2 Infection in Reconstituted Human Nasal Tissue In Vitro","abstract":"The COVID-19 pandemic is an enormous threat for healthcare systems and economies worldwide that urgently demands effective preventive and therapeutic strategies. Unlike the development of vaccines and new drugs specifically targeting SARS-CoV-2, repurposing of approved or clinically tested drugs can provide an immediate solution. We applied a novel computational approach to search among approved and clinically tested drugs from the DrugBank database. Candidates were selected based on Shannon entropy homology and predefined activity profiles of three small molecules with proven anti-SARS-CoV activity and a published data set. Antiviral activity of a predicted drug, azelastine, was tested in vitro in SARS-CoV-2 infection assays with Vero E6 monkey kidney epithelial cells and reconstituted human nasal tissue. The effect on viral replication was assessed by quantification of viral genomes by droplet digital PCR. The computational approach with four independent queries identified major drug families, most often and in overlapping fashion anti-infective, anti-inflammatory, anti-hypertensive, anti-histamine and neuroactive drugs. Azelastine, an histamine 1 receptor-blocker, was predicted in multiple screens, and based on its attractive safety profile and availability in nasal formulation, was selected for experimental testing. Azelastine significantly reduced cytopathic effect and SARS-CoV-2 infection of Vero E6 cells with an EC50 of \u223c6 \u03bcM both in a preventive and treatment setting. Furthermore, azelastine in a commercially available nasal spray tested at 5-fold dilution was highly potent in inhibiting viral propagation in SARS-CoV-2 infected reconstituted human nasal tissue. Azelastine, an anti-histamine, available in nasal sprays developed against allergic rhinitis may be considered as a topical prevention or treatment of nasal colonization with SARS-CoV-2. As such, it could be useful in reducing viral spread and prophylaxis of COVID-19. Ultimately, its potential benefit should be proven in clinical studies. provided by the Hungarian government to the National Laboratory of Virology and by CEBINA GmbH.","version":"1.1","doi":"10.1101/2020.09.15.296228","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.20193920","pub_date":"2020-09-15","title":"LOW BIRTH WEIGHT AS A RISK FACTOR FOR SEVERE COVID-19 IN ADULTS","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:p>The identification of factors predisposing to severe COVID-19 in young adults remains partially characterized. Low birth weight (LBW) alters cardiovascular and lung development and predisposes to adult disease. We hypothesized that LBW is a risk factor for severe COVID-19 in non-elderly subjects. We analyzed a prospective cohort of 397 patients (18-70y) with laboratory-confirmed SARS-CoV-2 infection attended in a tertiary hospital, where 15% required admission to Intensive Care Unit (ICU). Perinatal and current potentially predictive variables were obtained from all patients and LBW was defined as birth weight \u22642,500 g. Age (adjusted OR (aOR) 1.04 [1-1.07], P=0.012), male sex (aOR 3.39 [1.72-6.67], P&lt;0.001), hypertension (aOR 3.37 [1.69-6.72], P=0.001), and LBW (aOR 3.61 [1.55-8.43], P=0.003) independently predicted admission to ICU. The area under the receiver-operating characteristics curve (AUC) of this model was 0.79 [95% CI, 0.74-0.85], with positive and negative predictive values of 29.1% and 97.6% respectively. Results were reproduced in an independent cohort, from a web-based survey in 1,822 subjects who self-reported laboratory-positive SARS-CoV-2 infection, where 46 patients (2.5%) needed ICU admission (AUC 0.74 [95% CI 0.68-0.81]). LBW seems to be an independent risk factor for severe COVID-19 in non-elderly adults and might improve the performance of risk stratification algorithms.</jats:p>","version":null,"doi":"10.1101/2020.09.14.20193920","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.14.296491","pub_date":"2020-9-15","title":"Levels of genetic diversity of SARS-CoV-2 virus: reducing speculations about the genetic variability of the virus in South America","abstract":"In this work, we evaluated the levels of genetic diversity in 38 complete Genomes of SARS-CoV-2 from six countries in South America, using specific methodologies for paired FST, AMOVA, mismatch, demographic and spatial expansions, molecular diversity and for the time of evolutionary divergence. The analyses showed non-significant evolutionary divergences within and between the six countries, as well as a significant similarity to the time of genetic evolutionary divergence between all populations. Thus, it seems safe to affirm that we will find similar results for the other Countries of South America, reducing speculation about the existence of rapid and silent mutations that, although there are as we have shown in this work, do not increase, until this moment, the genetic variability of the Virus, a fact that would hinder the work with molecular targets for vaccines and drugs in general.","version":"1.1","doi":"10.1101/2020.09.14.296491","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.15.293100","pub_date":"2020-9-15","title":"LOX-1+ immature neutrophils predict severe COVID-19 patients at risk of thrombotic complications","abstract":"Lymphopenia and neutrophil/lymphocyte ratio may have prognostic value in coronavirus disease 2019 (COVID-19) severity. We sought to investigate the representation of neutrophil subsets in severe and critical COVID-19 patients based on Intensive Care Units (ICU) and non-ICU admission. We developed a multi-parametric neutrophil profiling strategy based on known neutrophil markers to distinguish COVID-19 phenotypes in critical and severe patients. Our results showed that 80% of ICU patients develop strong myelemia with CD10\u2212CD64+ immature neutrophils. Cellular profiling revealed two distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) or the Interleukin-3 receptor alpha (CD123), both significantly overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1-expressing immature neutrophils positively correlated with clinical severity, with the cytokine storm (IL-1\u03b2, IL-6, IL-8, TNF\u03b1), and with intravascular coagulation. Importantly, high proportions of LOX-1+-immature neutrophils are associated with high risks of severe thrombosis. Together these data suggest that point of care enumeration of LOX-1-immature neutrophils might help distinguish patients at risk of thrombosis complication and most likely to benefit from intensified anticoagulant therapy.","version":"1.1","doi":"10.1101/2020.09.15.293100","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.13.20193805","pub_date":"2020-09-14","title":"Genome sequencing of sewage detects regionally prevalent SARS-CoV-2 variants","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and near-complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant (SNV) calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the US or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside of CA, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.</jats:p>","version":null,"doi":"10.1101/2020.09.13.20193805","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.14.296327","pub_date":"2020-9-14","title":"The Coronavirus Network Explorer: Mining a large-scale knowledge graph for effects of SARS-CoV-2 on host cell function","abstract":"Building on recent work that identified human host proteins that interact with SARS-CoV-2 viral proteins in the context of an affinity-purification mass spectrometry screen, we use a machine learning-based approach to connect the viral proteins to relevant biological functions and diseases in a large-scale knowledge graph derived from the biomedical literature. Our aim is to explore how SARS-CoV-2 could interfere with various host cell functions, and also to identify additional drug targets amongst the host genes that could potentially be modulated against COVID-19. Results are presented in the form of interactive network visualizations, that allow exploration of underlying experimental evidence. A selection of networks is discussed in the context of recent clinical observations.","version":"1.1","doi":"10.1101/2020.09.14.296327","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.296178","pub_date":"2020-9-14","title":"SARS-CoV-2 protein Nsp1 alters actomyosin cytoskeleton and phenocopies arrhythmogenic cardiomyopathy-related PKP2 mutant","abstract":"Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic cardiomyopathy (ACM) and a common cause of sudden cardiac death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism to understand cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors, and demonstrate that the expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that the viral Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2, wildtype or mutant, induces the collapse of the actomyosin network, since shRNA-PKP2 knockdown maintains the cell structure, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and PKP2 mutant R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor survival prognosis. The specific cardiac isoform Plakophilin-2a (PKP2) interacts with Myh9 and Myh10. PKP2 delocalization alters actomyosin cytoskeleton component organization. SARS-CoV-2 Nsp1 protein hijacks PKP2 from the desmosome into the soluble fraction where it is downregulated. Viral Nsp1 collapses the actomyosin cytoskeleton and phenocopies the arrhythmogenic cardiomyopathy-related mutant R735X.","version":"1.1","doi":"10.1101/2020.09.14.296178","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.14.296806","pub_date":"2020-9-14","title":"Single-cell RNA Expression of SARS-CoV-2 Cell Entry Factors in Human Endometrium during Preconception","abstract":"We investigated potential SARS-CoV-2 tropism in human endometrium by single-cell RNA-sequencing of viral entry-associated genes in healthy women. Percentages of endometrial cells expressing ACE2, TMPRSS2, CTSB, or CTSL were <2%, 12%, 80%, and 80%, respectively, with 0.7% of cells expressing all four genes. Our findings imply low efficiency of SARS-CoV-2 infection in the endometrium before embryo implantation, providing information to assess preconception risk in asymptomatic carriers.","version":"1.1","doi":"10.1101/2020.09.14.296806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.13.295691","pub_date":"2020-9-14","title":"Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein\u2019s Receptor Binding Domain and Recombinant Human ACE2","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), enters the host cells through two main pathways, both involving key interactions between viral envelope-anchored spike glycoprotein of the novel coronavirus and the host receptor, angiotensin-converting enzyme 2 (ACE2). To date, SARS-CoV-2 has infected up to 26 million people worldwide; yet, there is no clinically approved drug or vaccine available. Therefore, a rapid and coordinated effort to re-purpose clinically approved drugs that prevent or disrupt these critical entry pathways of SARS-CoV-2 spike glycoprotein interaction with human ACE2, could potentially accelerate the identification and clinical advancement of prophylactic and/or treatment options against COVID-19, thus providing possible countermeasures against viral entry, pathogenesis and survival. Herein, we discovered that Ambroxol hydrochloride (AMB), and its progenitor, Bromhexine hydrochloride (BHH), both clinically approved drugs are potent effective modulators of the key interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and human ACE2. We also found that both compounds inhibited SARS-CoV-2 infection-induced cytopathic effect at micromolar concentrations. Therefore, in addition to the known TMPRSS2 activity of BHH; we report for the first time that the BHH and AMB pharmacophore has the capacity to target and modulate yet another key protein-protein interaction essential for the two known SARS-CoV-2 entry pathways into host cells. Altogether, the potent efficacy, excellent safety and pharmacologic profile of both drugs along with their affordability and availability, makes them promising candidates for drug repurposing as possible prophylactic and/or treatment options against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.09.13.295691","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.209288","pub_date":"2020-9-14","title":"SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signaling to induce analgesia","abstract":"Global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues unabated. Binding of SARS-CoV-2\u2019s Spike protein to host angiotensin converting enzyme 2 triggers viral entry, but other proteins may participate, including neuropilin-1 receptor (NRP-1). As both Spike protein and vascular endothelial growth factor-A (VEGF-A) \u2013 a pro-nociceptive and angiogenic factor, bind NRP-1, we tested if Spike could block VEGF-A/NRP-1 signaling. VEGF-A\u2013triggered sensory neuronal firing was blocked by Spike protein and NRP-1 inhibitor EG00229. Pro-nociceptive behaviors of VEGF-A were similarly blocked via suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A \u2018silencing\u2019 of pain via subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.","version":"1.3","doi":"10.1101/2020.07.17.209288","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.206680","pub_date":"2020-9-14","title":"Simulations support the interaction of the SARS-CoV-2 spike protein with nicotinic acetylcholine receptors","abstract":"Changeux et al. recently suggested that the SARS-CoV-2 spike (S) protein may interact with nicotinic acetylcholine receptors (nAChRs). Such interactions may be involved in pathology and infectivity. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the S protein, to investigate this \u2018nicotinic hypothesis\u2019. We examine the binding of the Y674-R685 loop of the S protein to three nAChRs, namely the human \u03b14\u03b22 and \u03b17 subtypes and the muscle-like \u03b1\u03b2\u03b3d receptor from Tetronarce californica. Our results indicate that Y674-R685 has affinity for nAChRs and the region responsible for binding contains the PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. In particular, R682 has a key role in the stabilisation of the complexes as it forms interactions with loops A, B and C in the receptor\u2019s binding pocket. The conformational behaviour of the bound Y674-R685 region is highly dependent on the receptor subtype, adopting extended conformations in the \u03b14\u03b22 and \u03b17 complexes and more compact ones when bound to the muscle-like receptor. In the \u03b14\u03b22 and \u03b1\u03b2\u03b3d complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation similar to other known nAChR antagonists. In contrast, in the \u03b17 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of the simulations of the full-length S protein show that the Y674-R685 region is accessible for binding, and suggest a potential binding orientation of the S protein with nAChRs.","version":"1.3","doi":"10.1101/2020.07.16.206680","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.13.20193722","pub_date":"2020-09-14","title":"Sensing of COVID-19 Antibodies in Seconds via Aerosol Jet Printed Three Dimensional Electrodes","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  <jats:italic>Rapid diagnosis is critical for the treatment and prevention of diseases. In this research, we report sensing of antibodies specific to SARS-CoV-2 virus in seconds via an electrochemical platform consisting of gold micropillar array electrodes decorated with reduced graphene oxide and functionalized with recombinant viral antigens. The array electrodes are fabricated by Aerosol Jet (AJ) nanoparticle 3D printing, where gold nanoparticles (3-5nm) are assembled in 3D space, sintered, and integrated with a microfluidic device. The device is shown to detect antibodies to SARS-CoV-2 spike S1 protein and its receptor-binding-domain (RBD) at concentrations down to 1pM via electrochemical impedance spectroscopy and read by a smartphone-based user interface. In addition, the sensor can be regenerated within a minute by introducing a low-pH chemistry that elutes the antibodies from the antigens, allowing successive testing of multiple antibody samples using the same sensor. The detection time for the two antibodies tested in this work is 11.5 seconds. S1 protein sensing of its antibodies is specific, which cross-reacts neither with other antibodies nor with proteins such as Nucleocapsid antibody and Interleukin-6 protein. The proposed sensing platform is generic and can also be used for the rapid detection of biomarkers for other infectious agents such as Ebola, HIV, and Zika, which will benefit the public health</jats:italic>\n                  .\n                </jats:p>","version":null,"doi":"10.1101/2020.09.13.20193722","journal":"medRxiv","score":null},{"id":"10.1101/596700","pub_date":"2020-9-14","title":"Infectious disease phylodynamics with occurrence data","abstract":"Phylodynamic models use pathogen genome sequence data to infer epidemiological dynamics. With the increasing genomic surveillance of pathogens, especially amid the SARS-CoV-2 outbreak, new practical questions about their use are emerging. One such question focuses on the inclusion of un-sequenced case occurrence data alongside sequenced data to improve phylodynamic analyses. This approach can be particularly valuable if sequencing efforts vary over time. Using simulations, we demonstrate that birth-death phylodynamic models can employ occurrence data to eliminate bias in estimates of the basic reproductive number due to misspecification of the sampling process. In contrast, the coalescent exponential model is robust to such sampling biases, but in the absence of a sampling model it cannot exploit occurrence data. Subsequent analysis of the SARS-CoV-2 epidemic in the northwest USA supports these results. We conclude that occurrence data are a valuable source of information in combination with birth-death models. These data should be used to bolster phylodynamic analyses of infectious diseases and other rapidly spreading species in the future.","version":"1.2","doi":"10.1101/596700","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.056085","pub_date":"2020-9-14","title":"Evolutionary Dynamics And Geographic Dispersal Of Beta Coronaviruses In African Bats","abstract":"Bats have been shown to serve as reservoir host of various viral agents including coronaviruses. They have also been associated with the novel coronavirus SARS-CoV-2. This has made them an all important agent for CoV evolution and transmission. Our objective in this study was to investigate the dispersal, phylogenomics and evolution of betacoronavirus (\u03b2CoV) among African bats. We retrieved sequence data from established databases such as GenBank and Virus Pathogen Resource, covering the partial RNA dependent RNA polymerase (RdRP) gene of Bat coronaviruses from eight African, three Asian, five European, two South American countries and Australia. We analyzed for Phylogeographic information relating to genetic diversity and evolutionary dynamics. Our study revealed that majority of the African strains fell within Norbecovirus subgenera, with an Evolutionary rate of 1.301 \u00d7 10\u22123, HPD (1.064 \u00d7 10\u22123 \u2013 1.434 \u00d7 10\u22123) subs/site/year. The African strains diversified into three main subgenera, Norbecovirus, Hibecovirus and Marbecovirus. The time to most common recent ancestor for Norbecovirus strains was 1968, and 2010, for the African Marbecovirus strains. There was evidence of inter species transmission of Norbecovirus among bats in Cameroun and DRC. Phlylogeography showed that there were inter-continental spread of Bt-CoV from Europe, China and Hong Kong into Central and Southern Africa, highlighting the possibility of long distance transmission. Our study has elucidated the possible evolutionary origins of \u03b2CoV among African bats, we therefore advocate for broader studies of whole genome sequences of BtCoV to further understand the drivers for their emergence and zoonotic spillovers into human population.","version":"1.2","doi":"10.1101/2020.05.14.056085","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.287508","pub_date":"2020-9-13","title":"Interaction network of SARS-CoV-2 with host receptome through spike protein","abstract":"Host cellular receptors are key determinants of virus tropism and pathogenesis. Virus utilizes multiple receptors for attachment, entry, or specific host responses. However, other than ACE2, little is known about SARS-CoV-2 receptors. Furthermore, ACE2 cannot easily interpret the multi-organ tropisms of SARS-CoV-2 nor the clinical differences between SARS-CoV-2 and SARS-CoV. To identify host cell receptors involved in SARS-CoV-2 interactions, we performed genomic receptor profiling to screen almost all human membrane proteins, with SARS-CoV-2 capsid spike (S) protein as the target. Twelve receptors were identified, including ACE2. Most receptors bind at least two domains on S protein, the receptor-binding-domain (RBD) and the N-terminal-domain (NTD), suggesting both are critical for virus-host interaction. Ectopic expression of ASGR1 or KREMEN1 is sufficient to enable entry of SARS-CoV-2, but not SARS-CoV and MERS-CoV. Analyzing single-cell transcriptome profiles from COVID-19 patients revealed that virus susceptibility in airway epithelial ciliated and secretory cells and immune macrophages highly correlates with expression of ACE2, KREMEN1 and ASGR1 respectively, and ACE2/ASGR1/KREMEN1 (ASK) together displayed a much better correlation than any individual receptor. Based on modeling of systemic SARS-CoV-2 host interactions through S receptors, we revealed ASK correlation with SARS-CoV-2 multi-organ tropism and provided potential explanations for various COVID-19 symptoms. Our study identified a panel of SARS-CoV-2 receptors with diverse binding properties, biological functions, and clinical correlations or implications, including ASGR1 and KREMEN1 as the alternative entry receptors, providing insights into critical interactions of SARS-CoV-2 with host, as well as a useful resource and potential drug targets for COVID-19 investigation.","version":"1.3","doi":"10.1101/2020.09.09.287508","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.13.276923","pub_date":"2020-9-13","title":"Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 disease severity","abstract":"Angiotensin-converting enzyme 2 (ACE2) maintains cardiovascular and renal homeostasis but also serves as the entry receptor for the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), the causal agent of novel coronavirus disease 2019 (COVID-19). COVID-19 disease severity, while highly variable, is typically lower in pediatric patients than adults (particularly the elderly), but increased rates of hospitalizations requiring intensive care are observed in infants than in older children. The reasons for these differences are unknown. To detect potential age-based correlates of disease severity, we measured ACE2 protein expression at the single cell level in human lung tissue specimens from over 100 donors from \u223c4 months to 75 years of age. We found that expression of ACE2 in distal lung epithelial cells generally increases with advancing age but exhibits extreme intra- and inter-individual heterogeneity. Notably, we also detected ACE2 expression on neonatal airway epithelial cells and within the lung parenchyma. Similar patterns were found at the transcript level: ACE2 mRNA is expressed in the lung and trachea shortly after birth, downregulated during childhood, and again expressed at high levels in late adulthood in alveolar epithelial cells. Furthermore, we find that apoptosis, which is a natural host defense system against viral infection, is also dynamically regulated during lung maturation, resulting in periods of heightened apoptotic priming and dependence on pro-survival BCL-2 family proteins including MCL-1. Infection of human lung cells with SARS-CoV-2 triggers an unfolded protein stress response and upregulation of the endogenous MCL-1 inhibitor Noxa; in juveniles, MCL-1 inhibition is sufficient to trigger apoptosis in lung epithelial cells \u2013 this may limit virion production and inflammatory signaling. Overall, we identify strong and distinct correlates of COVID-19 disease severity across lifespan and advance our understanding of the regulation of ACE2 and cell death programs in the mammalian lung. Furthermore, our work provides the framework for potential translation of apoptosis modulating drugs as novel treatments for COVID-19.","version":"1.1","doi":"10.1101/2020.09.13.276923","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.12.294066","pub_date":"2020-9-13","title":"SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.","version":"1.1","doi":"10.1101/2020.09.12.294066","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.286948","pub_date":"2020-9-13","title":"Real-time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles","abstract":"SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule F\u00f6rster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.","version":"1.2","doi":"10.1101/2020.09.10.286948","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.293035","pub_date":"2020-9-12","title":"Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat due to the lack of effective drugs or vaccines against SARS-CoV-2. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a relatively new antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry into host cells, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and SARS-CoV-2-infected Ad-ACE2-transduced Tmprss2 knockout (Tmprss2-KO) and wild-type (WT) mice. TMPRSS2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cancer cells (LNCaP) but not in human lung cancer cells or patient-derived lung organoids. Although Tmprss2 knockout effectively blocked SARS-CoV-2 infection in ACE2-transduced mice, enzalutamide showed no antiviral activity due to the AR independence of TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19.","version":"1.1","doi":"10.1101/2020.09.11.293035","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.12.294413","pub_date":"2020-9-12","title":"Inferring MHC interacting SARS-CoV-2 epitopes recognized by TCRs towards designing T cell-based vaccines","abstract":"The coronavirus disease 2019 (COVID-19) is triggered by severe acute respiratory syndrome mediated by coronavirus 2 (SARS-CoV-2) infection and was declared by WHO as a major international public health concern. While worldwide efforts are being advanced towards vaccine development, the structural modeling of TCR-pMHC (T Cell Receptor-peptide-bound Major Histocompatibility Complex) regarding SARS-CoV-2 epitopes and the design of effective T cell vaccine based on these antigens are still unresolved. Here, we present both pMHC and TCR-pMHC interfaces to infer peptide epitopes of the SARS-CoV-2 proteins. Accordingly, significant TCR-pMHC templates (Z-value cutoff > 4) along with interatomic interactions within the SARS-CoV-2-derived hit peptides were clarified. Also, we applied the structural analysis of the hit peptides from different coronaviruses to highlight a feature of evolution in SARS-CoV-2, SARS-CoV, bat-CoV, and MERS-CoV. Peptide-protein flexible docking between each of the hit peptides and their corresponding MHC molecules were performed, and a multi-hit peptides vaccine against the S and N glycoprotein of SARS-CoV-2 was designed. Filtering pipelines including antigenicity, and also physiochemical properties of designed vaccine were then evaluated by different immunoinformatics tools. Finally, vaccine-structure modeling and immune simulation of the desired vaccine were performed aiming to create robust T cell immune responses. We anticipate that our design based on the T cell antigen epitopes and the frame of the immunoinformatics analysis could serve as valuable supports for the development of COVID-19 vaccine.","version":"1.2","doi":"10.1101/2020.09.12.294413","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.265561","pub_date":"2020-9-12","title":"SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients","abstract":"Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19.","version":"1.2","doi":"10.1101/2020.08.25.265561","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.294330","pub_date":"2020-9-12","title":"A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2","abstract":"Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-\u03b1), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.","version":"1.1","doi":"10.1101/2020.09.11.294330","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.294231","pub_date":"2020-9-12","title":"Type I Interferon Limits Viral Dissemination-Driven Clinical Heterogeneity in a Native Murine Betacoronavirus Model of COVID-19","abstract":"Emerging clinical data demonstrates that COVID-19, the disease caused by SARS-CoV2, is a syndrome that variably affects nearly every organ system. Indeed, the clinical heterogeneity of COVID-19 ranges from relatively asymptomatic to severe disease with death resultant from multiple constellations of organ failures. In addition to genetics and host characteristics, it is likely that viral dissemination is a key determinant of disease manifestation. Given the complexity of disease expression, one major limitation in current animal models is the ability to capture this clinical heterogeneity due to technical limitations related to murinizing SARS-CoV2 or humanizing mice to render susceptible to infection. Here we describe a murine model of COVID-19 using respiratory infection with the native mouse betacoronavirus MHV-A59. We find that whereas high viral inoculums uniformly led to hypoxemic respiratory failure and death, lethal dose 50% (LD50) inoculums led to a recapitulation of most hallmark clinical features of COVID-19, including lymphocytopenias, heart and liver damage, and autonomic dysfunction. We find that extrapulmonary manifestations are due to viral metastasis and identify a critical role for type-I but not type-III interferons in preventing systemic viral dissemination. Early, but not late treatment with intrapulmonary type-I interferon, as well as convalescent serum, provided significant protection from lethality by limiting viral dissemination. We thus establish a Biosafety Level II model that may be a useful addition to the current pre-clinical animal models of COVID-19 for understanding disease pathogenesis and facilitating therapeutic development for human translation.","version":"1.1","doi":"10.1101/2020.09.11.294231","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.285445","pub_date":"2020-9-12","title":"Evaluation of Safety and Immunogenicity of an Adjuvanted, TH-1 Skewed, Whole Virion InactivatedSARS-CoV-2 Vaccine - BBV152","abstract":"We report the development and evaluation of safety and immunogenicity of a whole virion inactivated SARS-COV-2 vaccine (BBV152), adjuvanted with aluminium hydroxide gel (Algel), or a novel TLR7/8 agonist adsorbed Algel. We used a well-characterized SARS-CoV-2 strain and an established vero cell platform to produce large-scale GMP grade highly purified inactivated antigen, BBV152. Product development and manufacturing were carried out in a BSL-3 facility. Immunogenicity was determined at two antigen concentrations (3\u03bcg and 6\u03bcg), with two different adjuvants, in mice, rats, and rabbits. Our results show that BBV152 vaccine formulations generated significantly high antigen-binding and neutralizing antibody titers, at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation containing TLR7/8 agonist adjuvant-induced Th1 biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2 specific IFN-\u03b3+ CD4 T lymphocyte response. Our results support further development for Phase I/II clinical trials in humans.","version":"1.2","doi":"10.1101/2020.09.09.285445","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.294363","pub_date":"2020-9-12","title":"Ketogenesis restrains aging-induced exacerbation of COVID in a mouse model","abstract":"Increasing age is the strongest predictor of risk of COVID-19 severity. Unregulated cytokine storm together with impaired immunometabolic response leads to highest mortality in elderly infected with SARS-CoV-2. To investigate how aging compromises defense against COVID-19, we developed a model of natural murine beta coronavirus (mCoV) infection with mouse hepatitis virus strain MHV-A59 (mCoV-A59) that recapitulated majority of clinical hallmarks of COVID-19. Aged mCoV-A59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue and hypothalamus, including neutrophilia and loss of \u03b3\u03b4 T cells in lungs. Ketogenic diet increases beta-hydroxybutyrate, expands tissue protective \u03b3\u03b4 T cells, deactivates the inflammasome and decreases pathogenic monocytes in lungs of infected aged mice. These data underscore the value of mCoV-A59 model to test mechanism and establishes harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against COVID-19 in the elderly. \n\n-Natural MHV-A59 mouse coronavirus infection mimics COVID-19 in elderly.\n-Aged infected mice have systemic inflammation and inflammasome activation\n-Murine beta coronavirus (mCoV) infection results in loss of pulmonary \u03b3\u03b4 T cells.\n-Ketones protect aged mice from infection by reducing inflammation.\n\n Natural MHV-A59 mouse coronavirus infection mimics COVID-19 in elderly. Aged infected mice have systemic inflammation and inflammasome activation Murine beta coronavirus (mCoV) infection results in loss of pulmonary \u03b3\u03b4 T cells. Ketones protect aged mice from infection by reducing inflammation. Elderly have the greatest risk of death from COVID-19. Here, Ryu et al report an aging mouse model of coronavirus infection that recapitulates clinical hallmarks of COVID-19 seen in elderly. The increased severity of infection in aged animals involved increased inflammasome activation and loss of \u03b3\u03b4 T cells that was corrected by ketogenic diet.","version":"1.1","doi":"10.1101/2020.09.11.294363","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.290718","pub_date":"2020-9-11","title":"Daytime variation in SARS-CoV-2 infection and cytokine production","abstract":"S. Ray and A. Reddy recently anticipated the implication of circadian rhythm in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of the coronavirus disease (Covid-19). In addition to its key role in the regulation of biological functions, the circadian rhythm has been suggested as a regulator of viral infections. Specifically, the time of day of infection was found critical for illness progression, as has been reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. We analyzed circadian rhythm implication in SARS-CoV-2 virus infection of isolated human monocytes, key actor cells in Covid-19 disease, from healthy subjects. The circadian gene expression of Bmal1 and Clock genes was investigated with q-RTPCR. Monocytes were infected with SARS-CoV-2 virus strain and viral infection was investigated by One-Step qRT-PCR and immunofluorescence. Interleukin (IL)-6, IL-1\u03b2 and IL-10 levels were also measured in supernatants of infected monocytes. Using Cosinor analysis, we showed that Bmal1 and Clock transcripts exhibited circadian rhythm in monocytes with an acrophase and a bathyphase at Zeitgeber Time (ZT)6 and ZT17. After forty-eight hours, the amount of SARS-CoV-2 virus increased in the monocyte infected at ZT6 compared to ZT17. The high virus amount at ZT6 was associated with significant increased release in IL-6, IL-1\u03b2 and IL-10 compared to ZT17. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression. The implication of circadian rhythm (CR) in pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been recently anticipated. The time of day of infection is critical for illness progression as reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. In this study, we wondered if SARS-CoV-2 infection and cytokine production by human monocytes, innate immune cells affected by Covid-19, were regulated by CR. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression.","version":"1.1","doi":"10.1101/2020.09.09.290718","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.292318","pub_date":"2020-9-11","title":"A COVID-19 antibody curbs SARS-CoV-2 nucleocapsid protein-induced complement hyper-activation","abstract":"Although human antibodies elicited by severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-directed antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAb) from a quick recovery coronavirus disease-19 (COVID-19) convalescent, who had dominant antibody responses to SARS-CoV-2 N protein rather than to Spike protein. The complex structure of N protein RNA binding domain with the highest binding affinity mAb nCoV396 reveals the epitopes and antigen\u2019s allosteric changes. Functionally, a virus-free complement hyper-activation analysis demonstrates that nCoV396 specifically compromises N protein-induced complement hyper-activation, a risk factor for morbidity and mortality in COVID-19, thus paving the way for functional anti-N mAbs identification. B cell profiling, structural determination, and protease activity assays identify a functional antibody to N protein.","version":"1.1","doi":"10.1101/2020.09.10.292318","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.293183","pub_date":"2020-9-11","title":"Molecular Characterization, Phylogenetic and Variation Analyzes of SARS-CoV-2 strains in Turkey","abstract":"We present the sequence analysis for 47 complete genomes for SARS-CoV-2 isolates on Turkish patients. To identify their genetic similarity, phylogenetic analysis was performed by comparing the worldwide SARS-CoV-2 sequences, selected from GISAID, to the complete genomes from Turkish isolates. In addition, we focused on the variation analysis to show the mutations on SARS-CoV-2 genomes. Illumina MiSeq platform was used for sequencing the libraries. The raw reads were aligned to the known SARS-CoV-2 genome (GenBank: MN908947.3) using the Burrows-Wheeler aligner (v.0.7.1). The phylogenetic tree was constructer using Phylip v.3.6 with Neighbor-Joining and composite likelihood method. The variants were detected by using Genome Analysis Toolkit-HaplotypeCaller v.3.8.0 and were inspected on GenomeBrowse v2.1.2. All viral genome sequences of our isolates was located in lineage B under the different clusters such as B.1 (n=3), B.1.1 (n=28), and B.1.9 (n=16). According to the GISAID nomenclature, all our complete genomes were placed in G, GR and GH clades. Five hundred forty-nine total and 53 unique variants were detected. All 47 genomes exhibited different kinds of variants. The distinct variants consist of 274 missense, 225 synonymous, and 50 non-coding alleles. The results indicated that the SARS-CoV-2 sequences of our isolates have great similarity with all Turkish and European sequences. Further studies should be performed for better comparison of strains, after more complete genome sequences will be released. We also believe that collecting and sharing any data about SARS-CoV-2 virus and COVID-19 will be effective and may help the related studies.","version":"1.1","doi":"10.1101/2020.09.11.293183","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.292631","pub_date":"2020-9-11","title":"A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD","abstract":"The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients\u2019 plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.","version":"1.1","doi":"10.1101/2020.09.11.292631","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.291716","pub_date":"2020-9-11","title":"Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors","abstract":"The ongoing SARS-CoV-2 pandemic has devastated the global economy and claimed nearly one million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen four related coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43 and SARS-CoV-2) at two physiologically relevant temperatures (33 \u00b0C and 37 \u00b0C), allowing us to probe this interactome at a much higher resolution relative to genome scale studies. This approach yielded several new insights, including unexpected virus and temperature specific differences in Rab GTPase requirements and GPI anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating COVID-19, and help prepare for future coronavirus outbreaks. Focused CRISPR screens targeting host factors in the SARS-CoV-2 interactome were performed for SARS-CoV-2, HCoV-229E, HCoV-NL63, and HCoV-OC43 coronaviruses. Focused interactome CRISPR screens achieve higher resolution compared to genome-wide screens, leading to the identification of critical factors missed by the latter. Parallel CRISPR screens against multiple coronaviruses uncover host factors and pathways with pan-coronavirus and virus-specific functional roles. The number of host proteins that interact with a viral bait protein is not proportional to the number of functional interactors. Novel SARS-CoV-2 host factors are expressed in relevant cell types in the human airway.","version":"1.1","doi":"10.1101/2020.09.11.291716","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.292730","pub_date":"2020-9-11","title":"In silico prediction of COVID-19 test efficiency with DinoKnot","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus spreading across the world causing the disease COVID-19. The diagnosis of COVID-19 is done by quantitative reverse-transcription polymer chain reaction (qRT-PCR) testing which utilizes different primer-probe sets depending on the assay used. Using in silico analysis we aimed to determine how the secondary structure of the SARS-CoV-2 RNA genome affects the interaction between the reverse primer during qRT-PCR and how it relates to the experimental primer-probe test efficiencies. We introduce the program DinoKnot (Duplex Interaction of Nucleic acids with pseudoKnots) that follows the hierarchical folding hypothesis to predict the secondary structure of two interacting nucleic acid strands (DNA/RNA) of similar or different type. DinoKnot is the first program that utilizes stable stems in both strands as a guide to find the structure of their interaction. Using DinoKnot we predicted the interaction of the reverse primers used in four common COVID-19 qRT-PCR tests with the SARS-CoV-2 RNA genome. In addition, we predicted how 12 mutations in the primer/probe binding region may affect the primer/probe ability and subsequent SARS-CoV-2 detection. While we found all reverse primers are capable of interacting with their target area, we identified partial mismatching between the SARS-CoV-2 genome and some reverse primers. We predicted three mutations that may prevent primer binding, reducing the ability for SARS-CoV-2 detection. We believe our contributions can aid in the design of a more sensitive SARS-CoV-2 test. The current testing for the disease COVID-19 that is caused by the novel cornonavirus SARS-CoV-2 uses oligonucleotides called primers that bind to specific target regions on the SARS-CoV-2 genome to detect the virus. Our goal was to use computational tools to predict how the structure of the SARS-CoV-2 RNA genome affects the ability of the primers to bind to their target region. We introduce the program DinoKnot (Duplex interaction of nucleic acids with pseudoknots) that is able to predict the interactions between two DNA or RNA molecules. We used DinoKnot to predict the efficiency of four common COVID-19 tests, and the effect of mutations in the SARS-CoV-2 virus on ability of the COVID-19 tests in detecting those strains. We predict partial mismatching between some primers and the SARS-CoV-2 genome but that all primers are capable of interacting with their target areas. We also predict three mutations that prevent primer binding and thus SARS-CoV-2 detection. We discuss the limitations of the current COVID-19 testing and suggest the design of a more sensitive COVID-19 test that can be aided by our findings.","version":"1.1","doi":"10.1101/2020.09.11.292730","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.11.293449","pub_date":"2020-9-11","title":"Identifying zoonotic origin of SARS-CoV-2 by modeling the binding affinity between Spike receptor-binding domain and host ACE2","abstract":"Despite considerable research progress on SARS-CoV-2, the direct zoonotic origin (intermediate host) of the virus remains ambiguous. The most definitive approach to identify the intermediate host would be the detection of SARS-CoV-2-like coronaviruses in wild animals. However, due to the high number of animal species, it is not feasible to screen all the species in the laboratory. Given that the recognition of the binding ACE2 proteins is the first step for the coronaviruses to invade host cells, we proposed a computational pipeline to identify potential intermediate hosts of SARS-CoV-2 by modeling the binding affinity between the Spike receptor-binding domain (RBD) and host ACE2. Using this pipeline, we systematically examined 285 ACE2 variants from mammals, birds, fish, reptiles, and amphibians, and found that the binding energies calculated on the modeled Spike-RBD/ACE2 complex structures correlate closely with the effectiveness of animal infections as determined by multiple experimental datasets. Built on the optimized binding affinity cutoff, we suggested a set of 96 mammals, including 48 experimentally investigated ones, which are permissive to SARS-CoV-2, with candidates from primates, rodents, and carnivores at the highest risk of infection. Overall, this work not only suggested a limited range of potential intermediate SARS-CoV-2 hosts for further experimental investigation; but more importantly, it proposed a new structure-based approach to general zoonotic origin and susceptibility analyses that are critical for human infectious disease control and wildlife protection.","version":"1.1","doi":"10.1101/2020.09.11.293449","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.290932","pub_date":"2020-9-11","title":"SARS-CoV-2 NSP1 C-terminal region (residues 130-180) is an intrinsically disordered region","abstract":"Nonstructural protein 1 (NSP1) of SARS-CoV-2 plays a key role in downregulation of RIG-I pathways and interacts with 40 S ribosome. Recently, the cryo-EM structure in complex with 40S ribosome is deciphered. However, the structure of full length NSP1 without any partner has not been studies. Also, the conformation of NSP1-C terminal region in isolation is not been studied. In this study, we have investigated the conformational dynamics of NSP1C-terminal region (NSP1-CTR; amino acids 130-180) in isolation and under different solvent environments. The NSP1-CTR is found to be intrinsically disordered in aqueous solution. Further, we used alpha helix inducer, trifluoroethanol, and found induction of alpha helical conformation using CD spectroscopy. Additionally, in the presence of SDS, NSP1-CTR is showing a conformational change from disordered to ordered, possibly gaining alpha helix in part. But in presence of neutral lipid DOPC, a slight change in conformation is observed. This implies the possible role of hydrophobic interaction and electrostatic interaction on the conformational changes of NSP1. The changes in structural conformation were further studied by fluorescence-based studies, which showed significant blue shift and fluorescence quenching in the presence of SDS and TFE. Lipid vesicles also showed fluorescence-based quenching. In agreement to these result, fluorescence lifetime and fluorescence anisotropy decay suggests a change in conformational dynamics. The zeta potential studies further validated that the conformational dynamics is mostly because of hydrophobic interaction. In last, these experimental studies were complemented through Molecular Dynamics (MD) simulation which have also shown a good correlation and testify our experiments. We believe that the intrinsically disordered nature of the NSP1-CTR will have implications in disorder based binding promiscuity with its interacting proteins.","version":"1.1","doi":"10.1101/2020.09.10.290932","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.284737","pub_date":"2020-9-10","title":"Many bat species are not potential hosts of SARS-CoV and SARS-CoV-2: Evidence from ACE2 receptor usage","abstract":"Bats are the suggested natural hosts for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2, the latter of which caused the coronavirus disease 2019 (COVID-19) pandemic. The interaction of viral Spike proteins with their host receptor angiotensin-converting enzyme 2 (ACE2) is a critical determinant of potential hosts and cross-species transmission. Here we use virus-host receptor binding and infection assays to show that ACE2 orthologs from 24, 21, and 16 of 46 phylogenetically diverse bat species \u2013 including those in close and distant contact with humans \u2013 do not support entry of SARS-CoV, SARS-CoV-2, and both of these coronaviruses, respectively. Furthermore, we used genetic and functional analyses to identify genetic changes in bat ACE2 receptors associated with viral entry restrictions. Our study demonstrates that many \u2013 if not most \u2013 bat species are not potential hosts of SARS-CoV and SARS-CoV-2, and provides important insights into pandemic control and wildlife conservation.","version":"1.3","doi":"10.1101/2020.09.08.284737","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.288548","pub_date":"2020-9-10","title":"Susceptibility of domestic swine to experimental infection with SARS-CoV-2","abstract":"SARS-CoV-2, the agent responsible for COVID-19 has been shown to infect a number of species. The role of domestic livestock and the risk associated for humans in close contact remains unknown for many production animals. Determination of the susceptibility of pigs to SARS-CoV-2 is critical towards a One Health approach to manage the potential risk of zoonotic transmission. Here, pigs undergoing experimental inoculation are susceptible to SARS-CoV-2 at low levels. Viral RNA was detected in group oral fluids and nasal wash from at least two animals while live virus was isolated from a pig. Further, antibodies could be detected in two animals at 11 and 13 days post infection, while oral fluid samples at 6 days post inoculation indicated the presence of secreted antibodies. These data highlight the need for additional livestock assessment to better determine the potential role domestic animals may contribute towards the SARS-CoV-2 pandemic.","version":"1.1","doi":"10.1101/2020.09.10.288548","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.288720","pub_date":"2020-9-10","title":"Conserved interactions required for in vitro inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands\u2019 interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6,900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50\u03bcM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.09.10.288720","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.291757","pub_date":"2020-9-10","title":"Molecular basis for SARS-CoV-2 spike affinity for human ACE2 receptor","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused substantially more infections, deaths, and economic disruptions than the 2002-2003 SARS-CoV. The key to understanding SARS-CoV-2\u2019s higher infectivity may lie in its host receptor recognition mechanism. This is because experiments show that the human ACE2 protein, which serves as the primary receptor for both CoVs, binds to CoV-2\u2019s spike protein 5-20 fold stronger than SARS-CoV\u2019s spike protein. The molecular basis for this difference in binding affinity, however, remains unexplained and, in fact, a comparison of X-ray structures leads to an opposite proposition. To gain insight, we use all-atom molecular dynamics simulations. Free energy calculations indicate that CoV-2\u2019s higher affinity is due primarily to differences in specific spike residues that are local to the spike-ACE2 interface, although there are allosteric effects in binding. Comparative analysis of equilibrium simulations reveals that while both CoV and CoV-2 spike-ACE2 complexes have similar interfacial topologies, CoV-2\u2019s spike protein engages in greater numbers, combinatorics and probabilities of hydrogen bonds and salt bridges with ACE2. We attribute CoV-2\u2019s higher affinity to these differences in polar contacts, and these findings also highlight the importance of thermal structural fluctuations in spike-ACE2 complexation. We anticipate that these findings will also inform the design of spike-ACE2 peptide blockers that, like in the cases of HIV and Influenza, can serve as antivirals.","version":"1.1","doi":"10.1101/2020.09.10.291757","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.07.286344","pub_date":"2020-9-10","title":"Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease","abstract":"The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an \u03b1-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N\u03b4 (HD) and N\u03f5 (HE) protonation of His41 and His164, respectively, the \u03b1-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.","version":"1.1","doi":"10.1101/2020.09.07.286344","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.124966","pub_date":"2020-9-10","title":"coronapp: A Web Application to Annotate and Monitor SARS-CoV-2 Mutations","abstract":"The avalanche of genomic data generated from the SARS-CoV-2 virus requires the development of tools to detect and monitor its mutations across the world. Here, we present a webtool, coronapp, dedicated to easily processing user-provided SARS-CoV-2 genomic sequences and visualizing current worldwide status of SARS-CoV-2 mutations. The webtool allows users to highlight mutations and categorize them by frequency, country, genomic location and effect on protein sequences, and to monitor their presence in the population over time. The tool is available at http://giorgilab.unibo.it/coronapp/ for the worldwide dataset and at http://giorgilab.unibo.it/coronannotator/ for the annotation of user-provided sequences. The full code is freely shared at https://github.com/federicogiorgi/giorgilab/tree/master/coronapp The data that support the findings of this study derive from the GISAID consortium and are openly available in Github, in Rdata format for the R environment, in files results.rda and metadata.rda, at the following link: https://github.com/federicogiorgi/giorgilab/tree/master/coronapp/data","version":"1.3","doi":"10.1101/2020.05.31.124966","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.990242","pub_date":"2020-9-10","title":"Classification and Specific Primer Design for Accurate Detection of SARS-CoV-2 Using Deep Learning","abstract":"In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from available repositories, separating the genome of different virus strains from the Coronavirus family with considerable accuracy. The network\u2019s behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are first validated on samples from other repositories, and proven able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets on existing datasets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n=6 previously tested positive), delivering a sensibility similar to routine diagnostic methods, and 100% specificity. In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from NGDC, separating the genome of different virus strains from the Coronavirus family with accuracy 98.73%. The network\u2019s behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are validated on samples from NCBI and GISAID, and proven able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n=6 previously tested positive), delivering a sensibility similar to routine diagnostic methods, and 100% specificity. The proposed methodology has a substantial added value over existing methods, as it is able to both identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. Considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics.","version":"1.5","doi":"10.1101/2020.03.13.990242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.10.290841","pub_date":"2020-9-10","title":"CD8 T cell epitope generation toward the continually mutating SARS-CoV-2 spike protein in genetically diverse human population: Implications for disease control and prevention","abstract":"The ongoing pandemic of SARS-CoV-2 has brought tremendous crisis on global health care systems and industrial operations that dramatically affect the economic and social life of numerous individuals worldwide. Understanding anti-SARS-CoV-2 immune responses in population with different genetic backgrounds and tracking the viral evolution are crucial for successful vaccine design. In this study, we reported the generation of CD8 T cell epitopes by a total of 80 alleles of three major class I HLAs using NetMHC 4.0 algorithm for the spike protein of SARS-CoV-2, a key antigen that is targeted by both B cells and T cells. We found diverse capacities of S protein specific epitope presentation by different HLA alleles with very limited number of predicted epitopes for HLA-B*2705, HLA-B*4402 and HLA-B*4403 and as high as 132 epitopes for HLA-A*6601. Our analysis of 1000 S protein sequences from field isolates collected globally over the past few months identified three recurrent point mutations including L5F, D614G and G1124V. Differential effects of these mutations on CD8 T cell epitope generation by corresponding HLA alleles were observed. Finally, our multiple alignment analysis indicated the absence of seasonal CoV induced cross-reactive CD8 T cells to drive these mutations. Our findings provided molecular explanations for the observation that individuals with certain HLA alleles such as B*44 are more prone to SARS-CoV-2 infection. Studying anti-S protein specific CD8 T cell immunity in diverse genetic background is critical for better control and prevention of the SARS-CoV-2 pandemic.","version":"1.1","doi":"10.1101/2020.09.10.290841","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.196220","pub_date":"2020-9-10","title":"COVID-19 Biomarkers in research: Extension of the OncoMX cancer biomarker data model to capture biomarker data from other diseases","abstract":"Scientists, medical researchers, and health care workers have mobilized worldwide in response to the outbreak of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SCoV2). Preliminary data have captured a wide range of host responses, symptoms, and lingering problems post-recovery within the human population. These variable clinical manifestations suggest differences in influential factors, such as innate and adaptive host immunity, existing or underlying health conditions, co-morbidities, genetics, and other factors. As COVID-19-related data continue to accumulate from disparate groups, the heterogeneous nature of these datasets poses challenges for efficient extrapolation of meaningful observations, hindering translation of information into clinical applications. Attempts to utilize, analyze, or combine biomarker datasets from multiple sources have shown to be inefficient and complicated, without a unifying resource. As such, there is an urgent need within the research community for the rapid development of an integrated and harmonized COVID-19 Biomarker Knowledgebase. By leveraging data collection and integration methods, backed by a robust data model developed to capture cancer biomarker data we have rapidly crowdsourced the collection and harmonization of COVID-19 biomarkers. Our resource currently has 138 unique biomarkers. We found multiple instances of the same biomarker substance being suggested as multiple biomarker types during our extensive cross-validation and manual curation. As a result, our Knowledgebase currently has 265 biomarker type combinations. Every biomarker entry is made comprehensive by bringing in together ancillary data from multiple sources such as biomarker accessions (canonical UniProtKB accession, PubChem Compound ID, Cell Ontology ID, Protein Ontology ID, NCI Thesaurus Code, and Disease Ontology ID), BEST biomarker category, and specimen type (Uberon Anatomy Ontology) unified with ontology standards. Our preliminary observations show distinct trends in the collated biomarkers. Most biomarkers are related to the immune system (SAA,TNF-\u221d, and IP-10) or coagulopathies (D-dimer, antithrombin, and VWF) and a few have already been established as cancer biomarkers (ACE2, IL-6, IL-4 and IL-2). These trends align with proposed hypotheses of clinical manifestations compounding the complexity of COVID-19 pathobiology. We explore these trends as we put forth a COVID-19 biomarker resource that will help researchers and diagnosticians alike. All biomarker data are freely available from https://data.oncomx.org/covid19.","version":"1.1","doi":"10.1101/2020.09.09.196220","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.20190975","pub_date":"2020-09-10","title":"Vitamin D and COVID-19 susceptibility and severity in the COVID-19 Host Genetics Initiative: A Mendelian randomization study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>\n                    Increased vitamin D levels, as reflected by 25OHD measurements, have been proposed to protect against COVID-19 disease based on\n                    <jats:italic>in-vitro</jats:italic>\n                    , observational, and ecological studies. However, vitamin D levels are associated with many confounding variables and thus associations described to date may not be causal. Vitamin D Mendelian randomization (MR) studies have provided results that are concordant with large-scale vitamin D randomized trials. Here, we used two-sample MR to assess evidence supporting a causal effect of circulating 25OHD levels on COVID-19 susceptibility and severity.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods and findings</jats:title>\n                  <jats:p>Genetic variants strongly associated with 25OHD levels in a genome-wide association study (GWAS) of 443,734 participants of European ancestry (including 401,460 from the UK Biobank) were used as instrumental variables. GWASs of COVID-19 susceptibility, hospitalization, and severe disease from the COVID-19 Host Genetics Initiative were used as outcome GWASs. These included up to 14,134 individuals with COVID-19, and 1,284,876 without COVID-19, from 11 countries. SARS-CoV-2 positivity was determined by laboratory testing or medical chart review. Population controls without COVID-19 were also included in the control groups for all outcomes, including hospitalization and severe disease. Analyses were restricted to individuals of European descent when possible. Using inverse-weighted MR, genetically increased 25OHD levels by one standard deviation on the logarithmic scale had no significant association with COVID-19 susceptibility (OR = 0.97; 95% CI: 0.95, 1.10; P=0.61), hospitalization (OR = 1.11; 95% CI: 0.91, 1.35; P=0.30), and severe disease (OR = 0.93; 95% CI: 0.73, 1.17; P=0.53). We used an additional 6 meta-analytic methods, as well as sensitivity analyses after removal of variants at risk of horizontal pleiotropy and obtained similar results. These results may be limited by weak instrument bias in some analyses. Further, our results do not apply to individuals with vitamin D deficiency.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>In this two-sample MR study, we did not observe evidence to support an association between 25OHD levels and COVID-19 susceptibility, severity, or hospitalization. Hence, vitamin D supplementation as a means of protecting against worsened COVID-19 outcomes is not supported by genetic evidence. Other therapeutic or preventative avenues should be given higher priority for COVID-19 randomized controlled trials.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Author Summary</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>\n                        <jats:bold>Why was this study done?</jats:bold>\n                      </jats:p>\n                      <jats:list list-type='simple'>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>Vitamin D levels have been associated with COVID-19 outcomes in multiple observational studies, though confounders are likely to bias these associations.</jats:p>\n                        </jats:list-item>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>By using genetic instruments which limit such confounding, Mendelian randomization studies have consistently obtained results concordant with vitamin D supplementation randomized trials. This provides rationale to undertake vitamin D Mendelian randomization studies for COVID-19 outcomes.</jats:p>\n                        </jats:list-item>\n                      </jats:list>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>\n                        <jats:bold>What did the researchers do and find?</jats:bold>\n                      </jats:p>\n                      <jats:list list-type='simple'>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>We used the genetic variants obtained from the largest consortium of COVID-19 cases and controls, and the largest study on genetic determinants of vitamin D levels.</jats:p>\n                        </jats:list-item>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>We used Mendelian randomization to estimate the effect of increased vitamin D on COVID-19 outcomes, while limiting confounding.</jats:p>\n                        </jats:list-item>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>In multiple analyses, our results consistently showed no evidence for an association between genetically predicted vitamin D levels and COVID-19 susceptibility, hospitalization, or severe disease.</jats:p>\n                        </jats:list-item>\n                      </jats:list>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>\n                        <jats:bold>What do these findings mean?</jats:bold>\n                      </jats:p>\n                      <jats:list list-type='simple'>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>Using Mendelian randomization to reduce confounding that has traditionally biased vitamin D observational studies, we did not find evidence that vitamin D supplementation in the general population would improve COVID-19 outcomes</jats:p>\n                        </jats:list-item>\n                        <jats:list-item>\n                          <jats:label>-</jats:label>\n                          <jats:p>These findings, together with recent randomized controlled trial data, suggest that other therapies should be prioritized for COVID-19 trials.</jats:p>\n                        </jats:list-item>\n                      </jats:list>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.08.20190975","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.09.273268","pub_date":"2020-9-10","title":"Epigenetic Evolution of ACE2 and IL-6 Genes as Non-Canonical Interferon-Stimulated Genes Correlate to COVID-19 Susceptibility in Vertebrates","abstract":"Current novel coronavirus disease (COVID-19) has spread globally within a matter of months. The virus establishes a success in balancing its deadliness and contagiousness, and causes substantial differences in susceptibility and disease progression in people of different ages, genders and pre-existing comorbidities. Since these host factors are subjected to epigenetic regulation, relevant analyses on some key genes underlying COVID-19 pathogenesis were performed to longitudinally decipher their epigenetic correlation to COVID-19 susceptibility. The genes of host angiotensin-converting enzyme 2 (ACE2, as the major virus receptor) and interleukin (IL)-6 (a key immune-pathological factor triggering cytokine storm) were shown to evince active epigenetic evolution via histone modification and cis/trans-factors interaction across different vertebrate species. Extensive analyses revealed that ACE2 ad IL-6 genes are among a subset of non-canonical interferon-stimulated genes (non-ISGs), which have been designated recently for their unconventional responses to interferons (IFNs) and inflammatory stimuli through an epigenetic cascade. Furthermore, significantly higher positive histone modification markers and position weight matrix (PWM) scores of key cis-elements corresponding to inflammatory and IFN signaling, were discovered in both ACE2 and IL6 gene promoters across representative COVID-19-susceptible species compared to unsusceptible ones. Findings characterize ACE2 and IL-6 genes as non-ISGs that respond differently to inflammatory and IFN signaling from the canonical ISGs and their epigenetic properties may serve as biomarkers to longitudinally predict COVID-19 susceptibility in vertebrates and partially explain COVID-19 inequality in people of different subgroups.","version":"1.1","doi":"10.1101/2020.09.09.273268","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109124","pub_date":"2020-9-09","title":"Intestinal inflammation modulates the expression of ACE2 and TMPRSS2 and potentially overlaps with the pathogenesis of SARS-CoV-2 related disease","abstract":"The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication within enterocytes. Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation and IBD treatment. A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and non-biologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. Additionally, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD. These data generate a novel appreciation of the confluence of COVID-19- and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.05.21.109124","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.289850","pub_date":"2020-9-09","title":"Transcriptomic dysregulations associated with SARS-CoV-2 infection in human nasopharyngeal and peripheral blood mononuclear cells","abstract":"Over 24 million people have been infected globally with the novel coronavirus, SARS-CoV-2, with more than 820,000 succumbing to the resulting COVID-19 disease as of the end of August 2020. The molecular mechanisms underlying the pathogenesis of the disease are not completely elucidated. Thus, we aim to understand host response to SARS-CoV-2 infection by comparing samples collected from two distinct compartments (infection site and blood), obtained from COVID-19 subjects and healthy controls. We used two publicly available gene expression datasets generated via RNA sequencing in two different samples; nasopharyngeal swabs and peripheral blood mononuclear cells (PBMCs). We performed a differential gene expression analysis between COVID-19 subjects and healthy controls in the two datasets and then functionally profiled their differentially expressed genes (DEGs). The genes involved in innate immunity were also determined. We found a clear difference in the host response to SARS-CoV-2 infection between the two sample groups. In COVID-19 subjects, the nasopharyngeal sample group indicated upregulation of genes involved in cytokine activity and interferon signalling pathway, as well as downregulation of genes involved in oxidative phosphorylation and viral transcription. Host response in COVID-19 subjects for the PBMC group, involved upregulation of genes involved in the complement system and immunoglobulin mediated immune response. CXCL13, GABRE, IFITM3 were upregulated and HSPA1B was downregulated in COVID-19 subjects in both sample groups. Our results indicate the host response to SARS-CoV-2 is compartmentalized and suggests potential biomarkers of response to SARS-CoV-2 infection. Transcriptomic profiling from publicly available RNA-seq count data revealed a site-specific immune response in COVID-19. Host response was found cellular-mediated in nasopharyngeal samples and humoral-mediated in PBMCs samples. CXCL13, GABRE and IFITM3 commonly upregulated and HSPA1B downregulated in both sample groups highlights the potential of these molecules as markers of response to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.09.09.289850","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.289892","pub_date":"2020-9-09","title":"Initial insights into the genetic epidemiology of SARS-CoV-2 isolates from Kerala suggest local spread from limited introductions","abstract":"Coronavirus disease 2019 (COVID-19) rapidly spread from a city in China to almost every country in the world, affecting millions of individuals. Genomic approaches have been extensively used to understand the evolution and epidemiology of SARS-CoV-2 across the world. Kerala is a unique state in India well connected with the rest of the world through a large number of expatriates, trade, and tourism. The first case of COVID-19 in India was reported in Kerala in January 2020, during the initial days of the pandemic. The rapid increase in the COVID-19 cases in the state of Kerala has necessitated the understanding of the genetic epidemiology of circulating virus, evolution, and mutations in SARS-CoV-2. We sequenced a total of 200 samples from patients at a tertiary hospital in Kerala using COVIDSeq protocol at a mean coverage of 7,755X. The analysis identified 166 unique high-quality variants encompassing 4 novel variants and 89 new variants identified for the first time in SARS-CoV-2 samples isolated from India. Phylogenetic and haplotype analysis revealed that the circulating population of the virus was dominated (94.6% of genomes) by three distinct introductions followed by local spread, apart from identifying polytomies suggesting recent outbreaks. The genomes formed a monophyletic distribution exclusively mapping to the A2a clade. Further analysis of the functional variants revealed two variants in the S gene of the virus reportedly associated with increased infectivity and 5 variants that mapped to five primer/probe binding sites that could potentially compromise the efficacy of RT-PCR detection. To the best of our knowledge, this is the first and most comprehensive report of genetic epidemiology and evolution of SARS-CoV-2 isolates from Kerala.","version":"1.1","doi":"10.1101/2020.09.09.289892","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.288704","pub_date":"2020-9-09","title":"Small molecules inhibit SARS-COV-2 induced aberrant inflammation and viral replication in mice by targeting S100A8/A9-TLR4 axis","abstract":"The SARS-CoV-2 pandemic poses an unprecedented public health crisis. Accumulating evidences suggest that SARS-CoV-2 infection causes dysregulation of immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced by SARS-CoV-2 in animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could reduce inflammatory response and rescue the pneumonia with substantial reduction of viral titers in SASR-CoV-2 infected animals. Remarkably, Paquinimod treatment resulted in 100% survival of mice in a lethal model of mouse coronavirus (MHV) infection. A novel group of neutrophils that contributed to the uncontrolled inflammation and onset of COVID-19 were dramatically induced by coronavirus infections. Paquinimod treatment could reduce these neutrophils and regain antiviral responses, unveiling key roles of S100A8/A9 and noncanonical neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.","version":"1.1","doi":"10.1101/2020.09.09.288704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.286732","pub_date":"2020-9-09","title":"Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells","abstract":"Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/rcTA cells gave substantially better yields than the other methods. Different forms of the spike were expressed, including the wild-type SARS-CoV-2 sequence and a mutated/stabilized form (to favor expression of the full-length spike in prefusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.","version":"1.1","doi":"10.1101/2020.09.08.286732","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.288555","pub_date":"2020-9-09","title":"Enhanced SARS-CoV-2 Neutralization by Secretory IgA in vitro","abstract":"SARS-CoV-2 primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of the illness. Although potent IgG antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might impact the initial viral spread and transmissibility from the mucosa. Here we characterize the IgA response to SARS-CoV-2 in a cohort of 149 individuals. IgA responses in plasma generally correlate with IgG responses and clones of IgM, IgG and IgA producing B cells that are derived from common progenitors are evident. Plasma IgA monomers are 2-fold less potent than IgG equivalents. However, IgA dimers, the primary form in the nasopharynx, are on average 15 times more potent than IgA monomers. Thus, secretory IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.","version":"1.1","doi":"10.1101/2020.09.09.288555","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.287987","pub_date":"2020-9-09","title":"Near-Physiological-Temperature Serial Femtosecond X-ray Crystallography Reveals Novel Conformations of SARS-CoV-2 Main Protease Active Site for Improved Drug Repurposing","abstract":"The COVID19 pandemic has resulted in 25+ million reported infections and nearly 850.000 deaths. Research to identify effective therapies for COVID19 includes: i) designing a vaccine as future protection; ii) structure-based drug design; and iii) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determined two apo structures of Severe Acute Respiratory Syndrome CoronaVirus-2 main protease at ambienttemperature by Serial Femtosecond X-ray crystallography. We employed detailed molecular simulations of selected known main protease inhibitors with the structures and compared binding modes and energies. The combined structural biology and molecular modeling studies not only reveal the dynamics of small molecules targeting main protease but will also provide invaluable opportunities for drug repurposing and structure-based drug design studies against SARS-CoV-2. Radiation-damage-free high-resolution SARS-CoV-2 main protease SFX structures obtained at near-physiological-temperature offer invaluable information for immediate drug-repurposing studies for the treatment of COVID19.","version":"1.1","doi":"10.1101/2020.09.09.287987","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.09.290247","pub_date":"2020-9-09","title":"rSWeeP: A R/Bioconductor package deal with SWeeP sequences representation","abstract":"The rSWeeP package is an R implementation of the SWeeP model, designed to handle Big Data. rSweeP meets to the growing demand for efficient methods of heuristic representation in the field of Bioinformatics, on platforms accessible to the entire scientific community. We explored the implementation of rSWeeP using a dataset containing 31,386 viral proteomes, performing phylogenetic and principal component analysis. As a case study we analyze the viral strains closest to the SARS-CoV, responsible for the current pandemic of COVID-19, confirming that rSWeeP can accurately classify organisms taxonomically. rSWeeP package is freely available at https://bioconductor.org/packages/release/bioc/html/rSWeeP.html.","version":"1.1","doi":"10.1101/2020.09.09.290247","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.169946","pub_date":"2020-9-08","title":"Neuroinvasion of SARS-CoV-2 in human and mouse brain","abstract":"Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.06.25.169946","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.07.286567","pub_date":"2020-9-08","title":"Interaction of human ACE2 to membrane-bound SARS-CoV-1 and SARS-CoV-2 S glycoproteins","abstract":"A novel severe acute respiratory (SARS)-like coronavirus (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor binding domain of its trimeric Spike glycoprotein and the human angiotensin converting enzyme 2 (ACE2) receptor. A better understanding of the Spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both Spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restrains. Our findings can be of importance in the development of therapeutics that block the Spike/ACE2 interaction.","version":"1.1","doi":"10.1101/2020.09.07.286567","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.288191","pub_date":"2020-9-08","title":"Structural characterization of Nonstructural protein 1 from SARS-CoV-2","abstract":"Severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) is a single-stranded, enveloped RNA virus and the etiological agent of the current COVID-19 pandemic. Efficient replication of the virus relies on the activity of nonstructural protein 1 (Nsp1), a major virulence factor shown to facilitate suppression of host gene expression through promotion of host mRNA degradation and interaction with the 40S ribosomal subunit. Here, we report the crystal structure of the globular domain of SARS-CoV-2 Nsp1, encompassing residues 13 to 127, at a resolution of 1.65 \u00c5. Our structure features a six-stranded, capped \u03b2-barrel motif similar to Nsp1from SARS-CoV and reveals how variations in amino acid sequence manifest as distinct structural features. Through comparative analysis of structural homologues, we identified a topological signature associated with this protein fold that facilitated modeling of Nsp1 from MERS-CoV. Combining our high-resolution crystal structure with existing data on the C-terminus of Nsp1 from SARS-CoV-2, we propose a model of the full-length protein. Our results provide unparalleled insight into the molecular structure of a major pathogenic determinant of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.08.288191","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.07.286666","pub_date":"2020-9-08","title":"UVA radiation could be a significant contributor to sunlight inactivation of SARS-CoV-2","abstract":"Past experiments demonstrated SARS-CoV-2 inactivation by simulated sunlight; models have considered exclusively mechanisms involving UVB acting directly on RNA. However, UVA inactivation has been demonstrated for other enveloped RNA viruses, through indirect mechanisms involving the suspension medium. We propose a model combining UVB and UVA inactivation for SARS-CoV-2, which improves predictions by accounting for effects associated with the medium. UVA sensitivities deduced for SARS-CoV-2 are consistent with data for SARS-CoV-1 under UVA only. This analysis calls for experiments to separately assess effects of UVA and UVB in different media, and for including UVA in inactivation models. Recent experiments have demonstrated that SARS-CoV-2 is inactivated by simulated sunlight; however, there are still many unknowns, including the mechanism of action and which part of the light spectrum is principally responsible. Our analysis indicates the need for targeted experiments that can separately assess the effects of UVA and UVB on SARS-CoV-2, and that sunlight inactivation models may need to be expanded to also include the effect of UVA. A first UVA-inclusive model is also proposed here. These findings have implications for how to improve the safety of the built environment, and for the seasonality of COVID-19.","version":"1.1","doi":"10.1101/2020.09.07.286666","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.280818","pub_date":"2020-9-08","title":"A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates","abstract":"To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD \u2018up\u2019, two-RBD \u2018down\u2019 state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFN\u03b3+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).","version":"1.1","doi":"10.1101/2020.09.08.280818","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.02.280529","pub_date":"2020-9-08","title":"Mutation in position of 32 (G>U) of S2M differentiate human SARS-CoV2 from Bat Coronavirus","abstract":"The new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a zoonotic pathogen that has rapidly mutated and become transmissible to humans. There is little existing data on the mutations in SARS-CoV-2 and the impact of these polymorphisms on its transmission and viral load. In this study, the SARS-CoV-2 genomic sequence was analyzed to identify variants within the 3\u2019UTR region of its cis-regulatory RNA elements. A 43-nucleotide genetic element with a highly conserved stem-loop II-like motif (S2M), was discovered. The research revealed 32 G>U and 16 G>U/A mutations located within the S2M sequence in human SARS-CoV-2 models. These polymorphisms appear to make the S2M secondary and tertiary structures in human SARS-CoV-2 models less stable when compared to the S2M structures of bat/pangolin models. This grants the RNA structures more flexibility, which could be one of its escape mechanisms from host defenses or facilitate its entry into host proteins and enzymes. While this S2M sequence may not be omnipresent across all human SARS-CoV-2 models, when present, its sequence is always highly conserved. It may be used as a potential target for the development of vaccines and therapeutic agents.","version":"1.2","doi":"10.1101/2020.09.02.280529","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.063859","pub_date":"2020-9-08","title":"Role of 1\u2019-Ribose Cyano Substitution for Remdesivir to Effectively Inhibit Nucleotide Addition and Proofreading in SARS-CoV-2 Viral RNA Replication","abstract":"COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising but challenging drug target due to its intrinsic proofreading exoribonuclease (ExoN). Remdesivir targeting SARS-CoV-2 RdRp exerts high drug efficacy in vitro and in vivo. However, its underlying inhibitory mechanisms remain elusive. Here, we performed all-atom molecular dynamics simulations with an accumulated simulation time of 24 microseconds to elucidate the molecular mechanisms underlying the inhibitory effects of Remdesivir. We found that Remdesivir\u2019s 1\u2019-cyano group of possesses the dual role of inhibiting nucleotide addition and proofreading. The presence of its polar 1\u2019-cyano group at an upstream site in RdRp causes instability and hampers RdRp translocation. This leads to a delayed chain termination of RNA extension, which may also subsequently reduce the likelihood for Remdesivir to be cleaved by ExoN acting on the 3\u2019-terminal nucleotide. In addition, our simulations suggest that Remdesivir\u2019s 1\u2019-cyano group can also disrupt the cleavage active site of ExoN via steric interactions, leading to a further reduced cleavage efficiency. Our work provides plausible molecular mechanisms on how Remdesivir inhibits viral RNA replication and may guide rational design for new treatments of COVID-19 targeting viral replication.","version":"1.3","doi":"10.1101/2020.04.27.063859","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.08.287482","pub_date":"2020-9-08","title":"Antibody binding to SARS-CoV-2 S glycoprotein correlates with, but does not predict neutralization","abstract":"Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals as well as S-specific monoclonal antibodies were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to binding of S glycoprotein in the context of viral particles remains to be established. Here we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA and neutralization assays we observed a strong correlation between these two parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of viral particles is required but not sufficient to mediate neutralization. Altogether, our results highlights the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.","version":"1.1","doi":"10.1101/2020.09.08.287482","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.03.281774","pub_date":"2020-9-08","title":"Phylo-geo-network and haplogroup analysis of 611 novel Coronavirus (nCov-2019) genomes from India","abstract":"The novel Coronavirus from Wuhan China discovered in December 2019 (nCOV-2019) has since developed into a global epidemic with major concerns about the possibility of the virus evolving into something even more sinister. In the present study we constructed the phylo-geo-network of nCOV-2019 genomes from across India to understand the viral evolution in the country. A total of 611 full length genomes were extracted from different states of India from the EpiCov repository of GISAID initiative and NCBI. Their alignment uncovered 270 parsimony informative sites. Further, 339 genomes were divided into 51 haplogroups. The network revealed the core haplogroup as that of reference sequence NC_045512.2 (Haplogroup A1) with 157 identical sequences present across 16 states. The rest were having not more than ten identical sequences across not more than three locations. Interestingly, some locations with fewer samples have more haplogroups and most haplogroups (41) are localized exclusively to any one state only, suggesting the local evolution of viruses. The two most common lineages are B6 and B1 (Pangolin) whereas clade A2a (Covidex) appears to be the most predominant in India. However, since the pandemic is still emerging, the final outcome will be clear later only.","version":"1.2","doi":"10.1101/2020.09.03.281774","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.07.286120","pub_date":"2020-9-07","title":"The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells","abstract":"After the SARS-CoV outbreak in 2003, a second zoonotic coronavirus named SARS-CoV-2, emerged late 2019 in China and rapidly caused the COVID-19 pandemic leading to a public health crisis of an unprecedented scale. Despite the fact that SARS-CoV-2 uses the same receptor as SARS-CoV, transmission and pathogenesis of both viruses seem to be quite distinct. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site, which is absent in the SARS-CoV spike. The viral spike protein not only attaches to the entry receptor, but also mediates fusion after cleavage by host proteases. Here, we report that the SARS-CoV-2 spike multibasic cleavage site increases infectivity on differentiated organoid-derived human airway cells. Compared with SARS-CoV, SARS-CoV-2 entered faster into the lung cell line Calu-3, and more frequently formed syncytial cells in differentiated organoid-derived human airway cells. Moreover, the multibasic cleavage site increased entry speed and plasma membrane serine protease usage relative to endosomal entry using cathepsins. Blocking serine protease activity using the clinically approved drug camostat mesylate effectively inhibited SARS-CoV-2 entry and replication in differentiated organoid-derived human airway cells. Our findings provide novel information on how SARS-CoV-2 enters relevant airway cells and highlight serine proteases as an attractive antiviral target. Highly pathogenic coronaviruses have spilled from animals to humans three times in the past two decades. Late 2019, SARS-CoV-2 emerged in China and was declared a pandemic by March 2020. The other two highly pathogenic coronaviruses, SARS-CoV and MERS-CoV, emerged in 2002 and 2012, respectively, but did not attain sustained human-to-human transmission. Given the high diversity of coronaviruses in animals, urbanization and increased air travel, future coronavirus pandemics are likely to occur intermittently. Identifying which factors determine pandemic potential and pathogenicity are therefore of key importance to global health. Additionally, there is an urgent need to rapidly translate fundamental knowledge to the clinic, a process that is expedited through the use of relevant cell culture systems.","version":"1.1","doi":"10.1101/2020.09.07.286120","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.07.286088","pub_date":"2020-9-07","title":"Mutational Analysis of SARS-CoV-2 Genome in African Population","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly infectious and pathogenic virus has claimed lot of lives globally since its outbreak in December 2019 posing dire threat on public health, global economy, social and human interaction. At moderate rate, mutations in the SARS-CoV-2 genome are evolving which might have contributed to viral genome variability, transmission, replication efficiency and virulence in different regions of the world. The present study elucidated the mutational landscape in SARS-CoV-2 genome among the African population, which may have contributed to the virulence, pathogenicity and transmission observed in the region. Multiple sequence alignment of the SARS-CoV-2 genome (356 viral protein sequences) was performed using ClustalX version 2.1 and phylogenetic tree was built using Molecular Evolutionary Genetics Analysis (MEGA) X software. ORF1ab polyprotein, spike glycoprotein, ORF3, ORF8 and nucleocapsid phosphoprotein were observed as mutational hotspots in the African population and may be of keen interest in the adaptability of SARS-CoV-2 to the human host. While, there is conservation in the envelope protein, membrane glycoprotein, ORF6, ORF7a, ORF7b and ORF10. The accumulation of moderate mutations (though slowly) in the SARS-CoV-2 genome as revealed in our study, could be a promising strategy to develop drugs or vaccines with respect to the viral conserved domains and host cellular proteins and/or receptors involved in viral invasion and replication to avoid a new viral wave due to drug resistance and vaccine evasion.","version":"1.1","doi":"10.1101/2020.09.07.286088","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.20188631","pub_date":"2020-09-07","title":"Contact tracing efficiency, transmission heterogeneity, and accelerating COVID-19 epidemics","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Simultaneously controlling COVID-19 epidemics and limiting economic and societal impacts presents a difficult challenge, especially with limited public health budgets. Testing, contact tracing, and isolating/quarantining is a key strategy that has been used to reduce transmission of SARS-CoV-2, the virus that causes COVID-19. However, manual contact tracing is a time-consuming process and as case numbers increase it takes longer to reach each cases\u2019 contacts, leading to additional virus spread. Delays between symptom onset and being tested (and receiving results), and a low fraction of symptomatic cases being tested and traced can also reduce the impact of contact tracing on transmission. We examined the relationship between cases and delays and the pathogen reproductive number R\n                  <jats:sub>t</jats:sub>\n                  , and the implications for infection dynamics using a stochastic compartment model of SARS-CoV-2. We found that R\n                  <jats:sub>t</jats:sub>\n                  increases sigmoidally with the number of cases due to decreasing contact tracing efficacy. This relationship results in accelerating epidemics because R\n                  <jats:sub>t</jats:sub>\n                  increases, rather than declines, as infections increase. Shifting contact tracers from locations with high and low case burdens relative to capacity to locations with intermediate case burdens maximizes their impact in reducing R\n                  <jats:sub>t</jats:sub>\n                  (but minimizing total infections is more complicated). Contact tracing efficacy also decreased with increasing delays between symptom onset and tracing and with lower fraction of symptomatic infections being tested. Finally, testing and tracing reductions in R\n                  <jats:sub>t</jats:sub>\n                  can sometimes greatly delay epidemics due to the highly heterogeneous transmission dynamics of SARS-CoV-2. These results demonstrate the importance of having an expandable or mobile team of contact tracers that can be used to control surges in cases, and the value of easy access, high testing capacity and rapid turn-around of testing results, as well as outreach efforts to encourage symptomatic infections to be tested immediately after symptom onset.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary</jats:title>\n                  <jats:p>\n                    A key tool in the control of infectious diseases is contact tracing \u2013 the identification of individuals who have contacted the case and may have been infected by a newly detected case. However, to successfully contact and quarantine individuals requires time, and as cases rise, this can result in delays in reaching contacts during which time they may infect other people. Here we examine the quantitative relationships between increasing case numbers, contact tracing efficiency, and the pathogen reproductive number R\n                    <jats:sub>t</jats:sub>\n                    (the number of cases infected by each case) and how these relationships vary with delays and incomplete participation in the testing and tracing process. We built\n                  </jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.04.20188631","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.06.284976","pub_date":"2020-9-06","title":"Notable sequence homology of the ORF10 protein introspects the architecture of SARS-COV-2","abstract":"The global public health is endangered due to COVID-19 pandemic, which is caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Despite having similar pathology to MERS and SARS-CoV, the infection fatality rate of SARS-CoV-2 is likely lower than 1%. SARS-CoV-2 has been reported to be uniquely characterized by the accessory protein ORF10, which contains eleven cytotoxic T lymphocyte (CTL) epitopes of nine amino acids length each, across various human leukocyte antigen (HLA) subtypes. In this study, all missense mutations found in sequence databases were examined across twnety-two unique SARS-CoV-2 ORF10 variants that could possibly alter viral pathogenicity. Some of these mutations decrease the stability of ORF10, e.g. I4L and V6I were found in the MoRF region of ORF10 which may also possibly contribute to Intrinsic protein disorder. Furthermore, a physicochemical and structural comparative analysis was carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid homology. The high degree of physicochemical and structural similarity of ORF10 proteins of SARS-CoV-2 and Pangolin-CoV open questions about the architecture of SARS-CoV-2 due to the disagreement of these two ORF10 proteins over their sub-structure (loop/coil region), solubility, antigenicity and change from the strand to coil at amino acid position 26, where tyrosine is present. Altogether, SARS-CoV-2 ORF10 is a promising pharmaceutical target and a protein which should be monitored for changes which correlate to change pathogenesis and clinical course of COVID-19 infection.","version":"1.1","doi":"10.1101/2020.09.06.284976","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.06.284695","pub_date":"2020-9-06","title":"Long-term survival of salmon-attached SARS-CoV-2 at 4\u00b0C as a potential source of transmission in seafood markets","abstract":"Several outbreaks of COVID-19 were associated with seafood markets, raising concerns that fish-attached SARS-CoV-2 may exhibit prolonged survival in low-temperature environments. Here we showed that salmon-attached SARS-CoV-2 at 4\u00b0C could remain infectious for more than one week, suggesting that fish-attached SARS-CoV-2 may be a source of transmission.","version":"1.1","doi":"10.1101/2020.09.06.284695","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.256735","pub_date":"2020-9-06","title":"Identification of potential key genes for SARS-CoV-2 infected human bronchial organoids based on bioinformatics analysis","abstract":"There is an urgent need to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) that leads to COVID-19 and respiratory failure. Our study is to discover differentially expressed genes (DEGs) and biological signaling pathways by using a bioinformatics approach to elucidate their potential pathogenesis. The gene expression profiles of the GSE150819 datasets were originally produced using an Illumina NextSeq 500 (Homo sapiens). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) were utilized to identify functional categories and significant pathways. KEGG and GO results suggested that the Cytokine-cytokine receptor interaction, P53 signaling pathway, and Apoptosis are the main signaling pathways in SARS-CoV-2 infected human bronchial organoids (hBOs). Furthermore, NFKBIA, C3, and CCL20 may be key genes in SARS-CoV-2 infected hBOs. Therefore, our study provides further insights into the therapy of COVID-19.","version":"1.2","doi":"10.1101/2020.08.18.256735","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.06.284992","pub_date":"2020-9-06","title":"Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch Vaccines designed by reverse epitomics approach, shows potential to cover large ethnically distributed human population worldwide","abstract":"The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a positive-sense single-stranded RNA coronavirus responsible for the ongoing 2019-2020 COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. In present study we have designed and theoretically validated novel Multi-Patch Vaccines against SARS-CoV-2. A novel reverse epitomics approach, \u201coverlapping-epitope-clusters-to-patches\u201d method is utilized to identify multiple antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are here termed as \u201cAg-Patch or Ag-Patches\u201d, for Antigenic Patch or Patches. The identification of Ag-Patches is based on clusters of overlapping epitopes rising from a particular region of SARS-CoV-2 protein. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel methodology for vaccine design and development. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. We identified 73 CTL (Cytotoxic T-Lymphocyte), 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 (518 CTL and 250 HTL) overlapping epitopes targeting different HLA alleles. Such large number of epitope coverage is not possible for multi-epitope vaccines. The large number of epitopes covered implies large number of HLA alleles targeted, and hence large ethnically distributed human population coverage. The MPVs:Toll-Like Receptor ectodomain complex shows stable nature with numerous hydrogen bond formation and acceptable root mean square deviation and fluctuation. Further, the cDNA analysis favors high expression of the MPVs constructs in human cell line. Highly immunogenic novel Ag-Patches are identified from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach. We conclude that the novel Multi-Patch Vaccines could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. Multi-Patch Vaccine designing to combat SARS-CoV-2 infection by reverse epitomics approach, \u201cOverlapping-epitope-clusters-to-patches\u201d method.","version":"1.1","doi":"10.1101/2020.09.06.284992","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.283853","pub_date":"2020-9-06","title":"Pre-clinical studies of a recombinant adenoviral mucosal vaccine to prevent SARS-CoV-2 infection","abstract":"There is an urgent need to develop efficacious vaccines against SARS-CoV-2 that also address the issues of deployment, equitable access, and vaccine acceptance. Ideally, the vaccine would prevent virus infection and transmission as well as preventing COVID-19 disease. We previously developed an oral adenovirus-based vaccine technology that induces both mucosal and systemic immunity in humans. Here we investigate the immunogenicity of a range of candidate adenovirusbased vaccines, expressing full or partial sequences of the spike and nucleocapsid proteins, in mice. We demonstrate that, compared to expression of the S1 domain or a stabilized spike antigen, the full length, wild-type spike antigen induces significantly higher neutralizing antibodies in the periphery and in the lungs, when the vaccine is administered mucosally. Antigen-specific CD4+ and CD8+ T cells were induced by this leading vaccine candidate at low and high doses. This fulllength spike antigen plus nucleocapsid adenovirus construct has been prioritized for further clinical development.","version":"1.1","doi":"10.1101/2020.09.04.283853","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.05.284604","pub_date":"2020-9-06","title":"Genomic analysis reveals local transmission of SARS-CoV-2 in early pandemic phase in Peru","abstract":"The dissemination of cases of the new SAR-COV-2 coronavirus represents a serious public health problem for Latin America and Peru. For this reason, it is important to characterize the genome of the isolates that circulate in Latin America. To characterize the complete genome of first samples of the virus circulating in Peru, we amplified seven overlapping segments of the viral genome by RT-PCR and sequenced using Miseq platform. The results indicate that the genomes of the Peruvian SARS-COV-2 samples belong to the genetic groups G and S. Likewise, a phylogenetic and MST analysis of the isolates confirm the introduction of multiple isolates from Europe and Asia that, after border closing, were transmitted locally in the capital and same regions of the country. These Peruvian samples (56%) grouped into two clusters inside G clade and share B.1.1.1 lineage. The characterization of these isolates must be considered for the use and design of diagnostic tools, and effective treatment and vaccine formulations.","version":"1.1","doi":"10.1101/2020.09.05.284604","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.06.284901","pub_date":"2020-9-06","title":"Transmission of allosteric response within the homotrimer of SARS-CoV-2 spike upon recognition of ACE2 receptor by the receptor-binding domain","abstract":"The pathogenesis of novel SARS-CoV-2 virus initiates through recognition of ACE2 receptor (Angiotensin-converting enzyme 2) of the host cells by the receptor-binding domain (RBD) located at spikes of the virus. Following receptor-recognition, proteolytic cleavage between S1 and S2 subunits of the spike protein occurs with subsequent release of fusion peptide. Here, we report our study on allosteric communication within RBD that propagates the signal from ACE2-binding site towards allosteric site for the post-binding activation of proteolytic cleavage. Using MD simulations, we have demonstrated allosteric crosstalk within RBD in apo- and receptor-bound states where dynamic correlated motions and electrostatic energy perturbations contribute. While allostery, based on correlated motions, dominates inherent distal communication in apo-RBD, electrostatic energy perturbations determine favorable crosstalk within RBD upon binding to ACE2. Notably, allosteric path is constituted with evolutionarily conserved residues pointing towards their biological relevance. As revealed from recent structures, in the trimeric arrangement of spike, RBD of one copy interacts with S2 domain of another copy. Interestingly, the allosteric site identified is in direct contact (H-bonded) with a region in RBD that corresponds to the interacting region of RBD of one copy with S2 of another copy in trimeric constitution. Apparently, inter-monomer allosteric communication orchestrates concerted action of the trimer. Based on our results, we propose the allosteric loop of RBD as a potential drug target.","version":"1.1","doi":"10.1101/2020.09.06.284901","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.09.243444","pub_date":"2020-9-06","title":"Identification of key genes in SARS-CoV-2 patients on bioinformatics analysis","abstract":"The COVID-19 pandemic has infected millions of people and overwhelmed many health systems globally. Our study is to identify differentially expressed genes (DEGs) and associated biological processes of COVID-19 using a bioinformatics approach to elucidate their potential pathogenesis. The gene expression profiles of the GSE152075 datasets were originally produced by using the high-throughput Illumina NextSeq 500. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed to identify functional categories and biochemical pathways. GO and KEGG results suggested that several biological pathways such as \u201cFatty acid metabolism\u201d and \u201cCilium morphogenesis\u201d are mostly involved in the development of COVID-19. Moreover, several genes are critical for virus invasion and adhesion including FLOC, DYNLL1, FBXL3, and FBXW11 and show significant differences in COVID-19 patients. Thus, our study provides further insights into the underlying pathogenesis of COVID-19.","version":"1.2","doi":"10.1101/2020.08.09.243444","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.264465","pub_date":"2020-9-06","title":"Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism","abstract":"The N protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs\u2019 Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. N-NTD dsRNA destabilizing activity was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of \u03b22-\u03b23 and 2-\u03b25 loops, played a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTDs of dimeric N would act independently, increasing efficiency; (ii) two N-NTDs of dimeric N would bind to two different RNA sites, bridging distant regions of the genome; and (iii) monomeric N would be active, opening up the possibility of a regulatory dissociation event. Coronaviruses are among the largest positive-sense RNA viruses. They display a unique discontinous transcription mechanism, involving N protein as a major player. The N-NTD promote the dsRNA melting releasing the nascent sense negative strand via a poorly known mechanism of action. It specifically recognizes the body TRS conserved RNA motif located at the 5\u2019 end of each ORF. N protein has the ability to transfer the nascent RNA strand to the leader TRS. The mechanism is essential and one single mutation at the RNA binding site of the N-NTD impairs the viral replication. Here, we describe a counterintuitive mechanism of action of N-NTD based on molecular dynamics simulation and kinetic modelling of the experimental melting activity of N-NTD. This data impacts directly in the understanding of the way N protein acts in the cell and will guide future experiments.","version":"1.2","doi":"10.1101/2020.08.24.264465","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.06.285023","pub_date":"2020-9-06","title":"New insights into nCOVID-19 binding domain and its cellular receptors","abstract":"nCOVID-19 virus makes cellular entry using its spike protein protruding out on its surface. Angiotensin converting enzyme 2 receptor has been identified as a receptor that mediates the viral entry by binding with the receptor binding motif of spike protein. In the present study, we elucidate the significance of N-terminal domain of spike protein in spike-receptor interactions. Recent clinical reports indicate a link between nCOVID-19 infections with patient comorbidities. The underlying reason behind this relationship is not clear. Using molecular docking, we study the affinity of the nCOVID-19 spike protein with cell receptors overexpressed under disease conditions. Our results suggest that certain cell receptors such as DC/L-SIGN, DPP4, IL22R and ephrin receptors could act as potential receptors for the spike protein. The receptor binding domain of nCOVID-19 is more flexible than that of SARS-COV and has a high propensity to undergo phase separation. Higher flexibility of nCOVID-19 receptor binding domain might enable it to bind multiple receptor partners. Further experimental work on the association of these receptors with spike protein may help us to explain the severity of nCOVID-19 infection in patients with comorbidities.","version":"1.1","doi":"10.1101/2020.09.06.285023","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.06.282145","pub_date":"2020-9-06","title":"Cooperative efforts on developing vaccines and therapies for COVID-19 Cooperative efforts for COVID-19","abstract":"Health organizations have always sought partnership to join competencies in innovation, even with fierce competition in this sector. In this pandemic moment it is relevant to observe how organizations behave to seek quick and safe answers. The present research analyzes how the cooperation networks were set off considering the clinical trials on therapies and vaccines that were developed specifically to treat or prevent COVID-19. Social Network Analysis technique was used to build cooperation networks and apply metrics that characterize these connections. There was an evaluation of statistics of Strength of cooperation and Unilateral dependence of cooperation that identify the cooperation strength between two organizations, and the dependence of this relations. A total of 415 clinical trial were identified, of which 42% are in cooperation. From organizations that have partnership, firms are the first, followed by universities. We extracted the main categories that concentrate 74% of partnerships in the trials of antibody, and vaccine. Several organizations cooperate in multiple categories of trials, evidencing the efforts to focus on different strategies to treat the disease. We found high strength of cooperation and an assimetryc dependency between partners, which can be assigned to specialized models of partnership and it occurs in competitive enviroments like this pandemic moment. Cooperation were not limited to geographical proximity and the advent of Chinese players can represent a new change in the biotechnological development axis. Finally, the challenge of finding therapeutic or immunological solutions for COVID-19 demonstrates a clear composition of cooperation groups that complement their skills to manage organizational strategies to beat the pandemic. In this new paradigm, there can be partnerships not only in clinical trial but also in pre-competitive technologies development. This experience is expected to change the way of organizations define their R&D strategies and start to adopt more a collaborative innovation model.","version":"1.1","doi":"10.1101/2020.09.06.282145","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.03.20187062","pub_date":"2020-09-05","title":"College campuses and COVID-19 mitigation: clinical and economic value","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Decisions around US college and university operations will affect millions of students and faculty amidst the COVID-19 pandemic. We examined the clinical and economic value of different COVID-19 mitigation strategies on college campuses.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We used the Clinical and Economic Analysis of COVID-19 interventions (CEACOV) model, a dynamic microsimulation that tracks infections accrued by students and faculty, accounting for community transmissions. Outcomes include infections, $/infection-prevented, and $/quality-adjusted-life-year ($/QALY). Strategies included extensive social distancing (ESD), masks, and routine laboratory tests (RLT). We report results per 5,000 students (1,000 faculty) over one semester (105 days).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Mitigation strategies reduced COVID-19 cases among students (faculty) from 3,746 (164) with no mitigation to 493 (28) with ESD and masks, and further to 151 (25) adding\n                    <jats:italic>RLTq3</jats:italic>\n                    among asymptomatic students and faculty. ESD with masks cost $168/infection-prevented ($49,200/QALY) compared to masks alone. Adding\n                    <jats:italic>RLTq3</jats:italic>\n                    ($10/test) cost $8,300/infection-prevented ($2,804,600/QALY). If tests cost $1,\n                    <jats:italic>RLTq3</jats:italic>\n                    led to a favorable cost of $275/infection-prevented ($52,200/QALY). No strategies without masks were cost-effective.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Extensive social distancing with mandatory mask-wearing could prevent 87% of COVID-19 cases on college campuses and be very cost-effective. Routine laboratory testing would prevent 96% of infections and require low cost tests to be economically attractive.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.03.20187062","journal":"medRxiv","score":null},{"id":"10.1101/2020.02.08.939553","pub_date":"2020-9-04","title":"The immune vulnerability landscape of the 2019 Novel Coronavirus, SARS-CoV-2","abstract":"The outbreak of the 2019 Novel Coronavirus (SARS-CoV-2) rapidly spread from Wuhan, China to more than 150 countries, areas, or territories, causing staggering numbers of infections and deaths. In this study, bioinformatics analyses were performed on 5,568 complete genomes of SARS-CoV-2 virus to predict the T cell and B cell immunogenic epitopes of all viral proteins, which formed a systematic immune vulnerability landscape of SARS-CoV-2. The immune vulnerability and genetic variation profiles of SARS-CoV were compared with those of SARS-CoV and MERS-CoV. In addition, a web portal was developed to broadly share the data and results as a resource for the research community. Using this resource, we showed that genetic variations in SARS-CoV-2 are associated with loss of B cell immunogenicity, an increase in CD4+ T cell immunogenicity, and a minimum loss in CD8+ T cell immunogenicity, indicating the existence of a curious correlation between SARS-CoV-2 genetic evolutions and the immunity pressure from the host. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic/vaccine development and mechanistic research.","version":"1.3","doi":"10.1101/2020.02.08.939553","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.282616","pub_date":"2020-9-04","title":"Genomic diversity and evolution of coronavirus (SARS-CoV-2) in France from 309 COVID-19-infected patients","abstract":"The novel coronavirus (SARS-CoV-2) causes pandemic of viral pneumonia. The evolution and mutational events of the SARS-CoV-2 genomes are critical for controlling virulence, transmissibility, infectivity, severity of symptoms and mortality associated to this infectious disease. We collected and investigated 309 SARS-CoV-2 genomes from patients infected in France. Detailed genome cartography of all mutational events (SNPs, indels) was reported and correlated to clinical features of patients. A comparative analysis between our 309 SARS-CoV-2 genomes from French patients and the reference Wuhan coronavirus genome revealed 315 substitution mutations and six deletion events: ten were in 5\u2019/3\u2019 UTR, 178 were nonsynonymous, 126 were synonymous and one generated a stop codon. Six different deleted areas were also identified in nine viral variants. In particular, 30 substitution mutations (18 nonsynonymous) and one deletion (\u039421765-21770) concerned the spike S glycoprotein. An average of 7.8 mutational events (+/- 1.7 SD) and a median of 8 (range, 7-9) were reported per viral isolate. Comparative analyses and clustering of specific mutational signatures in 309 genomes disclose several divisions in groups and subgroups combining their geographical and phylogenetic origin. Clinical outcomes of the 309 COVID-19-infected patients were investigated according to the mutational signatures of viral variants. These findings highlight the genome dynamics of the coronavirus 2019-20 and shed light on the mutational landscape and evolution of this virus. Inclusion of the French cohort enabled us to identify 161 novel mutations never reported in SARS-CoV-2 genomes collected worldwide. These results support a global and continuing surveillance of the emerging variants of the coronavirus SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.09.04.282616","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.283077","pub_date":"2020-9-04","title":"Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore","abstract":"Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to \u00a310 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.","version":"1.1","doi":"10.1101/2020.09.04.283077","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.146522","pub_date":"2020-9-04","title":"Beyond Shielding: The Roles of Glycans in SARS-CoV-2 Spike Protein","abstract":"The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 15,000,000 infections and 600,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates the host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viruses, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans, and the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike\u2019s receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift towards the \u201cdown\u201d state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which may be exploited by therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.","version":"1.2","doi":"10.1101/2020.06.11.146522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.282640","pub_date":"2020-9-04","title":"Seroprevalence of SARS-CoV-2 specific IgG antibodies in District Srinagar, northern India \u2013 a cross-sectional study","abstract":"Prevalence of IgG antibodies against SARS-CoV-2 infection provides essential information for deciding disease prevention and mitigation measures. We estimate the seroprevalence of SARS-CoV-2 specific IgG antibodies in District Srinagar. 2906 persons >18 years of age selected from hospital visitors across District Srinagar participated in the study. We tested samples for the presence of SARS-CoV-2 specific IgG antibodies using a chemiluminescent microparticle immunoassay-based serologic test. Age- and gender-standardized seroprevalence was 3.6% (95% CI 2.9% to 4.3%). Age 30-69 years, a recent history of symptoms of an influenza-like-illness, and a history of being placed under quarantine were significantly related to higher odds of the presence of SARS-CoV-2 specific IgG antibodies. The estimated number of SARS-CoV-2 infections during the two weeks preceding the study, adjusted for test performance, was 32602 with an estimated (median) infection-to-known-case ratio of 46 (95% CI 36 to 57). The seroprevalence of SARS-CoV-2 specific IgG antibodies is low in the District. A large proportion of the population is still susceptible to the infection. A sizeable number of infections remain undetected, and a substantial proportion of people with symptoms compatible with COVID-19 are not tested.","version":"1.1","doi":"10.1101/2020.09.04.282640","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.280081","pub_date":"2020-9-04","title":"Monoclonal Antibodies Capable of Binding SARS-CoV-2 Spike Protein Receptor Binding Motif Specifically Prevent GM-CSF Induction","abstract":"A severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected more than 25.6 million and killed 852,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibody responses. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human monocyte and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited \u201ccytokine storm\u201d, and provided a potentially useful criteria for future assessment of innate immune-modulating properties of various SARS-CoV-2 vaccines. RBM-binding Antibodies Inhibit GM-CSF Induction.","version":"1.1","doi":"10.1101/2020.09.04.280081","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.03.282103","pub_date":"2020-9-04","title":"A SARS-CoV-2 \u2013 host proximity interactome","abstract":"Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.","version":"1.1","doi":"10.1101/2020.09.03.282103","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.282558","pub_date":"2020-9-04","title":"Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins","abstract":"Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.","version":"1.1","doi":"10.1101/2020.09.04.282558","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.282806","pub_date":"2020-9-04","title":"EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation","abstract":"Coagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1\u2212/\u2212 phenotype. EPAC1 regulated TNF\u03b1-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation. PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.","version":"1.1","doi":"10.1101/2020.09.04.282806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.04.283085","pub_date":"2020-9-04","title":"Nanoscopy on the Chea(i)p","abstract":"Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.","version":"1.1","doi":"10.1101/2020.09.04.283085","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.30.273235","pub_date":"2020-9-03","title":"The global landscape of SARS-CoV-2 genomes, variants, and haplotypes in 2019nCoVR","abstract":"On 22 January 2020, the National Genomics Data Center (NGDC), part of the China National Center for Bioinformation (CNCB), created the 2019 Novel Coronavirus Resource (2019nCoVR), an open-access SARS-CoV-2 information resource. 2019nCoVR features a comprehensive integration of sequence and clinical information for all publicly available SARS-CoV-2 isolates, which are manually curated with value-added annotations and quality evaluated by our in-house automated pipeline. Of particular note, 2019nCoVR performs systematic analyses to generate a dynamic landscape of SARS-CoV-2 genomic variations at a global scale. It provides all identified variants and detailed statistics for each virus isolate, and congregates the quality score, functional annotation, and population frequency for each variant. It also generates visualization of the spatiotemporal change for each variant and yields historical viral haplotype network maps for the course of the outbreak from all complete and high-quality genomes. Moreover, 2019nCoVR provides a full collection of SARS-CoV-2 relevant literature on COVID-19 (Coronavirus Disease 2019), including published papers from PubMed as well as preprints from services such as bioRxiv and medRxiv through Europe PMC. Furthermore, by linking with relevant databases in CNCB-NGDC, 2019nCoVR offers data submission services for raw sequence reads and assembled genomes, and data sharing with National Center for Biotechnology Information. Collectively, all SARS-CoV-2 genome sequences, variants, haplotypes and literature are updated daily to provide timely information, making 2019nCoVR a valuable resource for the global research community. 2019nCoVR is accessible at https://bigd.big.ac.cn/ncov/.","version":"1.2","doi":"10.1101/2020.08.30.273235","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.116020","pub_date":"2020-9-03","title":"Simeprevir potently suppresses SARS-CoV-2 replication and synergizes with remdesivir","abstract":"The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. Discovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.","version":"1.2","doi":"10.1101/2020.05.26.116020","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.03.280370","pub_date":"2020-9-03","title":"Development of SARS-CoV-2 Nucleocapsid Specific Monoclonal Antibodies","abstract":"The global COVID-19 pandemic has caused massive disruptions in every society around the world. To help fight COVID-19, new molecular tools specifically targeting critical components of the causative agent of COVID-19, SARS-Coronavirus-2 (SARS-CoV-2), are desperately needed. The SARS-CoV-2 nucleocapsid protein is a major component of the viral replication processes, integral to viral particle assembly, and is a major diagnostic marker for infection and immune protection. Currently available antibody reagents targeting the nucleocapsid protein were primarily developed against the related SARS-CoV virus and are not specific to SARS-CoV-2 nucleocapsid protein. Therefore, in this work we developed and characterized a series of new mouse monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein. The anti-nucleocapsid monoclonal antibodies were tested in ELISA, western blot, and immunofluorescence analyses. The variable regions from the heavy and light chains from five select clones were cloned and sequenced, and preliminary epitope mapping of the sequenced clones was performed. Overall, the new antibody reagents described here will be of significant value in the fight against COVID-19.","version":"1.1","doi":"10.1101/2020.09.03.280370","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214049","pub_date":"2020-9-03","title":"Intranasal Vaccination with a Lentiviral Vector Strongly Protects against SARS-CoV-2 in Mouse and Golden Hamster Preclinical Models","abstract":"To develop a vaccine candidate against COVID-19, we generated a Lentiviral Vector (LV), eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, conferred only partial protection, despite an intense serum neutralizing activity. However, targeting the immune response to the respiratory tract through an intranasal boost with this LV resulted in > 3 log10 decrease in the lung viral loads and avoided local inflammation. Moreover, both integrative and non-integrative LV platforms displayed a strong vaccine efficacy and inhibited lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and restitute the human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of the LV-based vaccination against SARS-CoV-2 and designate the intranasal immunization as a powerful approach against COVID-19. A lentiviral vector encoding for Spike predicts a promising COVID-19 vaccine Targeting the immune response to the upper respiratory tract is key to protection Intranasal vaccination induces protective mucosal immunity against SARS-CoV-2 Lung anti-Spike IgA responses correlate with protection and reduced inflammation","version":"1.2","doi":"10.1101/2020.07.21.214049","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.03.280727","pub_date":"2020-9-03","title":"The discovery of gene mutations making SARS-CoV-2 well adapted for humans: host-genome similarity analysis of 2594 genomes from China, the USA and Europe","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense single-stranded virus approximately 30 kb in length, causes the ongoing novel coronavirus disease-2019 (COVID-19). Studies confirmed significant genome differences between SARS-CoV-2 and SARS-CoV, suggesting that the distinctions in pathogenicity might be related to genomic diversity. However, the relationship between genomic differences and SARS-CoV-2 fitness has not been fully explained, especially for open reading frame (ORF)-encoded accessory proteins. RNA viruses have a high mutation rate, but how SARS-CoV-2 mutations accelerate adaptation is not clear. This study shows that the host-genome similarity (HGS) of SARS-CoV-2 is significantly higher than that of SARS-CoV, especially in the ORF6 and ORF8 genes encoding proteins antagonizing innate immunity in vivo. A power law relationship was discovered between the HGS of ORF3b, ORF6, and N and the expression of interferon (IFN)-sensitive response element (ISRE)-containing promoters. This finding implies that high HGS of SARS-CoV-2 genome may further inhibit IFN I synthesis and cause delayed host innate immunity. An ORF1ab mutation, 10818G>T, which occurred in virus populations with high HGS but rarely in low-HGS populations, was identified in 2594 genomes with geolocations of China, the USA and Europe. The 10818G>T caused the amino acid mutation M37F in the transmembrane protein nsp6. The results suggest that the ORF6 and ORF8 genes and the mutation M37F may play important roles in causing COVID-19. The findings demonstrate that HGS analysis is a promising way to identify important genes and mutations in adaptive strains, which may help in searching potential targets for pharmaceutical agents.","version":"1.1","doi":"10.1101/2020.09.03.280727","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.20184101","pub_date":"2020-09-03","title":"Rapid \u201cmix and read\u201d assay for scalable detection of SARS-CoV-2 antibodies in patient plasma","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  The human beta coronavirus SARS-CoV-2, causative virus of COVID-19, has infected more than 15 million people globally and continues to spread. Widespread, population level testing to detect active and past infections is critical to curb the COVID-19 pandemic. Antibody (serological) testing is the only option for detecting past infections outside the narrow window accessible to nucleic acid-based tests. However, currently available serological assays commonly lack scalability. Here, we describe the development of a rapid homogenous serological assay for the detection of antibodies to SARS-CoV-2 in patient plasma. We show that the fluorescence-based assay accurately detects seroconversion in COVID-19 patients from less than 1\n                  <jats:italic>\u03bc</jats:italic>\n                  L of plasma. Using a cohort of samples from COVID-19 infected or healthy individuals, we demonstrate detection with 100% sensitivity and specificity. This assay addresses an important need for a robust, low barrier to implementation, and scalable serological assay with complementary strengths to currently available serological platforms.\n                </jats:p>","version":null,"doi":"10.1101/2020.09.01.20184101","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.12.248823","pub_date":"2020-9-03","title":"Attenuated influenza virions expressing the SARS-CoV-2 receptor-binding domain induce neutralizing antibodies in mice","abstract":"An effective vaccine is essential to controlling the spread of SARS-CoV-2 virus. Here, we describe an influenza-virus-based vaccine for SARS-CoV-2. We incorporated a membrane-anchored form of the SARS-CoV-2 Spike receptor binding domain (RBD) in place of the neuraminidase (NA) coding sequence in an influenza virus also possessing a mutation that reduces the affinity of hemagglutinin for its sialic acid receptor. The resulting \u0394NA(RBD)-Flu virus can be generated by reverse genetics and grown to high titers in cell culture. A single-dose intranasal inoculation of mice with \u0394NA(RBD)-Flu elicits serum neutralizing antibody titers against SAR-CoV-2 comparable to those observed in humans following natural infection (\u223c1:200). Furthermore, \u0394NA(RBD)-Flu itself causes no apparent disease in mice. It might be possible to produce a vaccine similar to \u0394NA(RBD)-Flu at scale by leveraging existing platforms for production of influenza vaccines.","version":"1.2","doi":"10.1101/2020.08.12.248823","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.137935","pub_date":"2020-9-03","title":"Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody","abstract":"Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.","version":"1.2","doi":"10.1101/2020.06.09.137935","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.03.280446","pub_date":"2020-9-03","title":"A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice","abstract":"A self-transcribing and replicating RNA (STARR\u2122) based vaccine (LUNAR\u00ae-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolong SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell mediated immunity produced a strong viral antigen specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for IFN-\u03b3 and IL-4 positive CD4+ T helper lymphocytes as well as anti-spike glycoprotein IgG2a/IgG1 ratios supported a strong Th1 dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 \u03bcg and 10 \u03bcg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of Lunar-COV19 as a single dose vaccine.","version":"1.1","doi":"10.1101/2020.09.03.280446","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.02.276865","pub_date":"2020-9-03","title":"Longitudinal single-cell immune profiling revealed distinct innate immune response in asymptomatic COVID-19 patients","abstract":"Recent studies have characterized the single-cell immune landscape of host immune response of coronavirus disease 2019 (COVID-19), specifically focus on the severe condition. However, the immune response in mild or even asymptomatic patients remains unclear. Here, we performed longitudinal single-cell transcriptome sequencing and T cell/B cell receptor sequencing on 3 healthy donors and 10 COVID-19 patients with asymptomatic, moderate, and severe conditions. We found asymptomatic patients displayed distinct innate immune responses, including increased CD56briCD16\u2212 NK subset, which was nearly missing in severe condition and enrichment of a new Th2-like cell type/state expressing a ciliated cell marker. Unlike that in moderate condition, asymptomatic patients lacked clonal expansion of effector CD8+ T cells but had a robust effector CD4+ T cell clonal expansion, coincide with previously detected SARS-CoV-2-reactive CD4+ T cells in unexposed individuals. Moreover, NK and effector T cells in asymptomatic patients have upregulated cytokine related genes, such as IFNG and XCL2. Our data suggest early innate immune response and type I immunity may contribute to the asymptomatic phenotype in COVID-19 disease, which could in turn deepen our understanding of severe COVID-19 and guide early prediction and therapeutics.","version":"1.1","doi":"10.1101/2020.09.02.276865","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.02.20186528","pub_date":"2020-09-03","title":"Vaccination for some childhood diseases may impact the outcome of covid-19 infections","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>COVID-19 found the world in a state of unpreparedness. While research efforts to develop a vaccine are on-going, others have suggested the use of available vaccines to boost innate immunity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>We analysed three databases: UNICEF Immunization Coverage, Worldometer Corona Virus Updates and World Bank List of Economies to establish the association, if any, between vaccination for various diseases and COVID-19 death rates and recoveries across world economies.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Mean percentage death rates were lower in countries that vaccinated for Hepatitis-B birth dose (2.53% vs 3.79%, p = 0.001), Bacille Calmette-Gu\u00e9rin Vaccine (2.93% vs 5.10%, p = 0.025) and Inactivated Polio Vaccine 1\n                    <jats:sup>st</jats:sup>\n                    dose (2.8% vs 4.01%, p = 0.022) than those which did not report vaccination. In high income countries, a significant negative correlation with death rates was observed with vaccination for Measles-containing vaccine 2\n                    <jats:sup>nd</jats:sup>\n                    dose (r = \u20130.290, p = 0.032), Rubella-containing vaccine 1\n                    <jats:sup>st</jats:sup>\n                    dose (r = \u20130.325, p = 0.015), Hepatitis B 3\n                    <jats:sup>rd</jats:sup>\n                    dose (r = \u20130.562, p = 3.3 x10\n                    <jats:sup>\u22125</jats:sup>\n                    ), Inactivated Polio vaccine 1\n                    <jats:sup>st</jats:sup>\n                    dose (r = \u20130.720, p = 0.008). Inactivated Polio Vaccine 1st dose and Measles-containing vaccine 2\n                    <jats:sup>nd</jats:sup>\n                    dose also correlated with better recoveries. In Low Income countries, only Rubella-containing vaccine correlated with lower deaths while Yellow fever vaccine was associated with poorer recoveries.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Our analysis corroborates the potential benefit of vaccination and warrant further research to explore the rationale for repurposing other vaccines to fight COVID-19.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.09.02.20186528","journal":"medRxiv","score":null},{"id":"10.1101/2020.09.01.278952","pub_date":"2020-9-02","title":"Structural Variants in SARS-CoV-2 Occur at Template-Switching Hotspots","abstract":"The evolutionary dynamics of SARS-CoV-2 have been carefully monitored since the COVID-19 pandemic began in December 2019, however, analysis has focused primarily on single nucleotide polymorphisms and largely ignored the role of structural variants (SVs) as well as recombination in SARS-CoV-2 evolution. Using sequences from the GISAID database, we catalogue over 100 insertions and deletions in the SARS-CoV-2 consensus sequences. We hypothesize that these indels are artifacts of imperfect homologous recombination between SARS-CoV-2 replicates, and provide four independent pieces of evidence. (1) The SVs from the GISAID consensus sequences are clustered at specific regions of the genome. (2) These regions are also enriched for 5\u2019 and 3\u2019 breakpoints in the transcription regulatory site (TRS) independent transcriptome, presumably sites of RNA-dependent RNA polymerase (RdRp) template-switching. (3) Within raw reads, these structural variant hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these structural variants are both consequences of de novo recombination events within a host and artifacts of previous recombination. (4) Within the RNA secondary structure, the indels occur in \u201carms\u201d of the predicted folded RNA, suggesting that secondary structure may be a mechanism for TRS-independent template-switching in SARS-CoV-2 or other coronaviruses. These insights into the relationship between structural variation and recombination in SARS-CoV-2 can improve our reconstructions of the SARS-CoV-2 evolutionary history as well as our understanding of the process of RdRp template-switching in RNA viruses.","version":"1.1","doi":"10.1101/2020.09.01.278952","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.278689","pub_date":"2020-9-02","title":"Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility","abstract":"A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an \u201cup\u201d conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development. Understanding the evolution of SARS-CoV-2 during the COVID-19 pandemic is essential for disease control and prevention. A spike protein mutation D614G emerged and became dominant soon after the pandemic started. By engineering the D614G mutation into an authentic wild-type SARS-CoV-2 strain, we demonstrate the importance of this mutation to (i) enhanced viral replication on human lung epithelial cells and primary human airway tissues, (ii) improved viral fitness in the upper airway of infected hamsters, and (iii) increased susceptibility to neutralization. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.","version":"1.1","doi":"10.1101/2020.09.01.278689","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.277780","pub_date":"2020-9-02","title":"Modeling SARS-CoV-2 infection in vitro with a human intestine-on-chip device","abstract":"Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) has given rise to a global pandemic. The gastrointestinal symptoms of some COVID-19 patients are underestimated. There is an urgent need to develop physiologically relevant model that can accurately reflect human response to viral infection. Here, we report the creation of a biomimetic human intestine infection model on a chip system that allows to recapitulate the intestinal injury and immune response induced by SARS-CoV-2, for the first time. The microengineered intestine-on-chip device contains human intestinal epithelium (co-cultured human intestinal epithelial Caco-2 cells and mucin secreting HT-29 cells) lined in upper channel and vascular endothelium (human umbilical vein endothelial cells, HUVECs) in a parallel lower channel under fluidic flow condition, sandwiched by a porous PDMS membrane coated with extracellular matrix (ECM). At day 3 post-infection of SARS-CoV-2, the intestine epithelium showed high susceptibility to viral infection and obvious morphological changes with destruction of intestinal villus, dispersed distribution of mucus secreting cells and reduced expression of tight junction (E-cadherin), indicating the destruction of mucous layer and the integrity of intestinal barrier caused by virus. Moreover, the endothelium exhibited abnormal cell morphology with disrupted expression of adherent junction protein (VE-cadherin). Transcriptional analysis revealed the abnormal RNA and protein metabolism, as well as activated immune responses in both epithelial and endothelial cells after viral infection (e.g., up-regulated cytokine genes, TNF signaling and NF-kappa B signaling-related genes). This bioengineered in vitro model system can mirror the human relevant pathophysiology and response to viral infection at the organ level, which is not possible in existing in vitro culture systems. It may provide a promising tool to accelerate our understanding of COVID-19 and devising novel therapies.","version":"1.1","doi":"10.1101/2020.09.01.277780","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.278366","pub_date":"2020-9-02","title":"An intestinal cell type in zebrafish is the nexus for the SARS-CoV-2 receptor and the Renin-Angiotensin-Aldosterone System that contributes to COVID-19 comorbidities","abstract":"People with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with the coronavirus SARS-CoV-2. These COVID-19 comorbidities are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from rapidly dropping blood pressure or dehydration via the peptide Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II, thus counteracting its chronic effects. Ace2 is also the SARS-CoV-2 receptor. Ace, the coronavirus, and COVID-19 comorbidities all regulate Ace2, but we don\u2019t yet understand how. To exploit zebrafish (Danio rerio) as a disease model to understand mechanisms regulating the RAAS and its relationship to COVID-19 comorbidities, we must first identify zebrafish orthologs and co-orthologs of human RAAS genes, and second, understand where and when these genes are expressed in specific cells in zebrafish development. To achieve these goals, we conducted genomic analyses and investigated single cell transcriptomes. Results showed that most human RAAS genes have an ortholog in zebrafish and some have two or more co-orthologs. Results further identified a specific intestinal cell type in zebrafish larvae as the site of expression for key RAAS components, including Ace, Ace2, the coronavirus co-receptor Slc6a19, and the Angiotensin-related peptide cleaving enzymes Anpep and Enpep. Results also identified specific vascular cell subtypes as expressing Ang II receptors, apelin, and apelin receptor genes. These results identify specific genes and cell types to exploit zebrafish as a disease model for understanding the mechanisms leading to COVID-19 comorbidities. Genomic analyses identify zebrafish orthologs of the Renin-Angiotensin-Aldosterone System that contribute to COVID-19 comorbidities and single-cell transcriptomics show that they act in a specialized intestinal cell type.","version":"1.1","doi":"10.1101/2020.09.01.278366","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.230839","pub_date":"2020-9-02","title":"Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells","abstract":"COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1\u03b1, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.","version":"1.2","doi":"10.1101/2020.08.02.230839","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.130955","pub_date":"2020-9-02","title":"SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes","abstract":"Despite its overwhelming clinical importance, the SARS-CoV-2 gene set remains unresolved, hindering dissection of COVID-19 biology. Here, we use comparative genomics to provide a high-confidence protein-coding gene set, characterize protein-level and nucleotide-level evolutionary constraint, and prioritize functional mutations from the ongoing COVID-19 pandemic. We select 44 complete Sarbecovirus genomes at evolutionary distances ideally-suited for protein-coding and non-coding element identification, create whole-genome alignments, and quantify protein-coding evolutionary signatures and overlapping constraint. We find strong protein-coding signatures for all named genes and for 3a, 6, 7a, 7b, 8, 9b, and also ORF3c, a novel alternate-frame gene. By contrast, ORF10, and overlapping-ORFs 9c, 3b, and 3d lack protein-coding signatures or convincing experimental evidence and are not protein-coding. Furthermore, we show no other protein-coding genes remain to be discovered. Cross-strain and within-strain evolutionary pressures largely agree at the gene, amino-acid, and nucleotide levels, with some notable exceptions, including fewer-than-expected mutations in nsp3 and Spike subunit S1, and more-than-expected mutations in Nucleocapsid. The latter also shows a cluster of amino-acid-changing variants in otherwise-conserved residues in a predicted B-cell epitope, which may indicate positive selection for immune avoidance. Several Spike-protein mutations, including D614G, which has been associated with increased transmission, disrupt otherwise-perfectly-conserved amino acids, and could be novel adaptations to human hosts. The resulting high-confidence gene set and evolutionary-history annotations provide valuable resources and insights on COVID-19 biology, mutations, and evolution.","version":"1.2","doi":"10.1101/2020.06.02.130955","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.20185082","pub_date":"2020-09-02","title":"Outcome of SARS-CoV-2 infection linked to MAIT cell activation and cytotoxicity: evidence for an IL-18 dependent mechanism","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Immune system dysfunction is paramount in Coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in a cohort of 182 patients including patients at various stages of disease activity. A profound decrease of MAIT cell counts in blood of critically ill patients was observed. These cells showed a strongly activated and cytotoxic phenotype that positively correlated with circulating pro-inflammatory cytokines, notably IL-18. MAIT cell alterations markedly correlated with disease severity and patient mortality. SARS-CoV-2-infected macrophages activated MAIT cells in a cytokine-dependent manner involving an IFN\u03b1-dependent early phase and an IL-18-induced later phase. Therefore, altered MAIT cell phenotypes represent valuable biomarkers of disease severity and their therapeutic manipulation might prevent the inflammatory phase involved in COVID-19 aggravation.</jats:p>","version":null,"doi":"10.1101/2020.08.31.20185082","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.24.265827","pub_date":"2020-9-02","title":"Unique mutational changes in SARS-CoV-2 genome: A case study for the largest state of India","abstract":"COVID-19 is a global pandemic causing more than 8 million deaths till mid-August, 2020. In India, more than 3 million confirmed cases have been reported although with relatively low death rate of 1.8%. In this study, we sequenced 47 genomes of SARS-CoV-2 from the patients of 13 districts of Uttar Pradesh (UP), the largest state of India using third-generation sequencing technique. The phylogenetic clustering revealed that no UP sample was aligned with the previously defined USA clade, where the mortality was high. We identified 56 distinct SNP variations in the genomes of UP resulting in a unique mutation rate of 1.19% per sequence, which is greater than the value 0.88% obtained for the rest of India. The relatively less death rate in UP indicates that the mutation in the virus is deleterious. Further investigation is required with larger sample size to determine the degree of virulence vis-a-vis SNP variation.","version":"1.3","doi":"10.1101/2020.08.24.265827","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.02.280180","pub_date":"2020-9-02","title":"Induction of a regulatory myeloid program in bacterial sepsis and severe COVID-19","abstract":"A recent estimate suggests that one in five deaths globally are associated with sepsis. To date, no targeted treatment is available for this syndrome, likely due to substantial patient heterogeneity and our lack of insight into sepsis immunopathology. These issues are highlighted by the current COVID-19 pandemic, wherein many clinical manifestations of severe SARS-CoV-2 infection parallel bacterial sepsis. We previously reported an expanded CD14+ monocyte state, MS1, in patients with bacterial sepsis or non-infectious critical illness, and validated its expansion in sepsis across thousands of patients using public transcriptomic data. Despite its marked expansion in the circulation of bacterial sepsis patients, its relevance to viral sepsis and association with disease outcomes have not been examined. In addition, the ontogeny and function of this monocyte state remain poorly characterized. Using public transcriptomic data, we show that the expression of the MS1 program is associated with sepsis mortality and is up-regulated in monocytes from patients with severe COVID-19. We found that blood plasma from bacterial sepsis or COVID-19 patients with severe disease induces emergency myelopoiesis and expression of the MS1 program, which are dependent on the cytokines IL-6 and IL-10. Finally, we demonstrate that MS1 cells are broadly immunosuppressive, similar to monocytic myeloid-derived suppressor cells (MDSCs), and have decreased responsiveness to stimulation. Our findings highlight the utility of regulatory myeloid cells in sepsis prognosis, and the role of systemic cytokines in inducing emergency myelopoiesis during severe bacterial and SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2020.09.02.280180","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.278630","pub_date":"2020-9-02","title":"Prime-boost protein subunit vaccines against SARS-CoV-2 are highly immunogenic in mice and macaques","abstract":"SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assessed prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.","version":"1.1","doi":"10.1101/2020.09.01.278630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.29.20182899","pub_date":"2020-09-02","title":"Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and multiple organ involvement in individuals under 21 years following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To identify genes, pathways and cell types driving MIS-C, we sequenced the blood transcriptomes of MIS-C cases, pediatric cases of coronavirus disease 2019, and healthy controls. We define a MIS-C transcriptional signature partially shared with the transcriptional response to SARS-CoV-2 infection and with the signature of Kawasaki disease, a clinically similar condition. By projecting the MIS-C signature onto a co-expression network, we identified disease gene modules and found genes downregulated in MIS-C clustered in a module enriched for the transcriptional signatures of exhausted CD8\n                  <jats:sup>+</jats:sup>\n                  T-cells and CD56\n                  <jats:sup>dim</jats:sup>\n                  CD57\n                  <jats:sup>+</jats:sup>\n                  NK cells. Bayesian network analyses revealed nine key regulators of this module, including\n                  <jats:italic>TBX21</jats:italic>\n                  , a central coordinator of exhausted CD8\n                  <jats:sup>+</jats:sup>\n                  T-cell differentiation. Together, these findings suggest dysregulated cytotoxic lymphocyte response to SARS-Cov-2 infection in MIS-C.\n                </jats:p>","version":null,"doi":"10.1101/2020.08.29.20182899","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.31.276725","pub_date":"2020-9-01","title":"SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction","abstract":"Infections with SARS-CoV-2 lead to mild to severe coronavirus disease-19 (COVID-19) with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human alveolus-on-a-chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including the interferon expression. By contrast, the adjacent endothelial layer was no infected and did neither show productive virus replication or interferon release. With prolonged infection, both cell types are damaged, and the barrier function is deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokine release, which results in the damage of the alveolar barrier function and viral dissemination.","version":"1.1","doi":"10.1101/2020.08.31.276725","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.277152","pub_date":"2020-9-01","title":"Jumping back and forth: anthropozoonotic and zoonotic transmission of SARS-CoV-2 on mink farms","abstract":"The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by SARS-CoV-2. In addition, SARS-CoV-2 RNA has been detected in felids, mink and dogs in the field. Here, we describe an in-depth investigation of outbreaks on 16 mink farms and humans living or working on these farms, using whole genome sequencing. We conclude that the virus was initially introduced from humans and has evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period several weeks prior to detection. At the moment, despite enhanced biosecurity, early warning surveillance and immediate culling of infected farms, there is ongoing transmission between mink farms with three big transmission clusters with unknown modes of transmission. We also describe the first animal to human transmissions of SARS-CoV-2 in mink farms. SARS-CoV-2 transmission on mink farms.","version":"1.1","doi":"10.1101/2020.09.01.277152","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.274639","pub_date":"2020-9-01","title":"Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro","abstract":"There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.08.31.274639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.190413","pub_date":"2020-9-01","title":"Discovery of Drug-like Ligands for the Mac1 Domain of SARS-CoV-2 Nsp3","abstract":"Small molecules that bind the SARS-CoV-2 non-structural protein 3 Mac1 domain in place of ADP-ribose could be useful as molecular probes or scaffolds for COVID-19 antiviral drug discovery because Mac1 has been linked to coronavirus\u2019 ability to evade cellular detection. A high-throughput assay based on differential scanning fluorimetry (DSF) was therefore optimized and used to identify possible Mac1 ligands in small libraries of drugs and drug-like compounds. Numerous promising compounds included nucleotides, steroids, beta-lactams, and benzimidazoles. The main drawback to this approach was that a high percentage of compounds in some libraries were found to influence the observed Mac1 melting temperature. To prioritize DSF screening hits, the shapes of the observed melting curves and initial assay fluorescence were examined, and the results were compared with virtual screens performed using Autodock VINA. The molecular basis for alternate ligand binding was also examined by determining a structure of one of the hits, cyclic adenosine monophosphate, with atomic resolution.","version":"1.2","doi":"10.1101/2020.07.06.190413","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.274704","pub_date":"2020-9-01","title":"Engineered Trimeric ACE2 Binds and Locks \u201cThree-up\u201d Spike Protein to Potently Inhibit SARS-CoVs and Mutants","abstract":"SARS-CoV-2 enters cells via ACE-2, which binds the spike protein with moderate affinity. Despite a constant background mutational rate, the virus must retain binding with ACE2 for infectivity, providing a conserved constraint for SARS-CoV-2 inhibitors. To prevent mutational escape of SARS-CoV-2 and to prepare for future related coronavirus outbreaks, we engineered a de novo trimeric ACE2 (T-ACE2) protein scaffold that binds the trimeric spike protein with extremely high affinity (KD < 1 pM), while retaining ACE2 native sequence. T-ACE2 potently inhibits all tested pseudotyped viruses including SARS-CoV-2, SARS-CoV, eight naturally occurring SARS-CoV-2 mutants, two SARSr-CoVs as well as authentic SARS-CoV-2. The cryo-EM structure reveals that T-ACE2 can induce the transit of spike protein to \u201cthree-up\u201d RBD conformation upon binding. T-ACE2 thus represents a promising class of broadly neutralizing proteins against SARS-CoVs and mutants.","version":"1.1","doi":"10.1101/2020.08.31.274704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.276675","pub_date":"2020-9-01","title":"Characterization of neutralizing versus binding antibodies and memory B cells in COVID-19 recovered individuals from India","abstract":"India is one of the countries most affected by the recent COVID-19 pandemic. Characterization of humoral responses to SARS-CoV-2 infection, including immunoglobulin isotype usage, neutralizing activity and memory B cell generation, is necessary to provide critical insights on the formation of immune memory in Indian subjects. In this study, we evaluated SARS-CoV-2 receptor-binding domain (RBD)-specific IgG, IgM, and IgA antibody responses, neutralization of live virus, and RBD-specific memory B cell responses in pre-pandemic healthy versus convalescent COVID-19 individuals from India. We observed substantial heterogeneity in the formation of humoral and B cell memory post COVID-19 recovery. While a vast majority (38/42, 90.47%) of COVID-19 recovered individuals developed SARS-CoV-2 RBD-specific IgG responses, only half of them had appreciable neutralizing antibody titers. RBD-specific IgG titers correlated with these neutralizing antibody titers as well as with RBD-specific memory B cell frequencies. In contrast, IgG titers measured against SARS-CoV-2 whole virus preparation, which includes responses to additional viral proteins besides RBD, did not show robust correlation. Our results suggest that assessing RBD-specific IgG titers can serve as a surrogate assay to determine the neutralizing antibody response. These observations have timely implications for identifying potential plasma therapy donors based on RBD-specific IgG in resource-limited settings where routine performance of neutralization assays remains a challenge. Our study provides an understanding of SARS-CoV-2-specific neutralizing antibodies, binding antibodies and memory B cells in COVID-19 convalescent subjects from India. Our study highlights that PCR-confirmed convalescent COVID-19 individuals develop SARS-CoV-2 RBD-specific IgG antibodies, which correlate strongly with their neutralizing antibody titers. RBD-specific IgG titers, thus, can serve as a valuable surrogate measurement for neutralizing antibody responses. These finding have timely significance for selection of appropriate individuals as donors for plasma intervention strategies, as well as determining vaccine efficacy.","version":"1.1","doi":"10.1101/2020.08.31.276675","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.276683","pub_date":"2020-9-01","title":"A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2","abstract":"Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. Reporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.","version":"1.1","doi":"10.1101/2020.08.31.276683","journal":"bioRxiv","score":null},{"id":"10.1101/2020.09.01.277954","pub_date":"2020-9-01","title":"Angiotensin converting enzyme 2 is a novel target of the \u03b3-secretase complex","abstract":"Angiotensin converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system, but also the functional receptor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on structural similarity with other \u03b3-secretase (\u03b3S) targets, we hypothesized that ACE2 may be affected by \u03b3S proteolytic activity. We found that after ectodomain shedding, ACE2 is targeted for intramembrane proteolysis by \u03b3S, releasing a soluble ACE2 C-terminal fragment. Consistently, chemical or genetic inhibition of \u03b3S results in the accumulation of a membrane-bound fragment of ectodomain-deficient ACE2. Although chemical inhibition of \u03b3S does not alter SARS-CoV-2 cell entry, these data point to a novel pathway for cellular ACE2 trafficking.","version":"1.1","doi":"10.1101/2020.09.01.277954","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.20183442","pub_date":"2020-09-01","title":"Tocilizumab in Hospitalized Patients With COVID-19 Pneumonia","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>BACKGROUND</jats:title>\n                  <jats:p>COVID-19 is associated with immune dysregulation and hyperinflammation. Tocilizumab is an anti\u2013interleukin-6 receptor antibody.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS</jats:title>\n                  <jats:p>Patients hospitalized with severe COVID-19 pneumonia receiving standard care were randomized (2:1) to double-blinded intravenous tocilizumab 8 mg/kg or placebo. The primary outcome measure was clinical status on a 7-category ordinal scale at day 28 (1, discharged/ready for discharge; 7, death).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS</jats:title>\n                  <jats:p>Overall, 452 patients were randomized; the modified-intention-to-treat population included 294 tocilizumab-treated and 144 placebo-treated patients. Clinical status at day 28 was not statistically significantly improved for tocilizumab versus placebo (P=0.36). Median (95% CI) ordinal scale values at day 28: 1.0 (1.0 to 1.0) for tocilizumab and 2.0 (1.0 to 4.0) for placebo (odds ratio, 1.19 [0.81 to 1.76]). There was no difference in mortality at day 28 between tocilizumab (19.7%) and placebo (19.4%) (difference, 0.3% [95% CI, \u20137.6 to 8.2]; nominal P=0.94). Median time to hospital discharge was 8 days shorter with tocilizumab than placebo (20.0 and 28.0, respectively; nominal P=0.037; hazard ratio 1.35 [95% CI 1.02 to 1.79]). Median duration of ICU stay was 5.8 days shorter with tocilizumab than placebo (9.8 and 15.5, respectively; nominal P=0.045). In the safety population, serious adverse events occurred in 34.9% of 295 patients in the tocilizumab arm and 38.5% of 143 in the placebo arm.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS</jats:title>\n                  <jats:p>In this randomized placebo-controlled trial in hospitalized COVID-19 pneumonia patients, tocilizumab did not improve clinical status or mortality. Potential benefits in time to hospital discharge and duration of ICU stay are being investigated in ongoing clinical trials.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Trial registration</jats:title>\n                  <jats:p>ClinicalTrials.gov NCTG432G615</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.27.20183442","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.31.270736","pub_date":"2020-8-31","title":"ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas","abstract":"Diabetes is associated with increased mortality from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of the key molecule for SARS-CoV-2 infection of cells, angiotensin-converting enzyme-2 (ACE2). Specifically, we analyzed five public scRNAseq pancreas datasets and performed fluorescence in situ hybridization, Western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) patients. These in silico and ex vivo analyses demonstrated pancreatic expression of ACE2 is prominent in pancreatic ductal epithelium and the microvasculature, with rare endocrine cell expression of this molecule. Pancreata from COVID-19 patients demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression primarily limited to ducts. SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, appears an unlikely central pathogenic feature of COVID-19 as it relates to diabetes.","version":"1.1","doi":"10.1101/2020.08.31.270736","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.269118","pub_date":"2020-8-31","title":"Fractal signatures of SARS-CoV2 coronavirus, the indicator matrix, the fractal dimension and the 2D directional wavelet transform: A comparative study with SARS-CoV, MERS-CoV and SARS-like coronavirus","abstract":"The main goal of this paper is to show the 2D fractal signatures of SARS-CoV2 coronavirus, indicator matrixes maps showing the concentration of nucleotide acids are built form the RNA sequences, and then the fractal dimension and 2D Directional Wavelet Transform (DCWT) are calculated. Analysis of 21 RNA sequences downloaded from NCBI database shows that indicator matrixes and 2D DCWT exhibit the same patterns with different positions, while the fractal dimensions are oscillating around 1.60. A comparison with SARS-CoV, MERS-CoV and SARS-like Coronavirus shows slightly different fractal dimensions, however the indicator matrix and 2D DCWT exhibit the same patterns for the couple (SARS-CoV2, SARS-CoV) and (MERS-CoV, SARS-like) Coronavirus. Obtained results show that SARS-CoV2 is probably a result of SARS-CoV mutation process.","version":"1.2","doi":"10.1101/2020.08.26.269118","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.30.273979","pub_date":"2020-8-31","title":"Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies","abstract":"Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage - a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and anti-viral assays. In this study, we screened a collection of 23 ebselen derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Our work shows that ebselen constitutes a promising platform for development of new antiviral agents targeting both SARS-CoV-2 PLpro and Mpro.","version":"1.1","doi":"10.1101/2020.08.30.273979","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.31.275701","pub_date":"2020-8-31","title":"A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses","abstract":"There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.","version":"1.1","doi":"10.1101/2020.08.31.275701","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.29.273441","pub_date":"2020-8-31","title":"Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike \u2013 ACE2 Protein-Protein Interaction \u2013 A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19","abstract":"Due to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC50 = 3 \u03bcM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction with an activity similar, possibly slightly higher, than those found for it for other PPIs. This PPI inhibitory activity of methylene blue could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.","version":"1.1","doi":"10.1101/2020.08.29.273441","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.30.274506","pub_date":"2020-8-31","title":"From Face-to-Face to Online Modality: Implications for Undergraduate Learning While the World is Temporarily Closed in the Age of COVID-19","abstract":"The second half of the Spring 2020 semester has been an unprecedented time globally due to the ongoing coronavirus disease 2019 (COVID-19). COVID-19 pandemic has forced more than one billion students out of school, has disrupted the world and led all university courses switched to online instruction social distancing actions taken to limit the spread of the virus. The aim of the present study is to evaluate the pandemic related changes for undergraduate students, to assess their perspectives related to their learning, experiences in two courses, and to discuss the far-reaching potential implications for the upcoming summer and fall semesters. An electronic survey was conducted to gather data on the student perceptions and learning characteristics of this transition from face-to-face (F2F) to online at a medium-sized university in the Southeast in the Spring 2020 semester. Nearly 88% of the participants indicated that the COVID-19 pandemic impacted their education, while 19% indicated that they prefer online over F2F learning. Furthermore, the online modality significantly increased attendance in General Biology I. Our study also showed that the usage of live conferencing and digital applications increased due to the pandemic. The current research fills the gap in the existing literature by providing the first study on the effects of the ongoing COVID-19 pandemic on undergraduate learning and experiences in the most unique dual modality of the Spring 2020 semester.","version":"1.1","doi":"10.1101/2020.08.30.274506","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.30.274464","pub_date":"2020-8-30","title":"SARS-CoV-2 infects human pluripotent stem cell-derived cardiomyocytes, impairing electrical and mechanical function","abstract":"Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease. Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities. Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.","version":"1.1","doi":"10.1101/2020.08.30.274464","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.30.273920","pub_date":"2020-8-30","title":"Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies","abstract":"The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike show therapeutic promise and are being evaluated clincally. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to \u201cup\u201d RBDs, (2) ACE2-blocking hNAbs that bind both \u201cup\u201d and \u201cdown\u201d RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize \u201cup\u201d and \u201cdown\u201d RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only \u201cup\u201d RBDs. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent \u201cdown\u201d RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.30.273920","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.267781","pub_date":"2020-8-30","title":"Compositional Variability and Mutation Spectra of Monophyletic SARS-CoV-2 Clades","abstract":"COVID-19 and its causative pathogen SARS-CoV-2 have rushed the world into a staggering pandemic in a few months and a global fight against both is still going on. Here, we describe an analysis procedure where genome composition and its variables are related, through the genetic code, to molecular mechanisms based on understanding of RNA replication and its feedback loop from mutation to viral proteome sequence fraternity including effective sites on replicase-transcriptase complex. Our analysis starts with primary sequence information and identity-based phylogeny based on 22,051 SARS-CoV-2 genome sequences and evaluation of sequence variation patterns as mutation spectrum and its 12 permutations among organized clades tailored to two key mechanisms: strand-biased and function-associated mutations. Our findings include: (1) The most dominant mutation is C-to-U permutation whose abundant second-codon-position counts alter amino acid composition toward higher molecular weight and lower hydrophobicity albeit assumed most slightly deleterious. (2) The second abundance group includes: three negative-strand mutations U-to-C, A-to-G, G-to-A and a positive-strand mutation G-to-U generated through an identical mechanism as C-to-U. (3) A clade-associated and biased mutation trend is found attributable to elevated level of the negative-sense strand synthesis. (4) Within-clade permutation variation is very informative for associating non-synonymous mutations and viral proteome changes. These findings demand a bioinformatics platform where emerging mutations are mapped on to mostly subtle but fast-adjusting viral proteomes and transcriptomes to provide biological and clinical information after logical convergence for effective pharmaceutical and diagnostic applications. Such thoughts and actions are in desperate need, especially in the middle of the War against COVID-19.","version":"1.2","doi":"10.1101/2020.08.26.267781","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271965","pub_date":"2020-8-30","title":"Structure-Altering Mutations of the SARS-CoV-2 Frame Shifting RNA Element","abstract":"With the rapid rate of Covid-19 infections and deaths, treatments and cures besides hand washing, social distancing, masks, isolation, and quarantines are urgently needed. The treatments and vaccines rely on the basic biophysics of the complex viral apparatus. While proteins are serving as main drug and vaccine targets, therapeutic approaches targeting the 30,000 nucleotide RNA viral genome form important complementary approaches. Indeed, the high conservation of the viral genome, its close evolutionary relationship to other viruses, and the rise of gene editing and RNA-based vaccines all argue for a focus on the RNA agent itself. One of the key steps in the viral replication cycle inside host cells is the ribosomal frameshifting required for translation of overlapping open reading frames. The frameshifting element (FSE), one of three highly conserved regions of coronaviruses, includes an RNA pseudoknot considered essential for this ribosomal switching. In this work, we apply our graph-theory-based framework for representing RNA secondary structures, \u201cRAG\u201d (RNA-As Graphs), to alter key structural features of the FSE of the SARS-CoV-2 virus. Specifically, using RAG machinery of genetic algorithms for inverse folding adapted for RNA structures with pseudoknots, we computationally predict minimal mutations that destroy a structurally-important stem and/or the pseudoknot of the FSE, potentially dismantling the virus against translation of the polyproteins. Additionally, our microsecond molecular dynamics simulations of mutant structures indicate relatively stable secondary structures. These findings not only advance our computational design of RNAs containing pseudoknots; they pinpoint to key residues of the SARS-CoV-2 virus as targets for anti-viral drugs and gene editing approaches. Since the outbreak of Covid-19, numerous projects were launched to discover drugs and vaccines. Compared to protein-focused approaches, targeting the RNA genome, especially highly conserved crucial regions, can destruct the virus life cycle more fundamentally and avoid problems of viral mutations. We choose to target the small frame-shifting element (FSE) embedded in the Open Reading Frame 1a,b of SARS-CoV-2. This FSE is essential for translating overlapping reading frames and thus controlling the viral protein synthesis pathway. By applying graph-theory-based computational algorithms, we identify structurally crucial residues in the FSE as potential targets for anti-viral drugs and gene editing.","version":"1.1","doi":"10.1101/2020.08.28.271965","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.30.274241","pub_date":"2020-8-30","title":"Environmental stability of SARS-CoV-2 on different types of surfaces under indoor and seasonal climate conditions","abstract":"We report the stability of SARS-CoV-2 on various surfaces under indoor, summer and spring/fall conditions. The virus was more stable under the spring/fall condition with virus half-lives ranging from 17.11 to 31.82 hours, whereas under indoor and summer conditions the virus half-lives were 3.5\u201311.33 and 2.54\u20135.58 hours, respectively.","version":"1.1","doi":"10.1101/2020.08.30.274241","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.29.271015","pub_date":"2020-8-29","title":"Silicon Nitride Inactivates SARS-CoV-2 in vitro","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the COVID-19 pandemic, remains viable and therefore potentially infectious on several materials. One strategy to discourage the fomite-mediated spread of COVID-19 is the development of materials whose surface chemistry can spontaneously inactivate SARS-CoV-2. Silicon nitride (Si3N4), a material used in spine fusion surgery, is one such candidate because it has been shown to inactivate several bacterial species and viral strains. This study hypothesized that contact with Si3N4 would inactivate SARS-CoV-2, while mammalian cells would remain unaffected. SARS-CoV-2 virions (2\u00d7104 PFU/mL diluted in growth media) were exposed to 5, 10, 15, and 20% (w/v) of an aqueous suspension of sintered Si3N4 particles for durations of 1, 5, and 10 minutes, respectively. Before exposure to the virus, cytotoxicity testing of Si3N4 alone was assessed in Vero cells at 24 and 48 hour post-exposure times. Following each exposure to Si3N4, the remaining infectious virus was quantitated by plaque assay. Vero cell viability increased at 5% and 10% (w/v) concentrations of Si3N4 at exposure times up to 10 minutes, and there was only minimal impact on cell health and viability up to 20% (w/v). However, the SARS-CoV-2 titers were markedly reduced when exposed to all concentrations of Si3N4; the reduction in viral titers was between 85% - 99.6%, depending on the dose and duration of exposure. Si3N4 was non-toxic to the Vero cells while showing strong antiviral activity against SARS-CoV-2. The viricidal effect increased with increasing concentrations of Si3N4 and longer duration of exposure. Surface treatment strategies based on Si3N4 may offer novel methods to discourage SARS-CoV-2 persistence and infectivity on surfaces and discourage the spread of COVID-19.","version":"1.1","doi":"10.1101/2020.08.29.271015","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.23.263327","pub_date":"2020-8-29","title":"A negative feedback model to explain regulation of SARS-CoV-2 replication and transcription","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a preliminary understanding of the replication and transcription mechanisms of SARS-CoV-2 has recently emerged, their regulation remains unclear. Based on reanalysis of public data, we propose a negative feedback model to explain the regulation of replication and transcription in\u2014but not limited to\u2014SARS-CoV-2. The key step leading to new discoveries was the identification of the cleavage sites of nsp15\u2014an RNA uridylate-specific endoribonuclease, encoded by CoVs. According to this model, nsp15 regulates the synthesis of subgenomic RNAs (sgRNAs) and genomic RNAs (gRNAs) by cleaving transcription regulatory sequences in the body. The expression level of nsp15 determines the relative proportions of sgRNAs and gRNAs, which in turn change the expression level of nps15 to reach equilibrium between the replication and transcription of CoVs. The replication and transcription of CoVs are regulated by a negative feedback mechanism that influences the persistence of CoVs in hosts. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, and provide new clues for future studies. One important clue is that nsp15 may be an important and ideal target for the development of drugs (e.g. uridine derivatives) against CoVs.","version":"1.2","doi":"10.1101/2020.08.23.263327","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.22.262733","pub_date":"2020-8-29","title":"Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators","abstract":"Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism and energy homeostasis, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection modulates pathways of lipid synthesis and uptake, including CD36, SREBP-1, PPAR\u03b3 and DGAT-1 in monocytes and triggered LD formation in different human cells. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected cells. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of pro-inflammatory mediators. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.","version":"1.2","doi":"10.1101/2020.08.22.262733","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.29.272963","pub_date":"2020-8-29","title":"Establishment of murine hybridoma cells producing antibodies against spike protein of SARS-CoV-2","abstract":"In 2020 the world faced the pandemic of COVID-19 - severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing to investigate, diagnose and treat COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denaturized S protein in all tested applications including immunoblotting, immunofluorescence staining and enzyme-linked immunosorbent assay. In addition, we showed that the obtained mAbs decreased infection rate of human cells by SARS-CoV-2 pseudovirus particles in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow to use the corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however due to the restrictions caused by pandemic, it is imperative to have a local source of the antibodies suitable for researches and diagnostics of COVID-19. Moreover, as each mAb has a unique binding sequence, bigger sets of various antibodies will allow to detect SARS-CoV-2 proteins even if the virus acquires novel mutations.","version":"1.1","doi":"10.1101/2020.08.29.272963","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178384","pub_date":"2020-8-29","title":"A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2","abstract":"Management of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.06.29.178384","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052159","pub_date":"2020-8-29","title":"A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material","abstract":"The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly-trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point- of-care solution to increase the distribution of COVID-19 surveillance.","version":"1.3","doi":"10.1101/2020.04.20.052159","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.212860","pub_date":"2020-8-29","title":"Phylogenetic Analysis Of SARS-CoV-2 In The First Months Since Its Emergence","abstract":"During the first months of SARS-CoV-2 evolution in a new host, contrasting hypotheses have been proposed about the way the virus has evolved and diversified worldwide. The aim of this study was to perform a comprehensive evolutionary analysis to describe the human outbreak and the evolutionary rate of different genomic regions of SARS-CoV-2. The molecular evolution in nine genomic regions of SARS-CoV-2 was analyzed using three different approaches: phylogenetic signal assessment, emergence of amino acid substitutions, and Bayesian evolutionary rate estimation in eight successive fortnights since the virus emergence. All observed phylogenetic signals were very low and trees topologies were in agreement with those signals. However, after four months of evolution, it was possible to identify regions revealing an incipient viral lineages formation despite the low phylogenetic signal, since fortnight 3. Finally, the SARS-CoV-2 evolutionary rate for regions nsp3 and S, the ones presenting greater variability, was estimated to values of 1.37 \u00d7 10\u22123 and 2.19 \u00d7 10\u22123 substitution/site/year, respectively. In conclusion, results obtained in this work about the variable diversity of crucial viral regions and the determination of the evolutionary rate are consequently decisive to understand essential feature of viral emergence. In turn, findings may allow characterizing for the first time, the evolutionary rate of S protein that is crucial for vaccines development.","version":"1.2","doi":"10.1101/2020.07.21.212860","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.272955","pub_date":"2020-8-29","title":"Global BioID-based SARS-CoV-2 proteins proximal interactome unveils novel ties between viral polypeptides and host factors involved in multiple COVID19-associated mechanisms","abstract":"The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.","version":"1.1","doi":"10.1101/2020.08.28.272955","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.29.257360","pub_date":"2020-8-29","title":"The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans","abstract":"SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is a growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was not hampered. Also in vitro, the strains replicated similarly, as the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.","version":"1.1","doi":"10.1101/2020.08.29.257360","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.29.272864","pub_date":"2020-8-29","title":"A simplified cell-based assay to identify coronavirus 3CL protease inhibitors","abstract":"We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.","version":"1.1","doi":"10.1101/2020.08.29.272864","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.270835","pub_date":"2020-8-28","title":"Secondary analysis of transcriptomes of SARS-CoV-2 infection models to characterize COVID-19","abstract":"Knowledge about the molecular mechanisms driving COVID-19 pathophysiology and outcomes is still limited. To learn more about COVID-19 pathophysiology we performed secondary analyses of transcriptomic data from two in vitro (Calu-3 and Vero E6 cells) and one in vivo (Ad5-hACE2-sensitized mice) models of SARS-CoV-2 infection. We found 1467 conserved differentially expressed host genes (differentially expressed in at least two of the three model system transcriptomes compared) in SARS-CoV-2 infection. To find potential genetic factors associated with COVID-19, we analyzed these conserved differentially expressed genes using known human genotype-phenotype associations. Genome-wide association study enrichment analysis showed evidence of enrichment for GWA loci associated with platelet functions, blood pressure, body mass index, respiratory functions, and neurodegenerative and neuropsychiatric diseases, among others. Since human protein complexes are known to be directly related to viral infection, we combined and analyzed the conserved transcriptomic signature with SARS-CoV-2-host protein-protein interaction data and found more than 150 gene clusters. Of these, 29 clusters (with 5 or more genes in each cluster) had at least one gene encoding protein that interacts with SARS-CoV-2 proteome. These clusters were enriched for different cell types in lung including epithelial, endothelial, and immune cell types suggesting their pathophysiological relevancy to COVID-19. Finally, pathway analysis on the conserved differentially expressed genes and gene clusters showed alterations in several pathways and biological processes that could enable in understanding or hypothesizing molecular signatures inducing pathophysiological changes, risks, or sequelae of COVID-19.","version":"1.1","doi":"10.1101/2020.08.27.270835","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271569","pub_date":"2020-8-28","title":"A Transferable Deep Learning Approach to Fast Screen Potent Antiviral Drugs against SARS-CoV-2","abstract":"The COVID-19 pandemic calls for rapid development of effective treatments. Although various drug repurpose approaches have been used to screen the FDA-approved drugs and drug candidates in clinical phases against SARS-CoV-2, the coronavirus that causes this disease, no magic bullets have been found until now. We used directed message passing neural network to first build a broad-spectrum anti-beta-coronavirus compound prediction model, which gave satisfactory predictions on newly reported active compounds against SARS-CoV-2. Then we applied transfer learning to fine-tune the model with the recently reported anti-SARS-CoV-2 compounds. The fine-tuned model was applied to screen a large compound library with 4.9 million drug-like molecules from ZINC15 database and recommended a list of potential anti-SARS-CoV-2 compounds for further experimental testing. As a proof-of-concept, we experimentally tested 7 high-scored compounds that also demonstrated good binding strength in docking study against the 3C-like protease of SARS-CoV-2 and found one novel compound that inhibited the enzyme with an IC50 of 37.0 \u03bcM. Our model is highly efficient and can be used to screen large compound databases with billions or more compounds to accelerate the drug discovery process for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.08.28.271569","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.269175","pub_date":"2020-8-28","title":"A SARS-CoV-2 BioID-based virus-host membrane protein interactome and virus peptide compendium: new proteomics resources for COVID-19 research","abstract":"Key steps of viral replication take place at host cell membranes, but the detection of membrane-associated protein-protein interactions using standard affinity-based approaches (e.g. immunoprecipitation coupled with mass spectrometry, IP-MS) is challenging. To learn more about SARS-CoV-2 - host protein interactions that take place at membranes, we utilized a complementary technique, proximity-dependent biotin labeling (BioID). This approach uncovered a virus-host topology network comprising 3566 proximity interactions amongst 1010 host proteins, highlighting extensive virus protein crosstalk with: (i) host protein folding and modification machinery; (ii) membrane-bound vesicles and organelles, and; (iii) lipid trafficking pathways and ER-organelle membrane contact sites. The design and implementation of sensitive mass spectrometric approaches for the analysis of complex biological samples is also important for both clinical and basic research proteomics focused on the study of COVID-19. To this end, we conducted a mass spectrometry-based characterization of the SARS-CoV-2 virion and infected cell lysates, identifying 189 unique high-confidence virus tryptic peptides derived from 17 different virus proteins, to create a high quality resource for use in targeted proteomics approaches. Together, these datasets comprise a valuable resource for MS-based SARS-CoV-2 research, and identify novel virus-host protein interactions that could be targeted in COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2020.08.28.269175","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.272518","pub_date":"2020-8-28","title":"A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice","abstract":"Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (S\u0394C-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of S\u0394C-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with S\u0394C-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.","version":"1.1","doi":"10.1101/2020.08.28.272518","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.267567","pub_date":"2020-8-28","title":"A PCR amplicon\u2013based SARS-CoV-2 replicon for antiviral screening","abstract":"The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral screening; however, SARS-CoV-2 replicon has not been reported yet. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3,000 times higher luminescence than MOCK control cells at 24 hours post-electroporation, indicating robust viral translation and RNA replication. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 \u03bcM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 \u03bcM). Taken together, this system could be applied to the safe and effective antiviral screening without using infectious SARS-CoV-2. Because this is a transient replicon, further improvement including the establishment of stable cell line must be achieved.","version":"1.1","doi":"10.1101/2020.08.28.267567","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271601","pub_date":"2020-8-28","title":"Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutated receptor binding domain with ACE2 receptor","abstract":"SARS-CoV-2 is a RNA Coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). It has affected the whole world including Odisha, a state in eastern India. Many people migrated in the state from different countries as well as states during this SARS-CoV-2 pandemic. As per the protocol laid by ICMR and Health & Family welfare of India, all the suspected cases were tested for SARS-CoV-2 infection. The aim of this study was to analyze the RNA binding domain (RBD) sequence of spike protein from the isolates collected from the throat swab samples of COVID-19 positive cases and further to assess the RBD affinity with ACE2 of different species including human. Whole genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19 positive patients was performed using ARTIC amplicon based sequencing. Sequence analysis and phylogenetic analysis was carried out for the Spike and RBD region of all isolates. The interaction between the RBD and ACE2 receptor of five different species was also analysed. Except three isolates, spike region of 32 isolates showed one/multiple alterations in nucleotide bases in comparison to the Wuhan reference strain. One of the identified mutation at 1204 (Ref A, RMRC 22 C) in the RBD of spike protein was identified which depicted a stronger binding affinity with human ACE2 receptor compared to the wild type RBD. Furthermore, RBDs of all the Indian isolates are capable of binding to ACE2 of human, bat, hamster and pangolin. As mutated RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The study shows that RBDs of all the studied isolates have binding affinity for all the five species, which suggests that the virus can infect a wide variety of animals which could also act as natural reservoir for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.28.271601","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.255463","pub_date":"2020-8-28","title":"Resolvin D1 and D2 reduce SARS-Cov-2-induced inflammation in cystic fibrosis macrophages","abstract":"Resolvins (Rv) are endogenous lipid autacoids that mediate resolution of inflammation and bacterial infections. Their roles in SARS-CoV-2 and COVID-19 are of considerable interest in the context of cystic fibrosis (CF) given the paucity of data regarding the effect of this virus on immune cells from individuals with CF. Here, we provide evidence for Rv biosynthesis and regulatory actions on CF macrophage inflammatory responses.","version":"1.1","doi":"10.1101/2020.08.28.255463","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.271130","pub_date":"2020-8-28","title":"Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 \u00d7 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.27.271130","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271957","pub_date":"2020-8-28","title":"Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2","abstract":"A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with Mpro, 17 were chosen for evaluation in a kinetic assay for Mpro inhibition. Remarkably 14 of the compounds at 100-\u03bcM concentration were found to reduce the enzymatic activity and 5 provided IC50 values below 40 \u03bcM: manidipine (4.8 \u03bcM), boceprevir (5.4 \u03bcM), lercanidipine (16.2 \u03bcM), bedaquiline (18.7 \u03bcM), and efonidipine (38.5 \u03bcM). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1\u2019, and P2 pockets of Mpro. Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.","version":"1.1","doi":"10.1101/2020.08.28.271957","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271635","pub_date":"2020-8-28","title":"SARS-CoV-2 spike D614G variant exhibits highly efficient replication and transmission in hamsters","abstract":"SARS-CoV-2 causes disease varying in severity from asymptomatic infections to severe respiratory distress and death in humans. The viral factors which determine transmissibility and pathogenicity are not yet clearly characterized. We used the hamster infection model to compare the replication ability and pathogenicity of five SARS-CoV-2 strains isolated from early cases originating in Wuhan, China, in February, and infected individuals returning from Europe and elsewhere in March 2020. The HK-13 and HK-95 isolates showed distinct pathogenicity in hamsters, with higher virus titers and more severe pathological changes in the lungs observed compared to other isolates. HK-95 contains a D614G substitution in the spike protein and demonstrated higher viral gene expression and transmission efficiency in hamsters. Intra-host diversity analysis revealed that further quasi species were generated during hamster infections, indicating that strain-specific adaptive mutants with advantages in replication and transmission will continue to arise and dominate subsequent waves of SARS-CoV-2 dissemination.","version":"1.1","doi":"10.1101/2020.08.28.271635","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.244269","pub_date":"2020-8-28","title":"Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein","abstract":"Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and \u03b2-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue \u03b2-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.","version":"1.1","doi":"10.1101/2020.08.28.244269","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.271163","pub_date":"2020-8-28","title":"SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells","abstract":"The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.","version":"1.1","doi":"10.1101/2020.08.28.271163","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.28.270306","pub_date":"2020-8-28","title":"Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system","abstract":"Until now, no approved effective vaccine and antiviral therapeutic are available for treatment or prevention of SARS-coronavirus 2 (SCoV-2) virus infection. In this study, we established a SCoV-2 Spike glycoprotein (SP), including a SP mutant D614G, pseudotyped HIV-1-based vector system and tested their ability to infect ACE2-expressing cells. This study revealed that a C-terminal 17 amino acid deletion in SCoV-2 SP significantly increases the incorporation of SP into the pseudotyped viruses and enhanced its infectivity, which may be helpful in the design of SCoV2-SP-based vaccine strategies. Moreover, based on this system, we have demonstrated that an aqueous extract from the Chinese herb Prunella vulgaris (CHPV) and a compound, suramin, displayed potent inhibitory effects on both wild type and mutant (G614) SCoV-2 SP pseudotyped virus (SCoV-2-SP-PVs)-mediated infection. The 50% inhibitory concentration (IC50) for CHPV and suramin on SCoV-2-SP-PVs are 30, and 40 \u03bcg/ml, respectively. To define the mechanisms of their actions, we demonstrated that both CHPV and suramin are able to directly interrupt SCoV-2\u2013SP binding to its receptor ACE2 and block the viral entry step. Importantly, our results also showed that CHPV or suramin can efficiently reduce levels of cytopathic effect caused by SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) infection in Vero cells. Furthermore, our results demonstrated that the combination of CHPV/suramin with an anti-SARS-CoV-2 neutralizing antibody mediated more potent blocking effect against SCoV2-SP-PVs. Overall, this study provides evidence that CHPV and suramin has anti-SARS-CoV-2 activity and may be developed as a novel antiviral approach against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.08.28.270306","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.270819","pub_date":"2020-8-28","title":"Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics","abstract":"The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness. Prior clinical studies have identified adverse events, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.","version":"1.1","doi":"10.1101/2020.08.27.270819","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.261404","pub_date":"2020-8-27","title":"Functional and druggability analysis of the SARS-CoV-2 proteome","abstract":"The infectious coronavirus disease (COVID-19) pandemic, caused by the coronavirus SARS-CoV-2, appeared in December 2019 in Wuhan, China, and has spread worldwide. As of today, more than 22 million people have been infected, with almost 800,000 fatalities. With the purpose of contributing to the development of effective therapeutics, this work provides an overview of the viral machinery and functional role of each SARS-CoV-2 protein, and a thorough analysis of the structure and druggability assessment of the viral proteome. All structural, non-structural, and accessory proteins of SARS-CoV-2 have been studied, and whenever experimental structural data of SARS-CoV-2 proteins were not available, homology models were built based on solved SARS-CoV structures. Several potential allosteric or protein-protein interaction druggable sites on different viral targets were identified, knowledge that could be used to expand current drug discovery endeavors beyond the cysteine proteases and the polymerase complex. It is our hope that this study will support the efforts of the scientific community both in understanding the molecular determinants of this disease and in widening the repertoire of viral targets in the quest for repurposed or novel drugs against COVID-19.","version":"1.2","doi":"10.1101/2020.08.21.261404","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.267716","pub_date":"2020-8-27","title":"The immunodominant and neutralization linear epitopes for SARS-CoV-2","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes a tremendous threat to global health. Although vaccines against the virus are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulated the three-dimensional structures of SARS-CoV-2 proteins with high performance computer, predicted the B cell epitopes on spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches, and then validated the epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induced antibody production, six of which were immunodominant epitopes in patients identified via the binding of epitopes with the sera from domestic and imported COVID-19 patients, and 23 were conserved within SARS-CoV-2, SARS-CoV and bat coronavirus RaTG13. We also found that the immunodominant epitopes of domestic SARS-CoV-2 were different from that of the imported, which may be caused by the mutations on S (G614D) and N proteins. Importantly, we validated that eight epitopes on S protein elicited neutralizing antibodies that blocked the cell entry of both D614 and G614 pseudo-virus of SARS-CoV-2, three and nine epitopes induced D614 or G614 neutralizing antibodies, respectively. Our present study shed light on the immunodominance, neutralization, and conserved epitopes on SARS-CoV-2 which are potently used for the diagnosis, virus classification and the vaccine design tackling inefficiency, virus mutation and different species of coronaviruses.","version":"1.1","doi":"10.1101/2020.08.27.267716","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197889","pub_date":"2020-8-27","title":"A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV-2 main protease","abstract":"The SARS coronavirus type 2 (SARS-CoV-2) emerged in late 2019 as a zoonotic virus highly transmissible between humans that has caused the COVID-19 pandemic . This pandemic has the potential to disrupt healthcare globally and has already caused high levels of mortality, especially amongst the elderly. The overall case fatality rate for COVID-19 is estimated to be \u223c2.3% overall  and 32.3% in hospitalized patients age 70-79 years . Therapeutic options for treating the underlying viremia in COVID-19 are presently limited by a lack of effective SARS-CoV-2 antiviral drugs, although steroidal anti-inflammatory treatment can be helpful. A variety of potential antiviral targets for SARS-CoV-2 have been considered including the spike protein and replicase. Based upon previous successful antiviral drug development for HIV-1 and hepatitis C, the SARS-CoV-2 main protease (Mpro) appears an attractive target for drug development. Here we show the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of \u223c6,070 drugs with a previous history of use in humans for compounds that inhibit the activity of Mpro in vitro. In our primary screen we found \u223c50 compounds with activity against Mpro (overall hit rate <0.75%). Subsequent dose validation studies demonstrated 8 dose responsive hits with an IC50 \u2264 50 \u03bcM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including Boceprevir (IC50=0.95 \u03bcM), Ciluprevir (20.77\u03bcM). Narlaprevir (IC50=1.10\u03bcM), and Telaprevir (15.25\u03bcM). These results demonstrate that some existing approved drugs can inhibit SARS-CoV-2 Mpro and that screen saturation of all approved drugs is both feasible and warranted. Taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than Boceprevir and suitable for rapid repurposing.","version":"1.2","doi":"10.1101/2020.07.10.197889","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.270637","pub_date":"2020-8-27","title":"Structure of SARS-CoV-2 ORF8, a rapidly evolving coronavirus protein implicated in immune evasion","abstract":"The molecular basis for the severity and rapid spread of the COVID-19 disease caused by SARS-CoV-2 is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04 \u00c5 resolution by x-ray crystallography. The structure reveals a ~60 residue core similar to SARS-CoV ORF7a with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate non-covalent interface is formed by another SARS-CoV-2-specific sequence, 73YIDI76. Together the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.","version":"1.1","doi":"10.1101/2020.08.27.270637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.263988","pub_date":"2020-8-27","title":"Susceptibility of rabbits to SARS-CoV-2","abstract":"Transmission of severe acute respiratory coronavirus-2 (SARS-CoV-2) between livestock and humans is a potential public health concern. We demonstrate the susceptibility of rabbits to SARS-CoV-2, which excrete infectious virus from the nose and throat upon experimental inoculation. Therefore, investigations on the presence of SARS-CoV-2 in farmed rabbits should be considered.","version":"1.1","doi":"10.1101/2020.08.27.263988","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.269738","pub_date":"2020-8-27","title":"SARS-CoV-2 lineage B.6 is the major contributor to transmission in Malaysia","abstract":"As of June 30, 2020, Malaysia had confirmed 8,639 cases of COVID-19. About 39% of these were associated with a religious mass gathering event held in Kuala Lumpur between February 27 and March 1, 2020, which drove community transmission during Malaysia\u2019s main wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to understand the molecular epidemiology. We obtained whole genome sequences of SARS-CoV-2 from 58 COVID-19 patients in Kuala Lumpur, Malaysia, and performed phylogenetic analyses on these and a further 50 Malaysian sequences available in the GISAID database. Malaysian lineage B.6 sequences were further analysed with all available worldwide lineage B.6 sequences. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the mass gathering and became predominant (63%) despite being relatively rare (1.4%) among available global sequences. Increases in reported cases and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly associated with travel and showed limited onward transmission. There were also temporally-correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. We also report the presence of a nsp3-C6310A substitution found in 40.5% of global B.6 sequences which has associated with reduced sensitivity in a commercial assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the region. The COVID-19 pandemic in Malaysia was driven mainly by transmission following a religious mass gathering held in Kuala Lumpur at the end of February. To study the genetic epidemiology of SARS-CoV-2 in Malaysia, we analysed 50 available and 58 newly-generated Malaysian whole genome virus sequences. We found that lineage B.6, rare (1.4%) globally, first appeared after the mass gathering and became the most predominant (62.9%) in Malaysia. Increases in COVID-19 cases and locally-acquired B.6 strains were temporally linked. Non-B.6 viruses were mainly associated with travel and showed limited spread. Increases in B.6 viruses in Southeast Asian countries, India and Australia were linked to participants returning from this mass gathering. Altogether, 95.3% of global B.6 sequences originated in Asia or Australia. We also report a mutation in the virus nsP3 gene found in 40.5% of global B.6 sequences and associated with reduced detection by a commercial diagnostic test. In conclusion, the religious mass gathering in Kuala Lumpur was associated with the main wave of COVID-19 cases of predominantly B.6 lineage in Malaysia, and subsequent spread of B.6 viruses regionally. Genome sequence data provides valuable insight into virus spread and is important for monitoring continued accuracy of diagnostic kits.","version":"1.1","doi":"10.1101/2020.08.27.269738","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.27.269456","pub_date":"2020-8-27","title":"Designing of epitope-based vaccine from the conserved region of spike glycoprotein of SARS-CoV-2","abstract":"The emergence of COVID-19 as a pandemic with a high morbidity rate is posing serious global concern. There is an urgent need to design a suitable therapy or vaccine that could fight against SARS-CoV-2 infection. As spike glycoprotein of SARS-CoV-2 plays a crucial role in receptor binding and membrane fusion inside the host, it could be a suitable target for designing of an epitope-based vaccine. SARS-CoV-2 is an RNA virus and thus has a property to mutate. So, a conserved peptide region of spike glycoprotein was used for predicting suitable B cell and T cell epitopes. 4 T cell epitopes were selected based on stability, antigenicity, allergenicity and toxicity. Further, MHC-I were found from the immune database that could best interact with the selected epitopes. Population coverage analysis was also done to check the presence of identified MHC-I, in the human population of the affected countries. The T cell epitope that binds with the respective MHC-I with highest affinity was chosen. Molecular dynamic simulation results show that the epitope is well selected. This is an in-silico based study that predicts a novel T cell epitope from the conserved spike glycoprotein that could act as a target for designing of the epitope-based vaccine. Further, B cell epitopes have also been found but the main work focuses on T cell epitope as the immunity generated by it is long lasting as compared to B cell epitope.","version":"1.1","doi":"10.1101/2020.08.27.269456","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.269043","pub_date":"2020-8-27","title":"SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.","version":"1.1","doi":"10.1101/2020.08.26.269043","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.190074","pub_date":"2020-8-27","title":"The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia","abstract":"Coronavirus Disease 2019 (COVID-19) is transmitted through respiratory droplets containing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) particles. Once inhaled, SARS-CoV-2 particles gain entry into respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). It is known that ACE2 functions within the renin-angiotensin system to regulate blood pressure, fluid homeostasis and inflammation. However, it is largely unknown what roles ACE2 has in ciliated cells of the airway. Therefore, understanding the function and nature of ACE2 within airway tissue has become an essential element in combatting the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells isolated from nasal turbinates of donors and the air-liquid interface (ALI) model of differentiation. Using ALI tissue we cloned transcripts for three distinct variants of ACE2, one of which encodes the full-length ACE2 protein, the other two transcripts are truncated isoforms that had only been predicted to exist via sequence analysis software. We demonstrate that all three isoforms have the capacity to be glycosylated, a known modification of full-length ACE2. Immunofluorescence microscopy of individual ACE2 isoform transfected cells reveals distinct localization of variant 1 relative to X1 and X2. Double staining immunohistochemistry of ALI tissue using antibodies to either the N-term or C-term region of ACE2 revealed distinct and overlapping signals in the apical cytosol of ciliated cells. Most notably only the ACE2 C-term antibody displayed plasma-membrane localization in ciliated cells. We also observed a decrease in the total amount of ACE2 in ALI tissue derived from a 33 year-old male donor when compared to a 34 year-old female donor, thus there may be variation in the abundance of ACE2 protein in the airway among the population. Together, our data begins to highlight the dynamic status of the ACE2 protein in airway mucociliary tissue and we propose multiple ACE2 parameters that may impact an individual\u2019s susceptibility to SARS-CoV-2. These parameters include the balance of cytosolic versus membrane bound ACE2, isoform expression levels, maintenance of post-translational modifications and the impact of genetic, environmental and lifestyle factors on these processes.","version":"1.2","doi":"10.1101/2020.07.09.190074","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.269183","pub_date":"2020-8-27","title":"Serum Amyloid P inhibits single stranded RNA-induced lung inflammation, lung damage, and cytokine storm in mice","abstract":"SARS-CoV-2 is a single stranded RNA (ssRNA) virus and contains GU-rich sequences distributed abundantly in the genome. In COVID-19, the infection and immune hyperactivation causes accumulation of inflammatory immune cells, blood clots, and protein aggregates in lung fluid, increased lung alveolar wall thickness, and upregulation of serum cytokine levels. A serum protein called serum amyloid P (SAP) has a calming effect on the innate immune system and shows efficacy as a therapeutic for fibrosis in animal models and clinical trials. In this report, we show that aspiration of the GU-rich ssRNA oligonucleotide ORN06 into mouse lungs induces all of the above COVID-19-like symptoms. Men tend to have more severe COVID-19 symptoms than women, and in the aspirated ORN06 model, male mice tended to have more severe symptoms than female mice. Intraperitoneal injections of SAP starting from day 1 post ORN06 aspiration attenuated the ORN06-induced increase in the number of inflammatory cells and formation of clot-like aggregates in the mouse lung fluid, reduced ORN06-increased alveolar wall thickness and accumulation of exudates in the alveolar airspace, and attenuated an ORN06-induced upregulation of the inflammatory cytokines IL-1\u03b2, IL-6, IL-12p70, IL-23, and IL-27 in serum. Together, these results suggest that aspiration of ORN06 is a simple model for both COVID-19 as well as cytokine storm in general, and that SAP is a potential therapeutic for diseases with COVID-19-like symptoms as well as diseases that generate a cytokine storm.","version":"1.1","doi":"10.1101/2020.08.26.269183","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.268854","pub_date":"2020-8-26","title":"Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis","abstract":"SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (\u0394PRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the \u0394PRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the \u0394PRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. As COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.","version":"1.1","doi":"10.1101/2020.08.26.268854","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.267658","pub_date":"2020-8-26","title":"Global and Local Mutations in Bangladeshi SARS-CoV-2 Genomes","abstract":"Corona Virus Disease-2019 (COVID-19) warrants comprehensive investigations of publicly available Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) genomes to gain new insight about their epidemiology, mutations and pathogenesis. Nearly 0.4 million mutations were identified so far in \u223c60,000 SARS-CoV-2 genomic sequences. In this study, we compared 207 of SARS-CoV-2 genomes reported from different parts of Bangladesh and their comparison with 467 globally reported sequences to understand the origin of viruses, possible patterns of mutations, availability of unique mutations, and their apparent impact on pathogenicity of the virus in victims of Bangladeshi population. Phylogenetic analyses indicates that in Bangladesh, SARS-CoV-2 viruses might arrived through infected travelers from European countries, and the GR clade was found as predominant in this region. We found 2602 mutations including 1602 missense mutations, 612 synonymous mutations, 36 insertions and deletions with 352 other mutations types. In line with the global trend, D614G mutation in spike glycoprotein was predominantly high (95.6%) in Bangladeshi isolates. Interestingly, we found the average number of mutations in ORF1ab, S, ORF3a, M and N of genomes, having nucleotide shift at G614 (n=459), were significantly higher (p\u22640.001) than those having mutation at D614 (n=215). Previously reported frequent mutations such as P4715L, D614G, R203K, G204R and I300F were also prevalent in Bangladeshi isolates. Additionally, 87 unique amino acid changes were revealed and were categorized as originating from different cities of Bangladesh. The analyses would increase our understanding of variations in virus genomes circulating in Bangladesh and elsewhere and help develop novel therapeutic targets against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.25.267658","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.267328","pub_date":"2020-8-26","title":"A unique view of SARS-CoV-2 through the lens of ORF8 protein","abstract":"Immune evasion is one of the unique characteristics of COVID-19 attributed to the ORF8 protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protein is involved in modulating the host adaptive immunity through downregulating MHC (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the interferon mediated antiviral response of the host. To understand the immune perspective of the host with respect to the ORF8 protein, a comprehensive study of the ORF8 protein as well as mutations possessed by it, is performed. Chemical and structural properties of ORF8 proteins from different hosts, that is human, bat and pangolin, suggests that the ORF8 of SARS-CoV-2 and Bat RaTG13-CoV are very much closer related than that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 (SARS-CoV-2) are grouped into four classes based on their predicted effects. Based on geolocations and timescale of collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were endorsed upon sequence similarity and amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of rapid evolving SARS-CoV-2 through the ORF8.","version":"1.1","doi":"10.1101/2020.08.25.267328","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.216242","pub_date":"2020-8-26","title":"Fast Whole-Genome Phylogeny of the COVID-19 Virus SARS-CoV-2 by Compression","abstract":"We analyze the whole genome phylogeny and taxonomy of the SARS-CoV-2 virus using compression. This is a new fast alignment-free method called the \u201cnormalized compression distance\u201d (NCD) method. It discovers all effective similarities based on Kolmogorov complexity. The latter being incomputable we approximate it by a good compressor such as the modern zpaq. The results comprise that the SARS-CoV-2 virus is closest to the RaTG13 virus and similar to two bat SARS-like coronaviruses bat-SL-CoVZXC21 and bat-SL-CoVZC4. The similarity is quantified and compared with the same quantified similarities among the mtDNA of certain species. We treat the question whether Pangolins are involved in the SARS-CoV-2 virus. The compression method is simpler and possibly faster than any other whole genome method, which makes it the ideal tool to explore phylogeny.","version":"1.5","doi":"10.1101/2020.07.22.216242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.267831","pub_date":"2020-8-26","title":"SARS-CoV-2 genomic and quasispecies analyses in cancer patients reveal relaxed intrahost virus evolution","abstract":"Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (HCW; non-cancer controls) by deep sequencing and investigated the within-host viral quasispecies of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers (HCW) from the Brazilian Cancer Institute were collected in April\u2013May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants (iSNVs) were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%). Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to HCW (p = 0.009). Intrahost genetic diversity in cancer patients was independent of SARS-CoV-2 Ct values, and was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Such a feature may explain, at least in part, the more adverse outcomes to which cancer/COVID-19 patients experience. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. In this study, phylogenetic and variation analysis of SARS-CoV-2 genomes from cancer patients and non-cancer healthcare workers at the Brazilian National Cancer Institute were characterized by deep sequencing. Viral genomes showed signatures characteristic of Brazilian viruses, consistent with the hypothesis of local, community transmission rather than virus importation from abroad. Despite most genomes in patients and healthcare workers belonging to the same lineage, intrahost variability was higher in cancer patients when compared to non-cancer counterparts. The intrahost genomic diversity analysis presented in our study highlights the relaxed evolution of SARS-CoV-2 in a vulnerable population of cancer patients. The high number of minor variations can result in the selection of immune escape variants, resistance to potential drugs, and/or increased pathogenicity. The impact of this higher intrahost variability over time warrants further investigation.","version":"1.1","doi":"10.1101/2020.08.26.267831","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.266825","pub_date":"2020-8-26","title":"Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are validated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The newly established ELISA was validated using 59 sera of infected or vaccinated animals including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% was achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.","version":"1.1","doi":"10.1101/2020.08.26.266825","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.262188","pub_date":"2020-8-26","title":"Recombinant SARS-CoV-2 RBD molecule with a T helper epitope as a built in adjuvant induces strong neutralization antibody response","abstract":"Without approved vaccines and specific treatment, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading around the world with above 20 million COVID-19 cases and approximately 700 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 \u2013 Gly548 of Spike protein of SARS-CoV-2 linked with Gln830 \u2013 Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5), and prokaryotic expression, chromatography purification and the rational renaturation of the protein were performed. The antigenicity and immunogenicity of S1-4 protein was evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency of it was measured by virus neutralization test in Vero E6 cells with SARS-CoV-2. S1-4 protein was prepared to high homogeneity and purity by prokaryotic expression and chromatography purification. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity compared with S1-5 protein. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralization antibody GMT 256. The candidate subunit vaccine molecule could stimulate strong humoral and Th1 and Th2-type cellular immune response in mice, giving us solid evidence that S1-4 protein could be a promising subunit vaccine candidate.","version":"1.2","doi":"10.1101/2020.08.21.262188","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.265074","pub_date":"2020-8-26","title":"Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by a widely-used RT-PCR assay","abstract":"We identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection. A SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.","version":"1.1","doi":"10.1101/2020.08.25.265074","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.20180851","pub_date":"2020-08-26","title":"Age-specific mortality and immunity patterns of SARS-CoV-2 infection in 45 countries","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The number of COVID-19 deaths is often used as a key indicator of SARS-CoV-2 epidemic size. However, heterogeneous burdens in nursing homes and variable reporting of deaths in elderly individuals can hamper comparisons of deaths and the number of infections associated with them across countries. Using age-specific death data from 45 countries, we find that relative differences in the number of deaths by age amongst individuals aged &lt;65 years old are highly consistent across locations. Combining these data with data from 15 seroprevalence surveys we demonstrate how age-specific infection fatality ratios (IFRs) can be used to reconstruct infected population proportions. We find notable heterogeneity in overall IFR estimates as suggested by individual serological studies and observe that for most European countries the reported number of deaths amongst \u226565s are significantly greater than expected, consistent with high infection attack rates experienced by nursing home populations in Europe. Age-specific COVID-19 death data in younger individuals can provide a robust indicator of population immunity.</jats:p>","version":null,"doi":"10.1101/2020.08.24.20180851","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.25.267500","pub_date":"2020-8-26","title":"The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers","abstract":"Early in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.","version":"1.1","doi":"10.1101/2020.08.25.267500","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.267997","pub_date":"2020-8-26","title":"AI aided design of epitope-based vaccine for the induction of cellular immune responses against SARS-CoV-2","abstract":"The heavy burden imposed by the COVID-19 pandemic on our society triggered the race towards the development of therapies or preventive strategies. Among these, antibodies and vaccines are particularly attractive because of their high specificity, low probability of drug-drug interaction, and potentially long-standing protective effects. While the threat at hand justifies the pace of research, the implementation of therapeutic strategies cannot be exempted from safety considerations. There are several potential adverse events reported after the vaccination or antibody therapy, but two are of utmost importance: antibody-dependent enhancement (ADE) and cytokine storm syndrome (CSS). On the other hand, the depletion or exhaustion of T-cells has been reported to be associated with worse prognosis in COVID-19 patients. This observation suggests a potential role of vaccines eliciting cellular immunity, which might simultaneously limit the risk of ADE and CSS. Such risk was proposed to be associated with FcR-induced activation of proinflammatory macrophages (M1) by Fu et al. 2020 and Iwasaki et al. 2020. All aspects of the newly developed vaccine (including the route of administration, delivery system, and adjuvant selection) may affect its effectiveness and safety. In this work we use a novel in silico approach (based on AI and bioinformatics methods) developed to support the design of epitope-based vaccines. We evaluated the capabilities of our method for predicting the immunogenicity of epitopes. Next, the results of our approach were compared with other vaccine-design strategies reported in the literature. The risk of immuno-toxicity was also assessed. The analysis of epitope conservation among other Coronaviridae was carried out in order to facilitate the selection of peptides shared across different SARS-CoV-2 strains and which might be conserved in emerging zootic coronavirus strains. Finally, the potential applicability of the selected epitopes for the development of a vaccine eliciting cellular immunity for COVID-19 was discussed, highlighting the benefits and challenges of such an approach.","version":"1.1","doi":"10.1101/2020.08.26.267997","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.267351","pub_date":"2020-8-26","title":"Molecular dynamics reveals complex compensatory effects of ionic strength on the SARS-CoV-2 Spike/hACE-2 interaction","abstract":"The SARS-CoV-2 pandemic has already killed more than 800,000 people worldwide. To gain entry, the virus uses its spike protein to bind to host hACE-2 receptors on the host cell surface and mediate fusion between viral and cell membranes. As initial steps leading to virus entry involves significant changes in protein conformation as well as in the electrostatic environment in the vicinity of the spike-hACE-2 complex, we explored the sensitivity of the interaction to changes in ionic strength through computational simulations and surface plasmon resonance. We identified two regions in the receptor-binding domain (RBD), E1 and E2, which interact differently with hACE-2. At high salt concentration, E2-mediated interactions are weakened but are compensated by strengthening E1-mediated hydrophobic interactions. These results provide a detailed molecular understanding of spike RBD/hACE-2 complex formation and stability under a wide range of ionic strengths. \n\n","version":"1.1","doi":"10.1101/2020.08.25.267351","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.267625","pub_date":"2020-8-26","title":"MMGB/SA Consensus Estimate of the Binding Free Energy Between the Novel Coronavirus Spike Protein to the Human ACE2 Receptor","abstract":"The ability to estimate protein-protein binding free energy in a computationally efficient via a physics-based approach is beneficial to research focused on the mechanism of viruses binding to their target proteins. Implicit solvation methodology may be particularly useful in the early stages of such research, as it can offer valuable insights into the binding process, quickly. Here we evaluate the potential of the related molecular mechanics generalized Born surface area (MMGB/SA) approach to estimate the binding free energy \u0394Gbind between the SARS-CoV-2 spike receptor-binding domain and the human ACE2 receptor. The calculations are based on a recent flavor of the generalized Born model, GBNSR6. Two estimates of \u0394Gbind are performed: one based on standard bondi radii, and the other based on a newly developed set of atomic radii (OPT1), optimized specifically for protein-ligand binding. We take the average of the resulting two \u0394Gbind values as the consensus estimate. For the well-studied Ras-Raf protein-protein complex, which has similar binding free energy to that of the SARS-CoV-2/ACE2 complex, the consensus \u0394Gbind = \u221211.8 \u00b1 1 kcal/mol, vs. experimental \u22129.7 \u00b1 0.2 kcal/mol. The consensus estimates for the SARS-CoV-2/ACE2 complex is \u0394Gbind = \u22129.4 \u00b1 1.5 kcal/mol, which is in near quantitative agreement with experiment (\u221210.6 kcal/mol). The availability of a conceptually simple MMGB/SA-based protocol for analysis of the SARS-CoV-2 /ACE2 binding may be beneficial in light of the need to move forward fast.","version":"1.1","doi":"10.1101/2020.08.25.267625","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.26.266304","pub_date":"2020-8-26","title":"Inhibiting coronavirus replication in cultured cells by chemical ER stress","abstract":"Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. We found that the ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types, (partially) restores the virus-induced translational shut-down, and counteracts the CoV-mediated downregulation of IRE1\u03b1 and the ER chaperone BiP. Proteome-wide data sets revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including HERPUD1, an essential factor of ER quality control, and ER-associated protein degradation complexes. The data show that thapsigargin hits a central mechanism required for CoV replication, suggesting that thapsigargin (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs. Suppression of coronavirus replication through thapsigargin-regulated ER stress, ERQC / ERAD and metabolic pathways","version":"1.1","doi":"10.1101/2020.08.26.266304","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.265223","pub_date":"2020-8-25","title":"In Vitro Inactivation of Human Coronavirus by Titania Nanoparticle Coatings and UVC Radiation: Throwing Light on SARS-CoV-2","abstract":"The newly identified pathogenic human coronavirus, SARS-CoV-2, led to an atypical pneumonia-like severe acute respiratory syndrome (SARS) outbreak called coronavirus disease 2019 (COVID-19). Currently, nearly 23 million cases have been confirmed worldwide with the highest COVID-19 cases been confirmed in the United States. As there is no vaccine or any effective interventions, massive efforts to create a postential vaccine to combat COVID-19 is underway. In the meantime, safety precautions and effective disease control strategies appear to be vital for preventing the virus spread in the public places. Due to the longevity of the virus on smooth surfaces, photocatalytic properties of self-disinfecting/cleaning surfaces appear to be a promising tool to help guide disinfection policies to control infectious SAR-CoV-2 spread in high-traffic areas such as hospitals, grocery stores, airports, schools, and stadiums. Here, we explored the photocatalytic properties of nanosized TiO2 (TNPs) as induced by the UV radiation, towards virus deactivation. Our preliminary results using close genetic relative of SAR-CoV-2, HCoV-NL63, showed the virucidal efficacy of photoactive TNPs deposited on glass coverslips, as examined by quantitative RT-PCR and virus culture assays. Efforts to extrapolate the underlying concepts described in this study to SARS-CoV-2 are currently underway.","version":"1.1","doi":"10.1101/2020.08.25.265223","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.265496","pub_date":"2020-8-25","title":"Identification of SARS-CoV-2 induced pathways reveal drug repurposing strategies","abstract":"The global outbreak of SARS-CoV-2 necessitates the rapid development of new therapies against COVID-19 infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2-induced protein (SIP) network, based on disease signatures defined by COVID-19 multi-omic datasets(Bojkova et al., 2020; Gordon et al., 2020), and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2-induced pathways, 40 of which are already in COVID-19 clinical trials(Clinicaltrials.gov, 2020) testifying to the validity of the approach. Using artificial neural network analysis we classified these 200 drugs into 9 distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (130) and immune response (70). A subset of drugs implicated in viral replication were tested in cellular assays and two (proguanil and sulfasalazine) were shown to inhibit replication. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.","version":"1.1","doi":"10.1101/2020.08.24.265496","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.254995","pub_date":"2020-8-25","title":"Ferrets not infected by SARS-CoV-2 in a high-exposure domestic setting","abstract":"Ferrets (Mustela putorius furo) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to SARS-CoV-2 infection and onward transmission. Here, we report the results of a natural experiment where 29 ferrets in one home had prolonged, direct contact and constant environmental exposure to two humans with symptomatic COVID-19. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on RT-PCR and ELISA. To better understand this discrepancy in experimental and natural infection in ferrets, we compared SARS-CoV-2 sequences from natural and experimental mustelid infections and identified two surface glycoprotein (Spike) mutations associated with mustelids. While we found evidence that ACE2 provides a weak host barrier, one mutation only seen in ferrets is located in the novel S1/S2 cleavage site and is computationally predicted to decrease furin activity. These data support that host factors interacting with the novel S1/S2 cleavage site may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of natural infection from currently circulating SARS-CoV-2. This may be overcome in laboratory settings using concentrated viral inoculum, but the effects of ferret host-adaptations require additional investigation.","version":"1.2","doi":"10.1101/2020.08.21.254995","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.254474","pub_date":"2020-8-25","title":"Experimental infection of cattle with SARS-CoV-2","abstract":"Six cattle (Bos taurus) were intranasally inoculated with SARS-CoV-2 and kept together with three na\u00efve in-contact animals. Low-level virus replication and a specific sero-reactivity were observed in two inoculated animals, despite the presence of high antibody titers against a bovine betacoronavirus. The in-contact animals did not become infected.","version":"1.1","doi":"10.1101/2020.08.25.254474","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.25.266775","pub_date":"2020-8-25","title":"The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission","abstract":"The global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.","version":"1.1","doi":"10.1101/2020.08.25.266775","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.264333","pub_date":"2020-8-25","title":"Versatile, Multivalent Nanobody Cocktails Efficiently Neutralize SARS-CoV-2","abstract":"The outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered multiple elite Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration, more potent than some of the best human neutralizing antibodies. We determined a crystal structure of such an elite neutralizing Nb in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. Structural characterization facilitated the bioengineering of novel multivalent Nb constructs into multi-epitope cocktails that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization. These promising agents are readily translated into efficient, cost-effective, and convenient therapeutics to help end this once-in-a-century health crisis.","version":"1.3","doi":"10.1101/2020.08.24.264333","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.20178772","pub_date":"2020-08-25","title":"Hydroxychloroquine in the treatment of outpatients with mildly symptomatic COVID-19: A multi-center observational study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Hydroxychloroquine has not been associated with improved survival among hospitalized COVID-19 patients in the majority of observational studies and similarly was not identified as an effective prophylaxis following exposure in a prospective randomized trial. We aimed to explore the role of hydroxychloroquine therapy in mildly symptomatic patients diagnosed in the outpatient setting.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We examined the association between outpatient hydroxychloroquine exposure and the subsequent progression of disease among mildly symptomatic non-hospitalized patients with documented SARS-CoV-2 infection. The primary outcome assessed was requirement of hospitalization. Data was obtained from a retrospective review of electronic health records within a New Jersey USA multi-hospital network. We compared outcomes in patients who received hydroxychloroquine with those who did not applying a multivariable logistic model with propensity matching.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Among 1274 outpatients with documented SARS-CoV-2 infection 7.6% were prescribed hydroxychloroquine. In a 1067 patient propensity matched cohort, 21.6% with outpatient exposure to hydroxychloroquine were hospitalized, and 31.4% without exposure were hospitalized. In the primary multivariable logistic regression analysis with propensity matching there was an association between exposure to hydroxychloroquine and a decreased rate of hospitalization from COVID-19 (OR 0.53; 95% CI, 0.29, 0.95). Sensitivity analyses revealed similar associations. QTc prolongation events occurred in 2% of patients prescribed hydroxychloroquine with no reported arrhythmia events among those with data available.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>In this retrospective observational study of SARS-CoV-2 infected non-hospitalized patients hydroxychloroquine exposure was associated with a decreased rate of subsequent hospitalization. Additional exploration of hydroxychloroquine in this mildly symptomatic outpatient population is warranted.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Lay Summary</jats:title>\n                  <jats:p>In this observational study of 1,274 COVID-19 patients, hydroxychloroquine given as an outpatient treatment was associated with a 47% reduction in the hazard of hospitalization. Adverse events were not increased (2% QTc prolongation events, 0% arrhythmias). Further validation is required. Use of hydroxychloroquine to treat COVID-19 in the outpatient setting should be reserved for a clinical trial or after discussion with a physician regarding risks and benefits.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.20.20178772","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.24.264895","pub_date":"2020-8-24","title":"The influence of major S protein mutations of SARS-CoV-2 on the potential B cell epitopes","abstract":"SARS-CoV-2 has rapidly transmitted worldwide and results in the COVID-19 pandemic. Spike glycoprotein on surface is a key factor of viral transmission, and has appeared a lot of variants due to gene mutations, which may influence the viral antigenicity and vaccine efficacy. Here, we used bioinformatic tools to analyze B-cell epitopes of prototype S protein and its 9 common variants. 12 potential linear and 53 discontinuous epitopes of B-cells were predicted from the S protein prototype. Importantly, by comparing the epitope alterations between prototype and variants, we demonstrate that B-cell epitopes and antigenicity of 9 variants appear significantly different alterations. The dominant D614G variant impacts the potential epitope least, only with moderately elevated antigenicity, while the epitopes and antigenicity of some mutants(V483A, V367F, etc.) with small incidence in the population change greatly. These results suggest that the currently developed vaccines should be valid for a majority of SARS-CoV-2 infectors. This study provides a scientific basis for large-scale application of SARS-CoV-2 vaccines and for taking precautions against the probable appearance of antigen escape induced by genetic variation after vaccination. The global pandemic of SARS-CoV-2 has lasted for more than half a year and has not yet been contained. Until now there is no effective treatment for SARS-CoV-2 caused disease (COVID-19). Successful vaccine development seems to be the only hope. However, this novel coronavirus belongs to the RNA virus, there is a high mutation rate in the genome, and these mutations often locate on the Spike proteins of virus, the gripper of the virus entering the cells. Vaccination induce the generation of antibodies, which block Spike protein. However, the Spike protein variants may change the recognition and binding of antibodies and make the vaccine ineffective. In this study, we predict neutralizing antibody recognition sites (B cell epitopes) of the prototype S protein of SARS-COV2, along with several common variants using bioinformatics tools. We discovered the variability in antigenicity among the mutants, for instance, in the more widespread D614G variant the change of epitope was least affected, only with slight increase of antigenicity. However, the antigenic epitopes of some mutants change greatly. These results could be of potential importance for future vaccine design and application against SARS-CoV2 variants.","version":"1.1","doi":"10.1101/2020.08.24.264895","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.01.128058","pub_date":"2020-8-24","title":"Minimal system for assembly of SARS-CoV-2 virus like particles","abstract":"SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 \u00b1 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.","version":"1.3","doi":"10.1101/2020.06.01.128058","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.22.258459","pub_date":"2020-8-24","title":"The inhaled steroid ciclesonide blocks SARS-CoV-2 RNA replication by targeting viral replication-transcription complex in culture cells","abstract":"We screened steroid compounds to obtain a drug expected to block host inflammatory responses and MERS-CoV replication. Ciclesonide, an inhaled corticosteroid, suppressed replication of MERS-CoV and other coronaviruses, including SARS-CoV-2, the cause of COVID-19, in cultured cells. The effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 \u03bcM. Ciclesonide inhibited formation of double membrane vesicles, which anchor the viral replication-transcription complex in cells. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in non-structural protein 3 (nsp3) or nsp4. Of note, ciclesonide still suppressed replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients. The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. To identify the effective antiviral agents to combat the disease is urgently needed. In the present study, we found that an inhaled corticosteroid, ciclesonide suppresses replication of coronaviruses, including beta-coronaviruses (MHV-2, MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alpha-coronavirus (HCoV-229E) in cultured cells. The inhaled ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS, and COVID-19.","version":"1.1","doi":"10.1101/2020.08.22.258459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.265090","pub_date":"2020-8-24","title":"Induction of SARS-CoV-2 protein S-specific CD8+ T cells in the lungs of gp96-Ig-S vaccinated mice","abstract":"Given the aggressive spread of COVID-19-related deaths, there is an urgent public health need to support the development of vaccine candidates to rapidly improve the available control measures against SARS-CoV-2. To meet this need, we are leveraging our existing vaccine platform to target SARS-CoV-2. Here, we generated cellular heat shock chaperone protein, glycoprotein 96 (gp96), to deliver SARS-CoV-2 protein S (spike) to the immune system and to induce cell-mediated immune responses. We showed that our vaccine platform effectively stimulates a robust cellular immune response against protein S. Moreover, we confirmed that gp96-Ig, secreted from allogeneic cells expressing full-length protein S, generates powerful, protein S polyepitope-specific CD4+ and CD8+ T cell responses in both lung interstitium and airways. These findings were further strengthened by the observation that protein-S -specific CD8+ T cells were induced in human leukocyte antigen (HLA)-A2-02-01 transgenic mice thus providing encouraging translational data that the vaccine is likely to work in humans, in the context of SARS-CoV-2 antigen presentation.","version":"1.1","doi":"10.1101/2020.08.24.265090","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197913","pub_date":"2020-8-24","title":"A simple protein-based surrogate neutralization assay for SARS-CoV-2","abstract":"Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.","version":"1.2","doi":"10.1101/2020.07.10.197913","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.260901","pub_date":"2020-8-24","title":"The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a \u03b2-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely \u03b2-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other \u03b2-coronaviruses. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other \u03b2-coronaviruses.","version":"1.1","doi":"10.1101/2020.08.24.260901","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.264192","pub_date":"2020-8-24","title":"Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein","abstract":"SARS-CoV-2 contains a PRRA polybasic cleavage motif considered critical for efficient infection and transmission in humans. We previously reported that virus variants with spike protein S1/S2 junction deletions spanning this motif are attenuated. Here we characterize a further cell-adapted SARS-CoV-2 variant, Ca-DelMut. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut does not induce proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response. Ca-DelMut-immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, demonstrating sterilizing immunity.","version":"1.1","doi":"10.1101/2020.08.24.264192","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.24.264630","pub_date":"2020-8-24","title":"SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model","abstract":"Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.","version":"1.1","doi":"10.1101/2020.08.24.264630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.23.255364","pub_date":"2020-8-24","title":"Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro","abstract":"Influenza virus and coronavirus, belonging to enveloped RNA viruses, are major causes of human respiratory diseases. The aim of this study was to investigate the broad spectrum antiviral activity of a naturally existing sulfated polysaccharide, lambda-carrageenan (\u03bb-CGN), purified from marine red algae. Cell culture-based assays revealed that the macromolecule efficiently inhibited both influenza A and B viruses, as well as currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with EC50 values ranging from 0.3\u20131.4 \u03bcg/ml. No toxicity to host cells was observed at concentrations up to 300 \u03bcg/ml. Plaque titration and western blot analysis verified that \u03bb-CGN reduced expression of viral proteins in cell lysates and suppressed progeny virus production in culture supernatants in a dose-dependent manner. This polyanionic compound exerts antiviral activity by targeting viral attachment to cell surface receptors and preventing entry. Moreover, intranasal administration to mice during influenza A viral challenge not only alleviated infection-mediated reductions in body weight but also protected 60% of mice from virus-induced mortality. Thus, \u03bb-CGN could be a promising antiviral agent for preventing infection by several respiratory viruses.","version":"1.1","doi":"10.1101/2020.08.23.255364","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.20179317","pub_date":"2020-08-24","title":"Health Disparities and COVID-19: A Retrospective Study Examining Individual and Community Factors Causing Disproportionate COVID-19 Outcomes in Cook County, Illinois, March 16-May 31, 2020","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Early data from the COVID-19 pandemic suggests that the disease has had a disproportionate impact on communities of color causing higher infection and mortality rates within those communities.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>This study used demographic data from the 2018 US census estimates, mortality data from the Cook County Medical Examiner\u2019s office, and testing results from the Illinois Department of Public Health to perform both bivariate and multivariate regression analyses to explore the role race plays in COVID-19 outcomes at the individual and community levels.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Principal findings show that: 1) while Black Americans make up 22% of Cook County\u2019s population, they account for 36% of the county\u2019s COVID-19 related deaths; 2) the average age of death from COVID-19 is seven years younger for minorities compared to Non-Hispanic White (White) decedents; 3) minorities were more likely than Whites to have seven of the top 10 co-morbidities at death; 4) residents of predominantly minority areas were twice as likely to test positive for COVID-19 (p = 0.0001, IRR 1.94, 95% CI 1.50, 2.50) than residents of predominantly White areas; and 5) residents of predominantly minority areas were 1.43 times more likely to die of COVID-19 than those in predominantly White areas (p = 0.03).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>There are notable differences in COVID-19 related outcomes between racial and ethnic groups at individual and community levels. We hope that this study will scientifically illustrate the health disparities experienced by communities of color and help to address the underlying systemic inequalities still prevalent within our country.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.21.20179317","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.21.262295","pub_date":"2020-8-23","title":"Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein","abstract":"SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an immunofluorescence assay (IFA). The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.21.262295","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.181297","pub_date":"2020-8-23","title":"Pre-existing cutaneous conditions could increase the risk for SARS-COV-2 infection","abstract":"Since the end of 2019, COVID-19 pandemic caused by the SARS-CoV-2 emerged globally. The angiotensin-converting enzyme 2 (ACE2) on the cell surface is crucial for SARS-COV-2 entering into the cells. We use SARS-COV-2 pseudo virus and humanized ACE2 mice to mimic the possible transmitting of SARS-COV-2 through skin based on the data we found that skin ACE2 level is associated with skin pre-existing cutaneous conditions in human and mouse models and inflammatory skin disorders with barrier dysfunction increased the penetration of topical FITC conjugated spike protein into the skin. Our study indicated the possibility that the pre-existing cutaneous conditions could increase the risk for SARS-COV-2 infection.","version":"1.4","doi":"10.1101/2020.06.30.181297","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.191973","pub_date":"2020-8-23","title":"Spike protein fusion loop controls SARS-CoV-2 fusogenicity and infectivity","abstract":"Compared to the other human coronaviruses, SARS-CoV-2 has a higher reproductive number that is driving the COVID-19 pandemic. The high transmission of SARS-CoV-2 has been attributed to environmental, immunological, and molecular factors. The Spike protein is the foremost molecular factor responsible for virus fusion, entry and spread in the host, and thus holds clues for the rapid viral spread. The dense glycosylation of Spike, its high affinity of binding to the human ACE2 receptor, and the efficient priming by cleavage have already been proposed for driving efficient virus-host entry, but these do not explain its unusually high transmission rate. I have investigated the Spike from six \u03b2-coronaviruses, including the SARS-CoV-2, and find that their surface-exposed fusion peptides constituting the defined fusion loop are spatially organized contiguous to each other to work synergistically for triggering the virus-host membrane fusion process. The architecture of the Spike quaternary structure ensures the participation of the fusion peptides in the initiation of the host membrane contact for the virus fusion process. The SARS-CoV-2 fusion peptides have unique physicochemical properties, accrued in part from the presence of consecutive prolines that impart backbone rigidity which aids the virus fusogenicity. The specific contribution of these prolines shows significantly diminished fusogenicity in vitro and associated pathogenesis in vivo, inferred from comparative studies of their deletion-mutant in a fellow murine \u03b2-coronavirus MHV-A59. The priming of the Spike by its cleavage and subsequent fusogenic conformational transition steered by the fusion loop may be critical for the SARS-CoV-2 spread. The three proximal fusion peptides constituting the fusion loop in Spike protein are the membranotropic segments most suitable for engaging the host membrane surface for its disruption. Spike\u2019s unique quaternary structure architecture drives the fusion peptides to initiate the protein host membrane contact. The SARS-CoV-2 Spike trimer surface is relatively more hydrophobic among other human coronavirus Spikes, including the fusion peptides that are structurally more rigid owing to the presence of consecutive prolines, aromatic/hydrophobic clusters, a stretch of consecutive \u03b2-branched amino acids, and the hydrogen bonds. The synergy accrued from the location of the fusion peptides, their physicochemical features, and the fusogenic conformational transition appears to drive the virus fusion process and may explain the high spread of the SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.07.07.191973","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.24.219857","pub_date":"2020-8-23","title":"High Affinity Nanobodies Block SARS-CoV-2 Spike Receptor Binding Domain Interaction with Human Angiotensin Converting Enzyme","abstract":"There are currently no approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12-15 kDa single-domain antibody fragments that are amenable to inexpensive large-scale production and can be delivered by inhalation. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1-5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 micrograms/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.","version":"1.3","doi":"10.1101/2020.07.24.219857","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.262329","pub_date":"2020-8-23","title":"Impaired cytotoxic CD8+ T cell response in elderly COVID-19 patients","abstract":"SARS-CoV-2 infection induces a T cell response that most likely contributes to virus control in COVID-19 patients, but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B, as well as perforin within different effector CD8+ T cell subsets. PD-1 expressing CD8+ T cells also produced cytotoxic molecules during acute infection indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provides valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development. Cytotoxic T cells are responsible for the elimination of infected cells and are key players for the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group in comparison to younger patients.","version":"1.1","doi":"10.1101/2020.08.21.262329","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.244525","pub_date":"2020-8-23","title":"Ebselen Reacts with SARS Coronavirus-2 Main Protease Crystals","abstract":"The SARS coronavirus 2 main protease 3CLpro tailor cuts various essential virus proteins out of long poly-protein translated from the virus RNA. If the 3CLpro is inhibited, the functional virus proteins cannot form and the virus cannot replicate and assemble. Any compound that inhibits the 3CLpro is therefore a potential drug to end the pandemic. Here we show that the diffraction power of 3CLpro crystals is effectively destroyed by Ebselen. It appears that Ebselen may be a widely available, relatively cost effective way to eliminate the SARS coronavirus 2.","version":"1.2","doi":"10.1101/2020.08.10.244525","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.20177667","pub_date":"2020-08-22","title":"How early into the outbreak can surveillance of SARS-CoV-2 in wastewater tell us?","abstract":"<jats:p>There is increasing interest to use wastewater-based surveillance of SARS-CoV-2 as an early warning of the outbreak within a community. Despite successful detection of SARS-CoV-2 in wastewaters sampled from multiple locations, there is still no clear idea on the minimal number of cases needed in a community to result in a positive detection of the virus in wastewaters. To address this knowledge gap, we sampled wastewaters from a septic tank and biological activated sludge tank located on-site of a hospital. The hospital is providing treatment for SARS-CoV-2 infected patients, with the number of hospitalized patients per day known. It was observed that &gt; 253 positive cases out of 10,000 persons are required prior to detecting SARS-CoV-2 in wastewater. There was a weak correlation between N1 and N2 gene abundances in wastewater with the number of hospitalized cases. This correlation was however not observed for N3 gene. The occurrence frequency of SARS-CoV-2 is at least 5 times lower in the partially treated wastewater than in the septic tank. Furthermore, abundance of N1 and N3 genes in the activated sludge tank were 50 and 70% of the levels detected in septic tank, suggesting poor persistence of the SARS-CoV-2 gene fragments in wastewater.</jats:p>","version":null,"doi":"10.1101/2020.08.19.20177667","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.26.114033","pub_date":"2020-8-22","title":"Enantiomers of Chloroquine and Hydroxychloroquine Exhibit Different Activities Against SARS-CoV-2 in vitro, Evidencing S-Hydroxychloroquine as a Potentially Superior Drug for COVID-19","abstract":"In all of the clinical trials for COVID-19 conducted thus far and among those ongoing involving chloroquine or hydroxychloroquine, the drug substance used has invariably been chloroquine (CQ) diphosphate or hydroxychloroquine (HCQ) sulfate, i.e., the phosphoric or sulfuric acid salt of a racemic mixture of R- and S-enantiomer (50/50), respectively. As a result, the clinical outcome from previous CQ or HCQ trials were, in fact, the collective manifestation of both R and S- enantiomers with inherent different pharmacodynamic and pharmacokinetic properties, and toxicity liabilities. Our data for the first time demonstrated the stereoselective difference of CQ and HCQ against live SARS-CoV-2 virus in a Biosafety Level 3 laboratory. S-chloroquine (S-CQ) and S-hydroxychloroquine (S-HCQ) significantly more active against SARS-CoV-2, as compared to R-CQ and R-HCQ, respectively. In addition, Mpro, as one of the critical enzymes for viral transcription and replication, also exhibited an enantioselective binding affinity toward the S-enantiomers. The most significant finding from this study is the pronounced difference of the two enantiomers of CQ and HCQ observed in hERG inhibition assay. The IC50 value of S-HCQ was higher than 20 \u03bcM against hERG channel, which was much less active over all tested CQ and HCQ compounds. Moreover, S-HCQ alone did not prolong QT interval in guinea pigs after 3 days and 6 days of administration, indicating a much lower cardiac toxicity potential. With these and previous findings on the enantio-differentiated metabolism, we recommend that future clinical studies should employ S-HCQ, substantially free of the R-enantiomer, to potentially improve the therapeutic index for the treatment of COVID-19 over the racemic CQ and HCQ.","version":"1.4","doi":"10.1101/2020.05.26.114033","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.248351","pub_date":"2020-8-22","title":"Designed Variants of ACE2-Fc that Decouple Anti-SARS-CoV-2 Activities from Unwanted Cardiovascular Effects","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for SARS-CoV-2, and recombinant ACE2 decoys are being evaluated as new antiviral therapies. We designed and tested an antibody-like ACE2-Fc fusion protein, which has the benefit of long pharmacological half-life and the potential to facilitate immune clearance of the virus. Out of a concern that the intrinsic catalytic activity of ACE2 may unintentionally alter the balance of its hormonal substrates and cause adverse cardiovascular effects in treatment, we performed a mutagenesis screening for inactivating the enzyme. Three mutants, R273A, H378A and E402A, completely lost their enzymatic activity for either surrogate or physiological substrates. All of them remained capable of binding SARS-CoV-2 and could suppress the transduction of a pseudotyped virus in cell culture. This study established new ACE2-Fc candidates as antiviral treatment for SARS-CoV-2 without potentially harmful side effects from ACE2\u2019s catalytic actions toward its vasoactive substrates.","version":"1.2","doi":"10.1101/2020.08.13.248351","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.261727","pub_date":"2020-8-22","title":"The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera","abstract":"Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.","version":"1.1","doi":"10.1101/2020.08.21.261727","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.20178566","pub_date":"2020-08-22","title":"Development of antibodies to pan-coronavirus spike peptides in convalescent COVID-19 patients","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Coronaviruses are sharing several protein regions notable the spike protein (S) on their enveloped membrane surface, with the S1 subunit recognizing and binding to the cellular receptor, while the S2 subunit mediates viral and cellular membrane fusion. This similarity opens the question whether infection with one coronavirus will confer resistance to other coronaviruses? Investigating patient serum samples after SARS-CoV-2 infection in cross-reactivity studies of immunogenic peptides from Middle East respiratory syndrome coronavirus (MERS-CoV), we were able to detect the production of antibodies also recognizing MERS virus antigens. The cross-reactive peptide comes from the heptad repeat 2 (HR2) domain of the MERS virus spike protein. Indeed, the peptide of the HR2 domain of the MERS spike protein, previously proven to induce antibodies against MERS-CoV is sharing 74% homology with the corresponding sequence of SARS-CoV-19 virus. Sera samples of 47 convalescent SARS-CoV-2 patients, validated by RT-PCR-negative testes 30 days post-infection, and samples of 40 sera of control patients (not infected with SARS-CoV-2 previously) were used to establish eventual cross-bind reactivity with the MERS peptide antigen. Significantly stronger binding (p&lt; 0.0001) was observed for IgG antibodies in convalescent SARS-CoV-2 patients compared to the control group. If used as an antigen, the peptide of the HR2 domain of the MERS spike protein allows discrimination between post-Covid populations from non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection has the ability to recognize and probably decrease infectiveness of MERS-CoV infections as well as other coronaviruses. The high homology of the spike protein domain suggests in addition that the opposite effect can also be true: coronaviral infections producing cross-reactive antibodies affective against SARS-CoV-19. The collected data prove in addition that despite the core HR2 region being hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections.</jats:p>","version":null,"doi":"10.1101/2020.08.20.20178566","journal":"medRxiv","score":null},{"id":"10.1101/2020.01.21.914044","pub_date":"2020-8-22","title":"Host and infectivity prediction of Wuhan 2019 novel coronavirus using deep learning algorithm","abstract":"The recent outbreak of pneumonia in Wuhan, China caused by the 2019 Novel Coronavirus (2019-nCoV) emphasizes the importance of detecting novel viruses and predicting their risks of infecting people. In this report, we introduced the VHP (Virus Host Prediction) to predict the potential hosts of viruses using deep learning algorithm. Our prediction suggests that 2019-nCoV has close infectivity with other human coronaviruses, especially the severe acute respiratory syndrome coronavirus (SARS-CoV), Bat SARS-like Coronaviruses and the Middle East respiratory syndrome coronavirus (MERS-CoV). Based on our prediction, compared to the Coronaviruses infecting other vertebrates, bat coronaviruses are assigned with more similar infectivity patterns with 2019-nCoVs. Furthermore, by comparing the infectivity patterns of all viruses hosted on vertebrates, we found mink viruses show a closer infectivity pattern to 2019-nCov. These consequences of infectivity pattern analysis illustrate that bat and mink may be two candidate reservoirs of 2019-nCov.These results warn us to beware of 2019-nCoV and guide us to further explore the properties and reservoir of it. It is of great value to identify whether a newly discovered virus has the risk of infecting human. Guo et al. proposed a virus host prediction method based on deep learning to detect what kind of host a virus can infect with DNA sequence as input. Applied to the Wuhan 2019 Novel Coronavirus, our prediction demonstrated that several vertebrate-infectious coronaviruses have strong potential to infect human. This method will be helpful in future viral analysis and early prevention and control of viral pathogens.","version":"1.4","doi":"10.1101/2020.01.21.914044","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.147496","pub_date":"2020-8-22","title":"A Platform Incorporating Trimeric Antigens into Self-Assembling Nanoparticles Reveals SARS-CoV-2-Spike Nanoparticles to Elicit Substantially Higher Neutralizing Responses than Spike Alone","abstract":"Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer \u2013 stabilized in the prefusion conformation and fused with SpyCatcher \u2013 could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with the SARS-CoV-2 spike-LuS nanoparticles elicited ~25-fold higher neutralizing responses, weight-per-weight relative to spike alone. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.","version":"1.2","doi":"10.1101/2020.06.11.147496","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.20178319","pub_date":"2020-08-22","title":"Evaluating aerosol and splatter during orthodontic debonding: implications for the COVID-19 pandemic","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>Dental procedures often produce splatter and aerosol which have potential to spread pathogens such as SARS-CoV-2. Mixed guidance exists on the aerosol generating potential of orthodontic procedures. The aim of this study was to evaluate aerosol and/or splatter contamination during an orthodontic debonding procedure.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Material and Methods</jats:title>\n                  <jats:p>Fluorescein dye was introduced into the oral cavity of a mannequin. Orthodontic debonding was carried out in triplicate with filter papers placed in the immediate environment. Composite bonding cement was removed using a slow-speed handpiece with dental suction. A positive control condition included a high-speed air-turbine crown preparation. Samples were analysed using digital image analysis and spectrofluorometric analysis.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Contamination across the 8-metre experimental rig was 3% of the positive control on spectrofluorometric analysis and 0% on image analysis. There was contamination of the operator, assistant, and mannequin, representing 8%, 25%, and 28% of the positive control spectrofluorometric measurements, respectively.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Discussion</jats:title>\n                  <jats:p>Orthodontic debonding produces splatter within the immediate locality of the patient. Widespread aerosol generation was not observed.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Orthodontic debonding procedures are low risk for aerosol generation, but localised splatter is likely. This highlights the importance of personal protective equipment for the operator, assistant, and patient.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Three \u2018In brief\u2019 points</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>Orthodontic debonding, including removal of composite using a slow speed handpiece with dental suction, appears to be a low risk procedure for aerosol generation.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Splatter was produced during the debonding procedure, however this was mainly localised to the patient, operator and assistant.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>A single positive reading was identified 3.5 meters away from the patient, highlighting the need for suitable distancing and/or barriers in open clinical environments.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.19.20178319","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.20.259747","pub_date":"2020-8-21","title":"COVID-19 and Cholinergic Anti-inflammatory Pathway: In silico Identification of an Interaction between \u03b17 Nicotinic Acetylcholine Receptor and the Cryptic Epitopes of SARS-CoV and SARS-CoV-2 Spike Glycoproteins","abstract":"SARS-CoV-2 is the coronavirus that originated in Wuhan in December 2019 and has spread globally. The observation of a low prevalence of smokers among hospitalized COVID-19 patients has led to the development of a hypothesis that nicotine could have protective effects by enhancing the cholinergic anti-inflammatory pathway. Based on clinical data and on modelling and docking experiments we have previously presented the potential interaction between SARS-CoV-2 Spike glycoprotein and nicotinic acetylcholine receptors (nAChRs), due to a \u201ctoxin-like\u201d epitope on the Spike Glycoprotein, with homology to a sequence of a snake venom toxin. We here present that this epitope coincides with the well-described cryptic epitope for the human antibody CR3022 and with the epitope for the recently described COVA1-16 antibody. Both antibodies are recognizing neighboring epitopes, are not interfering with the ACE2 protein and are not able to inhibit SARS-CoV and SARS-CoV-2 infections. In this study we present the molecular complexes of both SARS-CoV and SARS-CoV-2 Spike Glycoproteins, at their open or closed conformations, with the molecular model of the human \u03b17 nAChR. We found that the interface of all studied protein complexes involves a large part of the \u201ctoxin-like\u201d sequences of SARS-CoV and SARS-CoV-2 Spike glycoproteins and toxin binding site of human \u03b17 nAChR.","version":"1.1","doi":"10.1101/2020.08.20.259747","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.225854","pub_date":"2020-8-21","title":"Iota-carrageenan and Xylitol inhibit SARS-CoV-2 in cell culture","abstract":"COVID-19 (coronavirus disease 2019) is a pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) infection affecting millions of persons around the world. There is an urgent unmet need to provide an easy-to-produce, affordable medicine to prevent transmission and provide early treatment for this disease. The nasal cavity and the rhinopharynx are the sites of initial replication of SARS-CoV-2. Therefore, a nasal spray may be a suitable dosage form for this purpose. The main objective of our study was to test the antiviral action of three candidate nasal spray formulations against SARS-CoV-2. We have found that iota-carrageenan in concentrations as low as 6 \u00b5g/ mL inhibits SARS-CoV-2 infection in Vero cell cultures. The concentrations found to be active in vitro against SARS-CoV-2 may be easily achieved by the application of nasal sprays already marketed in several countries. Xylitol at a concentration of 5 % m/V has proved to be viricidal on its own and the association with iota-carrageenan may be beneficial, as well.","version":"1.1","doi":"10.1101/2020.08.19.225854","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.257022","pub_date":"2020-8-21","title":"Targeting pentose phosphate pathway for SARS-CoV-2 therapy","abstract":"It becomes more and more obvious that deregulation of host metabolism play an important role in SARS-CoV-2 pathogenesis with implication for increased risk of severe course of COVID-19. Furthermore, it is expected that COVID-19 patients recovered from severe disease may experience long-term metabolic disorders. Thereby understanding the consequences of SARS-CoV-2 infection on host metabolism can facilitate efforts for effective treatment option. We have previously shown that SARS-CoV-2-infected cells undergo a shift towards glycolysis and that 2-deoxy-D-glucose (2DG) inhibits SARS-CoV-2 replication. Here, we show that also pentose phosphate pathway (PPP) is remarkably deregulated. Since PPP supplies ribonucleotides for SARS-CoV-2 replication, this could represent an attractive target for an intervention. On that account, we employed the transketolase inhibitor benfooxythiamine and showed dose-dependent inhibition of SARS-CoV-2 in non-toxic concentrations. Importantly, the antiviral efficacy of benfooxythiamine was further increased in combination with 2DG.","version":"1.1","doi":"10.1101/2020.08.19.257022","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.259838","pub_date":"2020-8-21","title":"Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay","abstract":"The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.","version":"1.1","doi":"10.1101/2020.08.20.259838","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.259937","pub_date":"2020-8-21","title":"SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier","abstract":"Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.","version":"1.1","doi":"10.1101/2020.08.20.259937","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.259770","pub_date":"2020-8-21","title":"Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation","abstract":"SARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1\u201340S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel. SARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how SARS-CoV-2 manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies.","version":"1.1","doi":"10.1101/2020.08.20.259770","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.253369","pub_date":"2020-8-21","title":"A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD","abstract":"Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.","version":"1.2","doi":"10.1101/2020.08.19.253369","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.259721","pub_date":"2020-8-21","title":"Temporal landscape of mutation accumulation in SARS-CoV-2 genomes from Bangladesh: possible implications from the ongoing outbreak in Bangladesh","abstract":"Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. Thus the analysis of genomic mutations of these circulatory strains may give an insight into the molecular basis of SARS-CoV-2 pathogenesis and evolution favoring the development of effective treatment and containment strategies. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA 7, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains in the country belong to B and/or L type among cluster A to Z and strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in Nonspecific protein 2 (NSP2), NSP3, RNA dependent RNA polymerase (RdRp), Helicase, Spike, ORF3a, and Nucleocapsid (N) protein were common in the circulating strains with varying degrees and the most unique mutations(UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, E (Envelope), NSP7a, ORF 6, and ORF 7b suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed an association with sex and age of the COVID-19 positive cases. So, identification of these mutational accumulation patterns may greatly facilitate drug/ vaccine development deciphering the age and the sex dependent differential susceptibility to COVID-19.","version":"1.1","doi":"10.1101/2020.08.20.259721","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.260745","pub_date":"2020-8-21","title":"Unraveling the molecular basis of host cell receptor usage in SARS-CoV-2 and other human pathogenic \u03b2-CoVs","abstract":"The recent emergence of the novel SARS-CoV-2 in China and its rapid spread in the human population has led to a public health crisis worldwide. Like in SARS-CoV, horseshoe bats currently represent the most likely candidate animal source for SARS-CoV-2. Yet, the specific mechanisms of cross-species transmission and adaptation to the human host remain unknown. Here we show that the unsupervised analysis of conservation patterns across the \u03b2-CoV spike protein family, using sequence information alone, can provide rich information on the molecular basis of the specificity of \u03b2-CoVs to different host cell receptors. More precisely, our results indicate that host cell receptor usage is encoded in the amino acid sequences of different CoV spike proteins in the form of a set of specificity determining positions (SDPs). Furthermore, by integrating structural data, in silico mutagenesis and coevolution analysis we could elucidate the role of SDPs in mediating ACE2 binding across the Sarbecovirus lineage, either by engaging the receptor through direct intermolecular interactions or by affecting the local environment of the receptor binding motif. Finally, by the analysis of coevolving mutations across a paired MSA we were able to identify key intermolecular contacts occurring at the spike-ACE2 interface. These results show that effective mining of the evolutionary records held in the sequence of the spike protein family can help tracing the molecular mechanisms behind the evolution and host-receptors adaptation of circulating and future novel \u03b2-CoVs. Unraveling the molecular basis for host cell receptor usage among \u03b2-CoVs is crucial to our understanding of cross-species transmission, adaptation and for molecular-guided epidemiological monitoring of potential outbreaks. In the present study, we survey the sequence conservation patterns of the \u03b2-CoV spike protein family to identify the evolutionary constraints shaping the functional specificity of the protein across the \u03b2-CoV lineage. We show that the unsupervised analysis of statistical patterns in a MSA of the spike protein family can help tracing the amino acid space encoding the specificity of \u03b2-CoVs to their cognate host cell receptors. We argue that the results obtained in this work can provide a framework for monitoring the evolution of SARS-CoV-2 specificity to the hACE2 receptor, as the virus continues spreading in the human population and differential virulence starts to arise.","version":"1.1","doi":"10.1101/2020.08.21.260745","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.261909","pub_date":"2020-8-21","title":"Limited window for donation of convalescent plasma with high live-virus neutralizing antibodies for COVID-19 immunotherapy","abstract":"The optimal timeframe for donating convalescent plasma to be used for COVID-19 immunotherapy is unknown. To address this important knowledge deficit, we determined in vitro live-virus neutralizing capacity and persistence of IgM and IgG antibody responses against the receptor-binding domain and S1 ectodomain of the SARS-CoV-2 spike glycoprotein in 540 convalescent plasma samples obtained from 175 COVID-19 plasma donors for up to 142 days post-symptom onset. Robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 persist, in the aggregate, for at least 100 days post-symptom onset. However, a notable acceleration in decline in virus neutralization titers \u2265160, a value suitable for convalescent plasma therapy, was observed starting 60 days after first symptom onset. Together, these findings better define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor, including age and COVID-19 disease severity score. Evaluation of SARS-CoV-2 anti-spike protein IgM, IgG, and live-virus neutralizing titer profiles reveals that the optimal window for donating convalescent plasma for use in immunotherapy is within the first 60 days of symptom onset.","version":"1.1","doi":"10.1101/2020.08.21.261909","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.259242","pub_date":"2020-8-21","title":"In Silico Modeling of Virus Particle Propagation and Infectivity along the Respiratory Tract: A Case Study for SARS-COV-2","abstract":"Respiratory viruses including Respiratory syncytial virus (RSV), influenza virus and cornaviruses such as Middle Eastern respiratory virus (MERS) and SARS-CoV-2 infect and cause serious and sometimes fatal disease in thousands of people annually. It is critical to understand virus propagation dynamics within the respiratory system because new insights will increase our understanding of virus pathogenesis and enable infection patterns to be more predictable in vivo, which will enhance targeting of vaccines and drug delivery. This study presents a computational model of virus propagation within the respiratory tract network. The model includes the generation network branch structure of the respiratory tract, biophysical and infectivity properties of the virus, as well as air flow models that aid the circulation of the virus particles. The model can also consider the impact of the immune response aim to inhibit virus replication and spread. The model was applied to the SARS-CoV-2 virus by integrating data on its life-cycle, as well as density of Angiotensin Converting Enzyme (ACE2) expressing cells along the respiratory tract network. Using physiological data associated with the respiratory rate and virus load that is inhaled, the model can improve our understanding of the concentration and spatiotemporal dynamics of virus.","version":"1.1","doi":"10.1101/2020.08.20.259242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.261289","pub_date":"2020-8-21","title":"Ubiquitous Forbidden Order in R-group classified protein sequence of SARS-CoV-2 and other viruses","abstract":"Each amino acid in a polypeptide chain has a distinctive R-group associated with it. We report here a novel method of species characterization based upon the order of these R-group classified amino acids in the linear sequence of the side chains associated with the codon triplets. In an otherwise pseudo-random sequence, we search for forbidden combinations of kth order. We applied this method to analyze the available protein sequences of various viruses including SARS-CoV-2. We found that these ubiquitous forbidden orders (UFO) are unique to each of the viruses we analyzed. This unique structure of the viruses may provide an insight into viruses\u2019 chemical behavior and the folding patterns of the proteins. This finding may have a broad significance for the analysis of coding sequences of species in general.","version":"1.1","doi":"10.1101/2020.08.21.261289","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.21.261347","pub_date":"2020-8-21","title":"Covidex: an ultrafast and accurate tool for virus subtyping","abstract":"Covidex is an open-source, alignment-free machine learning subtyping tool for viral species. It is a shiny app that allows a fast and accurate classification in pre-defined clusters for SARS-CoV-2 and FMDV genome sequences. The user can also build its own classification models with the Covidex model generator. Covidex is open-source, cross-platform compatible, and is available under the terms of the GNU General Public License v3 (http://www.gnu.org/licenses/gpl.txt). Covidex is available via SourceForge https://sourceforge.net/projects/covidex or the web application https://cacciabue.shinyapps.io/shiny2/ cacciabue.marco@inta.gob.ar; marcocacciabue@yahoo.com","version":"1.1","doi":"10.1101/2020.08.21.261347","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.256800","pub_date":"2020-8-20","title":"Susceptibility of raccoon dogs for experimental SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019, and became pandemic. The zoonotic virus most likely originated from bats, but definite intermediate hosts have not yet been identified. Raccoon dogs (Nyctereutes procyonoides) are kept for fur production, in particular in China, and were suspected as potential intermediate host for both SARS-CoV6 and SARS-CoV2. Here we demonstrate susceptibility of raccoon dogs for SARS-CoV-2 infection after intranasal inoculation and transmission to direct contact animals. Rapid, high level virus shedding, in combination with minor clinical signs and pathohistological changes, seroconversion and absence of viral adaptation highlight the role of raccoon dogs as a potential intermediate host. The results are highly relevant for control strategies and emphasize the risk that raccoon dogs may represent a potential SARS-CoV-2 reservoir. Our results support the establishment of adequate surveillance and risk mitigation strategies for kept and wild raccoon dogs. Raccoon dogs are susceptible to and efficiently transmit SARS-CoV2 and may serve as intermediate host","version":"1.1","doi":"10.1101/2020.08.19.256800","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222208","pub_date":"2020-8-20","title":"CAR Macrophages for SARS-CoV-2 Immunotherapy","abstract":"Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcR\u03b3 and CD3\u03b6 did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an \u2018immunologically silent\u2019 scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.","version":"1.2","doi":"10.1101/2020.07.26.222208","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.258244","pub_date":"2020-8-20","title":"An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2","abstract":"More than one hundred vaccines against SARS-CoV-2 have been developed and some of them have entered clinical trials, but the latest results revealed that these vaccines still face great challenges. Here, we developed a novel cell-based gp96-Ig-secreting chimeric vaccine which is composed of two viral antigens, the RBD of spike protein, and a truncated nucleocapsid protein that could induce epitope-specific cytotoxic T lymphocytes but low antibody response. Syrian hamsters immunized with the cell-based vaccine produced high level of SARS-CoV-2 specific NAbs and specific T cell immunity which could eliminate RBD-truncated N-expressing cells, without the induction of antibody against N protein and other observed toxicity. This study provides a proof of concept for clinical testing of this safe, effective and cost-effective vaccine against SARS-CoV2 infection.","version":"1.1","doi":"10.1101/2020.08.19.258244","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.258087","pub_date":"2020-8-20","title":"SARS-CoV-2 infection dynamics in lungs of African green monkeys","abstract":"Detailed knowledge about the dynamics of SARS-CoV-2 infection is important for unraveling the viral and host factors that contribute to COVID-19 pathogenesis. Old-World nonhuman primates recapitulate mild-moderate COVID-19 cases, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with SARS-CoV-2 or inactivated virus to study the dynamics of virus replication throughout the respiratory tract. RNA sequencing of single cells from the lungs and mediastinal lymph nodes allowed a high-resolution analysis of virus replication and host responses over time. Viral replication was mainly localized to the lower respiratory tract, with evidence of replication in the pneumocytes. Macrophages were found to play a role in initiating a pro-inflammatory state in the lungs, while also interacting with infected pneumocytes. Our dataset provides a detailed view of changes in host and virus replication dynamics over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.","version":"1.1","doi":"10.1101/2020.08.20.258087","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.258129","pub_date":"2020-8-20","title":"Development of a high-throughput homogeneous AlphaLISA drug screening assay for the detection of SARS-CoV-2 Nucleocapsid","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers the research field an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant NP and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 hours and 48 hours of infection. We simulated the viral infection by spiking in recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Anti-viral agents that inhibit either viral cell entry or replication will decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.08.20.258129","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.258376","pub_date":"2020-8-20","title":"SARS-CoV-2 Quasispecies provides insight into its genetic dynamics during infection","abstract":"A novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has been pandemic worldwide. The genetic dynamics of quasispecies afford RNA viruses a great fitness on cell tropism and host range. However, no quasispecies data of SARS-CoV-2 have been reported yet. To explore quasispecies haplotypes and its transmission characteristics, we carried out single-molecule real-time (SMRT) sequencing of the full-length of SARS-CoV-2 spike gene within 14 RNA samples from 2 infection clusters, covering first-to third-generation infected-patients. We observed a special quasispecies structure of SARS-CoV-2 (modeled as \u2018One-King\u2019): one dominant haplotype (mean abundance ~70.15%) followed by numerous minor haplotypes (mean abundance < 0.10%). We not only discovered a novel dominant haplotype of F1040 but also realized that minor quasispecies were also worthy of attention. Notably, some minor haplotypes (like F1040 and currently pandemic one G614) could potentially reveal adaptive and converse into the dominant one. However, minor haplotypes exhibited a high transmission bottleneck (~6% could be stably transmitted), and the new adaptive/dominant haplotypes were likely originated from genetic variations within a host rather than transmission. The evolutionary rate was estimated as 2.68-3.86 \u00d7 10\u22123 per site per year, which was larger than the estimation at consensus genome level. The \u2018One-King\u2019 model and conversion event expanded our understanding of the genetic dynamics of SARS-CoV-2, and explained the incomprehensible phenomenon at the consensus genome level, such as limited cumulative mutations and low evolutionary rate. Moreover, our findings suggested the epidemic strains may be multi-host origin and future traceability would face huge difficulties.","version":"1.1","doi":"10.1101/2020.08.20.258376","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.20.258772","pub_date":"2020-8-20","title":"What if we perceive SARS-CoV-2 genomes as documents? Topic modelling using Latent Dirichlet Allocation to identify mutation signatures and classify SARS-CoV-2 genomes","abstract":"Topic modeling is frequently employed for discovering structures (or patterns) in a corpus of documents. Its utility in text-mining and document retrieval tasks in various fields of scientific research is rather well known. An unsupervised machine learning approach, Latent Dirichlet Allocation (LDA) has particularly been utilized for identifying latent (or hidden) topics in document collections and for deciphering the words that define one or more topics using a generative statistical model. Here we describe how SARS-CoV-2 genomic mutation profiles can be structured into a \u2018Bag of Words\u2019 to enable identification of signatures (topics) and their probabilistic distribution across various genomes using LDA. Topic models were generated using ~47000 novel corona virus genomes (considered as documents), leading to identification of 16 amino acid mutation signatures and 18 nucleotide mutation signatures (equivalent to topics) in the corpus of chosen genomes through coherence optimization. The document assumption for genomes also helped in identification of contextual nucleotide mutation signatures in the form of conventional N-grams (e.g. bi-grams and tri-grams). We validated the signatures obtained using LDA driven method against the previously reported recurrent mutations and phylogenetic clades for genomes. Additionally, we report the geographical distribution of the identified mutation signatures in SARS-CoV-2 genomes on the global map. Use of the non-phylogenetic albeit classical approaches like topic modeling and other data centric pattern mining algorithms is therefore proposed for supplementing the efforts towards understanding the genomic diversity of the evolving SARS-CoV-2 genomes (and other pathogens/microbes).","version":"1.1","doi":"10.1101/2020.08.20.258772","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.255901","pub_date":"2020-8-20","title":"Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells","abstract":"Understanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we have profiled the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides revealed substantial trimming of glycan residues on the latter, likely introduced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region. Results from this study have application in vaccine design, and will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients.","version":"1.1","doi":"10.1101/2020.08.19.255901","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.19.257493","pub_date":"2020-8-20","title":"Exploring G and C-quadruplex structures as potential targets against the severe acute respiratory syndrome coronavirus 2","abstract":"In this paper we report the analysis of the 2019-nCoV genome and related viruses using an upgraded version of the open-source algorithm G4-iM Grinder. This version improves the functionality of the software, including an easy way to determine the potential biological features affected by the candidates found. The quadruplex definitions of the algorithm were optimized for 2019-nCoV. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, hundreds of potential quadruplex candidates were discovered. These sequences were evaluated by their in vitro formation probability, their position in the viral RNA, their uniqueness and their conservation rates (calculated in over three thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were compared sequentially to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire realm. Sequences found in common with other species were further analyzed and characterized. Sequences with high scores unique to the 2019-nCoV were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates was then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable theranostic targets against the 2019-nCoV.","version":"1.1","doi":"10.1101/2020.08.19.257493","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108506","pub_date":"2020-8-19","title":"No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2","abstract":"The COVID-19 pandemic is caused by the coronavirus SARS-CoV-2, which jumped into the human population in late 2019 from a currently uncharacterised animal reservoir. Due to this extremely recent association with humans, SARS-CoV-2 may not yet be fully adapted to its human host. This has led to speculations that some lineages of SARS-CoV-2 may be evolving towards higher transmissibility. The most plausible candidate mutations under putative natural selection are those which have emerged repeatedly and independently (homoplasies). Here, we formally test whether any of the recurrent mutations that have been observed in SARS-CoV-2 are significantly associated with increased viral transmission. To do so, we develop a phylogenetic index to quantify the relative number of descendants in sister clades with and without a specific allele. We apply this index to a carefully curated set of recurrent mutations identified within a dataset of 46,723 SARS-CoV-2 genomes isolated from patients worldwide. We do not identify a single recurrent mutation in this set convincingly associated with increased viral transmission. Instead, recurrent SARS-CoV-2 mutations currently in circulation appear to be evolutionary neutral. Recurrent mutations also seem primarily induced by the human immune system via host RNA editing, rather than being signatures of adaptation to the novel human host. In conclusion, we find no evidence at this stage for the emergence of significantly more transmissible lineages of SARS-CoV-2 due to recurrent mutations.","version":"1.5","doi":"10.1101/2020.05.21.108506","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.256446","pub_date":"2020-8-19","title":"Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world because of severe symptoms and relatively high mortality. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate the efficacy of prophylactics and therapeutics in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in cynomolgus macaques until 28 days after virus inoculation in the present study. Cynomolgus macaques showed body temperature rises after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening clinical signs of disease corresponding that approximately 80% of human patients did not show a critical disease in COVID-19. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-\u03b3 and interleukin (IL)-2 specifically for SARS-CoV-2 N protein were detected on day 14 in the macaque that showed viral pneumonia. On the other hand, in the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-\u03b3 specifically for SARS-CoV-2 N protein increased on day 7 to day 14 prior to an increase in the number of T-lymphocytes that produced IL-2. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-\u03b3 has a role in the elimination of SARS-CoV-2. Thus, because of the mild clinical signs of disease and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate their efficacy in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in a non-human primate model until 28 days after virus inoculation. Cynomolgus macaques showed a fever after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening symptoms. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-\u03b3 and interleukin (IL)-2 specifically for SARS-CoV-2 protein were detected on day 14 in the macaque that showed viral pneumonia. In the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-\u03b3 specifically for SARS-CoV-2 N protein increased on day 7 to day 14. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-\u03b3 has a role in the elimination of SARS-CoV-2. Thus, because of the mild symptoms and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development.","version":"1.1","doi":"10.1101/2020.08.18.256446","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.225581","pub_date":"2020-8-19","title":"Genome-wide bioinformatic analyses predict key host and viral factors in SARS-CoV-2 pathogenesis","abstract":"The novel betacoronavirus named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a worldwide pandemic (COVID-19) after initially emerging in Wuhan, China. Here we applied a novel, comprehensive bioinformatic strategy to public RNA sequencing and viral genome sequencing data, to better understand how SARS-CoV-2 interacts with human cells. To our knowledge, this is the first meta-analysis to predict host factors that play a specific role in SARS-CoV-2 pathogenesis, distinct from other respiratory viruses. We identified differentially expressed genes, isoforms and transposable element families specifically altered in SARS-CoV-2 infected cells. Well-known immunoregulators including CSF2, IL-32, IL-6 and SERPINA3 were differentially expressed, while immunoregulatory transposable element families were overexpressed. We predicted conserved interactions between the SARS-CoV-2 genome and human RNA-binding proteins such as hnRNPA1, PABPC1 and eIF4b, which may play important roles in the viral life cycle. We also detected four viral sequence variants in the spike, polymerase, and nonstructural proteins that correlate with severity of COVID-19. The host factors we identified likely represent important mechanisms in the disease profile of this pathogen, and could be targeted by prophylactics and/or therapeutics against SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.07.28.225581","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.256578","pub_date":"2020-8-19","title":"NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge","abstract":"There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988). Full-length SARS-CoV-2 prefusion spike with Matrix-M1\u2122 (NVX-CoV2373) vaccine. Induced hACE2 receptor blocking and neutralizing antibodies in macaques. Vaccine protected against SARS-CoV-2 replication in the nose and lungs. Absence of pulmonary pathology in NVX-CoV2373 vaccinated macaques.","version":"1.1","doi":"10.1101/2020.08.18.256578","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.256776","pub_date":"2020-8-19","title":"SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells","abstract":"Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. SARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.","version":"1.1","doi":"10.1101/2020.08.18.256776","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.072371","pub_date":"2020-8-19","title":"SARS-CoV-2 spike protein predicted to form complexes with host receptor protein orthologues from a broad range of mammals","abstract":"SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.","version":"1.6","doi":"10.1101/2020.05.01.072371","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.243303","pub_date":"2020-8-19","title":"Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.","version":"1.2","doi":"10.1101/2020.08.13.243303","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.039917","pub_date":"2020-8-19","title":"Human organ chip-enabled pipeline to rapidly repurpose therapeutics during viral pandemics","abstract":"The rising threat of pandemic viruses, such as SARS-CoV-2, requires development of new preclinical discovery platforms that can more rapidly identify therapeutics that are active in vitro and also translate in vivo. Here we show that human organ-on-a-chip (Organ Chip) microfluidic culture devices lined by highly differentiated human primary lung airway epithelium and endothelium can be used to model virus entry, replication, strain-dependent virulence, host cytokine production, and recruitment of circulating immune cells in response to infection by respiratory viruses with great pandemic potential. We provide a first demonstration of drug repurposing by using oseltamivir in influenza A virus-infected organ chip cultures and show that co-administration of the approved anticoagulant drug, nafamostat, can double oseltamivir\u2019s therapeutic time window. With the emergence of the COVID-19 pandemic, the Airway Chips were used to assess the inhibitory activities of approved drugs that showed inhibition in traditional cell culture assays only to find that most failed when tested in the Organ Chip platform. When administered in human Airway Chips under flow at a clinically relevant dose, one drug \u2013 amodiaquine - significantly inhibited infection by a pseudotyped SARS-CoV-2 virus. Proof of concept was provided by showing that amodiaquine and its active metabolite (desethylamodiaquine) also significantly reduce viral load in both direct infection and animal-to-animal transmission models of native SARS-CoV-2 infection in hamsters. These data highlight the value of Organ Chip technology as a more stringent and physiologically relevant platform for drug repurposing, and suggest that amodiaquine should be considered for future clinical testing.","version":"1.3","doi":"10.1101/2020.04.13.039917","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255927","pub_date":"2020-8-18","title":"Erythroid precursors and progenitors suppress adaptive immunity and get invaded by SARS-CoV-2","abstract":"SARS-CoV-2 infection is associated with lower blood oxygen levels even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients that have distinctive immunosuppressive properties. A subpopulation of abundant erythroid cells, CD45+CD71+cells, co-express ACE2, TMPRSS2, CD147, CD26 and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. Taken together, pathological abundance of erythroid cells might reflect stress erythropoiesis due to the invasion of erythroid progenitors by SARS-CoV-2. This may provide a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.08.18.255927","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255877","pub_date":"2020-8-18","title":"Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2","abstract":"Drug repurposing is a rapid approach to identifying therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drug and 49 investigational drugs. Among these confirmed compounds, the anti-SARS-CoV-2 activities of 230 compounds, including 38 approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set of drug repurposing screen for SARS-CoV-2 is useful for drug repurposing efforts including design of new drug combinations for clinical trials.","version":"1.1","doi":"10.1101/2020.08.18.255877","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197988","pub_date":"2020-8-18","title":"Identifying SARS-CoV-2 entry inhibitors through drug repurposing screens of SARS-S and MERS-S pseudotyped particles","abstract":"While vaccine development will hopefully quell the global pandemic of COVID-19 caused by SARS-CoV-2, small molecule drugs that can effectively control SARS-CoV-2 infection are urgently needed. Here, inhibitors of spike (S) mediated cell entry were identified in a high throughput screen of an approved drugs library with SARS-S and MERS-S pseudotyped particle entry assays. We discovered six compounds (cepharanthine, abemaciclib, osimertinib, trimipramine, colforsin, and ingenol) to be broad spectrum inhibitors for spike-mediated entry. This work should contribute to the development of effective treatments against the initial stage of viral infection, thus reducing viral burden in COVID-19 patients.","version":"1.2","doi":"10.1101/2020.07.10.197988","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.238444","pub_date":"2020-8-18","title":"Bacterial modification of the host glycosaminoglycan heparan sulfate modulates SARS-CoV-2 infectivity","abstract":"The human microbiota has a close relationship with human disease and it remodels components of the glycocalyx including heparan sulfate (HS). Studies of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike protein receptor binding domain suggest that infection requires binding to HS and angiotensin converting enzyme 2 (ACE2) in a codependent manner. Here, we show that commensal host bacterial communities can modify HS and thereby modulate SARS-CoV-2 spike protein binding and that these communities change with host age and sex. Common human-associated commensal bacteria whose genomes encode HS-modifying enzymes were identified. The prevalence of these bacteria and the expression of key microbial glycosidases in bronchoalveolar lavage fluid (BALF) was lower in adult COVID-19 patients than in healthy controls. The presence of HS-modifying bacteria decreased with age in two large survey datasets, FINRISK 2002 and American Gut, revealing one possible mechanism for the observed increase in COVID-19 susceptibility with age. In vitro, bacterial glycosidases from unpurified culture media supernatants fully blocked SARS-CoV-2 spike binding to human H1299 protein lung adenocarcinoma cells. HS-modifying bacteria in human microbial communities may regulate viral adhesion, and loss of these commensals could predispose individuals to infection. Understanding the impact of shifts in microbial community composition and bacterial lyases on SARS-CoV-2 infection may lead to new therapeutics and diagnosis of susceptibility.\n\nGraphical Abstract. Diagram of hypothesis for bacterial mediation of SARS-CoV-2 infection through heparan sulfate (HS).\nIt is well known that host microbes groom the mucosa where they reside. Recent investigations have shown that HS, a major component of mucosal layers, is necessary for SARS-CoV-2 infection. In this study we examine the impact of microbial modification of HS on viral attachment.\n\n\n It is well known that host microbes groom the mucosa where they reside. Recent investigations have shown that HS, a major component of mucosal layers, is necessary for SARS-CoV-2 infection. In this study we examine the impact of microbial modification of HS on viral attachment.","version":"1.1","doi":"10.1101/2020.08.17.238444","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.254979","pub_date":"2020-8-18","title":"The effect of whey protein on viral infection and replication of SARS-CoV-2 and pangolin coronavirus in vitro","abstract":"Since the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human breastmilk, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment, entry and even post-entry viral replication. Moreover, human whey protein inhibited infectious virus production proved by the plaque assay. We found that whey protein from different species such as cow and goat also showed anti-coronavirus properties. And commercial bovine milk also showed similar activity. Interestingly, the main antimicrobial components of breastmilk, such as Lactoferrin and IgA antibody, showed limited anti-coronavirus activity, indicating that other factors of breastmilk may play the important anti-coronavirus role. Taken together, we reported that whey protein inhibits SARS-CoV-2 and its related virus of GX_P2V. These results rule out whey protein as a direct-acting inhibitor of SARS-CoV-2 and GX_P2V infection and replication and further investigation of its molecular mechanism of action in the context of COVID-19.","version":"1.1","doi":"10.1101/2020.08.17.254979","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.254839","pub_date":"2020-8-18","title":"KG-COVID-19: a framework to produce customized knowledge graphs for COVID-19 response","abstract":"Integrated, up-to-date data about SARS-CoV-2 and coronavirus disease 2019 (COVID-19) is crucial for the ongoing response to the COVID-19 pandemic by the biomedical research community. While rich biological knowledge exists for SARS-CoV-2 and related viruses (SARS-CoV, MERS-CoV), integrating this knowledge is difficult and time consuming, since much of it is in siloed databases or in textual format. Furthermore, the data required by the research community varies drastically for different tasks - the optimal data for a machine learning task, for example, is much different from the data used to populate a browsable user interface for clinicians. To address these challenges, we created KG-COVID-19, a flexible framework that ingests and integrates biomedical data to produce knowledge graphs (KGs) for COVID-19 response. This KG framework can also be applied to other problems in which siloed biomedical data must be quickly integrated for different research applications, including future pandemics. An effective response to the COVID-19 pandemic relies on integration of many different types of data available about SARS-CoV-2 and related viruses. KG-COVID-19 is a framework for producing knowledge graphs that can be customized for downstream applications including machine learning tasks, hypothesis-based querying, and browsable user interface to enable researchers to explore COVID-19 data and discover relationships.","version":"1.1","doi":"10.1101/2020.08.17.254839","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.251728","pub_date":"2020-8-18","title":"A Lymph Node Targeted Amphiphile Vaccine Induces Potent Cellular and Humoral Immunity to SARS-CoV-2","abstract":"The SARS-CoV-2 pandemic has led to public health, economic, and social consequences that mandate urgent development of effective vaccines to contain or eradicate infection. To that end, we evaluated a novel amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid-modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain protein as a candidate vaccine (ELI-005) in mice. AMP immunogens are efficiently delivered to lymph nodes, where innate and adaptive immune responses are generated. Compared to alum, AMP immunization induced >25-fold higher antigen-specific T cells which produced multiple Th1 cytokines and trafficked into lung parenchyma and respiratory secretions. Antibody responses favored Th1 isotypes (IgG2bc, IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers 265-fold higher than the natural immune response from convalescent COVID-19 patients; responses were maintained despite 10-fold dose-reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.","version":"1.1","doi":"10.1101/2020.08.17.251728","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.20175489","pub_date":"2020-08-18","title":"Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast and cheap tools to detect viral particles in biological material so to identify the people capable to spread the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles \u2013 AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope and membrane) \u2013 is redshifted in few minutes when mixed to a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (\n                  <jats:italic>\n                    C\n                    <jats:sub>t</jats:sub>\n                  </jats:italic>\n                  ) of a Real Time-PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of RT-PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open new perspective not only in the context of the current and possible future pandemics, but also in microbiology as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.\n                </jats:p>","version":null,"doi":"10.1101/2020.08.15.20175489","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.18.158196","pub_date":"2020-8-18","title":"Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19","abstract":"The rapid geographic expansion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of Coronavirus Disease 2019 (COVID-19) pandemic, poses an immediate need for potent drugs. Enveloped viruses infect the host cell by cellular membrane fusion, a crucial mechanism required for virus replication. The SARS-CoV-2 spike glycoprotein, due to its primary interaction with the human angiotensin-converting enzyme 2 (ACE2) cell-surface receptor, is considered as a potential target for drug development. Based on in silico screening followed by in vitro studies, here we report that the existing FDA-approved Bcr-Abl tyrosine kinase inhibitor, imatinib, inhibits SARS-CoV-2 with an IC50 of 130 nM. We provide evidence that although imatinib binds to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein with an affinity at micromolar, i.e., 2.32 \u00b1 0.9 \u03bcM levels, imatinib does not directly inhibit the spike RBD:ACE2 interaction \u2013 suggesting a Bcr-Abl kinase-mediated fusion inhibition mechanism is responsible for the inhibitory action. We also show that imatinib inhibits other coronaviruses, SARS-CoV, and MERS-CoV via fusion inhibition. Based on promising in vitro results, we propose the Abl tyrosine kinase inhibitor (ATKI), imatinib, to be a viable repurposable drug against COVID-19.","version":"1.2","doi":"10.1101/2020.06.18.158196","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.18.255315","pub_date":"2020-8-18","title":"In-depth blood proteome profiling analysis revealed distinct functional characteristics of plasma proteins between severe and non-severe COVID-19 patients","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over ten million patients worldwide. Although most coronavirus disease 2019 (COVID-19) patients have a good prognosis, some develop severe illness. Markers that define disease severity or predict clinical outcome need to be urgently developed as the mortality rate in critical cases is approximately 61.5%. In the present study, we performed indepth proteome profiling of undepleted plasma from eight COVID-19 patients. Quantitative proteomic analysis using the BoxCar method revealed that 91 out of 1,222 quantified proteins were differentially expressed depending on the severity of COVID-19. Importantly, we found 76 proteins, previously not reported, which could be novel prognostic biomarker candidates. Our plasma proteome signatures captured the host response to SARS-CoV-2 infection, thereby highlighting the role of neutrophil activation, complement activation, platelet function, and T cell suppression as well as proinflammatory factors upstream and downstream of interleukin-6, interleukin-1B, and tumor necrosis factor. Consequently, this study supports the development of blood biomarkers and potential therapeutic targets to aid clinical decision-making and subsequently improve prognosis of COVID-19.","version":"1.1","doi":"10.1101/2020.08.18.255315","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.253484","pub_date":"2020-8-18","title":"Generalized linear models provide a measure of virulence for specific mutations in SARS-CoV-2 strains","abstract":"This study aims to highlight SARS-COV-2 mutations which are associated with increased or decreased viral virulence. We utilize, genetic data from all strains available from GISAID and countries\u2019 regional information such as deaths and cases per million as well as covid-19-related public health austerity measure response times. Initial indications of selective advantage of specific mutations can be obtained from calculating their frequencies across viral strains. By applying modelling approaches, we provide additional information that is not evident from standard statistics or mutation frequencies alone. We therefore, propose a more precise way of selecting informative mutations. We highlight two interesting mutations found in genes N (P13L) and ORF3a (Q57H). The former appears to be significantly associated with decreased deaths and cases per million according to our models, while the latter shows an opposing association with decreased deaths and increased cases per million. Moreover, protein structure prediction tools show that the mutations infer conformational changes to the protein that significantly alter its structure when compared to the reference protein.","version":"1.1","doi":"10.1101/2020.08.17.253484","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.249086","pub_date":"2020-8-18","title":"SARS-Cov-2-, HIV-1-, Ebola-neutralizing and anti-PD1 clones are predisposed","abstract":"Antibody repertoire refers to the totality of the superbly diversified antibodies within an individual to cope with the vast array of possible pathogens. Despite this extreme diversity, antibodies of the same clonotype, namely public clones, have been discovered among individuals. Although some public clones could be explained by antibody convergence, public clones in na\u00efve repertoire or virus-neutralizing clones from not infected people were also discovered. All these findings indicated that public clones might not occur by random and they might exert essential functions. However, the frequencies and functions of public clones in a population have never been studied. Here, we integrated 2,449 Rep-seq datasets from 767 donors and discovered 5.07 million public clones \u2013 ~10% of the repertoire are public in population. We found 38 therapeutic clones out of 3,390 annotated public clones including anti-PD1 clones in healthy people. Moreover, we also revealed clones neutralizing SARS-CoV-2, Ebola, and HIV-1 viruses in healthy individuals. Our result demonstrated that these clones are predisposed in the human antibody repertoire and may exert critical functions during particular immunological stimuli and consequently benefit the donors. We also implemented RAPID \u2013 a Rep-seq Analysis Platform with Integrated Databases, which may serve as a useful tool for others in the field.","version":"1.2","doi":"10.1101/2020.08.13.249086","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.252411","pub_date":"2020-8-17","title":"Multifractal Analysis of SARS-CoV-2 Coronavirus genomes using the wavelet transform","abstract":"In this paper, the 1D Wavelet Transform Modulus Maxima lines (WTMM) method is used to investigate the Long-Range Correlation (LRC) and to estimate the so-called Hurst exponent of 21 isolate RNA sequence downloaded from the NCBI database of patients infected by SARS-CoV-2, Coronavirus, the Knucleotidic, Purine, Pyramidine, Ameno, Keto and GC DNA coding are used. Obtained results show the LRC character in the most sequences; except some sequences where the anti-correlated or the Classical Brownian motion character is observed, demonstrating that the SARS-Cov2 coronavirus undergoes mutation from a country to another or in the same country, they reveals also the complexity and the heterogeneous genome structure organization far from the equilibrium and the self-organization.","version":"1.2","doi":"10.1101/2020.08.15.252411","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.16.252973","pub_date":"2020-8-17","title":"SARS-CoV-2 ORF9b Antagonizes Type I and III Interferons by Targeting Multiple Components of RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING Signaling Pathways","abstract":"Severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2), the etiologic agent of the coronavirus disease 2019 (COVID-19), has a catastrophic effect on human health and society. Clinical findings indicated that the suppression of innate antiviral immunity, especially the type I and III interferon (IFN) production, contributes to the pathogenesis of COVID-19. However, how SARS-CoV-2 evades antiviral immunity still needs further investigations. Here, we reported that the open reading frame 9b (ORF9b) protein encoded by the SARS-CoV-2 genome inhibits the activation of type I and III IFN response by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of type I and III IFNs by Sendai virus or the dsRNA mimic poly (I:C). SARS-CoV-2 ORF9b inhibits the activation of type I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKK\u03b5 rather than IRF3-5D, the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of type I and III IFNs by TRIF and STING, the adaptor protein of endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. Mechanistically, SARS-CoV-2 ORF9b protein interacts with RIG-I, MDA-5, MAVS, TRIF, STING, TBK1, and prevents TBK1 phosphorylation, thus impeding the phosphorylation and nuclear trans-localization of IRF3 activation. Overexpression of SARS-CoV-2 ORF9b facilitates the replication of the vesicular stomatitis virus. Therefore, SARS-CoV-2 ORF9b negatively regulates antiviral immunity, thus, facilitate virus replication. This study contributes to our understanding of the molecular mechanism of how SARS-CoV-2 impaired antiviral immunity and providing an essential clue to the pathogenesis of COVID-19.","version":"1.1","doi":"10.1101/2020.08.16.252973","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.218529","pub_date":"2020-8-17","title":"Immuno-informatics approach for multi-epitope vaccine designing against SARS-CoV-2","abstract":"The novel Corona Virus Disease 2019 (COVID-19) pandemic has set the fatality rates ablaze across the world. So, to combat this disease, we have designed a multi-epitope vaccine from various proteins of Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2) with an immuno-informatics approach, validated in silico to be stable, non-allergic and antigenic. Cytotoxic T-cell, helper T-cell, and B-cell epitopes were computationally predicted from six conserved protein sequences among four viral strains isolated across the world. The T-cell epitopes, overlapping with the B-cell epitopes, were included in the vaccine construct to assure the humoral and cell-mediated immune response. The beta-subunit of cholera toxin was added as an adjuvant at the N-terminal of the construct to increase immunogenicity. Interferon-gamma inducing epitopes were even predicted in the vaccine. Molecular docking and binding energetics studies revealed strong interactions of the vaccine with immune-stimulatory toll-like receptors (TLR) \u22122, 3, 4. Molecular dynamics simulation of the vaccine ensured in vivo stability in the biological system. The immune simulation of vaccine evinced elevated immune response. The efficient translation of the vaccine in an expression vector was assured utilizing in silico cloning approach. Certainly, such a vaccine construct could reliably be effective against COVID-19.","version":"1.3","doi":"10.1101/2020.07.23.218529","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.08.238469","pub_date":"2020-8-17","title":"An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation","abstract":"Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.","version":"1.2","doi":"10.1101/2020.08.08.238469","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.17.253682","pub_date":"2020-8-17","title":"Transcriptomic profiling of human corona virus (HCoV)-229E -infected human cells and genomic mutational analysis of HCoV-229E and SARS-CoV-2","abstract":"Human coronaviruses (HCoVs) cause mild to severe respiratory infection. Most of the common cold illnesses are caused by one of four HCoVs, namely HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43. Several studies have applied global transcriptomic methods to understand host responses to HCoV infection, with most studies focusing on the pandemic severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and the newly emerging SARS-CoV-2. In this study, Next Generation Sequencing was used to gain new insights into cellular transcriptomic changes elicited by alphacoronavirus HCoV-229E. HCoV-229E-infected MRC5 cells showed marked downregulation of superpathway of cholesterol biosynthesis and eIF2 signaling pathways. Moreover, upregulation of cyclins, cell cycle control of chromosomal replication, and the role of BRCA1 in DNA damage response, alongside downregulation of the cell cycle G1/S checkpoint, suggest that HCoV-229E favors S phase for viral infection. Intriguingly, a significant portion of key factors of cell innate immunity, interferon-stimulated genes (ISGs) and other transcripts of early antiviral response genes were downregulated early in HCoV-229E infection. On the other hand, early upregulation of the antiviral response factor Apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) was observed. APOBEC3B cytidine deaminase signature (C-to-T) was previously observed in genomic analysis of SARS-CoV-2 but not HCoV-229E. Higher levels of C-to-T mutations were found in countries with high mortality rates caused by SARS-CoV-2. APOBEC activity could be a marker for new emerging CoVs. This study will enhance our understanding of commonly circulating HCoVs and hopefully provide critical information about still-emerging coronaviruses. Human coronaviruses (HCoVs) generate respiratory tract infections. HCoV-229E is one of four known HCoV strains that circulate annually in the population for several decades. Beside these, three pandemic CoV emerged since year 2002, the Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2. These three strains attracted most attention for extensive research and less consideration has been given to the commonly infecting HCoVs. In this study we use Next generation sequencing analysis to understand global transcriptomic changes in human host cells following HCoV-229E infection. We found several cellular pathways that change during infection that involve cholesterol biosynthesis, cell cycle control, DNA replication, DNA repair, innate immune response and an interesting RNA editing enzyme which could be involve in CoVs pathogenesis.","version":"1.1","doi":"10.1101/2020.08.17.253682","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.20175844","pub_date":"2020-08-17","title":"IL-6 and IL-10 as predictors of disease severity in COVID-19 patients: results from meta-analysis and regression","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Aims</jats:title>\n                  <jats:p>SARS-CoV-2, an infectious agent behind the ongoing COVID-19 pandemic, induces high levels of cytokines such as IL-1, IL-2, IL-4, IL-6, IL-10, TNF-\u03b1, IFN-\u03b3 etc in infected individuals which contribute towards the underlying disease patho-physiology. Nonetheless, exact association and contribution of every cytokine towards COVID-19 pathology remains poorly understood. Delineation of the role of the cytokines during COVID-19 holds the key of efficient patient management in clinics. This study performed a comprehensive meta-analysis to establish association between induced cytokines and COVID-19 disease severity to help in prognosis and clinical care.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Main methods</jats:title>\n                  <jats:p>Scientific literature was searched to identify 13 cytokines (IL-1\u03b2, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-17, TNF-\u03b1 and IFN-\u03b3) from 18 clinical studies. Standardized mean difference (SMD) for selected 6 cytokines IL-2, IL-4, IL-6, IL-10, TNF-\u03b1 and IFN-\u03b3 between severe and non-severe COVID-19 patient groups were summarized using random effects model. A classifier was built using logistic regression model with cytokines having significant SMD as covariates.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Key findings</jats:title>\n                  <jats:p>Out of 13 cytokines, IL-6 and IL-10 showed statistically significant SMD across the studies synthesized. Classifier with mean values of both IL-6 and IL-10 as covariates performed well with accuracy of ~ 92% that was significantly higher than accuracy reported in literature with IL-6 and IL-10 as individual covariates.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Significance</jats:title>\n                  <jats:p>Simple panel proposed by us with only two cytokine markers can be used as predictors for fast diagnosis of patients with higher risk of COVID-19 disease deterioration and thus can be managed well for a favourable prognosis.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.15.20175844","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.12.148296","pub_date":"2020-8-17","title":"DeepEMhancer: a deep learning solution for cryo-EM volume post-processing","abstract":"Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to obtain much cleaner and more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.","version":"1.3","doi":"10.1101/2020.06.12.148296","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.221242","pub_date":"2020-8-17","title":"Integrated intra- and intercellular signaling knowledge for multicellular omics analysis","abstract":"Molecular knowledge of biological processes is a cornerstone in the analysis of omics data. Applied to single-cell data, such analyses can provide mechanistic insights into individual cells and their interactions. However, knowledge of intercellular communication is scarce, scattered across different resources, and not linked to intracellular processes. To address this gap, we combined over 100 resources in a single database. It covers the interactions and roles of proteins in inter- and intracellular signal transduction, as well as transcriptional and post-transcriptional regulation. We also provide a comprehensive collection of protein complexes and rich annotations on the properties of proteins, including function, localization, and role in diseases. The resource is available for human, and via homology translation for mouse and rat. The data is accessible via OmniPath\u2019s web service, a Cytoscape plugin, and packages in R/Bioconductor and Python, providing convenient access options for both computational and experimental scientists. Our resource provides a single access point to knowledge spanning intra- and intercellular processes for data analysis, as we demonstrate in applications to study SARS-CoV-2 infection and ulcerative colitis. \n\n\n","version":"1.2","doi":"10.1101/2020.08.03.221242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.252320","pub_date":"2020-8-16","title":"A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model","abstract":"The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 \u00c5 revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.","version":"1.1","doi":"10.1101/2020.08.15.252320","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.241349","pub_date":"2020-8-16","title":"Effects of SARS-CoV-2 Mutations on Protein Structures and Intraviral Protein-Protein Interactions","abstract":"Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has infected ten millions of people across the globe, and massive mutations in virus genome have occurred during the rapid spread of this novel coronavirus. Variance in protein sequence might lead to change in protein structure and interaction, then further affect the viral physiological characteristics, which could bring tremendous influence on the pandemic. In this study, we investigated 18 non-synonymous mutations in SARS-CoV-2 genome which incidence rates were all \u22651% as of July 15th, 2020, then modeled the mutated protein structures and compared them with the reference ones. The results showed that four types of mutations could cause dramatic changes in protein structures (RMSD \u22655.0 \u00c5), which were Q57H and G251V in open reading frames 3a (ORF3a), S194L and R203K/G204R in nucleocapsid (N). Next, we found that these mutations could affect the binding affinity of intraviral protein interactions. In addition, the hot spots within these docking complexes were altered, among which the mutation Q57H was involved in both Orf3a-Orf8 and Orf3a-S protein interactions. Besides, these mutations were widely distributed all over the world, and their occurrences fluctuated as time went on. Notably, the incidences of R203K/G204R in N and Q57H in Orf3a were both over 50% in some countries. Overall, our findings suggest that SARS-CoV-2 mutations can change viral protein structure, binding affinity and hot spots of the interface, thereby may have impacts on SARS-CoV-2 transmission, diagnosis and treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.08.15.241349","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.222836","pub_date":"2020-8-16","title":"Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine","abstract":"The Corona Virus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity against two currently circulating influenza A virus subtypes, an effect which was also observed upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract enveloped viruses, including SARS-CoV-2 and COVID 19.","version":"1.2","doi":"10.1101/2020.07.27.222836","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.252395","pub_date":"2020-8-16","title":"Susceptibility of swine cells and domestic pigs to SARS-CoV-2","abstract":"The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.","version":"1.1","doi":"10.1101/2020.08.15.252395","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.252353","pub_date":"2020-8-16","title":"High titers of multiple antibody isotypes against the SARS-CoV-2 spike receptor-binding domain and nucleoprotein associate with better neutralization","abstract":"Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Here, we determine the ability of sera from 101 recovered healthcare workers to neutralize both authentic SARS-CoV-2 and SARS-CoV-2 pseudotyped virus and address their antibody titers against SARS-CoV-2 nucleoprotein and spike receptor-binding domain. Interestingly, the majority of individuals have low neutralization capacity and only 6% of the healthcare workers showed high neutralizing titers against both authentic SARS-CoV-2 virus and the pseudotyped virus. We found the antibody response to SARS-CoV-2 infection generates antigen-specific isotypes as well as a diverse combination of antibody isotypes, with high titers of IgG, IgM and IgA against both antigens correlating with neutralization capacity. Importantly, we found that neutralization correlated with antibody titers as quantified by ELISA. This suggests that an ELISA assay can be used to determine seroneutralization potential. Altogether, our work provides a snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides evidence that possessing multiple antibody isotypes may play an important role in SARS-CoV-2 neutralization.","version":"1.1","doi":"10.1101/2020.08.15.252353","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.212563","pub_date":"2020-8-16","title":"In silico comparative genomics of SARS-CoV-2 to determine the source and diversity of the pathogen in Bangladesh","abstract":"The COVID19 pandemic caused by SARS-CoV-2 virus has severely affected most countries of the world including Bangladesh. We conducted comparative analysis of publicly available whole-genome sequences of 64 SARS-CoV-2 isolates in Bangladesh and 371 isolates from another 27 countries to predict possible transmission routes of COVID19 to Bangladesh and genomic variations among the viruses. Phylogenetic analysis indicated that the pathogen was imported in Bangladesh from multiple countries. The viruses found in the southern district of Chattogram were closely related to strains from Saudi Arabia whereas those in Dhaka were similar to that of United Kingdom and France. The 64 SARS-CoV-2 sequences from Bangladesh belonged to three clusters. Compared to the ancestral SARS-CoV-2 sequence reported from China, the isolates in Bangladesh had a total of 180 mutations in the coding region of the genome, and 110 of these were missense. Among these, 99 missense mutations (90%) were predicted to destabilize protein structures. Remarkably, a mutation that leads to an I300F change in the nsp2 protein and a mutation leading to D614G change in the spike protein were prevalent in SARS-CoV-2 genomic sequences, and might have influenced the epidemiological properties of the virus in Bangladesh.","version":"1.2","doi":"10.1101/2020.07.20.212563","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.251421","pub_date":"2020-8-16","title":"Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography","abstract":"The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5\u2032 terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2\u2032-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2\u2032-O Cap-1, where 2\u2032OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.","version":"1.2","doi":"10.1101/2020.08.14.251421","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167064","pub_date":"2020-8-16","title":"SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic \u03b2-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualized RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We found that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.","version":"1.2","doi":"10.1101/2020.06.23.167064","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.01.127019","pub_date":"2020-8-16","title":"INSIGHT: a population scale COVID-19 testing strategy combining point-of-care diagnosis with centralised high-throughput sequencing","abstract":"We present INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a barcoded isothermal NASBA reaction that combines point-of-care diagnosis with next generation sequencing, aiming to achieve population-scale COVID-19 testing. INSIGHT combines the advantages of near-patient with centralised testing. Stage 1 allows a quick decentralised readout for early isolation of pre-symptomatic or asymptomatic patients. The same reaction products can then be used in a highly multiplexed sequencing-based assay in Stage 2, confirming the near-patient testing results and facilitating centralised data collection. Based on experiments using commercially acquired human saliva with spiked-in viral RNA as input, the INSIGHT platform gives Stage 1 results within one to two hours, using either fluorescence detection or a lateral flow (dipstick) readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. INSIGHT Stage 2 can be performed by directly pooling and sequencing all post-amplification barcoded Stage 1 products from hundreds of thousands of samples with minimal sample preparation steps. The 95% limit of detection (LoD-95) for INSIGHT is estimated to be below 50 copies of viral RNA per 20 \u03bcl of reaction. Our two-stage testing strategy is suitable for further development into a rapid home-based and point-of-care assay, and is potentially scalable to the population level.","version":"1.2","doi":"10.1101/2020.06.01.127019","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.20175265","pub_date":"2020-08-16","title":"Assessment of physiological signs associated with COVID-19 measured using wearable devices","abstract":"<jats:p>\n                  Respiration rate, heart rate, and heart rate variability are some health metrics that are easily measured by consumer devices and which can potentially provide early signs of illness. Furthermore, mobile applications which accompany wearable devices can be used to collect relevant self-reported symptoms and demographic data. This makes consumer devices a valuable tool in the fight against the COVID-19 pandemic. We considered two approaches to assessing COVID-19 \u2013 a symptom-based approach, and a physiological signs based technique. Firstly, we trained a Logistic Regression classifier to predict the need for hospitalization of COVID-19 patients given the symptoms experienced, age, sex, and BMI. Secondly, we trained a neural network classifier to predict whether a person is sick on any specific day given respiration rate, heart rate, and heart rate variability data for that day and and for the four preceding days. Data on 1,181 subjects diagnosed with COVID-19 (active infection, PCR test) were collected from May 21 \u2013 July 14, 2020. 11.0% of COVID-19 subjects were asymptomatic, 47.2% of subjects recovered at home by themselves, 33.2% recovered at home with the help of someone else, 8.16% of subjects required hospitalization without ventilation support, and 0.448% required ventilation. Fever was present in 54.8% of subjects. Based on self-reported symptoms alone, we obtained an AUC of 0.77\n                  <jats:italic>\u00b1</jats:italic>\n                  0.05 for the prediction of the need for hospitalization. Based on physiological signs, we obtained an AUC of 0.77\n                  <jats:italic>\u00b1</jats:italic>\n                  0.03 for the prediction of illness on a specific day with 4 previous days of history. Respiration rate and heart rate are typically elevated by illness, while heart rate variability is decreased. Measuring these metrics can help in early diagnosis, and in monitoring the progress of the disease.\n                </jats:p>","version":null,"doi":"10.1101/2020.08.14.20175265","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.14.250258","pub_date":"2020-8-15","title":"Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 main protease by tafenoquine in vitro","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic, coronavirus disease 2019 (COVID-19), has taken a huge toll on human lives and the global economy. Therefore, effective treatments against this disease are urgently needed. Here, we established a fluorescence resonance energy transfer (FRET)-based high-throughput screening platform to screen compound libraries to identify drugs targeting the SARS-CoV-2 main protease (Mpro), in particular those which are FDA-approved, to be used immediately to treat patients with COVID-19. Mpro has been shown to be one of the most important drug targets among SARS-related coronaviruses as impairment of Mpro blocks processing of viral polyproteins which halts viral replication in host cells. Our findings indicate that the anti-malarial drug tafenoquine (TFQ) induces significant conformational change in SARS-CoV-2 Mpro and diminishes its protease activity. Specifically, TFQ reduces the \u03b1-helical content of Mpro, which converts it into an inactive form. Moreover, TFQ greatly inhibits SARS-CoV-2 infection in cell culture system. Hence, the current study provides a mechanistic insight into the mode of action of TFQ against SARS-CoV-2 Mpro. Moreover, the low clinical toxicity of TFQ and its strong antiviral activity against SARS-CoV-2 should warrant further testing in clinical trials.","version":"1.1","doi":"10.1101/2020.08.14.250258","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.15.252510","pub_date":"2020-8-15","title":"Rapid cost-effective viral genome sequencing by V-seq","abstract":"Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3\u2019 end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.","version":"1.1","doi":"10.1101/2020.08.15.252510","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.09.242867","pub_date":"2020-8-14","title":"A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential","abstract":"The outbreak of COVID-19 has emerged as a global pandemic. The unprecedented scale and severity call for rapid development of effective prophylactics or therapeutics. We here reported Nanobody (Nb) phage display libraries derived from four camels immunized with the SARS-CoV-2 spike receptor-binding domain (RBD), from which 381 Nbs were identified to recognize SARS-CoV-2-RBD. Furthermore, seven Nbs were shown to block interaction of human angiotensin converting enzyme 2 (ACE2) with SARS-CoV-2-RBD-variants, bat-SL-CoV-WIV1-RBD and SARS-CoV-1-RBD. Among the seven candidates, Nb11-59 exhibited the highest activity against authentic SARS-CoV-2 with ND50 of 0.55 \u03bcg/mL. Nb11-59 can be produced on a large-scale in Pichia pastoris, with 20 g/L titer and 99.36% purity. It also showed good stability profile, and nebulization did not impact its stability. Overall, Nb11-59 might be a promising prophylactic and therapeutic molecule against COVID-19, especially through inhalation delivery.","version":"1.2","doi":"10.1101/2020.08.09.242867","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.248872","pub_date":"2020-8-14","title":"SARS-CoV-2 manipulates the SR-B1-mediated HDL uptake pathway for its entry","abstract":"The recently emerged pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly, leading to a global COVID-19 pandemic. Binding of the viral spike protein (SARS-2-S) to cell surface receptor angiotensin-converting enzyme 2 (ACE2) mediates host cell infection. In the present study, we demonstrate that in addition to ACE2, the S1 subunit of SARS-2-S binds to HDL and that SARS-CoV-2 hijacks the SR-B1-mediated HDL uptake pathway to facilitate its entry. SR-B1 facilitates SARS-CoV-2 entry into permissive cells by augmenting virus attachment. MAb (monoclonal antibody)-mediated blocking of SARS-2-S-HDL binding and SR-B1 antagonists strongly inhibit HDL-enhanced SARS-CoV-2 infection. Notably, SR-B1 is co-expressed with ACE2 in human pulmonary and extrapulmonary tissues. These findings revealed a novel mechanism for SARS-CoV-2 entry and could provide a new target to treat SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.08.13.248872","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.251538","pub_date":"2020-8-14","title":"Differential methylation as a mediator of COVID-19 susceptibility","abstract":"The COVID-19 outbreak shows a huge variation in prevalence and mortality on geographical level but also within populations. The ACE2 gene, identified as the SARS-CoV2 receptor, has been shown to facilitate the viral invasion and people with higher ACE2 expression generally are more severely affected. As there is a lot of variability in ACE2 expression between individuals we hypothesized that differential DNA methylation profiles could be (one of) the confounding factors explaining this variability. Here we show that epigenetic profiling of host tissue, especially in the ACE2 promoter region and its homologue ACE1, may be important risk factors for COVID-19. Our results propose that variable methylation can explain (part of) the differential susceptibility, symptom severity and death rate for COVID-19. Our findings are a promising starting point to further evaluate the potential of ACE1/2 methylation and other candidates as a predictor for clinical outcome upon SARS-CoV2 infection.","version":"1.1","doi":"10.1101/2020.08.14.251538","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.071688","pub_date":"2020-8-14","title":"Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2","abstract":"The emergence of the COVID-19 pandemic caused by SARS-CoV-2 has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 human cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race, and pre-existing conditions of COVID-19 patients.","version":"1.2","doi":"10.1101/2020.05.01.071688","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.251496","pub_date":"2020-8-14","title":"Rational Design of SARS-CoV-2 Spike Glycoproteins To Increase Immunogenicity By T Cell Epitope Engineering","abstract":"The current COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of confirmed cases and thousands of deaths globally. Extensive efforts and progress have been made to develop effective and safe vaccines against COVID-19. A primary target of these vaccines is the SARS-CoV-2 spike (S) protein, and many studies utilized structural vaccinology techniques to either stabilize the protein or fix the receptor-binding domain at certain states. In this study, we extended an evolutionary protein design algorithm, EvoDesign, to create thousands of stable S protein variants without perturbing the surface conformation and B cell epitopes of the S protein. We then evaluated the mutated S protein candidates based on predicted MHC-II T cell promiscuous epitopes as well as the epitopes\u2019 similarity to human peptides. The presented strategy aims to improve the S protein\u2019s immunogenicity and antigenicity by inducing stronger CD4 T cell response while maintaining the protein\u2019s native structure and function. The top EvoDesign S protein candidate (Design-10705) recovered 31 out of 32 MHC-II T cell promiscuous epitopes in the native S protein, in which two epitopes were present in all seven human coronaviruses. This newly designed S protein also introduced nine new MHC-II T cell promiscuous epitopes and showed high structural similarity to its native conformation. The proposed structural vaccinology method provides an avenue to rationally design the antigen\u2019s structure with increased immunogenicity, which could be applied to the rational design of new COVID-19 vaccine candidates.","version":"1.1","doi":"10.1101/2020.08.14.251496","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.249433","pub_date":"2020-8-14","title":"Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: rapid induction, prolonged contraction, and formation of functional memory","abstract":"CD8+ T cells are critical for the elimination and long-lasting protection of many viral infections, but their role in the current SARS-CoV-2 pandemic is unclear. Emerging data indicates that SARS-CoV-2-specific CD8+ T cells are detectable in the majority of individuals recovering from SARS-CoV-2 infection. However, optimal virus-specific epitopes, the role of pre-existing heterologous immunity as well as their kinetics and differentiation program during disease control have not been defined in detail. Here, we show that both pre-existing and newly induced SARS-CoV-2-specific CD8+ T-cell responses are potentially important determinants of immune protection in mild SARS-CoV-2 infection. In particular, our results can be summarized as follows: First, immunodominant SARS-CoV-2-specific CD8+ T-cell epitopes are targeted in the majority of individuals with convalescent SARS-CoV-2 infection. Second, MHC class I tetramer analyses revealed the emergence of phenotypically diverse and functionally competent pre-existing and newly induced SARS-CoV-2-specific memory CD8+ T cells that showed similar characteristics compared to influenza-specific CD8+ T cells. Third, SARS-CoV-2-specific CD8+ T-cell responses are more robustly detectable than antibodies against the SARS-CoV-2-spike protein. This was confirmed in a longitudinal analysis of acute-resolving infection that demonstrated rapid induction of the SARS-CoV-2-specific CD8+ T cells within a week followed by a prolonged contraction phase that outlasted the waning humoral immune response indicating that CD8+ T-cell responses might serve as a more precise correlate of antiviral immunity than antibody measurements after convalescence. Collectively, these data provide new insights into the fine specificity, heterogeneity, and dynamics of SARS-CoV-2-specific memory CD8+ T cells, potentially informing the rational development of a protective vaccine against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.13.249433","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.250480","pub_date":"2020-8-14","title":"Discovery of Clioquinol and Analogues as Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, ACE2 and ACE2 - Spike Protein Interaction In Vitro","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease 2019 (COVID-19), has emerged as an ongoing global pandemic. Presently, there are no clinically approved vaccines nor drugs for COVID-19. Hence, there is an urgent need to accelerate the development of effective antivirals. Here in, we discovered Clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)), a FDA approved drug and two of its analogues (7-bromo-5-chloro-8-hydroxyquinoline (CLBQ14); and 5, 7-Dichloro-8-hydroxyquinoline (CLCQ)) as potent inhibitors of SARS-CoV-2 infection induced cytopathic effect in vitro. In addition, all three compounds showed potent anti-exopeptidase activity against recombinant human angiotensin converting enzyme 2 (rhACE2) and inhibited the binding of rhACE2 with SARS-CoV-2 Spike (RBD) protein. CLQ displayed the highest potency in the low micromolar range, with its antiviral activity showing strong correlation with inhibition of rhACE2 and rhACE2-RBD interaction. Altogether, our findings provide a new mode of action and molecular target for CLQ and validates this pharmacophore as a promising lead series for clinical development of potential therapeutics for COVID-19.","version":"1.1","doi":"10.1101/2020.08.14.250480","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.250928","pub_date":"2020-8-14","title":"SARS-CoV-2 Infection And Longitudinal Fecal Screening In Malayan Tigers (Panthera tigris jacksoni), Amur Tigers (Panthera tigris altaica), And African Lions (Panthera leo krugeri) At The Bronx Zoo, New York, USA","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019-2020. In March 2020 New York City became the USA epicenter for the pandemic. On March 27, 2020 a Malayan tiger (Panthera tigris jacksoni) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers (P. t. altaica) in the same building and three lions (Panthera leo krugeri) in a separate building also became ill. The index case was immobilized, and physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed peribronchial cuffing with bronchiectasis, and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1-5 days for most animals, though persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2, and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to animals in the USA, and is the first report of SARS-CoV-2 in non-domestic felids.","version":"1.1","doi":"10.1101/2020.08.14.250928","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.251090","pub_date":"2020-8-14","title":"Methylene Blue has a potent antiviral activity against SARS-CoV-2 in the absence of UV-activation in vitro","abstract":"Methylene blue is an FDA and EMA approved drug with an excellent safety profile. It displays broad-spectrum virucidal activity in the presence of UV light and has been shown to be effective in inactivating various viruses in blood products prior to transfusions. In addition, its use has been validated for methemoglobinemia and malaria treatment. Here we show the virucidal activity of methylene blue at low micromolar concentrations and in the absence of UV activation against SARS-CoV2.","version":"1.1","doi":"10.1101/2020.08.14.251090","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.234716","pub_date":"2020-8-14","title":"Structure of SARS-CoV-2 2\u2032-O-methyltransferase heterodimer with RNA Cap analog and sulfates bound reveals new strategies for structure-based inhibitor design","abstract":"There are currently no antiviral therapies specific against SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an X-ray crystallographic study of the nsp16/nsp10 2\u2032-O-methyltransferase complex that methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. Heterodimer structures are determined with the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH) or the SAH analog sinefungin (SFG). Furthermore, structures of nsp16/nsp10 with the methylated Cap-0 analog (m7GpppA) and SAM or SAH bound were obtained. Comparative analysis revealed flexible loops in open and closed conformations at the m7GpppA binding pocket. Bound sulfates in several structures suggested the location of the phosphates in the ribonucleotide binding groove. Additional nucleotide binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for development of antiviral inhibitors.","version":"1.2","doi":"10.1101/2020.08.03.234716","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.14.240093","pub_date":"2020-8-14","title":"Tissue-resident memory CD8 T-cell responses elicited by a single injection of a multi-target COVID-19 vaccine","abstract":"The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) which enters the body principally through the nasal and larynx mucosa and progress to the lungs through the respiratory tract. SARS-CoV-2 replicates efficiently in respiratory epithelial cells motivating the development of alternative and rapidly scalable vaccine inducing mucosal protective and long-lasting immunity. We have previously developed an immunologically optimized multi-neoepitopes-based peptide vaccine platform which has already demonstrated tolerance and efficacy in hundreds of lung cancer patients. Here, we present a multi-target CD8 T cell peptide COVID-19 vaccine design targeting several structural (S, M, N) and non-structural (NSPs) SARS-CoV-2 proteins with selected epitopes in conserved regions of the SARS-CoV-2 genome. We observed that a single subcutaneous injection of a serie of epitopes induces a robust immunogenicity in-vivo as measured by IFN\u03b3 ELIspot. Upon tetramer characterization we found that this serie of epitopes induces a strong proportion of virus-specific CD8 T cells expressing CD103, CD44, CXCR3 and CD49a, the specific phenotype of tissue-resident memory T lymphocytes (Trm). Finally, we observed broad cellular responses, as characterized by IFN\u03b3 production, upon restimulation with structural and non-structural protein-derived epitopes using blood T cells isolated from convalescent asymptomatic, moderate and severe COVID-19 patients. These data provide insights for further development of a second generation of COVID-19 vaccine focused on inducing lasting Th1-biased memory CD8 T cell sentinels protection using immunodominant epitopes naturally observed after SARS-CoV-2 infection resolution. Humoral and cellular adaptive immunity are different and complementary immune defenses engaged by the body to clear viral infection. While neutralizing antibodies have the capacity to block virus binding to its entry receptor expressed on human cells, memory T lymphocytes have the capacity to eliminate infected cells and are required for viral clearance. However, viruses evolve quickly, and their antigens are prone to mutations to avoid recognition by the antibodies (phenomenon named \u2018antigenic drift\u2019). This limitation of the antibody-mediated immunity could be addressed by the T-cell mediated immunity, which is able to recognize conserved viral peptides from any viral proteins presented by virus-infected cells. Thus, by targeting several proteins and conserved regions on the genome of a virus, T-cell epitope-based vaccines are less subjected to mutations and may work effectively on different strains of the virus. We designed a multi-target T cell-based vaccine containing epitope regions optimized for CD8+ T cell stimulation that would drive long-lasting cellular immunity with high specificity, avoiding undesired effects such as antibody-dependent enhancement (ADE) and antibody-induced macrophages hyperinflammation that could be observed in subjects with severe COVID-19. Our in-vivo results showed that a single injection of selected CD8 T cell epitopes induces memory viral-specific T-cell responses with a phenotype of tissue-resident memory T cells (Trm). Trm has attracted a growing interest for developing vaccination strategies since they act as immune sentinels in barrier tissue such as the respiratory tract and the lung. Because of their localization in tissues, they are able to immediately recognize infected cells and, because of their memory phenotypes, they rapidly respond to viral infection by orchestrating local protective immune responses to eliminate pathogens. Lastly, such multiepitope-based vaccination platform uses robust and well-validated synthetic peptide production technologies that can be rapidly manufactured in a distributed manner.","version":"1.1","doi":"10.1101/2020.08.14.240093","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.250076","pub_date":"2020-8-14","title":"In Silico Design of siRNAs Targeting Existing and Future Respiratory Viruses with VirusSi","abstract":"The COVID-19 pandemic has exposed global inadequacies in therapeutic options against both the COVID-19-causing SARS-CoV-2 virus and other newly emerged respiratory viruses. In this study, we present the VirusSi computational pipeline, which facilitates the rational design of siRNAs to target existing and future respiratory viruses. Mode A of VirusSi designs siRNAs against an existing virus, incorporating considerations on siRNA properties, off-target effects, viral RNA structure and viral mutations. It designs multiple siRNAs out of which the top candidate targets >99% of SARS-CoV-2 strains, and the combination of the top four siRNAs is predicted to target all SARS-CoV-2 strains. Additionally, we develop Greedy Algorithm with Redundancy (GAR) and Similarity-weighted Greedy Algorithm with Redundancy (SGAR) to support the Mode B of VirusSi, which pre-designs siRNAs against future emerging viruses based on existing viral sequences. Time-simulations using known coronavirus genomes as early as 10 years prior to the COVID-19 outbreak show that at least three SARS-CoV-2-targeting siRNAs are among the top 30 pre-designed siRNAs. Before-the-outbreak pre-design is also possible against the MERS-CoV virus and the 2009-H1N1 swine flu virus. Our data support the feasibility of pre-designing anti-viral siRNA therapeutics prior to viral outbreaks. We propose the development of a collection of pre-designed, safety-tested, and off-the-shelf siRNAs that could accelerate responses toward future viral diseases.","version":"1.1","doi":"10.1101/2020.08.13.250076","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.233064","pub_date":"2020-8-14","title":"Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery","abstract":"Development of accurate disease models and discovery of immune-modulating drugs is challenged by the immune system\u2019s highly interconnected and context-dependent nature. Here we apply deep-learning-driven analysis of cellular morphology to develop a scalable \u201cphenomics\u201d platform and demonstrate its ability to identify dose-dependent, high-dimensional relationships among and between immunomodulators, toxins, pathogens, genetic perturbations, and small and large molecules at scale. High-throughput screening on this platform demonstrates rapid identification and triage of hits for TGF-\u03b2- and TNF-\u03b1-driven phenotypes. We deploy the platform to develop phenotypic models of active SARS-CoV-2 infection and of COVID-19-associated cytokine storm, surfacing compounds with demonstrated clinical benefit and identifying several new candidates for drug repurposing. The presented library of images, deep learning features, and compound screening data from immune profiling and COVID-19 screens serves as a deep resource for immune biology and cellular-model drug discovery with immediate impact on the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.08.02.233064","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.164665","pub_date":"2020-8-14","title":"Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus","abstract":"The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating. The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. Results showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD =(7.31\u00b10.62)e\u22127 M and (4.82\u00b10.87)e\u22127 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.","version":"1.3","doi":"10.1101/2020.06.22.164665","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.249649","pub_date":"2020-8-14","title":"D-graph clusters flaviviruses and \u03b2-coronaviruses according to their hosts, disease type and human cell receptors","abstract":"There is a need for rapid and easy to use, alignment free methods to cluster large groups of protein sequence data. Commonly used phylogenetic trees based on alignments can be used to visualize only a limited number of protein sequences. DGraph, introduced here, is a dynamic programming application developed to generate 2D-maps based on similarity scores for sequences. The program automatically calculates and graphically displays property distance (PD) scores based on physico-chemical property (PCP) similarities from an unaligned list of FASTA files. Such \u201cPD-graphs\u201d show the interrelatedness of the sequences, whereby clusters can reveal deeper connectivities. PD-Graphs generated for flavivirus (FV), enterovirus (EV), and coronavirus (CoV) sequences from complete polyproteins or individual proteins are consistent with biological data on vector types, hosts, cellular receptors and disease phenotypes. PD-graphs separate the tick- from the mosquito-borne FV, clusters viruses that infect bats, camels, seabirds and humans separately and the clusters correlate with disease phenotype. The PD method segregates the \u03b2-CoV spike proteins of SARS, SARS-CoV-2, and MERS sequences from other human pathogenic CoV, with clustering consistent with cellular receptor usage. The graphs also suggest evolutionary relationships that may be difficult to determine with conventional bootstrapping methods that require postulating an ancestral sequence. DGraph is written in Java, compatible with the Java 5 runtime or newer. Source code and executable is available from the GitHub website (https://github.com/bjmnbraun/DGraph/releases). Documentation for installation and use of the software is available from the Readme.md file at (https://github.com/bjmnbraun/DGraph). bjmnbraun@gmail.com or webraun@utmb.edu Supplementary information Table S1 and Fig. S1 are online available.","version":"1.1","doi":"10.1101/2020.08.13.249649","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.148411","pub_date":"2020-8-13","title":"Prediction of non-canonical routes for SARS-CoV-2 infection in human placenta cells","abstract":"The SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The data available about COVID-19 during pregnancy have demonstrated placental infection; however, the intrauterine transmission of SARS-CoV-2 is still debated. Intriguingly, while canonical SARS-CoV-2 cell entry mediators are expressed at low levels in placental cells, the receptors for viruses that cause congenital infections such as the cytomegalovirus and Zika virus are highly expressed in these cells. Here we analyzed the transcriptional profile (microarray and single-cell RNA-Seq) of proteins potentially interacting with coronaviruses to identify non-canonical mediators of SARS-CoV-2 infection and replication in the placenta. We show that, despite low levels of the canonical cell entry mediators ACE2 and TMPRSS2, cells of the syncytiotrophoblast, villous cytotrophoblast, and extravillous trophoblast co-express high levels of the potential non-canonical cell-entry mediators DPP4 and CTSL. We also found changes in the expression of DAAM1 and PAICS genes during pregnancy, which are translated into proteins also predicted to interact with coronaviruses proteins. These results provide new insight into the interaction between SARS-CoV-2 and host proteins that may act as non-canonical routes for SARS-CoV-2 infection and replication in the placenta cells.","version":"1.2","doi":"10.1101/2020.06.12.148411","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.056127","pub_date":"2020-8-13","title":"Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract","abstract":"Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19. Previous studies of ACE2 mRNA transcript abundance in the human respiratory tract have suggested a possible association between ACE2 expression and age, sex, and the presence of comorbidities. However, these studies have provided conflicting results, as well as a lack of protein validation. Previous ACE2 protein studies have been limited by a paucity of lung tissue samples and reports that have produced contradictory results. Using a combination of single-cell RNA sequencing and immunohistochemistry, we describe ACE2 expression in the human respiratory tract. Staining protocols were optimized and validated to show consistent apical localization and avoid non-specific staining. We show ACE2 protein is found in subsets of airway cells and is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development for COVID-19, respectively. We show age, sex, and comorbidities do not increase ACE2 protein expression in the human respiratory tract. ACE2 protein abundance does not correlate with risk factors for severe clinical outcomes, but in some cases showed an inversed relationship. Features driving COVID-19 susceptibility and severity are complex, our data suggests factors other than ACE2 protein abundance as important determinants of clinical outcomes.","version":"1.3","doi":"10.1101/2020.04.22.056127","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.248211","pub_date":"2020-8-13","title":"Characterisation of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification","abstract":"The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3\u2019-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.08.13.248211","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.249953","pub_date":"2020-8-13","title":"Immunoreactive peptide maps of SARS-CoV-2 and other human coronaviruses","abstract":"Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity to the human coronaviruses (HCoV) SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 and other HCoV peptides that can be used to detect asymptomatic, mild and, severe SARS-CoV-2 infections, and may enable the discovery of biomarkers for immunity following infection or vaccination.","version":"1.1","doi":"10.1101/2020.08.13.249953","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.12.248732","pub_date":"2020-8-13","title":"Mutations of SARS-CoV-2 nsp14 exhibit strong association with increased genome-wide mutation load","abstract":"SARS-CoV-2 is a betacoronavirus responsible for human cases of COVID-19, a pandemic with global impact that first emerged in late 2019. Since then, the viral genome has shown considerable variance as the disease spread across the world, in part due to the zoonotic origins of the virus and the human host adaptation process. As a virus with an RNA genome that codes for its own genomic replication proteins, mutations in these proteins can significantly impact the variance rate of the genome, affecting both the survival and infection rate of the virus, and attempts at combating the disease. In this study, we analyzed the mutation densities of viral isolates carrying frequently observed mutations for four proteins in the RNA synthesis complex over time in comparison to wildtype isolates. Our observations suggest mutations in nsp14, an error-correcting exonuclease protein, have the strongest association with increased mutation load in both regions without selective pressure and across the genome, compared to nsp7, 8, and 12, which form the core polymerase complex. We propose nsp14 as a priority research target for understanding genomic variance rate in SARS-CoV-2 isolates, and nsp14 mutations as potential predictors for high mutability strains.","version":"1.1","doi":"10.1101/2020.08.12.248732","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.249177","pub_date":"2020-8-13","title":"The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer","abstract":"Large trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is \u2018locked\u2019 into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the \u2018locked\u2019 form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the \u2018locked\u2019 form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies. SARS-CoV-2 Spike can adopt a \u2018locked\u2019 conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting state This \u2018locked\u2019 conformation is compact and stable, braced by lipid bound within a potentially druggable pocket Key neutralization epitopes are shielded in the \u2018locked\u2019 form Loss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry","version":"1.1","doi":"10.1101/2020.08.13.249177","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.13.248575","pub_date":"2020-8-13","title":"Metaviromic identification of genetic hotspots of coronavirus pathogenicity using machine learning","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has become a major threat across the globe. Here, we developed machine learning approaches to identify key pathogenic regions in coronavirus genomes. We trained and evaluated 7,562,625 models on 3,665 genomes including SARS-CoV-2, MERS-CoV, SARS-CoV and other coronaviruses of human and animal origins to return quantitative and biologically interpretable signatures at nucleotide and amino acid resolutions. We identified hotspots across the SARS-CoV-2 genome including previously unappreciated features in spike, RdRp and other proteins. Finally, we integrated pathogenicity genomic profiles with B cell and T cell epitope predictions for enrichment of sequence targets to help guide vaccine development. These results provide a systematic map of predicted pathogenicity in SARS-CoV-2 that incorporates sequence, structural and immunological features, providing an unbiased collection of genetic elements for functional studies. This metavirome-based framework can also be applied for rapid characterization of new coronavirus strains or emerging pathogenic viruses.","version":"1.1","doi":"10.1101/2020.08.13.248575","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.247320","pub_date":"2020-8-12","title":"Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells","abstract":"SARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and \u2018off-the-shelf\u2019 CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particles in vitro. Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, \u2018off-the-shelf\u2019 CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.","version":"1.1","doi":"10.1101/2020.08.11.247320","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.12.247825","pub_date":"2020-8-12","title":"TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-\u03b3 (IFN-\u03b3)-induced ACE2 expression through a mechanism involving upregulating IFN-\u03b3 receptor 2 (IFNGR2) in both A549 and PAEpiCs. Importantly, the upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells but not PAEpiCs. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.","version":"1.1","doi":"10.1101/2020.08.12.247825","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.192104","pub_date":"2020-8-12","title":"Molecular architecture of the SARS-CoV-2 virus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins and the complexes of the spike (S) proteins with the cellular receptor ACE2 or neutralizing antibodies, detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in both pre- and postfusion conformations were determined to average resolutions of 8.7-11 \u00c5. Compositions of the N-linked glycans from the native spikes were analyzed by mass-spectrometry, which revealed highly similar overall processing states of the native glycans to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNP) and its higher-order assemblies were revealed. Overall, these characterizations have revealed the architecture of the SARS-CoV-2 virus in unprecedented detail, and shed lights on how the virus packs its \u223c30 kb long single-segmented RNA in the \u223c80 nm diameter lumen.","version":"1.2","doi":"10.1101/2020.07.08.192104","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.12.247338","pub_date":"2020-8-12","title":"Membrane Nanoparticles Derived from ACE2-rich Cells Block SARS-CoV-2 Infection","abstract":"The ongoing COVID-19 epidemic worldwide necessitates the development of novel effective agents against SARS-CoV-2. ACE2 is the main receptor of SARS-CoV-2 S1 protein and mediates viral entry into host cells. Herein, the membrane nanoparticles prepared from ACE2-rich cells are discovered with potent capacity to block SARS-CoV-2 infection. The membrane of human embryonic kidney-239T cell highly expressing ACE2 is screened to prepare nanoparticles. The nanomaterial termed HEK-293T-hACE2 NPs contains 265.1 ng mg\u22121 of ACE2 on the surface and acts as a bait to trap SARS-CoV-2 S1 in a dose-dependent manner, resulting in reduced recruitment of the viral ligand to host cells. Interestingly, SARS-CoV-2 S1 can translocate to the cytoplasm and affect the cell metabolism, which is also inhibited by HEK-293T-hACE2 NPs. Further studies reveal that HEK-293T-hACE2 NPs can efficiently suppress SARS-CoV-2 S pseudovirions entry into human proximal tubular cells and block viral infection with a low half maximal inhibitory concentration. Additionally, this biocompatible membrane nanomaterial is sufficient to block the adherence of SARS-CoV-2 D614G-S1 mutant to sensitive cells. Our study demonstrates a easy-to-acheive memrbane nano-antagonist for curbing SARS-CoV-2, which enriches the existing antiviral arsenal and provides new possibilities to treat COVID-19.\n\nGraphical Table of Contents\n\n Graphical Table of Contents","version":"1.1","doi":"10.1101/2020.08.12.247338","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.12.247767","pub_date":"2020-8-12","title":"Suppression of MDA5-mediated antiviral immune responses by NSP8 of SARS-CoV-2","abstract":"Melanoma differentiation-associated gene-5 (MDA5) acts as a cytoplasmic RNA sensor to detect viral dsRNA and mediates type I interferon (IFN) signaling and antiviral innate immune responses to infection by RNA viruses. Upon recognition of viral dsRNA, MDA5 is activated with K63-linked polyubiquitination and then triggers the recruitment of MAVS and activation of TBK1 and IKK, subsequently leading to IRF3 and NF-\u03baB phosphorylation. Great numbers of symptomatic and severe infections of SARS-CoV-2 are spreading worldwide, and the poor efficacy of treatment with type I interferon and antiviral agents indicates that SARS-CoV-2 escapes from antiviral immune responses via an unknown mechanism. Here, we report that SARS-CoV-2 nonstructural protein 8 (NSP8) acts as an innate immune suppressor and inhibits type I IFN signaling to promote infection of RNA viruses. It downregulates the expression of type I IFNs, IFN-stimulated genes and proinflammatory cytokines by binding to MDA5 and impairing its K63-linked polyubiquitination. Our findings reveal that NSP8 mediates innate immune evasion during SARS-CoV-2 infection and may serve as a potential target for future therapeutics for SARS-CoV-2 infectious diseases. The large-scale spread of COVID-19 is causing mass casualties worldwide, and the failure of antiviral immune treatment suggests immune evasion. It has been reported that several nonstructural proteins of severe coronaviruses suppress antiviral immune responses; however, the immune suppression mechanism of SARS-CoV-2 remains unknown. Here, we revealed that NSP8 protein of SARS-CoV-2 directly blocks the activation of the cytosolic viral dsRNA sensor MDA5 and significantly downregulates antiviral immune responses. Our study contributes to our understanding of the direct immune evasion mechanism of SARS-CoV-2 by showing that NSP8 suppresses the most upstream sensor of innate immune responses involved in the recognition of viral dsRNA.","version":"1.1","doi":"10.1101/2020.08.12.247767","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.12.247940","pub_date":"2020-8-12","title":"Coronacept \u2013 a potent immunoadhesin against SARS-CoV-2","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Computational analysis of mammalian ACE2 orthologues suggests various residues at the interface with the viral receptor binding domain that could facilitate tighter interaction compared to the human-ACE2. Introducing several mutations to the human-ACE2 resulted with significantly augmented affinity to the viral spike complex. This modified human-ACE2 fused to an Fc portion of an antibody makes a potent immunoadhesin that effectively targets SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.12.247940","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.247395","pub_date":"2020-8-12","title":"Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2","abstract":"A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.","version":"1.1","doi":"10.1101/2020.08.11.247395","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.207019","pub_date":"2020-8-11","title":"Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening","abstract":"The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emphasized the urgency to develop effective therapeutics. Drug repurposing screening is regarded as one of the most practical and rapid approaches for the discovery of such therapeutics. The 3C like protease (3CLpro), or main protease (Mpro) of SARS-CoV-2 is a valid drug target as it is a specific viral enzyme and plays an essential role in viral replication. We performed a quantitative high throughput screening (qHTS) of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds using a SARS-CoV-2 3CLpro assay. Twenty-three small molecule inhibitors of SARS-CoV-2 3CLpro have been identified with IC50s ranging from 0.26 to 28.85 \u03bcM. Walrycin B (IC50 = 0.26 \u00b5M), Hydroxocobalamin (IC50 = 3.29 \u00b5M), Suramin sodium (IC50 = 6.5 \u00b5M), Z-DEVD-FMK (IC50 = 6.81 \u00b5M), LLL-12 (IC50 = 9.84 \u00b5M), and Z-FA-FMK (IC50 = 11.39 \u00b5M) are the most potent 3CLpro inhibitors. The activities of anti-SARS-CoV-2 viral infection was confirmed in 7 of 23 compounds using a SARS-CoV-2 cytopathic effect assay. The results demonstrated a set of SARS-CoV-2 3CLpro inhibitors that may have potential for further clinical evaluation as part of drug combination therapies to treating COVID-19 patients, and as starting points for chemistry optimization for new drug development.","version":"1.2","doi":"10.1101/2020.07.17.207019","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.242834","pub_date":"2020-8-11","title":"AT-527 is a potent in vitro replication inhibitor of SARS-CoV-2, the virus responsible for the COVID-19 pandemic","abstract":"AT-527, an orally administered double prodrug of a guanosine nucleotide analog, has been shown previously to be highly efficacious and well tolerated in HCV-infected subjects. Herein we report the potent in vitro activity of AT-511, the free base form of AT-527, against several coronaviruses, including SARS-CoV-2, the causative agent of COVID-19. In normal human airway epithelial (HAE) cell preparations, the average concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC90) was 0.5 \u00b5M, very similar to the EC90 for AT-511 against HCoV-229E, HCoV-OC43 and SARS-CoV in Huh-7 cells. No cytotoxicity was observed for AT-511 in any of the antiviral assays up to the highest concentration tested (100 \u00b5M). Surprisingly, AT-511 was 30-fold less active against MERS-CoV. This differential activity may provide a clue to the apparent unique mechanism of action of the guanosine triphosphate analog formed from AT-527.","version":"1.2","doi":"10.1101/2020.08.11.242834","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.245704","pub_date":"2020-8-11","title":"Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19","abstract":"The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID50) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 \u03bcg of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease.","version":"1.1","doi":"10.1101/2020.08.11.245704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.157933","pub_date":"2020-8-11","title":"\u201cAcute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques\u201d","abstract":"SARS-CoV-2 induces a wide range of disease severity ranging from asymptomatic infection, to a life-threating illness, particularly in the elderly and persons with comorbid conditions. Among those persons with serious COVID-19 disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19 induced ARDS and evaluate therapeutic strategies. Here we report ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that demonstrated pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. We found dramatic increases in circulating cytokines in three of four infected, aged AGMs but not in infected RMs. All of the AGMs showed increased levels of plasma IL-6 compared to baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2 infection. Together, our results show that both RM and AGM are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations including ARDS.","version":"1.2","doi":"10.1101/2020.06.18.157933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.244863","pub_date":"2020-8-11","title":"Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15","abstract":"New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 \u00c5 resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.","version":"1.1","doi":"10.1101/2020.08.11.244863","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.245290","pub_date":"2020-8-11","title":"The selection of reference genome and the search for the origin of SARS-CoV-2","abstract":"The pandemic caused by SARS-CoV-2 has a great impact on the whole world. In a theory of the origin of SARS-CoV-2, pangolins were considered a potential intermediate host. To assemble the coronavirus found in pangolins, SARS-CoV-2 were used a reference genome in most of studies, assuming that pangolins CoV and SARS-CoV-2 are the closest neighbors in the evolution. However, this assumption may not be true. We investigated how the selection of reference genome affect the resulting CoV genome assembly. We explored various representative CoV as reference genome, and found significant differences in the resulting assemblies. The assembly obtained using RaTG13 as reference showed better statistics in total length and N50 than the assembly guided by SARS-CoV-2, indicating that RaTG13 maybe a better reference for assembling CoV in pangolin or other potential intermediate hosts.","version":"1.1","doi":"10.1101/2020.08.10.245290","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.246678","pub_date":"2020-8-11","title":"Opposing activities of IFITM proteins in SARS-CoV-2 infection","abstract":"Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. Here we show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by expression of human IFITM1, IFITM2, and IFITM3, using both gain- and loss-of-function approaches. Mechanistically, restriction of SARS-CoV-2 occurred independently of IFITM3 S-palmitoylation sites, indicating a restrictive capacity that is distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the human IFITM3 endocytosis-promoting Yxx\u03a6 motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which reportedly increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into strong enhancers of infection. In sum, these data uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal virus restriction. Indeed, the net effect of IFITM3 on SARS-CoV-2 infections may be a result of these opposing activities, suggesting that shifts in the balance of these activities could be coopted by viruses to escape this important first line innate defense mechanism.","version":"1.1","doi":"10.1101/2020.08.11.246678","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.245100","pub_date":"2020-8-11","title":"Ambroxol and Ciprofloxacin Show Activity Against SARS-CoV2 in Vero E6 Cells at Clinically-Relevant Concentrations","abstract":"We studied the activity of a range of weakly basic and moderately lipophilic drugs against SARS CoV2 in Vero E6 cells, using Vero E6 survival, qPCR of viral genome and plaque forming assays. No clear relationship between their weakly basic and hydrophobic nature upon their activity was observed. However, the approved drugs ambroxol and ciprofloxacin showed potent activity at concentrations that are clinically relevant and within their known safety profiles, and so may provide potentially useful agents for preclinical and clinical studies in COVID-19.","version":"1.1","doi":"10.1101/2020.08.11.245100","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.246314","pub_date":"2020-8-11","title":"K18-hACE2 mice develop respiratory disease resembling severe COVID-19","abstract":"SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Taken together, this suggests that this mouse model can be useful for studies of pathogenesis and medical countermeasure development. The disease manifestation of COVID-19 in humans range from asymptomatic to severe. While several mild to moderate disease models have been developed, there is still a need for animal models that recapitulate the severe and fatal progression observed in a subset of patients. Here, we show that humanized transgenic mice developed dose-dependent disease when inoculated with SARS-CoV-2, the etiological agent of COVID-19. The mice developed upper and lower respiratory tract infection, with virus replication also in the brain after day 3 post inoculation. The pathological and immunological diseases manifestation observed in these mice bears resemblance to human COVID-19, suggesting increased usefulness of this model for elucidating COVID-19 pathogenesis further and testing of countermeasures, both of which are urgently needed.","version":"1.1","doi":"10.1101/2020.08.11.246314","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.245993","pub_date":"2020-8-11","title":"Common low complexity regions for SARS-CoV-2 and human proteomes as potential multidirectional risk factor in vaccine development","abstract":"The rapid spread of the COVID-19 demands immediate response from the scientific communities. Appropriate countermeasures mean thoughtful and educated choice of viral targets (epitopes). There are several articles that discuss such choices in the SARS-CoV-2 proteome, other focus on phylogenetic traits and history of the Coronaviridae genome/proteome. However none consider viral protein low complexity regions (LCRs). Recently we created the first methods that are able to compare such fragments. We show that five low complexity regions (LCRs) in three proteins (nsp3, S and N) encoded by the SARS-CoV-2 genome are highly similar to regions from human proteome. As many as 21 predicted T-cell epitopes and 27 predicted B-cell epitopes overlap with the five SARS-CoV-2 LCRs similar to human proteins. Interestingly, replication proteins encoded in the central part of viral RNA are devoid of LCRs. Similarity of SARS-CoV-2 LCRs to human proteins may have implications on the ability of the virus to counteract immune defenses. The vaccine targeted LCRs may potentially be ineffective or alternatively lead to autoimmune diseases development. These findings are crucial to the process of selection of new epitopes for drugs or vaccines which should omit such regions. The outbreak of the COVID-19 disease affects humans all over the globe. More and more people get sick and many die because of the deadly SARS-CoV-2 virus. The whole machinery of this pathogen is enclosed in a short sequence of nucleotides, building blocks for both RNA and DNA strands. This RNA virus encodes less than 30 protein sequences that change the fate of our societies. Its proteins are composed of 20 amino acids (building bricks) that are usually used quite freely by proteins. However, there are fragments where only one or a few amino acids are used. We name those low complexity regions (LCRs). We invented the first programmes able to compare such LCRs. Using this new methodology we were able to show similarity of some viral proteins to human ones. This discovery has a serious implication when designing vaccines or drugs. It means that companies should not use these very LCRs as targets because it may trigger an autoimmune disease. On the other hand this specific similarity may suggest some kind of disguise of viral proteins into the machinery of human cells.","version":"1.1","doi":"10.1101/2020.08.11.245993","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.232520","pub_date":"2020-8-11","title":"A Comprehensive Classification of Coronaviruses and Inferred Cross-Host Transmissions","abstract":"In this work, we present a unified and robust classification scheme for coronaviruses based on concatenated protein clusters. This subsequently allowed us to infer the apparent \u201chorizontal gene transfer\u201d events via reconciliation with the corresponding gene trees, which we argue can serve as a marker for cross-host transmissions. The cases of SARS-CoV, MERS-CoV, and SARS-CoV-2 are discussed. Our study provides a possible technical route to understand how coronaviruses evolve and are transmitted to humans.","version":"1.1","doi":"10.1101/2020.08.11.232520","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.11.245696","pub_date":"2020-8-11","title":"A 3.4-\u00c5 cryo-EM structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles","abstract":"Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV and SARS-CoV-2. Here we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-\u00c5 resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a 3-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other amino acid positions as well as differences in glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies, or drugs, in the native state of the coronavirus particles.","version":"1.1","doi":"10.1101/2020.08.11.245696","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.244624","pub_date":"2020-8-11","title":"ViruSurf: an integrated database to investigate viral sequences","abstract":"ViruSurf, available at http://gmql.eu/virusurf/, is a large public database of viral sequences and integrated and curated metadata from heterogeneous sources (GenBank, COG-UK and NMDC); it also exposes computed nucleotide and amino acid variants, called from original sequences. A GISAID-specific ViruSurf database, available at http://gmql.eu/virusurf_gisaid/, offers a subset of these functionalities. Given the current pandemic outbreak, SARS-CoV-2 data are collected from the four sources; but ViruSurf contains other virus species harmful to humans, including SARS-CoV, MERS-CoV, Ebola, and Dengue. The database is centered on sequences, described from their biological, technological, and organizational dimensions. In addition, the analytical dimension characterizes the sequence in terms of its annotations and variants. The web interface enables expressing complex search queries in a simple way; arbitrary search queries can freely combine conditions on attributes from the four dimensions, extracting the resulting sequences. Several example queries on the database confirm and possibly improve results from recent research papers; results can be recomputed over time and upon selected populations. Effective search over large and curated sequence data may enable faster responses to future threats that could arise from new viruses.","version":"1.1","doi":"10.1101/2020.08.10.244624","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.09.243246","pub_date":"2020-8-10","title":"Discovery of COVID-19 Inhibitors Targeting the SARS-CoV2 Nsp13 Helicase","abstract":"The raging COVID-19 pandemic caused by SARS-CoV2 has infected millions of people and killed several hundred thousand patients worldwide. Currently, there are no effective drugs or vaccines available for treating coronavirus infections. In this study, we have focused on the SARS-CoV2 helicase (Nsp13), which is critical for viral replication and the most conserved non-structural protein within the coronavirus family. Using homology modeling and molecular dynamics approaches, we generated structural models of the SARS-CoV2 helicase in its apo- and ATP/RNA-bound conformations. We performed virtual screening of ~970,000 chemical compounds against the ATP binding site to identify potential inhibitors. Herein, we report docking hits of approved human drugs targeting the ATP binding site. Importantly, two of our top drug hits have significant activity in inhibiting purified recombinant SARS-CoV-2 helicase, providing hope that these drugs can be potentially repurposed for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.08.09.243246","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.242156","pub_date":"2020-8-10","title":"Sofosbuvir Terminated RNA is More Resistant to SARS-CoV-2 Proofreader than RNA Terminated by Remdesivir","abstract":"SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.","version":"1.1","doi":"10.1101/2020.08.07.242156","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.242677","pub_date":"2020-8-10","title":"High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing","abstract":"The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.10.242677","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.242271","pub_date":"2020-8-10","title":"SARS-CoV-2 neutralization and serology testing of COVID-19 convalescent plasma from donors with non-severe disease","abstract":"We determined the antigen binding activity of convalescent plasma units from 47 individuals with a history of non-severe COVID-19 using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. We compared these results with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. This revealed positive correlations of varying strength (Spearman r = 0.37-0.52) between binding and neutralization. Donors age 48-75 had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75-82%) for those with the highest levels of neutralization.","version":"1.1","doi":"10.1101/2020.08.07.242271","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.242263","pub_date":"2020-8-10","title":"Worldwide tracing of mutations and the evolutionary dynamics of SARS-CoV-2","abstract":"Understanding the mutational and evolutionary dynamics of SARS-CoV-2 is essential for treating COVID-19 and the development of a vaccine. Here, we analyzed publicly available 15,818 assembled SARS-CoV-2 genome sequences, along with 2,350 raw sequence datasets sampled worldwide. We investigated the distribution of inter-host single nucleotide polymorphisms (inter-host SNPs) and intra-host single nucleotide variations (iSNVs). Mutations have been observed at 35.6% (10,649/29,903) of the bases in the genome. The substitution rate in some protein coding regions is higher than the average in SARS-CoV-2 viruses, and the high substitution rate in some regions might be driven to escape immune recognition by diversifying selection. Both recurrent mutations and human-to-human transmission are mechanisms that generate fitness advantageous mutations. Furthermore, the frequency of three mutations (S protein, F400L; ORF3a protein, T164I; and ORF1a protein, Q6383H) has gradual increased over time on lineages, which provides new clues for the early detection of fitness advantageous mutations. Our study provides theoretical support for vaccine development and the optimization of treatment for COVID-19. We call researchers to submit raw sequence data to public databases.","version":"1.1","doi":"10.1101/2020.08.07.242263","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.244350","pub_date":"2020-8-10","title":"Human Embryonic Stem Cell-derived Lung Organoids: a Model for SARS-CoV-2 Infection and Drug Test","abstract":"The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we demonstrated that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids. Ciliated cells, alveolar type 2 (AT2) cells and rare club cells were virus target cells. Electron microscopy captured typical replication, assembly and release ultrastructures and revealed the presence of viruses within lamellar bodies in AT2 cells. Virus infection induced more severe cell death in alveolar organoids than in airway organoids. Additionally, RNA-seq revealed early cell response to SARS-CoV-2 infection and an unexpected downregulation of ACE2 mRNA. Further, compared to the transmembrane protease, serine 2 (TMPRSS2) inhibitor camostat, the nucleotide analog prodrug Remdesivir potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model for SARS-CoV-2 infection and drug discovery.","version":"1.1","doi":"10.1101/2020.08.10.244350","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.244756","pub_date":"2020-8-10","title":"The interplay of SARS-CoV-2 evolution and constraints imposed by the structure and functionality of its proteins","abstract":"Fast evolution of the SARS-CoV-2 virus provides us with unique information about the patterns of genetic changes in a single pathogen in the timescale of months. This data is used extensively to track the phylodynamic of the pandemic\u2019s spread and its split into distinct clades. Here we show that the patterns of SARS-CoV-2 virus mutations along its genome are closely correlated with the structural features of the coded proteins. We show that the foldability of proteins\u2019 3D structures and conservation of their functions are the universal factors driving evolutionary selection in protein-coding genes. Insights from the analysis of mutation distribution in the context of the SARS-CoV-2 proteins\u2019 structures and functions have practical implications including evaluating potential antigen epitopes or selection of primers for PCR-based COVID-19 tests.","version":"1.1","doi":"10.1101/2020.08.10.244756","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.243717","pub_date":"2020-8-10","title":"Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples","abstract":"A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 18 million cases of disease and 700,000 deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays aimed at detecting different classes of antibodies constitute the best surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population, in order to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to know the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict population immunity and possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, we performed a subtyping Immunoglobulin G ELISA. Our data showed an excellent statistical correlation between the neutralization titer and the IgG, IgM and IgA ELISA response against the receptor-binding domain of the spike protein, confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories which do not have Biosecurity level-3 facilities.","version":"1.1","doi":"10.1101/2020.08.10.243717","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.08.242511","pub_date":"2020-8-10","title":"Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike","abstract":"Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.","version":"1.1","doi":"10.1101/2020.08.08.242511","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.09.243451","pub_date":"2020-8-10","title":"Nonstructural protein 1 of SARS-CoV-2 is a potent pathogenicity factor redirecting host protein synthesis machinery toward viral RNA","abstract":"The COVID-19 pandemic affects millions of people worldwide with a rising death toll. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its nonstructural protein 1 (Nsp1) to redirect host translation machinery to the viral RNA by binding to the ribosome and suppressing cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the transcriptome in human cells. The changes include repression of major gene clusters in ribosomal RNA processing, translation, mitochondria function, cell cycle and antigen presentation; and induction of factors in transcriptional regulation. We further gained a mechanistic understanding of the Nsp1 function by determining the cryo-EM structure of the Nsp1-40S ribosomal subunit complex, which shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the cryo-EM structure of the 48S preinitiation complex (PIC) formed by Nsp1, 40S, and the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) RNA, which shows that this 48S PIC is nonfunctional due to the incorrect position of the 3\u2019 region of the mRNA. Results presented here elucidate the mechanism of host translation inhibition by SARS-CoV-2, provide insight into viral protein synthesis, and furnish a comprehensive understanding of the impacts from one of the most potent pathogenicity factors of SARS-CoV-2. ORF screen identified Nsp1 as a major cellular pathogenicity factor of SARS-CoV-2 Nsp1 broadly alters the gene expression programs in human cells Nsp1 inhibits translation by blocking mRNA entry channel Nsp1 prevents physiological conformation of the 48S PIC","version":"1.1","doi":"10.1101/2020.08.09.243451","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228890","pub_date":"2020-8-10","title":"EUAdb: a resource for COVID-19 test development","abstract":"Due to the sheer number of COVID-19 (coronavirus disease 2019) cases, the prevalence of asymptomatic cases and the fact that undocumented cases appear to be significant for transmission of the causal virus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), there is an urgent need for increased SARS-CoV-2 testing capability that is both efficient and effective. In response to the growing threat of the COVID-19 pandemic in February, 2020, the FDA (US Food and Drug Administration) began issuing Emergency Use Authorizations (EUAs) to laboratories and commercial manufacturers for the development and implementation of diagnostic tests. So far, the gold standard assay for SARS-CoV-2 detection is the RT-qPCR (real-time quantitative polymerase chain reaction) test. However, the authorized RT-qPCR test protocols vary widely, not only in the reagents, controls, and instruments they use, but also in the SARS-CoV-2 genes they target, what results constitute a positive SARS-CoV-2 infection, and their limit of detection (LoD). The FDA has provided a web site that lists most of the tests that have been issued EUAs, along with links to the authorization letters and summary documents describing these tests. However, it is very challenging to use this site to compare or replicate these tests for a variety of reasons. First, at least 12 of 18 tests for EUA submissions made prior to March 31, 2020, are not listed there. To our knowledge, no EUAs have been issued for these applications. Second, the data are not standardized and are only provided as longhand prose in the summary documents. Third, some details (e.g. primer sequences) are absent from several of the test descriptions. Fourth, for tests provided by commercial manufacturers, summary documents are completely missing. To address at least the first three issues, we have developed a database, EUAdb (EUAdb.org), that holds standardized information about EUA-issued tests and is focused on RT-qPCR diagnostic tests, or \u201chigh complexity molecular-based laboratory developed tests\u201d. By providing a standardized ontology and curated data in a relational architecture, we seek to facilitate comparability and reproducibility, with the ultimate goal of consistent, universal and high-quality testing nationwide. Here, we document the basics of the EUAdb data architecture and simple data queries. The source files can be provided to anyone who wants to modify the database for his/her own research purposes. We ask that the original source of the files be made clear and that the database not be used in its original or modified forms for commercial purposes.","version":"1.2","doi":"10.1101/2020.07.30.228890","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.242073","pub_date":"2020-8-10","title":"K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia","abstract":"The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.","version":"1.1","doi":"10.1101/2020.08.07.242073","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.09.242917","pub_date":"2020-8-10","title":"DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist","abstract":"The efficient spread of SARS-CoV-2 resulted in a pandemic that is unique in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in air mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. Thus, we described a mechanism potentiating viral capture and spreading of infection. Early involvement of APCs opens new avenues for understanding and treating the imbalanced innate immune response observed in COVID-19 pathogenesis","version":"1.1","doi":"10.1101/2020.08.09.242917","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.241653","pub_date":"2020-8-10","title":"Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France","abstract":"Through routine genomic surveillance of the novel SARS-CoV-2 virus (n=229 whole genome sequences), 2 different frameshifting deletions were newly detected in the open reading frame (ORF) 6, starting at the same position (27267). While the 26-nucleotide deletion variant was only found in one sample in March 2020, the 34-nucleotide deletion variant was found within a single geriatric hospital unit in 5/9 patients sequenced and one health care worker with samples collected between April 2nd and 9th, 2020. Both the presence of the 34-nucleotide deletion variant limited to this unit and the clustering of the corresponding whole genome sequences by phylogeny analysis strongly suggested a nosocomial transmission between patients. Interestingly, prolonged viral excretion of the 34-nucleotide deletion variant was identified in a stool sample 14 days after initial diagnosis for one patient. Clinical data revealed no significant difference in disease severity between patients harboring the wild-type or the 34-nucleotide deletion variants. The in vitro infection of the two deletion variants on primate endothelial kidney cells (BGM) and human lung adenocarcinoma cells (Calu-3) yielded comparable replication kinetics with the wild-type strain. Furthermore, high viral loads were found in vivo regardless of the presence or absence of the ORF6 deletion. Our study highlights the transmission and replication capacity of two newly described deletion variants in the same ORF6 region. While the SARS-CoV-2 genome has remained relatively stable since its emergence in the human population, genomic deletions are an evolutionary pattern previously described for the related SARS-CoV. Real-time genomic monitoring of the circulating variants is paramount to detect strain prevalence and transmission dynamics. Given the role of ORF6 in interferon modulation, further characterization, such as mechanistic interactions and interferon monitoring in patients, is crucial in understanding the viral-host factors driving disease evolution.","version":"1.1","doi":"10.1101/2020.08.07.241653","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.10.243980","pub_date":"2020-8-10","title":"A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against P. vivax antigens in a multiplexed bead-based assay using Luminex\u00ae technology (Bio-Plex\u00ae 200 or MAGPIX\u00ae)","abstract":"Multiplexed bead-based assays that use Luminex xMAP\u00ae technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data is lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex\u00ae 100/200\u2122 or Bio-Plex\u00ae 200), magnetic beads can be run on both these and the newer MAGPIX\u00ae instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex\u00ae 200, ii) magnetic beads run on the Bio-Plex\u00ae 200 versus MAGPIX\u00ae and iii) non-magnetic beads run on a Bio-Plex\u00ae 200 versus magnetic beads run on the MAGPIX\u00ae. We also performed an external validation of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were strongly correlated in all three of our comparisons, however higher amounts of protein were required for coupling to magnetic beads. Our external validation indicated that results generated in different laboratories using the same coupled beads are also highly comparable, particularly if a reference standard curve is used.","version":"1.1","doi":"10.1101/2020.08.10.243980","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.225912","pub_date":"2020-8-07","title":"Infection of human lymphomononuclear cells by SARS-CoV-2","abstract":"Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo. We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4+T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.","version":"1.2","doi":"10.1101/2020.07.28.225912","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.239574","pub_date":"2020-8-07","title":"Generation of tonsil organoids as an ex vivo model for SARS-CoV-2 infection","abstract":"Palatine tonsil (hereinafter referred to as \u201ctonsil\u201d) plays role in the immune system\u2019s first line of defense against foreign pathogens. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic since the infection was first reported in China in December 2019. The aim of this study was to establish tonsil epithelial cell-derived organoids and to examine their feasibility as an ex vivo model for SARS-CoV-2 infection. Using an optimized protocol, we achieved 3D tonsil organoid culture from human tonsil tissue that reflects the distinctive characteristics of the tonsil epithelium, such as its cellular composition, histologic properties, and molecular biological features. Notably, we verified that SARS-CoV-2 can infect tonsil organoids with a robust replication efficiency. Furthermore, treatment with remdesivir, an antiviral agent, effectively protected them from viral infection. Therefore, tonsil organoids could be available for investigation of SARS-CoV-2 infection-mediated pathology and for preclinical screening of novel antiviral drug candidates. This study established tonsil epithelial cell-derived organoids and demonstrated their feasibility as an ex vivo model for SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.08.06.239574","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.019463","pub_date":"2020-8-07","title":"Insights into The Codon Usage Bias of 13 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Isolates from Different Geo-locations","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of Coronavirus disease 2019 (COVID-19) which is an infectious disease that spread throughout the world and was declared as a pandemic by the World Health Organization (WHO). In this study, we performed a genome-wide analysis on the codon usage bias (CUB) of 13 SARS-CoV-2 isolates from different geo-locations (countries) in an attempt to characterize it, unravel the main force shaping its pattern, and understand its adaptation to Homo sapiens. Overall results revealed that, SARS-CoV-2 codon usage is slightly biased similarly to other RNA viruses. Nucleotide and dinucleotide compositions displayed a bias toward A/U content in all codon positions and CpU-ended codons preference, respectively. Eight common putative preferred codons were identified, and all of them were A/U-ended (U-ended: 7, A-ended: 1). In addition, natural selection was found to be the main force structuring the codon usage pattern of SARS-CoV-2. However, mutation pressure and other factors such as compositional constraints and hydrophobicity had an undeniable contribution. Two adaptation indices were utilized and indicated that SARS-CoV-2 is moderately adapted to Homo sapiens compared to other human viruses. The outcome of this study may help in understanding the underlying factors involved in the evolution of SARS-CoV-2 and may aid in vaccine design strategies.","version":"1.3","doi":"10.1101/2020.04.01.019463","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.238188","pub_date":"2020-8-07","title":"Alveolitis in severe SARS-CoV-2 pneumonia is driven by self-sustaining circuits between infected alveolar macrophages and T cells","abstract":"Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) [1]. Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia [2]. We collected bronchoalveolar lavage fluid samples from 86 patients with SARS-CoV-2-induced respiratory failure and 252 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single cell RNA-Seq in 5 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection at the onset of mechanical ventilation, the alveolar space is persistently enriched in alveolar macrophages and T cells without neutrophilia. Bulk and single cell transcriptomic profiling suggest SARS-CoV-2 infects alveolar macrophages that respond by recruiting T cells. These T cells release interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell recruitment. Our results suggest SARS-CoV-2 causes a slowly unfolding, spatially-limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 transcripts and T cells form a positive feedback loop that drives progressive alveolar inflammation. This manuscript is accompanied by an online resource: https://www.nupulmonary.org/covid-19/ SARS-CoV-2-infected alveolar macrophages form positive feedback loops with T cells in patients with severe COVID-19.","version":"1.2","doi":"10.1101/2020.08.05.238188","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.240192","pub_date":"2020-8-07","title":"Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors","abstract":"The number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60\u20132.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.08.06.240192","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.241810","pub_date":"2020-8-07","title":"SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for reverse zoonosis to New World rodents","abstract":"Coronavirus disease-19 (COVID-19) emerged in November, 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and likely underwent a recombination event in an intermediate host prior to entry into human populations. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 14 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood brain barrier. Despite this, no conspicuous signs of disease were observed and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, notably IFN\u03b1, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8\u03b2 expression in the lungs was concomitant with Tbx21, IFN\u03b3 and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources indicated the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 pathogenesis, and that they have the potential to serve as secondary reservoir hosts that could lead to periodic outbreaks of COVID-19 in North America.","version":"1.1","doi":"10.1101/2020.08.07.241810","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.240796","pub_date":"2020-8-07","title":"Expression of ACE2, TMPRSS2 and CTSL in human airway epithelial cells under physiological and pathological conditions: Implications for SARS-CoV-2 infection","abstract":"SARS-CoV-2 enters into human airway epithelial cells via membrane fusion or endocytosis, and this process is dependent on ACE2, TMPRSS2, and cathepsin L. In this study, we examined the expression profiles of the three SARS-CoV-2 entry-related genes in primary human airway epithelial cells isolated from donors with different physiological and pathological backgrounds such as smoking, COPD, asthma, lung cancer, allergic rhinitis, cystic fibrosis, or viral infections. By reanalyzing 54 GEO datasets comprising transcriptomic data of 3428 samples, this study revealed that i) smoking is associated with an increased expression of ACE2 and TMPRSS2 and a decreased expression of cathepsin L; ii) infection of rhinovirus as well as poly(I:C) stimulation leads to high expression of all three SARS-CoV-2 entry-related genes; iii) expression of ACE2 and cathepsin L in nasal epithelial cells are decreased in patients with asthma and allergic rhinitis. In conclusion, this study implicates that infection of respiratory viruses, cigarette smoking and allergic respiratory diseases might affect the susceptibility to and the development of COVID-19.","version":"1.1","doi":"10.1101/2020.08.06.240796","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.07.20169920","pub_date":"2020-08-07","title":"Wrong person, place and time: viral load and contact network structure predict SARS-CoV-2 transmission and super-spreading events","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  SARS-CoV-2 is difficult to contain because many transmissions occur during the pre-symptomatic phase of infection. Moreover, in contrast to influenza, while most SARS-CoV-2 infected people do not transmit the virus to anybody, a small percentage secondarily infect large numbers of people. We designed mathematical models of SARS-CoV-2 and influenza which link observed viral shedding patterns with key epidemiologic features of each virus, including distributions of the number of secondary cases attributed to each infected person (individual R\n                  <jats:sub>0</jats:sub>\n                  ) and the duration between symptom onset in the transmitter and secondarily infected person (serial interval). We identify that people with SARS-CoV-2 or influenza infections are usually contagious for fewer than one day congruent with peak viral load several days after infection, and that transmission is unlikely below a certain viral load. SARS-CoV-2 super-spreader events with over 10 secondary infections occur when an infected person is briefly shedding at a very high viral load and has a high concurrent number of exposed contacts. The higher predisposition of SARS-CoV-2 towards super-spreading events is not due to its 1-2 additional weeks of viral shedding relative to influenza. Rather, a person infected with SARS-CoV-2 exposes more people within equivalent physical contact networks than a person infected with influenza, likely due to aerosolization of virus. Our results support policies that limit crowd size in indoor spaces and provide viral load benchmarks for infection control and therapeutic interventions intended to prevent secondary transmission.\n                </jats:p>\n                <jats:sec>\n                  <jats:title>One Sentence Summary</jats:title>\n                  <jats:p>We developed a coupled within-host and between-host mathematical model to identify viral shedding levels required for transmission of SARS-CoV-2 and influenza, and to explain why super-spreading events occur more commonly during SARS-CoV-2 infection.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.07.20169920","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.07.241877","pub_date":"2020-8-07","title":"mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2","abstract":"The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.","version":"1.1","doi":"10.1101/2020.08.07.241877","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.225078","pub_date":"2020-8-07","title":"Serial co-expression analysis of host factors from SARS-CoV viruses highly converges with former high-throughput screenings and proposes key regulators and co-option of cellular pathways","abstract":"The current genomics era is bringing an unprecedented growth in the amount of gene expression data, only comparable to the exponential growth of sequences in databases during the last decades. This data now allows the design of secondary analyses that take advantage of this information to create new knowledge through specific computational approaches. One of these feasible analyses is the evaluation of the expression level for a gene through a series of different conditions or cell types. Based on this idea, we have developed ASACO, Automatic and Serial Analysis of CO-expression, which performs expression profiles for a given gene along hundreds of normalized and heterogeneous transcriptomics experiments and discover other genes that show either a similar or an inverse behavior. It might help to discover co-regulated genes, and even common transcriptional regulators in any biological model, including human diseases or microbial infections. The present SARS-CoV-2 pandemic is an opportunity to test this novel approach due to the wealth of data that is being generated, which could be used for validating results. In addition, new cell mechanisms identified could become new therapeutic targets. Thus, we have identified 35 host factors in the literature putatively involved in the infectious cycle of SARS-CoV and/or SARS-CoV-2 and searched for genes tightly co-expressed with them. We have found around 1900 co-expressed genes whose assigned functions are strongly related to viral cycles. Moreover, this set of genes heavily overlap with those identified by former laboratory high-throughput screenings (with p-value near 0). Some of these genes aim to cellular structures such as the stress granules, which could be essential for the virus replication and thereby could constitute potential targets in the current fight against the virus. Additionally, our results reveal a series of common transcription regulators, involved in immune and inflammatory responses, that might be key virus targets to induce the coordinated expression of SARS-CoV-2 host factors. All of this proves that ASACO can discover gene co-regulation networks with potential for proposing new genes, pathways and regulators participating in particular biological systems. ASACO identifies regulatory associations of genes using public transcriptomics data. ASACO highlights new cell functions likely involved in the infection of coronavirus. Comparison with high-throughput screenings validates candidates proposed by ASACO. Genes co-expressed with host\u2019s genes used by SARS-CoV-2 are related to stress granules.","version":"1.2","doi":"10.1101/2020.07.28.225078","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.238915","pub_date":"2020-8-06","title":"Peptide Antidotes to SARS-CoV-2 (COVID-19)","abstract":"The design of an immunogenic scaffold that serves a role in treating a pathogen, and can be rapidly and predictively modeled, has remained an elusive feat. Here, we demonstrate that SARS-BLOCK\u2122 synthetic peptide scaffolds act as antidotes to SARS-CoV-2 spike protein-mediated infection of human ACE2-expressing cells. Critically, SARS-BLOCK\u2122 peptides are able to potently and competitively inhibit SARS-CoV-2 S1 spike protein receptor binding domain (RBD) binding to ACE2, the main cellular entry pathway for SARS-CoV-2, while also binding to neutralizing antibodies against SARS-CoV-2. In order to create this potential therapeutic antidote-vaccine, we designed, simulated, synthesized, modeled epitopes, predicted peptide folding, and characterized behavior of a novel set of synthetic peptides. The biomimetic technology is modeled off the receptor binding motif of the SARS-CoV-2 coronavirus, and modified to provide enhanced stability and folding versus the truncated wildtype sequence. These novel peptides attain single-micromolar binding affinities for ACE2 and a neutralizing antibody against the SARS-CoV-2 receptor binding domain (RBD), and demonstrate significant reduction of infection in nanomolar doses. We also demonstrate that soluble ACE2 abrogates binding of RBD to neutralizing antibodies, which we posit is an essential immune-evasive mechanism of the virus. SARS-BLOCK\u2122 is designed to \u201cuncloak\u201d the viral ACE2 coating mechanism, while also binding to neutralizing antibodies with the intention of stimulating a specific neutralizing antibody response. Our peptide scaffolds demonstrate promise for future studies evaluating specificity and sensitivity of immune responses to our antidote-vaccine. In summary, SARS-BLOCK\u2122 peptides are a promising COVID-19 antidote designed to combine the benefits of a therapeutic and vaccine, effectively creating a new generation of prophylactic and reactive antiviral therapeutics whereby immune responses can be enhanced rather than blunted.","version":"1.2","doi":"10.1101/2020.08.06.238915","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.235549","pub_date":"2020-8-06","title":"Hypertonic saline solution inhibits SARS-CoV-2 in vitro assay","abstract":"We are facing an unprecedented global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At this date more than 680 thousand people have died due to coronavirus disease 2019 (COVID-19). Unfortunately, until now no effective treatment to combat the virus and vaccine are available. We performed experiments to test if hypertonic saline solution is able to inhibit virus replication in vitro. Our data shows that 260 mM NaCl (1.5%) inhibits 100% SARS-CoV-2 replication in Vero cells. Furthermore, our results suggest that the virus replication inhibition is due to an intracellular mechanism and not due to the dissociation between spike SARS-CoV-2 protein and its human receptor angiotensin-converting enzyme 2 interaction. NaCl depolarizes the plasma membrane supposedly associated with the inhibition of the SARS-CoV-2 life cycle. This observation could lead to simple, safe and low cost interventions at various stages of COVID-19 treatment, improving the prognosis of infected patients, thereby mitigating the social and economic costs of the pandemic.","version":"1.2","doi":"10.1101/2020.08.04.235549","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.236653","pub_date":"2020-8-06","title":"Pathogenetic Perspective of Missense Mutations of ORF3a Protein of SARS-CoV2","abstract":"One of the most important proteins for COVID-19 pathogenesis in SARS-CoV2 is the ORF3a protein which is the largest accessory protein among others accessory proteins coded by coronavirus genome. The major roles of the protein include virulence, infectivity, ion channel activity, morphogenesis and virus release. The coronavirus, SARS-CoV2 is continuously evolving naturally and thereby the encoded proteins are also mutating rapidly. Therefore, critical study of mutations in ORF3a is certainty important from the pathogenetic perspective. Here, a sum of 175 various non-synonymous mutations in the ORF3a protein of SARS-CoV2 are identified and their corresponding effects in structural stability and functions of the protein ORF3a are studied. Broadly three different classes of mutations, such as neutral, disease and mixed (neutral and disease) type mutations were observed. Consecutive mutations in some ORF3a proteins are established based on timeline of detection of mutations. Considering the amino acid compositions over the ORF3a primary protein sequences, twenty clusters are detected based on K-means clustering method. Our findings on 175 novel mutations of ORF3a proteins will extend our knowledge of ORF3a, a vital accessory protein in SARS-CoV2, which would assist to enlighten on the pathogenicity of this life-threatening COVID-19.","version":"1.2","doi":"10.1101/2020.08.04.236653","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.239798","pub_date":"2020-8-06","title":"IFN signaling and neutrophil degranulation transcriptional signatures are induced during SARS-CoV-2 infection","abstract":"The novel virus SARS-CoV-2 has infected more than 14 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Limited information on the underlying immune mechanisms that drive disease or protection during COVID-19 severely hamper development of therapeutics and vaccines. Thus, the establishment of relevant animal models that mimic the pathobiology of the disease is urgent. Rhesus macaques infected with SARS-CoV-2 exhibit disease pathobiology similar to human COVID-19, thus serving as a relevant animal model. In the current study, we have characterized the transcriptional signatures induced in the lungs of juvenile and old rhesus macaques following SARS-CoV-2 infection. We show that genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. We demonstrate that Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. In contrast, pathways involving VEGF are downregulated in lungs of old infected macaques. Using samples from humans with SARS-CoV-2 infection and COVID-19, we validate a subset of our findings. Finally, neutrophil degranulation, innate immune system and IFN gamma signaling pathways are upregulated in both tuberculosis and COVID-19, two pulmonary diseases where neutrophils are associated with increased severity. Together, our transcriptomic studies have delineated disease pathways to improve our understanding of the immunopathogenesis of COVID-19 to facilitate the design of new therapeutics for COVID-19.","version":"1.1","doi":"10.1101/2020.08.06.239798","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.20164061","pub_date":"2020-08-06","title":"Is Higher Viral Load in SARS-CoV-2 Associated With Death?","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>There is no proven prognostic marker or adequate number of studies in patients hospitalized for coronavirus disease 2019 (COVID-19).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted a retrospective cohort study of patients hospitalized with COVID-19 from March 14 to June 17, 2020, at S\u00e3o Paulo Hospital. SARS-CoV-2 viral load was assessed using the cycle threshold (Ct) values obtained from an RT-PCR assay applied to the nasopharyngeal swab samples. Disease severity and patient outcomes were compared.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Among the 875 patients, 50.1% (439/875) had mild, 30.4% (266/875) moderate, and 19.5% (170/875) severe disease. A Ct value of &lt;25 (472/875) indicated a high viral load, which was independently associated with mortality (OR: 0,34; 95% CI: 0,217\u20130,533;\n                    <jats:italic>p</jats:italic>\n                    &lt; 0.0001).\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>Admission SARS-CoV-2 viral load is an important surrogate biomarker of infectivity and is independently associated with mortality among patients hospitalized with COVID-19.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.08.04.20164061","journal":"medRxiv","score":null},{"id":"10.1101/2020.08.06.240333","pub_date":"2020-8-06","title":"Computational Hot-Spot Analysis of the SARS-CoV-2 Receptor Binding Domain / ACE2 Complex","abstract":"Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a Protein-Protein Interaction (PPI) between the SARS CoV-2 Receptor-Binding Domain (RBD) and the extracellular peptidase domain of ACE2, on the surface of cells in the lower respiratory tract, is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD / ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted \u2018hot-spot\u2019 residues in a positive control PPI (PMI / MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs\u2019 free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive control PPI and SARS CoV-2 / ACE2 PPI. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD / ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.","version":"1.1","doi":"10.1101/2020.08.06.240333","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.175695","pub_date":"2020-8-06","title":"SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response","abstract":"The most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed an antiviral dose-response to remdesivir and demonstrated the efficacy of TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a physiologically relevant platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.","version":"1.2","doi":"10.1101/2020.06.30.175695","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.06.234674","pub_date":"2020-8-06","title":"Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate","abstract":"Vaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel interactions between the spike trimers allowing formation of higher order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.","version":"1.1","doi":"10.1101/2020.08.06.234674","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.239046","pub_date":"2020-8-06","title":"Phylogenetic analysis of SARS-CoV-2 data is difficult","abstract":"Numerous studies covering some aspects of SARS-CoV-2 data analyses are being published on a daily basis, including a regularly updated phylogeny on nextstrain.org. Here, we review the difficulties of inferring reliable phylogenies by example of a data snapshot comprising all virus sequences available on May 5, 2020 from gisaid.org. We find that it is difficult to infer a reliable phylogeny on these data due to the large number of sequences in conjunction with the low number of mutations. We further find that rooting the inferred phylogeny with some degree of confidence either via the bat and pangolin outgroups or by applying novel computational methods on the ingroup phylogeny does not appear to be possible. Finally, an automatic classification of the current sequences into sub-classes based on statistical criteria is also not possible, as the sequences are too closely related. We conclude that, although the application of phylogenetic methods to disentangle the evolution and spread of COVID-19 provides some insight, results of phylogenetic analyses, in particular those conducted under the default settings of current phylogenetic inference tools, as well as downstream analyses on the inferred phylogenies, should be considered and interpreted with extreme caution.","version":"1.1","doi":"10.1101/2020.08.05.239046","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167544","pub_date":"2020-8-06","title":"An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19","abstract":"SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic. Consequently, much research has gone into the development of pre-clinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites (\u201cpriming\u201d) that causes a conformational change allowing for viral and host membrane fusion. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat and gabexate, clinically approved agents in Japan. Also, we profiled a camostat metabolite, FOY-251, and bromhexine hydrochloride, an FDA-approved mucolytic cough suppressant. The rank order potency for the compounds tested are: nafamostat (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM), FOY-251 (IC50 = 33.3 nM) and gabexate (IC50 = 130 nM). Bromhexine hydrochloride showed no inhibition of TMPRSS2. Further profiling of camostat, nafamostat and gabexate against a panel of recombinant proteases provides insight into selectivity and potency.","version":"1.2","doi":"10.1101/2020.06.23.167544","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.232975","pub_date":"2020-8-06","title":"Capturing and Recreating Diverse Antibody Repertoires as Multivalent Recombinant Polyclonal Antibody Drugs","abstract":"Plasma-derived polyclonal antibodies are polyvalent drugs used for many important clinical indications that require modulation of multiple drug targets simultaneously, including emerging infectious disease and transplantation. However, plasma-derived drugs suffer many problems, including low potency, impurities, constraints on supply, and batch-to-batch variation. In this study, we demonstrated proofs-of-concept for a technology that uses microfluidics and molecular genomics to capture diverse mammalian antibody repertoires as multivalent recombinant drugs. These \u201crecombinant hyperimmune\u201d drugs comprised thousands to tens of thousands of antibodies and were derived from convalescent human donors, or vaccinated human donors or immunized mice. Here we used our technology to build a highly potent recombinant hyperimmune for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2) in less than three months. We also validated a recombinant hyperimmune for Zika virus disease that abrogates antibody-dependent enhancement (ADE) through Fc engineering. For patients with primary immune deficiency (PID), we built high potency polyvalent recombinant hyperimmunes against pathogens that commonly cause serious lung infections. Finally, to address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated in vivo function against graft-versus-host disease (GVHD). Recombinant hyperimmunes are a novel class of drugs that could be used to target a wide variety of other clinical applications, including cancer and autoimmunity.","version":"1.1","doi":"10.1101/2020.08.05.232975","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.082909","pub_date":"2020-8-05","title":"The coding capacity of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic . In order to understand SARS-CoV-2 pathogenicity and antigenic potential, and to develop diagnostic and therapeutic tools, it is essential to portray the full repertoire of its expressed proteins. The SARS-CoV-2 coding capacity map is currently based on computational predictions and relies on homology to other coronaviruses. Since coronaviruses differ in their protein array, especially in the variety of accessory proteins, it is crucial to characterize the specific collection of SARS-CoV-2 proteins in an unbiased and open-ended manner. Utilizing a suite of ribosome profiling techniques , we present a high-resolution map of the SARS-CoV-2 coding regions, allowing us to accurately quantify the expression of canonical viral open reading frames (ORF)s and to identify 23 novel unannotated viral translated ORFs. These ORFs include upstream ORFs (uORFs) that are likely playing a regulatory role, several in-frame internal ORFs lying within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; rather, virus translation dominates host translation due to high levels of viral transcripts. Overall, our work reveals the full coding capacity of SARS-CoV-2 genome, providing a rich resource, which will form the basis of future functional studies and diagnostic efforts.","version":"1.3","doi":"10.1101/2020.05.07.082909","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.238394","pub_date":"2020-8-05","title":"The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines","abstract":"The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-CoV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally available compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS CoV-2 replication (EC50 range, 2.0 to 31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.","version":"1.1","doi":"10.1101/2020.08.05.238394","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.237867","pub_date":"2020-8-05","title":"Rapid GMP-compliant expansion of SARS-CoV-2-specific T cells from convalescent donors for use as an allogeneic cell therapy for COVID-19","abstract":"COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive cellular therapy using donor-derived virus-specific T cells. Here we show that SARS-CoV-2-exposed blood donations contain CD4 and CD8 memory T cells specific for SARS-CoV-2 spike, nucleocapsid and membrane antigens. These peptides can be used to isolate virus-specific T cells in a GMP-compliant process. These T cells can be rapidly expanded using GMP-compliant reagents for use as a therapeutic product. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 1010 to 1011 T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for treatment of COVID-19 patients CD4+ and CD8+ T cells specific for SARS-CoV-2 can be isolated from convalescent donors and rapidly expanded to therapeutic doses at GMP standard, maintaining the desired central memory phenotype required for protective immune responses against severe COVID-19 infections.","version":"1.1","doi":"10.1101/2020.08.05.237867","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.237651","pub_date":"2020-8-05","title":"Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity","abstract":"Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.","version":"1.1","doi":"10.1101/2020.08.05.237651","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.238618","pub_date":"2020-8-05","title":"Analysis of the potential impact of genomic variants in SARS-CoV-2 genomes from India on molecular diagnostic assays","abstract":"An isolated epidemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus Diseases (COVID-19) originating in Wuhan, China has now rapidly emerged into a global pandemic affecting millions of people worldwide. Molecular detection of SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) forms the mainstay in screening, diagnosis and epidemiology of disease. The virus has been evolving through base substitutions. The recent availability of genomes of SARS-CoV-2 isolates from different countries including India motivated us to assess the presence and potential impact of variations in target sites for the oligonucleotide primers and probes used in molecular diagnosis. We catalogued a total of 132 primers or probes sequences from the literature and the public domain. Our analysis revealed a total of 125 unique genetic variants in 80 either primers or probes binding sites. A total of 13 unique variants had allele frequency of \u2265 1% in Indian SARS-CoV-2 genomes mapped to the primers or probes binding sites. A total of 15 primers or probes binding sites had cumulative variant frequency of \u2265 1% in the SARS-CoV-2 genomes. These included primers or probes sites which are widely used in India and across the world for molecular diagnosis as well as approved by national and international agencies. This highlights the need for sequencing genomes of emerging pathogens to make evidence based policies for development and approval of diagnostics. To the best of our knowledge, ours is the most comprehensive analysis of genomic variants in genomes of SARS-CoV-2 isolates from India and their potential impact on efficacy of molecular diagnostics.","version":"1.1","doi":"10.1101/2020.08.05.238618","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.237404","pub_date":"2020-8-05","title":"Analysis of single nucleotide polymorphisms between 2019-nCoV genomes and its impact on codon usage","abstract":"The spread of COVID-19 is a global concern that has taken a toll on entire human health. Researchers across the globe has been working in devising the strategies to combat this dreadful disease. Studies focused on genetic variability helps design effective drugs and vaccines. Considering this, the present study entails the information regarding the genome-wide mutations detected in the 108 SARS CoV-2 genomes worldwide. We identified a few hypervariable regions localized in orf1ab, spike, and nucleocapsid gene. These nucleotide polymorphisms demonstrated their effect on both codon usage as well as amino acid usage pattern. Altogether the present study provides valuable information that would be helpful to ongoing research on 2019-nCoV vaccine development.","version":"1.1","doi":"10.1101/2020.08.05.237404","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.232645","pub_date":"2020-8-05","title":"Host metabolic reprogramming in response to SARS-Cov-2 infection","abstract":"Understanding the pathogenesis of SARS-CoV-2 is important for developing effective treatment strategies. Viruses hijack the host metabolism to redirect the resources for their replication and survival. How SARS-CoV-2 influences the host metabolism is still unclear. In this study, we analyzed transcriptomic data obtained from different human respiratory cell lines and patient samples (Swab, PBMC, lung biopsy, BALF) to understand the metabolic alterations in response to SARS-CoV-2 infection. For this purpose, the expression pattern of metabolic genes in the human genome-scale metabolic network model Recon3D was explored. We identified metabolic genes and pathways and reporter metabolites under each SARS-CoV-2-infected condition and compared them to identify common and unique changes in the metabolism. Our analysis revealed host-dependent dysregulation of glycolysis, mitochondrial metabolism, amino acid metabolism, glutathione metabolism, polyamine synthesis, and lipid metabolism. We observed different metabolic changes that are pro- and antiviral in nature. We generated hypotheses on how antiviral metabolism can be targeted/enhanced for reducing viral titers. These warrant further exploration with more samples and in vitro studies to test predictions.","version":"1.1","doi":"10.1101/2020.08.02.232645","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.230987","pub_date":"2020-8-05","title":"Global variation in the SARS-CoV-2 proteome reveals the mutational hotspots in the drug and vaccine candidates","abstract":"To accelerate the drug and vaccine development against the severe acute respiratory syndrome virus 2 (SARS-CoV-2), a comparative analysis of SARS-CoV-2 proteome has been performed in two phases by considering manually curated 31389 whole genome sequences from 84 countries. Among the 9 mutations that occur at a high significance (T85I-NPS2, L37F-NSP6, P323L-NSP12, D614G-spike, Q57H-ORF3a, G251V-ORF3a, L84S-ORF8, R203K-nucleocapsid and G204R-nucleocapsid), R203K-nucleocapsid and G204R-nucleocapsid are co-occurring (dependent) mutations and P323L-NSP12 and D614G-spike often appear simultaneously. Other notable variations that appear with a moderate to low significance are, M85-NSP1 deletion, D268-NSP2 deletion, 112 amino acids deletion in ORF8, a phenylalanine insertion amidst F34-F36 (NSP6) and several co-existing (dependent) substitution/deletion (I559V & P585S in NSP2, P504L & Y541C in NSP13, G82 & H83 deletions in NSP1 and K141, S142 & F143 deletions in NSP2) mutations. P323L-NSP12, D614G-spike, L37F-NSP6, L84S-ORF8 and the sequences deficient of the high significant mutations have led to 4 major SARS-CoV-2 clades. The top 5 countries bearing all the high significant and majority of the moderate significant mutations are: USA, England, Wales, Australia and Scotland. Further, the majority of the significant mutations have evolved in the first phase and have already transmitted around the globe indicating the positive selection pressure. Among the 26 SARS-CoV-2 proteins, nucleocapsid protein, ORF3a, ORF8, RNA dependent RNA polymerase and spike exhibit a higher heterogeneity compared with the rest of the proteins. However, NSP9, NSP10, NSP8, the envelope protein and NSP4 are highly resistant to mutations and can be exploited for drug/vaccine development.","version":"1.3","doi":"10.1101/2020.07.31.230987","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161620","pub_date":"2020-8-05","title":"Multi-pronged human protein mimicry by SARS-CoV-2 reveals bifurcating potential for MHC detection and immune evasion","abstract":"The hand of molecular mimicry in shaping SARS-CoV-2 evolution and immune evasion remains to be deciphered. We identify 33 distinct 8-mer/9-mer peptides that are identical between SARS-CoV-2 and human proteomes, along similar extents of viral mimicry observed in other viruses. Interestingly, 20 novel peptides have not been observed in any previous human coronavirus (HCoV) strains. Four of the total mimicked 8-mers/9-mers map onto HLA-B*40:01, HLA-B*40:02, and HLA-B*35:01 binding peptides from human PAM, ANXA7, PGD, and ALOX5AP proteins. This mimicry of multiple human proteins by SARS-CoV-2 is made salient by the targeted genes being focally expressed in arteries, lungs, esophagus, pancreas, and macrophages. Further, HLA-A*03 restricted 8-mer peptides are shared broadly by human and coronaviridae helicases with primary expression of the mimicked human proteins in the neurons and immune cells. This study presents the first comprehensive scan of peptide mimicry by SARS-CoV-2 of the human proteome and motivates follow-up research into its immunological consequences.","version":"1.2","doi":"10.1101/2020.06.19.161620","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.107011","pub_date":"2020-8-05","title":"SARS-CoV-2 ORF8 can fold into human factor 1 catalytic domain binding site on complement C3b: Predict functional mimicry","abstract":"Pathogens are often known to use host factor mimicry to take evolutionary advantage. As the function of the non-structural ORF8 protein of SARS-CoV-2 in the context of host-pathogen relationship is still obscure, we investigated its role in host factor mimicry using computational protein modelling techniques. Modest sequence similarity of ORF8 of SARS-CoV-2 with the substrate binding site within the C-terminus serine-protease catalytic domain of human complement factor 1 (F1; PDB ID: 2XRC), prompted us to verify their resemblance at the structural level. The modelled ORF8 protein was found to superimpose on the F1 fragment. Further, protein-protein interaction simulation confirmed ORF8 binding to C3b, an endogenous substrate of F1, via F1-interacting region on C3b. Docking results suggest ORF8 to occupy the binding groove adjacent to the conserved \u201carginine-serine\u201d (RS) F1-mediated cleavage sites on C3b. Comparative H-bond interaction dynamics indicated ORF8/C3b binding to be of higher affinity than the F1/C3b interaction. Hence, ORF8 is predicted to inhibit C3b proteolysis by competing with F1 for C3b binding using molecular mimicry with a possibility of triggering unregulated complement activation. This could offer a mechanistic premise for the unrestrained complement activation observed in large number of SARS-CoV-2 infected patients.","version":"1.2","doi":"10.1101/2020.06.08.107011","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.234872","pub_date":"2020-8-05","title":"Structure-Based Design of a Cyclic Peptide Inhibitor of the SARS-CoV-2 Main Protease","abstract":"This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. Synthesis and evaluation of a first-generation cyclic peptide inhibitor reveals that the inhibitor is active against Mpro in vitro and is non-toxic toward human cells in culture. The initial hit described in this manuscript, UCI-1, lays the groundwork for the development of additional cyclic peptide inhibitors against Mpro with improved activities.","version":"1.2","doi":"10.1101/2020.08.03.234872","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.237339","pub_date":"2020-8-05","title":"Evolutionary dynamics of SARS-CoV-2 nucleocapsid protein (N protein) and its consequences","abstract":"The emerging novel coronavirus SARS-CoV-2 has created a global confusing pandemic health crisis that warrants an accurate and detailed characterization of the rapidly evolving viral genome for understanding its epidemiology, pathogenesis and containment. We explored 61,485 sequences of the Nucleocapsid (N) protein, a potent diagnostic and prophylactic target, for identifying the mutations to review their roles in RT-PCR based diagnosis and observe consequent impacts. Compared to the Wuhan reference strain, a total of 1034 unique nucleotide mutations were identified in the mutant strains (49.15%, n=30,221) globally. Of these mutations, 367 occupy primer binding sites including 3\u2019-end mismatch to primer-pair of 11 well characterized primer sets. Noteworthy, CDC (USA) recommended N2 primer set contained lower mismatch than the other primer sets. Moreover, 684 amino acid (aa) substitutions located across 317 (75.66% of total aa) unique positions including 82, 21, and 83 of those in RNA binding N-terminal domain (NTD), SR-rich region, and C-terminal dimerization domain (CTD), respectively. Moreover, 11 in-frame deletions were revealed, mostly (n =10) within the highly flexible linker region, and the rest within the NTD region. Furthermore, we predicted the possible consequences of high-frequency mutations (\u2265 20) and deletions on the tertiary structure of the N protein. Remarkably, we observed that high frequency (67.94% of mutated sequences) coevolving mutations (R203K and G204R) destabilized and decreased overall structural flexibility. Despite being proposed as the alternate target to spike protein for vaccine and therapeutics, ongoing nonsynonymous evolution of the N protein may challenge the endeavors, thus need further immunoinformatics analyses. Therefore, continuous monitoring is required for tracing the ongoing evolution of the SARS-CoV-2 N protein in prophylactic and diagnostic interventions.","version":"1.1","doi":"10.1101/2020.08.05.237339","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.237313","pub_date":"2020-8-05","title":"Dynamics of the COVID -19 Related Publications","abstract":"This study aims to analyze the dynamics of the published articles and preprints of Covid-19 related literature from different scientific databases and sharing platforms. The PubMed, Elsevier, and Research Gate (RG) databases were under consideration in this study over a specific time. Analyses were carried out on the number of publications as (a) function of time (day), (b) journals and (c) authors. Doubling time of the number of publications was analyzed for PubMed \u201call articles\u201d and Elsevier published articles. Analyzed databases were (1A) PubMed \u201call articles\u201d (01/12/2019-12/06/2020) (1B) PubMed Review articles (01/12/2019-2/5/2020) and (1C) PubMed Clinical Trials (01/01/2020-30/06/2020) (2) Elsevier all publications (01/12/2019-25/05/2020) (3) RG (Article, Pre Print, Technical Report) (15/04/2020\u201330/4/2020). Total publications in the observation period for PubMed, Elsevier, and RG were 23000, 5898 and 5393 respectively. The average number of publications/day for PubMed, Elsevier and RG were 70.0 \u00b1128.6, 77.6\u00b1125.3 and 255.6\u00b1205.8 respectively. PubMed shows an avalanche in the number of publication around May 10, number of publications jumped from 6.0\u00b18.4/day to 282.5\u00b1110.3/day. The average doubling time for PubMed, Elsevier, and RG was 10.3\u00b14 days, 20.6 days, and 2.3\u00b12.0 days respectively. In PubMed average articles/journal was 5.2\u00b110.3 and top 20 authors representing 935 articles are of Chinese descent. The average number of publications per author for PubMed, Elsevier, and RG was 1.2\u00b11.4, 1.3\u00b10.9, and 1.1\u00b10.4 respectively. Subgroup analysis, PubMed review articles mean and median review time for each article were <0|17\u00b117|77> and 13.9 days respectively; and reducing at a rate of-0.21 days (count)/day. Although the disease has been known for around 6 months, the number of publications related to the Covid-19 until now is huge and growing very fast with time. It is essential to rationalize the publications scientifically by the researchers, authors, reviewers, and publishing houses. None","version":"1.1","doi":"10.1101/2020.08.05.237313","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.05.238360","pub_date":"2020-8-05","title":"IFN-\u03b3 and TNF-\u03b1 drive a CXCL10+ CCL2+ macrophage phenotype expanded in severe COVID-19 and other diseases with tissue inflammation","abstract":"Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies. Using an integrative strategy, we built a reference by meta-analyzing > 300,000 immune cells from COVID-19 and 5 inflammatory diseases including rheumatoid arthritis (RA), Crohn\u2019s disease (CD), ulcerative colitis (UC), lupus, and interstitial lung disease. Our cross-disease analysis revealed that an FCN1+ inflammatory macrophage state is common to COVID-19 bronchoalveolar lavage samples, RA synovium, CD ileum, and UC colon. We also observed that a CXCL10+ CCL2+ inflammatory macrophage state is abundant in severe COVID-19, inflamed CD and RA, and expresses inflammatory genes such as GBP1, STAT1, and IL1B. We found that the CXCL10+ CCL2+ macrophages are transcriptionally similar to blood-derived macrophages stimulated with TNF-\u03b1 and IFN-\u03b3 ex vivo. Our findings suggest that IFN-\u03b3, alongside TNF-\u03b1, might be a key driver of this abundant inflammatory macrophage phenotype in severe COVID-19 and other inflammatory diseases, which may be targeted by existing immunomodulatory therapies.","version":"1.1","doi":"10.1101/2020.08.05.238360","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.235002","pub_date":"2020-8-04","title":"SARS-CoV-2 infection, disease and transmission in domestic cats","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility and transmission studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, associated disease and transmission dynamics in domestic cats. Six 4- to 5-month-old cats were challenged with SARS-CoV-2 via intranasal and oral routes simultaneously. One day post challenge (DPC), two sentinel contact cats were co-mingled with the principal infected animals. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding throughout the 21 DPC observation period. Postmortem examinations were performed at 4, 7 and 21 DPC to investigate disease progression. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats on 4 and 7 DPC. Serology showed that both, principal and sentinel cats, developed SARS-CoV-2-specific and neutralizing antibodies to SARS-CoV-2 detectable at 7 DPC or 10 DPC, respectively. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels within 2 days of comingling. The results of this study are critical for our understanding of the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment of the maintenance of SARS-CoV-2 in felines and transmission to other animals and humans.","version":"1.1","doi":"10.1101/2020.08.04.235002","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.066951","pub_date":"2020-8-04","title":"Pervasive generation of non-canonical subgenomic RNAs by SARS-CoV-2","abstract":"SARS-CoV-2, a positive-sense RNA virus in the family Coronaviridae, has caused a worldwide pandemic of coronavirus disease 2019 or COVID-19 Coronaviruses generate a tiered series of subgenomic RNAs (sgRNAs) through a process involving homology between transcriptional regulatory sequences (TRS) located after the leader sequence in the 5\u2019 UTR (the TRS-L) and TRS\u2019 located near the start of structural and accessory proteins (TRS-B) near the 3\u2019 end of the genome. In addition to the canonical sgRNAs generated by SARS-CoV-2, non-canonical sgRNAs (nc-sgRNAs) have been reported. However, the consistency of these nc-sgRNAs across viral isolates and infection conditions is unknown. The comprehensive definition of SARS-CoV-2 RNA products is a key step in understanding SARS-CoV-2 pathogenesis. Here, we report an integrative analysis of eight independent SARS-CoV-2 transcriptomes generated using three sequencing strategies, five host systems, and seven viral isolates. Read-mapping to the SARS-CoV-2 genome was used to determine the 5\u2019 and 3\u2019 coordinates of all identified junctions in viral RNAs identified in these samples. Using junctional abundances, we show nc-sgRNAs make up as much as 33% of total sgRNAs in vitro, are largely consistent in abundance across independent transcriptomes, and increase in abundance over time during in vitro infection. By assessing the homology between sequences flanking the 5\u2019 and 3\u2019 junction points, we show that nc-sgRNAs are not associated with TRS-like homology. By incorporating read coverage information, we find strong evidence for subgenomic RNAs that contain only 5\u2019 regions of ORF1a. Finally, we show that non-canonical junctions change the landscape of viral open reading frames. We identify canonical and non-canonical junctions in SARS-CoV-2 sgRNAs and show that these RNA products are consistently generated across many independent viral isolates and sequencing approaches. These analyses highlight the diverse transcriptional activity of SARS-CoV-2 and offer important insights into SARS-CoV-2 biology.","version":"1.2","doi":"10.1101/2020.04.28.066951","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.230243","pub_date":"2020-8-04","title":"SARS-CoV-2 suppresses mRNA expression of selenoproteins associated with ferroptosis, endoplasmic reticulum stress and DNA synthesis","abstract":"A significant, positive association between selenium status and prognosis of SARS-CoV-2 infection has been identified among COVID-19 patients in China. Moreover, a German study revealed a pronounced deficit of serum selenium and SELENOP concentrations in COVID-19 patients, and selenium deficiency was associated with mortality risk from COVID-19. The present study investigated the influence of SARS-CoV-2 on gene expression of host selenoproteins which mediate many beneficial actions of selenium. We found that SARS-CoV-2 suppressed mRNA expression of selenoproteins associated with ferroptosis (GPX4), endoplasmic reticulum stress (SELENOF, SELENOK, SELENOM and SELENOS) and DNA synthesis (TXNRD3), while SARS-CoV-2 increased gene expression of IL-6 (an inflammatory cytokine positively correlated with severity of COVID-19), in Vero cells. These results provide a deeper insight into the connection between selenium and SARS-CoV-2 pathogenesis.","version":"1.2","doi":"10.1101/2020.07.31.230243","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.235689","pub_date":"2020-8-04","title":"Cold-adapted live attenuated SARS-CoV-2 vaccine completely protects human ACE2 transgenic mice from SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has infected more than 16,000,000 people and has caused the death of more than 650,000 individuals since December 2019. A safe and effective vaccine that can provide herd immunity against SARS-CoV-2 is urgently needed to stop the spread of this virus among humans. Many human viral vaccines are live attenuated forms of viruses that elicit humoral and cellular immunity. Here, we describe the development of a cold-adapted live attenuated vaccine (SARS-CoV-2/human/Korea/CNUHV03-CA22\u00b0C/2020) by gradually adapting the growth of SARS-CoV-2 from 37\u00b0C to 22\u00b0C in Vero cells. This vaccine can be potentially administered to humans through nasal spray. Its single dose was observed to strongly induce the neutralising antibody (>640), cellular immunity, and mucosal IgA antibody in intranasally immunised K18-hACE2 mice, which are very susceptible to SARS-CoV-2 and SARS-CoV infection. The one-dose vaccinated mice were completely protected from SARS-CoV-2 infection and did not show loss of body weight, death, and the presence of virus in tissues, such as the nasal turbinates, brain, lungs, and kidneys. Taken together, the cold-adapted live attenuated SARS-CoV-2 vaccine developed by us may contribute to saving of human lives from the threat of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.08.04.235689","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.234153","pub_date":"2020-8-04","title":"SARS-CoV-2 genome analysis of strains in Pakistan reveals GH, S and L clade strains at the start of the pandemic","abstract":"Pakistan has a high infectious disease burden with about 265,000 reported cases of COVID-19. We investigated the genomic diversity of SARS-CoV-2 strains and present the first data on viruses circulating in the country. We performed whole-genome sequencing and data analysis of SARS-CoV-2 eleven strains isolated in March and May. Strains from travelers clustered with those from China, Saudi Arabia, India, USA and Australia. Five of eight SARS-CoV-2 strains were GH clade with Spike glycoprotein D614G, Ns3 gene Q57H, and RNA dependent RNA polymerase (RdRp) P4715L mutations. Two were S (ORF8 L84S and N S202N) and three were L clade and one was an I clade strain. One GH and one L strain each displayed Orf1ab L3606F indicating further evolutionary transitions. This data reveals SARS-CoV-2 strains of L, G, S and I have been circulating in Pakistan from March, at the start of the pandemic. It indicates viral diversity regarding infection in this populous region. Continuing molecular genomic surveillance of SARS-CoV-2 in the context of disease severity will be important to understand virus transmission patterns and host related determinants of COVID-19 in Pakistan.","version":"1.1","doi":"10.1101/2020.08.04.234153","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.234880","pub_date":"2020-8-04","title":"SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing","abstract":"Here we provide technical data for amplifying the complete genome of SARS-CoV-2 from clinical samples using only seventeen pairs of primers. We demonstrate that the \u0421V2000bp primer panel successfully produces genomes when used with the residual total RNA extracts from positive clinical samples following diagnostic RT-PCRs (with Ct in the range from 13 to 20). The library preparation method reported here includes genome amplification of ~1750-2000 bp fragments followed by ultrasonic fragmentation combined with the introduction of Illumina compatible adapters. Using the SCV2000bp panel, 25 complete SARS-CoV-2 virus genome sequences were sequenced from clinical samples of COVID-19 patients from Moscow obtained in late March - early April.","version":"1.1","doi":"10.1101/2020.08.04.234880","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.235291","pub_date":"2020-8-04","title":"Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors","abstract":"We report the identification of three structurally diverse compounds \u2013 compound 4, GC376, and MAC-5576 \u2013 as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.","version":"1.1","doi":"10.1101/2020.08.03.235291","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.231746","pub_date":"2020-8-04","title":"Engineered ACE2 receptor traps potently neutralize SARS-CoV-2","abstract":"An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.","version":"1.3","doi":"10.1101/2020.07.31.231746","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228460","pub_date":"2020-8-04","title":"Phylogenomic analysis of SARS-CoV-2 genomes from western India reveals unique linked mutations","abstract":"India has become the third worst-hit nation by the COVID-19 pandemic caused by the SARS-CoV-2 virus. Here, we investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in western India, the most affected region of the country. A total of 90 genomes were sequenced. Four nucleotide variants, namely C241T, C3037T, C14408T (Pro4715Leu), and A23403G (Asp614Gly), located at 5\u2019UTR, Orf1a, Orf1b, and Spike protein regions of the genome, respectively, were predominant and ubiquitous (90%). Phylogenetic analysis of the genomes revealed four distinct clusters, formed owing to different variants. The major cluster (cluster 4) is distinguished by mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples. We thus report a newly emerging pattern of linked mutations. The predominance of these linked mutations suggests that they are likely a part of the viral fitness landscape. A novel and distinct pattern of mutations in the viral strains of each of the districts was observed. The Satara district viral strains showed mutations primarily at the 3\u2032 end of the genome, while Nashik district viral strains displayed mutations at the 5\u2032 end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients indicated an increased occurrence in symptomatic cases, which is more prominent in females. The age-wise specific pattern of mutation is observed. Mutations C18877T, G20326A, G24794T, G25563T, G26152T, and C26735T are found in more than 30% study samples in the age group of 10-25. Intriguingly, these mutations are not detected in the higher age range 61-80. These findings portray the prevalence of unique linked mutations in SARS-CoV-2 in western India and their prevalence in symptomatic patients. Elucidation of the SARS-CoV-2 mutational landscape within a specific geographical location, and its relationship with age and symptoms, is essential to understand its local transmission dynamics and control. Here we present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India, the worst affected region by the pandemic. Our analysis revealed three unique linked mutations, which are prevalent in most of the sequences studied. These may serve as a molecular marker to track the spread of this viral variant to different places.","version":"1.2","doi":"10.1101/2020.07.30.228460","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.235747","pub_date":"2020-8-04","title":"A valid protective immune response elicited in rhesus macaques by an inactivated vaccine is capable of defending against SARS-CoV-2 infection","abstract":"With the relatively serious global epidemic outbreak of SARS-CoV-2 infection, public concerns focus on not only clinical therapeutic measures and public quarantine for this disease but also the development of vaccines. The technical design of our SARS-CoV-2 inactivated vaccine provides a viral antigen that enables the exposure of more than one structural protein based upon the antibody composition of COVID-19 patients\u2019 convalescent serum. This design led to valid immunity with increasing neutralizing antibody titers and a CTL response detected post-immunization of this vaccine by two injections in rhesus macaques. Further, this elicited immunoprotection in macaques enables not only to restrain completely viral replication in tissues of immunized animals, compared to the adjuvant control and those immunized by an RBD peptide vaccine, but also to significantly alleviate inflammatory lesion in lung tissues in histo-pathologic detection, compared to the adjuvant control with developed interstitial pneumonia. The data obtained from these macaques immunized with the inactivated vaccine or RBD peptide vaccine suggest that immunity with a clinically protective effect against SARS-CoV-2 infection should include not only specific neutralizing antibodies but also specific CTL responses against at least the S and N antigens.","version":"1.1","doi":"10.1101/2020.08.04.235747","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.236521","pub_date":"2020-8-04","title":"Lymphopenia-induced T cell proliferation is a hallmark of severe COVID-19","abstract":"Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a broad clinical presentation ranging from asymptomatic infection to fatal disease. Different features associated with the immune response to SARS-CoV-2, such as hyperinflammation and reduction of peripheral CD8+ T cell counts are strongly associated with severe disease. Here, we confirm the reduction in peripheral CD8+ T cells both in relative and absolute terms and identify T cell apoptosis and migration into inflamed tissues as possible mechanisms driving peripheral T cell lymphopenia. Furthermore, we find evidence of elevated serum interleukin-7, thus indicating systemic T cell paucity and signs of increased T cell proliferation in patients with severe lymphopenia. Following T cell lymphopenia in our pseudo-longitudinal time course, we observed expansion and recovery of poly-specific antiviral T cells, thus arguing for lymphopenia-induced T cell proliferation. In summary, this study suggests that extensive T cell loss and subsequent T cell proliferation are characteristic of severe COVID-19.","version":"1.1","doi":"10.1101/2020.08.04.236521","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.04.236315","pub_date":"2020-8-04","title":"A distinct innate immune signature marks progression from mild to severe COVID-19","abstract":"Coronavirus disease 2019 (COVID-19) manifests with a range of severities, but immune signatures of mild and severe disease are still not fully understood. Excessive inflammation has been postulated to be a major factor in the pathogenesis of severe COVID-19 and innate immune mechanisms are likely to be central in the inflammatory response. We used 40-plex mass cytometry and targeted serum proteomics to profile innate immune cell populations from peripheral blood of patients with mild or severe COVID-19 and healthy controls. Sampling at different stages of COVID-19 allowed us to reconstruct a pseudo-temporal trajectory of the innate immune response. Despite the expected patient heterogeneity, we identified consistent changes during the course of the infection. A rapid and early surge of CD169+ monocytes associated with an IFN\u03b3+MCP-2+ signature quickly followed symptom onset; at symptom onset, patients with mild and severe COVID-19 had a similar signature, but over the course of the disease, the differences between patients with mild and severe disease increased. Later in the disease course, we observed a more pronounced re-appearance of intermediate/non-classical monocytes and mounting systemic CCL3 and CCL4 levels in patients with severe disease. Our data provide new insights into the dynamic nature of the early inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and identifies sustained pathological innate immune responses as a likely key mechanism in severe COVID-19, further supporting investigation of targeted anti-inflammatory interventions in severe COVID-19.","version":"1.1","doi":"10.1101/2020.08.04.236315","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.234989","pub_date":"2020-8-04","title":"Binding Ligands that Straddle an Important Contact Site on the RBD of the Covid-19 Spike Protein","abstract":"The receptor binding domain (RBD) of the spike protein of the Covid-19 virus is responsible for attachment to human ACE2. A number of recent articles have studied monoclonal antibody blocking [8-11] and peptide inhibitors [12-16] of the Covid-19 virus. Here we report virtual ligand-based screening that targets pockets on each side of an important binding site with residues 502-504 on the RBD that contact residues 353-357 [15] of hACE2. These ligands are intended as pre-exposure therapy for Covid-19 infection.","version":"1.1","doi":"10.1101/2020.08.03.234989","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.233320","pub_date":"2020-8-03","title":"REGN-COV2 antibody cocktail prevents and treats SARS-CoV-2 infection in rhesus macaques and hamsters","abstract":"An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.","version":"1.1","doi":"10.1101/2020.08.02.233320","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.234005","pub_date":"2020-8-03","title":"Similarity between mutation spectra in hypermutated genomes of rubella virus and in SARS-CoV-2 genomes accumulated during the COVID-19 pandemic","abstract":"Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella - another positive single stranded (ss) RNA virus. Each of the rubella isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2 - likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.","version":"1.1","doi":"10.1101/2020.08.03.234005","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.231340","pub_date":"2020-8-03","title":"De novo design of ACE2 protein decoys to neutralize SARS-CoV-2","abstract":"There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (\u223c18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.","version":"1.1","doi":"10.1101/2020.08.03.231340","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.111187","pub_date":"2020-8-03","title":"Age-determined expression of priming protease TMPRSS2 and localization of SARS-CoV-2 infection in the lung epithelium","abstract":"The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths around the globe. While the elderly appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1), and TMPRSS2 expression was increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases revealed SARS-CoV-2 RNA was detected most frequently in ciliated and secretory cells in the airway epithelium and AT1 cells in the peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressing TMPRSS2. Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in the lung epithelium, and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.","version":"1.2","doi":"10.1101/2020.05.22.111187","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.233536","pub_date":"2020-8-03","title":"Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity","abstract":"Most antibodies isolated from COVID-19 patients are specific to SARS-CoV-2. COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here we determined a crystal structure of COVA1-16 Fab with the SARS-CoV-2 RBD, and a negative-stain EM reconstruction with the spike glycoprotein trimer, to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long CDR H3, and competes with ACE2 binding due to steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with structural and functional rationale for the epitope conservation, provide a blueprint for development of more universal SARS-like coronavirus vaccines and therapies.","version":"1.1","doi":"10.1101/2020.08.02.233536","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.234559","pub_date":"2020-8-03","title":"SARS-CoV-2 ORF6 disrupts nucleocytoplasmic transport through interactions with Rae1 and Nup98","abstract":"RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be the major inhibitor of interferon production in both SARS-CoV and SARS-CoV-2. SARS-CoV-2 ORF6 was recently shown to co-purify with the host mRNA export factors Rae1 and Nup98. Here, we demonstrate SARS-CoV-2 ORF6 strongly represses protein expression of co-transfected reporter constructs and imprisons host mRNA in the nucleus, which is associated with its ability to co-purify with Rae1 and Nup98. These protein-protein interactions map to the C-terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. We further identify an ORF6 mutant containing a 9-amino acid deletion, ORF6 \u039422-30, in multiple SARS-CoV-2 clinical isolates that can still downregulate the expression of a co-transfected reporter and interact with Rae1 and Nup98. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly co-purifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and pre-symptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. SARS-CoV-2, the causative agent of COVID-19, is an RNA virus with a large genome that encodes accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. One of SARS-CoV-2\u2019s accessory proteins, ORF6, was recently shown to co-purify with two host proteins, Rae1 and Nup98, involved in mRNA nuclear export. We demonstrate SARS-CoV-2 ORF6 interaction with these proteins is associated with reduced expression of a reporter protein and accumulation of poly-A mRNA within the nucleus. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and co-purifies with Rae1 and Nup98 compared to SARS-CoV ORF6. The ability of SARS-CoV-2 ORF6 to more strongly disrupt nucleocytoplasmic transport than SARS-CoV ORF6 may partially explain critical differences in clinical presentation between the two viruses.","version":"1.1","doi":"10.1101/2020.08.03.234559","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.138826","pub_date":"2020-8-03","title":"SARS-CoV-2-specific T cells exhibit phenotypic features reflecting robust helper function, lack of terminal differentiation, and high proliferative potential","abstract":"Convalescing COVID-19 patients mount robust T cell responses against SARS-CoV-2, suggesting an important role for T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells, and predominantly Tcm with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can homeostatically proliferate, and can persist for over two months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection, and support an important role for SARS-CoV-2-specific T cells in host control of COVID-19.","version":"1.2","doi":"10.1101/2020.06.08.138826","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.233718","pub_date":"2020-8-03","title":"PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Reveals Important Insights into the Outbreak","abstract":"The PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Consortium has achieved its initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes from nasopharyngeal and oropharyngeal swabs collected from individuals testing positive for COVID-19 by Real Time PCR. The samples were collected across 10 states covering different zones within India. Given the importance of this information for public health response initiatives investigating transmission of COVID-19, the sequence data is being released in GISAID database. This information will improve our understanding on how the virus is spreading, ultimately helping to interrupt the transmission chains, prevent new cases of infection, and provide impetus to research on intervention measures. This will also provide us with information on evolution of the virus, genetic predisposition (if any) and adaptation to human hosts. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. Initial results indicate that multiple lineages of SARS-CoV-2 are circulating in India, probably introduced by travel from Europe, USA and East Asia. A2a (20A/B/C) was found to be predominant, along with few parental haplotypes 19A/B. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Additionally, mutations in important regions of the viral genome with significant geographical clustering have also been observed. The temporal haplotype diversities landscape in each region appears to be similar pan India, with haplotype diversities peaking between March-May, while by June A2a (20A/B/C) emerged as the predominant one. Within haplotypes, different states appear to have different proportions. Temporal and geographic patterns in the sequences obtained reveal interesting clustering of mutations. Some mutations are present at particularly high frequencies in one state as compared to others. The negative estimate Tajimas D (D = \u22122.26817) is consistent with the rapid expansion of SARS-CoV-2 population in India. Detailed mutational analysis across India to understand the gradual emergence of mutants at different regions of the country and its possible implication will help in better disease management.","version":"1.1","doi":"10.1101/2020.08.03.233718","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.234914","pub_date":"2020-8-03","title":"De novo design of picomolar SARS-CoV-2 miniprotein inhibitors","abstract":"We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC50 values ranging from 35 pM to 35 nM; the most potent of these \u2014 56 and 64 residue hyperstable proteins made using the second approach \u2014 are roughly six times more potent on a per mass basis (IC50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.","version":"1.1","doi":"10.1101/2020.08.03.234914","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.03.233866","pub_date":"2020-8-03","title":"SARS-CoV-2 genome sequences from late April in Stockholm, Sweden reveal a novel mutation in the spike protein","abstract":"Large research efforts are going into characterizing, mapping the spread, and studying the biology and clinical features of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report four complete SARS-CoV-2 genome sequences obtained from patients confirmed to have the disease in Stockholm, Sweden, in late April. A variant at position 23463 was found for the first time in one genome. It changes an arginine (R) residue to histidine (H) at position 364 in the S1 subunit of the spike protein. The genomes belonged to two different genetic groups, previously reported as two of the three main genetic groups found in Sweden. Three of them are from group B.1/G, corresponding to the Italian outbreak, reported by the Public Health Agency of Sweden to have declined in prevalence by late April, and more investigation is needed in order to ensure that the spread of different types of SARS-CoV-2 is fully characterized. Four near-complete genomes of SARS-CoV-2 were assembled from late April in Stockholm. A novel mutation in the spike protein were found. The phylogeny of the strains were discussed.","version":"1.1","doi":"10.1101/2020.08.03.233866","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.233510","pub_date":"2020-8-03","title":"Mechanism of duplex unwinding by coronavirus nsp13 helicases","abstract":"The current COVID-19 pandemic urges in-depth investigation into proteins encoded with coronavirus (CoV), especially conserved CoV replicases. The nsp13 of highly pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV exhibit the most conserved CoV replicases. Using single-molecule FRET, we observed that MERS-CoV nsp13 unwound DNA in discrete steps of approximately 9 bp when ATP was used. If another NTP was used, then the steps were only 4 to 5 bp. In dwell time analysis, we detected 3 or 4 hidden steps in each unwinding process, which indicated the hydrolysis of 3 or 4 dTTP. Based on crystallographic and biochemical studies of CoV nsp13 helicases, we modeled an unwinding mechanism similar to the spring-loaded mechanism of HCV NS3 helicase, although our model proposes that flexible 1B and stalk domains, by allowing a lag greater than 4 bp during unwinding, cause the accumulated tension on the nsp13-DNA complex. The hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible than in MERS-CoV nsp13 due to the difference of a single amino acid, which causes the former to induce significantly greater NTP hydrolysis. Our findings thus establish a blueprint for determining the unwinding mechanism of a unique helicase family. When dTTP was used as the energy source, 4 hidden steps in each individual unwinding step after 3 - 4 NTP hydrolysis were observed. An unwinding model of MERS-CoV-nsp13 which is similar to the spring-loaded mechanism of HCV NS3 helicase, except the accumulation of tension on nsp13/DNA complex is caused by the flexible 1B and stalk domains that allow a lag of 4-bp in unwinding. Comparing to MERS-CoV nsp13, the hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible due to a single amino acid difference, which contributes to the significantly higher NTP hydrolysis by SARS-CoV-2 nsp13.","version":"1.1","doi":"10.1101/2020.08.02.233510","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.01.232199","pub_date":"2020-8-02","title":"Rapid Inactivation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by Tungsten Trioxide-Based (WO3) Photocatalysis","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), is transmitted person-to-person via respiratory droplets and, likely, via smaller droplet nuclei light enough to remain suspended in the air for hours and contaminate surfaces particularly in indoor conditions. Thus, effective measures are needed to prevent SARS-CoV-2 transmission in indoor environments. In this regard, we have investigated whether a system based on a filter combining Tungsten Trioxide-Based (WO3) photocatalysis and an antiviral fabric treated-copper nanocluster could inactivate SARS-CoV-2. To this purpose, an infectious SARS-CoV-2 suspension was introduced in the upper opening of a closed cylinder containing a WO3 filter and a lightbased system that activates WO3 and the antiviral fabric. From the bottom exit, aliquots of fluid were collected every 10 min (up to 60 min) and tested for their infectivity by means of a viral plaque assay in Vero cells whereas, in parallel, the viral RNA content was measured by quantitative PCR (qPCR). As we have previously shown for SARS-CoV, a 1:1,000 ratio of plaque forming units (PFU) vs. viral RNA copies was observed also for SARS-CoV-2. After 10 min, the infectious viral content was already decreased by 98.2% reaching 100% inactivation after 30 min whereas the SARS-CoV-2 RNA load was decreased of 1.5 log10 after 30 min. Thus, in spite of only a partial decrease of viral RNA, SARS-CoV-2 infectivity was completely abolished by the WO3 photocatalysis system by 30 min. These results support the hypothesis that this system could be exploited to achieve SARS-CoV-2 inactivation in indoor environments.","version":"1.1","doi":"10.1101/2020.08.01.232199","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.232892","pub_date":"2020-8-02","title":"A New Model of SARS-CoV-2 Infection Based on (Hydroxy) Chloroquine Activity","abstract":"Chloroquine and hydroxychloroquine (H)CQ are well known anti-malarial drugs, while their use against COVID-19 is more controversial. (H)CQ activity was examined in tissue culture cells to determine if their anti-viral benefits or adverse effects might be due to altering host cell pathways. Metabolic analysis revealed (H)CQ inhibit oxidative phosphorylation in mitochondria, likely by sequestering protons needed to drive ATP synthase. This activity could cause cardiotoxicity because heart muscle relies on beta oxidation of fatty acids. However, it might also explain their therapeutic benefit against COVID-19. A new model of SARS-CoV-2 infection postulates virus enters host cell mitochondria and uses its protons for genome release. Oxidative phosphorylation is eventually compromised, so glycolysis is upregulated to maintain ATP levels. (H)CQ could prevent viral infection and/or slow its replication by sequestering these protons. In support of this model other potential COVID-19 therapeutics also targeted mitochondria, as did tobacco smoke, which may underlie smokers\u2019 protection. The mitochondria of young people are naturally more adaptable and resilient, providing a rationale for their resistance to disease progression. Conversely, obesity and diabetes could exacerbate disease severity by providing extra glucose to infected cells dependent on glycolysis. The description of (H)CQ function presented here, together with its implications for understanding SARS-CO-V2 infection, makes testable predictions about disease progression and identifies new approaches for treating COVID-19.","version":"1.1","doi":"10.1101/2020.08.02.232892","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.231282","pub_date":"2020-8-02","title":"Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay","abstract":"Serology testing for COVID-19 is highly attractive because of the relatively short diagnosis time and the ability to test for an active immune response against the SARS-CoV-2. In many types of serology tests, the sensitivity and the specificity are directly influenced by the quality of the antigens manufactured. Protein purification of these recombinantly expressed viral antigens [e.g., spike and its receptor binding domain (RBD)] is an important step in the manufacturing process. Simple and high-capacity protein purification schemes for spike, RBD, and CR3022 mAb, recombinantly expressed in CHO and HEK293 cells, are reported in this article. The schemes consist of an affinity chromatography step and a desalting step. Purified proteins were validated in ELISA-based serological tests. Interestingly, extracellular matrix proteins [most notably heparan sulfate proteoglycan (HSPG)] were co-purified from spike-expressing CHO culture with a long cultivation time. HSPG-spike interaction could play a functional role in the pathology and the pathogenesis of SARS-CoV-2 and other coronaviruses.","version":"1.1","doi":"10.1101/2020.07.31.231282","journal":"bioRxiv","score":null},{"id":"10.1101/2020.08.02.233023","pub_date":"2020-8-02","title":"A Systemic and Molecular Study of Subcellular Localization of SARS-CoV-2 Proteins","abstract":"Coronavirus possesses the largest RNA genome among all the RNA viruses. Its genome encodes about 29 proteins. Most of the viral proteins are non-structural proteins (NSP) except envelop (E), membrane (M), nucleocapsid (N) and Spike (S) proteins that constitute the viral nucleocapsid, envelop and surface. We have recently cloned all the 29 SARS-CoV-2 genes into vectors for their expressions in mammalian cells except NSP11 that has only 14 amino acids (aa). We are able to express all the 28 cloned SARS-CoV-2 genes in human cells to characterize their subcellular distributions. The proteins of SARS-CoV-2 are mostly cytoplasmic but some are both cytoplasmic and nuclear. Those punctate staining proteins were further investigated by immunofluorescent assay (IFA) using specific antibodies or by co-transfection with an organelle marker-expressing plasmid. As a result, we found that NSP15, ORF6, M and ORF7a are related to Golgi apparatus, and that ORF7b, ORF8 and ORF10 colocalize with endoplasmic reticulum (ER). Interestingly, ORF3a distributes in cell membrane, early endosome, endosome, late endosome and lysosome, which suggests that ORF3a might help the infected virus to usurp endosome and lysosome for viral use. Furthermore, we revealed that NSP13 colocalized with SC35, a protein standing for splicing compartments in the nucleus. Our studies for the first time visualized the subcellular locations of SARS-CoV-2 proteins and might provide novel insights into the viral proteins\u2019 biological functions.","version":"1.1","doi":"10.1101/2020.08.02.233023","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.230102","pub_date":"2020-8-01","title":"Hybrid capture-based sequencing enables unbiased recovery of SAR-CoV-2 genomes from fecal samples and characterization of the dynamics of intra-host variants","abstract":"In response to the current COVID-19 pandemic, it is crucial to understand the origin, transmission, and evolution of SARS-CoV-2, which relies on close surveillance of genomic diversity in clinical samples. Although the mutation at the population level had been extensively investigated, how the mutations evolve at the individual level is largely unknown, partly due to the difficulty of obtaining unbiased genome coverage of SARS-CoV-2 directly from clinical samples. Eighteen time series fecal samples were collected from nine COVID-19 patients during the convalescent phase. The nucleic acids of SARS-CoV-2 were enriched by the hybrid capture method with different rounds of hybridization. By examining the sequencing depth, genome coverage, and allele frequency change, we demonstrated the impeccable performance of the hybrid capture method in samples with Ct value < 34, as well as significant improvement comparing to direct metatranscriptomic sequencing in samples with lower viral loads. We identified 229 intra-host variants at 182 sites in 18 fecal samples. Among them, nineteen variants presented frequency changes > 0.3 within 1-5 days, reflecting highly dynamic intra-host viral populations. Meanwhile, we also found that the same mutation showed different frequency changes in different individuals, indicating a strong random drift. Moreover, the evolving of the viral genome demonstrated that the virus was still viable in the gastrointestinal tract during the convalescent period. The hybrid capture method enables reliable analyses of inter- and intra-host variants of SARS-CoV-2 genome, which changed dramatically in the gastrointestinal tract; its clinical relevance warrants further investigation.","version":"1.1","doi":"10.1101/2020.07.30.230102","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.20164921","pub_date":"2020-08-01","title":"Estrogen and COVID-19 symptoms: associations in women from the COVID Symptom Study","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Men and older women have been shown to be at higher risk of adverse COVID-19 outcomes. Animal model studies of SARS-CoV and MERS suggest that the age and sex difference in COVID-19 symptom severity may be due to a protective effect of the female sex hormone estrogen. Females have shown an ability to mount a stronger immune response to a variety of viral infections because of more robust humoral and cellular immune responses.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Objectives</jats:title>\n                  <jats:p>We sought to determine whether COVID-19 positivity increases in women entering menopause. We also aimed to identify whether premenopausal women taking exogenous hormones in the form of the combined oral contraceptive pill (COCP) and post-menopausal women taking hormone replacement therapy (HRT) have lower predicted rates of COVID-19, using our published symptom-based model.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>\n                    The COVID Symptom Study developed by King\u2019s College London and Zoe Global Limited was launched in the UK on 24\n                    <jats:sup>th</jats:sup>\n                    March 2020. It captured self-reported information related to COVID-19 symptoms. Data used for this study included records collected between 7\n                    <jats:sup>th</jats:sup>\n                    May - 15\n                    <jats:sup>th</jats:sup>\n                    June 2020.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Main outcome measures</jats:title>\n                  <jats:p>\n                    We investigated links between COVID-19 rates and 1) menopausal status, 2) COCP use and 3) HRT use, using symptom-based\n                    <jats:italic>predicted</jats:italic>\n                    COVID-19, tested COVID-19, and disease severity based on requirement for hospital attendance or respiratory support.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>Female users of the COVID Symptom Tracker Application in the UK, including 152,637 women for menopause status, 295,689 for COCP use, and 151,193 for HRT use. Analyses were adjusted for age, smoking and BMI.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    Post-menopausal women aged 40-60 years had a higher rate of\n                    <jats:italic>predicted</jats:italic>\n                    COVID (P=0.003) and a corresponding range of symptoms, with consistent, but not significant trends observed for tested COVID-19 and disease severity. Women aged 18-45 years taking COCP had a significantly lower\n                    <jats:italic>predicted</jats:italic>\n                    COVID-19 (P=8.03E-05), with a reduction in hospital attendance (P=0.023). Post-menopausal women using HRT or hormonal therapies did not exhibit consistent associations, including increased rates of\n                    <jats:italic>predicted</jats:italic>\n                    COVID-19 (P=2.22E-05) for HRT users alone.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>\n                    Our findings support a protective effect of estrogen on COVID-19, based on positive association between\n                    <jats:italic>predicted</jats:italic>\n                    COVID-19 and menopausal status, and a negative association with COCP use. HRT use was positively associated with COVID-19 symptoms; however, the results should be considered with caution due to lack of data on HRT type, route of administration, duration of treatment, and potential comorbidities.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Trial registration</jats:title>\n                  <jats:p>The App Ethics has been approved by KCL ethics Committee REMAS ID 18210, review reference LRs-19/20-18210</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.07.30.20164921","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.31.228486","pub_date":"2020-7-31","title":"SARS-CoV-2 protein subunit vaccination elicits potent neutralizing antibody responses","abstract":"The outbreak and spread of SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2), the cause of coronavirus disease 2019 (COVID-19), is a current global health emergency and a prophylactic vaccine is needed urgently. The spike glycoprotein of SARS-CoV-2 mediates entry into host cells, and thus is a target for neutralizing antibodies and vaccine design. Here we show that adjuvanted protein immunization with SARS-CoV-2 spike trimers, stabilized in prefusion conformation , results in potent antibody responses in mice and rhesus macaques with neutralizing antibody titers orders of magnitude greater than those typically measured in serum from SARS-CoV-2 seropositive humans. Neutralizing antibody responses were observed after a single dose, with exceptionally high titers achieved after boosting. Furthermore, neutralizing antibody titers elicited by a dose-sparing regimen in mice were similar to those obtained from a high dose regimen. Taken together, these data strongly support the development of adjuvanted SARS-CoV-2 prefusion-stabilized spike protein subunit vaccines.","version":"1.1","doi":"10.1101/2020.07.31.228486","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.230730","pub_date":"2020-7-31","title":"Biomechanical Characterization of SARS-CoV-2 Spike RBD and Human ACE2 Protein-Protein Interaction","abstract":"The current COVID-19 pandemic has led to a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD\u2013ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approach to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 110 pN, and are 30-50% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD\u2013ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After the removal of N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD\u2013ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics simulation approach to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 receptor-binding domain and human ACE2. The study reveals the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry.","version":"1.1","doi":"10.1101/2020.07.31.230730","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.172312","pub_date":"2020-7-31","title":"Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses","abstract":"An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 16,500,000 cases and 650,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the \u03b2-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (S37 and A267) were found to exist only in its N protein, along with an extended \u03b2-hairpin that bends towards the nucleotide binding site. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, these data will enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral and vaccine development.","version":"1.3","doi":"10.1101/2020.06.25.172312","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.163188","pub_date":"2020-7-31","title":"Genomic diversity and hotspot mutations in 30,983 SARS-CoV-2 genomes: moving toward a universal vaccine for the \u201cconfined virus\u201d?","abstract":"The COVID-19 pandemic has been ongoing since its onset in late November 2019 in Wuhan, China. Understanding and monitoring the genetic evolution of the virus, its geographical characteristics, and its stability are particularly important for controlling the spread of the disease and especially for the development of a universal vaccine covering all circulating strains. From this perspective, we analyzed 30,983 complete SARS-CoV-2 genomes from 79 countries located in the six continents and collected from December 24, 2019, to May 13, 2020, according to the GISAID database. Our analysis revealed the presence of 3,206 variant sites, with a uniform distribution of mutation types in different geographic areas. Remarkably, a low frequency of recurrent mutations has been observed; only 169 mutations (5.27%) had a prevalence greater than 1% of genomes. Nevertheless, fourteen non-synonymous hotspot mutations (> 10%) have been identified at different locations along the viral genome; eight in ORF1ab polyprotein (in nsp2, nsp3, transmembrane domain, RdRp, helicase, exonuclease, and endoribonuclease), three in nucleocapsid protein and one in each of three proteins: spike, ORF3a, and ORF8. Moreover, 36 non-synonymous mutations were identified in the RBD of the spike protein with a low prevalence (<1%) across all genomes, of which only four could potentially enhance the binding of the SARS-CoV-2 spike protein to the human ACE2 receptor. These results along with mutational frequency dissimilarity and intra-genomic divergence of SARS-CoV-2 could indicate that the SARS-CoV-2 is not yet adapted to its host. Unlike the influenza virus or HIV viruses, the low mutation rate of SARS-CoV-2 makes the development of an effective global vaccine very likely.","version":"1.2","doi":"10.1101/2020.06.20.163188","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.229120","pub_date":"2020-7-31","title":"A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine","abstract":"A successful SARS-CoV-2 vaccine must be not only safe and protective but must also meet the demand on a global scale at low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent COVID-19 vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections or significantly attenuated SARS-CoV-2 induced disease. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.","version":"1.1","doi":"10.1101/2020.07.30.229120","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.222562","pub_date":"2020-7-31","title":"Mutational analysis unveils the temporal and spatial distribution of G614 genotype of SARS-CoV-2in different Indian states and its association with case fatality rate of COVID-19","abstract":"Pan genomic analysis of the global SARS-CoV-2 isolates has resulted in the identification of several regions of increased genetic variation but there is absence of research on its association with the clinical outcome. The present study fills the vacuum and does mutational analysis of genomic sequence of Indian SARS-CoV-2 isolates. Results reveal the existence of non-synonymous G614 spike protein mutation in 61.45% of the total study genome along with three other mutations. Further, temporal variation in the frequencies of G614 genotype in the country is observed. The examination of the probable association of G614 genotype with COVID-19 severity shows that CFR G614 genotype in India is positively and strongly correlated. It appears that the clinical outcome of the COVID-19 cases in India are significantly and adversely affected by the increasing trend in the G614 genotype; which needs to be addressed combining both laboratory experiments and epidemiological investigations.","version":"1.2","doi":"10.1101/2020.07.27.222562","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228023","pub_date":"2020-7-31","title":"The SARS-CoV-2 Nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein","abstract":"The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein\u2019s C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.","version":"1.1","doi":"10.1101/2020.07.30.228023","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.075291","pub_date":"2020-7-31","title":"Low baseline pulmonary levels of cytotoxic lymphocytes as a predisposing risk factor for severe COVID-19","abstract":"COVID-19 is caused by the coronavirus SARS-CoV-2 and currently has detrimental human health, community and economic impacts around the world. It is unclear why some SARS-CoV-2-positive individuals remain asymptomatic, while others develop severe symptoms. Baseline pulmonary levels of anti-viral leukocytes, already residing in the lung prior to infection, may orchestrate an effective early immune response and prevent severe symptoms. Using \u201cin silico flow cytometry\u201d, we deconvoluted the levels of all seven types of anti-viral leukocytes in 1,927 human lung tissues. Baseline levels of CD8+ T cells, resting NK cells and activated NK cells, as well as cytokines that recruit these, are significantly lower in lung tissues with high expression of the SARS-CoV-2 entry receptor ACE2. We observe this in univariate analyses, in multivariate analyses, and in two independent datasets. Relevantly, ACE2 mRNA and protein levels very strongly correlate in human cells and tissues. Above findings also largely apply to the SARS-CoV-2 entry protease TMPRSS2. Both SARS-CoV-2-infected lung cells and COVID-19 lung tissues show upregulation of CD8+ T cell- and NK cell-recruiting cytokines. Moreover, tissue-resident CD8+ T cells and inflammatory NK cells are significantly more abundant in bronchoalveolar lavages from mildly affected COVID-19 patients, compared to severe cases. This suggests that these lymphocytes are important for preventing severe symptoms. Elevated ACE2 expression increases sensitivity to coronavirus infection. Thus, our results suggest that some individuals may be exceedingly susceptible to develop severe COVID-19 due to concomitant high pre-existing ACE2 and TMPRSS expression and low baseline cytotoxic lymphocyte levels in the lung.","version":"1.2","doi":"10.1101/2020.05.04.075291","journal":"bioRxiv","score":null},{"id":"10.1101/851543","pub_date":"2020-7-31","title":"ETV7 limits antiviral gene expression and control of SARS-CoV-2 and influenza viruses","abstract":"The type I interferon (IFN) response is an important component of the innate immune response to viral infection. Precise control of interferon responses is critical; insufficient levels of interferon-stimulated genes (ISGs) can lead to a failure to restrict viral spread while excessive ISG activation can result in interferon-related pathologies. While both positive and negative regulatory factors control the magnitude and duration of IFN signaling, it is also appreciated that a number of ISGs regulate aspects of the interferon response themselves. However, the mechanisms underlying these ISG regulatory networks remain incompletely defined. In this study, we performed a CRISPR activation screen to identify new regulators of the type I IFN response. We identified ETS variant transcription factor 7 (ETV7), a strongly induced ISG, as a protein that acts as a negative regulator of the type I IFN response; however, ETV7 did not uniformly suppress ISG transcription. Instead, ETV7 preferentially targeted a subset of known antiviral ISGs. Further, we showed the subset of ETV7-modulated ISGs was particularly important for IFN-mediated control of some viruses including influenza viruses and SARS-CoV-2. Together, our data assign a function for ETV7 as an IFN response regulator and also identify ETV7 as a therapeutic target to increase innate responses and potentiate the efficacy of interferon-based antiviral therapies. ETV7 is an interferon-induced, repressive transcription factor that negatively regulates antiviral interferon-stimulated genes essential for controlling influenza virus and SARS-CoV-2 infections.","version":"1.3","doi":"10.1101/851543","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.04.187989","pub_date":"2020-7-31","title":"Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains","abstract":"The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. Antibodies can engage RBD but some, such as CR3022, fail to inhibit entry despite nanomolar spike affinity. Here we show the SARS-CoV-2 spike to have low unfolding enthalpy at serological pH and up to 10-times more unfolding enthalpy at endosomal pH, where we observe significantly reduced CR3022 affinity. Cryo-EM structures \u2013at serological and endosomal pH\u2013 delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the \u2018up\u2019 conformation. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a locked all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning and spike shedding of antibodies like CR3022. An endosomal mechanism involving spike-conformational change can thus facilitate immune evasion from RBD-\u2018up\u2019-recognizing antibody. Reveal spike at serological pH to have only ~10% the unfolding enthalpy of a typical globular protein, explaining how antibodies like CR3022 can bind with avidity Define an endosomal mechanism whereby spike binds ACE2, but sheds CR3022, enabling immune evasion from potentially neutralizing antibody Determine cryo-EM structures of the SARS-CoV-2 spike along its endosomal entry pathway-at pH 5.5, 4.5, and 4.0, and in complexes with ACE2 receptor at pH 7.4 and 5.5 Show spike to exclusively adopt an all RBD-down conformation at the low pH of the late endosome-early lysosome Reveal structural basis by which a switch domain mediates RBD position in response to pH","version":"1.2","doi":"10.1101/2020.07.04.187989","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.231274","pub_date":"2020-7-31","title":"Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase","abstract":"The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.","version":"1.1","doi":"10.1101/2020.07.31.231274","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.224063","pub_date":"2020-7-31","title":"Multiomic Immunophenotyping of COVID-19 Patients Reveals Early Infection Trajectories","abstract":"Host immune responses play central roles in controlling SARS-CoV2 infection, yet remain incompletely characterized and understood. Here, we present a comprehensive immune response map spanning 454 proteins and 847 metabolites in plasma integrated with single-cell multi-omic assays of PBMCs in which whole transcriptome, 192 surface proteins, and T and B cell receptor sequence were co-analyzed within the context of clinical measures from 50 COVID19 patient samples. Our study reveals novel cellular subpopulations, such as proliferative exhausted CD8+ and CD4+ T cells, and cytotoxic CD4+ T cells, that may be features of severe COVID-19 infection. We condensed over 1 million immune features into a single immune response axis that independently aligns with many clinical features and is also strongly associated with disease severity. Our study represents an important resource towards understanding the heterogeneous immune responses of COVID-19 patients and may provide key information for informing therapeutic development.","version":"1.2","doi":"10.1101/2020.07.27.224063","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.231472","pub_date":"2020-7-31","title":"New Pathways of Mutational Change in SARS-CoV-2 Proteomes Involve Regions of Intrinsic Disorder Important for Virus Replication and Release","abstract":"The massive worldwide spread of the SARS-CoV-2 virus is fueling the COVID-19 pandemic. Since the first whole-genome sequence was published in January 2020, a growing database of tens of thousands of viral genomes has been constructed. This offers opportunities to study pathways of molecular change in the expanding viral population that can help identify molecular culprits of virulence and virus spread. Here we investigate the genomic accumulation of mutations at various time points of the early pandemic to identify changes in mutationally highly active genomic regions that are occurring worldwide. We used the Wuhan NC_045512.2 sequence as a reference and sampled 15,342 indexed sequences from GISAID, translating them into proteins and grouping them by month of deposition. The per-position amino acid frequencies and Shannon entropies of the coding sequences were calculated for each month, and a map of intrinsic disorder regions and binding sites was generated. The analysis revealed dominant variants, most of which were located in loop regions and on the surface of the proteins. Mutation entropy decreased between March and April of 2020 after steady increases at several sites, including the D614G mutation site of the spike (S) protein that was previously found associated with higher case fatality rates and at sites of the NSP 12 polymerase and the NSP13 helicase proteins. Notable expanding mutations include R203K and G204R of the nucleocapsid (N) protein inter-domain linker region and G251V of the viroporin encoded by ORF3a between March and April. The regions spanning these mutations exhibited significant intrinsic disorder, which was enhanced and decreased by the N-protein and viroporin 3a protein mutations, respectively. These results predict an ongoing mutational shift from the spike and replication complex to other regions, especially to encoded molecules known to represent major \u03b2-interferon antagonists. The study provides valuable information for therapeutics and vaccine design, as well as insight into mutation tendencies that could facilitate preventive control.","version":"1.1","doi":"10.1101/2020.07.31.231472","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.230870","pub_date":"2020-7-31","title":"A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the main entry point in the airways for SARS-CoV-2. ACE2 binding to SARS-CoV-2 protein Spike triggers viral fusion with the cell membrane, resulting in viral RNA genome delivery into the host. Despite ACE2\u2019s critical role in SARS-CoV-2 infection, an understanding of ACE2 expression, including in response to viral infection, remains unclear. Until now ACE2 was thought to encode five transcripts and one 805 amino acid protein. Here we identify a novel short isoform of ACE2. Short ACE2 is expressed in the airway epithelium, the main site of SARS-CoV-2 infection; it is substantially upregulated in response to interferon stimulation and RV infection, but not in response to SARS-CoV-2 infection, and it shows differential regulation in asthma patients. This short isoform lacks SARS-CoV-2 spike glycoprotein high-affinity binding sites and altogether, our data are consistent with a model where short ACE2 may influence host susceptibility to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.07.31.230870","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.31.230607","pub_date":"2020-7-31","title":"Biochemical and mathematical lessons from the evolution of the SARS-CoV-2 virus: paths for novel antiviral warfare","abstract":"In the fight against the spread of COVID-19 the emphasis is on vaccination or on reactivating existing drugs used for other purposes. The tight links that necessarily exist between the virus as it multiplies and the metabolism of its host are systematically ignored. Here we show that the metabolism of all cells is coordinated by the availability of a core building block of the cell\u2019s genome, cytidine triphosphate (CTP). This metabolite is also the key to the synthesis of the viral envelope and to the translation of its genome into proteins. This unique role explains why evolution has led to the early emergence in animals of an antiviral immunity enzyme, viperin, that synthesizes a toxic analogue of CTP. The constraints arising from this dependency guide the evolution of the virus. With this in mind, we explored the real-time experiment taking place before our eyes using probabilistic modelling approaches to the molecular evolution of the virus. We have thus followed, almost on a daily basis, the evolution of the composition of the viral genome to link it to the progeny produced over time, particularly in the form of blooms that sparked a firework of viral mutations. Some of those certainly increase the propagation of the virus. This led us to make out the critical role in this evolution of several proteins of the virus, such as its nucleocapsid N, and more generally to begin to understand how the virus ties up the host metabolism to its own benefit. A way for the virus to escape CTP-dependent control in cells would be to infect cells that are not expected to grow, such as neurons. This may account for unexpected body sites of viral development in the present epidemic.","version":"1.1","doi":"10.1101/2020.07.31.230607","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.229377","pub_date":"2020-7-30","title":"Cross-reactivity of SARS-CoV structural protein antibodies against SARS-CoV-2","abstract":"There is currently a lack of biological tools to study the replication cycle and pathogenesis of SARS-CoV-2, the etiological agent of COVID-19. Repurposing the existing tools, including antibodies of SARS-CoV, is an effective way to accelerate the development of therapeutics for COVID-19. Here, we extensively characterized antibodies of the SARS-CoV structural proteins for their cross-reactivity, experimental utility, and neutralization of SARS-CoV-2. We assessed a total of 10 antibodies (six for Spike, two for Membrane, and one for Nucleocapsid and Envelope viral protein). We evaluated the utility of these antibodies against SARS-CoV-2 in a variety of assays, including immunofluorescence, ELISA, biolayer interferometry, western blots, and micro-neutralization. Remarkably, a high proportion of the antibodies we tested showed cross-reactivity, indicating a potentially generalizable theme of cross-reactivity between SARS-CoV and SARS-CoV-2 antibodies. These antibodies should help facilitate further research into SARS-CoV-2 basic biology. Moreover, our study provides critical information about the propensity of SARS-CoV antibodies to cross-react with SARS-CoV-2 and highlights its relevance in defining the clinical significance of such antibodies to improve testing and guide the development of novel vaccines and therapeutics.","version":"1.1","doi":"10.1101/2020.07.30.229377","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.227470","pub_date":"2020-7-30","title":"Ad26-vector based COVID-19 vaccine encoding a prefusion stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses","abstract":"Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g. prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S1) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild type signal peptide was best suited for the correct cleavage needed for a natively-folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-\u03b3. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).","version":"1.1","doi":"10.1101/2020.07.30.227470","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.229187","pub_date":"2020-7-30","title":"A dynamic regulatory interface on SARS-CoV-2 RNA polymerase","abstract":"The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design. Since it was first discovered and reported in late 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by highly contagious SARS-CoV-2 virus is wreaking havoc around the world. Currently, no highly effective and specific antiviral drug is available for clinical treatment. Therefore, the threat of COVID-19 transmission necessitates the discovery of more effective antiviral strategies. Viral RNA-dependent RNA polymerase (RdRp) is an important antiviral drug target. Here, our cryo-EM structure of a SARS-CoV-2 RdRp/RNA replicating complex reveals a previously uncharacterized overall shift of the cofactor nsp8(I)/nsp7 interface, leading to its rearrangement. Through in vitro functional test, we found that the specific interactions on the interface are important to the efficient RNA polymerase activity of SARS-CoV-2 RdRp. These observations let us to suggest this interface as a potential new drug intervention site, outside of the canonical polymerase active center, in RdRp for antiviral design. Our findings would provide new insights into regulatory mechanism of this novel SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and benefit the global public health.","version":"1.1","doi":"10.1101/2020.07.30.229187","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.140244","pub_date":"2020-7-30","title":"Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike","abstract":"The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2020.06.08.140244","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.122366","pub_date":"2020-7-30","title":"Natural selection in the evolution of SARS-CoV-2 in bats, not humans, created a highly capable human pathogen","abstract":"RNA viruses are proficient at switching host species, and evolving adaptations to exploit the new host\u2019s cells efficiently. Surprisingly, SARS-CoV-2 has apparently required no significant adaptation to humans since the start of the COVID-19 pandemic, with no observed selective sweeps since genome sampling began. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related bat Sarbecoviruses. In contrast, our analysis detects significant positive episodic diversifying selection acting on the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in ancestral hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor \u223c1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. Collectively our results demonstrate the progenitor of SARS-CoV-2 was capable of near immediate human-human transmission as a consequence of its adaptive evolutionary history in bats, not humans.","version":"1.2","doi":"10.1101/2020.05.28.122366","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228478","pub_date":"2020-7-30","title":"Substrate specificity of SARS-CoV-2 nsp10-nsp16 methyltransferase","abstract":"The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2) encodes all viral enzymes. In this work, we focus on one particular methyltransferase (MTase), nsp16, which in complex with nsp10 is capable of methylating the first nucleotide of a capped RNA strand at the 2\u2032-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered five types of capped RNA (m7Gp3A(G)-, Gp3A(G)- and Gp4A-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for measurement of 2\u2032-O-MTase activity. Further, we determined the IC50 value of sinefungin (286 \u00b1 66 nM) to illustrate the value of our approach for inhibitor screening. In the future, this approach can be used for screening inhibitors of any type of 2\u2032-O-MTase.","version":"1.1","doi":"10.1101/2020.07.30.228478","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.227462","pub_date":"2020-7-30","title":"SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors","abstract":"The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are highly expressed in innate immune cells in tissues susceptible to SARS-CoV-2 infection. Binding of the above receptors to S is characterized by affinities in the picomolar range and consistent with S glycosylation analysis demonstrating a variety of N- and O-glycans as receptor ligands. These results indicate multiple routes for SARS-CoV-2 to interact with human cells and suggest alternative strategies for therapeutic intervention.","version":"1.1","doi":"10.1101/2020.07.29.227462","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.071290","pub_date":"2020-7-30","title":"Protoporphyrin IX and verteporfin prevent SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2","abstract":"The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two FDA-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 \u03bcM and 0.31 \u03bcM respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human ACE2. The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.04.30.071290","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.227785","pub_date":"2020-7-30","title":"Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins","abstract":"The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including unambiguous O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30 O-glycosites were identified in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.","version":"1.1","doi":"10.1101/2020.07.29.227785","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228221","pub_date":"2020-7-30","title":"Immuno-informatics Design of a Multimeric Epitope Peptide Based Vaccine Targeting SARS-CoV-2 Spike Glycoprotein","abstract":"Developing an efficacious vaccine to SARS-CoV-2 infection is critical to stem COVID-19 fatalities and providing the global community with immune protection. We have used a bioinformatic approach to aid in the design of an epitope peptide-based vaccine against the spike protein of the virus. Five antigenic B cell epitopes with viable antigenicity and a total of 27 discontinuous B cell epitopes were mapped out structurally in the spike protein for antibody recognition. We identified eight CD8+ T cell 9-mers along with 12 CD4+ T cell 14-15-mer as promising candidate epitopes putatively restricted by a large number of MHC-I and II alleles respectively. We used this information to construct an in silico chimeric peptide vaccine whose translational rate was highly expressed when cloned in pET28a (+) vector. The vaccine construct was predicted to elicit high antigenicity and cell-mediated immunity when given as a homologous prime-boost, with triggering of toll-like receptor 5 by the adjuvant linker. The vaccine was characterized by an increase in IgM and IgG and an array of Th1 and Th2 cytokines. Upon in silico challenge with SARS-CoV-2, there was a decrease in antigen levels using our immune simulations. We therefore propose that potential vaccine designs consider this approach.","version":"1.1","doi":"10.1101/2020.07.30.228221","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.226555","pub_date":"2020-7-30","title":"The genetic variant analyses of SARS-CoV-2 strains; circulating in Bangladesh","abstract":"Genomic mutation of the virus may impact the viral adaptation to the local environment, their transmission, disease manifestation, and the effectiveness of existing treatment and vaccination. The objectives of this study were to characterize genomic variations, non-synonymous amino acid substitutions, especially in target proteins, mutation events per samples, mutation rate, and overall scenario of coronaviruses across the country. To investigate the genetic diversity, a total of 184 genomes of virus strains sampled from different divisions of Bangladesh with sampling dates between the 10th of May 2020 and the 27th of June 2020 were analyzed. To date, a total of 634 mutations located along the entire genome resulting in non-synonymous 274 amino acid substitutions in 22 different proteins were detected with nucleotide mutation rate estimated to be 23.715 substitutions per year. The highest non-synonymous amino acid substitutions were observed at 48 different positions of the papain-like protease (nsp3). Although no mutations were found in nsp7, nsp9, nsp10, and nsp11, yet orf1ab accounts for 56% of total mutations. Among the structural proteins, the highest non-synonymous amino acid substitution (at 36 positions) observed in spike proteins, in which 9 unique locations were detected relative to the global strains, including 516E>Q in the boundary of the ACE2 binding region. The most dominated variant G614 (95%) based in spike protein is circulating across the country with co-evolving other variants including L323 (94%) in RNA dependent RNA polymerase (RdRp), K203 (82%) and R204 (82%) in nucleocapsid, and F120 (78%) in NSP2. These variants are mostly seen as linked mutations and are part of a haplotype observed in Europe. Data suggest effective containment of clade G strains (4.8%) with sub-clusters GR 82.4%, and GH clade 6.4%. We have sequenced 137 and analyzed 184 whole-genomes sequences of SARS-CoV-2 strains from different divisions of Bangladesh. A total of 634 mutation sites across the SARS-CoV-2 genome and 274 non-synonymous amino acid substitutions were detected. The mutation rate of SARS-CoV-2 estimated to be 23.715 nucleotide substitutions per year. Nine unique variants were detected based on non-anonymous amino acid substitutions in spike protein relative to the global SARS-CoV-2 strains.","version":"1.2","doi":"10.1101/2020.07.29.226555","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228213","pub_date":"2020-7-30","title":"No SARS-CoV-2 cross-neutralization by intravenous immunoglobulins produced from plasma collected before the 2020 pandemic","abstract":"The 2020 SARS-CoV-2 pandemic is caused by a zoonotic coronavirus transmitted to humans, similar to earlier events. Whether the other, seasonally circulating coronaviruses induce cross-reactive, potentially even cross-neutralizing antibodies to the new species in humans is unclear. The question is of particular relevance for people with immune deficiencies, as their health depends on treatment with immunoglobulin preparations that need to contain neutralizing antibodies against the pathogens in their environment. Testing 54 IVIG preparations, produced from plasma collected in Europe and the US, highly potent neutralization of a seasonal coronavirus was confirmed, yet no cross-neutralization of the new SARS-CoV-2 was seen. IVIG products manufactured from pre-pandemic plasma do not neutralize SARS-CoV-2 but contain high neutralizing titers for seasonal coronavirus hCoV-229E.","version":"1.1","doi":"10.1101/2020.07.30.228213","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.20163626","pub_date":"2020-07-30","title":"Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA-extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65\u00b0C for 15 minutes; 80\u00b0C for 5 minutes; 90\u00b0C for 5 minutes or 80\u00b0C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90\u00b0C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be \u223c40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.</jats:p>","version":null,"doi":"10.1101/2020.07.28.20163626","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.29.227595","pub_date":"2020-7-30","title":"A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses","abstract":"In response to the health crisis presented by the COVID-19 pandemic, rapid development of safe and effective vaccines that elicit durable immune responses is imperative. Recent reports have raised the concern that antibodies in COVID-19 convalescent patients may not be long lasting and thus even these individuals may require vaccination. Vaccine candidates currently in clinical testing have focused on the SARS-CoV-2 wild type spike (S) protein (S-WT) as the major antigen of choice and while pre-clinical and early clinical testing have shown that S elicits an antibody response, we believe the optimal vaccine candidate should be capable of inducing robust, durable T-cell responses as well as humoral responses. We report here on a next generation bivalent human adenovirus serotype 5 (hAd5) vaccine capable of inducing immunity in patients with pre-existing adenovirus immunity, comprising both an S sequence optimized for cell surface expression (S-Fusion) and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal subcellular compartment, with the potential to generate durable immune protection. Our studies suggest that this bivalent vaccine is optimized for immunogenicity as evidenced by the following findings: (i) The optimized S-Fusion displayed improved S receptor binding domain (RBD) cell surface expression compared to S-WT where little surface expression was detected; (ii) the expressed RBD from S-Fusion retained conformational integrity and recognition by ACE2-Fc; (iii) the viral N protein modified with an enhanced T-cell stimulation domain (ETSD) localized to endosomal/lysosomal subcellular compartments for MHC I/II presentation; and (iv) these optimizations to S and N (S-Fusion and N-ETSD) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T-cell responses in antigen-naive pre-clinical models. Both the T-cell and antibody immune responses to S and N demonstrated a T-helper 1 (Th1) bias. The antibody responses were neutralizing as demonstrated by two independent SARS-CoV-2 neutralization assays. Based on these findings, we are advancing this next generation bivalent hAd5 S-Fusion + N-ETSD vaccine as our lead clinical candidate to test for its ability to provide robust, durable cell-mediated and humoral immunity against SARS-CoV-2 infection. Further studies are ongoing to explore utilizing this vaccine construct in oral, intranasal, and sublingual formulations to induce mucosal immunity in addition to cell-mediated and humoral immunity. The ultimate goal of an ideal COVID-19 vaccine is to generate long-term T and B cell memory.","version":"1.1","doi":"10.1101/2020.07.29.227595","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.227553","pub_date":"2020-7-30","title":"Circulating Mitochondrial DNA is an Early Indicator of Severe Illness and Mortality from COVID-19","abstract":"Mitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether the appearance of cell-free MT-DNA is linked to poor COVID-19 outcomes remains undetermined. Here, we quantified circulating MT-DNA in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at the time of hospital presentation. Circulating MT-DNA were sharply elevated in patients who eventually died, required ICU admission or intubation. Multivariate regression analysis revealed that high circulating MT-DNA levels is an independent risk factor for all of these outcomes after adjusting for age, sex and comorbidities. Additionally, we found that circulating MT-DNA has a similar or superior area-under-the curve when compared to clinically established measures of systemic inflammation, as well as emerging markers currently of interest as investigational targets for COVID-19 therapy. These results show that high circulating MT-DNA levels is a potential indicator for poor COVID-19 outcomes.","version":"1.1","doi":"10.1101/2020.07.30.227553","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.30.228643","pub_date":"2020-7-30","title":"Perception of and anxiety about COVID-19 infection and risk behaviors for spreading infection: An international comparison","abstract":"To control the spread of the newly developed corona viral infection diseases (COVID-19), people\u2019s appropriate precautionary behaviors should be promoted. We conducted a series of online questionnaire survey, to gather a total of 8,000 citizen\u2019s responses on March 27\u201328, 2020 in Japan and April 17\u201321 in the UK and Spain. Compared to Japan, the knowledge and anxiety level and the frequency of precautionary behaviors were higher in the UK and Spain. Participants with infected acquaintances were more concerned about COVID-19. However, participants in the UK rarely wore a medical mask. Participants in the UK and Spain were eager to get information about COVID-19 compared to those in Japan. The participants in Spain tended not to trust official information and to believe specialists\\' comments instead. The urgency of the spread of COVID-19, cultural backgrounds, and recent political situations appear to contribute to the differences among countries revealed herein.","version":"1.1","doi":"10.1101/2020.07.30.228643","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.227249","pub_date":"2020-7-29","title":"Structural and functional analysis of female sex hormones against SARS-Cov2 cell entry","abstract":"Emerging evidence suggests that males are more susceptible to severe infection by the SARS-CoV-2 virus than females. A variety of mechanisms may underlie the observed gender-related disparities including differences in sex hormones. However, the precise mechanisms by which female sex hormones may provide protection against SARS-CoV-2 infectivity remains unknown. Here we report new insights into the molecular basis of the interactions between the SARS-CoV-2 spike (S) protein and the human ACE2 receptor. We further observed that glycosylation of the ACE2 receptor enhances SARS-CoV-2 infectivity. Importantly estrogens can disrupt glycan-glycan interactions and glycan-protein interactions between the human ACE2 and the SARS-CoV2 thereby blocking its entry into cells. In a mouse model, estrogens reduced ACE2 glycosylation and thereby alveolar uptake of the SARS-CoV-2 spike protein. These results shed light on a putative mechanism whereby female sex hormones may provide protection from developing severe infection and could inform the development of future therapies against COVID-19.","version":"1.1","doi":"10.1101/2020.07.29.227249","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.225649","pub_date":"2020-7-29","title":"Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model","abstract":"Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. Here, we assess immunogenicity and anamnestic protective efficacy in rhesus macaques of the intradermal (ID)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800. INO-4800 is an ID-delivered DNA vaccine currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and neutralizing antibody responses against both the D614 and G614 SARS-CoV-2 spike proteins. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T and B cell responses. These responses were associated with lower viral loads in the lung and with faster nasal clearance of virus. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against COVID-19 disease.","version":"1.1","doi":"10.1101/2020.07.28.225649","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.226092","pub_date":"2020-7-29","title":"Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stem cell-derived airway repair response","abstract":"Most demographic studies are now associating current smoking status with increased risk of severe COVID-19 and mortality from the disease but there remain many questions about how direct cigarette smoke exposure affects SARS-CoV-2 airway cell infection. We directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We found an increase in the number of infected airway cells after cigarette smoke exposure as well as an increased number of apoptotic cells. Cigarette smoke exposure alone caused airway injury that resulted in an increased number of ABSCs, which proliferate to repair the airway. But we found that acute SARS-CoV-2 infection or the combination of exposure to cigarette smoke and SARS-CoV-2 did not induce ABSC proliferation. We set out to examine the underlying mechanism governing the increased susceptibility of cigarette smoke exposed ALI to SARS-CoV-2 infection. Single cell profiling of the cultures showed that infected airway cells displayed a global reduction in gene expression across all airway cell types. Interestingly, interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in the ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response. Treatment of cigarette smoke-exposed ALI cultures with Interferon \u03b2-1 abrogated the viral infection, suggesting that the lack of interferon response in the cigarette smoke-exposed ALI cultures allows for more severe viral infection and cell death. In summary, our data show that acute smoke exposure allows for more severe proximal airway epithelial disease from SARS-CoV-2 by reducing the mucosal innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to cigarette smoke.","version":"1.1","doi":"10.1101/2020.07.28.226092","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.227389","pub_date":"2020-7-29","title":"On the molecular mechanism of SARS-CoV-2 retention in the upper respiratory tract","abstract":"Cell surface receptor engagement is a critical aspect of viral infection. At low pH, binding of SARS-CoV and its ACE2 receptor has a tight interaction that catalyzes the fusion of the spike and endosomal membranes followed by genome release. Largely overlooked has been the role of neutral pH in the respiratory tract, where we find that SARS-CoV stabilizes a transition state that enhances the off-rate from its receptor. An alternative pH-switch is found in CoV-2-like coronaviruses of tropical pangolins, but with a reversed phenotype where the tight interaction with ACE2 is at neutral pH. We show that a single point mutation in pangolin-CoV, unique to CoV-2, that deletes the last His residue in their receptor binding domain perpetuates this tight interaction independent of pH. This tight bond, not present in previous respiratory syndromes, implies that CoV-2 stays bound to the highly expressed ACE2 receptors in the nasal cavity about 100 times longer than CoV. This finding supports the unfamiliar pathology of CoV-2, observed virus retention in upper respiratory tract, longer incubation times and extended periods of shedding. Implications to combat pandemics that, like SARS-CoV-2, export evolutionarily successful strains via higher transmission rates due to retention in nasal epithelium and their evolutionary origin are discussed.","version":"1.1","doi":"10.1101/2020.07.29.227389","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.226761","pub_date":"2020-7-29","title":"Allosteric inhibition of the SARS-CoV-2 main protease \u2013 insights from mass spectrometry-based assays","abstract":"Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 \u00b1 0.03 \u03bcM, revealing MPro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively \u201cflash-frozen\u201d as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.","version":"1.1","doi":"10.1101/2020.07.29.226761","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.29.226217","pub_date":"2020-7-29","title":"Impact Analysis of SARS-CoV2 on Signaling Pathways during COVID19 Pathogenesis using Codon Usage Assisted Host-Viral Protein Interactions","abstract":"Understanding the molecular mechanism of COVID19 disease pathogenesis helps in the rapid development of therapeutic targets. Usually, viral protein targets host proteins in an organized fashion. The pathogen may target cell signaling pathways to disrupt the pathway genes\u2019 regular activities, resulting in disease. Understanding the interaction mechanism of viral and host proteins involved in different signaling pathways may help decipher the attacking mechanism on the signal transmission during diseases, followed by discovering appropriate therapeutic solutions. The expression of any viral gene depends mostly on the host translational machinery. Recent studies report the great significance of codon usage biases in establishing host-viral protein-protein interactions (PPI). Exploiting the codon usage patterns between a pair of co-evolved host and viral proteins may present novel insight into the host-viral protein interactomes during disease pathogenesis. Leveraging the codon usage pattern similarity (and dissimilarity), we propose a computational scheme to recreate the hostviral protein interaction network (HVPPI). We use seventeen (17) essential signaling pathways for our current work and study the possible targeting mechanism of SARS-CoV2 viral proteins on such pathway proteins. We infer both negatively and positively interacting edges in the network. We can find a relationship where one host protein may target by more than one viral protein. Extensive analysis performed to understand the network topologically and the attacking behavior of the viral proteins. Our study reveals that viral proteins, mostly utilize codons, rare in the targeted host proteins (negatively correlated interaction). Among non-structural proteins, NSP3 and structural protein, Spike (S) protein, are the most influential proteins in interacting multiple host proteins. In ranking the most affected pathways, MAPK pathways observe to be worst affected during the COVID-19 disease. A good number of targeted proteins are highly central in host protein interaction networks. Proteins participating in multiple pathways are also highly connected in their own PPI and mostly targeted by multiple viral proteins.","version":"1.1","doi":"10.1101/2020.07.29.226217","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.20162842","pub_date":"2020-07-29","title":"Association of D-dimer and fibrinogen magnitude with hypercoagulability by thromboelastography in severe COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>D-dimer concentration has been used to identify candidates for intensified anticoagulant treatment for both venous thromboembolism prevention and mitigation of the microthrombotic complications associated with COVID-19. Thromboelastography (TEG) maximum amplitude (MA) has been validated as an indicator of hypercoagulability and MA \u2265 68 mm has been utilized as a marker of hypercoagulability in other conditions. We evaluated the relationship between coagulation, inflammatory, and TEG parameters in patients with COVID-19 on extracorporeal membrane oxygenation (ECMO).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We performed a single center retrospective analysis of consecutive patients that received ECMO for the treatment of COVID-19. TEG, inflammatory, and coagulation markers were compared in patients with and without thrombotic complications. Correlation tests were performed to identify the coagulation and inflammatory markers that best predict hypercoagulability as defined by an elevated TEG MA.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>168 TEGs were available in 24 patients. C-reactive protein and fibrinogen were significantly higher in patients that developed a thrombotic event versus those that did not (p=0.038 and p=0.043 respectively). D-dimer was negatively correlated with TEG MA (p&lt;0.001) while fibrinogen was positively correlated (p&lt;0.001). A fibrinogen &gt; 441 mg/dL had a sensitivity of 91.2% and specificity of 85.7% for the detection of MA \u2265 68 mm.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>In critically ill patients with COVID-19, D-dimer concentration had an inverse relationship with hypercoagulability as measured by TEG MA. D-dimer elevation may reflect severity of COVID-19 related sepsis rather than designate patients likely to benefit from anticoagulation. Fibrinogen concentration may represent a more useful marker of hypercoagulability in this population.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.07.27.20162842","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.27.20161976","pub_date":"2020-07-28","title":"Exploratory analysis of immunization records highlights decreased SARS-CoV-2 rates in individuals with recent non-COVID-19 vaccinations","abstract":"<jats:p>Multiple clinical studies are ongoing to assess whether existing vaccines may afford protection against SARS-CoV-2 infection through trained immunity. In this exploratory study, we analyze immunization records from 137,037 individuals who received SARS-CoV-2 PCR tests. We find that polio, Hemophilus influenzae type-B (HIB), measles-mumps-rubella (MMR), varicella, pneumococcal conjugate (PCV13), geriatric flu, and hepatitis A / hepatitis B (HepA-HepB) vaccines administered in the past 1, 2, and 5 years are associated with decreased SARS-CoV-2 infection rates, even after adjusting for geographic SARS-CoV-2 incidence and testing rates, demographics, comorbidities, and number of other vaccinations. Furthermore, age, race/ethnicity, and blood group stratified analyses reveal significantly lower SARS-CoV-2 rate among black individuals who have taken the PCV13 vaccine, with relative risk of 0.45 at the 5 year time horizon (n: 653, 95% CI: (0.32, 0.64), p-value: 6.9e-05). These findings suggest that additional pre-clinical and clinical studies are warranted to assess the protective effects of existing non-COVID-19 vaccines and explore underlying immunologic mechanisms. We note that the findings in this study are preliminary and are subject to change as more data becomes available and as further analysis is conducted.</jats:p>","version":null,"doi":"10.1101/2020.07.27.20161976","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.26.221861","pub_date":"2020-7-28","title":"Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as vaccine candidate","abstract":"Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus. Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type or a pre-fusion membrane anchored format. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, we report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs. The spike (S) protein of the SARS-CoV-2 is the major antigen that notably induces neutralizing antibodies to block viral entry. Many COVID-19 vaccines are under development, among them viral vectors expressing the S protein of SARS-CoV-2 exhibit many benefits. Viral vector vaccines have the potential of being used as both live or inactivated vaccines and they can induce Th1 and Th2-based immune responses following different immunization regimens. Additionally, viral vector vaccines can be handled under BSL-2 conditions and they grow to high titers in cell cultures or other species restricted-hosts. For a SARS-CoV-2 vaccine, several viral vectors are being tested, such as adenovirus, measles virus and Modified vaccinia Ankara. The NDV vector vaccine against SARS-CoV-2 described in this study has advantages similar to those of other viral vector vaccines. But the NDV vector can be amplified in embryonated chicken eggs, which allows for high yields and low costs per dose. Also, the NDV vector is not a human pathogen, therefore the delivery of the foreign antigen would not be compromised by any pre-existing immunity in humans. Finally, NDV has a very good safety record in humans, as it has been used in many oncolytic virus trials. This study provides an important option for a cost-effective SARS-CoV-2 vaccine. This study informs of the value of a viral vector vaccine against SARS-CoV-2. Specifically, for this NDV based SARS-CoV-2 vaccine, the existing egg-based influenza virus vaccine manufactures in the U.S. and worldwide would have the capacity to rapidly produce hundreds of millions of doses to mitigate the consequences of the ongoing COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.07.26.221861","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.225151","pub_date":"2020-7-28","title":"Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism","abstract":"Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.07.28.225151","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.223784","pub_date":"2020-7-28","title":"A Speedy Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors","abstract":"The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain \u03b2-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 \u03bcM and A549 cells at 0.16-0.31 \u03bcM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.","version":"1.1","doi":"10.1101/2020.07.28.223784","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.225102","pub_date":"2020-7-28","title":"Pyronaridine and artesunate are potential antiviral drugs against COVID-19 and influenza","abstract":"Since the first human case was reported in Wuhan Province, China in December 2019, SARS-CoV-2 has caused millions of human infections in more than 200 countries worldwide with an approximately 4.01% case-fatality rate (as of 27 July, 2020; based on a WHO situation report), and COVID-19 pandemic has paralyzed our global community. Even though a few candidate drugs, such as remdesivir (a broad antiviral prodrug) and hydroxychloroquine, have been investigated in human clinical trials, their therapeutic efficacy needs to be clarified further to be used to treat COVID-19 patients. Here we show that pyronaridine and artesunate, which are the chemical components of anti-malarial drug Pyramax\u00ae, exhibit antiviral activity against SARS-CoV-2 and influenza viruses. In human lung epithelial (Calu-3) cells, pyronaridine and artesunate were highly effective against SARS-CoV-2 while hydroxychloroquine did not show any effect at concentrations of less than 100 \u03bcM. In viral growth kinetics, both pyronaridine and artesunate inhibited the growth of SARS-CoV-2 and seasonal influenza A virus in Calu-3 cells. Taken together, we suggest that artesunate and pyronaridine might be effective drug candidates for use in human patients with COVID-19 and/or influenza, which may co-circulate during this coming winter season.","version":"1.1","doi":"10.1101/2020.07.28.225102","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.224733","pub_date":"2020-7-28","title":"SARS-CoV-2 in-vitro neutralization assay reveals inhibition of virus entry by iota-carrageenan","abstract":"In the absence of a vaccine and other effective prophylactic or therapeutic countermeasures the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) remains a significant public health threat. Attachment and entry of coronaviruses including SARS-CoV-2 is mediated by the spike glycoprotein (SGP). Recently, a SARS-CoV-2 Spike Pseudotyped Lentivirus (SSPL) was developed that allows studying spike-mediated cell entry via luciferase reporter activity in a BSL2 environment. Here, we show that iota-carrageenan can inhibit the cell entry of SSPL in a dose dependent manner. SSPL particles were efficiently neutralized with an IC50 value of 2.6 \u00b5g/ml iota-carrageenan. In vitro data on iota-carrageenan against various Rhino- and Coronaviruses showed similar IC50 values and translated readily into clinical effectiveness when a nasal spray containing iota-carrageenan demonstrated a reduction in severity and duration of symptoms of common cold caused by various respiratory viruses. Accordingly, our in vitro data on SSPL suggest that administration of iota-carrageenan may be an effective and safe prophylaxis or treatment for SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2020.07.28.224733","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.140236","pub_date":"2020-7-28","title":"Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives","abstract":"Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 \u00b5g/L, 1.08 mg/L, 1.77 \u00b5g/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.","version":"1.2","doi":"10.1101/2020.06.08.140236","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.224089","pub_date":"2020-7-28","title":"Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries","abstract":"Antibodies with high affinity against the receptor binding domain (RBD) of the SARS-CoV-2 S1 ectodomain were identified from screens using the Retained Display\u2122 (ReD) platform employing a 1 \u00d7 1011 clone single-chain antibody (scFv) library. Numerous unique scFv clones capable of inhibiting binding of the viral S1 ectodomain to the ACE2 receptor in vitro were characterized. To maximize avidity, selected clones were reformatted as bivalent diabodies and monoclonal antibodies (mAb). The highest affinity mAb completely neutralized live SARS-CoV-2 virus in cell culture for four days at a concentration of 6.7 nM, suggesting potential therapeutic and/or prophylactic use. Furthermore, scFvs were identified that greatly increased the interaction of the viral S1 trimer with the ACE2 receptor, with potential implications for vaccine development.","version":"1.1","doi":"10.1101/2020.07.27.224089","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214064","pub_date":"2020-7-28","title":"DeepTracer: Fast Cryo-EM Protein Structure Modeling and Special Studies on CoV-related Complexes","abstract":"Information about macromolecular structure of protein complexes such as SARS-CoV-2, and related cellular and molecular mechanisms can assist the search for vaccines and drug development processes. To obtain such structural information, we present DeepTracer, a fully automatic deep learning-based method for fast de novo multi-chain protein complex structure determination from high-resolution cryo-electron microscopy (cryo-EM) density maps. We applied DeepTracer on a previously published set of 476 raw experimental density maps and compared the results with a current state of the art method. The residue coverage increased by over 30% using DeepTracer and the RMSD value improved from 1.29\u00c5 to 1.18\u00c5. Additionally, we applied DeepTracer on a set of 62 coronavirus-related density maps, among them 10 with no deposited structure available in EMDataResource. We observed an average residue match of 84% with the deposited structures and an average RMSD of 0.93\u00c5. Additional tests with related methods further exemplify DeepTracer\u2019s competitive accuracy and efficiency of structure modeling. DeepTracer allows for exceptionally fast computations, making it possible to trace around 60,000 residues in 350 chains within only two hours. The web service is globally accessible at https://deeptracer.uw.edu.","version":"1.2","doi":"10.1101/2020.07.21.214064","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.28.224576","pub_date":"2020-7-28","title":"Rapid host response to an infection with Coronavirus. Study of transcriptional responses with Porcine Epidemic Diarrhea Virus","abstract":"The transcriptional response in Vero cells (ATCC\u00ae CCL-81) infected with the coronavirus Porcine Epidemic Diarrhea Virus (PEDV) was measured by RNAseq analysis 4 and 6 hours after infection. Differential expressed genes (DEGs) in PEDV infected cells were compared to DEGs responding in Vero cells infected with Mammalian Orthoreovirus (MRV). Functional analysis of MRV and PEDV DEGs showed that MRV increased the expression level of several cytokines and chemokines (e.g. IL6, CXCL10, IL1A, CXCL8 [alias IL8]) and antiviral genes (e.g. IFI44, IFIT1, MX1, OASL), whereas for PEDV no enhanced expression was observed for these \u201challmark\u201d antiviral and immune effector genes. Pathway and Gene Ontology \u201cenrichment analysis\u201d revealed that PEDV infection did not stimulate expression of genes able to activate an acquired immune response, whereas MRV did so within 6h. Instead, PEDV down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene TMPRSS13 (alias MSPL) to support its own infection by virus-cell membrane fusion (Shi et al, 2017, Viruses, 9(5):114). PEDV also down-regulated expression of Ectodysplasin A, a cytokine of the TNF-family able to activate the canonical NFKB-pathway responsible for transcription of inflammatory genes like IL1B, TNF, CXCL8 and PTGS2. The only 2 cytokine genes found up-regulated by PEDV were Cardiotrophin-1, an IL6-type cytokine with pleiotropic functions on different tissues and types of cells, and Endothelin 2, a neuroactive peptide with vasoconstrictive properties. Furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of PEDV-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and \u201csynaptic clefts\u201d between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). The information in this study describing a \u201cvery early\u201d response of epithelial cells to an infection with a coronavirus may provide pharmacologists, immunological and medical specialists additional insights in the underlying mechanisms of coronavirus associated severe clinical symptoms including those induced by SARS-CoV-2. This may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat COVID-19 patients.","version":"1.1","doi":"10.1101/2020.07.28.224576","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222026","pub_date":"2020-7-27","title":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) Protein Inhibits Type I and III Interferon Production by Targeting RIG-I/MDA-5 Signaling","abstract":"The coronavirus disease 2019 (COVID-19) caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has quickly spread worldwide and has infected more than ten million individuals. One of the typical features of COVID-19 is that both type I and III interferon (IFN)-mediated antiviral immunity are suppressed. However, the molecular mechanism by which SARS-CoV-2 evades this antiviral immunity remains elusive. Here, we report that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway of RIG-I/MDA-5-MAVS signaling. The SARS-CoV2 M protein also dampens type I and III IFN induction stimulated by Sendai virus infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1 and prevents the formation of a multi-protein complex containing RIG-I, MAVS, TRAF3, and TBK1, thus impeding IRF3 phosphorylation, nuclear translocation, and activation. Consequently, the ectopic expression of the SARS-CoV2 M protein facilitates the replication of vesicular stomatitis virus (VSV). Taken together, the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of the SARS-CoV-2-induced antiviral immune suppression and sheds light on the pathogenic mechanism of COVID-19.","version":"1.1","doi":"10.1101/2020.07.26.222026","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.223495","pub_date":"2020-7-27","title":"Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals","abstract":"The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.","version":"1.1","doi":"10.1101/2020.07.27.223495","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.220954","pub_date":"2020-7-27","title":"Molecular mechanisms of Cardiac Injury associated with myocardial SARS-CoV-2 infection","abstract":"Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread around the world. Developing cardiac injury is a common condition in COVID-19 patients, but the pathogenesis remains unclear. The RNA-Seq dataset (GES150392) compared expression profiling of mock human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and SARS-cov-2-infected hiPSC-CMs were obtained from Gene Expression Omnibus (GEO). We identified the differentially expressed genes (DEGs) between those two groups. Through gene set enrichment analysis (GSEA), Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and CLINVAR human diseases analysis to identify the main effect of SARS-CoV-2 on cardiomyocytes. A protein-protein interaction (PPI) network was constructed to visualize interactions and functions of the hub genes. A total of 1554 DEGs were identified (726 upregulated genes and 828 downregulated genes). Gene enrichment analysis shown that SARS-CoV-2 activate immuno-inflammatory responses via multiple signal pathways, including TNF\u03b1, IL6-JAK-STAT3, IL2-STAT5, NF-\u03baB, IL17, and Toll-like receptor signaling pathway in hiPSC-CMs. Whereas, the muscle contraction, cellular respiration and cell cycle of hiPSC-CMs were inhibited by SARS-CoV-2. CLINVAR human diseases analysis shown SRAS-Cov-2 infection was associated with myocardial infarction, cardiomyopathy and Limb-girdle muscular dystrophy. 15 hub genes were identified based on PPI network. Function analysis revealed that 11 upregulated hub genes were mainly enriched in cytokine activity, chemokine activity, Inflammatory response, leukocyte chemotaxis, and lipopolysaccharide-mediated signaling pathway. Furthermore, 4 downregulated hub genes were related to cell cycle regulation. The present study elucidates that the SARS-CoV-2 infection induced a strong defensive response in cardiomyocyte, leading to excessive immune inflammation, cell hypoxia, functional contractility reduction and apoptosis, ultimately result in myocardial injury.","version":"1.1","doi":"10.1101/2020.07.27.220954","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.190561","pub_date":"2020-7-27","title":"Antiviral effects of miRNAs in extracellular vesicles against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mutations in SARS-CoV-2 RNA virus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 2019 (COVID-19). No treatment is available. Micro-RNAs (miRNAs) in mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are potential novel therapeutic agents because of their ability to regulate gene expression by inhibiting mRNA. Thus, they may degrade the RNA genome of SARS-CoV-2. EVs can transfer miRNAs to recipient cells and regulate conditions within them. MSC-EVs harbor major therapeutic miRNAs that play important roles in the biological functions of virus-infected host cells. Here, we examined their potential impact on viral and immune responses. MSC-EVs contained 18 miRNAs predicted to interact directly with the 3\u2019 UTR of SARS-CoV-2. These EVs suppressed SARS-CoV-2 replication in Vero E6 cells. In addition, five major miRNAs suppressed virus activity in a luciferase reporter assay by binding the 3\u2019 UTR. MSC-EVs showed strong regenerative effects and potent anti-inflammatory activity which may prevent lethal cytokine storms. We confirmed that EVs regulated inflammatory responses by several cell types, including human brain cells that express the viral receptor ACE2, suggesting that the brain may be targeted by SARS-CoV-2. miRNAs in MSC-EVs have several advantages as therapeutic agents against SARS-CoV-2: 1) they bind specifically to the viral 3\u2019 UTR, and are thus unlikely to have side effects; 2) because the 3\u2019 UTR is highly conserved and rarely mutates, MSC-EV miRNAs could be used against novel variants arising during viral replication; and 3) unique cargoes carried by MSC-EVs can have diverse effects, such as regenerating damaged tissue and regulating immunity.","version":"1.1","doi":"10.1101/2020.07.27.190561","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.23.112235","pub_date":"2020-7-27","title":"Bepridil is potent against SARS-CoV-2 In Vitro","abstract":"Guided by a computational docking analysis, about 30 FDA/EMA-approved small molecule medicines were characterized on their inhibition of the SARS-CoV-2 main protease (MPro). Of these tested small molecule medicines, six displayed an IC50 value in inhibiting MPro below 100 \u03bcM. Three medicines pimozide, ebastine, and bepridil are basic small molecules. Their uses in COVID-19 patients potentiate dual functions by both raising endosomal pH to slow SARS-CoV-2 entry into the human cell host and inhibiting MPro in infected cells. A live virus-based microneutralization assay showed that bepridil inhibited cytopathogenic effect induced by SARS-CoV-2 in Vero E6 cells completely at and dose-dependently below 5 \u03bcM and in A549 cells completely at and dose-dependently below 6.25 \u03bcM. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.","version":"1.2","doi":"10.1101/2020.05.23.112235","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.218198","pub_date":"2020-7-27","title":"Host transcriptional responses and SARS-CoV-2 isolates from the nasopharyngeal samples of Bangladeshi COVID-19 patients","abstract":"As the COVID-19 pandemic progresses, fatality and cases of new infections are also increasing at an alarming rate. SARS-CoV-2 follows a highly variable course and it is becoming more evident that individual\u2019s immune system has a decisive influence on the progression of the disease. However, the detailed underlying molecular mechanisms of the SARS-CoV-2 mediate disease pathogenesis are largely unknown. Only a few host transcriptional responses in COVID-19 have been reported so far from the Western world, but no such data has been generated from the South-Asian region yet to correlate the conjectured lower fatality around this part of the globe. In this context, we aimed to perform the transcriptomic profiling of the COVID-19 patients from Bangladesh along with the reporting of the SARS-CoV-2 isolates from these patients. Moreover, we performed a comparative analysis to demonstrate how differently the various SARS-CoV-2 infection systems are responding to the viral pathogen. We detected a unique missense mutation at 10329 position of ORF1ab gene, annotated to 3C like proteinase, which is found in 75% of our analyzed isolates; but is very rare globally. Upon the functional enrichment analyses of differentially modulated genes, we detected a similar host induced response reported earlier; this response was mainly mediated by the innate immune system, interferon stimulation, and upregulated cytokine expression etc. in the Bangladeshi patients. Surprisingly, we did not perceive the induction of apoptotic signaling, phagosome formation, antigen presentation and production, hypoxia response within these nasopharyngeal samples. Furthermore, while comparing with the other SARS-CoV-2 infection systems, we spotted that lung cells trigger the more versatile immune and cytokine signaling which was several folds higher compared to our reported nasopharyngeal samples. We also observed that lung cells did not express ACE2 in a very high amount as suspected, however, the nasopharyngeal cells are found overexpressing ACE2. But the amount of DPP4 expression within the nasal samples was significantly lower compared to the other cell types. Surprisingly, we observed that lung cells express a very high amount of integrins compared to the nasopharyngeal samples, which might suggest the putative reasons for an increased amount of viral infections in the lungs. From the network analysis, we got clues on the probable viral modulation for the overexpression of these integrins. Our data will provide valuable insights in developing potential studies to elucidate the roles of ethnicity effect on the viral pathogenesis, and incorporation of further data will enrich the search of an effective therapeutics.","version":"1.2","doi":"10.1101/2020.07.23.218198","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.082768","pub_date":"2020-7-27","title":"Mutation landscape of SARS-CoV-2 reveals five mutually exclusive clusters of leading and trailing single nucleotide substitutions","abstract":"The COVID-19 pandemic has spread across the globe at an alarming rate. However, unlike any of the previous global outbreaks the availability of a large number of SARS-CoV-2 sequences provides us with a unique opportunity to understand viral evolution in real time. We analysed 1448 full-length (>29000 nt) sequences available and identified 40 single-nucleotide substitutions occurring in >1% of the genomes. Majority of the substitutions were C to T or G to A. We identify C/Gs with an upstream TTT trinucleotide motif as hotspots for mutations in the SARS-CoV-2 genome. Interestingly, three of the 40 substitutions occur within highly conserved secondary structures in the 5\u2019 and 3\u2019 regions of the genomic RNA that are critical for the virus life cycle. Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. Of note, we observed several co-occurring mutations that almost never occur individually. We define five mutually exclusive lineages (A1, B1, C1, D1 and E1) of SARS-CoV-2 which account for about three quarters of the genomes analysed. We identify lineage-defining leading mutations in the SARS-CoV-2 genome which precede the occurrence of sub-lineage defining trailing mutations. The identification of mutually exclusive lineage-defining mutations with geographically restricted patterns of distribution has potential implications for diagnosis, pathogenesis and vaccine design. Our work provides novel insights on the temporal evolution of SARS-CoV-2. The SARS-CoV-2 / COVID-19 pandemic has spread far and wide with high infectivity. However, the severeness of the infection as well as the mortality rates differ greatly across different geographic areas. Here we report high frequency mutations in the SARS-CoV-2 genomes which show the presence of linage-defining, leading and trailing mutations. Moreover, we propose for the first time, five mutually exclusive clusters of SARS-CoV-2 which account for 75% of the genomes analysed. This will have implications in diagnosis, pathogenesis and vaccine design","version":"1.2","doi":"10.1101/2020.05.07.082768","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.223172","pub_date":"2020-7-27","title":"A-to-I RNA editing in SARS-COV-2: real or artifact?","abstract":"ADAR1-mediated deamination of adenosines in long double stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2 infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions and are, in the majority of cases, nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.","version":"1.1","doi":"10.1101/2020.07.27.223172","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222257","pub_date":"2020-7-27","title":"An antibody-dependent enhancement (ADE) activity eliminated neutralizing antibody with potent prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys","abstract":"Efficacious interventions are urgently needed for the treatment of COVID-19. Here, we report a monoclonal antibody (mAb), MW05, showing high SARS-CoV-2 neutralizing activity by disrupting the interaction of receptor binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) receptor. Crosslinking of Fc with Fc\u03b3RIIB mediates antibody-dependent enhancement (ADE) activity by MW05. This activity was eliminated by introducing the LALA mutation to the Fc region (MW05/LALA). Most importantly, potent prophylactic and therapeutic effects against SARS-CoV-2 were observed in rhesus monkeys. A single dose of MW05/LALA completely blocked the infection of SARS-CoV-2 in a study of its prophylactic effect and totally cleared SARS-CoV-2 in three days in a treatment setting. These results pave the way for the development of MW05/LALA as an effective strategy for combating COVID-19.","version":"1.1","doi":"10.1101/2020.07.26.222257","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.222943","pub_date":"2020-7-27","title":"Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV","abstract":"A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay (\u2018PepSeq\u2019) to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.","version":"1.1","doi":"10.1101/2020.07.27.222943","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.212076","pub_date":"2020-7-27","title":"Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures","abstract":"The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 \u201capical-out\u201d organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.","version":"1.1","doi":"10.1101/2020.07.27.212076","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.213405","pub_date":"2020-7-27","title":"Multi-site co-mutations and 5\u2019UTR CpG immunity escape drive the evolution of SARS-CoV-2","abstract":"The SARS-CoV-2 infected cases and the caused mortalities have been surging since the COVID-19 pandemic. Viral mutations emerge during the virus circulating in the population, which is shaping the viral infectivity and pathogenicity. Here we extensively analyzed 6698 SARS-CoV-2 whole genome sequences with specific sample collection dates in NCBI database. We found that four mutations, i.e., 5\u2019UTR_c-241-t, NSP3_c-3037-t, NSP12_c-14408-t, and S_a-23403-g, became the dominant variants and each of them represented nearly 100% of all virus sequences since the middle May, 2020. Notably, we found that co-occurrence rates of three significant multi-site co-mutational patterns, i.e., (i) S_a-23403-g, NSP12_c-14408-t, 5\u2019UTR_c-241-t, NSP3_c-3037-t, and ORF3a_c-25563-t; (ii) ORF8_t-28144-c, NSP4_c-8782-t, NSP14_c-18060-t, NSP13_a-17858-g, and NSP13_c-17747-t; and (iii) N_g-28881-a, N_g-28882-a, and N_g-28883-c, reached 66%, 90%, and nearly 100% of recent sequences, respectively. Moreover, we found significant decrease of CpG dinucleotide at positions 241(c)-242(g) in the 5\u2019UTR during the evolution, which was verified as a potential target of human zinc finger antiviral protein (ZAP). The four dominant mutations, three significant multi-site co-mutations, and the potential escape mutation of ZAP-target in 5\u2019UTR region contribute to the rapid evolution of SARS-CoV-2 virus in the population, thus shaping the viral infectivity and pathogenicity. This study provides valuable clues and frameworks to dissect the viral replication and virus-host interactions for designing effective therapeutics. Four dominant mutations, three significant multi-site co-mutations, and 5\u2019UTR CpG escape contribute to the rapid evolution of SARS-CoV-2 virus.","version":"1.3","doi":"10.1101/2020.07.21.213405","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.223578","pub_date":"2020-7-27","title":"Massive-scale biological activity-based modeling identifies novel antiviral leads against SARS-CoV-2","abstract":"The recent global pandemic caused by the new coronavirus SARS-CoV-2 presents an urgent need for new therapeutic candidates. While the importance of traditional in silico approaches such as QSAR in such efforts in unquestionable, these models fundamentally rely on structural similarity to infer biological activity and are thus prone to becoming trapped in the very nearby chemical spaces of already known ligands. For novel and unprecedented threats such as COVID-19 much faster and efficient paradigms must be devised to accelerate the identification of new chemical classes for rapid drug development. Here we report the development of a new biological activity-based modeling (BABM) approach that builds on the hypothesis that compounds with similar activity patterns tend to share similar targets or mechanisms of action. In BABM, compound activity profiles established on massive scale across multiple assays are used as signatures to predict compound activity in a new assay or against a new target. We first trained and validated this approach by identifying new antiviral lead candidates for Zika and Ebola based on data from ~0.5 million compounds screened against ~2,000 assays. BABM models were then applied to predict ~300 compounds not previously reported to have activity for SARS-CoV-2, which were then tested in a live virus assay with high (>30%) hit rates. The most potent compounds showed antiviral activities in the nanomolar range. These potent confirmed compounds have the potential to be further developed in novel chemical space into new anti-SARS-CoV-2 therapies. These results demonstrate unprecedented ability using BABM to predict novel structures as chemical leads significantly beyond traditional methods, and its application in rapid drug discovery response in a global public health crisis.","version":"1.1","doi":"10.1101/2020.07.27.223578","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.223727","pub_date":"2020-7-27","title":"Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L","abstract":"The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a \u03b3-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1\u2019 pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1\u2019 pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1\u2019, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the \u03b1-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.","version":"1.1","doi":"10.1101/2020.07.27.223727","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222232","pub_date":"2020-7-27","title":"An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain","abstract":"IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.","version":"1.1","doi":"10.1101/2020.07.26.222232","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.210096","pub_date":"2020-7-27","title":"Evaluation of diversity levels of the integrase gene sequences coming from HIV-1 virus, supporting the lack of target specificity of ivermectin versus the integrase-importin complex in SARS-CoV-2 infection","abstract":"Therapies with new drugs have been appearing in tests worldwide as potential inhibitors of sars-cov-2 virus replication. Recently, one of these drugs, Ivermectin, was reported as an inhibitor of the nuclear import of HIV-1 proteins in vitro, soon becoming the target of an international prospecting work (not yet published), with patients tested for COVID-19. However, understanding the evolutionary aspects of the biological components involved in the complex drug-nuclear import helps in understanding how these relationships exist in the deactivation of viral infections. Thus, 153 sequences of the HIV-1 integrase gene were analyzed for their genetic structure and molecular diversity and the presence of two distinct groups for the Gene and not only one, was detected; As well as different degrees of structuring for each of these groups. These results support the interpretation of the lack of conservation of the HIV-1 gene and that the number of existing polymorphisms, only for this structure of the complex, implies the non-efficiency of a drug at population levels. Thus, the molecular diversity found in HIV-1 can be extrapolated to other viruses, such as Including, SARS-CoV-2 and the functionality of the drug, interacting with the integrase-importin complex, can be further decreased.","version":"1.2","doi":"10.1101/2020.07.18.210096","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222117","pub_date":"2020-7-27","title":"Predicting the Emergence of SARS-CoV-2 Clades","abstract":"Evolution is a process of change where mutations in the viral RNA are selected based on their fitness for replication and survival. Given that current phylogenetic analysis of SARS-CoV-2 identifies new viral clades after they exhibit evolutionary selections, one wonders whether we can identify the viral selection and predict the emergence of new viral clades? Inspired by the Kolmogorov complexity concept, we propose a generative complexity (algorithmic) framework capable to analyze the viral RNA sequences by mapping the multiscale nucleotide dependencies onto a state machine, where states represent subsequences of nucleotides and state-transition probabilities encode the higher order interactions between these states. We apply computational learning and classification techniques to identify the active state-transitions and use those as features in clade classifiers to decipher the transient mutations (still evolving within a clade) and stable mutations (typical to a clade). As opposed to current analysis tools that rely on the edit distance between sequences and require sequence alignment, our method is computationally local, does not require sequence alignment and is robust to random errors (substitution, insertions and deletions). Relying on the GISAID viral sequence database, we demonstrate that our method can predict clade emergence, potentially aiding with the design of medications and vaccines.","version":"1.1","doi":"10.1101/2020.07.26.222117","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.24.217562","pub_date":"2020-7-27","title":"Identification, Mapping and Relative Quantitation of SARS-CoV-2 Spike Glycopeptides by Mass-Retention Time Fingerprinting","abstract":"We describe a novel analytical method for rapid and robust identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using any LC-TOF mass spectrometer, requires no specialised knowledge of glycan analysis and makes use of the differential resolving power of reversed phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. While this has previously been viewed as a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike. This database allows any ESI-TOF equipped lab to reliably identify and quantify spike glycans from a single overnight elastase protein digest in less than 90 minutes.","version":"1.2","doi":"10.1101/2020.07.24.217562","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.222265","pub_date":"2020-7-27","title":"Protein Surface Printer for Exploring Protein Domains","abstract":"The surface of proteins is vital in determining protein functions. Herein, a program, Protein Surface Printer(PSP), is built that performs multiple functions in quantifying protein surface domains. Two proteins, PETase and cytochrome P450, are used to validate that the program supports atomistic simulations with different combinations of programs and force fields. A case study is conducted on the structural analysis of the spike proteins of SARS-CoV-2 and SARS-CoV, and the human cell receptor ACE2. Although the surface domains of both spike proteins are highly similar, their receptor binding domains(RBDs) and the O-linked glycan domains are structurally different. Statistically, the outer surface of ACE2 displays less correlation with the RBD of SARS-CoV-2 than that of SARS-CoV. The O-linked glycan domain of SARS-CoV-2 is highly positively charged, which may promote binding to negatively charged human cells. Our program paves the way for an accurate understanding of protein binding for aggregation and ligand recognition.","version":"1.1","doi":"10.1101/2020.07.26.222265","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.27.222901","pub_date":"2020-7-27","title":"Evolution and Genetic Diversity of SARSCoV-2 in Africa Using Whole Genome Sequences","abstract":"The ongoing SARSCoV-2 pandemic was introduced into Africa on 14th February 2020 and has rapidly spread across the continent causing severe public health crisis and mortality. We investigated the genetic diversity and evolution of this virus during the early outbreak months using whole genome sequences. We performed; recombination analysis against closely related CoV, Bayesian time scaled phylogeny and investigated spike protein amino acid mutations. Results from our analysis showed recombination signals between the AfrSARSCoV-2 sequences and reference sequences within the N and S genes. The evolutionary rate of the AfrSARSCoV-2 was 4.133 \u00d7 10\u22124 high posterior density HPD (4.132 \u00d7 10\u22124 to 4.134 \u00d7 10\u22124) substitutions/site/year. The time to most recent common ancestor TMRCA of the African strains was December 7th 2019. The AfrSARCoV-2 sequences diversified into two lineages A and B with B being more diverse with multiple sub-lineages confirmed by both maximum clade credibility MCC tree and PANGOLIN software. There was a high prevalence of the D614-G spike protein amino acid mutation (82.61%) among the African strains. Our study has revealed a rapidly diversifying viral population with the G614 spike protein variant dominating, we advocate for up scaling NGS sequencing platforms across Africa to enhance surveillance and aid control effort of SARSCoV-2 in Africa.","version":"1.1","doi":"10.1101/2020.07.27.222901","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088500","pub_date":"2020-7-26","title":"Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 \u2013 implications for microvascular inflammation and hypercoagulopathy in COVID-19","abstract":"Accumulating clinical observations implicate vascular inflammation as an underlying cause of coagulopathy in severely ill COVID-19 patients and it was recently suggested that SARS-CoV-2 virus particles infect endothelial cells. Here, we show that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor. Instead, pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner. Pericyte deficiency leads to increased endothelial expression and release of Von Willebrand factor and intravascular platelet and fibrin aggregation, suggesting that pericytes limit endothelial pro-thrombotic responses. That pericytes and not endothelial cells express ACE2 may provide important clues to the pathology of COVID-19, as pericytes are normally shielded behind an endothelial barrier and may get infected only when this barrier is compromised by COVID-19 risk factors.","version":"1.2","doi":"10.1101/2020.05.11.088500","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.05.187344","pub_date":"2020-7-26","title":"N and O glycosylation of the SARS-CoV-2 spike protein","abstract":"Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of the SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage sites that differs from the SARS-CoV spike protein of 2002. We observe that the spike protein is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addition, we have identified eight additional O-glycopeptides on the spike glycoprotein and we confirmed that the spike protein is heavily N-glycosylated. Our recently developed LC-MS/MS methodology allowed us to identify LacdiNAc structural motives on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein\u2019s glycosylation and enables the investigation of the influence of the O-glycosylation on its proteolytic activation.","version":"1.2","doi":"10.1101/2020.07.05.187344","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.25.221291","pub_date":"2020-7-26","title":"North American deer mice are susceptible to SARS-CoV-2","abstract":"The zoonotic spillover of the pandemic SARS-coronavirus 2 (SARS-CoV-2) from an animal reservoir, currently presumed to be the Chinese horseshoe bat, into a na\u00efve human population has rapidly resulted in a significant global public health emergency. Worldwide circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive non-domesticated animals, potentially seeding new host reservoir species and geographic regions in which bat SARS-like coronaviruses have not historically been endemic. Here we report that North American deer mice (Peromyscus maniculatus) and some closely related members of the Cricetidae family of rodents possess key amino acid residues within the angiotensin-converting enzyme 2 (ACE2) receptor known to confer SARS-CoV-2 spike protein binding. Peromyscus rodent species are widely distributed across North America and are the primary host reservoirs of several emerging pathogens that repeatedly spill over into humans including Borrelia burgdorferi, the causative agent of Lyme disease, deer tick virus, and Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome (HPS). We demonstrate that adult deer mice are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to na\u00efve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 is unknown. Nevertheless, efforts to monitor wild, peri-domestic Peromyscus rodent populations are likely warranted as the SARS-CoV-2 pandemic progresses.","version":"1.1","doi":"10.1101/2020.07.25.221291","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.25.221036","pub_date":"2020-7-26","title":"No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor","abstract":"The spike protein of SARS-CoV-2 is known to enable viral invasion into human cells through direct binding to host receptors including ACE2. An alternate entry receptor for the virus was recently proposed to be basigin/CD147. These early studies have already prompted a clinical trial and multiple published hypotheses of the role of this host receptor in viral infection and pathogenesis. We sought to independently characterize the basigin-spike protein interaction. After conducting several lines of experiments, we report that we are unable to find evidence supporting the role of basigin as a putative spike-binding receptor. Recombinant forms of both the entire ectodomain and S1 domain of the SARS-CoV-2 spike protein that directly bind ACE2 do not interact with basigin expressed on the surface of human cells. Using specialized assays tailored to detect receptor interactions as weak or weaker than the proposed basigin-spike binding, we report no evidence for direct binding of the viral spike to either of the two common isoforms of basigin. Given the pressing need for clarity on which targets of SARS-CoV-2 may lead to promising therapeutics, we present these findings to allow more informed decisions about the translational relevance of this putative mechanism in the race to understand and treat COVID-19.","version":"1.1","doi":"10.1101/2020.07.25.221036","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.24.220715","pub_date":"2020-7-26","title":"CoVaccine HT\u2122 adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation","abstract":"The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT\u2122 or Alhydrogel. CoVaccine HT\u2122 induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralising antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT\u2122 can rapidly induce a comprehensive and protective immune response to SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.24.220715","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.219741","pub_date":"2020-7-26","title":"The SARS-CoV-2 Spike Variant D614G Favors an Open Conformational State","abstract":"The COVID-19 pandemic underwent a rapid transition with the emergence of a SARS-CoV-2 variant that carried the amino acid substitution D614G in the Spike protein that became globally prevalent. The G-form is both more infectious in vitro and associated with increased viral loads in infected people. To gain insight into the mechanism underlying these distinctive characteristics, we employed multiple replicas of microsecond all-atom simulations to probe the molecular-level impact of this substitution on Spike\u2019s closed and open states. The open state enables Spike interactions with its human cellular receptor, ACE2. Here we show that changes in the inter-protomer energetics due to the D614G substitution favor a higher population of infection-capable (open) states. The inter-protomer interactions between S1 and S2 subunits in the open state of the D-form are asymmetric. This asymmetry is resolved in the G-form due to the release of tensile hydrogen bonds resulting in an increased population of open conformations. Thus, the increased infectivity of the G-form is likely due to a higher rate of profitable binding encounters with the host receptor. It is also predicted to be more neutralization sensitive due to enhanced exposure of the receptor binding domain, a key target region for neutralizing antibodies.","version":"1.1","doi":"10.1101/2020.07.26.219741","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.26.221572","pub_date":"2020-7-26","title":"COVID-19 and Rheumatoid Arthritis share myeloid pathogenic and resolving pathways","abstract":"We recently delineated the functional biology of pathogenic and inflammation resolving synovial tissue macrophage clusters in rheumatoid arthritis (RA). Whilst RA is not a viral respiratory syndrome, it represents a pro-inflammatory cytokine-driven chronic articular condition often accompanied by cardiovascular and lung pathologies. We hypothesised that functionally equivalent macrophage clusters in the lung might govern inflammation and resolution of COVID-19 pneumonitis. To provide insight into the targetable functions of COVID-19 bronchoalveolar lavage (BALF) macrophage clusters, a comparative analysis of BALF macrophage single cell transcriptomics (scRNA-seq) with synovial tissue (ST) macrophage scRNA-seq and functional biology was performed. The function of shared BALF and ST MerTK inflammation-resolving pathway was confirmed with inhibitor in primary macrophage-synovial fibroblast co-cultures. Results. Distinct BALF FCNpos and FCNposSPP1pos macrophage clusters emerging in severe COVID-19 patients were closely related to ST CD48highS100A12pos and CD48posSPP1pos clusters driving synovitis in active RA. They shared transcriptomic profile and pathogenic mechanisms. Healthy lung resident alveolar FABP4pos macrophages shared a regulatory transcriptomic profile, including TAM (Tyro, Axl, MerTK) receptors pathway with synovial tissue TREM2pos macrophages that govern RA remission. This pathway was substantially altered in BALF macrophages of severe COVID-19. In vitro dexamethasone inhibited tissue inflammation via macrophages\u2019 MerTK function. Pathogenesis and resolution of COVID-19 pneumonitis and RA synovitis might be driven by similar macrophage clusters and pathways. The MerTK-dependent anti-inflammatory mechanisms of dexamethasone, and the homeostatic function of TAM pathways that maintain RA in remission advocate the therapeutic MerTK agonism to ameliorate the cytokine storm and pneumonitis of severe COVID-19.","version":"1.1","doi":"10.1101/2020.07.26.221572","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.24.205583","pub_date":"2020-7-25","title":"Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters through the airways and infects the lungs, causing lethal pulmonary damage in vulnerable patients. This virus contains spike proteins on its envelope that binds to human angiotensin-converting enzyme 2 (hACE2) expressed on the surface of airway cells, enabling entry of the virus for causing infection. In severe cases, the virus enters the circulatory system, contributing to multiorgan failure. Soluble form of hACE2 binds to SARS-CoV-2 spike protein and prevents viral entry into target cells. Moreover, soluble recombinant ACE2 ameliorates lung injury but its short half-life limits its therapeutic utility. Here, we engineered synthetic mRNA to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. Novel lipid nanoparticles (LNPs) were used to package mRNA and transfect mammalian cells for enhanced production of secreted proteins. Intravenously administered LNP led to hepatic delivery of the mRNA. This elicited secretion of hsACE2 into the blood circulation within 2 h, and levels of circulating hsACE2 peaked at 6 h and gradually decreased over several days. Since the primary site of entry and pathogenesis for SARS-CoV-2 is the lungs, we instilled LNPs into the lungs and were able to detect hsACE2 in the bronchoalveolar lavage fluid within 24 h and lasted for 48 h. Through co-immunoprecipitation, we found that mRNA-generated hsACE2 was able to bind with the receptor binding domain of the SARS-CoV-2 spike protein. Furthermore, hsACE2 was able to strongly inhibit (over 90%) SARS-CoV-2 pseudovirus infection. Our proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed for pulmonary and extrapulmonary neutralization of SARS-CoV-2 and open new treatment opportunities for COVID-19.","version":"1.1","doi":"10.1101/2020.07.24.205583","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.25.192310","pub_date":"2020-7-25","title":"\u03b2-Coronaviruses use lysosomal organelles for cellular egress","abstract":"\u03b2-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that \u03b2-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.","version":"1.1","doi":"10.1101/2020.07.25.192310","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.152157","pub_date":"2020-7-24","title":"Novel ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV2","abstract":"SARS-CoV2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. This fusion protein contained a LALA mutation that abrogates Fcr\u03b3 binding, but retains FcRN binding to prolong the half-life, as well as achieve therapeutic concentrations in the lung lavage. Interestingly, a mutation in the catalytic domain of ACE2, MDR504, completely abrogated catalytic activity, but significantly increased binding to SARS-CoV2 spike protein in vitro. This feature correlated with more potent viral neutralization in a plaque assay. Parental administration of the protein showed stable serum concentrations with a serum half-life of \u223c 145 hours with excellent bioavailability in the epithelial lining fluid of the lung. Prophylactic administration of MDR504 significantly attenuated SARS-CoV2 infection in a murine model. These data support that the MDR504 hACE2-Fc is an excellent candidate for pre or post-exposure prophylaxis or treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.06.15.152157","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153387","pub_date":"2020-7-24","title":"The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection","abstract":"Many efforts to design and screen therapeutics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) have focused on inhibiting viral cell entry by disrupting ACE2 binding with the SARS-CoV-2 spike protein. This work focuses on inhibiting SARS-CoV-2 entry through a hypothesized \u03b15\u03b21 integrin-based mechanism, and indicates that inhibiting the spike protein interaction with \u03b15\u03b21 integrin (+/\u2212 ACE2), and the interaction between \u03b15\u03b21 integrin and ACE2 using a molecule ATN-161 represents a promising approach to treat COVID-19.","version":"1.2","doi":"10.1101/2020.06.15.153387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.040204","pub_date":"2020-7-24","title":"Relevance of SARS-CoV-2 related factors ACE2 and TMPRSS2 expressions in gastrointestinal tissue with pathogenesis of digestive symptoms, diabetes-associated mortality, and disease recurrence in COVID-19 patients","abstract":"COVID-19 is caused by a new strain of coronavirus called SARS-coronavirus-2 (SARS-CoV-2), which is a positive sense single strand RNA virus. In humans, it binds to angiotensin converting enzyme 2 (ACE2) with the help a structural protein on its surface called the S-spike. Further, cleavage of the viral spike protein (S) by the proteases like transmembrane serine protease 2 (TMPRSS2) or Cathepsin L (CTSL) is essential to effectuate host cell membrane fusion and virus infectivity. COVID-19 poses intriguing issues with imperative relevance to clinicians. The pathogenesis of GI symptoms, diabetes-associated mortality, and disease recurrence in COVID-19 are of particular relevance because they cannot be sufficiently explained from the existing knowledge of the viral diseases. Tissue specific variations of SARS-CoV-2 cell entry related receptors expression in healthy individuals can help in understanding the pathophysiological basis the aforementioned collection of symptoms. The data were downloaded from the Human Protein Atlas available at (https://www.proteinatlas.org/humanproteome/sars-cov-2) and the tissue specific expressions (both mRNA and protein) of ACE2 and TMPRSS2 as yielded from the studies with RNA sequencing and immunohistochemistry (IHC) were analyzed as a function of the various components of the digestive tract. A digestive system specific functional enrichment map of ACE2 gene was created using g:profiler (https://biit.cs.ut.ee/gprofiler/gost) utility and the data were visualized using Cytoscape software, version 3.7.2 (https://cytoscape.org/). The correlated expression (transcriptomic and proteomic) of ACE2 (to which SARS-CoV-2 binds through the S-spike) was found to be enriched in the lower gastrointestinal tract (GIT) (highest in small intestine, followed by colon and rectum), and was undetectable in the upper GIT components: mouth cavity (tongue, oral mucosa, and salivary glands), esophagus, and stomach. High expression of ACE2 was noted in the glandular cells as well as in the enterocytes in the lining epithelium (including brush border epithelium). Among other digestive system organs, Gall bladder (GB) showed high expression of ACE2 in glandular cells, while any protein expression was undetectable in liver and pancreas. TMPRSS2 was found enhanced in GIT and exocrine glands of pancreas, and co-localized with ACE2 in enterocytes. Based on the findings of this study and supportive evidence from the literature we propose that a SARS-CoV-2 binding with ACE2 mediates dysregulation of the sodium dependent nutrient transporters and hence may be a plausible basis for the digestive symptoms in COVID-19 patients. ACE2 mediated dysregulation of sodium dependent glucose transporter (SGLT1 or SLC5A1) in the intestinal epithelium also links it to the pathogenesis of diabetes mellitus which can be a possible reason for the associated mortality in COVID-19 patients with diabetes. High expression of ACE2 in mucosal cells of the intestine and GB make these organs potential sites for the virus entry and replication. Continued replication of the virus at these ACE2 enriched sites may be a basis for the disease recurrence reported in some, thought to be cured, patients.","version":"1.2","doi":"10.1101/2020.04.14.040204","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.218784","pub_date":"2020-7-24","title":"Elucidation of cryptic and allosteric pockets within the SARS-CoV-2 protease","abstract":"The SARS-CoV-2 pandemic has rapidly spread across the globe, posing an urgent health concern. Many quests to computationally identify treatments against the virus rely on in silico small molecule docking to experimentally determined structures of viral proteins. One limit to these approaches is that protein dynamics are often unaccounted for, leading to overlooking transient, druggable conformational states. Using Gaussian accelerated molecular dynamics to enhance sampling of conformational space, we identified cryptic pockets within the SARS-CoV-2 main protease, including some within regions far from the active site and assed their druggability. These pockets can aid in virtual screening efforts to identify a protease inhibitor for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.07.23.218784","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.24.219097","pub_date":"2020-7-24","title":"PACIFIC: A lightweight deep-learning classifier of SARS-CoV-2 and co-infecting RNA viruses","abstract":"Viral co-infections occur in COVID-19 patients, potentially impacting disease progression and severity. However, there is currently no dedicated method to identify viral co-infections in patient RNA-seq data. We developed PACIFIC, a deep-learning algorithm that accurately detects SARS-CoV-2 and other common RNA respiratory viruses from RNA-seq data. Using in silico data, PACIFIC recovers the presence and relative concentrations of viruses with >99% precision and recall. PACIFIC accurately detects SARS-CoV-2 and other viral infections in 63 independent in vitro cell culture and patient datasets. PACIFIC is an end-to-end tool that enables the systematic monitoring of viral infections in the current global pandemic.","version":"1.1","doi":"10.1101/2020.07.24.219097","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.28.013789","pub_date":"2020-7-24","title":"Structural analysis of SARS-CoV-2 genome and predictions of the human interactome","abstract":"Specific elements of viral genomes regulate interactions within host cells. Here, we calculated the secondary structure content of >2000 coronaviruses and computed >100000 human protein interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic regions display different degrees of conservation. SARS-CoV-2 domain encompassing nucleotides 22500 \u2013 23000 is conserved both at the sequence and structural level. The regions upstream and downstream, however, vary significantly. This part codes for the Spike S protein that interacts with the human receptor angiotensin-converting enzyme 2 (ACE2). Thus, variability of Spike S may be connected to different levels of viral entry in human cells within the population. Our predictions indicate that the 5\u2019 end of SARS-CoV-2 is highly structured and interacts with several human proteins. The binding proteins are involved in viral RNA processing such as double-stranded RNA specific editases and ATP-dependent RNA-helicases and have strong propensity to form stress granules and phase-separated complexes. We propose that these proteins, also implicated in viral infections such as HIV, are selectively recruited by SARS-CoV-2 genome to alter transcriptional and post-transcriptional regulation of host cells and to promote viral replication.","version":"1.7","doi":"10.1101/2020.03.28.013789","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.213959","pub_date":"2020-7-24","title":"From people to Panthera: Natural SARS-CoV-2 infection in tigers and lions at the Bronx Zoo","abstract":"We describe the first cases of natural SARS-CoV-2 infection detected in animals in the United States. In March 2020, four tigers and three lions at the Bronx Zoo developed mild respiratory signs. SARS-CoV-2 RNA was detected by rRT-PCR in respiratory secretions and/or feces from all seven affected animals; viral RNA and/or antibodies were detected in their keepers. SARS-CoV-2 was isolated from respiratory secretions or feces from three affected animals; in situ hybridization co-localized viral RNA with cellular damage. Whole genome sequence and haplotype network analyses showed tigers and lions were infected with two different SARS-CoV-2 strains, suggesting independent viral introductions. The source of SARS-CoV-2 infection in the lions is unknown. Epidemiological data and genetic similarities between keeper and tiger viruses indicate human to animal transmission.","version":"1.2","doi":"10.1101/2020.07.22.213959","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.216770","pub_date":"2020-7-24","title":"Co-expression of Mitochondrial Genes and ACE2 in Cornea Involved in COVID-19 Infection","abstract":"The Coronavirus disease 2019 (COVID-19) pandemic severely challenges public health and necessitates the need for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and propagation. Here, the co-expression network was constructed by mapping the well-known ACE2, TMPRSS2 and host susceptibility genes implicated in COVID-19 GWAS onto a cornea, retinal pigment epithelium and lung. We found a significant co-expression module of these genes in the cornea, revealing that cornea is potential extra-respiratory entry portal of SARS-CoV-2. Strikingly, both co-expression and interaction networks show a significant enrichment in mitochondrial function, which are the hub of cellular oxidative homeostasis, inflammation and innate immune response. We identified a corneal mitochondrial susceptibility module (CMSM) of 14 mitochondrial genes by integrating ACE2 co-expression cluster and SARS-CoV-2 interactome. Gene ECSIT, as a cytosolic adaptor protein involved in inflammatory responses, exhibits the strongest correlation with ACE2 in CMSM, which has shown to be an important risk factor for SARS-CoV-2 infection and prognosis. Our co-expression and protein interaction network analysis uncover that the mitochondrial function related genes in cornea contribute to the dissection of COVID-19 susceptibility and potential therapeutic interventions.","version":"1.1","doi":"10.1101/2020.07.23.216770","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.24.217570","pub_date":"2020-7-24","title":"Adopting STING agonist cyclic dinucleotides as a potential adjuvant for SARS-CoV-2 vaccine","abstract":"A novel STING agonist CDGSF unilaterally modified with phosphorothioate and fluorine was synthesized. CDGSF displayed better STING activity over dithio CDG. Immunization of SARS-CoV-2 Spike protein with CDGSF as an adjuvant elicited an exceptional high antibody titer and a robust T cell response, which were better than the group using aluminium hydroxide as a adjuvant. These results highlighted the adjuvant potential of STING agonist in SARS-CoV-2 vaccine preparation for the first time.","version":"1.1","doi":"10.1101/2020.07.24.217570","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.172403","pub_date":"2020-7-24","title":"Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor","abstract":"The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.","version":"1.2","doi":"10.1101/2020.06.25.172403","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.20160762","pub_date":"2020-07-24","title":"Herd immunity thresholds for SARS-CoV-2 estimated from unfolding epidemics","abstract":"<jats:p>Variation in individual susceptibility or frequency of exposure to infection accelerates the rate at which populations acquire immunity by natural infection. Individuals that are more susceptible or more frequently exposed tend to be infected earlier and hence more quickly selected out of the susceptible pool, decelerating the incidence of new infections as the epidemic progresses. Eventually, susceptible numbers become low enough to prevent epidemic growth or, in other words, the herd immunity threshold (HIT) is reached. We have recently proposed a method whereby mathematical models, with gamma distributions of susceptibility or exposure to SARS-CoV-2, are fitted to epidemic curves to estimate coefficients of individual variation among epidemiological parameters of interest. In the initial study we estimated HIT around 25-29% for the original Wuhan virus in England and Scotland. Here we explore the limits of applicability of the method using Spain and Portugal as case studies. Results are robust and consistent with England and Scotland, in the case of Spain, but fail in Portugal due to particularities of the dataset. We describe failures, identify their causes, and propose methodological extensions.</jats:p>","version":null,"doi":"10.1101/2020.07.23.20160762","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.24.219139","pub_date":"2020-7-24","title":"Tissue-specific and interferon-inducible expression of non-functional ACE2 through endogenous retrovirus co-option","abstract":"Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), as well as a regulator of several physiological processes. ACE2 has recently been proposed to be interferon-inducible, suggesting that SARS-CoV-2 may exploit this phenomenon to enhance viral spread and questioning the efficacy of interferon treatment in Coronavirus disease 2019 (COVID-19). Using a recent de novo transcript assembly that captured previously unannotated transcripts, we describe a novel isoform of ACE2, generated by co-option of an intronic long terminal repeat (LTR) retroelement promoter. The novel transcript, termed LTR16A1-ACE2, exhibits specific expression patterns across the aerodigestive and gastrointestinal tracts and, importantly, is highly responsive to interferon stimulation. In stark contrast, expression of canonical ACE2 is completely unresponsive to interferon stimulation. Moreover, the LTR16A1-ACE2 translation product is a truncated, unstable ACE2 form, lacking domains required for SARS-CoV-2 binding and therefore unlikely to contribute to or enhance viral infection.","version":"1.1","doi":"10.1101/2020.07.24.219139","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.217703","pub_date":"2020-7-24","title":"Single-cell landscape of immunological responses in COVID-19 patients","abstract":"In COVID-19 caused by SARS-CoV-2 infection, the relationship between disease severity and the host immune response is not fully understood. Here we performed single-cell RNA sequencing in peripheral blood samples of five healthy donors and 13 COVID-19 patients including moderate, severe and convalescent cases. Through determining the transcriptional profiles of immune cells, coupled with assembled T cell receptor and B cell receptor sequences, we analyzed the functional properties of immune cells. Most cell types in COVID-19 patients showed a strong interferon-alpha response, and an overall acute inflammatory response. Moreover, intensive expansion of highly cytotoxic effector T cell subsets, such as CD4+ Effector-GNLY (Granulysin), CD8+ Effector-GNLY and NKT CD160, was associated with convalescence in moderate patients. In severe patients, the immune landscape featured a deranged interferon response, profound immune exhaustion with skewed T cell receptor repertoire and broad T cell expansion. These findings illustrate the dynamic nature of immune responses during the disease progression.","version":"1.1","doi":"10.1101/2020.07.23.217703","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.214254","pub_date":"2020-7-24","title":"The answer lies in the energy: how simple atomistic molecular dynamics simulations may hold the key to epitope prediction on the fully glycosylated SARS-CoV-2 spike protein","abstract":"Betacoronavirus SARS-CoV-2 is posing a major threat to human health and its diffusion around the world is having dire socioeconomical consequences. Thanks to the scientific community\u2019s unprecedented efforts, the atomic structure of several viral proteins has been promptly resolved. As the crucial mediator of host cell infection, the heavily glycosylated trimeric viral Spike protein (S) has been attracting the most attention and is at the center of efforts to develop antivirals, vaccines, and diagnostic solutions. Herein, we use an energy-decomposition approach to identify antigenic domains and antibody binding sites on the fully glycosylated S protein. Crucially, all that is required by our method are unbiased atomistic molecular dynamics simulations; no prior knowledge of binding properties or ad hoc combinations of parameters/measures extracted from simulations is needed. Our method simply exploits the analysis of energy interactions between all intra-protomer aminoacid and monosaccharide residue pairs, and cross-compares them with structural information (i.e., residueresidue proximity), identifying potential immunogenic regions as those groups of spatially contiguous residues with poor energetic coupling to the rest of the protein. Our results are validated by several experimentally confirmed structures of the S protein in complex with anti- or nanobodies. We identify poorly coupled sub-domains: on the one hand this indicates their role in hosting (several) epitopes, and on the other hand indicates their involvement in large functional conformational transitions. Finally, we detect two distinct behaviors of the glycan shield: glycans with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes; those with weaker coupling could themselves be poised for antibody recognition. Predicted Immunoreactive regions can be used to develop optimized antigens (recombinant subdomains, synthetic (glyco)peptidomimetics) for therapeutic applications.","version":"1.1","doi":"10.1101/2020.07.22.214254","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.083212","pub_date":"2020-7-24","title":"Coarse-grained molecular simulations of the binding of the SARS-CoV-2 spike protein RBD to the ACE2 receptor","abstract":"Since it was first observed, the COVID-19 pandemic has created a global emergency for national health systems due to millions of confirmed cases and hundreds of thousands of deaths. At a molecular level, the bottleneck for the infection is the binding of the receptor binding domain (RBD) of the viral spike protein to ACE2, an enzyme exposed on human cell membranes. Several experimental structures of the ACE2:RBD complex have been made available, however they offer only a static description of the arrangements of the molecules in either the free or bound states. In order to gain a dynamic description of the binding process that is key to infection, we use molecular simulations with a coarse grained model of the RBD and ACE2. We find that binding occurs in an all-or-none way, without intermediates, and that even in the bound state, the RBD exhibits a considerably dynamic behaviour. From short equilibrium simulations started in the unbound state we provide snapshots that result in a tentative mechanism of binding. Our findings may be important for the development of drug discovery strategies that target the RBD.","version":"1.2","doi":"10.1101/2020.05.07.083212","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.216275","pub_date":"2020-7-24","title":"MicrobeTrace: Retooling Molecular Epidemiology for Rapid Public Health Response","abstract":"Outbreak investigations use data from interviews, healthcare providers, laboratories and surveillance systems. However, integrated use of data from multiple sources requires a patchwork of software that present challenges in usability, interoperability, confidentiality, and cost. Rapid integration, visualization and analysis of data from multiple sources can guide effective public health interventions. We developed MicrobeTrace to facilitate rapid public health responses by overcoming barriers to data integration and exploration in molecular epidemiology. Using publicly available HIV sequences and other data, we demonstrate the analysis of viral genetic distance networks and introduce a novel approach to minimum spanning trees that simplifies results. We also illustrate the potential utility of MicrobeTrace in support of contact tracing by analyzing and displaying data from an outbreak of SARS-CoV-2 in South Korea in early 2020. MicrobeTrace is a web-based, client-side, JavaScript application (https://microbetrace.cdc.gov) that runs in Chromium-based browsers and remains fully-operational without an internet connection. MicrobeTrace is developed and actively maintained by the Centers for Disease Control and Prevention. The source code is available at https://github.com/cdcgov/microbetrace. ells@cdc.gov","version":"1.1","doi":"10.1101/2020.07.22.216275","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029488","pub_date":"2020-7-23","title":"Vulnerabilities of the SARS-CoV-2 virus to proteotoxicity \u2013 opportunity for repurposed chemotherapy of COVID-19 infection","abstract":"The global pandemic of COVID-19 disease caused by infection with the SARS-CoV-2 coronavirus, has produced an urgent requirement and search for improved treatments whilst effective vaccines are developed. A strategy for improved drug therapy is to increase levels of endogenous reactive metabolites for selective toxicity to SARS-CoV-2 by preferential damage to the viral proteome. Key reactive metabolites producing major quantitative damage to the proteome in physiological systems are: reactive oxygen species (ROS) and the reactive glycating agent methylglyoxal (MG); cysteine residues and arginine residues are their most susceptible targets, respectively. From sequenced-based prediction of the SARS-CoV-2 proteome, we found 0.8-fold enrichment or depletion of cysteine residues in functional domains of the viral proteome; whereas there was a 4.6-fold enrichment of arginine residues, suggesting SARS-CoV-2 is resistant to oxidative agents and sensitive to MG. For arginine residues of the SARS-CoV-2 predicted to be in functional domains, we examined which are activated towards modification by MG \u2013 residues with predicted or expected low pKa by neighbouring group in interactions. We found 25 such arginine residues, including 2 in the spike protein and 10 in the nucleoprotein. These sites were partially conserved in related coronaviridae: SARS-COV and MERS. Finally, we identified drugs which increase cellular MG concentration to virucidal levels: antitumor drugs with historical antiviral activity, doxorubicin and paclitaxel. Our findings provide evidence of potential vulnerability of SARS-CoV-2 to inactivation by MG and a scientific rationale for repurposing of doxorubicin and paclitaxel for treatment of COVID-19 disease, providing efficacy and adequate therapeutic index may be established.","version":"1.2","doi":"10.1101/2020.04.07.029488","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.216648","pub_date":"2020-7-23","title":"Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2","abstract":"Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.22.216648","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.095414","pub_date":"2020-7-23","title":"Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans","abstract":"Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.05.14.095414","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.212761","pub_date":"2020-7-23","title":"SARS-CoV-2-induced humoral immunity through B cell epitope analysis and neutralizing activity in COVID-19 infected individuals in Japan","abstract":"The aim of this study is to understand adaptive immunity to SARS-CoV-2 through the analysis of B cell epitope and neutralizing activity in coronavirus disease 2019 (COVID-19) patients. We obtained serum from thirteen COVID-19 patients. Most individuals revealed neutralizing activity against SARS-CoV-2 assessed by a pseudotype virus-neutralizing assay. The antibody production against the spike glycoprotein (S protein) or receptor-binding domain (RBD) of SARS-CoV-2 was elevated, with large individual differences, as assessed by ELISA. In the analysis of the predicted the linear B cell epitopes, two regions (671-690 aa. and 1146-1164 aa.), which were located in S1 and S2 but not in the RBD, were highly reactive with the sera from patients. In the further analysis of the B cell epitope within the S protein by utilizing a B cell epitope array, a hot spot in the N-terminal domain of the S protein but not the RBD was observed in individuals with neutralizing activity. Overall, the analysis of antibody production and B cell epitopes of the S protein from patient serum may provide a novel target for the vaccine development against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.22.212761","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.217174","pub_date":"2020-7-23","title":"Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat","abstract":"The 2019 novel coronavirus disease (COVID-19) is the disease that has been identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the prophylactic treatment of SARS-CoV-2 is still under investigation. The effective delivery of eukaryotic expression plasmids to the immune system\u2019s inductive cells constitutes an essential requirement for the generation of effective DNA vaccines. Here, we have explored the use of Salmonella typhimurium as vehicles to deliver expression plasmids orally. Attenuated Salmonella phoP harboring eukaryotic expression plasmids that encoded spike protein of SARS-CoV-2 was administered orally to Wistar rats. Rats were immunized orally with Salmonella that carried a eukaryotic expression plasmid once a week for three consecutive weeks. The efficiency of the vaccination procedure was due to the transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Our results showed that all immunized animals generated humoral immunity against the SARS-CoV-2 spike protein, indicating that a Salmonella-based vaccine carrying the Spike gene can elicit SARS-CoV-2-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.07.23.217174","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214346","pub_date":"2020-7-23","title":"Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy","abstract":"SARS-CoV-2 originated in animals and is now easily transmitted between people. Sporadic detection of natural cases in animals alongside successful experimental infections of pets, such as cats, ferrets and dogs, raises questions about the susceptibility of animals under natural conditions of pet ownership. Here we report a large-scale study to assess SARS-CoV-2 infection in 817 companion animals living in northern Italy, sampled at a time of frequent human infection. No animals tested PCR positive. However, 3.4% of dogs and 3.9% of cats had measurable SARS-CoV-2 neutralizing antibody titers, with dogs from COVID-19 positive households being significantly more likely to test positive than those from COVID-19 negative households. Understanding risk factors associated with this and their potential to infect other species requires urgent investigation. SARS-CoV-2 antibodies in pets from Italy.","version":"1.2","doi":"10.1101/2020.07.21.214346","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.217331","pub_date":"2020-7-23","title":"Development and validation of a clinical risk score to predict the risk of SARS-CoV-2 infection from administrative data: a population-based cohort study from Italy","abstract":"The novel coronavirus (SARS-CoV-2) pandemic spread rapidly worldwide increasing exponentially in Italy. To date, there is lack of studies describing clinical characteristics of the population most at risk of infection. Hence, we aimed to identify clinical predictors of SARS-CoV-2 infection risk and to develop and validate a score predicting SARS-CoV-2 infection risk comparing it with unspecific surrogates. Retrospective case/control study using administrative health-related database was carried out in Southern Italy (Campania region) among beneficiaries of Regional Health Service aged over than 30 years. For each subject with Covid-19 confirmed diagnosis (case), up to five controls were randomly matched for gender, age and municipality of residence. Odds ratios and 90% confidence intervals for associations between candidate predictors and risk of infection were estimated by means of conditional logistic regression. SARS-CoV-2 Infection Score (SIS), was developed by generating a total aggregate score obtained from assignment of a weight at each selected covariate using coefficients estimated from the model. Finally, the score was categorized by assigning increasing values from 1 to 4. SIS was validated by comparison with specific and unspecific predictors of SARS-CoV-2 infection. Subjects suffering from diabetes, anaemias, Parkinson\u2019s disease, mental disorders, cardiovascular and inflammatory bowel and kidney diseases showed increased risk of SARS-CoV-2 infection. Similar estimates were recorded for men and women and younger and older than 65 years. Fifteen conditions significantly contributed to the SIS. As SIS value increases, risk progressively increases, being odds of SARS-CoV-2 infection among people with the highest SIS value (SIS=4), 1.74 times higher than those unaffected by any SIS contributing conditions (SIS=1). This study identified conditions and diseases making individuals more vulnerable to SARS-CoV-2 infection. Our results are a decision-maker support tool for identifying population most at risk allowing adoption of preventive measures to minimize a potential new relapse damage.","version":"1.1","doi":"10.1101/2020.07.23.217331","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.136861","pub_date":"2020-7-23","title":"Insights on cross-species transmission of SARS-CoV-2 from structural modeling","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 14 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic, the question emerges whether human-to-animal transmission is possible and if so, which animal species are most at risk. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.","version":"1.2","doi":"10.1101/2020.06.05.136861","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.217083","pub_date":"2020-7-23","title":"An in-silico study on SARS-CoV-2: Its compatibility with human tRNA pool, and the polymorphism arising in a single lineage over a month","abstract":"SARS-CoV-2 has caused a global pandemic that has costed enormous human lives in the recent past. The present study is an investigation of the viral codon adaptation, ORFs\u2019 stability and tRNA co-adaptation with humans. We observed that for the codon usage bias in viral ssRNA, ORFs have near values of folding free energies and codon adaptation index with mRNAs of the human housekeeping CDS. However, the correlation between the stability of the ORFs in ssRNA and CAI is stronger than the mRNA stability and CAI of HKG, suggesting a greater expression capacity of SARS-CoV-2. Mutational analysis reflects polymorphism in the virus for ORF1ab, surface glycoprotein and nucleocapsid phosphoprotein ORFs. Non-synonymous mutations have shown non-polar substitutions. Out of the twelve mutations nine are for a higher t-RNA copy number. Viruses in general have high mutation rates. To understand the chances of survival for the mutated SARS-CoV-2 we did simulation for synonymous mutations. It resulted in 50% ORFs with higher stability than their native equivalents. Thus, considering only the synonymous mutations the virus can exhibit a lot of polymorphism. Collectively our data provides new insights for SARS-CoV-2 mutations and the human t-RNA compatibility. Survivability of SARS-CoV-2 in humans is essential for its spread. It has overlapping genes exhibiting a high codon optimization with humans even after a higher codon usage bias. They seem to possess cognizance for high copy number t-RNA (cognate or near-cognate) in humans, while mutating. Even though, it has been well established that native transcripts posses the highest stability, our in-silico studies show that SARS-CoV-2 under mutations give rise to ORFs with higher stability. These results significantly present the virus\u2019s ability and the credibility of survival for the mutants. Despite its focus on a geographical location it explains the ongoing behavior of SARS-CoV-2 for a steady existence in humans as all the different lineages have a common origin. Wuhan, China.","version":"1.1","doi":"10.1101/2020.07.23.217083","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.212357","pub_date":"2020-7-23","title":"Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2","abstract":"A safe and durable vaccine is urgently needed to tackle the COVID19 pandemic that has infected >15 million people and caused >620,000 deaths worldwide. As with other respiratory pathogens, the nasal compartment is the first barrier that needs to be breached by the SARS-CoV-2 virus before dissemination to the lung. Despite progress at remarkable speed, current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. We report the development of an intranasal subunit vaccine that contains the trimeric or monomeric spike protein and liposomal STING agonist as adjuvant. This vaccine induces systemic neutralizing antibodies, mucosal IgA responses in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA-sequencing confirmed the concomitant activation of T and B cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues (NALT), confirming its role as an inductive site that can lead to long-lasting immunity. The ability to elicit immunity in the respiratory tract has can prevent the initial establishment of infection in individuals and prevent disease transmission across humans.","version":"1.1","doi":"10.1101/2020.07.23.212357","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.217430","pub_date":"2020-7-23","title":"SARS-CoV2 genome analysis of Indian isolates and molecular modelling of D614G mutated spike protein with TMPRSS2 depicted its enhanced interaction and virus infectivity","abstract":"COVID-19 that emerged as a global pandemic is caused by SARS-CoV-2 virus. The virus genome analysis during disease spread reveals about its evolution and transmission. We did whole genome sequencing of 225 clinical strains from the state of Odisha in eastern India using ARTIC protocol-based amplicon sequencing. Phylogenetic analysis identified the presence of all five reported clades 19A, 19B, 20A, 20B and 20C in the population. The analyses revealed two major routes for the introduction of the disease in India i.e. Europe and South-east Asia followed by local transmission. Interestingly, 19B clade was found to be much more prevalent in our sequenced genomes (17%) as compared to other genomes reported so far from India. The haplogroup analysis for clades showed evolution of 19A and 19B in parallel whereas the 20B and 20C appeared to evolve from 20A. Majority of the 19A and 19B clades were present in cases that migrated from Gujarat state in India suggesting it to be one of the major initial points of disease transmission in India during month of March and April. We found that with the time 20A and 20B clades evolved drastically that originated from central Europe. At the same time, it has been observed that 20A and 20B clades depicted selection of four common mutations i.e. 241 C>T (5\u2019UTR), P323L in RdRP, F942F in NSP3 and D614G in the spike protein. We found an increase in the concordance of G614 mutation evolution with the viral load in clinical samples as evident from decreased Ct value of spike and Orf1ab gene in qPCR. Molecular modelling and docking analysis identified that D614G mutation enhanced interaction of spike with TMPRSS2 protease, which could impact the shedding of S1 domain and infectivity of the virus in host cells.","version":"1.1","doi":"10.1101/2020.07.23.217430","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.23.217372","pub_date":"2020-7-23","title":"The new silicone N99 half-piece respirator, VJR-NMU N99: A novel and effective tool to prevent COVID-19","abstract":"Filter facepiece respirators (FFRs) are critical for preventing the transmission of respiratory tract infection disease, especially the dreadful coronavirus 2 (SARs-CoV-2). The N95 mask is a prototype, high-efficiency protective device that can effectively protect against airborne pathogens of less than 0.3 \u03bcm. The N95 mask is tightly fitting and has high filtration capacity. The ongoing COVID-19 pandemic has led to a greater requirement for FFR. This rising demand greatly exceeds current production capabilities and stockpiles, resulting in shortages. To address this, our team has invented a new type of half-piece respirator made from silicone and assembled with HEPA or elastostatic filter. A variety of methods have been used to evaluate this new device, including a qualitative fit test with the Bitrex\u00ae test kit and filtration test. The preliminary results showed that the new N99 respirators pass the fit test. The filtration tests also confirmed the superiority of N99 over traditional N95 masks, with a mean performance of protection greater than 95%. For the filters, we used two types: SafeStar, which is a kind of HEPA filter; and CareStar, which is considered an elastostatic filler. CareStar was developed to filter virus and bacteria in the operating room, with a limit duration of use up to 24 h, while the safe star was designed for 72 h use and has the quality equivalent to a HEPA filter. Our study demonstrated superior filtration efficacy of both filters, more than 98% even after 24 h of use. CareStar has significantly more filtration efficacy than a safe star (p < 0.001). In conclusion, the development of our new N99 half-piece respirator should ultimately be applicable to healthcare workers with at least non-inferiority to the previously used N 95 respirators.. Currently, the adequate supply of such equipment is not feasible. The advent of the new protective device will help protect healthcare workers and replenish the shortage of N95 respirators during the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.07.23.217372","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.212704","pub_date":"2020-7-23","title":"Molecular determinants of vascular transport of dexamethasone in COVID-19 therapy","abstract":"Dexamethasone, a widely used corticosteroid, has recently been reported as the first drug to increase the survival chances of patients with severe COVID-19. Therapeutic agents, including dexamethasone, are mostly transported through the body by binding to serum albumin. Herein, we report the first structure of serum albumin in complex with dexamethasone. We show that it binds to Drug Site 7, which is also the binding site for commonly used nonsteroidal anti-inflammatory drugs and testosterone, suggesting potentially problematic binding competition. This study bridges structural findings with our analysis of publicly available clinical data from Wuhan and suggests that an adjustment of dexamethasone regimen should be considered for patients affected by two major COVID-19 risk-factors: low albumin levels and diabetes. Structure of serum albumin with dexamethasone reveals why the drug may not always help COVID-19 patients.","version":"1.1","doi":"10.1101/2020.07.21.212704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.17.995795","pub_date":"2020-7-23","title":"Scientometric Trends for Coronaviruses and Other Emerging Viral Infections","abstract":"COVID-19 is the most rapidly expanding coronavirus outbreak in the past two decades. To provide a swift response to a novel outbreak, prior knowledge from similar outbreaks is essential. Here, we study the volume of research conducted on previous coronavirus outbreaks, specifically SARS and MERS, relative to other infectious diseases by analyzing over 35 million papers from the last 20 years. Our results demonstrate that previous coronavirus outbreaks have been understudied compared to other viruses. We also show that the research volume of emerging infectious diseases is very high after an outbreak and drops drastically upon the containment of the disease. This can yield inadequate research and limited investment in gaining a full understanding of novel coronavirus management and prevention. Independent of the outcome of the current COVID-19 outbreak, we believe that measures should be taken to encourage sustained research in the field.","version":"1.3","doi":"10.1101/2020.03.17.995795","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.216358","pub_date":"2020-7-22","title":"Rescue of SARS-CoV-2 from a single bacterial artificial chromosome","abstract":"An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.","version":"1.1","doi":"10.1101/2020.07.22.216358","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.216150","pub_date":"2020-7-22","title":"Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2","abstract":"Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19. The novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two somewhat redundant pathways by which the SARS-CoV-2 spike (S) protein is activated to mediate infection. The first pathway is dependent on the endosomal protease cathepsin L and sensitive to hydroxychloroquine, whereas the second pathway is dependent on TMPRSS2, which is unaffected by this compound. We further show that SARS-CoV-2 is more reliant than SARS coronavirus (SARS-CoV-1) on the TMPRSS2 pathway, and that this difference is due to a furin cleavage site present in the SARS-CoV-2 S protein. Finally, we show that combinations of hydroxychloroquine and a clinically tested TMPRSS2 inhibitor work together to effectively inhibit SARS-CoV-2 entry. Thus TMPRSS2 expression on physiologically relevant SARS-CoV-2 target cells may bypass the antiviral activities of hydroxychloroquine, and explain its lack of in vivo efficacy.","version":"1.1","doi":"10.1101/2020.07.22.216150","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.215731","pub_date":"2020-7-22","title":"Elucidation of Genome Polymorphisms in Emerging SARS-CoV-2","abstract":"The COVID-19 pandemic is having a devastating effect on the healthcare system and the economy of the world. The unavailability of a specific treatment regime and a candidate vaccine yet opens up scope for new approaches and discoveries of drugs for mitigation of the sufferings of humankind due to the disease. The present isolated whole-genome sequences of SARS-CoV-2 from 11 different nations subjected to evolutionary study and genome-wide association study through in silico approaches including multiple sequence alignment, phylogenetic study through MEGA7 and have been analyzed through DNAsp respectively. These investigations recognized the nucleotide varieties and single nucleotide mutations/polymorphisms on the genomic regions as well as protein-coding regions. The resulted mutations have diversified the genomic contents of SARS-CoV-2 according to the altered nucleotides found in 11 genome sequences. India and Nepal have found to have progressively more distinct species of SARS-CoV-2 with variations in Spike protein and Nucleocapsid protein-coding sites. These genomic variations might be the explanation behind the less case fatality rate of India and Nepal dependent on the populaces. The anticipated idea of this investigation upgrades the information about genomic medication and might be useful in the planning of antibodies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.22.215731","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214932","pub_date":"2020-7-22","title":"The D614G mutation in the SARS-CoV2 Spike protein increases infectivity in an ACE2 receptor dependent manner","abstract":"The SARS-CoV2 coronavirus responsible for the current COVID19 pandemic has been reported to have a relatively low mutation rate. Nevertheless, a few prevalent variants have arisen that give the appearance of undergoing positive selection as they are becoming increasingly widespread over time. Most prominent among these is the D614G amino acid substitution in the SARS-CoV2 Spike protein, which mediates viral entry. The D614G substitution, however, is in linkage disequilibrium with the ORF1b P314L mutation where both mutations almost invariably co-occur, making functional inferences problematic. In addition, the possibility of repeated new introductions of the mutant strain does not allow one to distinguish between a founder effect and an intrinsic genetic property of the virus. Here, we synthesized and expressed the WT and D614G variant SARS-Cov2 Spike protein, and report that using a SARS-CoV2 Spike protein pseudotyped lentiviral vector we observe that the D614G variant Spike has >1/2 log10 increased infectivity in human cells expressing the human ACE2 protein as the viral receptor. The increased binding/fusion activity of the D614G Spike protein was corroborated in a cell fusion assay using Spike and ACE2 proteins expressed in different cells. These results are consistent with the possibility that the Spike D614G mutant increases the infectivity of SARS-CoV2.","version":"1.1","doi":"10.1101/2020.07.21.214932","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.215236","pub_date":"2020-7-22","title":"Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors","abstract":"The ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.","version":"1.1","doi":"10.1101/2020.07.22.215236","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.22.215962","pub_date":"2020-7-22","title":"Neonatal hyperoxia enhances age-dependent expression of SARS-CoV-2 receptors in mice","abstract":"The severity of COVID-19 lung disease is higher in the elderly and people with pre-existing co-morbidities. People who were born preterm may be at greater risk for COVID-19 because their early exposure to oxygen at birth increases their risk of being hospitalized when infected with RSV and other respiratory viruses. Our prior studies in mice showed how high levels of oxygen (hyperoxia) between postnatal days 0-4 increases the severity of influenza A virus infections by reducing the number of alveolar epithelial type 2 (AT2) cells. Because AT2 cells express the SARS-CoV-2 receptors angiotensin converting enzyme (ACE2) and transmembrane protease/serine subfamily member 2 (TMPRSS2), we expected their expression would decline as AT2 cells were depleted by hyperoxia. Instead, we made the surprising discovery that expression of Ace2 and Tmprss2 mRNA increases as mice age and is accelerated by exposing mice to neonatal hyperoxia. ACE2 is primarily expressed at birth by airway Club cells and becomes detectable in AT2 cells by one year of life. Neonatal hyperoxia increases ACE2 expression in Club cells and makes it detectable in 2-month-old AT2 cells. This early and increased expression of SARS-CoV-2 receptors was not seen in adult mice who had been administered the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia. Our finding that early life insults such as hyperoxia enhances the age-dependent expression of SARS-CoV-2 receptors in the respiratory epithelium helps explain why COVID-19 lung disease is greater in the elderly and people with pre-existing co-morbidities.","version":"1.1","doi":"10.1101/2020.07.22.215962","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214759","pub_date":"2020-7-22","title":"Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants","abstract":"Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.","version":"1.1","doi":"10.1101/2020.07.21.214759","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.213249","pub_date":"2020-7-21","title":"Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2","abstract":"The emergence of SARS-CoV-2 has led to the development of new serological assays that could aid in diagnosis and evaluation of seroprevalence to inform an understanding of the burden of COVID-19 disease. Many available tests lack rigorous evaluation and therefore results may be misleading. The aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against SARS-CoV-2 trimeric spike (S), spike receptor binding domain (RBD), spike N terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay. A multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery) was evaluated for the simultaneous detection of IgG binding to four SARS-CoV-2 antigens and the quantification of antibody-induced ACE-2 binding inhibition (pseudo-neutralisation assay). Sensitivity was evaluated with a total of 196 COVID-19 serum samples (169 confirmed PCR positive and 27 anti-nucleocapsid IgG positive) from individuals with mild symptomatic or asymptomatic disease. Specificity was evaluated with 194 control serum samples collected from adults prior to December 2019. The specificity and sensitivity of the binding IgG assay was highest for S protein with a specificity of 97.4% and sensitivity of 96.2% for samples taken 14 days and 97.9% for samples taken 21 days following the onset of symptoms. IgG concentration to S and RBD correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay. Excellent sensitivity for IgG detection was obtained over 14 days since onset of symptoms for three SARS-CoV-2 antigens (S, RBD and N) in this multiplexed assay which can also measure antibody functionality.","version":"1.1","doi":"10.1101/2020.07.20.213249","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.028522","pub_date":"2020-7-21","title":"Systemic analysis of tissue cells potentially vulnerable to SARS-CoV-2 infection by the protein-proofed single-cell RNA profiling of ACE2, TMPRSS2 and Furin proteases","abstract":"Single-cell RNA profiling of ACE2, the SARS-CoV-2 receptor, had proposed multiple tissue cells as the potential targets of SARS-CoV-2, the novel coronavirus causing the COVID-19 pandemic. However, most were not echoed by the patients\u2019 clinical manifestations, largely due to the lack of protein expression information of ACE2 and co-factors. Here, we incorporated the protein information to analyse the expression of ACE2, together with TMPRSS2 and Furin, two proteases assisting SARS-CoV-2 infection, at single cell level in situ, which we called protein-proofed single-cell RNA (pscRNA) profiling. Systemic analysis across 36 tissues revealed a rank list of candidate cells potentially vulnerable to SARS-CoV-2. The top targets are lung AT2 cells and macrophages, then cardiomyocytes and adrenal gland stromal cells, followed by stromal cells in testis, ovary and thyroid. Whereas, the polarized kidney proximal tubule cells, liver cholangiocytes and intestinal enterocytes are less likely to be the primary SARS-CoV-2 targets as ACE2 localizes at the apical region of cells, where the viruses may not readily reach. Actually, the stomach may constitute a physical barrier against SARS-CoV-2 as the acidic environment in normal stomach (pH < 2.0) could completely inactivate SARS-CoV-2 pseudo-viruses. These findings are in concert with the clinical characteristics of prominent lung symptoms, frequent heart injury, and uncommon intestinal symptoms and acute kidney injury. Together, we provide a comprehensive view on the potential SARS-CoV-2 targets by pscRNA profiling, and propose that, in addition to acute respiratory distress syndrome, attentions should also be paid to the potential injuries in cardiovascular, endocrine and reproductive systems during the treatment of COVID-19 patients.","version":"1.3","doi":"10.1101/2020.04.06.028522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.213298","pub_date":"2020-7-21","title":"S protein-reactive IgG and memory B cell production after human SARS-CoV-2 infection includes broad reactivity to the S2 subunit","abstract":"The high susceptibility of humans to SARS-CoV-2 infection, the cause of COVID-19, reflects the novelty of the virus and limited preexisting B cell immunity. IgG against the SARS-CoV-2 spike (S) protein, which carries the novel receptor binding domain (RBD), is absent or at low levels in unexposed individuals. To better understand the B cell response to SARS-CoV-2 infection, we asked whether virus-reactive memory B cells (MBCs) were present in unexposed subjects and whether MBC generation accompanied virus-specific IgG production in infected subjects. We analyzed sera and PBMCs from non-SARS-CoV-2-exposed healthy donors and COVID-19 convalescent subjects. Serum IgG levels specific for SARS-CoV-2 proteins (S, including the RBD and S2 subunit, and nucleocapsid [N]) and non-SARS-CoV-2 proteins were related to measurements of circulating IgG MBCs. Anti-RBD IgG was absent in unexposed subjects. Most unexposed subjects had anti-S2 IgG and a minority had anti-N IgG, but IgG MBCs with these specificities were not detected, perhaps reflecting low frequencies. Convalescent subjects had high levels of IgG against the RBD, S2, and N, together with large populations of RBD- and S2-reactive IgG MBCs. Notably, IgG titers against the S protein of the human coronavirus OC43 in convalescent subjects were higher than in unexposed subjects and correlated strongly with anti-S2 titers. Our findings indicate cross-reactive B cell responses against the S2 subunit that might enhance broad coronavirus protection. Importantly, our demonstration of MBC induction by SARS-CoV-2 infection suggests that a durable form of B cell immunity is maintained even if circulating antibody levels wane. Recent rapid worldwide spread of SARS-CoV-2 has established a pandemic of potentially serious disease in the highly susceptible human population. Key questions are whether humans have preexisting immune memory that provides some protection against SARS-CoV-2 and whether SARS-CoV-2 infection generates lasting immune protection against reinfection. Our analysis focused on pre- and post-infection IgG and IgG memory B cells (MBCs) reactive to SARS-CoV-2 proteins. Most importantly, we demonstrate that infection generates both IgG and IgG MBCs against the novel receptor binding domain and the conserved S2 subunit of the SARS-CoV-2 spike protein. Thus, even if antibody levels wane, long-lived MBCs remain to mediate rapid antibody production. Our study also suggests that SARS-CoV-2 infection strengthens preexisting broad coronavirus protection through S2-reactive antibody and MBC formation.","version":"1.1","doi":"10.1101/2020.07.20.213298","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.212639","pub_date":"2020-7-21","title":"SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-\u03baB","abstract":"SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to permanent lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses which can lead to systemic complications. We have evaluated transcriptional and cytokine secretion profiles from infected cell cultures and detected a distinct upregulation of inflammatory cytokines that parallels samples taken from infected patients. Building on these observations, we found a specific activation of NF-\u03baB and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-\u03baB response is mediated by cGAS-STING activation and could be attenuated through STING targeting drugs. Our results show that SARS-CoV-2 curates a cGAS-STING mediated NF-\u03baB driven inflammatory immune response in epithelial cells that likely contributes to inflammatory responses seen in patients and might be a target to suppress severe disease symptoms.","version":"1.1","doi":"10.1101/2020.07.21.212639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.135954","pub_date":"2020-7-21","title":"Assessing SARS-CoV-2 spatial phylogenetic structure: Evidence from RNA and protein sequences","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent RNA virus that spread around the planet in about 4 months. The consequences of this rapid dispersion are under investigation. In this work, we analyzed thousands of genomes and protein sequences from Africa, America, Asia, Europe, and Oceania. We show that the virus is a complex of slightly different variants that are unevenly distributed on Earth, and demonstrate that SARS-CoV-2 phylogeny is spatially structured. Remarkably, the virus phylogeographic patterns were associated with ancestral amino acidic mutations. We hypothesize that geographic structuring is the result of founder effects occurring as a consequence of, and local evolution occurring after, long-distance dispersal. Based on previous studies, the possibility that this could significantly affect the virus phenotype is not remote.","version":"1.2","doi":"10.1101/2020.06.05.135954","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.213280","pub_date":"2020-7-21","title":"The PRRA insert at the S1/S2 site modulates cellular tropism of SARS-CoV-2 and ACE2 usage by the closely related Bat raTG13","abstract":"Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting human and the presence of four residues (PRRA) at the S1/S2 site within the Spike protein may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species except mouse to infect 293T cells. Similarly, pseudoviruses bearing spike protein derived from either the bat raTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 Spike displayed distinct effects on pseudoviral entry into different cell types. Strikingly, insertion of PRRA into the raTG13 Spike selectively abrogated the usage of horseshoe bat and pangolin ACE2 but conferred usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and raTG13 spike proteins.","version":"1.1","doi":"10.1101/2020.07.20.213280","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.034967","pub_date":"2020-7-21","title":"Comparative ACE2 variation and primate COVID-19 risk","abstract":"The emergence of the novel coronavirus SARS-CoV-2, which in humans is highly infectious and leads to the potentially fatal disease COVID-19, has caused hundreds of thousands of deaths and huge global disruption. The viral infection may also represent an existential threat to our closest living relatives, the nonhuman primates, many of which are endangered and often reduced to small populations. The virus engages the host cell receptor, angiotensin-converting enzyme-2 (ACE2), through the receptor binding domain (RBD) on the spike protein. The contact surface of ACE2 displays amino acid residues that are critical for virus recognition, and variations at these critical residues are likely to modulate infection susceptibility across species. While infection studies are emerging and have shown that some primates, such as rhesus macaques and vervet monkeys, develop COVID-19-like symptoms when exposed to the virus, the susceptibility of many other nonhuman primates is unknown. Here, we show that all apes, including chimpanzees, bonobos, gorillas, and orangutans, and all African and Asian monkeys (catarrhines), exhibit the same set of twelve key amino acid residues as human ACE2. Monkeys in the Americas, and some tarsiers, lemurs and lorisoids, differ at significant contact residues, and protein modeling predicts that these differences should greatly reduce the binding affinity of the ACE2 for the virus, hence moderating their susceptibility for infection. Other lemurs are predicted to be closer to catarrhines in their susceptibility. Our study suggests that apes and African and Asian monkeys, as well as some lemurs are all likely to be highly susceptible to SARS-CoV-2, representing a critical threat to their survival. Urgent actions have been undertaken to limit the exposure of Great Apes to humans, and similar efforts may be necessary for many other primate species.","version":"1.3","doi":"10.1101/2020.04.09.034967","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.21.214098","pub_date":"2020-7-21","title":"Molecular mechanism of SARS-CoV-2 cell entry inhibition via TMPRSS2 by Camostat and Nafamostat mesylate","abstract":"The entry of the coronavirus SARS-CoV-2 into human cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations. In vitro assays confirm the hypothesis that both drugs act by inhibiting the human protein TMPRSS2. As no experimental structure is available, we provide a model of the TMPRSS2 equilibrium structure and its fluctuations by relaxing an initial homology structure with extensive 280 microseconds of all-atom molecular dynamics (MD) and Markov modeling. We describe the binding mode of both drugs with TMPRSS2 in a Michaelis complex (MC) state preceding the formation of a long-lived covalent inhibitory state. We find that nafamostat to has a higher MC population, which in turn leads to the more frequent formation of the covalent complex and thus higher inhibition efficacy, as confirmed in vitro and consistent with previous virus cell entry assays. Our TMPRSS2-drug structures are made public to guide the design of more potent and specific inhibitors.","version":"1.1","doi":"10.1101/2020.07.21.214098","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.213082","pub_date":"2020-7-21","title":"Discovery of potential imaging and therapeutic targets for severe inflammation in COVID-19 patients","abstract":"The COVID-19 pandemic has caused more than 540,000 deaths globally. Hyperinflammation mediated by dysregulated monocyte/macrophage function is considered to be the key factor that triggers severe illness in COVID-19. However, no specific targeting molecule has been identified for detecting or treating hyperinflammation related to dysregulated macrophages in severe COVID-19. Herein, we suggest candidate targets for imaging and therapy in severe COVID-19 by analyzing single-cell RNA-sequencing data based on bronchoalveolar lavage fluid of COVID-19 patients. We found that expression of SLC2A3, which can be imaged by [18F]fluorodeoxyglucose, was higher in macrophages from severe COVID-19 patients. Furthermore, by integrating the surface target database and drug-target binding database with RNA-sequencing data of severe COVID-19, we identified CCR1 and FPR1 as surface and druggable targets for drug delivery as well as molecular imaging. Our results provide a resource for candidate targets in the development of specific imaging and therapy for COVID-19-related hyperinflammation.","version":"1.1","doi":"10.1101/2020.07.20.213082","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.210534","pub_date":"2020-7-20","title":"SARS-CoV2 spike protein displays biologically significant similarities with paramyxovirus surface proteins; a bioinformatics study","abstract":"Recent emergence of SARS-CoV2 and associated COVID-19 pandemic has posed a great challenge for the scientific community. Understanding various aspects of SARS-CoV2 biology, virulence and pathogenesis as well as determinants of immune response have become a global research priority. In this study, we performed bioinformatic analyses on SAR-CoV2 protein sequences, trying to unravel biologically important similarities between this newly emerged virus with other RNA viruses. Comparing the proteome of SARS-CoV2 with major positive and negative strand ssRNA viruses showed significant homologies between SARS-CoV2 spike protein with pathogenic paramyxovirus fusion proteins. This \u2018spike-fusion\u2019 homology was not limited to SARS-CoV2 and it existed for some other pathogenic coronaviruses; nonetheless, SARS-CoV2 spike-fusion homology was orders of magnitude stronger than homologies observed for other known coronaviruses. Moreover, this homology did not seem to be a consequence of general ssRNA virus phylogenetic relations. We also explored potential immunological significance of this spike-fusion homology. Spike protein epitope analysis using experimentally verified data deposited in Immune Epitope Database (IEDB) revealed that the majority of spike\u2019s T cell epitopes as well as many B cell and MHC binding epitopes map within the spike-fusion homology region. Overall, our data indicate that there might be a relation between SARS-CoV2 and paramyxoviruses at the level of their surface proteins and this relation could be of crucial immunological importance.","version":"1.1","doi":"10.1101/2020.07.20.210534","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.212068","pub_date":"2020-7-20","title":"Computational optimization of the SARS-CoV-2 receptor-binding-motif affinity for human ACE2","abstract":"The coronavirus SARS-CoV-2, that is responsible for the COVID-19 pandemic, and the closely related SARS-CoV coronavirus enter cells by binding at the human angiotensin converting enzyme 2 (hACE2). The stronger hACE2 affinity of SARS-CoV-2 has been connected with its higher infectivity. In this work, we study hACE2 complexes with the receptor binding domains (RBDs) of the human SARS-CoV-2 and human SARS-CoV viruses, using all-atom molecular dynamics (MD) simulations and Computational Protein Design (CPD) with a physics-based energy function. The MD simulations identify charge-modifying substitutions between the CoV-2 and CoV RBDs, which either increase or decrease the hACE2 affinity of the SARS-CoV-2 RBD. The combined effect of these mutations is small, and the relative affinity is mainly determined by substitutions at residues in contact with hACE2. Many of these findings are in line and interpret recent experiments. Our CPD calculations redesign positions 455, 493, 494 and 501 of the SARS-CoV-2 RBM, which contact hACE2 in the complex and are important for ACE2 recognition. Sampling is enhanced by an adaptive importance sampling Monte Carlo method. Sequences with increased affinity replace CoV-2 glutamine by a negative residue at position 493, and serine by nonpolar, aromatic or a threonine at position 494. Substitutions at positions positions 455 and 501 have a smaller effect on affinity. Substitutions suggested by our design are seen in viral sequences encountered in other species, including bat and pangolin. Our results might be used to identify potential virus strains with higher human infectivity and assist in the design of peptide-based or peptidomimetic compounds with the potential to inhibit SARS-CoV-2 binding at hACE2. The coronavirus SARS-CoV-2 is responsible for the current COVID-19 pandemic. SARS-CoV-2 and the earlier, closely related SARS-CoV virus bind at the human angiotensin converting enzyme 2 (hACE2) receptor at the cell surface. The higher human infectivity of SARS-CoV-2 may be linked to its stronger affinity for hACE2. Here, we study by computational methods complexes of hACE2 with the receptor binding domains (RBDs) of viruses SARS-CoV-2 and SARS-CoV. We identify residues affecting the affinities of the two domains for hACE2. We also propose mutations at key SARS-CoV-2 positions, which might enhance hACE2 affinity. Such mutations may appear in viral strains with increased human infectivity and might assist the design of peptide-based compounds that inhibit infection of human cells by SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.20.212068","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.211789","pub_date":"2020-7-20","title":"A human disease model of SARS-CoV-2-induced lung injury and immune responses with a microengineered organ chip","abstract":"Coronavirus disease 2019 (COVID-19) is a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that seriously endangers human health. There is an urgent need to build physiological relevant human models for deep understanding the complex organ-level disease processes and facilitating effective therapeutics for COVID-19. Here, we first report the use of microengineered alveolus chip to create a human disease model of lung injury and immune responses induced by native SARS-CoV-2 at organ-level. This biomimetic system is able to reconstitute the key features of human alveolar-capillary barrier by co-culture of alveolar epithelial and microvascular endothelial cells under microfluidic flow. The epithelial cells on chip showed higher susceptibility to SARS-CoV-2 infection than endothelial cells identified by viral spike protein expression. Transcriptional analysis showed distinct responses of two cell types to SARS-CoV-2 infection, including activated type I interferon (IFN-I) signaling pathway in epithelium and activated JAK-STAT signaling pathway in endothelium. Notably, in the presence of circulating immune cells, a series of alveolar pathological changes were observed, including the detachment of endothelial cells, recruitment of immune cells, and increased production of inflammatory cytokines (IL-6, IL-8, IL-1\u03b2 and TNF-\u03b1). These new findings revealed a crucial role of immune cells in mediating lung injury and exacerbated inflammation. Treatment with antiviral compound remdesivir could suppress viral copy and alleviate the disruption of alveolar barrier integrity induced by viral infection. This bioengineered human organ chip system can closely mirror human-relevant lung pathogenesis and immune responses to SARS-CoV-2 infection, not possible by other in vitro models, which provides a promising and alternative platform for COVID-19 research and preclinical trials.","version":"1.1","doi":"10.1101/2020.07.20.211789","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.193193","pub_date":"2020-7-20","title":"Vinegar and Its Active Component Acetic Acid Inhibit SARS-CoV-2 Infection In Vitro and Ex Vivo","abstract":"Effective and economical measures are needed to either prevent or inhibit the replication of SARS-CoV-2, the causative agent of COVID-19, in the upper respiratory tract. As fumigation of vinegar at low concentration (0.34%) ameliorated the symptoms of mild SARS-CoV-2 infection, we tested in vitro the potential antiviral activity of vinegar and of its active component, acetic acid. We here demonstrate that both vinegar and acetic acid indeed strongly inactivate SARS-CoV-2 infectivity in Vero cells. Furthermore, vinegar treatment caused a 90% inhibition of the infectious titer when directly applied to a nasopharyngeal swab transfer medium of a COVID-19 patient. These effects were potentiated if conduced at a temperature of 45 \u00b0C vs. 37 \u00b0C, a condition that is transiently generated in the upper respiratory tract during fumigation. Our findings are consistent and extend the results of studies performed in the early and mid-20th century on the disinfectant capacity of organic acids and can provide an affordable home-made aid to prevent or contain SARS-CoV-2 infection of the upper respiratory tract.","version":"1.2","doi":"10.1101/2020.07.08.193193","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.19.210955","pub_date":"2020-7-20","title":"Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.","version":"1.1","doi":"10.1101/2020.07.19.210955","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.210013","pub_date":"2020-7-20","title":"The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models","abstract":"The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 \u03bcM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus. MTX is one of the earliest cancer drugs to be developed, giving rise to seven decades of clinical experience. It is on the World Health Organization\u2019s List of Essential Medicines, can be administered orally or parenterally, and its costs are at single digit \u20ac or $ amounts/day for standard treatment. In case of its successful further preclinical evaluation for treating SARS-CoV-2 infections, its repurposing to treat COVID-19 would thus be feasible, especially under low-resource conditions. Additional drugs exist to interfere with the synthesis of nucleotides, e.g. additional folate antagonists, inhibitors of GMP synthetase, or inhibitors of dihydroorotate dehydrogenase (DHODH). Such inhibitors have been approved as drugs for different purposes and might represent further therapeutic options against infections with SARS-CoV-2 Remdesivir is currently the most established drug for treating COVID-19. Our results argue that MTX and remdesivir, even at moderate concentrations, can act in a synergistic fashion to repress virus replication to a considerably greater extent than either drug alone. COVID-19, in its severe forms, is characterized by pneumonia and acute respiratory distress syndrome, and additional organ involvements. These manifestations are not necessarily a direct consequence of virus replication and cytopathic effects, but rather a result of an uncontrolled inflammatory and immune response. Anti-inflammatory drugs such as glucocorticoids are thus being evaluated for treating COVID-19. However, this bears the risk of re-activating virus spread by suppressing a sufficient and specific immune response. In this situation, it is tempting to speculate that MTX might suppress both excessive inflammation as well as virus replication at the same time, thus limiting both the pathogenesis of pneumonia and also the spread of virus within a patient.","version":"1.1","doi":"10.1101/2020.07.18.210013","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.092536","pub_date":"2020-7-20","title":"Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection","abstract":"Analyzing host transcriptional changes in response to SARS-CoV-2 infection will help delineate biological processes underlying viral pathogenesis. Comparison of expression profiles of lung cell lines A549 (infected with either SARS-CoV-2 (with ACE2 expression)) or Influenza A virus (IAV)) and Calu3 (infected with SARS-CoV-2 or MERS-CoV) revealed upregulation of the antiviral interferon signaling in all three viral infections. However, perturbations in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2 infected cells. Validation of findings from cell line data revealed perturbations in autophagy and mitochondrial processes in the infected human nasopharyngeal samples. Specifically, downregulation of mTOR expression, mitochondrial ribosomal, mitochondrial complex I, and lysosome acidification genes were concurrently observed in both infected cell lines and human datasets. Furthermore, SARS-CoV-2 infection impedes autophagic flux by upregulating GSK3B in lung cell lines, or by downregulating autophagy genes, SNAP29 and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Additionally, downregulation of MTFP1 (in cell lines) or SOCS6 (in human samples) results in hyperfused mitochondria and impede proper interferon response. Coexpression networks analysis identifies correlated clusters of genes annotated to inflammation and mitochondrial processes that are misregulated in SARS-CoV-2 infected cells. Finally, comparison of age stratified human gene expression data revealed impaired upregulation of chemokines, interferon stimulated and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.05.13.092536","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.105965","pub_date":"2020-7-20","title":"Computational Analysis of Dynamic Allostery and Control in the SARS-CoV-2 Main Protease","abstract":"The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has generated a global pandemic and no vaccine or antiviral drugs exist at the moment of writing. An attractive coronavirus drug target is the main protease (Mpro, also known as 3CLpro) because of its vital role in the viral cycle. A significant body of work has been focused on finding inhibitors which bind and block the active site of the main protease, but little has been done to address potential non-competitive inhibition which targets regions beyond the active site, partly because the fundamental biophysics of such allosteric control is still poorly understood. In this work, we construct an Elastic Network Model (ENM) of the SARS-CoV-2 Mpro homodimer protein and analyse the dynamics and thermodynamics of the main protease\u2019s ENM. We found a rich and heterogeneous dynamical structure in the correlated motions, including allosterically correlated motions between the homodimeric protease\u2019s active sites. Exhaustive 1-point and 2-point mutation scans of the ENM and their effect on fluctuation free energies confirm previously experimentally identified bioactive residues, but also suggest several new candidate regions that are distant from the active site for control of the protease function. Our results suggest new dynamically-driven control regions as possible candidates for non-competitive inhibiting binding sites in the protease, which may assist the development of current fragmentbased binding screens. The results also provide new insight into the protein physics of fluctuation allostery and its underpinning dynamical structure.","version":"1.2","doi":"10.1101/2020.05.21.105965","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.20.211623","pub_date":"2020-7-20","title":"Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism","abstract":"Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His8, the C-terminus with His8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.","version":"1.1","doi":"10.1101/2020.07.20.211623","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.209270","pub_date":"2020-7-20","title":"Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome","abstract":"Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 \u00c5, presents a topologically complex fold in which the 5\u2032 end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.","version":"1.1","doi":"10.1101/2020.07.18.209270","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.210211","pub_date":"2020-7-20","title":"Inhibitors of VPS34 and lipid metabolism suppress SARS-CoV-2 replication","abstract":"Therapeutics targeting replication of SARS coronavirus 2 (SARS-CoV-2) are urgently needed. Coronaviruses rely on host membranes for entry, establishment of replication centers and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we tested small molecule inhibitors that target membrane dynamics or lipid metabolism. Included were inhibitors of the PI3 kinase VPS34, which functions in autophagy, endocytosis and other processes; Orlistat, an inhibitor of lipases and fatty acid synthetase, is approved by the FDA as a treatment for obesity; and Triacsin C which inhibits long chain fatty acyl-CoA synthetases. VPS34 inhibitors, Orlistat and Triacsin C inhibited virus growth in Vero E6 cells and in the human airway epithelial cell line Calu-3, acting at a post-entry step in the virus replication cycle. Of these the VPS34 inhibitors exhibit the most potent activity.","version":"1.1","doi":"10.1101/2020.07.18.210211","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.19.179101","pub_date":"2020-7-20","title":"Haplotype Explorer: an infection cluster visualization tool for spatiotemporal dissection of the COVID-19 pandemic","abstract":"The worldwide eruption of COVID-19 that began in Wuhan, China in late 2019 reached 10 million cases by late June 2020. In order to understand the epidemiological landscape of the COVID-19 pandemic, many studies have attempted to elucidate phylogenetic relationships between collected viral genome sequences using haplotype networks. However, currently available applications for network visualization are not suited to understand the COVID-19 epidemic spatiotemporally, due to functional limitations That motivated us to develop Haplotype Explorer, an intuitive tool for visualizing and exploring haplotype networks. Haplotype Explorer enables people to dissect epidemiological consequences via interactive node filters to provide spatiotemporal perspectives on multimodal spectra of infectious diseases, including introduction, outbreak, expansion, and containment, for given regions and time spans. Here, we demonstrate the effectiveness of Haplotype Explorer by showing an example of its visualization and features. The demo using SARS-CoV-2 genome sequences is available at https://github.com/TKSjp/HaplotypeExplorer A lot of software for network visualization are available, but existing software have not been optimized to infection cluster visualization against the current worldwide invasion of COVID-19 started since 2019. To reach the spatiotemporal understanding of its epidemics, we developed Haplotype Explorer. It is superior to other applications in the point of generating HTML distribution files with metadata searches which interactively reflects GISAID IDs, locations, and collection dates. Here, we introduce the features and products of Haplotype Explorer, demonstrating the time-dependent snapshots of haplotype networks inferred from total of 4,282 SARS-CoV-2 genomes.","version":"1.1","doi":"10.1101/2020.07.19.179101","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.19.197129","pub_date":"2020-7-20","title":"Population genetic analysis of Indian SARS-CoV-2 isolates reveals a unique phylogenetic cluster","abstract":"The SARS-CoV-2 pandemic originated from Wuhan, China in December 2019 raised an alarming situation all over the globe. Sequencing of this novel virus provides an opportunity to evaluate the genetic polymorphism present in the viral population. Herein, we analysed 173 sequences isolated from Indian patients and performed SNP linkage, clustering and phylogenetic analysis to understand the local genetic diversity. We found that the SNP linkages that lead to the identification of some global clades, do not hold true for the local clade classification. In addition to the unique cluster, established by another Indian study, we identified a new cluster (I-20D) that encompasses 28% of the analysed sequences. This cluster is defined by two linked variations \u2013 C22444T and C28854T. A detailed study of such polymorphisms can be useful for drug and vaccine development.","version":"1.1","doi":"10.1101/2020.07.19.197129","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.206946","pub_date":"2020-7-20","title":"De novo design of modular and tunable allosteric biosensors","abstract":"Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications , but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest . Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 , we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.","version":"1.1","doi":"10.1101/2020.07.18.206946","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.19.211185","pub_date":"2020-7-20","title":"Probing the dynamic structure-function and structure-free energy relationships of the corona virus main protease with Biodynamics theory","abstract":"The SARS-CoV-2 Main protease (Mpro) is of major interest as an anti-viral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 Mpro crystal structures, are underway in many labs. However, little is known about the dynamic enzyme mechanism, which is needed to inform both structure-based design and assay development. Here, we apply Biodynamics theory to characterize the structural dynamics of substrate-induced Mpro activation, and explore the implications thereof for efficacious inhibition under non-equilibrium conditions. The catalytic cycle (including tetrahedral intermediate formation and hydrolysis) is governed by concerted dynamic structural rearrangements of domain 3 and the m-shaped loop (residues 132-147) on which Cys145 (comprising the thiolate nucleophile and one-half of the oxyanion hole) and Gly143 reside (comprising the other half of the oxyanion hole). In particular:\n\nDomain 3 undergoes dynamic rigid-body rotations about the domain 2-3 linker, alternately visiting two conformational states (denoted as ).\nThe Gly143-containing crest of the m-shaped loop (denoted as crest B) undergoes up and down translations in concert with the domain 3 rotations (denoted as , whereas the Cys145-containing crest (denoted as crest A) remains statically in the up position. The crest B translations are driven by conformational transitions within the rising leg of the loop (Lys137-Asn142).\n\n Domain 3 undergoes dynamic rigid-body rotations about the domain 2-3 linker, alternately visiting two conformational states (denoted as ). The Gly143-containing crest of the m-shaped loop (denoted as crest B) undergoes up and down translations in concert with the domain 3 rotations (denoted as , whereas the Cys145-containing crest (denoted as crest A) remains statically in the up position. The crest B translations are driven by conformational transitions within the rising leg of the loop (Lys137-Asn142). We propose that substrates associate to the  state, which promotes the  state, dimerization (denoted as -substrate), and catalysis. The structure resets to the dynamic monomeric form upon dissociation of the N-terminal product. We describe the energetics of the aforementioned state transitions, and address the implications of our proposed mechanism for efficacious Mpro inhibition under native-like conditions.","version":"1.1","doi":"10.1101/2020.07.19.211185","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.210179","pub_date":"2020-7-19","title":"Lethality of SARS-CoV-2 infection in K18 human angiotensin converting enzyme 2 transgenic mice","abstract":"Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.","version":"1.1","doi":"10.1101/2020.07.18.210179","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.210120","pub_date":"2020-7-19","title":"High expression of angiotensin-converting enzyme-2 (ACE2) on tissue macrophages that may be targeted by virus SARS-CoV-2 in COVID-19 patients","abstract":"Angiotensin-converting enzyme-2 (ACE2) has been recognized as the binding receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that infects host cells, causing the development of the new coronavirus infectious disease (COVID-19). To better understand the pathogenesis of COVID-19 and build up the host anti-viral immunity, we examined the levels of ACE2 expression on different types of immune cells including tissue macrophages. Flow cytometry demonstrated that there was little to no expression of ACE2 on most of the human peripheral blood-derived immune cells including CD4+ T, CD8+ T, activated CD4+ T, activated CD8+ T, CD4+CD25+CD127low/\u2212 regulatory T cells (Tregs), Th17 cells, NKT cells, B cells, NK cells, monocytes, dendritic cells (DCs), and granulocytes. Additionally, there was no ACE2 expression (< 1%) found on platelets. Compared with interleukin-4-treated type 2 macrophages (M2), the ACE2 expression was markedly increased on the activated type 1 macrophages (M1) after the stimulation with lipopolysaccharide (LPS). Immunohistochemistry demonstrated that high expressions of ACE2 were colocalized with tissue macrophages, such as alveolar macrophages found within the lungs and Kupffer cells within livers of mice. Flow cytometry confirmed the very low level of ACE2 expression on human primary pulmonary alveolar epithelial cells. These data indicate that alveolar macrophages, as the frontline immune cells, may be directly targeted by the SARS-CoV-2 infection and therefore need to be considered for the prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.07.18.210120","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.210161","pub_date":"2020-7-19","title":"An autocrine Vitamin D-driven Th1 shutdown program can be exploited for COVID-19","abstract":"Pro-inflammatory immune responses are necessary for effective pathogen clearance, but cause severe tissue damage if not shut down in a timely manner. Excessive complement and IFN-\u03b3-associated responses are known drivers of immunopathogenesis and are among the most highly induced immune programs in hyper-inflammatory SARS-CoV2 lung infection. The molecular mechanisms that govern orderly shutdown and retraction of these responses remain poorly understood. Here, we show that complement triggers contraction of IFN-\u03b3 producing CD4+ T helper (Th) 1 cell responses by inducing expression of the vitamin D (VitD) receptor (VDR) and CYP27B1, the enzyme that activates VitD, permitting T cells to both activate and respond to VitD. VitD then initiates the transition from pro-inflammatory IFN-\u03b3+ Th1 cells to suppressive IL-10+ Th1 cells. This process is primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating superenhancers and recruiting c-JUN and BACH2, a key immunoregulatory transcription factor. Accordingly, cells in psoriatic skin treated with VitD increased BACH2 expression, and BACH2 haplo-insufficient CD4+ T cells were defective in IL-10 production. As proof-of-concept, we show that CD4+ T cells in the bronchoalveolar lavage fluid (BALF) of patients with COVID-19 are Th1-skewed and that VDR is among the top regulators of genes induced by SARS-CoV2. Importantly, genes normally down-regulated by VitD were de-repressed in CD4+ BALF T cells of COVID-19, indicating that the VitD-driven shutdown program is impaired in this setting. The active metabolite of VitD, alfacalcidol, and cortico-steroids were among the top predicted pharmaceuticals that could normalize SARS-CoV2 induced genes. These data indicate that adjunct therapy with VitD in the context of other immunomodulatory drugs may be a beneficial strategy to dampen hyperinflammation in severe COVID-19.","version":"1.1","doi":"10.1101/2020.07.18.210161","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.18.204362","pub_date":"2020-7-18","title":"The landscape of SARS-CoV-2 RNA modifications","abstract":"In 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the first documented cases of severe lung disease COVID-19. Since then, SARS-CoV-2 has been spreading around the globe resulting in a severe pandemic with over 500.000 fatalities and large economical and social disruptions in human societies. Gaining knowledge on how SARS-Cov-2 interacts with its host cells and causes COVID-19 is crucial for the intervention of novel therapeutic strategies. SARS-CoV-2, like other coronaviruses, is a positive-strand RNA virus. The viral RNA is modified by RNA-modifying enzymes provided by the host cell. Direct RNA sequencing (DRS) using nanopores enables unbiased sensing of canonical and modified RNA bases of the viral transcripts. In this work, we used DRS to precisely annotate the open reading frames and the landscape of SARS-CoV-2 RNA modifications. We provide the first DRS data of SARS-CoV-2 in infected human lung epithelial cells. From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. Conservation of the RNA modification pattern during progression of the current pandemic suggests that this pattern is likely essential for the life cycle of SARS-CoV-2 and represents a possible target for drug interventions.","version":"1.1","doi":"10.1101/2020.07.18.204362","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.208959","pub_date":"2020-7-18","title":"Structural basis for the inhibition of the papain-like protease of SARS-CoV-2 by small molecules","abstract":"SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease has been implicated in virus maturation, dysregulation of host inflammation and antiviral immune responses. We showed that PLpro preferably cleaves the K48-ubiquitin linkage while also being capable of cleaving ISG15 modification. The multiple functions of PLpro render it a promising drug target. Therefore, we screened an FDA-approved drug library and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising IC50 of 2.1 \u03bcM. The co-crystal structure of SARS-CoV-2 PLpro-C111S in complex with GRL0617 suggests that GRL0617 is a non-covalent inhibitor. NMR data indicate that GRL0617 blocks the binding of ISG15 to PLpro. The antiviral activity of GRL0617 reveal that PLpro is a promising drug target for therapeutically treating COVID-19. Co-crystal structure of PLpro in complex with GRL0617 reveals the druggability of PLpro for SARS-CoV-2 treatment.","version":"1.1","doi":"10.1101/2020.07.17.208959","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.209304","pub_date":"2020-7-18","title":"Interplay of Monocytes and T Lymphocytes in COVID-19 Severity","abstract":"The COVID-19 pandemic represents an ongoing global crisis that has already impacted over 13 million people. The responses of specific immune cell populations to the disease remain poorly defined, which hinders improvements in treatment and care management. Here, we utilized mass cytometry (CyTOF) to thoroughly phenotype peripheral myeloid cells and T lymphocytes from 30 convalescent patients with mild, moderate, and severe cases of COVID-19. We identified 10 clusters of monocytes and dendritic cells and 17 clusters of T cells. Examination of these clusters revealed that both CD14+CD16+ intermediate and CD14dimCD16+ nonclassical monocytes, as well as CD4+ stem cell memory T (TSCM) cells, correlated with COVID-19 severity, coagulation factor levels, and/or inflammatory indicators. We also identified two nonclassical monocyte subsets distinguished by expression of the sugar residue 6-Sulfo LacNac (Slan). One of these subsets (Slanlo, nMo1) was depleted in moderately and severely ill patients, while the other (Slanhi, nMo2) increased with disease severity and was linked to CD4+ T effector memory (TEM) cell frequencies, coagulation factors, and inflammatory indicators. Intermediate monocytes tightly correlated with loss of naive T cells as well as an increased abundance of effector memory T cells expressing the exhaustion marker PD-1. Our data suggest that both intermediate and non-classical monocyte subsets shape the adaptive immune response to SARS-CoV-2. In summary, our study provides both broad and in-depth characterization of immune cell phenotypes in response to COVID-19 and suggests functional interactions between distinct cell types during the disease. Use of mass cytometry on peripheral blood mononuclear cells from convalescent COVID-19 patients allows correlation of distinct monocyte and T lymphocyte subsets with clinical factors.","version":"1.1","doi":"10.1101/2020.07.17.209304","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.208439","pub_date":"2020-7-17","title":"SARS-CoV-2 RNA shedding in recovered COVID-19 cases and the presence of antibodies against SARS-CoV-2 in recovered COVID-19 cases and close contacts","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 emerged in December 2019 and has spread globally. Although Thailand has been effective at controlling the spread of COVID-19, disease surveillance and information on antibody responses in infected cases and close contacts are needed because there is still no specific treatment or vaccine available. We investigated 217 recovered COVID-19 cases to monitor their viral RNA shedding and production of antibodies against SARS-CoV-2. The presence of antibodies in blood samples from 308 close contacts of COVID-19 cases was also determined. Viral RNA was still detectable in 6.6 % of recovered COVID-19 cases. The most prolonged duration of viral RNA shedding detected in this study was 105 days. IgM, IgG, and IgA antibodies against SARS-CoV-2 were detected in 13.82, 88.48, and 83.41 % of the recovered cases 4\u201312 weeks after disease onset, respectively. Although the patients had recovered from their illness, the levels of antibodies detected showed association with their symptoms during their stay in hospital. Fifteen of the 308 contacts (4.87 %) of COVID-19 cases tested positive for IgG antibodies. The presence of antibodies against SARS-CoV-2 suggested that there was viral exposure among close contacts. Viral clearance and the pattern of antibody responses in infected individuals are both crucial for effectively combatting SARS-CoV-2. Our study provides additional information on the natural history of this newly emerging disease related to both natural host defenses and a strategy for vaccine development.","version":"1.1","doi":"10.1101/2020.07.17.208439","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.206458","pub_date":"2020-7-17","title":"Identification of SARS-CoV2-mediated suppression of NRF2 signaling reveals a potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate","abstract":"Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular anti-viral program, which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2. NRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as replication of other human pathogenic viruses.","version":"1.1","doi":"10.1101/2020.07.16.206458","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.031807","pub_date":"2020-7-17","title":"Comparison of SARS-CoV-2 infections among 3 species of non-human primates","abstract":"COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. M. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus.","version":"1.2","doi":"10.1101/2020.04.08.031807","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.204339","pub_date":"2020-7-17","title":"Mutational dynamics and transmission properties of SARS-CoV-2 superspreading events in Austria","abstract":"Superspreading events shape the COVID-19 pandemic. Here we provide a national-scale analysis of SARS-CoV-2 outbreaks in Austria, a country that played a major role for virus transmission across Europe and beyond. Capitalizing on a national epidemiological surveillance system, we performed deep whole-genome sequencing of virus isolates from 576 samples to cover major Austrian SARS-CoV-2 clusters. Our data chart a map of early viral spreading in Europe, including the path from low-frequency mutations to fixation. Detailed epidemiological surveys enabled us to calculate the effective SARS-CoV-2 population bottlenecks during transmission and unveil time-resolved intra-patient viral quasispecies dynamics. This study demonstrates the power of integrating deep viral genome sequencing and epidemiological data to better understand how SARS-CoV-2 spreads through populations.","version":"1.1","doi":"10.1101/2020.07.15.204339","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.205088","pub_date":"2020-7-17","title":"A single intranasal dose of chimpanzee adenovirus-vectored vaccine confers sterilizing immunity against SARS-CoV-2 infection","abstract":"The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread.","version":"1.1","doi":"10.1101/2020.07.16.205088","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.206847","pub_date":"2020-7-17","title":"Longitudinal analysis of the humoral response to SARS-CoV-2 spike RBD in convalescent plasma donors","abstract":"H\u00e9ma-Qu\u00e9bec, the blood supplier in the Province of Quebec, Canada, collects and tests convalescent plasma used in a clinical trial to determine the clinical efficacy of this product for the treatment of hospitalized COVID-19 patients. So far, we have collected 1159 plasma units from 282 COVID-19 convalescent donors. The presence of antibodies to the receptor binding domain (RBD) of SARS-CoV-2 spike protein in convalescent donors was established at the first donation. Seropositive donors were asked to donate additional plasma units every six days. Until now, 15 donors have donated at least four times and, in some cases, up to nine times. This allowed us to perform a longitudinal analysis of the persistence of SARS-CoV-2 RBD-specific antibodies in these repeat donors, with the first donation occurring 33-77 days after symptoms onset and donations up to 71-114 days after symptoms onset thereafter. In all donors, the level of antibodies remained relatively stable up to about 76 days after symptoms onset but then started to decrease more rapidly to reach, in some convalescent donors, a seronegative status within 100-110 days after symptoms onset. The decline in anti-RBD antibodies was not related to the number of donations but strongly correlated with the numbers of days after symptoms onset (r = 0.821). This suggests that de novo secretion of SARS-CoV-2 RBD antibodies by short-lived plasma cells stopped about 2-3 months after disease onset, an observation that has important implications for convalescent plasma collection and seroprevalence studies undertaken several months after the peak of infection.","version":"1.1","doi":"10.1101/2020.07.16.206847","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.107870","pub_date":"2020-7-17","title":"ACE2-independent interaction of SARS-CoV-2 spike protein to human epithelial cells can be inhibited by unfractionated heparin","abstract":"The SARS-CoV-2 spike protein is known to bind to the receptor, ACE2, on the surface of target cells. The spike protein is processed by membrane proteases, including TMPRSS2, and either internalises or fuses directly with the cell, leading to infection. We have identified a human cell line that expresses both ACE2 and TMPRSS2, the RT4 urinary bladder transitional carcinoma, and used it to develop a proxy assay for viral interactions with host cells. A tagged recombinant form of the spike protein, containing both the S1 and S2 domains, interacted strongly with RT4 cells as determined by flow cytometry, whereas the S1 domain and the receptor binding domain (RBD) interacted weakly. S1S2 interaction was temperature dependent and increased sharply at 37\u00b0C, suggesting that processing of the intact spike protein is likely to be important in the interaction. S1S2 protein could associate with cells with a low dependence on ACE2 expression, while RBD required the presence of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we used a flow cytometric assay to determine the effect of heparin on spike protein interaction with RT4 cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05U/ml whereas two low molecular weight heparins were much less effective. A mutant form of the spike protein, lacking the Arg-rich region proposed to be a furin cleavage site, interacted very weakly with cells and had a lower affinity for unfractionated and lower molecular weight heparin than the wild type spike protein. This indicates that the furin cleavage site might also be a heparin binding site and potentially important in interactions with host cells. Taken together, our data suggest that heparin, particularly unfractionated forms, could be considered to reduce clinical manifestations of COVID-19 by inhibiting continuing viral infection. Since the emergence of SARS-CoV-2 in 2019, the world has faced a vast public health crisis. SARS-CoV-2 associates with human cells through interaction of the viral spike protein with the host receptor, ACE2. In the absence of a vaccine, new treatments are required to reduce the morbidity and mortality of SARS-CoV-2. Here, we use a novel technique to demonstrate spike protein interactions with human cells with low levels of ACE2 at the cell surface, suggesting a secondary receptor. We demonstrate the importance of a new heparin-binding site within the viral spike protein for these interactions. We also found that unfractionated heparin was able to bind to the viral spike protein and therefore, potently inhibit viral spike protein interactions with human cells. Our data demonstrate that ACE2 is not absolutely required for spike protein interactions with human cells and furthermore, that unfractionated heparin should be considered as a treatment to reduce SARS-CoV-2 viral infection.","version":"1.2","doi":"10.1101/2020.05.21.107870","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.205799","pub_date":"2020-7-17","title":"Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase","abstract":"Nucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are attracting antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by RdRp. We found that the incorporation efficiency of Remdesivir-TP is higher than ATP, and we did not observe chain termination or delayed chain termination caused by single Remdesivir-TP incorporation, while multiple incorporations of Remdesivir-TP caused chain termination in our assay condition. The incorporation efficiency of Ribavirin-TP and Favipiravir-TP is very low either as ATP or GTP analogs, which suggested that mutagenesis may not be the mechanism of action of those two drugs against SARS-CoV-2. Incorporation of Sofosbuvir-TP is also very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2\u2019-C-Methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2\u2019-C-Methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.","version":"1.1","doi":"10.1101/2020.07.16.205799","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.191775","pub_date":"2020-7-17","title":"Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model","abstract":"Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we used a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment was initiated before or simultaneously to infection, favipiravir had a strong dose effect, leading to dramatic reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlated with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. The antiviral efficacy observed in this study was achieved with plasma drug exposure comparable with those previously found during human clinical trials and was associated with weight losses in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.","version":"1.2","doi":"10.1101/2020.07.07.191775","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.207878","pub_date":"2020-7-17","title":"COVID-19 patients upregulate toll-like receptor 4-mediated inflammatory signaling that mimics bacterial sepsis","abstract":"Observational studies of the ongoing coronavirus disease 2019 (COVID-19) outbreak suggest that a cytokine storm is involved in the pathogenesis of severe illness. However, the molecular mechanisms underlying the altered pathological inflammation in COVID-19 are largely unknown. We report here that toll-like receptor (TLR) 4-mediated inflammatory signaling molecules are upregulated in peripheral blood mononuclear cells (PBMCs) from COVID-19 patients, compared with healthy controls. Among the most highly increased inflammatory mediators in severe/critically ill patients, S100A9, an alarmin and TLR4 ligand, was found as a noteworthy biomarker, because it inversely correlated with the serum albumin levels. These data support a link between TLR4 signaling and pathological inflammation during COVID-19 and contribute to develop therapeutic approaches through targeting TLR4-mediated inflammation.","version":"1.1","doi":"10.1101/2020.07.17.207878","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.17.208371","pub_date":"2020-7-17","title":"Modeling the Impact of Lock-down on COVID-19 Spread in Malaysia","abstract":"After a breakdown notified in Wuhan, China in December 2019, COVID-19 is declared as pandemic diseases. To the date more than 13 million confirmed cases and more than half a million are dead around the world. This virus also attached Malaysia in its immature stage where 8718 cases were confirmed and 122 were declared as death. Malaysia responsibly controlled the spread by enforcing MCO. Hence, it is required to visualize the pattern of Covid-19 spread. Also, it is necessary to estimate the impact of the enforced prevention measures. In this paper, an infectious disease dynamic modeling (SEIR) is used to estimate the epidemic spread in Malaysia. The main assumption is to update the reproduction number Rt with respect to the implemented prevention measures. For a time-frame of five month, the Rt was assumed to vary between 2.9 and 0.3. Moreover, the manuscript includes two possible scenarios: the first will be the extension of the stricter measures all over the country, and the second will be the gradual lift of the lock-down. After implementing several stages of lock-down we have found that the estimated values of the Rt with respect to the strictness degree varies between 0.2 to 1.1. A continuous strict lock-down may reduce the Rt to 0.2 and accordingly the estimated active cases will be reduced to 20 by the beginning of September 2020. In contrast, the second scenario considers a gradual lift of the enforced prevention measures by the end of June 2020, here we have considered three possible outcomes according to the MCO relaxation. Thus, the estimated values of Rt = 0.7, 0.9, 1.1, which shows a rapid increase in the number of active cases. The implemented SEIR model shows a close resemblance with the actual data recorded from 10, March till 7, July 2020. Conceptualization, A.A.A; methodology, A.A.A, N.M; validation, A.A.A, N.M; formal analysis, A.A.A; investigation, N.M, A.A.A; resources, G.E.M.A, L.T; data collection, L.T, N.M; writing\u2014original draft preparation, A.A.A, L.T, G.E.M.A, N.M; writing\u2014review and editing, V.S.A, S.C.D, B.S.G, P.S, S.A.B.M.Z, N.M; visualization, N.M; supervision, V.S.A; project administration, V.S.A. All authors have read and agreed to the published version of the manuscript","version":"1.1","doi":"10.1101/2020.07.17.208371","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.16.207951","pub_date":"2020-7-17","title":"DLPAlign: A Deep Learning based Progressive Alignment for Multiple Protein Sequences","abstract":"This paper proposed a novel and straightforward approach to improve the accuracy of progressive multiple protein sequence alignment. We trained a decision-making model based on the convolutional neural networks and bi-directional long short term memory networks, and based on this model, we progressively aligned the input sequences by calculating different posterior probability matrixes. To test the accuracy of this approach, we have implemented a multiple sequence alignment tool called DLPAlign and compared its performance with eleven leading alignment methods on three empirical alignment benchmarks (BAliBASE, OXBench and SABMark). Our results show that DLPAlign can get the best total-column scores on the three benchmarks. When evaluated against the 711 low similarity families with average PID \u2264 30%, DLPAlign improved about 2.8% over the second-best MSA software. Besides, we also compared the performance of DLPAlign and other alignment tools on a real-life application, namely protein secondary structure prediction on four protein sequences related to SARS-COV-2, and DLPAlign provides the best result in all cases.","version":"1.1","doi":"10.1101/2020.07.16.207951","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.153486","pub_date":"2020-7-16","title":"Potent Neutralizing Antibodies Directed to Multiple Epitopes on SARS-CoV-2 Spike","abstract":"The SARS-CoV-2 pandemic rages on with devasting consequences on human lives and the global economy. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this novel coronavirus. Here we report the isolation of 61 SARS-CoV-2-neutralizing monoclonal antibodies from 5 infected patients hospitalized with severe disease. Among these are 19 antibodies that potently neutralized the authentic SARS-CoV-2 in vitro, 9 of which exhibited exquisite potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng/mL. Epitope mapping showed this collection of 19 antibodies to be about equally divided between those directed to the receptor-binding domain (RBD) and those to the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that are overlapping with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody targeting RBD, a second targeting NTD, and a third bridging two separate RBDs revealed recognition of the closed, \u201call RBD-down\u201d conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.06.17.153486","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.20154005","pub_date":"2020-07-16","title":"A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  With the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, there is need for sensitive, specific and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR, with many commercial kits now available for this purpose. However, these are expensive and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (\n                  <jats:italic>RPP30</jats:italic>\n                  ) and a viral spike-in control (PhHV-1), which monitor sample quality and nucleic acid extraction efficiency respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate &gt;1,000-fold variability in material routinely collected by nose-and-throat swabbing, and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false negative rates. We demonstrate feasibility of establishing a robust RT-qPCR assay at \u223c10% of the cost of equivalent commercial assays, which could benefit low resource environments and make high volume testing more affordable.\n                </jats:p>","version":null,"doi":"10.1101/2020.07.14.20154005","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.05.079558","pub_date":"2020-7-16","title":"Development and simulation of fully glycosylated molecular models of ACE2-Fc fusion proteins and their interaction with the SARS-CoV-2 spike protein binding domain","abstract":"We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.","version":"1.2","doi":"10.1101/2020.05.05.079558","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.20154260","pub_date":"2020-07-16","title":"Neurological manifestations associated with COVID-19: a nationwide registry","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>The clinical description of the neurological manifestations in COVID-19 patients is still underway. This study aims to provide an overview of the spectrum, characteristics and outcomes of neurological manifestations associated with SARS-CoV-2 infection.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We conducted a nationwide, multicentric, retrospective study during the French COVID-19 epidemic in March-April 2020. All COVID-19 patients with\n                    <jats:italic>de novo</jats:italic>\n                    neurological manifestations were eligible.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>We included 222 COVID-19 patients with neurological manifestations from 46 centers throughout the country. Median age was 65 years (IQR 53-72), and 136 patients (61.3%) were male. COVID-19 was severe or critical in almost half of the patients (102, 45.2%). The most common neurological diseases were COVID-19 associated encephalopathy (67/222, 30.2%), acute ischemic cerebrovascular syndrome (57/222, 25.7%), encephalitis (21/222, 9.5%), and Guillain-Barr\u00e9 Syndrome (15/222, 6.8%). Neurological manifestations appeared after first COVID-19 symptoms with a median (IQR) delay of 6 (3-8) days in COVID-19 associated encephalopathy, 7 (5-10) days in encephalitis, 12 (7-18) days in acute ischemic cerebrovascular syndrome and 18 (15-28) days in Guillain-Barr\u00e9 Syndrome. Brain imaging was performed in 192 patients (86.5%), including 157 MRI (70.7%). Brain MRI of encephalitis patients showed heterogeneous acute non vascular lesion in 14/21 patients (66.7%) with associated small ischemic lesion or microhemorrhages in 4 patients. Among patients with acute ischemic cerebrovascular syndrome, 13/57 (22.8%) had multi territory ischemic strokes, with large vessel thrombosis in 16/57 (28.1%). Cerebrospinal fluid was analyzed in 97 patients (43.7%), with pleocytosis in 18 patients (18.6%). A SARS-CoV-2 PCR was performed in 75 patients and was positive only in 2 encephalitis patients. Among patients with encephalitis, ten out of 21 (47.6%) fully recovered, 3 of whom received corticosteroids (CS). Less common neurological manifestations included isolated seizure (8/222, 3.6%), critical illness neuropathy (8/222, 3.6%), transient alteration of consciousness (5/222, 2.3%), intracranial hemorrhage (5/222, 2.3%), acute benign lymphocytic meningitis (3/222, 1.4%), cranial neuropathy (3/222, 1.4%), single acute demyelinating lesion (2/222, 0.9%), Tapia syndrome (2/222, 0.9%), cerebral venous thrombosis (1/222, 0.5%), sudden paraparesis (1/222, 0.5%), generalized myoclonus and cerebellar ataxia (1/222, 0.5%), bilateral fibular palsy (1/222, 0.5%) and isolated neurological symptoms (headache, anosmia, dizziness, sensitive or auditive symptoms, hiccups, 15/222, 6.8%). The median (IQR) follow-up of the 222 patients was 24 (17-34) days with a high short-term mortality rate (28/222, 12.6%).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Neurological manifestations associated with COVID-19 mainly included CAE, AICS, encephalitis and GBS. Clinical spectrum and outcomes were broad and heterogeneous, suggesting different underlying pathogenic processes.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.07.15.20154260","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.15.20154773","pub_date":"2020-07-16","title":"Improved COVID-19 Serology Test Performance by Integrating Multiple Lateral Flow Assays using Machine Learning","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Mitigating transmission of SARS-CoV-2 has been complicated by the inaccessibility and, in some cases, inadequacy of testing options to detect present or past infection. Immunochromatographic lateral flow assays (LFAs) are a cheap and scalable modality for tracking viral transmission by testing for serological immunity, though systematic evaluations have revealed the low performance of some SARS-CoV-2 LFAs. Here, we re-analyzed existing data to present a proof-of-principle machine learning framework that may be used to inform the pairing of LFAs to achieve superior classification performance while enabling tunable False Positive Rates optimized for the estimated seroprevalence of the population being tested.</jats:p>","version":null,"doi":"10.1101/2020.07.15.20154773","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.15.20154815","pub_date":"2020-07-16","title":"The utility of established prognostic scores in COVID-19 hospital admissions: a multi-centre prospective evaluation of CURB-65, NEWS2, and qSOFA","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Introduction</jats:title>\n                  <jats:p>The COVID-19 pandemic is ongoing yet, due to the lack of a COVID-19 specific tool, clinicians must use pre-existing illness severity scores for initial prognostication. However, the validity of such scores in COVID-19 is unknown.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>The North West Collaborative Organization for Respiratory Research (NW-CORR) performed a multi-centre prospective evaluation of adult patients admitted to hospital with confirmed COVID-19 during a two-week period in April 2020. Clinical variables measured as part of usual care at presentation to hospital were recorded, including the CURB-65, NEWS2, and qSOFA scores. The primary outcome of interest was 30-day mortality.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Data were collected for 830 people with COVID-19 admitted across 7 hospitals. By 30 days, a total of 300 (36.1%) had died and 142 (17.1%) had been in ICU. All scores underestimated mortality compared to their original validation in non-COVID-19 populations, and overall prognostic performance was generally poor. Among the \u2018low risk\u2019 categories (CURB-65&lt;2, NEWS2&lt;5, qSOFA&lt;2) 30-day mortality was 16.7%, 32.9% and 21.4%, respectively. Multivariable logistic regression identified features of respiratory compromise rather than circulatory collapse as most relevant prognostic variables.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>All existing prognostic scores evaluated here underestimated adverse outcomes and performed sub-optimally in the COVID-19 setting. New prognostic tools including a focus on features of respiratory compromise rather than circulatory collapse are needed. We provide a baseline set of variables which are relevant to COVID-19 outcomes and may be used as a basis for developing a bespoke COVID-19 prognostication tool.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Key Messages</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>\n                        <jats:bold>What is the key question?</jats:bold>\n                      </jats:p>\n                      <jats:p>Do well-established illness severity scores have prognostic value in COVID-19?</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>\n                        <jats:bold>What is the bottom line?</jats:bold>\n                      </jats:p>\n                      <jats:p>All scores appeared to underestimate mortality in COVID-19 and prognostic performance was generally poor, and importantly could not discriminate those patients at very low risk of death within 30 days.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>\n                        <jats:bold>Why read on?</jats:bold>\n                      </jats:p>\n                      <jats:p>In this multi-centre prospective evaluation of CURB-65, NEWS2 and qSOFA we comprehensively evaluate score performance and also identify variables which may be of use in COVID-19 prognostication.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2020.07.15.20154815","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.07.137802","pub_date":"2020-7-15","title":"Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system","abstract":"The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Understanding how SARS-CoV-2 enters and spreads within human organs is crucial for developing strategies to prevent viral dissemination. For many viruses, tissue tropism is determined by the availability of virus receptors on the surface of host cells. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a host receptor, yet, their tropisms differ. Here, we found that the cellular receptor neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was inhibited by a monoclonal blocking antibody against the extracellular b1b2 domain of NRP1. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial cells and in the epithelial cells facing the nasal cavity. Neuropathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected NRP1-positive cells in the olfactory epithelium and bulb. In the olfactory bulb infection was detected particularly within NRP1-positive endothelial cells of small capillaries and medium-sized vessels. Studies in mice demonstrated, after intranasal application, NRP1-mediated transport of virus-sized particles into the central nervous system. Thus, NRP1 could explain the enhanced tropism and spreading of SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.06.07.137802","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.203059","pub_date":"2020-7-15","title":"The Effect of Famotidine on SARS-CoV-2 Proteases and Virus Replication","abstract":"The lack of coronavirus-specific antiviral drugs has instigated multiple drug repurposing studies to redirect previously approved medicines for the treatment of SARS-CoV-2, the coronavirus behind the ongoing COVID-19 pandemic. A recent, large-scale, retrospective clinical study showed that famotidine, when administered at a high dose to hospitalized COVID-19 patients, reduced the rates of intubation and mortality. A separate, patient-reported study associated famotidine use with improvements in mild to moderate symptoms such as cough and shortness of breath. While a prospective, multi-center clinical study is ongoing, two parallel in silico studies have proposed one of the two SARS-CoV-2 proteases, 3CLpro or PLpro, as potential molecular targets of famotidine activity; however, this remains to be experimentally validated. In this report, we systematically analyzed the effect of famotidine on viral proteases and virus replication. Leveraging a series of biophysical and enzymatic assays, we show that famotidine neither binds with nor inhibits the functions of 3CLpro and PLpro. Similarly, no direct antiviral activity of famotidine was observed at concentrations of up to 200 \u03bcM, when tested against SARS-CoV-2 in two different cell lines, including a human cell line originating from lungs, a primary target of COVID-19. These results rule out famotidine as a direct-acting inhibitor of SARS-CoV-2 replication and warrant further investigation of its molecular mechanism of action in the context of COVID-19.","version":"1.1","doi":"10.1101/2020.07.15.203059","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.19.048710","pub_date":"2020-7-15","title":"Broad and differential animal ACE2 receptor usage by SARS-CoV-2","abstract":"The COVID-19 pandemic has caused an unprecedented global public health and economy crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2 from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activity, respectively. Among the remaining species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models. SARS-CoV-2 uses human ACE2 as primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologues and found that wild type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models and molecular basis of receptor binding for SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.04.19.048710","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.097287","pub_date":"2020-7-15","title":"Recommendations for sample pooling on the Cepheid GeneXpert\u00ae system using the Cepheid Xpert\u00ae Xpress SARS-CoV-2 assay","abstract":"The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert\u00ae Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the Xpert\u00ae Xpress SARS-CoV-2 assay limit of detection was comparable to central laboratory reverse-transcription quantitative PCR tests with observed SARS-CoV-2 detection below 100 copies/mL. The Xpert\u00ae Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were diluted into six sample pools. Based on these data, we recommend the adoption of pooled testing with the Xpert\u00ae Xpress SARS-CoV-2 assay where warranted by population public health needs. The suggested number of samples per pool, or pooling depth, is unique for each point-of-care test site and should be determined by assessing positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.","version":"1.2","doi":"10.1101/2020.05.14.097287","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.204602","pub_date":"2020-7-15","title":"Evidence of SARS-CoV2 entry protein ACE2 in the human nose and olfactory bulb","abstract":"Usually, pandemic COVID-19 disease, caused by SARS-CoV2, presents with mild respiratory symptoms such as fever, cough but frequently also with anosmia and neurological symptom. Virus-cell fusion is mediated by Angiotensin-Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) with their organ expression pattern determining viral tropism. Clinical presentation suggests rapid viral dissemination to central nervous system leading frequently to severe symptoms including viral meningitis. Here, we provide a comprehensive expression landscape of ACE2 and TMPRSS2 proteins across human, post-mortem nasal and olfactory tissue. Sagittal sections through the human nose complemented with immunolabelling of respective cell types represent different anatomically defined regions including olfactory epithelium, respiratory epithelium of the nasal conchae and the paranasal sinuses along with the hardly accessible human olfactory bulb. ACE2 can be detected in the olfactory epithelium, as well as in the respiratory epithelium of the nasal septum, the nasal conchae and the paranasal sinuses. ACE2 is located in the sustentacular cells and in the glandular cells in the olfactory epithelium, as well as in the basal cells, glandular cells and epithelial cells of the respiratory epithelium. Intriguingly, ACE2 is not expressed in mature or immature olfactory receptor neurons and basal cells in the olfactory epithelium. Similarly ACE2 is not localized in the olfactory receptor neurons albeit the olfactory bulb is positive. Vice versa, TMPRSS2 can also be detected in the sustentacular cells and the glandular cells of the olfactory epithelium. Our findings provide the basic anatomical evidence for the expression of ACE2 and TMPRSS2 in the human nose, olfactory epithelium and olfactory bulb. Thus, they are substantial for future studies that aim to elucidate the symptom of SARS-CoV2 induced anosmia of via the olfactory pathway.","version":"1.1","doi":"10.1101/2020.07.15.204602","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.203489","pub_date":"2020-7-15","title":"Prophylactic and Therapeutic Inhibition of In Vitro SARS-CoV-2 Replication by Oleandrin","abstract":"With continued expansion of the COVID-19 pandemic, antiviral drugs are desperately needed to treat patients at high risk of life-threatening disease and even to limit spread if administered early during infection. Typically, the fastest route to identifying and licensing a safe and effective antiviral drug is to test those already shown safe in early clinical trials for other infections or diseases. Here, we tested in vitro oleandrin, derived from the Nerium oleander plant and shown previously to have inhibitory activity against several viruses. Using Vero cells, we found that prophylactic oleandrin administration at concentrations down to 0.05 \u03bcg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 \u03bcg/ml dose resulted in a greater than 3,000-fold reduction in infectious virus production. The EC50 values were 11.98ng/ml when virus output was measured at 24 hours post-infection, and 7.07ng/ml measured at 48 hours post-infection. Therapeutic (post-infection) treatment up to 24 hours after infection of Vero cells also reduced viral titers, with the 0.1 \u03bcg/ml dose causing greater than 100-fold reductions as measured at 48 hours, and the 0.05 \u03bcg/ml dose resulting in a 78-fold reduction. The potent prophylactic and therapeutic antiviral activities demonstrated here strongly support the further development of oleandrin to reduce the severity of COVID-19 and potentially also to reduce spread by persons diagnosed early after infection. COVID-19, a pandemic disease caused by infection with SARS-CoV-2, has swept around the world to cause millions of infections and hundreds-of-thousands of deaths due to the lack of vaccines and effective therapeutics. We tested oleandrin, derived from the Nerium oleander plant and shown previously to reduce the replication of several viruses, against SARS-CoV-2 infection of Vero cells. When administered both before and after virus infection, nanogram doses of oleandrin significantly inhibited replication by up to 3,000-fold, indicating the potential to prevent disease and virus spread in persons recently exposed to SARS-CoV-2, as well as to prevent severe disease in persons at high risk. These results indicate that oleandrin should be tested in animal models and in humans exposed to infection to determine its medical usefulness in controlling the pandemic.","version":"1.1","doi":"10.1101/2020.07.15.203489","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.201947","pub_date":"2020-7-15","title":"Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation","abstract":"Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct \u226525), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.","version":"1.1","doi":"10.1101/2020.07.14.201947","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.204610","pub_date":"2020-7-15","title":"Antigenic variation of SARS-CoV-2 in response to immune pressure","abstract":"The ongoing evolution of SARS-CoV-2 is expected to be at least partially driven by the selective pressure imposed by the human immune system. We exploited the availability of a large number of high-quality SARS-CoV-2 genomes, as well as of validated epitope predictions, to show that B cell epitopes in the spike glycoprotein (S) and in the nucleocapsid protein (N) have higher diversity than non-epitope positions. Similar results were obtained for other human coronaviruses. Conversely, in the SARS-CoV-2 population, epitopes for CD4+ and CD8+ T cells were not more variable than non-epitope positions. A significant reduction in epitope variability was instead observed for some of the most immunogenic proteins (S, N, ORF8, and ORF3a). Analysis over longer evolutionary time-frames indicated that this effect is not due to differential constraints. These data indicate that SARS-CoV-2 is evolving to elude the host humoral immune response, whereas recognition by T cells might benefit the virus.","version":"1.1","doi":"10.1101/2020.07.15.204610","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.204404","pub_date":"2020-7-15","title":"A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells","abstract":"SARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.","version":"1.1","doi":"10.1101/2020.07.15.204404","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.203414","pub_date":"2020-7-15","title":"Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization","abstract":"In vitro antibody selection against pathogens from na\u00efve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse na\u00efve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the \u201cup\u201d and the other in the \u201cdown\u201d position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.07.14.203414","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.203463","pub_date":"2020-7-15","title":"Comprehensive analysis of genomic diversity of SARS-CoV-2 in different geographic regions of India: An endeavour to classify Indian SARS-CoV-2 strains on the basis of co-existing mutations","abstract":"Accumulation of mutations within the genome is the primary driving force for viral evolution within an endemic setting. This inherent feature often leads to altered virulence, infectivity and transmissibility as well as antigenic shift to escape host immunity, which might compromise the efficacy of vaccines and antiviral drugs. Therefore, we aimed at genome-wide analyses of circulating SARS-CoV-2 viruses for the emergence of novel co-existing mutations and trace their spatial distribution within India. Comprehensive analysis of whole genome sequences of 441 Indian SARS-CoV-2 strains revealed the occurrence of 33 different mutations, 21 being distinctive to India. Emergence of novel mutations were observed in S glycoprotein (7/33), NSP3 (6/33), RdRp/NSP12 (4/33), NSP2 (2/33) and N (2/33). Non-synonymous mutations were found to be 3.4 times more prevalent than synonymous mutations. We classified the Indian isolates into 22 groups based on the co-existing mutations. Phylogenetic analyses revealed that representative strain of each group divided themselves into various sub-clades within their respective clades, based on the presence of unique co-existing mutations. India was dominated by A2a clade (55.60%) followed by A3 (37.38%) and B (7%), but exhibited heterogeneous distribution among various geographical regions. The A2a clade mostly predominated in East India, Western India and Central India, whereas A3 clade prevailed in South and North India. In conclusion, this study highlights the divergent evolution of SARS-CoV-2 strains and co-circulation of multiple clades in India. Monitoring of the emerging mutations would pave ways for vaccine formulation and designing of antiviral drugs.","version":"1.1","doi":"10.1101/2020.07.14.203463","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.04.187757","pub_date":"2020-7-15","title":"Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant","abstract":"The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and cells rendered permissive by ectopic expression of various mammalian ACE2 orthologs. Nonetheless, D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts a critical interprotomer contact and that this dramatically shifts the S protein trimer conformation toward an ACE2-binding and fusion-competent state. Consistent with the more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated. These results indicate that D614G adopts conformations that make virion membrane fusion with the target cell membrane more probable but that D614G retains susceptibility to therapies that disrupt interaction of the SARS-CoV-2 S protein with the ACE2 receptor.","version":"1.2","doi":"10.1101/2020.07.04.187757","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.205229","pub_date":"2020-7-15","title":"The S1 protein of SARS-CoV-2 crosses the blood-brain barrier: Kinetics, distribution, mechanisms, and influence of ApoE genotype, sex, and inflammation","abstract":"Evidence strongly suggests that SARS-CoV-2, the cause of COVID-19, can enter the brain. SARS-CoV-2 enters cells via the S1 subunit of its spike protein, and S1 can be used as a proxy for the uptake patterns and mechanisms used by the whole virus; unlike studies based on productive infection, viral proteins can be used to precisely determine pharmacokinetics and biodistribution. Here, we found that radioiodinated S1 (I-S1) readily crossed the murine blood-brain barrier (BBB). I-S1 from two commercial sources crossed the BBB with unidirectional influx constants of 0.287 \u00b1 0.024 \u03bcL/g-min and 0.294 \u00b1 0.032 \u03bcL/g-min and was also taken up by lung, spleen, kidney, and liver. I-S1 was uniformly taken up by all regions of the brain and inflammation induced by lipopolysaccharide reduced uptake in the hippocampus and olfactory bulb. I-S1 crossed the BBB completely to enter the parenchymal brain space, with smaller amounts retained by brain endothelial cells and the luminal surface. Studies on the mechanisms of transport indicated that I-S1 crosses the BBB by the mechanism of adsorptive transcytosis and that the murine ACE2 receptor is involved in brain and lung uptake, but not that by kidney, liver, or spleen. I-S1 entered brain after intranasal administration at about 1/10th the amount found after intravenous administration and about 0.66% of the intranasal dose entered blood. ApoE isoform or sex did not affect whole brain uptake, but had variable effects on olfactory bulb, liver, spleen, and kidney uptakes. In summary, I-S1 readily crosses the murine BBB, entering all brain regions and the peripheral tissues studied, likely by the mechanism of adsorptive transcytosis.","version":"1.1","doi":"10.1101/2020.07.15.205229","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.205211","pub_date":"2020-7-15","title":"An Antioxidant Enzyme Therapeutic for COVID-19","abstract":"The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. We report herein a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.","version":"1.1","doi":"10.1101/2020.07.15.205211","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.202887","pub_date":"2020-7-15","title":"Bioinformatic analysis of shared B and T cell epitopes amongst relevant coronaviruses to human health: Is there cross-protection?","abstract":"Within the last 30 years 3 coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have evolved and adapted to cause disease and spread amongst the human population. From the three, SARS-CoV-2 has spread world-wide and to July 2020 it has been responsible for more than 11 million confirmed cases and over half a million deaths. In the absence of an effective treatment or vaccine, social distancing has been the most effective measure to control the pandemic. However it has become evident that as the virus spreads the only tool that will allow us to fully control it is an effective vaccine. There are currently more than 150 vaccine candidates in different stages of development using a variety of viral antigens, with the S protein being the most targeted antigen. Although some new experimental evidence suggests cross-reacting responses between coronaviruses are present in the population, it remains unknown whether potential shared antigens between different coronaviruses could provide cross-protection. Given that coronaviruses are emerging pathogens and continue to represent a threat to global health, the development of a SARS-Cov-2 vaccine that could provide \u2018universal\u2019 protection against other coronaviruses should be pushed forward. Here we present a thorough review of reported B and T cell epitopes shared between SARS-CoV-2 and other relevant coronaviruses, in addition we used web-based tools to predict novel B and T cell epitopes that have not been reported before. Analysis of experimental evidence that is constantly emerging complemented with the findings of this study allow us support the hypothesis that cross-reactive responses, particularly those coming from T cells, might play a key role in controlling infection by SARS-CoV-2. We hope that with the evidence presented in this manuscript we provide arguments to encourage the study of cross-reactive responses in order to elucidate their role in immunity to SARS-CoV-2. Finally we expect our findings will aid targeted analysis of antigen-specific immune responses and guide future vaccine design aiming to develop a cross reactive effective vaccine against respiratory diseases caused by coronaviruses.","version":"1.1","doi":"10.1101/2020.07.14.202887","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.059527","pub_date":"2020-7-15","title":"Consensus transcriptional regulatory networks of coronavirus-infected human cells","abstract":"Establishing consensus around the transcriptional interface between coronavirus (CoV) infection and human cellular signaling pathways can catalyze the development of novel anti-CoV therapeutics. Here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in MERS-CoV (MERS), SARS-CoV-1 (SARS1) and SARS-CoV-2 (SARS2)-infected cells. Validating the CoV consensomes, we show that high confidence transcriptional targets (HCTs) of CoV infection intersect with HCTs of signaling pathway nodes with known roles in CoV infection. Among a series of novel use cases, we gather evidence for hypotheses that SARS2 infection efficiently represses E2F family target genes encoding key drivers of DNA replication and the cell cycle; that progesterone receptor signaling antagonizes SARS2-induced inflammatory signaling in the airway epithelium; and that SARS2 HCTs are enriched for genes involved in epithelial to mesenchymal transition. The CoV infection consensomes and HCT intersection analyses are freely accessible through the Signaling Pathways Project knowledgebase, and as Cytoscape-style networks in the Network Data Exchange repository.","version":"1.5","doi":"10.1101/2020.04.24.059527","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.15.176933","pub_date":"2020-7-15","title":"Alignment-free machine learning approaches for the lethality prediction of potential novel human-adapted coronavirus using genomic nucleotide","abstract":"A newly emerging novel coronavirus appeared and rapidly spread worldwide and World Health Organization declared a pandemic on March 11, 2020. The roles and characteristics of coronavirus have captured much attention due to its power of causing a wide variety of infectious diseases, from mild to severe on humans. The detection of the lethality of human coronavirus is key to estimate the viral toxicity and provide perspective for treatment. We developed alignment-free machine learning approaches for an ultra-fast and highly accurate prediction of the lethality of potential human-adapted coronavirus using genomic nucleotide. We performed extensive experiments through six different feature transformation and machine learning algorithms in combination with digital signal processing to infer the lethality of possible future novel coronaviruses using previous existing strains. The results tested on SARS-CoV, MERS-Cov and SARS-CoV-2 datasets show an average 96.7% prediction accuracy. We also provide preliminary analysis validating the effectiveness of our models through other human coronaviruses. Our study achieves high levels of prediction performance based on raw RNA sequences alone without genome annotations and specialized biological knowledge. The results demonstrate that, for any novel human coronavirus strains, this alignment-free machine learning-based approach can offer a reliable real-time estimation for its viral lethality.","version":"1.1","doi":"10.1101/2020.07.15.176933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.202028","pub_date":"2020-7-14","title":"Syncytia formation by SARS-CoV-2 infected cells","abstract":"Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2 infected cells express the viral Spike protein (S) at their surface and fuse with ACE2-positive neighbouring cells. Expression of S without any other viral proteins triggers syncytia formation. Type-I interferon (IFN)-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that the pathological effects of SARS-CoV-2 are modulated by cellular proteins that either inhibit or facilitate syncytia formation. Syncytia produced by SARS-CoV-2 infected cells and regulation of their\nformation by IFITMs and TMPRSS2.","version":"1.1","doi":"10.1101/2020.07.14.202028","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.201517","pub_date":"2020-7-14","title":"Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies","abstract":"Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and \u22122, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 \u2013 an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identifiy nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.","version":"1.1","doi":"10.1101/2020.07.13.201517","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.201616","pub_date":"2020-7-14","title":"SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2","abstract":"We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.","version":"1.1","doi":"10.1101/2020.07.14.201616","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.14.201954","pub_date":"2020-7-14","title":"The antibody response to the glycan \u03b1-Gal correlates with COVID-19 disease symptoms","abstract":"The coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. The characterization of the immunological mechanisms involved in disease symptomatology and protective response is important to advance in disease control and prevention. Humans evolved by losing the capacity to synthesize the glycan Gal\u03b11-3Gal\u03b21-(3)4GlcNAc-R (\u03b1-Gal), which resulted in the development of a protective response against pathogenic viruses and other microorganisms containing this modification on membrane proteins mediated by anti-\u03b1-Gal IgM/IgG antibodies produced in response to bacterial microbiota. In addition to anti-\u03b1-Gal antibody-mediated pathogen opsonization, this glycan induces various immune mechanisms that have shown protection in animal models against infectious diseases without inflammatory responses. In this study, we hypothesized that the immune response to \u03b1-Gal may contribute to the control of COVID-19. To address this hypothesis, we characterized the antibody response to \u03b1-Gal in patients at different stages of COVID-19 and in comparison with healthy control individuals. The results showed that while the inflammatory response and the anti-SARS-CoV-2 (Spike) IgG antibody titers increased, reduction in anti-\u03b1-Gal IgE, IgM and IgG antibody titers and alteration of anti-\u03b1-Gal antibody isotype composition correlated with COVID-19 severity. The results suggested that the inhibition of the \u03b1-Gal-induced immune response may translate into more aggressive viremia and severe disease inflammatory symptoms. These results support the proposal of developing interventions such as probiotics based on commensal bacteria with \u03b1-Gal epitopes to modify the microbiota and increase the \u03b1-Gal-induced protective immune response and reduce the severity of COVID-19.","version":"1.1","doi":"10.1101/2020.07.14.201954","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.201806","pub_date":"2020-7-14","title":"Peramivir, an anti-influenza virus drug, exhibits potential anti-cytokine storm effects","abstract":"Coronavirus Disease 2019 (COVID-19) infected by Severe Acute Respiratory Syndrome Coronavirus \u22122 (SARS-CoV-2) has been declared a public health emergency of international concerns. Cytokine storm syndrome (CSS) is a critical clinical symptom of severe COVID-19 patients, and the macrophage is recognized as the direct host cell of SARS-CoV-2 and potential drivers of CSS. In the present study, peramivir was identified to reduce TNF-\u03b1 by partly intervention of NF-\u03baB activity in LPS-induced macrophage model. In vivo, peramivir reduced the multi-cytokines in serum and bronchoalveolar lavage fluid (BALF), alleviated the acute lung injury and prolonged the survival time in mice. In human peripheral blood mononuclear cells (hPBMCs), peramivir could also inhibit the release of TNF-\u03b1. Collectively, we proposed that peramivir might be a candidate for the treatment of COVID-19 and other infections related CSS.","version":"1.1","doi":"10.1101/2020.07.13.201806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.20143339","pub_date":"2020-07-14","title":"Association between SSRI Antidepressant Use and Reduced Risk of Intubation or Death in Hospitalized Patients with Coronavirus Disease 2019: a Multicenter Retrospective Observational Study","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To examine the association between antidepressant use and the risk of intubation or death in hospitalized patients with COVID-19.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Design</jats:title>\n                  <jats:p>Multicenter observational retrospective cohort study.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Setting</jats:title>\n                  <jats:p>Greater Paris University hospitals, France.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Participants</jats:title>\n                  <jats:p>7,345 adults hospitalized with COVID-19 between 24 January and 1 April 2020, including 460 patients (6.3%) who received an antidepressant during the visit.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Data source</jats:title>\n                  <jats:p>Assistance Publique-H\u00f4pitaux de Paris Health Data Warehouse.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Main outcome measures</jats:title>\n                  <jats:p>The primary endpoint was a composite of intubation or death. We compared this endpoint between patients who received antidepressants and those who did not in time-to-event analyses adjusting for patient characteristics (such as age, sex, and comorbidities), disease severity and other psychotropic medications. The primary analyses were multivariable Cox models with inverse probability weighting.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>Over a mean follow-up of 18.5 days (SD=27.1), 1,331 patients (18.1%) had a primary end-point event. Unadjusted hazard ratio estimates of the association between antidepressant use and the primary outcome stratified by age (i.e., 18-50, 51-70, 71-80, and 81+) were non-significant (all p&gt;0.072), except in the group of patients aged 71-80 years (HR, 0.66; 95% CI, 0.45 to 0.98; p=0.041). Following adjustments, the primary analyses showed a significant association between use of any antidepressant (HR, 0.64; 95% CI, 0.51 to 0.80; p&lt;0.001), SSRI (HR, 0.56; 95% CI, 0.42 to 0.75; p&lt;0.001), and SNRI (HR, 0.57; 95% CI, 0.34 to 0.96; p=0.034), and reduced risk of intubation or death. Specifically, exposures to escitalopram, fluoxetine, and venlafaxine were significantly associated with lower risk of intubation or death (all p&lt;0.05). These associations remain significant in multiple sensitivity analyses, except for the association between SNRI use and the outcome.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>SSRI use could be associated with lower risk of death or intubation in hospitalized patients with COVID-19. Double-blind controlled randomized clinical trials of these medications for COVID-19 are needed.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>What is already known on this topic</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>\n                        A prior meta-analysis, mainly including studies on selective serotonin reuptake inhibitors (SSRIs), showed that antidepressant use in major depressive disorder was associated with reduced levels of several pro-inflammatory cytokines, including IL-6, TNF-\n                        <jats:italic>\u03b1</jats:italic>\n                        , and CCL-2, which have been suggested to be associated with severe COVID-19.\n                      </jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>A recent in-vitro study supports antiviral effects of the SSRI fluoxetine on SARS-CoV-2.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>To our knowledge, no study has examined the efficacy of antidepressants in patients with COVID-19.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>What this study adds</jats:title>\n                  <jats:list list-type='bullet'>\n                    <jats:list-item>\n                      <jats:p>In a multicenter observational retrospective study, we examined the association between antidepressant use and the risk of intubation or death in hospitalized patients with COVID-19, adjusting for patient characteristics, disease severity and other psychotropic medications.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Antidepressant use was significantly and substantially associated with reduced risk of intubation or death.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>At the level of antidepressant classes, SSRI use was significantly and substantially associated with reduced risk of intubation or death, but not other antidepressant classes.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>At the level of antidepressant medications, exposures to the SSRIs fluoxetine and escitalopram, and the SNRI venlafaxine were significantly associated with lower risk of intubation or death.</jats:p>\n                    </jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Double-blind controlled randomized clinical trials of these medications for COVID-19 are needed.</jats:p>\n                    </jats:list-item>\n                  </jats:list>\n                </jats:sec>","version":null,"doi":"10.1101/2020.07.09.20143339","journal":"medRxiv","score":null},{"id":"10.1101/2020.07.09.196337","pub_date":"2020-7-13","title":"Discovery of clinically approved drugs capable of inhibiting SARS-CoV-2 in vitro infection using a phenotypic screening strategy and network-analysis to predict their potential to treat covid-19","abstract":"The disease caused by SARS-CoV2, covid-19, rapidly spreads worldwide, causing the greatest threat to global public health in the last 100 years. This scenario has become catastrophic as there are no approved vaccines to prevent the disease, and the main measures to contain the virus transmission are confinement and social distancing. One priority strategy is based on drug repurposing by pursuing antiviral chemotherapy that can control transmission and prevent complications associated with covid-19. With this aim, we performed a high content screening assay for the discovery of anti-SARS-CoV-2 compounds. From the 65 screened compounds, we have found four drugs capable to selectively inhibit SARS-CoV-2 in vitro infection: brequinar, abiraterone acetate, neomycin, and the extract of Hedera helix. Brequinar and abiraterone acetate had higher inhibition potency against SARS-CoV-2 than neomycin and Hedera helix extract, respectively. Drugs with reported antiviral activity and in clinical trials for covid-19, chloroquine, ivermectin, and nitazoxanide, were also included in the screening, and the last two were found to be non-selective. We used a data mining approach to build drug-host molecules-biological function-disease networks to show in a holistic way how each compound is interconnected with host node molecules and virus infection, replication, inflammatory response, and cell apoptosis. In summary, the present manuscript identified four drugs with active inhibition effect on SARS-CoV-2 in vitro infection, and by network analysis, we provided new insights and starting points for the clinical evaluation and repurposing process to treat SARS-CoV-2 infection. Discovery of drug repurposing candidates, inhibitors of SARS-CoV-2 infection in vitro, using a phenotypic screening strategy and network analysis.","version":"1.2","doi":"10.1101/2020.07.09.196337","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.07.982264","pub_date":"2020-7-13","title":"Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV","abstract":"SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type-I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, while SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures (HAEC), we observe the absence of IFN-I stimulation by SARS-CoV-2 alone, but detect failure to counteract STAT1 phosphorylation upon IFN-I pretreatment resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment post infection and found SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame (ORF) 3b and changes to ORF6 suggest the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development. With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection. SARS-CoV-2 has similar replication kinetics to SARS-CoV, but demonstrates significant sensitivity to type I interferon treatment.","version":"1.4","doi":"10.1101/2020.03.07.982264","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.199562","pub_date":"2020-7-13","title":"Identifying Key Determinants of SARS-CoV-2/ACE2 Tight Interaction","abstract":"SARS-CoV-2 virus, the causative agent of Covid-19, has fired up a global pandemic. The virus interacts with the human receptor angiotensin-converting enzyme 2 (ACE2) for invasion via receptor binding domain (RBD) on its spike protein. To provide a deeper understanding of this interaction, we performed microsecond simulations of the RBD-ACE2 complex for SARS- CoV-2 and compared it with the closely related SARS-CoV discovered in 2003. We show residues in the RBD of SARS-CoV-2 that were mutated from SARS-CoV, collectively help make RBD anchor much stronger to the N-terminal part of ACE2 than the corresponding residues on RBD of SARS-CoV. This would result in reduced dissociation rate of SARS-CoV-2 from human recep- tor protein compared to SARS-CoV. This phenomenon was consistently observed in simulations beyond 500 ns and was reproducible across different force fields. Altogether, our study shed light on the key residues and their dynamics at the virus spike and human receptor binding interface and advance our knowledge for the development of diagnostics and therapeutics to combat the pandemic efficiently.","version":"1.1","doi":"10.1101/2020.07.13.199562","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.081695","pub_date":"2020-7-13","title":"Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium","abstract":"SARS-CoV-2, the causative agent of COVID-19, has tragically burdened individuals and institutions around the world. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon-stimulated genes in both infected and bystander cells. We observe similar gene expression changes from a COVID-19 patient ex vivo. In addition, we developed a new computational method termed CONditional DENSity Embedding (CONDENSE) to characterize and compare temporal gene dynamics in response to infection, which revealed genes relating to endothelin, angiogenesis, interferon, and inflammation-causing signaling pathways. In this study, we conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and a COVID-19 patient and revealed genes, cell types, and cell state changes associated with infection.","version":"1.2","doi":"10.1101/2020.05.06.081695","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.194084","pub_date":"2020-7-13","title":"Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex","abstract":"SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.","version":"1.2","doi":"10.1101/2020.07.08.194084","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199687","pub_date":"2020-7-13","title":"Modulating the transcriptional landscape of SARS-CoV-2 as an effective method for developing antiviral compounds","abstract":"To interfere with the biology of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, we focused on restoring the transcriptional response induced by infection. Utilizing expression patterns of SARS-CoV-2-infected cells, we identified a region in gene expression space that was unique to virus infection and inversely proportional to the transcriptional footprint of known compounds characterized in the Library of Integrated Network-based Cellular Signatures. Here we demonstrate the successful identification of compounds that display efficacy in blocking SARS-CoV-2 replication based on their ability to counteract the virus-induced transcriptional landscape. These compounds were found to potently reduce viral load despite having no impact on viral entry or modulation of the host antiviral response in the absence of virus. RNA-Seq profiling implicated the induction of the cholesterol biosynthesis pathway as the underlying mechanism of inhibition and suggested that targeting this aspect of host biology may significantly reduce SARS-CoV-2 viral load.","version":"1.1","doi":"10.1101/2020.07.12.199687","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197095","pub_date":"2020-7-13","title":"Genomic variations in SARS-CoV-2 genomes from Gujarat: Underlying role of variants in disease epidemiology","abstract":"Humanity has seen numerous pandemics during its course of evolution. The list includes many such as measles, Ebola, SARS, MERS, etc. Latest edition to this pandemic list is COVID-19, caused by the novel coronavirus, SARS-CoV-2. As of 4th July 2020, COVID-19 has affected over 10 million people from 170+ countries, and 5,28,364 deaths. Genomic technologies have enabled us to understand the genomic constitution of the pathogens, their virulence, evolution, rate of mutations, etc. To date, more than 60,000 virus genomes have been deposited in the public depositories like GISAID and NCBI. While we are writing this, India is the 3rd most-affected country with COVID-19 with 0.6 million cases, and >18000 deaths. Gujarat is the fourth highest affected state with 5.44 percent death rate compared to national average of 2.8 percent. Here, 361 SARS-CoV-2 genomes from across Gujarat have been sequenced and analyzed in order to understand its phylogenetic distribution and variants against global and national sequences. Further, variants were analyzed from diseased and recovered patients from Gujarat and the World to understand its role in pathogenesis. From missense mutations, found from Gujarat SARS-CoV-2 genomes, C28854T, deleterious mutation in nucleocapsid (N) gene was found to be significantly associated with mortality in patients. The other significant deleterious variant found in diseased patients from Gujarat and the world is G25563T, which is located in Orf3a and has a potential role in viral pathogenesis. SARS-CoV-2 genomes from Gujarat are forming distinct cluster under GH clade of GISAID.","version":"1.2","doi":"10.1101/2020.07.10.197095","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199521","pub_date":"2020-7-13","title":"Comparisons of the genome of SARS-CoV-2 and those of other betacoronaviruses","abstract":"The genome of SARS-CoV-2 virus causing the worldwide pandemic of COVID-19 is most closely related to viral metagenomes isolated from bats and, more distantly, pangolins. All are of sarbecoviruses of the genus Betacoronavirus. We have unravelled their recombinational and mutational histories. All showed clear evidence of recombination, most events involving the 3\u2019 half of the genomes. The 5\u2019 region of their genomes was mostly recombinant free, and a phylogeny calculated from this region confirmed that SARS-CoV-2 is closer to RmYN02 than RaTG13, and showed that SARS-CoV-2 diverged from RmYN02 at least 26 years ago, and both diverged from RaTG13 at least 37 years ago; recombinant regions specific to these three viruses provided no additional information as they matched no other Genbank sequences closely. Simple pairwise comparisons of genomes show that there are three regions where most non-synonymous changes probably occurred; the DUF3655 region of the nsp3, the S gene and ORF 8 gene. Differences in the last two of those regions have probably resulted from recombinational changes, however differences in the DUF3655 region may have resulted from selection. A hexamer of the proteins encoded by the nsp3 region may form the molecular pore spanning the double membrane of the coronavirus replication organelle (Wolff et al., 2020), and perhaps the acidic polypeptide encoded by DUF3655 lines it, and presents a novel target for pharmaceutical intervention.","version":"1.1","doi":"10.1101/2020.07.12.199521","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199505","pub_date":"2020-7-13","title":"PT150 is a modulator of glucocorticoid and androgen receptors with antiviral activity against SARS-CoV-2","abstract":"PT150 is a clinical stage molecule, taken orally, with a strong safety profile having completed Phase 1 and Phase 2 clinical trials for its original use as an anti-depressant. It has an active IND for COVID-19. Antiviral activities have been found for PT150 and other members of its class in a variety of virus families; thus, it was now tested against SARS-CoV-2 in human bronchial epithelial lining cells and showed effective 90% inhibitory antiviral concentration (EC90) of 5.55 \u03bcM. PT150 is a member of an extended platform of novel glucocorticoid receptor (GR) and androgen receptor (AR) binding molecules. In vivo, their predominant net effect is one of systemic glucocorticoid antagonism, but they also show direct downregulation of AR and minor GR agonism at the cellular level. We hypothesize that anti-SARS-CoV-2 activity depends in part on this AR downregulation through diminished TMPRSS2 expression and modulation of ACE2 activity. Given that hypercortisolemia is now suggested to be a significant co-factor for COVID-19 progression, we also postulate an additive role for its potent immunomodulatory effects through systemic antagonism of cortisol.","version":"1.1","doi":"10.1101/2020.07.12.199505","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.146332","pub_date":"2020-7-13","title":"Validation and performance of a quantitative IgG assay for the screening of SARS-CoV-2 antibodies","abstract":"The current COVID-19 epidemic imposed an unpreceded challenge to the scientific community in terms of treatment, epidemiology, diagnosis, social interaction, fiscal policies and many other areas. The development of accurate and reliable diagnostic tools (high specificity and sensitivity) is crucial in the current period, the near future and in the long term. These assays should provide guidance to identify immune presumptive protected persons, potential plasma, and/or B cell donors and vaccine development among others. Also, such assays will be contributory in supporting prospective and retrospective studies to identify the prevalence and incidence of COVID-19 and to characterize the dynamics of the immune response. As of today, only thirteen serological assays have received the Emergency Use Authorization (EUA) by the U.S. Federal Drug Administration (FDA). In this work we describe the development and validation of a quantitative IgG enzyme-linked immunoassay (ELISA) using the recombinant SARS-CoV-2 Spike Protein S1 domain, containing the receptor-binding domain (RBD), showing 98% sensitivity, 98.9% specificity and positive and negative predictive values of 100% and 99.2%, respectively. The assay showed to be useful to test for SARS-CoV-2 IgG antibodies in plasma samples from COVID-19-recovered subjects as potential donors for plasmapheresis. This assay is currently under review by the Federal Drug Administration for an Emergency Use Authorization request (Submission Number EUA201115).","version":"1.2","doi":"10.1101/2020.06.11.146332","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.189803","pub_date":"2020-7-13","title":"Animal Model Prescreening: Pre-exposure to SARS-CoV-2 impacts responses in the NHP model","abstract":"COVID-19 presents herculean challenges to research and scientific communities for producing diagnostic and treatment solutions. Any return to normalcy requires rapid development of countermeasures, with animal models serving as a critical tool in testing vaccines and therapeutics. Animal disease status and potential COVID-19 exposure prior to study execution may severely bias efficacy testing. We developed a toolbox of immunological and molecular tests to monitor countermeasure impact on disease outcome and evaluate pre-challenge COVID-19 status. Assay application showed critical necessity for animal pre-screening. Specifically, real-time PCR results documented pre-exposure of an African Green Monkey prior to SARS-CoV-2 challenge with sequence confirmation as a community-acquired exposure. Longitudinal monitoring of nasopharyngeal swabs and serum showed pre-exposure impacted both viral disease course and resulting immunological response. This study demonstrates utility in a comprehensive pre-screening strategy for animal models, which captured the first documented case of community-acquired, non-human primate infection. Pre-exposure to SARS-CoV-2 affects biomarker responses in animal models, highlighting a need for robust pre-screening protocols prior to medical countermeasure studies.","version":"1.2","doi":"10.1101/2020.07.06.189803","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.200188","pub_date":"2020-7-13","title":"Single-cell analysis reveals the function of lung progenitor cells in COVID-19 patients","abstract":"The high mortality of severe 2019 novel coronavirus disease (COVID-19) cases is mainly caused by acute respiratory distress syndrome (ARDS), which is characterized by increased permeability of the alveolar epithelial barriers, pulmonary edema and consequently inflammatory tissue damage. Some but not all patients showed full functional recovery after the devastating lung damage, and so far there is little knowledge about the lung repair process. Here by analyzing the bronchoalveolar lavage fluid (BALF) of COVID-19 patients through single cell RNA-sequencing (scRNA-Seq), we found that in severe (or critical) cases, there is remarkable expansion of TM4SF1+ and KRT5+ lung progenitor cells. The two distinct populations of progenitor cells could play crucial roles in alveolar cell regeneration and epithelial barrier re-establishment, respectively. In order to understand the function of KRT5+ progenitors in vivo, we transplanted a single KRT5+ cell-derived cell population into damaged mouse lung. Time-course single-cell transcriptomic analysis showed that the transplanted KRT5+ progenitors could long-term engrafted into host lung and differentiate into HOPX+ OCLN+ alveolar barrier cell which restored the epithelial barrier and efficiently prevented inflammatory cell infiltration. Similar barrier cells were also identified in some COVID-19 patients with massive leukocyte infiltration. Altogether this work uncovered the mechanism that how various lung progenitor cells work in concert to prevent and replenish alveoli loss post severe SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.07.13.200188","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.198630","pub_date":"2020-7-13","title":"Tobacco, but not nicotine and flavor-less electronic cigarettes, induces ACE2 and immune dysregulation","abstract":"COVID-19, caused by the virus SARS-CoV-2, has infected millions worldwide. This pandemic overlaps with the ongoing epidemics of cigarette smoking and electronic cigarette (e-cig) vaping, with over 1 billion smokers and vapers worldwide. However, there is scarce data relating COVID-19 risks and outcome with cigarette or e-cig use. In this study, we mined 3 independent RNA expression datasets from smokers and vapers to understand the potential relationship between vaping/smoking and the dysregulation of key genes and pathways related to COVID-19. We found that smoking, but not vaping, upregulates ACE2, the cellular receptor that SARS-CoV-2 requires for infection. Both smoking and use of nicotine and flavor-containing e-cig led to upregulations of pro-inflammatory cytokine production and expression of genes related to inflammasomes. Vaping flavor-less and nicotine-less e-cig, however, did not lead to significant cytokine dysregulation and inflammasome activation. Release of inflammasome products, such as IL-1B, and cytokine storms are hallmarks of COVID-19 infection, especially in severe cases. Therefore, our findings demonstrated that smoking or vaping, specifically use of flavored or nicotine-containing e-cigs, may critically exacerbate COVID-19-related inflammation or increase susceptibility to the disease. Further scientific and public health investigations should be undertaken to address these concerning links between COVID-19 and e-cig/smoking.","version":"1.1","doi":"10.1101/2020.07.13.198630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.13.190140","pub_date":"2020-7-13","title":"Anti-SARS-CoV-2 IgG from severely ill COVID-19 patients promotes macrophage hyper-inflammatory responses","abstract":"For yet unknown reasons, severely ill COVID-19 patients often become critically ill around the time of activation of adaptive immunity. Here, we show that anti-Spike IgG from serum of severely ill COVID-19 patients induces a hyper-inflammatory response by human macrophages, which subsequently breaks pulmonary endothelial barrier integrity and induces microvascular thrombosis. The excessive inflammatory capacity of this anti-Spike IgG is related to glycosylation changes in the IgG Fc tail. Moreover, the hyper-inflammatory response induced by anti-Spike IgG can be specifically counteracted in vitro by use of the active component of fostamatinib, an FDA- and EMA-approved therapeutic small molecule inhibitor of Syk. Anti-Spike IgG promotes hyper-inflammation.","version":"1.1","doi":"10.1101/2020.07.13.190140","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.10.087643","pub_date":"2020-7-13","title":"COVID-19 research in Wikipedia","abstract":"Wikipedia is one of the main sources of free knowledge on the Web. During the first few months of the pandemic, over 5,200 new Wikipedia pages on COVID-19 have been created and have accumulated over 400M pageviews by mid June 2020. At the same time, an unprecedented amount of scientific articles on COVID-19 and the ongoing pandemic have been published online. Wikipedia\u2019s contents are based on reliable sources such as scientific literature. Given its public function, it is crucial for Wikipedia to rely on representative and reliable scientific results, especially so in a time of crisis. We assess the coverage of COVID-19-related research in Wikipedia via citations to a corpus of over 160,000 articles. We find that Wikipedia editors are integrating new research at a fast pace, and have cited close to 2% of the COVID-19 literature under consideration. While doing so, they are able to provide a representative coverage of COVID-19-related research. We show that all the main topics discussed in this literature are proportionally represented from Wikipedia, after accounting for article-level effects. We further use regression analyses to model citations from Wikipedia and show that Wikipedia editors on average rely on literature which is highly cited, widely shared on social media, and has been peer-reviewed.","version":"1.3","doi":"10.1101/2020.05.10.087643","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199364","pub_date":"2020-7-12","title":"Flexibility and mobility of SARS-CoV-2-related protein structures","abstract":"The worldwide CoVid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. Existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. Here, we are using in silico modelling of SARS-CoV-2 drug targets, i.e. SARS-CoV-2 protein structures as deposited on the Protein Databank (PDB). We study their flexibility, rigidity and mobility, an important first step in trying to ascertain their dynamics for further drug-related docking studies. We are using a recent protein flexibility modelling approach, combining protein structural rigidity with possible motion consistent with chemical bonds and sterics. For example, for the SARS-CoV-2 spike protein in the open configuration, our method identifies a possible further opening and closing of the S1 subunit through movement of SB domain. With full structural information of this process available, docking studies with possible drug structures are then possible in silico. In our study, we present full results for the more than 200 thus far published SARS-CoV-2-related protein structures in the PDB.","version":"1.1","doi":"10.1101/2020.07.12.199364","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.12.199059","pub_date":"2020-7-12","title":"Tafenoquine inhibits replication of SARS-Cov-2 at pharmacologically relevant concentrations in vitro","abstract":"Tafenoquine [TQ] exhibited EC50/90s of ~ 2.6/5.1 \u03bcM against SARS-CoV-2 in VERO E6 cells and was 4-fold more potent than hydroxychloroquine [HCQ]. Time-of-addition experiments were consistent with a different mechanism for TQ v HCQ. Physiologically based pharmacokinetic (PBPK) modeling suggested that lung unbound concentrations of TQ in COVID-19 patients may exceed the EC90 for at least 8 weeks after administration. The therapeutic potential for TQ in management of COVID-19 should be further evaluated.","version":"1.1","doi":"10.1101/2020.07.12.199059","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.173476","pub_date":"2020-7-11","title":"In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges","abstract":"The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 \u00c5ngstrom, and is publicly available at EMPIAR-10453.","version":"1.2","doi":"10.1101/2020.06.26.173476","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.11.198291","pub_date":"2020-7-11","title":"A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine","abstract":"The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread world-wide with millions of cases and hundreds of thousands of deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8+ and CD4+ T cell responses with S protein-specific killing activity were detected. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines. CH performed research, analyzed data, and wrote the paper; CS performed research and analyzed data; AA performed research and analyzed data; AE performed research and analyzed data; SM performed research, analyzed data, and wrote the paper; MH developed the bioinformatics pipeline and analyzed data; BS contributed new reagents and concepts; MDM designed and supervised research, analyzed data and wrote the paper; all authors read, corrected and approved the final manuscript. The COVID-19 pandemic has caused hundreds of thousands of deaths, yet. Therefore, effective vaccine concepts are urgently needed. In search for such a concept, we have analysed a measles virus-based vaccine candidate targeting SARS-CoV-2. Using this well known, safe vaccine backbone, we demonstrate here induction of functional immune responses in both arms of adaptive immunity with the desired immune bias. Therefore, occurrence of immunopathologies such as antibody-dependent enhancement or enhanced respiratory disease is rather unlikely. Moreover, the candidate still induces immunity against the measles, recognized as a looming second menace, when countries are entrapped to stop routine vaccination campaigns in the face of COVID-19. Thus, a bivalent measles-based COVID-19 vaccine could be the solution for two significant public health threats.","version":"1.1","doi":"10.1101/2020.07.11.198291","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.11.198770","pub_date":"2020-7-11","title":"ACE2-expressing endothelial cells in aging mouse brain","abstract":"Angiotensin-converting enzyme 2 (ACE2) is a key receptor mediating the entry of SARS-CoV-2 into the host cell. Through a systematic analysis of publicly available mouse brain sc/snRNA-seq data, we found that ACE2 is specifically expressed in small sub-populations of endothelial cells and mural cells, namely pericytes and vascular smooth muscle cells. Further, functional changes in viral mRNA transcription and replication, and impaired blood-brain barrier regulation were most prominently implicated in the aged, ACE2-expressing endothelial cells, when compared to the young adult mouse brains. Concordant EC transcriptomic changes were further found in normal aged human brains. Overall, this work reveals an outline of ACE2 distribution in the mouse brain and identify putative brain host cells that may underlie the selective susceptibility of the aging brain to viral infection.","version":"1.1","doi":"10.1101/2020.07.11.198770","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.194027","pub_date":"2020-7-10","title":"Severely ill COVID-19 patients display augmented functional properties in SARS-CoV-2-reactive CD8+ T cells","abstract":"The molecular properties of CD8+ T cells that respond to SARS-CoV-2 infection are not fully known. Here, we report on the single-cell transcriptomes of >80,000 virus-reactive CD8+ T cells from 39 COVID-19 patients and 10 healthy subjects. COVID-19 patients segregated into two groups based on whether the dominant CD8+ T cell response to SARS-CoV-2 was \u2018exhausted\u2019 or not. SARS-CoV-2-reactive cells in the exhausted subset were increased in frequency and displayed lesser cytotoxicity and inflammatory features in COVID-19 patients with mild compared to severe illness. In contrast, SARS-CoV-2-reactive cells in the non-exhausted subsets from patients with severe disease showed enrichment of transcripts linked to co-stimulation, pro-survival NF-\u03baB signaling, and anti-apoptotic pathways, suggesting the generation of robust CD8+ T cell memory responses in patients with severe COVID-19 illness. CD8+ T cells reactive to influenza and respiratory syncytial virus from healthy subjects displayed polyfunctional features. Cells with such features were mostly absent in SARS-CoV-2 responsive cells from both COVID-19 patients and healthy controls non-exposed to SARS-CoV-2. Overall, our single-cell analysis revealed substantial diversity in the nature of CD8+ T cells responding to SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.09.194027","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.196386","pub_date":"2020-7-10","title":"Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects na\u00efve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.09.196386","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.196378","pub_date":"2020-7-10","title":"Molecular analysis of binding region of an ACE2 as a receptor for SARS-CoV-2 between humans and mammals","abstract":"In June 2020, a second wave of coronavirus disease-2019 (COVID-19) infections raised concern in Beijing, where salmon sold a fresh fish wholesale market was suspected of being the source of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. It has raised questions in the press and elsewhere about the scientific basis of salmon as a source of infection. With the number of cases growing, the surface of a salmon chopping board in the market was examined for the presence of SARS-CoV-2 and a positive reaction was observed. Following these test results, there has been debate over whether salmon can be infected with SARS-CoV-2. To find assess this, we investigated the structural homology of angiotensin-converting enzyme 2 (ACE2), a host-side receptor for SARS-CoV-2, between humans and other species including salmon and mink. As a result, a high structural homology between ACE2 and mink, which has reportedly transmitted SARS-CoV-2 to humans, was confirmed. However, a non-high structural homology of ACE2 between salmon and humans was observed. Further experiments are needed to find the source of SARS-CoV-2 transmission to the salmon.","version":"1.1","doi":"10.1101/2020.07.09.196378","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.196188","pub_date":"2020-7-10","title":"SARS-CoV-2 infection in the lungs of human ACE2 transgenic mice causes severe inflammation, immune cell infiltration, and compromised respiratory function","abstract":"Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) emerged in late 2019 and has spread worldwide resulting in the Coronavirus Disease 2019 (COVID-19) pandemic. Although animal models have been evaluated for SARS-CoV-2 infection, none have recapitulated the severe lung disease phenotypes seen in hospitalized human cases. Here, we evaluate heterozygous transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lung tissues with additional spread to other organs. Remarkably, a decline in pulmonary function, as measured by static and dynamic tests of respiratory capacity, occurs 4 days after peak viral titer and correlates with an inflammatory response marked by infiltration into the lung of monocytes, neutrophils, and activated T cells resulting in pneumonia. Cytokine profiling and RNA sequencing analysis of SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with prominent signatures of NF-kB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection recapitulates many features of severe COVID-19 infection in humans and can be used to define the mechanistic basis of lung disease and test immune and antiviral-based countermeasures.","version":"1.1","doi":"10.1101/2020.07.09.196188","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197079","pub_date":"2020-7-10","title":"Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms","abstract":"SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.","version":"1.1","doi":"10.1101/2020.07.10.197079","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030759","pub_date":"2020-7-10","title":"Genetic Grouping of SARS-CoV-2 Coronavirus Sequences using Informative Subtype Markers for Pandemic Spread Visualization","abstract":"We propose an efficient framework for genetic subtyping of SARS-CoV-2, the novel coronavirus that causes the COVID-19 pandemic. Efficient viral subtyping enables visualization and modeling of the geographic distribution and temporal dynamics of disease spread. Subtyping thereby advances the development of effective containment strategies and, potentially, therapeutic and vaccine strategies. However, identifying viral subtypes in real-time is challenging: SARS-CoV-2 is a novel virus, and the pandemic is rapidly expanding. Viral subtypes may be difficult to detect due to rapid evolution; founder effects are more significant than selection pressure; and the clustering threshold for subtyping is not standardized. We propose to identify mutational signatures of available SARS-CoV-2 sequences using a population-based approach: an entropy measure followed by frequency analysis. These signatures, Informative Subtype Markers (ISMs), define a compact set of nucleotide sites that characterize the most variable (and thus most informative) positions in the viral genomes sequenced from different individuals. Through ISM compression, we find that certain distant nucleotide variants covary, including non-coding and ORF1ab sites covarying with the D614G spike protein mutation which has become increasingly prevalent as the pandemic has spread. ISMs are also useful for downstream analyses, such as spatiotemporal visualization of viral dynamics. By analyzing sequence data available in the GISAID database, we validate the utility of ISM-based subtyping by comparing spatiotemporal analyses using ISMs to epidemiological studies of viral transmission in Asia, Europe, and the United States. In addition, we show the relationship of ISMs to phylogenetic reconstructions of SARS-CoV-2 evolution, and therefore, ISMs can play an important complementary role to phylogenetic tree-based analysis, such as is done in the Nextstrain [1] project. The developed pipeline dynamically generates ISMs for newly added SARS-CoV-2 sequences and updates the visualization of pandemic spatiotemporal dynamics, and is available on Github at https://github.com/EESI/ISM and via an interactive website at https://covid19-ism.coe.drexel.edu/. The novel coronavirus responsible for COVID-19, SARS-CoV-2, expanded to reportedly 8.7 million confirmed cases worldwide by June 21, 2020. The global SARS-CoV-2 pandemic highlights the importance of tracking viral transmission dynamics in real-time. Through June 2020, researchers have obtained genetic sequences of SARS-CoV-2 from over 47,000 samples from infected individuals worldwide. Since the virus readily mutates, each sequence of an infected individual contains useful information linked to the individual\u2019s exposure location and sample date. But, there are over 30,000 bases in the full SARS-CoV-2 genome\u2014so tracking genetic variants on a whole-sequence basis becomes unwieldy. We describe a method to instead efficiently identify and label genetic variants, or \u201csubtypes\u201d of SARS-CoV-2. Applying this method results in a compact, 11 base-long compressed label, called an Informative Subtype Marker or \u201cISM\u201d. We define viral subtypes for each ISM, and show how regional distribution of subtypes track the progress of the pandemic. Major findings include (1) covarying nucleotides with the spike protein which has spread rapidly and (2) tracking emergence of a local subtype across the United States connected to Asia and distinct from the outbreak in New York, which is found to be connected to Europe.","version":"1.5","doi":"10.1101/2020.04.07.030759","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.194613","pub_date":"2020-7-10","title":"Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives","abstract":"The COVID-19 pandemic has necessitated a rapid multi-faceted response by the scientific community, bringing researchers, health officials and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.","version":"1.1","doi":"10.1101/2020.07.08.194613","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.194498","pub_date":"2020-7-10","title":"Robust three-dimensional expansion of human adult alveolar stem cells and SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which is the cause of a present global pandemic, infects human lung alveolar cells (hACs). Characterising the pathogenesis is crucial for developing vaccines and therapeutics. However, the lack of models mirroring the cellular physiology and pathology of hACs limits the study. Here, we develop a feeder-free, long-term three-dimensional (3D) culture technique for human alveolar type 2 (hAT2) cells, and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and pro-inflammatory genes in infected hAT2 cells, indicating robust endogenous innate immune response. Further tracing of viral mutations acquired during transmission identifies full infection of individual cells effectively from a single viral entry. Our study provides deep insights into the pathogenesis of SARS-CoV-2, and the application of long-term 3D hAT2 cultures as models for respiratory diseases.","version":"1.1","doi":"10.1101/2020.07.10.194498","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.196618","pub_date":"2020-7-10","title":"A Strategy to Treat COVID-19 Disease with Targeted Delivery of Inhalable Liposomal Hydroxychloroquine: A Non-clinical Pharmacokinetic Study","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly identified pathogen causing coronavirus disease 2019 (COVID-19) pandemic. Hydroxychloroquine (HCQ), an antimalarial and anti-inflammatory drug, has been shown to inhibit SARS-CoV-2 infection in vitro and tested in clinical studies. However, lung concentration (6.7 \u00b5g/mL) to predict the in vivo antiviral efficacy might not be achievable with the currently proposed oral dosing regimen. Further, a high cumulative doses of HCQ may raise concerns of systemic toxicity, including cardiotoxicity. Here, we described a non-clinical study to investigate the pharmacokinetics of a novel formulation of liposomal HCQ administrated by intratracheal (IT) instillation in Sprague-Dawley (SD) rats which achieved 129.4 \u00b5g/g (Cmax) in the lung. Compared to unformulated HCQ administered intravenous (IV), liposomal HCQ with normalized dose showed higher (\u223c30-fold) lung exposure, longer (\u223c2.5-fold) half-life in lung, but lower blood exposure with \u223c20% of Cmax and 74% of AUC and lower heart exposure with 24% of Cmax and 58% of AUC. In conclusion, the pharmacokinetics results in an animal model demonstrate the proof of concept that inhalable liposomal HCQ may provide clinical benefit and serve as a potential treatment for COVID-19.","version":"1.1","doi":"10.1101/2020.07.09.196618","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.10.197814","pub_date":"2020-7-10","title":"Stabilizing the Closed SARS-CoV-2 Spike Trimer","abstract":"The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers, with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify two regions in the S-protein critical for the protein\u2019s stability: the heptad repeat region 1 of the S2 subunit and subunit domain 1 at the interface with S2. We combined a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.","version":"1.1","doi":"10.1101/2020.07.10.197814","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.194563","pub_date":"2020-7-10","title":"Which animals are at risk? Predicting species susceptibility to Covid-19","abstract":"In only a few months, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, leaving physicians, scientists, and public health officials racing to understand, treat, and contain this zoonotic disease. SARS-CoV-2 has made the leap from animals to humans, but little is known about variations in species susceptibility that could identify potential reservoir species, animal models, and the risk to pets, wildlife, and livestock. While there is evidence that certain species, such as cats, are susceptible, the vast majority of animal species, including those in close contact with humans, have unknown susceptibility. Hence, methods to predict their infection risk are urgently needed. SARS-CoV-2 spike protein binding to angiotensin converting enzyme 2 (ACE2) is critical for viral cell entry and infection. Here we identified key ACE2 residues that distinguish susceptible from resistant species using in-depth sequence and structural analyses of ACE2 and its binding to SARS-CoV-2. Our findings have important implications for identification of ACE2 and SARS-CoV-2 residues for therapeutic targeting and identification of animal species with increased susceptibility for infection on which to focus research and protection measures for environmental and public health.","version":"1.1","doi":"10.1101/2020.07.09.194563","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.196519","pub_date":"2020-7-10","title":"Discriminating Mild from Critical COVID-19 by Innate and Adaptive Immune Single-cell Profiling of Bronchoalveolar Lavages","abstract":"How innate and adaptive lung immune responses to SARS-CoV-2 synchronize during COVID-19 pneumonitis and regulate disease severity is poorly established. To address this, we applied single-cell profiling to bronchoalveolar lavages from 44 patients with mild or critical COVID-19 versus non-COVID-19 pneumonia as control. Viral RNA-tracking delineated the infection phenotype to epithelial cells, but positioned mainly neutrophils at the forefront of viral clearance activity during COVID-19. In mild disease, neutrophils could execute their antiviral function in an immunologically \u2018controlled\u2019 fashion, regulated by fully-differentiated T-helper-17 (TH17)-cells, as well as T-helper-1 (TH1)-cells, CD8+ resident-memory (TRM) and partially-exhausted (TEX) T-cells with good effector functions. This was paralleled by \u2018orderly\u2019 phagocytic disposal of dead/stressed cells by fully-differentiated macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, hence facilitating lung tissue repair. In critical disease, CD4+ TH1- and CD8+ TEX-cells were characterized by inflammation-associated stress and metabolic exhaustion, while CD4+ TH17- and CD8+ TRM-cells failed to differentiate. Consequently, T-cell effector function was largely impaired thereby possibly facilitating excessive neutrophil-based inflammation. This was accompanied by impaired monocyte-to-macrophage differentiation, with monocytes exhibiting an ATP-purinergic signalling-inflammasome footprint, thereby enabling COVID-19 associated fibrosis and worsening disease severity. Our work represents a major resource for understanding the lung-localised immunity and inflammation landscape during COVID-19.","version":"1.1","doi":"10.1101/2020.07.09.196519","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.193045","pub_date":"2020-7-09","title":"A single-dose live-attenuated YF17D-vectored SARS-CoV2 vaccine candidate","abstract":"The explosively expanding COVID-19 pandemic urges the development of safe, efficacious and fast-acting vaccines to quench the unrestrained spread of SARS-CoV-2. Several promising vaccine platforms, developed in recent years, are leveraged for a rapid emergency response to COVID-191. We employed the live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express the prefusion form of the SARS-CoV-2 Spike antigen. In mice, the vaccine candidate, tentatively named YF-S0, induces high levels of SARS-CoV-2 neutralizing antibodies and a favorable Th1 cell-mediated immune response. In a stringent hamster SARS-CoV-2 challenge model, vaccine candidate YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose confers protection from lung disease in most vaccinated animals even within 10 days. These results warrant further development of YF-S0 as a potent SARS-CoV-2 vaccine candidate.","version":"1.1","doi":"10.1101/2020.07.08.193045","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.194456","pub_date":"2020-7-09","title":"Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2","abstract":"During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped na\u00efve responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.","version":"1.1","doi":"10.1101/2020.07.08.194456","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.195040","pub_date":"2020-7-09","title":"Screening and testing for a suitable untransfected cell line for SARS-CoV-2 studies","abstract":"At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus. In the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.","version":"1.1","doi":"10.1101/2020.07.09.195040","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.194639","pub_date":"2020-7-09","title":"Decline of humoral responses against SARS-CoV-2 Spike in convalescent individuals","abstract":"In the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to COVID-19 patients. The therapy has been deemed safe and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of \u22651:160 have been recommended in some convalescent plasma trials for inclusion. Here we performed repeated analyses at one-month interval on 31 convalescent individuals to evaluate how the humoral responses against the SARS-CoV-2 Spike, including neutralization, evolve over time. We observed that receptor-binding domain (RBD)-specific IgG slightly decreased between six and ten weeks after symptoms onset but RBD-specific IgM decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing SARS-CoV-2 S wild-type or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after symptoms resolution.","version":"1.1","doi":"10.1101/2020.07.09.194639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.195230","pub_date":"2020-7-09","title":"Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease","abstract":"The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two different doses of SARS-CoV-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and the assessment of MCMs.","version":"1.1","doi":"10.1101/2020.07.09.195230","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.193722","pub_date":"2020-7-09","title":"Comprehensive variant and haplotype landscapes of 50,500 global SARS-CoV-2 isolates and accelerating accumulation of country-private variant profiles","abstract":"Understanding the genetic etiology of COVID-19 requires a comprehensive understanding of the variant and haplotype landscape of all reported genomes of SARS-COV-2, the causative virus of the disease. Country-, state/region- and possibly even city-private variant profiles may contribute to varied disease exemplifications and fatality rates observed across the globe along with host factors such as age, ethnicity and comorbidity. The Children\u2019s Hospital of Los Angeles (CHLA) COVID-19 Analysis Research Database (CARD) captures up-to-date fulllength SARS-CoV-2 sequences of ~50,500 isolates from GISAID, GenBank, CHLA Center for Personalized Medicine, and other sources (as of June 18, 2020). Among which, 49,637 isolates carry at least one variation from the reference genome NC_045512, a total of 6,070 variants and 2,513 haplotypes were detected in at least three isolates independently. Together, they constituted the most likely SARS-CoV-2 variant and haplotype landscapes world-wide currently. Evidence supporting positive (orf3a, orf8, S genes) and purifying (M gene) selections were detected, which warrants further investigation. Most interestingly, we identified 1,583 countryprivate variants from 10,238 isolates (20.6% overall) reported in 48 countries. 807 countryprivate haplotypes, defined as a haplotype shared by at least 5 isolates all of which came from the same country, were identified in in 8,656 isolates from 39 countries. United Kingdom, USA, and Australia had 464, 166 and 32 private haplotypes respectively, comprising 22.4%, 16.6% and 16.4% of the isolates from each country. Together with their descendent and private haplotypes with fewer members, 22,171 (45.8%) isolates carried country-private haplotypes globally. The percentage were 28.2-29.6% in January to March, and rapidly increased to 46.4% and 59.6% in April and May, co-occurring with global travel restrictions. The localization of the variant profiles appeared to be similarly accelerating from 14.2% in March and 28.4% in April to over 40% isolates carrying the country-private variants around May. In summary, a common pattern is seen world-wide in COVID-19 in which at the onset of disease there appeared to be a significant number of SARS-CoV-2 variants that accumulate quickly and then begin to rapidly coalesce into distinct haplotypes. This may be the result of localized outbreaks due to factors such as multiple points viral introduction, geographic separation and the introduction of policies such as travel restriction, social distancing and quarantine, resulting in the emergence of country-private haplotypes.","version":"1.1","doi":"10.1101/2020.07.09.193722","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.193680","pub_date":"2020-7-09","title":"Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike","abstract":"Binding of the spike protein of SARS-CoV-2 to the human angiotensin converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds into a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.","version":"1.1","doi":"10.1101/2020.07.09.193680","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.195263","pub_date":"2020-7-09","title":"Structures of potent and convergent neutralizing antibodies bound to the SARS-CoV-2 spike unveil a unique epitope responsible for exceptional potency","abstract":"Understanding the mechanism of neutralizing antibodies (NAbs) against SARS-CoV-2 is critical for effective vaccines and therapeutics development. We recently reported an exceptionally potent NAb, BD-368-2, and revealed the existence of VH3-53/VH3-66 convergent NAbs in COVID-19. Here we report the 3.5-\u00c5 cryo-EM structure of BD-368-2\u2019s Fabs in complex with a mutation-induced prefusion-state-stabilized spike trimer. Unlike VH3-53/VH3-66 NAbs, BD-368-2 fully blocks ACE2 binding by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their \u201cup\u201d and \u201cdown\u201d positions. BD-368-2 also triggers fusogenic-like structural rearrangements of the spike trimer, which could impede viral entry. Moreover, BD-368-2 completely avoids the common epitope of VH3-53/VH3-66 NAbs, evidenced by multiple crystal structures of their Fabs in tripartite complexes with RBD, suggesting a new way of pairing potent NAbs to prevent neutralization escape. Together, these results rationalize a unique epitope that leads to exceptional neutralization potency, and provide guidance for NAb therapeutics and vaccine designs against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.09.195263","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.194209","pub_date":"2020-7-09","title":"Assessment of SARS-CoV-2 Specific CD4(+) and CD8 (+) T Cell Responses Using MHC Class I and II Tetramers","abstract":"The success of SARS-CoV-2 (CoV-2) vaccines is measured by their ability to mount immune memory responses that are long-lasting. To achieve this goal, it is important to identify surrogates of immune protection, namely, CoV-2 MHC Class I and II immunodominant pieces/epitopes and methodologies to measure them. Here, we present results of flow cytometry-based MHC Class I and II QuickSwitch\u2122 platforms for assessing SARS-CoV-2 peptide binding affinities to various human alleles as well as the H-2 Kb mouse allele. Multiple SARS-CoV-2 potential MHC binders were screened and validated by QuickSwitch testing. While several predicted peptides with acceptable theoretical Kd showed poor MHC occupancies, fourteen MHC class II and a few MHC class I peptides showed promiscuity in that they bind with multiple MHC molecule types. With the peptide exchange generated MHC tetramers, scientists can assess CD4+ and CD8+ immune responses to these different MHC/peptide complexes. Results obtained with several SARS-CoV-2 MHC class I and II peptides are included and discussed.","version":"1.1","doi":"10.1101/2020.07.08.194209","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.195008","pub_date":"2020-7-09","title":"Air and surface contamination in non-health care settings among 641 environmental specimens of 39 COVID-19 cases","abstract":"Little is known about the SARS-CoV-2 contamination of environmental surfaces and air in non-health care settings among COVID-19 cases. We explored the SARS-CoV-2 contamination of environmental surfaces and air by collecting air and swabbing environmental surfaces among 39 COVID-19 cases in Guangzhou, China. The specimens were tested by RT-PCR testing. The information collected for COVID-19 cases included basic demographic, clinical severity, onset of symptoms, radiological testing, laboratory testing and hospital admission. A total of 641 environmental surfaces and air specimens were collected among 39 COVID-19 cases before disinfection. Among them, 20 specimens (20/641, 3.1%) were tested positive from 9 COVID-19 cases (9/39, 23.1%), with 5 (5/101, 5.0%) positive specimens from 3 asymptomatic cases, 5 (5/220, 2.3%) from 3 mild cases, and 10 (10/374, 2.7%) from 3 moderate cases. All positive specimens were collected within 3 days after diagnosis, and 10 (10/42, 23.8%) were found in toilet (5 on toilet bowl, 4 on sink/faucet/shower, 1 on floor drain), 4 (4/21, 19.0%) in anteroom (2 on water dispenser/cup/bottle, 1 on chair/table, 1 on TV remote), 1 (1/8, 12.5%) in kitchen (1 on dining-table), 1 (1/18, 5.6%) in bedroom (1 on bed/sheet pillow/bedside table), 1 (1/5, 20.0%) in car (1 on steering wheel/seat/handlebar) and 3 (3/20, 21.4%) on door knobs. Air specimens in room (0/10, 0.0%) and car (0/1, 0.0%) were all negative. SARS-CoV-2 was found on environmental surfaces especially in toilet, and could survive for several days. We provided evidence of potential for SARS-CoV-2 transmission through contamination of environmental surfaces.","version":"1.1","doi":"10.1101/2020.07.09.195008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.149427","pub_date":"2020-7-09","title":"Elucidation of the antiviral mechanism of cystine and theanine through transcriptome analysis of mice and comparison with COVID-19 gene set data","abstract":"We previously showed that oral administration of cystine and theanine (CT) to mice confers resistance to influenza virus infection. In human studies, CT prevented colds in healthy subjects and enhanced antibody production after influenza vaccination in elderly individuals with a poor nutritional status. The mechanism of action of CT is thought to be glutathione (GSH)-mediated regulation of intracellular redox, which might affect innate immune systems such as macrophages to exert physiological effects. The effect of CT on influenza is independent of viral type, and this treatment has a broad range of antiviral activities. To explore the mechanisms of CT in viral infection, we performed transcriptome profiling of spleen tissues isolated from influenza A virus (IAV)-infected mice. We identified unique gene signatures in response to CT in the IAV-infected mice. Genes upregulated by CT included redox-regulated genes such as GCLC/GCLM (subunits of glutamate cysteine ligase, a rate-limiting enzyme of GSH biosynthesis), TXN1, TXN2, TXNRD2, and SOD1, suggesting that the intracellular redox environment is substantially altered by CT. However, genes downregulated in response to CT included chemokine/chemokine receptor genes (CCL5, CCL19, CXCL9, CXCL12, CXCR3, CXCR4, and ACKR3), some of which are related to cytokine storm. A comparison with public COVID-19-related gene set data showed that the upregulated gene signature was highly similar to the downregulated gene sets of SARS-CoV/SARS-CoV-2-infected cells and the upregulated gene set of attenuated SARS-CoV-infected cells. In conclusion, the unique gene signatures observed in response to orally administered CT in IAV-infected mouse spleen tissues suggested that CT may attenuate viral infection, replication and associated symptoms such as cytokine storm.","version":"1.2","doi":"10.1101/2020.06.25.149427","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.09.195016","pub_date":"2020-7-09","title":"A rational approach to identifying effective combined anticoronaviral therapies against feline coronavirus","abstract":"Feline infectious peritonitis (FIP), caused by a genetic mutant of feline enteric coronavirus known as FIPV, is a highly fatal disease of cats with no currently available vaccine or FDA-approved cure. Dissemination of FIPV in affected cats results in a range of clinical signs including cavitary effusions, anorexia, fever and lesions of pyogranulomatous vasculitis and peri-vasculitis with or without central nervous system and/or ocular involvement. There is a critical need for effective and approved antiviral therapies against coronaviruses including FIPV and zoonotic coronaviruses such as SARS-CoV-2, the cause of COVID-19. With regards to SARS-CoV-2, preliminary evidence suggests that there may be potential clinical and pathological overlap with feline coronaviral disease including enteric and neurological involvement in some cases. We have screened 89 putative antiviral compounds and have identified 25 compounds with antiviral activity against FIPV, representing a variety of drug classes and mechanisms of antiviral action. Based upon successful combination treatment strategies for human patients with HIV or hepatitis C virus infections, we have identified combinations of drugs targeting different steps of the FIPV life cycle resulting in synergistic antiviral effect. Translationally, we suggest that a combined anticoronaviral therapy (cACT) with multiple mechanisms of action and penetration of all potential anatomic sites of viral infection should be applied towards other challenging to treat coronaviruses, like SARS-CoV-2. We have screened 89 compounds in vitro for antiviral activity against FIPV. The putative antiviral activity of these compounds was either purported to be a direct effect on viral proteins involved in viral replication or an indirect inhibitory effect on normal cellular pathways usurped by FIPV to aid viral replication. Twenty-five of these compounds were found to have significant antiviral activity. Certain combinations of these compounds were determined to be superior to monotherapy alone.","version":"1.1","doi":"10.1101/2020.07.09.195016","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.193946","pub_date":"2020-7-09","title":"Learning the language of viral evolution and escape","abstract":"Viral mutation that escapes from human immunity remains a major obstacle to antiviral and vaccine development. While anticipating escape could aid rational therapeutic design, the complex rules governing viral escape are challenging to model. Here, we demonstrate an unprecedented ability to predict viral escape by using machine learning algorithms originally developed to model the complexity of human natural language. Our key conceptual advance is that predicting escape requires identifying mutations that preserve viral fitness, or \u201cgrammaticality,\u201d and also induce high antigenic change, or \u201csemantic change.\u201d We develop viral language models for influenza hemagglutinin, HIV Env, and SARS-CoV-2 Spike that we use to construct antigenically meaningful semantic landscapes, perform completely unsupervised prediction of escape mutants, and learn structural escape patterns from sequence alone. More profoundly, we lay a promising conceptual bridge between natural language and viral evolution. Neural language models of semantic change and grammaticality enable unprecedented prediction of viral escape mutations.","version":"1.1","doi":"10.1101/2020.07.08.193946","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.130161","pub_date":"2020-7-08","title":"An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction","abstract":"SARS-CoV-2 is the etiologic agent of COVID-19, currently causing a devastating pandemic for which pharmacological interventions are urgently needed. The virus enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. The nanobody binds with high affinity in the low nM range to the RBD, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 \u00c5 resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the \u2018up\u2019 and \u2018down\u2019 conformations and that Ty1 sterically hinders RBD-ACE2 binding. This 12.8 kDa nanobody does not need an Fc domain to neutralize SARS-CoV-2, and can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.","version":"1.2","doi":"10.1101/2020.06.02.130161","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.192732","pub_date":"2020-7-08","title":"In vivo structural characterization of the whole SARS-CoV-2 RNA genome identifies host cell target proteins vulnerable to re-purposed drugs","abstract":"SARS-CoV-2 is an RNA virus of the Coronaviridae family that is the causal pathogen of the ongoing Coronavirus Disease 2019 pandemic. There are currently no antiviral drugs or vaccines to treat COVID-19, and the failure to identify effective interventions can be blamed on our incomplete understanding of the nature of this virus and its host cell infection process. Here, we experimentally determined structural maps of the SARS-CoV-2 RNA genome in infected human cells and also characterized in vitro refolded RNA structures for SARS-CoV-2 and 6 other coronaviruses. Our in vivo data confirms several structural elements predicted from theoretical analysis and goes much further in revealing many previously unknown structural features that functionally impact viral translation and discontinuous transcription in cells. Importantly, we harnessed our in vivo structure data alongside a deep-learning tool and accurately predicted several dozen functionally related host cell proteins that bind to the SARS-CoV-2 RNA genome, none of which were known previously. Thus, our in vivo structural study lays a foundation for coronavirus RNA biology and indicates promising directions for the rapid development of therapeutics to treat COVID-19. We mapped the in vivo structure and built secondary structural models of the SARS-CoV-2 RNA genome We discovered functionally impactful structural features in the RNA genomes of multiple coronaviruses We predicted and validated host cell proteins that bind to the SARS-CoV-2 RNA genome based on our in vivo RNA structural data using a deep-learning tool","version":"1.1","doi":"10.1101/2020.07.07.192732","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.193672","pub_date":"2020-7-08","title":"Extracellular vesicles containing ACE2 efficiently prevent infection by SARS-CoV-2 Spike protein-containing virus","abstract":"SARS-CoV-2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by TMPRRS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2-EVs are efficient decoys for SARS-CoV-2 S protein-containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is 500 to 1500 times more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2.","version":"1.1","doi":"10.1101/2020.07.08.193672","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.191007","pub_date":"2020-7-08","title":"SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques","abstract":"CD4 T follicular helper (Tfh) cells are important for the generation of long-lasting and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that, following infection with SARS-CoV-2, adult rhesus macaques exhibited transient accumulation of activated, proliferating Tfh cells in their peripheral blood on a transitory basis. The CD4 helper cell responses were skewed predominantly toward a Th1 response in blood, lung, and lymph nodes, reflective of the interferon-rich cytokine environment following infection. We also observed the generation of germinal center Tfh cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies but delayed or absent IgA antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.","version":"1.1","doi":"10.1101/2020.07.07.191007","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.191072","pub_date":"2020-7-08","title":"Continuous flexibility analysis of SARS-CoV-2 Spike prefusion structures","abstract":"With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. We show that in this dataset there are not well-defined, stable, spike conformations, but virtually a continuum of states moving in a concerted fashion. We obtained an improved resolution ensemble map with minimum bias, from which we model by flexible fitting the extremes of the change along the direction of maximal variance. Moreover, a high-resolution structure of a recently described biochemically stabilized form of the spike is shown to greatly reduce the dynamics observed for the wild-type spike. Our results provide new detailed avenues to potentially restrain the spike dynamics for structure-based drug and vaccine design and at the same time give a warning of the potential image processing classification instability of these complicated datasets, having a direct impact on the interpretability of the results.","version":"1.1","doi":"10.1101/2020.07.08.191072","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.08.193144","pub_date":"2020-7-08","title":"Enhanced COVID-19 data for improved prediction of survival","abstract":"The current COVID-19 pandemic, caused by the rapid world-wide spread of the SARS-CoV-2 virus, is having severe consequences for human health and the world economy. The virus effects individuals quite differently, with many infected patients showing only mild symptoms, and others showing critical illness. To lessen the impact of the pandemic, one important question is which factors predict the death of a patient? Here, we construct an enhanced COVID-19 dataset by processing two existing databases (from Kaggle and WHO) and using natural language processing methods to enhance the data by adding local weather conditions and research sentiment. In this study, we contribute an enhanced COVID-19 dataset, which contains 183 samples and 43 features. Application of Extreme Gradient Boosting (XGBoost) on the enhanced dataset achieves 95% accuracy in predicting patients survival, with country-wise research sentiment, and then age and local weather, showing the most importance. All data and source code are available at http://ab.inf.uni-tuebingen.de/publications/papers/COVID-19.","version":"1.1","doi":"10.1101/2020.07.08.193144","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.03.179028","pub_date":"2020-7-08","title":"Genes associated with liver damage signalling pathways may impact the severity of COVID-19 symptoms in Spanish and Italian populations","abstract":"The novel SARS-CoV-2 virus, which causes the COVID-19 disease, has infected more than 10 million people and caused 500K deaths worldwide. In Europe, over 2 million confirmed cases have been reported, while nearly 200K people have died from the disease. Despite strict containment measures in Spain and Italy after the first reported COVID-19 patient, these two countries have remained in the top five European nations with the highest mortality rate for over two months. We hypothesised that a genetic mechanism could partially explain the poor survival outcome observed in these two countries. An extensive literature search to identify human candidate genes linked to SARS-CoV infection, host immune evasion and disease aggressiveness was carried out. Pathway analysis (IPA) was performed to select the most significantly associated canonical signalling pathways with the genes of interest. The genetic variants\u2019 at these genes with \u00b11Mb flanking region was extracted (GRCh37/hg19 built). Over 80 million single nucleotide polymorphisms (SNPs) were analysed in genome-wide data of 2,504 individuals (1000 genomes, phase III, https://www.internationalgenome.org/). Principal component (PC) analysis was performed, ancestry by the whole genome was inferred and subsets of the regions of interest were extracted (PLINK v1.9b, http://pngu.mgh.harvard.edu/purcell/plink/). PC1 to PC20 values from five European ancestries, including the Spanish and Italian populations, were used for PC analysis. Gene function predictions were run with our genes of interest as a query to the GeneMANIA Cytoscape plugin (https://genemania.org/). A total of 437 candidate genes associated with SARS were identified, including 21 correlated with COVID-19 aggressiveness. The two most significant pathways associated with all 437 genes (Caveolar-mediated Endocytosis and MSP-RON Signalling) did not show any segregation at the population level. However, the most significant canonical pathway associated with genes linked to COVID-19 aggressiveness, the Hepatic Fibrosis and Hepatic Stellate Cell Activation, showed population-specific segregation. Both the Spanish and Italian populations clustered together from the rest of Europe. This was also observed for the Finnish population but in the opposite direction. These results suggest some of the severe COVID-19 cases reported in Spain and Italy could be partially explained by a pre-existing liver condition (especially liver cancer) and/or may lead to further COVID-19 related liver complications.","version":"1.1","doi":"10.1101/2020.07.03.179028","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.186122","pub_date":"2020-7-08","title":"Towards the design of multiepitope-based peptide vaccine candidate against SARS-CoV-2","abstract":"Coronavirus disease 2019 is a current pandemic health threat especially for elderly patients with comorbidities. This respiratory disease is caused by a beta coronavirus known as severe acute respiratory syndrome coronavirus 2. The disease can progress into acute respiratory distress syndrome that can be fatal. Currently, no specific drug or vaccine are available to combat this pandemic outbreak. Social distancing and lockdown have been enforced in many places worldwide. The spike protein of coronavirus 2 is essential for viral entry into host target cells via interaction with angiotensin converting enzyme 2. This viral protein is considered a potential target for design and development of a drug or vaccine. Previously, we have reported several potential epitopes on coronavirus 2 spike protein with high antigenicity, low allergenicity and good stability against specified proteases. In the current study, we have constructed and evaluated a peptide vaccine from these potential epitopes by using in silico approach. This construct is predicted to have a protective immunogenicity, low allergenicity and good stability with minor structural flaws in model build. The population coverage of the used T-cells epitopes is believed to be high according to the employed restricted alleles. The vaccine construct can elicit efficient and long-lasting immune response as appeared through simulation analysis. This multiepitope-based peptide vaccine may represent a potential candidate against coronavirus 2. However, further in vitro and in vivo verification are required.","version":"1.1","doi":"10.1101/2020.07.07.186122","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.182972","pub_date":"2020-7-07","title":"Unique transcriptional changes in coagulation cascade genes in SARS-CoV-2-infected lung epithelial cells: A potential factor in COVID-19 coagulopathies","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global pandemic. In addition to the acute pulmonary symptoms of COVID-19 (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients. Currently, the molecular pathogenesis underlying COVID-19 associated coagulopathies are unknown. While there are many theories for the cause of this pathology, including hyper inflammation and excess tissue damage, the cellular and molecular underpinnings are not yet clear. By analyzing transcriptomic data sets from experimental and clinical research teams, we determined that changes in the gene expression of genes important in the extrinsic coagulation cascade in the lung epithelium may be important triggers for COVID-19 coagulopathy. This regulation of the extrinsic blood coagulation cascade is not seen with influenza A virus (IAV)-infected NHBEs suggesting that the lung epithelial derived coagulopathies are specific to SARS-Cov-2 infection. This study is the first to identify potential lung epithelial cell derived factors contributing to COVID-19 associated coagulopathy. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global pandemic. In addition to the acute pulmonary symptoms of COVID-19 (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients. Currently, the molecular pathogenesis underlying COVID-19 associated coagulopathies are unknown. Understanding the molecular basis of dysregulated blood coagulation during SARS-CoV-2 infection may help promote new therapeutic strategies to mitigate these complications in COVID-19 patients. We analyzed three publicly available RNA sequencing datasets to identify possible molecular etiologies of COVID-19 associated coagulopathies. These data sets include sequencing libraries from clinically isolated samples of bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 positive patients and healthy controls. We also analyzed a publicly available RNA sequencing dataset derived from in vitro SARS-CoV-2 infected primary normal human bronchial epithelial (NHBE) cells and mock infected samples. Pathway analysis of both NHBE and BALF differential gene expression gene sets. We found that SARS-CoV-2 infection induces the activation of the extrinsic blood coagulation cascade and suppression of the plasminogen activation system in both NHBEs and cells isolated from the BALF. PBMCs did not differentially express genes regulating blood coagulation. Comparison with influenza A virus (IAV)-infected NHBEs revealed that the regulation of the extrinsic blood coagulation cascade is unique to SARS-CoV-2, and not seen with IAV infection. The hyper-activation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system in SARS-CoV-2 infected epithelial cells may drive diverse coagulopathies in the lung and distal organ systems. The gene transcription pattern in SARS-CoV-2 infected epithelial cells is distinct from IAV infected epithelial cells with regards to the regulation of blood coagulation.","version":"1.1","doi":"10.1101/2020.07.06.182972","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.190660","pub_date":"2020-7-07","title":"The global and local distribution of RNA structure throughout the SARS-CoV-2 genome","abstract":"SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ~30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to locate regions of high base pair content across this long genome and also identify well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity to stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most highly structured viral RNAs in nature. Furthermore, our analysis reveals varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts. The conclusions reported in this study provide a foundation for structure-function hypotheses in SARS-CoV-2 biology, and in turn, may guide the 3D structural characterization of potential RNA drug targets for COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2020.07.06.190660","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.192203","pub_date":"2020-7-07","title":"A rapidly adaptable biomaterial vaccine for SARS-CoV-2","abstract":"The global COVID-19 pandemic motivates accelerated research to develop safe and efficacious vaccines. To address this need, we leveraged a biomaterial vaccine technology that consists of mesoporous silica rods (MSRs) that provide a sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and adjuvants to concentrate and mature antigen-presenting cells at the vaccine site. Here we explored the humoral responses resulting from the use of monophosphoryl lipid A (MPLA) as the adjuvant and SARS-CoV-2 spike proteins S1, S2, the nucleocapsid (N) protein, and receptor binding domain (RBD) as the target antigens. The dose of antigen and impact of pre-manufacturing of vaccines as versus loading antigen just-in-time was explored in these studies. Single shot MSR vaccines induced rapid and robust antibody titers to the presented antigens, even without the use of a boost, and sera from vaccinated animals demonstrated neutralizing activity against a SARS-CoV-2 pseudovirus. Overall, these results suggest the MSR vaccine system may provide potent protective immunity when utilized to present SARS-CoV-2 antigens.","version":"1.1","doi":"10.1101/2020.07.07.192203","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.190967","pub_date":"2020-7-07","title":"A Small interfering RNA lead targeting RNA-dependent RNA-polymerase effectively inhibit the SARS-CoV-2 infection in Golden Syrian hamster and Rhesus macaque","abstract":"A small interfering RNA (siRNA) inhibitors have demonstrated the novel modality for suppressing infectious diseases. Sixty-one siRNA molecules, predicted by the bioinformatics programs, were screened for the possibility of treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using an in vitro plaque assay. Among six siRNA leads with the efficacy of reducing plaque number, the siRNA targeting RNA-dependent RNA polymerase (RdRp) showed a reduction in SARS-CoV-2 infection-induced fever and virus titer in the Golden Syrian hamster and rhesus macaque. These results suggest the potential for RdRp targeting siRNA as a new treatment for the coronavirus disease 2019 (COVID-19).","version":"1.1","doi":"10.1101/2020.07.07.190967","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.191247","pub_date":"2020-7-07","title":"Alignment of virus-host protein-protein interaction networks by integer linear programming: SARS-CoV-2","abstract":"Beside socio-economic issues, coronavirus pandemic COVID-19, the infectious disease caused by the newly discovered coronavirus SARS-CoV-2, has caused a deep impact in the scientific community, that has considerably increased its effort to discover the infection strategies of the new virus. Among the extensive and crucial research that has been carried out in the last few months, the analysis of the virus-host relationship plays an important role in drug discovery. Virus-host protein-protein interactions are the active agents in virus replication, and the analysis of virus-host protein-protein interaction networks is fundamental to the study of the virus-host relationship. We have adapted and implemented a recent integer linear programming model for protein-protein interaction network alignment to virus-host networks, and obtained a consensus alignment of the SARS-CoV-1 and SARS-CoV-2 virus-host protein-protein interaction networks. Despite the lack of shared human proteins in these virus-host networks and the low number of preserved virus-host interactions, the consensus alignment revealed aligned human proteins that share a function related to viral infection, as well as human proteins of high functional similarity that interact with SARS-CoV-1 and SARS-CoV-2 proteins, whose alignment would preserve these virus-host interactions.","version":"1.1","doi":"10.1101/2020.07.07.191247","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.191429","pub_date":"2020-7-07","title":"Genes Encoding ACE2, TMPRSS2 and Related Proteins Mediating SARS-CoV-2 Viral Entry are Upregulated with Age in Human Cardiomyocytes","abstract":"Age is an independent risk factor for adverse outcome in patients following COVID-19 infection. We hypothesised that differential expression of genes encoding proteins proposed to be required for entry of SARS-Cov-2 in aged compared to younger cardiomyocytes might contribute to the susceptibility of older individuals to COVID-19-associated cardiovascular complications. We generated strand-specific RNA-sequencing libraries from RNA isolated from flow-sorted cardiomyocyte nuclei from left ventricular tissue. RNASeq data were compared between five young (19-25yr) and five older (63-78yr) Caucasian males who had not been on medication or exhibited evidence of cardiovascular disease post-mortem. Expression of relevant genes encoding ACE2, TMPRSS2, TMPRS11D, TMPRS11E, FURIN, CTSL, CTSB and B0AT1/SLC6A19 were upregulated in aged cardiomyocytes and the combined relative cardiomyocyte expression of these genes correlated positively with age. Genes encoding proteins in the RAAS and interferon/interleukin pathways were also upregulated such as ACE, AGTR1, MAS1 and IL6R. Our results highlight SARS-CoV-2 related genes that have higher expression in aged compared with young adult cardiomyocytes. These data may inform studies using selective enzyme inhibitors/antagonists, available as experimental compounds or clinically approved drugs e.g. remdesivir that has recently been rapidly accepted for compassionate use, to further understand the contribution of these pathways in human cardiomyocytes to disease outcome in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.07.07.191429","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.191676","pub_date":"2020-7-07","title":"SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation","abstract":"The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5\u2019 untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes.","version":"1.1","doi":"10.1101/2020.07.07.191676","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.151357","pub_date":"2020-7-07","title":"The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types","abstract":"A novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis to introduce the D614G variant and show that in multiple cell lines, including human lung epithelial cells, that the D614G mutation is up to 8-fold more effective at transducing cells than wild-type. We demonstrate increased infection using both Spike-pseudotyped lentivirus and intact SARS-CoV-2 virus. Although there is minimal difference in ACE2 receptor binding between the Spike variants, we show that the G614 variant is more resistant to proteolytic cleavage in vitro and in human cells, suggesting a possible mechanism for the increased transduction. This result has important implications for the efficacy of Spike-based vaccines currently under development in protecting against this recent and highly-prevalent SARS-CoV-2 isolate.","version":"1.2","doi":"10.1101/2020.06.14.151357","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.07.192005","pub_date":"2020-7-07","title":"A Fluorescence-based High Throughput-Screening assay for the SARS-CoV RNA synthesis complex","abstract":"The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96% aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1,520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug repositioning or novel drug discovery against the SARS-CoV-2. - A new SARS-CoV non radioactive RNA polymerase assay is described - The robotized assay is suitable to identify RdRp inhibitors based on HTS","version":"1.1","doi":"10.1101/2020.07.07.192005","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.039941","pub_date":"2020-7-07","title":"High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes","abstract":"Isothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.","version":"1.3","doi":"10.1101/2020.04.13.039941","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.28.175802","pub_date":"2020-7-07","title":"Whole-Genome Sequences of the Severe Acute Respiratory Syndrome Coronavirus-2 obtained from Romanian patients between March and June of 2020","abstract":"Impact of mutations on the evolution of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) are needed for ongoing global efforts to track and trace the current pandemic, in order to enact effective prevention and treatment options. SARS-Co-V-2 viral genomes were detected and sequenced from 18 Romanian patients suffering from coronavirus disease-2019. Viral Spike S glycoprotein sequences were used to generate model structures and assess the role of mutations on protein stability. We integrated the phylogenetic tree within the available European SARS-Co-V-2 genomic sequences. We further provide an epidemiological overview of the pre-existing conditions that are lethal in relevant Romanian patients. Non-synonymous mutations in the viral Spike glycoprotein relating to infectivity are constructed in models of protein structures. Continuing search to limit and treat SARS-CoV-2 benefit from our contribution in delineating the viral Spike glycoprotein mutations, as well as from assessment of their role on protein stability or complex formation with human receptor angiotensin-converting enzyme 2. Our results help implement and extend worldwide genomic surveillance of coronavirus disease-2019.","version":"1.4","doi":"10.1101/2020.06.28.175802","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.20147272","pub_date":"2020-07-07","title":"Reopening universities during the COVID-19 pandemic: A testing strategy to minimize active cases and delay outbreaks","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>University campuses present an ideal environment for viral spread and are therefore at extreme risk of serving as a hotbed for a COVID-19 outbreak. While active surveillance throughout the semester such as widespread testing, contact tracing, and case isolation, may assist in detecting and preventing early outbreaks, these strategies will not be sufficient should a larger outbreak occur. It is therefore necessary to limit the initial number of active cases at the start of the semester. We examine the impact of pre-semester NAT testing on disease spread in a university setting.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>\n                    We implement simple dynamic transmission models of SARS-CoV-2 infection to explore the effects of pre-semester testing strategies on the number of active infections and occupied isolation beds throughout the semester. We assume an infectious period of 3 days and vary\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    to represent the effectiveness of disease mitigation strategies throughout the semester. We assume the prevalence of active cases at the beginning of the semester is 5%. The sensitivity of the NAT test is set at 90%.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>\n                    If no pre-semester screening is mandated, the peak number of active infections occurs in under 10 days and the size of the peak is substantial, ranging from 5,000 active infections when effective mitigation strategies (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 1.25) are implemented to over 15,000 active infections for less effective strategies (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 3). When one NAT test is mandated within one week of campus arrival, effective (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 1.25) and less effective (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 3) mitigation strategies delay the onset of the peak to 40 days and 17 days, respectively, and result in peak size ranging from 1,000 to over 15,000 active infections. When two NAT tests are mandated, effective (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 1.25) and less effective (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 3) mitigation strategies delay the onset of the peak through the end of fall semester and 20 days, respectively, and result in peak size ranging from less than 1,000 to over 15,000 active infections. If maximum occupancy of isolation beds is set to 2% of the student population, then isolation beds would only be available for a range of 1 in 2 confirmed cases (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 1.25) to 1 in 40 confirmed cases (\n                    <jats:italic>R</jats:italic>\n                    <jats:sub>\n                      <jats:italic>0</jats:italic>\n                    </jats:sub>\n                    = 3) before maximum occupancy is reached.\n                  </jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion</jats:title>\n                  <jats:p>Even with highly effective mitigation strategies throughout the semester, inadequate pre-semester testing will lead to early and large surges of the disease and result in universities quickly reaching their isolation bed capacity. We therefore recommend NAT testing within one week of campus return. While this strategy is sufficient for delaying the timing of the outbreak, pre-semester testing would need to be implemented in conjunction with effective mitigation strategies to reduce the outbreak size.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.07.06.20147272","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.20.161323","pub_date":"2020-7-06","title":"D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein that mediates SARS-CoV-2 entry into host cells is a major target for vaccines and therapeutics. Thus, insights into its sequence variations are key to understanding the infection and antigenicity of SARS-CoV-2. A dominant mutational variant at position 614 of the S protein (aspartate to glycine, D614G mutation) was observed in the SARS-CoV-2 genome sequence obtained from the Nextstrain database. Using a pseudovirus-based assay, we identified that S-D614 and S-G614 protein pseudotyped viruses share a common receptor, human angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 protein demonstrated functional differences. First, S-G614 protein could be cleaved by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than did the S-D614 pseudovirus, especially in the presence of elastase-2. Third, an elastase inhibitor approved for clinical use blocked elastase-enhanced S-G614 pseudovirus infection. Moreover, 93% (65/70) convalescent sera from patients with COVID-19 could neutralize both S-D614 and S-G614 pseudoviruses with comparable efficiencies, but about 7% (5/70) convalescent sera showed reduced neutralizing activity against the S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 transmission and immune interventions.","version":"1.2","doi":"10.1101/2020.06.20.161323","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.190066","pub_date":"2020-7-06","title":"Comparison of Transgenic and Adenovirus hACE2 Mouse Models for SARS-CoV-2 Infection","abstract":"Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2)(1-3). Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in both the lung and brain leading to lethality. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the lung, and no clinical signs of infection with a challenge dose of 104 plaque forming units. The K18-hACE2 model provides a stringent model for testing the ability of vaccines and antivirals to protect against disease, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.","version":"1.1","doi":"10.1101/2020.07.06.190066","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.188953","pub_date":"2020-7-06","title":"Identification of potent and safe antiviral therapeutic candidates against SARS-CoV-2","abstract":"COVID-19 pandemic has infected millions of people with mortality exceeding 300,000. There is an urgent need to find therapeutic agents that can help clear the virus to prevent the severe disease and death. Identifying effective and safer drugs can provide with more options to treat the COVID-19 infections either alone or in combination. Here we performed a high throughput screen of approximately 1700 US FDA approved compounds to identify novel therapeutic agents that can effectively inhibit replication of coronaviruses including SARS-CoV-2. Our two-step screen first used a human coronavirus strain OC43 to identify compounds with anti-coronaviral activities. The effective compounds were then screened for their effectiveness in inhibiting SARS-CoV-2. These screens have identified 24 anti-SARS-CoV-2 drugs including previously reported compounds such as hydroxychloroquine, amlodipine, arbidol hydrochloride, tilorone 2HCl, dronedarone hydrochloride, and merfloquine hydrochloride. Five of the newly identified drugs had a safety index (cytotoxic/effective concentration) of >600, indicating wide therapeutic window compared to hydroxychloroquine which had safety index of 22 in similar experiments. Mechanistically, five of the effective compounds were found to block SARS-CoV-2 S protein-mediated cell fusion. These FDA approved compounds can provide much needed therapeutic options that we urgently need in the midst of the pandemic.","version":"1.1","doi":"10.1101/2020.07.06.188953","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.190207","pub_date":"2020-7-06","title":"Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets","abstract":"To identify features in the genome of the SARS-CoV-2 pathogen responsible for the COVID-19 pandemic that may contribute to its viral replication, host pathogenicity, and vulnerabilities, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from other well-characterized human and animal coronavirus genomes. Our analyses suggest the presence of unique sequence signatures in the 3\u2019-untranslated region (UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2, SARS-CoV, as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host microRNA-mediated interaction between the 3\u2019-UTR and human microRNA hsa-miR-1307-3p based on predicted, yet extensive, complementary base-pairings and similar interactions involving the Influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3\u2019-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection, and the exploitation of conserved features in the 3\u2019-UTR as therapeutic targets warrant further investigation.","version":"1.1","doi":"10.1101/2020.07.06.190207","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.189860","pub_date":"2020-7-06","title":"Comparison of three TaqMan Real-Time Reverse Transcription-PCR assays in detecting SARS-CoV-2","abstract":"Quick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LOD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LOD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92% and 100%, respectively) than that of the WHO assays (with a detection rate of 60%), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64%) than those of the WHO assays and the CCDC assays (with detection rates of 48% and 20%, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.06.189860","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.06.182634","pub_date":"2020-7-06","title":"Gene Expression Network Analysis Provides Potential Targets Against SARS-CoV-2","abstract":"Cell entry of SARS-CoV-2, the novel coronavirus causing COVID-19, is facilitated by host cell angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We aimed to identify and characterize genes that are co-expressed with ACE2 and TMPRSS2, and to further explore their biological functions and potential as druggable targets. Using the gene expression profiles of 1,038 lung tissue samples, we performed a weighted gene correlation network analysis (WGCNA) to identify modules of co-expressed genes. We explored the biology of co-expressed genes using bioinformatics databases, and identified known drug-gene interactions. ACE2 was in a module of 681 co-expressed genes; 12 genes with moderate-high correlation with ACE2 (r>0.3, FDR<0.05) had known interactions with existing drug compounds. TMPRSS2 was in a module of 1,086 co-expressed genes; 15 of these genes were enriched in the gene ontology biologic process \u2018Entry into host cell\u2019, and 53 TMPRSS2-correlated genes had known interactions with drug compounds. Dozens of genes are co-expressed with ACE2 and TMPRSS2, many of which have plausible links to COVID-19 pathophysiology. Many of the co-expressed genes are potentially targetable with existing drugs, which may help to fast-track the development of COVID-19 therapeutics.","version":"1.1","doi":"10.1101/2020.07.06.182634","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.073387","pub_date":"2020-7-06","title":"PSGL-1 inhibits the virion incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins and impairs virus attachment and infectivity","abstract":"P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-\u03b3 (INF-\u03b3)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks virus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells.","version":"1.2","doi":"10.1101/2020.05.01.073387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.182964","pub_date":"2020-7-06","title":"Using iCn3D and the World Wide Web for structure-based collaborative research: Analyzing molecular interactions at the root of COVID-19","abstract":"The COVID-19 pandemic took us ill-prepared and tackling the many challenges it poses in a timely manner requires world-wide collaboration. Our ability to study the SARS-COV-2 virus and its interactions with its human host in molecular terms efficiently and collaboratively becomes indispensable and mission-critical in the race to develop vaccines, drugs, and neutralizing antibodies. There is already a significant corpus of 3D structures related to SARS and MERS coronaviruses, and the rapid generation of new structures demands the use of efficient tools to expedite the sharing of structural analyses and molecular designs and convey them in their native 3D context in sync with sequence data and annotations. We developed iCn3D (pronounced \u201cI see in 3D\u201d)  to take full advantage of web technologies and allow scientists of different backgrounds to perform and share sequence-structure analyses over the Internet and engage in collaborations through a simple mechanism of exchanging \u201clifelong\u201d web links (URLs). This approach solves the very old problem of \u201csharing of molecular scenes\u201d in a reliable and convenient manner. iCn3D links are sharable over the Internet and make data and entire analyses findable, accessible, and reproducible, with various levels of interoperability. Links and underlying data are FAIR  and can be embedded in preprints and papers, bringing a 3D live and interactive dimension to a world of text and static images used in current publications, eliminating at the same time the need for arcane supplemental materials. This paper exemplifies iCn3D capabilities in visualization, analysis, and sharing of COVID-19 related structures, sequence variability, and molecular interactions.","version":"1.2","doi":"10.1101/2020.07.01.182964","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.013342","pub_date":"2020-7-06","title":"A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production","abstract":"Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed a RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase / DNA polymerase (RTX). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use \u2013 (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs from Addgene or our lab and begin to develop their own assays, within whatever regulatory framework exists for them. We lay out protocols for dye-based and TaqMan probe-based assays, in order to best compare with \u2018gold standard\u2019 reagents. These protocols should form the basis of further modifications that can simplify the assay to the use of overexpressing cells themselves as reagents. Developing dye-based and TaqMan probe-based RT-qPCR assays with RTX","version":"1.5","doi":"10.1101/2020.03.29.013342","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.029769","pub_date":"2020-7-06","title":"Single-cell analysis of severe COVID-19 patients reveals a monocyte-driven inflammatory storm attenuated by Tocilizumab","abstract":"Despite the current devastation of the COVID-19 pandemic, several recent studies have suggested that the immunosuppressive drug Tocilizumab can powerfully treating inflammatory responses that occur in this disease. Here, by employing single-cell analysis of the immune cell composition of severe-stage COVID-19 patients and these same patients in post Tocilizumab-treatment remission, we have identified a monocyte subpopulation specific to severe disease that contributes to inflammatory storms in COVID-19 patients. Although Tocilizumab treatment attenuated the strong inflammatory immune response, we found that immune cells including plasma B cells and CD8+ T cells still exhibited an intense humoral and cell-mediated anti-virus immune response in COVID-19 patients after Tocilizumab treatment. Thus, in addition to providing a rich, very high-resolution data resource about the immune cell distribution at multiple stages of the COVID-19 disease, our work both helps explain Tocilizumab\u2019s powerful therapeutic effects and defines a large number of potential new drug targets related to inflammatory storms.","version":"1.2","doi":"10.1101/2020.04.08.029769","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.04.187435","pub_date":"2020-7-05","title":"Snapshot of the evolution and mutation patterns of SARS-CoV-2","abstract":"The COVID-19 pandemic is the most important public health threat in recent history. Here we study how its causal agent, SARS-CoV-2, has diversified genetically since its first emergence in December 2019. We have created a pipeline combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in a data set consisting of 4,894 SARS-CoV-2 complete genome sequences. Although the phylogenetic diversity of SARS-CoV-2 is low, the whole genome phylogenetic tree can be divided into five clusters/clades based on the tree topology and clustering of specific mutations, but its branches exhibit low genetic distance and bootstrap support values. We also identified 11 residues that are high-frequency substitutions, with four of them currently showing some signal for potential positive selection. These fast-evolving sites are in the non-structural proteins nsp2, nsp5 (3CL-protease), nsp6, nsp12 (polymerase) and nsp13 (helicase), in accessory proteins (ORF3a, ORF8) and in the structural proteins N and S. Temporal and spatial analysis of these potentially adaptive mutations revealed that the incidence of some of these sites was declining after having reached an (often local) peak, whereas the frequency of other sites is continually increasing and now exhibit a worldwide distribution. Structural analysis revealed that the mutations are located on the surface of the proteins that modulate biochemical properties. We speculate that this improves binding to cellular proteins and hence represents fine-tuning of adaptation to human cells. Our study has implications for the design of biochemical and clinical experiments to assess whether important properties of SARS-CoV-2 have changed during the epidemic.","version":"1.1","doi":"10.1101/2020.07.04.187435","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.123612","pub_date":"2020-7-05","title":"SARS-CoV-2 transmission chains from genetic data: a Danish case study","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus started in China in December 2019 and has since spread globally. Information about the spread of the virus in a country can inform the gradual reopening of a country and help to avoid a second wave of infections. Denmark is currently opening up after a lockdown in mid-March. We perform a phylogenetic analysis of 742 publicly available Danish SARS-CoV-2 genome sequences and put them into context using sequences from other countries. Our findings are consistent with several introductions of the virus to Denmark from independent sources. We identify several chains of mutations that occurred in Denmark and in at least one case find evidence that the virus spread from Denmark to other countries. A number of the mutations found in Denmark are non-synonymous, and in general there is a considerable variety of strains. The proportions of the most common haplotypes is stable after lockdown. Our work shows how genetic data can be used to identify routes of introduction of a virus into a region and provide alternative means for verifying existing assumptions. For example, our analysis supports the hypothesis that the virus was brought to Denmark by skiers returning from Ischgl. On the other hand, we identify transmission chains suggesting that Denmark was part of a network of countries among which the virus was being transmitted; thus challenging the common narrative that Denmark only got infected from abroad. Our analysis does not indicate that the major haplotypes appearing in Denmark have a different degree of virality. Our methods can be applied to other countries, regions or even highly localised outbreaks. When used in real-time, we believe they can serve to identify transmission events and supplement traditional methods such as contact tracing.","version":"1.2","doi":"10.1101/2020.05.29.123612","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.122648","pub_date":"2020-7-05","title":"Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding","abstract":"Rapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.","version":"1.3","doi":"10.1101/2020.05.28.122648","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.098079","pub_date":"2020-7-05","title":"Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Aluminum Hydroxide Induces Protective Immunity and Reduces Immune Enhancement","abstract":"We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel\u00ae, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel\u00ae were fully protected from lethal SARS-CoV challenge (0% mortality), compared to \u223c 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel\u00ae, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel\u00ae was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel\u00ae provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.05.15.098079","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.03.184846","pub_date":"2020-7-04","title":"The potential role of miR-21-3p in coronavirus-host interplay","abstract":"Host miRNAs are known as important regulators of virus replication and pathogenesis. They can interact with various viruses by several possible mechanisms including direct binding the viral RNA. Identification of human miRNAs involved in coronavirus-host interplay is becoming important due to the ongoing COVID-19 pandemic. In this work we performed computational prediction of high-confidence direct interactions between miRNAs and seven human coronavirus RNAs. In order to uncover the entire miRNA-virus interplay we further analyzed lungs miRNome of SARS-CoV infected mice using publicly available miRNA sequencing data. We found that miRNA miR-21-3p has the largest probability of binding the human coronavirus RNAs and being dramatically up-regulated in mouse lungs during infection induced by SARS-CoV. Further bioinformatic analysis of binding sites revealed high conservativity of miR-21-3p binding regions within RNAs of human coronaviruses and their strains.","version":"1.1","doi":"10.1101/2020.07.03.184846","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.04.187583","pub_date":"2020-7-04","title":"Gender, race and parenthood impact academic productivity during the COVID-19 pandemic: from survey to action","abstract":"While the Coronavirus disease 2019 (COVID-19) pandemic is altering academia dynamics, those juggling remote work and domestic demands \u2013 including childcare - have already felt the impacts on productivity. Female authors are facing a decrease in papers submission rates since the beginning of the pandemic period. The reasons for this decline in women productivity need to be further investigated. Here we show the influence of gender, parenthood and race in academics productivity during the pandemic period, based on a survey answered by 3,345 Brazilian academics from various knowledge areas and research institutions. Findings revealed that male academics - especially childless ones - were the least affected group, whereas female academics, especially Black women and mothers, were the most impacted group. This scenario will leave long-term effects on the career progression of the most affected groups. The results presented here are crucial for the development of actions and policies that aim to avoid further deepening the gender gap in science. This particular situation we are facing during the pandemic demands institutional flexibility and academia should foster the discussion about actions to benefit Black scientists and academics with families in the post-pandemic scenario.","version":"1.1","doi":"10.1101/2020.07.04.187583","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.03.186825","pub_date":"2020-7-03","title":"Map of SARS-CoV-2 spike epitopes not shielded by glycans","abstract":"The severity of the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, calls for the urgent development of a vaccine. The primary immunological target is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface to mediate viral entry into the host cell. To identify possible antibody binding sites not shielded by glycans, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for vaccine development. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics in epitope mapping.","version":"1.1","doi":"10.1101/2020.07.03.186825","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.03.185850","pub_date":"2020-7-03","title":"A CRISPR-based SARS-CoV-2 diagnostic assay that is robust against viral evolution and RNA editing","abstract":"Extensive testing is essential to break the transmission of the new coronavirus SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Recently, CRISPR-based diagnostics have emerged as attractive alternatives to quantitative real-time PCR due to their faster turnaround time and their potential to be used in point-of-care testing scenarios. However, existing CRISPR-based assays for COVID-19 have not considered viral genome mutations and RNA editing in human cells. Here, we present the VaNGuard (Variant Nucleotide Guard) test that is not only specific and sensitive for SARS-CoV-2, but can also detect the virus when its genome or transcriptome has evolved or has been edited by deaminases in infected human cells. We show that an engineered AsCas12a enzyme is more tolerant of mismatches than wildtype LbCas12a and that multiplexed Cas12a targeting can overcome the presence of single nucleotide variations. Our assay can be completed in 30 minutes with a dipstick for a rapid point-of-care test.","version":"1.1","doi":"10.1101/2020.07.03.185850","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.03.186296","pub_date":"2020-7-03","title":"The major genetic risk factor for severe COVID-19 is inherited from Neandertals","abstract":"A recent genetic association study (Ellinghaus et al. 2020) identified a gene cluster on chromosome 3 as a risk locus for respiratory failure in SARS-CoV-2. Recent data comprising 3,199 hospitalized COVID-19 patients and controls reproduce this and find that it is the major genetic risk factor for severe SARS-CoV-2 infection and hospitalization (COVID-19 Host Genetics Initiative). Here, we show that the risk is conferred by a genomic segment of ~50 kb that is inherited from Neandertals and occurs at a frequency of ~30% in south Asia and ~8% in Europe.","version":"1.1","doi":"10.1101/2020.07.03.186296","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.03.186304","pub_date":"2020-7-03","title":"Robust and sensitive detection of SARS-CoV-2 using PCR based methods","abstract":"The World Health Organization (WHO) has declared the Coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of viral RNA by RT-PCR significantly varied according to the sequence of primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be significantly higher than RT-qPCR. These findings suggest that ddPCR could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.","version":"1.1","doi":"10.1101/2020.07.03.186304","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.02.183764","pub_date":"2020-7-02","title":"Alpha-1 antitrypsin inhibits SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). To identify factors of the respiratory tract that suppress SARS-CoV-2, we screened a peptide/protein library derived from bronchoalveolar lavage, and identified \u03b11-antitrypsin (\u03b11-AT) as specific inhibitor of SARS-CoV-2. \u03b11-AT targets the viral spike protein and blocks SARS-CoV-2 infection of human airway epithelium at physiological concentrations. Our findings show that endogenous \u03b11-AT restricts SARS-CoV-2 and repurposes \u03b11-AT-based drugs for COVID-19 therapy.","version":"1.1","doi":"10.1101/2020.07.02.183764","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.182220","pub_date":"2020-7-02","title":"Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays","abstract":"Commercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies. However, there is limited published data associating the results from commercial assays with neutralizing antibodies. 67 specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys SARS-CoV-2, Abbott SARS-CoV-2 IgG, or EUROIMMUN SARS-CoV-2 IgG assays and 5 control specimens were analyzed for the presence of neutralizing antibodies to SARS-CoV-2. Correlation, concordance, positive percent agreement (PPA), and negative percent agreement (NPA) were calculated at several cutoffs. Results were compared in patients categorized by clinical outcomes. The correlation between SARS-CoV-2 neutralizing titer (EC50) and the Roche, Abbott, and EUROIMMUN assays was 0.29, 0.47, and 0.46 respectively. At an EC50 of 1:32, the concordance kappa with Roche was 0.49 (95% CI; 0.23-0.75), with Abbott was 0.52 (0.28-0.77), and with EUROIMMUN was 0.61 (0.4-0.82). At the same neutralizing titer, the PPA and NPA for the Roche was 100% (94-100) & 56% (30-80); Abbott was 96% (88-99) & 69% (44-86); and EUROIMMUN was 91% (80-96) & 81% (57-93) for distinguishing neutralizing antibodies. Patients who died, were intubated, or had a cardiac injury from COVID-19 infection had significantly higher neutralizing titers relative to those with mild symptoms. COVID-19 patients generate an antibody response to multiple viral proteins such that the calibrator ratios on the Roche, Abbott, and EUROIMMUN assays are all associated with SARS-CoV-2 neutralization. Nevertheless, commercial serological assays have poor NPA for SARS-CoV-2 neutralization, making them imperfect proxies for neutralization.","version":"1.1","doi":"10.1101/2020.07.01.182220","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.07.119032","pub_date":"2020-7-02","title":"Molecular mechanism of SARS-CoV-2 components caused ARDS in murine model","abstract":"COVID-19 has become a major challenge to global health, and until now, no efficient antiviral agents have been developed. The SARS-CoV-2 infection is characterized by pulmonary and systemic inflammation in severe patients, and acute respiratory distress syndrome (ARDS) caused respiratory failure contributes to most mortalities. There is an urgent need for developing effective drugs and vaccines against SARS-CoV-2 and COVID-19 caused ARDS. However, most researchers cannot perform SARS-CoV-2 related researches due to lacking P3 or P4 facility. We developed a non-infectious, highly safety, time-saving SARS-CoV-2 components induced murine model to study the SARS-CoV-2 caused ARDS and cytokine storm syndrome (CSS). We also investigated mAbs and inhibitors which potentially neutralize the pro-inflammatory phenotype of COVID-19, and found that anti-IL-1\u03b1, anti-IL-6, anti-TNF\u03b1, anti-GM-CSF mAbs, p38 inhibitor, and JAK inhibitor partially relieved CSS. Besides, anti-IL-6, anti-TNF\u03b1, anti-GM-CSF mAbs and inhibitors of p38, ERK, and MPO somewhat reduced neutrophilic alveolitis in the lung. In all, we established the murine model mimic of COVID-19, opening a biosafety and less time-consuming avenue for clarifying the mechanism of ARDS and CSS in COVID-19 and developing the therapeutic drugs.","version":"1.4","doi":"10.1101/2020.06.07.119032","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.183020","pub_date":"2020-7-02","title":"6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro protease activities","abstract":"A recently emerged betacoronavirus, SARS-CoV-2, has led to a global health crisis that calls for the identification of effective therapeutics for COVID-19 disease. Coronavirus papain-like protease (PLpro) is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG) inhibits SARS-CoV-2 PLpro-catalyzed viral polyprotein cleavage and ISG15 deconjugation in cells and inhibits replication of SARS-CoV-2 in Vero-E6 cells and Calu3 cells at submicromolar levels. As a well-characterized FDA-approved orally delivered drug, 6-TG represents a promising therapeutic for COVID-19 and other emerging coronaviruses. A repurposed drug that targets an essential enzymatic activity of SARS-CoV-2 represents a promising COVID-19 therapeutic.","version":"1.1","doi":"10.1101/2020.07.01.183020","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.02.184481","pub_date":"2020-7-02","title":"Hidden genomic diversity of SARS-CoV-2: implications for qRT-PCR diagnostics and transmission","abstract":"The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 clades that have spread throughout the world. In this study, we investigated over 7,000 SARS-CoV-2 datasets to unveil both intrahost and interhost diversity. Our intrahost and interhost diversity analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights iSNV and SNP similarity, albeit with high variability in C>T changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of small indels fuel the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.07.02.184481","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.056838","pub_date":"2020-7-02","title":"STAT2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in SARS-CoV-2 infected hamsters","abstract":"Since the emergence of SARS-CoV-2 causing COVID-19, the world is being shaken to its core with numerous hospitalizations and hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that productive SARS-CoV-2 infection in the lungs of mice is limited and restricted by early type I interferon responses. In contrast, we show that Syrian hamsters are highly permissive to SARS- CoV-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. Moreover, we identify an exuberant innate immune response as a key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Finally, we assess SARS-CoV- 2-induced lung pathology in hamsters by micro-CT alike used in clinical practice. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.","version":"1.2","doi":"10.1101/2020.04.23.056838","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.174557","pub_date":"2020-7-02","title":"Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population","abstract":"In six of seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients, VH clonotypes, encoded by either immunoglobin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobin heavy joining (IGHJ)6, were identified in IgG1, IgA1, and IgA2 subtypes, with minimal mutations, and could be paired with diverse light chains, resulting in binding to the SARS-CoV-2 receptor-binding domain (RBD). Because most human antibodies against the RBD neutralized the virus by inhibiting host cell entry, we selected one of these clonotypes and demonstrated that it could potently inhibit viral replication. Interestingly, these VH clonotypes pre-existed in six of 10 healthy individuals, predominantly as IgM isotypes, which could explain the expeditious and stereotypic development of these clonotypes among SARS-CoV-2 patients.","version":"1.2","doi":"10.1101/2020.06.26.174557","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.183236","pub_date":"2020-7-02","title":"Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine","abstract":"Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.","version":"1.1","doi":"10.1101/2020.07.01.183236","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.100537","pub_date":"2020-7-02","title":"Binding of the SARS-CoV-2 Spike Protein to Glycans","abstract":"The pandemic of SARS-CoV-2 has caused a high number of deaths in the world. To combat it, it is necessary to develop a better understanding of how the virus infects host cells. Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (HS) and sialic acid-containing oligosaccharides. In this study, we examined and compared the binding of the subunits and spike (S) proteins of SARS-CoV-2 and SARS-CoV, MERS-CoV to these glycans. Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner, the length of HS is not a critical factor for the binding, and no binding with sialic acid residues was detected. Overall, this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.","version":"1.2","doi":"10.1101/2020.05.17.100537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.02.184093","pub_date":"2020-7-02","title":"Interleukin-3 is a predictive marker for severity and outcome during SARS-CoV-2 infections","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. Here, we report that low plasma interleukin-3 (IL-3) levels were associated with increased severity and mortality during SARS-CoV-2 infections. IL-3 promoted the recruitment of antiviral circulating plasmacytoid dendritic cells (pDCs) into the airways by stimulating CXCL12 secretion from pulmonary CD123+ epithelial cells. This study identifies IL-3 as a predictive disease marker and potential therapeutic target for SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2020.07.02.184093","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.182550","pub_date":"2020-7-01","title":"Early postmortem mapping of SARS-CoV-2 RNA in patients with COVID-19 and correlation to tissue damage","abstract":"Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body in correlation to tissue damage can help understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of viral RNA in 61 tissues and organs of 11 deceased patients with the diagnosis COVID-19. The autopsies were performed within the (very) early postmortem interval (mean: 5.6 hours) to avoid bias due to viral RNA and tissue degradation. Viral loads, blood levels of cytokines, prothrombotic factors as well as macro- and micro-morphology were correlated. Very high (> 104 copies/ml) viral loads were detected in the lungs of most patients and then correlated to severe tissue damage. Intact viral particles could be verified in the lung tissue by transmission electron microscopy. Viral loads in the lymph nodes were associated with a loss of follicular architecture. Viral RNA was detected throughout further extra-pulmonary tissues and organs without visible tissue damage. Inflammatory cytokines as well as the prothrombotic factors were elevated in all patients. In conclusion, the dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis of a maladaptive host response with viremia and multi-organ dysfunction.","version":"1.1","doi":"10.1101/2020.07.01.182550","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.182659","pub_date":"2020-7-01","title":"High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity","abstract":"A novel coronavirus (SARS-CoV-2) has emerged to a global pandemic and caused significant damages to public health. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides Ang II to control vasodilatation and permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we wonder how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ~3-10 fold when fluorogenic caspase-1 substrate and Bradykinin-analog peptides were used to characterize ACE2 activity. In addition, the enhancement was mediated by ACE2 binding of RBD domain of SARS-CoV-2 spike. These results highlighted the altered activity of ACE2 during SARS-CoV-2 infection and would shed new lights on the pathogenesis of COVID-19 and its complications for better treatments.","version":"1.1","doi":"10.1101/2020.07.01.182659","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.093583","pub_date":"2020-7-01","title":"Detection of Viral RNA Fragments in Human iPSC-Cardiomyocytes following Treatment with Extracellular Vesicles from SARS-CoV-2 Coding-Sequence-Overexpressing Lung Epithelial Cells","abstract":"The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a worldwide pandemic. Early data suggest that the prevalence and severity of COVID-19 appear to be higher among patients with underlying cardiovascular risk factors. Despite the expression of angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV-2 infection, in cardiomyocytes, there has been no conclusive evidence of direct viral infection although the presence of inflammation and viral genome within the hearts of COVID-19 patients have been reported. Here we transduced A549 lung epithelial cells with lentivirus overexpressing selected genes of the SARS-CoV-2. We then isolated extracellular vesicles (EVs) from the supernatant of A549 cells and detected the presence of viral RNA within the purified EVs. Importantly, we observed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were able to actively uptake these EVs and viral genes were subsequently detected in the cardiomyocytes. Accordingly, uptake of EVs containing viral genes led to an upregulation of inflammation-related genes in hiPSC-CMs. Thus, our findings indicate that SARS-CoV-2 RNA-containing EVs represent an indirect route of viral RNA entry into cardiomyocytes.","version":"1.3","doi":"10.1101/2020.05.14.093583","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.182709","pub_date":"2020-7-01","title":"Genetic architecture of host proteins interacting with SARS-CoV-2","abstract":"Strategies to develop therapeutics for SARS-CoV-2 infection may be informed by experimental identification of viral-host protein interactions in cellular assays and measurement of host response proteins in COVID-19 patients. Identification of genetic variants that influence the level or activity of these proteins in the host could enable rapid \u2018in silico\u2019 assessment in human genetic studies of their causal relevance as molecular targets for new or repurposed drugs to treat COVID-19. We integrated large-scale genomic and aptamer-based plasma proteomic data from 10,708 individuals to characterize the genetic architecture of 179 host proteins reported to interact with SARS-CoV-2 proteins or to participate in the host response to COVID-19. We identified 220 host DNA sequence variants acting in cis (MAF 0.01-49.9%) and explaining 0.3-70.9% of the variance of 97 of these proteins, including 45 with no previously known protein quantitative trait loci (pQTL) and 38 encoding current drug targets. Systematic characterization of pQTLs across the phenome identified protein-drug-disease links, evidence that putative viral interaction partners such as MARK3 affect immune response, and establish the first link between a recently reported variant for respiratory failure of COVID-19 patients at the ABO locus and hypercoagulation, i.e. maladaptive host response. Our results accelerate the evaluation and prioritization of new drug development programmes and repurposing of trials to prevent, treat or reduce adverse outcomes. Rapid sharing and dynamic and detailed interrogation of results is facilitated through an interactive webserver (https://omicscience.org/apps/covidpgwas/).","version":"1.1","doi":"10.1101/2020.07.01.182709","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.182741","pub_date":"2020-7-01","title":"SARS-CoV-2 reactive T cells in uninfected individuals are likely expanded by beta-coronaviruses","abstract":"The current pandemic is caused by the SARS-CoV-2 virus and large progress in understanding the pathology of the virus has been made since its emergence in late 2019. Several reports indicate short lasting immunity against endemic coronaviruses, which contrasts repeated reports that biobanked venous blood contains SARS-CoV-2 reactive T cells even before the outbreak in Wuhan. This suggests there exists a preformed T cell memory in individuals not exposed to the pandemic virus. Given the similarity of SARS-CoV-2 to other members of the Coronaviridae family, the endemic coronaviruses appear likely candidates to generate this T cell memory. However, given the apparent poor immunological memory created by the endemic coronaviruses, other immunity against other common pathogens might offer an alternative explanation. Here, we utilize a combination of epitope prediction and similarity to common human pathogens to identify potential sources of the SARS-CoV-2 T cell memory. We find that no common human virus, other than beta-coronaviruses, can explain the pre-existing SARS-CoV-2 reactive T cells in uninfected individuals. Our study suggests OC43 and HKU1 are the most likely pathogens giving rise to SARS-CoV-2 preformed immunity.","version":"1.1","doi":"10.1101/2020.07.01.182741","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.168013","pub_date":"2020-7-01","title":"Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA","abstract":"Accurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.","version":"1.2","doi":"10.1101/2020.06.24.168013","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167098","pub_date":"2020-7-01","title":"RAAS blockade, kidney disease, and expression of ACE2, the entry receptor for SARS-CoV-2, in kidney epithelial and endothelial cells","abstract":"SARS-CoV-2, the coronavirus that causes COVID-19, binds to angiotensin-converting enzyme 2 (ACE2) on human cells. Beyond the lung, COVID-19 impacts diverse tissues including the kidney. ACE2 is a key member of the Renin-Angiotensin-Aldosterone System (RAAS) which regulates blood pressure, largely through its effects on the kidney. RAAS blockers such as ACE inhibitors (ACEi) and Angiotensin Receptor Blockers (ARBs) are widely used therapies for hypertension, cardiovascular and chronic kidney diseases, and therefore, there is intense interest in their effect on ACE2 expression and its implications for SARS-CoV-2 pathogenicity. Here, we analyzed single-cell and single-nucleus RNA-seq of human kidney to interrogate the association of ACEi/ARB use with ACE2 expression in specific cell types. First, we performed an integrated analysis aggregating 176,421 cells across 49 donors, 8 studies and 8 centers, and adjusting for sex, age, donor and center effects, to assess the relationship of ACE2 with age and sex at baseline. We observed a statistically significant increase in ACE2 expression in tubular epithelial cells of the thin loop of Henle (tLoH) in males relative to females at younger ages, the trend reversing, and losing significance with older ages. ACE2 expression in tLoH increases with age in females, with an opposite, weak effect in males. In an independent cohort, we detected a statistically significant increase in ACE2 expression with ACEi/ARB use in epithelial cells of the proximal tubule and thick ascending limb, and endothelial cells, but the association was confounded in this small cohort by the underlying disease. Our study illuminates the dynamics of ACE2 expression in specific kidney cells, with implications for SARS-CoV-2 entry and pathogenicity.","version":"1.2","doi":"10.1101/2020.06.23.167098","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.181305","pub_date":"2020-7-01","title":"Assessment of Proton-Coupled Conformational Dynamics of SARS and MERS Coronavirus Papain-like Proteases: Implication for Designing Broad-Spectrum Antiviral Inhibitors","abstract":"Broad-spectrum antiviral drugs are urgently needed to stop the COVID-19 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines and their protonation/deprotonation modulates catalysis and conformational plasticity. Here we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using a newly developed GPU-accelerated implicit-solvent continuous constant pH molecular dynamics method with an asynchronous replica-exchange scheme. The calculated pKa\u2019s support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH, among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 conformational dynamics is coupled to the titration of C271/270, in agreement with the crystal structures of SARS-CoV-2 PLpro. Simulations also revealed that BL2 in MERS-CoV PLpro is very flexible, sampling both open and closed states despite the lack of an analogous cysteine. Our work provides a starting point for more detailed mechanistic studies to assist structure-based design of broad-spectrum inhibitors against CoV PLpros.","version":"1.1","doi":"10.1101/2020.06.30.181305","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.176537","pub_date":"2020-7-01","title":"Attenuated Subcomponent Vaccine Design Targeting the SARS-CoV-2 Nucleocapsid Phosphoprotein RNA Binding Domain: In silico analysis","abstract":"The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has previously never been identified with humans, thereby creating devastation in public health. The need for an effective vaccine to curb this pandemic cannot be overemphasized. In view of this, we, therefore, designed a subcomponent antigenic peptide vaccine targeting the N-terminal (NT) and C-terminal (CT) RNA binding domains of nucleocapsid protein that aid in viral replication. Promising antigenic B-cells and T cell epitopes were predicted using computational pipelines. The peptides \u201cRIRGGDGKMKDL\u201d and \u201cAFGRRGPEQTQGNFG\u201d were the B cell linear epitopes with good antigenic index and non-allergenic property. Two CD8+ and Three CD4+ T-cell epitopes were also selected considering their safe immunogenic profiling such as allergenicity, antigen level conservancy, antigenicity, peptide toxicity, and putative restrictions to a number of MHC-I and II alleles. With these selected epitopes, a non-allergenic chimeric peptide vaccine incapable of inducing a Type II hypersensitivity reaction was constructed. The molecular interaction between the toll-like receptor-5 (TLR5) which was triggered by the vaccine was analyzed by molecular docking and scrutinized using dynamics simulation. Finally, in silico cloning was performed to ensure the expression and translation efficiency of the vaccine, utilizing pET-28a vector. This research, therefore, provides a guide for experimental investigation and validation.","version":"1.1","doi":"10.1101/2020.06.30.176537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.150805","pub_date":"2020-7-01","title":"Identification of peptide candidate against COVID-19 through reverse vaccinology: An immunoinformatics approach","abstract":"Novel corona virus disease 2019 (COVID-19) is emerging as a pandemic situation and declared as a global health emergency by WHO. Due to lack of specific medicine and vaccine, viral infection has gained a frightening rate and created a devastating state across the globe. Here the authors have attempted to design epitope based potential peptide as a vaccine candidate using immunoinformatics approach. As of evidence from literatures, SARS-CoV-2 Spike protein is a key protein to initiate the viral infection within a host cell thus used here as a reasonable vaccine target. We have predicted a 9-mer peptide as representative of both B-cell and T-cell epitopic region along with suitable properties such as antigenic and non-allergenic. To its support, strong molecular interaction of the predicted peptide was also observed with MHC molecules and Toll Like receptors. The present study may helpful to step forward in the development of vaccine candidates against COVID-19.","version":"1.1","doi":"10.1101/2020.07.01.150805","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.181446","pub_date":"2020-7-01","title":"Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2","abstract":"The detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples. A virus panel based on the principle of biotinylated RNA-baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2 positive samples. Libraries generated from complex samples were sequenced via generic HTS and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted. The VirBaits set consists of 177,471 RNA-baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also point to the risk of carryover between samples. The VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector or research question. The risk of carryover needs to be taken into consideration. The application of the RNA-baits principle turned out to be user-friendly, and even non-experts (without sophisticated bioinformatics skills) can easily use the VirBait workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible without any problems as shown for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.30.181446","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.170324","pub_date":"2020-7-01","title":"Saliva sampling is an excellent option to increase the number of SARS CoV2 diagnostic tests in settings with supply shortages","abstract":"As part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for SARS CoV-2. Detection of the viral genome through RT-qPCR is the golden standard for this test, however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR test has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is cheaper and can be as efficient as the classical protocol that involves column purification of the viral RNA. In addition, it surpasses the need for swab sampling, decreases the risk of the healthcare personnel involved in this process, and accelerates the diagnostic procedure.","version":"1.2","doi":"10.1101/2020.06.24.170324","journal":"bioRxiv","score":null},{"id":"10.1101/2020.07.01.180828","pub_date":"2020-7-01","title":"MORT, a locus for apoptosis in the human immunodeficiency virus-type 1 antisense gene: implications for AIDS, Cancer, and Covid-19","abstract":"Apoptosis, or programmed cell death, is a fundamental requirement for life in multicellular organisms, including humans, and a mechanism to maintain homeostasis and prevent unwarranted cellular proliferations such as cancer. An antisense gene in HIV-1 (Hap) induces apoptosis in human cells. Apoptotic T cell death following HIV-1 infection leads to a compromised immune system and eventually AIDS (acquired immunodeficiency syndrome). A review of several studies that focused on long-term survivors of HIV-1 reveals that these survivors had deletion-mutations in Hap. A subset of these survivors changed course and experienced CD4+ T cell death and progression to AIDS. These individuals had virus that regained Hap gene sequence that had previously been deleted. Analysis of the changes in the genetic sequences with in vivo progression of the revertant HIV-1 virus allowed identification of a specific region in Hap we are calling MORT. MORT, in Hap RNA forms a primary microRNA-like structure. Potential human mRNAs targeted by MORT mi/siRNAs include gene/RNA sequences of X-linked inhibitor of apoptosis (XIAP), survivin, and apollon, along with many other human gene sites/RNAs. Thus MORT may be acting as an RNA antagonist to cellular IAPs thereby inducing apoptotic cell death. Surprisingly, additional potential MORT targets include viral sites in human SARS-CoV-2, including the protease, nsp5 RNA. Future uses for RNA therapy and a hypothesis for an HIV intrinsic mechanism utilizing MORT for viral anti-viral (or anti-microbial) and HIV anti-immune cell defense are proposed.","version":"1.1","doi":"10.1101/2020.07.01.180828","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.179192","pub_date":"2020-6-30","title":"Validation and Comparison of a Modified CDC Assay with two Commercially Available Assays for the Detection of SARS-CoV-2 in Respiratory Specimen","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has spread rapidly around the globe since it was first identified in December of 2019 in Wuhan, China. In a race to contain the infection, researchers and healthcare officials have developed several assays to help diagnose individuals with COVID-19. To help laboratories in deciding what assay to bring into testing lines, factors such as assay availability, cost, throughput, and TAT should be considered. Here we validated a modified version of the CDC assay and used it as a reference to evaluate the performance of the NeuMoDx\u2122 SARS-CoV-2 and DiaSorin Simplexa\u2122 Covid-19 Direct assays. In silico analysis and clinical sample testing showed that the primesr/probes designed by the CDC were specific to the SARS-CoV-2 as they accurately detected all reactive samples with an assay LoD of 200 copies/ml. The performance of the three assays were analyzed using 161 nasopharyngeal swabs specimen tested within 24 hours or 5 days from routine testing. A 100% agreement was observed between the commercial assays and the modified CDC SARS-CoV-2 assay. A deeper look at the Ct values showed no significant difference between NeuMoDx and the modified CDC SARS-CoV-2 assay, whereas DiaSorin had lower overall Ct values than the modified CDC SARS-CoV-2 assay. NeuMoDx and DiaSorin workflows were much easier to perform. NeuMoDx has the highest throughput and shortest TAT, whereas although the modified CDC SARS-CoV-2 assay has comparable throughput to DiaSorin, it has the longest hands-on time, and highest TAT.","version":"1.1","doi":"10.1101/2020.06.29.179192","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.03.962332","pub_date":"2020-6-30","title":"Multi-epitope vaccine design using an immunoinformatics approach for 2019 novel coronavirus (SARS-CoV-2)","abstract":"A new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. An in silico approach based on the available virus genome was applied to identify 19 high immunogenic B-cell epitopes and 499 human-leukocyte-antigen (HLA) restricted T-cell epitopes. Thirty multi-epitope peptide vaccines were designed by iNeo Suite, and manufactured by solid-phase synthesis. Docking analysis showed stable hydrogen bonds of epitopes with their corresponding HLA alleles. When four vaccine peptide candidates from the spike protein of SARS-CoV-2 were selected to immunize mice, a significantly larger amount of IgG in serum as well as an increase of CD19+ cells in ILNs was observed in peptide-immunized mice compared to the control mice. The ratio of IFN-\u03b3-secreting lymphocytes in CD4+ or CD8+ cells in the peptides-immunized mice were higher than that in the control mice. There were also a larger number of IFN-\u03b3-secreting T cells in spleen in the peptides-immunized mice. This study screened antigenic B-cell and T-cell epitopes in all encoded proteins of SARS-CoV-2, and further designed multi-epitope based peptide vaccine against viral structural proteins. The obtained vaccine peptides successfully elicited specific humoral and cellular immune responses in mice. Primate experiments and clinical trial are urgently required to validate the efficacy and safety of these vaccine peptides. So far, a new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. Different from the development approaches for traditional vaccines, the development of our peptide vaccine is faster and simpler. In this study, we performed an in silico approach to identify the antigenic B-cell epitopes and human-leukocyte-antigen (HLA) restricted T-cell epitopes, and designed a panel of multi-epitope peptide vaccines. The resulting SARS-CoV-2 multi-epitope peptide vaccine could elicit specific humoral and cellular immune responses in mice efficiently, displaying its great potential in our fight of COVID-19.","version":"1.2","doi":"10.1101/2020.03.03.962332","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178889","pub_date":"2020-6-30","title":"Discovery of Synergistic and Antagonistic Drug Combinations against SARS-CoV-2 In Vitro","abstract":"COVID-19 is undoubtedly the most impactful viral disease of the current century, afflicting millions worldwide. As yet, there is not an approved vaccine, as well as limited options from existing drugs for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro. Overall, we identified 16 synergistic and 8 antagonistic combinations, 4 of which were both synergistic and antagonistic in a dose-dependent manner. Among the 16 synergistic cases, combinations of nitazoxanide with three other compounds (remdesivir, amodiaquine and umifenovir) were the most notable, all exhibiting significant synergy against SARS-CoV-2. The combination of nitazoxanide, an FDA-approved drug, and remdesivir, FDA emergency use authorization for the treatment of COVID-19, demonstrate a strong synergistic interaction. Notably, the combination of remdesivir and hydroxychloroquine demonstrated strong antagonism. Overall, our results emphasize the importance of both drug repurposing and preclinical testing of drug combinations for potential therapeutic use against SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2020.06.29.178889","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169268","pub_date":"2020-6-30","title":"Comparative transcriptome analyses reveal genes associated with SARS-CoV-2 infection of human lung epithelial cells","abstract":"Understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) is a scientific priority for 2020. Various research groups are working toward development of vaccines and drugs, and many have published genomic and transcriptomic data related to this viral infection. The power inherent in publicly available data can be demonstrated via comparative transcriptome analyses. In the current study, we collected high-throughput gene expression data related to human lung epithelial cells infected with SARS-CoV-2 or other respiratory viruses (SARS, H1N1, rhinovirus, avian influenza, and Dhori) and compared the effect of these viruses on the human transcriptome. The analyses identified fifteen genes specifically expressed in cells transfected with SARS-CoV-2; these included CSF2 (colony-stimulating factor 2) and S100A8 and S100A9 (calcium-binding proteins), all of which are involved in lung/respiratory disorders. The analyses showed that genes involved in the Type1 interferon signaling pathway and the apoptosis process are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein-protein interaction analyses were consistent with a functional role of CSF2 in COVID-19 disease. In conclusion, our analysis has revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.","version":"1.2","doi":"10.1101/2020.06.24.169268","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.178897","pub_date":"2020-6-30","title":"A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 \u00c5 resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design. Fab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike. 3.1 \u00c5 resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12. 2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike. Our studies suggest a common epitope for FDG antibodies centered around glycan 709.","version":"1.1","doi":"10.1101/2020.06.30.178897","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178616","pub_date":"2020-6-30","title":"A Targeted Vaccine against COVID-19: S1-Fc Vaccine Targeting the Antigen-Presenting Cell Compartment Elicits Protection against SARS-CoV-2 Infection","abstract":"Vaccination efficacy is enhanced by targeting the antigen-presenting cell compartment. Here, we show that S1-Fc antigen delivery targeting the Fc\u03b3R+ antigen-presenting cell compartment elicits anti-SARS-CoV-2 S1-antigen specific IgG production in vivo exerting biologically functional and protective activity against live virus infection, assessed in a stringent experimental virus challenge assay in vitro. The S1-domain of the SARS-CoV-2 spike protein was genetically fused to a human immunoglobulin Fc moiety, which contributes to mediate S1-Fc cellular internalization by Fc\u03b3R+ antigen-presenting cells. Immediately upon administration intramuscularly, our novel vaccine candidate recombinant rS1-Fc homes to lymph nodes in vivo where Fc\u03b3R+ antigen-presenting cells reside. Seroconversion is achieved as early as day 7, mounting considerably increased levels of anti-S1 IgGs in vivo. Interestingly, immunization at elevated doses with non-expiring S1-Fc encoding dsDNA favors the education of a desired antigen-specific adaptive T cell response. However, low-dose immunization, safeguarding patient safety, using recombinant rS1-Fc, elicits a considerably elevated protection amplitude against live SARS-CoV-2 infection. Our promising findings on rS1-Fc protein immunization prompted us to further develop an affordable and safe product for delivery to our communities in need for COVID-19 vaccinations.","version":"1.1","doi":"10.1101/2020.06.29.178616","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178509","pub_date":"2020-6-30","title":"SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice","abstract":"The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).","version":"1.1","doi":"10.1101/2020.06.29.178509","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178459","pub_date":"2020-6-30","title":"Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat (R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.","version":"1.1","doi":"10.1101/2020.06.29.178459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.172924","pub_date":"2020-6-30","title":"Comparative genomics provides an operational classification system and reveals early emergence and biased spatio-temporal distribution of SARS-CoV-2","abstract":"Effective systems for the analysis of molecular data are of fundamental importance for real-time monitoring of the spread of infectious diseases and the study of pathogen evolution. While the Nextstrain and GISAID portals offer widely used systems for the classification of SARS-CoV-2 genomes, both present relevant limitations. Here we propose a highly reproducible method for the systematic classification of SARS-CoV-2 viral types. To demonstrate the validity of our approach, we conduct an extensive comparative genomic analysis of more than 20,000 SARS-CoV-2 genomes. Our classification system delineates 12 clusters and 4 super-clusters in SARS-CoV-2, with a highly biased spatio-temporal distribution worldwide, and provides important observations concerning the evolutionary processes associated with the emergence of novel viral types. Based on the estimates of SARS-CoV-2 evolutionary rate and genetic distances of genomes of the early pandemic phase, we infer that SARS-CoV-2 could have been circulating in humans since August-November 2019. The observed pattern of genomic variability is remarkably similar between all clusters and super-clusters, being UTRs and the s2m element, a highly conserved secondary structure element, the most variable genomic regions. While several polymorphic sites that are specific to one or more clusters were predicted to be under positive or negative selection, overall, our analyses also suggest that the emergence of novel genome types is unlikely to be driven by widespread convergent evolution and independent fixation of advantageous substitutions. While, in the absence of rigorous experimental validation, several questions concerning the evolutionary processes and the phenotypic characteristics (increased/decreased virulence) remain open, we believe that the approach outlined in this study can be of relevance for the tracking and functional characterization of different types of SARS-CoV-2 genomes.","version":"1.2","doi":"10.1101/2020.06.26.172924","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.120105","pub_date":"2020-6-30","title":"Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5\u2019UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3\u2019UTR. Finally, five variants destabilize structured elements within the 3\u2019UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.","version":"1.2","doi":"10.1101/2020.05.27.120105","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.20142349","pub_date":"2020-06-30","title":"A handheld point-of-care system for rapid detection of SARS-CoV-2 in under 20 minutes","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (&lt; 20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 90.55% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 \u00b1 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n=40), showing average detection times of 12.89 \u00b1 2.59 min for positive samples (n=34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geo-localization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.</jats:p>\n                <jats:sec>\n                  <jats:title>One Sentence Summary</jats:title>\n                  <jats:p>We demonstrate isothermal detection of SARS-CoV-2 in under 20 minutes from extracted RNA samples with a handheld Lab-on-Chip platform.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.06.29.20142349","journal":"medRxiv","score":null},{"id":"10.1101/2020.04.24.058933","pub_date":"2020-6-30","title":"Comprehensive evolution and molecular characteristics of a large number of SARS-CoV-2 genomes revealed its epidemic trend and possible origins","abstract":"To reveal epidemic trend and possible origins of SARS-CoV-2 by exploring its evolution and molecular characteristics based on a large number of genomes since it has infected millions of people and spread quickly all over the world. Various evolution analysis methods were employed. The estimated Ka/Ks ratio of SARS-CoV-2 is 1.008 or 1.094 based on 622 or 3624 SARS-CoV-2 genomes, and the time to the most recent common ancestor (tMRCA) was inferred in late September 2019. Further 9 key specific sites of highly linkage and four major haplotypes H1, H2, H3 and H4 were found. The Ka/Ks, detected population size and development trends of each major haplotype showed H3 and H4 subgroups were going through a purify evolution and almost disappeared after detection, indicating H3 and H4 might have existed for a long time, while H1 and H2 subgroups were going through a near neutral or neutral evolution and globally increased with time. Notably the frequency of H1 was generally high in Europe and correlated to death rate (r>0.37). In this study, the evolution and molecular characteristics of more than 16000 genomic sequences provided a new perspective for revealing epidemiology of SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.04.24.058933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178707","pub_date":"2020-6-30","title":"Anti-frameshifting ligand active against SARS coronavirus-2 is resistant to natural mutations of the frameshift-stimulatory pseudoknot","abstract":"The coronavirus SARS-CoV-2 causing the COVID-19 pandemic uses \u22121 programmed ribosomal frameshifting (\u22121 PRF) to control the expression levels of key viral proteins. Because modulating \u22121 PRF can attenuate viral propagation, ligands binding to the viral RNA pseudoknot that stimulates \u22121 PRF may prove useful as therapeutics. Mutations in the pseudoknot have been observed over the course of the pandemic, but how they affect \u22121 PRF and the activity of inhibitors is unknown. Cataloguing natural mutations in all parts of the SARS-CoV-2 pseudoknot, we studied a panel of 6 mutations in key structural regions. Most mutations left the \u22121 PRF efficiency unchanged, even when base-pairing was disrupted, but one led to a remarkable three-fold decrease, suggesting that SARS-CoV-2 propagation may be less sensitive to modulation of \u22121 PRF efficiency than some other viruses. Examining the effects of one of the few small-molecule ligands known to suppress \u22121 PRF significantly in SARS-CoV, we found that it did so by similar amounts in all SARS-CoV-2 mutants tested, regardless of the basal \u22121 PRF efficiency, indicating that the activity of anti-frameshifting ligands can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of \u22121 PRF.","version":"1.1","doi":"10.1101/2020.06.29.178707","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.177097","pub_date":"2020-6-30","title":"Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM","abstract":"The recent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread pose a global health emergency. The trimeric spike (S) glycoprotein interacts with its receptor human ACE2 to mediate viral entry into host-cells. Here we present cryo-EM structures of an uncharacterized tightly closed SARS-CoV-2 S-trimer and the ACE2-bound-S-trimer at 2.7-\u00c5 and 3.8-\u00c5-resolution, respectively. The tightly closed S-trimer with inactivated fusion peptide may represent the ground prefusion state. ACE2 binding to the up receptor-binding domain (RBD) within S-trimer triggers continuous swing-motions of ACE2-RBD, resulting in conformational dynamics of S1 subunits. Noteworthy, SARS-CoV-2 S-trimer appears much more sensitive to ACE2-receptor than SARS-CoV S-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and residue Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2, and provided structural basis of the spike D614G-mutation induced enhanced infectivity. Our findings offer a thorough picture on the mechanism of ACE2-induced conformational transitions of S-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding.","version":"1.1","doi":"10.1101/2020.06.30.177097","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.179176","pub_date":"2020-6-30","title":"A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR","abstract":"The SARS-CoV-2 pandemic has created an urgent need for large amounts of diagnostic tests to detect viral RNA, which commercial suppliers are increasingly unable to deliver. In addition to the lack of availability, the current methods do not always fully inactivate the virus. Together, this calls for the development of safer methods for extraction and detection of viral RNA from patient samples that utilise readily available reagents and equipment present in most standard laboratories. We present a rapid and straightforward RNA extraction protocol for inactivating the SARS-CoV-2 virus that uses standard lab reagents. This protocol expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method circumvents the need for commercial RNA purification kits, takes about 30 minutes from swab to PCR-ready viral RNA, and enables downstream detection of SARS-CoV-2 by RT-qPCR with very high sensitivity (~4 viral RNA copies per RT-qPCR). In summary, we present a rapid, safe and sensitive method for high-throughput detection of SARS-CoV-2, that can be conducted in any laboratory equipped with a qPCR machine.","version":"1.1","doi":"10.1101/2020.06.29.179176","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.170688","pub_date":"2020-6-30","title":"Molecular Evolution of SARS-CoV-2 Structural Genes: Evidence of Positive Selection in Spike Glycoprotein","abstract":"SARS-CoV-2 caused a global pandemic in early 2020 and has resulted in more than 8,000,000 infections as well as 430,000 deaths in the world so far. Four structural proteins, envelope (E), membrane (M), nucleocapsid (N) and spike (S) glycoprotein, play a key role in controlling the entry into human cells and virion assembly of SARS-CoV-2. However, how these genes evolve during its human to human transmission is largely unknown. In this study, we screened and analyzed roughly 3090 SARS-CoV-2 isolates from GenBank database. The distribution of the four gene alleles is determined:16 for E, 40 for M, 131 for N and 173 for S genes. Phylogenetic analysis shows that global SARS-CoV-2 isolates can be clustered into three to four major clades based on the protein sequences of these genes. Intragenic recombination event isn\u2019t detected among different alleles. However, purifying selection has conducted on the evolution of these genes. By analyzing full genomic sequences of these alleles using codon-substitution models (M8, M3 and M2a) and likelihood ratio tests (LRTs) of codeML package, it reveals that codon 614 of S glycoprotein has subjected to strong positive selection pressure and a persistent D614G mutation is identified. The definitive positive selection of D614G mutation is further confirmed by internal fixed effects likelihood (IFEL) and Evolutionary Fingerprinting methods implemented in Hyphy package. In addition, another potential positive selection site at codon 5 in the signal sequence of the S protein is also identified. The allele containing D614G mutation has undergone significant expansion during SARS-CoV-2 global pandemic, implying a better adaptability of isolates with the mutation. However, L5F allele expansion is relatively restricted. The D614G mutation is located at the subdomain 2 (SD2) of C-terminal portion (CTP) of the S1 subunit. Protein structural modeling shows that the D614G mutation may cause the disruption of salt bridge among S protein monomers increase their flexibility, and in turn promote receptor binding domain (RBD) opening, virus attachment and entry into host cells. Located at the signal sequence of S protein as it is, L5F mutation may facilitate the protein folding, assembly, and secretion of the virus. This is the first evidence of positive Darwinian selection in the spike gene of SARS-CoV-2, which contributes to a better understanding of the adaptive mechanism of this virus and help to provide insights for developing novel therapeutic approaches as well as effective vaccines by targeting on mutation sites.","version":"1.2","doi":"10.1101/2020.06.25.170688","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.179663","pub_date":"2020-6-30","title":"Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex","abstract":"SARS-CoV-2, a highly transmittable pathogen has infected over 3.8 million people around the globe. The spike glycoprotein of SARS-CoV-2 engages host ACE2 for adhesion, TMPRSS2 for activation and entry. With the aid of whole-exome sequencing, we report a variant rs12329760 in TMPRSS2 gene and its mutant V160M, which might impede viral entry. Furthermore, we identified TMPRSS2 cleavage sites in S2 domain of spike glycoprotein and report the structure of TMPRSS2 in complex with spike glycoprotein. We also report the structures of protease inhibitors in complex with TMPRSS2, which could hamper the interaction with spike protein. These findings advance our understanding on the role of TMPRSS2 and in the development of potential therapeutics.","version":"1.1","doi":"10.1101/2020.06.30.179663","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167254","pub_date":"2020-6-30","title":"ViralLink: An integrated workflow to investigate the effect of SARS-CoV-2 on intracellular signalling and regulatory pathways","abstract":"The SARS-CoV-2 pandemic of 2020 has mobilised scientists around the globe to research all aspects of the coronavirus virus and its infection. For fruitful and rapid investigation of viral pathomechanisms, a collaborative and interdisciplinary approach is required. Therefore, we have developed ViralLink: a systems biology workflow which reconstructs and analyses networks representing the effect of viruses on intracellular signalling. These networks trace the flow of signal from intracellular viral proteins through their human binding proteins and downstream signalling pathways, ending with transcription factors regulating genes differentially expressed upon viral exposure. In this way, the workflow provides a mechanistic insight from previously identified knowledge of virally infected cells. By default, the workflow is set up to analyse the intracellular effects of SARS-CoV-2, requiring only transcriptomics counts data as input from the user: thus, encouraging and enabling rapid multidisciplinary research. However, the wide-ranging applicability and modularity of the workflow facilitates customisation of viral context, a priori interactions and analysis methods. Through a case study of SARS-CoV-2 infected bronchial/tracheal epithelial cells, we evidence the functionality of the workflow and its ability to identify key pathways and proteins in the cellular response to infection. The application of ViralLink to different viral infections in a cell-type specific manner using different available transcriptomics datasets will uncover key mechanisms in viral pathogenesis. The workflow is available on GitHub (https://github.com/korcsmarosgroup/ViralLink) in an easily accessible Python wrapper script, or as customisable modular R and Python scripts. Collaborative and multidisciplinary science provides increased value for experimental datasets and speeds the process of discovery. Such ways of working are especially important at present due to the urgency of the SARS-CoV-2 pandemic. Here, we present a systems biology workflow which models the effect of viral proteins on the infected host cell, to aid collaborative and multidisciplinary research. Through integration of gene expression datasets with context-specific and context-agnostic molecular interaction datasets, the workflow can be easily applied to different datasets as they are made available. Application to diverse SARS-CoV-2 datasets will increase our understanding of the mechanistic details of the infection at a cell type specific level, aid drug target discovery and help explain the variety of clinical manifestations of the infection.","version":"1.2","doi":"10.1101/2020.06.23.167254","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.28.013276","pub_date":"2020-6-30","title":"Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins","abstract":"The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunits. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, primarily of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.","version":"1.3","doi":"10.1101/2020.03.28.013276","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165936","pub_date":"2020-6-30","title":"Unsupervised cluster analysis of SARS-CoV-2 genomes indicates that recent (June 2020) cases in Beijing are from a genetic subgroup that consists of mostly European and South(east) Asian samples, of which the latter are the most recent","abstract":"Research efforts of the ongoing SARS-CoV-2 pandemic have focused on viral genome sequence analysis to understand how the virus spread across the globe. Here, we assess three recently identified SARS-CoV-2 genomes in Beijing from June 2020 and attempt to determine the origin of these genomes, made available in the GISAID database. The database contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Including the three new samples and excluding samples with missing annotations, we analyzed 7, 643 SARS-CoV-2 genomes. Using principal component analysis computed on a similarity matrix that compares all pairs of the SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index, we find that the newly discovered virus genomes from Beijing are in a genetic cluster that consists mostly of cases from Europe and South(east) Asia. The sequences of the new cases are most related to virus genomes from a small number of cases from China (March 2020), cases from Europe (February to early May 2020), and cases from South(east) Asia (May to June 2020). These findings could suggest that the original cases of this genetic cluster originated from China in March 2020 and were re-introduced to China by transmissions from samples from South(east) Asia between April and June 2020.","version":"1.1","doi":"10.1101/2020.06.22.165936","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.30.180380","pub_date":"2020-6-30","title":"A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic","abstract":"Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.","version":"1.1","doi":"10.1101/2020.06.30.180380","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.179184","pub_date":"2020-6-30","title":"Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2","abstract":"RT-PCR testing is crucial in the diagnostic of SARS-CoV-2 infection. The use of reliable and comparable PCR assays is a cornerstone to allow use of different PCR assays depending on the local equipment. In this work, we provide a comparison of the Cobas\u00ae (Roche) and the RealStar\u00ae assay (Altona). Assessment of the two assays was performed prospectively in three reference Parisians hospitals, using 170 clinical samples. They were tested with the Cobas\u00ae assay, selected to obtain a distribution of cycle threshold (Ct) as large as possible, and tested with the RealStar assay with three largely available extraction platforms: QIAsymphony (Qiagen), MagNAPure (Roche) and NucliSENS-easyMag (BioM\u00e9rieux). Overall, the agreement (positive for at least one gene) was 76%. This rate differed considerably depending on the Cobas Ct values for gene E: below 35 (n = 91), the concordance was 99%. Regarding the positive Ct values, linear regression analysis showed a determination correlation (R2) of 0.88 and the Deming regression line revealed a strong correlation with a slope of 1.023 and an intercept of -3.9. Bland-Altman analysis showed that the mean difference (Cobas\u00ae minus RealStar\u00ae) was + 3.3 Ct, with a SD of + 2.3 Ct. In this comparison, both RealStar\u00ae and Cobas\u00ae assays provided comparable qualitative results and a high correlation when both tests were positive. Discrepancies exist after 35 Ct and varied depending on the extraction system used for the RealStar\u00ae assay, probably due to a low viral load close to the detection limit of both assays.","version":"1.1","doi":"10.1101/2020.06.29.179184","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.173765","pub_date":"2020-6-30","title":"Glycans on the SARS-CoV-2 Spike Control the Receptor Binding Domain Conformation","abstract":"The glycan shield of the beta-coronavirus (\u03b2-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) \u2018up\u2019 and \u2018down\u2019 state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the \u2018up\u2019 state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in \u2018up\u2019 state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.","version":"1.2","doi":"10.1101/2020.06.26.173765","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.072942","pub_date":"2020-6-30","title":"Whole Genome Comparison of Pakistani Corona Virus with Chinese and US Strains along with its Predictive Severity of COVID-19","abstract":"Recently submitted 784 SARS-nCoV2 whole genome sequences from NCBI Virus database were taken for constructing phylogenetic tree to look into their similarities. Pakistani strain MT240479 (Gilgit1-Pak) was found in close proximity to MT184913 (CruiseA-USA), while the second Pakistani strain MT262993 (Manga-Pak) was neighboring to MT039887 (WI-USA) strain in the constructed cladogram in this article. Afterward, four whole genome SARS-nCoV2 strain sequences were taken for variant calling analysis, those who appeared nearest relative in the earlier cladogram constructed a week time ago. Among those two Pakistani strains each of 29,836 bases were compared against MT263429 from (WI-USA) of 29,889 bases and MT259229 (Wuhan-China) of 29,864 bases. We identified 31 variants in both Pakistani strains, (Manga-Pak vs USA=2del+7SNPs, Manga-Pak vs Chinese=2del+2SNPs, Gilgit1-Pak vs USA=10SNPs, Gilgit1-Pak vs Chinese=8SNPs), which caused alteration in ORF1ab, ORF1a and N genes with having functions of viral replication and translation, host innate immunity and viral capsid formation respectively. These novel variants are assumed to be liable for low mortality rate in Pakistan with 385 as compared to USA with 63,871 and China with 4,633 deaths by May 01, 2020. However functional effects of these variants need further confirmatory studies. Moreover, mutated N & ORF1a proteins in Pakistani strains were also analyzed by 3D structure modelling, which give another dimension of comparing these alterations at amino acid level. In a nutshell, these novel variants are assumed to be linked with reduced mortality of COVID-19 in Pakistan along with other influencing factors, these novel variants would also be useful to understand the virulence of this virus and to develop indigenous vaccines and therapeutics.","version":"1.3","doi":"10.1101/2020.05.01.072942","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.174623","pub_date":"2020-6-29","title":"SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery","abstract":"Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.","version":"1.1","doi":"10.1101/2020.06.29.174623","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.171173","pub_date":"2020-6-29","title":"An in vitro assessment of anti-SARS-CoV-2 activity of oral preparations of iodine complexes (RENESSANS)","abstract":"Since the emergence of CoVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In Vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule) of an Iodine-complex (Renessans). A monolayer of vero cells were exposed to SARS-CoV-2 in the presence and absence of different concentrations (equivalent to 50, 05 and 0.5 \u03bcg/ml of I2) of Renessans. Anti-SARS-CoV-2 activity of each of the formulation was assessed in the form of cell survival, SARS-CoV-2-specific cytopathic effect (CPE) and genome quantization. With varying concentrations of syrup and capsule, a varying rate of inhibition of CPE, cells survival and virus replication was observed. Compared to 0.5 \u03bcg/ml concentration of Renessans syrup, 5 and 50 \u03bcg/ml showed comparable results where there was a 100% cell survival, no CPEs and a negligible viral replication (\u0394CT= 0.11 and 0.13, respectively). This study indicates that Renessans, containing iodine, may have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.","version":"1.1","doi":"10.1101/2020.06.29.171173","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.177030","pub_date":"2020-6-29","title":"A mobile genetic element in the SARS-CoV-2 genome is shared with multiple insect species","abstract":"Unprecedented quantities of sequence data have been generated from the newly emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of COVID-19. We document here the presence of s2m, a highly conserved, mobile genetic element with unknown function, in both the SARS-CoV-2 genome and a large number of insect genomes. Although s2m is not universally present among coronaviruses and appears to undergo horizontal transfer, the high sequence conservation and universal presence of s2m among isolates of SARS-CoV-2 indicate that, when present, the element is essential for viral function.","version":"1.1","doi":"10.1101/2020.06.29.177030","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.172510","pub_date":"2020-6-29","title":"Nanotrap\u00ae particles improve detection of SARS-CoV-2 for pooled sample methods, extraction-free saliva methods, and extraction-free transport medium methods","abstract":"Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from transport medium and saliva using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus prior to RNA extraction, thus significantly improving detection of the virus using a real-time RT-PCR assay across a range of viral titers, from 100 to 1,000,000 viral copies/mL; in particular, detection of virus in low viral load samples is enhanced when using the method coupled with the IDT 2019-nCoV CDC EUA Kit. This method is compatible with commercially available nucleic acid extraction kits, as well with a simple heat and detergent method. Using transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2 using either the Abbott RealTime SARS-CoV-2 EUA Test (n=14) or the Cepheid Xpert Xpress SARS-CoV-2 EUA Test (n=35), we demonstrate that our method not only correctly identifies all positive samples (n = 17) but also significantly improves detection of the virus in low viral load samples. The average improvement in cycle threshold (Ct) value as measured with the IDT 2019-nCoV CDC EUA Kit was 3.1; n = 10. Finally, to demonstrate that the method could potentially be used to enable pooled testing, we spiked infectious virus or a confirmed positive diagnostic remnant sample into 5 mL and 10 mL of negative transport medium and observed significant improvement in the detection of the virus from those larger sample volumes.","version":"1.2","doi":"10.1101/2020.06.25.172510","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.177204","pub_date":"2020-6-29","title":"Perversely expressed long noncoding RNAs can alter host response and viral proliferation in SARS-CoV-2 infection","abstract":"Since December 2019, the world is experiencing an unprecedented crisis due to a novel coronavirus, SARS-CoV-2. Owing to poor understanding of pathogenicity, the virus is eluding treatment and complicating recovery. Regulatory roles of long non-coding RNAs (lncRNAs) during viral infection and associated antagonism of host antiviral immune responses has become more evident in last decade. To elucidate possible functions of lncRNAs in the COVID-19 pathobiology, we have utilized RNA-seq dataset of SARS-CoV-2 infected lung epithelial cells. Our analyses uncover 21 differentially expressed lncRNAs whose functions are broadly involved in cell survival and regulation of gene expression. By network enrichment analysis we find that these lncRNAs can directly interact with differentially expressed protein-coding genes ADAR, EDN1, KYNU, MALL, TLR2 and YWHAG; and also AKAP8L, EXOSC5, GDF15, HECTD1, LARP4B, LARP7, MIPOL1, UPF1, MOV10 and PRKAR2A, host genes that interact with SARS-CoV-2 proteins. These genes are involved in cellular signaling, metabolism, immune response and RNA homeostasis. Since lncRNAs have been known to sponge microRNAs and protect expression of upregulated genes, we also identified 9 microRNAs that are induced in viral infections; however, some lncRNAs are able to block their usual suppressive effect on overexpressed genes and consequently contribute to host defense and cell survival. Our investigation determines that deregulated lncRNAs in SARS-CoV-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome and this information could drive the search for novel RNA therapeutics as a treatment option.","version":"1.1","doi":"10.1101/2020.06.29.177204","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.174672","pub_date":"2020-6-29","title":"Role of Anti-SARS-CoV-2 antibodies in different cohorts: Can they provide clues for appropriate patient triaging?","abstract":"The emergence of coronavirus disease 2019 (COVID-19) has become a major global health crisis. Currently, diagnosis is based on molecular techniques, which detect the viral nucleic acids when present at detectable levels. The serum IgG response against SARS-CoV-2 was examined by using an ELISA-based assay. Serum samples, along with nasopharyngeal specimens were collected from various cohorts and analyzed by ELISA and rRT-PCR, respectively. A total of 167 serum samples were tested for serum IgG antibodies against SARS-CoV-2 in outpatient cohorts, 15 (8.9%) were positive by rRT-PCR and the remaining 152 (91%) were negative. We used these data to generate two different assay cutoffs for serum IgG assay and investigated percent concordance with rRT-PCR test results. The emergency department data revealed, out of 151 nasopharyngeal swabs, 4 (2.6%) were positive by rRT-PCR and 18 (11.9%) were positive for serum IgG assay. Among the 18 patients that were positive for serum IgG, 13 (72.2%) exhibited 1-3 symptoms of COVID-19 and 5 (27.7%) patients did not present with any COVID-19 related symptoms, per CDC criteria. All 4 (100%) patients that were positive by rRT-PCR had symptoms of COVID-19 disease. A longitudinal study from the inpatient population suggested there was a sharp increase in the serum IgG titers in 5 patients, a moderate increase in 1 patient and a plateau in 3 patients. Sero-prevalence of COVID-19 disease in pre-procedure patients was 5.5%. Our findings suggest serological tests can be used for appropriate patient triaging when performed as an adjunct to existing molecular testing.","version":"1.1","doi":"10.1101/2020.06.26.174672","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.175844","pub_date":"2020-6-29","title":"SARS-CoV-2 Spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity","abstract":"There is a well-known and established link between high lipopolysaccharide (LPS) levels in blood and the metabolic syndrome (MS). MS is a risk factor for developing severe COVID-19 and acute respiratory distress syndrome (ARDS). Here we define an interaction between SARS-CoV-2 Spike (S) protein and LPS and its link to aggravated inflammation in vitro and in vivo. Electrophoresis under native conditions demonstrated that SARS-CoV-2 S protein binds to Escherichia coli LPS, forming high molecular weight aggregates. Microscale thermophoresis analysis further defined the interaction, having a KD of ~47 nM, similar to the observed affinity between LPS and the human receptor CD14. Moreover, S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-\u03baB) and cytokine responses in monocytic THP-1 cells and human blood, respectively. In an experimental model of localized inflammation, employing NF-\u03baB reporter mice and in vivo bioimaging, S protein in conjunction with LPS significantly increased the inflammatory response when compared with S protein and LPS alone. Apart from providing information on LPS as a ligand for S protein, our results are of relevance for studies on comorbidities involving bacterial endotoxins, such as the MS, or co-existing acute and chronic infections in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.06.29.175844","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.174888","pub_date":"2020-6-29","title":"Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19","abstract":"SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. We systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in a large cohort of unexposed individuals as well as exposed family members and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative family members and individuals with a history of asymptomatic or mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits robust memory T cell responses akin to those observed in the context of successful vaccines, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19 also in seronegative individuals.","version":"1.1","doi":"10.1101/2020.06.29.174888","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.174896","pub_date":"2020-6-29","title":"Rational Design of the Remdesivir Binding Site in the RNA-dependent RNA Polymerase of SARS-CoV-2: Implications for Potential Resistance","abstract":"SARS-CoV-2 is rapidly evolving with the continuous emergence of new mutations. There is no specific antiviral therapy for COVID-19, and the use of Remdesivir for treating COVID-19 will likely continue before clinical trials are completed. Due to the lengthening pandemic and evolving nature of the virus, predicting potential residues prone to mutations is crucial for the management of Remdesivir resistance. We used a rational ligand-based interface design complemented with mutational mapping to generate a total of 100,000 mutations and provide insight into the functional outcome of mutations in the Remdesivir-binding site in nsp12. After designing 56 residues in the Remdesivir binding site of nsp12, the designs retained 96-98% sequence identity, which suggests that SARS-CoV-2 attains resistance and develops further infectivity with very few mutations in the nsp12. We also identified affinity-attenuating Remdesivir binding designs of nsp12. Several mutants acquired decreased binding affinity with Remdesivir, which suggested drug resistance. These hotspot residues had a higher probability of undergoing selective mutations in the future to develop Remdesivir and related drug-based resistance. A comparison of 21 nsp12 Remdesivir-bound designs to the 13 EIDD-2801-bound nsp12 designs suggested that EIDD-2801 would be more effective in preventing the emergence of resistant mutations and against Remdesivir-resistance strains due to the restricted mutational landscape. Combined with the availability of more genomic data, our information on mutation repertoires is critical to guide scientists to rational structure-based drug discovery. Knowledge of the potential residues prone to mutation improves our understanding and management of drug resistance and disease pathogenesis.","version":"1.1","doi":"10.1101/2020.06.27.174896","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.016931","pub_date":"2020-6-29","title":"Prioritization of SARS-CoV-2 epitopes using a pan-HLA and global population inference approach","abstract":"SARS-CoV-2 T cell response assessment and vaccine development may benefit from an approach that considers the global landscape of the human leukocyte antigen (HLA) proteins. We predicted the binding affinity between 9-mer and 15-mer peptides from the SARS-CoV-2 peptidome for 9,360 class I and 8,445 class II HLA alleles, respectively. We identified 368,145 unique combinations of peptide-HLA complexes (pMHCs) with a predicted binding affinity less than 500nM, and observed significant overlap between class I and II predicted pMHCs. Using simulated populations derived from worldwide HLA frequency data, we identified sets of epitopes predicted in at least 90% of the population in 57 countries. We also developed a method to prioritize pMHCs for specific populations. Collectively, this public dataset and accessible user interface (Shiny app: https://rstudio-connect.parkerici.org/content/13/) can be used to explore the SARS-CoV-2 epitope landscape in the context of diverse HLA types across global populations.","version":"1.2","doi":"10.1101/2020.03.30.016931","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.174953","pub_date":"2020-6-29","title":"SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damage and constitutes an antiviral target","abstract":"Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of the virus\u2019 excessively damaging abilities remains unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is sufficient to cause acute respiratory distress syndrome (ARDS)-like damage in vitro and in vivo. Overexpression of 2-E protein induced rapid pyroptosis-like cell death in various susceptible cells and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damage in lung and spleen. Overexpressed 2-E protein formed cation channels in host cell membranes, eventually leading to membrane rupture. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent protective effects against the 2-E-induced damage both in vitro and in vivo. Importantly, their channel inhibition, cell protection and antiviral activities were positively correlated with each other, supporting 2-E is a promising drug target against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.27.174953","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.177238","pub_date":"2020-6-29","title":"Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline","abstract":"In order to explore nonsynonymous mutations and deletions in the spike (S) protein of SARS-CoV-2, we comprehensively analyzed 35,750 complete S protein gene sequences from across six continents and five climate zones around the world, as documented in the GISAID database as of June 24th, 2020. Through a custom Python-based pipeline for analyzing mutations, we identified 27,801 (77.77 % of spike sequences) mutated strains compared to Wuhan-Hu-1 strain. 84.40% of these strains had only single amino-acid (aa) substitution mutations, but an outlier strain from Bosnia and Herzegovina (EPI_ISL_463893) was found to possess six aa substitutions. The D614G variant of the major G clade was found to be predominant across circulating strains in all climates. We also identified 988 unique aa substitution mutations distributed across 660 positions within the spike protein, with eleven sites showing high variability \u2013 these sites had four types of aa variations at each position. Besides, 17 in-frame deletions at four major regions (three in N-terminal domain and one just downstream of the RBD) may have possible impact on attenuation. Moreover, the mutational frequency differed significantly (p= 0.003, Kruskal\u2013Wallis test) among the SARS-CoV-2 strains worldwide. This study presents a fast and accurate pipeline for identifying nonsynonymous mutations and deletions from large dataset for any particular protein coding sequence and presents this S protein data as representative analysis. By using separate multi-sequence alignment with MAFFT, removing ambiguous sequences and in-frame stop codons, and utilizing pairwise alignment, this method can derive nonsynonymus mutations (Reference:Position:Strain). We believe this will aid in the surveillance of any proteins encoded by SARS-CoV-2, and will prove to be crucial in tracking the ever-increasing variation of many other divergent RNA viruses in the future.","version":"1.1","doi":"10.1101/2020.06.29.177238","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.177154","pub_date":"2020-6-29","title":"Tissue distribution of ACE2 protein in Syrian golden hamster (Mesocricetus auratus) and its possible implications in SARS-CoV-2 related studies","abstract":"Recently, the Syrian golden hamster (Mesocricetus auratus) has been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers optimal use of these models. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin\u25a1converting enzyme 2 (ACE2), a proven functional receptor for SARS-CoV-2 in different organs of the hamster. We have adapted immunoblot analysis, immunohistochemistry, and immunofluorescence analysis techniques to evaluate the ACE2 expression pattern in different tissues of the Syrian golden hamster. We found that kidney, small intestine, esophagus, tongue, brain, and liver express ACE2. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections for ACE2 showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine (caecum, colon, and rectum) were negative for ACE2 expression. Together, our findings corroborate some of the earlier reports related to ACE2 expression pattern in human tissues and also contradicts some others. We believe that the findings of this study will enable the appropriate use of the Syrian golden hamster to carryout SARS-CoV-2 related studies.","version":"1.1","doi":"10.1101/2020.06.29.177154","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.28.176248","pub_date":"2020-6-29","title":"Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein","abstract":"The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell. The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region is required for normal viral genome transcription, which occurs in a cytoplasmic structure called the replication transcription complex (RTC). It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.","version":"1.1","doi":"10.1101/2020.06.28.176248","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.29.178293","pub_date":"2020-6-29","title":"Angiotensin-(3\u20134) modulates angiotensin converting enzyme 2 (ACE2) downregulation in proximal tubule cells due to overweight and undernutrition: implications regarding the severity of renal lesions in Covid-19 infection","abstract":"The renal lesions \u2013 including severe acute kidney injury \u2013 are severe outcomes in SARS-CoV-2 infections. There are no reports regarding the influence of the nutritional status on the severity and progress of these lesions. Ageing is also an important risk factor. In the present communication we compare the influence of overweight and undernutrition in the levels of renal angiotensin converting enzymes 1 and 2. Since the renin-angiotensin-aldosterone system (RAAS) has been implicated in the progress of kidney failure during Covid-19, we also investigated the influence of Angiotensin-(3\u20134) (Ang-(3\u20134)) the shortest angiotensin-derived peptide, which is considered the physiological antagonist of several angiotensin II effects. We found that both overweight and undernutrition downregulate the levels of angiotensin converting enzyme 2 (ACE2) without influence on the levels of ACE1 in kidney rats. Administration of Ang-(3\u20134) recovers the control levels of ACE2 in overweight but not in undernourished rats. We conclude that chronic and opposite nutritional conditions play a central role in the pathophysiology of renal Covid-19 lesions, and that the role of RAAS is also different in overweight and undernutrition.","version":"1.1","doi":"10.1101/2020.06.29.178293","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.174961","pub_date":"2020-6-28","title":"Integrate Structural Analysis, Isoform Diversity, and Interferon-Inductive Propensity of ACE2 to Refine SARS-CoV2 Susceptibility Prediction in Vertebrates","abstract":"The current new coronavirus disease (COVID-19) has caused globally near 0.4/6 million confirmed deaths/infected cases across more than 200 countries. As the etiological coronavirus (a.k.a. SARS-CoV2) may putatively have a bat origin, our understanding about its intermediate reservoir between bats and humans, especially its tropism in wild and domestic animals, are mostly unknown. This constitutes major concerns in public health for the current pandemics and potential zoonosis. Previous reports using structural analysis of the viral spike protein (S) binding its cell receptor of angiotensin-converting enzyme 2 (ACE2), indicate a broad SARS-CoV2 susceptibility in wild and particularly domestic animals. Through integration of key immunogenetic factors, including the existence of S-binding-void ACE2 isoforms and the disparity of ACE2 expression upon early innate immune response, we further refine the SARS-CoV2 susceptibility prediction to fit recent experimental validation. In addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ACE2-related immunogenetic diversity to restrict SARS-CoV2 infections. Thus, we propose that domestic animals may be unlikely to play a role as amplifying hosts unless the virus has further species-specific adaptation. These findings may relieve relevant public concerns regarding COVID-19-like risk in domestic animals, highlight virus-host coevolution, and evoke disease intervention through targeting ACE2 molecular diversity and interferon optimization.","version":"1.1","doi":"10.1101/2020.06.27.174961","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.174698","pub_date":"2020-6-28","title":"A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC50 values were much higher than that required for HIV-1. Taken together, this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad number of RNA and DNA viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the COVID-19 pandemic, has quickly become a major global health problem causing immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here we developed a facile Q-RT-PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction, and is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA and remdesivir can substantially impede SARS-Cov-2 replication providing novel insight into viral entry and replication mechanisms. This facile approach will undoubtedly expedite basic SARS-CoV-2 research, be amenable to screening platforms to identify therapeutics against SARS-CoV-2 and can be adapted to numerous other RNA and DNA viruses.","version":"1.1","doi":"10.1101/2020.06.26.174698","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.173872","pub_date":"2020-6-28","title":"Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2","abstract":"SARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (\u2212) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme\u2019s active site, providing basis for the uracil scaffold-based drug development.","version":"1.2","doi":"10.1101/2020.06.26.173872","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.175166","pub_date":"2020-6-27","title":"Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response","abstract":"There is a great need for the development of vaccines for preventing SARS-CoV-2 infection and mitigating the COVID-19 pandemic. Here, we developed two modified vaccinia Ankara (MVA) based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the S1 region of the spike (MVA/S1) which forms trimers and is secreted. Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization. Following immunizations with MVA/S or MVA/S1, both spike protein recombinants induced strong IgG antibodies to purified full-length SARS-CoV-2 spike protein. The MVA/S induced a robust antibody response to purified RBD, S1 and S2 whereas MVA/S1 induced an antibody response to the S1 region outside of the RBD region. Both vaccines induced an antibody response in the lung and that was associated with induction of bronchus-associated lymphoid tissue. MVA/S but not MVA/S1 vaccinated mice generated robust neutralizing antibody responses against SARS-CoV-2 that strongly correlated with RBD antibody binding titers. Mechanistically, S1 binding to ACE-2 was strong but reduced following prolonged pre-incubation at room temperature suggesting confirmation changes in RBD with time. These results demonstrate MVA/S is a potential vaccine candidate against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.06.27.175166","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.175448","pub_date":"2020-6-27","title":"Critical Sequence Hot-spots for Binding of nCOV-2019 to ACE2 as Evaluated by Molecular Simulations","abstract":"The novel coronavirus (nCOV-2019) outbreak has put the world on edge, causing millions of cases and hundreds of thousands of deaths all around the world, as of June 2020, let alone the societal and economic impacts of the crisis. The spike protein of nCOV-2019 resides on the virion\u2019s surface mediating coronavirus entry into host cells by binding its receptor binding domain (RBD) to the host cell surface receptor protein, angiotensin converter enzyme (ACE2). Our goal is to provide a detailed structural mechanism of how nCOV-2019 recognizes and establishes contacts with ACE2 and its difference with an earlier coronavirus SARS-COV in 2002 via extensive molecular dynamics (MD) simulations. Numerous mutations have been identified in the RBD of nCOV-2019 strains isolated from humans in different parts of the world. In this study, we investigated the effect of these mutations as well as other Ala-scanning mutations on the stability of RBD/ACE2 complex. It is found that most of the naturally-occurring mutations to the RBD either strengthen or have the same binding affinity to ACE2 as the wild-type nCOV-2019. This may have implications for high human-to-human transmission of coronavirus in regions where these mutations have been found as well as any vaccine design endeavors since these mutations could act as antibody escape mutants. Furthermore, in-silico Ala-scanning and long-timescale MD simulations, highlight the crucial role of the residues at the interface of RBD and ACE2 that may be used as potential pharmacophores for any drug development endeavors. From an evolutional perspective, this study also identifies how the virus has evolved from its predecessor SARS-COV and how it could further evolve to become more infectious.","version":"1.1","doi":"10.1101/2020.06.27.175448","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.27.174979","pub_date":"2020-6-27","title":"Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.","version":"1.1","doi":"10.1101/2020.06.27.174979","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.173203","pub_date":"2020-6-26","title":"Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2","abstract":"The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. In this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.26.173203","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.171033","pub_date":"2020-6-26","title":"Evaluation of K18-hACE2 mice as a model of SARS-CoV-2 infection","abstract":"Murine models of SARS-CoV-2 infection are critical for elucidating the biological pathways underlying COVID-19 disease. Because human ACE2 is the receptor for SARS-CoV-2, mice expressing the human ACE2 gene have shown promise as a potential model for COVID-19. Five mice from the transgenic mouse strain K18-hACE2 were intranasally inoculated with SARS-CoV-2 Hong Kong/VM20001061/2020. Mice were followed twice daily for five days and scored for weight loss and clinical symptoms. Infected mice did not exhibit any signs of infection until day four, when weight loss, but no other obvious clinical symptoms were observed. By day five all infected mice had lost around 10% of their original body weight, but exhibited variable clinical symptoms. All infected mice showed high viral titers in the lungs as well as altered lung histology associated with proteinaceous debris in the alveolar space, interstitial inflammatory cell infiltration and alveolar septal thickening. Overall, these results show that symptomatic SARS-CoV-2 infection can be established in the K18-hACE2 transgenic background and should be a useful mouse model for COVID-19 disease.","version":"1.1","doi":"10.1101/2020.06.26.171033","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.151779","pub_date":"2020-6-26","title":"Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry","abstract":"The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the angiotensin-converting enzyme-2 (ACE2) receptor and is cleaved by transmembrane protease serine 2 (TMPRSS2). However, whether S mutations affect SARS-CoV-2 infectivity remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural observations. Nevertheless, the D614G mutant remains susceptible to neutralization by antisera against prototypic viruses. Taken together, these data indicate that the D614G mutation enhances viral infectivity while maintaining neutralization susceptibility.","version":"1.2","doi":"10.1101/2020.06.15.151779","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.173146","pub_date":"2020-6-26","title":"Air and surface measurements of SARS-CoV-2 inside a bus during normal operation","abstract":"Transmission pathways of SARS-CoV-2 are through aerosol, droplet and touching infected material. Indoor locations are more likely environments for the diffusion of the virus contagion among people, but direct detection of SARS-CoV-2 in air or on surfaces is quite sparse, especially regarding public transport. In fact, an important demand is to know how and if it is safe to use them. To understand the possible spreading of COVID-19 inside a city bus during normal operation and the effectiveness of the protective measures adopted for transportation, we analysed the air and the surfaces most usually touched by passengers. The measurements were carried out across the last week of the lockdown and the first week when gradually all the travel restrictions were removed.","version":"1.1","doi":"10.1101/2020.06.26.173146","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.174193","pub_date":"2020-6-26","title":"A cytosine-to-uracil change within the programmed -1 ribosomal frameshift signal of SARS-CoV-2 results in structural similarities with the MERS-CoV signal","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing COVID-19 pandemic, like many other viruses, uses programmed ribosomal frameshifting (PRF) to enable synthesis of multiple proteins from its compact genome. In independent analyses, we evaluated the PRF regions of all SARS-CoV-2 sequences available in GenBank and from the Global Initiative on Sharing All Influenza Data for variations. Of the 5,156 and 27,153 sequences analyzed, respectively, the PRF regions were identical in 95.7% and 97.2% of isolates. The most common change from the reference sequence was from C to U at position 13,536, which lies in the three-stemmed pseudoknot known to stimulate frameshifting. With the conversion of the G13493-C13536 Watson-Crick pair to G-U, the SARS-CoV-2 PRF closely resembles its counterpart in the Middle East respiratory syndrome coronavirus. The occurrence of this change increased from 0.5 to 3% during the period of March to May 2020.","version":"1.1","doi":"10.1101/2020.06.26.174193","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.174128","pub_date":"2020-6-26","title":"Development of a Coronavirus Disease 2019 Nonhuman Primate Model Using Airborne Exposure","abstract":"Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comprehensive and comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to define parameters critical to disease progression and the extent to which they correlate with human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation. Nonhuman primates develop COVID-19 following airborne virus exposure.","version":"1.1","doi":"10.1101/2020.06.26.174128","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.152520","pub_date":"2020-6-26","title":"Ag nanoparticles-based antimicrobial polycotton fabrics to prevent the transmission and spread of SARS-CoV-2","abstract":"Pathogens (bacteria, fungus and virus) are becoming a potential threat to the health of human beings and environment worldwide. They widely exist in the environment, with characteristics of variety, spreading quickly and easily causing adverse reactions. In this work, an Ag-based material is used to be incorporated and functionalized in polycotton fabrics using pad-dry-cure method. This composite proved to be effective for inhibiting the SARS-CoV-2 virus, decreasing the number of replicates in 99.99% after an incubation period of 2 minutes. In addition, it caused 99.99% inhibition of the pathogens S. aureus, E. coli and C. albicans, preventing cross-infections and does not cause allergies or photoirritation processes, demonstrating the safety of its use.","version":"1.1","doi":"10.1101/2020.06.26.152520","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.172528","pub_date":"2020-6-26","title":"Resource optimization in COVID-19 diagnosis","abstract":"The emergence and rapid dissemination worldwide of a novel Coronavirus (SARS-CoV-2) results in decrease of swabs availability for clinical samples collection, as well as, reagents for RT-qPCR diagnostic kits considered a confirmatory test for COVID-19 infection. This scenario, showed the requirement of improve de diagnostic capacity, so the aim of this study were to verify the possibility of reducing the reaction volume of RT-qPCR and to test cotton swabs as alternative for sample collection. RT-qPCR volumes and RNA sample concentration were optimized without affecting the sensitivity of assays, using both probe-based and intercalation dyes methods. Although rayon swabs showed better performance, cotton swabs could be used as alternative type for clinical sample collection. COVID-19 laboratory diagnosis is important to isolate and restrict the dissemination of virus, so seek for alternatives to decrease the coast of assays improve the control of disease.","version":"1.1","doi":"10.1101/2020.06.25.172528","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.172734","pub_date":"2020-6-26","title":"Assessment of the use and quick preparation of saliva for rapid microbiological diagnosis of COVID-19","abstract":"The objective of this study was to assess the performance of direct real time RT-PCR detection of SARS-CoV-2 in heated saliva samples, avoiding the RNA isolation step. Oropharyngeal and nasopharyngeal swabs together with saliva samples were obtained from 51 patients clinically diagnosed as potentially having COVID-19. Two different methods were compared: 1. RNA was extracted from 500 \u03bcl of sample using a MagNA Pure Compact Instrument with an elution volume of 50\u03bcl and 2. 700\u00b5L of saliva were heat-inactivated at 96\u00b0C for 15 minutes, and directly subjected to RT-PCR. One step real time RT-PCR was performed using 5 \u03bcl of extracted RNA or directly from 5 \u03bcl of heated sample. RT-PCR was performed targeting the SARS-CoV-2 envelope (E) gene region. Diagnostic performance was assessed using the results of the RT-PCR from nasopharyngeal and oropharyngeal swabs as the gold standard. The overall sensitivity, specificity, positive and negative predictive values were 81.08%, 92.86%, 96.77% and 65.00%, respectively when RNA extraction was included in the protocol with saliva, whereas sensitivity, specificity, positive and negative predictive values were 83.78%, 92.86%, 68.42% and 96.88%, respectively, for the heat-inactivation protocol. However, when the analysis was performed exclusively on saliva samples with a limited time from the onset of symptoms (<9 days, N=28), these values were 90%, 87.5%, 44% and 98.75% for the heat-inactivation protocol. The study showed that RT-PCR can be performed using saliva in an RNA extraction free protocol, showing good sensitivity and specificity.","version":"1.1","doi":"10.1101/2020.06.25.172734","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.26.173567","pub_date":"2020-6-26","title":"Adhesive Contact Between Cylindrical (Ebola) and Spherical (SARS-CoV-2) Viral Particles and a Cell Membrane","abstract":"A critical event during the process of cell infection by a viral particle is attachment, which is driven by adhesive interactions and resisted by bending and tension. The biophysics of this process has been studied extensively but the additional role of externally applied force or displacement has generally been neglected. In this work we study the adhesive force-displacement response of viral particles against a cell membrane. We have built two models: one in which the viral particle is cylindrical (say, representative of filamentous virus such as Ebola) and another in which it is spherical (such as SARS-CoV-2 and Zika). Our interest is in initial adhesion, in which case deformations are small and the mathematical model for the system can be simplified considerably. The parameters that characterize the process combine into two dimensionless groups that represent normalized membrane bending stiffness and tension. In the limit where bending dominates, for sufficiently large values of normalized bending stiffness, there is no adhesion between viral particles and the cell membrane without applied force. (The zero-external-force contact width and pull-off force are both zero.) For large values of normalized membrane tension, the adhesion between virus and cell membrane is weak but stable. (The contact width at zero external force has a small value.) Our results for pull-off force and zero force contact width help to quantify conditions that could aid the development of therapies based on denying the virus entry into the cell by blocking its initial adhesion.","version":"1.1","doi":"10.1101/2020.06.26.173567","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.171744","pub_date":"2020-6-25","title":"Chasing the origin of SARS-CoV-2 in Canada\u2019s COVID-19 cases: A genomics study","abstract":"The emergence and global spread of SARS-CoV-2 has had profound social and economic consequences and has shed light on the importance of continued and additional investment in global health and infectious disease surveillance. Identifying changes in viral genomes provides key insights into viral diversity, how viruses spread within populations, and viral strategies for evasion of host immune systems. Here we report twenty-five SARS-CoV-2 genome sequences collected from some of the first COVID-19 cases in eastern Ontario, Canada (March 18-30, 2020). The reported genomes belong to the S-clade (n=2) and G-clade (n=23) of SARS-CoV-2 and contain 45 polymorphic sites including one shared missense and three unique synonymous variants in the gene encoding the spike protein. A phylogenetic analysis enabled the tracing of viral origin and potential transmission into and within Canada. There may be as many as sixteen unique infection events represented in these samples, including at least three that were likely introduced from Europe and seven from the USA. In addition, four separate genomes are each shared by multiple patients, suggesting a common origin or community spread even during this early stage of infection. These results demonstrate how molecular epidemiology and evolutionary phylogenetics can help local health units track origins and vectors of spread for emerging diseases like SARS-CoV-2. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to prevent future pandemics.","version":"1.1","doi":"10.1101/2020.06.25.171744","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.165902","pub_date":"2020-6-25","title":"Genetic Diversity and Genomic Epidemiology of SARS-COV-2 in Morocco","abstract":"COVID-A9 is an infection disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), declared as a pandemic due to its rapid expansion worldwide. In this study we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 using 22 virus genome sequences reported by three different laboratories in Morocco till the date 07/06/2020 as well as (40366) virus genomes from all around the world. The SARS-CoV-2 genomes from Moroccan patients revealed 62 mutations of which 30 were missense mutations. The mutations Spike_D614G and NSP12_P323L were present in all the 22 analyzed sequences, followed by N_G204R and N_R203K which occurred in 9 among the 22 sequences. The mutations NSP10_R134S, NSP15_D335N, NSP16_I169L, NSP3_L431H, NSP3_P1292L and Spike_V6F occurred one time in our sequences with no record in other sequence worldwide. These mutations should be investigated to figure out their potential effects on all around the world virulence. Phylogenetic analyses revealed that Moroccan SARS-CoV-2 genomes included 9 viruses pertaining to clade 20A, 9 to clade 20B and 2 to clade 20C. This finding suggest that the epidemic spread in Morocco did not show a predominant SARS-CoV-2 route. For multiple and unrelated introductions of SARS-CoV-2 into Morocco via different routes have occurred, giving rise to the diversity of virus genomes in the country. Furthermore, very likely, the SARS-CoV-2 virus circulated in cryptic way in Morocco starting from the fifteen January before the discovering of the first case the second of March.","version":"1.2","doi":"10.1101/2020.06.23.165902","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167072","pub_date":"2020-6-25","title":"Molecular features similarities between SARS-CoV-2, SARS, MERS and key human genes could favour the viral infections and trigger collateral effects","abstract":"In December 2019 rising pneumonia cases caused by a novel \u03b2-coronavirus (SARS-CoV-2) occurred in Wuhan, China, which has rapidly spread worldwide causing thousands of deaths. The WHO declared the SARS-CoV-2 outbreak as a public health emergency of international concern therefore several scientists are dedicated to the study of the new virus. Since human viruses have codon usage biases that match highly expressed proteins in the tissues they infect and depend on host cell machinery for replication and co-evolution, we selected the genes that are highly expressed in the tissue of human lungs to perform computational studies that permit to compare their molecular features with SARS, SARS-CoV-2 and MERS genes. In our studies, we analysed 91 molecular features for 339 viral genes and 463 human genes that consisted of 677873 codon positions. Hereby, we found that A/T bias in viral genes could propitiate the viral infection favoured by a host dependant specialization using the host cell machinery of only some genes. The envelope protein E, the membrane glycoprotein M and ORF7 could have been further benefited by a high rate of A/T in the third codon position. Thereby, the mistranslation or de-regulation of protein synthesis could produce collateral effects, as a consequence of viral occupancy of the host translation machinery due tomolecular similarities with viral genes. Furthermore, we provided a list of candidate human genes whose molecular features match those of SARS-CoV-2, SARSand MERS genes, which should be considered to be incorporated into genetic population studies to evaluate thesusceptibility to respiratory viral infections caused by these viruses. The results presented here, settle the basis for further research in the field of human genetics associated with the new viral infection, COVID-19, caused by SARS-CoV-2 and for the development of antiviral preventive methods.","version":"1.1","doi":"10.1101/2020.06.23.167072","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.170704","pub_date":"2020-6-25","title":"Binding Ability Prediction between Spike Protein and Human ACE2 Reveals the Adaptive Strategy of SARS-CoV-2 in Humans","abstract":"SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus causing an outbreak of COVID-19 globally in the past six months. A relatively higher divergence on the spike protein of SASR-CoV-2 enables it to transmit across species efficiently. We particularly believe that the adaptive mutations of the receptor-binding domain (RBD) of spike protein in SARS-CoV-2 might be essential to its high transmissibility among humans. Thus here we collected 2,142 high-quality genome sequences of SARS-CoV-2 from 160 regions in over 50 countries and reconstructed their phylogeny, and also analyzed the interaction between the polymorphisms of spike protein and human ACE2 (hACE2). Phylogenetic analysis of SARS-CoV-2 and coronavirus in other hosts show SARS-CoV-2 is highly possible originated from Bat-CoV (RaTG13) found in horseshoe bat and a recombination event may occur on the spike protein of Pangolin-CoV to imbue it the ability to infect humans. Moreover, compared to the S gene of SARS-CoV-2, it is more conserved in the direct-binding sites of RBD and we noticed that spike protein of SARS-CoV-2 may under a consensus evolution to adapt to human hosts better. 3,860 amino acid mutations in spike protein RBD (T333-C525) of SARS-CoV-2 were simulated and their stability and affinity binding to hACE2 (S19-D615) were calculated. Our analysis indicates SARS-CoV-2 could infect humans from different populations with no preference, and a higher divergence in the spike protein of SARS-CoV-2 at the early stage of this pandemic may be a good indicator that could show the pathway of SARS-CoV-2 transmitting from the natural reservoir to human beings.","version":"1.1","doi":"10.1101/2020.06.25.170704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.150326","pub_date":"2020-6-25","title":"Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication","abstract":"Emergence of a highly contagious novel coronavirus, SARS-CoV-2 that causes COVID-19, has precipitated the current global health crisis with over 479,000 deaths and more than 9.3 million confirmed cases. Currently, our knowledge of the mechanisms of COVID-19 disease pathogenesis is very limited which has hampered attempts to develop targeted antiviral strategies. Therefore, we urgently need an effective therapy for this unmet medical need. Viruses hijack and dysregulate cellular machineries in order for them to replicate and infect more cells. Thus, identifying and targeting dysregulated signaling pathways that have been taken over by viruses is one strategy for developing an effective antiviral therapy. We have developed a high-throughput drug screening system to identify potential antiviral drugs targeting SARS-CoV-2. We utilized a small molecule library of 430 protein kinase inhibitors, which are in various stages of clinical trials. Most of the tested kinase antagonists are ATP competitive inhibitors, a class of nucleoside analogs, which have been shown to have potent antiviral activity. From the primary screen, we have identified 34 compounds capable of inhibiting viral cytopathic effect in epithelial cells. Network of drug and protein relations showed that these compounds specifically targeted a limited number of cellular kinases. More importantly, we have identified mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-Damage Response (DDR) pathways as key cellular signaling pathways critical for SARS-CoV-2 infection. Subsequently, a secondary screen confirmed compounds such as Berzosertib (VE-822), Vistusertib (AZD2014), and Nilotinib with anti SARS-CoV-2 activity. Finally, we found that Berzosertib, an ATR kinase inhibitor in the DDR pathway, demonstrated potent antiviral activity in a human epithelial cell line and human induced pluripotent stem cell (hIPSC)-derived cardiomyocytes. These inhibitors are already in clinical trials of phase 2 or 3 for cancer treatment, and can be repurposed as promising drug candidates for a host-directed therapy of SARS-CoV-2 infection. In conclusion, we have identified small molecule inhibitors exhibiting anti SARS-CoV-2 activity by blocking key cellular kinases, which gives insight on important mechanism of host-pathogen interaction. These compounds can be further evaluated for the treatment of COVID-19 patients following additional in vivo safety and efficacy studies. None declared.","version":"1.1","doi":"10.1101/2020.06.24.150326","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.170639","pub_date":"2020-6-25","title":"The discovery of potential natural products for targeting SARS-CoV-2 spike protein by virtual screening","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters into the cells through its spike proteins binding to human angiotensin-converting enzyme 2 (ACE2) protein and causes virus infection in host cells. Until now, there are no available antiviral drugs have been reported that can effectively block virus infection. The study aimed to discover the potential compounds to prevent viral spike proteins to bind to the human ACE2 proteins from Taiwan Database of Extracts and Compounds (TDEC) by structure-based virtual screening. In this study, to rapidly discover potential inhibitors against SARS-CoV-2 spike proteins, the molecular docking calculation was performed by AutoDock Vina program. Herein, we found that 39 potential compounds may have good binding affinities and can respectively bind to the viral receptor-binding domain (RBD) of spike protein in the prefusion conformation and spike-ACE2 complex protein in silico. Among those compounds, especially natural products thioflexibilolide A and candidine that were respectively isolated from the soft corals Sinularia flexibilis and Phaius mishmensis may have better binding affinity than others. This study provided the predictions that these compounds may have the potential to prevent SARS-CoV-2 spike protein from entry into cells.","version":"1.1","doi":"10.1101/2020.06.25.170639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.149153","pub_date":"2020-6-25","title":"Ionophore antibiotic X-206 is a potent and selective inhibitor of SARS-CoV-2 infection in vitro","abstract":"Pandemic spread of emerging human pathogenic viruses such as the current SARS-CoV-2, poses both an immediate and future challenge to human health and society. Currently, effective treatment of infection with SARS-CoV-2 is limited and broad spectrum antiviral therapies to meet other emerging pandemics are absent leaving the World population largely unprotected. Here, we have identified distinct members of the family of polyether ionophore antibiotics with potent ability to inhibit SARS-CoV-2 replication and cytopathogenicity in cells. Several compounds from this class displayed more than 100-fold selectivity between viral-induced cytopathogenicity and inhibition of cell viability, however the compound X-206 displayed >500-fold selectivity and was furthermore able to inhibit viral replication even at sub-nM levels. The antiviral mechanism of the polyether ionophores is currently not understood in detail. We demonstrate, through unbiased bioactivity profiling, that their effects on the host cells differ from those of cationic amphiphiles such as hydroxychloroquine. Collectively, our data suggest that polyether ionophore antibiotics should be subject to further investigations as potential broad-spectrum antiviral agents.","version":"1.2","doi":"10.1101/2020.06.14.149153","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.25.170936","pub_date":"2020-6-25","title":"Cellular exocytosis gene (EXOC6/6B): a potential molecular link for the susceptibility and mortality of COVID-19 in diabetic patients","abstract":"Diabetes is one of the most critical comorbidities linked to an increased risk of severe complications in the current coronavirus disease 2019 (COVID-19) pandemic. A better molecular understanding of COVID-19 in people with type diabetes mellitus (T2D) is mandatory, especially in countries with a high rate of T2D, such as the United Arab Emirates (UAE). Identification of the cellular and molecular mechanisms that make T2D patients prone to aggressive course of the disease can help in the discovery of novel biomarkers and therapeutic targets to improve our response to the disease pandemic. Herein, we employed a system genetics approach to explore potential genomic, transcriptomic alterations in genes specific to lung and pancreas tissues, affected by SARS-CoV-2 infection, and study their association with susceptibility to T2D in Emirati patients. Our results identified the Exocyst complex component, 6 (EXOC6/6B) gene (a component for docks insulin granules to the plasma membrane) with documented INDEL in 3 of 4 whole genome sequenced Emirati diabetic patients. Publically available transcriptomic data showed that lung infected with SARS-CoV-2 showed significantly lower expression of EXOC6/6B compared to healthy lungs. In conclusion, our data suggest that EXOC6/6B might be an important molecular link between dysfunctional pancreatic islets and ciliated lung epithelium that makes diabetic patients more susceptible to severe SARS-COV-2 complication.","version":"1.1","doi":"10.1101/2020.06.25.170936","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.170332","pub_date":"2020-6-25","title":"Comparison of 12 molecular detection assays for SARS-CoV-2","abstract":"Molecular testing for SARS-CoV-2 is the mainstay for accurate diagnosis of the infection, but the diagnostic performances of available assays have not been defined. We compared 12 molecular diagnostic assays, including 8 commercial kits using 155 respiratory samples (65 nasopharyngeal swabs, 45 oropharyngeal swabs, and 45 sputum) collected at 2 Japanese hospitals. Sixty-eight samples were positive for more than one assay and one genetic locus and were defined as true positive samples. All the assays showed a specificity of 100% (95% confidence interval, 95.8 to 100). The N2 assay kit of the US Centers for Disease Control and Prevention (CDC) and the N2 assay of the Japanese National Institute of Infectious Disease (NIID) were the most sensitive assays with 100% sensitivity (95% confidence interval, 94.7 to 100), followed by the CDC N1 kit, E assay by Corman, and NIID N2 assay multiplex with internal control reactions. These assays are reliable as first-line molecular assays in laboratories when combined with appropriate internal control reactions.","version":"1.1","doi":"10.1101/2020.06.24.170332","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091983","pub_date":"2020-6-25","title":"CoV3D: A database and resource for high resolution coronavirus protein structures","abstract":"SARS-CoV-2, the etiologic agent behind COVID-19, exemplifies the general threat to global health posed by coronaviruses. The urgent need for effective vaccines and therapies is leading to a rapid rise in the number of high resolution structures of SARS-CoV-2 proteins that collectively reveal a map of virus vulnerabilities. To assist structure-based design of vaccines and therapeutics against SARS-CoV-2 and other coronaviruses, we have developed CoV3D, a database and resource for coronavirus protein structures, which is updated on a weekly basis. CoV3D provides users with comprehensive sets of structures of coronavirus proteins and their complexes with antibodies, receptors, and small molecules. Integrated molecular viewers allow users to visualize structures of the spike glycoprotein, which is the major target of neutralizing antibodies and vaccine design efforts, as well as sets of spike-antibody complexes, spike sequence variability, and known polymorphisms. In order to aid structure-based design and analysis of the spike glycoprotein, CoV3D permits visualization and download of spike structures with modeled N-glycosylation at known glycan sites, and contains structure-based classification of spike conformations, generated by unsupervised clustering. CoV3D can serve the research community as a centralized reference and resource for spike and other coronavirus protein structures, and is available at: https://cov3d.ibbr.umd.edu.","version":"1.3","doi":"10.1101/2020.05.12.091983","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169730","pub_date":"2020-6-25","title":"Rapid Single Cell Evaluation of Human Disease and Disorder Targets Using REVEAL: SingleCell\u2122","abstract":"Single-cell (sc) sequencing performs unbiased profiling of individual cells and enables evaluation of less prevalent cellular populations, often missed using bulk sequencing. However, the scale and the complexity of the sc datasets poses a great challenge in its utility and this problem is further exacerbated when working with larger datasets typically generated by consortium efforts. As the scale of single cell datasets continues to increase exponentially, there is an unmet technological need to develop database platforms that can evaluate key biological hypothesis by querying extensive single-cell datasets. Large single-cell datasets like human cell atlas and COVID-19 cell atlas (collection of annotated sc datasets from various human organs) are excellent resources for profiling target genes involved in human diseases and disorders ranging from oncology, auto-immunity, as well as infectious diseases like COVID-19 caused by SARS-CoV-2 virus. SARS-CoV-2 infections have led to a worldwide pandemic with massive loss of lives, infections exceeding 7 million cases. The virus uses ACE2 and TMPRSS2 as key viral entry associated proteins expressed in human cells for infections. Evaluating the expression profile of key genes in large single-cell datasets can facilitate testing for diagnostics, therapeutics and vaccine targets; as the world struggles to cope with the on-going spread of COVID-19 infections. In this manuscript we describe, REVEAL: SingleCell which enables storage, retrieval and rapid query of single-cell datasets inclusive of millions of cells. The analytical database described here enables selecting and analyzing cells across multiple studies. Cells can be selected using individual metadata tags, more complex hierarchical ontology filtering, and gene expression threshold ranges, including co-expression of multiple genes. The tags on selected cells can be further evaluated for testing biological hypothesis. One such example includes identifying the most prevalent cell type annotation tag on returned cells. We used REVEAL: SingleCell to evaluate expression of key SARS-CoV-2 entry associated genes, and queried the current database (2.2 Million cells, 32 projects) to obtain the results in <60 seconds. We highlighted cells expressing COVID-19 associated genes are expressed on multiple tissue types, thus in part explains the multi-organ involvement in infected patients observed worldwide during the on-going COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.06.24.169730","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.166421","pub_date":"2020-6-25","title":"Modeling the effect of COVID-19 disease on the cardiac function: a computational study","abstract":"The effect of COVID-19 on the cardiac function and on the vascular system increases the morbidity and mortality of infected subjects with cardiovascular diseases. To provide preliminary results on cardiac global outcomes (such as cardiac output, ventricular pressures) obtained by means of computational models in plausible scenarios characterized by COVID-19. We considered a lumped parameters computational model of the cardiovascular system, which models, from the mechanical point of view, the systemic and pulmonary circulations, the four cardiac valves and the four heart chambers, through mathematical equations of the underlying physical processes. To study the effect of COVID-19, we varied the heart rate, the contractility and the pulmonary resistances in suitable ranges. Our computations on individuals with both otherwise normal and impaired cardiac functions revealed that COVID-19 worsen cardiac function, as shown by a decrease of some cardiac biomarkers values such as cardiac output and ejection fraction. In the case of existing impaired cardiac function, the presence of COVID-19 lead to values outside the normal ranges. Computational models revealed to be an effective tool to study the effect of COVID-19 on the cardiovascular system. Such effect could be significant for patients with impaired cardiac function. This is especially useful to perform a sensitivity analysis of the hemodynamics for different conditions. Emerging studies address how COVID-19 infection might impact the cardiovascular system. This relates particularly to the development of myocardial injury, acute coronary syndrome, myocarditis, arrhythmia, and heart failure. Prospective treatment approach is advised for these patients. By the assessment of conventional important biomarkers obtained with new sources as a 0-dimentional computational model, we propose a new study protocol as an effective method to evaluate short-term prognosis. The clinical protocol proposed will help to rapidly identify which patients require intensive monitoring, diagnostic strategy and most adequate therapy.","version":"1.2","doi":"10.1101/2020.06.23.166421","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.167049","pub_date":"2020-6-24","title":"SARS-CoV-2 infection and replication in human fetal and pediatric gastric organoids","abstract":"Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global public health emergency. COVID-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (GI) symptoms. This is particularly true in children in whom GI symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. By contrast, fetuses seem to be rarely affected by COVID-19, although the virus has been detected in placentas of affected women. These observations raise the question of whether the virus can infect and replicate within the stomach once ingested. Moreover, it is not yet clear whether active replication of SARS-CoV-2 is possible in the stomach of children or in fetuses at different developmental stages. Here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (PCW) fetuses, and from pediatric biopsies, to be used as an in vitro model for SARS-CoV-2 gastric infection. Gastric organoids recapitulate human stomach with linear increase of gastric mucin 5AC along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin A. In order to investigate SARS-CoV-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (RP-GOs) in suspension. In this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ACE2). The pediatric RP-GOs are fully susceptible to infection with SARS-CoV-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. The RP-GOs derived from pediatric patients show sustained robust viral replication of SARS-CoV-2, compared with organoids derived from fetal stomachs. Transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. Collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to SARS-CoV-2 infection, especially in early stages of development. However, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.24.167049","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169565","pub_date":"2020-6-24","title":"Development of a fluorescence based, high-throughput SARS-CoV-2 3CLpro reporter assay","abstract":"In late 2019 a human coronavirus, now known as SARS-CoV-2, emerged, likely from a zoonotic reservoir. This virus causes COVID-19 disease, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro. 3CLpro is an attractive target for antiviral therapeutics as it is essential for processing newly translated viral proteins, and the viral lifecycle cannot be completed without protease activity. In this work, we present a new assay to identify inhibitors of the SARS-CoV-2 main protease, 3CLpro. Our reporter is based on a GFP-derived protein that only fluoresces after cleavage by 3CLpro. This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. The COVID-19 pandemic has already led to more than 400,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro. This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.06.24.169565","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.168534","pub_date":"2020-6-24","title":"Lung expression of genes encoding SARS-CoV-2 cell entry molecules and antiviral restriction factors: interindividual differences are associated with age and germline variants","abstract":"Germline variants in genes involved in SARS-CoV-2 cell entry (i.e. ACE2 and TMPRSS2) may influence the susceptibility to infection, as may polymorphisms in genes involved in the innate host response to viruses (e.g. APOBEC3 family). We searched for polymorphisms acting, in lung tissue, as expression quantitative trait loci (eQTLs) for 15 candidate COVID-19 susceptibility genes, selected for their roles in virus cell entry and host antiviral responses. No significant eQTLs were identified for ACE2 and TMPRSS2 genes, whose expression levels did not associate with either sex or age of the 408 patients whose non-diseased lung tissue was analyzed. Instead, we identified seven cis-eQTLs (FDR<0.05) for APOBEC3D and APOBEC3G (rs139296, rs9611092, rs139331, rs8177832, rs17537581, rs61362448, and rs738469). The genetic control of the expression of APOBEC3 genes, which encode enzymes that interfere with virus replication, may explain interindividual differences in risk or severity of viral infections. Future studies should investigate the role of host genetics in COVID-19 patients using a genome-wide approach, to identify other genes whose expression levels are associated with susceptibility to SARS-CoV-2 infection or COVID-19 severity. Identification of expression quantitative trait loci (eQTLs) has become commonplace in functional studies on the role of individual genetic variants in susceptibility to diseases. In COVID-19, it has been proposed that individual variants in SARS-CoV-2 cell entry and innate host response genes may influence the susceptibility to infection. We searched for polymorphisms acting, in non-diseased lung tissue of 408 patients, as eQTLs for 15 candidate COVID-19 susceptibility genes, selected for their roles in virus cell entry and host antiviral responses. Seven cis-eQTLs were detected for APOBEC3D and APOBEC3G genes, which encode enzymes that interfere with virus replication. No significant eQTLs were identified for ACE2 and TMPRSS2 genes. Therefore, the identified eQTLs may represent candidate loci modulating interindividual differences in risk or severity of SARS-CoV-2 virus infection.","version":"1.1","doi":"10.1101/2020.06.24.168534","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167916","pub_date":"2020-6-24","title":"In silico identification of conserved cis-acting RNA elements in the SARS-CoV-2 genome","abstract":"The aim of this study was to computationally predict conserved RNA sequences and structures known as cis-acting RNA elements (CREs) located within the SARS-CoV-2 genome. Bioinformatics tools were used to analyse and predict cis-acting regulatory elements by obtaining viral sequences from available databases. Computational analysis prediction revealed the presence of RNA stem-loop structures within the 3\u2019 end of the ORF1ab region that are analogous to the previously identified SARS-CoV genomic packaging signals. Alignment-based RNA secondary structures prediction of the 5\u2019 end of the SARS-CoV-2 genome identified also conserved CREs. These CREs could be used as potential targets for a vaccine and/or antiviral therapeutics developments; however, further studies would be required to confirm their roles in the SARS-CoV-2 life cycle.","version":"1.1","doi":"10.1101/2020.06.23.167916","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.168252","pub_date":"2020-6-24","title":"The SARS-CoV-2 receptor, Angiotensin converting enzyme 2 (ACE2) is required for human endometrial stromal cell decidualization","abstract":"Is SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE 2) expressed in the human endometrium during the menstrual cycle, and does it participate in endometrial decidualization? ACE2 protein is highly expressed in human endometrial stromal cells during the secretory phase and is essential for human endometrial stromal cell decidualization. ACE2 is expressed in numerous human tissues including the lungs, heart, intestine, kidneys and placenta. ACE2 is also the receptor by which SARS-CoV-2 enters human cells. Proliferative (n = 9) and secretory (n = 6) phase endometrium biopsies from healthy reproductive-age women and primary human endometrial stromal cells from proliferative phase endometrium were used in the study. ACE2 expression and localization were examined by qRT-PCR, Western blot, and immunofluorescence in both human endometrial samples and mouse uterine tissue. The effect of ACE2 knockdown on morphological and molecular changes of human endometrial stromal cell decidualization were assessed. Ovariectomized mice were treated with estrogen or progesterone to determine the effects of these hormones on ACE2 expression. In human tissue, ACE2 protein is expressed in both endometrial epithelial and stromal cells in the proliferative phase of the menstrual cycle, and expression increases in stromal cells in the secretory phase. The ACE2 mRNA (P < 0.0001) and protein abundance increased during primary human endometrial stromal cell (HESC) decidualization. HESCs transfected with ACE2-targeting siRNA were less able to decidualize than controls, as evidenced by a lack of morphology change and lower expression of the decidualization markers PRL and IGFBP1 (P < 0.05). In mice during pregnancy, ACE2 protein was expressed in uterine epithelial and stromal cells increased through day six of pregnancy. Finally, progesterone induced expression of Ace2 mRNA in mouse uteri more than vehicle or estrogen (P < 0.05). N/A. Experiments assessing the function of ACE2 in human endometrial stromal cell decidualization were in vitro. Whether SARS-CoV-2 can enter human endometrial stromal cells and affect decidualization have not been assessed. Expression of ACE2 in the endometrium allow SARS-CoV-2 to enter endometrial epithelial and stromal cells, which could impair in vivo decidualization, embryo implantation, and placentation. If so, women with COVID-19 may be at increased risk of early pregnancy loss. This study was supported by National Institutes of Health / National Institute of Child Health and Human Development grants R01HD065435 and R00HD080742 to RK and Washington University School of Medicine start-up funds to RK. The authors declare that they have no conflicts of interest.","version":"1.1","doi":"10.1101/2020.06.23.168252","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.167791","pub_date":"2020-6-24","title":"N-glycosylation network construction and analysis to modify glycans on the spike S glycoprotein of SARS-CoV-2","abstract":"The spike S-protein of SARS-CoV-2 is N-glycosylated. The N-glycan structure and composition of this glycoprotein influence how the virus interacts with host cells. To identify a putative N-glycan biosynthesis pathway of SARS-CoV-2 (HEK293 cell recombinant) from previously published mass spectrometric studies, and to identify what effect blocking some enzymes has on the overall glycoprotein profile. Finally, our goal was to provide the biosynthesis network, and glycans in easy-to-use format for further glycoinformatics work. We reconstructed the glycosylation network based on previously published empirical data using GNAT, a glycosylation network analysis tool. Our compilation of the network tool had 23 glycosyltransferase and glucosidase enzymes, and could infer the pathway of glycosylation machinery based on glycans identified in the virus spike protein. Once the glycan biosynthesis pathway was generated, we simulated the effect of blocking specific enzymes - Mannosidase-II and alpha-1,6-fucosyltransferase to see how they would affect the biosynthesis network. Of the 23 enzymes, a total of 12 were involved in glycosylation of SARS-CoV-2 - Man-Ia, MGAT1, MGAT2, MGAT4, MGAT5, B4GalT, B4GalT, Man II, SiaT, ST3GalI, ST3GalVI and FucT8. Blocking enzymes resulted in a substantially modified glycan profile of the protein. A network analysis of N-glycan biosynthesis of SARS-CoV-2 spike protein shows an elaborate enzymatic pathway with several intermediate glycans, along with the ones identified by mass spectrometric studies. Variations in the final N-glycan profile of the virus, given its site-specific microheterogeneity, could be a factor in the host response to the infection and response to antibodies. Here we provide all the resources generated - the glycans derived from mass spectrometry and intermediate glycans in glycoCT xml format, and the biosynthesis network for future drug and vaccine development work.","version":"1.1","doi":"10.1101/2020.06.23.167791","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.162941","pub_date":"2020-6-24","title":"Stroke increases the expression of ACE2, the SARS-CoV-2 binding receptor, in murine lungs","abstract":"The newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused a worldwide pandemic of human respiratory disease. Angiotensin-converting enzyme (ACE) 2 is the key receptor on lung epithelial cells to facilitate initial binding and infection of SARS-CoV-2. The binding to ACE2 is mediated via the spike glycoprotein present on the virus surface. Recent clinical data have demonstrated that patients suffering from stroke are particularly susceptible to severe courses of SARS-CoV-2 infection, thus forming a defined risk group. However, a mechanistic explanation for this finding is lacking. Sterile tissue injuries including stroke induce lymphocytopenia and systemic inflammation that might modulate the expression levels of surface proteins in distant organs. Whether systemic inflammation following stroke can specifically modulate ACE2 expression in the lung has not been investigated. Mice were subjected to transient middle cerebral artery occlusion (MCAO) for 45 min and sacrificed after 24 h and 72 h for analysis of brain and lung tissues. Gene expression and protein levels of ACE2, ACE, IL-6 and IL1\u03b2 were measured by quantitative PCR and Western blot, respectively. Immune cell populations in lymphoid organs were analyzed by flow cytometry. Strikingly, 24 h after stroke, we observed a substantial increase in the expression of ACE2 both on the transcriptional and protein levels in the lungs of MCAO mice compared to sham-operated mice. This increased expression persisted until day 3 after stroke. In addition, MCAO increased the expression of inflammatory cytokines IL-6 and IL-1\u03b2 in the lungs. Higher gene expression of cytokines IL-6 and IL-1\u03b2 was found in ischemic brain hemispheres and a reduced number of T-lymphocytes were present in the blood and spleen as an indicator of sterile tissue injury-induced immunosuppression. We demonstrate significantly augmented ACE2 levels and inflammation in murine lungs after experimental stroke. These pre-clinical findings might explain the clinical observation that patients with pre-existing stroke represent a high-risk group for the development of severe SARS-CoV-2 infections. Our studies call for further investigations into the underlying signaling mechanisms and possible therapeutic interventions. Brain tissue injury increases ACE2 levels in the lungs Brain injury induces pro-inflammatory cytokine expression in the lungs Brain injury causes parenchymal inflammation and systemic lymphopenia","version":"1.1","doi":"10.1101/2020.06.24.162941","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169383","pub_date":"2020-6-24","title":"Intestinal receptor of SARS-CoV-2 in inflamed IBD tissue is downregulated by HNF4A in ileum and upregulated by interferon regulating factors in colon","abstract":"Patients with IBD are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the effect of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2, in IBD. We collected (un)inflamed mucosal biopsies from CD (n=193) and UC (n=158) patients, and 51 matched non-IBD controls for RNA sequencing, differential gene expression and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell (sc) sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-TNF therapy, were analyzed. In inflamed CD ileum, ACE2 was significantly decreased compared to control ileum (p=4.6E-07), whereas colonic ACE2 expression was higher in inflamed colon of CD/UC compared to control (p=8.3E-03; p=1.9E-03). Sc-RNA sequencing confirmed this ACE2 dysregulation, and exclusive epithelial ACE2 expression. Network analyses highlighted HNF4A as key regulator of ileal ACE2, while pro-inflammatory cytokines and interferon regulating factors regulated colonic ACE2. Inflammatory stimuli upregulated ACE2 in UC organoids (p=1.7E-02), not in non-IBD controls (p=9.1E-01). Anti-TNF therapy restored colonic ACE2 dysregulation in responders. Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing effects in ileum and colon. HNF4A, an IBD susceptibility gene, is an important upstream regulator of ACE2 in ileum, whereas interferon signaling dominates in colon. Our data support the importance of adequate control of IBD in order to reduce risk of (complicated) COVID-19.","version":"1.1","doi":"10.1101/2020.06.24.169383","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169144","pub_date":"2020-6-24","title":"Integration of viral transcriptome sequencing with structure and sequence motifs predicts novel regulatory elements in SARS-CoV-2","abstract":"In the last twenty years, three separate coronaviruses have left their typical animal hosts and became human pathogens. An area of research interest is coronavirus transcription regulation that uses an RNA-RNA mediated template-switching mechanism. It is not known how different transcriptional stoichiometries of each viral gene are generated. Analysis of SARS-CoV-2 RNA sequencing data from whole RNA transcriptomes identified TRS dependent and independent transcripts. Integration of transcripts and 5\u2019-UTR sequence motifs identified that the pentaloop and the stem-loop 3 were also located upstream of spliced genes. TRS independent transcripts were detected as likely non-polyadenylated. Additionally, a novel conserved sequence motif was discovered at either end of the TRS independent splice junctions. While similar both SARS viruses generated similar TRS independent transcripts they were more abundant in SARS-CoV-2. TRS independent gene regulation requires investigation to determine its relationship to viral pathogenicity.","version":"1.1","doi":"10.1101/2020.06.24.169144","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.24.169094","pub_date":"2020-6-24","title":"Modelling donor screening strategies to reduce the risk of SARS-CoV-2 via fecal microbiota transplantation","abstract":"The potential for transmission of SARS-CoV-2 shed in stool via fecal microbiota transplantation is not yet known, and the effectiveness of various testing strategies to prevent FMT-based transmission has also not yet been quantified. Here we use a mathematical model to simulate the utility of different testing strategies.","version":"1.1","doi":"10.1101/2020.06.24.169094","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.166900","pub_date":"2020-6-23","title":"Biochemical evidence of furin specificity and potential for phospho-regulation at Spike protein S1/S2 cleavage site in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV","abstract":"The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR\u2193SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous insertion was present in the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR\u2193SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin\u2019s ability to cleave the site. Although phosphoregulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.","version":"1.1","doi":"10.1101/2020.06.23.166900","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165712","pub_date":"2020-6-23","title":"A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19","abstract":"A high-throughput platform would greatly facilitate COVID-19 serological testing and antiviral screening. Here we report a nanoluciferase SARS-CoV-2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. We demonstrate that the optimized reporter virus assay in Vero E6 cells can be used to measure neutralizing antibody activity in patient sera and produces results in concordance with a plaque reduction neutralization test (PRNT). Compared with the low-throughput PRNT (3 days), the SARS-CoV-2-Nluc assay has substantially shorter turnaround time (5 hours) with a high-throughput testing capacity. Thus, the assay can be readily deployed for large-scale vaccine evaluation and neutralizing antibody testing in humans. Additionally, we developed a high-throughput antiviral assay using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2). When tested against this reporter virus, remdesivir exhibited substantially more potent activity in A549-hACE2 cells compared to Vero E6 cells (EC50 0.115 vs 1.28 \u03bcM), while this difference was not observed for chloroquine (EC50 1.32 vs 3.52 \u03bcM), underscoring the importance of selecting appropriate cells for antiviral testing. Using the optimized SARS-CoV-2-Nluc assay, we evaluated a collection of approved and investigational antivirals and other anti-infective drugs. Nelfinavir, rupintrivir, and cobicistat were identified as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 \u03bcM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 \u03bcM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.22.165712","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.155457","pub_date":"2020-6-23","title":"Loss of pH switch unique to SARS-CoV2 supports unfamiliar virus pathology","abstract":"Cell surface receptor engagement is a critical aspect of viral infection. This paper compares the dynamics of virus-receptor interactions for SARS-CoV (CoV1) and CoV2. At low (endosomal) pH, the binding free energy landscape of CoV1 and CoV2 interactions with the angiotensin-converting enzyme 2 (ACE2) receptor is almost the same. However, at neutral pH the landscape is different due to the loss of a pH-switch (His445Lys) in the receptor binding domain (RBD) of CoV2 relative to CoV1. Namely, CoV1 stabilizes a transition state above the bound state. In situations where small external strains are applied by, say, shear flow in the respiratory system, the off rate of the viral particle is enhanced. As a result, CoV1 virions are expected to detach from cell surfaces in time scales that are much faster than the time needed for other receptors to reach out and stabilize virus attachment. On the other hand, the loss of this pH-switch, which sequence alignments show is unique to CoV2, eliminates the transition state and allows the virus to stay bound to the ACE2 receptor for time scales compatible with the recruitment of additional ACE2 receptors diffusing in the cell membrane. This has important implications for viral infection and its pathology. CoV1 does not trigger high infectivity in the nasal area because it either rapidly drifts down the respiratory tract or is exhaled. By contrast, this novel mutation in CoV2 should not only retain the infection in the nasal cavity until ACE2-rich cells are sufficiently depleted, but also require fewer particles for infection. This mechanism explains observed longer incubation times, extended period of viral shedding, and higher rate of transmission. These considerations governing viral entry suggest that number of ACE2-rich cells in human nasal mucosa, which should be significantly smaller for children (and females relative to males), should also correlate with onset of viral load that could be a determinant of higher virus susceptibility. Critical implications for the development of new vaccines to combat current and future pandemics that, like SARS-CoV2, export evolutionarily successful strains via higher transmission rates by viral retention in nasal epithelium are also discussed.","version":"1.1","doi":"10.1101/2020.06.16.155457","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.165324","pub_date":"2020-6-23","title":"Systemic analysis of putative SARS-CoV-2 entry and processing genes in cardiovascular tissues identifies a positive correlation of BSG with age in endothelial cells","abstract":"COVID-19, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread throughout the world with unprecedented global healthcare and socio-economic consequences. There is now an established secondary syndrome of COVID-19 characterised by thrombosis, vascular dysfunction and hypertension, seen in those most severely affected. Advancing age in adults is the single most significant risk factor for hospitalisation and death with COVID-19. In light of the cardiovascular/thrombotic sequalae associated with severe COVID-19 disease and the overwhelming risk that increased age carries, in this study, our aim was to obtain mechanistic insight by interrogating gene expression profiles in cardiovascular tissues and cells. Our focus was on the two putative receptors for SARS-CoV-2, ACE2 and BSG along with a selected range of genes thought to be involved in virus binding/processing. In this study we have made four important observations: (i)Cardiovascular tissues and/or endothelial cells express the required genes for SARS-CoV-2 infection, (ii) SASR-CoV-2 receptor pathways, ACE2/TMPRSS2 and BSG/PPIB(A) polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of SARS-CoV-2 host genes are, on the whole, relatively stable with age and (iv) notable exceptions were ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells. Our data support the idea that that BSG is the dominate pathway utilised by SARS-CoV-2 in endothelial cells and are the first to demonstrate a positive correlation with age. We suggest BSG expression in the vasculature is a critical driver which explains the heightened risk of severe disease and death observed in those >40 years of age. Since BSG is utilised by other pathogens our findings have implications beyond the current pandemic. Finally, because BSG is functions in a range of cardiovascular diseases and fibrosis, our observations may have relevance to our understanding of the diseases associated with aging.","version":"1.1","doi":"10.1101/2020.06.23.165324","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.165415","pub_date":"2020-6-23","title":"Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2","abstract":"The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 \u00b5g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the \u2018up\u2019 ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.","version":"1.1","doi":"10.1101/2020.06.23.165415","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165035","pub_date":"2020-6-23","title":"Oral epithelial expression of angiotensin converting enzyme-2: Implications for COVID-19 diagnosis and prognosis","abstract":"The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) uses the angiotensin converting enzyme (ACE)-2 as the host receptor for target cell entry. The extent and distribution of ACE-2 has been associated with the clinical symptoms of coronavirus disease (COVID)-19. Here we show by immunofluorescence analysis that the ACE2 is abundantly expressed in oral mucosa, particularly in the surface epithelial cells suggesting that these cells could represent sites of entry for SARS-CoV-2. Further, together with the reports on ACE2 ectodomain shedding, we discuss the rationale for the hypothesis that the ACE-2 measurement in saliva could be a marker for COVID-19 infection during early phase following SARS-CoV-2 exposure.","version":"1.1","doi":"10.1101/2020.06.22.165035","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165464","pub_date":"2020-6-23","title":"Accommodating individual travel history, global mobility, and unsampled diversity in phylogeography: a SARS-CoV-2 case study","abstract":"Spatiotemporal bias in genome sequence sampling can severely confound phylogeographic inference based on discrete trait ancestral reconstruction. This has impeded our ability to accurately track the emergence and spread of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Despite the availability of unprecedented numbers of SARS-CoV-2 genomes on a global scale, evolutionary reconstructions are hindered by the slow accumulation of sequence divergence over its relatively short transmission history. When confronted with these issues, incorporating additional contextual data may critically inform phylodynamic reconstructions. Here, we present a new approach to integrate individual travel history data in Bayesian phylogeographic inference and apply it to the early spread of SARS-CoV-2, while also including global air transportation data. We demonstrate that including travel history data for each SARS-CoV-2 genome yields more realistic reconstructions of virus spread, particularly when travelers from undersampled locations are included to mitigate sampling bias. We further explore methods to ameliorate the impact of sampling bias by augmenting the phylogeographic analysis with lineages from undersampled locations in the analyses. Our reconstructions reinforce specific transmission hypotheses suggested by the inclusion of travel history data, but also suggest alternative routes of virus migration that are plausible within the epidemiological context but are not apparent with current sampling efforts. Although further research is needed to fully examine the performance of our travel-aware phylogeographic analyses with unsampled diversity and to further improve them, they represent multiple new avenues for directly addressing the colossal issue of sample bias in phylogeographic inference.","version":"1.1","doi":"10.1101/2020.06.22.165464","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.150482","pub_date":"2020-6-23","title":"SARS-CoV-2 mutations altering regulatory properties: deciphering host\u2019s and virus\u2019s perspectives","abstract":"Since the first recorded case of the SARS-CoV-2, it has acquired several mutations in its genome while spreading throughout the globe. However, apart from some changes in protein coding, functional importance of these mutations in disease pathophysiology are still largely unknown. In this study, we investigated the significance of these mutations both from the host\u2019s and virus\u2019s perspective by analyzing the host miRNA binding and virus\u2019s internal ribosome entry site (IRES), respectively. Strikingly, we observed that due to the acquired mutations, host miRNAs bind differently compared to the reference; where few of the miRNAs lost and few gained the binding affinity for targeting the viral genome. Moreover, functional enrichment analysis suggests that targets of both of these gained and lost miRNAs might be involved in various host immune signaling pathways. Also, we sought to shed some insights on the impacts of mutations on the IRES structure of SARS-CoV-2. Remarkably, we detected that three particular mutations in the IRES can disrupt its secondary structure which can further make the virus less functional. These results could be valuable in exploring the functional importance of the mutations of SARS-CoV-2 and could provide novel insights into the differences observed different parts of the world.","version":"1.1","doi":"10.1101/2020.06.15.150482","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.162701","pub_date":"2020-6-23","title":"Green tea and Spirulina extracts inhibit SARS, MERS, and SARS-2 spike pseudotyped virus entry in vitro","abstract":"Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike (S) protein to bind to a host cell surface receptor. The rapid outbreak of emerging novel coronavirus, SARS-CoV 2 in China inculcates the need for the development of hasty and effective intervention strategies. Medicinal plants and natural compounds have been traditionally used to treat viral infections. Here, we generated VSV based pseudotyped viruses (pvs) of SARS-, MERS-, and SARS-2 CoVs to screen entry inhibitors from natural products. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind on their receptors and enter into the cells. SARS and MERS polyclonal antibodies neutralize SARSpv and SARS-2pv, and MERSpv respectively. Incubation of soluble ACE2 inhibited entry of SARS and SARS-2 pvs but not MERSpv. In addition, expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARSpv, SARS-2pv, and MERSpv respectively. Next, we showed the antiviral properties of known enveloped virus entry inhibitors, Spirulina and Green tea extracts against CoVpvs. SARSpv, MERSpv, and SARS-2pv entry were blocked with higher efficiency when preincubated with either green tea or spirulina extracts. Green tea provided a better inhibitory effect than the spirulina extracts by binding to the S1 domain of spike and blocking the interaction of spike with its receptor. Further studies are required to understand the exact mechanism of viral inhibition. In summary, we demonstrate that pseudotyped virus is an ideal tool for screening viral entry inhibitors. Moreover, spirulina and green tea could be promising antiviral agents against emerging viruses.","version":"1.1","doi":"10.1101/2020.06.20.162701","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.166033","pub_date":"2020-6-23","title":"Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes","abstract":"Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from \u223c0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.","version":"1.1","doi":"10.1101/2020.06.22.166033","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.164335","pub_date":"2020-6-23","title":"Expression of Ace2, Tmprss2, and Furin in mouse ear tissue","abstract":"Intracellular entry of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) depends on the interaction between its spike protein to a cellular receptor named angiotensin-converting enzyme 2 (ACE2) and depends on Furin-mediated spike 23 protein cleavage and spike protein priming by host cell proteases including 24 transmembrane protease serine 2 (TMPRSS2). Tmprss1, Tmprss3, and Tmprss5 are expressed in the spiral ganglion neurons and the organ of Corti in the inner ear; however, Ace2, Tmprss2, and Furin expression profiles in the middle ear remain unclear. Therefore, this study aimed to analyze Ace2, Tmprss2, and Furin expression in the middle and inner ear of mice. Animal research. Department of Otolaryngology and Head and Neck Surgery, University of Tokyo. We performed immunohistochemical analysis to examine the distribution of Ace2, Tmprss2, and Furin in the eustachian tube, middle ear space, and cochlea of mice. Ace2 was expressed in the cytoplasm in the middle ear epithelium, eustachian tube epithelium, stria vascularis, and spiral ganglion. Tmprss2 and Furin were widely expressed in the middle ear spaces and the cochlea. Co-expression of Ace2, Tmprss2, and Furin in the middle ear indicates that the middle ear is susceptible to SARS-CoV-2 infections, thus warranting the use of personal protective equipment during mastoidectomy for coronavirus disease (COVID-19) patients.","version":"1.1","doi":"10.1101/2020.06.23.164335","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.23.164947","pub_date":"2020-6-23","title":"A graph-based approach identifies dynamic H-bond communication networks in spike protein S of SARS-CoV-2","abstract":"Corona virus spike protein S is a large homo-trimeric protein embedded in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, has a relatively large H-bond cluster at the receptor binding domain, and a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.","version":"1.1","doi":"10.1101/2020.06.23.164947","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.163097","pub_date":"2020-6-22","title":"Female reproductive tract has low concentration of SARS-CoV2 receptors","abstract":"There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2. Our findings suggest that COVID-19 is unlikely to contribute to pregnancy-related adverse outcomes such as preterm birth, transmission of COVID-19 through breast milk, oogenesis and female fertility.","version":"1.1","doi":"10.1101/2020.06.20.163097","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.154930","pub_date":"2020-6-22","title":"SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients","abstract":"SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.","version":"1.2","doi":"10.1101/2020.06.19.154930","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.164442","pub_date":"2020-6-22","title":"Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins","abstract":"SARS-CoV-2 is having severe impact on public health at a global scale. Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. We show that CT scans of virus-positive pangolins reveal bilateral ground-glass opacities in lungs in similar manner to COVID-19 patients. The virus infected multiple organs in pangolins, with the lungs being the major target. Histological expression showed that ACE2 and TMPRSS2 are co-expressed with viral RNA. Transcriptome analysis revealed an inadequate interferon response, with different dysregulated chemokines and cytokines responses in pregnant and non-pregnant adults and fetuses. Viral RNA and protein were detected in three fetuses providing evidence for vertical virus transmission. In sum, our study identifies the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.","version":"1.1","doi":"10.1101/2020.06.22.164442","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.165225","pub_date":"2020-6-22","title":"In vivo antiviral host response to SARS-CoV-2 by viral load, sex, and age","abstract":"Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA-sequencing profiles of nasopharyngeal swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with upregulation of antiviral factors such as OAS1-3 and IFIT1-3, and Th1 chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial cultures replicated the in vivo antiviral host response. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of Th1 chemokines CXCL9/10/11 and their cognate receptor, CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B and NK cell-specific transcripts and an increase in inhibitors of NF-\u03baB signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.","version":"1.1","doi":"10.1101/2020.06.22.165225","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.21.162396","pub_date":"2020-6-22","title":"Calcitriol, the active form of vitamin D, is a promising candidate for COVID-19 prophylaxis","abstract":"COVID-19, the disease caused by SARS-CoV-2 (1), was declared a pandemic by the World Health Organization (WHO) in March 2020 (2). While awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4). To expand this arsenal, we screened 4 compound libraries: a United States Food and Drug Administration (FDA) approved drug library, an angiotensin converting enzyme-2 (ACE2) targeted compound library, a flavonoid compound library as well as a natural product library. Of the 121 compounds identified with activity against SARS-CoV-2, 7 were shortlisted for validation. We show for the first time that the active form of Vitamin D, calcitriol, exhibits significant potent activity against SARS-CoV-2. This finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients.","version":"1.1","doi":"10.1101/2020.06.21.162396","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.22.164681","pub_date":"2020-6-22","title":"Generation of SARS-CoV-2 S1 spike glycoprotein putative antigenic epitopes in vitro by intracellular aminopeptidases","abstract":"Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2 and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.","version":"1.1","doi":"10.1101/2020.06.22.164681","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.21.163550","pub_date":"2020-6-22","title":"Mathematical modeling explains differential SARS CoV-2 kinetics in lung and nasal passages in remdesivir treated rhesus macaques","abstract":"Remdesivir was recently demonstrated to decrease recovery time in hospitalized patients with SARS-CoV-2 infection. In rhesus macaques, early initiation of remdesivir therapy prevented pneumonia and lowered viral loads in the lung, but viral loads increased in the nasal passages five days after therapy. We developed mathematical models to explain these results. We identified that 1) drug potency is slightly higher in nasal passages than in lungs, 2) viral load decrease in lungs relative to nasal passages during therapy because of infection-dependent generation of refractory cells in the lung, 3) incomplete drug potency in the lung that decreases viral loads even slightly may allow substantially less lung damage, and 4) increases in nasal viral load may occur due to a slight blunting of peak viral load and subsequent decrease of the intensity of the innate immune response, as well as a lack of refractory cells. We also hypothesize that direct inoculation of the trachea in rhesus macaques may not recapitulate natural infection as lung damage occurs more abruptly in this model than in human infection. We demonstrate with sensitivity analysis that a drug with higher potency could completely suppress viral replication and lower viral loads abruptly in the nasal passages as well as the lung. We developed a mathematical model to explain why remdesivir has a greater antiviral effect on SARS CoV-2 in lung versus nasal passages in rhesus macaques.","version":"1.1","doi":"10.1101/2020.06.21.163550","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.163162","pub_date":"2020-6-21","title":"An Analysis of SARS-CoV-2 Using ViReport","abstract":"The ongoing outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of cases and hundreds of thousands of deaths. Given the current lack of treatments or vaccines available, it may be useful to trace the evolu-tion and spread of the virus to better develop methods of preventative intervention. In this study, we analyzed over 4,000 full genome sequences of human SARS-CoV-2 using novel tool ViReport [13], an automated workflow for performing phylogenetic analyses on viral sequences and generating comprehensive molecular epidemiologi-cal reports. The complete ViReport output can be found at https://github.com/mirandajsong/ViReport-SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.20.163162","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.21.163444","pub_date":"2020-6-21","title":"Characterization of 100 sequential SARS-CoV-2 convalescent plasma donations","abstract":"Transfusion of SARS-CoV-2 convalescent plasma is a promising treatment for severe COVID-19 cases, with success of the intervention based on neutralizing antibody content. Measurement by serological correlates without biocontainment needs, and an understanding of donor characteristics that may allow for targeting of more potent donors would greatly facilitate effective collection.","version":"1.1","doi":"10.1101/2020.06.21.163444","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.151761","pub_date":"2020-6-21","title":"Cumulative effect of aging and SARS-CoV2 infection on poor prognosis in the elderly: Insights from transcriptomic analysis of lung and blood","abstract":"The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) has affected millions of people worldwide and with notable heterogeneity in its clinical presentation. Probability of contracting this highly contagious infection is similar across age groups but disease severity and fatality among aged patients with or without comorbidities are higher. We hypothesized that SARS-CoV-2 infection may augment aging-related gene expression alterations resulting in severe outcomes in elderly patients. We performed a comparative analysis of publicly available transcriptome data from Broncho Alveolar Lavage Fluid (BALF)/lung/blood of healthy aging group with i) COVID-19 patients; and ii) data of host genes interacting with SARS-CoV-2 proteins. We observed i) a significant overlap of gene expression profiles of patients\u2019 BALF and blood with lung and blood of the healthy group respectively; ii) a more pronounced overlap in blood compared to lung; and iii) a similar overlap between host genes interacting with SARS-CoV-2 and aging blood transcriptome. Pathway enrichment analysis of overlapping gene sets suggests that infection alters expression of genes already dysregulated in the elderly, which together may lead to poor prognosis. eQTLs in these genes may also confer poor outcome in young patients worsening with age and co-morbidities. Furthermore, the pronounced overlap observed in blood may explain clinical symptoms including blood clots, strokes, heart attack, multi-organ failure etc. in severe cases. This model based on a limited patient dataset seems robust and holds promise for testing larger tissue specific datasets from patients with varied severity and across populations.","version":"1.2","doi":"10.1101/2020.06.15.151761","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.137687","pub_date":"2020-6-21","title":"SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and prolonged shedding of infectious virus from both respiratory and gastrointestinal tracts","abstract":"Vaccines are urgently needed to combat the global coronavirus disease 2019 (COVID-19) pandemic, and testing of candidate vaccines in an appropriate non-human primate (NHP) model is a critical step in the process. Infection of African green monkeys (AGM) with a low passage human isolate of SARS-CoV-2 by aerosol or mucosal exposure resulted in mild clinical infection with a transient decrease in lung tidal volume. Imaging with human clinical-grade 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) co-registered with computed tomography (CT) revealed pulmonary lesions at 4 days post-infection (dpi) that resolved over time. Infectious virus was shed from both respiratory and gastrointestinal (GI) tracts in all animals in a biphasic manner, first between 2-7 dpi followed by a recrudescence at 14-21 dpi. Viral RNA (vRNA) was found throughout both respiratory and gastrointestinal systems at necropsy with higher levels of vRNA found within the GI tract tissues. All animals seroconverted simultaneously for IgM and IgG, which has also been documented in human COVID-19 cases. Young AGM represent an excellent species to study mild/subclinical COVID-19 disease and have shed light on unknown aspects of long-term virus shedding. They are ideally suited for preclinical evaluation of candidate vaccines and therapeutic interventions. Subclinical infection of African green monkeys infected with SARS-CoV-2 results in prolonged shedding of infectious virus from both respiratory and gastrointestinal tracts.","version":"1.1","doi":"10.1101/2020.06.20.137687","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.21.163592","pub_date":"2020-6-21","title":"RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir","abstract":"We combine sequence analysis, molecular dynamics and hybrid quantum mechanics/molecular mechanics simulations to obtain the first description of the mechanism of reaction of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and of the inhibition of the enzyme by Remdesivir. Despite its evolutionary youth, the enzyme is highly optimized to have good fidelity in nucleotide incorporation and a good catalytic efficiency. Our simulations strongly suggest that Remdesivir triphosphate (the active form of drug) is incorporated into the nascent RNA replacing ATP, leading to a duplex RNA which is structurally very similar to an unmodified one. We did not detect any reason to explain the inhibitory activity of Remdesivir at the active site. Displacement of the nascent Remdesivir-containing RNA duplex along the exit channel of the enzyme can occur without evident steric clashes which would justify delayed inhibition. However, after the incorporation of three more nucleotides we found a hydrated Serine which is placed in a perfect arrangement to react through a Pinner\u2019s reaction with the nitrile group of Remdesivir. Kinetic barriers for crosslinking and polymerization are similar suggesting a competition between polymerization and inhibition. Analysis of SARS-CoV-2 mutational landscape and structural analysis of polymerases across different species support the proposed mechanism and suggest that virus has not explored yet resistance to Remdesivir inhibition.","version":"1.1","doi":"10.1101/2020.06.21.163592","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.162933","pub_date":"2020-6-21","title":"A path towards SARS-CoV-2 attenuation: metabolic pressure on CTP synthesis rules the virus evolution","abstract":"Fighting the COVID-19 epidemic summons deep understanding of the way SARS-CoV-2 taps into its host cell metabolic resources. We describe here the singular metabolic background that creates a bottleneck constraining coronaviruses to evolve towards likely attenuation in the long term. Cytidine triphosphate (CTP) is at the crossroad of the biosynthetic processes that allow the virus to multiply. This is because CTP is in demand for three essential steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope and, finally, it is a critical building block of the host transfer RNAs synthesis. The CCA 3\u2019-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate (UTP) that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. We also highlight and provide a raison d\u2019\u00eatre to viperin, an enzyme of innate antiviral immunity, which synthesizes 3\u2019-deoxy-3\u2032,4\u2019-didehydro-CTP (ddhCTP) as an extremely efficient antiviral nucleotide.","version":"1.1","doi":"10.1101/2020.06.20.162933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.21.163410","pub_date":"2020-6-21","title":"Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination","abstract":"Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently co-located with TRS-B sites than expected.","version":"1.1","doi":"10.1101/2020.06.21.163410","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.159715","pub_date":"2020-6-20","title":"Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19","abstract":"Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.","version":"1.1","doi":"10.1101/2020.06.20.159715","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.163006","pub_date":"2020-6-20","title":"Transcriptional response of signalling pathways to SARS-CoV-2 infection in normal human bronchial epithelial cells","abstract":"SARS-CoV-2 virus, the pathogen that causes Covid-19 disease, emerged in Wuhan region in China in 2019, infected more than 4M people and is responsible for death of at least 300K patients globally as of May 2020. Identification of the cellular response mechanisms to viral infection by SARS-CoV-2 may shed light on progress of the disease, indicate potential drug targets, and make design of new test methods possible. In this study, we analysed transcriptomic response of normal human bronchial epithelial cells (NHBE) to SARS-CoV-2 infection and compared the response to H1N1 infection. Comparison of transcriptome of NHBE cells 24 hours after mock-infection and SARS-CoV-2 infection demonstrated that most genes that respond to infection were upregulated (320 genes) rather than being downregulated (115 genes).While upregulated genes were enriched in signalling pathways related to virus response, downregulated genes are related to kidney development. We mapped the upregulated genes on KEGG pathways to identify the mechanisms that mediate the response. We identified canonical NF\u03baB, TNF and IL-17 pathways to be significantly upregulated and to converge to NF\u03baB pathway via positive feedback loops. Although virus entry protein ACE2 has low expression in NHBE cells, pathogen response pathways are strongly activated within 24 hours of infection. Our results also indicate that immune response system is activated at the early stage of the infection and orchestrated by a crosstalk of signalling pathways. Finally, we compared transcriptomic SARS-CoV-2 response to H1N1 response in NHBE cells to elucidate the virus specificity of the response and virus specific extracellular proteins expressed by NHBE cells.","version":"1.1","doi":"10.1101/2020.06.20.163006","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.160499","pub_date":"2020-6-20","title":"Analysis of SARS-CoV-2 specific T-cell receptors in ImmuneCode reveals cross-reactivity to immunodominant Influenza M1 epitope","abstract":"Adaptive Biotechnologies and Microsoft have recently partnered to release ImmuneCode, a database containing SARS-CoV-2 specific T-cell receptors derived through MIRA, a T-cell receptor (TCR) sequencing based sequencing approach to identify antigen-specific TCRs. Herein, we query the extent of cross reactivity between these derived SARS-CoV-2 specific TCRs and other known antigens present in McPas-TCR, a manually curated catalogue of pathology-associated TCRs. We reveal cross reactivity between SARS-CoV-2 specific TCRs and the immunodominant Influenza GILGFVFTL M1 epitope, suggesting the importance of further work in characterizing the implications of prior Influenza exposure or co-exposure to the pathology of SARS-CoV-2 illness.","version":"1.1","doi":"10.1101/2020.06.20.160499","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.159970","pub_date":"2020-6-20","title":"Rapid Inactivation of SARS-CoV-2 by Silicon Nitride, Copper, and Aluminum Nitride","abstract":"Viral disease spread by contaminated commonly touched surfaces is a global concern. Silicon nitride, an industrial ceramic that is also used as an implant in spine surgery, has known antibacterial activity. The mechanism of antibacterial action relates to the hydrolytic release of surface disinfectants. It is hypothesized that silicon nitride can also inactivate the coronavirus SARS-CoV-2. SARS-CoV-2 virions were exposed to 15 wt.% aqueous suspensions of silicon nitride, aluminum nitride, and copper particles. The virus was titrated by the TCD50 method using VeroE6/TMPRSS2 cells, while viral RNA was evaluated by real-time RT-PCR. Immunostaining and Raman spectroscopy were used as additional probes to investigate the cellular responses to virions exposed to the respective materials. All three tested materials showed >99% viral inactivation at one and ten minutes of exposure. Degradation of viral RNA was also observed with all materials. Immunofluorescence testing showed that silicon nitride-treated virus failed to infect VeroE6/TMPRSS2 cells without damaging them. In contrast, the copper-treated virus suspension severely damaged the cells due to copper ion toxicity. Raman spectroscopy indicated differential biochemical cellular changes due to infection and metal toxicity for two of the three materials tested. Silicon nitride successfully inactivated the SARS-CoV-2 in this study. The mechanism of action was the hydrolysis-mediated surface release of nitrogen-containing disinfectants. Both aluminum nitride and copper were also effective in the inactivation of the virus. However, while the former compound affected the cells, the latter compound had a cytopathic effect. Further studies are needed to validate these findings and investigate whether silicon nitride can be incorporated into personal protective equipment and commonly touched surfaces, as a strategy to discourage viral persistence and disease spread.","version":"1.1","doi":"10.1101/2020.06.19.159970","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.156224","pub_date":"2020-6-20","title":"Structural variability, expression profile and pharmacogenetics properties of TMPRSS2 gene as a potential target for COVID-19 therapy","abstract":"The human serine protease TMPRSS2 gene is involved in the priming of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins being one of the possible targets for COVID-19 therapy. TMPRSS2 gene is possibly co-expressed with SARS-CoV-2 cell receptor genes ACE2 and BSG, but only TMPRSS2 demonstrates tissue-specific expression in alveolar cells according to single cell RNA sequencing data. Our analysis of the structural variability of the TMPRSS2 gene based on genome-wide data of 76 human populations demonstrates that functionally significant missense mutation in exon 6/7 in TMPRSS2 gene, was found in many human populations in relatively high frequency, featuring region-specific distribution patterns. The frequency of the missense mutation encoded by the rs12329760, which previously was found to be associated with prostate cancer, is ranged between 10% and 63% being significantly higher in populations of Asian origin compared to European populations. In addition to SNPs, two copy numbers variants (CNV) were detected in the TMPRSS2 gene. Number of microRNAs have been predicted to regulate TMPRSS2 and BSG expression levels, but none of them is enriched in lung or respiratory tract cells. Several well studied drugs can downregulate the expression of TMPRSS2 in human cells, including Acetaminophen (Paracetamol) and Curcumin. Thus TMPRSS2 interaction with the SARS-CoV-2, its structural variability, gene-gene interactions, and expression regulation profiles, and pharmacogenomics properties characterize this gene as a potential target for COVID-19 therapy.","version":"1.1","doi":"10.1101/2020.06.20.156224","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.20135491","pub_date":"2020-06-20","title":"Inhaled corticosteroid use and risk COVID-19 related death among 966,461 patients with COPD or asthma: an OpenSAFELY analysis","abstract":"<jats:title>Abstract</jats:title>\n                <jats:sec>\n                  <jats:title>Background</jats:title>\n                  <jats:p>Early descriptions of the coronavirus outbreak showed a lower prevalence of asthma and COPD than was expected for people diagnosed with COVID-19, leading to speculation that inhaled corticosteroids (ICS) may protect against infection with SARS-CoV-2, and development of serious sequelae. We evaluated the association between ICS and COVID-19 related death using linked electronic health records in the UK.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>We conducted cohort studies on two groups of people (COPD and asthma) using the OpenSAFELY platform to analyse data from primary care practices linked to national death registrations. People receiving an ICS were compared to those receiving alternative respiratory medications. Our primary outcome was COVID-19 related death.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Findings</jats:title>\n                  <jats:p>We identified 148,588 people with COPD and 817,973 people with asthma receiving relevant respiratory medications in the four months prior to 01 March 2020. People with COPD receiving ICS were at a greater risk of COVID-19 related death compared to those receiving a long-acting beta agonist (LABA) and a long-acting muscarinic antagonist (LAMA) (adjusted HR = 1.38, 95% CI = 1.08 \u2013 1.75). People with asthma receiving high dose ICS were at an increased risk of death compared to those receiving a short-acting beta agonist (SABA) only (adjusted HR = 1.52, 95%CI = 1.08 \u2013 2.14); the adjusted HR for those receiving low-medium dose ICS was 1.10 (95% CI = 0.82 \u2013 1.49). Quantitative bias analyses indicated that an unmeasured confounder of only moderate strength of association with exposure and outcome could explain the observed associations in both populations.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Interpretation</jats:title>\n                  <jats:p>These results do not support a major role of ICS in protecting against COVID-19 related deaths. Observed increased risks of COVID-19 related death among people with COPD and asthma receiving ICS can be plausibly explained by unmeasured confounding due to disease severity.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>This work was supported by the Medical Research Council MR/V015737/1.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.06.19.20135491","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.19.162529","pub_date":"2020-6-20","title":"The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus","abstract":"Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3\u2019-to-5\u2019 exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless \u2013 in some cases \u2013 reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV. The bifunctional nsp14 subunit of the coronavirus replicase contains 3\u2019-to-5\u2019 exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.","version":"1.1","doi":"10.1101/2020.06.19.162529","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.20.163030","pub_date":"2020-6-20","title":"Engineered human mesenchymal stem cells as new vaccine platform for COVID-19","abstract":"Recently, there are several routes for COVID-19 vaccine research, yet their weaknesses lie in low efficiency, tolerability, immune adaptability and safety. We describe a new approach to COVID-19 based on engineered human mesenchymal stem cells(hu-MSC), which is like a small protein antigen response device, but will be gradually cleared and degraded by body\u2019s immune system among recognization process. The antibody response results show that this is effective and fast. Furthermore, after several antibody response tests, we obtained an injection of a set of cocktail-like modified human mesenchymal stem cell line. This strategy opened a new avenue for vaccine design against COVID-19.","version":"1.1","doi":"10.1101/2020.06.20.163030","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.160655","pub_date":"2020-6-19","title":"A single dose of recombinant VSV-\u0394G-spike vaccine provides protection against SARS-CoV-2 challenge","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-\u0394G-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-\u0394G-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-\u0394G-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters\u2019 lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-\u0394G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.06.18.160655","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.160614","pub_date":"2020-6-19","title":"Mechanism and inhibition of SARS-CoV-2 PLpro","abstract":"Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.","version":"1.1","doi":"10.1101/2020.06.18.160614","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.160879","pub_date":"2020-6-19","title":"Cross-neutralization activity against SARS-CoV-2 is present in currently available intravenous immunoglobulins","abstract":"There is a crucial need for effective therapies that are immediately available to counteract COVID-19 disease. Recently, ELISA binding cross-reactivity against components of human epidemic coronaviruses with currently available intravenous immunoglobulins (IVIG) Gamunex-C and Flebogamma DIF (5% and 10%) have been reported. In this study, the same products were tested for neutralization activity against SARS-CoV-2, SARS-CoV and MERS-CoV and their potential as an antiviral therapy. The neutralization capacity of six selected lots of IVIG was assessed against SARS-CoV-2 (two different isolates), SARS-CoV and MERS-CoV in cell cultures. Infectivity neutralization was measured by determining the percent reduction in plaque-forming units (PFU) and by cytopathic effects for two IVIG lots in one of the SARS-CoV-2 isolates. Neutralization was quantified using the plaque reduction neutralization test 50 (PRNT50) in the PFU assay and the half maximal inhibitory concentration (IC50) in the cytopathic/cytotoxic method (calculated as the minus log10 dilution which reduced the viral titer by 50%). All IVIG preparations showed neutralization of both SARS-CoV-2 isolates, ranging from 79 to 89.5% with PRNT50 titers from 4.5 to >5 for the PFU method and ranging from 47.0%-64.7% with an IC50 ~1 for the cytopathic method. All IVIG lots produced neutralization of SARS-CoV ranging from 39.5 to 55.1 % and PRNT50 values ranging from 2.0 to 3.3. No IVIG preparation showed significant neutralizing activity against MERS-CoV. In cell culture neutralization assays, the tested IVIG products contain antibodies with significant cross-neutralization capacity against SARS-CoV-2 and SARS-CoV. However, no neutralization capacity was demonstrated against MERS-CoV. These preparations are currently available and may be immediately useful for COVID-19 management.","version":"1.1","doi":"10.1101/2020.06.19.160879","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.139329","pub_date":"2020-6-19","title":"The hypothalamus as a hub for SARS-CoV-2 brain infection and pathogenesis","abstract":"Most patients with COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), display neurological symptoms, and respiratory failure in certain cases could be of extra-pulmonary origin. Hypothalamic neural circuits play key roles in sex differences, diabetes, hypertension, obesity and aging, all risk factors for severe COVID-19, besides being connected to olfactory/gustative and brainstem cardiorespiratory centers. Here, human brain gene-expression analyses and immunohistochemistry reveal that the hypothalamus and associated regions express angiotensin-converting enzyme 2 and transmembrane proteinase, serine 2, which mediate SARS-CoV-2 cellular entry, in correlation with genes or pathways involved in physiological functions or viral pathogenesis. A post-mortem patient brain shows viral invasion and replication in both the olfactory bulb and the hypothalamus, while animal studies indicate that sex hormones and metabolic diseases influence this susceptibility.","version":"1.2","doi":"10.1101/2020.06.08.139329","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.160648","pub_date":"2020-6-19","title":"Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates","abstract":"The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.","version":"1.1","doi":"10.1101/2020.06.18.160648","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161612","pub_date":"2020-6-19","title":"Disruption of Adaptive Immunity Enhances Disease in SARS-CoV-2 Infected Syrian Hamsters","abstract":"Animal models recapitulating human COVID-19 disease, especially with severe disease, are urgently needed to understand pathogenesis and evaluate candidate vaccines and therapeutics. Here, we develop novel severe disease animal models for COVID-19 involving disruption of adaptive immunity in Syrian hamsters. Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters were exposed to SARS-CoV-2 by the respiratory route. Both the CyP-treated and RAG2 KO hamsters developed clinical signs of disease that were more severe than in immunocompetent hamsters, notably weight loss, viral loads, and fatality (RAG2 KO only). Disease was prolonged in transiently immunosuppressed hamsters and uniformly lethal in RAG2 KO hamsters. We evaluated the protective efficacy of a neutralizing monoclonal antibody and found that pretreatment, even in immunosuppressed animals, limited infection. Our results suggest that functional B and/or T cells are not only important for the clearance of SARS-CoV-2, but also play an early role in protection from acute disease. An antibody targeting the spike protein of SARS-CoV-2 limits infection in immunosuppressed Syrian hamster models.","version":"1.1","doi":"10.1101/2020.06.19.161612","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.159053","pub_date":"2020-6-19","title":"Antiviral treatment of SARS-CoV-2-infected hamsters reveals a weak effect of favipiravir and a complete lack of effect for hydroxychloroquine","abstract":"SARS-CoV-2 rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus was able to infect millions of people. To date, close to half a million patients succumbed to the viral disease, COVID-19. The high need for treatment options, together with the lack of small animal models of infection has led to clinical trials with repurposed drugs before any preclinical in vivo evidence attesting their efficacy was available. We used Syrian hamsters to establish a model to evaluate antiviral activity of small molecules in both an infection and a transmission setting. Upon intranasal infection, the animals developed high titers of SARS-CoV-2 in the lungs and pathology similar to that observed in mild COVID-19 patients. Treatment of SARS-CoV-2-infected hamsters with favipiravir or hydroxychloroquine (with and without azithromycin) resulted in respectively a mild or no reduction in viral RNA and infectious virus. Micro-CT scan analysis of the lungs showed no improvement compared to non-treated animals, which was confirmed by histopathology. In addition, both compounds did not prevent virus transmission through direct contact and thus failed as prophylactic treatments. By modelling the PK profile of hydroxychloroquine based on the trough plasma concentrations, we show that the total lung exposure to the drug was not the limiting factor. In conclusion, we here characterized a hamster infection and transmission model to be a robust model for studying in vivo efficacy of antiviral compounds. The information acquired using hydroxychloroquine and favipiravir in this model is of critical value to those designing (current and) future clinical trials. At this point, the data here presented on hydroxychloroquine either alone or combined with azithromycin (together with previously reported in vivo data in macaques and ferrets) provide no scientific basis for further use of the drug in humans.","version":"1.1","doi":"10.1101/2020.06.19.159053","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161141","pub_date":"2020-6-19","title":"High-density amplicon sequencing identifies community spread and ongoing evolution of SARS-CoV-2 in the Southern United States","abstract":"SARS-CoV-2 is constantly evolving. Prior studies have focused on high case-density locations, such as the Northern and Western metropolitan areas in the U.S. This study demonstrates continued SARS-CoV-2 evolution in a suburban Southern U.S. region by high-density amplicon sequencing of symptomatic cases. 57% of strains carried the spike D614G variant. The presence of D614G was associated with a higher genome copy number and its prevalence expanded with time. Four strains carried a deletion in a predicted stem loop of the 3\u2019 untranslated region. The data are consistent with community spread within the local population and the larger continental U.S. No strain had mutations in the target sites used in common diagnostic assays. The data instill confidence in the sensitivity of current tests and validate \u201ctesting by sequencing\u201d as a new option to uncover cases, particularly those not conforming to the standard clinical presentation of COVID-19. This study contributes to the understanding of COVID-19 by providing an extensive set of genomes from a non-urban setting and further informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the U.S.","version":"1.1","doi":"10.1101/2020.06.19.161141","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161000","pub_date":"2020-6-19","title":"Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: a proof-of-concept focused on a 3 min mass spectrometry window","abstract":"Rapid but yet sensitive, specific and high-throughput detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is key to diagnose infected people and to better control the spread of the virus. Alternative methodologies to PCR and immunodiagnostic that would not require specific reagents are worth to investigate not only for fighting the COVID-19 pandemic, but also to detect other emergent pathogenic threats. Here, we propose the use of tandem mass spectrometry to detect SARS-CoV-2 marker peptides in nasopharyngeal swabs. We documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. Simili nasopharyngeal swabs spiked with different quantities of purified SARS-CoV-2 viral material were used to develop a nanoLC-MS/MS acquisition method, which was then successfully applied on COVID-19 clinical samples. We argue that peptides ADETQALPQR and GFYAQGSR from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a 3 min window in the tested conditions. These results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry.","version":"1.1","doi":"10.1101/2020.06.19.161000","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.109702","pub_date":"2020-6-19","title":"SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva","abstract":"We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7\u00d7106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.","version":"1.2","doi":"10.1101/2020.05.29.109702","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161802","pub_date":"2020-6-19","title":"An in-silico based clinical insight on the effect of noticeable CD4 conserved residues of SARS-CoV-2 on the CD4-MHC-II interactions","abstract":"The study is aimed to unveil the conserved residues of CD4 in the context of its purposeful interaction with MHC-II at the receptor-binding domain (RBD) of SARS-CoV-2 compared with the envelope (Env) glycoprotein (gp) 120 of HIV-1. The paired CD4 conserved residues, including the matched CD4 interacting MHC-II epitopes of the structural viral protein domains, were chosen for the protein modelling using the SWISS-MODEL online server. Energy minimization and structural validation of the modelled viral protein domains, including the CD4 and MHC-II protein were achieved by CHIMERA and PROCHECK-Ramachandran Plot respectively. Protein-protein docking was performed by the HADDOCK online tool. The binding affinity score was measured using the PRODIGY online server. As per our docking report, the Env gp120 of HIV-1 with three identical and three conserved residues of CD4 exhibited the highest binding affinity (\u221213.9 kcal/mol) with MHC-II than the second-highest RBD-S1-SARS-CoV-2 (\u221212.5 kcal/mol) with three identical and a single conserved residue of CD4. With a noticeable single salt bridge formation identified at the interacting residues Lys305 (of Env gp120-HIV-1) and Glu139 (of MHC-II); the Env gp120 interaction with MHC-II occupied the crucial His144 and Glu194 (salt-bridge) interacting residues of CD4 with the measured buried surface area 2554.8\u00b140.8 \u00c52. Similarly, the RBD-S1-SARS-CoV-2-MHC-II complex showed two salt bridge formations at the residue sites: 1) Arg567 (of SARS-CoV-2)-Glu194 (of MHC-II) 2) 2) Asp568(of SARS-CoV-2)-Arg165 (of MHC-II) with the increased buried surface area of 1910.9\u00b197.1 \u00c52 over the SARS-CoV score 1708.2\u00b150.8 \u00c52; that camouflaged all crucial CD4 interacting residues of MHC-II. In conclusion, the noticeable conserved residues of CD4 at the RBD-S1 sites of SARS-CoV-2 could interrupt the aspired CD4-MHC-II interactions of adaptive immune activation.","version":"1.1","doi":"10.1101/2020.06.19.161802","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.158105","pub_date":"2020-6-19","title":"Dog Savior: Immediate Scent-Detection of SARS-COV-2 by Trained Dogs","abstract":"Molecular tests for viral diagnostics are essential to confront the COVID-19 pandemic, but their production and distribution cannot satisfy the current high demand. Early identification of infected people and their contacts is the key to being able to isolate them and prevent the dissemination of the pathogen; unfortunately, most countries are unable to do this due to the lack of diagnostic tools. Dogs can identify, with a high rate of precision, unique odors of volatile organic compounds generated during an infection; as a result, dogs can diagnose infectious agents by smelling specimens and, sometimes, the body of an infected individual. We trained six dogs of three different breeds to detect SARS-CoV-2 in respiratory secretions of infected patients and evaluated their performance experimentally, comparing it against the gold standard (rRT-PCR). Here we show that viral detection takes one second per specimen. After scent-interrogating 9,200 samples, our six dogs achieved independently and as a group very high sensitivity, specificity, predictive values, accuracy, and likelihood ratio, with very narrow confidence intervals. The highest metric was the negative predictive value, indicating that with a disease prevalence of 7.6%, 99.9% of the specimens indicated as negative by the dogs did not carry the virus. These findings demonstrate that dogs could be useful to track viral infection in humans, allowing COVID-19 free people to return to work safely.","version":"1.1","doi":"10.1101/2020.06.17.158105","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.161042","pub_date":"2020-6-19","title":"Drug repurposing screens reveal FDA approved drugs active against SARS-Cov-2","abstract":"There are an urgent need for antivirals to treat the newly emerged SARS-CoV-2. To identify new candidates we screened a repurposing library of ~3,000 drugs. Screening in Vero cells found few antivirals, while screening in human Huh7.5 cells validated 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we found that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH-independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we found 9 drugs are antiviral in lung cells, 7 of which have been tested in humans, and 3 are FDA approved including Cyclosporine which we found is targeting Cyclophilin rather than Calcineurin for its antiviral activity. These antivirals reveal essential host targets and have the potential for rapid clinical implementation.","version":"1.1","doi":"10.1101/2020.06.19.161042","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.145086","pub_date":"2020-6-19","title":"Investigation of the effect of temperature on the structure of SARS-Cov-2 Spike Protein by Molecular Dynamics Simulations","abstract":"Statistical and epidemiological data imply temperature sensitivity of the SARS-CoV-2 coronavirus. However, the molecular level understanding of the virus structure at different temperature is still not clear. Spike protein is the outermost structural protein of the SARS-CoV-2 virus which interacts with the Angiotensin Converting Enzyme 2 (ACE2), a human receptor, and enters the respiratory system. In this study, we performed an all atom molecular dynamics simulation to study the effect of temperature on the structure of the Spike protein. After 200ns of simulation at different temperatures, we came across some interesting phenomena exhibited by the protein. We found that the solvent exposed domain of Spike protein, namely S1, is more mobile than the transmembrane domain, S2. Structural studies implied the presence of several charged residues on the surface of N-terminal Domain of S1 which are optimally oriented at 10-30 \u00b0C. Bioinformatics analyses indicated that it is capable of binding to other human receptors and should not be disregarded. Additionally, we found that receptor binding motif (RBM), present on the receptor binding domain (RBD) of S1, begins to close around temperature of 40 \u00b0C and attains a completely closed conformation at 50 \u00b0C. The closed conformation disables its ability to bind to ACE2, due to the burying of its receptor binding residues. Our results clearly show that there are active and inactive states of the protein at different temperatures. This would not only prove beneficial for understanding the fundamental nature of the virus, but would be also useful in the development of vaccines and therapeutics. Statistical and epidemiological evidence show that external climatic conditions influence the SARS-CoV infectivity, but we still lack a molecular level understanding of the same. Here, we study the influence of temperature on the structure of the Spike glycoprotein, the outermost structural protein, of the virus which binds to the human receptor ACE2. Results show that the Spike\u2019s S1 domain is very sensitive to external atmospheric conditions compared to the S2 transmembrane domain. The N-terminal domain comprises of several solvent exposed charged residues that are capable of binding to human proteins. The region is specifically stable at temperatures ranging around 10-30\u00b0 C. The Receptor Binding Motif adopts a closed conformation at 40\u00b0C and completely closes at higher temperatures making it unsuitable of binding to human receptors","version":"1.2","doi":"10.1101/2020.06.10.145086","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.160671","pub_date":"2020-6-19","title":"Identification of a critical horseshoe-shaped region in the nsp5 (Mpro, 3CLpro) protease interdomain loop (IDL) of coronavirus mouse hepatitis virus (MHV)","abstract":"Human coronaviruses are enveloped, positive-strand RNA viruses which cause respiratory diseases ranging in severity from the seasonal common cold to SARS and COVID-19. Of the 7 human coronaviruses discovered to date, 3 emergent and severe human coronavirus strains (SARS-CoV, MERS-CoV, and SARS-CoV-2) have recently jumped to humans in the last 20 years. The COVID-19 pandemic spawned by the emergence of SARS-CoV-2 in late 2019 has highlighted the importance for development of effective therapeutics to target emerging coronaviruses. Upon entry, the replicase genes of coronaviruses are translated and subsequently proteolytically processed by virus-encoded proteases. Of these proteases, nonstructural protein 5 (nsp5, Mpro, or 3CLpro), mediates the majority of these cleavages and remains a key drug target for therapeutic inhibitors. Efforts to develop nsp5 active-site inhibitors for human coronaviruses have thus far been unsuccessful, establishing the need for identification of other critical and conserved non-active-site regions of the protease. In this study, we describe the identification of an essential, conserved horseshoe-shaped region in the nsp5 interdomain loop (IDL) of mouse hepatitis virus (MHV), a common coronavirus replication model. Using site-directed mutagenesis and replication studies, we show that several residues comprising this horseshoe-shaped region either fail to tolerate mutagenesis or were associated with viral temperature-sensitivity. Structural modeling and sequence analysis of these sites in other coronaviruses, including all 7 human coronaviruses, suggests that the identified structure and sequence of this horseshoe regions is highly conserved and may represent a new, non-active-site regulatory region of the nsp5 (3CLpro) protease to target with coronavirus inhibitors. In December 2019, a novel coronavirus (SARS-CoV-2) emerged in humans and triggered a pandemic which has to date resulted in over 8 million confirmed cases of COVID-19 across more than 180 countries and territories (June 2020). SARS-CoV-2 represents the third emergent coronavirus in the past 20 years and the future emergence of new coronaviruses in humans remains certain. Critically, there remains no vaccine nor established therapeutics to treat cases of COVID-19. The coronavirus nsp5 protease is a conserved and indispensable virus-encoded enzyme which remains a key target for therapeutic design. However, past attempts to target the active site of nsp5 with inhibitors have failed stressing the need to identify new conserved non-active-site targets for therapeutic development. This study describes the discovery of a novel conserved structural region of the nsp5 protease of coronavirus mouse hepatitis virus (MHV) which may provide a new target for coronavirus drug development.","version":"1.1","doi":"10.1101/2020.06.18.160671","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.158717","pub_date":"2020-6-19","title":"No evidence of coronaviruses or other potentially zoonotic viruses in Sunda pangolins (Manis javanica) entering the wildlife trade via Malaysia","abstract":"The legal and illegal trade in wildlife for food, medicine and other products is a globally significant threat to biodiversity that is also responsible for the emergence of pathogens that threaten human and livestock health and our global economy. Trade in wildlife likely played a role in the origin of COVID-19, and viruses closely related to SARS-CoV-2 have been identified in bats and pangolins, both traded widely. To investigate the possible role of pangolins as a source of potential zoonoses, we collected throat and rectal swabs from 334 Sunda pangolins (Manis javanica) confiscated in Peninsular Malaysia and Sabah between August 2009 and March 2019. Total nucleic acid was extracted for viral molecular screening using conventional PCR protocols used to routinely identify known and novel viruses in extensive prior sampling (>50,000 mammals). No sample yielded a positive PCR result for any of the targeted viral families \u2013 Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae and Paramyxoviridae. In light of recent reports of coronaviruses including a SARS-CoV-2 related virus in Sunda pangolins in China, the lack of any coronavirus detection in our \u2018upstream\u2019 market chain samples suggests that these detections in \u2018downstream\u2019 animals more plausibly reflect exposure to infected humans, wildlife or other animals within the wildlife trade network. While confirmatory serologic studies are needed, it is likely that Sunda pangolins are incidental hosts of coronaviruses. Our findings further support the importance of ending the trade in wildlife globally.","version":"1.1","doi":"10.1101/2020.06.19.158717","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.19.160606","pub_date":"2020-6-19","title":"Phylogenetic pattern of SARS-CoV-2 from COVID-19 patients from Bosnia and Herzegovina: lessons learned to optimize future molecular and epidemiological approaches","abstract":"Whole Genome Sequence of four samples from COVID-19 outbreaks was done in two laboratories in Bosnia and Herzegovina (Veterinary Faculty Sarajevo and Alea Genetic Center). All four BiH sequences cluster mainly with European ones (Italy, Austria, France, Sweden, Cyprus, England). The constructed phylogenetic tree indicates probable multiple independent introduction events. The success of future containment measures concernig new introductions will be highly challenging for country due to the significant proportion of BH population living abroad.","version":"1.1","doi":"10.1101/2020.06.19.160606","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.157081","pub_date":"2020-6-19","title":"Prefusion spike protein stabilization through computational mutagenesis","abstract":"A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has emerged as a human pathogen, causing global pandemic and resulting in over 400,000 deaths worldwide. The surface spike protein of SARS-CoV-2 mediates the process of coronavirus entry into human cells by binding angiotensin-converting enzyme 2 (ACE2). Due to the critical role in viral-host interaction and the exposure of spike protein, it has been a focus of most vaccines\u2019 developments. However, the structural and biochemical studies of the spike protein are challenging because it is thermodynamically metastable. Here, we develop a new pipeline that automatically identifies mutants that thermodynamically stabilize the spike protein. Our pipeline integrates bioinformatics analysis of conserved residues, motion dynamics from molecular dynamics simulations, and other structural analysis to identify residues that significantly contribute to the thermodynamic stability of the spike protein. We then utilize our previously developed protein design tool, Eris, to predict thermodynamically stabilizing mutations in proteins. We validate the ability of our pipeline to identify protein stabilization mutants through known prefusion spike protein mutants. We finally utilize the pipeline to identify new prefusion spike protein stabilization mutants.","version":"1.1","doi":"10.1101/2020.06.17.157081","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.151845","pub_date":"2020-6-19","title":"Predicting mammalian hosts in which novel coronaviruses can be generated","abstract":"Novel pathogenic coronaviruses \u2013 including SARS-CoV and SARS-CoV-2 \u2013 arise by homologous recombination in a host cell. This process requires a single host to be infected with more than one type of coronavirus, which recombine to form novel strains of virus with unique combinations of genetic material. Identifying possible sources of novel coronaviruses requires identifying hosts (termed recombination hosts) of more than one coronavirus type, in which recombination might occur. However, the majority of coronavirus-host interactions remain unknown, and therefore the vast majority of recombination hosts for coronaviruses cannot be identified. Here we show that there are 11.5-fold more coronavirus-host associations, and over 30-fold more potential SARS-CoV-2 recombination hosts, than have been observed to date. We show there are over 40-fold more host species with four or more different subgenera of coronaviruses. This underestimation of both number and novel coronavirus generation in wild and domesticated animals. Our results list specific high-risk hosts in which our model predicts homologous recombination could occur, our model identifies both wild and domesticated mammals including known important and understudied species. We recommend these species for coronavirus surveillance, as well as enforced separation in livestock markets and agriculture.","version":"1.2","doi":"10.1101/2020.06.15.151845","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.147470","pub_date":"2020-6-19","title":"Dissecting the common and compartment-specific features of COVID-19 severity in the lung and periphery with single-cell resolution","abstract":"As the global COVID-19 pandemic continues to escalate, no effective treatment has yet been developed for the severe respiratory complications of this disease. This may be due in large part to the unclear immunopathological basis for the development of immune dysregulation and acute respiratory distress syndrome (ARDS) in severe and critical patients. Specifically, it remains unknown whether the immunological features of the disease that have been identified so far are compartment-specific responses or general features of COVID-19. Additionally, readily detectable biological markers correlated with strata of disease severity that could be used to triage patients and inform treatment options have not yet been identified. Here, we leveraged publicly available single-cell RNA sequencing data to elucidate the common and compartment-specific immunological features of clinically severe COVID-19. We identified a number of transcriptional programs that are altered across the spectrum of disease severity, few of which are common between the lung and peripheral immune environments. In the lung, comparing severe and moderate patients revealed severity-specific responses of enhanced interferon, A20/I\u03baB, IL-2, and IL-6 pathway signatures along with broad signaling activity of IFNG, SPP1, CCL3, CCL8, and IL18 across cell types. These signatures contrasted with features unique to ARDS observed in the blood compartment, which included depletion of interferon and A20/I\u03baB signatures and a lack of IL-6 response. The cell surface marker S1PR1 was strongly upregulated in patients diagnosed with ARDS compared to non-ARDS patients in \u03b3\u03b4 T cells of the blood compartment, and we nominate S1PR1 as a potential marker for immunophenotyping ARDS in COVID-19 patients using flow cytometry. COVID-19 disease severity is associated with a number of compositional shifts in the cellular makeup of the blood and lung environments. Transcriptional data suggest differentially expressed cell surface proteins as markers for COVID-19 immunophenotyping from BALF and PBMC samples. Severity-specific features COVID-19 manifest at the pathway level, suggesting distinct changes to epithelia and differences between local and systemic immune dynamics. Immune-epithelial cellular communication analysis identifies ligands implicated in transcriptional regulation of proto-oncogenes in the lung epithelia of severe COVID-19 patients. Network analysis suggests broadly-acting dysregulatory ligands in the pulmonary microenvironment as candidate therapeutic targets for the treatment of severe COVID-19.","version":"1.2","doi":"10.1101/2020.06.15.147470","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088013","pub_date":"2020-6-19","title":"Local computational methods to improve the interpretability and analysis of cryo-EM maps","abstract":"Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, \u2018over-sharpening\u2019 due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.","version":"1.2","doi":"10.1101/2020.05.11.088013","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.159202","pub_date":"2020-6-18","title":"Divergent SARS-CoV-2-specific T and B cell responses in severe but not mild COVID-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding both the immunological processes providing specific immunity and potential immunopathology underlying the pathogenesis of this disease may provide valuable insights for potential therapeutic interventions. Here, we quantified SARS-CoV-2 specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. The observed disparate T and B cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.","version":"1.1","doi":"10.1101/2020.06.18.159202","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.156471","pub_date":"2020-6-18","title":"The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins","abstract":"SARS-CoV-2 emerged in late 2019, leading to the COVID-19 pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus we demonstrate that, in addition to human ACE2, the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the twenty-two different hosts we investigated, ACE2 proteins from dog, cat and rabbit were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the three genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Interestingly, while SARS-CoV-2 pseudoparticle entry was inefficient in cells bearing the ACE2 receptor from bats or birds the live virus was still able to enter these cells, albeit with markedly lower efficiency. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock and wildlife.","version":"1.1","doi":"10.1101/2020.06.17.156471","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.159434","pub_date":"2020-6-18","title":"Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction","abstract":"Convenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations.","version":"1.1","doi":"10.1101/2020.06.18.159434","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.158527","pub_date":"2020-6-18","title":"SARS-CoV-2 assays to detect functional antibody responses that block ACE2 recognition in vaccinated animals and infected patients","abstract":"SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a global pandemic of COVID-19 resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >8 million people worldwide with >2 million cases in the US (June 17th, 2020). There is an urgent need for vaccines and therapeutics to combat the spread of this coronavirus. Similarly, the development of diagnostic and research tools to determine infection and vaccine efficacy are critically needed. Molecular assays have been developed to determine viral genetic material present in patients. Serological assays have been developed to determine humoral responses to the spike protein or receptor binding domain (RBD). Detection of functional antibodies can be accomplished through neutralization of live SARS-CoV2 virus, but requires significant expertise, an infectible stable cell line, a specialized BioSafety Level 3 (BSL-3) facility. As large numbers of people return from quarantine, it is critical to have rapid diagnostics that can be widely adopted and employed to assess functional antibody levels in the returning workforce. This type of surrogate neutralization diagnostic can also be used to assess humoral immune responses induced in patients from the large number of vaccine and immunotherapy trials currently on-going. Here we describe a rapid serological diagnostic assay for determining antibody receptor blocking and demonstrate the broad utility of the assay by measuring the antibody functionality of sera from small animals and non-human primates immunized with an experimental SARS-CoV-2 vaccine and using sera from infected patients.","version":"1.1","doi":"10.1101/2020.06.17.158527","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.06.138339","pub_date":"2020-6-18","title":"SARS-CoV-2 Whole Genome Amplification and Sequencing for Effective Population-Based Surveillance and Control of Viral Transmission","abstract":"With the gradual reopening of economies and resumption of social life, robust surveillance mechanisms should be implemented to control the ongoing COVID-19 pandemic. Unlike RT-qPCR, SARS-CoV-2 Whole Genome Sequencing (cWGS) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. However, practical and cost considerations of cWGS should be addressed before it can be widely implemented. We performed shotgun transcriptome sequencing using RNA extracted from nasopharyngeal swabs of patients with COVID-19, and compared it to targeted SARS-CoV-2 full genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. To track virus origin, we used open-source multiple sequence alignment and phylogenetic tools to compare the assembled SARS-CoV-2 genomes to publicly available sequences. We show a significant improvement in whole genome sequencing data quality and viral detection using amplicon-based target enrichment of SARS-CoV-2. With enrichment, more than 99% of the sequencing reads mapped to the viral genome compared to an average of 0.63% without enrichment. Consequently, a dramatic increase in genome coverage was obtained using significantly less sequencing data, enabling higher scalability and significant cost reductions. We also demonstrate how SARS-CoV-2 genome sequences can be used to determine their possible origin through phylogenetic analysis including other viral strains. SARS-CoV-2 whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the COVID-19 pandemic.","version":"1.2","doi":"10.1101/2020.06.06.138339","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.158006","pub_date":"2020-6-18","title":"Genomic surveillance of SARS-CoV-2 reveals community transmission of a major lineage during the early pandemic phase in Brazil","abstract":"Despite all efforts to control the COVID-19 spread, the SARS-CoV-2 reached South America within three months after its first detection in China, and Brazil became one of the hotspots of COVID-19 in the world. Several SARS-CoV-2 lineages have been identified and some local clusters have been described in this early pandemic phase in Western countries. Here we investigated the genetic diversity of SARS-CoV-2 during the early phase (late February to late April) of the epidemic in Brazil. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 in Brazil and the community transmission of a major B.1.1 lineage defined by two amino acid substitutions in the Nucleocapsid and ORF6. This SARS-CoV-2 Brazilian lineage was probably established during February 2020 and rapidly spread through the country, reaching different Brazilian regions by the middle of March 2020. Our study also supports occasional exportations of this Brazilian B.1.1 lineage to neighboring South American countries and to more distant countries before the implementation of international air travels restrictions in Brazil.","version":"1.1","doi":"10.1101/2020.06.17.158006","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.158584","pub_date":"2020-6-18","title":"Unexpected free fatty acid binding pocket in the cryo-EM structure of SARS-CoV-2 spike protein","abstract":"COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2. A direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.","version":"1.1","doi":"10.1101/2020.06.18.158584","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.156810","pub_date":"2020-6-18","title":"Variant analysis of SARS-CoV-2 strains in Middle Eastern countries","abstract":"SARS-CoV-2 is diverging from the initial Wuhan serotype, and different variants of the virus are reported. Mapping the variant strains and studying their pattern of evolution will provide better insights into the pandemic spread Data on different SARS-CoV2 for WHO EMRO countries were obtained from the Chinese National Genomics Data Center (NGDC), Genbank and the Global Initiative on Sharing All Influenza Data (GISAID). Multiple sequence alignments (MSA) was performed to study the evolutionary relationship between the genomes. Variant calling, genome and variant alignment were performed to track the strains in each country. Evolutionary and phylogenetic analysis is used to explore the evolutionary hypothesis. Of the total 50 samples, 4 samples did not contain any variants. Variant calling identified 379 variants. Earliest strains are found in Iranian samples. Variant alignment indicates Iran samples have a low variant frequency. Saudi Arabia has formed an outgroup. Saudi Arabia, Qatar and Kuwait were the most evolved genomes and are the countries with the highest number of cases per million. Iran was exposed to the virus earlier than other countries in the Eastern Mediterranean Region. None","version":"1.1","doi":"10.1101/2020.06.18.156810","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.18.158329","pub_date":"2020-6-18","title":"Polymorphism and selection pressure of SARS-CoV-2 vaccine and diagnostic antigens: implications for immune evasion and serologic diagnostic performance","abstract":"The ongoing SARS-CoV-2 pandemic has triggered multiple efforts for serological tests and vaccine development. Most of these tests and vaccines are based on the Spike glycoprotein (S) or the Nucleocapsid (N) viral protein. Conservation of these antigens among viral strains is critical to ensure optimum diagnostic test performance and broad protective efficacy, respectively. We assessed N and S antigen diversity from 17,853 SARS-CoV-2 genome sequences and evaluated selection pressure. Up to 6-7 incipient phylogenetic clades were identified for both antigens, confirming early variants of the S antigen and identifying new ones. Significant diversifying selection was detected at multiple sites for both antigens. Some sequence variants have already spread in multiple regions, in spite of their low frequency. In conclusion, the N and S antigens of SARS-CoV-2 are well conserved antigens, but new clades are emerging and may need to be included in future diagnostic and vaccine formulations.","version":"1.1","doi":"10.1101/2020.06.18.158329","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.137331","pub_date":"2020-6-18","title":"Effects of Renin-Angiotensin Inhibition on ACE2 and TMPRSS2 Expression: Insights into COVID-19","abstract":"Angiotensin-converting enzyme 2 (ACE2), a component of the renin-angiotensin system, is a receptor for SARS-CoV-2, the virus that causes COVID-19. To determine whether the renin-angiotensin inhibition regulates ACE2 expression, either enalapril (an angiotensin-converting enzyme inhibitor) or losartan (an AT1 receptor blocker) was infused subcutaneously to male C57BL/6J mice for two weeks. Neither enalapril nor losartan changed abundance of ACE2 mRNA in lung, ileum, kidney, and heart. Viral entry also depends on transmembrane protease serine 2 (TMPRSS2) to prime the S protein. TMPRSS2 mRNA was abundant in lungs and ileum, modest in kidney, but barely detectable in heart. TMPRSS2 mRNA abundance was not altered by either enalapril or losartan in any of the 4 tissues. Next, we determined whether depletion of angiotensinogen (AGT), the unique substrate of the renin-angiotensin system, changes ACE2 and TMPRSS2 mRNA abundance. AGT antisense oligonucleotides (ASO) were injected subcutaneously to male C57BL/6J mice for 3 weeks. Abundance of ACE2 mRNA was unchanged in any of the 4 tissues, but TMPRSS2 mRNA was significantly decreased in lungs. Our data support that the renin-angiotensin inhibition does not regulate ACE2 and hence are not likely to increase risk for COVID-19.","version":"1.3","doi":"10.1101/2020.06.08.137331","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.118729","pub_date":"2020-6-17","title":"Expansion of SARS-CoV-2-specific Antibody-secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients","abstract":"Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific antibodies are typically a major predictor of protective immunity, yet B cell and antibody responses during COVID-19 are not fully understood. Here, we analyzed antibody-secreting cell (ASC) and antibody responses in twenty hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19, and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific ASCs in all twenty COVID-19 patients using a multicolor FluoroSpot assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing antibodies by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG and IgM antibody levels positively correlated with SARS-CoV-2-neutralizing antibody titers, suggesting that SARS-CoV-2-specific antibody levels may reflect the titers of neutralizing antibodies in COVID-19 patients during the acute phase of infection. Lastly, we showed that interleukin 6 (IL-6) and C-reactive protein (CRP) concentrations were higher in serum of patients who were hospitalized for longer, supporting the recent observations that IL-6 and CRP could be used to predict COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in twenty COVID-19 patients, with a focus on B cell and antibody responses, and provides tools to study immune responses to SARS-CoV-2 infection and vaccination.","version":"1.2","doi":"10.1101/2020.05.28.118729","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.156166","pub_date":"2020-6-17","title":"Evaluation of the performance of SARS-CoV-2 serological tools and their positioning in COVID-19 diagnostic strategies","abstract":"Rapid and accurate diagnosis is crucial for successful outbreak containment. During the current coronavirus disease 2019 (COVID-19) public health emergency, the gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnosis is the detection of viral RNA by reverse transcription (RT)-PCR. Additional diagnostic methods enabling the detection of current or past SARS-CoV-2 infection would be highly beneficial to ensure the timely diagnosis of all infected and recovered patients. Here, we investigated several serological tools, i.e., two immunochromatographic lateral flow assays (LFA-1 (Biosynex COVID-19 BSS) and LFA-2 (COVID-19 Sign IgM/IgG)) and two enzyme-linked immunosorbent assays (ELISAs) detecting IgA (ELISA-1 Euroimmun), IgM (ELISA-2 EDI) and/or IgG (ELISA-1 and ELISA-2) based on well-characterized panels of serum samples from patients and healthcare workers with PCR-confirmed COVID-19 and from SARS-CoV-2-negative patients. A total of 272 serum samples were used, including 62 serum samples from hospitalized patients (panel 1 and panel 3), 143 serum samples from healthcare workers (panel 2) diagnosed with COVID-19 and 67 serum samples from negative controls. Diagnostic performances of each assay were assessed according to days after symptom onset (dso) and the antigenic format used by manufacturers. We found overall sensitivities ranging from 69% to 93% on panels 1 and 2 and specificities ranging from 83% to 98%. The clinical sensitivity varied greatly according to the panel tested and the dso. The assays we tested showed poor mutual agreement. A thorough selection of serological assays for the detection of ongoing or past infections is advisable.","version":"1.1","doi":"10.1101/2020.06.16.156166","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.147819","pub_date":"2020-6-17","title":"Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel","abstract":"To battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy. We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as \u201c2019-nCoV not detected\u201d, whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as \u201cInvalid Result\u201d and the procedure should be repeated. In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.","version":"1.2","doi":"10.1101/2020.06.12.147819","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.155101","pub_date":"2020-6-17","title":"Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection","abstract":"Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-\u03b2 signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.","version":"1.1","doi":"10.1101/2020.06.16.155101","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.150607","pub_date":"2020-6-17","title":"Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis","abstract":"Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F\u2122 and ExpiCHO-S\u2122 cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S\u2122 cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.06.14.150607","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.19.999318","pub_date":"2020-6-17","title":"Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD","abstract":"Coronavirus disease 19 (COVID-19) is an emerging global health crisis. With over 7 million confirmed cases to date, this pandemic continues to expand, spurring research to discover vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact interface. Using molecular dynamics simulations based on the recently solved cryo-EM structure of ACE2 in complex with SARS-CoV-2-RBD, we observed that the ACE2 peptidase domain (PD) \u03b11 helix is important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we chemically synthesized a 23-mer peptide fragment of the ACE2 PD \u03b11 helix (SBP1) composed entirely of proteinogenic amino acids. Chemical synthesis of SBP1 was complete in 1.5 hours, and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer interferometry (BLI) revealed that SBP1 associates with micromolar affinity to insect-derived SARS-CoV-2-RBD protein obtained from Sino Biological. Association of SBP1 was not observed to an appreciable extent to HEK cell-expressed SARS-CoV-2-RBD proteins and insect-derived variants acquired from other vendors. Moreover, competitive BLI assays showed SBP1 does not outcompete ACE2 binding to Sino Biological insect-derived SARS-CoV-2-RBD. Further investigations are ongoing to gain insight into the molecular and structural determinants of the variable binding behavior to different SARS-CoV-2-RBD protein variants.","version":"1.2","doi":"10.1101/2020.03.19.999318","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.155580","pub_date":"2020-6-17","title":"High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform","abstract":"The outbreak and rapid spread of SARS-CoV-2 virus has led to a dire global pandemic with millions of people infected and ~ 400,000 deaths thus far. Highly accurate detection of antibodies for COVID-19 is an indispensable part of the effort to combat the pandemic. Here we developed two-plex antibody detection against SARS-CoV-2 spike proteins (the S1 subunit and receptor binding domain RBD) in human serum and saliva on a near-infrared nano-plasmonic gold (pGOLD) platform. By testing nearly 600 serum samples, pGOLD COVID-19 assay achieved ~ 99.78 % specificity for detecting both IgG and IgM with 100 % sensitivity in sera collected > 14 days post disease symptom onset, with zero cross-reactivity to other diseases. Two-plex correlation analysis revealed higher binding of serum IgM to RBD than to S1. IgG antibody avidity toward multiple antigens were measured, shedding light on antibody maturation in COVID-19 patients and affording a powerful tool for differentiating recent from remote infections and identifying re-infection by SARS-CoV-2. Just as important, due to high analytical sensitivity, the pGOLD COVID-19 assay detected minute amounts of antibodies in human saliva, offering the first non-invasive detection of SARS-CoV-2 antibodies.","version":"1.1","doi":"10.1101/2020.06.16.155580","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.155200","pub_date":"2020-6-17","title":"Pervasive RNA secondary structure in the genomes of SARS-CoV-2 and other coronaviruses \u2013 an endeavour to understand its biological purpose","abstract":"The ultimate outcome of the COVID-19 pandemic is unknown and is dependent on a complex interplay of its pathogenicity, transmissibility and population immunity. In the current study, SARS coronavirus 2 (SARS-CoV-2) was investigated for the presence of large scale internal RNA base pairing in its genome. This property, termed genome scale ordered RNA structure (GORS) has been previously associated with host persistence in other positive-strand RNA viruses, potentially through its shielding effect on viral RNA recognition in the cell. Genomes of SARS-CoV-2 were remarkably structured, with minimum folding energy differences (MFEDs) of 15%, substantially greater than previously examined viruses such as HCV (MFED 7-9%). High MFED values were shared with all coronavirus genomes analysed created by several hundred consecutive energetically favoured stem-loops throughout the genome. In contrast to replication-association RNA structure, GORS was poorly conserved in the positions and identities of base pairing with other sarbecoviruses \u2013 even similarly positioned stem-loops in SARS-CoV-2 and SARS-CoV rarely shared homologous pairings, indicative of more rapid evolutionary change in RNA structure than in the underlying coding sequences. Sites predicted to be base-paired in SARS-CoV-2 showed substantially less sequence diversity than unpaired sites, suggesting that disruption of RNA structure by mutation imposes a fitness cost on the virus which is potentially restrictive to its longer evolution. Although functionally uncharacterised, GORS in SARS-CoV-2 and other coronaviruses represent important elements in their cellular interactions that may contribute to their persistence and transmissibility.","version":"1.1","doi":"10.1101/2020.06.17.155200","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.150490","pub_date":"2020-6-17","title":"The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2","abstract":"To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications \u201coff-label\u201d against SARS-CoV-2. In these screenings, Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8\u00b5g/ml significantly, and the EC50 was determined with 387ng/ml. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus. Fluoxetine treatment resulted in a decrease in viral protein expression. Furthermore, Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. We see the role of Fluoxetine in the early treatment of SARS-CoV-2 infected patients of risk groups.","version":"1.2","doi":"10.1101/2020.06.14.150490","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.155887","pub_date":"2020-6-17","title":"Detection of SARS-CoV-2 RNA by multiplex RT-qPCR","abstract":"The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United\nStates requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the CDC, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.","version":"1.1","doi":"10.1101/2020.06.16.155887","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.155689","pub_date":"2020-6-17","title":"Holder Pasteurization Inactivates SARS-CoV-2 in Human Breast Milk","abstract":"SARS-CoV-2 RNA has been detected in the human breast milk of infected mothers, raising concerns regarding the safety of breastfeeding upon infection. We here show that holder pasteurization inactivates SARS-CoV-2 and provides an alternative and safe option for infected mothers to continue feeding breast milk to their infants.","version":"1.1","doi":"10.1101/2020.06.17.155689","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.152835","pub_date":"2020-6-17","title":"A thermostable, closed, SARS-CoV-2 spike protein trimer","abstract":"The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.","version":"1.1","doi":"10.1101/2020.06.15.152835","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.103325","pub_date":"2020-6-17","title":"Developing a Fully-glycosylated Full-length SARS-CoV-2 Spike Protein Model in a Viral Membrane","abstract":"This technical study describes all-atom modeling and simulation of a fully-glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB:6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently-determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully-glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19), so researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.05.20.103325","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.17.157982","pub_date":"2020-6-17","title":"Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding","abstract":"The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor, and is a major determinant of host range and a dominant target of neutralizing antibodies. Here we experimentally measure how all amino-acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD\u2019s surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.","version":"1.1","doi":"10.1101/2020.06.17.157982","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.155812","pub_date":"2020-6-17","title":"Crystal structure of SARS-CoV-2 main protease in complex with a Chinese herb inhibitor shikonin","abstract":"Main protease (Mpro, also known as 3CLpro) has a major role in the replication of coronavirus life cycle and is one of the most important drug targets for anticoronavirus agents. Here we report the crystal structure of main protease of SARS-CoV-2 bound to a previously identified Chinese herb inhibitor shikonin at 2.45 angstrom resolution. Although the structure revealed here shares similar overall structure with other published structures, there are several key differences which highlight potential features that could be exploited. The catalytic dyad His41-Cys145 undergoes dramatic conformational changes, and the structure reveals an unusual arrangement of oxyanion loop stabilized by the substrate. Binding to shikonin and binding of covalent inhibitors show different binding modes, suggesting a diversity in inhibitor binding. As we learn more about different binding modes and their structure-function relationships, it is probable that we can design more effective and specific drugs with high potency that can serve as effect SARS-CoV-2 anti-viral agents.","version":"1.1","doi":"10.1101/2020.06.16.155812","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.151282","pub_date":"2020-6-17","title":"Time-series analyses of directional sequence changes in SARS-CoV-2 genomes and an efficient search method for advantageous mutations for growth in human cells","abstract":"We first conducted time-series analysis of mono- and dinucleotide composition for over 10,000 SARS-CoV-2 genomes, as well as over 1500 Zaire ebolavirus genomes, and found clear time-series changes in the compositions on a monthly basis, which should reflect viral adaptations for efficient growth in human cells. We next developed a sequence alignment free method that extensively searches for advantageous mutations and rank them in an increase level for their intrapopulation frequency. Time-series analysis of occurrences of oligonucleotides of diverse lengths for SARS-CoV-2 genomes revealed seven distinctive mutations that rapidly expanded their intrapopulation frequency and are thought to be candidates of advantageous mutations for the efficient growth in human cells.","version":"1.1","doi":"10.1101/2020.06.16.151282","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.150243","pub_date":"2020-6-16","title":"Catch and kill airborne SARS-CoV-2 to control spread of COVID-19 by a heated air disinfection system","abstract":"Airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via air-conditioning systems poses a significant threat for the continued escalation of the current coronavirus disease (COVID-19) pandemic. Considering that SARS-CoV-2 cannot tolerate temperatures above 70 \u00b0C, here we designed and fabricated efficient air disinfection systems based on heated nickel (Ni) foam to catch and kill SARS-CoV-2. Virus test results revealed that 99.8% of the aerosolized SARS-CoV-2 was caught and killed by a single pass through a Ni-foam-based filter when heated up to 200 \u00b0C. Additionally, the same filter was also used to catch and kill 99.9% of Bacillus anthracis, an airborne spore. This study paves the way for preventing transmission of SARS-CoV-2 and other highly infectious airborne agents in closed environments. Heated Ni-foam filters are capable of effectively catching and killing airborne SARS-CoV-2 and Bacillus anthracis spores.","version":"1.1","doi":"10.1101/2020.06.13.150243","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153064","pub_date":"2020-6-16","title":"Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients","abstract":"Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.","version":"1.1","doi":"10.1101/2020.06.15.153064","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154559","pub_date":"2020-6-16","title":"in-silica Analysis of SARS-CoV-2 viral strain using Reverse Vaccinology Approach: A Case Study for USA","abstract":"The recent pandemic of COVID19 that has struck the world is yet to be battled by a potential cure. Countless lives have been claimed due to the existing pandemic and the societal normalcy has been damaged permanently. As a result, it becomes crucial for academic researchers in the field of bioinformatics to combat the existing pandemic. The study involved collecting the virulent strain sequence of SARS-nCoV19 for the country USA against human host through publically available bioinformatics databases. Using in-silica analysis and reverse vaccinology, two leader proteins were identified to be potential vaccine candidates for development of a multi-epitope drug. The results of this study can provide further researchers better aspects and direction on developing vaccine and immune responses against COVID19. This work also aims at promoting the use of existing bioinformatics tools to faster streamline the pipeline of vaccine development. A new infection respiratory disease was first observed in the month of December 2019, in Wuhan, situated in the Hubei province, China. Studies have indicated that the reason of this disease was the emergence of a genetically-novel coronavirus closely related to SARS-CoV. This coronavirus, now named as nCoV-19, is the reason behind the spread of this fatal respiratory disease, now named as COVID-19. The initial group of infections is supposedly linked with the Huanan seafood market, most likely due to animal contact. Eventually, human-to-human interaction occurred and resulted in the transmission of the virus to humans. [13]. Since then, nCoV-19 has been rapidly spreading within China and other parts of World. At the time of writing this article (mid-March 2020), COVID-19 has spread across 146 countries. A count of 164,837 cases have been confirmed of being diagnosed with COVID-19, and a total of 6470 deaths have occurred. The cumulative cases have been depicting a rising trend and the numbers are just increasing. WHO has declared COVID-19 to be a \u201cglobal health emergency\u201d. [14]. Currently, research is being conducted on a massive level to understand the immunology and genetic characteristics of the disease. However, no cure or vaccine of nCoV-19 has been developed at the time of writing this article. Though, nCoV-19 and SARS-CoV are almost genetically similar, the respiratory syndrome caused by both of them, COVID-19 and SARS respectively, are completely different. Studies have indicated that \u2013 \u201cSARS was more deadly but much less infectious than COVID-19\u201d. -World Health Organization","version":"1.1","doi":"10.1101/2020.06.16.154559","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154765","pub_date":"2020-6-16","title":"Machine Learning Models Identify Inhibitors of SARS-CoV-2","abstract":"With the ongoing SARS-CoV-2 pandemic there is an urgent need for the discovery of a treatment for the coronavirus disease (COVID-19). Drug repurposing is one of the most rapid strategies for addressing this need and numerous compounds have been selected for in vitro testing by several groups already. These have led to a growing database of molecules with in vitro activity against the virus. Machine learning models can assist drug discovery through prediction of the best compounds based on previously published data. Herein we have implemented several machine learning methods to develop predictive models from recent SARS-CoV-2 in vitro inhibition data and used them to prioritize additional FDA approved compounds for in vitro testing selected from our in-house compound library. From the compounds predicted with a Bayesian machine learning model, CPI1062 and CPI1155 showed antiviral activity in HeLa-ACE2 cell-based assays and represent potential repurposing opportunities for COVID-19. This approach can be greatly expanded to exhaustively virtually screen available molecules with predicted activity against this virus as well as a prioritization tool for SARS-CoV-2 antiviral drug discovery programs. The very latest model for SARS-CoV-2 is available at www.assaycentral.org.","version":"1.1","doi":"10.1101/2020.06.16.154765","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.134403","pub_date":"2020-6-16","title":"AGE IS ASSOCIATED WITH INCREASED EXPRESSION OF PATTERN RECOGNITION RECEPTOR GENES AND ACE2, THE RECEPTOR FOR SARS-COV-2: IMPLICATIONS FOR THE EPIDEMIOLOGY OF COVID-19 DISEASE","abstract":"Older aged adults and those with pre-existing conditions are at highest risk for severe COVID-19 associated outcomes. Using a large dataset of genome-wide RNA-seq profiles derived from human dermal fibroblasts (GSE113957) we investigated whether age affects the expression of pattern recognition receptor (PRR) genes and ACE2, the receptor for SARS-CoV-2. Older age was associated with increased expression of PRR genes, ACE2 and four genes that encode proteins that have been shown to interact with SAR2-CoV-2 proteins. Assessment of PRR expression might provide a strategy for stratifying the risk of severe COVID-19 disease at both the individual and population levels.","version":"1.1","doi":"10.1101/2020.06.15.134403","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153916","pub_date":"2020-6-16","title":"Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species","abstract":"The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to many countries around the world. However, where is it origin and which animals are sensitive to cross-species transmission is unclear. The interaction of virus and cell receptor is a key determinant of host range for the novel coronavirus. Angiotensin-converting enzyme 2 (ACE2) is demonstrated as the primary entry receptor for SARS-CoV-2. In this study, we evaluated the SARS-CoV-2 entry mediated by ACE2 of 11 different species of animals, and discovered that ACE2 of Rhinolophus sinicus (Chinese horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (pig), Capra hircus (goat) and especially Manis javanica (Malayan pangolin) were able to render SARS-CoV-2 entry in non-susceptible cells. This is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the presume that SARS-CoV-2 may have a pangolin origin. However, none of the ACE2 proteins from Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (chicken), Notechis scutatus (mainland tiger snake), Mus musculus (house mouse) rendered SARS-CoV-2 entry. Specifically, a natural isoform of Macaca mulatta (Rhesus monkey) ACE2 with a mutation of Y217N was resistance to infection, which rises the possible impact of this type of ACE2 during monkey studies of SARS-CoV-2. Overall, these results clarify that SARS-CoV-2 could engage receptors of multiple species of animals and it is a perplexed work to track SARS-CoV-2 origin and its intermediate hosts. In this study, we illustrated that SARS-CoV-2 is able to engage receptors of multiple species of animals. This indicated that it may be a perplexed work to track SARS-CoV-2 origin and discover its intermediate hosts. This feature of virus is considered to potentiate its diverse cross-species transmissibility. Of note, here is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the possibility that SARS-CoV-2 may have a pangolin origin. And we also demonstrated that not all species of bat were sensitive to SARS-CoV-2 infection. At last, it is also important to detect the expression ratio of the Y217N ACE2 to the prototype in Rhesus monkeys to be recruited for studies on SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.06.15.153916","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.151704","pub_date":"2020-6-16","title":"Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters","abstract":"Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-COV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.","version":"1.1","doi":"10.1101/2020.06.16.151704","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.151290","pub_date":"2020-6-16","title":"Genomic modeling as an approach to identify surrogates for use in experimental validation of SARS-CoV-2 and HuNoVs inactivation by UV-C treatment","abstract":"Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) is responsible for the COVID-19 pandemic that continues to pose significant public health concerns. While research to deliver vaccines and antivirals are being pursued, various effective technologies to control its environmental spread are also being targeted. Ultraviolet light (UV-C) technologies are effective against a broad spectrum of microorganisms when used even on large surface areas. In this study, we developed a pyrimidine dinucleotide frequency based genomic model to predict the sensitivity of select enveloped and non-enveloped viruses to UV-C treatments in order to identify potential SARS-CoV-2 and human noroviruses surrogates. The results revealed that this model was best fitted using linear regression with r2=0.90. The predicted UV-C sensitivity (D90 - dose for 90% inactivation) for SARS-CoV-2 and MERS-CoV was found to be 21 and 28 J/m2, respectively (with an estimated 18 J/m2 as published for SARS-CoV-1), suggesting that coronaviruses are highly sensitive to UV-C light compared to other ssRNA viruses used in this modeling study. Murine hepatitis virus (MHV) A59 strain with a D90 of 21 J/m2 close to that of SARS-CoV-2 was identified as a suitable surrogate to validate SARS-CoV-2 inactivation by UV-C treatment. Furthermore, the non-enveloped human noroviruses (HuNoVs), had predicted D90 values of 69.1, 89 and 77.6 J/m2 for genogroups GI, GII and GIV, respectively. Murine norovirus (MNV-1) of GV with a D90 = 100 J/m2 was identified as a potential conservative surrogate for UV-C inactivation of these HuNoVs. This study provides useful insights for the identification of potential nonpathogenic surrogates to understand inactivation kinetics and their use in experimental validation of UV-C disinfection systems. This approach can be used to narrow the number of surrogates used in testing UV-C inactivation of other human and animal ssRNA viral pathogens for experimental validation that can save cost, labor and time.","version":"1.1","doi":"10.1101/2020.06.14.151290","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154658","pub_date":"2020-6-16","title":"Lack of susceptibility of poultry to SARS-CoV-2 and MERS-CoV","abstract":"Chickens, turkeys, ducks, quail and geese were challenged with SARS-CoV-2 or MERS-CoV. No disease was observed, no virus replication was detected, and antibodies were not detected in serum. Neither virus replicated in embryonating chicken\u2019s eggs. Poultry are unlikely to serve a role in the maintenance of either virus.","version":"1.1","doi":"10.1101/2020.06.16.154658","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153239","pub_date":"2020-6-16","title":"Mutation density changes in SARS-CoV-2 are related to the pandemic stage but to a lesser extent in the dominant strain with mutations in spike and RdRp","abstract":"Since its emergence in Wuhan, China in late 2019, the origin and evolution of SARS-CoV-2 have been among the most debated issues related to COVID-19. Throughout its spread around the world, the viral genome continued acquiring new mutations and some of them became widespread. Among them, 14408 C>T and 23403 A>G mutations in RdRp and S, respectively, became dominant in Europe and the US, which led to debates regarding their effects on the mutability and transmissibility of the virus. In this study, we aimed to investigate possible differences between time-dependent variation of mutation densities (MDe) of viral strains that carry these two mutations and those that do not. Our analyses at the genome and gene level led to two important findings: First, time-dependent changes in the average MDe of circulating SARS-CoV-2 genomes showed different characteristics before and after the beginning of April, when daily new case numbers started levelling off. Second, this pattern was much delayed or even non-existent for the \u201cmutant\u201d (MT) strain that harbored both 14408 C>T and 23403 A>G mutations. Although these differences were not limited to a few hotspots, it is intriguing that the MDe increase is most evident in two critical genes, S and Orf1ab, which are also the genes that harbor the defining mutations of the MT genotype. The nature of these unexpected relationships warrant further research.","version":"1.1","doi":"10.1101/2020.06.15.153239","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.111237","pub_date":"2020-6-16","title":"Biophysical modeling of the SARS-CoV-2 viral cycle reveals ideal antiviral targets","abstract":"Effective therapies for COVID-19 are urgently needed. Presently there are more than 800 COVID-19 clinical trials globally, many with drug combinations, resulting in an empirical process with an enormous number of possible combinations. To identify the most promising potential therapies, we developed a biophysical model for the SARS-CoV-2 viral cycle and performed a sensitivity analysis for individual model parameters and all possible pairwise parameter changes (162 = 256 possibilities). We found that model-predicted virion production is fairly insensitive to changes in most viral entry, assembly, and release parameters, but highly sensitive to some viral transcription and translation parameters. Furthermore, we found a cooperative benefit to pairwise targeting of transcription and translation, predicting that combined targeting of these processes will be especially effective in inhibiting viral production.","version":"1.2","doi":"10.1101/2020.05.22.111237","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.147025","pub_date":"2020-6-16","title":"COVID-19-related coagulopathy \u2013 Is transferrin a missing link?","abstract":"SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. Since the risk of severe COVID-19 disease is higher in males than in females and increases with age, we combined proteomics data from SARS-CoV-2-infected cells with human gene expression data from the Genotype-Tissue Expression (GTEx) database to identify gene products involved in coagulation that change with age, differ in their levels between females and males, and are regulated in response to SARS-CoV-2 infection. This resulted in the identification of transferrin as a candidate coagulation promoter, whose levels increases with age and are higher in males than in females and that is increased upon SARS-CoV-2 infection. A systematic investigation of gene products associated with the GO term \u201cblood coagulation\u201d did not reveal further high confidence candidates, which are likely to contribute to COVID-19-related coagulopathy. In conclusion, the role of transferrin should be considered in the course of COVID-19 disease and further examined in ongoing clinic-pathological investigations.","version":"1.1","doi":"10.1101/2020.06.11.147025","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154591","pub_date":"2020-6-16","title":"Antigenic evolution on global scale reveals potential natural selection of SARS-CoV-2 by pre-existing cross-reactive T cell immunity","abstract":"The mutation pattern of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is constantly changing with the places of transmission, but the reason remains to be revealed. Here, we presented the study that comprehensively analyzed the potential selective pressure of immune system restriction, which can drive mutations in circulating SARS-CoV-2 isolates. The results showed that the most common mutation sites of SARS-CoV-2 proteins were located on the non-structural protein ORF1ab and the structural protein Spike. Further analysis revealed mutations in cross-reactive epitopes between SARS-CoV-2 and seasonal coronavirus may help SARS-CoV-2 to escape cellular immunity under the long-term and large-scale community transmission. Meanwhile, the mutations on Spike protein may enhance the ability of SARS-CoV-2 to enter the host cells and escape the recognition of B-cell immunity. This study will increase the understanding of the evolutionary direction and warn about the potential immune escape ability of SARS-CoV-2, which may provide important guidance for the potential vaccine design.","version":"1.1","doi":"10.1101/2020.06.16.154591","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154286","pub_date":"2020-6-16","title":"Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform","abstract":"Genomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.","version":"1.1","doi":"10.1101/2020.06.16.154286","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153197","pub_date":"2020-6-16","title":"Comparative analysis of coronavirus genomic RNA structure reveals conservation in SARS-like coronaviruses","abstract":"Coronaviruses, including SARS-CoV-2 the etiological agent of COVID-19 disease, have caused multiple epidemic and pandemic outbreaks in the past 20 years. With no vaccines, and only recently developed antiviral therapeutics, we are ill equipped to handle coronavirus outbreaks. A better understanding of the molecular mechanisms that regulate coronavirus replication and pathogenesis is needed to guide the development of new antiviral therapeutics and vaccines. RNA secondary structures play critical roles in multiple aspects of coronavirus replication, but the extent and conservation of RNA secondary structure across coronavirus genomes is unknown. Here, we define highly structured RNA regions throughout the MERS-CoV, SARS-CoV, and SARS-CoV-2 genomes. We find that highly stable RNA structures are pervasive throughout coronavirus genomes, and are conserved between the SARS-like CoV. Our data suggests that selective pressure helps preserve RNA secondary structure in coronavirus genomes, suggesting that these structures may play important roles in virus replication and pathogenesis. Thus, disruption of conserved RNA secondary structures could be a novel strategy for the generation of attenuated SARS-CoV-2 vaccines for use against the current COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.06.15.153197","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154211","pub_date":"2020-6-16","title":"TMPRSS2, a SARS-CoV-2 internalization protease is downregulated in head and neck cancer patients","abstract":"Two of the main target tissues of SARS-coronavirus 2 are the oral cavity pharynx-larynx epithelium, the main virus entry site, and the lung epithelium. The virus enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. Herein we aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity-pharynx-larynx and lung tissues as well as neoplastic tissues from the same histological areas. The information provided in this study may contribute to better understanding of SARS-coronavirus 2 ability to interact with different biological systems and contributes to cumulative knowledge on potential mechanisms to inhibit its diffusion. The study has been conducted using The Cancer Genome Atlas (TCGA) and the Regina Elena Institute (IRE) databases and validated by experimental model in HNSCC and Lung cancer cells. Data from one COVID19 positive patient who was operated on for HNSCC was also included. We have analyzed 478 tumor samples and 44 normal samples from TCGA HNSCC cohort for whom both miRNA and mRNA sequencing was available. The dataset included 391 HPV- and 85 HPV+ cases, with 331 P53 mutated and 147 P53 wild type cases respectively. 352 out of 478 samples were male and 126 female. In IRE cohort we analyzed 66 tumor samples with matched normal sample for miRNA profiling and 23 tumor\\normal matched samples for mRNA profiling. 45 out of 66 tumors from IRE cohort were male and 21 female, 38 were P53 mutated and 27 wild type. Most patients (63 of 66) in IRE cohort were HPV negative. Normalized TCGA HNSCC gene expression and miRNA expression data were obtained from Broad Institute TCGA Genome Data Analysis Center (http://gdac.broadinstitute.org/). mRNA expression data from IRE cohort used in this study has been deposited to NCBI\u2019s Gene Expression Omnibus and is accessible through GEO series accession number GSE107591. In order to inference about potential molecular modulation of TMPRSS2, we also included miRNAs expression for the 66 IRE cohort matched tumor and normal samples from Agilent platform. DNA methylation data for TCGA tumors were obtained from Wanderer (http://maplab.imppc.org/wanderer/). We used miRWalk and miRNet web tools for miRNA-target interaction prediction and pathway enrichment analysis. The correlation and regression analyses as well as the miRNA and gene modulation and the survival analysis were conducted using Matlab R2019. TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues and had a prognostic value in HNSCC patients. Reduction of TMPRSS2 expression was more evident in women than in men, in TP53 mutated versus wild TP53 tumors as well as in HPV negative patients compared to HPV positive counterparts. Functionally, we assessed the multivariate effect on TMPRSS2 in a single regression model. We observed that all variables had an independent effect on TMPRSS2 in HNSCC patients with HPV negative, TP53 mutated status and with elevated TP53-dependent Myc-target genes associated with low TMPRSS2 expression. Investigation of the molecular modulation of TMPRSS2 in both HNSCC and lung cancers revealed that expression of microRNAs targeting TMPRSS2 anti-correlated in both TCGA and IRE HNSCC datasets, while there was not evidence of TMPRSS2 promoter methylation in both tumor cohorts. Interestingly, the anti-correlation between microRNAs and TMPRSS2 expression was corroborated by testing this association in a SARS-CoV-2 positive HNSCC patient. Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. The protective mechanism might occur, at least partially, through the aberrant activation of TMPRSS2 targeting microRNAs; thereby providing strong evidence on the role of non-coding RNA molecule in host viral infection. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.","version":"1.1","doi":"10.1101/2020.06.16.154211","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.153817","pub_date":"2020-6-16","title":"The IMEx Coronavirus interactome: an evolving map of Coronaviridae-Host molecular interactions","abstract":"The current Coronavirus Disease 2019 (COVID-19) pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions enables studying fine-grained resolution of the mechanisms behind the virus biology and the human organism response. Here we present a curated dataset of physical molecular interactions, manually extracted by IMEx Consortium curators focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family. Currently, the dataset comprises over 2,200 binarized interactions extracted from 86 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website (www.ebi.ac.uk/intact), and will be continuously updated as research on COVID-19 progresses.","version":"1.1","doi":"10.1101/2020.06.16.153817","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.153403","pub_date":"2020-6-16","title":"Oral drug repositioning candidates and synergistic remdesivir combinations for the prophylaxis and treatment of COVID-19","abstract":"The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. A high-throughput, high-content imaging assay of human HeLa cells expressing the SARS-CoV-2 receptor ACE2 was used to screen ReFRAME, a best-in-class drug repurposing library. From nearly 12,000 compounds, we identified 66 compounds capable of selectively inhibiting SARS-CoV-2 replication in human cells. Twenty-four of these drugs show additive activity in combination with the RNA-dependent RNA polymerase inhibitor remdesivir and may afford increased in vivo efficacy. We also identified synergistic interaction of the nucleoside analog riboprine and a folate antagonist 10-deazaaminopterin with remdesivir. Overall, seven clinically approved drugs (halofantrine, amiodarone, nelfinavir, simeprevir, manidipine, ozanimod, osimertinib) and 19 compounds in other stages of development may have the potential to be repurposed as SARS-CoV-2 oral therapeutics based on their potency, pharmacokinetic and human safety profiles.","version":"1.1","doi":"10.1101/2020.06.16.153403","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.16.154708","pub_date":"2020-6-16","title":"Targeting ACE2-RBD interaction as a platform for COVID19 therapeutics: Development and drug repurposing screen of an AlphaLISA proximity assay","abstract":"The COVID-19 pandemic, caused by SARS-CoV-2, is a pressing public health emergency garnering rapid response from scientists across the globe. Host cell invasion is initiated through direct binding of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Disrupting the spike-ACE2 interaction is a potential therapeutic target for treating COVID-19. We have developed a proximity-based AlphaLISA assay to measure binding of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) to ACE2. Utilizing this assay platform, a drug-repurposing screen against 3,384 small molecule drugs and pre-clinical compounds was performed, yielding 25 high-quality, small-molecule hits that can be evaluated in cell-based models. This established AlphaLISA RBD-ACE2 platform can facilitate evaluation of biologics or small molecules that can perturb this essential viral-host interaction to further the development of interventions to address the global health pandemic.","version":"1.1","doi":"10.1101/2020.06.16.154708","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.152983","pub_date":"2020-6-16","title":"Virus survival in evaporated saliva microdroplets deposited on inanimate surfaces","abstract":"The novel coronavirus respiratory syndrome (COVID-19) has now spread worldwide. The relative contribution of viral transmission via fomites is still unclear. SARS-CoV-2 has been shown to survive on inanimate surfaces for several days, yet the factors that determine its survival on surfaces are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for human respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Although saliva microdroplets dried out rapidly at all tested RH levels (unlike SM that remained hydrated at RH \u2265 57%), survival of all three viruses in dry saliva microdroplets was significantly higher than in water or SM. Thus, RH and hydration conditions are not sufficient to explain virus survival, indicating that the suspended medium, and association with saliva components in particular, likely affect physicochemical properties that determine virus survival. The observed high virus survival in dry saliva deposited on surfaces, under a wide range of RH levels, can have profound implications for human public health, specifically the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.06.15.152983","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.153692","pub_date":"2020-6-16","title":"Bio-JOIE: Joint Representation Learning of Biological Knowledge Bases","abstract":"The widespread of Coronavirus has led to a worldwide pandemic with a high mortality rate. Currently, the knowledge accumulated from different studies about this virus is very limited. Leveraging a wide-range of biological knowledge, such as gene on-tology and protein-protein interaction (PPI) networks from other closely related species presents a vital approach to infer the molecular impact of a new species. In this paper, we propose the transferred multi-relational embedding model Bio-JOIE to capture the knowledge of gene ontology and PPI networks, which demonstrates superb capability in modeling the SARS-CoV-2-human protein interactions. Bio-JOIE jointly trains two model components. The knowledge model encodes the relational facts from the protein and GO domains into separated embedding spaces, using a hierarchy-aware encoding technique employed for the GO terms. On top of that, the transfer model learns a non-linear transformation to transfer the knowledge of PPIs and gene ontology annotations across their embedding spaces. By leveraging only structured knowledge, Bio-JOIE significantly outperforms existing state-of-the-art methods in PPI type prediction on multiple species. Furthermore, we also demonstrate the potential of leveraging the learned representations on clustering proteins with enzymatic function into enzyme commission families. Finally, we show that Bio-JOIE can accurately identify PPIs between the SARS-CoV-2 proteins and human proteins, providing valuable insights for advancing research on this new disease.","version":"1.1","doi":"10.1101/2020.06.15.153692","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.151738","pub_date":"2020-6-16","title":"Mapping RNA dependent RNA polymerase activity and immune gene expression using PRO-seq","abstract":"Positive strand, single strand RNA viruses ((+)ssRNA viruses) are viruses with an RNA genome that have broad impacts on a wide range of hosts, including SARS-CoV-2 human respiratory infections. Their replication and gene expression are driven by RNA dependent RNA polymerases (RdRp). Detecting active RNA synthesis by RdRp is critical for assessing the infectivity and pathogenicity of (+)ssRNA viruses. Current approaches rely on viral RNA detection, which cannot distinguish viral titer from RdRp activity. Precision Run-On sequencing (PRO-seq) is a nuclear run-on based nascent RNA sequencing method, widely used to map eukaryotic RNA polymerases by using labeled nucleotide analogues. Here we provide evidence that PRO-seq also detects RdRp activity and can serve as a highly sensitive RdRp mapping method. Coupled to PRO-seq in human blood samples, we propose to use PRO-seq as a single package method to detect (+)ssRNA virus RdRp activity and its interaction with host immune response through transcriptome-wide profiling of leukocyte gene expressions at once.","version":"1.1","doi":"10.1101/2020.06.15.151738","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.053058","pub_date":"2020-6-15","title":"Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2","abstract":"Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.04.21.053058","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.149039","pub_date":"2020-6-15","title":"Inhaled corticosteroids downregulate the SARS-CoV-2 receptor ACE2 in COPD through suppression of type I interferon","abstract":"Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is a new rapidly spreading infectious disease. Early reports of hospitalised COVID-19 cases have shown relatively low frequency of chronic lung diseases such as chronic obstructive pulmonary disease (COPD) but increased risk of adverse outcome. The mechanisms of altered susceptibility to viral acquisition and/or severe disease in at-risk groups are poorly understood. Inhaled corticosteroids (ICS) are widely used in the treatment of COPD but the extent to which these therapies protect or expose patients with a COPD to risk of increased COVID-19 severity is unknown. Here, using a combination of human and animal in vitro and in vivo disease models, we show that ICS administration attenuates pulmonary expression of the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme (ACE)-2. This effect was mechanistically driven by suppression of type I interferon as exogenous interferon-\u03b2 reversed ACE2 downregulation by ICS. Mice deficient in the type I interferon-\u03b1/\u03b2 receptor (Ifnar1\u2212/\u2212) also had reduced expression of ACE2. Collectively, these data suggest that use of ICS therapies in COPD reduces expression of the SARS-CoV-2 entry receptor ACE2 and this effect may thus contribute to altered susceptibility to COVID-19 in patients with COPD.","version":"1.2","doi":"10.1101/2020.06.13.149039","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.991083","pub_date":"2020-6-15","title":"Structural and functional conservation of the programmed \u22121 ribosomal frameshift signal of SARS-CoV-2","abstract":"17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called \u22121 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of \u22121 PRF, and that it also harbors a \u22121 PRF attenuation element. Altering \u22121 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of \u22121 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated \u22121 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit \u22121 PRF was similarly effective against \u22121 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.","version":"1.2","doi":"10.1101/2020.03.13.991083","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.150862","pub_date":"2020-6-15","title":"An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection","abstract":"SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID50 of virus stocks, antiviral efficiencies (IC50 values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research. Determination of SARS-CoV-2 infection by enzymatically quantifying the expression of viral spike protein in bulk cell cultures Targeting a highly conserved region in the S2 subunit of the S protein allows broad detection of several SARS-CoV-2 isolates in different cell lines Screening of antivirals in microtiter format and determining the antiviral activity as inhibitory concentrations 50 (IC50)","version":"1.1","doi":"10.1101/2020.06.14.150862","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030650","pub_date":"2020-6-15","title":"The spatial and cell-type distribution of SARS-CoV-2 receptor ACE2 in human and mouse brain","abstract":"By engaging angiotensin-converting enzyme 2 (ACE2 or Ace2), the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) may invade host cells in many organs, including the brain. However, the distribution of ACE2 in the brain is still obscure. Here we investigated the ACE2 expression in the brain by analyzing data from publicly available brain transcriptome databases. According to our spatial distribution analysis, ACE2 was relatively highly expressed in some brain locations, such as the choroid plexus and paraventricular nuclei of the thalamus. According to cell-type distribution analysis, nuclear expression of ACE2 was found in many neurons (both excitatory and inhibitory neurons) and some non-neuron cells (mainly astrocytes, oligodendrocytes, and endothelial cells) in human middle temporal gyrus and posterior cingulate cortex. A few ACE2-expressing nuclei were found in a hippocampal dataset, and none were detected in the prefrontal cortex. Except for the additional high expression of Ace2 in the olfactory bulb areas for spatial distribution as well as in the pericytes and endothelial cells for cell-type distribution, the distribution of Ace2 in mouse brain was similar to that in the human brain. Thus, our results reveal an outline of ACE2/Ace2 distribution in the human and mouse brain, which indicates the brain infection of SARS-CoV-2 may be capable of inducing central nervous system symptoms in coronavirus disease 2019 (COVID-19) patients. Potential species differences should be considered when using mouse models to study the neurological effects of SARS-CoV-2 infection.","version":"1.3","doi":"10.1101/2020.04.07.030650","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.106609","pub_date":"2020-6-15","title":"Tiger team: a panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes","abstract":"The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug, specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.05.20.106609","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.151647","pub_date":"2020-6-15","title":"Genome-wide mapping of therapeutically-relevant SARS-CoV-2 RNA structures","abstract":"SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome of \u223c30 kb, whose outbreak caused the still ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally conserved structural elements within coronavirus RNA genomes have been identified to date. Here, we performed RNA structure probing by SHAPE-MaP to obtain a single-base resolution secondary structure map of the full SARS-CoV-2 coronavirus genome. The SHAPE-MaP probing data recapitulate the previously described coronavirus RNA elements (5\u2032 UTR, ribosomal frameshifting element, and 3\u2032 UTR), and reveal new structures. Secondary structure-restrained 3D modeling of highly-structured regions across the SARS-CoV-2 genome allowed for the identification of several putative druggable pockets. Furthermore, \u223c8% of the identified structure elements show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. In addition, we identify a set of persistently single-stranded regions having high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.","version":"1.1","doi":"10.1101/2020.06.15.151647","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.150912","pub_date":"2020-6-15","title":"The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood\u2013brain barrier","abstract":"As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.06.15.150912","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.147868","pub_date":"2020-6-15","title":"A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy","abstract":"In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provides a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain (NTD) of the viral S glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain (RBD) of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic virus infection in a standard assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.","version":"1.1","doi":"10.1101/2020.06.14.147868","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.15.152892","pub_date":"2020-6-15","title":"Interfacial Water Molecules Make RBD of SPIKE Protein and Human ACE2 to Stick Together","abstract":"A novel coronavirus (SARS-CoV-2; COVID-19) that initially originates from Wuhan province in China has emerged as a global pandemic, an outbreak that started at the end of 2019 which claims 431,192 (Date: 15th June 2020 (https://covid19.who.in) life till now. Since then scientists all over the world are engaged in developing new vaccines, antibodies, or drug molecules to combat this new threat. Here in this work, we performed an in-silico analysis on the protein-protein interactions between the receptor-binding (RBD) domain of viral SPIKE protein and human angiotensin-converting enzyme 2 (hACE2) receptor to highlight the key alteration that happened from SARS-CoV to SARS-CoV-2. We analyzed and compared the molecular differences between these two viruses by using various computational approaches such as binding affinity calculations, computational alanine, and molecular dynamics simulations. The binding affinity calculations show SARS-CoV-2 binds little more firmly to the hACE2 receptor than that of SARS-CoV. Analysis of simulation trajectories reveals that enhanced hydrophobic contacts or the van der Waals interaction play a major role in stabilizing the protein-protein interface. The major finding obtained from molecular dynamics simulations is that the RBD-ACE2 interface is populated with water molecules and interacts strongly with both RBD and ACE2 interfacial residues during the simulation periods. We also emphasize that the interfacial water molecules play a critical role in binding and maintaining the stability of the RBD/hACE2 complex. The water-mediated hydrogen bond by the bridge water molecules is crucial for stabilizing the RBD and ACE2 domains. The structural and dynamical features presented here may serve as a guide for developing new drug molecules, vaccines, or antibodies to combat the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.06.15.152892","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.151381","pub_date":"2020-6-15","title":"Pulmonary toxicity and inflammatory response of e-cigarettes containing medium-chain triglyceride oil and vitamin E acetate: Implications in the pathogenesis of EVALI but independent of SARS-COV-2 COVID-19 related proteins","abstract":"Recently, there has been an outbreak associated with the use of e-cigarette or vaping products, associated lung injury (EVALI). The primary components of vaping products, vitamin E acetate (VEA) and medium-chain triglycerides (MCT) may be responsible for acute lung toxicity. Currently, little information is available on the physiological and biological effects of exposure to these products. We hypothesized that these e-cig cartridges and their constituents (VEA and MCT) induce pulmonary toxicity, mediated by oxidative damage and inflammatory responses, leading to acute lung injury. We studied the potential mechanisms of cartridge aerosol induced inflammatory response by evaluating the generation of reactive oxygen species by MCT, VEA, and cartridges, and their effects on the inflammatory state of pulmonary epithelium and immune cells both in vitro and in vivo. Cells exposed to these aerosols generated reactive oxygen species, caused cytotoxicity, induced epithelial barrier dysfunction, and elicited an inflammatory response. Using a murine model, the parameters of acute toxicity to aerosol inhalation were assessed. Infiltration of neutrophils and lymphocytes was accompanied by significant increases in IL-6, eotaxin, and G-CSF in the bronchoalveolar lavage fluid (BALF). In mouse plasma, eicosanoid inflammatory mediators, leukotrienes, were significantly increased. Plasma from e-cig users also showed increased levels of hydroxyeicosatetraenoic acid (HETEs) and various eicosanoids. Exposure to e-cig cartridge aerosols showed the most significant effects and toxicity compared to MCT and VEA. In addition, we determined at SARS-COV-2 related proteins and found no impact associated with aerosol exposures from these tested cartridges. Overall, this study demonstrates acute exposure to specific e-cig cartridges induces in vitro cytotoxicity, barrier dysfunction, and inflammation and in vivo mouse exposure induces acute inflammation with elevated pro-inflammatory markers in the pathogenesis of EVALI.","version":"1.1","doi":"10.1101/2020.06.14.151381","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.143990","pub_date":"2020-6-14","title":"Dynamics of the ACE2 - SARS-CoV/SARS-CoV-2 spike protein interface reveal unique mechanisms","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major public health concern. A handful of static structures now provide molecular insights into how SARS-CoV-2 and SARS-CoV interact with its host target, which is the angiotensin converting enzyme 2 (ACE2). Molecular recognition, binding and function are dynamic processes. To evaluate this, multiple all atom molecular dynamics simulations of at least 500 ns each were performed to better understand the structural stability and interfacial interactions between the receptor binding domain of the spike protein of SARS-CoV-2 and SARS-CoV bound to ACE2. Several contacts were observed to form, break and reform in the interface during the simulations. Our results indicate that SARS-CoV and SARS-CoV-2 utilizes unique strategies to achieve stable binding to ACE2. Several differences were observed between the residues of SARS-CoV-2 and SARS-CoV that consistently interacted with ACE2. Notably, a stable salt bridge between Lys417 of SARS-CoV-2 spike protein and Asp30 of ACE2 as well as three stable hydrogen bonds between Tyr449, Gln493, and Gln498 of SARS-CoV-2 and Asp38, Glu35, and Lys353 of ACE2 were observed, which were absent in the SARS-CoV-ACE2 interface. Some previously reported residues, which were suggested to enhance the binding affinity of SARS-CoV-2, were not observed to form stable interactions in these simulations. Stable binding to the host receptor is crucial for virus entry. Therefore, special consideration should be given to these stable interactions while designing potential drugs and treatment modalities to target or disrupt this interface.","version":"1.1","doi":"10.1101/2020.06.10.143990","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.140657","pub_date":"2020-6-14","title":"Genotypic and antigenic study of SARS-CoV-2 from an Indian isolate","abstract":"Coronaviruses (CoVs) are one of the largest groups of positive-sense RNA virus families within the Nidovirales order, which are further classified into four genera: alpha, beta, gamma, and delta. Coronaviruses have an extensive range of natural hosts and are known to be responsible for a broad spectrum of diseases in multiple species. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) that has unleashed a global threat to public health and the economy. Coronaviruses are extensively present in birds and mammals, with horseshoe bats (Rhinolophus affinis), being the reservoir for the ongoing SARS-CoV-2 that seems to have resulted from a zoonotic spillover to the human host, causing respiratory infections, lung injury and Acute Respiratory Distress Syndrome(ARDS). About six coronavirus serotypes are linked with the disease in humans, namely HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, SARS-CoV-2, and MERS-CoV. SARS-CoV-2 is the seventh CoV to infect humans. We analyzed the genome sequence of CoV-2 from isolates derived from China as well from India and encountered minute variations in their sequence. A cladogram analysis revealed the predominant strain circulating in India belongs to the A2a clad. We took one such strain (MT012098) and performed a rigorous in-silico genotypic and antigenic analysis to identify its relatedness to other strains. Further, we also performed a detailed prediction for B and T cell epitopes using BepiPred 2.0 server and NetCTL 1.2 server (DTU Bioinformatics), respectively. We hope this information may assist in an effective vaccine designing program against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.10.140657","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.149690","pub_date":"2020-6-14","title":"Single-cell screening of SARS-CoV-2 target cells in pets, livestock, poultry and wildlife","abstract":"A few animals have been suspected to be intermediate hosts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a large-scale single-cell screening of SARS-CoV-2 target cells on a wide variety of animals is missing. Here, we constructed the single-cell atlas for 11 representative species in pets, livestock, poultry, and wildlife. Notably, the proportion of SARS-CoV-2 target cells in cat was found considerably higher than other species we investigated and SARS-CoV-2 target cells were detected in multiple cell types of domestic pig, implying the necessity to carefully evaluate the risk of cats during the current COVID-19 pandemic and keep pigs under surveillance for the possibility of becoming intermediate hosts in future coronavirus outbreak. Furthermore, we screened the expression patterns of receptors for 144 viruses, resulting in a comprehensive atlas of virus target cells. Taken together, our work provides a novel and fundamental strategy to screen virus target cells and susceptible species, based on single-cell transcriptomes we generated for domesticated animals and wildlife, which could function as a valuable resource for controlling current pandemics and serve as an early warning system for coping with future infectious disease threats.","version":"1.1","doi":"10.1101/2020.06.13.149690","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.150250","pub_date":"2020-6-14","title":"SARS-CoV-2 Neutralizing Antibody Responses Are More Robust in Patients with Severe Disease","abstract":"We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ~5-fold and ~7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.","version":"1.1","doi":"10.1101/2020.06.13.150250","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.14.150458","pub_date":"2020-6-14","title":"Calreticulin co-expression supports high level production of a recombinant SARS-CoV-2 spike mimetic in Nicotiana benthamiana","abstract":"An effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 infection. The viral spike, which mediates entry into cells by interacting with the host angiotensin-converting enzyme 2, is the primary target of most vaccines in development. These vaccines aim to elicit protective immunity against the glycoprotein by use of inactivated virus, vector-mediated delivery of the antigen in vivo, or by direct immunization with the purified antigen following expression in a heterologous system. These approaches are mostly dependent on the growth of mammalian or insect cells, which requires a sophisticated infrastructure that is not generally available in developing countries due to the incumbent costs which are prohibitive. Plant-based subunit vaccine production has long been considered as a cheaper alternative, although low expression yields and differences along the secretory pathway to mammalian cells have posed a challenge to producing certain viral glycoproteins. Recent advances that have enabled many of these constraints to be addressed include expressing the requisite human proteins in plants to support the maturation of the protein of interest. In this study we investigated these approaches to support the production of a soluble and putatively trimeric SARS-CoV-2 spike mimetic in Nicotiana benthamiana via transient Agrobacterium-mediated expression. The co-expression of human calreticulin dramatically improved the accumulation of the viral spike, which was barely detectable in the absence of the co-expressed accessory protein. The viral antigen was efficiently processed even in the absence of co-expressed furin, suggesting that processing may have occurred at the secondary cleavage site and was mediated by an endogenous plant protease. In contrast, the spike was not efficiently processed when expressed in mammalian cells as a control, although the co-expression of furin improved processing considerably. This study demonstrates the feasibility of molecular engineering to improve the production of viral glycoproteins in plants, and supports plant-based production of SARS-CoV-2 spike-based vaccines and reagents for serological assays.","version":"1.1","doi":"10.1101/2020.06.14.150458","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.143545","pub_date":"2020-6-14","title":"In silico multi-epitope vaccine against covid19 showing effective interaction with HLA-B*15:03","abstract":"The recent outbreak of severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 (SARS-CoV-2) causing coronavirus disease (covid19) has posed a great threat to human health. Previous outbreaks of SARS-CoV and Middle East respiratory Syndrome CoV (MERS-CoV) from the same CoV family had posed similar threat to human health and economic growth. To date, not even a single drug specific to any of these CoVs has been developed nor any anti-viral vaccine is available for the treatment of diseases caused by CoVs. Subunits present in spike glycoproteins of SARS-CoV and SARS-CoV-2 are involved in binding to human ACE2 Receptor which is the primary method of viral invasion. As it has been observed in the previous studies that there are very minor differences in the spike glycoproteins of SARS-CoV and SARS-CoV-2. SARS-CoV-2 has an additional furin cleavage site that makes it different from SARS-CoV (Walls et al., 2020). In this study, we have analyzed spike glycoproteins of SARS-CoV-2 and SARS-CoV phylogenetically and subjected them to selection pressure analysis. Selection pressure analysis has revealed some important sites in SARS-CoV-2 and SARS-CoV spike glycoproteins that might be involved in their pathogenicity. Further, we have developed a potential multi-epitope vaccine candidate against SARS-CoV-2 by analyzing its interactions with HLA-B*15:03 subtype. This vaccine consists of multiple T-helper (TH) cells, B-cells, and Cytotoxic T-cells (CTL) epitopes joined by linkers and an adjuvant to increase its immunogenicity. Conservation of selected epitopes in SARS, MERS, and human hosts, suggests that the designed vaccine could provide cross-protection. The vaccine is designed in silico by following a reverse vaccinology method acknowledging its antigenicity, immunogenicity, toxicity, and allergenicity. The vaccine candidate that we have designed as a result of this work shows promising result indicating its potential capability of simulating an immune response.","version":"1.1","doi":"10.1101/2020.06.10.143545","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.143271","pub_date":"2020-6-14","title":"Differential expression of COVID-19-related genes in European Americans and African Americans","abstract":"The Coronavirus disease 2019 (COVID-19) pandemic has affected African American populations disproportionately in regards to both morbidity and mortality. A multitude of factors likely account for this discrepancy. Gene expression represents the interaction of genetics and environment. To elucidate whether levels of expression of genes implicated in COVID-19 vary in African Americans as compared to European Americans, we re-mine The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) RNA-Seq data. Multiple genes integral to infection, inflammation and immunity are differentially regulated across the two populations. Most notably, F8A2 and F8A3, which encode the HAP40 protein that mediates early endosome movement in Huntington\u2019s Disease, are more highly expressed by up to 24-fold in African Americans. Such differences in gene expression can establish prognostic signatures and have critical implications for precision treatment of diseases such as COVID-19. We advocate routine inclusion of information such as postal code, education level, and profession (as a proxies for socioeconomic condition) and race in the metadata about each individual sampled for sequencing studies. This relatively simple change would enable large-scale data-driven approaches to dissect relationships among race, socio-economic factors, and disease.","version":"1.3","doi":"10.1101/2020.06.09.143271","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.146878","pub_date":"2020-6-13","title":"Colon Cancer and SARS-CoV-2: Impact of ACE2 Expression in Susceptibility to COVID-19","abstract":"Novel coronavirus disease (COVID-19) pandemic has become a global health emergency. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with angiotensin-converting enzyme 2 (ACE2) to enter the cells and infects diverse human tissues. It has been reported that a few conditions, including cancer, predispose individuals to SARS-CoV-2 infection and severe form of COVID-19. These findings led us to evaluate the susceptibility of colon adenocarcinoma (COAD) patients to SARS-CoV-2 infection by investigation of ACE2 expression in their tumor tissues. The expression analysis revealed that both mRNA and protein levels of ACE2 had increased in colon cancer samples than normal group. Next, the prognosis analysis has indicated that the upregulation of ACE2 was not correlated with patient survival outcomes. Further assessment displayed the hypomethylation of the ACE2 gene promoter in COAD patients. Surprisingly, this methylation status has a strong negative correlation with ACE2 gene expression. The functional enrichment analysis of the genes that had similar expression patterns with ACE2 in colon cancer tissues demonstrated that they mainly enriched in Vitamin digestion and absorption, Sulfur relay system, and Fat digestion and absorption pathways. Finally, we found that ACE2 gene expression had a significant association with the immune cell infiltration levels in COAD patients. In conclusion, it has plausible that COAD patients are more likely to be infected with SARS-CoV-2 and experience severe injuries. Moreover, COVID-19 would bring unfavorable survival outcomes of patients with colon cancer by the way of immune cell infiltration linked process. The present study highlights the importance of preventive actions for COAD patients during the COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.06.11.146878","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.149930","pub_date":"2020-6-13","title":"Receptor utilization of angiotensin converting enzyme 2 (ACE2) indicates a narrower host range of SARS-CoV-2 than that of SARS-CoV","abstract":"Coronavirus pandemics have become a huge threat to the public health worldwide in the recent decades. Typically, SARS-CoV caused SARS pandemic in 2003 and SARS-CoV-2 caused the COVID-19 pandemic recently. Both viruses have been reported to originate from bats. Thus, direct or indirect interspecies transmission from bats to humans is required for the viruses to cause pandemics. Receptor utilization is a key factor determining the host range of viruses which is critical to the interspecies transmission. Angiotensin converting enzyme 2 (ACE2) is the receptor of both SARS-CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of SARS-CoV-2. Meanwhile, pangolin CoV, another SARS-related coronavirus highly homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile with SARS-CoV rather than SARS-CoV-2. To clarify the mechanism underlying the receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 20th and 42nd amino acids that may determine the different receptor utilization of SARS-CoV, SARS-CoV-2 and pangolin CoV. Our studies promote the understanding of receptor utilization of pandemic coronaviruses, potentially contributing to the virus tracing, intermediate host screening and epidemic prevention for pathogenic coronaviruses.","version":"1.1","doi":"10.1101/2020.06.13.149930","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.148916","pub_date":"2020-6-13","title":"Single-cell transcriptomic analysis of SARS-CoV-2 reactive CD4+ T cells","abstract":"The contribution of CD4+ T cells to protective or pathogenic immune responses to SARS-CoV-2 infection remains unknown. Here, we present large-scale single-cell transcriptomic analysis of viral antigen-reactive CD4+ T cells from 32 COVID-19 patients. In patients with severe disease compared to mild disease, we found increased proportions of cytotoxic follicular helper (TFH) cells and cytotoxic T helper cells (CD4-CTLs) responding to SARS-CoV-2, and reduced proportion of SARS-CoV-2 reactive regulatory T cells. Importantly, the CD4-CTLs were highly enriched for the expression of transcripts encoding chemokines that are involved in the recruitment of myeloid cells and dendritic cells to the sites of viral infection. Polyfunctional T helper (TH)1 cells and TH17 cell subsets were underrepresented in the repertoire of SARS-CoV-2-reactive CD4+ T cells compared to influenza-reactive CD4+ T cells. Together, our analyses provide so far unprecedented insights into the gene expression patterns of SARS-CoV-2 reactive CD4+ T cells in distinct disease severities.","version":"1.1","doi":"10.1101/2020.06.12.148916","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.149377","pub_date":"2020-6-13","title":"WHOLE-GENOME SEQUENCING AND DE NOVO ASSEMBLY OF A 2019 NOVEL CORONAVIRUS (SARS-COV-2) STRAIN ISOLATED IN VIETNAM","abstract":"The pandemic COVID-19 caused by the zoonotic virus SARS-CoV-2 has devastated countries worldwide, infecting more than 4.5 million people and leading to more than 300,000 deaths. Whole genome sequencing (WGS) is an effective tool to monitor emerging strains and provide information for intervention, thus help to inform outbreak control decisions. Here, we reported the first effort to sequence and de novo assemble the whole genome of SARS-CoV-2 using PacBio\u2019s SMRT sequencing technology in Vietnam. We also presented the annotation results and a brief analysis of the variants found in our SARS-CoV-2 strain, which was isolated from a Vietnamese patient. The sequencing was successfully completed and de novo assembled in less than 30 hours, resulting in one contig with no gap and a length of 29,766 bp. All detected variants as compared to the NCBI reference were highly accurate as confirmed by Sanger sequencing. The results have shown the potential of long read sequencing to provide high quality WGS data to support public health responses, and advance understanding of this and future pandemics.","version":"1.1","doi":"10.1101/2020.06.12.149377","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.148387","pub_date":"2020-6-13","title":"Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient","abstract":"The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6\u00c5 crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.","version":"1.1","doi":"10.1101/2020.06.12.148387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.149880","pub_date":"2020-6-13","title":"Multi-epitope Based Peptide Vaccine Design Using Three Structural Proteins (S, E, and M) of SARS-CoV-2: An In Silico Approach","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the novel coronavirus responsible for the ongoing pandemic of coronavirus disease (COVID-19). No sustainable treatment option is available so far to tackle such a public health threat. Therefore, designing a suitable vaccine to overcome this hurdle asks for immediate attention. In this study, we targeted for a design of multi-epitope based vaccine using immunoinformatics tools. We considered the structural proteins S, E and, M of SARS-CoV-2, since they facilitate the infection of the virus into host cell and using different bioinformatics tools and servers, we predicted multiple B-cell and T-cell epitopes having potential for the required vaccine design. Phylogenetic analysis provided insight on ancestral molecular changes and molecular evolutionary relationship of S, E, and M proteins. Based on the antigenicity and surface accessibility of these proteins, eight epitopes were selected by various B cell and T cell epitope prediction tools. Molecular docking was executed to interpret the binding interactions of these epitopes and three potential epitopes WTAGAAAYY, YVYSRVKNL, and GTITVEELK were selected for their noticeable higher binding affinity scores \u22129.1, \u22127.4, and \u22127.0 kcal/mol, respectively. Targeted epitopes had 91.09% population coverage worldwide. In summary, we identified three epitopes having the most significant properties of designing the peptide-based vaccine against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.13.149880","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.140285","pub_date":"2020-6-13","title":"ROBOCOV: An affordable open-source robotic platform for SARS-CoV-2 testing by RT-qPCR","abstract":"Current global pandemic due to the SARS-CoV-2 has struggled and pushed the limits of global health systems. Supply chain disruptions and scarce availability of commercial laboratory reagents have motivated worldwide actors to search for alternative workflows to cope with the demand. We have used the OT-2 open-source liquid-handling robots (Opentrons, NY), RNA extraction and RT-qPCR reagents to set-up a reproducible workflow for RT-qPCR SARS-CoV-2 testing. We developed a framework with a template and several functions and classes that allow the creation of customized RT-qPCR automated circuits. As a proof of concept, we provide data obtained by a fully-functional tested code using the MagMax\u2122 Pathogen RNA/DNA kit and the MagMax\u2122 Viral/Pathogen II kit (Thermo Fisher Scientific, MA) on the Kingfisher\u2122 Flex instrument (Thermo Fisher Scientific, MA). With these codes available is easy to create new stations or circuits from scratch, adapt existing ones to changes in the experimental protocol, or perform fine adjustments to fulfil special needs. The affordability of this platform makes it accessible for most laboratories and hospitals with a bioinformatician, democratising automated SARS-CoV-2 PCR testing and increasing, temporarily or not, the capacity of carrying out tests. It also confers flexibility, as this platform is not dependant on any particular commercial kit and can be quickly adapted to protocol changes or other special needs.","version":"1.3","doi":"10.1101/2020.06.11.140285","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030734","pub_date":"2020-6-13","title":"Analysis of Infected Host Gene Expression Reveals Repurposed Drug Candidates and Time-Dependent Host Response Dynamics for COVID-19","abstract":"The repurposing of existing drugs offers the potential to expedite therapeutic discovery against the current COVID-19 pandemic caused by the SARS-CoV-2 virus. We have developed an integrative approach to predict repurposed drug candidates that can reverse SARS-CoV-2-induced gene expression in host cells, and evaluate their efficacy against SARS-CoV-2 infection in vitro. We found that 13 virus-induced gene expression signatures computed from various viral preclinical models could be reversed by compounds previously identified to be effective against SARS- or MERS-CoV, as well as drug candidates recently reported to be efficacious against SARS-CoV-2. Based on the ability of candidate drugs to reverse these 13 infection signatures, as well as other clinical criteria, we identified 10 novel candidates. The four drugs bortezomib, dactolisib, alvocidib, and methotrexate inhibited SARS-CoV-2 infection-induced cytopathic effect in Vero E6 cells at < 1 \u00b5M, but only methotrexate did not exhibit unfavorable cytotoxicity. Although further improvement of cytotoxicity prediction and bench testing is required, our computational approach has the potential to rapidly and rationally identify repurposed drug candidates against SARS-CoV-2. The analysis of signature genes induced by SARS-CoV-2 also revealed interesting time-dependent host response dynamics and critical pathways for therapeutic interventions (e.g. Rho GTPase activation and cytokine signaling suppression).","version":"1.2","doi":"10.1101/2020.04.07.030734","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.13.149591","pub_date":"2020-6-13","title":"Coronavirus genomes carry the signatures of their habitats","abstract":"Coronaviruses such as SARS-CoV-2 regularly infect host tissues that express antiviral proteins (AVPs) in abundance. Understanding how they evolve to adapt or evade host immune responses is important in the effort to control the spread of COVID-19. Two AVPs that may shape viral genomes are the zinc finger antiviral protein (ZAP) and the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 protein (APOBEC3). The former binds to CpG dinucleotides to facilitate the degradation of viral transcripts while the latter deaminates C into U residues leading to dysfunctional transcripts. We tested the hypothesis that both APOBEC3 and ZAP may act as primary selective pressures that shape the genome of an infecting coronavirus by considering a comprehensive number of publicly available genomes for seven coronaviruses (SARS-CoV-2, SARS-CoV, MERS, Bovine CoV, Murine MHV, Porcine HEV, and Canine CoV). We show that coronaviruses that regularly infect tissues with abundant AVPs have CpG-deficient and U-rich genomes; whereas viruses that do not infect tissues with abundant AVPs do not share these sequence hallmarks. In SARS-CoV-2, CpG is most deficient in the S protein region to evaded ZAP-mediated antiviral defense during cell entry. Furthermore, over four months of SARS-CoV-2 evolutionary history, we observed a marked increase in C to U substitutions in the 5\u2019 UTR and ORF1ab regions. This suggests that the two regions could be under constant C to U deamination by APOBEC3. The evolutionary pressures exerted by host immune systems onto viral genomes may motivate novel strategies for SARS-CoV-2 vaccine development.","version":"1.1","doi":"10.1101/2020.06.13.149591","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.146290","pub_date":"2020-6-12","title":"Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients","abstract":"The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and world economy. Since approved drugs and vaccines are not available, new options for COVID-19 treatment and prevention are highly demanded. To identify SARS-CoV-2 neutralizing antibodies, we analysed the antibody response of 12 COVID-19 patients from 8 to 69 days post diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days post diagnosis. Among these, 28 potently neutralized authentic SARS-CoV-2 (IC100 as low as 0.04 \u03bcg/ml), showing a broad spectrum of V genes and low levels of somatic mutations. Interestingly, potential precursors were identified in na\u00efve B cell repertoires from 48 healthy individuals that were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2 neutralizing antibodies are readily generated from a diverse pool of precursors, fostering the hope of rapid induction of a protective immune response upon vaccination.","version":"1.1","doi":"10.1101/2020.06.12.146290","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.131755","pub_date":"2020-6-12","title":"In Silico design and characterization of multi-epitopes vaccine for SARS-CoV2 from its spike proteins","abstract":"COVID 19 is disease caused by novel corona virus, SARS-CoV2 originated in China most probably of Bat origin. Till date, no specific vaccine or drug has been discovered to tackle the infections caused by SARS-CoV2. In response to this pandemic, we utilized bioinformatics knowledge to develop efficient vaccine candidate against SARS-CoV2. Designed vaccine was rich in effective BCR and TCR epitopes screened from the sequence of S-protein of SARS-CoV2. Predicted BCR and TCR epitopes were antigenic in nature non-toxic and probably non-allergen. Modelled and refined tertiary structure was predicted as valid for further use. Protein-Protein interaction prediction of TLR2/4 and designed vaccine indicates promising binding. Designed multiepitope vaccine has induced cell mediated and humoral immunity along with increased interferon gamma response. Macrophages and dendritic cells were also found increased over the vaccine exposure. In silico codon optimization and cloning in expression vector indicates that vaccine can be efficiently expressed in E. coli. In conclusion, predicted vaccine is a good antigen, probable no allergen and has potential to induce cellular and humoral immunity.","version":"1.3","doi":"10.1101/2020.06.03.131755","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.19.048991","pub_date":"2020-6-12","title":"CoV2ID: Detection and Therapeutics Oligo Database for SARS-CoV-2","abstract":"The ability to detect the SARS-CoV-2 in a widespread epidemic is crucial for screening of carriers and for the success of quarantine efforts. Methods based on real-time reverse transcription polymerase chain reaction (RT-qPCR) and sequencing are being used for virus detection and characterization. However, RNA viruses are known for their high genetic diversity which poses a challenge for the design of efficient nucleic acid-based assays. The first SARS-CoV-2 genomic sequences already showed novel mutations, which may affect the efficiency of available screening tests leading to false-negative diagnosis or inefficient therapeutics. Here we describe the CoV2ID (http://covid.portugene.com/), a free database built to facilitate the evaluation of molecular methods for detection of SARS-CoV-2 and treatment of COVID-19. The database evaluates the available oligonucleotide sequences (PCR primers, RT-qPCR probes, etc.) considering the genetic diversity of the virus. Updated sequences alignments are used to constantly verify the theoretical efficiency of available testing methods. Detailed information on available detection protocols are also available to help laboratories implementing SARS-CoV-2 testing.","version":"1.2","doi":"10.1101/2020.04.19.048991","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.148692","pub_date":"2020-6-12","title":"Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation","abstract":"SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain (RBD). The structure reveals CV30\u2019s epitope overlaps with the human ACE2 receptor binding site thus providing the structural basis for its neutralization by preventing ACE2 binding.","version":"1.1","doi":"10.1101/2020.06.12.148692","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.147199","pub_date":"2020-6-12","title":"Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate","abstract":"A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5\u2019-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5\u2019-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.11.147199","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.082834","pub_date":"2020-6-12","title":"SARS-CoV-2 sequence typing, evolution and signatures of selection using CoVa, a Python-based command-line utility","abstract":"The current global pandemic COVID-19, caused by SARS-CoV-2, has resulted in millions of infections worldwide in a few months. Global efforts to tackle this situation have produced a tremendous body of genomic data, which can be used for tracing transmission routes, characterization of isolates, and monitoring variants with potential for unusual virulence. Several groups have analyzed these genomes using different approaches. However, as new data become available, the research community needs a pipeline to perform a set of routine analyses, that can quickly incorporate new genome sequences and update the analysis reports. We developed a programmatic tool, CoVa, with this objective. It is a fast, accurate and user-friendly utility to perform a variety of genome analyses on hundreds of SARS-CoV-2 sequences. Using CoVa, we define a modified sequence typing nomenclature and identify sites under positive selection. Further analysis identified some peptides and sites showing geographical patterns of selection. Specifically, we show differences in sequence type distribution between sequences from India and those from the rest of the world. We also show that several sites show signatures of positive selection uniquely in sequences from India. Preliminary evolutionary analysis, using features that will be incorporated into CoVa in the near future, show a mutation rate of 7.4 \u00d7 10\u22124 substitutions/site/year, confirm a temporal signal with a November 2019 origin of SARS-CoV-2, and a heterogeneity in the geographical distribution of Indian samples.","version":"1.2","doi":"10.1101/2020.06.09.082834","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.147363","pub_date":"2020-6-12","title":"Highly sensitive and specific multiplex antibody assays to quantify immunoglobulins M, A and G against SARS-CoV-2 antigens","abstract":"Reliable serological tests are required to determine the prevalence of antibodies against SARS-CoV-2 antigens and to characterise immunity to the disease in order to address key knowledge gaps in the context of the COVID-19 pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and ELISA with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. We developed three qSAT assays to detect IgM, IgA and IgG to a panel of eight SARS-CoV-2 antigens including spike (S), nucleoprotein (N) and membrane (M) protein constructs. The assays were optimized to minimize processing time and maximize signal to noise ratio. We evaluated the performance of the assays using 128 plasmas obtained before the COVID-19 pandemic (negative controls) and 115 plasmas from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 8 were asymptomatic, 58 had mild symptoms and 49 were hospitalized. Pre-existing IgG antibodies recognizing N, M and S2 proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. The best performing antibody isotype/antigen signatures had specificities of 100% and sensitivities of 94.94% at \u226514 days since the onset of symptoms and 96.08% at \u226521 days since the onset of symptoms, with AUC of 0.992 and 0.999, respectively. Combining multiple antibody markers as assessed by qSAT assays has the highest efficiency, breadth and versatility to accurately detect low-level antibody responses for obtaining reliable data on prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity required for vaccine development to combat pandemics like the COVID-19.","version":"1.1","doi":"10.1101/2020.06.11.147363","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.134585","pub_date":"2020-6-12","title":"Global cataloguing of variations in untranslated regions of viral genome and prediction of key host RNA binding protein-microRNA interactions modulating genome stability in SARS-CoV2","abstract":"The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5\u2019 and 3\u2019 untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. To have an idea of the global changes in 5\u2019 and 3\u2019-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by \u2018RBPmap\u2019 and \u2018RNA22 v2\u2019 respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. We identified 28 unique variants in SARS-CoV2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5\u2019-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3\u2019UTR among most of the continents. Furthermore, we found that despite of the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. Our results, shows for the first time in SARS-Cov2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.","version":"1.2","doi":"10.1101/2020.06.09.134585","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.097410","pub_date":"2020-6-12","title":"The impact of super-spreaders in COVID-19: mapping genome variation worldwide","abstract":"The human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the major pandemic of the 21st century. We analyzed >4,700 SARS-CoV-2 genomes and associated meta-data retrieved from public repositories. SARS-CoV-2 sequences have a high sequence identity (>99.9%), which drops to >96% when compared to bat coronavirus. We built a mutation-annotated reference SARS-CoV-2 phylogeny with two main macro-haplogroups, A and B, both of Asian origin, and >160 sub-branches representing virus strains of variable geographical origins worldwide, revealing a uniform mutation occurrence along branches that could complicate the design of future vaccines. The root of SARS-CoV-2 genomes locates at the Chinese haplogroup B1, with a TMRCA dating to 12 November 2019 - thus matching epidemiological records. Sub-haplogroup A2a originates in China and represents the major non-Asian outbreak. Multiple founder effect episodes, most likely associated with super-spreader hosts, explain COVID-19 pandemic to a large extent.","version":"1.3","doi":"10.1101/2020.05.19.097410","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142760","pub_date":"2020-6-12","title":"Hidden in plain sight: The effects of BCG vaccination in COVID-19 pandemic","abstract":"To investigate the relationship between BCG vaccination and SARS-CoV-2 by bioinformatic approach. Two datasets for Sars-CoV-2 infection group and BCG-vaccinated group were downloaded. Differentially Expressed Genes were identified. Gene ontology and pathways were functionally enriched, and networking was constructed in NetworkAnalyst. Lastly, correlation between post-BCG vaccination and COVID-19 transcriptome signatures were established. A total of 161 DEGs (113 upregulated DEGs and 48 downregulated genes) were identified in the Sars-CoV-2 group. In the pathway enrichment analysis, cross-reference of upregulated KEGG pathways in Sars-CoV-2 with downregulated counterparts in the BCG-vaccinated group, resulted in the intersection of 45 common pathways, accounting for 86.5% of SARS-CoV-2 upregulated pathways. Of these intersecting pathways, a vast majority were immune and inflammatory pathways with top significance in IL-17, TNF, NOD-like receptors, and NF-\u03baB signaling pathways. Our data suggests BCG-vaccination may incur a protective role in COVID-19 patients until a targeted vaccine is developed. (https://drive.google.com/open?id=15Na738L282XNaQAJUh0cZf1WoG9jJfzJ)","version":"1.1","doi":"10.1101/2020.06.09.142760","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.148577","pub_date":"2020-6-12","title":"Interactions of SARS-CoV-2 infection with chronic obesity inflammation: a complex network phenomenon","abstract":"Obesity is one of the biggest public health problems in the world, and its pathophysiological characteristics include chronic inflammation with an increase in various circulating inflammatory markers, such as acute inflammatory cytokines. Complications in the respiratory tract are related to bodily problems, which lead to a restriction of lung function due to reduced volume, inducing an increase in respiratory work. SARS-CoV-2 has a high potential for contamination by respiratory secretions and, therefore, obesity is one of the main risk factors for complications due to the association established between obesity, chronic inflammation and respiratory infection. The objective was to analyze the complex relationships between obesity and COVID-19 in a meta-analysis study using complex network modeling and the theoretical knockouts technique. Here, we identify and justify through a mathematical analysis the relationships between all the immunological agents added to the proposed immunological networks, considered as a simple evident interaction, relationship, influence, response, activation, based on our quantifiers. They performed the knockouts of all 52 vertices in the COVID-19 network and obesity - regardless of the environment, which would result in nonsense - and the COVID-19 infection network without considering obesity. The stationary flow vector (flow profile), for some knockouts of immunological interest in COVID-19 infections, was chosen IFN\u03b1, IL-6, IL-10, IL-17 and TNF\u03b1. This initial study pointed out the importance of chronic inflammation in the obese individual as an important factor in potentiating the disease caused by covid-19 and, in particular, the importance on IL-17.","version":"1.1","doi":"10.1101/2020.06.12.148577","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.12.148726","pub_date":"2020-6-12","title":"The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity","abstract":"SARS coronavirus 2 (SARS-CoV-2) isolates encoding a D614G mutation in the viral spike (S) protein predominate over time in locales where it is found, implying that this change enhances viral transmission. We therefore compared the functional properties of the S proteins with aspartic acid (SD614) and glycine (SG614) at residue 614. We observed that retroviruses pseudotyped with SG614 infected ACE2-expressing cells markedly more efficiently than those with SD614. This greater infectivity was correlated with less S1 shedding and greater incorporation of the S protein into the pseudovirion. Similar results were obtained using the virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, SG614 did not bind ACE2 more efficiently than SD614, and the pseudoviruses containing these S proteins were neutralized with comparable efficiencies by convalescent plasma. These results show SG614 is more stable than SD614, consistent with epidemiological data suggesting that viruses with SG614 transmit more efficiently.","version":"1.1","doi":"10.1101/2020.06.12.148726","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142125","pub_date":"2020-6-12","title":"Unravelling the debate on heme effects in COVID-19 infections","abstract":"The SARS-CoV-2 outbreak was recently declared a worldwide pandemic. Infection triggers the respiratory tract disease COVID-19, which is accompanied by serious changes of clinical biomarkers such as hemoglobin and interleukins. The same parameters are altered during hemolysis, which is characterized by an increase in labile heme. We present two approaches that aim at analyzing a potential link between available heme and COVID-19 pathogenesis. Four COVID-19 related proteins, i.e. the host cell proteins ACE2 and TMPRSS2 as well as the viral protein 7a and S protein, were identified as potential heme binders. We also performed a detailed analysis of the common pathways induced by heme and SARS-CoV-2 by superimposition of knowledge graphs covering heme biology and COVID-19 pathophysiology. Herein, focus was laid on inflammatory pathways, and distinct biomarkers as the linking elements. Finally, the results substantially improve our understanding of COVID-19 infections and disease progression of patients with different clinical backgrounds and expand the diagnostic and treatment options.","version":"1.2","doi":"10.1101/2020.06.09.142125","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142372","pub_date":"2020-6-12","title":"One-Step Rapid Quantification of SARS-CoV-2 Virus Particles via Low-Cost Nanoplasmonic Sensors in Generic Microplate Reader and Point-of-Care Device","abstract":"The spread of SARS-CoV-2 virus in the ongoing global pandemics has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemics. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. We demonstrate that we can detect as few as 30 virus particles in one step within 15 minutes and can quantify the virus concentration linearly in the range of 103 vp/ml to 106 vp/ml. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.","version":"1.2","doi":"10.1101/2020.06.09.142372","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.147389","pub_date":"2020-6-12","title":"Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early prognostic marker for severe COVID-19","abstract":"SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients revealed a dramatic increase in the number of immature neutrophils. This increase strongly correlated with disease severity and was associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts was observed for CD8 T-cells and VD2 \u03b3\u03b4 T-cells, which both exhibited increased differentiation and activation. ROC analysis revealed that the count ratio of immature neutrophils to CD8 or VD2 T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.","version":"1.1","doi":"10.1101/2020.06.11.147389","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.145425","pub_date":"2020-6-12","title":"Machine Learning Maps Research Needs in COVID-19 Literature","abstract":"Manually assessing the scope of the thousands of publications on the COVID-19 (coronavirus disease 2019) pandemic is an overwhelming task. Shortcuts through metadata analysis (e.g., keywords) assume that studies are properly tagged. However, machine learning approaches can rapidly survey the actual text of coronavirus abstracts to identify research overlap between COVID-19 and other coronavirus diseases, research hotspots, and areas warranting exploration. We propose a fast, scalable, and reusable framework to parse novel disease literature. When applied to the COVID-19 Open Research Dataset (CORD-19), dimensionality reduction suggested that COVID-19 studies to date are primarily clinical-, modeling- or field-based, in contrast to the vast quantity of laboratory-driven research for other (non-COVID-19) coronavirus diseases. Topic modeling also indicated that COVID-19 publications have thus far focused primarily on public health, outbreak reporting, clinical care, and testing for coronaviruses, as opposed to the more limited number focused on basic microbiology, including pathogenesis and transmission.","version":"1.1","doi":"10.1101/2020.06.11.145425","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.145292","pub_date":"2020-6-11","title":"COVID-19 Variants Database: A repository for Human SARS-CoV-2 Polymorphism Data","abstract":"COVID-19 is a newly communicable disease with a catastrophe outbreak that affects all over the world. We retrieved about 8,781 nucleotide fragments and complete genomes of SARS-CoV-2 reported from sixty-four countries. The CoV-2 reference genome was obtained from the National Genomics Data Center (NGDC), GISAID, and NCBI Genbank. All the sequences were aligned against reference genomes using Clustal Omega and variants were called using in-house built Python script. We intend to establish a user-friendly online resource to visualize the variants in the viral genome along with the Primer Infopedia. After analyzing and filtering the data globally, it was made available to the public. The detail of data available to the public includes mutations from 5688 SARS-CoV-2 sequences curated from 91 regions. This database incorporated 39920 mutations over 3990 unique positions. According to the translational impact, these mutations include 11829 synonymous mutations including 681 synonymous frameshifts and 21701 nonsynonymous mutations including 10 nonsynonymous frameshifts. Development of SARS-CoV-2 mutation genome browsers is a fundamental step obliging towards the virus surveillance, viral detection, and development of vaccine and therapeutic drugs. The SARS-COV-2 mutation browser is available at http://covid-19.dnageography.com.","version":"1.1","doi":"10.1101/2020.06.10.145292","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.06.112474","pub_date":"2020-6-11","title":"Quantitative PCR for cannabis flower containing SARs-CoV-2","abstract":"In January of 2020, COVID-19 became a worldwide pandemic. As many industries shutdown to comply with social distancing measures, the cannabis industry was deemed an essential business in most U.S. jurisdictions. Cannabis is manually farmed, trimmed and packaged. Employees and trimmers in cannabis grows have been reported to test qPCR positive for SARs-CoV-2 and as a result cannabis flower can be a potential inhaled SARs-CoV-2 fomite. Many of the comorbidities described in COVID-19 are also qualifying conditions for medical cannabis access. Bat guano has been identified as a rich source for novel coronavirus discovery and it is also a common fertilizer in the cannabis field. To better assess cannabis fomite risk we developed a SARs-CoV-2 quantitative PCR assay optimized to operate with a hemp flower background matrix. This assay was utilized to estimate the stability of gamma irradiated SARs-CoV-2 as a hemp flower fomite.","version":"1.1","doi":"10.1101/2020.06.06.112474","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.144816","pub_date":"2020-6-11","title":"Remdesivir but not famotidine inhibits SARS-CoV-2 replication in human pluripotent stem cell-derived intestinal organoids","abstract":"Gastrointestinal symptoms in COVID-19 are associated with prolonged symptoms and increased severity. We employed human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) to analyze SARS-CoV-2 pathogenesis and to validate efficacy of specific drugs in the gut. Certain, but not all cell types in PSC-HIOs express SARS-CoV-2 entry factors ACE2 and TMPRSS2, rendering them susceptible to SARS-CoV-2 infection. Remdesivir, a promising drug to treat COVID-19, effectively suppressed SARS-CoV-2 infection of PSC-HIOs. In contrast, the histamine-2-blocker famotidine showed no effect. Thus, PSC-HIOs provide an interesting platform to study SARS-CoV-2 infection and to identify or validate drugs.","version":"1.1","doi":"10.1101/2020.06.10.144816","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.145144","pub_date":"2020-6-11","title":"Hydroxychloroquine Proves Ineffective in Hamsters and Macaques Infected with SARS-CoV-2","abstract":"We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in two animal models. When used for prophylaxis or treatment neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Similarly, HCQ prophylaxis/treatment (6.5 mg/kg) did not significantly benefit clinical outcome nor reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. In conclusion, our preclinical animal studies do not support the use of HCQ in prophylaxis/treatment of COVID-19. Hydroxychloroquine prophylaxis/treatment showed no beneficial effect in SARS-CoV-2 hamster and macaque disease models.","version":"1.1","doi":"10.1101/2020.06.10.145144","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.11.145920","pub_date":"2020-6-11","title":"SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness","abstract":"A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.","version":"1.1","doi":"10.1101/2020.06.11.145920","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.141150","pub_date":"2020-6-11","title":"Modeling the structure of the frameshift stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers","abstract":"The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses \u22121 programmed ribosomal frameshifting (\u22121 PRF) to control the relative expression of viral proteins. As modulating \u22121 PRF can inhibit viral replication, the RNA pseudoknot stimulating \u22121 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by \u03bcs-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through the junction formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting \u22121 PRF in SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.08.141150","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.144964","pub_date":"2020-6-11","title":"Hijacking SARS-Cov-2/ACE2 receptor interaction by natural and semi-synthetic steroidal agents acting on functional pockets on receptor binding region","abstract":"The coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the severe acute respiratory syndrome coronavirus (SARS)-CoV-2. In the light of the urgent need to identify novel approaches to be used in the emergency phase, a largely explored strategy has been the repurpose of clinically available drugs as new antivirals, by targeting different viral proteins. In this paper, we describe a drug repurposing strategy based on a virtual screening of druggable pockets located in the central \u03b2-sheet core of the SARS-CoV-2 Spike protein RBD supported by in vitro tests identifying several steroidal derivatives as SARS-CoV-2 entry inhibitors. Our results demonstrate that several potential binding sites exist in the SARS CoV-2 S protein, and that the occupancy of these pockets reduces the ability of the S protein RBD to bind to the ACE2 consensus in vitro. In particular, natural occurring and clinically available steroids as glycyrrhetinic and oleanolic acids, as well as the bile acids derivatives glyco-UDCA and obeticholic acid have been shown to be effective in preventing virus entry in the case of low viral load. All together, these results might help to define novel approaches to reduce the viral load by using SARS-CoV-2 entry inhibitors.","version":"1.1","doi":"10.1101/2020.06.10.144964","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.20126862","pub_date":"2020-06-11","title":"Bayesian investigation of SARS-CoV-2-related mortality in France","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>The SARS-CoV-2 epidemic in France has focused a lot of attention as it has had one of the largest death tolls in Europe. It provides an opportunity to examine the effect of the lockdown and of other events on the dynamics of the epidemic. In particular, it has been suggested that municipal elections held just before lockdown was ordered may have helped spread the virus. In this manuscript we use Bayesian models of the number of deaths through time to study the epidemic in 13 regions of France. We found that the models accurately predict the number of deaths 2 to 3 weeks in advance, and recover estimates that are in agreement with recent models that rely on a different structure and different input data. In particular, the lockdown reduced the viral reproduction number by \u2248 80%. However, using a mixture model, we found that the lockdown had had different effectiveness depending on the region, and that it had been slightly more effective in decreasing the reproduction number in denser regions. The mixture model predicts that 2.08 (95% CI: 1.85-2.47) million people had been infected by May 11, and that there were 2567 (95% CI: 1781-5182) new infections on May 10. We found no evidence that the reproduction numbers differ between week-ends and week days, and no evidence that the reproduction numbers increased on the election day. Finally, we evaluated counterfactual scenarios showing that ordering the lockdown 1 to 7 days sooner would have resulted in 19% to 76% fewer deaths, but that ordering it 1 to 7 days later would have resulted in 21% to 266% more deaths. Overall, the predictions of the model indicate that holding the elections on March 15 did not have a detectable impact on the total number of deaths, unless it motivated a delay in imposing the lockdown.</jats:p>","version":null,"doi":"10.1101/2020.06.09.20126862","journal":"medRxiv","score":null},{"id":"10.1101/2020.06.09.143412","pub_date":"2020-6-11","title":"Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests","abstract":"DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centers for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.","version":"1.1","doi":"10.1101/2020.06.09.143412","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.143289","pub_date":"2020-6-11","title":"Navigating Chemical Space By Interfacing Generative Artificial Intelligence and Molecular Docking","abstract":"Here we report the testing and application of a simple, structure-aware framework to design target-specific screening libraries for drug development. Our approach combines advances in generative artificial intelligence (AI) with conventional molecular docking to rapidly explore chemical space conditioned on the unique physiochemical properties of the active site of a biomolecular target. As a proof-of-concept, we used our framework to construct a focused library for cyclin-dependent kinase type-2 (CDK2). We then used it to rapidly generate a library specific to the active site of the main protease (Mpro) of the SARS-CoV-2 virus, which causes COVID-19. By comparing approved and experimental drugs to compounds in our library, we also identified six drugs, namely, Naratriptan, Etryptamine, Panobinostat, Procainamide, Sertraline, and Lidamidine, as possible SARS-CoV-2 Mpro targeting compounds and, as such, potential drug repurposing candidates. To complement the open-science COVID-19 drug discovery initiatives, we make our SARS-CoV-2 Mpro library fully accessible to the research community (https://github.com/atfrank/SARS-CoV-2).","version":"1.1","doi":"10.1101/2020.06.09.143289","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.136887","pub_date":"2020-6-11","title":"Protein covariance networks reveal interactions important to the emergence of SARS coronaviruses as human pathogens","abstract":"SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21st century and that have likely emerged from animal reservoirs based on genomic similarities to bat and other animal viruses. Here we report the analysis of conserved interactions between amino acid residues in proteins encoded by SARS-CoV-related viruses. We identified pairs and networks of residue variants that exhibited statistically high frequencies of covariance with each other. While these interactions are likely key to both protein structure and other protein-protein interactions, we have also found that they can be used to provide a new computational approach (CoVariance-based Phylogeny Analysis) for understanding viral evolution and adaptation. Our data provide evidence that the evolutionary processes that converted a bat virus into human pathogen occurred through recombination with other viruses in combination with new adaptive mutations important for entry into human cells.","version":"1.2","doi":"10.1101/2020.06.05.136887","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.129098","pub_date":"2020-6-10","title":"Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus has resulted in an unprecedented public health crisis. There are no approved vaccines or therapeutics for treating COVID-19. Here we reported a humanized monoclonal antibody, H014, efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nM level by engaging the S receptor binding domain (RBD). Importantly, H014 administration reduced SARS-CoV-2 titers in the infected lungs and prevented pulmonary pathology in hACE2 mouse model. Cryo-EM characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a novel conformational epitope, which is only accessible when the RBD is in open conformation. Biochemical, cellular, virological and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncover broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19. A potent neutralizing antibody conferred protection against SARS-CoV-2 in an hACE2 humanized mouse model by sterically blocking the interaction of the virus with its receptor.","version":"1.2","doi":"10.1101/2020.06.02.129098","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.137349","pub_date":"2020-6-10","title":"Synthetic antibodies neutralize SARS-CoV-2 infection of mammalian cells","abstract":"Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.06.05.137349","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.144188","pub_date":"2020-6-10","title":"Age-dependent progression of SARS-CoV-2 infection in Syrian hamsters","abstract":"In late 2019, an outbreak of a severe respiratory disease caused by an emerging coronavirus, SARS-CoV-2, resulted in high morbidity and mortality in infected humans. Complete understanding of COVID-19, the multi-faceted disease caused by SARS-CoV-2, requires suitable small animal models, as does the development and evaluation of vaccines and antivirals. Because age-dependent differences of COVID-19 were identified in humans, we compared the course of SARS-CoV-2 infection in young and aged Syrian hamsters. We show that virus replication in the upper and lower respiratory tract was independent of the age of the animals. However, older hamsters exhibited more pronounced and consistent weight loss. In situ hybridization in the lungs identified viral RNA in bronchial epithelium, alveolar epithelial cells type I and II, and macrophages. Histopathology revealed clear age-dependent differences, with young hamsters launching earlier and stronger immune cell influx than aged hamsters. The latter developed conspicuous alveolar and perivascular edema, indicating vascular leakage. In contrast, we observed rapid lung recovery at day 14 after infection only in young hamsters. We propose that comparative assessment in young versus aged hamsters of SARS-CoV-2 vaccines and treatments may yield valuable information as this small-animal model appears to mirror age-dependent differences in human patients.","version":"1.1","doi":"10.1101/2020.06.10.144188","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.144642","pub_date":"2020-6-10","title":"Ultrastructural analysis of SARS-CoV-2 interactions with the host cell via high resolution scanning electron microscopy","abstract":"SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 hours after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2. We used high resolution scanning electron microscopy to investigate Vero cells infected with SARS-CoV-2 at 2 and 48 hours post-infection. The central conclusions of this work include:\n\nInfected cells display polarization of their cytosol forming a restricted viroplasm-like zone dedicated to virus production and morphogenesis.\nThis is the first demonstration of SARS-CoV-2 attachment by scanning electron microscopy.\nThis is the first scanning electron microscopy images of the interior of SARS-CoV-2 infected cells and exploration of their vacuole contents.\nPerspective-viewing of bordering vesicles in close association with vacuoles.\nObservation of membrane ruffles and structures suggestive of exocytosis on the surface of infected cells.\nThe first demonstration of viral surfing in cell-to-cell communication on SARS-CoV-2 infection.\n\n Infected cells display polarization of their cytosol forming a restricted viroplasm-like zone dedicated to virus production and morphogenesis. This is the first demonstration of SARS-CoV-2 attachment by scanning electron microscopy. This is the first scanning electron microscopy images of the interior of SARS-CoV-2 infected cells and exploration of their vacuole contents. Perspective-viewing of bordering vesicles in close association with vacuoles. Observation of membrane ruffles and structures suggestive of exocytosis on the surface of infected cells. The first demonstration of viral surfing in cell-to-cell communication on SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.06.10.144642","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.095794","pub_date":"2020-6-10","title":"Phylogenetic Analysis of SARS-CoV-2 Genomes in Turkey","abstract":"COVID-19 has effectively spread worldwide. As of May 2020, Turkey is among the top ten countries with the most cases. A comprehensive genomic characterization of the virus isolates in Turkey is yet to be carried out. Here, we built a phylogenetic tree with globally obtained 15,277 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. We identified the subtypes based on the phylogenetic clustering in comparison with the previously annotated classifications. We performed a phylogenetic analysis of the first thirty SARS-CoV-2 genomes isolated and sequenced in Turkey. We suggest that the first introduction of the virus to the country is earlier than the first reported case of infection. Virus genomes isolated from Turkey are dispersed among most types in the phylogenetic tree. We find two of the seventeen sub-clusters enriched with the isolates of Turkey, which likely have spread expansively in the country. Finally, we traced virus genomes based on their phylogenetic placements. This analysis suggested multiple independent international introductions of the virus and revealed a hub for the inland transmission. We released a web application to track the global and interprovincial virus spread of the isolates from Turkey in comparison to thousands of genomes worldwide.","version":"1.2","doi":"10.1101/2020.05.15.095794","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142877","pub_date":"2020-6-10","title":"The Distal Polybasic Cleavage Sites of SARS-CoV-2 Spike Protein Enhance Spike Protein-ACE2 Binding","abstract":"The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD-ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein-ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD-ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD-ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD-ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection. The SARS-CoV-2 spike protein-ACE2 complex showing the polybasic cleavage sites","version":"1.1","doi":"10.1101/2020.06.09.142877","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142315","pub_date":"2020-6-10","title":"Designed peptides as potential fusion inhibitors against SARA-CoV-2 coronavirus infection","abstract":"Inspired by fusion-inhibitory peptides from heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains from human immuno-deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 and severe acute respiratory syndrome-coronavirus (SARS-CoV) based on viral fusogenic mechanism in the present work, we provided a similar approach to design the synthesized peptides against the entry into host cells of SARA-CoV-2 virus that causes 2019 novel coronavirus disease (COVID-19). These peptides derived from HR1 and HR2 of SARA-CoV-2 spike protein were further tested for their interaction and potential fusion possibility through circular dichroism spectrum. Here we used the peptide COVID-2019-HR1P1 (40 amino acids) as the target, which was derived from HR1 of SARA-CoV-2 spike protein, while the designed peptides including COVID-2019-HR2P1 (37 amino acids), COVID-2019-HR2P2 (32 amino acids) and others derived from HR2 of SARA-CoV-2 were tested for their binding to COVID-2019-HR1P1. Interestingly, results showed that both COVID-2019-HR2P1 and COVID-2019-HR2P2 can form the complex with COVID-2019-HR1P1, respectively. This implied that these designed peptides could play an important role in blocking SARA-CoV-2 entry into mammalian host cells via viral fusogenic mechanism, and thus could be used for preventing SARA-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.06.09.142315","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.144196","pub_date":"2020-6-10","title":"Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction","abstract":"SARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies support the elimination of the extraction step. Herein, we assessed the RNA extraction necessity, by comparing RT-qPCR efficacy in several direct approaches vs. the gold standard RNA extraction, in detection of SARS-CoV-2 from laboratory samples as well as clinical Oro-nasopharyngeal SARS-CoV-2 swabs. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified up to 70% of positive clinical specimens.","version":"1.1","doi":"10.1101/2020.06.10.144196","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142794","pub_date":"2020-6-10","title":"The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor","abstract":"The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over six million cases and nearly four hundred thousand deaths reported world-wide by the end of May 2020. A rush to find the cures prompted re-evaluation of a range of existing therapeutics vis-\u00e0-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the non-covalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease of its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.","version":"1.1","doi":"10.1101/2020.06.09.142794","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.134460","pub_date":"2020-6-10","title":"A Rare Deletion in SARS-CoV-2 ORF6 Dramatically Alters the Predicted Three-Dimensional Structure of the Resultant Protein","abstract":"The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong. The genome sequence shows a 27 nt in-frame deletion (\u039427,264-27,290) within ORF6, predicted to result in a 9 aa deletion (\u0394FKVSIWNLD) from the central portion of p6. This deletion is predicted to result in a dramatic alteration in the three-dimensional structure of the resultant protein (p6\u039422-30), possibly with significant functional implications. Analysis of the original clinical sample indicates that the deletion was not present, while sequencing of subsequent passages of the strain identifies the deletion as a majority variant. This suggests that the deletion originated ab initio during passaging and subsequently propagated into the majority, possibly due to the removal of selective pressure through the IFN-deficient Vero E6 cell line. The specific function of the SARS-CoV-2 p6 N-terminus, if any, has not yet been determined. However, this deletion is predicted to cause a shift from N-endo to N-ecto in the transmembrane localization of the SARS-CoV-2 p6\u039422-30 N-terminus, possibly leading to the ablation of its native function.","version":"1.1","doi":"10.1101/2020.06.09.134460","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015685","pub_date":"2020-6-10","title":"Recombination and purifying selection preserves covariant movements of mosaic SARS-CoV-2 protein S","abstract":"In depth evolutionary and structural analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from bats, pangolins, and humans are necessary to assess the role of natural selection and recombination in the emergence of the current pandemic strain. The SARS-CoV-2 S glycoprotein unique features have been associated with efficient viral spread in the human population. Phylogeny-based and genetic algorithm methods clearly show that recombination events between viral progenitors infecting animal hosts led to a mosaic structure in the S gene. We identified recombination coldspots in the S glycoprotein and strong purifying selection. Moreover, although there is little evidence of diversifying positive selection during host-switching, structural analysis suggests that some of the residues emerged along the ancestral lineage of current pandemic strains may contribute to enhanced ability to infect human cells. Interestingly, recombination did not affect the long-range covariant movements of SARS-CoV-2 S glycoprotein monomer in pre-fusion conformation but, on the contrary, could contribute to the observed overall viral efficiency. Our dynamic simulations revealed that the movements between the host cell receptor binding domain (RBD) and the novel furin-like cleavage site are correlated. We identified threonine 333 (under purifying selection), at the beginning of the RBD, as the hinge of the opening/closing mechanism of the SARS-CoV-2 S glycoprotein monomer functional to hACE2 binding. Our findings support a scenario where ancestral recombination and fixation of amino acid residues in the RBD of the S glycoprotein generated a virus with unique features, capable of extremely efficient infection of the human host.","version":"1.2","doi":"10.1101/2020.03.30.015685","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.10.144212","pub_date":"2020-6-10","title":"Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic","abstract":"The COVID-19 pandemic spread very fast around the world. A few days after the first detected case in South Africa, an infection started a large hospital outbreak in Durban, KwaZulu-Natal. Phylogenetic analysis of SARS-CoV-2 genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. In this manuscript, we outline the obstacles we encountered in order to genotype SARS-CoV-2 in real-time during an urgent outbreak investigation. In this process, we encountered problems with the length of the original genotyping protocol, reagent stockout and sample degradation and storage. However, we managed to set up three different library preparation methods for sequencing in Illumina. We also managed to decrease the hands on library preparation time from twelve to three hours, which allowed us to complete the outbreak investigation in just a few weeks. We also fine-tuned a simple bioinformatics workflow for the assembly of high-quality genomes in real-time. In order to allow other laboratories to learn from our experience, we released all of the library preparation and bioinformatics protocols publicly and distributed them to other laboratories of the South African Network for Genomics Surveillance (SANGS) consortium.","version":"1.1","doi":"10.1101/2020.06.10.144212","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091090","pub_date":"2020-6-10","title":"Static All-Atom Energetic Mappings of the SARS-Cov-2 Spike Protein with Potential Latch Identification of the Down State Protomer","abstract":"The SARS-Cov-2 virion responsible for the current world-wide pandemic Covid-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD). The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-Cov-2 Spike protein in its static prefusion state based on recent structural information. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called \u201cglue\u201d points) between residues in the Up and Down state protomers. In general, the S2 or fusion machinery domain of S is relatively rigid with strong noncovalent interactions facilitated by helical secondary structures, whereas the S1 domain, which contains the RBD and N-terminal domain (NTD), is relatively more flexible and characterized by beta strand structural motifs. The S2 domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer\u2019s intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. Surprisingly, the Up-Down, RBD-NTD interactions were overall stronger and more numerous than the Down-Down cross chain interactions, including the appearance of the three residue sequence ALA520-PRO521-ALA522 associated with a turn structure in the RBD of the Up state protomer. Additionally, our intrachain dominant energy mappings within each protomer, identified a significant \u201cglue\u201d point or possible \u201clatch\u201d for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-Cov-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. To help verify the potential latch for the Down state protomer, we conducted some preliminary molecular dynamic simulations that effectively turn off this specific latch glue point via a single point mutation of GLN564. Interestingly, the single point mutation lead to the latch releasing in less than a few nanoseconds, but the latch remained fixed in the wild state protomer for up to 0.1 microseconds that were simulated. Many more detailed studies are needed to understand the dynamics of the Up and Down states of the Spike protein, including the stabilizing chain-chain interactions and the mechanisms of transition from Down to Up state protomers. Nonetheless, static dominant energy landscape mappings and preliminary molecular dynamic studies given here may represent a useful starting point for more detailed dynamic analyses and hopefully an improved understanding of the structure-function relationship of this highly complex protein associated with COVID-19.","version":"1.2","doi":"10.1101/2020.05.12.091090","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.141101","pub_date":"2020-6-10","title":"SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins","abstract":"Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.","version":"1.1","doi":"10.1101/2020.06.09.141101","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.142323","pub_date":"2020-6-10","title":"The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 and other viruses","abstract":"During the 2020 SARS-Cov-2 pandemic, there has been an acute shortage of viral transport medium. Many different products have been used to meet the demands of large-scale diagnostic and surveillance testing. The stability of SARS-Cov-2 RNA was assessed in several commercially produced transport media and an in-house solution. Coronavirus RNA was rapidly destroyed in the commercial transport media though the deleterious effects on intact virus were limited. Similar results were obtained for a Type A influenza virus. There was reduced detection of both virus and nucleic acid when a herpesvirus sample and purified DNA were tested. Collectively these data showed that the commercial viral transport media contained nucleases or similar substances and may seriously compromise diagnostic and epidemiological investigations. Recommendations to include foetal bovine serum as a source of protein to enhance the stabilising properties of viral transport media are contraindicated. Almost all commercial batches of foetal bovine serum contain pestiviruses and at times other bovine viruses. In addition to the potential for there to be nucleases in the transport medium, the presence of these viruses and other extraneous nucleic acid in samples may compromise the interpretation of sequence data. The inclusion of foetal bovine serum presents a biosecurity risk for the movement of animal pathogens and renders these transport media unsuitable for animal disease diagnostic applications. While these transport media may be suitable for virus culture purposes, there could be misleading results if used for nucleic acid-based tests. Therefore, these products should be evaluated to ensure fitness for purpose.","version":"1.1","doi":"10.1101/2020.06.09.142323","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.099788","pub_date":"2020-6-10","title":"Nrf2 Activator PB125\u00ae as a Potential Therapeutic Agent Against COVID-19","abstract":"Nrf2 is a transcription factor that regulates cellular redox balance and the expression of a wide array of genes involved in immunity and inflammation, including antiviral actions. Nrf2 activity declines with age, making the elderly more susceptible to oxidative stress-mediated diseases, which include type 2 diabetes, chronic inflammation, and viral infections. Published evidence suggests that Nrf2 activity may regulate important mechanisms affecting viral susceptibility and replication. We examined gene expression levels by GeneChip microarray and by RNA-seq assays. We found that the potent Nrf2 activating composition PB125\u00ae downregulates ACE2 and TMPRSS2 mRNA expression in human liver-derived HepG2 cells. ACE2 is a surface receptor and TMPRSS2 activates the spike protein for SARS-Cov-2 entry into host cells. Furthermore, in endotoxin-stimulated primary human pulmonary artery endothelial cells we report the marked downregulation by PB125 of 36 genes encoding cytokines. These include IL1-beta, IL6, TNF-\u03b1, the cell adhesion molecules ICAM1, VCAM1, and E-selectin, and a group of IFN-\u03b3-induced genes. Many of these cytokines have been specifically identified in the \u201ccytokine storm\u201d observed in fatal cases of COVID-19, suggesting that Nrf2 activation may significantly decrease the intensity of the storm.","version":"1.2","doi":"10.1101/2020.05.16.099788","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.017657","pub_date":"2020-6-09","title":"Molecular Docking Analysis Of Some Phytochemicals On Two SARS-CoV-2 Targets: Potential Lead Compounds Against Two Target Sites of SARS-CoV-2 Obtained from Plants","abstract":"COV spike (S) glycoprotein and Mpro are two key targets that have been identified for vaccines and drug development against the COVID-19 disease. Virtual screening of some compounds of plants origin that have shown antiviral activities were carried out on the two targets, 6lu7 and 6vsb by docking with the PyRx software. The binding affinities were compared with other compounds and drugs already identified as potential ligands for 6lu7 and 6vsb as well as Chloroquine and hydroxychloroquine. The docked compounds with best binding affinities were also filtered for drug likeness using the SwissADME and PROTOX platforms on the basis of Physicochemical properties and toxicity respectively. The docking results revealed that scopodulcic acid and dammarenolic acid had the best binding affinity on the s-glycoprotein and Mpro protein targets respectively. Silybinin also demonstrated a good binding affinity to both protein targets making it a potential candidate for further evaluation as repurposed candidate for SARS COV2 with likelihood of having a multitarget activity.","version":"1.2","doi":"10.1101/2020.03.31.017657","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.141127","pub_date":"2020-6-09","title":"Stability of SARS-CoV-2 Phylogenies","abstract":"The SARS-CoV-2 pandemic has led to unprecedented, nearly real-time genetic tracing due to the rapid community sequencing response. Researchers immediately leveraged these data to infer the evolutionary relationships among viral samples and to study key biological questions, including whether host viral genome editing and recombination are features of SARS-CoV-2 evolution. This global sequencing effort is inherently decentralized and must rely on data collected by many labs using a wide variety of molecular and bioinformatic techniques. There is thus a strong possibility that systematic errors associated with lab-specific practices affect some sequences in the repositories. We find that some recurrent mutations in reported SARS-CoV-2 genome sequences have been observed predominantly or exclusively by single labs, co-localize with commonly used primer binding sites and are more likely to affect the protein coding sequences than other similarly recurrent mutations. We show that their inclusion can affect phylogenetic inference on scales relevant to local lineage tracing, and make it appear as though there has been an excess of recurrent mutation and/or recombination among viral lineages. We suggest how samples can be screened and problematic mutations removed. We also develop tools for comparing and visualizing differences among phylogenies and we show that consistent clade- and tree-based comparisons can be made between phylogenies produced by different groups. These will facilitate evolutionary inferences and comparisons among phylogenies produced for a wide array of purposes. Building on the SARS-CoV-2 Genome Browser at UCSC, we present a toolkit to compare, analyze and combine SARS-CoV-2 phylogenies, find and remove potential sequencing errors and establish a widely shared, stable clade structure for a more accurate scientific inference and discourse. We wish to thank all groups that responded rapidly by producing these invaluable and essential sequence data. Their contributions have enabled an unprecedented, lightning-fast process of scientific discovery---truly an incredible benefit for humanity and for the scientific community. We emphasize that most lab groups with whom we associate specific suspicious alleles are also those who have produced the most sequence data at a time when it was urgently needed. We commend their efforts. We have already contacted each group and many have updated their sequences. Our goal with this work is not to highlight potential errors, but to understand the impacts of these and other kinds of highly recurrent mutations so as to identify commonalities among the suspicious examples that can improve sequence quality and analysis going forward.","version":"1.1","doi":"10.1101/2020.06.08.141127","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.140871","pub_date":"2020-6-09","title":"Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses","abstract":"The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1) and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.","version":"1.1","doi":"10.1101/2020.06.08.140871","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.141267","pub_date":"2020-6-09","title":"Structural basis of a public antibody response to SARS-CoV-2","abstract":"Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We determined crystal structures of two IGHV3-53 neutralizing antibodies +/- Fab CR3022 ranging from 2.33 to 3.11 \u00c5 resolution. The germline-encoded residues of IGHV3-53 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 epitope. Moreover, IGHV3-53 is used in combination with a very short CDR H3 and different light chains. Overall, IGHV3-53 represents a versatile public VH in neutralizing SARS-CoV-2 antibodies, where their specific germline features and minimal affinity maturation provide important insights for vaccine design and assessing outcomes.","version":"1.1","doi":"10.1101/2020.06.08.141267","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.141077","pub_date":"2020-6-09","title":"Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion","abstract":"Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG) that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an ISG screen against VSV-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of virus replication. Mechanistically, internalized 25HC accumulates in the late endosomes and blocks cholesterol export, thereby restricting SARS-CoV-2 spike protein catalyzed membrane fusion. Our results highlight a unique antiviral mechanism of 25HC and provide the molecular basis for its possible therapeutic development.","version":"1.1","doi":"10.1101/2020.06.08.141077","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.138990","pub_date":"2020-6-09","title":"Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma","abstract":"Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer \u2265160 was 80% or greater with anti-RBD or anti-ECD titers of \u22651:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers \u2265160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of \u22651:1350, and evidence of VN \u22651:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of \u22651:1350 may provide critical information about protection against COVID-19 disease.","version":"1.1","doi":"10.1101/2020.06.08.138990","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.09.141580","pub_date":"2020-6-09","title":"Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins","abstract":"Coronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. However, no commercial vaccines against the coronaviruses that can infect humans are available. The spike (S) homotrimers bind to their receptors through the receptor-binding domain (RBD), which is believed to be a major target to block viral entry. In this study, we selected Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs were observed to adopt two different conformational states (lying or standing). Then, structural and immunological analyses were used to explore differences in the immune response with RBDs among these coronaviruses. Our results showed that more RBD-specific antibodies were induced by the S trimer with the RBD in the \u201cstanding\u201d state (SARS-CoV and SARS-CoV-2) than the S trimer with the RBD in the \u201clying\u201d state (HCoV-229E), and the affinity between the RBD-specific antibodies and S trimer was also higher in the SARS-CoV and SARS-CoV-2. In addition, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was much lower and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus. Our findings will provide important implications for future development of coronavirus vaccines. Outbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in lying and standing state, respectively. Here, we tested the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that Beta-CoVs RBDs are in a standing state, and their S proteins can induce more neutralizing antibodies targeting RBD. However, HCoV-229E RBD is in a lying state, and its S protein induces a low level of neutralizing antibody targeting RBD. Our results indicate that Alpha-coronavirus is more conducive to escape host immune recognition, and also provide novel ideas for the development of vaccines targeting S protein.","version":"1.1","doi":"10.1101/2020.06.09.141580","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.085308","pub_date":"2020-6-09","title":"VIRTUS: a pipeline for comprehensive virus analysis from conventional RNA-seq data","abstract":"The possibility that RNA transcripts from clinical samples contain plenty of virus RNAs has not been pursued actively so far. We here developed a new tool for analyzing virus-transcribed mRNAs, not virus copy numbers, in the data of conventional and single-cell RNA-sequencing of human cells. Our pipeline, named VIRTUS (VIRal Transcript Usage Sensor), was able to detect 763 viruses including herpesviruses, retroviruses, and even SARS-CoV-2 (COVID-19), and quantify their transcripts in the sequence data. This tool thus enabled simultaneously detecting infected cells, the composition of multiple viruses within the cell, and the endogenous host gene expression profile of the cell. This bioinformatics method would be instrumental in addressing the possible effects of covertly infecting viruses on certain diseases and developing new treatments to target such viruses. VIRTUS is implemented using Common Workflow Language and Docker under a CC-NC license. VIRTUS is freely available at https://github.com/yyoshiaki/VIRTUS. Supplementary data are available at Bioinformatics online.","version":"1.2","doi":"10.1101/2020.05.08.085308","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.033522","pub_date":"2020-6-09","title":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein harbors a conserved BH3-like sequence","abstract":"The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions further. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.3","doi":"10.1101/2020.04.09.033522","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.07.138677","pub_date":"2020-6-08","title":"Structure Basis for Inhibition of SARS-CoV-2 by the Feline Drug GC376","abstract":"The pandemic of SARS-CoV-2 coronavirus disease-2019 (COVID-19) caused by SARS-COV-2 continues to ravage many countries in the world. Mpro is an indispensable protein for viral translation in SARS-CoV-2 and a potential target in high-specificity anti-SARS-CoV-2 drug screening. In this study, to explore potential drugs for treating COVID-19, we elucidated the structure of SARS-CoV-2 Mpro and explored the interaction between Mpro and GC376, an antiviral drug used to treat a range of coronaviruses in Feline via inhibiting Mpro. The availability and safety of GC376 were proved by biochemical and cell experiments in vitro. We determined the structure of an important protein, Mpro, in SARS-CoV-2, and revealed the interaction of GC376 with the viral substrate and inhibition of the catalytic site of SARS-CoV-2 Mpro.","version":"1.1","doi":"10.1101/2020.06.07.138677","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.07.981928","pub_date":"2020-6-08","title":"Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging","abstract":"In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.","version":"1.2","doi":"10.1101/2020.03.07.981928","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.140459","pub_date":"2020-6-08","title":"Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide","abstract":"The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10 - 25 January) 2020. We also applied structural bioinformatics to assess D614G potential impact on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.","version":"1.1","doi":"10.1101/2020.06.08.140459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.134551","pub_date":"2020-6-08","title":"Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients","abstract":"COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN\u03b3 based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.","version":"1.1","doi":"10.1101/2020.06.05.134551","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.100685","pub_date":"2020-6-08","title":"Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein","abstract":"The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 \u00c5 resolution crystal structure of this protein\u2019s N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an \u03b1-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein\u2019s N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.","version":"1.2","doi":"10.1101/2020.05.17.100685","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.134379","pub_date":"2020-6-08","title":"The Zinc Finger Antiviral Protein restricts SARS-CoV-2","abstract":"Recent evidence shows that the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly sensitive to interferons (IFNs). However, the underlying antiviral effectors remain to be defined. Here, we show that Zinc finger antiviral protein (ZAP) that specifically targets CpG dinucleotides in viral RNA sequences restricts SARS-CoV-2. We demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Strikingly, SARS-CoV-2 and its closest relatives from bats show the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, knock-down of ZAP significantly increased SARS-CoV-2 production in lung cells, particularly upon treatment with IFN-\u03b1 or IFN-\u03b3. Thus, our results identify ZAP as an effector of the IFN response against SARS-CoV-2, although this pandemic pathogen may be preadapted to the low CpG environment in humans. SARS-CoV-2 and its closest bat relatives show strong CpG suppression IFN-\u03b2, -\u03b3 and -\u03bb inhibit SARS-CoV-2 with high efficiency ZAP restricts SARS-CoV-2 and contributes to the antiviral effect of IFNs","version":"1.2","doi":"10.1101/2020.06.04.134379","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.25.115618","pub_date":"2020-6-08","title":"Human H-ferritin presenting RBM of spike glycoprotein as potential vaccine of SARS-CoV-2","abstract":"The outbreak of COVID-19 has so far inflicted millions of people all around the world and will have a long lasting effect on every aspect of everyone\u2019s life. Yet there is no effective approved treatment for the disease. In an effort of utilizing human ferritin as nanoplatform for drug delivery, we engineered a fusion protein by presenting receptor-binding motif (RBM) of SARS-CoV-2 virus spike glycoprotein on the N-terminus of ferritin subunits. The designed fusion protein with a cage-like structure, similar to that of corona virus, is a potential anti-SARS-CoV-2 vaccine. We hereby show the construction, preparation, and characterization of the fusion protein RBM-HFtn. Our initial affinity study confirmed its biological activity towards ACE2 receptor which suggests its mode of action against SARS-CoV-2 could be either through vaccine therapy or blocking the cellular entry of virus as antagonist of ACE2 receptor.","version":"1.2","doi":"10.1101/2020.05.25.115618","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.129585","pub_date":"2020-6-08","title":"Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients","abstract":"The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site, generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We propose that natural selection has favored a \u201cDon\u2019t burn down the house\u201d strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.","version":"1.2","doi":"10.1101/2020.06.03.129585","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.139055","pub_date":"2020-6-08","title":"Rapid whole genome sequence typing reveals multiple waves of SARS-CoV-2 spread","abstract":"As the pandemic SARS-CoV-2 virus has spread globally its genome has diversified to an extent that distinct clones can now be recognized, tracked, and traced. Identifying clonal groups allows for assessment of geographic spread, transmission events, and identification of new or emerging strains that may be more virulent or more transmissible. Here we present a rapid, whole genome, allele-based method (GNUVID) for assigning sequence types to sequenced isolates of SARS-CoV-2 sequences. This sequence typing scheme can be updated with new genomic information extremely rapidly, making our technique continually adaptable as databases grow. We show that our method is consistent with phylogeny and recovers waves of expansion and replacement of sequence types/clonal complexes in different geographical locations. GNUVID is available as a command line application (https://github.com/ahmedmagds/GNUVID).","version":"1.1","doi":"10.1101/2020.06.08.139055","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.140152","pub_date":"2020-6-08","title":"D936Y and Other Mutations in the Fusion Core of the SARS-Cov-2 Spike Protein Heptad Repeat 1 Undermine the Post-Fusion Assembly","abstract":"The iconic \u201cred crown\u201d of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is made of its spike (S) glycoprotein. The S protein is the Trojan horse of coronaviruses, mediating their entry into the host cells. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On April 21st, the GISAID resource had collected 10,823 SARS-CoV-2 genomic sequences. We extracted from them all the complete S protein sequences and identified point mutations thereof. Six mutations were located on a 14-residue segment (929-943) in the \u201cfusion core\u201d of the heptad repeat 1 (HR1). Our modeling in the pre- and post-fusion S protein conformations revealed, for three of them, the loss of interactions stabilizing the post-fusion assembly. On May 29th, the SARS-CoV-2 genomic sequences in GISAID were 34,805. An analysis of the occurrences of the HR1 mutations in this updated dataset revealed a significant increase for the S929I and S939F mutations and a dramatic increase for the D936Y mutation, which was particularly widespread in Sweden and Wales/England. We notice that this is also the mutation causing the loss of a strong inter-monomer interaction, the D936-R1185 salt bridge, thus clearly weakening the post-fusion assembly.","version":"1.1","doi":"10.1101/2020.06.08.140152","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.139477","pub_date":"2020-6-08","title":"An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance","abstract":"With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.","version":"1.1","doi":"10.1101/2020.06.08.139477","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.08.139907","pub_date":"2020-6-08","title":"Structural Motifs, Disorder, and the Efficacy of Viral Vaccines","abstract":"We demonstrate that it is possible to draw direct numerical correlations between virus particles and effective virus-like particle (VLP) derived vaccines through extraction of a Bragg-Williams order parameter from electron microscopy. The method has its roots in studies of disorder in metal alloys, and is adapted to describe the type and occurrence of structural motifs within the arrangement of viral coat proteins, captured by the value of the order parameter as a measure of disorder. A conventional approach to viral vaccine design consists of replicating select proteins to create a VLP designed to trigger an immune response while remaining non-infectious. Understanding variations between viruses and vaccine strains therefore tends to focus on differences between proteins, which can be characterized through genetic analysis. While such an approach provides vital information about the functioning and interactions of the proteins, it does not yet yield an early-stage pathway towards predicting the efficacy of a vaccine, and so large-scale clinical trials are required to obtain critical information. With the urgency associated with pandemics, including Coronavirus Disease-2019 (COVID-19) originating from the SARS-CoV-2 virus, there is a need for earlier indications of whether a vaccine has the necessary characteristics. Application of the methodology to Dengue and influenza virus particles indicates that temperature and pH during incubation could potentially be exploited to fine-tune the order parameter of VLP-based vaccines to match the corresponding virus. Additionally, utilization of an Ising model plot reveals a clear relationship between case fatality rate and order parameter for distinct virus families.","version":"1.1","doi":"10.1101/2020.06.08.139907","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.025635","pub_date":"2020-6-08","title":"LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding","abstract":"The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.","version":"1.2","doi":"10.1101/2020.04.06.025635","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.06.138149","pub_date":"2020-6-07","title":"Rapid inactivation of SARS-CoV-2 with Deep-UV LED irradiation","abstract":"The spread of novel coronavirus disease 2019 (COVID-19) infections worldwide has raised concerns about the prevention and control of SARS-CoV-2. Devices that rapidly inactivate viruses can reduce the chance of infection through aerosols and contact transmission. This in vitro study demonstrated that irradiation with a deep ultraviolet light-emitting diode (DUV-LED) of 280 \u00b15 nm wavelength rapidly inactivates SARS-CoV-2 obtained from a COVID-19 patient. Development of devices equipped with DUV-LED is expected to prevent virus invasion through the air and after touching contaminated objects.","version":"1.1","doi":"10.1101/2020.06.06.138149","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.07.138800","pub_date":"2020-6-07","title":"Genetic analysis of SARS-CoV-2 isolates collected from Bangladesh: insights into the origin, mutation spectrum, and possible pathomechanism","abstract":"As the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rages across the world, killing hundreds of thousands and infecting millions, researchers are racing against time to elucidate the viral genome. Some Bangladeshi institutes are also in this race, sequenced a few isolates of the virus collected from Bangladesh. Here, we present a genomic analysis of 14 isolates. The analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and the Middle East, respectively. Our analysis identified a total of 42 mutations, including three large deletions, half of which were synonymous. Most of the missense mutations in Bangladeshi isolates found to have weak effects on the pathogenesis. Some mutations may lead the virus to be less pathogenic than the other countries. Molecular docking analysis to evaluate the effect of the mutations on the interaction between the viral spike proteins and the human ACE2 receptor, though no significant interaction was observed. This study provides some preliminary insights into the origin of Bangladeshi SARS-CoV-2 isolates, mutation spectrum and its possible pathomechanism, which may give an essential clue for designing therapeutics and management of COVID-19 in Bangladesh.","version":"1.1","doi":"10.1101/2020.06.07.138800","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.06.137604","pub_date":"2020-6-07","title":"Identification of unique mutations in SARS-CoV-2 strains isolated from India suggests its attenuated pathotype","abstract":"Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first reported in Wuhan, China in November 2019 has developed into a pandemic since March 2020, causing substantial human casualties and economic losses. Studies on SARS-CoV-2 are being carried out at an unprecedented rate to tackle this threat. Genomics studies, in particular, are indispensable to elucidate the dynamic nature of the RNA genome of SARS-CoV-2. RNA viruses are marked by their unique ability to undergo high rates of mutation in their genome, much more frequently than their hosts, which diversifies their strengths qualifying them to elude host immune response and amplify drug resistance. In this study, we sequenced and analyzed the genomic information of the SARS-CoV-2 isolates from two infected Indian patients and explored the possible implications of point mutations in its biology. In addition to multiple point mutations, we found a remarkable similarity between relatively common mutations of 36-nucleotide deletion in ORF8 of SARS-CoV-2. Our results corroborate with the earlier reported 29-nucleotide deletion in SARS, which was frequent during the early stage of human-to-human transmission. The results will be useful to understand the biology of SARS-CoV-2 and itsattenuation for vaccine development.","version":"1.1","doi":"10.1101/2020.06.06.137604","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.06.137513","pub_date":"2020-6-06","title":"Cross-neutralization antibodies against SARS-CoV-2 and RBD mutations from convalescent patient antibody libraries","abstract":"The emergence of coronavirus disease 2019 (COVID-19) pandemic led to an urgent need to develop therapeutic interventions. Among them, neutralizing antibodies play crucial roles for preventing viral infections and contribute to resolution of infection. Here, we describe the generation of antibody libraries from 17 different COVID-19 recovered patients and screening of neutralizing antibodies to SARS-CoV-2. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Then the positive clones were sequenced and reconstituted into whole human IgG for epitope binning assays. After that, all 19 IgG were classified into 6 different epitope groups or Bins. Although all these antibodies were shown to have ability to bind RBD, the antibodies in Bin2 have more superiority to inhibit the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 can also strongly bind with mutant RBDs (W463R, R408I, N354D, V367F and N354D/D364Y) derived from SARS-CoV-2 strain with increased infectivity, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, these neutralizing antibodies strongly restrict the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, these antibodies can even block the entry of live SARS-CoV-2 into cells at only 12.5 nM. These results suggest that these neutralizing human antibodies from the patient-derived antibody libraries have the potential to become therapeutic agents against SARS-CoV-2 and its mutants in this global pandemic.","version":"1.1","doi":"10.1101/2020.06.06.137513","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.134114","pub_date":"2020-6-05","title":"Neuropilin-1 is a host factor for SARS-CoV-2 infection","abstract":"SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2(. For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19.","version":"1.1","doi":"10.1101/2020.06.05.134114","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.135749","pub_date":"2020-6-05","title":"Whole genome identification of potential G-quadruplexes and analysis of the G-quadruplex binding domain for SARS-CoV-2","abstract":"The Coronavirus Disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) quickly become a global public health emergency. G-quadruplex, one of the non-canonical secondary structures, has shown potential antiviral values. However, little is known about G-quadruplexes on the emerging SARS-CoV-2. Herein, we characterized the potential G-quadruplexes both in the positive and negative-sense viral stands. The identified potential G-quadruplexes exhibits similar features to the G-quadruplexes detected in the human transcriptome. Within some bat and pangolin related beta coronaviruses, the G-quartets rather than the loops are under heightened selective constraints. We also found that the SUD-like sequence is retained in the SARS-CoV-2 genome, while some other coronaviruses that can infect humans are depleted. Further analysis revealed that the SARS-CoV-2 SUD-like sequence is almost conserved among 16,466 SARS-CoV-2 samples. And the SARS-CoV-2 SUDcore-like dimer displayed similar electrostatic potential pattern to the SUD dimer. Considering the potential value of G-quadruplexes to serve as targets in antiviral strategy, we hope our fundamental research could provide new insights for the SARS-CoV-2 drug discovery.","version":"1.1","doi":"10.1101/2020.06.05.135749","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.135806","pub_date":"2020-6-05","title":"A novel in-cell ELISA assay allows rapid and automated quantification of SARS-CoV-2 to analyse neutralizing antibodies and antiviral compounds","abstract":"The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. Constituting important correlates of protection, determination of virus-neutralizing antibodies (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. In contrast to standard serology ELISAs, plaque reduction neutralization tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell ELISA (icELISA) approach. After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source for quantitative icELISA. Using commercially available nucleocapsid protein-specific antibodies, viral infection could easily be quantified in human and highly permissive Vero E6 cells by icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose-dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the icNT was superior to PRNT in discriminating convalescent sera with high from those with intermediate neutralizing capacities. The SARS-CoV-2 icELISA test allows rapid (<48h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of NAbs as well as antiviral drugs, using reagents and equipment present in most routine diagnostics departments. We propose the icELISA and the icNT for COVID-19 research and diagnostics.","version":"1.1","doi":"10.1101/2020.06.05.135806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.135608","pub_date":"2020-6-05","title":"Design of a Novel Multiplex Real Time RT-PCR Assay for SARS-CoV-2 Detection","abstract":"Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 386,000 deaths globally as of June 4, 2020. In this study, we developed a novel multiplex real time reverse transcription (RT)-PCR test for detection of SARS-CoV-2, with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) of SARS-CoV-2 and a hydrolysis Taqman probe designed to specifically detect SARS-CoV-2. Following our design, we evaluated the Limit of detection (LOD) and clinical performance of this laboratory-developed test (LDT). A LOD study with inactivated whole virus exhibited equal performance to that seen in the modified CDC assay with a final LOD of 1,301 \u00b1 13 genome equivalents/ml for our assay vs 1,249 \u00b1 14 genome equivalents/ml for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal (NP) swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement with the modified CDC assay. The multiplex design of this assay allows the testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents during a time when both of these parameters have been critical. Our results demonstrate that our multiplex assay performs as well as the modified CDC assay, but is more efficient and cost effective and is therefore adequate for use as a diagnostic assay and for epidemiological surveillance and clinical management of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.06.04.135608","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.135293","pub_date":"2020-6-05","title":"Characterization of the substitution hotspots in SARS-CoV-2 genome using BioAider and detection of a SR-rich region in N protein providing further evidence of its animal origin","abstract":"The novel human coronavirus (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic worldwide. The increasing sequencing data have shown abundant single nucleotide variations in SARS-CoV-2 genome. However, it is difficult to quickly analyze genomic variation and screen key mutations of SARS-CoV-2. In this study, we developed a visual program, named BioAider, for quick and convenient sequence annotation and mutation analysis on multiple genome-sequencing data. Using BioAider, we conducted a comprehensive genome variation analysis on 3,240 sequences of SARS-CoV-2 genome. Herein, we detected 14 substitution hotspots within SARS-CoV-2 genome, including 10 non-synonymous and 4 synonymous ones. Among these hotspots, NSP13-Y541C was predicted to be a crucial substitution which might affect the unwinding activity of NSP13, a key protein for viral replication. Besides, we also found 3 groups of potentially linked substitution hotspots which were worth further study. In particular, we discovered a SR-rich region (aa 184-204) on the N protein of SARS-CoV-2 distinct from SARS-CoV, indicating more complex replication mechanism and unique N-M interaction of SARS-CoV-2. Interestingly, the quantity of SRXX repeat fragments in the SR-rich region well reflected the evolutionary relationship among SARS-CoV-2 and SARS-CoV-2 related animal coronaviruses, providing further evidence of its animal origin. Overall, we developed an efficient tool for rapid identification of mutations, identified substitution hotspots in SARS-CoV-2 genomes, and detected a distinctive polymorphism SR-rich region in N protein. This tool and the detected hotspots could facilitate the viral genomic study and may contribute for screening antiviral target sites.","version":"1.1","doi":"10.1101/2020.06.04.135293","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.135616","pub_date":"2020-6-05","title":"Comparative study of four SARS-CoV-2 Nucleic Acid Amplification Test (NAAT) platforms demonstrates that ID NOW performance is impaired substantially by patient and specimen type","abstract":"The advent of the COVID-19 pandemic in the United States created a unique situation where multiple molecular diagnostic assays with various indications for use in the detection of SARS-CoV-2 rapidly received Emergency Use Authorization by the FDA, were validated by laboratories and utilized clinically, all within a period of a few weeks. We compared the performance of four of these assays that were being evaluated for use at our institution: Abbott RealTime m2000 SARS-CoV-2 Assay, DiaSorin Simplexa COVID-19 Direct, Cepheid Xpert Xpress SARS-CoV-2 and Abbott ID NOW COVID-19. Nasopharyngeal and nasal specimens were collected from 88 ED and hospital-admitted patients and tested by the four methods in parallel to compare performance. ID NOW performance stood out as significantly worse than the other three assays despite demonstrating comparable analytic sensitivity. Further study determined that the use of a foam nasal swab compared to a nylon flocked nasopharyngeal swab, as well as use in a population chronically vs. acutely positive for SARS-CoV-2, were significant factors in the poor comparable performance.","version":"1.1","doi":"10.1101/2020.06.04.135616","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.135996","pub_date":"2020-6-05","title":"Several FDA-approved drugs effectively inhibit SARS-CoV-2 infection in vitro","abstract":"To identify drugs that are potentially used for the treatment of COVID-19, the potency of 1403 FDA-approved drugs were evaluated using a robust pseudovirus assay and the candidates were further confirmed by authentic SARS-CoV-2 assay. Four compounds, Clomiphene (citrate), Vortioxetine, Vortioxetine (hydrobromide) and Asenapine (hydrochloride), showed potent inhibitory effects in both pseudovirus and authentic virus assay. The combination of Clomiphene (citrate), Vortioxetine and Asenapine (hydrochloride) is much more potent than used alone, with IC50 of 0.34 \u03bcM.","version":"1.1","doi":"10.1101/2020.06.05.135996","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.131748","pub_date":"2020-6-05","title":"Ultra-sensitive nanozyme-based chemiluminescence paper test for rapid diagnosis of SARS-CoV-2 infection","abstract":"The recently emerged coronavirus disease COVID-19 has now evolved into a global pandemic. Early detection is crucial for its effective control. Nucleic acid testing for viral pathogen and serological testing for host antibodies are playing important roles in current COVID-19 diagnosis. However, while nucleic acid testing is complicated, facility-restricted and time-consuming, antibody testing may result in high rates of false-negative diagnoses, especially during the early stages of viral infection. Thus, a more rapid and reliable test for both early COVID-19 diagnosis and whole-population screening is urgently needed. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and high-sensitive testing of SARS-CoV-2 spike antigen. Our paper test uses a newly established peroxidase-mimic Co-Fe@hemin nanozyme instead of natural HRP that catalytically amplifies the chemiluminescent signal, allowing for target concentrations to be as low as 0.1 ng/ml. Furthermore, our nanozyme-based chemiluminescence test exhibits a linear range that is 32-fold wider compared to ELISA tests. Importantly, testing is completed in less than 16 min, compared to 1-2 h required for ELISA or nucleic acid tests. Critically, signal detection is feasible using a smartphone camera. Ingredients for our test are simple and readily available, rendering overall cost considerably lower than those used in current diagnoses. In conclusion, our novel test provides a high-sensitive, point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly increase current early screening capacities for suspected infections, and considerably lower demand for national healthcare resources.","version":"1.1","doi":"10.1101/2020.06.05.131748","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.136481","pub_date":"2020-6-05","title":"SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species","abstract":"There are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.","version":"1.1","doi":"10.1101/2020.06.05.136481","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.135699","pub_date":"2020-6-05","title":"SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts","abstract":"The genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type- I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19. Finally, we discovered that one particular species of bats, David\u2019s Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.","version":"1.1","doi":"10.1101/2020.06.05.135699","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.132134","pub_date":"2020-6-05","title":"Detection dogs as a help in the detection of COVID-19 Can the dog alert on COVID-19 positive persons by sniffing axillary sweat samples ? Proof-of-concept study","abstract":"The aim of this study is to evaluate if the sweat produced by COVID-19 persons (SARS-CoV-2 PCR positive) has a different odour for trained detection dogs than the sweat produced by non COVID-19 persons. The study was conducted on 3 sites, following the same protocol procedures, and involved a total of 18 dogs. A total of 198 armpits sweat samples were obtained from different hospitals. For each involved dog, the acquisition of the specific odour of COVID-19 sweat samples required from one to four hours, with an amount of positive samples sniffing ranging from four to ten. For this proof of concept, we kept 8 dogs of the initial group (explosive detection dogs and colon cancer detection dogs), who performed a total of 368 trials, and will include the other dogs in our future studies as their adaptation to samples scenting takes more time. The percentages of success of the dogs to find the positive sample in a line containing several other negative samples or mocks (2 to 6) were 100p100 for 4 dogs, and respectively 83p100, 84p100, 90p100 and 94p100 for the others, all significantly different from the percentage of success that would be obtained by chance alone. We conclude that there is a very high evidence that the armpits sweat odour of COVID-19+ persons is different, and that dogs can detect a person infected by the SARS-CoV-2 virus.","version":"1.1","doi":"10.1101/2020.06.03.132134","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.05.135194","pub_date":"2020-6-05","title":"Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia","abstract":"Regulatory T (Treg) cells orchestrate resolution and repair of acute lung inflammation and injury following viral pneumonia. Compared with younger patients, older individuals experience impaired recovery and worse clinical outcomes after severe viral infections, including influenza and the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Whether age is a key determinant of Treg cell pro-repair function following lung injury remains unknown. Here, we show that aging results in a cell-autonomous impairment of reparative Treg cell function following experimental influenza pneumonia. Transcriptional and DNA methylation profiling of sorted Treg cells provide insight into the mechanisms underlying their age-related dysfunction, with Treg cells from aged mice demonstrating both loss of reparative programs and gain of maladaptive programs. Novel strategies that restore youthful Treg cell functional programs could be leveraged as therapies to improve outcomes among older individuals with severe viral pneumonia.","version":"1.1","doi":"10.1101/2020.06.05.135194","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.135012","pub_date":"2020-6-04","title":"Olfactory transmucosal SARS-CoV-2 invasion as port of Central Nervous System entry in COVID-19 patients","abstract":"The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease presenting with fever, cough, and often pneumonia. Moreover, thromboembolic events throughout the body including the central nervous system (CNS) have been described. Given first indication for viral RNA presence in the brain and cerebrospinal fluid and in light of neurological symptoms in a large majority of COVID-19 patients, SARS-CoV-2-penetrance of the CNS is likely. By precisely investigating and anatomically mapping oro- and pharyngeal regions and brains of 32 patients dying from COVID-19, we not only describe CNS infarction due to cerebral thromboembolism, but also demonstrate SARS-CoV-2 neurotropism. SARS-CoV-2 enters the nervous system via trespassing the neuro-mucosal interface in the olfactory mucosa by exploiting the close vicinity of olfactory mucosal and nervous tissue including delicate olfactory and sensitive nerve endings. Subsequently, SARS-CoV-2 follows defined neuroanatomical structures, penetrating defined neuroanatomical areas, including the primary respiratory and cardiovascular control center in the medulla oblongata.","version":"1.1","doi":"10.1101/2020.06.04.135012","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091165","pub_date":"2020-6-04","title":"Potential antiviral options against SARS-CoV-2 infection","abstract":"As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6,5 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified most potent sera from recovered patients for treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that combinations of virus-directed nelfinavir along with host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.","version":"1.3","doi":"10.1101/2020.05.12.091165","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.23.111385","pub_date":"2020-6-04","title":"COVIDep platform for real-time reporting of vaccine target recommendations for SARS-CoV-2: Description and connections with COVID-19 immune responses and preclinical vaccine trials","abstract":"We introduce COVIDep (https://COVIDep.ust.hk), a web-based platform that provides immune target recommendations for guiding SARS-CoV-2 vaccine development. COVIDep implements a protocol that pools together publicly-available genetic data for SARS-CoV-2 and epitope data for SARS-CoV to identify B cell and T cell epitopes that present potential immune targets for SARS-CoV-2. Correspondences between outputs of COVIDep and immune responses recorded in COVID-19 patients and preclinical vaccine trials are also indicated. The platform is user-friendly, flexible, and based on up-to-date data. It may help guide vaccine designs and associated experimental studies for SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.05.23.111385","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.117440","pub_date":"2020-6-04","title":"Impact of Comorbidities on SARS-CoV-2 Viral Entry-Related Genes","abstract":"Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2). The distribution of expression of these genes across cell types representing multiple organ systems in healthy individuals has been recently demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remain unclear. Here we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes are modulated in target organs of select disease states. In tissues such as heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities vs healthy individuals. Additionally, we found increased expression of viral entry-related genes in the settings of hypertension, cancer or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and suggest new therapeutic targets to improve outcomes in vulnerable patient populations.","version":"1.2","doi":"10.1101/2020.05.26.117440","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.128751","pub_date":"2020-6-04","title":"Integrated genomic view of SARS-CoV-2 in India","abstract":"India first detected SARS-CoV-2, causal agent of COVID-19 in late January-2020, imported from Wuhan, China. March-2020 onwards; importation of cases from rest of the countries followed by seeding of local transmission triggered further outbreaks in India. We used ARTIC protocol based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT from Oxford Nanopore Technology to understand introduction and local transmission. The analyses revealed multiple introductions of SARS-CoV-2 from Europe and Asia following local transmission. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, here, proposed as A4-clade based on its divergence within A-cluster. The viral haplotypes may link their persistence to geo-climatic conditions and host response. Despite the effectiveness of non-therapeutic interventions in India, multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.","version":"1.1","doi":"10.1101/2020.06.04.128751","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.132357","pub_date":"2020-6-04","title":"Pitfalls in SARS-CoV-2 PCR diagnostics","abstract":"To combat the COVID-19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive values of the test. Here, we report the occurrence of contaminations of commercial primers/probe sets with the SARS-CoV-2 target sequence of the RT-qPCR as an example for pitfalls during PCR diagnostics affecting diagnostic specificity. In several purchased in-house primers/probe sets, quantification cycle values as low as 17 were measured for negative control samples. However, there were also primers/probe sets that displayed very low-level contaminations, which were detected only during thorough internal validation. Hence, it appears imperative to pre-test each batch of reagents extensively before use in routine diagnosis, to avoid false-positive results and low positive predictive value in low-prevalence situations. As such, contaminations may have happened more widely, COVID-19 diagnostic results should be re-assessed retrospectively to validate the epidemiological basis for control measures.","version":"1.1","doi":"10.1101/2020.06.03.132357","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.04.135004","pub_date":"2020-6-04","title":"Landscape and Selection of Vaccine Epitopes in SARS-CoV-2","abstract":"There is an urgent need for a vaccine with efficacy against SARS-CoV-2. We hypothesize that peptide vaccines containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation would drive both humoral and cellular immunity with high specificity, potentially avoiding undesired effects such as antibody-dependent enhancement (ADE). Additionally, such vaccines can be rapidly manufactured in a distributed manner. In this study, we combine computational prediction of T cell epitopes, recently published B cell epitope mapping studies, and epitope accessibility to select candidate peptide vaccines for SARS-CoV-2. We begin with an exploration of the space of possible T cell epitopes in SARS-CoV-2 with interrogation of predicted HLA-I and HLA-II ligands, overlap between predicted ligands, protein source, as well as concurrent human/murine coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, viral source protein abundance, sequence conservation, coverage of high frequency HLA alleles and co-localization of CD4+ and CD8+ T cell epitopes. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering to select regions with surface accessibility, high sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. From 58 initial candidates, three B cell epitope regions were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we propose a set of SARS-CoV-2 vaccine peptides for use in subsequent murine studies and clinical trials.","version":"1.1","doi":"10.1101/2020.06.04.135004","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.132639","pub_date":"2020-6-04","title":"Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors","abstract":"Development of an anti-SARS-CoV-2 therapeutic is hindered by the lack of physiologically relevant model systems that can recapitulate host-viral interactions in human cell types, specifically the epithelium of the lung. Here, we compare induced pluripotent stem cell (iPSC)-derived alveolar and airway epithelial cells to primary lung epithelial cell controls, focusing on expression levels of genes relevant for COVID-19 disease modeling. iPSC-derived alveolar epithelial type II-like cells (iAT2s) and iPSC-derived airway epithelial lineages express key transcripts associated with lung identity in the majority of cells produced in culture. They express ACE2 and TMPRSS2, transcripts encoding essential host factors required for SARS-CoV-2 infection, in a minor subset of each cell sub-lineage, similar to frequencies observed in primary cells. In order to prepare human culture systems that are amenable to modeling viral infection of both the proximal and distal lung epithelium, we adapt iPSC-derived alveolar and airway epithelial cells to two-dimensional air-liquid interface cultures. These engineered human lung cell systems represent sharable, physiologically relevant platforms for SARS-CoV-2 infection modeling and may therefore expedite the development of an effective pharmacologic intervention for COVID-19.","version":"1.1","doi":"10.1101/2020.06.03.132639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.131144","pub_date":"2020-6-04","title":"SARS-CoV2 Testing: The Limit of Detection Matters","abstract":"Resolving the COVID-19 pandemic requires diagnostic testing to determine which individuals are infected and which are not. The current gold standard is to perform RT-PCR on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of ~100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss more infected patients, resulting in more false negatives. However, the false-negative rate for a given LoD remains unknown. Here we address this question using over 27,500 test results for patients from across our healthcare network tested using the Abbott RealTime SARS-CoV-2 EUA. These results suggest that each 10-fold increase in LoD is expected to increase the false negative rate by 13%, missing an additional one in eight infected patients. The highest LoDs on the market will miss a majority of infected patients, with false negative rates as high as 70%. These results suggest that choice of assay has meaningful clinical and epidemiological consequences. The limit of detection matters.","version":"1.1","doi":"10.1101/2020.06.02.131144","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.20119925","pub_date":"2020-06-04","title":"Early phylodynamics analysis of the COVID-19 epidemic in France","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  France was one of the first countries to be reached by the COVID-19 pandemic. Here, we analyse 196 SARS-Cov-2 genomes collected between Jan 24 and Mar 24 2020, and perform a phylodynamics analysis. In particular, we analyse the doubling time, reproduction number (\n                  <jats:italic>\u211b</jats:italic>\n                  <jats:sub>t</jats:sub>\n                  ) and infection duration associated with the epidemic wave that was detected in incidence data starting from Feb 27. Different models suggest a slowing down of the epidemic in Mar, which would be consistent with the implementation of the national lock-down on Mar 17. The inferred distributions for the effective infection duration and\n                  <jats:italic>\n                    \u211b\n                    <jats:sub>t</jats:sub>\n                  </jats:italic>\n                  are in line with those estimated from contact tracing data. Finally, based on the available sequence data, we estimate that the French epidemic wave originated between mid-Jan and early Feb. Overall, this analysis shows the potential to use sequence genomic data to inform public health decisions in an epidemic crisis context and calls for further analyses with denser sampling.\n                </jats:p>","version":null,"doi":"10.1101/2020.06.03.20119925","journal":"medRxiv","score":null},{"id":"10.1101/2020.04.05.026336","pub_date":"2020-6-04","title":"A protocol for adding knowledge to Wikidata, a case report","abstract":"Pandemics, even more than other medical problems, require swift integration of knowledge. When caused by a new virus, understanding the underlying biology may help finding solutions. In a setting where there are a large number of loosely related projects and initiatives, we need common ground, also known as a \u201ccommons\u201d. Wikidata, a public knowledge graph aligned with Wikipedia, is such a commons and uses unique identifiers to link knowledge in other knowledge bases However, Wikidata may not always have the right schema for the urgent questions. In this paper, we address this problem by showing how a data schema required for the integration can be modelled with entity schemas represented by Shape Expressions. As a telling example, we describe the process of aligning resources on the genomes and proteomes of the SARS-CoV-2 virus and related viruses as well as how Shape Expressions can be defined for Wikidata to model the knowledge, helping others studying the SARS-CoV-2 pandemic. How this model can be used to make data between various resources interoperable, is demonstrated by integrating data from NCBI Taxonomy, NCBI Genes, UniProt, and WikiPathways. Based on that model, a set of automated applications or bots were written for regular updates of these sources in Wikidata and added to a platform for automatically running these updates. Although this workflow is developed and applied in the context of the COVID-19 pandemic, to demonstrate its broader applicability it was also applied to other human coronaviruses (MERS, SARS, Human Coronavirus NL63, Human coronavirus 229E, Human coronavirus HKU1, Human coronavirus OC4).","version":"1.2","doi":"10.1101/2020.04.05.026336","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.132894","pub_date":"2020-6-04","title":"Enhancing Colorimetric LAMP Amplification Speed and Sensitivity with Guanidine Chloride","abstract":"Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated technique and enabling accurate and high-throughput diagnostics.","version":"1.1","doi":"10.1101/2020.06.03.132894","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.130849","pub_date":"2020-6-03","title":"A \u201cDeep Dive\u201d into the SARS-Cov-2 Polymerase Assembly: Identifying Novel Allosteric Sites and Analyzing the Hydrogen Bond Networks and Correlated Dynamics","abstract":"Replication of the SARS-CoV-2 genome is a fundamental step in the virus life cycle and inhibiting the SARS-CoV2 replicase machinery has been proven recently as a promising approach in combating the virus. Despite this recent success, there are still several aspects related to the structure, function and dynamics of the CoV-2 polymerase that still need to be addressed. This includes understanding the dynamicity of the various polymerase subdomains, analyzing the hydrogen bond networks at the active site and at the template entry in the presence of water, studying the binding modes of the nucleotides at the active site, highlighting positions for acceptable nucleotides\u2019 substitutions that can be tolerated at different positions within the nascent RNA strand, identifying possible allosteric sites within the polymerase structure and studying their correlated dynamics relative to the catalytic site. Here, we combined various cutting-edge modelling tools with the recently resolved SARS-CoV-2 cryo-EM polymerase structures to fill this gap in knowledge. Our findings provide a detailed analysis of the hydrogen bond networks at various parts of the polymerase structure and suggest possible nucleotides\u2019 substitutions that can be tolerated by the polymerase complex. We also report here three \u201cdruggable\u201d allosteric sites within the nsp12 RdRp that can be targeted by small molecule inhibitors. Our correlated motion analysis shows that the dynamics within one of the newly identified sites are linked to the active site, indicating that targeting this site can significantly impact the catalytic activity of the SARS-CoV-2 polymerase.","version":"1.1","doi":"10.1101/2020.06.02.130849","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.131474","pub_date":"2020-6-03","title":"Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow","abstract":"The ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65\u00b0C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.","version":"1.1","doi":"10.1101/2020.06.03.131474","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.131102","pub_date":"2020-6-03","title":"Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies","abstract":"SARS-CoV-2 causes ongoing infections worldwide, and identifying people with immunity is becoming increasingly important. Available point-of-care diagnostic systems as lateral flow assays have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity or possibility for quantitative measurements. Here, a new point-of-care approach for SARS-CoV-2 specific antibody detection in human serum based on magnetic immuno-detection is described and compared to standard ELISA. For magnetic immuno-detection, immunofiltration columns were coated with a SARS-CoV-2 spike protein peptide. SARS-CoV-2 peptide reactive antibodies, spiked at different concentrations into PBS and human serum, were rinsed through immunofiltration columns. Specific antibodies were retained within the IFC and labelled with an isotype specific biotinylated antibody. Streptavidin-functionalized magnetic nanoparticles were applied to label the secondary antibodies. Enriched magnetic nanoparticles were then detected by means of frequency magnetic mixing detection technology, using a portable magnetic read-out device. Measuring signals corresponded to the amount of SARS-CoV-2 specific antibodies in the sample. Our preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader detection range and was four times faster than ELISA. Further optimizations could reduce assay times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-of-care measurements without expensive lab equipment.","version":"1.1","doi":"10.1101/2020.06.02.131102","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.130591","pub_date":"2020-6-03","title":"Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping","abstract":"The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq (\u201ccRASL-seq\u201d), which enables analysis of any targeted pathogen-(and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to \u223c1-100 pfu/ml or cfu/ml) and highly multiplexed (up to \u223c10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.","version":"1.1","doi":"10.1101/2020.06.03.130591","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.129817","pub_date":"2020-6-03","title":"Closing coronavirus spike glycoproteins by structure-guided design","abstract":"The recent spillover of SARS-CoV-2 in the human population resulted in the ongoing COVID-19 pandemic which has already caused 4.9 million infections and more than 326,000 fatalities. To initiate infection the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface, determining host and tissue tropism, and fusion of the viral and host membranes. Although SARS-CoV- 2 S is the main target of neutralizing antibodies and the focus of vaccine design, its stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a prefusion SARS-CoV-2 S ectodomain trimer construct covalently stabilized in the closed conformation. Structural and antigenicity analysis showed we successfully shut S in the closed state without otherwise altering its architecture. Finally, we show that this engineering strategy is applicable to other \u03b2-coronavirus S glycoproteins and might become an important tool for vaccine design, structural biology, serology and immunology studies.","version":"1.1","doi":"10.1101/2020.06.03.129817","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.126615","pub_date":"2020-6-03","title":"An exploration of the SARS-CoV-2 spike receptor binding domain (RBD) \u2013 a complex palette of evolutionary and structural features","abstract":"SARS-CoV-2 spike protein (S) is associated with the entry of virus inside the host cell by recruiting its loop dominant receptor binding domain (RBD) and interacting with the host ACE2 receptor. Our study deploying a two-tier approach encompassing evolutionary and structural analysis provides a comprehensive picture of the RBD, which could be of potential use for better understanding the RBD and address its druggability issues. Resorting to an ensemble of sequence space exploratory tools including co-evolutionary analysis and deep mutational scans we provide a quantitative insight into the evolutionarily constrained subspace of the RBD sequence space. Guided by structure network analysis and Monte Carlo simulation we highlight regions inside the RBD, which are critical for providing structural integrity and conformational flexibility of the binding cleft. We further deployed fuzzy C-means clustering by plugging the evolutionary and structural features of discrete structure blocks of RBD to understand which structure blocks share maximum overlap based on their evolutionary and structural features. Deploying this multi-tier interlinked approach, which essentially distilled the evolutionary and structural features of RBD, we highlight discrete region, which could be a potential druggable pocket thereby destabilizing the structure and addressing evolutionary routes.","version":"1.1","doi":"10.1101/2020.05.31.126615","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.03.132506","pub_date":"2020-6-03","title":"The inhibitory effect of a Corona virus spike protein fragment with ACE2","abstract":"In this paper, we investigate the molecular assembly processes of a Coronavirus Spike protein fragment, the hexapeptide YKYRYL on the ACE2 receptor and its inhibitory effect on the aggregation and activation of the CoV-2 spike receptor protein at the same receptor protein. In agreement with an experimental study, we find a high affinity of the hexapeptide to the binding interface between the spike receptor protein and ACE2, which we investigate using 20 independent equilibrium MD simulations over a total of 1 \u03bcs and a 200 ns enhanced MD simulation. We then evaluate the effect of the hexapeptide on the aggregation process of the spike receptor protein to ACE2 in long-time enhanced MD simulations. In that set of simulations, we find that the spike receptor protein does not bind to ACE2 with the binding motif shown in experiments, but it rotates due to an electrostatic repulsion and forms a hydrophobic interface with ACE2. Surprisingly, we observe that the hexapeptide binds to the spike receptor domain, which has the effect that this protein only weakly attaches to ACE2, so that the activation of the spike protein receptor might be inhibited in this case. Our results indicate that the hexapeptide might be a possible treatment option which prevents the viral activation through the inhibition of the interaction between ACE2 and the spike receptor protein. A novel coronavirus, CoV-19 and a later phenotype CoV-2 were identified as primary cause for a severe acute respiratory syndrome (SARS CoV-2). The spike (S) protein of CoV-2 is one target for the development of a vaccine to prevent the viral entry into human cells. The inhibition of the direct interaction between ACE2 and the S-protein could provides a suitable strategy to prevent the membrane fusion of CoV-2 and the viral entry into human cells. Using MD simulations, we investigate the assembly process of a Coronavirus Spike protein fragment, the hexapeptide YKYRYL on the ACE2 receptor and its inhibitzory effect on the aggregation and activation of the CoV-2 spike receptor protein at the same receptor protein.","version":"1.1","doi":"10.1101/2020.06.03.132506","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.111526","pub_date":"2020-6-02","title":"In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19","abstract":"Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).","version":"1.3","doi":"10.1101/2020.05.22.111526","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.071175","pub_date":"2020-6-02","title":"Computational Electrostatics Predict Variations in SARS-CoV-2 Spike and Human ACE2 Interactions","abstract":"SARS-CoV-2 is a novel virus that is presumed to have emerged from bats to crossover into humans in late 2019. As the global pandemic ensues, scientist are working to evaluate the virus and develop a vaccine to counteract the deadly disease that has impacted lives across the entire globe. We perform computational electrostatic simulations on multiple variants of SARS-CoV-2 spike protein s1 in complex with human angiotensin-converting enzyme 2 (ACE2) variants to examine differences in electrostatic interactions across the various complexes. Calculations are performed across the physiological pH range to also examine the impact of pH on these interactions. Two of six spike protein s1 variations having greater electric forces at pH levels consistent with nasal secretions and significant variations in force across all five variants of ACE2. Five out of six spike protein s1 variations have relatively consistent forces at pH levels of the lung, and one spike protein s1 variant that has low potential across a wide range of pH. These predictions indicate that variants of SARS-CoV-2 spike proteins and human ACE2 in certain combinations could potentially play a role in increased binding efficacy of SARS-CoV-2 in vivo.","version":"1.4","doi":"10.1101/2020.04.30.071175","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093088","pub_date":"2020-6-02","title":"Rapid selection of a human monoclonal antibody that potently neutralizes SARS-CoV-2 in two animal models","abstract":"Effective therapies are urgently needed for the SARS-CoV-2/COVID19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. One high affinity mAb, IgG1 ab1, specifically neutralized replication competent SARS-CoV-2 with exceptional potency as measured by two different assays. There was no enhancement of pseudovirus infection in cells expressing Fc\u03b3 receptors at any concentration. It competed with human angiotensin-converting enzyme 2 (hACE2) for binding to RBD suggesting a competitive mechanism of virus neutralization. IgG1 ab1 potently neutralized mouse ACE2 adapted SARS-CoV-2 in wild type BALB/c mice and native virus in hACE2 expressing transgenic mice. The ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 days) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.","version":"1.2","doi":"10.1101/2020.05.13.093088","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.129312","pub_date":"2020-6-02","title":"System Dynamics Modeling of Within-Host Viral Kinetics of Coronavirus (SARS CoV-2)","abstract":"Mathematical models are being used extensively in the study of SARS-CoV-2 transmission dynamics, becoming an essential tool for decision making concerning disease control. It is now required to understand the mechanisms involved in the interaction between the virus and the immune response effector cells, both innate and adaptive, in order to support lines of research related to the use of drugs, production of protective antibodies and of course, vaccines against SARS-CoV-2. The present study, using a system dynamic approach, hypothesizes over the conditions that characterize the fraction of the population which get infected by SARS-CoV-2 as the asymptomatic patients, the mild symptomatic, acute symptomatic, and also super-spreaders, in terms of innate immune response, the initial virus load, the virus burden with shedding events, and the cytokine levels.","version":"1.1","doi":"10.1101/2020.06.02.129312","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.02.130484","pub_date":"2020-6-02","title":"HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing","abstract":"The recent outbreak of a new coronavirus that causes a Severe Acute Respiratory Syndrome in humans (SARS-CoV-2) has developed into a global pandemic with over 6 million reported cases and more than 375,000 deaths worldwide. Many countries have faced a shortage of diagnostic kits as well as a lack of infrastructure to perform necessary testing. Due to these limiting factors, only patients showing symptoms indicating infection were subjected to testing, whilst asymptomatic individuals, who are widely believed to be responsible for the fast dispersion of the virus, were largely omitted from the testing regimes. The inability to implement high throughput diagnostic and contact tracing strategies has forced many countries to institute lockdowns with severe economic and social consequences. The World Health Organization (WHO) has encouraged affected countries to increase testing capabilities to identify new cases, allow for a well-controlled lifting of lockdown measures, and prepare for future outbreaks. Here, we propose HiDRA-seq, a rapidly implementable, high throughput, and scalable solution that uses NGS lab infrastructure and reagents for population-scale SARS-CoV-2 testing. This method is based on the use of indexed oligo-dT primers to generate barcoded cDNA from a large number of patient samples. From this, highly multiplexed NGS libraries are prepared targeting SARS-CoV-2 specific regions and sequenced. The low amount of sequencing data required for diagnosis allows the combination of thousands of samples in a sequencing run, while reducing the cost to approximately 2 CHF/EUR/USD per RNA sample. Here, we describe in detail the first version of the protocol, which can be further improved in the future to increase its sensitivity and to identify other respiratory viruses or analyze individual genetic features associated with disease progression.","version":"1.1","doi":"10.1101/2020.06.02.130484","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.25.115600","pub_date":"2020-6-01","title":"Generation of human bronchial organoids for SARS-CoV-2 research","abstract":"Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that can reproduce the viral life cycle and evaluate the drug efficacy of anti-viral drugs is essential. In this study, we established a method to generate human bronchial organoids (hBO) from commercially available cryopreserved human bronchial epithelial cells and examined whether they could be used as a model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research. Our hBO contain basal, club, ciliated, and goblet cells. Angiotensin-converting enzyme 2 (ACE2), which is a receptor for SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an essential serine protease for priming spike (S) protein of SARS-CoV-2, were highly expressed. After SARS-CoV-2 infection, not only the intracellular viral genome, but also progeny virus, cytotoxicity, pyknotic cells, and moderate increases of the type I interferon signal could be observed. Treatment with camostat, an inhibitor of TMPRSS2, reduced the viral copy number to 2% of the control group. Furthermore, the gene expression profile in SARS-CoV-2-infected hBO was obtained by performing RNA-seq analysis. In conclusion, we succeeded in generating hBO that can be used for SARS-CoV-2 research and COVID-19 drug discovery.","version":"1.2","doi":"10.1101/2020.05.25.115600","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.01.127605","pub_date":"2020-6-01","title":"SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes","abstract":"The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality in COVID-19 patients. It is unclear whether cardiac injury may have been caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here we investigate whether human cardiomyocytes are permissive for SARS-CoV-2 infection. Infection was induced by two strains of SARS-CoV-2 (FFM1 and FFM2) in human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CM) and in two models of human cardiac tissue. We show that SARS-CoV-2 infects hiPS-CM as demonstrated by detection of intracellular double strand viral RNA and viral spike glycoprotein protein expression. Increasing concentrations of virus RNA are detected in supernatants of infected cardiomyocytes, which induced infections in CaCo-2 cell lines documenting productive infections. SARS-COV-2 infection induced cytotoxic and pro-apoptotic effects and abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signaling, apoptosis and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a iPS-derived human 3D cardiosphere tissue models. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. The demonstration that cardiomyocytes are permissive for SARS-CoV-2 infection in vitro warrants the further in depth monitoring of cardiotoxic effects in COVID-19 patients. This study demonstrates that human cardiac myocytes are permissive for SARS-CoV-2 infection. The study documents that SARS-CoV-2 undergoes a full replicatory circle and induces a cytotoxic response in cardiomyocytes. Infection was confirmed in two cardiac tissue models, including living human heart slices. The study may provide a rational to explain part of the cardiotoxicity observed in COVID-19 patients The demonstration of direct cardiotoxicity induced by SARS-CoV-2 warrants an in depth further analysis of cardiac tissue of COVID-19 patients and a close monitoring for putative direct cardiomyocyte injury. The established models can be used to test novel therapeutic approaches targeting COVID-19.","version":"1.1","doi":"10.1101/2020.06.01.127605","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.126524","pub_date":"2020-6-01","title":"A Scalable Topical Vectored Vaccine Candidate Against SARS-CoV-2","abstract":"The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) caused an ongoing unprecedented global public health crises of coronavirus disease in 2019 (CoVID-19). The precipitously increased death rates, its impact on livelihood and trembling economies warrant the urgent development of SARS-CoV-2 vaccine which would be safe, efficacious and scalable. Owing to unavailability of the vaccine, we propose a de novo synthesised avian orthoavulavirus 1 (AOaV-1)-based topical respiratory vaccine candidate against CoVID-19. Avirulent strain of Newcastle disease virus, proto-type virus of AOaV-1, was engineered to express full length spike (S) glycoprotein which is highly neutralizing and major protective antigen of the SARS-CoV-2. Broad-scale in vitro characterization of recombinant vaccine candidate demonstrated efficient co-expression of the hemagglutinin-neuraminidase (HN) of AOaV-1 and S protein of SARS-CoV-2, and comparable replication kinetics were observed in cell culture model. The recombinant vaccine candidate virus actively replicated and spread within cells independently of exogenous trypsin. Interestingly, incorporation of S protein of SARS-CoV-2 into the recombinant AOaV-1 particles attributed the sensitivity to anti-SARS-CoV-2 antiserum and more prominently to anti-AOaV-1 antiserum. Finally, our results demonstrated that the recombinant vaccine vector stably expressed S protein after multiple propagation in chicken embryonated eggs, and this expression did not significantly impact the in vitro growth characteristics of the recombinant. Taken together, the presented respiratory vaccine candidate is highly attenuated in primates per se, safe and lacking pre-existing immunity in human, and carries the potential for accelerated vaccine development against CoVID-19 for clinical studies.","version":"1.1","doi":"10.1101/2020.05.31.126524","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.124107","pub_date":"2020-6-01","title":"Synergistic effects of anionic surfactants on coronavirus (SARS-CoV-2) virucidal efficiency of sanitizing fluids to fight COVID-19","abstract":"Our surrounding environment, especially often-touched contaminated surfaces, plays an important role in the transmission of pathogens in society. The shortage of effective sanitizing fluids, however, became a global challenge quickly after the coronavirus disease-19 (COVID-19) outbreak in December 2019. In this study, we present the effect of surfactants on coronavirus (SARS-CoV-2) virucidal efficiency in sanitizing fluids. Sodium dodecylbenzenesulfonate (SDBS), sodium laureth sulfate (SLS), and two commercial dish soap and liquid hand soap were studied with the goal of evaporation rate reduction in sanitizing liquids to maximize surface contact time. Twelve fluids with different recipes composed of ethanol, isopropanol, SDBS, SLS, glycerin, and water of standardized hardness (WSH) were tested for their evaporation time and virucidal efficiency. Evaporation time increased by 17-63% when surfactant agents were added to the liquid. In addition, surfactant incorporation enhanced the virucidal efficiency between 15-27% according to the 4-field test in the EN 16615:2015 European Standard method. Most importantly, however, we found that surfactant addition provides a synergistic effect with alcohols to inactivate the SARS-CoV-2 virus. This study provides a simple, yet effective solution to improve the virucidal efficiency of commonly used sanitizers.","version":"1.1","doi":"10.1101/2020.05.29.124107","journal":"bioRxiv","score":null},{"id":"10.1101/420737","pub_date":"2020-6-01","title":"Ozone Treatment for Elimination of Bacteria and SARS-CoV-2 for Medical Environments","abstract":"Pathogenic bacteria and viruses in medical environments can lead to treatment complications and hospital-acquired infections (HAIs), and current cleaning protocols do not address hard-to-access areas or that may be beyond line-of-sight treatment such as with ultraviolet radiation. At the time of writing, the ongoing pandemic of the novel coronavirus known as novel coronavirus (2019-nCoV) has claimed over 4 million cases worldwide and is expected to have multiple peaks, with possible resurgences throughout 2020. It is therefore imperative that disinfection methods in the meantime be employed to keep up with the supply of personal protective equipment (PPE) and sterilize a wide array of surfaces as quarantine lockdowns begin to be lifted. Here, we tested the efficacy of Sani Sport ozone devices as a means to treat hospital equipment and surfaces for killing bacteria, degrading synthetic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, and RNA from non-replicative capsid enclosed SARS-CoV-2. We observed a rapid killing of medically-relevant and environmental bacteria (Escherichia coli, Enterococcus faecalis, Bacillus subtlis, and Deinococcus radiodurans) across four surfaces (blankets, catheter, remotes, and syringes) within 30 minutes, and up to a 99% reduction in viable bacteria at the end of 2-hour treatment cycles. Significant RNA degradation of synthetic SARS-CoV-2 RNA was seen an hour into the ozone treatment as compared to non-treated controls and a non-replicative form of the virus was shown to have significant RNA degradation at 30 minutes compared to a no treatment control and RNA degradation could be reliably detected at 10,000 and 1,000 copies of virus per sample. These results show the strong promise of ozone treatment for reducing risk of infection and HAIs.","version":"1.3","doi":"10.1101/420737","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.01.127829","pub_date":"2020-6-01","title":"Targeted Intracellular Degradation of SARS-CoV-2 RBD via Computationally-Optimized Peptide Fusions","abstract":"The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has elicited a global health crisis of catastrophic proportions. With no approved cure or vaccine currently available, there is a critical need for effective antiviral strategies. In this study, we report a novel antiviral platform, through computational design of ACE2-derived peptides which both target the viral spike protein receptor binding domain (RBD) and recruit E3 ubiquitin ligases for subsequent intracellular degradation of SARS-CoV-2 in the proteasome. Our engineered peptide fusions demonstrate robust RBD degradation capabilities in human cells, thus prompting their further experimental characterization and therapeutic development.","version":"1.1","doi":"10.1101/2020.06.01.127829","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.10.985150","pub_date":"2020-6-01","title":"Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network\u2019s multiplex PCR","abstract":"Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample\u2019s viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network\u2019s original (V1) and modified (V3) primer set.","version":"1.4","doi":"10.1101/2020.03.10.985150","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.126813","pub_date":"2020-6-01","title":"Covid-19 pandemic and the unprecedented mobilisation of scholarly efforts prompted by a health crisis: Scientometric comparisons across SARS, MERS and 2019-nCov literature","abstract":"During the current century, each major coronavirus outbreak has triggered a quick and immediate surge of academic publications on this topic. The spike in research publications following the 2019 Novel Coronavirus (Covid-19) outbreak, however, has been like no other. The global crisis caused by the Covid-19 pandemic has mobilised scientific efforts in an unprecedented way. In less than five months, more than 12,000 research items have been indexed while the number increasing every day. With the crisis affecting all aspects of life, research on Covid-19 seems to have become a focal point of interest across many academic disciplines. Here, scientometric aspects of the Covid-19 literature are analysed and contrasted with those of the two previous major Coronavirus diseases, i.e. Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). The focus is on the co-occurrence of key-terms, bibliographic coupling and citation relations of journals and collaborations between countries. Certain recurring patterns across all three literatures were discovered. All three outbreaks have commonly generated three distinct and major cohort of studies: (i) studies linked to the public health response and epidemic control, (ii) studies associated with the chemical constitution of the virus and (iii) studies related to treatment, vaccine and clinical care. While studies affiliated with the category (i) seem to have been the first to emerge, they overall received least numbers of citations compared to those of the two other categories. Covid-19 studies seem to have been distributed across a broader variety of journals and subject areas. Clear links are observed between the geographical origins of each outbreak or the local geographical severity of each outbreak and the magnitude of research originated from regions. Covid-19 studies also display the involvement of authors from a broader variety of countries compared to SARS and MRS.","version":"1.1","doi":"10.1101/2020.05.31.126813","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.126342","pub_date":"2020-6-01","title":"Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs","abstract":"Rigorous testing is the way forward to fight the Covid-19 pandemic. Here we show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer and economical by bypassing the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore, can be used for mass screening. Our method takes about half the time and is cheaper by about 40% compared to current most widely used method. We also provide a variant of the new method that increases the efficiency of detection by about 20% compared to the currently used method. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection.","version":"1.1","doi":"10.1101/2020.05.31.126342","journal":"bioRxiv","score":null},{"id":"10.1101/2020.06.01.127381","pub_date":"2020-6-01","title":"The knowledge and practice towards COVID-19 pandemic prevention among residents of Ethiopia: An Online Cross-Sectional Study","abstract":"The Novel coronavirus disease-2019 (COVID-19) is now the international concerns and a pandemic, since the World Health Organization declared as the outbreaks. The objective of this study to assess the prevention knowledge and practices towards the COVID-19 among the residents of Ethiopia. An online cross-sectional study was conducted among the sample of Ethiopian residents via social platforms of the author\u2019s network with the popular media such as Facebook, in Ethiopia from the April 15-22, 2020 and successfully recruited 341 responses. The snowball sampling was employed to recruit the participants. The data were analyzed using STATA version 14. Descriptive statistics were used to summarize the level of knowledge and practices. The majority of the respondents (80.35%) were male. The overall prevention knowledge of the participants towards the novel coronavirus (COVID-19) was high. About 91.2% of the participant was heard about the novel coronavirus disease and Social Medias\u2019 were the main source of the information. About 90% of the participants had a good prevention knowledge of maintaining social distance and frequent hand washing. The practices of the participants towards the COVID-19 prevention were very low. Out of 341 participants, only 61% and 84% of the participants were practicing social distance and frequent hand washing, respectively. Majority of the participants had knew the ways of protecting themselves from the novel coronavirus. But, there was deficiencies of changing these prevention knowledge to practice. This indicates that there was gap between knowledge and implementation of prevention strategies in the community. The concerned body should focus on providing education for the community regarding the implementation of the prevention knowledge to practice.","version":"1.1","doi":"10.1101/2020.06.01.127381","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.30.125856","pub_date":"2020-5-31","title":"Sofosbuvir protects human brain organoids against SARS-CoV-2","abstract":"COVID-19 was rapidly declared a pandemic by the World Health Organization, only three months after the initial outbreak in Wuhan, China. Early clinical care mainly focused on respiratory illnesses. However, a variety of neurological manifestations in both adults and newborns are also emerging. To determine whether SARS-CoV-2 could target the human brain, we infected iPSC-derived human brain organoids. Our findings show that SARS-CoV-2 was able to infect and kill neural cells, including cortical neurons. This phenotype was accompanied by impaired synaptogenesis. Finally, Sofosbuvir, an FDA-approved antiviral drug, was able to rescue these alterations. Given that there are currently no vaccine or antiviral treatments available, urgent therapies are needed. Our findings put Sofosbuvir forward as a potential treatment to alleviate COVID-19-related neurological symptoms. SARS-CoV-2 infection causes neuronal death and impaired synaptogenesis, both rescued by Sofosbuvir treatment.","version":"1.1","doi":"10.1101/2020.05.30.125856","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.126136","pub_date":"2020-5-31","title":"A distinct phylogenetic cluster of Indian SARS-CoV-2 isolates","abstract":"From an isolated epidemic, COVID-19 has now emerged as a global pandemic. The availability of genomes in the public domain following the epidemic provides a unique opportunity to understand the evolution and spread of the SARS-CoV-2 virus across the globe. The availability of whole genomes from multiple states in India prompted us to analyse the phylogenetic clusters of genomes in India. We performed whole-genome sequencing for 64 genomes making a total of 361 genomes from India, followed by phylogenetic clustering, substitution analysis, and dating of the different phylogenetic clusters of viral genomes. We describe a distinct phylogenetic cluster (Clade I / A3i) of SARS-CoV-2 genomes from India, which encompasses 41% of all genomes sequenced and deposited in the public domain from multiple states in India. Globally 3.5% of genomes, which till date could not be mapped to any distinct known cluster fall in this newly defined clade. The cluster is characterized by a core set of shared genetic variants \u2013 C6312A (T2016K), C13730T (A88V/A97V), C23929T, and C28311T (P13L). Further, the cluster is also characterized by a nucleotide substitution rate of 1.4 \u00d7 10\u22123 variants per site per year, lower than the prevalent A2a cluster, and predominantly driven by variants in the E and N genes and relative sparing of the S gene. Epidemiological assessments suggest that the common ancestor emerged in the month of February 2020 and possibly resulted in an outbreak followed by countrywide spread, as evidenced by the low divergence of the genomes from across the country. To the best of our knowledge, this is the first comprehensive study characterizing the distinct and predominant cluster of SARS-CoV-2 in India.","version":"1.1","doi":"10.1101/2020.05.31.126136","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.30.125740","pub_date":"2020-5-31","title":"Synonymous sites in SARS-CoV-2 genes display trends affecting translational efficiency","abstract":"A novel coronavirus, SARS-CoV-2, has caused a pandemic of COVID-19. The evolutionary trend of the virus genome may have implications for infection control policy but remains obscure. We introduce an estimation of fold change of translational efficiency based on synonymous variant sites to characterize the adaptation of the virus to hosts. The increased translational efficiency of the M and N genes suggests that the population of SARS-CoV-2 benefits from mutations toward favored codons, while the ORF1ab gene has slightly decreased the translational efficiency. In the coding region of the ORF1ab gene upstream of the \u22121 frameshift site, the decreasing of the translational efficiency has been weakening parallel to the growth of the epidemic, indicating inhibition of synthesis of RNA-dependent RNA polymerase and promotion of replication of the genome. Such an evolutionary trend suggests that multiple infections increased virulence in the absence of social distancing.","version":"1.1","doi":"10.1101/2020.05.30.125740","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.120642","pub_date":"2020-5-31","title":"Molecules inhibit the enzyme activity of 3-chymotrypsin-like cysteine protease of SARS-CoV-2 virus: the experimental and theory studies","abstract":"SARS-CoV-2 has emerged as a world public health threat. Herein, we report that the clinical approved auranofin could perfectly inhibit the activity of 3-chymotrypsin-like cysteine protease (Mpro or 3CLpro) of SARS-CoV-2. Gold cluster could significantly inhibit 3CLpro of SARS-COV-2. Phenyl isothiocyanate and Vitamin K3 could well suppress the activity of 3CLpro. For Mpro inhibition, IC50 of auranofin, Vitamin K3, phenyl isothiocyanate, gold cluster are about 0.51\u03bcM, 7.96\u03bcM, 10.13\u03bcM, 1.61\u03bcM, respectively. These compounds may be with potentials for treatment SARS-CoV-2 virus replication. Especially for FDA approved auranofin, it is an anti-inflammation drug in clinic, thus it may with strong potential to inhibit virus replication and suppress the inflammation damage in COVID-19 patients. Gold cluster is with better safety index and well anti-inflammation in vitro/vivo, therefore it is with potential to inhibit virus replication and suppress the inflammation damage caused by COVID-19 virus. As Au(I) ion is active metabolism specie derived from gold compounds or gold clusters in vivo, further computational studies revealed Au ion could tightly bind thiol group of Cys145 residue of 3CLpro thus inhibit enzyme activity. Also, phenyl isothiocyanate and Vitamin K3 may interact with thiol group of Cys145 via Michael addition reaction, molecular dynamic (MD) theory studied are applied to confirmed these small molecules are stable in the pocket and inhibit Mpro activity.","version":"1.2","doi":"10.1101/2020.05.28.120642","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.116061","pub_date":"2020-5-31","title":"Origin and cross-species transmission of bat coronaviruses in China","abstract":"Bats are presumed reservoirs of diverse coronaviruses (CoVs) including progenitors of Severe Acute Respiratory Syndrome (SARS)-CoV and SARS-CoV-2, the causative agent of COVID-19. However, the evolution and diversification of these coronaviruses remains poorly understood. We used a Bayesian statistical framework and sequence data from all known bat-CoVs (including 630 novel CoV sequences) to study their macroevolution, cross-species transmission, and dispersal in China. We find that host-switching was more frequent and across more distantly related host taxa in alpha-than beta-CoVs, and more highly constrained by phylogenetic distance for beta-CoVs. We show that inter-family and -genus switching is most common in Rhinolophidae and the genus Rhinolophus. Our analyses identify the host taxa and geographic regions that define hotspots of CoV evolutionary diversity in China that could help target bat-CoV discovery for proactive zoonotic disease surveillance. Finally, we present a phylogenetic analysis suggesting a likely origin for SARS-CoV-2 in Rhinolophus spp. bats.","version":"1.1","doi":"10.1101/2020.05.31.116061","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.31.125302","pub_date":"2020-5-31","title":"Assignment of coronavirus spike protein site-specific glycosylation using GlycReSoft","abstract":"Widely-available LC-MS instruments and methods allow users to acquire glycoproteomics data. Complex glycans, however, add a dimension of complexity to the data analysis workflow. In a sense, complex glycans are post-translationally modified post-translational modifications, reflecting a series of biosynthetic reactions in the secretory pathway that are spatially and temporally regulated. One problem is that complex glycan is micro-heterogeneous, multiplying the complexity of the proteome. Another is that glycopeptide glycans undergo dissociation during tandem MS that must be considered for tandem MS interpretation algorithms and quantitative tools. Fortunately, there are a number of algorithmic tools available for analysis of glycoproteomics LC-MS data. We summarize the principles for glycopeptide data analysis and show use of our GlycReSoft tool to analyze SARS-CoV-2 spike protein site-specific glycosylation.","version":"1.1","doi":"10.1101/2020.05.31.125302","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.124776","pub_date":"2020-5-30","title":"Validation and Performance Comparison of Three SARS-CoV-2 Antibody Assays","abstract":"Serology testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly being used during the current pandemic of Coronavirus Disease 2019 (COVID-19). The clinical and epidemiologic utilities of antibody-based SARS-CoV-2 testing are under debate. Characterizing these assays helps to understand the disease and provides scientific basis for deciding how to best use these assays. The study assessed one chemiluminescent assay (Abbott COVID-2 IgG) and two lateral flow assays (STANDARD Q [SQ] IgM/IgG Duo and Wondfo Total Antibody Test). Validation included 113 blood samples from 71 PCR-confirmed COVID-19 patients and 1182 samples from negative controls with potential interferences/cross-reactions, including 1063 pre-pandemic samples. IgM antibodies against SARS-CoV-2 were detected as early as post-symptom onset days 3-4. IgG antibodies were first detected post-onset days 5-6 by SQ assays. The detection rates increased gradually, and SQ IgG, Abbott IgG and Wondfo Total detected antibodies from all the PCR-confirmed patients 14 days after symptom onset. Overall agreements between SQ IgM/IgG and Wondfo Total was 88.5% and between SQ IgG and Abbott IgG was 94.6% (Kappa = 0.75, 0.89). No cross-reaction with other endemic coronavirus infections were identified. Viral hepatitis and autoimmune samples were the main cross-reactions observed. However, the interferences/cross-reactions were low. The specificities were 100% for SQ IgG and Wondfo Total and 99.62% for Abbott IgG and 98.87% for SQ IgM. These findings demonstrate high sensitivity and specificity of appropriately validated antibody-based SARS-CoV-2 assays with implications for clinical use and epidemiological seroprevalence studies.","version":"1.1","doi":"10.1101/2020.05.29.124776","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.30.125484","pub_date":"2020-5-30","title":"Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes","abstract":"The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to \u223c10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 \u00c5-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.30.125484","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.038752","pub_date":"2020-5-30","title":"Bioinformatic characterization of angiotensin-converting enzyme 2, the entry receptor for SARS-CoV-2","abstract":"The World Health Organization declared the COVID-19 epidemic a public health emergency of international concern on March 11th, 2020, and the pandemic is rapidly spreading worldwide. COVID-19 is caused by a novel coronavirus SARS-CoV-2, which enters human target cells via angiotensin converting enzyme 2 (ACE2). We used a number of bioinformatics tools to computationally characterize ACE2 by determining its cell-specific expression in trachea, lung, and small intestine, derive its putative functions, and predict transcriptional regulation. The small intestine expressed higher levels of ACE2 than any other organ. The large intestine, kidney and testis showed moderate signals, whereas the signal was weak in the lung. Single cell RNA-Seq data from trachea indicated positive signals along the respiratory tract in key protective cell types including club, goblet, proliferating, and ciliary epithelial cells; while in lung the ratio of ACE2-expressing cells was low in all cell types (<2.6%), but was highest in vascular endothelial and goblet cells. Gene ontology analysis suggested that, besides its classical role in renin-angiotensin system, ACE2 may be functionally associated with angiogenesis/blood vessel morphogenesis. Using a novel tool for the prediction of transcription factor binding sites we identified several putative binding sites within two tissue-specific promoters of the ACE2 gene. Our results also confirmed that age and gender play no significant role in the regulation of ACE2 mRNA expression in the lung. Vaccines and new medicines are urgently needed to prevent spread of COVID-19 pandemic, reduce the symptoms, shorten the duration of disease, prevent virus spread in the body, and most importantly to save lives. One of the key drug targets could be angiotensin-converting enzyme 2 (ACE2), which is a crucial receptor for the corona virus (SARS-CoV-2). It is known that SARS coronavirus infections lead to worse outcome in the elderly and in males. Therefore, one aim of the present study was to investigate whether age or sex could contribute to the regulation of ACE2 expression. We also decided to explore the transcriptional regulation of ACE2 gene expression. Since data on ACE2 distribution is still conflicting, we aimed to get a more comprehensive view of the cell types expressing the receptor of SARS-CoV-2. Finally, we studied the coexpression of ACE2 with other genes and explored its putative functions using gene ontology enrichment analysis.","version":"1.2","doi":"10.1101/2020.04.13.038752","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.124610","pub_date":"2020-5-30","title":"COVID-3D: An online resource to explore the structural distribution of genetic variation in SARS-CoV-2 and its implication on therapeutic development","abstract":"The emergence of the COVID-19 pandemic has spurred a global rush to uncover basic biological mechanisms, to inform effective vaccine and drug development. Despite viral novelty, global sequencing efforts have already identified genomic variation across isolates. To enable easy exploration and spatial visualization of the potential implications of SARS-CoV-2 mutations on infection, host immunity and drug development we have developed COVID-3D (http://biosig.unimelb.edu.au/covid3d/).","version":"1.1","doi":"10.1101/2020.05.29.124610","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.013268","pub_date":"2020-5-30","title":"SARS-CoV-2 receptor and entry genes are expressed by sustentacular cells in the human olfactory neuroepithelium","abstract":"Various reports indicate an association between COVID-19 and anosmia, suggesting an infection of the olfactory sensory epithelium, and thus a possible direct virus access to the brain. To test this hypothesis, we generated RNA-seq libraries from human olfactory neuroepithelia, in which we found substantial expression of the genes coding for the virus receptor angiotensin-converting enzyme-2 (ACE2), and for the virus internalization enhancer TMPRSS2. We analyzed a human olfactory single-cell RNA-seq dataset and determined that sustentacular cells, which maintain the integrity of olfactory sensory neurons, express ACE2 and TMPRSS2. We then observed that the ACE2 protein was highly expressed in a subset of sustentacular cells in human and mouse olfactory tissues. Finally, we found ACE2 transcripts in specific brain cell types, both in mice and humans. Sustentacular cells thus represent a potential entry door for SARS-CoV-2 in a neuronal sensory system that is in direct connection with the brain.","version":"1.2","doi":"10.1101/2020.03.31.013268","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.115923","pub_date":"2020-5-30","title":"T-cell hyperactivation and paralysis in severe COVID-19 infection revealed by single-cell analysis","abstract":"Severe COVID-19 patients can show respiratory failure, T-cell reduction, and cytokine release syndrome (CRS), which can be fatal in both young and aged patients and is a major concern of the pandemic. However, the pathogenetic mechanisms of CRS in COVID-19 are poorly understood. Here we show single cell-level mechanisms for T-cell dysregulation in severe SARS-CoV-2 infection, and thereby demonstrate the mechanisms underlying T-cell hyperactivation and paralysis in severe COVID-19 patients. By in silico sorting CD4+ T-cells from a single cell RNA-seq dataset, we found that CD4+ T-cells were highly activated and showed unique differentiation pathways in the lung of severe COVID-19 patients. Notably, those T-cells in severe COVID-19 patients highly expressed immunoregulatory receptors and CD25, whilst repressing the expression of the transcription factor FOXP3 and interestingly, both the differentiation of regulatory T-cells (Tregs) and Th17 was inhibited. Meanwhile, highly activated CD4+ T-cells express PD-1 alongside macrophages that express PD-1 ligands in severe patients, suggesting that PD-1-mediated immunoregulation was partially operating. Furthermore, we show that CD25+ hyperactivated T-cells differentiate into multiple helper T-cell lineages, showing multifaceted effector T-cells with Th1 and Th2 characteristics. Lastly, we show that CD4+ T-cells, particularly CD25-expressing hyperactivated T-cells, produce the protease Furin, which facilitates the viral entry of SARS-CoV-2. Collectively, CD4+ T-cells from severe COVID-19 patients are hyperactivated and FOXP3-mediated negative feedback mechanisms are impaired in the lung, while activated CD4+ T-cells continue to promote further viral infection through the production of Furin. Therefore, our study proposes a new model of T-cell hyperactivation and paralysis that drives pulmonary damage, systemic CRS and organ failure in severe COVID-19 patients.","version":"1.2","doi":"10.1101/2020.05.26.115923","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.120238","pub_date":"2020-5-30","title":"One enzyme reverse transcription qPCR using Taq DNA polymerase","abstract":"Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase, and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/\u00b5L of input viral genomic RNA.","version":"1.1","doi":"10.1101/2020.05.27.120238","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.122671","pub_date":"2020-5-29","title":"Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein","abstract":"The SARS-COV2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS-COV2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals. When working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here we describe a series of modifications that result in the production of relatively high titer SARS-COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.","version":"1.1","doi":"10.1101/2020.05.28.122671","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.123810","pub_date":"2020-5-29","title":"Dose-dependent response to infection with SARS-CoV-2 in the ferret model: evidence of protection to re-challenge","abstract":"In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions. Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5\u00d7106 pfu) and medium (5\u00d7104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5\u00d7102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.","version":"1.1","doi":"10.1101/2020.05.29.123810","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.123513","pub_date":"2020-5-29","title":"CD147 (BSG) but not ACE2 expression is detectable in vascular endothelial cells within single cell RNA sequencing datasets derived from multiple tissues in healthy individuals","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is associated with a wide range of systemic manifestations. Several observations support a role for vascular endothelial dysfunction in the pathogenesis including an increased incidence of thrombotic events and coagulopathy and the presence of vascular risk factors as an independent predictor of poor prognosis. It has recently been reported that endothelitis is associated with viral inclusion bodies suggesting a direct role for SARS-CoV-2 in the pathogenesis. The ACE2 receptor has been shown to mediate SARS-CoV-2 uptake and it has been proposed that CD147 (BSG) can function as an alternative cell surface receptor. To define the endothelial cell populations that are susceptible to infection with SARS-CoV-2, we investigated the expression of ACE2 as well as other genes implicated in the cellular entry of SARS-Cov-2 in the vascular endothelium through the analysis of single cell sequencing data derived from multiple human tissues (skin, liver, kidney, lung and intestine). We found that CD147 (BSG) but not ACE2 is detectable in vascular endothelial cells within single cell sequencing datasets derived from multiple tissues in healthy individuals. This implies that either ACE2 is not expressed in healthy tissue but is instead induced in response to SARS-Cov2 or that SARS-Cov2 enters endothelial cells via an alternative receptor such as CD147.","version":"1.1","doi":"10.1101/2020.05.29.123513","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.122374","pub_date":"2020-5-29","title":"ACE2 expression in human dorsal root ganglion sensory neurons: implications for SARS-CoV-2 virus-induced neurological effects","abstract":"SARS-CoV-2 has created a global crisis. COVID-19, the disease caused by the virus, is characterized by pneumonia, respiratory distress and hypercoagulation and is often fatal. An early sign of infection is loss of smell, taste and chemesthesis - loss of chemical sensation. Other neurological effects of the disease have been described, but not explained. We show that human dorsal root ganglion (DRG) neurons express the SARS-CoV-2 receptor, ACE2. ACE2 mRNA is expressed by a subset of nociceptors that express MRGPRD mRNA suggesting that SARS-CoV-2 may gain access to the nervous system through entry into neurons that form free-nerve endings at the outer-most layers of skin and luminal organs. Therefore, sensory neurons are a potential target for SARS-CoV-2 invasion of the nervous system.","version":"1.1","doi":"10.1101/2020.05.28.122374","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.122358","pub_date":"2020-5-29","title":"Comparison of Different Kits for SARS-CoV-2 RNA Extraction Marketed in Brazil","abstract":"December 2019 marked the begining of the greatest pandemic since Spanish Flu, the disease named Covid-19 that cause severe pneumonia. Until May 19, 2020 more than 4 million and 700 thousand cases were oficially notified with about 316 thousand deaths. Etiological agent of the disease was identified as being a new coronavirus, Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2). In this study we compared four different manual methods for RNA isolation and purification for detection of SARS-CoV-2 through qRT-PCR, as well as the extraction quality itself through detection of RNAse P. Magnetic beads-based (MagMax\u2122) and silica column-based (Biopur\u00ae) methods presented the better performances. Concerning to the mean delay in CT values when compared to MagMax\u2122, TRIzol\u2122, Biopur\u00ae and EasyExtract presented 0,39, 0,95 and 5,23 respectively. Agreement between positive and negative results of different methods when compared with the one with better performance MagMax\u2122 was 94,44% for silica column-based method (Biopur\u00ae), 88,89% for phenol-chroloform-based method (TRIzol\u2122) and 77,78% for EasyExtract. We aimed to evaluate how reliable each method is for diagnostic purposes and to propose alternatives when usual methods are not available. In this regard, magnectic beads and silica column-based methods are convenient and reliable choices and phenol-chloroform-based method could also be chosen as an alternative.","version":"1.1","doi":"10.1101/2020.05.29.122358","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.089896","pub_date":"2020-5-29","title":"Performance of Abbott ID NOW COVID-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution","abstract":"The recent emergence of the SARS-CoV-2 pandemic has posed formidable challenges for clinical laboratories seeking reliable laboratory diagnostic confirmation. The swift advance of the crisis in the United States has led to Emergency Use Authorization (EUA) facilitating the availability of molecular diagnostic assays without the more rigorous examination to which tests are normally subjected prior to FDA approval. Our laboratory currently uses two real time RT-PCR platforms, the Roche Cobas SARS-CoV2 and the Cepheid Xpert Xpress SARS-CoV-2. Both platforms demonstrate comparable performance; however, the run times for each assay are 3.5 hours and 45 minutes, respectively. In search for a platform with shorter turnaround time, we sought to evaluate the recently released Abbott ID NOW COVID-19 assay which is capable of producing positive results in as little as 5 minutes. We present here the results of comparisons between Abbott ID NOW COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media and comparisons between Abbott ID NOW COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media for Cepheid and dry nasal swabs for Abbott ID NOW. Regardless of method of collection and sample type, Abbott ID NOW COVID-19 had negative results in a third of the samples that tested positive by Cepheid Xpert Xpress when using nasopharyngeal swabs in viral transport media and 45% when using dry nasal swabs.","version":"1.2","doi":"10.1101/2020.05.11.089896","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.121533","pub_date":"2020-5-29","title":"Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies","abstract":"Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1A and RBD epitopes. A 3.4\u00c5 cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on \u201cup\u201d RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.","version":"1.1","doi":"10.1101/2020.05.28.121533","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.122986","pub_date":"2020-5-29","title":"Risk factors associated with mortality in hospitalized patients with SARS-CoV-2 infection. A prospective, longitudinal, unicenter study in Reus, Spain","abstract":"Spain is one of the countries that has suffered the most from the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the strain that causes coronavirus disease 2019 (COVID-19). However, there is a lack of information on the characteristics of this disease in the Spanish population. The objective of this study has been to characterize our patients from an epidemiological point of view and to identify the risk factors associated with mortality in our geographical area. We performed a prospective, longitudinal study on 188 hospitalized cases of SARS-Cov-2 infection in Hospital Universitari de Sant Joan, in Reus, Spain, admitted between 15th March 2020 and 30th April 2020. We recorded demographic data, signs and symptoms and comorbidities. We also calculated the Charlson and McCabe indices. A total of 43 deaths occurred during the study period. Deceased patients were older than the survivors (77.7 \u00b1 13.1 vs. 62.8 \u00b1 18.4 years; p < 0.001). Logistic regression analyses showed that fever, pneumonia, acute respiratory distress syndrome, diabetes mellitus and cancer were the variables that showed independent and statistically significant associations with mortality. The Charlson index was more efficient than the McCabe index in discriminating between deceased and survivors. This is one of the first studies to describe the factors associated with mortality in patients infected with SARS-CoV-2 in Spain, and one of the few in the Mediterranean area. We identified the main factors independently associated with mortality in our population. Further studies in are needed to complete and confirm our findings.","version":"1.1","doi":"10.1101/2020.05.29.122986","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.120998","pub_date":"2020-5-29","title":"Pathogenesis, transmission and response to re-exposure of SARS-CoV-2 in domestic cats","abstract":"The pandemic caused by SARS-CoV-2 has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is likely that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to na\u00efve cats. We report that cats are highly susceptible to subclinical infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats develop a robust neutralizing antibody response that prevented re-infection to a second viral challenge. Conversely, we found that dogs do not shed virus following infection, but do mount an anti-viral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human exposure; however, reverse zoonosis is possible if infected owners expose their domestic pets during acute infection. Resistance to re-exposure holds promise that a vaccine strategy may protect cats, and by extension humans, to disease susceptibility.","version":"1.1","doi":"10.1101/2020.05.28.120998","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.123190","pub_date":"2020-5-29","title":"Impact of emerging mutations on the dynamic properties the SARS-CoV-2 main protease: an in silico investigation","abstract":"The new coronavirus (SARS-CoV-2) is a global threat to world health and its economy. Its main protease (Mpro), which functions as a dimer, cleaves viral precursor proteins in the process of viral maturation. It is a good candidate for drug development owing to its conservation and the absence of a human homolog. An improved understanding of the protein behaviour can accelerate the discovery of effective therapies in order to reduce mortality. 100 ns all-atom molecular dynamics simulations of 50 homology modelled mutant Mpro dimers were performed at pH 7 from filtered sequences obtained from the GISAID database. Protease dynamics were analysed using RMSD, RMSF, Rg, the averaged betweenness centrality and geometry calculations. Domains from each Mpro protomer were found to generally have independent motions, while the dimer-stabilising N-finger region was found to be flexible in most mutants. A mirrored interprotomer pocket was found to be correlated to the catalytic site using compaction dynamics, and can be a potential allosteric target. The high number of titratable amino acids of Mpro may indicate an important role of pH on enzyme dynamics, as previously reported for SARS-CoV. Independent coarse-grained Monte Carlo simulations suggest a link between rigidity/mutability and enzymatic function.","version":"1.1","doi":"10.1101/2020.05.29.123190","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.124123","pub_date":"2020-5-29","title":"Development Optimization and Validation of RT-LAMP based COVID-19 Facility in Pakistan","abstract":"The pandemic SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has created a widespread panic across the globe especially in the developing countries like Pakistan. The lack of resources and technical staff are causing havoc challenges in the detection and prevention of this global outbreak. Therefore, a less expensive and massive screening of suspected individuals for COVID-19 is required. In this study, a user-friendly technique of reverse transcription-loop mediated isothermal amplification (RT-LAMP) was designed and validated to suggest a potential RT-qPCR alternate for rapid testing of COVID-19 suspected individuals. A total of 12 COVID-19 negative and 72 COVID-19 suspected individuals were analyzed. Both RT-qPCR and RT-LAMP assays were performed for all the individuals using open reading frame (ORF 1ab), nucleoprotein (N) and Spike (S) genes. All 12 specimens which were negative using RT-qPCR were also found negative using RT-LAMP assay. Overall 62 out of 72 positive samples (detected using RT-qPCR) were found COVID-19 positive using RT-LAMP assay. Interestingly all samples (45) having Ct values less than 30 showed 100% sensitivity. However, samples with weaker Ct values (i.e., => 35) showed 54% concordance, suggesting potential false negatives or false positives in RT-LAMP or RT-qPCR results, respectively. Overall comparative assessment showed that RT-LAMP assay showed strong sensitivity and specificity and can be used as an alternative strategy for rapid COVID-19 testing. Hence, based on fast processing time, minimal risk of specimens transfer and utilizing available resources, LAMP based detection of COVID-19 is strongly advocated especially for developing countries.","version":"1.1","doi":"10.1101/2020.05.29.124123","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.118059","pub_date":"2020-5-29","title":"An enhanced isothermal amplification assay for viral detection","abstract":"Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available. Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.","version":"1.1","doi":"10.1101/2020.05.28.118059","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.29.123455","pub_date":"2020-5-29","title":"The representation of women as authors of submissions to ecology journals during the COVID-19 pandemic","abstract":"Observations made from papers submitted to preprint servers, and the speculation of editors on social media platforms, suggest that women are submitting fewer papers to scholarly journals than are men during the COVID-19 pandemic. Here I examine whether submissions by men and women to six ecology journals (all published by the British Ecological Society) have changed since the start of COVID disruptions. At these six ecology journals there is no evidence of a decline in the proportion of submissions that are authored by women (as either first or submitting author) since the start of the COVID-19 disruptions; the proportion of papers authored by women in the post-COVID period of 2020 has increased relative to the same period in 2019, and is higher than in the period pre-COVID in 2020. There is also no evidence of a change in the geographic pattern of submissions from across the globe.","version":"1.1","doi":"10.1101/2020.05.29.123455","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.121640","pub_date":"2020-5-28","title":"Single-dose replicating RNA vaccine induces neutralizing antibodies against SARS-CoV-2 in nonhuman primates","abstract":"The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.05.28.121640","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.120410","pub_date":"2020-5-28","title":"Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification","abstract":"Rapid detection of SARS-CoV-2 is critical for the diagnosis of coronavirus disease 2019 (COVID-19) and preventing the spread of the virus. A novel \u201c2019 Novel Coronavirus Detection Kit (nCoV-DK)\u201d halves detection time by eliminating the steps of RNA extraction and purification. We evaluated concordance between the nCoV-DK and direct PCR. The virus was detected in 53/71 fresh samples by the direct method and 55/71 corresponding frozen samples by the nCoV-DK. The overall concordance rate of the virus detection between the two methods was 94.4% (95% CI, 86.2-98.4). Concordance rates were 95.2% (95% CI, 83.8-99.4), 95.5% (95% CI, 77.2-99.9), 85.7% (95% CI, 42.1-99.6) in nasopharyngeal swab, saliva, and sputum samples, respectively. These results indicate that the nCoV-DK effectively detects SARS-CoV-2 in all types of the samples including saliva, while reducing time required for detection, labor, and risk of human error.","version":"1.1","doi":"10.1101/2020.05.27.120410","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.120444","pub_date":"2020-5-28","title":"Assessment of Inactivation Procedures for SARS-CoV-2","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), presents a challenge to laboratorians and healthcare workers around the world. Handling of biological samples from individuals infected with the SARS-CoV-2 virus requires strict biosafety and biosecurity measures. Within the laboratory, non-propagative work with samples containing the virus requires, at minimum, Biosafety Level-2 (BSL-2) techniques and facilities. Therefore, handling of SARS-CoV-2 samples remains a major concern in areas and conditions where biosafety and biosecurity for specimen handling is difficult to maintain, such as in rural laboratories or austere field testing sites. Inactivation through physical or chemical means can reduce the risk of handling live virus and increase testing ability worldwide. Herein we assess several chemical and physical inactivation techniques employed against SARS-CoV-2 isolates from Cambodian COVID-19 patients. This data demonstrates that all chemical (AVL, inactivating sample buffer and formaldehyde) and heat treatment (56\u00b0C and 98\u00b0C) methods tested completely inactivated viral loads of up to 5 log10.","version":"1.1","doi":"10.1101/2020.05.28.120444","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.091520","pub_date":"2020-5-28","title":"The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators","abstract":"SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus\u2019 pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 \u03bcM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.","version":"1.2","doi":"10.1101/2020.05.16.091520","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.120162","pub_date":"2020-5-28","title":"Plasmin cascade mediates thrombolytic events in SARS-CoV-2 infection via complement and platelet-activating systems","abstract":"Recently emerged beta-coronavirus, SARS-CoV-2 has resulted in the current pandemic designated COVID-19. COVID-19 manifests as severe illness exhibiting systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), thrombotic events, and shock, exacerbated further by co-morbidities and age. Recent clinical reports suggested that the pulmonary failure seen in COVID-19 may not be solely driven by acute ARDS, but also microvascular thrombotic events, likely driven by complement activation. However, it is not fully understood how the SARS-CoV-2 infection mechanisms mediate thrombotic events, and whether such mechanisms and responses are unique to SARS-CoV-2 infection, compared to other respiratory infections. We address these questions here, in the context of normal lung epithelia, in vitro and in vivo, using publicly available data. Our results indicate that plasmin is a crucial mediator which primes interactions between complement and platelet-activating systems in lung epithelia upon SARS-CoV-2 infection, with a potential for therapeutic intervention.","version":"1.1","doi":"10.1101/2020.05.28.120162","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.23.004580","pub_date":"2020-5-28","title":"Identify potent SARS-CoV-2 main protease inhibitors via accelerated free energy perturbation-based virtual screening of existing drugs","abstract":"Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global crisis. There is no therapeutic treatment specific for COVID-19. It is highly desirable to identify potential antiviral agents against SARS-CoV-2 from existing drugs available for other diseases and, thus, repurpose them for treatment of COVID-19. In general, a drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. Here we report a new virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions and its use in identifying drugs targeting SARS-CoV-2 main protease (Mpro). The accurate FEP-ABFE predictions were based on the use of a new restraint energy distribution (RED) function designed to accelerate the FEP-ABFE calculations and make the practical FEP-ABFE-based virtual screening of the existing drug library possible for the first time. As a result, out of twenty-five drugs predicted, fifteen were confirmed as potent inhibitors of SARS-CoV-2 Mpro. The most potent one is dipyridamole (Ki=0.04 \u03bcM) which has showed promising therapeutic effects in subsequently conducted clinical studies for treatment of patients with COVID-19. Additionally, hydroxychloroquine (Ki=0.36 \u03bcM) and chloroquine (Ki=0.56 \u03bcM) were also found to potently inhibit SARS-CoV-2 Mpro for the first time. We anticipate that the FEP-ABFE prediction-based virtual screening approach will be useful in many other drug repurposing or discovery efforts. Drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. It has been demonstrated that a new virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions can reach an unprecedently high hit rate, leading to successful identification of 16 potent inhibitors of SARS-CoV-2 main protease (Mpro) from computationally selected 25 drugs under a threshold of Ki = 4 \u03bcM. The outcomes of this study are valuable for not only drug repurposing to treat COVID-19, but also demonstrating the promising potential of the FEP-ABFE prediction-based virtual screening approach.","version":"1.2","doi":"10.1101/2020.03.23.004580","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.23.104919","pub_date":"2020-5-28","title":"In silico Proteome analysis of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2019-nCoV), is a positive-sense, single-stranded RNA coronavirus. The virus is the causative agent of coronavirus disease 2019 (COVID-19) and is contagious through human-to-human transmission. The present study reports sequence analysis, complete coordinate tertiary structure prediction and in silico sequence-based and structure-based functional characterization of full SARS-CoV-2 proteome based on the NCBI reference sequence NC_045512 (29903 bp ss-RNA) which is identical to GenBank entry MN908947 and MT415321. The proteome includes 12 major proteins namely orf1ab polyprotein (includes 15 proteins), surface glycoprotein, ORF3a protein, envelope protein, membrane glycoprotein, ORF6 protein, ORF7a protein, orf7b, ORF8, Nucleocapsid phosphoprotein and ORF10 protein. Each protein of orf1ab polyprotein group has been studied separately. A total of 25 polypeptides have been analyzed out of which 15 proteins are not yet having experimental structures and only 10 are having experimental structures with known PDB IDs. Out of 15 newly predicted structures six (6) were predicted using comparative modeling and nine (09) proteins having no significant similarity with so far available PDB structures were modeled using ab-initio modeling. Structure verification using recent tools QMEANDisCo 4.0.0 and ProQ3 for global and local (per-residue) quality estimates indicate that the all-atom model of tertiary structure of high quality and may be useful for structure-based drug designing targets. The study has identified nine major targets (spike protein, envelop protein, membrane protein, nucleocapsid protein, 2\u2019-O-ribose methyltransferase, endoRNAse, 3\u2019-to-5\u2019 exonuclease, RNA-dependent RNA polymerase and helicase) for which drug design targets could be considered. There are other 16 nonstructural proteins (NSPs), which may also be percieved from the drug design angle. The protein structures have been deposited to ModelArchive. Tunnel analysis revealed the presence of large number of tunnels in NSP3, ORF 6 protein and membrane glycoprotein indicating a large number of transport pathways for small ligands influencing their reactivity.","version":"1.2","doi":"10.1101/2020.05.23.104919","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.104117","pub_date":"2020-5-28","title":"Human IgG neutralizing monoclonal antibodies block SARS-CoV-2 infection","abstract":"The coronavirus induced disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide threat to human lives, and neutralizing antibodies present a great therapeutic potential in curing affected patients. We purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain) antigens from 11 convalescent COVID-19 patients, and a total of 729 naturally paired heavy and light chain fragments were obtained by single B cell cloning technology. Among these, 178 recombinant monoclonal antibodies were tested positive for antigen binding, and the top 13 binders with Kd below 0.5 nM are all RBD binders. Importantly, all these 13 antibodies could block pseudoviral entry into HEK293T cells overexpressing ACE2, with the best ones showing IC50s around 2-3 nM. We further identified 8 neutralizing antibodies against authentic virus with IC50s within 10 nM. Among these, 414-1 blocked authentic viral entry at IC50 of 1.75 nM and in combination with 105-38 could achieve IC50 as low as 0.45 nM. Meanwhile, we also found that 3 antibodies could cross-react with the SARS-CoV spike protein. Altogether, our study provided a panel of potent human neutralizing antibodies for COVID19 as therapeutics candidates for further development.","version":"1.3","doi":"10.1101/2020.05.19.104117","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.120402","pub_date":"2020-5-28","title":"Genomic analysis of early SARS-CoV-2 strains introduced in Mexico","abstract":"The COVID-19 pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to implement effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, the SARS-CoV-2 lineage A/G, containing mostly sequences from North America, and the lineage B/S containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized fourteen independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represent local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described in the country. Within this Mexican cluster, we also identified an H49Y amino acid change in the spike protein. This mutation is a homoplasy occurring independently through time and space, and may function as a molecular marker to follow on any further spread of these viral variants throughout the country. Our results depict the general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts. This work is the result of the collaboration of five institutions into one research consortium: three public health institutes and two universities. From the beginning of this work, it was agreed that the experimental leader of each institution would share the first authorship. Those were the criteria followed to assign first co-first authorship in this manuscript. The order of the other authors was randomly assigned. Understanding the introduction, spread and establishment of SARS-CoV-2 within distinct human populations is crucial to implement effective control strategies as well as the evolution of the pandemics. In this work, we describe that the initial virus strains introduced in Mexico came from Europe and the United States and the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains having the mutation H49Y in the Spike protein, that could be further used as a molecular marker to follow viral spread within the country and the region.","version":"1.1","doi":"10.1101/2020.05.27.120402","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108563","pub_date":"2020-5-28","title":"SARS-CoV-2 mutations and where to find them: An in silico perspective of structural changes and antigenicity of the Spike protein","abstract":"The recent emergence of a novel coronavirus (SARS-CoV-2) is causing a severe global health threat characterized by severe acute respiratory syndrome (Covid-19). At the moment, there is no specific treatment for this disease, and vaccines are still under development. The structural protein Spike is essential for virus infection and has been used as the main target for vaccine and serological diagnosis test development. We analysed 2363 sequences of the Spike protein from SARS-CoV-2 isolates and identified variability in 44 amino acid residues and their worldwide distribution in all continents. We used the three-dimensional structure of the homo-trimer model to predict conformational epitopes of B-cell, and sequence of Spike protein Wuhan-Hu-1 to predict linear epitopes of T-Cytotoxic and T-Helper cells. We identified 45 epitopes with amino acid variations. Finally, we showed the distribution of mutations within the epitopes. Our findings can help researches to identify more efficient strategies for the development of vaccines, therapies, and serological diagnostic tests based on the Spike protein of Sars-Cov-2.","version":"1.2","doi":"10.1101/2020.05.21.108563","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.121889","pub_date":"2020-5-28","title":"Comprehensive Transcriptomic Analysis of COVID-19 Blood, Lung, and Airway","abstract":"SARS-CoV2 is a previously uncharacterized coronavirus and causative agent of the COVID-19 pandemic. The host response to SARS-CoV2 has not yet been fully delineated, hampering a precise approach to therapy. To address this, we carried out a comprehensive analysis of gene expression data from the blood, lung, and airway of COVID-19 patients. Our results indicate that COVID-19 pathogenesis is driven by populations of myeloid-lineage cells with highly inflammatory but distinct transcriptional signatures in each compartment. The relative absence of cytotoxic cells in the lung suggests a model in which delayed clearance of the virus may permit exaggerated myeloid cell activation that contributes to disease pathogenesis by the production of inflammatory mediators. The gene expression profiles also identify potential therapeutic targets that could be modified with available drugs. The data suggest that transcriptomic profiling can provide an understanding of the pathogenesis of COVID-19 in individual patients.","version":"1.1","doi":"10.1101/2020.05.28.121889","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.120204","pub_date":"2020-5-28","title":"Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays","abstract":"The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots. Improved yields of SARS-CoV-2 spike protein through modification of expression and purification parameters Yields of greater than 5 mg/l obtained for VRC spike under optimal conditions Spike protein quality was validated by QC methods to ensure utility in serology assays","version":"1.1","doi":"10.1101/2020.05.27.120204","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.119131","pub_date":"2020-5-28","title":"Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2","abstract":"The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINE\u2019s results can be visualized with an in-tube fluorescent readout \u2014 reducing contamination risk as amplification reaction tubes remain sealed \u2014 and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.","version":"1.1","doi":"10.1101/2020.05.28.119131","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.122143","pub_date":"2020-5-28","title":"Phylogenetic clustering of the Indian SARS-CoV-2 genomes reveals the presence of distinct clades of viral haplotypes among states","abstract":"The first Indian cases of COVID-19 caused by SARS-Cov-2 were reported in February 29, 2020 with a history of travel from Wuhan, China and so far above 4500 deaths have been attributed to this pandemic. The objectives of this study were to characterize Indian SARS-CoV-2 genome-wide nucleotide variations, trace ancestries using phylogenetic networks and correlate state-wise distribution of viral haplotypes with differences in mortality rates. A total of 305 whole genome sequences from 19 Indian states were downloaded from GISAID. Sequences were aligned using the ancestral Wuhan-Hu genome sequence (NC_045512.2). A total of 633 variants resulting in 388 amino acid substitutions were identified. Allele frequency spectrum, and nucleotide diversity (\u03c0) values revealed the presence of higher proportions of low frequency variants and negative Tajima\u2019s D values across ORFs indicated the presence of population expansion. Network analysis highlighted the presence of two major clusters of viral haplotypes, namely, clade G with the S:D614G, RdRp: P323L variants and a variant of clade L [Lv] having the RdRp:A97V variant. Clade G genomes were found to be evolving more rapidly into multiple sub-clusters including clade GH and GR and were also found in higher proportions in three states with highest mortality rates namely, Gujarat, Madhya Pradesh and West Bengal.","version":"1.1","doi":"10.1101/2020.05.28.122143","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.120709","pub_date":"2020-5-28","title":"\u201cMonoclonal-type\u201d plastic antibodies for SARS-CoV-2 based on Molecularly Imprinted Polymers","abstract":"Our idea is focused on the development of \u201cmonoclonal-type\u201d plastic antibodies based on Molecularly Imprinted Polymers (MIPs) able to selectively bind a portion of the novel coronavirus SARS-CoV-2 spike protein to block its function and, thus, the infection process. Molecular Imprinting, indeed, represents a very promising and attractive technology for the synthesis of MIPs characterized by specific recognition abilities for a target molecule. Given these characteristics, MIPs can be considered tailor-made synthetic antibodies obtained by a templating process. In the present study, the developed imprinted polymeric nanoparticles were characterized in terms of particles size and distribution by Dynamic Light Scattering (DLS) and the imprinting effect and selectivity were investigated by performing binding experiments using the receptor-binding domain (RBD) of the novel coronavirus and the RBD of SARS-CoV spike protein, respectively. Finally, the hemocompatibility of the prepared MIP-based plastic antibodies was also evaluated.","version":"1.1","doi":"10.1101/2020.05.28.120709","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.118190","pub_date":"2020-5-28","title":"Comparison of the NeuMoDX, Diasorin Simplexa, Cepheid and Roche CDC SARS-CoV 2 EUA assays using nasopharyngeal/nasal swabs in universal transport media (UTM) and sputum and tracheal aspirates","abstract":"In March 2019 the outbreak of SARS-CoV 2 was officially defined as a pandemic by the World Health Organization and shortly after, the United States Food and Drug Administration (FDA) granted Emergency Use Authorization (EUA) to the Centers for Disease Control (CDC) for reverse transcription polymerase chain reaction (rtPCR) molecular testing for the detection of the SARS-CoV-2 virus from NP swabs. Since then, EUA with relaxed regulations were granted to numerous manufacturers and clinical microbiology laboratories to implement in-house testing assays with nasopharyngeal swabs (NP) and subsequently additional specimen types. Because of supply chain shortages leading to competition for reagents, sustaining any significant volume of testing soon became problematic. As a countermeasure, within several weeks the Henry Ford Microbiology Laboratory validated 4 different rtPCR assays and multiple specimen types using NeuMoDX, Diasorin Simplexa, Cepheid and Roche platforms. The purpose of this study was to analyze the analytic sensitivity of these rtPCR assays with NP/nasal swabs and sputum/tracheal aspirates. Qualitative analytic agreement between the 4 platforms for NP/nasal swabs ranged 95% - 100% overall with no statistically significant difference in threshold cT values. Similar results were obtained with the sputum/tracheal aspirates. These data demonstrate the high accuracy and reproducibility in detection of SARS-CoV 2 between the rtPCR assays performed on 4 different platforms with numerous specimen types.","version":"1.1","doi":"10.1101/2020.05.26.118190","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.071027","pub_date":"2020-5-28","title":"Insurgence and worldwide diffusion of genomic variants in SARS-CoV-2 genomes","abstract":"The SARS-CoV-2 pandemic that we are currently experiencing is exerting a massive toll both in human lives and economic impact. One of the challenges we must face is to try to understand if and how different variants of the virus emerge and change their frequency in time. Such information can be extremely valuable as it may indicate shifts in aggressiveness, and it could provide useful information to trace the spread of the virus in the population. In this work we identified and traced over time 7 amino acid variants that are present with high frequency in Italy and Europe, but that were absent or present at very low frequencies during the first stages of the epidemic in China and the initial reports in Europe. The analysis of these variants helps defining 6 phylogenetic clades that are currently spreading throughout the world with changes in frequency that are sometimes very fast and dramatic. In the absence of conclusive data at the time of writing, we discuss whether the spread of the variants may be due to a prominent founder effect or if it indicates an adaptive advantage.","version":"1.2","doi":"10.1101/2020.04.30.071027","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.118208","pub_date":"2020-5-28","title":"Characterization of accessory genes in coronavirus genomes","abstract":"The Covid19 infection is caused by the SARS-CoV-2 virus, a novel member of the coronavirus (CoV) family. CoV genomes code for a ORF1a / ORF1ab polyprotein and four structural proteins widely studied as major drug targets. The genomes also contain a variable number of open reading frames (ORFs) coding for accessory proteins that are not essential for virus replication, but appear to have a role in pathogenesis. The accessory proteins have been less well characterized and are difficult to predict by classical bioinformatics methods. We propose a computational tool GOFIX to characterize potential ORFs in virus genomes. In particular, ORF coding potential is estimated by searching for enrichment in motifs of the X circular code, that is known to be over-represented in the reading frames of viral genes. We applied GOFIX to study the SARS-CoV-2 and related genomes including SARS-CoV and SARS-like viruses from bat, civet and pangolin hosts, focusing on the accessory proteins. Our analysis provides evidence supporting the presence of overlapping ORFs 7b, 9b and 9c in all the genomes and thus helps to resolve some differences in current genome annotations. In contrast, we predict that ORF3b is not functional in all genomes. Novel putative ORFs were also predicted, including a truncated form of the ORF10 previously identified in SARS-CoV-2 and a little known ORF overlapping the Spike protein in Civet-CoV and SARS-CoV. Our findings contribute to characterizing sequence properties of accessory genes of SARS coronaviruses, and especially the newly acquired genes making use of overlapping reading frames.","version":"1.1","doi":"10.1101/2020.05.26.118208","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.28.120667","pub_date":"2020-5-28","title":"Evaluation of commercial qPCR kits for detection of SARS-CoV-2 in pooled samples","abstract":"Due to the current pandemic, global shortage of reagents has drawn interest in developing alternatives to increase the number coronavirus tests. One such alternative is sample pooling. Here we compared commercial kits that are used in COVID-19 diagnostics, in terms of sensitivity and feasibility for use in pooling. We showed that pooling of up to 60 samples did not affect the efficiency of the kits. Also, the RNA dependent RNA polymerase (RdRp) is a more suitable target in pooled samples than the Envelope (E) protein. This approach could provide an easy method of screening large number of samples and help adjust different government regulations.","version":"1.1","doi":"10.1101/2020.05.28.120667","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.26.063032","pub_date":"2020-5-28","title":"Coronavirus, as a source of pandemic pathogens","abstract":"The coronavirus and the influenza virus have similarities and differences. In order to comprehensively compare them, their genome sequencing data were examined by principal component analysis. Variations in coronavirus were smaller than those in a subclass of the influenza virus. In addition, differences among coronaviruses in a variety of hosts were small. These characteristics may have facilitated the infection of different hosts. Although many of the coronaviruses were more conservative, those repeatedly found among humans showed annual changes. If SARS-CoV-2 changes its genome like the Influenza H type, it will repeatedly spread every few years. In addition, the coronavirus family has many other candidates for subsequent pandemics. The genome data of coronavirus were compared to influenza virus, to investigate its spreading mechanism and future status. Coronavirus would repeatedly spread every few years. In addition, the coronavirus family has many other candidates for subsequent pandemics.","version":"1.3","doi":"10.1101/2020.04.26.063032","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108605","pub_date":"2020-5-28","title":"Mechanistic insights into ventricular arrhythmogenesis of hydroxychloroquine and azithromycin for the treatment of COVID-19","abstract":"We investigate mechanisms for potential pro-arrhythmic effects of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM), in Covid-19 management supplementing the limited available experimental cardiac safety data. We integrated patch-clamp studies utilizing In Vitro ProArrhythmia Assay (CiPA) Schema IC50 paradigms, molecular modelling, cardiac multi-electrode array and voltage (RH237) mapping, ECG studies, and Ca2+ (Rhod-2 AM) mapping in isolated Langendorff-perfused guinea-pig hearts with human in-silico ion current modelling. HCQ blocked IKr and IK1 with IC50s (10\u00b10.6 and 34\u00b15.0 \u03bcM) within clinical therapeutic ranges, INa and ICaL at higher IC50s, leaving Ito and IKs unaffected. AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, sparing IK1 and Ito. HCQ+AZM combined inhibited IKr and IK1 with IC50s of 7.7\u00b10.8 \u03bcM and 30.4\u00b13.0 \u03bcM, sparing INa, ICaL and Ito. Molecular modelling confirmed potential HCQ binding to hERG. HCQ slowed heart rate and ventricular conduction. It prolonged PR, QRS and QT intervals, and caused prolonged, more heterogeneous, action potential durations and intracellular Ca2+ transients. These effects were accentuated with combined HCQ+AZM treatment, which then elicited electrical alternans, re-entrant circuits and wave break. Modelling studies attributed these to integrated HCQ and AZM actions reducing IKr and IK1, thence altering cell Ca2+ homeostasis. Combined HCQ+AZM treatment exerts pro-arrhythmic ventricular events by synergetically inhibiting IKr, IKs with resulting effects on cellular Ca2+ signalling, and action potential propagation and duration. These findings provide an electrophysiological basis for recent FDA cardiac safety guidelines cautioning against combining HCQ/AZM when treating Covid-19.","version":"1.3","doi":"10.1101/2020.05.21.108605","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.118893","pub_date":"2020-5-27","title":"Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling","abstract":"Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate. Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies. Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds. This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19.","version":"1.1","doi":"10.1101/2020.05.27.118893","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.115261","pub_date":"2020-5-27","title":"Angiotensin-converting enzyme 2, a SARS-CoV-2 receptor, is upregulated by interleukin-6 via STAT3 signaling in rheumatoid synovium","abstract":"Detected in December 2019, the coronavirus disease 2019 (COVID-19) has since spread all over the world, resulting in a global pandemic. The disease is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), and its symptoms usually include cough, fever, and gastrointestinal problems. Although the prevalence of rheumatoid arthritis (RA) is about 1 % of the global population and RA patients naturally have a chance of acquiring COVID-19 in this pandemic, no studies have considered the expression of angiotensin-converting enzyme 2 (ACE2) (a receptor for SARS-CoV-2) in synovial tissues. Our presenting data revealed that ACE2 expression was elevated in active rheumatoid synovium, and siRNA against STAT3 was able to downregulate ACE2 expression, which was in turn induced by IL-6 signaling.","version":"1.1","doi":"10.1101/2020.05.26.115261","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.115832","pub_date":"2020-5-27","title":"Different pattern of pre-existing SARS-COV-2 specific T cell immunity in SARS-recovered and uninfected individuals","abstract":"Memory T cells induced by previous infections can influence the course of new viral infections. Little is known about the pattern of SARS-CoV-2 specific pre-existing memory T cells in human. Here, we first studied T cell responses to structural (nucleocapsid protein, NP) and non-structural (NSP-7 and NSP13 of ORF1) regions of SARS-CoV-2 in convalescent from COVID-19 (n=24). In all of them we demonstrated the presence of CD4 and CD8 T cells recognizing multiple regions of the NP protein. We then show that SARS-recovered patients (n=23), 17 years after the 2003 outbreak, still possess long-lasting memory T cells reactive to SARS-NP, which displayed robust cross-reactivity to SARS-CoV-2 NP. Surprisingly, we observed a differential pattern of SARS-CoV-2 specific T cell immunodominance in individuals with no history of SARS, COVID-19 or contact with SARS/COVID-19 patients (n=18). Half of them (9/18) possess T cells targeting the ORF-1 coded proteins NSP7 and 13, which were rarely detected in COVID-19- and SARS-recovered patients. Epitope characterization of NSP7-specific T cells showed recognition of protein fragments with low homology to \u201ccommon cold\u201d human coronaviruses but conserved among animal betacoranaviruses. Thus, infection with betacoronaviruses induces strong and long-lasting T cell immunity to the structural protein NP. Understanding how pre-existing ORF-1-specific T cells present in the general population impact susceptibility and pathogenesis of SARS-CoV-2 infection is of paramount importance for the management of the current COVID-19 pandemic.","version":"1.1","doi":"10.1101/2020.05.26.115832","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.118117","pub_date":"2020-5-27","title":"Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease","abstract":"COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.","version":"1.1","doi":"10.1101/2020.05.27.118117","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.119255","pub_date":"2020-5-27","title":"Head-to-head comparison of four antigen-based rapid detection tests for the diagnosis of SARS-CoV-2 in respiratory samples","abstract":"In the context of the Covid-19 pandemic, the development and validation of rapid and easy-to-perform diagnostic methods are of high priority. We compared the performance of four rapid antigen detection tests for SARS-CoV-2 in respiratory samples. Immunochromatographic SARS-CoV-2 assays from RapiGEN, Liming bio, Savant, and Bioeasy were evaluated using universal transport medium containing naso-oropharyngeal swabs from suspected Covid-19 cases. The diagnostic accuracy was determined in comparison to SARS-CoV-2 RT-PCR. A total of 111 samples were included; 80 were RT-PCR positive. Median patients\u2019 age was 40 years, 55% were female, and 88% presented within the first week after symptom onset. The evaluation of the Liming bio assay was discontinued due to insufficient performance. The overall sensitivity values of RapiGEN, Liming bio, and Bioeasy tests were 62.0% (CI95% 51.0\u201371.9), 16.7% (CI95% 10.0\u201326.5), and 85.0% (CI95% 75.6\u201391.2), respectively, with specificities of 100%. Sensitivity was significantly higher in samples with high viral loads (RapiGEN, 84.9%; Bioeasy, 100%). The study highlighted the significant heterogeneity of test performance among evaluated assays, which might have been influenced by the use of a non-validated sample material. The high sensitivity of some tests demonstrated that rapid antigen detection has the potential to serve as an alternative diagnostic method, especially in patients presenting with high viral loads in early phases of infection. This is particularly important in situations with limited access to RT-PCR or prolonged turnaround time. Further comparative evaluations are necessary to select products with high performance among the growing market of diagnostic tests for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.27.119255","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.117549","pub_date":"2020-5-27","title":"Development and validation of IMMUNO-COV\u2122: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2","abstract":"We here describe the development and validation of IMMUNO-COV\u2122, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-\u039419CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-\u039419CT infection was blocked by monoclonal \u03b1-SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of \u03b1-SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNTEC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV\u2122 assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.","version":"1.1","doi":"10.1101/2020.05.26.117549","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.118752","pub_date":"2020-5-27","title":"SARS-CoV-2 envelope protein topology in eukaryotic membranes","abstract":"Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and establish the basis to tackle the pathogenesis of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.27.118752","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.119610","pub_date":"2020-5-27","title":"Assessment of ACE2, CXCL10 and Their co-expressed Genes: An In-silico Approach to Evaluate the Susceptibility and Fatality of Lung Cancer Patients towards COVID-19 Infection","abstract":"COVID-19 is a recent pandemic that started to spread out worldwide from Wuhan, China. This disease is caused by a newly discovered strain of the coronavirus, namely SARS CoV-2. Lung cancer patients are reported to be more susceptible to COVID-19 infection. To evaluate the probable reasons behind the excessive susceptibility and fatality of lung cancer patients to COVID-19 infection, we targeted two most crucial biomarkers of COVID-19, ACE2 and CXCL10. ACE2 plays a vital role in the SARS CoV-2 entry into the host cell while CXCL10 is a cytokine mainly responsible for the lung cell damage involving in a cytokine storm. Firstly, we used the TIMER, UALCAN and GEPIA2 databases to analyze the expression and correlation of ACE2 and CXCL10 in LUAD and LUSC. After that, using the cBioPortal database, we performed an analytical study to determine the genetic changes in ACE2 and CXCL10 protein sequences that are responsible for lung cancer development. Finally, we analyzed different functional approaches of ACE2, CXCL10 and their co-expressed genes associated with lung cancer and COVID-19 development by using the PANTHER database. Initially, we observed that ACE2 and CXCL10 are mostly overexpressed in LUAD and LUSC. We also found the functional significance of ACE2 and CXCL10 in lung cancer development by determining the genetic alteration frequency in their amino acid sequences. Lastly, by doing the functional assessment of the targeted genes, we identified that ACE2 and CXCL10 along with their commonly co-expressed genes are respectively involved in the binding activity and immune responses in case of lung cancer and COVID-19 infection. Finally, on the basis of this systemic analysis, we came to the conclusion that ACE2 and CXCL10 are possible biomarkers responsible for the higher susceptibility and fatality of lung cancer patients towards the COVID-19.","version":"1.1","doi":"10.1101/2020.05.27.119610","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.097501","pub_date":"2020-5-27","title":"Traffic-derived particulate matter and angiotensin-converting enzyme 2 expression in human airway epithelial cells","abstract":"The mechanism for the association between traffic-derived particulate matter less than 10 microns (PM10) and cases of COVID-19 disease reported in epidemiological studies is unknown. To infect cells, the spike protein of SARS-CoV-2 interacts with angiotensin-converting enzyme 2 (ACE2) on host airway cells. Increased ACE2 expression in lower airway cells in active smokers, suggests a potential mechanism whereby PM10 increases vulnerability to COVID-19 disease. To assess the effect of traffic-derived PM10 on human airway epithelial cell ACE2 expression in vitro. PM10 was collected from Marylebone Road (London) using a kerbside impactor. A549 and human primary nasal epithelial cells were cultured with PM10 for 2 h, and ACE2 expression (median fluorescent intensity; MFI) assessed by flow cytometry. We included cigarette smoke extract as a putative positive control. Data were analysed by either Mann-Whitney test, or Kruskal-Wallis with Dunn\u2019s multiple comparisons test. PM10 at 10 \u03bcg/mL, and 20 \u03bcg/mL increased ACE2 expression in A549 cells (P<0.05, 0.01 vs. medium control, respectively). Experiments using a single PM10 concentration (10 \u03bcg/mL), found increased ACE2 expression in both A549 cells (control vs. PM10, median (IQR) MFI; 470 (0.1 to 1114) vs 6217 (5071 to 8506), P<0.01), and in human primary epithelial cells (0 (0 to 591) vs. 4000 (2610 to 7853), P<0.05). Culture of A549 cells with 5% cigarette smoke extract increased ACE2 expression (n=4, 0 (0 to 28) vs. 9088 (7557 to 15831, P<0.05). Traffic-related PM10 increases the expression of the receptor for SARS-CoV-2 in human respiratory epithelial cells.","version":"1.2","doi":"10.1101/2020.05.15.097501","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029447","pub_date":"2020-5-27","title":"Role of RNA Guanine Quadruplexes in Favoring the Dimerization of SARS Unique Domain in Coronaviruses","abstract":"Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible of a global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting SARS viruses to bypass the host protective response. This work also constitutes a first step of the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.","version":"1.2","doi":"10.1101/2020.04.07.029447","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.118133","pub_date":"2020-5-27","title":"Assessment of sample pooling for clinical SARS-CoV-2 testing","abstract":"Accommodating large increases in sample workloads has presented one of the biggest challenges to clinical laboratories during the COVID-19 pandemic. Despite the implementation of new automated detection systems, and previous efficiencies such as barcoding, electronic data transfer and extensive robotics, throughput capacities have struggled to meet the demand. Sample pooling has been suggested as an additional strategy to further address this need. The greatest concern with this approach in a clinical setting is the potential for reduced sensitivity, particularly the risk of false negative results when weak positive samples are pooled. To investigate this possibility, detection rates in pooled samples were evaluated, with extensive assessment of pools containing weak positive specimens. Additionally, the frequency of occurrence of weak positive samples across ten weeks of the pandemic were reviewed. Weak positive specimens were detected in all five-sample pools but failed to be detected in four of the 24 nine-sample pools tested. Weak positive samples comprised an average 16.5% of the positive specimens tested during the pandemic thus far, slightly increasing in frequency during later weeks. Other aspects of the testing process should be considered, however, such as accessioning and reporting, which are not streamlined and may be complicated by pooling procedures. Therefore, the impact on the entire laboratory process needs to be carefully assessed prior to implementing such a strategy.","version":"1.1","doi":"10.1101/2020.05.26.118133","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.27.118414","pub_date":"2020-5-27","title":"Finding disease modules for cancer and COVID-19 in gene co-expression networks with the Core&Peel method","abstract":"Diseases imply dysregulation of cell\u2019s functions at several levels. The study of differentially expressed genes in case-control cohorts of patients is often the first step in understanding the details of the cell\u2019s dysregulation. A further level of analysis is introduced by noticing that genes are organized in functional modules (often called pathways), thus their action and their dysregulation may be better understood by the identification of the modules most affected by the disease (aka disease modules, or active subnetworks). We describe how an algorithm based on the Core&Peel method developed originally for detecting protein complexes in PPI networks, can be adapted to detect disease modules in co-expression networks of genes. We first validate Core&Peel for the easier general task of functional module detection by comparison with 42 methods participating in the Disease Module Identification DREAM challenge of 2019. Next, we use four specific disease test cases (colorectal cancer, prostate cancer, asthma and rheumatoid arthritis), four state-of-the-art algorithms (ModuleDiscoverer, Degas, KeyPathwayMiner and ClustEx), and several pathway databases to validate the proposed algorithm. Core&Peel is the only method able to find significant associations of the predicted disease module with known validated relevant pathways for all four diseases. Moreover for the two cancer data sets, Core&Peel detects further nine relevant pathways enriched in the predicted disease module, not discovered by the other methods used in the comparative analysis. Finally we apply Core&Peel, along with other methods, to explore the transcriptional response of human cells to SARS-CoV-2 infection, at a modular level, aiming at finding supporting evidence for drug repositioning efforts.","version":"1.1","doi":"10.1101/2020.05.27.118414","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.116780","pub_date":"2020-5-26","title":"Study of cell to cell transmission of SARS CoV 2 virus particle using gene network from microarray data","abstract":"Microarray data from SARS CoV2 patients helps to construct a gene network relating to this disease. Analysis of these genes, present in network, highlights their biological functions and related cellular pathways. With the assistance of these information, a drug(s) can be identified to treat COVID-19. A detailed analysis of the human host response to SARS CoV 2 with expression profiling by high throughput sequencing has executed with primary human lung epithelium cell lines. Clustered genes annotation and gene network construction are performed with the help of String database. Among four cluster of genes from microarray data, cluster 1 is identified as basal cells with p= 1.37e\u221205 with 44 genes. Functional enrichment analysis of genes present in gene network has completed using String database, ToppFun online tool and NetworkAnalyst tool. For SARS CoV2 virus particles, keratin proteins, which are part of cytoskeleton structure of host cell, play a major role in cell to cell virus particle transmission. Among three types of cell- cell communication, only anchoring junction between basal cell membrane and basal lamina, is involved in this virus transmission. In this junction point, hemidesmosome structure play a vital role in virus spread from one cell to basal lamina in respiratory tract. In this protein complex structure, keratin, integrin and laminin proteins of host cell is used to promote the spread of virus infection into extracellular matrix. So, small molecular blockers of different anchoring junction proteins i.e. ITGA3, ITGA2 can provide efficient protection against this deadly virus disease. Understanding the human host response against this virus is very important to develop novel therapeutics for the treatment of SARS-CoV 2.","version":"1.1","doi":"10.1101/2020.05.26.116780","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.117069","pub_date":"2020-5-26","title":"SARS-CoV-2 genome evolution exposes early human adaptations","abstract":"The set of mutations observed at the outset of the SARS-CoV-2 pandemic may illuminate how the virus will adapt to humans as it continues to spread. Viruses are expected to quickly acquire beneficial mutations upon jumping to a new host species. Advantageous nucleotide substitutions can be identified by their parallel occurrence in multiple independent lineages and are likely to result in changes to protein sequences. Here we show that SARS-CoV-2 is acquiring mutations more slowly than expected for neutral evolution, suggesting purifying selection is the dominant mode of evolution during the initial phase of the pandemic. However, several parallel mutations arose in multiple independent lineages and may provide a fitness advantage over the ancestral genome. We propose plausible reasons for several of the most frequent mutations. The absence of mutations in other genome regions suggests essential components of SARS-CoV-2 that could be the target of drug development. Overall this study provides genomic insights into how SARS-CoV-2 has adapted and will continue to adapt to humans. In this study we sought signals of evolution to identify how the SARS-CoV-2 genome has adapted at the outset of the COVID-19 pandemic. We find that the genome is largely undergoing purifying selection that maintains its ancestral sequence. However, we identified multiple positions on the genome that appear to confer an adaptive advantage based on their repeated evolution in independent lineages. This information indicates how SARS-CoV-2 will evolve as it diversifies in an increasing number of hosts.","version":"1.1","doi":"10.1101/2020.05.26.117069","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.25.115774","pub_date":"2020-5-26","title":"Alveolar early progenitors in the aged human lung have increased expression of ACE2 accompanied with genes involved in beta-amyloid clearance: Indication of SARS-CoV-2 also using soluble ACE2 in aged-lungs to enter ACE2-negative cells","abstract":"COVID-19 is the current pandemic caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2) that uses ACE2 protein on the cell surface. By analyzing publicly available datasets, I uncovered that alveolar early progenitors (AEP), a subset of the type-2 pneumocytes, showed increased ACE2 expression in the older lungs. AEPs co-express TMPRSS2, CTSL. Aged AEP-gene expression signature suggested an active response to beta-amyloid-induced ACE2 shedding, to limit the intercellular beta-amyloid accumulation in otherwise healthy human lungs. Susceptibility of AEP to SARS-CoV2 and ACE2 secretory capacity of these cells makes aged human lung sensitive for rapid-infection, by a possible in-solution ACE2 binding and entry into ACE2-negative cells, thereby increasing the target cell diversity and numbers. Single-cell analysis of COVID19 patients with moderate and severe infections, clearly showed that severe infections showed SARS-CoV-2 transcript in ACE2-negative TMPRSS-negative but CTSL-positive cell types in their bronchoalveolar lavage fluid, validating in-solution ACE2-binding enabling infection.","version":"1.1","doi":"10.1101/2020.05.25.115774","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.25.060947","pub_date":"2020-5-26","title":"MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples","abstract":"The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for the understanding of viral spread and viral evolution, as well as for vaccine development. Existing sample preparation methods for viral genome sequencing are demanding on user technique and time, and thus not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We have developed MetagenomIc RNA EnRichment VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. This approach uses direct tagmentation of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library construction process, while subsequent targeted enrichment can generate viral genomes with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA is compatible with clinical nucleic extracts containing carrier RNA. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations during outbreaks, both current and future.","version":"1.2","doi":"10.1101/2020.04.25.060947","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.25.114199","pub_date":"2020-5-26","title":"The novel Coronavirus enigma: Phylogeny and mutation analyses of SARS-CoV-2 viruses circulating in India during early 2020","abstract":"This is a comprehensive analysis of 46 Indian SARS-CoV-2 genome sequences available from the NCBI and GISAID repository during early 2020. Evolutionary dynamics, gene-specific phylogeny and emergence of the novel co-evolving mutations in nine structural and non-structural genes among circulating SARS-CoV-2 strains in ten states of India have been assessed. 46 SARS-CoV-2 nucleotide sequences submitted from India were downloaded from the GISAID (39/46) or from NCBI (7/46) database. Phylogenetic study and analyses of mutation were based on the nine structural and non-structural genes of SARS-CoV-2 strains. Secondary structure of RdRP/NSP12 protein was predicted with respect to the novel A97V mutation. Phylogenetic analyses revealed the evolution of \u201cgenome-type clusters\u201d and adaptive selection of \u201cL\u201d type SARS-CoV-2 strains with genetic closeness to the bat SARS-like coronaviruses than pangolin or MERS-CoVs. With regards to the novel co-evolving mutations, 2 groups are seen to circulate in India at present: the \u201cmajor group\u201d (52.2%) and the \u201cminor group\u201d (30.4%), harboring four and five co-existing mutations, respectively. The \u201cmajor group\u201d mutations fall in the A2a clade. All the minor group mutations, except 11083G>T (L37F, NSP6) were unique to the Indian isolates. The study highlights rapidly evolving SARS-CoV-2 virus and co-circulation of multiple clades and sub-clades, driving this pandemic worldwide. This comprehensive study is a potential resource for monitoring the novel mutations in the viral genome, changes in viral pathogenesis, for designing vaccines and other therapeutics.","version":"1.1","doi":"10.1101/2020.05.25.114199","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.115790","pub_date":"2020-5-26","title":"An Immunoinformatics Study to Predict Epitopes in the Envelope Protein of SARS-COV-2","abstract":"COVID-19 is a new viral emergent human disease caused by a novel strain of Coronavirus. This virus has caused a huge problem in the world as millions of the people are affected with this disease in the entire world. We aimed to design a peptide vaccine for COVID-19 particularly for the envelope protein using computational methods to predict epitopes inducing the immune system and can be used later to create a new peptide vaccine that could replace conventional vaccines. A total of available 370 sequences of SARS-CoV-2 were retrieved from NCBI for bioinformatics analysis using Immune Epitope Data Base (IEDB) to predict B and T cells epitopes. Then we docked the best predicted CTL epitopes with HLA alleles. CTL cell epitopes namely interacted with MHC class I alleles and we suggested them to become universal peptides based vaccine against COVID-19. Potentially continuous B cell epitopes were predicted using tools from IEDB. The Allergenicity of predicted epitopes was analyzed by AllerTOP tool and the coverage was determined throughout the worlds. We found these CTL epitopes to be T helper epitopes also. The B cell epitope, SRVKNL and T cell epitope, FLAFVVFLL were suggested to become a universal candidate for peptide-based vaccine against COVID-19. We hope to confirm our findings by adding complementary steps of both in vitro and in vivo studies to support this new universal predicted candidate.","version":"1.1","doi":"10.1101/2020.05.26.115790","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.066977","pub_date":"2020-5-26","title":"Controlling the SARS-CoV-2 outbreak, insights from large scale whole genome sequences generated across the world","abstract":"SARS-CoV-2 most likely evolved from a bat beta-coronavirus and started infecting humans in December 2019. Since then it has rapidly infected people around the world, with more than 4.5 million confirmed cases by the middle of May 2020. Early genome sequencing of the virus has enabled the development of molecular diagnostics and the commencement of therapy and vaccine development. The analysis of the early sequences showed relatively few evolutionary selection pressures. However, with the rapid worldwide expansion into diverse human populations, significant genetic variations are becoming increasingly likely. The current limitations on social movement between countries also offers the opportunity for these viral variants to become distinct strains with potential implications for diagnostics, therapies and vaccines. We used the current sequencing archives (NCBI and GISAID) to investigate 15,487 whole genomes, looking for evidence of strain diversification and selective pressure. We used 6,294 SNPs to build a phylogenetic tree of SARS-CoV-2 diversity and noted strong evidence for the existence of two major clades and six sub-clades, unevenly distributed across the world. We also noted that convergent evolution has potentially occurred across several locations in the genome, showing selection pressures, including on the spike glycoprotein where we noted a potentially critical mutation that could affect its binding to the ACE2 receptor. We also report on mutations that could prevent current molecular diagnostics from detecting some of the sub-clades. The worldwide whole genome sequencing effort is revealing the challenge of developing SARS-CoV-2 containment tools suitable for everyone and the need for data to be continually evaluated to ensure accuracy in outbreak estimations.","version":"1.3","doi":"10.1101/2020.04.28.066977","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.26.116608","pub_date":"2020-5-26","title":"Structure, function and variants analysis of the androgen-regulated TMPRSS2, a drug target candidate for COVID-19 infection","abstract":"SARS-CoV-2 is a novel virus causing mainly respiratory, but also gastrointestinal symptoms. Elucidating the molecular processes underlying SARS-CoV-2 infection, and how the genetic background of an individual is responsible for the variability in clinical presentation and severity of COVID-19 is essential in understanding this disease. Cell infection by the SARS-CoV-2 virus requires binding of its Spike (S) protein to the ACE2 cell surface protein and priming of the S by the serine protease TMPRSS2. One may expect that genetic variants leading to a defective TMPRSS2 protein can affect SARS-CoV-2 ability to infect cells. We used a range of bioinformatics methods to estimate the prevalence and pathogenicity of TMPRSS2 genetic variants in the human population, and assess whether TMPRSS2 and ACE2 are co-expressed in the intestine, similarly to what is observed in lungs. We generated a 3D structural model of the TMPRSS2 extracellular domain using the prediction server Phyre and studied 378 naturally-occurring TMPRSS2 variants reported in the GnomAD database. One common variant, p.V160M (rs12329760), is predicted damaging by both SIFT and PolyPhen2 and has a MAF of 0.25. Valine 160 is a highly conserved residue within the SRCS domain. The SRCS is found in proteins involved in host defence, such as CD5 and CD6, but its role in TMPRSS2 remains unknown. 84 rare variants (53 missense and 31 leading to a prematurely truncated protein, cumulative minor allele frequency (MAF) 7.34\u00d710\u22124) cause structural destabilization and possibly protein misfolding, and are also predicted damaging by SIFT and PolyPhen2 prediction tools. Moreover, we extracted gene expression data from the human protein atlas and showed that both ACE2 and TMPRSS2 are expressed in the small intestine, duodenum and colon, as well as the kidneys and gallbladder. The implications of our study are that: i. TMPRSS2 variants, in particular p.V160M with a MAF of 0.25, should be investigated as a possible marker of disease severity and prognosis in COVID-19 and ii. in vitro validation of the co-expression of TMPRSS2 and ACE2 in gastro-intestinal is warranted.","version":"1.1","doi":"10.1101/2020.05.26.116608","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.24.113043","pub_date":"2020-5-25","title":"Mass Spectrometric detection of SARS-CoV-2 virus in scrapings of the epithelium of the nasopharynx of infected patients via Nucleocapsid N protein","abstract":"Detection of viral RNA by PCR is currently the main diagnostic tool for COVID-19 [1]. The PCR-based test, however, shows limited sensitivity, especially at early and late stages of the disease development [2,3], and is relatively time consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemia conditions. Mass-spectrometry is one of such possibilities. We have developed a mass-spectrometry based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs, based on the detection of the viral nucleocapsid N protein. The N protein of the SARS-COV-2 virus, the most abundant protein in the virion, is the best candidate for mass-spectrometric detection of the infection, and MS-based detection of several peptides from the SARS-COoV-2 nucleoprotein has been reported earlier by the Sinz group [4]. Our approach shows confident identification of the N protein in patient samples even with the lowest viral loads and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and adding of isopropanol, and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.","version":"1.1","doi":"10.1101/2020.05.24.113043","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.24.096164","pub_date":"2020-5-25","title":"COVIEdb: A database for potential immune epitopes of coronaviruses","abstract":"2019 novel coronavirus (2019-nCoV) has caused large-scale pandemic COVID-19 all over the world. It\u2019s essential to find out which parts of the 2019-nCoV sequence are recognized by human immune system for vaccine development. And for the prevention of the potential outbreak of similar coronaviruses in the future, vaccines against immunogenic epitopes shared by different human coronaviruses are essential. Here we predict all the potential B/T-cell epitopes for SARS-CoV, MERS-CoV, 2019-nCoV and RaTG13-CoV based on the protein sequences. We found YFKYWDQTY in ORF1ab protein, VYDPLQPEL and TVYDPLQPEL in spike (S) protein might be pan-coronavirus targets for vaccine development. All the predicted results are stored in a database COVIEdb (http://biopharm.zju.edu.cn/coviedb/).","version":"1.1","doi":"10.1101/2020.05.24.096164","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.109900","pub_date":"2020-5-25","title":"Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses","abstract":"Viruses are the major aetiological agents of acute and chronic severe human diseases that place a tremendous burden on global public health and economy; however, for most viruses, effective prophylactics and therapeutics are lacking, in particular, broad-spectrum antiviral agents. Herein, we identified 2 secreted bacterial lipases from a Chromobacterium bacterium, named Chromobacterium antiviral effector-1 (CbAE-1) and CbAE-2, with a broad-spectrum virucidal activity against dengue virus (DENV), Zika virus (ZIKV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The CbAEs potently blocked viral infection in the extracellular milieu through their lipase activity. Mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. A mutation of CbAE-1 in its lipase motif fully abrogated the virucidal ability. Furthermore, CbAE-2 presented low toxicity in vivo and in vitro, highlighting its potential as a broad-spectrum antiviral drug.","version":"1.2","doi":"10.1101/2020.05.22.109900","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.25.114884","pub_date":"2020-5-25","title":"COVID-Align: Accurate online alignment of hCoV-19 genomes using a profile HMM","abstract":"The first cases of the COVID-19 pandemic emerged in December 2019. Until the end of February 2020, the number of available genomes was below 1,000, and their multiple alignment was easily achieved using standard approaches. Subsequently, the availability of genomes has grown dramatically. Moreover, some genomes are of low quality with sequencing/assembly errors, making accurate re-alignment of all genomes nearly impossible on a daily basis. A more efficient, yet accurate approach was clearly required to pursue all subsequent bioinformatics analyses of this crucial data. hCoV-19 genomes are highly conserved, with very few indels and no recombination. This makes the profile HMM approach particularly well suited to align new genomes, add them to an existing alignment and filter problematic ones. Using a core of \u223c2,500 high quality genomes, we estimated a profile using HMMER, and implemented this profile in COVID-Align, a user-friendly interface to be used online or as standalone via Docker. The alignment of 1,000 genomes requires less than 20mn on our cluster. Moreover, COVID-Align provides summary statistics, which can be used to determine the sequencing quality and evolutionary novelty of input genomes (e.g. number of new mutations and indels). https://covalign.pasteur.cloud, hub.docker.com/r/evolbioinfo/covid-align olivier.gascuel@pasteur.fr, frederic.lemoine@pasteur.fr Supplementary information is available at Bioinformatics online.","version":"1.1","doi":"10.1101/2020.05.25.114884","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.24.111823","pub_date":"2020-5-24","title":"The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I","abstract":"SARS-CoV-2 infection have caused global pandemic and claimed over 5,000,000 tolls. Although the genetic sequences of their etiologic viruses are of high homology, the clinical and pathological characteristics of COVID-19 significantly differ from SARS. Especially, it seems that SARS-CoV-2 undergoes vast replication in vivo without being effectively monitored by anti-viral immunity. Here, we show that the viral protein encoded from open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all the viral proteins, can directly interact with MHC-I molecules and significantly down-regulates their surface expression on various cell types. In contrast, ORF8a and ORF8b of SARS-CoV do not exert this function. In the ORF8-expressing cells, MHC-I molecules are selectively target for lysosomal degradation by an autophagy-dependent mechanism. As a result, CTLs inefficiently eliminate the ORF8-expressing cells. Our results demonstrate that ORF8 protein disrupts antigen presentation and reduces the recognition and the elimination of virus-infected cells by CTLs. Therefore, we suggest that the inhibition of ORF8 function could be a strategy to improve the special immune surveillance and accelerate the eradication of SARS-CoV-2 in vivo.","version":"1.1","doi":"10.1101/2020.05.24.111823","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.24.113175","pub_date":"2020-5-24","title":"Structural basis of SARS-CoV-2 spike protein induced by ACE2","abstract":"The recent emergence of the novel SARS-coronavirus 2 (SARS-CoV-2) and its international spread pose a global health emergency. The viral spike (S) glycoprotein binds the receptor (angiotensin-converting enzyme 2) ACE2 and promotes SARS-CoV-2 entry into host cells. The trimeric S protein binds the receptor using the distal receptor-binding domain (RBD) causing conformational changes in S protein that allow priming by host cell proteases. Unravelling the dynamic structural features used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal novel therapeutic targets. Using structures determined by X-ray crystallography and cryo-EM, we performed structural analysis and atomic comparisons of the different conformational states adopted by the SARS-CoV-2-RBD. Here, we determined the key structural components induced by the receptor and characterized their intramolecular interactions. We show that \u03ba-helix (also known as polyproline II) is a predominant structure in the binding interface and in facilitating the conversion to the active form of the S protein. We demonstrate a series of conversions between switch-like \u03ba-helix and \u03b2-strand, and conformational variations in a set of short \u03b1-helices which affect the proximal hinge region. This conformational changes lead to an alternating pattern in conserved disulfide bond configurations positioned at the hinge, indicating a possible disulfide exchange, an important allosteric switch implicated in viral entry of various viruses, including HIV and murine coronavirus. The structural information presented herein enables us to inspect and understand the important dynamic features of SARS-CoV-2-RBD and propose a novel potential therapeutic strategy to block viral entry. Overall, this study provides guidance for the design and optimization of structure-based intervention strategies that target SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.24.113175","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108308","pub_date":"2020-5-23","title":"Systemic and mucosal antibody secretion specific to SARS-CoV-2 during mild versus severe COVID-19","abstract":"Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an acute illness termed coronavirus disease 2019 (COVID-19). Humoral immune responses likely play an important role in containing SARS-CoV-2, however, the determinants of SARS-CoV-2-specific antibody responses are unclear. Using immunoassays specific for the SARS-CoV-2 spike protein, we determined SARS-CoV-2-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) in sera and mucosal fluids of two cohorts, including patients with quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR)-confirmed SARS-CoV-2 infection (n = 56; median age 61 years) with mild versus severe COVID-19, and SARS-CoV-2-exposed healthcare workers (n = 109; median age 36 years) with or without symptoms and tested negative or positive by RT-qPCR. On average, SARS-CoV-2-specific serum IgA titers in mild COVID-19 cases became positive eight days after symptom onset and were often transient, whereas serum IgG levels remained negative or reached positive values 9\u201310 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers as a function of duration since symptom onset, independent of patient age and comorbidities. Very high levels of SARS-CoV-2-specific serum IgA correlated with severe acute respiratory distress syndrome (ARDS). Interestingly, some of the SARS-CoV-2-exposed healthcare workers with negative SARS-CoV-2-specific IgA and IgG serum titers had detectable SARS-CoV-2-specific IgA antibodies in their nasal fluids and tears. Moreover, SARS-CoV-2-specific IgA levels in nasal fluids of these healthcare workers were inversely correlated with patient age. These data show that systemic IgA and IgG production against SARS-CoV-2 develops mainly in severe COVID-19, with very high IgA levels seen in patients with severe ARDS, whereas mild disease may be associated with transient serum titers of SARS-CoV-2-specific antibodies but stimulate mucosal SARS-CoV-2-specific IgA secretion. The findings suggest four grades of antibody responses dependent on COVID-19 severity.","version":"1.1","doi":"10.1101/2020.05.21.108308","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108035","pub_date":"2020-5-23","title":"Methods of inactivation of SARS-CoV-2 for downstream biological assays","abstract":"The scientific community has responded to the COVID-19 pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious SARS-CoV-2 is undertaken in high containment laboratories, however, it is often desirable to work with samples at lower containment levels. To facilitate the transfer of infectious samples from high containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80\u00b0C, and a range of detergents and UV energies were successful at inactivating a high titre of SARS-CoV-2. These protocols can provide a framework for in house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower containment levels.","version":"1.1","doi":"10.1101/2020.05.21.108035","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.089763","pub_date":"2020-5-23","title":"Children\u2019s Hospital Los Angeles COVID-19 Analysis Research Database (CARD) - A Resource for Rapid SARS-CoV-2 Genome Identification Using Interactive Online Phylogenetic Tools","abstract":"Effective response to the Coronavirus Disease 2019 (COVID-19) pandemic requires genomic resources and bioinformatics tools for genomic epidemiology and surveillance studies that involve characterizing full-length viral genomes, identifying origins of infections, determining the relatedness of viral infections, performing phylogenetic analyses, and monitoring the continuous evolution of the SARS-CoV-2 viral genomes. The Children\u2019s Hospital, Los Angeles (CHLA) COVID-19 Analysis Research Database (CARD) (https://covid19.cpmbiodev.net/) is a comprehensive genomic resource that provides access to full-length SARS-CoV-2 viral genomes and associated meta-data for over 30,000 (as of May 20, 2020) isolates collected from global sequencing repositories and the sequencing performed at the Center for Personalized Medicine (CPM) at CHLA. Reference phylogenetic trees of global and USA viral isolates were constructed and are periodically updated using selected high quality SARS-CoV-2 genome sequences. These provide the baseline and analytical context for identifying the origin of a viral infection, as well as the relatedness of SARS-CoV-2 genomes of interest. A web-based and interactive Phylogenetic Tree Browser supports flexible tree manipulation and advanced analysis based on keyword search while highlighting time series animation, as well as subtree export for graphical representation or offline exploration. A Virus Genome Tracker accepts complete or partial SARS-CoV-2 genome sequence, compares it against all available sequences in the database (>30,000 at time of writing), detects and annotates the variants, and places the new viral isolate within the global or USA phylogenetic contexts based upon variant profiles and haplotype comparisons, in a few seconds. The generated analysis can potentially aid in genomic surveillance to trace the transmission of any new infection. Using CHLA CARD, we demonstrate the identification of a candidate outbreak point where 13 of 31 CHLA internal isolates may have originated. We also discovered multiple indels of unknown clinical significance in the orf3a gene, and revealed a number of USA-specific variants and haplotypes.","version":"1.2","doi":"10.1101/2020.05.11.089763","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109835","pub_date":"2020-5-23","title":"Systemic Effects of Missense Mutations on SARS-CoV-2 Spike Glycoprotein Stability and Receptor Binding Affinity","abstract":"The spike (S) glycoprotein of SARS-CoV-2 is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18,354 mutations in S protein were analyzed and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3,705 mutations in SARS-CoV-2 RBD and 11,324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.","version":"1.2","doi":"10.1101/2020.05.21.109835","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109322","pub_date":"2020-5-23","title":"The emergence of SARS-CoV-2 in Europe and the US","abstract":"Accurate understanding of the global spread of emerging viruses is critically important for public health response and for anticipating and preventing future outbreaks. Here, we elucidate when, where and how the earliest sustained SARS-CoV-2 transmission networks became established in Europe and the United States (US). Our results refute prior findings erroneously linking cases in January 2020 with outbreaks that occurred weeks later. Instead, rapid interventions successfully prevented onward transmission of those early cases in Germany and Washington State. Other, later introductions of the virus from China to both Italy and Washington State founded the earliest sustained European and US transmission networks. Our analyses reveal an extended period of missed opportunity when intensive testing and contact tracing could have prevented SARS-CoV-2 from becoming established in the US and Europe.","version":"1.1","doi":"10.1101/2020.05.21.109322","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.111518","pub_date":"2020-5-23","title":"A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing","abstract":"To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100x more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.","version":"1.1","doi":"10.1101/2020.05.22.111518","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.23.112797","pub_date":"2020-5-23","title":"High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Viruses in vitro","abstract":"Influenza and other respiratory viruses represent a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as the current COVID-19 crisis. One of the greatest barriers to the development of effective therapeutic agents to treat influenza, coronaviruses, and many other infections of the respiratory tract is the absence of a robust preclinical model. Preclinical studies currently rely on high-throughput, low-fidelity in vitro screening with cell lines and/or low-throughput animal models that often provide a poor correlation to human clinical responses. Here, we introduce a human primary airway epithelial cell-based model integrated into a high-throughput platform where tissues are cultured at an air-liquid interface (PREDICT96-ALI). We present results on the application of this platform to influenza and coronavirus infections, providing multiple readouts capable of evaluating viral infection kinetics and potentially the efficacy of therapeutic agents in an in vitro system. Several strains of influenza A virus are shown to successfully infect the human primary cell-based airway tissue cultured at an air-liquid interface (ALI), and as a proof-of-concept, the effect of the antiviral oseltamivir on one strain of Influenza A is evaluated. Human coronaviruses NL63 (HCoV-NL63) and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for protein priming, and we confirm expression of both in our ALI model. We also demonstrate coronavirus infection in this system with HCoV-NL63, observing sufficient viral propagation over 96 hours post-infection to indicate successful infection of the primary cell-based model. This new capability has the potential to address a gap in the rapid assessment of therapeutic efficacy of various small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.","version":"1.1","doi":"10.1101/2020.05.23.112797","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.111005","pub_date":"2020-5-22","title":"Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals","abstract":"The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity. From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type (wt) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD. Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.","version":"1.1","doi":"10.1101/2020.05.22.111005","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109298","pub_date":"2020-5-22","title":"Recombinant SARS-CoV-2 spike proteins for sero-surveillance and epitope mapping","abstract":"The newly emergent SARS-CoV-2 coronavirus is closely related to SARS-CoV which emerged in 2002. Studies on coronaviruses in general, and SARS in particular, have identified the virus spike protein (S) as being central to virus tropism, to the generation of a protective antibody response and to the unambiguous detection of past infections. As a result of this centrality SARS-CoV-2 S protein has a role in many aspects of research from vaccines to diagnostic tests. We describe a number of recombinant forms of SARS-CoV-2 S expressed in commonly available expression systems and their preliminary use in diagnostics and epitope mapping. These sources may find use in the current and future analysis of the virus and the Covid-19 disease it causes.","version":"1.1","doi":"10.1101/2020.05.21.109298","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.092619","pub_date":"2020-5-22","title":"Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals","abstract":"During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.","version":"1.2","doi":"10.1101/2020.05.13.092619","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.22.110932","pub_date":"2020-5-22","title":"Pooling nasopharyngeal swab specimens to increase testing capacity for SARS-CoV-2","abstract":"The recent emergence of SARS-CoV-2 has lead to a global pandemic of unprecedented proportions. Current diagnosis of COVID-19 relies on the detection of SARS-CoV-2 RNA by RT-PCR in upper and lower respiratory specimens. While sensitive and specific, these RT-PCR assays require considerable supplies and reagents, which are often limited during global pandemics and surge testing. Here, we show that a nasopharyngeal swab pooling strategy can detect a single positive sample in pools of up to 10 samples without sacrificing RT-PCR sensitivity and specificity. We also report that this pooling strategy can be applied to rapid, moderate complexity assays, such as the BioFire COVID-19 test. Implementing a pooling strategy can significantly increase laboratory testing capacity while simultaneously reducing turnaround times for rapid identification and isolation of positive COVID-19 cases in high risk populations.","version":"1.1","doi":"10.1101/2020.05.22.110932","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109546","pub_date":"2020-5-22","title":"A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation","abstract":"Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. Our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.","version":"1.1","doi":"10.1101/2020.05.21.109546","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109637","pub_date":"2020-5-22","title":"Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2","abstract":"The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays. Rapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps\u2014factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.","version":"1.1","doi":"10.1101/2020.05.21.109637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.107243","pub_date":"2020-5-22","title":"A modular framework for the development of targeted Covid-19 blood transcript profiling panels","abstract":"Covid-19 morbidity and mortality are associated with a dysregulated immune response. Tools are needed to enhance existing immune profiling capabilities in affected patients. Here we aimed to develop an approach to support the design of focused blood transcriptome panels for profiling the immune response to SARS-CoV-2 infection. We designed a pool of candidates based on a pre-existing and well-characterized repertoire of blood transcriptional modules. Available Covid-19 blood transcriptome data was also used to guide this process. Further selection steps relied on expert curation. Additionally, we developed several custom web applications to support the evaluation of candidates. As a proof of principle, we designed three targeted blood transcript panels, each with a different translational connotation: therapeutic development relevance, SARS biology relevance and immunological relevance. Altogether the work presented here may contribute to the future expansion of immune profiling capabilities via targeted profiling of blood transcript abundance in Covid-19 patients.","version":"1.1","doi":"10.1101/2020.05.20.107243","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108043","pub_date":"2020-5-22","title":"Open source 3D printed Ventilation Device","abstract":"COVID-19 is an acute respiratory tract infection caused by a coronavirus known as SARS-CoV-2. The common signs of infection include respiratory symptoms such as shortness of breath, breathing difficulties, dry cough, fever, and in some patients, severe acute respiratory syndrome, kidney failure, and death. 312,009 deaths from COVID-19 has been reported as of today. While respiratory symptoms are commonly caused by the infection, the use of mechanical ventilation is required for some patients. The following is intended to review the development and testing of a 3D printed and open-source mechanical ventilation device that is capable of adjusting breathing rate, volume, and pressure simultaneously and was designed according to the latest clinical observations of the current pandemic. The intuitive design of this device along with the use of primarily 3D printed or readily available components allow the rapid manufacturing and transportation of this ventilation device to the impacted regions.","version":"1.1","doi":"10.1101/2020.05.21.108043","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.107565","pub_date":"2020-5-21","title":"Immunization with the receptor\u2013binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement","abstract":"Recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Currently, there is no vaccine available for preventing SARS-CoV-2 infection. Like closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses its receptor-binding domain (RBD) on the spike (S) protein to engage the host receptor, human angiotensin-converting enzyme 2 (ACE2), facilitating subsequent viral entry. Here we report the immunogenicity and vaccine potential of SARS-CoV-2 RBD (SARS2-RBD)-based recombinant proteins. Immunization with SARS2-RBD recombinant proteins potently induced a multi-functional antibody response in mice. The resulting antisera could efficiently block the interaction between SARS2-RBD and ACE2, inhibit S-mediated cell-cell fusion, and neutralize both SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection. In addition, the anti-RBD sera also exhibited cross binding, ACE2-blockade, and neutralization effects towards SARS-CoV. More importantly, we found that the anti-RBD sera did not promote antibody-dependent enhancement of either SARS-CoV-2 pseudovirus entry or authentic virus infection of Fc receptor-bearing cells. These findings provide a solid foundation for developing RBD-based subunit vaccines for SARS-CoV2.","version":"1.1","doi":"10.1101/2020.05.21.107565","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109272","pub_date":"2020-5-21","title":"An insertion unique to SARS-CoV-2 exhibits superantigenic character strengthened by recent mutations","abstract":"Multisystem Inflammatory Syndrome in Children (MIS-C) associated with Coronavirus Disease 2019 (COVID-19) is a newly recognized condition in which children with recent SARS-CoV-2 infection present with a constellation of symptoms including hypotension, multiorgan involvement, and elevated inflammatory markers. These symptoms and the associated laboratory values strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to MHCII molecules and T cell receptors (TCRs). Here, we used structure-based computational models to demonstrate that the SARS-CoV-2 spike (S) exhibits a high-affinity motif for binding TCR, interacting closely with both the \u03b1- and \u03b2-chains variable domains\u2019 complementarity-determining regions. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in any other SARS coronavirus), which is highly similar in both sequence and structure to bacterial superantigens. Further examination revealed that this interaction between the virus and human T cells is strengthened in the context of a recently reported rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from a motif shared between the SARS viruses from the 2003 and 2019 pandemics, which has intracellular adhesion molecule (ICAM)-like character. These data suggest that the SARS-CoV-2 S may act as a superantigen to drive the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches. Although children have been largely spared from severe COVID-19 disease, a rare hyperinflammatory syndrome has been described in Europe and the East Coast of the United States, termed Multisystem Inflammatory Syndrome in Children (MISC). The symptoms and diagnostic lab values of MIS-C resemble those of toxic shock, typically caused by pathogenic superantigens stimulating excessive activation of the adaptive immune system. We show that SARS-CoV-2 spike has a sequence and structure motif highly similar to those of bacterial superantigens, and may directly bind to the T cell receptors. This sequence motif, not present in other coronaviruses, may explain the unique potential for SARS-CoV-2 to cause both MIS-C and the cytokine storm observed in adult COVID-19 patients.","version":"1.1","doi":"10.1101/2020.05.21.109272","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.107912","pub_date":"2020-5-21","title":"Analyzing the impact of SARS CoV-2 on the human proteome","abstract":"The COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2, which uses the enzyme ACE2 to entry human cells. This disease is characterized by important damages at multi-organ level, partially due to the abundant expression of ACE2 in practically all human tissues. However, not every organ in which ACE2 is abundant is affected by SARS CoV-2, which suggests the existence of other multi-organ routes for transmitting the perturbations produced by the virus. We consider here diffusive processes through the protein-protein interaction (PPI) network of proteins targeted by SARS CoV-2 as such alternative route. We found a subdiffusive regime that allows the propagation of virus perturbations through the PPI network at a significant rate. By following the main subdiffusive routes across the PPI network we identify proteins mainly expressed in the heart, cerebral cortex, thymus, testis, lymph node, kidney, among others of the organs reported to be affected by COVID-19.","version":"1.1","doi":"10.1101/2020.05.21.107912","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.074567","pub_date":"2020-5-21","title":"Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations","abstract":"In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variant sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 38 recurrent non-synonymous mutations, including ten hotspot mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed certain genotypes specific to the geographic location. We also found co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets We have also created an inclusive unified database (http://genoma.ma/covid-19/) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.","version":"1.2","doi":"10.1101/2020.05.03.074567","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.107052","pub_date":"2020-5-21","title":"Discovery of potent inhibitors of PLproCoV2 by screening a library of selenium-containing compounds","abstract":"A collection of twelve organoselenium compounds, structural analogues of antioxidant drug ebselen were screened for inhibition of the papain-like protease (PLpro) from the acute respiratory syndrome coronavirus 2 (SARS-CoV-2, CoV2). This cysteine protease, being responsible for the hydrolysis of peptide bonds between specific amino acids, plays a critical role in CoV2 replication and in assembly of new viral particles within human cells. The activity of the PLpro CoV2 is essential for the progression of coronavirus disease 2019 (COVID-19) and it constitutes a key target for the development of anti-COVID-19 drugs. Here, we identified four strong inhibitors that bind favorably to the PLpro CoV2 with the IC50 in the nanomolar range.","version":"1.2","doi":"10.1101/2020.05.20.107052","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.108381","pub_date":"2020-5-21","title":"Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2","abstract":"The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/\u03bcL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/\u03bcL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests. An important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.","version":"1.1","doi":"10.1101/2020.05.21.108381","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.099242","pub_date":"2020-5-21","title":"Capillary Electrophoresis of PCR fragments with 5\u2019-labelled primers for testing the SARS-Cov-2","abstract":"Due to the huge demand for SARS-Cov-2 determination, alternatives to the standard qtPCR tests are potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples. We isolated the naso-pharingeal RNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5\u2019-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks. The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. We describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 hours. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR (lack of reactive and real-time PCR equipment) and increase the testing capacity.","version":"1.1","doi":"10.1101/2020.05.16.099242","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.21.109157","pub_date":"2020-5-21","title":"Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity","abstract":"A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction. Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2-accessible \u201cup\u201d conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology.","version":"1.1","doi":"10.1101/2020.05.21.109157","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.096081","pub_date":"2020-5-21","title":"Crystal structures of SARS-CoV-2 ADP-ribose phosphatase (ADRP): from the apo form to ligand complexes","abstract":"Among 15 nonstructural proteins (Nsps), the newly emerging SARS-CoV-2 encodes a large, multidomain Nsp3. One of its units is ADP-ribose phosphatase domain (ADRP, also known as macrodomain) which is believed to interfere with the host immune response. Such a function appears to be linked to the protein\u2019s ability to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remains unknown. Here, we have determined five, high resolution (1.07 - 2.01 \u00c5) crystal structures corresponding to the apo form of the protein and complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADPr. We show that the protein undergoes conformational changes to adapt to the ligand in a manner previously observed before for in close homologs from other viruses. We also identify a conserved water molecule that may participate in hydrolysis. This work builds foundations for future structure-based research of the ADRP, including search for potential antiviral therapeutics.","version":"1.2","doi":"10.1101/2020.05.14.096081","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.016048","pub_date":"2020-5-21","title":"The protein expression profile of ACE2 in human tissues","abstract":"The novel SARS-coronavirus 2 (SARS-CoV-2) poses a global challenge on healthcare and society. For understanding the susceptibility for SARS-CoV-2 infection, the cell type-specific expression of the host cell surface receptor is necessary. The key protein suggested to be involved in host cell entry is Angiotensin I converting enzyme 2 (ACE2). Here, we report the expression pattern of ACE2 across >150 different cell types corresponding to all major human tissues and organs based on stringent immunohistochemical analysis. The results were compared with several datasets both on the mRNA and protein level. ACE2 expression was mainly observed in enterocytes, renal tubules, gallbladder, cardiomyocytes, male reproductive cells, placental trophoblasts, ductal cells, eye and vasculature. In the respiratory system, the expression was limited, with no or only low expression in a subset of cells in a few individuals, observed by one antibody only. Our data constitutes an important resource for further studies on SARS-CoV-2 host cell entry, in order to understand the biology of the disease and to aid in the development of effective treatments to the viral infection.","version":"1.2","doi":"10.1101/2020.03.31.016048","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.107490","pub_date":"2020-5-21","title":"METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies - Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses","abstract":"We present METATRYP version-2 software that identifies shared peptides across organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include: ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the Least Common Ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine database confirms low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed for ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A Coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa, sharing 0.1% peptides or less (1 peptide) with the Influenza A & B pan-proteomes, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches. When assigning taxonomic attribution in bottom-up metaproteomics, the potential for shared tryptic peptides among organisms in mixed communities should be considered. The software program METATRYP v 2 and associated interactive web portals enables users to identify the frequency of shared tryptic peptides among taxonomic groups and evaluate the occurrence of specific tryptic peptides within complex communities. METATRYP facilitates phyloproteomic studies of taxonomic groups and supports the identification and evaluation of potential metaproteomic biomarkers.","version":"1.1","doi":"10.1101/2020.05.20.107490","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.097352","pub_date":"2020-5-21","title":"Understanding olfactory dysfunction in COVID-19: Expression of ACE2, TMPRSS2 and Furin in the nose and olfactory bulb in human and mice","abstract":"Anosmia is a frequent symptom in coronavirus disease 2019 (COVID-19) patients that generally resolves within weeks. In contrast, the anosmia caused by other upper respiratory infections affects a small proportion of patients and may take months to resolve or never resolve. The mechanisms behind COVID-19-induced olfactory dysfunction remain unknown. Here, we address the unique pathophysiology of COVID-19-associated olfactory dysfunction. The expression of ACE2 (virus binding receptor) and TMPRSS2 and Furin (host cell proteases facilitating virus entry) was examined in the nasal mucosa, composed of respiratory mucosa (RM), olfactory mucosa (OM), and olfactory bulb (OB) of mouse and human tissues using immunohistochemistry and gene analyses. Co-expression of ACE2, TMPRSS2, and Furin was observed in the RM and in the OM, especially in the supporting cells of the olfactory epithelium and the Bowman\u2019s glands. Notably, the olfactory receptor neurons (ORNs) in the OM were positive for ACE2 but almost negative for TMPRSS2 and Furin. Cells in the OB expressed ACE2 strongly and Furin weakly, and did not express TMPRSS2. All three gene expressions were confirmed in the nasal mucosa and OB. ACE2 was widely expressed in all tissues, whereas TMPRSS2 and Furin were expressed only in certain types of cells and were absent in the ORNs. These findings, together with clinical reports, suggest that COVID-19-related anosmia occurs mainly through sensorineural and central dysfunction and, to some extent, conductive olfactory dysfunction. The expression of ACE2, but not TMPRSS2 or Furin, in ORNs may explain the early recovery from anosmia.","version":"1.2","doi":"10.1101/2020.05.15.097352","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.106575","pub_date":"2020-5-20","title":"SARS-CoV-2 targets cortical neurons of 3D human brain organoids and shows neurodegeneration-like effects","abstract":"COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a D\u00fcsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within two days of exposure. Using COVID-19 convalescent serum, we identified that SARS-CoV-2 preferably targets soma of cortical neurons but not neural stem cells, the target cell type of ZIKA virus. Imaging cortical neurons of organoids reveal that SARS-CoV-2 exposure is associated with missorted Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Surprisingly, SARS-CoV-2 co-localizes specifically with Tau phosphorylated at Threonine-231 in the soma, indicative of early neurodegeneration-like effects. Our studies, therefore, provide initial insights into the impact of SARS-CoV-2 as a neurotropic virus and emphasize that brain organoids could model CNS pathologies of COVID-19. COVID-19 modeling in human brain organoids","version":"1.1","doi":"10.1101/2020.05.20.106575","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.103549","pub_date":"2020-5-20","title":"Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients","abstract":"As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.","version":"1.1","doi":"10.1101/2020.05.20.103549","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.105437","pub_date":"2020-5-20","title":"Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures","abstract":"The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNF\u03b1, CXCL8. We also identified NF-\u03baB and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.","version":"1.1","doi":"10.1101/2020.05.19.105437","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.105247","pub_date":"2020-5-20","title":"A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition","abstract":"There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.","version":"1.1","doi":"10.1101/2020.05.20.105247","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.103184","pub_date":"2020-5-20","title":"In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations","abstract":"To curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.","version":"1.1","doi":"10.1101/2020.05.18.103184","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.104885","pub_date":"2020-5-20","title":"RdRp mutations are associated with SARS-CoV-2 genome evolution","abstract":"COVID-19, caused by the novel SARS-CoV-2 virus, started in China in late 2019, and soon became a global pandemic. With the help of thousands of viral genome sequences that have been accumulating, it has become possible to track the evolution of viral genome over time as it spread across the world. An important question that still needs to be answered is whether any of the common mutations affect the viral properties, and therefore the disease characteristics. Therefore, we sought to understand the effects of mutations in RNA-dependent RNA polymerase (RdRp), particularly the common 14408C>T mutation, on mutation rate and viral spread. By focusing on mutations in the slowly evolving M or E genes, we aimed to minimize the effects of selective pressure. Our results indicate that 14408C>T mutation increases the mutation rate, while the third-most common RdRp mutation, 15324C>T, has the opposite effect. It is possible that 14408C>T mutation may have contributed to the dominance of its co-mutations in Europe and elsewhere.","version":"1.1","doi":"10.1101/2020.05.20.104885","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.107292","pub_date":"2020-5-20","title":"CD8+ T cell cross-reactivity against SARS-CoV-2 conferred by other coronavirus strains and influenza virus","abstract":"While individuals infected with coronavirus disease 2019 (COVID-19) manifested a broad range in susceptibility and severity to the disease, the pre-existing immune memory of related pathogens can influence the disease outcome. Here, we investigated the potential extent of T cell cross-reactivity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be conferred by other coronaviruses and influenza virus, and generated a map of public and private predicted CD8+ T cell epitopes between coronaviruses. Moreover, to assess the potential risk of self-reactivity and/or diminished T cell response for peptides identical or highly similar to the host, we identified predicted epitopes with high sequence similarity with human proteome. Lastly, we compared predicted epitopes from coronaviruses with epitopes from influenza virus deposited in IEDB to support vaccine development against different virus strains. We believe the comprehensive in silico profile of private and public predicted epitopes across coronaviruses and influenza viruses will facilitate design of vaccines capable of protecting against various viral infections.","version":"1.1","doi":"10.1101/2020.05.20.107292","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.105445","pub_date":"2020-5-20","title":"Clinical And Analytical Performance Of An Automated Serological Test That Identifies S1/S2 Neutralizing IgG In Covid-19 Patients Semiquantitatively","abstract":"In the Covid-19 pandemic, highly selective serological testing is essential to define exposure to SARS-CoV-2 virus. Many tests have been developed, yet with variable speed to first result, and of unknown quality, particularly when considering the prediction of neutralizing capacity. The LIAISON\u00ae SARS-CoV-2 S1/S2 IgG assay was designed to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized automated chemiluminescent assay. Clinical and analytical performance of the test were validated in an observational study using residual samples (>1500) with positive or negative Covid-19 diagnosis. The LIAISON\u00ae SARS-CoV-2 S1/S2 IgG assay proved highly selective and specific, and offers semiquantitative measures of serum or plasma levels of anti-S1/S2 IgG with neutralizing activity. The diagnostic sensitivity was 91.3% and 95.7% at >5 or \u226515 days from diagnosis respectively, and 100% when assessed against a neutralizing assay. The specificity ranged between 97% and 98.5%. The average imprecision of the assay was <5 % coefficient of variation. Assay performance at 2 different cut-offs was evaluated to optimize predictive values in settings with different % disease prevalence. CONCLUSIONS. The automated LIAISON\u00ae SARS-CoV-2 S1/S2 IgG assay brings efficient, sensitive, specific, and precise serological testing to the laboratory, with the capacity to test large amounts of samples per day: first results are available within 35 minutes with a throughput of 170 tests/hour. The test also provides a semiquantitative measure to identify samples with neutralizing antibodies, useful also for a large scale screening of convalescent plasma for safe therapeutic use. With the worldwide advance of the COVID-19 pandemic, efficient, reliable and accessible diagnostic tools are needed to support public health officials and healthcare providers in their efforts to deliver optimal medical care, and articulate sound demographic policy. DiaSorin has developed an automated serology based assay for the measurement of IgG specific to SARS CoV-2 Spike protein, and tested its clinical performance in collaboration with Italian health care professionals who provided access to large numbers of samples from infected and non-infected individuals. The assay delivers excellent sensitivity and specificity, and is able to identify samples with high levels of neutralizing antibodies. This will provide guidance in assessing the true immune status of subjects, as well as meeting the pressing need to screen donors for high titer convalescent sera for subsequent therapeutic and prophylactic use.","version":"1.1","doi":"10.1101/2020.05.19.105445","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.106294","pub_date":"2020-5-20","title":"Deep sequencing of B cell receptor repertoires from COVID-19 patients reveals strong convergent immune signatures","abstract":"Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 19 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients and may be a positive indicator of disease outcome. Clonal expansion of the B cell memory response is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 777 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralising antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand and predict positive patient responses.","version":"1.1","doi":"10.1101/2020.05.20.106294","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.106625","pub_date":"2020-5-20","title":"CONTAIN: An open-source shipping container laboratory optimised for automated COVID-19 diagnostics","abstract":"The COVID-19 pandemic has challenged diagnostic systems globally. Expanding testing capabilities to conduct population-wide screening for COVID-19 requires innovation in diagnostic services at both the molecular and industrial scale. No report to-date has considered the complexity of laboratory infrastructure in conjunction with the available molecular assays to offer a standardised solution to testing. Here we present CONTAIN. A modular biosafety level 2+ laboratory optimised for automated RT-qPCR COVID-19 testing based on a standard 40ft shipping container. Using open-source liquid-handling robots and RNA extraction reagents we demonstrate a reproducible workflow for RT-qPCR COVID-19 testing. With five OT2 liquid handlers, a single CONTAIN unit reaches a maximum daily testing capacity of 2400 tests/day. We validate this workflow for automated RT-qPCR testing, using both synthetic SARS-CoV-2 samples and patient samples from a local NHS hospital. Finally, we discuss the suitability of CONTAIN and its flexibility in a range of diagnostic testing scenarios including high-density urban environments and mobile response units.","version":"1.1","doi":"10.1101/2020.05.20.106625","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.104513","pub_date":"2020-5-20","title":"Prediction of the virus incubation period for COVID-19 and future outbreaks","abstract":"A crucial factor in mitigating respiratory viral outbreaks is early determination of the duration of the incubation period and, accordingly, the required quarantine time for potentially exposed individuals. Here, we explore different genomic features of RNA viruses that correlate with the incubation times and provide a predictive model that accurately estimates the upper limit incubation time for diverse viruses including SARS-CoV-2, and thus, could help control future outbreaks.","version":"1.1","doi":"10.1101/2020.05.19.104513","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.20.106658","pub_date":"2020-5-20","title":"Kidney and Lung ACE2 expression after an ACE inhibitor or an Ang II receptor blocker: implications for COVID-19","abstract":"There have been concerns that ACE inhibitors and Ang II receptor blockers may cause an increase in ACE2, the main receptor for SARs-CoV-2. Kidneys from two genetic models of kidney ACE ablation and mice treated with captopril or telmisartan were used to examine ACE2 in isolated kidney and lung membranes. In a global ACE KO mice, ACE2 protein abundance in kidney membranes was reduced to 42 % of wild type, p < 0.05. In ACE 8/8 mice that over-expresses cardiac ACE protein but also has no kidney ACE expression, ACE2 protein in kidney membranes was also decreased (38 % of the WT, p<0.01). In kidney membranes from mice that received captopril or telmisartan for 2 weeks there was a reduction in ACE2 protein (37% in captopril treated p<0.01) and 76% in telmisartan treated p <0.05). In lung membranes the expression of ACE2 was very low and not detected by western blotting but no significant differences in terms of ACE2 activity could be detected in mice treated with captopril (118% of control) or telmisartan (93% of control). Genetic kidney ACE protein deficiency, suppressed enzymatic activity by Captopril or blockade of the AT1 receptor with telmisartan are all associated with a decrease in ACE2 in kidney membranes. ACE2 protein in kidney or lungs is decreased or unaffected by RAS blockers indicating that these medications can not pose a risk for SARS-CoV-2 infection related to amplification of ACE2 at these two target sites for viral entry.","version":"1.1","doi":"10.1101/2020.05.20.106658","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.103101","pub_date":"2020-5-20","title":"Cell cycle-gated feedback control mediates desensitization to interferon stimulation","abstract":"Cells use sophisticated molecular circuits to interpret and respond to extracellular signal factors, such as hormones and cytokines, which are often released in a temporally varying fashion. In this study, we focus on type I interferon (IFN) signaling in human epithelial cells and combine microfluidics, time-lapse microscopy, and computational modeling to investigate how the IFN-responsive regulatory network operates in single cells to process repetitive IFN stimulation. We found that IFN-\u03b1 pretreatments lead to opposite effects, priming versus desensitization, depending on the input durations. These effects are governed by a regulatory network composed of a fast-acting positive feedback loop and a delayed negative feedback loop, mediated by upregulation of ubiquitin-specific peptidase 18 (USP18). We further revealed that USP18 upregulation can only be initiated at the G1 and early S phases of cell cycle upon the treatment onset, resulting in heterogeneous and delayed induction kinetics in single cells. This cell cycle gating provides a temporal compartmentalization of feedback control processes, enabling duration-dependent desensitization to repetitive stimulations. Moreover, our results, highlighting the importance of IFN dynamics, may suggest time-based strategies for enhancing the effectiveness of IFN pretreatment in clinical applications against viruses, such as SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.18.103101","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.095661","pub_date":"2020-5-19","title":"Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication","abstract":"SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinder therapy development. We employed a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. We identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways were activated. Drug-protein network analyses revealed GFR signaling as key pathway targetable by approved drugs. Inhibition of GFR downstream signaling by five compounds prevented SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as central pathway essential for SARS-CoV-2 replication. It provides with novel strategies for COVID-19 treatment.","version":"1.2","doi":"10.1101/2020.05.14.095661","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.103846","pub_date":"2020-5-19","title":"Emerging phylogenetic structure of the SARS-CoV-2 pandemic","abstract":"Since spilling over into humans, SARS-CoV-2 has rapidly spread across the globe, accumulating significant genetic diversity. The structure of this genetic diversity, and whether it reveals epidemiological insights, are fundamental questions for understanding the evolutionary trajectory of this virus. Here we use a recently developed phylodynamic approach to uncover phylogenetic structures underlying the SARS-CoV-2 pandemic. We find support for three SARS-CoV-2 lineages co-circulating, each with significantly different demographic dynamics concordant with known epidemiological factors. For example, Lineage C emerged in Europe with a high growth rate in late February, just prior to the exponential increase in cases in several European countries. Mutations that characterize Lineage C in particular are non-synonymous and occur in functionally important gene regions responsible for viral replication and cell entry. Even though Lineages A and B had distinct demographic patterns, they were much more difficult to distinguish. Continuous application of phylogenetic approaches to track the evolutionary epidemiology of SARS-CoV-2 lineages will be increasingly important to validate the efficacy of control efforts and monitor significant evolutionary events in the future.","version":"1.1","doi":"10.1101/2020.05.19.103846","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.103283","pub_date":"2020-5-19","title":"Antiviral activity of Glucosylceramide synthase inhibitors against SARS-CoV-2 and other RNA virus infections","abstract":"The need for antiviral drugs is real and relevant. Broad spectrum antiviral drugs have a particular advantage when dealing with rapid disease outbreaks, such as the current COVID-19 pandemic. Since viruses are completely dependent on internal cell mechanisms, they must cross cell membranes during their lifecycle, creating a dependence on processes involving membrane dynamics. Thus, in this study we examined whether the synthesis of glycosphingolipids, biologically active components of cell membranes, can serve as an antiviral therapeutic target. We examined the antiviral effect of two specific inhibitors of GlucosylCeramide synthase (GCS); (i) Genz-123346, an analogue of the FDA-approved drug Cerdelga\u00ae, (ii) GENZ-667161, an analogue of venglustat which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit the replication of four different enveloped RNA viruses of different genus, organ-target and transmission route: (i) Neuroinvasive Sindbis virus (SVNI), (ii) West Nile virus (WNV), (iii) Influenza A virus, and (iv) SARS-CoV-2. Moreover, GCS inhibitors significantly increase the survival rate of SVNI-infected mice. Our data suggest that GCS inhibitors can potentially serve as a broad-spectrum antiviral therapy and should be further examined in preclinical and clinical trial. Analogues of the specific compounds tested have already been studied clinically, implying they can be fast-tracked for public use. With the current COVID-19 pandemic, this may be particularly relevant to SARS-CoV-2 infection. An analogue of Cerdelga\u00ae, an FDA-approved drug, is effective against a broad range of RNA-viruses including the newly emerging SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.18.103283","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.103630","pub_date":"2020-5-19","title":"Transcriptional profiling of immune and inflammatory responses in the context of SARS-CoV-2 fungal superinfection in a human airway epithelial model","abstract":"Superinfections of bacterial/fungal origin are known to affect the course and severity of respiratory viral infections. An increasing number of evidence indicate a relatively high prevalence of superinfections associated with COVID-19, including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection we sought to determine and compare the host transcriptional response to SARS-CoV-2 versus Aspergillus superinfection, using a model of reconstituted humain airway epithelium. Our analyses reveal that both simple infection and superinfection induce a strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2 Aspergillus superinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte- and neutrophil associated chemokines, that could be useful for the understanding of Aspergillus-associated COVID-19 and but also management of severe forms of aspergillosis in this specific context.","version":"1.1","doi":"10.1101/2020.05.19.103630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.099234","pub_date":"2020-5-19","title":"Multi-Antigenic Virus-like Particle of SARS CoV-2 produced in Saccharomyces cerevisiae as a vaccine candidate","abstract":"Spike, Envelope and Membrane proteins from the SARS CoV-2 virus surface coat are important vaccine targets. We hereby report recombinant co-expression of the three proteins (Spike, Envelope and Membrane) in a engineered Saccharomyces cerevisiae platform (D-Crypt\u2122) and their self-assembly as Virus-like particle (VLP). This design as a multi-antigenic VLP for SARS CoV-2 has the potential to be a scalable vaccine candidate. The VLP is confirmed by transmission electron microscopy (TEM) images of the SARS CoV-2, along with supportive HPLC, Dynamic Light Scattering (DLS) and allied analytical data. The images clearly outline the presence of a \u201cCorona\u201d like morphology, and uniform size distribution.","version":"1.1","doi":"10.1101/2020.05.18.099234","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.104042","pub_date":"2020-5-19","title":"High Throughput Designing and Mutational Mapping of RBD-ACE2 Interface Guide Non-Conventional Therapeutic Strategies for COVID-19","abstract":"Considering the current status of the SARS-CoV-2 pandemic, sequence variations and possibly structural changes in the rapidly evolving SARS-CoV-2 is highly expected in the coming months. The SARS-CoV-2 spike (S) protein is responsible for mediating viral attachment and fusion with cell membranes. Mutations in the receptor-binding domain (RBD) of the S-protein occur at the most variable part of the SARS-CoV-2 genome, and specific sites of S-protein have undergone positive selection impacting the viral pathogenicity. In the present work, we used high-throughput computation to design 100,000 mutants in RBD interfacial residues and identify novel affinity-enhancing and affinity-weakening mutations. Our data suggest that SARS-CoV-2 can establish a higher rate of infectivity and pathogenesis when it acquires combinatorial mutations at the interfacial residues in RBD. Mapping of the mutational landscape of the interaction site suggests that a few of these residues are the hot-spot residues with a very high tendency to undergo positive selection. Knowledge of the affinity-enhancing mutations may guide the identification of potential cold-spots for this mutation as targets for developing a possible therapeutic strategy instead of hot-spots, and vice versa. Understanding of the molecular interactions between the virus and host protein presents a detailed systems view of viral infection mechanisms. The applications of the present research can be explored in multiple antiviral strategies, including monoclonal antibody therapy, vaccine design, and importantly in understanding the clinical pathogenesis of the virus itself. Our work presents research directions for the exploitation of non-conventional solutions for COVID-19.","version":"1.1","doi":"10.1101/2020.05.19.104042","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.19.104281","pub_date":"2020-5-19","title":"Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Na\u00efve scFv Phage Display Library","abstract":"SARS-CoV-2 (Covid-19) has caused currently ongoing global plague and imposed great challenges to health managing systems all over the world, with millions of infections and hundreds of thousands of deaths. In addition to racing to develop vaccines, neutralizing antibodies (nAbs) to this virus have been extensively sought and are expected to provide another prevention and therapy tool against this frantic pandemic. To offer fast isolation and shortened early development, a large human na\u00efve phage display antibody library, was built and used to screen specific nAbs to the receptor-binding domain, RBD, the key for Covid-19 virus entry through a human receptor, ACE2. The obtained RBD-specific antibodies were characterized by epitope mapping, FACS and neutralization assay. Some of the antibodies demonstrated spike-neutralizing property and ACE2-competitiveness. Our work proved that RBD-specific neutralizing binders from human na\u00efve antibody phage display library are promising candidates to for further Covid-19 therapeutics development.","version":"1.1","doi":"10.1101/2020.05.19.104281","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.050534","pub_date":"2020-5-19","title":"ACE2 Homo-dimerization, Human Genomic variants and Interaction of Host Proteins Explain High Population Specific Differences in Outcomes of COVID19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive single-stranded RNA virus that causes a highly contagious Corona Virus Disease (COVID19). Entry of SARS-CoV-2 in human cells depends on binding of the viral spike (S) proteins to cellular receptor Angiotensin-converting enzyme 2 (ACE2) and on S-protein priming by host cell serine protease TMPRSS2. Recently, COVID19 has been declared pandemic by World Health Organization (WHO) yet high differences in disease outcomes across countries have been seen. We provide evidences to explain these population-level differences. One of the key factors of entry of the virus in host cells presumably is because of differential interaction of viral proteins with host cell proteins due to different genetic backgrounds. Based on our findings, we conclude that a higher expression of ACE2 is facilitated by natural variations, acting as Expression quantitative trait loci (eQTLs), with different frequencies in different populations. We suggest that high expression of ACE2 results in homo-dimerization, proving disadvantageous for TMPRSS2 mediated cleavage of ACE2; whereas, the monomeric ACE2 has higher preferential binding with SARS-CoV-2 S-Protein vis-a-vis its dimerized counterpart. Further, eQTLs in TMPRSS2 and natural structural variations in the gene may also result in differential outcomes towards priming of viral S-protein, a critical step for entry of the Virus in host cells. In addition, we suggest that several key host genes, like SLC6A19, ADAM17, RPS6, HNRNPA1, SUMO1, NACA, BTF3 and some other proteases as Cathepsins, might have a critical role. To conclude, understanding population specific differences in these genes may help in developing appropriate management strategies for COVID19 with better therapeutic interventions.","version":"1.4","doi":"10.1101/2020.04.24.050534","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.20101253","pub_date":"2020-05-19","title":"The infection fatality rate of COVID-19 inferred from seroprevalence data","abstract":"<jats:title>ABSTRACT</jats:title>\n                <jats:sec>\n                  <jats:title>Objective</jats:title>\n                  <jats:p>To estimate the infection fatality rate of coronavirus disease 2019 (COVID-19) from data of seroprevalence studies.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Methods</jats:title>\n                  <jats:p>Population studies with sample size of at least 500 and published as peer-reviewed papers or preprints as of July 11, 2020 were retrieved from PubMed, preprint servers, and communications with experts. Studies on blood donors were included, but studies on healthcare workers were excluded. The studies were assessed for design features and seroprevalence estimates. Infection fatality rate was estimated from each study dividing the number of COVID-19 deaths at a relevant time point by the number of estimated people infected in each relevant region. Correction was also attempted accounting for the types of antibodies assessed. Secondarily, results from national studies were also examined from preliminary press releases and reports whenever a country had no other data presented in full papers of preprints.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Results</jats:title>\n                  <jats:p>36 studies (43 estimates) were identified with usable data to enter into calculations and another 7 preliminary national estimates were also considered for a total of 50 estimates. Seroprevalence estimates ranged from 0.222% to 47%. Infection fatality rates ranged from 0.00% to 1.63% and corrected values ranged from 0.00% to 1.31%. Across 32 different locations, the median infection fatality rate was 0.27% (corrected 0.24%). Most studies were done in pandemic epicenters with high death tolls. Median corrected IFR was 0.10% in locations with COVID-19 population mortality rate less than the global average (&lt;73 deaths per million as of July 12, 2020), 0.27% in locations with 73-500 COVID-19 deaths per million, and 0.90% in locations exceeding 500 COVID-19 deaths per million. Among people &lt;70 years old, infection fatality rates ranged from 0.00% to 0.57% with median of 0.05% across the different locations (corrected median of 0.04%).</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions</jats:title>\n                  <jats:p>The infection fatality rate of COVID-19 can vary substantially across different locations and this may reflect differences in population age structure and case-mix of infected and deceased patients as well as multiple other factors. Estimates of infection fatality rates inferred from seroprevalence studies tend to be much lower than original speculations made in the early days of the pandemic.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.05.13.20101253","journal":"medRxiv","score":null},{"id":"10.1101/2020.05.18.101717","pub_date":"2020-5-18","title":"Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection","abstract":"Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified broad changes in neutrophils, NK cells, and monocytes during severe COVID-19, suggesting excessive mobilization of innate lineages. We found marked activation within T and B cells, highly oligoclonal B cell populations, profound plasmablast expansion, and SARS-CoV-2-specific antibodies in many, but not all, severe COVID-19 cases. Despite this heterogeneity, we found selective clustering of severe COVID-19 cases through unbiased analysis of the aggregated immunological phenotypes. Our findings demonstrate broad immune perturbations spanning both innate and adaptive leukocytes that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation. Broad immune perturbations in severe COVID-19","version":"1.1","doi":"10.1101/2020.05.18.101717","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.101485","pub_date":"2020-5-18","title":"Does the human placenta express the canonical cell entry mediators for SARS-CoV-2?","abstract":"The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected over 3.8 million people, including pregnant women. To date, no consistent evidence of vertical transmission for SARS-CoV-2 exists. This new coronavirus canonically utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and the serine protease TMPRSS2 for cell entry. Herein, building upon our previous single cell study of the placenta (Pique-Regi, 2019), another study, and new single-cell/nuclei RNA-sequencing data, we investigated the expression of ACE2 and TMPRSS2 throughout pregnancy as well as in third-trimester chorioamniotic membranes. We report that co-transcription of ACE2 and TMPRSS2 is negligible, thus not a likely path of vertical transmission for SARS-CoV-2 at any stage of pregnancy. In contrast, receptors for Zika virus and cytomegalovirus which cause congenital infections are highly expressed by placental cell types. These data suggest that SARS-CoV-2 is unlikely to infect the human placenta through the canonical cell entry mediators; yet, other interacting proteins could still play a role in the viral infection.","version":"1.1","doi":"10.1101/2020.05.18.101485","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.009084","pub_date":"2020-5-18","title":"Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia","abstract":"Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) \u2013 the causal agent in COVID-19 \u2013 affects olfaction directly by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing revealed that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing and immunostaining demonstrated ACE2 expression in support cells, stem cells, and perivascular cells; in contrast, neurons in both the olfactory epithelium and bulb did not express ACE2 message or protein. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.","version":"1.4","doi":"10.1101/2020.03.25.009084","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.102467","pub_date":"2020-5-18","title":"Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2","abstract":"SARS-CoV-2 is the causative agent of the current COVID-19 pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1) which suppresses host gene expression by ribosome association via an unknown mechanism. Here, we show that Nsp1 from SARS-CoV-2 binds to 40S and 80S ribosomes, resulting in shutdown of capped mRNA translation both in vitro and in cells. Structural analysis by cryo-electron microscopy (cryo-EM) of in vitro reconstituted Nsp1-40S and of native human Nsp1-ribosome complexes revealed that the Nsp1 C-terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks RIG-I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.18.102467","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.102038","pub_date":"2020-5-18","title":"Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2","abstract":"Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune \u03b3-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.","version":"1.1","doi":"10.1101/2020.05.18.102038","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.101691","pub_date":"2020-5-18","title":"Repurposing of Miglustat to inhibit the coronavirus Severe Acquired Respiratory Syndrome SARS-CoV-2","abstract":"Repurposing clinically available drugs to treat the new coronavirus disease COVID-19 is an urgent need in these early stages of the SARS-CoV-2 pandemic, when very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated for SARS-CoV-2 at concentrations achievable in the plasma by current clinical regimens without cytotoxicity. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious virus. The mechanism resides in the inhibitory activity towards \u03b1-glucosidases that are involved in early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. The wealth of available data on the clinical use of Miglustat for the treatment of lysosomal storage disorders and the antiviral properties against SARS-CoV-2 make it an ideal candidate for drug repurposing.","version":"1.1","doi":"10.1101/2020.05.18.101691","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.12.988246","pub_date":"2020-5-18","title":"Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing","abstract":"Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.","version":"1.3","doi":"10.1101/2020.03.12.988246","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.101493","pub_date":"2020-5-18","title":"SARS-CoV2 infection in farmed mink, Netherlands, April 2020","abstract":"In April 2020, respiratory disease and increased mortality were observed in farmed mink on two farms in the Netherlands. In both farms, at least one worker had been found positive for SARS-CoV-2. Necropsies of the mink revealed interstitial pneumonia, and organ and swab samples tested positive for SARS-CoV-2 RNA by qPCR. Variations in viral genomes point at between-mink transmission on the farms and lack of infection link between the farms. Inhalable dust in the mink houses contained viral RNA, indicating possible exposure of workers.","version":"1.1","doi":"10.1101/2020.05.18.101493","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.18.102087","pub_date":"2020-5-18","title":"Controlling the SARS-CoV-2 Spike Glycoprotein Conformation","abstract":"The coronavirus (CoV) viral host cell fusion spike (S) protein is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available \u03b2-CoV S-protein structures. We found that despite overall similarity in domain organization, different \u03b2-CoV strains display distinct S-protein configurations. Based on this analysis, we developed two soluble ectodomain constructs in which the highly immunogenic and mobile receptor binding domain (RBD) is locked in either the all-RBDs \u2018down\u2019 position or is induced to display a previously unobserved in SARS-CoV-2 2-RBDs \u2018up\u2019 configuration. These results demonstrate that the conformation of the S-protein can be controlled via rational design and provide a framework for the development of engineered coronavirus spike proteins for vaccine applications.","version":"1.1","doi":"10.1101/2020.05.18.102087","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.096107","pub_date":"2020-5-17","title":"Molecular conservation and Differential mutation on ORF3a gene in Indian SARS-CoV2 genomes","abstract":"A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory proteins in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2\u2019s virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservations of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2.","version":"1.1","doi":"10.1101/2020.05.14.096107","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.099143","pub_date":"2020-5-17","title":"Computational Study of Ions and Water Permeation and Transportation Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel","abstract":"Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus (SARS-CoV-2) and represents the causative agent of a potentially fatal disease that is of public health emergency of international concern. Coronaviruses, including SARS-CoV-2, encode an envelope (E) protein, which is a small, hydrophobic membrane protein; the E protein of SARS-CoV-2 has high homology with that of severe acute respiratory syndrome coronavirus. (SARS-CoV) In this study, we provide insights into the function of the SARS-CoV-2 E protein channel and the ion and water permeation mechanisms on the basis of combined in silico methods. Our results suggest that the pentameric E protein promotes the penetration of monovalent ions through the channel. Analysis of the potential mean force (PMF), pore radius and diffusion coefficient reveals that Leu10 and Phe19 are the hydrophobic gates of the channel. In addition, the pore demonstrated a clear wetting/dewetting transition with monovalent cation selectivity under transmembrane voltage, which indicates that it is a hydrophobic voltage-dependent channel. Overall, these results provide structural-basis insights and molecular-dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric SARS-CoV-2 E protein channel.","version":"1.1","doi":"10.1101/2020.05.17.099143","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.099747","pub_date":"2020-5-17","title":"REMBRANDT: A high-throughput barcoded sequencing approach for COVID-19 screening","abstract":"The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A still-debated percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions that includes diabetes, COPD and pregnancy. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. As communities re-open, large numbers of people will need to be tested and contact-tracing of positive patients will be required to prevent additional waves of infections and enable the continuous monitoring of the viral loads COVID-19 positive patients. A number of molecular assays are currently in clinical use to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening, a major bottleneck for testing samples from a large patient population for COVID-19.","version":"1.1","doi":"10.1101/2020.05.16.099747","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.100404","pub_date":"2020-5-17","title":"Identification of five antiviral compounds from the Pandemic Response Box targeting SARS-CoV-2","abstract":"With currently over 4 million confirmed cases worldwide, including more than 300\u2019000 deaths, the current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has a major impact on the economy and health care system. Currently, a limited amount of prophylactic or therapeutic intervention options are available against SARS-CoV-2. In this study, we screened 400 compounds from the antimicrobial \u2018Pandemic Response Box\u2019 library for inhibiting properties against SARS-CoV-2. We identified sixteen compounds that potently inhibited SARS-CoV-2 replication, of which five compounds displayed equal or even higher antiviral activity compared to Remdesivir. These results show that five compounds should be further investigated for their mode of action, safety and efficacy against SARS-CoV-2. 400 compounds from the pandemic response box were tested for antiviral activity against SARS-CoV-2. 5 compounds had an equal or higher antiviral efficacy towards SARS-CoV-2, compared to the nucleoside analogue Remdesivir.","version":"1.1","doi":"10.1101/2020.05.17.100404","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.100768","pub_date":"2020-5-17","title":"Ebselen as a highly active inhibitor of PLProCoV2","abstract":"Since December 2019 a novel a coronavirus identified as SARS-CoV-2 or COV2 has been spreading around the world. On the 16th of May around 4.5 million people got infected and over 300,000 died due to the infection of COV2. The effective treatment remains a challenge. Targeted therapeutics are still under investigation. The papain-like protease (PLPro) from the human SARS-CoV-2 coronavirus is a cysteine protease that plays a critical role in virus replication. Its activity is required to process the viral polyprotein into functional, mature subunits. Moreover, COV2 uses this enzyme to modulate the host\u2019s immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity in mammalian cells and cytotoxicity in lower organisms, is a highly active inhibitor of PLProCoV2. We proved that ebselen is a covalent, fast-binding inhibitor of PLProCoV2 exhibiting a low micromolar potency. Furthermore, we identified a difference between PLPro from SARS-CoV-1 (the corona virus which caused the 2002\u20132004 outbreak, SARS) and SARS-CoV-2 that allows to explain the difference in dynamics of the replication, and, thus, the disease progression. Namely, we present that they show differences in the binding affinity of substrates that we observed through kinetics and molecular docking studies. Using a novel Approximate Bayesian Computation method we were able to find kinetic constants for both enzymes. Molecular modeling study on the structure of the active site and binding mode of the ebselen with SARS and COV2 showed also significant differences that could explain our observation that ebselen is less active and slower bounding with SARS than COV2. In conclusion, we show that ebselen inhibits the activity of the essential viral enzyme papain-like protease (PLpro) from SARS-COV-2 in low micromolar range.","version":"1.1","doi":"10.1101/2020.05.17.100768","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.100289","pub_date":"2020-5-17","title":"Establishment of an African green monkey model for COVID-19","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to screen candidate vaccines and treatments. Nonhuman primates (NHP) are considered the gold standard model for many infectious pathogens as they usually best reflect the human condition. Here, we show that African green monkeys support a high level of SARS-CoV-2 replication and develop pronounced respiratory disease that may be more substantial than reported for other NHP species including cynomolgus and rhesus macaques. In addition, SARS-CoV-2 was detected in mucosal samples of all animals including feces of several animals as late as 15 days after virus exposure. Importantly, we show that virus replication and respiratory disease can be produced in African green monkeys using a much lower and more natural dose of SARS-CoV-2 than has been employed in other NHP studies.","version":"1.1","doi":"10.1101/2020.05.17.100289","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.099317","pub_date":"2020-5-17","title":"Distinct conformational states of SARS-CoV-2 spike protein","abstract":"The ongoing SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic has created urgent needs for intervention strategies to control the crisis. The spike (S) protein of the virus forms a trimer and catalyzes fusion between viral and target cell membranes - the first key step of viral infection. Here we report two cryo-EM structures, both derived from a single preparation of the full-length S protein, representing the prefusion (3.1\u00c5 resolution) and postfusion (3.3\u00c5 resolution) conformations, respectively. The spontaneous structural transition to the postfusion state under mild conditions is independent of target cells. The prefusion trimer forms a tightly packed structure with three receptor-binding domains clamped down by a segment adjacent to the fusion peptide, significantly different from recently published structures of a stabilized S ectodomain trimer. The postfusion conformation is a rigid tower-like trimer, but decorated by N-linked glycans along its long axis with almost even spacing, suggesting possible involvement in a mechanism protecting the virus from host immune responses and harsh external conditions. These findings advance our understanding of how SARS-CoV-2 enters a host cell and may guide development of vaccines and therapeutics.","version":"1.1","doi":"10.1101/2020.05.16.099317","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.100206","pub_date":"2020-5-17","title":"Epitope-Based Peptide Vaccine Against Severe Acute Respiratory Syndrome-Coronavirus-2 Nucleocapsid Protein: An in silico Approach","abstract":"With an increasing fatality rate, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a promising threat to human health worldwide. SARS-CoV-2 is a member of the Coronaviridae family, which is transmitted from animal to human and because of being contagious, further it transmitted human to human. Recently, the World Health Organization (WHO) has announced the infectious disease caused by SARS-CoV-2, which is known as coronavirus disease-2019 (COVID-2019) as a global pandemic. But, no specific medications are available for the treatment of COVID-19 so far. As a corollary, there is a need for a potential vaccine to impede the progression of the disease. Lately, it has been documented that the nucleocapsid (N) protein of SARS-CoV-2 is responsible for viral replication as well as interferes with host immune responses. We have comparatively analyzed the sequences of N protein of SARS-CoV-2 for the identification of core attributes and analyzed the ancestry through phylogenetic analysis. Subsequently, we have predicted the most immunogenic epitope for T-cell as well as B-cell. Importantly, our investigation mainly focused on major histocompatibility complex (MHC) class I potential peptides and NTASWFTAL interacted with most human leukocyte antigen (HLA) that are encoded by MHC class I molecules. Further, molecular docking analysis unveiled that NTASWFTAL possessed a greater affinity towards HLA and also available in a greater range of the population. Our study provides a consolidated base for vaccine design and we hope that this computational analysis will pave the way for designing novel vaccine candidates.","version":"1.1","doi":"10.1101/2020.05.16.100206","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.088989","pub_date":"2020-5-17","title":"Robust computational design and evaluation of peptide vaccines for cellular immunity with application to SARS-CoV-2","abstract":"We present a combinatorial machine learning method to evaluate and optimize peptide vaccine formulations, and we find for SARS-CoV-2 that it provides superior predicted display of viral epitopes by MHC class I and MHC class II molecules over populations when compared to other candidate vaccines. Our method is robust to idiosyncratic errors in the prediction of MHC peptide display and considers target population HLA haplotype frequencies during optimization. To minimize clinical development time our methods validate vaccines with multiple peptide presentation algorithms to increase the probability that a vaccine will be effective. We optimize an objective function that is based on the presentation likelihood of a diverse set of vaccine peptides conditioned on a target population HLA haplotype distribution and expected epitope drift. We produce separate peptide formulations for MHC class I loci (HLA-A, HLA-B, and HLA-C) and class II loci (HLA-DP, HLA-DQ, and HLA-DR) to permit signal sequence based cell compartment targeting using nucleic acid based vaccine platforms. Our SARS-CoV-2 MHC class I vaccine formulations provide 93.21% predicted population coverage with at least five vaccine peptide-HLA hits on average in an individual (\u2265 1 peptide 99.91%) with all vaccine peptides perfectly conserved across 4,690 geographically sampled SARS-CoV-2 genomes. Our MHC class II vaccine formulations provide 90.17% predicted coverage with at least five vaccine peptide-HLA hits on average in an individual with all peptides having observed mutation probability \u2264 0.001. We evaluate 29 previously published peptide vaccine designs with our evaluation tool with the requirement of having at least five vaccine peptide-HLA hits per individual, and they have a predicted maximum of 58.51% MHC class I coverage and 71.65% MHC class II coverage given haplotype based analysis. We provide an open source implementation of our design methods (OptiVax), vaccine evaluation tool (EvalVax), as well as the data used in our design efforts.","version":"1.1","doi":"10.1101/2020.05.16.088989","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.099499","pub_date":"2020-5-17","title":"SARS-CoV-2 amino acid substitutions widely spread in the human population are mainly located in highly conserved segments of the structural proteins","abstract":"The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic offers a unique opportunity to study the introduction and evolution of a pathogen into a completely na\u00efve human population. We identified and analysed the amino acid mutations that gained prominence worldwide in the early months of the pandemic. Eight mutations have been identified along the viral genome, mostly located in conserved segments of the structural proteins and showing low variability among coronavirus, which indicated that they might have a functional impact. At the moment of writing this paper, these mutations present a varied success in the SARS-CoV-2 virus population; ranging from a change in the spike protein that becomes absolutely prevalent, two mutations in the nucleocapsid protein showing frequencies around 25%, to a mutation in the matrix protein that nearly fades out after reaching a frequency of 20%.","version":"1.1","doi":"10.1101/2020.05.16.099499","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.084806","pub_date":"2020-5-17","title":"A single-cell RNA expression map of human coronavirus entry factors","abstract":"To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell RNA-sequencing data from a wide range of healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Among adult organs, enterocytes and goblet cells of the small intestine and colon, kidney proximal tubule cells, and gallbladder basal cells appear most permissive to SARS-CoV-2, consistent with clinical data. Our analysis also suggests alternate entry paths for SARS-CoV-2 infection of the lung, central nervous system, and heart. We predict spermatogonial cells and prostate endocrine cells, but not ovarian cells, to be highly permissive to SARS-CoV-2, suggesting male-specific vulnerabilities. Early stages of embryonic and placental development show a moderate risk of infection. The nasal epithelium looks like another battleground, characterized by high expression of both promoting and restricting factors and a potential age-dependent shift in SCARF expression. Lastly, SCARF expression appears broadly conserved across human, chimpanzee and macaque organs examined. Our study establishes an important resource for investigations of coronavirus biology and pathology.","version":"1.2","doi":"10.1101/2020.05.08.084806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091256","pub_date":"2020-5-17","title":"The Host Cell ViroCheckpoint: Identification and Pharmacologic Targeting of Novel Mechanistic Determinants of Coronavirus-Mediated Hijacked Cell States","abstract":"Most antiviral agents are designed to target virus-specific proteins and mechanisms rather than the host cell proteins that are critically dysregulated following virus-mediated reprogramming of the host cell transcriptional state. To overcome these limitations, we propose that elucidation and pharmacologic targeting of host cell Master Regulator proteins\u2014whose aberrant activities govern the reprogramed state of infected-coronavirus cells\u2014presents unique opportunities to develop novel mechanism-based therapeutic approaches to antiviral therapy, either as monotherapy or as a complement to established treatments. Specifically, we propose that a small module of host cell Master Regulator proteins (ViroCheckpoint) is hijacked by the virus to support its efficient replication and release. Conventional methodologies are not well suited to elucidate these potentially targetable proteins. By using the VIPER network-based algorithm, we successfully interrogated 12h, 24h, and 48h signatures from Calu-3 lung adenocarcinoma cells infected with SARS-CoV, to elucidate the time-dependent reprogramming of host cells and associated Master Regulator proteins. We used the NYS CLIA-certified Darwin OncoTreat algorithm, with an existing database of RNASeq profiles following cell perturbation with 133 FDA-approved and 195 late-stage experimental compounds, to identify drugs capable of virtually abrogating the virus-induced Master Regulator signature. This approach to drug prioritization and repurposing can be trivially extended to other viral pathogens, including SARS-CoV-2, as soon as the relevant infection signature becomes available.","version":"1.1","doi":"10.1101/2020.05.12.091256","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.17.100388","pub_date":"2020-5-17","title":"Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by virus-like particle","abstract":"With sequencing as a standard frontline protocol to identify emerging viruses such zika virus and SARS-CoV2, direct utilization of sequence data to program antivirals against the viruses could accelerate drug development to treat their infections. CRISPR-Cas effectors are promising candidates that could be programmed to inactivate viral genetic material based on sequence data but several challenges such as delivery and design of effective crRNA need to be addressed to realize practical use. Here, we showed that virus-like particle (VLP) could deliver PspCas13b-crRNA ribonucleoprotein (RNP) in nanomolar range to efficiently suppress dengue virus infection in primary human target cells. Shortening spacer length could significantly enhance RNA-targeting efficiency of PspCas13b in mammalian cells compared to the natural length of 30 nucleotides without compromising multiplex targeting by a crRNA array. Our results demonstrate the potentials of applying PspCas13b RNP to suppress RNA virus infection, with implications in targeting host RNA as well.","version":"1.1","doi":"10.1101/2020.05.17.100388","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.099176","pub_date":"2020-5-16","title":"A comprehensive germline variant and expression analyses of ACE2, TMPRSS2 and SARS-CoV-2 activator FURIN genes from the Middle East: Combating SARS-CoV-2 with precision medicine","abstract":"The severity of the new COVID-19 pandemic caused by the SARS-CoV-2 virus is strikingly variable in different global populations. SARS-CoV-2 uses ACE2 as a cell receptor, TMPRSS2 protease, and FURIN peptidase to invade human cells. Here, we investigated 1,378 whole-exome sequences of individuals from the Middle Eastern populations (Kuwait, Qatar, and Iran) to explore natural variations in the ACE2, TMPRSS2, and FURIN genes. We identified two activating variants (K26R and N720D) in the ACE2 gene that are more common in Europeans than in the Middle Eastern, East Asian, and African populations. We postulate that K26R can activate ACE2 and facilitate binding to S-protein RBD while N720D enhances TMPRSS2 cutting and, ultimately, viral entry. We also detected deleterious variants in FURIN that are frequent in the Middle Eastern but not in the European populations. This study highlights specific genetic variations in the ACE2 and FURIN genes that may explain SARS-CoV-2 clinical disparity. We showed structural evidence of the functionality of these activating variants that increase the SARS-CoV-2 aggressiveness. Finally, our data illustrate a significant correlation between ACE2 variants identified in people from Middle Eastern origins that can be further explored to explain the variation in COVID-19 infection and mortality rates globally.","version":"1.1","doi":"10.1101/2020.05.16.099176","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.045419","pub_date":"2020-5-16","title":"Sybodies targeting the SARS-CoV-2 receptor-binding domain","abstract":"The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike ectodomain. Among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of RBD interaction with human angiotensin-converting enzyme 2 (ACE2). Additionally, we identified a pair of anti-RBD sybodies that can simultaneously bind to the RBD. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource.","version":"1.2","doi":"10.1101/2020.04.16.045419","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.098947","pub_date":"2020-5-16","title":"Implications of SARS-CoV-2 mutations for genomic RNA structure and host microRNA targeting","abstract":"The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2 permissive host cells, we identified twelve separate target sequences in the SARS-CoV-2 genome; eight of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host could be constrained by host miRNA defence. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineered viral attenuation and antigen presentation.","version":"1.1","doi":"10.1101/2020.05.15.098947","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.16.097238","pub_date":"2020-5-16","title":"Mechanistic modeling of the SARS-CoV-2 and immune system interplay unravels design principles for diverse clinicopathological outcomes","abstract":"The disease caused by SARS-CoV-2 is a global pandemic that threatens to bring long-term changes worldwide. Approximately 80% of infected patients are asymptomatic or have mild symptoms such as fever or cough, while rest of the patients have varying degrees of severity of symptoms, with 3-4% mortality rate. Severe symptoms such as pneumonia and Acute Respiratory Distress Syndrome can be caused by tissue damage mostly due to aggravated and unresolved innate and adaptive immune response, often resulting from a cytokine storm. However, the mechanistic underpinnings of such responses remain elusive, with an incomplete understanding of how an intricate interplay among infected cells and cells of innate and adaptive immune system can lead to such diverse clinicopathological outcomes. Here, we use a dynamical systems approach to dissect the emergent nonlinear intra-host dynamics among virally infected cells, the immune response to it and the consequent immunopathology. By mechanistic analysis of cell-cell interactions, we have identified key parameters affecting the diverse clinical phenotypes associated with COVID-19. This minimalistic yet rigorous model can explain the various phenotypes observed across the clinical spectrum of COVID-19, various co-morbidity risk factors such as age and obesity, and the effect of antiviral drugs on different phenotypes. It also reveals how a fine-tuned balance of infected cell killing and resolution of inflammation can lead to infection clearance, while disruptions can drive different severe phenotypes. These results will help further the case of rational selection of drug combinations that can effectively balance viral clearance and minimize tissue damage simultaneously. The SARS-CoV-2 pandemic has already infected millions of people, and thousands of lives have been lost to it. The pandemic has already tested the limits of our public healthcare systems with a wide spectrum of clinicopathological symptoms and outcomes. The mechanistic underpinnings of the resultant immunopathology caused by the viral infection still remains to be elucidated. Here we propose a minimalistic but rigorous description of the interactions of the virus infected cells and the core components of the immune system that can potentially explain such diversity in the observed clinical outcomes. Our proposed framework could enable a platform to determine the efficacy of various treatment combinations and can contributes a conceptual understanding of dynamics of disease pathogenesis in SARS-CoV-2 infections.","version":"1.1","doi":"10.1101/2020.05.16.097238","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.096511","pub_date":"2020-5-16","title":"Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein","abstract":"Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.","version":"1.2","doi":"10.1101/2020.05.15.096511","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.092163","pub_date":"2020-5-16","title":"A Computational Toolset for Rapid Identification of SARS-CoV-2, other Viruses, and Microorganisms from Sequencing Data","abstract":"In this paper, we present a toolset and related resources for rapid identification of viruses and microorganisms from short-read or long-read sequencing data. We present fastv as an ultra-fast tool to detect microbial sequences present in sequencing data, identify target microorganisms, and visualize coverage of microbial genomes. This tool is based on the k-mer mapping and extension method. K-mer sets are generated by UniqueKMER, another tool provided in this toolset. UniqueKMER can generate complete sets of unique k-mers for each genome within a large set of viral or microbial genomes. For convenience, unique k-mers for microorganisms and common viruses that afflict humans have been generated and are provided with the tools. As a lightweight tool, fastv accepts FASTQ data as input, and directly outputs the results in both HTML and JSON formats. Prior to the k-mer analysis, fastv automatically performs adapter trimming, quality pruning, base correction, and other pre-processing to ensure the accuracy of k-mer analysis. Specifically, fastv provides built-in support for rapid SARS-CoV-2 identification and typing. Experimental results showed that fastv achieved 100% sensitivity and 100% specificity for detecting SARS-CoV-2 from sequencing data; and can distinguish SARS-CoV-2 from SARS, MERS, and other coronaviruses. This toolset is available at: https://github.com/OpenGene/fastv.","version":"1.2","doi":"10.1101/2020.05.12.092163","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.098731","pub_date":"2020-5-15","title":"Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase","abstract":"The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.05.15.098731","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.068890","pub_date":"2020-5-15","title":"Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design","abstract":"In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.","version":"1.2","doi":"10.1101/2020.04.29.068890","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091082","pub_date":"2020-5-15","title":"Androgen Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a global health crisis, and yet our understanding of the disease pathophysiology and potential treatment options remains limited. SARS-CoV-2 infection occurs through binding and internalization of the viral spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane. Lethal complications are caused by damage and failure of vital organs that express high levels of ACE2, including the lungs, the heart and the kidneys. Here, we established a high-throughput drug screening strategy to identify therapeutic candidates that reduce ACE2 levels in human embryonic stem cell (hESC) derived cardiac cells. Drug target analysis of validated hit compounds, including 5 alpha reductase inhibitors, revealed androgen signaling as a key modulator of ACE2 levels. Treatment with the 5 alpha reductase inhibitor dutasteride reduced ACE2 levels and internalization of recombinant spike receptor binding domain (Spike-RBD) in hESC-derived cardiac cells and human alveolar epithelial cells. Finally, clinical data on coronavirus disease 2019 (COVID-19) patients demonstrated that abnormal androgen states are significantly associated with severe disease complications and cardiac injury as measured by blood troponin T levels. These findings provide important insights on the mechanism of increased disease susceptibility in male COVID-19 patients and identify androgen receptor inhibition as a potential therapeutic strategy.","version":"1.2","doi":"10.1101/2020.05.12.091082","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.098616","pub_date":"2020-5-15","title":"SARS-CoV2 (COVID-19) Structural/Evolution Dynamicome: Insights into functional evolution and human genomics","abstract":"The SARS-CoV-2 pandemic, starting in 2019, has challenged the speed at which labs perform science, ranging from discoveries of the viral composition to handling health outcomes in humans. The small ~30kb single-stranded RNA genome of Coronaviruses makes them adept at cross species spread and drift, increasing their probability to cause pandemics. However, this small genome also allows for a robust understanding of all proteins coded by the virus. We employed protein modeling, molecular dynamic simulations, evolutionary mapping, and 3D printing to gain a full proteome and dynamicome understanding of SARS-CoV-2. The Viral Integrated Structural Evolution Dynamic Database (VIStEDD) has been established (prokoplab.com/vistedd), opening future discoveries and educational usage. In this paper, we highlight VIStEDD usage for nsp6, Nucleocapsid (N), and Spike (S) surface glycoprotein. For both nsp6 and N we reveal highly conserved surface amino acids that likely drive protein-protein interactions. In characterizing viral S protein, we have developed a quantitative dynamics cross correlation matrix insight into interaction with the ACE2/SLC6A19 dimer complex. From this quantitative matrix, we elucidated 47 potential functional missense variants from population genomic databases within ACE2/SLC6A19/TMPRSS2, warranting genomic enrichment analyses in SARS-CoV-2 patients. Moreover, these variants have ultralow frequency, but can exist as hemizygous in males for ACE2, which falls on the X-chromosome. Two noncoding variants (rs4646118 and rs143185769) found in ~9% of African descent individuals for ACE2 may regulate expression and be related to increased susceptibility of African Americans to SARS-CoV-2. This powerful database of SARS-CoV-2 can aid in research progress in the ongoing pandemic.","version":"1.1","doi":"10.1101/2020.05.15.098616","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.096719","pub_date":"2020-5-15","title":"IgA MAb blocks SARS-CoV-2 Spike-ACE2 interaction providing mucosal immunity","abstract":"COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.","version":"1.1","doi":"10.1101/2020.05.15.096719","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.095885","pub_date":"2020-5-15","title":"Predicting the Immunogenicity of T cell epitopes: From HIV to SARS-CoV-2","abstract":"We describe a physics-based learning model for predicting the immunogenicity of Cytotoxic T Lymphocyte (CTL) epitopes derived from diverse pathogens, given a Human Leukocyte Antigen (HLA) genotype. The model was trained and tested on experimental data on the relative immunodominance of CTL epitopes in Human Immunodeficiency Virus infection. The method is more accurate than publicly available models. Our model predicts that only a fraction of SARS-CoV-2 epitopes that have been predicted to bind to HLA molecules is immunogenic. The immunogenic CTL epitopes across all SARS-CoV-2 proteins are predicted to provide broad population coverage, but the immunogenic epitopes in the SARS-CoV-2 spike protein alone are unlikely to do so. Our model predicts that several immunogenic SARS-CoV-2 CTL epitopes are identical to those contained in low-pathogenicity coronaviruses circulating in the population. Thus, we suggest that some level of CTL immunity against COVID-19 may be present in some individuals prior to SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.05.14.095885","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.093054","pub_date":"2020-5-15","title":"A single dose SARS-CoV-2 simulating particle vaccine induces potent neutralizing activities","abstract":"Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for which a vaccine is urgently needed to control its spreading. To facilitate the representation of a native-like immunogen without being infectious, here, we reported a SARS-CoV-2 vaccine candidate (designated ShaCoVacc) by incorporating spike-encoding mRNA inside and decorating spike protein on the surface of the virus simulating particles (VSPs) derived from lentiviral particles. We characterized the mRNA copy number, glycosylation status, transduction efficiency, and innate immune property of the new vaccine platform. Importantly, we showed the ShaCoVacc induced strong spike-specific humoral immune responses and potent neutralizing activities by a single injection. Additionally, we disclosed the epitopes of spike-specific antibodies using peptide microarray and revealed epitopes susceptible to specific neutralizing antibodies. These results support further development of ShaCoVacc as a candidate vaccine for COVID-19 and VSP may serve as a new vaccine platform for emerging infectious diseases.","version":"1.1","doi":"10.1101/2020.05.14.093054","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.097311","pub_date":"2020-5-15","title":"Analytical and Clinical Comparison of Three Nucleic Acid Amplification Tests for SARS-CoV-2 Detection","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first identified in December 2019 and has quickly become a worldwide pandemic. In response, many diagnostic manufacturers have developed molecular assays for SARS-CoV-2 under the Food and Drug Administration (FDA) Emergency Use Authorization (EUA) pathway. This study compared three of these assays: the Hologic Panther Fusion SARS-CoV-2 assay (Fusion), the Hologic Aptima SARS-CoV-2 assay (Aptima) and the BioFire Diagnostics COVID-19 test (BioFire), to determine analytical and clinical performance, as well as workflow. All three assays showed a similar limit of detection (LOD) using inactivated virus, with 100% detection ranging from 500-1,000 genome equivalents/ml, whereas use of a quantified RNA transcript standard showed the same trend, but had values ranging from 62.5 to 125 copies/ml, confirming variability in absolute quantification of reference standards. The clinical correlation found that the Fusion and BioFire assays had a positive percent agreement (PPA) of 98.7%, followed by the Aptima assay at 94.7% when compared to the consensus result. All three assays exhibited 100% negative percent agreement (NPA). Analysis of discordant results revealed that all four samples missed by the Aptima assay had Ct values >37 on the Fusion assay. In conclusion, while all three assays showed similar relative LODs, we showed differences in absolute LODs depending on which standard was employed. In addition, the Fusion and BioFire assays showed better clinical performance, while the Aptima assay showed a modest decrease in overall PPA. These findings should be kept in mind when making platform testing decisions.","version":"1.1","doi":"10.1101/2020.05.14.097311","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088674","pub_date":"2020-5-15","title":"Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model","abstract":"The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.","version":"1.2","doi":"10.1101/2020.05.11.088674","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.097170","pub_date":"2020-5-15","title":"SARS-CoV-2 transcriptome analysis and molecular cataloguing of immunodominant epitopes for multi-epitope based vaccine design","abstract":"SARS-CoV-2 is a single-stranded RNA virus that has caused more than 0.29 million deaths worldwide as of May 2020, and influence of COVID-19 pandemic is increasing continuously in the absence of approved vaccine and drug. Moreover, very limited information is available about SARS-CoV-2 expressed regions and immune responses. In this paper an effort has been made, to facilitate vaccine development by proposing multiple epitopes as potential vaccine candidates by utilising SARS-CoV-2 transcriptome data. Here, publicly available RNA-seq data of SARS-CoV-2 infection in NHBE and A549 human cell lines were used to construct SARS-CoV-2 transcriptome to understand disease pathogenesis and immune responses. In the first step, epitope prediction, MHC class I and II gene identification for epitopes, population coverage, antigenicity, immunogenicity, conservation and crossreactivity analysis with host antigens were performed by using SARS-CoV-2 transcriptome, and in the second step, structural compatibility of identified T-and B-cell epitopes were evaluated with MHC molecules and B-cell receptors through molecular docking studies. Quantification of MHC gene expression was also performed that indicated high variation in allele types and expression level of MHC genes with respect to cell lines. In A549 cell line, HLA-A*30:01:01:01 and HLA-B*44:03:01:01 were highly expressed, whereas 92 variants of HLA-A*24 genes such as HLA-A*24:02:01:01, HLA-A*24:286, HLA-A*24:479Q, HLA-A*24:02:134 and HLA-A*24:02:116 were highly expressed in NHBE cell lines. Prevalence of HLA-A*24 alleles was suggested as risk factors for H1N1 infection, and associated with type-1 diabetes. HLA-C*03:03, linked with male infertility factors was also highly expressed in SARS-CoV-2 infected NHBE cell lines. Finally, three potential T-cell and five B-cell epitopes were selected for molecular docking studies with twenty-two MHC molecules and two B-cell receptors respectively. The results of in silico analysis indicated that proposed epitopes have high potential to recognize immune response of SARS-CoV-2 infection. This study will facilitate in vitro and in vivo vaccine related research studies.","version":"1.1","doi":"10.1101/2020.05.14.097170","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.097741","pub_date":"2020-5-15","title":"Analysis of SARS-CoV-2 RNA-Sequences by Interpretable Machine Learning Models","abstract":"We present an approach to investigate SARS-CoV-2 virus sequences based on alignment-free methods for RNA sequence comparison. In particular, we verify a given clustering result for the GISAID data set, which was obtained analyzing the molecular differences in coronavirus populations by phylogenetic trees. For this purpose, we use alignment-free dissimilarity measures for sequences and combine them with learning vector quantization classifiers for virus type discriminant analysis and classification. Those vector quantizers belong to the class of interpretable machine learning methods, which, on the one hand side provide additional knowledge about the classification decisions like discriminant feature correlations, and on the other hand can be equipped with a reject option. This option gives the model the property of self controlled evidence if applied to new data, i.e. the models refuses to make a classification decision, if the model evidence for the presented data is not given. After training such a classifier for the GISAID data set, we apply the obtained classifier model to another but unlabeled SARS-CoV-2 virus data set. On the one hand side, this allows us to assign new sequences to already known virus types and, on the other hand, the rejected sequences allow speculations about new virus types with respect to nucleotide base mutations in the viral sequences. The currently emerging global disease COVID-19 caused by novel SARS-CoV-2 viruses requires all scientific effort to investigate the development of the viral epidemy, the properties of the virus and its types. Investigations of the virus sequence are of special interest. Frequently, those are based on mathematical/statistical analysis. However, machine learning methods represent a promising alternative, if one focuses on interpretable models, i.e. those that do not act as black-boxes. Doing so, we apply variants of Learning Vector Quantizers to analyze the SARS-CoV-2 sequences. We encoded the sequences and compared them in their numerical representations to avoid the computationally costly comparison based on sequence alignments. Our resulting model is interpretable, robust, efficient, and has a self-controlling mechanism regarding the applicability to data. This framework was applied to two data sets concerning SARS-CoV-2. We were able to verify previously published virus type findings for one of the data sets by training our model to accurately identify the virus type of sequences. For sequences without virus type information (second data set), our trained model can predict them. Thereby, we observe a new scattered spreading of the sequences in the data space which probably is caused by mutations in the viral sequences.","version":"1.1","doi":"10.1101/2020.05.15.097741","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.090332","pub_date":"2020-5-15","title":"Distribution of ACE2, CD147, cyclophilins, CD26 and other SARS-CoV-2 associated molecules in human tissues and immune cells in health and disease","abstract":"Morbidity and mortality from COVID-19 caused by novel coronavirus SARS-CoV-2 is accelerating worldwide and novel clinical presentations of COVID-19 are often reported. The range of human cells and tissues targeted by SARS-CoV-2, its potential receptors and associated regulating factors are still largely unknown. The aim of our study was to analyze the expression of known and potential SARS-CoV-2 receptors and related molecules in the extensive collection of primary human cells and tissues from healthy subjects of different age and from patients with risk factors and known comorbidities of COVID-19. We performed RNA sequencing and explored available RNA-Seq databases to study gene expression and co-expression of ACE2, CD147 (BSG), CD26 (DPP4) and their direct and indirect molecular partners in primary human bronchial epithelial cells, bronchial and skin biopsies, bronchoalveolar lavage fluid, whole blood, peripheral blood mononuclear cells (PBMCs), monocytes, neutrophils, DCs, NK cells, ILC1, ILC2, ILC3, CD4+ and CD8+ T cells, B cells and plasmablasts. We analyzed the material from healthy children and adults, and from adults in relation to their disease or COVID-19 risk factor status. ACE2 and TMPRSS2 were coexpressed at the epithelial sites of the lung and skin, whereas CD147 (BSG), cyclophilins (PPIA and PPIB), CD26 (DPP4) and related molecules were expressed in both, epithelium and in immune cells. We also observed a distinct age-related expression profile of these genes in the PBMCs and T cells from healthy children and adults. Asthma, COPD, hypertension, smoking, obesity, and male gender status generally led to the higher expression of ACE2- and CD147-related genes in the bronchial biopsy, BAL or blood. Additionally, CD147-related genes correlated positively with age and BMI. Interestingly, we also observed higher expression of ACE2- and CD147-related genes in the lesional skin of patients with atopic dermatitis. Our data suggest different receptor repertoire potentially involved in the SARS-CoV-2 infection at the epithelial barriers and in the immune cells. Altered expression of these receptors related with age, gender, obesity and smoking, as well as with the disease status might contribute to COVID-19 morbidity and severity patterns.","version":"1.1","doi":"10.1101/2020.05.14.090332","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.094441","pub_date":"2020-5-15","title":"Genome Analysis of SARS-CoV-2 Isolate from Bangladesh","abstract":"Recently the first genome sequence for a Severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 isolate from Bangladesh became available. The sequencing was carried out by the Child Health Research Foundation and provided the first insight into the genetic details of the viral strain responsible for the SARS-CoV-2 infections in Bangladesh. Here we carried out a comparative study were we explored the phylogenetic relationship between the Bangladeshi isolate with other isolates from different parts of the world. Afterwards we identified single nucleotide variants in the Bangladeshi isolate, using the Wuhan virus reference sequence. We found a total of 9 variants in the Bangladeshi isolate using 2 separate tools. Barring 2, the rest of these variants were also observed in other isolates from different countries. Most of the variants occurred in the ORF1ab gen. Another noteworthy finding was a sequence of three consecutive variants in the N protein gene that were observed in other isolates as well. Lastly the phylogenetic analysis revealed a close relationship between the Bangladeshi isolate and those from Taiwan, Kazakhstan, Greece, California, Spain, Israel, and Sri Lanka.","version":"1.2","doi":"10.1101/2020.05.13.094441","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.097980","pub_date":"2020-5-15","title":"Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation","abstract":"COVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2\u2019-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.","version":"1.1","doi":"10.1101/2020.05.15.097980","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.097295","pub_date":"2020-5-15","title":"High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening","abstract":"SARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.","version":"1.1","doi":"10.1101/2020.05.14.097295","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.097493","pub_date":"2020-5-15","title":"Sequence characterization and molecular modeling of clinically relevant variants of the SARS-CoV-2 main protease","abstract":"The SARS-CoV-2 main protease (Mpro) is essential to viral replication and cleaves highly specific substrate sequences, making it an obvious target for inhibitor design. However, as for any virus, SARS-CoV-2 is subject to constant selection pressure, with new Mpro mutations arising over time. Identification and structural characterization of Mpro variants is thus critical for robust inhibitor design. Here we report sequence analysis, structure predictions, and molecular modeling for seventy-nine Mpro variants, constituting all clinically observed mutations in this protein as of April 29, 2020. Residue substitution is widely distributed, with some tendency toward larger and more hydrophobic residues. Modeling and protein structure network analysis suggest differences in cohesion and active site flexibility, revealing patterns in viral evolution that have relevance for drug discovery.","version":"1.1","doi":"10.1101/2020.05.15.097493","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.094839","pub_date":"2020-5-15","title":"Patient DNA cross-reactivity of the CDC SARS-CoV-2 extraction control leads to an inherent potential for false negative results","abstract":"Testing for RNA viruses such as SARS-CoV-2 requires careful handling of inherently labile RNA during sample collection, clinical processing, and molecular analysis. Tests must include fail-safe controls that affirmatively report the presence of intact RNA and demonstrate success of all steps of the assay. A result of \u201cno virus signal\u201d is insufficient for clinical interpretation: controls must also say \u201cThe reaction worked as intended and would have found virus if present.\u201d Unfortunately, a widely used test specified by the US Centers for Disease Control and Prevention (CDC) incorporates a control that does not perform as intended and claimed. Detecting SARS-CoV-2 with this assay requires both intact RNA and successful reverse transcription. The CDC-specified control does not require either of these, due to its inability to differentiate human genomic DNA from reverse-transcribed RNA. Patient DNA is copurified from nasopharyngeal swabs during clinically-approved RNA extraction and is sufficient to return an \u201cextraction control success\u201d signal using the CDC design. As such, this assay fails-unsafe: truly positive patient samples return a false-negative result of \u201cno virus detected, control succeeded\u201d following any of several readily-encountered mishaps. This problem affects tens-of-millions of patients worth of shipped assays, but many of these flawed reagents have not yet been used. There is an opportunity to improve this important diagnostic tool. As demonstrated here, a re-designed transcript-specific control correctly monitors sample collection, extraction, reverse transcription, and qPCR detection. This approach can be rapidly implemented and will help reduce truly positive patients from being incorrectly given the all-clear. A widely-used COVID-19 diagnostic is mis-designed and generates false-negative results, dangerously confusing \u201cNo\u201d with \u201cDon\u2019t know\u201d \u2013 but it\u2019s fixable","version":"1.2","doi":"10.1101/2020.05.13.094839","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.097204","pub_date":"2020-5-15","title":"Expression of ACE2 and TMPRSS2 proteins in the upper and lower aerodigestive tracts of rats","abstract":"Patients with coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibit not only respiratory symptoms but also symptoms of chemo-sensitive disorders and kidney failure. Cellular entry of SARS-CoV-2 depends on the binding of its spike protein to a cellular receptor named angiotensin-converting enzyme 2 (ACE2), and the subsequent spike protein-priming by host cell proteases, including transmembrane protease serine 2 (TMPRSS2). Thus, high expression of ACE2 and TMPRSS2 are considered to enhance the invading capacity of SARS-CoV-2. To elucidate the underlying histological mechanisms of the aerodigestive disorders caused by SARS-CoV-2, we investigated the expression of ACE2 and TMPRSS2 proteins in the aerodigestive tracts of the tongue, hard palate with partial nasal tissue, larynx with hypopharynx, trachea, esophagus, lung, and kidney of rats through immunohistochemistry. Strong co-expression of ACE2 and TMPRSS2 proteins was observed in the nasal respiratory epithelium, trachea, bronchioles, alveoli, kidney, and taste buds of the tongue. Remarkably, TMPRSS2 expression was much stronger in the peripheral alveoli than in the central alveoli. These results coincide with the reported clinical symptoms of COVID-19, such as the loss of taste, loss of olfaction, respiratory dysfunction, and acute nephropathy. A wide range of organs have been speculated to be affected by SARS-CoV-2 depending on the expression levels of ACE2 and TMPRSS2. Differential distribution of TMPRSS2 in the lung indicated the COVID-19 symptoms to possibly be exacerbated by TMPRSS2 expression. This study might provide potential clues for further investigation of the pathogenesis of COVID-19. NA","version":"1.1","doi":"10.1101/2020.05.14.097204","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.095620","pub_date":"2020-5-15","title":"Unveiling diffusion pattern and structural impact of the most invasive SARS-CoV-2 spike mutation","abstract":"Starting in Wuhan, China, SARS-CoV-2 epidemics quickly propagated worldwide in less than three months, geographically sorting genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries and/or actual advantage in virus transmissibility could explain the high frequency reached by some genomic variants during the course of the outbreak. Using a suite of statistical, population genetics, and theoretical approaches, we show that the globally most represented spike protein variant (i.e., the G clade, A \u2192 G nucleotide change at genomic position 23,403; D \u2192 G amino acid change at spike protein position 614) i) underwent a significant demographic expansion in most countries not explained by stochastic effects or enhanced pathogenicity; ii) affects the spike S1/S2 furin-like site increasing its conformational plasticity (short range effect), and iii) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our study unambiguously links the spread of the G614 with a non-random process, and we hypothesize that this process is related to the selective advantage produced by a specific structural modification of the spike protein. We conclude that the different conformation of the S1/S2 proteolytic site is at the basis of the higher transmission rate of this invasive SARS-CoV-2 variant, and provide structural information to guide the design of selective and efficient drugs.","version":"1.1","doi":"10.1101/2020.05.14.095620","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.095224","pub_date":"2020-5-15","title":"Two mutations P/L and Y/C in SARS-CoV-2 helicase domain exist together and influence helicase RNA binding","abstract":"RNA helicases play pivotal role in RNA replication by catalysing the unwinding of complex RNA duplex structures into single strands in ATP/NTP dependent manner. SARS coronavirus 2 (SARS-CoV-2) is a single stranded positive sense RNA virus belonging to the family Coronaviridae. The viral RNA encodes non structural protein Nsp13 or the viral helicase protein that helps the viral RNA dependent RNA polymerase (RdRp) to execute RNA replication by unwinding the RNA duplexes. In this study we identified a novel mutation at position 541of the helicase where the tyrosine (Y) got substituted with cytosine (C). We found that Y541C is a destabilizing mutation increasing the molecular flexibility and leading to decreased affinity of helicase binding with RNA. Earlier we had reported a mutation P504L in the helicase protein for which had not performed RNA binding study. Here we report that P504L mutation leads to increased affinity of helicase RNA interaction. So, both these mutations have opposite effects on RNA binding. Moreover, we found a significant fraction of isolate population where both P504L and Y541C mutations were co-existing.","version":"1.1","doi":"10.1101/2020.05.14.095224","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.15.077313","pub_date":"2020-5-15","title":"CoV-AbDab: the Coronavirus Antibody Database","abstract":"The emergence of a novel strain of betacoronavirus, SARS-CoV-2, has led to a pandemic that has been associated with hundreds of thousands of deaths. Research is ongoing around the world to create vaccines and therapies to minimise rates of disease spread and mortality. Crucial to these efforts are molecular characterisations of neutralising antibodies to SARS-CoV-2. Such antibodies would be valuable for measuring vaccine efficacy, diagnosing exposure, and developing effective biotherapeutics. Here, we describe our new database, CoV-AbDab, which already contains data on over 380 published/patented antibodies and nanobodies known to bind to at least one betacoronavirus. This database is the first consolidation of antibodies known to bind SARS-CoV-2 and other betacoronaviruses such as SARS-CoV-1 and MERS-CoV. We supply relevant metadata such as evidence of cross-neutralisation, antibody/nanobody origin, full variable domain sequence (where available) and germline assignments, epitope region, links to relevant PDB entries, homology models, and source literature. Our preliminary analysis exemplifies a spectrum of potential applications for the database, including identifying characteristic germline usage biases in receptor-binding domain antibodies and contextualising the diagnostic value of the SARS-CoV binding CDRH3s through comparison to over 500 million antibody sequences from SARS-CoV serologically naive individuals. Community submissions are invited to ensure CoV-AbDab is efficiently updated with the growing body of data analysing SARS-CoV-2. CoV-AbDab is freely available and downloadable on our website at http://opig.stats.ox.ac.uk/webapps/coronavirus.","version":"1.1","doi":"10.1101/2020.05.15.077313","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.095133","pub_date":"2020-5-14","title":"Identification of conserved epitopes in SARS-CoV-2 spike and nucleocapsid protein","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first occurred in Wuhan (China) in December 2019, is a novel virus that causes a severe acute respiratory disease. The virus spike glycoproteins and nucleocapsid proteins are the main targets for the development of vaccines and antiviral drugs, to control the disease spread. We herein study the structural order-disorder propensity and the rates of evolution of these two proteins to characterize their B cell and T cell epitopes, previously suggested to contribute to immune response caused by SARS-CoV-2 infections. We first analyzed the rates of evolution along the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes. For this purpose, we compared orthologs from seven human coronaviruses: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. We then focus on the local, structural order-disorder propensities of the protein regions where the SARS-CoV-2 epitopes are located. We show that the vast majority of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains and some of them are characterized by low rates of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well-conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by high rates of evolution, probably to overcome host immunity. In conclusion, our results provide evidence for conserved epitopes that may help to develop long-lasting, broad-spectrum SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2020.05.14.095133","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.096727","pub_date":"2020-5-14","title":"Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis)","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6\u201312 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.","version":"1.1","doi":"10.1101/2020.05.14.096727","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.092478","pub_date":"2020-5-14","title":"Novel ACE2-Independent Carbohydrate-Binding of SARS-CoV-2 Spike Protein to Host Lectins and Lung Microbiota","abstract":"The immediate call for translational research in the field of coronavirus disease (COVID-19) pandemic, needs new and unexplored angles to support and contribute to this important worldwide health problem. The aim of this study is to better understand the pathogenic mechanisms underlying COVID-19, deciphering the carbohydrate-mediated interactions of the SARS-CoV-2 spike protein. We studied the carbohydrate-binding receptors that could be important for viral entry and for immune-modulatory responses, and we studied the interactions of the spike protein with the host lung microbiota. Exploring solid-phase immunoassays, we evaluated the interactions between the SARS-CoV-2 spike protein and a library of 12 different human carbohydrate-binding proteins (C-type lectins and Siglecs) involved in binding, triggering and modulation of innate and adaptive immune-responses. We revealed a specific binding of the SARS-CoV-2 spike protein to the receptors DC-SIGN, MGL, Siglec-9 and Siglec-10 that are all expressed on myeloid immune cells. In addition, because the lung microbiota can promote or modulate viral infection, we studied the interactions between the SARS-CoV-2 spike protein and a library of Streptococcus pneumoniae capsular polysaccharides, as well as other bacterial glyco-conjugates. We show specific binding of the spike protein to different S. pneumoniae capsular polysaccharides (serotypes 19F and 23F but not to serotype 14). Moreover we demonstrated a specific binding of SARS-CoV-2 spike protein to the lipopolysaccharide from the opportunistic human pathogen Pseudomonas aeruginosa, one of the leading cause of acute nosocomial infections and pneumonia. Interestingly, we identified rhamnosylated epitopes as one of the discriminating structures in lung microbiota to bind SARS-CoV-2 spike protein. In conclusion, we revealed novel ACE2-independent carbohydrate-mediated interactions with immune modulating lectins expressed on myeloid cells, as well as host lung microbiota glyco-conjugates. Our results identified new molecular pathways using host lectins and signalling, that may contribute to viral infection and subsequent immune exacerbation. Moreover we identified specific rhamnosylated epitopes in lung microbiota to bind SARS-CoV-2, providing a hypothetical link between the presence of specific lung microbiota and SARS-CoV-2 infection and severity.","version":"1.1","doi":"10.1101/2020.05.13.092478","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.092767","pub_date":"2020-5-14","title":"ACE2-Variants Indicate Potential SARS-CoV-2-Susceptibility in Animals: An Extensive Molecular Dynamics Study","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late 2019 and since evolved into a global threat with nearly 4.4 million infected people and over 290,000 confirmed deaths worldwide. SARS-CoV-2 is an enveloped virus presenting spike (S) glycoproteins on its outer surface. Binding of S to host cell angiotensin converting enzyme 2 (ACE2) is thought to be critical for cellular entry. The host range of the virus extends far beyond humans and non-human primates. Natural and experimental infections have confirmed high susceptibility of cats, ferrets, and hamsters, whereas dogs, mice, rats, pigs, and chickens seem refractory to SARS-CoV-2 infection. To investigate the reason for the variable susceptibility observed in different species, we have developed molecular descriptors to efficiently analyze our dynamic simulation models of complexes between SARS-CoV-2 S and ACE2. Based on our analyses we predict that: (i) the red squirrel is likely susceptible to SARS-CoV-2 infection and (ii) specific mutations in ACE2 of dogs, rats, and mice render them susceptible to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.05.14.092767","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.14.093757","pub_date":"2020-5-14","title":"Immunoinformatic identification of B cell and T cell epitopes in the SARS-CoV-2 proteome","abstract":"A novel coronavirus (SARS-CoV-2) emerged from China in late 2019 and rapidly spread across the globe, infecting millions of people and generating societal disruption on a level not seen since the 1918 influenza pandemic. A safe and effective vaccine is desperately needed to prevent the continued spread of SARS-CoV-2; yet, rational vaccine design efforts are currently hampered by the lack of knowledge regarding viral epitopes targeted during an immune response, and the need for more in-depth knowledge on betacoronavirus immunology. To that end, we developed a computational workflow using a series of open-source algorithms and webtools to analyze the proteome of SARS-CoV-2 and identify putative T cell and B cell epitopes. Using increasingly stringent selection criteria to select peptides with significant HLA promiscuity and predicted antigenicity, we identified 41 potential T cell epitopes (5 HLA class I, 36 HLA class II) and 6 potential B cell epitopes, respectively. Docking analysis and binding predictions demonstrated enrichment for peptide binding to HLA-B (class I) and HLA-DRB1 (class II) molecules. Overlays of predicted B cell epitopes with the structure of the viral spike (S) glycoprotein revealed that 4 of 6 epitopes were located in the receptor-binding domain of the S protein. To our knowledge, this is the first study to comprehensively analyze all 10 (structural, non-structural and accessory) proteins from SARS-CoV-2 using predictive algorithms to identify potential targets for vaccine development. The novel coronavirus SARS-CoV-2 recently emerged from China, rapidly spreading and ushering in a global pandemic. Despite intensive research efforts, our knowledge of SARS-CoV-2 immunology and the proteins targeted by the immune response remains relatively limited, making it difficult to rationally design candidate vaccines. We employed a suite of bioinformatic tools, computational algorithms, and structural modeling to comprehensively analyze the entire SARS-CoV-2 proteome for potential T cell and B cell epitopes. Utilizing a set of stringent selection criteria to filter peptide epitopes, we identified 41 T cell epitopes (5 HLA class I, 36 HLA class II) and 6 B cell epitopes that could serve as promising targets for peptide-based vaccine development against this emerging global pathogen.","version":"1.1","doi":"10.1101/2020.05.14.093757","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088781","pub_date":"2020-5-14","title":"Structural insight into the putative role of novel SARS CoV-2 E protein in viral infection: a potential target for LAV development and therapeutic strategies","abstract":"The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases. Using the bioinformatics and structural modeling approach, we modeled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS-CoV-1, and is highly conserved in the N-terminal regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modeling, we found that the most favorable structure could function as a gated proton channel. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds. Additionally, two distinct \u201ccore\u201d structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral proton channeling activity which may play a critical role in viral infection. In addition, it provides a structural basis and additional avenues for LAV development and generating therapeutic interventions against the virus. Structural modeling of the novel SARS-CoV-2 envelope protein (E-protein) demonstrating its possible proton channeling activity","version":"1.2","doi":"10.1101/2020.05.11.088781","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.09.085613","pub_date":"2020-5-14","title":"Potential modes of COVID-19 transmission from human eye revealed by single-cell atlas","abstract":"There is a pressing urgency to understand the entry route of SARS-CoV-2 viruses into the human body. SARS-CoV-2 viruses enter through ACE2 receptors after the S proteins of the virus are primed by proteases such as TMPRSS2. Most studies focused on the airway epithelial and lung alveolar cells as the route of infection, while the mode of transmission through the ocular route is not well established. Here, we profiled the presence of SARS-CoV-2 receptors and receptor-associated enzymes at single-cell resolution of thirty-three human ocular cell types. We identified unique populations of corneal cells with high ACE2 expression, among which the conjunctival cells co-expressed both ACE2 and TMPRSS2, suggesting that they could serve as the entry points for the virus. Integrative analysis further models the signaling and transcription regulon networks involved in the infection of distinct corneal cells. Our work constitutes a unique resource for the development of new treatments and management of COVID-19.","version":"1.3","doi":"10.1101/2020.05.09.085613","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079848","pub_date":"2020-5-14","title":"Repurposing low\u2013molecular-weight drugs against the main protease of severe acute respiratory syndrome coronavirus 2","abstract":"The coronavirus disease (COVID-19) pandemic caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the global healthcare system. Drug repurposing is a feasible method for emergency treatment. As low\u2013molecular-weight drugs have high potential to completely match interactions with essential SARS-CoV-2 targets, we propose a strategy to identify such drugs using the fragment-based approach. Herein, using ligand- and protein-observed fragment screening approaches, we identified niacin and hit 1 binding to the catalytic pocket of the main protease of the SARS-CoV-2 (Mpro), thereby modestly inhibiting the enzymatic activity of Mpro. Chemical shift perturbations induced by niacin and hit 1 indicate a partial overlap of their binding sites, i.e., the catalytic pocket of Mpro may accommodate derivatives with large molecular sizes. Therefore, we searched for drugs containing niacin or hit 1 pharmacophores and identified carmofur, bendamustine, triclabendazole, and emedastine; these drugs are highly capable of inhibiting protease activity. Our study demonstrates that the fragment-based approach is a feasible strategy for identifying low\u2013molecular-weight drugs against the SARS-CoV-2 and other potential targets lacking specific drugs.","version":"1.2","doi":"10.1101/2020.05.05.079848","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093195","pub_date":"2020-5-13","title":"ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques","abstract":"Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 2019 and is responsible for the COVID-19 pandemic. Vaccines are an essential countermeasure urgently needed to control the pandemic. Here, we show that the adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, is immunogenic in mice, eliciting a robust humoral and cell-mediated response. This response was not Th2 dominated, as demonstrated by IgG subclass and cytokine expression profiling. A single vaccination with ChAdOx1 nCoV-19 induced a humoral and cellular immune response in rhesus macaques. We observed a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated rhesus macaques. Importantly, no evidence of immune-enhanced disease following viral challenge in vaccinated animals was observed. ChAdOx1 nCoV-19 is currently under investigation in a phase I clinical trial. Safety, immunogenicity and efficacy against symptomatic PCR-positive COVID-19 disease will now be assessed in randomised controlled human clinical trials.","version":"1.1","doi":"10.1101/2020.05.13.093195","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093971","pub_date":"2020-5-13","title":"Insights into molecular evolution recombination of pandemic SARS-CoV-2 using Saudi Arabian sequences","abstract":"The recently emerged SARS-CoV-2 (Coronaviridae; Betacoronavirus) is the underlying cause of COVID-19 disease. Here we assessed SARS-CoV2 from the Kingdom of Saudi Arabia alongside sequences of SARS-CoV, bat SARS-like CoVs and MERS-CoV, the latter currently detected in this region. Phylogenetic analysis, natural selection investigation and genome recombination analysis were performed. Our analysis showed that all Saudi SARS-CoV-2 sequences are of the same origin and closer proximity to bat SARS-like CoVs, followed by SARS-CoVs, however quite distant to MERS-CoV. Moreover, genome recombination analysis revealed two recombination events between SARS-CoV-2 and bat SARS-like CoVs. This was further assessed by S gene recombination analysis. These recombination events may be relevant to the emergence of this novel virus. Moreover, positive selection pressure was detected between SARS-CoV-2, bat SL-CoV isolates and human SARS-CoV isolates. However, the highest positive selection occurred between SARS-CoV-2 isolates and 2 bat-SL-CoV isolates (Bat-SL-RsSHC014 and Bat-SL-CoVZC45). This further indicates that SARS-CoV-2 isolates were adaptively evolved from bat SARS-like isolates, and that a virus with originating from bats triggered this pandemic. This study thuds sheds further light on the origin of this virus. The emergence and subsequent pandemic of SARS-CoV-2 is a unique challenge to countries all over the world, including Saudi Arabia where cases of the related MERS are still being reported. Saudi SARS-CoV-2 sequences were found to be likely of the same or similar origin. In our analysis, SARS-CoV-2 were more closely related to bat SARS-like CoVs rather than to MERS-CoV (which originated in Saudi Arabia) or SARS-CoV, confirming other phylogenetic efforts on this pathogen. Recombination and positive selection analysis further suggest that bat coronaviruses may be at the origin of SARS-CoV-2 sequences. The data shown here give hints on the origin of this virus and may inform efforts on transmissibility, host adaptation and other biological aspects of this virus.","version":"1.1","doi":"10.1101/2020.05.13.093971","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.094482","pub_date":"2020-5-13","title":"Propagation, inactivation, and safety testing of SARS-CoV-2","abstract":"In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. Because work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable safe study of RNA, DNA and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.","version":"1.1","doi":"10.1101/2020.05.13.094482","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093781","pub_date":"2020-5-13","title":"In Vitro Inhibition of SARS-CoV-2 Infection by Bovine Lactoferrin","abstract":"Since its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been posing a serious threat to public health worldwide as the causative agent of coronavirus disease 2019 (COVID-19). Now distributed in a pandemic pattern, this disease still lacks an effective drug treatment with low toxicity, leading pharmaceutical companies and research labs to work against time to find a candidate molecule to efficiently treat the affected patients. Due to the well-known broad-spectrum antimicrobial activity of the lactoferrin protein, we sought to verify whether its bovine form (bLf) would also be effective in vitro against SARS-CoV-2. Using an antiviral assay based on quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found that bLf reduced progeny virus yield by up to \u223c84,6% in African green monkey kidney epithelial cells (Vero E6) and \u223c68,6% in adenocarcinomic human alveolar basal epithelial cells (A549) at 1 mg/mL, a concentration previously shown to have low cytotoxicity. Therefore, our preliminary data suggest that bLf has the potential to constitute a biochemical approach to fight the new coronavirus pandemic.","version":"1.1","doi":"10.1101/2020.05.13.093781","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091918","pub_date":"2020-5-13","title":"Antibody repertoire induced by SARS-CoV-2 spike protein immunogens","abstract":"Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody response generated following vaccination by these vaccine modalities. To better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various SARS-CoV-2 spike protein antigens: S-ectodomain (S1+S2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (aa 16-685), the receptor-binding domain (RBD) (aa 319-541), and the S2 domain (aa 686-1213 as control). Antibody response was analyzed by ELISA, Surface Plasmon Resonance (SPR) against different Spike proteins in native conformation, and a pseudovirion neutralization assay to measure the quality and function of the antibodies elicited by the different Spike antigens. All three antigens (S1+S2 ectodomain, S1 domain, and RBD) generated strong neutralizing antibodies against SARS-CoV-2. Vaccination induced antibody repertoire was analyzed by SARS-CoV-2 spike Genome Fragment Phage Display Libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD and S2 domains. Furthermore, these analyses demonstrated that surprisingly the RBD immunogen elicited a higher antibody titer with 5-fold higher affinity antibodies to native spike antigens compared with other spike antigens. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease. SARS-CoV-2 Spike induced immune response","version":"1.1","doi":"10.1101/2020.05.12.091918","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.090324","pub_date":"2020-5-13","title":"Infection Groups Differential (IGD) Score Reveals Infection Ability Difference between SARS-CoV-2 and Other Coronaviruses","abstract":"The Corona Virus Disease 2019 (COVID-19) pandemic that began in late December 2019 has resulted in millions of cases diagnosed worldwide. Reports have shown that SARS-CoV-2 shows extremely higher infection rates than other coronaviruses. This study conducted a phylogenetics analysis of 91 representative coronaviruses and found that the functional spike protein of SARS-CoV-2, which interacts with the human receptor ACE2, is actually not undergoing distinct selection pressure compared to other coronaviruses. Furthermore, we define a new measurement, infection group differential (IGD) score, in assessing the infection ability of two human coronavirus groups. There are nine extremely high IGD (ehIGD) sites in the receptor-binding domain (RBD) out of 40 high IGD (hIGD) sites that exhibit a unique infection-related pattern from the haplotype network and docking energy comparison. These 40 hIGD sites are basically conserved among the SARS-CoV-2, i.e. there are only two hIGD sites mutated in four out of 1,058 samples, defined as rare-mutation hIGD (rhIGD) sites. In conclusion, ehIGD and rhIGD sites might be of great significance to the development of vaccines.","version":"1.1","doi":"10.1101/2020.05.12.090324","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.089862","pub_date":"2020-5-13","title":"Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies","abstract":"As the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 57 samples from COVID-19 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 53 tested negative and 4 tested positive for IgA (93.0% agreement) while 56 tested negative and 1 tested positive for IgG (98.2% agreement). For COVID-19 PCR-positive patients, 29 of 30 (96.7%) samples collected \u22653 days after positive PCR were positive for IgA, and 28 of 28 samples collected \u22654 days after positive PCR were positive for IgG. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrates excellent sensitivity for detection of IgA and IgG antibodies from samples collected \u22653 days and \u22654 days, respectively, after COVID-19 diagnosis by PCR. This assay did not demonstrate cross reaction in any of the 28 samples from patients with common human coronaviruses, including types HKU1, NL63, CV229E, and OC43.","version":"1.1","doi":"10.1101/2020.05.11.089862","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.092171","pub_date":"2020-5-13","title":"Structure of SARS-CoV-2 main protease in the apo state reveals the inactive conformation","abstract":"Mpro is of considerable interest as a drug target in the treatment of COVID-19 since the proteolytic activity of this viral protease is essential for viral replication. Here we report the first insight of the structure Mpro for SARS-CoV-2 in the inactive conformation under conditions close to the physiological state (pH 7.5) to an overall resolution of 1.9 \u00c5. The comparisons of Mpro in different states reveal that substrate binding site and the active site are more flexible in the inactive conformation than that in the active conformations. Notably, compared with the active conformation of the apo state structure in pH7.6 of SARS, the SARS-CoV-2 apo state is in the inactive conformation under condition close to physiological state (pH7.5). Two water molecules are present in the oxyanion hole in our apo state structure, whereas in the ligand-bound structure, water molecular is absence in the same region. This structure provides novel and important insights that have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational, structure-based, approaches for the design of specific SARS-CoV-2 ligands as new therapeutic agents.","version":"1.1","doi":"10.1101/2020.05.12.092171","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.094490","pub_date":"2020-5-13","title":"A rapid, point of care red blood cell agglutination assay for detecting antibodies against SARS-CoV-2","abstract":"The COVID-19 pandemic has brought the world to a halt, with cases observed around the globe causing significant mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess the prevalence of infection, as well as ascertain individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which may lack scalability to meet the demand of hundreds of millions of antibody tests in the coming year. Herein, we present an alternative method of antibody testing that just depends on one protein reagent being added to patient serum/plasma or whole blood and a short five-minute assay time. A novel fusion protein was designed that binds red blood cells (RBC) via a single-chain variable fragment (scFv) against the H antigen and displays the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Upon mixing of the fusion protein, RBD-scFv with recovered COVID-19 patient serum and RBCs, we observed agglutination of RBCs, indicating the patient developed antibodies against SARS-CoV-2 RBD. Given that the test uses methods routinely used in hospital clinical labs across the world, we anticipate the test can be rapidly deployed with only the protein reagent required at projected manufacturing cost at U.S. cents per test. We anticipate our agglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.","version":"1.1","doi":"10.1101/2020.05.13.094490","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.092387","pub_date":"2020-5-13","title":"Comparison of SARS-CoV-2 Indirect and Direct Detection Methods","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies \u2013 2.1\u00d7105 copies of viral Orf1ab/\u03bcl. However, the BGI detection system is \u223c4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were \u223c10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.","version":"1.1","doi":"10.1101/2020.05.12.092387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.092056","pub_date":"2020-5-13","title":"Tracing two causative SNPs reveals SARS-CoV-2 transmission in North America population","abstract":"During the COVID-19 pandemic, precisely tracing the route of the SARS-CoV-2 transmission in human population remains challenging. Because this RNA virus can mutate massively without a specifically tracing maker. Herein, using a geographic stratified genome-wide association study (GWAS) of 2599 full-genome sequences, we identified that two SNPs (i.e., 1059.C>T and 25563.G>T) of linkage disequilibrium were presented in approximately half of North America SARS-CoV-2 population (p = 2.44 x 10\u2212212 and p = 2.98 x 10\u2212261), resulting two missense mutations (i.e., Thr 265 Ile and Gln 57 His) in ORF1ab and ORF3a, respectively. Interestingly, these two SNPs exclusively occurred in the North America dominated clade 1, accumulated during mid to late March, 2020. We did not find any of these two SNPs by retrospectively tracing the two SNPs in bat and pangolin related SARS-CoV-2 and human SARS-CoV-2 from the first epicenter Wuhan or other regions of China mainland. This suggested that the SARS-CoV-2 population of Chinese mainland were different from the prevalent strains of North America. Time-dependently, we found that these two SNPs first occurred in Europe SARS-CoV-2 (26-Feb-2020) which was 3 days early than the occurring date of North America isolates and 17 days early for Asia isolates (Taiwan China dominated). Collectively, this population genetic analysis highlights a well-confidential transmission route of the North America isolates and the two SNPs we newly identified are possibly novel diagnosable or druggable targets for surveillance and treatment.","version":"1.1","doi":"10.1101/2020.05.12.092056","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.092379","pub_date":"2020-5-13","title":"Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens","abstract":"Several point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. The aim of this study was to assess test performance of the Accula SARS-CoV-2 test. The performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen\u2019s kappa coefficient. Overall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). Compared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.","version":"1.1","doi":"10.1101/2020.05.12.092379","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093690","pub_date":"2020-5-13","title":"Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection","abstract":"New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.05.13.093690","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091462","pub_date":"2020-5-13","title":"Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein","abstract":"Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.","version":"1.1","doi":"10.1101/2020.05.12.091462","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.092577","pub_date":"2020-5-13","title":"Lung Disease Network Reveals the Impact of Comorbidity on SARS-CoV-2 infection","abstract":"Higher mortality of COVID19 patients with comorbidity is the formidable challenge faced by the health care system. In response to the present crisis, understanding the molecular basis of comorbidity is essential to accelerate the development of potential drugs. To address this, we have measured the genetic association between COVID19 and various lung disorders and observed a remarkable resemblance. 141 lung disorders directly or indirectly linked to COVID19 result in a high-density disease-disease association network that shows a small-world property. The clustering of many lung diseases with COVID19 demonstrates a greater complexity and severity of SARS-CoV-2 infection. Furthermore, our results show that the functional protein-protein interaction modules involved RNA and protein metabolism, substantially hijacked by SARS-CoV-2, are connected to several lung disorders. Therefore we recommend targeting the components of these modules to inhibit the viral growth and improve the clinical conditions in comorbidity.","version":"1.1","doi":"10.1101/2020.05.13.092577","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093609","pub_date":"2020-5-13","title":"Evaluation Of SYBR Green Real Time PCR For Detecting SARS-CoV-2 From Clinical Samples","abstract":"The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since all these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low and middle income countries. In the interest of economy, time and availability of chemicals and consumables, the SYBR Green-based detection was implemented to establish a convenient assay. Therefore, we adapted one of WHO recommended Taqman-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results suggest that SYBR-Green detection represents a reliable cost-effective alternative to increase the testing capacity.","version":"1.1","doi":"10.1101/2020.05.13.093609","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.13.093658","pub_date":"2020-5-13","title":"Evolutionary arms race between virus and host drives genetic diversity in bat SARS related coronavirus spike genes","abstract":"The Chinese horseshoe bat (Rhinolophus sinicus), reservoir host of severe acute respiratory syndrome coronavirus (SARS-CoV), carries many bat SARS-related CoVs (SARSr-CoVs) with high genetic diversity, particularly in the spike gene. Despite these variations, some bat SARSr-CoVs can utilize the orthologs of human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), for entry. It is speculated that the interaction between bat ACE2 and SARSr-CoV spike proteins drives diversity. Here, we have identified a series of R. sinicus ACE2 variants with some polymorphic sites involved in the interaction with the SARS-CoV spike protein. Pseudoviruses or SARSr-CoVs carrying different spike proteins showed different infection efficiency in cells transiently expressing bat ACE2 variants. Consistent results were observed by binding affinity assays between SARS- and SARSr-CoV spike proteins and receptor molecules from bats and humans. All tested bat SARSr-CoV spike proteins had a higher binding affinity to human ACE2 than to bat ACE2, although they showed a 10-fold lower binding affinity to human ACE2 compared with their SARS-CoV counterpart. Structure modeling revealed that the difference in binding affinity between spike and ACE2 might be caused by the alteration of some key residues in the interface of these two molecules. Molecular evolution analysis indicates that these residues were under strong positive selection. These results suggest that the SARSr-CoV spike protein and R. sinicus ACE2 may have coevolved over time and experienced selection pressure from each other, triggering the evolutionary arms race dynamics. It further proves that R. sinicus is the natural host of SARSr-CoVs. Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoV in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS- and SARSr-CoV infection but with different binding affinity to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity of spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests a long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease.","version":"1.1","doi":"10.1101/2020.05.13.093658","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.089961","pub_date":"2020-5-13","title":"Global view on virus infection in non-human primates and implication for public health and wildlife conservation","abstract":"The pandemic outbreak and rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not only a threat for humans, but potentially also for many animals. Research has revealed that SARS-CoV-2 and other coronaviruses have been transmitted from animals to humans and vice versa, and across animal species, and hence, attracted public attention concerning host-virus interactions and transmission ways. Non-human primates (NHPs), as our evolutionary closest relatives, are susceptible to human viruses, and a number of pathogens are known to circulate between humans and NHPs. Here we generated global statistics of virus infection in NHPs (VI-NHPs). In total, 121 NHP species from 14 families have been reported to be infected by 139 DNA and RNA viruses from 23 virus families; 74.8 percent of viruses in NHPs have also been found in humans, indicative of the high potential for cross species transmission of these viruses. The top ten NHP species with high centrality in the NHP-virus network are two apes (Pan troglodytes, Pongo pygmaeus), seven Old World monkeys (Macaca mulatta, M. fascicularis, Papio cynocephalus, Lophocebus albigena, Chlorocebus aethiops, Cercopithecus ascanius, C. nictitans) and a lemur (Propithecus diadema). Besides apes, there is a high risk of virus circulation between humans and Old World monkeys, given the wide distribution of many Old World monkey species and their frequent contact with humans. We suggest epidemiological investigations in NHPs, specifically in Old World monkeys with close contact to humans, and other effective measures to prevent this potential circular transmission.","version":"1.1","doi":"10.1101/2020.05.12.089961","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.071050","pub_date":"2020-5-12","title":"CoV-Seq: SARS-CoV-2 Genome Analysis and Visualization","abstract":"COVID-19 has become a global pandemic not long after its inception in late 2019. SARS-CoV-2 genomes are being sequenced and shared on public repositories at a fast pace. To keep up with these updates, scientists need to frequently refresh and reclean datasets, which is ad hoc and labor-intensive. Further, scientists with limited bioinformatics or programming knowledge may find it difficult to analyze SARS-CoV-2 genomes. In order to address these challenges, we developed CoV-Seq, a webserver to enable simple and rapid analysis of SARS-CoV-2 genomes. Given a new sequence, CoV-Seq automatically predicts gene boundaries and identifies genetic variants, which are presented in an interactive genome visualizer and are downloadable for further analysis. A command-line interface is also available for high-throughput processing. CoV-Seq is implemented in Python and Javascript. The webserver is available at http://covseq.baidu.com/ and the source code is available from https://github.com/boxiangliu/covseq. jollier.liu@gmail.com Supplementary information are available at bioRxiv online.","version":"1.2","doi":"10.1101/2020.05.01.071050","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091298","pub_date":"2020-5-12","title":"Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual","abstract":"B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design. SARS-CoV-2 infection leads to expansion of diverse B cells clones against the viral spike glycoprotein (S). The antibodies bind S with high affinity despite being minimally mutated. Thus, the development of neutralizing antibody responses by vaccination will require the activation of certain na\u00efve B cells without requiring extensive somatic mutation. Analysis of early B cell response to SARS-CoV-2 spike protein Most antibodies target non-neutralizing epitopes Potent neutralizing mAb blocks the interaction of the S protein with ACE2 Neutralizing antibodies are minimally mutated","version":"1.1","doi":"10.1101/2020.05.12.091298","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.091314","pub_date":"2020-5-12","title":"Meta-analysis of transcriptomes of SARS-Cov2 infected human lung epithelial cells identifies transmembrane serine proteases co-expressed with ACE2 and biological processes related to viral entry, immunity, inflammation and cellular stress","abstract":"The COVID-19 pandemic resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in December 2019 in the Chinese city of Wuhan in the province Hubei has placed immense burden on national economies and global health. At present neither vaccination nor therapies are available although several antiviral agents such as remdesivir, originally an Ebola drug, nelfinavir, an HIV-1 protease inhibitor and other drugs such as lopinavir have been evaluated. Here, we performed a meta-analysis of RNA-sequencing data from three studies employing human lung epithelial cells. Of these one focused on lung epithelial cells infected with SARS-CoV-2. We aimed at identifying genes co-expressed with angiotensin I converting enzyme 2 (ACE2) the human cell entry receptor of SARS-CoV-2, and unveiled several genes correlated or inversely correlated with high significance, among the most significant of these was the transmembrane serine protease 4 (TMPRSS4). Serine proteases are known to be involved in the infection process by priming the virus spike protein. Pathway analysis revealed papilloma virus infection amongst the most significantly correlated pathways. Gene Ontologies revealed regulation of viral life cycle, immune responses, pro-inflammatory responses-several interleukins such as IL6, IL1, IL20 and IL33, IFI16 regulating the interferon response to a virus, chemo-attraction of macrophages, last and not least cellular stress resulting from activated Reactive Oxygen Species. We believe that this dataset will aid in a better understanding of the molecular mechanism(s) underlying COVID-19.","version":"1.1","doi":"10.1101/2020.05.12.091314","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.088716","pub_date":"2020-5-12","title":"Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability","abstract":"The rapid spread of SARS-CoV-2 has a significant impact on global health, travel and economy. Therefore, preventative and therapeutic measures are urgently needed. Here, we isolated neutralizing antibodies from convalescent COVID-19 patients using a SARS-CoV-2 stabilized prefusion spike protein. Several of these antibodies were able to potently inhibit live SARS-CoV-2 infection at concentrations as low as 0.007 \u00b5g/mL, making them the most potent human SARS-CoV-2 antibodies described to date. Mapping studies revealed that the SARS-CoV-2 spike protein contained multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as previously undefined non-RBD epitopes. In addition to providing guidance for vaccine design, these mAbs are promising candidates for treatment and prevention of COVID-19.","version":"1.1","doi":"10.1101/2020.05.12.088716","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.090035","pub_date":"2020-5-12","title":"Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells: Nafamostat is the most potent antiviral drug candidate","abstract":"Drug repositioning represents an effective way to control the current COVID-19 pandemic. Previously, we identified 24 FDA-approved drugs which exhibited substantial antiviral effect against SARS-CoV-2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. Comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC50 = 0.0022\u00b5M).","version":"1.1","doi":"10.1101/2020.05.12.090035","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.12.088088","pub_date":"2020-5-12","title":"A putative new SARS-CoV protein, 3a*, encoded in an ORF overlapping ORF3a","abstract":"Identification of the full complement of genes in SARS-CoV-2 is a crucial step towards gaining a fuller understanding of its molecular biology. However, short and/or overlapping genes can be difficult to detect using conventional computational approaches, whereas high throughput experimental approaches \u2013 such as ribosome profiling \u2013 cannot distinguish translation of functional peptides from regulatory translation or translational noise. By studying regions showing enhanced conservation at synonymous sites in alignments of SARS-CoV and related viruses (subgenus Sarbecovirus), and correlating with the conserved presence of an open reading frame and plausible translation mechanism, we identified a putative new gene, ORF3a*, overlapping ORF3a in an alternative reading frame. A recently published ribosome profiling study confirmed that ORF3a* is indeed translated during infection. ORF3a* is conserved across the subgenus Sarbecovirus, and encodes a 40\u201341 amino acid predicted transmembrane protein.","version":"1.1","doi":"10.1101/2020.05.12.088088","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.20092965","pub_date":"2020-05-12","title":"Predicted success of prophylactic antiviral therapy to block or delay SARS-CoV-2 infection depends on the drug\u2019s mechanism of action","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Repurposed drugs that are immediately available and safe to use constitute a first line of defense against new viral infections. Despite limited antiviral activity against SARS-CoV-2, several drugs are being tested as medication or as prophylaxis to prevent infection. Using a stochastic model of early phase infection, we find that a critical efficacy above 87% is needed to block viral establishment. This can be improved by combination therapy. Below the critical efficacy, establishment of infection can sometimes be prevented, most effectively with drugs blocking viral entry into cells or enhancing viral clearance. Even when a viral infection cannot be prevented, antivirals delay the time to detectable viral loads. This delay flattens the within-host viral dynamic curve, possibly reducing transmission and symptom severity. Thus, antiviral prophylaxis, even with reduced efficacy, could be efficiently used to prevent or alleviate infection in people at high risk.</jats:p>","version":null,"doi":"10.1101/2020.05.07.20092965","journal":"medRxiv","score":null},{"id":"10.1101/2020.04.21.053017","pub_date":"2020-5-11","title":"Enisamium is a small molecule inhibitor of the influenza A virus and SARS-CoV-2 RNA polymerases","abstract":"Influenza A virus and coronavirus strains cause a mild to severe respiratory disease that can result in death. Although vaccines exist against circulating influenza A viruses, such vaccines are ineffective against emerging pandemic influenza A viruses. Currently, no vaccine exists against coronavirus infections, including pandemic SARS-CoV-2, the causative agent of the Coronavirus Disease 2019 (COVID-19). To combat these RNA virus infections, alternative antiviral strategies are needed. A key drug target is the viral RNA polymerase, which is responsible for viral RNA synthesis. In January 2020, the World Health Organisation identified enisamium as a candidate therapeutic against SARS-CoV-2. Enisamium is an isonicotinic acid derivative that is an inhibitor of multiple influenza B and A virus strains in cell culture and clinically approved in 11 countries. Here we show using in vitro assays that enisamium and its putative metabolite, VR17-04, inhibit the activity of the influenza virus and the SARS-CoV-2 RNA polymerase. VR17-04 displays similar efficacy against the SARS-CoV-2 RNA polymerase as the nucleotide analogue remdesivir triphosphate. These results suggest that enisamium is a broad-spectrum small molecule inhibitor of RNA virus RNA synthesis, and implicate it as a possible therapeutic option for treating SARS-CoV-2 infection. Unlike remdesivir, enisamium does not require intravenous administration which may be advantageous for the development of COVID-19 treatments outside a hospital setting. Influenza A virus and SARS-CoV-2 are respiratory viruses capable of causing pandemics, and the latter is responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Both viruses encode RNA polymerases which transcribe their RNA genomes and are important targets for antiviral drugs including remdesivir. Here, we show that the antiviral drug enisamium inhibits the RNA polymerases of both influenza A virus and SARS-CoV-2. Furthermore, we show that a putative metabolite of enisamium is a more potent inhibitor, inhibiting the SARS-CoV-2 RNA polymerase with similar efficiency to remdesivir. Our data offer insight into the mechanism of action for enisamium, and implicate it as a broad-spectrum antiviral which could be used in the treatment of SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.04.21.053017","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.04.976662","pub_date":"2020-5-11","title":"Genome-wide data inferring the evolution and population demography of the novel pneumonia coronavirus (SARS-CoV-2)","abstract":"As the highly risk and infectious diseases, the outbreak of coronavirus disease 2019 (COVID-19) poses unprecedent challenges to global health. Up to March 3, 2020, SARS-CoV-2 has infected more than 89,000 people in China and other 66 countries across six continents. In this study, we used 10 new sequenced genomes of SARS-CoV-2 and combined 136 genomes from GISAID database to investigate the genetic variation and population demography through different analysis approaches (e.g. Network, EBSP, Mismatch, and neutrality tests). The results showed that 80 haplotypes had 183 substitution sites, including 27 parsimony-informative and 156 singletons. Sliding window analyses of genetic diversity suggested a certain mutations abundance in the genomes of SARS-CoV-2, which may be explaining the existing widespread. Phylogenetic analysis showed that, compared with the coronavirus carried by pangolins (Pangolin-CoV), the virus carried by bats (bat-RaTG13-CoV) has a closer relationship with SARS-CoV-2. The network results showed that SARS-CoV-2 had diverse haplotypes around the world by February 11. Additionally, 16 genomes, collected from Huanan seafood market assigned to 10 haplotypes, indicated a circulating infection within the market in a short term. The EBSP results showed that the first estimated expansion date of SARS-CoV-2 began from 7 December 2019.","version":"1.3","doi":"10.1101/2020.03.04.976662","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088179","pub_date":"2020-5-11","title":"SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant","abstract":"One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays revealed that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of more than 15,000 SARS-CoV-2 sequences identified a natural variant, in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients, but also describe a possibility of the emergence of natural SARS-CoV-2 quasispecies with extended ORF3b that may exacerbate COVID-19 symptoms. ORF3b of SARS-CoV-2 and related bat and pangolin viruses is a potent IFN antagonist SARS-CoV-2 ORF3b suppresses IFN induction more efficiently than SARS-CoV ortholog The anti-IFN activity of ORF3b depends on the length of its C-terminus An ORF3b with increased IFN antagonism was isolated from two severe COVID-19 cases","version":"1.1","doi":"10.1101/2020.05.11.088179","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088724","pub_date":"2020-5-11","title":"A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2","abstract":"The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.","version":"1.1","doi":"10.1101/2020.05.11.088724","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.083147","pub_date":"2020-5-11","title":"Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with Angiotensin Converting Enzyme 2 Receptor","abstract":"The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an ongoing pandemic of coronavirus disease (COVID-19), which started in 2019. This is a member of Coronaviridae family in the genus Betacoronavirus, which also includes SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). The angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV and SARS-CoV-2 to enter the host cells. In particular, the interaction of viral spike proteins with ACE2 is a critical step in the viral replication cycle. The receptor binding domain of the viral spike proteins and ACE2 have several cysteine residues. In this study, the role of thiol-disulfide balance on the interactions between SARS-CoV/CoV-2 spike proteins and ACE2 was investigated using molecular dynamic simulations. The study revealed that the binding affinity was significantly impaired when all the disulfide bonds of both ACE2 and SARS-CoV/CoV-2 spike proteins were reduced to thiol groups. The impact on the binding affinity was less severe when the disulfide bridges of only one of the binding partners were reduced to thiols. This computational finding provides a molecular basis for the severity of COVID-19 infection due to the oxidative stress.","version":"1.2","doi":"10.1101/2020.05.07.083147","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079939","pub_date":"2020-5-11","title":"Identification of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2 and their implications on its protein structure","abstract":"The rapid development of SARS-CoV-2 mediated COVID-19 pandemic has been the cause of significant health concern, highlighting the immediate need for the effective antivirals. SARS-CoV-2 is an RNA virus that has an inherent high mutation rate. These mutations drive viral evolution and genome variability, thereby, facilitating viruses to have rapid antigenic shifting to evade host immunity and to develop drug resistance. Viral RNA-dependent RNA polymerases (RdRp) perform viral genome duplication and RNA synthesis. Therefore, we compared the available RdRp sequences of SARS-CoV-2 from Indian isolates and \u2018Wuhan wet sea food market virus\u2019 sequence to identify, if any, variation between them. We report seven mutations observed in Indian SARS-CoV-2 isolates and three unique mutations that showed changes in the secondary structure of the RdRp protein at region of mutation. We also studied molecular dynamics using normal mode analyses and found that these mutations alter the stability of RdRp protein. Therefore, we propose that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents.","version":"1.2","doi":"10.1101/2020.05.05.079939","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.034868","pub_date":"2020-5-11","title":"De novo design of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike protein","abstract":"The emergence of SARS-CoV-2 is responsible for the pandemic of respiratory disease known as COVID-19, which emerged in the city of Wuhan, Hubei province, China in late 2019. Both vaccines and targeted therapeutics for treatment of this disease are currently lacking. Viral entry requires binding of the viral spike receptor binding domain (RBD) with the human angiotensin converting enzyme (hACE2). In an earlier paper, we report on the specific residue interactions underpinning this event. Here we report on the de novo computational design of high affinity antibody variable regions through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike protein using the software tool OptMAVEn-2.0. Subsequently, we carry out computational affinity maturation of the designed prototype variable regions through point mutations for improved binding with the target epitope. Immunogenicity was restricted by preferring designs that match sequences from a 9-mer library of \u201chuman antibodies\u201d based on H-score (human string content, HSC). We generated 106 different designs and report in detail on the top five that trade-off the greatest affinity for the spike RBD epitope (quantified using the Rosetta binding energies) with low H-scores. By grafting the designed Heavy (VH) and Light (VL) chain variable regions onto a human framework (Fc), high-affinity and potentially neutralizing full-length monoclonal antibodies (mAb) can be constructed. Having a potent antibody that can recognize the viral spike protein with high affinity would be enabling for both the design of sensitive SARS-CoV-2 detection devices and for their deployment as therapeutic antibodies.","version":"1.2","doi":"10.1101/2020.04.09.034868","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015891","pub_date":"2020-5-11","title":"Biophysical characterization of the SARS-CoV-2 spike protein binding with the ACE2 receptor and implications for infectivity","abstract":"SARS-CoV-2 is a novel highly virulent pathogen which gains entry to human cells by binding with the cell surface receptor \u2013 angiotensin converting enzyme (ACE2). We computationally contrasted the binding interactions between human ACE2 and coronavirus spike protein receptor binding domain (RBD) of the 2002 epidemic-causing SARS-CoV-1, SARS-CoV-2, and bat coronavirus RaTG13 using the Rosetta energy function. We find that the RBD of the spike protein of SARS-CoV-2 is highly optimized to achieve very strong binding with human ACE2 (hACE2) which is consistent with its enhanced infectivity. SARS-CoV-2 forms the most stable complex with hACE2 compared to SARS-CoV-1 (23% less stable) or RaTG13 (11% less stable) while occupying the greatest number of residues in the ATR1 binding site. Notably, the SARS-CoV-2 RBD out-competes the angiotensin 2 receptor type I (ATR1) which is the native binding partner of ACE2 by 35% in terms of the calculated binding affinity. Strong binding is mediated through strong electrostatic attachments with every fourth residue on the N-terminus alpha-helix (starting from Ser19 to Asn53) as the turn of the helix makes these residues solvent accessible. By contrasting the spike protein SARS-CoV-2 Rosetta binding energy with ACE2 of different livestock and pet species we find strongest binding with bat ACE2 followed by human, feline, equine, canine and finally chicken. This is consistent with the hypothesis that bats are the viral origin and reservoir species. These results offer a computational explanation for the increased infectivity of SARS-CoV-2 and allude to therapeutic modalities by identifying and rank-ordering the ACE2 residues involved in binding with the virus.","version":"1.3","doi":"10.1101/2020.03.30.015891","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.083410","pub_date":"2020-5-11","title":"Characterization of SARS-CoV-2 viral diversity within and across hosts","abstract":"In light of the current COVID-19 pandemic, there is an urgent need to accurately infer the evolutionary and transmission history of the virus to inform real-time outbreak management, public health policies and mitigation strategies. Current phylogenetic and phylodynamic approaches typically use consensus sequences, essentially assuming the presence of a single viral strain per host. Here, we analyze 621 bulk RNA sequencing samples and 7,540 consensus sequences from COVID-19 patients, and identify multiple strains of the virus, SARS-CoV-2, in four major clades that are prevalent within and across hosts. In particular, we find evidence for (i) within-host diversity across phylogenetic clades, (ii) putative cases of recombination, multi-strain and/or superinfections as well as (iii) distinct strain profiles across geographical locations and time. Our findings and algorithms will facilitate more detailed evolutionary analyses and contact tracing that specifically account for within-host viral diversity in the ongoing COVID-19 pandemic as well as future pandemics.","version":"1.2","doi":"10.1101/2020.05.07.083410","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.089045","pub_date":"2020-5-11","title":"Activation of the SARS-CoV-2 receptor Ace2 by cytokines through pan JAK-STAT enhancers","abstract":"ACE2, in concert with the protease TMPRSS2, binds the novel coronavirus SARS-CoV-2 and facilitates its cellular entry. The ACE2 gene is expressed in SARS-CoV-2 target cells, including Type II Pneumocytes (Ziegler, 2020), and is activated by interferons. Viral RNA was also detected in breast milk (Wu et al., 2020), raising the possibility that ACE2 expression is under the control of cytokines through the JAK-STAT pathway. Here we show that Ace2 expression in mammary tissue is induced during pregnancy and lactation, which coincides with the establishment of a candidate enhancer. The prolactin-activated transcription factor STAT5 binds to tandem sites that coincide with activating histone enhancer marks and additional transcription components. The presence of pan JAK-STAT components in mammary alveolar cells and in Type II Pneumocytes combined with the autoregulation of both STAT1 and STAT5 suggests a prominent role of cytokine signaling pathways in cells targeted by SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.11.089045","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.11.088112","pub_date":"2020-5-11","title":"Evidence for strong mutation bias towards, and selection against, T/U content in SARS-CoV2: implications for attenuated vaccine design","abstract":"Large-scale re-engineering of synonymous sites is a promising strategy to generate attenuated viruses for vaccines. Attenuation typically relies on de-optimisation of codon pairs and maximization of CpG dinculeotide frequencies. So as to formulate evolutionarily-informed attenuation strategies, that aim to force nucleotide usage against the estimated direction favoured by selection, here we examine available whole-genome sequences of SARS-CoV2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias towards T with concomitant selection against T. Accounting for dinucleotide effects reinforces this conclusion, observed TT content being a quarter of that expected under neutrality. A significantly different mutational profile at CDS sites that are not 4-fold degenerate is consistent with contemporaneous selection against T mutations more widely. Although selection against CpG dinucleotides is expected to drive synonymous site G+C content below mutational equilibrium, observed G+C content is slightly above equilibrium, possibly because of selection for higher expression. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV2 genes. We propose an evolutionarily informed gene-bespoke approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.","version":"1.1","doi":"10.1101/2020.05.11.088112","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.16.994236","pub_date":"2020-5-11","title":"The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2","abstract":"The rapid and escalating spread of SARS coronavirus 2 (SARS-CoV-2) poses an immediate public health emergency. The viral spike protein S binds ACE2 on host cells to initiate molecular events that release the viral genome intracellularly. Soluble ACE2 inhibits entry of both SARS and SARS-2 coronaviruses by acting as a decoy for S binding sites, and is a candidate for therapeutic, prophylactic and diagnostic development. Using deep mutagenesis, variants of ACE2 are identified with increased binding to the receptor binding domain of S. Mutations are found across the interface, in the N90-glycosylation motif, and at buried sites where they are predicted to enhance local folding and presentation of the interaction epitope. When single substitutions are combined, large increases in binding can be achieved. The mutational landscape offers a blueprint for engineering high affinity proteins and peptides that block receptor binding sites on S to meet this unprecedented challenge.","version":"1.3","doi":"10.1101/2020.03.16.994236","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.059204","pub_date":"2020-5-11","title":"eCovSens-Ultrasensitive Novel In-House Built Printed Circuit Board Based Electrochemical Device for Rapid Detection of nCovid-19 antigen, a spike protein domain 1 of SARS-CoV-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or nCovid-19) outbreak has become a huge public health issue due to its rapid transmission and global pandemic. Currently, there are no vaccines or drugs available for nCovid-19, hence early detection is crucial to help and manage the outbreak. Here, we report an in-house built biosensor device (eCovSens) and compare it with a commercial potentiostat for the detection of nCovid-19 spike antigen (nCovid-19Ag) in spiked saliva samples. A potentiostat based sensor was fabricated using fluorine doped tin oxide electrode (FTO) with gold nanoparticle (AuNPs) and immobilized with nCovid-19 monoclonal antibody (nCovid-19Ab) to measure change in the electrical conductivity. Similarly, eCovSens was used to measure change in electrical conductivity by immobilizing nCovid-19 Ab on screen printed carbon electrode (SPCE). The performances of both sensors were recorded upon interaction of nCovid-19Ab with its specific nCovid-19Ag. Under optimum conditions, the FTO based immunosensor and eCovSens displayed high sensitivity for detection of nCovid-19Ag, ranging from 1 fM to 1 \u03bcM. Our in-house developed device can successfully detect nCovid-19Ag at 10 fM concentration in standard buffer that is in close agreement with FTO/AuNPs sensor. The limit of detection (LOD) was found to be 90 fM with eCovSens and 120 fM with potentiostst in case of spiked saliva samples. The proposed portable eCovSens device can be used as a diagnostic tool for the rapid (within 10-30 s) detection of nCovid-19Ag traces directly in patient saliva in a non-invasive manner.","version":"1.3","doi":"10.1101/2020.04.24.059204","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.085506","pub_date":"2020-5-10","title":"Clinical performance of SARS-CoV-2 IgG antibody tests and potential protective immunity","abstract":"As the current SARS-CoV-2 pandemic continues, serological assays are urgently needed for rapid diagnosis, contact tracing and for epidemiological studies. So far, there is little data on how commercially available tests perform with real patient samples and if detected IgG antibodies provide protective immunity. Focusing on IgG antibodies, we demonstrate the performance of two ELISA assays (Euroimmun SARS-CoV-2 IgG & Vircell COVID-19 ELISA IgG) in comparison to one lateral flow assay ((LFA) FaStep COVID-19 IgG/IgM Rapid Test Device) and two in-house developed assays (immunofluorescence assay (IFA) and plaque reduction neutralization test (PRNT)). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. Most of the SARS-CoV-2 samples were from individuals with moderate to severe clinical course, who required an in-patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5-9) and from 93.8 to 100% for the later period (days 10-18) after PCR-diagnosed with COVID-19. With exception of one sample, all positive tested samples in the analysed cohort, using the commercially available assays examined (including the in-house developed IFA), demonstrated neutralizing (protective) properties in the PRNT, indicating a potential protective immunity to SARS-CoV-2. Regarding specificity, there was evidence that samples of endemic coronavirus (HCoV-OC43, HCoV-229E) and Epstein Barr virus (EBV) infected individuals cross-reacted in the ELISA assays and IFA, in one case generating a false positive result (may giving a false sense of security). This need to be further investigated.","version":"1.1","doi":"10.1101/2020.05.08.085506","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.09.086223","pub_date":"2020-5-10","title":"Multiple introductions, regional spread and local differentiation during the first week of COVID-19 epidemic in Montevideo, Uruguay","abstract":"After its emergence in China in December 2019, the new coronavirus disease (COVID-19) caused by SARS-CoV-2, has rapidly spread infecting more than 3 million people worldwide. South America is among the last regions hit by COVID-19 pandemic. In Uruguay, first cases were detected on March 13 th 2020 presumably imported by travelers returning from Europe. We performed whole-genome sequencing of 10 SARS-CoV-2 from patients diagnosed during the first week (March 16th to 19th) of COVID-19 outbreak in Uruguay. Then, we applied genomic epidemiology using a global dataset to reconstruct the local spatio-temporal dynamics of SARS-CoV-2. Our phylogeographic analysis showed three independent introductions of SARS-CoV-2 from different continents. Also, we evidenced regional circulation of viral strains originally detected in Spain. Introduction of SARS-CoV-2 in Uruguay could date back as early as Feb 20th. Identification of specific mutations showed rapid local genetic differentiation. We evidenced early independent introductions of SARS-CoV-2 that likely occurred before first cases were detected. Our analysis set the bases for future genomic epidemiology studies to understand the dynamics of SARS-CoV-2 in Uruguay and the Latin America and the Caribbean region.","version":"1.1","doi":"10.1101/2020.05.09.086223","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.020941","pub_date":"2020-5-10","title":"LxxIxE-like Motif in Spike Protein of SARS-CoV-2 that is Known to Recruit the Host PP2A-B56 Phosphatase Mimics Artepillin C, an Immunomodulator, of Brazilian Green Propolis","abstract":"SARS-CoV-2 is highly contagious and can cause acute respiratory distress syndrome (ARDS) and multiple organ failure that are largely attributed to the cytokine storm. The surface coronavirus spike (S) glycoprotein is considered as a key factor in host specificity because it mediates infection by receptor-recognition and membrane fusion. Here, the analysis of SARS-CoV-2 S protein revealed two B56-binding LxxIxE-like motifs in S1 and S2 subunits that could recruit the host protein phosphatase 2A (PP2A). The motif in S1 subunit is absent in SARS-CoV and MERS-CoV. Phosphatases and kinases are major players in the regulation of pro-inflammatory responses during pathogenic infections. Moreover, studies have shown that viruses target PP2A in order to manipulate host\u2019s antiviral responses. Recent researches have indicated that SARS-CoV-2 is involved in sustained host inflammation. Therefore, by controlling acute inflammation, it is possible to eliminate its dangerous effects on the host. Among efforts to fight COVID-19, the interaction between LxxIxE-like motif and the PP2A-B56-binding pocket could be a target for the discovery and/or development of a bioactive ligand inhibitor for therapeutic purposes. Indeed, a small molecule called Artepillin C (ArtC), a main compound in Brazilian honeybee green propolis, mimics the side chains of LxxLxE motif. Importantly, ArtC is known, among other effects, to have anti-inflammatory activity that makes it an excellent candidate for future clinical trials in COVID-19 patients.","version":"1.2","doi":"10.1101/2020.04.01.020941","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.09.085811","pub_date":"2020-5-10","title":"Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses","abstract":"Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91\u00b10.25 \u03bcM to 1.88\u00b10.34 \u03bcM. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the formation of the negative curvature required for fusion and blocking the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.","version":"1.1","doi":"10.1101/2020.05.09.085811","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.071985","pub_date":"2020-5-09","title":"On spatial molecular arrangements of SARS-CoV2 genomes of Indian patients","abstract":"A pandemic caused by the SARS-CoV2 is being experienced by the whole world since December, 2019. A thorough understanding beyond just sequential similarities among the protein coding genes of SARS-CoV2 is important in order to differentiate or relate to the other known CoVs of the same genus. In this study, we compare three genomes namely MT012098 (India-Kerala), MT050493 (India-Kerala), MT358637 (India-Gujrat) from India with NC_045512 (China-Wuhan) to view the spatial as well as molecular arrangements of nucleotide bases of all the genes embedded in these four genomes. Based on different features extracted for each gene embedded in these genomes, corresponding phylogenetic relationships have been built up. Differences in phylogenetic tree arrangement with individual gene suggest that three genomes of Indian origin have come from three different origins or the evolution of viral genome is very fast process. This study would also help to understand the virulence factors, disease pathogenicity, origin and transmission of the SARS-CoV2.","version":"1.2","doi":"10.1101/2020.05.01.071985","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.083139","pub_date":"2020-5-09","title":"Pre-treatment of the clinical sample with Proteinase K allows detection of SARS-CoV-2 in the absence of RNA extraction","abstract":"COVID-19 (Coronavirus Disease 2019) outbreak was declared a pandemic, by World Health Organization, on March 11, 2020. Viral detection using RT-qPCR has been among the most important factors helping to control local spread of SARS-CoV-2 and it is considered the \u201cgold standard\u201d for diagnosis. Nevertheless, the RNA extraction step is both laborious and expensive, thus hampering the diagnosis in many places where there are not laboratory staff of funds enough to contribute for diagnosis efforts. Thus, the need to simplify procedures, reduce costs of the techniques used, and expand the capacity of the number of diagnostics of COVID-19 is imperative. In this study, detection of SARS-CoV-2 in the absence of RNA extraction has been successfully achieved through pre-treatment of the clinical sample with Proteinase K. The results show that only the use of proteinase K, without the need to perform the whole standard protocol for sample extraction and purification, can be an efficient technique for the diagnosis of COVID-19, since 91% of the samples matched the results with the standard procedure, with an average increase of 5.64 CT in the RT-qPCR.","version":"1.1","doi":"10.1101/2020.05.07.083139","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.084996","pub_date":"2020-5-09","title":"Elevated ACE2 expression in the olfactory neuroepithelium: implications for anosmia and upper respiratory SARS-CoV-2 entry and replication","abstract":"The site of SARS-CoV-2 entry and replication critically impacts strategies for COVID-19 diagnosis, transmission mitigation, and treatment. We determined the cellular location of the SARS-CoV-2 target receptor protein, ACE2, in the human upper airway, finding striking enrichment (200-700 folds) in the olfactory neuroepithelium relative to nasal respiratory or tracheal epithelial cells. This cellular tropism of SARS-CoV-2 may underlie its high transmissibility and association with olfactory dysfunction, while suggesting a viral reservoir potentially amenable to intranasal therapy.","version":"1.1","doi":"10.1101/2020.05.08.084996","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.09.086165","pub_date":"2020-5-09","title":"ACE2 and TMPRSS2 are expressed on the human ocular surface, suggesting susceptibility to SARS-CoV-2 infection","abstract":"Conjunctival signs and symptoms are observed in a subset of patients with COVID-19, and SARS-CoV-2 has been detected in tears, raising concerns regarding the eye both as a portal of entry and carrier of the virus. The purpose of this study was to determine whether ocular surface cells possess the key factors required for cellular susceptibility to SARS-CoV-2 entry/infection. We analyzed human post-mortem eyes as well as surgical specimens for the expression of ACE2 (the receptor for SARS-CoV-2) and TMPRSS2, a cell surface-associated protease that facilitates viral entry following binding of the viral spike protein to ACE2. Across all eye specimens, immunohistochemical analysis revealed expression of ACE2 in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface. Surgical conjunctival specimens also showed expression of ACE2 in the conjunctival epithelium, especially prominent in the superficial epithelium, as well as the substantia propria. All eye and conjunctival specimens also expressed TMPRSS2. Finally, western blot analysis of protein lysates from human corneal epithelium obtained during refractive surgery confirmed expression of ACE2 and TMPRSS2. Together, these results indicate that ocular surface cells including conjunctiva are susceptible to infection by SARS-CoV-2, and could therefore serve as a portal of entry as well as a reservoir for person-to-person transmission of this virus. This highlights the importance of safety practices including face masks and ocular contact precautions in preventing the spread of COVID-19 disease.","version":"1.1","doi":"10.1101/2020.05.09.086165","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.078154","pub_date":"2020-5-09","title":"Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients","abstract":"In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study. Neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.","version":"1.2","doi":"10.1101/2020.05.05.078154","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.084236","pub_date":"2020-5-09","title":"Knowledge, attitude and practice of secondary school students toward COVID-19 epidemic in Italy: a cross selectional study","abstract":"The coronavirus disease (COVID-19) is a highly transmittable and pathogenic viral infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which emerges in December 2019 in Wuhan, China and spreads around the world at the beginning of 2020. The World Health Organization declares the outbreak a Public Health Emergency of International Concern on the 30th of January, and a pandemic on the 11th of March. On the 4th of March, the Italian government orders the full closure of all schools and universities nationwide. The aim of this study is to investigate the knowledge, practice and attitudes (KAP) of secondary school at the time of COVID-19 pandemic in Italy. In this cross-sectional, web-based survey, conducted among the high school student population with age ranging from 14 to 19 years old, a questionnaire with 19 items regarding the KAP toward COVID-19 is asked. Study participants are recruited from several secondary schools of different areas. Frequencies and histograms are computed for descriptive purposes. Statistical analysis is computed with Chi square test, utilized to depict relevant difference between geender. Among a total of 2380 students who answers the questionnaire, 40.7% are male and 59.3% are female. Level of knowledge about generical characteristics of COVID-19 is quite similar among gender. Students present a good level of knowledge about the clinical presentation of the disease, the basic hygiene principles, the modes of transmission and the method of protection against virus transmission. The knowledge about number of this pandemia and easy scientific correlation with COVID-19 is quite confused. The most frequently reported source of knowledge about COVID-19 is television, whereas the less is the school. Our findings suggest that student population shows appropriate practice, and positive attitude towards COVID-19 at the time of its outbreak. More emphasis should be placed on education of the student partecipants about biological meaning of this infection and relative preventive or future measures.","version":"1.1","doi":"10.1101/2020.05.08.084236","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.20092098","pub_date":"2020-05-09","title":"Using viral genomics to estimate undetected infections and extent of superspreading events for COVID-19","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Asymptomatic infections and limited testing capacity have led to under-reporting of SARS-CoV-2 cases. This has hampered the ability to ascertain true infection numbers, evaluate the effectiveness of surveillance strategies, determine transmission dynamics, and estimate reproductive numbers. Leveraging both viral genomic and time series case data offers methods to estimate these parameters.</jats:p>\n                <jats:p>Using a Bayesian inference framework to fit a branching process model to viral phylogeny and time series case data, we estimated time-varying reproductive numbers and their variance, the total numbers of infected individuals, the probability of case detection over time, and the estimated time to detection of an outbreak for 12 locations in Europe, China, and the United States.</jats:p>\n                <jats:p>The median percentage of undetected infections ranged from 13% in New York to 92% in Shanghai, China, with the length of local transmission prior to two cases being detected ranging from 11 days (95% CI: 4-21) in California to 37 days (9-100) in Minnesota. The probability of detection was as low as 1% at the start of local epidemics, increasing as the number of reported cases increased exponentially. The precision of estimates increased with the number of full-length viral genomes in a location. The viral phylogeny was informative of the variance in the reproductive number with the 32% most infectious individuals contributing 80% of total transmission events.</jats:p>\n                <jats:p>This is the first study that incorporates both the viral genomes and time series case data in the estimation of undetected COVID-19 infections. Our findings suggest the presence of undetected infections broadly and that superspreading events are contributing less to observed dynamics than during the SARS epidemic in 2003. This genomics-informed modeling approach could estimate in near real-time critical surveillance metrics to inform ongoing COVID-19 response efforts.</jats:p>\n                <jats:sec>\n                  <jats:title>Funding</jats:title>\n                  <jats:p>AWS provided computational credit via the Diagnostic Development Initiative.</jats:p>\n                </jats:sec>","version":null,"doi":"10.1101/2020.05.05.20092098","journal":"medRxiv","score":null},{"id":"10.1101/2020.04.15.043364","pub_date":"2020-5-08","title":"Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray","abstract":"The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.","version":"1.2","doi":"10.1101/2020.04.15.043364","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.051581","pub_date":"2020-5-08","title":"Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease","abstract":"A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 \u03bcM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 \u00c5 resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.","version":"1.2","doi":"10.1101/2020.04.20.051581","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.080960","pub_date":"2020-5-08","title":"The transcriptomic profiling of COVID-19 compared to SARS, MERS, Ebola, and H1N1","abstract":"COVID-19 pandemic is a global crisis that threatens our way of life. As of April 29, 2020, COVID-19 has claimed more than 200,000 lives, with a global mortality rate of ~7% and recovery rate of ~30%. Understanding the interaction of cellular targets to the SARS-CoV2 infection is crucial for therapeutic development. Therefore, the aim of this study was to perform a comparative analysis of transcriptomic signatures of infection of COVID-19 compared to different respiratory viruses (Ebola, H1N1, MERS-CoV, and SARS-CoV), to determine unique anti-COVID1-19 gene signature. We identified for the first time molecular pathways for Heparin-binding, RAGE, miRNA, and PLA2 inhibitors, to be associated with SARS-CoV2 infection. The NRCAM and SAA2 that are involved in severe inflammatory response, and FGF1 and FOXO1 genes, which are associated with immune regulation, were found to be associated with a cellular gene response to COVID-19 infection. Moreover, several cytokines, most significantly the IL-8, IL-6, demonstrated key associations with COVID-19 infection. Interestingly, the only response gene that was shared between the five viral infections was SERPINB1. The PPI study sheds light on genes with high interaction activity that COVID-19 shares with other viral infections. The findings showed that the genetic pathways associated with Rheumatoid arthritis, AGE-RAGE signaling system, Malaria, Hepatitis B, and Influenza A were of high significance. We found that the virogenomic transcriptome of infection, gene modulation of host antiviral responses, and GO terms of both COVID-19 and Ebola are more similar compared to SARS, H1N1, and MERS. This work compares the virogenomic signatures of highly pathogenic viruses and provides valid targets for potential therapy against COVID-19.","version":"1.1","doi":"10.1101/2020.05.06.080960","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.023887","pub_date":"2020-5-08","title":"Topological Analysis of SARS CoV-2 Main Protease","abstract":"There is an urgent necessity of effective medication against SARS CoV-2, which is producing the COVID-19 pandemic across the world. Its main protease (Mpro) represents an attractive pharmacological target due to its involvement in essential viral functions. The crystal structure of free Mpro shows a large structural resemblance with the main protease of SARS CoV (nowadays known as SARS CoV-1). Here we report that as average SARS CoV-2 Mpro is 1900% more sensitive than SARS CoV-1 Mpro in transmitting tiny structural changes across the whole protein through long-range interactions. The largest sensitivity of Mpro to structural perturbations is located exactly around the catalytic site Cys-145, and coincides with the binding site of strong inhibitors. These findings, based on a simplified representation of the protein as a residue network, may help in designing potent inhibitors of SARS CoV-2 Mpro. The main protease of the new coronavirus SARS CoV-2 represents one of the most important targets for the antiviral pharmacological actions againsts COVID-19. This enzyme is essential for the virus due to its proteolytic processing of polyproteins. Here we discover that the main protease of SARS CoV-2 is topologically very similar to that of the SARS CoV-1. This is not surprising taking into account that both proteases differ only in 12 amino acids. However, we remarkable found a topological property of SARS CoV-2 that has increased in more than 1900% repect to its SARS CoV-1 analogue. This property reflects the capacity of the new protease of transmitting perturbations across its domains using long-range interactions. Also remarkable is the fact that the amino acids displaying such increased sensitivity to perturbations are around the binding site of the new protease, and close to its catalytic site. We also show that this sensititivy to perturbations is related to the effects of powerful protease inhibitors. In fact, the strongest inhibitors of the SARS CoV-2 main protease are those that produce the least change of this capacity of transmitting perturbations across the protein. We think that these findings may help in the design of new potent anti-SARS CoV-2 inhibitors.","version":"1.2","doi":"10.1101/2020.04.03.023887","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.083964","pub_date":"2020-5-08","title":"A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability","abstract":"The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.","version":"1.1","doi":"10.1101/2020.05.08.083964","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.082701","pub_date":"2020-5-08","title":"Efficacy of a novel iodine complex solution, CupriDyne, in inactivating SARS-CoV-2","abstract":"The coronavirus known as SARS-CoV-2, which causes COVID-19 disease, is presently responsible for a global pandemic wherein more than 3.5 million people have been infected and more than 250,000 killed to-date. There is currently no vaccine for COVID-19, leaving governments and public health agencies with little defense against the virus aside from advising or enforcing best practices for virus transmission prevention, which include hand-washing, physical distancing, use of face covers, and use of effective disinfectants. In this study, a novel iodine complex called CupriDyne\u00ae was assessed for its ability to inactivate SARS-CoV-2. CupriDyne was shown to be effective in inactivating the virus in a time-dependent manner, reducing virus titers by 99% (2 logs) after 30 minutes, and reducing virus titers to below the detection limit after 60 minutes. The novel iodine complex tested herein offers a safe and gentle alternative to conventional disinfectants for use on indoor and outdoor surfaces.","version":"1.1","doi":"10.1101/2020.05.08.082701","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.083816","pub_date":"2020-5-08","title":"Intra-genome variability in the dinucleotide composition of SARS-CoV-2","abstract":"CpG dinucleotides are under-represented in the genomes of single stranded RNA viruses, and coronaviruses, including SARS-CoV-2, are no exception to this. Artificial modification of CpG frequency is a valid approach for live attenuated vaccine development, and if this is to be applied to SARS-CoV-2, we must first understand the role CpG motifs play in regulating SARS-CoV-2 replication. Accordingly, the CpG composition of the newly emerged SARS-CoV-2 genome was characterised in the context of other coronaviruses. CpG suppression amongst coronaviruses does not significantly differ according to genera of virus, but does vary according to host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. Although SARS-CoV-2 exhibits overall strong CpG suppression, this varies considerably across the genome, and the Envelope (E) open reading frame (ORF) and ORF10 demonstrate an absence of CpG suppression. While ORF10 is only present in the genomes of a subset of coronaviruses, E is essential for virus replication. Across the Coronaviridae, E genes display remarkably high variation in CpG composition, with those of SARS and SARS-CoV-2 having much higher CpG content than other coronaviruses isolated from humans. Phylogeny indicates that this is an ancestrally-derived trait reflecting their origin in bats, rather than something selected for after zoonotic transfer. Conservation of CpG motifs in these regions suggests that they have a functionality which over-rides the need to suppress CpG; an observation relevant to future strategies towards a rationally attenuated SARS-CoV-2 vaccine.","version":"1.1","doi":"10.1101/2020.05.08.083816","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.082297","pub_date":"2020-5-08","title":"Lung biopsy cells transcriptional landscape from COVID-19 patient stratified lung injury in SARS-CoV-2 infection through impaired pulmonary surfactant metabolism","abstract":"Clinical management of COVID-19 is still complicated due to the lack of therapeutic interventions to reduce the breathing problems, respiratory complications and acute lung injury \u2013 which are the major complications of most of the mild to critically affected patients and the molecular mechanisms behind these clinical features are still largely unknown. In this study, we have used the RNA-seq gene expression pattern in the COVID-19 affected lung biopsy cells and compared it with the effects observed in typical cell lines infected with SARS-CoV-2 and SARS-CoV. We performed functional overrepresentation analyses using these differentially expressed genes to signify the processes/pathways which could be deregulated during SARS-CoV-2 infection resulting in the symptomatic impairments observed in COVID-19. Our results showed that the significantly altered processes include inflammatory responses, antiviral cytokine signaling, interferon responses, and interleukin signaling etc. along with downmodulated processes related to lung\u2019s functionality like-responses to hypoxia, lung development, respiratory processes, cholesterol biosynthesis and surfactant metabolism. We also found that the viral protein interacting host\u2019s proteins involved in similar pathways like: respiratory failure, lung diseases, asthma, and hypoxia responses etc., suggesting viral proteins might be deregulating the processes related to acute lung injury/breathing complications in COVID-19 patients. Protein-protein interaction networks of these processes and map of gene expression of deregulated genes revealed that several viral proteins can directly or indirectly modulate the host genes/proteins of those lung related processes along with several host transcription factors and miRNAs. Surfactant proteins and their regulators SPD, SPC, TTF1 etc. which maintains the stability of the pulmonary tissue are found to be downregulated through viral NSP5, NSP12 that could lead to deficient gaseous exchange by the surface films. Mitochondrial dysfunction owing to the aberration of NDUFA10, NDUFAF5, SAMM50 etc. by NSP12; abnormal thrombosis in lungs through atypical PLAT, EGR1 functions by viral ORF8, NSP12; dulled hypoxia responses due to unusual shift in HIF-1 downstream signaling might be the causative elements behind the acute lung injury in COVID-19 patients. Our study put forward a distinct mechanism of probable virus induced lung damage apart from cytokine storm and advocate the need of further research for alternate therapy in this direction.","version":"1.1","doi":"10.1101/2020.05.07.082297","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.083618","pub_date":"2020-5-08","title":"Multiple expression assessments of ACE2 and TMPRSS2 SARS-CoV-2 entry molecules in the urinary tract and their associations with clinical manifestations of COVID-19","abstract":"Since December 2019, the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first spread quickly in Wuhan, China, then globally. From previously published evidence, ACE2 and TMPRSS2, are both pivotal entry molecules that enable cellular infection by SARS-CoV-2. Meanwhile, increased expression of pro-inflammatory cytokines, or a \u201ccytokine storm,\u201d is associated with multiple organ dysfunction syndrome that is often observed in critically ill patients. We investigated the expression pattern of ACE2 and TMPRSS2 in major organs in the human body, especially under specific disease conditions. Multiple sequence alignment of ACE2 in different species was used to explain animal susceptibility. Moreover, the cell-specific expression patterns of ACE2 and cytokine receptors in the urinary tract were assessed using single-cell RNA sequencing (scRNA-seq). Additional biological relevance was determined through Gene Set Enrichment Analysis (GSEA) using an ACE2 specific signature. Our results revealed that ACE2 and TMPRSS2 were highly expressed in genitourinary organs. ACE2 was highly and significantly expressed in the kidney among individuals with chronic kidney diseases or diabetic nephropathy. In single cells, ACE2 was primarily enriched in gametocytes in the testis, and renal proximal tubules. The receptors for pro-inflammatory cytokines, especially IL6ST, were remarkably concentrated in endothelial cells, macrophages, and spermatogonial stem cells in the testis, and renal endothelial cells, which suggested the occurrence of alternative damaging mechanisms via autoimmune attacks. This study provided new insights into the pathogenicity mechanisms of SARS-CoV-2 that underlie the clinical manifestations observed in the human testis and kidney. These observations might substantially facilitate the development of effective treatments for this rapidly spreading disease.","version":"1.1","doi":"10.1101/2020.05.08.083618","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.050260","pub_date":"2020-5-08","title":"SARS-CoV-2 proteins exploit host\u2019s genetic and epigenetic mediators for the annexation of key host signaling pathways that confers its immune evasion and disease pathophysiology","abstract":"The constant rise of the death toll and cases of COVID-19 has made this pandemic a serious threat to human civilization. Understanding of host-SARS-CoV-2 interaction in viral pathogenesis is still in its infancy. In this study we aimed to correlate how SARS-CoV-2 utilizes its proteins for tackling the host immune response; parallelly, how host epigenetic factors might play a role in this pathogenesis was also investigated. We have utilized a blend of computational and knowledgebase approach to elucidate the interplay between host and SARS-CoV-2. Integrating the experimentally validated host interactome proteins and differentially expressed host genes due to SARS-CoV-2 infection, we have taken a blend of computational and knowledgebase approach to delineate the interplay between host and SARS-CoV-2 in various signaling pathways. Also, we have shown how host epigenetic factors are involved in the deregulation of gene expression. Strikingly, we have found that several transcription factors and other epigenetic factors can modulate some immune signaling pathways, helping both host and virus. We have identified miRNA hsa-miR-429 whose transcription factor was also upregulated and targets were downregulated and this miRNA can have pivotal role in suppression of host immune responses. While searching for the pathways in which viral proteins interact with host proteins, we have found pathways like-HIF-1 signaling, autophagy, RIG-I signaling, Toll-like receptor signaling, Fatty acid oxidation/degradation, Il-17 signaling etc significantly associated. We observed that these pathways can be either hijacked or suppressed by the viral proteins, leading to the improved viral survival and life-cycle. Moreover, pathways like-Relaxin signaling in lungs suggests aberration by the viral proteins might lead to the lung pathophysiology found in COVID-19. Also, enrichment analyses suggest that deregulated genes in SARS-CoV-2 infection are involved in heart development, kidney development, AGE-RAGE signaling pathway in diabetic complications etc. might suggest why patients with comorbidities are becoming more prone to SARS-CoV-2 infection. Our results suggest that SARS-CoV-2 integrates its proteins in different immune signaling pathway and other cellular signaling pathways for developing efficient immune evasion mechanisms, while leading the host to more complicated disease condition. Our findings would help in designing more targeted therapeutic interventions against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.06.050260","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.084384","pub_date":"2020-5-08","title":"ACE2 coding variants in different populations and their potential impact on SARS-CoV-2 binding affinity","abstract":"The susceptibility of different populations to the SARS-CoV-2 infection is not yet understood. A deeper analysis of the genomes of individuals from different populations might explain their risk for infection. In this study, a combined analysis of ACE2 coding variants in different populations and computational chemistry calculations are conducted in order to probe the potential effects of ACE2 coding variants on SARS-CoV-2/ACE2 binding affinity. Our study reveals novel interaction data on the variants and SARS-CoV-2. We could show that ACE2-K26R; which is more frequent in the Ashkenazi Jewish population decrease the electrostatic attraction between SARS-CoV-2 and ACE2. On the contrary, ACE2-I468V, R219C, K341R, D206G, G211R were found to increase the electrostatic attraction and increase the binding to SARS-CoV-2; ordered by the strength of binding from weakest to strongest. I468V, R219C, K341R, D206G and G211R were more frequent in East Asian, South Asian, African and African American, European and European and South Asian populations, respectively. SARS-CoV-2/ACE2 interface in the WT protein and corresponding variants is showed to be a dominated by van der Waals (vdW) interactions. All the mutations except K341R induce an increase in the vdW attractions between the ACE2 and the SARS-CoV-2. The largest increase of is observed for the R219C mutant.","version":"1.1","doi":"10.1101/2020.05.08.084384","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.084103","pub_date":"2020-5-08","title":"AI334 and AQ806 antibodies recognize the spike S protein from SARS-CoV-2 by ELISA","abstract":"We tested 10 recombinant antibodies directed against the spike S protein from SARS-CoV-1. Among them, antibodies AI334 and AQ806 detect by ELISA the spike S protein from SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.08.084103","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.08.084061","pub_date":"2020-5-08","title":"Comparison of SARS-CoV-2 spike protein binding to human, pet, farm animals, and putative intermediate hosts ACE2 and ACE2 receptors","abstract":"The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used recently released X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptor allows to define residues important for binding. From the 20 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S-binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites located near the S-interface. Of note, pigs and dogs which are not or not effectively infected, respectively, have only a few changes in the binding site have relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with viruses from bat and pangolin revealed that the latter contains only one substitution, whereas the bat virus exhibits five. However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-COV-2. SARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the species barrier might be low. However, the lower expression of ACE2 in the upper respiratory tract of some pets and livestock means more research and monitoring should be done to explore the animal source of infection and the risk of potential cross-species transmission. Finally, the analysis also showed that SARS-CoV-2 may not specifically adapted to any of its putative intermediate hosts.","version":"1.1","doi":"10.1101/2020.05.08.084061","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.066761","pub_date":"2020-5-08","title":"Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin","abstract":"The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 \u03bcg.ml\u22121, which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the consequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.","version":"1.2","doi":"10.1101/2020.04.28.066761","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079400","pub_date":"2020-5-08","title":"Massive Multiplexing Can Deliver a $1 Test for COVID-19","abstract":"The severe acute respiratory syndrome virus, SARS-CoV-2 (hereafter COVID-19), rapidly achieved global pandemic status, provoking large-scale screening programs in many nations. Their activation makes it imperative to identify methods that can deliver a diagnostic result at low cost. This paper describes an approach which employs sequence variation in the gene coding for its envelope protein as the basis for a scalable, inexpensive test for COVID-19. It achieves this by coupling a simple RNA extraction protocol with low-volume RT-PCR, followed by E-Gel screening and sequencing on high-throughput platforms to analyze 10,000 samples in a run. Slight modifications to the protocol could support screening programs for other known viruses and for viral discovery. Just as the $1,000 genome is transforming medicine, a $1 diagnostic test for viral and bacterial pathogens would represent a major advance for public health.","version":"1.1","doi":"10.1101/2020.05.05.079400","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.067983","pub_date":"2020-5-07","title":"Evaluation of 19 antiviral drugs against SARS-CoV-2 Infection","abstract":"The global pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) has prompted multiple clinical trials to jumpstart search for anti-SARS-CoV-2 therapies from existing drugs, including those with reported in vitro efficacies as well as those ones that are not known to inhibit SARS-CoV-2, such as ritonavir/lopinavir and favilavir. Here we report that after screening 19 antiviral drugs that are either in clinical trials or with proposed activity against SARS-CoV-2, remdesivir was the most effective. Chloroquine only effectively protected virus-induced cytopathic effect at around 30 \u00b5M with a therapeutic index of 1.5. Our findings also suggest that velpatasvir, ledipasvir, ritonavir, litonavir, lopinavir, favilavir, sofosbuvir, danoprevir, and pocapavir do not have direct antiviral effect.","version":"1.3","doi":"10.1101/2020.04.29.067983","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.081497","pub_date":"2020-5-07","title":"A mouse-adapted SARS-CoV-2 model for the evaluation of COVID-19 medical countermeasures","abstract":"Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic. Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs). SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2. We used reverse genetics to remodel the S and mACE2 binding interface resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro, and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of IFN lambda-1a treatment in human COVID-19 infections.","version":"1.1","doi":"10.1101/2020.05.06.081497","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.081026","pub_date":"2020-5-07","title":"Epigenetic regulator miRNA pattern differences among SARS-CoV, SARS-CoV-2 and SARS-CoV-2 world-wide isolates delineated the mystery behind the epic pathogenicity and distinct clinical characteristics of pandemic COVID-19","abstract":"Detailed molecular mechanism of SARS-CoV-2 pathogenesis is still elusive to address its deadlier nature and to design effective theraputics. Here, we present our study elucidating the interplay between the SARS-CoV and SARS-CoV-2 viruses\u2019; and host\u2019s miRNAs, an epigenetic regulator, as a mode of pathogenesis, and enlightened how the SARS-CoV and SARS-CoV-2 infections differ in terms of their miRNA mediated interactions with host and its implications in the disease complexity. We have utilized computational approaches to predict potential host and viral miRNAs, and their possible roles in different important functional pathways. We have identified several putative host antiviral miRNAs that can target the SARS viruses, and also SARS viruses\u2019 encoded miRNAs targeting host genes. In silico predicted targets were also integrated with SARS infected human cells microarray and RNA-seq gene expression data. Comparison of the host miRNA binding profiles on 67 different SARS-CoV-2 genomes from 24 different countries with respective country\u2019s normalized death count surprisingly uncovered some miRNA clusters which are associated with increased death rates. We have found that induced cellular miRNAs can be both a boon and a bane to the host immunity, as they have possible roles in neutralizing the viral threat, parallelly, they can also function as proviral factors. On the other hand, from over representation analysis, interestingly our study revealed that although both SARS-CoV and SARS-CoV-2 viral miRNAs could target broad immune signaling pathways; only some of the SARS-CoV-2 miRNAs are found to uniquely target some immune signaling pathways like-autophagy, IFN-I signaling etc, which might suggest their immune-escape mechanisms for prolonged latency inside some hosts without any symptoms of COVID-19. Further, SARS-CoV-2 can modulate several important cellular pathways which might lead to the increased anomalies in patients with comorbidities like-cardiovascular diseases, diabetes, breathing complications, etc. This might suggest that miRNAs can be a key epigenetic modulator behind the overcomplications amongst the COVID-19 patients. Our results support that miRNAs of host and SARS-CoV-2 can indeed play a role in the pathogenesis which can be further concluded with more experiments. These results will also be useful in designing RNA therapeutics to alleviate the complications from COVID-19.","version":"1.1","doi":"10.1101/2020.05.06.081026","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.04.976027","pub_date":"2020-5-07","title":"Interaction of the spike protein RBD from SARS-CoV-2 with ACE2: similarity with SARS-CoV, hot-spot analysis and effect of the receptor polymorphism","abstract":"The spread of COVID-19 caused by the SARS-CoV-2 outbreak has been growing since its first identification in December 2019. The publishing of the first SARS-CoV-2 genome made a valuable source of data to study the details about its phylogeny, evolution, and interaction with the host. Protein-protein binding assays have confirmed that Angiotensin-converting enzyme 2 (ACE2) is more likely to be the cell receptor through which the virus invades the host cell. In the present work, we provide an insight into the interaction of the viral spike Receptor Binding Domain (RBD) from different coronavirus isolates with host ACE2 protein. By calculating the binding energy score between RBD and ACE2, we highlighted the putative jump in the affinity from a progenitor form of SARS-CoV-2 to the current virus responsible for COVID-19 outbreak. Our result was consistent with previously reported phylogenetic analysis and corroborates the opinion that the interface segment of the spike protein RBD might be acquired by SARS-CoV-2 via a complex evolutionary process rather than a progressive accumulation of mutations. We also highlighted the relevance of Q493 and P499 amino acid residues of SARS-CoV-2 RBD for binding to human ACE2 and maintaining the stability of the interface. Moreover, we show from the structural analysis that it is unlikely for the interface residues to be the result of genetic engineering. Finally, we studied the impact of eight different variants located at the interaction surface of ACE2, on the complex formation with SARS-CoV-2 RBD. We found that none of them is likely to disrupt the interaction with the viral RBD of SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.03.04.976027","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.057380","pub_date":"2020-5-07","title":"Tracing Back the Temporal Change of SARS-CoV-2 with Genomic Signatures","abstract":"The coronavirus disease (COVID-19) outbreak starting from China at the end of 2019 and its subsequent spread in many countries have given rise to thousands of coronavirus samples being collected and sequenced till date. To trace back the initial temporal change of SARS-CoV-2, the coronavirus implicated in COVID-19, we study the limited genomic sequences that were available within the first couple of months of its spread. These samples were collected under varying circumstances and highlight wide variations in their genomic compositions. In this paper, we explore whether these variations characterize the initial temporal change of SARS-CoV-2 sequences. We observe that n-mer distributions in the SARS-CoV-2 samples, which were collected at an earlier period of time, predict its collection timeline with approximately 78% accuracy. However, such a distinctive pattern disappears with the inclusion of samples collected at a later time. We further observe that isolation sources (e.g., oronasopharynx, saliva, feces, etc.) could not be predicted by the n-mer patterns in these sequences. Finally, the phylogenetic and protein-alignment analyses highlight interesting associations between SARS-CoV-2 and other coronaviruses.","version":"1.3","doi":"10.1101/2020.04.24.057380","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.077016","pub_date":"2020-5-07","title":"The SARS-CoV-2-like virus found in captive pangolins from Guangdong should be better sequenced","abstract":"Viruses closely related to SARS-CoV-2, which is the virus responsible of the Covid-19 pandemic, were sequenced in several Sunda pangolins (Manis javanica) seized in the Guangdong and Guangxi provinces of China between 2017 and 2019. These viruses belong to two lineages: one from Guangdong (GD/P) and the other from Guangxi (GX/P). The GD/P viruses are particularly intriguing as the amino-acid sequence of the receptor binding domain of the spike protein is very similar to that of the human SARS-CoV-2 virus (97.4%). This characteristic suggests that GD/P viruses are capable of binding human ACE2 receptor and may therefore be able to mediate infection of human cells. Whereas all six GX/P genomes were deposited as annotated sequences in GenBank, none of the two GD/P genomes assembled in previous studies are currently available. To overcome this absence, I assembled these genomes from the Sequence Read Archive (SRA) data available for SARS-CoV-2-like viruses detected in five captive pangolins from Guangdong. I found the genome assemblies of GD/P virus of poor quality, having high levels of missing data. Additionally, unexpected reads in the Illumina sequencing data were identified. The GD/P2S dataset contains reads that are identical to SARS-CoV-2, suggesting either the coexistence of two SARS-CoV-2-like viruses in the same pangolin or contamination by the human virus. In the four other GD/P datasets many mitochondrial reads from pangolin were identified, as well as from three other species, namely, human, mouse and tiger. Importantly, I only identified three polymorphic nucleotide sites between the five GD/P sequences. Such low levels of polymorphism may reasonably be accounted for by sequencing errors alone, thus raising the possibility that the five pangolins seized in Guangdong in March 2019 were infected by the same virus strain, most probably during their captivity.","version":"1.1","doi":"10.1101/2020.05.07.077016","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.082487","pub_date":"2020-5-07","title":"COVID-19: Viral-host interactome analyzed by network based-approach model to study pathogenesis of SARS-CoV-2 infection","abstract":"Epidemiological, virological and pathogenetic characteristics of SARS-CoV-2 infection are under evaluation. A better understanding of the pathophysiology associated with COVID-19 is crucial to improve treatment modalities and to develop effective prevention strategies. Transcriptomic and proteomic data on the host response against SARS-CoV-2 still have anecdotic character; currently available data from other coronavirus infections are therefore a key source of information. We investigated selected molecular aspects of three human coronavirus (HCoV) infections, namely SARS-CoV, MERS-CoV and HCoV-229E, through a network based-approach. A functional analysis of HCoV-host interactome was carried out in order to provide a theoretic host-pathogen interaction model for HCoV infections and in order to translate the results in prediction for SARS-CoV-2 pathogenesis. The 3D model of S-glycoprotein of SARS-CoV-2 was compared to the structure of the corresponding SARS-CoV, HCoV-229E and MERS-CoV S-glycoprotein. SARS-CoV, MERS-CoV, HCoV-229E and the host interactome were inferred through published protein-protein interactions (PPI) as well as gene co-expression, triggered by HCoV S-glycoprotein in host cells. Although the amino acid sequences of the S-glycoprotein were found to be different between the various HCoV, the structures showed high similarity, but the best 3D structural overlap shared by SARS-CoV and SARS-CoV-2, consistent with the shared ACE2 predicted receptor. The host interactome, linked to the S-glycoprotein of SARS-CoV and MERS-CoV, mainly highlighted innate immunity pathway components, such as Toll Like receptors, cytokines and chemokines. In this paper, we developed a network-based model with the aim to define molecular aspects of pathogenic phenotypes in HCoV infections. The resulting pattern may facilitate the process of structure-guided pharmaceutical and diagnostic research with the prospect to identify potential new biological targets.","version":"1.1","doi":"10.1101/2020.05.07.082487","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.082925","pub_date":"2020-5-07","title":"A Combined approach of MALDI-TOF Mass Spectrometry and multivariate analysis as a potential tool for the detection of SARS-CoV-2 virus in nasopharyngeal swabs","abstract":"Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid, sensitive and specific diagnosis of SARS-CoV-2 by fast and unambiguous testing is widely recognized to be critical in responding to the ongoing outbreak. Since the current testing capacity of RT-PCR-based methods is being challenged due to the extraordinary demand of supplies, such as RNA extraction kits and PCR reagents worldwide, alternative and/or complementary testing assays should be developed. Here, we exploit the potential of mass spectrometry technology combined with machine learning algorithms as an alternative fast tool for SARS-CoV-2 detection from nasopharyngeal swabs samples. According to our preliminary results, mass spectrometry-based methods combined with multivariate analysis showed an interesting potential as a complementary diagnostic tool and further steps should be focused on sample preparation protocols and the improvement of the technology applied.","version":"1.1","doi":"10.1101/2020.05.07.082925","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.081968","pub_date":"2020-5-07","title":"Suramin inhibits SARS-CoV-2 infection in cell culture by interfering with early steps of the replication cycle","abstract":"The SARS-CoV-2 pandemic that originated from Wuhan, China, in December 2019 has impacted public health, society and economy and the daily lives of billions of people in an unprecedented manner. There are currently no specific registered antiviral drugs to treat or prevent SARS-CoV-2 infections. Therefore, drug repurposing would be the fastest route to provide at least a temporary solution while better, more specific drugs are being developed. Here we demonstrate that the antiparasitic drug suramin inhibits SARS-CoV-2 replication, protecting Vero E6 cells with an EC50 of \u223c20 \u00b5M, which is well below the maximum attainable level in human serum. Suramin also decreased the viral load by 2-3 logs when Vero E6 cells or cells of a human lung epithelial cell line (Calu-3) were treated. Time of addition and plaque reduction assays showed that suramin acts on early steps of the replication cycle, possibly preventing entry of the virus. In a primary human airway epithelial cell culture model, suramin also inhibited the progression of infection. The results of our preclinical study warrant further investigation and suggest it is worth evaluating whether suramin provides any benefit for COVID-19 patients, which obviously requires well-designed, properly controlled randomized clinical trials.","version":"1.1","doi":"10.1101/2020.05.06.081968","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.082230","pub_date":"2020-5-07","title":"A Modified ACE2 peptide mimic to block SARS-CoV2 entry","abstract":"A 23-residue peptide fragment that forms a part of the \u03b1-1 helix of the ACE2 peptidase domain, the recognition domain for SARS-CoV2 on the ACE2 receptor, holds the potential as a drug to block the viral receptor binding domain (RBD) from forming a complex with ACE2. The peptide has recently been shown to bind the viral RBD with good efficiency. Here, we present a detailed analysis of the energetics of binding of the peptide to the SARS-CoV2 RBD. We use equilibrium molecular dynamics simulation to study the dynamics of the complex. We perform end-state binding energy calculations to gain a residue-level insight into the binding process and use the information to incorporate point mutations into the peptide. We demonstrate using binding energy calculations that the peptide with certain point mutations, especially E17L, shows a stronger binding to the RBD as compared to the wild type peptide. We propose that the modified peptide will thus be more efficient in blocking RBD-ACE2 binding.","version":"1.1","doi":"10.1101/2020.05.07.082230","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.26.062422","pub_date":"2020-5-07","title":"Quality control of low-frequency variants in SARS-CoV-2 genomes","abstract":"During the current outbreak of COVID-19, research labs around the globe submit sequences of the local SARS-CoV-2 genomes to the GISAID database to provide a comprehensive analysis of the variability and spread of the virus during the outbreak. We explored the variations in the submitted genomes and found a significant number of variants that can be seen only in one submission (singletons). While it is not completely clear whether these variants are erroneous or not, these variants show lower transition/transversion ratio. These singleton variants may influence the estimations of the viral mutation rate and tree topology. We suggest that genomes with multiple singletons even marked as high-covered should be considered with caution. We also provide a simple script for checking variant frequency against the database before submission.","version":"1.2","doi":"10.1101/2020.04.26.062422","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.080630","pub_date":"2020-5-07","title":"In Silico Trial to test COVID-19 candidate vaccines: a case study with UISS platform","abstract":"SARS-CoV-2 is a severe respiratory infection that infects humans. Its outburst entitled it as a pandemic emergence. To get a grip on this outbreak, specific preventive and therapeutic interventions are urgently needed. It must be said that, until now, there are no existing vaccines for coronaviruses. To promptly and rapidly respond to pandemic events, the application of in silico trials can be used for designing and testing medicines against SARS-CoV-2 and speed-up the vaccine discovery pipeline, predicting any therapeutic failure and minimizing undesired effects. Here, we present an in silico platform that showed to be in very good agreement with the latest literature in predicting SARS-CoV-2 dynamics and related immune system host response. Moreover, it has been used to predict the outcome of one of the latest suggested approach to design an effective vaccine, based on monoclonal antibody. Universal Immune System Simulator (UISS) in silico platform is potentially ready to be used as an in silico trial platform to predict the outcome of vaccination strategy against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.06.080630","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.15.907873","pub_date":"2020-5-07","title":"The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal respiratory infection by influenza A virus at epithelial surfaces","abstract":"Respiratory viruses such as influenza A virus (IAV) and SARS-CoV-2 (Covid-19) cause pandemic infections where cytokine storm syndrome, lung inflammation and pneumonia lead to high mortality. Given the high social and economic cost of these viruses, there is an urgent need for a comprehensive understanding of how the airways defend against virus infection. Viruses entering cells by endocytosis are killed when delivered to lysosomes for degradation. Lysosome delivery is facilitated by non-canonical autophagy pathways that conjugate LC3 to endo-lysosome compartments to enhance lysosome fusion. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that non-canonical autophagy protects mice from lethal IAV infection of the airways. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity murine-adapted IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV infection was controlled within epithelial barriers where non-canonical autophagy slowed fusion of IAV with endosomes and reduced activation of interferon signalling. This was consistent with conditional mouse models and ex vivo analysis showing that protection against IAV infection of lung was independent of phagocytes and other leukocytes. This establishes non-canonical autophagy pathways in airway epithelial cells as a novel innate defence mechanism that can restrict IAV infection and lethal inflammation at respiratory surfaces.","version":"1.2","doi":"10.1101/2020.01.15.907873","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.07.075093","pub_date":"2020-5-07","title":"Holobiont Urbanism: sampling urban beehives reveals cities\u2019 metagenomes","abstract":"Over half of the world\u2019s population lives in urban areas with, according to the United Nations (UN), nearly 70% expected to live in cities by 2050 (United Nations, 2019). Our cities are built by and for humans, but are also complex, adaptive biological systems involving a diversity of other living species. The majority of these species are invisible and constitute the city\u2019s microbiome. Our design decisions for the built environment shape these invisible populations, and we interact with them on a constant basis. A growing body of evidence shows us that our health and well-being are dependent on these interactions. Indeed, multicellular organisms owe meaningful aspects of their development and phenotype to interactions with the microorganisms\u2014bacteria or fungi\u2014with which they live in continual exchange and symbiosis. While the processing and sequencing of samples can be high-throughput, gathering samples is still very expensive, labor intensive, and can require mobilizing large numbers of volunteers to get a snapshot of the microbial landscape of a city, such as City Sampling Day (metasub.org). Here we postulate that honeybees may be effective collaborators in the sampling process, as they daily forage within a 2-mile radius of their hive. We describe the results of a pilot study conducted with 3 rooftop beehives in Brooklyn, NY, where we evaluated the potential of various hive materials (beeswax, honey, debris, pollen, propolis) to reveal information as to the surrounding metagenomic landscape, and where we conclude that the bee debris are the richest substrate. Based on these results, we profiled 4 additional cities in this manner: Sydney, Melbourne, Venice and Tokyo. While the molecular and computational methods used here were based on DNA analysis, it is possible they could be used to monitor RNA-based viruses such as Sars-Cov-2. Here we present the results of this study, and discuss them in terms of architectural implications, as well as the potential of this method for epidemic surveillance.","version":"1.1","doi":"10.1101/2020.05.07.075093","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079608","pub_date":"2020-5-06","title":"Inhibition of the replication of SARS-CoV-2 in human cells by the FDA-approved drug chlorpromazine","abstract":"Urgent action is needed to fight the ongoing COVID-19 pandemic by reducing the number of infected people along with the infection contagiousness and severity. Chlorpromazine (CPZ), the prototype of typical antipsychotics from the phenothiazine group, is known to inhibit clathrin-mediated endocytosis and acts as an antiviral, in particular against SARS-CoV-1 and MERS-CoV. In this study, we describe the in vitro testing of CPZ against a SARS-CoV-2 isolate in monkey and human cells. We evidenced an antiviral activity against SARS-CoV-2 with an IC50 of \u223c10\u03bcM. Because of its high biodistribution in lung, saliva and brain, such IC50 measured in vitro may translate to CPZ dosage used in clinical routine. This extrapolation is in line with our observations of a higher prevalence of symptomatic and severe forms of COVID-19 infections among health care professionals compared to patients in psychiatric wards. These preclinical findings support the repurposing of CPZ, a largely used drug with mild side effects, in COVID-19 treatment.","version":"1.1","doi":"10.1101/2020.05.05.079608","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079202","pub_date":"2020-5-06","title":"Neutralization of SARS-CoV-2 by destruction of the prefusion Spike","abstract":"There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 \u00c5 of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. CR3022 neutralises SARS-CoV-2 Neutralisation is by destroying the prefusion SPIKE conformation This antibody may have therapeutic potential alone or with one blocking receptor attachment","version":"1.1","doi":"10.1101/2020.05.05.079202","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.080119","pub_date":"2020-5-06","title":"Multiple SARS-CoV-2 introductions shaped the early outbreak in Central Eastern Europe: comparing Hungarian data to a worldwide sequence data-matrix","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019 the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of the 21th of April, 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.","version":"1.1","doi":"10.1101/2020.05.06.080119","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.06.079798","pub_date":"2020-5-06","title":"Applying Lexical Link Analysis to Discover Insights from Public Information on COVID-19","abstract":"SARS-Cov-2, the deadly and novel virus, which has caused a worldwide pandemic and drastic loss of human lives and economic activities. An open data set called the COVID-19 Open Research Dataset or CORD-19 contains large set full text scientific literature on SARS-CoV-2. The Next Strain consists of a database of SARS-CoV-2 viral genomes from since 12/3/2019. We applied an unique information mining method named lexical link analysis (LLA) to answer the call to action and help the science community answer high-priority scientific questions related to SARS-CoV-2. We first text-mined the CORD-19. We also data-mined the next strain database. Finally, we linked two databases. The linked databases and information can be used to discover the insights and help the research community to address high-priority questions related to the SARS-CoV-2\u2019s genetics, tests, and prevention. In this paper, we show how to apply an unique information mining method lexical link analysis (LLA) to link unstructured (CORD-19) and structured (Next Strain) data sets to relevant publications, integrate text and data mining into a single platform to discover the insights that can be visualized, and validated to answer the high-priority questions of genetics, incubation, treatment, symptoms, and prevention of COVID-19.","version":"1.1","doi":"10.1101/2020.05.06.079798","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079087","pub_date":"2020-5-06","title":"SARS-CoV-2 codon usage bias downregulates host expressed genes with similar codon usage","abstract":"Severe acute respiratory syndrome is quickly spreading throughout the world and was declared as a pandemic by the World Health Organisation (WHO). The pathogenic agent is a new coronavirus (SARS-CoV-2) that infects pulmonary cells with great effectiveness. In this study we focus on the codon composition for the viral proteins synthesis and its relationship with the proteins synthesis of the host. Our analysis reveals that SARS-CoV-2 preferred codons have poor representation of G or C nucleotides in the third position, a characteristic which could conduct to an unbalance in the tRNAs pools of the infected cells with serious implications in host protein synthesis. By integrating this observation with proteomic data from infected cells, we observe a reduced translation rate of host proteins associated with highly expressed genes, and that they share the codon usage bias of the virus. The functional analysis of these genes suggests that this mechanism of epistasis contributes to understand some deleterious collateral effect as result of the viral replication. In this manner, our finding contribute to the understanding of the SARS-CoV-2 pathogeny and could be useful for the design of a vaccine based on the live attenuated strategy.","version":"1.1","doi":"10.1101/2020.05.05.079087","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.078956","pub_date":"2020-5-05","title":"Selectomic and Evolvability Analyses of the Highly Pathogenic Betacoronaviruses SARS-CoV-2, SARS-CoV, and MERS-CoV","abstract":"SARS-CoV-2, the causative agent of COVID-19, is widespread in several countries around the world following its late-2019 emergence in the human population. Rapid development of molecular diagnostic tests and subunit vaccines have been prioritized, and as such evaluating the SARS-CoV-2 genomic plasticity and evolutionary dynamics is an urgent need. We determined the SARS-CoV-2 selectome by calculating rates of pervasive and episodic diversifying selection for every amino acid coding position in the SARS-CoV-2 genome. To provide context for evolutionary dynamics of a highly pathogenic betacoronavirus following a zoonotic spillover into human hosts, we also determined the selectomes of SARS-CoV and MERS-CoV, and performed evolvability calculations for SARS-CoV-2 based on SARS-CoV. These analyses identify the amino acid sites within each coding sequence that have been subjected to pervasive diversifying selection or episodic diversifying selection, and report significantly evolvable sites in the ORF1a polyprotein, the spike protein, and the membrane protein of SARS-CoV-2. These findings provide a comprehensive view of zoonotic, highly pathogenic betacoronavirus evolutionary dynamics that can be directly applied to diagnostic assay and vaccine design for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.05.078956","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.073080","pub_date":"2020-5-05","title":"Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication","abstract":"The COVID-19 pandemic, attributed to the SARS-CoV-2 coronavirus infection, resulted in millions infected worldwide and an immediate need for antiviral treatments. The main protease (Mpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide and subsequent viral replication. Feline infectious peritonitis, a fatal infection in cats caused by a coronavirus, was successfully treated previously with a dipeptide-based protease inhibitor. Here we show this drug, GC376, and its analog GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of the SARS-CoV and SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 in cell culture, with EC50 values near one micromolar and little to no toxicity. These protease inhibitors are soluble, non-toxic, and bind reversibly. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals (cats). The work here lays the framework for their use in human trials for the treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.05.03.073080","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079194","pub_date":"2020-5-05","title":"Bulk and single-cell gene expression profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for therapeutic intervention","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health threat with more than two million infected people since its emergence in late 2019. Detailed knowledge of the molecular biology of the infection is indispensable for understanding of the viral replication, host responses, and disease progression. We provide gene expression profiles of SARS-CoV and SARS-CoV-2 infections in three human cell lines (H1299, Caco-2 and Calu-3 cells), using bulk and single-cell transcriptomics. Small RNA profiling showed strong expression of the immunity and inflammation-associated microRNA miRNA-155 upon infection with both viruses. SARS-CoV-2 elicited approximately two-fold higher stimulation of the interferon response compared to SARS-CoV in the permissive human epithelial cell line Calu-3, and induction of cytokines such as CXCL10 or IL6. Single cell RNA sequencing data showed that canonical interferon stimulated genes such as IFIT2 or OAS2 were broadly induced, whereas interferon beta (IFNB1) and lambda (IFNL1-4) were expressed only in a subset of infected cells. In addition, temporal resolution of transcriptional responses suggested interferon regulatory factors (IRFs) activities precede that of nuclear factor-\u03baB (NF-\u03baB). Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 charperone activity by Tanespimycin/17-N-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in a reduction of viral replication, and of TNF and IL1B mRNA levels. In summary, our study established in vitro cell culture models to study SARS-CoV-2 infection and identified HSP90 protein as potential drug target for therapeutic intervention of SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.05.05.079194","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.077842","pub_date":"2020-5-05","title":"SARS-CoV-2 Spike Glycoprotein Receptor Binding Domain is Subject to Negative Selection with Predicted Positive Selection Mutations","abstract":"COVID-19 is a highly contagious disease caused by a novel coronavirus SARS-CoV-2. The interaction between SARS-CoV-2 spike protein and the host cell surface receptor ACE2 is responsible for mediating SARS-CoV-2 infection. By analyzing the spike-hACE2 interacting surface, we predicted many hot spot residues that make major contributions to the binding affinity. Mutations on most of these residues are likely to be deleterious, leading to less infectious virus strains that may suffer from negative selection. Meanwhile, several residues with mostly advantageous mutations have been predicted. It is more probable that mutations on these residues increase the transmission ability of the virus by enhancing spike-hACE2 interaction. So far, only a limited number of mutations has been reported in this region. However, the list of hot spot residues with predicted downstream effects from this study can still serve as a tracking list for SARS-CoV-2 evolution studies. Coincidentally, one advantageous mutation, p.476G>S, started to surge in the last couple of weeks based on the data submitted to the public domain, indicating that virus strains with increased transmission ability may have already spread.","version":"1.1","doi":"10.1101/2020.05.04.077842","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.079095","pub_date":"2020-5-05","title":"Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids","abstract":"The SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.","version":"1.1","doi":"10.1101/2020.05.05.079095","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.069054","pub_date":"2020-5-05","title":"Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2","abstract":"We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified thirteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; it began spreading in Europe in early February, and when introduced to new regions it rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.","version":"1.2","doi":"10.1101/2020.04.29.069054","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.075911","pub_date":"2020-5-05","title":"Global Spread of SARS-CoV-2 Subtype with Spike Protein Mutation D614G is Shaped by Human Genomic Variations that Regulate Expression of TMPRSS2 and MX1 Genes","abstract":"COVID-19 pandemic is a major human tragedy. Worldwide, SARS-CoV-2 has already infected over 3 million and has killed about 230,000 people. SARS-CoV-2 originated in China and, within three months, has evolved to an additional 10 subtypes. One particular subtype with a non-silent (Aspartate to Glycine) mutation at 614th position of the Spike protein (D614G) rapidly outcompeted other pre-existing subtypes, including the ancestral. We assessed that D614G mutation generates an additional serine protease (Elastase) cleavage site near the S1-S2 junction of the Spike protein. We also identified that a single nucleotide deletion (delC) at a known variant site (rs35074065) in a cis-eQTL of TMPRSS2, is extremely rare in East Asians but is common in Europeans and North Americans. The delC allele facilitates entry of the 614G subtype into host cells, thus accelerating the spread of 614G subtype in Europe and North America where the delC allele is common. The delC allele at the cis-eQTL locus rs35074065 of TMPRSS2 leads to overexpression of both TMPRSS2 and a nearby gene MX1. The cis-eQTL site, rs35074065 overlaps with a transcription factor binding site of an activator (IRF1) and a repressor (IRF2). IRF1 activator can bind to variant delC allele, but IRF2 repressor fails to bind. Thus, in an individual carrying the delC allele, there is only activation, but no repression. On viral entry, IRF1 mediated upregulation of MX1 leads to neutrophil infiltration and processing of 614G mutated Spike protein by neutrophil Elastase. The simultaneous processing of 614G spike protein by TMPRSS2 and Elastase serine proteases facilitates the entry of the 614G subtype into host cells. Thus, SARS-CoV-2, particularly the 614G subtype, has spread more easily and with higher frequency to Europe and North America where the delC allele regulating expression of TMPRSS2 and MX1 host proteins is common, but not to East Asia where this allele is rare.","version":"1.1","doi":"10.1101/2020.05.04.075911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.065383","pub_date":"2020-5-05","title":"In silico design and validation of commercial kit GPS\u2122 CoVID-19 dtec-RT-qPCR Test under criteria of UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012","abstract":"The Corona Virus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has become a serious infectious disease affecting human health worldwide and rapidly declared a pandemic by WHO. Early, several RT-qPCR were designed by using only the first SARS-CoV-2 genome sequence. A few days later, when additional SARS-CoV-2 genome were retrieved, the kit GPS\u2122 CoVID-19 dtec-RT-qPCR Test was designed to provide a highly specific detection method and commercially available worldwide. The kit was validated following criteria recommended by the UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012. The present study approached the in silico specificity of the GPS\u2122 CoVID-19 dtec-RT-qPCR Test and RT-qPCR designs currently published. The empirical validation parameters specificity (inclusivity/exclusivity), quantitative phase analysis (10-106 copies), reliability (repeatability/reproducibility) and sensitivity (detection/quantification limits) were evaluated for a minimum of 10-15 assays. Diagnostic validation was achieved by two independent reference laboratories, the Instituto de Salud Carlos III (ISCIII), (Madrid, Spain) and the Public Health England (PHE; Colindale, London, UK). The GPS\u2122 RT-qPCR primers and probe showed the highest number of mismatches with the closet related non-SARS-CoV-2 coronavirus, including some indels. The kits passed all parameters of validation with strict acceptance criteria. Results from reference laboratories 100% correlated with these obtained by suing reference methods and received an evaluation with 100% of diagnostic sensitivity and specificity. The GPS\u2122 CoVID-19 dtec-RT-qPCR Test, available with full analytical and diagnostic validation, represents a case of efficient transfer of technology being successfully used since the pandemic was declared. The analysis suggested the GPS\u2122 CoVID-19 dtec-RT-qPCR Test is the more exclusive by far.","version":"1.2","doi":"10.1101/2020.04.27.065383","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.078238","pub_date":"2020-5-05","title":"Translation-associated mutational U-pressure in the first ORF of SARS-CoV-2 and other coronaviruses","abstract":"Within four months of the ongoing COVID-19 pandemic caused by SARS-CoV-2, more than 250 nucleotide mutations have been detected in the ORF1 of the virus isolated from different parts of the globe. These observations open up an obvious question about the rate and direction of mutational pressure for further vaccine and therapeutics designing. In this study, we did a comparative analysis of ORF1a and ORF1b by using the first isolate (Wuhan strain) as the parent sequence. We observed that most of the nucleotide mutations are C to U transitions. The rate of synonymous C to U transitions is significantly higher than the rate of nonsynonymous ones, indicating negative selection on amino acid substitutions. Further, trends in nucleotide usage bias have been investigated in 49 coronaviruses species. A strong bias in nucleotide usage in fourfold degenerated sites towards uracil residues is seen in ORF1 of all the studied coronaviruses. A more substantial mutational U pressure is observed in ORF1a than in ORF1b owing to the translation of ORF1ab via programmed ribosomal frameshifting. Unlike other nucleotide mutations, mutational U pressure caused by cytosine deamination, mostly occurring in the RNA-plus strand, cannot be corrected by the proof-reading machinery of coronaviruses. The knowledge generated on the direction of mutational pressure during translation of viral RNA-plus strands has implications for vaccine and nucleoside analogue development for treating covid-19 and other coronavirus infections.","version":"1.1","doi":"10.1101/2020.05.05.078238","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.078501","pub_date":"2020-5-05","title":"Test Agreement Between Roche Cobas 6800 and Cepheid GeneXpert Xpress SARS-CoV-2 Assays at High Cycle Threshold Ranges","abstract":"The SARS-CoV-2 pandemic has changed the face of the globe and upended the daily lives of billions. In an effort to bring mass-testing to as many as possible, multiple diagnostic tests including molecular, antigen detection and serological assays have been rapidly developed. However, there is very little information on positive test agreement across modalities, especially for lower viral loads. Thirty-five nasopharyngeal samples that had cycle threshold (Ct) values greater than 30.0 from the Roche cobas 6800 assay were run on the Cepheid GeneXpert Xpress SARS-CoV-2 assay. Ct values ranged from 30.1 to 37.9 (mean 36.7 \u00b1 1.9) on the Roche cobas 6800 assay and 24.6 to 42.4 (mean 32.8\u00b14.1) on the Cepheid assay. There was a bias of 0.33 \u00b1 3.21, (mean difference \u22121.59, 95% limits of agreement \u22125.97, 6.63) signifying close agreement between the 2 instruments with a high standard deviation. The close test agreement between the cobas 6800 and GeneXpert at high Ct values allows for utilization of both assays interchangeable in accordance with testing algorithms.","version":"1.1","doi":"10.1101/2020.05.05.078501","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.05.077867","pub_date":"2020-5-05","title":"Type III interferons disrupt the lung epithelial barrier upon viral recognition","abstract":"Lower respiratory tract infections are a leading cause of mortality driven by infectious agents. RNA viruses such as influenza virus, respiratory syncytial virus and the new pandemic coronavirus SARS-CoV-2 can be highly pathogenic. Clinical and experimental evidence indicate that most severe and lethal cases do not depend on the viral burden and are, instead, characterized by an aberrant immune response. In this work we assessed how the innate immune response contributes to the pathogenesis of RNA virus infections. We demonstrate that type III interferons produced by dendritic cells in the lung in response to viral recognition cause barrier damage and compromise the host tissue tolerance. In particular, type III interferons inhibit tissue repair and lung epithelial cell proliferation, causing susceptibility to lethal bacterial superinfections. Overall, our data give a strong mandate to rethink the pathophysiological roles of this group of interferons and their possible use in the clinical practice against endemic as well as emerging viral infections.","version":"1.1","doi":"10.1101/2020.05.05.077867","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.074781","pub_date":"2020-5-04","title":"Missense variants in ACE2 are predicted to encourage and inhibit interaction with SARS-CoV-2 Spike and contribute to genetic risk in COVID-19","abstract":"SARS-CoV-2 invades host cells via an endocytic pathway that begins with the interaction of the SARS-CoV-2 Spike glycoprotein (S-protein) and human Angiotensin-converting enzyme 2 (ACE2). Genetic variability in ACE2 may be one factor that mediates the broad-spectrum severity of SARS-CoV-2 infection and COVID-19 outcomes. We investigated the capacity of ACE2 variation to influence SARS-CoV-2 infection with a focus on predicting the effect of missense variants on the ACE2 SARS-CoV-2 S-protein interaction. We validated the mCSM-PPI2 variant effect prediction algorithm with 26 published ACE2 mutant SARS-CoV S-protein binding assays and found it performed well in this closely related system (True Positive Rate = 0.7, True Negative Rate = 1). Application of mCSM-PPI2 to ACE2 missense variants from the Genome Aggregation Consortium Database (gnomAD) identified three that are predicted to strongly inhibit or abolish the S-protein ACE2 interaction altogether (p.Glu37Lys, p.Gly352Val and p.Asp355Asn) and one that is predicted to promote the interaction (p.Gly326Glu). The S-protein ACE2 inhibitory variants are expected to confer a high degree of resistance to SARS-CoV-2 infection whilst the S-protein ACE2 affinity enhancing variant may lead to additional susceptibility and severity. We also performed in silico saturation mutagenesis of the S-protein ACE2 interface and identified a further 38 potential missense mutations that could strongly inhibit binding and one more that is likely to enhance binding (Thr27Arg). A conservative estimate places the prevalence of the strongly protective variants between 12-70 per 100,000 population but there is the possibility of higher prevalence in local populations or those underrepresented in gnomAD. The probable interplay between these ACE2 affinity variants and ACE2 expression polymorphisms is highlighted as well as gender differences in penetrance arising from ACE2\u2019s situation on the X-chromosome. It is also described how our data can help power future genetic association studies of COVID-19 phenotypes and how the saturation mutant predictions can help design a mutant ACE2 with tailored S-protein affinity, which may be an improvement over a current recombinant ACE2 that is undergoing clinical trial. \n1 ACE2 gnomAD missense variant (p.Gly326Glu) and one unobserved missense mutation (Thr27Arg) are predicted to enhance ACE2 binding with SARS-CoV-2 Spike protein, which could result in increased susceptibility and severity of COVID-19\n3 ACE2 missense variants in gnomAD plus another 38 unobserved missense mutations are predicted to inhibit Spike binding, these are expected to confer a high degree of resistance to infection\nThe prevalence of the strongly protective variants is estimated between 12-70 per 100,000 population but higher prevalence may exist in local populations or those underrepresented in gnomAD\nA strategy to design a recombinant ACE2 with tailored affinity towards Spike and its potential therapeutic value is presented\nThe predictions were extensively validated against published ACE2 mutant binding assays for SARS-CoV Spike protein 1 ACE2 gnomAD missense variant (p.Gly326Glu) and one unobserved missense mutation (Thr27Arg) are predicted to enhance ACE2 binding with SARS-CoV-2 Spike protein, which could result in increased susceptibility and severity of COVID-19 3 ACE2 missense variants in gnomAD plus another 38 unobserved missense mutations are predicted to inhibit Spike binding, these are expected to confer a high degree of resistance to infection The prevalence of the strongly protective variants is estimated between 12-70 per 100,000 population but higher prevalence may exist in local populations or those underrepresented in gnomAD A strategy to design a recombinant ACE2 with tailored affinity towards Spike and its potential therapeutic value is presented The predictions were extensively validated against published ACE2 mutant binding assays for SARS-CoV Spike protein","version":"1.1","doi":"10.1101/2020.05.03.074781","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.074971","pub_date":"2020-5-04","title":"Pathogen Reduction of SARS-CoV-2 Virus in Plasma and Whole Blood using Riboflavin and UV Light","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products. SARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero cells. Each plasma pool (n=9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n=3). Riboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60-100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were \u2265 4.79 \u00b1 0.15 Logs in plasma and 3.30 \u00b1 0.26 in whole blood units. Riboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 \u00b1 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories.","version":"1.1","doi":"10.1101/2020.05.03.074971","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.075473","pub_date":"2020-5-04","title":"Computational analysis on the ACE2-derived peptides for neutralizing the ACE2 binding to the spike protein of SARS-CoV-2","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19, is spreading globally and has infected more than 3 million people. It has been discovered that SARS-CoV-2 initiates the entry into cells by binding to human angiotensin-converting enzyme 2 (hACE2) through the receptor binding domain (RBD) of its spike glycoprotein. Hence, drugs that can interfere the SARS-CoV-2-RBD binding to hACE2 potentially can inhibit SARS-CoV-2 from entering human cells. Here, based on the N-terminal helix \u03b11 of human ACE2, we designed nine short peptides that have potential to inhibit SARS-CoV-2 binding. Molecular dynamics simulations of peptides in the their free and SARS-CoV-2 RBD-bound forms allow us to identify fragments that are stable in water and have strong binding affinity to the SARS-CoV-2 spike proteins. The important interactions between peptides and RBD are highlighted to provide guidance for the design of peptidomimetics against the SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.05.03.075473","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.075945","pub_date":"2020-5-04","title":"The UCSC SARS-CoV-2 Genome Browser","abstract":"Researchers are generating molecular data pertaining to the SARS-CoV-2 RNA genome and its proteins at an unprecedented rate during the COVID-19 pandemic. As a result, there is a critical need for rapid and continuously updated access to the latest molecular data in a format in which all data can be quickly cross-referenced and compared. We adapted our genome browser visualization tool to the viral genome for this purpose. Molecular data, curated from published studies or from database submissions, are mapped to the viral genome and grouped together into \u201cannotation tracks\u201d where they can be visualized along the linear map of the viral genome sequence and programmatically downloaded in standard format for analysis. The UCSC Genome Browser for SARS-CoV-2 (https://genome.ucsc.edu/covid19.html) provides continuously updated access to the mutations in the many thousands of SARS-CoV-2 genomes deposited in GISAID and the international nucleotide sequencing databases, displayed alongside phylogenetic trees. These data are augmented with alignments of bat, pangolin, and other animal and human coronavirus genomes, including per-base evolutionary rate analysis. All available annotations are cross-referenced on the virus genome, including those from major databases (PDB, RFAM, IEDB, UniProt) as well as up-to-date individual results from preprints. Annotated data include predicted and validated immune epitopes, promising antibodies, RT-PCR and sequencing primers, CRISPR guides (from research, diagnostics, vaccines, and therapies), and points of interaction between human and viral genes. As a community resource, any user can add manual annotations which are quality checked and shared publicly on the browser the next day. We invite all investigators to contribute additional data and annotations to this resource to accelerate research and development activities globally. Contact us at genome-www@soe.ucsc.edu with data suggestions or requests for support for adding data. Rapid sharing of data will accelerate SARS-CoV-2 research, especially when researchers take time to integrate their data with those from other labs on a widely-used community browser platform with standardized machine-readable data formats, such as the SARS-CoV-2 Genome Browser.","version":"1.1","doi":"10.1101/2020.05.04.075945","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.074914","pub_date":"2020-5-04","title":"Neutralizing Antibodies Isolated by a site-directed Screening have Potent Protection on SARS-CoV-2 Infection","abstract":"Neutralizing antibody is one of the most effective interventions for acute pathogenic infection. Currently, over three million people have been identified for SARS-CoV-2 infection but SARS-CoV-2-specific vaccines and neutralizing antibodies are still lacking. SARS-CoV-2 infects host cells by interacting with angiotensin converting enzyme-2 (ACE2) via the S1 receptor-binding domain (RBD) of its surface spike glycoprotein. Therefore, blocking the interaction of SARS-CoV-2-RBD and ACE2 by antibody would cause a directly neutralizing effect against virus. In the current study, we selected the ACE2 interface of SARS-CoV-2-RBD as the targeting epitope for neutralizing antibody screening. We performed site-directed screening by phage display and finally obtained one IgG antibody (4A3) and several domain antibodies. Among them, 4A3 and three domain antibodies (4A12, 4D5, and 4A10) were identified to act as neutralizing antibodies due to their capabilities to block the interaction between SARS-CoV-2-RBD and ACE2-positive cells. The domain antibody 4A12 was predicted to have the best accessibility to all three ACE2-interfaces on the spike homotrimer. Pseudovirus and authentic SARS-CoV-2 neutralization assays showed that all four antibodies could potently protect host cells from virus infection. Overall, we isolated multiple formats of SARS-CoV-2-neutralizing antibodies via site-directed antibody screening, which could be promising candidate drugs for the prevention and treatment of COVID-19.","version":"1.2","doi":"10.1101/2020.05.03.074914","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.066266","pub_date":"2020-5-04","title":"Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission","abstract":"The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has a capability of gaining rapid mutations as the virus spreads. Whole genome sequencing data offers a wide range of opportunities to study the mutation dynamics. The advantage of increasing amount of whole genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity and to investigate the implications of those mutations in protein stability and viral transmission. Four proteins, surface glycoprotein, nucleocapsid, ORF1ab and ORF8 showed frequent mutations, while envelop, membrane, ORF6 and ORF7a proteins showed conservation in terms of amino acid substitutions. Some of the mutations across different proteins showed co-occurrence, suggesting their functional cooperation in stability, transmission and adaptability. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Analysis of protein structure stability of surface glycoprotein mutants indicated viability of specific variants and are more prone to be temporally and spatially distributed across the globe. Similar empirical analysis of other proteins indicated existence of important functional implications of several variants. Analysis of co-occurred mutants indicated their structural and/or functional interaction among different SARS-COV-2 proteins. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing and containment of the disease.","version":"1.1","doi":"10.1101/2020.05.03.066266","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030544","pub_date":"2020-5-04","title":"Revealing variants in SARS-CoV-2 interaction domain of ACE2 and loss of function intolerance through analysis of >200,000 exomes","abstract":"Our researchers took a look at a sequence of DNA known as the ACE2 gene. This gene is most well known for its role in regulating blood pressure. But in recent times, it\u2019s drawn a lot of attention from the scientific community because it may also serve as a doorway of sorts, enabling viruses like SARS-CoV-2 to infect cells. Our researchers looked at the ACE2 gene in more than 200,000 people, comparing their exact DNA sequences to see where there are differences among people. Variation in the DNA sequence of a gene is common and is sometimes meaningless. But other times, small changes in the DNA sequence can alter the protein that is made from that gene. In this case the ACE2 gene makes the ACE2 protein, which is what the SARS-CoV-2 virus interacts with. We found a lot of variation between individuals and checked to see if that variation coincided with any traits (i.e., people with variant X tend to have high blood pressure more often than people without variant X). All of the traits we looked at were non-COVID-19-related traits, meaning we haven\u2019t asked these people anything about COVID-19 yet (this is because these DNA sequences were collected before the pandemic). We found that there are a number of variations observed among people in a specific part of the ACE2 gene. These variations are expected to alter the shape or functionality of a specific part of the ACE2 protein: The part that interacts with the SARS-CoV-2 virus. We don\u2019t yet know what the real-life significance of this variation is, but it\u2019s possible that these variants decrease the protein\u2019s ability to interact with the SARS-CoV-2 virus, thus decreasing the person\u2019s likelihood of being infected. We can speculate that there will be a spectrum of vulnerability to COVID-19 among people, where some people are more vulnerable than others, and that variants in this part of the ACE2 gene may be one of the reasons. The research we presented here shines a light on this part of the ACE2 gene and may give future researchers a direction to go in as they try to figure out what makes people vulnerable to COVID-19 and similar viruses.","version":"1.3","doi":"10.1101/2020.04.07.030544","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.077511","pub_date":"2020-5-04","title":"Complexity in SARS-CoV-2 genome data: Price theory of mutant isolates","abstract":"SARS-CoV-2 is a highly virulent and deadly RNA virus causing the Covid-19 pandemic and several deaths across the world. The pandemic is so fast that any concrete theory of sudden widespread of this disease is still not known. In this work, we studied and analyzed a large number of publicly available SARS-CoV-2 genomes across the world using the multifractal approach. The mutation events in the isolates obey the Markov process and exhibit very high mutational rates, which occur in six specific genes and highest in orf1ab gene, leading to virulent nature. f (\u03b1) analysis indicated that the isolates are highly asymmetric (left-skewed), revealing the richness of complexity and dominance by large fluctuations in genome structure organization. The values of Hq and Dq are found to be significantly large, showing heterogeneous genome structure self-organization, strong positive correlation in organizing the isolates, and quite sensitive to fluctuations in and around it. We then present multiple-isolates hosts-virus interaction models, and derived Price equation for the model. The phase plane analysis of the model showed asymptotic stability type bifurcation. The competition among the mutant isolates drives the trade-off of the dominant mutant isolates, otherwise confined to the present hosts.","version":"1.1","doi":"10.1101/2020.05.04.077511","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.04.077735","pub_date":"2020-5-04","title":"Temporal signal and the phylodynamic threshold of SARS-CoV-2","abstract":"The ongoing SARS-CoV-2 outbreak marks the first time that large amounts of genome sequence data have been generated and made publicly available in near real-time. Early analyses of these data revealed low sequence variation, a finding that is consistent with a recently emerging outbreak, but which raises the question of whether such data are sufficiently informative for phylogenetic inferences of evolutionary rates and time scales. The phylodynamic threshold is a key concept that refers to the point in time at which sufficient molecular evolutionary change has accumulated in available genome samples to obtain robust phylodynamic estimates. For example, before the phylodynamic threshold is reached, genomic variation is so low that even large amounts of genome sequences may be insufficient to estimate the virus\u2019s evolutionary rate and the time scale of an outbreak. We collected genome sequences of SARS-CoV-2 from public databases at 8 different points in time and conducted a range of tests of temporal signal to determine if and when the phylodynamic threshold was reached, and the range of inferences that could be reliably drawn from these data. Our results indicate that by February 2nd 2020, estimates of evolutionary rates and time scales had become possible. Analyses of subsequent data sets, that included between 47 to 122 genomes, converged at an evolutionary rate of about 1.1\u00d710\u22123 subs/site/year and a time of origin of around late November 2019. Our study provides guidelines to assess the phylodynamic threshold and demonstrates that establishing this threshold constitutes a fundamental step for understanding the power and limitations of early data in outbreak genome surveillance.","version":"1.1","doi":"10.1101/2020.05.04.077735","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.071506","pub_date":"2020-5-04","title":"Computational methods to develop potential neutralizing antibody Fab region against SARS-CoV-2 as therapeutic and diagnostic tool","abstract":"SARS-CoV-2, a global pandemic originated from Wuhan city of China in the month of December 2019. There is an urgency to identify potential antibodies to neutralize the virus and also as a diagnostic tool candidate. At present palliative treatments using existing antiviral drugs are under trails to treat SARS-CoV-2.Whole Genome sequence of Wuhan market sample of SARS-CoV-2 was obtained from NCBI Gene ID MN908947.3.Spike protein sequence PDB ID 6VSB obtained from RCSB database. Spike protein sequence had shown top V gene match with IGLV1-44*01, IGLV1-47*02 and has VL type chain. Whole Genome sequence had shown top V gene match with IGHV1-38-4*01 and has VH type chain. VD chain had shown link to allele HLA-A0206 80%, HLA-A0217 80%, HLA-A2301 75%, HLA-A0203 75%, HLA-A0202 70% and HLA-A0201 55% of binding levels. Some conserved regions of spike protein had shown strong binding affinity with HLA-A-0*201, HLA-A24, HLA-B-5701 and HLA-B-5703 alpha chains. Synthetic Fab construct BCR type antibody IgG (CR5840) had shown Polyspecific binding activity with spike glycoprotein when compared with available Anti-SARS antibody CR3022.Thus we propose CR5840 Fab constructed antibody as potential neutralizing antibody for SARS-CoV-2. Based on germline analysis we also propose cytotoxic T lymphocyte epitope peptide selective system as effective tool for the development of SARS-CoV-2 vaccine.","version":"1.1","doi":"10.1101/2020.05.02.071506","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.043554","pub_date":"2020-5-04","title":"Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease","abstract":"Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.","version":"1.1","doi":"10.1101/2020.05.02.043554","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.03.075549","pub_date":"2020-5-04","title":"COVIDier: A Deep-learning Tool For Coronaviruses Genome And Virulence Proteins Classification","abstract":"COVID-19, caused by SARS-CoV-2 infection, has already reached pandemic proportions in a matter of a few weeks. At the time of writing this manuscript, the unprecedented public health crisis caused more than 2.5 million cases with a mortality range of 5-7%. The SARS-CoV-2, also called novel Coronavirus, is related to both SARS-CoV and bat SARS. Great efforts have been spent to control the pandemic that has become a significant burden on the health systems in a short time. Since the emergence of the crisis, a great number of researchers started to use the AI tools to identify drugs, diagnosing using CT scan images, scanning body temperature, and classifying the severity of the disease. The emergence of variants of the SARS-CoV-2 genome is a challenging problem with expected serious consequences on the management of the disease. Here, we introduce COVIDier, a deep learning-based software that is enabled to classify the different genomes of Alpha coronavirus, Beta coronavirus, MERS, SARS-CoV-1, SARS-CoV-2, and bronchitis-CoV. COVIDier was trained on 1925 genomes, belonging to the three families of SARS retrieved from NCBI Database to propose a new method to train deep learning model trained on genome data using Multi-layer Perceptron Classifier (MLPClassifier), a deep learning algorithm, that could blindly predict the virus family name from the genome of by predicting the statistically similar genome from training data to the given genome. COVIDier able to predict how close the emerging novel genomes of SARS to the known genomes with accuracy 99%. COVIDier can replace tools like BLAST that consume higher CPU and time.","version":"1.1","doi":"10.1101/2020.05.03.075549","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.072652","pub_date":"2020-5-03","title":"Molecular Architecture of Early Dissemination and Evolution of the SARS-CoV-2 Virus in Metropolitan Houston, Texas","abstract":"We sequenced the genomes of 320 SARS-CoV-2 strains from COVID-19 patients in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. These genomes were from the viruses causing infections in the earliest recognized phase of the pandemic affecting Houston. Substantial viral genomic diversity was identified, which we interpret to mean that the virus was introduced into Houston many times independently by individuals who had traveled from different parts of the country and the world. The majority of viruses are apparent progeny of strains derived from Europe and Asia. We found no significant evidence of more virulent viral types, stressing the linkage between severe disease, underlying medical conditions, and perhaps host genetics. We discovered a signal of selection acting on the spike protein, the primary target of massive vaccine efforts worldwide. The data provide a critical resource for assessing virus evolution, the origin of new outbreaks, and the effect of host immune response. COVID-19, the disease caused by the SARS-CoV-2 virus, is a global pandemic. To better understand the first phase of virus spread in metropolitan Houston, Texas, we sequenced the genomes of 320 SARS-CoV-2 strains recovered from COVID-19 patients early in the Houston viral arc. We identified no evidence that a particular strain or its progeny causes more severe disease, underscoring the connection between severe disease, underlying health conditions, and host genetics. Some amino acid replacements in the spike protein suggest positive immune selection is at work in shaping variation in this protein. Our analysis traces the early molecular architecture of SARS-CoV-2 in Houston, and will help us to understand the origin and trajectory of future infection spikes.","version":"1.2","doi":"10.1101/2020.05.01.072652","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.066985","pub_date":"2020-5-03","title":"Analyses of spike protein from first deposited sequences of SARS-CoV2 from West Bengal, India","abstract":"India has recently started sequencing SARS-CoV2 genome from clinical isolates. Currently only few sequences are available from three states in India. Kerala was the first state to deposit complete sequence from two isolates followed by one from Gujarat. On April 27, 2020, the first five sequences from the state of West Bengal (Eastern India) were deposited on \u2018a global initiative on sharing avian flu data\u2019 (GISAID) platform. In this paper we have analysed the spike protein sequences from all these five isolates and also compared for their similarities or differences with other sequences reported in India and with isolates of Wuhan origin. We report one unique mutation at position 723 and the other at 1124 in the S2 domain of spike protein of the isolates from West Bengal only and one mutation downstream of the receptor binding domain at position 614 in S1 domain which was common with the sequence from Gujarat (a state of western part of India). Mutation in the S2 domain showed changes in the secondary structure of the spike protein at region of mutation. We also studied molecular dynamics using normal mode analyses and found that this mutation decreases the flexibility of S2 domain. Since both S1 and S2 are important in receptor binding followed by entry in the host cells, such mutations may define the affinity or avidity of receptor binding.","version":"1.2","doi":"10.1101/2020.04.28.066985","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.067843","pub_date":"2020-5-03","title":"A study\u2019s got to know its limitations","abstract":"All research has room for improvement, but authors do not always clearly acknowledge the limitations of their work. In this brief report, we sought to identify the prevalence of limitations statements in the medRxiv COVID-19 SARS-CoV-2 dataset. We combined automated methods with manual review to analyse manuscripts for the presence, or absence, either of a defined limitations section in the text, or as part of the general discussion. We identified a structured limitations statement in 28% of the manuscripts, and overall 52% contained at least one mention of a study limitation. Over one-third of manuscripts contained none of the terms that might typically be associated with reporting of limitations. Overall our method performed with precision of 0.97 and recall of 0.91. The presence or absence of limitations statements can be identified with reasonable confidence using automated tools. We suggest that it might be beneficial to require a defined, structured statement about study limitations, either as part of the submission process, or clearly delineated within the manuscript.","version":"1.2","doi":"10.1101/2020.04.29.067843","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.073411","pub_date":"2020-5-02","title":"Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy","abstract":"Coronavirus disease 2019 (COVID-19) threatens global public health and economy. In order to develop safe and effective vaccines, suitable animal models must be established. Here we report the rapid adaption of SARS-CoV-2 in BALB/c mice, based on which a convenient, economical and effective animal model was developed. Specifically, we found that mouse-adapted SARS-CoV-2 at passage 6 (MACSp6) efficiently infected both aged and young wild-type BALB/c mice, resulting in moderate pneumonia as well as inflammatory responses. The elevated infectivity of MACSp6 in mice could be attributed to the substitution of a key residue (N501Y) in the receptorbinding domain (RBD). Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge. This novel mouse model is convenient and effective in evaluating the in vivo protective efficacy of SARS-CoV-2 vaccine. This study describes a unique mouse model for SARS-CoV-2 infection and confirms protective efficacy of a SARS-CoV-2 RBD subunit vaccine.","version":"1.1","doi":"10.1101/2020.05.02.073411","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.073262","pub_date":"2020-5-02","title":"SARS-CoV-2 is well adapted for humans. What does this mean for re-emergence?","abstract":"In a side-by-side comparison of evolutionary dynamics between the 2019/2020 SARS-CoV-2 and the 2003 SARS-CoV, we were surprised to find that SARS-CoV-2 resembles SARS-CoV in the late phase of the 2003 epidemic after SARS-CoV had developed several advantageous adaptations for human transmission. Our observations suggest that by the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV. However, no precursors or branches of evolution stemming from a less human-adapted SARS-CoV-2-like virus have been detected. The sudden appearance of a highly infectious SARS-CoV-2 presents a major cause for concern that should motivate stronger international efforts to identify the source and prevent near future re-emergence. Any existing pools of SARS-CoV-2 progenitors would be particularly dangerous if similarly well adapted for human transmission. To look for clues regarding intermediate hosts, we analyze recent key findings relating to how SARS-CoV-2 could have evolved and adapted for human transmission, and examine the environmental samples from the Wuhan Huanan seafood market. Importantly, the market samples are genetically identical to human SARS-CoV-2 isolates and were therefore most likely from human sources. We conclude by describing and advocating for measured and effective approaches implemented in the 2002-2004 SARS outbreaks to identify lingering population(s) of progenitor virus.","version":"1.1","doi":"10.1101/2020.05.01.073262","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.073320","pub_date":"2020-5-02","title":"Identification of Drugs Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colonic Organoids","abstract":"The current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to screen 1280 FDA-approved drugs against viral infection. Mycophenolic acid and quinacrine dihydrochloride were found to block the infection of SARS-CoV-2 pseudo-entry virus in COs both in vitro and in vivo, and confirmed to block infection of SARS-CoV-2 virus. This study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified drugs that blocks SARS-CoV-2 infection, suitable for rapid clinical testing.","version":"1.1","doi":"10.1101/2020.05.02.073320","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.072439","pub_date":"2020-5-02","title":"Release of potential pro-inflammatory peptides from SARS-CoV-2 spike glycoproteins in neutrophil-extracellular traps","abstract":"COVID-2019 has progressed in around 10-15% of patients to an acute respiratory distress syndrome characterized by extensive pulmonary inflammation and elevated production of pro-inflammatory cytokines. Neutrophil activation seems to be crucial in the initiation and perpetuation of this exacerbated lung inflammation. However, the precise mechanisms by which this activation occurs remain yet elusive. To this end, this in silico study tried to identify potential proinflammatory inducing peptides (PIPs) produced by the action of the elastase released in neutrophil-extracellular traps over SARS-CoV-2 particles. We found nine potential PIPs exclusive from the SARS-CoV-2, showing homology against T cell recognition epitopes. Moreover, 78 percent of these exclusive PIPs were found produced by the enzymatic cleavage on the spike glycoproteins, suggesting that high PIP concentrations might be released following SARS-CoV-2 huge replication rate. Therefore, these PIPs might play a role in the exacerbated inflammatory response observed in some patients. Nine potential PIPs were predicted exclusive from the SARS-CoV-2. SARS-CoV-2 PIPs showed homology against T cell recognition epitopes. Most of PIPs were produced by enzymatic cleavage of the spike glycoproteins. The release of these PIPs might be related to the increased inflammatory response observed in the patients.","version":"1.1","doi":"10.1101/2020.05.02.072439","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.02.074021","pub_date":"2020-5-02","title":"An artificial intelligence system reveals liquiritin inhibits SARS-CoV-2 by mimicking type I interferon","abstract":"The pandemic COVID-19 has spread to all over the world and greatly threatens safety and health of people. COVID-19 is highly infectious and with high mortality rate. As no effective antiviral treatment is currently available, new drugs are urgently needed. We employed transcriptional analysis to uncover potential antiviral drugs from natural products or FDA approved drugs. We found liquiritin significantly inhibit replication of SARS-CoV-2 in Vero E6 cells with EC50 = 2.39 \u03bcM. Mechanistically, we found liquiritin exerts anti-viral function by mimicking type I interferon. Upregulated genes induced by liquiritin are enriched in GO categories including type I interferon signaling pathway, negative regulation of viral genome replication and etc. In toxicity experiment, no death was observed when treated at dose of 300 mg/kg for a week in ICR mice. All the organ indexes but liver and serum biochemical indexes were normal after treatment. Liquiritin is abundant in licorice tablet (~0.2% by mass), a traditional Chinese medicine. Together, we recommend liquiritin as a competitive candidate for treating COVID-19. We also expect liquiritin to have a broad and potent antiviral function to other viral pathogens, like HBV, HIV and etc.","version":"1.1","doi":"10.1101/2020.05.02.074021","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.069922","pub_date":"2020-5-01","title":"Broad-spectrum antiviral activity of naproxen: from Influenza A to SARS-CoV-2 Coronavirus","abstract":"There is an urgent need for specific antiviral drugs directed against SARS-CoV-2 both to prevent the most severe forms of COVID-19 and to reduce viral excretion and subsequent virus dissemination; in the present pandemic context, drug repurposing is a priority. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus in order to inhibit its association with viral RNA could be a strategy to impeding viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, belonging to the NSAID family, previously demonstrated against Influenza A virus, were evaluated against SARS-CoV-2. Naproxen binding to the nucleoprotein of SARS-CoV2 was shown by molecular modeling. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2 induced-damage. The benefit of naproxen addition to the standard of care is tested in an on-going clinical study.","version":"1.1","doi":"10.1101/2020.04.30.069922","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.990226","pub_date":"2020-5-01","title":"Lack of Reinfection in Rhesus Macaques Infected with SARS-CoV-2","abstract":"A global pandemic of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is ongoing spread. It remains unclear whether the convalescing patients have a risk of reinfection. Rhesus macaques were rechallenged with SARS-CoV-2 during an early recovery phase from initial infection characterized by weight loss, interstitial pneumonia and systemic viral dissemination mainly in respiratory and gastrointestinal tracts. The monkeys rechallenged with the identical SARS-CoV-2 strain have failed to produce detectable viral dissemination, clinical manifestations and histopathological changes. A notably enhanced neutralizing antibody response might contribute the protection of rhesus macaques from the reinfection by SARS-CoV-2. Our results indicated that primary SARS-CoV-2 infection protects from subsequent reinfection. Neutralizing antibodies against SARS-CoV-2 might protect rhesus macaques which have undergone an initial infection from reinfection during early recovery days.","version":"1.2","doi":"10.1101/2020.03.13.990226","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.071654","pub_date":"2020-5-01","title":"Mutational spectra of SARS-CoV-2 orf1ab polyprotein and Signature mutations in the United States of America","abstract":"Pandemic COVID-19 outbreak has been caused due to SARS-COV2 pathogen, resulting millions of infection and death worldwide, USA being on top at the present moment. The long, complex orf1ab polyproteins of SARS-COV2 play an important role in viral RNA synthesis. To assess the impact of mutations in this important domain, we analyzed 1134 complete protein sequences of orf1ab polyprotein from NCBI Virus database from affected patients across various states of USA from December 2019 to 25th April, 2020. Multiple sequence alignment using Clustal Omega followed by statistical significance was calculated. Four significant mutations T265I (nsp 2), P4715L (nsp 12) and P5828L and Y5865C (both at nsp 13) were identified in important non-structural proteins, which function either as replicase or helicase. A comparative analysis shows 265T>I, 5828P>L and 5865Y>C are unique to USA and not reported from Europe or Asia; while one, 4715P>L is predominant in both Europe and USA. Mutational changes in amino acids are predicted to alter structure and function of corresponding proteins, thereby it is imperative to consider the mutational spectra while designing new antiviral therapeutics targeting viral orf1ab.","version":"1.1","doi":"10.1101/2020.05.01.071654","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.072728","pub_date":"2020-5-01","title":"SARS-CoV2 quantification using RT-dPCR: a faster and safer alternative to assist viral genomic copies assessment using RT-qPCR","abstract":"In this study, serial dilutions of SARS-CoV 2 RNA extract were tested using RT-dPCR using three different primer-probe assays aiming SARS-CoV 2 nucleocapsid coding region. Narrower confidence intervals, indicating high quantification precision were obtained in 100 and 1000-fold serial dilution and RT-dPCR results were equivalent between different assays in the same dilution. High accuracy of this test allowed conclusions regarding the ability of this technique to evaluate precisely the amount of genomic copies present in a sample. We believe that this fast and safe method can assist other researchers in titration of SARS-CoV2 controls used in RT-qPCR without the need of virus isolation.","version":"1.1","doi":"10.1101/2020.05.01.072728","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.071274","pub_date":"2020-5-01","title":"Comparative transcriptome analysis reveals the intensive early-stage responses of host cells to SARS-CoV-2 infection","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a widespread outbreak of highly pathogenic COVID-19. It is therefore important and timely to characterize interactions between the virus and host cell at the molecular level to understand its disease pathogenesis. To gain insights, we performed high-throughput sequencing that generated time-series data simultaneously for bioinformatics analysis of virus genomes and host transcriptomes implicated in SARS-CoV-2 infection. Our analysis results showed that the rapid growth of the virus was accompanied by an early intensive response of host genes. We also systematically compared the molecular footprints of the host cells in response to SARS-CoV-2, SARS-CoV and MERS-CoV. Upon infection, SARS-CoV-2 induced hundreds of up-regulated host genes hallmarked by a significant cytokine production followed by virus-specific host antiviral responses. While the cytokine and antiviral responses triggered by SARS-CoV and MERS-CoV were only observed during the late stage of infection, the host antiviral responses during the SARS-CoV-2 infection were gradually enhanced lagging behind the production of cytokine. The early rapid host responses were potentially attributed to the high efficiency of SARS-CoV-2 entry into host cells, underscored by evidence of a remarkably up-regulated gene expression of TPRMSS2 soon after infection. Taken together, our findings provide novel molecular insights into the mechanisms underlying the infectivity and pathogenicity of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.30.071274","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.070383","pub_date":"2020-5-01","title":"Dysregulation in mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells","abstract":"How Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remain largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected HuH7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. Unlike other human respiratory viruses, we found a significant inhibition of HIF-1\u03b1 through the entire time course of the infection, suggesting a crosstalk between the SARS-CoV-2 and the mTOR/HIF-1 signaling. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe COVID-19 patients.","version":"1.1","doi":"10.1101/2020.04.30.070383","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.048066","pub_date":"2020-5-01","title":"Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions","abstract":"The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in interferon, ACE, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health, and new therapeutic targets.","version":"1.5","doi":"10.1101/2020.04.20.048066","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.010694","pub_date":"2020-5-01","title":"The Nucleocapsid Protein of SARS-CoV-2 Abolished Pluripotency in Human Induced Pluripotent Stem Cells","abstract":"The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging across the world, leading to a global mortality rate of 3.4% (estimated by World Health Organization in March 2020). As a potential vaccine and therapeutic target, the nucleocapsid protein of SARS-CoV-2 (nCoVN) functions in packaging the viral genome and viral self-assembly. To investigate the biological effects of nCoVN to human stem cells, genetically engineered human induced pluripotent stem cells (iPSC) expressing nCoVN (iPSC-nCoVN) were generated by lentiviral expression systems, in which the expression of nCoVN could be induced by the doxycycline. The proliferation rate of iPSC-nCoVN was decreased. Unexpectedly, the morphology of iPSC started to change after nCoVN expression for 7 days. The pluripotency marker TRA-1-81 were not detectable in iPSC-nCoVN after a four-day induction. Meanwhile, iPSC-nCoVN lost the ability for differentiation into cardiomyocytes with a routine differentiation protocol. The RNA-seq data of iPSC-nCoVN (induction for 30 days) and immunofluorescence assays illustrated that iPSC-nCoVN were turning to fibroblast-like cells. Our data suggested that nCoVN disrupted the pluripotent properties of iPSC and turned them into other types of cells, which provided a new insight to the pathogenic mechanism of SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.03.26.010694","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.071357","pub_date":"2020-5-01","title":"Distinct Inductions of and Responses to Type I and Type III Interferons Promote Infections in Two SARS-CoV-2 Isolates","abstract":"The recent emerging coronavirus, SARS-CoV-2, has been rapidly and widely spread and causing an ongoing viral pneumonia outbreak worldwide. It has been observed that SARS-CoV-2 patients show a rather long and asymptomatic incubation time. We characterized the abilities to induce and to response to IFN\u03b2/IFN\u03bb1 of two or our clinical isolates, SARS-CoV-2/NTU01/TWN/human/2020 and SARS-CoV-2/NTU02/TWN/human/2020, which exhibit only two amino acid differences over the \u223c30kb viral genome. We found that both isolates may infect Huh7, A549 and Calu-3 cells, yet the RIG-I-like receptor-dependent antiviral signaling was poorly induced in these cells in the early infections. Unexpectedly, we found that the intracellular vRNA levels of these isolates were sustained upon to type I/III IFN treatments, and this phenotype was more pronounced in the Taiwan/NTU01/2020 isolate. The type I/III IFN responses are antiviral but partially proviral in the case of SARS-CoV-2 infections. Poor induction and response to innate immunity may contribute to destitute neutralization index of the antibody produced, and indeed we found that the patient serum could not efficiently neutralize SARS-CoV-2 virions. With better understandings of the interplay between SARS-CoV-2 and the host antiviral innate immunity, our report may provide new insights for the regimen of therapies for SARS-CoV-2 infected patients.","version":"1.1","doi":"10.1101/2020.04.30.071357","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.072330","pub_date":"2020-5-01","title":"Rampant C->U hypermutation in the genomes of SARS-CoV-2 and other coronaviruses \u2013 causes and consequences for their short and long evolutionary trajectories","abstract":"The pandemic of SARS coronavirus 2 (SARS-CoV-2) has motivated an intensive analysis of its molecular epidemiology following its worldwide spread. To understand the early evolutionary events following its emergence, a dataset of 985 complete SARS-CoV-2 sequences was assembled. Variants showed a mean 5.5-9.5 nucleotide differences from each other, commensurate with a mid-range coronavirus substitution rate of 3\u00d710\u22124 substitutions/site/year. Almost half of sequence changes were C->U transitions with an 8-fold base frequency normalised directional asymmetry between C->U and U->C substitutions. Elevated ratios were observed in other recently emerged coronaviruses (SARS-CoV and MERS-CoV) and to a decreasing degree in other human coronaviruses (HCoV-NL63, -OC43, -229E and -HKU1) proportionate to their increasing divergence. C->U transitions underpinned almost half of the amino acid differences between SARS-CoV-2 variants, and occurred preferentially in both 5\u2019U/A and 3\u2019U/A flanking sequence contexts comparable to favoured motifs of human APOBEC3 proteins. Marked base asymmetries observed in non-pandemic human coronaviruses (U>>A>G>>C) and low G+C contents may represent long term effects of prolonged C->U hypermutation in their hosts. The evidence that much of sequence change in SARS-CoV-2 and other coronaviruses may be driven by a host APOBEC-like editing process has profound implications for understanding their short and long term evolution. Repeated cycles of mutation and reversion in favoured mutational hotspots and the widespread occurrence of amino acid changes with no adaptive value for the virus represents a quite different paradigm of virus sequence change from neutral and Darwinian evolutionary frameworks that are typically used in molecular epidemiology investigations.","version":"1.1","doi":"10.1101/2020.05.01.072330","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030445","pub_date":"2020-5-01","title":"Analysis of the SARS-CoV-2 spike protein glycan shield: implications for immune recognition","abstract":"Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight (17% for the HEK293 glycoform) the level of surface shielding is disproportionately high at 42%.","version":"1.2","doi":"10.1101/2020.04.07.030445","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.043273","pub_date":"2020-5-01","title":"Investigating the genomic landscape of novel coronavirus (2019-nCoV) to identify non-synonymous mutations for use in diagnosis and drug design","abstract":"Novel coronavirus has wrecked medical and health care facilities claiming ~5% death tolls globally. All efforts to contain the pathogenesis either using inhibitory drugs or vaccines largely remained futile due to a lack of better understanding of the genomic feature of this virus. In the present study, we compared the 2019-nCoV with other coronaviruses, which indicated that bat-SARS like coronavirus could be a probable ancestor of the novel coronavirus. The protein sequence similarity of pangolin-hCoV and bat-hCoV with human coronavirus was higher as compared to their nucleotide similarity denoting the occurrence of more synonymous mutations in the genome. Phylogenetic and alignment analysis of 591 novel coronaviruses of different clades from Group I to Group V revealed several mutations and concomitant amino acid changes. Detailed investigation on nucleotide substitution unfolded 100 substitutions in the coding region of which 43 were synonymous and 57 were of non-synonymous type. The non-synonymous substitutions resulting into 57 amino acid changes were found to be distributed over different hCoV proteins with maximum on spike protein. An important diamino acid change RG to KR was observed in ORF9 protein. Additionally, several interesting features of the novel coronavirus genome have been highlighted in respect to various other human infecting viruses which may explain extreme pathogenicity, infectivity and simultaneously the reason behind failure of the antiviral therapies. This study presents a comprehensive phylogenetic analysis of SARS-CoV2 isolates to understand discrete mutations that are occurring between patient samples. This analysis will provide an explanation for varying treatment efficacies of different inhibitory drugs and a future direction towards a combinatorial treatment therapies based on the kind of mutation in the viral genome.","version":"1.2","doi":"10.1101/2020.04.16.043273","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.069039","pub_date":"2020-5-01","title":"SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms","abstract":"Genomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.","version":"1.1","doi":"10.1101/2020.04.30.069039","journal":"bioRxiv","score":null},{"id":"10.1101/2020.05.01.067769","pub_date":"2020-5-01","title":"Heat inactivation of the Severe Acute Respiratory Syndrome Coronavirus 2","abstract":"Supernatants of cells infected with SARS-CoV-2, nasopharyngeal and sera samples containing SARS-CoV-2 were submitted to heat inactivation for various periods of time, ranging from 30 seconds to 60 minutes. Our results showed that SARS-CoV-2 could be inactivated in less than 30 minutes, 15 minutes and 3 minutes at 56\u00b0C, 65\u00b0C and 95\u00b0C respectively. These data could help laboratory workers to improve their protocols with handling of the virus in biosafety conditions.","version":"1.1","doi":"10.1101/2020.05.01.067769","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.067637","pub_date":"2020-5-01","title":"Empowering Virus Sequences Research through Conceptual Modeling","abstract":"The pandemic outbreak of the coronavirus disease has attracted attention towards the genetic mechanisms of viruses. We hereby present the Viral Conceptual Model (VCM), centered on the virus sequence and described from four perspectives: biological (virus type and hosts/sample), analytical (annotations and variants), organizational (sequencing project) and technical (experimental technology). VCM is inspired by GCM, our previously developed Genomic Conceptual Model, but it introduces many novel concepts, as viral sequences significantly differ from human genomes. When applied to SARS-CoV2 virus, complex conceptual queries upon VCM are able to replicate the search results of recent articles, hence demonstrating huge potential in supporting virology research. In addition to VCM, we also illustrate the data dictionary for patient\u2019s phenotype used by the COVID-19 Host Genetic Initiative. Our effort is part of a broad vision: availability of conceptual models for both human genomics and viruses will provide important opportunities for research, especially if interconnected by the same human being, playing the role of virus host as well as provider of genomic and phenotype information.","version":"1.3","doi":"10.1101/2020.04.29.067637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.029736","pub_date":"2020-4-30","title":"Susceptibility of tree shrew to SARS-CoV-2 infection","abstract":"Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39\u00b0 C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches.","version":"1.1","doi":"10.1101/2020.04.30.029736","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.30.070771","pub_date":"2020-4-30","title":"Origin of imported SARS-CoV-2 strains in The Gambia identified from Whole Genome Sequences","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a positive-sense single stranded RNA virus with high human transmissibility. This study generated Whole Genome data to determine the origin and pattern of transmission of SARS-CoV-2 from the first six cases tested in The Gambia. Total RNA from SARS-CoV-2 was extracted from inactivated nasopharyngeal-oropharyngeal swabs of six cases and converted to cDNA following the ARTIC COVID-19 sequencing protocol. Libraries were constructed with the NEBNext ultra II DNA library prep kit for Illumina and Oxford Nanopore Ligation sequencing kit and sequenced on Illumina MiSeq and Nanopore GridION, respectively. Sequencing reads were mapped to the Wuhan reference genome and compared to eleven other SARS-CoV-2 strains of Asian, European and American origins. A phylogenetic tree was constructed with the consensus genomes for local and non-African strains. Three of the Gambian strains had a European origin (UK and Spain), two strains were of Asian origin (Japan). In The Gambia, Nanopore and Illumina sequencers were successfully used to identify the sources of SARS-CoV-2 infection in COVID-19 cases.","version":"1.1","doi":"10.1101/2020.04.30.070771","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.069476","pub_date":"2020-4-30","title":"SARS-CoV-2 selectively mimics a cleavable peptide of human ENaC in a strategic hijack of host proteolytic machinery","abstract":"Molecular mimicry of host proteins is an evolutionary strategy adopted by viruses to evade immune surveillance and exploit host cell systems. We report that SARS-CoV-2 has evolved a unique S1/S2 cleavage site (RRARSVAS), absent in any previous coronavirus sequenced, that results in mimicry of an identical FURIN-cleavable peptide on the human epithelial sodium channel \u03b1-subunit (ENaC-\u03b1). Genetic truncation at this ENaC-\u03b1 cleavage site causes aldosterone dysregulation in patients, highlighting the functional importance of the mimicked SARS-CoV-2 peptide. Single cell RNA-seq from 65 studies shows significant overlap between the expression of ENaC-\u03b1 and ACE2, the putative receptor for the virus, in cell types linked to the cardiovascular-renal-pulmonary pathophysiology of COVID-19. Triangulating this cellular fingerprint with amino acid cleavage signatures of 178 human proteases shows the potential for tissue-specific proteolytic degeneracy wired into the SARS-CoV-2 lifecycle. We extrapolate that the evolution of SARS-CoV-2 into a global coronavirus pandemic may be in part due to its targeted mimicry of human ENaC and hijack of the associated host proteolytic network.","version":"1.1","doi":"10.1101/2020.04.29.069476","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.067363","pub_date":"2020-4-30","title":"Validation of a single-step, single-tube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA","abstract":"The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. We used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 \u03bcL of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65\u00b0C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD \u00b17 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL\u22121), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. With a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.","version":"1.2","doi":"10.1101/2020.04.28.067363","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.069591","pub_date":"2020-4-30","title":"A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings","abstract":"Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. RT-qPCR requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers, which are not available in many low resource settings and developing countries. As a pandemic, COVID-19 has quickly spread to the rest of the world. Many underdeveloped and developing counties do not have the means for fast and accurate COVID-19 detection to control this outbreak. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 minutes using untreated samples, heat-inactivated samples, or extracted RNA templates. Rapid RT-PCR was achieved using thin-walled PCR tubes and a setup including sous vide immersion heaters/circulators. Our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in situations where sophisticated laboratory instruments are not available.","version":"1.1","doi":"10.1101/2020.04.29.069591","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.059576","pub_date":"2020-4-29","title":"Introductions and early spread of SARS-CoV-2 in France","abstract":"Following the emergence of coronavirus disease (COVID-19) in Wuhan, China in December 2019, specific COVID-19 surveillance was launched in France on January 10, 2020. Two weeks later, the first three imported cases of COVID-19 into Europe were diagnosed in France. We sequenced 97 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from samples collected between January 24 and March 24, 2020 from infected patients in France. Phylogenetic analysis identified several early independent SARS-CoV-2 introductions without local transmission, highlighting the efficacy of the measures taken to prevent virus spread from symptomatic cases. In parallel, our genomic data reveals the later predominant circulation of a major clade in many French regions, and implies local circulation of the virus in undocumented infections prior to the wave of COVID-19 cases. This study emphasizes the importance of continuous and geographically broad genomic sequencing and calls for further efforts with inclusion of asymptomatic infections.","version":"1.2","doi":"10.1101/2020.04.24.059576","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.067728","pub_date":"2020-4-29","title":"An engineered stable mini-protein to plug SARS-Cov-2 Spikes","abstract":"The novel betacoronavirus SARS-CoV-2 is the etiological agent of the current pandemic COVID-19. Like other coronaviruses, this novel virus relies on the surface Spike glycoprotein to access the host cells, mainly through the interaction of its Receptor Binding Domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to interfere with binding of the SARS-CoV-2 Spike protein to ACE2 have a great potential to inhibit viral entry. Starting from the available structural data on the interaction between SARS-CoV-2 Spike protein and the host ACE2 receptor, we here engineered a mini-protein with the aim of creating a soluble and stable Spike interactor. This mini-protein, which was recombinantly produced in high yields, possesses a stable \u03b1 helical conformation and is able to interact with the RBD of glycosylated Spike protein from SARS-CoV-2 with nanomolar affinity, as measured by microscale thermophoresis. By plugging the Spike protein, our mini-protein constitutes a valid tool for the development of treatments against different types of coronavirus.","version":"1.1","doi":"10.1101/2020.04.29.067728","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.26.062471","pub_date":"2020-4-29","title":"Emergence of multiple variants of SARS-CoV-2 with signature structural changes","abstract":"This study explores the divergence pattern of SARS-CoV-2 using whole genome sequences of the isolates from various COVID-19 affected countries. The phylogenomic analysis indicates the presence of at least four distinct groups of the SARS-CoV-2 genomes. The emergent groups have been found to be associated with signature structural changes in specific proteins. Also, this study reveals the differential levels of divergence patterns for the protein coding regions. Moreover, we have predicted the impact of structural changes on a couple of important viral proteins via structural modelling techniques. This study further advocates for more viral genetic studies with associated clinical outcomes and hosts\u2019 response for better understanding of SARS-CoV-2 pathogenesis enabling better mitigation of this pandemic situation.","version":"1.2","doi":"10.1101/2020.04.26.062471","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.014639","pub_date":"2020-4-29","title":"Molecular Basis for ADP-ribose Binding to the Macro-X Domain of SARS-CoV-2 Nsp3","abstract":"The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymers, helicase and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those seen in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses, and potential therapeutic target.","version":"1.3","doi":"10.1101/2020.03.31.014639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.051789","pub_date":"2020-4-29","title":"A collection of designed peptides to target SARS-Cov-2 \u2013 ACE2 interaction: PepI-Covid19 database","abstract":"The angiotensin-converting enzyme 2 is the cellular receptor used by SARS coronavirus SARS-CoV and SARS-CoV-2 to enter the cell. Both coronavirus use the receptor-binding domain (RBD) of their viral spike protein to interact with ACE2. The structural basis of these interactions are already known, forming a dimer of ACE2 with a trimer of the spike protein, opening the door to target them to prevent the infection. Here we present PepI-Cov19 database, a repository of peptides designed to target the interaction between the RDB of SARS-CoV-2 and ACE2 as well as the dimerization of ACE2 monomers. The peptides were modelled using our method PiPreD that uses native elements of the interaction between the targeted protein and cognate partner that are subsequently included in the designed peptides. These peptides recapitulate stretches of residues present in the native interface plus novel and highly diverse conformations that preserve the key interactions on the interface. PepI-Covid19 database provides an easy and convenient access to this wealth of information to the scientific community with the view of maximizing its potential impact in the development of novel therapeutic agents.","version":"1.1","doi":"10.1101/2020.04.28.051789","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.29.971093","pub_date":"2020-4-29","title":"The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding","abstract":"Many pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals.","version":"1.2","doi":"10.1101/2020.02.29.971093","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.068767","pub_date":"2020-4-29","title":"Glycosaminoglycans induce conformational change in the SARS-CoV-2 Spike S1 Receptor Binding Domain","abstract":"The glycosaminoglycan (GAG) class of polysaccharides are utilised by a plethora of microbial pathogens as receptors for adherence and invasion. The GAG heparin prevents infection by a range of viruses when added exogenously, including the S-associated coronavirus strain HSR1 and more recently we have demonstrated that heparin can block cellular invasion by SARS-CoV-2. Heparin has found widespread clinical use as anticoagulant drug and this molecule is routinely used as a proxy for the GAG, heparan sulphate (HS), a structural analogue located on the cell surface, which is a known receptor for viral invasion. Previous work has demonstrated that unfractionated heparin and low molecular weight heparins binds to the Spike (S1) protein receptor binding domain, inducing distinct conformational change and we have further explored the structural features of heparin with regard to these interactions. In this article, previous research is expanded to now include a broader range of GAG family members, including heparan sulphate. This research demonstrates that GAGs, other than those of heparin (or its derivatives), can also interact with the SARS-CoV-2 Spike S1 receptor binding domain and induce distinct conformational changes within this region. These findings pave the way for future research into next-generation, tailor-made, GAG-based antiviral agents, against SARS-CoV-2 and other members of the Coronaviridae.","version":"1.1","doi":"10.1101/2020.04.29.068767","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.068486","pub_date":"2020-4-29","title":"SARS-CoV-2 Spike S1 Receptor Binding Domain undergoes Conformational Change upon Interaction with Low Molecular Weight Heparins","abstract":"The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses when added exogenously, including S-associated coronavirus strain HSR1 and inhibits cellular invasion by SARS-CoV-2. We have previously demonstrated that unfractionated heparin binds to the Spike (S1) protein receptor binding domain, induces a conformational change and have reported the structural features of heparin on which this interaction depends. Furthermore, we have demonstrated that enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. Here we expand upon these studies, to a wide range of low molecular weight heparins and demonstrate that they induce a variety of conformational changes in the SARS-CoV-2 RBD. These findings may have further implications for the rapid development of a first-line therapeutic by repurposing low molecular weight heparins, as well as for next-generation, tailor-made, GAG-based antiviral agents, against SARS-CoV-2 and other members of the Coronaviridae.","version":"1.1","doi":"10.1101/2020.04.29.068486","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.29.068098","pub_date":"2020-4-29","title":"Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike S protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications","abstract":"The pneumonia-causing COVID-19 pandemia has prompt worldwide efforts to understand its biological and clinical traits of newly identified HCoV-19 virus. In this study, post-translational modification (PTM) of recombinant HCoV-19 S and hACE2 were characterized by LC-MSMS. We revealed that both proteins were highly decorated with specific proportions of N-glycan subtypes. Out of 21 possible glycosites in HCoV-19 S protein, 20 were confirmed completely occupied by N-glycans, with oligomannose glycans being the most abundant type. All 7 possible glycosylation sites in hACE2 were completely occupied mainly by complex type N-glycans. However, we showed that glycosylation did not directly contribute to the binding affinity between SARS-CoV spike protein and hACE2. Additionally, we also identified multiple sites methylated in both proteins, and multiple prolines in hACE2 were converted to hydroxylproline. Refined structural models were built by adding N-glycan and PTMs to recently published cryo-EM structure of the HCoV-19 S and hACE2 generated with glycosylation sites in the vicinity of binding surface. The PTM and glycan maps of both HCoV-19 S and hACE2 provide additional structural details to study mechanisms underlying host attachment, immune response mediated by S protein and hACE2, as well as knowledge to develop remedies and vaccines desperately needed nowadays.","version":"1.1","doi":"10.1101/2020.04.29.068098","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.065409","pub_date":"2020-4-29","title":"Safety, pharmacokinetics, and liver-stage Plasmodium cynomolgi effect of high-dose ivermectin and chloroquine in Rhesus Macaques","abstract":"Previously, ivermectin (1\u201310 mg/kg) was shown to inhibit liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (IC50 = 10.42 \u03bcM) and hypnozoites (IC50 = 29.24 \u03bcM) in primary macaque hepatocytes when administered in high-dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for seven consecutive days was evaluated for prophylaxis or radical cure of Plasmodium cynomolgi liver-stages in Rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for seven days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.","version":"1.1","doi":"10.1101/2020.04.27.065409","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.28.065201","pub_date":"2020-4-28","title":"RNA-GPS Predicts SARS-CoV-2 RNA Localization to Host Mitochondria and Nucleolus","abstract":"The SARS-CoV-2 coronavirus is driving a global pandemic, but its biological mechanisms are less well understood. SARS-CoV-2 is an RNA virus whose multiple genomic and sub-genomic RNA (sgRNA) transcripts hijack the host cell\u2019s machinery, located across distinct cytotopic locations. Subcellular localization of its viral RNA could play important roles in viral replication and host antiviral immune response. Here we perform computational modeling of SARS-CoV-2 viral RNA localization across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes to the human transcriptome and other coronaviruses and perform systematic sub-sequence analyses to identify the responsible signals. Using state-of-the-art machine learning models, we predict that the SARS-CoV-2 RNA genome and all sgRNAs are enriched in the host mitochondrial matrix and nucleolus. The 5\u2019 and 3\u2019 viral untranslated regions possess the strongest and most distinct localization signals. We discuss the mitochondrial localization signal in relation to the formation of double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus.","version":"1.1","doi":"10.1101/2020.04.28.065201","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.033233","pub_date":"2020-4-28","title":"Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug Carmofur","abstract":"The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease (Mpro). Here the X-ray crystal structure of Mpro in complex with Carmofur reveals that the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 \u03bcM) and it is a promising lead compound to develop new antiviral treatment for COVID-19.","version":"1.2","doi":"10.1101/2020.04.09.033233","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.052225","pub_date":"2020-4-28","title":"Characterising the epidemic spread of Influenza A/H3N2 within a city through phylogenetics","abstract":"Infecting large portions of the global population, seasonal influenza is a major burden on societies around the globe. While the global source sink dynamics of the different seasonal influenza viruses have been studied intensively, it\u2019s local spread remains less clear. In order to improve our understanding of how influenza is transmitted on a city scale, we collected an extremely densely sampled set of influenza sequences alongside patient metadata. To do so, we sequenced influenza viruses isolated from patients of two different hospitals, as well as private practitioners in Basel, Switzerland during the 2016/2017 influenza season. The genetic sequences reveal that repeated introductions into the city drove the influenza season. We then reconstruct how the effective reproduction number changed over the course of the season. We find trends in transmission dynamics correlated positively with trends in temperature, but not relative humidity nor school holidays. Alongside the genetic sequence data that allows us to see how individual cases are connected, we gathered patient information, such as the age or household status. Zooming into the local transmission outbreaks suggests that the elderly were to a large extent infected within their own transmission network, while school children likely drove the spread within the remaining transmission network. These patterns will be valuable to plan interventions combating the spread of respiratory diseases within cities given that similar patterns are observed for other influenza seasons and cities. As shown with the current SARS-CoV-2 pandemic, respiratory diseases can quickly spread around the globe. While it can be hugely important to understand how diseases spread around the globe, local spread is most often the main driver of novel infections of respiratory diseases such as SARS-CoV-2 or influenza. We here use genetic sequence data alongside patient information to better understand what the drives the local spread of influenza by looking at the 2016/2017 influenza season in Basel, Switzerland as an example. The genetic sequence data allows us to reconstruct the how the transmission dynamics changed over the course of the season, which we correlate to changes, but not humidity or school holidays. Additionally, the genetic sequence data allows us to see how individual cases are connected. Using patient information, such as age and household status our analyses suggest that the elderly mainly transmit within their own transmission network. Additionally, they suggest that school aged children, but not pre-school aged children are important drivers of the local spread of influenza.","version":"1.1","doi":"10.1101/2020.04.27.052225","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.056853","pub_date":"2020-4-27","title":"Mapping the Immunodominance Landscape of SARS-CoV-2 Spike Protein for the Design of Vaccines against COVID-19","abstract":"The ongoing coronavirus disease 2019 (COVID-19) pandemic is a serious threat to global public health, and imposes severe burdens on the entire human society. The severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can cause severe respiratory illness and death. Currently, there are no specific antiviral drugs that can treat COVID-19. Several vaccines against SARS-CoV-2 are being actively developed by research groups around the world. The surface S (spike) protein and the highly expressed internal N (nucleocapsid) protein of SARS-CoV-2 are widely considered as promising candidates for vaccines. In order to guide the design of an effective vaccine, we need experimental data on these potential epitope candidates. In this study, we mapped the immunodominant (ID) sites of S protein using sera samples collected from recently discharged COVID-19 patients. The SARS-CoV-2 S protein-specific antibody levels in the sera of recovered COVID-19 patients were strongly correlated with the neutralising antibody titres. We used epitope mapping to determine the landscape of ID sites of S protein, which identified nine linearized B cell ID sites. Four out of the nine ID sites were found in the receptor-binding domain (RBD). Further analysis showed that these ID sites are potential high-affinity SARS-CoV-2 antibody binding sites. Peptides containing two out of the nine sites were tested as vaccine candidates against SARS-CoV-2 in a mouse model. We detected epitope-specific antibodies and SARS-CoV-2-neutralising activity in the immunised mice. This study for the first time provides human serological data for the design of vaccines against COVID-19.","version":"1.2","doi":"10.1101/2020.04.23.056853","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.064279","pub_date":"2020-4-27","title":"Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 \u03bcM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 \u03bcM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.04.27.064279","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.008482","pub_date":"2020-4-27","title":"Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo","abstract":"SARS-CoV-2 emerged in China at the end of 2019 and has rapidly become a pandemic with roughly 2.7 million recorded COVID-19 cases and greater than 189,000 recorded deaths by April 23rd, 2020 (www.WHO.org). There are no FDA approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA approved drugs. Rapid development and human testing of potential antivirals is greatly needed. A quick way to test compounds with potential antiviral activity is through drug repurposing. Numerous drugs are already approved for human use and subsequently there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV. We found that 17 of these inhibit SARS-CoV-2 at a range of IC50 values at non-cytotoxic concentrations. We directly follow up with seven of these to demonstrate all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we have evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found both drugs protect mice from clinical disease.","version":"1.3","doi":"10.1101/2020.03.25.008482","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.063180","pub_date":"2020-4-27","title":"Structure of replicating SARS-CoV-2 polymerase","abstract":"The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged \u2018sliding poles\u2019 that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19).","version":"1.1","doi":"10.1101/2020.04.27.063180","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.27.064774","pub_date":"2020-4-27","title":"A SARS-CoV-2 vaccine candidate would likely match all currently circulating strains","abstract":"The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Here we analyzed SARS-CoV-2 sequence diversity across 5,700 sequences sampled since December 2019. The Spike protein, which is the target immunogen of most vaccine candidates, showed 93 sites with shared polymorphisms; only one of these mutations was found in more than 1% of currently circulating sequences. The minimal diversity found among SARS-CoV-2 sequences can be explained by drift and bottleneck events as the virus spread away from its original epicenter in Wuhan, China. Importantly, there is little evidence that the virus has adapted to its human host since December 2019. Our findings suggest that a single vaccine should be efficacious against current global strains. The limited diversification of SARS-CoV-2 reflects drift and bottleneck events rather than adaptation to humans as the virus spread.","version":"1.1","doi":"10.1101/2020.04.27.064774","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.26.063024","pub_date":"2020-4-27","title":"Specific mutations in SARS-CoV2 RNA dependent RNA polymerase and helicase alter protein structure, dynamics and thus function: Effect on viral RNA replication","abstract":"The open reading frame (ORF) 1ab of SARS-CoV2 encodes non-structural proteins involved in viral RNA functions like translation and replication including nsp1-4; 3C like proteinase; nsp6-10; RNA dependent RNA polymerase (RdRp); helicase and 3\u2019-5\u2019 exonuclease. Sequence analyses of ORF1ab unravelled emergence of mutations especially in the viral RdRp and helicase at specific positions, both of which are important in mediating viral RNA replication. Since proteins are dynamic in nature and their functions are governed by the molecular motions, we performed normal mode analyses of the SARS-CoV2 wild type and mutant RdRp and helicases to understand the effect of mutations on their structure, conformation, dynamics and thus function. Structural analyses revealed that mutation of RdRp (at position 4715 in the context of the polyprotein/ at position 323 of RdRp) leads to rigidification of structure and that mutation in the helicase (at position 5828 of polyprotein/ position 504) leads to destabilization increasing the flexibility of the protein structure. Such structural modifications and protein dynamics alterations might alter unwinding of complex RNA stem loop structures, the affinity/ avidity of polymerase RNA interactions and in turn the viral RNA replication. The mutation analyses of proteins of the SARS-CoV2 RNA replication complex would help targeting RdRp better for therapeutic intervention.","version":"1.1","doi":"10.1101/2020.04.26.063024","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.02.973255","pub_date":"2020-4-27","title":"Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2","abstract":"The COVID-19 outbreak has become a global health risk and understanding the response of the host to the SARS-CoV-2 virus will help to contrast the disease. Editing by host deaminases is an innate restriction process to counter viruses, and it is not yet known whether it operates against Coronaviruses. Here we analyze RNA sequences from bronchoalveolar lavage fluids derived from infected patients. We identify nucleotide changes that may be signatures of RNA editing: Adenosine-to-Inosine changes from ADAR deaminases and Cytosine-to-Uracil changes from APOBEC ones. A mutational analysis of genomes from different strains of human-hosted Coronaviridae reveals mutational patterns compatible to those observed in the transcriptomic data. Our results thus suggest that both APOBECs and ADARs are involved in Coronavirus genome editing, a process that may shape the fate of both virus and patient. Just to make a few things clear:\n\n- RNA editing and DNA editing are PHYSIOLOGICAL processes. Organisms uses them to (a) try to fight viruses, (b) increase heterogeneity inside cells (on many levels), (c) recognise their own RNA.\n- our work suggests that: (a) cells use RNA editing in trying to deal with Coronaviruses. We don\\'t know to what extent they succeed (and it would be nice if we could help them). (b) Whatever happens, mutations inserted by RNA editing fuel viral evolution. We don\\'t know whether viruses actively exploit this.\n- If you (scientist or not) think our work suggests ANYTHING ELSE, contact us. It can be a first step to help fight these !@#$ coronavirus, or towards a Nobel prize - but we need to discuss it thoroughly.\n- If you think these cellular processes are fascinating, join the club and contact us. We can have a nice cup of tea while chatting how wondrous nature is at coming up with extraordinary solutions\u2026 - RNA editing and DNA editing are PHYSIOLOGICAL processes. Organisms uses them to (a) try to fight viruses, (b) increase heterogeneity inside cells (on many levels), (c) recognise their own RNA. - our work suggests that: (a) cells use RNA editing in trying to deal with Coronaviruses. We don\\'t know to what extent they succeed (and it would be nice if we could help them). (b) Whatever happens, mutations inserted by RNA editing fuel viral evolution. We don\\'t know whether viruses actively exploit this. - If you (scientist or not) think our work suggests ANYTHING ELSE, contact us. It can be a first step to help fight these !@#$ coronavirus, or towards a Nobel prize - but we need to discuss it thoroughly. - If you think these cellular processes are fascinating, join the club and contact us. We can have a nice cup of tea while chatting how wondrous nature is at coming up with extraordinary solutions\u2026","version":"1.2","doi":"10.1101/2020.03.02.973255","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.047498","pub_date":"2020-4-26","title":"The crystal structure of nsp10-nsp16 heterodimer from SARS-CoV-2 in complex with S-adenosylmethionine","abstract":"SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2\u2019-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 \u00c5. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.","version":"1.2","doi":"10.1101/2020.04.17.047498","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.26.061705","pub_date":"2020-4-26","title":"Structural Basis of RNA Cap Modification by SARS-CoV-2 Coronavirus","abstract":"The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5\u2019-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2\u2019-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2\u2019-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.","version":"1.1","doi":"10.1101/2020.04.26.061705","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.28.013672","pub_date":"2020-4-26","title":"Cigarette smoke exposure and inflammatory signaling increase the expression of the SARS-CoV-2 receptor ACE2 in the respiratory tract","abstract":"The factors mediating fatal SARS-CoV-2 infections are poorly understood. Here, we show that cigarette smoke causes a dose-dependent upregulation of Angiotensin Converting Enzyme 2 (ACE2), the SARS-CoV-2 receptor, in rodent and human lungs. Using single-cell sequencing data, we demonstrate that ACE2 is expressed in a subset of secretory cells in the respiratory tract. Chronic smoke exposure triggers the expansion of this cell population and a concomitant increase in ACE2 expression. In contrast, quitting smoking decreases the abundance of these secretory cells and reduces ACE2 levels. Finally, we demonstrate that ACE2 expression is responsive to inflammatory signaling and can be upregulated by viral infections or interferon treatment. Taken together, these results may partially explain why smokers are particularly susceptible to severe SARS-CoV-2 infections. Furthermore, our work identifies ACE2 as an interferon-stimulated gene in lung cells, suggesting that SARS-CoV-2 infections could create positive-feedback loops that increase ACE2 levels and facilitate viral dissemination.","version":"1.2","doi":"10.1101/2020.03.28.013672","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.26.062406","pub_date":"2020-4-26","title":"How did SARS-CoV-19 spread in India from Italy, Iran and China? Genetic surveillance of early cases and virus demography","abstract":"SARS-CoV-19 after emerging from Wuhan, drastically devastated all sectors of human life by crushing down the global economy and increased psychological burden on public, government, and healthcare professionals. We manifested by analyzing 35 early coronavirus cases of India, that virus introduction in India, occurred from Italy, Iran and China and population demography apparently revealed a rapid population expansion after the outbreak with a present steady growth. We depicted nucleotide substitutions in structural genes, drove for the adaptive selection and plead for sequencing more genomes to facilitate identification of new emerged mutants, genetic evolution and disease transmission caused by coronavirus.","version":"1.1","doi":"10.1101/2020.04.26.062406","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.057307","pub_date":"2020-4-26","title":"Open Access and Altmetrics in the pandemic age: Forescast analysis on COVID-19 literature","abstract":"We present an analysis on the uptake of open access on COVID-19 related literature as well as the social media attention they gather when compared with non OA papers. We use a dataset of publications curated by Dimensions and analyze articles and preprints. Our sample includes 11,686 publications of which 67.5% are openly accessible. OA publications tend to receive the largest share of social media attention as measured by the Altmetric Attention Score. 37.6% of OA publications are bronze, which means toll journals are providing free access. MedRxiv contributes to 36.3% of documents in repositories but papers in BiorXiv exhibit on average higher AAS. We predict the growth of COVID-19 literature in the following 30 days estimating ARIMA models for the overall publications set, OA vs. non OA and by location of the document (repository vs. journal). We estimate that COVID-19 publications will double in the next 20 days, but non OA publications will grow at a higher rate than OA publications. We conclude by discussing the implications of such findings on the dissemination and communication of research findings to mitigate the coronavirus outbreak.","version":"1.1","doi":"10.1101/2020.04.23.057307","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.25.060350","pub_date":"2020-4-25","title":"SARS-CoV-2 Productively Infects Human Gut Enterocytes","abstract":"COVID-19, caused by SARS-CoV-2, is an influenza-like disease with a respiratory route of transmission, yet clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids, enterocytes were readily infected by SARS-CoV and SARS-CoV-2 as demonstrated by confocal- and electron-microscopy. Consequently, significant titers of infectious viral particles were measured. mRNA expression analysis revealed strong induction of a generic viral response program. We conclude that intestinal epithelium supports SARS-CoV-2 replication. SARS-CoV-2 infection of enterocytes in human small intestinal organoids","version":"1.1","doi":"10.1101/2020.04.25.060350","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.055608","pub_date":"2020-4-25","title":"Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered COVID-19 patients","abstract":"The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle as a vaccine and demonstrate induction of robust neutralization of a pseudo-virus, proportional to quantity of specific IgG and of higher quantities than recovered COVID-19 patients. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.","version":"1.1","doi":"10.1101/2020.04.22.055608","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052258","pub_date":"2020-4-25","title":"Optimized qRT-PCR approach for the detection of intra- and extra-cellular SARS-CoV-2 RNAs","abstract":"The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.","version":"1.1","doi":"10.1101/2020.04.20.052258","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.057323","pub_date":"2020-4-25","title":"Validation of a SARS-CoV-2 spike protein ELISA for use in contact investigations and sero-surveillance","abstract":"Since emergence of SARS-CoV-2 in late 2019, there has been a critical need to understand prevalence, transmission patterns, to calculate the burden of disease and case fatality rates. Molecular diagnostics, the gold standard for identifying viremic cases, are not ideal for determining true case counts and rates of asymptomatic infection. Serological detection of SARS-CoV-2 specific antibodies can contribute to filling these knowledge gaps. In this study, we describe optimization and validation of a SARS-CoV-2-specific-enzyme linked immunosorbent assay (ELISA) using the prefusion-stabilized form of the spike protein [1]. We performed receiver operator characteristic (ROC) analyses to define the specificities and sensitivities of the optimized assay and examined cross reactivity with immune sera from persons confirmed to have had infections with other coronaviruses. These assays will be used to perform contact investigations and to conduct large-scale, cross sectional surveillance to define disease burden in the population.","version":"1.2","doi":"10.1101/2020.04.24.057323","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.058776","pub_date":"2020-4-25","title":"A Library of Nucleotide Analogues Terminate RNA Synthesis Catalyzed by Polymerases of Coronaviruses Causing SARS and COVID-19","abstract":"SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of additional nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2\u2019 or 3\u2019 modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses\u2019 exonuclease activity. We examined these nucleotide analogues with regard to their ability to be incorporated by the RdRps in the polymerase reaction and then prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (Carbovir triphosphate, Ganciclovir triphosphate, Stavudine triphosphate, Entecavir triphosphate, 3\u2019-O-methyl UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2\u2019-O-methyl UTP), and 3 did not terminate the polymerase reaction (2\u2019-fluoro-dUTP, 2\u2019-amino-dUTP and Desthiobiotin-16-UTP). The coronavirus genomes encode an exonuclease that apparently requires a 2\u2019 -OH group to excise mismatched bases at the 3\u2019-terminus. In this study, all of the nucleoside triphosphate analogues we evaluated form Watson-Cricklike base pairs. All the nucleotide analogues which demonstrated termination either lack a 2\u2019-OH, have a blocked 2\u2019-OH, or show delayed termination. These nucleotides may thus have the potential to resist exonuclease activity, a property that we will investigate in the future. Furthermore, prodrugs of five of these nucleotide analogues (Brincidofovir/Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA approved for other viral infections, and their safety profile is well known. Thus, they can be evaluated rapidly as potential therapies for COVID-19.","version":"1.1","doi":"10.1101/2020.04.23.058776","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.050633","pub_date":"2020-4-25","title":"Rapid SARS-CoV-2 whole genome sequencing for informed public health decision making in the Netherlands","abstract":"SARS-CoV-2 is a novel coronavirus that has rapidly spread across the globe. In the Netherlands, the first case of SARS-CoV-2 has been notified on the 27th of February. Here, we describe the first three weeks of the SARS-CoV-2 outbreak in the Netherlands, which started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also, outside the South of the Netherlands. The timely generation of whole genome sequences combined with epidemiological investigations facilitated early decision making in an attempt to control local transmission of SARS-CoV-2 in the Netherlands.","version":"1.1","doi":"10.1101/2020.04.21.050633","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.25.061507","pub_date":"2020-4-25","title":"Insight towards the effect of the multibasic cleavage site of SARS-CoV-2 spike protein on cellular proteases","abstract":"Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents an immense global health problem. Spike (S) protein of coronavirus is the primary determinant of its entry into the host as it consists of both receptor binding and fusion domain. While tissue tropism, host range, and pathogenesis of coronavirus are primarily controlled by the interaction of S protein with the cell receptor, it is possible that proteolytic activation of S protein by host cell proteases also plays a decisive role. The host-cell proteases have shown to be involved in the proteolysis of S protein and cleaving it into two functional subunits, S1 and S2, during the maturation process. In the present study, the interaction of S protein of SARS-CoV-2 with different host proteases like furin, cathepsin B, and plasmin has been analyzed. Incorporation of the furin cleavage site (R-R-A-R) in the S protein in SARS-CoV-2 has been studied by mutating the individual amino acid. Our results suggest the polytropic nature of the S protein of SARS-CoV-2. Our analysis indicated that a single amino acid substitution in the polybasic cleavage site of S protein perturb the binding of cellular proteases. This mutation study might help to generate an attenuated SARS-CoV-2. Besides, targeting of host proteases by inhibitors may result in a practical approach to stop the cellular spread of SARS-CoV-2 and to develop its antiviral.","version":"1.1","doi":"10.1101/2020.04.25.061507","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.056259","pub_date":"2020-4-25","title":"Expression of ACE2, the SARS-CoV-2 receptor, and TMPRSS2 in prostate epithelial cells","abstract":"The COVID-19 pandemic has spread across more than 200 countries and resulted in over 170,000 deaths. For unclear reasons, higher mortality rates from COVID-19 have been reported in men compared to women. While the SARS-CoV-2 receptor ACE2 and serine protease TMPRSS2 have been detected in lung and other tissues, it is not clear what sex differences may exist. We analyzed a publicly-available normal human prostate single-cell RNA sequencing dataset and found TMPRSS2 and ACE2 co-expressing cells in epithelial cells, with a higher proportion in club and hillock cells. Then we investigated datasets of lung epithelial cells and also found club cells co-expressing TMPRSS2 and ACE2. A comparison of ACE2 expression in lung tissue between males and females showed higher expression in males and a larger proportion of ACE2+ cells in male type II pneumocytes, with preliminary evidence that type II pneumocytes of all lung epithelial cell types showed the highest expression of ACE2. These results raise the possibility that sex differences in ACE2 expression and the presence of double-positive cells in the prostate may contribute to the observed disparities of COVID-19.","version":"1.2","doi":"10.1101/2020.04.24.056259","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.055327","pub_date":"2020-4-25","title":"Comparison of Cepheid Xpert Xpress and Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2","abstract":"The SARS-CoV-2 pandemic has created an urgent and unprecedented need for rapid large-scale diagnostic testing to inform timely patient management. This study compared two recently-authorized rapid tests, Cepheid Xpert Xpress SARS-CoV-2 and Abbott ID Now SARS-CoV-2 to the Roche cobas SARS-CoV-2 assay. A total of 113 nasopharyngeal swabs were tested, including 88 positives spanning the full range of observed Ct values on the cobas assay. Compared to cobas, the overall positive agreement was 73.9% with ID Now and 98.9% with Xpert. Negative agreement was 100% and 92.0% for ID Now and Xpert, respectively. Both ID Now and Xpert showed 100% positive agreement for medium and high viral concentrations (Ct value <30). However, for Ct values >30, positive agreement was 34.3% for ID Now and 97.1% for Xpert. These findings highlight an important limitation of ID Now for specimens collected in viral or universal transport media with low viral concentrations. Further studies are needed to evaluate the performance of ID Now for direct swabs.","version":"1.1","doi":"10.1101/2020.04.22.055327","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.25.061200","pub_date":"2020-4-25","title":"Elevated expression of ACE2 in tumor-adjacent normal tissues of cancer patients","abstract":"The rapidly developing COVID-19 pandemic has raised a concern that cancer patients may have increased susceptibility to SARS-CoV-2 infection. This discussion has mostly focused on therapy-induced immune suppression. Here, we examined the expression patterns of ACE2, the receptor through which SARX-CoV2 enters human cells, and found that ACE2 mRNA levels are elevated in tumor-adjacent normal tissues of cancer patients, including in normal-adjacent lung tissues of lung cancer patients. These observations raise the possibility that the elevated COVID-19 risk of cancer patients may not be limited to those undergoing immune-suppressing treatment.","version":"1.1","doi":"10.1101/2020.04.25.061200","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.056309","pub_date":"2020-4-24","title":"SARS-CoV-2 Isolation and Propagation from Turkish COVID-19 patients","abstract":"The novel coronavirus pneumonia, which was named later as Coronavirus Disease 2019 (COVID-19), is caused by the Severe Acute Respiratory Syndrome Coronavirus 2, namely SARS-CoV-2. It is a positive-strand RNA virus that is the seventh coronavirus known to infect humans. The COVID-19 outbreak presents enormous challenges for global health behind the pandemic outbreak. The first diagnosed patient in Turkey has been reported by the Republic of Turkey Ministry of Health on March 11, 2020. Today, over ninety thousand cases in Turkey, and two million cases around the world have been declared. Due to the urgent need for vaccine and anti-viral drug, isolation of the virus is crucial. Here, we report one of the first isolation and characterization studies of SARS-CoV-2 from nasopharyngeal and oropharyngeal specimens of diagnosed patients in Turkey. This study provides an isolation and replication methodology, and cell culture tropism of the virus that will be available to the research communities. Scientists have isolated virus from Turkish COVID-19 patients. The isolation, propagation, and plaque and immune response assays of the virus described here will serve in following drug discovery and vaccine testing.","version":"1.1","doi":"10.1101/2020.04.23.056309","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.19.048751","pub_date":"2020-4-24","title":"Revealing COVID-19 Transmission by SARS-CoV-2 Genome Sequencing and Agent Based Modelling","abstract":"Community transmission of the new coronavirus SARS-CoV-2 is a major public health concern that remains difficult to assess. We present a genomic survey of SARS-CoV-2 from a during the first 10 weeks of COVID-19 activity in New South Wales, Australia. Transmission events were monitored prospectively during the critical period of implementation of national control measures. SARS-CoV-2 genomes were sequenced from 209 patients diagnosed with COVID-19 infection between January and March 2020. Only a quarter of cases appeared to be locally acquired and genomic-based estimates of local transmission rates were concordant with predictions from a computational agent-based model. This convergent assessment indicates that genome sequencing provides key information to inform public health action and has improved our understanding of the COVID-19 evolution from outbreak to epidemic.","version":"1.2","doi":"10.1101/2020.04.19.048751","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.055863","pub_date":"2020-4-24","title":"Genomic, geographic and temporal distributions of SARS-CoV-2 mutations","abstract":"The COVID-19 pandemic is the most significant public health issue in recent history. Its causal agent, SARS-CoV-2, has evolved rapidly since its first emergence in December 2019. Mutations in the viral genome have critical impacts on the adaptation of viral strains to the local environment, and may alter the characteristics of viral transmission, disease manifestation, and the efficacy of treatment and vaccination. Using the complete sequences of 1,932 SARS-CoV-2 genomes, we examined the genomic, geographic and temporal distributions of aged, new, and frequent mutations of SARS-CoV-2, and identified six phylogenetic clusters of the strains, which also exhibit a geographic preference in different continents. Mutations in the form of single nucleotide variations (SNVs) provide a direct interpretation for the six phylogenetic clusters. Linkage disequilibrium, haplotype structure, evolutionary process, global distribution of mutations unveiled a sketch of the mutational history. Additionally, we found a positive correlation between the average mutation count and case fatality, and this correlation had strengthened with time, suggesting an important role of SNVs on disease outcomes. This study suggests that SNVs may become an important consideration in virus detection, clinical treatment, drug design, and vaccine development to avoid target shifting, and that continued isolation and sequencing is a crucial component in the fight against this pandemic. Mutation is the driving force of evolution for viruses like SARS-CoV-2, the causal agent of COVID-19. In this study, we discovered that the genome of SARS-CoV-2 is changing rapidly from the originally isolated form. These mutations have been spreading around the world and caused more than 2.5 million of infected cases and 170 thousands of deaths. We found that fourteen frequent mutations identified in this study can characterize the six main clusters of SARS-CoV-2 strains. In addition, we found the mutation burden is positively correlated with the fatality of COVID-19 patients. Understanding mutations in the SARS-CoV-2 genome will provide useful insight for the design of treatment and vaccination.","version":"1.1","doi":"10.1101/2020.04.22.055863","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.057042","pub_date":"2020-4-24","title":"ACE2 polymorphisms and individual susceptibility to SARS-CoV-2 infection: insights from an in silico study","abstract":"The current SARS covid-19 epidemic spread appears to be influenced by ethnical, geographical and sex-related factors that may involve genetic susceptibility to diseases. Similar to SARS-CoV, SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells, notably type II alveolar epithelial cells. Importantly, ACE2 gene is highly polymorphic. Here we have used in silico tools to analyze the possible impact of ACE2 single-nucleotide polymorphisms (SNPs) on the interaction with SARS-CoV-2 spike glycoprotein. We found that S19P (common in African people) and K26R (common in European people) were, among the most diffused SNPs worldwide, the only two SNPs that were able to potentially affect the interaction of ACE2 with SARS-CoV-2 spike. FireDock simulations demonstrated that while S19P may decrease, K26R might increase the ACE2 affinity for SARS-CoV-2 Spike. This finding suggests that the S19P may genetically protect, and K26R may predispose to more severe SARS-CoV-2 disease.","version":"1.1","doi":"10.1101/2020.04.23.057042","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.057190","pub_date":"2020-4-24","title":"Genetic analysis of the novel SARS-CoV-2 host receptor TMPRSS2 in different populations","abstract":"The infection coronavirus disease 2019 (COVID-19) is caused by a virus classified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At cellular level, virus infection initiates with binding of viral particles to the host surface cellular receptor angiotensin converting enzyme 2 (ACE2). SARS-CoV-2 engages ACE2 as the entry receptor and employs the cellular serine protease 2 (TMPRSS2) for S protein priming. TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host. Understanding how TMPRSS2 protein expression in the lung varies in the population could reveal important insights into differential susceptibility to influenza and coronavirus infections. Here, we systematically analyzed coding-region variants in TMPRSS2 and the eQTL variants, which may affect the gene expression, to compare the genomic characteristics of TMPRSS2 among different populations. Our findings suggest that the lung-specific eQTL variants may confer different susceptibility or response to SARS-CoV-2 infection from different populations under the similar conditions. In particular, we found that the eQTL variant rs35074065 is associated with high expression of TMPRSS2 but with a low expression of the interferon (IFN)-\u03b1/\u03b2-inducible gene, MX1, splicing isoform. Thus, these subjects could account for a more susceptibility either to viral infection or to a decrease in cellular antiviral response.","version":"1.1","doi":"10.1101/2020.04.23.057190","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.059667","pub_date":"2020-4-24","title":"Critical role of type III interferon in controlling SARS-CoV-2 infection, replication and spread in primary human intestinal epithelial cells","abstract":"SARS-CoV-2 is an unprecedented worldwide health problem that requires concerted and global approaches to better understand the virus in order to develop novel therapeutic approaches to stop the COVID-19 pandemic and to better prepare against potential future emergence of novel pandemic viruses. Although SARS-CoV-2 primarily targets cells of the lung epithelium causing respiratory infection and pathologies, there is growing evidence that the intestinal epithelium is also infected. However, the importance of the enteric phase of SARS-CoV-2 for virus-induced pathologies, spreading and prognosis remains unknown. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of SARS-CoV-2 lifecycle in human intestinal epithelial cells. Our results demonstrate that human intestinal epithelial cells fully support SARS-CoV-2 infection, replication and production of infectious de-novo virus particles. Importantly, we identified intestinal epithelial cells as the best culture model to propagate SARS-CoV-2. We found that viral infection elicited an extremely robust intrinsic immune response where, interestingly, type III interferon mediated response was significantly more efficient at controlling SARS-CoV-2 replication and spread compared to type I interferon. Taken together, our data demonstrate that human intestinal epithelial cells are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing in increasing patient viremia and by fueling an exacerbated cytokine response.","version":"1.1","doi":"10.1101/2020.04.24.059667","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.060376","pub_date":"2020-4-24","title":"The anti-HIV Drug Nelfinavir Mesylate (Viracept) is a Potent Inhibitor of Cell Fusion Caused by the SARS-CoV-2 Spike (S) Glycoprotein Warranting further Evaluation as an Antiviral against COVID-19 infections","abstract":"Coronaviruses belong to a group of enveloped, positive-single stranded RNA viruses that are known to cause severe respiratory distress in animals and humans. The current SARS coronavirus-2 (SARS CoV-2) pandemic has caused more than 2,000,000 infections globally and nearly 200,000 deaths. Coronaviruses enter susceptible cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after endocytosis of the virus into endosomes. Previous results with SARS and MERS CoV have shown that the Spike (S) glycoprotein is a major determinant of virus infectivity and immunogenicity. Herein, we show that expression of SARS CoV-2 S (S-n) glycoprotein after transient transfection of African green monkey kidney (Vero) cells caused extensive cell fusion in comparison to limited cell fusion caused by the SARS S (S-o) glycoprotein. S-n expression was detected intracellularly and on transfected Vero cell surfaces and caused the formation of very large multinucleated cells (syncytia) by 48 hours post transfection. These results are in agreement with published pathology observations of extensive syncytial formation in lung tissues of COVID-19 patients. This differential S-n versus S-o-mediated cell fusion suggests that SARS-CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular spaces and neutralizing antibodies. A systematic screening of several drugs for ability to inhibit S-n and S-o cell fusion revealed that the FDA approved HIV-protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n and S-o-mediated cell fusion in a dose-dependent manner. Complete inhibition of cell fusion was observed at a 10 micromolar concentration. Computational modeling and in silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n and S-o-mediated membrane fusion. Also, it is possible that nelfinavir mesylate acts on cellular processes to inhibit S proteolytic processing. These results warrant further investigations of the potential of nelfinavir mesylate as an antiviral drug, especially at early times after SARS-CoV-2 symptoms appear.","version":"1.1","doi":"10.1101/2020.04.24.060376","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.042002","pub_date":"2020-4-24","title":"A transcription regulatory network within the ACE2 locus may promote a pro-viral environment for SARS-CoV-2 by modulating expression of host factors","abstract":"A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was recently identified as the pathogen responsible for the COVID-19 outbreak. SARS-CoV-2 triggers severe pneumonia, which leads to acute respiratory distress syndrome and death in severe cases. As reported, SARS-CoV-2 is 80% genetically identical to the 2003 SARS-CoV virus. Angiotensin-converting enzyme 2 (ACE2) has been identified as the main receptor for entry of both SARS-CoV and SARS-CoV-2 into human cells. ACE2 is normally expressed in cardiovascular and lung type II alveolar epithelial cells, where it positively modulates the RAS system that regulates blood flow, pressure, and fluid homeostasis. Thus, virus-induced reduction of ACE2 gene expression is considered to make a significant contribution to severe acute respiratory failure. Chromatin remodeling plays a significant role in the regulation of ACE2 gene expression and the activity of regulatory elements within the genome. Here, we integrated data on physical chromatin interactions within the genome organization (captured by Hi-C) with tissue-specific gene expression data to identify spatial expression quantitative trait loci (eQTLs) and thus regulatory elements located within the ACE2 gene. We identified regulatory elements within ACE2 that control the expression of PIR, CA5B, and VPS13C in the lung. The gene products of these genes are involved in inflammatory responses, de novo pyrimidine and polyamine synthesis, and the endoplasmic reticulum, respectively. Our study, although limited by the fact that the identification of the regulatory interactions is putative until proven by targeted experiments, supports the hypothesis that viral silencing of ACE2 alters the activity of gene regulatory regions and promotes an intra-cellular environment suitable for viral replication.","version":"1.2","doi":"10.1101/2020.04.14.042002","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.24.060418","pub_date":"2020-4-24","title":"Treating Influenza and SARS-CoV-2 via mRNA-encoded Cas13a","abstract":"Here, Cas13a has been used to target and mitigate influenza virus A (IAV) and SARS-CoV-2 using a synthetic mRNA-based platform. CRISPR RNAs (crRNA) against PB1 and highly conserved regions of PB2 were screened in conjunction with mRNA-encoded Cas13a. Screens were designed such that only guides that decreased influenza RNA levels in a Cas13-mediated fashion, were valid. Cas13a mRNA and validated guides, delivered post-infection, simulating treatment, were tested in combination and across multiplicities of infection. Their function was also characterized over time. Similar screens were performed for guides against SARS-CoV-2, yielding multiple guides that significantly impacted cytopathic effect. Last, the approach was utilized in vivo, demonstrating the ability to degrade influenza RNA in a mouse model of infection, using polymer-formulated, nebulizer-based mRNA delivery. Our findings demonstrate the applicability of Cas13a in mitigating respiratory infections both in vitro and in a mouse model, paving the way for future therapeutic use.","version":"1.1","doi":"10.1101/2020.04.24.060418","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.056747","pub_date":"2020-4-24","title":"Comparison of commercial RT-PCR diagnostic kits for COVID-19","abstract":"The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was \u226596% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.","version":"1.1","doi":"10.1101/2020.04.22.056747","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.057786","pub_date":"2020-4-24","title":"The coronavirus proofreading exoribonuclease mediates extensive viral recombination","abstract":"Coronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and generates defective viral genomes (DVGs) of unknown function. However, the determinants and patterns of CoV recombination are unknown. Here, we show that divergent \u03b2-CoVs SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV) perform extensive RNA recombination in culture, generating similar patterns of recombination junctions and diverse populations of DVGs and sgmRNAs. We demonstrate that the CoV proofreading nonstructural protein (nsp14) 3\u2019-to-5\u2019 exoribonuclease (nsp14-ExoN) is required for normal CoV recombination and that its genetic inactivation causes significantly decreased frequency and altered patterns of recombination in both infected cells and released virions. Thus, nsp14-ExoN is a key determinant of both high fidelity CoV replication and recombination, and thereby represents a highly-conserved and vulnerable target for virus inhibition and attenuation.","version":"1.1","doi":"10.1101/2020.04.23.057786","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.054973","pub_date":"2020-4-24","title":"Deep Sentiment Classification and Topic Discovery on Novel Coronavirus or COVID-19 Online Discussions: NLP Using LSTM Recurrent Neural Network Approach","abstract":"Internet forums and public social media, such as online healthcare forums, provide a convenient channel for users (people/patients) concerned about health issues to discuss and share information with each other. In late December 2019, an outbreak of a novel coronavirus (infection from which results in the disease named COVID-19) was reported, and, due to the rapid spread of the virus in other parts of the world, the World Health Organization declared a state of emergency. In this paper, we used automated extraction of COVID-19\u2013related discussions from social media and a natural language process (NLP) method based on topic modeling to uncover various issues related to COVID-19 from public opinions. Moreover, we also investigate how to use LSTM recurrent neural network for sentiment classification of COVID-19 comments. Our findings shed light on the importance of using public opinions and suitable computational techniques to understand issues surrounding COVID-19 and to guide related decision-making.","version":"1.2","doi":"10.1101/2020.04.22.054973","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.054122","pub_date":"2020-4-23","title":"Population genomics insights into the recent evolution of SARS-CoV-2","abstract":"The current coronavirus disease 2019 (COVID-19) pandemic is caused by the SARS-CoV-2 virus and is still spreading rapidly worldwide. Full-genome-sequence computational analysis of the SARS-CoV-2 genome will allow us to understand the recent evolutionary events and adaptability mechanisms more accurately, as there is still neither effective therapeutic nor prophylactic strategy. In this study, we used population genetics analysis to infer the mutation rate and plausible recombination events that may have contributed to the evolution of the SARS-CoV-2 virus. Furthermore, we localized targets of recent and strong positive selection. The genomic regions that appear to be under positive selection are largely co-localized with regions in which recombination from non-human hosts appeared to have taken place in the past. Our results suggest that the pangolin coronavirus genome may have contributed to the SARS-CoV-2 genome by recombination with the bat coronavirus genome. However, we find evidence for additional recombination events that involve coronavirus genomes from other hosts, i.e., Hedgehog and Sparrow. Even though recombination events within human hosts cannot be directly assessed, due to the high similarity of SARS-CoV-2 genomes, we infer that recombinations may have recently occurred within human hosts using a linkage disequilibrium analysis. In addition, we employed an Approximate Bayesian Computation approach to estimate the parameters of a demographic scenario involving an exponential growth of the size of the SARS-CoV-2 populations that have infected European, Asian and Northern American cohorts, and we demonstrated that a rapid exponential growth in population size can support the observed polymorphism patterns in SARS-CoV-2 genomes.","version":"1.1","doi":"10.1101/2020.04.21.054122","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.054015","pub_date":"2020-4-23","title":"TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes","abstract":"Both gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA have been frequently observed in COVID-19 patients. However, whether SARS-CoV-2 replicate in the human intestine and its clinical relevance to potential fecal-oral transmission remain unclear. Here, we demonstrate productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. In addition to TMPRSS2, another mucosa-specific serine protease, TMPRSS4, also enhanced SARS-CoV-2 spike fusogenic activity and mediated viral entry into host cells. However, newly synthesized viruses released into the intestinal lumen were rapidly inactivated by human colonic fluids and no infectious virus was recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.","version":"1.1","doi":"10.1101/2020.04.21.054015","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.053199","pub_date":"2020-4-23","title":"Bioinformatics Study on Structural Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) For Better Understanding the Vaccine Development","abstract":"Novel coronavirus 2019 (2019-nCoV), also known as SARS-CoV-2), leads high morbidity and mortality in global epidemics. Four structural proteins (surface glycoprotein (QIQ22760.1), envelop glycoprotein (QIQ22762.1), nucleocapsid phosphoprotein (QIQ22768.1) and membrane glycoprotein (QIQ22763.1)) of SARS-CoV-2 are extracted from the NCBI database and further analyzed with ExPASy ProtParam tool. Lucien is the highest in envelope, surface and membrane glycoprotein that is an optimal environment for rapid virus fixation on host cell\\'s surface to the receptor molecule. Transmembrane region prediction was performed by SOSUI server. For all structural proteins, except nucleocapsid Phosphoprotein, the trans-membrane prediction indicates that the virus can enter the host easily. Domain analysis was done by SMART tool. Domain information helps in the function of the viral protein. Lastly, the 3D structure prediction was carried out by Swiss Model and the result validation was achieved by PROCHECK. Such models are the starting point of the community for structural drug and vaccine designs as well as virtual computational screening.","version":"1.2","doi":"10.1101/2020.04.21.053199","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.054981","pub_date":"2020-4-23","title":"The anticoagulant nafamostat potently inhibits SARS-CoV-2 infection in vitro: an existing drug with multiple possible therapeutic effects","abstract":"Although infection by SARS-CoV-2, the causative agent of COVID-19, is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked MERS-CoV S protein-initiated cell fusion by targeting TMPRSS2, and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on SARS-CoV-2 S protein, ACE2 and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an EC50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. These findings, together with accumulated clinical data regarding its safety, make nafamostat a likely candidate drug to treat COVID-19.","version":"1.1","doi":"10.1101/2020.04.22.054981","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.052209","pub_date":"2020-4-23","title":"Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates","abstract":"The COVID-19 outbreak has become a global pandemic responsible for over 2,000,000 confirmed cases and over 126,000 deaths worldwide. In this study, we examined the immunogenicity of CHO-expressed recombinant SARS-CoV-2 S1-Fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a COVID-19 vaccine. We demonstrate that the S1-Fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day 7. Strong virus neutralizing activity was observed on day 14 in rabbits immunized with the S1-Fc fusion protein using a pseudovirus neutralization assay. Most importantly, in less than 20 days and three injections of the S1-Fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered COVID-19 patient in a live SARS-CoV-2 infection assay. Our data strongly suggests that the CHO-expressed SARS-CoV-2 S1-Fc recombinant protein could be a strong candidate for vaccine development against COVID-19. CHO-expressed S1-Fc protein is very immunogenic in various animals and can rapidly induce strong antibody production S1-Fc protein solicits strong neutralizing activities against live virus Stable CHO cell line expressing 50 mg/L of S1-Fc and a 3,000 L Bioreactor can produce 3 million doses of human COVID-19 vaccine every 10 days, making it an accessible and affordable option for worldwide vaccination","version":"1.1","doi":"10.1101/2020.04.21.052209","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.054387","pub_date":"2020-4-23","title":"Identification of potential treatments for COVID-19 through artificial intelligence-enabled phenomic analysis of human cells infected with SARS-CoV-2","abstract":"To identify potential therapeutic stop-gaps for SARS-CoV-2, we evaluated a library of 1,670 approved and reference compounds in an unbiased, cellular image-based screen for their ability to suppress the broad impacts of the SARS-CoV-2 virus on phenomic profiles of human renal cortical epithelial cells using deep learning. In our assay, remdesivir is the only antiviral tested with strong efficacy, neither chloroquine nor hydroxychloroquine have any beneficial effect in this human cell model, and a small number of compounds not currently being pursued clinically for SARS-CoV-2 have efficacy. We observed weak but beneficial class effects of \u03b2-blockers, mTOR/PI3K inhibitors and Vitamin D analogues and a mild amplification of the viral phenotype with \u03b2-agonists.","version":"1.1","doi":"10.1101/2020.04.21.054387","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.12.038216","pub_date":"2020-4-23","title":"Detection of spreader nodes and ranking of interacting edges in Human-SARS-CoV protein interaction network","abstract":"The entire world has recently witnessed the commencement of coronavirus disease 19 (COVID-19) pandemic. It is caused by a novel coronavirus (n-CoV) generally distinguished as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). It has exploited human vulnerabilities to coronavirus outbreak. SARS-CoV-2 promotes fatal chronic respiratory disease followed by multiple organ failure which ultimately puts an end to human life. No proven vaccine for n-CoV is available till date in spite of significant research efforts worldwide. International Committee on Taxonomy of Viruses (ICTV) has reached to a consensus that the virus SARS-CoV-2 is highly genetically similar to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) outbreak of 2003. It has been reported that SARS-CoV has \u223c89% genetic similarities with n-CoV. With this hypothesis, the current work focuses on the identification of spreader nodes in SARS-CoV protein interaction network. Various network characteristics like edge ratio, neighborhood density and node weight have been explored for defining a new feature spreadability index by virtue of which spreader nodes and edges are identified. The selected top spreader nodes having high spreadability index have been also validated by Susceptible-Infected-Susceptible (SIS) disease model. Initially, the proposed method is applied on a synthetic protein interaction network followed by SARS-CoV-human protein interaction network. Hence, key spreader nodes and edges (ranked edges) are unmasked in SARS-CoV proteins and its connected level 1 and level 2 human proteins. The new network attribute spreadability index along with generated SIS values of selected top spreader nodes when compared with the other network centrality based methodologies like Degree centrality (DC), Closeness centrality (CC), Local average centrality (LAC) and Betweeness centrality (BC) is found to perform relatively better than the existing-state-of-art.","version":"1.2","doi":"10.1101/2020.04.12.038216","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.050138","pub_date":"2020-4-23","title":"Interplay of host regulatory network on SARS-CoV-2 binding and replication machinery","abstract":"We dissect the mechanism of SARS-CoV-2 in human lung host from the initial phase of receptor binding to viral replication machinery. We constructed two independent lung protein interactome to reveal the signaling process on receptor activation and host protein hijacking machinery in the pathogenesis of virus. Further, we test the functional role of the hubs derived from both interactome. Most hubs proteins were differentially regulated on SARS-CoV-2 infection. Also, the proteins of viral replication hubs were related with cardiovascular disease, diabetes and hypertension confirming the vulnerability and severity of infection in the risk individual. Additionally, the hub proteins were closely linked with other viral infection, including MERS and HCoVs which suggest similar infection pattern in SARS-CoV-2. We identified five interconnecting cascades between hubs of both networks that show the preparation of optimal environment in the host for viral replication process upon receptor attachment. Interestingly, we propose that seven potential miRNAs, targeting the intermediate phase that connects receptor and viral replication process a better choice as a drug for SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.04.20.050138","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.054221","pub_date":"2020-4-23","title":"The genomic variation landscape of globally-circulating clades of SARS-CoV-2 defines a genetic barcoding scheme","abstract":"We describe fifteen major mutation events from 2,058 high-quality SARS-CoV-2 genomes deposited up to March 31st, 2020. These events define five major clades (G, I, S, D and V) of globally-circulating viral populations, representing 85.7% of all sequenced cases, which we can identify using a 10 nucleotide genetic classifier or barcode. We applied this barcode to 4,000 additional genomes deposited between March 31st and April 15th and classified successfully 95.6% of the clades demonstrating the utility of this approach. An analysis of amino acid variation in SARS-CoV-2 ORFs provided evidence of substitution events in the viral proteins involved in both host-entry and genome replication. The systematic monitoring of dynamic changes in the SARS-CoV-2 genomes of circulating virus populations over time can guide therapeutic and prophylactic strategies to manage and contain the virus and, also, with available efficacious antivirals and vaccines, aid in the monitoring of circulating genetic diversity as we proceed towards elimination of the agent. The barcode will add the necessary genetic resolution to facilitate tracking and monitoring of infection clusters to distinguish imported and indigenous cases and thereby aid public health measures seeking to interrupt transmission chains without the requirement for real-time complete genomes sequencing.","version":"1.1","doi":"10.1101/2020.04.21.054221","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.022103","pub_date":"2020-4-23","title":"Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19","abstract":"Stopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans. We generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19. ACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2. ACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities. We unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry.","version":"1.2","doi":"10.1101/2020.04.10.022103","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.22.055897","pub_date":"2020-4-23","title":"Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2)","abstract":"SARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.","version":"1.1","doi":"10.1101/2020.04.22.055897","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.23.057265","pub_date":"2020-4-23","title":"Structural and biochemical characterization of nsp12-nsp7-nsp8 core polymerase complex from COVID-19 virus","abstract":"The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus. The viral polymerase is a promising antiviral target. However, the structure of COVID-19 virus polymerase is yet unknown. Here, we describe the near-atomic resolution structure of its core polymerase complex, consisting of nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases, and suggests the mechanism for activation by cofactors. Biochemical studies revealed reduced activity of the core polymerase complex and lower thermostability of individual subunits of COVID-19 virus as compared to that of SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate a well adaptation of COVID-19 virus towards humans with relatively lower body temperatures than the natural bat hosts.","version":"1.1","doi":"10.1101/2020.04.23.057265","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.12.902452","pub_date":"2020-4-23","title":"Programmable low-cost DNA-based platform for viral RNA detection","abstract":"Viral detection is critical for controlling disease spread and progression. Recent emerging viral threats including Zika, Ebola, and the current COVID-19 outbreak highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches designed to mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show non-enzymatic detection of viral RNA to the attomole level, with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with a sample preparation step using either RNA extraction or isothermal pre-amplification. Our assay can be performed with minimal or no lab infrastructure, and is readily adaptable to detect other viruses. We demonstrate the adaptability of our method by quickly developing and testing DNA nanoswitches for detecting a fragment of SARS-CoV-2 RNA in human saliva. Given this versatility, we expect that further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.","version":"1.2","doi":"10.1101/2020.01.12.902452","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.15.043166","pub_date":"2020-4-22","title":"Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2","abstract":"Effective therapeutics to treat COVID-19 are urgently needed. Remdesivir is a nucleotide prodrug with in vitro and in vivo efficacy against coronaviruses. Here, we tested the efficacy of remdesivir treatment in a rhesus macaque model of SARS-CoV-2 infection. To evaluate the effect of remdesivir treatment on SARS-CoV-2 disease outcome, we used the recently established rhesus macaque model of SARS-CoV-2 infection that results in transient lower respiratory tract disease. Two groups of six rhesus macaques were infected with SARS-CoV-2 and treated with intravenous remdesivir or an equal volume of vehicle solution once daily. Clinical, virological and histological parameters were assessed regularly during the study and at necropsy to determine treatment efficacy. In contrast to vehicle-treated animals, animals treated with remdesivir did not show signs of respiratory disease and had reduced pulmonary infiltrates on radiographs. Virus titers in bronchoalveolar lavages were significantly reduced as early as 12hrs after the first treatment was administered. At necropsy on day 7 after inoculation, lung viral loads of remdesivir-treated animals were significantly lower and there was a clear reduction in damage to the lung tissue. Therapeutic remdesivir treatment initiated early during infection has a clear clinical benefit in SARS-CoV-2-infected rhesus macaques. These data support early remdesivir treatment initiation in COVID-19 patients to prevent progression to severe pneumonia.","version":"1.2","doi":"10.1101/2020.04.15.043166","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.18.047951","pub_date":"2020-4-22","title":"Significant expression of FURIN and ACE2 on oral epithelial cells may facilitate the efficiency of SARS-CoV-2 entry","abstract":"Leading to a sustained epidemic spread with >2,000,000 confirmed human infections, including >100,000 deaths, COVID-19 was caused by SARS-CoV-2 and resulted in acute respiratory distress syndrome (ARDS) and sepsis, which brought more challenges to the patient\u2019s treatment. The S-glycoprotein, which recognized as the key factor for the entry of SARS-CoV-2 into the cell, contains two functional domains: an ACE2 receptor binding domain and a second domain necessary for fusion of the coronavirus and cell membranes. FURIN activity, exposes the binding and fusion domains, is essential for the zoonotic transmission of SARS-CoV-2. Moreover, it has been reported that ACE2 is likely to be the receptor for SARS-CoV-2. In addition, FURIN enzyme and ACE2 receptor were expressed in airway epithelia, cardiac tissue, and enteric canals, which considered as the potential target organ of the virus. However, report about the expression of FURIN and ACE2 in oral tissues was limited. In order to investigate the potential infective channel of new coronavirus in oral cavity, we analyze the expression of ACE2 and FURIN that mediate the new coronavirus entry into host cells in oral mucosa using the public single-cell sequence datasets. Furthermore, immunohistochemical staining experiment was performed to confirm the expression of ACE2 and FURIN in the protein level. The bioinformatics results indicated the differential expression of ACE2 and FURIN on epithelial cells of different oral mucosal tissues and the proportion of FURIN-positive cells was obviously higher than that of ACE2-positive cells. IHC experiments revealed that both the ACE2-positive and FURIN-positive cells in the target tissues were mainly positioned in the epithelial layers, partly expressed in fibroblasts, which further confirm the bioinformatics results. Based on these findings, we speculated that SARS-CoV-2 could effectively invade oral mucosal cells though two possible routes: binding to the ACE2 receptor and fusion with cell membrane activated by FURIN protease. Our results indicated that oral mucosa tissues are susceptible to SARS-CoV-2, which provides valuable information for virus-prevention strategy in clinical care as well as daily life.","version":"1.2","doi":"10.1101/2020.04.18.047951","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.042911","pub_date":"2020-4-21","title":"Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than one million cases and tens of thousands of deaths. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.","version":"1.1","doi":"10.1101/2020.04.21.042911","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.051912","pub_date":"2020-4-21","title":"Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection","abstract":"Coronavirus disease 2019 (COVID-19) is a viral pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is predominantly defined by respiratory symptoms, but cardiac complications including arrhythmias, heart failure, and viral myocarditis are also prevalent. Although the systemic ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well-understood. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. Microscopy and immunofluorescence demonstrated that SARS-CoV-2 can enter and replicate within hiPSC-CMs, localizing at perinuclear locations within the cytoplasm. Viral cytopathic effect induced hiPSC-CM apoptosis and cessation of beating after 72 hours of infection. These studies show that SARS-CoV-2 can infect hiPSC-CMs in vitro, establishing a model for elucidating the mechanisms of infection and potentially a cardiac-specific antiviral drug screening platform.","version":"1.1","doi":"10.1101/2020.04.21.051912","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.053009","pub_date":"2020-4-21","title":"Elucidating the differences in the molecular mechanism of receptor binding between 2019-nCoV and the SARS-CoV viruses using computational tools","abstract":"The outbreak of the 2019-nCoV coronavirus causing severe acute respiratory syndrome which can be fatal, especially in elderly population, has been declared a pandemic by the World Health Organization. Many biotechnology laboratories are rushing to develop therapeutic antibodies and antiviral drugs for treatment of this viral disease. The viral CoV spike (S) glycoprotein is one of the main targets for pharmacological intervention. Its receptor-binding domain (RBD) interacts with the human ACE2 receptor ensuring the entry of the viral genomes into the host cell. In this work, we report on the differences in the binding of the RBD of the previous coronavirus SARS-CoV and of the newer 2019-nCoV coronavirus to the human ACE2 receptor using atomistic molecular dynamics techniques. Our results show major mutations in the 2019-nCoV RBD with respect to the SARS-CoV RBD occurring at the interface of RBD-ACE2 complex. These mutations make the 2019-nCoV RBD protein backbone much more flexible, hydrophobic interactions are reduced and additional polar/charged residues appear at the interface. We observe that higher flexibility of the 2019-nCoV RBD with respect to the SARS-CoV RBD leads to a bigger binding interface between the 2019-nCoV RBD and ACE2 and to about 20% more contacts between them in comparison with SARS-CoV. Taken together, the 2019-nCoV RBD shows more stable binding interface and higher binding affinity for the ACE2 receptor. The mutations not only stabilize the binding interface, they also lead to overall more stable 2019-nCoV RBD protein structure, even far from the binding interface. Our results on the molecular differences in the binding between the two viruses can provide important inputs for development of appropriate antiviral treatments of the new viruses, addressing the necessity of ongoing pandemics.","version":"1.1","doi":"10.1101/2020.04.21.053009","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052126","pub_date":"2020-4-21","title":"Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals","abstract":"The current COVID-19 pandemic, caused by a novel coronavirus SARS-CoV-2, poses serious threats to public health and social stability, calling for urgent need for vaccines and therapeutics. SARS-CoV-2 is genetically close to SARS-CoV, thus it is important to define the between antigenic cross-reactivity and neutralization. In this study, we firstly analyzed 20 convalescent serum samples collected from SARS-CoV infected individuals during the 2003 SARS outbreak. All patient sera reacted strongly with the S1 subunit and receptor-binding domain (RBD) of SARS-CoV, cross-reacted with the S ectodomain, S1, RBD, and S2 proteins of SARS-CoV-2, and neutralized both SARS-CoV and SARS-CoV-2 S protein-driven infections. Multiple panels of antisera from mice and rabbits immunized with a full-length S and RBD immunogens of SARS-CoV were also characterized, verifying the cross-reactive neutralization against SARS-CoV-2. Interestingly, we found that a palm civet SARS-CoV-derived RBD elicited more potent cross-neutralizing responses in immunized animals than the RBD from a human SARS-CoV strain, informing a strategy to develop a universe vaccine against emerging CoVs. Serum antibodies from SARS-CoV infected patients and immunized animals cross-neutralize SARS-CoV-2 suggests strategies for universe vaccines against emerging CoVs.","version":"1.1","doi":"10.1101/2020.04.20.052126","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.052639","pub_date":"2020-4-21","title":"Structural Basis of SARS-CoV-2 Spike Protein Priming by TMPRSS2","abstract":"Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational details of complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike and to point candidate drug targets. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provide structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.","version":"1.1","doi":"10.1101/2020.04.21.052639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.052084","pub_date":"2020-4-21","title":"Artificial intelligence predicts the immunogenic landscape of SARS-CoV-2: toward universal blueprints for vaccine designs","abstract":"The global population is at present suffering from a pandemic of Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The goals of this study were to use artificial intelligence (AI) to predict blueprints for designing universal vaccines against SARS-CoV-2, that contain a sufficiently broad repertoire of T-cell epitopes capable of providing coverage and protection across the global population. To help achieve these aims, we profiled the entire SARS-CoV-2 proteome across the most frequent 100 HLA-A, HLA-B and HLA-DR alleles in the human population, using host-infected cell surface antigen presentation and immunogenicity predictors from the NEC Immune Profiler suite of tools, and generated comprehensive epitope maps. We then used these epitope maps as input for a Monte Carlo simulation designed to identify statistically significant \u201cepitope hotspot\u201d regions in the virus that are most likely to be immunogenic across a broad spectrum of HLA types. We then removed epitope hotspots that shared significant homology with proteins in the human proteome to reduce the chance of inducing off-target autoimmune responses. We also analyzed the antigen presentation and immunogenic landscape of all the nonsynonymous mutations across 3400 different sequences of the virus, to identify a trend whereby SARS-COV-2 mutations are predicted to have reduced potential to be presented by host-infected cells, and consequently detected by the host immune system. A sequence conservation analysis then removed epitope hotspots that occurred in less-conserved regions of the viral proteome. Finally, we used a database of the HLA genotypes of approximately 22 000 individuals to develop a \u201cdigital twin\u201d type simulation to model how effective different combinations of hotspots would work in a diverse human population, and used the approach to identify an optimal constellation of epitopes hotspots that could provide maximum coverage in the global population. By combining the antigen presentation to the infected-host cell surface and immunogenicity predictions of the NEC Immune Profiler with a robust Monte Carlo and digital twin simulation, we have managed to profile the entire SARS-CoV-2 proteome and identify a subset of epitope hotspots that could be harnessed in a vaccine formulation to provide a broad coverage across the global population.","version":"1.1","doi":"10.1101/2020.04.21.052084","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.19.049254","pub_date":"2020-4-21","title":"Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells","abstract":"The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.","version":"1.2","doi":"10.1101/2020.04.19.049254","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.036335","pub_date":"2020-4-21","title":"A Computational Approach to Design Potential siRNA Molecules as a Prospective Tool for Silencing Nucleocapsid Phosphoprotein and Surface Glycoprotein Gene of SARS-CoV-2","abstract":"An outbreak, caused by a RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature and efficacy prediction process 8 siRNA molecules were selected which are proposed to exerts the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2","version":"1.3","doi":"10.1101/2020.04.10.036335","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052233","pub_date":"2020-4-21","title":"Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples","abstract":"COVID-19 pandemic has caused tens of thousands of deaths and is now a severe threat to global health. Clinical practice has demonstrated that the SARS-CoV-2 S1 specific antibodies and viral antigens can be used as diagnostic and prognostic markers of COVID-19. However, the popular point-of-care biomarker detection technologies, such as the lateral-flow test strips, provide only yes/no information and have very limited sensitivities. Thus, it has a high false negative rate and cannot be used for the quantitative evaluation of patient\u2019s immune response. Conventional ELISA (enzyme-linked immunosorbent assay), on the other hand, can provide quantitative, accurate, and sensitive results, but it involves complicated and expensive instruments and long assay time. In addition, samples need to be sent to centralized labs, which significantly increases the turn-around time. Here, we present a microfluidic ELISA technology for rapid (15-20 minutes), quantitative, sensitive detection of SARS-CoV-2 biomarkers using SARS-CoV-2 specific IgG and viral antigen \u2013 S protein in serum. We also characterized various humanized monoclonal IgG, and identified a candidate with a high binding affinity towards SARS-CoV-2 S1 protein that can serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. Furthermore, we demonstrated that our microfluidic ELISA platform can be used for rapid affinity evaluation of monoclonal anti-S1 antibodies. The microfluidic ELISA device is highly portable and requires less than 10 \u03bcL of samples for each channel. Therefore, our technology will greatly facilitate rapid and quantitative analysis of COVID-19 patients and vaccine recipients at point-of-care.","version":"1.1","doi":"10.1101/2020.04.20.052233","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.052290","pub_date":"2020-4-21","title":"Dynamical asymmetry exposes 2019-nCoV prefusion spike","abstract":"The novel coronavirus (2019-nCoV) spike protein is a smart molecular machine that instigates the entry of coronavirus to the host cell causing the COVID-19 pandemic. In this study, a structural-topology based model Hamiltonian of C3 symmetric trimeric spike is developed to explore its complete conformational energy landscape using molecular dynamic simulations. The study finds 2019-nCoV to adopt a unique strategy by undertaking a dynamic conformational asymmetry induced by a few unique inter-chain interactions. This results in two prevalent asymmetric structures of spike where one or two spike heads lifted up undergoing a dynamic transition likely to enhance rapid recognition of the host-cell receptor turning on its high-infectivity. The crucial interactions identified in this study are anticipated to potentially affect the efficacy of therapeutic targets. Inter-chain-interaction driven rapid symmetry breaking strategy adopted by the prefusion trimeric spike protein likely to make 2019-nCoV highly infective.","version":"1.1","doi":"10.1101/2020.04.20.052290","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.21.050922","pub_date":"2020-4-21","title":"A New Resource for Genomics and Precision Health Information and Publications on the Investigation and Control of COVID-19 and other Coronaviruses","abstract":"We developed a new online database that contains the most updated published scientific literature, online news and reports, CDC and National Institutes of Health (NIH) resources. The tool captures emerging discoveries and applications of genomics, molecular, and other precision medicine and precision public health tools in the investigation and control of coronavirus diseases, including COVID-19, MERS-CoV, and SARS. Coronavirus Disease Portal (CDP) can be freely accessed via https://phgkb.cdc.gov/PHGKB/coVInfoStartPage.action. wyu@cdc.gov","version":"1.1","doi":"10.1101/2020.04.21.050922","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.050039","pub_date":"2020-4-20","title":"Phylodynamics of SARS-CoV-2 transmission in Spain","abstract":"SARS-CoV-2 whole-genome analysis has identified three large clades spreading worldwide, designated G, V and S. This study aims to analyze the diffusion of SARS-CoV-2 in Spain/Europe. Maximum likelihood phylogenetic and Bayesian phylodynamic analyses have been performed to estimate the most probable temporal and geographic origin of different phylogenetic clusters and the diffusion pathways of SARS-CoV-2. Phylogenetic analyses of the first 28 SARS-CoV-2 whole genome sequences obtained from patients in Spain revealed that most of them are distributed in G and S clades (13 sequences in each) with the remaining two sequences branching in the V clade. Eleven of the Spanish viruses of the S clade and six of the G clade grouped in two different monophyletic clusters (S-Spain and G-Spain, respectively), with the S-Spain cluster also comprising 8 sequences from 6 other countries from Europe and the Americas. The most recent common ancestor (MRCA) of the SARS-CoV-2 pandemic was estimated in the city of Wuhan, China, around November 24, 2019, with a 95% highest posterior density (HPD) interval from October 30-December 17, 2019. The origin of S-Spain and G-Spain clusters were estimated in Spain around February 14 and 18, 2020, respectively, with a possible ancestry of S-Spain in Shanghai. Multiple SARS-CoV-2 introductions have been detected in Spain and at least two resulted in the emergence of locally transmitted clusters, with further dissemination of one of them to at least 6 other countries. These results highlight the extraordinary potential of SARS-CoV-2 for rapid and widespread geographic dissemination.","version":"1.1","doi":"10.1101/2020.04.20.050039","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.040782","pub_date":"2020-4-20","title":"The global population of SARS-CoV-2 is composed of six major subtypes","abstract":"The World Health Organization characterized the COVID-19 as a pandemic in March 2020, the second pandemic of the 21st century. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-stranded RNA betacoronavirus of the family Coronaviridae. Expanding virus populations, as that of SARS-CoV-2, accumulate a number of narrowly shared polymorphisms imposing a confounding effect on traditional clustering methods. In this context, approaches that reduce the complexity of the sequence space occupied by the SARS-CoV-2 population are necessary for a robust clustering. Here, we proposed the subdivision of the global SARS-CoV-2 population into sixteen well-defined subtypes by focusing on the widely shared polymorphisms in nonstructural (nsp3, nsp4, nsp6, nsp12, nsp13 and nsp14) cistrons, structural (spike and nucleocapsid) and accessory (ORF8) genes. Six virus subtypes were predominant in the population, but all sixteen showed amino acid replacements which might have phenotypic implications. We hypothesize that the virus subtypes detected in this study are records of the early stages of the SARS-CoV-2 diversification that were randomly sampled to compose the virus populations around the world, a typical founder effect. The genetic structure determined for the SARS-CoV-2 population provides substantial guidelines for maximizing the effectiveness of trials for testing the candidate vaccines or drugs.","version":"1.2","doi":"10.1101/2020.04.14.040782","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.051656","pub_date":"2020-4-20","title":"Exceptional diversity and selection pressure on SARS-CoV and SARS-CoV-2 host receptor in bats compared to other mammals","abstract":"Pandemics originating from pathogen transmission between animals and humans highlight the broader need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection. Here, we investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2. We find that the ACE2 gene is under strong selection pressure in bats, the group in which the progenitors of SARS-CoV and SARS-CoV-2 are hypothesized to have evolved, particularly in residues that contact SARS-CoV and SARS-CoV-2. We detect positive selection in non-bat mammals in ACE2 but in a smaller proportion of branches than in bats, without enrichment of selection in residues that contact SARS-CoV or SARS-CoV-2. Additionally, we evaluate similarity between humans and other species in residues that contact SARS-CoV or SARS-CoV-2, revealing potential susceptible species but also highlighting the difficulties of predicting spillover events. This work increases our understanding of the relationship between mammals, particularly bats, and coronaviruses, and provides data that can be used in functional studies of how host proteins are bound by SARS-CoV and SARS-CoV-2 strains.","version":"1.1","doi":"10.1101/2020.04.20.051656","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.051011","pub_date":"2020-4-20","title":"Limited SARS-CoV-2 diversity within hosts and following passage in cell culture","abstract":"Since the first reports of pneumonia associated with a novel coronavirus (COVID-19) emerged in Wuhan, Hubei province, China, there have been considerable efforts to sequence the causative virus, SARS-CoV-2 (also referred to as hCoV-19) and to make viral genomic information available quickly on shared repositories. As of 30 March 2020, 7,680 consensus sequences have been shared on GISAID, the principal repository for SARS-CoV-2 genetic information. These sequences are primarily consensus sequences from clinical and passaged samples, but few reports have looked at diversity of virus populations within individual hosts or cultures. Understanding such diversity is essential to understanding viral evolutionary dynamics. Here, we characterize within-host viral diversity from a primary isolate and passaged samples, all originally deriving from an individual returning from Wuhan, China, who was diagnosed with COVID-19 and subsequently sampled in Wisconsin, United States. We use a metagenomic approach with Oxford Nanopore Technologies (ONT) GridION in combination with Illumina MiSeq to capture minor within-host frequency variants \u22651%. In a clinical swab obtained from the day of hospital presentation, we identify 15 single nucleotide variants (SNVs) \u22651% frequency, primarily located in the largest gene \u2013 ORF1a. While viral diversity is low overall, the dominant genetic signatures are likely secondary to population size changes, with some evidence for mild purifying selection throughout the genome. We see little to no evidence for positive selection or ongoing adaptation of SARS-CoV-2 within cell culture or in the primary isolate evaluated in this study. Within-host variants are critical for addressing molecular evolution questions, identifying selective pressures imposed by vaccine-induced immunity and antiviral therapeutics, and characterizing interhost dynamics, including the stringency and character of transmission bottlenecks. Here, we sequenced SARS-CoV-2 viruses isolated from a human host and from cell culture on three distinct Vero cell lines using Illumina and ONT technologies. We show that SARS-CoV-2 consensus sequences can remain stable through at least two serial passages on Vero 76 cells, suggesting SARS-CoV-2 can be propagated in cell culture in preparation for in-vitro and in-vivo studies without dramatic alterations of its genotype. However, we emphasize the need to deep-sequence viral stocks prior to use in experiments to characterize sub-consensus diversity that may alter outcomes.","version":"1.1","doi":"10.1101/2020.04.20.051011","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.042366","pub_date":"2020-4-20","title":"Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler","abstract":"There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.","version":"1.1","doi":"10.1101/2020.04.17.042366","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.051219","pub_date":"2020-4-20","title":"Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays","abstract":"SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.","version":"1.1","doi":"10.1101/2020.04.20.051219","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.049924","pub_date":"2020-4-20","title":"SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology","abstract":"The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative \u2018furin-like cleavage site\u2019 in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.","version":"1.1","doi":"10.1101/2020.04.20.049924","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.19.049643","pub_date":"2020-4-20","title":"Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy","abstract":"The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a \u201csandwich\u201d complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.19.049643","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.051557","pub_date":"2020-4-20","title":"Design an efficient multi-epitope peptide vaccine candidate against SARS-CoV-2: An in silico analysis","abstract":"To date, no specific vaccine or drug has been proven to be effective for SARS-CoV-2 infection. Therefore, we implemented immunoinformatics approach to design an efficient multi-epitopes vaccine against SARS-CoV-2. The designed vaccine construct has several immunodominant epitopes from structural proteins of Spike, Nucleocapsid, Membrane and Envelope. These peptides promote cellular and humoral immunity and Interferon gamma responses. In addition, these epitopes have antigenicity ability and no allergenicity probability. To enhance the vaccine immunogenicity, we used three potent adjuvants; Flagellin, a driven peptide from high mobility group box 1 as HP-91 and human beta defensin 3 protein. The physicochemical and immunological properties of the vaccine structure were evaluated. Tertiary structure of the vaccine protein was predicted and refined by I-Tasser and galaxi refine and validated using Rampage and ERRAT. Results of Ellipro showed 242 residues from vaccine might be conformational B cell epitopes. Docking of vaccine with Toll-Like Receptors 3, 5 and 8 proved an appropriate interaction between the vaccine and receptor proteins. In silico cloning demonstrated that the vaccine can be efficiently expressed in Escherichia coli. The designed multi epitope vaccine is potentially antigenic in nature and has the ability to induce humoral and cellular immune responses against SARS-CoV-2. This vaccine can interact appropriately with the TLR3, 5, and 8. Also, this vaccine has high quality structure and suitable characteristics such as high stability and potential for expression in Escherichia coli.","version":"1.1","doi":"10.1101/2020.04.20.051557","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.11.036855","pub_date":"2020-4-20","title":"Evaluation of heating and chemical protocols for inactivating SARS-CoV-2","abstract":"Clinical samples collected in COVID-19 patients are commonly manipulated in BSL-2 laboratories for diagnostic purpose. We used the French norm NF-EN-14476+A2 derived from the European standard EN-14885. To avoid the risk of exposure of laboratory workers, we showed that Triton-X100 must be added to guanidinium thiocyanate-lysis buffers to obtain a 6-log reduction of infectious virus. Although heating protocol consisting of 92\u00b0C-15min was more effective rather than 56\u00b0C-30min and 60\u00b0C-60min to achieve 6-log reduction, it is not amenable for molecular detection on respiratory specimens because of important decrease of detectable RNA copies in the treated sample vs untreated sample. The 56\u00b0C-30min and 60\u00b0C-60min should be used for inactivation of serum / plasma samples for serology because of the 5log10 reduction of infectivity and low viral loads in blood specimens.","version":"1.2","doi":"10.1101/2020.04.11.036855","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.20.050088","pub_date":"2020-4-20","title":"\u201cIdentification and enrichment of SECReTE cis-acting RNA elements in the Coronaviridae and other (+) single-strand RNA viruses\u201d","abstract":"cis-acting RNA motifs play a major role in regulating many aspects of RNA biology including posttranscriptional processing, nuclear export, RNA localization, translation and degradation. Here we analyzed the genomes of SARS-CoV-2 and other single-strand RNA (ssRNA) viruses for the presence of a unique cis RNA element called SECReTE. This motif consists of 10 or more consecutive triplet nucleotide repeats where a pyrimidine nucleotide (C or U) in present every third base, and which we identified in mRNAs encoding secreted proteins in bacteria, yeast, and humans. This motif facilitates mRNA localization to the endoplasmic reticulum (ER), along with the enhanced translation and secretion of translated protein. We now examined for SECReTE presence in Group IV and V RNA viruses, the former including the Coronaviridae, like SARS-CoV-2 and other positive (+)ssRNA viruses, and the latter consisting of negative (-) ssRNA viruses. Interestingly, the SARS-CoV-2 genome contains 40 SECReTE motifs at an abundance of ~1.3 SECReTEs/kilobase (kb). Moreover, all ssRNA viruses we examined contain multiple copies of this motif and appears in (+)ssRNA viruses as non-random in occurrence and independent of genome length. Importantly, (+)ssRNA viruses (e.g. Coronaviruses and Hepaciviruses), which utilize ER membranes to create double membrane vesicles to serve as viral replication centers (VRCs), contain more SECReTE motifs per kb as compared to (\u2212)ssRNA viruses (e.g. Rabies, Mumps, and Influenza), that replicate in the nucleus or the cytoplasm, or other (+)ssRNA viruses (e.g. Enteroviruses and Flaviviruses) which employ different organellar membranes. As predicted by our earlier work, SECReTE sequences are mostly found in membranal or ER-associated/secreted proteins. Thus, we propose that SECReTE motifs could be important for the efficient translation and secretion of secreted viral proteins, as well as for VRC formation. Future studies of SECReTE function and identification of SECReTE-binding proteins could provide new drug targets to treat COVID-19 and other (+)ssRNA related diseases.","version":"1.1","doi":"10.1101/2020.04.20.050088","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.041301","pub_date":"2020-4-20","title":"Phylogenetic Analysis of the Novel Coronavirus Reveals Important Variants in Indian Strains","abstract":"Recently classified as a pandemic by WHO, novel Corononavirus 2019 has affected almost every corner of the globe causing human deaths in a range of hundred thousands. The virus having its roots in Wuhan (China) has been spread over the world by its own property to change itself accordingly. These changes correspond to its transmission and pathogenicity due to which the concept of social distancing appeared into the picture. In this paper, a few findings from the whole genome sequence analysis of viral genome sequences submitted from India are presented. The data used for analysis comprises 440 collective genome sequences of virus submitted in GenBank, GISAID, and SRA projects, from around the world as well as 28 viral sequences from India. Multiple sequence alignment of all genome sequences was performed and analysed. A novel non-synonymous mutation 4809C>T (S1515F) in NSP3 gene of SARS-CoV2 Indian strains is reported along with other frequent and important changes from around the world: 3037C>T, 14408C>T, and 23403A>G. The novel change was observed in samples collected in the month of March, whereas was found to be absent in samples collected in January with the respective persons\u2019 travel history to China. Phylogenetic analysis clustered the sequences with this change as one separate clade. Mutation was predicted as stabilising change by insilco tool DynaMut. A second patient in the world to our knowledge with multiple (Wuhan and USA) strain contraction was observed in this study. The infected person is among the two early infected patients with travel history to China. Strains sequenced in Iran stood out to have different variants, as most of the reported frequent variants were not observed. The objective of this paper is to highlight the similarities and changes observed in the submitted Indian viral strains. This helps to keep track on the activity, that how virus is changing into a new subtype. Major strains observed were European with the novel change in India and other being emergent clade of Iran. Its important to observe the changes in NSP3 gene, as this gene has been reported with extensive positive selection as well as potential drug target. Extensive Positive Selection Drives the Evolution of Nonstructural Proteins. With the limited number of sequences this was the only frequent novel non-synonymous change observed from Indian strains, thereby making this change vulnerable for investigation in future. This paper has a special focus on tracking of Indian viral sequences submitted in public domain.","version":"1.2","doi":"10.1101/2020.04.14.041301","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.18.047878","pub_date":"2020-4-19","title":"Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients","abstract":"Mass spectrometry (MS) can deliver valuable diagnostic data that complements genomic information and allows us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. Our protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Image credit (left): Gerd Altmann, Pixabay License, https://pixabay.com/illustrations/corona-coronavirus-virus-covid-19-4959447","version":"1.1","doi":"10.1101/2020.04.18.047878","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.046375","pub_date":"2020-4-19","title":"Rapid development of an inactivated vaccine for SARS-CoV-2","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3\u03bcg or 6 \u03bcg per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans. A purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology","version":"1.1","doi":"10.1101/2020.04.17.046375","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.047118","pub_date":"2020-4-19","title":"Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes","abstract":"We aim to test four one-step RT real-time mastermix options for use in SARS-CoV2 real-time PCR, with three primer/probe assays targeting the N gene. The lower limit of detection is determined using a SARS CoV2 N gene RNA transcript dilution series (to 1 copy/\u00b5l) and verified using 74 nose and throat swabs. The N2 assay demonstrates the most sensitive detection of SARS-Cov-2 RNA. Three of the four mastermixes performed well, with the Takara One Step PrimeScript\u2122 III RT-PCR Kit mastermix demonstrating improved performance at the lower limit of detection.","version":"1.1","doi":"10.1101/2020.04.17.047118","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.046086","pub_date":"2020-4-19","title":"A dynamic nomenclature proposal for SARS-CoV-2 to assist genomic epidemiology","abstract":"The ongoing pandemic spread of a novel human coronavirus, SARS-COV-2, associated with severe pneumonia disease (COVID-19), has resulted in the generation of thousands of virus genome sequences. The rate of genome generation is unprecedented, yet there is currently no coherent nor accepted scheme for naming the expanding phylogenetic diversity of SARS-CoV-2. We present a rational and dynamic virus nomenclature that uses a phylogenetic framework to identify those lineages that contribute most to active spread. Our system is made tractable by constraining the number and depth of hierarchical lineage labels and by flagging and declassifying virus lineages that become unobserved and hence are likely inactive. By focusing on active virus lineages and those spreading to new locations this nomenclature will assist in tracking and understanding the patterns and determinants of the global spread of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.17.046086","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026146","pub_date":"2020-4-19","title":"CovProfile: profiling the viral genome and gene expressions of SARS-COV-2","abstract":"The SARS-CoV-2 virus has infected more than one million people worldwide to date. Knowing its genome and gene expressions is essential to understand the virus\u2019 mechanism. Here, we propose a computational tool CovProfile to detect the viral genomic variations as well as viral gene expressions from the sequences obtained from Nanopore devices. We applied CovProfile to 11 samples, each from a terminally ill patient, and discovered that all the patients are infected by multiple viral strains, which might affect the reliability of phylogenetic analysis. Moreover, the expression of viral genes ORF1ab gene, S gene, M gene, and N gene are high among most of the samples. While performing the tests, we noticed a consistent abundance of transcript segments of MUC5B, presumably from the host, across all the samples.","version":"1.2","doi":"10.1101/2020.04.05.026146","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.047324","pub_date":"2020-4-19","title":"Exploring Conformational Transition of 2019 Novel Coronavirus Spike Glycoprotein Between Its Closed and Open States Using Molecular Dynamics Simulations","abstract":"Since its first recorded appearance in December 2019, a novel coronavirus (SARS-CoV-2) causing the disease COVID-19 has resulted in more than 2,000,000 infections and 128,000 deaths. Currently there is no proven treatment for COVID-19 and there is an urgent need for the development of vaccines and therapeutics. Coronavirus spike glycoproteins play a critical role in coronavirus entry into the host cells, as they provide host cell recognition and membrane fusion between virus and host cell. Thus, they emerged as popular and promising drug targets. Crystal structures of spike protein in its closed and open states were resolved very recently in March 2020. These structures comprise 77% of the sequence and provide almost the complete protein structure. Based on down and up positions of receptor binding domain (RBD), spike protein can be in a receptor inaccessible closed or receptor accessible open state, respectively. Starting from closed and open state crystal structures, and also 16 intermediate conformations, an extensive set of all-atom molecular dynamics (MD) simulations in the presence of explicit water and ions were performed. Simulations show that in its down position, RBD has significantly lower mobility compared to its up position; probably caused by the 6 interdomain salt bridges of RBD in down position compared to 3 in up position. Free energy landscapes based on MD simulations revealed a semi-open state located between closed and open states. Minimum energy pathway between down and up positions comprised a gradual salt bridge switching mechanism. Furthermore, although significantly lower than open state, ACE2 binding surface of RBD contained a partial solvent accessibility in its closed state.","version":"1.1","doi":"10.1101/2020.04.17.047324","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.045302","pub_date":"2020-4-18","title":"Broad Host Range of SARS-CoV-2 Predicted by Comparative and Structural Analysis of ACE2 in Vertebrates","abstract":"The novel coronavirus SARS-CoV-2 is the cause of Coronavirus Disease-2019 (COVID-19). The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of 410 vertebrates, including 252 mammals, to study cross-species conservation of ACE2 and its likelihood to function as a SARS-CoV-2 receptor. We designed a five-category ranking score based on the conservation properties of 25 amino acids important for the binding between receptor and virus, classifying all species from very high to very low. Only mammals fell into the medium to very high categories, and only catarrhine primates in the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 binding, and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (<0.1%) variants in 10/25 binding sites. In addition, we observed evidence of positive selection in ACE2 in multiple species, including bats. Utilized appropriately, our results may lead to the identification of intermediate host species for SARS-CoV-2, justify the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.","version":"1.1","doi":"10.1101/2020.04.16.045302","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.045799","pub_date":"2020-4-18","title":"Potential microenvironment of SARS-CoV-2 infection in airway epithelial cells revealed by Human Protein Atlas database analysis","abstract":"The outbreak of COVID-19 has caused serious epidemic events in China and other countries. With the rapid spread of COVID-19, it is urgent to explore the pathogenesis of this novel coronavirus. However, the foundational research of COVID-19 is very weak. Although angiotensin converting enzyme 2 (ACE2) is the reported receptor of SARS-CoV-2, information about SARS-CoV-2 invading airway epithelial cells is very limited. Based on the analysis of the Human Protein Atlas database, we compared the virus-related receptors of epithelial-derived cells from different organs and found potential key molecules in the local microenvironment for SARS-CoV-2 entering airway epithelial cells. In addition, we found that these proteins were associated with virus reactive proteins in host airway epithelial cells, which may promote the activation of the immune system and the release of inflammatory factors. Our findings provide a new research direction for understanding the potential microenvironment required by SARS-CoV-2 infection in airway epithelial, which may assist in the discovery of potential drug targets against SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.04.16.045799","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.046185","pub_date":"2020-4-18","title":"Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies","abstract":"The recent severe acute respiratory syndrome, known as Corona Virus Disease 2019 (COVID-19) has spread so much rapidly and severely to induce World Health Organization (WHO) to declare state of emergency over the new coronavirus SARS-CoV-2 pandemic. While several countries have chosen the almost complete lock-down for slowing down SARS-CoV-2 spread, scientific community is called to respond to the devastating outbreak by identifying new tools for diagnosis and treatment of the dangerous COVID-19. With this aim we performed an in silico comparative modeling analysis, which allows to gain new insights about the main conformational changes occurring in the SARS-CoV-2 spike protein, at the level of the receptor binding domain (RBD), along interactions with human cells angiotensin converting enzyme 2 (ACE2) receptor, that favour human cell invasion. Furthermore, our analysis provides i) an ideal pipeline to identify already characterized antibodies that might target SARS-CoV-2 spike RBD, for preventing interactions with the human ACE2, and ii) instructions for building new possible neutralizing antibodies, according to chemical/physical space restraints and complementary determining regions (CDR) mutagenesis of the identified existing antibodies. The proposed antibodies show in silico a high affinity for SARS-CoV-2 spike RBD and can be used as reference antibodies also for building new high affinity antibodies against present and future coronavirus able to invade human cells through interactions of their spike proteins with the human ACE2. More in general, our analysis provides indications for the set-up of the right biological molecular context for investigating spike RBD-ACE2 interactions for the development of new vaccines, diagnosis kits and other treatments based on the usage or the targeting of SARS-CoV-2 spike protein.","version":"1.1","doi":"10.1101/2020.04.17.046185","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.047548","pub_date":"2020-4-18","title":"Distinct Structural Flexibility within SARS-CoV-2 Spike Protein Reveals Potential Therapeutic Targets","abstract":"The emergence and rapid worldwide spread of the novel coronavirus disease, COVID-19, has prompted concerted efforts to find successful treatments. The causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its spike (S) protein to gain entry into host cells. Therefore, the S protein presents a viable target to develop a directed therapy. Here, we deployed an integrated artificial intelligence with molecular dynamics simulation approach to provide new details of the S protein structure. Based on a comprehensive structural analysis of S proteins from SARS-CoV-2 and previous human coronaviruses, we found that the protomer state of S proteins is structurally flexible. Without the presence of a stabilizing beta sheet from another protomer chain, two regions in the S2 domain and the hinge connecting the S1 and S2 subunits lose their secondary structures. Interestingly, the region in the S2 domain was previously identified as an immunodominant site in the SARS-CoV-1 S protein. We anticipate that the molecular details elucidated here will assist in effective therapeutic development for COVID-19.","version":"1.1","doi":"10.1101/2020.04.17.047548","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.045161","pub_date":"2020-4-18","title":"An in silico map of the SARS-CoV-2 RNA Structurome","abstract":"SARS-CoV-2 is a positive-sense single-stranded RNA virus that has exploded throughout the global human population. This pandemic coronavirus strain has taken scientists and public health researchers by surprise and knowledge of its basic biology (e.g. structure/function relationships in its genomic, messenger and template RNAs) and modes for therapeutic intervention lag behind that of other human pathogens. In this report we used a recently-developed bioinformatics approach, ScanFold, to deduce the RNA structural landscape of the SARS-CoV-2 transcriptome. We recapitulate known elements of RNA structure and provide a model for the folding of an essential frameshift signal. Our results find that the SARS-CoV-2 is greatly enriched in unusually stable and likely evolutionarily ordered RNA structure, which provides a huge reservoir of potential drug targets for RNA-binding small molecules. Our results also predict regions that are accessible for intermolecular interactions, which can aid in the design of antisense therapeutics. All results are made available via a public database (the RNAStructuromeDB) where they may hopefully drive drug discovery efforts to inhibit SARS-CoV-2 pathogenesis.","version":"1.1","doi":"10.1101/2020.04.17.045161","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.046938","pub_date":"2020-4-18","title":"Regulation of angiotensin converting enzyme 2 (ACE2) in obesity: implications for COVID-19","abstract":"The ongoing COVID-19 pandemic is caused by the novel coronavirus SARS-CoV-2. Age, smoking, obesity, and chronic diseases such as cardiovascular disease and diabetes have been described as risk factors for severe complications and mortality in COVID-19. Obesity and diabetes are usually associated with dysregulated lipid synthesis and clearance which can initiate or aggravate pulmonary inflammation and injury. It has been shown that for viral entry into the host cell, SARS-CoV-2 utilizes the angiotensin converting enzyme 2 (ACE2) receptors present on the cells. We aimed to characterize how SARS-CoV-2 dysregulates lipid metabolism pathways in the host and the effect of dysregulated lipogenesis on the regulation of ACE2, specifically in obesity. In our study, through the re-analysis of publicly available transcriptomic data, we first found that lung epithelial cells infected with SARS-CoV-2 showed upregulation of genes associated with lipid metabolism, including the SOC3 gene which is involved in regulation of inflammation and inhibition of leptin signaling. This is of interest as viruses may hijack host lipid metabolism to allow completion of their viral replication cycles. Furthermore, a mouse model of diet-induced obesity showed a significant increase in Ace2 expression in the lungs which negatively correlated with the expression of genes that code for sterol response element binding proteins 1 and 2 (SREBP). Suppression of Srebp1 showed a significant increase in Ace2 expression in the lung. Together our results suggest that the dysregulated lipogenesis and the subsequently high ACE2 expression in obese patients might be the mechanism underlying the increased risk for severe complications in those patients when infected by SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.17.046938","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.045690","pub_date":"2020-4-18","title":"The scRNA-seq expression profiling of the receptor ACE2 and the cellular protease TMPRSS2 reveals human organs susceptible to COVID-19 infection","abstract":"COVID-19 caused by SARA-CoV-2 is a disaster sweeping over 200 countries, and more than 2,150,000 people are suffering from the disease and 140,000 people died. ACE2 is a receptor protein of SARS- CoV-2, and TMPRSS2 promotes virus proliferation and transmission. Some patients developed multiple organ dysfunction syndromes other than lungs. Therefore, studying the viral susceptibility of other organs is important for a deeper understanding of viral pathogenesis. The advantage of scRNA-seq data is the identification of cell types by clustering the gene expression of cells. ACE2 and TMPRSS2 are highly expressed in AT2 of lungs, we compared the ACE2 and TMPRSS2 expression levels of cell types from 31 organs, with AT2 of lungs to evaluate the risk of the viral infection using scRNA-seq data. For the first time, we found the brain, gall bladder, and fallopian tube are vulnerable to COVID-19 infection. Besides, the nose, heart, small intestine, large intestine, esophagus, testis and kidney are also identified to be high-risk organs with high expression levels of ACE2 and TMPRSS2. Moreover, the susceptible organs are grouped into three risk levels based on the TMPRSS2 expression. As a result, the respiratory system, digestive system and reproductive system are at the top-risk level to COVID-19 infection. This study provides evidence for COVID-19 infection in the human nervous system, digestive system, reproductive system, respiratory system, circulatory system and urinary system using scRNA-seq data, which helps for the clinical diagnosis and treatment of patients.","version":"1.1","doi":"10.1101/2020.04.16.045690","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.045054","pub_date":"2020-4-18","title":"Immune modulation to improve survival of respiratory virus infections in mice","abstract":"Viral pneumonia remains a global health threat requiring novel treatment strategies, as strikingly exemplified in the SARS-CoV-2 pandemic of 2019-2020. We have reported that mice treated with a combination of inhaled Toll-like receptor (TLR) 2/6 and TLR 9 agonists (Pam2-ODN) to stimulate innate immunity are broadly protected against respiratory pathogens, but the mechanisms underlying this protection remain incompletely elucidated. Here, we show in a lethal paramyxovirus model that Pam2-ODN-enhanced survival is associated with robust virus inactivation by reactive oxygen species (ROS), which occurs prior to internalization by lung epithelial cells. However, we also found that mortality in sham-treated mice temporally corresponded with CD8+ T cell-enriched lung inflammation that peaks on days 11-12 after viral challenge, when the viral burden has waned to a scarcely detectable level. Pam2-ODN treatment blocked this injurious inflammation by reducing the viral burden, and alternatively, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. These findings reveal opportunities for targeted immunomodulation to protect susceptible individuals against the morbidity and mortality of respiratory viral infections.","version":"1.1","doi":"10.1101/2020.04.16.045054","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.18.045963","pub_date":"2020-4-18","title":"Pandemic Publishing: Medical journals drastically speed up their publication process for Covid-19","abstract":"In times of public crises, including the current Covid-19 pandemic, rapid dissemination of relevant scientific knowledge is of paramount importance. The duration of scholarly journals\u2019 publication process is one of the main factors hindering quick delivery of new information. While proper editorial assessment and peer review obviously require some time, turnaround times for medical journals can be up to several months, which is undesirable in the era of a crisis. Following initiatives of medical journals and scholarly publishers to accelerate their publication process, this study assesses whether medical journals have indeed managed to speed up their publication process for Covid-19 related articles. It studies the duration of 14 medical journals\u2019 publication process both during and prior to the current pandemic. Assessing a total of 669 articles, the study concludes that medical journals have indeed drastically accelerated the publication process for Covid-19 related articles since the outbreak of the pandemic. Compared to articles published in the same journals before the pandemic, turnaround times have decreased on average by 49%. The largest decrease in number of days between submission and publication of articles was due to a decrease in the number of days required for peer review. For articles not related to Covid-19, no acceleration of the publication process is found. While the acceleration of journals\u2019 publication process is laudable from the perspective of quick information dissemination, it also raises concerns relating to the quality of the peer review process and the quality of the resulting publications.","version":"1.1","doi":"10.1101/2020.04.18.045963","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.041319","pub_date":"2020-4-17","title":"A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities","abstract":"The COVID-19 pandemic is expanding at an unprecedented rate. As a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. Academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. Here, we describe the process of developing a SARS-Cov2 diagnostic workflow in a conventional academic Containment Level 2 (CL2) laboratory. Our outline includes simple SARS-Cov2 deactivation upon contact, the methods for a quantitative real-time reverse transcriptase PCR (qRT-PCR) detecting SARS-Cov2, a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. This was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. Within 14 days, we created a validated COVID-19 diagnostics service for healthcare workers in our local hospital. The described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame.","version":"1.1","doi":"10.1101/2020.04.14.041319","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.044016","pub_date":"2020-4-17","title":"A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals","abstract":"The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.","version":"1.1","doi":"10.1101/2020.04.16.044016","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.044503","pub_date":"2020-4-17","title":"SARS-CoV-2 is transmitted via contact and via the air between ferrets","abstract":"SARS-CoV-2, a coronavirus that newly emerged in China in late 2019  and spread rapidly worldwide, caused the first witnessed pandemic sparked by a coronavirus. As the pandemic progresses, information about the modes of transmission of SARS-CoV-2 among humans is critical to apply appropriate infection control measures and to slow its spread. Here we show that SARS-CoV-2 is transmitted efficiently via direct contact and via the air (via respiratory droplets and/or aerosols) between ferrets. Intranasal inoculation of donor ferrets resulted in a productive upper respiratory tract infection and long-term shedding, up to 11 to 19 days post-inoculation. SARS-CoV-2 transmitted to four out of four direct contact ferrets between 1 and 3 days after exposure and via the air to three out of four independent indirect recipient ferrets between 3 and 7 days after exposure. The pattern of virus shedding in the direct contact and indirect recipient ferrets was similar to that of the inoculated ferrets and infectious virus was isolated from all positive animals, showing that ferrets were productively infected via either route. This study provides experimental evidence of robust transmission of SARS-CoV-2 via the air, supporting the implementation of community-level social distancing measures currently applied in many countries in the world and informing decisions on infection control measures in healthcare settings .","version":"1.1","doi":"10.1101/2020.04.16.044503","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.17.046193","pub_date":"2020-4-17","title":"Shotgun proteomics of SARS-CoV-2 infected cells and its application to the optimisation of whole viral particle antigen production for vaccines","abstract":"Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread quickly around the globe. Currently, no effective vaccine is available to prevent COVID-19 and an intense global development activity is in progress. In this context, the different technology platforms face several challenges resulting from the involvement of a new virus still not fully characterised. Finding of the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles is among them. Here, we describe the establishment of a workflow based on shotgun tandem mass spectrometry data to guide the optimisation of the conditions for viral amplification. In parallel, we analysed the dynamic of the host cell proteome following SARS-CoV-2 infection providing a global overview of biological processes modulated by the virus and that could be further explored to identify drug targets to address the pandemic.","version":"1.1","doi":"10.1101/2020.04.17.046193","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.16.045617","pub_date":"2020-4-17","title":"Single-cell analysis of human lung epithelia reveals concomitant expression of the SARS-CoV-2 receptor ACE2 with multiple virus receptors and scavengers in alveolar type II cells","abstract":"The novel coronavirus SARS-CoV-2 was identified as the causative agent of the ongoing pandemic COVID 19. COVID-19-associated deaths are mainly attributed to severe pneumonia and respiratory failure. Recent work demonstrated that SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE2) in the lung. To better understand ACE2 abundance and expression patterns in the lung we interrogated our in-house single-cell RNA-sequencing dataset containing 70,085 EPCAM+ lung epithelial cells from paired normal and lung adenocarcinoma tissues. Transcriptomic analysis revealed a diverse repertoire of airway lineages that included alveolar type I and II, bronchioalveolar, club/secretory, quiescent and proliferating basal, ciliated and malignant cells as well as rare populations such as ionocytes. While the fraction of lung epithelial cells expressing ACE2 was low (1.7% overall), alveolar type II (AT2, 2.2% ACE2+) cells exhibited highest levels of ACE2 expression among all cell subsets. Further analysis of the AT2 compartment (n = 27,235 cells) revealed a number of genes co-expressed with ACE2 that are important for lung pathobiology including those associated with chronic obstructive pulmonary disease (COPD; HHIP), pneumonia and infection (FGG and C4BPA) as well as malarial/bacterial (CD36) and viral (DMBT1) scavenging which, for the most part, were increased in smoker versus light or non-smoker cells. Notably, DMBT1 was highly expressed in AT2 cells relative to other lung epithelial subsets and its expression positively correlated with ACE2. We describe a population of ACE2-positive AT2 cells that co-express pathogen (including viral) receptors (e.g. DMBT1) with crucial roles in host defense thus comprising plausible phenotypic targets for treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.04.16.045617","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.006213","pub_date":"2020-4-16","title":"Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Viruses Reveal Mosaic Pattern of Phylogeographical Distribution","abstract":"The Coronavirus Disease-2019 (COVID-19) that started in Wuhan, China in December 2019 has spread worldwide emerging as a global pandemic. The severe respiratory pneumonia caused by the novel SARS-CoV-2 has so far claimed more than 60,000 lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 displays high transmission rates, their underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries suggesting that the outbreak is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nsp13-16 (ORF1b polyprotein) and S-Protein within 10 viral isolates from the USA. These viral proteins are involved in RNA replication, indicating highly evolved viral strains circulating in the population of USA than other countries. Furthermore, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of the USA isolate (MT188341) leading to shift in amino acid frame from position 2540 onwards. Through the construction of SARS-CoV-2-human interactome, we further revealed that multiple host proteins (PHB, PPP1CA, TGF-\u03b2, SOCS3, STAT3, JAK1/2, SMAD3, BCL2, CAV1 & SPECC1) are manipulated by the viral proteins (nsp2, PL-PRO, N-protein, ORF7a, M-S-ORF3a complex, nsp7-nsp8-nsp9-RdRp complex) for mediating host immune evasion. Thus, the replicative machinery of SARS-CoV-2 is fast evolving to evade host challenges which need to be considered for developing effective treatment strategies.","version":"1.2","doi":"10.1101/2020.03.25.006213","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029017","pub_date":"2020-4-16","title":"Currently available intravenous immunoglobulin (Gamunex\u00ae-C and Flebogamma\u00ae DIF) contains antibodies reacting against SARS-CoV-2 antigens","abstract":"There is a critical need for effective therapies that are immediately available to control the spread of COVID-19 disease. In this study, we assessed currently marketed intravenous immunoglobulin (IVIG) products for antibodies against human common coronaviruses that may cross-react with the SARS-CoV-2 virus. Gamunex\u00ae-C and Flebogamma\u00ae DIF (Grifols) IVIG were tested against several betacoronaviruses antigens using ELISA techniques: HCoV (undetermined antigen), HCoV-HKU1 (N protein), SARS-CoV (culture lysate), MERS-CoV (N protein; S1 protein/RBD; S protein), and SARS-CoV-2 (S1 protein). Both IVIG products showed consistent reactivity to components of the tested viruses. Positive cross-reactivity was seen in SARS-CoV, MERS-CoV, and SARS-CoV-2. For SARS-CoV-2, positive reactivity was observed at IVIG concentrations ranging from 100 \u03bcg/mL with Gamunex-C to 1 mg/mL with Flebogamma 5% DIF. Gamunex-C and Flebogamma DIF IVIG contain antibodies reacting against SARS-CoV-2 antigens. These preparations may be useful for immediate treatment of COVID-19 disease.","version":"1.2","doi":"10.1101/2020.04.07.029017","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.019299","pub_date":"2020-4-16","title":"Structural basis to design multi-epitope vaccines against Novel Coronavirus 19 (COVID19) infection, the ongoing pandemic emergency: an in silico approach","abstract":"The 2019 novel coronavirus (COVID19 / Wuhan coronavirus), officially named as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a positive-sense single-stranded RNA coronavirus. SARS-CoV-2 causes the contagious COVID19 disease also known as 2019-nCoV acute respiratory disease and has led to the ongoing 2019\u201320 pandemic COVID19 outbreak. The effective counter measures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidate. In the present study, we have screened and shortlisted 38 CTL, 33 HTL and 12 B cell epitopes from the eleven Protein sequences of SARS-CoV-2 by utilizing different in silico tools. The screened epitopes were further validated for their binding with their respective HLA allele binders and TAP (Transporter associated with antigen processing) molecule by molecular docking. The shortlisted screened epitopes were further utilized to design novel two multi-epitope vaccines (MEVs) composed of CTL, HTL and B cell epitopes overlaps with potential to elicit humoral as well as cellular immune response against SARS-CoV-2. To enhance the immune response for our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has been utilized as an adjuvant at N terminal of both the MEVs. Further molecular models for both the MEVs were prepared and validated for their stable molecular interactions with Toll-Like Receptor 3 (TLR 3). The codon-optimized cDNA of both the MEVs were further analyzed for their potential of high level of expression in a human cell line. The present study is very significant in terms of molecular designing of prospective CTL and HTL vaccine against SARS-CoV-2 infection with the potential to elicit cellular as well as humoral immune response. (SARS-CoV-2), Coronavirus, Human Transporter associated with antigen processing (TAP), Toll-Like Receptor (TLR), Epitope, Immunoinformatics, Molecular Docking, Molecular dynamics simulation, Multi-epitope Vaccine The designed CTL (Cytotoxic T lymphocyte) and HTL (Helper T lymphocyte) multi-epitope vaccines (MEV) against COVID19 infection. Both the CTL and HTL MEV models show a very stable and well fit conformational complex formation tendency with the Toll like receptor 3. CTL and HTL MEVs: ribbon; Toll like receptor 3: gray cartoon; Adjuvant [truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1]: orange ribbon regions; Epitopes: cyan ribbons regions; 6xHis Tag: magenta ribbon regions.","version":"1.2","doi":"10.1101/2020.04.01.019299","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.036020","pub_date":"2020-4-16","title":"Analysis of Ten Microsecond simulation data of SARS-CoV-2 dimeric main protease","abstract":"The authors have withdrawn this manuscript because of a violation of research ethics. Unknown to the first author, the corresponding author - the main designer of the project - did not obtain consent for the use of a data-set. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author\u201d","version":"1.2","doi":"10.1101/2020.04.10.036020","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030684","pub_date":"2020-4-15","title":"The aging transcriptome and cellular landscape of the human lung in relation to SARS-CoV-2","abstract":"Since the emergence of SARS-CoV-2 in December 2019, Coronavirus Disease-2019 (COVID-19) has rapidly spread across the globe. Epidemiologic studies have demonstrated that age is one of the strongest risk factors influencing the morbidity and mortality of COVID-19. Here, we interrogate the transcriptional features and cellular landscapes of the aging human lung through integrative analysis of bulk and single-cell transcriptomics. By intersecting these age-associated changes with experimental data on host interactions between SARS-CoV-2 or its relative SARS-CoV, we identify several age-associated factors that may contribute to the heightened severity of COVID-19 in older populations. We observed that age-associated gene expression and cell populations are significantly linked to the heightened severity of COVID-19 in older populations. The aging lung is characterized by increased vascular smooth muscle contraction, reduced mitochondrial activity, and decreased lipid metabolism. Lung epithelial cells, macrophages, and Th1 cells decrease in abundance with age, whereas fibroblasts, pericytes and CD4+ Tcm cells increase in abundance with age. Several age-associated genes have functional effects on SARS-CoV replication, and directly interact with the SARS-CoV-2 proteome. Interestingly, age-associated genes are heavily enriched among those induced or suppressed by SARS-CoV-2 infection. These analyses illuminate potential avenues for further studies on the relationship between the aging lung and COVID-19 pathogenesis, which may inform strategies to more effectively treat this disease.","version":"1.2","doi":"10.1101/2020.04.07.030684","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.15.042085","pub_date":"2020-4-15","title":"TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets","abstract":"In December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines. In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2\u2019 site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.04.15.042085","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.041459","pub_date":"2020-4-15","title":"Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry","abstract":"Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 \u03bcM, 0.12 \u03bcM, and 26.4 \u03bcM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.","version":"1.1","doi":"10.1101/2020.04.14.041459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.15.040618","pub_date":"2020-4-15","title":"Designing a multi-epitope peptide-based vaccine against SARS-CoV-2","abstract":"COVID-19 pandemic has resulted so far 14,395,16 confirmed cases with 85,711 deaths from the 212 countries, or territories. Due to multifacet issues and challenges in implementation of the safety & preventive measures, inconsistent coordination between societies-governments and most importanly lack of specific vaccine to SARS-CoV-2, the spread of Wuhan originated virus is still uprising after taking a heavy toll on human life. In the present study, we mapped several immunogenic epitopes (B-cell, T-cell, and IFN-gamma) over the entire structural proteins of SARS-CoV-2 and by applying various computational and immunoinformatics approaches, we designed a multi-epitope peptide based vaccine that predicted high immunogenic response in the largest proportion of world\u2019s human population. To ensure high expression of the recombinant vaccine in E. coli, codon optimization and in-silico cloning were also carried out. The designed vaccine with high molecular affinity to TLR3 and TLR4, was found capable to initiate effective innate and adaptive immune response. The immune simulation also suggested uprising high levels of both B-cell and T-cell mediated immunity which on subsequent exposure cleared antigen from the system. The proposed vaccine found promising by yielding desired results and hence, should be tested by practical experimentations for its functioning and efficacy to neutralize SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.15.040618","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.15.036285","pub_date":"2020-4-15","title":"Genome based Evolutionary study of SARS-CoV-2 towards the Prediction of Epitope Based Chimeric Vaccine","abstract":"SARS-CoV-2 is known to infect the neurological, respiratory, enteric, and hepatic systems of human and has already become an unprecedented threat to global healthcare system. COVID-19, the most serious public condition caused by SARS-CoV-2 leads the world to an uncertainty alongside thousands of regular death scenes. Unavailability of specific therapeutics or approved vaccine has made the recovery of COVI-19 more troublesome and challenging. The present in silico study aimed to predict a novel chimeric vaccines by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein (S), membrane protein (M), envelope protein and nucleocapsid protein (N) were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins (S, E, M and N) reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and Normal Mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. The development of preventive measures to combat COVID-19 infections might be aided the present study. However, the in vivo and in vitro validation might be ensured with wet lab trials using model animals for the implementation of the presented data.","version":"1.1","doi":"10.1101/2020.04.15.036285","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.039925","pub_date":"2020-4-15","title":"Multidrug treatment with nelfinavir and cepharanthine against COVID-19","abstract":"Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.","version":"1.1","doi":"10.1101/2020.04.14.039925","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.042010","pub_date":"2020-4-15","title":"Humanized Single Domain Antibodies Neutralize SARS-CoV-2 by Targeting Spike Receptor Binding Domain","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across more than 200 countries and regions, leading to an unprecedented medical burden and live lost. SARS-CoV-2 specific antivirals or prophylactic vaccines are not available. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Using SARS-CoV-2 spike RBD as a bait, we have discovered a panel of humanized single domain antibodies (sdAbs). These sdAbs revealed binding kinetics with the equilibrium dissociation constant (KD) of 0.7~33 nM. The monomeric sdAbs showed half maximal inhibitory concentration (IC50) of 0.003~0.3 \u03bcg/mL in pseudotyped particle neutralization assay, and 0.23~0.50 \u03bcg/mL in authentic SARS-CoV-2 neutralization assay. Competitive ligand-binding data suggested that the sdAbs either completely blocked or significantly inhibited the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Finally, we showed that fusion of the human IgG1 Fc to sdAbs improved their neutralization activity by tens of times. These results reveal the novel SARS-CoV-2 RBD targeting sdAbs and pave a road for antibody drug development.","version":"1.1","doi":"10.1101/2020.04.14.042010","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.15.997254","pub_date":"2020-4-15","title":"Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an acute threat to public health and the world economy, especially because no approved specific drugs or vaccines are available. Pharmacological modulation of metabolism-dependent cellular pathways such as autophagy reduced propagation of highly pathogenic Middle East respiratory syndrome (MERS)-CoV. Here we show that SARS-CoV-2 infection limits autophagy by interfering with multiple metabolic pathways and that compound-driven interventions aimed at autophagy induction reduce SARS-CoV-2 propagation in vitro. In-depth analyses of autophagy signaling and metabolomics indicate that SARS-CoV-2 reduces glycolysis and protein translation by limiting activation of AMP-protein activated kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1). Infection also downregulates autophagy-inducing spermidine, and facilitates AKT1/SKP2-dependent degradation of autophagy-initiating Beclin-1 (BECN1). Targeting of these pathways by exogenous administration of spermidine, AKT inhibitor MK-2206, and the Beclin-1 stabilizing, antihelminthic drug niclosamide inhibited SARS-CoV-2 propagation by 85, 88, and >99%, respectively. In sum, SARS-CoV-2 infection causally diminishes autophagy. A clinically approved and well-tolerated autophagy-inducing compound shows potential for evaluation as a treatment against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.15.997254","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.04.025080","pub_date":"2020-4-15","title":"Computational analysis suggests putative intermediate animal hosts of the SARS-CoV-2","abstract":"The authors have withdrawn their manuscript whilst they wish to perform additional experiments to validate their conclusions further. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author for more details.","version":"1.2","doi":"10.1101/2020.04.04.025080","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.039263","pub_date":"2020-4-14","title":"Comparative in vitro transcriptomic analyses of COVID-19 candidate therapy hydroxychloroquine suggest limited immunomodulatory evidence of SARS-CoV-2 host response genes","abstract":"Hydroxychloroquine (HCQ) has emerged as a potential and controversial antiviral candidate therapy for COVID-19. While many clinical trials are underway to test the efficacy of HCQ as a treatment for COVID-19, underlying mechanisms of HCQ in the setting of COVID-19 remain unclear. Hence, we examined differential gene expression signatures of HCQ exposure, in vitro SARS-CoV-2 infection, and host signatures of COVID-19 in blood, bronchoalveolar lavage, and postmortem lung to evaluate whether HCQ transcriptome signatures associate with restoration of SARS-CoV-2-related host transcriptional responses. Here, we show that 24 hours of in vitro treatment of peripheral blood mononuclear cells(PBMC) with HCQ significantly impacted transcription of 16 genes involved in immune regulation and lipid metabolism. Using transcriptome data from in vitro SARS-CoV-2 infected NHBE and A549 cells and PBMC derived from confirmed COVID-19 infected patients, we determined that only 0.24% of the COVID-19 PBMC differentially expressed gene set and 0.39% of the in vitro SARS-CoV-2 cells differentially expressed gene set overlapped with HCQ-related differentially expressed genes. Moreover, we observed that HCQ treatment significantly impacted transcription of 159 genes in human primary monocyte-derived macrophages involved in cholesterol biosynthetic process and chemokine activity. Notably, when we compared the macrophage HCQ-related gene lists with genes transcriptionally altered during SARS-CoV-2 infection and in bronchoalveolar lavage of COVID-19+ patients, the CXCL6 gene was impacted in all three transcriptional signatures revealing evidence in favor of chemokine modulation. HCQ-related transcriptional changes minimally overlapped with host genes altered in postmortem lung biopsies from COVID-19 participants. These results may provide insight into the immunomodulation mechanisms of HCQ treatment in the setting of COVID-19 and suggest HCQ is not a panacea to SARS-CoV-2 infection.","version":"1.1","doi":"10.1101/2020.04.13.039263","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.12.038554","pub_date":"2020-4-14","title":"The origin and underlying driving forces of the SARS-CoV-2 outbreak","abstract":"The spread of SARS-CoV-2 since December 2019 has become a pandemic and impacted many aspects of human society. Here, we analyzed genetic variation of SARS-CoV-2 and its related coronavirus and found the evidence of intergenomic recombination. After correction for mutational bias, analysis of 137 SARS-CoV-2 genomes as of 2/23/2020 revealed the excess of low frequency mutations on both synonymous and nonsynonymous sites which is consistent with recent origin of the virus. In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China was two time higher than those derived from the rest of the world. In addition, in network analysis, haplotypes collected from Wuhan city were at interior and have more mutational connections, both of which are consistent with the observation that the outbreak of cov-19 was originated from China. In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated.","version":"1.1","doi":"10.1101/2020.04.12.038554","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.038687","pub_date":"2020-4-14","title":"Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro","abstract":"Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a \u201cshield\u201d in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.","version":"1.1","doi":"10.1101/2020.04.13.038687","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.027698","pub_date":"2020-4-14","title":"Genetic variability in the expression of the SARS-CoV-2 host cell entry factors across populations","abstract":"The entry of SARS-CoV-2 into host cells is dependent upon angiotensin-converting enzyme 2 (ACE2), which serves as a functional attachment receptor for the viral spike glycoprotein, and the serine protease TMPRSS2 which allows fusion of the viral and host cell membranes. We devised a quantitative measure to estimate genetic determinants of ACE2 and TMPRSS2 expression and applied this measure to >2,500 individuals. Our data show significant variability in genetic determinants of ACE2 and TMPRSS2 expression among individuals and between populations, and demonstrate a genetic predisposition for lower expression levels of both key viral entry genes in African populations. These data suggest that genetic factors might lead to lower susceptibility for SARS-CoV-2 infection in African populations and that host genetics might help explain inter-individual variability in disease susceptibility and severity of COVID-19.","version":"1.1","doi":"10.1101/2020.04.06.027698","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.036343","pub_date":"2020-4-14","title":"CoV Genome Tracker: tracing genomic footprints of Covid-19 pandemic","abstract":"Genome sequences constitute the primary evidence on the origin and spread of the 2019-2020 Covid-19 pandemic. Rapid comparative analysis of coronavirus SARS-CoV-2 genomes is critical for disease control, outbreak forecasting, and developing clinical interventions. CoV Genome Tracker is a web portal dedicated to trace Covid-19 outbreaks in real time using a haplotype network, an accurate and scalable representation of genomic changes in a rapidly evolving population. We resolve the direction of mutations by using a bat-associated genome as outgroup. At a broader evolutionary time scale, a companion browser provides gene-by-gene and codon-by-codon evolutionary rates to facilitate the search for molecular targets of clinical interventions. CoV Genome Tracker is publicly available at http://cov.genometracker.org and updated weekly with the data downloaded from GISAID (http://gisaid.org). The website is implemented with a custom JavaScript script based on jQuery (https://jquery.com) and D3-force (https://github.com/d3/d3-force). weigang@genectr.hunter.cuny.edu, City University of New York, Hunter College All supporting scripts developed in JavaScript, Python, BASH, and PERL programming languages are available as Open Source at the GitHub repository https://github.com/weigangq/cov-browser.","version":"1.1","doi":"10.1101/2020.04.10.036343","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026633","pub_date":"2020-4-14","title":"ACE 2 Coding Variants: A Potential X-linked Risk Factor for COVID-19 Disease","abstract":"Viral genetic variants are widely known to influence disease progression among infected humans. Given the recent and rapid emergence of pandemic SARS-CoV-2 infection, the cause of COVID-19 disease, viral protein variants have attracted research interest. However, little has yet been written about genetic risk factors among human hosts. Human genetic variation has proven to affect disease progression and outcome for important diseases such as HIV infection and malaria infestation. The fact that the human ACE2 protein is encoded on the X chromosome means that males who carry rare ACE2 coding variants will express those variants in all ACE2-expressing cells, whereas females will typically express those variants in a mosaic distribution determined by early X-inactivation events. This sex-based difference in ACE2 expression has unique implications for epidemiological studies designed to assess host genetic factors influencing progression from asymptomatic SARS-coV-2 infection to COVID-19. Here we present theoretical modelling of rare ACE2 coding variants documented to occur naturally in several human superpopulations and subpopulations, and show that rare variants predicted to affect the binding of ACE2 to the SARS-CoV-2 spike protein exist in people. Though the rs4646116 (p.Lys26Arg) allele is found in 1 in 70 Ashkenazi Jewish males, and in 1 in 172 non-Finnish European males, this allele is found at higher frequencies in females. Furthermore, the class of missense ACE2 alleles predicted to affect SARS-CoV-2 binding are found in aggregate among 1.43% and 2.16% of Ashkenazi males and females, respectively, as well as in 0.58% and 1.24% of European males and females outside of Finland. These alleles are rarer in other population groups, and almost absent from East Asians genotyped to date. Though we are aware that full genome-wide and exome-wide sequencing studies may ultimately be required to assess human genetic susceptibility to SARS-CoV-2 fully, we argue on the basis of strong prior probabilities that genotyping of this class of alleles is justified in cases of atypical SARS-CoV-2 diseases, such as asymptomatic super-spreaders (if any are identified), and in neonatal/paediatric-onset COVID-19 disease. Even relatively rare susceptibility factors (1% or fewer carriers) may become quantitatively important in the context of hundreds of thousands of infections. A small number of asymptomatic carriers, or a small number of super-spreaders, or a small segment of the population that is disproportionately likely to require intensive care, can magnify the medical, social and economic impacts of a pandemic of this size. The speed of the pandemic and the large number of affected cases worldwide justify efforts to identify all possible risk factors for adverse outcomes, including efforts to identify genetic susceptibility factors in human hosts.","version":"1.1","doi":"10.1101/2020.04.05.026633","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.041228","pub_date":"2020-4-14","title":"The FDA- approved gold drug Auranofin inhibits novel coronavirus (SARS-COV-2) replication and attenuates inflammation in human cells","abstract":"SARS-COV-2 has recently emerged as a new public health threat. Herein, we report that the FDA-approved gold drug, auranofin, inhibits SARS-COV-2 replication in human cells at low micro molar concentration. Treatment of cells with auranofin resulted in a 95% reduction in the viral RNA at 48 hours after infection. Auranofin treatment dramatically reduced the expression of SARS-COV-2-induced cytokines in human cells. These data indicate that auranofin could be a useful drug to limit SARS-CoV-2 infection and associated lung injury due to its anti-viral, anti-inflammatory and anti-ROS properties. Auranofin has a well-known toxicity profile and is considered safe for human use.","version":"1.1","doi":"10.1101/2020.04.14.041228","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.12.037580","pub_date":"2020-4-14","title":"Single Nucleus Multiomic Profiling Reveals Age-Dynamic Regulation of Host Genes Associated with SARS-CoV-2 Infection","abstract":"Respiratory failure is the leading cause of COVID-19 death and disproportionately impacts adults more than children. Here, we present a large-scale snATAC-seq dataset (90,980 nuclei) of the human lung, generated in parallel with snRNA-seq (46,500 nuclei), from healthy donors of ~30 weeks, ~3 years and ~30 years of age. Focusing on genes implicated in SARS-CoV-2 cell entry, we observed an increase in the proportion of alveolar epithelial cells expressing ACE2 and TMPRSS2 in adult compared to young lungs. Consistent with expression dynamics, 10 chromatin peaks linked to TMPRSS2 exhibited significantly increased activity with age and harbored IRF and STAT binding sites. Furthermore, we identified 14 common sequence variants in age-increasing peaks with predicted regulatory function, including several associated with respiratory traits and TMPRSS2 expression. Our findings reveal a plausible contributor to why children are more resistant to COVID-19 and provide an epigenomic basis for transferring this resistance to older populations.","version":"1.1","doi":"10.1101/2020.04.12.037580","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.041434","pub_date":"2020-4-14","title":"Genetic Variability of Human Angiotensin-Converting Enzyme 2 (hACE2) Among Various Ethnic Populations","abstract":"There appears to be large regional variations for susceptibility, severity and mortality for Covid-19 infections. We set out to examine genetic differences in the human angiotensin-converting enzyme 2 (hACE2) gene, as its receptor serves as a cellular entry for SARS- CoV-2. By comparing 56,885 Non-Finnish European and 9,197 East Asians (including 1,909 Koreans) four missense mutations were noted in the hACE2 gene. Molecular dynamic demonstrated that two of these variants (K26R and I468V) may affect binding characteristics between S protein of the virus and hACE2 receptor. We also examined hACE2 gene expression in eight global populations from the HapMap3 and noted marginal differences in expression for some populations as compared to the Chinese population. However, for both of our studies, the magnitude of the difference was small and the significance is not clear in the absence of further in vitro and functional studies.","version":"1.1","doi":"10.1101/2020.04.14.041434","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.035683","pub_date":"2020-4-14","title":"Sphingolipid Biosynthesis Inhibition As A Host Strategy Against Diverse Pathogens","abstract":"Chloroquine is an anti-malarial and immunosuppressant drug that has cationic amphipathic chemical properties. We performed genome-wide screens in human cells with chloroquine and several other widely used cationic amphipathic drugs (CADs) including the anti-depressants, sertraline (Zoloft) and fluoxetine (Prozac), the analgesic nortriptyline (Pamelor), the anti-arrhythmic amiodarone (Cordarone), and the anti-hypertensive verapamil (Calan) to characterize their molecular similarities and differences. Despite CADs having different disease indications but consistent with them sharing key chemical properties, we found CADs to have remarkably similar phenotypic profiles compared with non-CADs we and others have previously screened (1\u20135). The most significant genetic interaction for all CADs was the initiating step in sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT). A comparison of genome-wide screens performed with diverse pathogens from viruses, bacteria, plants, and parasites including Ebola (6), adeno-associated virus AAV2 (7), HIV (8), Rotavirus (9), Influenza A (10), Zika virus (11), Picornavirus (12), Exotoxin A (13), Cholera toxin (14), Type III secretion system and Shiga toxin (15, 16), Ricin toxin (17), and Toxoplasma gondii (18) showed SPT as a top common host factor and 80% overlap overall in top hits specifically with CADs. Potential sphingolipid-mediated mechanisms for the host response- and virulence-modulating effects of CADs involve autophagy and SERPINE1/PAI-1 (plasminogen activator inhibitor-1). Chloroquine has recently shown potential as an anti-viral agent for the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease (19, 20). Our study demonstrates that numerous readily available drugs molecularly function highly similar to chloroquine, which suggests they might be considered for further pre-clinical investigation in the context of SARS-CoV-2. More generally, our work suggests the diverse pathogen mitigating potential of drugs that inhibit host sphingolipid biosynthesis such as CADs. Our study demonstrates that numerous readily available drugs molecularly function highly similar to chloroquine, which suggests they might be considered for further pre-clinical investigation in the context of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.10.035683","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.040196","pub_date":"2020-4-14","title":"Tissue-resident CD8+ T cells drive age-associated chronic lung sequelae following viral pneumonia","abstract":"Lower respiratory viral infections, such as influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infections, often cause severe viral pneumonia in aged individuals. Here, we report that influenza viral pneumonia leads to chronic non-resolving lung pathology and exaggerated accumulation of CD8+ tissue-resident memory T cells (TRM) in the respiratory tract of aged hosts. TRM accumulation relies on elevated TGF-\u03b2 present in aged tissues. Further, we show that TRM isolated from aged lungs lack a subpopulation characterized by expression of molecules involved in TCR signaling and effector function. Consequently, TRM cells from aged lungs were insufficient to provide heterologous protective immunity. Strikingly, the depletion of CD8+ TRM cells dampens persistent chronic lung inflammation and ameliorates tissue fibrosis in aged, but not young, animals. Collectively, our data demonstrate that age-associated TRM cell malfunction supports chronic lung inflammatory and fibrotic sequelae following viral pneumonia in aged hosts.","version":"1.1","doi":"10.1101/2020.04.13.040196","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.13.036079","pub_date":"2020-4-14","title":"Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection","abstract":"The CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3\u2019- or 5\u2019-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes.","version":"1.1","doi":"10.1101/2020.04.13.036079","journal":"bioRxiv","score":null},{"id":"10.1101/402370","pub_date":"2020-4-14","title":"PathoLive \u2013 Real-time pathogen identification from metagenomic Illumina datasets","abstract":"Over the past years, NGS has become a crucial workhorse for open-view pathogen diagnostics. Yet, long turnaround times result from using massively parallel high-throughput technologies as the analysis can only be performed after sequencing has finished. The interpretation of results can further be challenged by contaminations, clinically irrelevant sequences, and the sheer amount and complexity of the data. We implemented PathoLive, a real-time diagnostics pipeline for the detection of pathogens from clinical samples hours before sequencing has finished. Based on real-time alignment with HiL-ive2, mappings are scored with respect to common contaminations, low-entropy areas, and sequences of widespread, non-pathogenic organisms. The results are visualized using an interactive taxonomic tree that provides an easily interpretable overview of the relevance of hits. For a human plasma sample that was spiked in vitro with six pathogenic viruses, all agents were clearly detected after only 40 of 200 sequencing cycles. For a real-world sample from Sudan the results correctly indicated the presence of Crimean-Congo hemorrhagic Fever Virus. In a second real-world dataset from the 2019 SARS-CoV-2 outbreak in Wuhan, we found the presence of a SARS Coronavirus as the most relevant hit without the novel virus reference genome being included in the database. For all samples, clinically irrelevant hits were correctly de-emphasized. Our approach is valuable to obtain fast and accurate NGS-based pathogen identifications and correctly prioritize and visualize them based on their clinical significance. PathoLive is open source and available on GitLab (https://gitlab.com/rkibioinformatics/PathoLive) and BioConda (conda install \u2013c bioconda patholive). Bernhard.Renard@hpi.de, NitscheA@rki.de","version":"1.2","doi":"10.1101/402370","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.14.040667","pub_date":"2020-4-14","title":"COVID-19 Knowledge Graph: a computable, multi-modal, cause-and-effect knowledge model of COVID-19 pathophysiology","abstract":"The past few weeks have witnessed a worldwide mobilization of the research community in response to the novel coronavirus (COVID-19). This global response has led to a burst of publications on the pathophysiology of the virus, yet without coordinated efforts to organize this knowledge, it can remain hidden away from individual research groups. By extracting and formalizing this knowledge in a structured and computable form, as in the form of a knowledge graph, researchers can readily reason and analyze this information on a much larger scale. Here, we present the COVID-19 Knowledge Graph, an expansive cause-and-effect network constructed from scientific literature on the new coronavirus that aims to provide a comprehensive view of its pathophysiology. To make this resource available to the research community and facilitate its exploration and analysis, we also implemented a web application and released the KG in multiple standard formats. The COVID-19 Knowledge Graph is publicly available under CC-0 license at https://github.com/covid19kg and https://bikmi.covid19-knowledgespace.de. alpha.tom.kodamullil@scai.fraunhofer.de Supplementary data are available online.","version":"1.1","doi":"10.1101/2020.04.14.040667","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.994756","pub_date":"2020-4-13","title":"SARS-CoV-2 proteome microarray for mapping COVID-19 antibody interactions at amino acid resolution","abstract":"COVID-19 has quickly become a worldwide pandemic, which has significantly impacted the economy, education, and social interactions. Understanding the humoral antibody response to SARS-CoV-2 proteins may help identify biomarkers that can be used to detect and treat COVID-19 infection. However, no immuno-proteomics platform exists that can perform such proteome-wide analysis. To address this need, we created a SARS-CoV-2 proteome microarray to analyze antibody interactions at amino acid resolution by spotting peptides 15 amino acids long with 5-amino acid offsets representing full-length SARS-CoV-2 proteins. Moreover, the array processing time is short (1.5 hours), the dynamic range is ~2 orders of magnitude, and the lowest limit of detection is 94 pg/mL. Here, the SARS-CoV-2 proteome array reveals that antibodies commercially available for SARS-CoV-1 proteins can also target SARS-CoV-2 proteins. These readily available reagents could be used immediately in COVID-19 research. Second, IgM and IgG immunogenic epitopes of SARS-CoV-2 proteins were profiled in the serum of ten COVID-19 patients. Such epitope biomarkers provide insight into the immune response to COVID-19 and are potential targets for COVID-19 diagnosis and vaccine development. Finally, serological antibodies that may neutralize viral entry into host cells via the ACE2 receptor were identified. Further investigation into whether these antibodies can inhibit the propagation of SARS-CoV-2 is warranted. Antibody and epitope profiling in response to COVID-19 is possible with our peptide-based SARS-COV-2 proteome microarray. The data gleaned from the array could provide invaluable information to the scientific community to understand, detect, and treat COVID-19.","version":"1.3","doi":"10.1101/2020.03.26.994756","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.026948","pub_date":"2020-4-13","title":"Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells","abstract":"The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.","version":"1.2","doi":"10.1101/2020.04.08.026948","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026435","pub_date":"2020-4-13","title":"Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer","abstract":"The COVID-19 pandemic has resulted in increased need for diagnostic testing using reverse transcriptase real-time PCR (RT-PCR). An exponential increase in demand has resulted in a shortage of numerous reagents in particular those associated with the lysis buffer required to extract the viral RNA. Herein, we describe a rapid collective effort by hospital laboratory scientists, academic researchers and the biopharma industry to generate a validated lysis buffer. We have formulated a 4M Guanidinium thiocyanate (GITC)/ Triton X-100 Lysis buffer which provides comparable results with the recommended reagents. This buffer will ease the burden on hospital labs in their heroic efforts to diagnose a large population of patients.","version":"1.2","doi":"10.1101/2020.04.05.026435","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.11.037382","pub_date":"2020-4-12","title":"Isolation, sequence, infectivity and replication kinetics of SARS-CoV-2","abstract":"SARS-CoV-2 emerged in December 2019 in Wuhan, China and has since infected over 1.5 million people, of which over 107,000 have died. As SARS-CoV-2 spreads across the planet, speculations remain about the range of human cells that can be infected by SARS-CoV-2. In this study, we report the isolation of SARS-CoV-2 from two cases of COVID-19 in Toronto, Canada. We determined the genomic sequences of the two isolates and identified single nucleotide changes in representative populations of our virus stocks. More importantly, we tested a wide range of human immune cells for productive infection with SARS-CoV-2. Here we confirm that human primary peripheral blood mononuclear cells (PBMCs) are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype using in vitro and in vivo infection models.","version":"1.2","doi":"10.1101/2020.04.11.037382","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026005","pub_date":"2020-4-12","title":"Immuno-informatics Characterization SARS-CoV-2 Spike Glycoprotein for Prioritization of Epitope based Multivalent Peptide Vaccine","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 is a public-health emergency of international concern and thus calling for the development of safe and effective therapeutics and prophylactics particularly a vaccine to protect against the infection. SARS-CoV-2 spike glycoprotein is an attractive candidate for vaccine, antibodies and inhibitor development because of many roles it plays in attachment, fusion and entry into the host cell. In this study, we characterized the SARS-CoV-2 spike glycoprotein by immune-informatics techniques to put forward potential B and T cell epitopes, followed by the use of epitopes in construction of a multi-epitope peptide vaccine construct (MEPVC). The MEPVC revealed robust host immune system simulation with high production of immunoglobulins, cytokines and interleukins. Stable conformation of the MEPVC with a representative innate immune TLR3 receptor was observed involving strong hydrophobic and hydrophilic chemical interactions, along with enhanced contribution from salt-bridges towards inter-molecular stability. Molecular dynamics simulation in solution aided further in interpreting strong affinity of the MEPVC for TLR3. This stability is the attribute of several vital residues from both TLR3 and MEPVC as shown by radial distribution function (RDF) and a novel analytical tool axial frequency distribution (AFD). Comprehensive binding free energies estimation was provided at the end that concluded major domination by electrostatic and minor from van der Waals. Summing all, the designed MEPVC has tremendous potential of providing protective immunity against COVID-19 and thus has the potential to be considered in experimental studies.","version":"1.1","doi":"10.1101/2020.04.05.026005","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.12.025577","pub_date":"2020-4-12","title":"Mechanistic modeling of the SARS-CoV-2 disease map","abstract":"Here we present a web interface that implements a comprehensive mechanistic model of the SARS-CoV-2 disease map in which the detailed activity of the human signaling circuits related to the viral infection and the different antiviral responses, including immune and inflammatory activities, can be inferred from gene expression experiments. Moreover, given to the mechanistic properties of the model, the effect of potential interventions, such as knock-downs, over-expression or drug effects (currently the system models the effect of more than 8000 DrugBank drugs) can be studied in specific conditions. By providing a holistic, systems biology approach to the understanding of the complexities of the viral infection process, this tool will become an important asset in the search for efficient antiviral treatments. The tool is freely available at: http://hipathia.babelomics.org/covid19/","version":"1.1","doi":"10.1101/2020.04.12.025577","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.034462","pub_date":"2020-4-12","title":"Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions","abstract":"Here we aim to describe early mutational events across samples from publicly available SARS-CoV-2 sequences from the sequence read archive repository. Up until March 27, 2020, we downloaded 53 illumina datasets, mostly from China, USA (Washington DC) and Australia (Victoria). Of 30 high quality datasets, 27 datasets (90%) contain at least a single founder mutation and most of the variants are missense (over 63%). Five-point mutations with clonal (founder) effect were found in USA sequencing samples. Sequencing samples from USA in GenBank present this signature with 50% allele frequencies among samples. Australian mutation signatures were more diverse than USA samples, but still, clonal events were found in those samples. Mutations in the helicase and orf1a coding regions from SARS-CoV-2 were predominant, among others, suggesting that these proteins are prone to evolve by natural selection. Finally, we firmly urge that primer sets for diagnosis be carefully designed, since rapidly occurring variants would affect the performance of the reverse transcribed quantitative PCR (RT-qPCR) based viral testing.","version":"1.1","doi":"10.1101/2020.04.09.034462","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.032342","pub_date":"2020-4-11","title":"Potential host range of multiple SARS-like coronaviruses and an improved ACE2-Fc variant that is potent against both SARS-CoV-2 and SARS-CoV-1","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a currently uncontrolled pandemic and the etiological agent of coronavirus disease 2019 (COVID-19). It is important to study the host range of SARS-CoV-2 because some domestic species might harbor the virus and transmit it back to humans. In addition, insight into the ability of SARS-CoV-2 and SARS-like viruses to utilize animal orthologs of the SARS-CoV-2 receptor ACE2 might provide structural insight into improving ACE2-based viral entry inhibitors. Here we show that ACE2 orthologs of a wide range of domestic and wild animals support entry of SARS-CoV-2, as well as that of SARS-CoV-1, bat coronavirus RaTG13, and a coronavirus isolated from pangolins. Some of these species, including camels, cattle, horses, goats, sheep, pigs, cats, and rabbits may serve as potential intermediate hosts for new human transmission, and rabbits in particular may serve as a useful experimental model of COVID-19. We show that SARS-CoV-2 and SARS-CoV-1 entry could be potently blocked by recombinant IgG Fc-fusion proteins of viral spike protein receptor-binding domains (RBD-Fc) and soluble ACE2 (ACE2-Fc). Moreover, an ACE2-Fc variant, which carries a D30E mutation and has ACE2 truncated at its residue 740 but not 615, outperforms all the other ACE2-Fc variants on blocking entry of both viruses. Our data suggest that RBD-Fc and ACE2-Fc could be used to treat and prevent infection of SARS-CoV-2 and any new viral variants that emerge over the course of the pandemic.","version":"1.1","doi":"10.1101/2020.04.10.032342","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.033910","pub_date":"2020-4-11","title":"Integrative Network Biology Framework Elucidates Molecular Mechanisms of SARS-CoV-2 Pathogenesis","abstract":"COVID-19 (Coronavirus disease 2019) is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the pathophysiology of this deadly virus is complex and largely unknown, we employ a network biology-fueled approach and integrated multiomics data pertaining to lung epithelial cells-specific coexpression network and human interactome to generate Calu-3-specific human-SARS-CoV-2 Interactome (CSI). Topological clustering and pathway enrichment analysis show that SARS-CoV-2 target central nodes of host-viral network that participate in core functional pathways. Network centrality analyses discover 28 high-value SARS-CoV-2 targets, which are possibly involved in viral entry, proliferation and survival to establish infection and facilitate disease progression. Our probabilistic modeling framework elucidates critical regulatory circuitry and molecular events pertinent to COVID-19, particularly the host modifying responses and cytokine storm. Overall, our network centric analyses reveal novel molecular components, uncover structural and functional modules, and provide molecular insights into SARS-CoV-2 pathogenicity.","version":"1.1","doi":"10.1101/2020.04.09.033910","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.023358","pub_date":"2020-4-11","title":"SARS-CoV-2 detection with CRISPR diagnostics","abstract":"The novel coronavirus (CoV) disease termed COVID-19 (Coronavirus Disease-19) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) is causing a massive pandemic worldwide, threatening public health systems across the globe. During this ongoing COVID-19 outbreak, nucleic acid detection has played an important role in early diagnosis. Here we report a SARS-CoV-2 detection protocol using a CRISPR-based CRISPR diagnostic platform - CDetection (Cas12b-mediated DNA detection). By combining sample treatment protocols and nucleic acid amplification methods with CDetection, we have established an integrated viral nucleic acid detection platform - CASdetec (CRISPR-assisted detection). The detection limit of CASdetec for SARS-CoV-2 pseudovirus is 1 \u00d7 104 copies/mL, with no cross reactivity observed. Our assay design and optimization process can provide guidance for future CRISPR-based nucleic acid detection assay development and optimization.","version":"1.1","doi":"10.1101/2020.04.10.023358","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.035964","pub_date":"2020-4-11","title":"Positive selection of ORF3a and ORF8 genes drives the evolution of SARS-CoV-2 during the 2020 COVID-19 pandemic","abstract":"In this study, we analyzed full-length SARS-CoV-2 genomes from multiple countries to determine early trends in the evolutionary dynamics of the novel COVID-19 pandemic. Results indicated SARS-CoV-2 evolved early into at least three phylogenetic groups, characterized by positive selection at specific residues of the accessory proteins OFR3a and ORF8a. We also report evidence of epistatic interactions among sites in the genome that may be important in the generation of variants adapted to humans. These observations might impact not only public health, but also suggest more studies are needed to understand the genetic mechanisms that may affect the development of therapeutic and preventive tools, like antivirals and vaccines.","version":"1.1","doi":"10.1101/2020.04.10.035964","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030742","pub_date":"2020-4-11","title":"Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue","abstract":"In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilizes ACE2 and TMPRSS2 host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147, and GRP78 may also function as receptors for SARS-CoV-2. To determine the expression and in situ localization of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analyzed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples. We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung. We present confirmatory evidence for the presence of TMPRSS2, CD147, and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 in the respiratory mucosa. Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection.","version":"1.2","doi":"10.1101/2020.04.07.030742","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.035824","pub_date":"2020-4-11","title":"Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro","abstract":"COVID-19 has become a global pandemic that threatens millions of people worldwide. There is an urgent call for developing effective drugs against the virus (SARS-CoV-2) causing this disease. The main protease of SARS-CoV-2, 3C-like protease (3CLpro), is highly conserved across coronaviruses and is essential for the maturation process of viral polyprotein. Scutellariae radix (Huangqin in Chinese), the root of Scutellaria baicalensis has been widely used in traditional Chinese medicine to treat viral infection related symptoms. The extracts of S. baicalensis have exhibited broad spectrum antiviral activities. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredient compounds. We found that the ethanol extract of S. baicalensis inhibits SARS-CoV-2 3CLpro activity in vitro and the replication of SARS-CoV-2 in Vero cells with an EC50 of 0.74 \u03bcg/ml. Among the major components of S. baicalensis, baicalein strongly inhibits SARS-CoV-2 3CLpro activity with an IC50 of 0.39 \u03bcM. We further identified four baicalein analogue compounds from other herbs that inhibit SARS-CoV-2 3CLpro activity at microM concentration. Our study demonstrates that the extract of S. baicalensis has effective anti-SARS-CoV-2 activity and baicalein and analogue compounds are strong SARS-CoV-2 3CLpro inhibitors.","version":"1.1","doi":"10.1101/2020.04.10.035824","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.11.034603","pub_date":"2020-4-11","title":"Cell-Type-Specific Expression of Renin-Angiotensin-System Components in the Human Body and Its Relevance to SARS-CoV-2 Infection","abstract":"We have analyzed the cell-type-specific expression of the renin-angiotensin system (RAS) components across 141 cell types or subtypes as defined by single-cell RNA-seq (scRNA-seq) analysis. ACE2, one of the components of RAS, also facilitates SARS-CoV-2 entry into cells in cooperation with its associated protease TMPRSS2. Therefore, our analysis also contributes to the understanding of SARS-CoV-2 infection, spreading of the virus throughout the body, and potential viral interference with RAS in COVID-19 patients.","version":"1.1","doi":"10.1101/2020.04.11.034603","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030924","pub_date":"2020-4-11","title":"Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: a computational biology approach","abstract":"The newly identified SARS-CoV-2 has now been reported from around 183 countries with more than a million confirmed human cases including more than 68000 deaths. The genomes of SARS-COV-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins have gotten substantial attention recently. Spike glycoprotein is widely considered as a possible target to be explored because of its role during the entry of coronaviruses into host cells. We analyzed 320 whole-genome sequences and 320 spike protein sequences of SARS-CoV-2 using multiple sequence alignment tools. In this study, 483 unique variations have been identified among the genomes including 25 non-synonymous mutations and one deletion in the spike protein of SARS-CoV-2. Among the 26 variations detected, 12 variations were located at the N-terminal domain and 6 variations at the receptor-binding domain (RBD) which might alter the interaction with receptor molecules. In addition, 22 amino acid insertions were identified in the spike protein of SARS-CoV-2 in comparison with that of SARS-CoV. Phylogenetic analyses of spike protein revealed that Bat coronavirus have a close evolutionary relationship with circulating SARS-CoV-2. The genetic variation analysis data presented in this study can help a better understanding of SARS-CoV-2 pathogenesis. Based on our findings, potential inhibitors can be designed and tested targeting these proposed sites of variation.","version":"1.1","doi":"10.1101/2020.04.07.030924","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.036418","pub_date":"2020-4-11","title":"The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement","abstract":"The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates entry of SARS-CoV-2 into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. Antibodies to the RBD domain of SARS-CoV (SARS-CoV-1), a closely related coronavirus which emerged in 2002-2003, have been shown to potently neutralize SARS-CoV-1 S-protein-mediated entry, and the presence of anti-RBD antibodies correlates with neutralization in SARS-CoV-2 convalescent sera. Here we show that immunization with the SARS-CoV-2 RBD elicits a robust neutralizing antibody response in rodents, comparable to 100 \u00b5g/ml of ACE2-Ig, a potent SARS-CoV-2 entry inhibitor. Importantly, anti-sera from immunized animals did not mediate antibody-dependent enhancement (ADE) of S-protein-mediated entry under conditions in which Zika virus ADE was readily observed. These data suggest that an RBD-based vaccine for SARS-CoV-2 could be safe and effective.","version":"1.1","doi":"10.1101/2020.04.10.036418","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.036533","pub_date":"2020-4-11","title":"Structural interactions between pandemic SARS-CoV-2 spike glycoprotein and human Furin protease","abstract":"The SARS-CoV-2 pandemic is an urgent global public health emergency and warrants investigating molecular and structural studies addressing the dynamics of viral proteins involved in host cell adhesion. The recent comparative genomic studies highlight the insertion of Furin protease site in the SARS-CoV-2 spike glycoprotein alerting possible modification in the viral spike protein and its eventual entry to host cell and presence of Furin site implicated to virulence. Here we structurally show how Furin interacts with the SARS-CoV-2 spike glycoprotein homotrimer at S1/S2 region, which underlined the mechanism and mode of action, which is a key for host cell entry. Unravelling the structural features of biding site opens the arena in rising bonafide antibodies targeting to block the Furin cleavage and have great implications in the development of Furin inhibitors or therapeutics.","version":"1.1","doi":"10.1101/2020.04.10.036533","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.031252","pub_date":"2020-4-11","title":"In-depth Bioinformatic Analyses of Human SARS-CoV-2, SARS-CoV, MERS-CoV, and Other Nidovirales Suggest Important Roles of Noncanonical Nucleic Acid Structures in Their Lifecycles","abstract":"Noncanonical nucleic acid structures play important roles in the regulation of molecular processes. Considering the importance of the ongoing coronavirus crisis, we decided to evaluate genomes of all coronaviruses sequenced to date (stated more broadly, the order Nidovirales) to determine if they contain noncanonical nucleic acid structures. We discovered much evidence of putative G-quadruplex sites and even much more of inverted repeats (IRs) loci, which in fact are ubiquitous along the whole genomic sequence and indicate a possible mechanism for genomic RNA packaging. The most notable enrichment of IRs was found inside 5\u2032UTR for IRs of size 12+ nucleotides, and the most notable enrichment of putative quadruplex sites (PQSs) was located before 3\u2032UTR, inside 5\u2032UTR, and before mRNA. This indicates crucial regulatory roles for both IRs and PQSs. Moreover, we found multiple G-quadruplex binding motifs in human proteins having potential for binding of SARS-CoV-2 RNA. Noncanonical nucleic acids structures in Nidovirales and in novel SARS-CoV-2 are therefore promising druggable structures that can be targeted and utilized in the future.","version":"1.1","doi":"10.1101/2020.04.09.031252","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029132","pub_date":"2020-4-11","title":"A virus that has gone viral: Amino acid mutation in S protein of Indian isolate of Coronavirus COVID-19 might impact receptor binding and thus infectivity","abstract":"Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the recent outbreak of SARS-CoV-2 late in 2019 (also named as COVID-19 or novel coronavirus 2019 or nCoV2019. Spike(S) protein, one of the structural proteins of this virus plays key role in receptor (ACE2) binding and thus virus entry. Thus, this protein has attracted scientists for detailed study and therapeutic targeting. As the 2019 novel coronavirus takes its course throughout the world, more and more sequence analyses are been done and genome sequences getting deposited in various databases. From India two clinical isolates have been sequenced and the full genome deposited in GenBank. We have performed sequence analyses of the spike protein of the Indian isolates and compared with that of the Wuhan, China (where the outbreak was first reported). While all the sequences of Wuhan isolates are identical, we found point mutations in the Indian isolates. Out of the two isolates one was found to harbour a mutation in its Receptor binding domain (RBD) at position 407. At this site arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic amino acid that is also a C-beta branched amino acid). This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor ding of the virus. Although this finding needs further validation and more sequencing, the information might be useful in rational drug designing and vaccine engineering.","version":"1.1","doi":"10.1101/2020.04.07.029132","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.028712","pub_date":"2020-4-11","title":"Functional Immune Deficiency Syndrome via Intestinal Infection in COVID-19","abstract":"Using a Systems Biology approach, we integrated genomic, transcriptomic, proteomic, and molecular structure information to provide a holistic understanding of the COVID-19 pandemic. The expression data analysis of the Renin Angiotensin System indicates mild nasal, oral or throat infections are likely and that the gastrointestinal tissues are a common primary target of SARS-CoV-2. Extreme symptoms in the lower respiratory system likely result from a secondary-infection possibly by a comorbidity-driven upregulation of ACE2 in the lung. The remarkable differences in expression of other RAS elements, the elimination of macrophages and the activation of cytokines in COVID-19 bronchoalveolar samples suggest that a functional immune deficiency is a critical outcome of COVID-19. We posit that using a non-respiratory system as a major pathway of infection is likely determining the unprecedented global spread of this coronavirus. A Systems Approach Indicates Non-respiratory Pathways of Infection as Key for the COVID-19 Pandemic","version":"1.2","doi":"10.1101/2020.04.06.028712","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.10.036640","pub_date":"2020-4-11","title":"Python nidoviruses, more than respiratory pathogens","abstract":"In recent years nidoviruses have emerged as an important respiratory pathogen of reptiles, affecting especially captive python populations. In pythons, nidovirus infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe and often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full post mortem examinations on 30 nidovirus RT-PCR positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether the atypical tissue tropism associates with previously unknown nidoviruses or changes in the nidovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and to obtain virus isolates. Immunohistochemistry and immunofluorescence staining against nidovirus nucleoprotein demonstrated that the virus not only infects a broad spectrum of epithelial (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts etc.), but also intravascular monocytes, intralesional macrophages and endothelial cells. By next-generation sequencing we obtained full length genome for a novel nidovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing nidovirus infection-associated respiratory or systemic disease did not explain the observed phenotypes. The results indicate that python nidoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated systemic spread of the virus. During the last years, python nidoviruses have become a primary cause of fatal disease in pythons. Nidoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previously evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that compromise important zoonotic pathogens such as MERS-CoV and SARS-CoV, as well as the recently emerged SARS-CoV-2. Python nidoviruses belong to the same subfamily as the mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order nidovirales.","version":"1.1","doi":"10.1101/2020.04.10.036640","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.032763","pub_date":"2020-4-10","title":"Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir","abstract":"The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 \u00c5. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.","version":"1.2","doi":"10.1101/2020.04.08.032763","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026377","pub_date":"2020-4-10","title":"On the interactions of the receptor-binding domain of SARS-CoV-1 and SARS-CoV-2 spike proteins with monoclonal antibodies and the receptor ACE2","abstract":"A new betacoronavirus named SARS-CoV-2 has emerged as a new threat to global health and economy. A promising target for both diagnosis and therapeutics treatments of the new disease named COVID-19 is the coronavirus (CoV) spike (S) glycoprotein. By constant-pH Monte Carlo simulations and the PROCEEDpKa method, we have mapped the electrostatic epitopes for four monoclonal antibodies and the angiotensin-converting enzyme 2 (ACE2) on both SARS-CoV-1 and the new SARS-CoV-2 S receptor binding domain (RBD) proteins. We also calculated free energy of interactions and shown that the S RBD proteins from both SARS viruses binds to ACE2 with similar affinities. However, the affinity between the S RBD protein from the new SARS-CoV-2 and ACE2 is higher than for any studied antibody previously found complexed with SARS-CoV-1. Based on physical chemical analysis and free energies estimates, we can shed some light on the involved molecular recognition processes, their clinical aspects, the implications for drug developments, and suggest structural modifications on the CR3022 antibody that would improve its binding affinities for SARS-CoV-2 and contribute to address the ongoing international health crisis.","version":"1.1","doi":"10.1101/2020.04.05.026377","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.024885","pub_date":"2020-4-10","title":"Rapid in silico design of antibodies targeting SARS-CoV-2 using machine learning and supercomputing","abstract":"Rapidly responding to novel pathogens, such as SARS-CoV-2, represents an extremely challenging and complex endeavor. Numerous promising therapeutic and vaccine research efforts to mitigate the catastrophic effects of COVID-19 pandemic are underway, yet an efficacious countermeasure is still not available. To support these global research efforts, we have used a novel computational pipeline combining machine learning, bioinformatics, and supercomputing to predict antibody structures capable of targeting the SARS-CoV-2 receptor binding domain (RBD). In 22 days, using just the SARS-CoV-2 sequence and previously published neutralizing antibody structures for SARS-CoV-1, we generated 20 initial antibody sequences predicted to target the SARS-CoV-2 RBD. As a first step in this process, we predicted (and publicly released) structures of the SARS-CoV-2 spike protein using homology-based structural modeling. The predicted structures proved to be accurate within the targeted RBD region when compared to experimentally derived structures published weeks later. Next we used our in silico design platform to iteratively propose mutations to SARS-CoV-1 neutralizing antibodies (known not to bind SARS-Cov-2) to enable and optimize binding within the RBD of SARS-CoV-2. Starting from a calculated baseline free energy of \u221248.1 kcal/mol (\u00b1 8.3), our 20 selected first round antibody structures are predicted to have improved interaction with the SARS-CoV-2 RBD with free energies as low as \u221282.0 kcal/mole. The baseline SARS-CoV-1 antibody in complex with the SARS-CoV-1 RBD has a calculated interaction energy of \u221252.2 kcal/mole and neutralizes the virus by preventing it from binding and entering the human ACE2 receptor. These results suggest that our predicted antibody mutants may bind the SARS-CoV-2 RBD and potentially neutralize the virus. Additionally, our selected antibody mutants score well according to multiple antibody developability metrics. These antibody designs are being expressed and experimentally tested for binding to COVID-19 viral proteins, which will provide invaluable feedback to further improve the machine learning\u2013driven designs. This technical report is a high-level description of that effort; the Supplementary Materials includes the homology-based structural models we developed and 178,856 in silico free energy calculations for 89,263 mutant antibodies derived from known SARS-CoV-1 neutralizing antibodies.","version":"1.1","doi":"10.1101/2020.04.03.024885","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.09.034454","pub_date":"2020-4-10","title":"Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium","abstract":"Coronavirus disease 2019 (COVID-19) outcomes vary from asymptomatic infection to death. This disparity may reflect different airway levels of the SARS-CoV-2 receptor, ACE2, and the spike protein activator, TMPRSS2. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci (eQTL) for both ACE2 and TMPRSS2, that vary in frequency across world populations. Importantly, we find TMPRSS2 is part of a mucus secretory network, highly upregulated by T2 inflammation through the action of interleukin-13, and that interferon response to respiratory viruses highly upregulates ACE2 expression. Finally, we define airway responses to coronavirus infections in children, finding that these infections upregulate IL6 while also stimulating a more pronounced cytotoxic immune response relative to other respiratory viruses. Our results reveal mechanisms likely influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.","version":"1.1","doi":"10.1101/2020.04.09.034454","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.024752","pub_date":"2020-4-10","title":"Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease (COVID-19) that has resulted in a global pandemic. It is a highly contagious positive strand RNA virus and its clinical presentation includes severe to critical respiratory disease that appears to be fatal in \u223c3-5% of the cases. The viral spike (S) coat protein engages the human angiotensin-converting enzyme2 (ACE2) cell surface protein to invade the host cell. The SARS-CoV-2 S-protein has acquired mutations that increase its affinity to human ACE2 by \u223c10-15-fold compared to SARS-CoV S-protein, making it highly infectious. In this study, we assessed if ACE2 polymorphisms might alter host susceptibility to SARS-CoV-2 by affecting the ACE2 S-protein interaction. Our comprehensive analysis of several large genomic datasets that included over 290,000 samples representing >400 population groups identified multiple ACE2 protein-altering variants, some of which mapped to the S-protein-interacting ACE2 surface. Using recently reported structural data and a recent S-protein-interacting synthetic mutant map of ACE2, we have identified natural ACE2 variants that are predicted to alter the virus-host interaction and thereby potentially alter host susceptibility. In particular, human ACE2 variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R are predicted to increase susceptibility. The T92I variant, part of a consensus NxS/T N-glycosylation motif, confirmed the role of N90 glycosylation in immunity from non-human CoVs. Other ACE2 variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y are putative protective variants predicted to show decreased binding to SARS-CoV-2 S-protein. Overall, ACE2 variants are rare, consistent with the lack of selection pressure given the recent history of SARS-CoV epidemics, however, are likely to play an important role in altering susceptibility to CoVs.","version":"1.1","doi":"10.1101/2020.04.07.024752","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.028647","pub_date":"2020-4-10","title":"ACE2 fragment as a decoy for novel SARS-Cov-2 virus","abstract":"Novel SARS-Cov-2 enters human cells via interaction between the surface spike (S) glycoprotein and the cellular membrane receptor angiotensin-converting enzyme 2 (ACE2). Using a combination of comparative structural analyses of the binding surface of the S protein to ACE2, docking experiments, and molecular dynamics simulations we computationally identified a minimal, stable fragment of ACE2. This fragment binds to the S protein, is soluble, and appears not to bind to the physiological ligand angiotensinII. These results suggest a possible use of the ACE2 fragment as a decoy that could interfere with viral binding by competition.","version":"1.1","doi":"10.1101/2020.04.06.028647","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029199","pub_date":"2020-4-10","title":"A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing","abstract":"There is currently an urgent unmet need to increase coronavirus disease 2019 (COVID-19) testing capability to effectively respond to the COVID-19 pandemic. However, the current shortage in RNA extraction reagents as well as limitations in qPCR protocols have resulted in bottlenecks in testing capacity. Herein, we describe a novel molecular diagnostic for COVID-19 based on Sanger sequencing. This assay uses the addition of a frame-shifted spike-in, a modified PCR master mix, and custom Sanger sequencing data analysis to detect and quantify SARS-CoV-2 RNA at a limit of detection comparable to existing qPCR-based assays, at 10-20 genome copy equivalents. Crucially, our assay was able to detect SARS-CoV-2 RNA from viral particles suspended in transport media that was directly added to the PCR master mix, suggesting that RNA extraction can be skipped entirely without any degradation of test performance. Since Sanger sequencing instruments are widespread in clinical laboratories and commonly have built-in liquid handling automation to support up to 3840 samples per instrument per day, the widespread adoption of qSanger COVID-19 diagnostics can unlock more than 1,000,000 tests per day in the US.","version":"1.1","doi":"10.1101/2020.04.07.029199","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.08.980383","pub_date":"2020-4-10","title":"In silico approach toward the identification of unique peptides from viral protein infection: Application to COVID-19","abstract":"We describe a method for rapid in silico selection of diagnostic peptides from newly described viral pathogens and applied this approach to SARS-CoV-2/COVID-19. This approach is multi-tiered, beginning with compiling the theoretical protein sequences from genomic derived data. In the case of SARS-CoV-2 we begin with 496 peptides that would be produced by proteolytic digestion of the viral proteins. To eliminate peptides that would cause cross-reactivity and false positives we remove peptides from consideration that have sequence homology or similar chemical characteristics using a progressively larger database of background peptides. Using this pipeline, we can remove 47 peptides from consideration as diagnostic due to the presence of peptides derived from the human proteome. To address the complexity of the human microbiome, we describe a method to create a database of all proteins of relevant abundance in the saliva microbiome. By utilizing a protein-based approach to the microbiome we can more accurately identify peptides that will be problematic in COVID-19 studies which removes 12 peptides from consideration. To identify diagnostic peptides, another 7 peptides are flagged for removal following comparison to the proteome backgrounds of viral and bacterial pathogens of similar clinical presentation. By aligning the protein sequences of SARS-CoV-2 field isolates deposited to date we can identify peptides for removal due to their presence in highly variable regions that may lead to false negatives as the pathogen evolves. We provide maps of these regions and highlight 3 peptides that should be avoided as potential diagnostic or vaccine targets. Finally, we leverage publicly deposited proteomics data from human cells infected with SARS-CoV-2, as well as a second study with the closely related MERS-CoV to identify the two proteins of highest abundance in human infections. The resulting final list contains the 24 peptides most unique and diagnostic of SARS-CoV-2 infections. These peptides represent the best targets for the development of antibodies are clinical diagnostics. To demonstrate one application of this we model peptide fragmentation using a deep learning tool to rapidly generate targeted LCMS assays and data processing method for detecting CoVID-19 infected patient samples.","version":"1.2","doi":"10.1101/2020.03.08.980383","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.013516","pub_date":"2020-4-10","title":"Understanding the B and T cells epitopes of spike protein of severe respiratory syndrome coronavirus-2: A computational way to predict the immunogens","abstract":"The 2019 novel severe respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak has caused a large number of deaths with thousands of confirmed cases worldwide. The present study followed computational approaches to identify B- and T-cell epitopes for spike glycoprotein of SARS-CoV-2 by its interactions with the human leukocyte antigen alleles. We identified twenty-four peptide stretches on the SARS-CoV-2 spike protein that are well conserved among the reported strains. The S protein structure further validated the presence of predicted peptides on the surface. Out of which twenty are surface exposed and predicted to have reasonable epitope binding efficiency. The work could be useful for understanding the immunodominant regions in the surface protein of SARS-CoV-2 and could potentially help in designing some peptide-based diagnostics.","version":"1.1","doi":"10.1101/2020.04.08.013516","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.023903","pub_date":"2020-4-10","title":"Structural and functional analysis of a potent sarbecovirus neutralizing antibody","abstract":"SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than one million infections and 73,000 deaths. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of a SARS survivor infected in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.","version":"1.3","doi":"10.1101/2020.04.07.023903","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.031963","pub_date":"2020-4-10","title":"BioLaboro: A bioinformatics system for detecting molecular assay signature erosion and designing new assays in response to emerging and reemerging pathogens","abstract":"Emerging and reemerging infectious diseases such as the novel Coronavirus disease, COVID-19 and Ebola pose a significant threat to global society and test the public health community\u2019s preparedness to rapidly respond to an outbreak with effective diagnostics and therapeutics. Recent advances in next generation sequencing technologies enable rapid generation of pathogen genome sequence data, within 24 hours of obtaining a sample in some instances. With these data, one can quickly evaluate the effectiveness of existing diagnostics and therapeutics using in silico approaches. The propensity of some viruses to rapidly accumulate mutations can lead to the failure of molecular detection assays creating the need for redesigned or newly designed assays. Here we describe a bioinformatics system named BioLaboro to identify signature regions in a given pathogen genome, design PCR assays targeting those regions, and then test the PCR assays in silico to determine their sensitivity and specificity. We demonstrate BioLaboro with two use cases: Bombali Ebolavirus (BOMV) and the novel Coronavirus 2019 (SARS-CoV-2). For the BOMV, we analyzed 30 currently available real-time reverse transcription-PCR assays against the three available complete genome sequences of BOMV. Only two met our in silico criteria for successful detection and neither had perfect matches to the primer/probe sequences. We designed five new primer sets against BOMV signatures and all had true positive hits to the three BOMV genomes and no false positive hits to any other sequence. Four assays are closely clustered in the nucleoprotein gene and one is located in the glycoprotein gene. Similarly, for the SARS-CoV-2, we designed five highly specific primer sets that hit all 145 whole genomes (available as of February 28, 2020) and none of the near neighbors. Here we applied BioLaboro in two real-world use cases to demonstrate its capability; 1) to identify signature regions, 2) to assess the efficacy of existing PCR assays to detect pathogens as they evolve over time, and 3) to design new assays with perfect in silico detection accuracy, all within hours, for further development and deployment. BioLaboro is designed with a user-friendly graphical user interface for biologists with limited bioinformatics experience.","version":"1.1","doi":"10.1101/2020.04.08.031963","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029090","pub_date":"2020-4-09","title":"Absence of SARS-CoV-2 infection in cats and dogs in close contact with a cluster of COVID-19 patients in a veterinary campus","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, China, in 2019, is responsible for the COVID-19 pandemic. It is now accepted that the wild fauna, probably bats, constitute the initial reservoir of the virus, but little is known about the role pets can play in the spread of the disease in human communities, knowing the ability of SARS-CoV-2 to infect some domestic animals. We tested 21 domestic pets (9 cats and 12 dogs) living in close contact with their owners (belonging to a veterinary community of 20 students) in which two students tested positive for COVID-19 and several others (n = 11/18) consecutively showed clinical signs (fever, cough, anosmia, etc.) compatible with COVID-19 infection. Although a few pets presented many clinical signs indicative for a coronavirus infection, no animal tested positive for SARS-CoV-2 by RT-PCR and no antibodies against SARS-CoV-2 were detectable in their blood using an immunoprecipitation assay. These original data can serve a better evaluation of the host range of SARS-CoV-2 in natural environment exposure conditions.","version":"1.1","doi":"10.1101/2020.04.07.029090","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.028902","pub_date":"2020-4-09","title":"Optimization of SARS-CoV-2 detection by RT-QPCR without RNA extraction","abstract":"Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study we evaluated the reliability and the efficiency of a direct RT-QPCR method (without RNA extraction) using SeeGene Allplex\u2122 2019-nCoV RT-QPCR and the influence of swab storage media composition on further viral detection. We show that SeeGene\u2019s assay provides similar efficiency as the RealStar\u00ae SARS-CoV-2 RT-PCR kit (Altona Diagnostics), and that RNA extraction is not necessary nor advantageous if samples are stored in UTM or molecular water but is recommended if samples are stored in saline solution and in Hanks medium.","version":"1.1","doi":"10.1101/2020.04.06.028902","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.26.919985","pub_date":"2020-4-09","title":"Single-cell RNA expression profiling of ACE2, the receptor of SARS-CoV-2","abstract":"A novel coronavirus SARS-CoV-2 was identified in Wuhan, Hubei Province, China in December of 2019. According to WHO report, this new coronavirus has resulted in 76,392 confirmed infections and 2,348 deaths in China by 22 February, 2020, with additional patients being identified in a rapidly growing number internationally. SARS-CoV-2 was reported to share the same receptor, Angiotensin-converting enzyme 2 (ACE2), with SARS-CoV. Here based on the public database and the state-of-the-art single-cell RNA-Seq technique, we analyzed the ACE2 RNA expression profile in the normal human lungs. The result indicates that the ACE2 virus receptor expression is concentrated in a small population of type II alveolar cells (AT2). Surprisingly, we found that this population of ACE2-expressing AT2 also highly expressed many other genes that positively regulating viral entry, reproduction and transmission. This study provides a biological background for the epidemic investigation of the COVID-19, and could be informative for future anti-ACE2 therapeutic strategy development.","version":"1.2","doi":"10.1101/2020.01.26.919985","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029884","pub_date":"2020-4-09","title":"Immunoglobulin fragment F(ab\u2019)2 against RBD potently neutralizes SARS-CoV-2 in vitro","abstract":"COVID-19 caused by the emerging human coronavirus, SARS-CoV-2, has become a global pandemic, leading a serious threat to human health. So far, there is none vaccines or specific antiviral drugs approved for that. Therapeutic antibodies for SARS-CoV-2, was obtained from hyper immune equine plasma in this study. Herein, SARS-CoV-2 RBD with gram level were obtained through Chinese hamster ovary cells high-density fermentation. The binding of RBD to SARS-CoV-2 receptor, human ACE2, was verified and the efficacy of RBD in vivo was tested on mice and then on horses. As a result, RBD triggered high-titer neutralizing antibodies in vivo, and immunoglobulin fragment F(ab\u2019)2 was prepared from horse antisera through removing Fc. Neutralization test demonstrated that RBD-specific F(ab\u2019)2 inhibited SARS-CoV-2 with EC50 at 0.07 \u03bcg/ml, showing a potent inhibitory effect on SARS-CoV-2. These results highlights as RBD-specific F(ab\u2019)2 as therapeutic candidate for SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.07.029884","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.028316","pub_date":"2020-4-09","title":"Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic","abstract":"Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19), a respiratory tract infection. The standard molecular diagnostic test is a multistep process involving viral RNA extraction and real-time quantitative reverse transcriptase PCR (qRT-PCR). Laboratories across the globe face constraints on equipment and reagents during the COVID-19 pandemic. We have developed a simplified qRT-PCR assay that removes the need for an RNA extraction process and can be run on a real-time thermal cycler. The assay uses custom primers and probes, and maintains diagnostic sensitivity within 98.0% compared to the assay run on a high-throughput, random-access automated platform, the Panther Fusion (Hologic). This assay can be used to increase capacity for COVID-19 testing for national programmes worldwide.","version":"1.2","doi":"10.1101/2020.04.06.028316","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.029934","pub_date":"2020-4-09","title":"Room-temperature-storable PCR Mixes for SARS-CoV-2 Detection","abstract":"A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] in Wuhan, China, which then rapidly spread globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. We found that freeze-dried PCR mixes with \u223c1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18\u223c25\u00b0C) for 28 days, and for 14 and 10 days when stored at 37\u00b0C and 56\u00b0C, respectively. The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories. This method can also be applied to the generation of freeze-dried PCR mixes for the detection of other pathogens.","version":"1.1","doi":"10.1101/2020.04.07.029934","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.030767","pub_date":"2020-4-09","title":"Prediction of SARS-CoV interaction with host proteins during lung aging reveals a potential role for TRIB3 in COVID-19","abstract":"COVID-19 is prevalent in the elderly. Old individuals are more likely to develop pneumonia and respiratory failure due to alveolar damage, suggesting that lung senescence may increase the susceptibility to SARS-CoV-2 infection and replication. Considering that human coronavirus (HCoVs; SARS-CoV-2 and SARS-CoV) require host cellular factors for infection and replication, we analyzed Genotype-Tissue Expression (GTEx) data to test whether lung aging is associated with transcriptional changes in human protein-coding genes that potentially interact with these viruses. We found decreased expression of the gene tribbles homolog 3 (TRIB3) during aging in male individuals, and its protein was predicted to interact with HCoVs nucleocapsid protein and RNA-dependent RNA polymerase. Using publicly available lung single-cell data, we found TRIB3 expressed mainly in alveolar epithelial cells that express SARS-CoV-2 receptor ACE2. Functional enrichment analysis of age-related genes, in common with SARS-CoV-induced perturbations, revealed genes associated with the mitotic cell cycle and surfactant metabolism. Given that TRIB3 was previously reported to decrease virus infection and replication, the decreased expression of TRIB3 in aged lungs may help explain why older male patients are related to more severe cases of the COVID-19. Thus, drugs that stimulate TRIB3 expression should be evaluated as a potential therapy for the disease.","version":"1.1","doi":"10.1101/2020.04.07.030767","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.028589","pub_date":"2020-4-09","title":"The IMPDH inhibitor merimepodib suppresses SARS-CoV-2 replication in vitro","abstract":"The ongoing COVID-19 pandemic continues to pose a major public health burden around the world. The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected over one million people worldwide as of April, 2020, and has led to the deaths of nearly 300,000 people. No approved vaccines or treatments in the USA currently exist for COVID-19, so there is an urgent need to develop effective countermeasures. The IMPDH inhibitor merimepodib (MMPD) is an investigational antiviral drug that acts as a noncompetitive inhibitor of IMPDH. It has been demonstrated to suppress replication of a variety of emerging RNA viruses. We report here that MMPD suppresses SARS-CoV-2 replication in vitro. After overnight pretreatment of Vero cells with 10 \u03bcM of MMPD, viral titers were reduced by 4 logs of magnitude, while pretreatment for 4 hours resulted in a 3-log drop. The effect is dose-dependent, and concentrations as low as 3.3 \u03bcM significantly reduced viral titers when the cells were pretreated prior to infection. The results of this study provide evidence that MMPD may be a viable treatment option for COVID-19.","version":"1.1","doi":"10.1101/2020.04.07.028589","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.08.033001","pub_date":"2020-4-09","title":"Relative Abundance of SARS-CoV-2 Entry Genes in the Enterocytes of the Lower Gastrointestinal Tract","abstract":"COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread throughout the world and was declared a pandemic by the World Health Organization, thus leading to a rapid surge in the efforts to understand the mechanisms of transmission, methods of prevention, and potential therapies. While COVID-19 frequently manifests as a respiratory infection, there is evidence for infection of the gastrointestinal (GI) tract with documented viral RNA shedding in the stool of infected patients. In this study, we aimed to investigate the expression of ACE2 and TMPRSS2, which are required for SARS-CoV-2 entry into mammalian cells, from single-cell RNA sequencing (scRNA-seq) datasets of five different parts of the GI tract: esophagus, stomach, pancreas, small intestine, and colon/rectum.","version":"1.1","doi":"10.1101/2020.04.08.033001","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026450","pub_date":"2020-4-09","title":"Genomic determinants of pathogenicity in SARS-CoV-2 and other human coronaviruses","abstract":"SARS-CoV-2 poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and MERS-CoV, from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial elements of coronavirus pathogenicity and possible targets for diagnostics, prognostication and interventions.","version":"1.2","doi":"10.1101/2020.04.05.026450","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.027854","pub_date":"2020-4-09","title":"Decoding the lethal effect of SARS-CoV-2 (novel coronavirus) strains from global perspective: molecular pathogenesis and evolutionary divergence","abstract":"COVID-19 is a disease with global public health emergency that have shook the world since its\u2019 first detection in China in December, 2019. Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is the pathogen responsible behind this pandemic. The lethality of different viral strains is found to vary in different geographical locations but the molecular mechanism is yet to be known. Available data of whole genome sequencing of different viral strains published by different countries were retrieved and then analysed using Multiple Sequence Alignment and Pair-wise Sequence Alignment leading to Phylogenetic tree construction. Each location and the corresponding genetic variations were screened in depth. Then the variations are analysed at protein level giving special emphasis on Non Synonymous amino acid substitutions. The fatality rates in different countries were matched against the mutation number, rarity of the nucleotide alterations and functional impact of the Non Synonymous changes at protein level, separately and in combination. All the viral strains have been found to evolve from the viral strain of Taiwan (MT192759) which is 100% identical with the ancestor SARS-CoV-2 sequences of Wuhan (NC 045512.2; submitted on 5th Jan, 2020). Transition from C to T (C>T) is the most frequent mutation in this viral genome and mutations A>T, G>A, T>A are the rarest ones, found in countries with maximum fatality rate i.e Italy, Spain and Sweden. 20 Non Synonymous mutations are located in viral genome spanning Orf1ab polyprotein, Surface glycoprotein, Nucleocapsid protein etc. The functional effect on the structure and function of the protein can favourably or unfavourably interact with the host body. The fatality outcome depends on three important factors (a) number of mutation (b) rarity of the allelic variation and (c) functional consequence of the mutation at protein level. The molecular divergence, evolved from the ancestral strain (S) lead to extremely lethal (E), lethal(L) and non lethal (N) strains with the involvement of an Intermediate strain(I).","version":"1.1","doi":"10.1101/2020.04.06.027854","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.07.024455","pub_date":"2020-4-09","title":"JAK1 inhibition blocks lethal sterile immune responses: implications for COVID-19 therapy","abstract":"Cytokine storms are drivers of pathology and mortality in myriad viral infections affecting the human population. In SARS-CoV-2-infected patients, the strength of the cytokine storm has been associated with increased risk of acute respiratory distress syndrome, myocardial damage, and death. However, the therapeutic value of attenuating the cytokine storm in COVID-19 remains to be defined. Here, we report results obtained using a novel mouse model of lethal sterile anti-viral immune responses. Using a mouse model of Down syndrome (DS) with a segmental duplication of a genomic region encoding four of the six interferon receptor genes (Ifnrs), we demonstrate that these animals overexpress Ifnrs and are hypersensitive to IFN stimulation. When challenged with viral mimetics that activate Toll-like receptor signaling and IFN anti-viral responses, these animals overproduce key cytokines, show exacerbated liver pathology, rapidly lose weight, and die. Importantly, the lethal immune hypersensitivity, accompanying cytokine storm, and liver hyperinflammation are blocked by treatment with a JAK1-specific inhibitor. Therefore, these results point to JAK1 inhibition as a potential strategy for attenuating the cytokine storm and consequent organ failure during overdrive immune responses. Additionally, these results indicate that people with DS, who carry an extra copy of the IFNR gene cluster encoded on chromosome 21, should be considered at high risk during the COVID-19 pandemic. Inhibition of the JAK1 kinase prevents pathology and mortality caused by a rampant innate immune response in mice.","version":"1.1","doi":"10.1101/2020.04.07.024455","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.16.993386","pub_date":"2020-4-09","title":"Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target","abstract":"A novel coronavirus (2019-nCoV) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12), which catalyzes the synthesis of viral RNA, is a key component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. Here we report the cryo-EM structure of 2019-nCoV full-length nsp12 in complex with cofactors nsp7 and nsp8 at a resolution of 2.9-\u00c5. Additional to the conserved architecture of the polymerase core of the viral polymerase family and a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain featured in coronaviral RdRp, nsp12 possesses a newly identified \u03b2-hairpin domain at its N-terminal. Key residues for viral replication and transcription are observed. A comparative analysis to show how remdesivir binds to this polymerase is also provided. This structure provides insight into the central component of coronaviral replication/transcription machinery and sheds light on the design of new antiviral therapeutics targeting viral RdRp. Structure of 2019-nCov RNA polymerase.","version":"1.2","doi":"10.1101/2020.03.16.993386","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.027805","pub_date":"2020-4-08","title":"Sequence-based prediction of vaccine targets for inducing T cell responses to SARS-CoV-2 utilizing the bioinformatics predictor RECON","abstract":"The ongoing COVID-19 pandemic has created an urgency to identify novel vaccine targets for protective immunity against SARS-CoV-2. Consistent with observations for SARS-CoV, a closely related coronavirus responsible for the 2003 SARS outbreak, early reports identify a protective role for both humoral and cell-mediated immunity for SARS CoV-2. In this study, we leveraged HLA-I and HLA-II T cell epitope prediction tools from RECON\u00ae (Real-time Epitope Computation for ONcology), our bioinformatic pipeline that was developed using proteomic profiling of individual HLA-I and HLA-II alleles to predict rules for peptide binding to a diverse set of such alleles. We applied these binding predictors to viral genomes from the Coronaviridae family, and specifically to identify SARS-CoV-2 T cell epitopes. To test the suitability of these tools to identify viral T cell epitopes, we first validated HLA-I and HLA-II predictions on Coronaviridae family epitopes deposited in the Virus Pathogen Database and Analysis Resource (ViPR) database. We then use our HLA-I and HLA-II predictors to identify 11,776 HLA-I and 7,991 HLA-II candidate binding peptides across all 12 open reading frames (ORFs) of SARS-CoV-2. This extensive list of identified candidate peptides is driven by the length of the ORFs and the significant number of HLA-I and HLA-II alleles that we are able to predict (74 and 83, respectively), providing over 99% coverage for the US, European and Asian populations, for both HLA-I and HLA-II. From our SARS-CoV-2 predicted peptide-HLA-I allele pairs, 368 pairs identically matched previously reported pairs in the ViPR database, originating from other forms of coronaviruses. 320 of these pairs (89.1%) had a positive MHC-binding assay result. This analysis reinforces the validity our predictions. Using this bioinformatic platform, we identify multiple putative epitopes for CD4+ and CD8+ T cells whose HLA binding properties cover nearly the entire population and thus may be effective when included in prophylactic vaccines against SARS-CoV-2 to induce broad cellular immunity.","version":"1.1","doi":"10.1101/2020.04.06.027805","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.026476","pub_date":"2020-4-08","title":"Nelfinavir inhibits replication of severe acute respiratory syndrome coronavirus 2 in vitro","abstract":"In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, Hubei Province, China. No specific treatment has been established against coronavirus disease-2019 (COVID-19) so far. Therefore, it is urgently needed to identify effective antiviral agents for the treatment of this disease, and several approved drugs such as lopinavir have been evaluated. Here, we report that nelfinavir, an HIV-1 protease inhibitor, potently inhibits replication of SARS-CoV-2. The effective concentrations for 50% and 90% inhibition (EC50 and EC90) of nelfinavir were 1.13 \u00b5M and 1.76 \u00b5M respectively, the lowest of the nine HIV-1 protease inhibitors including lopinavir. The trough and peak serum concentrations of nelfinavir were three to six times higher than EC50 of this drug. These results suggest that nelfinavir is a potential candidate drug for the treatment of COVID-19 and should be assessed in patients with COVID-19.","version":"1.1","doi":"10.1101/2020.04.06.026476","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.028811","pub_date":"2020-4-08","title":"The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction Step","abstract":"Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. With many PCR-based molecular assays, an extraction step is routinely used as part of the protocol. This step can take up a significant amount of time and labor, especially if the extraction is performed manually. Long assay time, partly caused by slow sample preparation steps, has created a large backlog when testing patient samples suspected of COVID-19. Using flu and RSV clinical specimens, we have collected evidence that the RT-qPCR assay can be performed directly on patient sample material from a nasal swab immersed in virus transport medium (VTM) without an RNA extraction step. We have also used this approach to test for the direct detection of SARS-CoV-2 reference materials spiked in VTM. Our data, while preliminary, suggest that using a few microliters of these untreated samples still can lead to sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests and the backlog we currently experience can be reduced drastically. Next, we will confirm our findings using patient samples.","version":"1.1","doi":"10.1101/2020.04.06.028811","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.027557","pub_date":"2020-4-07","title":"Translational adaptation of human viruses to the tissues they infect","abstract":"Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of over 500 human-infecting viruses and determine that tropism explains changes in codon usage. Using an in silico model of translational efficiency, we validate the correspondence of the viral codon usage with the translational machinery of their tropism. In particular, we propose that the improved translational adaptation to the upper respiratory airways of the pandemic agent SARS-CoV-2 coronavirus could enhance its transmissibility. Furthermore, this correspondence is specifically defined in early viral proteins, as upon infection cells undergo reprogramming of tRNA pools that favors the translation of late counterparts.","version":"1.1","doi":"10.1101/2020.04.06.027557","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.20.000794","pub_date":"2020-4-06","title":"COVID-19 Vaccine Candidates: Prediction and Validation of 174 SARS-CoV-2 Epitopes","abstract":"The recent outbreak of SARS-CoV-2 (2019-nCoV) virus has highlighted the need for fast and efficacious vaccine development. Stimulation of a proper immune response that leads to protection is highly dependent on presentation of epitopes to circulating T-cells via the HLA complex. SARS-CoV-2 is a large RNA virus and testing of all overlapping peptides in vitro to deconvolute an immune response is not feasible. Therefore HLA-binding prediction tools are often used to narrow down the number of peptides to test. We tested 19 epitope-HLA-binding prediction tools, and using an in vitro peptide MHC stability assay, we assessed 777 peptides that were predicted to be good binders across 11 MHC allotypes. In this investigation of potential SARS-CoV-2 epitopes we found that current prediction tools vary in performance when assessing binding stability, and they are highly dependent on the MHC allotype in question. Designing a COVID-19 vaccine where only a few epitope targets are included is therefore a very challenging task. Here, we present 174 SARS-CoV-2 epitopes with high prediction binding scores, validated to bind stably to 11 HLA allotypes. Our findings may contribute to the design of an efficacious vaccine against COVID-19.","version":"1.4","doi":"10.1101/2020.03.20.000794","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.27.012906","pub_date":"2020-4-06","title":"RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses","abstract":"As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences reported to date from the current COVID-19 outbreak, and we present a curated list of 30 \u2018SARS-related-conserved\u2019 regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 \u2018SARS-CoV-2-conserved-structured\u2019 regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the 5\u2019 UTR, frame-shifting element, and 3\u2019 UTR. Last, we predict regions of the SARS-CoV-2 viral genome have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 \u2018SARS-CoV-2-conserved-unstructured\u2019 genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.","version":"1.2","doi":"10.1101/2020.03.27.012906","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.04.020925","pub_date":"2020-4-06","title":"Atazanavir inhibits SARS-CoV-2 replication and pro-inflammatory cytokine production","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors, such as atazanavir (ATV). ATV is of high interest because of its bioavailability within the respiratory tract. Our results show that ATV could dock in the active site of SARS-CoV-2 Mpro, with greater strength than LPV. ATV blocked Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells, human pulmonary epithelial cell line and primary monocytes, impairing virus-induced enhancement of IL-6 and TNF-\u03b1 levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.","version":"1.2","doi":"10.1101/2020.04.04.020925","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.06.027318","pub_date":"2020-4-06","title":"The potential genetic network of human brain SARS-CoV-2 infection","abstract":"The literature reports several symptoms of SARS-CoV-2 in humans such as fever, cough, fatigue, pneumonia, and headache. Furthermore, patients infected with similar strains (SARS-CoV and MERS-CoV) suffered testis, liver, or thyroid damage. Angiotensin-converting enzyme 2 (ACE2) serves as an entry point into cells for some strains of coronavirus (SARS-CoV, MERS-CoV, SARS-CoV-2). Our hypothesis was that as ACE2 is essential to the SARS-CoV-2 virus invasion, then brain regions where ACE2 is the most expressed are more likely to be disturbed by the infection. Thus, the expression of other genes which are also over-expressed in those damaged areas could be affected. We used mRNA expression levels data of genes provided by the Allen Human Brain Atlas (ABA), and computed spatial correlations with the LinkRbrain platform. Genes whose co-expression is spatially correlated to that of ACE2 were then clustered into 16 groups, depending on the organ in which they are the most expressed (as described by the NCBI genes database). The list of organs where genes sharing local over-expression with the ACE2 gene are the most expressed is astonishingly similar to the organs affected by Covid-19.","version":"1.1","doi":"10.1101/2020.04.06.027318","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.20.001008","pub_date":"2020-4-06","title":"DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP","abstract":"The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.","version":"1.2","doi":"10.1101/2020.03.20.001008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.006197","pub_date":"2020-4-06","title":"In-Silico evidence for two receptors based strategy of SARS-CoV-2","abstract":"We propose a novel numerical method able to determine efficiently and effectively the relationship of complementarity between portions of protein surfaces. This innovative and general procedure, based on the representation of the molecular iso-electron density surface in terms of 2D Zernike polynomials, allows the rapid and quantitative assessment of the geometrical shape complementarity between interacting proteins, that was unfeasible with previous methods. We first tested the method with a large dataset of known protein complexes obtaining an overall area under the ROC curve of 0.76 in the blind recognition of binding sites and then applied it to investigate the features of the interaction between the Spike protein of SARS-CoV-2 and human cellular receptors. Our results indicate that SARS-CoV-2 uses a dual strategy: its spike protein could also interact with sialic acid receptors of the cells in the upper airways, in addition to the known interaction with Angiotensin-converting enzyme 2.","version":"1.2","doi":"10.1101/2020.03.24.006197","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.020966","pub_date":"2020-4-06","title":"Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2","abstract":"The current emergence of the novel coronavirus pandemic caused by SARS-CoV-2 demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor binding domain (RBD) of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation is strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research towards the development of a suitable vaccine candidate.","version":"1.2","doi":"10.1101/2020.04.01.020966","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.27.968008","pub_date":"2020-4-06","title":"Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is an Inconvenient Target for Small-Molecule Inhibitors Design","abstract":"The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar SARS Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins show major differences in both shape and size indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site\u2019s conformational changes during the simulation time indicates its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicates that the virus\u2019 mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.","version":"1.3","doi":"10.1101/2020.02.27.968008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.25.963546","pub_date":"2020-4-06","title":"An Effective CTL Peptide Vaccine for Ebola Zaire Based on Survivors\u2019 CD8+ Targeting of a Particular Nucleocapsid Protein Epitope with Potential Implications for COVID-19 Vaccine Design","abstract":"The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus.","version":"1.4","doi":"10.1101/2020.02.25.963546","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.008805","pub_date":"2020-4-06","title":"An artificial intelligence-based first-line defence against COVID-19: digitally screening citizens for risks via a chatbot","abstract":"To combat the pandemic of the coronavirus disease (COVID-19), numerous governments have established phone hotlines to prescreen potential cases. These hotlines have struggled with the volume of callers, leading to wait times of hours or, even, an inability to contact health authorities. Symptoma is a symptom-to-disease digital health assistant that can differentiate more than 20,000 diseases with an accuracy of more than 90%. We tested the accuracy of Symptoma to identify COVID-19 using a set of diverse clinical cases combined with case reports of COVID-19. We showed that Symptoma can accurately distinguish COVID-19 in 96.32% of clinical cases. When considering only COVID-19 symptoms and risk factors, Symptoma identified 100% of those infected when presented with only three signs. Lastly, we showed that Symptoma\u2019s accuracy far exceeds that of simple \u201cyes-no\u201d questionnaires widely available online. In summary, Symptoma provides unparalleled accuracy in systematically identifying cases of COVID-19 while also considering over 20,000 other diseases. Furthermore, Symptoma allows free text input, furthered with disease-specific follow up questions, in 36 languages. Combined, these results and accessibility give Symptoma the potential to be a key tool in the global fight against COVID-19. The Symptoma predictor is freely available online at https://www.symptoma.com.","version":"1.3","doi":"10.1101/2020.03.25.008805","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.022764","pub_date":"2020-4-05","title":"Potent Antiviral Activities of Type I Interferons to SARS-CoV-2 Infection","abstract":"The ongoing historic outbreak of COVID-19 not only constitutes a global public health crisis, but also carries a devastating social and economic impact. The disease is caused by a newly identified coronavirus, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). There is an urgent need to identify antivirals to curtail the COVID-19 pandemic. Herein, we report the remarkable sensitivity of SARS-CoV-2 to recombinant human interferons \u03b1 and \u03b2 (IFN\u03b1/\u03b2). Treatment with IFN-\u03b1 at a concentration of 50 international units (IU) per milliliter drastically reduces viral titers by 3.4 log or over 4 log, respectively, in Vero cells. The EC50 of IFN-\u03b1 and IFN-\u03b2 treatment is 1.35 IU/ml and 0.76 IU/ml, respectively, in Vero cells. These results suggest that SARS-CoV-2 is more sensitive than many other human pathogenic viruses, including SARS-CoV. Overall, our results demonstrate the potent efficacy of human Type I IFN in suppressing SARS-CoV-2 infection, a finding which could inform future treatment options for COVID-19.","version":"1.2","doi":"10.1101/2020.04.02.022764","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.023846","pub_date":"2020-4-05","title":"In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication","abstract":"A novel coronavirus, named SARS-CoV-2, emerged in 2019 from Hubei region in China and rapidly spread worldwide. As no approved therapeutics exists to treat Covid-19, the disease associated to SARS-Cov-2, there is an urgent need to propose molecules that could quickly enter into clinics. Repurposing of approved drugs is a strategy that can bypass the time consuming stages of drug development. In this study, we screened the Prestwick Chemical Library\u00ae composed of 1,520 approved drugs in an infected cell-based assay. 90 compounds were identified. The robustness of the screen was assessed by the identification of drugs, such as Chloroquine derivatives and protease inhibitors, already in clinical trials. The hits were sorted according to their chemical composition and their known therapeutic effect, then EC50 and CC50 were determined for a subset of compounds. Several drugs, such as Azithromycine, Opipramol, Quinidine or Omeprazol present antiviral potency with 2<EC50<20\u00b5M. By providing new information on molecules inhibiting SARS-CoV-2 replication in vitro, this study could contribute to the short-term repurposing of drugs against Covid-19.","version":"1.1","doi":"10.1101/2020.04.03.023846","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.024257","pub_date":"2020-4-05","title":"SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles","abstract":"SARS-CoV-2 is a novel coronavirus currently causing a pandemic. We show that the majority of amino acid positions, which differ between SARS-CoV-2 and the closely related SARS-CoV, are differentially conserved suggesting differences in biological behaviour. In agreement, novel cell culture models revealed differences between the tropism of SARS-CoV-2 and SARS-CoV. Moreover, cellular ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (enables virus entry via S protein cleavage) levels did not reliably indicate cell susceptibility to SARS-CoV-2. SARS-CoV-2 and SARS-CoV further differed in their drug sensitivity profiles. Thus, only drug testing using SARS-CoV-2 reliably identifies therapy candidates. Therapeutic concentrations of the approved protease inhibitor aprotinin displayed anti-SARS-CoV-2 activity. The efficacy of aprotinin and of remdesivir (currently under clinical investigation against SARS-CoV-2) were further enhanced by therapeutic concentrations of the proton pump inhibitor omeprazole (aprotinin 2.7-fold, remdesivir 10-fold). Hence, our study has also identified anti-SARS-CoV-2 therapy candidates that can be readily tested in patients.","version":"1.1","doi":"10.1101/2020.04.03.024257","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.022194","pub_date":"2020-4-05","title":"Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus disease 2019 (COVID-19) which is currently negatively affecting the population and disrupting the global economy. SARS-CoV-2 belongs to the +RNA virus family that utilize single-stranded positive-sense RNA molecules as genomes. SARS-CoV-2, like other coronaviruses, has an unusually large genome for a +RNA virus that encodes four structural proteins \u2013 the matrix (M), small envelope (E), spike (S) and nucleocapsid phosphoprotein (N) - and sixteen nonstructural proteins (nsp1-16) that together ensure replication of the virus in the host cell. The nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Its N-terminal RNA binding domain (N-NTD) captures the RNA genome while the C-terminal domain anchors the ribonucleoprotein complex to the viral membrane via its interaction with the M protein. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD lined with arginine residues suggesting a putative RNA binding site. Next, we performed an NMR titration experiment using an RNA duplex. The observed changes in positions of signals in the N-NTD NMR spectra allowed us to construct a model of the N-NTD in complex with RNA.","version":"1.1","doi":"10.1101/2020.04.02.022194","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.022939","pub_date":"2020-4-05","title":"Triphosphates of the Two Components in DESCOVY and TRUVADA are Inhibitors of the SARS-CoV-2 Polymerase","abstract":"SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. We previously demonstrated that four nucleotide analogues (specifically, the active triphosphate forms of Sofosbuvir, Alovudine, AZT and Tenofovir alafenamide) inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Tenofovir and emtricitabine are the two components in DESCOVY and TRUVADA, the two FDA-approved medications for use as pre-exposure prophylaxis (PrEP) to prevent HIV infection. This is a preventative method in which individuals who are HIV negative (but at high-risk of contracting the virus) take the combination drug daily to reduce the chance of becoming infected with HIV. PrEP can stop HIV from replicating and spreading throughout the body. We report here that the triphosphates of tenofovir and emtricitabine, the two components in DESCOVY and TRUVADA, act as terminators for the SARS-CoV-2 RdRp catalyzed reaction. These results provide a molecular basis to evaluate the potential of DESCOVY and TRUVADA as PrEP for COVID-19.","version":"1.1","doi":"10.1101/2020.04.03.022939","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.021725","pub_date":"2020-4-05","title":"Energetics based epitope screening in SARS CoV-2 (COVID 19) spike glycoprotein by Immuno-informatic analysis aiming to a suitable vaccine development","abstract":"The recent outbreak by SARS-CoV-2 has generated a chaos in global health and economy and claimed/infected a large number of lives. Closely resembling with SARS CoV, the present strain has manifested exceptionally higher degree of spreadability, virulence and stability possibly due to some unidentified mutations. The viral spike glycoprotein is very likely to interact with host Angiotensin-Converting Enzyme 2 (ACE2) and transmits its genetic materials and hijacks host machinery with extreme fidelity for self propagation. Few attempts have been made to develop a suitable vaccine or ACE2 blocker or virus-receptor inhibitor within this short period of time. Here, attempt was taken to develop some therapeutic and vaccination strategies with a comparison of spike glycoproteins among SARS-CoV, MERS-CoV and the SARS-CoV-2. We verified their structure quality (SWISS-MODEL, Phyre2, Pymol) topology (ProFunc), motifs (MEME Suite, GLAM2Scan), gene ontology based conserved domain (InterPro database) and screened several epitopes (SVMTrip) of SARS CoV-2 based on their energetics, IC50 and antigenicity with regard to their possible glycosylation and MHC/paratopic binding (Vaxigen v2.0, HawkDock, ZDOCK Server) effects. We screened here few pairs of spike protein epitopic regions and selected their energetic, IC50, MHC II reactivity and found some of those to be very good target for vaccination. A possible role of glycosylation on epitopic region showed profound effects on epitopic recognition. The present work might be helpful for the urgent development of a suitable vaccination regimen against SARS CoV-2.","version":"1.1","doi":"10.1101/2020.04.02.021725","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.019877","pub_date":"2020-4-05","title":"Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo","abstract":"SARS-CoV-2 has rapidly become a pandemic worldwide; therefore, an effective vaccine is urgently needed. Recently, messenger RNAs (mRNAs) have emerged as a promising platform for vaccination. Here, we systematically investigated the untranslated regions (UTRs) of mRNAs in order to enhance protein production. Through a comprehensive analysis of endogenous gene expression and de novo design of UTRs, we identified the optimal combination of 5\u2019 and 3\u2019 UTR, termed as NASAR, which was five to ten-fold more efficient than the tested endogenous UTRs. More importantly, NASAR mRNAs delivered by lipid-derived nanoparticles showed dramatic expression of potential SARS-CoV-2 antigens both in vitro and in vivo. These NASAR mRNAs merit further development as alternative SARS-CoV-2 vaccines.","version":"1.1","doi":"10.1101/2020.04.01.019877","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.022723","pub_date":"2020-4-05","title":"Prediction and Evolution of B Cell Epitopes of Surface Protein in SARS-CoV-2","abstract":"The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. The sequences of the surface protein of SARS-CoV-2 and its proximal sequences were obtained by BLAST, the sequences of the whole genome of SARS-CoV-2 were obtained from the GenBank. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed. 7 epitopes were predicted, including 5 linear epitopes and 2 conformational epitopes, one of the linear and one of the conformational were coincide. The epitope D mutated easily, but the other epitopes were very conservative and the epitope C was the most conservative. It is worth mentioning that all of the 6 dominated epitopes were absolutely conservative in nearly 1000 SARS-CoV-2 genomes, and they deserved further study. The findings would facilitate the vaccine development, had the potential to be directly applied on the treatment in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.","version":"1.1","doi":"10.1101/2020.04.03.022723","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.017624","pub_date":"2020-4-05","title":"Indomethacin has a potent antiviral activity against SARS CoV-2 in vitro and canine coronavirus in vivo","abstract":"The outbreak of SARS CoV-2 has caused ever-increasing attention and public panic all over the world. Currently, there is no specific treatment against the SARS CoV-2. Therefore, identifying effective antiviral agents to combat the disease is urgently needed. Previous studies found that indomethacin has the ability to inhibit the replication of several unrelated DNA and RNA viruses, including SARS-CoV. SARS CoV-2 pseudovirus-infected African green monkey kidney VERO E6 cells treated with different concentrations of indomethacin or aspirin at 48 hours post infection (p.i). The level of cell infection was determined by luciferase activity. Anti-coronavirus efficacy in vivo was confirmed by evaluating the time of recovery in canine coronavirus (CCV) infected dogs treated orally with 1mg/kg body weight indomethacin. We found that indomethacin has a directly and potently antiviral activity against the SARS CoV-2 pseudovirus (reduce relative light unit to zero). In CCV-infected dogs, recovery occurred significantly sooner with symptomatic treatment + oral indomethacin (1 mg/kg body weight) daily treatments than with symptomatic treatment + ribavirin (10-15 mg/kg body weight) daily treatments (P =0.0031), but was not significantly different from that with symptomatic treatment + anti-canine coronavirus serum + canine hemoglobin + canine blood immunoglobulin + interferon treatments (P =0.7784). The results identify indomethacin as a potent inhibitor of SARS CoV-2.","version":"1.1","doi":"10.1101/2020.04.01.017624","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.020594","pub_date":"2020-4-05","title":"A snapshot of SARS-CoV-2 genome availability up to 30th March, 2020 and its implications","abstract":"The SARS-CoV-2 pandemic has been growing exponentially, affecting nearly 900 thousand people and causing enormous distress to economies and societies worldwide. A plethora of analyses based on viral sequences has already been published, in scientific journals as well as through non-peer reviewed channels, to investigate SARS-CoV-2 genetic heterogeneity and spatiotemporal dissemination. We examined full genome sequences currently available to assess the presence of sufficient information for reliable phylogenetic and phylogeographic studies in countries with the highest toll of confirmed cases. Although number of-available full-genomes is growing daily, and the full dataset contains sufficient phylogenetic information that would allow reliable inference of phylogenetic relationships, country-specific SARS-CoV-2 datasets still present severe limitations. Studies assessing within country spread or transmission clusters should be considered preliminary at best, or hypothesis generating. Hence the need for continuing concerted efforts to increase number and quality of the sequences required for robust tracing of the epidemic. Although genome sequences of SARS-CoV-2 are growing daily and contain sufficient phylogenetic information, country-specific data still present severe limitations and should be interpreted with caution.","version":"1.1","doi":"10.1101/2020.04.01.020594","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.022186","pub_date":"2020-4-05","title":"Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods","abstract":"The COVID-19 global pandemic is an unprecedented health emergency. Insufficient access to testing has hampered effective public health interventions and patient care management in a number of countries. Furthermore, the availability of regulatory-cleared reagents has challenged widespread implementation of testing. We rapidly developed a qRT-PCR SARS-CoV-2 detection assay using a 384-well format and tested its analytic performance across multiple nucleic acid extraction kits. Our data shows robust analytic accuracy on residual clinical biospecimens. Limit of detection sensitivity and specificity was confirmed with currently available commercial reagents. Our methods and results provide valuable information for other high-complexity laboratories seeking to develop effective, local, laboratory-developed procedures with high-throughput capability to detect SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.04.02.022186","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.021469","pub_date":"2020-4-05","title":"LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2","abstract":"C3A is a sub-clone of human hepatoblastoma HepG2 cell line with the strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ADAP2, GILT and LY6E, three cellular proteins with known activity of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a distinct mechanism. Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.","version":"1.1","doi":"10.1101/2020.04.02.021469","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.05.026187","pub_date":"2020-4-05","title":"\u201cAmantadine disrupts lysosomal gene expression; potential therapy for COVID19\u201d","abstract":"SARS-coronavirus 2 is the causal agent of the COVID-19 outbreak. SARS-Cov-2 entry into a cell is dependent upon binding of the viral spike (S) protein to cellular receptor and on cleavage of the spike protein by the host cell proteases such as Cathepsin L and Cathepsin B. CTSL/B are crucial elements of lysosomal pathway and both enzymes are almost exclusively located in the lysosomes.CTSL disruption offers potential for CoVID-19 therapies. The mechanisms of disruption include: decreasing expression of CTSL, direct inhibition of CTSL activity and affecting the conditions of CTSL environment (increase pH in lysosomes). We have conducted a high throughput drug screen gene expression analysis to identify compounds that would downregulate the expression of CTSL/CTSB. One of the top significant results shown to downregulate the expression of the CTSL gene is Amantadine. Amantadine was approved by the US Food and Drug Administration in 1968 as a prophylactic agent for influenza and later for Parkinson\u2019s disease. It is available as a generic drug.. Amantadine in addition to downregulating CTSL appears to further disrupt lysosomal pathway, hence interfering with the capacity of the virus to replicate. It acts as a lysosomotropic agent altering the CTSL functional environment. We hypothesize that Amantadine could decrease the viral load in SARS-CoV-2 positive patients and as such it may serve as a potent therapeutic decreasing the replication and infectivity of the virus likely leading to better clinical outcomes. Clinical studies will be needed to examine the therapeutic utility of amantadine in COVID-19 infection.","version":"1.2","doi":"10.1101/2020.04.05.026187","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.02.021451","pub_date":"2020-4-05","title":"Decrease in ACE2 mRNA expression in aged mouse lung","abstract":"Angiotensin-converting enzyme 2 (ACE2) has been identified as a critical receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has led to extensive speculation on the role of ACE2 in disease severity, and in particular, whether variation in its expression can explain higher mortality in older individuals. We examine this question in mouse lung and show that 24-month old mice have significantly reduced ACE2 mRNA expression relative to 3-month old mice. The differences appear to be localized to ciliated cells.","version":"1.1","doi":"10.1101/2020.04.02.021451","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.03.024216","pub_date":"2020-4-05","title":"Increasing testing throughput and case detection with a pooled-sample Bayesian approach in the context of COVID-19","abstract":"Rapid and widespread implementation of infectious disease surveillance is a critical component in the response to novel health threats. Molecular assays are the preferred method to detect a broad range of pathogens with high sensitivity and specificity. The implementation of molecular assay testing in a rapidly evolving public health emergency can be hindered by resource availability or technical constraints. In the context of the COVID-19 pandemic, the applicability of a pooled-sample testing protocol to screen large populations more rapidly and with limited resources is discussed. A Bayesian inference analysis in which hierarchical testing stages can have different sensitivities is implemented and benchmarked against early COVID-19 testing data. Optimal pool size and increases in throughput and case detection are calculated as a function of disease prevalence. Even for moderate losses in test sensitivity upon pooling, substantial increases in testing throughput and detection efficiency are predicted, suggesting that sample pooling is a viable avenue to circumvent current testing bottlenecks for COVID-19.","version":"1.1","doi":"10.1101/2020.04.03.024216","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.018499","pub_date":"2020-4-04","title":"In-silico analysis of SARS-CoV-2 genomes: Insights from SARS encoded non-coding RNAs","abstract":"Recently a novel coronavirus (SARS-CoV-2) emerged from Wuhan, China and has infected more than 571000 people leading to more than 26000 deaths. Since SARS-CoV-2 genome sequences show similarity with those of SARS, we sought to analyze all the available SARS-CoV-2 genomes based on the insights obtained from SARS genome specifically focusing on non-coding RNAs. Here, results are presented from the dual approach i.e identifying host encoded miRNAs that might regulate viral pathogenesis as well as identifying viral encoded miRNAs that might regulate host cell signaling pathways and aid in viral pathogenesis. Analysis utilizing first approach resulted in the identification of 10 host encoded miRNAs that could target the genome of both the viruses (SARS-CoV-2 and SARS reference genome). Interestingly our analysis revealed that there is significantly higher number of host miRNAs that could target SARS-CoV-2 genome as compared to the SARS reference genome. Results from second approach involving SARS-CoV-2 and SARS reference genome identified a set of virus encoded miRNAs which might regulate host signaling pathways. Our analysis further identified a similar \u201cGA\u201d rich motif in SARS-CoV-2 genome that was shown to play a vital role in lung pathogenesis during severe SARS infections. Hence, we successfully identified human and virus encoded miRNAs that might regulate pathogenesis of both these coronaviruses and the fact that more number of host miRNAs could target SARS-CoV-2 genomes possibly reveal as to why this virus follows mild pathogenesis in healthy individuals. We identified non-coding sequences in SARS-CoV-2 genomes that were earlier reported to contribute towards SARS pathology. The study provides insights into the overlapping sequences among these viruses for their effective inhibition as well as identifying new drug targets that could be used for development of new antivirals.","version":"1.1","doi":"10.1101/2020.03.31.018499","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.016790","pub_date":"2020-4-04","title":"Comparative genomics suggests limited variability and similar evolutionary patterns between major clades of SARS-CoV-2","abstract":"Phylogenomic analysis of SARS-CoV-2 as available from publicly available repositories suggests the presence of 3 prevalent groups of viral episomes (super-clades), which are mostly associated with outbreaks in distinct geographic locations (China, USA and Europe). While levels of genomic variability between SARS-CoV-2 isolates are limited, to our knowledge, it is not clear whether the observed patterns of variability in viral super-clades reflect ongoing adaptation of SARS-CoV-2, or merely genetic drift and founder effects. Here, we analyze more than 1100 complete, high quality SARS-CoV-2 genome sequences, and provide evidence for the absence of distinct evolutionary patterns/signatures in the genomes of the currently known major clades of SARS-CoV-2. Our analyses suggest that the presence of distinct viral episomes at different geographic locations are consistent with founder effects, coupled with the rapid spread of this novel virus. We observe that while cross species adaptation of the virus is associated with hypervariability of specific protein coding regions (including the RDB domain of the spike protein), the more variable genomic regions between extant SARS-CoV-2 episomes correspond with the 3\u2019 and 5\u2019 UTRs, suggesting that at present viral protein coding genes should not be subjected to different adaptive evolutionary pressures in different viral strains. Although this study can not be conclusive, we believe that the evidence presented here is strongly consistent with the notion that the biased geographic distribution of SARS-CoV-2 isolates should not be associated with adaptive evolution of this novel pathogen.","version":"1.2","doi":"10.1101/2020.03.30.016790","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.990598","pub_date":"2020-4-03","title":"When Darkness Becomes a Ray of Light in the Dark Times: Understanding the COVID-19 via the Comparative Analysis of the Dark Proteomes of SARS-CoV-2, Human SARS and Bat SARS-Like Coronaviruses","abstract":"Recently emerged coronavirus designated as SARS-CoV-2 (also known as 2019 novel coronavirus (2019-nCoV) or Wuhan coronavirus) is a causative agent of coronavirus disease 2019 (COVID-19), which is rapidly spreading throughout the world now. More than 9,00,000 cases of SARS-CoV-2 infection and more than 47,000 COVID-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. World Health Organization (WHO) has declared the SARS-CoV-2 spread as a global public health emergency and admitted that the COVID-19 is a pandemic now. The multiple sequence alignment data correlated with the already published reports on the SARS-CoV-2 evolution and indicated that this virus is closely related to the bat Severe Acute Respiratory Syndrome-like coronavirus (bat SARS-like CoV) and the well-studied Human SARS coronavirus (SARS CoV). The disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. Therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of SARS-CoV-2, bat SARS-like, and human SARS CoVs by analysing the prevalence of intrinsic disorder in their proteins. According to our findings, SARS-CoV-2 proteome contains very significant levels of structural order. In fact, except for Nucleocapsid, Nsp8, and ORF6, the vast majority of SARS-CoV-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (IDPRs). However, IDPRs found in SARS-CoV-2 proteins are functionally important. For example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all SARS-CoV-2 proteins were shown to contain molecular recognition features (MoRFs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. The results of our extensive investigation of the dark side of the SARS-CoV-2 proteome will have important implications for the structural and non-structural biology of SARS or SARS-like coronaviruses. The infection caused by a novel coronavirus (SARS-CoV-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current COVID-19 pandemic. No in-depth information on structures and functions of SARS-CoV-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. Our study provides the first comparative analysis of the order- and disorder-based features of the SARS-CoV-2 proteome relative to human SARS and bat CoV that may be useful for structure-based drug discovery.","version":"1.2","doi":"10.1101/2020.03.13.990598","journal":"bioRxiv","score":null},{"id":"10.1101/2020.04.01.021196","pub_date":"2020-4-03","title":"SARS-CoV-2 neutralizing serum antibodies in cats: a serological investigation","abstract":"Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, and rapidly spread worldwide. Previous studies suggested cat could be a potential susceptible animal of SARS-CoV-2. Here, we investigated the infection of SARS-CoV-2 in cats by detecting specific serum antibodies. A cohort of serum samples were collected from cats in Wuhan, including 102 sampled after COVID-19 outbreak, and 39 prior to the outbreak. 15 of 102 (14.7%) cat sera collected after the outbreak were positive for the receptor binding domain (RBD) of SARS-CoV-2 by indirect enzyme linked immunosorbent assay (ELISA). Among the positive samples, 11 had SARS-CoV-2 neutralizing antibodies with a titer ranging from 1/20 to 1/1080. No serological cross-reactivity was detected between the SARS-CoV-2 and type I or II feline infectious peritonitis virus (FIPV). Our data demonstrates that SARS-CoV-2 has infected cat population in Wuhan during the outbreak.","version":"1.1","doi":"10.1101/2020.04.01.021196","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.05.976167","pub_date":"2020-4-03","title":"Direct RNA sequencing and early evolution of SARS-CoV-2","abstract":"Fundamental aspects of SARS-CoV-2 biology remain to be described, having the potential to provide insight to the response effort for this high-priority pathogen. Here we describe the first native RNA sequence of SARS-CoV-2, detailing the coronaviral transcriptome and epitranscriptome, and share these data publicly. A data-driven inference of viral genetic features and evolutionary rate is also made. The rapid sharing of sequence information throughout the SARS-CoV-2 pandemic represents an inflection point for public health and genomic epidemiology, providing early insights into the biology and evolution of this emerging pathogen.","version":"1.2","doi":"10.1101/2020.03.05.976167","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.017459","pub_date":"2020-4-03","title":"In silico approach for designing of a multi-epitope based vaccine against novel Coronavirus (SARS-COV-2)","abstract":"A novel Coronavirus (SARS-COV-2) has now become a global pandemic. Considering the severity of infection and the associated mortalities, there is an urgent need to develop an effective preventive measure against this virus. In this study, we have designed a novel vaccine construct using computational strategies. Spike (S) glycoprotein is the major antigenic component that trigger the host immune responses. Detailed investigation of S protein with various immunoinformatics tools enabled us to identify 5 MHC I and 5 MHC II B-cell derived T-cell epitopes with VaxiJen score > 1 and IC50 value < 100nM. These epitopes were joined with a suitable adjuvant and appropriate linkers to form a multi-epitope based vaccine construct. Further, in silico testing of the vaccine construct for its antigenicity, allergenicity, solubility, and other physicochemical properties showed it to be safe and immunogenic. Suitable tertiary structure of the vaccine protein was generated using 3Dpro of SCRATCH suite, refined with GalaxyRefine, and validated with ProSA, PROCHECK, and ERRAT server. Finally, molecular docking studies were performed to ensure a favorable binding affinity between the vaccine construct and TLR3 receptor. The designed multi-epitope vaccine showed potential to elicit specific immune responses against the SARS-COV-2. However, further wet lab validation is necessary to confirm the actual effectiveness, safety and immunogenic potency of the vaccine construct against derived in this study.","version":"1.1","doi":"10.1101/2020.03.31.017459","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.019216","pub_date":"2020-4-02","title":"Virus-host interactome and proteomic survey of PMBCs from COVID-19 patients reveal potential virulence factors influencing SARS-CoV-2 pathogenesis","abstract":"The ongoing coronavirus disease (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a global public health concern due to relatively easy person-to-person transmission and the current lack of effective antiviral therapy. However, the exact molecular mechanisms of SARS-CoV-2 pathogenesis remain largely unknown. We exploited an integrated proteomics approach to systematically investigate intra-viral and virus-host interactomes for the identification of unrealized SARS-CoV-2 host targets and participation of cellular proteins in the response to viral infection using peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Using this approach, we elucidated 251 host proteins targeted by SARS-CoV-2 and more than 200 host proteins that are significantly perturbed in COVID-19 derived PBMCs. From the interactome, we further identified that non-structural protein nsp9 and nsp10 interact with NKRF, a NF-\u041aB repressor, and may precipitate the strong IL-8/IL-6 mediated chemotaxis of neutrophils and overexuberant host inflammatory response observed in COVID-19 patients. Our integrative study not only presents a systematic examination of SARS-CoV-2-induced perturbation of host targets and cellular networks to reflect disease etiology, but also reveals insights into the mechanisms by which SARS-CoV-2 triggers cytokine storms and represents a powerful resource in the quest for therapeutic intervention.","version":"1.1","doi":"10.1101/2020.03.31.019216","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.017889","pub_date":"2020-4-02","title":"Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia","abstract":"In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the lack of biologically relevant pre-clinical experimental models of SARS-CoV-2 infection as a complement of classic cell lines represents a major barrier for scientific and medical progress. Here, we advantageously used human reconstituted airway epithelial models of nasal or bronchial origin to characterize viral infection kinetics, tissue-level remodeling of the cellular ultrastructure and transcriptional immune signatures induced by SARS-CoV-2. Our results underline the relevance of this model for the preclinical evaluation of antiviral candidates. Foremost, we provide evidence on the antiviral efficacy of remdesivir and the therapeutic potential of the remdesivir-diltiazem combination as a rapidly available option to respond to the current unmet medical need imposed by COVID-19. New insights on SARS-CoV-2 biology and drug combination therapies against COVID-19.","version":"1.1","doi":"10.1101/2020.03.31.017889","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.015941","pub_date":"2020-4-02","title":"Identification of a common deletion in the spike protein of SARS-CoV-2","abstract":"Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.","version":"1.1","doi":"10.1101/2020.03.31.015941","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.018978","pub_date":"2020-4-02","title":"A SARS-CoV-2 Vaccination Strategy Focused on Population-Scale Immunity","abstract":"Here we propose a vaccination strategy for SARS-CoV-2 based on identification of both highly conserved regions of the virus and newly acquired adaptations that are presented by MHC class I and II across the vast majority of the population, are highly dissimilar from the human proteome, and are predicted B cell epitopes. We present 65 peptide sequences that we expect to result in a safe and effective vaccine which can be rapidly tested in DNA, mRNA, or synthetic peptide constructs. These include epitopes that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor, and within a novel furin cleavage site thought to increase membrane fusion. This vaccination strategy specifically targets unique vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the human population.","version":"1.2","doi":"10.1101/2020.03.31.018978","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.009209","pub_date":"2020-4-01","title":"Scrutinizing the SARS-CoV-2 protein information for the designing an effective vaccine encompassing both the T-cell and B-cell epitopes","abstract":"Novel SARS coronavirus (SARS-CoV-2) has caused a pandemic condition world-wide and has been declared as public health emergency of International concern by WHO in a very short span of time. The community transmission of this highly infectious virus has severely affected various parts of China, Italy, Spain and USA among others. The prophylactic solution against SARS-CoV-2 infection is challenging due to the high mutation rate of its RNA genome. Herein, we exploited a next generation vaccinology approach to construct a multi-epitope vaccine candidate against SARS-CoV-2 with high antigenicity, safety and efficacy to combat this deadly infectious agent. The whole proteome was scrutinized for the screening of highly conserved, antigenic, non-allergen and non-toxic epitopes having high population coverage that can elicit both humoral and cellular mediated immune response against COVID-19 infection. These epitopes along with four different adjuvants were utilized to construct a multi-epitope vaccine candidate that can generate strong immunological memory response having high efficacy in humans. Various physiochemical analyses revealed the formation of a stable vaccine product having a high propensity to form a protective solution against the detrimental SARS-CoV-2 strain with high efficacy. The vaccine candidate interacted with immunological receptor TLR3 with high affinity depicting the generation of innate immunity. Further, the codon optimization and in silico expression show the plausibility of the high expression and easy purification of the vaccine product. Thus, this present study provides an initial platform of the rapid generation of an efficacious protective vaccine for combating COVID-19.","version":"1.1","doi":"10.1101/2020.03.26.009209","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015008","pub_date":"2020-3-31","title":"Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic","abstract":"There are outstanding evolutionary questions on the recent emergence of coronavirus SARS-CoV-2/hCoV-19 in Hubei province that caused the COVID-19 pandemic, including (1) the relationship of the new virus to the SARS-related coronaviruses, (2) the role of bats as a reservoir species, (3) the potential role of other mammals in the emergence event, and (4) the role of recombination in viral emergence. Here, we address these questions and find that the sarbecoviruses \u2013 the viral subgenus responsible for the emergence of SARS-CoV and SARS-CoV-2 \u2013 exhibit frequent recombination, but the SARS-CoV-2 lineage itself is not a recombinant of any viruses detected to date. In order to employ phylogenetic methods to date the divergence events between SARS-CoV-2 and the bat sarbecovirus reservoir, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Bayesian evolutionary rate and divergence date estimates were consistent for all three recombination-free alignments and robust to two different prior specifications based on HCoV-OC43 and MERS-CoV evolutionary rates. Divergence dates between SARS-CoV-2 and the bat sarbecovirus reservoir were estimated as 1948 (95% HPD: 1879-1999), 1969 (95% HPD: 1930-2000), and 1982 (95% HPD: 1948-2009). Despite intensified characterization of sarbecoviruses since SARS, the lineage giving rise to SARS-CoV-2 has been circulating unnoticed for decades in bats and been transmitted to other hosts such as pangolins. The occurrence of a third significant coronavirus emergence in 17 years together with the high prevalence and virus diversity in bats implies that these viruses are likely to cross species boundaries again. The Betacoronavirus SARS-CoV-2 is a member of the sarbecovirus subgenus which shows frequent recombination in its evolutionary history. We characterize the extent of this genetic exchange and identify non-recombining regions of the sarbecovirus genome using three independent methods to remove the effects of recombination. Using these non-recombining genome regions and prior information on coronavirus evolutionary rates, we obtain estimates from three approaches that the most likely divergence date of SARS-CoV-2 from its most closely related available bat sequences ranges from 1948 to 1982. RaTG13 is the closest available bat virus to SARS-CoV-2; a sub-lineage of these bat viruses is able to infect humans. Two sister lineages of the RaTG13/SARS-CoV-2 lineage infect Malayan pangolins. The sarbecoviruses show a pattern of deep recombination events, indicating that there are high levels of co-infection in horseshoe bats and that the viral pool can generate novel allele combinations and substantial genetic diversity; the sarbecoviruses are efficient \u2018explorers\u2019 of phenotype space. The SARS-CoV-2 lineage is not a recent recombinant, at least not involving any of the bat or pangolin viruses sampled to date. Non-recombinant regions of the sarbecoviruses can be identified, allowing for phylogenetic inference and dating to be performed. We constructed three such regions using different methods. We estimate that RaTG13 and SARS-CoV-2 diverged 40 to 70 years ago. There is a diverse unsampled reservoir of generalist viruses established in horseshoe bats. While an intermediate host responsible for the zoonotic event cannot be ruled out, the relevant evolution for spillover to humans very likely occurred in horseshoe bats.","version":"1.1","doi":"10.1101/2020.03.30.015008","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015990","pub_date":"2020-3-31","title":"Fully human single-domain antibodies against SARS-CoV-2","abstract":"The COVID-19 pandemic is spreading rapidly, highlighting the urgent need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We describe here the development of a phage-displayed single-domain antibody library by grafting na\u00efve CDRs into framework regions of an identified human germline IGHV allele. This enabled the isolation of high-affinity single-domain antibodies of fully human origin. The panning using SARS-CoV-2 RBD and S1 as antigens resulted in the identification of antibodies targeting five types of neutralizing or non-neutralizing epitopes on SARS-CoV-2 RBD. These fully human single-domain antibodies bound specifically to SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of them were found to potently neutralize pseudotyped and live virus, and therefore may represent promising candidates for prophylaxis and therapy of COVID-19. This study also reports unique immunogenic profile of SARS-CoV-2 RBD compared to that of SARS-CoV and MERS-CoV, which may have important implications for the development of effective vaccines against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.03.30.015990","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015347","pub_date":"2020-3-31","title":"Susceptibility of ferrets, cats, dogs, and different domestic animals to SARS-coronavirus-2","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19, which was first reported in Wuhan, China in December, 2019. Despite the tremendous efforts to control the disease, COVID-19 has now spread to over 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are completely unknown. Here, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but efficiently in ferrets and cats. We found that the virus transmits in cats via respiratory droplets. Our study provides important insights into the animal reservoirs of SARS-CoV-2 and animal management for COVID-19 control.","version":"1.1","doi":"10.1101/2020.03.30.015347","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.014407","pub_date":"2020-3-31","title":"Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro","abstract":"While the SARS-CoV-2 pandemic is hardly hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 cells. This strongly suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.","version":"1.1","doi":"10.1101/2020.03.29.014407","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015461","pub_date":"2020-3-31","title":"Potent neutralizing antibodies in the sera of convalescent COVID-19 patients are directed against conserved linear epitopes on the SARS-CoV-2 spike protein","abstract":"The ongoing SARS-CoV-2 pandemic demands rapid identification of immunogenic targets for the design of efficient vaccines and serological detection tools. In this report, using pools of overlapping linear peptides and functional assays, we present two immunodominant regions on the spike glycoprotein that were highly recognized by neutralizing antibodies in the sera of COVID-19 convalescent patients. One is highly specific to SARS-CoV-2, and the other is a potential pan-coronavirus target.","version":"1.1","doi":"10.1101/2020.03.30.015461","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015644","pub_date":"2020-3-31","title":"Atlas of ACE2 gene expression in mammals reveals novel insights in transmisson of SARS-Cov-2","abstract":"COVID-19 has become a worldwide pandemic. It is caused by a novel coronavirus named SARS-CoV-2 with elusive origin. SARS-CoV-2 infects mammalian cells by binding to ACE2, a transmembrane protein. Therefore, the conservation of ACE2 and its expression pattern across mammalian species, which are yet to be comprehensively investigated, may provide valuable insights into tracing potential hosts of SARS-CoV-2. We analyzed gene conservation of ACE2 across mammals and collected more than 140 transcriptome datasets from human and common mammalian species, including presumed hosts of SARS-CoV-2 and other animals in close contact with humans. In order to enable comparisons across species and tissues, we used a unified pipeline to quantify and normalize ACE2 expression levels. We first found high conservation of ACE2 genes among common mammals at both DNA and peptide levels, suggesting that a broad range of mammalian species can potentially be the hosts of SARS-CoV-2. Next, we showed that high level of ACE2 expression in certain human tissues is consistent with clinical symptoms of COVID-19 patients. Furthermore, we observed that ACE2 expressed in a species-specific manner in the mammals examined. Notably, high expression in skin and eyes in cat and dog suggested that these animals may play roles in transmitting SARS-CoV-2 to humans. Through building the first atlas of ACE2 expression in pets and livestock, we identified species and tissues susceptible to SARS-CoV-2 infection, yielding novel insights into the viral transmission.","version":"1.1","doi":"10.1101/2020.03.30.015644","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.014381","pub_date":"2020-3-31","title":"Computational Prediction of the Comprehensive SARS-CoV-2 vs. Human Interactome to Guide the Design of Therapeutics","abstract":"Understanding the disease pathogenesis of the novel coronavirus, denoted SARS-CoV-2, is critical to the development of anti-SARS-CoV-2 therapeutics. The global propagation of the viral disease, denoted COVID-19 (\u201ccoronavirus disease 2019\u201d), has unified the scientific community in searching for possible inhibitory small molecules or polypeptides. Given the known interaction between the human ACE2 (\u201cAngiotensin-converting enzyme 2\u201d) protein and the SARS-CoV virus (responsible for the coronavirus outbreak circa. 2003), considerable focus has been directed towards the putative interaction between the SARS-CoV-2 Spike protein and ACE2. However, a more holistic understanding of the SARS-CoV-2 vs. human inter-species interactome promises additional putative protein-protein interactions (PPI) that may be considered targets for the development of inhibitory therapeutics. To that end, we leverage two state-of-the-art, sequence-based PPI predictors (PIPE4 & SPRINT) capable of generating the comprehensive SARS-CoV-2 vs. human interactome, comprising approximately 285,000 pairwise predictions. Of these, we identify the high-scoring subset of human proteins predicted to interact with each of the 14 SARS-CoV-2 proteins by both methods, comprising 279 high-confidence putative interactions involving 225 human proteins. Notably, the Spike-ACE2 interaction was the highest ranked for both the PIPE4 and SPRINT predictors, corroborating existing evidence for this PPI. Furthermore, the PIPE-Sites algorithm was used to predict the putative subsequence that might mediate each interaction and thereby inform the design of inhibitory polypeptides intended to disrupt the corresponding host-pathogen interactions. We hereby publicly release the comprehensive set of PPI predictions and their corresponding PIPE-Sites landscapes in the following DataVerse repository: 10.5683/SP2/JZ77XA. All data and metadata are released under a CC-BY 4.0 licence. The information provided represents theoretical modeling only and caution should be exercised in its use. It is intended as a resource for the scientific community at large in furthering our understanding of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.03.29.014381","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.28.013607","pub_date":"2020-3-31","title":"Computational Design of Peptides to Block Binding of the SARS-CoV-2 Spike Protein to Human ACE2","abstract":"The outbreak of COVID-19 has now become a global pandemic and it continues to spread rapidly worldwide, severely threatening lives and economic stability. Making the problem worse, there is no specific antiviral drug that can be used to treat COVID-19 to date. SARS-CoV-2 initiates its entry into human cells by binding to angiotensin-converting enzyme 2 (hACE2) via the receptor binding domain (RBD) of its spike protein. Therefore, molecules that can block SARS-CoV-2 from binding to hACE2 may potentially prevent the virus from entering human cells and serve as an effective antiviral drug. Based on this idea, we designed a series of peptides that can strongly bind to SARS-CoV-2 RBD in computational experiments. Specifically, we first constructed a 31-mer peptidic scaffold by linking two fragments grafted from hACE2 (a.a. 22-44 and 351-357) with a linker glycine, and then redesigned the peptide sequence to enhance its binding affinity to SARS-CoV-2 RBD. Compared with several computational studies that failed to identify that SARS-CoV-2 shows higher binding affinity for hACE2 than SARS-CoV, our protein design scoring function, EvoEF2, makes a correct identification, which is consistent with the recently reported experimental data, implying its high accuracy. The top designed peptide binders exhibited much stronger binding potency to hACE2 than the wild-type (\u221253.35 vs. \u221246.46 EvoEF2 energy unit for design and wild-type, respectively). The extensive and detailed computational analyses support the high reasonability of the designed binders, which not only recapitulated the critical native binding interactions but also introduced new favorable interactions to enhance binding. Due to the urgent situation created by COVID-19, we share these computational data to the community, which should be helpful to develop potential antiviral peptide drugs to combat this pandemic.","version":"1.1","doi":"10.1101/2020.03.28.013607","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.015164","pub_date":"2020-3-31","title":"Epitope-based chimeric peptide vaccine design against S, M and E proteins of SARS-CoV-2 etiologic agent of global pandemic COVID-19: an in silico approach","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19), a public health emergency of international concern declared by the World Health Organization (WHO). An immuno-informatics approach along with comparative genomic was applied to design a multi-epitope-based peptide vaccine against SARS-CoV-2 combining the antigenic epitopes of the S, M and E proteins. The tertiary structure was predicted, refined and validated using advanced bioinformatics tools. The candidate vaccine showed an average of \u2265 90.0% world population coverage for different ethnic groups. Molecular docking of the chimeric vaccine peptide with the immune receptors (TLR3 and TLR4) predicted efficient binding. Immune simulation predicted significant primary immune response with increased IgM and secondary immune response with high levels of both IgG1 and IgG2. It also increased the proliferation of T-helper cells and cytotoxic T-cells along with the increased INF-\u03b3 and IL-2 cytokines. The codon optimization and mRNA secondary structure prediction revealed the chimera is suitable for high-level expression and cloning. Overall, the constructed recombinant chimeric vaccine candidate demonstrated significant potential and can be considered for clinical validation to fight against this global threat, COVID-19.","version":"1.1","doi":"10.1101/2020.03.30.015164","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.014183","pub_date":"2020-3-31","title":"HTCC as a highly effective polymeric inhibitor of SARS-CoV-2 and MERS-CoV","abstract":"The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the healthcare measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of previously developed by us HTCC compound (N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride), which may be used as potential inhibitor of currently circulating highly pathogenic coronaviruses \u2013 SARS-CoV-2 and MERS-CoV.","version":"1.1","doi":"10.1101/2020.03.29.014183","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.016832","pub_date":"2020-3-31","title":"Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design","abstract":"As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.","version":"1.1","doi":"10.1101/2020.03.30.016832","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.013490","pub_date":"2020-3-31","title":"Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2","abstract":"The burgeoning epidemic caused by novel coronavirus 2019 (2019-nCoV) is currently a global concern. Angiotensin-converting enzyme-2 (ACE2) is a receptor of 2019-nCoV spike 1 protein (S1) and mediates viral entry into host cells. Despite the abundance of ACE2 in small intestine, few digestive symptoms are observed in patients infected by 2019-nCoV. Herein, we investigated the interactions between ACE2 and human defensins (HDs) specifically secreted by intestinal Paneth cells. The lectin-like HD5, rather than HD6, bound ACE2 with a high affinity of 39.3 nM and weakened the subsequent recruitment of 2019-nCoV S1. The cloak of HD5 on the ligand-binding domain of ACE2 was confirmed by molecular dynamic simulation. A remarkable dose-dependent preventive effect of HD5 on 2019-nCoV S1 binding to intestinal epithelial cells was further evidenced by in vitro experiments. Our findings unmasked the innate defense function of lectin-like intestinal defensin against 2019-nCoV, which may provide new insights into the prevention and treatment of 2019-nCoV infection.","version":"1.1","doi":"10.1101/2020.03.29.013490","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.014290","pub_date":"2020-3-31","title":"Sequence variation among SARS-CoV-2 isolates in Taiwan","abstract":"Taiwan experienced two waves of imported cases of coronavirus disease 2019 (COVID-19), first from China in January to late February, followed by those from other countries starting in early March. Additionally, several cases could not be traced to any imported cases and were suspected as sporadic local transmission. Twelve full viral genomes were determined in this study by Illumina sequencing either from virus isolates or directly from specimens, among which 5 originated from clustered infections. Phylogenetic tree analysis revealed that these sequences were in different clades, indicating that no major strain has been circulating in Taiwan. A deletion in open reading frame 8 was found in one isolate. Only a 4-nucleotide difference was observed among the 5 genomes from clustered infections.","version":"1.1","doi":"10.1101/2020.03.29.014290","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.30.016485","pub_date":"2020-3-31","title":"The target landscape of N4-hydroxycytidine based on its chemical neighborhood","abstract":"N4-hydroxycytidine (NHC) has been recently reported to have promising antiviral activity against SARS-CoV-2. To join worldwide efforts in identifying potential drug targets against this pandemic, the target landscape of NHC was defined by extracting all known targets of its chemical neighborhood, including drugs, analogues, and metabolites, and by performing target predictions from two independent platforms, following the recent Public Health Assessment via Structural Evaluation (PHASE) protocol. The analysis provides a list of over 30 protein targets that could be useful in future design activities of new COVID-19 antivirals. The relevance for existing drugs within the same chemical space, such as remdesivir, is also discussed.","version":"1.1","doi":"10.1101/2020.03.30.016485","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.31.018606","pub_date":"2020-3-31","title":"Detecting tropical selective logging with SAR data requires a time series approach","abstract":"Selective logging is the primary driver of forest degradation in the tropics and reduces the capacity of forests to harbour biodiversity, maintain key ecosystem processes, sequester carbon, and support human livelihoods. While the preceding decade has seen a tremendous improvement in the ability to monitor forest disturbances from space, advances in forest monitoring have almost universally relied on optical satellite data from the Landsat program, whose effectiveness is limited in tropical regions with frequent cloud cover. Synthetic aperture radar (SAR) data can penetrate clouds and have been utilized in forest mapping applications since the early 1990s, but no study has exclusively used SAR data to map tropical selective logging. A detailed selective logging dataset from three lowland tropical forest regions in the Brazilian Amazon was used to assess the effectiveness of SAR data from Sentinel-1, RADARSAT-2 and PALSAR-2 for monitoring tropical selective logging. We built Random Forest models in an effort to classify pixel-based differences in logged and unlogged areas. In addition, we used the BFAST algorithm to assess if a dense time series of Sentinel-1 imagery displayed recognizable shifts in pixel values after selective logging. Random Forest classification with SAR data (Sentinel-1, RADARSAT-2, and ALOS-2 PALSAR-2) performed poorly, having high commission and omission errors for logged observations. This suggests little to no difference in pixel-based metrics between logged and unlogged areas for these sensors. In contrast, the Sentinel-1 time series analyses indicated that areas under higher intensity selective logging (> 20 m3 ha\u22121) show a distinct spike in the number of pixels that included a breakpoint during the logging season. BFAST detected breakpoints in 50% of logged pixels and exhibited a false alarm rate of approximately 10% in unlogged forest. Overall our results suggest that SAR data can be used in time series analyses to detect tropical selective logging at high intensity logging locations within the Amazon (> 20 m3 ha\u22121). These results have important implications for current and future abilities to detect selective logging with freely available SAR data from SAOCOM 1A, the planned continuation missions of Sentinel-1 (C and D), ALOS PALSAR-1 archives (expected to be opened for free access in 2020), and the upcoming launch of NISAR.","version":"1.1","doi":"10.1101/2020.03.31.018606","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.28.013508","pub_date":"2020-3-30","title":"SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction","abstract":"The ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of an RNA extraction step should be considered in SARS-CoV-2 diagnosis.","version":"1.1","doi":"10.1101/2020.03.28.013508","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.990036","pub_date":"2020-3-30","title":"Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in Rhesus macaques","abstract":"The outbreak of Corona Virus Disease 2019 caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly transmitted. The potential extra-respiratory transmission routes remain uncertain. Five rhesus macaques were inoculated with 1\u00d7106 TCID50 of SARS-CoV-2 via conjunctival (CJ), intratracheal (IT), and intragastric (IG) routes, respectively. Remarkably, the CJ inoculated-macaques developed mild interstitial pneumonia and viral load was detectable in the conjunctival swabs at 1 days post-inoculation (dpi). Only via IT inoculation, viral load was detected in the anal swab at 1-7 dpi and macaque showed weight loss. However, viral load was undetectable after IG inoculation. Comparatively, viral load was higher in the nasolacrimal system but lesions of lung were relatively mild and local via CJ inoculation compared with that via IT inoculation, demonstrating distinct characteristics of virus dispersion. Both the two routes affected the alimentary tract. Therefore the clinicians need to protect eye while working with patients.","version":"1.2","doi":"10.1101/2020.03.13.990036","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.991455","pub_date":"2020-3-30","title":"SARS-CoV-2 receptor ACE2 and TMPRSS2 are predominantly expressed in a transient secretory cell type in subsegmental bronchial branches","abstract":"The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.","version":"1.3","doi":"10.1101/2020.03.13.991455","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.28.013920","pub_date":"2020-3-30","title":"Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9","abstract":"Many of the proteins produced by SARS-CoV-2 have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate both viral replication and virulence. Current understanding suggests that Nsp9 is involved in viral genomic RNA reproduction. Nsp9 is thought to bind RNA via a fold that is unique to this class of betacoronoaviruses although the molecular basis for this remains ill-defined. We sought to better characterise the SARS-CoV-2 Nsp9 protein and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The structure of the SARS-CoV-2 Nsp9 revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted on the relative juxtaposition of the monomers within the homodimer. Together we have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that may warrant further study from the perspective of understanding Nsp9 function.","version":"1.1","doi":"10.1101/2020.03.28.013920","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.007898","pub_date":"2020-3-30","title":"Glycoinformatics approach for identifying target positions to inhibit initial binding of SARS-CoV-2 S1 protein to the host cell","abstract":"COVID-19 outbreak is still threatening the public health. Therefore, in the middle of the pandemic, all kind of knowledge on SARS-CoV-2 may help us to find the solution. Determining the 3D structures of the proteins involved in host-pathogen interactions are of great importance in the fight against infection. Besides, post-translational modifications of the protein on 3D structure should be revealed in order to understand the protein function since these modifications are responsible for the host-pathogen interaction. Based on these, we predicted O-glycosylation and phosphorylation positions using full amino acid sequence of S1 protein. Candidate positions were further analyzed with enzyme binding activity, solvent accessibility, surface area parameters and the positions determined with high accuracy rate were used to design full 3D glycoprotein structure of the S1 protein using carbohydrate force field. In addition, the interaction between the C-type lectin CD209L and \u03b1-mannose residues was examined and carbohydrate recognition positions were predicted. We suggest these positions as a potential target for the inhibition of the initial binding of SARS-CoV-2 S1 protein to the host cell.","version":"1.2","doi":"10.1101/2020.03.25.007898","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.29.014209","pub_date":"2020-3-30","title":"Orthogonal genome-wide screenings in bat cells identify MTHFD1 as a target of broad antiviral therapy","abstract":"Bats are responsible for the zoonotic transmission of several major viral diseases including the 2003 SARS outbreak and the ongoing COVID-19 pandemic. While bat genomic sequencing studies have revealed characteristic adaptations of the innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the tolerance of viral infections in bats. Here we report the establishment and screening of genome-wide RNAi library and CRISPR library for the model megabat, Pteropus Alecto. We used the complementary RNAi and CRISPR libraries to interrogate Pteropus Alecto cells for infection with two different viruses, mumps virus and Influenza A virus, respectively. Screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C-1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells as well as in human cells. MTHFD1 inhibitor carolacton potently blocked replication of several RNA viruses including SARS-CoV-2. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad spectrum antiviral therapy.","version":"1.1","doi":"10.1101/2020.03.29.014209","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.010975","pub_date":"2020-3-29","title":"Comparative performance of four nucleic acid amplification tests for SARS-CoV-2 virus","abstract":"Coronavirus disease 2019 (COVID-19) can be screened and diagnosed through the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing during the pandemic. However, studies evaluating the performance of these NAAT assays are limited. We evaluated the performance of four NAATs, which were marked by the Conformit\u00e9 Europ\u00e9enne and widely used in China during the pandemic. Results showed that the analytical sensitivity of the four assays was significantly lower than that claimed by the NAAT manufacturers. The limit of detection (LOD) of Daan, Sansure, and Hybribio NAATs was 3000 copies/mL, whereas the LOD of Bioperfectus NAATs was 4000 copies/mL. The results of the consistency test using 46 samples showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16 /16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity 81.25% (13/16). The sensitivity of Bioperfectus NAAT was lower than that of the three other NAATs; this finding was consistent with the result that Bioperfectus NAAT had a higher LOD than the three other kinds of NAATs. The four above mentioned reagents presented high specificity; however, for the detection of the samples with low virus concentration, Bioperfectus reagent had the risk of missing detection. Therefore, the LOD should be considered in the selection of SARS-CoV-2 NAATs.","version":"1.1","doi":"10.1101/2020.03.26.010975","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.006569","pub_date":"2020-3-29","title":"Codon usage and evolutionary rates of the 2019-nCoV genes","abstract":"Severe acute respiratory syndrome coronavirus 2 (2019-nCoV), which first broke out in Wuhan (China) in December of 2019, causes a severe acute respiratory illness with a mortality ranging from 3% to 6%. To better understand the evolution of the newly emerging 2019-nCoV, in this paper, we analyze the codon usage pattern of 2019-nCoV. For this purpose, we compare the codon usage of 2019-nCoV with that of other 30 viruses belonging to the subfamily of orthocoronavirinae. We found that 2019-nCoV shows a rich composition of AT nucleotides that strongly influences its codon usage, which appears to be not optimized to human host. Then, we study the evolutionary pressures influencing the codon usage and evolutionary rates of the sequences of five conserved genes that encode the corresponding proteins (viral replicase, spike, envelope, membrane and nucleocapsid) characteristic of coronaviruses. We found different patterns of both mutational bias and nature selection that affect the codon usage of these genes at different extents. Moreover, we show that the two integral membrane proteins proteins (matrix and envelope) tend to evolve slowly by accumulating nucleotide mutations on their genes. Conversely, genes encoding nucleocapsid (N), viral replicase and spike proteins are important targets for the development of vaccines and antiviral drugs, tend to evolve faster as compared to other ones. Taken together, our results suggest that the higher evolutionary rate observed for these two genes could represent a major barrier in the development of antiviral therapeutics 2019-nCoV.","version":"1.2","doi":"10.1101/2020.03.25.006569","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.005702","pub_date":"2020-3-29","title":"Knowledge synthesis from 100 million biomedical documents augments the deep expression profiling of coronavirus receptors","abstract":"The COVID-19 pandemic demands assimilation of all available biomedical knowledge to decode its mechanisms of pathogenicity and transmission. Despite the recent renaissance in unsupervised neural networks for decoding unstructured natural languages, a platform for the real-time synthesis of the exponentially growing biomedical literature and its comprehensive triangulation with deep omic insights is not available. Here, we present the nferX platform for dynamic inference from over 45 quadrillion possible conceptual associations extracted from unstructured biomedical text, and their triangulation with Single Cell RNA-sequencing based insights from over 25 tissues. Using this platform, we identify intersections between the pathologic manifestations of COVID-19 and the comprehensive expression profile of the SARS-CoV-2 receptor ACE2. We find that tongue keratinocytes, airway club cells, and ciliated cells are likely underappreciated targets of SARS-CoV-2 infection, in addition to type II pneumocytes and olfactory epithelial cells. We further identify mature small intestinal enterocytes as a possible hotspot of COVID-19 fecal-oral transmission, where an intriguing maturation-correlated transcriptional signature is shared between ACE2 and the other coronavirus receptors DPP4 (MERS-CoV) and ANPEP (\u03b1-coronavirus). This study demonstrates how a holistic data science platform can leverage unprecedented quantities of structured and unstructured publicly available data to accelerate the generation of impactful biological insights and hypotheses. The nferX Platform Single-cell resource - https://academia.nferx.com/","version":"1.1","doi":"10.1101/2020.03.24.005702","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.26.964882","pub_date":"2020-3-29","title":"Structure of Mpro from COVID-19 virus and discovery of its inhibitors","abstract":"A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 \u03bcM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.","version":"1.3","doi":"10.1101/2020.02.26.964882","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.005561","pub_date":"2020-3-28","title":"Computational simulations reveal the binding dynamics between human ACE2 and the receptor binding domain of SARS-CoV-2 spike protein","abstract":"A novel coronavirus (the SARS-CoV-2) has been identified in January 2020 as the causal pathogen for COVID-19 pneumonia, an outbreak started near the end of 2019 in Wuhan, China. The SARS-CoV-2 was found to be closely related to the SARS-CoV, based on the genomic analysis. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS-CoV as a receptor was found to facilitate the infection of SARS-CoV-2, initiated by the binding of the spike protein to the human ACE2. Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The predicted model is highly consistent with the experimentally determined complex structures. Besides the binding interface reported in the crystal structures, new plausible binding poses were revealed from all-atom MD simulations. The simulation data helped identify critical residues at the complex interface and provided more details about the interactions between the SARS-CoV-2 RBD and human ACE2. Two mutants mimicking rat ACE2 were modeled to study the mutation effects on RBD binding to ACE2. The simulations showed that the N-terminal helix and the K353 are very important for the tight binding of the complex, the mutants were found to alter the binding modes of the CoV2-RBD to ACE2.","version":"1.2","doi":"10.1101/2020.03.24.005561","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.010322","pub_date":"2020-3-28","title":"Site-specific analysis of the SARS-CoV-2 glycan shield","abstract":"The emergence of the betacoronavirus, SARS-CoV-2 that causes COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, that mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in immune evasion and occluding immunogenic protein epitopes. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.","version":"1.1","doi":"10.1101/2020.03.26.010322","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.27.012013","pub_date":"2020-3-28","title":"SARS-CoV-2 and ORF3a: Non-Synonymous Mutations and Polyproline Regions","abstract":"The effect of the rapid accumulation of non-synonymous mutations on the pathogenesis of SARS-CoV-2 is not yet known. To predict the impact of non-synonymous mutations and polyproline regions identified in ORF3a on the formation of B-cell epitopes and their role in evading the immune response, nucleotide and protein sequences of 537 available SARS-CoV-2 genomes were analyzed for the presence of non-synonymous mutations and polyproline regions. Mutations were correlated with changes in epitope formation. A total of 19 different non-synonymous amino acids substitutions were detected in ORF3a among 537 SARS-CoV-2 strains. G251V was the most common and identified in 9.9% (n=53) of the strains and was predicted to lead to the loss of a B-cell like epitope in ORF3a. Polyproline regions were detected in two strains (EPI_ISL_410486, France and EPI_ISL_407079, Finland) and affected epitopes formation. The accumulation of non-synonymous mutations and detected polyproline regions in ORF3a of SARS-CoV-2 could be driving the evasion of the host immune response thus favoring viral spread. Rapid mutations accumulating in ORF3a should be closely monitored throughout the COVID-19 pandemic. At the surge of the COVID-19 pandemic and after three months of the identification of SARS-CoV-2 as the disease-causing pathogen, nucleic acid changes due to host-pathogen interactions are insightful into the evolution of this virus. In this paper, we have identified a set of non-synonymous mutations in ORF3a and predicted their impact on B-cell like epitope formation. The accumulation of non-synonymous mutations in ORF3a could be driving protein changes that mediate immune evasion and favoring viral spread.","version":"1.1","doi":"10.1101/2020.03.27.012013","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.20.999730","pub_date":"2020-3-28","title":"Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs","abstract":"COVID-19 is an emerging infectious disease and was recently declared as a pandemic by WHO. Currently, there is no vaccine or therapeutic available for this disease. Drug repositioning represents the only feasible option to address this global challenge and a panel of 48 FDA-approved drugs that have been pre-selected by an assay of SARS-CoV was screened to identify potential antiviral drug candidates against SARS-CoV-2 infection. We found a total of 24 drugs which exhibited antiviral efficacy (0.1 \u03bcM < IC50 < 10 \u03bcM) against SARS-CoV-2. In particular, two FDA-approved drugs - niclosamide and ciclesonide \u2013 were notable in some respects. These drugs will be tested in an appropriate animal model for their antiviral activities. In near future, these already FDA-approved drugs could be further developed following clinical trials in order to provide additional therapeutic options for patients with COVID-19.","version":"1.3","doi":"10.1101/2020.03.20.999730","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.27.009480","pub_date":"2020-3-28","title":"SARS-CoV-2 exhibits intra-host genomic plasticity and low-frequency polymorphic quasispecies","abstract":"In December 2019, an outbreak of atypical pneumonia (Coronavirus disease 2019 - COVID-19) associated with a novel coronavirus (SARS-CoV-2) was reported in Wuhan city, Hubei province, China. The outbreak was traced to a seafood wholesale market and human to human transmission was confirmed. The rapid spread and the death toll of the new epidemic warrants immediate intervention. The intra-host genomic variability of SARS-CoV-2 plays a pivotal role in the development of effective antiviral agents and vaccines, but also in the design of accurate diagnostics. We analyzed NGS data derived from clinical samples of three Chinese patients infected with SARS-CoV-2, in order to identify small- and large-scale intra-host variations in the viral genome. We identified tens of low- or higher-frequency single nucleotide variations (SNVs) with variable density across the viral genome, affecting 7 out of 10 protein-coding viral genes. The majority of these SNVs corresponded to missense changes. The annotation of the identified SNVs but also of all currently circulating strain variations revealed colocalization of intra-host but also strain specific SNVs with primers and probes currently used in molecular diagnostics assays. Moreover, we de-novo assembled the viral genome, in order to isolate and validate intra-host structural variations and recombination breakpoints. The bioinformatics analysis disclosed genomic rearrangements over poly-A / poly-U regions located in ORF1ab and spike (S) gene, including a potential recombination hot-spot within S gene. Our results highlight the intra-host genomic diversity and plasticity of SARS-CoV-2, pointing out genomic regions that are prone to alterations. The isolated SNVs and genomic rearrangements, reflect the intra-patient capacity of the polymorphic quasispecies, which may arise rapidly during the outbreak, allowing immunological escape of the virus, offering resistance to anti-viral drugs and affecting the sensitivity of the molecular diagnostics assays.","version":"1.1","doi":"10.1101/2020.03.27.009480","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.009233","pub_date":"2020-3-28","title":"Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity","abstract":"The coronavirus disease COVID-19, caused by emerging SARS-CoV-2, has posed serious threats to global public health, economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we firstly verified that SARS-CoV-2 uses human ACE2 as a cell receptor and its spike (S) protein mediates high membrane fusion activity. Comparing to that of SARS-CoV, the heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased \u03b1-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR1) site. Then, we designed a HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly poent activities in inibibiting the SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus infection. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the strcuture and activity relationship (SAR) of IPB02 were characterzized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the presented results have provided important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.","version":"1.1","doi":"10.1101/2020.03.26.009233","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.23.004176","pub_date":"2020-3-28","title":"Structure-based modeling of SARS-CoV-2 peptide/HLA-A02 antigens","abstract":"As a first step toward the development of diagnostic and therapeutic tools to fight the Coronavirus disease (COVID-19), it is important to characterize CD8+ T cell epitopes in the SARS-CoV-2 peptidome that can trigger adaptive immune responses. Here, we use RosettaMHC, a comparative modeling approach which leverages existing high-resolution X-ray structures from peptide/MHC complexes available in the Protein Data Bank, to derive physically realistic 3D models for high-affinity SARS-CoV-2 epitopes. We outline an application of our method to model 439 9mer and 279 10mer predicted epitopes displayed by the common allele HLA-A*02:01, and we make our models publicly available through an online database (https://rosettamhc.chemistry.ucsc.edu). As more detailed studies on antigen-specific T cell recognition become available, RosettaMHC models of antigens from different strains and HLA alleles can be used as a basis to understand the link between peptide/HLA complex structure and surface chemistry with immunogenicity, in the context of SARS-CoV-2 infection.","version":"1.2","doi":"10.1101/2020.03.23.004176","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.996348","pub_date":"2020-3-28","title":"Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease","abstract":"SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 \u03bcM and 0.04 \u03bcM respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 \u03bcM and 0.33 \u03bcM, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 \u00c5 resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.","version":"1.1","doi":"10.1101/2020.03.25.996348","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.009605","pub_date":"2020-3-28","title":"Re-analysis of SARS-CoV-2 infected host cell proteomics time-course data by impact pathway analysis and network analysis. A potential link with inflammatory response","abstract":"The disease known as coronavirus disease 19 (COVID-19), potentially caused by an outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in Wuhan, China, has hit the world hard, and has led to an unprecedent health and economic crisis. In order to develop treatment options able to stop or ameliorate SARS-CoV-2 effects, we need to understand the biology of the virus inside cells, but this kind of studies are still scarce. A recent study investigated translatome and proteome host cell changes induced in vitro by SARS-CoV-2. In the present study, we use the publicly available proteomics data from this study to re-analyze the mechanisms altered by the virus infection by impact pathways analysis and network analysis. Proteins linked to inflammatory response, but also proteins related to chromosome segregation during mitosis, were found to be regulated. The up-regulation of the inflammatory-related proteins observed could be linked to the propagation of inflammatory reaction and lung injury that is observed in advanced stages of COVID-19 patients.","version":"1.1","doi":"10.1101/2020.03.26.009605","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.25.008904","pub_date":"2020-3-28","title":"Modeling of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Proteins by Machine Learning and Physics-Based Refinement","abstract":"Protein structures are crucial for understanding their biological activities. Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need to understand the biological behavior of the virus and provide a basis for developing effective therapies. Since the proteome of the virus was determined, some of the protein structures could be determined experimentally, and others were predicted via template-based modeling approaches. However, tertiary structures for several proteins are still not available from experiment nor they could be accurately predicted by template-based modeling because of lack of close homolog structures. Previous efforts to predict structures for these proteins include efforts by DeepMind and the Zhang group via machine learning-based structure prediction methods, i.e. AlphaFold and C-I-TASSER. However, the predicted models vary greatly and have not yet been subjected to refinement. Here, we are reporting new predictions from our in-house structure prediction pipeline. The pipeline takes advantage of inter-residue contact predictions from trRosetta, a machine learning-based method. The predicted models were further improved by applying molecular dynamics simulation-based refinement. We also took the AlphaFold models and refined them by applying the same refinement method. Models based on our structure prediction pipeline and the refined AlphaFold models were analyzed and compared with the C-I-TASSER models. All of our models are available at https://github.com/feiglab/sars-cov-2-proteins.","version":"1.1","doi":"10.1101/2020.03.25.008904","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.010165","pub_date":"2020-3-28","title":"Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies","abstract":"The pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.","version":"1.1","doi":"10.1101/2020.03.26.010165","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.22.002386","pub_date":"2020-3-27","title":"A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing","abstract":"An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 67 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.","version":"1.3","doi":"10.1101/2020.03.22.002386","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.006007","pub_date":"2020-3-27","title":"Automatic Identification of SARS Coronavirus using Compression-Complexity Measures","abstract":"Finding vaccine or specific antiviral treatment for global pandemic of virus diseases (such as the ongoing COVID-19) requires rapid analysis, annotation and evaluation of metagenomic libraries to enable a quick and efficient screening of nucleotide sequences. Traditional sequence alignment methods are not suitable and there is a need for fast alignment-free techniques for sequence analysis. Information theory and data compression algorithms provide a rich set of mathematical and computational tools to capture essential patterns in biological sequences. In 2013, our research group (Nagaraj et al., Eur. Phys. J. Special Topics 222(3-4), 2013) has proposed a novel measure known as Effort-To-Compress (ETC) based on the notion of compression-complexity to capture the information content of sequences. In this study, we propose a compression-complexity based distance measure for automatic identification of SARS coronavirus strains from a set of viruses using only short fragments of nucleotide sequences. We also demonstrate that our proposed method can correctly distinguish SARS-CoV-2 from SARS-CoV-1 viruses by analyzing very short segments of nucleotide sequences. This work could be extended further to enable medical practitioners in automatically identifying and characterizing SARS coronavirus strain in a fast and efficient fashion using short and/or incomplete segments of nucleotide sequences. Potentially, the need for sequence assembly can be circumvented. The main ideas and results of this research were first presented at the International Conference on Nonlinear Systems and Dynamics (CNSD-2013) held at Indian Institute of Technology, Indore, December 12, 2013. In this manuscript, we have extended our preliminary analysis to include SARS-CoV-2 virus as well.","version":"1.1","doi":"10.1101/2020.03.24.006007","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.005900","pub_date":"2020-3-27","title":"Topoisomerase III-\u00df is required for efficient replication of positive-sense RNA viruses","abstract":"Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-\u00df (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.","version":"1.1","doi":"10.1101/2020.03.24.005900","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.26.009803","pub_date":"2020-3-26","title":"The potential SARS-CoV-2 entry inhibitor","abstract":"Outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan and has rapidly spread to almost all parts of world. In coronaviruses, the receptor binding domain (RBD) in the distal part of S1 subunit of SARS-CoV-2 spike protein can directly bind to angiotensin converting enzyme 2 (ACE2). RBD promote viral entry into the host cells and is an important therapeutic target. In this study, we discovered that theaflavin showed the lower idock score (idock score: \u22127.95 kcal/mol). To confirm the result, we discovered that theaflavin showed FullFitness score of \u2212991.21 kcal/mol and estimated \u0394G of \u22128.53 kcal/mol for the most favorable interaction with contact area of SARS-CoV-2 RBD by SwissDock service. Regarding contact modes, hydrophobic interactions contribute significantly in binding and additional hydrogen bonds were formed between theaflavin and Arg454, Phe456, Asn460, Cys480, Gln493, Asn501 and Val503 of SARS-CoV-2 RBD, near the direct contact area with ACE2. Our results suggest that theaflavin could be the candidate of SARS-CoV-2 entry inhibitor for further study.","version":"1.1","doi":"10.1101/2020.03.26.009803","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.21.990770","pub_date":"2020-3-26","title":"Potent human neutralizing antibodies elicited by SARS-CoV-2 infection","abstract":"The pandemic caused by emerging coronavirus SARS-CoV-2 presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. SARS-CoV-2 cellular entry depends on binding between the viral Spike protein receptor-binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) target cell receptor. Here, we report on the isolation and characterization of 206 RBD-specific monoclonal antibodies (mAbs) derived from single B cells of eight SARS-CoV-2 infected individuals. These mAbs come from diverse families of antibody heavy and light chains without apparent enrichment for particular families in the repertoire. In samples from one patient selected for further analyses, we found coexistence of germline and germline divergent clones. Both clone types demonstrated impressive binding and neutralizing activity against pseudovirus and live SARS-CoV-2. However, the antibody neutralizing potency is determined by competition with ACE2 receptor for RBD binding. Surprisingly, none of the SARS-CoV-2 antibodies nor the infected plasma cross-reacted with RBDs from either SARS-CoV or MERS-CoV although substantial plasma cross-reactivity to the trimeric Spike proteins from SARS-CoV and MERS-CoV was found. These results suggest that antibody response to RBDs is viral species-specific while that cross-recognition target regions outside the RBD. The specificity and neutralizing characteristics of this plasma cross-reactivity requires further investigation. Nevertheless, the diverse and potent neutralizing antibodies identified here are promising candidates for prophylactic and therapeutic SARS-CoV-2 interventions.","version":"1.2","doi":"10.1101/2020.03.21.990770","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.006544","pub_date":"2020-3-25","title":"Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray","abstract":"The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.","version":"1.1","doi":"10.1101/2020.03.24.006544","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.23.002832","pub_date":"2020-3-25","title":"scRNA-seq reveals ACE2 and TMPRSS2 expression in TROP2+ Liver Progenitor Cells: Implications in COVID-19 associated Liver Dysfunction","abstract":"The recent pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 was first reported in China (December 2019) and now prevalent in \u223c170 countries across the globe. Entry of SARS-CoV-2 into mammalian cells require the binding of viral Spike (S) proteins to the ACE2 (angiotensin converting enzyme 2) receptor. Once entered the S protein is primed by a specialised serine protease, TMPRSS2 (Transmembrane Serine Protease 2) in the host cell. Importantly, beside respiratory symptoms, consistent with other common respiratory virus infection when patients become viraemic, a significant number of COVID-19 patients also develop liver comorbidities. We explored if specific target cell-type in the mammalian liver, could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here we employed single-cell RNA-seq (scRNA-seq) to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We report the co-expression of ACE2 and TMPRSS2 in a TROP2+ liver progenitor population. Importantly, we fail to detect the expression of ACE2 in hepatocyte or any other liver (immune and stromal) cell types. These results indicated that in COVID-19 associated liver dysfunction and cell death, viral infection of TROP2+ progenitors in liver may significantly impaired liver regeneration and could lead to pathology. - EPCAM+ Liver progenitors co-express ACE2 and TMPRSS2 - ACE2 and TMPRSS2 expression is highest in TROP2high progenitors - ACE2 and TMPRSS2 cells express cholangiocyte biased fate markers - ACE2 and TMPRSS2 positive cells are absent in human fetal liver","version":"1.1","doi":"10.1101/2020.03.23.002832","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.23.002931","pub_date":"2020-3-25","title":"Ultra-Low-Cost Integrated Silicon-based Transducer for On-Site, Genetic Detection of Pathogens","abstract":"Rapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention e.g., drug therapy, quarantine, no action etc. when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2 (the pathogen causing COVID-19), and shows no or similar symptoms to other common infections. We report a silicon-based integrated Point-of-Need (PoN) transducer (TriSilix) that can chemically-amplify and detect pathogen-specific sequences of nucleic acids (NA) quantitatively in real-time. Unlike other silicon-based technologies, TriSilix can be produced at wafer-scale in a standard laboratory; we have developed a series of methodologies based on metal-assisted chemical (wet) etching, electroplating, thermal bonding and laser-cutting to enable a cleanroom-free low-cost fabrication that does not require processing in an advanced semiconductor foundry. TriSilix is, therefore, resilient to disruptions in the global supply chain as the devices can be produced anywhere in the world. To create an ultra-low-cost device, the architecture proposed exploits the intrinsic properties of silicon and integrates three modes of operation in a single chip: i) electrical (Joule) heater, ii) temperature sensor (i.e. thermistor) with a negative temperature coefficient that can provide the precise temperature of the sample solution during reaction and iii) electrochemical sensor for detecting target NA. Using TriSilix, the sample solution can be maintained at a single, specific temperature (needed for isothermal amplification of NA such as Recombinase Polymerase Amplification (RPA) or cycled between different temperatures (with a precision of \u00b11.3\u00b0C) for Polymerase Chain Reaction (PCR) while the exact concentration of amplicons is measured quantitatively and in real-time electrochemically. A single 4-inch Si wafer yields 37 TriSilix chips of 10\u00d710\u00d70.65 mm in size and can be produced in 7 hours, costing ~US $0.35 per device. The system is operated digitally, portable and low power \u2013 capable of running up to 35 tests with a 4000 mAh battery (a typical battery capacity of a modern smartphone). We were able to quantitatively detect a 563-bp fragment (Insertion Sequence IS900) of the genomic DNA of M. avium subsp. paratuberculosis (extracted from cultured field samples) through PCR in real-time with a Limit-of-Detection of 20 fg, equivalent to a single bacterium, at the 30th cycle. Using TriSilix, we also detected the cDNA from SARS-CoV-2 (1 pg), through PCR, with high specificity against SARS-CoV (2003).","version":"1.1","doi":"10.1101/2020.03.23.002931","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.006270","pub_date":"2020-3-25","title":"Fibrinolytic niche is requested for alveolar type 2 cell-mediated alveologenesis and injury repair","abstract":"COVID-19, SARS, and MERS are featured by fibrinolytic dysfunction. To test the role of the fibrinolytic niche in the regeneration of alveolar epithelium, we compared the self-renewing capacity of alveolar epithelial type 2 (AT2) cells and its differentiation to AT1 cells between wild type (wt) and fibrinolytic niche deficient mice (Plau\u2212/\u2212 and Serpine1Tg). A significant reduction in both proliferation and differentiation of deficient AT2 cells was observed in vivo and in 3D organoid cultures. This decrease was mainly restored by uPA derived A6 peptide, a binding fragment to CD44 receptors. The proliferative and differential rate of CD44+ AT2 cells was greater than that of CD44\u2212 controls. There was a reduction in transepithelial ion transport in deficient monolayers compared to wt cells. Moreover, we found a marked suppression in total AT2 cells and CD44+ subpopulation in lungs from brain dead patients with acute respiratory distress syndrome (ARDS) and a mouse model infected by influenza viruses. Thus, we demonstrate that the fibrinolytic niche can regulate AT2-mediated homeostasis and regeneration via a novel uPA-A6-CD44+-ENaC cascade.","version":"1.1","doi":"10.1101/2020.03.24.006270","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.20.000885","pub_date":"2020-3-24","title":"Emergence of SARS-CoV-2 through Recombination and Strong Purifying Selection","abstract":"COVID-19 has become a global pandemic caused by a novel coronavirus SARS-CoV-2. Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis and for drug discovery and vaccine development. We show evidence of strong purifying selection around the receptor binding motif (RBM) in the spike gene and in other genes among bat, pangolin and human coronaviruses, indicating similar strong evolutionary constraints in different host species. We also demonstrate that SARS-CoV-2\u2019s entire RBM was introduced through recombination with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2\u2019s ability to infect humans. Similar purifying selection in different host species and frequent recombination among coronaviruses suggest a common evolutionary mechanism that could lead to new emerging human coronaviruses. Extensive Recombination and Strong Purifying Selection among coronaviruses from different hosts facilitate the emergence of SARS-CoV-2","version":"1.2","doi":"10.1101/2020.03.20.000885","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.09.983064","pub_date":"2020-3-24","title":"Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2","abstract":"Epidemics of Coronavirus Disease 2019 (COVID-19) now have more than 100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by RT-qPCR methods, but the capacity of RT-qPCR methods is limited by its requirement of high-level facilities and instruments. Here, we developed and evaluated RT-LAMP assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human Coronaviruses was not observed. We also adapted a colorimetric detection method for our RT-LAMP assay so that the tests potentially performed in higher throughput.","version":"1.2","doi":"10.1101/2020.03.09.983064","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.004655","pub_date":"2020-3-24","title":"SARS-CoV-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems","abstract":"One of the greatest threats to humanity is the emergence of a pandemic virus. Among those with the greatest potential for such an event include influenza viruses and coronaviruses. In the last century alone, we have observed four major influenza A virus pandemics as well as the emergence of three highly pathogenic coronaviruses including SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic. As no effective antiviral treatments or vaccines are presently available against SARS-CoV-2, it is important to understand the host response to this virus as this may guide the efforts in development towards novel therapeutics. Here, we offer the first in-depth characterization of the host transcriptional response to SARS-CoV-2 and other respiratory infections through in vitro, ex vivo, and in vivo model systems. Our data demonstrate the each virus elicits both core antiviral components as well as unique transcriptional footprints. Compared to the response to influenza A virus and respiratory syncytial virus, SARS-CoV-2 elicits a muted response that lacks robust induction of a subset of cytokines including the Type I and Type III interferons as well as a numerous chemokines. Taken together, these data suggest that the unique transcriptional signature of this virus may be responsible for the development of COVID-19.","version":"1.1","doi":"10.1101/2020.03.24.004655","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.22.002204","pub_date":"2020-3-24","title":"Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site","abstract":"Direct RNA sequencing using an Oxford Nanopore MinION characterised the transcriptome of SARS-CoV-2 grown in Vero E6 cells. This cell line is being widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. This revealed the pattern of viral transcripts, (i.e. subgenomic mRNAs), generally fitted the predicted replication and transcription model for coronaviruses. A 24 nt in-frame deletion was detected in subgenomic mRNAs encoding the spike (S) glycoprotein. This feature was identified in over half of the mapped transcripts and was predicted to remove a proposed furin cleavage site from the S glycoprotein. This motif directs cleavage of the S glycoprotein into functional subunits during virus entry or exit. Cleavage of the S glycoprotein can be a barrier to zoonotic coronavirus transmission and affect viral pathogenicity. Allied to this transcriptome analysis, tandem mass spectrometry was used to identify over 500 viral peptides and 44 phosphopeptides, covering almost all of the proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein reinforces the point that this and other regions of SARS-CoV-2 proteins may readily mutate. This is of clear significance given the interest in the S glycoprotein as a potential vaccine target and the observation that the furin cleavage site likely contributes strongly to the pathogenesis and zoonosis of this virus. The viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.","version":"1.1","doi":"10.1101/2020.03.22.002204","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.24.005298","pub_date":"2020-3-24","title":"A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis","abstract":"Zoonotic coronavirus (CoV) infections, like those responsible for the current SARS-CoV-2 epidemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). While double-membrane vesicles (DMVs) appear to be a pan-coronavirus RO element, studies to date describe an assortment of additional coronavirus-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labelling of newly-synthesized viral RNA followed by quantitative EM autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs MERS-CoV and SARS-CoV, and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in coronavirus infection.","version":"1.1","doi":"10.1101/2020.03.24.005298","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.21.001933","pub_date":"2020-3-23","title":"SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability","abstract":"The emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.","version":"1.1","doi":"10.1101/2020.03.21.001933","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.28.970343","pub_date":"2020-3-22","title":"Designing of a next generation multiepitope based vaccine (MEV) against SARS-COV-2: Immunoinformatics and in silico approaches","abstract":"Coronavirus disease 2019 (COVID-19) associated pneumonia caused by severe acute respiratory coronavirus 2 (SARS-COV-2) was first reported in Wuhan, China in December 2019. Till date, no vaccine or completely effective drug is available to cure COVID-19. Therefore, an effective vaccine against SARS-COV-2 is crucially needed. This study was conducted to design an effective multiepitope based vaccine (MEV) against SARS-COV-2. Seven antigenic proteins were taken as targets and different epitopes (B-cell, T-cell and IFN-\u03b3 inducing) were predicted. Highly antigenic and overlapping epitopes were shortlisted. Selected epitopes indicated significant interactions with the HLA-binding alleles and 99.29% coverage of the world\u2019s population. Finally, 505 amino acids long MEV was designed by connecting sixteen MHC class I and eleven MHC class II epitopes with suitable linkers and adjuvant. Linkers and adjuvant were added to enhance the immunogenicity response of the MEV. The antigenicity, allergenicity, physiochemical properties and structural details of MEV were analyzed in order to ensure safety and immunogenicity. MEV construct was non-allergenic, antigenic, stable and flexible. Molecular docking followed by molecular dynamics (MD) simulation analysis, demonstrated a stable and strong binding affinity of MEV with human pathogenic toll-like receptors (TLR), TLR3 and TLR8. Codon optimization and in silico cloning of MEV ensured increased expression in the Escherichia coli K-12 system. Designed MEV in present study could be a potential candidate for further vaccine production process against COVID-19. However, to ensure its safety and immunogenic profile, the proposed MEV needs to be experimentally validated.","version":"1.2","doi":"10.1101/2020.02.28.970343","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.20.000141","pub_date":"2020-3-22","title":"COVID-19 coronavirus vaccine design using reverse vaccinology and machine learning","abstract":"To ultimately combat the emerging COVID-19 pandemic, it is desired to develop an effective and safe vaccine against this highly contagious disease caused by the SARS-CoV-2 coronavirus. Our literature and clinical trial survey showed that the whole virus, as well as the spike (S) protein, nucleocapsid (N) protein, and membrane (M) protein, have been tested for vaccine development against SARS and MERS. However, these vaccine candidates might lack the induction of complete protection and have safety concerns. We then applied the Vaxign reverse vaccinology tool and the newly developed Vaxign-ML machine learning tool to predict COVID-19 vaccine candidates. By investigating the entire proteome of SARS-CoV-2, six proteins, including the S protein and five non-structural proteins (nsp3, 3CL-pro, and nsp8-10), were predicted to be adhesins, which are crucial to the viral adhering and host invasion. The S, nsp3, and nsp8 proteins were also predicted by Vaxign-ML to induce high protective antigenicity. Besides the commonly used S protein, the nsp3 protein has not been tested in any coronavirus vaccine studies and was selected for further investigation. The nsp3 was found to be more conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV than among 15 coronaviruses infecting human and other animals. The protein was also predicted to contain promiscuous MHC-I and MHC-II T-cell epitopes, and linear B-cell epitopes localized in specific locations and functional domains of the protein. By applying reverse vaccinology and machine learning, we predicted potential vaccine targets for effective and safe COVID-19 vaccine development. We then propose that an \u201cSp/Nsp cocktail vaccine\u201d containing a structural protein(s) (Sp) and a non-structural protein(s) (Nsp) would stimulate effective complementary immune responses.","version":"1.2","doi":"10.1101/2020.03.20.000141","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.21.001586","pub_date":"2020-3-21","title":"Comparative analyses of SAR-CoV2 genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis","abstract":"The ongoing pandemic of the coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). We have performed an integrated sequence-based analysis of SARS-CoV2 genomes from different geographical locations in order to identify its unique features absent in SARS-CoV and other related coronavirus family genomes, conferring unique infection, facilitation of transmission, virulence and immunogenic features to the virus. The phylogeny of the genomes yields some interesting results. Systematic gene level mutational analysis of the genomes has enabled us to identify several unique features of the SARS-CoV2 genome, which includes a unique mutation in the spike surface glycoprotein (A930V (24351C>T)) in the Indian SARS-CoV2, absent in other strains studied here. We have also predicted the impact of the mutations in the spike glycoprotein function and stability, using computational approach. To gain further insights into host responses to viral infection, we predict that antiviral host-miRNAs may be controlling the viral pathogenesis. Our analysis reveals nine host miRNAs which can potentially target SARS-CoV2 genes. Interestingly, the nine miRNAs do not have targets in SARS and MERS genomes. Also, hsa-miR-27b is the only unique miRNA which has a target gene in the Indian SARS-CoV2 genome. We also predicted immune epitopes in the genomes","version":"1.1","doi":"10.1101/2020.03.21.001586","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.19.998179","pub_date":"2020-3-21","title":"Molecular characterization of SARS-CoV-2 in the first COVID-19 cluster in France reveals an amino-acid deletion in nsp2 (Asp268Del)","abstract":"We present the first genetic characterization of a COVID-19 cluster in Europe using metagenomic next-generation sequencing (mNGS). Despite low viral loads, the mNGS workflow used herein allowed to characterize the whole genome sequences of SARS-CoV2 isolated from an asymptomatic patient, in 2 clinical samples collected 1 day apart. Comparison of these sequences suggests viral evolution with development of quasispecies. In addition, the present workflow identified a new deletion in nsp2 (Asp268Del) which was found in all 3 samples originating from this cluster as well as in 37 other viruses collected in England and in Netherlands, suggesting the spread of this deletion in Europe. The impact of Asp268Del on SARS-CoV-2 transmission and pathogenicity, as well as on PCR performances and anti-viral strategy should be rapidly evaluated in further studies.","version":"1.1","doi":"10.1101/2020.03.19.998179","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.21.001628","pub_date":"2020-3-21","title":"Respiratory disease and virus shedding in rhesus macaques inoculated with SARS-CoV-2","abstract":"An outbreak of a novel coronavirus, now named SARS-CoV-2, causing respiratory disease and a \u223c2% case fatality rate started in Wuhan, China in December 2019. Following unprecedented rapid global spread, the World Health Organization declared COVID-19 a pandemic on March 11, 2020. Although data on disease in humans are emerging at a steady pace, certain aspects of the pathogenesis of SARS-CoV-2 can only be studied in detail in animal models, where repeated sampling and tissue collection is possible. Here, we show that SARS-CoV-2 causes respiratory disease in infected rhesus macaques, with disease lasting 8-16 days. Pulmonary infiltrates, a hallmark of human disease, were visible in lung radiographs of all animals. High viral loads were detected in swabs from the nose and throat of all animals as well as in bronchoalveolar lavages; in one animal we observed prolonged rectal shedding. Taken together, the rhesus macaque recapitulates moderate disease observed in the majority of human cases. The establishment of the rhesus macaque as a model of COVID-19 will increase our understanding of the pathogenesis of this disease and will aid development and testing of medical countermeasures.","version":"1.1","doi":"10.1101/2020.03.21.001628","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.20.999029","pub_date":"2020-3-21","title":"Replication of SARS-CoV-2 in human respiratory epithelium","abstract":"SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost importance to understand the virus biology and to rapidly assess the potential of existing drugs and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in the Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2. Further, we also provide basic characteristics of the system.","version":"1.1","doi":"10.1101/2020.03.20.999029","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.19.998724","pub_date":"2020-3-21","title":"All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus","abstract":"A recent outbreak of novel coronavirus (SARS-CoV-2), the causative agent of COVID-19, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and reduce disease transmission risk. Here, we present an All-In-One Dual CRISPR-Cas12a (termed \u201cAIOD-CRISPR\u201d) assay method for simple, rapid, ultrasensitive, one-pot, and visual detection of coronavirus SARS-CoV-2 and HIV virus. In our AIOD CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a detection and improve detection sensitivity. The AIOD-CRISPR assay system was successfully utilized to detect nucleic acids (DNA and RNA) of SARS-CoV-2 and HIV with a sensitivity of few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR method. Thus, our method has a great potential for developing next-generation point-of-care molecular diagnostics.","version":"1.1","doi":"10.1101/2020.03.19.998724","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.18.997585","pub_date":"2020-3-20","title":"Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase","abstract":"SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). Here, using polymerase extension experiments, we have demonstrated that the active triphosphate form of Sofosbuvir (a key component of the FDA approved hepatitis C drug EPCLUSA), is incorporated by SARS-CoV-2 RdRp, and blocks further incorporation. Using the same molecular insight, we selected the active triphosphate forms of three other anti-viral agents, Alovudine, AZT (an FDA approved HIV/AIDS drug) and Tenofovir alafenamide (TAF, an FDA approved drug for HIV and hepatitis B) for evaluation as inhibitors of SARS-CoV-2 RdRp. We demonstrated the ability of these three viral polymerase inhibitors, 3\u2019-fluoro-3\u2019-deoxythymidine triphosphate, 3\u2019-azido-3\u2019-deoxythymidine triphosphate and Tenofovir diphosphate (the active triphosphate forms of Alovudine, AZT and TAF, respectively) to be incorporated by SARS-CoV-2 RdRp, where they also terminate further polymerase extension. These results offer a strong molecular basis for these nucleotide analogues to be evaluated as potential therapeutics for COVID-19.","version":"1.1","doi":"10.1101/2020.03.18.997585","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.19.997890","pub_date":"2020-3-20","title":"An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus","abstract":"Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog \u03b2-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5\u2019-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses.","version":"1.1","doi":"10.1101/2020.03.19.997890","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.16.993816","pub_date":"2020-3-19","title":"Evidence of the Recombinant Origin and Ongoing Mutations in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)","abstract":"The recent global outbreak of viral pneumonia designated as Coronavirus Disease 2019 (COVID-19) by coronavirus (SARS-CoV-2) has threatened global public health and urged to investigate its source. Whole genome analysis of SARS-CoV-2 revealed ~96% genomic similarity with bat CoV (RaTG13) and clustered together in phylogenetic tree. Furthermore, RaTGl3 also showed 97.43% spike protein similarity with SARS-CoV-2 suggesting that RaTGl3 is the closest strain. However, RBD and key amino acid residues supposed to be crucial for human-to-human and cross-species transmission are homologues between SARS-CoV-2 and pangolin CoVs. These results from our analysis suggest that SARS-CoV-2 is a recombinant virus of bat and pangolin CoVs. Moreover, this study also reports mutations in coding regions of 125 SARS-CoV-2 genomes signifying its aptitude for evolution. In short, our findings propose that homologous recombination has been occurred between bat and pangolin CoVs that triggered cross-species transmission and emergence of SARS-CoV-2, and, during the ongoing outbreak, SARS-CoV-2 is still evolving for its adaptability.","version":"1.2","doi":"10.1101/2020.03.16.993816","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.16.993584","pub_date":"2020-3-19","title":"Multiple approaches for massively parallel sequencing of HCoV-19 (SARS-CoV-2) genomes directly from clinical samples","abstract":"COVID-19 has caused a major epidemic worldwide, however, much is yet to be known about the epidemiology and evolution of the virus. One reason is that the challenges underneath sequencing HCoV-19 directly from clinical samples have not been completely tackled. Here we illustrate the application of amplicon and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of HCoV-19 from clinical samples covering a range of sample types and viral load. We also examine and compare the bias, sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. This is, to date, the first work systematically implements amplicon and capture approaches in sequencing HCoV-19, as well as the first comparative study across methods. Our work offers practical solutions for genome sequencing and analyses of HCoV-19 and other emerging viruses.","version":"1.2","doi":"10.1101/2020.03.16.993584","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.16.994152","pub_date":"2020-3-18","title":"Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation","abstract":"Pandemic coronavirus disease 2019 (COVID-19) is caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there are no efficacious vaccines or therapeutics that are urgently needed. We expressed three versions of spike (S) proteins\u2014receptor binding domain (RBD), S1 subunit and S ectodomain\u2014in insect cells. RBD appears monomer in solutions, whereas S1 and S associate into homotrimer with substantial glycosylation. The three proteins confer excellent antigenicity with six convalescent COVID-19 patient sera. Cryo-electron microscopy (cryo-EM) analyses indicate that the SARS-CoV-2 S trimer dominate in a unique conformation distinguished from the classic prefusion conformation of coronaviruses by the upper S1 region at lower position ~15 \u00c5 proximal to viral membrane. Such conformation is proposed as an early prefusion state for the SARS-CoV-2 spike that may broaden the knowledge of coronavirus and facilitate vaccine development.","version":"1.2","doi":"10.1101/2020.03.16.994152","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.19.955484","pub_date":"2020-3-18","title":"Potential T-cell and B-cell Epitopes of 2019-nCoV","abstract":"As of early March, 2019-nCoV has infected more than one hundred thousand people and claimed thousands of lives. 2019-nCoV is a novel form of coronavirus that causes COVID-19 and has high similarity with SARS-CoV. No approved vaccine yet exists for any form of coronavirus. Here we use computational tools from structural biology and machine learning to identify 2019-nCoV T-cell and B-cell epitopes based on viral protein antigen presentation and antibody binding properties. These epitopes can be used to develop more effective vaccines and identify neutralizing antibodies. We identified 405 viral peptides with good antigen presentation scores for both human MHC-I and MHC-II alleles, and two potential neutralizing B-cell epitopes near the 2019-nCoV spike protein receptor binding domain (440-460 and 494-506). Analyzing mutation profiles of 68 viral genomes from four continents, we identified 96 coding-change mutations. These mutations are more likely to occur in regions with good MHC-I presentation scores (p=0.02). No mutations are present near the spike protein receptor binding domain. Based on these findings, the spike protein is likely immunogenic and a potential vaccine candidate. We validated our computational pipeline with SARS-CoV experimental data. The novel coronavirus 2019-nCoV has affected more than 100 countries and continues to spread. There is an immediate need for effective vaccines that contain antigens which trigger responses from human T-cells and B-cells (known as epitopes). Here we identify potential T-cell epitopes through an analysis of human antigen presentation, as well as B-cell epitopes through an analysis of protein structure. We identify a list of top candidates, including an epitope located on 2019-nCoV spike protein that potentially triggers both T-cell and B-cell responses. Analyzing 68 samples, we observe that viral mutations are more likely to happen in regions with strong antigen presentation, a potential form of immune evasion. Our computational pipeline is validated with experimental data from SARS-CoV.","version":"1.2","doi":"10.1101/2020.02.19.955484","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.15.993097","pub_date":"2020-3-17","title":"Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections","abstract":"The World Health Organization has recently declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, as pandemic. There is currently a lack of knowledge in the antibody response elicited from SARS-CoV-2 infection. One major immunological question is concerning the antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by using plasma from patients infected by SARS-CoV-2 or SARS-CoV, and plasma obtained from infected or immunized mice. Our results show that while cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses is rare, indicating the presence of non-neutralizing antibody response to conserved epitopes in the spike. Whether these non-neutralizing antibody responses will lead to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding the antigenicity differences between SARS-CoV-2 and SARS-CoV, but also has important implications in vaccine development.","version":"1.1","doi":"10.1101/2020.03.15.993097","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.10.986711","pub_date":"2020-3-17","title":"Efficient inactivation of SARS-CoV-2 by WHO-recommended hand rub formulations and alcohols","abstract":"The recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 is a major burden for health care systems worldwide. It is important to address if the current infection control instructions based on active ingredients are sufficient. We therefore determined the virucidal activity of two alcohol-based hand rub solutions for hand disinfection recommended by the World Health Organization (WHO), as well as commercially available alcohols. Efficient SARS-CoV-2 inactivation was demonstrated for all tested alcohol-based disinfectants. These findings show the successful inactivation of SARS-CoV-2 for the first time and provide confidence in its use for the control of COVID-19. The current COVID-19 outbreak puts a huge burden on the world\u2019s health care systems. Without effective therapeutics or vaccines being available, effective hygiene measure are of utmost importance to prevent viral spreading. It is therefore crucial to evaluate current infection control strategies against SARS-CoV-2. We show the inactivation of the novel coronavirus for the first time and endorse the importance of disinfectant-based hand hygiene to reduce SARS-CoV-2 transmission.","version":"1.1","doi":"10.1101/2020.03.10.986711","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.17.995639","pub_date":"2020-3-17","title":"Comparative Pathogenesis Of COVID-19, MERS And SARS In A Non-Human Primate Model","abstract":"A novel coronavirus, SARS-CoV-2, was recently identified in patients with an acute respiratory syndrome, COVID-19. To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or MERS-CoV and compared with historical SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in absence of clinical signs, and detected in type I and II pneumocytes in foci of diffuse alveolar damage and mucous glands of the nasal cavity. In SARS-CoV-infection, lung lesions were typically more severe, while they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 can cause a COVID-19-like disease, and suggest that the severity of SARS-CoV-2 infection is intermediate between that of SARS-CoV and MERS-CoV. SARS-CoV-2 infection in macaques results in COVID-19-like disease with prolonged virus excretion from nose and throat in absence of clinical signs.","version":"1.1","doi":"10.1101/2020.03.17.995639","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.15.992925","pub_date":"2020-3-17","title":"Master Regulator Analysis of the SARS-CoV-2/Human interactome","abstract":"the recent epidemic outbreak of a novel human coronavirus called SARS-CoV-2 and causing the respiratory tract disease COVID-19 has reached worldwide resonance and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. In this paper, we set out to shed light on the SARS-CoV-2/host receptor recognition, a crucial factor for successful virus infection. Based on the current knowledge of the interactome between SARS-CoV-2 and host cell proteins, we performed Master Regulator Analysis to detect which parts of the human interactome are most affected by the infection. We detected, amongst others, affected apoptotic and mitochondrial mechanisms, and a downregulation of the ACE2 protein receptor, notions that can be used to develop specific therapies against this new virus.","version":"1.1","doi":"10.1101/2020.03.15.992925","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.15.992883","pub_date":"2020-3-17","title":"A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein","abstract":"SARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease\u2014COVID-19\u2014 within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronavirus\u2019s surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 \u00c5. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the \u201cclosed\u201d prefusion S structure, but is accessible in \u201copen\u201d conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses. High resolution structure of the SARS-CoV-2 Receptor-Binding-Domain (RBD). Recognition of the SARS-CoV-2 RBD by SARS-CoV antibodies. Structure of the SARS-COV-2 RBD in complex with antibody CR3022. Identification of a cryptic site of vulnerability on the SARS-CoV-2 Spike.","version":"1.1","doi":"10.1101/2020.03.15.992883","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.16.990317","pub_date":"2020-3-17","title":"Recapitulation of SARS-CoV-2 Infection and Cholangiocyte Damage with Human Liver Organoids","abstract":"The newly emerged pandemic coronavirus, SARS-CoV-2, has posed a significant public health threat worldwide. However, the mode of virus transmission and tissue tropism is not well established yet. Recent findings of substantial liver damage in patients and ACE2+ cholangiocytes in healthy liver tissues prompted us to hypothesize that human liver ductal organoids could serve as a model to determine the susceptibility and mechanisms underlining the liver damage upon SARS-CoV-2 infection. By single-cell RNA sequencing, we found that long-term liver ductal organoid culture preserved the human specific ACE2+ population of cholangiocytes. Moreover, human liver ductal organoids were permissive to SARS-CoV-2 infection and support robust replication. Notably, virus infection impaired the barrier and bile acid transporting functions of cholangiocytes through dysregulation of genes involved in tight junction formation and bile acid transportation, which could explain the bile acid accumulation and consequent liver damage in patients. These results indicate that control of liver damage caused directly by viral infection should be valued in treating COVID-19 patients. Our findings also provide an application of human organoids in investigating the tropism and pathogenesis of SARS-CoV-2, which would facilitate novel drug discovery.","version":"1.1","doi":"10.1101/2020.03.16.990317","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.15.992818","pub_date":"2020-3-17","title":"An emergent clade of SARS-CoV-2 linked to returned travellers from Iran","abstract":"The SARS-CoV-2 epidemic has rapidly spread outside China with major outbreaks occurring in Italy, South Korea and Iran. Phylogenetic analyses of whole genome sequencing data identified a distinct SARS-CoV-2 clade linked to travellers returning from Iran to Australia and New Zealand. This study highlights potential viral diversity driving the epidemic in Iran, and underscores the power of rapid genome sequencing and public data sharing to improve the detection and management of emerging infectious diseases.","version":"1.1","doi":"10.1101/2020.03.15.992818","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.15.992438","pub_date":"2020-3-17","title":"Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection","abstract":"MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that have been found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs that would target their own genes or alter the host gene expression, there are examples of miRNAs functioning as an antiviral defense mechanism. In the case of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA \u2013 based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Our PANTHER gene function analysis results indicate that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription. For instance, a transcriptional regulator, STAT1 and transcription machinery might be targeted by virus-derived miRNAs. In addition, many known human miRNAs appear to be able to target viral genes. Considering the fact that miRNA-based therapies have been successful before, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.","version":"1.1","doi":"10.1101/2020.03.15.992438","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.12.946087","pub_date":"2020-3-16","title":"Candidate targets for immune responses to 2019-Novel Coronavirus (nCoV): sequence homology- and bioinformatic-based predictions","abstract":"Effective countermeasures against the recent emergence and rapid expansion of the 2019-Novel Coronavirus (2019-nCoV) require the development of data and tools to understand and monitor viral spread and immune responses. However, little information about the targets of immune responses to 2019-nCoV is available. We used the Immune Epitope Database and Analysis Resource (IEDB) resource to catalog available data related to other coronaviruses, including SARS-CoV, which has high sequence similarity to 2019-nCoV, and is the best-characterized coronavirus in terms of epitope responses. We identified multiple specific regions in 2019-nCoV that have high homology to SARS virus. Parallel bionformatic predictions identified a priori potential B and T cell epitopes for 2019-nCoV. The independent identification of the same regions using two approaches reflects the high probability that these regions are targets for immune recognition of 2019-nCoV. We identified potential targets for immune responses to 2019-nCoV and provide essential information for understanding human immune responses to this virus and evaluation of diagnostic and vaccine candidates.","version":"1.3","doi":"10.1101/2020.02.12.946087","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.12.988865","pub_date":"2020-3-15","title":"The architecture of SARS-CoV-2 transcriptome","abstract":"SARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3\u2032 tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2. We provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing. The transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs. We discover at least 41 potential RNA modification sites with an AAGAA motif.","version":"1.2","doi":"10.1101/2020.03.12.988865","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.990267","pub_date":"2020-3-15","title":"Differential Antibody Recognition by Novel SARS-CoV-2 and SARS-CoV Spike Protein Receptor Binding Domains: Mechanistic Insights and Implications for the Design of Diagnostics and Therapeutics","abstract":"The appearance of the novel betacoronavirus SARS-CoV-2 represents a major threat to human health, and its diffusion around the world is causing dramatic consequences. The knowledge of the 3D structures of SARS-CoV-2 proteins can facilitate the development of therapeutic and diagnostic molecules. Specifically, comparative analyses of the structures of SARS-CoV-2 proteins and homologous proteins from previously characterized viruses, such as SARS-CoV, can reveal the common and/or distinctive traits that underlie the mechanisms of recognition of cell receptors and of molecules of the immune system. Herein, we apply our recently developed energy-based methods for the prediction of antibody-binding epitopes and protein-protein interaction regions to the Receptor Binding Domain (RBD) of the Spike proteins from SARS-CoV-2 and SARS-CoV. Our analysis focusses only on the study of the structure of RBDs in isolation, without making use of any previous knowledge of binding properties. Importantly, our results highlight structural and sequence differences among the regions that are predicted to be immunoreactive and bind/elicit antibodies. These results provide a rational basis to the observation that several SARS-CoV RDB-specific monoclonal antibodies fail to appreciably bind the SARS-CoV-2 counterpart. Furthermore, we correctly identify the region of SARS-CoV-2 RBD that is engaged by the cell receptor ACE2 during viral entry into host cells. The data, sequences and structures we present here can be useful for the development of novel therapeutic and diagnostic interventions.","version":"1.2","doi":"10.1101/2020.03.13.990267","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.991570","pub_date":"2020-3-14","title":"A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV","abstract":"The outbreak of COVID-19, which is caused by SARS-CoV-2 virus, continues to spread globally, but there is currently very little understanding of the epitopes on the virus. In this study, we have determined the crystal structure of the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein in complex with CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient. CR3022 targets a highly conserved epitope that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding site can only be accessed when at least two RBDs on the trimeric S protein are in the \u201cup\u201d conformation. Overall, this study provides structural and molecular insight into the antigenicity of SARS-CoV-2. Structural study of a cross-reactive SARS antibody reveals a conserved epitope on the SARS-CoV-2 receptor-binding domain.","version":"1.1","doi":"10.1101/2020.03.13.991570","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.14.988345","pub_date":"2020-3-14","title":"SARS-CoV-2 invades host cells via a novel route: CD147-spike protein","abstract":"Currently, COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely spread around the world; nevertheless, so far there exist no specific antiviral drugs for treatment of the disease, which poses great challenge to control and contain the virus. Here, we reported a research finding that SARS-CoV-2 invaded host cells via a novel route of CD147-spike protein (SP). SP bound to CD147, a receptor on the host cells, thereby mediating the viral invasion. Our further research confirmed this finding. First, in vitro antiviral tests indicated Meplazumab, an anti-CD147 humanized antibody, significantly inhibited the viruses from invading host cells, with an EC50 of 24.86 \u03bcg/mL and IC50 of 15.16 \u03bcg/mL. Second, we validated the interaction between CD147 and SP, with an affinity constant of 1.85\u00d710\u22127M. Co-Immunoprecipitation and ELISA also confirmed the binding of the two proteins. Finally, the localization of CD147 and SP was observed in SARS-CoV-2 infected Vero E6 cells by immuno-electron microscope. Therefore, the discovery of the new route CD147-SP for SARS-CoV-2 invading host cells provides a critical target for development of specific antiviral drugs.","version":"1.1","doi":"10.1101/2020.03.14.988345","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.12.989186","pub_date":"2020-3-14","title":"Nucleotide Analogues as Inhibitors of SARS-CoV Polymerase","abstract":"SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved heptatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the activated triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the activated triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected two other anti-viral agents, Alovudine and AZT (an FDA approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two HIV reverse transcriptase inhibitors, 3\u2019-fluoro-3\u2019-deoxythymidine triphosphate and 3\u2019-azido-3\u2019-deoxythymidine triphosphate (the active triphosphate forms of Alovudine and AZT), to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.","version":"1.1","doi":"10.1101/2020.03.12.989186","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.13.991307","pub_date":"2020-3-14","title":"Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza","abstract":"The outbreak of the coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.","version":"1.1","doi":"10.1101/2020.03.13.991307","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.11.987958","pub_date":"2020-3-12","title":"A human monoclonal antibody blocking SARS-CoV-2 infection","abstract":"The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV). This cross-neutralizing antibody targets a communal epitope on these viruses and offers potential for prevention and treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.03.11.987958","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.09.983247","pub_date":"2020-3-12","title":"Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion","abstract":"The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed plasma membrane fusion capacity superior to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. We then generated a series of lipopeptides and found that the EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than that of EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, potently inhibiting replication of 4 live human coronaviruses, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs.","version":"1.1","doi":"10.1101/2020.03.09.983247","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.10.986398","pub_date":"2020-3-12","title":"The SARS-CoV-2 exerts a distinctive strategy for interacting with the ACE2 human receptor","abstract":"The COVID-19 disease has plagued over 110 countries and has resulted in over 4,000 deaths within 10 weeks. We compare the interaction between the human ACE2 receptor and the SARS-CoV-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. SARS-CoV, SARS-CoV-2, and HCoV-NL63 recognize ACE2 as the natural receptor but present a distinct binding interface to ACE2 and a different network of residue-residue contacts. SARS-CoV and SARS-CoV-2 have comparable binding affinities achieved by balancing energetics and dynamics. The SARS-CoV-2\u2013ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to SARS-CoV. These findings expose an exceptional evolutionary exploration exerted by coronaviruses toward host recognition. We postulate that the versatility of cell receptor binding strategies has immediate implications on therapeutic strategies. Molecular dynamics simulations reveal a temporal dimension of coronaviruses interactions with the host receptor.","version":"1.1","doi":"10.1101/2020.03.10.986398","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.10.986190","pub_date":"2020-3-12","title":"Rigidity, normal modes and flexible motion of a SARS-CoV-2 (COVID-19) protease structure","abstract":"The rigidity and flexibility of two recently reported crystal structures (PDB entries 6Y2E and 6LU7) of a protease from the SARS-CoV-2 virus, the infectious agent of the COVID-19 respiratory disease, has been investigated using pebble-game rigidity analysis, elastic network model normal mode analysis, and all-atom geometric simulations. This computational investigation of the viral protease follows protocols that have been effective in studying other homodimeric enzymes. The protease is predicted to display flexible motions in vivo which directly affect the geometry of a known inhibitor binding site and which open new potential binding sites elsewhere in the structure. A database of generated PDB files representing natural flexible variations on the crystal structures has been produced and made available for download from an institutional data archive. This information may inform structure-based drug design and fragment screening efforts aimed at identifying specific antiviral therapies for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.03.10.986190","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.11.987222","pub_date":"2020-3-12","title":"Discovery of a 382-nt deletion during the early evolution of SARS-CoV-2","abstract":"To date, the SARS-CoV-2 genome has been considered genetically more stable than SARS-CoV or MERS-CoV. Here we report a 382-nt deletion covering almost the entire open reading frame 8 (ORF8) of SARS-CoV-2 obtained from eight hospitalized patients in Singapore. The deletion also removes the ORF8 transcription-regulatory sequence (TRS), which in turn enhances the downstream transcription of the N gene. We also found that viruses with the deletion have been circulating for at least four weeks. During the SARS-CoV outbreak in 2003, a number of genetic variants were observed in the human population [1], and similar variation has since been observed across SARS-related CoVs in humans and bats. Overwhelmingly these viruses had mutations or deletions in ORF8, that have been associated with reduced replicative fitness of the virus [2]. This is also consistent with the observation that towards the end of the outbreak sequences obtained from human SARS cases possessed an ORF8 deletion that may be associated with host adaptation [1]. We therefore hypothesise that the major deletion revealed in this study may lead to an attenuated phenotype of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.03.11.987222","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.11.986836","pub_date":"2020-3-12","title":"A data-driven drug repositioning framework discovered a potential therapeutic agent targeting COVID-19","abstract":"The global spread of SARS-CoV-2 requires an urgent need to find effective therapeutics for the treatment of COVID-19. We developed a data-driven drug repositioning framework, which applies both machine learning and statistical analysis approaches to systematically integrate and mine large-scale knowledge graph, literature and transcriptome data to discover the potential drug candidates against SARS-CoV-2. The retrospective study using the past SARS-CoV and MERS-CoV data demonstrated that our machine learning based method can successfully predict effective drug candidates against a specific coronavirus. Our in silico screening followed by wet-lab validation indicated that a poly-ADP-ribose polymerase 1 (PARP1) inhibitor, CVL218, currently in Phase I clinical trial, may be repurposed to treat COVID-19. Our in vitro assays revealed that CVL218 can exhibit effective inhibitory activity against SARS-CoV-2 replication without obvious cytopathic effect. In addition, we showed that CVL218 is able to suppress the CpG-induced IL-6 production in peripheral blood mononuclear cells, suggesting that it may also have anti-inflammatory effect that is highly relevant to the prevention immunopathology induced by SARS-CoV-2 infection. Further pharmacokinetic and toxicokinetic evaluation in rats and monkeys showed a high concentration of CVL218 in lung and observed no apparent signs of toxicity, indicating the appealing potential of this drug for the treatment of the pneumonia caused by SARS-CoV-2 infection. Moreover, molecular docking simulation suggested that CVL218 may bind to the N-terminal domain of nucleocapsid (N) protein of SARS-CoV-2, providing a possible model to explain its antiviral action. We also proposed several possible mechanisms to explain the antiviral activities of PARP1 inhibitors against SARS-CoV-2, based on the data present in this study and previous evidences reported in the literature. In summary, the PARP1 inhibitor CVL218 discovered by our data-driven drug repositioning framework can serve as a potential therapeutic agent for the treatment of COVID-19.","version":"1.1","doi":"10.1101/2020.03.11.986836","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.11.983056","pub_date":"2020-3-12","title":"Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2","abstract":"Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of coronavirus SARS-CoV-2. Existing direct-acting antiviral (DAA) drugs cannot be applied immediately to new viruses because of virus-specificity, and the development of new DAA drugs from the beginning is not timely for outbreaks. Thus, host-targeting antiviral (HTA) drugs have many advantages to fight against a broad spectrum of viruses, by blocking the viral replication and overcoming the potential viral mutagenesis simultaneously. Herein, we identified two potent inhibitors of DHODH, S312 and S416, with favorable drug-like and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus (H1N1, H3N2, H9N2), Zika virus, Ebola virus, and particularly against the recent novel coronavirus SARS-CoV-2. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knocking-out cells. We also proposed the drug combination of DAA and HTA was a promising strategy for anti-virus treatment and proved that S312 showed more advantageous than Oseltamivir to treat advanced influenza diseases in severely infected animals. Notably, S416 is reported to be the most potent inhibitor with an EC50 of 17nM and SI value >5882 in SARS-CoV-2-infected cells so far. This work demonstrates that both our self-designed candidates and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-repression may have clinical potentials not only to influenza but also to COVID-19 circulating worldwide, no matter such viruses mutate or not.","version":"1.1","doi":"10.1101/2020.03.11.983056","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.11.987016","pub_date":"2020-3-12","title":"The inhaled corticosteroid ciclesonide blocks coronavirus RNA replication by targeting viral NSP15","abstract":"Steroid compounds, which are expected to have dual functions in blocking host inflammation and MERS-CoV replication, were screened from a chemical library. Within this library, ciclesonide, an inhaled corticosteroid, suppressed human coronavirus replication in cultured cells, but did not suppress replication of respiratory syncytial virus or influenza virus. The effective concentration of ciclesonide to block SARS-CoV-2 (the cause of COVID-19) replication (EC90) was 6.3 \u03bcM. After the eleventh consecutive MERS-CoV passage in the presence of ciclesonide, a resistant mutation was generated, which resulted in an amino acid substitution (A25V) in nonstructural protein (NSP) 15, as identified using reverse genetics. A recombinant virus with the mutation was also resistant to ciclesonide suppression of viral replication. These observations suggest that the effect of ciclesonide was specific to coronavirus, suggesting this is a candidate drug for treatment of patients suffering MERS or COVID-19.","version":"1.1","doi":"10.1101/2020.03.11.987016","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.06.977876","pub_date":"2020-3-11","title":"Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites","abstract":"The outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 \u00c5 crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the \u03b2-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.03.06.977876","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.27.967760","pub_date":"2020-3-11","title":"The SARS-CoV-2 receptor ACE2 expression of maternal-fetal interface and fetal organs by single cell transcriptome study","abstract":"Recent studies have demonstrated that SARS-CoV-2 cell entry depends on both ACE2 and TMPRSS2 genes (DOI: 10.1016/j.cell.2020.02.052), but our current work only focus on ACE2, which is insufficient to support the conclusion of this paper. So the authors have withdrawn their manuscript whilst they perform additional experiments and analysis to test some of their conclusions further. Therefore, the authors do not wish this work to be cited as reference for the project.","version":"1.3","doi":"10.1101/2020.02.27.967760","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.04.976258","pub_date":"2020-3-11","title":"Cryo-electron microscopy structure of the SADS-CoV spike glycoprotein provides insights into an evolution of unique coronavirus spike proteins","abstract":"The current outbreak of Coronavirus Disease 2019 (COVID-19) by a novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has aroused great public health concern. Coronavirus has a history of causing epidemics in human and animals. In 2017 an outbreak in piglets by a novel coronavirus was emerged designated as swine acute diarrhea syndrome coronavirus (SADS-CoV) which is originated from the same genus of horseshoe bats (Rhinolophus) as Severe Acute Respiratory Syndrome CoV (SARS-CoV) having a broad species tropism. In addition to human cells, it can also infect cell lines from diverse species. Coronavirus host range is determined by its spike glycoprotein (S). Given the importance of S protein in viral entry to cells and host immune responses, here we report the cryo-EM structure of the SADS-CoV S in the prefusion conformation at a resolution of 3.55 \u00c5. Our study reveals that SADS-CoV S structure takes an intra-subunit quaternary packing mode where the NTD and CTD from the same subunit pack together by facing each other. The comparison of NTD and CTD with that of the other four genera suggests the evolutionary process of the SADS-CoV S. Moreover, SADS-CoV S has several characteristic structural features, such as more compact architecture of S trimer, and masking of epitopes by glycan shielding, which may facilitate viral immune evasion. These data provide new insights into the evolutionary relationships of SADS-CoV S and would extend our understanding of structural and functional diversity, which will facilitate to vaccine development.","version":"1.2","doi":"10.1101/2020.03.04.976258","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.08.982637","pub_date":"2020-3-10","title":"Aerodynamic Characteristics and RNA Concentration of SARS-CoV-2 Aerosol in Wuhan Hospitals during COVID-19 Outbreak","abstract":"The ongoing outbreak of COVID-19 has spread rapidly and sparked global concern. While the transmission of SARS-CoV-2 through human respiratory droplets and contact with infected persons is clear, the aerosol transmission of SARS-CoV-2 has been little studied. Thirty-five aerosol samples of three different types (total suspended particle, size segregated and deposition aerosol) were collected in Patient Areas (PAA) and Medical Staff Areas (MSA) of Renmin Hospital of Wuhan University (Renmin) and Wuchang Fangcang Field Hospital (Fangcang), and Public Areas (PUA) in Wuhan, China during COVID-19 outbreak. A robust droplet digital polymerase chain reaction (ddPCR) method was employed to quantitate the viral SARS-CoV-2 RNA genome and determine aerosol RNA concentration. The ICU, CCU and general patient rooms inside Renmin, patient hall inside Fangcang had undetectable or low airborne SARS-CoV-2 concentration but deposition samples inside ICU and air sample in Fangcang patient toilet tested positive. The airborne SARS-CoV-2 in Fangcang MSA had bimodal distribution with higher concentration than those in Renmin during the outbreak but turned negative after patients number reduced and rigorous sanitization implemented. PUA had undetectable airborne SARS-CoV-2 concentration but obviously increased with accumulating crowd flow. Room ventilation, open space, proper use and disinfection of toilet can effectively limit aerosol transmission of SARS-CoV-2. Gathering of crowds with asymptomatic carriers is a potential source of airborne SARS-CoV-2. The virus aerosol deposition on protective apparel or floor surface and their subsequent resuspension is a potential transmission pathway and effective sanitization is critical in minimizing aerosol transmission of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.03.08.982637","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.03.972133","pub_date":"2020-3-08","title":"AI-aided design of novel targeted covalent inhibitors against SARS-CoV-2","abstract":"The focused drug repurposing of known approved drugs (such as lopinavir/ritonavir) has been reported failed for curing SARS-CoV-2 infected patients. It is urgent to generate new chemical entities against this virus. As a key enzyme in the life-cycle of coronavirus, the 3C-like main protease (3CLpro or Mpro) is the most attractive for antiviral drug design. Based on a recently solved structure (PDB ID: 6LU7), we developed a novel advanced deep Q-learning network with the fragment-based drug design (ADQN-FBDD) for generating potential lead compounds targeting SARS-CoV-2 3CLpro. We obtained a series of derivatives from those lead compounds by our structure-based optimization policy (SBOP). All the 47 lead compounds directly from our AI-model and related derivatives based on SBOP are accessible in our molecular library at https://github.com/tbwxmu/2019-nCov. These compounds can be used as potential candidates for researchers in their development of drugs against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.03.03.972133","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.06.980037","pub_date":"2020-3-07","title":"Monoclonal antibodies for the S2 subunit of spike of SARS-CoV cross-react with the newly-emerged SARS-CoV-2","abstract":"The emergence of a novel coronavirus, SARS-CoV-2, at the end of 2019 has resulted in widespread human infections across the globe. While genetically distinct from SARS-CoV, the etiological agent that caused an outbreak of severe acute respiratory syndrome (SARS) in 2003, both coronaviruses exhibit receptor binding domain (RBD) conservation and utilize the same host cell receptor, angiotensin-converting enzyme 2 (ACE2), for virus entry. Therefore, it will be important to test the cross-reactivity of antibodies that have been previously generated against the surface spike (S) glycoprotein of SARS-CoV in order to aid research on the newly emerged SARS-CoV-2. Here, we show that an immunogenic domain in the S2 subunit of SARS-CoV S is highly conserved in multiple strains of SARS-CoV-2. Consistently, four murine monoclonal antibodies (mAbs) raised against this immunogenic SARS-CoV fragment were able to recognise the S protein of SARS-CoV-2 expressed in a mammalian cell line. Importantly, one of them (mAb 1A9) was demonstrated to detect S in SARS-CoV-2-infected cells. To our knowledge, this is the first study showing that mAbs targeting the S2 domain of SARS-CoV can cross-react with SARS-CoV-2 and this observation is consistent with the high sequence conservation in the S2 subunit. These cross-reactive mAbs may serve as tools useful for SARS-CoV-2 research as well as for the development of diagnostic assays for its associated coronavirus disease COVID-19.","version":"1.1","doi":"10.1101/2020.03.06.980037","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.02.972935","pub_date":"2020-3-07","title":"Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient","abstract":"The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. Scientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.","version":"1.2","doi":"10.1101/2020.03.02.972935","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.29.965418","pub_date":"2020-3-07","title":"The within-host viral kinetics of SARS-CoV-2","abstract":"In this work, we use a within-host viral dynamic model to describe the SARS-CoV-2 kinetics in host. Chest radiograph score data are used to estimate the parameters of that model. Our result shows that the basic reproductive number of SARS-CoV-2 in host growth is around 3.79. Using the same method we also estimate the basic reproductive number of MERS virus is 8.16 which is higher than SARS-CoV-2. The PRCC method is used to analyze the sensitivities of model parameters and the drug effects on virus growth are also implemented to analyze the model.","version":"1.2","doi":"10.1101/2020.02.29.965418","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.04.977736","pub_date":"2020-3-07","title":"Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis Mechanisms of SARS-Related Coronaviruses","abstract":"A novel coronavirus (SARS-CoV-2) is the causative agent of an emergent severe respiratory disease (COVID-19) in humans that is threatening to result in a global health crisis. By using genomic, sequence, structural and evolutionary analysis, we show that Alpha- and Beta-CoVs possess several novel families of immunoglobulin (Ig) domain proteins, including ORF8 and ORF7a from SARS-related coronaviruses and two protein groups from certain Alpha-CoVs. Among them, ORF8 is distinguished in being rapidly evolving, possessing a unique insert and a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover many Ig proteins from several metazoan viruses which are distinct in sequence and structure but share an architecture comparable to that of CoV Ig domain proteins. Hence, we propose that deployment of Ig domain proteins is a widely-used strategy by viruses, and SARS-CoV-2 ORF8 is a potential pathogenicity factor which evolves rapidly to counter the immune response and facilitate the transmission between hosts.","version":"1.1","doi":"10.1101/2020.03.04.977736","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.05.979260","pub_date":"2020-3-07","title":"LY6E impairs coronavirus fusion and confers immune control of viral disease","abstract":"Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.","version":"1.1","doi":"10.1101/2020.03.05.979260","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.17.952895","pub_date":"2020-3-06","title":"Functional pangenome analysis suggests inhibition of the protein E as a readily available therapy for COVID-2019","abstract":"The spread of the novel coronavirus (SARS-CoV-2) has triggered a global emergency, that demands urgent solutions for detection and therapy to prevent escalating health, social and economic impacts. The spike protein (S) of this virus enables binding to the human receptor ACE2, and hence presents a prime target for vaccines preventing viral entry into host cells. The S proteins from SARS-CoV-1 and SARS-CoV-2 are similar, but structural differences in the receptor binding domain (RBD) preclude the use of SARS-CoV-1\u2013specific neutralizing antibodies to inhibit SARS-CoV-2. Here we used comparative pangenomic analysis of all sequenced Betacoronaviruses to reveal that, among all core gene clusters present in these viruses, the envelope protein E shows a variant shared by SARS and SARS-Cov2 with two completely-conserved key functional features, an ion-channel and a PDZ-binding Motif (PBM). These features trigger a cytokine storm that activates the inflammasome, leading to increased edema in lungs causing the acute respiratory distress syndrome (ARDS), the leading cause of death in SARS-CoV-1 and SARS-CoV-2 infection. However, three drugs approved for human use may inhibit SARS-CoV-1 and SARS-CoV-2 Protein E, either acting upon the ion channel (Amantadine and Hexamethylene amiloride) or the PBM (SB203580), thereby potentially increasing the survival of the host, as already demonstrated for SARS-CoV-1in animal models. Hence, blocking the SARS protein E inhibits development of ARDS in vivo. Given that our results demonstrate that the protein E subcluster for the SARS clade is quasi-identical for the key functional regions of SARS-CoV-1 and SARS-CoV-2, we conclude that use of approved drugs shown to act as SARS E protein inhibitors can help prevent further casualties from COVID-2019 while vaccines and other preventive measures are being developed.","version":"1.2","doi":"10.1101/2020.02.17.952895","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.05.935072","pub_date":"2020-3-06","title":"The Essential Facts of Wuhan Novel Coronavirus Outbreak in China and Epitope-based Vaccine Designing against COVID-19","abstract":"Wuhan Novel Coronavirus disease (COVID-19) outbreak has become a global outbreak which has raised the concern of scientific community to design and discover a definitive cure against this deadly virus which has caused deaths of numerous infected people upon infection and spreading. To date, no antiviral therapy or vaccine is available which can effectively combat the infection caused by this virus. This study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against SARS-CoV-2. Later, molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. Hopefully, this study will contribute to uphold the present efforts of the researches to secure a definitive treatment against this lethal virus.","version":"1.2","doi":"10.1101/2020.02.05.935072","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.02.972927","pub_date":"2020-3-05","title":"Viral Architecture of SARS-CoV-2 with Post-Fusion Spike Revealed by Cryo-EM","abstract":"Since December 2019, the outbreak of Coronavirus Disease 2019 (COVID-19) spread from Wuhan, China to the world, it has caused more than 87,000 diagnosed cases and more than 3,000 deaths globally. To fight against COVID-19, we carried out research for the near native SARS-CoV-2 and report here our preliminary results obtained. The pathogen of the COVID-19, the native SARS-CoV-2, was isolated, amplified and purified in a BSL-3 laboratory. The whole viral architecture of SARS-CoV-2 was examined by transmission electron microscopy (both negative staining and cryo-EM). We observed that the virion particles are roughly spherical or moderately pleiomorphic. Spikes have nail-like shape towards outside with a long body embedded in the envelope. The morphology of virion observed in our result indicates that the S protein of SARS-CoV-2 is in post-fusion state, with S1 disassociated. This state revealed by cryo-EM first time could provide an important information for the identification and relevant clinical research of this new coronavirus.","version":"1.1","doi":"10.1101/2020.03.02.972927","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.02.968818","pub_date":"2020-3-05","title":"Rapid metagenomic characterization of a case of imported COVID-19 in Cambodia","abstract":"Rapid production and publication of pathogen genome sequences during emerging disease outbreaks provide crucial public health information. In resource-limited settings, especially near an outbreak epicenter, conventional deep sequencing or bioinformatics are often challenging. Here we successfully used metagenomic next generation sequencing on an iSeq100 Illumina platform paired with an open-source bioinformatics pipeline to quickly characterize Cambodia\u2019s first case of COVID-2019.","version":"1.1","doi":"10.1101/2020.03.02.968818","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.04.975995","pub_date":"2020-3-04","title":"Strong evolutionary convergence of receptor-binding protein spike between COVID-19 and SARS-related coronaviruses","abstract":"Coronavirus Disease 2019 (COVID-19) and severe acute respiratory syndrome (SARS)-related coronaviruses (e.g., 2019-nCoV and SARS-CoV) are phylogenetically distantly related, but both are capable of infecting human hosts via the same receptor, angiotensin-converting enzyme 2, and cause similar clinical and pathological features, suggesting their phenotypic convergence. Yet, the molecular basis that underlies their phenotypic convergence remains unknown. Here, we used a recently developed molecular phyloecological approach to examine the molecular basis leading to their phenotypic convergence. Our genome-level analyses show that the spike protein, which is responsible for receptor binding, has undergone significant Darwinian selection along the branches related to 2019-nCoV and SARS-CoV. Further examination shows an unusually high proportion of evolutionary convergent amino acid sites in the receptor binding domain (RBD) of the spike protein between COVID-19 and SARS-related CoV clades, leading to the phylogenetic uniting of their RBD protein sequences. In addition to the spike protein, we also find the evolutionary convergence of its partner protein, ORF3a, suggesting their possible co-evolutionary convergence. Our results demonstrate a strong adaptive evolutionary convergence between COVID-19 and SARS-related CoV, possibly facilitating their adaptation to similar or identical receptors. Finally, it should be noted that many observed bat SARS-like CoVs that have an evolutionary convergent RBD sequence with 2019-nCoV and SARS-CoV may be pre-adapted to human host receptor ACE2, and hence would be potential new coronavirus sources to infect humans in the future.","version":"1.1","doi":"10.1101/2020.03.04.975995","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.02.968388","pub_date":"2020-3-03","title":"Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2","abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report the high-resolution crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 \u2013 a virus causing current world-wide epidemics. We compare this structure with previously reported models of Nsp15 from SARS and MERS coronaviruses.","version":"1.1","doi":"10.1101/2020.03.02.968388","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.27.969006","pub_date":"2020-3-02","title":"Comparative genomic analysis revealed specific mutation pattern between human coronavirus SARS-CoV-2 and Bat-SARSr-CoV RaTG13","abstract":"The novel coronavirus SARS-CoV-2 (2019-nCoV) is a member of the family coronaviridae and contains a single-stranded RNA genome with positive-polarity. To reveal the evolution mechanism of SARS-CoV-2 genome, we performed comprehensive genomic analysis with newly sequenced SARS-CoV-2 strains and 20 closely related coronavirus strains. Among 98 nucleotide mutations at 93 sites of the genome among different SARS-CoV-2 strains, 58 of them caused amino acid change, indicating a result of neutral evolution. However, the ratio of nucleotide substitutions to amino acid substitutions of spike gene (9.07) between SARS-CoV-2 WIV04 and Bat-SARSr-CoV RaTG13 was extensively higher than those from comparisons between other coronaviruses (range 1.29 - 4.81). The elevated synonymous mutations between SARS-CoV-2 and RaTG13, suggesting they underwent stronger purifying selection. Moreover, their nucleotide substitutions are enriched with T:C transition, which is consistent with the mutation signature caused by deactivity of RNA 3\u2019-to-5\u2019 exoribonuclease (ExoN). The codon usage was similar between SARS-CoV-2 and other strains in beta-coronavirus lineage B, suggesting it had small impact on the mutation pattern. In comparison of SARS-CoV-2 WIV04 with Bat-SARSr-CoV RaTG13, the ratios of non-synonymous to synonymous substitution rates (dN/dS) was the lowest among all performed comparisons, reconfirming the evolution of SARS-CoV-2 under stringent selective pressure. Moreover, some sites of spike protein might be subjected to positive selection. Therefore, our results will help understanding the evolutionary mechanisms contribute to viral pathogenicity and its adaptation with hosts.","version":"1.1","doi":"10.1101/2020.02.27.969006","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.29.971101","pub_date":"2020-3-02","title":"Mutations, Recombination and Insertion in the Evolution of 2019-nCoV","abstract":"The 2019 novel coronavirus (2019-nCoV or SARS-CoV-2) has spread more rapidly than any other betacoronavirus including SARS-CoV and MERS-CoV. However, the mechanisms responsible for infection and molecular evolution of this virus remained unclear. We collected and analyzed 120 genomic sequences of 2019-nCoV including 11 novel genomes from patients in China. Through comprehensive analysis of the available genome sequences of 2019-nCoV strains, we have tracked multiple inheritable SNPs and determined the evolution of 2019-nCoV relative to other coronaviruses. Systematic analysis of 120 genomic sequences of 2019-nCoV revealed co-circulation of two genetic subgroups with distinct SNPs markers, which can be used to trace the 2019-nCoV spreading pathways to different regions and countries. Although 2019-nCoV, human and bat SARS-CoV share high homologous in overall genome structures, they evolved into two distinct groups with different receptor entry specificities through potential recombination in the receptor binding regions. In addition, 2019-nCoV has a unique four amino acid insertion between S1 and S2 domains of the spike protein, which created a potential furin or TMPRSS2 cleavage site. Our studies provided comprehensive insights into the evolution and spread of the 2019-nCoV. Our results provided evidence suggesting that 2019-nCoV may increase its infectivity through the receptor binding domain recombination and a cleavage site insertion. Novel 2019-nCoV sequences revealed the evolution and specificity of betacoronavirus with possible mechanisms of enhanced infectivity.","version":"1.1","doi":"10.1101/2020.02.29.971101","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.26.961938","pub_date":"2020-3-02","title":"Predictions for the binding domain and potential new drug targets of 2019-nCoV","abstract":"An outbreak of new SARS-like viral in Wuhan, China has been named 2019-nCoV. The current state of the epidemic is increasingly serious, and there has been the urgent necessity to develop an effective new drug. In previous studies, it was found that the conformation change in CTD1 was the region where SARS-CoV bound to human ACE2. Although there are mutations of the 2019-nCoV, the binding energy of ACE2 remains high. The surface glycoprotein of 2019-nCoV was coincident with the CTD1 region of the S-protein by comparing the I-TASSER prediction model with the actual SARS model, which suggests that 2019-nCoV may bind to the ACE2 receptor through conformational changes. Furthermore, site prediction on the surface glycoprotein of 2019-nCoV suggests some core amino acid area may be a novel drug target against 2019-nCoV.","version":"1.1","doi":"10.1101/2020.02.26.961938","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.29.971127","pub_date":"2020-3-02","title":"An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received global attention due to the recent outbreak in China. In this work, we report a CRISPR-Cas12 based diagnostic tool to detect synthetic SARS-CoV-2 RNA sequences in a proof-of-principle evaluation. The test proved to be sensitive, rapid, and potentially portable. These key traits of the CRISPR method are critical for virus detection in regions that lack resources to use the currently available methods.","version":"1.1","doi":"10.1101/2020.02.29.971127","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.26.967026","pub_date":"2020-3-02","title":"CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design","abstract":"The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at https://adapt.sabetilab.org/.","version":"1.2","doi":"10.1101/2020.02.26.967026","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.04.934232","pub_date":"2020-3-02","title":"Design of multi epitope-based peptide vaccine against E protein of human COVID-19: An immunoinformatics approach","abstract":"New endemic disease has been spread across Wuhan City, China on December 2019. Within few weeks, the World Health Organization (WHO) announced a novel coronavirus designated as coronavirus disease 2019 (COVID-19). In late January 2020, WHO declared the outbreak of a \u201cpublic-health emergency of international concern\u201d due to the rapid and increasing spread of the disease worldwide. Currently, there is no vaccine or approved treatment for this emerging infection; thus the objective of this study is to design a multi epitope peptide vaccine against COVID-19 using immunoinformatics approach. Several techniques facilitating the combination of immunoinformatics approach and comparative genomic approach were used in order to determine the potential peptides for designing the T cell epitopes-based peptide vaccine using the envelope protein of 2019-nCoV as a target. Extensive mutations, insertion and deletion were discovered with comparative sequencing in COVID-19 strain. Additionally, ten peptides binding to MHC class I and MHC class II were found to be promising candidates for vaccine design with adequate world population coverage of 88.5% and 99.99%, respectively. T cell epitopes-based peptide vaccine was designed for COVID-19 using envelope protein as an immunogenic target. Nevertheless, the proposed vaccine is rapidly needed to be validated clinically in order to ensure its safety, immunogenic profile and to help on stopping this epidemic before it leads to devastating global outbreaks.","version":"1.2","doi":"10.1101/2020.02.04.934232","journal":"bioRxiv","score":null},{"id":"10.1101/2020.03.01.971499","pub_date":"2020-3-02","title":"Kallikrein 13: a new player in coronaviral infections","abstract":"Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host specificity, but virus entry is a complex process requiring a concerted action of multiple cellular elements. Here, we show that KLK13 is required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that protease distribution and specificity predetermines the tissue and cell specificity of the virus and may also regulate interspecies transmission. It is also of importance that presented data may be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand the differences in their zoonotic potential.","version":"1.1","doi":"10.1101/2020.03.01.971499","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.07.939389","pub_date":"2020-2-28","title":"The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice","abstract":"Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) caused the Corona Virus Disease 2019 (COVID-19) cases in China has become a public health emergency of international concern (PHEIC). Based on angiotensin converting enzyme 2 (ACE2) as cell entry receptor of SARS-CoV, we used the hACE2 transgenic mice infected with SARS-CoV-2 to study the pathogenicity of the virus. Weight loss and virus replication in lung were observed in hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes and monocytes in alveolar interstitium, and accumulation of macrophages in alveolar cavities. Viral antigens were observed in the bronchial epithelial cells, alveolar macrophages and alveolar epithelia. The phenomenon was not found in wild type mice with SARS-CoV-2 infection. The pathogenicity of SARS-CoV-2 in hACE2 mice was clarified and the Koch\u2019s postulates were fulfilled as well, and the mouse model may facilitate the development of therapeutics and vaccines against SARS-CoV-2.","version":"1.3","doi":"10.1101/2020.02.07.939389","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.22.951178","pub_date":"2020-2-27","title":"Spike protein binding prediction with neutralizing antibodies of SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19) is a new emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV), originated in Wuhan seafood and animal market, China. Since December 2019, more than 69,000 cases of COVID-19 have been confirmed in China and quickly spreads to other counties. Currently, researchers put their best efforts to identify effective drugs for COVID-19. The neutralizing antibody, which binds to viral capsid in a manner that inhibits cellular entry of virus and uncoating of the genome, is the specific defense against viral invaders. In this study, we investigate to identify neutralizing antibodies that can bind to SARS-CoV-2 Sipke (S) protein and interfere with the interaction between viral S protein and a host receptor by bioinformatic methods. The sequence analysis of S protein showed two major differences in the RBD region of the SARS-CoV-2 S protein compared to SARS-CoV and SARS-CoV related bat viruses (btSARS-CoV). The insertion regions were close to interacting residues with the human ACE2 receptor. Epitope analysis of neutralizing antibodies revealed that SARS-CoV neutralizing antibodies used conformational epitopes, whereas MERS-CoV neutralizing antibodies used a common linear epitope region, which contributes to form the \u03b2-sheet structure in MERS-CoV S protein and deleted in SARS-CoV-2 S protein. To identify effective neutralizing antibodies for SARS-CoV-2, the binding affinities of neutralizing antibodies with SARS-CoV-2 S protein were predicted and compared by antibody-antigen docking simulation. The result showed that CR3022 neutralizing antibody from human may have higher binding affinity with SARS-CoV-2 S protein than SARS-CoV S protein. We also found that F26G19 and D12 mouse antibodies could bind to SARS-CoV S protein with high affinity. Our findings provide crucial clues towards the development of antigen diagnosis, therapeutic antibody, and the vaccine against SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.02.22.951178","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.25.964775","pub_date":"2020-2-27","title":"Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2","abstract":"Coronavirus disease 2019 (COVID-19) is newly emerging human infectious diseases, which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within two months of the outbreak, more than 80,000 cases of COVID-19 have been confirmed worldwide. Since the human to human transmission occurred easily and the human infection is rapidly increasing, the sensitive and early diagnosis is essential to prevent the global outbreak. Recently, World Health Organization (WHO) announced various primer and probe sets for SARS-CoV-2 previously developed in China, Germany, Hong Kong, Japan, Thailand, and USA. In this study, we compared the ability to detect SARS-CoV-2 RNA among the seven primer-probe sets for N gene and the three primer-probe sets for Orf1 gene. The result of the comparative analysis represented that the \u20182019-nCoV_N2, N3\u2019 of USA and the \u2018ORF1ab\u2019 of China are the most sensitive primer-probe sets for N and Orf1 genes, respectively. Therefore, the appropriate combination from ORF1ab (China), 2019-nCoV_N2, N3 (USA), and NIID_2019-nCOV_N (Japan) sets should be selected for the sensitive and reliable laboratory confirmation of SARS-CoV-2.","version":"1.1","doi":"10.1101/2020.02.25.964775","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.24.963348","pub_date":"2020-2-27","title":"Increasing Host Cellular Receptor\u2014Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019-nCoV Infection","abstract":"The ongoing outbreak of a new coronavirus (2019-nCoV) causes an epidemic of acute respiratory syndrome in humans. 2019-nCoV rapidly spread to national regions and multiple other countries, thus, pose a serious threat to public health. Recent studies show that spike (S) proteins of 2019-nCoV and SARS-CoV may use the same host cell receptor called angiotensin-converting enzyme 2 (ACE2) for entering into host cells. The affinity between ACE2 and 2019-nCoV S is much higher than ACE2 binding to SARS-CoV S protein, explaining that why 2019-nCoV seems to be more readily transmitted from the human to human. Here, we reported that ACE2 can be significantly upregulated after infection of various viruses including SARS-CoV and MERS-CoV. Basing on findings here, we propose that coronavirus infection can positively induce its cellular entry receptor to accelerate their replication and spread, thus drugs targeting ACE2 expression may be prepared for the future emerging infectious diseases caused by this cluster of viruses.","version":"1.1","doi":"10.1101/2020.02.24.963348","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.22.961268","pub_date":"2020-2-27","title":"A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2","abstract":"The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) originating from Wuhan, China, draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based molecular diagnosis techniques are being widely applied for clinical diagnosis currently, timely and accurate diagnosis are still limited due to labour intensive and time-consuming operations of these techniques. To address the issue, herein we report the synthesis of poly (amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs), and the development of pcMNPs-based viral RNA extraction method for the sensitive detection of COVID-19 causing virus, the SARS-CoV-2. This method combines the lysis and binding steps into one step, and the pcMNPs-RNA complexes can be directly introduced into subsequent RT-PCR reactions. The simplified process can purify viral RNA from multiple samples within 20 min using a simple manual method or an automated high-throughput approach. By identifying two different regions (ORFlab and N gene) of viral RNA, a 10-copy sensitivity and a strong linear correlation between 10 and 105 copies of SARS-CoV-2 pseudovirus particles are achieved. Benefitting from the simplicity and excellent performances, this new extraction method can dramatically reduce the turn-around time and operational requirements in current molecular diagnosis of COVID-19, in particular for the early clinical diagnosis.","version":"1.1","doi":"10.1101/2020.02.22.961268","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.25.965434","pub_date":"2020-2-27","title":"Epitope-based peptide vaccine design and target site characterization against novel coronavirus disease caused by SARS-CoV-2","abstract":"The outbreak of the 2019 novel coronavirus (SARS-CoV-2) has infected thousands of people with a large number of deaths across 26 countries. The sudden appearance of the virus leads to the limited existing therapies for SARS-CoV-2. Therefore, vaccines and antiviral medicines are in desperate need. This study took immune-informatics approaches to identify B- and T-cell epitopes for surface glycoprotein (S) of SARS-CoV-2, followed by estimating their antigenicity and interactions with the human leukocyte antigen (HLA) alleles. We identified four B cell epitopes, two MHC class-I and nine MHC class-II binding T-cell epitopes, which showed highly antigenic features. Allergenicity, toxicity and physiochemical properties analysis confirmed the specificity and selectivity of epitopes. The stability and safety of epitopes were confirmed by digestion analysis. No mutations were observed in all the selected B- and T-cell epitopes across all isolates from different locations worldwide. Epitopes were thus identified and some of them can be potential candidates for vaccine development.","version":"1.2","doi":"10.1101/2020.02.25.965434","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.15.950568","pub_date":"2020-2-25","title":"Multivariate Analyses of Codon Usage of SARS-CoV-2 and other betacoronaviruses","abstract":"Coronavirus disease 2019 (COVID-19) is a global health concern as it continues to spread within China and beyond. The causative agent of this disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus Betacoronavirus which also includes severe acute respiratory syndrome related coronavirus (SARSr-CoV) and Middle East respiratory syndrome related coronavirus (MERSr-CoV). Codon usage of viral genes are believed to be subjected to different selection pressures in different host environments. Previous studies on codon usage of influenza A viruses can help identify viral host origins and evolution trends, however, similar studies on coronaviruses are lacking. In this study, global correspondence analysis (CA), within-group correspondence analysis (WCA) and between-group correspondence analysis (BCA) were performed among different genes in coronavirus viral sequences. The amino acid usage pattern of SARS-CoV-2 was generally found similar to bat and human SARSr-CoVs. However, we found greater synonymous codon usage differences between SARS-CoV-2 and its phylogenetic relatives on spike and membrane genes, suggesting these two genes of SARS-CoV-2 are subjected to different evolutionary pressures.","version":"1.3","doi":"10.1101/2020.02.15.950568","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.21.959973","pub_date":"2020-2-25","title":"No more business as usual: agile and effective responses to emerging pathogen threats require open data and open analytics","abstract":"The current state of much of the Wuhan pneumonia virus (COVID-19) research shows a regrettable lack of data sharing and considerable analytical obfuscation. This impedes global research cooperation, which is essential for tackling public health emergencies, and requires unimpeded access to data, analysis tools, and computational infrastructure. Here we show that community efforts in developing open analytical software tools over the past ten years, combined with national investments into scientific computational infrastructure, can overcome these deficiencies and provide an accessible platform for tackling global health emergencies in an open and transparent manner. Specifically, we use all COVID-19 genomic data available in the public domain so far to (1) underscore the importance of access to raw data and to (2) demonstrate that existing community efforts in curation and deployment of biomedical software can reliably support rapid, reproducible research during global health crises. All our analyses are fully documented at https://github.com/galaxyproject/SARS-CoV-2.","version":"1.2","doi":"10.1101/2020.02.21.959973","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.20.958785","pub_date":"2020-2-22","title":"Inactivating porcine coronavirus before nuclei acid isolation with the temperature higher than 56 \u00b0C damages its genome integrity seriously","abstract":"2019-Novel Coronavirus (2019-nCoV) is the pathogen of Corona Virus Disease 2019. Nucleic acid detection of 2019-nCoV is one of the key indicators for clinical diagnosis. However, the positive rate is only 30-50%. Currently, fluorescent quantitative RT-PCR technology is mainly used to detect 2019-nCoV. According to \u201cThe Laboratory Technical Guidelines for Detection 2019-nCoV (Fourth Edition)\u201d issued by National Health and Commission of China and \u201cThe Experts\u2019 Consensus on Nucleic Acid Detection of 2019-nCoV\u201d released by Chinese Society of Laboratory Medicine, the human samples must be placed under 56\u00b0C or higher to inactivate the viruses in order to keep the inspectors from virus infection before the nucleic acids were isolated as the template of qRT-PCR. In this study, we demonstrated that the virus inactivation treatment disrupts its genome integrity seriously when using porcine epidemic diarrhea virus (vaccine), a kind of coronavirus, as a model. Our results showed that only 50.11% of the detectable viral templates left after the inactivation of 56 \u00b0C for 30 minutes and only 3.36% left after the inactivation of 92 \u00b0C for 5 minutes when the samples were preserved by Hank\u2019s solution, one of an isotonic salt solutions currently suggested. However, the detectable templates of viral nucleic acids can be unchanged after the samples were incubated at 56 \u00b0C or higher if the samples were preserved with an optimized solution to protect the RNA from being disrupted. We therefore highly recommend to carry out systematic investigation on the impact of high temperature inactivation on the integrity of 2019-nCoV genome and develop a sample preservation solution to protect the detectable templates of 2019-nCoV nucleic acids from high temperature inactivation damage.","version":"1.1","doi":"10.1101/2020.02.20.958785","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.17.952903","pub_date":"2020-2-21","title":"Molecular mechanism of evolution and human infection with the novel coronavirus (2019-nCoV)","abstract":"Since December, 2019, an outbreak of pneumonia caused by the new coronavirus (2019-nCoV) has hit the city of Wuhan in the Hubei Province. With the continuous development of the epidemic, it has become a national public health crisis and calls for urgent antiviral treatments or vaccines. The spike protein on the coronavirus envelope is critical for host cell infection and virus vitality. Previous studies showed that 2019-nCoV is highly homologous to human SARS-CoV and attaches host cells though the binding of the spike receptor binding domain (RBD) domain to the angiotensin-converting enzyme II (ACE2). However, the molecular mechanisms of 2019-nCoV binding to human ACE2 and evolution of 2019-nCoV remain unclear. In this study, we have extensively studied the RBD-ACE2 complex, spike protein, and free RBD systems of 2019-nCoV and SARS-CoV using protein-protein docking and molecular dynamics (MD) simulations. It was shown that the RBD-ACE2 binding free energy for 2019-nCoV is significantly lower than that for SARS-CoV, which is consistent with the fact that 2019-nCoV is much more infectious than SARS-CoV. In addition, the spike protein of 2019-nCoV shows a significantly lower free energy than that of SARS-CoV, suggesting that 2019-nCoV is more stable and able to survive a higher temperature than SARS-CoV. This may also provide insights into the evolution of 2019-nCoV because SARS-like coronaviruses are thought to have originated in bats that are known to have a higher body-temperature than humans. It was also revealed that the RBD of 2019-nCoV is much more flexible especially near the binding site and thus will have a higher entropy penalty upon binding ACE2, compared to the RBD of SARS-CoV. That means that 2019-nCoV will be much more temperature-sensitive in terms of human infection than SARS-CoV. With the rising temperature, 2019-nCoV is expected to decrease its infection ability much faster than SARS-CoV, and get controlled more easily. The present findings are expected to be helpful for the disease prevention and control as well as drug and vaccine development of 2019-nCoV.","version":"1.1","doi":"10.1101/2020.02.17.952903","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.21.959817","pub_date":"2020-2-21","title":"Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform","abstract":"Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-2 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.","version":"1.1","doi":"10.1101/2020.02.21.959817","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.17.951939","pub_date":"2020-2-21","title":"Protection of Rhesus Macaque from SARS-Coronavirus challenge by recombinant adenovirus vaccine","abstract":"A recombinant adenovirus vaccine against the SARS Coronavirus (SARS-CoV) was constructed, which contains fragments from the S, N, and Orf8 genes. Rhesus Macaques immunized with the recombinant adenovirus generated antigen-specific humoral and cellular response. Furthermore, the vaccine provided significant protection against subsequent live SARS-CoV challenge. In contrast, three out of four monkeys immunized with placebo suffered severe alveolar damage and pulmonary destruction.","version":"1.1","doi":"10.1101/2020.02.17.951939","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.16.951913","pub_date":"2020-2-21","title":"Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses","abstract":"The new coronavirus (2019-nCoV) outbreak from December 2019 in Wuhan, Hubei, China, has been declared a global public health emergency. Angiotensin I converting enzyme 2 (ACE2), is the host receptor by 2019-nCov to infect human cells. Although ACE2 is reported to be expressed in lung, liver, stomach, ileum, kidney and colon, its expressing levels are rather low, especially in the lung. 2019-nCoV may use co-receptors/auxiliary proteins as ACE2 partner to facilitate the virus entry. To identify the potential candidates, we explored the single cell gene expression atlas including 119 cell types of 13 human tissues and analyzed the single cell co-expression spectrum of 51 reported RNA virus receptors and 400 other membrane proteins. Consistent with other recent reports, we confirmed that ACE2 was mainly expressed in lung AT2, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileum ECs, rectum ECs, stomach epithelial cells, and kidney proximal tubules. Intriguingly, we found that the candidate co-receptors, manifesting the most similar expression patterns with ACE2 across 13 human tissues, are all peptidases, including ANPEP, DPP4 and ENPEP. Among them, ANPEP and DPP4 are the known receptors for human CoVs, suggesting ENPEP as another potential receptor for human CoVs. We also conducted \u201cCellPhoneDB\u201d analysis to understand the cell crosstalk between CoV-targets and their surrounding cells across different tissues. We found that macrophages frequently communicate with the CoVs targets through chemokine and phagocytosis signaling, highlighting the importance of tissue macrophages in immune defense and immune pathogenesis.","version":"1.1","doi":"10.1101/2020.02.16.951913","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.20.957472","pub_date":"2020-2-21","title":"Vulnerabilities in coronavirus glycan shields despite extensive glycosylation","abstract":"Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development has focussed on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein, which mediates receptor recognition and membrane fusion. Coronavirus S proteins are extensively glycosylated viral fusion proteins, encoding around 69-87 N-linked glycosylation sites per trimeric spike. Using a multifaceted structural approach, we reveal a specific area of high glycan density on MERS S that results in the formation of under-processed oligomannose-type glycan clusters, which was absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Consistent with the ability of the antibody-mediated immune response to effectively target and neutralize coronaviruses, we demonstrate that the glycans of coronavirus spikes are not able to form an efficacious high-density global shield to thwart the humoral immune response. Overall, our data reveal how differential organisation of viral glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the viral protein surface.","version":"1.1","doi":"10.1101/2020.02.20.957472","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.18.954628","pub_date":"2020-2-20","title":"Are pangolins the intermediate host of the 2019 novel coronavirus (2019-nCoV) ?","abstract":"The outbreak of 2019-nCoV pneumonia (COVID-19) in the city of Wuhan, China has resulted in more than 70,000 laboratory confirmed cases, and recent studies showed that 2019-nCoV (SARS-CoV-2) could be of bat origin but involve other potential intermediate hosts. In this study, we assembled the genomes of coronaviruses identified in sick pangolins. The molecular and phylogenetic analyses showed that pangolin Coronaviruses (pangolin-CoV) are genetically related to both the 2019-nCoV and bat Coronaviruses but do not support the 2019-nCoV arose directly from the pangolin-CoV. Our study also suggested that pangolin be natural host of Betacoronavirus, with a potential to infect humans. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. Conservation of wildlife and limits of the exposures of humans to wildlife will be important to minimize the spillover risks of coronaviruses from wild animals to humans.","version":"1.1","doi":"10.1101/2020.02.18.954628","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.19.956946","pub_date":"2020-2-20","title":"Structural basis for the recognition of the 2019-nCoV by human ACE2","abstract":"Angiotensin-converting enzyme 2 (ACE2) has been suggested to be the cellular receptor for the new coronavirus (2019-nCoV) that is causing the coronavirus disease 2019 (COVID-19). Like other coronaviruses such as the SARS-CoV, the 2019-nCoV uses the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) to engage ACE2. We most recently determined the structure of the full-length human ACE2 in complex with a neutral amino acid transporter B0AT1. Here we report the cryo-EM structure of the full-length human ACE2 bound to the RBD of the 2019-nCoV at an overall resolution of 2.9 \u00c5 in the presence of B0AT1. The local resolution at the ACE2-RBD interface is 3.5 \u00c5, allowing analysis of the detailed interactions between the RBD and the receptor. Similar to that for the SARS-CoV, the RBD of the 2019-nCoV is recognized by the extracellular peptidase domain (PD) of ACE2 mainly through polar residues. Pairwise comparison reveals a number of variations that may determine the different affinities between ACE2 and the RBDs from these two related viruses.","version":"1.1","doi":"10.1101/2020.02.19.956946","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.19.956581","pub_date":"2020-2-20","title":"Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein","abstract":"The recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.","version":"1.1","doi":"10.1101/2020.02.19.956581","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.16.951723","pub_date":"2020-2-20","title":"SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development","abstract":"SARS-CoV-2 and SARS-CoV share a common human receptor ACE2. Protein-protein interaction structure modeling indicates that spike-RBD of the two viruses also has similar overall binding conformation and binding free energy to ACE2. In vitro assays using recombinant ACE2 proteins and ACE2 expressing cells confirmed the two coronaviruses\u2019 similar binding affinities to ACE2. The above studies provide experimental supporting evidences and possible explanation for the high transmissibility observed in the SARS-CoV-2 outbreak. Potent ACE2-blocking SARS-CoV neutralizing antibodies showed limited cross-binding and neutralizing activities to SARS-CoV-2. ACE2-non-blocking SARS-CoV RBD antibodies, though with weaker neutralizing activities against SARS-CoV, showed positive cross-neutralizing activities to SARS-CoV-2 with an unknown mechanism. These findings suggest a trade-off between the efficacy and spectrum for therapeutic antibodies to different coronaviruses, and hence highlight the possibilities and challenges in developing broadly protecting antibodies and vaccines against SARS-CoV-2 and its future mutants.","version":"1.1","doi":"10.1101/2020.02.16.951723","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.19.956235","pub_date":"2020-2-20","title":"Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor","abstract":"A novel and highly pathogenic coronavirus (2019-nCoV) has caused an outbreak in Wuhan city, Hubei province of China since December 2019, and soon spread nationwide and spilled over to other countries around the world. To better understand the initial step of infection at atomic-level, we determined the crystal structure of the 2019-nCoV spike receptor-binding domain (RBD) bound with the cell receptor ACE2 at 2.45 \u00c5 resolution. The overall ACE2-binding mode of the 2019-nCoV RBD is nearly identical to that of the SARS-CoV RBD, which also utilizes ACE2 as the cell receptor. Structural analysis identified residues in 2019-nCoV RBD critical for ACE2 binding, and majority of which are either highly conserved or shared similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly argue for a convergent evolution between 2019-nCoV and SARS-CoV RBD for improved binding to ACE2 despite of being segregated in different genetic lineages in the betacoronavirus genus. The epitopes of two SARS-CoV antibodies targeting the RBD are also analyzed with the 2019-nCoV RBD, providing insights into future identification of cross-reactive antibodies.","version":"1.1","doi":"10.1101/2020.02.19.956235","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.19.950253","pub_date":"2020-2-20","title":"Pangolin homology associated with 2019-nCoV","abstract":"To explore potential intermediate host of a novel coronavirus is vital to rapidly control continuous COVID-19 spread. We found genomic and evolutionary evidences of the occurrence of 2019-nCoV-like coronavirus (named as Pangolin-CoV) from dead Malayan Pangolins. Pangolin-CoV is 91.02% and 90.55% identical at the whole genome level to 2019-nCoV and BatCoV RaTG13, respectively. Pangolin-CoV is the lowest common ancestor of 2019-nCoV and RaTG13. The S1 protein of Pangolin-CoV is much more closely related to 2019-nCoV than RaTG13. Five key amino-acid residues involved in the interaction with human ACE2 are completely consistent between Pangolin-CoV and 2019-nCoV but four amino-acid mutations occur in RaTG13. It indicates Pangolin-CoV has similar pathogenic potential to 2019-nCoV, and would be helpful to trace the origin and probable intermediate host of 2019-nCoV.","version":"1.1","doi":"10.1101/2020.02.19.950253","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.17.951335","pub_date":"2020-2-20","title":"Isolation and Characterization of 2019-nCoV-like Coronavirus from Malayan Pangolins","abstract":"The outbreak of 2019-nCoV in the central Chinese city of Wuhan at the end of 2019 poses unprecedent public health challenges to both China and the rest world. The new coronavirus shares high sequence identity to SARS-CoV and a newly identified bat coronavirus. While bats may be the reservoir host for various coronaviruses, whether 2019-nCoV has other hosts is still ambiguous. In this study, one coronavirus isolated from Malayan pangolins showed 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in the E, M, N and S genes, respectively. In particular, the receptor-binding domain of the S protein of the Pangolin-CoV is virtually identical to that of 2019-nCoV, with one amino acid difference. Comparison of available genomes suggests 2019-nCoV might have originated from the recombination of a Pangolin-CoV-like virus with a Bat-CoV-RaTG13-like virus. Infected pangolins showed clinical signs and histopathological changes, and the circulating antibodies reacted with the S protein of 2019-nCoV. The isolation of a coronavirus that is highly related to 2019-nCoV in the pangolins suggests that these animals have the potential to act as the intermediate host of 2019-nCoV. The newly identified coronavirus in the most-trafficked mammal could represent a continuous threat to public health if wildlife trade is not effectively controlled.","version":"1.1","doi":"10.1101/2020.02.17.951335","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.12.945576","pub_date":"2020-2-20","title":"Aberrant pathogenic GM-CSF+ T cells and inflammatory CD14+CD16+ monocytes in severe pulmonary syndrome patients of a new coronavirus","abstract":"Pathogenic human coronavirus infections, such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV), cause high morbidity and mortality . Recently, a severe pneumonia-associated respiratory syndrome caused by a new coronavirus was reported at December 2019 (2019-nCoV) in the city Wuhan, Hubei province, China, which was also named as pneumonia-associated respiratory syndrome (PARS). Up to 9th of February 2020, at least 37, 251 cases have been reported with 812 fatal cases according to the report from China CDC. However, the immune mechanism that potential orchestrated acute mortality from patients of 2019-nCoV is still unknown. Here we show that after the 2019-nCoV infection, CD4+T lymphocytes are rapidly activated to become pathogenic T helper (Th) 1 cells and generate GM-CSF etc. The cytokines environment induces inflammatory CD14+CD16+ monocytes with high expression of IL-6 and accelerates the inflammation. These aberrant and excessive immune cells may enter the pulmonary circulation in huge numbers and play an immune damaging role to causing lung functional disability and quick mortality. Our results demonstrate that excessive non-effective host immune responses by pathogenic T cells and inflammatory monocytes may associate with severe lung pathology. Therefore, we suggest that monoclonal antibody that targets the GM-CSF or interleukin 6 receptor may potentially curb immunopathology caused by 2019-nCoV and consequently win more time for virus clearance.","version":"1.1","doi":"10.1101/2020.02.12.945576","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.19.957118","pub_date":"2020-2-20","title":"Mucin 4 Protects Female Mice from Coronavirus Pathogenesis","abstract":"Using incipient lines of the Collaborative Cross (CC), a murine genetic reference population, we previously identified a quantitative trait loci (QTL) associated with low SARS-CoV titer. In this study, we integrated sequence information and RNA expression of genes within the QTL to identify mucin 4 (Muc4) as a high priority candidate for controlling SARS-CoV titer in the lung. To test this hypothesis, we infected Muc4-/- mice and found that female, but not male, Muc4-/- mice developed more weight loss and disease following infection with SARS-CoV. Female Muc4-/- mice also had more difficulty breathing despite reduced lung pathology; however, no change in viral titers was observed. Comparing across viral families, studies with chikungunya virus, a mosquito-borne arthralgic virus, suggests that Muc4\u2019s impact on viral pathogenesis may be widespread. Although not confirming the original titer QTL, our data identifies a role for Muc4 in the SARS-CoV disease and viral pathogenesis. Given the recent emergence of SARS-CoV-2, this work suggest that Muc4 expression plays a protective role in female mice not conserved in male mice following SARS-CoV infection. With the SARS-CoV-2 outbreak continuing, treatments that modulate or enhance Muc4 activity may provide an avenue for treatment and improved outcomes. In addition, the work highlights the importance of studying host factors including host genetics and biological sex as key parameters influencing infection and disease outcomes.","version":"1.1","doi":"10.1101/2020.02.19.957118","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.03.932350","pub_date":"2020-2-20","title":"Machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: COVID-19 case study","abstract":"As of February 20, 2020, the 2019 novel coronavirus (renamed to COVID-19) spread to 30 countries with 2130 deaths and more than 75500 confirmed cases. COVID-19 is being compared to the infamous SARS coronavirus, which resulted, between November 2002 and July 2003, in 8098 confirmed cases worldwide with a 9.6% death rate and 774 deaths. Though COVID-19 has a death rate of 2.8% as of 20 February, the 75752 confirmed cases in a few weeks (December 8, 2019 to February 20, 2020) are alarming, with cases likely being under-reported given the comparatively longer incubation period. Such outbreaks demand elucidation of taxonomic classification and origin of the virus genomic sequence, for strategic planning, containment, and treatment. This paper identifies an intrinsic COVID-19 genomic signature and uses it together with a machine learning-based alignment-free approach for an ultra-fast, scalable, and highly accurate classification of whole COVID-19 genomes. The proposed method combines supervised machine learning with digital signal processing for genome analyses, augmented by a decision tree approach to the machine learning component, and a Spearman\u2019s rank correlation coefficient analysis for result validation. These tools are used to analyze a large dataset of over 5000 unique viral genomic sequences, totalling 61.8 million bp. Our results support a hypothesis of a bat origin and classify COVID-19 as Sarbecovirus, within Betacoronavirus. Our method achieves high levels of classification accuracy and discovers the most relevant relationships among over 5,000 viral genomes within a few minutes, ab initio, using raw DNA sequence data alone, and without any specialized biological knowledge, training, gene or genome annotations. This suggests that, for novel viral and pathogen genome sequences, this alignment-free whole-genome machine-learning approach can provide a reliable real-time option for taxonomic classification.","version":"1.3","doi":"10.1101/2020.02.03.932350","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.17.952879","pub_date":"2020-2-20","title":"X-ray Structure of Main Protease of the Novel Coronavirus SARS-CoV-2 Enables Design of \u03b1-Ketoamide Inhibitors","abstract":"A novel coronavirus has been identified as the causative agent of a massive outbreak of atypical pneumonia originating at Wuhan, Hubei province, China. Involved in the formation of the coronavirus replication complex, the viral main protease (Mpro, also called 3CLpro) represents an attractive target for therapy. We determined the crystal structure of the unliganded Mpro at 1.75 \u00c5 resolution and used this structure to guide optimization of a series of alpha-ketoamide inhibitors. The main goal of the optimization efforts was improvement of the pharmacokinetic properties of the compounds. We further describe 1.95- and 2.20-\u00c5 crystal structures of the complex between the enzyme and the most potent alpha-ketoamide optimized this way. These structures will form the basis for further development of these compounds to antiviral drugs.","version":"1.1","doi":"10.1101/2020.02.17.952879","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.10.942185","pub_date":"2020-2-18","title":"Structural modeling of 2019-novel coronavirus (nCoV) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to SARS-CoV and related SARS-like coronaviruses","abstract":"The 2019 novel coronavirus (2019-nCoV) is currently causing a widespread outbreak centered on Hubei province, China and is a major public health concern. Taxonomically 2019-nCoV is closely related to SARS-CoV and SARS-related bat coronaviruses, and it appears to share a common receptor with SARS-CoV (ACE-2). Here, we perform structural modeling of the 2019-nCoV spike glycoprotein. Our data provide support for the similar receptor utilization between 2019-nCoV and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains, which we predict to be proteolytically-sensitive. We suggest this loop confers fusion activation and entry properties more in line with MERS-CoV and other coronaviruses, and that the presence of this structural loop in 2019-nCoV may affect virus stability and transmission.","version":"1.1","doi":"10.1101/2020.02.10.942185","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.31.929695","pub_date":"2020-2-18","title":"Molecular Modeling Evaluation of the Binding Effect of Ritonavir, Lopinavir and Darunavir to Severe Acute Respiratory Syndrome Coronavirus 2 Proteases","abstract":"Three anti-HIV drugs, ritonavir, lopinavir and darunavir, might have therapeutic effect on coronavirus disease 2019 (COVID-19). In this study, the structure models of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteases, coronavirus endopeptidase C30 (CEP_C30) and papain like viral protease (PLVP), were built by homology modeling. Ritonavir, lopinavir and darunavir were then docked to the models, respectively, followed by energy minimization of the protease-drug complexes. In the simulations, ritonavir can bind to CEP_C30 most suitably, and induce significant conformation changes of CEP_C30; lopinavir can also bind to CEP_C30 suitably, and induce significant conformation changes of CEP_C30; darunavir can bind to PLVP suitably with slight conformation changes of PLVP. It is suggested that the therapeutic effect of ritonavir and lopinavir on COVID-19 may be mainly due to their inhibitory effect on CEP_C30, while ritonavir may have stronger efficacy; the inhibitory effect of darunavir on SARS-CoV-2 and its potential therapeutic effect may be mainly due to its inhibitory effect on PLVP.","version":"1.2","doi":"10.1101/2020.01.31.929695","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.08.939892","pub_date":"2020-2-18","title":"Single-cell Analysis of ACE2 Expression in Human Kidneys and Bladders Reveals a Potential Route of 2019-nCoV Infection","abstract":"Since December 2019, a novel coronavirus named 2019 coronavirus (2019-nCoV) has emerged in Wuhan of China and spread to several countries worldwide within just one month. Apart from fever and respiratory complications, acute kidney injury has been observed in some patients with 2019-nCoV. In a short period of time, angiotensin converting enzyme II (ACE2), have been proposed to serve as the receptor for the entry of 2019-nCoV, which is the same for severe acute respiratory syndrome coronavirus (SARS). To investigate the possible cause of kidney damage in 2019-nCoV patients, we used both published kidney and bladder cell atlas data and an independent unpublished kidney single cell RNA-Seq data generated in-house to evaluate ACE2 gene expressions in all cell types in healthy kidneys and bladders. Our results showed the enriched expression of all subtypes of proximal tubule cells of kidney and low but detectable levels of expression in bladder epithelial cells. These results indicated the urinary system is a potential route for 2019-nCoV infection, along with the respiratory system and digestion system. Our findings suggested the kidney abnormalities of SARS and 2019-nCoV patients may be due to proximal tubule cells damage and subsequent systematic inflammatory response induced kidney injury. Beyond that, laboratory tests of viruses and related indicators in urine may be needed in some special patients of 2019-nCoV.","version":"1.1","doi":"10.1101/2020.02.08.939892","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.13.945485","pub_date":"2020-2-18","title":"Identification of 2019-nCoV related coronaviruses in Malayan pangolins in southern China","abstract":"The ongoing outbreak of viral pneumonia in China and beyond is associated with a novel coronavirus, provisionally termed 2019-nCoV. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection. Although bats are likely reservoir hosts for 2019-nCoV, the identity of any intermediate host facilitating transfer to humans is unknown. Here, we report the identification of 2019-nCoV related coronaviruses in pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin associated CoVs that belong to two sub-lineages of 2019-nCoV related coronaviruses, including one very closely related to 2019-nCoV in the receptor-binding domain. The discovery of multiple lineages of pangolin coronavirus and their similarity to 2019-nCoV suggests that pangolins should be considered as possible intermediate hosts for this novel human virus and should be removed from wet markets to prevent zoonotic transmission.","version":"1.1","doi":"10.1101/2020.02.13.945485","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.17.951848","pub_date":"2020-2-18","title":"Structure of dimeric full-length human ACE2 in complex with B0AT1","abstract":"Angiotensin-converting enzyme 2 (ACE2) is the surface receptor for SARS coronavirus (SARS-CoV), directly interacting with the spike glycoprotein (S protein). ACE2 is also suggested to be the receptor for the new coronavirus (2019-nCoV), which is causing a serious epidemic in China manifested with severe respiratory syndrome. B0AT1 (SLC6A19) is a neutral amino acid transporter whose surface expression in intestinal cells requires ACE2. Here we present the 2.9 \u00c5 resolution cryo-EM structure of full-length human ACE2 in complex with B0AT1. The complex, assembled as a dimer of ACE2-B0AT1 heterodimers, exhibits open and closed conformations due to the shifts of the peptidase domains (PDs) of ACE2. A newly resolved Collectrin-like domain (CLD) on ACE2 mediates homo-dimerization. Structural modelling suggests that the ACE2-B0AT1 complex can bind two S proteins simultaneously, providing important clues to the molecular basis for coronavirus recognition and infection.","version":"1.1","doi":"10.1101/2020.02.17.951848","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.08.926006","pub_date":"2020-2-16","title":"The insert sequence in SARS-CoV-2 enhances spike protein cleavage by TMPRSS","abstract":"At the end of 2019, the SARS-CoV-2 induces an ongoing outbreak of pneumonia in China, even more spread than SARS-CoV infection. The entry of SARS-CoV into host cells mainly depends on the cell receptor (ACE2) recognition and spike protein cleavage-induced cell membrane fusion. The spike protein of SARS-CoV-2 also binds to ACE2 with a similar affinity, whereas its spike protein cleavage remains unclear. Here we show that an insertion sequence in the spike protein of SARS-CoV-2 enhances the cleavage efficiency, and besides pulmonary alveoli, intestinal and esophagus epithelium were also the target tissues of SARS-CoV-2. Compared with SARS-CoV, we found a SPRR insertion in the S1/S2 protease cleavage sites of SARS-CoV-2 spike protein increasing the cleavage efficiency by the protein sequence aligment and furin score calculation. Additionally, the insertion sequence facilitates the formation of an extended loop which was more suitable for protease recognition by the homology modeling and molicular docking. Furthermore, the single-cell transcriptomes identified that ACE2 and TMPRSSs are highly coexpressed in AT2 cells of lung, along with esophageal upper epithelial cells and absorptive enterocytes. Our results provide the bioinformatics evidence for the increased spike protein cleavage of SARS-CoV-2 and indicate its potential target cells.","version":"1.3","doi":"10.1101/2020.02.08.926006","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.11.944462","pub_date":"2020-2-15","title":"Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation","abstract":"The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development we determined a 3.5 \u00c5-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.","version":"1.1","doi":"10.1101/2020.02.11.944462","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.10.942136","pub_date":"2020-2-14","title":"Structural genomics and interactomics of 2019 Wuhan novel coronavirus, 2019-nCoV, indicate evolutionary conserved functional regions of viral proteins","abstract":"During its first month, the recently emerged 2019 Wuhan novel coronavirus (2019-nCoV) has already infected many thousands of people in mainland China and worldwide and took hundreds of lives. However, the swiftly spreading virus also caused an unprecedentedly rapid response from the research community facing the unknown health challenge of potentially enormous proportions. Unfortunately, the experimental research to understand the molecular mechanisms behind the viral infection and to design a vaccine or antivirals is costly and takes months to develop. To expedite the advancement of our knowledge we leverage the data about the related coronaviruses that is readily available in public databases, and integrate these data into a single computational pipeline. As a result, we provide a comprehensive structural genomics and interactomics road-maps of 2019-nCoV and use these information to infer the possible functional differences and similarities with the related SARS coronavirus. All data are made publicly available to the research community at http://korkinlab.org/wuhan","version":"1.1","doi":"10.1101/2020.02.10.942136","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.08.939660","pub_date":"2020-2-14","title":"Identification of a Pangolin Niche for a 2019-nCoV-like Coronavirus via an Extensive Meta-metagenomic Search","abstract":"In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic datasets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic dataset can be readily queried using web-based tools, meta-searches through all such datasets are less accessible. In this brief communication, we demonstrate such a meta-meta-genomic approach, examining close matches to the Wuhan coronavirus 2019-nCoV in all high-throughput sequencing datasets in the NCBI Sequence Read Archive accessible with the keyword \u201cvirome\u201d. In addition to the homology to bat coronaviruses observed in descriptions of the 2019-nCoV sequence (F. Wu et al. 2020, Nature, doi.org/10.1038/s41586-020-2008-3; P. Zhou et al. 2020, Nature, doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in a metavirome dataset generated from the lungs of deceased Pangolins reported by Liu et al. (Viruses 11:11, 2019, http://doi.org/10.3390/v11110979). Our observations are relevant to discussions of the derivation of 2019-nCoV and illustrate the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences. Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.","version":"1.2","doi":"10.1101/2020.02.08.939660","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.05.936013","pub_date":"2020-2-13","title":"Potentially highly potent drugs for 2019-nCoV","abstract":"The World Health Organization (WHO) has declared the 2019 novel coronavirus (2019-nCoV) infection outbreak a global health emergency. Currently, there is no effective anti-2019-nCoV medication. The sequence identity of the 3CL proteases of 2019-nCoV and SARS is 96%, which provides a sound foundation for structural-based drug repositioning (SBDR). Based on a SARS 3CL protease X-ray crystal structure, we construct a 3D homology structure of 2019-nCoV 3CL protease. Based on this structure and existing experimental datasets for SARS 3CL protease inhibitors, we develop an SBDR model based on machine learning and mathematics to screen 1465 drugs in the DrugBank that have been approved by the U.S. Food and Drug Administration (FDA). We found that many FDA approved drugs are potentially highly potent to 2019-nCoV.","version":"1.1","doi":"10.1101/2020.02.05.936013","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.07.939207","pub_date":"2020-2-13","title":"Evidence of recombination in coronaviruses implicating pangolin origins of nCoV-2019","abstract":"A novel coronavirus (nCoV-2019) was the cause of an outbreak of respiratory illness detected in Wuhan, Hubei Province, China in December of 2019. Genomic analyses of nCoV-2019 determined a 96% resemblance with a coronavirus isolated from a bat in 2013 (RaTG13); however, the receptor binding motif (RBM) of these two genomes share low sequence similarity. This divergence suggests a possible alternative source for the RBM coding sequence in nCoV-2019. We identified high sequence similarity in the RBM between nCoV-2019 and a coronavirus genome reconstructed from a viral metagenomic dataset from pangolins possibly indicating a more complex origin for nCoV-2019.","version":"1.1","doi":"10.1101/2020.02.07.939207","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.25.919787","pub_date":"2020-2-13","title":"Time-varying transmission dynamics of Novel Coronavirus Pneumonia in China","abstract":"Several studies have estimated basic production number of novel coronavirus pneumonia (NCP). However, the time-varying transmission dynamics of NCP during the outbreak remain unclear. We aimed to estimate the basic and time-varying transmission dynamics of NCP across China, and compared them with SARS. Data on NCP cases by February 7, 2020 were collected from epidemiological investigations or official websites. Data on severe acute respiratory syndrome (SARS) cases in Guangdong Province, Beijing and Hong Kong during 2002-2003 were also obtained. We estimated the doubling time, basic reproduction number (R0) and time-varying reproduction number (Rt) of NCP and SARS. As of February 7, 2020, 34,598 NCP cases were identified in China, and daily confirmed cases decreased after February 4. The doubling time of NCP nationwide was 2.4 days which was shorter than that of SARS in Guangdong (14.3 days), Hong Kong (5.7 days) and Beijing (12.4 days). The R0 of NCP cases nationwide and in Wuhan were 4.5 and 4.4 respectively, which were higher than R0 of SARS in Guangdong (R0=2.3), Hongkong (R0=2.3), and Beijing (R0=2.6). The Rt for NCP continuously decreased especially after January 16 nationwide and in Wuhan. The R0 for secondary NCP cases in Guangdong was 0.6, and the Rt values were less than 1 during the epidemic. NCP may have a higher transmissibility than SARS, and the efforts of containing the outbreak are effective. However, the efforts are needed to persist in for reducing time-varying reproduction number below one. Since December 29, 2019, pneumonia infection with 2019-nCoV, now named as Novel Coronavirus Pneumonia (NCP), occurred in Wuhan, Hubei Province, China. The disease has rapidly spread from Wuhan to other areas. As a novel virus, the time-varying transmission dynamics of NCP remain unclear, and it is also important to compare it with SARS. We compared the transmission dynamics of NCP with SARS, and found that NCP has a higher transmissibility than SARS. Time-varying production number indicates that rigorous control measures taken by governments are effective across China, and persistent efforts are needed to be taken for reducing instantaneous reproduction number below one.","version":"1.2","doi":"10.1101/2020.01.25.919787","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.03.933226","pub_date":"2020-2-12","title":"Preliminary identification of potential vaccine targets for the COVID-19 coronavirus (SARS-CoV-2) based on SARS-CoV immunological studies","abstract":"The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the available SARS-CoV-2 sequences (as of 9 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.","version":"1.4","doi":"10.1101/2020.02.03.933226","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.07.937862","pub_date":"2020-2-11","title":"Severe acute respiratory syndrome-related coronavirus: The species and its viruses \u2013 a statement of the Coronavirus Study Group","abstract":"The present outbreak of lower respiratory tract infections, including respiratory distress syndrome, is the third spillover, in only two decades, of an animal coronavirus to humans resulting in a major epidemic. Here, the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the official classification of viruses and taxa naming (taxonomy) of the Coronaviridae family, assessed the novelty of the human pathogen tentatively named 2019-nCoV. Based on phylogeny, taxonomy and established practice, the CSG formally recognizes this virus as a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus and designates it as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To facilitate communication, the CSG further proposes to use the following naming convention for individual isolates: SARS-CoV-2/Isolate/Host/Date/Location. The spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined. The independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying the entire (virus) species to complement research focused on individual pathogenic viruses of immediate significance. This research will improve our understanding of virus-host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.","version":"1.1","doi":"10.1101/2020.02.07.937862","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.07.939124","pub_date":"2020-2-11","title":"Exploring the coronavirus epidemic using the new WashU Virus Genome Browser","abstract":"Since its debut in mid-December, 2019, the novel coronavirus (2019-nCoV) has rapidly spread from its origin in Wuhan, China, to several countries across the globe, leading to a global health crisis. As of February 7, 2020, 44 strains of the virus have been sequenced and uploaded to NCBI\u2019s GenBank [1], providing insight into the virus\u2019s evolutionary history and pathogenesis. Here, we present the WashU Virus Genome Browser, a web-based portal for viewing virus genomic data. The browser is home to 16 complete 2019-nCoV genome sequences, together with hundreds of related viral sequences including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and Ebola virus. In addition, the browser features unique customizability, supporting user-provided upload of novel viral sequences in various formats. Sequences can be viewed in both a track-based representation as well as a phylogenetic tree-based view, allowing the user to easily compare sequence features across multiple strains. The WashU Virus Genome Browser inherited many features and track types from the WashU Epigenome Browser, and additionally incorporated a new type of SNV track to address the specific needs of viral research. Our Virus Browser portal can be accessed at https://virusgateway.wustl.edu, and documentation is available at https://virusgateway.readthedocs.io/.","version":"1.1","doi":"10.1101/2020.02.07.939124","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.04.933135","pub_date":"2020-2-08","title":"Protein structure and sequence re-analysis of 2019-nCoV genome does not indicate snakes as its intermediate host or the unique similarity between its spike protein insertions and HIV-1","abstract":"As the infection of 2019-nCoV coronavirus is quickly developing into a global pneumonia epidemic, careful analysis of its transmission and cellular mechanisms is sorely needed. In this report, we re-analyzed the computational approaches and findings presented in two recent manuscripts by Ji et al. (https://doi.org/10.1002/jmv.25682) and by Pradhan et al. (https://doi.org/10.1101/2020.01.30.927871), which concluded that snakes are the intermediate hosts of 2019-nCoV and that the 2019-nCoV spike protein insertions shared a unique similarity to HIV-1. Results from our re-implementation of the analyses, built on larger-scale datasets using state-of-the-art bioinformatics methods and databases, do not support the conclusions proposed by these manuscripts. Based on our analyses and existing data of coronaviruses, we concluded that the intermediate hosts of 2019-nCoV are more likely to be mammals and birds than snakes, and that the \u201cnovel insertions\u201d observed in the spike protein are naturally evolved from bat coronaviruses.","version":"1.1","doi":"10.1101/2020.02.04.933135","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.02.931162","pub_date":"2020-2-05","title":"Genomic variance of the 2019-nCoV coronavirus","abstract":"There is rising global concern for the recently emerged novel Coronavirus (2019-nCov). Full genomic sequences have been released by the worldwide scientific community in the last few weeks in order to understand the evolutionary origin and molecular characteristics of this virus. Taking advantage of all the genomic information currently available, we constructed a phylogenetic tree including also representatives of other coronaviridae, such as Bat coronavirus (BCoV) and SARS. We confirm high sequence similarity (>99%) between all sequenced 2019-nCoVs genomes available, with the closest BCoV sequence sharing 96.2% sequence identity, confirming the notion of a zoonotic origin of 2019-nCoV. Despite the low heterogeneity of the 2019-nCoV genomes, we could identify at least two hyper-variable genomic hotspots, one of which is responsible for a Serine/Leucine variation in the viral ORF8-encoded protein. Finally, we perform a full proteomic comparison with other coronaviridae, identifying key aminoacidic differences to be considered for antiviral strategies deriving from previous anti-coronavirus approaches.","version":"1.2","doi":"10.1101/2020.02.02.931162","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.30.927889","pub_date":"2020-2-04","title":"Machine intelligence design of 2019-nCoV drugs","abstract":"Wuhan coronavirus, called 2019-nCoV, is a newly emerged virus that infected more than 9692 people and leads to more than 213 fatalities by January 30, 2020. Currently, there is no effective treatment for this epidemic. However, the viral protease of a coronavirus is well-known to be essential for its replication and thus is an effective drug target. Fortunately, the sequence identity of the 2019-nCoV protease and that of severe-acute respiratory syndrome virus (SARS-CoV) is as high as 96.1%. We show that the protease inhibitor binding sites of 2019-nCoV and SARS-CoV are almost identical, which means all potential anti-SARS-CoV chemotherapies are also potential 2019-nCoV drugs. Here, we report a family of potential 2019-nCoV drugs generated by a machine intelligence-based generative network complex (GNC). The potential effectiveness of treating 2019-nCoV by using some existing HIV drugs is also analyzed.","version":"1.1","doi":"10.1101/2020.01.30.927889","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.01.930537","pub_date":"2020-2-04","title":"Fast assessment of human receptor-binding capability of 2019 novel coronavirus (2019-nCoV)","abstract":"The outbreaks of 2002/2003 SARS, 2012/2015 MERS and 2019/2020 Wuhan respiratory syndrome clearly indicate that genome evolution of an animal coronavirus (CoV) may enable it to acquire human transmission ability, and thereby to cause serious threats to global public health. It is widely accepted that CoV human transmission is driven by the interactions of its spike protein (S-protein) with human receptor on host cell surface; so, quantitative evaluation of these interactions may be used to assess the human transmission capability of CoVs. However, quantitative methods directly using viral genome data are still lacking. Here, we perform large-scale protein-protein docking to quantify the interactions of 2019-nCoV S-protein receptor-binding domain (S-RBD) with human receptor ACE2, based on experimental SARS-CoV S-RBD-ACE2 complex structure. By sampling a large number of thermodynamically probable binding conformations with Monte Carlo algorithm, this approach successfully identified the experimental complex structure as the lowest-energy receptor-binding conformations, and hence established an experiment-based strength reference for evaluating the receptor-binding affinity of 2019-nCoV via comparison with SARS-CoV. Our results show that this binding affinity is about 73% of that of SARS-CoV, supporting that 2019-nCoV may cause human transmission similar to that of SARS-CoV. Thus, this study presents a method for rapidly assessing the human transmission capability of a newly emerged CoV and its mutant strains, and demonstrates that post-genome analysis of protein-protein interactions may provide early scientific guidance for viral prevention and control.","version":"1.2","doi":"10.1101/2020.02.01.930537","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.03.931766","pub_date":"2020-2-04","title":"Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection","abstract":"A newly identified coronavirus, 2019-nCoV, has been posing significant threats to public health since December 2019. ACE2, the host cell receptor for severe acute respiratory syndrome coronavirus (SARS), has recently been demonstrated in mediating 2019-nCoV infection. Interestingly, besides the respiratory system, substantial proportion of SARS and 2019-nCoV patients showed signs of various degrees of liver damage, the mechanism and implication of which have not yet been determined. Here, we performed an unbiased evaluation of cell type specific expression of ACE2 in healthy liver tissues using single cell RNA-seq data of two independent cohorts, and identified specific expression in cholangiocytes. The results indicated that virus might directly bind to ACE2 positive cholangiocytes but not necessarily hepatocytes. This finding suggested the liver abnormalities of SARS and 2019-nCoV patients may not be due to hepatocyte damage, but cholangiocyte dysfunction and other causes such as drug induced and systemic inflammatory response induced liver injury. Our findings indicate that special care of liver dysfunction should be installed in treating 2019-nCoV patients during the hospitalization and shortly after cure.","version":"1.1","doi":"10.1101/2020.02.03.931766","journal":"bioRxiv","score":null},{"id":"10.1101/2020.02.01.929976","pub_date":"2020-2-03","title":"Potent neutralization of 2019 novel coronavirus by recombinant ACE2-Ig","abstract":"2019-nCoV, which is a novel coronavirus emerged in Wuhan, China, at the end of 2019, has caused at least infected 11,844 as of Feb 1, 2020. However, there is no specific antiviral treatment or vaccine currently. Very recently report had suggested that novel CoV would use the same cell entry receptor, ACE2, as the SARS-CoV. In this report, we generated a novel recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. An ACE2 mutant with low catalytic activity was also used in the study. The fusion proteins were then characterized. Both fusion proteins has high affinity binding to the receptor-binding domain (RBD) of SARS-CoV and 2019-nCoV and exerted desired pharmacological properties. Moreover, fusion proteins potently neutralized SARS-CoV and 2019-nCoV in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they could have potential applications for diagnosis, prophylaxis, and treatment of 2019-nCoV.","version":"1.2","doi":"10.1101/2020.02.01.929976","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.31.928796","pub_date":"2020-2-02","title":"Genome Detective Coronavirus Typing Tool for rapid identification and characterization of novel coronavirus genomes","abstract":"Genome Detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present The Genome Detective Coronavirus Typing Tool that can accurately identify novel coronavirus (2019-nCoV) sequences isolated in China and around the world. The tool can accept up to 2,000 sequences per submission and the analysis of a new whole genome sequence will take approximately one minute. The tool has been tested and validated with hundreds of whole genomes from ten coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for 2019-nCoV. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines. Available online: https://www.genomedetective.com/app/typingtool/cov koen@emweb.be and deoliveira@ukzn.ac.za Supplementary data is available online.","version":"1.1","doi":"10.1101/2020.01.31.928796","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.31.929547","pub_date":"2020-2-02","title":"Predicting commercially available antiviral drugs that may act on the novel coronavirus (2019-nCoV), Wuhan, China through a drug-target interaction deep learning model","abstract":"The infection of a novel coronavirus found in Wuhan of China (2019-nCoV) is rapidly spreading, and the incidence rate is increasing worldwide. Due to the lack of effective treatment options for 2019-nCoV, various strategies are being tested in China, including drug repurposing. In this study, we used our pretrained deep learning-based drug-target interaction model called Molecule Transformer-Drug Target Interaction (MT-DTI) to identify commercially available drugs that could act on viral proteins of 2019-nCoV. The result showed that atazanavir, an antiretroviral medication used to treat and prevent the human immunodeficiency virus (HIV), is the best chemical compound, showing a inhibitory potency with Kd of 94.94 nM against the 2019-nCoV 3C-like proteinase, followed by efavirenz (199.17 nM), ritonavir (204.05 nM), and dolutegravir (336.91 nM). Interestingly, lopinavir, ritonavir, and darunavir are all designed to target viral proteinases. However, in our prediction, they may also bind to the replication complex components of 2019-nCoV with an inhibitory potency with Kd < 1000 nM. In addition, we also found that several antiviral agents, such as Kaletra, could be used for the treatment of 2019-nCoV, although there is no real-world evidence supporting the prediction. Overall, we suggest that the list of antiviral drugs identified by the MT-DTI model should be considered, when establishing effective treatment strategies for 2019-nCoV.","version":"1.1","doi":"10.1101/2020.01.31.929547","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.31.929497","pub_date":"2020-2-02","title":"Highly Distinguished Amino Acid Sequences of 2019-nCoV (Wuhan Coronavirus)","abstract":"Using a method for pathogen screening in DNA synthesis orders, we have identified a number of amino acid sequences that distinguish 2019-nCoV (Wuhan Coronavirus) from all other known viruses in Coronaviridae. We find three main regions of unique sequence: two in the 1ab polyprotein QHO60603.1, one in surface glycoprotein QHO60594.1.","version":"1.1","doi":"10.1101/2020.01.31.929497","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.31.929042","pub_date":"2020-1-31","title":"The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells","abstract":"The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum form a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention. The novel 2019 coronavirus and the SARS-coronavirus share central biological properties which can guide risk assessment and intervention.","version":"1.1","doi":"10.1101/2020.01.31.929042","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.30.927806","pub_date":"2020-1-31","title":"The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes","abstract":"Since December 2019, a newly identified coronavirus (2019 novel coronavirus, 2019-nCov) is causing outbreak of pneumonia in one of largest cities, Wuhan, in Hubei province of China and has draw significant public health attention. The same as severe acute respiratory syndrome coronavirus (SARS-CoV), 2019-nCov enters into host cells via cell receptor angiotensin converting enzyme II (ACE2). In order to dissect the ACE2-expressing cell composition and proportion and explore a potential route of the 2019-nCov infection in digestive system infection, 4 datasets with single-cell transcriptomes of lung, esophagus, gastric, ileum and colon were analyzed. The data showed that ACE2 was not only highly expressed in the lung AT2 cells, esophagus upper and stratified epithelial cells but also in absorptive enterocytes from ileum and colon. These results indicated along with respiratory systems, digestive system is a potential routes for 2019-nCov infection. In conclusion, this study has provided the bioinformatics evidence of the potential route for infection of 2019-nCov in digestive system along with respiratory tract and may have significant impact for our healthy policy setting regards to prevention of 2019-nCoV infection.","version":"1.1","doi":"10.1101/2020.01.30.927806","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.30.927574","pub_date":"2020-1-31","title":"Nucleotide Analogues as Inhibitors of Viral Polymerases","abstract":"Coronaviruses such as the newly discovered virus from Wuhan, China, 2019-nCoV, and the viruses that cause SARS and MERS, have resulted in regional and global public health emergencies. Based on our molecular insight that the hepatitis C virus and the coronavirus use a similar viral genome replication mechanism, we reasoned that the FDA-approved drug EPCLUSA (Sofosbuvir/Velpatasvir) for the treatment of hepatitis C will also inhibit the above coronaviruses, including 2019-nCoV. To develop broad spectrum anti-viral agents, we further describe a novel strategy to design and synthesize viral polymerase inhibitors, by combining the ProTide Prodrug approach used in the development of Sofosbuvir with the use of 3\u2019-blocking groups that we have previously built into nucleotide analogues that function as polymerase terminators.","version":"1.1","doi":"10.1101/2020.01.30.927574","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.25.919688","pub_date":"2020-1-31","title":"MRCA time and epidemic dynamics of the 2019 novel coronavirus","abstract":"The 2019 novel coronavirus (2019-nCoV) have emerged from Wuhan, China. Studying the epidemic dynamics is crucial for further surveillance and control of the outbreak. We employed a Bayesian framework to infer the time-calibrated phylogeny and the epidemic dynamics represented by the effective reproductive number (Re) changing over time from 33 genomic sequences available from GISAID. The time of the most recent common ancestor (MRCA) was December 17, 2019 (95% HPD: December 7, 2019 \u2013 December 23, 2019). The median estimate of Re shifted from 1.6 to 1.1 on around January 1, 2020. This study provides an early insight of the 2019-nCoV epidemic. However, due to limited amount of data, one should be cautious when interpreting the results at this stage.","version":"1.3","doi":"10.1101/2020.01.25.919688","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.28.922922","pub_date":"2020-1-30","title":"Therapeutic Drugs Targeting 2019-nCoV Main Protease by High-Throughput Screening","abstract":"2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of the outbreak of pneumonia first detected in Wuhan, China. Investigations on the transmissibility, severity, and other features associated with this virus are ongoing. Currently, there is no vaccine or therapeutic antibody to prevent the infection, and more time is required to develop an effective immune strategy against the pathogen. In contrast, specific inhibitors targeting the key protease involved in replication and proliferation of the virus are the most effective means to alleviate the epidemic. The main protease of SARS-CoV is essential for the life cycle of the virus, which showed 96.1% of similarity with the main proteaseof 2019-nCoV, is considered to be an attractive target for drug development. In this study, we have identified 4 small molecular drugs with high binding capacity with SARS-CoV main protease by high-throughput screening based on the 8,000 clinical drug libraries, all these drugs have been widely used in clinical applications with guaranteed safety, which may serve as promising candidates to treat the infection of 2019-nCoV.","version":"1.2","doi":"10.1101/2020.01.28.922922","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.30.926477","pub_date":"2020-1-30","title":"Evolution and variation of 2019-novel coronavirus","abstract":"The current outbreak caused by novel coronavirus (2019-nCoV) in China has become a worldwide concern. As of 28 January 2020, there were 4631 confirmed cases and 106 deaths, and 11 countries or regions were affected. We downloaded the genomes of 2019-nCoVs and similar isolates from the Global Initiative on Sharing Avian Influenza Database (GISAID and nucleotide database of the National Center for Biotechnology Information (NCBI). Lasergene 7.0 and MEGA 6.0 softwares were used to calculate genetic distances of the sequences, to construct phylogenetic trees, and to align amino acid sequences. Bayesian coalescent phylogenetic analysis, implemented in the BEAST software package, was used to calculate the molecular clock related characteristics such as the nucleotide substitution rate and the most recent common ancestor (tMRCA) of 2019-nCoVs. An isolate numbered EPI_ISL_403928 showed different phylogenetic trees and genetic distances of the whole length genome, the coding sequences (CDS) of ployprotein (P), spike protein (S), and nucleoprotein (N) from other 2019-nCoVs. There are 22, 4, 2 variations in P, S, and N at the level of amino acid residues. The nucleotide substitution rates from high to low are 1\u00b705 \u00d7 10\u22122 (nucleotide substitutions/site/year, with 95% HPD interval being 6.27 \u00d7 10\u22124 to 2.72 \u00d7 10\u22122) for N, 5.34 \u00d7 10\u22123 (5.10 \u00d7 10\u22124, 1.28 \u00d7 10\u22122) for S, 1.69 \u00d7 10\u22123 (3.94 \u00d7 10\u22124, 3.60 \u00d7 10\u22123) for P, 1.65 \u00d7 10\u22123 (4.47 \u00d7 10\u22124, 3.24 \u00d7 10\u22123) for the whole genome, respectively. At this nucleotide substitution rate, the most recent common ancestor (tMRCA) of 2019-nCoVs appeared about 0.253-0.594 year before the epidemic. Our analysis suggests that at least two different viral strains of 2019-nCoV are involved in this outbreak that might occur a few months earlier before it was officially reported.","version":"1.1","doi":"10.1101/2020.01.30.926477","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.27.922443","pub_date":"2020-1-30","title":"Breaking down of healthcare system: Mathematical modelling for controlling the novel coronavirus (2019-nCoV) outbreak in Wuhan, China","abstract":"A novel coronavirus pneumonia initially identified in Wuhan, China and provisionally named 2019-nCoV has surged in the public. In anticipation of substantial burdens on healthcare system following this human-to-human spread, we aim to scrutinise the currently available information and evaluate the burden of healthcare systems during this outbreak in Wuhan. We applied a modified SIR model to project the actual number of infected cases and the specific burdens on isolation wards and intensive care units (ICU), given the scenarios of different diagnosis rates as well as different public health intervention efficacy. Our estimates suggest, assuming 50% diagnosis rate if no public health interventions were implemented, that the actual number of infected cases could be much higher than the reported, with estimated 88,075 cases (as of 31st January, 2020), and projected burdens on isolation wards and ICU would be 34,786 and 9,346 respectively The estimated burdens on healthcare system could be largely reduced if at least 70% efficacy of public health intervention is achieved. The health system burdens arising from the actual number of cases infected by the novel coronavirus appear to be considerable if no effective public health interventions were implemented. This calls for continuation of implemented anti-transmission measures (e.g., closure of schools and facilities, suspension of public transport, lockdown of city) and further effective large-scale interventions spanning all subgroups of populations (e.g., universal facemask wear) aiming at obtaining overall efficacy with at least 70% to ensure the functioning of and to avoid the breakdown of health system.","version":"1.2","doi":"10.1101/2020.01.27.922443","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.23.916395","pub_date":"2020-1-29","title":"Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak","abstract":"An ongoing outbreak of a novel coronavirus (2019-nCoV) pneumonia hit a major city of China, Wuhan, December 2019 and subsequently reached other provinces/regions of China and countries. We present estimates of the basic reproduction number, R0, of 2019-nCoV in the early phase of the outbreak. Accounting for the impact of the variations in disease reporting rate, we modelled the epidemic curve of 2019-nCoV cases time series, in mainland China from January 10 to January 24, 2020, through the exponential growth. With the estimated intrinsic growth rate (\u03b3), we estimated R0 by using the serial intervals (SI) of two other well-known coronavirus diseases, MERS and SARS, as approximations for the true unknown SI. The early outbreak data largely follows the exponential growth. We estimated that the mean R0 ranges from 2.24 (95%CI: 1.96-2.55) to 3.58 (95%CI: 2.89-4.39) associated with 8-fold to 2-fold increase in the reporting rate. We demonstrated that changes in reporting rate substantially affect estimates of R0. The mean estimate of R0 for the 2019-nCoV ranges from 2.24 to 3.58, and significantly larger than 1. Our findings indicate the potential of 2019-nCoV to cause outbreaks.","version":"1.2","doi":"10.1101/2020.01.23.916395","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.29.924100","pub_date":"2020-1-29","title":"Potential inhibitors for 2019-nCoV coronavirus M protease from clinically approved medicines","abstract":"Starting from December 2019, a novel coronavirus, named 2019-nCoV, was found to cause Severe Acute Respiratory (SARI) symptoms and rapid pandemic in China. With the hope to identify candidate drugs for 2019-nCoV, we adopted a computational approach to screen for available commercial medicines which may function as inhibitors for the Mpro of 2019-nCoV. Up to 10 commercial medicines that may form hydrogen bounds to key residues within the binding pocket of 2019-nCoV Mpro were identified, which may have higher mutation tolerance than lopinavir/ritonavir and may also function as inhibitors for other coronaviruses with similar Mpro binding sites and pocket structures.","version":"1.1","doi":"10.1101/2020.01.29.924100","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.28.923011","pub_date":"2020-1-28","title":"Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody","abstract":"The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 800 laboratory-confirmed human infections, including 25 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. Therefore, CR3022 has the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g., m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, indicating that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.","version":"1.1","doi":"10.1101/2020.01.28.923011","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.27.921627","pub_date":"2020-1-28","title":"Nelfinavir was predicted to be a potential inhibitor of 2019-nCov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation","abstract":"2019-nCov has caused more than 80 deaths as of 27 January 2020 in China, and infection cases have been reported in more than 10 countries. However, there is no approved drug to treat the disease. 2019-nCov Mpro is a potential drug target to combat the virus. We built homology models based on SARS Mpro structures, and docked 1903 small molecule drugs to the models. Based on the docking score and the 3D similarity of the binding mode to the known Mpro ligands, 4 drugs were selected for binding free energy calculations. Both MM/GBSA and SIE methods voted for nelfinavir, with the binding free energy of \u221224.69\u00b10.52 kcal/mol and \u22129.42\u00b10.04 kcal/mol, respectively. Therefore, we suggested that nelfinavir might be a potential inhibitor against 2019-nCov Mpro.","version":"1.1","doi":"10.1101/2020.01.27.921627","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.28.923169","pub_date":"2020-1-28","title":"Beware of asymptomatic transmission: Study on 2019-nCoV prevention and control measures based on extended SEIR model","abstract":"The 2019 new coronavirus, \u201c2019-nCoV\u201d, was discovered from Wuhan Viral Pneumonia cases in December 2019, and was named by the World Health Organization on January 12, 2020. In the early stage, people knows little about the 2019-nCoV virus was not clear, and the spread period was encountering China\u2019s annual spring migration, which made the epidemic spread rapidly from Wuhan to almost all provinces in China. This study builds a SEIRD model that considers the movement of people across regions, revealing the effects of three measures on controlling the spread of the epidemic.Based on MATLAB R2017a, computational experiments were performed to simulate the epidemic prevention and control measures. The research results show that current prevention and control measures in China are very necessary. This study further validates the concerns of international and domestic experts regarding asymptomatic transmission (E-status). The results of this study are applicable to explore the impact of the implementation of relevant measures on the prevention and control of epidemic spread, and to identify key individuals that may exist during the spread of the epidemic.","version":"1.1","doi":"10.1101/2020.01.28.923169","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.24.919241","pub_date":"2020-1-28","title":"From SARS-CoV to Wuhan 2019-nCoV Outbreak: Similarity of Early Epidemic and Prediction of Future Trends","abstract":"This manuscript has been withdrawn as it was submitted without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.","version":"1.4","doi":"10.1101/2020.01.24.919241","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.26.920249","pub_date":"2020-1-27","title":"Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event","abstract":"A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus. Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods. Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat-SARS-like coronavirus sequences. Specifically, in the 5\u2019-part spanning the first 11,498 nucleotides and the last 3\u2019-part spanning 24,341-30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat-SARS-like coronavirus sequences, whereas in the middle region spanning the 3\u2019-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch. The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.","version":"1.1","doi":"10.1101/2020.01.26.920249","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.23.916726","pub_date":"2020-1-25","title":"Modelling the epidemic trend of the 2019 novel coronavirus outbreak in China","abstract":"We present a timely evaluation of the Chinese 2019-nCov epidemic in its initial phase, where 2019-nCov demonstrates comparable transmissibility but lower fatality rates than SARS and MERS. A quick diagnosis that leads to case isolation and integrated interventions will have a major impact on its future trend. Nevertheless, as China is facing its Spring Festival travel rush and the epidemic has spread beyond its borders, further investigation on its potential spatiotemporal transmission pattern and novel intervention strategies are warranted.","version":"1.1","doi":"10.1101/2020.01.23.916726","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.24.919159","pub_date":"2020-1-25","title":"2019-20 Wuhan coronavirus outbreak: Intense surveillance is vital for preventing sustained transmission in new locations","abstract":"The outbreak of pneumonia originating in Wuhan, China, has generated 830 confirmed cases, including 26 deaths, as of 24 January 2020. The virus (2019-nCoV) has spread elsewhere in China and to other countries, including South Korea, Thailand, Japan and USA. Fortunately, there has not yet been evidence of sustained human-to-human transmission outside of China. Here we assess the risk of sustained transmission whenever the coronavirus arrives in other countries. Data describing the times from symptom onset to hospitalisation for 47 patients infected in the current outbreak are used to generate an estimate for the probability that an imported case is followed by sustained human-to-human transmission. Under the assumptions that the imported case is representative of the patients in China, and that the 2019-nCoV is similarly transmissible to the SARS coronavirus, the probability that an imported case is followed by sustained human-to-human transmission is 0.37. However, if the mean time from symptom onset to hospitalisation can be halved by intense surveillance, then the probability that an imported case leads to sustained transmission is only 0.005. This emphasises the importance of current surveillance efforts in countries around the world, to ensure that the ongoing outbreak will not become a large global epidemic.","version":"1.1","doi":"10.1101/2020.01.24.919159","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.24.915157","pub_date":"2020-1-24","title":"The 2019-new coronavirus epidemic: evidence for virus evolution","abstract":"There is concern about a new coronavirus, the 2019-nCoV, as a global public health threat. In this article, we provide a preliminary evolutionary and molecular epidemiological analysis of this new virus. A phylogenetic tree has been built using the 15 available whole genome sequence of 2019-nCoV and 12 whole genome sequences highly similar sequences available in gene bank (5 from SARS, 2 from MERS and 5 from Bat SARS-like Coronavirus). FUBAR analysis shows that the Nucleocapsid and the Spike Glycoprotein has some sites under positive pressure while homology modelling helped to explain some molecular and structural differences between the viruses. The phylogenetic tree showed that 2019.nCoV significantly clustered with Bat SARS-like Coronavirus sequence isolated in 2015, whereas structural analysis revealed mutation in S and nucleocapsid proteins. From these results, 2019nCoV could be considered a coronavirus distinct from SARS virus, probably transmitted from bats or another host where mutations conferred upon it the ability to infect humans.","version":"1.1","doi":"10.1101/2020.01.24.915157","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.23.917351","pub_date":"2020-1-24","title":"Pattern of early human-to-human transmission of Wuhan 2019-nCoV","abstract":"On December 31, 2019, the World Health Organization was notified about a cluster of pneumonia of unknown aetiology in the city of Wuhan, China. Chinese authorities later identified a new coronavirus (2019-nCoV) as the causative agent of the outbreak. As of January 23, 2020, 655 cases have been confirmed in China and several other countries. Understanding the transmission characteristics and the potential for sustained human-to-human transmission of 2019-nCoV is critically important for coordinating current screening and containment strategies, and determining whether the outbreak constitutes a public health emergency of international concern (PHEIC). We performed stochastic simulations of early outbreak trajectories that are consistent with the epidemiological findings to date. We found the basic reproduction number, R0, to be around 2.2 (90% high density interval 1.4\u20143.8), indicating the potential for sustained human-to-human transmission. Transmission characteristics appear to be of a similar magnitude to severe acute respiratory syndrome-related coronavirus (SARS-CoV) and the 1918 pandemic influenza. These findings underline the importance of heightened screening, surveillance and control efforts, particularly at airports and other travel hubs, in order to prevent further international spread of 2019-nCoV.","version":"1.1","doi":"10.1101/2020.01.23.917351","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.22.914952","pub_date":"2020-1-23","title":"Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin","abstract":"Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans. Here we report the identification and characterization of a novel coronavirus (nCoV-2019) which caused an epidemic of acute respiratory syndrome in humans, in Wuhan, China. The epidemic, started from December 12th, 2019, has caused 198 laboratory confirmed infections with three fatal cases by January 20th, 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that nCoV-2019 is 96% identical at the whole genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The nCoV-2019 virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients. Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV.","version":"1.2","doi":"10.1101/2020.01.22.914952","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.22.915660","pub_date":"2020-1-22","title":"Functional assessment of cell entry and receptor usage for lineage B \u03b2-coronaviruses, including 2019-nCoV","abstract":"Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS- CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these novel viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent 2019-nCoV, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells and confirm that human ACE2 is the receptor for the recently emerging 2019-nCoV.","version":"1.1","doi":"10.1101/2020.01.22.915660","journal":"bioRxiv","score":null},{"id":"10.1101/2020.01.20.913368","pub_date":"2020-1-22","title":"Genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China","abstract":"Detailed genomic and structure-based analysis of a new coronavirus, namely 2019-nCoV, showed that the new virus is a new type of bat coronavirus and is genetically fairly distant from the human SARS coronavirus. Structure analysis of the spike (S) protein of this new virus showed that its S protein only binds weakly to the ACE2 receptor on human cells whereas the human SARS coronavirus exhibits strongly affinity to the ACE receptor. These findings suggest that the new virus does not readily transmit between humans and should theoretically not able to cause very serious human infection. These data are important to guide design of infection control policy and inform the public on the nature of threat imposed by 2019-nCov when results of direct laboratory tests on this virus are not expected to be available in the near future.","version":"1.2","doi":"10.1101/2020.01.20.913368","journal":"bioRxiv","score":null}]